Processes for the preparation of GalNAc acid derivatives

Abstract

The invention comprises a new process for the preparation of GalNAc derivatives of the formula I ##STR00001## wherein n is an integer between 0 and 10 and its salts, corresponding enantiomers and/or optical isomers thereof. The GalNAc acid derivative of formula I can be used for the preparation of therapeutically valuable GalNAc oligonucleotide conjugates.

Claims

1. A process for the preparation of a GalNAc acid derivative of the formula I: ##STR00034## or a salt, enantiomer or optical isomer thereof, wherein n is an integer between 0 and 10, comprising the steps: a) reacting a triamine salt of formula II: ##STR00035## wherein R.sup.1 is an ester protecting group and X is an anion of an acid, with a tetrahydropyran acid of formula III: ##STR00036## wherein R.sup.2 is a hydroxy protecting group and n is as defined above, in the presence of a peptide coupling agent, an amine base and an organic solvent, to form a GalNAc ester of formula IV: ##STR00037## wherein R.sup.1, R.sup.2 and n are as defined above; b) removing the ester protecting group R.sup.1 and the hydroxy protecting groups R.sup.2 in the presence of a mineral base to form a GalNAc acid salt of formula V: ##STR00038## wherein n is as defined above and M is a metal cation; and c) optionally transforming the GalNAc acid salt of formula V into the GalNAc acid derivative of formula I.

2. The process of claim 1, wherein n is an integer between 0 and 5, the ester protecting group R.sup.1 is C.sub.1-7 alkyl or phenyl-C.sub.1-7 alkyl, wherein the phenyl group is optionally substituted with halogen or C.sub.1-7 alkyl, the hydroxy protecting group R.sup.2 is acetyl, and X is an anion of a sulfonic acid.

3. The process of claim 1, wherein the peptide coupling agent is n-propylphosphonic acid anhydride and the amine base is a tertiary amine.

4. The process of claim 1, wherein the coupling step a) takes place in an organic solvent at a reaction temperature from 20 C. to 70 C., wherein the organic solvent is a polar aprotic solvent.

5. The process of claim 1, wherein the mineral base in step b) is an alkali hydroxide.

6. The process of claim 1, wherein the steps a) and b) are combined and performed in one step without isolating the GalNAc ester of formula V.

7. The process of claim 1, wherein the optional step of transforming the GalNAc acid salt of formula V into the GalNAc acid derivative of formula I is performed via cation exchange or via treatment with an acid.

8. The process of claim 1, wherein the GalNAc acid derivative of formula I is of the formula Ia: ##STR00039## or a salt or enantiomer thereof.

9. The process of claim 1, wherein the process further comprises producing the triamine salt of formula II comprising the steps: a1) transforming a carboxylic acid of formula X: ##STR00040## wherein R .sup.3 and R.sup.4 are different and independently amino protecting groups, into an ester of formula XI: ##STR00041## wherein R.sup.1 is an ester protecting group and R.sup.3 and R.sup.4 are as defined above; b1) removing the amino protecting group R.sup.4 in the ester of formula XI, and subsequently forming an amine salt of formula XII: ##STR00042## wherein R.sup.1 and R.sup.3 are as defined above and X.sup. is an acid anion; c1) coupling the amine salt of formula XII with a hexanoic acid derivative of formula XIII: ##STR00043## wherein R.sup.3 and R.sup.3 are amino protecting groups to form the protected triamine of formula XIV: ##STR00044## wherein R.sup.3, R.sup.3, R.sup.3 and R.sup.1 are as above; and d1) converting the protected triamine of formula XIV with an acid into the triamine salt of formula II.

10. Process of claim 9, wherein R.sup.3, R.sup.3 and R.sup.3 are the same and protecting groups which are cleavable under acidic conditions and R.sup.4 is a protecting group which is cleavable under basic conditions or by way of hydrogenolysis.

