JP2008545697A - Glycyl-prolyl-glutamate analogues - Google Patents

Abstract

  Embodiments of the invention include novel analogs of glycyl-prolyl-glutamate (GPE) and compositions containing such analogs of GPE. Of these, some analogs have modified proline residues. Other embodiments of the invention include the use of GPE analogs to protect neurons from degeneration and / or death in response to injury or disease. Diseases that can be treated with the compounds and compositions of the present invention include chronic neurodegenerative disorders including hypoxia / ischemia, toxic injury, and Parkinson's disease.

Description

  The present invention relates to novel analogs of glycyl-prolyl-glutamate (GPE) and methods for their use. The invention also relates to the use of these compounds and their pharmaceutical compositions in the treatment of diseases and conditions characterized by neurodegeneration and / or death.

The tripeptide glycyl-L-prolyl-L-glutamate (Gly-Pro-Glu or GPE) is a naturally occurring peptide that is proteolytically cleaved from insulin-like growth factor-1 (IGF-1). The IGF-1 is a potent neurotrophic factor that is endogenously generated in areas that have been damaged in the brain. Although some neuroprotective action of IGF-1 is required to be mediated by GPE, the exact mechanism of action remains unclear. GPE has a different mode of action on IGF- when GPE does not bind to the IGF-1 receptor. Rather, GPE has been shown to bind to the N-methyl-D-aspartate (NMDA) receptor with low affinity and also elicits a biological response through other mechanisms. GPE promotes dopamine release by interacting with the NMDA receptor, but GPE-stimulated acetylcholine release is via an unknown, non-NMDA pathway.
GPE has been shown to be able to act as a neuronal rescuer following hypoxic ischemic brain injury, NMDA challenge, brain injury or disease including chemical toxins and in animal models of Parkinson's disease and Alzheimer's disease. GPE analogs are of particular interest in the development of new drugs for central nervous system (CNS) injury and neurodegenerative disorders.
The inventors have identified several GPE analogs that have been modified with glycine and / or glutamate residues in an effort to study structure activity relationships and in an attempt to improve properties such as metabolic stability and oral bioavailability. It is disclosed in advance. Some of these include US Pat. No. 7,041,314; PCT International Patent Application No: PCT / US02 / 16361 (filed May 24, 2002); US Patent Application No. 11 / 314,424 (December 2005) U.S. Patent Application No. 11 / 315,784 (filed on Dec. 21, 2005); U.S. Patent Application No. 11 / 398,032 (filed on Apr. 4, 2006) and U.S. Provisional Patent Application No. 60 / 782,148 (Filed March 14, 2006). Each of the aforementioned patents and patent applications is fully incorporated herein by reference. Also, certain analogs are Trotter et al. Bioorg. Med. Chem. 2005, 13, 501; Lai et al. Bioorg. Med. Chem. 2005, 13, 533; Brimble et al. Bioorg. Med. Chem. 2005 , 13, 519).
However, there is a continuing need for other GPE analogs and compositions and agents for treating other types of diseases for the treatment of diseases of the nervous system, including diseases involving neurodegeneration and neural cell death. There is.

Summary In some aspects, the present invention provides novel GPE analogs.
In some embodiments of the invention, the proline residue is modified.
Some embodiments of the present invention provide molecules having the following structural formula and substituents:

Formula 1

Some embodiments of the present invention comprise a compound of formula 1 or a pharmaceutically acceptable salt or hydrate thereof, wherein:
The bond between X ¹ and X ² is saturated or unsaturated;
X ¹ is selected from the group consisting of CH ₂ , S, C (OH) H and is CH when the bond between X ¹ and X ² is unsaturated;
X ² is selected from the group consisting of CH ₂ , CH ₂ CH ₂ and is CH when the bond between X ¹ and X ² is unsaturated;
R ¹ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ² is CH ₃ or COOH;
R ³ is selected from the group consisting of H, alkyl, or NR ³ R ³ is collectively pyrrolidino or piperidino;
R ⁴ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ⁵ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
n is an integer of 0-2.

Formula 2

  Another embodiment includes a compound of formula 2 or a pharmaceutically acceptable salt or hydrate thereof, wherein R is H or OH.

Formula 3

Additional embodiments include compounds of Formula 3 or pharmaceutically acceptable salts or hydrates thereof, wherein:
R ¹ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ² is CH ₃ or COOH;
R ³ is selected from the group consisting of H, alkyl, or NR ³ R ³ is collectively pyrrolidino or piperidino;
R ⁴ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
n is an integer of 0-2.

Formula 4

A further embodiment comprises a compound of formula 4 or a pharmaceutically acceptable salt or hydrate thereof, wherein R ¹ is H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl. , Substituted aryl or substituted arylalkyl.
In another aspect of the invention, pharmaceutically acceptable salts of the compounds described in Formulas 1-4 are provided.
It should be understood that the compounds of the present invention include stereoisomers, optical isomers, conformers and the like of the structural formulas represented. It should also be understood that the compounds of the present invention may exist as racemic mixtures of different stereoisomers. All such stereoisomers are contemplated as part of the present invention.
In yet another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of at least one compound of the present invention. These compounds have found use as anti-apoptotic agents and anti-necrosis agents and for other conditions including neurodegeneration and injury.
In a further aspect, the present invention provides a method of treating an animal having a disease or injury treatable by administration of a stable compound, optionally with at least one other conventional therapeutic agent for the disease to be treated Also provided is a method comprising administering to the animal at least one compound of the invention.
In a further aspect, the animal to be treated is a human.
In a further aspect, the present invention provides a method for making the compounds of formulas 1-4 of the present invention.
In yet another aspect, the present invention provides methods for synthesizing, formulating and manufacturing pharmaceutical formulations comprising the compounds of formulas 1-4 of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be described in connection with specific embodiments thereof. Other characteristics, features and embodiments of the invention can be better understood with reference to the figures.
FIG. 1 shows the effect of analog 49 (glycyl-L-thia-5,5-dimethylprolyl-L-glutamic acid) on neuronal survival after administration of apoptosis-inducing toxin (100 nM okadaic acid) in cortical cell culture. It is a graph.
FIG. 2 shows analog 50 (glycyl- (D, L) -5,5-dimethylprolyl-L- in neuronal survival after excitotoxic oxidative stress in cortical cell cultures induced by 100 μM H ₂ O _2. It is a graph which shows the effect of glutamic acid.
FIG. 3 shows the effect of analog 60 (glycyl-trans-4-hydroxy-L-prolyl-L-glutamic acid) on neuronal survival after administration of apoptosis-inducing toxin (30 nM okadaic acid) in striatal cell culture. It is a graph.
FIG. 4 shows the effect of analog 64 (aminoisobutryl-L-prolyl-L-glutamic acid) on neuronal survival after administration of apoptosis-inducing toxin (30 nM okadaic acid) in striatal cell culture. It is a graph.
FIG. 5 is a graph showing the effect of analog 65 (glycyl-L-prolyl-L-norvaline) on neuronal survival after administration of apoptosis-inducing toxin (100 nM okadaic acid) in cortical cell culture.
FIG. 6 is a graph showing the effect of analog 66 (glycyl-D, L-pipecolinyl-L-glutamic acid) on neuronal survival after administration of apoptosis-inducing toxin (okadaic acid) in striatal cell culture.
FIG. 7 shows analog 67 (pyrrolidinoglycyl-L-2-methyl-proline-) in neuronal survival after excitotoxic / oxidative stress (induced by 0.5 mM 3-NP glutamate) in cerebellar cell cultures. It is a graph which shows the effect of (L-glutamic acid).

DETAILED DESCRIPTION OF THE INVENTION Definitions “Alkenyl” means an unsaturated branched, straight chain or cyclic hydrocarbon group having at least one carbon-carbon double bond. The group may be in either cis or trans conformation with respect to the double bond. Typical alkyl groups include allyl, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, cyclopentenyl and the like. In some embodiments, the alkenyl group is (C _2-6 ) alkenyl and in other embodiments, allyl may be particularly useful.
“Alkyl” means a saturated branched, straight chain or cyclic hydrocarbon group. Typical alkyl groups include methyl, ethyl, isopropyl, cyclopropyl, t-butyl, cyclopropylmethyl, hexyl and the like. In some embodiments, the alkyl group is (C _1-6 ) alkyl.
“Alkynyl” means an unsaturated branched, straight chain or cyclic hydrocarbon group having at least one carbon-carbon triple bond. Typical alkynyl groups include ethynyl, propynyl, butynyl, isobutynyl and the like. In some embodiments, the alkynyl group is (C _2-6 ) alkynyl.
“Aryl” means an unsaturated cyclic hydrocarbon group having a conjugated π-electron system. Typical aryl groups include phenyl, naphthyl and the like. In some embodiments, the aryl group is (C _5-20 ) aryl.
“Arylalkyl” means a straight chain alkyl, alkenyl or alkynyl group in which one of the hydrogen atoms bonded to the terminal carbon is replaced by an aryl group. Typical arylalkyl groups include benzyl, naphthylmethyl, benzylidene, and the like.
The term “glycyl-prolyl-glutamate” or “Gly-Pro-Glu” means a tripeptide containing the designated amino acid. The nominated compounds include the C-terminal acid form of the peptide, also referred to as “glycyl-prolyl-glutamic acid”.
“Growth factor” means an extracellular polypeptide signaling molecule that stimulates a cell to grow or proliferate.
“Heteroalkyl” means an alkyl group in which one or more carbon atoms have been replaced by another atom, for example, N, P, O, S, and the like. Typical heteroalkyl groups include pyrrolidine, morpholine, piperidine, piperazine, imidazolidine, pyrazolidine, terahydrofuran, (C _1-10 ) substituted amines, (C _2-6 ) thioethers, and the like.
“Heteroaryl” refers to an aryl group in which one or more carbon atoms have been replaced with another atom, such as N, P, O, S, and the like. Typical heteroaryl groups include carbazole, furan, imidazole, indazole, indole, isoquinoline, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrrole, thiazole, thiophene, triazole and the like.

“Injury” includes acute or chronic damage to an animal resulting in degeneration or death of cells in the nervous system. Such cells include nerve cells and non-neuronal cells. Injuries include stroke, non-hemorrhagic stroke, traumatic brain injury, fetal asphyxia, e.g., perinatal asphyxia associated with subsequent abruption, cord occlusion or associated with intrauterine growth retardation, Perinatal asphyxia associated with proper resuscitation or respiratory disease, near miss drowning, sudden death of near miss infants, carbon monoxide inhalation, ammonia or other gas poisoning, cardiac arrest, coma, meninges Serious central nervous system trauma associated with inflammation, hypoglycemia and status epilepticus, coronary bypass surgery, hypotensive episodes and brain asphyxia associated with hypotensive crisis, and brain Trauma. The above examples are illustrative and are not intended to be a complete list of injuries that can be treated by the compounds and methods of the present invention.
“Pharmaceutically acceptable excipient” means an excipient that is generally safe, non-toxic and useful in producing the desired pharmaceutical composition, and is used in domestic animals and in humans. Contains excipients that are acceptable for use. Such excipients may be solid, liquid, semi-solid, or in the case of an aerosol composition, a gas.
“Pharmaceutically acceptable salt” means a salt that is pharmaceutically acceptable and has the desired pharmacological properties. Such salts include salts that can be formed such that acidic protons present in the compound can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals such as sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include those formed with organic bases such as amine bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. Such salts also include inorganic acids such as hydrochloric acid and hydrobromic acid and organic acids such as acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids such as methanesulfonic acid and Acid addition salts formed with benzenesulfonic acid). If two acidic groups are present, the pharmaceutically acceptable salt may be a mono-acid mono-salt or di-salt; similarly, if more than two acidic groups are present, such Some or all of the gas may be salified.

“Protecting group” refers to the meaning conventionally associated with it in organic synthesis, ie, one or more reactive sites in a multifunctional compound such that a chemical reaction can occur selectively at other unprotected reactive sites. Means a blocking group (the tree can be easily removed after the selective reaction is complete).
“Substituted” means that one or more hydrogen atoms in an alkyl, heteroalkyl, alkenyl, alkynyl, aryl, heteroaryl or arylalkyl group are independently replaced with another substituent. Examples of substituents, -R ', - OR', - SR ', - NR'R', - NO 2, -CN, -C (O) R ', - C (O) OR', - C (O) NR'R ', -C (NR') NR'R ', -NR'-C (NR')-OR ', -NR'-C (NR')-SR ', NR'-C (NR ′) — NR ′ R ′, trihalomethyl and halogen, wherein each R ′ is independently —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl or Heteroarylalkyl.
A “therapeutically effective amount” is an amount that, when administered to an animal for treating a disease, is sufficient to affect the treatment for the disease; Meaning an amount that promotes a desired sign or outcome and / or treats and / or treats the underlying disorder.
“Treatment” of a disease includes preventing disease (preventive treatment) that occurs in an animal that is susceptible to the disease but still not experiencing or showing signs of the disease (preventive treatment), or suppressing the disease (delaying its development) Or providing) relief from signs or side effects of the disease (including elective treatment), and good for the disease (causing regression of the disease).
An implicit hydrogen atom (eg, hydrogen in a pyrrole ring, etc.) is omitted from the formula for clarity, but should be understood to be present.
Compounds of the Invention One aspect of the invention includes molecules having the following structural formula and substituents:

Formula 1

Some embodiments of the present invention include a compound of Formula 1 or a pharmaceutically acceptable salt or hydrate thereof, in which:
X ¹ is selected from the group consisting of CH ₂ , S, C (OH) H and is CH when the bond between X ¹ and X ² is unsaturated;
X ² is selected from the group consisting of CH ₂ , CH ₂ CH ₂ and is CH when the bond between X ¹ and X ² is unsaturated;
R ¹ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ² is CH ₃ or COOH;
R ³ is selected from the group consisting of H, alkyl, or NR ³ R ³ is collectively pyrrolidino or piperidino;
R ⁴ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ⁵ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl; and n is an integer from 0-2.

In a further embodiment of the invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is CH ₂ —CH _3. R ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is H; and n is 0 (analogue 2: glycyl-L- 2-ethylprolyl-L-glutamic acid ("G-2 EthylPE")).
In yet another embodiment of the present invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is CH ₂ —CH. be ₂ -CH _3; R ² is located at COOH; R ³ is is H; R ⁴ is is H; R ⁵ is is H; and n is 0 (analogue 3: Glycyl-L-2-propylprolyl-L-glutamic acid ("G-2PropylPE")).
In a further embodiment of the invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is CH ₂ —CH═ be CH _2; R ² is located at COOH; R ³ is is H; R ⁴ is is H; R ⁵ is is H; and n is 0 (analogue 4: glycyl - L-2-allylprolyl-L-glutamic acid trifluoroacetate ("G-2AllylPE")).
In another embodiment of the present invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is benzyl; ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is H; and n is 0 (analogue 5: glycyl-L-2-benzylpro) Ryl-L-glutamic acid trifluoroacetate ("G-2benzylPE")).
In yet another embodiment of the present invention, the compound is a compound of formula 1, wherein X ¹ is S; X ² is CH ₂ ; R ¹ is H; ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is H; and n is 0 (analog 48: glycyl-L-4-thiaprolyl- L-glutamic acid trifluoroacetate).
In a further embodiment of the invention, the compound is a compound of formula 1, wherein X ¹ is S; X ² is CH ₂ ; R ¹ is H; R ² Is COOH; R ³ is H; R ⁴ is CH ₃ ; R ⁵ is H; and n is 0 (analog 49: glycyl-L-thia-5, 5-Dimethylprolyl-L-glutamic acid ("G-thiadiMePE")).
In a further embodiment of the invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is H; ² is COOH; R ³ is H; R ⁴ is CH ₃ ; R ⁵ is H; and n is 0 (analog 50: glycyl- (D, L) -5,5-dimethylprolyl-L-glutamic acid).
In another embodiment of the invention, the compound is a compound of formula 1, wherein X ¹ is C (OH) H; X ² is CH ₂ ; R ¹ is H R ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is H; and n is 0 (analogue 60: glycyl-trans-4) -Hydroxy-L-prolyl-L-glutamic acid ("GHypE")).

In yet another embodiment of the present invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is H; and n is 1 (analog 62: glycyl-L-homoprolyl-L -Glutamic acid ("GHomoPE")).
In a further embodiment of the invention, the compound is a compound of formula 1, wherein the bond between the X ¹ and X ² be unsaturated; X ¹ is located in CH; X ² is, R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is H; and n is 0 (Analog 63: glycyl-L-3,4-dehydroprolyl-L-glutamic acid trifluoroacetate ("G-3, 4-dehydroPE.TFA")).
In a further embodiment of the invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is H; ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is CH ₃ ; and n is 0 (analog 64: aminoisobutryl-L- Prolyl-L-glutamic acid (“AibPE”)).
In a further embodiment of the invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is H; ² is CH ₃ ; R ³ is H; R ⁴ is H; R ⁵ is H; and n is 0 (analog 65: glycyl-L-prolyl-L -Norvaline ("GPnorvaline")).
In another embodiment of the present invention, the compound is a compound of formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ CH ₂ ; R ¹ is H R ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is H; and n is 0 (analog 66: glycyl-D, L- Pipecolinyl-L-glutamic acid (“G (D, L) PipE”)).
In yet another embodiment of the present invention, the compound is a compound of Formula 1, wherein X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is CH ₃ R ² is COOH; NR ³ R ³ is collectively pyrrolidino; R ⁴ is H; R ⁵ is H; and n is 0 (analog 67: pylori) Dinoglycyl-L-2-methyl-proline-L-glutamic acid ("PyrrolidinoG-2MePE")).

Formula 2

In some embodiments, the invention includes a compound of Formula 2 or a pharmaceutically acceptable salt or hydrate thereof, wherein R is H or OH.
In a further embodiment of the invention, the compound is a compound of formula 2, wherein R is H (analog 35: (2S, 5′R)-[1 ′-(2 ″ -amino -Acetyl) -6'-oxo-1 ', 7'-diazaspiro [4.4] non-7'-yl] -1,5-pentanedioic acid ("GP-5,5-spirolactam E" )).
In a further embodiment of the invention, the compound is a compound of formula 2, wherein R is H (analog 36: (2S, 5′R, 8′R) — and (2S, 5 'R, 8'S)-[1'-(2 "-Amino-acetyl) -8'-hydroxy-6'-oxo-1 ', 7'-diazaspiro [4.4] non-7'-yl] -1,5-pentanedioic acid ("GP-5,5-hydroxyspirolactam E").

Formula 3

In some embodiments, the invention includes a compound of Formula 3 or a pharmaceutically acceptable salt or hydrate thereof, wherein R ¹ is H, alkyl, alkenyl, aryl, arylalkyl , Substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ² is CH ₃ or COOH;
R ³ is selected from the group consisting of H, alkyl, or NR ³ R ³ is collectively pyrrolidino or piperidino;
R ⁴ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
n is an integer of 0-2.
In a further embodiment of the invention, the compound is a compound of formula 3, wherein R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is , H; and n is 0 (analog 61: glycyl-L-2-pyroglutamyl-L-glutamate hydrochloride (“GpyroE.HCl”)).

Formula 4

In some embodiments, the present invention includes a compound of Formula 4 or a pharmaceutically acceptable salt or hydrate thereof, wherein R ¹ is H, alkyl, alkenyl, aryl, arylalkyl. , Substituted alkyl, substituted alkenyl, substituted aryl, or substituted arylalkyl.
In a further embodiment of the invention, the compound is a compound of formula 4, wherein R ¹ is CH ₃ (analog 68: glycyl-L-2-methylproline (“G-2MeP”) ).
In some embodiments, the present invention includes compounds of Formula 4 or a pharmaceutically acceptable salt or hydrate thereof wherein R ¹ is methyl.
Another aspect of the present invention provides pharmaceutically acceptable salts of the compounds described in Formulas 1-4.
In yet another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of at least one compound of the present invention. These compositions have found use as anti-apoptotic and anti-necrotic agents and for other conditions involving neurodegeneration or injury.
In a further aspect, the invention provides a method of treating an animal having a disease or injury treatable by administering a suitable compound of formulas 1-4, wherein said compound is a compound of the invention. A method is provided comprising administering at least one, optionally together with at least one other conventional therapeutic agent for the disease to be treated.
In a further embodiment, the animal to be treated is a human.
In a further aspect, the present invention provides methods for synthesizing, formulating and manufacturing pharmaceutical formulations comprising the compounds of Formulas 1-4 of the present invention.
One skilled in the art will appreciate that the above structural formula includes chiral centers, the number of which depends on different substituents. Chirality is simply shown for several centers. The chirality may be either R or S at each center. Formula (formulae drawing) represents only one of the possible tautomers, conformers or enantiomers, and the present invention may be used as a biological or pharmaceutical agent as described herein. It should be understood to include tautomers, conformers or enantiomers that exhibit physical activity.

Pharmacology and Utility Some aspects of the present invention include the use of compounds of the present invention in the treatment or prevention of cell damage, degeneration and / or death in an animal responsive to injury or disease. Some embodiments include delivering a composition comprising a compound of the invention to an animal afflicted with a condition involving neurodegeneration and, in some cases, apoptosis and necrotic cell death. In some embodiments, the composition is desirable for treating CNS injuries or diseases that affect or tend to affect brain cells. Compositions are also provided that also include one or more other agents that promote nerve regeneration, reduce cell degeneration or death, or have a neuroprotective action.
Such other agents include, for example, growth factors and related derivatives such as insulin-like growth factor-I (IGF-1), insulin-like growth factor-II (IGF-II), tripeptide GPE, transforming growth factor -Β1, activin, growth hormone, nerve growth factor, growth hormone binding protein, and / or IGF-binding protein.
Other aspects of the invention include compositions and methods that promote axon fasiculation. By promoting the formation of nerve bundles, the compounds of the present invention allow neurites (axons and / or dendrites) to be severed, eg, sharp force injury, necrosis or local disease. Or may be useful in treating conditions that result in other localized damage to the neurites.
In yet another embodiment, compositions and methods for treating or preventing damage and death in response to damage and disease, including CNS damage and disease, may comprise therapeutic low doses of the compounds of the invention alone or in other Including in combination with a drug after an attack. These embodiments may be particularly desirable in situations such as unexpected injury, eg, heart failure, trauma, eg, head injury, head injury due to a car accident, etc.
In a further embodiment, the compounds of the invention can be used alone or in combination with other agents to prevent the adverse effects of planned head injury. Such conditions include CABG or other planned surgery, such as brain surgery, vascular surgery, or other practice that may result in reduced reflux of the nervous system. By treating animals, such as humans, prior to and / or simultaneously with and / or after surgery, adverse neurological effects can be improved.
As indicated above, the present invention broadly relates to the inventor that the compounds of the present invention can protect cells, particularly neurons, from damage, neurite loss, and / or apoptosis or necrotic cell death. Based on knowledge.
It has been demonstrated herein that the compounds of the present invention exhibit neuroprotection in a cell culture model of neurodegenerative disease and can therefore be an effective additive or alternative to conventional treatments for neurodegeneration. The
Although the mechanism of protective action is not known, one possible mechanism involves protecting cells from apoptosis and necrotic cell death. However, regardless of the mechanism of action, the compounds of the invention can be used as an effective treatment for a variety of neurological diseases including hypoxia, ischemia and neurotoxin-induced nerve damage. Furthermore, the compounds of the present invention can be used without the specific neurological deficiencies that promote neurite outgrowth and neural facilitation. Thus, in situations where cell death is not necessarily associated with a neurological disorder (eg, axonal damage caused by, for example, spinal cord injury), administration of a compound of the invention can be an effective method of promoting axonal regeneration.

Therapeutic Applications The compositions and methods of the present invention have found use in the treatment of animals suffering from nerve injury or disease, such as human patients. More generally, the compositions and methods of the present invention find use in the treatment of mammals, eg, human patients, suffering from nerve damage or potential apoptosis and / or necrotic cell death (due to injury and disease). It was.
General observations have been made in the study of GPE and other GPE analogs G-2 methyl PE, G-2 AllylPE, diketopiperazine derivatives of GPE, macrocyclic analogs of GPE and L-Ala-PE. For compounds that are neuroprotective as assessed using in vitro systems, such as those described herein and described in other patents and patent applications of the inventor, the presence of neuroprotective Independent of the type of damage being done. Thus, the neurodegenerative effects of hypoxia / ischemia and toxic injury can be mitigated or prevented by the previously disclosed GPE and GPE analogs. Moreover, the parallel neuroprotective action of the GPE analogs previously disclosed above is also seen in the in vivo system. Thus, compounds that exhibit neuroprotective properties in vitro may also inhibit neurodegeneration associated with stroke, hypoxia / ischemic traumatic brain injury and animal models of Parkinson's disease and Alzheimer's disease. Furthermore, in in vivo studies using pre-disclosed GPE analogs, functional defects and histologically observed neurodegeneration are also at least partially in a parallel fashion with GPE and pre-disclosed GPE analogs. To be relaxed. Such functional disadvantages include gait, motor nerve and memory impairments. Some of these studies and interrelationships can be found in US Pat. No. 7,041,314, PCT International Patent Application No: PCT / US02 / 16361 (filed May 24, 2002), US Patent Application No: 11 / 314,424 (December 2005). U.S. Patent Application No. 11 / 315,784 (filed on Dec. 21, 2005), U.S. Patent Application No. 11 / 398,032 (filed on Apr. 4, 2006) and U.S. Provisional Patent Application No. 60 / 782,148 (Filed on Mar. 14, 2006). Each of the foregoing patents and patent applications is fully incorporated herein by reference. Thus, in vitro studies as described herein can be useful and effective tools for assessing the effects of GPE analogs for in vivo applications and are obtained using such in vitro systems. The results have a high degree of predictability of effects observed in humans suffering from disorders associated with neurodegeneration and in vivo.
Specific conditions and diseases characterized by neurodegeneration, apoptosis and / or necrosis include, but are not limited to: Alzheimer's disease, Parkinson's disease, multiple sclerosis, muscle atrophy. Lateral sclerosis, spinal muscular atrophy, peripheral neuropathy, Creutzfeldt-Jakob disease, AIDS dementia, progressive supranuclear palsy, myelinopathia centralis diffusa (vanishing) white matter Disease), chronic neurodegenerative disease, Huntington's disease, stroke, ischemic injury, hypoxia injury, reperfusion injury, head injury, invasive brain injury, CNS trauma, epilepsy, cerebral ischemia, glaucoma, retinal disease, optic nerve Disease, optic neuritis, Down syndrome, encephalomyelitis, meningitis, panencephalitis, neuroblastoma, schizophrenia and depression. Each of the above conditions presents pathophysiological findings and symptoms that are manifested by neurotoxicity associated with glutamate toxicity, where GPE and GPE analogs may have therapeutic value for them.
The compounds of the invention can also be applied in the induction of nerve bundle formation after injury in the form of trauma, toxic exposure, respiratory arrest or hypoxic ischemia. The compounds of the invention are also applied in the treatment or prevention of cancer, viral infections, autoimmune diseases, nervous system diseases and injuries and other forms of neurodegeneration, necrosis or damage in response to cardiovascular diseases or apoptosis. be able to.

Treatment can be performed prior to injury, for example, prior to elective surgery. Examples of relevant alternative means are neurosurgery (in which retraction of the lobe can lead to cerebral edema), or cardiac surgery, eg valve replacement (in which rice small small) Emboli (invitable small emboli) is said to cause detectable dysfunction of brain function in 75% of cases).
It is also known that after the initial injury, there is a combination of an “early” or rapid phase of neurodegeneration associated with the injury or disease and a “slow” or delayed phase of neurodegeneration. In some cases, late phase neurodegeneration can be effectively treated by administering a neuroprotective dose of a GPE or GPE analog of the invention from 0 to about 100 hours after the initial injury. Administration of the GPE analogs of the present invention can alleviate late phase neurodegeneration, even hours after the initial injury, even during the time after early phase neurodegeneration. Thus, late phase neurodegeneration may be prevented or suppressed by administering the GPE analogs of the present invention after the initial injury, after which it may be necessary to prevent early phase neurodegeneration.

Measurement effect The neuroprotective activity of the compounds of the present invention can be determined by the method of Klempt et al. It can be measured in vivo using cell counts by methods known to those skilled in the art including neuronal cell counts. The effects of the compounds of the invention can also be measured in vitro using assays for neurite outgrowth, mass spectrometry, immunological or chromatographic methods known in the art. These methods are well characterized and validated in various test systems and are therefore expected to be neuroprotective in animal cells including human cells and humans.
CNS damage can be measured clinically, for example, by the degree of persistent neuropathy cognitive function and / or tendency to seizure disorders. Disclosed herein are histological techniques suitable for measuring in vivo effects. Several methods suitable for in vivo evaluation are described in US Pat. No. 7,041,314, PCT International Patent Application No: PCT / US02 / 16361 (filed May 24, 2002), US Patent Application No: 11 / 314,424 (2005 12 U.S. Patent Application No. 11 / 315,784 (filed on Dec. 21, 2005), U.S. Patent Application No. 11 / 398,032 (filed on Apr. 4, 2006) and U.S. Provisional Patent Application No. 60 / 782,148 (filed on Mar. 14, 2006). Each of the foregoing patents and patent applications is fully incorporated herein by reference.
It is understood that the treatable ratio of a compound is (2) the average dose that causes the desired therapeutic effect, and (1) the ratio of the dose that causes side effects. Thus, for compounds with therapeutic effects at relatively low doses and unintended side effects at high doses, the treatable ratio is> 1. The treatable ratio compares, for example, the amount that produces significant mass loss (or other observable side effects) divided by the amount that produces anti-apoptotic and anti-necrotic activity in an appropriate in vivo animal species, eg, rat or mouse. Can be measured. Suitable models include hypoxic ischemic injury (Sirimanne et al., 1994 Journal of Neuroscience Methods: 55: 7-14) and experimental immune encephalomyelitis (Mendel et al., 1995 Eur. J. Immunol .: 1951-1959). Including.

Pharmaceutical Compositions and Administration The compounds of the present invention can be administered as part of a drug or pharmaceutical formulation. This includes combining a compound of the present invention with a pharmaceutically suitable carrier, adjuvant or excipient. The choice of carrier, adjuvant or excipient will of course usually depend on the route of administration used.
In general, the compounds of the present invention will be administered in a therapeutically effective amount, by any conventional mode known in the art, alone or in combination with other conventional therapeutic agents for the disease being treated. Let's go. A therapeutically effective amount can vary widely depending on the disease or injury, its severity, age and the relevant health of the animal being treated, the potency of the compound and other factors. Therapeutically effective amounts of the compounds of the invention as anti-apoptotic and anti-necrotic agents can vary from 0.001 to 100 milligrams per kg of animal mass, and low doses such as 0.001 to 0.1 mg / kg Suitable for administration through cerebrospinal fluid, for example by intracerebroventricular administration, and high doses, for example 1-1 mg / kg, are oral, systemic (for example transdermal), or parenteral Suitable for administration (eg intravenous). One of ordinary skill in the art can determine a therapeutically effective amount of a compound of the present invention for a given disease and injury without undue experimentation and with reference to that technique and the disclosure herein. It will be possible.
The compounds of the present invention can be administered peripherally via the peripheral routes known in the art. These are parenteral routes such as injection into the peripheral circulation, subcutaneous, intraorbital, ophthalmic, intrathecal, intracapsular, topical, injection (eg delayed release devices or minipumps such as osmotic pumps or skin Can be used), implants, aerosols, inhalation, scarification, intraperitoneal, intracapsular, intramuscular, intranasal, oral, buccal, subcutaneous, pulmonary, rectal or vaginal routes. The composition may be administered parenterally to a human or other mammal in a therapeutically effective amount (eg, an amount that eliminates or reduces the pathological condition of the patient) to provide treatment for the neurological disorder. Can be blended.
Desirably, if possible, the compounds of the invention will be administered orally when administered as an anti-apoptotic and anti-necrotic agent. The amount of the compound of the invention in the composition can vary widely depending on the type of composition, the size of the unit dosage, the type of excipient, and other factors well known to those skilled in the art. . In general, the final composition may contain 0.0001 to 10% by weight (% w), preferably 0.001 to 1% w, of the compound of the invention, with the remainder being excipients.
Other conventional routes of administration include subcutaneous injection (eg, dissolved in a physiologically compatible carrier such as 0.9% sodium chloride) or direct administration to the CNS. Using an accurate map of the animal's CNS and a stereotaxic device, the compound is injected directly into the site of nerve damage. Such a route of administration may be desirable, particularly in situations where perfusion at that location is compromised by either reduced vascular perfusion or reduced cerebrospinal fluid (CSF) flow to the area. . Examples include intravenous, surgically inserted shunt, direct injection into the desired location, or administration via other routes, by lateral ventricular injection or into the lateral ventricle of the patient's brain. Can be mentioned.

An effective amount of a compound within the CNS can be increased by administration of a prodrug form of the compound, which includes a compound of the present invention and a carrier, wherein the carrier includes the compound of the present invention within the division or digestion within a patient. It is connected by a bond that is susceptible to damage. Any suitable linkage that will be split or digested after administration may be used.
However, it is not intended to exclude other forms of administration.
In a further embodiment of the invention, inhibiting neurodegeneration and / or restoring nerve function in an animal combines a therapeutic amount of a compound of the invention with other neuroprotective agents selected from: US Patent No. 7,041,314, PCT International Patent Application No: PCT / US02 / 16361 (filed May 24, 2002), US Patent Application No: 11 / 314,424 (December 20, 2005) US Patent Application No. 11 / 315,784 (filed December 21, 2005), US Patent Application No. 11 / 398,032 (filed April 4, 2006) and US Provisional Patent Application No. 60 / 782,148 (2006). GPE, GPE analogs, US Patent Application No. 10 / 976,699 (filed October 29, 2004), PCT International Patent Application No. PCT / US02 / 026782 (2002) Filed on Aug. 22, 2000) or PCT / US2004 / 036203 The disclosed neuroregenerative peptides, macrocyclic analogs of GPE described in PCT International Patent Application No: PCT / US2004 / 008108 (filed on March 16, 2004), PCT International Patent Application No: PCT / US2004 / GPE bicyclic analogs, growth factors and related derivatives (insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF- II), transforming growth factor-β1, activin, growth hormone, nerve growth factor, growth hormone binding protein, IGF-binding protein (particularly IGFBP-3), basic fibroblast growth factor, acidic fibroblast growth factor Hst / Kfgk gene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor, androgen-induced growth factor, an additional member of the FGF family. Examples of the bar include int-2, fibroblast growth factor homologous factor (FHF-1), FHF-2, FHF-3 and FHF-4, keratinocyte growth factor 2, glial activator, FGF-10 and FGF -16, ciliary neurotrophic factor, brain-derived growth factor, neurotrophin 3, neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial cell line-derived neurotrophic factor, activity-dependent neurotrophic factor , Cytokine leukemia inhibitory factor, oncostatin M, interleukin), α-, β-, γ- or consensus interferon, and TNF-α. Other forms of neuroprotective therapeutic agents include, for example, chromethiazole, kynurenic acid, Semax, tacrolimus, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, andreno Corticotropin- (4-9) analogs [ORG2766] and dizolcipine (MK-801), selegiline; glutamate antagonists such as NPS1506, GV1505260, MK-801, GV150526; AMPA antagonists such as 2,3-dihydroxy Anti-inflammatory against -6-nitro-7-sulfamoylbenzo (f) quinoxaline (NBQX), LY303070 and LY300164; addressin MAdCAM-1 and / or its integrin α4 receptor (α4β1 and α4β7) , For example, anti -MAdCAM-1mab MECA-367 (ATCC Accession No. no.HB-9478) can be mentioned.

The compound can be administered by a sustained release system. Suitable examples of sustained release compositions include semipermeable polymer matrices, such as films, or macrocapsules in the form of shaped articles. Sustained release matrices include polyactides (US Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers: 22: 547-56), poly (2-hydroxy Ethyl methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res .: 15: 267), ethylene vinyl acetate (Langer et al., 1981, J. Biomed. Mater. Res .: 15: 267), or poly-D -(-)-3-hydroxybutyric acid (EP 133,988) is included. Sustained release compositions also include liposomally entrapped compounds. Liposomes containing the compounds are produced by methods known per se: DE 3,218,121, EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142,641, Japanese patent application 83-118008, US Pat. Nos. 4,485,045 and 4,544,545. And EP 102,324. Liposomes are usually small (about 200-800 cm) monolayer types, in which the lipid content is higher than about 30 mol% cholesterol, and the selected proportion is adjusted for the most effective treatment Is done.
For parenteral administration, in certain embodiments, the compounds are generally pharmaceutically or parenterally, each in a unit dose injectable form (solution, suspension, or emulsion) in the desired degree of purity. Formulated with a pharmaceutically acceptable carrier, ie, one that is non-toxic at the dosage and concentration used by the recipient and compatible with the other ingredients of the formulation.
In general, formulations are prepared by contacting the compound uniformly and intimately with liquid carriers or finely divided solid carriers or both. If necessary, the product is then shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, buffer, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein.
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations used, and buffers, phosphates, citrates, succinates, acetic acids, and other organic acids or their salts; antioxidants such as Ascorbic acid; low molecular weight (less than about 10 residues) polypeptide, such as polyarginine or tripeptide; protein, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; glycine; For example, glutamic acid, aspartic acid, histidine, or arginine; other carbohydrates including monosaccharides, disaccharides, cellulose or derivatives thereof, glucose, mannose, trehalose, or dextrin; chelating agents such as EDTA; sugar alcohols such as Mannitol or sorbitol Counterions such as sodium; including and / or neutral salts, for example, NaCl, KCl, a and MgCl _2, CaCl _2; nonionic surfactants, such as polysorbate, poloxamer or polyethylene glycol (PEG).
The compound is typically formulated in such vehicles at a pH of about 4.5-8. It will be appreciated that some use of the aforementioned excipients, carriers or stabilizers will result in the salt form of the compound. The final formulation may be a stable liquid or a lyophilized solid.

