OA21080A

OA21080A - Adjuvant with TLR4 agonist activity.

Info

Publication number: OA21080A
Application number: OA1202200537
Authority: OA
Inventors: Hitoshi Ban; Yusuke Imazaki; Yosuke Takanashi; Akihisa Fukushima
Original assignee: Sumitomo Pharma Co., Ltd.
Priority date: 2020-06-22
Filing date: 2021-06-21
Publication date: 2023-10-09

Abstract

The present invention relates to a compound that is useful as a vaccine adjuvant, a method for producing the same, a pharmaceutical composition that contains the compound, and use of the compound as vaccine adjuvant.

Description

ADJUVANT WITH TLR4 AGONIST ACTIVITY

TECHNICAL FIELD [0001]

The present invention relates to a compound useful as vaccine adjuvant, a manufacturing process thereof, a pharmaceutical composition comprising the compound, and use of the compound as vaccine adjuvant.

BACKGROUND ART [0002]

A vaccine comprising a partial protein/peptide derived from some protein that microorganisms produce is advantageous in terms of the safety and the preparing 15 process since it can be prepared through Chemical synthesis or genetic engineering technique. On the other hand, such vaccine tends to hâve weaker efficacy than a live or inactivated vaccine prepared from raw bacteria, and sometimes an adjuvant is added to the vaccine to enhance 20 immunogenicity. As an adjuvant for vaccine, alum has been often used, and recently a vaccine comprising 3-desacyl-4 ' monophosphoryl lipid A (MPL) which is an agonist for Toll like receptor 4 (TLR4) as an adjuvant has been commercially approved (Non-Patent Literature 1) .

[0003]

It is known that TLR4 can préparé a heterodimer with MD-2 (myeloid différentiation factor 2) , and activate the TLR4 pathway. Lipopolysaccharide (LPS) which is known as one of TLR4 agonists has a lipid A structure which is composed of phosphorylated disaccharide and fatty acid side chain (Non-Patent Literature 2) , and it has been reported that the lipid A interacts with both of TLR4 and MD-2 to play an important rôle to form the heterodimer (Non-Patent Literature 3) . [0004]

LPS can be transformée! to the above-mentioned MPL by détoxification (Non-Patent Literature 1) , and thus MPL is a mixture of plural compounds composed of phosphorylated disaccharide and fatty acid side chain, like LPS (NonPatent Literature 4). The X-ray crystal structure analysis shows that the phosphate group interacts with both of TLR4 and MD-2 (Non-Patent Literature 3) , and it is also known that the activity of MPL greatly decreases when the phosphate group is removed from the lipid A (Non-Patent Literature 2). And, it has been reported that the structure of fatty acid therein is also important on the TLR4 agonistic effect of MPL, and sometimes the effect of MPL may change to antagonistic effect depending on the structure of fatty acid side chain (Non-Patent Literature 2) .

[0005]

Compounds derived from biological components such as MPL hâve manufacturing challenges, and thus some synthetic TLR4 agonists mimicking MPL hâve been studied. For example, Patent Literatures 1 and 2 reported that phosphorylated disaccharide structure which is a basic structure in aminoalkyl glucosaminide phosphate (AGP) was transformed to its phosphorylated monosaccharide structure, and then the phosphate group was an essential structure in AGP. And, Non-Patent Literature 5 also reported detailed studies about fatty acid side chain in AGP, in which the TLR4 agonistic effect of AGP was deactivated by transforming the fatty acid side chain, like the case of lipid A.

It is thought that synthetic TLR4 agonist which has a structure equipped with phosphate group such as AGP is disadvantageous in terms of manufacturing cost or storage stability, but there has been no report of vaccine adjuvants that are TLR4 agonist having saccharide and fatty acid side chain as main structures, but having no phosphate group, which still retain TLR4 agonistic effect, in the past research reports.

PRIOR ART

[Patent Reference] [0006]

[Patent Literature] WO 1998/050399

[Patent Literature 2] WO 2001/034617

[Non-patent Reference] [0007]

[Non-Patent Literature 1] Cervarix interview form

[Non-Patent Literature 2] Microbes Infect, 2002 Jul ; 4(8): 837-51.

[Non-Patent Literature 3] Nature. 2009 Apr 30; 458 (7242): 1191-5.

[Non-Patent Literature 4] Regul Toxicol Pharmacol. 2002 Jun; 35(3): 398-413.

[Non-Patent Literature 5] J Biol Chem. 2004 Feb 6;

279 (6) : 4440-9

Summary of Invention

[0008] (Technical Problem)

The purpose of the present invent ion may be to provide a TLR4 agonist having a high adjuvant effect even having no phosphate group.

[0009] (Solution to Problem)

The present inventors hâve extensively studied to find a TLR4 agonist having a high adjuvant effect, and then hâve found some compounds which retain TLR4 agonistic effect by suitably transforming their fatty acid side chain even having no phosphate group on the sugar structure. Further, the present inventors hâve found that these compounds hâve excellent adjuvant effect. Based upon the findings, the present invention has been achieved. According to the present invention, a TLR4 agonist dérivative of the foîlowing formula (1) (hereinafter, also referred to as the present compound) is provided.

[0010]

The present invention is as described below.

[0011] (Item 1)

A compound of formula ( 1 ) :

OH

Q or a pharmaceutically acceptable sait thereof, wherein

A and A ’ are independently hydrogen, hydroxy, or (CH₂)_m-COOH, provided that at least one of A or A' is (CH₂)_m-COOH,

R¹ is -C(O) (CH₂)n-X or -CH₂-(CH₂) _n-X,

R² is -C(0) (CH₂)_o-Y or -CH₂-(CH₂) _O-Y,

R³ is -C(0) (CH₂)_P-Z or -CH₂- (CH₂) _P-Z,

X, Y, and Z are independently methyl, Ce-io aryl (said Cs-io aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and alkoxy), or 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1-4 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-6 alkoxy) , provîded that at least one of X, Y, or Z is C₆-io aryl (said C₆-io aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-₆ alkyl, halogen, cyano, and Ci-₆ alkoxy) or 5~ to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1-4 substituents selected independently from hydroxy, Ci-e alkyl, halogen, cyano, and Ci-e alkoxy) , provîded that when A is -COOH and the stereochemistry of * is S-configuration, Y is methyl,

R⁴, R⁵, and R⁶ are independently CiQ-20 alkyl, m is independently an integer of 0-6, and n, o, and p are independently an integer of 5 ~ 20. [0012] (Item 2)

The compound of Item 1, or a pharmaceutically acceptable sait thereof, wherein m is 0.

[0013] (Item 3)

The compound of Item 1 or 2, or a pharmaceutically acceptable sait thereof, wherein R¹ is -C(O) (CH2) n-X, R² is -C(0) (CH2)_o-Y, and R³ is -C(0) (CH2)p~Z. [0014] (Item 4)

The compound of any one of Items 1 to 3, or a pharmaceutically acceptable sait thereof, wherein A is COOH. [0015] (Item 5)

The compound of any one of Items 1 to 4, or a pharmaceutically acceptable sait thereof, wherein A is COOH, and A' is hydroxy. [0016] (Item 6)

The compound of any one of Items 1 to 5, or a pharmaceutically acceptable sait thereof, wherein R⁴, R⁵, and R⁶ are independently C10-12 alkyl. [0017] (Item 7)

The compound of Item 1 which is represented by formula (2) :

wherein

Ri is -C(0) (CH₂)_n-X,

R² is -C(O) (CH₂)o-Y,

R³ is -C(0) (CH₂)_P-Z,

X, Y, and Z are independently methyl, C₆-io aryl, or 5to 10-membered heteroaryl, provided that at least one of X, Y, or Z is Cê-io aryl or 5- to 10-membered heteroaryl, provided that when the stereochemistry of * is ΒΙΟ configuration, Y is methyl,

R⁴, R⁵, and R⁶ are independently Cio-i₂ alkyl, and n, o, and p are independently an integer of 6 - 10, or a pharmaceutically acceptable sait thereof.

[0018] (Item 8)

The compound of Item 1 which is represented by formula (3) :

wherein

R¹ is -C (O) (CH₂) n-X,

R² is -C (O) (CH₂) o-Y,

R³ is -C(0) (CH₂)_P-Z,

X, Y, and Z are independently methyl, Ce-io aryl, or 5to 10-membered heteroaryl, provided that at least one of X, Y, or Z is Ce-io aryl or 5- to 10-membered heteroaryl, provided that when the stereochemistry of * is S10 configuration, Y is methyl, and n, o, and p are independently an integer of 6 - 10, or a pharmaceutically acceptable sait thereof.

[0019] (Item 9)

The compound of Item 1 which is represented by formula (4) or formula (5) :

wherein

Ri is -C(O) (CH₂)n-X,

R² is -C (O) (CH₂)o-Y,

R³ is -C(O) (CH₂)_P-Z,

R²' is -C(O) (CH₂)_O-CH₃,

X, Y, and Z are independently methyl, Ce-io aryl, or 5to 10-membered heteroaryl, provided that at least one of X, Y, or Z in formula (4) is Cê-io aryl or 5- to 10-membered 10 heteroaryl, and at least one of X or Z in formula ( 5 ) is Ce-io aryl or 5- to 10-membered heteroaryl, and n, o, and p are independently an integer of 7 - 9, or a pharmaceutically acceptable sait thereof.

[0020] (Item 10)

The compound of any one of Items 1 to 9, or a pharmaceutically acceptable sait thereof, wherein

X, Y, and Z are independently methyl or phenyl, provided that at least one of X, Y, or Z is phenyl, provided that when A is -COOH and the stereochemistry of * is S-configuration, Y is methyl. [0021] (Item 11)

The compound of Item 1 which is selected from the following compound group:

(2R)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-3{[3—{[(3R)~3-(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)— 4 —({ (3R)-3-[(9phenylnonanoyl)oxy]tetradecanoyl}oxy)oxan-2yl]oxy}propanoic acid (Example 1) , (2 S)-2-{ [(3F)^-3-(decanoyloxy)tetradecanoyl]amino} -3{[3—{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4-({(3R)-3-[(9phenylnonanoyl) oxy ]tetradecanoyl}oxy)oxan-2yl]oxy}propanoic acid (Exemple 2), (2R) -2-{[( 3R)-3-(decanoyloxy)tetradecanoyl]amino} -3{ [4 — { [(3R)-3-(decanoyloxy)tetradecanoyl]oxy)-5-hydroxy-6(hydroxymethyl)—3—({(3R)-3-[(9phenylnonanoyl)oxy]tetradecanoyl}amino)oxan-2yl]oxy}propanoic acid (Example 3), (2R)-2-({ [ (3R)-3-(9phenylnonanoyl)oxy]tetradecanoyl}amino)-3-([ 3- {[(3R)-321080 (decanoyloxy)tetradecanoyl]amino(-5-hydroxy-6(hydroxymethyl)-4-({(3R)-3( decanoyloxy)tetradecanoyl(oxy)oxan-2-yl]oxy(propanoic acid (Example 4), and (2S) -2- ( { [ (3R)-3-(9phenylnonanoyl)oxy]tetradecanoyl}amino)-3-{[3-{[(3R)-3(decanoyloxy)tetradecanoyl]amino}-5-hydroxy~6(hydroxymethyl)-4-({(3R) -3(decanoyloxy)tetradecanoyl}oxy)oxan-2-yl]oxy(propanoic acid (Example 5), or a pharmaceutically acceptable sait thereof.

[0022] (Item 12)

A pharmaceutical composition comprising the compound of any one of Items 1 to 11, or a pharmaceutically acceptable sait thereof. [0023] (Item 13)

The pharmaceutical composition of Item 12, which is a lipid formulation. [0024] (Item 14)

The pharmaceutical composition of Item 12 or 13, wherein the lipid formulation is a liposome formulation including phospholipid.

[0025] (Item 15)

The pharmaceutical composition of Item 14 , wherein the phospholipid is 1,2~dimyristoyl-sn-glycero-3-phosphocholine and egg yolk phosphatidylglycerol. [0026] (Item 16)

The pharmaceutical composition of Item 14 or 15, wherein the lipid formulation comprises at least one additive selected form the group consisting of an inorganic acid, an inorganic acid sait, an organic acid, an organic acid sait, sugars, a buffering agent, an antioxidant, and polymers. [0027] (Item 17)

The pharmaceutical composition of any one of Items 12 to 16, which further comprises an antigen. [0028] (Item 18)

The pharmaceutical composition of Item 17, wherein the antigen is a pathogen-derived antigen. [0029] (Item 19)

A vaccine adj uvant comprising the compound of any one of Items 1 to 11, or a pharmaceutically acceptable sait thereof.

[0030] (Item 20)

The vaccine adjuvant of Item 19, which is an adjuvant for infection vaccine. [0031] (Item 21)

A médicament for treating or preventing infection, comprising the compound of any one of Items 1 to 11, or a pharmaceutically acceptable sait thereof, which is used in combination with a pathogen-derived antigen. [0032] (Item 22)

The compound of any one of Items 1 to 11, or a pharmaceutically acceptable sait thereof, which is used as a vaccine adjuvant. [0033] (Item 23)

A method for enhancing spécifie immune response in a mammal to an antigen, comprising administering the compound of any one of Items 1 to 11 or a pharmaceutically acceptable sait thereof to the mammal. [0034] (Item 24)

Use of the compound of any one of Items 1 to 11 or a pharmaceutically acceptable sait thereof in the préparation of a vaccine adjuvant.

[0035] (Item 25)

A kit comprising

a) the compound of Item 1 or a pharmaceutically acceptable sait thereof, or a pharmaceutical composition comprising the compound of Item 1 or a pharmaceutically acceptable sait thereof; and

b) a pharmaceutical composition comprising an antigen. [0036] (Item 26)

The kit of Item 25, wherein the antigen is a pathogenderived antigen.

[0037] (Effect of the Invention)

The present compound has acquired TLR4 agonistic effect by suitably transforming the fatty acid side chain even having no phosphate group on the sugar structure, which has a high adjuvant effect. The present compound has a high storage stability since it has no phosphate group on the sugar structure, and the manuf acturing cost can be lowered since there is no process of introducing a phosphate group. Thus, the present compound is very useful as a vaccine adjuvant.

