TW201311636A - Compounds for use in the treatment of autoimmune inflammatory disease - Google Patents

Abstract

Compounds particularly for use in an autoimmune inflammatory disease and especially the treatment of inflammatory bowel disease have the formula: wherein R is selected from one or more of the same or different of hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted aryl, alkoxy, aryloxy, thiol, and optionally substituted amino, and wherein R1 is selected from one or more of the same or different of hydrogen, acetyl, optionally substituted alkyl, optionally substituted aryl, and an amino acid selected from leucine, valine, isoleucine, and glycine.

Description

Compound for the treatment of autoimmune inflammatory diseases

The present invention relates to compounds which are particularly useful for autoimmune inflammatory diseases and in particular for the treatment of inflammatory bowel diseases.

Cytokines can be produced by a variety of cell populations and have been shown to potentiate or limit immune responses to pathogens and affect autoimmune responses. Has been a family of cytokines (which use the common receptor gamma chain (cc) (a type of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21 Body components)) are generally defined as growth and survival factors.

IL-2 production induces an immune response by promoting proliferation and production of CD4+ Th1, CD4+ Th2 and CD8+ CTL effector cells. Many immunosuppressive drugs (such as corticosteroids) and immunosuppressive drugs (cyclosporine, tacrolimus) used in the treatment of autoimmune diseases and organ transplant rejection by inhibiting the production of IL-2 by antigen-activated T cells And it works. Other drugs (sirolimus) block IL-2R signaling, thereby preventing the expansion and function of antigen-selective T cells. [Reference: Opposing functions of IL-2 and IL-7 in the Regulation of immune responses Shoshana D. Katzman, Katrina K. Hoyer, Hans Dooms, Iris K. Gratz, Michael D. Rosenblum, Jonathan S. Paw, Sara H. Isakson, Abul K. Abbas. Cytokine 56 (2011) 116-121 ].

In contrast, IL-2 promotes the induction and production of induced (peripheral) Tregs by promoting the survival and function of natural (thymus) regulatory T-cells (Tregs). Inhibition of immune response by the production of CD4+ Th17 effector cells [Reference: IL-2 and autoimmune disease. Anneliese Schimpl, A., Berberich, I, Kneitz, B., Krämer, S., Santner-Nanan, B., Wagner, S ., Wolf, M., Hünig, T. Cytokine & Growth Factor Reviews 13 (2002) 369-378]. Defects of interleukin-2/IL-2R over time will result in inflammation of multiple organs and the formation of multiple specific autoantibodies. Depending on the genetic background, death will occur in weeks to months, with most dying from autoimmune hemolytic anemia or inflammatory bowel disease (IBD) [Reference: Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 1993; 75: 253-61].

IL-2 signaling has been shown to be important in the initiation and regulation of immune responses. In these dual and antagonistic roles, IL-2 acts to balance the immune response, promoting both immune cell activation and subsequent reduction. The potential clinical applications for enhancing or inhibiting IL-2 mediated signals are significant and include cancer, autoimmune inflammatory diseases, organ transplantation, and HIV.

Inflammatory bowel disease (IBD) is an autoimmune inflammatory disease that includes two congenital inflammatory diseases: ulcerative colitis (UC) and Crohn's disease (CD). The biggest difference between UC and CD is the extent of inflamed bowel tissue. In the CD, the inflammatory system is discontinuously segmented, called local enteritis, and UC is superficial inflammation, extending proximally and continuously from the rectum. Currently, the exact cause of IBD is not known. The disease appears to be associated with an over-immune response of the mucosa to intestinal epithelial cell infection because pro-inflammatory molecules are not balanced with immunomodulatory molecules. The genetic pattern of IBD suggests that complex genetic components of pathology can include several combinatorial gene mutations. With the pathology machine The understanding of the system has improved, and there is still no specific diagnostic test for IBD. The corresponding test method is the same. Treatment of IBD involves induction and maintenance of remission. IBD patients can be relieved by the use of 5-aminosalicylate. However, although the use of aminosalicylate in UC provides significant benefits in inducing both mild to moderate disease remission and prevention of relapse, the effectiveness of these drugs in maintaining CD remission is not reliable and is no longer Recommended. The treatment of active disease mainly relies on corticosteroids, which are often used to relieve UC and CD patients within a limited time, although the systemic absorption of budesonide designed for local administration is limited, but in terms of maintaining remission No benefit. Alternatives, such as the immunosuppressive drugs azathioprine and guanidinium, have limited efficacy with methotrexate and cyclosporine and can cause serious side effects. Anti-TNFα antibodies, such as unfliximab and adalimubab, can be used in patients who do not respond to standard immunosuppressive therapies. However, many patients do not respond to anti-TNFa therapy due to their specific phenotype or the production of autoantibodies.

According to the present invention, there are provided compounds for the treatment of autoimmune inflammatory diseases and inflammatory bowel diseases which specifically include Crohn's disease and ulcerative colitis.

In one aspect of the invention, a compound having a relative stereochemistry and a formula is provided:

Wherein R is selected from one or more of the following or different hydrogen, hydroxy, optionally substituted alkyl, optionally substituted aryl, alkoxy, aryloxy, thiol, and optionally a substituted amine group, and wherein the R ₁ group is selected from the group consisting of the same or different ones or more of hydrogen, an ethyl hydrazide group, an optionally substituted alkyl group, an optionally substituted aryl group, and one selected from the group consisting of leucine In the present case, the compounds herein include the pharmaceutically acceptable salts, esters, guanamines, solvates and prodrugs thereof, the amino acids of proline, isoleucine and glycine.

The invention also provides a compound having absolute stereochemistry and the following formula:

Wherein R is selected from one or more of the following or different hydrogen, hydroxy, optionally substituted alkyl, optionally substituted aryl, alkoxy, aryloxy, thiol, and optionally a substituted amine group, and wherein the R ₁ group is selected from the group consisting of the same or different ones or more of hydrogen, an ethyl hydrazide group, an optionally substituted alkyl group, an optionally substituted aryl group, and one selected from the group consisting of leucine , amidic acid, isoleucine and amino acid of glycine.

In one embodiment, the alkyl group contains from 1 to 10 carbon atoms in a straight or branched chain, and may be saturated or unsaturated, or a cycloalkyl group containing from 3 to 8 carbon atoms, which may be saturated or Unsaturated.

In some embodiments, the alkyl group is substituted with one or more of the same or different ones: alkyl, alkoxy, alkylamino, decyl, amine, aryl, aralkyl, aryloxy, Carboxy, halo, hydroxy, nitrile, nitro or pendant oxy group.

In one embodiment, the aryl group is substituted with one or more of the following or different: alkyl, alkoxy, alkylamino, decylamino, amine, anhydride, aryl, Aralkyl, aryloxy, carboxy, halo, hydroxy, nitrile, nitro or pendant oxy group.

In one embodiment, the amine group is substituted with one or more of the same or different ones: an alkyl group, a hydroxyalkyl group, an aryl group, and a substituted aryl group. In some cases, the amine group is substituted with an aryl group substituted with one or more of OH, NH ₂ and COOH.

In an embodiment, R is OH.

In an embodiment, R ₁ is H.

The present invention also provides a compound having the following absolute stereochemistry and the following formula Wherein R is selected from the group consisting of: OH, OCH ₃ , OCH ₂ CH ₃ , OCH ₂ CH ₂ CH ₃ , NH ₂ , NHCH ₂ CH ₂ OH, NHCH ₃ , N(CH ₃ ) ₂ , NH(4-OH-3- benzoic acid).

Compounds with absolute stereochemistry and the following formula are also available Wherein R ¹ is H or leucine, and R is selected from the group consisting of: OH, OCH ₃ , OCH ₂ CH ₃ , OCH ₂ CH ₂ CH ₃ , NH ₂ , NHCH ₂ CH ₂ OH, NHCH ₃ , N(CH ₃ ) ₂ , NH (4-OH-3-benzoic acid).

In some aspects, R may be selected from one or more of the following or different: H, hydroxy, alkoxy, alkylcarbonyl, aryloxy, anhydride, substituted anhydride, amine, substituted amine Base, decylamine, alkylamine, nitro, nitrite, nitrile, aryl group of mono and polybenzoic acid, substituted aryl group, thiol, thioureyl, benzene thiol a group, a sulfonic acid group, an anthracene group, a fluorenyl group, an alkyl group having 1 to 10 carbon atoms or a cycloalkyl group having 3 to 8 carbon atoms (which may be saturated or unsaturated), And substituted alkyl or cycloalkyl (which may be saturated and unsaturated).

In some aspects, R ₁ may be selected from one or more of the following or different: H, alkoxy, alkylcarbonyl, aryl, ethoxylated, alkyl having from 1 to 10 carbon atoms or a cycloalkyl group having 3 to 8 carbon atoms which may be saturated or unsaturated, a substituted alkyl group, and an amine selected from the group consisting of leucine, valine, isoleucine and glycine Base acid.

Also provided herein are pharmaceutically acceptable salts, esters, guanamines and solvates.

In one embodiment, the alkyl or cycloalkyl group is substituted by one or more of the following: halo, pendant oxy, hydroxy, alkoxy, aryloxy, carboxy, amine, decylamine, alkylamino, nitro, nitrate, nitrite, nitro group, a nitrile, a heterocyclic group, an aryl group, an aralkyl group, by OH, COOH and / or NH ₂ substituted alkyl of.

In one embodiment, the amine group is substituted with one or more of the same or different ones: a hydroxyl group, an alkylhydroxy group, an aryl group, and an aryl group substituted with one or more of OH, NH ₂ and COOH.

In one case, the anhydride is substituted with an aryl group or an aryl group substituted by one or more of OH, NH ₂ and COOH.

In one embodiment, R is OH and R ₁ is H.

The invention also provides a compound of formula I: Also provided are pharmaceutically acceptable isomers and salts of the compound of formula (I) (Compound 1 ).

In particular, the present invention provides compounds of relative stereochemistry as shown in Structural Formula II: Also provided are pharmaceutically acceptable salts of the compound of formula (II) (Compound 2 ).

The active enantiomeric system is spectrally identified based on its physical and chemical properties, along with standard and palmitic HPLC retention data.

It has been found that a particular enantiomeric form is particularly effective in the treatment of IBD.

The invention also provides N-methyl-(D)-glucosamine salts of the compounds of formula III:

The invention further provides a pharmaceutical composition comprising an effective amount of a compound of the invention and a pharmaceutically acceptable carrier.

The invention also provides a method of preventing or treating inflammatory bowel disease comprising administering to an individual an effective amount of a compound of the invention.

The invention also provides a method of preventing or treating ulcerative colitis comprising administering to an individual an effective amount of a compound of the invention.

Also provided is a method of preventing or treating Crohn's disease comprising administering to an individual an effective amount of a compound of the invention.

The compounds of the invention are also effective in enhancing or inhibiting IL-2 mediated signaling. Clinical uses include treatment of cancer, autoimmune inflammatory conditions, organ transplantation, and HIV.

Specifically, the present invention provides a compound as exemplified below.

The invention also provides a compound of the formula:

The compound includes two diastereomers Preferred isomer is

The invention further provides a pharmaceutical composition comprising any of the above compounds.

The active compound may be presented in a suitable amount in a pharmaceutical formulation in a suitable dosage to achieve the desired effect. For example, the final dose can be between 0.1 and 10 mg/kg.

The compounds of the invention can be administered in bulk active chemical form. However, it is preferred to administer such compounds in the form of a pharmaceutical formulation or composition. Such formulations may include one or more pharmaceutically acceptable excipients, carriers or diluents.

The compounds of the invention can be administered in a number of different ways. These compounds can be administered orally. Preferred pharmaceutical formulations for oral administration include lozenges, capsules, caplets, solutions, suspensions or syrups.

Pharmaceutical formulations may be provided in a modified release form, such as a timed release capsule or lozenge.

The agent can be administered orally, parenterally, intranasally, transdermally or by inhalation.

The invention also provides a method of preventing or treating inflammatory bowel disease comprising administering to an individual an effective amount of a compound of the invention.

The invention further provides a method of preventing or treating ulcerative colitis comprising administering to an individual an effective amount of a compound of the invention.

The invention also provides a method of preventing or treating Crohn's disease comprising administering to an individual an effective amount of a compound of the invention.

As described herein, the compounds of the invention may optionally be substituted with one or more substituents, such as generally described above, or as indicated by the particular classes, subcategories, and species of the invention. In general, the term substituted refers to the replacement of a hydrogen atom in a given structure using a particular substituent. Unless otherwise indicated, optionally substituted groups may have a substituent at each substitutable position of the group, and when more than one position in any given position structure may be selected from more than one When a substituent of a particular group is substituted, the substituents at each position may be the same or different. Preferably, combinations of substituents contemplated by the present invention are those which result in the formation of stable or chemically feasible compounds. The term "stable" as used herein refers to a compound that does not substantially change when subjected to conditions of manufacture, detection, and better recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, the stabilizing compound or chemically feasible compound is a compound that does not substantially change when kept at a temperature of 40 ° C or less, free of moisture or other chemical reaction conditions for at least one week.

The present invention is described in terms that are known and understood by those skilled in the art. For ease of reference, several terms are defined below, but are not limited to clarifying the terms defined.

The term alkyl refers to a straight or branched chain hydrocarbon, which preferably has from one to ten carbon atoms. Typical alkyl groups are methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, t-butyl, isopentyl, n-pentyl and the like.

The term unsaturated means a group having one or more units of unsaturation.

The term cycloalkyl refers to a non-aromatic cyclic hydrocarbon ring. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term "cycloalkyl" includes fused ring systems in which, for example, a cycloalkyl ring is fused to an aromatic ring.

The term aryl, used alone or as part of a larger group, refers to a monocyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one of the rings in the system is an aromatic ring, and wherein the system is Each ring contains 3 to 7 ring members. In particular, the term aryl refers to a benzene ring or a fused benzene ring system such as a fluorene, phenanthrene or naphthalene ring system. Examples of the aryl group include a phenyl group, a 2-naphthyl group, a 1-naphthyl group and the like.

The term alkoxy refers to the group --OX, wherein X is alkyl as defined herein.

The term aryloxy, alone or as part of another group, includes any of the above-described aryl groups attached to an oxygen atom.

As used herein, alkoxycarbonyl refers to the group -C(O)OX, wherein X is alkyl as defined herein.

The term amine refers to a nitrogen group substituted with hydrogen, an alkyl group, an aryl group or a combination thereof. Examples of the amine group include -NHmethyl, -NH ₂ , -N(methyl) 2, -N-benzyl, -NHphenyl, -Nethylmethyl, and the like. "Alkylamino" refers to a nitrogen group substituted with at least one alkyl group. Examples of the alkylamino group include -NH methyl group, -N(methyl) 2, -N propylmethyl group, -NH butyl group, -Nethyl methyl group, -N phenyl group Base and so on. "Aromatic amine group" means a nitrogen atom substituted with at least one aryl group.

The term "halogen" means fluoro, chloro, bromo or iodo.

The term "hydroxy" refers to the group --OH.

The term "thiol" refers to the group --SH.

The compounds of the invention may be crystallized in more than one form. This property is referred to as a polymorphism, and such polymorphic forms ("polymorphs") fall within the scope of the invention. Polymorphisms typically occur with a reaction to temperature, pressure, or both. The difference in crystallization methods can also cause polymorphism. Polymorphs can be distinguished by various physical properties known in the art, such as x-ray diffraction patterns, solubility, and melting point.

Several of the compounds described herein may exist as stereoisomers. The scope of the invention includes mixtures of stereoisomers and purified or concentrated mixtures. Also included within the scope of the invention are individual isomers of the compounds of the invention and any fully or partially balanced mixtures thereof. Several compounds of the invention comprise one or more pairs of palmar centers. Accordingly, the invention includes racemates, purified enantiomers and enantiomerically concentrated mixtures of the compounds of the invention. The compounds of the invention include racemic and palmitic 1,2-dihydroindole dimers. Preferably, the salt of the invention is a pharmaceutically acceptable salt. Terminology medicine Salts encompassed by the above acceptable salts refer to the non-toxic salts of the compounds of the invention. Salts of the compounds of the invention may include acid addition salts.

Solvate refers to a variable stoichiometric complex formed by a solute (in the present invention, a compound of the invention or a salt thereof or a physiologically functional derivative thereof) and a solvent. For the purposes of the present invention, such solvents should not interfere with the biological activity of the solute. Non-limiting examples of suitable solvents include, but are not limited to, water, methanol, ethanol, and acetic acid. Preferably, the solvent used is a pharmaceutically acceptable solvent. Non-limiting examples of suitable pharmaceutically acceptable solvents include water, ethanol, and acetic acid. Most preferably, the solvent used is water.

Prodrug means any pharmaceutically acceptable derivative of a compound of the invention which, when administered to a mammal, provides (directly or indirectly) a compound of the invention or an active metabolite thereof. Such derivatives (e.g., esters and guanamine) will be readily apparent to those skilled in the art.

The pharmaceutical formulation can be modified for administration by any suitable route, such as orally (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including Subcutaneous, intramuscular, intravenous or intradermal) pathways. Such formulations may be prepared by incorporating the active ingredient with carriers or excipients.

Pharmaceutical formulations suitable for oral administration may be discrete units (such as capsules or lozenges; powders or granules; solutions or suspensions, each having an aqueous or non-aqueous liquid; edible foam; or an oil-in-water liquid emulsion or water-in-oil) Liquid emulsion) is presented. For oral administration in the form of a lozenge or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Powder can be crushed by compounding It is suitably micronized and prepared by mixing with a suitable pharmaceutical carrier such as an edible carbohydrate such as starch or mannitol. Flavoring agents, preservatives, dispersing agents, coloring agents, and the like can also be included.

Capsules can be made by preparing a powder, liquid, or suspension mixture and sealing into gelatin or other suitable shell material. A lubricant such as colloidal cerium oxide, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the mixture. When the capsule is ingested, a disintegrating or solubilizing agent such as calcium carbonate or sodium carbonate may be added to enhance the utilization of the agent. Other formulations may also be incorporated into the mixture, such as binding agents, lubricants, disintegrants, and color formers. Examples of suitable binding agents include starch, gelatin, natural sugars, corn sweeteners, natural or synthetic gums, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, and the like. Lubricants suitable for such dosage forms include, for example, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Suitable disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Tablets can be prepared by preparing a powder mixture, granulating the mixture, adding a lubricant and a disintegrant, and compressing into a tablet. The powder mixture can be prepared by mixing the compound with a diluent or a base as described above, suitably pulverizing. Optional ingredients include binders (such as carboxymethylcellulose, alginic acid, gelatin or polyvinylpyrrolidone), solvents such as paraffin, absorption enhancers (such as quaternary salts) and/or absorbents (bentonite, kaolin) Or similar). The powder mixture can be wet granulated using a binder such as a syrup, starch paste or a solution of cellulose or polymeric material and passed through a sieve.

The compounds of the invention may also be combined with a free flowing inert carrier and directly The tablet is compressed without the need for other steps such as granulation. A transparent or opaque protective coating consisting of a sealing layer of a suitable material such as shellac, sugar or polymeric material, and a polishing coating such as a wax may be provided. If appropriate, colorants are added to the coatings to distinguish between different unit doses.

Oral solutions (such as solutions, syrups and elixirs) can be presented in unit dosage form such that a given amount comprises a predetermined amount of compound. A syrup can be prepared, for example, by dissolving the compound in an aqueous solution of a suitable taste, and the elixirs are prepared by using a non-toxic alcohol vehicle. The suspension can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers (such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether), preservatives; flavor additives (such as peppermint oil) or natural sweeteners, saccharin, or other artificial Sweeteners and more.

Dosage unit formulations for oral administration may be microencapsulated, where appropriate. Formulations can also be prepared, for example, by coating or embedding particulate material in a suitable polymer, wax, or the like, for extended or sustained release.

The compounds of formula (I), and salts, solvates and physiologically functional derivatives thereof, may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be prepared from a variety of phospholipids, such as cholesterol, stearylamine or phospholipid choline.

The compounds of the invention, as well as salts, solvates and physiologically functional derivatives thereof, can also be administered by the use of monoclonal antibodies as a single carrier to which the molecules of the compound are coupled.