11. Process of claim 10, wherein R.sup.3, R.sup.3 and R.sup.3 are Boc and R.sup.4 is FMOC.

12. Process of any one of claims 9, wherein the transformation in step a1) takes place with benzyl alcohol in the presence of an activating agent, an amine catalyst and an aprotic organic solvent at a reaction temperature of 20 C. to 50 C.

13. Process of any one of claims 9, wherein the amino protecting group R.sup.4 is FMOC and its removal in step b1) is performed with a secondary aliphatic amine in a polar aprotic solvent at a reaction temperature of 20 C. to 50 C.

14. Process of any one of claims 9, wherein the subsequent formation of the amine salt of formula XII in step b1) is effected with a sulfonic acid.

15. Process of any one of claims 9, wherein the coupling in step c1) is performed with n-propylphosphonic acid anhydride as coupling agent in the presence of a tertiary amine and a polar aprotic solvent at a reaction temperature from 20 C. to 50 C.

16. The process of claim 9, wherein in step d1) the triamine salt of formula II is formed with a sulfonic acid in a polar aprotic solvent at a reaction temperature of 20 C. to 80 C.

17. The process of claim 16, wherein a polar aprotic solvent is selected which prevents the triamine salt of formula II to crystallize.

18. The process of claim 1, wherein the process further comprises producing the tetrahydropyran acid of formula III comprising the steps: a2) transforming a diol of formula XX: ##STR00045## wherein n is as defined in claim 1, into an alcohol ester of formula XXI: ##STR00046## wherein n is as defined in claim 1 and R.sup.5 is an ester protecting group; b2) coupling the alcohol ester of formula XXI with a tetrahydropyran derivative of formula XXII: ##STR00047## wherein each R.sup.2 and R.sup.6 are independently hydroxy protecting groups, to form a tetrahydropyran ester of formula XXIII: ##STR00048## wherein n, R.sup.2 and R.sup.5 are as defined above; and c2) removing the ester protecting group R.sup.5 to form the tetrahydropyran acid of formula III.

19. The process of claim 18, wherein the hydroxy protecting group R.sup.2 is acetyl, the ester protecting group R.sup.5 is benzyl and the hydroxy protecting group R.sup.6 is acetyl.

20. The process of claim 18, wherein in a first step of step a2) the diol of formula XX is deprotonated with an alkali metal alcoholate in the presence of a polar protic or polar aprotic solvent at a reaction temperature from 50 C. to 120 C.

21. The process of claim 18, wherein in a second step of step a2) an acetic acid moiety is introduced with a halogen acetic acid or with a salt thereof in the presence of a polar protic or polar aprotic solvent at a reaction temperature from 50 C. to 120 C.

22. The process of claim 18, wherein in a third step of step a2) the alcohol ester of formula XXI wherein R.sup.5 is benzyl is formed with a benzyl halogenide or a benzyl sulfonylester in a polar aprotic solvent at a reaction temperature from 20 C. to 120 C.

23. The process of claim 18, wherein in step b2) the alcohol ester of formula XXI is coupled with the tetrahydropyran derivative of formula XXII in the presence of a halogenated sulfonic acid in the presence of a polar aprotic solvent at a reaction temperature from 0 C. to 140 C.

24. The process of claim 18, wherein in step c2) the benzylester group is removed by a catalytic hydrogenation with hydrogen in the presence of a hydrogenation catalyst.

25. The process of claim 18, wherein the step of transforming the diol of formula XX into the alcohol ester of formula XXI comprises the steps of: a3) reacting a 2-amino acetate of formula XXV: ##STR00049## wherein R.sup.5 is as defined in claim 18 and X is a halogen atom, with a nitrite salt to form a 2-diazo compound of formula XXVI: ##STR00050## wherein R.sup.5 is as defined in claims 18; and b3) reacting the 2-diazo compound of formula XXVI with the diol of formula XX.

