JP2008214296A - Modulator for autoinducer-2 receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound useful as an antagonist to autoinducer-2 receptor. <P>SOLUTION: The compound is represented by formula (1) (wherein R<SP>1</SP>is hydrogen, a 1-8C alkyl group or the like and R<SP>2</SP>is hydrogen, a formyl group, an acetyl group, a phenylalkylcarbonyl group having a 1-4C alkyl group, an N-[(naphthyl)alkyl]amide group having a 1-3C alkyl group or the like). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to novel compounds useful as autoinducer-2 receptor antagonists.

  Bacteria have a mechanism to control collective behavior by sensing cell density. This cell density-dependent mechanism of bacterial behavior control is called quorum sensing. Bacteria that perform quorum sensing produce a substance called an autoinducer. Autoinducers have the function of promoting the synthesis of specific proteins in bacterial cells. In an environment where only a small number of bacteria are present, the autoinducer concentration is low and transcription is not promoted. Specific substances will be produced.

Substances produced by quorum sensing vary depending on the species, but typical examples include Pseudomonas aeruginosa (Pseudomonas aeruginosa) biofilm, Vibrio fisheri luminescent substance, Serratia marcescens (Serratia marcescens) red pigment and the like.
Gram-negative bacteria use homoserine lactone derivatives collectively called autoinducer-1 (AI-1) as signal molecules, and Gram-positive bacteria use oligopeptide derivatives generically called autoinducer peptides (AIP) as signal molecules. These have slightly different structures depending on the individual bacteria, and are thought to be involved in the transmission of information between the same species.

  On the other hand, another signal molecule regardless of the type of bacteria has been identified and has been called autoinducer-2 (hereinafter sometimes referred to as “AI-2”). The entity of AI-2 is 4,5-dihydroxy-2,3-pentanedione (DPD), but its form changes when it binds to the target bacterial receptor. For example, when AI-2 binds to the signal molecule receptor LuxP of the marine microorganism Vibrio harveyi, it forms a cyclic structure and further forms a cyclic diester (S-) condensed with one molecule of boric acid. THMF-borate) (Patent Document 1, Non-Patent Document 1). In addition, for example, when binding to the signal molecule receptor LsrB of Salmonella typhimurium, which is the main causative agent of equine Salmonella diarrhea, the stereochemistry of ring formation is reversed, and boric acid It becomes a structure R-THMF without any involvement (Patent Document 2, Non-Patent Document 2). FIG. 1 shows the structure of DPD that is the substance of AI-2 and its receptor binding structure.

As a result of quorum sensing, Pseudomonas aeruginosa, which is a causative bacterium of severe infections, forms a biofilm mainly composed of alginic acid and is resistant to antibiotics. Quorum sensing mechanism can be disturbed against the background of the formation of dental plaque (dental plaque) on the surface of teeth, which causes periodontal disease that affects many adults. Drug development is in progress. Although there is no example of practical application, drug development targeting the inhibition of the signal transduction system from AI-1 has progressed considerably. For example, Patent Document 3 discloses a compound that becomes an agonist or antagonist of AI-1. Yes.
On the other hand, since AI-2 is chemically unstable, elucidation of the signal transduction system has been delayed, and no development example of an effective antagonist or agonist has been reported.
WO2005 / 005598 Publication US2006 / 0063721A US2004 / 0115732 Nature, 415, 545 (2002) Mol. Cell, 15, 677 (2004)

  The main object of the present invention is to provide a compound useful as an antagonist of an autoinducer-2 receptor.

The inventors of the present invention have made researches to solve the above problems, and have obtained the following knowledge.
AI-2 analog compound represented by the following general formula (1) (hereinafter also referred to as “the first compound of the present invention”), and AI-2 analog represented by the following general formula (2) The compounds (hereinafter sometimes referred to as “second compounds of the present invention”) include Porphyromonas gingivalis and Eikenella corrodens, which are representative causative agents of periodontal disease. Inhibits growth and biofilm formation and functions as an antagonist of AI-2 receptor.
(In the formula, R ¹ is a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, a phenyl group, a naphthyl group, a phenylalkyl group having 1 to 6 carbon atoms, a naphthyl group, or an alkyl having 1 to 3 carbon atoms. R ² represents a hydrogen atom, a formyl group, an acetyl group, a propionyl group, a butyryl group, a benzoyl group, an alkyl group having 1 to 8 carbon atoms, or a phenyl group having an alkyl group having 1 to 4 carbon atoms. An alkylcarbonyl group, a phenyloxycarbonyl group, an N-phenylamide group, an N- (naphthyl) amide group, an alkyl group having 1 to 3 carbon atoms, and the cycloalkyl group has 4 to 13 carbon atoms (cycloalkyl) ) N- (cycloalkylalkyl) amido group having an alkyloxycarbonyl group, an alkyl group having 1 to 3 carbon atoms, and a cycloalkyl group having 4 to 13 carbon atoms, carbon number An alkyloxycarbonyl group having an alkyl group of ˜6, an N-alkylamide group having an alkyl group of 1 to 6 carbon atoms, a fluorenylalkyloxycarbonyl group having an alkyl group of 1 to 3 carbon atoms, or a carbon number of 1 A phenylalkyloxycarbonyl group having an alkyl group of ˜3, an N- (phenylalkyl) amide group having an alkyl group of 1 to 3 carbon atoms, a (naphthyl) alkyloxycarbonyl group having an alkyl group of 1 to 3 carbon atoms, N-[(naphthyl) alkyl] amido group having an alkyl group having 1 to 3 carbon atoms.In R ¹ and R ² , the phenyl group, naphthyl group, and fluorenyl group may have a substituent.)
(Wherein R ¹ and R ² are the same as above)
The present invention has been completed based on the above findings, and provides the following compounds.

Item 1. The compound represented by the following general formula (1).
(In the formula, R ¹ is a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, a phenyl group, a naphthyl group, a phenylalkyl group having 1 to 6 carbon atoms, a naphthyl group, or an alkyl having 1 to 3 carbon atoms. R ² represents a hydrogen atom, a formyl group, an acetyl group, a propionyl group, a butyryl group, a benzoyl group, an alkyl group having 1 to 8 carbon atoms, or a phenyl group having an alkyl group having 1 to 4 carbon atoms. An alkylcarbonyl group, a phenyloxycarbonyl group, an N-phenylamide group, an N- (naphthyl) amide group, an alkyl group having 1 to 3 carbon atoms, and the cycloalkyl group has 4 to 13 carbon atoms (cycloalkyl) ) N- (cycloalkylalkyl) amido group having an alkyloxycarbonyl group, an alkyl group having 1 to 3 carbon atoms, and a cycloalkyl group having 4 to 13 carbon atoms, carbon number An alkyloxycarbonyl group having an alkyl group of ˜6, an N-alkylamide group having an alkyl group of 1 to 6 carbon atoms, a fluorenylalkyloxycarbonyl group having an alkyl group of 1 to 3 carbon atoms, or a carbon number of 1 A phenylalkyloxycarbonyl group having an alkyl group of ˜3, an N- (phenylalkyl) amide group having an alkyl group of 1 to 3 carbon atoms, a (naphthyl) alkyloxycarbonyl group having an alkyl group of 1 to 3 carbon atoms, N-[(naphthyl) alkyl] amido group having an alkyl group having 1 to 3 carbon atoms.In R ¹ and R ² , the phenyl group, naphthyl group, and fluorenyl group may have a substituent.)

Item 2. A compound represented by the following general formula (2).
(In the formula, R ¹ and R ² are the same as above.)
Item 3. An autoinducer-2 receptor antagonist comprising a compound represented by the following general formula (1) or general formula (2).
(In the formula, R ¹ and R ² are the same as above.)

Item 4. The biofilm formation inhibitor containing the compound represented by following General formula (1) or General formula (2).
(In the formula, R ¹ and R ² are the same as above.)
Item 5. A plaque formation inhibitor comprising a compound represented by the following general formula (1) or general formula (2).
(In the formula, R ¹ and R ² are the same as above.)

Item 6. A biofilm formation inhibition method comprising a step of adding a compound represented by the following general formula (1) or general formula (2) into a wastewater pipe, a wastewater tank, a circulation bath, a swimming tank, or an aquarium.
(In the formula, R ¹ and R ² are the same as above.)

The compounds of the present invention function as antagonists of the AI-2 receptor.
For example, Pseudomonas aeruginosa forms a biofilm when the number of bacteria increases, but antibiotics become ineffective. However, the first and second compounds of the present invention, which are AI-2 antagonists, inhibit the formation of biofilm. In combination with antibiotics, the effect can be increased.

In addition, there are many kinds of bacteria in the oral cavity, and when the number of these bacteria increases, plaque is formed with metabolites, causing periodontal disease. Therefore, even if an autoinducer AI-1 antagonist that functions between specific bacterial species is used, it can only prevent plaque formation by specific bacteria, and thus cannot prevent or treat periodontal disease. In this regard, since the first and second compounds of the present invention which are antagonists of the AI-2 receptor are antagonists of autoinducer receptors that act beyond bacterial species, plaque formation by multiple bacterial species Can be effectively inhibited, and periodontal disease can be effectively prevented or treated. In addition, resident bacteria in the oral cavity are present in a well-balanced manner and thus fulfill a biological defense function, so it is not preferable to sterilize resident bacteria. In this regard, if the plaque formation is inhibited by using the first and second compounds of the present invention which are antagonists of the AI-2 receptor, periodontal disease can be prevented or treated without disturbing the biological defense function. it can. Therefore, the first and second compounds of the present invention are effective for periodontal disease not only in combination with antibiotics but also as a single agent.
In addition, a large number of bacteria adhere to wastewater pipes and the like, and biofilms are formed, causing dirt and odor. The formation of biofilm is hindered and it becomes easier to remove such dirt with a drug.

Hereinafter, the present invention will be described in detail.
(I) Compound of the present invention
First and second compounds of the present invention The first and second compounds of the present invention are novel compounds not described in any literature, and are represented by the above general formula (1) and general formula (2), respectively. The compound of the general formula (1) and the compound of the general formula (2) are in an enantiomeric relationship with each other with respect to the 5-position carbon atom, the compound of the general formula (1) is a 5S form, and the compound of the general formula (2) is In each compound of the general formulas (1) and (2) which are 5R isomers, R ¹ is a hydrogen atom, a linear or branched alkyl group having 1 to 8 carbon atoms, a phenyl group, a naphthyl group, or a carbon number of 1 A phenylalkyl group having a linear or branched alkyl group of -6 or a naphthylalkyl group having a linear or branched alkyl group having 1 to 3 carbon atoms.

The linear or branched alkyl group having 1 to 8 carbon atoms is preferably linear, and the carbon number is preferably 1 to 6, and more preferably 1 to 4.
The phenyl group may have 1 to 5 substituents, such as methyl, methoxy, nitro, cyano, trifluoromethyl, hydroxyl, amino, fluorine, chlorine. Group, iodine group, bromine group and the like.
In the phenylalkyl group having a linear or branched alkyl group having 1 to 6 carbon atoms, the alkyl group is preferably linear, and the carbon number is preferably 1 to 4, and more preferably 1. The phenyl group of the phenylalkyl group may have 1 to 5 substituents, such as a methyl group, a methoxy group, a nitro group, a cyano group, a trifluoromethyl group, a hydroxyl group, and an amino group. , Fluorine group, chlorine group, iodine group, bromine group and the like.

The naphthyl group may have 1 to 7 substituents, and examples of such substituents include a methyl group, a methoxy group, a nitro group, a cyano group, a trifluoromethyl group, a hydroxyl group, an amino group, and a fluorine group. , Chlorine group, iodine group, bromine group and the like.
In the naphthylalkyl group having a linear or branched alkyl group having 1 to 3 carbon atoms, the alkyl group is preferably linear, and the alkyl group preferably has 1 to 2 carbon atoms, more preferably 1 preferable. The naphthyl group of this naphthylalkyl group may have 1 to 7 substituents, such as a methyl group, a methoxy group, a nitro group, a cyano group, a trifluoromethyl group, a hydroxyl group, an amino group, Examples include a fluorine group, a chlorine group, an iodine group, and a bromine group.

R ² is a hydrogen atom, a formyl group, an acetyl group, a propionyl group, a butyryl group, a benzoyl group, a linear or branched alkyl group having 1 to 8 carbon atoms, or a linear or branched chain having 1 to 4 carbon atoms. A phenylalkylcarbonyl group having a chain-like alkyl group, a phenyloxycarbonyl group, an N-phenylamide group, an N- (naphthyl) amide group, a linear or branched alkyl group having 1 to 3 carbon atoms, The cycloalkyl group has a (cycloalkyl) alkyloxycarbonyl group having 4 to 13 carbon atoms, a linear or branched alkyl group having 1 to 3 carbon atoms, and the cycloalkyl group has 4 to 4 carbon atoms. N- (cycloalkylalkyl) amide group which is 13, an alkyloxycarbonyl group having a linear or branched alkyl group having 1 to 6 carbon atoms, a linear or branched chain having 1 to 6 carbon atoms Alkyl group N-alkylamide group, a fluorenylalkyloxycarbonyl group having a linear or branched alkyl group having 1 to 3 carbon atoms, or a linear or branched alkyl group having 1 to 3 carbon atoms A phenylalkyloxycarbonyl group having 1 to 3 carbon atoms, an N- (phenylalkyl) amide group having a linear or branched alkyl group having 1 to 3 carbon atoms, and a (naphthyl) alkyloxy having an alkyl group having 1 to 3 carbon atoms An N-[(naphthyl) alkyl] amido group having a carbonyl group or a linear or branched alkyl group having 1 to 3 carbon atoms is shown.

The phenyl group in the benzoyl group may have 1 to 5 substituents, such as a methyl group, a methoxy group, a nitro group, a cyano group, a trifluoromethyl group, a hydroxyl group, an amino group, Examples include a fluorine group, a chlorine group, an iodine group, and a bromine group.
Among the alkyl groups having 1 to 8 carbon atoms, an alkyl group having 1 to 6 carbon atoms is preferable, and a butyl group, a pentyl group, and a hexyl group are more preferable.
In the phenylalkylcarbonyl group having a linear or branched alkyl group having 1 to 4 carbon atoms, the alkyl group is preferably linear, and the alkyl group preferably has 1 to 2 carbon atoms. The phenyl group of this phenylalkylcarbonyl group may have 1 to 5 substituents, such as a methyl group, a methoxy group, a nitro group, a cyano group, a trifluoromethyl group, a hydroxyl group, an amino group. Group, fluorine group, chlorine group, iodine group, bromine group and the like.