BRIEF DESCRIPTION OF DRAWINGS

[0038]

FIG. 1 is a graph showing the results of measuring OVA-specific IgG2c in immunized mouse sérum in mice to which the formulation prepared in Example 6 or 7 was intramuscularly administered, by ELISA in Test 3. The vertical axis indicates the antibody titer of OVA-specific IgG2c in sérum. The horizontal axis indicates the administered sample (administered dose in parenthèses). 1: négative control group (phosphate buffered saline), 2. Example 6 administration group (the compound of Example 1, 10 pg/mouse) , 3: Example 6 administration group (the compound of Example 1, 100 pg/mouse) , 4: Example 7 administration group (the compound of Example 2, 10 pg/mouse), 5: Example 7 administration group (the compound of Example 2, 100 pg/mouse).

FIG. 2 is a graph showing the results of measuring OVA-specific IgG2c in immunized mouse sérum in mice to which the formulation prepared in Reference Example 16 or Example 6 was intramuscularly administered, by ELISA in Test 3. The vertical axis indicates the antibody titer of

OVA-specific IgG2c in sérum. The horizontal axis indicates the administered sample (administered dose in parenthèses).

1: négative control group (phosphate buffered saline), 2:

Reference example 16 administration group (the compound of Reference example 9, 10 pg/mouse)_f 3 : Reference example 16 administration group (the compound of Reference example 9, 100 pg/mouse), 4: Example 6 administration group (the compound of Example 1, 10 pg/mouse), 5: Example 6 administration group (the compound of Example 1, 100 pg/mouse).

FIG. 3 is a graph showing the results of the évaluation performed in Test 4, showing the percentage of type 1 helper T cell in the spleen cell of the mouse to which the formulation prepared in Example 6 or 7 was intramuscularly administered. The horizontal axis is the same as FIG. 1.

FIG. 4 is a graph showing the results of the évaluation performed in Test 4, showing the percentage of type 1 helper T cell in the spleen cell of the mouse to which the formulation prepared in Reference example 16 or Example 6 was intramuscularly administered. The horizontal axis is the same as FIG. 2.

FIG. 5 is a graph showing the results of the évaluation performed in Test 4, showing the percentage of OVA tetramer-positive CD8T cell in the spleen cell of the mouse to which the formulation prepared in Example 6 or Example 1 was intramuscularly administered. The horizontal axis is the same as FIG. 1.

FIG. 6 is a graph showing the results of the évaluation performed in Test 4, showing the percentage of OVA tetramer-positive CD8T cell in the spleen cell of the mouse to which the formulation prepared in Reference Example 16 or Example 6 was intramuscularly administered. The horizontal axis is the same as FIG. 2.

FIG. 7 is a graph showing the results of the évaluation performed in Test 4, showing the percentage of effector memory CD8 T cell in the spleen cell of the mouse to which the formulation prepared in Example 6 or 7 was intramuscularly administered. The horizontal axis is the same as FIG. 1.

FIG. 8 is a graph showing the results of the évaluation performed in Test 4, showing the percentage of effector memory CD8 T cell in the spleen cell of the mouse to which the formulation prepared in Reference Example 16 or Example 6 was intramuscularly administered. The horizontal axis is the same as FIG. 2.

DESCRIPTION OF EMBODIMENTS

[0039]

Hereinafter, terms used herein are explained as follows. [0040]

The number of substituents that are defined postenor to optionally-substituted or substituted should not be limited, if it is possible to be substituted. Unless otherwise specified, the définition of each substituent group also extends over the case of partially-including the substituent group or the case of the substituent group existing on another substituent group. [0041]

The halogen used herein includes, for example, fluorine, chlorine, bromine, and iodine. It is preferably fluorine or chlorine, more preferably fluorine. [0042]

The Ci-6 alkyl means straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. The Ci-6 alkyl includes preferably C1-4 alkyl, more preferably C1-3 alkyl. The C1-6 alkyl includes, for example, methyl, ethyl, propyl, 1-methylethy1, butyl, 2methylpropyl, l-methylpropyl, 1,1-dimethylethyl, pentyl, 3methylbutyl, 2-methylbutyl, 2,2-dimethylpropyl, 1ethylpropyl, 1,1-dimethylpropyl, hexyl, 4-methylpentyl, 3methylpentyl, 2-methylpentyl, and 1-methylpentyl, and the C1-4 alky includes the examples of the C1-6 alkyl provided that the number of carbon atoms is 1 - 4 . The Ci-3 alkyl includes the examples of the C1-6 alkyl provided that the number of carbon atoms is 1 - 3. [0043]

The Ci-6 alkoxy means Ci-6 alkyloxy, and the part Ci-6 alkyl is as defined in the said Ci-β alkyl. The ’’Ci-6 alkoxy includes preferably Ci-4 alkoxy, more preferably Cx-3 alkoxy. The Ci-₆ alkoxy includes, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 2-methylpropoxy, 1—methylpropoxy, 1,1-dimethylethoxy, pentyloxy, 3-methylbutoxy, 2-methylbutoxy, 2,2~ dimethylpropoxy, l~ethylpropoxy, 1,1-dimethylpropoxy, hexyloxy, 4-methylpentyloxy, 3-methylpentyloxy, ²“ methylpentyloxy, 1-methylpentyloxy, 3,3-dlmethylbutoxy,

2,2-dimethylbutoxy, 1,1-dimethylbutoxy, · and 1,2dimethyIbutoxy, and the C1-4 alkoxy includes the examples of the ”Ci-6 alkoxy provided that the number of carbon atoms is 1 - 4. The C1-3 alkoxy includes the examples of the C1-6 alkoxy provided that the number of carbon atoms is 1 - 3. [0044]

The C10-20 alkyl means straight or branched chain saturated hydrocarbon group having 10 — 20 carbon atoms. The Cio-ΞΟ alkyl includes preferably ”Cio-i5 alkyl, more preferably C10-12 alkyl, and even more preferably straight chain C10-12 alkyl. The straight chain C10-20 alkyl includes, for example, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl, and the C10-15 alkyl includes the above examples of C 10-20 alkyl wherein the number of carbon atoms are 10 - 15. The C10-12 alkyl includes the above examples of C10-20 alkyl wherein the number of carbon atoms are 10 - 12.

[0045]

The Ce-io aryl means aromatic hydrocarbon having 6 10 carbon atoms. The Cg-io aryl includes, for example, phenyl, 1-naphthyl, and 2-naphthyl, and more preferably phenyl. [0046]

The 5- to 10-membered heteroaryl means a monocyclic 5- to 7-membered aromatic or bicyclic 8- to 10-membered aromatic heterocyclyl group comprising 1 to 4 atoms selected independently from nitrogen atom, oxygen atom, and sulfur atom. The 5- to 10-membered heteroaryl includes preferably 5- to 7-membered heteroaryl, more preferably 5- to 7-membered aromatic heterocyclyl having one or more nitrogen atom in the ring ( 5- to 7-membered nitrogencontaining heteroaryl).

The 5- to 10-membered heteroaryl includes, for example, pyridyl, pyridazinyl, isothiazolyl, pyrrolyl, furyl, thienyl, thiazolyl, imidazolyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrazinyl, triazinyl, triazolyl, imidazolidinyl, oxadiazolyl, triazolyl, tetrazolyl, indolyl, indazolyl, quinolyl.

isoquinolyl, benzofuranyl, benzothienyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, benzotriazolyl, benzimidazolyl, and 6,11-dihydrodibenzo [b, e]thiepinyl, preferably pyridyl, pyrimidinyl, quinolyl, and isoquinolyl, and more preferably pyridyl.

The 5- to 7-membered heteroaryl includes, for example, monocyclic groups in the above examples of 5- to 10-membered heteroaryl. The 5- to 7-membered nitrogencontaining heteroaryl includes, for example, nitrogencontaining monocyclic groups in the above examples of 5to 10-membered heteroaryl.

[0047]

The Ce-io aryl used herein may hâve 1-5 substituents selected independently from hydroxy, Ci-₆ alkyl, halogen, cyano, and Ci-₆ alkoxy. The 5- to 10-membered heteroaryl may hâve 1-4 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-₆ alkoxy. The number of the substituents which may be on the Ce-io aryl or the 5- to 10-membered heteroaryl is preferably 1-3, more preferably 1-2, and even more preferably 1. [0048]

In the present compounds of formulae (1) - (5), preferred A, A' , R¹, R², R²', R³, X, Y, 2, R⁴, R⁵, R⁶, m, n, o, and p are shown below, but the technical scope of the present invention is not limited to the scope of compounds listed below. [0049]

A includes preferably hydrogen, hydroxy, and -{CHzïmCOOH, more preferably - (CH2) m-COOH, and even more preferably -COOH.

[0050]

A^r includes preferably hydrogen, hydroxy, and -(CHaimCOOH, more preferably hydroxy. [0051]

The combination of A and A ' includes preferably a combination of A and A' wherein at least one of A and A' is - (CH2) m-COOH. More preferably, the combination includes a combination wherein A is - (CH₂ ) m-COOH, and A' is hydroxy. Even more preferably, the combination includes a combination wherein A is -COOH, and A' is hydroxy. [0052]

R¹ includes preferably -C(O) (CH2)_n-X and -CH2-(CH2 ) n~X, more preferably -C(O) (CH₂)_n-X. [0053]

R² includes preferably -C (O) (CH2) _O-Y and -CH2- (CH₂) _O-Y, more preferably -C(0) (CH2)o-Y. [0054]

R²’ includes preferably -C (O) (CH₂ ) 0-CH3. [0055]

R³ includes preferably -C (O) (CH₂) _P~Z and -CH2- (CH₂) _P-Z, more preferably -C(0)(CHa)p-Z. [0056]

X includes preferably methyl, Ce-io aryl (said Ce-io aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-s alkoxy), and 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1-4 substituents selected independently from hydroxy, Ci-₆ alkyl, halogen, cyano, and Ci-₆ alkoxy), more preferably methyl and Ce-ίο aryl, more preferably methyl and phenyl, and even more preferably phenyl.

[0057]

Y includes preferably methyl, Ce-io aryl (said Ce-ίο aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-e alkyl, halogen, cyano, and Ci-6 alkoxy), and 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be subst ituted with 1 — 4 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Cj-₆ alkoxy), more preferably methyl and Ce-io aryl, even more preferably methyl and phenyl, and even much more preferably methyl.

[0058]

Z includes preferably methyl, Ce-io aryl (said Ce-io aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and

Ci-6 alkoxy), and 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1 - 4 substituents selected independently from hydroxy, Ci-e alkyl, halogen, cyano, and Ci-e alkoxy), more preferably methyl and Cç-io aryl, even more preferably methyl and phenyl, and even much more preferably methyl.

[0059]

The combination of X, Y, and Z includes preferably a combination of X, Y, and Z wherein at least one of X, Y, and Z is Ce-io aryl (said Ce-io aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-6 alkoxy) or 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1 - 4 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-e alkoxy), provided that when A is -COOH and the stereochemistry of * is S-configuration, the combination includes a combination of X, Y, and Z wherein Y is methyl, and at least one of X and Z is Cê-io aryl (said Ce-io aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-β alkoxy) or 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1-4 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-e alkoxy) .

More preferably, the combination of X, Y, and Z includes a combination of X, Y, and Z wherein at least one of X, Y, and Z is C₆-io aryl or 5- to 10-membered heteroaryl, provided that when A is -COOH and the stereochemistry of is S-configuration, the combination includes a combination of X, Y, and Z wherein Y is methyl, and at least one of X and Z is C₆-io aryl or 5- to 10-membered heteroaryl.

Even more preferably, the combination of X, Y, and Z includes a combination of X, Y, and Z wherein at least one of X, Y, and Z is phenyl, provided that when A is -COOH and the stereochemistry of * is S-configuration, the combination includes a combination of X, Y, and Z wherein Y is methyl, and at least one of X and Z is phenyl.

Even much more preferably, the combination of X, Y, and Z includes a combination of X, Y, and Z wherein X is phenyl, and Y and Z are methyl.

[0060]

R⁴, R⁵, and R⁶ include independently, preferably C102O alkyl, more preferably C10-15 alkyl, more preferably C10-12 alkyl, and even more preferably Cu alkyl.

[0061] m includes preferably an integer of 0 - 6, more preferably an integer of 0 - 1, and even more preferably 0. [0062] n, o, and p include independently, preferably an integer of 5 - 20, more preferably an integer of 6 - 10, even more preferably 7-9, and even much more preferably 8. [0063]

Preferred embodiments of the compounds of formula (1) include the following compounds or a pharmaceutically acceptable sait thereof. [0064]

In an embodiment, the présent compound of formula (1) includes the following (A). (A)

A compound of formula (1) or a pharmaceutically acceptable sait thereof, wherein

A is hydrogen, hydroxy, or - (CH₂)_m-COOH;

A' is hydrogen, hydroxy, or -(CH₂)m-COOH;

provided that at least one ofAorA' is-(CH₂) m-COOH ;

R¹ is -C(0) {CH₂)n-X or -CH₂-(CH₂)_n-X;

R² is -C (O) (CH₂)_O-Y or -CH₂- (CH₂ ) _O-Y ;

R³ is -C(O) (CH₂)_P-Z or -CH₂- (CH₂) _P-Z ;

X, Y, and Z are independently methyl, Cê-io aryl (said Ce-io aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-e alkyl, halogen, cyano, and Ci-6 alkoxy), or 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1-4 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-6 alkoxy) ;

provided that at least one of X, Y, or Z is Ce-io aryl (said C₆-in aryl may be substituted with 1-5 substituents selected independently from hydroxy, Ci-₆ alkyl, halogen, cyano, and Ci-₆ alkoxy) or 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1-4 substituents selected independently from hydroxy, Ci-e alkyl, halogen, cyano, and Ci-6 alkoxy), provided that when A is -COOH and the stereochemistry of * is S-configuration, Y is methyl;

R⁴, R⁵, and R⁶ are independently C10-20 alkyl; m is independently an integer of 0 - 6; and n, o, and p are independently an integer of 5 - 20.

[0065]

In an embodiment, the present compound of formula (1) includes the following (B).