The compound can also be coupled to a soluble polymer as a targeted delivery vehicle. Such polymers include, for example, polyvinylpyrrolidone (PVP). The compound can also be coupled to a biodegradable polymer to achieve controlled release of the drug. These polymers include block copolymers of polylactic acid, polycyanoacrylate, and hydrogels.

Pharmaceutical formulations suitable for transdermal administration can be presented as discrete patches to maintain long-term intimate contact with the patient's skin/skin. For example, the active ingredient can be delivered by ion penetration from the patch.

Pharmaceutical formulations suitable for topical administration may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. For the treatment of extracorporeal tissue, the formulation can be administered as a topical ointment or cream.

For topical administration to the oral cavity, the formulation may include buccal tablets, tablets, and mouthwashes.

For nasal administration, a powder having a particle size of, for example, between 20 and 500 microns can be used. The powder can be administered by rapid inhalation through the nasal passages from a powder container held close to the nose. Suitable formulations for administration as a nasal spray or nasal drops wherein the carrier is a liquid include aqueous or oily solutions of the active ingredient.

Pharmaceutical formulations suitable for administration by inhalation include finely divided powders or mists which may be produced by means of pressurized quantitative aerosols, nebulizers or insufflators and the like.

For rectal administration, the formulation can be presented as a suppository or as an enema.

For vaginal administration, the formulation can be administered in the form of a pessary, a hemostatic tampon, a cream, a gel, a spray, or the like.

For parenteral administration, the formulation may be an aqueous and non-aqueous sterile injectable solution which may contain various additives such as an antioxidant, a buffer, a bacteriostatic agent, and a solute which renders the formulation isotonic with the blood of the intended recipient; And aqueous or non-aqueous sterile suspensions which may include suspending and thickening agents. Formulations may be presented in single or multiple dose containers, such as encapsulated ampoules and vials, and may be stored under lyophilization (lyophilization) conditions, which require the addition of a sterile liquid carrier (eg, water for injection) prior to use. . The ready-to-use injections and suspensions can be prepared from sterile powders, granules and the like.

The compounds of the present invention, and salts, solvates and physiologically functional derivatives thereof, may also be used alone or in combination with other therapeutic agents. The compounds of the invention and other pharmaceutically active agents may be administered together or separately. If administered separately, the administration can be done simultaneously or sequentially (in any order). The amount of the compound of the invention and other pharmaceutically active agents, as well as the relative timing of administration, will be selected to achieve the desired combination of therapeutic effects. The combination of a compound of the present invention and a salt, solvate or physiologically functional derivative thereof with other treating agents can be administered by a single pharmaceutical composition comprising the two compounds, or as a separate pharmaceutical comprising one of the compounds, respectively. The compositions are combined and administered in combination. In some cases, the combination of drugs can be administered separately in a continuous manner, with one formulation being administered for the first time and the second formulation being administered a second time, or vice versa. Such administration can be performed in the same time frame or over a longer period of time.

The invention will be more fully understood from the following description, which is provided by way of example only.

Compound 1 represents a pair of diastereomers which are produced by the reduction and demethylation of ketone compound A, which have a palmitic center at C-2 and are therefore a pair of enantiomers.

Reduction of the compound using LiAlH ₄ to produce a compound of the formula This compound includes two diastereomers: Hydrolysis of diastereomer B produces compounds 2 and 3 Diastereoisomer C hydrolysis produces compounds 4 and 5 These diastereomers can be resolved chemically or chromatically to their constituent enantiomers.

The absolute stereochemistry of compound 4 has been established by single crystal X-ray of compound 4(R)-(+)-methylbenzylamine salt (compound 9 ) (Fig. 1).

Chemistry by absolute stereochemistry of compound 2 Compound 2 (S) - (-) - methylbenzylamine salt (Compound 8) of the single crystal X- ray is determined (FIGS. 2 and 2A).

The invention also relates to compounds having the formula: These compounds include two diastereomers Preferred diastereomers having the following relative stereochemistry are presented below This diastereomer consists of two enantiomers. The absolute stereochemistry of the preferred enantiomer is presented below

General reaction procedure

A general synthetic procedure for the coupling of enantiomeric mixtures as exemplified below is described in WO 9720806 A, the entire contents of which is incorporated herein by reference.

General preparation of acid-derived compound A

Addition of (4-bromomethyl)benzoic acid methyl ester (6 mmol, 1.41 g) to a solution of the title compound (4 mmol, 1.00 g) in tert-butanol (5 mL) and diethyl ether (30 mL) ) in the stirring solution. A solution of potassium t-butoxide in tert-butanol (30 mL) and diethyl ether (5 mL) was slowly added dropwise thereto. Each time a drop is added, the mixture turns yellow and then returns to its original gray color. The mixture was further stirred for 3 hours until TLC (80:20, hexanes: ethyl acetate) showed no starting material. By the addition of saturated NH ₄ Cl The reaction was quenched. The layers were separated and the aqueous layer was extracted with diethyl ether (2×120 mL). The combined organic layers were washed with water and brine, dried over MgSO ₄ and evaporated. The solid product precipitated from the crude product upon removal of most of the solvent. The solid was filtered off and washed with cold diethyl ether to give <RTIgt;

Reduction of methyl benzoate compound

Tris-tris-butoxyaluminum hydride (1.9 mmol, 0.48 g) was added portionwise to a stirred solution of methyl benzoate (1.27 mmol, 0.50 g) in THF (15 mL). The reaction was monitored by TLC (80:20, hexanes: ethyl acetate) and after 3 hours, all starting material was consumed.

The reaction was quenched by pouring onto ice, and ethyl acetate was used, and the crude mixture was extracted from ethyl acetate by stirring for 10-15 minutes, then poured into a separating funnel and then separated. With water, the organic layers were washed with brine, dried over MgSO ₄ and evaporated to yield 0.34 g (68%) tan solid emulsions. This product was isolated as a mixture of two diastereomers in a ratio of about 2:1.

Analysis of the mixture of two diastereomers

Purity (HPLC): 94.9% (diastereoisomer in a ratio of 2:1)

δ _H (300 MHz, CDCl ₃ ): 2.77-3.60 (6H, m , 3 × C H ₂ ), 3.85 (3H, s , C H ₃ ), [5.02 (1H, s, C H -OH)] 5.18 (1H, s , C H -OH), [6.23(1H, s , C H =C)]6.43(1H, s ,C H =C), 6.90-6.98(2H, m ,Ar- H ),7.11 -7.21(1H, m ,Ar- H ), 7.22-7.31(5H, m ,Ar- H ), 7.36-7.42(2H, m ,Ar- H ), 7.78-7.84(2H, m ,Ar- H ) .

If possible, provide a small amount of diastereomer in parentheses.

δ _C (75.5 MHz, CDCl ₃ ): 38.3 ( C H ₂ ), 38.4 ( C H ₂ ), 38.6 ( C H ₂ ), 39.9 ( C H ₂ ), 40.3 ( C H ₂ ), 43.4 ( C H ₂ ), 51.9 (COO C H ₃ ), 52.0 (COO C H ₃ ), 55.9 (fourth C ), 56.3 (fourth C ), 82.0 ( C H-OH), 82.8 ( C H-OH), 120.5 ( Third C ), 120.7 (third C ), 123.5 (third C ), 123.6 (third C ), 124.0 (third C ), 124.2 (third C ), 124.5 (third C ), 124.6 (p. C ), 124.8 (third C ), 124.9 (third C ), 125.1 (third C ), 125.2 (third C ), 126.1 (third C ), 126.4 (third C ), 127.0 (fourth C ), 127.1 (fourth C ), 128.0 (third C ), 128.2 (third C ), 128.5 (third C ), 128.8 (third C ), 129.0 (third C ), 129.2 (third C ), 129.5 (third C ), 2 × 130.0 (2 × third C ), 2 × 130.2 (2 × third C ), 130.7 (third C ), 140.4 (fourth C ), 141.5 (fourth C ), 142.8 (fourth C ), 143.2 (fourth C ), 143.5 (fourth C ), 143.6 (fourth C ), 143.7 (fourth C ), 144.2 (fourth C ), 144.3 (fourth C ) , 144.5 (fourth C ), 150.4 (fourth C ), 152.6 (fourth C ), 167.0 ( C = O), 167.2 ( C = O).

Hydrolysis of methyl benzoate

The ester was placed in a round bottom flask and a 10% aqueous NaOH solution (1 mL) was added, followed by a sufficient amount of methanol to form a solution (6 mL). The solution was heated at 40 ° C and monitored by TLC (80:20, hexane:EtOAc). After standing for about 4 hours, there was no more ester.

The mixture was cooled, and saturated NH ₄ Cl (solution pH 12). Dilute HCl was added to acidify the pH (pH 2). The product was extracted from turbid solution into ethyl acetate (3×10 mL). The combined extracts were evaporated in vacuo, and dried over MgSO ₄ to yield 0.15 g (quantitative) as a solid emulsions. This product was isolated as a mixture of two diastereomers in a ratio of about 2:1.

Analysis of the mixture of two diastereomers

Purity (HPLC): 95.2% (diastereoisomer in a ratio of 2:1)

δ _H (400 MHz, CDCl ₃ ): 2.81-3.59 (6H, m , 3 × C H ₂ ), [5.05(1H, s , C H -OH)], 5.23 (1H, s , C H -OH) , 6.46 (1H, s , C H = C), [6.66 (1H, s , C H = C)], 6.95-7.03 (2H, m , Ar- H ), 7.12-7.17 (1H, m , Ar- H ), 7.21-7.29 (5H, m , Ar- H ), 7.37-7.43 (2H, m , Ar- H ), 7.85-7.91 (2H, m , Ar- H ).

If possible, provide a small amount of diastereomer in parentheses.

δ _C (100 MHz, CDCl ₃ ): 37.9 ( C H ₂ ), 38.1 ( C H ₂ ), 38.2 ( C H ₂ ), 39.5 ( C H ₂ ), 39.9 ( C H ₂ ), 43.1 ( C H ₂ ), 55.5 (fourth C ), 55.9 (fourth C ), 81.6 ( C H-OH), 82.4 ( C H-OH), 120.2 (third C ), 120.3 (third C ), 123.1 (third C ), 123.2 (third C ), 123.5 (third C ), 123.9 (third C ), 124.1 (third C ), 124.4 (third C ), 124.5 (third C ), 124.7 (third C ), 125.9 (third C ), 126.0 (third C ), 126.5 (third C ), 2 x 126.7 (fourth C & third C ), 126.9 (fourth C ), 128.1 (third C ), 128.2 (third C ), 128.4 (third C ), 2 x 129.2 (2 x third C ), 2 x 129.4 (2 x third C ), 2 x 129.8 (2 x third C ), 2 x 129.9 (2×third C ), 130.4 (third C ), 140.0 (fourth C ), 141.0 (fourth C ), 142.3 (fourth C ), 142.7 (fourth C ), 143.0 (fourth C ), 143.2 (fourth C ), 143.8 (fourth C ), 144.0 (fourth C ), 144.1 (fourth C ), 144.7 (fourth C ), 150.0 (fourth C ), 152.0 (fourth C ), 170.8 ( C = O), 171.1 ( C = O).

Chemical separation of enantiomers

Preparation of N-BOC D-phenylalanine derivatives of methyl benzoate diastereomers and/or isolation of subsequent diastereomers α1 and α2 (or β1 and β2).

Note: The process can be applied to two diastereomers, but the given example is the first diastereomer.

Non diastereomer A (2.5 mmol, 1.0 g) and N-BOC D- phenylalanine (3.1 mmol, 0.8 g) placed in a round bottom flask equipped with a condenser of and suspended under nitrogen in CH ₃ In CN (25 mL). Pyridine (3.1 mmol, 0.3 mL) was added to the suspension, followed by the addition in (2 mL) in the _{DCC CH 3 CN (3.1 mmol,} 0.7 g) and DMAP (10% mol, 0.25 mmol , 0.05 g) of Solution. The mixture was stirred at 50 ° C for 20 hours and then reached room temperature.

The white solid was filtered off and the solvent was removed in vacuo. Ethyl acetate was added using 10% H ₂ SO _4, the resulting solution was washed with saturated NaHCO _3, dried over MgSO ₄ and evaporated to yield 2.1 g as a yellow oil (detected via HPLC purity 83%, yield: quantitative).

The diastereomers α1 and α2 were separated by flash chromatography (90 g of cerium oxide/g product) using hexane/MTBE 90:10. From the 4.17 g mixture, 1.3 g of the α2 derivative (and 1.71 g of the α1 derivative, and 0.3 g of a mixture of both) were obtained.

Hydrolysis of N-BOC D-phenylalanine derivatives of methyl benzoate compounds (α1, α2, β1 or β2)

The diastereomers a2 (2.3 mmol, 1.45 g) were dissolved in MeOH (25 mL) and EtOAc (11.5 mmol, 0.45 g), and the mixture was stirred at reflux temperature and monitored by TLC. After 20 hours, the starting material was consumed.

The reaction was cooled to room temperature, and by the addition of saturated NH ₄ Cl suspended. The methanol was removed in vacuo and the aqueous solution was acidified to pH 1 using concentrated HCl. The product was extracted with EtOAc, dried over MgSO ₄ and evaporated tolulululululululululululululululululululululululululululululululululu 0.44 g of acidic derivative compound 5 (yield 50%) was obtained, and the purity by HPLC was 97.2%.

Note: Alternative hydrolysis was also carried out at 40-50 ° C using 10% aqueous NaOH in methanol. The process takes about 5 days to complete.

Analysis results of enantiomers α1, α2, β1, β2 Enantiomers of diastereomer B, β1-compound 3

Description: Amorphous solid milk

Melting point 195-196°C

[α] _D : +98.51 (1.07%, MeOH)

Purity: 99.0%

δ _H (400 MHz, CDCl ₃ ): 2.87 (1H, d , J = 13.28 Hz, C H ₂ ), 3.00-3.09 (2H, m , C H ₂ ), 3.29 (1H, d , J = 13.36 Hz, C H ₂ ), 3.43 - 3.61 (2H, m , C H ₂ ), 5.27 (1H, s , C H -OH), 6.49 (1H, s , C H = C), 7.00 (2H, d , J = 7.88 Hz, Ar- H ), 7.16-7.32 (6H, m , Ar- H ), 7.44 (2H, d , J = 7.24 Hz, Ar- H ), 7.90 (2H, d , J = 7.92 Hz, Ar- H ).

Enantiomers of diastereomer B, β2-compound 2

Description: Amorphous solid milk

Melting point 184-185 ° C

[α] _D :-114.44 (0.18%, MeOH)

Purity: 99.8%

δ _H (400 MHz, CDCl ₃ ): 2.87 (1H, d , J = 13.32 Hz, C H ₂ ), 3.00-3.09 (2H, m , C H ₂ ), 3.29 (1H, d , J = 13.28 Hz, C H ₂ ), 3.46 (1H, d , J = 22.64 Hz, C H ₂ ), 3.58 (1H, d , J = 22.56 Hz, C H ₂ ), 5.27 (1H, s , C H -OH), 6.49 (1H, s , C H = C), 7.00 (2H, d , J = 8.04 Hz, Ar- H ), 7.15-7.34 (6H, m , Ar- H ), 7.44 (2H, d, J = 7.20 Hz , Ar- H ), 7.90 (2H, d, J = 8.04 Hz, Ar- H ).

Enantiomers of diastereomer C, alpha 1-compound 4

Description: solid milk

Melting point 136-140 ° C

[α] _D : -39.3 (0.66%, MeOH)

Purity: 94.0%

δ _H (400 MHz, CDCl ₃ ): 2.90-3.59 (6H, m , 3 × C H ₂ ), 5.08 (1H, s , C H -OH), 6.70 (1H, s , C H = C), 7.05 (2H, d , J = 8.08 Hz, Ar- H ), 7.19 (1H, t , J = 7.34 Hz, Ar- H ), 7.26-7.47 (7H, 2 × m , Ar- H ), 7.93 (2H, d , J = 8.08 Hz, Ar- H ).

Enantiomers of diastereomer C, α2-compound 5

Description: Amorphous solid milk

Melting point 195-196°C

[α] _D : +32.1 (1.18%, MeOH)

Purity: 97.2%

δ _H (400 MHz, CDCl ₃ ): 2.94 - 3.59 (6H, m , 3 × C H ₂ ), 5.08 (1H, s , C H -OH), 6.70 (1H, s , C H = C), 7.05 (2H, d , J = 8.12 Hz, Ar- H ), 7.19 (1H, t , J = 7.34 Hz, Ar- H ), 7.26-7.47 (7H, 2 × m , Ar- H ), 7.93 (2H, d , J = 8.12 Hz, Ar- H ).

HPLC method

A non-pivoling and palm-wise HPLC method for the qualitative and quantitative separation of enantiomer compounds 2 , 3 , 4 , 5 has been established.

HPLC analysis of enantiomers

Salt formation

The salt is prepared by dissolving the free acid of compounds 2 , 3 , 4 and 5 in an aqueous or aqueous organic solvent in the presence of a suitable base and isolating the salt by evaporation of the solvent.

Compound 6 : N-methyl-(D)-glucosamine salt of compound 2 (NMDG)

Physiological and chemical properties of Compound 6 : Appearance: off-white solid

Molecular weight: 577 (free acid: 382)

Molecular formula: C ₃₃ H ₃₉ O ₈ N (free acid: C ₂₆ H ₂₂ O ₃ )

Melting point: 165-167 ° C

Compound 6: [α] _D : -76.5 (sample concentration: 200 mg/10 ml in water)

Quality (Da): ES+ only visible in [NMDG+Na]

Elemental analysis: Calculated values: C (68.61), H (6.80), N (2.42), O (22.16). Experimental values: C (68.44), H (6.80), N (2.50), O (21.98)

δ _H (400 MHz, DMSO-d6): 2.48 (3H, apparent s , NC H ₃ ), 2.65 (1H, d , J = 13.56 Hz, HC H ), 2.84-3.02 (4H, m ), 3.16 ( 1H, d , J=13.60 Hz, HC H ), 3.40-3.70(7H, m ), 3.85-3.92(1H, m ), 5.06(1H, s ,C H -OH), 5.93 (1H, width s , CH-O H ), 6.41 (1H, s , C H = C), 6.80 (2H, d , J = 7.92 Hz, Ar- H ), 7.06-7.41 (8H, m , Ar- H ), 7.64 (2H , d , J = 7.80 Hz, Ar- H ).

δ _C (100 MHz, DMSO): 33.8 ( C H ₃ ), 37.9 ( C H ₂ ), 38.2 ( C H ₂ ), 39.5 ( C H ₂ ), 51.6 ( C H ₂ -N), 55.8 (fourth C ), 63.5 ( C H ₂ -O), 69.0 ( C H-O), 70.3 ( C H-O), 70.6 ( C H-O), 71.3 ( C H-O), 81.1 ( C H-OH), 120.1 (third C ) , 123.4 (third C ), 123.7 (third C ), 124.3 (third C ), 124.4 (third C ), 126.1 (third C ), 126.3 (third C ), 127.0 (third C ), 127.5 (third C ), 2 × 128.5 (2 × third C ), 2 × 129.1 (2 × third C ), 140.4 (fourth C ), 141.1 (fourth C ), 142.9 (fourth C ), 144.5 (fourth C ), 145.2 (fourth C ), 154.3 (fourth C ), 170.4 ( C = O).

X-ray study

The absolute stereochemistry of Compound 2 was constructed by single crystal X-ray analysis of its ( S )-(-)-methylbenzylamine salt (Compound 8 ). The results are provided in Appendix 2. The results are consistent with the stereochemistry shown in Figure 2. The absolute stereochemistry of compounds 4 and 5 was constructed by converting the alcohol (compound 2-5 ) to its ketone and by comparing its optical rotation.

Synthesis of 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl) Methyl)benzoic acid methyl ester (10):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol) and a solution of K ₂ CO ₃ (72 mg, 0.52 mmol) in DMF (2.5 mL). The reaction mixture was diluted with 1.5 N EtOAc (50 mL) andEtOAc. Using 10% NaHCO ₃ solution (25 mL), brine (25 mL) The washed organic layer was separated, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS (-OH): measurement value of 379.3, calc. 396.17, molecular formula C ₂₇ H ₂₄ O _3.

