KR101327797B1 - Glycosylated Tricyclo compounds derivatives - Google Patents

Abstract

The present invention relates to a technique of attaching a sugar to a tricyclo compound structure to form tricyclo compound derivatives of a novel structure, and to glycated derivatives synthesized by the technique. .

Description

Glycosylated Tricyclo compounds derivatives

The present invention relates to a technique for attaching a sugar to the structure of a tricyclo compound including tacrolimus (used herein as "FK506") to form tricyclo compound derivatives of a novel structure and to glycated derivatives synthesized by the technique. will be.

About 70% of the leading drugs currently being developed are estimated to be derived from natural substances, the majority of which are sugar modified (Poiter, P. Actual. Chim. 11: 9 (1999)). The attachment of sugars has a great effect on the regulation of the absorption, distribution, metabolism or excretion of the drug. Depending on the type and number of sugars, the affinity for the target can also vary greatly.

Tricyclo compounds are microbial secondary metabolites that show antifungal and immunosuppressive activity mainly produced by microorganisms, especially actinomycetes. These substances are generally very toxic and have very high pharmacological activity, and have been developed as immunosuppressants, anticancer agents, etc., and are sold as expensive medicines such as Prograf ^TM and Rapamune ^TM .

Tacrolimus (FK506), ascomycin (FK520), rapamycin (sirolimus), meridamycin and the like are known as representative tricyclo compounds. Tacrolimus, ascomycin, rapamycin and their structural analogs have similar structural sites, indicating the activity of inhibiting the activation of immune cells, T cells, in vivo and in vitro . These compounds have a pyranose-pipecolinyl region, a structure similar to the leucine-proline peptide, which is a peptidyl prolyl cis / trans. isomerase) has been reported to exhibit various physiological activities (Rosen MK et al., 1990).

No sugar is attached to these tricyclo compounds. Therefore, most tricyclo compound drugs are very nonpolar and insoluble in water-soluble solvents. This is very disadvantageous for the formulation of the drug. Attaching sugars to these drugs not only provides an opportunity to provide new novel active substances, but also increases the water solubility which may allow for the development of new drugs that are more effective.

Tacrolimus (FK506) is an immunosuppressive agent developed by Fujisawa Co., Ltd. of Japan in 1984. It is a macrolide structural small molecule secondary metabolite with a molecular weight of about 803 m / z produced by the Gram-positive bacterium Soil Actinomycete Streptomyces tsukubaensis . Tacrolimus is mainly used to reduce and resolve rejection after extensive transplantation including liver, kidney, heart, spinal cord, small intestine, pancreas, lung, trachea, skin, cornea and limbs. It is also used.

Tacrolimus trade name is Prograf ^™ , which is developed as an injection and ointment for capsule and intravascular diffusion. Tacrolimus is a white powder and shows low solubility in water, hexane, heptane, etc., and high solubility in alcoholic solvents. In particular, tacrolimus has a rapid decrease in solubility due to the incorporation of water. Due to these physical properties, tacrolimus is not easy to develop formulations such as injectables, and in particular, pharmacokinetics have very low bioavailability. For this reason, tacrolimus is likely to form precipitates or crystals when rapidly dissolved in organic solvents due to the nature of water-insoluble drugs, and thus, localization of local drugs is likely to occur, and various solutions, powders, etc. It is very difficult to apply it to various clinical uses as preparation of suspension, oral administration solution, topical dispersion, ophthalmic charge agent, etc.

If the solubility of tacrolimus in the aqueous solution is increased, various formulations and formulations can be studied, and the permeability of the drug can be greatly improved, thereby increasing the absorbency of the drug, thereby significantly improving the pharmacokinetic properties of the drug.

Accordingly, an object of the present invention is to provide a method for increasing the solubility of a tricyclo compound.

Moreover, an object of this invention is to provide the tricyclo compound derivative with high solubility.

The present inventors prepared a tricyclo compound glycosylated derivative having a high solubility by modifying sugar to synthesize a new structure while studying a method of increasing the solubility of the tricyclo compound.

The present invention is a glycosylated derivatives of tricyclo compounds having high solubility and various physiological activities, more specifically, water solubility is greatly improved, thereby providing a tricyclo compound having excellent body dynamics and safety. The present invention also provides a method of interposing a linker between a tricyclo compound and a sugar to overcome steric hindrance to attach the sugar to the tricyclo compound.

The sample used in the present invention is a tricyclo compound, specifically, tacrolimus, ascomycin, rapamycin, meridamycin, and the like, more preferably tacrolimus. In the tricyclo compound, a functional group for modifying a sugar may include a hydroxyl group, a methyl group, a methoxyl group, and preferably a hydroxyl group. For example, tacrolimus may be a hydroxyl group at 32 or 24 positions, and may be modified at both sites. One of the two sites may be modified. In the present invention, a linker may be used to link the sugar with the compound, and the linker may be linked by using an ester or amide bond, and may be a formyl group, an acetyl group, or propionyl as a functional group. A propionyl group, a butyl group, an acryl group, an ethylsuccinyl group, a succinyl group, an aminohexyl group, etc. may be attached, preferably a succinyl group , Aminohexyl groups may be used alone or in combination. The sugars to be attached may be monosaccharides, disaccharides, trisaccharides and polysaccharides, and the number of sugars attached is preferably 1 to 5, and each sugar is usually an aldose or ketose composed of C3 to C7. Sugar may be included. There is no particular limitation on the type of sugar, and preferably, the monosaccharide is glucose, galactose, fructose, fucose, mannose, rhamnose, galactosamine, glucosamine, N-acetylgalactosamine, N-acetylglucosamine, vancosamine, Epi-vancosamine, glucuronic acid, sialic acid, deoxyglucose, deoxygalactose, and more preferably glucose, monosaccharide, lactose and trisaccharide sialic lactose. .

The method of attaching a sugar may attach a sugar to a linker and then attach a tricyclo compound, or attach a linker to a tricyclo compound and attach a sugar to it.

The linker-sugar complex may extend to disaccharides, trisaccharides after attaching monosaccharides to the linker, or to trisaccharides after attaching disaccharides to linkers. At this time, the disaccharide, trisaccharide expansion can be synthesized through a chemical reaction, or can be extended by enzymatic reaction. Enzymatic reaction may be used galactosyltransferase for the transformation from glucose to lactose, and sialyltransferase is suitable for the conversion from lactose to sialyllactose.

Some specific examples of the tacrolimus glycation derivatives of the present invention are described below.

The present invention provides a macrolide glycosylated derivative compound represented by the formula (1) made from a macrolide compound as a raw material, a pharmaceutically acceptable salt thereof, or a solvate thereof.

≪ Formula 1 >

Wherein Rx is a hydroxyl group, a methoxy group or H, and at least one of R ₁ and R ₂ is an aldose or ketose consisting of C 3 to C 7 linked to a linker of C 1 to C 12 by an ester bond or an amide bond ) 1 to 5 sugar chains are bonded, and R ₁ or R ₂ with no sugar chains is H)

In addition, the present invention is a trisite having the structure of formula (I) to (1) to (24) which is a compound having a linker attached to R ₁ and R ₂ in the compound skeleton, or a sugar residue attached to the linker To provide a compound.