The formulation of the compound in the pharmaceutical composition may also include an adjuvant. Typical adjuvants that can be introduced into tablets, capsules, etc. are binders such as acacia, corn starch, or gelatin; excipients such as microcrystalline cellulose; disintegrants such as corn starch or alginic acid; lubricants such as stearin Magnesium acid; sweeteners such as sucrose or lactose; flavorings such as peppermint, winter green, or cherries. Where the dosage form is a capsule, in addition to the above materials, it may also contain a liquid carrier such as a fatty oil. Various other types of materials can be used as coatings in the physical form of the dosage unit or as improvers. A syrup or elixir may contain the active compound, sweeteners such as sucrose, preservatives such as propylparaben, colorants, and flavoring agents such as cherries. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice. For example, a solution or suspension of the active compound in a vehicle such as water or a naturally occurring vegetable oil such as sesamin oil, peanut oil or cottonseed oil or a synthetic fat vehicle such as ethyl oleate may be desirable. Buffers, preservatives, antioxidants and the like can be introduced according to accepted pharmaceutical practice.
For infusion, intracerebroventricular administration, and other invasive routes of administration, the compounds used must be sterile. Sterilization can be achieved by methods known in the art, such as filtration through sterile filtration membranes (eg, 0.2 micron membranes). The therapeutic composition is generally placed in a container having a sterile access port, eg, a solution bag or vial for intravenous injection having a stopper that can be penetrated by a hypodermic injection needle.
The pharmaceutical formulation will generally be stored as an aqueous solution or as a lyophilized formulation for reconstitution in one or more dosing containers, such as sealed ampoules or vials. As an example of a lyophilized formulation, a 10 mL vial is filled with 5 mL of a sterile filtered 1% (w / v) aqueous solution of the compound and the remaining mixture is lyophilized. Injection solutions are prepared by reconstitution of the lyophilized compound with bacteriostatic water for injection. It is readily understood that other dosage forms and types of formulations can be used, and all are part of the present invention.

Preparation of Compounds of the Invention Starting materials and reagents used to prepare these compounds are commercially available, for example, from Aldrich Chemical Company (Milwaukee, Wis), Bachem (Torrance, Calif.), Sigma (St. Louis, Mo.), available from Strem (Newburyport, Ma.) Or manufactured by procedures well known to those skilled in the art and described in the following references: : Fieser and Fieser's Reagents for Organic Synthesis, vols 1-17, John Wiley and Sons, New York, NY, 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supplements, Elsevier Science Publishers, 1989; Organic Reactions, vols .. 1-40, John Wiley and Sons , New York, NY, 1991; March J; Advanced Organic Chemistry, 4 th ed John Wiley and Sons, New York, NY, 1992; and Larock: Comprehensive Organic Transformations, VCH Publishers, 1989. In most cases, amino acids and their esters or amides, and protected amino acids are widely commercially available; modified amino acid and their amide or ester formulations are extensively described in the chemical and biochemical literature. And therefore well known to those skilled in the art.
The starting materials, intermediates and compounds of the present invention can be isolated and purified using conventional techniques including filtration, distillation, crystallization, chromatography and the like. They can be characterized using conventional methods including physical constants and spectral data.

Examples Example 1: Synthesis of novel analogs of GPE:
Analog 2 (Glycyl-L-2-ethylpropyl-L-glutamic acid), Analog 3 (Glycyl-L-2-propylpropyl-L-glutamic acid), Analog 4 (Glycyl-L-2-allylprolyl-L-) Glutamic acid trifluoroacetic acid), analog 5 (glycyl-L-2-benzylprolyl-L-glutamic acid trifluoroacetic acid), analog 35 ((2S, 5′R)-[1 ′-(2 ″ -amino-) Acetyl) -6'-oxo-1, '7'-diazaspiro [4.4] non-7'-yl] -1,5-pentanedioic acid), analog 36 ((2S, 5'R, 8' R)-and (2S, 5'R, 8'S)-[1 '-(2 "-amino-acetyl) -8'-hydroxy-6'-oxo-1,'7'-diazaspiro [4.4 Non-7′-yl] -1,5-pentanedioic acid), analog 48 (glycyl-L-4-thiapro) Ru-L-glutamic acid trifluoroacetic acid), analog 49 (glycyl-L-thia-5,5-dimethylprolyl-L-glutamic acid) and analog 50 (glycyl- (D, L) -5,5-dimethyl) The synthesis of (prolyl-L-glutamic acid) is described herein.
To examine the remaining importance of proline in GPE, analogs modified with either Pro or Pro-Glu linkages were synthesized. In particular, conformationally fixed analogs were produced to gain insight into the receptor binding conformation. The general synthetic method used then involved the rest of the preparation of the individual modified prolines attached to the glycine derivative and glutamic acid di-ester (Scheme 1).

Scheme 1. General reverse synthesis of GP ^* E analogs

The presence of (S) -2-methylproline (2-MePro) is known to stabilize rotation (Bisang, C et al. J. Am. Chem. Soc. 1995, 117, 7904; Welsh et al. A. FEBS Lett. 1992, 297, 216) and also peptidases that recognize Pro-Glu amide bonds that confer resistance to proteolysis can be prevented (Thaisrivongs et al. Med. Chem. 1987, 30, 536). Therefore, glycyl-L-2-methylpropyl-L-glutamic acid (G-2MePE) 1 was synthesized. In addition, to study the effect of modification at this position, four other 2-alkylproline analogs 2-5 were also synthesized (Scheme 2).
2-alkylproline derivatives were synthesized using the Wang and Germanas modifications of the self-regenerative chiral Savebach method (Seebach et al. J. Am. Chem. Soc. 1983, 105, 5390; Beck et al J Org Synth. 1992, 72, 62). Condensation of L-proline 6 with chloral (trichloroacetaldehyde) gave the oxazolidinone 7 used for the synthesis of five 2-alkylproline modified tripeptides 1-5 (Amedjkouth et al. Tetrahedron: Asymmetry 2002, 13,2229). Treatment of 7 with LDA to effect enolate formation, followed by alkylation with iodomethane, iodoethane, allyl bromide or benzyl bromide, respectively, provided alkylated oxazolidinone 8-11. Esterification with thionyl chloride (for 8, 9) or acetyl chloride (for 10, 11) in methanol is carried out by N-benzyloxycarbonyl-glycine 16 (for 12-14) or Nt -Methyl ester hydrochloride 12-15 coupled with butyloxycarbonyl-glycine 17 (for 14 and 15) was given to give dipeptide 18-22.

Optimal conditions for amide bond formation were investigated in detail using a bond between 12 and 16; bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BoPCl) was found to be superior ( 92% yield compared to 66% with DCC) and used for all subsequent coupling reactions. Hydrolysis of methyl esters 18, 19, 20 (NaOH in dioxane) to carboxylic acids 23, 24, 25, followed by conjugation with dibenzyl glutamate 28 (BoPCl) results in benzyl protected tripeptides 30, 31, 32. Provided. Eventually, extensive deprotection of the benzyl group gave tripeptides 1 and 2, while simultaneous hydrogenolysis of the allyl group at 25 gave tripeptide 3.
Boc and t-butyl protecting groups were used for the synthesis of tripeptides 4 and 5, which incorporate 2-allylproline and 2-benzylproline units, respectively. In these cases, coupling acids 26 and 27 with di-t-butylglutamate 29 gives tripeptides 33 and 34 which provide tripeptides 4 and 5 as trifluoroacetate after deprotection with TFA. It was.

Scheme 2. Reagents, status and yield: (i) chloral, CHCl ₃ , reflux, 6h (77%); (ii) LDA, THF, -78 ° C, MeI, EtI, CH ₃ CH ₂ = CH ₂ Br or PhCH ₂ Br , -78-> -30 ° C, 4h, 8, 63%, 9, 46%, 10, 60%, 11, 2.5h, 32%; (iii) SOCl ₂ , CH ₃ OH, reflux, 3h, 12, 100%, 13, 71%, AcCl, CH ₃ OH, reflux, 24h, 14, 63%, 15, 48%; (iv) For 18, 19, 20: Et ₃ N, BoPCl, 16, CH ₂ Cl ₂ , rt, 18, 20.5h, 92%, 19, 19.5h, 46%, 20, 20h, 30%; About 21, 22: Et ₃ N, BoPCl, 17, CH ₂ Cl ₂ , rt, 19.5h, 21 , 45%, 22, 18.5h, 22%; (v) Dioxane, 1 M aq.NaOH, rt, 15-20h, 23, 90%, 24, 95%, 25, 92%, 26, 83%, 27 , 95%; (vi) About 30, 31, 32: Et ₃ N, BoPCl, 28, CH ₂ Cl ₂ , rt, 17h, 30, 89%, 31, 17.5h, 70%, 32, 19.5h, 76 %; About 33, 34: Et ₃ N, BoPCl, 29, CH ₂ Cl ₂ , rt, 17.5h, 33, 77%, 34, 17h, 68%; (vii) H ₂ , 10% Pd / C, CH ₃ OH / H ₂ O (90:10), rt, 23h, 1, 86%, 2, 20h, 99%, 3, 19h, 100%; (viii) CF ₃ CO ₂ H, CH ₂ Cl ₂ , rt , 6.5h, 4, 96%, 5, 3.5h, 100%.

In contrast to most peptide bonds that exclusively select the trans conformation, the Xaa-Pro amide bond can exist as a mixture of cis and trans isomers (Dugave et al. Chem. Rev. 2003, 103 , 2475). The conformational nature of this bond can affect the biological activity of the peptide and is evidence that some proteases only recognize transpeptide bonds (Vanhoof et al. FASEB J. 1995, 9, 736; Lin et al. Biochemistry 1983, 22, 4480). The presence of a specific peptidyl-prolyl cis-trans isomerase would seem to support this evidence (Fanghanel Angew. Chem Int. Ed. 2003, 42, 490). GPE, as evidenced by NMR analysis, D ₂ O solution of isomers of 20:80 cis - exist as trans mixture. The trans conformation is preferred when the alkyl group is substituted at the 2-position of the proline. Compounds 1-4 selected all trans conformations and the trans population was also increased in compound 5 with only 10% selecting the cis conformation. The prevalence of trans isomers in 2-methylproline compounds has been attributed to large methyl groups that destabilize the cis conformation (Delaney et al. J. Am. Chem. Soc. 1982, 104, 6635).

Another way to conformally limit peptides is to synthesize peptidomimetics that contain a spirolactam ring system. Spirolactam ring systems suggested that the compound could be anchored primarily within one conformation and that different ring systems mimic both b- and g-rotations (Hinds et al J. Med Chem. 1991, 43, 1777; Fernandez et al. J. Org. Chem. 2002, 67, 7587; Kang J. Phys. Chem. 2002, 106, 2074). Spirocyclic g-lactam bridges can be formed between the remaining nitrogen of glutamate and the remaining 2-position of proline in GPE, and thus to investigate the effects of such conformational restriction in GPE analogs. Give an opportunity.
The synthesis of GP- [5.5] spirolactam E 35 and the corresponding GP- [5.5] hydroxyspirolactam E 36 is summarized in Scheme 3.

Scheme 3. Reagents, status and yield: (i) O ₃ , MeOH / CH ₂ Cl ₂ (1: 1), 15 min then PPh ₃ , 24 h, then silica gel, 63%; (ii) CF ₃ CO ₂ H / Et ₃ SiH / CH ₂ Cl ₂ (1: 1: 1), rt, 45 min, 96%; (iii) 10% Pd / C, CH ₃ OH / H ₂ O (88:12), 18h, 35, 78% , 36, 99%.

Ozonolysis of alkene 32, followed by treatment with triphenylphosphine, began via aldehyde 37 mediation to give alcohol 38 as a 1: 1 mixture of diastereoisomers. Direct hydrogenation of 38 gave hydroxyspirolactam 36, while the initial reduction of hydroxy groups (trifluoroacetic acid-triethylsilane-dichloromethane) relative to 39 prior to the hydrocracking step provided spirolactam 35. Both spirolactams exclusively selected the trans conformation for the proline ring.
The pyrrolidine ring of proline can select two reliable conformations. These down- and up-puckered conformations occur when the proline C ^g and carbonyl groups are on the same and opposite sides of the plane defined by C ^d , N and C ^a, respectively. Is defined as The presence of a sulfur atom in the proline ring can affect the bond angle and bond length in some cases that alter the proline ring conformation. Kang shows that the remaining resubstitution of proline in AcProNHMe with 4-thiaproline 40 [(R) -thiozolidine-4-carboxylic acid (Thz)] results in a more Packard conformation (Kan 2002). I found it. Further conformational changes in the proline ring can be facilitated by the addition of a methyl group at the 5-position of proline or Thz. The next set of analogs incorporated such a pseudo-proline moiety in which the Pro g-CH ₂ was replaced by a C ^d with a sulfur and / or a dimethyl substituent.

  Pseudo-proline: 4-thiaproline 40 [(R) -thiozolidine-4-carboxylic acid (Thz)] and 2,2-dimethylthiazolidine-4-carboxylic acid 41 can be simply converted to cysteine 42 and formaldehyde (Pellegrini et al. Chem. Bull. 1999, 47, 950) or 2,2-dimethoxypropane (Lewis et al. J. Med. Chem. Pharm. 1978, 21, 1071; Kemp et al. J. Org. Chem. 1989, 54, 3640) Used by reaction with each (Scheme 4).

Scheme 4. Reagents, status and yield: (i) 37% aq. HCHO, H ₂ O, rt, 22h, then pyridine, 56%; (ii) dimethoxypropane, acetone, reflux, 2h, 58%.

Boc-protected methyl D, L-5,5-dimethylprolinate 43 was prepared from nitrile 44 (Scheme 5). Nitrile 44 is prepared as described in the literature (Ono et al. J. Org. Chem., 1985, 50 3692), however, subsequent hydrolysis of the nitrile moiety and hydrogenation of the intermediate N-oxide (described (Magaard et al. Tetrahedron Lett. 1993, 34, 381) occurred simultaneously with acid-catalyzed methyl transfer yielding a mixture of 45 and 46. (6: 4 ratio, ¹ H NMR). Extensive modification of the hydrolysis reaction failed to overcome the formation of N-methylated 45. This unwanted reaction has been described by the respective research group (An et al. J. Am. Chem Soc. 1999, 121, 11588) and has not been recorded during the synthesis of 5,5-dimethylproline. Interesting.
Protection of both acid and amine functionality, such as methyl ester and t-butyloxycarbamate (Boc), respectively, protected 5,5-dimethylproline 43 (22% yield over 4 steps) and N-methyl byproduct 47 ( Easy separation and characterization of 42% over 4 steps). The desired protected 5,5-dimethylproline 43 was present as a mixture of epimers (55:45), exclusively as the cis conformer.

Scheme 5. Reagents, status and yield: (i) 32% aqueous HCl, 50 ° C 5h; (ii) H ₂ (44 psi), 10% Pd / C, MeOH / H ₂ O (1: 1), 20h; (iii ) SOCl ₂ , MeOH, 0 ° C. to rt, overnight then Boc ₂ O, N-methylmorpholine, CH ₂ Cl ₂ , reflux, 48 h [43 (22%) 47 (42%)] More than 4 steps.

Tripeptide 48-50 was synthesized in a similar manner to a 2-alkylproline analog (Scheme 6). Binding of 4-thia-proline building block 40 to Boc-glycine 17 was performed using a mixed anhydrous activation procedure, while hindered 5,5-dimethyl-4-thia-proline 41 provided 53 to provide The use of reactive acid fluid 51 was required. In the case of 5,5-dimethylproline 43, the Boc group was removed with trifluoroacetic acid to provide 54 before the BoPCl was coupled to Boc-glycine 17. Hydrolysis of the methyl ester then provided acid 55 in preparation for the second peptide bond.
Binding of pseudo-dipeptides 52, 53 and 55 with either dibenzyl glutamate 28 or di-t-butyl glutamate 29 using either a mixed anhydrous protocol or BoPCl gives the desired peptides 56, 57 and 58. It was. The nature of the final deprotection step depending on the protecting group used in the synthesis thus provided tripeptide 49 for the removal of benzyloxycarbonyl and benzyl groups by hydrogenation. The Boc and t-butyl ester groups at 56 were removed using trifluoroacetic acid to give the tripeptide 48 as the trifluoroacetate salt, while treatment with trifluoroacetic acid for 58 deprotection followed by Hydrogenation provided the tripeptide 50.

  The presence of a sulfur atom at C-4 in the pyrrolidine ring of proline alone did not appear to significantly change the peptide conformation for the Gly-Pro bond. In GPE analog 48, the cis: trans ratio was established to be 20:80, unchanged from natural peptides. The presence of two methyl groups at C-5 had a dramatic effect on the conformation with a cis: trans ratio that favorably dramatically changed the cis conformer. The population of cis-conformers in 5,5-dimethylated peptide 50 increased to 72% compared to 20% seen with GPE. A further significant effect is the presence of a sulfur atom at C-4 combined with two methyl groups at C-5 in the proline ring, an important role in determining the ratio of cis: trans isomers for the Gly-Pro bond. Was observed with analog 49 showing a cis: trans ratio of 85:15. The high population of cis conformers in the relevant 5,5-dimethylproline was considered to be due to steric hindrance due to the methyl group when the compound selected the trans conformation.

Scheme 6. Reagents, Status and Yield: (i) 17, i-BuOCOCl, Et ₃ N, THF, 0 ° C to rt, then 40, Et ₃ N, H ₂ O, rt, 2h, 52, 81%; (ii) 51, 41, i-Pr ₂ EtN, DMF, rt, 18h and then MeOH, Me ₃ SiCl, rt, 15h, 53, 65%; (iii) 43, CF ₃ CO ₂ H, CH ₂ Cl ₂ , rt, 2h Then, 17, BoPCl, i-Pr ₂ EtN, CH ₂ Cl ₂ , rt, 15h, 54, 52%; (iv) Dioxane, 1 M aq.NaOH, rt, 21h, 55, 94%; (v) About 56 : EtOCOCl, Et ₃ N, CH ₂ Cl ₂ , 0 ° C, 35 min, then 29, Et ₃ N, CH ₂ Cl ₂ , 0 ° C to rt, 15h, 56, 54%; About 57, 58: BoPCl, i- Pr ₂ EtN, CH ₂ Cl ₂ , 28, rt, 7h, 57, 68%, 58, 24h, 67%; (vi) CF ₃ CO ₂ H, Et ₃ SiH, CH ₂ Cl ₂ , rt, 4h, 48 , 61%; (vii) H ₂ (42 psi), 10% Pd / C, CH ₃ OH / H ₂ O (80:20), 24h, 49, 48%; (viii) CF ₃ CO ₂ H, CH ₂ Cl ₂ , rt, 75 min, then H ₂ , 10% Pd / C, CH ₃ OH / H ₂ O (80:20), 15 h, 50, 93%.

1.1 General details All reactions were carried out in a flame-dried or oven-dried glassware under a dry nitrogen atmosphere unless otherwise stated. All reagents were used as supplied. Tetrahydrofuran was dried over sodium / benzophenone and distilled before use. Flash chromatography was performed using Merck Kieselgel 60 (230-400 mesh) with the indicated solvents. Thin layer chromatography (TLC) is performed on precoated silica plates (Merck Kieselgel 60F ₂₅₄ ) and the compound is heated in UV fluorescence and a plate soaked in anisaldehyde or alkaline potassium permanganate solution in ethanolic sulfuric acid. It was visualized by. Melting points at Celsius temperature (° C.) were measured with an Electrothermal® melting point instrument and not corrected. Infrared spectra were recorded on a Perkin Elmer 1600 series Fourier transform infrared spectrometer, such as a thin film between sodium chloride plates. Absorption maxima are stated in wavenumbers (cm ⁻¹ ) in the following abbreviations: s = strong, m = medium, w = weak and br = broad nuclear magnetic resonance (NMR) spectra, Bruker AVANCE DRX400 ( ¹ H, 400 MHz; ¹³ C, 100 MHz), Bruker AVANCE 300 ( ¹ H, 300 MHz; ¹³ C, 75 MHz) or Bruker AC200 ( ¹ H, 200 MHz; ¹³ C, 50 MHz) spectrometer, recorded at 298K. ^{For 1} H NMR data, the chemical shift is tetramethylsilane (δ0.00), DOH (δ4.75), CHD ₂ OD (δ3.30) or CHD ₂ S (O) CD ₃ (δ2.50). In terms of parts per million (ppm) and position (δ _H ), relative integral, multiple (s = singlet), d = doublet, t = triplet, q = quartet, p = pentet, q = pentlet, s = hexet, dd = doublet doublet, m = multiplet and br = broad), coupling constant (J / Hz) and continuous as configuration And recorded. ^{For 13} C NMR data, the chemical shift (ppm) is internal to CDCl ₃ (δ77.0), CD ₃ OD (δ49.1) and (CD ₃ ) ₂ S (O) (δ39.4) or 3- (Trimethylsilyl) -1-propanesulfonic acid sodium salt (DSS) is referred to externally and position (δc), recorded continuously as the degree and configuration of mating. The asterisk ^* indicates the resonance assigned to the smallest conformer. High resolution mass spectra were recorded using a VG70-SE spectrometer operating at a nominal acceleration voltage of 70 eV. Chemical ionization (CI) mass spectra were obtained with ammonia as reagent gas. Light intensity is measured at 20 ° C. with a Perkin Elmer 341 polarimeter using a 10 cm path cell and is given in units of 10 ⁻¹ degcm ² g ⁻¹ . Samples were prepared in the solvents indicated at specific concentrations (measured in g / 100 cm ³ ).

1.1.1 (2R, 5S) -2-trichloromethyl-1-aza-3-oxabicyclo [3.3.0] octan-4-one
A suspension of L-proline (10.0 g, 86.8 mmol) and chloral hydrate (21.6 g, 130 mmol) was heated in a reverse Dean-Stark trap in chloroform (100 cm ³ ) for 6 hours under reflux. The solution was washed with water (2 × 30 cm ³ ) and the water wash was extracted with chloroform (50 cm ³ ). The combined organic layers were dried (MgSO ₄ ), filtered and the solvent removed in vacuo to provide a light brown solid (19.8 g). The parent product was recrystallized from ethanol (80 cm ³ ) at 40 ° C. to form oxazolidinone 7 (16.1 g, 77%) as a white solid: mp 107-109 ^o C (lit. Amedjkouh et al. Tetrahydrofuran: Asymmetric 2002, 13, 2229) ethanol, 107.6 ° C); [α] _D +34.2 (c 2 in C ₆ H ₆ ), (lit. Wang Synlett 1999,1,33) [α] _D +33 (C ₆ C 2.0 in H ₆ ): ^TM _H (200 MHz; CDCl ₃ ) 1.67-2.29 (4H, m, Proβ-H ₂ and Proγ-H ₂ ), 3.08-3.20 (1H, m, Proβ-H _A H _B ), 3.37-3.49 (1H, m, Proβ-H _A H _B ), 4.09-4.15 (1H, m, Proα-H) and 5.17 (1H, s, NCH); ^TM _C (50 MHz; CDCl ₃ ) 25.3 (CH ₂ , Proγ-C), 29.9 (CH ₂ , Proβ-C), 57.9 (CH ₂ , Proδ-C), 62.4 (CH, Proα-C), 100.6 [quat., C (Cl ₃ )], 103.6 (CH, NCH) and 175.5 (quat., CO); m / z (EI +) 244 (MH ⁺ 244).

1.1.2. (2R, 5S) -5-Methyl-2-trichloromethyl-1-aza-3-oxabicyclo [3.3.0] octan-4-one
n-Butyllithium (1.31M, 4.68 cm ³ , 6.14 mmol) was added to a stirred solution of diisopropylamine (0.86 cm ³ , 6.14 mmol) in dry tetrahydrofuran (10 cm ³ ) at −78 ° C. Added dropwise under an atmosphere of nitrogen. The solution was stirred for 5 minutes, warmed to 0 ° C. and stirred for 15 minutes. The solution was added dropwise to a solution of oxazolidinone 7 (1.00 g, 4.09 mmol) in dry tetrahydrofuran (20 cm ³ ) at −78 ° C. over 20 minutes (thickly changed reaction mixture) for an additional 30 minutes. With stirring, iodomethane (0.76 cm ³ , 12.3 mmol) was then added dropwise over 5 minutes. The solution was warmed to −50 ° C. over 2 hours. Water (15 cm ³ ) was added, the solution was warmed to room temperature and extracted with chloroform (3 × 40 cm ³ ). The combined organic extracts were dried (MgSO ₄ ), filtered and evaporated in vacuo to dryness to give a dark brown semi-solid. The remaining purification by flash column chromatography (15% ethyl acetate-hexane) provided oxazolidinone 8 (0.67 g, 63%) as a pale yellow solid: mp 55-57 ° C. (lit. Wang 57-60 ° C.) : δ _H (300 MHz; CDCl ₃ ) 1.53 (3H, s, CH ₃ ), 1.72-2.02 (3H, m, Proβ-H and Proγ-H ₂ ), 2.18-2.26 (1H, m, Proβ-H) , 3.15-3.22 (1H, m, Proδ-H), 3.35-3.44 (1H, m, Proδ-H) and 4.99 (1H, s, NCH).

1.1.3. (2R, 5S) -5-Ethyl-2-trichloromethyl-1-aza-3-oxabicyclo [3.3.0] octan-4-one
The reaction was carried out according to a procedure similar to that described for the preparation of oxazolidinone 8, n-butyllithium (1.31M, 28.3 cm ³ , 37.1 mmol), diisopropylamine (5.2 cm ³ , 37.1 mmol). ), Oxazolidinone 7 (6.0 g, 24.7 mmol) and iodoethane (5.9 cm ³ , 73.8 mmol) to solidify oxazolidinone 9 (3.05 g, 46%) as a light brown solid. Served as a bright red oil: mp 76-77 ° C: [α] _D +18.5 (c 0.25 in CHCl ₃ ): δ _H (300 MHz; CDCl ₃ ) 1.04 (3 H, t, J 7.5, CH ₃ ), 1.60-1.80 (1H, m, CH _A H _B CH ₃ ), 1.72-1.99 (4H, m, CH _A H _B CH ₃ , Proβ-H _A H _B and Proγ-H ₂ ), 2.20- 2.30 (1H, m, Proβ-H _A H _B ), 3.22-3.29 (2H, m, Proδ-H ₂ ) and 5.00 (1 H, s, NCH); δ _c (75 MHz; CDCl ₃ ) 8.4 (CH ₃ , CH ₃ ), 25.5 (CH ₂ , CH ₂ CH ₃ ), 30.9 (CH ₂ , Proγ-C), 35.6 (CH ₂ , Proβ-C), 58.6 (CH ₂ , Proδ-C), 72.5 (quat., Proα-C), 100.9 (quat., C (Cl ₃ )], 102.5 ( CH, NCH) and 176.9 (quat., CO); m / z (EI +) 272.0014 (MH ⁺ . C ₉ H ₁₃ ³⁵ ClN ₃ O ₂ requires 272.0012).

1.1.4. (2R, 5S) -5-allyl-2-trichloromethyl-1-aza-3-oxabicyclo [3.3.0] octan-4-one
(Wang et al. Synlett 1999, 1,33) The reaction was carried out according to a procedure similar to that described for the preparation of oxazolidinone 8, n-butyllithium (1.31 M, 9.93 cm ³ , 13.0 mmol). , Diisopropylamine (1.82 cm ³ , 13.0 mmol), oxazolidinone 7 (2.10 g, 8.7 mmol) and allyl bromide (2.25 cm ³ , 26.0 mmol) to give oxazolidinone 10 (1. 48g, 60%) was provided as a bright orange oil with NMR data consistent with the literature.

1.1.5. (2R, 5S) -5-Benzyl-2-trichloromethyl-1-aza-3-oxabicyclo [3.3.0] octan-4-one
(Wang et al. Synlett 1999, 1,33) The reaction was carried out according to a procedure similar to that described for the preparation of oxazolidinone 8, n-butyllithium (1.31 M, 5.53 cm ³ , 7.2 mmol). , Diisopropylamine (1.01 cm ³ , 7.24 mmol), oxazolidinone 7 (1.18 g, 4.8 mmol) and benzyl bromide (1.72 cm ³ , 14.5 mmol) to give oxazolidinone 11 (0. 52 g, 32%) was provided as a colorless crystalline solid: mp 75-77 ° C. (lit. Wang 72-77): δ _H (400 MHz; CDCl ₃ ) 1.32-1.55 (2H, m, Proγ-H ₂ ), 1.93-2.13 (2H, m, Proβ-H ₂ ), 2.58-2.65 (1H, m, Proδ-H ₂ ), 2.92 (1H, d, J 13.6, PhCH _A H _B ), 2.98-3.03 ( 1H, m, Proδ-H ₂ ), 3.32 (1H, d, J 13.6, PhCH _A H _B ), 4.99 (1H, s, NCH) and 7.21-7.35 (5H, m, PhH); δ _C (100 MHz ; CDCl ₃ ) 24.8 (CH ₂ , Proγ-C), 34.6 (CH ₂ , Proβ-C), 41.6 (CH ₂ , Proδ-C), 58.4 (CH ₂ , PhCH ₂ ), 72.3 (quat., Proα-C), 100.6 (quat., CCl ₃ ), 102.8 (CH, NCH), 127.0 (CH, Ph), 128.2 ( CH, Ph), 130.9 (CH, Ph), 135.5 (quat., Ph) and 176.6 (quat., C = O); m / z (EI +) 333.0081 [(M + H) ⁺ .C ₁₄ H ₁₄ ³⁵ Cl ₃ NO ₂ requires 333.0090], 335.0069 [(M + H) ⁺ . C ₁₄ H ₁₄ ³⁵ Cl ₂ ³⁷ ClNO ₂ requires 335.0061], 337.0014 [(M + H) ⁺ . C ₁₄ H ₁₄ ³⁵ Cl ³⁷ Cl ₂ NO ₂ requires 337.0031] and 339.0009 [(M + H) ⁺ . C ₁₄ H ₁₄ ³⁷ Cl ₃ NO ₂ requires 339.0002].

1.1.6. 11. methyl L-methylprolinate hydrochloride
Thionyl chloride (4.30 mL, 58.9 mmol) was carefully added dropwise to a stirred solution of 8 (7.57 g, 29.4 mmol) at 0 ° C. under a nitrogen atmosphere. The cooling layer was removed and the solution stirred at room temperature for 20 minutes was then heated to reflux for 3 hours. Volatiles were removed in vacuo and the residue was suspended in toluene (20 mL) and concentrated at 50 ° C. to remove traces of thionyl chloride. Trituration with dry ethanol yielded a brown solid. The yellow / orange ether was decanted and the solid was shaken with dry ether, decanted and the procedure was repeated until the ether was colorless. Removal of traces of ether at 50 ° C. in vacuo provided 12 (about 5.0 g, 100%) as a free-flowing, hygroscopic brown solid that was used without further purification: mp 107 -109 ° C (lit. (Lewis et al. J. Cem. Soc. Perkin Trans. 1 1998, 3777) 106-108 ° C).

1.1.7. 12. methyl L-2-ethylprolinate hydrochloride
An ice-cooled solution of oxazolidinone 9 (2.86 g, 10.6 mmol) in dry methanol (35 cm ³ ) was treated dropwise with a solution of thionyl chloride (2.3 cm ³ , 31.5 mmol). The solution was heated under reflux for 3 hours, cooled and the solvent removed under reduced pressure. The resulting brown oil was purified by flash column chromatography (10% methanol / dichloromethane) to provide hydrochloride 13 (1.45 g, 71%) as a light brown semi-solid: [α] _D 61.1 ( C 0.3 in CHCl ₃ ): δ _H (300 MHz; CDCl ₃ ) 1.07 (3H, t, J 7.3, CH ₃ ), 1.95-2.33 (5H, m, CH ₂ CH ₃ , Proγ-H ₂ and Proβ- H _A H _B ), 2.43-2.47 (1 H, Proβ-H _A H _B ), 3.63 (3 H, s, OCH ₃ ) and 6.98-7.35 (2 H, br s, NH ₂ ); δ _c (75 MHz; CDCl ₃ ) 9.9 (CH ₃ , CH ₃ ), 22.8 (CH ₂ , CH ₂ CH ₃ ), 28.9 (CH ₂ , Proγ-C), 35.1 (CH ₂ , Proβ-C), 45.7 (CH ₂ , Proδ-C), 53.8 (CH ₃ , OCH ₃ ), 73.9 (quat., Proα-C) and 171.0 (quat., CO) .m / z (EI +) 158.1181 (MH ⁺ .C ₈ H ₁₆ NO ₂ is , 158.1181 is required).

1.1.8. Methyl L-2-allylprolinate hydrochloride '14.
(Wang et al. Synlett 1999, 1,33, Hoffmann et al. Angew. Chem. Int. Ed. Engl. 2001, 40, 3361). An ice-cooled solution of oxazolidinone 10 (0.64 g, 2.24 mmol) in dry methanol (15 cm ³ ) was added to acetyl chloride (0.36 cm ³ , 5.0 mmol) in methanol (5 cm ³ ). Treated dropwise with solution. The solution was heated under reflux for 24 hours, cooled and the solvent removed under reduced pressure. The resulting brown oil was dissolved in toluene (40 cm ³ ) and concentrated to dryness to remove residual thionyl chloride and methanol, then flash column chromatography (5-10% CH ₃ OH—CH ₂ Purification by Cl ₂ ; gradient elution) provided hydrochloride 14 (0.29 g, 63%) as a solid whose NMR data was consistent with that recorded in the literature. δ _H (300 MHz, CDCl ₃ ) 1.72-2.25 (3H, m, Proβ-H _A H _B and Proγ-H ₂ ), 2.32-2.52 (1H, m, Proβ-H _A H _B ), 2.72-3.10 ( 2H, m, Proδ-H ₂ ), 3.31-3.78 (2H, m, CH ₂ CH = CH ₂ ), 3.84 (3H, s, CO ₂ CH ₃ ), 5.20-5.33 (2H, m, CH = CH ₂ ), 5.75-5.98 (1H, m, CH = CH ₂ ) and 8.06 (1H, br s, NH); m / z (CI +) 170.1183 ((M + H) ⁺ .C ₉ H ₁₆ NO ₂ is 170.1181 Need].

1.1.9. 14. methyl L-2-benzylprolinate hydrochloride
(Yasuo et al. Chem. Pharm. Bull. 1979, 27, 1931) An ice-cooled solution of oxazolidinone 11 (1.03 g, 3.07 mmol) in dry methanol (10 cm ³ ) was added to methanol (10 cm ³ ) A solution of acetyl chloride in 0.7) (0.71 m ³ , 10.0 mmol) was treated dropwise. The solution was heated under reflux for 24 hours, cooled and the solvent removed under reduced pressure. The resulting brown solid was dissolved in toluene (80 m ³ ) and concentrated to dryness to remove residual thionyl chloride and methanol, then flash column chromatography (5% CH ₃ OH—CH ₂ Cl ₂ ). To give hydrochloride 15 (0.38 g, 48%) as a beige solid: δ _H (400 MHz; D ₂ O) 1.92-2.01 (1H, m, Proγ-H _A H _B ) , 2.11-2.23 (2H, m, Proβ-H _A H _B and Proγ-H _A H _B ), 2.52-2.60 (1H, m, Proβ-H _A H _B ), 3.19 (1H, d, J 14.3, PhCH _A H _B ), 3.24-3.31 (1H, m, Proδ-H _A H _B ), 3.37-3.43 (1H, m, Proδ-H _A H _B ), 3.53 (1H, d, J 14.3, PhCH _A H _B ), 3.83 (3H, s, CO ₂ CH ₃ ) and 7.26-7.47 (5H, m, PhH); δ _C (100 MHz; D ₂ O) 24.4 (CH ₂ , Proγ-C), 36.8 (CH ₂ , Proβ-C), 43.8 (CH ₂ , PhCH ₂ ), 47.6 (CH ₂ , Proδ-C), 56.0 (CH ₃ , OCH ₃ ), 75.9 (quat., Proα-C), 130.4 (CH, Ph), 131.5 (CH, Ph), 131.7 (CH, Ph), 137.1 (quat., Ph) and 175.8 ( quat., C = O); m / z (CI +) 220.1340 [(M + H) ⁺ . C ₁₃ H ₁₈ NO ₂ requires 220.1338].

1.1.10. Methyl N-benzyloxycarbonyl-glycyl-L-2-methylprolinate 18.
Dry triethylamine (0.27 cm ³ , 1.96 mmol) was added to hydrochloride 12 (0.11 g, 0.61 mmol) and N-benzyloxycarbonylglycine 16 (0.17 g, 0. 01 mmol) in dry dichloromethane (35 cm ³ ). 79 mmol) was added dropwise at room temperature under an atmosphere of nitrogen and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.196 g, 0.77 mmol) was added and the resulting colorless solution was stirred for 20.5 hours. The solution was subsequently washed with 10% aqueous hydrochloric acid (30 m ³ ) and saturated aqueous sodium bicarbonate (30 m ³ ), dried (MgSO ₄ ), filtered and evaporated in vacuo to dryness. The remaining purification obtained by flash column chromatography (50-80% ethyl acetate-hexanes; gradient elution) yielded ester 18 (0.18 g, 92%) as a colorless oil: [α] _D − 33.0 (c 1.0 in MeOH); ν _max (film) / cm ^-1 3406, 2952, 1732, 1651, 1521, 1434, 1373, 1329, 1310, 1284, 1257, 1220, 1195, 1172, 1135, 1107, 1082, 1052, 1029, 986, 965, 907, 876, 829, 775, 738 and 699; δ _H (300 MHz, CDCl ₃ ) 1.49 (3H, s, CH ₃ ), 1.77-2.11 (4H, m, Proβ -H ₂ and Proγ-H ₂ ), 3.43-3.48 (2H, m, Proδ-H ₂ ), 3.61 (3H, s, OCH ₃ ), 3.85-3.89 (2H, m, Glyα-H ₂ ), 5.04 ( 2H, s, PhCH ₂ ), 5.76 (1H, br s, NH) and 7.21-7.28 (5H, s, ArH); δ _C (75 MHz, CDCl ₃ ), 21.1 (CH ₃ , Proα-CH ₃ ), 23.5 (CH ₂ , Proγ-C), 38.0 (CH ₂ , Proβ-C), 43.3 (CH ₂ , Glyα-C), 46.6 (CH ₂ , Proδ-C), 52.1 (CH ₃ , OCH ₃ ), 66.0 (quat., Proα-C), 66.3 (CH ₂ , PhCH ₂ ), 127.5 (CH, Ph), 127.6 (CH, Ph), 128.1 (CH, Ph), 136.2 (quat., Ph), 155 .9 (quat., NCO ₂ ), 166.0 (quat., Gly-CON) and 173.6 (quat., CO ₂ CH ₃ ); m / z (EI +) 334.1535 (M ⁺ .C ₁₇ H ₂₂ N ₂ O ₅ Requires 334.1529).