(Cycloalkyl) alkyloxycarbonyl group or N- (cycloalkylalkyl) amide group having a linear or branched alkyl group having 1 to 3 carbon atoms and a cycloalkyl group having 4 to 13 carbon atoms In the above, examples of the cycloalkyl group include a cyclohexyl group, a cyclopentyl group, and a cyclopropyl group. The alkyl group of the (cycloalkyl) alkyloxycarbonyl group and the N- (cycloalkylalkyl) amide group is preferably linear, and preferably has 1 to 3 carbon atoms, more preferably 1.
In the alkyloxycarbonyl group and N-alkylamide group or fluorenylalkyloxycarbonyl group having a linear or branched alkyl group having 1 to 5 carbon atoms, the alkyl group is preferably linear. The number of carbon atoms is preferably 1 to 2, and more preferably 1. The fluorenyl group may have 1 to 8 substituents, such as methyl, methoxy, nitro, cyano, trifluoromethyl, hydroxyl, amino, fluorine, chlorine Group, iodine group, bromine group and the like.

In the phenylalkyloxycarbonyl group having an alkyl group having 1 to 3 carbon atoms and the N- (phenylalkyl) amide group, the alkyl group is preferably linear, the carbon number is preferably 1 to 2, and 2 is preferably More preferred. The phenyl group in the phenylalkyloxycarbonyl group and N- (phenylalkyl) amide group may have 1 to 5 substituents, such as a methyl group, a methoxy group, a nitro group, and a cyano group. Group, trifluoromethyl group, hydroxyl group, amino group, fluorine group, chlorine group, iodine group, bromine group and the like.
The naphthyl group may have 1 to 7 substituents, and examples of such substituents include a methyl group, a methoxy group, a nitro group, a cyano group, a trifluoromethyl group, a hydroxyl group, an amino group, and a fluorine group. , Chlorine group, iodine group, bromine group and the like.

  In the (naphthyl) alkyloxycarbonyl group and N-[(naphthyl) alkyl] amide group having a linear or branched alkyl group having 1 to 3 carbon atoms, the alkyl group is preferably linear. 1-2 are preferable and, as for carbon number of an alkyl group, 1 is more preferable. The naphthyl group of this naphthylalkyl group may have 1 to 7 substituents, such as a methyl group, a methoxy group, a nitro group, a cyano group, a trifluoromethyl group, a hydroxyl group, an amino group, Examples include a fluorine group, a chlorine group, an iodine group, and a bromine group.

In each compound of the general formulas (1) and (2), a preferable combination of R ¹ and R ² includes combinations shown in Table 1 below. Also in Table 1, the phenyl group, the naphthyl group, the phenyl group in the benzoyl group, and the fluorenyl group may have a substituent. Further, the alkyl group may be linear or branched.

(II) Method for producing the compound of the present invention
Production method of first and second compounds of the present invention The production steps of the compounds of the general formula (1) and the general formula (2) are shown in FIGS. Referring to FIG. 2, Compound 1 as a starting material is commercially available and can be purchased from, for example, biochem.
First, compound 1 is reacted in iBuOCOCl and DME in the presence of NMM, and after filtration, an aqueous NaBH ₄ solution is added to the filtrate to reduce the reaction product to obtain the target alcohol. Subsequently, aldehyde 2 was obtained by Swern oxidation, and then reacted with HFIPA in DMF in the presence of a catalytic amount of QD-4 (Hatakeyama et al. Tetrahedron Lett. 2001, 42, 7867.) by the method of Hatakeyama et al. Fluoroisopropyl ester is obtained and transesterified with sodium methoxide to give compound 3.

Compound 5 is obtained by protecting the hydroxyl group of compound 3 with TBS and then oxidizing the olefin with a catalytic amount of OsO ₄ . A method for synthesizing compound 5 from compound 1 is described in Tetrahedron Lett. 2001, 42, 7867.
After compound 5 is acetonide protected, the ester is reduced with LiBH ₄ to give compound 7. Here, the Cbz group of compound 7 can be deprotected by hydrogenation, and a benzoyl group can be introduced as R ² using Bz ₂ O. Thereby, compound 8 is obtained.
The compound having a benzyloxycarbonyl group or a benzoyl group of compound 7 or 8 is refluxed in the presence of Et ₃ N in an isocyanate having the R ¹ group exemplified above (O═C═N—R ¹ ) and toluene. Gives compound 9.

Compound 11 is obtained by removing the acetonide of compound 9 with 10% TFA-CH ₂ Cl ₂ and then oxidizing the resulting diol with NaIO ₄ .
Compound 11 is then reacted with TBAF in THF, passed through a short column, and the residue is stirred in TFA to obtain a mixture of compounds of general formulas (1) and (2) of the present invention. The combination of R ¹ and R ² in the compound obtained here is as described above.
Introduction of R ² in the compounds of general formula (1) and general formula (2) was performed as follows. As shown in FIG. 3, in the compound of the general formula (1) or (2), for example, the benzyloxycarbonyl group of the compound in which R ¹ is a methyl group and R ² is a benzyloxycarbonyl group is deprotected by hydrogenation. And then reacted with pentafluorophenylformate and an acid anhydride in THF, THF / acetone or THF / methanol at 0 ° C. for 1-2 hours to obtain the desired R ² (formyl group, acetyl group, propionyl group). , Butyryl group, etc.) can be introduced. Similarly, compound 15 or compound 17 is reacted with a fluorenylalkyloxycarbonyl N-hydroxysuccinimide ester such as Fmoc N-hydroxysuccinimide ester and fluorenylmethyloxycarbonyl (Fmoc) as R ² A fluorenylalkyloxycarbonyl group such as the group can be introduced.

Further, compound 15 or compound 17 is reacted with phenylalkanoyl chloride {Ph (CH ₂ ) _n COCl} or a substituted phenyl derivative thereof in THF at 0 ° C. for 1-2 hours in the presence of Et ₂ N, and R ² has 1 carbon atom. A phenylalkylcarbonyl group {Ph (CH ₂ ) _n CO—} having ˜4 alkyl groups or a substituted phenyl derivative thereof can be introduced.
Similarly, compound 15 or compound 17 can be reacted with phenyl chloroformate (PhOCOCl) or a substituted phenyl derivative thereof to introduce a phenyloxycarbonyl group {PhOCO-} or a substituted phenyl derivative thereof as R ² .

A (cycloalkyl) alkyloxycarbonyl group having an alkyl group having 1 to 3 carbon atoms and a cycloalkyl group having 4 to 13 carbon atoms can be introduced.
Similarly, an alkyloxycarbonyl group {(CH ₂ ) _n OCO—} having an alkyl group having 1 to 5 carbon atoms can be introduced as R ² .
Similarly reacted with phenylalkyl chloroformate _{{Ph (CH 2) n OCOCl} } or a substituted phenyl derivatives, phenyl alkyloxycarbonyl group having an alkyl group having 1 to 3 carbon atoms other than benzyloxycarbonyl group as R ² {Ph (CH ₂ ) _n OCO—} or a substituted phenyl derivative thereof can be introduced.

In addition, in the compound of the general formula (2) in FIG. 2, a compound in which R ¹ is a paramethoxybenzyl group and R ² is a phenylalkyloxycarbonyl group having an alkyl group having 1 to 3 carbon atoms in the presence of CAN ₃ A mixture of a compound of general formula (1) and a compound of formula (2) in which R ¹ is a hydrogen atom and R ² is the above phenylalkyloxycarbonyl group by reacting in CN / water at room temperature for 10 hours Is obtained.

(III) Use of the Compound of the Present Invention The first and second compounds of the present invention are useful as antagonists of the AI-2 receptor.
Therefore, the biofilm formation inhibitor of the present invention contains the first compound or the second compound of the present invention as an active ingredient. The biofilm formation inhibitor may also contain an antibiotic. The antibiotic may be any substance that exhibits antibacterial activity against bacteria that form biofilms, and a substance having antibacterial activity against Pseudomonas aeruginosa is particularly preferable.
When a biofilm formation inhibitor is used as a medicine, it is a solid preparation for internal use such as tablets, pills, capsules, powders, or granules for oral administration; water solution, suspension, emulsion, syrup Or a liquid for internal use such as an elixir, or an injection for parenteral administration (subcutaneous, intravenous, intramuscular, intraperitoneal injection, etc.).

The biofilm formation inhibitor as a pharmaceutical only needs to contain, for example, about 0.0001 to 30% by weight of the first or second compound of the present invention.
In addition, when the biofilm formation inhibitor is applied to an environment such as a water tank for storing a waste water pipe or waste water, the first or second compound of the present invention is used in a solvent such as water or ethanol. It may take a dissolved liquid form or a solid form in which the first or second compound of the present invention is contained together with excipients such as simple syrup, vegetable resin, aromatic powder, honey and various elixirs. it can.
Moreover, the 1st and 2nd compound of this invention may be contained as an active ingredient of a plaque formation inhibitor. This plaque formation inhibitor may also contain antibiotics. The antibiotic may be any substance that exhibits antibacterial activity against bacteria that form plaque, such as Streptococcus mutans, lactic acid bacteria, and filamentous fungi. Plaque formation inhibitors can take the form of lozenges, chewables, external preparations for gargles and the like.

The first biofilm formation inhibition method of the present invention includes a step of introducing the first or second compound of the present invention into a wastewater pipe, a wastewater tank, a circulation bath, a swimming tank, or an aquarium. It is the method of including. Specifically, the compound of the present invention may be introduced into a wastewater pipe, a treatment tank of a sewage treatment plant, a circulation bath, a swimming tank, an aquarium, or the like. Thereby, it is prevented that a biofilm is formed on the waste water pipe wall or the drain tank wall and the dirt is fixed. The amount of the compound of the present invention used may be such that the concentration in water is about 0.0001 to 10 w / v%.
Moreover, the 2nd biofilm formation inhibition method of this invention is a method of administering the 1st or 2nd compound of this invention to a human or an animal. The administration method may be a known method such as oral administration, subcutaneous injection, intravenous injection, and intramuscular injection. The dose may be about 0.01 to 500 mg per day.
Further, the method for inhibiting plaque formation of the present invention is a method of administering the first or second compound of the present invention into the oral cavity of a human or animal. The administration method may be a method according to the dosage form. The dose may be about 0.01 to 500 mg per day.

  EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated in detail, this invention is not limited to these.

Examples of 3,6-dioxa-1-azaspiro [4.4] nonan-2-one compounds
<Step 1>
Synthesis of benzyl N-[(1R) -2- (t-butoxy) -1- (hydroxymethyl) ethyl] carbamate (compound 1 in FIG. 2)

Z-Ser (tBu) -OH [Na-CBZ-Ot-butyl-L-serine] (novabiochem) (13.3 g, 44.9 mmol) in 1,2-dimethylethane (DME, 40 mL) solution (-15 N-methylmorpholine (5.92 mL, 53.8 mmol) and isobutyl chloroformate (7.26 mL, 53.8 mmol) were sequentially added to the cold solution at 0 ° C. Then, after stirring for 2 hours at the same temperature, the precipitated N-methylmorpholine hydrochloride was removed by filtration, washed with DME, and the filtrate and washings were combined in a 1 L flask and placed in an ice-salt bath. placed. While stirring the reaction mixture, a solution of sodium borohydride (3.39 g, 89.7 mmol) in water (20 mL, 0 ° C.) was added in one portion to generate a strong gas, and 300 mL (0 ° C.) of water was immediately added. After stirring for 5 minutes, the aqueous layer was extracted with AcOEt, the organic layer was washed with saturated brine, dried over MgSO ₄ and evaporated under reduced pressure. The residue was purified by silica gel chromatography (AcOEt: hexane (1: 2)) and concentrated under reduced pressure to obtain alcohol as a milky yellow oil (12.2 g, yield 96%).
The analysis results of this compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) d 27.6, 51.6, 63.4, 64.5, 66.8, 73.7, 128.1, 136.3, 156.3 ppm.

<Step 2>
Synthesis of benzyl N-[(1S) -2- (t-butoxy) -1-formylethyl] carbamate (compound 2 in FIG. 2)
Oxalyl chloride (5.56 mL, 64.9 mmol) was added to CH ₂ Cl ₂ (100 mL) at −78 ° C. under a nitrogen atmosphere, and then DMSO (10.1 mL, 130 mmol) was added dropwise, followed by stirring at the same temperature. Ten minutes later, a solution of the alcohol obtained in Step 1 (12.2 g, 43.3 mmol) in CH ₂ Cl ₂ (25 mL) was added dropwise over 10 minutes using dirinji. The reaction mixture was stirred at −78 ° C. for 40 minutes and at −45 ° C. for 40 minutes, and then DIEA (45.2 mL, 260 mmol) was added. After stirring at 0 ° C. for 20 minutes, the mixture was quenched by adding saturated aqueous NH ₄ Cl (200 mL) and extracted 3 times with CH ₂ Cl ₂ (300 mL). The organic layer was washed with 1N HCl aqueous solution and saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 9-1: 4)) to obtain the aldehyde as a milky yellow oil. (9.79 g, 81%).
The analysis results of this compound are as follows.
¹ H NMR (400 MHz, CDCl ₃ ) δ 1.14 (s, 9H), 3.62 (dd, J = 3.6, 9.3 MHz, 1H), 3.95 (dd, J = 3.2, 9.3 MHz, 1H), 4.31-4.35 ( m, 1H), 5.14 (s, 2H), 5.63 (d, J = 7.4 MHz, 1H), 7.33-7.38 (m, 5H), 9.62 (s, 1H).

<Step 3>
Synthesis of methyl 2-[(1S, 2S) -2-[(benzyloxy) carbonyl] amino-3- (t-butoxy) -1-hydroxypropyl 1] acrylate (compound 3 in FIG. 2)
Aldehyde (9.28 g, 32.3 mmol) and QD-4 (978 mg, 3.30 mmol) with the structure shown in the above process diagram in DMF (75 mL) solution (? 55 ° C cold solution) 1,1,1 3,3,3-hexafluoroisopropyl acrylate (HFIPA, 7.17 mL, 42.9 mmol) was added. After stirring for 48 hours at the same temperature, the reaction mixture was quenched by the addition of 0.1 N aqueous HCl and extracted with AcOEt. The organic layer was washed with saturated aqueous NaHCO ₃ solution and saturated brine, dried over MgSO ₄ and evaporated under reduced pressure. To a solution of the residue in MeOH (100 mL) (0 ° C) was added sodium methoxide (178 mg, 3.30 mmol) in methanol (10 mL), 30 minutes later the cooling bath was removed and the reaction mixture was allowed to warm to room temperature. It was. After stirring for 30 minutes, the reaction mixture was quenched by addition of 0.1 N HCl water and evaporated under reduced pressure. The aqueous layer was extracted with CH ₂ Cl ₂ , then the organic layer was washed with brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 3)), and the methyl ester was converted to a yellow oil (12.1 g, 65%).
The analysis results of the product are as follows.
¹ H NMR (400 MHz, CDCl ₃ ) δ1.23 (s, 9H), 3.71 (dd, J = 3.1, 9.3 Hz, 1H), 3.76 (dd, J = 2.4, 9.3 Hz, 1H), 3.78 (s , 3H), 4.06-4.13 (m, 1H), 4.86 (s, 1H), 5.06 (s, 2H), 5.93 (s, 1H), 6.97 (s, 1H), 7.26-7.34 (m, 5H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 27.4, 51.9, 65.3, 66.7, 72.1, 72.6, 74.4, 126.3, 128.1, 128.5, 136.4, 138.7, 156.3, 166.1 ppm. MS (FAB) calcd for C ₁₉ H ₂₇ NO ₆ , m / z = 365. Obsd, m / z = 365.

