Methods of preparing activated polymers having alpha nitrogen groups
View Patent ↗Methods for preparing substantially non-antigenic polymers having lone electron pair-containing moieties in high purity are disclosed. The polymers are useful as intermediates for synthesis of amine-based polymers and in the formation of activated polymers for conjugation with nucleophiles. Conjugates and methods of preparation and treatment with the conjugates are also disclosed. The resultant polymer-amines are of sufficient purity so that expensive and time consuming purification steps required for pharmaceutical grade polymers are avoided.
1. A method of preparing a polymer having a lone electron pair-containing moiety thereon, comprising:
reacting a polymer of formula (I)
R 2 -R 1 -(L 1 ) n -LG (I)
wherein
R 1 is a substantially non-antigenic polymer;
R 2 is a capping group, LG or LG-(L 1 ) n -;
L 1 is a bifunctional linker;
LG is a leaving group selected from the group consisting of
wherein R 3 is methyl, halogen, nitro, fluoromethyl, difluoromethyl, trifluoromethyl, or substituted carboxyl; a multi-halogen substituted benzenesulfonyl;
mesylate; brosylate; tresylate, and nosylate; and
n is 0 or positive integer,
with a compound of Formula (III) in an aqueous base at a temperature of from about 45 to about 50° C. and for a reaction time of less than about 18 hours,
wherein the compound of Formula (III) is
wherein
Z is NR 7 ;
X is O, S or NR 8 ;
m is a positive integer;
R 4 is H;
R 5 , R 6 , R 7 and R 8 are independently selected from the group consisting of H, C 1-6 alkyls, C 3-12 branched alkyls, C 3-8 cycloalkyls, C 1-6 substituted alkyls, C 3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C 1-6 heteroalkyls, substituted C 1-6 heteroalkyls, C 1-6 alkoxy, phenoxy and C 1-6 heteroalkoxy,
provided that R 4 and R 7 are both not H; and
the aqueous base is selected from the group consisting of NaOH, KOH, LiOH, Mg(OH) 2 , Ca(OH) 2 and mixtures thereof,
wherein
10 molar equivalent of compound of Formula (III) is employed for each equivalent of LG; and
the purity of the polymer having the lone electron pair-containing moiety thereon formed by said method is greater than 95%.
2. The method of claim 1 , wherein the reaction time is less than about 12 hours.
3. The method of claim 1 , wherein R 2 is methoxy.
4. The method of claim 3 , wherein LG is
5. The method of claim 1 , wherein X is O, m is 1, R 5 and R 6 are each H, and R 7 is methyl.
6. The method of claim 1 , wherein the compound of formula (III) is N-methyl-glycine (sarcosine).
7. The method of claim 1 , wherein R 1 is a polyalkylene oxide.
8. The method of claim 1 , wherein the polymer of Formula (I) is selected from the group consisting of
wherein
j is an integer from about 10 to about 340;
x is an integer from about 10 to about 2,300;
L 1 , L 2 and L 3 are bifunctional linkers; and
z is an integer from 1 to about 120.
9. The method of claim 7 , wherein said polyalkylene oxide is a polyethylene glycol of the formula: —CH 2 CH 2 —O—(CH 2 CH 2 O) x —CH 2 CH 2
wherein x is an integer from about 10 to about 2,300.
10. The method of claim 1 , wherein the substantially non-antigenic polymer has a weight average molecular weight from about 200 to about 100,000 Daltons.
11. The method of claim 10 , wherein the substantially non-antigenic polymer has a weight average molecular weight from about 2,000 to about 48,000 Daltons.
12. The method of claim 1 , wherein the polymer of formula (I) is:
wherein
R 2 is methoxy; and
R 1 is a polyalkylene oxide.
13. The method of claim 12 , wherein R 1 is a polyethylene glycol.
14. The method of claim 1 , wherein the aqueous base is NaOH.
15. The method of claim 1 , wherein the polymer having the lone electron pair-containing moiety thereon is
wherein
X is O or S;
m is 1;
n is 0 or 1;
R 5 and R 6 are independently selected from the group consisting of H, C 1-6 alkyls, C 3-12 branched alkyls, C 3-8 cycloalkyls, C 1-6 substituted alkyls, C 3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C 1-6 alkoxy, and phenoxy; and
R 7 is selected from the group consisting of C 1-6 alkyls, C 3-12 branchedalkyls, C 3-8 cycloalkyls, C 1-6 substituted alkyls, C 3-8 substituted cycloalkyls, aryls, and substituted aryls.
16. The method of claim 1 , further comprising reacting the polymer having the lone electron pair-containing moiety thereon with a compound having an activating group thereon to form an activated polymer having a lone electron pair-containing moiety thereon.
17. The method of claim 16 , wherein the activated polymer having the lone electron pair-containing moiety thereon is
18. The method of claim 16 , wherein the compound having the activating group thereon has a functional group capable of reacting with a nucleophile.
19. The method of claim 1 , wherein the purity of the polymer the polymer having the lone electron pair-containing moiety thereon formed by said method is greater than 98%.
20. The method of claim 17 , further comprising reacting
with asparaginase under conditions sufficient to form conjugates.
21. The method of claim 20 , wherein said asparaginase is derived from a recombinant source.
22. The method of claim 1 , wherein R 1 is a linear, branched or multi-armed polymer.