PL221526B1 - Prodrugs of excitatory amino acids - Google Patents

Abstract

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

Description

The invention relates to a compound of formula I, a method for its preparation and its use in therapy, especially in the treatment of psychiatric and neurological disorders.

The subject compounds are synthetic prodrugs of excitatory amino acids.

State of the art

Treatment of neurological or psychiatric disorders, such as anxiety, involves the selective activation of metabotropic receptors for excitatory amino acids. For example, in US Patent No. US No. 5,688,826 (abbreviated '826), issued November 18, 1997 discloses that (+) - 4-amino-2-sulfonylbicyclo [3.1.0] hexane-4,6-dicarboxylic acid is an active agonist for the mGluR2 receptor. In addition, U.S. Patent No. US No. 5,958,960 (abbreviated '960), issued September 28, 1999, discloses that (+) - 2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid is an active agonist for the mGluR2 receptor.

The present invention provides prodrug forms of the mGluR2 receptor agonists which enhance the in vivo potency of the respective parent compounds and provide better oral exposure of the parent compound. The compounds of the present invention are the most advantageous in terms of maintaining safety and efficacy compared to previously disclosed mGluR2 receptor agonists with enhanced oral bioavailability.

Synthetic excitatory amino acid prodrugs and methods for their preparation are disclosed in International Application Nos. PCT / US01 / 45866 and PCT / US02 / 00488.

The essence of the invention

The invention relates to a compound of formula I

wherein A is H- (Q) p-; Q is selected from the group of amino acyls derived from amino acids selected from the group consisting of natural amino acids and unnatural amino acids selected from D-isomers of natural a-amino acids, p being an integer from 1 to 10;

X is SO ₂ ; R is hydrogen or fluoro; and R is hydrogen, fluoro or hydroxy; and which compound includes the following compounds;

(1R, 4S, 5S, 6S) -4- (2'S-aminopropionyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid;

(1R, 4S, 5S, 6S) -4- (2'S-2'-aminopropionyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid;

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'-hydroxypropionyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4.6 acid -dicarboxylic;

(1R, 4S, 5S, 6S) -4 - [(pyrrolidine-2'S-carbonyl) -amino] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6- acid dicarboxylic;

acid [1R, 4S, 5S, 6S) -4- (2'S-Amino-4'-methyl-sulfanylobutyloamino) -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1.0.] hexane-4,6- -dicarboxylic;

acid (1R, 4S, 5S, 6S) -4- [2'S-amino-3 '- (1H-indol-3-yl) -propionylamino] -2,2-dioxo-2X ⁶ -thia-bicyclo- [3.1. 0.] hexane-4,6-dicarboxylic;

acid (1R, 4S, 5S, 6S) -4- [2'S-amino-3 '- (4-hydroxyphenyl) propionylamino] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane- 4,6-dicarboxylic;

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'-phenylpropionylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid ;

GB 221 526 B1 (1R, 4S, 5S, 6S) -4- (2'S-amino-3'S-metylopentanonyloamino) -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1.0.] - hexan-4 6-dicarboxylic;

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'-methylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid ;

(1R, 4S, 5S, 6S) -4- (2'S-amino-4'-methylpentanoylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid ;

(1R, 4S, 5S, 6S) -4- (2'S, 6'-diaminohexanoylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid;

(1R, 4S, 5S, 6S) -4- (2'S-amino-4'-carbamoylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid , and pharmaceutically acceptable salts thereof.

A particularly preferred pharmaceutically acceptable salt is the hydrochloride of (1R, 4S, 5S, 6S) -4- (2'S-aminopropionyl) amino] -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1.0.] Hexane-4, 6-dicarboxylic acid, or (1R, 4S, 5S, 6S) -4- (2'S-2'-aminopropionyl) amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] Hexane-4 , 6-dicarboxylic acid.

Another aspect of the present invention is a process for preparing a compound of Formula I or a pharmaceutically acceptable salt thereof, comprising acylating a compound of Formula (ii):

using the appropriate aminoacyl of formula III Pg ^N -A- (III), wherein A is H- (Q) p-; Q is selected from the group of amino acyls derived from amino acids selected from the group consisting of natural amino acids and unnatural amino acids selected from D-isomers of natural a-amino acids, p being an integer from 1 to 10;

P _g N is a nitrogen protecting group such as, for example, tert-butoxycarbonyl; then, when the functional group is protected with a protecting group, the protecting group is removed; then, when a pharmaceutically acceptable salt of a compound of formula I is required, the base form of such compound of formula I is reacted with an acid to form a pharmaceutically acceptable counterion; or for a compound of formula I that contains an acidic group, the acidic form of such compound of formula I is reacted with a base which produces a pharmaceutically acceptable cation; or for a zwitterionic compound of formula I, neutralizing the acid addition salt form or the base addition salt form of said compound of formula I; or using any other conventional procedure.

The invention also relates to the use of a compound according to claim 1. 1-2, for the manufacture of a medicament for administering an effective amount of a compound of Formula II,

wherein X and R are as defined above in the treatment of a neurological disorder in a patient. The subject compound of formula I finds use in therapy, particularly in the treatment of neurological and psychiatric disorders in a patient.

PL 221 526 B1

Neurological disorders are selected from the group consisting of cerebral deficiencies following coronary bypass implantation of the heart and transplant; cerebral ischemia; spinal cord injury; head trauma; Alzheimer's disease; Huntington's Chorea; amyotrophic lateral sclerosis; AIDS-induced dementia; perinatal hypoxia; neuronal damage from hypoglycemia; eyeball damage and retinopathy; cognitive impairment; idiopathic and drug-induced disorders Parkinson's disease; muscle cramps; migraine headaches; urinary incontinence; tolerance, withdrawal, discontinuation and drug dependence; nicotine withdrawal; vomiting; swelling of the brain; chronic pain; sleep disturbance; seizures; Tourette's syndrome; attention deficit disorder; and tardive dyskinesia.

Particularly preferably, the subject compound finds use as a medicament for the treatment of a neurological disorder selected from the group consisting of tolerance, withdrawal, interruption and drug dependence; or nicotine withdrawal.

The subject compound finds use as a medicament for the treatment of psychiatric disorders which are selected from the group consisting of schizophrenia, anxiety and related disorders, depression, bipolar disorders, psychosis and obsessions, and compulsions.

Particularly preferably, the subject compound is used as a medicament for the treatment of psychiatric disorders selected from the group consisting of anxiety and related disorders.

Particularly preferably, the subject compound is used for the treatment of psychiatric disorders where the psychiatric disorder is schizophrenia.

Most preferably, the compound of formula (I) is used as a medicament for the treatment of schizophrenia.

The pages of the graphs show the pharmacological activity of the compounds in question.

Appendix 1 shows the pharmacological profiles of action of the monohydrate of the compound of formula (I) (LY2140023) and the parent compound, ie the compound of formula II (LY404039), when dosed orally. The graphs show that the plasma concentration of the compound of formula II (LY404039) following an oral dose of the monohydrate of the compound of formula (I) (LY2140023) is significantly higher (left graph) than the plasma concentration of the compound of formula II (LY404039) ) after an oral dose of a compound of formula II, at the same time.

Appendix 2 provides plots of the plasma concentration of the compound of formula (II) (LY404039) versus the administered dose of both compounds (monohydrate and parent compound). The graphs show that the AUC concentration in the plasma of the compound of formula (II) (LY404039) after administration of the monohydrate prodrug of the compound of formula (I) (LY2140023) is 10-fold higher than the concentration of the compound of formula (II) (LY404039) after administration. direct administration of a compound of formula (II).

Appendix 3 provides graphs showing that the C max of the compound of formula (II) (LY404039) in plasma is 17-fold greater after administration of the monohydrate prodrug (LY2140023) than after direct administration of the compound of formula (II).

These graphs show that the effects obtained after administration of the monohydrate of the compound of formula (I) to form the parent compound (formula II) in vivo are significantly better than the direct administration of the parent compound (formula II).

Detailed Description of the Invention

It has been found that the compounds of the invention are useful prodrugs of compounds which exhibit metabotropic glutamate receptor agonist activity and are therefore useful for the treatment of central nervous system diseases such as neurological diseases, e.g. neurological degenerations, and as antipsychotic, anxiolytic, anti-anxiety agents. Relieve drug withdrawal symptoms, with antidepressants, anticonvulsants, analgesics and antiemetics.

In the compound of formula (I), p is preferably from to 3, R ³ and R ⁴ are preferably hydrogen, and A is preferably alanyl.

The amino acid group is preferably in the natural configuration, i.e. the L configuration with respect to the D-glycerol aldehyde.

The pharmaceutically acceptable salts of the compound of formula I are within the scope of the present invention. These salts may be present in combination with the acid or base moieties of the molecule and may take the form of an acid, primary, secondary, tertiary or quaternary ammonium, alkali metal or alkaline earth metal addition salt. In general, acid addition salts can be obtained by reacting an acid with a compound of Formula I. Alternatively, acid addition salts can be prepared by reacting the penultimate compound (protected intermediate) with appropriate acid equivalents to form

The corresponding salt, which in turn can be converted into a compound of formula I or another salt. The alkali metal and alkaline earth metal salts are generally prepared by reacting the hydroxide of the desired metal with a compound of Formula I.

Certain specific salts give the formulation certain advantages due to their crystalline form. The non-crystalline amorphous forms of the compounds can be hygroscopic. Crystalline forms of pharmaceutical compounds are sometimes more desirable because they exhibit favorable solid state properties.

Acids commonly used in the preparation of such salts include inorganic acids, e.g., hydrochloric, hydrobromic, nitric, sulfuric or phosphoric acid, or organic acids, such as organic carboxylic acids, e.g., glycolic, maleic, hydroxymaleic, fumaric, malic, tartaric, citric, salicylic, o-acetoxybenzoic or organic sulfonic acids including 2-hydroxyethanesulfonic acid, toluene-p-sulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid or ethanesulfonic acid.

It has been shown that disorders of many physiological functions are caused by excessive or inappropriate stimulation of excitatory amino acid conduction. Compounds of Formula 1 of the present invention are believed to have the ability to treat a variety of mammalian neurological disorders associated with the condition, including acute neurological disorders such as cerebral deficiencies following coronary bypass surgery and graft, stroke, ischemia. brain, spinal cord injury, head trauma, perinatal hypoxia, cardiac arrest and neuronal damage from hypoglycemia. Compounds of Formula I are believed to have the ability to treat a variety of chronic neurological disorders such as Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, AIDS-induced dementia, eyeball injury and retinopathy, cognitive and idiopathic disorders, and drug-induced parkinsonian disorders. . The present invention also relates to methods of treating these disorders which include administering to a patient in need thereof an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

The compounds of formula I of the present invention are active against many other neurological disorders associated with glutamate abnormalities including muscle spasms, seizures, migraine headaches, urinary incontinence, pain, premenstrual syndrome (PMS), psychosis (such as schizophrenia), tolerance , withdrawal, discontinuation and addiction to drugs (such as nicotine, opiates, cocaine, benzodiazepines and ethanol), anxiety and related disorders, vomiting, brain edema, chronic pain and tardive dyskinesia. The compounds of formula I are also useful as antidepressants and analgesics. Thus, the present invention also relates to methods of treating these disorders which include administering to a patient in need thereof an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

The following definitions are provided to clarify the meaning and scope of various terms used in this specification. General terms used in this specification have their usual meanings.

The term "acting on" in connection with a compound of Formula II includes agonist activity at the excitatory amino acid receptor. The term "excitatory amino acid receptor" refers to a metabotropic glutamate receptor that binds to cellular effectors by binding to a GTP protein. The term "cAMP-associated metabotropic glutamate receptor" refers to a metabotropic receptor that is coupled to inhibition of adenylate cyclase activity.

The term "neurological disorder" refers to both acute and chronic states of neurological degeneration, including cerebral deficiencies following coronary bypass surgery and transplant, cerebral ischemia (e.g., cardiac arrest stroke), spinal cord injury spinal cord injury, head trauma, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, AIDS-induced dementia, perinatal hypoxia, hypoglycemic neuronal damage, eye injury and retinopathy, cognitive, idiopathic and drug-induced disorders, and Parkinson's disease. This term also includes other neurological conditions caused by glutamate abnormalities including muscle spasms, migraine headaches, urinary incontinence, tolerance, withdrawal, discontinuation and drug dependence (i.e. opiates, benzodiazepines, nicotine, cocaine or ethanol), nicotine withdrawal, vomiting, brain edema, chronic pain, sleep disturbance, seizures, Tourette's syndrome, attention deficit disorder and tardive dyskinesia.

The term "psychiatric disorder" refers to both acute and chronic psychiatric conditions, including schizophrenia, anxiety and related disorders (e.g., panic attacks and stress-induced cardiovascular disorders), depression, bipolar disorder,

Psychosis, obsessions and compulsions, generalized anxiety states, sudden stress disorders and panic states.

The term "effective amount" as used herein refers to the amount or dose of a compound that, upon single or multiple administration to a patient, provides the desired diagnostic or therapeutic effect.

The effective amount can be easily determined by the attending physician skilled in the art by employing known techniques and analyzing the results obtained in similar circumstances. In determining the effective amount or dose of a compound to be administered, many factors should be taken into account by the attending physician, including, but not limited to: the species of mammal; its size, age and general health; the specific disease to be treated; the severity or severity of the disease being treated; the patient's individual response; the specific compound administered; the manner of its administration; the bioavailability of the administered preparation; selected dosing regimen; other medications used in parallel; and other relevant circumstances. For example, a typical daily dose may contain from about 5 mg to about 300 mg of the active ingredient. The compounds can be administered by a variety of methods including orally, rectally, transdermally, subcutaneously, intravenously, intramuscularly, buccally, or intranasally. The compound may alternatively be administered by continuous infusion.

The term "patient" as used herein refers to a mammal such as a mouse, guinea pig, rat, dog or human. It is understood that preferably the patient is a human being.

The term "treating" (or "treating") as used herein includes the generally used meaning which includes preventing, reducing, and slowing, arresting, or reversing the progression of a disease. As such, the methods of the invention encompass administration for both therapeutic and prophylactic purposes.

General chemical terms used herein have their usual meanings. For example, the term "(1-6C) alkyl" means a straight or branched group. Examples of the meanings for (1-6C) alkyl include (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) alkynes" includes (2-4C) alkynyl such as propynyl.

The term "nitrogen-protecting group", as used herein, refers to those groups which protect or block a nitrogen atom against undesirable reactions during synthesis. The choice of the appropriate nitrogen protecting group to be used will depend on the conditions employed in the subsequent reaction steps where protection is required as well as on the knowledge of those skilled in the art. Nitrogen protecting groups described by T. W. Greene and P. G. M. Wuts, in "Protective Groups in Organic Synthesis", 3rd edition (John Wiley & Sons, New York (1999)), are commonly used. A preferred nitrogen protecting group is tert-butoxycarbonyl.

The term "carboxy protecting group" as used herein, designated by "PgN", refers to carboxylic acid ester derivatives commonly used to block or protect a carboxyl group while carrying out reactions on other functional groups on the compound. Particular examples include, for example, methyl, ethyl, tert-butyl, benzyl, methoxymethyl, trimethylsilyl and the like. Further examples of such groups can be found in T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis" 3rd edition (John Wiley & Sons, New York (1999)). Preferred carboxyl protecting groups are methyl and ethyl. The ester is degraded using standard procedures that do not affect other parts of the molecule.

The term "hydroxyl protecting group" refers to groups known to those skilled in the art of organic chemistry of the types described in Chapter 2 of Greene and Wuts. Representative hydroxyl protecting groups include, for example, ether groups, ethyl substituted ether groups, isopropyl ether groups, phenyl and substituted phenyl ether groups, benzyl and substituted benzyl ether groups, alkylsilyl ether groups, ester protecting groups etc. Type used the hydroxyl protecting group is not critical as long as the resulting derivative of the hydroxyl group is stable to the conditions of the subsequent reaction (s) occurring at other positions in the intermediate molecule and can be selectively removed at the appropriate point without damaging the remainder of the molecule including any other protecting group hydroxy (hydroxyl protecting groups).

The term "aminoacyl" refers to an aminoacyl 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. The term "neutral amino acid" refers to an uncharged containing amino acid

Side chain substituents. Exemplary neutral amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, glutamine, and asparagine. The term "positive amino acid" refers to an amino acid whose side chain substituents are positively charged at physiological pH. Exemplary positive amino acids include lysine, arginine, and histidine. The term "negative amino acid" refers to an amino acid whose side chain substituents are negatively charged at physiological pH. Exemplary amine negative acids include aspartic acid and glutamic acid. Preferred amino acids include α-amino acids. Most preferred amino acids include α-amino acids with L-stereochemistry at the α-carbon. Exemplary natural α-amino acids include valine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine , histidine, aspartic acid, and glutamic acid.

The term "unnatural amino acid" refers to an amino acid for which there is no codon in the nucleic acid. Examples of unnatural amino acids include, eg, D-isomers of the natural α-amino acids indicated above; Aib (aminobutyric acid), 3Aib (3-aminoisobutyric acid), Nva (norvaline), β-Ala, Aad (2-aminoadipic acid), 3Aad (3-aminoadipic acid), Abu (2-aminobutyric acid), Gaba (γ-aminobutyric acid), Acp (6-aminobutyric acid), Dbu (2,4-di-aminobutyric acid), a-aminopimelic acid, TMSA (trimethylsilyl-Ala), alle (allo-isoleucine), Nle (norleucine) , tert-Leu, Cit (citrulline), Om, Dpm (2,2'-diaminopimelic acid), Dpr (2,3-diaminopropionic acid), a- or β-Nal, Cha (cyclohexyl-Ala), hydroxyproline, Sar (sarcosine), O-methyltyrosine, phenylglycine and the like; cyclic amino acids; ^{N α} -alkylated amino acids, wherein the N ^α -alkylated amino acid includes N ^α - (1-10C) alkyl ^{amino acid such as MeGly (N α} -methylglycine), EtGly (N ^α -ethylglycine) and EtAsn (N ^α -ethyl aspartate) and amino acids with two substituents at carbon a. Examples of unnatural α-amino acids include D-alanine, D-leucine, and phenylglycine. The names of natural and unnatural amino acids and their residues used in this description meet the requirements of the nomenclature convention suggested by IUPAC-1UB Joint Commission on Biochemical Nomenclature (JCBN) presented in the publication "Nomenclature and Symbolism for Amino Acids and Peptides (Recommendations, 1983)" European Journal of Biochemistry, 138,9-37 (1984). The extent to which the amino acid names and abbreviations of their residues used in this specification and the appended claims differ from those indicated is indicated, where appropriate, hereinafter.

While all compounds of Formula I are useful as active agonists of the mGluR2 receptor, some compounds are especially preferred. The preferred classes are defined below.

A) Q is glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl, phenylalanyl, tyrosyl, tryptophyl, methionyl, lysyl or serinyl.

B) Q is alanyl.

C) Q is methionyl.

D) p is the number 1.

E) p is the number 2.

F) X is SO ₂ .

G) X is CR ³ R ⁴ .

H) is fluoro and R ⁴ is hydrogen.

L) R ³ is hydroxy and R ⁴ is hydrogen.

J) R ³ and R ⁴ together are = O.

K) R ¹⁰ is hydrogen.

L) R is fluoro.

M) R ¹¹ is hydrogen.

N) Relationship is a free rule.

O) The compound is in the form of a salt.

P) The compound exists in the form of the hydrochloride.

Q) The compound is in the form of a mesylate salt.

R) The compound is in the form of the esylate salt.

S) The compound is in the form of a tosylate salt.

The listed features may be combined to define additional preferred classes of compounds.

The compounds of the present invention may be prepared using a variety of methods, some of which are illustrated in the Schemes below. The sequence of individual steps required to obtain the compounds of Formula I depends on the particular compound, compound, being synthesized

The starting material and the relative lability of the substituted groups. Some substituents have been omitted in the following schemes for the sake of clarity, but this does not limit the applicability of the schemes in any way. It will be appreciated by those skilled in the art that R ¹⁵ and R ¹⁶ represent a side chain suitable for making the desired aminoacyl.

If not commercially available, necessary starting materials in the following schemes can be obtained by methods including conventional techniques of organic and heterocyclic chemistry, techniques of analogous syntheses, known structurally similar compounds, and methods described in the syntheses and examples, including novel methods.

Compounds of formula I are converted by an in vivo enzymatic or hydrolytic process to give compounds of formula II as shown above in Scheme 1. In particular, the crystalline form of a compound of formula I can be prepared using the pathway shown below in Scheme 2.

PL 221 526 B1

Hydrolysis of the protected di-ester peptide compound of formula (iii) with a suitable base such as lithium hydroxide or sodium hydroxide in a suitable solvent such as THF or THF / water gives the protected di-acid peptide compound of formula (iv ). A compound of formula (iv) can be deprotected using the appropriate acid in a suitable solvent. These conditions allow the formation of the corresponding acid salt of the diacid peptide compound, shown as the salt in Formula I, as an amorphous solid or, directly, a crystalline solid in which X is the corresponding anion. In the case of an amorphous solid, crystallization can then be carried out from suitable solvents. Carboxylate salts can be formed by introducing cationic molecules using a reagent such as sodium acetate.

A zwitterionic compound can be obtained by treating a crystalline salt of the compound using an appropriate base.

For example, the protected di-acid peptide compound of formula (iv) is converted into the deprotected hydrochloride as an amorphous solid after treatment of the acid with hydrogen chloride gas in a suitable solvent. The amorphous hydrochloride can then be crystallized from acetone and water to yield the crystalline hydrochloride salt. In the case of a crystalline solid that forms directly, filtration of the reaction mixture yields a crystalline salt. The zwitterionic compound was obtained by treating crystalline hydrochloride with sodium hydroxide; alternatively, treatment of the compound's mesylate salt or the compound's tosylate salt with sodium hydroxide also provides a zwitterionic compound. It will be appreciated by those skilled in the art that the compound of formula I can be prepared using a procedure in which the intermediates indicated are not isolated.

The di-ester of formula (ii) is acylated with a compound of formula III using a suitable coupling agent to provide the protected di-ester peptide compound of formula (iii). Alternatively, this transformation can be performed using an acid chloride of a compound of formula III.

Suitable peptide coupling reagents 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) phosphine chloride and benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate.

PL 221 526 B1

Scheme 4

In Scheme 4 above, a compound of formula II, a diacid, is treated with a suitable carboxyl protecting agent such as a catalytic amount of hydrochloric acid or thionyl chloride and methanol or ethanol to provide the corresponding di-ester of formula (ii). Alternatively, a compound of formula II may first be treated with a nitrogen protecting agent such as BOC ₂ O to provide a nitrogen protected compound of formula (I). Thereafter, a compound of formula (I) may be treated with a carboxyl protecting agent such as methyl iodide in the presence of a base such as potassium carbonate followed by a nitrogen deprotecting agent such as hydrochloric acid or trifluoroacetic acid to provide a compound of the formula (ii).

Moreover, those skilled in the art will appreciate that depending on X, an appropriate safener may be necessary. For example, if X is CR ³ R ⁴ , R ³ is hydroxy, and R ⁴ is hydrogen, then those skilled in the art will appreciate that it may be necessary to use a suitable hydroxyl protecting group prior to initiating any of the above schemes.

Compounds of formula II are known in the art. For example, formulations of these compounds are disclosed in US Pat. US Nos. 5,688,826 ('826) and 5,958,960 (' 960 for short).

Various improvements to the synthetic pathways for compounds of Formula II have been made in the methods previously disclosed. The improvements include sulfur and alcohol oxidation as well as the separation of various intermediates using optical properties as described below.

The first improvement relates to the conversion disclosed in the '826 Patent at column 8 lines 22-34 and at column 7 starting at column 33 (Formula V) and includes the oxidation of the compound of Formula VII of the' 826 Patent

Formula (VII) of the '826 Patent to provide the compound of Formula V of the' 826 Patent

PL 221 526 B1

Formula (V) according to the '826 patent

Sulfur trioxide-pyridine complex or trifluoroacetic anhydride in combination with DMSO has been found to be advantageous in many oxidation methods conducted in the art.

Second, with regard to the isolation of the compound of Formula III according to the '826 Patent

Formula (III) of the '826 Patent ₂ wherein R is carboxyl as described in column 8, lines 3-7, and column 6, starting with line 1 (Formula, (R) -a-methylbenzylamine has been found to be preferred and quinine. (R) -a-methylbenzylamine is particularly preferred.

Further, it was found that upon oxidation of the sulfide of Formula III of the '826 Patent where X is sulfur to give a compound of Formula III of the' 826 Patent where X is sulfonyl as described in the '826 Patent at column 8, lines 39-53, it is preferable to use a basic aqueous system and hydrogen peroxide in combination with a catalyst.

The following examples illustrate the compounds of the present invention and methods of synthesizing them. The examples are not intended to limit the scope of the present invention in any way and should not be construed as so. All experiments were carried out under an atmosphere of dry nitrogen or argon. Unless otherwise indicated, all solvents and reagents were purchased from commercially known sources and used as received. Dry tetrahydrofuran (THF) can be obtained by distillation from sodium or sodium benzophenone ketyl before use. ₁ spectra were proton nuclear magnetic resonance (H NMR) were obtained using a Bruker Avance device II bay-500 at 500 MHz, a Bruker Avance I bay-200 at 200 MHz or a Varian Inova / Varian 300 / Varian 400 at 500 MHz. Electrospray mass spectroscopy (ESI) was performed on an Agilent MSD / B instrument using acetonitrile / aqueous ammonium acetate as the mobile phase. Free Atom Bombardment Mass Spectroscopy (FABMS) is performed using the VG ZAB-2SE apparatus. Field Desorption Mass Spectroscopy (FDMS) is performed using either a VG 70SE instrument or a Varian MAT 731 instrument. Optical rotation was measured using a Perkin-Elmer 241 polarimeter. Chromatographic separations were performed on a Waters Prep 500 LC instrument, generally using a linear solvent gradient of the indicated solvents in description. The reactions are generally monitored for completion point using thin layer chromatography (TLC). Thin layer chromatography is performed using E. Merck Kieselgel 60 F254 plates, 5 cm x 10 cm, 0.25 mm thick. Spots are observed using a combination of UV and chemical development (plates are immersed in a solution of cerium ammonium molybdate [75 g ammonium molybdate and 4 g cerium (IV) sulfate in 500 ml 10% aqueous sulfuric acid] and then heated on a hot plate) . Flash chromatography is performed as described by Still, et al. Still, Kahn, and Mitra, J. Org. Chem. 43, 2923 (1978). Elemental analyzes for carbon, hydrogen and nitrogen content were performed on a Control Equipment Corporation 440 Elemental Analyzer or were performed at the Universidad Complutense Analytical Center (Facultad de Farmacia, Madrid, Spain). Melting points were determined in open glass capillaries using a Gallenkamp apparatus, a hot air bath, or a melting point apparatus and the values were uncorrected.