Purity (HPLC): 94%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.73 (1H, d, J = 13.48 Hz, C H ₂ ), 2.96 (2H, s, C H ₂ ), 3.20 (1H, d, J = 13.52) Hz, C H ₂ ), 3.44 (1H, d, J = 23.16 Hz, C H ₂ ), 3.58 (1H, d, J = 23.00 Hz, C H ₂ ), 3.78 (3H, s, OC H ₃ ), 5.06 (1H, d, J = 6.76 Hz, C H OH), 5.85 (1H, d, J = 6.88 Hz, O H ), 6.40 (1H, s, C H = C), 6.98 (2H, d, J =8.16 Hz, Ar- H ), 7.08 (1H, t, J = 7.32 Hz, Ar- H ), 7.16 (1H, t, J = 7.40 Hz, Ar- H ), 7.20-7.23 (4H, m, Ar - H ), 7.34-7.36 (1H, m, Ar- H ), 7.39 (1H, d, J = 7.36 Hz, Ar- H ), 7.71 (2H, d, J = 8.16 Hz, Ar- H ).

Synthesis of 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoic acid Ester (11):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol) and a solution of K ₂ CO ₃ (72 mg, 0.52 mmol) in DMF (2.5 mL). The reaction mixture was diluted with 1.5 N EtOAc (50 mL) andEtOAc. Using 10% NaHCO ₃ solution (25 mL), brine (25 mL) The organic layer was washed, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS (-OH): measurement value of 393.3, calc. 410.19, molecular formula C ₂₈ H ₂₆ O _3.

Purity (HPLC): 92%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.27 (3H, t, J = 7.08 Hz, C H ₃ ), 2.72 (1H, d, J = 13.52 Hz, C H ₂ ), 2.96 (2H, s, C H ₂ ), 3.20 (1H, d, J = 13.48 Hz, C H ₂ ), 3.45 (1H, d, J = 23.08 Hz, C H ₂ ), 3.59 (1H, d, J = 22.84 Hz, _{C H 2), 4.24 (2H} , q, J = 7.08 Hz, OC H 2), 5.05 (1H, d, J = 6.84 Hz, C H OH), 5.85 (1H, d, J = 6.92 Hz, O H ), 6.40 (1H, s, C H = C), 6.97 (2H, d, J = 8.24 Hz, Ar- H ), 7.08 (1H, td, J = 1.16, 7.30 Hz, Ar- H ), 7.16 ( 1H, t, J = 6.84 Hz, Ar- H ), 7.19-7.25 (4H, m, Ar- H ), 7.34-7.36 (1H, m, Ar- H ), 7.40 (1H, d, J = 7.36 Hz , Ar- H ), 7.70 (2H, d, J = 8.20 Hz, Ar- H ).

Synthesis of 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoate Ester (12):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol) and _{K 2 CO 3 (72 mg,} 0.52 mmol) in DMF solution (2.5 mL) was added in the n -PrI (90 mg, 0.52 mmol ), and then stirred at room temperature for 4 hours. The reaction mixture was diluted with 1.5 N EtOAc (50 mL) andEtOAc. Using 10% NaHCO ₃ solution (25 mL), brine (25 mL) The organic layer was washed, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS (-OH): measurement value of 407.3, calc. 424.20, molecular formula C ₂₉ H ₂₈ O _3.

Purity (HPLC): 95%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.93 (3H, t, J = 7.40 Hz, C H ₃ ), 1.67 (2H, q, J = 6.92 Hz, OCH ₂ C H ₂ ), 2.72 ( 1H,d,J=13.56 Hz, C H ₂ ), 2.96 (2H, s, C H ₂ ), 3.20 (1H, d, J = 13.52 Hz, C H ₂ ), 3.46 (1H, d, J = 22.84) Hz, C H ₂ ), 3.59 (1H, d, J = 22.96 Hz, C H ₂ ), 4.16 (2H, t, J = 6.56 Hz, O CH ₂ ), 5.05 (1H, d, J = 6.88 Hz, C H OH), 5.85 (1H, d, J = 6.92 Hz, O H ), 6.40 (1H, s, C H = C), 6.98 (2H, d, J = 8.16 Hz, Ar- H ), 7.10 ( 1H, dt, J = 1.00 Hz, Ar- H ), 7.16 (1H, t, J = 7.32 Hz, Ar- H ), 7.21-7.25 (4H, m, Ar- H ), 7.35-7.36 (1H, m , Ar- H ), 7.40 (1H, d, J = 7.28 Hz, Ar- H ), 7.71 (2H, d, J = 8.16 Hz, Ar- H ).

Synthesis of 4-{[(1' R , 2' R )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- Benzoylamine (13)

4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2- in DMSO (5 mL) To a solution of methyl)benzoic acid (100 mg, 0.26 mmol) was added Boc anhydride (69 mg, 0.31 mmol), followed by pyridine (24 mg, 0.26 mmol) and stirred at room temperature for 5 min. Ammonium bicarbonate (62 mg, 0.78 mmol) was added and stirred for an additional 1 hour. The reaction mixture was poured into water (25 mL). Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS ⁽⁺ H +): measurement value of 382.4, calc. 381.17, molecular formula C ₂₆ H ₂₃ NO _2.

Purity (HPLC): 99%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.68 (1H, d, J = 13.60 Hz, C H ₂ ), 2.94 (1H, d J = 16.92 Hz, C H ₂ ), 2.99 (1H, d , J=16.00 Hz, C H ₂ ), 3.19 (1H, d, J=13.60 Hz, C H ₂ ), 3.47 (1H, d, J=23.08 Hz, C H ₂ ), 3.61 (1H, d, J =23.00 Hz, C H ₂ ), 5.05 (1H, d, J = 6.92 Hz, C H OH), 5.84 (1H, d, J = 6.96 Hz, O H ), 6.41 (1H, s, C H = C ), 6.89 (2H, d, J = 8.20 Hz, Ar- H ), 7.09 (1H, td, J = 1.28, 7.28 Hz, Ar- H ), 7.17 (1H, t, J = 7.32 Hz, Ar- H ), 7.22-7.27 (5H, m, Ar- H and N H), 7.35-7.37 (1H, m, Ar- H), 7.41 (1H, d, J = 7.32 Hz, Ar- H), 7.63 (2H , d, J = 8.24 Hz, Ar- H ), 7.82 (1H, br s, N H ).

Synthesis of 4-{[(1' R , 2' R )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- }}- N- (2-hydroxyethyl)benzamide (14):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol), Et 3 N (157 mg, 1.56 mmol) and 2-aminoethanol (40 mg, 0.65 mmol) in DCM (5 mL) was added in the T ₃ P (0.33 mL, in acetic acid A 50 wt% solution in the ester, 0.52 mmol), and then stirred at room temperature for 12 hours. The reaction mixture was quenched with water (25 mL)EtOAc Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS ⁽⁺ H +): measurement value of 426.4, calc. 425.20, molecular formula C ₂₈ H ₂₇ NO _3.

Purity (HPLC): 99%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.68 (1H, d, J = 13.56 Hz, C H ₂ ), 2.94 (1H, d, J = 15.64 Hz, C H ₂ ), 2.98 (1H, d, J = 15.84 Hz, C H ₂ ), 3.19 (1H, d, J = 13.56 Hz, C H ₂ ), 3.26-3.29 (2H, m, CONHC H ₂ ), 3.43 - 3.49 (3H, m, C 2H of H ₂ OH and 1H) of C H ₂ , 3.61 (1H, d, J = 23.16 Hz, C H ₂ ), 4.68 (1H, t, J = 5.60 Hz, CH ₂ O H ), 5.05 (1H, d, J = 6.88 Hz, C H OH), 5.84 (1H, d, J = 6.96 Hz, CHO H ), 6.40 (1H, s, C H = C), 6.89 (2H, d, J = 8.20 Hz, Ar- H ), 7.09 (1H, dt, J = 1.20, 10.13 Hz, Ar- H ), 7.17 (1H, t, J = 7.40 Hz, Ar- H ), 7.21-7.28 (4H, m, Ar- H ), 7.35-7.37 (1H, m, Ar- H ), 7.41 (1H, d, J = 7.28 Hz, Ar- H ), 7.61 (2H, d, J = 8.20 Hz, Ar- H ), 8.28 (1H) , t, J = 5.64 Hz, N H ).

Synthesis of 4-{[(1' R , 2' R )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- }}-N-methylbenzamide (15):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol), Et 3 N (157 mg, 1.56 mmol) and methylamine (0.32 mL, 2.0 M in THF, the solution of the 0.65 mmol) in DCM (5 mL) was added T ₃ P (0.33 mL, 50 wt% solution in ethyl acetate, 0.52 mmol), and then stirred at room temperature for 12 h. The reaction mixture was quenched with water (25 mL)EtOAc Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS ⁽⁺ H +): measurement value of 396.4, calc. 395.19, molecular formula C ₂₇ H ₂₅ NO _2.

Purity (HPLC): 100%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.67 (1H, d, J = 13.60 Hz, C H ₂ ), 2.72 (3H, d, J = 4.48 Hz, CONHC H ₃ ), 2.93 (1H, d, J = 15.64 Hz, C H ₂ ), 2.98 (1H, d, J = 15.68 Hz, C H ₂ ), 3.18 (1H, d, J = 13.60 Hz, C H ₂ ), 3.44 (1H, d, J=23.12 Hz, C H ₂ ), 3.59 (1H, d, J=23.04 Hz, C H ₂ ), 5.05 (1H, d, J = 6.88 Hz, C H OH), 5.83 (1H, d, J = 6.92 Hz, O H ), 6.40 (1H, s, C H = C), 6.89 (2H, d, J = 8.12 Hz, Ar- H ), 7.06-7.10 (1H, m, Ar- H ), 7.16 ( 1H,t,J=7.32 Hz,Ar- H ), 7.23-7.27(4H,m,Ar- H ),7.34-7.36(1H,m,Ar- H ), 7.40(1H,d,J=7.24 Hz , Ar- H ), 7.57 (2H, d, J = 8.16 Hz, Ar- H ), 8.26-8.28 (1H, m, N H ).

Synthesis of 4-{[(1' R , 2' R )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- Base} -N , N -dimethylbenzamide (16):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Add T ₃ P (100 mg, 0.26 mmol), Et ₃ N (157 mg, 1.56 mmol) and dimethylamine (0.33 mL, 2.0 M in THF, 0.65 mmol) in DCM (5 mL) 0.33 mL, a 50 wt% solution in ethyl acetate, 0.52 mmol), and stirred at room temperature for 12 h. The reaction mixture was quenched with water (25 mL)EtOAc Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS ⁽⁺ H +): measurement value of 410.4, calc. 409.20, molecular formula C ₂₈ H ₂₇ NO _2.

Purity (HPLC): 100%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.69 (1H, d, J = 13.60 Hz, C H ₂ ), 2.83 (3H, s, CONC H ₃ ), 2.93 (3H, s, CONC H _{3 )} ), 2.98 (2H, s, C H ₂ ), 3.16 (1H, d, J = 13.52 Hz, C H ₂ ), 3.45 (1H, d, J = 23.44 Hz, C H ₂ ), 3.59 (1H, d , J=23.04 Hz, C H ₂ ), 5.06 (1H, d, J = 6.84 Hz, C H OH), 5.85 (1H, d, J = 6.96 Hz, O H ), 6.45 (1H, s, C H =C), 6.89 (2H, d, J = 8.04 Hz, Ar- H ), 7.07-7.24 (8H, m, Ar- H ), 7.34-7.36 (1H, m, Ar- H ), 7.40 (1H, d, J = 7.24 Hz, Ar- H ).

Synthesis of 4-(((1 S , 2 S )-1-hydroxy-2,3-dihydro-1 H ,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid Methyl ester (17):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol) and a solution of K ₂ CO ₃ (72 mg, 0.52 mmol) in DMF (2.5 mL). The reaction mixture was diluted with 1.5 N EtOAc (50 mL) andEtOAc. Using an aqueous solution of 10% NaHCO3 (25 mL), brine (25 mL) The organic layer was washed, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS (-OH): measured 379.2, calc. 396.17, molecular formula C ₂₇ H ₂₄ O _3.

Purity (HPLC): 97%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 2.84 (1H, d, J = 13.28 Hz, C H ₂ ), 3.00 (1H, d, J = 15.64 Hz, C H ₂ ), 3.05 (1H, d, J = 15.56 Hz, C H ₂ ), 3.27 (1H, d, J = 13.32 Hz, C H ₂ ), 3.45 (1H, d, J = 22.52 Hz, C H ₂ ), 3.57 (1H, d, J = 22.60 Hz, C H ₂ ), 3.89 (3H, s, OC H ₃ ), 5.25 (1H, s, C H OH), 6.47 (1H, s, C H = C), 6.96 (2H, d, J = 8.24 Hz, Ar- H ), 7.17 (1H, dt, J=2.04, 9.88 Hz, Ar- H ), 7.24-7.33 (5H, m, Ar- H ), 7.43 (2H, d, J = 7.60 Hz, Ar- H ), 7.83 (2H, dd, J = 1.76, 6.60 Hz, Ar- H ).

Synthesis of 4-(((1 S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid Ester (18):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol) and a solution of K ₂ CO ₃ (72 mg, 0.52 mmol) in DMF (2.5 mL). The reaction mixture was diluted with 1.5 N EtOAc (50 mL) andEtOAc. Using 10% NaHCO ₃ solution (25 mL), brine (25 mL) The organic layer was washed, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS (-OH): measurement value of 393.4, calc. 410.19, molecular formula C ₂₈ H ₂₆ O _3.

Purity (HPLC): 92%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 1.38 (3H, t, J = 7.12 Hz, C H ₃ ), 2.84 (1H, d, J = 13.28 Hz, C H ₂ ), 3.00 (1H, d, J = 15.60 Hz, C H ₂ ), 3.05 (1H, d, J = 15.60 Hz, C H ₂ ), 3.27 (1H, d, J = 13.28 Hz, C H ₂ ), 3.45 (1H, d, J = 22.52 Hz, C H ₂ ), 3.58 (1H, d, J = 22.52 Hz, C H ₂ ), 4.35 (2H, q, J = 7.12 Hz, OC H ₂ ), 5.25 (1H, s, C H OH) , 6.48 (1H, s, C H = C), 6.96 (2H, d, J = 8.28 Hz, Ar- H ), 7.17 (1H, dt, J = 2.00, 9.92 Hz, Ar- H ), 7.25-7.34 (5H, m, Ar- H ), 7.44 (2H, d, J = 7.72 Hz, Ar- H ), 7.85 (2H, dd, J = 1.72, 6.56 Hz, Ar- H ).

Synthesis of 4-(((1 S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoate Ester (19):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( in the 100 mg, 0.26 mmol) and _{K 2 CO 3 (72 mg,} 0.52 mmol) in DMF (2.5 mL) was added n -PrI (89 mg, 0.52 mmol ), and stirred at room temperature for 4 hours. The reaction mixture was diluted with 1.5 N EtOAc (50 mL) andEtOAc. Using 10% NaHCO ₃ solution (25 mL), brine (25 mL) The organic layer was washed, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS (-OH): measurement value of 407.2, calc. 424.20, molecular formula C ₂₉ H ₂₈ O _3.

Purity (HPLC): 90%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 1.03 (3H, t, J = 7.48 Hz, C H ₃ ), 1.73-1.82 (2H, m, C H ₂ ), 2.84 (1H, d, J = 13.32) Hz, C H ₂ ), 3.00 (1H, d, J = 15.04 Hz, C H ₂ ), 3.06 (1H, d, J = 15.52 Hz, C H ₂ ), 3.27 (1H, d, J = 13.32 Hz, C H ₂ ), 3.46 (1H, d, J = 22.56 Hz, C H ₂ ), 3.58 (1H, d, J = 22.68 Hz, C H ₂ ), 4.26 (2H, t, J = 6.64 Hz, OC H ₂ ), 5.25 (1H, s, C H OH), 6.48 (1H, s, C H = C), 6.96 (2H, d, J = 8.24 Hz, Ar- H ), 7.18 (1H, dt, J = 1.96, 9.96 Hz, Ar- H ), 7.24-7.33 (5H, m, Ar- H ), 7.44 (2H, d, J = 7.60 Hz, Ar- H ), 7.85 (2H, dd, J = 1.68, 6.60 Hz, Ar- H ).

Synthesis of 4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- Benzoylamine (20):

4-(((1 S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2- in DMSO (5 mL) To a solution of methyl)benzoic acid (100 mg, 0.26 mmol) was added Boc anhydride (69 mg, 0.31 mmol), followed by pyridine (24 mg, 0.26 mmol) and stirred at room temperature for 5 min. Ammonium bicarbonate (62 mg, 0.78 mmol) was added and stirred for an additional 1 hour. The reaction mixture was poured into water (25 mL)EtOAc. Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS (-OH): measurement value of 364.2, calc. 381.17, molecular formula C ₂₆ H ₂₃ NO _2.

Purity (HPLC): 98%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 2.84 (1H, d, J = 13.32 Hz, C H ₂ ), 3.00 (1H, d, J = 15.68 Hz, C H ₂ ), 3.05 (1H, d, J = 15.56 Hz, C H ₂ ), 3.26 (1H, d, J = 13.32 Hz, C H ₂ ), 3.46 (1H, d, J = 22.60 Hz, C H ₂ ), 3.58 (1H, d, J = 22.68 Hz, C H ₂ ), 5.25 (1H, s, C H OH), 5.65 and 6.07 (2H, 2 × br s, N H and CHO H ), 6.48 (1H, s, C H = C), 6.97 (2H, d, J = 8.16 Hz, Ar- H ), 7.17 (1H, dt, J = 2.00, 9.95 Hz, Ar- H ), 7.24-7.33 (5H, m, Ar- H ), 7.44 (2H, d, J = 7.56 Hz, Ar- H ), 7.60 (2H, d, J = 8.24 Hz, Ar- H ).

Synthesis of 4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- }}- N- (2-hydroxyethyl)benzamide (21):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol), Et 3 N (157 mg, 1.56 mmol) and 2-aminoethanol (40 mg, 0.65 mmol) in DCM (5 mL) was added in the T ₃ P (0.33 mL, in acetic acid A 50 wt% solution in the ester, 0.52 mmol), and stirred at room temperature for 12 hours. The reaction mixture was quenched with water (25 mL)EtOAc Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS ⁽⁺ H +): measurement value of 426.5, calc. 425.20, molecular formula C ₂₈ H ₂₇ NO _3.

Purity (HPLC): 99%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.68 (1H, d, J = 13.44 Hz, C H ₂ ), 2.94 (1H, d, J = 15.04 Hz, C H ₂ ), 2.98 (1H, d, J = 16.28 Hz, C H ₂ ), 3.19 (1H, d, J = 13.32 Hz, C H ₂ ), 3.25-3.30 (2H, m, C H ₂ ), 3.44 - 3.49 (3H, m, 2 ×C H ₂ of 3H), 3.61 (1H, d, J=23.16 Hz, C H ₂ ), 4.68 (1H, t, J = 5.48 Hz, CH ₂ O H ), 5.05 (1H, d, J = 6.84 Hz, C H -OH), 5.84 (1H, d, J = 6.88 Hz, CH-O H ), 6.40 (1H, s, C H = C), 6.89 (2H, d, J = 8.20 Hz, Ar- H ), 7.09 (1H, dt, J = 1.16, 10.12 Hz, Ar- H ), 7.17 (1H, t, J = 7.32 Hz, Ar- H ), 7.22-7.28 (4H, m, Ar- H ), 7.36 (1H, t, J = 3.48 Hz, Ar- H ), 7.41 (1H, d, J = 7.20 Hz, Ar- H ), 7.61 (2H, d, J = 8.20 Hz, Ar- H ), 8.28 ( 1H, t, J = 5.52 Hz, N H ).

Synthesis of 4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- }}- N -methylbenzamide (22):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( 100 mg, 0.26 mmol), Et 3 N (157 mg, 1.56 mmol) and methylamine (0.32 mL, 2.0 M in THF, the solution of the 0.65 mmol) in DCM (5 mL) was added T ₃ P (0.33 mL, 50 wt% solution in ethyl acetate, 0.52 mmol), and stirred at room temperature for 12 h. The reaction mixture was quenched with water (25 mL)EtOAc Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS ⁽⁺ H +): measurement value of 396.4, calc. 395.19, molecular formula C ₂₇ H ₂₅ NO _2.