<Step 4>
Methyl 2-((1S, 2S) -2-[(benzyloxy) carbonyl] amino-3- (tert-butoxy) -1- [1- (tert-butyl) -1,1-dimethylsilyl] oxypropyl) Synthesis of acrylate (compound 4 in FIG. 2)
A solution of methyl ester (7.78 g, 21.29 mmol) obtained in step 3 in 80 mL of CH ₂ Cl ₂ was cooled to 18 ° C., and 2,6-lutidine (7.29 mL, 63.87 mmol) and TBDMS-OTf (7.31 mL, 31.94 mmol) was added. After stirring at -18 ° C for 1.5 hours, the mixture was extracted with AcOEt. The organic layer was washed with (water, saturated aqueous KHSO ₄ solution and saturated brine), dried over MgSO ₄ and purified by silica gel chromatography (AcOEt-hexane (1:10)) to obtain the target compound as a white solid. (10.25 g, 100% yield). The analysis results of the product are as follows. ¹³ C NMR (100 MHz, CDCl ₃ ) δ -5.2, -4.6, 18.1, 25.9, 27.5, 51.5 and 51.8, 54.5 and 54.8, 60.9 and 61.6, 66.6, 68.3, 73.2, 126.4 and 127.0, 127.6 and 127.8, 128.1 and 128.2, 128.3 and 128.5, 136.6, 140.7, 156.3, 166.0 ppm.

<Step 5>
Methyl (3R, 4S) -4-[(benzyloxy) carbonyl] amino-5- (t-butoxy) -3- [1- (t-butyl) -1,1-dimethylsilyl] oxy-2-hydroxy- Synthesis of 2- (hydroxymethyl) pentanoate (compound 5 in Figure 2)
A solution of methyl ester (10.21 g, 21.28 mmol) obtained in step 4 in acetone (50 mL) was cooled to 0 ° C., and N-methylmorpholine-N-oxide (4.99 g, 42.56 mmol) in acetonitrile (50 mL). After the solution and an osmium tetroxide (500 mg, 1.97 mmol) aqueous solution (25 mL) were added, the reaction solution was allowed to reach room temperature. After stirring at room temperature for 12 hours, the reaction mixture was cooled to 0 ° C. and then quenched with saturated aqueous Na ₂ S ₂ O ₃ solution. After stirring at the same temperature for 30 minutes, the mixture was extracted with AcOEt, the organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 3)). Of alcohol as an oil (10.76 g, 98% yield). The analysis results of the product are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ -4.8 and -4.6, -4.2, 18.3, 25.9, 27.2 and 27.5, 51.3, 52.9, 60.4, 65.4, 66.9, 71.9, 80.0, 128.1, 128.2, 128.5, 136.4, 155.9, 174.1 ppm.

<Step 6>
Methyl 4-((1R, 2S) -2-[(benzyloxy) carbonyl] amino-3- (t-butoxy) -1- [1- (t-butyl) -1,1-dimethylsilyl] oxypropyl) Synthesis of -2,2-dimethyl-1,3-dioxolane-4-carboxylate (Compound 6 in FIG. 2)
To a solution of the diol obtained in Step 5 (10.93 g, 21.93 mmol) in acetone (30 mL) and DMF (70 mL), 2,2-dimethoxypropane (52.26 mL, 425.6 mmol) and PPTS (10.68 g, 42.55 mmol) Of DMF (20 mL) was added. After stirring at room temperature for 24 hours, the mixture was concentrated under reduced pressure and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated aqueous NaHCO ₃ solution and saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 6)) to obtain the desired acetonide (11.71 g, 99%) as milky yellow Obtained as an oil. The analysis results of the product are as follows. ¹³ C NMR (100 MHz, CDCl ₃ ) δ -4.6 and -4.4, -4.3 and -4.1, 18.3 and 18.4, 25.4 and 25.7, 25.9 and 26.0, 26.4 and 26.5, 27.5 and 27.6, 50.9 and 51.3, 52.2 and 52.4 , 59.9 and 60.5, 66.5 and 66.8, 68.9 and 69.4, 71.4 and 71.7, 73.3, 85.5, 111.2, 127.9 and 128.0, 128.2 and 128.4, 128.4, 136.5, 155.6, 172.2 ppm.

<Step 7>
Benzyl N- (1S, 2R) -1- (t-butoxymethyl) -2- [1- (t-butyl) -1,1-dimethylsilyl] oxy-2- [4- (hydroxymethyl) -2, Synthesis of 2-dimethyll-1,3-dioxolan-4-yl] ethylcarbamate (compound 7 in FIG. 2)
Water (3 mL) was added to a solution of the ester obtained in Step 6 (2.24 g, 4.04 mmol) in 2-methoxyethyl ether (100 mL). After stirring for 1 hour, the mixture was heated to 70 ° C., and a solution of LiBH ₄ (2 M) in THF (4 mL, 8.08 mmol) was added dropwise to this solution over 5 minutes. After stirring at the same temperature for 15 minutes, the mixture was cooled to 0 ° C., then quenched with saturated aqueous NH ₄ Cl and 1 N HCl and extracted with ether. The organic layer was washed with saturated aqueous NaHCO ₃ solution and saturated brine, and dried over MgSO ₄ . The solution was evaporated under reduced pressure (45 ° C / 0.2 mmHg) and purified by silica gel chromatography (AcOEt-hexane (1: 3-2: 3)) to obtain the target alcohol as a colorless oil (1.60 g, 75% ). The analysis results of the product are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ -4.9, -3.8, 18.2, 25.9, 26.8, 27.0, 27.6, 41.8, 49.7, 61.8, 62.3, 66.8, 66.9, 72.9, 73.3, 84.4, 110.1, 128.0, 128.1 , 128.5, 136.4, 156.2 ppm.

<Step 8>
N1- (1S, 2R) -1- (t-butoxymethyl) -2- [1- (t-butyl) -1,1-dimethylsilyl] oxy-2- [4- (hydroxymethyl-l) -2, Synthesis of 2-dimethyl-1,3-dioxolan-4-yl] ethylbenzamide (Compound 8 in FIG. 2)
The amine having a protecting group obtained in Step 7 (1.63 g, 3.10 mmol) was hydrogenated in the presence of Pd (OH) ₂ .2H ₂ O / C in a methanol (50 mL) solution at room temperature. After stirring for 10 minutes, the mixture was filtered and washed with MeOH. The washing solution and the filtrate were combined and concentrated.
To a solution of the residue in CH ₂ Cl ₂ (25 mL) was added a solution of Bz ₂ O (1.33 g, 5.88 mmol) in CH ₂ Cl ₂ (5 mL), stirred at room temperature for 1.5 hours, and then the mixture was concentrated under reduced pressure. Purification by silica gel chromatography (AcOEt-hexane (2: 3)) gave the desired benzamide as a colorless oil (1.35 g, 88%).
The analysis results of this compound are as follows.
¹ H NMR (400 MHz, CDCl ₃ ) δ0.15 (s, 3H), 0.18 (s, 3H), 0.90 (s, 9H), 1.22 (s, 9H), 1.41 (s, 3H), 1.46 (s , 3H) ,, OH), 3.30 (t, J = 8.5 Hz), 3.60 (dd, J = 3.9, 8.5 Hz), 3.65 (dd, J = 11.9 Hz, 7.9 Hz, 1H, C H HOH), 3.83 (dd, J = 3.0, 11.9 Hz, 1H), 3.96 (d, J = 8.7 Hz, 1H), 4.03 (d, J = 8.7 Hz, 1H), 4.25 (d, J = 1.9 Hz, 1H), 4.26 -4.28 (m, 1H), 7.41-7.52 (m, 3H), 7.72-7.78 (m, 2H).

<Step 9>
N, N'-disubstituted [4-{(1R, 2S) -2-amino-3- (t-butoxy) -1- [1- (t-butyl) -1,1-dimethylsilyl] oxy} propyl General Method for the Synthesis of] -2,2-Dimethyl-1,3-dioxolan-4-yl] methylcarbamate (Compound 9 of FIG. 2) In Step 9, various R ¹ groups were introduced. Specific conditions after step 9 will be described later.
Et ₃ N (2 equivalents) and isocyanate (5 equivalents) were added to a toluene solution of the alcohol obtained in step 8 (1 equivalent), refluxed for 10-24 hours, and then the reaction mixture was extracted with Et ₂ O and organic. The layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt / hexane) to give the desired carbamate. The yield was 87 to 92% as described later, although it varies depending on the type of isocyanate.

<Step 10>
N, N'-disubstituted (3R, 4S) -4-amino-5- (t-butoxy) -3-{[1- (t-butyl) -1,1-
General method for the synthesis of dimethylsilyl] oxy} -2-hydroxy-2- (hydroxymethyl) pentylcarbamate (compound 10 of FIG. 2)
CH ₂ Cl ₂ was added a solution of TFA in acetonide and triethylsilyl obtained in Step 9 in CH ₂ Cl ₂ (? 15 ° C. cold solution) of (ca. 10%). After stirring for 48 hours at the same temperature, the reaction mixture was quenched by the addition of 6N aqueous NaOH and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (ethyl acetate / hexane) to give the desired diol. The yield was 46-87% as described later.

<Step 11>
N, N'-disubstituted (3R, 4S) -4-amino-5- (t-butoxy) -3-{[1- (t-butyl) -1,1-dimethylsilyl] oxy} -2-oxo General method for the synthesis of pentylcarbamate (compound 11 of FIG. 2)
An aqueous NaIO ₄ (6 equivalent) solution was added to a THF solution (0 ° C. cold solution) of the diol (1 equivalent) obtained in step 10. After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt: hexane) to obtain the desired ketone. The yield was 76-99% as described later.

<Step 12 and Step 13>
N1-substituted [(5R / S, 8S, 9R) -9-hydroxy-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide (compound 13 of FIG. 2 (general formula General method for the synthesis of compound (2) and compound 14 (compound of general formula (1)) in FIG.
To a THF solution of carbamate 11 obtained in step 12 (? 18 ° C cold solution) was added a solution of THF in TBAF (1.5 equivalents). The reaction mixture was stirred at the same temperature for 1.5 h and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. TFA cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (ethyl acetate: hexane) to separate the 5R and 5S forms. The yield was 51 to 68% as described later.

Example 1
The following steps were performed after steps 1 to 8 described above.
Benzyl N-[(5R / S, 8S, 9R) -1-benzyl-9-hydroxy-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] carbamate (13a / 14a) (Synthesis of compound 13 (general formula (2)) and compound 14 (general formula (1) in FIG. 2 wherein R ¹ is a benzoyl group and R ² is a benzyloxycarbonyl group))

<Step 9>
Et ₃ N (719 μL, 5.2 mmol) and benzyl isocyanate (1.6 mL, 13 mmol) were added to a solution of alcohol 7 (1.37 g, 2.6 mmol) obtained in Step 7 in CH ₂ Cl ₂ (40 mL). After refluxing for 19 hours, the mixture was extracted with CH ₂ Cl ₂ . The organic layer was washed with 0.1 N HCl aqueous solution, saturated NaHCO ₃ aqueous solution and saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (ethyl acetate: hexane (1: 4)) to obtain the desired carbamate 9a in yellow Obtained as an oil (1.59 g, 93% yield). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ -4.6, -3.7, 18.4, 26.0, 26.3, 26.5, 26.9, 27.6, 44.9, 61.7, 63.4, 66.5, 70.1, 72.5, 73.2, 83.1, 110.5, 127.2, 127.4 , 127.7, 127.9, 128.4, 128.5, 136.7, 138.8, 156.3, 156.5 ppm.

<Step 10>
A solution of compound 9a and TES (0.5 mL) obtained in step 9 in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution) in TFA (3 mL) in CH ₂ Cl ₂ (3 mL) (? 15 C. cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added to the reaction mixture, and the mixture was extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (ethyl acetate: hexane (3: 2)) to give the desired diol 10a (122 mg, 81% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.8, -4.0, 18.5, 26.2, 27.5, 45.0, 50.9, 61.2, 61.3, 65.0, 66.5, 73.4, 73.5, 75.9, 127.4, 127.5, 127.8, 128.1, 128.5 , 128.6, 136.4, 138.2, 156.1, 157.4 ppm.

<Step 11>
NaIO ₄ (193 mg, 0.9 mmol) aqueous solution (2 mL) was added to a THF (6 mL) solution of diol 10a (93 mg, 0.15 mmol) obtained in step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with ethyl acetate, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ , purified by silica gel chromatography (AcOEt: hexane (2: 3)) and the desired ketone 11a (97 mg, 96% yield) was obtained.
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.1, 18.0, 25.7, 27.2, 45.1, 54.4, 58.8, 66.8, 67.7, 73.6, 73.9, 74.8, 127.4, 127.5, 128.1, 128.5, 128.6, 136.3, 138.1, 155.7, 155.9, 206.0 ppm.

<Step 12 and Step 13>
To a THF (2 mL) solution of ketone 11a (47.1 mg, 0.080 mmol) obtained in Step 11 (? 18 ° C cold solution), a THF 1 M solution of TBAF (120 μL, 0.12 mmol) was added. The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (ethyl acetate: hexane (3: 2)) to give the 5R form (compound 13a) and the 5S form (compound). The yield of compound 13a was 19.9 mg and the yield was 63%, and the yield of compound 14a was 6.7 mg and the yield was 21%.
The analysis results of the obtained compound are as follows.
Compound 13a: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 43.7, 55.8, 67.2, 67.4, 69.2, 74.4, 97.9, 127.0, 127.6, 128.2, 128.4, 128.6, 128.7, 137.0, 156.9, 157.9 ppm.
Compound 14a: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 46.2, 56.8, 67.6, 68.2, 72.1, 79.8, 97.2, 127.4, 127.6, 128.5, 128.7, 135.6, 137.2, 157.1, 158.3 ppm.