PL 221 526 B1

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

Ac = acetyl

Anal. = elemental analysis

ATR = Attenuated Global Internal Reflection

Bn or Bzl = benzyl

Bu = butyl

BOC = t-butoxycarbonyl calcd = calculated

D ₂ O = deuterium oxide

DCC = dicyclohexylcarbodiimide

DCM = 1,2-dichloromethane

DIBAL-H = diisobutylaluminum hydride

DMAP = 4-dimethylaminopyridine

DMF = dimethylformamide

DMSO = dimethyl sulfoxide

DSC = Differential Scanning Calorimetry

EDC = N-ethyl-N'N'-dimethylaminopropylcarbodiimide hydrochloride

ES = Electrospray

Et = ethyl

EtOH = ethanol

FAB = Fast Atom Bombardment (Mass Spectroscopy)

FDMS = Field Desorption Mass Spectrum

FTIR = Fourier transform of infrared spectrometry

HOAt = 1-hydroxyl-7-azabenzotriazole

HOBt = 1-hydroxybenzotriazole

HPLC = High Pressure 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 centrifugal thin layer chromatography

Ph = phenyl

after. = oral administration i-Pr = isopropyl

Rochelle salt = sodium potassium tartrate rt = room temperature

SM = starting material

IBS = tert-butyldimethylsilyl

TEA = triethylamine

Temp. = temperature

TFA = trifluoroacetic acid

THF = tetrahydrofuran

TLC = thin layer chromatography t-BOC = tert-butoxycarbonyl

General procedure A

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

The starting amino-dialkyl ester (compound of formula II, Scheme 3) (1.0 eq.) Is suspended in dry dichloromethane under nitrogen atmosphere. The appropriate N-Boc- (L) -amino acid (1.5-2.0 eq.), EDC (1.5-2.0 eq.), HOBt (1.5-2.0 eq.) Are added sequentially.

PL 221 526 B1 and dimethylaminopyridine (DMAP, 0.1-0.2 eq.). The reaction mixture is stirred at room temperature until the reaction is complete as judged by TLC unless otherwise indicated. The reaction mixture is diluted with ethyl acetate and washed successively with saturated aqueous NaHCO ₃ solution and / or aqueous NaHSO ₄ solution and brine. After drying over sodium sulfate and concentration in vacuo, the crude residue (compound of formula iii) is purified by silica gel chromatography using an appropriate eluent (typically ethyl acetate / hexanes).

General procedure B

Coupling of amine anhydrides and N-BOC- (L) -amino acid isobutyl

To a solution of the appropriate N-Boc- (L) -amino acid (1.5 eq.) In dry dichloromethane (10 ml) at -20 ° C under nitrogen is added N-methylmorpholine (NMM, 1.5 eq, in 1 ml CH ₂ Cl ₂ ), then isobutyl chloroformate (IBCF, 1.5 eq, in 5 ml CH 2 Cl 2) is added dropwise at such a rate that the internal temperature of the reaction mixture does not exceed -15 ° C. The resulting reaction mixture is stirred at -20 ° C for 30 minutes, then a solution of (1S, 2S, 4S, 5R, 6R) -2- (2'-aminopropionylamino) -4-hydroxybicyclo [3.1] acid diethyl ester hydrochloride is added. .0] hexane-2,6-dicarboxylic acid (1.0 eq.) In dichloromethane (10 mL) at -20 ° C, at a rate such that the internal temperature of the reaction mixture does not exceed -15 ° C. After the addition is complete, the cooling bath is removed and the reaction mixture is stirred at room temperature until complete as judged by TLC. The reaction mixture is diluted with ethyl acetate and washed sequentially with saturated aqueous NaHCO3, aqueous NaHSO4, and brine. After drying over magnesium sulfate and concentration in vacuo, the crude residue is purified by silica gel chromatography using an appropriate eluent (typically hexanes / ethyl acetate).

General procedure C

N-Boc and ester protecting group removal sequence

The appropriate N-Boc peptide diester derivative (compound of formula III, Scheme 2) (1.0 eq.) Is stirred in a mixture of THF / 2.5N LiOH, 1: 1 (10-20 eq.) At room temperature for up to 4 eq. hours. The reaction mixture is diluted with H ₂ O and washed with ethyl acetate. The organic layer is discarded. The pH of the aqueous phase is adjusted with a 1 N HCl solution (if necessary, NaCl is added to the aqueous phase to increase the extraction capacity) and the product, N-boc dicarboxylic acid (compound of formula iv) is extracted thoroughly with ethyl acetate. Combine all organics, wash with brine, dry over MgSO4, and concentrate to dryness in vacuo to afford the desired carboxylate product as a foamed solid. The product is dissolved in ethyl acetate and cooled to 0 ° C. The reaction mixture is purged with anhydrous HCl gas until saturated with HCl. The resulting reaction mixture is stirred at 0 ° C for up to 4 hours. The fully deprotected peptide derivative (compound of Formula I) is isolated as its hydrochloride salt by filtration under nitrogen atmosphere or by concentration of the reaction mixture to dryness followed by trituration with ethyl acetate or Et ₂ O and concentration to a white powder. Optionally, to remove residual solvent and excess HCl, the products are taken up in H ₂ O, frozen, and then lyophilized to give the hydrochloride salt of the desired products.

Synthesis 1

(1R, 4S, 5S, 6S) -4-Amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester

For a suspension of (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (10 g, mmol, disclosed in the specification in US Patent No. 5,688,826) in 100 mL of 2B ethanol at room temperature was added dropwise thionyl chloride (15.5 mL, 212.6 mmol) over 20 minutes, followed by a rinse with 40 mL of ethanol. The slurry was heated to reflux and stirred overnight. Analysis by 500 MHz ¹ H NMR (CD 3 OD) of the concentrate

The samples showed complete consumption of the starting material and the monoester intermediate. The resulting solution was allowed to cool to room temperature, then concentrated to a gelatinous residue. EtOAc (50 mL) was added to the jelly, then the mixture was concentrated to a solid and then diluted with an additional 94 mL of EtOAc. A 15% aqueous sodium carbonate solution (70 ml) was slowly added to the mixture with hand stirring to obtain gradual dissolution, resulting in a final pH of 7.95. The resulting sodium carbonate precipitate was filtered before extraction. The aqueous layer was extracted again with EtOAc (2 x 100 mL). The combined organic extracts were washed with brine (1 x 100 mL), dried (MgSO), filtered and concentrated in vacuo to give a faint yellow oil that 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 where it dissolved. After stirring for 15 minutes at 56 ° C, heptane (1 mL) was added dropwise to the solution, the heating was turned off. The solution was allowed to cool to 52 ° C whereupon precipitation occurred. Upon cooling and additional dilution with heptane (600 µL), a suspension formed. The resulting suspension was stirred at room temperature for 1 hour before being filtered, washed with heptane (2 x 500 µL) and dried at 45 ° C overnight to provide 145 mg (73% recovery) of the title compound as a white solid. Melting point 80- 83 ° C.

[a] ²⁵ D -57.7 ° (c 1.04, CH 3 OH).

500 MHz ¹ H NMR (CD3CI3) δ 4.31 (q, 2H, J = 7.0 Hz), 4.20 (m, 2H), 3.78 (d, 1H, J = 15.0 Hz), 3.36 (dd, 1H, J = 4.0, 7.0Hz), 2.93 (dd, 1H, J = 4.0, 7.0Hz), 2.81 (d, 1H, J = 15.0 Hz), 2.46 (t, 1H, J = 4.0), 1.34 (t, 3H, J = 7.0), 1.30 (t, 3H, J = 7.0) .

¹³ C NMR (125 MHz, CDaCla) δ 171.68, 168.57, 63.26, 62.42, 59.96, 56.06, 43.78, 32.25, 22.49, 14.31, 14.25.

FTIR (ATR) 3364.15 (s), 1725.95 (s), 1304.91 (s), 1259.24 (s), 1200.84 (s), 1104.91 (s), 1022.99 ( s), 896.45 (s), 851.21 (s) cm ^-1 .

Analysis calculated for C11H17NO6S: C-45.35; H, 5.88; N, 4.81. Found C, 45.02; H, 5.75; N, 4.82. Synthesis 2

Acid diethyl ester, (1R, 4S, 5S, 6S) -4- (2'S-tert-Butoxycarbonylamino-propionylamino) -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1.0] hexane-4,6-dicarboxylic acid

To a solution of N-Boc-L-alanine (43.52 g, 230 mmol) and N-methylmorpholine (25.5 ml, 232 mmol) in 457 ml of methylene chloride at -30 ° C under nitrogen, isobutyl chloroformate ( 30.4 ml, 234 mmol) for 10 minutes. The resulting weak suspension was stirred at -25 to -30 ° C for 30 minutes, then a solution of (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2X ⁶ -thia-bicyclo acid diethyl ester was added. [3.1.0] hexane-4,6-dicarboxylic acid (63.90 g, 219 mmol, Synthesis 1) in 213 ml of methylene chloride over 25 minutes so that the temperature of the reaction mixture did not exceed -25 ° C. After the addition was complete, the cooling bath was removed and allowed to stir at ambient temperature for 60 minutes after which the temperature of the reaction mixture reached 19 ° C and this color turned faintly orange. The reaction mixture was treated with 350 mL of 1 N HCl and the layers were separated. The organic layer was washed with saturated aqueous NaHCO3 (1 x 350 mL) and brine (1 x 350 mL), dried (Na2SO4), filtered, and concentrated in vacuo to a white foam (105.2 g, 104%).

¹ H NMR (300 MHz, CDCl 3) δ: 7.62 (brs, 1H), 4.90 (brd, 1H, J = 7.1 Hz), 4.34-4.10 (m, 6H), 3 , 39 (ddd, 1H, J = 7.2, 3.9, 1.0 Hz), 3.00 (dd, 1H, J = 7.1, 3.9 Hz), 2.90 (brd, 1H , J = 14.9 Hz), 2.43 (t, 1H,

J = 4.1Hz), 1.46 (s, 9H), 1.31 (m, 9H).

GB 221 526 B1 ¹³ C NMR (75 MHz, CDCl3) δ: 173.0, 168.6, 167.6, 80.9, 76.5, 63.3, 62.3, 59.9, 55.7 , 42.8, 31.5, 28.2, 22.7, 16.6, 14.0, 13.9. MS (ES) m / z 461.0 [MH] ^- .

Synthesis 3

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylaminopropionylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid

To a solution of (1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylaminopropionylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester ( 181.4 g, 392 mmol theoretical, Synthesis 2) in 292 ml of THF at room temperature, 490 ml (980 mmol) of a 2N sodium hydroxide solution were added. The biphasic mixture was vigorously stirred at room temperature for 1.25 hours after which time the reaction mixture became homogeneous. The mixture was diluted with 490 mL of ethyl acetate and the layers were 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 re-extracted using 245 mL of ethyl acetate. The combined organic layers were dried (Na ₂ SO ₄ ), filtered and concentrated to yield 167.9 g (105%) of the title compound as a white foam. This material was used without identification in Examples 1 and 2.

Synthesis 4

(1R, 4S, 5S, 6S) -4-Amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid dimethyl ester

For a vigorously stirred suspension of (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (10.0 g, 42.5 mmol, US Patent No. 5,688,826) in MeOH (170 ml, 5 ° C) was added dropwise thionyl chloride (6.2 ml, 85.0 mmol). After all was added, the reaction mixture was allowed to warm slowly to room temperature, then heated to reflux for 48 hours. The volatiles were removed under reduced pressure and the residue was partitioned between saturated NaHCO 3 solution (200 ml) and ethyl acetate (400 ml). The layers were separated and the aqueous layer was extracted with ethyl acetate (2 x 400 ml each). The combined organic layers were dried over K ₂ CO ₃ and concentrated in vacuo to give 8.10 g (30.8 mmol) of the title compound, a yield of 72%.

[α] ²³ o = -84 ° (C = 0.5, MeOH).

Anal. calcd for C9H13NO6S: C, 41.06; H, 4.98; N, 5.32. Found: C, 40.94; H, 4.93; N, 5.30.

MS (ES) m / z 264.0 [M + H] +.

PL 221 526 B1

Synthesis 5

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'-phenylpropionylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4 acid dimethyl ester , 6-dicarboxylic acid

The process was carried out according to General Procedure A using commercially available N-BOC- (I) -phenylalanine and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2X ⁶ -thia-bicyclo acid dimethyl ester - [3.1.0] hexane-4,6-dicarboxylic acid (Synthesis 4). The reaction mixture was heated to melting point overnight. Purification was by PC-TLC, 4 mm SiO ₂ rotor, (10% ethyl acetate / hexanes to 100% ethyl acetate). 0.85 g of product (88%, 1.67 mmol) was obtained as a white foam.

[a] ²⁵ o = -35.2 ° (c = 0.45, CHCl3).

¹ H NMR (300 MHz, CDCl 3) δ 1.43 (9H, s), 2.38-2.40 (1H, m), 2.86 (1H, d, J = 15.0 Hz), 2, 91 (1H, dd, J = 4.4, 7.3Hz), 3.04 (2H, d, J = 7.3Hz), 3.35-3.39 (1H, m), 3.77 (3H, s), 3.84 (3H, s), 4.11 (1H, d, J = 14.3), 4.30 (1H, apparent q, J = 7.3), 4.96 (1H, broad d, J = 6.6Hz), 6.96 (1H, broads), 7.22-7.36 (5H, m).

Anal. Calcd for _C2 3H3 ₀ N ₂ OGS-0.1H ₂ O: C, 53.92; H, 5.94; N, 5.47. Found: C, 53.62; H, 5.90; N, 5.28.

MS (ES) m / z 509.16 [MH] ^- ; 411.2 [M-Boc] +.

Synthesis 6

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'S-methylpentanoylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4.6- acid dimethyl ester dicarboxylic acid

The process was carried out according to general procedure A using commercially available N-BOC- (L) -isoleucine and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2X ⁶ -thia-bicyclo acid dimethyl ester [3.1.0] -hexane-4,6-dicarboxylic acid (Synthesis 4). The reaction mixture was refluxed overnight. Purification was by PC-TLC, 4mm SiO2 rotor, (10% ethyl acetate / hexanes to 100% ethyl acetate). 0.75 g of product (83%, 1.57 mmol) was obtained as a white foam.

[a] ²³ o = -32.65 ° (c = 0.49, CHCl3).

¹ H NMR (300 MHz, CDCl 3) δ 0.91 (3H, t, J = 7.3 Hz), 0.93 (3H, d, J = 6.6 Hz), 1.10-1.18 ( 1H, m), 1.46 (9H, s), 1.42-1.52 (1H, m), 1.81-1.86 (1H, broad m), 2.51 (1H, t, J = 4.0Hz), 2.95 (1H, d, J = 15.0Hz), 3.06 (1H, dd, J = 4.4, 7.3Hz), 3.43 (3H, dd , J = 3.7, 7.0 Hz), 3.78 (3H, s), 3.85 (3H, s), 3.82-3.90 (1H, m), 4.20 (1H, d, J = 14.7 Hz), 4.94 (1H, d, J = 8.4 Hz), 7.19 (1H, broad s).

Anal. Calcd for C ₂₀ H3 ₂ N ₂ OgS: C, 50.41; H, 6.77; N, 5.88. Found C, 50.32; H, 6.92; N, 5.76.

MS (ES) m / z 475.1 [MH] ^- .

PL 221 526 B1

Synthesis 7

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'-methylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4.6 acid dimethyl ester -dicarboxylic acid

HjC NHBoc

The process was carried out according to General Procedure A using commercially available N-BOC- (L) -valine and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2X ⁶ -thia-bicyclo acid dimethyl ester [3.1.0] hexane-4,6-dicarboxylic acid (Synthesis 4). The reaction mixture was refluxed overnight. Purification was by PC-TLC, 4mm SiO ₂ rotor, (10% ethyl acetate / hexanes to 100% ethyl acetate). 0.41 g of the product (47%, 0.89 mmol) was obtained as a white foam.

[a] ²³ D = -35.36 ° (c = 0.51, CHCl3).

¹ H NMR (300 MHz, CDCl 3) δ 0.93 (3H, d, J = 7.0 Hz), 0.96 (3H, d, J = 6.6 Hz), 1.46 (9H, s) , 2.06-2.13 (1H, m), 2.50 (1H, t, J = 4.0Hz), 2.94 (1H, d, J = 15.0Hz), 3.04 ( 1H, dd, J = 4.4, 7.3 Hz), 3.43 (1H, dd, J = 3.3, 6.6), 3.78 (3H, s), 3.80-3, 86 (1H, m), 3.86 (3H, s), 4.24 (1H, d, J = 15.0Hz), 4.94 (1H, d, J = 8.1Hz), 7, 15 (1H, broad s).

Anal. Calcd for C1gH3 OGS ₀ N _2: C, 49.34; H, 6.54; N, 6.06. Found C, 49.33; H, 6.44; N, 6.05.

MS (ES) m / z 461.2 [MH] ^- .

Synthesis 8

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

The process was carried out according to General Procedure A using commercially available N-BOC- monohydrate (L) -leucine and dimethyl (1R, 4S, 5S, 6S) ^{-4-amino-2,2-dioxo-6} 2X -tia- bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (Synthesis 4). The reaction mixture was refluxed overnight. Purification was by PC-TLC, 4mm SiO2 rotor, (10% ethyl acetate / hexanes to 100% ethyl acetate). 0.85 g of product (94%, 1.78 mmol) was obtained as a white foam.

[α] ²³ D = -46.15 ° (c = 1.04, CHCl 3).

¹ H NMR (300 MHz, CDCl 3) δ 0.92 (3H, d, J = 6.2 Hz), 0.95 (3H, d, J = 6.6 Hz), 1.47 (9H, s) , 1.42-1.47 (1H, m), 1.63-1.67 (1H, m), 2.46 (1H, t, J = 3.7Hz), 2.87 (1H, d , J = 15.0 Hz), 3.04 (1H, dd, J = 4.4, 7.3 Hz), 3.41 (1H, dd, J = 3.7, 7.0), 3, 78 (3H, s), 3.86 (3H, s), 4.00-4.05 (1H, m), 4.20 (1H, d, J = 15.0Hz), 4.75 (1H , d, J = 6.6 Hz), 7.43 (1H, broad s).

Anal. Calcd for C ₂₀ H3 ₂ N ₂ OgS: C, 50.41; H, 6.77; N, 5.88. Found: C, 50.30; H, 6.82; N, 5.75.

MS (ES) m / z 475.2 [MH] ^- .

PL 221 526 B1

Synthesis 9

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

The process was carried out according to General Procedure A using commercially available N-BOC-Lys (BOC) -OH and dimethyl (1R, 4S, 5S, 6S) ^{-4-amino-2,2-dioxo-6} 2X -tia- bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (Synthesis 4). The reaction mixture was refluxed overnight. Purification was by PC-TLC, 4mm SiO2 rotor, (10% ethyl acetate / hexanes to 100% ethyl acetate). 1.04 g of product (93%, 1.76 mmol) was obtained in the form of a white foam.

[a] ²³ o = -32.0 ° (c = 0.5, CHCl3).

¹ H NMR (300 MHz, CDCl 3) δ 1.44 (9H, s), 1.46 (9H, s), 1.39-1.53 (3H, m), 1.56-1.65 (1H , m), 1.77-1.84 (2H, m), 2.50 (1H, t, J = 4.4Hz), 2.98-3.20 (4H, m), 3.42 ( 1H, dd, J = 3.7, Hz), 3.76 (3H, s), 3.86 (3H, s), 4.01 (1H, dd, J = 7.7, 13.2Hz) , 4.09-4.19 (1H, m), 4.71 (1H, t, J = 7.3Hz), 5.13 (1H, broad s), 7.59 (1H, broad s).

Anal. Calcd for C25H41N3O11S: C-50.75; H, 6.98; N- 7.10. Found C, 50.36; H, 6.99; N, 6.87.

MS (ES) m / z 590.2 [MH] ^- .

Synthesis 10

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

The process was carried out according to General Procedure A using commercially available N-BOC- (1) -glutamine (Trt) -OH and (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2X acid dimethyl ester ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (Synthesis 4). The reaction mixture was refluxed overnight. Purification was by PC-TLC, 4mm SiO2 rotor, (10% ethyl acetate / hexanes to 100% ethyl acetate). 0.53 g of the product (48%, 0.72 mmol) was obtained as a white foam.

[a] ²³ o = -8.0 ° (c = 0.50, MeOH).

¹ H NMR (300 MHz, CDCl 3) δ 1.42 (9H, s), 1.83-1.88 (1H, m), 2.18 (1H, m), 2.16 (1H, t, J = 4.0 Hz),

2.57-2.64 (1H, m), 2.60 (1H, d, J = 15.0Hz), 2.64-2.80 (1H, m), 2.88 (1H, dd, J = 4.4 Hz), 3.26 (1H, dd,

J = 4.0, 7.0 Hz), 3.47 (3H, s), 3.76-3.90 (1H, m), 3.81 (3H, s), 4.05 (1H, d , J = 15.0 Hz), 5.47 (1H, broad s),

7.02 (1H, broad s), 7.20-7.35 (15H, m), 8.68 (1H, broad s).

Anal. Calcd for Cas ^ a ^ O ^: C, 62.20; H, 5.91; N, 5.73. Found: C, 61.83; H-6.09; N-5.57.

PL 221 526 B1

MS (ES) m / z 731.9 [MH] ^- ;

HRMS calcd for C ₃₈ H _{4 3} N 3 O ₁₀ S [M + Na] +, 756.2567. Found: 756.2585.

Synthesis 11

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

The process was carried out according to general procedure A using commercially available Boc- (L) -pyroline (0.61 g, 2.9 mmol) and acid dimethyl ester (1R, 4S, 5S, 6S) -4-amino-2.2 -Dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (0.5 g, 1.9 mmol, Synthesis 4). Purification was performed using PC-TLC (ethyl acetate / hexanes) to obtain 0.87 grams (99.2%) of the title compound.

[a] ²³ D = -61.2 (c = 0.49, CHCl 3).

¹ H NMR (300 MHz, CDCl 3) δ 1.51 (9H, s), 1.75-1.98 (3H, m), 2.35-2.5 (2H, m), 2.84 (1H , d, J = 14.7Hz), 2.9-3.03 (1H, m), 3.25-3.4 (1H, m), 3.3-4.1 (2H, m), 3.76 (3H, s), 3.86 (3H, s), 4.14-4.31 (2H, m), 8.66 (1H, s).

MS (ES) m / z 459.2 [M-1] ^- .

Synthesis 12

(1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-4'-methylsulfanylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4.6 acid dimethyl ester -dicarboxylic acid

The process was carried out according to general procedure A using commercially available Boc- (L) -methionine (0.71 g, 2.9 mmol) and acid dimethyl ester (1R, 4S, 5S, 6S) -4-amino-2.2 -Dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (0.5 g, mmol, Synthesis 4). Purification was performed using PC-TLC (ethyl acetate / hexanes) to obtain 0.85 grams (90.5%) of the title compound.

[a] ²³ D = -20.4 (c = 0.49, CHCl 3).

¹ H NMR (300 MHz, CDCl 3) δ 1.46 (9H, s), 1.69 (1H, s), 1.8-2.05 (2H, m), 1.9-2.0 (1H , m), 1.95-2.3 (3H, broad s), 2.0-2.2 (1H, m), 2.4-2.8 (2H, m), 2.48 (1H, t, J = 4.0 Hz), 2.58 (1H, broad s), 2.92 (1H, d, J = 14.7 Hz), 3.01 (1H, dd, 3 = 4A, 7, 0Hz), 3.42 (1H, dd, J = 3.7, 7.3Hz), 3.78 (3H, s), 3.87 (3H, s), 4.22-4.24 ( 2H, m), 5.06 (1H, d, J = 7.7Hz), 7.27 (IH, s).

MS (ES) m / z 493.1 [M-1] ^- .

PL 221 526 B1

Synthesis 13

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

Boc

The process was carried out according to general procedure A using commercially available Boc- (L) -Tryptophan (Boc) (1.1 g, 2.8 mmol) and acid dimethyl ester (1R, 4S, 5S, 6S) -4-amino- 2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (0.5 g, mmol, Synthesis 4). Purification was performed by PC-TLC (ethyl acetate / hexanes) to obtain 0.7 g (56.7%) of the title compound.

¹ H NMR (300 MHz, CDCl 3) δ 1.44 (9H, s), 1.67 (9H, s), 2.37 (1H, broad s), 2.86 (1H, d, J = 15, 0Hz), 2.88 (1H, t, J = 4.4Hz), 3.15 (2H, d, J = 6.6Hz), 3.39 (1H, dd, J = 3.7, Hz), 3.73 (3H, s), 3.83 (3H, s), 4.18 (1H, d, J = 14.7Hz), 4.37-4.44 (1H, m), 5.01 (1H, broad d, J = 8.1Hz), 7.11 (1H, broad s), 7.25-7.59 (4H, m), 8.14 (1H, broad d, J = 8.4 Hz).

[a] ²³ o = -19.6 (c = 0.51, CHCl3).

Analysis: values calculated for C ₃₀ H ₃₉ N ₃ O ₁₁ S -1.0 C ₄ H ₈ O ₂ : C, 55.35; H, 6.42; N, 5.70. Found: C-54.98; H-6.09; N, 6.07.

HRMS calcd for C30H39N3O11Na1S, 672.2203.

Found: 672.2180.

Synthesis 14

(1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-3 '- (4-tert-butoxycarbony] oxyphenyl) -propionylamino] -2,2-dioxo-2X ^{6-thia acid dimethyl ester} -bicyclo [3.1.0] hexane-4,6-dicarboxylic acid

The process was carried out according to general procedure A using commercially available 2S-tert-butoxycarbonylamino-3- (4-tert-butoxycarbonyloxyphenyl) propionic acid (1.1 g, 2.9 mmol) and acid dimethyl ester (1R, 4S, 5S , 6S) -4-Amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (0.5 g, mmol, Synthesis 4). Purification was performed by PC-TLC (ethyl acetate / hexanes) to obtain 0.94 g (79.0%) of the title compound.

[a] ²³ o = +4 (c = 1.00, CH3OH).

¹ H NMR (300 MHz, CDCl 3) δ 1.44 (9H, s), 1.56 (9H, s), 2.44 (1H, t, J = 4.0 Hz), 2.88 (1H, d, J = 14.7Hz), 2.98 (1H, dd, J = 4.4, 7.3Hz), 3.04 (2H, d, J = 7.3Hz), 3.38 ( 1H, dd, J = 4.0, 4.22-4.29 (1H, apparent q, J = 7.3Hz), 4.92 (1H, broad d, J = 7.7Hz), 7, 07 (1H, broad s), 7.1-7.26 (4H, m).

HRMS Obl. for C ₂₃ H3 ₃ N ₂ O1 ₂ SNa, 649.2043. Found: 649.2001.

PL 221 526 B1

Synthesis 15

(1R, 4S, 5S, 6S) -4- (3'-acetoxy-2'S-tert-butoxycarbonylaminopropionyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4 acid dimethyl ester , 6-dicarboxylic acid

The process was carried out according to general procedure A using 3-acetoxy-2S- (tert-butoxycarbonylamino) propionic acid (0.25 g, 1.0 mmol, Synthesis 44) and the acid dimethyl ester (1R, 4S, 5S, 6S) -4 -amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (0.2 g, 0.8 mmol, Synthesis 4) and no DMAP was used. Purification was performed by PC-TLC (ethyl acetate / hexanes) to obtain 0.19 g (48.2%) of the title compound.

[a] ²³ o = -24 (c = 1.0, CH 3 OH).

¹ H NMR (300 MHz, CDCl 3) δ 1.47 (9H, s), 2.10 (3H, s), 2.51 (1H, t, J = 4.4 Hz), 2.99-3, 07 (2H, m), 3.43 (1H, dd, J = 4.0, Hz), 3.77 (3H, s), 3.86 (3H, s), 4.14-4.40 ( 4H, m), 5.29 (1H, broad d, J = 7.3Hz), 7.64 (1H, broad s).

HRMS calcd for C ₁₉ H ₂₈ N ₂ O ₁₁ SNa, 515.1312. Found: 515, 1305.