1.1.11. 18. methyl N-benzyloxycarbonyl-glycyl-L-2-methylprolinate
Dry triethylamine (2.88 m ³ , 20.7 mmol) was added to hydrochloride 13 (1.14 g, 5.9 mmol) and N-benzyloxycarbonylglycine 16 (2.47 g, 11.7 mmol) in dry dichloromethane (100 cm ³ ). 8 mmol) was added dropwise at 0 ° C. under a nitrogen atmosphere and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (3.00 g, 11.8 mmol) was added and the solution was stirred for 2 hours, warmed to room temperature and stirred for an additional 17.5 hours. Dichloromethane (50 cm ³ ) was added and subsequently washed with 0.5 M aqueous hydrochloric acid (2 × 50 cm ³ ) and saturated aqueous sodium bicarbonate (2 × 50 cm ³ ), dried (MgSO ₄ ), filtered and vacuum Evaporated to give a bright orange gum. The remaining purification obtained by flash column chromatography (40% ethyl acetate / hexane) yielded ester 19 (0.95 g, 46%) as a clear oil: [α] _D 9.2 (c in CHCl ₃ 0.13); δ _H (300 MHz; CDCl ₃ ) 0.81 (3 H, t, J 7.5, CH ₃ ), 1.85-2.09 (5 H, m, CH ₂ CH ₃ , Proγ-H ₂ and Proβ-H _A H _B ), 2.38 (1 H, hexat, J 7.5, Proβ-H _A H _B ), 3.43-3.47 (1 H, m, Proδ-H _A H _B ), 3.61-3.67 (1 H, m, Proδ -H _A H _B ), 3.70 (3 H, s, OCH ₃ ), 4.10-4.13 (2 H, m, Glyα-H ₂ ) 5.11 (2 H, s, OCH ₂ Ph), 5.71 (1 H, br s, Gly-NH) and 7.27-7.35 (5 H, m, Ph); δ _c (75 MHz; CDCl ₃ ) 8.3 (CH ₃ , CH ₃ ), 24.1 (CH ₂ , CH ₂ CH ₃ ), 26.5 ( CH ₂ , Proγ-C), 35.3 (CH ₂ , Proβ-C), 44.1 (CH ₂ , Glyα-C), 48.2 (CH ₂ , Proδ-C), 52.9 (CH ₃ , OCH ₃ ), 67.0 (CH ₂ , OCH ₂ Ph), 70.2 (quat., Proα-C), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.7 (CH, Ph), 136.8 (quat., Ph), 156.5 (quat. , NCO), 166.8 (quat., Gly-CON) and 174.5 (quat., CO ₂ CH ₃ ); m / z (EI +) 348.1688 (MH ⁺ . C ₁₈ H ₂₄ N ₂ O ₅ requires 348.1685).

1.1.12. Methyl N-benzyloxycarbonyl-glycyl-L-2-allylprolinate20.
Dry triethylamine (1.07 m ³ , 7.70 mmol) was added to hydrochloride 14 (0.50 g, 2.41 mmol) and N-benzyloxycarbonyl-glycine 16 (0.65 g, 3 cm) in dry dichloromethane (80 cm ³ ). .13 mmol) was added dropwise at room temperature under an atmosphere of nitrogen and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.772 g, 3.03 mmol) is added and the solution is stirred for 20 hours, then 10% aqueous hydrochloric acid (80 cm ³ ) and saturated aqueous sodium bicarbonate Subsequently washed with (80 cm ³ ), dried (MgSO ₄ ), filtered and evaporated in vacuo to dryness. The remaining purification obtained by flash column chromatography (60% ethyl acetate / hexane, 7 drops of Et ₃ N for all 200 cm ³ ) gave ester 20 (0.26 g, 30%) as a yellow oil. Yield: [α] _D +46.0 (c 0.50 in CH ₂ Cl ₂ ); ν _max (film) / cm ^-1 3405, 3066, 3032, 2953, 2877, 1723, 1655, 1586, 1507, 1434, 1373 , 1333, 1309, 1248, 1169, 1121, 1083, 1047, 1027, 1002, 919, 866, 827, 776, 737 and 699; δ _H (300 MHz; CDCl ₃ ) 1.92-2.17 (4H, m, Proβ- H ₂ and Proγ-H ₂ ), 2.60-2.67 (1H, m, CH _A H _B CH = CH ₂ ), 3.09-3.16 (1H, m, CH _A H _B CH = CH ₂ ), 3.35-3.42 (1H , m, Proδ-H _A H _B ), 3.56-3.63 (1H, m, Proδ-H _A H _B ), 3.70 (3H, s, OCH ₃ ), 3.96 (2H, d, J 4.4, Glyα-H ₂ ), 5.07-5.12 (4H, m, PhCH ₂ and CH = CH ₂ ), 5.58-5.70 (1H, m, CH = CH ₂ ) and 7.27-7.35 (5H, s, PhH); δ _C (75 MHz; CDCl ₃ ) 23.6 (CH ₂ , Proγ-C), 34.9 (CH ₂ , Proβ-C), 37.6 (CH ₂ , CH ₂ CH = CH ₂ ), 43.6 (CH ₂ , Gly α-C), 47.5 (CH ₂ , Proδ-C), 52.5 (CH ₃ , OCH ₃ ), 66.7 (CH ₂ , PhCH ₂ ), 68.8 (quat., Proα-C), 119.4 (CH ₂ , CH = CH ₂ ), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.4 (CH, Ph), 132.8 (CH, CH = CH ₂ ), 136.4 (quat., Ph), 156.1 (quat., NCO ₂ ), 166.4 (quat., Gly-CON) and 173.7 (quat., CO ₂ CH ₃ ); m / z (EI +) 360.1682 (M ⁺ .C ₁₉ H ₂₄ N ₂ O ₅ requires 360.1685) .

1.1.13. Methyl Nt-butyloxycarbonyl-glycyl-L-2-allylprolinate 21.
Dry triethylamine (0.28 m ³ , 2.02 mmol) was added to hydrochloride 14 (0.13 g, 0.63 mmol) and Nt-butyloxycarbonylglycine 17 (0.14 g, 0.15 g, in dry dichloromethane (35 cm ³ )). 0.82 mmol) was added dropwise at room temperature under an atmosphere of nitrogen and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.20 g, 0.80 mmol) is added and the solution is stirred for 19.5 hours, then 10% aqueous hydrochloric acid (35 cm ³ ) and saturated aqueous carbonate Subsequent washing with sodium hydride (35 cm ³ ), drying (MgSO ₄ ), filtration and evaporation in vacuo to dryness. The remaining purification obtained by flash column chromatography (40% ethyl acetate / hexane) yielded ester 21 (0.09 g, 45%) as a light yellow oil: [α] _D +33.8 (CH ₂ Cl C 0.83 in ₂ ); ν _maximum (film) / cm ^-1 3419, 3075, 2977, 2930, 2874, 1739, 1715, 1656, 1499, 1434, 1392, 1366, 1332, 1268, 1248, 1212, 1168, 1122, 1051, 1026, 1003, 943, 919, 867, 830, 779, 739, 699 and 679; δ _H (300 MHz; CDCl ₃ ) 1.42 [9H, s, C (CH ₃ ) ₃ ], 1.93-2.08 (4H, m, Proβ-H ₂ and Proγ-H ₂ ), 2.59-2.67 (1H, m, CH _A H _B CH = CH ₂ ), 3.09-3.16 (1H, m, CH _A H _B CH = CH ₂ ), 3.35-3.44 (1H, m, Proδ-H _A H _B ), 3.56-3.62 (1H, m, Proδ-H _A H _B ), 3.70 (3H, s, OCH ₃ ), 3.89 (2H, d, J 4.2, Glyα-H ₂ ), 5.06-5.11 (2H, m, CH = CH ₂ ), 5.42 (1H, br s, Gly-NH) and 5.58-5.72 (1H, m, CH = CH ₂ ); δ _C (75 MHz; CDCl ₃ ) 23.7 (CH ₂ , Proγ-C), 28.3 [CH ₃ , C (CH ₃ ) ₃ ], 35.0 (CH ₂ , Proβ-C), 37.6 (CH ₂ , CH ₂ CH = CH ₂ ), 43.3 (CH ₂ , G lyα-C), 47.5 (CH ₂ , Proδ-C), 52.5 (CH ₃ , OCH ₃ ), 68.8 (quat., Proα-C), 79.5 (quat., C (CH ₃ ) ₃ ], 119.4 (CH ₂ , CH = CH ₂ ), 132.9 (CH, CH = CH ₂ ), 155.7 (quat., NCO ₂ ), 166.9 (quat., Gly-CON) and 173.8 (quat., CO ₂ CH ₃ ); m / z (EI +) 326.1845 (M ⁺ . C ₁₆ H ₂₆ N ₂ O ₅ requires 326.1842).

1.1.14. Methyl Nt-butyloxycarbonyl-glycyl-L-2-benzylprolinate 22.
Dry triethylamine (0.59 m ³ , 4,22 mmol) was added to hydrochloride 15 (0.34 g, 1.32 mmol) and Nt-butyloxycarbonylglycine 17 (0.30 g, in dry dichloromethane (70 cm ³ )). 1.71 mmol) was added dropwise at room temperature under an atmosphere of nitrogen and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.42 g, 1.66 mmol) is added and the solution is stirred for 18.5 hours, then 10% aqueous hydrochloric acid (70 cm ³ ) and saturated aqueous carbonate Subsequent washing with sodium hydride (70 cm ³ ), drying (MgSO ₄ ), filtration and evaporation in vacuo to dryness. The remaining purification obtained by flash column chromatography (50% ethyl acetate / hexane) yielded ester 22 (0.11 g, 22%) as a pale yellow oil: [α] _D +105.3 (CH ₂ Cl C 0.99 in ₂ ); ν _max (film) / cm ^-1 3419, 3061, 3028, 2977, 2873, 1799, 1739, 1715, 1655, 1582, 1497, 1454, 1432, 1392, 1366, 1330, 1250, 1167, 1121, 1093, 1049, 1026, 948, 915, 865, 819, 765, 736, 706 and 653; δ _H (300 MHz; CDCl ₃ ) 1.08-1.12 (1H, m, Proγ-H _A H _B ) , 1.47 [9H, s, C (CH ₃ ) ₃ ], 1.67-1.72 (1H, m, Proγ-H _A H _B ), 2.01-2.17 (2H, m, Proβ-H ₂ ), 2.86-2.92 (1H , m, Proδ-H _A H _B ), 3.08 (1H, d, J 13.8, PhCH _A H _B ), 3.36-3.42 (1H, m, Proδ-H _A H _B ), 3.75 (3H, s, OCH ₃ ), 3.83 (1H, m, PhCH _A H _B ), 3.90 (2 H, d, J 3.6, Glyα-CH ₂ ), 5.54 (1H, br s, NH) and 7.06-7.30 (5H, m, PhH) ; δ _C (100 MHz; CDCl ₃ ) 23.4 (CH ₂ , Proγ-C), 28.4 [CH ₃ , C (CH ₃ ) ₃ ], 34.7 (CH ₂ , Proβ-C), 37.8 (CH ₂ , PhCH ₂ ), 43.6 (CH ₂ , Gl yα-C), 47.4 (CH ₂ , Proδ-C), 52.6 (CH ₃ , OCH ₃ ), 69.6 (quat., Proα-C), 79.6 [quat., C (CH ₃ ) ₃ ], 126.8 (CH , Ph), 128.3 (CH, Ph), 130.5 (CH, Ph), 136.7 (quat., Ph), 155.8 (quat., NCO ₂ ), 167.2 (quat., Gly-CON) and 174.0 (quat., CO ₂ CH ₃ ); m / z (EI +) 376.2001 (M ⁺ . C ₂₀ H ₂₈ N ₂ O ₅ requires 376.1998).

1.1.15. N-benzyloxycarbonyl-glycyl-L-2-methylproline23.
To a solution of methyl ester 18 (0.56 g, ca. 1.67 mmol) in 1,4-dioxane (33 cm ³ ) 1M aqueous sodium hydroxide (10 cm ³ , 10 mmol) was added dropwise and the mixture was added for 19 hours. Stir at room temperature. Dichloromethane (100 cm ³ ) was then added and the organic layer was extracted with saturated aqueous sodium bicarbonate (2 × 100 cm ³ ). The combined aqueous layers are carefully acidified with dilute hydrochloric acid, extracted with dichloromethane (2 × 100 cm ³ ), and the combined organic layers are dried (MgSO ₄ ), filtered, and concentrated in vacuo to dryness. did. Purification of the next remainder (0.47 g) by flash column chromatography (50% ethyl acetate-hexanes to 30% methanol-dichloromethane; gradient elution) gave acid 23 (0.68 g, 90%) as a white foam. : [Α] _D -62.3 (c 0.20 in CH ₂ Cl ₂ ); ν _max (film) / cm ^-1 3583, 3324 br, 2980, 2942, 1722, 1649, 1529, 1454, 1432, 1373, 1337 , 1251, 1219, 1179, 1053, 1027, 965, 912, 735 and 698; δ _H (300 MHz; CDCl ₃ ) 1.59 (3H, s, Proα-CH ₃ ), 1.89 (1H, 6 lines, J 18.8, 6.2 and 6.2, Proβ-H _A H _B ), 2.01 (2H, dtt, J 18.7, 6.2 and 6.2, Proγ-H ₂ ), 2.25-2.40 (1H, m, Proβ-H _A H _B ), 3.54 (2H , t, J 6.6, Proδ-H ₂ ), 3.89 (1H, dd, J 17.1 and 3.9, Glyα-H _A H _B ), 4.04 (1H, dd, J 17.2 and 5.3, Glyα-H _A H _B ), 5.11 (2H, s, OCH ₂ Ph), 5.84 (1H, br t, J 4.2, NH), 7.22-7.43 (5H, m, Ph) and 7.89 (1H, br s, -COOH); δ _C (75 MHz; CDCl ₃ ) 21.3 (CH ₃ , Proα-CH ₃ ), 23.8 (CH ₂ , Proγ-C), 38.2 (CH ₂ , Proβ-C), 43.6 (CH ₂ , Glyα-C), 47.2 (CH ₂ , Proδ-C), 66.7 (quat, Proα-C), 66.8 (CH ₂ , OCH ₂ Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.4, (CH, Ph), 136.4 (quat., Ph), 156.4 (quat., NCO ₂ ), 167.5 (quat., Gly-CON) and 176.7 ( quat., CO); m / z (EI +) 320.1368 (M ⁺ . C ₁₆ H ₂₀ N ₂ O ₅ requires 320.1372).

1.1.16. N-benzyloxycarbonyl-glycyl-L-2-ethylproline 24.
To a solution of methyl ester 19 (0.39 g, ca. 1.11 mmol) in dioxane (15 cm ³ ) 1M NaOH (7.5 cm ³ , 7.50 mmol) was added dropwise and the mixture was stirred for 20 hours at room temperature. did. The solution was acidified with 1M HCl and evaporated in vacuo. The resulting aqueous layer is extracted with dichloromethane (2 × 30 cm ³ ), dried (MgSO ₄ ), filtered and evaporated in vacuo to form a clear gum that is erected with acid 24 ( 0.35 g, 95%) as a colorless solid, which was used without further purification: [α] _D 9.9 (c 0.11 in MeOH); δ _H (300 MHz; CDCl ₃ ) 0.83 ( 3H, t, J 7.4, CH ₃ ), 1.93-2.22 (5H, m, CH ₂ CH ₃ , Proγ-H ₂ and Pro-βH _A H _B ), 2.35-2.40 (1H, m, Pro-βH _A H _B ), 3.43-3.46 (1H, m, Proδ-H _A H _B ), 3.57-3.62 (1H, m, Proδ-H _A H _B ), 4.01 (2H, dd, J 4.3 and 17.3, Glyα-H ₂ ), 5.11 (2H, s, OCH ₂ Ph), 5.82 (1H, br s, Gly-NH), 7.26-7.36 (5H, m, Ph) and 7.70 (1H, br s, CO ₂ H); δ _C (75 MHz; CDCl ₃ ) 8.5 (CH ₃ , CH ₃ ), 23.9 (CH ₂ , CH ₂ CH ₃ ), 26.7 (CH ₂ , Proγ-C), 34.8 (CH ₂ , Proβ-C), 44.1 (CH ₂ , Glyα-C), 48.7 (CH ₂ , Proδ-C), 67.3 (CH ₂ , OCH ₂ Ph), 71.9 (quat., Proα-C), 127.3 (CH, Ph), 127.9 (CH, Ph) , 128.3 (CH, P h), 128.4 (CH, Ph), 128.9 (CH, Ph), 136.7 (quat., Ph), 156.7 (quat., NCO ₂ ), 168.9 (quat., Gly-CON) and 175.8 (quat., CO ₂ H); m / z (EI ⁺ ) 334.1523 (M ⁺ , C ₁₇ H ₂₂ N ₂ O ₅ requires 334.1529).

1.1.17. N-benzyloxycarbonyl-glycyl-L-2-allylproline 25.
To a solution of ester 20 (0.12 g, 0.34 mmol) in dioxane (7 cm ³ ), 1M aqueous NaOH (2.06 cm ³ , 2.06 mmol) was added dropwise and the mixture was stirred for 20 hours at room temperature. . Dichloromethane (25 cm ³ ) was then added and the organic layer was extracted with saturated aqueous sodium bicarbonate (3 × 25 cm ³ ). Careful acidification of the combined aqueous layers with dilute hydrochloric acid, extraction with dichloromethane (3 × 25 cm ³ ), drying of the combined organic layers (MgSO ₄ ), filtration and concentration to dryness gave acid 25 (0 .11 g, 92%) as a yellow oil: [α] _D −3.16 (c 0.95 in CH ₂ Cl ₂ ); ν _max (film) / cm ⁻¹ 3408, 2957, 2923, 2850, 2622, 1715, 1650, 1531, 1453, 1375, 1333, 1259, 1214, 1173, 1121, 1083, 1056, 1028, 1002, 916, 823, 737 and 698; δ _H (300 MHz; CDCl ₃ ) 1.93-2.07 (3H , m, Proβ-H _A H _B and Proγ-H ₂ ), 2.22-2.26 (1H, m, Proβ-H _A H _B ), 2.62-2.69 (1H, m, CH _A H _B CH = CH ₂ ), 3.04-3.11 (1H, m, CH _A H _B CH = CH ₂ ), 3.31-3.62 (2H, m, Proδ-H ₂ ), 3.91-4.05 (2H, m, Glyα-H ₂ ), 5.08-5.13 ( 3H, m, PhCH ₂ and CH = CH _A H _B ), 5.55-5.72 (1H, m, CH = CH _A H _B ), 5.89 (1H, m, CH = CH ₂ ), 7.29-7.45 (5H, m , PhH) and 8.12 (1H, br s, NH); δ _C (75 MHz; CDCl ₃ ) 23.5 (CH ₂ , Proγ-C), 34.6 (CH ₂ , Proβ- C), 37.5 (CH ₂ , CH ₂ CH = CH ₂ ), 43.7 (CH ₂ , Glyα-C), 48.1 (CH ₂ , Proδ-C), 66.9 (CH ₂ , PhCH ₂ ), 69.9 (quat., Proα-C), 119.8 (CH ₂ , CH = CH ₂ ), 127.97 (CH, Ph), 128.04 (CH, Ph), 128.4 (CH, Ph), 132.3 (CH, CH = CH ₂ ), 136.4 (quat , Ph), 156.4 (quat., NCO ₂ ), 168.2 (quat., Gly-CON) and 176.1 (quat., CO ₂ H); m / z (EI +) 346.1526 (M ⁺ .C ₁₈ H ₂₂ N ₂ O ₅ requires 346.1529).

1.1.18. Nt-butyloxycarbonyl-glycyl-L-2-allylproline 26.
To a solution of ester 21 (0.039 g, 0.12 mmol) in dioxane (2.4 cm ³ ), 1M aqueous NaOH (0.72 cm ³ , 0.72 mmol) was added dropwise and the mixture was stirred for 16 hours at room temperature. Stir. Dichloromethane (10 cm ³ ) was then added and the organic layer was extracted with saturated aqueous sodium bicarbonate (3 × 25 cm ³ ). Careful acidification of the combined aqueous layers with dilute hydrochloric acid, extraction with dichloromethane (3 × 10 cm ³ ), drying of the combined organic layers (MgSO ₄ ), filtration and concentration to dryness gave acid 26 (0 031 g, 83%) as a yellow oil: [α] _D -15.8 (c 0.89 in CH ₂ Cl ₂ ); ν _max (film) / cm ^-1 3414, 3076, 2978, 2931, 2620, 1713, 1654, 1510, 1454, 1434, 1392, 1367, 1268, 1250, 1213, 1169, 1121, 1056, 1028, 920, 869, 779, 736 and 701; δ _H (300 MHz; CDCl ₃ ) 1.43 (9H , s, C (CH ₃ ) ₃ ], 1.95-2.15 (3H, m, Proβ-H _A H _B and Proγ-CH ₂ ), 2.21-2.35 (1H, m, Proβ-H _A H _B ), 2.63- 2.70 (1H, m, CH _A H _B CH = CH ₂ ), 3.04-3.11 (1H, m, CH _A H _B CH = CH ₂ ), 3.35-3.47 (1H, m, Proδ-H _A H _B ), 3.53-3.62 (1H, m, Proδ-H _A H _B ), 3.92 (2H, m, Glyα-H ₂ ), 5.08-5.13 (2H, m, CH = CH ₂ ), 5.52 (1H, br s, Gly -NH), 5.56-5.71 (1H, m, CH = CH ₂ ) and 8.31 (1 H, br s, OH); δ _C (75 MHz; CDCl ₃ ) 23.5 (CH ₂ , Pr oγ-C), 28.3 [CH ₃ , C (CH ₃ ) ₃ ], 34.6 (CH ₂ , Proβ-C), 37.5 (CH ₂ , CH ₂ CH = CH ₂ ), 43.4 (CH ₂ , Glyα-C) , 48.1 (CH ₂ , Proδ-C), 69.8 (quat., Proα-C), 79.8 [quat., C (CH ₃ ) ₃ ], 119.8 (CH ₂ , CH = CH ₂ ), 132.3 (CH, CH = CH ₂ ), 155.8 (quat., NCO ₂ ), 168.5 (quat., Gly-CON) and 175.9 (quat., CO ₂ H); m / z (EI +) 312.1692 (M ⁺ .C ₁₅ H ₂₄ N ₂ O ₅ requires 312.1685).

1.1.19. Nt-butyloxycarbonyl-glycyl-L-benzylproline27.
To a solution of ester 22 (0.098 g, 0.26 mmol) in dioxane (5.2 cm ³ ), 1 aqueous NaOH (1.56 cm ³ , 1.56 mmol) was added dropwise and the mixture was stirred for 15 hours at room temperature. Stir. Dichloromethane (20 cm ³ ) was then added and the organic layer was extracted with saturated aqueous sodium bicarbonate (3 × 20 cm ³ ). Careful acidification of the aqueous layer combined with dilute hydrochloric acid, extraction with dichloromethane (3 × 20 cm ³ ), drying of the combined organic layer (MgSO ₄ ), filtration and concentration to dryness gave acid 27 (0.09 g , 95%) as a colorless glassy solid: mp 74-77 ° C .; [α] _D +69.8 (c 0.99 in CH ₂ Cl ₂ ); ν _max (CH ₂ Cl ₂ ) / cm ⁻¹ 3415, 3060, 3028, 2978, 2879, 2621, 1711, 1655, 1497, 1454, 1392, 1367, 1252, 1167, 1120, 1092, 1081, 1049, 1029, 988, 942, 915, 887, 872, 814, 764, 736, 705 and 653; δ _H (400 MHz; CDCl ₃ ) 1.14-1.21 (1H, m, Proγ-H _A H _B ), 1.50 [9H, s, C (CH ₃ ) ₃ ], 1.72-1.78 (1H, m, Proγ-H _A H _B ), 2.11-2.29 (2H, m, Proβ-H ₂ ), 2.94-3.00 (1H, m, Proδ-H _A H _B ), 3.13 (1H, d, J 13.9, PhCH _A H _B ), 3.42-3.48 (1H, m, Proδ-H _A H _B ), 3.83 (1H, d, J 13.9, PhCH _A H _B ), 4.13 (2H, m, Glyα-H ₂ ) , 5.69 (1H, br s, Gly-NH), 7.10-7.33 (5H, m, PhH) and 8.37 (1 H, br s, OH); δ _C (100 MHz; CDCl ₃ ) 23.3 (CH ₂ , Proγ-C), 28.3 [CH ₃ , C (CH ₃ ) ₃ ], 34.5 (CH ₂ , Proβ-C), 37.8 (CH ₂ , PhCH ₂ ), 43.7 (CH ₂ , Glyα-C), 47.8 (CH ₂ , Proδ-C), 70.3 (quat., Proα-C), 79.8 [quat., C (CH ₃ ) ₃ ], 126.9 (CH, Ph), 128.4 (CH, Ph), 130.5 (CH, Ph), 136.3 (quat., Ph), 156.0 (quat., NCO ₂ ), 168.6 (quat., Gly-CON) and 176.9 (quat., CO ₂ H); m / z (EI +) 362.1834 (M ⁺ . C ₁₉ H ₂₆ N ₂ O ₅ requires 362.1842).

1.1.20. Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-methylprolyl-L-glutamate30.
Triethylamine (0.50 cm ³ , 3.59 mmol) was added to dipeptide 23 (0.36 g, 1.12 mmol) and L-glutamic acid dibenzyl ester p-toluenesulfonate 28 (0.73 g, 1.30 mmol) in dichloromethane (60 cm ³ ). 46 mmol) was added dropwise at room temperature under nitrogen and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.37 g, 1.41 mmol) was added and the colorless solution was stirred for 17 hours. The dichloromethane solution was subsequently washed with 10% aqueous hydrochloric acid (50 cm ³ ) and saturated aqueous sodium bicarbonate (50 cm ³ ), dried (MgSO ₄ ), filtered and evaporated in vacuo to dryness. The remaining purification obtained by repeated flash column chromatography (30-70% ethyl acetate-hexanes; elution gradient) yielded protected tripeptide 30 (0.63 g, 89%) as a colorless oil. Tripeptide 30 is shown to select exclusively the trans conformer by NMR: R _f 0.55 (EtOAc); [α] _D -41.9 (c 0.29 in CH ₂ Cl ₂ ); v _max (film ) / cm ^-1 3583, 3353 br, 2950, 1734, 1660, 1521, 1499, 1454, 1429, 1257, 1214, 1188, 1166, 1051, 911, 737 and 697; δ _H (400 MHz; CDCl ₃ ; Me ₄ Si) 1.64 (3H, s, Proα-CH ₃ ), 1.72 (1H, dt, J 12.8, 7.6 and 7.6, Proβ-H _A H _B ), 1.92 (2H, 5-wire, J 6.7, Proγ-H ₂ ), 2.04 (1H, 6 lines, J 7.3 Gluβ-H _A H _B ), 2.17-2.27 (1H, m, Gluβ-H _A H _B ), 2.35-2.51 (3H, m, Proβ-H _A H _B and Gluγ-H ₂ ), 3.37-3.57 (2H, m, Proδ-H ₂ ), 3.90 (1H, dd, J 17.0 and 3.6, Glyα-H _A H _B ), 4.00 (1H, dd, J 17.1 and 5.1, Glyα-H _A H _B ), 4.56 (1H, td, J 7.7 and 4.9, Gluα-H), 5.05-5.20 (6H, m, 3 x OCH ₂ Ph), 5.66-5.72 (1H, br m, Gly- NH), 7.26-7.37 (15H, m, 3 x Ph) and 7.44 (1H, d, J 7.2, Glu-NH); δ _C (100 MHz; CDCl ₃ ) 21.9 (CH ₃ , Proα-CH ₃ ), 23.4 (CH ₂ , Proγ-C), 26.6 (CH ₂ , Gluβ-C), 30.1 (CH ₂ , Gl uγ-C), 38.3 (CH ₂ , Proβ-C), 43.9 (CH ₂ , Glyα-C), 47.6 (CH ₂ , Proδ-C), 52.2 (CH, Gluα-C), 66.4 (CH ₂ , OCH ₂ Ph), 66.8 (CH ₂ , OCH ₂ Ph), 67.1 (CH ₂ , OCH ₂ Ph), 68.2 (quat, Proα-C), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.1, ( CH, Ph), 128.2, (CH, Ph), 128.2, (CH, Ph), 128.3, (CH, Ph), 128.4, (CH, Ph), 128.5, (CH, Ph), 128.5, (CH, Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 136.4 (quat., Ph), 156.1 (quat., NCO ₂ ), 167.3 (quat., Gly-CO), 171.4 (quat. , CO), 172.9 (quat., CO) and 173.4 (quat., CO); m / z (FAB +) 630.2809 (MH ⁺ . C ₃₅ H ₄₀ N ₃ O ₈ requires 630.2815).

1.1.21. Dibenzyl N-butyloxycarbonyl-glycyl-L-2-ethylprolyl-L-glutamate 31.
Dry triethylamine (0.44 cm ³ , 3.16 mmol) was added to acid 24 (0.30 g, 0.91 mmol) and L-glutamic acid dibenzyl ester p-toluenesulfonate 28 (0.57 g, in dry dichloromethane (50 cm ³ )). 1.13 mmol) was added dropwise at 0 ° C. under a nitrogen atmosphere and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.29 g, 1.14 mmol) was added and the solution was stirred for 2 hours, warmed to room temperature and further stirred for 17.5 hours. The solution was subsequently washed with 0.5 M aqueous hydrochloric acid (10 cm ³ ) and saturated aqueous sodium bicarbonate (10 cm ³ ), dried (MgSO ₄ ), filtered and evaporated in vacuo to form a bright orange gum. . The remaining purification obtained by flash column chromatography (30% ethyl acetate-hexane) yielded protected tripeptide 31 (0.41 g, 70%) as a clear oil: [α] _D 52.7 (c in MeOH 0.16); δ _H (300 MHz; CDCl ₃ ) 0.78 (3 H, t, J 7.4, CH ₃ ), 1.25-2.24 (7 H, m, CH ₂ CH ₃ , Gluβ-H ₂ , Proγ-H ₂ and Proβ-H _A H _B ), 2.34-2.40 (2H, m, Gluγ-H ₂ ), 2.50-2.60 (1 H, m, Proβ-H _A H _B ), 3.34 (1 H, q, J 9.4, Proδ -H _A H _B ), 3.49-3.53 (1 H, m, Proδ-H _A H _B ), 3.96 (2 H, ddd, J 4.9 and 17.3, Glyα-H ₂ ), 4.51-4.54 (1 H, td , J 5.4 and 7.8, Gluα-H), 5.06-5.19 (6 H, m, 3 x OCH ₂ Ph), 5.70 (1H, br s, Gly-NH), 7.26-7.36 (15 H, 3 x Ph) And 8.09 (1H, d, J 7.3, Glu-NH); δ _C (75 MHz; CDCl ₃ ) 8.8 (CH ₃ , CH ₃ ), 23.6 (CH ₂ , CH ₂ CH ₃ ), 27.2 (CH ₂ , Proγ -C), 27.7 (CH ₂ , Gluβ-C), 30.6 (CH ₂ , Gluγ-C), 34.3 (CH ₂ , Proβ-C), 44.5 (CH ₂ , Glyα-C), 49.0 (CH ₂ , Proδ -C), 52.6 (CH, Gluα-C), 66.9 (CH ₂ , OCH ₂ Ph), 67.3 (CH ₂ , OCH ₂ Ph), 67.5 (CH ₂ , OCH ₂ Ph), 73.9 (quat., Proα-C), 128.4 (CH, Ph), 128.5 (CH, Ph), 128.6 ( CH, Ph), 128.7 (CH, Ph), 128.8 (CH, Ph), 128.9 (CH, Ph), 135.7 (quat., Ph), 136.1 (quat., Ph), 136.8 (quat., Ph), 156.6 (quat., NCO ₂ ), 168.7 (quat., Gly-CON), 171.8 (quat., Pro-CON), 172.9 (quat., Gluα-CO) and 173.6 (quat., Gluγ-CO); m / z (FAB +) 644.2981 (MH ⁺ .C ₃₆ H ₄₂ N ₃ O ₈ requires 644.2972).

1.1.22. Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-allylprolyl-L-glutamate32.
Dry triethylamine (0.07 cm ³ , 0.49 mmol) was added to acid 25 (0.05 g, 0.15 mmol) and L-glutamic acid dibenzyl ester p-toluenesulfonate 28 (0. 0. 0,3 in dry dichloromethane (8.2 cm ³ )). 10 g, 0.20 mmol) was added dropwise at room temperature under an atmosphere of nitrogen and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.05 g, 0.19 mmol) was added and the solution was stirred for 19.5 hours. The solution was subsequently washed with 10% aqueous hydrochloric acid (7 cm ³ ) and saturated aqueous sodium bicarbonate (7 cm ³ ), dried (MgSO ₄ ), filtered and evaporated to dryness. The remaining purification obtained by flash column chromatography (50-80% ethyl acetate-hexanes; gradient elution) yielded protected tripeptide 32 (0.08 g, 76%) as a colorless oil: [α] _D − 27.8 (c 0.79 in CH ₂ Cl ₂ ); ν _max (film) / cm ^-1 3943, 3583, 3413, 3055, 3032, 2982, 2956, 2880, 2685, 2411, 2305, 1955, 1732, 1668, 1586 , 1499, 1454, 1423, 1388, 1329, 1265, 1214, 1169, 1170, 1081, 1058, 1028, 994, 924, 897, 824, 737 and 701; δ _H (400 MHz; CDCl ₃ ) 1.85 (3H, m, Proβ-H _A H _B and Proγ-H ₂ ), 1.99-2.08 (1H, m, Gluβ-H _A H _B ), 2.17-2.25 (1H, m, Gluβ-H _A H _B ), 2.32-2.49 (3H, m, Proβ-H _A H _B and Gluγ-H ₂ ), 2.71-2.76 (1H, m, CH _A H _B CH = CH ₂ ), 3.05-3.10 (1H, m, CH _A H _B CH = CH ₂ ), 3.33 (1H, m, Proδ-H _A H _B ), 3.51 (1H, m, Proδ-H _A H _B ), 3.96 (2H, d, J 3.9, Glyα-H ₂ ), 4.55 (1H , dd, J 7.6 and 5.1, Gluα-H), 5.07-5.19 (8H, m, 3 x PhCH ₂ and CH = CH ₂ ), 5.53-5.63 (1H, m, CH = CH ₂ ), 5.71 (1H, br s, Gly-NH), 7.32-7.36 (15H, m, 3 x Ph) and 7.90 (1H, d, J 7.2, Glu-NH); δ _C (100 MHz; CDCl ₃ ) 23.1 (CH ₂ , Proγ-C), 26.7 (CH ₂ , Gluβ-C), 30.2 (CH ₂ , Gluγ-C), 34.2 (CH ₂ , Proβ-C), 37.9 (CH ₂ , CH ₂ CH = CH ₂ ), 44.1 (CH ₂ , Glyα-C), 48.5 (CH ₂ , Proδ-C), 52.2 (CH, Gluα-C), 66.4 (CH ₂ , PhCH ₂ ), 66.9 (CH ₂ , PhCH ₂ ), 67.2 (CH ₂ , PhCH ₂ ), 71.9 (quat., Proα-C), 119.9 (CH ₂ , CH = CH ₂ ), 127.9 (CH, Ph), 128.05 (CH, Ph), 128.1 (CH, Ph) , 128.2 (CH, Ph), 128.3 (CH, Ph), 128.4 (CH, Ph), 128.45 (CH, Ph), 128.5 (CH, Ph), 132.1 (CH, CH = CH ₂ ), 135.3 (quat. , Ph), 135.7 (quat., Ph), 136.4 (quat., Ph), 156.2 (quat., NCO ₂ ), 168.1 (quat., Gly-CO), 171.3 (quat., Gluα-CO), 172.7 (quat., Gluγ-CO) and 173.0 (quat., Pro-CON); m / z (FAB +) 656.2970 (MH ⁺ . C ₃₇ H ₄₂ N ₃ O ₈ requires 656.2992).

