MACROCYCLIC COMPOUNDS
20200017454 ยท 2020-01-16
Inventors
- Anthony Davis (Bristol, GB)
- Robert Tromans (Bristol, GB)
- Miriam Ruth Wilson (Bristol, GB)
- Michael Glen Orchard (Marlborough, GB)
- Andrew Michael Chapman (Bristol, GB)
- Michael Roger Tomsett (Bristol, GB)
- Johnathan Vincent Matlock (Bristol, GB)
Cpc classification
G01N2800/042
PHYSICS
G01N2400/00
PHYSICS
C07D259/00
CHEMISTRY; METALLURGY
International classification
C07D259/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to macrocyclic compounds which are capable of selective binding to a target saccharide (e.g. glucose), making them particularly well suited for use in saccharide sensing applications. The present invention also relates to processes for the preparation of said compounds, to compositions and devices comprising them, and to their use in the detection of a target saccharide.
Claims
1. A compound of Formula (I), or a salt, hydrate or solvate thereof, as shown below: ##STR00252## wherein: bonds b.sub.1 and b.sub.2 are independently selected from a single bond or double bond; R.sub.1a, R.sub.1b, R.sub.2a and R.sub.2b are independently selected from hydrogen, carbonyl, (1-8C)alkyl, (3-10C)cycloalkyl, aryl, heteroaryl and heterocyclyl, each of which, other than hydrogen and carbonyl, is optionally substituted by one or more substituent groups selected from (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxyl, carboxy, carbamoyl, sulfamoyl, mercapto and a hydrophilic substituent group; or R.sub.1a and R.sub.1b are linked so as to form a group of the formula: ##STR00253## and/or R.sub.2a and R.sub.2b are linked so as to form a group of the formula: ##STR00254## wherein: denotes the point of attachment; bonds b.sub.1 and b.sub.2 are as described above; Rings A and B are independently selected from aryl, heteroaryl, heterocyclyl, cycloalkyl and cycloalkenyl; R.sub.1 and R.sub.2 are independently selected from (1-6C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-6C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxyl, carboxy, carbamoyl, sulfamoyl and mercapto; a and b are integers independently selected from 0 to 2; m and n are integers independently selected from 0 to 2; Z.sub.1 and Z.sub.2 are independently selected from a hydrophilic substituent group; C and D are independently selected from aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl and a group of the formula: ##STR00255## wherein: s, t and v are integers independently selected from 1 or 2;
denotes the point of attachment; R.sub.3 and R.sub.4 are independently selected from halo, (1-4C)alkyl, (1-4C)alkoxy, amino, nitro, (1-4C)alkylamino, (1-4C)dialkylamino, (1-4C)haloalkyl, (1-4C)haloalkoxy, cyano, (2-4C)alkenyl, (2-4C)alkynyl and a group of the formula:
-L.sup.1-Y.sup.1-Q.sup.1 wherein: L.sup.1 is absent or a (1-5C)alkylene optionally substituted by one or more substituents selected from (1-2C)alkyl and oxo; Y.sup.1 is absent or selected from a one of the following groups; O, S, SO, SO.sub.2, N(R.sub.a), C(O), C(O)O, OC(O), C(O)N(R.sub.a), N(R.sub.a)C(O), N(Rb)C(O)N(R.sub.a), N(R.sub.a)C(O)O, OC(O)N(R.sub.a), S(O).sub.2N(R.sub.a), and N(R.sub.a)SO.sub.2, wherein R.sub.a and Rb are each independently selected from hydrogen and (1-4C)alkyl; and Q.sup.1 is hydrogen, (1-8C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-10C)cycloalkyl, (3-10C)cycloalkenyl, heteroaryl and heterocyclyl; wherein Q.sup.1 is optionally further substituted by one or more substituent groups independently selected from (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, amino, (1-4C)aminoalkyl, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, oxy, NR.sub.cR.sub.d, OR.sub.c, C(O)R.sub.d, C(O)OR.sub.c, OC(O)R.sub.c, C(O)N(R.sub.d)R.sub.c, N(R.sub.d)C(O)R.sub.c, S(O).sub.yR.sub.c (where y is 0, 1 or 2), SO.sub.2N(R.sub.d)R.sub.c, N(R.sub.d)SO.sub.2R.sub.c, Si(Re)(R.sub.d)R.sub.c and (CH.sub.2).sub.zNR.sub.dR.sub.c (where z is 1, 2 or 3); wherein R.sub.c, R.sub.d and Re are each independently selected from hydrogen, (1-6C)alkyl and (3-6C)cycloalkyl; and R.sub.c and R.sub.d can be linked such that, together with the nitrogen atom to which they are attached, they form a 4-7 membered heterocyclic ring which is optionally substituted by one or more substituents selected from (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano or hydroxyl; and wherein two R.sub.3 and/or two R.sub.4 groups taken together may form a group of the formula: ##STR00256## wherein: R.sub.x is selected from hydrogen and (1-6C)alkyl optionally substituted by one or more substituent groups selected from halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, cyano, hydroxy, sulfamoyl, mercapto, ureido, NR.sub.fR.sub.g, OR.sub.f, C(O)R.sub.f, C(O)OR.sub.f, OC(O)R.sub.f, C(O)N(R.sub.g)R.sub.f and N(R.sub.g)C(O)R.sub.f, wherein R.sub.f and R.sub.g are selected from hydrogen and (1-4C)alkyl; and the dashed lines represent the points of attachment to C and/or D; W.sub.1, W.sub.2, W.sub.3 and W.sub.4 are independently selected from CR.sub.hR.sub.i, wherein R.sub.h and R.sub.i are selected from hydrogen and (1-2C)alkyl; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are independently selected from a group of the formula: ##STR00257## wherein: denotes the point of attachment; W.sub.x is selected from O or NH; and Q is selected from O, S and NR.sub.j, wherein R.sub.j