(B)

A is -COOH;

A’ is hydroxy;

Ri is -C(0) (ΟΗ_Ξ)_η-Χ;

R² is -C (O) (CH₂) o~Y;

R³ is -C(0) (CH₂)p-Z;

X, Y, and Z are independently methyl, Ce-io aryl, or 5to 10-membered heteroaryl;

provided that at least one of X, Y, or Z is Ce-io aryl or 5- to 10-membered heteroaryl, provided that when the stereochemistry of * is Sconfiguration, Y is methyl;

R⁴, R⁵, and R⁶ are independently C10-12 alkyl; and n, o, and p are independently an integer of 6 - 10. [0066]

In an embodiment, the present compound of formula (1) includes the following (C).

(C)

A is -COOH;

A' is hydroxy;

Ri is -C(O) (CH₂)_n-X;

R² is -C (O) (CH₂) o-Y;

R³ is -C (0) (CH₂)p-Z;

X, Y, and Z are independently methyl, Ce-10 aryl, or 5to 10-membered heteroaryl;

provided that at least one of X, Y, or Z is Ce-io aryl or 5- to 10-membered heteroaryl, provided that when the stereochemistry of * is Sconfiguration, Y is methyl, R⁴, R⁶, and R⁶ are independently undecyl; and n, o, and p are independently an integer of 6 - 10. [0067]

In an embodiment, the present compound of formula (1) includes the following (D) .

(D)

A is -COOH;

A' is hydroxy;

Ri is -C(0) (CH₂)n-X;

R2 is -C(0) (CH₂)o-Y;

r³ is -C(0) (CH₂)p-Z;

X, Y, and Z are independently methyl, Cê-io aryl, or Βίο 10-membered heteroaryl;

provided that at least one of X, Y, or Z is Cg-io aryl and 5- to 10-membered heteroaryl, provided that when the stereochemistry of * is Ξconfiguration, Y is methyl;

R⁴, R⁵, and R⁶ are independently undecyl; and n, o, and p are independently an integer of 7 - 9. [0068]

In an embodiment, the compound of formula (1) includes the following compound group:

₍₂J?) -2-{ [ ( 3R) -3- (decanoyloxy) tetradecanoyl]amino]-3{[3—{[ ( 3R)“3^_(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4-({(3R)-3-[(925 phenyInonanoyl)oxy]tetradecanoyl}oxy)oxan 2 yl]oxy}propanoic acid (Example 1);

(2S)-2-{[(32?)-3-(decanoyloxy)tetradecanoyl]amino} -3{[3—{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4- ( {(37?)-3-[(9phenylnonanoyl)oxy]tetradecanoyl}oxy)oxan-2yl]oxylpropanoic acid (Example 2);

(2 R}-2-{[(3^)-3-(decanoyloxy)tetradecanoyl]amino}-3{ [ 4 - { [ (37?)-3-(decanoyloxy)tetradecanoyl]oxy}-5-hydroxy-6(hydroxymethyl)-3-({( 32?) -3-[ ( 9phenylnonanoyl)oxy]tetradecanoyl)amino)oxan-2yl]oxy}propanoic acid (Example 3);

(2R)-2-({ [(3^)-3-(9phenylnonanoyl ) oxy] tetradecanoyl} amino) -3-{ [3 - { [ ( 32?) -3(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4- ( { (37?) -3(decanoyloxy)tetradecanoyl}oxy)oxan-2-yl]oxy}propanoic acid (Example 4); and (2S)-2-({[(32?)-3-(9phenylnonanoyl ) oxy] tetradecanoyl}amino ) -3 - { [ 3 - { [ ( 37?) -3(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4-( { (37?) -3(decanoyloxy)tetradecanoyl}oxy)oxan-2-yl]oxy}propanoic acid (Example 5).

[0069]

Hereinafter, the processes to préparé the compound of the present invention are explained, but the processes of the present invention should not be limited thereto.

The present compound of formula (1) can be prepared, for example, according to Processes 1 to 3 described below.

[0070]

Process 1

The compound of formula (1) can be prepared, for example, by the following process.

Step 1-1

Condensation reaction

Deprotection

Step 1-2

wherein R¹, R², R³, R⁴, R⁵, R⁶, A, A' and m are as defined in Item 1; PA and PA¹ are independently hydrogen hydroxy, group.

[0071]

O-PG⁴, or (CH₂)m-C (O) O-PG⁵; PG¹ and PG² are independently an amino-protecting group; PG³ and PG⁴ are independently a hydroxy-protecting group; and PG⁵ is a carboxyl-protecting

The protecting groups represented by the above PG¹, PG², PG³, PG⁴, and PG⁵ may include protecting groups described in Protective Groups in Organic Synthesis (edited by Theodora W. Greene, Peter G. M. Wuts, issued by John Wiley & Sons, Inc., 1999). Compound al can be prepared, for example, by the process described in WO 98/50399. Compounds a2, a5, and a8 can be prepared by Processes 2 and 3 which are described later. [0072] (Step 1-1)

Compound a3 can be prepared by reacting Compound al with Compound a2 in a suitable solvent in the presence of a suitable condensation agent. The condensation agent used herein may be selected from the exemplified condensation agents listed later, which includes preferably carbodiimides, more preferably 1-[3—(dimethylamino)propyl] 3-ethylcarbodiimide methiodide. Preferably, the condensation reaction may be do ne along with N,N-dimethyl 4-aminopyridine (DMAP) or 4-pyrrolidinopyridine which is a promoter in condensation reaction. The solvent used herein may be selected from the exemplified solvents listed later, which includes preferably chloroform and dichloromethane. The reaction time is generally 5 minutes to 48 hours, preferably 1 to 24 hours. The reaction température is generally -78°C to 100°C, preferably 0°C to 5Û°C.

[0073] (Step 1-2)

Compound ai can be prepared by deprotecting the aminoprotecting group PG¹ of Compound a3. The step can be done according to a known method described in Protective Groups in Organic Synthesis (edited by Theodora W. Greene, Peter G. M. Wuts, issued by John Wiley & Sons, Inc., 1999) and the like. [0074] (Step 1-3)

Compound a6 can be prepared by reacting Compound a4 with Compound a5 in a suitable solvent in the presence of a suitable condensation agent. The condensation agent used herein may be selected from the exemplified reagents listed later, which includes preferably l-ethoxycarbonyl-2-ethoxy1,2-dihydroquinoline. The solvent used herein may be selected from the exemplified solvents listed later, which includes preferably chloroform and dichloromethane. The reaction time is générally 5 minutes to 48 hours, preferably 1 to 24 hours. The reaction température is generally -78°C to 100°C, preferably 0°C to 50°C. [0075] (Step 1-4)

Compound a7 can be prepared by deprotecting the aminoprotecting group PG² of Compound a6. The step can be done according to a known method described in Protective Groups in Organic Synthesis (edited by Theodora W. Greene, Peter G. M. Buts, issued by John Wiley & Sons, Inc. , 1999) and the like.

[0076] (Step 1-5)

Compound a9 can be prepared by reacting Compound al with Compound a8 by the method according to the above Step 1-3. [0077] (Step 1-6)

The compound of formula (1) can be prepared by deprotecting the protecting group PG³ in Compound a9, and by deprotecting PG⁴ and/or PG⁵ when PA and PA' hâve O-PG⁴and/or C(O)OPG⁵. The step can be done according to a known method described in Protective Groups in Organic Synthesis (edited by Theodora W. Greene, Peter G. M. Wuts, issued by John Wiley & Sons, Inc., 1999) and the like. [0078]

The above condensation reactions with Compounds a2, a5, and a8 may be done simultaneously or in a different order, depending on the substituents. For example, when R²and R³ are the same and R⁵ and R^e are the same, Compound a9 can be prepared from Compound a3 in two steps of Step 1-2 and

Step 1-3 as shown below, by using the same protecting group in PG¹ and PG².

a9

[0079]

Process 2

Compound a2 drawn in Process 1 wherein R¹ in formula (1) is -C (0) (CH₂)n-X can be prepared, for example, by the following process. Compound a5 wherein R² is —C(0)(CH2)O-Y and Compound a8 wherein R³ is -C(0) (CH2)_P-Z can be also prepared in the same process.

Nucleophilic substitution reaction

Step 2-1

b2 k ' . ^r b3

Wittig reaction

Step 2-2

Réduction o reaction X

Step 2-3 b5

Condenstaion reaction

Step 2-4

O

Deprotection O

Step 2-5 a2 and n are as defined in Item 1; j is an integer of 0 - 18; k is n-j-2; and PG⁶ is a carboxylprotecting group. [0080]

Step 2-1 - Step 2-3 are, for example, a known method according to the process described in US2008/0188566. And, Step 2-4 and Step 2-5 are, for example, a known method according to the process described in WO2004/062599.

(1) is -CH₂- (CH₂) _n~X can be prepared, for example, by the following process. Compound a5 wherein R² is CH₂(CH₂)o Y

[0081]

Process 3

Compound a2 drawn in Process 1 wherein R¹ in formula and Compound a8 wherein R³ is -CH₂(CH₂)_P-Z can be also prepared in the same process.

Réduction reaction

Step 3-1

Nucleophilic substitution reaction

Step 3-2 b6 c1 c2

OH O r4'^^xxOPG⁶c4

Condensation reaction

Step 3-3

Deprotection

Step 3-4

wherein X, R⁴, and n are as defined in Item 1; PG⁶ is a carboxyl-protecting group.

[0082]

Step 3-1 is a réduction reaction from carboxylic acid to alcohol, which can be dons, for example, according to the process disclosed in Sériés of Experimental Chemistry 10 5th édition, Vol. 14, pli- 16 (Jikken Kagaku Kouza, edited by the Chemical Society of Japan, 2005). And, Step 3-2 Step 3-4 are, for example, a known method according to the process described in WO 01/36433. [0083]

Compound a2 can be also prepared by the method described in Bioorg Med Chem Lett. 2015 Feb 1; 25(3): 54721080

53. [0084]

The base used in each step of the above processes should be suitably selected based on the reaction, the start ing compound, e t c. , which includes alkaline bicarbonates such as sodium bicarbonate, and potassium bicarbonate; alkaline carbonate such as sodium carbonate, and potassium carbonate; metallic hydrides such as sodium hydride, and potassium hydride; alkaline métal hydroxides such as sodium hydroxide, and potassium hydroxide; alkaline métal alkoxides such as sodium methoxide, and sodium tbutoxide; organic meta! bases such as butyllithium, and lithium diisopropylamide; and organic bases such as triethylamine, diisopropylethylamine, pyridine, 4dimethylaminopyridine (DMA?), and 1,8diazabicyclo[5.4.0]undec-7-ene (DBU). [0085]

The condensation agent should be suitably selected depending on starting compounds, etc., which includes, for example, phosphates such as diethyl cyanophosphate and diphenylphosphoryl azide; carbodiimides such as l-ethyl·^-3( 3-dimethylaminopropyl)~carbodiimide hydrochloride (WSC’HCl) and dicyclohexylcarbodiimide (DCC), and 1- [3(dimethylamino)propyl]-3-ethylcarbodiimide methiodide, combinations of a disulfïde such as 2,2’-dipyridyldisulfide and a phosphine such as triphenylphosphine; phosphorus halides such as N,N'-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOPC1); combinations of an azodicarboxylate diester such as diethyl azodicarboxylate and a phosphine such as triphenylphosphine; 2-halo-l-lower alkylpyridinium halides such as 2-chloro-l-methylpyridinium iodide; 1,1'carbonyldiimidazole (CDI); l-ethoxycarbonyl-2-ethoxy-l,2dihydroquinoline (EEDQ); diphenylphosphoryl azide (DPPA); diethylphosphoryl cyanide (DEPC); tetrafluoroborates such as 2- (IH-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TETU) and 2-chloro-l,3dimethylimidazolidinium tetrafluoroborate (CIE); phosphates such as 2-( IH-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTÜ), benzotriazol-1yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-l-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PYBOP), and 2-(7-aza-lH-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU). [0086]

The solvent used in each step of the above processes should be suitably selected based on the reaction, the starting compound, etc., which includes, for example, alcohol solvents such as methanol, éthanol, and isopropanol; ketone solvents such as acetone and methylketone; halogenated hydrocarbon solvents such as methylene chloride and chloroform; ether solvents such as tetrahydrofuran (THF) and dioxane; aromatic hydrocarbon solvents such as toluene and benzene; aliphatic hydrocarbon solvents such as hexane and heptane; ester solvents such as ethyl acetate and propyl acetate; amide solvents such as N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone;

sulfoxide solvents such as dimethylsulfoxide (DMSO); nitrile solvents such as acetonitrîle. The solvent used herein may be one of these solvent s or a mixture of two or more solvents selected from these solvents. And, if possible in the reaction, an organic base or an organic acid may be used as a solvent used herein. [0087]

In addition, each intermedîate or each final product în the above préparation processes can be also transformed to another compound of the present invention by suitably modifying its functional group, especially extending various side^-chains from amino, hydroxy, carbonyl, halogen, etc.; and optionally making the above-mentioned protection and deprotection if necessary. The modification of functional group and the extension of s ide-chain can be done by a conventional method (for example, see Comprehensive Organic Transformations, R. C. Larock, John

Wiley & Sons Inc. (1999), etc.).

[0088]

The pharmaceutically acceptable sait includes an acid addition sait and a base addition sait. For example, the acid addition sait includes an inorganic acid sait such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate; and an organic acid sait such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, and camphorsulfonate; and the base addition sait includes an inorganic base sait such as sodium sait, potassium sait, calcium sait, magnésium sait, barium sait, and aluminium sait ; and an organic base sait such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tertbutylamine, cyclohexylamine, dicyclohexylamine, and N,Ndibenzylethylamine. Furthermore, it includes a basic or acidic amino acid sait such as arginine, lysine, ornithine, aspartate, and glutamate.

The suitabie salts of starting compounds or desired compounds, and the pharmaceutically acceptable salts are conventional non-toxic salts, which include an acid addition sait such as an organic acid sait (e.g. acetate, trifluoroacetate, maleate, fumarate, citrate, tartrate, methanesulfonate, benzenesulfonate, formate, para toluenesulfonate, etc.) and an Inorganic acid sait (e.g. hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, etc.); a sait with an amino acid (e.g. arginine, aspartate, glutamate, etc.); a metallic sait such as an alkaline métal sait (e.g. sodium sait, potassium sait, etc.) and an alkaline-earth métal sait (e.g. calcium sait, magnésium sait, etc.); ammonium sait; and an organic base sait (e.g. trimethylamine sait, triethylamine sait, pyridine sait, picoline sait, dicyclohexylamine sait, N,N'dibenzylethylenediamine sait, etc.); and furthermore, what a skilled person selects suitably.