Purity (HPLC): 93%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.68 (1H, d, J = 13.64 Hz, C H ₂ ), 2.72 (3H, d, J = 4.48 Hz, C H ₃ ), 2.94 (1H, d, J = 16.28 Hz, C H ₂ ), 2.99 (1H, d, J = 15.84 Hz, C H ₂ ), 3.19 (1H, d, J = 13.56 Hz, C H ₂ ), 3.45 (1H, d, J = 22.92 Hz, C H ₂ ), 3.60 (1H, d, J = 23.40 Hz, C H ₂ ), 5.05 (1H, d, J = 6.88 Hz, C H OH), 5.84 (1H, d, J = 6.92 Hz, CHO H ), 6.40 (1H, s, C H = C), 6.90 (2H, d, J = 8.12 Hz, Ar- H ), 7.09 (1H, t, J = 7.24 Hz, Ar- H ) , 7.15-7.26 (5H, m, Ar- H ), 7.35-7.37 (1H, m, Ar- H ), 7.41 (1H, d, J = 7.24 Hz, Ar- H ), 7.58 (2H, d, J = 8.16 Hz, Ar- H ), 8.27 (1H, d, J = 4.36 Hz, N H ).

Synthesis of 4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- }}- N , N - dimethyl benzyl amide (23):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Add T ₃ P (100 mg, 0.26 mmol), Et ₃ N (157 mg, 1.56 mmol) and dimethylamine (0.33 mL, 2.0 M in THF, 0.65 mmol) in DCM (5 mL) 0.33 mL, 50 wt% in ethyl acetate, 0.52 mmol), and then stirred at room temperature for 12 hours. The reaction mixture was quenched with water (25 mL)EtOAc Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS ⁽⁺ H +): measurement value of 410.4, calc. 409.20, molecular formula C ₂₈ H ₂₇ NO _2.

Purity (HPLC): 97%.

^{1 H NMR (400 MHz, DMSO} -d 6): δ 2.70 (1H, d, J = 13.64 Hz, C H 2), 2.83-2.98 (8H, m, C H 2 of 2H, and 2 × C H ₃ of 6H), 3.16 (1H, d, J = 13.60 Hz, C H ₂ ), 3.45 (1H, d, J = 22.92 Hz, C H ₂ ), 3.59 (1H, d, J = 23.20 Hz, C H ₂ ) , 5.06 (1H, s, C H OH), 6.45 (1H, s, C H = C), 6.89 (2H, d, J = 8.08 Hz, Ar- H ), 7.09 (1H, dt, J = 1.20, 10.12 Hz, Ar- H ), 7.13-7.17 (3H, m, Ar- H ), 7.19-7.25 (4H, m, Ar- H ), 7.34-7.36 (1H, m, Ar- H ), 7.40 (1H , d, J = 7.36 Hz, Ar- H ).

Synthesis of 4-{[(1' R , 2' R )-1'-(L-Acetylmethoxy)-1',3'-dihydro-1 H , 2' H -2, 2'-茚-2'-yl]methyl}benzoic acid (24):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Add N -[(9 H -茀-9-ylmethoxy) to a solution of 200 mg, 0.52 mmol), DCC (129 mg, 0.62 mmol) and DMAP (6 mg, 0.052 mmol) in ethyl acetate (10 mL) The carbonyl]-L-leucine (183 mg, 0.52 mmol) was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure, and the residue 1 (210 mg) was dissolved in dry THF (5 mL) then cooled to 0 ℃. Under an atmosphere of N ₂ was added dropwise dimethylamine (5 mL, 20% in THF), and stirred slowly at room temperature the reaction mixture for 1 hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjjj

LCMS ⁽⁺ H +): measurement value of 496.4, calc. 495.24, molecular formula C ₃₂ H ₃₃ NO _4.

Purity (HPLC): 93%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.90 (3H, d, J = 8.00 Hz, C H ₃ ), 0.93 (3H, d, J = 8.00 Hz, C H ₃ ), 1.58-1.66 ( 1H, m, C H), 1.68-1.76 (2H, m, C H 2), 3.13-3.37 (5H, m, 3 × C H 2 of 5H), 3.49 (1H, d , J = 24.00 Hz, C H ₂ ), 4.05 (1H, t, J = 4.00 Hz, C H -N), 6.39 (1H, s, C H -O), 6.53 (1H, s, C H = C), 7.06-7.11 (3H , m, Ar- H ), 7.16-7.23 (3H, m, Ar- H ), 7.29-7.39 (4H, m, Ar- H ), 7.73 (2H, d, J = 8.00 Hz, Ar- H ).

^{13 C NMR (100 MHz, DMSO} -d 6): δ 22.36 (C H 3), 22.75 (C H 3), 24.36-25.80 (C H), 39.36-40.61 (4 × C H 2), 51.59 and 54.71 (Fourth C and C H-N), 84.38 ( C H-O), 120.93, 123.92, 124.72, 125.27, 126.24, 126.72, 127.13, 129.20, 2×129.29, 129.51, 129.77, 2×130.35 (13×third C and 1) × fourth C ), 139.90 (fourth C ), 142.99 (fourth C ), 143.23 (fourth C ), 143.90 (fourth C ), 144.36 (fourth C ), 151.55 (fourth C ), 167.65 ( O- C = O), 170.70 ( C = O).

Synthesis of 4-{[(1' R , 2' R )-1'-(L-nonylaminooxy)-1',3'-dihydro-1 H , 2' H -2,2'-茚-2'-yl]methyl}benzoic acid (25):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Addition of N -[(9 H -茀-9-ylmethoxy) to a solution of 230 mg, 0.60 mmol), DCC (148 mg, 0.72 mmol) and DMAP (7 mg, 0.059 mmol) in ethyl acetate (12 mL) The carbonyl]-L-proline (193 mg, 0.60 mmol) was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and the residue 1 (250 mg) was dissolved in anhydrous THF (7 mL) and then cooled to 0 °C. Under an atmosphere of N ₂ was added dropwise dimethylamine (7 mL, 20% in THF), and stirred slowly at room temperature the reaction mixture for 1 hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjjj

LCMS ⁽⁺ H +): measurement value of 482.6, calc. 481.23, molecular formula C ₃₁ H ₃₁ NO _4.

Purity (HPLC): 95%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.97-1.05 (6H, m, (C H ₃ ) ₂ ), 2.30-2.35 (1H, m, C H ), 3.12 (1H, d, J = 12.84 Hz, C H ₂ ), 3.16 (1H, d, J = 5.48 Hz, C H ₂ ), 3.21-3.24 (2H, m, C H ₂ ), 3.41-3.49 (2H, m, C H ₂ ), 4.05 (1H, d, J = 4.28 Hz, C H -N), 6.40 (1H, s, C H -O), 6.56 (1H, s, C H = C), 7.08 (1H, dt, J = 1.24) , 10.15 Hz, Ar- H ), 7.10-7.21 (5H, m, Ar- H ), 7.28-7.40 (4H, m, Ar- H ), 7.73 (2H, d, J = 4.48 Hz, Ar- H ) .

^{13 C NMR (100 MHz, DMSO} -d 6): δ 18.14 (C H 3), 18.43 (C H 3), 29.89 (C H), 39.36-40.61 (3 × C H 2), 54.76 and 58.16 (first Four C and C H-N), 84.73 ( C H-O), 120.91, 123.89, 124.72, 125.27, 126.39, 126.71, 127.08, 129.15, 2×129.26, 129.69, 129.83, 2×130.42 (13×third C and 1× four C), 139.93 (IV C), 142.97 (IV C), 143.53 (IV C), 143.97 (IV C), 144.36 (IV C), 151.42 (IV C), 167.66 (O- C = O), 169.09 ( C = O).

Synthesis of 4-{[(1' R , 2' R )-1'-(L-iso-aramidinomethoxy)-1',3'-dihydro-1 H , 2' H -2,2'- Diterpene-2'yl]methyl}benzoic acid (26):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Add N -[(9 H -茀-9-ylmethoxy) to a solution of 250 mg, 0.65 mmol), DCC (160 mg, 0.78 mmol) and DMAP (8 mg, 0.06 mmol) in ethyl acetate (15 mL) The carbonyl]-L-isoleucine (228 mg, 0.65 mmol) was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and the residue 1 (275 mg) was dissolved in anhydrous THF (10 mL) and then cooled to 0 °C. Under an atmosphere of N ₂ was added dropwise dimethylamine (10 mL, 20% in THF), and stirred slowly at room temperature the reaction mixture for 1 hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjj

LCMS ⁽⁺ H +): measurement value of 496.6, calc. 495.24, molecular formula C ₃₂ H ₃₃ NO _4.

Purity (HPLC): 91%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.90 (3H, t, J = 7.40 Hz, C H ₃ ), 0.93 (3H, d, J = 6.96 Hz, C H ₃ ), 1.26-1.36 ( 1H, m, C H ₂ -CH ₃ ), 1.44-1.52 (1H, m, C H ₂ -CH ₃ ), 2.02-2.08 (1H, m, C H -CH ₃ ), 3.09-3.25 (4H, m , 2 × C H ₂ ), 3.35-3.48 (2H, m, C H ₂ ), 4.11 (1H, d, J = 7.12 Hz, C H -N), 6.40 (1H, s, C H -O), 6.54 (1H, s, C H = C), 7.05 - 7.21 (6H, m, Ar- H ), 7.27-7.38 (4H, m, Ar- H ), 7.72 (2H, d, J = 8.24 Hz, Ar - H ), 8.58 (2H, br s, N H ₂ ).

^{13 C NMR (100 MHz, DMSO} -d 6): δ 11.98 (C H 3), 14.95 (C H 3), 25.20 (C H 2), 36.42 (C H), 36.35-40.60 (3 × C H 2 ), 54.77 ( C H-N or fourth C ), 57.26 (fourth C or C H-N), 84.77 ( C HO), 120.91, 123.89, 124.72, 125.23, 126.39, 126.72, 127.05, 129.15, 2×129.24, 129.65, 129.78, 2 × 130.45 (13 × third C and 1 × fourth C ), 139.91 (fourth C), 142.97 (fourth C ), 144.33 (fourth C ), 143.99 (fourth C ), 144.36 (the first Four C ), 151.46 (fourth C ), 167.66 ( C =O), 168.83 ( C =O).

Synthesis of 4-{[(1' R , 2' R )-1'-(glycine decyloxy)-1',3'-dihydro-1 H , 2' H -2,2'-diindole- 2'-yl]methyl}benzoic acid (27):

To 4-(((1 R , 2 R )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Add N -[(9 H -茀-9-ylmethoxy) to a solution of 220 mg, 0.57 mmol), DCC (142 mg, 0.69 mmol) and DMAP (7 mg, 0.059 mmol) in ethyl acetate (10 mL) Glycol]glycine (171 mg, 0.57 mmol) was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and the residue 1 (250 mg) was dissolved in anhydrous THF (8 mL) and then cooled to 0 °C. Under an atmosphere of N ₂ was added dropwise dimethylamine (8 mL, 20% in THF), and stirred slowly at room temperature the reaction mixture for 1 hour. The reaction mixture was evaporated under reduced EtOAcqqqqqm

LCMS (-H ^+): 438.2 measured, calcd 439.18, molecular formula C ₂₈ H ₂₅ NO _4.

Purity (HPLC): 97%.

^{1 H NMR (400 MHz, DMSO} -d 6): δ 3.05-3.28 (5H, m, 3 × C H 2 of the 5H), 3.45-3.50 (1H, m , C H 2), 3.60-3.76 (2H, m, NC H ₂ ), 6.32 (1H, s, C H -O), 6.48 (1H, s, C H = C), 7.02 (2H, d, J = 8.12 Hz, Ar- H ), 7.05-7.09 (1H, m, Ar- H ), 7.15-7.22 (3H, m, Ar- H ), 7.28-7.36 (4H, m, Ar- H ), 7.70 (2H, d, J = 8.08 Hz, Ar- H ).

^{13 C NMR (100 MHz, DMSO} -d 6): δ 39.36-40.61 (4 × C H 2), 54.86 ( IV C), 82.96 (C HO) , 120.91,123.94,124.65,125.24,125.87,126.69, 127.14, 2×129.21, 129.32, 129.52, 129.76, 2×130.33 (13×third C and 1×fourth C ), 140.42 (fourth C ), 142.96 (fourth C ), 144.04 (fourth C ), 143.61 (fourth C ), 144.43 (fourth C ), 151.89 (fourth C ), 167.89 ( C = O).

Synthesis of 4-{[(1' S , 2' S )-1'-(L-Acetylmethoxy)-1',3'-dihydro-1 H , 2' H -2, 2'-茚-2'-yl]methyl}benzoic acid (28):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Add N -[(9 H -茀-9-ylmethoxy) to a solution of 200 mg, 0.52 mmol), DCC (129 mg, 0.62 mmol) and DMAP (6 mg, 0.052 mmol) in ethyl acetate (10 mL) The carbonyl]-L-leucine (183 mg, 0.52 mmol) was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure, and the residue 1 (210 mg) was dissolved in dry THF (5 mL) then cooled to 0 ℃. Under an atmosphere of N ₂ was added dropwise dimethylamine (5 mL, 20% in THF), and stirred slowly at room temperature the reaction mixture for 1 hour. The reaction mixture was evaporated under reduced EtOAcqqqqqqm

LCMS ⁽⁺ H +): measurement value of 496.5, calc. 495.24, molecular formula C ₃₂ H ₃₃ NO _4.

Purity (HPLC): 98%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.84 (3H, d, J = 6.24 Hz, C H ₃ ), 0.86 (3H, d, J = 6.28 Hz, C H ₃ ), 1.37-1.71 ( 3H, 2 × m, C H - C H ₂ of the 1H and 2H), 3.04-3.53 (7H, m , 3 × C H 2 and C H -N 6H of the 1H), 6.26 (1H, s , C H -O), 6.48 (1H, s, C H = C), 7.01 (2H, d, J = 8.04 Hz, Ar- H ), 7.08 (1H, dt, J = 1.04, 10.12 Hz, Ar- H ) , 7.14 - 7.24 (4H, m, Ar- H ), 7.25-7.36 (3H, m, Ar- H ), 7.69 (2H, d, J = 8.12 Hz, Ar- H ).

Synthesis of 4-{[(1' S , 2' S )-1'-(L-nonylaminooxy)-1',3'-dihydro-1 H , 2' H -2,2'-茚-2'-yl]methyl}benzoic acid (29):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Addition of N -[(9 H -茀-9-ylmethoxy) to a solution of 230 mg, 0.60 mmol), DCC (148 mg, 0.72 mmol) and DMAP (7 mg, 0.06 mmol) in ethyl acetate (12 mL) The carbonyl]-L-proline (193 mg, 0.60 mmol) was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 mg) was evaporated. Under an atmosphere of N ₂ was added dropwise dimethylamine (8 mL, 20% in THF), and stirred slowly at room temperature the reaction mixture for 1 hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjjj

LCMS ⁽⁺ H +): measurement value of 482.5, calc. 481.23, molecular formula C ₃₁ H ₃₁ NO _4.

Purity (HPLC): 96%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.80 (3H, d, J = 6.80 Hz, C H ₃ ), 0.87 (3H, d, J = 6.80 Hz, C H ₃ ), 1.88-1.90 ( 1H, m, C H ), 3.05-3.47 (1H, m, 3 × C H ₂ and 1 H of C H -N), 6.27 (1H, s, C H -O), 6.49 (1H, s, C H =C), 6.95 (2H, d, J = 8.00 Hz, Ar- H ), 7.07 (1H, t, J = 14.40 Hz, Ar- H ), 7.14-7.22 (3H, m, Ar- H ), 7.25 - 7.29 (2H, m, Ar- H ), 7.32-7.35 (2H, m, Ar- H ), 7.67 (2H, d, J = 8.00 Hz, Ar- H ).

Synthesis of 4-{[(1' S , 2' S )-1'-(L-iso-aramidinomethoxy)-1',3'-dihydro-1 H , 2' H -2,2'- Diterpene-2'-yl]methyl}benzoic acid (PH30):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Add N -[(9 H -茀-9-ylmethoxy) to a solution of 250 mg, 0.65 mmol), DCC (160 mg, 0.78 mmol) and DMAP (8 mg, 0.06 mmol) in ethyl acetate (10 mL) The carbonyl]-L-isoleucine (228 mg, 0.65 mmol) was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (275 mg) was dissolved in anhydrous THF (8 mL) and then cooled to 0. Under an atmosphere of N ₂ was added dropwise dimethylamine (8 mL, 20% in THF), and stirred slowly at room temperature the reaction mixture for 1 hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjj

LCMS ⁽⁺ H +): measurement value of 496.4, calc. 495.24, molecular formula C ₃₂ H ₃₃ NO _4.

Purity (HPLC): 95%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.77 (3H, t, J = 8.00 Hz, C H ₃ ), 0.82 (3H, d, J = 4.00 Hz, C H ₃ ), 1.02-1.12 ( 1H, m, C H ₂ -CH ₃ ), 1.28-1.38 (1H, m, C H ₂ -CH ₃ ), 1.62-1.68 (1H, m, C H -CH ₃ ), 3.10-3.50 (7H, m 3H of 3×C H ₂ and 1H of C H -N, 6.26 (1H, s, C H -O), 6.49 (1H, s, C H = C), 7.01 (2H, d, J = 8.00 Hz, Ar- H ), 7.07 (1H, t, J = 8.00 Hz, Ar- H ), 7.13-7.21 (3H, m, Ar- H ), 7.25-7.29 (2H, m, Ar- H ), 7.33 (2H, t, J = 8.00 Hz, Ar- H ), 7.70 (2H, d, J = 8.00 Hz, Ar- H ).

^{13 C NMR (100 MHz, DMSO} -d 6): δ 11.94 (C H 3), 15.88 (C H 3), 24.90 (C H 2 CH 3), 39.35-40.60 (3 × C H 2 and 1 × C H), 54.77 (fourth C or C H-N), 58.69 ( C H-N or fourth C ), 82.53 ( C HO), 120.86, 123.91, 124.60, 125.22, 125.84, 126.66, 127.02, 2×129.21, 2×129.32 , 129, 44, 2 × 130.05 (13 × third C and 1 × fourth C ), 140.79 (fourth C ), 2 × 143.01 (2 × fourth C ), 143.11 (fourth C ), 144.45 (the first Four C ), 151.95 (fourth C ), 175.25 ( C =O).

Synthesis of 4-{[(1' S , 2' S )-1'-(glycine decyloxy)-1',3'-dihydro-1 H , 2' H -2,2'-diindole- 2'-yl]methyl}benzoic acid (31):

To 4-((( S S , 2 S )-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ( Add N -[(9 H -茀-9-ylmethoxy) to a solution of 220 mg, 0.57 mmol), DCC (142 mg, 0.69 mmol) and DMAP (7 mg, 0.057 mmol) in ethyl acetate (10 mL) Glycol]glycine (171 mg, 0.57 mmol) was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 mg) was evaporated. Under an atmosphere of N ₂ was added dropwise dimethylamine (8 mL, 20% in THF), and stirred slowly at room temperature the reaction mixture for 1 hour. The reaction mixture was evaporated under reduced EtOAcqqqqqm

LCMS (-H ^+): measurement value of 438.4, calc. 439.18, molecular formula C ₂₈ H ₂₅ NO _4.

Purity (HPLC): 95%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.84-3.57 (8H, 2×m, 4×C H ₂ ), 6.30 (1H, s, C H -O), 6.48 (1H, s, C H = C), 7.00 (2H, d, J = 8.00 Hz, Ar- H ), 7.09 (1H, t, J = 7.20 Hz, Ar- H ), 7.14-7.22 (3H, m, Ar- H ), 7.28-7.37 (4H, m, Ar- H ), 7.69 (2H, d, J = 8.00 Hz, Ar- H ).

Synthesis of 4-(((1 R , 2 R )-1-((())-2-amino-4-methylpentyl)oxy)-2,3-dihydro-1H,1'H -[2,2'-Dioxa]-2-yl)methyl)benzoic acid methyl ester (32):

To 4-((1R,2R)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoic acid methyl ester ( 10, 180 mg, 0.45 mmol) , DCC (112 mg, 0.54 mmol) and DMAP (6 mg, 0.045 mmol) in ethyl acetate (10 mL) was added in the Fmoc leucine (158 mg, 0.45 mmol), It was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 mg) was dissolved in anhydrous THF (5 mL) and then cooled to 0 °C. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified EtOAcjjjjjjjj

LCMS ⁽⁺ H +): measurement value of 510.0, calc. 509.26, molecular formula C ₃₃ H ₃₅ NO _4.