is selected from hydrogen, (1-4C)alkyl, aryl, heteroaryl and sulfonyl; Z.sub.3 and Z.sub.4 are independently selected from a hydrophilic substituent group; L is absent or a linker, which optionally bears a hydrophilic substituent group Z.sub.5; c and d are integers independently selected from 0 to 4; and o and p are integers independently selected from 0 to 2; and wherein: i) the compound of Formula I is optionally attached to a displaceable reporter molecule via one or more of the substituent groups associated with R.sub.1, R.sub.2, R.sub.3, R.sub.4, Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4 and/or Z.sup.5; and/or ii) the compound of Formula I is optionally attached to a substituent group of Formula A1 shown below at a position associated with one or more of the substituent groups R.sub.1a, R.sub.1b, R.sub.2a, R.sub.2b, R.sub.1, R.sub.2, R.sub.3, R.sub.4, Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4 and/or Z.sub.5:
X.sub.2a-L.sub.2a-Z.sub.2a(Formula A1) wherein: X.sub.2a is absent or selected from O, S, SO, SO.sub.2, N(R.sup.x2), C(O), C(O)O, OC(O), C(O)N(R.sup.x2), N(R.sup.x2)C(O), N(R.sup.x2)C(O)N(R.sup.x3), N(R.sup.x2)C(O)O, OC(O)N(R.sup.x2), S(O).sub.2N(R.sup.x2) and N(R.sup.x2)SO.sub.2, wherein R.sup.x2 and R.sup.x3 are each independently selected from hydrogen and (1-4C)alkyl; L.sub.2a is absent or selected from (1-20C)alkylene, (1-20C)alkylene oxide, (1-20C)alkenyl and (1-20C)alkynyl, each of which being optionally substituted by one or more substituents selected from (1-2C)alkyl, aryl and oxo; and Z.sub.2a is selected from carboxy, carbamoyl, sulphamoyl, mercapto, amino, azido, (1-4C)alkenyl, (1-4C)alkynyl, NR.sup.xcR.sup.xd, OR.sup.xc, ONR.sup.xcR.sup.xd, C(O)X.sub.a, C(Q.sup.z)OR.sup.xf, NCO, NR.sup.xcC(O)CH.sub.2X.sub.b, C(O)N(R.sup.xe)NR.sup.XcR.sup.Xd, S(O).sub.yX.sub.a (where y is 0, 1 or 2), SO.sub.2N(R.sup.xe)NR.sup.xcR.sup.xd, Si(R.sup.xg)(R.sup.xh)R.sup.xi, SSX.sub.c an amino acid and ##STR00258## wherein: X.sub.a is a leaving group (e.g. halo or CF.sub.3); X.sub.b is a halo (e.g. iodo); X.sub.c is an aryl or heteroaryl, optionally substituted with one or more substituents selected from halo, cyano and nitro; R.sup.xc, R.sup.xd and R.sup.xe are each independently selected from hydrogen and (1-6C)alkyl; R.sup.xf is selected from hydrogen and (1-6C)alkyl, or R.sup.xf is a substituent group that renders C(O)OR.sup.xf, when taken as a whole, to be an activated ester (e.g a hydroxysuccinimide ester, a hydroxy-3-sulfo-succinimide ester or a pentafluorophenyl ester); Q.sup.Z is selected from O or .sup.+NR.sup.Q1R.sup.Q2, where R.sup.Q1 and R.sup.Q2 are independently selected from hydrogen and methyl; and R.sup.xg, R.sup.xh and R.sup.xi are each independently selected from (1-4C)alkyl, hydroxy, halo and (1-4C)alkoxy; with the proviso that the compound of Formula I comprises at least one hydrophilic substituent group (e.g. Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4 or Z.sub.5).
2. A compound according to claim 1, wherein W.sub.1, W.sub.2, W.sub.3 and W.sub.4 are CH.sub.2.
3. A compound according to claim 1, wherein Q is selected from O or S.
4. A compound according to claim 3, wherein Q is O.
5. A compound, salt, hydrate or solvate thereof, according to claim 1, wherein the compound has the structural Formula Ib shown below: ##STR00259## wherein, each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4, a, b, c, d, m, n, o, p, L, C, D and Rings A and B are as defined in any preceding claim.
6. A compound according to claim 1, wherein Rings A and B are independently selected from phenyl, pyridyl, naphthyl and pyrrolidinyl.
7. A compound according to claim 6, wherein Rings A and B are phenyl.
8. A compound according to claim 1, wherein L is a linker selected from a group of the formula: ##STR00260## wherein: denotes the point of attachment; W.sub.5 and W.sub.6 are independently selected from CR.sub.kR.sub.l, wherein R.sub.k and R.sub.l are selected from hydrogen and (1-2C)alkyl; X.sub.5 and X.sub.6 are independently selected from a group of the formula: ##STR00261## wherein:
denotes the point of attachment; and Q.sub.2 is selected from O, S and NR.sub.m, wherein R.sub.m, is selected from hydrogen, (1-4C)alkyl, aryl, heteroaryl and sulfonyl; bond b.sub.3 is a single or double bond; Ring E is selected from aryl, heteroaryl, heterocyclyl, cycloalkyl and cycloalkenyl; R.sub.5 is selected from (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxyl, carboxy, carbamoyl, sulfamoyl and mercapto; Z.sub.5 is a hydrophilic substituent group; q is an integer from 0 to 2; and e is an integer from 0 to 2.
9. A compound according to claim 8, wherein W.sub.5 and W.sub.6 are CH.sub.2.
10. A compound according to claim 8, wherein Q.sub.2 is O.
11. A compound according to claim 8, wherein Ring E is selected from phenyl, pyridyl, naphthyl and pyrrolidinyl.
12. A compound according to claim 11, wherein Ring E is phenyl.
13. A compound according to claim 8, wherein integers a and b are 0.
14. A compound according to claim 8, wherein integers m and n are 1.
15. A compound, salt, hydrate or solvate thereof, according to claim 8, wherein the compound has the structural formula Id shown below: ##STR00262## wherein, each of C, D, R.sub.3, R.sub.4, Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4, Z.sub.5, c, d, o and p are as defined in any preceding claim.