Example 2
Synthesis of N1-[(5R / S, 8S, 9R) -9-hydroxy-1-methyll-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide (13b / 14b) (Synthesis of compound 13 (general formula (2)) and compound 14 (general formula (1) in FIG. 2 wherein R ¹ is a methyl group and R ² is a benzoyl group))
The following steps were performed after steps 1 to 8 described above.

<Step 9>
Et ₃ N (90 μL, 0.65 mmol) and methyl isocyanate (O═C═N—R ¹ : R ¹ = methyl group) were added to a solution of alcohol 8 (161 mg, 0.33 mol) obtained in Step 8 in toluene (8 ml). (96 μL, 1.62 mmol) was added and refluxed for 12 hours, after which the reaction mixture was extracted with Et ₂ O, the organic layer was washed with saturated brine, dried over MgSO ₄ and silica gel chromatography (ethyl acetate / hexanes). The desired carbamate 9b was obtained by purification from (2: 3)) (172 mg, yield 96%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.7, 3.6, 18.5, 26.0 and 26.2, 26.9 and 27.4, 27.7, 48.6, 61.6, 63.4, 70.4, 72.8, 73.3, 82.9, 110.7, 126.4, 128.6, 131.4, 157.0, 167.0 ppm.

<Step 10>
9b (171 mg, 0.31 mmol) obtained in Step 9 and a solution of TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution) were added to TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added to the reaction mixture, and the mixture was extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt / hexane (3: 2)) to obtain the desired diol 10b (114 mg, yield 72%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.9, 3.9, 18.6, 26.2, 27.5, 27.6, 49.6, 60.5, 61.4, 64.8, 73.4, 73.6, 75.9, 126.6, 128.7, 131.7, 134.2, 157.8, 166.5 ppm .

<Step 11>
NaIO ₄ (268 mg, 1.25 mmol) aqueous solution (2 mL) was added to a THF (6 mL) solution of diol 10b (107 mg, 0.21 mmol) obtained in step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt: hexane (2: 3)) to obtain the desired ketone 11b. Obtained (97 mg, yield 96%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.3, -5.0, 18.0, 25.7, 27.2, 27.6, 53.0, 58.6, 67.7, 73.6, 74.5, 127.0, 128.5, 131.5, 134.2, 156.3, 166.7, 207.6 ppm.

<Step 12 '>
This step is different from the general method of step 12 and step 13 described above.
To the ketone 11b obtained in Step 11 (127 mg, 0.26 mmol), TFA (3 mL) cooled to 0 ° C. was added. After stirring at room temperature for 1 hour, water (0.3 mL) was added and stirred for 12 hours. The reaction mixture was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (AcOEt: hexane (3: 1-4: 1)) to separate the 5R form (compound 13b) and the 5S form (compound 14b). The yield of compound 13b was 40 mg and the yield was 49%, and the yield of compound 14b was 19 mg and the yield was 23%.
The analysis results of the obtained compound are as follows.
Compound 13b: ¹ H NMR (400MHz, Acetone-d6) δ2.82 (s, 3H), 3,87 (t, J = 8.7 Hz, 1H), 4.18 (d, J = 9.6 Hz, 1H), 4.24 ( dd, J = 8.1, 8.7 Hz, 1H), 4.66 (dd, J = 4.0, 9.0 Hz, 1H), 4.72 (d, J = 9.6 Hz, 1H), 7.47-7.49 (m, 2H), 7.54-7.57 (m, 1H), 7.90-7.92 ( m, 2H). 13 C NMR (100MHz, Acetone-d6) δ25.8, 56.0, 68.4, 70.6, 73.2, 98.8, 128.4, 132.9, 135.2, 159.7, 170.9 ppm.
Compound 14b: ¹ H NMR (400MHz, Acetone-d6) δ2.99 (s, 3H), 3,87 (t, J = 8.8 Hz, 1H), 4.27 (d, J = 9.8 Hz, 1H), 4.24 ( d, J = 9.8 Hz, 1H), 4.30 (d, J = 9.8 Hz, 1H), 4.42 (dd, J = 8.4, 8.8 Hz, 1H), 4.44 (s, 1H), 4.69-4.75 (m, 1H ), 7.45-7.49 (m, 2H) , 7.53-7.57 (m, 1H), 7.90-7.92 (m, 2H). 13 C NMR (100MHz, CD 3 OD) δ28.3, 56.4, 69.3, 73.4, 78.2 , 97.8, 128.4, 129.6, 135.2, 171.0 ppm

Example 3
N1-[(5R / S, 8S, 9R) -9-hydroxy-2-oxo-1-phenyl-3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide (13c / 14c) Composition
(Synthesis of compound 13 (general formula (2)) and compound 14 (general formula (1) in FIG. 2) wherein R ¹ is a phenyl group and R ² is a benzoyl group)
The following steps were performed after steps 1 to 8 described above.

<Step 9>
Et ₃ N (87iL, 0.63 mmol) and phenyl isocyanate (O═C═N—R ¹ : R ¹ = phenyl group) in a toluene (8 ml) solution of alcohol 8 (155 mg, 0.31 mol) obtained in Step 8 (169iL, 1.56 mmol) was added and after refluxing for (12) hours, the reaction mixture was extracted with Et ₂ O and the organic layer was washed with saturated brine, dried over MgSO ₄ and chromatographed on silica gel (AcOEt / Purification with hexane (1: 3)) gave the desired carbamate 9c (190 mg, 99% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.9, -4.0 and? 3.5, 25.9 and 26.0, 26.6, 26.7, 27.6 and 27.8, 48.5, 62.3, 63.1, 71.0, 73.0, 73.3, 82.3, 111.0, 118.1, 122.5, 126.6, 128.7, 128.8, 131.6, 134.7, 138.7, 153.1, 167.8 ppm.

<Step 10>
9c (181 mg, 0.29 mmol) obtained in Step 9 and TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (~ 15 ° C cold solution) were added to TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added to the reaction mixture, and the mixture was extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt / hexane (2: 3)) to obtain the desired diol 10c (142 mg, 84% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.9, -4.1 and -3.9, 18.5, 26.1 and 26.2, 27.5 and 27.6, 48.6 and 49.4, 60.9, 61.4 and 61.9, 63.3 and 64.1, 64.2, 73.4, 73.6, 75.7, 118.6, 123.2, 126.7, 128.7, 128.2, 131.8, 134.0 and 134.2, 137.8, 154.0, 166.9 ppm.

<Step 11>
NaIO ₄ (118 mg, 0.55 mmol) aqueous solution (1 mL) was added to a THF (3 mL) solution of diol 10c (53 mg, 0.09 mmol) obtained in Step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 2)) to obtain the desired ketone 11c. Obtained (45 mg, 82% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.2, 4.9, 25.7, 27.3, 53.0, 58.7, 67.9, 73.7, 74.6, 118.7, 123.7, 127.0, 128.6, 129.0, 131.6, 134.2, 137.5, 166.7, 207.0 ppm .

<Step 12 and Step 13>
A THF 1 M solution (140 μL, 0.14 mmol) of TBAF was added to a THF (2 mL) solution of ketone 10c (30.4 mg, 0.056 mmol) obtained in Step 11 (? 18 ° C cold solution). The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (2: 1) to give 5R form (compound 13c) and 5S form (compound 14c). The yield of compound 13c was 10.9 mg and the yield was 55%, and the yield of compound 14c was 3.6 mg and the yield was 18%.
The analysis results of the obtained compound are as follows.
Compound 13c: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 56.5, 67.5, 70.0, 75.8, 99.3, 127.1, 128.5, 128.8, 129.2, 129.5, 132.2, 132.6, 133.8, 156.7, 169.3 ppm.
Compound 14c: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 53.3, 54.5, 68.4, 71.6, 98.0, 127.0, 128.4, 128.9, 129.1, 130.1, 131.8, 133.0, 135.0, 157.4, 169.4 ppm.

Example 4
N1-[(5R / S, 8S, 9R) -1-Benzyl-9-hydroxy-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide (13d / 14d) Synthesis Synthesis of compounds in which R ¹ is a benzyl group and R ² is a benzoyl group in compound 13 (general formula (2)) and compound 14 (general formula (1)) in FIG.
The following steps were performed after steps 1 to 8 described above.

<Step 9>
Et ₃ N (223 μL, 1.61 mmol) and benzyl isocyanate (O═C═N—R ¹ : R ¹ = benzoyl group) were added to a solution of alcohol 8 (100 mg, 0.81 mol) obtained in Step 8 in toluene (15 ml). (498 μL, 4.04 mmol) was added and refluxed for 12 hours, after which the reaction mixture was extracted with Et ₂ O, the organic layer was washed with saturated brine, dried over MgSO ₄ and silica gel chromatography (AcOEt / hexane ( 1: 3)) to obtain the desired carbamate 9d (559 mg, yield 98%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.8, -3.6. 18.4, 26.0, 26.5, 26.8, 27.6, 44.7, 48.5, 61.6, 63.4, 70.5, 72.7, 73.3, 82.7, 110.7, 126.6, 127.0, 127.3 , 128.4, 128.6, 131.4, 134.7, 138.6, 156.4, 167.1, 183.9 ppm.

<Step 10>
9d (199 mg, 0.32 mmol) obtained in Step 9 and TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution) were added to TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (2: 3)) to give the desired diol 10d (135 mg, 73% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.0, -3.9, 18.5, 26.2, 27.6, 45.0, 49.4, 60.5, 61.1, 64.8, 73.4, 73.5, 75.9, 126.7, 127.2, 128.5, 128.7, 131.6, 134.1 , 138.0, 157.3, 166.4 ppm.

<Step 11>
NaIO ₄ (284 mg, 1.33 mmol) aqueous solution (2 mL) was added to a THF (6 mL) solution of diol 10d (130 mg, 0.22 mmol) obtained in Step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 3)) to obtain the desired ketone 11d. Obtained (118 mg, 91% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.3, -5.0, 18.0, 25.7, 27.2, 45.2, 53.0, 58.6, 67.9, 73.6, 74.5, 127.0, 127.5, 128.5, 128.6, 131.5, 134.2, 138.1, 155.7 , 166.7, 207.3 ppm.

<Step 12 and Step 13>
1M TBAF in THF (220 μL, 0.22 mmol) was added to a solution of ketone 11d (56.1 mg, 0.10 mmol) obtained in step 11 in THF (2 mL) (cold solution at 18 ° C.). The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. Then TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (2: 1)) to give 5R (compound 13d) and 5S (compound) 14d). The yield of compound 13d was 20.4 mg and the yield was 55%, and the yield of compound 14d was 5.1 mg and the yield was 14%.
The analysis results of the obtained compound are as follows.
Compound 13d: ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.65 (dd, J = 6.8, 8.7 Hz, 1H), 4.22-4.31 (m, 2H), 4.30 (d, J = 9.8 Hz, 1H), 4.98 (d, J = 9.8 Hz, 1H), 7.23-7.29 (m, 1H), 7.32-7.40 (m, 6H), 7.50-7.56 (m, 3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ43.5 , 55.6, 67.3, 69.3, 73.6, 98.0, 126.8, 126.9, 127.1, 127.2, 127.3, 127.4, 128.4, 131.9, 133.1, 136.9, 158.3, 169.5 ppm.
Compound 14d: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 46.4, 56.9, 68.2, 72.4, 79.4, 97.3, 127.2, 127.4, 128.5, 128.7, 132.2, 137.3, 158.5, 169.7 ppm.

Example 5
N1-[(5R / S, 8S, 9R) -9-hydroxy-2-oxo-1-phenethyl-3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide (13e / 14e) Synthesis Synthesis of compound 13 (general formula (2)) and compound 14 (general formula (1)) in Fig. 2 wherein R ¹ is a phenylethyl group and R ² is a benzoyl group.
The following steps were performed after steps 1 to 8 described above.

<Step 9>
Et ₃ N (85 mL, 0.61 mmol) and phenylethyl isocyanate (O═C═N—R ¹ : R ¹ = phenyl) were added to a solution of alcohol 8 (152 mg, 0.31 mmol) obtained in Step 8 in toluene (8 ml). (Ethyl group) (212 μL, 1.43 mmol) was added and the mixture was refluxed for 12 hours. The reaction mixture was extracted with Et ₂ O, and the organic layer was washed with saturated brine, dried over MgSO ₄ , and chromatographed on silica gel ( AcOEt: xan (1: 3)) to obtain the desired carbamate 9e (180 mg, yield 91%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.8, -3.7, 18.4, 26.0, 26.5, 26.9, 27.6, 36.0, 42.2, 48.5, 61.5, 63.2, 70.2, 72.7, 73.3, 82.8, 110.6, 126.2, 126.6 , 128.4, 128.6, 128.7, 131.4, 134.9, 138.9, 156.2, 167.0 ppm.

<Step 10>
9e (155 mg, 0.31 mmol) obtained in Step 9 and TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution) were added to TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt / hexane) (2: 3) to obtain the desired diol 10e (126 mg, yield 87%).
The analysis results of the obtained compound are as follows:
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.9, -3.9, 18.5, 26.2, 27.6, 35.9, 42.3, 49.6, 60.4, 61.3, 64.7, 73.4, 73.55, 73.58, 75.9, 126.4, 126.7, 128.5, 128.6 , 128.7, 131.7, 134.1, 138.5, 157.2, 166.4 ppm.

<Step 11>
An aqueous NaIO ₄ (2 mL) solution was added to a THF (6 mL) solution of diol 10e (120 mg, 0.12 mmol) obtained in step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt: hexane (2: 3)) to obtain the desired ketone 11e. Obtained (113 mg, 99% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.26, -5.00, 18.0, 25.7, 27.2, 36.0, 42.3, 53.0, 58.6, 67.9, 73.6, 67.9, 73.6, 74.5, 126.5, 127.0, 128.5, 128.6, 128.8 , 131.5, 134.2, 138.6, 155.6, 166.7, 207.4 ppm.

