Methods of Producing Sulfated Oligosaccharide Derivatives and Intermediates Thereof
20240218010 ยท 2024-07-04
Inventors
- Paul Newton HANDLEY (Darra, AU)
- Tomislav KAROLI (Darra, AU)
- Jessica Anne ROWLEY (Darra, AU)
- Helen FRANKS (Reading Brookshire, GB)
- Alexander WEYMOUTH-WILSON (Reading Berkshire, GB)
- Robert CLARKSON (Reading Berkshire, GB)
- Laura WALLIS (Reading Berkshire, GB)
- Aileen WHITE (Reading Berkshire, GB)
- Andrew TYRRELL (Reading Berkshire, GB)
- Helen GALE (Reading Berkshire, GB)
- Phillip GREENWOOD (Reading Berkshire, GB)
Cpc classification
International classification
C07J31/00
CHEMISTRY; METALLURGY
Abstract
The invention relates inter alia to methods of producing sulfated oligosaccharide derivatives and intermediates thereof. The sulfated oligosaccharide derivatives may be represented by the following formula (I):
[X].sub.nY?ZR.sup.1R.sup.2Formula (I)
wherein: X and Y are any D- or L-hexose or pentose, wherein each hydroxyl group not involved in a glycosidic linkage is substituted by a group W, and Y has an anomeric carbon atom; W is SO.sub.3M, and M is any pharmaceutically acceptable cation; n is an integer from 2 to 6; Z is O, and is linked to the anomeric carbon atom of Y; and R.sup.1R.sup.2 together form a lipophilic moiety.
Claims
1. A method of producing a compound of Formula (I),
[X].sub.nY?ZR.sup.1R.sup.2Formula (I) wherein: X and Y are any D- or L-hexose or pentose, wherein each hydroxyl group not involved in a glycosidic linkage is substituted by a group W, and Y has an anomeric carbon atom; W is SO.sub.3M, and M is any pharmaceutically acceptable cation; n is an integer from 2 to 6; Z is O, and is linked to the anomeric carbon atom of Y; R.sup.1 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, R.sup.10CONHR.sup.11, R.sup.10NHCOR.sup.11, R.sup.10CSNHR.sup.11, R.sup.10NHCSR.sup.11, R.sup.10COR.sup.11, or is a bond; R.sup.2 is selected from the group consisting of optionally substituted terpenoidyl, optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, R.sup.8CONHR.sup.9, R.sup.8NHCOR.sup.9, R.sup.8CSNHR.sup.9, R.sup.8NHCSR.sup.9, R.sup.8COR.sup.9, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, and optionally substituted heteroaryl; R.sup.8 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and a bond; R.sup.9 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and hydrogen; R.sup.10 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; and R.sup.11 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; the method comprising: (A1) preparing a mixture comprising a compound of Formula (II), a reaction liquid, and a sulfur trioxide complex,
[X].sub.nY?ZR.sup.1R.sup.2Formula (II) wherein: n, Z, R.sup.1 and R.sup.2 are as defined in the compound of Formula (I), and X and Y are any D- or L-hexose or pentose, wherein each hydroxyl group not involved in a glycosidic linkage is substituted by a group W, Y has an anomeric carbon atom, and W is H; (A2) separating a solid from the mixture; and (A3) washing the solid with a dipolar aprotic wash solvent to produce the compound of Formula (I).
2. The method of claim 1, wherein the dipolar aprotic wash solvent comprises dimethylformamide.
3. A method of producing a compound of Formula (II),
[X].sub.nY?ZR.sup.1R.sup.2Formula (II) wherein: X and Y are any D- or L-hexose or pentose, wherein each hydroxyl group not involved in a glycosidic linkage is substituted by a group W, Y has an anomeric carbon atom, and W is H; n is an integer from 2 to 6; Z is O, and is linked to the anomeric carbon atom of Y; R.sup.1 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, R.sup.10CONHR.sup.11, R.sup.10NHCOR.sup.11, R.sup.10CSNHR.sup.11, R.sup.10NHCSR.sup.11, R.sup.10COR.sup.11, or is a bond; R.sup.2 is selected from the group consisting of optionally substituted terpenoidyl, optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, R.sup.8CONHR.sup.9, R.sup.8NHCOR.sup.9, R.sup.8CSNHR.sup.9, R.sup.8NHCSR.sup.9, R.sup.8COR.sup.9, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, and optionally substituted heteroaryl; R.sup.8 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and a bond; R.sup.9 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and hydrogen; R.sup.10 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; and R.sup.11 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; the method comprising: (B1) mixing a compound of Formula (III) and a deprotecting agent to form the compound of Formula (II),
[X].sub.nY?ZR.sup.1R.sup.2Formula (III) wherein: n, Z, R.sup.1 and R.sup.2 are as defined in the compound of Formula (II), and X and Y are any D- or L-hexose or pentose, wherein each hydroxyl group not involved in a glycosidic linkage is substituted by a group W, Y has an anomeric carbon atom, and W is a protecting group; (B2) performing a membrane filtration to separate the compound of Formula (II) from one or more impurity; wherein the membrane filtration uses a membrane with a pore size which is at least twice the molecular weight of the compound of Formula (II).
4. The method of claim 3, wherein the membrane filtration uses a membrane with a pore size which is at least five times the molecular weight of the compound of Formula (II).
5. A method of producing a compound of Formula (III),
[X].sub.nY?ZR.sup.1R.sup.2Formula (III) wherein: X and Y are any D- or L-hexose or pentose, wherein each hydroxyl group not involved in a glycosidic linkage is substituted by a group W, Y has an anomeric carbon atom, and W is a protecting group; n is an integer from 2 to 6; Z is O, and is linked to the anomeric carbon atom of Y; R.sup.1 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, R.sup.10CONHR.sup.11, R.sup.10NHCOR.sup.11, R.sup.10CSNHR.sup.11, R.sup.10NHCSR.sup.11, R.sup.10COR.sup.11, or is a bond; R.sup.2 is selected from the group consisting of optionally substituted terpenoidyl, optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, R.sup.8CONHR.sup.9, R.sup.8NHCOR.sup.9, R.sup.8CSNHR.sup.9, R.sup.8NHCSR.sup.9, R.sup.8COR.sup.9, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, and optionally substituted heteroaryl; R.sup.8 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and a bond; R.sup.9 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and hydrogen; R.sup.10 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; and R.sup.11 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; the method comprising: (C.sub.1) reacting a compound of Formula (IV), with a compound of Formula (V) to form a compound of Formula (III);
[X].sub.nY?R.sup.3Formula (IV) wherein: X, Y and n are as defined in the compound of Formula (III), and R.sup.3 is an optionally substituted thioaryl group, and is linked to the anomeric carbon atom of Y;
HOR.sup.1R.sup.2Formula (V) wherein: R.sup.1 and R.sup.2 are as defined in the compound of Formula (III).
6. The method of any one of claims 3 to 5, wherein the protecting group is an acetate or benzoate group.
7. The method of any one of claims 1 to 6, wherein R.sup.1 is a bond, and R.sup.2 is selected from the group consisting of optionally substituted steroidyl, optionally substituted C.sub.1-C.sub.10 alkyl-NHCOC.sub.1-C.sub.26 alkyl, and optionally substituted C.sub.1-C.sub.10 alkyl-CONHC.sub.1-C.sub.26 alkyl; wherein said optional substituents are selected from the group consisting of C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, and halogen.
8. The method of any one of claims 1 to 7, wherein R.sup.1 is a bond, and R.sup.2 is cholestanyl or propyl stearamide.
9. A method of producing a compound of Formula (IV),
[X].sub.nY?R.sup.3Formula (IV) wherein: X and Y are any D- or L-hexose or pentose, wherein each hydroxyl group not involved in a glycosidic linkage is substituted by a group W, Y has an anomeric carbon atom, and W is a protecting group; n is an integer from 2 to 6; Z is O, and is linked to the anomeric carbon atom of Y; and R.sup.3 is an optionally substituted thioaryl group, and is linked to the anomeric carbon atom of Y; the method comprising: (D1) reacting a compound of Formula (VI), with a thioaryl compound to form a compound of Formula (IV);
[X].sub.nY?V Formula (VI) wherein: X, Y and n are as defined in the compound of Formula (IV), and V is W, and is linked to the anomeric carbon atom of Y.
10. The method of claim 9, wherein R.sup.3 is thiotolyl.
11. The method of any one of claims 1 to 10, wherein X and Y are glucose monosaccharide units.
12. The method of any one of claims 1 to 11, wherein X and Y are glucose monosaccharide units linked together with ?-1,4 glycosidic linkages.