(Rx is OH or methoxy)

, R₂=H (Compound 1); R ₁ =

, R ₂ = H

(Compound 2); R ₁ = H, R ₂ =

(Compound 3); R ₁ , R ₂ =

, R₂=H (Compound 4); R ₁ =

, R ₂ = H

(Compound 5); R ₁ = H, R ₂ =

(Compound 6); R _1, R ₂ =

, R₂=H (Compound 7); R ₁ =

, R ₂ = H

(Compound 8); R ₁ = H, R ₂ =

(Compound 9); R ₁ , R ₂ =

, R₂=H (Compound 10); R ₁ =

, R ₂ = H

(Compound 11); R ₁ = H, R ₂ =

(Compound 12); R ₁ , R ₂ =

, R₂=H (Compound 13); R ₁ =

, R ₂ = H

(Compound 14); R ₁ = H, R ₂ =

(Compound 15); R ₁ , R ₂ =

, R₂=H (Compound 16); R ₁ =

, R ₂ = H

(Compound 17); R ₁ = H, R ₂ =

(Compound 18); R ₁ , R ₂ =

, R₂=H (Compound 19); R ₁ =

, R ₂ = H

(Compound 20); R ₁ = H, R ₂ =

(Compound 21); R ₁ , R ₂ =

,R₂=H (Compound 22); R ₁ =

, R ₂ = H

(Compound 23); R ₁ = H, R ₂ =

(Compound 24); R ₁ , R ₂ =

Lt; / RTI >

Moreover, this invention also provides the process shown to the following (25)-(35) by the manufacturing method of the compound as described in said (1)-(24).

(25) Process of introducing succinyl group into 1-OH position using glucose as raw material and protecting group into other -OH group

(R ₃ and R ₄ may be the same or different, and is a protecting group of a conventional chemical synthetic OH group that can be easily dissociated by treatment with an acid or a base to be transformed into H, preferably a MMTr (Monomethoxytrityl) group. Or a TMS (Tetramethylsilane) group, more preferably R ₃ is an MMTr group, and R ₄ is a TMS group.

(26) Process for preparing 1-O-aminohexyl glucose by introducing aminohexyl groups into glucose or lactose (steps 1 and 2 described below) or making 1-O-aminohexyl lactose (steps below) 1, 3 or 1, 2, 4 to perform sequentially, or 1-O-aminohexyl sialylactose to make a process (step 1, 3, 5 or 1, 2, 4, 5 to perform sequentially Process, or a process of sequentially performing 1 or 2 processes and simultaneously performing 4 and 5), each step may be repeated 1 to 4 times.

Process 26-1

R is an amino group protecting group, Preferably it is MMTr group.

Process 26-2

에 초산 브롬 글루코오스 (acetyl bromo glucose)를 부착한 후 당에 부착된 아세틸기 및 R기를 제거하여 1-O-아미노헥실 글루코오스를 만드는 공정이다. Generated in step 1

After attaching acetyl bromoglucose (acetyl bromo glucose) to the acetyl group and R groups attached to the sugar to make 1-O-aminohexyl glucose.

Process 26-3

에 초산 브롬 락토오스 (acetyl bromo lactose)를 부착한 후 당에 부착된 초산기 및 R기를 제거하여 1-O-아미노헥실 락토오스를 만드는 공정이다. Generated in step 1

After attaching acetyl bromo lactose to acetyl bromo lactose, it removes the acetic acid groups and R groups attached to the sugar to make 1-O-aminohexyl lactose.

Process 26-4

It is a step of producing l-O-aminohexyl lactose by introducing lactose by performing a saccharification reaction using enzyme on 1-O-aminohexyl glucose.

Process 26-5

It is a step of making 1-O-aminohexyl 3'-sialyl lactose by introducing sialic acid by performing a saccharification reaction using an enzyme on 1-O-aminohexyl lactose.

(27) Compound (25) prepared by step (25) is added to the compound of marcrolide in FIG. 1, followed by removal of R1 and R2, which are OH protecting groups, to form compound 7, 8, or 9. .

(R ₃ and R ₄ may be the same or different, and are a protecting group of a conventional chemical synthetic OH group that can be easily dissociated by treatment with an acid or a base and transformed into H, preferably MMTr group, TMS group More preferably, R ₃ is an MMTr group, and R ₄ is a TMS group).

(R ₁ and R ₂ are

(화합물 7)

(Compound 7)

(화합물 8)

(Compound 8)

(화합물 9)이며, Rx는 수산화기 또는 메톡시기 또는 H임)

(Compound 9), and Rx is hydroxyl group or methoxy group or H)

(28) A step of synthesizing the compound 10, 11 or 12 to the compound 7, 8 or 9 obtained in the step (27) using galactosyltransferase (galactosyltransferase).

(R1 and R2 are respectively

, R₂=H (화합물 10),R ₁ =

, R ₂ = H (compound 10),

(화합물 11) 또는R ₁ = H, R ₂ =

(Compound 11) or

(화합물 12)임, Rx는 수산화기, 메톡시기 또는 H임)
R ₁ , R ₂ =

(Compound 12), Rx is hydroxyl, methoxy or H)

(29) the following steps

A process for synthesizing compound 13, 14 or 15 by transferring sialic acid to an enzymatic method using sialyltransferase to compound 10, 11 or 12 prepared in the step (28).

(R ₁ and R ₂ are

, R₂=H (화합물 13),R ₁ =

, R ₂ = H (compound 13),

(화합물 14) 또는R ₁ = H, R ₂ =

(Compound 14) or

(화합물 15)임,R ₁ , R ₂ =

(Compound 15),

Rx is hydroxyl, methoxy or H)

30 or less steps

A process of ester-bonding dicarboxylate with dicarboxylate to the OH group of the macrolide compound of FIG. 1 and adding alkyl chlorocarbonates or aralkyl chlorocarbonates to form an active ester.

If you divide the process below

Process 30-1

X may be added as a linker capable of ester bonding to the hydroxyl group of the macrolide compound of FIG. 1. X is dicarboxylic acid or its anhydride, salts or solvates thereof, preferably oxalic acid, malonic acid, succinic acid, glutaric acid, Adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid (dodecanedioic acid), phthalic acid, isophthalic acid, terephthalic acid, maleic acid, maleic acid, fumaric acid, glutaconic acid, tromatic acid ), And muconic acid, more preferably malonic acid, succinic acid, maleic acid, phthalic acid, glutaric acid , Adipic acid, pimelic acid, pew Acid is one selected from among (fumaric acid) or the group consisting of its anhydrate.

(Rx is a hydroxyl group, a methoxy group or H) (X is a diacetic acid group of C2 to C12, preferably succinic anhydride)

(화합물 1),

(Compound 1),

(화합물 2) 또는

(Compound 2) or

(화합물 3)임, Rx는 수산화기 메톡시기 또는 H임)

(Compound 3), Rx is hydroxyl methoxy group or H)

Process 30-2

Compound 1, 2 or 3 is selected from Y (alkyl chlorocarbonate, aralkyl chlorocarbonate, salts or solvates thereof, chlorocarbonate is methyl chloroformate, preferably alkyl chloroformate, ethyl chloro Ethyl chlorocarbonate, isobutyl chloroformate, isopropyl chloroformate, beznyl chloroformate, nitrophenyl chloroformate, More preferably nitrophenyl chloroformate may be selected) to make compounds 4, 5 or 6, respectively.