<Step 12 and Step 13>
TBAF in THF 1M (220 μL, 0.22 mmol) was added to a solution of ketone 11e obtained in step 11 (55.1 mg, 0.096 mmol) in THF (2 mL) (cold solution at 18 ° C.). The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. Then TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (2: 1)) to obtain the 5R form (compound 13e) and the 5S form (compound). 14e). The yield of compound 13e is 21.2 mg and the yield is 58%, and the yield of compound 14e is 5.5 mg and the yield is 15%.
The analysis results of the obtained compound are as follows.
Compound 13e: ¹³ C NMR (100 MHz, CD ₃ OD) δ 36.6, 43.3, 56.0, 68.0, 70.7, 73.9, 82.6, 99.3, 127.5, 128.4, 129.6, 129.8, 133.0, 135.2, 139.9, 159.6, 170.9 ppm.
Compound 14e: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 35.1, 45.0, 56.8, 68.1, 72.9, 78.8, 97.6, 126.4, 127.3, 128.6, 128.7, 129.0, 132.2, 133.3, 158.2, 169.7 ppm

Example 6
N1-[(5R / S, 8S, 9R) -9-hydroxy-2-oxo-1- (3-phenylpropyl) -3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide ( Synthesis of 13f / 14f) In compound 13 (general formula (2)) and compound 14 (general formula (1)) in FIG. 2, R ¹ is a phenylpropyl group and R ² is a benzoyl group Compound synthesis
The following steps were performed after steps 1 to 8 described above.

<Step 9>
To a solution of alcohol 8 (145 mg, 0.30 mmol) obtained in step 8 in toluene (8 ml), Et ₃ N (82 μL, 0.59 mmol) and phenylpropyl isocyanate (O═C═N—R ¹ : R ¹ = phenylpropiate) The reaction mixture was extracted with Et ₂ O, and the organic layer was washed with saturated brine, dried over MgSO ₄ , and chromatographed on silica gel (AcOEt) (229 μL, 1.48 mmol). / Hexane (1: 3)) to obtain the desired carbamate 9f (184 mg, yield 95%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.8, -3.6, 18.4, 26.0, 26.6, 26.9, 27.7, 31.4, 32.9, 40.3, 48.5, 61.7, 63.3, 70.5, 72.8, 73.3, 82.7, 110.7, 125.7 , 126.6, 128.3, 128.4, 128.6, 131.4, 134.9, 141.7, 156.3, 167.2 ppm.

<Step 10>
9f (159 mg, 0.24 mmol) obtained in Step 9 and TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution) were added to TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt / hexane) (2: 3) to obtain the desired diol 10f (122 mg, 82% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.0, -3.8, 18.6, 26.2, 27.6, 31.3, 32.8, 40.5, 49.5, 60.4, 61.0, 64.4, 73.9, 73.6, 76.0, 125.8, 126.4, 128.3, 128.7 , 131.7, 134.1, 141.3, 157.3, 166.4 ppm.

<Step 11>
NaIO ₄ (241 mg, 1.13 mmol) aqueous solution (2 mL) was added to a THF (6 mL) solution of diol 10f (116 mg, 0.2 mmol) obtained in Step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 2)) to obtain the desired ketone 11f. Obtained (98 mg, yield 89%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.3, -5.0, 17.9, 25.7, 27.2, 31.4, 32.9, 40.7, 53.0, 58.6, 67.1, 70.4, 73.6, 74.5, 125.9, 127.0, 128.3, 128.5, 131.5 , 134.1, 141.3, 155.6, 166.7, 207.5 ppm.

<Step 12 and Step 13>
To a THF (2 mL) solution of ketone 11f (53.9 mg, 0.092 mmol) obtained in Step 11 (? 18 ° C cold solution) was added a THF 1 M solution of TBAF (230 μL, 0.23 mmol). The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. Then TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (2: 1)) to obtain the 5R form (compound 13f) and the 5S form (compound). 8f). (The yield of Compound 13f was 18.8 mg and the yield was 52%, and the yield of Compound 14f was 6.6 mg and the yield was 18%.
The analysis results of the obtained compound are as follows.
Compound 13f: ¹³ C NMR (100 MHz, CDCl ₃ ) δ30.5, 32.8, 40.0, 55.7, 66.9, 74.0, 98.1, 125.8, 127.1, 128.3, 128.5, 132.1, 133.0, 141.2, 157.8, 169.5 ppm.
Compound 14f: ¹³ C NMR (100 MHz, CDCl ₃ ) δ30.1, 33.1, 42.4, 57.2, 67.6, 72.3, 80.0, 98.0, 125.9, 127.1, 128.4, 128.5, 128.8, 132.4, 132.7, 141.5, 157.9, 169.8 ppm

Example 7
N1-[(5R / S, 8S, 9R) -1-Butyl-9-hydroxy-2-oxo-3,6-ddioxa-1-azaspiro [4.4] non-8-yl] benzamide (13g / 14g) Synthesis of compound of compound 13 (general formula (2)) and compound 14 (general formula (1)) in FIG. 2 wherein R ¹ is an n-butyl group and R ² is a benzoyl group
The following steps were performed after steps 1 to 8 described above.

<Step 9>
Et ₃ N (86 μL, 0.62 mmol) and butyl isocyanate (O═C═N—R ¹ : R ¹ = butyl group) were added to a solution of alcohol 8 (154 mg, 0.31 mmol) obtained in Step 8 in toluene (8 ml). (173 μL, 1.55 mmol) was added and refluxed for 15 hours, after which the reaction mixture was extracted with Et ₂ O, the organic layer was washed with saturated brine, dried over MgSO ₄ and silica gel chromatography (AcOEt: hexane ( 1: 3)) to obtain 9 g of the desired carbamate (173 mg, yield 97%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.8, -3.7, 13.7, 18.4, 19.8, 26.0, 26.6, 26.9, 27.6, 31.7, 40.3, 40.5, 48.5, 61.6, 63.2, 70.4, 73.3, 82.8, 110.7 , 126.6, 128.6, 131.4, 134.9, 156.3, 167.0 ppm.

<Step 10>
To 9 g (154 mg, 0.26 mmol) obtained in Step 9 and a solution of TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution), TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt / hexane) (2: 3) to give 10 g of the desired diol (111 mg, 77% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.1, -3.8, 13.6, 18.5, 19.7, 26.2, 27.6, 31.7, 40.7, 49.6, 60.4, 61.0, 64.4, 73.5, 73.6, 76.0, 126.7, 128.7, 131.7 , 134.1, 157.3, 166.3 ppm.

<Step 11>
NaIO ₄ (256 mg, 1.19 mmol) aqueous solution (2 mL) was added to a THF (6 mL) solution of 10 g (105 mg, 0.19 mmol) of the diol obtained in Step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (2: 3)) to obtain 11 g of the desired ketone. Obtained (113 mg, 99% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.3, -5.0, 13.7, 17.9, 19.8, 25.7, 27.2, 31.9, 40.9, 53.0, 58.6, 67.7, 73.6, 74.5, 127.0, 128.5, 131.5, 134.2, 155.6 , 166.7, 207.6 ppm.

<Step 12 and Step 13>
A THF 1 M solution (173 μL, 0.17 mmol) of TBAF was added to a THF (2 mL) solution of 11 g (36.2 mg, 0.069 mmol) of the ketone obtained in step 12 (? 18 ° C cold solution). The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. Then TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (2: 1)) to give 5R form (compound 13g) and 5S form (compound). 14 g). The yield of Compound 13g was 12.4 mg and the yield was 54%, and the yield of Compound 14g was 3.2 mg and the yield was 14%.
The analysis results of the obtained compound are as follows.
Compound 13g: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 13.6, 20.0, 31.3, 40.6, 56.1, 66.9, 69.1, 74.4, 98.3, 127.1, 128.6, 132.2, 133.0, 157.9, 169.6 ppm.
Compound 14g: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 13.8, 20.3, 30.9, 43.1, 57.4, 67.7, 72.3, 80.1, 97.9, 127.1, 128.8, 132.4, 132.7, 157.9, 169.8 ppm

Example 8
N1-[(5R / S, 8S, 9R) -9-hydroxy-1- (2-naphthyl) -2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide (13h / 14h)
(Synthesis of Compound 13 (General Formula (2)) and Compound 14 (General Formula (1) in FIG. 2) wherein R ¹ is a 2-naphthyl group and R ² is a benzoyl group)
The following steps were performed after steps 1 to 8 described above.

<Step 9>
To a solution of alcohol 8 (152 mg, 0.31 mmol) obtained in Step 8 in toluene (8 ml), Et ₃ N (85 μL, 0.61 mmol) and 2-naphthyl isocyanate (O═C═N—R ¹ : R ¹ = Naphthyl group) (259 mg, 1.53 mmol) was added and the mixture was refluxed for 10 hours. The reaction mixture was extracted with Et ₂ O, the organic layer was washed with saturated brine, dried over MgSO ₄ , and chromatographed on silica gel ( AcOEt: hexane (1: 3)) to obtain the desired carbamate 9h (186 mg, 92% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.9, 4.0 and? 3.5, 18.4 and 18.5, 26.0 and 26.1, 26.6, 27.5 and 27.6, 48.5 and 49.4, 61.4 and 62.4, 61.8 and 63.3, 67.9 and 71.1, 73.1 , 73.4, 82.3 and 83.5, 110.5 and 111.1, 113.6, 118.9 and 119.1, 124.1, 126.1, 126.6, 127.4, 127.5, 128.5, 128.6 and 128.7, 128.5, 128.6 and 128.7, 129.8, 131.6, 134.0, 134.7, 136.3, 153.2 , 168.0 ppm.

<Step 10>
9h (189 mg, 0.28 mmol) obtained in Step 9 and a solution of TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution) were added to TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt / hexane) (2: 3) to give the desired diol 10h (118 mg, 76% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.9, -3.9, 18.5, 26.2, 27.6, 49.4, 60.9, 61.4, 64.1, 73.5, 73.6, 75.8, 114.8, 119.2, 124.5, 126.3, 126.7, 127.4, 127.5 , 128.6, 128.8, 130.1, 131.8, 133.8, 134.0, 135.3, 154.1, 167.1 ppm.

<Step 11>
NaIO4 (207 mg, 0.97 mmol) aqueous solution (2 mL) was added to a THF (6 mL) solution of diol 10h (101 mg, 0.16 mmol) obtained in step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt: hexane (1: 2)) to obtain the desired ketone 11h. Obtained (73 mg, yield 76%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.2, -4.9, 18.0, 25.7, 27.3, 53.0, 58.7, 68.0, 73.8, 74.6, 114.9, 119.0, 124.7, 126.5, 127.0, 127.5, 128.6, 128.8, 130.3 , 11.6, 133.8, 134.1, 135.0, 152.7, 166.7, 207.0 ppm.

<Step 12 and Step 13>
To a THF (2 mL) solution of ketone 11h (33 mg, 0.053 mmol) obtained in Step 11 (? 18 ° C cold solution), a THF 1 M solution of TBAF (120 μL, 0.12 mmol) was added. The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. Then TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (2: 1)) to give 5R form (compound 13h) and 5S form (compound). 14h). The yield of compound 13h was 12.4 mg and the yield was 58%, and the yield of compound 14h was 2.2 mg and the yield was 11%.
The analysis results of the obtained compound are as follows.
Compound 13h: ¹³ C NMR (100 MHz, acetone-d ₆ ) δ 55.1, 67.7, 70.5, 73.9, 99.3, 127.4, 127.5, 128.0, 128.5, 128.9, 129.0, 129.6, 132.2, 133.2, 133.5, 134.4, 134.9 , 156.5, 168.3 ppm.
Compound 14h: ¹³ C NMR (100 MHz, DMF-d ₇ ) δ12.6, 19.1, 23.1, 54.2, 57.8, 69.2, 71.5, 76.1, 96.9, 126.0, 126.3, 127.1, 127.3, 127.6, 127.8, 128.0, 128.8 , 130.6, 132.5, 133.0, 133.7, 133.9, 156.4, 166.5 ppm.

Example 9
N1-[(5R / S, 8S, 9R) -9-Hydroxy-1- (2-naphthylmethyl) -2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide (13i / 14i) Synthesis In compound 13 (general formula (2)) and compound 14 (general formula (1)) in FIG. 2, R ¹ is a 2-naphthylmethyl group, R ² is a benzoyl group Synthesis of certain compounds
The following steps were performed after steps 1 to 8 described above.

<Step 9>
Et ₃ N (404 μL, 2.21 mmol) and 2-naphthylmethyl isocyanate (O═C═N—R ¹ : R ¹ ) were added to a toluene (8 ml) solution of alcohol 8 (219 mg, 0.44 mmol) obtained in Step 8. = Naphthylmethyl group) (404 mg, 2.21 mmol) and refluxed for 10 hours, the reaction mixture was extracted with Et ₂ O, the organic layer was washed with saturated brine, dried over MgSO ₄ , and chromatographed on silica gel. Purification by chromatography (AcOEt: hexane (1: 3)) gave the desired carbamate 9i (298 mg, 99% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.8, -3.6, 18.4, 26.0, 26.6, 26.8, 27.6, 44.9, 48.5, 61.7, 63.4, 70.6, 72.8, 73.3, 82.7, 110.8, 125.6, 125.7, 125.9 , 126.6, 127.6, 127.8, 128.1, 128.5, 131.4, 132.6, 133.3, 134.7, 136.2, 156.5, 167.2 ppm.
The analysis results of 2-naphthylmethyl isocyanate are as follows. ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.67 (s, 2H), 7.41 (dm, J = 8.5 Hz, 1H), 7.48-7.54 (m, 2H), 7.78 (br-s, 1H), 7.84- 7.88 (m, 3H). IR: v 2253 cm-1 (NCO).
In addition, Ref. Kurita, K .; Matsumura, T .; Iwakura, YJ Org. Chem., 1976, 41, 2070. describes a method for synthesizing phenyl isocyanate. Isocyanates were synthesized.

<Step 10>
9i (226 mg, 0.33 mmol) obtained in Step 9 and TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution) were added to TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added and extracted with CH2Cl2. The organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt / hexane) (2: 1) to give the desired diol 10i (168 mg, 74% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.0, -3.8, 18.5, 26.2, 27.6, 45.2, 49.4, 60.5, 61.1, 64.7, 73.5, 73.6, 76.0, 125.5, 125.7, 126.0, 126.5, 127.6, 127.8 , 128.4, 128.6, 131.6, 132.7, 133.2, 133.9, 135.5, 157.5, 166.4 ppm.

<Step 11>
NaIO4 (321 mg, 1.5 mmol) aqueous solution (2 mL) was added to a THF (6 mL) solution of diol 10i (160 mg, 0.25 mmol) obtained in Step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ solution and brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 3)) to obtain the desired ketone 11i. Obtained (150 mg, 99% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.2, 5.0, 18.0, 25.7, 27.3, 45.3, 53.0, 58.7, 68.0, 73.7, 74.5, 125.6, 125.9, 126.0, 126.2, 127.0, 127.6, 127.7, 128.5, 128.6, 131.6, 132.7, 133.3, 134.2, 135.5, 155.8, 166.7, 207.4 ppm.