13. The method of any one of claims 1 to 10, wherein X and Y are mannose monosaccharide units.
14. The method of any one of claims 1 to 13, wherein n is 3 or 4.
15. A method of producing a compound of Formula (VII), ##STR00050## R.sup.4 is SO.sub.3M, and M is any pharmaceutically acceptable cation; R.sup.5 is OR.sup.1R.sup.2; R.sup.1 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, R.sup.10CONHR.sup.11, R.sup.10NHCOR.sup.11, R.sup.10CSNHR.sup.11, R.sup.10NHCSR.sup.11, R.sup.10COR.sup.11, or is a bond; R.sup.2 is selected from the group consisting of optionally substituted terpenoidyl, optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, R.sup.8CONHR.sup.9, R.sup.8NHCOR.sup.9, R.sup.8CSNHR.sup.9, R.sup.8NHCSR.sup.9, R.sup.8COR.sup.9, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, and optionally substituted heteroaryl; R.sup.8 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and a bond; R.sup.9 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and hydrogen; R.sup.10 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; and R.sup.11 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; the method comprising: (E1) providing a starting material comprising a mixture of maltooligosaccharides, said starting material comprising from 50% w/w to 95% w/w of maltotetraose on a dry weight basis; (E2) reacting the starting material with an acyl halide, acyl anhydride, aroyl halide, or aroyl anhydride to form a compound of Formula (VIII), ##STR00051## wherein: R.sup.4 is an acyl or aroyl group, and R.sup.5 is O-acyl or O-aroyl; (E3) converting the compound of Formula (VIII) to a glycosyl donor of Formula (IX), ##STR00052## wherein: R.sup.4 is as defined in the compound of Formula (VIII), and R.sup.5 is a leaving group; (E4) glycosylating a compound of Formula (X) with the glycosyl donor of Formula (IX) to form a compound of Formula (XI),
HOR.sup.1R.sup.2Formula (X) wherein R.sup.1 and R.sup.2 are as defined in the compound of Formula (VII), ##STR00053## wherein: R.sup.4 is as defined in the compound of Formula (VIII), and R.sup.5 is as defined in the compound of Formula (VII); (E5) removing acyl or aroyl protecting groups from the compound of Formula (XI) to form a first mixture comprising a compound of Formula (XII); ##STR00054## wherein: R.sup.4 is OH; R.sup.5 is as defined in the compound of Formula (VII); (E6) subjecting the first mixture to membrane filtration to form a first purified composition comprising a compound of Formula (XII); (E7) reacting the first purified composition with a sulfur trioxide complex in a reaction liquid to form a second mixture comprising a compound of Formula (VII); and (E8) washing the second mixture with a dipolar aprotic wash solvent to form a second purified composition comprising a compound of Formula (VII).
16. The method of claim 15, which does not include a chromatography step.
17. The method of claims 15 or 16, wherein R.sup.1 is a bond, and R.sup.2 is selected from the group consisting of optionally substituted steroidyl, optionally substituted C.sub.1-C.sub.10 alkyl-NHCOC.sub.1-C.sub.26 alkyl, and optionally substituted C.sub.1-C.sub.10 alkyl-CONHC.sub.1-C.sub.26 alkyl; wherein said optional substituents are selected from the group consisting of C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, and halogen.
18. The method of any one of claims 15 to 17, wherein R.sup.1 is a bond, and R.sup.2 is cholestanyl or propyl stearamide.
19. A compound of Formula (VII) produced according to the method of any one of claims 15 to 17.
20. A compound of Formula (I) produced according to the method of claim 1 or 2.
21. A compound of Formula (XIII), or a salt thereof ##STR00055## wherein: R.sup.6 is an acyl or aroyl group; and R.sup.7 is an optionally substituted thioaryl group.
22. The compound or salt thereof of claim 20, wherein R.sup.6 is benzoyl, and R.sup.7 is thiotolyl.
23. Use of the compound or salt thereof of claim 21 or 22 in the manufacture of a compound of Formula (VII); ##STR00056## wherein: R.sup.4 is SO.sub.3M, and M is any pharmaceutically acceptable cation; R.sup.5 is OR.sup.1R.sup.2; R.sup.1 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, R.sup.10CONHR.sup.11, R.sup.10NHCOR.sup.11, R.sup.10CSNHR.sup.11, R.sup.10NHCSR.sup.11, R.sup.10COR.sup.11, or is a bond; R.sup.2 is selected from the group consisting of optionally substituted terpenoidyl, optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, R.sup.8CONHR.sup.9, R.sup.8NHCOR.sup.9, R.sup.8CSNHR.sup.9, R.sup.8NHCSR.sup.9, R.sup.8COR.sup.9, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, and optionally substituted heteroaryl; R.sup.8 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and a bond; R.sup.9 is selected from the group consisting of optionally substituted C.sub.1-C.sub.36 alkyl, optionally substituted C.sub.2-C.sub.36 alkenyl, optionally substituted C.sub.2-C.sub.36 alkynyl, optionally substituted C.sub.4-C.sub.36 cycloalkyl, optionally substituted C.sub.4-C.sub.36 cycloalkenyl, optionally substituted C.sub.4-C.sub.36 cycloalkynyl, optionally substituted aryl, optionally substituted C.sub.1-C.sub.36 heteroalkyl, optionally substituted C.sub.2-C.sub.36 heteroalkenyl, optionally substituted C.sub.2-C.sub.36 heteroalkynyl, optionally substituted heteroaryl, and hydrogen; R.sup.10 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; and R.sup.11 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond.
24. A compound of Formula (XIV), or a salt thereof ##STR00057## wherein: R.sup.6 is an acyl or aroyl group; and R.sup.7 is an optionally substituted thioaryl group.
25. The compound or salt thereof of claim 24, wherein R.sup.6 is acetyl, and R.sup.7 is thiotolyl.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0364]
[0365]
[0366]
[0367]
[0368]
[0369]
[0370] Preferred features, embodiments and variations of the invention may be discerned from the following Examples which provides sufficient information for those skilled in the art to perform the invention. The following Examples are not to be regarded as limiting the scope of the preceding Summary of the Invention in any way.
Abbreviations
[0371] AcOHacetic acid; BzClbenzoyl chloride; CEcapillary electrophoresis; DCMdichloromethane; DMA N,N-dimethylacetamide; DMAP N,N-dimethyl aminopyridine; DMF N,N-dimethylformamide; DMI1,3-dimethyl-2-imidazolidinone; DMSOdimethyl sulfoxide; DMPUN,N-dimethylpropyleneurea; EtOAcethyl acetate; EtOHethanol; HMPA-hexamethyl phosphoramide; HSQCHeteronuclear Single Quantum Coherence; IMSindustrial methylated spirits (ethanol with about 5% methanol); MeCNacetonitrile; MeOHmethanol; MWCOmolecular weight cut-off, NaOMesodium methoxide; NIS N-iodosuccinimide; NMPN-methyl-2-pyrrolidone; pypyridine; SFCAsurfactant-free cellulose acetate; STPsulfur trioxide pyridine complex; TCAtrichloroacetimidate; THFtetrahydrofuran; TBMEmethyl tertiary butyl ether; TMSOTfTMS triflate, or trimethylsilyl trifluoromethanesulfonate; TPPAtripyrrolidinophosphoric acid; UFultrafiltration.
Definitions
[0372] As used herein, the term about, is relative to the actual value stated, as will be appreciated by those of skill in the art, and allows for approximations, inaccuracies and limits of measurement under the relevant circumstances. Depending on context, it may allow a variation from the stated value of ?10%, +5%, +2%, +1%, +0.5%, +0.2%, +0.1%, +0.05%, +0.02%, or 0.01%.
[0373] As used herein, the term comprising indicates the presence of the specified integer(s), but allows for the possibility of other integers, unspecified. This term does not imply any particular proportion of the specified integers. Variations of the word comprising, such as comprise and comprises, have correspondingly similar meanings.
[0374] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as would be commonly understood by those of ordinary skill in the art to which this invention belongs.
[0375] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases in one embodiment or in an embodiment in various places throughout this specification are not necessarily all referring to the same embodiment.
[0376] Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.
[0377] As used herein, the term aroyl, means a group selected from the group consisting of CO-aryl, and CO-heteroaryl, each of which may be optionally substituted with one or more groups independently selected from C.sub.1-6 alkyl, C.sub.1-6 alkenyl, nitro, cyano, NHR14, N(R.sup.14).sub.2, NHCOR.sup.14, CF.sub.3, aryl, heteroaryl, and halogen; wherein R.sup.14 is independently selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 heteroalkyl, and C.sub.2-C.sub.6 heteroalkenyl.
[0378] As used herein, the term acyl, means a group selected from the group consisting of CO-alkyl, CO-alkenyl, CO-heteroalkyl, and CO-heteroalkenyl, each of which may be optionally substituted with one or more groups independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, and halogen.
[0379] The term alkyl refers to a straight-chain or branched alkyl substituent containing from, for example, 1 to about 36 carbon atoms. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, 2-methylbutyl, 3-methylbutyl, hexyl, heptyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.
[0380] The term alkenyl refers to a straight-chain or branched alkenyl substituent containing from, for example, 2 to about 36 carbon atoms. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl and the like. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.
[0381] The term alkynyl refers to a straight-chain or branched alkynyl substituent containing from, for example, 2 to about 36 carbon atoms. Examples of suitable alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, butadienyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl and the like. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.
[0382] The term cycloalkyl refers to a saturated non-aromatic cyclic hydrocarbon. The cycloalkyl ring may include a specified number of carbon atoms. For example, a 3 to 8 membered cycloalkyl group includes 3, 4, 5, 6, 7 or 8 carbon atoms. The cycloalkyl group may be monocyclic, bicyclic or tricyclic. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). Non-limiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
[0383] The term cycloalkenyl or cycloalkene refers to a cyclic hydrocarbon having at least one double bond, which is not aromatic. The cycloalkenyl ring may include a specified number of carbon atoms. The cycloalkenyl group may be monocyclic, bicyclic or tricyclic. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). For example, a 5 membered cycloalkenyl group includes 5 carbon atoms. Non-limiting examples may include cyclopentenyl and cyclopenta-1,3-dienyl.