(Y is alkyl chlorocarbonate or aralkyl chlorocarbonate, preferably p-nitrophenyl chloroformate)

(R ₁ and R ₂ are

, R₂=H (화합물 4),R ₁ =

, R ₂ = H (compound 4),

(화합물 5) 또는R ₁ = H, R ₂ =

(Compound 5) or

(화합물 6)임, Rx는 수산화기, 메톡시기 또는 H임)
R ₁ = R ₂ =

(Compound 6), Rx is hydroxyl, methoxy or H)

(31) the following steps

A process for producing compound 16, 17 or 18 by adding compound 1-O-aminohexyl glucose produced in step (26) to compound 4, 5 or 6 prepared in step (30), respectively.

(R ₁ and R ₂ are

(화합물 16),

(Compound 16),

(화합물 17) 또는

(Compound 17) or

(화합물 18)임, Rx는 수산화기, 메톡시기 또는 H임)

(Compound 18), Rx is hydroxyl, methoxy or H)

(32) the following steps

Compound (16), Compound (17) or Compound (18) produced in step (31) is prepared by adding galactose using enzyme galactosyltransferase to produce compound 19, 20 or 21.

(R ₁ and R ₂ are

, R₂=H (화합물 19),R ₁ =

, R ₂ = H (compound 19),

(화합물 20) 또는R ₁ = H, R ₂ =

(Compound 20) or

(화합물 21)임, Rx는 수산화기, 메톡시기 또는 H임)
R ₁ , R ₂ =

(Compound 21), Rx is hydroxyl, methoxy or H)

(33) the following steps

Adding 1-O-aminohexyl lactose made in step (26) to compound 4, 5, or 6 made by step (30) to make compound 19, 20, or 21, respectively.

(R ₁ and R ₂ are

, R₂=H (화합물 19),R ₁ =

, R ₂ = H (compound 19),

(화합물 20) 또는R ₁ = H, R ₂ =

(Compound 20) or

(화합물 21)임, Rx는 수산화기, 메톡시기 또는 H임)
R ₁ , R ₂ =

(Compound 21), Rx is hydroxyl, methoxy or H)

(34) the following steps

Synthesis of sialic acid by enzymatic method using sialyltransferase to compound 19, 20 or 21 produced by step (32) or (33) to synthesize compound 22, 23 or 24

(R ₁ and R ₂ are

, R₂=H (화합물 22),R ₁ =

, R ₂ = H (compound 22),

(화합물 23) 또는 R ₁ = H, R ₂ =

(Compound 23) or

(화합물 24)임, Rx는 수산화기, 메톡시기 또는 H임)
R ₁ , R ₂ =

(Compound 24), Rx is hydroxyl, methoxy or H)

(35) the following steps

To compound 4, 5 or 6 made by step (30), 1-O-aminohexyl sialylactose made in step (28) is added to form compound 22, 23 or 24, respectively. fair

(R ₁ and R ₂ are

, R₂=H (화합물 22),R ₁ =

, R ₂ = H (compound 22),

(화합물 23) 또는, R ₁ = H, R ₂ =

(Compound 23) or,

(화합물 24)임, Rx는 수산화기, 메톡시기 또는 H임)
R ₁ , R ₂ =

(Compound 24), Rx is hydroxyl, methoxy or H)

The FK506 glycosylated derivative compounds prepared according to the present invention had up to 10,000-fold increase in water solubility compared to FK506, and showed excellent immunosuppressive activity.

1 shows the results of water solubility test of FK506 and FK506 glycosylated derivative compounds.
Figure 2a shows the results of immunosuppressive activity of FK506 and FK506 glycosylated derivatives FK506-M32-LS, FK506-D-LS, FK506-M32-LS-Glc compounds.
2B shows the results of immunosuppressive activity test of FK506 glycosylated derivatives FK506-M32-LS-Lac, FK506-M32-LS-SL, FK506-M32-LA-Glc, and FK506-M24-LA-Glc compounds.
Figure 2c is the result of immunosuppressive activity test of FK506 glycosylated derivatives FK506-M32-LA-Lac, FK506-M24-LA-Lac, FK506-M32-LA-SL, FK506-M24-LA-SL compounds.

Hereinafter, the configuration of the present invention through examples and test examples in more detail. However, it is apparent to those skilled in the art that the present invention is not limited to the scope of the Examples and Test Examples. Although the examples described specifically describe the preparation of saccharified derivatives of tacrolimus (FK506) among tricyclo compounds, it is possible to prepare saccharified derivatives in the same manner as other tricyclo compounds. Self-evident to those who have

Abbreviations used in the examples are as follows.

MMTr-Cl; Monomethoxytrityl chloride

MC; Methylene Chloride

TEA; Triethylamine

DMAP; 4-Dimethylaminopyridine

TMS; Tetramethylsilane

CMP; Cytidine 5 'monophosphate

DCC; N, N-dicyclohexylcarbodiimide

Et3N; Triethylamine

Hex; Hexane

EA; Ethylacetate

The structures, names, and molecular weights of the compounds 1 to 24 of the present invention are shown in Tables 1 to 6.

Example rescue designation mass
(MW) Confirm One

FK506-M32-LS
(Compound 1) 904.0
909 ¹ H-NMR 2

FK506-M24-LS
(2) 904.0
909 ¹ H-NMR 3

FK506-D-LS
(3) 1004.1637 1026.5
[M + Na] ⁺ 4

FK506-M32-LS-N
(4) 1069.1939 ¹ H-NMR

5

FK506-M24-LS-N
(5) 1069.1939 ¹ H-NMR 6

FK506-D-LS-N
(6) 1334.3697 ¹ H-NMR 7

FK506-M32-LS-Glc
(7) 1066.2315 1088.5
[M + Na] ⁺ 8

FK506-M24-LS-Glc
(8) 1066.2315 1088.5
[M + Na] ⁺

9

FK506-D-LS-Glc
(9) 1328.4449 1350.4
[M + Na] ⁺ 10

FK506-M32-LS-Lac
(10) 1228.3721 1250.5
[M + Na] ⁺ 11

FK506-M24-LS-Lac
(11) 1228.3721 1250.5
[M + Na] ⁺ 12

FK506-D-LS-Lac
(12) 1652.7261 1674.8
[M + Na]

13

FK506-M32-LS-SL
(13) 1519.6267 1564.3
[M + 2Na] ²⁺ 14

FK506-M24-LS-SL
(14) 1519.6267 1564.3
[M + 2Na] ²⁺ 15

FK506-D-LS-SL
(15) 2235.2352 2234.2
[MH] ^- 16

FK506-M32-LA-Glc
(16) 1165.4056 1187.6
[M + Na] ⁺

17

FK506-M24-LA-Glc
(17) 1165.4056 1187.6
[M + Na] ⁺ 18

FK506-D-LA-Glc
(18) 1526.7931 1549.7
[M + Na] ⁺ 19,22

FK506-M32-LA-Lac
(19) 1327.5462 1349.7
[M + Na] ⁺ 20,23

FK506-M24-LA-Lac
(20) 1327.5462 1349.7
[M + Na] ⁺

21,24

FK506-D-LA-Lac
(21) 1851.0743 1872.8
[M + Na] ⁺ 25,28

FK506-M32-LA-SL
(22) 1618.8008 1616.8
[M-2H] ^2- 26,29

FK506-M24-LA-SL
(23) 1618.8008 1616.8
[M-2H] ^2- 27,30

FK506-D-LA-SL
(24) 2433.5835 2432.2
[MH] ^-

As a typical example of the preparation method of these compounds, the compound of Example 1, Example 4, Example 7, Example 10, Example 13, Example 16, Example 19, Example 22, Example 25, Example 28 The manufacturing method is illustrated.