Purity (HPLC): 94%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.98 (3H, d, J = 6.56 Hz, C H ₃ ), 1.01 (3H, d, J = 6.64 Hz, C H ₃ ), 1.56-1.96 (3H, 3 × m, C H ₂ -CH of 2H and -C H - the 1H), 3.06-3.37 (6H, m , 3 × C H 2 of the 6H), 3.64 (1H, dd , J = 5.40,8.90 Hz, C H -N), 3.89 (3H, s, OC H ₃ ), 6.45 (1H, s, C H -O), 6.54 (1H, s, C H = C), 7.03 (2H, d, J = 8.28) Hz, Ar- H ), 7.12-7.16 (1H, m, Ar- H ), 7.19-7.30 (5H, m, Ar- H ), 7.32-7.35 (2H, m, Ar- H ), 7.85 (2H, d, J = 8.28 Hz, Ar- H ).

^{13 C NMR (100 MHz, CDCl} 3): δ 21.74 (C H 3), 23.15 (C H 3), 24.85 (C H), 39.67 (C H 2), 40.45 (C H 2), 40.81 (C H ₂ ), 43.77( C H ₂ ), 52.03 (O C H ₃ or C H-N), 53.19 (fourth C ), 54.38 ( C H-N or O C H ₃ ), 83.48 ( C HO), 120.70, 123.50, 124.49 , 124.62, 125.76, 126.46, 127.07, 128.30, 3 × 129.25, 129.79, 2 × 129.94 (13 × third C and 1 × fourth C ), 140.21 (fourth C ), 142.16 (fourth C ), 142.54 ( Fourth C ), 143.65 (fourth C ), 144.22 (fourth C ), 150.99 (fourth C ), 167.03 ( C =O), 175.90 ( C =O).

Synthesis of 4-(((1 R , 2 R )-1-((())-2-amino-4-methylpentyl)oxy)-2,3-dihydro-1H,1'H -[2,2'-Dioxa]-2-yl)methyl)benzoic acid ethyl ester (33):

To 4-(((1R,2R)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoic acid ethyl ester ( Fmoc lysine (168 mg, 0.48 mmol) was added to a solution of 11 mg (200 mg, 0.48 mmol), DCC (120 mg, 0.58 mmol) and DMAP (6 mg, 0.048 mmol) in ethyl acetate (10 mL). It was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 mg) was dissolved in anhydrous THF (5 mL) and then cooled to 0 °C. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified EtOAcqqqqqqq

LCMS ⁽⁺ H +): measurement value of 524.2, calc. 523.27, molecular formula C ₃₄ H ₃₇ NO _4.

Purity (HPLC): 98%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.97 (3H, d, J = 6.56 Hz, C H ₃ ), 1.01 (3H, d, J = 6.64 Hz, C H ₃ ), 1.37 (3H, t, J=7.08 Hz, C H ₃ ), 1.56-1.60 (1H, m, C H ₂ -CH), 1.66-1.73 (1H, m, C H ₂ -CH), 1.88-1.93 (1H, m, -C _{H - (CH 3) 2)} , 3.06-3.36 (6H, m, C H 2 and 4H of the allylic C H ₂ 2H), 3.62 (1H, dd , J = 5.36,8.92 Hz, C H -N ), 4.35 (2H, q, J = 7.12 Hz, OC H ₂ ), 6.44 (1H, s, C H -O), 6.53 (1H, s, C H = C), 7.02 (2H, d, J = 8.20 Hz, Ar- H ), 7.10-7.15 (1H, m, Ar- H ), 7.18-7.29 (5H, m, Ar- H ), 7.31-7.35 (2H, m, Ar- H ), 7.85 (2H , d, J = 8.20 Hz, Ar- H ).

^{13 C NMR (100 MHz, CDCl} 3): δ 14.32 (C H 3), 21.74 (C H 3), 23.16 (C H 3), 24.85 (C H), 39.67 (C H 2), 40.47 (C H ₂ ), 40.79 ( C H ₂ ), 43.85 ( C H ₂ ), 53.22 (fourth C ), 54.38 ( C H-N), 60.86 (O C H ₂ ), 83.45 ( C H-O), 120.70 (third C ) , 123.50 (third C ), 124.48 (third C ), 124.62 (third C ), 125.75 (third C ), 126.45 (third C ), 127.06 (third C ), 128.65 (fourth C ), 3×129.23 (3×third C ), 129.77 (third C ), 2×129.88 (2×third C ), 140.25 (fourth C ), 142.17 (fourth C ), 142.55 (fourth C ), 143.51 (fourth C ), 144.24 (fourth C ), 151.03 (fourth C ), 166.55 ( C = O), 176.02 ( C = O).

Synthesis of 4-(((1 R , 2 R )-1-((())-2-amino-4-methylpentyl)oxy)-2,3-dihydro-1H,1'H -[2,2'-dioxin]-2-yl)methyl)benzoic acid propyl ester (34):

To 4-(((1R,2R)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoic acid propyl ester ( 12, 190 mg, 0.44 mmol) , DCC (111 mg, 0.54 mmol) and DMAP (6 mg, 0.045 mmol) in ethyl acetate (10 mL) was added in the Fmoc leucine (158 mg, 0.45 mmol), It was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 g) was dissolved in anhydrous THF (6 mL) and then cooled to 0 °C. Dimethylamine (6 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced EtOAcqqqqqqqqq

LCMS ⁽⁺ H +): measurement value of 538.2, calc. 537.29, molecular formula C ₃₅ H ₃₉ NO _4.

Purity (HPLC): 89%.

^{1 H NMR (400 MHz, CDCl} 3): δ 0.97-1.04 (9H, m, 3 × C H 3), 1.56-1.96 (5H, 3 × m, 2 × C H 2 and C H of the 4H 1H) , 3.07-3.38 (6H, m, 3 × C H 2 of the 6H), 3.62-3.68 (1H, m , C H -N), 4.26 (2H, t, J = 6.60 Hz, OC H 2), 6.45 ( 1H, s, C H -O), 6.54 (1H, s, C H = C), 7.03 (2H, d, J = 8.28 Hz, Ar- H ), 7.12-7.16 (1H, m, Ar- H ) , 7.18-7.32 (5H, m, Ar- H ), 7.34 (2H, dd, J = 3.32, 7.34 Hz, Ar- H ), 7.86 (2H, d, J = 8.24 Hz, Ar- H ).

^{13 C NMR (100 MHz, CDCl} 3): 10.52 (C H 3), 21.74,22.10,23.13 (2 × C H 3 and 1 × C H), 24.84 ( C H 2), 39.66 (C H 2), 40.46 ( C H ₂ ), 40.77 ( C H ₂ ), 43.67 ( C H ₂ ), 53.15 (fourth C ), 54.36 ( C H-N), 66.46 ( C H ₂ -O), 83.56 ( C HO), 120.70 (third C), 123.51 (third C), 124.48 (third C), 124.61 (third C), 125.78 (third C), 126.45 (third C), 127.07 (third C), 128.67 ( Fourth C ), 3×129.23 (3×third C ), 129.79 (third C ), 2×129.89 (2×third C ), 140.18 (fourth C ), 142.19 (fourth C ), 142.56 ( Fourth C ), 143.50 (fourth C ), 144, 24 (fourth C ), 151.02 (fourth C ), 166.60 ( C =O), 175.69 ( C =O).

Synthesis of (1 R , 2 R )-2-(4-Aminomethylbenzyl)-2,3-dihydro-1 H , 1' H -2,2'-diin-1-yl L-white Amine (35):

To 4-{[(1' R , 2' R )-1'-hydroxy-1',3'-dihydro-1H,2'H-2,2'-dioxa-2'-yl]methyl Addition of Fmoc leucine (126) to a solution of benzamide ( 13 , 140 mg, 0.36 mmol), DCC (90 mg, 0.44 mmol) and DMAP (4.3 mg, 0.036 mmol) in ethyl acetate (10 mL) Mg, 0.36 mmol), and then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 mg) was dissolved in anhydrous THF (5 mL) and then cooled to 0 °C. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjjj

LCMS ⁽⁺ H +): measurement value of 495.5, calc. 494.26, molecular formula _{C 32 H 34 N 2 O 3} .

Purity (HPLC): 91%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.87 (3H, d, J = 6.56 Hz, C H ₃ ), 0.91 (3H, d, J = 6.64 Hz, C H ₃ ), 1.42-1.48 ( 1H, m, C H ₂ - CH), 1.52-1.58 (1H, m, C H ₂ -CH), 1.72-1.82 (1H, m, C H ), 3.04-3.56 (7H, 3×m, 3× 6H of C H ₂ and 1H of C H -N, 6.30 (1H, s, C H -O), 6.49 (1H, s, C H = C), 7.00 (2H, d, J = 8.20 Hz, Ar - H ), 7.08 (1H, dt, J = 1.16, 10.15 Hz, Ar- H ), 7.16 (1H, t, J = 7.28 Hz, Ar- H ), 7.21 (2H, t, J = 6.48 Hz, Ar - H ), 7.25-7.30 (2H, m, Ar- H ), 7.34 (2H, t, J = 7.80 Hz, Ar- H ), 7.65 (2H, d, J = 8.20 Hz, Ar- H ), 7.84 (1H, br s, N H ₂ ).

^{13 C NMR (100 MHz, DMSO} -d 6): δ 22.33 (C H 3), 23.27 (C H 3), 24.66 (C H), 39.36-40.62 (3 × C H 2), 43.51 (C H 2 ), 53.27 (fourth C ), 54.75 ( C H-N), 82.79 ( C H-O), 120.89 (third C ), 123.93 (third C ), 124.65 (third C ), 125.23 (third C ), 125.81 (third C ), 126.70 (third C ), 127.14 (third C ), 2 x 127.45 (2 x third C ), 129.21 (third C ), 129.44 (third C ), 2 x 129.98 (2 × third C ), 132.49 (fourth C ), 140.62 (fourth C ), 142.08 (fourth C ), 142.86 (fourth C ), 143.03 (fourth C ), 144.44 (fourth C ), 152.06 ( Fourth C ), 168.04 (O- C = O), 175.44 ( C = O).

Synthesis of (1 R , 2 R )-2-{4-[(2-hydroxyethyl)amine-carbamoyl]benzyl}-2,3-dihydro-1 H ,1' H -2,2'- Dinon-1-yl L-leucine (36):

To 4-{[(1' R , 2' R )-1'-(L-Acetylmethoxy)-1',3'-dihydro-1 H , 2' H -2, 2'- inden-2'-yl] methyl} benzoic acid (24, 130 mg, 0.26 mmol ), Et 3 N (78 mg, 0.78 mmol) and 2-aminoethanol (24 mg, 0.39 mmol) in DCM (5 mL ) was added in the _{T 3 P (0.33 mL, 50} wt% solution in ethyl acetate, 0.52 mmol), and then stirred at room temperature for 12 hours. The reaction mixture was quenched with water (25 mL)EtOAc Brine (25 mL) The washed organic layer was separated, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified with EtOAc (EtOAc) elute

LCMS ⁽⁺ H +): measurement value of 539.6, calc. 538.28, molecular formula _{C 34 H 38 N 2 O 4} .

Purity (HPLC): 92%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.87 (3H, d, J = 6.52 Hz, C H ₃ ), 0.92 (3H, d, J = 6.64 Hz, C H ₃ ), 1.38-1.46 ( _{1H, m, C H 2 -CH} -), 1.50-1.56 (1H, m, C H 2 -CH -), 1.74-1.82 (1H, m, CH 2 -C H -), 3.03-3.56 (11H, 5H of 5×C H ₂ and 1H of C H -N, 4.70 (1H, br s, CH ₂ -O H ), 6.29 (1H, s, C H -O), 6.49 (1H, s, C H =C), 7.00 (2H, d, J = 8.24 Hz, Ar- H ), 7.09 (1H, dt, J = 1.20, 10.16 Hz, Ar- H ), 7.15-7.23 (3H, m, Ar- H ) , 7.27-7.37 (4H, m, Ar- H ), 7.64 (2H, d, J = 8.24 Hz, Ar- H ), 8.31 (1H, t, J = 5.64 Hz, N H ).

^{13 C NMR (100 MHz, DMSO} -d 6): 22.33 (C H 3), 23.38 (C H 3), 24.72 (CH), 39.36-40.62 (3 × C H 2), 42.55 (C H 2), 44.19 ( C H ₂ ), 53.63 ( C H-N or fourth C ), 54.79 (fourth C or C H-N), 60.20 ( C H ₂ OH), 82.46 ( C H-O), 120.88 (third C ), 123.94 ( Third C ), 124.63 (third C ), 125.21 (third C ), 125.72 (third C ), 126.69 (third C ), 3 x 127.12 (3 x third C ), 129.17, 129.37, 2× 130.01, 132.77 (4 × third C and 1 × fourth C ), 140.78 (fourth C ), 141.91 (fourth C ), 142.76 (fourth C ), 144.04 (fourth C ), 144.45 (fourth C ), 152.12 (fourth C ), 166.44 ( C = O), 176.37 ( C = O).

Synthesis of (1 R , 2 R )-2-[4-(methylamine-mercapto)benzyl]-2,3-dihydro-1 H ,1' H -2,2'-diindole-1- L-lysamine (37):

To 4-{[(1' R , 2' R )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- Addition of a solution of N -methylbenzamide ( 15 , 150 mg, 0.38 mmol), DCC (94 mg, 0.45 mmol) and DMAP (5 mg, 0.038 mmol) in ethyl acetate (10 mL) Fmoc leucine (133 mg, 0.38 mmol) was then stirred at room temperature for 12 h. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 mg) was dissolved in anhydrous THF (5 mL) and then cooled to 0 °C. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced EtOAcqqqqqm

LCMS ⁽⁺ H +): measurement value of 509.5, calc. 508.27, molecular formula _{C 33 H 36 N 2 O 3} .

Purity (HPLC): 99%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.95 (3H, d, J = 6.52 Hz, C H ₃ ), 0.98 (3H, d, J = 6.52 Hz, C H ₃ ), 1.60-1.76 (2H, m, C H ₂ -CH), 1.86-1.96 (1H, m, C H ), 2.97 (3H, d, J = 4.80 Hz, NH-C H ₃ ), 3.04-3.36 (6H, m, 3×C H ₂ ), 3.62-3.68 (1H, m, C H -N), 6.08-6.14 (1H, m, N H -CH ₃ ), 6.45 (1H, s, C H -O), 6.51 (1H, s , C H = C), 7.00 (2H, d, J = 8.20 Hz, Ar- H ), 7.09-7.19 (2H, m, Ar- H ), 7.21-7.29 (4H, m, Ar- H ), 7.32 (2H, d, J = 6.84 Hz, Ar- H ), 7.50-7.56 (2H, m, Ar- H ).

¹³ C NMR (100 MHz, CDCl ₃ ): δ 21.74 ( C H ₃ ), 23.14 ( C H ₃ ), 24.84 ( C H ₃ -N or C H), 26.80 ( C H or C H ₃ -N), 39.67( C H ₂ ),40.46( C H ₂ ),40.64( C H ₂ ),43.74( C H ₂ ),53.17(fourth C ),54.38( C H-N),83.48( C HO),120.68 C ), 123.51 (third C ), 124.47 (third C ), 124.63 (third C ), 125.75 (third C ), 126.45 (third C ), 2 x 126.52 (2 x third C ), 127.06 (third C ), 129.25 (third C ), 129.75 (third C ), 2×130.06 (2×third C ), 132.66 (fourth C ), 140.22 (fourth C ), 141.84 (fourth C ), 142.21 (fourth C ), 142.56 (fourth C ), 144.25 (fourth C ), 151.10 (fourth C ), 168.03 ( C =O).

Synthesis of (1 R , 2 R )-2-[4-(dimethylaminocarbamimidyl)benzyl]-2,3-dihydro-1 H ,1' H -2,2'-diindole-1 -yl L-leucine (38):

To 4-{[(1' R , 2' R )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- benzoyl dimethyl amine (16, 185 mg, 0.45 mmol ), DCC (112 mg, 0.54 mmol) and DMAP (6 mg, 0.045 mmol) in ethyl acetate (10 mL) - yl} - N, N A solution of Fmoc leucine (158 mg, 0.45 mmol) was added, followed by stirring at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 mg) was dissolved in anhydrous THF (5 mL) and then cooled to 0 °C. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjj

LCMS ⁽⁺ H +): measurement value of 523.2, calc. 522.29, molecular formula _{C 34 H 38 N 2 O 3} .

Purity (HPLC): 95%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.97 (3H, d, J = 6.52 Hz, C H ₃ ), 1.01 (3H, d, J = 6.56 Hz, C H ₃ ), 1.60-1.94 (3H, 3 × m, C H's and 1H of C H ₂ -CH 2H), 2.92 (3H, s , NC H 3), 3.05-3.36 (9H, m, 3 × C H 2 NC H ₃ of 6H and 3H of ), 3.63 - 3.67 (1H, m, C H -N), 6.43 (1H, s, C H -O), 6.53 (1H, s, C H = C), 6.97 (2H, d, J = 8.00 Hz) , Ar- H ), 7.10-7.14 (1H, m, Ar- H ), 7.18-7.28 (7H, m, Ar- H ), 7.30-7.35 (2H, m, Ar- H ).

¹³ C NMR (100 MHz, CDCl ₃ ): δ 21.76 ( C H ₃ ), 23.14 ( C H ₃ ), 24.85 ( C H), 39.70, 40.29 and 40.57 (2 x C H ₂ and 1 x N- C H ₃ ), 43.61 ( C H ₂ ), 53.13 (fourth C ), 54.42 ( C H-N), 83.62 ( C H-O), 120.66 (third C ), 123.48 (third C ), 124.42 (third C ), 124.61 (third C ), 125.72 (third C ), 124.63 (third C ), 2×126.89 (2×third C ), 127.01 (third C ), 129.20 (third C ), 129.72 (fourth C ), 2 × 129.82 (2 × third C ), 134.25 (fourth C ), 139.75 (fourth C ), 140.19 (fourth C ), 142.26 (fourth C ), 142.60 (fourth C ), 144.31 (Fourth C ), 151.23 (Fourth C ), 171.54 ( C = O).

Synthesis of 4-(((1S,2S)-1-(((S)-2-amino-4-methylpentyl)oxy)-2,3-dihydro-1H,1'H-[ Methyl 2,2'-dioxa]-2-yl)methyl)benzoate (39):

To 4-(((1S,2S)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoic acid methyl ester ( 17, 200 mg, 0.50 mmol) , DCC (125 mg, 0.60 mmol) and DMAP (6 mg, 0.045 mmol) in ethyl acetate (10 mL) was added in the Fmoc leucine (176 mg, 0.50 mmol), It was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure, and the residue 1 (220 mg) was dissolved in dry THF (5 mL) then cooled to 0 ℃. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified EtOAcqqqqqqq

LCMS ⁽⁺ H +): measurement value of 510.2, calc. 509.26, molecular formula C ₃₃ H ₃₅ NO _4.

Purity (HPLC): 96%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.93 (3H, d, J = 6.44 Hz, C H ₃ ), 0.94 (3H, d, J = 5.20 Hz, C H ₃ ), 1.54-1.80 (3H, ₂ × m, C H 2 -CH and 2H of CH ₂ C H of 1H), 3.05-3.39 (6H, m , 3 × C H 2), 3.70-3.74 (1H, m, C H -N), 3.89 (3H, s, OC H ₃ ), 6.42 (1H, s, C H -O), 6.51 (1H, s, C H = C), 7.01 (2H, d, J = 8.32 Hz, Ar- H ), 7.11-7.16(1H,m,Ar- H ), 7.18-7.31(6H,m,Ar- H ), 7.36(1H,d,J=7.28 Hz,Ar- H ),7.84(2H,d,J= 8.28 Hz, Ar- H ).