[0384] The term cycloalkynyl or cycloalkyne refers to a cyclic hydrocarbon having at least one triple bond, which is not aromatic. The cycloalkynyl ring may include a specified number of carbon atoms. The cycloalkynyl group may be monocyclic, bicyclic or tricyclic. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). For example, a 5 membered cycloalkynyl group includes 5 carbon atoms. Non-limiting examples may include cyclopentynyl.
[0385] The term aryl refers to an aromatic carbocyclic substituent, as commonly understood in the art. It is understood that the term aryl applies to cyclic substituents in which at least one ring is planar and comprises 4?+2 ?r electrons, according to H?ckel's Rule. Aryl groups may be monocyclic, bicyclic or tricyclic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and 1,2,3,4-tetrahydronaphthyl. An aryl group may be monocyclic, bicyclic or tricyclic, provided that at least one ring is aromatic. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings).
[0386] The term heteroalkyl refers to a straight-chain or branched alkyl substituent in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. It may contain from, for example, 1 to about 36 carbon atoms. For example, between 1 and 4 carbon atoms may be replaced by heteroatoms independently selected from N, S and O. Examples of suitable heteroalkyl groups include, but are not limited to, methoxy, ethoxy, propyloxy, isopropyloxy, and the like. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.
[0387] The term heteroalkenyl refers to a straight-chain or branched alkenyl substituent in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. It may contain from, for example, 2 to about 36 carbon atoms. For example, between 1 and 4 carbon atoms may be replaced by heteroatoms independently selected from N, S and O. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.
[0388] The term heteroalkynyl refers to a straight-chain or branched alkynyl substituent in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. It may contain from, for example, 2 to about 36 carbon atoms. For example, between 1 and 4 carbon atoms may be replaced by heteroatoms independently selected from N, S and O. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.
[0389] The term heterocyclic or heterocyclyl as used herein, refers to a cycloalkyl or cycloalkenyl group in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. For example, between 1 and 4 carbon atoms in each ring may be replaced by heteroatoms independently selected from N, S and O. The heterocyclyl group may be monocyclic, bicyclic or tricyclic in which at least one ring includes a heteroatom. When more than one ring is present the rings may be fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). Each of the rings of a heterocyclyl group may include, for example, between 5 and 7 atoms. Examples of heterocyclyl groups include tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolinyl, dithiolyl, 1,3-dioxanyl, dioxinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyranyl, 1,4-dithiane, and decahydroisoquinoline. In one embodiment, heterocyclyl may be optionally substituted by ?O.
[0390] The term heteroaryl, as used herein, refers to a monocyclic, bicyclic or tricyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and said at least one ring contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). Consideration must be provided to tautomers of heteroatom containing ring systems containing carbonyl groups, for example, when determining if a ring is a heterocyclyl or heteroaryl ring. Heteroaryl includes, but is not limited to, 5-membered heteroaryls having one hetero atom (e.g., thiophenes, pyrroles, furans); 5 membered heteroaryls having two heteroatoms in 1,2 or 1,3 positions (e.g., oxazoles, pyrazoles, imidazoles, thiazoles, purines); 5-membered heteroaryls having three heteroatoms (e.g., triazoles, thiadiazoles); 5-membered heteroaryls having four heteroatoms (e.g., tetrazoles); 6-membered heteroaryls with one heteroatom (e.g., pyridine, quinoline, isoquinoline); 6-membered heteroaryls with two heteroatoms (e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines, quinazolines, quinoxalinone, quinazolinone); 6-membered heteroaryls with three heteroatoms (e.g., 1,3,5-triazine); and 6-membered heteroaryls with four heteroatoms. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, furan, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, and phenoxazine. Further exemplary heteroaryl groups may include, for example, indoline or 2,3-dihydrobenzofuran. In one embodiment, heteroaryl may be optionally substituted by ?O.
[0391] The term terpenoid, or terpenoidyl as used herein, refers to an organic chemical derived from one or more isoprene units. Steroids are a sub-class of terpenoid.
[0392] The term steroid, or steroidyl as used herein, refers to a carbocyclic moiety comprising a backbone having four fused rings having the following arrangement:
##STR00030##
[0393] The rings may be saturated carbocycles, or they may comprise one or more double bonds. One or more of their ring carbon atoms may be substituted with one or more R.sup.15 groups, wherein R.sup.15 is selected from the group consisting of?O, ?S, OH, SH, NH.sub.2, C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, aryl, C.sub.1-C.sub.12 heteroalkyl, C.sub.2-C.sub.12 heteroalkenyl, C.sub.2-C.sub.12 heteroalkynyl, and heteroaryl. A steroid or steroidyl group may be bonded to another group through one or more substituents attached to its backbone. For example, cholesterol/cholesteryl or cholestanol/cholestanyl may be connected to another moiety through its OH substituent group.
[0394] Whenever a range of the number of atoms in a structure is indicated (e.g., a C.sub.1-C.sub.12, C.sub.1-C.sub.6 alkyl, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used. Thus, for instance, the recitation of a range of 1-12 carbon atoms (e.g., C.sub.1-C.sub.12), 1-6 carbon atoms (e.g., C.sub.1-C.sub.6) as used with respect to any chemical group (e.g., alkyl, etc.) referenced herein encompasses and specifically describes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12 carbon atoms, as appropriate, as well as any sub-range thereof (e.g., 1-2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms, 1-11 carbon atoms, 1-12 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms, 2-10 carbon atoms, 2-11 carbon atoms, 2-12 carbon atoms, 3-4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-11 carbon atoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbon atoms, 4-11 carbon atoms, and/or 4-12 carbon atoms, etc., as appropriate).
[0395] As used herein, halo refers to a halogen atom, especially F, C.sub.1 or Br; more especially F or C.sub.1; most especially F.
[0396] As used herein, the term optionally substituted means that any number of hydrogen atoms on the optionally substituted group are replaced with another moiety. Unless defined otherwise, said moiety is independently selected from the group consisting of C.sub.1-C.sub.12 alkyl (or C.sub.1-C.sub.6 alkyl), C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, R.sup.12CONHR.sup.13, R.sup.12NHCOR.sup.13, R.sup.12-CSNHR.sup.13, R.sup.12NHCSR.sup.13, R.sup.12COR.sup.13, =0, ?S, cyano, CF.sub.3, and halogen; [0397] wherein: [0398] R.sup.12 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and a bond; and [0399] R.sup.13 is selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 heteroalkenyl, C.sub.2-C.sub.6 heteroalkynyl, heteroaryl, and hydrogen.
[0400] Preferred features, embodiments and variations of the invention may be discerned from the following Examples which provides sufficient information for those skilled in the art to perform the invention. The following Examples are not to be regarded as limiting the scope of the preceding Summary of the Invention in any way.
EXAMPLES
Synthesis of Compound 1
[0401] Overall synthesis
[0402] The synthesis of Compound 1 was performed according to the five-stage synthesis outlined in Scheme 1. Each stage of the synthesis is discussed in further detail in the following sections.
##STR00031## ##STR00032##
Stage 1: Synthesis of G4Bz
[0403] The synthesis of G4Bz from G4 is outlined in Scheme 2 below. Briefly, the hydroxyl groups of G4 (maltotetraose) were benzoylated using benzoyl chloride in pyridine.
##STR00033##
Materials
[0404]
TABLE-US-00004 Reagents Quantity (wts.) Quantity (g) G4 syrup (~50% w/w by 1 wt. 200 g dry weight) Pyridine 5.84 wts. + 2.0 wts 1168 g + 400 g Benzoyl chloride 2.66 wts. 532 g 2-Propanol 11.88 wts. + 2.0 wts 2376 g + 400 g Ethyl acetate 7 wts. 1400 g Water 1.0 wts. 200 g 10% aq. NaCl 2 ? 5.0 wts 2 ? 1000 g 2M aq. HCl 3 ? 5.0 wts 3 ? 1000 g TBME-Heptane (9:1 v/v) 2.0 wts 400 g
Procedure
[0405] 1. A solution of G4 syrup (comprising maltotetraose at ?50% (w/w) by dry weight) in pyridine (200 g in 1168 g) in a reaction vessel was azeotropically dried by distilling ca. 3.0 wts. of solvent at ? 100? C./250 mbar (?600 mL); the solution was dried to a moisture content of <133% (w/w) as determined by Karl Fischer (KF) titration.
[0406] 2. Pyridine (400 g) was charged into the reaction vessel and the solution was heated to 90? C.
[0407] 3. Benzoyl chloride (532 g) was added slowly, maintaining the reaction at 100-110? C.
[0408] 4. The reaction was stirred under Argon at 105-115? C. for 16-20 h.
[0409] 5. The reaction was cooled to 90? C., then quenched by the addition of H.sub.2O (200 g); maintaining the reaction mixture at <110? C. during the quench.
[0410] 6. The reaction was cooled to 30? C. and ethyl acetate (EtOAc) (1400 g) was charged into the reaction vessel.
[0411] 7. Brine (10% aq. NaCl, 1000 g) was charged into the reaction vessel and the phases mixed for >5 minutes. The aqueous phase was separated and drained.
[0412] 8. 2M aq. HCl (1000 g) was charged into the reaction vessel and the phases were mixed for >5 minutes. The aqueous phase was separated and drained. The 2M aq. HCl (1000 g) washes were repeated until the pH of the aqueous phase was <2.
[0413] 9. Brine (10% aq. NaCl, 1000 g) was charged into the reaction vessel and the phases were mixed for >5 minutes. The aqueous phase was separated and drained.