[0089]

If it is désirable to fix the compound of the présent invention as a sait, when the compound of the present invention is obtained as a sait, it may be purified without further réaction, and when it is obtained in a free form, it may be dissolved or suspended in an approprîate organic solvent and an acid or base may be added therein to form a sait in a general manner.

The compound of the present invention or a pharmaceutically acceptable sait thereof may sometimes exist in form of solvaté with water or various solvents. Such solvatés are also included in the present invention.

[0090]

The température for forming a sait is selected from the range of generally -50°C to boiling point of a solvent used herein, preferably 0°C to the boiling point, and more preferably room température to the boiling point. In order to enhance the optical purity, it is désirable to make the température raised to around boiling point of a solvent used herein. In collecting a precipitated crystal on a filter, an optîonal cooling can make the yield increased. The amount of an optically active acid or amine used herein is suitably about 0.5 - about 2.0 équivalents against that of the substance compound, preferably around one équivalent. If appropriate, the obtained crystal may be recrystallized in an inert solvent (for example, an alcohol solvent such as methanol, éthanol, and 2-propane1 ; an ether solvent such as diethyl ether; an ester solvent such as ethyl acetate; a hydrocarbon solvent such as toluene; an aprotic solvent such as acetonitrile; or a mixed solvent thereof) to obtain its highly pure sait thereof. And, if appropriate, the optically-resolved sait can be also treated with an acid or a base to obtain its free form. [0091]

The compound of formula (1) in which any one or more ^ΤΗ atoms are replaced by ²H(D) atoms is also within the scope of the present invention of formula (1).

[0092]

The present invention .encompasses the compound of formula (1) or a pharmaceutically acceptable sait thereof. In addition, the present invention encompasses a hydrate thereof and a solvaté thereof such as ethanolate thereof. Furthermore, the present invention encompasses ail tautomers, stereoisomers, and crystal forms thereof.

[0093]

The present compound (1) also includes an optical isomer which is based on chiral center, an atropisomer which is based on axiality caused by intramolecular rotational hindrance or planar-chirality, other stereoisomers, tautomer, and géométrie isomer, ail possible isomers of which and a mixture thereof are encompassed in the present invention.

[0094]

The optical isomer mixture of the present compounds can be prepared in a conventions1 manner. The compounds having an asymmetric structure can be prepared, for example, by using a starting material having an asymmetric center or by introducing an asymmetric structure anywhere along the process. For example, in case of optical isorners, optical isomers can be obtained by using an optically active starting material or resolving a mixture of optical isomers at an appropriate step. In case that the compound of formula or its intermediate has a basic functional group, the optical resolution thereof includes, for example, diastereomer method, wherein the compound is transformed to a sait thereof by reacting with an optically active acid (for example, a monocarboxylic acid such as mandelic acid, N-benzyloxyalanine, and lactic acid; dicarboxylic acid such as tartaric acid, o-diisopropylidene-tartaric acid, and malic acid; or a sulfonic acid such as camphorsulfonic acid and bromocamphorsulfonic acid), in an inert solvent (for example, an alcohols such as methanol, éthanol, and 2propanol; an ether solvent such as diethyl ether; an ester solvent such as ethyl acetate; a hydrocarbon solvent such as toluene; an aprotic solvent such as acetonitrîle; or a mixed solvent thereof ) . In case that the compound of formula (1) or its intermedîate has an acidic functional group such as carboxyl group, the compound can be also optically resolved after forming its sait with an optically active amine (for example, an organic amine such as 1phenylethylamine, kinin, quinidine, cinchonidine, cinchonine, and strychnine). [0095]

The present compounds of formula (1) and their intermediates can be isolated and purified in a manner known by a skilled person. It includes, for example, extraction, partition, reprecipitation, column chromatography (e.g. silica gel column chromatography, ion exchange column chromatography, and préparative liquid chromatography), and recrystallization.

The solvent for recrystallization used herein includes, for example, an alcohols solvent such as me thanoï, éthanol, and 2-propanol; an ether solvent such as diethyl ether; an ester solvent such as ethyl acetate; an aromat ic hydrocarbon solvent such as benzene and toluene; a ketone solvent such as acetone; a halogenated solvent such as dichloromethane and chloroform; a hydrocarbon solvent such as hexane; an aprotic solvent such as dimethylformamide and acetonitrile; water; and a mixed solvent thereof. As other methods for purification, for example, methods described in Sériés of Experimental Chemistry (Jikken Kagaku Kouza, edited by the Chemical Society of Japan, Maruzen) Vol. 1 can be used. And, the structural détermination of the present compounds can be easily done by spectroscopic analytical method such as nuclear magnetic résonance method, infrared absorption technigue, and circuler dichroism spectra analysis, and mass spectrometry, considering the structure of each starting compound. [0096]

Among the starting materials and the intermediates in each préparation process mentioned above, the compounds that are not described in each process are commercially available or can be prepared by a skilled person with a commercially available material in a known manner or a similar manner thereto.

[0097]

The present invention provides the above-defined compound of formula (1) or a pharmaceutically acceptable sait thereof which is useful as vaccine adjuvant, preferably vaccine adjuvant for infection vaccine. [0098]

In addition, the present invention provides a pharmaceutical composition comprising the above-defined compound of formula (1) or a pharmaceutically acceptable sait thereof in combination with a pharmaceutically acceptable diluent or carrier (hereinafter, referred to as the present pharmaceutical composition). [0099]

The present compound or a pharmaceutically acceptable sait thereof may be used as an adjuvant for maintaining or enhancing the immunostimulatory of an active ingrédient having an immunostimulating activity.

Namely, the present compound or a pharmaceutically acceptable sait thereof has an activity for inducing or enhancing antigen-specific antibody, specifically antigenspecific IgG, and in more detail Thl-type antigen-specific IgG (e.g. IgG2c).

And, the present compound or a pharmaceutically acceptable sait thereof has an activity for increasing cytotoxic T-lymphocyte (CTL). Or, the present compound or a pharmaceutically acceptable sait thereof has an activity for inducing CTL in mammal or enhancing the CTL induction in mammal.

And, the present compound or a pharmaceutically acceptable sait thereof has an activity for enhancing CD4positive (i.e., MHC class II-restricted) and/or CD8positive (i.e., MHC Class I-restricted) T-cell.

And, the present compound or a pharmaceutically acceptable sait thereof has an activity for increasing antigen-specific T-cell.

And, the present compound or a pharmaceutically acceptable sait thereof has an activity for increasing memory T-cell, specifically, CD8-positive effector memory T-cell.

And, the present compound or a pharmaceutically acceptable sait thereof has a character that the action of increasing CTL is almost the same level as the case that the same moles of an adj uvant having phosphate st ructure which has TLR4 agonistic action when administered to mammal.

And, the present compound or a pharmaceutically acceptable sait thereof has an activity for activating immunocompétent cells.

[0100]

The pharmaceutical composition of the present invention may comprise an antigen, said antigen includes a tumor antigen or a pathogen-derived antigen. The tumor antigen includes, for example, a tumor antigen protein and a partial peptide derived from its tumor antigen protein. And, a complex of the antigen and carrier, etc. is included in the scope of the antigen in the present invention. And, the pathogen-derived antigen includes, for example, a pathogen (such as virus and bacterium) antigen protein and a partial peptide derived from its pathogen antigen protein. And, a complex of the antigen and carrier, etc. is included in the scope of the antigen in the present invention. The complex includes an antigen (including protein and peptide, but not limited thereto) bridged to a protein which is a carrier via a linker which is well known by a skilled person, and an antigen contained in virus-like particle (VLP) . Thus, the present compound or a pharmaceutically acceptable sait thereof is useful as a médicament for treating or preventing infection of virus or bacterium or cancer by using in combination with the above-mentioned antigen. And, the present compound or a pharmaceutically acceptable sait thereof is useful as an adjuvant for activating the therapeutic or préventive effect for infection of virus or bacterium or cancer by using in combination with the above-mentioned antigen.

[0101]

Examples of the administration route of the pharmaceutical composition of the présent invention includes parenteral administration, specifically int ravascular (e.g., intravenous), subcutaneous, intradermal, intramuscular, transnasal, lymph node, and transdermal administrations. [0102]

In one embodiment, the pharmaceutical composition of the présent invention may comprise the compound of formula (1) or a pharmaceutically acceptable sait thereof and a pharmaceutically acceptable diluent or carrier. [0103]

The drug formulation of the présent pharmaceutical composition includes a liquid formulation. [0104]

The liquid formulation of the présent invention includes an aqueous solution formulation/an aqueous suspension formulation, an oily solution formulation/an oily suspension formulation, a lipid formulation, and an émulsion formulation.

The aqueous solution formulation or the aqueous suspension formulation includes, for example, a formulation prepared by dissolving or dispersing an antigen (tumor antigen or pathogen-derived antigen), and/or the compound of formula (1) or a pharmaceutically acceptable sait thereof in water.

The oily solution formulation or the oily suspension formulation includes, for example, a formulation prepared by dissolving or dispersing an antigen (tumor antigen or pathogen-derived antigen), and/or the compound of formula (1) or a pharmaceutically acceptable sait thereof in an oily ingrédient.

The lipid formulation includes, for example, a liposome formulation comprising an antigen (tumor antigen or pathogen-derived antigen), and/or the compound of formula (1) or a pharmaceutically acceptable sait thereof.

The émulsion formulation includes, for example, a formulation including an aqueous solution and an oily composition, which comprises an antigen (tumor antigen or pathogen-derived antigen) , and/or the compound of formula (1) or a pharmaceutically acceptable sait thereof.

The additive used in the present aqueous solution formulation or aqueous suspension formulation includes, for example, purified water, water for injection, a buffering agent, a pH adjusting agent, a stabiliser, an isotonizing agent, a solubilizer, and a solubilizing agent.

The additive used in the present oily solution formulation or oily suspension formulation includes, for example, a buffering agent, a pH adjusting agent, a stabilizer, an isotonizing agent, animal or vegetable oil and fat, hydrocarbons, a fatty acid, fatty acid esters, a solubilizer, and a solubilizing agent.

[0105]

The present émulsion formulation used herein includes oil-in-water émulsion (also refered to as 0/W émulsion), water-in-oil émulsion (also refered to as W/0 émulsion), water-in-oil-in-water émulsion (also refered to as W/O/W émulsion), and oil-in-water-in-oil émulsion (also refered to as 0/W/0 émulsion). The present émulsion formulation includes, preferably water-in-oil émulsion (W/O émulsion) and oil-in-water émulsion (0/W émulsion), and more preferably oil-in-water émulsion (O/W émulsion).

The present water-in-oil émulsion formulation can be prepared by emulsifying an agueous phase and an oil phase in a general manner. As for the present water-in-oil émulsion formulation, an antigen (tumor antigen or pathogen-derived antigen), and/or the compound of formula (1) or a pharmaceutically acceptable sait thereof may be contained in an oil phase and/or an aqueous phase.

[0106]

The present oil-in-water émulsion formulation can be prepared by emulsifying an aqueous phase and an oil phase in a general manner. As for the present oil-in-water émulsion formulation, an antigen (tumor antigen or pathogen-derived antigen) , and/or the compound of formula (1) or a pharmaceutically acceptable sait thereof may be contained in an oil phase and/or an aqueous phase. [0107]

In the liposome formulation of the present invention, the liposome means a microvesicle composed of lipid multiple layers such as bilayer membrane of amphiphilic lipid molécule (lipid bilayer), which has an internai phase. The preferred lipid multiple layer is lipid bilayer. [0108]

The present liposome formulation includes amphiphilic lipid molécule. The amphiphilic lipid molécule includes, preferably one or more phospholipid. The phospholipid includes, for example, phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin. The phospholipid includes, preferably phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine. The phospholipid includes, more preferably phosphatidylcholine, sphingomyelin, phosphatidylserine, and phosphatidylglycerol. [0109]

The liposome internally-including the present compound may contain sterols. The sterols includes cholestérol, β~ sitosterol, stigmasterol, campesterol, brassicasterol, ergosterol, and fucosterol, and preferably cholestérol. [0110]

The liposome internally-including the present compound may contain a pharmaceutically acceptable additive. The additive includes, for example, an înorganic acid, an înorganic acid sait, an organic acid, an organic acid sait, sugars, a buffering agent, an antioxidant, and polymers. The Inorganic acid includes, for example, phosphoric acid, hydrochloric acid, and sulfuric acid. The inorganic acid sait includes, for example, disodium hydrogen phosphate, sodium chloride, ammonium sulfate, and magnésium sulfate.

The organic acid includes, for example, citric acid, acetic acid, succinic acid, and tartaric acid. The organic acid sait includes, for example, sodium citrate, sodium acetate, disodium succinate, and sodium tartrate. The sugar includes, for example, glucose, sucrose, mannitol, sorbitol, and trehalose. The buffering agent includes, for example, p _ arginine, L—histidine, t rometamo1 (trishydroxymethylaminomethane, Tris), and a sait thereof. The antioxidant includes, for example, sodium sulfite, Lcysteine, sodium thioglycolate, sodium thiosulfate, ascorbic acid, and tocopherol. The polymers includes, for example, polyvinyl alcohol, polyvinylpyrrolidone, carboxy vinyl polymer, and carboxymethylcellulose sodium.

[OUI]

The compound of formula (1), or a pharmaceutically acceptable sait thereof, or a pharmaceutical composition of the present invention may be used in combination with further another médicament besides the above pathogenderived antigen. [0112]

The pharmaceutical composition of the present invention may further contain other additives, and examples of such additives include surfactant, antioxidants, preservatives, and soothing agents. [0113]

The compound of formula (1) or a pharmaceutically acceptable sait thereof may be administered simultaneously with the antigenic substance (immunogen) or at any interval before or after the administration of the antigenic substance in a unit dose ranging from generally about 0.1 ng/kg to 100 mg/kg to warm-blooded animal, which provides an effective dose for vaccine adjuvant. The unit dosage form for injections generally contains, for example, 1 ng to 250 mg of the active ingrédient, and preferably, used at a dose ranging from 1 ng to 50 mg/kg of the active ingrédient per day. However, the daily dose may vary depending on the host to be treated, the route of administration and the severity of the disease being treated. Thus, the optimal dose can be determined by a practitioner who treats individual patient or warm-blooded animal.