<Examples 1, 2, 3>

Example 1 relates to the preparation of Compound 1, and Examples 2 and 3 relate to the preparation of Compound 2 and Compound 3, respectively, in the same manner as in Example 1.

FK506 (10 g) and DMAP (4-Dimethylaminopyridine, 0.3 g) were dissolved in anhydrous MC (Methylene Chloride anhydride, 100 ml), and Et3N (Triethylamine, 3.5 ml) was added thereto and stirred. Succinic anhydride (1.5 g) was added and stirred overnight. After confirming the thin layer chromatography with MC: MeOH (9: 1), the reaction solution was washed with 0.5 M citric acid aqueous solution. Compounds 1, 2, and 3 were mixed in the reaction solution, and the normal column was filled with normal hexane in a silica column, followed by normal hexane: ethyl acetate (1: 1 → 0: 1) mixed solution and MC: MeOH ( 9: 1) The mixed solution was sequentially developed to obtain Compound 1, Compound 2, Mixture of Compound 1 and Compound 2, or Compound 3.

<Examples 4, 5, 6>

Example 4 describes a method for preparing Compound 4, and Compounds 5 and 6 of Examples 5 and 6 can be prepared by the same method. In this case, compound 5 may be prepared from compound including compound 2, compound 6 including compound 3.

The mixture containing Compound 1 prepared in Example 1 was dissolved in anhydrous pyridine (22 ml), and then DMAP (0.1 equivalent, 0.016 g) was added thereto and stirred. p-nitrophenyl chloroformate (p-nitrophenyl chloroformate, 1.5 equiv., 0.398 g) was added thereto, followed by stirring for 1 hour. The reaction was confirmed by thin layer chromatography (methylene chloride: MeOH, 95: 5) and concentrated. The organic layer was concentrated after fractionation with methylene chloride and 5% aqueous citric acid solution. A light yellow compound 4 was obtained.

Compound 1, 2 or 3

Compound 4, 5 or 6

<Example 7, 8, 9>

Example 7 describes a method of preparing Compound 7, and Compound 8 and Compound 9 of Examples 8 and 9 can be prepared by the same method. The following steps are divided and explained.

First Step: Intermediate Compound 6-MMTr Glucose Synthesis

Glucose (60 g), dried in a vacuum oven at 35 ° C. for 3 hours, was dissolved in 600 ml of anhydrous pyridine and stirred. After stirring for about 10 minutes, the mixture was stirred at 0 ° C. for about 30 minutes. MMTr-Cl (1.1 equiv, 113.3 g) was dissolved in 150 ml of anhydrous methylene chloride and slowly added to the reaction solution (0 ° C.). Stirred at room temperature for 1 hour. The reaction was completed by thin layer chromatography (MC: MeOH 9: 1). After the reaction was completed, the reaction solution was concentrated. The organic layer was concentrated after fractionation with 800 ml of methylene chloride and 800 ml of distilled water. The silica column (12 x 31 cm) was filled with methylene chloride and then developed in the order of MC: MeOH (98: 2), MC: MeOH (95: 5), and MC: MeOH (9: 1). MMTr glucose (74.5 g, 49.3%) was obtained.

Second Process: Intermediate Compound 6-MMTr 1-O-succinyl Glucose Synthesis

6-MMTr glucose (74.5 g) was dissolved in 740 ml of anhydrous methylene chloride and stirred. After stirring for about 10 minutes, anhydrous triethyleneamine (2 equiv, 46 ml), DMAP (0.2 equiv, 4 g) and succinic anhydride (1.2 equiv, 19.8 g) were added thereto. Stir at room temperature for 2 hours. The reaction was terminated by thin layer chromatography (MC: MeOH 5: 1). After the reaction, 60 ml of methylene chloride was added to the reaction solution, and the mixture was partitioned into 800 ml of 10% citric acid aqueous solution. The organic layer (MC) was received and filtered through a glass filter after Na ₂ SO ₄ treatment. The filtrate was concentrated and dried overnight in high vacuum. Light ocher 6-MMTr 1-O-succinyl glucose was obtained (95.9 g, 105.5%).

Third Step: Intermediate Compound 6-MMTr 2,3,4-tritetramethylsilane 1-O-succinyl Glucose Synthesis

6-MMTr 1-O-succinyl glucose (46.78 g) was dissolved in 230 ml of anhydrous methylene chloride. 230 ml of anhydrous pyridine was added and stirred. Stirred at 0 ° C. for about 15-20 minutes. TMS-Cl (6 equiv, 64.2 ml) was added slowly (0 ° C.). After stirring at 0 ° C. for about 20-30 minutes, the mixture was stirred at room temperature for 1-2 hours. The reaction was terminated by TLC (MC: MeOH 9: 1). 570 ml of methylene chloride was added to the reaction mixture, and the mixture was partitioned into 800 ml of 5% aqueous NaHCO ₃ solution. At this time, it was confirmed whether the pH of the organic layer (MC) was 6-7. After checking the pH, the organic layer was treated with Na ₂ SO ₄ . The filtrate was concentrated after filtration with a glass filter. After filling a silica column (8 x 36 cm) with hexane, it was developed in the order of hexane: ethyl acetate (5: 1 ~ 2: 1) and light yellow 6-MMTr 2,3,4-tritetramethylsilane 1- O-succinyl glucose was obtained (23.34 g, 35.8%).

Fourth Step: Synthesis of Intermediate Compound FK506-LS-Glucose (6-MMTr, 2,3,4-tri TMS)

인 경우 FK506-M32-LS-glc(6-MMTr, 2,3,4-tri TMS)이며,

인 경우 FK506-M24-LS-glc(6-MMTr, 2,3,4-tri TMS)이고,

인 경우 FK506-D-LS-glc(6-MMTr, 2,3,4-tri TMS)이다. At this time

Is FK506-M32-LS-glc (6-MMTr, 2,3,4-tri TMS),

FK506-M24-LS-glc (6-MMTr, 2,3,4-tri TMS),

FK506-D-LS-glc (6-MMTr, 2,3,4-tri TMS).

Fully dried FK506 (12 g) was dissolved in 120 ml of anhydrous methylene chloride and stirred. After stirring for about 10 minutes, DMAP (1.5 equivalents, 4.6 g) and DCC (1.5 equivalents, 2.7 g) were added thereto and stirred at 0 ° C. for 15-20 minutes. 6-MMTr 2,3,4-tri TMS 1-O-succinyl glucose (2 equivalents, 23 g) prepared in step 3 was dissolved in 120 ml of anhydrous methylene chloride and slowly added at 0 ° C. While stirring at room temperature for 3-4 hours, the reaction was confirmed by developing with TLC (MC: MeOH 95: 5). 260 ml of methylene chloride was added to the reaction solution, and the mixture was partitioned into 500 ml of 10% citric acid aqueous solution. The organic layer was filtered with a glass filter after Na ₂ SO ₄ treatment and the filtrate was concentrated. The concentrate was loaded on a silica column (8 x 36 cm) packed in methylene chloride solvent and then developed into MC: MeOH (99: 1), MC: MeOH (99: 1) + (98: 2) in light yellow color. A mixture FK506-LS-glc (6-MMTr, 2,3,4-tri TMS) was obtained (18.52 g, 79.9%).