[0414] 10. The reaction mixture was concentrated to approximately 4-5 volumes by distilling EtOAc at ? 70? C./200 mbar.
[0415] 11. The crude solution of G4Bz in EtOAc was cooled to 20? C. and drained into a bottle (labelled Crude G4Bz in EtOAc). The crude solution was <5 wts. EtOAc to ensure that the material did not form a gummy aggregate but not too concentrated as it would tend to form more granular solids during precipitation.
[0416] 12. Isopropyl alcohol (WA) (11.88 wts, 2376 g) was charged to the reactor and cooled to 15? C.
[0417] 13. Crude G4Bz in EtOAc was charged slowly to the reactor using a peri-pump. The G4Bz solution was charged such that it easily dissipated into the WA and did not form clumps. The resulting suspension was stirred at 15? C. for 30-60 minutes.
[0418] 14. The suspension was drained portion-wise from the reactor and filtered via vacuum filtration through a filter cloth.
[0419] 15. The filter cake was washed with 1?2.0 wts of IPA (400 g) and 1?2.0 wts of (methyl tert-butyl ether) TBME-Heptane (1:9 w/w, 400 g).
[0420] 16. The solids were transferred into a tared flask and dried to a constant mass by rotary evaporation.
Step 1: Drying of Maltotetraose Syrups
[0421] Maltotetraose is available as either a high-purity solid, or as various oligosaccharide mixtures in the form of syrups containing ?25% water. Whether a >70% purity (on dried basis) syrup is used, or the >50% syrup as used in the present example, the water must be removed as it interferes with the addition of the protecting groups in the next step.
[0422] The syrups may be dried by lyophilization. Other methods of drying such as spray drying or acetone precipitation may be used. The pyridine azeotrope method was successfully employed on scale-up (?2 kg of >50% syrup) for production of API (active pharmaceutical ingredient) for clinical trial use. The residual water after the pyridine azeotrope step was measured using Karl Fischer titration. Experiments on spiking with known quantities of water established that >2% residual water leads to incomplete benzoylation (Step 4). Residual water at 1.35% gave a complete reaction.
Step 2: Charging Additional Pyridine
[0423] Pyridine was added back in the reaction vessel after the azeotropic distillation so that the benzoylation reaction was performed in about 5 weights of pyridine in total. This prevented precipitation of solid during the benzoylation that otherwise could clump up and interfere with the stirring.
Steps 2-4: Benzoylation Conditions
[0424] ##STR00034##
[0425] Benzoylation of alcohols is known in the art. However, the ?/?-selectivity of the protection chemistry (shown in Scheme 3) as distinct from the ?/?-selectivity of the glycosylation step, is important, because only the ?-anomer of perbenzoate G4Bz is observed to react in the next chemical step and therefore the unreacted ?-anomer is an undesired impurity. Formation of the undesired ?-anomer may be minimized by performing the reaction at elevated temperature (typically greater than 100? C.).
Step 5: Quench
[0426] The unreacted benzoyl chloride was quenched at the end of the reaction. This prevented further uncontrolled/undesired reaction during workup, and prevents exposure of workers to this hazardous compound. The reaction was quenched with water so that the benzoic acid by-product could be removed in the aqueous workup. Quenching with IPA also worked, with the resulting isopropyl benzoate by-product removed in the precipitation/wash steps.
Steps 7-9: Acid Wash
[0427] The isolation of the product G4Bz from the reaction mixture was based on washing to remove pyridine residues, followed by precipitation using a mixture of solvents. Suitable conditions were those that controlled the level of residual pyridine or other bases, and water, in the product, since these can interfere with the subsequent step, while leaving the product as a filterable solid for easy isolation and handling. Levels of pyridine in the product were measured using HPLC analysis, or by UV/Visible spectroscopy (e.g. at a wavelength of 290 nm). Residual water was quantified e.g. by Karl Fischer titration.
[0428] An initial brine wash (Step 7) was used to reduce the number of acid washes required to remove all of the pyridine, from ?5 to ?3 washes, and prevented the formation of emulsions where aqueous and organic layers were difficult to separate. The final brine wash (Step 9) reduced residual HCl which is corrosive.
[0429] Continuing acid-washes until the pH of the aqueous layer remains under 2 yields product (after final drying) with a preferred pyridine content of <1% by HPLC. Higher amounts can be tolerated in the following Stage e.g. <5% pyridine by HPLC, but require that additional BF.sub.3 is added to compensate.
Steps 10-13: Precipitation Conditions
[0430] The perbenzoylated products throughout the example syntheses of Compound 1 and of Compound 4 could be precipitated in a range of forms from a viscous oil to a glassy solid. Under suitable conditions, the products can be reliably precipitated in the form of glassy, amorphous beads (see, e.g.,
[0431] The steps above demonstrate removal of pyridine by an acid-wash, followed by precipitation incorporating only minimal quantities of water as retained in the organic layer after washing. This method is preferred since it minimises the amounts of both residual pyridine and residual water that must be removed from the final product by drying (Step 16). Alternatively, bulk pyridine may be removed in the precipitation process. In this case, the presence of large quantities of pyridine in the strong solvent required large quantities of water in the anti-solvent for correct formation of the filterable solid. The resulting filter cake contained appreciable quantities of both pyridine and water, requiring extended drying time (days). The acid wash procedure, while adding repeated wash steps, yielded a precipitated product that was much more easily dried, and thus is more efficient overall.
Step 14: Isolation by Filtration
[0432] On kilogram scale, the filter cakes former in the filtration step were noted to be compressible solids. This meant that too high a pressure differential during filtration, whether by application of vacuum below the filter plate or air/nitrogen above, caused the cakes to compress and greatly slow the flow rate of the filtrate. Ideally, the bulk of the mother liquor was allowed to proceed under gravity at atmospheric pressure, then application of pressure was used for the final deliquoring/drying step.
Stage 2: Synthesis of G4BzSTol
[0433] The synthesis of G4BzSTol from G4Bz is outlined in Scheme 4 below. Briefly, the anomeric group of G4Bz is thiolated using para-thiocresol and BF.sub.3.Math.OEt.sub.2 in dichloromethane.
##STR00035##
Materials
[0434]
TABLE-US-00005 Reagents Quantity (wts.) Quantity (g) G4Bz 1 wt. 275 g Thiocresol 0.09 wt. 24.75 g DCM 2.91 wts. 800.25 g BF.sub.3Et.sub.2O 0.12 wt. 34 g Et.sub.3N 0.10 wt. 26.62 g Ethyl acetate 5.82 wts. 1600 g 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3 2 ? 5.0 wts. 2 ? 1375 g 10% aq. NaCl 2 ? 5.0 wts. 2 ? 1375 g Water 4.0 wts. 1100 g Propanol 7.02 wts. 1939 g Heptane 4.72 wts. 1298 g
Procedure
[0435] 1. G4Bz (1 wt.) and thiocresol (0.09 wt.) were charged to the reactor and dissolved in DCM (2.65 wts). The solution was then warmed to 35? C.
[0436] 2. BF.sub.3.Math.OEt.sub.2 was charged to the reactor slowly, maintaining the reaction at <
[0437] 40? C. during the addition. A line rinse was performed with DCM (0.26 wts).
[0438] 3. The reaction was stirred at 35? C.?5? C. and monitored by HPLC for reaction completion (NMT (not more than) 1.0% G4Bz).
[0439] 4. The reaction was cooled to 20? C.?5? C. and quenched with the addition of Et.sub.3N (0.10 wts.) in EtOAc (5.82 wts.), maintaining the reaction temperature at <40? C. during the quench.
[0440] 5. The organic phase was washed with the following: 1?5 wts. 10% aq. NaCl, 2 ?5 wts. 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3, 1?5 wts. 10% aq. NaCl.
[0441] 6. The aqueous phases were separated and drained from the reactor. The organic phase was concentrated to 2.5 volumes by vacuum distillation and then drained from the reactor.
[0442] 7. Water (4 wts.) and WA (7 wts.) were charged to the reactor and cooled to 15? C. 5? C.
[0443] 8. The organic phase was charged slowly into the reactor, ensuring that it dissipated quickly into the WAwater solution upon contact.
[0444] 9. The suspension was stirred at 15? C.?5? C. for 1 hour?30 minutes.
[0445] 10. The suspension was drained from the reactor and filtered through a filter cloth (105 ?m PP(polypropylene)) via vacuum filtration. The filter cake was washed with WA (2 wts.).
[0446] 11. Heptane (4.72 wts.) was charged to the reactor and cooled to 15? C.?5? C. The filter cake from step 10 was charged to the reactor, and the suspension was stirred for 30 minutes?15 minutes.
[0447] 12. The suspension was drained from the reactor and filtered through a filter cloth (105 ?m PP) via vacuum filtration. The filter cake was washed with heptane (2 wts.).
[0448] 13. The solids were transferred to a tared evaporating flask and dried to a constant mass.
Step 2: Conditions for Formation of the Thioglycoside
[0449] Significant optimization of the conditions for formation of thioglycoside G4BzSTol was performed, and the above process is considered suitable for scale-up. Boron trifluoride was used as a Lewis acid catalyst, although a skilled worker would understand that this may be substituted with other promoters.
[0450] Somewhat surprisingly, the presence of ethereal solvents has a significant effect on reaction rate. The use of boron trifluoride diethyl ether complex in THE solvent completely inhibits the reaction, while the same complex inDCM solvent is successful. Use of boron trifluoride acetic acid complex in DCM solution, where no ethers are present, gives a large enhancement of both the desired reaction as well as side reactions, such that the starting material G4Bz is consumed within a few minutes, and impurities begin to accumulate. Accordingly, it may be possible to modulate the reactivity by the addition of ethers.