[0114]

The term treatment as used herein means aileviating some or ail of the symptoms of disease, in whole or in part, or preventing or delaying the progression of disease. [0115]

The term prévention as used herein means primary prévention of disease (prévention of onset of disease) or secondary prévention of disease (prévention of relapse in a patient whose symptom has been alleviated or disease has been cured after the onset of the disease, prévention of récurrence) . Vihen there are infection stage and onset stage to pathogens (such as bacteria, fungi, protozoa, and viruses) in an infections disease, prévention includes both prévention of pathogen infection and prévention of post-infection onset. In addition, the prévention used herein includes the meaning of preventing transmission of a pathogen from humans to other vector organisms. [0116]

Since the compound of the present invention or a pharmaceutically acceptable sait thereof has an immune adjuvant activity in vitro or in vivo _f it is useful as a adjuvant for maintaining or enhancing the immunogenicity of tumor antigen or pathogen-derived antigen. [0117]

The compound of the present invention or a pharmaceutically acceptable sait thereof has an adjuvant activity for cellular immunity in vitro or in vivo, and thus it is useful as a vaccine adjuvant for maintaining or enhancing the immunogenicity of tumor antigen or pathogenderived antigen.

[0118]

The compound of the present invention or a pharmaceutically acceptable sait thereof can be used for maintaining or enhancing the effect of an immunostimulant for treating or preventing a disease, that is a substance inducing tumor antigen-specific immune reaction or pathogen-derived antigen-specific immune reaction.

The pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable sait thereof, and a substance enhancing the spécifie immune response for tumor or pathogen (also referred to as tumor antigen-derived antigen or pathogen-derived antigen, respectively) is also included in one embodiment of the present invention. The tumor antigen or pathogen-derived antigen includes, but not limited to, a tumor-derived antigen protein, a pathogen-derived antigen protein, a tumor-derived antigen peptide derived from said tumor21080 derived antigen protein, a pathogen-derived antigen peptide (partial peptide) derived from said pathogen-derived antigen protein, and a complex thereof with a carrier.

[0119]

In a spécifie embodiment of the present invention, the present compound or a pharmaceutically acceptable sait thereof can treat or prevent infection by the administration together with a pathogen-derived antigen protein or a pathogen-derived antigen peptide for preventing infection. And, the effect of the present compound or a pharmaceutically acceptable sait thereof for treating or preventing infection can be enhanced by the administration in combination with a tumor antigen protein or a tumor antigen peptide for treating or preventing infection. The preventable infectious disease includes, for example, virus diseases such as génital wart, common wart, plantar wart, hepatitis B, hepatitis C, herpes simplex virus, molluscum contagiosum, smallpox, human immunodeficiency virus (HIV), human papilloma virus (HPV), rs virus, norovirus, cytomégalovirus (CMV), varicella zoster virus (VZV), rhinovirus, adenovirus, coronavirus, influenza, and parainfluenza; bacterial diseases such as tuberculosis, mycobacterium avium, and Hansen's disease; infections such as mycosis, chlamydia, Candida, Aspergillus, cryptococcal meningitis, Pneumocystis carini, cryptosporidiosis, histoplasmosis, toxoplasmosis, malaria, Trypanosoma infection, and leishmaniasis, but should not be limited thereto. Examples of the active ingrédient of the vaccine for preventing infectious include, but not limited to, substances derived from microorganisms/pathogens including bacteria, fungi, protozoa, and viruses which cause infectious diseases, such as antigenic protein, antigen peptide (partial peptide) from said antigenic protein, polysaccharide, lipid, and a combination thereof or a combination of the substance derived from said microorganisms/pathogen and a carrier. [0120]

Examples of the viral antigenic peptide derived from the viral antigen include, but not limited to, influenza matrix protein peptide 58-66 (Jager E et al., Int. J. Cancer 67: 54 (1996)), HPV16 E7 peptide 86-93 (van Driel WJ et al., Eur. J. Cancer 35:946 (1999)), HPV E7 peptide 12-20 (Scheibenbogen C et al., J. Immunother 23: 275 (2000)), HPV16 E7 peptide 11-20 (Smith JWI et al., J. Clin. Oncol. 21: 1562 (2003)), HSV2 gD (Berman PW et al., Science 227: 1490 (1985)), CMV gB (Frey SE et al., Infect Dis. 180: 1700 (1999), Gonczol E. et al., Exp. Opin. Biol. Ther. 1: 401 (2001)), and CMV pp65 (Rosa CL et al., Blood 100: 3681 (2002), Gonczol E. et al., Exp. Opin. Biol. Ther. 1: 401 (2001) ) .

[0121]

In a spécifie embodiment of the present invention, the present compound or a pharmaceutically acceptable sait thereof can treat or prevent cancer by the administration 5 in combination with a tumor antigen protein or a tumor antigen peptide for cancer immunotherapy. And, the effect of the present compound or a pharmaceutically acceptable sait thereof for treating or preventing cancer can be enhanced by the administration in combination with a tumor 10 antigen protein or a tumor antigen peptide for cancer immunotherapy. The cancer includes, for example, leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, 15 bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, brain tumor, bone cancer, pancreatic cancer, head and neck cancer, skin or intraorbital malignant melanoma, rectal cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endométrial 20 carcinoma, uterocervical carcinoma, vaginal carcinoma, vulval carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestinal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, uréthral cancer, pemle cancer, 25 acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic or acute leukemia including chronic lymphocytic leukemia, children solid cancer, lymphocytic lymphoma, renal/ureter cancer, rénal pelvic carcinoma, central nervous System (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, pontine glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, planocellular carcinoma, T —cell lymphoma, polytypic glioblastoma, malignant melanoma, non-small-cell lung cancer, rénal cell cancer, and asbestos-induced cancer. The treatment or prévention of cancer includes preventing metastatic disease and tumor récurrence, and preventing and treating paraneoplastic syndrome. [0122]

The carrier as used herein is a substance, such as protein and lipid, to which an antigenic protein or an antigenic peptide is bound chemically and/or physically, and the examples include, but not limited to, CRM 197 (Vaccine. 2013 Oct 1; 31(42):4827-33), KLH (Cancer Immunol Immunother. 2003 Oct; 52 (10) : 608-16), virus-like particles (PLoS ONE 5(3): e9809) and liposomes (J Liposome Res. 2004; 14 (3-4) :175-89) . [0123]

The antigenic protein may be prepared by cloning cDNA which encodes the antigenic protein, and expressing it in a host cell, according to a textbook such as Molecular

Cloning 2nd ed., Cold Spring Harbor Laboratory Press (1989). [0124]

The synthesis of the antigenîc peptide can be carried out according to a method generally used in peptide chemistry, for example, as described in literatures (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academie Press Inc., New York, 1976). [0125]

In an embodiment, the present invention further provides a kit comprising:

a) a compound of the formula (1) or a pharmaceutically acceptable sait thereof, or a pharmaceutical composition comprising a compound of the formula (1) or a pharmaceutically acceptable sait thereof; and

b) a pharmaceutical composition comprising a tumorderived antigen or a pathogen-derived antigen.

In an embodiment, the present invention further provides a kit comprising:

b) a pharmaceutical composition comprising a pathogenderived antigen.

The antigen is not limited so long as it is an antigen that may be used as an active ingrédient of vaccines, which includes antigenic proteins as mentioned above, antigenic peptides (partial peptides) derived from such antigenic proteins, and a complex thereof with a carrier.

[0126]

In one embodiment of the present invention, the present invention provides use of a compound of the formula (1) or a pharmaceutically acceptable sait thereof in the préparation of a vaccine adjuvant.

Further in one embodiment of the present invention, the present invention provides use of a compound of the formula (1) or a pharmaceutically acceptable sait thereof as a vaccine adjuvant in the préparation of a vaccine for treating infection. [0127]

Further, one embodiment of the present invention provides a method for the treatment or prévention of cancer or infection, or the prévention of the progress thereof, comprising a step of administering a compound of the formula (I) as defined above, or a pharmaceutically acceptable sait thereof, together with a tumor-derived antigen or a pathogen-derived antigen, to a patient.

One embodiment of the present invention provides a method for the treatment or prévention of infection, or the prévention of the progress thereof, comprising a step of administering a compound of the formula (I) as defined above, or a pharmaceutically acceptable sait thereof, together with a pathogen-derived antigen, to a patient. EXAMPLES [0128]

The present invention will be further described with reference to the following examples which should not be regarded as limiting in any respect. [0129] Troc : 2,2,2-trichloroethoxycarbonyl group TES: tert-butyldimethylsilyl group Bn: benzyl group Fmoc: 9-fluorenylmethyloxycarbonyl DBU: diazabicycloundecene Boc: tert-butoxycarbonyl Alko: p-alkoxybenzyl alcohol PEG: polyethylene glycol tBu : tert-butyl HBTU: 0-(benzotriazol-l-yl)~N,N,N', N'-tetramethyluronium hexafluorophosphate DIPEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide TFA: trifluoroacetic acid TIS: triisopropylsilane

THF: tetrahydrofuran

TBDPS: tert-butyldiphenylsilyl group [0130]

The analysis conditions of high performance liquid chromatograph-mass spectrometer (LCMS) are shown below. LCMS Condition A

MS detector: LCMS-IT-TOF

HPLC: Shimadzu Nexera X2 LC 3ÛAD

Column: Kinetex 1.7 μ C18 100A New column 50 χ 2.1 mm

Flow rate: 1.2 ml/min

Wave length: 254/220 nm

Mobile phase : A: 0.1% formic acid/water

B: acetonitrile

Time program:

Step Time (min)

0.01-1.40 A:B = 90:10 - 5:95

1.40-1.60 A:B =5:95

1.61-2.00 A:B = 99:1

[0131]

LCMS Condition B

MS detector: ACQUITY™ SQ detector (Waters)

HPLC: ACQUITY™ system

Column: Waters ACQUITY™ UPLC BEH C18 (1.7 pm, 2.1 mm χ 30 mm)

Flow rate: 0.8 ml/min

Wave length: 254/220 nm

Mobile phase: A: 0.06 % formic acid/acetonitrile

B: 0.06 % formic acid/water

Time program: 0.0-1.30 A:B = 2:98 - 96:4

Column température: 25°C

[0132]

LCMS Condition C

MS detector: LCMS-1T-TOF

HPLC: Shimadzu Nexera X2 LC 30AD

Column: none

Flow rate : 1.2 ml/min

Wave length: 254/220 nm

Mobile phase: A: 0.1 % formic acid/water B : acetonitrile

Time program:

Step Time (min)

0.01-1.40 A:B = 90:10 - 5:95

1.40-1.60 A:B = 5:95

1.61-2.00 A:B = 99:1

[0133]

Reference example 1

2- ( 4-Bromophenyl)-2-oxoethyl (3R)-3-[(9phenylnonanoyl)oxy]tetradecanoate

To a solution of 9-phenylnonanoic acid (398 mg ) in chloroform (5 mL) were added oxalyl dichloride (0.15 mL) and DMF (1 drop), and the solution was stirred at room température for 3 hours. Then, 2-(4-bromophenyl)-2oxoethyl ( 3R)-3-hydroxytetradecanoate (500 mg) which is a known compound and pyridine (2 mL) were added thereto, and the solution was stirred at room température for 18 hours. The reaction solution was extracted with water and ethyl acetate, and the organic layer was dried over anhydrous magnésium sulfate and concentrated. The obtained residue was purified by silica gel column chromatography (eluting solvent; hexane:ethyl acetate) to give the titled compound (676 mg). iH-NMR (400 MHz, CDC1₃) : 7.73 (2H, dt, J = 9.0, 2.1 Hz),

7.60 (2H, dt, J = 9.0, 2.1 Hz), 7.27-7.22 (2H, m), 7.177.12 (3H, m), 5.29-5.23 (3H, m), 2.77-2.66 (2H, m), 2.57 (2H, t, J = 7.9 Hz), 2.28 (2H, t, J = 7.9 Hz), 1.70-1.50 (6H, m) , 1.35-1.20 (26H, m), 0.86 (3H, dd, J = 7.9, 5.5 Hz) . [0134] Reference example 2 ç 3K)— 3—[(9-Phenylnonanoyl)oxy]tetradecanoic acid

To a solution of Reference example 1 (676 mg) in acetic acid (15 mL) was added zinc powder (672 mg), and the mixture was stirred at 60^oC for 4 hours. The reaction jnlxture was filtered with Celite, and the fïltrate was concentrated. The obtained residue was purified by silica gel column chromatography (eluting solvent; chloroform:methanol) to give the titled compound (451 mg). ¹H-NMR (400 MHz, CDCla) : 7.28-7.23 (2H, m) , 7.16-7.11 (3H, m), 5.22-5.15 (IH, m), 2.64-2.52 (4H, m), 2.25 (2H, t, J = 7.6 Hz), 1.60-1.50 (6H, m) , 1.32-1.20 (26H, m) , 0.86 (3H, t, J = 7.0 Hz) . [0135] Reference example 3 (2R)-3-(Benzyloxy)-2-{[(3R) -3(decanoyloxy)tetradecanoyl]amino]-3-oxopropyl 6-0-[tertbuty 1 (dimethyl)silyl]-2-{ [ ( 3R)-3(decanoyloxy)tetradecanoyl]amino}-2-deoxy-3-O-{(3R)-3-[(9phenylnonanoyl)oxy]tetradecanoyl)hexopyranoside

OTBS ^Η$^^ο^^ΟΟΒπ

NHTroc^ NHTroc

[0136]

OTBS

a) Préparation of (22?)-3-(benzyloxy )-2-( { [ ( 9H-f luoren-9yl)methoxy]carbonyl}amino)-3-oxopropyl

6-0- [ tert5 butyl (dimethyl ) silyl]-3-O-[ ( 32? ) -3(decanoyloxy)tetradecanoyl]-2-deoxy-2-{[(2,2,2trichloroethoxy)carbonyl]amino}hexopyranoside (Compound Ql)

Benzyl (2R)-3-{[6-({[tertbutyl (dimethyl)silyl]oxy[methyl)-4,5-dihydroxy-3-{[(2,2,210 trichloroethoxy)carbonyl]amino}oxan-2-yl]oxy}-2-{[(2,2,2trichloroethoxy)carbonyl]amino}propanoate (4 64 mg) ,

Reference example 2 (853 mg), and 4-pyrrolidinopyridine (14 mg) were dissolved in dichloromethane (40 mL), and then 1[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (550 mg) was added to the solution at 0°C. The reaction solution was stirred at room température for 18 hours, and then extracted with water/chloroform. The organic layer was dried over anhydrous magnésium sulfate and concentrated. The obtained residue was purified by silica gel column chromatography (eluting solvent; hexane:ethyl acetate) to give the titled Compound Q1 (1.62 g).

iH-NMR (400 MHz, CDC1₃) : 7.30 (5H, m) , 7.24-7.15 (2H, m) , 7.12-7.06 (3H, m), 5.95 (IH, d, J = 9.8 Hz), 5.26 (IH, d, J = 12.2 Hz), 5.12-5.02 (IH, m), 4.97 (IH, d, J = 11.7 Hz), 4.84-4.60 (5H, m), 4.50-4.40 (2H, m), 4.00-3.87 (2H, m), 3.78 (2H, qd, J = 10.8, 4.6 Hz), 3.60-3.36 (3H, m), 3.203.10 (IH, m), 2.57-2.42 (5H, m), 2.20 (2H, t, J = 7.6 Hz), 1.56-1.40 (6H, m) , 1.39-1.20 (26H, m) , 0.83-0.75 (12H, m), 0.00 (6H, s) .