26. The process of claim 25, wherein the diazotization in step a3) is performed with an alkali nitrite in the presence of a solvent mixture of water and a non-polar aprotic solvent at a reaction temperature of 10 C. to 10 C.

27. The process of claim 25, wherein the transformation of the 2-diazo compound with the diol of formula XX in step b3) is performed in the presence of a Lewis acid and a non-polar aprotic solvent at a reaction temperature of 10 C. to 10 C.

28. A process for the preparation of a GalNAc oligonucleotide conjugate comprising the steps: a3) preparing a GalNAc acid derivative of formula I or a GalNAc acid salt of formula V according to claim 1; and b3) conjugating the GalNAc acid derivative of formula I or the GalNAc acid salt of formula V under peptide coupling conditions with an oligonucleotide.

29. The process of claim 28, wherein the GalNAc acid salt of formula V is used.

Description

EXAMPLES

(1) Abbreviations

(2) AcOH acetic acid DMAP 4-(dimethylamino)-pyridine DMF N, N-dimethylformamide EtOH ethanol MeOH methanol rt room temperature THF tetrahydrofuran TBME tert.-butyl methyl ether

Building Block A

Example 1

Benzyl (2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoate

(3) ##STR00020##

(4) 234.0 g (500.0 mmol) (2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid was suspended in 500 ml dichloromethane, 62.0 ml (600 mmol, 1.2 eq) benzyl alcohol and 3.05 g DMAP (25.0 mmol, 0.05 eq) were added. The solution was cooled to 0-5 C. in the course of 40 min, a solution of 108.0 g (525.0 mmol, 1.05 eq) N,N-dicyclohexyl carbodiimide in 500 ml dichloromethane, was added dropwise. The white suspension was stirred for 1 h at 0-5 C. and then for 15 h at room temperature. The suspension was filtered over a G3 glass filter, the white filter cake was washed portion-wise with total 250 ml dichloromethane. The filtrate was evaporated at 650-10 mbar/1 h to obtain a yellow oil, which was in dissolved in 2.0 L ethyl acetate, extracted with 2.0 L 0.5M hydrochloric acid, 2.0 L 1M NaHCO.sub.3 and 1.0 L brine, the organic layer was evaporated to dryness at 40 C./150-10 mbar/5 h to obtain 291.1 g crude benzyl (2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino) hexanoate as white solid in 104% yield and 96.4% purity (HPLC area-%; contains ca. 5% benzyl alcohol). The material was used in the next step without further purification. MS: m/z=459.22735 (M-boc+H).sup.+.

Example 2

Benzyl (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoate methanesulfonic acid salt

(5) ##STR00021##

(6) 291.1 g Benzyl (500.0 mmol) (2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino) hexanoate (HPLC purity; 95.8%; contains ca. 5% benzyl alcohol) were dissolved in 1.4 L THF at room temperature. Within 10 min, 1.04 L diethylamine (10.0 mol, 20 eq) were added, the light yellow solution was stirred for 2 h at room temperature and then evaporated at 40 C./200-10 mbar, 200 ml acetonitrile was added and evaporated again to efficiently remove diethylamine at 40 C./100-10 mbar/1 h. Finally, 268.1 g of a yellow oil was obtained, which was dissolved in 2.5 L acetonitrile, stirred for 10 min at room temperature. Insoluble particles were filtered over a glass fiber filter and washed with 500 ml acetonitrile. The filtrate was treated dropwise in the course of 10 min with 34.0 ml methanesulfonic acid at 20 C.-25 C. The formed white suspension was stirred for 17 h at room temperature and filtered over a G3 glass filter. The filter cake was washed portion-wise with 500 ml acetonitrile. The white crystals were dried at 40 C./15 mbar/4 h to obtain 195.8 g benzyl (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoate methanesulfonic acid salt as white crystals in 91% yield (2 steps) and 99.3% purity (HPLC area-%). MS: m/z=337.2149 (M+H).sup.+.