1.1.23. Di-t-butyl Nt-butyloxycarbonyl-glycyl-L-2-allylprolyl-L-glutamate 33.
Dry triethylamine (0.044cm ^3, 0.32mmol) in dry dichloromethane (5.30cm ³⁾ acid in 26 (0.031 g, 0.10 mmol) and L- glutamic acid di -t- butyl ester hydrochloride 29 (0 0.038 g, 0.129 mmol) solution (under a nitrogen atmosphere at room temperature) and the reaction mixture stirred for 10 minutes was added dropwise. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.032 g, 0.13 mmol) and a stirred solution for 17.5 hours were added. The solution was subsequently washed with 10% aqueous hydrochloric acid (5 cm ³ ) and saturated aqueous sodium bicarbonate (5 cm ³ ), dried (MgSO ₄ ), filtered and evaporated to dryness in vacuo. The remaining purification obtained by flash column chromatography (50% ethyl acetate-hexane) produced the protected tripeptide 33 (0.53 g, 77%) as a pale yellow oil: [α] _D -29.9 (CH ₂ Cl C 0.90 in) in ₂ ; ν _max (film) / cm ^-1 3583, 3417, 3076, 2978, 2931, 1728, 1664, 1522, 1453, 1426, 1392, 1367, 1329, 1251, 1158, 1056, 1028 , 949, 919, 846 and 735; δ _H (300 MHz; CDCl ₃ ) 1.26 [9H, s, C (CH ₃ ) ₃ ], 1.42 [9H, s, C (CH ₃ ) ₃ ], 1.43 [9H, s, C (CH ₃ ) ₃ ], 1.85-1.94 (4H, m, Proβ-H _A H _B , Gluβ-H _A H _B and Proγ-H ₂ ), 2.02-2.12 (1H, m, Gluβ-H _A H _B ), 2.16-2.33 (2H, m, Gluγ-H ₂ ), 2.48-2.53 (1H, m, Proβ-H _A H _B ), 2.69-2.77 (1H, m, 0.5 CH _A H _B CH = CH ₂ ), 3.08-3.15 (1H, m, CH _A H _B CH = CH ₂ ), 3.29-3.38 (1H, m, Proδ-H _A H _B ), 3.53-3.56 (1H, m, Proδ-H _A H _B ), 3.91 (2H, d, J 4.0, Glyα-H ₂ ), 4.33 (1H, dd, J 7.5 and 5.2, Gluα-H), 5.08-5.14 (2H, m, CH = CH ₂ ), 5.47 ( 1H, br s, GlyNH), 5.52-5.66 (1H, m, CH = CH ₂ ) and 7.77 (1H, d, J 7.4, GluNH); δ _C (75 MHz; CDCl ₃ ) 23.1 (CH ₂ , Proγ-C) , 27.4 (CH ₂ , Gluβ-C), 27.9 [CH ₃ , C (CH ₃ ) ₃ ], 28.0 [CH ₃ , C (CH ₃ ) ₃ ], 28.3 [CH ₃ , C (CH ₃ ) ₃ ], 31.5 (CH ₂ , Gluγ-C), 34.2 (CH ₂ , Proβ-C), 38.0 (CH ₂ , CH ₂ CH = CH ₂ ), 43.7 (CH ₂ , Glyα-C), 48.4 (CH ₂ , Proδ- C), 52.7 (CH, Gluα-C), 71.8 (quat., Proα-C), 79.5 [quat., C (CH ₃ ) ₃ ], 80.6 [quat., C (CH ₃ ) ₃ ], 81.9 [ quat., C (CH ₃ ) ₃ ], 119.8 (CH ₂ , CH = CH ₂ ), 132.3 (CH, CH = CH ₂ ), 155.7 (quat., NCO ₂ ), 168.4 (quat., GlyCO), 170.8 (quat., Gluα-CO), 172.3 (quat., Gluγ-CO) and 172.8 (quat., ProCON); m / z (EI +) 553.3359 (M ⁺ .C ₂₈ H ₄₇ N ₃ O ₈ I need).

1.1.24. Di-t-butyl Nt-butyloxycarbonyl-glycyl-L-2-benzylprolyl-L-glutamate 34.
Dry triethylamine (0.11 cm ³ , 0.77 mmol) was added to acid 27 (0.09 g, 0.24 mmol) L-glutamic acid di-t-butyl ester hydrochloride 29 (0.09 g, in dry dichloromethane (13 cm ³ ). 0.31 mmol) solution (under an atmosphere of nitrogen at room temperature) and the reaction mixture stirred for 10 minutes was added dropwise. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.08 g, 0.30 mmol) and a solution stirred for 17 hours were added followed by 10% aqueous hydrochloric acid (12 cm ³ ) and saturated aqueous Washed with sodium bicarbonate (12 cm ³ ), dried (MgSO ₄ ), filtered and evaporated to dryness in vacuo. The remaining purification obtained by flash column chromatography (40% ethyl acetate-hexane) produced the protected tripeptide 34 (0.10 g, 68%) as a colorless oil: [α] _D +15.7 (CH ₂ Cl ₂ in c 1.15); ν _max (film) / cm ^-1 3418, 3357, 3060, 3028, 2978, 2933, 2875, 1727, 1663, 1582, 1519, 1497, 1454, 1426, 1392, 1367, 1330, 1251, 1158, 1093, 1051, 1029, 949, 914, 846, 736, 705 and 651; δ _H (400 MHz; CDCl ₃ ) 1.28-1.39 (1H, m, Proγ-H _A H _B ), 1.43 (27H , s, C (CH ₃ ) ₃ ], 1.47 [27H, s, C (CH ₃ ) ₃ ], 1.48 [27H, s, C (CH ₃ ) ₃ ], 1.69-1.74 (1H, m, Proγ-H _A H _B ), 1.89-1.99 (2H, m, Proβ-H ₂ ), 2.08-2.17 (1H, m, Gluβ-H _A H _B ), 2.23-2.41 (3H, m, Gluβ-H _A H _B and Gluγ-H ₂ ), 2.89-2.95 (1H, m, Proδ-H _A H _B ), 3.13 (1H, d, J 13.4, PhCH _A H _B ), 3.40-3.46 (1H, m, Proδ-H _A H _B ), 3.85 (1H, d, J 13.4, PhCH _A H _B ), 3.92 (2H, d, J 3.8, Glyα-H ₂ ), 4.33 (1H, td, J 7.5 and 5.2, Gluα-H ), 5.58 (1H, br s, GlyNH), 7.07-7.28 (5H, m, PhH) and 7.71 (1H, d, J 7.2, GluNH); δ _C (100 MHz; CDCl ₃ ) 23.0 (CH ₂ , Proγ -C), 27.4 (CH ₂ , Gluβ-C), 28.0 [CH ₃ , C (CH ₃ ) ₃ ], 28.1 [CH ₃ , C (CH ₃ ) ₃ ], 28.3 [CH ₃ , C (CH ₃ ) ₃ ], 31.5 (CH ₂ , Gluγ-C), 34.2 (CH ₂ , Proβ-C), 38.0 (CH ₂ , PhCH ₂ ), 44.1 (CH ₂ , Glyα-C), 48.2 (CH ₂ , Proδ-C ), 52.9 (CH, Gluα-C), 72.4 (quat., Proα-C), 79.6 [quat., C (CH ₃ ) ₃ ], 80.7 [quat., C (CH ₃ ) ₃ ], 82.1 [quat ., C (CH ₃ ) ₃ ], 126.8 (CH, Ph), 128.3 (CH, Ph), 130.4 (CH, Ph), 136.3 (quat., Ph), 155.8 (quat., NCO ₂ ), 168.6 ( quat., GlyCO), 171.0 (quat., Gluα-CO), 172.5 (quat., Gluγ-CO) and 173.1 (quat., ProCON); m / z (FAB +) 604.3592 (MH ⁺ .C ₃₂ H ₅₀ N ₃ O ₈ requires 604.3598).

1.1.25. Glycyl-L-2-methylprolyl-L-glutamic acid
A mixture of protected tripeptide 30 (0.63 g, 1.00 mmol) and 10% palladium on activated carbon (0.32 g, 0.30 mmol) in 90:10 methanol to water (22 cm ³ ) under an atmosphere of hydrogen. At room temperature and protected from light (23 hours). The reaction mixture was filtered through a pad washed with Celite® pad and 75:25 methanol-water (200 cm ³ ). The filtrate was concentrated to dryness under reduced pressure and the remainder was triturated with anhydrous diethyl ether to provide tripeptide 1 (0.27 g, 86%) as a hygroscopic colorless solid. Tripeptide 1 was shown to adopt the trans conformation by NMR analysis: mp 144 °; [α] _D -52.4 (c 0.19 in H ₂ O); δ _H (400 MHz; D ₂ O) 1.62 (3H, s, Proα-CH ₃ ), 1.97-2.25 (6H, m, Proβ-H ₂ , Proγ-H ₂ and Gluβ-H ₂ ), 2.45 (2H, t, J 7.3, Gluγ-H ₂ ), 3.62-3.70 (2H, m, Proδ-H ₂ ), 3.96 (1H, d, J 16.5, Glyα-H _A H _B ), 4.02 (1H, d, J 16.4, Glyα-H _A H _B ) and 4.28 ( 1H, dd, J 8.4 and 4.7, Gluα-H); δ _C (100 MHz; D ₂ O) 19.9 (CH ₃ , Proα-CH ₃ ), 23.0 (CH ₂ , Proγ-C), 26.9 (CH ₂ , Gluβ-C), 30.9 (CH ₂ , Gluγ-C), 38.8 (CH ₂ , Proβ-C), 40.7 (CH ₂ , Glyα-C), 47.5 (CH ₂ , Proδ-C), 54.4 (CH, Gluα -C), 67.8 (quat, Proα-C), 164.6 (quat., GlyCO), 175.3 (quat., ProCON), 177.2 (quat., Gluα-CO), and 178.5 (quat., Gluγ-CO); m / z (FAB +) 316.1508 (MH ⁺ . C ₁₃ H ₂₂ N ₃ O ₆ requires 316.1509).

1.1.26. 1. Glycyl-L-2-ethylprolyl-L-glutamic acid
A mixture of protected tripeptide 31 (0.51 g, 0.80 mmol) and 10% palladium on activated carbon (0.09 g, 0.08 mmol) in 90:10 methanol-water (50 cm ³ ) under an atmosphere of hydrogen. And stirred at room temperature for 20 hours. The solution was filtered through a pad washed with Celite® pad, methanol (2 × 30 cm ³ ) and the filtrate was evaporated to dryness to give a clear gum. The gum was placed under vacuum for 30 minutes and then triturated with anhydrous diethyl ether to tripeptide 2 (0.26 g, 99%) as a hygroscopic colorless solid. Tripeptide 2 was shown to be the trans conformer exclusively by ¹ H and ¹³ C NMR analysis: mp 82-85 ° C .; [α] _D 43.8 (c 0.1 in MeOH); δ _H (400 MHz; D ₂ O) 0.86 (3 H, t, J 7.4, CH ₃ ), 1.94-2.40 (8 H, m, CH ₂ CH ₃ , Gluβ-H ₂ , Proβ-H ₂ and ProγH ₂ ), 2.52-2.56 (2H, m, Gluγ-H ₂ ), 3.55-3.61 (1 H, td, J 6.9 and 9.7, Proδ-H _A H _B ), 3.75-3.80 (1 H, m, Proδ-H _A H _B ), 4.08 (2H, q, J 16.6, Glyα-H ₂ ) and 4.44 (1 H, q, J 4.9, Gluα-H); δ _C (100 MHz; CDCl ₃ ) 6.9 (CH ₃ , CH ₃ ) , 22.8 (CH ₂ , CH ₂ CH ₃ ), 25.3 (CH ₂ , Proγ-C), 25.5 (CH ₂ , Gluβ-C), 30.11 (CH ₂ , Gluγ-C), 35.0 (CH ₂ , Proβ-C ), 40.7 (CH ₂ , Glyα-C), 48.6 (CH ₂ , Proδ-C), 52.6 (CH, Gluα-C), 71.7 (quat., Proα-C), 164.9 (quat., GlyCON), 175.2 (quat., ProCON) 176.0 (quat., Gluα-CO) and 177.3 (quat., Gluγ-CO); m / z (FAB +) 330.1667 (MH ⁺ .C ₁₄ H ₂₄ N ₃ O ₆ requires 330.1665 And).

1.1.27 Glycyl-L-2-propylprolyl-L-glutamic acid
Protected tripeptide 32 (64 mg, 0.097 mmol) in 90:10 methanol-water (9.8 cm ³ ) and 10% palladium on activated carbon (20 mg, 0.19 mmol) at room temperature under an atmosphere of hydrogen. Stir and protect from light (19 hours). The reaction mixture was filtered through a pad washed with Celite® pad and 75:25 methanol-water (50 cm ³ ). The filtrate was concentrated to dryness under reduced pressure to give tripeptide 3 (33 mg, 100%) as a colorless solid. Tripeptide 3 was exclusively shown to be a conformer by NMR analysis: mp 278-280 ° C. (dec.); [Α] _D -16.7 (c 0.18 in H ₂ O); δ _H (300 MHz; D ₂ O) 0.98 (3H, t, CH ₂ CH ₃ ), 1.09 (1H, m, CH _A H _B CH ₃ ), 1.44 (1H, m, CH _A H _B CH ₃ ), 1.82 -2.31 (8H, m, Proβ-H ₂ , Proγ-H ₂ , Gluβ-H ₂ and CH ₂ CH ₂ CH ₃ ), 2.40 (1H, m, Gluγ-H ₂ ), 3.55-3.63 (1H, m, Proδ-H _A H _B ), 3.80 (1H, m, Proδ-H _A H _B ), 4.03 (1H, d, J 16.6, Glyα-H _A H _B ), 4.15 (1H, d, J 16.6, Glyα- H _A H _B ) and 4.27 (1H, dd, J 8.2 and 4.8, Gluα-H); δ _C (100 MHz; D ₂ O) 16.0 (CH ₃ , CH ₂ CH ₃ ), 19.0 (CH ₂ , CH ₂ CH ₃ ), 25.4 (CH ₂ , Proγ-C), 30.9 (CH ₂ , Gluβ-C), 36.3 (CH ₂ , Gluγ-C), 37.4 (CH ₂ , CH ₂ CH ₂ CH ₃ ), 38.3 (CH ₂ , Proβ-C), 43.3 (CH ₂ , Glyα-C), 51.2 (CH ₂ , Proδ-C), 58.1 (CH, Gluα-C), 74.1 (quat., Proα-C), 167.7 (quat. , NCO), 177.8 (quat., ProCON), 180.9 (quat., Gluα-CO) and 184.7 (quat., Gluγ-CO); m / z (FAB +) 344.1827 (MH ⁺ .C ₁₅ H ₂₆ N ₃ O ₆ is 344. 1822 is required).

1.1.28. 3. Glycyl-2-allylprolyl-L-glutamic acid trifluoroacetate
To a solution of protected tripeptide 33 (41 mg, 0.073 mmol) in dichloromethane (4.5 cm ³ ) at room temperature, trifluoroacetic acid (0.75 cm ³ , 9.74 mmol) was added dropwise and the reaction mixture was Stir for 6.5 hours. The solution was evaporated under reduced pressure to form tripeptide 4 (32 mg, 96%) as a pale yellow solid. Tripeptide 4 was shown to exist exclusively as a trans conformer by NMR analysis: [α] _D -7.64 (c 0.39 in H ₂ O); δ _H (400 MHz; D ₂ O ) 1.90-2.02 (1H, m, Proγ-H _A H _B ), 2.03-2.14 (2H, m, Proγ-H _A H _B and Gluβ-H _A H _B ), 2.19-2.32 (3H, m, Proβ- H ₂ and Gluβ-H _A H _B ), 2.54 (2H, ddd, J 8.1, 7.3, 2.0, Gluγ-H ₂ ), 2.74 (1H, dd, J 13.7 and 7.3, CH _A H _B CH = CH ₂ ) , 3.12 (1H, dd, J 13.7 and 7.3, 0.5 CH _A H _B CH = CH ₂ ), 3.48-3.55 (1H, m, Proδ-H _A H _B ), 3.72-3.77 (1H, m, Proδ-H _A H _B ), 3.98 (1H, d, J 16.7, Glyα-H _A H _B ), 4.10 (1H, d, J 16.7, Glyα-H _A H _B ), 4.46 (1H, dd, J 4.9 and 9.5, Gluα-H) 5.20-5.26 (2H, m, CH = CH ₂ ) and 5.73-5.82 (1H, m, CH = CH ₂ ); δ _C (100 MHz; D ₂ O) 25.4 (CH ₂ , Proγ-C ), 28.1 (CH ₂ , Gluβ-C), 32.7 (CH ₂ , Gluγ-C), 38.1 (CH ₂ , Proβ-C), 39.1 (CH ₂ , CH ₂ CH = CH ₂ ), 43.3 (CH ₂ , Glyα-C), 51.0 (CH ₂ , Proδ-C), 55.1 (CH, Gluα-C), 73.2 (quat., Proα-C), 120.3 (quat., CF ₃ ), 122.6 (CH ₂ , CH = C H ₂ ), 134.4 (CH, CH = CH ₂ ), 165.4 (quat., CF ₃ CO ₂ ), 167.5 (quat., GlyCO), 177.5 (quat., ProCON), 178.0 (quat., Glyα-CO) And 179.8 (quat., Gluγ-CO); m / z (EI +) 342.1653 (M ⁺ -CF ₃ CO ₂ .C ₁₅ H ₂₄ N ₃ O ₆ requires 342.1665).

1.1.29. 4. Glycyl-L-2-benzylprolyl-L-glutamic acid trifluoroacetate
To a solution of protected tripeptide 34 (98 mg, 0.16 mmol) in dichloromethane (4 cm ³ ) at room temperature, trifluoroacetic acid (0.75 cm ³ ) was added dropwise and the reaction mixture was stirred for 3.5 hours. . The solution was evaporated under reduced pressure to give tripeptide 5 (82 mg, 100%) as a hygroscopic colorless solid. Tripeptide 5 was shown to be a 90:10 trans: cis mixture of conformers by ¹ H NMR analysis (ratio doublet and multiplet at δ4.51 and 4.33). From the related intensities of and individually given to the large and small conformer glu α-H protons: mp 73-82 ° C; [α] _D +41.0 (c 1.61 in MeOH); δ _H (300 MHz; D ₂ O) 1.27-1.39 (1H, m, Proγ-H _A H _B ), 1.68-1.83 (1H, m, Proγ-H _A H _B ), 2.07-2.42 (4H, m, Proβ- H ₂ and Gluβ-H ₂ ), 2.57 (2H, t, J 7.1, Gluγ-H ₂ ), 2.82-2.92 (1H, m, Proδ-H _A H _B ), 3.24 (1H, d, J 13.3, PhCH _A H _B ), 3.50-3.59 (1H, m, Proδ-H _A H _B ), 3.70 (1H, d, J 13.3, PhCH _A H _B ), 3.91 (1H, d, J 16.7, Glyα-H _A H _B ), 4.09 (1H, d, J 16.7, Glyα-H _A H _B ), 4.33 * (0.1H, m, Gluα-H), 4.51 (0.9H, dd, J 4.8 and 9.6, Gluα-H) and 7.21-7.44 (5H, m, PhH); δ _C (100 MHz; D ₂ O) 25.0 (CH ₂ , Proγ-C), 27.9 (CH ₂ , Gluβ-C), 32.5 (CH ₂ , Gluγ-C) , 37.7 (C H ₂ , Proβ-C), 39.2 (CH ₂ , PhCH ₂ ), 43.5 (CH ₂ , Glyα-C), 50.6 (CH ₂ , Proδ-C), 54.9 (CH, Gluα-C), 56.0 * (CH , Gluα-C), 73.8 (quat., Proα-C), 117.0 (quat., CF ₃ ), 129.6 (CH, Ph), 131.1 (CH, Ph), 132.9 (CH, Ph), 138.3 (quat. , Ph), 165.4 (quat., CF ₃ CO ₂ ), 167.7 (quat., GlyCO), 177.2 (quat., ProCON), 178.0 (quat., Gluα-CO) and 179.7 (quat., Gluγ-CO) m / z (FAB +) 392.1817 (M ⁺ -CF ₃ CO ₂ .C ₁₉ H ₂₆ N ₃ O ₆ requires 392.1822).

1.1.30. Dibenzyl (2S, 5′R, 8′R)-and (2S, 5′R, 8 ′S)-[1 ′-(2 ″ -benzyloxycarbonylamino-acetyl) -8′-hydroxy-6 ′ -Oxo-1 ', 7'-diazaspiro [4.4] non-7'-yl] -1,5-pentanedioate 38.
Alkene 32 (96 mg, 0.15 mmol) was dissolved in dry dichloromethane-methanol (6 cm ³ , 1: 1) and the solution was cooled to −78 ° C. A slow stream of ozone was bubbled through the solution for 15 minutes and then O ₂ to remove excess ozone. Triphenylphosphine (57.5 mg, 0.22 mmol) is added and the resulting mixture is stirred vigorously for 24 hours, then a small amount of silica is added to the reaction mixture (containing aldehyde 37) and The solvent was removed under reduced pressure. The resulting residue was purified by flash column chromatography (100% ethyl acetate) to provide hydroxyspirolactam 38 (61 mg, 63%) as a pale yellow oil. Hydroxyspirolactam 38 was shown to be a 7: 3 mixture of diastereomers by ¹ H NMR analysis. The ratios are measured from the multiple intensities at δ 4.76 and 4.79 and the doubly related strengths at δ 5.00 and given to the large and small isomeric 2-H protons, and the isomer is [Α] _D -44.4 (c 0.90 in MeOH); ν _max (film) / cm ⁻¹ 3410, 3064, 3034, 2953, 2881, 2083, 1718, 1649, 1498, 1454, 1332, 1267, 1215, 1170, 1121, 1082, 1048, 1028, 1003, 984, 909, 776, 736 and 698; δ _H (400 MHz; CDCl ₃ ) 1.74-1.77 ^* (0.3H, m, 9'- H _A H _B ), 1.93-2.04 (1.3H, m, 9'-H _A H _B and 3'-H _A ^* H _B ), 2.11-2.39 (4.4H, m, 4'-H _A H _B , _{3'-H A H B, 3} -H A H B and _{3-H A} B H (high)], 2.42-2.66 (3.3H, m , 3-H A H B *, 4-H 2 and 4 ' -H _A H _B ), 2.76 (0.7H, dd, J 12.9 and 6.1, 9'-H _A H _B ), 3.46-3.58 (2H, m, 2'-H ₂ ), 3.80-3.85 ^* (0.3H , obsc., 2``-H <sub>A</sub> H <sub>B</sub> ), 3.87 (0.7H, dd, J 17.5 and 3.3, 2 ''-H <sub>A</sub> H <sub>B</sub> ), 4.02 (0.7H, dd, J 17.5 and 5.1, 2 ''-H <sub>A</sub> H <sub>B</sub> ), 3.99-4.05 <sup>*</sup> (0.3H, obsc., 2``-H _A H _B ), 4.69 (1H, s, OH), 4. 76-4.79 ^* (0.3H, m, 2-H), 5.00 (0.7H, dd, J 10.8 and 4.8, 2-H), 5.09-5.21 (6H, m, 3 x OCH ₂ Ph), 5.25 ^* ( 0.3H, dd, J 12.3 and 7.1, 8'-H), 5.42 (0.7H, dd, J 6.0 and 2.4, 8'-H), 5.48 ^* (0.3H, m, NH), 5.58-5.59 (0.7 H, m, NH) and 7.27-7.39 (15H, m, 3 x Ph); δ _C (100 MHz; CDCl ₃ ) 24.1 ^* (CH ₂ , 3-C), 24.2 (CH ₂ , 3-C), 24.5 (CH ₂ , 3'-C), 25.3 ^* (CH ₂ , 3'-C), 30.2 (CH ₂ , 4'-C), 30.7 ^* (CH ₂ , 4'-C), 37.9 (CH ₂ , 4-C), 39.4 (CH ₂ , 9-C), 39.7 ^* (CH ₂ , 9-C), 43.0 ^* (CH ₂ , 2``-C), 43.7 (CH <sub>2</sub> , 2 ''-C ), 47.0 (CH <sub>2</sub> , 2-C), 47.7 <sup>*</sup> (CH <sub>2</sub> , 2-C), 54.3 (CH, 2'-C), 54.7 <sup>*</sup> (CH, 2'-C), 65.6 <sup>*</sup> (quat., <sup>5-C), 66.2 * (</sup> CH 2, OCH 2 Ph), 66.4 (CH 2, OCH 2 Ph), 66.8 (CH 2, OCH 2 Ph), 67.0 * (CH 2, OCH 2 Ph), 67.2 * ( CH <sub>2</sub> , OC <sub>2</sub> Ph), 67.3 (CH <sub>2</sub> , OCH <sub>2</sub> Ph), 68.1 (quat., 5-C), 77.7 (CH, 8-C), 80.6 <sup>*</sup> (CH, 8-C), 128.05 (CH , Ph), 128.09 (CH, Ph), 128.2 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 128.6 (CH, Ph), 128.7 (CH, Ph), 134.5 (quat. , Ph), 135.3 <sup>*</sup> (quat., Ph), 135.8 (quat., Ph), 136.0 <sup>*</sup> (quat., Ph) , 136.4 (quat., Ph), 156.1 (quat., NCO <sub>2</sub> ), 166.0 (quat., 1``-C), 167.1 ^* (quat., 1 ''-C), 170.1 ^* (quat., 1 '-C), 172.5 (quat., 1'-C), 172.7 ^* (quat., 5'-C), 173.6 (quat., 5'-C), 173.8 ^* (quat., 6-C) and 175.1 (quat., 6-C); m / z (FAB +) 658.2749 (MH ⁺ . C ₃₆ H ₄₀ N ₃ O ₉ requires 658.2765).

1.1.31. Dibenzyl (2S, 5′R)-[1 ′-(2 ″ -benzyloxycarbonylamino-acetyl) -6′-oxo-1 ′, 7′-diazaspiro [4.4] non-7′-yl]- 1,5-pentanedioate 39.
A solution of hydroxyspirolactam 38 (33 mg, 0.05 mmol) in trifluoroacetic acid-triethylsilane-dichloromethane (1.0 cm ³ , 1: 1: 1) at room temperature was stirred for 45 minutes and then in vacuo. Concentration gave an opaque white oil purified by flash column chromatography (100% ethyl acetate) to provide spirolactam 39 (31 mg, 96%) as a colorless oil: [α] _D -18.0 ( C 0.67 in CH ₂ Cl ₂ ); ν _max (film) / cm ^-1 3583, 3412, 3032, 2952, 1734, 1654, 1497, 1454, 1432, 1289, 1259, 1215, 1171, 1048, 982, 738 And 698; δ _H (300 MHz; CDCl ₃ ) 1.76-2.19 (6H, m, 9'-H _A H _B , 4'-H ₂ , 3'-H _A H _B and 3-H ₂ ), 2.31- 2.63 (4H, m, 9'-H _A H _B , 3'-H _A H _B and 4-H ₂ ), 3.31 (1H, dd, J 16.8 and 8.1, 8'-H _A H _B ), 3.40- 3.47 (1H, m, 8'-H _A H _B ), 3.51-3.55 (2H, m, 2'-H ₂ ), 3.86 (1H, dd, J 17.1 and 3.3, 2``-H <sub>A</sub> H <sub>B</sub> ) , 4.05 (1H, dd, J 17.1 and 5.4, 2``-H _A H _B ), 4.90 (1H, dd, J 11.3 and 4.5, 2-H), 5.07-5.18 (6H, m, 3 x OCH ₂ Ph), 5.63 (1H, br s, NH) and 7.27-7.34 (15H, m, 3 x Ph); δ _C (75 MHz; CDCl ₃ ) 23.4 (CH ₂ , 3'-C), 24.0 (CH ₂ , 3-C), 29.8 ( CH 2, 9'-C), 30.4 (CH 2, 4-C), 36.2 (CH 2, 4'-C), 39.8 (CH 2, 8'-C), 43.7 (CH ₂ , 2``-C), 47.1 (CH ₂ , 2'-C), 53.8 (CH, 2-C), 66.3 (CH ₂ , OCH ₂ Ph), 66.9 (CH ₂ , OCH ₂ Ph) , 67.2 (CH ₂ , OCH ₂ Ph), 68.2 (quat., 5-C), 128.0 (CH, Ph), 128.05 (CH, Ph), 128.1 (CH, Ph), 128.3 (CH, Ph), 128.49 (CH, Ph), 128.5 (CH, Ph), 128.7 (CH, Ph), 135.2 (quat., Ph), 136.0 (quat., Ph), 136.5 (quat., Ph), 156.2 (quat., NCO ₂ ), 166.1 (quat., 1 ''-C), 170.1 (quat., 1-C), 172.7 (quat., 5-C) and 174.4 (quat., 6'-C); m / z ( FAB +) 642.2802 (MH ⁺ . C ₃₆ H ₄₀ N ₃ O ₈ requires 642.2815).

1.1.31. (2S, 5′R)-[1 ′-(2 ″ -Amino-acetyl) -6′-oxo-1 ′, 7′-diazaspiro [4.4] non-7′-yl] -1,5- Pentanedioic acid 35.
Protected spirolactam 39 (30 mg, 0.047 mmol) and 10% palladium on activated carbon (9.6 mg, 0.09 mmol) in 88:12 methanol-water (6.6 cm ³ ) under an atmosphere of hydrogen. Stir at room temperature and protect from light (18 hours). The reaction mixture was filtered through a Celite® pad with 75:25 methanol-water (30 cm ³ ), and the filtrate was concentrated to dryness under reduced pressure to reverse phase C18 flash column chromatography ( A yellow solid purified by H ₂ O) was obtained to provide spirolactam 35 (12 mg, 78%) as a colorless solid: mp 238-239 ° C (dec.); [Α] _D -23.5 [ C 1.15] in MeOH-H ₂ O (1: 1); δ _H (400 MHz; D ₂ O) 1.97-2.61 (10H, m, 9'-H ₂ , 4'-H ₂ , 3'-H ₂ , 3-H ₂ and 4-H ₂ ), 3.44-3.78 (4H, br m, 8'-H ₂ and 2'-H ₂ ) 3.81-4.12 (2H, br m, 2``-H <sub>2</sub> ) And 4.41 (1H, m, 2-H); δ <sub>C</sub> (100 MHz; D <sub>2</sub> O) 25.8 (CH <sub>2</sub> , 3'-C), 27.8 (CH <sub>2</sub> , 3-C), 31.4 (CH <sub>2</sub> , 9 ' -C), 37.3 (2 x CH 2, 4'-C and 4-C), 43.1 (2 x CH 2, 8'-C and 2 '' - C), 50.1 (CH 2, 2'-C) , 60.2 (CH, 2-C), 72.5 (quat., 5'-C), 173.6 (quat., 1``-C), 178.9 (quat., 6'-C) 179.1 (quat., 1- C) and 184.9 (quat., 5-C); m / z (FAB +) 32 8.1521 (MH ⁺ . C ₁₄ H ₂₁ N ₃ O ₆ requires 328.1509).

1.1.32. (2S, 5′R, 8′R)-and (2S, 5′R, 8 ′S)-[1 ′-(2 ″ -amino-acetyl) -8′-hydroxy-6′-oxo-1 ′ , 7'-Diazaspiro [4.4] non-7'-yl] -1,5-pentanedioic acid 36.
A mixture of protected hydroxyspirolactam 38 (27 mg, 0.041 mmol) and 10% palladium on activated carbon (8.4 mg, 0.079 mmol) in 88:12 methanol-water (5.8 cm ³ ) was added to an atmosphere of hydrogen. Under stirring at room temperature, protected from light (18 hours). The reaction mixture was filtered through a Celite® pad with 75:25 methanol-water (30 cm ³ ) and the filtrate was concentrated to dryness under reduced pressure to give spirolactam 36 (14 mg, 99 %) As a colorless solid. Spirolactam 36 was shown by ¹ H NMR analysis to be a 7: 3 mixture of two diastereomers (ratio was determined from the relevant intensity of the doublet doublet of δ 4.47 and 4.53). Measured and given individually to the large and small isomer 2-H: [α] _D +0.86 [c 0.35 in MeOH-H ₂ O (1: 1)]; δ _H (400 MHz; D ₂ O) 2.10-2.46 (9H, m, 9'-H _A H _B , 4'-H ₂ , 3'-H ₂ , 3-H ₂ and 4-H ₂ ), 2.65 * (0.3H, dd, J 13.8 and 7.0, 9'-H _A H _B ), 2.75 (0.7H, dd, J 13.6 and 6.2, 9'-H _A H _B ), 3.55-3.61 (1H, m, 2'-H _A H _B ) , 3.69-3.73 (1H, m, 2'-H _A H _B ), 3.94-4.09 (2H, d, J 16.5, 2``-H <sub>2</sub> ), 4.47 * (0.3H, dd, J 9.9 and 5.8, 2-H), 4.53 (0.7H, dd, J 10.4 and 5.0, 2-H), 5.49 * (0.3H, dd, J 6.8 and 5.1, 8'-H) and 5.59 (0.7H, d, J 6.1 , 8'-H); δ <sub>C</sub> (100 MHz; D <sub>2</sub> O) 26.0 * (CH <sub>2</sub> , 3'-C), 26.1 (CH <sub>2</sub> , 3'-C), 27.4 (CH <sub>2</sub> , 3-C), <sub>29.6 * (CH 2, 3-</sub> C), 39.3 * (CH 2, 4-C), 40.1 (CH 2, 4-C), 40.7 (2 x CH 2, 4'-C and 9'-C), 42.2 * (CH <sub>2</sub> , 9'-C), 43 .1 (CH <sub>2</sub> , 2``-C), 49.8 (CH ₂ , 2'-C), 49.9 * (CH ₂ , 2'-C), 60.2 * (CH, 2-C), 60.7 (CH, 2-C), 70.1 * (quat., 5'-C), 70.9 (quat., 5'-C), 80.6 (CH, 8'-C), 83.2 * (CH, 8'-C), 166.9 (quat., 1``-C), 167.1 * (quat., 1 ''-C), 178.2 * (quat., 6'-C), 179.7 (2 x quat., 1-C and 6'- C), 180.4 (quat., 1-C), 182.5 (quat., 5-C) and 182.8 * (quat., 5-C); m / z (FAB +) 344.1467 (MH ⁺ .C ₁₄ H ₂₂ N ₃ O ₇ requires 344.1458).

1.1.33. Methyl Nt-butyloxycarbonyl- (D, L) -5,5-dimethylprolinate 43.
(Magaard et al. Tetrahedron Lett. 1993, 34, 381) Nitrile 44 (Magaard et al. Tetrahedron Lett. 1993, 34, 381; Bonnet et al. J. Chem. Soc. 1959, 34, 381) (2 g, 14.3 mmol), 32% Dissolved in hydrochloric acid (6 cm ³ ) and heated to 50 ° C. for 5 hours. Solvent evaporation was dissolved in methanol: water (1: 1, 30 cm ³ ) and 44 p. s. Provided the remainder hydrogenated with 10% palladium on activated carbon for 20 hours under i hydrogen. The catalyst is removed by filtration through Celite® and the solvent is removed in vacuo to give a 6: 4 mixture of N-methyl-5-methylproline ^* 45 and the desired 5,5-dimethylproline. 46 was obtained: δ _H (300 MHz; D ₂ O) 1.34-1.40 (8.6H, m, 4 x CH ₃ ), 1.67-1.74 (1.3H, m) 1.91 (1.6H, t, J 12.2), 2.07-2.32 (3.5H, m), 2.40-2.54 (2H, m), 2.90 * (3.6H, s, N-CH ₃ ), 3.45-3.53 (1.3H, m), 4.23 * (1.3H, dd , J 9.7 and 7.5, Proα-H) and 4.47 (1H, d, J 8.4, Proα-H); δ _C (75 MHz; D ₂ O) 14.6 * (CH ₃ ), 24.5 (CH ₃ ), 24.7 ( CH ₃ ), 25.9 * (CH ₂ ), 27.2 (CH ₂ ), 29.9 * (CH ₂ ), 37.0 (CH ₂ ), 39.1 * (CH ₃ , N-CH ₃ ), 58.9 * (CH, Proδ-C ), 67.1 (quat., Proδ-C), 67.6 * (CH, Proα-C), 69.2 (CH, Proα-C), 171.5 * (quat., Proα-CO) and 172.4 (quat., Proα-CO) ).

The mixture was subsequently dissolved in dry methanol (60 cm ³ ), cooled to 0 ° C. and thionyl chloride (4.2 cm ³ , 57.2 mmol) was added dropwise over 5 minutes. The reaction was warmed to room temperature and stirred overnight. The solvent was removed and the remainder was dissolved in a product extracted with saturated sodium bicarbonate solution and chloroform to produce a mixture of unseparated methyl ester (1.53 g) dissolved in dry dichloromethane (20 cm ³ ). N-methylmorpholine (1.56 cm ³ , 14.2 mmol) and di-t-butyl dicarbonate (3.1 g, 14.2 mmol) were added and the reaction was heated at reflux under nitrogen for 48 hours. . After cooling to room temperature, the reaction mixture was washed with water, 1M aqueous hydrochloric acid (2 × 30 cm ³ ), brine and dried (MgSO ₄ ). The aqueous layer was concentrated in vacuo to give methyl N-methyl-5-methylprolinate 47 (1.093 g, 42%, 4 steps) as its hydrochloride salt. This was neutralized with saturated sodium bicarbonate and purified by chromatography (silica gel, 4: 1, dichloromethane: ethyl acetate): δ _H (200 MHz; CDCl ₃ ; Me ₄ Si) 1.06 (3H, d, J 6.0 , Proδ-CH ₃ ), 1.35-1.55 (1H, m), 1.70-2.01 (3H, m), 2.17-2.27 (1H, m), 2.24 (3H, s, N-CH ₃ ), 2.90 (1H, t, J 7.8, Proα-H) and 3.65 (3H, s, 2-CO ₂ CH ₃ ); δ _C (75 MHz; CDCl ₃ ) 18.2 (CH ₃ , Proδ-CH ₃ ), 26.4 (CH ₂ ), 31.3 (CH ₂ ), 38.9 (CH ₃ , N-CH ₃ ), 51.3 (CH ₃ , Proα-CO ₂ CH ₃ ) 61.8 (CH, Proδ-C), 68.4 (CH, Proα-C) and 173.6 (quat M / z (CI +) 158.1176 (MH ⁺ . C ₈ H ₁₆ NO ₂ requires 158.1181); the organic layer is concentrated in vacuo and chromatographed (silica gel, dichloromethane then chloroform) And purified by methyl Nt-butyloxycarbonyl- (D, L) -5,5-dimethylprolinate 43 (0.795 g, 22%, 4 steps) It was obtained as a yellow oil. This compound existed almost exclusively as a cis conformer as a mixture of epimers (55:45): δ _H (300 MHz; CDCl ₃ ) 1.26-1.53 [15H, m, C (CH ₃ ) ₂ , C (CH ₃ ) ₃ ], 1.60-1.90 (4H, m, Proβ-H ₂ and Proγ-H ₂ ), 3.66 (3H, CO ₂ CH ₃ ), 4.24 (0.55H, dd J 8.9 and 3.5 , Proα-H) and 4.35 (0.45H, dd J 8.5 and 2.6, Proα-H); δ _C (75 MHz; CDCl ₃ ) 25.7 (CH ₃ , Proδ-CH ₃ ), 25.9 (CH ₂ , Proγ-C ), 26.0 (CH ₃ , Proδ-CH ₃ ), 26.5 (CH ₂ , Proγ-C), 26.6 (CH ₃ , Proδ-CH ₃ ), 27.2 (CH ₃ , Proδ-CH ₃ ), 28.2 (CH ₃ , C (CH ₃ ) ₃ ], 28.3 [CH ₃ , C (CH ₃ ) ₃ ], 39.8 (CH ₂ , Proβ-C), 40.7 (CH ₂ , Proβ-C), 51.6 (CH ₃ , Proα-CO ₂ CH ₃ ), 51.7 (CH ₃ , Proα-CO ₂ CH ₃ ), 60.5 (quat., Proδ-C), 61.16 (CH, Proα-C), 61.2 (CH, Proα-C), 61.3 (quat., Proδ-C), 79.1 [quat., C (CH ₃ ) ₃ ], 79.6 [quat., C (CH ₃ ) ₃ ], 152.4 (quat., NCO ₂ ), 154.0 (quat., NCO ₂ ), 173.4 (quat., Proα-CO) and 173.8 (quat., Proα-CO); m / z (EI +) 257.1624 (M ⁺ .C ₁₃ H ₂₃ NO ₄ requires 257.1627).