Fifth Process: Synthesis of FK506-M32-LS-glc

The compound prepared in the fourth step (18.52 g) was dissolved in 120 ml of MeOH and stirred. 420 ml of acetic acid was added and stirred at room temperature overnight. The reaction was completed by TLC (MC: MeOH 9: 1) to confirm the completion of the reaction, and then concentrated. Ethanol was added and vacuum concentration was repeated 2-3 times. The concentrate was loaded onto a silica column (8 x 27 cm) packed in methylene chloride solvent and sequentially followed by MC: MeOH (99: 1), (98: 2), (95: 5), (92: 8). Expanded to give light yellow FK506-M32-LS-glc (5.14 g, 40.4%).

<Examples 10, 11, 12>

Example 10 describes a method of preparing Compound 10, and Compounds 11 and 12 of Examples 11 and 12 can be prepared by the same method. At this time, in Example 11, Compound 8 was used, and in Example 12, Compound 9 was used as a reaction sample.

FK506-M32-LS-Lac was synthesized using 1,4 galactosyltransferase (LgtB), which transfers galactose to the glucose position of compound (7), FK506-M32-LS-glc glucose. .

4.06 g of MgCl ₂ 6H ₂ O, 12 g of UDP-D-galactose and Tris HCl buffer (pH 7.5) were added to 25 to 50 mM, and the volume was adjusted to distilled water to 2.4 L. 1,4 galactosyltransferase was added to prepare a reaction. 4.3 g of the reaction starting material, FK506-M32-LS-Glc, was dissolved in 1.2 L of DMSO and slowly injected into the reactor to initiate the reaction. The reaction conditions were maintained at pH 7.5 and 37 ℃, and the degree of conversion was confirmed by HPLC analysis to terminate the reaction at a conversion rate of about 90%. The reaction solution (4.3 g, about 4 L) was extracted twice with 4 L of ethyl acetate to recover FK506-M32-LS-Lac. The ethyl acetate layer was concentrated and the concentrate contained DMSO, which was loaded onto a silica column (4 x 26 cm) filled with methylene chloride solvent conditions to remove DMSO, MC: MeOH (10: 1), (8: 1), (6: 1), (5: 1) was developed to obtain light yellow FK506-M32-LS-Lac, compound 10 (1.49 g, 30%).

<Examples 13, 14, 15>

Example 13 describes a method for preparing Compound 13. Compounds 14 and 15 of Examples 14 and 15 may be prepared by the same method. Instead of compound 10 of Example 13, compound 11 was used in Example 14 and compound 12 was used as a reaction sample in Example 15.

Sial from FK506-M32-LS-Lac and CMP-sialic acid precursors using sialyltransferase (SialT), which transfers sialic acid to galactose of compound 10 FK506-M32-LS-Lac prepared in Example 10 FK506-M32-LS-SL as a compound 13 with an acid added was synthesized.

4.2 g of CMP-sialic acid (purity approx. 60%) and Tris HCl buffer (pH 7.5) of the reactor were added to 25-50 mM, and the volume was adjusted to 1.4 L with distilled water. Approximately 3 g of the reaction starting material FK506-M32-LS-Lac was dissolved in 0.4 L of DMSO and slowly added to the reactor. The reaction was started by adding α2,3 sialyltransferase to the opaque reaction solution and the reaction proceeded at room temperature. The pH of the reaction was maintained at 7.5, and the degree of conversion was confirmed by HPLC analysis. When the conversion was about 90%, the same amount of MeOH (2 L) was added to terminate the reaction. After the reaction was completed, the reaction solution was concentrated. The reaction solution (about 1.2 L) was extracted with 1.2 L of ethyl acetate to remove FK506-M32-LS-Lac remaining in the reaction. Extraction is repeated twice in the same way. Compound 13, ie FK506-M32-LS-SL, was identified in the reaction liquid layer. The reaction solution layer was concentrated by drying under reduced pressure with EtOH. Concentration had residual DMSO and developed on a silica column (8 x 33 cm) filled with MC. After DMSO was removed to some extent, MC: MeOH (2: 1) was developed to obtain compound 13, FK506-M32-LS-SL, in the form of a yellow oil. In order to increase the purity was further purified using Prep-HPLC. Further buffer (A), purified water in a reverse phase column (50 × 500 mm) for high purity purification; (B), purification was carried out with acetonitrile with time varying. Drying under reduced pressure drying and lyophilization yielded final recovery (1.1 g, 29.5%).

<Examples 16, 17, 18>

In Example 16, a method of preparing Compound 16 is described. Example 17 and Example 18 are the methods for preparing Compound 17 and Compound 18, which are the same as in Example 16, with different reaction samples. That is, in place of compound 4, compound 5 was used in Example 17 and compound 6 was used as a reaction sample in Example 18.

The following steps are divided and explained.

Process 1: Intermediate Compound MMTr-Amino-hexanol Synthesis

50 ml of methylene chloride anhydride was added to 3 g of 6-amino-1-hexanol dried at 40 ° C. for 2 hours in a vacuum dryer, and the inlet was plugged with a stopper, and an argon balloon was inserted and dissolved. Dried triethyleneamine (2 equivalents 7.14 ml) and MMTr-Cl (1.1 equivalents 8.7 g) were added and stirred for about 3 hours. The reaction was confirmed by the ninhydrin coloring reagent under TLC conditions (n-Hex: EA = 1: 1). After the reaction, methylene chloride and 5% NaHCO ₃ were added and the layers were separated. After confirming TLC, the methylene chloride layer was treated with Na ₂ SO ₄ and filtered with a glass filter. The organic solvent layer was concentrated and purified by silica column (5.5 × 25 cm). Purification conditions were followed by 2.4 L development after filling with n-Hex: ethyleneamine (5: 1), 1.6 L of n-Hex: EA (3: 1), and 1.8 L of n-Hex: EA (2: 1) Developed. The resulting fractions were concentrated and dried overnight in a vacuum dryer to give MMTr-amino-hexanol (9.29 g, 93%).

Process 2: Intermediate MMTr 1-O-Aminohexyl l 2,3,4,6-Tetra Acetyl Glucose Synthesis

A magnet rod, molecular sieve, and Ag₂CO₃6.2 g were added to 3.1 g of acetyl bromo glucose and dried at 40 ° C. for 2 hours in a vacuum oven. 31 ml of methylene chloride anhydride was added thereto, the inlet was closed with a stopper, and an argon balloon was inserted and stirred. 8.81 g of MMTr-amino-hexanol was added thereto, and the mixture was stirred overnight. The reaction was hydrolyzed in small amounts to confirm the reaction under TLC conditions (n-Hex: EA = 1: 2). After the reaction was filtered with a glass filter. Methylene chloride and 5% NaHCO₃ were added, and the layers were separated. After TLC confirmation, the methylene chloride layer was treated with Na₂SO₄, filtered through a glass filter, and concentrated to form MMTr 1-O-aminohexyl 2,3,4,6-tetraacetylacetylglucose. (10.14 g).