^{13 C NMR (100 MHz, CDCl} 3): δ 21.95 (C H 3), 22.78 (C H 3), 24.77 (C H), 39.62 (C H 2), 40.37 (C H 2), 40.73 (C H ₂ ), 43.69 ( C H ₂ ), 52.02, 52.96 and 54.40 (O C H ₃ , fourth C and C H-N), 83.54 ( C HO), 120.71, 123.54, 124.49, 124.64, 125.59, 126.45, 127.04, 128.28 , 3 × 129.21, 129.77, 2 × 130.00 (13 × third C and 1 × fourth C ), 140.06 (fourth C ), 142.06 (fourth C ), 142.56 (fourth C ), 143.62 (fourth C ), 144.22 (fourth C ), 150.94 (fourth C ), 167.04 ( C = O), 175.51 ( C = O).

Synthesis of 4-(((1S,2S)-1-(((S)-2-amino-4-methylpentyl)oxy)-2,3-dihydro-1H,1'H-[ Ethyl 2,2'-dioxa]-2-yl)methyl)benzoate (40):

To 4-(((1S,2S)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ethyl ester ( 18, 200 mg, 0.48 mmol) , DCC (120 mg, 0.58 mmol) and DMAP (6 mg, 0.048 mmol) in ethyl acetate (10 mL) was added in the Fmoc leucine (161 mg, 0.48 mmol), It was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (250 g) was dissolved in anhydrous THF (6 mL) and then cooled to 0 °C. Dimethylamine (6 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjj

LCMS ⁽⁺ H +): measurement value of 524.2, calc. 523.27, molecular formula C ₃₄ H ₃₇ NO _4.

Purity (HPLC): 98%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.89-0.94 (6H, m, (C H ₃ ) ₂ ), 1.37 (3H, t, J = 6.80 Hz, C H ₃ ), 1.64-1.80 (3H, m, C H's and 1H of C H ₂ -CH 2H), 3.02-3.16 (2H, m , C H 2), 3.20-3.36 (4H, m, 2 × C H 2), 3.82-3.86 (1H, m, C H -N), 4.34 (2H, q, J = 7.20 Hz, OC H ₂ ), 6.41 (1H, s, C H -O), 6.49 (1H, s, C H = C), 6.99 ( 2H,d,J=8.00 Hz,Ar- H ), 7.11-7.15(1H,m,Ar- H ),7.16-7.30(6H,m,Ar- H ),7.36(1H,d,J=7.60 Hz , Ar- H ), 7.83 (2H, d, J = 8.40 Hz, Ar- H ).

^{13 C NMR (100 MHz, CDCl} 3): δ 14.31 (C H 3), 21.95 (C H 3), 22.80 (C H 3), 24.78 (C H), 39.62 (C H 2), 40.39 (C H ₂ ), 40.70 ( C H ₂ ), 43.86 ( C H ₂ ), 52.99 (fourth C ), 54.40 ( C H-N), 60.87 ( C H ₂ -O), 83.49 ( C H-O), 120.71 (third C ), 123.54 (third C), 124.48 (third C), 124.64 (third C), 125.57 (third C), 126.45 (third C), 127.03 (third C), 128.62 (IV C) , 3 × 129.18 (3 × third C ), 129.74 (third C ), 2 × 129.94 (2 × third C ), 140.10 (fourth C ), 142.06 (fourth C ), 142.57 (fourth C ) , 143.49 (fourth C ), 144.24 (fourth C ), 150.99 (fourth C ), 166.58 ( C = O), 175.80 ( C = O).

Synthesis of 4-(((1S,2S)-1-(((S)-2-amino-4-methylpentyl)oxy)-2,3-dihydro-1H,1'H-[ 2,2'-Dioxa]-2-yl)methyl)benzoic acid propyl ester (41):

To 4-(((1S,2S)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoic acid propyl ester ( Fmoc leucine (148 mg, 0.42 mmol) was added to a solution of 19 , 180 mg, 0.42 mmol), DCC (105 mg, 0.50 mmol) and DMAP (6 mg, 0.042 mmol) in ethyl acetate (10 mL). It was then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure, and the residue 1 (210 mg) was dissolved in dry THF (5 mL) then cooled to 0 ℃. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified EtOAcjjjjjjjj

LCMS ⁽⁺ H +): measurement value of 538.5, calc. 537.29, molecular formula C ₃₅ H ₃₉ NO _4.

Purity (HPLC): 95%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.93 (3H, d, J = 4.12 Hz, C H ₃ ), 0.94 (3H, d, J = 4.12 Hz, (C H ₃ ), 1.03 (3H, t , J = 8.00 Hz, C H 3), 1.49-1.81 (5H, m, C H 's and 1H of ₂ × C H 2 4H), 3.07-3.41 (6H, m , 3 × C H 2), 3.64- 3.67 (1H, m, C H -N), 4.26 (2H, t, J = 8.00 Hz, OC H ₂ ), 6.42 (1H, s, C H -O), 6.53 (1H, s, C H = C ), 7.01 (2H, d, J = 8.00 Hz, Ar- H ), 7.13-7.17 (1H, m, Ar- H ), 7.20-7.32 (6H, m, Ar- H ), 7.37 (1H, d, J = 4.00 Hz, Ar- H ), 7.86 (2H, d, J = 8.00 Hz, Ar- H ).

^{13 C NMR (100 MHz, CDCl} 2): δ 10.53 (C H 3), 21.93 (C H 3), 22.09 (C H 3), 22.87 (C H), 24.80 (C H 2), 39.61 (C H ₂ ), 40.43 ( C H ₂ ), 40.68 ( C H ₂ ), 44.24 ( C H ₂ ), 53.12 (fourth C ), 54.39 ( C H-N), 66.47 ( C H ₂ -O), 83.31 ( C HO ), 120.72 (third C), 123.53 (third C), 124.48 (third C), 124.64 (third C), 125.54 (third C), 126.46 (third C), 127.02 (third C) , 128.65 (fourth C ), 129.15 (third C ), 2 × 129.19 (2 × third C ), 129.71 (third C ), 2 × 129.93 (2 × third C ), 140.18 (fourth C ) , 142.01 (fourth C ), 142.77 (fourth C ), 143.51 (fourth C ), 144.26 (fourth C ), 151.05 (fourth C ), 166.64 (O- C = O), 176.39 ( C = O ).

Synthesis of (1 S , 2 S )-2-(4-Aminomethylbenzyl)-2,3-dihydro-1 H , 1' H -2,2'-diin-1-yl L-white Amine (42):

To 4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- Addition of Fmoc leucine to a solution of benzamide ( 20 , 150 mg, 0.26 mmol), DCC (64 mg, 0.31 mmol) and DMAP (3 mg, 0.026 mmol) in ethyl acetate (10 mL) 91 mg, 0.26 mmol), and then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (180 mg) was dissolved in anhydrous THF (5 mL). Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjj

LCMS ⁽⁺ H +): measurement value of 495.2, calc. 494.26, molecular formula _{C 32 H 34 N 2 O 3} .

Purity (HPLC): 97%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.85 (3H, d, J = 6.20 Hz, C H ₃ ), 0.87 (3H, d, J = 6.20 Hz, C H ₃ ), 1.40-1.74 ( 3H, 2 × m, C H 2 -CH 2H and of the C H -CH ₂ 1H), 3.04 (1H, d , J = 13.44 Hz, C H 2), 3.11-3.28 (4H, m, 2 × C _{H 2), 3.48-3.56 (2H,} m, C H 2 and 1H of C H -N), 6.27 (1H , s, C H -O), 6.49 (1H, s, C H = C), 6.99 ( 2H,d,J=8.20 Hz, Ar- H ), 7.09 (1H, dt, J=1.20, 10.16 Hz, Ar- H ), 7.15-7.38 (8H, m, 7H×Ar- H and N H ₂ 1H), 7.66 (2H, d, J = 8.24 Hz, Ar- H ), 7.84 (1H, br s, N H ₂ ).

^{13 C NMR (100 MHz, DMSO} -d 6): 22.50 (C H 3), 23.18 (C H 3), 24.71 (C H), 39.35-40.60 (2 × C H 2), 44.30 (C H 2) , 53.26 (fourth C ), 54.80 ( C H-N), 82.43 ( C H-O), 120.89 (third C ), 123.95 (third C ), 124.62 (third C ), 125.22 (third C ), 125.57 ( Third C ), 126.69 (third C ), 127.14 (fourth C ), 2×127.41 (2×third C ), 129.14 (third C ), 129.34 (third C ), 2×130.05 (2× Third C ), 132.47 (third C ), 140.70 (fourth C ), 142.10 (fourth C ), 142.73 (fourth C ), 143.08 (fourth C ), 144.47 (fourth C ), 152.17 (first Four C ), 168.06 (N- C = O), 176.56 ( C = O).

Synthesis of (1 S , 2 S )-2-{4-[(2-hydroxyethyl)amine-methylmethyl]benzyl}-2,3-dihydro-1 H ,1' H -2,2'- Diterpene-1-yl leucine (43):

4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diindole-2 at -78 °C '- yl] methyl} - N - (2- hydroxyethyl) benzoyl amine (21, 300 mg, 0.79 mmol ) and 2,4,6-collidine (85 mg, 0.79 mmol) in DCM Acetyl chloride (62 mg, 0.79 mmol) was added to the solution in (10 mL) and then slowly warmed to room temperature over a period of 1 hour. The reaction mixture was quenched with EtOAc (EtOAc)EtOAc. Brine (25 mL) the organic layer was washed, dried over anhydrous evaporated Na ₂ SO _4, and under reduced pressure. Used in the 20% ethyl acetate in chloroform as the solvent the residue was purified by CombiFlash purification from agent, to yield 250 mg (68%) of an off-white solid intermediate.

LCMS ⁽⁺ H +): measurement value of 468.4, calc. 467.56, molecular formula C ₃₀ H ₂₉ NO _4.

Purity (UPLC): 99%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 2.10 (3H, s, C H ₃ ), 2.83 (1H, d, J = 13.20 Hz, C H ₂ ), 3.01-3.03 (2H, mC H ₂ ), 3.26 (1H, d, J = 13.20 Hz, C H ₂ ), 3.46 (1H, d, J = 22.40 Hz, C H ₂ ), 3.58 (1H, d, J = 22.80 Hz, C H ₂ ), 3.69- 3.73 (2H, m, CONHC H ₂ ), 4.28 (2H, t, J = 5.60 Hz, C H ₂ OCH ₃ ), 5.25 (1H, d, J = 7.60 Hz, C H -OH), 6.44 (1H, m,CH-O H or N H ), 6.47 (1H, s, C H = C), 6.97 (2H, d, J = 8.00 Hz, Ar- H ), 7.17 (1H, dt, J = 2.00, 9.87 Hz, Ar- H ), 7.26-7.32 (5H, m, Ar- H ), 7.44 (2H, d, J = 6.40 Hz, Ar- H ), 7.57 (2H, d, J = 8.40 Hz, Ar- H ).

To 2-[(4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diindole-2' -ethyl]methyl}benzhydryl)amino]acetate (intermediate 1 , 250 mg, 0.53 mmol), DCC (142 mg, 0.62 mmol) and DMAP (7 mg, 0.053 mmol) in ethyl acetate (10 mL) was added in the N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] leucine (226 mg, 0.53 mmol), and then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure, and a 20% ethyl acetate in chloroform as the solvent of the separating agent residue was purified by CombiFlash to yield 320 mg (76%) of intermediate 2 an off-white solid.

LCMS ⁽⁺ H +): measurement value of 803.4, calc. 802.95, molecular formula _{C 51 H 50 N 2 O 7} .

Purity (UPLC): 96%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.93 (6H, d, J = 5.53 Hz, CH(C H ₃ ) ₂ ), 1.46-1.78 (3H, 3×m, C H ₂ -C H -) , 2.07 (3H, s, COC H ₃ ), 3.06-3.41 (6H, m, 3 × C H ₂ ), 3.66-3.70 (2H, m, CONHC H ₂ ), 4.22-4.27 (3H, m, C H ₂ OCOCH ₃ and C H -CH ₂ O), 4.42-4.46 (2H, m, CH-C H ₂ O), 4.49-4.54 (1H, m, C H -NH), 5.21 (1H, d, J = 8.92 Hz, CH-N H ), 6.41-6.43 (2H, m, C H -O and CH ₂ N H CO), 6.51 (1H, s, C H = C), 6.98 (2H, d, J = 8.20) Hz, Ar- H ), 7.11-7.14 (1H, m, Ar- H ), 7.19-7.25 (4H, m, Ar- H ), 7.30-7.34 (5H, m, Ar- H ), 7.71 (2H, t, J = 7.68 Hz, Ar- H ), 7.54 (2H, d, J = 8.32 Hz, Ar- H ), 7.59-7.63 (2H, m, Ar- H ), 7.78 (2H, d, J = 7.24) Hz, Ar- H ).

Take (1 S , 2 S )-2-(4-{[2-(ethyloxy)ethyl]amine-methylmethyl}benzyl)-2,3-dihydro-1 H ,1' H - 2,2'-inden-1-yl N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] melam ester (intermediate 2, 320 mg, 0.40 mmol) was dissolved in dry THF (5 In mL) and cool to 0 °C. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure, and the residue was purified by CombiFlash using 10% methanol in chloroform as a solvent to yield 160 mg of solid material (38% product mass (581.5) as determined by UPLC). The solid was dissolved in THF:H ₂ O (1:1, 10 mL), and lithium hydroxide dihydrate (6 mg, 0.27 mmol) was added at 0 ° C and then stirred at room temperature for 1 hour. The reaction mixture was neutralized with 1.5 N HCl (pH 7.0), then extracted with ethyl acetate (3×10 mL), washed with 10% NaHCO ₃ (10 mL) and then brine (10 mL). Over anhydrous Na ₂ SO ₄ the organic layer was dried, evaporated under reduced pressure, and used in 10% methanol in chloroform as the solvent of the separating agent residue was purified by CombiFlash to yield 35 mg (21%) of the title compound an off-white solid .

LCMS ⁽⁺ H +): measurement value of 540.3, calc. 538.68, molecular formula _{C 34 H 38 N 2 O 4} .

Purity (HPLC): 95%.

^{1 H NMR (400 MHz, CDCl} 3): δ 0.92-0.94 (6H, m, CH (C H 3) 2), 1.46-1.78 (3H, 3 × m, C H 2 -C H -), 3.06- 3.13 (2H, m, C H 2), 3.18-3.27 (3H, m, C H 2 of the allyl group and 1H of C H ₂ 2H), 3.37 (1H, d , J = 22.40 Hz, C H 2) , 3.58-3.65 (3H, m, CONHC H 2 and _{C H -NH 2), 3.82 (} 2H, t, J = 5.20 Hz, C H 2 OH), 6.41 (1H, s, C H -O), 6.52 (1H, s, C H = C), 6.61 (1H, t, J = 5.20 Hz, CON H or CH ₂ O H ), 7.00 (2H, d, J = 8.40 Hz, Ar- H ), 7.12-7.19 (1H, m, Ar- H ), 7.15-7.31 (6H, m, Ar- H ), 7.36 (1H, d, J = 7.20 Hz, Ar- H ), 7.59 (2H, d, J = 8.40 Hz, Ar- H ).

^{13 C NMR (100 MHz, CDCl} 3): 21.94 (C H 3), 22.85 (C H 3), 24.79 (C H), 29.27 (C H 2), 39.61 (C H 2), 40.59 (C H 2 ), 42.82 ( C H ₂ ), 44.19 ( C H ₂ ), 53.08 (fourth C ), 54.39 ( C H-N), 62.44 ( C H ₂ OH), 83.26 ( C H-O), 120.72 (third C ), 123.54 (third C ), 124.49 (third C ), 124.65 (third C ), 125.54 (third C ), 126.47 (third C ), 2×126.66 (2×third C ), 127.03 (third C), 129.18 (third C), 129.70 (third C), 2 × 130.13 (2 × third C), 132.18 (IV C), 140.18 (IV C), 142.01 (IV C), 142.24 (Fourth C ), 142.55 (Fourth C ), 144.25 (Fourth C ), 151.77 (Fourth C ), 168.36 ( C =O), 176.27 ( C =O).

Synthesis of (1 S , 2 S )-2-[4-(methylamine-mercapto)benzyl]-2,3-dihydro-1 H ,1' H -2,2'-diindole-1- L-lysamine (44):

To 4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- Addition of a solution of N -methylbenzamide ( 22 , 150 mg, 0.38 mmol), DCC (94 mg, 0.45 mmol) and DMAP (5 mg, 0.038 mmol) in ethyl acetate (10 mL) Fmoc leucine (134 mg, 0.38 mmol) was then stirred at room temperature for 12 h. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (180 mg) was dissolved in anhydrous THF (5 mL). Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjj

LCMS ⁽⁺ H +): measurement value of 509.5, calc. 508.27, molecular formula _{C 33 H 36 N 2 O 3} .

Purity (HPLC): 97%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.85 (3H, d, J = 6.00 Hz, C H ₃ ), 0.87 (3H, d, J = 6.40 Hz, C H ₃ ), 1.39-1.72 ( 3H, 2 × m, C H 2 -CH 2H and of the C H -CH ₂ 1H), 2.73 (3H, d , J = 4.40 Hz, C H 3), 3.04 (1H, d, J = 13.60 Hz, _{C H 2), 3.15 (1H} , t, J = 26.80 Hz, C H 2), 3.23-3.27 (3H, m, 2 × C H 2 of 3H), 3.49-3.55 (2H, m , C H 2 of 1H) of 1H and C H -N, 6.27 (1H, s, C H -O), 6.48 (1H, s, C H = C), 6.99 (2H, d, J = 8.40 Hz, Ar- H ), 7.09 (1H, dt, J = 1.20, 10.27 Hz, Ar- H ), 7.15-7.25 (4H, m, Ar- H ), 7.27-7.37 (3H, m, Ar- H ), 7.61 (2H, d, J = 8.40 Hz, Ar- H ), 8.31 (1H, d, J = 4.40 Hz, N H ).

Synthesis of (1 S , 2 S )-2-[4-(dimethylaminocarbamimidyl)benzyl]-2,3-dihydro-1 H ,1' H -2,2'-diindole-1 -yl L-leucine (45):

To 4-{[(1' S , 2' S )-1'-hydroxy-1',3'-dihydro-1 H , 2' H -2,2'-diin-2'-yl]- benzoyl dimethyl amine (23, 100 mg, 0.24 mmol ), DCC (56 mg, 0.28 mmol) and DMAP (3 mg, 0.024 mmol) in ethyl acetate (5 mL) - yl} - N, N The solution was added with Fmoc leucine (84 mg, 0.24 mmol) and then stirred at room temperature for 12 hours. The solid was filtered off, ethyl acetate (25 ml) wash, and using 1.5 N HCl (25 mL), water (25 mL), brine (10 mL) and the combined filtrate was washed, dried over anhydrous Na ₂ SO _4. The organic layer was evaporated under reduced pressure and residue 1 (140 mg) was dissolved in anhydrous THF (5 mL) and then cooled to 0 °C. Dimethylamine (5 mL, 20% in THF) was added dropwise and the mixture was slowly stirred at room temperature for one hour. The reaction mixture was evaporated under reduced pressure and purified titled mjjjjjjjj

LCMS ⁽⁺ H +): measurement value of 523.5, calc. 522.29, molecular formula _{C 34 H 38 N 2 O 3} .

Purity (HPLC): 97%.

^{1 H NMR (400 MHz, DMSO} -d 6): δ 0.82-0.87 (6H, m, 2 × C H 3), 1.58-1.66 (3H, m, C H 2 CH of 2H and 1H of CH ₂ C H ), 2.83 (3H, s, NC H ₃ ), 2.93 (3H, s, NC H ₃ ), 3.11 (1H, d, J = 13.20 Hz, C H ₂ ), 3.18-3.30 (4H, m, 2× C H ₂ ), 3.45 (1H, d, J = 22.80 Hz, C H ₂ ), 4.26-4.30 (1H, m, C H -N), 6.36 (1H, s, C H -O), 6.55 (1H , s, C H = C), 7.00 (2H, d, J = 8.00 Hz, Ar- H ), 7.09 (1H, dt, J = 1.20, 10.00 Hz, Ar- H ), 7.15-7.23 (5H, m , Ar- H ), 7.27-7.37 (4H, m, Ar- H ), 8.47 (2H, br s, N H ₂ ).