16. A compound according to claim 8, wherein C and D are independently selected from phenyl and pyridyl.
17. A compound according to claim 8, wherein C and D are phenyl.
18. A compound according to claim 8, wherein integers o and p are 0.
19. A compound, salt, hydrate or solvate thereof, according to claim 8, wherein the compound has the structural Formula Ie shown below: ##STR00263## wherein: Z.sup.1, Z.sup.2 and Z.sup.5 are as defined in claim 1; and R.sup.3a, R.sup.3b, R.sup.3c, R.sup.4a, R.sup.4b and R.sup.4c are independently selected from hydrogen, halo, (1-4C)alkyl, (1-4C)alkoxy, amino, nitro, (1-4C)alkylamino, (1-4C)dialkylamino, (1-4C)haloalkyl, (1-4C)haloalkoxy, cyano, (2-4C)alkenyl, (2-4C)alkynyl and a group of the formula:
-L.sup.1a-Y.sup.1a-Q.sup.1a wherein: L.sup.1a is absent or (1-2C)alkylene optionally substituted by one or more substituents selected from (1-2C)alkyl or oxo; Y.sup.1a is absent or selected from a one of the following groups; O, S, SO, SO.sub.2, N(R.sub.n), C(O), C(O)O, OC(O), C(O)N(R.sub.n) and N(R.sub.n)C(O), wherein R.sub.n is selected from hydrogen and (1-4C)alkyl; and Q.sup.1a is hydrogen, (1-8C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-10C)cycloalkyl, (3-10C)cycloalkenyl, heteroaryl and heterocyclyl; wherein Q.sup.1a is optionally further substituted by one or more substituent groups independently selected from (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, amino, (1-4C)aminoalkyl, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, oxy, NR.sub.oR.sub.p, OR.sub.o, C(O)R.sub.o, C(O)OR.sub.o, OC(O)R.sub.o, C(O)N(R.sub.p)R.sub.o, N(R.sub.p)C(O)R.sub.o, S(O).sub.y1R.sub.o (where y1 is 0, 1 or 2), SO.sub.2N(R.sub.p)R.sub.o, N(R.sub.p)SO.sub.2R.sub.o, Si(R.sub.q)(R.sub.p)R.sub.o and (CH.sub.2).sub.z1NR.sub.oR.sub.p (where z.sub.1 is 1, 2 or 3); wherein R.sub.o, R.sub.p and R.sub.q are each independently selected from hydrogen and (1-6C)alkyl.
20. A compound according to claim 19, wherein R.sup.3a, R.sup.3b, R.sup.3c, R.sup.4a, R.sup.4b and R.sup.4c are independently selected from hydrogen, halo, (1-4C)alkyl, (1-4C)alkoxy, amino, nitro, (1-4C)alkylamino, (1-4C)dialkylamino, (1-4C)haloalkyl, (1-4C)haloalkoxy, cyano, (2-4C)alkenyl, (2-4C)alkynyl and a group of the formula:
-L.sup.1a-Y.sup.1a-Q.sup.1a wherein: L.sup.1a is absent or (1-2C)alkylene; Y.sup.1a is absent or selected from a one of the following groups; O, S, SO, SO.sub.2, N(R.sub.i), C(O), C(O)O, OC(O), C(O)N(R.sub.n) and N(R.sub.n)C(O), wherein R.sub.n is selected from hydrogen and (1-4C)alkyl; and Q.sup.1a is hydrogen, (1-8C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-10C)cycloalkyl, (3-10C)cycloalkenyl, heteroaryl and heterocyclyl; wherein Q.sup.1a is optionally further substituted by one or more substituent groups independently selected from (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, amino, (1-4C)aminoalkyl, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl and mercapto.
21. A compound according to claim 19, wherein R.sup.3a, R.sup.3b, R.sup.3c, R.sup.4a, R.sup.4b and R.sup.4c are independently selected from hydrogen, halo, (1-4C)alkyl, (1-4C)alkoxy, amino, nitro, (1-4C)alkylamino, (1-4C)dialkylamino, (1-4C)haloalkyl, (1-4C)haloalkoxy, cyano, (2-4C)alkenyl and (2-4C)alkynyl.
22. A compound according to claim 1, wherein L is absent.
23. A compound according to claim 22, wherein C and D are independently selected from naphthenyl or anthracenyl.
24. A compound according to claim 23, wherein C and D are anthracenyl.
25. A compound according to claim 22, wherein the compound has the structural Formula Ig, shown below: ##STR00264## wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4, a, b, c, d, m, n, o and p are as defined in claim 1.
26. A compound according to claim 22, wherein integers m and n are 0.
27. A compound according to claim 22, wherein integers a and b are 0.
28. A compound according to claim 1, wherein Z.sub.1, Z.sub.2, Z.sub.3 and Z.sub.4 and Z.sub.5 are independently selected from a hydrophilic substituent group comprising one or more hydrophilic functional groups selected from carboxylic acids, carboxylate ions, carboxylate esters, hydroxyl, amines, amides, ethers, ketone and aldehyde groups, nitro groups, sulphates, sulphonates, phosphates, phosphonates, and combinations thereof.
29. A compound according to claim 1, wherein Z.sub.1, Z.sub.2, Z.sub.3 and Z.sub.4 and Z.sub.5 are independently selected from a hydrophilic substituent group, wherein said hydrophilic substituent group is a hydrophilic polymer or hydrophilic dendritic group.