<Step 12>
To a solution of ketone 11i obtained in step 11 (100 mg, 0.17 mmol) in THF (2 mL) (? 18 ° C cold solution) was added a THF 1 M solution of TBAF (363 μL, 0.36 mmol). The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. Then TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (2: 1)) to give 5R (compound 13i) and 5S (compound) 14i). The yield of compound 7i was 42.1 mg and the yield was 61%, and the yield of compound 8i was 10.9 mg and the yield was 16%.
The analysis results of the obtained compound are as follows.
Compound 13i: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 43.8, 56.1, 69.5, 74.2, 98.2, 124.9, 125.4, 125.9, 126.2, 127.1, 127.5, 127.7, 128.3, 128.5, 132.0, 132.6, 132.9, 133.1, 134.4, 158.3, 169.3 ppm.
Compound 14i: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 46.4, 56.5, 68.1, 72.4, 78.9, 97.0, 125.5, 125.8, 125.9, 126.0, 127.1, 128.1, 128.5, 132.0, 132.6, 133.1, 134.8, 158.6, 170.0 ppm.

Example 10
N1-[(5R / S, 8S, 9R) -1- (cyclohexylmethyl) -9-hydroxy-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] benzamide (13j / 14j) Synthesis of compound 13 (general formula (2)) and compound 14 (general formula (1)) in FIG. 2 wherein R ¹ is a cyclohexanemethyl group and R ² is a benzoyl group Composition
The following steps were performed after steps 1 to 8 described above.

<Step 9>
Et ₃ N (117 μL, 0.85 mmol) and cyclohexylmethyl isocyanate (O═C═N—R ¹ : R ¹ = cyclohexylmethyl) were added to a solution of alcohol 8 (210 mg, 0.42 mmol) obtained in Step 8 in toluene (8 ml). Group) (371 μL, 2.17 mmol) was added and refluxed for (24) hours, after which the reaction mixture was extracted with Et ₂ O, the organic layer was washed with saturated brine, dried over MgSO ₄ and chromatographed on silica gel ( AcOEt: hexane (1: 3)) to obtain the desired carbamate 9j (258 mg, yield 96%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.8, -3.6, 18.4, 25.8, 26.0, 26.4, 26.6, 26.9, 27.6, 30.5, 30.9, 38.0, 47.1, 48.5, 61.6, 63.1, 70.5, 72.7, 73.3 , 82.7, 110.7, 126.7, 128.6, 131.4, 134.8, 156.5, 167.1 ppm.

<Step 10>
To 9j (231 mg, 0.36 mmol) obtained in Step 9 and a solution of TES (0.5 mL) in CH ₂ Cl ₂ (7 mL) (? 15 ° C cold solution), TFA (3 mL) in CH ₂ Cl ₂ (3 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt / hexane) (2: 3) to give the desired diol 10j (186 mg, 86% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.1, -3.7, 18.6, 25.6, 25.7, 26.3, 27.6, 30.5, 38.0, 47.4, 49.5, 60.4, 60.9, 64.4, 73.5, 73.7, 76.1, 126.7, 128.7 , 131.7, 134.1, 157.5, 166.3 ppm.

<Step 11>
NaIO4 (384 mg, 1.79 mmol) aqueous solution (2 mL) was added to a THF (6 mL) solution of diol 10j (178 mg, 0.30 mmol) obtained in Step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt: hexane (2: 3)) to obtain the desired ketone 11j. Obtained (165 mg, yield 98%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-5.3, -5.0, 18.0, 25.7, 26.3, 27.2, 30.6, 38.1, 47.4, 53.0, 58.7, 67.7, 73.6, 74.5, 127.0, 128.5, 131.5, 134.2, 155.8 , 166.7, 207.6 ppm.

<Step 12 and Step 13>
To a THF (2 mL) solution of ketone 11j (100 mg, 0.18 mmol) obtained in Step 11 (? 18 ° C cold solution), a THF 1 M solution of TBAF (391 μL, 0.39 mmol) was added. The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. Then TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (2: 1)) to give 5R (compound 13j) and 5S (compound) 14j). The yield of compound 13j was 80 mg and the yield was 51%, and the yield of compound 14j was 6.3 mg and the yield was 13%.
The analysis results of the obtained compound are as follows.
Compound 13j: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 25.7, 26.3, 30.7, 30.8, 37.1, 49.9, 56.3, 66.8, 69.0, 74.5, 98.3, 127.3, 128.7, 132.3, 132.8, 158.4, 169.8 ppm.
Compound 14j: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 25.9, 26.4, 30.8, 31.1, 37.1, 49.4, 56.8, 67.4, 68.1, 72.4, 80.0, 97.4, 127.2, 127.3, 128.7, 132.3, 132.8, 158.3, 169.7 ppm.

Example 11
Benzyl N-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] carbamate (13k / 14k) Synthesis of compound in compound 13 (general formula (2)) and compound 14 (general formula (1)) in FIG. 2 wherein R ¹ is a methyl group and R ² is a benzyloxycarbonyl group
The following steps were performed after steps 1 to 8 described above.

<Step 9>
To a toluene (160 ml) solution of alcohol 7 (2.95 g, 5.6 mmol) obtained in Step 7, Et ₃ N (1.55 mL, 11.2 mmol) and methyl isocyanate (O═C═N—R ¹ : R ¹ = methyl group) ) (2.19 mL, 28.0 mmol) was added and refluxed for 12 hours, then the reaction mixture was extracted with Et ₂ O, the organic layer was washed with brine, dried over MgSO ₄ , and silica gel chromatography (AcOEt: Purification with hexane (1: 3)) gave the desired carbamate 9k (3.13 g, 96% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ -4.6, -3.8, 26.0 and 26.1, 26.5, 26.8, 27.4, 27.6, 49.9, 61.6, 63.2, 66.5, 69.9, 72.4, 73.2, 83.1, 110.3, 127.6, 127.7 , 127.9, 128.4, 136.7, 156.2, 157.0 ppm.

<Step 10>
A solution of 9i (1.41 g, 2.42 mmol) obtained in step 9 in CH ₂ Cl ₂ (70 mL) (? 15 ° C cold solution) in TFA (15 mL) in CH ₂ Cl ₂ (30 mL) (? 15 C. cold solution) was added. After stirring at the same temperature for 48 hours, the reaction solution was poured into a 500 mL eggplant flask containing 6N NaOH aqueous solution (60 mL) cooled to 0 ° C., and stirred for 3 minutes. Extracted with CH ₂ Cl ₂ , the organic layer was washed with 0.1 N HCl aqueous solution and saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt / hexane) (2: 3) to obtain the desired diol 10k. (1.02 g, 78% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.86, -4.03, 18.5, 26.2, 27.4, 27.5, 50.9, 61.2, 64.8, 66.6, 73.3, 73.4, 75.9, 76.6, 127.7, 128.1, 128.6, 136.5, 156.1 , 157.9 ppm.

<Step 11>
NaIO4 (1.23 g, 5.73 mmol) aqueous solution (8 mL) was added to a THF (24 mL) solution of diol 10k (519 mg, 0.96 mmol) obtained in Step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 2)) to obtain the desired ketone 11k. Obtained (468 mg, yield 96%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ -5.3, 17.9, 25.6, 27.1, 27.5, 54.2, 58.7, 66.7, 67.4, 73.4, 74.7, 127.9, 128.0, 136.2, 155.8, 156.2, 206.2 ppm.

<Step 12>
A THF 1 M solution (3.26 mL, 3.26 mmol) of TBAF was added to a THF (100 mL) solution of ketone 11k (1.11 g, 2.17 mmol) obtained in Step 11 (? 18 ° C cold solution). The reaction mixture was stirred at the same temperature for 1.5 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. TFA (40 mL) cooled to 0 ° C. was then added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (3: 2)). 14k). The yield of compound 13k was 347 mg and the yield was 53%, and the yield of compound 14k was 225 mg and the yield was 34%.
The analysis results of the obtained compound are as follows.
Compound 13k: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 29.2, 55.8, 67.3, 67.7, 69.3, 73.4, 128.1, 128.3, 128.5, 135.8, 156.9, 157.8 ppm.
Compound 14k: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 28.0, 56.5, 67.5, 68.6, 72.0, 79.2, 96.8, 128.2, 128.5, 128.6, 135.7, 157.1, 158.0 ppm

Example 12
Benzyl N-[(5R / S, 8S, 9R) -9-hydroxy-1 (4-methoxybenzyl) -2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] carbamate ( (13l / 14l)
Synthesis of a compound in which R ¹ is a paramethoxybenzyl group and R ² is a benzyloxycarbonyl group in compound 13 (general formula (2)) and compound 14 (general formula (1)) in FIG.
The following steps were performed after steps 1 to 8 described above.

<Step 9>
To a toluene (8 ml) solution of alcohol 7 (150 mg, 0.29 mmol) obtained in Step 8, Et ₃ N (79 μL, 0.57 mmol) and 4-methoxybenzyl isocyanate (O═C═N—R ¹ : R ¹ = Methoxybenzyl group) (204 μL, 1.43 mmol) was added and the mixture was refluxed for 12 hours. The reaction mixture was extracted with Et ₂ O, the organic layer was washed with saturated brine, dried over MgSO ₄ , and chromatographed on silica gel ( AcOEt: hexane (1: 3)) to obtain the desired carbamate 9l (187 mg, 95% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ -4.6, -3.7, 18.4, 26.1, 26.6, 26.9, 27.5, 27.6, 44.4, 49.9, 55.2, 61.7, 63.3, 66.4, 70.0, 72.5, 73.2, 83.0, 110.5 , 113.9, 127.7, 127.9, 128.4, 128.8, 130.9, 136.7, 156.2, 156.4, 158.8 ppm.

<Step 10>
9L (200 mg, 0.29 mmol) obtained in Step 9 and a solution of TES (1 mL) in CH ₂ Cl ₂ (14 mL) (? 15 ° C cold solution) were added to TFA (6 mL) in CH ₂ Cl ₂ (6 mL) solution (? 15 ° C cold solution) was added. After stirring at the same temperature for 48 hours, 6N NaOH aqueous solution (9 mL) was added and extracted with CH ₂ Cl ₂ . The organic layer was washed with saturated brine, dried over MgSO ₄ , and purified by silica gel chromatography (AcOEt / hexane) (1: 2) to obtain the desired diol 10l (86 mg, yield 46%).
The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ-4.9, -4.0, 18.5, 26.2, 27.5, 44.5, 50.8, 55.2, 61.2, 61.3, 64.9, 66.6, 73.4, 75.9, 114.0, 127.8, 128.0, 128.8, 130.3 , 136.4, 156.1, 157.4, 159.0 ppm.

<Step 11>
NaIO ₄ (75 mg, 0.35 mmol) aqueous solution (1 mL) was added to a THF (3 mL) solution of the diol 10l (38 mg, 0.06 mmol) obtained in Step 10 (cold solution at 0 ° C.). After stirring at the same temperature for 6 hours, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was extracted with AcOEt, washed with saturated aqueous Na ₂ S ₂ O ₃ and saturated brine, dried over MgSO ₄ and purified by silica gel chromatography (AcOEt: hexane (1: 2)) to obtain the desired ketone 11l. Obtained (38 mg, 94% yield).
The analysis results of the obtained compound are as follows.
¹³ C NMR (600 MHz, CDCl ₃ ) δ-5.1, 18.0, 25.7, 27.3, 31.0, 44.7, 54.4, 55.3, 58.9, 66.9, 67.6, 73.6, 74.9, 114.1, 128.1, 128.5, 128.9, 130.3, 136.4, 155.7, 156.0, 159.0, 206.1 ppm.

<Step 12>
A THF 1 M solution (258 μL, 0.26 mmol) of TBAF was added to a THF (10 mL) solution of ketone 11l (106 mg, 0.17 mmol) obtained in Step 11 (? 18 ° C cold solution). The reaction mixture was stirred at the same temperature for 3 hours and then extracted with AcOEt. The mixture was washed with saturated brine, dried over MgSO ₄ and evaporated in vacuo. The residue was passed through a short column and then concentrated under reduced pressure. Then TFA (2 mL) cooled to 0 ° C. was added to the residue and the reaction mixture was allowed to warm to room temperature. After stirring at the same temperature for 1 hour, the mixture was concentrated under reduced pressure, and the spirodiastereomer was purified by silica gel chromatography (AcOEt: hexane (3: 2)) to give 5R form (compound 13l) and 5S form (compound). 14l). The yield of Compound 13l was 49 mg and the yield was 69%, and the yield of Compound 14l was 14 mg and the yield was 20%.
The analysis results of the obtained compound are as follows.
Compound 13l : ¹³ C NMR (100 MHz, CDCl ₃ ) δ43.2, 55.2, 55.7, 67.2, 67.4, 69.2, 74.3, 97.9, 114.0, 114.1, 128.2, 128.4, 128.5, 128.6, 129.0, 135.6, 156.9, 157.9, 159.0 ppm.
Compound 14l: ¹³ C NMR (100 MHz, CDCl ₃ ) δ 47.6, 55.3, 56.7, 67.5, 68.3, 72.2, 79.6, 97.1, 114.1, 128.2, 128.5, 128.7, 128.8, 129.2, 135.7, 157.0, 158.4, 159.0 ppm.

Example 13
Phenyl N-[(5R / S, 8S, 9R) -9-hydroxy-2-oxo-3,6-dioxa-1-
Synthesis of azaspiro [4.4] non-8-yl] carbamate In compound 13 (general formula (2)) and compound 14 (general formula (1)) in FIG. 2, R ¹ is a hydrogen atom, R Synthesis of compounds in which ² is a benzyloxycarbonyl group
Cerium (IV) ammonium nitrite (CAN) (4.5 mg, 0.084 mmol) was added to a solution of 13l (14.3 mg, 0.033 mmol) obtained in Example 12 in CH ₃ CN (0.5 mL) / water (0.25 mL). . After stirring at room temperature for 10 hours, CAN (4.5 mg, 0.084 mmol) was added again to the mixture and stirred at the same temperature for 5 hours, then quenched by the addition of saturated NaHCO ₃ . The mixture was extracted with AcOEt, washed with saturated brine, and dried over MgSO ₄ . The mixture of spirocyanasteromers was purified by silica gel chromatography (AcOEt: hexane (3: 2-3: 1)) to obtain compounds 13m (2.4 mg, 24%) and 14m (3.0 mg, 29%) as colorless oils, respectively. Obtained. The analysis results of the obtained compound are as follows.
Compound (13m): ¹³ C NMR (600 MHz, acetone-d ₆ ) δ 58.5, 66.9, 68.9, 71.5, 79.1, 96.8, 128.7, 129.2, 138.0, 157.1, 158.1 ppm.
Compound (14m): ¹³ C NMR (600 MHz, acetone-d ₆ ) δ 57.2, 66.8, 68.7, 73.3, 77.0, 79.0, 79.1, 95.6, 128.7, 129.2, 138.0, 157.2, 158.0 ppm.