Synthesis 16

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

For a suspension of (1R, 2S, 4R, 5R, 6R) -2-amino-4-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid (14.45 g, 71.12 mmol, St No. 5,958,960) in 202 mL of absolute ethanol at room temperature, thionyl chloride (26 mL, 356 mmol) was added dropwise over 20 minutes. The slurry was heated to reflux and allowed to stir for 3 hours. It was allowed to cool to room temperature and stirred overnight. The resulting solution was concentrated under reduced pressure, then the residue was diluted with 136 mL of ethyl acetate and treated with 306 mL of 10% aqueous sodium carbonate for 15 minutes with manual stirring until a final pH was 10. The layers were separated and the aqueous layer was washed with ethyl acetate. (1 x 136 ml). The combined organic extracts were washed with brine (1 x 136 mL), dried (MgSO4), filtered, and concentrated in vacuo to give 17.07 g (93%) of the title compound as a white solid.

[a] ²³ o = + 20.37 ° (c = 1.1, MeOH).

Melting point = 64-66 ° C.

¹ H NMR (400 MHz, CDCl3) δ 1.28 (3H, t, J = 7.3 Hz), 1.31 (3H, t, J = 6.8 Hz), 1.34-1.45 ( 1H, m), 1.85 (2H, broad s), 2.17-2.21 (2H, m), 2.32-2.34 (1H, m), 2.49 (1H, dd, J = 7.8, 14.1 Hz), 4.24 (2H, dq, J = 1.5, 7.3 Hz), 5.33-5.52 (1H, m).

Analysis: calcd for C C H ^NO C, 55.59; H, 7.00; N, 5.40. Found C, 55.29; H, 6.75; N, 5.45.

MS (ES) m / z found 260.3 [M + H] +.

PL 221 526 B1

Synthesis 17

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

To a solution of N-Boc-L-alanine (38.62 g, 204 mmol) in 396 ml of methylene chloride at -22 ° C under nitrogen atmosphere, was added N-methylmorpholine (22.44 ml, 204 mmol) followed by dropwise isobutyl chloroformate (26.48 mL, 204 mmol) over a period of 15 minutes so that the temperature of the reaction mixture does not exceed -18 ° C. The resulting slurry was allowed to stir at -20 ° C for 30 minutes, then a solution of (1R, 2S, 4R, 5R, 6R) -2-amino-4-fluorobicyclo [3.1.0] hexane-2 was added. , 6-dicarboxylic acid (49.46 g, 191 mmol, Synthesis 16) in 247 mL of methylene chloride over 40 minutes so that the temperature of the reaction mixture does not exceed -16 ° C. After the addition was complete, the reaction mixture was removed from the cooling bath and allowed to stir at ambient temperature for 70 minutes, whereupon the temperature of the reaction mixture reached 15 ° C and the color turned faintly orange. The reaction mixture was treated with 408 mL of 1 N HCl solution and stirred for 5 minutes, then the layers were separated. The organic layer was washed with a saturated aqueous sodium bicarbonate solution (1 x 408 ml), dried (Na2SO4), filtered and concentrated in vacuo to a white foam (88.16 g).

Anal. calcd for C ₂₀ H _{31 FN 2} O _r 0.1 CH ₂ Cl ₂ : C, 55.00; H, 7.16; N, 6.38. Found: C, 55.18; H, 7.18; N, 6.49.

MS (ES) m / z 431.3 [M + H] +, 331.2 [M + H-Boc] +.

Synthesis 18

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

To a solution of (1R, 2S, 4R, 5R, 6R) -2- [2'S- (tert-butoxycarbonylamino) -propionyl] amino-4-flurobicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (88, 16 g, 191 mmol, Synthesis 17) in 238 ml of THF were added at room temperature 238 ml (477 mmol) of 2N sodium hydroxide solution. The biphasic mixture was stirred vigorously at room temperature for 2.5 hours after which time the reaction mixture became homogeneous. The mixture was diluted with 238 ml of methyl t-butyl ether, then stirred and the layers were separated. The aqueous layer was then diluted with 238 mL of water and filtered to remove the solid. The solution was treated with concentrated HCl (42.9 mL, 515 mmol) for 30 minutes, optionally seeded with crystals of the title compound, and stirred for 1 hour. The resulting suspension was filtered, washed with water (2 x 100 mL) and dried in high vacuum at 45 ° C for 40 hours to provide the title compound as a white solid. Part of the solid (69.5 g) was stirred with 490 mL of acetone for 1 hour to form a cloudy solution; filtered and washed with acetone (2 x 100 ml). The filtrate was concentrated in vacuo to a white foam which was then dried in high vacuum at 45 ° C for 16 hours to give 61.8 g (86%,

Corrected for 12% w / w acetone) of the title compound. This material was used in Examples 14-18 without identification.

Synthesis 19

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

To a solution of (1R, 2S, 4R, 5R, 6R) -2- (tert-butoxycarbonyl) amino-4-hydroxybicic [3.1.0] hexane-6-dicarboxylic acid diethyl ester (1.50 g, 4.20 mmol, US 5,958,960), pyridine (0.365 ml, 4.62 mmol) and DMAP (0.513 g, 4.20 mmol) in dichloromethane (40 ml) were added acetic anhydride (0.514 ml, 5.0 mmol) under nitrogen. The mixture was stirred at room temperature for 16 hours, diluted with dichloromethane and poured into 10% aqueous citric acid (50 ml). The organic layer was washed with water (50 ml) and brine (50 ml). Dried over MgSO4, filtered, and concentrated under reduced pressure to yield the title compound as a white solid (1.295 g, 75%).

LCMS: m / z 400 [M + H] + and m / z 300 [M + H-CO ₂ ¹ Bu] + R t 1.39 minutes Synthesis 20

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

(1R, 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 Synthesis 19) was dissolved in 95% TFA in dichloromethane (60 ml) and stirred under nitrogen at room temperature for 5 minutes. At this time, the TFA / dichloromethane mixture was removed under reduced pressure. The crude product was dissolved in a suspension of NaHCO3 (1.00 g) in dichloromethane (50 ml) and stirred for 30 minutes. The slurry was filtered, washed with dichloromethane (3 x 25 ml) and concentrated in vacuo to give 916 mg (98%) of the product as a yellow oil.

LCMS: m / z 300 [M + H] + R t 0.92 min

Synthesis 21

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

For a suspension of (1S, 2S, 4S, 5R, 6R) -4-acetyloxy-2-aminobicyclo [3.1.0] -hexane-2,6-dicarboxylic acid diethyl ester (0.80 g, 1.94 mmol, Synthesis 20) and Boc-Z-Ala (0.477 g,

PL 221 526 B1

2.52 mmol) in dry dichloromethane (50 ml) under nitrogen atmosphere successively added EDC (0.519 g, 2.72 mmol), HOBt (0.314 g, 2.32 mmol), a catalytic amount of DMAP (0.024 g, 0.19 mmol) ) and triethylamine (1.08 mL, 7.76 mmol). After stirring at room temperature for about 15 minutes, the initial white solid was completely dissolved. The reaction mixture was stirred for 16 hours, diluted with dichloromethane and washed with saturated aq. NaHCO ₃ (50 ml), 1.0N aq. HCl (3 x 20 ml) and saturated brine (40 ml). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. Purification of the resulting crude amide by column chromatography eluting with a mixture of ethyl acetate and hexanes (4: 1) gave 638 mg (75%) of the product as a white solid.

¹ H NMR (CD3OD) *: δ 5.05 (1H, d, 5.7 Hz), 4.09 (2H, q, 7.3 Hz), 4.02 (2H, q, 7.3 Hz) , 4.01-3.94 (1H, m), 2.65-2.59 (2H, m), 2.38 (1H, d, 14Hz), 2.10-2.03 (3H, m ), 1.91 (3H, s), 1.78 (1H, dd, 5.9Hz, 16Hz), 1.59 (1H, broad s), 1.34 (9H, s), 1.16 (6H, 2xt, 7.3Hz);

* N.B. Number of exchangeable protons not observed by NMR = 2.

LCMS: m / z 471 [M + H] + and m / z 371 [M + H-CO ₂ ¹ -Bu] + R t 1.30 minutes. R f 0.50 (80% ethyl acetate: heptane). Synthesis 22

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

Solution of (1R, 2S, 4S, 5R, 6R) -2-tert-butoxycarbonylamino-4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (5.0 g, 14.0 mmol, US 5,958,960 ) in ethyl acetate, cooled to 0 ° C. Anhydrous HCl (g) was passed through the solution until saturated and stirred for 0.5 hours. The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. 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 organics were dried with K2CO3 and concentrated to give 2.2 g (59.7%) of a white solid.

[a] ²³ D = -30.8 (c = 0.52, CH 3 OH).

¹ H NMR (300 MHz, CDCl 3) δ 1.26 (3H, t, J = 7.33 Hz), 1.36 (3H, t, 7.33 Hz), 1.60 (1H, dd, J = 5.87, 15.40Hz), 1.64 (1H, t, J = 2.93Hz), 2.27 (2H, broads), 2.15-2.29 (3H, m), 3 , 93 (1H, broad s), 4.13 (2H, q, J = 6.97Hz), 4.27-4.36 (3H, m).

Analysis: calculated values for C12H19NO5: C, 56.02; H, 7.44; N, 5.44. Found: C, 55.75; H, 7.36; N, 5.40.

MS (ES) m / z 258.1 [M + H] +.

Synthesis 23

(1R, 2S, 4S, 5R, 6R) -2- (2'-tert-butoxycarbonylaminoacetylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure A using Boc-glycine (458 mg, 2.62 mmol, Aldrich) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1.0] acid diethyl ester. hexane-2,6-dicarboxylic acid (450 mg, 1.75 mmol, Synthesis 22). Purification was carried out on 35 g of silica,

Eluting with a hexanes / ethyl acetate gradient from 50/50 to 20/80. Yield: 560 mg (77%).

¹ H NMR (400 MHz, CDCl 3) δ 1.28 (3H, t, J = 7.3 Hz), 1.29 (3H, t, J = 7.3 Hz), 1.48 (9H, s) , 1.57 (2H, m), 2.21 (1H, m), 2.41 (1H, dd, J = 2.9, 5.8Hz), 2.81 (1H, d, J = 15 , 6Hz), 3.76 (1H, d, J = 5.8Hz), 4.10-4.18 (3H, m), 4.26 (2H, q, J = 7.3Hz), 4.33 (1H, m), 5.10 (1H, broad s), 6.86 (1H, broad s).

Synthesis 24

(1R, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-3'-methylbutyrylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure A using Boc-L-valine (569 mg, 2.62 mmol, Sigma) and acid diethyl ester (1R, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1. O] -hexane-2,6-dicarboxylic acid (450 mg, 1.75 mmol, Synthesis 22). The crude material was purified over 35 g of silica; eluted with a gradient of hexanes / ethyl acetate from 70/30 to 20/80. Yield: 694 mg (87%) of the product as a white foam.

¹ H NMR (400 MHz, CDCl 3) δ 0.95 (3H, d, J = 6.8 Hz), 1.00 (3H, d, J = 6.8 Hz), 1.29 (6H, t, J = 7.3Hz), 1.45 (9H, s), 1.52-1.60 (2H, m), 2.08 (1H, m), 2.20 (1H, m), 2, 42 (1H, m), 2.81 (1H, d, J = 15.6 Hz), 3.86 (1H, dd, J = 6.3, 8.8 Hz), 4.14 (2H, q , J = 7.3Hz), 4.21-4.32 (4H, m), 5.00 (1H, d, J = 8.3Hz), 6.63 (1H, s).

Anal. Calcd for C22H36N2O8: C, 57.88; H, 7.95; N, 6.14. Found: C, 57.87; H, 8.03; N, 6.12. MS (ES) m / z 457.2 [M + H] +, 479.2 [M + Na] +.

Synthesis 25

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

The process was carried out according to general procedure A using Boc-L-leucine (606 mg, 2.62 mmol, Chemlog) and acid diethyl ester (1R, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1. O] -hexane-2,6-dicarboxylic acid (450 mg, 1.75 mmol, Synthesis 22). The crude material was purified over 35 g of silica; eluted with a gradient of hexanes / ethyl acetate from 70/30 to 20/80. 689 mg (84%) of the product was obtained as a white foam.

¹ H NMR (400 MHz, CDCl 3) δ 0.96 (6H, m), 1.28 (3H, t, J = 7.3 Hz), 1.29 (3H, t, J = 7.3 Hz) , 1.46 (9H, s), 1.45-1.73 (5H, m), 2.20 (1H, m), 2.42 (1H, m), 2.80 (1H, d, J = 16.1 Hz), 4.07-4.35 (7H, m), 4.81 (1H, broad d), 6.86 (1H, broad s).

Anal. Calcd for C23H38N2O ₈ 0.1 H ₂ O: C, 58.48; H, 8.15; N, 5.93. Found: C, 58.22;

H, 7.94; N, 5.92. MS (ES) m / z [M + H] +, 493.2 [M + Na] +.

PL 221 526 B1

Synthesis 26

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylamino-3'S-methylpentanoylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure A using Boc-L-isoleucine (606 mg, 2.62 mmol, Aldrich) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1. O] hexane-2,6-dicarboxylic acid (450 mg, 1.75 mmol, Synthesis 22). The crude material was purified over 35 g of silica eluting with a hexanes / ethyl acetate gradient from 70/30 to 20/80. Yield: 731 mg (89%) of the product.

¹ H NMR (400 MHz, CDCl 3) δ 0.92 (3H, t, J = 7.3 Hz), 0.97 (3H, d, J = 6.8 Hz), 1.17 (1H, m) , 1.29 (6H, t, J = 6.8Hz), 1.46 (9H, s), 1.53 (1H, dd, J = 5.8, 15.6Hz), 1.58 ( 1H, t, J = 2.4Hz), 1.74 (1H, m), 1.83 (1H, m), 2.20 (1H, m), 2.43 (1H, m), 2, 80 (1H, d, J = 15.6 Hz), 3.88 (1H, dd, J = 7.3, 8.8 Hz), 4.12-4.32 (6H, m), 4.98 (1H, d, J = 7.3Hz), 6.60 (IH, s).

Anal. Calculated for C _{2 3} H 38 N 2 O ₈ -0.2 H ₂ O: C-58.25; H, 8.16; N, 5.91. Found: C, 58.17; H, 8.11; N, 5.91.

MS (ES) m / z 471.2 [M + H] +, 493.2 [M + Na] +.

Synthesis 27

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

The process was carried out according to general procedure A using Boc-Gly-Gly (474 mg, 2.04 mmol) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo-hexane-2-acid diethyl ester hydrochloride. , 6-dicarboxylic acid (400 mg, 1.36 mmol, Synthesis 22 without basic treatment) with the following changes. DMAP was not used. One equivalent of triethylamine was added. The crude material was purified over 35 g of silica; eluted with ethyl acetate. Yield: 517 mg (81%).

[a] ²³ o = -18.18 (c = 0.55, MeOH).

¹ H NMR (300 MHz, CDCl 3) δ 1.26 (3H, t, J = 6.8 Hz), 1.27 (1H, t, J = 7.3 Hz), 1.28 (3H, t, J = 6.8Hz), 1.46 (9H, s), 1.59 (1H, t, J = 3.4Hz), 1.62 (1H, dd, J = 6.3.16.1 Hz), 2.21 (1H, dd, 3 = 2.9, 5.8 Hz), 2.46 (1H, dd, J = 2.4, 5.8 Hz), 2.73 (1H, d , J = 15.6Hz), 3.78-3.91 (3H, m), 4.01-4.15 (3H, m), 4.24 (2H, q, J = 6.8Hz) , 4.32 (1H, d, J = 5.8Hz), 5.28 (1H, b), 6.87 (1H, bt, J = 4.9Hz), 7.39 (1H, broad s ).

HRMS calcd for C21H34N3O9, 472, 2295. Found: 472, 2303.

HPLC: 16.755 minutes. Column: Symmetry C18, 3.5 µm, 4.6 x 150 mm. λ = 230 nM. Flow rate: 1 ml / minute Gradient: 10% to 50% ACN / water containing 0.1% TFA over 25 minutes.

PL 221 526 B1

Synthesis 28

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

The process was carried out according to general procedure A using Boc-Ala-Gly (502 mg, 2.04 mmol) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo-hexane-2-acid diethyl ester hydrochloride. , 6-dicarboxylic acid (400 mg, 1.36 mmol, Synthesis 22 without basic treatment) with the following changes. DMAP was not used. One equivalent of triethylamine was added. The crude material was purified over 35 g of silica eluting with ethyl acetate. Yield: 500 mg (76%).

[a] ²³ o = -31.37 (c = 0.55, MeOH).

¹ H NMR (300 MHz, CDCl 3) δ 1.27 (3H, t, J = 6.8 Hz), 1.28 (3H, t, J = 6.8 Hz), 1.42 (3H, d, J = 6.8Hz), 1.45 (9H, s), 1.58 (1H, t, J = 3.4Hz), 1.64 (1H, dd, J = 5.8, 15.6 Hz), 2.23 (1H, dd, J = 3.4, 5.8 Hz), 2.50 (1H, dd, 3 = 2.9, 6.3 Hz), 2.69 (1H, d , J = 15.6 Hz), ABq of doublets (2H, I = 3.87, Jb = 3.98, Jab = 17.1 Hz, J _d = 6.3 Hz), 3.92 (1H, m) , 4.07-4.16 (3H, m), 4.24 (2H, q, J = 7.3Hz), 4.32 (1H, b), 5.08 (1H, b), 6, 84 (1H, bt, J = 4.88Hz), 7.14 (1H, broad s).

HRMS calcd for C22H36N3O9, 486, 2452.

Found: 486, 2451.

Synthesis 29

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

The process was carried out according to general procedure A using Boc-L-phenylalanine (772 mg, 2.91 mmol) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1.0] acid diethyl ester. hexane-2,6-dicarboxylic acid (480 mg, 1.94 mmol, Synthesis 22). The crude reaction mixture was concentrated, the residue was dissolved in dichloromethane, flash chromatography performed through a short bed of silica gel eluting with ethyl acetate / DCM (1: 1) and concentrated in vacuo to yield a yellow oil. The material was then purified by silica gel chromatography eluting with a gradient of 30% ethyl acetate / hexanes to 80% ethyl acetate / hexanes to provide 852 mg (87%) of the title compound.

[a] ²³ o = -23.66 (c = 0.93, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 1.22-1.30 (6H, m), 1.35 (9H, s), 1.59 (1H, t, J = 2.9Hz), 1.70 (1H, dd,

J = 5.5, 15.4Hz), 2.06 (1H, m), 2.40 (1H, d, J = 15.4Hz), 2.63 (1H, dd, J = 2.9 , 5.9 Hz), 2.76 (1H, dd,

J = 9.2, 13.9 Hz), 4.08-4.33 (6H, m), 7.20-7.29 (5H, m).

MS found 505.0 [M + H] ⁺ .

HRMS calcd for C26H36N2O8, 505, 2550.

Found: 505, 2559.

PL 221 526 B1

Synthesis 30

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

The process was carried out according to general procedure A using Boc-L-glutamino (trityl) -OH (1.40 g, 2.86 mmol) and acid diethyl ester (1S, 2S, 4S, 5R, 6R) -2-amino-4 -hydroxybicyclohexane-2,6-dicarboxylic acid (490 mg, 1.90 mmol, Synthesis 22) except that DMAP was not used. The crude reaction mixture was concentrated, the residue was dissolved in dichloromethane, flash chromatographed through a short bed of silica gel, eluted with ethyl acetate and concentrated in vacuo to yield a yellow oil. Purification was then performed by silica gel chromatography eluting with a gradient of 30% ethyl acetate / hexanes to 100% ethyl acetate / hexanes to provide 1.3 g (94%) of the title compound.

[a] ²³ o = -21.28 (c = 0.94, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 1,18-1,26 (6H, m), 1.44 (9H, s), 1,62-1,74 (3H, m), 1,91-2 .00 (1H, m), 2.05 (1H, m), 2.38-2.48 (3H, m), 2.54 (1H, m), 4.00-4.27 (6H, m ), 7.18-7.30 (15H, m).

MS found 728.2 [M + H] ⁺ .

HRMS calcd for C ₄₁ H ₄₉ N ₃ O ₉ , 728, 3547.

Found: 728, 3533.

Synthesis 31

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

The process was carried out according to general procedure A using Boc-L-lysine (Boc) -OH (910 mg, 2.63 mmol) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo acid diethyl ester [3.1.0.] Hexane-2,6-dicarboxylic acid (450mg, 1.75mmol, Synthesis 22) except that no DMAP was added to the reaction mixture. The crude reaction mixture was concentrated, the residue was dissolved in dichloromethane, flash chromatographed through a short bed of silica gel, eluted with ethyl acetate and concentrated in vacuo to yield a yellow oil. The material was then purified by silica gel chromatography eluting with ethyl acetate / hexanes (1: 1) to give 800 mg (78%) of the title compound.

[a] ²³ D = -30.19 (c = 0.53, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 1,21-1,24 (6H, m), 1,26-1,17 (27H, m), 2.07 (1H, m), 2.42 (1 H , d

J = 15.8 Hz), 2.59 (1H, dd, J = 2.6, 5.5 Hz), 3.03 (1H, t, J = 6.2 Hz), 3.99 (1H, m), 4.07-4.28 (5H, m).

PL 221 526 B1

MS found 586.1 [M + H] ⁺ .

HRMS calcd for C28H47N3O10, 586.3340 Found: 586, 3348.

Synthesis 32

(1S, 2S, 4S, 5R, 6R) -2- [2'-tert-butoxycarbonylamino-3 '- (tritylcarbamoylpropionylamino] -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure A using Boc-L-asparagino (trityl) -OH (1.35 g, 2.84 mmol) and acid diethyl ester (1S, 2S, 4S, 5R, 6R) -2-amino-4 -hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid (476 mg, 1.85 mmol, Synthesis 22) except that no DMAP was added to the reaction mixture. The crude reaction mixture was concentrated, the residue was dissolved in dichloromethane, flash chromatographed through a short bed of silica gel, eluted with ethyl acetate and concentrated in vacuo to yield a yellow oil. The material was then purified by silica gel chromatography eluting with a gradient of 20% ethyl acetate / hexanes to 50% ethyl acetate / hexanes to provide 1.07 g (81%) of the title compound.

[a] ²³ o = -25.45 (b = 0.55, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 1,19-1,26 (6H, m), 1.45 (9H, s), 1.61-1.69 (2H, m), 2.05 (1 H , m), 2.44 (1H, d, J = 15.4Hz), 2.51-2.72 (3H, m), 4.02-4.21 (4H, m), 4.25 ( 1H, d, J = 5.5 Hz), 4.36 (1H, dd, J = 4.8, 8.8 Hz), 7.18-7.30 (15H, m).

MS found 714.1 [M + H] +.

HRMS calcd for C40H47N3O9, 714, 3391.

Found: 714, 3380.

Synthesis 33

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

The process was carried out according to general procedure A using Boc-L-tryptophan (Boc) -OH (1.18 g,

2.91 mmol) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1.0.] Hexane-2,6-dicarboxylic acid diethyl ester (520 mg, 1.90 mmol, Synthesis 22). The crude reaction mixture was concentrated, the residue was dissolved in dichloromethane, flash chromatography was performed through a short bed of silica gel, eluted with ethyl acetate and concentrated in vacuo.

With a yellow oil. The material was then purified by chromatography on silica gel, eluting with ethyl acetate / DCM (1: 1), to give 1.2 g (92%) of the title compound.

[α] ²³ ο = -12.5 (c = 0.96, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 1,18-1,27 (6H, m), 1.34 (9H, s), 1.62 (1H, t, J = 2.9 Hz), 1. 67 (9H, s), 1.74 (1H, dd, J = 5.9, 15.2 Hz), 2.07 (1H, m), 2.46 (1H, d, J = 15.7 Hz ), 2.60 (1H, d, J = 2.9, Hz), 2.89 (1H, dd, J = 9.3, 15.0 Hz), 3.16 (1H, dd, J = 4 , 9, 15.2Hz), 4.04-4.12 (2H, m), 4.18-4.24 (2H, m), 4.27 (1H, d, J = 5.4Hz) , 4.43 (1H, dd, J = 5.4, 9.3Hz), 7.26 (2H, m), 7.52 (1H, s), 7.64 (1H, d, J = 7 , 3 Hz), 8.10 (1H, d, J = 8.3 Hz).

MS found 644.8 [M + H] +, 666.8 [M + Na] +.

HRMS calcd for C ₃₃ H ₄₄ N ₃ O ₁₀ , 666, 3002.

Found: 666, 2988.

Synthesis 34

(1S, 2S, 4S, 5R, 6R) -2- (1'-tert-butoxycarbonylpyrrolidin-2'S-carbonyl) -4-hydroxybicyclo [3.1.0] -hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure A using Boc-L-proline and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1.0.] Hexane-2,6-dicarboxylic acid diethyl ester ( Synthesis 22). The crude material was purified on 110 g ISCO silica eluting with a gradient of 80% ethyl acetate / hexanes to 100% ethyl acetate to provide 699 mg (84%) of the title compound.

[a] ²³ o = -52.53 (c = 0.99, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 1.26 (6H, m), 1.44 (9H, s), 1.62 (1H, m), 1.73 (1H, dd, J = 5.4 , 15.3 Hz), 1.84 (1H, m), 1.97 (1H, m), 2.07 (4H, m), 2.41 (1H, d, J = 15.3 Hz), 2.50 (1H, m), 2.59 (1H, m), 4.12 (2H, m), 4.20 (1H, q, J = 7.4Hz), 4.27 (1H, d , J = 5.4 Hz).

MS found 454.9 [M + H] +, 476.8 [M + Na] +.

HRMS calcd for C 22 H ₃ 4N2O ₈ Na: 477, 2213.

Found: 477, 2210.

Synthesis 35

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

PL 221 526 B1

The title compound was prepared according to general procedure A using 2S-tert-butoxycarbonylamino-3- (4-tert-butoxycarbonyloxy-phenyl) propionic acid and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [ 3.1.0.] Hexanedicarboxylic acid (Synthesis 22). The crude material was purified on 110 g ISCO silica eluting with a gradient of 60% ethyl acetate / hexanes to 90% ethyl acetate / hexanes to provide 1.09 g (91%) of the title compound.

[a] ²³ D = -12 (c = 1.0, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 1.25 (6H, m), 1.37 (9H, s), 1.52 (9H, s), 1.68 (1H, dd, J = 5.4 , 15.3Hz), 2.06 (1H, m), 2.38 (1H, d, J = 15.3Hz), 2.64 (1H, m), 2.79 (1H, dd, J = 8.4, 13.9 Hz), 3.05 (1H, dd, J = 5.4, 13.9 Hz), 4.20 (6H, m), 7.04 (2H, d, J = 8.4 Hz), 7.26 (2H, d, J = 8.4 Hz).

MS found 621.8 [M + H] +, 643.8 [M + Na] +.

HRMS calcd for C ₃₁ H ₄₄ N ₂ O ₁₁ Na, 643, 2843. Found: 643, 2845.

Synthesis 36

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

The title compound was prepared according to general procedure A using (Boc-L-methionine) and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1.0.] Hexane-2,6- acid diethyl ester. dicarboxylic acid (Synthesis 22). The crude material was purified on 110 g ISCO silica eluting with a gradient of 80% ethyl acetate / hexanes to 100% ethyl acetate to provide 1.0 g (92%) of the title compound.

[a] ²³ D = -30 (c = 1.0, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 1.26 (6H, m), 1.43 (9H, s), 1.84 (6H, m), 2.06 (1H, m), 2.2 ( 1H, m), 2.48 (1H, m), 2.61 (1H, m), 3.37 (1H, m), 2.49 (1H, m), 4.2 (6H, m).

MS found 488.8 [M + H] +, 510.8 [M + Na] +.

HRMS calcd for C22H36N2O8SNa, 511, 2090.

Found: 511, 2071.