Step 3: Synthesis of Intermediate MMTr 1-O-Aminohexyl Glucose

To MMTr 1-O-aminohexyl 2,3,4,6-tetra acetyl glucose (10.14 g) was added 89.5 ml of methanol and 89.5 ml of 1 M NaOH and stirred for 1 hour. The reaction was confirmed by TLC conditions (n-Hex: EA = 1: 2) and (MC: MeOH = 9: 1) and concentrated after the reaction. Ethylene amine and distilled water were added, the layers were separated, and after confirming TLC, the ethylene amine layer was filtered with a glass filter after Na₂SO₄ treatment. The filtrate was concentrated and purified by silica column. Purification conditions were developed after filling with MC: MeOH (95: 5) followed by MC: MeOH (9: 1). The resulting fractions were concentrated and then dried in a vacuum oven overnight to give MMTr 1-O-aminohexyl glucose (1.57 g, 39.5%).

Step 4: Synthesis of Intermediate 1-O-Aminohexyl Glucose

64.65 ml of MeOH and 432.36 ml of acetic acid were added to MMTr 1-O-aminohexyl glucose (1.55 g) prepared in Step 3, followed by stirring for about 8 hours. The reaction was confirmed under TLC conditions (n-pro: NH₄OH = 6: 10). After concentration, the layers were separated by methylene chloride and water, and after confirming TLC, the water layer was concentrated to obtain 1-O-aminohexyl glucose (1.04 g, 100%).

Process 5: FK506-M-LA-Glc synthesis

Compound 4, FK506-M32-LA (1.406 g), prepared in Example 4, was dissolved in anhydrous pyridine (10 ml) and triethanolamine (10 equiv, 1.833 ml), followed by 1-O-aminohexyl glucose (1.5 equiv, 0.551 g) was added and stirred. After overnight reaction, the reaction was confirmed by TLC (methylene chloride: MeOH 5: 1), followed by silica column (4.5x24 cm) MC: MeOH (9: 1, 8: 1, 6: 1) to obtain a mixed product. . The mixture was then separated through Prep HPLC purification to give compound 16, FK506-M32-LA-Glc (1.113 g).

<Examples 19, 20, 21>

Example 19 describes a method for preparing Compound 19, and Compound 20 and Compound 21 of Examples 20 and 21 can be prepared by the same method. In this case, instead of compound 16, compound 17 was used in Example 20, and compound 18 was used as a reaction sample in Example 21.

1,4 galactosyltransferase (LgtB), which transfers galactose to compound 4, FK506-M-LA-glc, synthesized by Example 16 from glucose-galactose precursor in glycosyltransferase to position 4 of glucose ) 19 FK506-M-LA-Lac was synthesized.

0.15 g of UDP-D-galactose, MgCl ₂ 6H ₂ O and Tris HCl buffer (pH 7.5) were added to 25-50 mM in 20 ml of distilled water of the reactor, and the substrates were dissolved well and calibrated to pH 7.5. 1,4 galactosyltransferase was added to this solution, and 0.13 g of FK506-M-LA-Lac was dissolved in 13 ml of DMSO, and slowly added to the reactor. Maintaining the pH of the reaction condition 7.5 and reacting at 37 ℃, the degree of conversion was confirmed through HPLC analysis. When the conversion was 90% or more, the reaction was terminated and the reaction product Compound 19, FK506-M32-LA-Lac was purified using Prep-HPLC. Purification conditions included buffer (A), distilled water in a reversed phase column (50 × 500 mm); (B), and purified with acetonitrile with time-dependent change, and the active fractions were dried using a vacuum dryer and a lyophilizer to obtain compound 19, FK506-M32-LA-Lac (0.016g).

<Examples 22, 23, 24>

Example 22 describes a method of preparing Compound 19, and Compounds 20 and 21 of Examples 23 and 24 may be prepared by the same method. At this time, instead of compound 4, compound 5 was used in Example 23, and compound 6 was used as reaction sample in Example 24.

The following steps are divided and explained.

Process 1: MMTr-1-O-aminohexyl 2,3,6,2'3'4'6'-hepta acetyl lactose synthesis

45 g of acetyl bromo lactose was added to a magnet rod, molecular sieve, and 35.5 g of Ag₂CO₃, and dried at 40 ° C. for 2 hours in a vacuum dryer. 450 ml of anhydrous methylene chloride was added, the inlet was closed with a stopper, and an argon balloon was inserted and stirred. 30 g of MMTr-amino-hexanol was added thereto and stirred overnight. In the TLC condition (n-Hex: EA = 1: 2), since the starting material and the reactant were in the same position, the reaction was confirmed by a small amount of hydrolysis. After the reaction was filtered with a glass filter. Methylene chloride and 5% NaHCO₃ were added, and the layers were separated. After confirming TLC, the methylene chloride layer was treated with Na₂SO₄, and then filtered through a glass filter and concentrated. This gave MMTr-1-O-aminohexyl 2,3,6,2'3'4'6'-hepta acetyl lactose (60 g).

Process 2: MMTr-1-O-Aminohexyl Lactose Synthesis

To MMTr-1-O-aminohexyl 2,3,6,2'3'4'6'-hepta acetyl lactose (60 g) was added with a minimum amount of MeOH and 457.5 ml of 1M NaOH. The reaction was confirmed by n-Hex: EA = 1: 2) and (MC: MeOH = 9: 1) and concentrated after the reaction. Ethyleneamine and distilled water were added, the layers were separated and confirmed by TLC. The ethyleneamine layer was treated with Na2SO3 and filtered through a glass filter. The filtrate was concentrated and then loaded onto a silica column and purified. Purification conditions were developed with MC: MeOH (9: 1) after filling MC: MeOH (95: 5). The obtained active ingredient was concentrated and dried overnight in a vacuum dryer to obtain MMTr-1-O-aminohexyl lactose (32 g).

Step 3: 1-O-aminohexyl lactose synthesis

To MMTr-1-O-aminohexyl lactose (32 g), a minimum amount of MeOH and 1.1 L of acetic acid were added, followed by stirring for about 8 hours. The reaction was confirmed by TLC conditions (methylene chloride: MeOH = 2: 1), and the reactants were concentrated, and then the layers were separated by methylene chloride and water to recover and concentrate the water layer. It was dried to give 1-O-aminohexyl lactose (5.3 g).

Process 4: synthesis of FK506-LA-Lac

Compound 4, FK506-M32-LA (1.8 g), obtained in Example 4, was dissolved in anhydrous pyridine (70 ml) and triethanolamine (10 equiv, 2.3 ml), followed by 1-O-aminohexyl lactose (1.2 equiv, 0.89). g) was added and stirred. After overnight reaction, the reaction was confirmed by TLC (MC: MeOH 5: 1) and developed on a silica column with methylene chloride: MeOH (9: 1, 8: 1, 5: 1) to give a compound 16, FK506-M32-LA-Lac Was obtained (1.5 g).