30. A compound according to claim 1, wherein Z.sub.1, Z.sub.2, Z.sub.3 and Z.sub.4 and Z.sub.5 are independently selected from a hydrophilic polymer or a dendritic group comprising between 1 and 5 generations of building units and a terminal functional group T.sub.1, and wherein each building unit is independently selected from a group of Formula A:
-L.sup.2-L.sup.2a-V(Formula A) wherein: L.sup.2 is selected from O, C(O), C(O)O, OC(O), C(O)N(R.sub.r), N(R.sub.r)C(O), N(R.sub.s)C(O)N(R.sub.r), N(R.sub.r)C(O)O, OC(O)N(R.sub.r), S(O).sub.2N(R.sub.r), and N(R.sub.r)SO.sub.2, wherein R.sub.r and R.sub.s are each independently selected from hydrogen and (1-4C)alkyl; L.sup.2a is a bond or a (1-4C)alkylene; V is absent or a group of the formula: ##STR00265## wherein: V.sub.1, V.sub.2, V.sub.3, V.sub.4 and V.sub.5 are independently selected from a (1-6C)alkylene optionally interrupted by one or more groups selected from O, S and NR.sub.t, wherein R.sub.t is selected from hydrogen and (1-2C)alkyl; # denotes the point of attachment to one of Rings A, B, C, D or E; denotes the point of attachment to either another group of Formula A or a terminal functional group T.sub.1; and the terminal functional group T.sub.1 is selected from OH, C(O)OM.sub.x, C(O)OR.sub.u and C(O)NHR.sub.u, wherein R.sub.u is selected from hydrogen, (1-4C)alkyl, (1-4C)alkoxy, hydroxy(1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl, ethylene glycol and polyethylene glycol, and wherein M.sub.x is a cation (e.g. Na, Li, NH.sub.4).
31. A compound according to claim 30, wherein the dendritic group comprises between 1 and 4 generations of building units and a terminal functional group T.sub.1, and wherein each building unit is independently selected from a group of Formula A:
-L.sup.2-L.sup.2a-V(Formula A) wherein: L.sup.2 is selected from O, C(O), C(O)O and C(O)N(R.sub.r), wherein R.sub.r is selected from hydrogen and (1-4C)alkyl; L.sup.2a is a bond or a (1-4C)alkylene; V is absent or a group of the formula: ##STR00266## wherein: V.sub.1, V.sub.2, and V.sub.3 are independently selected from a (1-6C)alkylene optionally interrupted by one or more groups selected from oxygen atoms; # denotes the point of attachment to one of Rings A, B, C, D or E; denotes the point of attachment to either another group of Formula A or a terminal functional group T.sub.1; and the terminal functional group T.sub.1 is selected from OH, C(O)OM.sub.x, C(O)OR.sub.u and C(O)NHR.sub.u, wherein R.sub.u is selected from hydrogen, (1-4C)alkoxy and hydroxy(1-4C)alkyl, wherein M.sub.x is a cation (e.g. Na, Li, NH.sub.4).
32. A compound according to claim 30, wherein the dendritic group comprises between 1 and 3 generations of building units and a terminal functional group T.sub.1, and wherein each building unit is independently selected from a group of Formula A:
-L.sup.2-L.sup.2a-V(Formula A) wherein: L.sup.2 is C(O)N(R.sub.r), wherein R.sub.r is selected from hydrogen and (1-4C)alkyl; L.sup.2a is a bond or a (1-2C)alkylene; V is a group of the formula: ##STR00267## wherein: V.sub.1, V.sub.2, and V.sub.3 are independently selected from a (1-4C)alkylene optionally interrupted by one or more groups selected from oxygen atoms; # denotes the point of attachment to one of Rings A, B, C, D or E; denotes the point of attachment to either another group of Formula A or a terminal functional group T.sub.1; and the terminal functional group T.sub.1 is selected is C(O)OM.sub.x, wherein M.sub.x is a cation (e.g. Na, Li, NH.sub.4).
33. A compound selected from any one of the following: ##STR00268## ##STR00269## ##STR00270## ##STR00271## ##STR00272## ##STR00273## ##STR00274## ##STR00275## ##STR00276## ##STR00277##
34. A compound of the formula shown below: ##STR00278## wherein each of Z.sub.1, Z.sub.2 and Z.sub.5 is a group of the formula: ##STR00279## wherein denotes the point of attachment.
35. A compound of the formula shown below: ##STR00280## wherein each of Z.sub.1 and Z.sub.2 is a group of the formula: ##STR00281## wherein denotes the point of attachment.
36. A compound of the formula shown below: ##STR00282## wherein each of Z.sub.3, and Z.sub.4 is a group of the formula: ##STR00283## wherein denotes the point of attachment.
37. A compound according to claim 1, which is immobilised on or in a solid or semi-solid support.
38. A compound according to claim 37, wherein the solid or semi-solid support is a polymeric matrix and/or a gel, such as a hydrogel.
39. A compound according to claim 38, wherein the polymeric matrix and/or gel is a polymer selected from cross-linked polyethylene glycol and/or polyacrylamide.
40. A compound according to claim 38, wherein the compounds are chemically linked to the polymeric matrix and/or gel.
41. A compound according to claim 38, wherein the compounds are physically incorporated within the polymeric matrix and/or gel via non-covalent interactions.
42. A complex comprising a compound according to claim 1 in association with a displaceable reporter molecule.
43. A composition comprising a compound according to claim 1 and a displaceable reporter molecule.
44. A complex according to claim 42, wherein the displaceable reporter molecule is an aromatic molecule or dye or a fluorescent aromatic molecule.
45. A composition according to claim 43, wherein the displaceable reporter molecule is a fluorescent aromatic molecule or an aromatic molecule or dye.
46. A complex comprising a compound according to claim 15 in association with a displaceable reporter molecule, wherein the displaceable reporter molecule is an aromatic molecule or dye or a fluorescent aromatic molecule.
47-54. (canceled)
55. A complex comprising a compound according to claim 1, covalently attached to insulin.
56. A composition comprising a compound according to claim 15 and a displaceable reporter molecule, wherein the displaceable reporter molecule is a fluorescent aromatic molecule or an aromatic molecule or dye.
57. A saccharide detection device comprising a complex according to claim 42.
58. A saccharide detection device comprising a composition according to claim 43.
59. A saccharide detection device comprising a compound according to claim 1.
60. A method for detecting a target saccharide in an aqueous environment, comprising using a compound according to claim 1.
61. A method of detecting a target saccharide in an aqueous environment, comprising using a saccharide detection device according to claim 59.