Synthesis 37

(1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylaminopropionylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure A using commercially available N-BOC- (L) -alanine and (1S, 2S, 4S, 5R, 6R) -2-amino-4-hydroxybicyclo [3.1.0.] Hexanedicarboxylic acid diethyl ester (Synthesis 22). The reaction mixture was refluxed overnight. Purification was done by preparative HPLC, 1 x 500 g SiO ₂ column, (10% ethyl acetate / hexanes to 100% ethyl acetate). 3.0 g of product (90%, 7.00 mmol) were obtained in the form of a white foam.

[a] ²³ D = -32.31 ° (c = 0.37, CHCl 3).

¹ H NMR (300 MHz, CDCl 3) δ 1.28 (6H, t, J = 7.3 Hz), 1.34 (3H, d, J = 7.3 Hz), 1.46 ( 9H, s), 1.51-1.58 (2H, m), 1.64 (1H, s), 2.19 (1H, dd, J = 3.3,6.2Hz), 2.40 (1H, dd, J = 2.9, Hz), 2.80 (1H, d, J = 15.8 Hz), 4.10-4.36 (6H, m), 4.92 (1H, broad s), 6.94 (1H, broad s).

Anal. Calcd for C2 H32N2O _{0 8} 0.1 H ₂ O: C, 55 83; H, 7.54; N, 6.51. Found: C, 55.57; H, 7.64; N, 6.44.

MS (ES) m / z 429.2 [M + H] +, 329.1 [M-Boc] +.

Synthesis 38

(1S, 2S, 4S, 5R, 6R) -2- [2'S-aminopropionylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid, hydrochloride

Solution of (1S, 2S, 4S, 5R, 6R) -2- (2'S-tert-butoxycarbonylaminopropionylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester (2.95 g, 6.88 mmol, Synthesis 37) in ethyl acetate (30 mL) at 0 ° C was purged with anhydrous HCl gas until the HCl solution was saturated. The resulting reaction mixture was stirred at 0 ° C until the reaction was complete as judged by TLC. The reaction mixture was purged with N ₂ for 30 minutes to remove excess HCl gas. The resulting slurry was concentrated to dryness under reduced pressure to provide 2.47 g (98%, 6.78 mmol) of the desired amine hydrochloride. No additional clean-up was necessary.

[a] ²³ D = -28.0 ° (c = 0.50, MeOH).

¹ H NMR (300 MHz, CDCla) δ 1.24 (3H, t, J = 7.0 Hz), 1.28 (3H, t, J = 7.3 Hz), 1.50 (3H, d, J = 7.3 Hz), 1.61 (1H, t, J = 2.9 Hz), 76 (1H, dd, J = 5.9, 15.8 Hz), 2.10 (1H, dd, J = 3.3, 5.9 Hz), 2.43 (1H, d, J = 15.4 Hz), 2.60 (1H, dd, J = 2.9, 6.2 Hz), 3, 90 (1H, q, J-7.0, 13.9Hz), 4.15 (2H, q, J = 7.3Hz), 4.14-4.31 (3H, m).

Anal. Calcd for C1 _{5 2} H24N O6-HCl-0.7 H ₂ O: C, 47.73; H, 7.05; N, 7.42. Found: C, 47.96; H, 6.91; N, 7.04.

MS (ES) m / z 329.1 [M + H] +.

Synthesis 39

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2S-tert-butoxycarbonylaminopropionylamino) propionylamino] -4-hydroxybicyclo [3.1.0.] Hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure A using (1S, 2S, 4S, 5R, 6R) -2- (2'S-aminopropionylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride (0, 2 g, 0.55 mmol, Synthesis 38) and Boc- (L) -alanine (0.16 g, 0.82 mmol), except that no DMAP was used. Purification was performed by PC-TLC (ethyl acetate / hexanes) to obtain 0.13 g (47.3%) of the title compound.

[α] ²³ ο = -46.2 (c = 0.52, CHCl3).

¹ H NMR (300 MHz, CDCl 3) δ 1.27 (3H, t, J = 7.0 Hz), 1.28 (3H, t, J = 7.0 Hz), 1.36 (3H, d, J = 7.0Hz), 1.37 (3H, d, J = 7.0Hz), 1.44 (9H, s), 1.58-1.65 (2H, m), 2.19 ( 1H, dd, J = 3.0Hz), 2.46 (1H, dd, J = 2.6, 5.9Hz), 2.70 (1H, d, J = 15.4Hz), 4 . 09-4.33 (7H, m), 4.48 (1H, apparent p, J = 7.0Hz), 5.05 (1H, broad d, J = 6.6Hz), 6.79 ( 1H, broad d, J = 7.7Hz), 7.26 (IH, s).

HRMS calcd for C23H38N3O9, 500, 2608.

Found: 500, 2598.

Synthesis 40

(1S, 2S, 4S, 5R, 6R) -2- [2'S- (2-tert-butoxycarbonylaminoacetylamino) propionylamino] -4-hydroxybicyclo [3.1.0.] Hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure A using Boc-glycine (0.29 g, 1.6 mmol) and (1S, 2S, 4S, 5R, 6R) -2- (2'S-aminopropionylamino) -4-hydroxybicyclo acid diethyl ester hydrochloride [3.1.0] hexane-2,6-dicarboxylic acid (0.4 g, 1.1 mmol, Synthesis 38) except that no DMAP was used. Purification was performed by PC-TLC (ethyl acetate / hexanes) to obtain 0.14 g (26.2%) of the title compound.

[a] ²³ O = -14 (c = 1.00, CDCl 3).

¹ H NMR (300 MHz, CDCl 3) δ 1.27 (3H, t, J = 7.3 Hz), 1.29 (3H, t, J = 7.3 Hz), 1.38 (3H, d, J = 7.0Hz), 1.45 (9H, s), 1.57-1.65 (2H, m), 2.19 (1H, dd, J = 3.3, 5.9Hz), 2.44 (1H, dd, J = 2.9, 5.9 Hz), 2.74 (1H, d, J = 15.8 Hz) 3.70-3.86 (2H, m), 4. 08-4.34 (6H, m), 4.56 (1H, apparent p, J = 7.0Hz), 5.31 (1H, broad s), 6.88 (1H, broad d, J = 7 , 0 Hz), 7.50 (1H, s).

HRMS calcd for C ₂₂ H 36 N ₃ O9, 486, 2452.

Found: 486, 2444.

Synthesis 41

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

The process was carried out according to general procedure B using commercially available N-BOC- (L) -leucine monohydrate and (1S, 2S, 4S, 5R, 6R) -2- (2'S-aminopropionylamino) -4-hydroxybicyclo [3.1] diethyl ester hydrochloride. .0] hexane-2,6-dicarboxylic acid (0.2 g, 0.55 mmol, Synthesis 38).

PL 221 526 B1

Purify by PC-TLC, 4 mm _SiO2 rotor, (10% ethyl acetate / hexanes to 100% ethyl acetate). 0.54 g of the product (62%, 1.00 mmol) was obtained as a white foam.

[α] ²³ D = -61.2 ° (c = 0.49, MeOH).

¹ H NMR (300 MHz, CDCl 3) δ 0.93 (3H, d, J = 6.2 Hz), 0.94 (3H, d, J = 6.2 Hz), 1.25 (3H, t, J = 7.3Hz), 1.27 (3H, t, J = 7.3Hz), 1.36 (3H, d, J = 7.0Hz), 1.43 (9H, s), 1 , 53 (1H, d, J = 9.5Hz), 1.55-1.69 (5H, m), 2.18 (1H, dd, J = 2.9, 5.9Hz), 2. 44 (1H, dd, 3 = 2.9, 6.2Hz), 2.70 (1H, d, J = 15.8Hz), 4.06 (1H, broad s), 4.13 (4H, q, J = 7.3Hz), 4.18-4.28 (2H, m), 4.31 (1H, d, J = 5.9Hz), 4.41-4.45 (1H, m ), 4.85 (1H, broad s), 6.57 (1H, d, J = 7.3Hz), 6.97 (1H, broad s).

Anal. Calcd for C _{2 6 H 43} N 3 O 9 -0.1 H 2 O: C, 57.46; H, 8.01; N, 7.73. Found C, 57.18; H, 8.00; N, 7.64.

HRMS calcd for C ₂₆ H _{43 N 3 O 9} [M + Na] +; 564.2897. Found: 564, 2922.

Synthesis 42

(1S, 2S, 4S, 5R, 6R) -2- [2 '- (2S-tert-butoxycarbonylamino-3-methylbutyrylamino) propionylammo] -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

The process was carried out according to general procedure B using commercially available N-BOC- (L) -valine and (1S, 2S, 4S, 5R, 6R) -2- (2'S-aminopropionyl-amino) -4-hydroxybicyclo [acid diethyl ester hydrochloride] 3.1.0] hexane-2,6-dicarboxylic acid (0.2 g, 0.55 mmol, Synthesis 38). Purification was by PC-TLC, 4 mm SiO ₂ rotor, (10% ethyl acetate / hexanes to 67% ethyl acetate). 0.36 g of the product (43%, 0.68 mmol) was obtained in the form of a white foam.

[a] ²³ D = -65.5 ° (c = 0.58, MeOH).

¹ H NMR (300 MHz, CDCl 3) δ 0.91 (3H, d, J = 7.0 Hz), H 0.97 (3H, d, J = 6.6 Hz), 1.27 (3H, t , J = 7.3Hz), 1.28 (3H, t, J = 7.0Hz), 1.37 (3H, d, J = 1 7.3Hz), 1.44 (9H, s) , 1.54- 1.61 (3H, m), 2.12-2.20 (2H, m), 2.42 (1H, dd, J = 2.6, 5.9Hz), 2.70 (1H, d, J = 15.4Hz), 3.90 (1H, t, J = 6.6Hz), 4.13 (2H, q, J = 7.0Hz), 4, 20-4 . 29 (2H, m), 4.31 (1H, d, J = 5.9Hz), 4.40-4.48 (1H, m), 4.93 (1H, broads), 6.41 (1H, d, J = 7.3Hz), 6.91 (1H, broad s).

Anal. Calcd for C ₂₅ H41N3O ₉ 0.2 CH ₂ Cl _2: C, 55.58; H, 7.66; N, 7.72. Found: C, 55.76; H, 7.27; N, 7.49.

MS (ES) m / z 528.3 [M + H] +.

Synthesis 43

(1R, 4S, 5R, 6R) -2- (2'S-acetylaminopropionylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester

PL 221 526 B1

Solution of (1S, 2S, 4S, 5R, 6R) -2- (2'S-aminopropionylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid, hydrochloride (0.30 g, 0.82 mmol The synthesis of 38) in CH ₂ Cl ₂ (30 mL) was stirred at 0 ° C then triethylamine (0.16 g, 1.6 mmol) was added sequentially, followed by acetyl chloride (0.10 g, 1.18 mmoles). The reaction mixture was allowed to warm with stirring overnight. The reaction mixture was diluted with ethyl acetate (700 ml) and washed with an aqueous NaHSO4 solution and brine. The organic layer was dried over magnesium sulfate. Purification was by PC-TLC, 4mm SiO2 rotor, (10% ethyl acetate / hexanes to 100% ethyl acetate) to give 0.24 g (79%, 0.65 mmol) of the product as a white foam.

[a] ²³ o = -48 ° (c = 0.5, MeOH).

¹ H NMR (300 MHz, CDCl 3) δ 1.27 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz), 1.37 (3H, d, J = 7.0 Hz), 1.55-1.62 (2H, m), 1.99 (3H, s), 2.19 (1H, dd, J = 3.3, 6.6 Hz), 2.40 (1H, dd, 3 = 2.6, 5.9Hz), 2.77 (1H, d, J = 15.8Hz), 4.07 (2H, q, J = 7.0Hz ), 4.21-4.31 (2H, m), 4.33 (1H, d, J = 6.2Hz), 4.43-4.47 (1H, m), 6.13 (1H, broad s), 7.06 (1H, broad s).

Anal. Calculated for C17H26N2O7: C-55.13; H, 7.08; N, 7.56.

Found: C, 55.05; H, 7.12; N, 7.29.

HRMS calcd for C17H26N2O [M + Na] +, 393, 1638. Found: 393, 1644.

Synthesis 44

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

N-methylmorpholine (2.8 g, 27.2 mmol) and di-tert-butyl dicarbonate were added to a 1: 1 solution of O-acetyl-L-serine (3.7 g, 25.2 mmol) in dioxane: water. (5.8 g, 25.2 mmol). The reaction mixture was stirred for 24 hours and then partitioned between ethyl acetate and water. The aqueous layer was extracted with EtOAc + and the organics discarded, the pH was adjusted to 0-1 with aq. NaHSO4. The aqueous layers were extracted with ethyl acetate, dried over Na ₂ SO _4, filtered and concentrated under reduced pressure. Purification by flash chromatography gave 2.6 g (41.7%) of the product. The substances were used in Synthesis 15 without identification.

Synthesis of 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) was added to a suspension of LiH (14.9 g, 1.875 mol) in THF (900 ml) and N, N-dimethylpropylene urea (DMPU, 155 g, 1.2 mol). in THF (385 mL) over 30 minutes, keeping the temperature at 0-5 ° C. The reaction mixture was heated to 65 ° C and a solution of cis-1,4-dichloro-2-butene (95%, 100 g, 0.8 mol, 1.08 eq.) In THF (100 mL) was added over an hour while maintaining temperature at the level of 63-67 ° C. The reaction mixture was stirred for 4 hours at 65 ° C. Work-up of the reaction mixture with a water / MTBE mixture gave 1- (methoxycarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene.

¹ H NMR (300 MHz, CDCl3) δ 5.57 (s, 2H, CH = CH), 3.71 (s, 3H, CH3), 2.95 (s, 4H, 2CH _2), 1.42 ( s, 9H, C (CH3) 3).

IR (foil) 1734 (C = O), 1646, 1268, 1149 cm ^-1 .

The reaction solution containing 1- (methoxycarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene was cooled to 10-15 ° C and added to the cooled solution (10-15 ° C) 1N NaOH (1.3L, 1 , 3 mol) in 30 minutes. The reaction solution was stirred at 25 ° C for 24 hours and the course of the reaction was monitored by GC test. When the hydrolysis reaction was complete, MTBE (645 ml) was added to the reaction mixture and the solution was stirred for 5 minutes. Layers have been created and separated. The organic layer was discarded. 1.5 M was added to acidify the aqueous layer

NaHSO4 solution (1470 ml) (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 a 5% LiCl solution. The organic layer was concentrated under reduced pressure and diluted with heptane (780 mL). The solution was concentrated to a volume of approximately 500 mL and the resulting suspension was stirred 1 hour at ambient temperature. The solid was filtered, washed with heptane (250 ml) and dried under high vacuum at 35 ° C to give 103.29 g (61% yield) of 1- (carboxylic acid) -1- (tert-butoxycarbonyl) cyclopent-3- enu in the form of a white solid.

Melting point = 119 ° C.

¹ H NMR (300 MHz, CDCl 3), δ 5.61 (s, 2H, CH = CH), 3.00 (s, 4H, 2CH2), 1.46 (s, 9H, C (CHs) s).

IR: 3800-3000 (broad, COOH), 1741 (CO ₂ R), 1705 (CO ₂ H), 1283), 1149 cm ^-1 .

1- (carboxylic acid) -1- (tert-butoxycarbonyl) cyclopent-3-ene (50 g, 236 mmol) was dissolved in 850 ml of toluene: MTBE 70:30 under nitrogen in a 1 L flask equipped with a mechanical stirrer . Thionyl chloride (33.6 g, 283 mmol, 1.2 eq.) Was added and the reaction mixture was stirred while the temperature was kept at 23 ° C. The reaction solution was cooled to 0-5 ° C, and triethylamine (32.2 g, 318 mmol, 1.35 eq.) Was added dropwise over 30 minutes. The reaction mixture was warmed to 23 ° C and stirred for 1 hour. The reaction mixture was added quickly dropwise to deionized water (625 ml) maintaining the temperature at 20-25 ° C. The layers were separated and the organic layer was washed with 500 ml of 1M NaHCO3 solution. Concentration of the organic layer allowed to isolate 1- (chlorocarbonyl) -1- (tert-butoxycarbonyl) -cyclopent-3-ene as a light yellow liquid.

¹ H NMR (300 MHz, CDCl3) δ 5.61 (s, 2H, CH = CH), 3.04 (apparent q, J = 15.1 Hz, 4H, 2CH _2), 1.49 (s, 9H , C (CHa) a).

IR (foil) 1796 (COCl), 1743 (CO ₂ R), 1274, 1233, 1158 cm ^-1 .

A solution of tetrabutylammonium hydrogen sulfate (0.81 g, 2.4 mmol) in deionized water (700 mL) was added to sodium azide (20.16 g, 310 mmol). To the azide solution was added a solution containing 1- (chlorocarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene in MTBE / toluene mixture over 45 minutes. The reaction mixture was stirred for 3 hours at 23 ° C until 1- (chlorocarbonyl) -1- (tert-butoxycarbonyl) cyclopent-3-ene was consumed by GC analysis of the reaction solution. The layers were separated and the organic layer was washed with 1M NaHCO3 (540 ml) and deionized water (540 ml then 270 ml). The organic layer was dried with Na ₂ SO ₄ and filtered. The solution was concentrated under reduced pressure to yield 1- (acyl azide) -1- (tert-butoxycarbonyl) cyclopent-3-ene as an oil.

¹ H NMR (300 MHz, CDCl3) δ 5.58 (s, 2H, CH = CH), 2.96 (apparent t, J = 2.3 Hz, 4H, 2CH _2), 1.46 (s, 9H , C (CH3) 3).

IR (foil) 2137 (CON3), 1720 (CO ₂ R), 1246 (s, 1185, 1136 cm ^-1 ).

To 110 ml of toluene at 95 ° C was added the solution of (acyl azide) -1- (tert-butoxycarbonyl) cyclopent-3-ene over 1 hour. The evolution of nitrogen gas was controlled by the addition rate under these conditions. MTBE was distilled from the reaction mixture during the addition. The reaction mixture was stirred for 1 hour at 95 ° C and allowed to cool to 23 ° C overnight. Evaporation of the solvent under reduced pressure gave 1- (isocyanate) -1- (tert-butoxycarbonyl) cyclopent-3-ene as an oil.

¹ H NMR (300 MHz, CDCl 3) δ 5.67 (s, 2H, CH = CH), 3.01 (d, J = 15.6 Hz, 2H), 2.61 (d, J = 15.6 Hz, 2H), 1.50 (s, 9H, C (CHs) s).

IR (foil) 2258 (-NCO), 1732 (-CO ₂ R), 1157 cm ^-1 .

To a solution of potassium t-butoxide (1M in THF, 471 mL, 471 mmol) was added t-butanol (35 g, 471 mmol) under a nitrogen atmosphere. The reaction solution was cooled to 0-5 ° C and a toluene solution containing 1- (isocyanate) -1- (tert-butoxycarbonyl) cyclopent-3-ene was added over 60 minutes, maintaining the temperature at 0-10 ° C. The reaction mixture was warmed to 23 ° C, stirred for 2 hours and examined by GC for disappearance of the isocyanate starting material. 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 were separated. The organic layer was washed with 20% brine (250 ml) and the layers were separated. The organic layer was concentrated by distillation to a volume of approximately 250 mL. Heptane (500 ml) was added and the solution was concentrated to a total volume of 250 ml. The resulting thin slurry was cooled to 0 ° C, stirred for hours and filtered. The filter cake was washed with cold heptane (2 x 100 ml) and dried in vacuo to give 34.54 g (52% yield relative to 1- (carboxylic acid) -1- (tert-butycarbonyl) cyclopent- 3-ene) -1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) cyclopent-3-ene as a white solid.

Melting point 87-89 ° C.

¹ H NMR (500 MHz, CDCl 3) δ 5.63 (s, 2H, CH = CH), 5.1 (broad s, 1H, NH), 2.99 (d, J = 17.2 Hz, 2H) , 2.57 (d, J = 16.0 Hz, 2H), 1.46 (s, 9H), 1.44 (s, 9H).

¹³ CNMR (CDCl3) δ 173.3, 154.9, 127.7, 81.1.64.5, 44.8, 27.8.

IR (KBr): 3451, 2981, 2932, 1712, 1489, 1369, 1154 cm ^-1 .

MS (FIA) m / e (% relative intensity) 284.2 (M ++ 1.56), 228.2 (73), 172.1 (97), 128.0 (100).

To a mixture of tributylammonium bisulfate (4.08 g, 10 mmol, 0.17 eq), magnesium monoperoxyphthalate hydrate (MMPA, 52.4 g, 4.76% active oxygen = 2.1 eq) and deionized water (100 ml) was added a solution of 1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) cyclopent-3-ene (20.00 g, 71 mmol) in 100 mL of EtOAc. The reaction mixture was stirred with a mechanical stirrer for 19 hours. 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 were separated. The organic layer was washed with deionized H ₂ O (100 mL), 2N NaOH (2 x 100 mL), and deionized H ₂ O (2 x 100 mL). The organic layer was distilled at 75-90 ° C at atmospheric pressure until a volume of 40 ml was reached. 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 suspension was allowed to cool to ambient temperature and then cooled further in an ice bath for 1 hour. The product was filtered and washed with 80 ml of cold heptane: EtOAc 4: 1, then dried under high vacuum at 40 ° C to give 15.45 g (73% yield) of the title compound as a white solid. The compound can be further purified by recrystallization from isopropanol: water 2: 1. Melting point 130-133 ° C.

¹ H NMR (500 MHz, CDCl 3) δ 5.0 (broad s, 1H), 3.58 (s, 2H), 2.41 (d, J = 15.3 Hz, 2H), 2.20 (d , J = 15.2 Hz, 2H), 1.43 (s, 9H), 1.40 (s, 9H).

¹³ C NMR (CDCl 3) g 171.3, 154.3, 81.2, 79.5, 62.8, 57.0, 38.6, 28.3, 27.7.

IR (KBr): 3453, 2982, 2932, 1726, 1708, 1489, 1369, 1293, 1156, 840 cm ^-1 .

MS (FIA) m / e (% relative intensity) 300.3 (M ++ 1.65, 244.4 (68), 188.2 (100) 144.1 (99).

Synthesis 46

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

(R) -a-methylbenzylamine (98% enantiomeric excess, 121 g, 1 mol) was heated to 100 ° C under a nitrogen atmosphere. 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 solid, and the mixture was allowed to cool to room temperature over 30 minutes. MTBE (100 ml) was added. The precipitate was dissolved by adding 100 ml of water, then the layers were separated. The organic layer was washed with 50 mL of brine, dried (Na ₂ SO ₄ ), and concentrated in vacuo to yield 126.0 g of a light yellow oil. The oil was vacuum distilled at 70 ° C (head temperature) using a 12 inch column to remove excess (R) -α-methylbenzylamine. The pot residue (68 g of crude diamine) was dissolved in 1 L i-PrOH in a 2 L flask with a mechanical stirrer. Concentrated HCl (12 M, 45 mL, 540 mmol) was added dropwise over 10 minutes. An additional 100 mL of i-PrOH was added to ensure thorough mixing of the thick slurry. The mixture was stirred for 90 minutes and filtered. The filter cake was washed with i-PrOH and dried in high vacuum at 40 ° C to give 64.32 g (72% yield based on dibromopropane) of the title compound as a white solid.

¹ H NMR (500 MHz, DMSO-d6) δ 9.93 (broad s, 2H), 9.47 (broad s, 2H), 7.56 (d, J = 7.0 Hz, 4H), 7, 37 (m, 6H), 4.27 (m, 2H), 2.84 (m, 2H) 2.48 (m, 2H), 2.1 (m, 2H), 1.56 (d, J = 6.7 Hz, 6H).

PL 221 526 B1

Synthesis 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, Synthesis 46) was dissolved in 150 mL of water with stirring. 35 ml (175 mmol) of 5N were added to the solution. 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 of brine, dried with 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-necked flask equipped with an external stirrer, 20.59 g (73.5 mmol) of 1R, 3R-di (methylbenzylamino) propane were dissolved in a mixture of 30 ml of dry MTBE and THF each under a stream of nitrogen. The solution was cooled to -45 ° C and 59 mL (147 mmol) of n-BuLi solution (2.5M in hexanes) was added dropwise over 17 minutes. The pale yellow solution was stirred for 2 hours at -45 ° C. A solution of (1S, 3S, 5R) -3- (tert-butoxycarbonylamino) -3- (tert-butoxycarbonyl) -6-oxabicyclo [3.1.0] hexane (10.00 g, 33.4 mmol) in 55 ml was added dropwise. dry THF, for 28 minutes, keeping the temperature below -40 ° C. The dropping funnel was rinsed with an additional 5 mL of THF. The reaction mixture was stirred for 18 hours at -45 ° C, then quenched by the dropwise addition of 4N aqueous sulfuric acid (75 ml). The cooling bath was removed after all the acid had been added. The layers were separated and the aqueous layer was extracted with 50 ml of MTBE. The combined organic layers were washed with 50 mL portions of water and brine, dried with Na ₂ SO _4, and concentrated in vacuo to yield 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 portionwise. The mixed solution was allowed to cool to room temperature, then the mixture was stirred for 1 hour at 0 ° C. The solid was filtered, washed with 2 x 10 ml of cold heptane: MTBE 2: 1 and dried under high vacuum for 4 hours at 35 ° C, yielding 8.52 g (85% yield) (1S, 3R) -1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) -3-hydroxycyclopent-4-ene as a white solid.

Melting point 111-112 ° C.

Chiral HPLC analysis: 99.7% enantiomeric excess.

[a] ²³ D = +114 (c 1, MeOH).

¹ H NMR (500 MHz, CDCl3) δ 6.1 (br s, 1H), 5.9 (br s, 1H), 5.55 (d, J = 5.0 Hz, 1H), 4.8 ( m, 1H), 4.44 (d, J = 10.5 Hz, 1H), 2.87 (dd, J = 14.5, 7.5 Hz, 1H), 2.00 (d, J = 14 , 5 Hz, 1H), 1.45 (s, 9H), 1.42 (s, 9H).

IR (KBr): 3413, 2983, 1703, 1491, 1370, 1309, 1255, 1155, 1055 cm ^-1 .

MS (FIA) m / e (% relative intensity) 300.3 (M ++ 1.15), 226.2 (29), 170.1 (100), 126.1 (89), 108.3 (20 ).

To a solution of (1S, 3R) -1- (tert-butoxycarbonylamino) - (tert-butoxycarbonyl) -3-hydroxycyclopent-4-ene (20 g, mmol) in MTBE (200 ml) was added 2.2,6,6- tetramethylpiperidinyloxy (free radical) (TEMPO) (0.84 g, 5.3 mmol) and KBr (0.63 g, 5.3 mmol, in 2 mL of water). The reaction mixture was cooled to 0 ° C and a NaOCl solution (3.14%, 240 g, 100 mmol) containing NaHCO 3 (8.4 g) was added dropwise keeping the temperature below 5 ° C. The reaction mixture was stirred 1 hour at 0 ° C and allowed to warm to room temperature. The layers were separated and the aqueous layer was extracted with MTBE (2 x 200 ml). The combined organic layers were washed with 200 ml of 1N HCl solution containing 2.21 g of KI followed by 10% Na2SO3 solution (200 ml). The organic layer was washed with water (2 x 200 ml) and concentrated to dryness under reduced pressure. The crude product was dissolved in MTBE (60 ml) at 50 ° C and crystallized by adding heptane (200 ml) over 1 hour. 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 under high vacuum to give 16.99 g (89% yield) of the title compound as a white solid.

PL 221 526 B1

Melting point 116-18 ° C.

[α] ²³ D = +123 (c1, MeOH).

¹ H NMR (500 MHz, CDCl ₃ ) δ 7.4 (br s, 1H), 6.32 (d, J = 5.5 Hz, 1H), 5.6 (br s, 1H), 2.87 (d, J = 18.2 Hz, 1H), 2.9 (d, J = 18.2 Hz, 1H), 1.43 (s, 18H).