1.1.34. Nt-butyloxyglycyl-L-thiaproline52.
Iso-butyl chloroformate (0.154 g, 1.12 mmol) was added to a stirred solution of Nt-butyloxycarbonylglycine 17 and triethylamine (0.215 g, 1.15 mmol) in tetrahydrofuran (6 cm ³ ). Added at 0 ° C. A white precipitate was observed, the cooling bath was removed and the mixture was stirred at room temperature for 10 minutes. A solution of 4-thiaproline hydrochloride 40 (pellegrini above (0.150 g, 1.12 mmol) and triethylamine (0.215 g, 1.15 mmol) in water (2 cm ³ ) was added and the resulting solution The mixture was acidified with 2 M HCl and extracted with ethyl acetate The combined organic extracts were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo and flash chromatographed. A purified oil (0.39 g) was produced by (hexane: ethyl acetate: acetic acid 2: 1: 0.3 then 1: 1: 0.2) to give dipeptide 52 (0.264 g, 81%) the .52 was obtained as a hygroscopic white foam, ¹ H NMR analysis by 62:38 of conformers trans: indicated a mixture of cis (ratio, Deruta5.65 and 5. 5 measured from the intensity of glycidyl N-H protons, the large and small conformers, individually, _{gave: [α] D -93.5 (c} 0.25 in _{CH 2 Cl 2); δ H} (400 MHz ; CDCl ₃ ) 1.43 ^* [3.4H, s, C (CH ₃ ) ₃ ], 1.44 [5.6H, s, C (CH ₃ ) ₃ ], 3.31 (1.24H, d, J 5.1, 3-H _A H _B ), 3.37 ^* (0.38H, dd, J 11.8 and 5.2, 3-H _A H _B ), 3.49 ^* (0.38H, dd, J 11.7 and 1.4, 3-H _A H _B ), 3.90-4.20 (2H , m, Glyα-H ₂ ), 4.55-4.62 (1.62H, m 5-H ₂ , ^* 5-H _A H _B ), 4.79 ^* (0.38H, d, J 9.7, 5-H _A H _B ), 4.86 ^* (0.38H, d, J 5.7, 2-H), 5.11 (0.62H, t, J 4.9, 2-H), 5.65 (0.62H, br s, GlyN-H) and 5.75 ^* (0.38H, br s, GlyN-H); δ _C (100 MHz; CDCl ₃ ) 28.2 [CH ₃ , C (CH ₃ ) ₃ ], 32.3 (CH ₂ , 3-C), 34.4 ^* (CH ₂ , 3-C) , 43.1 (CH ₂ , Glyα-C), 43.4 ^* (CH ₂ , Glyα-C), 47.6 (CH ₂ , 5-C), 48.9 ^* (CH ₂ , 5-C), 60.7 ^* (CH, 2- C), 61.6 (CH, 2-C), 80.3 ^* (quat., C (CH ₃ ) ₃ ], 80.7 [quat., C (CH ₃ ) ₃ ], 156.1 (quat., NCO ₂ ), 156.4 ^* (quat., NCO ₂ ), 167.8 (quat., GlyCO), 168.0 ^* (quat., GlyCO), 171.6 (qua t., 2-CO) and 172.0 ^* (quat., 2-CO); m / z (EI +) 290.0938 (M ⁺ . C ₁₁ H ₁₈ N ₂ O ₅ S requires 290.0936).

1.1.35. Di-t-butyl Nt-butyloxycarbonylglycyl-L-4-thioprolyl-L-glutamate 56.
Ethyl chlorofomate (0.048 g, 0.445 mmol) was added to a solution of acid 52 (0.129 g, 0.445 mmol) and triethylamine (0.050 g, 0.49 mmol) in dichloromethane (3 cm ³ ). Was added dropwise at 0 ° C. The solution was stirred for 35 minutes at 0 ° C. and then di-t-butyl glutamate hydrochloride 29 (0.132 g, 0.445 mmol) and triethylamine (0.050 g, 0.49 mmol) in dichloromethane (3 cm ³ ). ) Was added. The mixture was stirred overnight and subsequently washed with 2M aqueous hydrochloric acid, saturated sodium bicarbonate, dried (Na ₂ SO ₄ ), filtered and the solvent removed. Purification by flash chromatography (dichloromethane: ethyl acetate, 3: 1) provided the protected tripeptide 56 (0.128 g, 54%) as a colorless solid. 56 was shown to be a 66:34 trans: cis mixture of conformers by ¹ H NMR analysis (ratio was determined from the intensity of thiapro N—H protons at δ 7.20 and 7.43). , Large and small conformers, given individually): mp 99-100 ° C; [α] _D -70 (c 0.6 in CH ₂ Cl ₂ ); δ _H (400 MHz; CDCl ₃ ) 1.25 -1.45 [27H, m, C (CH ₃ ) ₃ ], 1.80-1.95 (1H, m, Gluβ-H _A H _B ), 1.97-2.14 (1H, m, Gluβ-H _A H _B ), 2.18-2.35 (2H, m, Gluγ-H ₂ ), 3.14 (0.66H, dd, J 11.2 and 7.0, 3-H _A H _B ), 3.22-3.35 ^* (0.34H, br m, 3-H _A H _B ), 3.38 (1H, dd, J 12.3 and 3.6, 3-H _A H _B ), 3.85-4.05 (2H, m, Glyα-H ₂ ), 4.38 (1H, br t, J 4.8, Gluα-H), 4.49- 4.57 (1.66H, 5-H _A H _B and ^* 5-H _A H _B ), 4.69-4.77 ^* (0.72H, ^* 5-H _A H _B and ^* 2-H), 4.98 (0.66H, br s , 2-H), 5.51 (1H, br s, GlyN-H), 7.20 (0.66H, d, J 7.2, thiaProN-H), 7.20 ^* (0.34H, br s, thiaProN-H); δ _C ( 100 MHz; CDCl ₃ ) 26.4 ^* (CH ₂ , Gluβ-C), 27.1 (CH ₂ , Gl uβ-C), 27.8 [CH ₃ , C (CH ₃ ) ₃ ], 27.9 [CH ₃ , C (CH ₃ ) ₃ ], 28.2 [CH ₃ , C (CH ₃ ) ₃ ], 31.3 (CH ₂ , Gluγ -C), 32.2 (CH ₂ , 3-C), 35.4 ^* (CH ₂ , 3-C), 43.2 (CH ₂ , Glyα-C), 43.5 ^* (CH ₂ , Glyα-C), 48.2 (CH ₂ , 5-C), 49.7 ^* (CH ₂ , 5-C), 52.5 (CH, Gluα-C), 62.3 ^* (CH, 2-C), 62.5 (CH, 2-C), 79.7 (quat., C (CH ₃ ) ₃ ], 80.6 [quat., C (CH ₃ ) ₃ ], 80.9 ^* [quat., C (CH ₃ ) ₃ ], 82.1 [quat., C (CH ₃ ) ₃ ], 82.2 ^* [quat., C (CH ₃ ) ₃ ], 155.7 (quat., NCO ₂ ), 167.9 (quat., GlyCO), 168.6 ^* (quat., 2-CO), 168.9 (quat., 2-CO), 170.4 (quat., Gluα-CO) and 172.4 (quat., Gluγ-CO); m / z (FAB +) 532.2628 (MH ⁺ .C ₂₄ H ₄₂ N ₃ O ₈ S requires 53.2693).

1.1.36. 51. Glycyl-L-4-thiaprolyl-L-glutamic acid trifluoroacetate
Trifluoroacetic acid (1 cm ³ ) was added to a stirred solution of protected tripeptide 56 (0.128 g, 0.241) and triethylsilane (0.084 g, 0.723 mmol) in dichloromethane (3 cm ³ ). The resulting solution was stirred for 4 hours at room temperature and the volatility was removed in vacuo. Chromatography (reverse phase C ₁₈ , water, 10-20% acetonitrile: water) and the remaining purification by lyophilization gave 48 (0.066 g, 61%) as a hygroscopic off-white solid. 48 was shown to be a 80:20 trans: cis mixture of conformers by ¹ H NMR analysis (ratio measured from the intensity of the glyca-alpha proton at δ3.80 and 3.58, large And small conformers given individually): non-mp due to hygroscopic sample; [α] _D -88.6 (c 0.203 in H ₂ O); δ _H (400 MHz; D ₂ O ) 1.73-1.82 (1H, m, Gluβ-H _A H _B ), 1.96-2.06 (1H, m, Gluβ-H _A H _B ), 2.28 (2H, t, J 6.9, Gluγ-H ₂ ), 2.96 ( 0.8H, dd, J 12.4 and 3.7, 3-H _A H _B ), 3.13 ^* (0.2H, d, 12.2, 3-H _A H _B ), 3.20 (0.8H, dd, J 12.4 and 7.3, 3- H _A H _B ), 3.27 ^* (0.2H, dd, J 12.4 and 6.9, 3-H _A H _B ), 3.58 (0.2H, d, J 16.4, Glyα-H _A H _B ), 3.80-3.92 (1.8 H, m, Glyα-H ₂ and ^* Glyα-H _A H _B ), 4.25 (0.8H, dd, 9.4 and 4.8, Gluα-H), 4.30 ^* (0.2H, dd, 10.0 and 4.8, Gluα-H) , 4.35-4.49 (2H, m, 5-H ₂ ) and 4.67 (1H, dd, J 6.9 and 3.8, 2-H); δ _C (100 MHz; CDCl ₃ ) 24.8 ^* (CH ₂ , Gluβ-C) , 2 5.3 (CH ₂ , Gluβ-C), 29.5 (CH ₂ , Gluγ-C), 29.8 ^* (CH ₂ , Gluγ-C), 32.8 (CH ₂ , 3-C), 34.9 ^* (CH ₂ , 3-C ), 40.3 (CH ₂ , Glyα-C), 40.5 ^* (CH ₂ , Glyα-C), 48.4 (CH ₂ , 5-C), 49.6 ^* (CH ₂ , 5-C), 51.7 (CH, Gluα- C), 51.9 ^* (CH, Gluα-C), 61.6 ^* (CH, 2-C), 62.6 (CH, 2-C), 115.7 (quat., Q, J 290.7, CF ₃ CO ₂ H), 161.9 (quat., q, J 36.2, CF ₃ CO ₂ H), 165.0 (quat., GlyCO), 165.6 ^* (quat., GlyCO), 171.0 ^* (quat., 2-CO), 171.5 (quat., 2 -CO), 174.0 ^* (quat., Gluα-CO), 174.1 ^* (quat., Gluα-CO) and 176.7 (quat., Gluγ-CO); m / z (FAB +) 320.0921 [M (free base) H ⁺ . C ₁₁ H ₁₈ N ₃ O ₆ S requires 320.0916].

1.1.37. N-benzyloxycarbonylglycyl-L-thia-5,5-dimethylproline
A stirred solution of 5,5-dimethyl-4-thiaproline hydrochloride 41 in dry dimethylformamide (35 cm ³ ) under nitrogen (Lewis et al. J. Med. Chem. 1978, 21, 1071; Kemp et al. J. Org. Chem. 1989, 54, 3640) (0.354 g, 1.79 mmol), diisopropylethylamine (0.594 cm ³ , 1.9 mmol) and acid fluoride 51 (Bertho et al. Tetrahedron Lett. 1991, 32,1303; Carpino et al. J. Org. Chem. 1991, 56, 2611) (0,341 g, 1.61 mmol) was added. The solution was stirred for 18 hours, the solvent removed in vacuo and the remainder redissolved in ethyl acetate, washed with 10% citric acid solution, brine and dried (MgSO ₄ ). The solvent was removed and the residue was purified by flash chromatography (4: 1: 0.5, ethyl acetate: hexane: acetic acid then 3: 1.0.4) to give N-benzyloxycarbonylglycine 16 ( The desired compound contaminated with 14%, ¹ H NMR) was obtained. This mixture was dissolved in methanol, trimethylsilyl chloride (0.07 cm ³ ) was added and the solution was stirred overnight. Removal of the solvent in vacuo and subsequent flash chromatography (3: 1, ethyl acetate: hexane [to remove N-benzyloxycarbonylglycine methyl ester] then 3: 1.0.4 ethyl acetate: hexane: acetic acid ) Afforded dipeptide 53 (0.320 g, 65% based on the amount of reacted acid fluoride) as a white solid. This compound existed purely as a cis conformer: mp 130-132 ° C .; [α] _D -51.7 (c 0.116 in dichloromethane); δ _H (300 MHz; CDCl ₃ ) 1.85 (3H, s, ^Ψ Pδ-CH ₃ ) 1.91 (3H, s, Pδ-CH ₃ ), 3.30 (1H, dd, J 12.1 and 5.6, Pβ-H _A H _B ), 3.40 (1H, d, J 12.1, Pβ-H _A H _B ), 3.87 (1H, dd, J 16.6 and 3.7, Glyα-H _A H _B ), 4.09 (1H, dd, J16.6 and 3.7, Glyα-H _A H _B ), 4.82 (1H, d, J 5.2, Pα-H), 5.13 (2H, s, OCH ₂ Ph), 6.10 (1H, br t, J 3.8, GlyN-H) and 7.29-7.39 (5H, m, Ph); δ _C (75 MHz; CDCl ₃ ) 27.0 (CH ₃ , Pδ-CH ₃ ) 29.4 (CH ₃ , Pδ-CH ₃ ), 31.6 (CH ₂ , Pβ-C), 44.8 (CH ₂ , Glyα-C), 64.3 (CH, Pα- C), 67.3 (CH ₂ , OCH ₂ Ph), 74.2 (quat., Pδ-C), 127.9 (CH, Ph), 128.1 (CH, Ph), 128.4 (CH, Ph), 135.8 (quat., Ph ), 156.8 (quat., NCO ₂ ), 166.6 (quat., GlyCO) and 172.0 (quat., Pα-CO); m / z (EI +) 352.1088 (M ⁺ .C ₁₆ H ₂₀ N ₂ O ₅ S , 352.11093).
^Ψ P relates to the proline analog part in question.

1.1.38. Dibenzyl N-benzyloxycarbonyl-glycyl-L-thia-5,5-dimethylprolyl-L-glutamate 57.
Dry dichloromethane (40 cm ³⁾ solution of dipeptide 53 (0.398g, 1.11mol), L- glutamic acid dibenzyl ester p- toluenesulfonate 28 (0.670g, 1.34mol) and diisopropylethylamine (0.51 cm ^3, To a stirred solution of 2.90 mmol) was added a portion of bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.370 g, 1.45 mmol). The solution was stirred for 7 hours under nitrogen and the solvent was removed. The remainder was suspended in ethyl acetate, washed with 10% citric acid solution, saturated sodium bicarbonate, brine, and dried (MgSO ₄ ). Removal of solvent and subsequent chromatography (2: 1, hexane: ethyl acetate, then 1: 1) gave protected tripeptide 57 (0.5 g, 68%) as a colorless oil. The protected tripeptide 57 was shown by ¹ H NMR analysis to be a 90:10 cis: trans mixture of conformers (ratio from a wide range of singlet intensities at δ 5.69 and 5.80). Measured and given individually to the large and small conformational glyc NH protons :) [α] _D -35.6 (c 0.399 in dichloromethane); δ _H (300 MHz; CDCl ₃ ) 1.85 ( 3H, Pδ-CH ₃ ) 1.97 (3H, Pδ-CH ₃ ), 2.08-2.15 (1H, m, Gluβ-H _A H _B ), 2.29-2.38 (1H, m, Gluβ-H _A H _B ), 2.47 (2H, t, J 5.4, Gluγ-H ₂ ), 3.32 (2H, m, Pβ-H ₂ ), 3.85 (1H, d, J 16.6, Glyα-H _A H _B ), 3.91 (1H, dd, J 17.0 and 8.0, Glyα-H _A H _B ), 4.72 (2H, br s, Pα-H, Gluα-H), 5.20-5.08 (6H, m, 3 x OCH ₂ Ph), 5.69 (0.9H, br s , GlyNH), 5.80 ^* (0.1H, br s, GlyNH) and 7.33-7.39 (15H, m, Ph); δ _C (75 MHz; CDCl ₃ ) 26.3 (CH ₂ , Gluβ-C), 26.45 ^* (CH ₂ , Gluβ-C), 27.21 ^* (CH ₃ , Pδ-CH ₃ ), 27.43 (CH ₃ , Pδ-CH ₃ ), 28.4 (CH ₃ , Pδ-CH ₃ ), 29. 91 (CH ₂ , Gluγ-C), 30.09 ^* (CH ₂ , Gluγ-C), 32.3 (CH ₂ , Pβ-C), 44.7 (CH ₂ , Glyα-C), 45.1 ^* (CH ₂ , Glyα-C ), 52.1 (CH, Gluα-C), 52.3 ^* (CH, Gluα-C), 65.8 (CH, Pα-H), 66.4 (CH ₂ , OCH ₂ Ph), 66.8 (CH ₂ , OCH ₂ Ph), 67.4 (CH ₂ , OCH ₂ Ph), 74.5 (quat., Pδ-C), 127.8 (CH, Ph), 127.9 (CH, Ph), 128.1, (CH, Ph), 128.14 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.5 (CH, Ph), 134.8 (quat., Ph), 135.5 (quat., Ph), 136.2 (quat., Ph), 156.4 (quat., NCO ₂ ), 167.1 (quat., GlyCO), 169.6 (quat., P-CON), 170.9 (quat., Gluα-CO) and 172.7 (quat., Gluγ-CO); m / z (FAB +) 662.2536 (MH ⁺ . C ₃₅ H ₄₀ N ₃ O ₈ S requires 662.2536).

1.1.39. 48. Glycyl-L-thia-5,5-dimethylprolyl-L-glutamic acid
Protected tripeptide 57 (0.44 g, 0.66 mmol) was dissolved in methanol: water (4: 1, 50 cm ³ ) and placed in a Parr bottle. The vessel was flushed with nitrogen, 10% palladium on activated carbon (70 mg, 0.066 mmol) was added and the mixture was added 40 p. s. Pressurized i and vibrated for 3 hours. Additional 10% palladium on activated carbon (70 mg) was added and the reaction continued for 21 hours. The reaction mixture was filtered through Celite®, washed with methanol: water (4: 1) and the solvent removed to remove t. l. The oil was produced by c and ¹ H NMR analysis contained the product containing the desired product and varying the amount of debenzylation. The mixture was dissolved in water and passed through a _C18 column eluting with water then 10% methanol: water. The relevant fractions were combined, the solvent was removed and the remainder was triturated with dry ether to produce tripeptide 49 (0.110 g, 48%) as a white solid. Tripeptide 49 was shown by ¹ H NMR analysis to be a 85:15 cis: trans mixture of conformers (ratio measured from doublet intensity at δ4.95 and 5.02). , Large and small conformer Pα-H protons, given individually): mp 145-150 ° C .; C; [α] _D -75 (c 0.064 in water); δ _H (300 MHz; D ₂ O) 1.84 (2.55H, s, Pδ-CH ₃ ). 1.90 ^* (0.45H, s, Pδ-CH ₃ ) 1.93 (3H, s, Pδ-CH ₃ ), 1.96-2.05 (1H, m, Gluβ- H _A H _B ), 2.18-2.27 (1H, m, Gluβ-H _A H _B ), 2.42 (2H, t, J 7.5, Gluγ-H ₂ ), 3.36 (1H, d, J 12.7, Pβ-H _A H _B ) 3.59 (1H, dd, J 12.8 and 6.4, Pβ-H _A H _B ), 3.70 (1H, d, J 16.2, Glyα-H _A H _B ), 4.01 (1H, d, J 16.3, Glyα -CH _A H _B ), 4.23 ^* (0.15H, dd, J 9.1 and 4.9, Gluα-H), 4.32 (0.85H, dd, J 9.1 and 4.9, Gluα-H), 4.95 (0.85H, d, J 6.2, Pα-H) and 5.02 ^* (0.15H, d, J 6.0, Pα-H); δ _C (75 MHz; D ₂ O) 26.1 ^* (CH ₃ , Pδ-CH ₃ ), 26.3 (CH ₃ , Pδ- CH ₃ ), 26.56 (CH ₂ , Gluβ-C), 27.6 (CH ₃ , Pδ-CH ₃ ), 27.9 ^* (CH ₃ , Pδ-CH ₃ ), 31.1 (CH ₂ , Gluγ-C), 30.3 ^* ( CH ₂ , Gluγ-C), 32.1 (CH ₂ , Pβ-C), 32.3 ^* (CH ₂ , Pβ-C), 41.1 ^* (CH ₂ , Glyα-C), 41.6 (CH ₂ , Glyα-C), 54.4 (CH, Gluα-C), 55.0 ^* (CH, Gluα-C), 65.3 ^* (CH, Pα-H), 65.5 (CH, Pα-H), 74.3 ^* (quat., Pδ-C), 74.6 (quat., Pδ-C), 164.4 ^* (quat., GlyCO), 164.6 (quat., GlyCO), 170.5 (quat., P-CON), 170.8 ^* (quat., P-CON), 176.9 (quat ., Gluα-CO) and 178.3 (quat., Gluγ-CO); m / z (FAB +) 348.1249 (MH ⁺ . C ₁₂ H ₂₂ N ₃ O ₆ S requires 348.1229).

1.1.40. Methyl Nt-butyloxycarbonylglycyl- (D, L) -5,5-dimethylprolinate;
Trifluoroacetic acid (1 cm ³ ) was added to a stirred solution of carbamate 43 (0.584 g, 2.27 mmol) in dichloromethane (6 cm ³ ). The solution was stirred for 2 hours, after which time the volatility was removed in vacuo and traces of trifluoroacetic acid were removed by placing the sample on an oil pump for 2 hours. The salt is then dissolved in dichloromethane (20 cm ³ ) and diisopropylethylamine (1.3 cm ³ , 7.49 mmol), then Nt-butyloxycarbonylglycine 17 (0.477 g, 2.73 mmol) and bis ( 2-Oxo-3-oxazolidinyl) phosphinic chloride (0.691 g, 2.28 mmol) was added. Additional dichloromethane (5 cm ³ ) was added and the solution was stirred overnight under nitrogen. The solvent was then removed in vacuo and the remainder was dissolved in ethyl acetate and subsequently washed with 2M aqueous hydrochloric acid, saturated sodium bicarbonate and dried (MgSO ₄ ). Removal of the solvent gave an oil (0.440 g) purified by chromatography (silica gel, hexane: ethyl acetate, 2: 1 then 1: 1) to give dipeptide 54 (0.368 g, 52%) Obtained as a colorless oil. Dipeptide 54 was shown to be an 80:20 cis: trans mixture of conformers by ¹ H NMR analysis (ratio measured from the intensity of chemical shift at δ 4.28 and 4.46, large And a small conformer proα-H proton, given individually): δ _H (300 MHz; CDCl ₃ ) 1.26 (2.4H, s, Proδ-CH ₃ ), 1.28 [9H, s, C (CH ₃ ) ₃ ], 1.31 ^* (0.6H, s, Proδ-CH ₃ ), 1.44 ^* (0.6H, s, Proδ-CH ₃ ), 1.46 (2.4H, s, Proδ-CH ₃ ), 1.59- 2.15 (4H, m, Proβ-H ₂ , Proγ-H ₂ ), 3.37 (0.8H, dd, J 16.7 and 3.3, Glyα-H _A H _B ), 3.57 ^* (0.6H, s, Proα-CO ₂ CH ₃ ), 3.61 (2.4H, s, Proα-CO ₂ CH ₃ ), 3.74 (0.8H, dd, J 16.7 and 3.3, Glyα-H _A H _B ), 3.84 ^* (0.2H, dd, J 16.9 and 3.9 , Glyα-H _A H _B ), 3.39-4.01 ^* (0.2H, m, Glyα-H _A H _B ), 4.28 (0.8H, d, J 8.3, Proα-H), 4.46 ^* (0.2H, dd, J 8.0 and 2.2, Proα-H) and 5.40 (1H, br s, 1H, GlyNH); δ _H (75 MHz; CDCl ₃ ) 24.5 (CH ₃ , Proδ-CH ₃ ), 25.0 ^* (CH ₂ , Proβ-C), 26.1 (CH ₃ , Proδ-CH ₃ ), 26.9 ^* (CH ₃ , Proδ-CH ₃ ), 27.2 ^* (CH ₃ , Proδ-CH ₃ ), 27.4 (CH ₂ , Proβ-C) , 27.9 [CH ₃ , C (CH ₃ ) ₃ ], 38.9 (CH ₂ , Proγ-C), 41.7 ^* (CH ₂ , Proγ-C), 42.8 ^* (CH ₂ , Glyα-C), 43.1 (CH ₂ , Glyα-C), 51.7 ^* (CH ₃ , Proα-CO ₂ CH ₃ ), 52.3 (CH ₃ , Proα-CO ₂ CH ₃ ), 60.1 (CH, Proα-C), 60.9 ^* (quat., Proδ- C), 61.7 ^* (CH, Proα-C), 63.8 (CH, Proα-C), 78.0 (quat., C (CH ₃ ) ₃ ], 155.3 (quat., NCO ₂ ), 155.4 ^* (quat., NCO ₂ ), 166.5 (quat., GlyCO), 167.7 ^* (quat., GlyCO), 171.9 (quat., Proα-CO) and 172.4 ^* (quat., Proα-CO); m / z (CI +) 315.1927 ( MH ⁺ . C ₁₅ H ₂₇ N ₂ O ₅ requires 315.1920).

1.1.41. Nt-butyloxycarbonylglycyl- (D, L) -5,5-dimethylproline55.
To a solution of dipeptide 54 (0.362 g, 1.16 mmol) in dioxane (12 cm ³ ) was added 1M aqueous sodium hydroxide (5.91 cm ³ , 5.91 mmol) and the mixture was stirred for 21 hours. The reaction was acidified with solid citric acid and the product was extracted with ethyl acetate. The combined organic extracts are washed with brine, dried (MgSO ₄ ) and the solvent removed in vacuo and chromatographed (silica gel, hexane: ethyl acetate, 2: 1, 1: 1, 4: 6). Produced an oil 55 (0.324 g, 94%) as a rapidly liquefied white foam. The acid 55 was shown to be an 80:20 cis: trans mixture of conformers by ¹ H NMR analysis (ratio measured from a wide range of singlet intensities at δ5.81 and 5.66). , Large and small conformer glyc N—H protons, given individually): δ _H (300 MHz; CDCl ₃ ) 1.40 (2.4H, s, Proδ-CH ₃ ), 1.43 [7.2H, s, C (CH ₃ ) ₃ ], 1.44 ^* [1.8H, s, C (CH ₃ ) ₃ ], 1.47 ^* (0.6H, s, Proδ-CH ₃ ), 1.58 ^* (0.6H, s, Proδ- CH ₃ ), 1.61 (2.4H, s, Proδ-CH ₃ ), 1.74-2.33 (4H, m, Proβ-H ₂ , Proγ-H ₂ ), 3.37 (0.8H, dd, J 16.7 and 3.3, Glyα- H _A H _B ), 3.65 (0.8H, dd, J 16.8 and 3.6, Glyα-H _A H _B ), 3.96 (1H, dd, J 16.9 and 3.9, Glyα-H _A H _B , Glyα-H _A H _B ^* (Partially unclear)), 4.21 ^* (0.2H, dd, J 17.0 and 5.9, Gyα-H _A H _B ), 4.42 (0.8H, d, J 7.2, Proα-H), 4.67 ^* ( 0.2H, d, J 7.9 Proα-H), 5.66 ^* (0.2H, br s, GlyNH), 5.81 (0.8H, br s, GlyNH) and 6.2 (1H, br s, OH); δ _H (75 MHz ; CDCl ₃ ) 24.8 (CH ₃ , Proδ-CH ₃ ), 25.1 ^* (CH ₂ , Proβ-C), 26.3 (CH ₃ , Proδ-CH ₃ ), 27.1 ^* (CH ₃ , Proδ-CH ₃ ), 27.6 ^* (CH ₃ , Proδ-CH ₃ ), 28.0 (CH ₂ , Proβ-C), 28.2 [CH ₃ , C (CH ₃ ) ₃ ], 39.2 (CH ₂ , Proγ-C), 42.0 ^* (CH ₂ , Proγ -C), 43.0 ^* (CH ₂ , Glyα-C), 43.5 (CH ₂ , Glyα-C), 60.5 (CH, Proα-C), 61.8 ^* (quat., Proδ-C), 62.2 ^* (CH, Proα-C), 64.3 (CH, Proα-C), 79.7 ^* (quat., C (CH ₃ ) ₃ ], 80.2 [quat., C (CH ₃ ) ₃ ], 156.0 ^* (quat., NCO ₂ ) , 156.4 (quat., NCO ₂ ), 166.8 (quat., GlyCO), 169.5 ^* (quat., GlyCO), 173.9 (quat., Pro) α-CO) and 174.5 ^* (quat., Proα-CO); m / z (CI +) 315.1927 (MH ⁺ . C ₁₅ H ₂₇ N ₂ O ₅ requires 315.1920).

1.1.42. Dibenzyl Nt-butyloxycarbonylglycyl- (D, L) -5,5-dimethylprolyl-L-glutamate 58.
A stirred solution of acid 55 (0.298 g, 1 mmol) in dry dichloromethane (40 cm ³ ) is followed by diisopropylethylamine (0.453 cm ³ , 2.6 mmol), L-glutamic acid dibenzyl ester p-toluenesulfonate 28. (0.648 g, 1.3 mmol) and bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.330 g, 1.3 mmol) were added. The resulting solution was stirred overnight at room temperature under nitrogen and the solvent was removed. The remainder was dissolved in ethyl acetate, washed with 10% aqueous citric acid solution, saturated sodium bicarbonate, brine and dried (MgSO ₄ ). The solvent was evaporated and the product purified by chromatography (silica gel, hexane: ethyl acetate, 1: 1) to give the protected tripeptide 58 (0.408 g, 67%) as a yellow oil (pro-α-C epimer of Obtained as a 1: 1 mixture). Tripeptide 58 was shown by ¹ H NMR analysis to be a 85:15 cis: trans mixture of conformers (ratio of chemical shift intensity at δ 5.31-5.38 and 5.48). Large and small conformational glyc-NH protons, given individually): δ _H (300 MHz; CDCl ₃ ) 1.41 [9H, s, C (CH ₃ ) ₃ ], 1.44 ( 2.55H, s, Proδ-CH ₃ ), 1.52 ^* (0.45H, s, Proδ-CH ₃ ), 1.54 ^* (0.45H, s, Proδ-CH ₃ ), 1.64 (2.55H, s, Proδ-CH ₃ ), 1.67 (2.55H, s, Proδ-CH ₃ ), 1.70-2.24 (6H, m, Proβ-H ₂ , Proγ-H ₂ , Gluβ-H ₂ ), 2.35-2.45 (2H, m, Gluγ-H ₂ ), 3.58-3.69 (0.85H, m, Glyα-H _A H _B ), 3.85 (0.85H, m, Glyα-H _A H _B ), 3.88-3.98 ^* (0.15H, m, Glyα-H _A H _B ), 4.12-4.17 ^* (0.15H, m, Glyα-H _A H _B ), 4.20-4.31 (1H, m, Proα-H), 4.59-4.71 (1H, m, Gluα-H), 5.10-5.22 (m, 4H, 2 x OCH ₂ Ph), 5.31-5.38 (0.85H, m, GlyNH) and 5.48 ^* (0.15H, m, GlyNH); δ _C (75 MHz; CDCl ₃ ) 24.38 (CH ₃ , Proδ -CH ₃ ), 24.46 (CH ₃ , Proδ-CH ₃ ), 25.1 ^* (CH ₂ , Proβ-C), 25.2 ^* (CH ₂ , Proβ-C), 26.2 (CH ₂ , Gluβ-C), 26.3 (CH ₂ , Gluβ-C), 26.6 (CH ₃ , Proδ-CH ₃ ), 26.8 ^* (CH ₂ , Proβ-C), 27.0 ^* (CH ₃ , Proδ-CH ₃ ), 27.1 ^* (CH ₃ , Proδ-CH ₃ ), 27.9 ^* (CH ₃ , Proδ-CH ₃ ), 28.2 [CH ₃ , C (CH ₃ ) ₃ ], 28.5 (CH ₂ , Proβ-C), 28.8 (CH ₂ , Proβ-C), 30.05 ^* ( CH ₂ , Gluγ-C), 30.1 ^* (CH ₂ , Gluγ-C), 30.2 (CH ₂ , Gluγ-C), 30.25 (CH ₂ , Gluγ-C), 38.9 (CH ₂ , Proγ-C), 39.1 (CH ₂ , Proγ-C), 42.4 ^** (CH ₂ , Proγ-C), 43.1 ^* (CH ₂ , Glyα-C), 43.3 ^* (CH ₂ , Glyα-C), 43.5 (CH ₂ , Glyα- C), 43.6 (CH ₂ , Glyα-C), 51.7 ^* (CH, Gluα-C), 52.0 (CH, Gluα-C), 52.1 (CH, Gluα-C), 61.5 ^* (quat., Proδ-C ), 61.6 ^* (quat., Proδ-C), 61.9 (CH, Proα-C), 62.0 (CH, Proα-C), 63.0 ^* (CH, Proα-C), 63.05 ^* (CH, Proα-C) , 64.3 (quat., Proδ-C), 64.3 (quat., Proδ-C), 66.3 ^* (CH ₂ , OCH ₂ Ph), 66.4 ^* (CH ₂ , OCH ₂ Ph), 66.41 (CH ₂ , OCH ₂ Ph), 66.5 (CH ₂ , OCH ₂ Ph), 67.0 ^* (CH ₂ , OCH ₂ Ph), 67.1 ^* (CH ₂ , OCH ₂ P h), 67.21 (CH ₂ , OCH ₂ Ph), 67.24 (CH ₂ , OCH ₂ Ph), 79.3 (quat., C (CH ₃ ) ₃ ], 128.1 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.4 ^* (CH, Ph), 128.43 (CH, Ph), 128.5 (CH, Ph), 135.03 (quat., Ph), 135.07, (quat., Ph), 153.13 ^* (quat , Ph), 135.18 ^* (quat., Ph), 135.6 (quat., Ph), 135.7 ^* (quat., Ph), 155.8 ^* (quat., NCO ₂ ), 155.9 (quat., NCO ₂ ), 156.0 (quat., NCO ₂ ), 167.6 (quat., GlyCO), 167.7 (quat., GlyCO), 168.8 ^* (quat., GlyCO), 169.3 ^* (quat., GlyCO), 171.1 (quat., ProCON) , 171.3 ^* (quat., ProCON), 171.4 ^* (quat., ProCON), 171.6 (quat., Gluα-CO), 171.8 (quat., Gluα-CO), 172.4 (quat., Gluγ-CO), 172.5 ^* (quat., Gluγ-CO) and 172.6 (quat., Gluγ-CO); mz / (EI +) 609.3035 (M ⁺ . C ₃₃ H ₄₃ N ₃ O ₈ requires 609.3050).

1.1.43. 50. Glycyl- (D, L) -5,5-dimethylprolyl-L-glutamic acid
Dichloromethane (10 cm ³⁾ solution of protected tripeptide 58 in (0.276 g, 0.454 mmol) to a stirred solution of was added trifluoroacetic acid (1 cm ³⁾ and the mixture was stirred for 75 minutes. The solvent was removed in vacuo, the remainder was dissolved in saturated sodium bicarbonate and the product was extracted with ethyl acetate. The combined organic layers were washed with brine, dried (MgSO ₄ ) and the solvent removed to yield an oil (0.244 g) dissolved in methanol: water (4: 1, 50 cm ³ ). The flask was flushed with nitrogen, 10% palladium on activated carbon (0.048 mg, 0.454 mmol) was added, and the mixture was then stirred overnight under one atmosphere of hydrogen. Filtration through Celite® followed by solvent removal yielded an oil ground with dry diethyl ether to yield tripeptide 50 (0.139 g, 93%) as an off-white solid. Tripeptide 50 was shown by ¹ H NMR analysis to be a 72:28 cis: trans mixture of conformers (ratio of intensity of chemical shift at δ3.57 and 4.15-4.16). Large and small conformational gly α-H protons, given individually): about 10% of the final product was given experimentally as hydrochloride ^** : mp 145-150 ° C. ; δ _H (400 MHz; D ₂ O) 1.43 (2.16H, s, Proδ-CH ₃ ), 1.49 ^* (0.84H, s, Proδ-CH ₃ ), 1.57 ^* (0.84H, s, Proδ-CH ₃ ), 1.58 ^* (0.84H, s, Proδ-CH ₃ ), 1.60 (2.16H, s, Proδ-CH ₃ ), 1.61 (2.16H, s, Proδ-CH ₃ ), 1.90-2.48 (8H, m, Proβ-H ₂ , Proγ-H ₂ , Gluβ-H ₂ , Gluγ-H ₂ ), 3.57 (0.72H, dd, J 16.1 and 2.8, Glyα-H _A H _B ), 3.75-3.82 ^** (0.2H, m, Glyα-H _A H _B , Gluα-H), 3.94 (0.72H, dd, J 16.1 and 6.5, Glyα-H _A H _B ), 4.11 ^** (0.1H, d, J 2.5, Glyα-H _A H _B ), 4.15-4.16 ^* (0.56H, m, Glyα-H ₂ ), 4.24-4.30 (1H, m, Gluα-H), 4.46-4.51 ^** (0.1H, m, Proα-H), 4.60 (0.72H, t, J 10.1, Proα-H) and 4.68 ^* (0.28H, dd, J 13.5 and 4.3, Proα -H); δ _C (400 MHz; D ₂ O) 23.56 (CH ₃ , Proδ-CH ₃ ), 23.76 (CH ₃ , Proδ-CH ₃ ), 24.1 ^** (CH ₃ , Proδ-CH ₃ ), 25.0 (CH ₂ , Gluβ-C), 25.2 (CH ₃ , Proδ-CH ₃ ), 25.6 (CH ₂ , Gluβ-C), 25.7 ^** (CH ₃ , Proδ-CH ₃ ), 26.0 (CH ₂ , Gluβ- C), 26.1 ^* (CH ₃ , Proδ-CH ₃ ), 26.2 ^* (CH ₃ , Proδ-CH ₃ ), 26.4 (CH ₂ , Gluβ-C), 26.8 ^* (CH ₂ , Gluβ-C), 28.4, (CH ₂ , Proβ-C), 28.7, (CH ₂ , Proβ-C), 30.8 ^* (CH ₂ , Gluγ-C), 31.0 ^* (CH ₂ , Gluγ-C), 31.2 (CH ₂ , Gluγ-C ), 38.7 (CH ₂ , Proγ-C), 38.8 (CH ₂ , Proγ-C), 40.6 ^* (CH ₂ , Glyα-C), 40.7 (CH ₂ , Glyα-C), 40.8 (CH ₂ , Glyα- C), 41.1 ^* (CH ₂ , Proγ-C), 41.2 ^* (CH ₂ , Proγ-C), 46.1 ^** (CH ₂ , Glyα-C), 54.1 (CH, Gluα- * C), 54.5 (CH , Gluα-C), 59.7 ^* (CH, Proα-C), 61.7 (CH, Proα-C), 61.8 (CH, Proα-C), 62.6 ^** (quat., Proδ-C), 62.7 ^* (quat ., Proδ-C), 63.4 ^* (CH, Proα-C), 63.9 ^** (quat., Proδ-C), 65.0 (quat. , Proδ-C), 65.1 (quat., Proδ-C), 164.8 (quat., GlyCO), 164.9 (quat., GlyCO), 165.6 ^* (quat., GlyCO), 165.8 ^* (quat., GlyCO), 166.0 ^** (quat., GlyCO), 172.3 ^** (quat., ProCON), 172.8 (quat., ProCON), 173.1 (quat., ProCON), 173.3 ^* (quat., ProCON), 173.6 ^* (quat. , ProCON), 176.9 (quat., Gluα-CO), 177.3 (quat., Gluα-CO) and 178.1 (quat., Gluγ-CO); m / z (FAB +) 330.1666 (MH ⁺ .C ₁₄ H ₂₄ N ₃ O ₆ requires 330.1666).