Example 14
(5R / S, 8S, 9R) -8-Amino-9-hydroxy-1-methyl-3,6-dioxa-1-azaspiro [4.4]
Synthesis of nonan-2-one (15/17) Synthesis of compound 15 (general formula (2)) and compound 17 (general formula (1)) of Fig. 3
Hereinafter, a description will be given with reference to FIG.
Benzyl N-[(5R, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] carbamate obtained in Example 11 13k (49.8 mg, 0.16 mmol) was hydrogenated at room temperature in the presence of Pd (OH) ₂ .2H ₂ O / C in a methanol (50 mL) solution. After 40 minutes, the mixture was filtered and washed with MeOH. The washing solution and the filtrate were combined and concentrated to obtain Compound 15 as a white solid (27.8 mg, yield 96%).
In addition, benzyl N-[(5S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl obtained in Example 11 Compound 17 was similarly obtained as a white solid from carbamate 14k (51.0 mg, 0.16 mmol) (30.1 mg, yield 100%).
The analysis results of the obtained compound are as follows.
Compound 15: ¹³ C NMR (100 MHz, CD ₃ OD) δ 25.7, 56.3, 70.6, 70.8, 75.9, 98.8, 159.7 ppm.
Compound 17: ¹³ C NMR (100 MHz, CD ₃ OD) δ 28.2, 56.9, 71.5, 73.6, 81.4, 98.1, 160.1 ppm.

Example 15
N-[(5R / S, 8S, 9R) -9-Hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] formamide (16a / 18a) Synthesis of compound of compound 16 (general formula (2)) and compound 18 (general formula (1)) of FIG. 3 wherein R ² is a formyl group
Pentafluorophenyl formate (30 μM, 0.15 mmol) was added to a solution of compound 15 (14.3 mg, 0.076 mmol) obtained in Example 14 in THF (1.5 mL) / acetone (0.5 mL) (cold solution at 0 ° C.). . The reaction mixture was stirred at the same temperature for 2 hours, a small amount of silica gel (basic, ca. 200 mg) was added, and the mixture was evaporated under reduced pressure. The obtained powder was purified by silica gel column chromatography (acetone / hexane (10: 3) to obtain the desired formamide 16a (13.4 mg, yield 82%) as a white solid. Analysis of the obtained compound The results are as follows.
¹³ C NMR (100 MHz, CD ₃ OD) δ25.6, 25.8, 54.3, 57.4, 67.9, 68.4, 70.5, 73.3, 73.5, 98.8, 159.7, 164.2, 166.9 ppm
Similarly, Compound 18a (15.6 mg) was obtained as a colorless oil from 17 (16.4 mg, 0.087 mmol) in a yield of 83%. The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CD ₃ OD) δ 28.1, 28.3, 29.5, 68.5, 69.2, 73.2, 73.4, 78.5, 78.6, 97.7, 160.0, 164.3, 170.0 ppm

Example 16
N1-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro
[4.4] Synthesis of non-8-yl] acetamide (16b / 18b) In compound 16 (general formula (2)) and compound 18 (general formula (1)) of Fig. 3, R ² is an acetyl group. Synthesis of certain compounds
Ac ₂ O (7.7 μM, 0.081 mmol) was added to a solution of compound 15 (13.9 mg, 0.074 mmol) in THF (1.5 mL) / MeOH (0.5 mL) (cold solution at 0 ° C.). After the reaction mixture was stirred at the same temperature for 1 hour, a small amount of silica gel (basic, ca. 200 mg) was added to the mixture and evaporated under reduced pressure. The obtained powder was subjected to silica gel column chromatography (acetone / hexane (10: The product was purified by 3)) to obtain the desired acetamide 16b (15.4 mg, 91%) as an oil. The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CD ₃ OD) δ22.5, 25.8, 55.6, 68.6, 70.6, 73.5, 98.8, 159.7, 174.0 ppm.
In a similar manner, Compound 18b (18.6 mg) was obtained as an oil in 92% yield from Compound 17 (16.5 mg, 0.088 mmol). The analysis results of the obtained compound are as follows.
¹³ C NMR (100MHz, CD ₃ OD) δ22.5, 28.3, 55.9, 69.4, 73.4, 78.5, 97.7, 160.0, 174.0 ppm.

Example 17
N1-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] -2-phenylacetamide Synthesis of (16c / 18c) Synthesis of compound 16 (general formula (2)) and compound 18 (general formula (1)) of Fig. 3 wherein R ² is a phenylmethylcarbonyl group
Et ₃ N (6.8 μL, 0.05 mmol) and phenylacetyl chloride (6.3 μL, 0.05 mmol) were added to a solution of compound 15 (4.5 mg, 0.024 mmol) (1 equivalent) in THF (1.5 mL) (cold solution at 0 ° C.). Added. After stirring the reaction mixture at the same temperature for 1 hour, a small amount of silica gel (ca. 200 mg) was added and evaporated under reduced pressure, and the resulting powder was purified by silica gel column chromatography (AcOEt) to give compound 16c (5.4 mg) was obtained as a white solid (74% yield). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CD ₃ OD) δ 28.3, 43.7, 55.9, 68.6, 73.6, 78.5, 99.0, 128.0, 130.1, 136.6, 159.7, 174.7 ppm.
Similarly, compound 18c (7.7 mg) was obtained as a white solid in 70% yield from compound 17 (6.7 mg, 0.036 mmol). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, acetone-d6) δ 28.1, 43.3, 56.5, 68.9, 72.4, 78.9, 97.2, 127.4, 129.1, 130.0, 136.7, 157.9, 172.7 ppm.

Example 18
N1-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] -3-phenylpropanamide ( 16d / 18d)
Synthesis of compounds in which R ² is a phenylethylcarbonyl group in compound 16 (general formula (2)) and compound 18 (general formula (1)) in FIG.
Et ₃ N (2 equivalents) and hydrocinnamoyl chloride (8.7 μL, 0.059 mmol) were added to a solution of compound 9a (5.5 mg, 0.029 mmol) in THF (1.5 mL) (cold solution at 0 ° C.). The reaction mixture was stirred at the same temperature for 1 hour, a small amount of silica gel (ca. 200 mg) was added, and the reaction solution was concentrated. The resulting powder was purified by silica gel column chromatography (AcOEt) to give compound 16d ( 7.2 mg) was obtained as a white solid (77% yield). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, acetone-d6) δ 25.6, 32.1, 38.2, 55.9, 68.1, 69.5, 74.0, 79.0, 98.3, 126.8, 129.1, 129.2, 142.2, 157.6, 173.8 ppm.
Similarly, Compound 18d (7.9 mg) was obtained as a white solid from Compound 17 (6.4 mg, 0.034 mmol) in a yield of 72%. The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, acetone-d6) δ 28.1, 32.1, 38.2, 56.4, 69.0, 72.4, 79.0, 97.3, 126.8, 129.1, 129.2, 142.1, 157.9, 174.0 ppm.

Example 19
Phenyl N-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] carbamate (16e / 18e) Synthesis of compound of compound 16 (general formula (2)) and compound 18 (general formula (1)) of FIG. 3 wherein R ² is a phenyloxycarbonyl group
Et ₃ N (10 μL, 0.072 mmol) and phenyl chloroformate (9 μL, 0.072 mmol) were added to a THF (1.5 mL) solution of Compound 15 (6.8 mg, 0.036 mmol) (cold solution at 0 ° C.). After stirring the reaction mixture at the same temperature for 2 hours, a small amount of silica gel (ca. 200 mg) was added, and the reaction solution was concentrated to obtain a powder obtained by silica gel column chromatography (AcOEt / hexane (3: 1)). To give compound 16e (9.3 mg) as a white solid (yield 85%). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ 25.6, 56.3, 67.5, 69.2, 73.6, 97.8, 121.5, 126.0, 129.5, 150.4, 155.4, 157.3 ppm.
Similarly, Compound 18e (9.0 mg) was obtained as a white solid from Compound 17 (7.7 mg, 0.041 mmol) in a yield of 71%. The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ 28.1, 56.8, 68.4, 72.1, 79.1, 96.9, 121.4, 126.0, 129.5, 150.4, 155.6, 158.0 ppm.

Example 20
Of cyclohexylmethyl N-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-azaspiro [4.4] non-8-yl] carbamate (16f / 18f) Synthesis Synthesis of compound 16 (general formula (2)) and compound 18 (general formula (1)) of Fig. 3 in which R ² is a cyclohexylmethyloxycarbonyl group.
Et ₃ N (2 eq) and chloroformate (2 eq) were added to a solution of compound 15 (6.5 mg, 0.035 mmol) (1 eq) in THF (1.5 mL) (cold solution at 0 ° C.). The reaction mixture was stirred at the same temperature for 2 hours, then a small amount of silica gel (ca. 200 mg) was added, and the powder obtained after distillation under reduced pressure was purified by silica gel column chromatography (AcOEt / hexane (3: 1)). Compound 16f (6.0 mg) was obtained as a white solid (52% yield). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ25.6, 26.2, 29.5, 29.7, 37.2, 56.4, 67.4, 69.0, 71.0, 74.5, 98.0, 157.5, 157.7 ppm.
Similarly, Compound 18f (10.7 mg) was obtained as a white solid in 63% yield from Compound 17 (9.7 mg, 0.052 mmol). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ 25.6, 26.2, 28.0, 29.5, 37.3, 56.6, 68.5, 71.0, 72.0, 79.5, 96.9, 157.7, 157.9 ppm.

Example 21
2,3,4,5,6-pentafluorobenzyl N-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] Synthesis of non-8-yl] carbamate (16g / 18g) In compound 16 (general formula (2)) and compound 18 (general formula (1)) in Fig. 3, R ² is 2,3,4, Synthesis of compounds that are 5,6-pentafluorobenzyloxycarbonyl groups
Compound 15 (7.4 mg, 0.04 mmol) in THF (1.5 mL) (0 ° C cold solution) Et ₃ N (11 μL, 0.080 mmol) and 2,3,4,5,6-pentafluorobenzyl chloroformate (13 μL, 0.08 mmol) was added. After stirring the reaction mixture at room temperature for 1 hour, a small amount of silica gel (ca. 200 mg) was added, and the powder obtained after distillation under reduced pressure was purified by silica gel column chromatography (AcOEt / hexane (3: 1)). Compound 16g (13.4 mg) was obtained as a white solid (81% yield). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ25.4, 54.3, 55.9, 67.6, 69.2, 73.5, 97.6, 109.3, 136.2, 138.7, 142.4, 144.3, 155.9, 157.8 ppm.
Similarly, Compound 18g (12.3 mg) was obtained as a white solid from Compound 17 (7.2 mg, 0.04 mmol) (yield 78%). The analysis results of the obtained compound are as follows. ¹³ C NMR (100 MHz, CDCl ₃ ) δ28.0, 54.4, 56.6, 67.9, 68.5, 72.1, 79.1, 96.7, 109.3, 138.7, 144.4, 144.5, 156.1, 158.1 ppm.

Example 22
2-Chlorobenzyl N-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] carbamate (16h / 18h) Synthesis of compound 16 (general formula (2)) and compound 18 (general formula (1)) in Fig. 3 wherein R ² is a 2-chlorobenzyloxycarbonyl group
Et ₃ N (11 μL, 0.079 mmol) and 2-chlorobenzyl chloroformate (12 μL, 0.079 mmol) were added to a THF (1.5 mL) solution of Compound 15 (7.4 mg, 0.04 mmol) (cold solution at 0 ° C.). Added. After stirring the reaction mixture at room temperature for 1 hour, a small amount of silica gel (ca. 200 mg) was added and evaporated under reduced pressure, and the resulting powder was purified by silica gel column chromatography (AcOEt / hexane (3: 1)). Compound 16h (11.0 mg) was obtained as a white solid (yield 79%). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ25.6, 56.2, 64.8, 67.6, 69.2, 74.0, 97.8, 127.0, 129.6, 129.8, 130.1, 133.4, 133.8, 156.8, 157.7 ppm.
Similarly, Compound 18h (10.5 mg) was obtained as a white solid from Compound 17 (6.8 mg, 0.04 mmol) (yield 81%). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ28.0, 56.6, 64.8, 68.6, 72.1, 79.3, 96.8, 127.0, 129.6, 129.8, 130.1, 133.4, 133.8, 156.9, 158.0 ppm.

Example 23
4,5-Dimethoxy-2-nitrobenzyl N-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8 Synthesis of -yl] carbamate (16i / 18i) In compound 16 (general formula (2)) and compound 18 (general formula (1)) of Fig. 3, R ² 4,5-dimethoxy-2-nitrobenzyl Synthesis of compounds that are oxycarbonyl groups
Compound 15 (7.3 mg, 0.039 mmol) in THF (1.5 mL) (0 ° C. cold solution) was added Et ₃ N (11 μL, 0.078 mmol) and 4,5-dimethoxy-2-nitrobenzyl chloroformate (21 mg , 0.078 mmol) was added. After stirring the reaction mixture at room temperature for 1 hour, a small amount of silica gel (ca. 200 mg) was added, and after distilling under reduced pressure, the resulting powder was purified by silica gel column chromatography (AcOEt / hexane (6: 1)). Compound 16i (12.4 mg) was obtained as a yellow solid (yield 74%). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, CDCl ₃ ) δ 25.5, 56.4, 56.5, 64.3, 67.6, 69.2, 73.5, 97.6, 108.2, 110.9, 126.7, 140.0, 148.4, 153.5, 156.5, 157.8 ppm.
Similarly, Compound 18i (10.5 mg) was obtained as a yellow solid from Compound 17 (6.8 mg, 0.04 mmol) (yield 81%). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 28.0, 56.4, 56.5, 64.4, 68.5, 72.1, 79.0, 96.7, 108.2, 111.0, 126.7, 140.0, 148.4, 153.5, 156.4, 158.1 ppm.

Example 24
9H-9-Fluorenylmethyl N-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl ] Synthesis of carbamate (16j / 18j) In the compound 16 (general formula (2)) and compound 18 (general formula (1)) of Fig. 3, R ² is a fluorenylmethyloxycarbonyl group. Composition
Fmoc N-hydroxysuccinimide ester (27 mg, 0.04 mmol) was added to a solution of compound 15 (7.5 mg, 0.04 mmol) in THF (1.5 mL) (cold solution at 0 ° C.). After stirring the reaction mixture at room temperature for 2 hours, a small amount of silica gel (ca. 200 mg) was added, and after distilling under reduced pressure, the resulting powder was purified by silica gel column chromatography (AcOEt / hexane (3: 1)). Compound 16j (15.8 mg) was obtained as a white solid (yield 96%). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, acetone-d6) δ 25.6, 48.0, 56.4, 67.0, 68.3, 69.6, 73.2, 98.0, 120.8, 125.9, 127.9, 128.5, 142.1, 144.9, 157.2, 157.6 ppm.
Similarly, Compound 18j (11.3 mg) was obtained as a white solid from Compound 17 (5.5 mg, 0.04 mmol) (yield 95%). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, acetone-d6) δ 28.1, 48.0, 57.2, 67.0, 69.0, 72.5, 78.4, 97.0, 120.8, 126.0, 127.9, 128.5, 142.1, 145.0, 157.3, 158.0 ppm.