Example 3

Benzyl (2S)-2-[[(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoate

(7) ##STR00022##

(8) 190.0 g (439.0 mmol) Benzyl (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoate methanesulfonic acid salt were suspended in 1.9 L THF at room temperature. 335 ml (1.98 mol, 4.5 eq)N-ethyldiisopropylamine were added whereby the temperature slightly decreased to 15 C. Next, 213 g (615 mmol, 1.4 eq) (S)-2,6-bis((tert-butoxycarbonyl)amino)hexanoic acid were added and the white suspension was stirred at room temperature for 20 min. 390 ml n-propylphosphonic acid anhydride (T3P as cyclic trimer 50% in ethyl acetate, 659 mmol, 1.5 eq) were added dropwise in the course of 20 min at 20-25 C. (cooled in a cool water bath). The resulting light yellow, cloudy solution was stirred at room temperature for 1.5 h, transferred to a separating funnel, diluted with 1.9 L TBME and extracted with 1.9 L water, 1.9 L 0.5M hydrochloric acid, 1.9 L0.5M NaOH, 1.9 L water and 1.9 L brine. The separated, still cloudy organic layer was filtered over a glass fiber filter, the filter was washed with 100 ml TBME and the combined filtrates were evaporated at 40 C./100 mbar/1 h, 1.0 L TBME (to aceotropic remove water) were added again and evaporated at 40 C./250-10 mbar/1 h to obtain crude 296.4 g as white solid residue.

(9) The crude solid was treated with 500 ml acetonitrile and the cloudy solution was heated to 60-65 C. for 10 min. The mixture was cooled to 20-25 C., stirred for 10 min, filtered over a glass fiber filter and washed with 50 ml acetonitrile. The light yellow solution was evaporated at 40 C./100-10 mbar/4 h to obtain 295 g benzyl (2S)-2-[[(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoate as off-white solid in a yield of 101% (HPLC purity: 100%, diastereomer purity (SS) 98.6%) which was used without further purification in the next step. MS: m/z=565.3741 (M-boc+H).sup.+.

Example 4

Benzyl (2S)-6-amino-2-[[(2S)-2,6-diaminohexanoyl]amino]hexanoate tri-methanesulfonic acid salt

(10) ##STR00023##

(11) 124.0 g (187 mmol) benzyl (2S)-2-[[(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoate was suspended in 1.25 L acetonitrile. 61.0 ml (935.0 mmol, 5.0 eq) methanesulfonic acid was added at 20-25 C. in the course of 10 min (gas evolution). The resulting orange suspension was heated in 40 min to 55-60 C. and stirred for another 1 h at 55-60 C. The orange-red emulsion was cooled to room temperature (debocation was controlled by .sup.1H-NMR) and used without further purification in the A+B assembly step, example 8. MS: m/z=365.2558 (M+H).sup.+.

Building Block B

Example 5a

Benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate

(12) ##STR00024##

(13) 30.0 g (200.0 mmol), 2-[2-(2-Hydroxyethoxy)ethoxy]ethanol were dissolved in 50 ml DMF, at 20-25 C., then, 46.0 ml sodium methoxide 25% (200.0 mmol, 1.0 eq) in methanol were added. The formed solution was evaporated at 40 C./50 mbar/0.5 h (remove of 40 ml solvent), 50 ml DMF was added again and evaporated at 40 C./20 mbar/0.5 h (remove of 15 ml solvent), To the slightly jellylike suspension a solution of 13.9 g bromoacetic acid (100 mmol, 0.5 eq) in 50 ml DMF was added at 20-25 C. and the mixture was stirred for 6 h. 11.9 ml benzyl bromide (100 mmol, 0.5 eq) was added and the mixture stirred for another 16 h at 20-25 C. The reaction mixture was then treated with 200 ml brine and extracted with 200 ml TBME. The separated TBME layer was extracted with 200 ml brine, the separated TBME layer was then dried with anhydrous sodium sulfate, filtered and evaporated at 40 C./300-10 mbar/1 h to obtain crude 23.9 g benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate.