[0451] The use of triethylamine as a Lewis base to quench the reaction on completion neutralised the Lewis acidic boron trifluoride and enabled the reaction mixture to be held overnight if required, before the workup was commenced, without further reaction leading to the formation of impurities.
Step 1: Stoichiometry of Thiocresol
[0452] Although a larger excess of thiocresol helped ensure that the reaction was driven to completion, this excess reagent needed to be purged during the reaction workup or else it would interfere with the glycosylation reaction (Stage 3). Charging the reactor with 0.09 weights of thiocresol resulted in an easier workup compared with using a larger excess (e.g. 0.11 weights) while still giving complete conversion (<1% starting material G4Bz remaining by HPLC).
Step 2: Stoichiometry of BF.sub.3.Math.OEt.sub.2
[0453] An excess of boron trifluoride helped to drive the reaction to completion. However, too much BF.sub.3.Math.OEt.sub.2 resulted in problems with the aqueous workup. Quenching boron trifluoride in water can yield metaboric acid which is only slightly soluble in water, interfering with the separation of aqueous and organic phases in the extraction.
[0454] The conditions chosen represents a slight excess of BF.sub.3 with respect to thiocresol (e.g. 0.2 molar equivalents excess). A larger excess (e.g. 0.8 molar equivalents in excess) was problematic.
Step 5: Wash Procedure
[0455] The initial NaCl wash removed BF.sub.3.Math.OEt.sub.2 as a suitable water soluble boron derivative. If significant amounts of boron were present in the subsequent KOH wash, the formation of troublesome metaboric acid seemed to be favoured.
[0456] The wash with 10% NaOH was important to remove excess thiocresol, as otherwise the subsequent glycosylation stage did not go to completion. A weaker base (sodium bicarbonate) did not completely remove the thiocresol residues by itself. Thiosulfate was added as a reducing agent to clean up oxidised forms of thiocresol (which was important for the subsequent glycosylation stage where free iodine was present as an oxidising agent, but also was included in this step also).
Steps 11-12: Drying Procedure
[0457] Residual water used in the precipitation step was removed to prevent it from quenching the TMSOTf promoter in the subsequent glycosylation reaction (Stage 3). Slurrying the precipitated product in heptane (Step 11) yielded a slightly different, more powdery form that could more easily be dried (Step 12). Note that simply washing the filter cake on the filter with heptane was not sufficient, and the slurrying procedure was required to properly alter the solid form.
Stage 3: Synthesis of545Bz
[0458] The synthesis of 545Bz from G4BzSTol is outlined in Scheme 5 below. Briefly, cholestanol is glycosylated using G4BzSTol, NIS and TMSOTf in dichloromethane.
##STR00036##
Materials
[0459]
TABLE-US-00006 Reagents Quantity (wts.) Quantity (g) G4BzSTol 1 wt. 250 g 5?-cholestan-3?-ol 0.20 wt. 50.27 g DCM 7.96 wts. + 0.10 wt. 1990 g + 25 g NIS 0.21 wt. 52.91 g TMSOTf 0.07 wt. 16.25 g 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3 2 ? 5.0 wts. 2 ? 1250 g 5% aq. NaHCO.sub.3 1 ? 5.0 wts. 1 ? 1250 g 10% aq. NaCl 1 ? 5.0 wts. 1 ? 1250 g EtOAc 2.70 wts. 675 g Heptane 13.70 wts. 3425
Procedure
[0460] 1. A solution of G4BzSTol (1.0 wts) and 5?-cholestan-3?-ol (0.20 wts) in DCM (7.96 wts) was stirred under N.sub.2 for 30-60 min.
[0461] 2. The mixture was cooled to 5? C.?5? C. and NIS (0.21 wts.) was added. TMSOTf (0.07 wts) was added slowly, maintaining the reaction temperature at <10? C. during addition, followed by a DCM line rinse (0.10 wts).
[0462] 3. The mixture was stirred at 5? C.?5? C. for 2 h?15 min, until HPLC confirmed that G4BzSTol was NMT 4.0%.
[0463] 4. The reaction was quenched by adding a solution of 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3 (5 wts.), maintaining the reaction temperature at <20? C. during addition.
[0464] 5. The phases were separated, and the organic phase was washed with the following: 1?5 wts. 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3 solution, 1?5% aq. NaHCO.sub.3, 1?10% aq. NaCl.
[0465] 6. A solvent swap into EtOAc was performed by adding EtOAc (2.7 wts) then concentrating the solution down to 2.5 volumes by vacuum distillation at 45? C.
[0466] 7. The solution of 545Bz in EtOAc was added slowly to heptane (13.7 wts) at 15-25? C. to precipitate the product.
[0467] 8. The product was filtered off, washed with heptane (2 wts.) and dried to constant mass by rotary evaporation at 45? C.
Steps 1-2: Reaction Stoichiometry
[0468] An excess of cholestanol was used, otherwise the reaction did not go to completion and the hemiacetal side-product G4BzOH was produced. A slight excess of NIS was also required, otherwise the conversion would stall and unreacted thioglycoside G4BzSTol remain. The TMSOTf promoter was used catalytically (i.e. sub-stoichiometrically). The amount of solvent DCM was important; at Step 1 the mixture forms a gel-like suspension that can be difficult to stir. Adding more DCM allows efficient stirring, but surprisingly once the TMSOTf is added the suspension dissolved anyway (possibly the hydroxyl group of the cholestanol was converted to a TMS ether).
Steps 2-3: Reaction Temperature
[0469] The reaction conditions employed required careful optimisation to ensure the reaction went to completion. The conditions used maximized the formation of the desired ?-anomer ?545Bz (Scheme 6), including conducting the reaction at ice temperature and monitoring the reaction by HPLC. The desired ?-anomer ?545Bz appears to be the initially formed kinetic product, which rearranges to the thermodynamically favoured ?545Bz upon prolonged reaction.
[0470] Lower temperatures favoured the formation of the correct ?-anomeric configuration, 5? C. (chilled water) was convenient for large-scale manufacture.
##STR00037##
Step 4-5: Quench Wash Solutions
[0471] Thiosulfate was used to remove free iodine (from the reaction of NIS) by reduction to water-soluble iodide, and also to reduce any disulfide-linked thiocresol dimers back to the corresponding thiol, so that they could be removed by washing with KOH base.
Step 6-7: Precipitation
[0472] Small amounts of DCM interfered with formation of the easily-filterable solid form, and accordingly DCM was replaced with EtOAc. Addition into the correct quantity of non-solvent (heptane here, but IPA would also work) resulted in product precipitation. Small amounts of water seem to be necessary for formation of the solid form, so no drying of the EtOAc solution was performed.
Step 8: Heptane Slurry
[0473] Once correctly precipitated, the solid product was treated by slurrying it with heptane in order to convert it to a finer, more powdery solid for easier drying.
Stage 4: Synthesis of 5450H
[0474] Global deprotection of perbenzoate 545Bz was accomplished using standard Zemplen transesterification with methanol, catalysed by methoxide as shown in Scheme 7. The deprotection chemistry has a major effect on the properties of the molecule: perbenzoate 545Bz is insoluble in water, while polyol 545OH is soluble in water but poorly soluble in almost all organic solvents. Purification or manipulation of polyol 545OH therefore is easiest in aqueous solution, but the water must then be removed as it interferes with the final sulfation step.
[0475] Previously disclosed workups for this reaction included quenching the methoxide catalyst using resin-supported acid, then filtering and evaporating the reaction mixture to dryness. However, both the use of resins and evaporation to dryness may be impractical on scale-up as they can lead to cleaning issues and safety issues, respectively. For example, resin beads are typically insoluble in standard cleaning solvents and as such can be difficult to remove from reactor vessels, filtration equipment and pipes. Further, the use of THF, which can be contaminated with peroxides, may result in an explosion if this solvent is evaporated to dryness.
##STR00038##
Materials
[0476]
TABLE-US-00007 Reagents Quantities (wts.) Quantities (g) 545Bz 1 wt 200 g THF 1.78 wts (2 vol) 356 g MeOH 3.16 wts (4 vol) 632 g 25% NaOMe in MeOH 0.31 wts 62.0 g AcOH As req. ~19 g EtOAc 9.02 wts + 2 ? 0.9 wts 1804 g + 2 ? 180 g
Procedure
[0477] 1. A suspension of 545Bz in THE (1.78 wts) and MeOH (3.16 wts) was treated with 25% w/w NaOMe in MeOH (0.31 wts) under N.sub.2.
[0478] 2. The mixture was stirred at 50-60? C. for 3-3.5 h then cooled to 15-25? C. and stirred for a further 16-24 h.
[0479] 3. The reaction pH was adjusted to 6-8 by the addition of glacial AcOH.
[0480] 4. The mixture was distilled under vacuum at 45? C. to 2.0 volumes.
[0481] 5. The mixture was added slowly to EtOAc (9.02 wts) at 15-25? C. to precipitate the product.
[0482] 6. The suspension was filtered, and the filter cake was washed with EtOAc (2?0.9 wts).
[0483] 7. The product was dried to a constant mass at 45? C. under vacuum to give crude product (60% 545OH by mass).
Purification by ultrafiltration and precipitation
[0484] 8. Crude 545OH (1 wt calculated from active mass) in water (20 wts) was filtered through a 0.22 ?m filter and charged to the retentate tank.