In summary, we synthesized a new alanogue of GPE. In five of these analogs, the trans-conformation for glypro ^* binding is due to the presence of a hydrophobic alkyl group at C-2 on proline (compound 1-5) or to the 2-position of proline and the nitrogen of glutamate. Between spirolactam bridges (compounds 35 and 36). In contrast, dimethylation at C-5 on proline destabilizes the trans conformation (compounds 49 and 50) provided in an increased population of cis conformers. These GP ^* E mimetics are valuable tools for providing information about the remaining effects of proline on the biological activity of the parent peptide GPE.
Example 2: Synthesis of analog 60 (Glycyl-trans-4-hydroxy-L-prolyl-L-glutamic acid (GhypE))

Experimental Flash chromatography was performed using Scharlau 60 (40-60 μm mesh) silica gel. Analytical thin layer chromatography was performed on 0.20 mm pre-coated silica gel plates (ALUGRAM® SIL G / UV ₂₅₄ ) and the compounds were processed using UV fluorescence or permanganese in alkaline solution. Plates soaked in potassium acid were visualized by heating.
The melting point at Celsius temperature (° C.) is determined and not corrected with an electrothermal® melting point instrument.
Light intensity is measured at 20 ° C. with a Perkin Elmer 341 polarimeter using 10 cm path length cells and is obtained in units of 10 ⁻¹ degcm ² g ⁻¹ . Samples were prepared in the solvents indicated at specific concentrations (measured in g / 100 cm ³ ).

NMR spectra were recorded at ambient temperature on a Bruker AVANCE DRX400 ( ¹ H, 400 MHz; ¹³ C, 100 MHz) or Bruker AVANCE 300 ( ¹ H, 300 MHz; ¹³ C, 75 MHz) spectrometer. ^{For 1} H NMR data, chemical shifts are listed in parts per million downfield from SiMe ₄ and sequentially, position (δ _H ), relative integral, variety (s = singlet, d = doublet, Record as t = triplet, dd = doublet, m = multiplet, br = broad), coupling constant (J / Hz) and configuration. ^{For 13} C NMR data, chemical shifts are stated in parts per million in relation to CDCl ₃ and sequentially as position (δc), degree of mating as determined by DEPT experiments, and configuration Record. ¹³ H NMR spectra were referenced internally using SiMe ₄ (δ0.00) or CDCl ₃ (δ7.26). ¹³ H NMR spectra were referenced internally on CDS (3- (trimethylsilyl) -1-propanesulfonic acid, sodium salt) using CDCl ₃ (δ77.0) or externally. When two sets of peaks occur in the NMR spectrum due to different agents surrounding the glycine-prolinamide bond, the chemical shift for the small cis conformer is an asterisk ( ^* ).

  Accurate mass measurements were recorded on a VG-70SE mass spectrometer. Hexane and dichloromethane were distilled before use. Methanol was dried using magnesium rotation and iodine and distilled under nitrogen. Triethylamine was dried over calcium hydride and distilled under nitrogen. L-glutamic acid dibenzyl ester p-toluenesulfonate 4 was obtained from Bachem. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BoPCl) (97%) was obtained from Fluka. Trans-4-hydroxy-L-proline 1 and 10% palladium on activated carbon were obtained from Aldrich Chemical Company.

2.1 N-benzyloxycarbonylglycyl-trans-4-hydroxy-L-proline 3:
Trans-4-hydroxy-L-proline 1 (0.28 g, 2.13 mmol) and sodium bicarbonate (0.18 g, 2.13 mmol) were dissolved in water (3 cm ³ ) at room temperature. N-benzyloxycarbonyl-glycyl-N-hydroxysuccinimide 2 (0.44 g, 1.44 mmol) was added dropwise over a period of 5 minutes. The solution was stirred for 2.5 and then acidified to pH 1 with concentrated hydrochloric acid (38%). The solution was placed in the refrigerator overnight. Ethyl acetate (2 × 20 cm ³ ) was added and the resulting organic layer was dried (MgSO ₄ ), filtered and evaporated under reduced pressure to produce a light pink foam, which was a flash column Purification by chromatography (10% methanol / methylene chloride) produced acid 3 (0.32 g, 70%) as a clear foam. Acid 3 was shown by ¹ H NMR analysis to be a 77:23 trans: cis mixture of conformers (ratio signals at δ 4.33-4.38 and 4.52-4.54). Large and small conformers pro-α-H, given individually): [α] _D 75.5 (c 0.08 in MeOH); δ _H (300 MHz; CD ₃ OD) 1.89-1.94 (0.77H, m, Proβ-H _A H _B ), 2.08-2.14 (1H, m, Proβ-H _A H _B ), 2.14-2.16 ^* (0.23H, m, Proβ-H _A H _B ) , 3.17-3.20 (1H, m, Proδ-H _A H _B ), 3.53-3.57 (1H, m, Proδ-H _A H _B ), 3.73-3.78 (1H, d, J 17.1, Glyα-H _A H _B ), 3.87-3.93 (1H, d, J 17.1, Glyα-H _A H _B ), 4.20-4.30 ^* (0.23H, m, Proγ-H), 4.33-4.38 (1.54H, m, Proγ-H and Proα -H), 4.52-4.54 ^* (0.23H, t, J 7.0, Proα-H) 4.94 (2H, s, OCH ₂ Ph) and 7.11-7.23 (5H, m, Ph); δ _C (75 MHz, CD ₃ OD) 39.0 (CH ₂ , Proβ-C), 41.3 ^* (CH ₂ , Proβ-C), 44.2 ^* (CH ₂ , Proδ-C), 44.8 (CH ₂ , Proδ-C), 55.9 (CH ₂ , Glyα-C), 56.2 ^* (CH ₂ , Gl yα-C), 59.5 ^* (CH, Proα-C), 60.1 (CH, Proα-C), 68.5 (CH ₂ , OCH ₂ Ph), 68.7 ^* (CH ₂ , OCH ₂ Ph), 69.7 ^* (CH, Proγ-C), 71.7 (CH, Proγ-C), 129.6 (CH, Ph), 129.7 (CH, Ph), 130.2 (CH, Ph), 138.9 (quat., Ph), 159.6 (quat., NCO ₂ ), 170.1 (quat., GlyCO), 171.6 ^* (quat., GlyCO), 175.9 ^* (quat., CO ₂ H) and 176.3 (quat., CO ₂ H) ,; m / z (FAB +) 323.1240 (M ⁺ .C ₁₆ H ₂₀ N ₂ O ₅ requires 320.1243).

2.2 Dibenzyl-N-benzyloxycarbonylglycyl-trans-4-hydroxy-L-prolyl-L-glutamate 5:
Acid 3 (0.70 g, 2.18 mmol), L-glutamic acid dibenzyl ester p-toluenesulfonate 4 (1.19 g, 2.40 mmol), 1-hydroxybenzotriazole hydrate (0.37 g, 2.40 mmol) And 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.46 g, 2.40 mmol) were dissolved in dichloromethane (60 cm ³ ) under nitrogen and cooled to 0 ° C. Triethylamine (0.60 cm ³ , 4.30 mmol) was added dropwise over a period of 5 minutes. The solution was stirred for 1.5 hours, then warmed to room temperature and stirred overnight. Dichloromethane (40 cm ³ ) was added and the organic layer was subsequently washed with saturated sodium bicarbonate (25 cm ³ ) and aqueous 2M citric acid (25 cm ³ ), then dried (MgSO ₄ ), filtered and under reduced pressure Evaporate to form a white sticky solid that is purified by flash column chromatography (10% methanol / ethyl acetate) to provide sufficient protected tripeptide 5 (0.97 g, 71%) Prepared as a white solid. Tripeptide 5 was shown by ¹ H NMR analysis to be a 91: 9 trans: cis mixture of conformers (the ratio was determined from a wide range of signal intensities at δ5.89 and 6.51). Large and small conformers of glyc-NH, given individually): m. p. [Α] _D 57.7 (c 0.07 in MeOH); δ _H (300 MHz; CDCl ₃ ; Me ₄ Si) 2.08-2.18 (4H, m, Gluβ-H ₂ , and Proβ-H ₂ ) , 2.44-2.46 (2H, m, Gluγ-H ₂ ), 3.43-3.62 (2H, m, Proδ-H ₂ ), 3.75-4.03 (2H, m, Glyα-H ₂ ), 4.40-4.50 (1H, m , Gluα-H), 4.53-4.56 (2H, m, Proα-H and Proγ-H), 5.04 (2H, s, OCH ₂ Ph), 5.05 (2H, s, OCH ₂ Ph), 5.10 (2H, s , OCH ₂ Ph), 5.89 (0.91 H, br s, GlyNH), 6.51 ^* (0.09H, br s, GlyNH), 7.30-7.40 (15H, m, 3 x Ph), 7.51 (0.91H, d, J 7.4, GluNH) and 7.64 ^* (0.09H, br s, GluNH); δ _C (75 MHz, CDCl ₃ ) 27.3 (CH ₂ , Gluβ-C), 30.4 (CH ₂ , Proβ-C), 30.7 ^* (CH ₂ , Proβ-C), 37.5 (CH ₂ , Gluγ-C), 43.7 (CH ₂ , Glyα-C), 52.2 (CH, Gluα-C), 54.8 (CH ₂ , Proδ-C), 55.8 ^* (CH ₂ , Proδ-C), 59.0 ^* (CH, Proα-C), 59.3 (CH, Proα-C), 66.8 (CH ₂ , OCH ₂ Ph), 67.3 (CH ₂ , OCH ₂ Ph) ,, 67.7 (CH ₂ , OCH ₂ Ph), 68.7 ^* (CH, Proγ-C), 70.5 (CH, Proγ-C), 128.3 (CH, Ph), 128.5 (CH, Ph) ,, 128.6 (CH, Ph), 128.8 ( CH, Ph), 128.9 (CH, Ph), 135.6 (quat., Ph) ,, 13 5.9 (quat., Ph) ,, 136.1 (quat., Ph), 157.0 (quat., NCO ₂ ), 168.7 (quat., GlyCO), 169.6 ^* (quat., GlyCO), 171.8 (quat., ProCON ) And 173.2 (quat., Gluγ-CO and Gluα-CO); m / z (FAB +) 632.2613 (M ⁺ .C ₃₄ H ₃₈ N ₃ O ₉ requires 632.2608).

2.3 Glycyl-trans-4-hydroxy-L-prolyl-glutamic acid (GHypE) 60:
A mixture of protected tripeptide 5 (0.50 g, 0.79 mmol) and 10% palladium on activated carbon in 20% water / methanol (100 cm ³ ) was stirred at room temperature for 20 hours under an atmosphere of hydrogen. The solution was filtered through a Celite® pad, washed with methanol (60 cm ³ ) and the filtrate evaporated to form a clear gum. The rubber was dissolved in methanol (30 cm ³ ) and refiltered through a Celit® pad. The solution was evaporated to dryness to form a clear gum. The rubber was placed in a vacuum line for 15 hours and then triturated with anhydrous diethyl ether to form GHypE (0.20 g, 80%) as a white solid. GHypE was shown by ¹³ C NMR analysis to be a 80:20 trans: cis mixture of conformers (ratio measured from signal intensity at δ70.2 and 68.5, large and small Conformer pro-C was given individually); [α] _D 58.1 (c 0.1 in MeOH); δ _H (300 MHz; D ₂ O) 1.96-2.23 (4H, m, Gluβ- H ₂ , and Proβ-H ₂ ), 2.48-2.56 (2H, m, Gluγ-H ₂ ), 3.62 (2H, d, J 10.0, Glyα-H ₂ ), 3.80 (1H, dd, J 8.8 and 4.9, Gluα-H), 4.08 (1H, dd, J 10.4 and 2.6, Proα-H), 4.30-4.35 (2H, m, Proδ-H ₂ ) and 4.63-4.69 (1 H, m, Proγ-H); δ _C (75 MHz, D ₂ O) 29.2 (CH ₂ , Gluβ-C), 31.7 (CH ₂ , Proβ-C), 32.3 ^* (CH ₂ , Proβ-C), 37.6 (CH ₂ , Gluγ-C), 40.0 ^* (CH ₂ , Glyα-C), 41.0 (CH ₂ , Glyα-C), 54.8 (CH ₂ , Proδ-C), 55.3 (CH, Gluα-C), 59.1 ^* (CH, Proα-C), 59.7 (CH, Proα-C), 68.5 ^* (CH, Proγ-C), 70.2 (CH, Proγ-C), 166.4 (quat., GlyCO), 167.1 ^* (quat., GlyCO), 173.0 ^* (quat. , P roCON), 173.4 (quat., ProCON), 177.9 (quat., Gluα-CO) and 179.2 (quat., Gluγ-CO); m / z (FAB +) 318.1329 (MH ⁺ .C ₁₇ H ₂₀ NO ₅ 318.1342 is required).

Example 3 Synthesis of Analog 61 (Glycyl-L-2-pyroglutamyl-L-glutamic acid hydrochloride (GpyroE / HCl) Description of a general experiment as in Example 2 above. L-pyroglutamic acid Acquired from Acros Organic L-glutamic acid dibenzyl ester p-toluenesulfonate was obtained from Bachem, and t-butyl acetate was obtained from Lancaster Chemical Co. Bis (2- Oxo-3-oxazolidinyl) phosphinic chloride (97%) was obtained from Fluka and 10% palladium on activated carbon was obtained from Aldrich Chemical Company.

3.1 t-Butyl-L-pyroglutamate 2:
70% perchloric acid (0.85 cm ³ , 9.90 mmol) was added to a solution of L-pyroglutamic acid 1 (1.70 g, 13.20 mmol) in t-butyl acetate (17.0 cm ³ , 126.20 mmol). Was added dropwise at room temperature over a period of 5 minutes. The solution was stirred for 20 hours and then solid sodium carbonate was slowly added dropwise until pH 7 was reached. The aqueous layer was extracted with diethyl ether (40 cm ³ ) and ethyl acetate (40 cm ³ ). The combined extracts are dried (MgSO ₄ ), filtered and evaporated under reduced pressure to produce a clear oil that is purified by flash column chromatography (ethyl acetate) to give ester 2 (1 .33 g, 55%) was produced as a white solid. : Mp101-103 ° C. , Lit. ¹ mp 102 ° C .; [α] _D + 9.7 (c 0.14 in MeOH), lit. ¹ +11 (c 3 in MeOH); δ _H (200 MHz; CD ₃ OD) 1.44 [9H, s C (CH ₃ ) ₃ ], 2.30-2.39 (4H, m, pyro β-H ₂ and pyro γ-H ₂ ), 4.10-4.13 (1H, m, pyro α-H) and 6.71 (1H, br s, NH); δ _C (50 MHz; CD ₃ OD) 24.8 (CH ₂ , pyro β-C), 27.9 [CH ₃ , C (CH ₃ ) ₃ ], 29.4 (CH ₂ , pyro γ-C), 56.1 (CH, pyro α- C), 82.2 [quat., C (CH ₃ ) ₃ ], 171.2 (quat. Pyro-CO) and 178.3 (quat., Pyro-CONH).

3.2 t-Butyl-N-benzyloxycarbonylglycyl-L-pyroglutamate 4:
Ester 2 (0.61 g, 3.30 mmol) was dissolved in tetrahydrofuran (20 cm ³ ) under nitrogen and cooled to -78 ° C. Rutium hexamethyldisilazide (1.06M, 3.30 cm ³ , 3.50 mmol) was added dropwise over a period of 5 minutes. The solution was stirred for 15 minutes, then N-benzyloxycarbonyl-glycyl-N-hydroxysuccinimide ⁴ 3 (DC89.52) (1.23 g, 4.01 mmol) in tetrahydrofuran (30 cm ³ ) was added and The solution was stirred for 45 minutes. Water (50 cm ³ ) was added and the reaction was warmed to room temperature. The resulting aqueous solution is extracted with ethyl acetate (2 × 50 cm ³ ), dried (MgSO ₄ ), filtered and evaporated under reduced pressure to form a white sticky solid that is Purification by flash column chromatography (ethyl acetate) produced carbamate 4 (1.04 g, 83%) as a white solid: mp 132-135 ° C., lit ² . [Α] _D 45.5 (c 0.1 in EtOH), lit. ² 61.7 (c 1.01 in EtOH) ,; δ _H (300 MHz; CDCl ₃ ; Me ₄ Si) 1.47 [9 H, s , C (CH ₃ ) ₃ ], 2.08-2.13 (1H, m, Pyroβ-H _A H _B ), 2.31-2.39 (1H, m, Pyroβ-H _A H _B ), 2.56 (1H, ddd, J 3.1, 9.2 and 12.3, Pyroγ-H _A H _B ), 2.64-2.73 (1H, m, Pyroγ-H _A H _B ), 4.48 (1H, dd, J 4.8 and 19.6, Glyα-H _A H _B ), 4.61-4.71 (2H, m, Glyα-H _A H _B and Pyroα-H), 5.08 (2H, s, OCH ₂ Ph), 5.47 (1H, s br, GlyNH) and 7.33 (5H, s, Ph); δ _C ( 75 MHz, CDCl ₃ ) 22.3 (CH ₂ , Pyroβ-C), 28.2 [CH ₃ , C (CH ₃ ) ₃ ], 31.8 (CH ₂ , Pyroγ-C), 46.7 (CH ₂ , Glyα-C), 58.7 (CH, Pyroα-C), 67.3 (CH ₂ , OCH ₂ Ph), 83.1 [quat., C (CH ₃ ) ₃ ], 128.4 (CH, Ph), 128.6 (CH, Ph), 129.0 (CH, Ph ), 136.8 (quat., Ph), 156.6 (quat., NCO ₂ ), 170.1 (quat., GlyCON), 170.5 (quat., PyroCONH) and 175.1 (quat., PyroCO); m / z (FAB +) 377.1698 (MH ⁺ .C ₁₉ H ₂₅ N ₂ O ₆ requires 377.1713).

3.3 t-Butyl-N-benzyloxycarbonylglycyl-L-pyroglutamic acid 5:
Carbamate 4 (0.74 g, 1.97 mmol) was dissolved in dichloromethane (12 cm ³ ) under nitrogen and cooled to 0 ° C. Trifluoroacetic acid (6 cm ³ ) was added dropwise over a period of 5 minutes and the solution was stirred for 1.5 hours. The solution was evaporated under reduced pressure to give an orange gum which was dissolved in toluene and evaporated under reduced pressure (2 × 10 cm ³ ) to give the grand acid 5 as an orange gum. . The material was used without further purification.

3.4 Dibenzyl-N-benzyloxycarbonylglycyl-L-pyroglutamyl-L-glutamate 7:
Anhydrous acid 5 (0.63 g, 1.97 mmol), L-glutamic acid dibenzyl ester p-toluenesulfonate 6 (1.18 g, 2.36 mmol) and bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0. 60 g, 2.36 mmol) was dissolved in dichloromethane (20 cm ³ ) under nitrogen and cooled to 0 ° C. Triethylamine (0.64 cm ³ , 4.60 mmol) was added dropwise over a period of 5 minutes. The solution was stirred for 1.5 hours, then warmed to room temperature and stirred overnight. Dichloromethane (40 cm ³ ) is added and the organic layer is subsequently washed with saturated sodium bicarbonate solution (30 cm ³ ) and aqueous 2M citric acid (30 cm ³ ), then dried (MgSO ₄ ), filtered and Evaporated under reduced pressure to form a white sticky solid. Purification by flash column chromatography (ethyl acetate) provided a white solid. This white solid was washed with diethyl ether (2 × 15 cm ³ ) and filtered to produce fully protected tripeptide 7 (0.98 g, 78%) as a white solid. : M. p. 109-111 ° C; [α] _D 55.5 (c 0.25 in MeOH); δ _H (300 MHz; CDCl ₃ ; Me ₄ Si) 1.80-2.24 (7H, m, Gluβ-H ₂ , Gluγ-H ₂ , Pyroβ -H ₂ and Pyroγ-H _A H _B ), 2.74-2.80 (1H, m, Pyroγ-H _A H _B ), 4.38 (1H, dd, J 4.2 and 19.5, Glyα-H _A H _B ), 4.58-4.68 (3H, m, Glyα-H _A H _B , Proα-H and Gluα-H), 5.08-5.19 (6H, m, 3 x OCH ₂ Ph), 5.38 (1H, s br, GlyNH), 6.84 (1H, d, J 7.7, GluNH) and 7.30 (15H, s, 3 x Ph); δc (75 MHz, CDCl ₃ ) 22.2 (CH ₂ , Pyroβ-C), 26.6 (CH ₂ , Gluβ-C), 30.0 (CH ₂ , Gluγ-C), 33.9 (CH ₂ , Pyroγ-C), 46.2 (CH ₂ , Glyα-C), 52.0 (CH, Gluα-C), 58.6 (CH, Pyroα-C), 66.7 (CH ₂ , OCH ₂ Ph), 66.9 (CH ₂ , OCH ₂ Ph), 67.4 (CH ₂ , OCH ₂ Ph), 128.0 (CH, Ph), 128.1 (CH, Ph), 128.2 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 128.6 (CH, Ph), 135.0 (quat., Ph), 135.3 (quat., Ph), 135.6 (quat., Ph), 156.3 (quat., NCO ₂ ), 170.2 (quat., GlyCO), 170.6 (quat., PyroCO), 171.2 (quat., PyroCO), 172.9 (quat., GluCO) and 175.2 (quat., GluCO); m / z (FAB +) 630.2460 (MH ⁺ .C ₃₄ H ₃₆ N ₃ O ₉ requires 630.2452).

3.5 Glycyl-L-pyroglutamyl-L-glutamic acid hydrochloride (GpyroE.HCl) 61:
Protected tripeptide 7 (0.45 g, 0.80 mmol) in 20% water / tetrahydrofuran (60 cm ³ ), concentrated hydrochloric acid (38%, 0.13 cm ³ ) and 10% palladium on activated carbon (0.08 g, .0. (08 mmol) was stirred at room temperature under an atmosphere of hydrogen for 4 hours. The solution was filtered through a Celite® pad, washed with 20% water / tetrahydrofuran (2 × 60 cm ³ ) and the filtrate was evaporated to dryness to give GpyroE. HCl (0.19 g, 74%) was formed as a solid. GpyroE. HCl was shown to be exclusively the trans conformer by ¹ H and ¹³ C NMR analysis ⁵ : mp 42-44 ° C. [α] _D 57.3 (c 0.09 in MeOH); δ _H (400 MHz; D ₂ O) 2.07-2.14 (1H, m, Gluβ-H _A H _B ), 2.20-2.25 (1H, m, Gluβ- H _A H _B ), 2.30-2.34 (1H, m, Pyroβ-H _A H _B ), 2.60-2.65 (3H, m, Pyroβ-H _A H _B and Gluγ-H ₂ ), 2.74-2.89 (2H, m , Pyroγ-H ₂ ), 4.48-4.59 (3H, Glyα-H ₂ and Gluα-H) and 4.93-4.95 (1H, m, Pyroα-H); δ _C (75 MHz, D ₂ O) 22.5 (CH ₂ , Pyroβ-C), 26.4 (CH ₂ , Gluβ-C), 30.5 (CH ₂ , Gluγ-C), 31.8 (CH ₂ , Pyroγ-C), 44.3 (CH ₂ , Glyα-C), 52.7 (CH, Gluα-C), 59.3 (CH, Pyroα-C), 168.5 (quat., GlyCO), 173.6 (quat., PyroCO), 175.2 (quat., PyroCO), 177.6 (quat., GluCO) and 178.9 (quat. , GluCO); m / z (FAB +) 316.1138 (MH ⁺ .C ₁₂ H ₁₈ N ₃ O ₇ requires 316.1145).

Reference: 1. Besson et al. Chem. Eur.j., 2000, 6, 949. 2. Johnson et al. J. Med. Chem. 1985, 28, 1596. 3. Li et al. Synth. Comm. 1995 , 25, 4045. Succinimide 3 was prepared according to the experimental procedure given in the synthesis of analog 66 (G (D, L) PipE). 5. Galardyet al. Int. J. Pept. Protein Res 1982, 19, 123.
Example 4: Synthesis of analog 62 (glycyl-L-homopropyl-L-glutamic acid (GHomoPE)) General experimental details are described in Example 2 above. N-benzyloxycarbonyl-glycine, 10% palladium on activated carbon and bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BoPCl, 97%) were obtained from Aldrich Chemco. L-glutamate dibenzyl p-toluenesulfonate was obtained from Bachem.

Scheme 1: Reagents, Status and Yield: (i) Thionyl chloride, CH ₃ OH, RT, N ₂ , 24 h (97%); (ii) Et ₃ N, BoPCl, CH ₂ Cl ₂ , RT, N ₂ , 19.5 h (78%); (iii) Dioxane, 1 M NaOH (aq), RT, 16 h (93%); (iv) Et ₃ N, BoPCl, CH ₂ Cl ₂ , RT, N ₂ , 17.5 h (74%); (v) H 2, 10% Pd / C, 10% H 2 O / MeOH, RT, 20 h (93%).

4.1 Methyl L-pyrrolidin-2-yl acetate hydrochloride ¹ 2:
An ice-cooled solution (0.25 g, 1.50 mmol) of a hydrochloride-cooled hydrochloride of hydrochloride 1 in an atmosphere of nitrogen in dry methanol (3 cm ³ ) was added to thionyl chloride (0. 13 cm ³ , 1.78 mmol). The solution was stirred overnight and the solvent was removed under reduced pressure. The resulting light green gum was purified by flash column chromatography (10% methanol / dichloromethane) to provide hydrochloride 2 (0.26 g, 97%) as a pale yellow, which was Solidified to a semi-solid: [α] _D +44.3 (c 0.05 in MeOH): δ _H (300 MHz; CDCl ₃ ) 1.73-1.79 (1H, m, Proγ-H _A H _B ), 2.00-2.11 (2H , m, Proγ-H _A H _B and Proβ-H _A H _B ), 2.26-2.31 (1H, m, Proβ-H _A H _B ), 2.86 (1 H, dd, J 6.9, 17.2, CH _A H _B CO ₂ ), 3.22 (1 H, dd, J 6.9, 17.2, CH _A H _B CO ₂ ), 3.29 (2 H, t, J 6.8, Proδ-H ₂ ), 3.44 (3 H, s, OCH ₃ ) , 3.92 (1 H, m, Proα-H) and 8.15 (2 H, br s, NCH); δ _C (75 MHz; CDCl ₃ ) 23.8 (CH ₂ , Proγ-C), 30.8 (CH ₂ , Proβ- C), 36.4 (CH ₂ , CH ₂ CO ₂ ), 45.3 (CH ₂ , Proδ-C), 52.6 (CH ₃ , CH ₃ O), 56.3 (CH, Proα-C) and 171.0 (quat., CO) m / z (FAB +) 323.1737 [M ₂ .H ³⁵ Cl.H ⁺ ] (C ₇ H ₁₃ ³⁵ ClNO ₂ ) _2. H ³⁵ Cl.H requires 323.1738].

4.2 Methyl-N-benzyloxycarbonyl-glycyl-L-pyrrolidin-2-yl acetate 4:
Dry triethylamine (0.70 cm ³ , 5.00 mmol) was added to hydrochloride 2 (0.26 g, 1.45 mmol) and N-benzyloxycarbonyl-glycine 3 (0.32 g, 1.50 mmol) in dry dichloromethane (8 cm ³ ). 53 mmol) was added dropwise at 0 ° C. under a nitrogen atmosphere and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (0.39 g, 1.53 mmol) was added and the solution was stirred for 2 hours, warmed to room temperature and stirred for an additional 20 hours. Dichloromethane (40 cm ³ ) was added and the solution was subsequently washed with 0.5 M aqueous hydrochloric acid (2 × 10 cm ³ ) and saturated aqueous sodium bicarbonate (2 × 10 cm ³ ), dried (MgSO ₄ ) and filtered. And evaporated in vacuo to give a bright orange gum. The remaining purification obtained by flash column chromatography (ethyl acetate) produced ester 4 (0.38 g, 78%) as a colorless oil. Ester 4 was shown by ¹ H NMR analysis to be a 85:15 trans: cis mixture of conformers (ratio signals at δ 4.21-4.30 and 4.37-4.47). The small and large conformer pro-α-H, measured from the intensity of, were given individually: [α] _D 36.0 (c 0.13 in MeOH); δ _H (300 MHz; CDCl ₃ ) 1.81- 2.07 (4 H, m, Proγ-H ₂ and Proβ-H ₂ ), 2.36 (0.85 H, dd, J 9.2 and 15.5, CH _A H _B CO ₂ ), 2.47 * (0.15 H, d, J 10.1, CH _A H _B CO ₂ ), 2.60 * (0.15 H, d, J 10.1, CH _A H _B CO ₂ ), 2.92 (0.85 H, dd, J 9.2 and 15.5, CH _A H _B CO ₂ ), 3.43-3.44 ( 2 H, m, Proδ-H ₂ ), 3.66 (2.55 H, s, OCH ₃ ), 3.70 * (0.45 H, s, OCH ₃ ), 3.92 (1.7 H, d, J 4.1, Glyα-H ₂ ), 3.95 * (0.3 H, m, Glyα-H ₂ ), 4.21-4.30 * (0.15 H, m, Proα-H), 4.37-4.47 (0.85 H, m, Proα-H), 5.11 (2 H, s, OCH ₂ Ph), 5.76 (1 H, br s, GlyNH) and 7.27-7.36 (5 H, m, Ph); δ _c (75 MHz; CDCl ₃ ) 21.4 * (CH ₂ , Proγ-C), 23.9 ( CH ₂ , Proγ-C), 30.0 (CH ₂ , Proβ-C), 31.5 * (CH ₂ , Proβ-C), 37.5 (CH ₂ , CH ₂ CO ₂ ), 43.4 * (CH ₂ , Proδ-C), 43.8 (CH ₂ , Proδ-C), 46.0 (CH ₂ , Glyα-C), 51.9 (CH ₃ , OCH ₃ ), 52.2 * (CH ₃ , OCH ₃ ), 53.9 * (CH, Proα-C), 54.7 (CH, Proα-C) 67.1 (CH ₂ , OCH ₂ Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.7 (CH, Ph), 136.4 (quat., Ph), 156.2 (quat. , NCO), 166.6 (quat., GlyCON) and 171.6 (quat., CO ₂ CH ₃ ); m / z (EI +) 334.1532 (M ⁺ .C ₁₇ H ₂₂ N ₂ O ₅ requires 334.1529) .

4.3 N-benzyloxycarbonyl-glycyl-L-pyrrolidin-2-ylacetic acid 5:
To a solution of (methyl ester 4 (0.36 g, 1.08 mmol) in dioxane (10 cm ³ ), 1M aqueous NaOH (5 cm ³ , 5.00 mmol) was added dropwise and the mixture was stirred at room temperature for 20 hours. The solution was acidified with 1M HCl and evaporated in vacuo The resulting aqueous layer was extracted with dichloromethane (2 × 25 cm ³ ), dried (NaSO ₄ ), filtered and evaporated in vacuo to clear rubber to form such, it upright, the acid 5 (0.32g, 93%), solidified as a clear gum, which was used without further purification. the acid 5, ¹ H NMR analysis Showed a 76:24 trans: cis mixture of conformers (ratio measured from broad signal intensities at δ5.97 and 6.10; large and small conformers Gri-NH, individual In, _{gave: [α] D 33.3 (c} 0.03 in _{MeOH); δ H (300 MHz} ; CDCl 3) 1.81-2.04 (4 H, m, Proγ-H 2 and _{Proβ-H 2), 2.38 (} 0.76 H, dd, J 8.9, 15.7, CH _A H _B CO ₂ ), 2.45 * (0.24 H, d, J 10.6, CH _A H _B CO ₂ ), 2.58 * (0.24 H, d, J 10.6, CH _A H _B CO ₂ ), 2.92 (0.76 H, dd, J 3.8 and 15.7, CH _A H _B CO ₂ ), 3.36-3.47 (2 H, m, Proδ-H ₂ ), 3.92 (1.52 H, m, Glyα-H ₂ ), 3.95 * (0.48 H, m, Glyα-H ₂ ), 4.21-4.30 * (0.76 H, m, Proα-H), 4.37-4.69 (0.24 H, m, Proα-H), 5.11 (2 H , s, OCH ₂ Ph), 5.97 (0.76 H, br s, GlyNH), 6.10 * (0.24 H, br s, GlyNH), 7.27-7.35 (5 H, m, Ph) and 7.72 (1 H, br s , CO ₂ H); δ _c (75 MHz; CDCl ₃ ) 21.4 * (CH ₂ , Proγ-C), 24.0 (CH ₂ , Proγ-C), 30.4 (CH ₂ , Proβ-C), 31.7 * (CH ₂ , Proβ-C), 37.9 (CH ₂ , CH ₂ CO ₂ ), 39.1 * (CH ₂ , CH ₂ CO ₂ ), 43.5 * (CH ₂ , Proδ-C), 43.9 (CH ₂ , Proδ-C) , 46.4 (CH ₂ , Glyα-C), 53.8 * (CH, Proα-C), 54.1 (CH, Proα-C) 67.3 (CH ₂ , OCH ₂ Ph), 128.3 (CH, Ph), 128.4 (CH, Ph), 128.8 (CH, Ph), 136.6 (quat., Ph), 156.8 (quat., NCO), 167 .8 (quat., GlyCON), 175.0 * (quat., CO ₂ H) and 175.3 (quat., CO ₂ H); m / z (EI ⁺ ) 320.1369 (M ⁺ .C ₁₆ H ₂₀ N ₂ O ₅ Requires 320.1372).

4.4 Dibenzyl N-benzyloxycarbonyl-glycyl-L-pyrrolidin-2-yl-acetyl-L-glutamate 7:
Dry triethylamine (0.38 cm ³ , 2.73 mmol) was added to acid 6 (0.29 g, 0.91 mmol) and L-glutamic acid dibenzyl ester p-toluenesulfonate 6 (0.50 g, in dry dichloromethane (50 cm ³ )). Was added at 0 ° C. under a nitrogen atmosphere and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BoPCl, 97%) (0.25 g, 1.00 mmol) was added and the solution was stirred for 2 hours, warmed to room temperature and further stirred for 20 hours. The solution is subsequently washed with 0.5M aqueous hydrochloric acid (10 cm ³ ) and saturated aqueous sodium hydrogen carbonate (10 cm ³ ), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo to give a bright orange A rubber was formed. The remaining purification obtained by flash column chromatography (ethyl acetate) produced fully protected tripeptide 7 (0.42 g, 74%) as a clear oil: [α] _D 26.6 (c 0.13 in MeOH) ); δ _H (300 MHz; CDCl ₃ ) 1.95-2.47 (9 H, m, Proγ-H ₂ , Proβ-H ₂ , Gluβ-H ₂ , Gluγ-H ₂ and CH _A H _B CO ₂ ), 2.75 ( 1 H, dd, J 3.5 and 13.9, CH _A H _B CO ₂ ), 3.35-3.42 (2 H, m, Proδ-H ₂ ), 3.94 (2 H, d, J 4.1, Glyα-H ₂ ), 4.33 -4.34 (1 H, m, Gluα-H), 4.63-4.67 (1 H, m, Proα-H), 5.11-5.26 (6 H, s, 3 x OCH ₂ Ph), 5.80 (1 H, br s , GlyNH), 7.00 (1 H, d, J 7.8, GluNH) and 7.31-7.35 (15 H, m, 3 x Ph); δ _c (75 MHz; CDCl ₃ ) 23.8 (CH ₂ , Proγ-C), 27.1 (CH ₂ , Gluβ-C), 30.0 (CH ₂ , Gluγ-C), 30.3 (CH ₂ , Proβ-C), 40.2 (CH ₂ , CH ₂ CO ₂ ), 43.5 (CH ₂ , Proδ-C) , 45.9 (CH ₂ , Glyα-C), 51.6 (CH, Gluα-C), 55.5 (CH, Proα-C), 66.4 (CH ₂ , OCH ₂ Ph), 66.8 (CH ₂ , OCH ₂ Ph), 67.2 (CH ₂ , OCH ₂ Ph), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 136.4 (quat., Ph) , 156.2 (quat., NCO ₂ ), 166.9 (quat., GlyCON), 170.7 (quat., ProCON), 171.5 (quat., Gluα-CO) and 172.4 (quat., Gluγ-CO); m / z ( FAB +) 630.2809 (MH ⁺ .C ₃₅ H ₄₀ N ₃ O ₈ requires 630.2815).