[0137]

b) Préparation of Reference example 3

Compound Q1 (1.62 g) was dissolved in acetic acid (18 mL) , and zinc powder (2.79 g) was added to the solution. The reaction mixture was stirred at room température for 2 hours, and filtered with Celite. The filtrate was neutralized with sodium hydrogen carbonate and extracted with chloroform. The organic layer was dried over anhydrous magnésium sulfate, filtered, and concentrated. The obtained residue (1.15 g) and (3R)-321080 (decanoyloxy)tetradecanoic acid (1.10 g) were dissolved in dîchloromethane (70 mL), and l-ethoxycarbonyl-2-ethoxy-1,2dihydroquinoline (746 mg) was added to the solution. The reaction solution was stirred at room température for 16 hours. The reaction solution was purified by silica gel column chromatography (eluting solvent; hexane:ethyl acetate) to give the titled compound (1.38 g).

^-NMR (400 MHz, CDC1₃) : 7.35-7.32 (5H, m), 7.21-7.17 (2H, m) , 7.12-7.07 (3H, m), 6.65 (IH, d, J = 8.5 Hz), 5.44 (IH, d, J = 8.5 Hz), 5.22-4.94 (5H, m) , 4.69-4.61 (2H, m), 3.91-

3.73 (5H, m), 3.65 (1H, q, J = 9.5 Hz), 3.49 (IH, t, J =

11.6 Hz), 3.39 (IH, d, J = 2.4 Hz), 3.11-3.06 (IH, m),

2.54-2.16 (14H, m) , 1.51 (14H, s), 1.2Û (86H, m), 0.82-0.78 (24H, m), 0.00 (6H, s). [0138]

Reference example 4 (2R)-3-(Benzyloxy)-3-oxo-2-({(3R)-3-[(9phenylnonanoyl) oxy] tetradecanoyl} amino)propyl 6-0 [ tertbutyl(dimethyl)silyl]-3-O-[(3R)-3(decanoyloxy)tetradecanoyl]-2-{[(3R)-3(decanoyloxy)tetradecanoyl]amino}-2-deoxyhexopyranoside

[0139]

a) Préparation of (2R)-3-(benzyloxy)-2-({[(9V-fluoren-921080 yl)methoxy]carbonyl}amino)-3-oxopropyl 6-0-[tertbutyl (dimethyl)silyl]-3-O-[(3R) -3(decanoyloxy)tetradecanoyl]~2-deoxy-2-{[(2,2,2trichloroethoxy)carbonyl]amino)hexopyranoside (Compound Q2)

The titled compound Q2 was prepared in a similar reaction and treatment to Step a) described in Reference example 3.

LCMS (Condition A): 1271.5 [M+Na⁺] , 1.26 min [0140]

b) Préparation of (2R)-3-(benzyloxy )-2-( { [ ( 9Jf-f luoren-9yl)methoxy]carbonyl}amino)-3-oxopropyl 2-amino-6-O- [tertbutyl (dimethyl)silyl]-3-0-[(3R)-3- (decanoyloxy)tetradecanoyl]-2-deoxyhexopyranoside (Compound Q3)

Compound Q2 (220 mg) was dissolved in acetic acid (2 mL) , and zinc powder (346 mg) was added to the solution. The reaction mixture was stirred at room température for 2.5 hours, and filtered with Celite. The filtrate was extracted with water/ethyl acetate. The organic layer was dried over anhydrous magnésium sulfate and concentrated. The obtained residue was purified by silica gel column chromatography (eluting solvent ; hexane:ethyl acetate) to give the titled Compound Q3 (114 mg).

1H-NMR (CDC13) Ô: 7.73 (2H, d, J = 7.9 Hz), 7.57 (2H, d, J = 6.7 Hz) , 7.38-7.25 (9H, m) , 5.88 (IH, d, J = 8.5 Hz) ,

5.22- 5.08 (3H, m) , 4.73 (IH, t, J = 9.4 Hz), 4.54 (IH, d, J = 8.5 Hz), 4.40 (IH, dd, J = 10.4, 6.7 Hz), 4.29 (IH, dd, J = 10.7, 7.6 Hz), 4.20 (IH, t, J = 7.3 Hz), 4.10-3.95 (3H, m), 3.90-3.74 (2H, m), 3.55 (IH, t, J= 9-1 Hz), 3.40-3.20 (2H, m), 2.71-2.58 (3H, m), 2.25 (2H, t, J = 7.3 Hz), 1.651.30 (6H, m) , 1.30-1.10 (30H, m) , 0.86-0.80 (15H, m) , 0.01 (6H, d, J = 3.0 Hz). [0141]

c) Préparation of (2R)-3-(benzyloxy)-2-({[(9H-tluoren~9 yl)methoxy]carbonyl}amino)~3-oxopropyl 6-O- [tert- butyl (dimethyl)silyl]-3-O-[(3R) -3(decanoyloxy)tetradecanoyl]-2-{[(3R) -3(decanoyloxy)tetradecanoyl]amino}-2-deoxyhexopyranoside (Compound Q4)

Compound Q3 (114 mg) and (3R)-3(decanoyloxy)tetradecanoic acid (46.6 mg) were dissolved in dichloromethane (3 mL) , and l-ethoxycarbonyl-2-ethoxy-l,2dihydroquinoline (31.5 mg) was added to the solution. The reaction solution was stirred at room température for 16 hours. The reaction solution was purified by silica gel column chromatography (eluting solvent; hexane:ethyl acetate) to give the titled Compound Q4 (107 mg).

¹H-NMR (400 MHz, CDCI3) : 7.70 (2H, d, J = 7.9 Hz), 7.59 (2H, d, J = 7.3 Hz), 7.36-7.21 (9H, m) , 5.94 (IH, d, J =

9.1 Hz), 5.49 (IH, d, J = 8.5 Hz)

5.25 ( IH, d, J

12.2

Hz), 5.15-5.01 (3H, m) , 4.74 (IH, t, J = 10.1 Hz),4.43 (IH, d, J = 9.1 Hz), 4.33 (IH, dd, J = 10.4, 7.3 Hz),4.25 (IH, t, J = 8.8 Hz), 4.18 (IH, t, J = 7.3 Hz), 4.03-3.92 (3H, m) , 3.83-3.69 (3H, m) , 3.53 (IH, t, J = 9.1 Hz),3.41 (IH, d, J = 2.4 Hz), 3.18-3.10 (IH, m) , 2.57-2.37 (3H, m), 2.2 6-2.21 (5H, m), 1.60-1.40 (8H, m) , 1.30-1.10 (60H, m) , 0.86-0.79 (21H, m), 0.00 (6H, d, J - 2.4 Hz).

[0142]

d) Préparation of (2R)-2-amino-3-(benzyloxy)-3-oxopropyl 6O— [tert-butyl(dimethyl)silyl]-3-O-[(3R)-3- (decanoyloxy)tetradecanoyl] -2- {[(3R)-3(decanoyloxy)tetradecanoyl]amino}-2-deoxyhexopyranoside (Compound Q5)

To a solution of Compound Q4 (107 mg) in dichloromethane (3 mL) was added a solution of DBU (4.48 mg ) in dichloromethane (0.1 mL) , and the solution was stirred at room température for 30 minutes. Then, the reaction solution was directly loaded on silica gel and purified by silica gel column chromatography (eluting solvent; chloroform:methanol) to give Compound Q5 (89 mg). 1H-NMR (400 MHz, CDC1₃) : 7.34-7.27 (5H, m) , 5.68 (IH, d, J = 9.2 Hz), 5.16 (IH, d, J = 12.2 Hz), 5.10-5.01 (3H, m) , 4.75 (IH, dd, J = 11.0, 9.2 Hz), 4.16 (IH, d, J = 7.9 Hz), 3.98 (IH, dd, J = 9.8, 3.1 Hz), 3.82-3.38 (7H, m), 3.203.15 (IH, m) , 2.54-2.34 (3H, m) , 2.24-2.18 (5H, m) , 1.6021080

1.40 (8H, m) , 1.28-1-10 (60H, m), 0.83-0.76 (21H, m) , 0.00 (6H, d, J = 2.4 Hz) . [0143]

e) Préparation of Reference example 4

To a solution of Reference example Q5 (89 mg) and

Reference example 2 (36.5 mg) in dichloromethane (3 mL) was added l-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (21.4 mg), and the reaction solution was stirred at room température for 18 hours. The reaction solution was .0 concentrated and the residue was purified by silica gel column chromatography (eluting solvent; hexane:ethyl acetate) to give Reference example 4 (97 mg).

iH-NMR (400 MHz, CDCla) : 7.37-7.17 (7H, m) , 7.12-7.07 (3H, m) , 6.68 (IH, d, J = 8.5 Hz), 5.45 (IH, d, J = 8.5 Hz), 15 5.22-4.94 (5H, m) , 4,70-4.60 (2H, m) , 3.90-3.70 (5H, m) ,

3.65 (IH, td, J = 9.3, 7.3 Hz), 3.49 (IH, t, J = 9.5 Hz), 3.41 (IH, d, J = 2.4 Hz), 3.12-3.07 (IH, m), 2.53-2.36 (7H, m), 2.24-2.15 (7H, m) , 1.60-1.40 (14H, m) , 1.30-1,10 (86H, d, J = 9.8 Hz), 0.82-0.78 (24H, m), 0.00 (6H, s).

[0144]

Reference examples 5-8

Each compound shown in Table 1 was prepared from each corresponding starting compound in a similar reaction and treatment to the method described in Reference example 3 or

4.

Table 1
Reference example	Structure	1H-NMR (400 MHz, CDC1₃)
5	OTBS F⁰⁰⁶” fl 1	Ί.30-7.18 (7H, m) , 7.11-7.08 (3H, m) , 6.98 (IH, d, J = 7.9 Hz) , 6.20 (IH, d, J = 7.9 Hz) , 5.09-5.02 (5H, m) , 4.90 (IH, t, J = 9.8 Hz), 4.674.60 (IH, m) , 4.53 (IH, d, J = 8.5 Hz), 4.14 (IH, dd, J = 10.7, 3.4 Hz), 3.85-3.56 (5H, m) , 3.41-3,33 (IH, m) , 3.293.20 (IH, m), 2.62-2.19 (14H, m) , 1.49 (14H, brs), 1.25 (86H, m), 0.82-0,78 (24H, m) , 0.00 (6H, s) .
6	OTBS p ÇOOBn o=< NH	7.4 0-7.31 (7H, m) , 7.10-7.08 (3H, m) , 6.70-6.67 (IH, m) , 5.45 (IH, d, J = 8.4 Hz), 5.21-5.16 (3H, m), 5.10-4.95 (4H, m), 4.69-4.62 (2H, m), 3.91-3.83 (4H, m), 3.80-3.61 (4H, m), 3.54-3.46 (2H, m), 3.12-3.07 (IH, m), 2.56-2.36 (14H, m) , 1.50 (14H, brs), 1.18 (86H, m), 0.86-0.79 (24H, m), 0.00 (6H, s).
7	OTBS o=< NH p-< °=$ °A ° \ / °\ ofl / \°Λ / o< ς Ph Ç ^X X	7.36-7.32 (7H, m) , 7.10-7.08 (3H, m), 6.70-6.67 (IH, m) , 5.49 (IH, d, J = 8.0 Hz), 5.21-4.95 (5H, m), 4.79-4.62 (2H, m) , 3.92-3.61 (6H, m), 3.52-3.43 (2H, m), 3.12-3.08 (IH, m), 2.54-2.36 (8H, m), 2.23-2.10 (6H, m) , 1.52 (14H, brs) , 1.18 (86H, m), 0.85- 0.79 (24H, m), 0.00 (6H, s).

8

OTBS H8^^O^°^OBn°=i NH NH ) O=< O=K °=\ / o-< o< < )^o5 î s X X / >h\

7,30-7.18 (7H, m) , 7.11-7.08 (3H, m), 6.96 (IH, t, J = 11.0 Hz), 6.18 (IH, d, J = 7.9 Hz), 5.12-5.00 (5H, m)_r 4.90 (IH, dd, J = 11.0, 9.1 Hz), 4.66-4.63 (IH, m), 4.53 (IH, d, J = 7.9 Hz), 4.14 (IH, dd, J = 11.0, 3.0 Hz), 3.85-3.56 (5H, m) , 3.40 (IH, s) , 3.29-3.24 (IH, m) , 2.612.19 (14H, m), 1.60-1.42 (14H, m) , 1.30-1.10 (86H, m) , 0.84-0.78 (24H, m) , 0.00 (6H, s) __

[0145]

Example 1 {2R)-2~{[(3F)-3-(Decanoyloxy)tetradecanoyl]amino}-3-{[3{[(3F)-3-(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-65 (hydroxymethyl)-4 - ( ((3R)-3-[ ( 9phenylnonanoyl)oxy]tetradecanoyl}oxy)oxan-2yl]oxy}propanoic acid

OH

Reference example 3 (33 mg) and 10 % palladium carbon (25.9 mg) were added to THF (2 mL) , and the mixture was stirred at room température at one atm under hydrogen atmosphère for 8 hours. The reaction mixture was filtered with Celite, and the filtrate was concentrated. To the residue were added THF (1 mL) and TFA (0.1 mL) , and the mixture was stirred at room température for 3 hours. After the reaction was compteted, the reaction mixture was neutralized with sodium hydrogen carbonate and extracted with chloroform. The organic layer was dried over anhydrous magnésium sulfate, and concentrated. The obtained residue was purified by silica gel column chromatography (eluting solvent; chloroform:methanol) to give the titled compound (5.1 mg).