<Examples 25, 26, 27>

In Example 25, a method of preparing Compound 22 is described, and Compounds 23 and 24 of Examples 26 and 27 may be prepared by the same method. Instead of compound 19, compound 20 was used in Example 26 and compound 21 was used as a reaction sample in Example 27.

FK506-M-LA-Lac and CMP- using sialyltransferase (SialT), which transfers sialic acid to galactose of Compound 19, FK506-M32-LA-Lac, prepared by Example 19 or Example 22 Compound 22, FK506-M32-LA-SL, to which sialic acid was added, was synthesized from the sialic acid precursor.

1.52 g of CMP-sialic acid and Tris HCl buffer (pH 7.5) were dissolved in 0.47 L of distilled water to 25 to 50 mM, and calibrated to pH 7.5. After the a2,3 sialyltransferase was added to the reaction solution, 1.35 g of the reaction starting material FK506-M32-LA-Lac was dissolved in 25 ml of DMSO, and the reaction was slowly added dropwise to the reactor. The reaction was carried out while maintaining pH 7.5 at room temperature, and the degree of conversion to HPLC analysis was confirmed. When the conversion was 90% or more, the same amount of MeOH (0.5 L) was added to terminate the reaction, and the reaction solution was concentrated using a reduced pressure dryer. The concentrate was loaded onto a silica column (8 x 33 cm) filled with methylene chloride and fully developed to 100% MC to remove DMSO. After some removal of DMSO, it was developed with MC: MeOH (2: 1) to obtain FK506-M32-LA-SL as a yellow oil. In order to increase the purity was further purified using Prep-HPLC. Purification conditions included buffer (A), 0.01% TFA in reverse phase column (50 × 500 mm); (B), the purification was carried out with a change in time with a solvent of acetonitrile. The final reaction product, Compound 22, FK506-M32-LA-SL fractions, was dried using a vacuum dryer and a reduced pressure freeze dryer (0.5 g).

<Examples 28, 29, 30>

In Example 28, a method of preparing Compound 22 is described, and Compounds 23 and 24 of Examples 29 and 30 can be prepared by the same method. At this time, instead of compound 4, compound 5 was used in Example 29, and compound 6 was used as reaction sample in Example 30.

The following steps are divided and explained.

Process 1: 1-O-aminohexyl 3'-sialyl lactose synthesis

2.6 g of 1-O-aminohexyl lactose, 5.76 g of CMP-sialic acid, and Tris HCl buffer (pH 7.5) were added to 25 to 50 mM in a reactor and dissolved in 0.11 L of distilled water. Correction was made to 7.5. The reaction was started by adding a2,3 sialyltransferase. The reaction proceeded while maintaining pH 7.5 at room temperature, and the reaction was terminated when TLC analysis showed more than 90% conversion. Methanol equivalent amount (0.12 L) was added to terminate the reaction, followed by centrifugation to remove solids, and methanol was removed by concentration. The concentrated reaction solution was loaded on a column filled with an ion exchange resin (Acros, DoweX 1x2, 50-100 mesh, 5 x 45 cm) to sequentially change the NaCl concentration of the eluent to separate the reaction product. Thereafter, high purity 1-O-aminohexyl 3'-sialylactose was purified using a column filled with exclusion resin (Bio-RAD, Bio-Gel P2 gel, 5 x 100 cm) ( 2 g).

Process 2: synthesis of FK506-M32-LA-SL

Compound 4, FK506-M32-LA (0.94 g) prepared as in Example 4, was dissolved in anhydrous pyridine (70 ml) and triethanolamine (10 equiv, 1.2 ml), and then 1-O-aminohexyl obtained in step 1 3'-sialylactose (1.2 equiv, 0.776 g) was added and stirred. After overnight reaction, the reaction was confirmed by TLC (MC: MeOH 3: 1) and loaded onto a silica column. MC: MeOH (5: 1, 2: 1) gave compound 22, FK506-M32-LA-SL (1.24 g).

Analysis Example 1 Analysis of Solubility of FK506 Glycosylated Derivatives

In order to measure and compare the solubility in the aqueous solution of the FK506 glycosylated derivatives prepared according to the above example, a calibration curve for the area (A) of HPLC (UV) to the weight of the FK506 standard was prepared and the relative water solubility was confirmed. . Each sample was dissolved in a certain amount of distilled water in a supersaturated state and left for 30 minutes in a constant temperature water bath at 37 ° C., and the supernatant was analyzed by HPLC analysis to obtain the area (A) of the peak appearing at a UV wavelength of 260 nM. The amount of was obtained. As a result, when the solubility of FK506 is 1, the solubility of FK506-M32-LA-glc is 11, FK506-M32-LA-Lac is 160, FK506-M32-LA-SL is> 5000, and FK506-D-LA- SL is> 5000, FK506-D-LA-glc is> 5000, FK506-D-LA-Lac is> 5000, and FK506-M32-LS-glc is 2.5, FK506-M32-LS-Lac is 76.3, FK506- M32-LS-SL showed high solubility, such as> 10000.

Example 31: Validation of FK506 Glycosylated Derivatives

In Vitro cell based assay was performed to determine the usefulness of the 12 FK506 glycosylated derivative compounds synthesized in the Examples.

Spleens were extracted from C57BL / 6 mice (magnetic, 6 week old) and placed in Petri dishes to release spleen cells using a syringe plug. The suspension containing the cells was transferred to a 15 ml tube, allowed to stand for 5 minutes, the supernatant was taken, and centrifuged to separate the cell precipitate with RPMI complete medium containing 10% FBS (Hyclone, Logan, UT, USA) (Invitrogen Life Technologies, Carlsbad, Splenocytes were prepared by resuspension in CA, USA). Spleen lymphocytes were adjusted to a concentration of 1 × 10 ⁶ cells / ml and then divided into 200 μl per well in 96-well plates. One hour after the sample to be evaluated, Concanavalin A (1 μg / ml, Sigma-Aldrich, St. Louis, MO, USA) was treated and incubated for 3 days in a 37 ° C., 5% CO ₂ incubator. During the last 18 hours, 1 μCi [3H] thymidine was added to each well, and the cells were harvested using an automatic cell collection device (Inotech, Dottikon, Switzerland) and the Wallac Microbeta scintillation counter (Wallac, Turku, Finland) The amount of [3 H] thymidine inserted into the DNA was measured.

Samples used in this experiment were FK506, FK506-M32-LS (Compound 1), FK506-D-LS (Compound 3), FK506-M32-LS-Glc (Compound 7), FK506-M32-LS-Lac (Compound 10), FK506-M32-LS-SL (Compound 13), FK506-M32-LA-Glc (Compound 16), FK506-M24-LA-Glc (Compound 17), FK506-M32-LA-Lac (Compound 19) , FK506-M24-LA-Lac (Compound 20), FK506-M32-LA-SL (Compound 22), FK506-M24-LA-SL (Compound 23). Experiments were carried out under various concentration conditions (pM ~ nM level) and the results are shown in Figures 2a, b, c.