(14) After chromatography (600 g silica 60 (0.063-0.2 mm), mobile phase: ethyl acetate) a total of 7.85 g benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate as colorless oil was isolated in 13% yield and 99.0% purity (HPLC area-%). MS: m/z=299.1517 (M+H).sup.+.

Example 5b

Benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate

(15) ##STR00025##

(16) 11.2 g potassium tert.-butylate (100.0 mmol, 0.5 eq) was suspended in 70 ml 2-methyl-2-butanol (light exothermic 35 C.), then 30.0 g (200.0 mmol) 2-[2-(2-Hydroxyethoxy)ethoxy]ethanol were added dropwise in the course of 5 min. the dropping funnel were rinsed with 10 ml 2-methyl-2-butanol (temp. increase to 45 C.), the solution was heated to 60-65 C., 11.6 g (100 mmol, 0.5 eq) sodium chloroacetate were added and stirred for 16 h at 60-65 C., then 11.9 ml benzyl bromide (100 mmol, 0.5 eq) were added and the mixture stirred for another 16 h at 60-65 C. The reaction mixture was cooled to rt, then treated with 50 ml water and extracted with 80 ml TBME and 40 ml TBME. The combined TBME layer was washed with 50 ml half saturated brine, the organic layer were evaporated at 40 C./300-10 mbar/1 h to obtain crude 27.0 g benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate.

(17) After chromatography (270 g silica 60 (0.063-0.2 mm), mobile phase: start with ethyl acetate/n-heptane 1/1, when pure product are visible, mobile phase were changed to 100% ethyl acetate, total 11.4 g benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate as nearby colorless oil was isolated in 19% yield (38% from sodium chloroacetate) and 99.0% purity (HPLC area-%)

Example 5c

Benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate

(18) ##STR00026##
40.3 g (200.0 mmol) Benzyl 2-aminoacetate hydrochloride was dissolved in 340 ml water and 340 ml toluene cooled to 0-5 C., in the course of 60 min a solution of 16.5 g (240 mmol, 1.2 eq) sodium nitrite in 50 ml water was added dropwise at 0-5 C. under vigorous stirring. The reaction mixture was stirred for 3 hour at 0-5 C. The yellow toluene-layer was separated and washed with 340 ml 1M NaHCO.sub.3 and 340 ml brine, the separated toluene layer was treated with 60 g sodium sulfate and stirred for 1 hour at 20-25 C. The yellow suspension was filtered and washed with 50 ml toluene. The clear yellow toluene solution contain in maximum 200.0 mmol benzyl 2-diazoacetate (8.5% in toluene). This solution was added dropwise in the course of 60 min to a cooled 0-5 C. and well stirred mixture of 60.0 g (400 mmol) triethylen glycol and 465 l (3.67 mmol, 0.02 eq) boron trifluoride diethyl etherate in 170 ml toluene under evolving of nitrogen gas. The yellow reaction mixture was stirred for 90 min at 20-25 C. at which a colorless solution was formed. The solution was extracted with 250 ml brine, the separated organic layer was dried with 60 g sodium sulfate, filtered, washed with 100 ml toluene and evaporated at 40 C./40-10 mbar/1 h to obtain crude 49.9 g benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate. Chromatography was performed with a Teledyne Isco CombiFlash (330 g silica 60 (0.035-0.070 mm Teledyne Isco Cat. No. 69-2203-330), mobile phase: gradient with 15% acetone 85% n-heptane in 45 min to 30% and 70%, fraction size 20 ml. The combined fraction gave 33.88 g benzyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate as colorless oil and with an overall yield of 57% and 99.0% purity (HPLC area-%).