[0485] 9. Ultrafiltration/diafiltration in water was performed using a 30 kDa MWCO filter (160 wts, 8 diavolumes).
[0486] 10. The product solution was concentrated to 8.0 volumes then slowly added to acetonitrile (7.37 wts. with respect to (wrt) the aqueous solution; 59 wts. wrt the contained 5450H).
[0487] 11. The precipitated product was isolated using a filter under N.sub.2 and was washed with acetonitrile (3.14 wts)/EtOAc (3.61 wts), and then EtOAc (3.61 wts).
[0488] 12. The solid was slurried in EtOAc (3.16 wts) and then filtered. The filter cake was washed with EtOAc (3.16 wts?2).
[0489] 13. The product was dried to constant mass at 45? C. under vacuum.
Step 1: Solvent Ratio
[0490] The polarity and solubility of the product changes significantly during the debenzoylation reaction (when compared with the starting material). The perbenzoate 545Bz is soluble in THE (2 volumes) but insoluble in MeOH, while the polyol 545OH is slightly soluble in MeOH, but more soluble when THE is added. The ratio of THF:MeOH is therefore important to balance solubility of both starting material and product, and maintain adequate stirring during reaction.
Step 5: Precipitation Washing
[0491] Although the polyol 545OH was poorly soluble in most organic solvents, the form it takes on completion of debenzoylation could be converted to a solid by precipitation with MeCN, optionally mixed with EtOAc. This solid form was suitable for isolation by filtration under inert atmosphere, but was hygroscopic and not suitable for storage. Slurry washing with EtOAc converted the solid to a non-hygroscopic form that could be more easily handled.
Purification by Diafiltration Ultrafiltration
[0492] The inventors of the present invention have surprising found that polyol 545OH is strongly retained by a 30 kDa MWCO regenerated cellulose membrane, allowing low-molecular weight contaminants to pass through and be removed. As the molecular weight of 545OH is only 1037.23 Da, such strong retention by a large pore-size membrane was unexpected.
[0493] Without being bound by theory, the amphiphilic nature of the polyol 545OH may cause it to form large micellular structures in aqueous solution that enable it to be retained by the 30 kDa MWCO membrane.
Removal of Non-Coupled Oligosaccharides and Low-Molecular Weight Impurities
[0494] The major benefit of ultrafiltration as a purification technique is that the purification is dependent on size, rather than on the chemical nature of each impurity. Only molecules which form part of the micellar structure are retained by the membrane, and all other components below the molecular weight cut-off are removed, whether they be solvents, or inorganic or organic impurities. In particular, the ?-anomer of perbenzoate G4Bz is chemically unreactive, and is carried through the glycosylation reaction unchanged. This impurity is converted back to maltotetraose G4 in the global deprotection (Scheme 8), and is efficiently removed by ultrafiltration as it lacks a cholestanol group, and hence is not incorporated into micelles.
##STR00039##
Stage 5: Synthesis of Compound 1
[0495] The synthesis of Compound 1 uses a combination of sulfur trioxide pyridine complex (?3 eq. per hydroxyl group) in DMF solution as outlined in Scheme 9.
##STR00040##
Materials
[0496]
TABLE-US-00008 Reagents Quantities (wts.) 545OH 1 eq. SO.sub.3py 5.37 wts DMF 18.9 wts + 3 ? 4.72 wts 10% NaOH As req. EtOH 4.00 wts IPA 2.00 wts + 6 wts + 2 wts
Procedure
[0497] 1. 545OH (1 wt.) and SO.sub.3.Py (5.37 wts) in DMF (18.9 wts) was stirred under N.sub.2 at 15-25? C. for 35-30 min then at 40-45? C. for 16-24 h.
[0498] 2. The suspension was allowed to settle, and the supernatant was decanted and discarded.
[0499] 3. The solid was slurried three times in DMF (3?4.72 wts), decanting the supernatant to waste.
[0500] 4. Chilled water (2.0 wts.) was added and the mixture was slowly warmed until the product dissolved. The pH was adjusted to 8.0-10.0 using 10% w/w aq. NaOH, maintaining the temperature below 25? C.
[0501] 5. EtOH (4.00 wts) was added at 15-25? C. and the suspension was aged overnight at 5? C.?5? C.
[0502] 6. The suspension was filtered, and the filter cake was washed with WA (2wts).
[0503] 7. The product was re-slurried in WA (6 wts) at 15-25? C. then filtered again. The filter cake was washed with WA (2 wts.)
[0504] 8. The solid was dissolved in 1.39 w/w NaHCO.sub.3 in H.sub.2O (18.0 wts), filtered through a 0.22 ?m SFCA filter and charged to a UF retentate tank.
[0505] 9. Ultrafiltration/diafiltration was performed over a 2 kDa MWCO filter against 1.39% w/w NaHCO.sub.3 in H.sub.2O.
[0506] 10. The retentate was concentrated to ca. 5 volumes and charged slowly to cooled WA (20 wts.).
[0507] 11. The suspension was aged overnight, filtered over Grade 1 filter paper, and the filter cake was dried to a constant mass.
Step 2: Precipitation of the Crude Compound 1 Product
[0508] The previously disclosed sulfation procedure for Compound 1 in WO2009049370A1 was unsuitable for scale-up, as it required evaporation of the DMF solvent to dryness to yield the crude product. However, during an initial scale-up of Compound 1 synthesis, the crude product was noted to precipitate as a finely divided solid, facilitating the slurry purification discussed above. This precipitation was generally more reliable when a certain minimum quantity of polyol 545OH was employed, for example 20 g or more. A further precipitation step was then carried out after basification, which removed impurities including residual pyridine. The use of precipitation rather than evaporation of high-boiling DMF solvent enabled efficient manufacture of Compound 1 on a large scale.
Steps 2-3: Extractive Removal of Low-Molecular Weight Impurities During Workup
[0509] A major issue in the synthesis of Compound 1 is the removal of the undesired homologous oligosaccharide impurities, present in the original maltotetraose starting material. As purified maltotetraose G4 is expensive, the cost effective use of impure G4 syrups without chromatographic purification is a key improvement in the previously disclosed process to make Compound 1. Unable to remove ester-protected derivatives of G2 (Compound 2) and G3 (Compound 3) by crystallization or precipitation during the synthesis, the inventors of the present invention surprisingly discovered that the impurities could be carried through and removed after the final sulfation reaction.
[0510] The pyridinium salts of trisaccharide impurity Compound 3 and disaccharide impurity Compound 2 were surprisingly discovered to have an unexpectedly high solubility in the DMF reaction solvent. Thus, the sulfation conditions were developed to encourage precipitation of Compound 1 while keeping Compound 2 and Compound 3 in solution. This difference in solubility was particularly surprising when compared to previously reported sulfonation conditions for other oligosaccharides, in particular oligosaccharide derivatives not having a hydrophobic aglycon, which separated as a thick oil under the sulfation conditions. In contrast, the method presented herein can yield the initial crude Compound 1 as a filterable solid (Step 2), suitable for slurry washing. The previously reported conditions for the sulfation of Compound 1 did not give a filterable solid, but instead required evaporation of the DMF reaction solvent to dryness, which may be impractical on a manufacturing scale. However, in the present method a lower volume of DMF reaction solvent was used to encourage precipitation of Compound 1 while keeping the undesired lower homologues in solution. Further, a less aggressive temperature profile was used during the sulfation reaction to encourage slower precipitation of Compound 1 as a filterable solid rather than a thick oil. Previous sulfation reactions to prepare Compound 1 used 60? C. throughout the sulfation reaction, whereas the present method used a reaction temperature of 15-25? C. for 35-30 min followed by 40-45? C. for 16-24 h.
##STR00041##
Final Purification by Ultrafiltration
[0511] The use of dialysis/ultrafiltration for de-salting and final purification of Compound 1 has been disclosed previously [Yu, G. et al. Eur. J. Med. Chem. 2002, 37, 783-791; and Ferro, V. et al. J. Med. Chem. 2012, 55, 3804-3813]. This purification is considered extremely efficient, as it allows purging of all impurities that pass through the UF membrane (2000 Da MWCO).
[0512] Diafiltration against a solution containing a pharmaceutically acceptable cation is preferred to enable the product to be isolated in a pharmaceutically acceptable salt form of the product. In certain embodiments, the pharmaceutically acceptable cation is sodium, which can be formed, for example, by diafiltration of the product against a solution of sodium chloride in water.
[0513] The stability of Compound 1 requires that the compound is maintained at basic pH, or more conveniently at the pH of human blood (7.24). Optionally then, diafiltration against a pharmaceutically acceptable buffer solution at basic pH can yield a product with increased stability and shelf-life. A number of non-toxic buffers are available for this purpose, such as phosphate and citrate. The inventors of the present invention have used bicarbonate as it is volatile under HPLC analysis conditions when evaporative light scattering (ELS) detection is used. Performing the diafiltration/ultrafiltration against sodium bicarbonate (isotonic, 1.39%) in Water for Injection (Step 9 above) yields a product suitable for parenteral administration into humans, once it has been suitably sterilised. This was desirable due to the cost and limited availability of lyophilisation for bulk Compound 1 product, previously required for isolation of the final product as a solid.
[0514] Alternatively, if isolation of Compound 1 as a solid is required, the material may be precipitated by continuing on to Step 10 and Step 11 in the procedure above.