62. The method of claim 60, wherein the target saccharide is glucose.
63. The method of claim 61, wherein the target saccharide is glucose.
64. The method of claim 62, wherein the aqueous environment is blood or blood plasma.
65. The method of claim 63, wherein the aqueous environment is blood or blood plasma.
66. The method of claim 62, wherein the aqueous environment is a fermentation medium.
67. The method of claim 63, wherein the aqueous environment is a fermentation medium.
68. A method for the diagnosis of a condition which results in, or is otherwise associated with, an abnormal concentration of, and/or a change in the concentration of, a target saccharide, comprising using a compound according to claim 1.
69. The method of claim 68, wherein the condition is diabetes.
Description
EXAMPLES
[0755] Embodiments of the invention will be described, by way of example only, with reference to the accompanying drawings, in which:
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MATERIALS AND METHODS
[0833] Commercial reagents were purchased from Sigma-Aldrich, Alfa-Aesar or Acros Organics and were used without further purification unless otherwise specified. All air and moisture sensitive manipulations were carried out using standard vacuum line and Schlenk techniques, or in a drybox containing a purified argon atmosphere. Solvents for air and moisture sensitive manipulations were obtained from an Anhydrous Engineering Solvent Purification System or distilled and dried over activated molecular sieves.
[0834] Column chromatography was performed using silica gel 60 (Sigma Aldrich) and a suitable eluent. TLC was performed using aluminium backed TLC plates (Merck-Keiselgel 60 F254) and visualised using UV fluorescence and/or developed using ninhydrin, potassium permanagante, EtOH/H.sub.2SO.sub.4, vanillin, Pd(OAc).sub.2/H.sub.2O or iodine.
[0835] HPLC chromatography was performed using a Waters 600 Controller with a Waters 2998 Photodiode Array Detector. For analytical runs a XSELECT CSH C18 5 m (4.6150 mm) column was used and for preparative runs a XSELECT CSH Prep C18 5 m OBD (19250 mm) column was used, normally with an acetone-water solvent mixture.
[0836] .sup.1H and .sup.13C NMR spectra were recorded on Varian VNMR 400 MHz, Jeol Eclipse 400 MHz, Varian VNMR 500 MHz, Bruker cryogenically cooled 500 MHz and Varian VNMR cryogenically cooled S600 MHz spectrometers. All spectra were obtained at ambient temperature unless stated otherwise. All .sup.1H and .sup.13C NMR chemical shifts are reported relative to tetramethylsilane as an internal standard and in CDCl.sub.3 unless otherwise stated, with H (residual) and .sup.13C chemical shifts of the solvent as a secondary standard.
[0837] IR spectra were recorded on Perkin-Elmer Spectrum One FT-IR spectrometer with an ATR accessory and frequencies reported in wavenumbers (cm.sup.1). ESI-LRMS (electrospray ionisation low resolution mass spectrometry) was performed on a VG Analytical Quattro, ESI-HRMS (electrospray ionisation high resolution mass spectrometry) was performed on a Bruker Daltonics Apex IV and MALDI-MS (matrix-assisted laser desorption/ionisation) was performed on an Applied Biosystems 4700. Elemental analysis was performed on a EuroVector EA3000 Elemental Analyser.
[0838] .sup.1H-NMR titrations were performed on a Varian VNMR cryogenically cooled S600 spectrometer. Solutions of saccharides in D.sub.2O (99.9%), containing receptor at a known concentration to be used in the experiment, were prepared and allowed to equilibrate overnight before use if necessary. Aliquots were then added to an NMR tube containing a known concentration of receptor solution (typically 100 M-400 M). The receptor concentration was therefore held constant while the carbohydrate concentration was increased. The sample tube was shaken after each addition and .sup.1H-NMR spectra were acquired at 298 K.
[0839] Isothermal Titration (Micro)Calorimetry (ITC) experiments were performed on a MicroCal iTC200 microcalorimeter and/or a MicroCali VP-ITC. ITC experiments were carried out at 298 K. Saccharide solutions were prepared in HPLC-grade water and allowed to equilibrate overnight if necessary. The sample cell was charged with a known concentration of receptor solution in HPLC-grade water (typically 50 M-200 M). Then, aliquots (typically 1.0 L) of carbohydrate solution were added and the evolution of heat was followed as a function of time. Heats of dilution were measured by injecting the same carbohydrate solution into HPLC-grade water, using identical conditions. For every addition, the heat of dilution was subtracted from the heat of binding using a MicroCal software programme implemented in ORIGIN 7.0. This gave an XY matrix of heat vs. total guest concentration. This matrix was then imported into a specially written Excel programme to fit the data to a 1:1 binding model to give a Ka. G can be derived from Ka, and S can be derived from H and G using common thermodynamic equations. This method of analysis was used in conjunction with fits for K.sub.a calculated using the MicroCal software to corroborate the results obtained.