¹H-NMR (400 MHz, CDCI3/CD3OD ( 30:1 ) ) : 7.19 (2H, d, J = 7.6 Hz), 7.11-7.08 (3 H, m) , 5.18-5.14 (IH, m) , 5.04 (2H,s),

4.83 (IH, t, J = 9.1 Hz), 4.51 (IH, s), 4.32 (IH, d,J =

8.5 Hz), 3.95-3.60 (4H, m), 3.49 (IH, t, J = 9.4 Hz), 3.353.26 (2H, m), 2.54-2.37 (6H, m) , 2.26-2.18 (8H, m) ,1.51 (14H, s), 1.18 (86H, s), 0.81 (15H, t, J = 6.7 Hz).

[0146]

Examples 2-5

Each compound shown in Table 2 was prepared from each corresponding starting compound in a similar reaction and treatment to the method described in Example 1.

Table 2

OH -Ο ΰθθΗ NH NH ^o=^ °Λ o\ / / o=< / o< o\ / ? o< ς ^RÎ x < S U ^x > < ^RS \ _____
Example		R^e	R⁹	Stereochemistry of *	iH-NMR (400 MHz, CDC1₃/CD₃OD (30:1))
2	Ph	Me	Me	S	7.19 (2H, d, J = 6.7 Hz), 7.11-7.08 (3 H, m), 5.195.00 (3H, m), 4.83 (IH, t, J = 9.8 Hz), 4.37 (2H, d, J = 7.9 Hz), 4.07 (IH, d, J = 9.1 Hz), 3.86-3.60 (4H, m), 3.47 (IH, t, J = 9.4 Hz), 3.35-3.26 (2H, m) , 2.57-2.36 (6H, m) , 1.53 (14H, s), 1.19 (86H, s), 0.81 (15H, t, J = 7.2 Hz) . _____________________________
3	Me	Ph	Me	R	7.18 (2H, d, J = 7.2 Hz), 7.10-7.08 (3H, m) , 5.135.06 (3H, m) , 4.89 (IH, t, J = 8.0 Hz), 4.44-4.35 (2H, d, J = 3.6 Hz), 4.29 (IH, s), 3.93 (IH, br), 3.86-3.62 (4H, m) , 3.383.35 (2H, m) , 2.54-2.44 (6H, m), 2.33-2.19 (8H, m), 1.57-1.44 (14H, m), 1.30-1.12 (86H, m) , 0.81 (15H, t, J - 6.7 Hz).
4	Me	Me	Ph	R	7.18 (2H, d, J = 7,3 Hz), 7.11-7.07 (3H, m) , 5.205.00 (3H, m) , 4.86 (IH, t, J = 9.8 Hz), 4.42-4.35

					(2H, m) , 3.93 (IH, d, J = 7.9 Hz), 3.84-3.64 (4H, m), 3.46 (IH, t, J = 9.5 Hz) , 3.34-3.25 (2H, m) , 2.55-2.42 (6H, m) , 2.302.10 (8H, m), 1.58-1.45 (14H, m), 1.28-1.15 (86H, m) , 0.81 (15H, t, J = 6.7 Hz) .
5	Me	Me	Ph	S	7.18 (2H, d, J = 7.3 Hz), 7.11-7.07 (3H, m) , 5.14- 5.02 (3H, m) , 4.85 (IH, t, J = 10.1 Hz), 4.38 (IH, d, J - 8,5 Hz), 4.30 (IH, s), 4.08 (IH, d, J = 11.0 Hz) _f 3.83-3.64 (4H, m) , 3.44 (IH, t, J = 9.5 Hz), 3.34-3.28 (3H, m), 2.50-2.10 (14H, m) , 1.601.40 (14H, m) , 1.28-1.12 (86H, m) , 0.81 (15H, t, J = 7.0 Hz).

[0147]

Reference example 9 (2 R)-2-{ [(32?)-3-(Decanoyloxy)tetradecanoyl]amino}-3-{ [3{[(32?)-3-(decanoyloxy)tetradecanoyl]amino} -65 ( hydroxymethyl) -4 - ( { ( 32?) -3- [ ( 9— phenylnonanoyl)oxy]tetradecanoyl}oxy)-5-(phosphonooxy )oxan2-yl]oxy}propanoic acid

Q6

[0148]

a) Préparation of (2R)-3-(benzyloxy)-2-{[(3R)-3(decanoyloxy)tetradecanoyl]amino}-3-oxopropyl 4-0-

[bis(benzyloxy)phosphoryl]-6-0-[tert-butyl(dimethyl)silyl]2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino}-2-deoxy-3-O{(3P)-3-[(9-phenylnonanoyl)oxy]tetradecanoyl}hexopyranoside (Compound Q6)

To a solution of Reference example 3 (400 mg) in diohloromethane (8 mL) were added dibenzyl N,Ndiisopropylphosphoramidite (231 mg) and 4,5dicyanoimidazole (79 mg) , and the solution was stirred at room température for 18 hours. Then, 3-chloroperoxybenzoic acid (190 mg) was added to the reaction solution, and the mixture was stirred at 0°C for an hour. Aqueous sodium hydrogen carbonate was added to the reaction mixture, and the mixture was stirred at room température for 15 minutes and then extracted with chloroform. The organic layer was dried over anhydrous magnésium sulfate and concentrated. The obtained residue was purified by silica gel column chromatography (eluting solvent; hexane:ethyl acetate) to give Compound Q6 (365 mg) .

¹H-NMR (400 MHz, CDCla) : 7.43-7.24 (17H, m) , 7.19-7.13 (3H,

m) , 6.79 (IH, d, J = 8.2 Hz), 5.81 (IH, d, J = 8.2 Hz),

5.29-5.05 (6H, m) , 4.96 (4H, dd, J = 18.3, 7.8 Hz), 4.7525

4.68 (IH

m) ,

4.31-4.25 (2H, m), 4.00-3.85 (3H, m) ,

3.68 (IH, dd, J = 11.4, 5.5 Hz), 3.55 (IH, dt, J = 14.5, 5.6 Hz), 3.31-3.26 (IH, m), 2.61-2.18 (14H, m), 1.70-1.50 (14H, m), 1.35-1.18 (86H, dd, J = 34.3, 27.4 Hz), 0.88 (15H, td, J = 6.7, 2.4 Hz), 0.85 (9H, s), 0.00 (6H, d, J = 5.5 Hz). [0149]

b) Préparation of Reference example 9

To a solution of Compound Q6 (365 mg) in THF (20 mL) were added 10 % palladium carbon (248 mg) and 2 mol/L hydrochloric acid (0.5 mL), and the mixture was stirred at room température under hydrogen atmosphère ( 4 atm) for 8 hours. The reaction mixture was filtered with Celite, and the fîltrate was concentrated. The obtained residue was purified by silica gel column chromatography (eluting solvent; chloroform:inethanol (the methanol contained 2.5 % water and 2.5 % triethylamine)) to give the titled compound (140 mg).

iH-NMR (400 MHz, CDCI3/CD3OD ( 30 :1 ) ) : 7.19 (2H, d, J = 7.3 Hz), 7.11-7.07 (3H, m), 5.22-4.99 (4H, m), 4.61-4.52 (IH, m) , 4.42-4.32 (IH, m) , 4.21-4.10 (IH, m) , 3.95-3.80 (2H, m) , 3.80-3.60 (2H, m) , 3.34-3.20 (2H, m) , 2.97 (4H, q, J = 7.3 Hz), 2.60-2.30 (6H, m) , 2.30-2.10 (8H, m) , 1.60-1.40 (14H, m), 1.30-1.10 (86H, m), 0.80 (15H, t, J = 6.7 Hz). [0150]

Reference examples 10 - 14

Each compound shown in Table 3 was prepared from each corresponding starting compound in a similar reaction and treatment to the method described in Reference example 9. Table 3

Reference example	R⁷	R⁸	R⁹	Stereochemistry of *	ïH-NMR (400 MHz, CDCI3/CD3OD (30:1))
10	Ph	Me	Me	S	7.18 (2H, d, J = 6.7 Hz), 7.11-7.07 (3H, m), 5.18- 5.05 (4H, m), 4.51-4.40 (2H, m), 4.20-4.05 (2H, m) , 3.78-3.60 (4H, m) , 3.4-3.38 (2H, m), 2.65- 2.10 (14H, m), 1.60-1.40 (14H, m), 1.30-1.10 (86H, m) , 0.81 (15H, t, J = 6.7 Hz) . _______________________
11	Me	Ph	Me	R	7.18 (2H, d, J = 7.2 Hz), 7.11-7.09 (3H, m) , 5.17- 5.00 (4H, m) , 4.47 (IH, d, J = 3.6 Hz), 4.32 (IH, s) , 4.14-3.84 (4H, m) , 3.68-3.57 (2H, m) , 3.34- 3.25 (3H, m), 2.63-2.35 (8H, m), 2.30-2.12 (6H, m) 1.60-1.40 (14H, m) , 1.25-1.10 (86H, m), 0.81 (15H, t, J - 6.7 Hz) ._______
12	Me	Ph	Me	S	7.18 (2H, d, J = 7.2 Hz), 7.11-7.09 (3H, m) , 5.27- 5.13 (4H, m), 3.66-3.35

					(4H, m), 3.08-3.04 (2H, m), 2.51-2.50 (3H, m) , 2.20-1.99 (24H, m), 1.601.40 (14H, m), 1.31-1.18 (86H, m) , 0.78 (15H, t, J =6.7 Hz).
13	Me	Me	Ph	R	7.20-7.05 (5H, m) , 5.20- 4.98 (4H, m) , 4.43 (IH, d, J = 8.5 Hz), 4.35 (IH, s), 4.12 (IH, q, J = 9.1 Hz), 3.92-3.80 (3H, m), 3.72-3.60 (2H, m) , 3.34- 3.10 (2H, m), 2.60-2.10 (14H, m) , 1.58-1.40 (14H, m) , 1.25-1.10 (86H, m), 0.80 (15H, t, J = 6.7 Hz) .
14	Me	Me	Ph	S	7.18 (2H, d, J = 7.3 Hz), 7.11-7.07 (3H, m) , 5.10- 5.02 (4H, m) , 4.53 (IH, d, J = 8.5 Hz), 4.33 (IH, s) , 4.15-3.90 (4H, m), 3.67-3.59 (2H, m) , 3.35- 3.33 (2H, m), 3.24-3.20 (IH, m) , 2.60-2.15 (14H, m) , 1.60-1.40 (14H, m) , 1.25-1,10 (86H, m) , 0.81 (15H, t, J - 6.7 Hz).

[0151]

Reference example 15

Each compound shown in Table 4 was prepared from each corresponding starting compound in a similar reaction and treatment to the method described in Example 1.

Table 4

OH

Reference example	R⁷	R⁸	R⁹	Stereochemistry of *	¹H-NMR (400 MHz, CDCI3/CD3OD (30:1))
15	Me	Ph	Me	S	7.18 (2H, d, J = 7.2 Hz), 7. 10-7.08 (3H, m), 5.13- 5.06 (3H, m), 4.85 (IH, t, J = 8.8 Hz), 4.41-4.11 (3H, m), 3.77-3.64 (4H, m), 3.38-3.35 (2H, m), 2.57-2.42 (6H, m) , 2.282.11 (8H, m), 1.57-1.44 (14H, m) , 1.33-1.08 (86H, m) , 0.81 (15H, t, J = 6.7 Hz) . ___________________________

[0152]

Test 1

Human TLR4 reporter gene assay

HEK-Blue™ hTLR4 cell line (Tnvivogen Corporation) is 5 a stably co-transfected cell line which expresses human TLR4, MD2, CD14, and secretory alkaline phosphatase (SEAP) reporter gene under the transcriptional régulation of an NF-κΒ response element. The TLR4 expression of the cell line has been already tested by RT-PCR, flow cytometry.

Transfectants with stable expression were selected using the antibiotic HEK-Blue™ Sélection. TLR signaling leads to the translocation of NF-κΒ and the activation of the promoter results in expression of the SEAP gene. TLR4specific activation was assessed by determining the level of SEAP produced following incubation of the cells at 37 °C for 16 - 20 hours with each compound prepared in Examples and Reference examples in the presence of 0.1 % (v/v) DMSO. The human TLR4 activity for the present compound was assessed by human TLR4 reporter gene assay, and the concentration of the compound concentration which produced half of the maximal level of SEAP induced with lipopolysaccharide (LPS) was determined as ECso[0153] Test 2

Mouse TLR4 reporter gene assay

HEK-Blue™ mTLR4 cell line (Invivogen) is a stably cotransfected cell line which expresses mouse TLR4, MD2, CD14 and secretory SEAP reporter gene under the transcriptional régulation of an NF-κΒ response element. The TLR4 expression of the cell line has been already tested by RTPCR, flow cytometry. Transfectants with stable expression were selected using the antibiotic HEK-Blue™ Sélection. TLR signaling leads to the translocation,of NF-κΒ and the activation of the promoter results in expression of the SEAP gene. TLR4-spécifie activation was assessed by determining the level of SEAP produced following incubation of the cells at 37°C for 16 - 20 hours with each compound prepared in Examples and Reference examples in the presence of 0.1 % (v/v) DMSO. The mouse TLR4 activity for the present compound was assessed by mouse TLR4 reporter gene assay, and the concentration of the compound concentration which produced half of the maximal level of SEAP induced with LPS was determined as ECso-

[0154]

1q The results of Tests 1 and 2 are shown in Tables 5 and

6.