Synthesis of Compound 4
[0515] The synthesis above can be adapted to give an improved convergent synthesis of Compound 4 as shown in Scheme 10. The use of the potentially hazardous azide building block used in WO2009049370A1 is avoided in favour of preparing the required propylstearamide aglycon by acylation of aminopropanol with stearoyl chloride. Glycosylation with G4BzSTol as for Compound 1 proceeds smoothly to yield glycoside 562Bz, which is then reacted on to form Compound 4 in a manner analogous to the synthesis of Compound 1 discussed above.
##STR00042##
Synthesis of Compound 5
Overall Synthesis
[0516] The synthesis of Compound 5 was performed according to the four-stage synthesis outlined in Scheme 11. Each stage of the synthesis is discussed in further detail in the following sections. Importantly, the M5Ac starting material is depicted in Scheme 11 as a single pentasaccharide component for convenience, however it is actually a mixture of oligosaccharides from disaccharide to pentasaccharide, as shown in the structures below.
##STR00043##
[0517] The ultimate source of the M5Ac starting material was the neutral oligosaccharide fraction isolated as a by-product of the manufacture of phosphomannan (Ferro, V., Fewings, K., Palermo, M. C., & Li, C. (2001). Large-scale preparation of the oligosaccharide phosphate fraction of Pichia holstii NRRL Y-2448 phosphomannan for use in the manufacture of PI-88. Carbohydrate Research, 332(2), 183-189. doi:10.1016/s0008-6215(01)00061-1). The neutral oligosaccharide fraction was shown therein to be comprised of a mixture of homologous mannooligosaccharides. The mixture was isolated by lyophilisation, and acetylated using acetic anhydride and pyridine as described previously (see WO2009049370A1).
[0518] The synthesis outlined in Scheme 11 enables enhancement in the amount of the pentasaccharide product (compound 5), which is the component with the higher molecular weight, as compared with the lower oligosaccharide homologues (i.e. di, tri and tetra-saccharide).
##STR00044## ##STR00045##
Stage 1: Synthesis of M5AcSTol
[0519] The synthesis of M5AcSTo1 from M5Ac is outlined in Scheme 12 below. Briefly, the anomeric group of M5Ac is thiolated using para-thiocresol and BF.sub.3.Math.OEt.sub.2 in dichloromethane.
##STR00046##
Materials
[0520]
TABLE-US-00009 Reagents Quantity (wts.) Quantity (g) M5Ac 1 wt. 5 g Thiocresol 1.54 wt. 0.62 g DCM 1 ? 2 wt., 1 ? 0.2 wt. 10 mL, 1 mL BF.sub.3Et.sub.2O 1.8 wt. 0.72 mL Et.sub.3N 2.10 wt. 0.95 mL Ethyl acetate 8 wts. 40 mL 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3 2 ? 5.0 wts. 2 ? 25 mL 10% aq. NaCl 2 ? 5.0 wts. 2 ? 25 mL
Procedure
[0521] 1. M5Ac (1 wt.) and thiocresol (451 mg, 1.54 equiv.) were charged to a three necked round bottom flask and dissolved in DCM (10 mL, 2 vol.). The solution was then warmed to 34? C. using a metal heating block.
[0522] 2. BF.sub.3.Math.OEt.sub.2 (0.5 mL) was added dropwise, maintaining the reaction at <36? C. during the addition.
[0523] 3. The reaction was stirred at 35? C. for 5 hours, then cooled to ambient temperature and aged for a further 11 hours. The reaction was further warmed to 36? C. and stirred for a further 2 h 45 mins.
[0524] 4. BF.sub.3.Math.OEt.sub.2 (0.28 mL) was added dropwise.
[0525] 5. The reaction was stirred at 36? C. for a further 17 hours, when DCM (6 mL) was added.
[0526] 6. Thiocresol (94.5 mg) was added, followed by the dropwise addition of BF.sub.3.Et.sub.2O (0.20 mL). The reaction was heated at 36? C. for a further 4 hours 30 minutes.
[0527] 7. The reaction was quenched with the addition of Et.sub.3N (1.2 mL) in EtOAc (40 mL).
[0528] 8. The organic phase was successively washed with the following: 1?25 mL 10% aq. NaCl, 2?25 mL 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3, and 1?25 mL 10% aq. NaCl.
[0529] 9. The aqueous phases were separated. The organic phase was concentrated under reduced pressure to give the product M5AcSTol as a beige foam (5.44 g). The .sup.1H NMR spectrum of the product is shown at
Stage 2: Synthesis of M5AcChol
[0530] The synthesis of M5AcChol from M5AcSTol is outlined in Scheme 13 below. Briefly, cholestanol is glycosylated using M5AcSTol, NIS and TMSOTf in dichloromethane.
##STR00047##
Materials
[0531]
TABLE-US-00010 Reagents Quantity (wts.) Quantity (g) M5AcSTol 1 wt. 5.21 g 5?-cholestan-3?-ol 1.47 wt. 1.85 g DCM 8 wt. + 0.11 wt. 41.68 mL + 0.573 mL NIS 2 wt. 1.458 g TMSOTf 0.67 wt. 0.483 mL 10% aq. KOH/5% aq. 2 ? 5.0 wts. 2 ? 26.05 mL Na.sub.2S.sub.2O.sub.3 5% aq. NaHCO.sub.3 1 ? 5.0 wts. 1 ? 26.05 mL 10% aq. NaCl 1 ? 5.0 wts. 1 ? 26.05 mL EtOAc 3 wts. 15.63 mL Heptane 20 wts. 104.20 mL
Procedure
[0532] 1. A solution of M5AcSTo1 (1.0 wts) and 5?-cholestan-3?-ol (1.80 g, 1.47 equiv.) in DCM (40 mL) was stirred under N.sub.2 for 30-60 min.
[0533] 2. The mixture was cooled to 6? C. and NIS (1.40 g) was added to give a dark orange/red partial suspension. TMSOTf (0.48 mL) in DCM (0.5 mL) was added dropwise, and stirred for 20 mins.
[0534] 3. The mixture was stirred at 5? C. overnight.
[0535] 4. After 16 hours, the reaction was quenched by adding a solution of 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3 (5 wts.) at 0-5? C. during addition.
[0536] 5. The phases were separated, and the organic phase was washed successively with the following: 1?25 mL 10% aq. KOH/5% aq. Na.sub.2S.sub.2O.sub.3 solution, 1?25 mL 10% aq. NaHCO.sub.3, and 1?25 mL 10% aq. NaCl.
[0537] 6. The organic phase was concentrated under reduced pressure to give the crude material as a dark brown oil (7.87 g).
[0538] 7. The crude material was dissolved in EtOAc (15 mL) and stored overnight at 5? C.
[0539] 8. Heptane (100 mL) was cooled to 5? C. (ice/water bath) and the crude EtOAc solution was added dropwise to give a suspension which was aged at 5? C. for 1 hour.
[0540] 9. The solid was isolated by filtration (Whatman #1 paper), the solids and mother liquors were analysed by TLC (visualised using 10% H.sub.2SO.sub.4 in IMS).
[0541] 7. The solids were dried under reduced pressure to give a light tan powder M5AcChol product (4.66 g, 0.93 wts.) which was analysed by NMR, see
Stage 3: Synthesis of M50HChol
[0542] Global deprotection of peracetate M5AcChol was accomplished using standard Zempl?n transesterification with methanol, catalysed by methoxide as shown in Scheme 14.
##STR00048##
Materials
[0543]
TABLE-US-00011 Reagents Quantities (wts.) Quantities (g) M5AcChol 1 wt. 4.62 g THF 2 vol. 9.5 mL MeOH 4 vol. 19 mL 25% NaOMe in MeOH 3.45 wt. 2 mL AcOH As req. EtOAc 10 vol. + 2 ? 1 vol. 47 g + 2 ? 4.7 g H.sub.2O As req. Acetonitrile 59 wt.
Procedure
[0544] 1. To a 100 mL 3-neck round bottom flask (fitted with N.sub.2 bubbler, magnetic stirrer and thermometer) was charged M5AcChol (4.62 g, 1 equiv., 1 wts.), THE (9.5 mL, 2.0 vol.) and MeOH (19 mL, 4 vol.).
[0545] 2. The dark brown solution was heated to 36? C. using a heating block.
[0546] 3. 25% Sodium methoxide in methanol (2.0 mL) was added, monitoring the temperature.
[0547] 4. The resultant suspension was warmed to 51? C. and stirred for 4 hours.
[0548] 5. The reaction was allowed to cool to ambient temperature, and stirred overnight.
[0549] 6. Glacial acetic acid (400 ?L) was added (portion-wise) until the pH was neutral and a sample was analysed by TLC (visualised with 10% H.sub.2SO.sub.4 in IMS).
[0550] 7. A sample of the reaction mixture was filtered, and the resulting solid and mother liquors were analysed by TLC (visualised with 10% H.sub.2SO.sub.4 in IMS).
[0551] 8. The reaction mixture was concentrated to a solution in water under reduced pressure.
[0552] 9. The crude product material was precipitated by the addition of MeOH and EtOAc and the supernatant was decanted.
[0553] 10. The solids were dried under reduced pressure to give a tan solid (3.67 g, 122% yield, 0.79 wts.) containing crude M50HChol.
Purification by Ultrafiltration (Diafiltration)
[0554] 11. The crude solids were dissolved in water (50 mL) and passed through a 0.22 m SFCA filter rinsing with water (10 mL+5 mL) to give a dark brown solution.
[0555] 12. The product solution underwent diafiltration against water for 7 diavolumes using a 30 kDa size exclusion membrane (vivaflow unit, 1 diavolume=65 mL).