Synthetic Procedures
Bicyclic Receptor Synthesis
[0840] ##STR00119##
1,3,5-triethyl-2,4,6-tris(aminomethyl)benzene (Compound 106)
[0841] ##STR00120##
[0842] Under an inert N.sub.2 atmosphere, 1,3,5-tris(bromomethyl)-2,4,6-triethylbenzene 104 (324 mg, 0.74 mmol) was dissolved in anhydrous DMF (4.5 mL) and NaN.sub.3 (157 mg, 2.42 mmol) added. The reaction was heated to 60 C. for 16 hours. The reaction mixture as then diluted with ethyl acetate (20 mL) and washed with water (320 mL), dried (MgSO.sub.4) and filtered. DMF (4 mL) was added to the filtrate and the solvent removed under vacuum to a volume of 4 mL. Conversion to tris-azide 105 was confirmed by .sup.1H NMR (220 mg, 0.68 mmol, 92%). The resultant DMF solution was transferred to a degassed anhydrous solution of THF (22 mL) and PMe.sub.3 (1M in THF, 4.1 mL) under an inert N.sub.2 atmosphere. The reaction mixture was stirred at room temperature for 1 hour and degassed H.sub.2O (5 mL) added, with the reaction mixture stirred for a further 16 hours. The solvent and excess PMe.sub.3 was then evaporated by bubbling N.sub.2 through the solution, and the crude residue suspended in H.sub.2O (10 mL). The suspension was then freeze dried to afford 106 (148 mg, 0.61 mmol, 90%) as a white solid. .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.24 (t, J=7.5 Hz, 9H, C(1)H), 2.83 (q, J=7.5 Hz, 6H, C(2)H), 3.88 (s, 6H, C(5)H.sub.2); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 16.8 (C(1).sub.2), 22.6 (C(2)), 39.7 (C(5)H), 137.4 (C3), 140.4 (C4); LRMS: (ESI.sup.+) Found [M+Na].sup.+: 272.2
1,3,5-triethyl-2,4,6-tris(isocyanatomethyl)benzene (Compound 103)
[0843] ##STR00121##
Method A
[0844] A flask was charged with 106 (30 mg, 0.12 mmol) and NaHCO.sub.3 (20 mg, 0.24 mmol). CH.sub.2Cl.sub.2 (5 mL) and H.sub.2O (5 mL) were added, the mixture cooled to 0 C. and rapidly stirred. Triphosgene (40 mg, 0.13 mmol) was added and the reaction mixture vigorously stirred at room temperature for 1 hour. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (20 mL) and brine (10 mL), and the organic layer separated, dried (MgSO.sub.4) and the solvent removed under vacuum to afford 103 (50 mg, 0.166 mmol, 78%) as a colourless oil.
Method B
[0845] Under an inert N.sub.2 atmosphere, a flask was charged with triphosgene (1.81 g, 6.1 mmol). Anhydrous toluene (70 mL) was added. A solution of 106 (500 mg, 2.0 mmol) in anhydrous toluene (40 mL) was added dropwise over a period of 7 minutes. The reaction mixture was heated to reflux and stirred for a further 75 min. The reaction mixture was allowed to cool down and the solvent removed under high vacuum. The residue was then redissolved in about 40 mL toluene and filtered on cotton wool. The solvent was removed under high vacuum to afford 103 (630 mg, 1.9 mmol, 95%) as an oil that slowly crystallised into a light yellow solid.
[0846] .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.26 (t, J=7.6 Hz, 9H, C(1)H), 2.84 (q, J=7.6 Hz, 6H, C(2)H), 4.49 (s, 6H, C(5)H.sub.2); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 16.1 (C(1)H.sub.2), 22.8 (C(2)H), 40.4 (C(5)H), 123.1 (C6), 132.4 (C3), 143.1 (C4); V.sub.max 2973, 2933, 2875, 2243, 1495, 1453, 1335, 1042, 856, 577 cm.sup.1; LRMS: (ESI) Found [M+Na].sup.+: 350.1. HRMS: (ESI.sup.+) calculated for C.sub.18H.sub.21N.sub.3O.sub.3Na.sup.+: 350.1475, found [M+Na].sup.+: 350.1474.
Fmoc Protected Tert-Butyl G2 Linker (Compound 84)
[0847] ##STR00122##
Method A
[0848] Under an inert N.sub.2 atmosphere, Fmoc-amino benzoic acid 67 (983 mg, 2.63 mmol), HBTU (996 mg, 2.63 mmol) and HOBt (355 mg, 2.63 mmol) were suspended in anhydrous THF (30 mL). DIPEA (1.2 mL, 6.57 mmol) was added and the reaction stirred at room temperature for 10 minutes. Second generation dendritic amine 82 (3.1 g, 2.19 mmol) was then added and the reaction stirred for 24 hours. The solvent was then removed under vacuum and the crude residue purified by flash column chromatography (3% MeOH:CH.sub.2Cl.sub.2) to afford 84 (3.49 g, 2.01 mmol, 92%) as an off-white solid.
Method B
[0849] Under an inert N.sub.2 atmosphere, 67 (5 g, 15.3 mmol), HBTU (5.03 g, 15.3 mmol) and HOBt.H.sub.2O (2.06 g mg, 15.3 mmol) were suspended in anhydrous THF (60 mL). DIPEA (7.2 mL, 38.3 mmol) was added and the reaction stirred at room temperature for 1 hour. The solvent was removed under vacuum and the crude residue dissolved in ethyl acetate (100 mL) and then poured into water (300 mL). The precipitate was then filtered and air dried to afford the crude HOBt ester/tetramethyl urea complex (1:1, 4.36 g, 7.19 mmol, Mw=607.67 g mol.sup.1) which was used without further purification. The crude HOBt ester complex was then suspended in anhydrous THF (60 mL) and 82 (9.4 g, 6.54 mmol) and DIPEA (2.1 mL, 12.3 mmol) added. The reaction mixture was stirred at room temperature for 24 hours and the solvent removed under vacuum. The crude residue was then purified by flash column chromatography (5% MeOH:CH.sub.2Cl.sub.2) to afford 84 (9.02 g, 5.19 mmol, 86%) as an off white solid.
[0850] .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.43 (s, 81H, C(26)H.sub.3), 1.95 (m, 18H, C(23)H.sub.2), 2.11 (t, J=7.2 Hz, 6H, C(18)H.sub.2), 2.17 (m, 18H, C(22)H.sub.2), 2.25 (t, J=7.2 Hz, 6H, C(19)H.sub.2), 4.27 (m, 3H, C(7)H and NH.sub.2), 4.47 (d, J=7.4 Hz, 2H, C(8)H.sub.2), 6.08 (s, 3H, NH), 6.76 (d, J=8.4 Hz, 1H, C(13)H), 7.26-7.31 (m, 2H, C(4)H), 7.38 (t, J=7.4 Hz, 2H, C(3)H), 7.57-7.69 (m, 2H, C(5)H), 7.71 (d, J=8.7 Hz, 1H, C(2)H), 7.75 (d, J=7.6 Hz, 3H, C(2)H), 7.78 (d, J=2.1 Hz, 1H, C(15)H), 8.54 (s, 1H, NH); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 28.0 (C26), 29.8 (C22), 29.9 (C23), 31.8 (C19), 32.2 (C18), 47.2 (C7), 53.4 (C17), 57.4 (C21), 67.3 (C8), 80.6 (C25) 116.6 (C12), 119.9 (C2), 122.6 (C10), 124.6 (C14), 125.3 (C4), 126.0 (C15), 126.8 (C13), 127.0 (C5), 127.6 (C3), 141.3 (C1), 143.8 (C6), 145.3 (C11), 154.9 (C9), 166.6 (C16), 172.7 (C24), 173.1 (C20); V.sub.max 2977, 2963, 1752, 1723, 1689, 1637, 1535, 1367, 1242, 1151, 1098, 844 cm.sup.1; HRMS: (ESI.sup.+) Found [M+2Na].sup.2+: 921.0252.