¹³ C NMR (CDCl3) 8206.1, 170.2, 160.8, 154.9, 136,1,84,5, 66,1,46,6, 28.9, 28.4.

IR (KBr): 3419, 2983, 1722, 1487, 1730, 1300, 1259, 1151, 1012 cm ^-1 .

MS (FIA) m / e (% relative intensity) 254.2 (M ++ 1.11), 242.3 (18), 228.2 (13), 186.1 (76), 143.2 (11), 242.3 (100).

Synthesis 48

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

To a solution of tetrahydrothiophene (2.19 L, 24.8 mol, eq.) In acetone (11.38 L) in a 22 L flask was added t-Butyl bromoacetate (2.44 L, 16.52 mol, 1 eq.) for 30-60 minutes while maintaining the temperature between 15-25 ° C using a water bath. The reaction mixture was stirred for ₁ hour, and the sample was examined by ¹ H NMR to confirm completion of the reaction. The precipitate was filtered, washed with acetone (2 L) and dried under high vacuum for 3 days at 28-33 ° C to give 4.328 kg (92.5% yield) of the title compound.

¹ H NMR (500 MHz, DMSO d-6) δ 4.40 (s, 1H), 3.51 (m, 2H), 3.48 (m, 2H), 2.23 (m, 2H), 2 , 13 (M, 2H), 1.42 (s, 9H).

Synthesis 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) was added to a solution of 1- (2-tert-butoxy-2-oxoethyl) tetrahydrothiophene bromide (11.9 g, 42 mmol, 2.5 eq. Synthesis 48) in acetonitrile (30 ml) at 0 ° C. , 42 mmol, 1M solution in THF, 2.5 eq) under a N ₂ atmosphere for 10 minutes, keeping the temperature below 5 ° C. The milky solution was stirred with cooling for 1.5 hours. Trifluoroethanol (6.9 g, 69 mmol, 4.1 eq.) Was added dropwise. A solution of (1S) -1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) cyclopent-4-en-3-one (5 g, mmol, Synthesis 47) and trifluoroethanol (13.3 g, 132 mmol, equivalent) in acetonitrile (20 mL) over 5 minutes, keeping the temperature at 35 ° C. The solution was stirred for 4.5 hours at 0-5 ° C. MTBE (155 ml) and H 2 O (80 ml) were added to the cold reaction mixture. The layers were separated and the organic layer was washed with H 2 O (50 mL) followed by 20% brine (50 mL). The organic layer was concentrated by atmospheric distillation to a volume of approximately 30 mL. Heptane (100 ml) was added and the solution was concentrated. Additional heptane was added as needed until the distillate vapor temperature was 93 ° C. THF (65 ml) was added to give a solution of the title compound.

Synthesis 50

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

To a solution of 1- (2-tert-butoxy-2-oxoethyl) -tetrahydrothiophene bromide (757 g, 2.67 mol, Synthesis 48) in 2.2 L CH ₂ Cl ₂ at 0 ° C, 1.7 1 of saturated aqueous K ₂ CO _{3 solution} , keeping the temperature below 10 ° C. After the biphasic mixture was stirred for 1.5 hours, 223 ml of 50% NaOH solution was added portionwise keeping the temperature below 5 ° C. The mixture was stirred for 3 hours and filtered. The precipitate was washed with CH2Cl2. 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 under reduced pressure

It was pressurized to give 533.3 g (98% yield) of (2-tert-butoxy-2-oxoethyl) tetrahydrothiophene as a light yellow oil. Upon storage in the freezer, the oil crystallizes to give an off-white solid.

Melting point 48-50 ° C.

¹ H NMR (500 MHz, CDCl 3) δ 3.12 (m, 2H), 3.00 (m, 2H), 2.85 (s, 1H), 2.41 (m, 2H), 1.84 ( m, 2H). 1.38 (s, 9H).

To a solution of (1S) -1- (tert-butoxycarbonylamino) -1- (tert-butoxycarbonyl) cyclopent-4-en-3-one (194 g, 653 mmol) in 650 ml CH ₂ Cl ₂ at 0 ° C , 474 mL (6.5 mol, 10 eq.) of trifluoroethanol was added. A solution of (2-tert-butoxy-2-oxoethyl) tetrahydrothiophene (396 g, 1.96 mol) in 325 ml CH ₂ Cl _{2 was added} dropwise over 40 minutes keeping the temperature below 10 ° C. The ice bath was removed after 1 hour. Deionized water (680 ml) was added and the layers were separated. The aqueous layer was extracted with 400 mL of CH2Cl2. The combined organic layers were washed with 500 mL of brine, dried with Na ₂ SO _4, and concentrated in vacuo to give 587 g of an amber oily solid. The solid was dissolved in 400 ml of CH ₂ Cl ₂ and eluted through a bed of 1.6 kg of silica gel, eluting with hexanes: methyl t-butyl ether: CH ₂ Cl ₂ 5: 1: 1, yielding a total of 13.2 liters of eluent. . The eluent was concentrated to give 398.7 g of a white solid. Dissolve the solid in 3 L of boiling hexanes: MTBE 70:30. The solution was allowed to cool to room temperature overnight then cooled in an ice bath for 1 hour. The solid was filtered, washed with cold solvent (approximately 700 ml) and dried in high vacuum at 35 ° C to give 173 g (64% yield) acid di-tert-butyl ester (1S, 2S, 5R, 6R) -2 - (tert-butoxycarbonylamino) -4-oxo-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid as a white solid.

Melting point 144-46 ° C.

[a] ²³ D = + 30.5 (c1, CHCl3).

¹ H NMR (500 MHz, CDCl 3) δ 5.36 (d, 1H), 2.88 (m, 1H), 2.64 (dd, J = 5.2, 3.2 Hz, 1H), 2, 37 (d, J = 2.7Hz, 1H), 2.23 (broad s, 1H), 1.45 (s, 9H), 1.43 (s, 18H).

¹³ C NMR (125 MHz, CDCl 3) δ 206.2, 171.2, 168.5, 155.3, 82.7, 80.7, 61.2, 43.2, 36.0, 34.328, 4, 28.2, 28.0, 25.3.

IR (CHCl): 2982, 1744, 1719, 1485, 1394, 1309 cm ^-1 .

MS (ES +) m / e (% relative intensity) 412.2 (M ++ 1.79), 356.2 (50), 300.1 (97), 276.1 (68), 244.1 (100 ).

Analysis: Values calculated for C ₂₁ H ₃₃ NO ₇ (411.29): C, 61.30; H, 8.08; N, 3.40.

Found C, 61.32; H, 8.04; N-3.51.

Synthesis 51

(1S, 2S, 4S, 5R, 6R) -2-tert-butoxycarbonyl] amino) -4-hydroxybicyclo [3.1.0] hexane-6-dicarboxylic acid di-tert-butyl ester

The synthesis solution 49 was cooled to 0 ° C under nitrogen and lithium tri-sec-butyl borohydride (L-Selectride ^TM , 1M solution in THF, 21 mL, mmol) was added dropwise. The reaction mixture was stirred for 45 minutes at 0 ° C. 2M sodium carbonate solution (31 ml) was added dropwise 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 keeping 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 Na2SO3 solution (40 ml) and 1M NaHSO4 solution (40 ml). The organic layer was concentrated by distillation at atmospheric pressure to a volume of approximately 90 ml. Distillation was continued and heptane was added to keep the volume constant. When the distillate vapor temperature 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 high vacuum at 50 ° C to give 4.68 g (67% yield) of the acid di-tert-butyl ester (1S, 2S, 4S, 5R, 6R). ) -2- (tert-butoxycarbonylamino) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid as a white solid.

GB ¹ 221 526 B1 H NMR (CDCl3, 500 MHz) δ 5.31 (br s, 1H), 4.38 (d, JAB = 10.5 Hz, 1H), 4.30 (dd, J = 11. 0, 6.0Hz, 1H), 2.68 (d, J = 15.3Hz, 1H), 2.17 (m, 1H), 2.07 (m, 1H), 1.58 (m, J = 15.2 Hz, 1H), 1.45 (s, 9H), 1.44 (s, 9H), 1.43 (s, 9H).

¹³ CNMR (75 MHz, CDCl3) δ 175.2, 170.5, 155, 83.2, 81.3, 73.7, 66.9, 43.1, 36.0, 34.4, 29.3 , 28.3, 28.1,27.4, 22.1.

IR (CHCl3): 3445, 2982, 1714, 1485, 1361 cm ^-1 .

MS (ES +) m / e (% relative intensity) 414.2 (M ++ 1.58), 358.1 (75), 302.1 (78), 246.0 (100).

[a] ²³ o = -28.6 (c1, MeOH).

Synthesis 52

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

To a solution of Deoxofluor ^TM (16.05 g, 72.6 mmol, 1.2 eq.) In CH 2 Cl 2 (105 mL) at -78 ° C, a solution of di-tert-butyl acid (1S, 2S, 4S, 5R, 6R) -2- (tert -butoxycarbonylamino) -4-hydroxybicyclo [3.1.0] hexane-dicarboxylic acid (25.00 g, 60.5 mmol, Synthesis 51) in CH ₂ Cl ₂ (230 mL) over time 2 hours. The reaction mixture was stirred for 1.5 hours and an additional 1.66 g of Deoxofluor ™ was added. The solution was stirred for 30 minutes and allowed to warm to -10 ° C. Saturated NaHCO ₃ solution (105 ml) was added dropwise over 20 minutes keeping the temperature below 5 ° C. The pH of the aqueous layer was adjusted to 7 by the addition of 160 ml of saturated NaHCO3 solution. The mixture was allowed to warm to ambient temperature and the layers were separated. The aqueous layer was extracted with 100 mL CH ₂ Cl ₂ . The combined organic layers were washed with 250 ml of brine then dried with Na2SO4. The solvent was removed under reduced pressure and heptane (75 mL) was added to the crude product. The resulting mixture was heated to 50 ° C until all solids dissolved and the mixture was stirred at ambient 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 to give 20.23 g (80% yield) of the di-tert-butyl acid (1R, 2S, 4R, 5R, 6R) -2- (tert-butoxycarbonylamino) -4- fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid as a white solid.

Melting point 140-143 ° C.

[a] ²³ o = +20.6 (c1, CHCl3).

¹ H NMR (CDCl3, 500 MHz) δ 5.45 (dd, JH-F = 56, JH-H = 4.8 Hz, 1H), 5.28 (br s, 1H), 3.00 (m, 1H), 2.23 (broad s, 1H), 2.11 (m, 1H), 2.08 (m, 1H), 1.46 (s, 9H), 1.46 (s, 9H), 1 . 43 (s, 9H), 1.37 (m, 1H).

¹³ CNMR (CDCl3, 75 MHz) δ 177.7, 170.5, 155.0, 94.4, 92.0, 82.2, 81.5, 80.2, 64.7, 37.7, 33 , 2, 29.7, 29.3, 28.3, 28.1, 27.8, 20.4.

MS (ES +) m / e (% relative intensity) 416.2 (M ++ 1.66), 360.1 (67), 304.1 (100), 248.0 (60).

IR (CHCl3): 3444, 2981, 1715, 1485, 1369 cm ^-1 .

For a solution of (1R, 2S, 4R, 5R, 6R) -2- (tert-butoxycarbonylamino) -4-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid di-tert-butyl ester (14.87 g, 0.036 mol) in EtOH (abs, 149 ml) was added dropwise SOCl ₂ (21.29 g, 13.05 ml, 0.179 mol, 5 eq.) over 10 minutes without cooling to obtain a gently boiling solution. The solution was heated to reflux and the solution was heated overnight. The solvent was removed from the reaction mixture under reduced pressure. The residue was dissolved in EtOAc (150 ml) and a solution of 10% Na2CO3 (75 ml) was added dropwise with stirring over 5-10 minutes. The layers were separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organic extracts were washed with brine (1 x 50 ml), dried

After Na2SO4, filtered and concentrated the product, (1R, 2S, 4R, 5R, 6R) -2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid diethyl ester under reduced pressure to a thick liquid that solidified on standing (11.02 g).

N-Methylmorpholine (22.44 ml, 204 mmol) was added to a solution of N-Boc-L-alanine (38.62 g, 204 mmol) in 396 ml of methylene chloride at -22 ° C under nitrogen atmosphere followed by dropwise addition of isobutyl chloroformate (26.48 mL, 204 mmol) over a period of 15 minutes so that the temperature of the reaction mixture exceeded -18 ° C. The resulting slurry was stirred at -20 ° C for 30 minutes, then a solution of (1R, 2S, 4R, 5R, 6R) -2-amino-4-fluorobicyclo [3.1.0] hexane-2,6- was added. dicarboxylic acid (49.46 g, 191 mmol) in 247 mL of methylene chloride over 40 minutes so that the temperature of the reaction mixture does not exceed -16 ° C. The reaction mixture was removed from the cooling bath and stirred at ambient temperature for 70 minutes. 408 ml of 1N hydrochloric acid solution was added, the mixture was stirred for 5 minutes and the layers were separated.

The organic layer was washed with saturated aqueous sodium bicarbonate (1 x 408 ml), dried (Na ₂ SO ₄ ), filtered and the product, acid diethyl ester (1R, 2S, 4R, 5R, 6R) -2- (2'S-2 ' - (tert-butoxycarbonylamino) propionyl) amino-4-fluoro-bicyclo [3.1.0] hexane-2,6-dicarboxylic acid, concentrated under reduced pressure to a white foam (88.16 g).

To a solution of the crude acid diethyl ester (1R, 2S, 4R, 5R, 6R) -2- (2'S'-2 '- (tert-butoxycarbonylamino) propionyl) -amino-4-fluoro-bicyclo [3.1.0] hexane-2 Of 6-dicarboxylic acid (17.5 g, 37.3 mmol theoretical) in 46.6 mL of tetrahydrofuran at room temperature, 46.7 mL (93.4 mmol) of 2N sodium hydroxide solution was added. The biphasic mixture was stirred vigorously at room temperature until it was homogeneous and then stirred for an additional hour (three hours total). The mixture was diluted with 46 mL of methyl t-butyl ether, then stirred for 10 minutes and the layers were separated. In a separate flask, 93 mL of water was prepared, then 8.4 mL (101 mmol) of concentrated HCl was added. Finally, the acid solution was seeded with acid crystals (1R, 2S, 4R, 5R, 6R) -2- (2'S-2 '- (tert-butoxycarbonylamino) propionyl) amino-4-fluoro-bicyclo [3.1.0] hexane-2, 6-dicarboxylic acid, then the aqueous layer was collected. First, the aqueous layer was added slowly so as to form a moderately thick slurry. At this time, the addition rate was increased (40 minutes total addition time). The dropping funnel was washed with water (16 ml). The resulting slurry was stirred for 2 hours, filtered, washed with water (2 x 32 mL), and dried in high vacuum at 45 ° C to constant weight to give 13.9 g (99%) of the title compound as a white solid. .

Synthesis 53

(1R, 4S, 5S, 6S) -4-Amino-2,2-dioxo-2/7-thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester

For a suspension of (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2/ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (10 g, mmol, patent No. 5,688,826) in 100 mL of 2B ethanol at room temperature (15.5 mL, 212.6 mmol) was added dropwise with thionyl chloride over 20 minutes, then the mixture was rinsed 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 a gelatinous residue. Ethyl acetate (50 mL) was added to this residue, and then the mixture was diluted with an additional 94 mL of ethyl acetate. A 15% aqueous sodium carbonate solution (70 mL) was slowly added to the mixture with manual stirring to obtain gradual dissolution to give a final pH of 7.95. The mixture was filtered and the layers were separated. The aqueous layer was extracted with ethyl acetate (2 x 100 ml). The combined organic extracts were washed with brine (1 x 100 mL), dried (MgSO ₄ ), filtered, and concentrated in vacuo to give the title compound as a faint yellow oil that solidified to an off-white solid (11.71 g, 95% yield) ).

Melting point 80-83 ° C.

[a] ²⁵ o = -57.7 (c 1.04, CH 3 OH).

PL 221 526 B1

500 MHz NMR (CDCl3) δ 4.31 (q, 2H, J = 7.0 Hz), 4.20 (m, 2H), 3.78 (d, 1H, J = 15.0 Hz), 3, 36 (dd, 1H, J = 4.0, 7.0Hz), 2.93 (dd, 1H, J = 4.0, 7.0Hz), 2.81 (d, 1H, J = 15, 0Hz), 2.46 (t, 1H, J = 4.0), 1.34 (t, 3H, J = 7 = 0), 1.30 (t, 3H, J = 7.0).

¹³ C NMR (125 MHz, CD3Cl3) δ 171.68, 168.57, 63.26, 62.42, 59.96, 56.06, 43.78, 32.25, 22.49, 14.31, 14.25.

FTIR (ATR) 3364.15 (s), 1725.95 (s), 1304.91 (s), 1259.24 (s), 1200.84 (s), 1104.91 (s), 1022.99 ( s), 896.45 (s), 851.21 (s) cm ^-1 .

Analysis calculated for C11H17NO6S: C, 45.35; H, 5.88; N, 4.81. Found C, 45.02; H, 5.75; N, 4.82. Synthesis 54

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '- (tert-butoxycarbonyl) aminobutanonyl) amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1. O] hexane-4,6-dicarboxylic acid

To a clear solution of N-Boc-L-methionine (32.64 g, mmol) in 110 mL of methylene chloride was added N-methylmorpholine (14.4 mL, 130.9 mmol) at -22 ° C under nitrogen and then iso-butyl chloroformate (17 mL, 130.9 mmol) was added dropwise over 7 minutes while maintaining the temperature of the reaction mixture at -22 ° C. After the addition was complete, a thin slurry formed. This mixture was stirred at -22 to -26 ° C for 30 minutes. A solution of (1R, 4S, 5S, 6S) -4-amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid diethyl ester (35.65 g, 122 , 4 mmol, Synthesis 53) in 107 mL of methylene chloride, over 15 minutes, then the mixture was washed with 36 mL of methylene chloride. The reaction mixture was removed from the cooling bath and stirred at room temperature for 70 minutes. 51 ml of 5N hydrochloric acid was added to the solution, 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 x 107 ml), dried (MgSO ₄ ), filtered and concentrated in vacuo to give 65.82 g (103% wt) of the title compound as a white foam.

[a] ²⁵ D = -12.7 (c 1.2, CH 3 OH).

500 MHz ¹ H NMR (CDCl3) δ 7.53 (s, 1H), 5.06 (d, 1H, J = 8.0 Hz), 4,34-4,20 (m, 6H), 3.41 (dd, 1H, J = 4.0, 7.0), 2.97-2.89 (m, 2H), 2.64-2.59 (m, 2H, J = 4.0), 2, 12-1.89 (m, 5H), 1.47 (s, 9H), 1.32 (t, 6H, J = 7.0).

¹³ C NMR (125 MHz, CD3OD) δ 172.53, 169.03, 167.88, 156.00, 80.62, 63.45, 62.56, 60.20, 55.33, 52.78, 42.81.31.52, 31.38, 30.12, 28.49, 22.69, 15.44, 14.23, 14.143.

FTIR (ATR) 3341.88 (w), 2979.38 (s), 1733.03 (s), 1674.92 (s), 1514.58 (s), 1315.80 (s), 1255, 15 ( s), 1161.47 (s), 1142.63 (s), 1025.68 (s), 854.85 (s), 763.53 (s) cm ^-1 .

Synthesis 55 (1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '- (tert-butoxycarbonyl) aminobutanonyl) amino-2,2-dioxo-2X ⁶ -thia-bicyclo [acid monosodium salt 3.1.0] hexane-4,6-dicarboxylic acid

For a solution of (1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '- (tert-butoxycarbonyl) aminobutanonyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo acid diethyl ester [3.1.0] hexane-4,6-dicarboxylic acid (58.95 g, 112.8 mmol, theory, Synthesis 54) in 141 ml of tetrahydrofuran was added 141 ml (282 mmol) of 2N sodium hydroxide solution at room temperature. The mixture was stirred vigorously at room temperature for two minutes. The solution was diluted with 141 mL of methyl tert-butyl ether and the layers were separated. The aqueous layer was then diluted with 141 ml of water, and concentrated hydrochloric acid was added dropwise to lower the pH to 4.46. The mixture was stirred for 10 minutes resulting in a thin slurry. More concentrated hydrochloric acid was added to the slurry to lower the pH to 1.4 (total of 17 mL concentrated HCl, 204 mmol) was used. After stirring for 2 hours, the suspension was filtered. The filter cake was washed with water (2 x 118 mL) and dried in vacuo at 45 ° C for 1 hour before being transferred to a dish. The filter cake was dried again under reduced pressure

At 45 ° C for 16 hours and 58 ° C for hours, to give 52.96 g (96% w / w) of the title compound as a white solid.

Melting point (decomposition) 258 ° C.

[a] ²⁵ D = -25.2 (c 1.03, H ₂ O).

500 MHz ^1H NMR (D20) δ 4.07-4.01 (m, 2H), 3, 45-3, 43 (m, 1H), 3.11 (d, 1H, J = 15.0 Hz) , 2.85 (m, 1H), 2.71 (s, 3H), 2.47-2.35 (m, 3H), 1.96-1.90 (m, 4H), 1.78-1 . 72 (m, 1H), 1.28 (s, 9H).

¹³ C NMR (125 MHz, CD3OD) δ 174.45, 173.58, 172.80, 157.46, 81.71, 61.41, 55.04, 53.24, 42.29, 31.71, 30.85, 29.41.27.79, 23.65, 14.41.

FTIR (ATR) 3287.62 (s), 1698.00 (s), 1528.91 (s), 1327.36 (s), 1283.74 (s), 1245.90 (s), 1174.81 ( s), 1109.06 (s), 1053.05 (s), 874.27 (s), 808.95 (s) cm ^-1 .

Synthesis 56

(1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '- (tert-butoxycarbonyl) aminobutanonyloamino-2,2-dioxo-2 _and ^{6-thia-bicyclo} [3.1.0] hexane -4,6-dicarboxylic acid

For a solution of (1R, 4S, 5S, 6S) -4- (2'S-4'-methylthio-2 '- (tert-butoxycarbonyl) aminobutanonyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo acid diethyl ester [3.1.0] hexane-4,6-dicarboxylic acid (166.15 g, 318 mmol, Synthesis 54) in 480 mL of tetrahydrofuran was added 397 mL (795 mmol) of 2N sodium hydroxide solution at room temperature. The mixture was stirred vigorously at room temperature for two minutes and the reaction mixture became homogeneous to give a clear slightly yellow / green solution. The mixture was stirred for an additional two hours at room temperature. The solution was diluted with 480 mL of methyl tert-butyl ether, then the layers were 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 the remainder of the aqueous layer to give an emulsion, and then the mixture was diluted with ethyl acetate (460 mL). The emulsion was stirred for 40 minutes, filtered and washed with water (2 x 250 ml). The layers of the filtrate were separated and the aqueous layer was re-extracted with ethyl acetate (500 ml). The combined organic layers were washed with brine (75 mL), dried (MgSO4), filtered and concentrated in vacuo to give 125.26 g (corrected yield 84%) of the title compound as a white foam.

Synthesis of 57 bromo-2-fluoro-2- (3-oxocyclopentyl) ethyl acetate

To a suspension of 2.99 g of activated Zn (45.8 mmol) in dry acetonitrile (100 ml) was added 8.45 ml (50.4 mmol) of triethylsilyl chloride at -20 ° C and the mixture was stirred for 5 minutes. 8.0 ml (57.3 mmol) of ethyl 2,2-dibromo-2-fluoracetate were added and the mixture was stirred for 90 minutes at -20 ° C. 1.86 ml (22.9 mmol) of cyclopenten-1-one was added and the reaction mixture was stirred overnight, allowing the temperature to slowly rise to room temperature. 1N HCl (125 ml) and EtOAc (100 ml) were added. The organic layer was washed with saturated NaHCO3 (2 x 150 ml), water (2 x 150 ml) and brine (2 x 150 ml), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. Purification of the residue by column chromatography using EtOAc / hexane (1: 8) as eluent afforded the title compound (5.36 g, 88% overall yield) as a colorless oil as a mixture of diastereoisomers.

¹ H-NMR (300 MHz, CDCl 3): 1.34-1.41 (m, 6H), 1.91-1.95 (m, 1H), 2.04-2.68 (m , 11H), 3.12-3.31 (m, 2H), 4.33-4.43 (m, 4H).

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

Synthesis of 58 ethyl (1RS, 5RS, 6RS) -6-fluoro-2-oxobicyclo [3.1.0] hexane carboxylate

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

¹ H-NMR (300 MHz, CDCl ₃ ): 1.33 (t, 3H, J = 7.1 Hz), 2.19-2.34 (m, 3H), 2.41-2.49 (m , 1H), 2.59 (d, 1H, J = 6.6Hz), 2.71-2.76 (m, 1H), 4.29 (q, 2H, J = 7.1Hz).

Synthesis 59

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

A mixture of ethyl (1RS, 5RS, 6RS) -6-fluoro-2-oxobicyclo [3.1.0] hexane-6-carboxylate (0.1 g, 0.54 mmol, Synthesis 58) and 1N NaOH solution (0.55 ml , 0.55 mmol) in EtOH (1 mL) was stirred for 10 minutes with ice cooling. 1 N 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 MgSO4 then concentrated under reduced pressure. A mixture of the residue, (NH4) 2CO3 (0.31 g, 3.2 mmol) and KCN (0.11 g, 1.62 mmol) in a mixture of EtOH and water (1: 1) (2 ml) was stirred at 60 ° C. C overnight. The mixture was cooled in an ice bath and acidified by treatment with a 1N KHSO4 solution. The solvent was removed under reduced pressure and the residue was redissolved in MeOH, filtered and concentrated under reduced pressure. The crude product was used without further purification.

PL 221 526 B1

Synthesis of 60 (1R, 2S, 5R, 6R) -2- (2'R-tert-butoxycarbonylaminopropionylamino) -6-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylate and diethyl (1S, 2R, 5S, 6S) -diethyl-2- (2'R-tert-butoxycarbonylaminopropionylamino) -6-fluoro-bicyclo [3.1.0] hexane dicarboxylate

Isomer A

Isomer B

To a solution of 0.32 g (1.5 mmol) of (1R *, 2S *, 5R *, 6R *) - 2-spiro-5'-hydantoin-6-fluorobicyclo [3.1.0] hexane-6-carboxylic acid ( Synthesis 59) in water (10 ml), 1.59 g (5.0 mmol) of Ba (OH) _{2 was added} and the mixture was stirred at 105 ° C overnight. The solution was acidified to pH 1 with 1 N HCl at 0 ° C, then the mixture was concentrated under reduced pressure. The residue was redissolved in absolute EtOH and concentrated in vacuo several times until the solid was very thoroughly dry. To the residue dissolved in absolute EtOH (20 ml) was added 0.37 ml (5.0 mmol) of SOCl ₂ at 0 ° C, then the mixture was stirred at reflux for 5 hours. The solution was basified with saturated NaHCO ₃ solution and EtOAc (25 mL) was added. The organic layer was separated and the aqueous layer was extracted with additional EtOAc (2 x 25 mL). The combined organic layers were dried over anhydrous MgSO ₄ , filtered, and concentrated in vacuo. The residue was dissolved in 20 ml of a DCM-DMF mixture (4: 1), 0.73 g (1.9 mmol) of HATU, 0.26 g (1.9 mmol) of HoAt, 0.35 g (1.8 mmol) of 1 -Ala. 2.7 ml (15.3 mmol) of diisopropylethylamine was added and the mixture was stirred under argon Ar overnight. DCM (15 ml) was added, the layers were separated and washed with saturated NaHCO3 (2 x 25 ml), water (2 x 25 ml) and brine (2 x 25 ml). The organic layer was washed over anhydrous MgSO4, filtered, and concentrated under reduced pressure. Purification of the residue by column chromatography using EtOAc / hexane (1: 2) as eluent gave the title compound (0.33 g, 51% overall yield) as a 1: 1 mixture of diastereomers as a colorless oil.