Example 6

Benzyl 2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetate

(19) ##STR00027##

(20) 268.0 g Benzyl 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)acetate (900 mol) were dissolved in 2.4 L dichloromethane. 385.0 g (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyltriacetate (990 mmol, 1.1 eq) and 12.0 ml trifluoromethanesulfonic acid (135 mmol, 0.15 eq) were added. The suspension was heated to reflux with a dean-stark separator (50 ml, to remove AcOH). After 1 h, 4.50 ml trifluoromethanesulfonic acid (50.7 mmol, 0.05 eq) and 50 ml dichloromethane were added to the orange suspension, the solvent (50 ml) from the dean-stark separator was discharged. Every half hour this procedure was repeated, total 6 times (3 h). After a total of 4.5 h, the red solution was cooled to 10-15 C. and added within 30 min at 20-25 C. to a solution of 1.8 L 1M sodium hydrogen carbonate (1.8 mol, 2.0 eq) (CO.sub.2 evolution, pH 7-8). The yellow organic layer was separated and evaporated at 40 C./600-10 mbar/3 h to obtain 585.4 g of crude benzyl 2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetate as yellow oil (HPLC purity: 87%). The crude product was dissolved in 700 ml acetone and charged to a preloaded silica column (3.0 kg silica 60; 0.063-0.2 mm). The chromatography was conducted using n-heptane/acetone as mobile phase (gradient from 5:1 to 1:2). The combined collected fractions were evaporated at 40 C./600-10 mbar and dried at 20-25 C./0.3 mbar/3 h to obtain 465.0 g benzyl 2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-pyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetate as yellow oil in 83% yield and 100% purity (HPLC area-%). MS: m/z=628.2627 (M+H).sup.+.

Example 7

2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetic acid

(21) ##STR00028##

(22) Under argon atmosphere, 456.0 g Benzyl 2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-pyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetate (727 mmol) were dissolved in 1.4 L THF. 4.56 g Pd/C 10% were added and the argon atmosphere was replaced with hydrogen (1 bar). The black suspension was hydrogenated at 20-25 C. for 2 h. The hydrogen atmosphere was replaced with argon, the black suspension was filtered and the filter cake was washed portion-wise with total of 400 ml THF. The colorless filtrate (HPLC purity: 71% and 27% toluene) was used without any purification in the A+B assembly step, example 8. MS: m/z=538.2191 (M+H).sup.+.

Assembly of Building Block A and B

Example 8a

Benzyl (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate

(23) ##STR00029##

(24) The red-orange solution (1.4 L) of benzyl (2S)-6-amino-2-[[(2S)-2,6-diaminohexanoyl]amino]hexanoate tri-methanesulfonate (180.0 mmol) from Example 4 was diluted with 3.60 L acetonitrile. At 20-25 C., 365.0 ml N-ethyldiisopropylamine (2.16 mol, 12.0 eq) were added within 5 min. To the formed sticky slurry, a solution (2.25 L) of 2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetic acid (720 mmol, 4.0 eq) from Example 7 was added at 20-25 C. in within 10 min, whereby the temperature slightly increased to 40 C. At 45-50 C., a solution of 425 ml n-propylphosphonic acid anhydride (T3P, trimer 50% in ethyl acetate, 720 mmol, 4.0 eq) was added within 10 min. The reaction solution was stirred for 1 h at 45-50 C. The light yellow solution was cooled to 20-25 C. and evaporated at 40 C./10 mbar/6 h to obtain crude 1.06 kg benzyl (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate (HPLC purity: 24.1%). The crude product was precipitated in three portions to remove methansulfonic acid N-ethyldiisopropylamine and residual T3P. 353 g crude product was dissolved in 7.0 L 2-propanol, cooled in 1 h to 25 C., stirred for 1 h at 25 C., filtered over a precooled (25 C.) G3-glass-filter (no rinse), a part from the precipitated product deposited on the glass-wall from the reactor. All precipitates were dissolved portion-wise from the filter and glass-wall with a total of 1.0 L THF. The combined solutions were evaporated at 40 C./20 mbar/6 h to obtain 390.0 g benzyl (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate (HPLC purity: 71.9%), which was used without further purification in the next step. MS: m/z=1923.8438 (M+H).sup.+