4.5 Glycyl-L-homoproyl-L-glutamic acid (GHomoPE) 62:
Protected tripeptide 7 (0.39 g, 0.61 mmol) in 10% water / methanol (40 cm ³ ) and 10 wt% palladium on activated carbon (0.007 g, 0.07 mmol) at room temperature under nitrogen atmosphere. Stir for 2 hours. The solution was filtered through a Celite® pad, washed with methanol (2 × 30 cm ³ ) and the filtrate was evaporated to dryness to give a clear gum. The gum was placed under vacuum for 30 minutes and then triturated with anhydrous diethyl ether to form GHomoPE (0.19 g, 93%) as a white solid. GHomoPE was shown to be an 80:20 trans: cis mixture of conformers by ¹ H NMR analysis (ratio measured from signal intensity at δ 4.08 and 4.11, large and small The conformer glu α-H was given individually: mp 48-50 ° C .; [α] _D 36.6 (c 0.06 in H ₂ O); δ _H (400 MHz; D ₂ O) 1.84-2.22 (6 H, m, Gluβ-H ₂ , Proβ-H ₂ and ProγH ₂ ), 2.41-2.62 (3.2 H, m, Gluγ-H ₂ , CH _A H _B CO ₂ and 2 x * CH ₂ CO ₂ ), 2.77 (0.8 H, dd, J 5.4 and 13.9, CH _A H _B CO ₂ ), 3.45-3.55 (1 H, m, Proδ-H _A H _B ), 3.51-3.60 (1 H, m, Proδ-H _A H _B ), 4.08 (1.6 H, q, J 16.9, Glyα-H ₂ ), 4.11 * (0.4 H, q, J 16.9, Glyα-H ₂ ), 4.34 (1 H, dd, J 8.4 and 13.7, Gluα -H) and 4.45-4.47 (1 H, m, Proα-H); δ _c (100 MHz; CDCl ₃ ) 21.1 * (CH ₂ , Proγ-C), 23.4 (CH ₂ , Proγ-C), 26.0 * (CH ₂ , Gluβ-C), 26.7 (CH ₂ , Gluβ-C), 29.5 (CH ₂ , Gluγ-C), 31.0 (CH ₂ , Proβ-C), 31.1 * (CH ₂ , Proβ- C), 39.1 (CH ₂ , Glyα-C), 39.9 * (CH ₂ , Glyα-C), 40.6 * (CH ₂ , CH ₂ CO ₂ ), 40.9 (CH ₂ , CH ₂ CO ₂ ), 46.2 * ( CH ₂ , Proδ-C), 46.4 (CH ₂ , Proδ-C), 53.8 (CH, Gluα-C), 54.2 * (CH, Gluα-C), 55.4 * (CH, Proα-C), 55.9 (CH , Proα-C), 165.4 (quat., GlyCON), 165.5 * (quat., GlyCON), 173.0 * (quat., ProCON), 173.6 (quat., ProCON), 177.2 (quat., Gluα-CO) and 178.1 (quat., Gluγ-CO); m / z (FAB +) 316.1503 (MH ⁺ . C ₁₃ H ₂₂ N ₃ O ₆ requires 316.1509).

Reference: 1. Wanner et al. Ger.Offen., 2000, 36pp, DE 98-19840611 19980905 (Chem Abstr., 132: 194656 AN 2000: 157915).
Example 5: Synthesis of Analog 63 (Glycyl-L-3,4-dehydroprolyl-L-glutamate trifluoroacetic acid (G-3,4-dehydroPE / TFA)) As described in Example 2. L-3,4-dehydroproline 1 was obtained from Fluka. L-glutamic acid di-t-butyl ester hydrochloride 4 was obtained from Bachem.

5.1 t-Butyloxycarbonylglycyl-L-3.4-dehydroproline 2:
3,4-Dehydroproline 1 (0.011 g, 0.097 mmol) and sodium bicarbonate (0.0082 g, 0.097 mmol) were dissolved in water (1 cm ³ ). A solution of Nt-butyloxycarbonylglycine N-hydroxysuccinimide 2 (0.024 g, 0.088 mmol) in dioxane (1 cm ³ ) was added dropwise and stirred overnight. The reaction mixture was diluted with water, acidified with solid citric acid and extracted with dichloromethane (3x). The combined organic layers were dried (MgSO ₄ ), filtered and the solvent removed to provide the grand acid 3 (0.0242 g, ˜100%) used without further purification. Acid 3 was shown by ¹ H NMR analysis to be a 75:25 trans: cis mixture of conformers (ratio, δ 5.21 and 5. 13 from the intensity of the pro-alpha proton: δ _H (300 MHz; CDCl ₃ ; Me ₄ Si) 1.41 ¹ [2.25H, s, C (CH ₃ ) ₃ ], 1.42 [6.75H, s, C (CH ₃ ) ₃ ], 3.83-4.08 (2H, m, Glyα-H ₂ ), 4.25-4.45 (2H, m, Proδ-H ₂ ), 5.13 ^* (0.25H, br d, J 2.8, Proα-H), 5.21 (0.75H, br d, J 3.1, Proα-H), 5.55-5.80 (2H, br m, OH and NH) and 5.86-6.02 (2H, m, Proβ-H and Proγ-H).

5.2 Di-t-butyl Nt-butyloxycarbonylglycyl-L-3,4-dehydroprolyl-L-glutamate 5:
Triethylamine (0.041 cm ^3, 0.291 mmol), and acid 3 in dichloromethane (3cm ³⁾ (0.0242g, approximately 0.097 mmol), L-glutamic acid di -t- butyl ester hydrochloride 4 (0.029 g, 0.097 mmol) and benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BoP) (0.043 g, 0.097 mmol). The solution is washed overnight with 2M aqueous hydrochloric acid, saturated sodium bicarbonate solution, dried (MgSO ₄ ), filtered and the solvent removed and chromatographed (SiO ₂ , hexane: ethyl acetate, 1: 1, then 1: 2) provided the purified oil (0.0622 g) to give protected tripeptide 5 (0.0310 g, 59%) as a colorless oil. 5 was shown by ¹ H NMR analysis to be a 79:21 trans: cis mixture of conformers (the ratios were δ5.07 and 5.17 for the large and small conformers, respectively). Measured from the intensity of the pro-alpha proton at −5.18: δ _H (300 MHz; CDCl ₃ ; Me ₄ Si) 1.45-1.48 [27H, m, 3 × C (CH ₃ ) ₃ ], 1.90-1.98 (1H , m, Gluβ-H _A H _B ), 2.06-2.14 (1H, m, Gluβ-H _A H _B ), 2.24-2.35 (2H, m, Gluγ-H ₂ ), 3.82 ^* (0.21H, br d, J 17.2, Glyα-H _A H _B ), 3.95 (1H, dd, J 17.1 and 2.0, Glyα-H and ^* Glyα-H _A H _B ), 4.04 (0.79H, dd, J 17.2 and 4.4, Glyα-H ), 4.30 (0.79H, dd, J 14.0 and 2.0, Proδ-H _A H _B ), 4.38-4.43 (2H, m, Proδ-H and Gluα-H), 4.54 ^* (0.21H, br d, J 15.2 , Proδ-H _A H _B ), 5.07 ^* (0.21H, br s, Proα-H), 5.17-5.18 (0.79H, m, Proα-H), 5.14 ^* (0.21H, br s, NH), 5.52 (0.79H, br s, NH), 5.88-6.03 (2H, m, Proβ-H and Proγ-H) and 7.11 (1H, d, J 7.6, NH).

5.3 Glycyl-L-3,4-dehydroprolyl-L-glutamic acid trifluoroacetic acid (G-3,4-dehydroPE) 63:
Dichloromethane (3 cm ³⁾ in the protective tripeptide 5 (0.0301g, 0.0575mmol) To a solution of the added and the solution trifluoroacetic acid (1.5 cm ^3), and stirred at room temperature for 3 hours. Volatiles were removed in vacuo and the remainder was purified by chromatography (C ₁₈ , water) and G-3,4-dehydroPE (0.0224 g, 94%) was obtained as a hygroscopic pale yellow solid. It was. G-dehydroPE was shown by ¹ H NMR analysis to be a 84:16 trans: cis mixture of conformers (the ratios were individually measured for the large and small conformers δ4.07 and Measured from the intensity of Gly α proton at 3.78: non-mp due to hygroscopic sample; [α] _D -139.5 (c 0.2 in MeOH); δ _H (300 MHz; D ₂ O) 2.02-2.14 (1H, m, Gluβ-H _A H _B ), 2.25-2.61 (1H, m, Gluβ-H _A H _B ), 2.53-2.61 (2H, m, Gluγ-H ₂ ), 3.78 ^* (0.16H, d , J 16.2 Glyα-H), 4.03 ^* (0.16H, d, J 16.5 Glyα-H), 4.07 (1.68H, s, Glyα-H ₂ ), 4.38-4.45 (1H, m, Gluα-H), 4.53 (1H, dd, J 9.3 and 5.1, Proδ-H ₂ ), 5.28-5.32 (1H, m, Proα-H), 5.95 (1H, dd, J 6.2 and 2.2, Proβ-H or Proγ-H) and 6.16 -6.19 (1H, m, Proβ-H or Proγ-H); δ _C (75 MHz; D ₂ O) 25.2 ^* (CH ₂ , Gluβ-C), 25.7 (CH ₂ , Gluβ-C), 29.8 (CH ₂ , Gluγ-C), 30.2 ^* (CH ₂ , Gluγ-C), 40.0 ^* (CH ₂ , Glyα-C), 40.2 ^* (CH ₂ , Glyα-C), 52.1 ^* (CH , Gluα-C), 52.3 (CH, Gluα-C), 53.3 (CH ₂ , Proδ-C), 54.5 ^* (CH ₂ , Proδ-C), 67.0 ^* (CH, Proα-C), 67.7 (CH, Proα-C), 124.3 (CH), 128.4 ^* (CH), 128.5 (CH), 165.4 (quat., GlyCO), 171.7 (quat., ProCO), 174.6 (quat., Proα-CO) and 177.0 (quat ., Proγ-CO) (CF ₃ CO ₂ H is not determined); m / z (FAB +) 300.1196 [MH (free base) ⁺ . C ₁₂ H ₁₈ N ₃ O ₆ requires 300.1196].

Example 6 Analog 64 (Aminoisobutryl-L-prolyl-L-glutamic acid (AibPE)
General experimental details were as described in Example 2 above.

6.1 N-benzyloxycarbonylaminoisoacetic acid ^1,2 1:
Aminoisobutyric acid (2.00 g, 19.40 mmol) and sodium carbonate (6.16 g, 58.12 mmol) were dissolved in water (70 cm ³ ) and the solution was cooled to 0 ° C. Benzyl chloroformate (3.05 cm ³ , 21.4 mmol) was added dropwise over a period of 15 minutes. The solution was stirred at 0 ° C. for 1.5 hours and warmed to room temperature. Stirring was continued for 20 hours and the aqueous layer was extracted with diethyl ether (100 cm ³ ), acidified to pH 1 with hydrochloric acid (32%) and extracted with ethyl acetate (2 × 75 cm ³ ). The organic layers are combined, dried (MgSO ₄ ), filtered and evaporated under reduced pressure to give a clear oil that is erected to the original carbamate 1 (4.59 g, 100%). Solidified as a solid and used without further purification: δ _H (200 MHz; CDCl ₃ ; Me ₄ Si) 1.57 (6H, s, 2 × CH ₃ ), 5.10 (2H, s, OCH ₂ Ph ), 5.55 (1H, br s, NH), 7.26-7.33 (5H, m, Ph) and 10.53 (1H, br s, CO ₂ H); δ _c (50 MHz; CDCl ₃ ) 25.0 (CH ₃ , 2 × CH ₃ ), 56.3 (quat., C (CH ₃ ) ₂ ], 66.9 (CH ₂ , OCH ₂ Ph), 128.0 (CH, Ph), 128.4 (CH, Ph), 136.1 (quat., Ph), 155.2 (quat., NCO ₂ ) and 179.6 (quat., CO ₂ H); m / z (EI ⁺ ) 237 (M ⁺ . 12%).

6.2 Dibenzyl-N-benzyloxycarbonylaminoisobutyryl-L-prolyl-L-glutamate 3:
Amine ^{3 2 (DC64.10) (1.00g,} 2.36mmol), the acid 1 (0.56 g, 2.36 mmol) and 1-hydroxybenzotriazole hydrate (0.36 g, 2.26 mmol) in tetrahydrofuran (10 cm ³ ) at room temperature. 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.45 g, 2.36 mmol) was added and the solution was stirred for 3 days. The solvent is removed to dryness under reduced pressure and the remainder is dissolved in ethyl acetate (100 cm ³ ) and subsequently washed with saturated sodium bicarbonate solution (25 cm ³ ), brine (25 cm ³ ), water (25 cm ³ ). And then dried (MgSO ₄ ). The solvent was removed under reduced pressure to give an orange gum. Purification of the gum by flash column chromatography (20% hexane / ethyl acetate) produced the fully protected tripeptide 3 (0.40 g, 26%) as a clear gum: [α] _D 36.5 (c in MeOH 0.05): δ _H (200 MHz; CDCl ₃ ; Me ₄ Si) 1.41 (3H, s, CH ₃ ), 1.51 (3H, s, CH ₃ ), 1.68-2.29 (6H, m, Proβ-H ₂ , Proγ -H ₂ and Gluβ-H ₂ ), 2.44 (2H, t, J 7.5, Gluβ-H ₂ ), 3.20-3.25 (1H, m, Proδ-H), 3.55-3.60 (1H, m, Proδ-H) , 4.44-4.51 (1H, m, Gluα-H) 4,54-4.61 (1H, m, Proα-H), 4.97 (2H, s, OCH ₂ Ph), 5.06 (2H, s, OCH ₂ Ph), 5.15 (2H, s, OCH ₂ Ph) 5.48 (1H, br s, NHCO ₂ ), 7.28-7.33 (15H, m, Ph) and 7.58-7.62 (1H, m, NHCO); δc (50 MHz; CDCl ₃ ) 24.8 [CH ₃ , (CH ₃ ) ₂ C], 25.6 (CH ₂ , Proγ-C), 26.1 [CH ₃ , C (CH ₃ ) ₂ ], 28.0 (CH ₂ , Gluβ-C and Proβ-C) , 30.1 (CH ₂ , Gluγ-C), 48.0 (CH ₂ , Proδ-C), 51.8 (CH, Gluα-C), 57.0 (quat, C (CH ₃ ) ₂ ), 62.2 (CH, Proα-C) , 66.1 (CH ₂ , OCH ₂ Ph), 66.9 (CH ₂ , OCH ₂ Ph), 128.0 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 136.0 (quat., Ph), 155.1 (quat., NCO ₂ ), 171.0 (quat., GluCO), 172.0 (quat. , GluCO) and 172.7 (quat., ProCON); m / z (EI ⁺ ) 644.2969 (M ⁺ . C ₃₆ H ₄₂ N ₃ O ₈ requires 644.2972).

6.3 Aminoisobutyryl-L-prolyl-L-glutamic acid (AibPE) 64:
Protected tripeptide 3 (0.44 g, 0.68 mmol) in methanol (40 cm ³ ) and 10 wt% palladium on activated carbon (0.07 g, 0.07 mmol) at room temperature under an atmosphere of nitrogen and then hydrogen. Stir for 20 hours. The solution was filtered through a Celite® pad and washed with methanol (2 × 20 cm ³ ). The filtrate was evaporated under reduced pressure to form a clear rubber. The gum was dissolved in methanol (20 cm ³ ) and refiltered through a Celite® pad. The solution was evaporated under reduced pressure until dry to form a bright orange gum. The rubber was placed in a vacuum line for 15 minutes and then triturated with anhydrous diethyl ether to form AibPE (0.14 g, 63%) as a pale yellow solid. AibPE and by ¹ H NMR analysis 65:35 trans conformers: indicated a mixture of _{cis: mp190-192 ℃: [α] D} 35.5 (c 0.06 in MeOH): [delta] _H ( 200 MHz; D ₂ O) 1.47 * (1.05H, s, CH ₃ ), 1.49 * (1.05H, s, CH ₃ ), 1.73 (1.95H, s, CH ₃ ), 1.76 (1.95H, s, CH ₃ ), 1.96-2.34 (6H, m, Proβ-H ₂ , Proγ-H ₂ and Gluβ-H ₂ ), 2.54-2.60 (1.3H, m, Gluβ-H ₂ ) 2.60-2.64 * (0.7H, m , Gluβ-H ₂ ), 3.55-3.59 (1H, m, Proδ-H), 3.70-3.80 (1H, m, Proδ-H), 4.46-4.58 (1H, m, Gluα-H), 4.55-4.57 ( 1H, m, Proα-H); δc (50 MHz; D ₂ O) 21.3 * [CH ₃ , (CH ₃ ) C], 21.5 [CH ₃ , (CH ₃ ) C], 22.5 * (CH ₃ , ( CH ₃ ) C), 23.8 (CH ₃ , (CH ₃ ) C), 24.8 (CH ₂ , Proγ-C), 25.0 (CH ₂ , Gluβ-C), 25.5 * (CH ₂ , Proγ-C), 27.9 (CH ₂ , Proβ-C), 29.3 * (CH ₂ , Proβ-C), 29.6 (CH ₂ , Gluγ-C), 45.5 * (CH ₂ , Proδ-C), 48.5 (CH ₂ , Proδ-C) , 51.9 (CH, Gluα-C), 52.7 * (CH, Gluα-C), 57.0 * [quat., C (CH ₃ ) ₂ ], 57.9 [quat., C (CH ₃ ) ₂ ], 58.5 * ( CH, Proα-C ), 62.1 (CH, Proα-C), 171.0 (quat., Aib-CO), 174.0 (quat., ProCON), 174.8 (quat., Gluα-CO ₂ ) and 176.8 (quat, Gluγ-CO ₂ ); m / z (FAB +) 330.1664 (MH ⁺ .C ₁₄ H ₂₄ N ₃ O ₆ requires 330.1665)

References: 1. Wipf et al. Helv.Chim.Acta, 1988, 71, 140; 2.Kent et al. J.Sol.Chem., 1985, 14, 101; 3. Gillesse et al. Helv.Chim.Acta , 1970, 53, 63.
Example 7: Synthesis of analog 65 (Glycyl-L-prolyl-L-norvaline) General experimental details were as described in Example 2 above. L-norvaline 1 was obtained from Sigma.
Glycyl-L-prolyl-L-norvaline (GPnorval)

7.1 L-norvaline benzyl ester p-toluenesulfonate 2:
A suspension of L-norvaline 1 (1.0 g, 8.53 mmol) and p-toluenesulfonic acid (1.70 g, 8.96 mmol) in benzyl alcohol (5 cm ³ ) was refluxed in benzene (25 cm ³ ). Heated for 17 hours with removal of water (Dean-Stark trap) (solution obtained after 5 minutes). The solution was cooled to room temperature and dry ether was added. Upon cooling to 0 ° C., a white solid precipitated and was collected by filtration, washed with dry ether and dried to give p-toluenesulfonate 2 (3.07 g, 95%) as a white solid: mp142 [Α] _D -1.45 (c 1 in dichloromethane); δ _H (200 MHz; CDCl ₃ ; Me ₄ Si) 0.7 (3H, t, J 7.1, 5-H ₃ ), 1.09-1.37, (2H, m, 4-H ₂ ), 1.74, (2H, app.quartet, J 7.9, 3-H ₂ ), 2.30 (3H, s, Ar-CH ₃ ), 3.99 (1H, t, J 6.2, 2-H ₂ ), 4.98 (1H, d, J 12.3, OCH _A H _B Ph), 5.11 (1H, d, J 12.3, OCH _A H _B Ph), 7.07 (2H, d, J 8.0, 3'- H); 7.22-7.30 (5H, m, Ph-H) and 7.74 (2H, d, J 8.0, 2'-H); δ _C (50 MHz; CDCl ₃ ) 13.4 (CH ₃ , 5-C), 17.9 (CH ₂ , 4-C), 21.2 (CH ₃ , Ar-CH ₃ ) 32.3 (CH ₂ , 3-C), 53.1 (CH, 2-C), 67.6 (CH ₂ , OCH ₂ Ph), 126.0 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.4 (CH, Ph), 128.7 (CH, Ph), 134.8 (quat., Ph), 140.2 (quat., Ph), 141.4 (quat., Ph) and 169.4, (quat., 1-C); m / z (FAB) 587.2777 [M ₂ .pTsOH.H ⁺ : ( C ₁₂ H ₁₇ NO ₂ ) ₂ .C ₇ H ₈ O ₃ SH requires 587.2791].

7.2 N-benzyloxylcarbonyl-glycyl-L-norvaline benzyl ester 4:
To a stirred solution of benzyl N-benzyloxycarbonyl-glycyl-L-proline 3 (0.4 g, 1.3 mmol) in dry dichloromethane (20 cm ³ ) under nitrogen was added diisopropylethylamine (0.31 cm ³ , 1. 76 mmol) was added. The suspension was stirred for 5 minutes (to obtain a solution), cooled to 0 ° C. and ethyl chloroformate (0.166 cm ³ , 1.73 mmol) was added. After 45 minutes, a solution of diisopropylethylamine (0.31 cm ³ , 1.76 mmol) and p-toluenesulfonate 2 (0.644 g, 1.70 mmol) in dry dichloromethane (20 cm ³ ) was added dropwise over 5 minutes. did. The cooling bath was removed and the resulting solution was stirred overnight. The solvent was removed in vacuo and the remainder was chromatographed (silica gel, 50-100% ethyl acetate-hexanes) to give fully protected tripeptide 4 (0.502 g, 78%) as a colorless oil. Tripeptide 4 was shown by ¹³ C NMR analysis to be a 84:16 trans: cis mixture of conformers (ratio was determined individually for the large and small conformers δ34.0 and 33). .6 measured from the intensity of Eγ-C carbon at ^Ψ Eβ-C and δ 18.5 and 18.8): [α] _D -70.3 (c 0.8 in dichloromethane); δ _H (300 MHz; CDCl ₃ ; Me ₄ Si) 0.87 (3H, d, J 6.0, Eγ-CH ₃ ), 1.26-1.33 (2H, m, Eγ-H ₂ ), 1.58-1.68 (1H, m, Eβ-H _A H _B ), 1.70 -2.26 (5H, m, Eβ-H _A H _B , Proβ-H ₂ , and Proγ-H ₂ ), 3.38 (0.84H, d, J 7.6, Proδ-H _A H _B ), 3.51-3.56 (0.84H , m, Proδ-H _A H _B ), 3.60-3.65 ^* (0.32 H, m, Proδ-H ₂ ), 3.76 ^* (0.16H, d, J 16.5, Glyα-H _A H _B ), 3.91 (1.84H , m, Glyα-H ₂ ), 4.37 ^* (0.16H, br s, Proα-H), 4.49-4.62 (2H, m, Proα-H, Eα-H), 5.09-5.21 (4H, m, 2 x OCH ₂ Ph), 5.85 (1H, br s, GlyNH), 7.2 (1H, d, J 5.5, Eα-NH) and 7.34 (10H, s, 2 x Ph); δ _C (75 MHz; CDCl ₃ ) 13.4 (CH _{3 , Eγ-CH 3), 13.5} * (CH 3, Eγ-CH 3), 18.5 (CH 2, Eγ-C), 18.8 * (CH 2, Eγ-C), 22.2 * (CH 2, Proγ-C) , 24.7 (CH ₂ , Proγ-C), 27.7, (CH ₂ , Proβ-C) 31.9 ^* (CH ₂ , Proβ-C) 33.6 ^* (CH ₂ , Eβ-C), 34.0 (CH ₂ , Eβ-C ), 43.3 (CH ₂ , Glyα-C), 46.2 (CH, Proδ-C), 47.0 ^* (CH, Proδ-C), 52.1 ^* (CH, Eα-C), 52.3 (CH, Eα-C), 59.9 (CH, Proα-C), 66.8 (CH ₂ , OCH ₂ Ph), 67.1 ^* (CH ₂ , OCH ₂ Ph), 127.9 (CH, Ph), 127.99 (CH, Ph), 128.0 (CH, Ph) , 128.1 ^* (CH, Ph), 128.2 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 135.4 (quat., Ph), 136.3 (quat., Ph), 156.2 (quat. , NCO ₂ ), 156.4 ^* (quat., NCO ₂ ), 167.9 (quat., GlyCO), 168.2 ^* (quat., Gly-CO), 170.7 (quat., ProCON), 171.1 ^* (quat., ProCON) And 172.1 (quat., Eα-CO); m / z (FAB +) 495.2381 (MH ⁺ . C ₂₇ H ₃₃ N ₃ O ₆ requires 495.2370).
^Ψ E relates to the proline analog part in question.
^* Indicates a resonance located in a small conformer.

7.3 Glycyl-L-prolyl-L-norvaline (GPNorval) 65:
Tripeptide 4 (0.308 g, 0.62 mmol) was dissolved in methanol (30 cm ³ ). The reaction flask was flushed with nitrogen and 10 wt% palladium on activated carbon (66 mg, 0.062 mmol) was added and the mixture was hydrogenated with one atmosphere of hydrogen. After 30 minutes, a white solid precipitated. Water (15 cm ³ ) was added to dissolve the solid and the reaction was stirred for 18 hours. The reaction was filtered through Celite®, washed with methanol / water and the solvent removed to produce an oil combined with palladium. The filtration was repeated again and the solvent was removed to produce a white solid suspended in methanol and placed in the refrigerator overnight. The solid was filtered, washed with ice-cold methanol and dried to produce GPnorval (0.142 g, 84%) as white flakes. GPNorval was shown by ¹³ C NMR analysis to be a 78:22 trans: cis mixture of conformers (ratio was determined individually for the large and small conformers δ29.4 and 31.6 Measured from the intensity of pro-β-C carbon and pro-γ-C carbon at δ24.1 and 21.9): [α] _D -79 (c 0.1 in water); δ _H (300 MHz; D ₂ O) 0.85 (3H, d, J 7.4, Eγ-CH ₃ ), 1.29 (2H, hexat, J 7.0, Eγ-H ₂ ), 1.53-1.75 (2H, m, Eβ-H ₂ ) 1.88-2.05 (3H , m, Proβ-H _A H _B , Proγ-H ₂ ) 2.09-2.27 (1H, m, Proβ-H _A H _B ), 3.51-3.82 (4H, m, Proδ-H ₂ , Glyα-H ₂ ), 4.09 (0.78H, dd, J 7.8 5.0, Eα-H), 4.14 ^* (0.22H, dd, J 9 5.0, Eα-H) and 4.41 (1H, m, Proα-H); δ _C (75 MHz; D ₂ O) 12.8 ^* (CH ₃ , Eγ-CH ₃ ), 12.9 (CH ₃ , Eγ-CH ₃ ), 18.5 (CH ₂ , Eγ-C), 18.7 ^* (CH ₂ , Eγ-C), 21.9 ^* (CH ₂ , Proγ-C), 24.1 (CH ₂ , Proγ-C), 29.4, (CH ₂ , Proβ-C), 31.6 ^* (CH ₂ , Proβ-C), 33.3 ^* (CH ₂ , Eβ-C ), 33.8 (CH ₂ , Eβ -C), 40.6 (CH ₂ , Glyα-C), 46.8 (CH, Proδ-C), 47.5 ^* (CH, Proδ-C), 55.1 (CH, Eα-C), 60.0 ^* (CH, Proα-C ), 60.4 (CH, Proα-C), 166.6 (quat., GlyCO), 173.1 (quat., ProCON), 178.9 ^* (quat., Eα-CO) and 179.0 (quat., Eα-CO); m / z (FAB +) 272.1641 (MH ⁺ . C ₁₂ H ₂₂ N ₃ O ₄ requires 272.1610).

Example 8 Synthesis of Analog 66 (Glycyl-D, L-pipecolinyl-L-glutamic acid (G (D, L) PipE)) General experimental details are described in Example 1 above. It was something like that. N-benzyloxycarbonyl-glycine 1, N-hydroxysuccinimide 2 (97%) (NHS), D, L-pipecolic acid 4 (98%) and 10% palladium on activated carbon were obtained from Aldrich Chemical Company. N, N′-dicyclohexylcarbodiimide (DCC) was obtained from Riedel-de-Haen. L-glutamic acid dibenzyl ester p-toluenesulfonate 6 was obtained from Bachem. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (97%) (BoPCl) was obtained from Fluka.

8.1 N-benzyloxycarbonyl-glycylsuccinimide ^1,2 3:
N-benzyloxycarbonyl-glycine 1 (7.50 g, 35.9 mmol) and N-hydroxysuccinimide 2 (4.13 g, 35.9 mmol) were dissolved in 1,2-dimethoxyethane (60 cm ³ ) and 0 ° C. Cool to (ice / water) under nitrogen. N, N′-dicyclohexylcarbodimide (8.14 g, 39.5 mmol) was added and the solution was stirred for 1.5 hours and then placed in a refrigerator at −5 ° C. for 20 hours. The solution was filtered and evaporated under reduced pressure to form a yellow oil. The oil was triturated with diethyl ether (30 cm ³ ) to form succinimide 3 as a white solid. This solid was subsequently used without further purification.

8.2 N-benzyloxycarbonyl-glycyl-D, L-pipecolic acid ³ 5:
D, L-pipecolic acid 4 (0.20 g, 1.55 mmol) and N-benzyloxycarbonyl-glycyl N-hydroxysuccinimide 3 (0.47 g, 1.5 mmol) were added to dimethylformamide (2 cm ³ ) at room temperature. Added below. Triethylamine (0.22 cm ³ , 1.58 mmol) was then added dropwise over a period of 5 minutes. The solution was heated at 80 ° C. for 4 hours and then cooled to room temperature and stirred for 2 days. Ethyl acetate (40 cm ³ ) was added and the resulting solution was washed with 1M hydrochloric acid (2 × 5 cm ³ ), dried (MgSO ₄ ), filtered and evaporated under reduced pressure to produce a clear gum. It was triturated with diethyl ether (5 cm ³ ) to produce acid 5 (0.33 g, 67%) as a white solid. Acid 5 was shown by ¹ H NMR analysis to be a 75:25 trans: cis mixture of conformers (ratio measured from doublet intensity at δ 3.76 and 3.81). , Individually given to the small and large conformers of glycine α- _HA H _B ): δ _H (300 MHz; CD ₃ OD) 1.05-1.76 (5H, m, pipβ-H, pipγ-H ₂ and pipδ-H ₂ ), 2.19 (0.75H, t, J 12.6, pipβ-H), 2.62 ^* (0.25H, t, J 15.4, pipβ-H), 3.14-3.25 (1H, m, pipε-H _A H _B ), 3.65-3.70 (1H, pipε-H _A H _B ), 3.76 ^* (0.25H, d, J 16.7, Glyα-H _A H _B ), 3.81 (0.75H, d, J 16.9, Glyα-H _A H _B ,), 4.06 ^* (0.25H, d, J 16.6, Glyα-H _A H _B ,), 4.08 (0.75H, d, J 17.0, Glyα-H _A H _B ,), 4.22 ^* (0.25H , d, J 13.6, pipε-H), 4.60 ^* (0.25H, br s, pipα-H), 4.83 (2H, s, OCH ₂ Ph), 5.14 (0.75H, br s, pipα-H) and 7.20 -7.31 (5H, m, Ph); δc (75 MHz, CD ₃ OD) 22.5 (CH ₂ , pipγ-C), 26.2 ^* (CH ₂ , pipγ-C), 26.6 (CH ₂ , pipβ-C), 27.3 ^* (CH ₂ , pipβ-C), 28.2 (CH ₂ , pipδ-C), 28.5 ^* (CH ₂ , pipδ-C), 35.3 (CH ₂ , pipε-C), 41.8 ^* (CH ₂ , Glyα-C), 44.2 (CH ₂ , Glyα-C), 54.3 (CH, pipα-C), 57.1 ^* (CH, pipα-C), 68.3 (CH ₂ , OCH ₂ Ph), 129.5 (CH, Ph), 129.6 (CH, Ph), 130.1 (CH, Ph), 138.8 (quat., Ph), 159.5 (quat., NCO ₂ ), 171.5 (quat., GlyCO), 174.1 ^* (quat., CO ₂ H) and 174.7 (quat., CO ₂ H); m / z (EI +) 320.1369 (M ⁺ .C ₁₆ H ₂₀ N ₂ O ₅ requires 320.1372).

8.3 Benzyl-N-benzyloxycarbonylglycyl-D, L-pipecolinyl-L-glutamate 7:
Acid 5 (0.25 g, 0.78 mmol), L-glutamic acid dibenzyl ester p-toluenesulfonate 6 (0.47 g, 0.94 mmol) and bis (2-oxo-3-oxazolidinyl) phosphinic chloride (97%) (0.24 g, 0.94 mmol) was dissolved in dichloromethane (10 cm ³ ) under nitrogen and cooled to 0 ° C. Triethylamine (0.25 cm ³ , 1.80 mmol) was added dropwise over a period of 5 minutes. The solution was stirred for 1.5 hours, then warmed to room temperature and stirred overnight. Dichloromethane (30 cm ³ ) was added and the organic layer was subsequently washed with saturated sodium bicarbonate solution (10 cm ³ ) and aqueous 2M citric acid (10 cm ³ ), then dried (MgSO ₄ ), filtered and under reduced pressure Evaporate to form a white gummy solid that is purified by flash column chromatography (ethyl acetate) to produce fully protected tripeptide 7 (0.37 g, 74%) as a clear gum. did. Tripeptide 7 was shown by ¹ H NMR analysis to be an 87:13 trans: cis mixture of two equimolar diastereomeric conformers (ratio δ4.05-4.11). And 4.20-4.30, measured from singlet intensities and individually given the large and small conformer glycerine α-H ₂ protons): [α] _D 9.2 (c 0.07 in MeOH) ; δ _H (300 MHz; CDCl ₃ ; Me ₄ Si) 1.11-2.22 (8H, m, pipβ-H ₂ , pipγ-H ₂ pipδ-H ₂ and Gluβ-H ₂ ), 2.30-2.41 (2H, m, Gluγ-H ₂ ), 3.00-3.20 (1 H, m, pipε-H), 3.48 (1 H, t, J 10.6, pipε-H), 4.05-4.11 (1.74 H, m, Glyα-H ₂ ), 4.20-4.30 ^* (0.26 H, m, Glyα-H ₂ ), 4.55-4.59 (1 H, m, Gluα-H), 5.07-5.23 (7H, m, 3 x OCH ₂ Ph and pipα-H), 5.84 (1 H, br s, GlyNH), 6.97 ^* (0.13 H, d, J 7.2, GluNH), 7.19 (0.87 H, d, J 7.2, GluNH) and 7.31-7.32 (15H, m, 3 x Ph); _{δ c (75 MHz, CDCl 3} ) 20.1 (CH 2, pipγ-C), 20.4 (CH 2, pipγ-C), 25.3 (CH 2, Glu _{-C), 25.4 (CH 2,} Gluβ-C), 25.7 (CH 2, pipβ-C), 25.9 (CH 2, pipβ-C), 26.1 (CH 2, pipδ-C), 26.7 (CH 2, pipδ -C), 30.8 (CH ₂ , Gluγ-C), 30.9 (CH ₂ , Gluγ-C), 34.2 (CH ₂ , pipε-C), 40.6 ^* (CH ₂ , Glyα-C), 43.0 (CH ₂ , Glyα-C), 43.2 (CH ₂ , Glyα-C), 49.4 ^* (CH, pipα-C), 52.5 (CH, pipα-C), 52.6 (CH, pipα-C), 52.7 (CH, Gluα-C ), 53.0 (CH, Gluα-C), 67.0 (CH ₂ , OCH ₂ Ph), 67.1 (CH ₂ , OCH ₂ Ph), 67.2 (CH ₂ , OCH ₂ Ph), 67.6 (CH ₂ , OCH ₂ Ph) , 67.7 (CH ₂ , OCH ₂ Ph), 69.4 (CH ₂ , OCH ₂ Ph), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.2 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 135.1 (quat., Ph), 135.5 (quat., Ph), 136.4 (quat., Ph), 156.2 (quat., NCO ₂ ), 168.3 (quat., GlyCO), 168.4 (quat ., GlyCO), 170.3 (quat., GluCO), 170.5 (quat., GluCO), 171.1 (quat., GluCO), 171.3 (quat., GluCO), 173.0 (quat., Pip-CONH) and 173.4 (quat , pip-CONH); m / z (FAB +) 630.2821 (M ⁺ .C ₃₅ H ₄₀ N ₃ O ₈ requires 630.2815).