As a result of activity measurement, the immunosuppressive activity of FK506 was confirmed that the IC ₅₀ value is very strong in the 1-10 pM region. Compared to such potent activity, FK506 -polysaccharide derivatives of the present invention showed a level of 1-100 nM activity, but was lower than FK506, it was confirmed that the immunosuppressive activity is maintained to a considerable extent. This is a high immunosuppressive activity comparable to the activity of cyclosporin A (CsA) reported to be active by the same mechanism as FK506. Aminohexyl linker is more active than succinyl linker, and sugar is attached at 32-OH position more than that modified at 24-OH position. It showed activity, but the difference was not large. In particular, there was no significant difference in the decrease in activity depending on the degree of sugar addition. When siallylactose is attached, water solubility was improved by more than 10,000-fold by the increase of sugar chains, and it was confirmed that the immunosuppressive activity was also significantly maintained.

Claims (13)

Tricyclo saccharified derivative compounds represented by the formula (1) made from tricyclo compounds as a raw material, pharmaceutically acceptable salts or solvates thereof.
&Lt; Formula 1 >

Wherein Rx is a hydroxyl group, a methoxy group or H, and at least one of R ₁ and R ₂ is an aldose or ketose consisting of C 3 to C 7 linked to a linker of C 1 to C 12 by an ester bond or an amide bond ) 1 to 5 sugar chains are bonded, and R ₁ or R ₂ with no sugar chains is H)
The method of claim 1,
And said tricyclo compound is one selected from tacrolimus, ascomycin, rapamycin and meridamycin, pharmaceutically acceptable salts or solvates thereof.
The method of claim 1,
The linker may be a formyl group, an acetyl group, a propionyl group, a butyl group, an acryl group, an ethylsuccinyl group, a succinyl group, or Compounds, pharmaceutically acceptable salts or solvates thereof, characterized in that they comprise aminohexyl groups.
The method of claim 1,
The aldose or ketose is glucose, galactose, fructose, fucose, mannose, rhamnose, galactosamine, glucosamine, N-acetylgalactosamine, N-acetylglucosamine, vancosamine, epi-vancosamine, and glue A compound, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the compound is one selected from the group consisting of curonic acid, sialic acid, deoxyglucose, and deoxygalactose.
The method of claim 1,
The compound
(Compound 7); R ₁ =

, R2 = H,
(Compound 8); R ₁ = H, R ₂ =

,
(Compound 9); R ₁ , R ₂ =

,
(Compound 10); R ₁ =

, R ₂ = H,
(Compound 11); R ₁ = H, R ₂ =

,
(Compound 12); R ₁ , R ₂ =

,
(Compound 13); R ₁ =

, R ₂ = H,
(Compound 14); R ₁ = H, R ₂ =

,
(Compound 15); R ₁ , R ₂ =

,
(Compound 16); R ₁ =

, R ₂ = H,
(Compound 17); R ₁ = H, R ₂ =

,
(Compound 18); R ₁ , R ₂ =

,
(Compound 19); R ₁ =

, R ₂ = H,
(Compound 20); R ₁ = H, R ₂ =

,
(Compound 21); R ₁ , R ₂ =

,
(Compound 22); R ₁ =

, R ₂ = H,
(Compound 23); R ₁ = H, R ₂ =

And
(Compound 24); R ₁ , R ₂ =

A compound, a pharmaceutically acceptable salt thereof, or a solvate thereof, characterized in that one kind selected from among them.
(a) introducing succinyl group into 1 -OH position using glucose as a raw material and introducing a protecting group to another -OH group; And

(The protecting groups R ₃ and R ₄ are the same or different and are protecting groups of OH groups that can be dissociated by acid or base treatment and transformed into H.)
(b) removing the protecting groups R ₃ and R ₄ of compound 25 after adding the macrolide compound to compound 25;
And a tricyclo saccharified derivative compound represented by the following Compound 7, Compound 8, or Compound 9, a pharmaceutically acceptable salt thereof, or a solvate thereof.
{Wherein Compound 7 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ =

, R2 = H),
Compound 8

Where Rx is hydroxyl, methoxy or H, R ₁ = H, R ₂ =

),
Compound 9 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ and R ₂ =

being}
Tricyclo glycosylated derivative compounds represented by compounds 10, 11 or 12, respectively, including the process of adding galactosyltransferase and UDP-D-galactose to compounds 7, 8 or 9 shown in claim 6, pharmaceutically acceptable salts thereof Or a process for preparing solvates thereof.
{Compound 10 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ =

, R ₂ = H),
Compound 11 is

Where Rx is hydroxyl, methoxy or H, R ₁ = H, R ₂ =

),
Compound 12 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ and R ₂ =

)being}
Tricyclo glycosylated derivative compounds represented by compounds 13, 14 or 15, pharmaceutically acceptable salts thereof or solvents thereof, including the steps of adding CMP-sialic acid and sialyltransferase to compounds 10, 11 or 12 of claim 7 How to manufacture a cargo.
{Wherein Compound 13 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ =

, R ₂ = H),
Compound 14 is

Where Rx is hydroxyl, methoxy or H, R ₁ = H, R ₂ =

),
Compound 15 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ and R ₂ =

)being}
1-o-aminohexyl glucose is added to the following compounds 4, 5 or 6 to prepare tricyclo glycated derivative compounds represented by compounds 16, 17 or 18, pharmaceutically acceptable salts or solvates thereof.
{Compound 4 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ =

, R ₂ = H),
Compound 5

Where Rx is hydroxyl, methoxy or H, R ₁ = H, R ₂ =

),
Compound 6 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ and R ₂ =

),
Compound 16 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ =

, R ₂ = H),
Compound 17 is

Where Rx is hydroxyl, methoxy or H, R ₁ = H, R ₂ =

),
Compound 18 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ and R ₂ =

being)}
Tricyclo glycosylated derivative compounds represented by compounds 19, 20 or 21, respectively, by adding UDP-D-galactose and galactosyltransferase to the tricyclo glycated derivative compounds represented by compounds 16, 17 or 18 of claim 9, pharmaceutically Process for preparing acceptable salts or solvates thereof.
{Compound 19 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ =

, R ₂ = H),
Compound 20

Where Rx is hydroxyl, methoxy or H, R ₁ = H, R ₂ =

),
Compound 21 is

Wherein Rx is hydroxyl, methoxy or H, R ₁ and R ₂ =

)being}
Adding 1-o-aminohexyl lactose to compound 4, 5 or 6 of claim 9 to prepare a tricyclo glycosylated derivative compound of compound 19, 20 or 21 represented by claim 10, a pharmaceutically acceptable salt thereof or solvate thereof Way.
Sialyltransferase and CMP-sialic acid are added to compound 19, 20 or 21 shown in claim 10 to prepare tricyclo glycated derivative compounds represented by compound 22, 23 or 24, pharmaceutically acceptable salts or solvates thereof. How to.
{Compound 22 is

Wherein Rx is hydroxyl, methoxy or H,
R ₁ =

, R ₂ = H),
Compound 23 is

Wherein Rx is hydroxyl, methoxy or H,
R ₁ = H, R ₂ =

),
Compound 24 is

Wherein Rx is hydroxyl, methoxy or H,
R ₁ and R ₂ =

)being}
Tricyclo glycosylated derivative compounds represented by compounds 22, 23 or 24 represented by claim 12 by adding 1-O-aminohexyl sialylactose to compounds 4, 5 or 6 shown in claim 9, or a pharmaceutically acceptable salt thereof Method for preparing solvates.

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