[0556] 13. After the diafiltration was completed, the retentate was concentrated to 20-30 mL.
[0557] 14. The retentate was flushed from the unit and the membrane rinsed with water (5 mL+20 mL).
[0558] 15. The combined retentate was concentrated under reduced pressure to 3.44 g, water (1.5 mL) was added to give a final retentate weight of ?5 g.
[0559] 16. The material was precipitated by adding the retentate dropwise to cold acetonitrile (120 mL) (ice/water bath).
[0560] 17. The resulting suspension was aged for 1 hour, then filtered (cloth) under a blanket of inert gas.
[0561] 18. The isolated solids were washed with EtOAc.
[0562] 19. The polyol was dried under reduced pressure to yield M5OHChol as a tan solid (1.72 g, 0.58 wts.), which was analysed by NMR, see
Stage 4: Synthesis of Compound 5
[0563] The synthesis of Compound 5 uses a combination of sulfur trioxide pyridine complex (?3 eq. per hydroxyl group) in DMF solution as outlined in Scheme 15.
##STR00049##
Materials
[0564]
TABLE-US-00012 Reagents Quantities (wts.) M5OHChol 1 eq. SO.sub.3py 39.10 wts DMF 57.2 wts + 40.2 wts 10% NaOH As required EtOH 9 vol. IPA 2.5 vol. + 16 vol. H.sub.2O 8 vol. NaCl 2.84 wt.
Procedure
[0565] 1. To a 250 mL 3-neck round bottom flask (fitted with N.sub.2 bubbler, large magnetic stirrer and thermometer) was charged M5OHChol (1.672 g, 1 equiv., 1 wts.) and DMF (95 mL, 57 vol.).
[0566] 2. The resulting mixture was stirred until a brown hazy solution formed. The reaction mixture was cooled to 5? C. (?5? C.) by use of an ice bath.
[0567] 3. SO.sub.3-py (9.44 g, 39 equiv.) was added in one portion to the cold mixture.
[0568] 4. The reaction was stirred for a further 30 min before the ice/water bath was removed. The reaction mixture was subsequently heated to 45? C.
[0569] 5. The reaction was left to stir overnight at 45? C.
[0570] 6. The reaction was allowed to cool to ambient temperature; the mixture still appeared as a hazy solution.
[0571] 7. A sample of the reaction mixture was analysed by TLC to confirm full consumption of starting material.
[0572] 8. To a ?60 mL portion of the reaction mixture; EtOAc (18 mL) was added without stirring.
[0573] 9. Stirring was started to facilitate full mixing of the layers and then stopped after (5-10 seconds) to allow the precipitated material to settle.
[0574] 10. The solid was allowed to settle and the supernatant was removed by vacuum transfer through a dip tube with a sintered tip.
[0575] 11. A solution of DMF (40 mL) and EtOAc (9 mL) was added, and the mixture stirred vigorously.
[0576] 12. Once the material was completely suspended, the stirring was stropped and left to settle over 1-2 hrs.
[0577] 13. The supernatant was removed by vacuum transfer.
[0578] 14. The resultant slurry was dissolved in water (10 mL).
[0579] 15. 10% NaOH solution (4 mL) was added (resulting in a pH of 6-7). Then 10% NaOH solution (1 mL) was added, (resulting in a pH of 14). Following this, 2M HCl solution (0.2 mL) was added, (resulting in a pH of 7). Finally, 10% NaOH solution (0.1 mL) was added, (resulting in a pH of pH 8-9).
[0580] 16. The supernatant was removed by vacuum transfer.
[0581] 17. The solids were redissolved in water (14 mL)
[0582] 18. Ethanol (5.5 mL) was added dropwise to cause precipitation.
[0583] 19. The solids were filtered using a Buchner filter and washed with IPA (4 mL).
Filtration was Slow Due to a Partial Blockage; However, this was Manageable on this Small Scale.
[0584] 20. The solids were dried under reduced pressure.
[0585] 21. The obtained dry solid was a fine powder containing larger clumps with a beige/light brown colour (1.95 g 72% yield, 1.82 wts.).
Diafiltration
[0586] 22. The obtained solids (1.87 g) were dissolved in 1.39% aq. NaHCO.sub.3 solution (100 mL)
[0587] 23. The solution was filtered through a 0.2 ?m SFCA filter. An additional 50 mL portion of 1.39% NaHCO.sub.3 was used to rinse the flask and filter and to dilute the solution to the desired concentration (12.4 mg/mL).
[0588] 24. The product solution underwent diafiltration against 1.39% aq. NaHCO.sub.3 solution for 7 diavolumes with a 2 kDa size exclusion membrane (vivaflow unit, 1 diavolume=150 mL).
[0589] 25. After 7 diavolumes, the retentate was concentrated to 20-30 mL
[0590] 26. The retentate was flushed from the unit and the membrane rinsed with an additional 2?20 mL, 1.39% aq. NaHCO.sub.3 solution.
[0591] 27. The combined retentate and flushes (65 mL) were transferred to a stirred Amicon ultrafiltration cell fitted with a 1 kDa size exclusion membrane to further concentrate the material to 10-15 mL total volume.
[0592] 28. The material was precipitated by adding the concentrated mixture dropwise into cold IPA (80 mL) (ice/water bath).
[0593] 29. The solids were removed by filtration washing with IPA (20 mL). The collected solids were dried under reduced pressure.
[0594] 30. The obtained dry solid was a fine powder containing larger clumps with a beige/light brown colour (1.64g 61% yield, 1.72 wts.)
Precipitation
[0595] 31. EtOAc was added in 0.1 mL portions up to a total of 0.6 mL. Further additions of a few drops showed minimal amounts of further precipitation. The thick flocculant suspension settled into a paste like solid.
[0596] 32. The supernatant was removed by syringe and the remaining solids were washed with DMF (5 mL)/EtOAc (0.6 mL)
[0597] 33. The wash resulted in a finely dispersed suspension which slowly settled to a paste like solid.
[0598] 34. The supernatant was removed by syringe, yielding Compound 5 as a mixture with other mannooligosaccharide homologues, which was analysed by NMR, see
HPLC comparison of the sulfated product with the unsulfated polyol precursor
[0599] M5OHChol showed that levels of Compound 5 had been enriched by partial removal of the lower homologues according to the inventive method. Table 1 shows the relative abundances of oligosaccharide homologues from di- to pentasaccharide before and after sulfation. The relative abundance of the pentasaccharide Compound 5 increased from 30.9% to 42.8% during the sulfation process, while the relative abundances of the lower homologues (di- to tetrasaccharides) all decreased to varying degrees.
TABLE-US-00013 TABLE 1 Relative abundance of mannooligosaccharides before and after sulfation. Absolute abundances (HPLC area/area) reported above have been normalised to total 100% for the selected chain lengths. Relative Composition of Relative Composition of Homologue Polyol Sulfated Mixture Disaccharide 3.0 0.7 Trisaccharide 16.0 11.8 Tetrasaccharide 50.1 44.7 Pentasaccharide 30.9 42.8 Total 100.0 100.0
[0600] Although the procedure for Compound 5 was not as efficient as that illustrated for Compound 1 above, the skilled person will appreciate that the absolute abundance of the required pentasaccharide fraction of mixture M5OHChol was only 28% before sulfation (area/area by HPLC), whereas the process illustrated above for Compound 1 began with G4 syrup containing at least 50% of the required tetrasaccharide (area/area by HPLC on dried basis). In addition, while the preparation of Compound 1 was extensively optimised for GMP manufacture, the preparation of Compound 5 was carried out with minimal optimisation on a laboratory scale, and in view of this, the purity achieved for Compound 5 was not as high as that reported for Compound 1. However, a person of skill in the art would realise that the preparation of Compound 5 could be further optimised according to the methods illustrated above. Without being limited, further improvements to the preparation of Compound 5 might result from changing some of the factors and conditions described above. These may include but should not be limited to: [0601] beginning with M5Ac starting material containing a higher proportion of pentasaccharide; [0602] optimising the quantities of sulfur trioxide pyridine complex and polar aprotic solvent (e.g. DMF) used in the sulfation reaction based on the relative solubilities of the different homologues; and/or [0603] optimising the composition of the wash steps using a dipolar aprotic solvent.
[0604] The skilled person will appreciate that the examples disclosed herewith with respect to the synthesis of Compound 1, Compound 4, and Compound 5 could be adapted without undue burden to the synthesis of other sulfated oligosaccharides having a hydrophobic aglycon, in particular those having a maltotetraose or manno-oligosaccharide backbone.
[0605] Further illustration of the utility of the inventive methods can be gained from the comparison of these methods with similar methods from the literature. For example, the preparation of PI-88 on a scale suitable for clinical trial use has been reported previously. The phosphomannan starting material for the preparation of PI-88 is based on the same mannooligosaccharide backbone used herein for the preparation of Compound 5 (indeed, the two oligosaccharide mixtures are ultimately derived from the same biological source as mentioned above). However, there are important differences in that the M5Ac oligosaccharides used here do not contain phosphate groups, but instead feature a hydrophobic aglycon. It follows that the method reported here for sulfation of Compound 5 must necessarily have some similarities to the literature methods for the production of PI-88, as they are chemically related products. However, the literature methods for PI-88 generally do not result in the useful enrichment of the higher oligosaccharides, and the purging of the lower homologues, in contrast with the inventive method.
[0606] It will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention as defined in the following claims.