[0851] Fmoc-amino benzoic acid (67) was prepared according to literature procedure as described in Angew. Chem., 2008, 120, 6957.
[0852] Second generation dendritic amine (82) was prepared according to literature procedure as described in Angew. Chem. Int. Ed., 2015, 54, 2057
Triamino G2MM Tert-Butyl Protected Triethylbenzene Half Receptor (Compound 108)
[0853] ##STR00123##
[0854] Under an inert N.sub.2 atmosphere, 84 (600 mg, 0.35 mmol) was dissolved in a solution of 103 (28 mg, 0.086 mmol) in anhydrous dichloromethane (5 mL). Anhydrous pyridine (40 L) was added and the reaction heated to reflux for 16 hours, after which it was cooled to room temperature and the solvent removed under vacuum. The crude residue was purified by flash column chromatography (1:1, EtOAc:CH.sub.2Cl.sub.2.fwdarw.4% MeOH:CH.sub.2Cl.sub.2) to afford 107 (400 mg, 0.072 mmol, 84%) as a white solid. Conversion to 107 was confirmed by limited NMR studies* and high resolution mass spectrometry (ESI.sup.+): m/z calculated for [M+3Na].sup.3+ 2869.6877, found 1928.1169, calculated for [M+4Na].sup.4+: 1451.8339, found 1451.8320. Under an inert N.sub.2 atmosphere, 107 (300 mg, 0.052 mmol) was dissolved in anhydrous dichloromethane (8 mL) and cooled to 0 C. DBU (50 L, 0.30 mmol) was added dropwise and the reaction mixture warmed to room temperature and stirred for 1 hour. The solvent was removed under vacuum and the crude product purified by flash column chromatography (4% MeOH:CH.sub.2Cl.sub.2) to afford 108 (238 mg, 0.047 mmol, 91%) as a white solid. .sup.1H NMR: (400 MHz, (CD.sub.3OD): 1.24 (t, J=7.6 Hz, C(1)H.sub.3), 1.43 (s, 243H, C(23)H.sub.3), 1.94 (m, 54H, C(20)H.sub.2), 2.09 (m, 18H, C(15)H.sub.2), 2.18 (m, 54H, C(19)H.sub.2), 2.23 (m, 18H, C(16)H.sub.2), 2.86 (m, 6H, C(2)H.sub.2), 4.51 (s, 6H, C(5)H.sub.2), 7.21 (dd, J=2.1, 8.4 Hz, 3H, C(9)H), 7.29 (d, J=2.1 Hz, 3H, C(11)H), 7.4 (d, J=8.4 Hz, 3H, C(8)H), 7.4 (s, 9H, NH), 7.91 (s, 3H, NH); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 17.0 (C1), 22.5 (C2), 28.5 (C23), 30.5 (C19), 30.7 (C20), 32.3 (C15), 32.6 (C16), 37.9 (C5), 58.9 (C18), 59.4 (C14), 81.6 (C22), 117.4 (C11), 118.9 (C9) 124.5 (C8), 130.0 (C10), 132.9 (C3), 133.8 (C7), 141.4 (C12), 145.1 (C4), 157.9 (C6), 170.1 (C13), 174.4 (C21), 175.6 (C17); HRMS: (ESI.sup.+) Found [M+3Na].sup.3+: 1706.0490. * Limited NMR studies were only possible due to slow conformational exchange of 107 resulting in very broad signals of low intensity.
Tert-Butyl Protected Triethylbenzene Receptor (Compound 1a)
[0855] ##STR00124##
Method A
[0856] Under an inert N.sub.2 atmosphere, 108 (124 mg, 0.024 mmol) was dissolved in anhydrous dichloromethane (50 ml) and heated to reflux. A solution of 103 (8 mg, 0.024 mmol) in anhydrous dichloromethane (3 mL) was added and the reaction stirred at reflux for 3 days. The reaction mixture was cooled to room temperature and the solvent removed under vacuum. The crude product was purified by reverse-phase HPLC and then freeze dried to Ia (20 mg, 0.004 mmol, 15%) as a white solid.
Method B
[0857] Under an inert N.sub.2 atmosphere, 108 (200 mg, 0.04 mmol), Octyl-glucoside (23 mg, 0.08 mmol) and 4-dimethylaminopyridine (14 mg, 0.12 mmol) were dissolved in anhydrous dichloromethane (35 mL). A solution of 103 (13 mg, 0.04 mmol) in anhydrous dichloromethane (5 mL) was added and the reaction stirred at reflux for 2 days. The reaction mixture as cooled to room temperature and the solvent removed under vacuum. The crude product was purified by reverse phase HPLC and then freeze dried to Ia (85 mg, 0.016 mmol, 40%) as a white solid.