Synthesis of 2-hydroxy-5-(4-((1R,2R)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl) A Benzobenzamide)benzoic acid (46):

To 4-(((1R,2R)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl)methyl)benzoic acid ethyl ester ( Add 11 mg, 0.5 mg, 0.48 mmol), 5-amino-2-hydroxybenzoic acid (75 mg, 0.48 mmol) and THF (1.0 mL) in a 5 mL microwave vial. A 20% solution in toluene) and the mixture was irradiated at 100 ° C for 5 minutes. The reaction mixture was quenched with EtOAc (3×15 mL). With water (25 mL), brine (25 mL) The organic layer was washed, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified by preparative HPLC [Kromasil C18 (250×50 mm), flow rate: 40 mL/min, 0.1% TFA in water/MeOH, run time: 40 min) to yield 75 mg (30) %) the title compound as an off-white solid.

LCMS ⁽⁺ H +): measurement value of 518.4, calc. 517.19, molecular formula C ₃₃ H ₂₇ NO _5.

Purity (HPLC): 90%.

^{1 H NMR (400 MHz, DMSO} -d 6): δ 2.72 (1H, d, J = 13.64 Hz, -C H 2 -), 2.97 (1H, d, J = 17.20 Hz, -C H 2), 3.01 (1H, d, J = 16.36 Hz, -C H ₂ -), 3.23 (1H, d, J = 13.64 Hz, -C H ₂ ), 3.50 (1H, d, J = 23.16 Hz, C H ₂ ), 3.65 (1H, d, J = 23.16 Hz, C H 2), 5.07 (1H, s, C H -OH), 5.87 (1H, br s, CH-O H), 6.44 (1H, s, C H = C), 6.87 (1H, d, J = 8.88 Hz, Ar- H ), 6.97 (2H, d, J = 8.20 Hz, Ar- H ), 7.10 (1H, t, J = 7.28 Hz, Ar- H ) , 7.16-7.21 (1H, m, Ar- H ), 7.23-7.29 (4H, m, Ar- H ), 7,37 (1H, t, J = 3.52 Hz, Ar- H ), 7.42 (1H, d , J=7.28 Hz, Ar- H ), 7.73 (2H, d, J=8.20 Hz, Ar- H ), 7.77 (1H, dd, J=2.68, 8.90 Hz, Ar- H ), 8.18 (1H, d , J = 2.64 Hz, Ar- H ), 10.05 (1H, s, Ar-O H or O = CO H ).

^{13 C NMR (100 MHz, DMSO} -d 6): δ 39.34-40.59 (3 × C H 2), 56.23 ( IV C), 81.49 (C H- OH), 117.10,120.64,122.41,123.94,124.27, 124.80,124.92,126.58,126.81,2×127.38,127.67,128.05,128.30,3×130.21,130.81,132.74 (16×third C and 3×fourth C ), 140.77 (fourth C ), 143.39 (fourth C ), 143.42 (fourth C ), 144.88 (fourth C ), 145.53 (fourth C ), 154.46 (fourth C ), 158.14 (fourth C ), 165.36 (N- C = O), 172.15 (O - C = O).

Synthesis of 2-hydroxy-5-(4-((1S,2S)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxin]-2-yl) A Benzobenzamide)benzoic acid (47):

To 4-(((1S,2S)-1-hydroxy-2,3-dihydro-1H,1'H-[2,2'-dioxa]-2-yl)methyl)benzoic acid ethyl ester ( Add 18 mg, 200 mg, 0.48 mmol), 5-amino-2-hydroxybenzoic acid (75 mg, 0.48 mmol) and THF (1.0 mL) in a 5 mL microwave vial. A 20% solution in toluene) and the mixture was irradiated at 100 ° C for 5 minutes. The reaction mixture was quenched with EtOAc (3×15 mL). With water (25 mL), brine (25 mL) The organic layer was washed, dried over anhydrous Na ₂ SO _4, and evaporated under reduced pressure. The residue was purified by preparative HPLC [Kromasil C18 (250×50 mm), flow rate: 40 mL/min, 0.1% TFA in water/MeOH, run time: 40 min) to yield 90 mg (36) %) the title compound as an off-white solid.

LCMS (-H ^+): measurement value of 516.0, calc. 517.19, molecular formula C ₃₃ H ₂₇ NO _5.

Purity (HPLC): 89%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 2.72 (1H, d, J = 13.64 Hz, C H ₂ ), 2.97 (1H, d, J = 16.92 Hz, C H ₂ ), 3.01 (1H, d, J = 16.24 Hz, -C H ₂ -), 3.23 (1H, d, J = 13.60 Hz, C H ₂ ), 3.49 (1H, d, J = 23.12 Hz, C H ₂ ), 3.64 (1H, d, J = 23.12 Hz, C H 2), 5.06 (1H, s, C H -OH), 5.85 (1H, br s, CH-O H), 6.43 (1H, s, C H = C), 6.93 (1H, d, J = 8.96 Hz, Ar- H ), 6.97 (2H, d, J = 8.04 Hz, Ar- H ), 7.07 (1H, t, J = 7.28 Hz, Ar- H ), 7.17 (1H , t, J = 7.40 Hz, Ar- H ), 7.22-7.28 (4H, m, Ar- H ), 7.39 (1H, t, J = 3.44 Hz, Ar- H ), 7.41 (1H, d, J = 7.28 Hz, Ar- H ), 7.72 (2H, d, J = 8.20 Hz, Ar- H ), 7.82 (1H, dd, J = 2.60, 8.94 Hz, Ar- H ), 8.24 (1H, d, J = 2.48 Hz, Ar- H ), 10.09 (1H, s, Ar-O H or O = CO H ).

^{13 C NMR (100 MHz, DMSO} -d 6): δ 38.47-40.58 (3 × C H 2), 56.23 ( IV C), 81.48 (C H- OH), 112.82,117.46,120.64,122.15,123.94, 124.27, 124.81, 124.91, 126.58, 122.82, 2×127.39, 127.68, 128.06, 129.00, 2×130.23, 131.35, 132.64 (16×third C and 3×fourth C ), 140.76 (fourth C ), 143.38 ( Fourth C ), 143.53 (fourth C ), 144.87 (fourth C ), 145.52 (fourth C ), 154.45 (fourth C ), 157.77 (fourth C ), 165.47 (N- C = O), 172.22 (O- C = O).

IL2 and autoimmune inflammatory diseases.

Cytokines can be produced by a variety of cell populations and have been shown to potentiate or limit immune responses to pathogens and affect autoimmune responses. Has been a family of cytokines (which use the common receptor gamma chain (cc) (a type of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21 Body components)) are generally defined as growth and survival factors.

IL-2 production induces an immune response by promoting proliferation and production of CD4+ Th1, CD4+ Th2 and CD8+ CTL effector cells. Many immunosuppressive drugs (such as corticosteroids) and immunosuppressive drugs (cyclosporine, tacrolimus) used in the treatment of autoimmune diseases and organ transplant rejection by inhibiting the production of IL-2 by antigen-activated T cells And it works. Other drugs (sirolimus) block IL-2R signaling, thereby preventing the expansion and function of antigen-selective T cells. [Reference: Opposing functions of IL-2 and IL-7 in the Regulation of immune responses Shoshana D. Katzman, Katrina K. Hoyer, Hans Dooms, Iris K. Gratz, Michael D. Rosenblum, Jonathan S. Paw, Sara H. Isakson, Abul K. Abbas. Cytokine 56 (2011) 116-121 ].

In contrast, IL-2 can promote the induction of (peripheral) Tregs production by inhibiting the survival and function of natural (thymus) regulatory T-cells (Tregs) and inhibiting the production of CD4+ Th17 effector cells [Reference: IL-2 and autoimmune disease.Anneliese Schimpl, A., Berberich, I, Kneitz, B., Krämer, S., Santner-Nanan, B., Wagner, S., Wolf, M., Hünig, T. Cytokine & Growth Factor Reviews 13 (2002) 369-378]. Defects of interleukin-2/IL-2R over time will result in inflammation of multiple organs and the formation of multiple specific autoantibodies. Depending on the genetic background, death will occur in weeks to months, with most dying from autoimmune hemolytic anemia or inflammatory bowel disease (IBD) [Reference: Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 1993; 75: 253-61].

IL-2 signaling has been shown to be important in the initiation and regulation of immune responses. In these dual and antagonistic roles, IL-2 acts to balance the immune response, promoting both immune cell activation and subsequent reduction. The potential clinical applications for enhancing or inhibiting IL-2 mediated signals are significant and include cancer, autoimmune inflammatory diseases, organ transplantation, and HIV.

Effect of compounds on IL-2 secretion in T lymphocytes

Methodology

The T cell line Jurkat 6.1 (ATCC) was used. Cells were pretreated with 1 μM or 10 μM of each compound for 30 minutes and then stimulated with anti-CD3 (BD Pharmingen) and anti-CD28 (AnCell) conjugate plates. DMSO was used as a vehicle control group. The immunosuppressive cyclosporin A was used as a control group for inhibiting IL-2 production. After 24 hours, the supernatant was collected and IL-2 secretion was measured by ELISA.

2. Preparation of compounds (raw materials)

Compounds were prepared by dissolving in DMSO to give final concentrations of 1 and 10 μg/ml.

3. Results

Absolute value of IL-2 production

Compound breeding results

The effects of synthetic compounds 2-5 , 10-16 , 18-38 and 39-47 on the release of IL2 from Jurkat cells are shown in Figures 21 and 22. Compounds were evaluated at concentrations of 1 and 10 μm. Most compounds showed inhibition of release relative to the negative control group. However, compounds 40 (12.71 pg/ml), 43 (12.79 pg/ml), 44 (12.43 pg/ml), 45 (14.29 pg/ml) and 46 (72.07 pg/ml) (Figure 22) are the most effective. Compared with the negative control group (326.67 pg/ml), the IL-2 release was significantly reduced, and the efficacy of cyclosporine (101.00 pg/ml) was close to 1 μm. In addition, Compound 10 (122.22 pg/ml), 32 (74.88 pg/ml), 33 (74.38 pg/ml), and 34 (71.38 pg/ml) (Figure 21) were also effective compared to the negative control group (320.67). Pg/ml) significantly reduced IL-2 release. In contrast, Compound 27 potentiated IL2 release (350.13 pg/ml) at 1 μm, which was stronger than the negative control group (338.25 pg/ml). IL-2 signaling has been shown to be important in the initiation and regulation of immune responses. In these dual and antagonistic roles, IL-2 acts to balance the immune response, promoting both immune cell activation and subsequent reduction. The potential clinical applications for enhancing or inhibiting IL-2 mediated signals are significant and include cancer, autoimmune inflammatory diseases, organ transplantation, and HIV.

Inflammatory bowel disease (IBD)

Inflammatory bowel disease (IBD) is an inflammatory immune disease that includes two congenital inflammatory diseases: ulcerative colitis (UC) and Crohn's disease. (Crohn's disease) (CD). The biggest difference between CD and UC is the extent of inflamed bowel tissue. In the CD, the inflammatory system is discontinuously segmented, called local enteritis, and UC is superficial inflammation, extending continuously from the proximal rectum. Currently, the exact cause of IBD is not known. The disease appears to be associated with an over-immune response of the mucosa to intestinal epithelial cell infection because pro-inflammatory molecules are not balanced with immunomodulatory molecules. The genetic pattern of IBD suggests that complex genetic components of pathology can include several combinatorial gene mutations. There is still no specific diagnosis for IBD, but as the understanding of pathological mechanisms increases, the test methods also improve. Treatment of IBD involves induction and maintenance of remission. IBD patients can be relieved by the use of 5-aminosalicylate. However, although the use of aminosalicylate in UC provides significant benefits, not only can mild to moderate disease be alleviated, but also relapse can be prevented, but the effectiveness of these drugs in maintaining CD remission is not reliable, and It is no longer recommended. The treatment of active disease mainly relies on corticosteroids, which are often used to relieve UC and CD patients within a limited time, although the systemic absorption of budesonide designed for local administration is limited, but in terms of maintaining remission No benefit. Alternatives, such as the immunosuppressive drugs azathioprine and mercaptopurine, have limited efficacy with methotrexate and cyclosporine and can cause serious side effects. Anti-TNFα antibodies, such as infliximab and adalimumab, can be used in patients who do not respond to standard immunosuppressive therapies. However, many patients do not respond to anti-TNFa therapy due to their specific phenotype or the production of autoantibodies.

Acute murine DSS colitis model

The dextran sulfate sodium (DSS) colitis model is an experimental mouse model that shows many of the symptoms observed in human UC, such as diarrhea, blood in the stool, mucosal ulcers, colon shortening, weight loss, and several changes in colon cytokines. This study is widely used as a model for the study of UC pathology and screening for novel therapeutic interventions for the treatment of UC.

In these studies, 5% DSS was administered to BALB/c mice drinking water using the acute DSS colitis model. This feeding regimen induced severe acute colitis, which showed significant bleeding in the rectum and a significant weight loss on days 7-8; all mice will die on days 10-12 unless sacrificed in advance.

Experiment 1: About Compound 2-6 Mouse

No specific pathogen BALB/c mice were obtained from commercial suppliers (Harlan UK), 6-8 weeks old. Irradiated foods were fed to mice and housed in an independent ventilator (Tecniplast UK) under positive pressure.

DSS processing

Dissolve DSS (5%) in drinking water. The compound was orally administered at a dose of 10 mg/kg or 30 mg/kg on days 0-7, and mice were selected on the 8th or 9th day depending on the severity of the disease. The incidence of mice was checked daily and the body weight of individual mice was recorded. The induction of colitis was determined based on autopsy, colon length, and histology. The colon was recovered and stored at -20 °C for immunoassay. All compounds and experimental groups were randomly labeled in alphabetical order. All data for the entire experiment was recorded in a blinded manner. The box/group code is not revealed until the data is analyzed, that is, the box marked A, B, C, etc. is determined to be not in place. Treatment, DSS treatment or treatment with DSS+ compounds.

The disease activity index (DAI) was determined based on weight loss, blood in the stool, and stool hardness to quantify the severity of colitis. Give each parameter a rating and the sum of the scores used as the DAI. n=8 for each treatment group.

definition

¹ soft stool - feces are not formed, but into a paste.

² diarrhea - not feces, sticking around the anus.

³ obvious bleeding - blood in the feces exceeded the standard, blood around the anus.

Administration of compounds

All compounds were prepared as a suspension in 0.5% carboxymethylcellulose/2% Tween 80 (0.1 mL orally (p.o.) per 10 g body weight) and orally administered at a dose of 3-30 mg/kg. Initially, the compound was dissolved as free acid in absolute alcohol and diluted with 14+1 and 0.5% carboxymethylcellulose/2% Tween 80, which resulted in fine precipitation in the suspension, but only N-methyl -(D)-glucosamine salt is soluble in this vehicle.

Effect of single enantiomer compounds 2, 3, 4 and 5 on 5% DSS murine colitis

Compounds 2 , 3 , 4, and 5 were administered to BALB/c fed 5% DSS in drinking water, and 30 mg/kg orally per day as 0.5% carboxymethylcellulose/2% Tween 80 The suspension was administered for 7 days. DAI measures the severity of the disease in this model. Compound 4 was not active in this variable and there was no significant (P > 0.05) difference in DAI at any time point (Figure 3). On day 7, both Compound 2 and Compound 5 significantly reduced DAI (P < 0.5) from 9.0 ± 0.53 in the vehicle control group to 3.2 ± 0.73 in Compound 5 and 2.5 ± 0.71 in Compound 2 , both There were no significant differences between the two (Figure 4). In contrast, Compound 3 only reduced DAI to 5.3 ± 0.6. This apparent (P>0.05) is less affected than either Compound 2 or Compound 5 . In addition, on day 7, although the effect of DAI in mice treated with compound 3 was statistically lower (P < 0.05) than in the vehicle control group (Fig. 4), on day 6, compound 3 and vehicle There was no statistically significant (P > 0.05) difference (Figure 3). In summary, in the four enantiomers of compounds 2 , 3 , 4 and 5 , both compounds 2 and 5 were highly active in this model at 30 mg/kg. Compound 3 had the lowest activity and was significantly (P < 0.05) lower than Compound 2 and Compound 5 . Compound 4 was almost inactive in this 5% DSS murine colitis model.

Select the salts of compounds 2 and 5

Since the water solubility of enantiomers 2 and 5 is limited, we have attempted to synthesize five salts of compound 5 . Synthesis of sodium salt, potassium salt, calcium salt, α-methylbenzylamine salt and N-methyl-(D)-glucosamine salt. Sodium and calcium salts were not successful. Three salts of Compound 5 , namely potassium salt, alpha-methylbenzylamine salt and N-methyl-(D)-glucosamine salt, were used for solubility and partition coefficient (logP) studies.

Determine the solubility of four compounds:

Surprisingly, it was found the compound N- methyl-5 - (D) - glucosamine salt (Compound 7) The maximum solubility, much higher than the set of similar compounds, the solubility of> 60,000 μg / mL (in Milli-RO water) 0.14 μg/mL (in pH 4 buffer), >60,000 μg/mL (in pH 7.0 buffer) and >3,000 μg/mL (in pH 9.0 buffer). The compound 2-methyl-N- - (D) - glucosamine salt (Compound 6) obtained in substantially the same value, the solubility of> 60,000 μg / mL (in Milli-RO water), 0.5 μg / mL (4 buffer at pH ), >60,000 μg/mL (in pH 7.0 buffer) and >3,000 μg/mL (pH 9.0 buffer).

The partition coefficients of Compound 5 and related analogs at neutral, acidic and basic pH were investigated using an HPLC method (reverse phase C18 HPLC column).

The partition coefficients of each of the salts of Compound 5 were found to be similar. This phenomenon is shown to occur because when the salt is in solution, the compound dissociates into parent compound 5 and related salt ions. Therefore, the measured partition coefficient is derived from the parent ion rather than the salt molecule.

Success was measured compound of N- methyl-2 - (D) - glucosamine salt (Compound 6) in neutral, alkaline and acidic conditions of the partition coefficient (Log10 POW) were 3.5,4.3 and 2.6.

N-methyl-(D)-glucosamine was selected as a candidate salt for Compound 2 and Compound 5 .

Effect of 10 mg/kg enantiomer compound 2 and compound 5 and their N-methyl-(D)-glucosamine salt (compounds 6 and 7) on 5% DSS murine colitis

At 30 mg/kg, compounds 2 and 5 were known to show better activity in the 5% DSS model, and we tested it again at a lower dose of 10 mg/kg and its N-methyl-(D)-glucosamine. The salt activity is administered daily in a suspension or solution in 0.5% carboxymethylcellulose/2% Tween 80 for 7 days. The salt dose is not adjusted to compensate for the increase in molecular weight. At 10 mg/kg, neither Compounds 5 and 7 had a significant (P > 0.05) effect on DAI in the 5% DSS murine colitis model compared to the vehicle control group (see Figure 5). In contrast, on day 7, 10 mg/kg of compound 2 and 6 , N-methyl-(D)-glucosamine salt were significantly (P < 0.05) and effectively made DAI from 9.3 ± 0.51 (vehicle). Reduced to 2.1 ± 0.7 and 3.3 ± 0.52, respectively (Figure 6).

In conclusion, Compound 2 (and its N-methyl-(D)-glucosamine salt, Compound 6 ) is clearly the most potent of the four enantiomers and is the only lower dose of 10 mg/kg. The active enantiomer is kept underneath.

The effect of the dose range of Compound 6 on 5% DSS murine colitis and comparison with dehydrocortisol

Compound 6 was chosen as the best enantiomer. The activity of Compound 6 at various dose levels in 5% DSS murine model colitis was determined to determine the presence of a dose/response relationship and was associated with an effective oral steroid (dehydrocortisol, commonly used to treat patients suffering from acute IBD episodes) Return to ease) comparison.

Compound 6 was administered to mice at dose levels of 3, 10 and 30 mg/Kg (equivalent to 6.6-20 mg/Kg of Compound 2 ). A group of DSS-treated mice were also treated with dehydrocortisol (5 mg/Kg). Dehydrocortisol is a corticosteroid that is clinically used to treat human IBD, and the amount used in this study is the optimal dose for dehydrocortisol used in this model. The colitis condition was evident after 3 days of treatment of BALB/c mice with 5% DSS in drinking water. It was characterized by weight loss (Figure 7) and increased disease DAI (Figure 8). However, after 7 days of daily oral administration, Compound 6 at three doses (3, 10, and 30 mg/Kg) did not significantly respond to mice. After acute DSS treatment, Compound 6 attenuated the severity of colitis in various parameters of the disease being tested. The ability of Compound 6 to attenuate disease in the DSS model was dose dependent. At 30 mg / Kg of compound 6 pairs DSS model-based curative efficacy less than or even more than 5 mg / Kg of dehydroepiandrosterone cortisol.