Example 8b

Sodium; (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl) tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate

(25) ##STR00030##

(26) The red-orange solution (95 ml) of benzyl (2S)-6-amino-2-[[(2S)-2,6-diaminohexanoyl]amino]hexanoate tri-methanesulfonate (12.2 mmol) was diluted with 240 ml acetonitrile. At 20-25 C., 30.0 ml N-ethyldiisopropylamine (2.16 mol, 14.5 eq) were added within 5 min. To the formed sticky slurry, a solution (150 ml) of 2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetic acid (48.8 mmol, 4.0 eq) was added at 20-25 C. in within 10 min, whereby the temperature slightly increased to 40 C. At 45-50 C., a solution of 28.8 ml n-propylphosphonic acid anhydride (T3P, trimer 50% in ethyl acetate, 48.8 mmol, 4.0 eq) was added within 10 min. The reaction solution was stirred for 1 h at 45-50 C. The light yellow solution was cooled to 20-25 C. and evaporated at 40 C./10 mbar/6 h to obtain crude 73.6 g benzyl (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate (HPLC purity: 32% area).

(27) 68.0 g (11.0 mmol) of the crude product was dissolved in 340 ml methanol 20.0 ml (220 mmol, 20 eq) NaOH 10.8M was added to the light yellow solution, the temperature increased to 32 C., the reaction mixture was stirred for 2.5 h at rt, whereby a suspension was formed (pH 12.0). The suspension was filtered and the filter cake was washed with 100.0 ml methanol, the filtrate was evaporated at 40 C./250-10 mbar/2 h to obtain 41.5 g sodium (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate, which was then purified by preparative reversed phase chromatography, conditions see experiment 9.

Example 9

Sodium; (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate

(28) ##STR00031##

(29) 378.0 g (197.0 mmol, crude) Benzyl (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate was dissolved in 1.9 L methanol. Within 10 min, 200.0 mL 10.8 M sodium hydroxide solution (2.16 mol, 11.0 eq) were added at 20-25 C. Thereby the temperature increased to 31 C. The light yellow solution was stirred for 2 h at 20-25 C. (pH 13.4), then 80.0 mL 5M ammonium chloride solution were added (pH 10.7). The light yellow solution was then evaporated at 20-25 C./100-20 mbar/5 h and dried at 20-0.5 mbar/1 h to obtain crude 543 g sodium (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate (HPLC purity: 40.1%), which was then purified by preparative reversed phase chromatography.

(30) Column: Triart C18-120 2615 cm; 10 um;

(31) Mobile phase: A: 2 mM NaHCO.sub.3/B: Acetonitrile;

(32) Gradient:

(33) TABLE-US-00001 Minutes A B Flow(ml/Min) 0 94 6 700 2 94 6 700 20 88 12 700 20.1 10 90 750 26 10 90 750 26.1 94 6 700 36 94 6 700
Thermostatization: room temperature
Detection: 220 nm
Solution: 543 g dissolved in 4500 ml 2 mM NaHCO.sub.3 and filtered (GF5)(=5000 ml (109 mg/ml)
Sample solution/Injection: Per run 200 ml sample=21.8 g (25 runs) Concentration: The combined fractions (46 L) were diluted with 110 L water, this solution were pumped in 3 portions to a RP C18 column and washed with water/MeOH 98/2, then with MeOH eluted and concentrated on a rotary evaporator to obtain 1.18 kg methanolic solution. A quarter of the 1.18 kg methanolic solution of the preparative HPLC purification step, i.e. 295 g were evaporated at 40 C./20 mbar/1 h and then at 20-25 C./0.35 mbar/14 h to dryness to obtain 43.5 g sodium; (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate as amorphous white powder, 99.88% HPLC purity. The remaining three-quarters of the above solution (885 g) were used in the next step. MS: m/z=1452.684 (MH).sup..