The diastereomeric mixture was separated by chiral HPLC using the following analytical kit: Chiralpak AD 10m, 4.6 x 250mm; Eluent: 10% IPA in hexane; Flow: ml / min; UV: 215 nm. A-isomer hold time = minutes. B-isomer hold time = 9.2 minutes.

Isomer A: (1R, 2S, 5R, 6R, 2'R) -2- (2'-tert-Butoxycarbonylamino-propionylamino) -6-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylate ¹ H-NMR (300 MHz, CDCl3): 1.18 (t, 3H, J = 7.1Hz), 1.23 (t, 1H, J = 7.1Hz), 1.36 (s, 9H), 1.56- 1.68 (m, 1H), 2.03-2.11 (m, 2H), 2.21-2.31 (m, 1H), 2.37-2.45 (m, 1H), 2, 61-2.63 (m, 1H), 4.09-4.16 (m, 5H), 5.10 (broad d, 1H, J = 6.6Hz), 7.11 (broad s, 1H) .

¹³ C-NMR (75 MHz, CDCl 3): 13.9, 14.0, 17.7, 24.9 (d, J = 1.0 Hz), 28.1, 31.5 (d, J = 10 , 9 Hz), 34.9 (d, J = 9.4 Hz), 36.4 (d, J = 8.8 Hz), 49.1, 61.5, 61.8, 67.4, 77 , 2.81.5 (d, J = 242.6 Hz), 168.6 (d, J = 25.1 Hz), 171.8 and 172.7 ppm.

Isomer B: (1S, 2R, 5S, 6S, 2'R) -2- (2'-tert-Butoxycarbonylamino-propionylamino) -6-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylate ¹ H NMR (300 MHz , CDCl3): 1.13 (t, 3H, J = 7.1 HZ), 1.18 (t, 1H, J = 7.1Hz), 1.31 (s, 9H), 1.51-1 , 60 (m, 1H), 1.98-2.07 (m, 2H), 2.15-2.22 (m, 1H), 2.26-2.39 (m, 1H), 2.55 -2.60 (m, 1H), 4.07-4.12 (m, 5H), 5.20 (sd, 1H), 7.22 (broad s, 1H).

¹³ CNMR (75 MHz, CDCl3): 13.7, 13.8, 18.0, 24.7, 28.0, 31.4 (d, J = 10.9 Hz), 34.5 (d, J = 8.9 Hz), 36.3 (d, J = 9.4 Hz), 61.4, 61.7, 67.3, 77.2, 81.4 (d, J = 242.6 Hz) , 155.2, 168.5 (d, J = 25.1 Hz), 171.7 and 172.8 ppm.

PL 221 526 B1

Synthesis 61

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

A solution of 30 mg (0.07 mmol) of Isomer A from Synthesis 60 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 room temperature overnight. The solvent was decanted and the residue was washed with ether and dried under Ar stream to give the title compound (12 mg, 85%) as a white solid.

[a] D = -25.0 (c = 0.80 mg / ml, H ₂ O).

Synthesis 62

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

Starting with the solution of isomer B obtained in the synthesis of 60, the title compound was in fact prepared by a method analogous to that in the synthesis of 61.

[a] D = 21.7 (c = 0.46 mg / ml, H ₂ O).

Synthesis 63 Ethyl (6S) -4-thiabicyclo [3.1.0] hex-2-ene-6-carboxylate

A 5-liter three-neck flask equipped with a stirrer, thermocouple, Teflon dosing tube and N ₂ inlet was charged with 2000 ml of thiophene (d = 1.05 g / ml, 25.0 moles at the beginning and increasing to 45 moles by adding the diazoacetate solution) of ethyl diazoacetate, 17.1 equivalents based on the total amount of ethyl diazoacetate added and uncorrected for EDA purity, 19.0 equivalents based on the adjusted purity of ethyl diazoacetate). To this was added, under nitrogen atmosphere, 0.968 g of Rh ₂ (octoate) (1.24 mmol, 0.0472 mol% based on g of ethyl diazoacetate, not corrected for purity, 0.052 mol% based on moles of pure ethyl diazoacetate, Johnson Mathey: Lot # 059255001). The slurry was heated to 46 ° C and stirred at 46 ° C for 10 minutes, dissolving into a green solution. To the solution was added via a positive displacement pump, a solution of 300 g of ethyl diazoacetate (90% pure, 2.63 moles, 2.37 moles of pura, equiv, Aldrich: Lot No. 17603PI) was dissolved in 1600 ml of thiophene. The rate of addition was such that the total addition time was 8 hours; the slow addition rate inhibits the formation of maleate and fumarate ethyl esters. When the ethyl diazoacetate was exhausted (approximately thirty minutes), the dark amber reaction mixture (3.985 g) was cooled to 23 ° C. The reaction mixture was aliquoted and the major part (3.240 g with 985 g total = 81.3%) was concentrated directly to an oil. The crude product was passed through a membrane distillation unit at 1.5 torr and 23 ° C to degass the product and to remove residual thiophene. The product was then distilled at 120 ° C and 1.3 torr. The title compound (faintly yellow) was collected in two fractions, 155.0 g and 32.2 g. Analysis by HPLC revealed a purity of 78% and 76% for the two portions respectively. Crystallization of the distillate from the above product was carried out by dissolving it in methanol (2 ml per 1 g of distillate) and cooling to -10 ° C, and the solution was seeded as

Crystal growth occurred. When crystal growth began, the mixture was cooled to -45 ° C and stirred for 2-3 hours, filtered and washed with cold (-45 ° C) methanol (1 x 1 mL per 1 g of distillate). The material was dried in vacuo at 24 ° C to afford the title compound as a white to off white solid with 80-85% recovery and> 98% purity.

Synthesis of 64 ethyl (4S, 6S) -4-hydroxyl-2-thiabicyclo [3.1.0] hexane-6-carboxylate

Borane-THF complex was added to a solution of ethyl (6S) -4-thiabicyclo [3.1.0] hex-2-ene-6-carboxylate (22.2 g, 131 mmol) in 136 ml of tetrahydrofuran under nitrogen at 0 ° C. (98 mL, 98 mmol) over 15-20 minutes. After stirring at 0 < 0 > C for 30 minutes, the reaction mixture was allowed to warm to 15 < 0 > C and was stirred until the reaction was complete as confirmed by HPLC (1.5-2 hours). The reaction mixture was cooled to 0 ° C and transferred over 10-15 minutes to 111 ml of pre-cooled (0 ° C) 1 N buffer, pH 7, keeping the temperature at 0 ° C. Sodium perborate monohydrate (15.6 g, 157 mmol) was added as a solid in five portions to the mixture so that the temperature was kept below 20 ° C. The solution was allowed to warm to room temperature and stirred for 1 hour, then 222 mL of water was added. After stirring for 2 hours, the peroxide reaction was stopped by adding sodium thiosulfate pentahydrate (9.7 g) dissolved in 24 ml of water, followed by stirring for 10 minutes. The mixture was extracted with ethyl acetate (2 x 222 ml). The combined organic extracts were washed with a saturated aqueous sodium bicarbonate solution (1 x 222 ml) followed by brine (1 x 222 ml) then concentrated under reduced pressure to dryness. The crude product was dissolved in 1,2-dichloroethane (1 ml per 1 g crude oil) and applied to a silica gel column (4 g silica gel per 1 g crude oil, suspended and packed in 15% ethyl acetate-heptane). The column was eluted with 15% ethyl acetate-heptane until product was found by TLC and the solvent was changed to 50% ethyl acetate-heptane. All fractions containing the title product were combined and concentrated in vacuo to an oil. The overall yield was in the range 55-65%.

Synthesis of 65 Ethyl (6S) -4-oxo-2-thiabicyclo [3.1.0] hexane-6-carboxylate

To a solution of dimethyl sulfoxide (33.4 mL, 471 mmol) in 194 mL of methylene chloride at -70 ° C, a solution of trifluoracetic anhydride (33.2 mL, 235 mmol) in 73 mL of methylene chloride was slowly added over 30 minutes (temperature kept below -66 ° C). After stirring for 20 minutes, a solution of ethyl (4S, 6S) -4-hydroxyl-2-thiabicyclo [3.1.0] hexane-6-carboxylate (34.1 g, 181 mmol) in 194 mL of methylene chloride was added over 60 minutes. so that the temperature is kept below -60 ° C. After stirring for one hour, the reaction mixture was treated with triethylamine (75.7 mL, 543 mmol) for 35 minutes such that the temperature remained below -50 ° C. The reaction mixture was allowed to stir for an additional 1 hour, then the cooling bath was removed and 400 ml of 2N hydrochloric acid solution was added. After warming to 0 ° C, the layers were separated and the organic layer was washed with 2N hydrochloric acid (1 x 300 ml), 1N aqueous sodium bicarbonate (1 x 670 ml) and water (1 x 300 ml), then dried over sodium sulfate , filtered and concentrated under reduced pressure to a red oil which solidified on standing. The crude product was passed through a pad of silica gel (2 g per 1 g of starting alcohol packed with methylene chloride) and eluted with methylene chloride (200-300 mL).

PL 221 526 B1

All product fractions were collected and concentrated to give the title compound as an orange / brown solid. Typical corrected efficiency is in the range 85-90%.

Synthesis 66

(6S, 11S) -8,10-dioxo-2-thiaspyro [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 allowed to stir 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 of methanol and warmed the reaction mixture to 30 ° C. C and stirred for hours. Volatiles were evaporated and the residue was dissolved in 2.75N sodium hydroxide solution (13.1 ml) and stirred for 1 hour. After diluting with 13.1 ml of water, the pH was lowered to 3.1 with concentrated hydrochloric acid and the mixture was seeded with (6S, 11R) -8.10-dioxo-2-thia-spiro [bicyclo [3.1.0] -hexane-4 , 5'-imidazolidine] -6-carboxylic acid. The pH was lowered to 1.0 and the slurry was cooled to 0 ° C and stirred for 1.25 hours. The brown solid was collected, washed with cold water (2.3 ml and 0.8 ml) and dried overnight in vacuo at 40 ° C to give 2.00 g (55% purity corrected) of the acid (6S, 11R ) -8,10-dioxo-2-thiaspiro [bicyclo [3.1.0] hexane-4,5'-imidazolidine] -6-carboxylic acid. 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 then the aqueous layer was washed with ethyl acetate (2 x 50 ml). The combined organic extracts were dried with sodium sulfate, filtered and concentrated in vacuo to a slurry (about 5 mL) to which was added methyl tert-butyl ether (25 mL) then stirred overnight. The solid was collected, washed with methyl tert-butyl ether and dried in vacuo at 40 ° C for 2 hours to yield 0.64 g (10% corrected for purity) of the title compound as a 1: 1 mixture of hydantoin diastereomers. This second part of the hydantoin was combined with the first part and passed through to the next step.

Separating

To a racemic acid suspension (15 g, 65.7 mmol, approximately 6: 1 hydantoin diastereomeric ratio) in 300 mL of ethanol and 75 mL of water was added (R) -phenylglycinol (9.0 g, 65.7 mmol). The mixture was heated to about 80 ° C to effect dissolution. The dark solution was allowed to cool slowly and precipitation was observed at 40-45 ° C. The slurry was then cooled to 0 ° C and held for 1-1.5 hours. The solid was collected, washed (with stirring) with ethanol: water 4: 1 (1 x 60 ml, pre-cooled to 0 ° C) and dried in vacuo at 65 ° C for 12-24 hours. Typical salt separation efficiencies are in the range 37-45% with> 98% de and> 98% enantiomeric excess. The separated salt was dissolved in volumes (mL per g) of water then treated with an equivalent of concentrated HCl. The slurry was cooled to 0 ° C and allowed to stir for 1 hour, then filtered, rinsed with 1 volume of cold water and dried under vacuum at 60 ° C. Typical yields of the title compound were> 90% with% de and% ee> 99%.

PL 221 526 B1

Synthesis 67

(6S, 11S) -2,2,8,10-Tetraoxo-2-thiaspyrbicyclo [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 solution and 186 mg (0.74 mmol) of tungsten acid was added (6S, 11S) -8.10-dioxo-2-thiaspyro [bicyclo [3.1 .0] -hexane-4,5'-imidazolidine] -6-carboxylic acid (3.4 g, 14.9 mmol). The resulting solution was heated to 50 ° C and treated with 35% hydrogen peroxide (1.7 mL, 74.5 mmol) slowly over 66 minutes. The reaction mixture was then allowed to stir at 47-48 ° C for 5 hours, then cooled to 0 ° C, filtered through a thin layer of Celite and rinsed with cold water (1 x 2 ml). The filtrate was heated to 50 ° C and treated with concentrated hydrochloric acid to pH = 1.5. The slurry was allowed to cool to room temperature and stirred overnight. After cooling to 0 ° C, the slurry was filtered, washed with cold water (2 x 2 ml) and dried under high vacuum at 55 ° C until constant weight to give 3.19 g (82%) of the title compound as a white solid body:

[a] ²⁵ o = -4 s, 6 (c, 1.19, 1N NaOH).

Mp 275 ° C (gray), 295 ° C (brown).

500 MHz ¹ H NMR (DMSO-d6) 613.15 (br s, 1H), 10.99 (s, 1H), 8.13 (s, 1H), 3.85 (d, 1H, J = 15. 0 Hz), 3.74 (dd, 1H, J = 7.0, 4.0 Hz), 3.03 (d, 1H, J = 15.5 Hz) 2.80 (dd, 1H, 7.0 , 4.0 Hz) 2.39 (t, 1H, J = 4.0 Hz) ¹³ C NMR (125 MHz, DMSO-d6) δ 174.39, 169.87, 156.35, 62.67, 52 , 59.44.16, 31.69, 21.92;

FTIR (KBr) 3317 (s), 3250 (s), 3211 (s), 3086 (w), 1791 (s), 1742 (s), 1713 (s), 1327 (s), 1192 (s), 1140 (s) cm ^-1 .

Analysis Calculated for C ₈ H ₈ N ₂ O 6S: C, 36.93; H-3.10; N, 10.77. Found C, 36.76; H-3.07; N, 10.60.

Synthesis 68

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

(6S, 11S) -2,2,8,10-tetraoxo-2-thiaspyro- [bicyclo [3.1.0] hexane-4,5'-imidazolidine] -6-carboxylic acid was added to a stainless steel Parr reactor (2.50 g, 9.60 mmol) and 2N sodium hydroxide solution (24.0 mL, 48.0 mmol). After heating the mixture to 95 ° C and stirring for 21 hours, the mixture was cooled to room temperature and treated with activated charcoal (1.25 g). The mixture was filtered through celite and the filtrate was concentrated to 17 g and diluted with H ₂ O to give a weight of 24 g. The pH was lowered to 6.5 using concentrated HCl and the mixture was heated to 62 ° C. Upon lowering the pH value to 2.5 with concentrated HCl, crystallization occurred. The slurry was allowed to cool to 30 ° C before the pH was adjusted to 1.7 and its temperature was lowered to 5 ° C. The slurry was left at this temperature for 18 hours, the solid was collected and washed with cold H ₂ O (2 x 2.9 mL). The white solid was dried in vacuo at 45 ° C to give the title compound (1.81 g, 80%). The title compound was suspended in 10 volumes of water and heated to 85 ° C for 4 hours, cooled to room temperature, stirred for 2-3 hours, filtered and washed with water (1 x 1 volume). Recovery is> 95%.

Synthesis 69 (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylate

To a suspension of (ethoxycarbonylmethyl) dimethylsulfonium bromide (134 g, 585 mmol) in 486 ml of acetonitrile at room temperature was added dropwise 87.4 ml (585 mmol) 1,8-diazabicyclo [5.4.0] -undec-7-ene in 15 minutes. After stirring for 1 hour, the yellow mixture was treated with 40 g (487 mmol) of 2-cyclopenten-1-one over 10 minutes. The mixture was allowed to stir overnight, then 480 mL of methyl tert-butyl ether was added, followed by washing with 1N hydrochloric acid (1 x 240 mL). The aqueous layer was washed with methyl tert-butyl ether (1 x 240 ml). The combined organic extracts were washed with brine (1 x 400 mL), dried (MgSO4), filtered, and concentrated in vacuo to provide crude ethyl (6S) -2-oxo-bicyclohexane-6-carboxylate as an orange solid (84.8 g ). The crude material was purified by distillation (~ 138 ° C, 10 mm Hg) followed by suspending the solidified distillate in heptane, filtering and drying.

Synthesis 70

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

To a solution of crude ethyl (6S) -2-oxobicyclo [3.1.0] hexane carboxylate (30.2 g, 180 mmol, uncorrected) in 30 mL of ethanol at room temperature, 89 mL (178 mmol) of 2N sodium hydroxide solution was added. After stirring for 80 minutes, the reaction mixture was washed with methyl tert-butyl ether (1 x 90 ml) and the aqueous layer was treated with concentrated hydrochloric acid (18 ml) to reach pH = 1.0. The mixture was treated with 15 g of sodium chloride then washed with ethyl acetate (3 x 90 ml). The combined organic extracts were dried (Na2SO4), filtered, and concentrated in vacuo to give 23.8 g (94%, uncorrected) of the title compound as an off-white solid.

Synthesis 71

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

To a solution of the crude acid of (±) (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylic acid (11.9 g, 84.9 mmol, assumed 100% purity) in 119 ml of ethyl acetate: ethanol 6: 1 at reflux, 18 g (85.1 mmol) of (S) -N-benzyl-α-methylbenzylamine were added. After dissolution, the mixture was allowed to cool and optionally seeded at 52 ° C. After cooling to room temperature and stirring an additional 13.5 hours, the crystals were collected and washed with ethyl acetate: ethanol 6: 1 (2 x 48 ml). Drying in vacuo gave 10.8 g (36%, 77% de) of the separated salt as a solid.

PL 221 526 B1

The de-salt value was determined by chiral GC analysis of the resulting methyl ester, prepared as follows: 150 mg of the salt to be separated was dissolved in 5 ml of methylene chloride and washed with 1N sulfuric acid (2 x 1 ml). The organic layer was dried, filtered, diluted with 2 mL of methanol and treated with 1 mL of a 2M solution of trimethylsilyl diazomethane in hexanes. After stirring at room temperature for minutes, the mixture was concentrated under reduced pressure to provide the methyl ester suitable for chiral GC analysis. GC conditions: 30m x 0.25mm x 0.25u, P-DEX 325 column, 140 ° C oven temperature, helium gas carrier @ 1ml / min, FID detection at 250 ° C, 1 µl, split 1: 100, 1 mg / mL sample in methylene chloride.

Synthesis of 72 (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylate

To a suspension of 46.3 g (132 mmol) of (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylic acid N-benzyl-α-methylbenzylamine salt in 200 ml of ethyl acetate was added 198 ml (198 mmol) of a 2N solution. sodium hydroxide. After thorough mixing, the layers were separated and the aqueous layer was washed with ethyl acetate (1 x 200 ml). The aqueous layer was treated with 18 ml (211 mmol) of concentrated hydrochloric acid and 100 g of sodium chloride. The mixture was allowed to stir for 30 minutes then washed with ethyl acetate (2 x 200 ml). The combined organics were dried (MgSO4), filtered, and concentrated in vacuo to give 18.3 g (99%) of the separated [(+) (6S) -2-oxobicyclo [3.1.0] hexane-6-carboxylic acid as a white solid.

Then, 10 g (71 mmol) of the crude resolved acid obtained above was dissolved in 42 ml of ethanol and treated dropwise with 4 ml (71 mmol) of concentrated sulfuric acid. The mixture was heated to 45 ° C and allowed to stir for 75 minutes. After cooling to room temperature, 42 ml of water were added along with 20 ml of ethyl acetate and 12 g of sodium bicarbonate. After stirring for several minutes, the mixture was washed with ethyl acetate (2 x 50 ml). The combined organics were dried (MgSO ₃ ), 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. Crystallization from 6: 1 heptane: methyl tert-butyl ether (3.5 ml per g substrate) gave the title compound in approximately 80% yield and> 98% ee as determined by chiral GC analysis.

Synthesis 73

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

A mixture 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) was heated to reflux with a Dean-Stark apparatus for 2.5 hours at which time GC analysis showed a mixture of the title compound and (65 ') - 2-oxobicyclo [3.1.0] hexane-6-carboxylate ethyl 9: 1. After removing 950 ml of solvent by distillation over 1 hour, the GC indicates that the product / starting material ratio is 17: 1. Additional isopropenyl acetate (900 mL) and concentrated H ₂ SO ₄ (3.15 mL) were added and the mixture was stirred at reflux for an additional 15 hours at which time GC indicated a product / starting material ratio of 21: 1. After an additional 1.35 L of solvent was distilled off, the mixture was cooled to room temperature before it was diluted with MTBE (2 L), H 2 O (250 mL) and a saturated aqueous NaHCO 3 solution (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 (Na2SO4), filtered and concentrated to a dark red / brown oil (540 g). The crude oil was divided into two equal portions and SiO ₂ flash chromatography (713 g for each feed) was performed, eluting with 10: 1 heptane: ethyl acetate. Product containing fractions from both beds were combined and concentrated to afford the title compound as a yellow oil (460 g, 97%; 90% solvent corrected by NMR). Column chromatography on silica gel, eluting with ethyl acetate / hexanes (1: 5) gave an analytically pure sample of the title compound as a colorless oil.

[a] ²⁵ D +185 (c 1.48, CHCl3).

500 MHz ¹ H NMR (CDCl3) δ 5,19-5,18 (m, 1H), 4.12 (q, 1H, J = 7.0 Hz), 4.11 (q, 1H, J = 7. 0Hz), 2.74-2.69 (m, 1H), 2.48-2.43 (m, 2H), 2.22-2.19 (m, 1H), 2.16 (s, 3H ), 1.39 (dd, 1H, J = 2.5, 2.5Hz), 1.25 (t, 3H, J = 7.0Hz).

¹³ C NMR (125 MHz, CDCl 3) δ 173.37, 169.01, 152.26, 111.56, 61.28, 32.47, 32.40, 29.72, 24.97, 21.67, 14.95.

FTIR (CHCl3) 3026 (m), 2985 (m), 1724 (s), 1272 (s), 1187 (s) cm ^-1 .

ES HRMS calcd for C 11 H 21 NOA [M + NH ₄ ] + 228, 1236,

Found 228, 1252.

Synthesis of 74 (3S, 1R, 6R) -oxa-5-oxotricyclo [4.1.0.0 <2.4>] heptane-3-carboxylate

A mixture of (6S) -6- (ethoxycarbonyl) bicyclo [3.1.0] hex-2-en-2-yl acetate (212.2 g, 1.01 mol) and 2,3-dichlorodicyano-1,4- The benzoquinone (252.0 g, 1.11 mol) in 2.02 L of 1,4-dioxane was heated to reflux and stirred for 17 hours, whereupon GC analysis indicated complete conversion of (6S) -4-oxobicyclo [ 3.1.0] hex-2-ene-6-carboxylate. The mixture was cooled to room temperature and diluted with THF (564 mL). After cooling the mixture to 8 ° C, 1,8-diazabicyclo [5.4.0] undec-7-ene (377 ml, 2.52 mol) was added over 30 minutes so that the temperature of the reaction mixture was kept below 10 ° C. . The mixture was then cooled to 5 ° C and tert-butyl hydroxide (70 wt% in water, 210 ml, 1.51 mol) was added over 50 minutes keeping the temperature of the reaction mixture below 9 ° C. After stirring the mixture for an additional 50 minutes, it was filtered and the brown cake was washed with MTBE (2 x 800 mL). 1.20 L of 1N HCl solution was added to the filtrate, and after mixing thoroughly, the layers were separated. The organic layer was washed sequentially with an aqueous saturated NaHCO3 solution (1.20 L), an aqueous saturated Nat2S2O3 solution (1.20 L), and brine (600 mL). The solution was then dried (Na2SO4), concentrated to an orange solid which was diluted with 200 mL of heptane. Volatiles were evaporated to yield an orange solid which was triturated with 350 mL of heptane and filtered, the filter cake was washed with additional heptane (2 x 175 mL). The collected solid was dried in vacuo at room temperature for 17 hours to provide 138.7 g (75%) of the title compound as a tan-yellow solid.

Crystallization from MTBE gave an analytically pure sample of the title compound as a white solid.

[a] ²⁵ D = +2.3 (c 1.20, CHCl 3), + 8.4 ° (c 1.28, acetone);

Melting point 129-130 ° C.

500 MHz ¹ H NMR (CDCl3) δ 4.16 (q, 2H, J = 7.0 Hz), 3.99 (t, ^1H, J = 2.5 Hz), 3,24-3,23 ( m, 1H), 2.96-2.94 (m, 1H), 2.21-2.19, (m, 1H), 2.08 (t, 1H, J = 3.0Hz), 1, 26 (t, 3H, J = 7.0Hz).

¹³ C NMR (125 MHz, CDCl 3) δ 201.19, 168.84, 62.42, 57.04, 51.25, 31.16, 30.54, 29.60, 14.79.

FTIR (KBr) 3087 (w), 3059 (w), 3051 (w), 3007 (w), 2993 (w), 2963 (w), 1753 (s), 1719 (s), 1273 (s), 1191 (s), 1009 (m), 848 (m) cm ^-1 .

Analysis Calculated for C ₉ H ₁₀ O ₄ : C, 59.34; H-5.53.

Found: C, 59.32; H, 5.43.

PL 221 526 B1

Synthesis of 75 ethyl (6S) -4-oxobicyclo [3.1.0] hex-2-ene-6-carboxylate

Although the title compound is typically used in situ in the preparation of ethyl (3S, 1R, 6R) -7-oxa-5-oxotricyclo [4.1.0.0 <2.4>] heptane-3-carboxylate, an analytically pure sample of the title the compound was obtained by filtering the reaction mixture containing this compound and concentrating the filtrate to obtain a brown solid. The solid was re-suspended in ethyl acetate, the suspension was filtered and the filtrate was concentrated. Chromatography of the residue on silica gel, eluting with ethyl acetate / hexanes (1: 5 to 1: 2) gave the title compound which was crystallized from hot ethyl acetate and rechromatographed using the previous conditions to give the title compound as a white solid.

[a] ²⁵ D -268 (c 1.17, CHCl 3).

Melting point 97-98 ° C.

500 MHz ¹ H NMR (CDCl3) δ 7.60 (ddd, 1H, J = 5.5, 2.5, 0.75 Hz), 5.73 (dd, 1H, J = 5.0, 0.5 Hz), 4.15 (q, 2H, J = 7.0Hz), 2.96-2.94 (m, 1H), 2.63-2.61 (m, 1H), 2.60 (t , 1H, J = 2.5Hz), 1.26 (t, 3H, J = 7.0Hz).

¹³ C NMR (125 MHz, CDCl 3) δ 203.96, 168.61, 160.33, 130.29, 62.03, 46.53, 30.72, 29.62, 14.82.

FTIR (KBr) 3080 (m), 2996 (m), 1717 (s), 1695 (s), 1266 (s), 1291 (m), 1191 (s), 1179 (s) cm ^-1 .

Analysis: calcd for C9H10O3: C, 65.05; H, 6.07. Found: C, 64.97; H, 6.01.