8.4 Glycyl-D, L-pipecolinyl-L-glutamic acid [G (D, L) PipE] 66:
Protected tripeptide 7 (0.72 g, 1.14 mmol) and 10% palladium on activated carbon (0.12 g, 0.11 mmol) in methanol (70 cm ³ ) were stirred at room temperature under an atmosphere of hydrogen for 20 hours. The solution was filtered through a Celite® pad, washed with methanol (60 cm ³ ) and the filtrate was evaporated to dryness to form a clear gum. The gum was dissolved in methanol (30 cm ³ ) and refiltered through a Celite® pad. The solution was evaporated to dryness to form a clear gum. The rubber was placed in a vacuum line for 15 minutes and then triturated with anhydrous diethyl ether to form G (D, L) PipE (0.34 g, 95%) as a white solid. G (D, L) PipE was shown by ¹ H NMR analysis to be an 80:20 trans: cis mixture of the two equimolar diastereomeric conformers (ratio was δ2.91 and Measured from the triplet intensity at 3.34 and given individually to the small and large conformers, pi ε-H _A H _B ): mp 67-69 ° C. [α] _D +7.9 (c 0.09 in MeOH); δ _H (400 MHz; CD ₃ OD) 1.37-1.65 (4H, Gluβ-H ₂ and pipγ-H ₂ ), 1.89-2.52 (6H, pipδ- H ₂ , pipβ-H ₂ and Gluγ-H ₂ ), 2.78 ^* (0.1H, t, J 10.5, pipε-H _A H _B ), 2.91 ^* (0.1H, t, J 10.5, pipε-H _A H _B ), 3.18 (0.4H, t, J 10.5, pipε-H _A H _B ), 3.34 (0.4H, t, J 10.5, pipε-H _A H _B ), 3.52-3.58 (1H, m, pipε-H _A H _B ), 3.81-3.88 (1.6H, m, Glyα-H ₂ ), 4.00-4.10 ^* (0.2H, m, Glyα-H ₂ ), 4.16 (0.8H, t, J 6.4, Gluα-H), 4.40-4.50 ^* (0.2H, m, Glyα-H ₂ ), 4.46-4.50 ^* (0.2H, m, pipα-H) and 5.05-5.09 (0.8H, m, pipα-H); δ _C (75 MHz , CD ₃ OD) 21.8 (CH ₂ , pipγ-C), 25.7 (CH ₂ , Gluβ-C), 26.0 ^* (CH ₂ , Gluβ-C), 26.5 (CH ₂ , Gluβ-C), 27.0 (CH ₂ , pipβ-C), 27.4 ^* (CH ₂ , pipβ-C), 27.6 (CH ₂ , pipβ-C), 28.1 (CH ₂ , pipδ-C), 28.3 ^* (CH ₂ , pipδ-C), 28.7 ( CH ₂ , pipδ-C), 32.2 (CH ₂ , Gluγ-C), 33.1 (CH ₂ , Gluγ-C), 33.2 ^* (CH ₂ , Gluγ-C), 33.5 ^* (CH ₂ , Gluγ-C), 41.9 (CH ₂ , pipε-C), 44.1 (CH ₂ , Glyα-C), 44.6 ^* (CH ₂ , Glyα-C), 44.8 ^* (CH ₂ , Glyα-C), 45.6 (CH ₂ , Glyα-C), 55.0 (CH, pipα-C), 56.1 (CH, pipα-C), 56.2 (CH, Gluα-C), 57.7 ^* (CH, Gluα -C), 57.9 ^* (CH, Gluα-C), 60.5 (CH, Gluα-C), 168.3 (quat., GlyCO), 169.8 (quat., GlyCO), 172.5 (quat., Pip-CON), 174.6 (quat., pip-CON), 177.2 (quat., Gluα-CO), 178.5 (quat., Gluα-CO), 179.3 (quat., Gluγ-CO) and 180.0 (quat., Gluγ-CO); m / z (FAB +) 316.1509 (MH ⁺ . C ₁₃ H ₂₂ N ₃ O ₆ requires 316.1509).

See: 1. Detsiet al. J. Chem. Soc. Perkin Trans1, 1998, 15, 2443; 2. Scudder et al. Tet. Lett. 1995, 36, 2105; 3. Nishitani et al. J. Org. Chem. 1982, 47, 1706.
Example 9: Synthesis of analog 67 (pyrrolidinoglycyl-L-2-methyl-proline-L-glutamic acid (PyrrolidinoG-2MePE)) Details of the general experiment are described in Example 2 above. It was like that. Hydrochloride (1) and pyrrolidinoacetic acid (2) were synthesized individually as described in the synthesis of DC54 (CyclicG-2EtP) and DC68 (PyrrolidinoGPE). Glutamic acid dibenzyl ester p-toluenesulfonate (5) and bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BoPCl, 97%) were obtained individually from bachem and fluka.

9.1 Methylpyrrolidinoglycyl-L-2-methylprolinate (3):
Dry triethylamine (1.9 cm ³ , 13.7 mmol) was added to a solution of acid 2 (0.39 g, 3.32 mmol) and hydrochloride 1 (0.40 g, 2.21 mmol) in dry dichloromethane (10 cm ³ ). It was added dropwise at room temperature under an atmosphere of nitrogen and the reaction mixture was stirred for 1 hour. Bis (2-oxo-3-oxazolidinyl) phosphonic chloride (BoPCl, 97%) (0.70 g, 2.75 mmol) was added and the solution was stirred for 2 hours at room temperature and then heated to 100 ° C. for 3 hours And cooled to room temperature and stirred for 3 days. The solution was evaporated to dryness in vacuo to form a dark brown solid dissolved in ethyl acetate (50 cm ³ ) and then washed with saturated sodium bicarbonate (20 cm ³ ). The aqueous layer was re-extracted with ethyl acetate (2 × 20 cm ³ ). The combined organic layers were dried (MgSO ₄ ) and evaporated under reduced pressure. The remaining purification obtained by flash column chromatography (5% methanol-dichloromethane) gave amide 3 (0.11 g, 19%) as an orange oil: δ _H (400 MHz, CDCl ₃ ) 1.56 (3H , s, Proα-CH ₃ ), 1.77-1.80 (4H, m, 2 x Pyrβ-H ₂ ), 1.85-1.90 (1H, m, Proγ-H _A H _B ), 2.00-2.04 (2H, m, Proβ -H ₂ ), 2.10-2.13 (1H, m, Proγ-H _A H _B ), 2.58-2.63 (4H, m, 2 x Pyrα-H ₂ ), 3.25 (2H, s, Glyα-H ₂ ) and 3.66 -3.73 (5H, m, Proδ-H ₂ and OCH ₃ ); δ _C (100 MHz, CDCl ₃ ) 21.8 (CH ₃ , Proα-CH ₃ ), 24.0 (CH ₂ , 2 x Pyrβ-C), 24.5 ( CH ₂ , Proγ-C), 38.6 (CH ₂ , Proβ-C), 47.9 (CH ₂ , Proδ-C), 52.6 (CH ₃ , OCH ₃ ), 54.4 (CH ₂ , 2 x Pyrα-C), 59.3 (CH ₂ , Glyα-C), 66.2 (quat., Proα-C), 168.6 (quat., GlyCO) and 174.8 (quat., CO ₂ CH ₃ ).

9.2 Pyrrolidinoglycyl-L-2-methylproline (4):
To a solution of amide 3 (0.08 g, 0.31 mmol) in dioxane (5 cm ³ ), 1M aqueous NaOH (2.5 cm ³ , 2.5 mmol) was added dropwise and the mixture was stirred for 2 days at room temperature. . The reaction mixture was acidified with 1M hydrochloric acid and evaporated to dryness under reduced pressure. The resulting suspension was redissolved in ethanol, filtered and evaporated to dryness in vacuo to provide acid 4 (0.05 g, 66%) as a dark brown gum: δ _H (400 MHz, CD ₃ OD) 1.56 (3H, s, Proα-CH ₃ ), 1.94-2.22 (8H, m, Proβ-H ₂ , Proγ-H ₂ and 2 x Pyrβ-H ₂ ), 3.10-3.13 (2H, m, Pyrα- H ₂ ), 3.54-3.63 (2H, m, Proδ-H ₂ ), 3.67-3.70 (2H, m, Pyrα-H ₂ ), and 4.27 (2H, s, Glyα-H ₂ ); δ _C (100 MHz , CD ₃ OD) 22.1 (CH ₃ , Proα-CH ₃ ), 24.6 (CH ₂ , Pyrβ-C), 24.7 (CH ₂ , Pyrβ-C), 25.3 (CH ₂ , Proβ-C), 40.3 (CH ₂ , Proγ-C), 49.1 (CH ₂ , Proδ-C), 56.6 (CH ₂ , Pyrα-C), 56.8 (CH ₂ , Pyrα-C), 57.9 (CH ₂ , Glyα-C), 68.5 (quat. , Proα-C), 164.7 (quat., GlyCO) and 177.2 (quat., CO ₂ H).

9.3 Dibenzylpyrrolidinoglycyl-L-2-methyl-prolyl-L-glutamate (6):
Dry triethylamine (0.09 cm ³ , 0.62 mmol) was added to acid 4 (0.05 g, 0.15 mmol) and L-glutamic acid diethyl ester p-toluenesulfonate 5 (0.11 g, in dry dichloromethane (8 cm ³ )). 0.22 mmol) was added dropwise at room temperature under an atmosphere of nitrogen and the reaction mixture was stirred for 10 minutes. Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BoPCl, 97%) (0.06 g, 0.22 mmol) was added and the solution was stirred for 3 days. The solution was subsequently washed with 10% aqueous hydrochloric acid (7 cm ³ ) and saturated aqueous sodium hydrogen carbonate (7 cm ³ ), dried (MgSO ₄ ), filtered and evaporated to dryness in vacuo. The remaining purification obtained by flash column chromatography (10-20% methanol-dichloromethane; gradient elution) produced the fully protected tripeptide 4 (0.038 g, 40%) as a colorless oil: δ _H ( 400 MHz, CDCl ₃ ) 1.62 (3H, s, Proα-CH ₃ ), 1.83-2.44 (12H, m, Proβ-H ₂ , Proγ-H ₂ , Gluβ-H ₂ , Gluγ-H ₂ and 2 × Pyrβ- H ₂ ), 2.80-2.90 (4H, m, 2 × Pyrα-H ₂ ), 3.43-3.72 (4H, m, NCH ₂ CO and Proδ-H ₂ ), 4.52-4.54 (1H, m, Gluα-H) , 5.08 (2H, s, OCH ₂ Ph), 5.08-5.14 (2H, m, OCH ₂ Ph), 7.27-7.37 (10H, m, 2 × Ph) and 7.77 (1H, d, J 7.3, GluNH). δ _C (100 MHz, CDCl ₃ ) 21.4 (CH _3, Proα-C), 23.1 (CH ₂ , Pyrβ-C and Proγ-C), 26.3 (CH ₂ , Gluβ-C), 29.8 (CH ₂ , Gluγ- C), 38.1 Proγ-C), 47.8 (CH ₂ , Proδ-C), 51.7 (CH, Gluα-C), 53.9 (CH ₂ , Pyrα-C), 57.9 (CH ₂ , NCH ₂ CO), 65.9 ( CH ₂ , OCH ₂ Ph), 66.6 (CH ₂ , OCH ₂ Ph), 67.7 (quat., Proα-C), 128.1 (CH, Ph), 128.3 (CH, Ph), 128.5 (CH, Ph), 134.9 (quat., Ph), 135.4 (quat., Ph), 167.5 (quat., Pyr-CONH), 171.3 (quat., GluCO), 172.3 (quat., GluCO) and 173.4 (quat., Pro-CON ).

9.4 Pyrrolidinoglycyl-L-2-methyl-proline-L-glutamic acid (Pyrrolidino G-2-MePE) 67:
A mixture of protected peptide 6 (0.038 g, 0.07 mmol) and 10 wt% palladium on activated carbon (0.073 g, 0.07 mmol) in 10% water-ethanol (8 cm ³ ) at room temperature under an atmosphere of hydrogen. For 20 hours. The solution was filtered through a Celite® pad and the filtrate was evaporated to dryness to form a clear gum. The gum was dissolved in 10% water-ethanol (10 cm ³ ) and refiltered through a Celite® pad. The filtrate was evaporated to dryness to give a clear gum that was placed in a vacuum line for 15 minutes and then triturated with anhydrous diethyl ether to give Pyrrolidino G-2MePE (0.024 g, 98%) to the clear gum. Formed as. ; δ _H (300 MHz; CD ₃ OD) 1.57 (3H, s,, Proα-CH ₃ ), 1.87-2.20 (10H, m, Proβ-H ₂ , Proγ-H ₂ , Gluβ-H ₂ and 2 × Pyrβ -H ₂ ), 2.30-2.33 (2H, m, Gluγ-H ₂ ), 3.35-3.45 (4H, m, 2 x Pyrα-H ₂ ), 3.59-3.62 (4H, m, Proδ-H ₂ and NCH ₂ CO) and 4.18-4.25 (1H, m, Gluα-H); δ _C (75 MHz; CD ₃ OD) 21.9 (CH _3, Proα-C), 24.8 (CH ₂ , Pyrβ-C), 25.8 (CH ₂ , Proγ-C), 29.3 (CH ₂ , Gluβ-C), 32.6 (CH ₂ , Gluγ-C), 40.9 (CH ₂ , Proβ-C), 49.3 (CH ₂ , Proδ-C), 56.0 (CH, Gluα-C), 56.5 (CH ₂ , Pyrα-C), 70.0 (quat., Proα-C), 165.3 (quat, Pyr-CONH), 175.9 (quat, ProCONH) and 179.0 (quat, Gluγ-CO ₂ H ).

Example 10: Study of in vitro activity of GPE analogs Synthetic analogs of GPE were subjected to in vitro evaluation of their effects in preventing neuronal cell death.
a) Striatal cell culture methods and materials Two pregnant wistar rats (gestation day 18) underwent cesarean sections according to approved procedures from the Animal Ethics Committee of the University of Auckland. Wistar dam (wistar dam), CO ₂ - it was sacrificed by anesthesia and subsequent spinal cord metastasis in concentrated atmosphere. The fetuses were removed from their fetal sac and the head was excised. After incision in the skull with thin scissors, the fetal brain was removed with a thin spatula. Striatal tissue was separated from the neocortical tissue under the microscope and placed in serum-free DMEM / F-12 medium supplemented with penicillin / streptomycin (100 μ / mL). The tissue was subjected to 15 grinding steps using a P1000 dispenser to obtain dissociated cells, which were centrifuged at 250 G (4 ° C.) for 5 minutes and the supernatant was discarded. Cells were re-suspended in 1 mL DMEM / F-12 medium and kept on ice. Cell suspension (400,000 cells / cm ² ) was applied to 0.1 mg / mL poly-L-lysine pre-coated in 96-well plates (3 hours at 37 ° C.) and the volume was adjusted to DMEM / F. Increased up to 100 μL with −12 + 5% FBS. Cells were cultured at 37 ° C. and 100% humidity in a 5% CO ₂ atmosphere. After 24 hours, the medium was changed to neural basis / B27 without serum. Cell media changed every 3 days and was maintained in vitro for up to 8 days (DIV).

Cytotoxicity, drug administration and cell survival analysis Striatal cells were injured in vitro after 2, 3, 7 or 8 days. The disability paradigm included 30 nM okadaic acid treatment with co-administration of the GPE analogs of the present invention for 24 hours. 1 μL of 3 μM okadaic acid stock solution was incubated with striatal cells for 24 hours along with 1 μL of GPE analog dissolved in PBS (vehicle receiving 2 μL of PBS). Subsequently, 20 μL of MTT (5 mg / mL in PBS) was added for 4 hours. Reaction
Finished by adding 100 μL of 4% sodium dodecyl sulfate (SDS) solution. After a culture time of 16-24 hours, the extinction value is shown at 595 nm. Differences between vehicle and okadaic acid damage conditions were calculated. This value was set as the theoretical 100% recovery value. An unpaired student t-test was used for statistical analysis.

b) Cerebellar cell culture methods and materials Wistar rats at birth date 3, 4 or 8 were used for the study. Rats were sacrificed and placed in ice for 1 minute, the head was cut off and the cerebellum was removed and placed on ice. Cerebellar tissue was placed in 1 ml 0.65% glucose-capture PBS (10 μl 65% stock D (+) glucose / 1 ml PBS) in a large petri dish, minced into small pieces and 23 standards (0.4 mm ) Triturated with a 1 ml insulin syringe via a syringe needle and then sprayed into the glucose solution in a Petri dish. Tissues are sieved (125 μm pore size) and centrifuged (2 minutes at 60 × g) (twice), medium exchange in serum free BSA-capture START V medium (Biochrom, Germany) did. A second centrifugation step was performed with 1 ml of START V media. Micrografts were reconstituted in 500 μl START V media and placed on ice.

Production of culture cover slips and culture of cerebellar cells After 2 hours of PDL-coating (100 μg / ml), the slides were washed with Millipore H ₂ O and air dried. Each slide was placed in a small petri dish (diameter: 35 mm) and 40 μl of START V / cell suspension was added. The tissue was incubated for 2 hours at 34 ° C. (settlement phase). START V-medium (1 ml) was then added to the Petri dish and incubated at 34 ° C. in the presence of 5% CO ₂ in air at 100% humidity for 48 hours.

Cell damage, drug administration and cell survival analysis 10 μl of toxin 1 (L-glutamate in Millipore water—100 mM; final concentration: 0.5 mM) and 10 μl of toxin 2 (3-nitropropionic acid in Millipore water—50 mM pH 7— Final concentration: 0.5 mM) was simultaneously applied with the compound and tested (10 mM stock solution prepared in PBS and diluted to a final concentration between 1-100 nM). In each case, the drug was left for transplantation during the study.
After transplantation was exposed to the GPE analogs of the invention for the duration of the study, the cells were then rinsed in PBS and then fixed in increasing concentrations of paraformaldehyde (500 μl 0.4% PFA, Then 1.2% PFA; then 3% PFA and finally 4% PFA were applied (each solidification step: 2-3 minutes) Finally, the micrografts were rinsed with PBS The nerves in the transplant were then evaluated for morphology (presence of neurites) and counted as live cells through a microscopic field.The four areas displaying the highest cell density were covered slips. Counts and data were expressed as meaning (SEM); n = mean of 4 each ± standard error Statistical significance was assessed by using a non-versus Student t-test.

c) Rat cortical dissociated cell culture material Fetal E18 / 19 Wistar rats were used for the generation of dissociated cortical cell cultures. The posterior part of the cortical plate (occipital cortex of visual cortex—area 17/18) is used for the production of cultures. Cells were grown for 3-10 DIV in a portion of the serum-free medium and a portion of rat cortex-derived cultured astrological medium (ACM).
Methods 96-well tissue culture plates were coated with 0.2 mg / ml poly-L-lysine followed by 2 μg / ml laminin. Thereafter, 60 μl of ACM was added per well and the plate was stored at 37 ° C. until plated with cortical cells.

Production of Astral-Condition Medium (ACM ()) The whole cortex of 1/2 Wistar pup was collected in a separation tube containing 4 ml DMEM-1 cortex per tube. The pipette was crushed several times to break them into small pieces, each tube was poured separately into a reagent container and crushed once with a 5 ml syringe and an 18 gauge drafting syringe. Filtered through a cell strainer into a 50 ml centrifuge tube, washed with 5 ml DMEM, expanded the cell solution to a volume of 50 ml with DMEN, then centrifuged at 350 g for 5 minutes at RT. It was subjected to separation. cells were resuspended in 40ml of DMEM + 10% FBS. cells were then (tehn), was reconstituted in 75 cm ² cell flasks and At _{7 ℃ / 10% CO 2/} 100% humidity and incubated in the presence of 5nM okadaic acid. The toxin kills nerve cells and enrich the stellate entities in culture. The medium, fresh after 1 day DMEM + 10% FBS was replaced Cell growth was monitored and vehicle + FBS was replaced twice weekly until cells were fused (-10-14 days) As soon as confluency was reached, the cells were Washed once with 20 ml sterile PBS, cells were incubated for 3 days in 30 ml neural basal medium + B27 capture and cells were maintained under formation-free conditions Cell suspension was then 0.22 μm Through a sterile filter and frozen in aliquots at -80 ° C.

Cortical cell dissection and plating Visual cortex from fetus was collected and dissected-all steps performed in PBS + 0.65% glucose. Tissue was stored in PBS + glucose solution on ice until all dissections were complete. Tissue material was centrifuged at 350 g for 5 minutes at RT. The supernatant was removed and the cell pellet was incubated in 5 ml trypsin / EDTA solution (Invitrogen) for 8 minutes at 37 ° C. Proteolysis was stopped by the addition of a portion of DMEM + 10% FBS. The tissue was then centrifuged at 350 g for 5 minutes at RT. The pellet was collected, washed twice in NB / B27 medium (Invitrogen) (centrifugation) and resuspended in 1 ml NB / B27 medium. The tissue was transferred into a petri dish and crushed twice with a glass Pasteur pipette coupled to the pipette to break up large pieces of tissue. The tissue was then crushed twice using a 1 ml syringe and a 22 gauge syringe and the remaining large tissue piece removed through a 100 μm cell container. Cells were washed with 1 ml NB / B27. The number of viable neurons was counted and the percentage of cell viability determined. The cell solution was diluted to some degree with a final cell count of 50,000 cells (833,333 cells / ml) per 60 μl. Cortical cells were incubated at 37 ° C./10% CO ₂ /100% humidity.

Disorder-neuronal rescue agent and neuroprotective action analysis To produce nerve damage, the phosphatase 1 / 2A antagonist okadaic acid is added for 24 hours. At the same time, an analog of the GPE of the present invention was administered. After 24 hours, metabolic activity was measured by addition of MTT for 2-4 hours, followed by addition of SDS-solution and photometric endpoint measurement at 595 nm.
Student unpaired t-test is used for testing for statistical significance when comparing intact control with a condition-containing damaged nerve rescue agent.
Results In the absence of GPE analogs, okadaic acid treatment resulted in an approximately 30% reduction in viable cells (FIG. 1; second column from left) compared to untreated control cells (left column; open bar). It was. In other studies represented herein, okadaic acid reduced the number of viable cells from about 10% to about 50% (FIGS. 2-6).

FIG. 1 also shows that GPE treatment (hatched bar) reversed okadaic acid that caused loss of cell viability. Similarly, analog 49 (Glycyl-L-thia-5,5-dimethylprolyl-L-glutamic acid) is neuroprotective. Analog 49 protected the cells from okadaic acid-induced loss of cell survival in a concentration-dependent fashion. In the concentration range of 10 nM to 10 μM, treatment with analog 49 produced 100% protection against the loss of neuronal survival caused by okadaic acid.
FIG. 2 shows that analog 50 (glycyl- (D, L) -5,5-dimethylprolyl-L-glutamic acid), like analog 49, is also neuroprotective (FIG. 2). . In the concentration range of 10 nM to 100 nM, treatment with analog 50 produced 100% protection from the loss of neuronal survival caused by okadaic acid.
FIG. 3 shows that treatment with analog 60 (glycyl-trans-4-hydroxy-L-prolyl-L-glutamic acid) is also associated with statistically significant effects observed at concentrations of 100 nM and 100 μM and 1 mM. Indicates a protective action.

FIG. 4 shows that analog 64 (aminoisobutryl-L-propyl-L-glutamic acid) AibPe is neuroprotective. In the concentration range from 100 uM to 1 mM, analog 64 was more effective than GPE. At a concentration of 1 mM, treatment with analog 64 resulted in complete protection from the loss of neuronal survival induced by okadaic acid.
FIG. 5 shows that analog 65 (glycyl-L-prolyl-L-norvaline) is neuroprotective over a wide range of concentrations from 10 nM to 1 mM. At almost all concentrations tested, cells treated with analog 65 showed 100% protection against the loss of cell survival induced by okadaic acid.
FIG. 6 shows that analog 66 (glycyl-D, L-pipecolinyl-L-glutamic acid) is a neuroprotective effect with statistically significant effects observed at concentrations of 100 nM, 100 mM and 1 mM.
FIG. 7 shows that analog 67 (pyrrolidinoglycyl-L-2-methyl-proline-L-glutamic acid) protected cerebellar cells from neurite loss caused by okadaic acid. A statistically significant effect was observed at a concentration of 10 pM-10 nM.

One can be assessed from the comparison of FIGS. 1-6 and 7 where the damage caused by okadaic acid can be determined more finely by measuring neurite outgrowth than by cell survival. Okadaic acid reduced survival by about 10% to about 50% (FIGS. 1-6). In contrast, okadaic acid reduced the number of cells with neurites by more than 80%. Regardless of how the damage was assessed, each GPE analog tested had a statistically significant neuroprotective effect.
We conclude from these studies that the GPE analogs of the present invention can protect neurons from loss of survival and / or loss of neurites in response to the well-known neurotoxin, okadaic acid. . Because okadaic acid toxins make histological changes similar to those made by other causes of nerve damage, including hypoxia / ischemia, stroke, Parkinson's disease, etc., and study neuronal cell protection We conclude that this system for predicting the effects that would be observed using other types of neurodegenerative stimuli. Furthermore, the neuroprotective effects observed in these studies appear therefore to be highly relevant to other systems for studying neuroprotective effects, including in vivo studies in laboratory animals such as rats. In addition, because compounds related to GPE have been shown to protect animals in vivo against functional neurological defects and neuronal loss associated with a variety of different conditions, these studies have shown that We conclude that the inventive GPE analogs may be useful in treating various conditions associated with neurological deficits and neurological disorders.

It can be appreciated that the present invention is described with respect to specific examples thereof. Those skilled in the art will be able to make variations and other embodiments herein based on the disclosure and techniques without undue experimentation and with a reasonable chance of success. All these other embodiments are considered part of this invention.
FIG. 1 also shows that GPE treatment (hatched bar) reversed okadaic acid that caused loss of cell viability. Similarly, analog 49 (Glycyl-L-thia-5,5-dimethylprolyl-L-glutamic acid) is neuroprotective. Analog 49 protected the cells from okadaic acid-induced loss of cell survival in a concentration-dependent fashion. In the concentration range of 10 nM to 10 μM, treatment with analog 49 produced 100% protection against the loss of neuronal survival caused by okadaic acid.
FIG. 2 shows that analog 50 (glycyl- (D, L) -5,5-dimethylprolyl-L-glutamic acid), like analog 49, is also neuroprotective (FIG. 2). . In the concentration range of 10 nM to 100 nM, treatment with analog 50 produced 100% protection from the loss of neuronal survival caused by okadaic acid.
FIG. 3 shows that treatment with analog 60 (glycyl-trans-4-hydroxy-L-prolyl-L-glutamic acid) is also associated with statistically significant effects observed at concentrations of 100 nM and 100 μM and 1 mM. Indicates a protective action.
FIG. 4 shows that analog 64 (aminoisobutryl-L-propyl-L-glutamic acid) AibPe is neuroprotective. In the concentration range from 100 uM to 1 mM, analog 64 was more effective than GPE. At a concentration of 1 mM, treatment with analog 64 resulted in complete protection from the loss of neuronal survival induced by okadaic acid.
FIG. 5 shows that analog 65 (glycyl-L-prolyl-L-norvaline) is neuroprotective over a wide range of concentrations from 10 nM to 1 mM. At almost all concentrations tested, cells treated with analog 65 showed 100% protection against the loss of cell survival induced by okadaic acid.
FIG. 6 shows that analog 66 (glycyl-D, L-pipecolinyl-L-glutamic acid) is a neuroprotective effect with statistically significant effects observed at concentrations of 100 nM, 100 mM and 1 mM.
FIG. 7 shows that analog 67 (pyrrolidinoglycyl-L-2-methyl-proline-L-glutamic acid) protected cerebellar cells from neurite loss caused by okadaic acid. A statistically significant effect was observed at a concentration of 10 pM-10 nM.

FIG. 6 is a graph showing the effect of analog 49 (glycyl-L-thia-5,5-dimethylprolyl-L-glutamic acid) on neuronal survival after administration of apoptosis-inducing toxin (100 nM okadaic acid) in cortical cell culture. Effect of analog 50 (glycyl- (D, L) -5,5-dimethylprolyl-L-glutamic acid) on neuronal survival after excitotoxic oxidative stress in cortical cell cultures induced by 100 μM H ₂ O ₂ It is a graph which shows. FIG. 6 is a graph showing the effect of analog 60 (glycyl-trans-4-hydroxy-L-prolyl-L-glutamic acid) on neuronal survival after administration of apoptosis-inducing toxin (30 nM okadaic acid) in striatal cell culture. FIG. 6 is a graph showing the effect of analog 64 (aminoisobutryl-L-prolyl-L-glutamic acid) on neuronal survival after administration of apoptosis-inducing toxin (30 nM okadaic acid) in striatal cell culture. FIG. 6 is a graph showing the effect of analog 65 (glycyl-L-prolyl-L-norvaline) on neuronal survival after administration of apoptosis-inducing toxin (100 nM okadaic acid) in cortical cell culture. It is a graph which shows the effect of the analog 66 (Glycyl-D, L-pipecolinyl-L-glutamic acid) in survival of the neuron after administration of the apoptosis-inducing toxin (okadaic acid) in the striatal cell culture. Analog 67 (Pyrrolidinoglycyl-L-2-methyl-proline-L-glutamic acid) in neuronal survival after excitotoxicity / oxidative stress (induced by 0.5 mM 3-NP glutamate) in cerebellar cell cultures It is a graph which shows the effect of.

Claims (41)

A compound of the following formula or a pharmaceutically acceptable salt or hydrate thereof:
Wherein the bond between X ¹ and X ² is either saturated or unsaturated;
X ¹ is selected from the group consisting of CH ₂ , S, C (OH) H, and is CH when the bond between X ¹ and X ² is unsaturated;
X ² is selected from the group consisting of CH ₂ , CH ₂ CH ₂ and is CH when the bond between X ¹ and X ² is unsaturated;
R ¹ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ² is CH ₃ or COOH;
R ³ is selected from the group consisting of H, alkyl, or NR ³ R ³ is collectively pyrrolidino or piperidino;
R ⁴ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ⁵ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl, or substituted arylalkyl; and n is an integer from 0-2. ) X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is CH ₂ —CH ₃ ; R ² is COOH; R ³ is H; R ⁴ is The compound of claim 1 (Glycyl-L-2-ethylprolyl-L-glutamic acid ("G-2 EthylPE")) wherein H is H; R ⁵ is H; and n is 0. X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is CH ₂ —CH ₂ —CH ₃ ; R ² is COOH; R ³ is H; R The compound of claim 1 wherein ⁴ is H; R ⁵ is H; and n is 0 (glycyl-L-2-propylprolyl-L-glutamic acid ("G-2PropylPE")) . X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is CH ₂ —CH═CH ₂ ; R ² is COOH; R ³ is H; R ⁴ The compound according to claim 1, wherein R ⁵ is H; and n is 0 (Glycyl-L-2-allylprolyl-L-glutamic acid trifluoroacetate (“G-2AllylPE”)) . X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is benzyl; R ² is COOH; R ³ is H; R ⁴ is H; A compound according to claim 1 wherein R ⁵ is H; and n is 0 (glycyl-L-2-benzylprolyl-L-glutamic acid trifluoroacetate ("G-2benzylPE")). X ¹ is S; X ² is CH ₂ ; R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is H; R The compound according to claim 1, wherein ⁵ is H; and n is 0 (glycyl-L-4-thiaprolyl-L-glutamic acid trifluoroacetate). X ¹ is S; X ² is CH ₂ ; R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is CH ₃ ; The compound of claim 1 wherein R ⁵ is H; and n is 0 (glycyl-L-thia-5,5-dimethylprolyl-L-glutamic acid (“G-thiadiMePE”)). X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is CH ₃ ; R ⁵ is is H; a compound of claim 1 and n is 0 (glycyl - (D, L)-5,5-dimethyl-prolyl -L- glutamic acid). X ¹ is C (OH) H; X ² is CH ₂ ; R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is H The compound of claim 1 wherein R ⁵ is H; and n is 0 (glycyl-trans-4-hydroxy-L-prolyl-L-glutamic acid (“GHypE”)). X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is is H; a compound of claim 1 and n is 1 (glycyl -L- Homopuroriru -L- glutamic acid ( "GHomoPE")). The bond between X ¹ and X ² is unsaturated; X ¹ is CH; X ² is CH; R ¹ is H; R ² is COOH; R ³ is And R ⁴ is H; R ⁵ is H; and n is 0. The compound of claim 1 (glycyl-L-3,4-dehydroprolyl-L-glutamic acid) Trifluoroacetate, ie "G-3,4-dehydroPE.TFA"). X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is H; R ⁵ is, be CH _3; a compound according to claim 1 and n is 0 (amino isobutoxide tolyl -L- prolyl -L- glutamic acid ( "AibPE")). X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is H; R ² is CH ₃ ; R ³ is H; R ⁴ is H ; R ⁵ is is H; a compound of claim 1 and n is 0 (glycyl -L- prolyl -L- norvaline ( "GPNorvaline")). X ¹ is CH ₂ ; X ² is CH ₂ CH ₂ ; R ¹ is H; R ² is COOH; R ³ is H; R ⁴ is H A compound according to claim 1, wherein R ⁵ is H; and n is 0 (glycyl-D, L-pipecolinyl-L-glutamic acid (“G (D, L) PipE”)). X ¹ is CH ₂ ; X ² is CH ₂ ; R ¹ is CH ₃ ; R ² is COOH; NR ³ R ³ are collectively pyrrolidino; R ⁴ 2 is H; R ⁵ is H; and n is 0. The compound of claim 1 (pyrrolidinoglycyl-L-2-methyl-proline-L-glutamic acid (“PyrrolidinoG-2MePE”) )). A compound of the following formula or a pharmaceutically acceptable salt or hydrate thereof:
(In the formula, R is H or OH.)   The compound according to claim 16, wherein R is H ((2S, 5'R)-[1 '-(2 "-amino-acetyl) -6'-oxo-1', 7'-diazaspiro [4.4 ] Non-7'-yl] -1,5-pentanedioic acid ("GP-5,5-spirolactam E").   The compound according to claim 16, wherein R is OH ((2S, 5'R, 8'R)-and (2S, 5'R, 8'S)-[1 '-(2 "-amino-acetyl) -8′-hydroxy-6′-oxo-1 ′, 7′-diazaspiro [4.4] non-7′-yl] -1,5-pentanedioic acid (“GP-5,5-hydroxyspirolactam E”)) . A compound of the following formula or a pharmaceutically acceptable salt or hydrate thereof:
Wherein R ¹ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
R ² is CH ₃ or COOH;
R ³ is selected from the group consisting of H, alkyl, or NR ³ R ³ is collectively pyrrolidino or piperidino;
R ⁴ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl;
n is an integer of 0-2. ) R ¹ is is H; R ² is located at COOH; R ³ is is H; R ⁴ is is H; a compound of claim 19 and n is 0 (glycyl -L- 2-pyroglutamyl-L-glutamate hydrochloride (“GpyroE.HCl”)). A compound of the following formula or a pharmaceutically acceptable salt or hydrate thereof:
(Wherein R ¹ is selected from the group consisting of H, alkyl, alkenyl, aryl, arylalkyl, substituted alkyl, substituted alkenyl, substituted aryl or substituted arylalkyl.) A compound according to claim 21 R ¹ is CH ₃ (glycyl -L-2-methyl proline ( "G-2MeP")) .   23. A pharmaceutical composition comprising a compound according to any one of claims 1 to 22 and a pharmaceutically acceptable excipient.   23. A pharmaceutical composition comprising a compound according to any one of claims 1 to 22, a pharmaceutically acceptable excipient, and a binder.   23. A pharmaceutical composition comprising a compound according to any one of claims 1 to 22, a pharmaceutically acceptable excipient, and a capsule.   23. A method of treating an animal to protect nervous system cells from death or degeneration as a result of injury or disease, comprising an effective amount of compound 1 or 2 according to any one of claims 1-22. And administering a pharmaceutically acceptable excipient to the patient.   27. The method of claim 26, wherein the animal is a human.   27. The method of claim 26, wherein the injury or disease is characterized by apoptotic neuronal cell death.   27. The method of claim 26, wherein the injury or disease is characterized by necrotic neuronal cell death.   27. The method of claim 26, wherein the injury or disease is characterized by neuronal degeneration.   Disease is Huntington's disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, peripheral neuropathy, spinal muscular atrophy, Creutzfeldt-Jakob disease, dementia due to AIDS, progressive nuclear 27. The method of claim 26, selected from the group consisting of paralysis, myelinopathia centralis diffusa (burnishing white matter disease), chronic neurodegenerative disease, Down's syndrome, leukoencephalopathy and Schilder's disease.   Injury or disease is neuroblastoma, head trauma, traumatic brain injury, stroke, ischemic injury, hypoxia injury, reperfusion injury, epilepsy, heart disease arterial bypass graft surgery, poison injury, radiation injury and breathing 27. The method of claim 26, wherein the method is the result of one or more states selected from the group consisting of stops.   27. The method of claim 26, wherein the injury or disease is a result of an inflammatory condition.   Injury or disease from chronic or acute encephalomyelitis, encephalitis, optic neuritis, transversal myelitis, meningitis, panencephalitis, Devic's disease, progressive multifocal leukoencephalopathy, Hashi center myelination and optic neuromyelitis 27. The method of claim 26, wherein the method is the result of one or more states selected from the group consisting of:   27. The method of claim 26, wherein the disease is the result of schizophrenia or depression.   27. The method of claim 26, wherein at least one other anti-apoptotic agent, anti-necrosis agent or neuroprotective agent is administered.   Other apoptotic or neuroprotective agents include GPE, GPE diketopiperazine analogs, GPE macrocyclic analogs, GPE bicyclic analogs, neuroregenerative peptides, insulin-like growth factor-I (IGF-1) , Insulin-like growth factor-II (IGF-II), transforming growth factor-β1, activin, growth hormone, nerve growth factor, growth hormone binding protein, IGF-binding protein, IGFBP-3, basic fibroblast growth factor Acidic fibroblast growth factor, hst / Kfgk gene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor, androgen inducible growth factor, int-2, fibroblast growth factor homologous factor-1 ( FHF-1), FHF-2, FHF-3 and FHF-4, caratinocyte growth factor 2, glial activator, FGF-1 And FGF-16, ciliary neurotrophic factor, brain-derived growth factor, neurotrophin 3, neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial cell line-derived neurotrophic factor, activity-dependent neurotrophic Factor, cytokine leukemia inhibitory factor, oncostatin M, interleukin, α-interferon, β-interferon, γ-interferon, consensus interferon, TNF-α, chromethiazole; kynurenic acid, semax, tacrolimus, L-threo-1-phenyl From 2-decanoylamino-3-morpholino-1-propanol, adrenocorticotropin- (4-9) analog ORG2766, disorcipin (MK-801), selegiline, glutamate antagonist, AMPA antagonist, and anti-inflammatory agent Claims selected from the group The method according to 26.   38. The method of claim 37, wherein the glutamate antagonist is selected from the group consisting of NPS1506, GV1505260, MK-801, and GV150526.   38. The method of claim 37, wherein the AMPA antagonist is selected from the group consisting of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (f) quinoxaline (NBQX), LY303070 and LY300164.   38. The method of claim 37, wherein the anti-inflammatory agent is selected from the group consisting of anti-MAdCAM-1 antibodies and antibodies to integrin α4β1 receptor and integrin α4β7 receptor.   41. The method of claim 40, wherein the anti-MAdCAM-1 antibody is MECA-367.