The severity of these symptoms was progressive, and on day 7, the DSS-treated mice had lost up to 15% of their body weight and all mice had bleeding in the rectum. The DAI values for the day of autopsy showed that mice treated with Compound 6 (3-30 mg/kg) had significant (P < 0.05 - P < 0.01) at each dose level than the DAI of the vehicle control group. Dehydrocortisol (5 mg/kg) is also evident (P < 0.01; ANOVA; Dunnett's multiple comparison test) reduced the DAI score (Figure 9).

On the 7th day of autopsy, the colon length of all DSS-treated groups was significantly shorter than that of non-DSS-treated mice (P<0.05-P<0.01;ANOVA;Dunnett's multiple comparison test) 10). When compared with the vehicle control group, the lowest dose of 3 mg/kg of Compound 6 had no significant effect on inhibiting colon shortening, while the 10 and 30 mg/kg group did have a significant effect on the dehydrocortisol group. Compound 6 at 30 mg/kg was significantly better than dehydrocortisol (P <0.05;ANOVA;Dunnett's multiple comparison test) (Figure 10).

After DSS treatment, histological sections of the terminal colon showed extensive damage to the glandular fossa and cellular infiltration (Figure 11).

The degree of colonic damage was quantified using an arbitrary scoring system. Compound 6 at 10 and 30 mg/Kg resulted in a dose-dependent and highly statistically significant reduction in colon pathology relative to the vehicle group (P <0.01; Kruksal-Wallis ANOVA; Dunnett's multiple comparison test). In contrast, the dehydrocortisol (5 mg/Kg) treated group showed no significant improvement in histological score relative to vehicle treated mice.

Inflammation of the colon in mice was consistent with histological findings, and colonic bone marrow peroxidase (MPO) activity was significantly greater in DSS-treated mice administered with vehicle alone (P <0.001; Kruksal-Wallis ANOVA; Dunnett's multiple comparison test) Upgrade. On day 7, colonic bone marrow peroxidase activity (MPO), which represents the level of neutrophil infiltration into the intestinal wall, increased approximately 8-fold due to DSS treatment, but due to compound 6 at 30 mg/kg Significantly (P < 0.05) decreased by 63% and 54% compared with dehydrocortisol (Figure 13).

Quantitative concentration of colonic cytokines showed that DS1 treatment caused IL1β (Fig. 14(a)), TNFα (Fig. 14(b)), and IL6 (Fig. 14(c)) to increase to 0.744±0.076 ng/mg, 1.478±, respectively. 0.378 ng/mg and 1.057±0.1784 ng/mg. In each case, Compound 6 caused a significant (P < 0.05, 30 mg/kg) and dose-dependent decrease in the concentration of these cytokines. Dehydrocortisol (5 mg/kg) also reduced (p<0.05) the increase in cytokine concentration; on day 7, dehydrocortisol (5 mg/kg) and compound 6 (30 for each cytokine) There was no significant difference in the effect of the higher dose levels of mg/kg.

In summary, three doses (3, 10 and 30 mg/Kg) of Compound 6 did not cause a significant response to mice after 7 days of daily oral administration. After acute 5% DSS treatment, Compound 6 attenuated the severity of colitis in a variety of parameters of the disease being detected, and the ability to reduce the disease was dose dependent. In addition, 30 mg/Kg of Compound 6 is effective against the DSS model and is no less effective than or better than dehydrocortisol (5 mg/Kg).

Chronic IL10 ^--/-- model

Mice that have deleted the IL10 ^--/-- gene naturally develop chronic colitis. The age of onset and the severity of the disease depend on the conditions of the background mouse strain and the captive animal. In the captive conditions used in this study, the incidence of colitis in IL10 ^--/- mice also depends on the strain. In terms of mortality, BALB/c strains are younger than C57BL/6 strains and more serious. In this experiment, animals received oral treatment with the MWF regimen within nine weeks. Initially, the two groups of mice gradually gained weight (Figure 15). The body weight of the vehicle-treated mice stopped growing from the 5th week of treatment, while the body weight of the mice treated with Compound 6 continued to increase until the 8th week. At week 9, the animals lost weight significantly, and on day 60, a dying animal was humanely killed in each group. As other mice continued to lose weight and clinical symptoms of the disease appeared, both groups were selected at Week 9 (Day 63) and analyzed. Although by Kaplan-Meier analysis showed that vehicle-treated group (25%) of Compound 6 was treated mice than (9.2%) higher mortality at 9 weeks but IL10 ^{- / -} mice survival was not statistically difference.

Serum was recovered from mice and analyzed for serum amyloid A (SAA) as a marker of the severity of colitis. The SAA concentration of the mice treated with Compound 6 was significantly lower than that of the vehicle-treated IL10 ^-/- mice ( P <0.05;Stuart's t-test) (Fig. 16).

Histological sections of IL10 ^-/- mice treated with vehicle or Compound 6 are shown in Figure 17.

Histological sections of the colon of IL10 ^-/- mice treated with vehicle or Compound 6 were scored. The extent of colonic pathology in IL10 ^-/- mice receiving Compound 6 was significantly lower than that in vehicle-treated patients ( P <0.05;Stuart's t-test) (Figure 18).

In conclusion, using the MWF regimen within 9 weeks, oral administration of Compound 6 (300 mg/kg/week) in IL10 ^--/-- BALB/c strains was more likely to delay the colon than the vehicle-treated mice. Weight loss due to inflammation and reduced death. In this model of chronic colitis, Compound 6 significantly reduced the disease index in terms of serum markers of inflammation of the colon and the degree of inflammation and damage to the colon. The plasma half-life (t _1/2 ) of Compound 6 in rats is particularly noteworthy for 3 hours. Due to the standard MWF administration protocol, mice will not be affected by Compound 6 for a considerable period of time during the experiment.

Experiment 2: About Compounds 31 and 47 Procedure 2.1 Animals and captivity

A total of 90 BDF1 (H. Pylori- free, no-norovirus mice) male mice (Harlan Laboratories, UK) were used in this study. It is available for 8-10 weeks old animals and is used at 10-12 weeks old. All mice were housed individually in an independent ventilation cage (IVC) of the SPF (no specific pathogen) barrier unit. Animals are identified by labeling cages and ear piercings.

2.2 Diet and animal welfare

The animals were fed a B&K rat and mouse puffed feed. Feed and water (from drinking water bottles) are free to use. The constant room temperature was 21 ± 2 ° C and the average relative humidity was 55 ± 10%. The day and night cycle is constant, and the light and dark periods are 12 hours. Monitor animal health daily and clean the cage regularly.

2.3 grouping, dosing, administration and formulation

Mice were randomly divided into experimental groups. All mice in any cage received the same treatment and ear perforation for identification purposes. The daily body weight measurements are used to calculate the volume of test or vehicle administered in the applicable group.

2.4 Preparation and administration of DSS and test substances DSS

DSS (MP Biomedicals 0216011090, lot # M2709) was prepared as a 5% (w/v) solution in drinking water and was prepared daily on days 0 to 6 (including the first and last days). The DSS was administered from day 0 to day 7.

Compounds 31 and 47

The test article was stored at -20 ° C until the start of the study. One day prior to the study, each test substance was formulated into a homogeneous suspension in 0.5% carboxymethylcellulose (CMC, Sigma C4888) using an Ultraturax homogenizer (in sterile water (Sigma W3500, lot # RNBC 1419)) . Compound 31 was formulated to 2 mg/ml, and compound 47 was formulated to 4 mg/ml. After the blending, the test article and the vehicle material are anonymized using an alphabetic code. All solutions were stored at 4 °C during the study and 3.8 ml of each suspension was dispensed daily. From the 0th day of the study to the 6th day of the study, the test article was administered by oral gavage at 10 ml/kg every day at 09.00 hours.

5-ASA

5-ASA (5-aminosalicylic acid, Sigma A3537, lot # 051M1878V) was aliquoted into pre-weighed weights between 71 and 84 mg. The aliquots were anonymized by letter code as in the test suspension. On each dosing day, each aliquot was formulated into a 10 mg/ml suspension (in sterile water (Sigma W3500, lot # RNBC 1419)) in 0.5% CMC (Sigma C4888). From study day 0 to study day 6, 5-ASA was administered by oral gavage at 10 ml/kg (to produce a dose of 100 mg/kg) daily at 09.00 hours.

2.5 clinical examination

Any animal showing a weight loss of more than 15% is considered uncomfortable and may stop processing. If the weight loss is more than 20%, any animal will be removed. Monitor animal health daily. From day 0 until the end of the study, all mice were weighed and assessed for fecal stiffness, and significant bleeding in the feces and around the anus was assessed according to the criteria of 2.1 once daily.

At the time of autopsy, observe the feces in the large intestine to supplement the observation of the living body. If the large intestine is hollow and has not been observed in vivo, the pre-set score for fecal hardness is 1. In addition to the standard observations detailed above, mice were housed individually for up to 30 minutes to increase the likelihood of fecal observations; this was only done for mice in the primary study group. According to the manufacturer's instructions, the fecal occult blood test was also performed on the feces observed by the main research group using the occult blood test card (Becton Coulter).

The effect of a single dose of Compounds 47 and 31 and the effect on murine DSS (Model 2) on Day 7.

Initial results showed that Compound 47 did not cause an increase in weight loss in the DSS murine colitis model compared to the vehicle control group and was comparable to 5ASA (Figure 19). In addition, both Compound 47 and Compound 31 reduced DAI compared to vehicle, comparable or equal to 5ASA (Figure 20). This shows that the activity of these compounds on these parameters of the disease model is comparable to that of 5ASA and has utility for the treatment of IBD.

Figure 21 is a bar graph showing the effect of compounds on the release of IL2 from Jurkat cells.

Figure 22 is a bar graph showing the effect of compounds on the release of IL2 from Jurkat cells.

The invention is not limited to the embodiments in which the details described above may vary.

Appendix 1 List of chemical indentations used

Biological use List of abbreviations

Appendix 2 X-ray study

Single crystal X-ray analysis of compound 2( S )-(-)-methylbenzylamine salt (Compound 8 ) using SuperNova, dual, zero degree Cu, Altas diffractometer and the parameters listed in Table 1.

Actuarial overview:

单晶 Single crystal X-ray data confirmed that the structure of compound 8 was monoclinic, space group P 2 ₁ , and one compound 8 molecule was in the asymmetric unit (Table 2).

绝对 The absolute stereochemistry of compound 2 at C9 and C10 is determined as S , S , and the methylbenzylamine cation is S at C33. This assignment was made taking into account the prior knowledge of the Fleck parameters and the salt-forming stereochemistry determined to be 0.04 (14).

亦可 You can also use the Bayesian statistics for Bijvoet to determine the absolute stereochemistry. The probability that the stereochemical centers C9, C10 and C33 are S , S and S are 1.000, respectively, and R , R and R are the same . The probability is 0.000. From the Fleck parameter measurement, this result supports the allocation of S , S and S for C9, C10 and C33 respectively.

X The X-ray powder diffraction pattern calculated from the single crystal X-ray structure is consistent with the stereochemistry shown in Figure 2 (or the following figure).

Figure 1 is an X-ray crystal structure showing the absolute stereochemistry of the enantiomeric compound 4(R)-(+)-methylbenzylamine salt (Compound 9 ); Figure 2 is an X-ray crystal structure showing The absolute stereochemistry of the 2(S)-(-)-methylbenzylamine salt (Compound 8 ); Figure 2A is a crystal structure view of the molecule 8 of the compound numbering scheme shown. An anisotropic atomic displacement ellipsoid showing a non-hydrogen atom at a 50% probability level. Hydrogen atoms are shown at any small radius. Only the major disordered components are shown; Figure 3 is a graph of the effects of 30 mg/kg of Compounds 2 , 3 , 4, and 5 on 5% DSS colitis disease activity index (DAI) over 7 days; Figure 4 on Day 7. Bar graph of the effects of 30 mg/kg of Compounds 2 , 3 , 4, and 5 on 5% DSS colitis disease activity index (DAI); Figure 5 shows 10 mg/kg of compound 5 , 7 , 2 in 7 days. and six pairs graph showing the effect of 5% DSS colitis disease activity index (DAI) of; FIG. 6 is a day 7 10 mg / 5 kg of a compound, 7, 2, and six pairs of 5% DSS colitis disease activity index ( Bar chart of the impact of DAI). Asterisks indicate significant differences from the vehicle control group (P < 0.05) (single factor variance analysis); Figure 7 is a graph showing the effect of Compound 6 on body weight loss in 5% DSS treated mice. Data are mean ± SEM from 6-7 mice per group; Figure 8 is a graph showing the effect of Compound 6 on 5% DSS treated mouse DAI. Data are mean ± SEM from 6-7 mice per group; Figure 9 is a bar graph of the effect of Compound 6 on 5% DSS treated mouse DAI on day 7. Data are mean ± SEM. Asterisks indicate significant differences from the vehicle control group (P < 0.05) (single factor variance analysis); Figure 10 is a bar effect of compound 6 on colon length of 5% DSS treated mice on day 7 Figure. Asterisks indicate significant differences from the vehicle control group (P < 0.05) (single factor variance analysis); Figure 11 shows representative hematoxylin and eosin stained sections of the mouse terminal colon. A higher magnification (X10) is shown; Figure 12 is a bar graph showing the effect of Compound 6 on the histological score of the DSS-treated mouse colon. Data are mean ± SEM from 5-6 mice. The asterisk indicates a significant difference (P<0.05) from the vehicle control group (single factor analysis). Note that the maximum score is 10; Figure 13 shows the activity of bone marrow peroxidase (MPO) in the colon of compound 6 treated with untreated or vehicle, dehydrocortisol and compound 2 (exposed to 5% DSS) Bar chart of the impact. Data are mean ± SEM from 5-6 mice. Asterisks indicate significant differences from the vehicle control group (P<0.05) (single factor variance analysis); Figures 14 (A) to (C) show compound 6 against cytokines in mice treated with DSS (IL1β(A) Bar graph of the effects of TNFα (B) and IL6 (C) concentrations. Data are mean ± SEM from 5-6 mice. Asterisks indicate significant differences from the vehicle control group (P < 0.05) (single factor variance analysis); Figure 15 is a graph showing weight loss of IL10 ^-/- mice treated with vehicle or Compound 6 . Mice were orally administered Compound 6 (300 mg/kg/week) or vehicle on a Monday/Wednesday/Friday (MWF) dosing schedule. At the beginning of the experiment, the mice were ~4 weeks old and treated for 9 weeks. Mice were weighed weekly and data were expressed as mean ± SEM from 9-12 mice per group. The mice were monitored for obvious disease, rectal prolapse, and humans were sacrificed humanely; Figure 16 is a scatter plot representing serum amyloid A (SAA) concentrations and mean values from a single mouse surviving animals at week 9. (horizontal line) (11 and 9 mice, respectively, in the compound 6 or vehicle treated group). Statistical differences between groups were tested using the Student's t-test; Figure 17 is a representative hematoxylin in the end-column of IL10 ^-/-- mice treated with vehicle or compound 6 for 9 weeks. And eosin stained sections; Figure 18 is the histological score of the terminal colon of IL10 ^-/-- mice treated with vehicle or compound 6 . Scatter plots represent histological scores and mean (horizontal lines) from single mice surviving animals at week 9 (11 and 9 mice, respectively, in compound 6 or vehicle treated groups). Statistical differences between groups were tested using the Stuart's t-test; Figure 19 is a scatter plot showing weight loss in mice treated with 5% DSS on day 7. Data are mean ± SEM from 6-7 mice per group; DSS murine colitis for Compounds 31 and 47 (Method 2); Figure 20 shows mice treated with 5% DSS on Day 7 DAI scatter plot. Data are mean ± SEM from 6-7 mice per group; DSS murine colitis for compound 31 and 47 (Method 2); Figure 21 is a bar graph showing compound 10-16 and 18-38 pairs The effect of IL2 released from Jurkat cells; and Figure 22 is a bar graph showing the effect of compounds 2-5 and 39-47 on IL2 released from Jurkat cells.

Claims (19)

A compound having a relative stereochemistry and the following formula: Wherein R is selected from one or more of the following or different hydrogen, hydroxy, optionally substituted alkyl, optionally substituted aryl, alkoxy, aryloxy, thiol, and optionally a substituted amine group, wherein R ₁ is selected from the group consisting of the same or different ones or more of hydrogen, an ethyl hydrazide group, optionally substituted alkyl group, optionally substituted aryl group, and one selected from leucine, Amino acids of proline, isoleucine and glycine, or pharmaceutically acceptable salts, esters, guanamines, solvates and prodrugs thereof. A compound having absolute stereochemistry and the following formula: Wherein R is selected from one or more of the following or different hydrogen, hydroxy, optionally substituted alkyl, optionally substituted aryl, alkoxy, aryloxy, thiol, and optionally a substituted amine group, and wherein the R ₁ group is selected from the group consisting of the same or different ones or more of hydrogen, an ethyl hydrazide group, an optionally substituted alkyl group, an optionally substituted aryl group, and one selected from the group consisting of leucine Amino acids of proline, isoleucine and glycine, or pharmaceutically acceptable salts, esters, guanamines, solvates and prodrugs thereof. A compound according to claim 1 or 2, wherein the alkyl group contains 1 to 10 carbon atoms in a straight or branched chain, and may be saturated or unsaturated, or a cycloalkyl group containing 3 to 8 carbon atoms. It can be saturated or unsaturated. The compound of any one of claims 1 to 3, wherein the alkyl group is substituted by one or more of the same or different ones: an alkyl group, an alkoxy group, an alkylamino group, a decylamino group, an amine group, an aryl group, Aralkyl, aryloxy, carboxy, halo, hydroxy, nitrile, nitro or pendant oxy group. The compound according to any one of claims 1 to 4, wherein the aryl group is substituted by one or more of the following or different: alkyl group, alkoxy group, alkylamino group, decylamino group, amine group, anhydride group, aromatic group A aryl, aralkyl, aryloxy, carboxy, halo, hydroxy, nitrile, nitro or pendant oxy group. The compound of any one of claims 1 to 5, wherein the amine group is substituted by one or more of the same or different ones: an alkyl group, a hydroxyalkyl group, an aryl group, and a substituted aryl group. The compound of claim 6, wherein the amine group is substituted with an aryl group substituted with one or more of OH, NH ₂ and COOH. A compound of claim 1 or 2 wherein R is OH. The compound of claim 1 or 2, wherein R ₁ is H. A compound having the following absolute stereochemistry and the following formula Wherein R is selected from the group consisting of: OH, OCH ₃ , OCH ₂ CH ₃ , OCH ₂ CH ₂ CH ₃ , NH ₂ , NHCH ₂ CH ₂ OH, NHCH ₃ , N(CH ₃ ) ₂ , NH(4-OH-3- benzoic acid). a compound having absolute stereochemistry and the following formula Wherein R ₁ is H or leucine, and R is selected from the group consisting of: OH, OCH ₃ , OCH ₂ CH ₃ , OCH ₂ CH ₂ CH ₃ , NH ₂ , NHCH ₂ CH ₂ OH, NHCH ₃ , N(CH ₃ ) ₂ , NH (4-OH-3-benzoic acid). A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier. A method of preventing or treating inflammatory bowel disease comprising administering to an individual an effective amount of a compound according to any one of claims 1 to 11. A method of preventing or treating ulcerative colitis comprising administering to an individual an effective amount of a compound according to any one of claims 1 to 11. A method of preventing or treating Crohn's Disease, which comprises administering to an individual an effective amount of a compound according to any one of claims 1 to 11. The compound according to any one of claims 1 to 11, which is for use in the prevention or treatment of inflammatory bowel disease. A compound according to any one of claims 1 to 11 for use in prevention or treatment Treat ulcerative colitis. A compound according to any one of claims 1 to 11 for use in the prevention or treatment of Crohn's disease. A compound according to any one of claims 1 to 11 for use in the prevention of autoimmune inflammatory diseases.