Table 5 _______________________________________
Example	human TLR4 EC50 (ng/ mL)	mouse TLR4 EC50 (ng/ mL)
1	64	21_____
2	22	21_________
3	237	202
4	77	57__________
5	____________148____________	_____________73_____________

Table 6 __
Reference example	human TLR4 ECso (ng/ mL)	mouse TLR4 ECso (ng/ mL)________
9	68	44
10	20	20 ____
11	708	78
12	>1000	>1000 ____
13	53	21
14	22	_____________22_____________

[0155]

The results in Table 5 hâve clarified that the example compounds of the present invention hâve TLR4 agonistic effect, which are almost equal or higher than those of Reference example 9-14 shown in Table 6 which hâve phosphate group. [0156] Préparation of liposome formulation (Exemples 6 - 7,

Reference examples 16 - 17)

1,2-Dimyristoyl-sn-glycero-3“phosphocholine (35.45 mg ) , egg yolk phosphatidylglycerol (24.45 mg) , each compound shown in Table 7 (6 mg) were dissolved in t-butyl alcohol, and the solution was lyophilized. Phosphate buffered saline ( 3 mL) was added to the lyophilized product. The obtained solution was allowed to pass through 0.1 pm polycarbonate membrane with an extruder heated at about 65°C (Mini-Extruder, Avant! Polar Lipids) to préparé a liposome formulation. The préparation scale was optionally changed if necessary. In the liposome formulation of Reference exemple 17, the compound of Reference example 10 was not detected under LCMS Condition C. It was supposed that the compound of Reference example 10 was not formed to liposomes, i.e., it was trapped with the membrane of the extruder.

The above liposome was diluted 10 times with purified water, and the mean particle size, polydisperse index, and zêta potential of the diluted liposome were measured with a dynamic light scattering (ZETASIZER Nano-ZS, Malvern). And, the formulations of Examples 6 and 7 and Reference example 16 were measured about the content with high-performance liquid chromatography (HPLC).

HPLC Condition

Detector: UV (205 nm)

HPLC: Shimadzu LC 20AD

Column: XSelect CSH Phenyl-Hexyl 2.5 pm 75 χ 4.6 mm

Flow rate: 0.8 ml/min

Mobile phase: A: 0.1 % trifluoroacetic acid/water

B: 2-propanol

Time program:

Step	Time (min)
1	0.0-7.0 A:B = 20 : 80
2	7.0-7.5 A:B = 20: 80 - 5: 95
3	7.5-11.0 A:B = 5: 95
4	11.0-11.1 A:B =5: 95 - 20: 80
5	11.1-15.0 A:B = 20: 80

The results are shown in Table 7.

Table 7___________—
Example/ Reference example	Compound	Content (mg/ mL)	mean particle size (Z- average, nm)	polydisperse index (PDI)	zêta potential (mV)
Example 6	Example 1	2.1	98.4	0.229	-74.7
Example 7	Example 2	2.2	84.6	0.212	-71.4
Reference example 16	Reference example 9	2.0	123.0	0.122	-64.8
Reference	Reference	___________impossible to measure___________

example [example 10 | 17 I

[0157]

Test 3

An equal mixture of ovalbumin (OVA) (2 mg/ mL) as an antigen and the formulation prepared in Example 6, Example 7, or Reference example 16 (containing 0.2 or 2 mg/mL the compound of Example 1, Example 2, or Reference example 9) was intramuscularly administered to the gastrocnemius of a 7-week-old C57BL/6 male mouse (100 pL/mouse), which was defined as the first immunization. Two weeks later, the equal mixture was intramuscularly administered to the gastrocnemius again, which was defined as the additional immunization. One week after the additional immunization, the heart blood was collected under inhalation anesthésia, and the sérum was collected from the blood by centrifugation. The OVA-specific IgG2c value in each sérum was measured by the following ELISA method. To 96-well plate, OVA solution (SIGMA) was added, 1 % Skim Milk (Wako) was added for blocking, the sérum sample which was diluted with phosphate buffer solution was added, caprine antimouse IgG2c (Southern Bio) as the secondary antibody was added, SureBlue™ TMB Micrewell Peroxidase Substrate (KPL) was added, and then the product of the enzymatic reaction was quantified with a microplate reader. The results are shown in Figures 1 and 2.

Example 1, Example 2, and Reference example 9 exhibited significantly strong OVA-specific IgG2c induction, compared with the négative control group. [0158]

Test 4

The equal mixture prepared in Test 3 of ovalbumin (OVA) (2 mg/mL) and the formulation prepared in Example 6, Example 7, or Reference example 16 (containing 0.2 or 2 mg/mL the compound of Example 1, Example 2, or Reference example 9) was administered to a mouse (100 pL/mouse). The spleen cell of the mouse was prepared, OVA and Brefeldin A (eBioscience) were added the cell, and the cell was cultured overnight. The collected cell was stained with ARC-labeled antimouse CD3e antibody (Invitrogen), PerCPlabeled antimouse CD4 antibody (BioLegend), and Fixable Viability Dye eFluor™ 520 (invitorgen), and fixed with Fixation/Permeabilization buffer (Invitrogen). The cell was treated with Permeabilization buffer (Invitrogen), and then stained with antibody cocktail BV421-labeled anti-IFNy antibody (BioLegend), PE-Cy7-labeled anti-IL-2 antibody (eBioscience), and PE-labeled TNF-α (BioLegend). The data were taken and analyzed with FACS Cant II (BD Biosciences) and FLOWJO software (TreeStar). The results are shown in

Figures 3 and 4.

Separately, the spleen cell was stained with V450 labled antimouse CD3e antibody (invitrogen), Alexa Fluor™ 647-labeled antimouse CD8 antibody (MBL), PE-labeled H-2Kb OVA Tetramer-SIINFEKL (MBL), and Fixable Viability dye eFluor 520 (invitrogen). The data were taken and analyzed with FACS Cant II (BD Biosciences) and FLOWJO software (TreeStar). The results are shown in Figures 5 and 6.

Further separately, the spleen cell was stained with V450-labeled antimouse CD3e antibody (invitrogen), Alexa Fluor™ 647-labeled antimouse CD8 antibody (MBL), PE-Cy7labeled antimouse CD44 antibody (Invitrogen), PerCP-Cy5.5labeled antimouse CD62L antibody (Invitrogen), and Fixable Viability dye eFluor 520 (Invitrogen). The data were taken and analyzed with FACS Cant II (BD Biosciences) and FLOWJO software (TreeStar). The results are shown in Figures 7 and 8.

By using the compound of Example 1, Example 2, or Reference example 9, the percentage of OVA-specific type 1 helper T-cell, especially OVA-specific multifunctional CD4positive T-lymphocyte, the percentage of MHC-restricted OVA-specific CD8-positive T-lymphocyte (OVA tetramerpositive CD8T cell in Figures 5 and 6) , and the percentage of CD8-positive effector memory T-lymphocyte were significantly increased, compared with the négative control group. [0159]

The results in Tests 3 and 4 hâve clarified that the example compounds of the present invention hâve adjuvant effect.

INDUSTRIAL APPLICABILITY [0160]

The compounds of the present invention are usefui as adjuvant for enhancing immunostimulating activity in a vaccine formulation.

Claims

1. A compound of formula (1):

or a pharmaceutically acceptable sait thereof, wherein

A and A' are independently hydrogen, hydroxy, or (CH₂)_m-COOH, provided that at least one of A or A' is (CHzJm-COOH,

R¹ is -C(0) (CH₂)_n-X or -CH₂- (CH₂)_n-X,

R² is -C (0) (CH₂)_O-Y or -CH₂- (CH₂) _O-Y,

R³ is -C (0) (CH₂)_P-Z or -CH₂- (CH₂) _P-Z,

X, Y, and Z are independently methyl, Ce-ίο aryl (said Ce-io aryl may be substituted with 1 - 5 substituents selected independently from hydroxy, Ci-6 alkyl, halogen, cyano, and Ci-e alkoxy), or 5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1-4 substituents selected independently from hydroxy, Ci-ç alkyl, halogen, cyano, and Ci-6 alkoxy), provided that at least one of X, Y, or Z is Ce-io aryl (said Ce-ίο aryl may be substituted with 1 - 5 substituents selected independently from hydroxy, Ci-β alkyl, halogen, cyano, and Ci-e alkoxy) or

5- to 10-membered heteroaryl (said 5- to 10-membered heteroaryl may be substituted with 1-4 substituents selected independently from hydroxy, Ci-₆ alkyl, halogen, cyano, and Ci-6 alkoxy) ,

5 provided that when A is -COOH and the stereochemistry of * is S-configuration, Y is methyl,

R⁴, R⁵, and R⁶ are independently C10-20 alkyl, m is independently an integer of 0 - 6, and n, o, and p are independently an integer of 5 - 20.

2. The compound of claim 1, or a pharmaceutically acceptable sait thereof, wherein m is 0.

3. The compound of claim 1 or 2, or a pharmaceutically 15 acceptable sait thereof, wherein R¹ is -C (0) (CH₂) _n-X, R² is

-C(0) (CH₂)o-Y, and R³ is -C(O) (CH2)_P~Z.

4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable sait thereof, wherein A is COOH.

5. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable sait thereof, wherein A is COOH, and A' is hydroxy.

25 6. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable sait thereof, wherein R⁴, R⁵, and R⁶ are independently C10-12 alkyl.

Ί. The compound of claim 1 which is represented by formula (2):

OH ^-0 wherein

R¹ is -C (0) (CH₂)_n-X,

R² is -C (0) (CH2) _O-Y,

R³ is -C (0) (CH₂)_P-Z,

X, Y, and Z are independently methyl, Ce-10 aryl, or 5to 10-membered heteroaryl, provided that at least one of X, Y, or Z is Ce-ίο aryl or 5- to 10-membered heteroaryl, provided that when the stereochemistry of * is Sconfiguration, Y is methyl,

R⁴, R⁵, and R⁶ are independently C10-12 alkyl, and n, o, and p are independently an integer of 6 - 10, or a pharmaceutically acceptable sait thereof,

8. The compound of claim 1 which is represented by formula ( 3) :

100

R¹ is —C (O) (CH2)n-X/

R2 is “C (O) (CH₂)oY,

5 R³ is -C(O) (CH₂)_P-Z,

X, Y, and Z are independently methyl, Ce-io aryl, or 5to 10-membered heteroaryl, provided that at least one of X, Y, or Z is Cê-io aryl or 5- to 10-membered heteroaryl, provided that when the stereochemistry of * is S10 configuration, Y is methyl, and n, o, and p are independently an integer of 6 - 10, or a pharmaceutically acceptable sait thereof.

9. The compound of claim 1 which is represented by

15 formula (4) or formula (5):

101

wherein

R¹ is -C(O) (CH₂)n-X,

R² is -C (0) (CH₂) o-Y,

5 R³ is -C (0) (CH₂)_P-Z,

R²' is -C (0) (CH₂) _o-CH₃,

X, Y, and Z are independently methyl, Ce-io aryl, or 5to 10-membered heteroaryl, provided that at least one of X, Y, or Z in formula (4 ) is Ce-io aryl or 5- to 10-membered 10 heteroaryl, and at least one of X or Z in formula (5) is Ce-ίο aryl or 5- to 10-membered heteroaryl, and n, o, and p are independently an integer of 7 - 9, or a pharmaceutically acceptable sait thereof.

15

10. The compound of any one of claims 1 to 9, or a pharmaceutically acceptable sait thereof, wherein

X, Y, and Z are independently methyl or phenyl.

102 provided that at least one of X, Y, or Z is phenyl, provided that when A is -COOH and the stereochemistry of * is S-configuration, Y is methyl.

11. The compound of claim 1 which is selected from the following compound group:

(2R)-2-{ [ (3R)-3-(decanoyloxy)tetradecanoyl]amino}-3{[3-{ [(3R)-3-(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4-({(3R)-3-[ ( 9phenylnonanoyl)oxy]tetradecanoyl}oxy)oxan-2yl]oxy}propanoic acid, ( 2S) -2- {[(3R)-3-(decanoyloxy)tetradecanoyl]amino) - 3- { [3-{ [ (3R)-3-(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4-(((3R)-3-[(9phenyInonanoyl)oxy]tetradecanoy1}oxy)oxan-2yl]oxy}propanoic acid, (2R)-2-{[(3R)-3-(decanoyloxy)tetradecanoyl]amino)-3{[4-{[(3R)-3-(decanoyloxy)tetradecanoyl]oxy}~5-hydroxy-6(hydroxymethyl)-3-({(3R)-3-[(9phenyInonanoyl)oxy]tetradecanoyl}amino)oxan-2— yl]oxy}propanoic acid, (2R)-2-({[(3R)-3-(9phenylnonanoyl)oxy]tetradecanoyl}amino)-3-{[3-{[(3R)-3(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4-({(3R)-321080

103 (decanoyloxy)tetradecanoyl}oxy)oxan-2-yl]oxy}propanoic acid, and (2S) -2-({ [ (3R) -3-(9phenylnonanoyl)oxy]tetradecanoyl}amino)-3-{[3-{[( 3R)-3(decanoyloxy)tetradecanoyl]amino}-5-hydroxy-6(hydroxymethyl)-4-({(3R)-3(decanoyloxy)tetradecanoyl}oxy)oxan-2-yl]oxy}propanoic acid.

12. A pharmaceutical composition comprising the compound of any one of claims 1 to 11, or a pharmaceutically acceptable sait thereof.

13. The pharmaceutical composition of claim 12, which is a lipid formulation.

14. The pharmaceutical composition of claim 12 or 13, wherein the lipid formulation is a liposome formulation including phospholipid.

15. The pharmaceutical composition of claim 14, wherein the phospholipid is 1,2-dimyristoyl-sn-glycero-3phosphocholine and egg yolk phosphatidylglycerol.

16. The pharmaceutical composition of claim 14 or 15, wherein the lipid formulation comprises at least one

104 additive selected form the group consisting of an inorganic acid, an inorganic acid sait, an organic acid, an organic acid sait, sugars, a buffering agent, an antioxidant, and polymers.

17. The pharmaceutical composition of any one of claims 12 to 16, which further comprises an antigen.

18. The pharmaceutical composition of claim 17, wherein the antigen is a pathogen-derived antigen.

19. A vaccine adjuvant comprising the compound of any one of claims 1 to 11, or a pharmaceutically acceptable sait thereof.

20. The vaccine adjuvant of claim 19, which is an adjuvant for infection vaccine.

21. A kit comprising

a) the compound of claim 1 or a pharmaceutically acceptable sait thereof, or a pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable sait thereof; and

b) a pharmaceutical composition comprising an antigen.

105

22. The kit of claim 21, wherein the antigen is a pathogen-derived antigen.