Synthesis of 76 ethyl (4S, 6S) -4-hydroxyl-2-oxobicyclo [3.1.0] hexane-6-carboxylate

Mixed solution of ethyl (3S, 1R, 6R) -7-oxa-5-oxotricyclo [4.1.0.0 <2.4>] heptane-3-carboxylate (36.3 g, 0.20 mol) in 667 mL of 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). The mixture was allowed to stir at 30 ° C for 15 hours before removing the acetone in vacuo to give a tan 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 x 323 ml). The combined organics were washed sequentially with an aqueous saturated Na ₂ S ₂ O _{3 solution} (364 ml) and an aqueous saturated NaHCO ₃ solution (364 ml). Each aqueous phase was re-extracted with ethyl acetate (323 ml). The combined organics were dried (Na ₂ SO ₄ ), filtered and concentrated to a red / brown oil which was dissolved in 300 mL of ethanol. Evaporation of the volatiles gave the title product as a reddish brown oil (41.8 g, 114%). Column chromatography on silica gel using ethyl acetate / hexanes (1: 2 to 2: 1) followed by crystallization from hot MTBE gave a pure sample of the title compound as a white solid.

[a] ²⁵ D +3.9 (c 1.39, CHCl 3), + 6.0 ° (c 1.69, MeOH).

Melting point 81-82 ° C.

500 MHz ¹ H NMR (CDCl 3) δ 4.60 (broad s, 1H), 4.16 (q, 2H, J = 7.0 Hz), 2.66 (dd, 1H, J = 5.0.4 , 0Hz), 2.42-2.40 (m, 1H), 2.34 (dd, 1H, J = 19.0, 5.5Hz), 2.24, (broad d, 1H, J = 3.0Hz), 2.07 (d, 1H, J = 19.0Hz), 1.91 (t, 1H, J = 3.0Hz), 1.27 (t, 3H, J = 7, 0 Hz).

¹³ C NMR (125 MHz, CDCl 3) δ 209.74, 170.07, 69.04, 62.32, 43.47, 36.89, 34.95, 26.14, 14.83.

FTIR (CHCl3) 3607 (w), 3447 (w), 3025 (m), 2985 (w), 1739 (s), 1728 (s), 1270 (s). 187 (s) cm ^-1 .

Analysis Calculated for C ₉ H ₁₂ O ₄ : C, 58.69; H, 6.57. Found: C, 58.48; H, 6.63.

PL 221 526 B1

Synthesis 77

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

To a solution of ethyl (4S ', 6S) -4-hydroxyl-2-oxobicyclo [3.1.0] hexane-6-carboxylate (68.2 g corrected to 60.0 g for ethanol contamination, 0.326 mol) in ethanol (332 mL) and H ₂ O (332 mL) were added (R) -methylbenzylamine (46.3 mL, 0.359 mol) and NaCN (20.8 g, 0.424 mol) keeping the temperature between 20 and 25 ° C. Concentrated HCl (35.3 mL, 0.424 mol) was then added over 10 minutes while maintaining the temperature of the reaction mixture above. The dark brown mixture was stirred for 1 hour before seeding with the title compound to start crystallization. The suspension was stirred for 1 hour before H ₂ O (664 mL) was added. The suspension was then stirred for another 1.75 hours, the title compound was collected as a brown solid which was washed with H ₂ O (332 mL). Air was blown through the wet cake on the filter for 25 minutes before using the substance directly for nitrile hydrolysis (wet cake weight 145 g). Although the title compound decomposes rapidly on drying in vacuo at temperatures above 25 ° C, it is possible to dry small samples in vacuo at room temperature without decomposing.

[a] ²⁵ D + 81.6 (cl. 18, CHCl ₃ ).

Mp 70-72 ° C (decomposition).

500 MHz ¹ H NMR (CDCl 3) δ 7.39 (d, 2H, J = 7.0 Hz), 7.26-7.16 (m, 3H), 4.31 (d, 1H, J = 5, 0Hz), 4.22 (q, 1H, J = 6.5Hz), 3.93-3.85 (m, 2H), 2.33 (d, 1H, J = 15.0Hz), 2 .01 (broad t, 1H, J = 4.5Hz), 1.64 (dd, 1H, J = 15.0, 5.0Hz), 1.55-1.54 (m, 1H), 1 , 40-1.39 (m, 4H), 1.17 (t, 3H, J = 7.0Hz).

¹³ C NMR (125 MHz, CDCl3) δ 170.54, 144.85, 128.61, 127.45, 127.38, 121.88, 72.17, 61.02, 60.66, 56.57, 45.82, 36.70, 34.45, 25.83, 21.75, 14.22.

FTIR (KBr) 3568 (m), 3489 (m, 3285 (m), 2923 (m), 2228 (w), 1712 (s), 1298 (m), 1197 (m) cm ^-1 .

FAB HRMS calcd for C 12 H 16 O [M + H] + 315, 1709, found 315, 1704.

Synthesis 78

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

To a solution of ethyl 2 - [((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2-cyano-4-hydroxybicyclo [3.1.0] hexane-6-carboxylate from wet filter cake (theory 0.326 mmol ) in DMSO (220 ml) 30% H ₂ O ₂ (44.5 ml, 0.426 mol) was added slowly keeping the temperature below 21 ° C. The temperature was lowered to 19 ° C and 5N NaOH (52.3 mL, 0.262 mol) was carefully and slowly added, first over 15 minutes, keeping the temperature between 22 and 27 ° C. An ice bath of adequate capacity is required to remove excess heat from this reaction. After stirring the brown, heterogeneous mixture for 20 minutes in the above temperature range, HPLC indicated the starting materials were consumed yielding the intermediate amide. After stirring the reaction mixture 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 and the mixture was free from peroxides using starch iodide paper. After adding 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 concentrated HCl was added to bring the pH down to 3.6 while maintaining the temperature between 30 and 35 ° C. After crystallization had begun at a pH value of 3.6, the suspension was stirred for 15 minutes until the water dropped to a r56.

PH values up to 2.5. After agitating the mixture for an additional 10 minutes, it was cooled to 2 ° C and stirred for 2 hours before collecting the gray solid, then it was 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, yielding 42.9 g (43% from the start in synthesis of 13) of the title compound. In order to recover all the title compound produced in the reaction from the process, it was recovered from the mother liquors as follows. The ethanolic portions of the mother liquor were concentrated and the residue was combined with the aqueous portions of the mother liquor. After distillation of H ₂ O (485 ml) under reduced pressure, the pH of the mother liquor was adjusted to 12.9 with 70 ml of 5N NaOH and 5 ml of 50% NaOH. After washing the solutions with n-BuOH (3 x 800 ml), its pH was adjusted to concentrated HCl and the solution was concentrated. The residue was diluted with EtOH (100 mL) and the volatiles were evaporated (2X). The residue was diluted with EtOH (150 mL) and the brown solid containing more title compound and salt was washed with EtOH (75 mL) and dried at 50 ° C in vacuo to a weight of 102 g. Both portions of the title compound were used in the subsequent esterification.

[α] ²⁵ ο +4.5 (c 1.41, 1N NaOH).

Mp 220 ° C (gray to off white), 280 ° C (brown).

500 MHz ¹ H NMR (D ₂ O, CODE) δ 7.39 (d, 2H, J = 7.0 Hz), 7.19-7.04 (m, 5H), 3.92 (d, 1H, J = 5.0Hz), 3.67 (q, 1H, J = 7.0Hz), 1.76 (d, 1H, J = 15.0Hz), 1.54-152 (m, 1H) , 1.37 (dd, 1H, J = 15.0, Hz), 1.15 (d, 3H, J = 6.5 Hz), 1.12 (dd, 1H, J = 6.0, 3, 0 Hz), 0.92 (t, 1H, J = 3.3Hz).

¹³ C NMR (125 MHz, D ₂ O, KOD) δ 185.82, 182.96, 148.01, 131.31, 129.97, 129.78, 74.99, 73.84, 58.78, 46.91.38.05, 35.02, 27.34, 27.15.

FTIR (KBr) 3366 (m), 3072 (s), 2886 (s), 1696 (m), 1611 (m), 1560 (m), 1455 (m), 1377 (m), 1278 (m), 1202 (m), 1188 (m) cm ^-1 .

Calcd for C16H19NO5: C, 62.94; H, 6.27; N, 4.59. Found: C, 62.70; H, 6.21; N, 4.67. Synthesis 79

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

Although the title compound is typically used in situ in the preparation of 2 - [((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid, the compound can be isolated at least with some loss in yield, due to the accompanying ester hydrolysis during nitrile hydrolysis. During isolation, the nitrile hydrolysis reaction mixture was partitioned between CH ₂ Cl ₂ and H ₂ O shortly after consumption of 2 - [((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2-cyano- Ethyl 4-hydroxybicyclo [3.1.0] hexane-6-carboxylate. After drying the organic layer (MgSO4) and concentrating, the residue was purified by column chromatography on silica gel using EtOAc / hexanes (2: 1) to EtOAc to afford the title compound as a white foam.

[a] ²⁵ ° +61.3 (c 1.20, CHCl 3).

500 MHz ¹ H NMR (CDCl3) δ 7.32-7.20 (m, 5H), 7.19 (br d, 1H, J = 4.0 Hz), 5.49 (br d, 1H, J = 4.0 Hz), 4.88 (d, 1H, J = 11.5 Hz), 4.24 (dd, 1H, J = 11.5, 6.0 Hz), 4.06-4.00 ( m, 2H), 3.77 (q, 1H, J = 7.0Hz), 2.21 (d, 1H, J = 15.0Hz), 2.18-2.15 (m, 2H), 1.71 (broad s, 1H), 1.54 (dd, 1H, J = 14.5, 6.0Hz), 1.38, (d, 3H, J = 6.5Hz), 1.32 (t, 1H, J = 3.3Hz), 1.24 (t, 3H, J = 7.0Hz).

¹³ C NMR (125 MHz, CDCl 3) δ 180.42, 171.47, 146.05, 127.43, 126.48, 73.16, 70.76, 61.08, 56.00, 42.82, 35, 67, 26.13, 21.53, 14.34.

FTIR (CHCl3) 3441 (m), 3345 (m), 2975 (w), 1725 (s), 1665 (s), 1288, 1186 (m) cm ^-1 .

Analysis Calculated for C ₁₈ H ₂₄ N ₂ O ₄ : C, 65.04; H, 7.28; N, 8.43. Found: C, 65.41; H, 7.58; N, 8.32.

PL 221 526 B1

Synthesis of 80

Ethyl 2 - [((1R) -1-phenylethanino] (2S, 4S, 6R) -4- (ethoxycarbonyl) hydroxybicyclo [3.1.0] hexane-6-carboxylate

For a suspension of 2 - [((1R) -1-phenylethyl) amino] - (2S, 4S, 6R) -4-hydroxybicyclo [3.1.0] hexane-2,6-dicarboxylic acid (4 g, 13 mmol) in 48 mL of ethanol at room temperature, acetyl chloride (11.2 mL, 157 mmol) was added via an addition funnel so as to maintain a gentle boil. The resulting mixture was allowed to stir for additional hours at reflux and, after cooling to room temperature, concentrated under reduced pressure to a solid residue. The solid was treated slowly with a solution of sodium bicarbonate (6.6 g) in 100 ml of water then washed with ethyl acetate (2 x 100 ml). The combined organics were dried (MgSO ₄ ), filtered, and concentrated in vacuo to yield 4.7 g (99%) of the title compound as a solid. Column chromatography on silica gel, elution with CH ₂ Cl ₂ / MeOH (95: 5) followed by recrystallization from Et ₂₀ gave an analytically pure sample of the title compound as a white solid.

[a] ²⁵ by +52.5 (c 1.30, CHCl3).

Melting point 73-74 ° C.

500 MHz ¹ H NMR (CDCl 3) δ 7.29-7.14 (m, 5H), 4.25 (dq, 1H, 11.0, 7.0 Hz), 4.18 (dd, 1H, J = 9.5

5.5 Hz), 4.10 (dq, 1H, J = 11.0, 7.0 Hz), 3.92 (dq, 1H, J = 11.0, 7.0 Hz) 3.82 (dq , 1H, J = 11.0 Hz, 7.0 Hz), 3.67 (q, 1H, J = 7.0 Hz), 2.73 (d, 1H, J = 9.5 Hz), 2, 15-2.12 (m, 2H), 2.01-1.99 (m, 1H), 1.89 (dd, 1H, J = 6.0, Hz), 1.61 (dd, 1H, J = 15.0, 6.0Hz), 1.36 (t, 1H, J = 3.5Hz), 1.33-1.30 (m, 6H), 1.18 (t, 3H, J = 7.0 Hz).

¹³ C NMR (125 MHz, CDCl3) δ 178.11, 171.59, 146.32, 128.41, 127.07, 126.85, 73.33, 70.15, 62.07, 60.75, 56.66, 44.72, 36.78, 33.61.26.24, 20.07, 14.37, 14.23.

FTIR (KBr) 3492 (s), 3303 (m), 3055 (w), 2981 (w), 2896 (w), 1722 (s), 1705 (s), 1289 (m), 1251 (m), 1171 (m) cm ^-1 .

Analysis Calculated for C ₂₀ H ₂₇ NO ₅ : C, 66.46; H, 7.52; N, 3.88. Found C, 66.42; H, 7.44; N, 3.92. Synthesis 81

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

To a solution of ethyl 2 - [((1R) -1-phenylethyl) amino] (2S, 4S, 6R) -2- (ethoxycarbonyl) -4-hydroxybicyclo [3.1.0] hexane-6-carboxylate (59.0 g crude, 0.163 mol) in CH ₂ Cl ₂ (690 ml) at -20 ° C was added Deoxo-fluoro (45.1 ml, 0.245 mol) over 15 minutes keeping the temperature between -15 and -20 ° C. The mixture was stirred for 20 minutes at this temperature and at 0 ° C for 15 minutes then 15% aqueous Na ₂ CO _{3 solution} (650 ml) was slowly added keeping the temperature below 10 ° C. The layers were separated and the aqueous layer was re-extracted with CH ₂ Cl ₂ (150 mL). The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated to a brown oil (73 g). The oil was purified over a pad of silica gel (400 g) eluting with EtOAc / heptane (1: 6) to give the title compound as a yellow oil (49.7 g, 84%).

[a] ²⁵ o +36.2 (c 1.30, CHCl3).

500 MHz ¹ H NMR (CDCl3) δ 7,29-7,14 (m, 5H), 5.22 (ddt, 1H, J = 8.0, 4.5 Hz, JHF = 56.0 Hz), 4 , 16 (dq, 1H, J = 11.0, 7.0Hz), 4.05 (dq, 1H, 11.0, 7.0Hz), 3.96 (dq, 1H, 10.5, 7 , 0 Hz), 3.85 (dq, 10.5, 7.0 Hz), 3.66 (q, 1H, 6.5 Hz), 2.45 (dd, 1H, J = 14.0.8 , 0Hz), 2.16-2.12 (m, 1H), 1.95 (t, 1H, J = 3.5Hz), 1.81

PL 221 526 B1 (dt, 1H, J = 3.5 Hz, Jhf = 3.5 Hz), 1.51 (ddd, 1H, J = 14.0 Hz, Jhf = 22.0 Hz), 1. 32 (d, 3H, J-6.5Hz), 1.27 (t, 3H, J = 7.0Hz), 1.21 (t, 3H, J = 7.0Hz).

¹³ C NMR (125 MHz, CDCl 3) δ 175.29, 171.66, 146.21, 128.45, 127.03, 126.90, 92.65 (d, J _C f = 182 Hz), 68, 68 (d, Jcf = 4.9Hz), 61.70, 60.92, 56.13, 38.60 (d, J _C f = 23.0 Hz), 33.07 (d, J _C f = 7.6 Hz), 32.23 (d, Jcf = 22.0 Hz), 26.26, 20.22 (d, Jcf = 3.9 Hz), 14.41, 14.24.

FTIR (CHCl3) 3028 (w), 2983 (w), 1724 (s), 1705 (s), 1293 (m), 1242 (m), 1190 (m), 1037 (m), 1013 (m) cm ^{- 1} .

Analysis Calculated for C20H26FNO4: C, 66.10; H, 7.21; N, 3.85. Found C, 66.02; H, 7.00; N, 3.95. Synthesis 82

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

A mixture of ethyl 2 - [((1jR) -1-phenylethyl) amino] (2S, 4R, 6R) -2- (ethoxycarbonyl) -4-fluorobicyclo [3.1.0] -hexane-6-carboxylate (68.4 g, 0.188 mol), concentrated HCl (15.7 mL, 0.188 mol), and 10% Pd / C (dry, 13.7 g) in EtOH (400 mL) was placed under a hydrogen atmosphere (50 psi) for 18 hours. The catalyst was filtered off and the filtrate was concentrated to give the title compound as an off-white foam (59.2 g, 106% corrected to 97% due to EtOH contamination). Crystallization from EtO-Ac / MTBE gave an analytically pure sample of the title compound as a white solid.

[a] ²⁵ D +55.6 (c 1.17, CHCl 3).

Melting point 86-88 ° C.

500 MHz ¹ H NMR (CDCl3) δ 9.20 (br s, 2H), 5.50 (ddt, 1H, J = 8.0, 4.5 Hz, JHF = 56.0 Hz), 4.31 ( q, 1H, J = 7.0Hz), 4.20-4.07 (m, 3H), 2.88 (t, 1H, J = 3.0Hz), 2.71 (dd, 1H, J = 14.5, 8.0 Hz), 2.482.43 (m, 2H), 2.16 (ddd, 1H, J = 14.5, 7.5 Hz, Jhf = 22.0 Hz), 1.34 (t, 3H, J = 7.0Hz), 1.25 (t, 3H, J = 7.0Hz) ^13C NMR (125MHz, CDCl3) δ 171, 12, 169, 41, 91, 94 ( d, Jcf = 1S9 Hz), 63.85, 63.66 (d, Jcf = 3.8 Hz), 61.73, 34.55 (d, Jcf = 26.4 Hz), 31.58 (d, Jcf = 7.8Hz), 30.80 (d, Jcf = 24.1Hz), 20.22, 14.31, 14.21.

FTIR (KBr) 3353 (m), 3173 (w), 2745 (m), 1729 (s), 1547 (m), 1294 (m), 1269 (m), 1195 (m), 1011 (m) cm ^{- 1} .

Analysis Calculated for C12H18FNO4: C-48, 74; H, 6.48; N, 4.74. Found: C, 48.80; H, 6.41; N, 4.76. Synthesis 83

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

A solution of 3N NaOH (251 mL, 0.753 mol) was slowly added to 1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride (59.2 g crude , theoretically 0.188 mol), keeping the temperature below 26 ° C. After stirring the mixture for 10 minutes, it became homogeneous. The mixture was stirred for 1.25 hours at room temperature, then the pH was slowly decreased to 2.8 using concentrated HCl, keeping the temperature between 20 and 26 ° C. At a pH of 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 a further 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 in vacuo at 40 ° C for 18 hours to provide the title compound as a white solid (33.1 g, 87% from synthesis start 23).

PL 221 526 B1

Synthesis 84

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

A stirred suspension 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) was heated to 89 ° C over 1 hour and added i-PrOH (41 mL). The mixture was then stirred for 5 minutes at reflux (83 ° C) before being allowed to cool to room temperature with stirring 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 in vacuo at 40 ° C for 18 hours to give the title compound as a white solid ( 30.6 g, 93%).

Synthesis 85

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

For a suspension 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 absolute ethanol at at room temperature, thionyl chloride (26 mL, 356 mmol) was added dropwise over 20 minutes. The slurry was heated to reflux and allowed to stir for hours then cooled to room temperature overnight. The resulting solution was concentrated under reduced pressure to 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 stirring to give a final pH of 10. The layers were separated and the aqueous layer was washed with ethyl acetate. (1 x 136 ml). The combined organic extracts were washed with brine (1 x 136 mL), dried (MgSO4), filtered, and concentrated in vacuo to give 17.07 g (93%) of the title compound as a white solid.

FDMS: M ⁺ +1 = 260.

Anal. Calculated for C ₂ H ₁₈ FNO4-0.1 H ₂ O: C, 55.21; H, 7.03; N, 5.37. Found: C, 55.10; H, 6.96; N, 5.22.

Melting point = 64-66 ° C.

[a] ²⁵ D = +20 (c = 0.96, MeOH), [a] ²⁵ D = +15 (c = 1.21, DMSO)

Synthesis 86

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

To a solution of N-Boc-L-alanine (38.62 g, 204 mmol) in 396 ml of methylene chloride at -22 ° C under nitrogen atmosphere was added N-methylmorpholine (22.44 ml, 204 mmol) followed by dropwise addition of iso-butyl chloroformate (26.48 mL, 204 mmol) over a period of 15 minutes so that the temperature of the reaction mixture does not exceed -18 ° C. The resulting slurry was allowed to stir at -20 ° C for 30 minutes, then a solution of 1R, 2S, 4R, 5R, 6R-2-amino-4-fluorobicyclo [3.1.0] hexane-2,6 acid ethyl ester was added. -dicarboxylic acid (49.46 g, 191 mmol) in 247 mL of methylene chloride over 40 minutes so that the temperature of the reaction mixture does not exceed -16 ° C. After the addition was complete, the reaction mixture was removed from the cooling bath and allowed to stand

After stirring at ambient temperature for 70 minutes, the temperature of the reaction mixture reached 15 ° C and the color turned faintly orange in color. The reaction mixture was treated with 408 ml of 1 N hydrochloric acid solution, then stirred for 5 minutes and the layers were separated. The organic layer was washed with a saturated aqueous sodium bicarbonate solution (1 x 408 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to a white foam (88.16 g). FDMS: M ++ 1 = 260.

Anal. calculated for C1 ₂ H13FNO4 · 0.1 H ₂ O: C, 55.21; H, 7.03; N, 5.37. Found: C, 55.10; H, 6.96; N, 5.22.

Melting point = 64-66 ° C.

[a] ²⁵ D = +20 (c = 0.96, MeOH), [a] ²⁵ D = +15 (c = 1.21, DMSO).

Synthesis 87

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

For a solution of 1R, 2S, 4R, 5R, 6R-2- [2'S-2 '- (tert-butoxycarbonylamino) -propionyl] amino-4-flurobicyclo [3.1.0] hexane-2,6-dicarboxylic acid ethyl ester (88 , 16 g, 191 mmol) in 238 ml of tetrahydrofuran were added at room temperature 238 ml (477 mmol) of 2N sodium hydroxide solution. The two-phase mixture was allowed to stir vigorously at room temperature for

2.5 hours, whereupon the reaction became homogeneous. The mixture was diluted with 238 mL of methyl butyl ether, mixed and the layers were separated. The aqueous layer was then diluted with 238 mL of water and filtered to remove the solid. The solution was treated with concentrated HCl (42.9 mL, 515 mmol) for 30 minutes, seeded with the title compound, and stirred for 1 hour. The resulting suspension was filtered, washed with water (2 x 100 ml) and dried in high vacuum at 45 ° C for 40 hours to give 72.2 g of the title compound as a white solid. A portion of the solid (69.5 g) was allowed to stir for 1 hour with 490 ml of acetone to form a cloudy solution which was filtered and washed with acetone (2 x 100 ml). The filtrate was concentrated in vacuo to a white foam which was then dried in high vacuum at 45 ° C for 16 hours to give 61.8 g (corrected for 12% w / w acetone) of the title compound.

P r z k ł a d 1

(1R, 4S, 5S, 6S) -4- (2'S-Aminopropionyl) -amino-2,2-dioxo-2X- ⁶ -thia-bicyclo- [3.1.0.] Hexane-4,6-dicarboxylic acid hydrochloride

To a suspension of (1R, 4S, 5S, 6S) -4- (2'S-tert-Butoxycarbonylamino-propionylamino) -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1.0] hexane-4,6-dicarboxylic acid (110, 0 g, 271 mmol, Synthesis 3) in 563 mL of ethyl acetate was added a solution of hydrogen chloride in ethyl acetate (3.7 M, 514 mL) over 20 minutes. After stirring the suspension for 2.5 hours, it was filtered and the filter cake was washed with ethyl acetate (1 x 200 ml, 1 x 115 ml). After vacuum drying at 46 ° C for 18 hours, the title compound was collected as a white solid (85.77 g, 92%).

GB ¹ 221 526 B1 H NMR (300 MHz, Methanol-d) δ: 4.12 (br d, 1H, J = 14.6 Hz), 3.94 (q, 1H, J = 7.1 Hz), 3 , 52 (ddd, 1H, J = 7.0, 3.9, 0.9Hz), 3.16 (d, 1H, J = 14.6Hz), 3.02 (dd, 1H, J = 7 , 0.4Hz), 2.49 (t, 1H, J = 4.1Hz), 1.52 (d, 3H, J = 7.1Hz).

P r z k ł a d 2

(1R, 4S, 5S, 6S) -4- (2'S-2'-aminopropionyl) amino-dioxo-2X ⁶ 6-thia-bicyclo [3.1.0.] Hexane-4,6-dicarboxylic acid tosylate

A suspension of (1R, 4S, 5S, 6S) -4- (2'S-tert-Butoxycarbonylamino-propionylamino) ^{-2,2-dioxo-6-thia-2X} bicykloheksano-4,6-dicarboxylic acid (300 mg, 0.738 mmol, 3 Synthesis) and toluenesulfonic acid monohydrate (140 mg, 0.738 mmol) in toluene (3 mL) was heated to 75 ° C and stirred for 45 minutes before being allowed to cool to room temperature and stirring was continued for 16 hours. The slurry was filtered and the filter cake was washed with toluene (2 x 1 mL). After drying in vacuo at 45 ° C for 1 hour, 307 mg (87%) of the title compound was collected as a white solid.

Melting point (DSC) 233 ° C.

¹ H NMR (500 MHz, CD3OD) δ 7.70 (d, 2H, J = 8.5 Hz), 7.24 (d, 2H, J = 8.0 Hz), 4.11 (d, 1H, J = 15Hz), 3.94 (q, 1H, J = 7.0Hz), 3.53 (dd, 1H, J = 7.0, 4.0Hz), 3.13 (dd, 1H, J = 14.1.0 Hz), 3.02 (dd, 1H, J = 7.0,

4.5 Hz), 2.48 (t, 1H, J = 4.5 Hz), 2.37 (s, 3H), 1.52 (d, 3H, J = 7.5 Hz).

¹³ C NMR (125 MHz, CD3OD) δ 170.70, 170.32, 169, 80, 142.04, 140.78, 128.79, 125.79, 60.20, 54.73, 48.77, 42.44, 30.84, 22.22, 20.20, 16.09.

P r z k ł a d 3

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'-hydroxypropionyl) amino-2,2-dixoxo2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

The process was carried out according to General Procedure C using (1R, 4S, 5S, 6S) -4- (3'-acetoxy-2'S-tert-butoxycarbonylaminopropionyl) amino-2,2-dioxo-2X- ⁶ -thia-bicyclo [acid dimethyl ester 3.1.0] -hexane-4,6-dicarboxylic acid (380 mg, 0.77 mmol, Synthesis 15).

[a] ²³ D = -19.23 (c = 0.52, MeOH).

¹ H NMR (400 MHz, CD3OD) δ 2.48 (1H, t, J = 4.0 Hz), 3.04 (1H, dd, J = 4.4, 7.0 Hz), 3.17 ( 1H, d, J = 14.7 Hz), 3.51 (1H, dd, J = 4.4, 7.0 Hz), 3.77 (1H, dd, J = 7.0, 10.6 Hz ), 3.94-4.12 (3H, m).

Anal. Calculated for C 19 H 27 Oe S-HCl 2 O: C, 31.88; H-4.55; N, 7.44; Cl, 9.41. Found: C, 31.53; H, 4.40; N, 7.32; Cl, 9.24.

HRMS calcd for C10H15N2O8S, 323.0549. Found 323, 0533.