Example 10

(2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoic acid

(34) ##STR00032##

(35) The methanol solution (885 g) from Example 9 was treated at 20-25 C. with 47.9 g Dowex (508 kation-exchanger; H.sub.3O.sup.+ conc. 2.57 mmol/g) stirred for 1 h (pH 3.1), filtered and washed with 200 mL methanol. The filtrate was evaporated at 20-25 C./15-50 mbar and dried at 20-25 C./0.01 mbar/2 h to obtain 128.0 g (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoic acid as a white amorphous powder, 99.77% HPLC purity. MS: m/z=1452.684 (MH).sup..

Example 11

Calcium; (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoate

(36) ##STR00033##

(37) 0.10 g (0.068 mmol), (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoic acid, was dissolved in 3.0 ml methanol and 0.30 ml water, 2.60 mg (0.034 mmol, 0.5 eq) calcium hydroxide was added and the mixture was stirred for 1 h at room temperature. The light cloudy solution was evaporated at 40 C./200-10 mbar/1 h to obtain 0.11 g as white solid. 99.60% HPLC purity. MS: m/z=1452.684 (MH).sup..

(38) Conjugation to Oligonucleotide

Example 11

(in Accordance with Example 15 of US Patent Application Publication 2011/0207799)

(39) (20 mg, 0.014 mmol) (2S)-6-[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-2-[[(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoic acid (GalNAc acid) was co-evaporated with pyridine and dichloromethane. The residue was dissolved in dry DMF (0.9 ml) and a solution of N-Hydroxysuccinimide (HOSu) in DMF (1.6 mg, 0.014 mmol) was added while stirring under an argon atmosphere. At 0 C. a solution of N,N-Dicyclohexylcarbodiimide (DCC) in DMF (3.2 mg, 0.016 mmol) was slowly added. The reaction was allowed to warm to room temperature and stirred overnight. The formed GalNAc N-hydroxysuccinimid ester was used without further purification for conjugation to RNA.

(40) The RNA used was an amino-modified RNA having the sequence: 5-(NH.sub.2C.sub.6)GGAAUCuuAuAuuuGAUCcAsA-3 (SEQ ID 1) wherein u and c are the respective 2-O-methyl nucleotides of the corresponding bases and s means phosphorothioate.

(41) The RNA (2.54 mol) equipped with a C-6 amino linker at the 5-end was lyophilized and dissolved in 250 L sodium borate buffer (0.1 mol/L sodium borate, pH 8.5, 0.1 mol/L KCl) and 1.1 mL DMSO. After addition of 8 L N,N-Diisopropylethylamine (DIPEA), a solution of the GalNAc N-hydroxysuccinimid ester (theoretical 0.014 mmol) in DMF was slowly added under continuous stirring to the RNA solution. The reaction mixture was agitated at 35 C. overnight. The reaction was monitored using RP-HPLC (Resource RPC 3 ml, buffer: A: 100 mM Triethylammonium acetate (TEAA, 2.0 M, pH 7.0) in water, B: 100 mM TEAA in 95% acetonitrile, gradient: 5% B to 22% B in 20 CV). After precipitation of RNA using sodium acetate (3 M) in EtOH at 20 C., the RNA conjugate was purified using the conditions described above. The pure fractions were pooled, and the desired conjugate was precipitated using sodium acetate/EtOH to give the pure RNA conjugate. The conjugate has been isolated in 59% yield (1.50 mol). The purity of conjugate was analyzed by anion exchange HPLC (purity: 85.5%) and identity was confirmed by ESI-MS ([M+H].sup.1+.sub.calculated: 8374.4; [M+H].sup.1+.sub.measured: 8376.0.