Example 25
4-methoxybenzyl N-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] carbamate (16k / 18k) Synthesis of compound 16 (general formula (2)) and compound 18 (general formula (1)) in Fig. 3 wherein R ² is a 4-methoxybenzyloxycarbonyl group
A solution of compound 15 (8.2 mg, 0.044 mmol) in THF (1.5 mL) (cold solution at 0 ° C.) with Et ₃ N (9 μL, 0.078 mmol) and Sp-methoxybenzylcarbonyl-4,6-dimethyl-2-mercapto Pyrimidine Sp-Methoxybenzyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine (MZ-SDP) was added. After the reaction mixture was stirred at room temperature for 3 hours, a small amount of silica gel (ca. 200 mg) was added and evaporated under reduced pressure, and the resulting powder was purified by silica gel column chromatography (AcOEt / hexane (4: 1)). Compound 16k (12.1 mg) was obtained as a white solid (yield 78%). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, acetone-d ₆ ) δ 25.6, 55.5, 56.5, 66.7, 68.2, 69.6, 73.2, 97.2, 114.5, 129.8, 130.7, 157.3, 157.6, 160.5 ppm.
Similarly, Compound 18k (10.5 mg) was obtained as a white solid from Compound 17 (8.0 mg, 0.043 mmol) (yield 70%).
¹³ C NMR (100 MHz, acetone-d ₆ ) δ 28.0, 55.5, 57.0, 66.7, 68.9, 72.4, 78.3, 96.9, 114.5, 129.8, 130.7, 157.4, 157.9, 160.5 ppm.

Example 26
N-benzyl-N '-[(5R / S, 8S, 9R) -9-hydroxy-1-methyl-2-oxo-3,6-dioxa-1-azaspiro [4.4] non-8-yl] urea ( Synthesis of 16l / 18l) Synthesis of compound 16 (general formula (2)) and compound 18 (general formula (1)) of Fig. 3 wherein R ² is an N-benzylamide group
Benzyl isocyanate (4.9 μL, 0.040 mmol) was added to a solution of compound 15 (7.5 mg, 0.040 mmol) in THF (1.5 mL). The reaction mixture was stirred at room temperature for 2 hours, a small amount of silica gel (ca. 200 mg) was added, and the powder obtained after distillation under reduced pressure was purified by silica gel column chromatography (AcOEt / hexane (15: 1)). Compound 16l (10.2 mg) was obtained as a white solid (yield 80%). The analysis results of the obtained compound are as follows.
¹³ C NMR (100 MHz, acetone-d ₆ ) δ 25.6, 44.3, 56.7, 68.4, 69.6, 74.7, 74.8, 98.4, 127.6, 128.0, 129.1, 141.3, 157.6, 159.8 ppm.
Similarly, Compound 18l (11.7 mg) was obtained as a white solid from Compound 17 (8.4 mg, 0.045 mmol) (yield 82%).
¹³ C NMR (100 MHz, acetone-d ₆ ) δ 28.1, 44.3, 57.1, 69.2, 72.5, 79.8, 79.9, 97.4, 127.6, 128.0, 129.1, 141.3, 158.0, 159.9 ppm.

Evaluation of activity against AI-2 receptor Vibrio Harvey BB170 strain (available from Prof. Bonnie El Basler, Princeton University) was cultured in AB Grouse medium (2 ml) at 30 ° C for 13-15 hours, and AB Grouse medium Diluted 5,000 times in. In a black 96-well microplate, add 90 μl of culture solution and 10 μl of a mixture of known AI-2 agonist compound solution (30 μM), test AI-2 antagonist compound solution (10-100 μM) and B (OH) ₃ (20 μM). Added. After incubation at 30 ° C. for 5-6 hours, the plate was measured for luminescence with a luminescence counter (Luminescencer JNRII, ATTO corp.). The temperature of the counting chamber was set at 20 ° C.
A mixture of AI-2 antagonist compound solution (30 μM), test AI-2 antagonist compound solution (10-100 μM) and B (OH) ₃ (20 μM) was prepared as follows.

<Final antagonist concentration>
The final concentration of antagonist in the reaction solution is adjusted by diluting the initial concentration to 1/100.
(Example 1) Final concentration of antagonist (100 μM): First, prepare a 10 mM DMSO solution of antagonist, take 20 μL of this solution and mix with agonist, B (OH) ₃ (2 μL) and AB medium (176 μL). Set to 200 μL. Here, the antagonist is diluted 1/10 to 1 mM. Next, 10 μL of this is mixed with AB medium (90 μL) in the well to make a total volume of 100 μL. Here the antagonist is further diluted 1/10 to a final concentration of 100 μM. The final concentrations of other antagonists (50 μM, 10 μM) are prepared similarly. 5 mM and 1 mM DMSO solutions are finally diluted 1/100 by the above procedure to 50 μM and 10 μM, respectively.

<Final agonist concentration>
The final concentration of the agonist in the reaction is adjusted by diluting the initial agonist concentration by 1/1000.
(Example 2) Final concentration of antagonist (30 μM): First, prepare a 30 mM aqueous solution of agonist, take 2 μL of this and mix with antagonist (20 μL), B (OH) ₃ (2 μL) and AB medium (176 μL). Make the total volume 200 μL. Here, the agonist is diluted 1/100 to 0.3 mM. Next, 10 μL of this is mixed with AB medium (90 μL) in a well to make a total volume of 100 μL. Here the agonist is further diluted 1/10 to a final concentration of 30 μM.

< Final concentration of B (OH) ₃ >
The final concentration of B (OH) _{3 in} the reaction solution is adjusted by diluting the initial concentration of B (OH) ₃ by 1/1000, as with the agonist.
(Example 3) Final concentration of B (OH) ₃ (20 μM): First, a 20 mM aqueous solution of B (OH) ₃ is prepared. Hereinafter, the same as the item of “2. Final concentration of agonist” above.
By adding both an antagonist and an agonist in one well, it can be observed how much the antagonist suppresses the physiological activity of the AI-2 agonist. The Vibrio Harvey BB170 strain used here has the property of incorporating an AI-2 agonist and emitting light. In the presence of an antagonist, the activity of the agonist is inhibited and the intensity of luminescence observed is reduced. By observing the intensity, the activity intensity as an antagonist can be evaluated. The luminescence intensity when no antagonist is added is used as a control.
The purpose of adding B (OH) ₃ is as follows. As shown in FIG. 4, it is considered that DPD (AI-2 precursor) or agonist interacts with the receptor of Vibrio Harvey BB170 by binding to B (OH) ₃ and exhibits physiological activity. The left figure in FIG. 4 shows the binding between DPD, which is an AI-2 precursor, and B (OH) _3, and the right figure shows how the agonist and B (OH) ₃ are bound. B (OH) ₃ is present in nature, but without the addition of B (OH) ₃ emission is observed, by adding B (OH) _3, easily observed increases overall emission intensity In addition, it is considered that the influence of the concentration gradient (bias) of natural B (OH) ₃ is reduced and stable experimental results can be obtained.

1. Preparation of AI-2 analog solution
Prepare 10 mM test AI-2 analog (about 1.5-2 mg) in DMSO solution (about 500-800 μl), then dilute 1: 2 and 1:10 in DMSO to give 5 mM and 1 mM antagonist solutions. Prepared. The agonist was prepared as a 100 mM aqueous solution.
2. Preparation of the mixture To the AB medium (176 μM) in a test tube, add the agonist (2 μl, 30 mM aqueous solution), antagonist (20 μl, 1-10 mM DMSO solution), and B (OH) ₃ ( 2 μl, 20 mM aqueous solution) was added to prepare 200 μl of a mixture ([agonist] = 300 μM, [antagonist] = 100 to 1000 μM, [B (OH) ₃ ] = 200 μM.) As a control solution, DMSO (20 μl ) Was added in place of the antagonist. 10 μl of this solution was combined with 90 μl of AB medium to make the final concentration of the substance 1/10 of these values).

The structure of the agonist used is as follows.
5 to 11 show the luminescence of the test substance as a relative value when the luminescence of the control is 100%. The symbol of the test antagonist compound displayed in FIGS. 5 to 11 is the symbol shown in FIGS. 2 and 3. The left side of each bar graph shows the results when the final concentration of the test antagonist is 10 μM, the middle is 50 μM, and the right is 100 μM. A substance that suppresses luminescence in a concentration-dependent manner has an AI-2 receptor antagonist activity.
5 to 11, it can be seen that the compounds 13b-k, 13m, 14b-k, 14m, 16c-i, 161, 18a, 18c-h, and 18l exhibit antagonist activity.

Assessment of the impact on biofilm formation
Using a 96-well plate, a culture solution in which P. gingivalis 381 strain or compound 13k (see FIG. 2) as a test AI-2 analog was added to a final concentration of 100 μM was cultured for 1 week ( 37 ℃, N ₂ 85%, H ₂ 5%, CO ₂ 10%). The bacterial solution on the plate was changed every 2 days, and the plate was further washed 3 times with PBS to remove floating bacteria. As a control, only the culture broth was used, and the test AI-2 analog compound 13k was not added.
The biofilm (adherent bacteria) was then stained with 0.1% crystal violet for 30 minutes and washed with distilled water. Subsequently, it was decolored with 70% ethanol for 15 minutes, and the absorbance (OD 595 nm) of the supernatant was measured. Six sets of experiments were performed.
The results are shown in FIG. It can be seen that the presence of 100 μM compound 13K significantly suppressed biofilm formation by P. gingivalis strain 381.

It is a figure which shows the structure at the time of DPD and its receptor coupling | bonding. It is a figure which shows the manufacturing method of the compound of General formula (1) and (2) of this invention. It is a figure which shows the manufacturing method of the compound of General formula (1) and (2) of this invention. It is a figure explaining the objective which uses B (OH) ₃ in an Example. It is a figure which shows the strength of the antagonist activity with respect to AI-2 receptor of the compound synthesize | combined in the Example. The left side of each bar graph shows the results when the final concentration of the test antagonist is 10 μM, the middle is 50 μM, and the right is 100 μM. It is a figure which shows the strength of the antagonist activity with respect to AI-2 receptor of the compound synthesize | combined in the Example. The left of each bar graph shows the results when the final concentration of the test antagonist is 10 μM, the middle is 50 μM, and the right is 100 μM. It is a figure which shows the strength of the antagonist activity with respect to AI-2 receptor of the compound synthesize | combined in the Example. The left side of each bar graph shows the results when the final concentration of the test antagonist is 10 μM, the middle is 50 μM, and the right is 100 μM. It is a figure which shows the strength of the antagonist activity with respect to AI-2 receptor of the compound synthesize | combined in the Example. The left side of each bar graph shows the results when the final concentration of the test antagonist is 10 μM, the middle is 50 μM, and the right is 100 μM. It is a figure which shows the strength of the antagonist activity with respect to AI-2 receptor of the compound synthesize | combined in the Example. The left side of each bar graph shows the results when the final concentration of the test antagonist is 10 μM, the middle is 50 μM, and the right is 100 μM. It is a figure which shows the strength of the antagonist activity with respect to AI-2 receptor of the compound synthesize | combined in the Example. The left of each bar graph shows the results when the final concentration of the test antagonist is 10 μM, the middle is 50 μM, and the right is 100 μM. It is a figure which shows the strength of the antagonist activity with respect to AI-2 receptor of the compound synthesize | combined in the Example. The left of each bar graph shows the results when the final concentration of the test antagonist is 10 μM, the middle is 50 μM, and the right is 100 μM. It is a figure which shows the biofilm formation inhibitory effect of the compound 13k synthesize | combined in the Example.

Claims (6)

The compound represented by the following general formula (1).
(In the formula, R ¹ is a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, a phenyl group, a naphthyl group, a phenylalkyl group having 1 to 6 carbon atoms, a naphthyl group, or an alkyl having 1 to 3 carbon atoms. R ² represents a hydrogen atom, a formyl group, an acetyl group, a propionyl group, a butyryl group, a benzoyl group, an alkyl group having 1 to 8 carbon atoms, or a phenyl group having an alkyl group having 1 to 4 carbon atoms. An alkylcarbonyl group, a phenyloxycarbonyl group, an N-phenylamide group, an N- (naphthyl) amide group, an alkyl group having 1 to 3 carbon atoms, and the cycloalkyl group has 4 to 13 carbon atoms (cycloalkyl) ) N- (cycloalkylalkyl) amido group having an alkyloxycarbonyl group, an alkyl group having 1 to 3 carbon atoms, and a cycloalkyl group having 4 to 13 carbon atoms, carbon number An alkyloxycarbonyl group having an alkyl group of ˜6, an N-alkylamide group having an alkyl group of 1 to 6 carbon atoms, a fluorenylalkyloxycarbonyl group having an alkyl group of 1 to 3 carbon atoms, or a carbon number of 1 A phenylalkyloxycarbonyl group having an alkyl group of ˜3, a (naphthyl) alkyloxycarbonyl group having an alkyl group of 1 to 3 carbon atoms, an N- (phenylalkyl) amide group having an alkyl group of 1 to 3 carbon atoms, N-[(naphthyl) alkyl] amido group having an alkyl group having 1 to 3 carbon atoms.In R ¹ and R ² , the phenyl group, naphthyl group, and fluorenyl group may have a substituent.) A compound represented by the following general formula (2).
(In the formula, R ¹ and R ² are the same as above.) An autoinducer-2 receptor antagonist comprising a compound represented by the following general formula (1) or general formula (2).
(In the formula, R ¹ and R ² are the same as above.) The biofilm formation inhibitor containing the compound represented by following General formula (1) or General formula (2).
(In the formula, R ¹ and R ² are the same as above.) A plaque formation inhibitor comprising a compound represented by the following general formula (1) or general formula (2).
(In the formula, R ¹ and R ² are the same as above.) A biofilm formation inhibition method comprising a step of adding a compound represented by the following general formula (1) or general formula (2) into a wastewater pipe, a wastewater tank, a circulation bath, a swimming tank, or an aquarium.
(In the formula, R ¹ and R ² are the same as above.)