PL 221 526 B1

P r z k ł a d 4

(1R, 4S, 5S, 6S) -4 - [(pyrrolidine-2'S-carbonyl) amino] -2,2-dioxo-2X ^6- thiabicyclo [3.1.0.] Hexane-4,6-dicarboxylic acid hydrochloride

The process was performed according to general procedure C using the dimethyl ester of (1R, 4S, 5S, 6S) -4 - [(1'-tert-Butoxycarbonyl-2'S-carbonyl) -amino] ^-2,2-dioxo-6 2X -tia- bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid (0.8 g, 1.7 mmol, Synthesis 11) to give 0.42 g (67.0%) of the title compound

[a] ²³ D = -32 (c = 1.00, CH 3 OH).

¹ H NMR (300 MHz, CD3OD) δ 1.98-2.12 (3H, m), 2.40-2.50 (1H, m), 2.52 (1H, t, J = 4.4Hz) , 2.99 (1H, dd, J = 4.4, 7.0 Hz), 3.2 (1H, d, J = 14.7 Hz), 3.29-3.42 (3H, m), 3.54 (1H, m), 4.13 (1H, d, J = 15.8 Hz), 4.30 (1H, dd, J = 6.2, 9.2 Hz).

HRMS calcd for C12H17N2O7S, 333.0756.

Found: 333.0740.

P r z k ł a d 5

The hydrochloride of (1R, 4S, 5S, 6S) -4- (2'S-Amino-4'-methylsulfanyl-butylamino) -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1.0] hexane-4,6- -dicarboxylic acid

The process was carried out according to general procedure C using (1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-4'-methylsulfanylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1 .0.] - hexane-4,6-dicarboxylic acid (0.77 g, mmol, Synthesis 12) to give 0.41 g (54.9%) of the title compound.

[a] ²³ D = +4 (c = 1.00, MeOH).

¹ H NMR (300 MHz, CD3OD) δ 2.1-2.2 (2H, m), 2.13 (3H, s), 2.47 (1H, t, J = 4.4 Hz), 2, 58-2.63 (2H, m), 3.02 (1H, dd, J = 4.0, Hz), 3.12 (1H, d, J = 14.7 Hz), 3.52 (1H, dd, J = 3.3, 6.6 Hz), 3.98 (1H, t, J = 6.2 Hz), 4.14 (1H, d, J = 14.7 Hz).

HRMS calculated for C12H19N2O7S2, 367.0634. Found: 367.0634.

P r z k ł a d 6

(1R, 4S, 5S, 6S) -4- [2'S-amino-3 '- (1H-indol-3-yl) -propionylamino] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1. O] hexane-4,6-dicarboxylic acid

PL 221 526 B1

The process was carried out according to the general procedure C using (1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-3 '- (1-tert-butoxycarbonyl-1H-indol-3-yl) -propionylamino acid dimethyl ester ] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] Hexane-dicarboxylic acid (0.54 g, 0.83 mmol, Synthesis 13) to give 0.28 g (73.6%) of the title relationship.

[a] ²³ D = +7.8 (c = 1.02, CH 3 OH).

¹ H NMR (300 MHz, CD3OD) δ 2.47-2.53 (1H, m), 3.05-3.18 (3H, m), 3.44-3.57 (2H, m), 4 , 13-4.23 (2H, m), 7.07-7.41 (3H, m), 7.71-7.78 (2H, m), 8.22 (1H, apparent d, J = 7 , 7 Hz).

Anal. Calculated for C21H203O / S-HCl: C, 47.22; H, 4.40; N, 9.18. Found: C, 46.51; H, 3.96; N, 8.54.

MS (ES) m / z 420.1 [M-1] ^- .

P r z k ł a d 7

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

The process was carried out according to the general procedure C using (1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-3 '- (4-tert-butoxycarbonyloxy-phenyl) -pionylamino] -2,2-dioxo-2X acid dimethyl ester ⁶ -thia-bicyclo [3.1.0.] Hexane-4,6-dicarboxylic acid (0.33 g, 0.53 mmol, Synthesis 14) to give 0.13 g (56.4%) of the title compound.

[a] ²³ D = -6 (c = 1.00, H2O).

¹ H NMR (300 MHz, CD3OD) δ 2.47 (1H, t, J = 4.0 Hz), 2.87 (1H, dd, J = 9.2, 14.7 Hz), 3.05- 3.12 (2H, m), 3.23 (1H, dd, J = 5.1, 14.7 Hz), 3.55 1H, dd, J = 4.0, 7.0 Hz), 4, 00 (1H, dd, J = 4.8, 9.2 Hz), 4.13 (1H, d, J = 14.7 Hz), 6.80 (2H, d, J = 8.4 Hz), 7.13 (2H, d, 8.8Hz).

HRMS calcd for C ₁₆ H ₁₉ N ₂ O ₈ S, 399.0862.

Found: 399.0844.

P r z k ł a d 8

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'-phenylpropionylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

The process was performed according to general procedure C using the dimethyl ester of (1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'-phenyl-propionylamino) -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1 .0] hexane-4,6-dicarboxylic acid (Synthesis 5). 0.55 g of the product (85%, 1.31 mmol), a white solid, was obtained.

[a] ²³ D = +4.17 (c = 0.48, MeOH).

¹ H NMR (300 MHz, CD3OD) δ 2, 45 (1H, t, J = 4.0 Hz), 2.98 (1H, dd, 3 = 9.2, 14.7 Hz), 3.06 ( 1H, dd, 3 = 4.4, 7.0Hz), 3.10 (1H, d, J = 14.6Hz), 3.33 (1H, dd, J = 4.8, 13.6Hz ), 3.52 (1H, dd, J = 3.3, 7.3Hz), 4.08 (1H, dd, J = 5.1.8.8Hz), 4.10 (1H, d, J = 15.4 Hz), 7.31-7.42 (5H, m).

¹³ C NMR (300 MHz, D ₂ O w / 1,4-dioxane): δ 172.55, 171.94, 170.01, 134.31, 130.39, 130.27, 130, 01, 129.00, 61.38, 54.76, 54.37, 43.07, 37.36, 31.57, 23.18.

Anal. Calcd for C16H18N O7Sd _2, 5 HCI: C, 43.97; H-4.50; N, 6.41. Found: C, 43.59; H-4.17; M-6.46.

MS (ES) m / z found 383.1 [M + H] ⁺ .

HRMS calcd for C16H19N2O7S [M + H] +: 383.0913. Found: 383.0923.

P r z k ł a d 9

(1R, 4S, 5S, 6S) -4- (2'S-amino-3'S-methylpentanonylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

The process was performed according to general procedure C using the dimethyl ester of (1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'S-metylopentanoiloamino) -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1. O] -hexane-4,6-dicarboxylic acid (Synthesis 6). 0.43 g of the product (80%, 1.12 mmol), a white solid, was obtained.

[a] ²³ o = +4.08 (c = 0.49, MeOH).

¹ H NMR (300 MHz, CD3OD) δ 0.98 (3H, t, J = 7.3 Hz), 1.07 (3H, d, J = 7.0 Hz), 1.15-1.24 ( 1H, m), 1.53-1.62 (1H, m), 1.95-2.04 (1H, m), 2.44 (1H, t, J = 4.0Hz), 3.06 (1H, dd, J = 4.4, Hz), 3.14 (1H, d, J = 15.0 Hz), 3.52 (1H, dd, J = 3.7, 7.0 Hz), 3.73 (1H, d, J = 5.1 Hz), 4.10 (1H, d, J = 14.7 Hz).

Anal. Calcd for C13H ₂₀ N ₂ O7S-HCl-0.5H ₂ O: C, 39.64; H, 5.63; N, 7.11. Found C, 39.38; H 5.39; N, 7.04.

MS (ES) m / z found 349.1 [M + H] ⁺ .

HRMS (ES) calcd for C ₁₁ H 21 N ₂ O 7 S [M + H] +, 349, 1069. Found: 349, 1086.

P r z k ł a d 10

(1R, 4S, 5S, 6S) -4- (2'S-Amino-3'-methylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4.6- acid hydrochloride dicarboxylic acid

The process was carried out according to the general procedure C using (1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-3'-methyl-butyrylamino) -2,2-dioxo-2X- ⁶ -thia-bicyclo acid dimethyl ester [3.1.0] hexane-4,6-dicarboxylic acid (Synthesis 1). 0.18 g of the product (88%, 0.49 mmol), a white solid in the form of the hydrochloride, was obtained.

[a] ²³ o = +1.84 (c = 0.51, MeOH).

¹ H NMR (300 MHz, CD3OD) δ 1.04 (3H, d, J = 6.6 Hz), 1.09 (3H, d, J = 7.0 Hz), 2.22-2.29 ( 1H, m), 2.44 (1H, t, J = 4.0Hz), 3.05 (1H, dd, J = 4.4, 7.0Hz), 3.10 (1H, d, J = 14.7 Hz), 3.52 (1H, dd, J = 4.0, 7.3 Hz), 3.67 (1H, d, J = 5.5 Hz), 4.10 (1H, d , J = 14.7 Hz).

PL 221 526 B1

Anal. Calcd for C12H1 ₈ N 2 O ₇ S-HCl-0,4H2O: C, 38.12; H, 5.20; N, 7.41. Found: C, 37.78; H, 4.90; N, 7.15.

MS (ES) m / z found 335.1 [M + H] +.

P r x l a d 11

(1R, 4S, 5S, 6S) -4- (2'S-amino-4'-methyl-pentanoylamino) -2.2-dioxo-2/ ⁶ -thia-bicyclo [3.1.0] hexane-4.6, hydrochloride salt -dicarboxylic acid

The process was carried out according to general procedure C using (1R, 4S, 5S, 6S) -4- (2'S-tert-butoxycarbonylamino-4'-methyl-pentanoylamino) -2,2-dioxo-2/6-thia- acid dimethyl ester bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (Synthesis 8). 0.50 g of the product (76%, 1.30 mmol), a white solid in the form of the hydrochloride, was obtained.

[a] ²³ D = -4.0 (c = 0.50, MeOH).

¹ H NMR (300 MHz, CD3OD) δ 1.00 (3H, d, J = 5.5 Hz), 1.02 (3H, d, J = 5.9 Hz), 1.62-1.79 ( 3H, m), 2.42 (1H, t, J = 4.0Hz), 3.04 (1H, dd, J = 4.4, 7.3Hz), 3.13 (1H, d, J = 15.0 Hz), 3.52 (1H, dd, J = 3.3, 7.0 Hz), 3.84-3.89 (1H, m), 4.10 (1H, d, J = 15.0 Hz).

Anal. Calcd for C13H2 ₀ N 2 O ₇ S-HCl-0,3H2O: C, 40.01; H-5.58; N, 7.18. Found C, 39.66; H-5.57; N, 6.99.

MS (ES) m / z found 349.1 [M + H] ⁺ .

P r z k ł a d 12

(1R, 4S, 5S, 6S) -4- (2'S, 6'-diaminohexanoylamino) -2,2-dioxo-2/ ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride bis

The process was carried out according to the general procedure C using (1R, 4S, 5S, 6S) -4- (2'S, 6'-bis-tert-butoxycarbonylamino-hexanamino) -2,2-dioxo-2/ ^6- thia- acid dimethyl ester bicyclo [3.1.0] hexane-4,6-dicarboxylic acid (Synthesis 9). 0.56 g of the product (86%, 1.28 mmol), a white solid in the form of the bis hydrochloride, was obtained.

[a] ²³ D = -4.0 (c = 0.50, MeOH).

¹ H NMR (300 MHz, CD3OD) δ 1.48-1.55 (2H, m), 1.67-74 (2H, m), 1.89-1.97 (2H, m), 2.47 (1H, t, J = 4.0Hz), 2.97 (2H, apparent t, J = 4.0), 3.08 (1H, dd, 3 = 4.4,7.0Hz), 3 , 20 (1H, d, J = 15.0Hz), 3.53 (1H, dd, J = 3.7,7.0Hz), 3.93 (1H, apparent, t, 3 = 6.2 Hz), 4.08 (1H, d, 3 = 14.1 Hz).

Anal. Calcd for C, 3H21N3O7S _1-0,2H2O 2HCl: C, 35.49; H 5.36; N, 9.55. Found: C, 35.30; H, 5.48; N, 9.42.

PL 221 526 B1

MS (ES) m / z found 364.1 [M + H] +.

P r x l a d 13

(1R, 4S, 5S, 6S) -4- (2'S-amino-4'-carbamoylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane-4,6-dicarboxylic acid hydrochloride

(1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-4 '- [tritylcarbamoyl) butyrylamino] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0] hexane- acid dimethyl ester The 4,6-dicarboxylic acid (0.48 g, 0.65 mmol, Synthesis 10) was stirred in a mixture of 2.5N LiOH and THF 1: 1 (6 ml total volume) at room temperature for 4 hours. The pH of the reaction mixture was adjusted to = 2 with 1 N HCl and the product was extracted with ethyl acetate. All organics were combined, washed with brine, dried over MgSO4 and concentrated to give 0.46 g of the acid (1R, 4S, 5S, 6S) -4- [2'S-tert-butoxycarbonylamino-4 '- (tritylcarbamoyl) butyrylamino] -2. 2 ^{2X-dioxo-6-thia-bicyclo} [3.1.0] hexane-4,6-dicarboxylic acid as a white foam. The diacid was dissolved in DCM and stirred at room temperature as anisole (0.28 g, 2.6 mmol) followed by the addition of TFA (3.70 g, 32.5 mmol). The resulting reaction mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. Yellow oil obtained. Triturated with Et2O to form a free flowing white precipitate. The TFA salt was collected by vacuum filtration under a nitrogen blanket. The product was dissolved in 1 ml of 1N HCl solution and subjected to lyophilization to obtain the desired product in the form of the hydrochloride. 0.16 g of the product (62%, 0.40 mmol), a white solid, was obtained.

[a] ²³ D = +8.0 (c = 1.0, H ₂ O) ¹ H NMR (300 MHz, CD3OD) δ 2.00-2.08 (2H, m), 2.37-2, 41 (3H, m), 2.93 (1H, dd, J = 4.4, 7.3Hz), 3.04 (1H, d, J = 14.7Hz), 3.45 (1H, dd , J = 3.7, 7.0 Hz), 3.86 (1H, apparent, t, J = 5.9 Hz), 4.05 (1H, d, J = 14.7 Hz).

Anal. Calculated for C1 ₂ H17N3O3S · HCl · 0.2H ₂ O: C-33.07; H-5.09; N, 9.64. Found C, 33.37; H, 4.69; N, 9.39.

MS (ES) m / z found 363.9 [M + H] +.

HRMS (ES) calcd for C1 ₂ H13N3O3S [M + H] +: 364.0815. Found: 364.0825.

The prodrug compounds of the present invention can be compared to the corresponding parent compound using various cellular uptake assays. As a result of these tests, comparative data can be obtained allowing those skilled in the art to identify compounds that readily enter the cell, providing better exposure. Two of these assays include the Gly-Sar Uptake Assay and the Caco-2 Assay described below.

Gly-Sar Capture Assay

Some orally administered peptidomimetics have been found to be absorbed via the enteric peptide transport system. Yang et al., Pharm. Res. 16 (9) (1999). In particular, the intestinal peptide transporter hPepT1 was investigated for the expression of peptide uptake inhibition and the corresponding level of its recognition in the cell. Meredith, et al., Eur. J. Biochem., 267, 3723-3728 (2000). Moreover, by characterizing the intestinal amino acid absorption mechanism, the hPepT1 transporter has been set as a target as an effective strategy for identifying an increase in drug absorption after oral administration. Han et al., Polim. Prepr. (Am. Chem. Soc., Div. Polim. Chem.) 40 (1): 259-260 (1999); Sawada, et al., J. Pharmacol. Exp. Ther., 291 (2): 705-709 (1999).

In U.S. Patent No. US No. 5,849,525 discloses methods for measuring the affinity level of compounds of the present invention for the hPepT 1 transporter.

PL 221 526 B1

For example, stably transfected Chinese hamster ovary (CHO) cells overexpressing the hPepT1 transporter can be used to test the compounds of the present invention. Gly-Sar uptake is then monitored in CHO cells, which, in the presence of the prodrug compounds of the present invention, if more potent than when the cell does not contain the prodrug compounds of the present invention, indicates agonist activity; and when the uptake of the prodrug compounds of the present invention is less than the uptake in the absence of the prodrug compounds of the present invention, it is indicative of inhibitory activity. The compounds of the present invention generally have EC ₅₀ values less than 5 mM.

Caco-2 test

Another method of measuring the cell uptake of compounds of the present invention is by testing the carrier for peptide transport of the human intestinal Caco-2 cell line. Human adenocarcinoma cells (Memorial Sloan-Kettering Cancer Centrum, Rye, NY, and / or ATCC, Rockville, MD) are passaged in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% non-essential amino acid solution in a minimal medium basic without the addition of sodium pyruvate or antibiotics. These cells do not contain mycoplasma and are selected from between passage 28 and 40. For flow measurements, 5 to 10 × 10 ⁴ cells are grown in collagen-coated multiwell plates for 13-18 days, changing the medium every two to three days.

Drug uptake is measured at 37 ° C with a test compound using a plate grouping technique (see Gazzola et al., Anal. Biochem. 115, 368-74 (1981)). The flow test buffer is Earle's bicarbonate-free equilibrated salt solution containing 25 mM Mes, titrated to pH 6.0 using KOH, and choline chloride instead of sodium chloride. The osmolality of the flow test buffer is adjusted to 300 ± 5 moles / kg ₃ using choline chloride. [H] inulin is used as a marker for extracellular fluid and adheres to the cells during the washing procedure, allowing a zero time to be established for determining the uptake rate. Fresh solutions of test compounds and dipeptides are prepared daily. At the end of the experiment, cells are lysed in water and test compounds can be detected in cell lysates using LC / MS / MS. Protein content is measured as described by Smith et al., Anal. Biochem. 150, 76-85 (1985).

The uptake is measured over 40 minutes. The initial uptake rate is calculated in the linear region of the regression curve and time zero is determined as described above using linear regression. Percent inhibition is calculated considering the control uptake rate measured in the absence of the dipeptide. Examples of the Caco-2 test can be found in Dantzig & Bergin, Biochim. Biophys. Acta 1027, 211-17 (1990).

In vivo exposure using rat plasma concentration measurements

To test the in vivo exposure of compounds of formula II after oral administration of compounds of formula I compared to compounds of formula II, measurements of the rat plasma concentrations of the corresponding compounds of formula II are carried out. Mature male Fischer 344 rats (190-270 grams) purchased from Harlan Sprague-Dawley, Cumberland, IN, USA are acclimated for 3 days prior to the start of the study. On the day, test compounds were dissolved in buffered water (1 mg / ml = test compound / 20 mM potassium dihydrogen phosphate, pH = 2) and administered orally in a single dose of 5 mg / kg. Blood samples are taken at 0.5 and 1 hour, or alternatively at 1 and 3 hours, via the orbital sinus or cardiac puncture (last time point). Plasma samples are stored at -20 ° C in the presence of phenylmethylsulfonyl fluoride, a protease inhibitor prior to analysis. Plasma samples and internal standard compounds are pre-subjected to solid phase extraction (SAX carrier, methanol / water / dilute acetic acid).

As shown below in Tables 1A and 1B, the plasma concentrations (ng / ml) of the corresponding compound of Formula II for each test compound are determined by LC / MS / MS and are presented as the sum of the concentrations at 0.5 and 1 hour or, alternatively, after 1 and 3 hours, in samples taken at these time points.

PL 221 526 B1

Table 1A In vivo exposure test Relationship Rat exposure (ng / ml (1R, 4S, 5S, 6S) -4- (2'S-aminopropionyl) amino] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] Hexane- acid hydrochloride 4,6-dicarboxylic acid) Example 1 2251 ng / ml (after 10 mg / kg orally) Non-prodrug form of the compound of Example 1 1521 ng / ml (after administration of 5 mg / kg orally) 3981 ng / ml (after administration of 10 mg / kg orally)

Table 1B In vivo exposure test Relationship Rat exposure (ng / ml (1R, 2S, 4R, 5R, 6R) -2- (2'S-aminopropionyl) amino-4-fluorobicyclo [3.1.0] hexane-2,6-dicarboxylic acid hydrochloride) Example 14 5271 ng / ml (after administration of 5 mg / kg orally) Non-prodrug form of Example 14 1162 ng / ml (after administration of 5 mg / kg orally) 1342 ng / ml (after 10 mg / kg orally)

As shown above in Tables 1A and 1B, when administered orally to rats, the compounds of the invention show comparable plasma concentrations of the parent compound. This is evidence that the compounds of the present invention convert in vivo to the parent compounds of formula II.

The compounds of the present invention are preferably in the form of a pharmaceutical composition. Thus, another aspect of the present invention includes a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient. Pharmaceutical compositions can be prepared by methods well known to those skilled in the art. For the preparation of the compositions of the present invention, the active ingredient is usually mixed with a carrier or diluted with a carrier, or enclosed within a carrier, and may be in the form of a capsule, sachet, bag or other container. When the carrier serves as a diluent, it can be solid, semi-solid, or liquid and act as an excipient, diluent or medium for the active ingredient. The compositions may take the form of tablets, pills, powders, lozenges, sachets, wafer capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments containing e.g. up to 10% by weight of the active ingredient, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile powders for solution preparation.

Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gums, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, aqueous cellulose, methylcellulose, methyl and propyl hydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations can additionally contain lubricants, wetting agents, suspending and emulsifying agents, preservatives, sweetening agents or flavoring agents. The compositions of the invention may be formulated so as to provide immediate, sustained or delayed release of the active ingredient after administration to the patient by employing methods well known in the art.

The compositions are preferably presented in unit dosage form, each dose containing from about mg to about 500 mg of the active ingredient, preferably about 25 mg to about 300 mg of the active ingredient. The term "active ingredient" as used herein refers to a compound encompassed by Formula I.

The term "unit dosage form" refers to a physically discrete unit that is a single dose of medicament that can be administered to humans and other mammals, each unit containing a predetermined quantity of active ingredient to provide the desired therapeutic effect in association with a suitable pharmaceutical carrier, diluent or agent. the excipient.

Claims (12)

A compound of formula I wherein A is H- (Q) p-; Q is selected from the group of amino acyls derived from amino acids selected from the group consisting of natural amino acids and unnatural amino acids selected from D-isomers of natural a-amino acids, p being an integer from 1 to 10; X is SO ₂ ; R ₁₀ is hydrogen or fluoro; and R ₁₁ is hydrogen, fluoro or hydroxy; and which compound includes the following compounds; (1R, 4S, 5S, 6S) -4- (2'S-aminopropionyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid; (1R, 4S, 5S, 6S) -4- (2'S-2'-aminopropionyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid; (1R, 4S, 5S, 6S) -4- (2'S-amino-3'-hydroxypropionyl) -amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4.6 acid -dicarboxylic; (1R, 4S, 5S, 6S) -4 - [(pyrrolidine-2'S-carbonyl) -amino] -2,2-dioxo-2X ⁶ -thia-bicyclo- [3.1.0.] hexane-4,6- acid dicarboxylic; (1R, 4S, 5S, 6S) -4- (2'S-amino-4'-methylsulfanylbutylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo- [3.1.0.] hexane-4.6 -dicarboxylic; (1R, 4S, 5S, 6S) -4- [2'S-amino-3 '- (1H-indol-3-yl) -propionylamino] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0 .] hexane-4,6-carboxylic; acid (1R, 4S, 5S, 6S) -4- [2'S-amino-3 '- (4-hydroxyphenyl) propionylamino] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane- 4,6-dicarboxylic; (1R, 4S, 5S, 6S) -4- (2'S-amino-3'-phenylpropionylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid ; acid, (1R, 4S, 5S, 6S) -4- (2'S-amino-3'S-metylopentanonyloamino) -2,2-dioxo-2X ^{6-thia-bicyclo} [3.1.0.] hexane-4,6-dicarboxylic acid; (1R, 4S, 5S, 6S) -4- (2'S-amino-3'-methylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid ; (1R, 4S, 5S, 6S) -4- (2'S-amino-4'-methylpentanoylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid ; (1R, 4S, 5S, 6S) -4- (2'S, 6'-diamino-hexanoylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] hexane-4,6-dicarboxylic acid ; (1R, 4S, 5S, 6S) -4- (2'S, -amino-4'-carbamoylbutyrylamino) -2,2-dioxo-2X ⁶ -thia-bicyclo- [3.1.0.] hexane-4,6- acid dicarboxylic acid, and pharmaceutically acceptable salts thereof. 2. The pharmaceutically acceptable salt of Claim 1 1 which is (1R, 4S, 5S, 6S) -4- (2'S-aminopropionyl) amino] -2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] Hexanedicarboxylic acid hydrochloride, or the acid tosylate 1R, 4S, 5S, 6S) -4- (2'S-2'-aminopropionyl) amino-2,2-dioxo-2X ⁶ -thia-bicyclo [3.1.0.] Hexane-4,6-dicarboxylic acid. 3. A process for the preparation of a compound of formula I or a pharmaceutically acceptable salt thereof as defined in claim 1 1-2, characterized in that the compound of formula (ii) Is acylated with the appropriate aminoacyl of formula P _g NA- (III) wherein A is H- (Q) p-; Q is selected from the group of amino acyls derived from amino acids selected from the group consisting of natural amino acids and unnatural amino acids selected from D-isomers of natural a-amino acids, p being an integer from 1 to 10; P _g N is a nitrogen protecting group such as, for example, tert-butoxycarbonyl; then, when the functional group is protected with a protecting group, the protecting group is removed; then, when a pharmaceutically acceptable salt of a compound of formula I is required, the base form of such compound of formula I is reacted with an acid to form a pharmaceutically acceptable counterion; or for a compound of formula I that contains an acidic group, the acidic form of such compound of formula I is reacted with a base which gives a pharmaceutically acceptable cation; or for a zwitterionic compound of formula I, neutralizing the acid addition salt form or the base addition salt form of said compound of formula I; or using any other conventional procedure. 4. Use of a compound as defined in claim 1 For the manufacture of a medicament for administering an effective amount of a compound of formula II, wherein X and R are as defined in claims 1-2, 1 for the treatment of a neurological disorder in a patient. 5. A compound according to any one of claims 1 to 5 1 or 2 for use in therapy. 6. A compound according to any one of claims 1-7 1 or 2, for use in treating a neurological disorder in a patient. 7. The compound of claim 1 For use in treating a neurological disorder selected from the group consisting of cerebral deficiencies following coronary bypass implantation of the heart muscle and transplant; cerebral ischemia; spinal cord injury; head trauma; Alzheimer's disease; Huntington's Chorea; amyotrophic lateral sclerosis; AIDS-induced dementia; perinatal hypoxia; neuronal damage as a result of hypoglycemia; eyeball damage and retinopathy; cognitive impairment; idiopathic and drug-induced disorders Parkinson's disease; muscle cramps; migraine headaches; urinary incontinence; tolerance, withdrawal, discontinuation and drug dependence; nicotine withdrawal; vomiting; swelling of the brain; chronic pain; sleep disturbance; seizures; Tourette's syndrome; attention deficit disorder; and tardive dyskinesia. 8. The compound of claim 1 7, for use as a medicament for the treatment of a neurological disorder selected from the group consisting of tolerance, withdrawal, interruption, and drug addiction; or nicotine withdrawal. 9. A compound according to any one of claims 1-7 1-2, for use as a medicament for treating a psychiatric disorder in a patient. 10. The compound of claim 1 9, for use as a medicament for the treatment of psychiatric disorders selected from the group consisting of schizophrenia, anxiety and related disorders, depression, bipolar disorders, psychosis and obsessions, and compulsive acts. PL 221 526 B1 11. The compound of claim 1 10, for use as a medicament for the treatment of psychiatric disorders selected from the group consisting of anxiety and related disorders. 12. The compound of claim 1 responses to 10, for use as a medicament for treating psychiatric disorders, wherein the psychiatric disorder is schizophrenia.