[0858] .sup.1H NMR: (400 MHz, (CD.sub.3OD): 1.24 (m, 18H C(1)H.sub.3), 1.43 (s, 243H, C(23)H.sub.3), 1.95 (m, 54H, C(20)H.sub.2), 2.13 (m, 18H, C(15)H.sub.2), 2.20 (m, 54H, C(19)H.sub.2), 2.25 (m, 18H, C(16)H.sub.2), 2.74, 2.84 (br m, 6H, C(2)H.sub.2), 4.40, 4.49 (br s, 6H, C(5)H.sub.2), 7.43 (s, 9H, NH), 7.63 (d, J=8.7 Hz, 3H, C(10)H), 8.03 (d, J=8.7 Hz, 3H, C(11)H), 8.07 (s, 3H, C(8)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 15.5, 15.6 (C1), 22.3, 22.5 (C2), 27.1 (C23), 29.1 (C19), 29.3 (C20), 30.8 (C15), 31.0 (C16), 37.5 (C5), 57.3 (C18), 58.1 (C14), 80.2 (C22), 120.3 (C11), 123.5 (C8), 124.1 (C10), 127.6 (C7), 129.4 (C9), 132.0, 132.6 (C3), 135.0 (C12), 143.0, 143.2 (C4), 155.8, 156.6 (C6), 168.2 (C13), 173.0 (C21), 174.1 (C17); HRMS: (ESI.sup.+) Found [M+3Na].sup.3+: 1816.1093.
Triethylbenzene Receptor (Receptor 1)
[0859] ##STR00125##
[0860] 1a (3.5 mg, 0.65 mol) was dissolved in HPLC grade dichloromethane (1.6 ml). Trifluoroacetic acid (0.4 mL) was added and the reaction stirred at room temperature for 16 hours. The solvent was then removed under a flow of nitrogen, the crude product suspended in water and freeze dried. The resultant solid was then suspended in water, neutralised to pH 7 with NaOH (aq) and then freeze dried to afford receptor 1 (3.3 mg, 0.62 mol, 95%) as a white solid. .sup.1H NMR: (400 MHz, (D.sub.2O): 1.17 (m, 18H C(1)H.sub.3), 1.94 (m, 54H, C(20)H.sub.2), 2.12 (m, 18H, C(15)H.sub.2), 2.18 (m, 54H, C(19)H.sub.2), 2.31 (m, 18H, C(16)H.sub.2), 2.76 (br m, 6H, C(2)H.sub.2), 4.46 (br s, 6H, C(5)H.sub.2), 7.61 (d, J=9.6 Hz, 3H, C(10)H), 7.82 (br m, 6H, C(11)H, C(8)H);); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 18.0, 18.1 (C1), 25.0, 25.2 (C2), 32.6 (C15), 32.7 (C19), 32.9 (C16), 33.3 (C20), 40.1, 40.3 (C5), 60.7 (C18), 61.3 (C14), 120.0 (C11), 121.6 (C10), 122.8 (C8), 125.7 (C7), 130.0 (C9), 134.7, 135.0 (C3), 135.6 (C12), 146.7, 146.9 (C4), 159.6, 159.9 (C6), 170.3 (C13), 177.8 (C17), 184.1 (C17).
##STR00126##
[0861] A schlenk flask equipped with a magnetic stirrer was charged with 108 (200.0 mg, 0.04 mmol), DMAP (14.5 mg, 0.12 mmol) and n-octyl glucoside (23.2 mg, 0.08 mmol) were added to the flask and placed under nitrogen. Anhydrous DCM (40 mL) was added and the reactive was warmed to 34 C. A solution of compound 5 (12.5 mg, 0.051 mmol) in toluene (ca. 85% purity) was added to the flask and the reaction was allowed to stir for 16 hours. The solvent was removed completely and the crude product was purified by reverse phase MPLC on a C18 SNAP Ultra 60 g cartridge eluting (10% acetone:water for 1 CV, a gradient of 10-95% acetone:water for 10 CV and 95% acetone:water for 2 CV). White solid (67 mg, 0.012 mmol, 32%).
##STR00127##
[0862] Method A:
[0863] Compound 8d was dissolved in anhydrous DCM (Vol: 6 mL) and TFA (1.74 mL, 22.67 mmol) at RT. The resulting off yellow solution was stirred for 2 h at room temperature. Volatiles were removed under vacuum to give a yellow solid. The solid which was purified by reverse phase MPLC on a C18 SNAP Ultra 60 g cartridge by loading the sample in 1:1 MeOH/H.sub.2O+0.1% formic acid). The resulting white solid was neutralised using 100 mM NaOH solution to pH 7 and the resulting solution concentrated to dryness under vacuum. White crystalline solid (37 mg, 0.008 mmol, 74%).
[0864] Method B:
[0865] The solid obtained from Compound 44 was dissolved in anhydrous CH.sub.2Cl.sub.2 (5.9 mL) and TFA (1.7 mL, 23 mmol) was added at RT. The resulting off yellow solution was stirred for 2 h at RT or until complete by TLC (product on the baseline in 60% EtOAc/CH.sub.2Cl.sub.2). The solvent/TFA were removed under vacuum (rotary evaporator then high vacuum) to give a yellow solid. The solid was purified by reverse phase chromatography (loading sample in 1:1 MeOH/H.sub.2O+0.1% formic acid). Fr 2-7 taken and concentrated under vacuum. The resulting white solid was neutralised using 100 mM NaOH solution to pH 7 and the resulting solution concentrated under vacuum (rotary evaporator/cold finger with liquid N.sub.2) to give Compound 45 as a white crystalline solid (37 mg, 75%).
[0866] .sup.1H NMR: (500 MHz, D.sub.2O, 298 K): 7.74 (br d, J=7.1 Hz, 3H, H.sub.9), 7.59 (d, J=8.6 Hz, 3H, H.sub.8), 7.48 (s, 3H, NH), 7.40 (s, 3H, H.sub.6), 7.01 (s, 3H, H.sub.1), 4.40 (br s, 6H, H.sub.12), 3.93 (br s, 6H, H.sub.3), 2.70 (br s, 6H, H.sub.15), 2.20 (br s, 18H, H.sub.20), 2.07 (m, 72H, H.sub.19+H.sub.24), 1.83 (m, 54H, H.sub.23), 1.07 (br t, 9H, H.sub.16).
[0867] .sup.13C NMR (126 MHz, D.sub.2O) 173.2, 166.0, 160.0, 149.4, 148.1, 135.5, 131.1, 128.9, 122.8, 121.6, 119.6, 118.0, 117.4, 114.5, 113.9, 49.5, 49.0, 48.9, 43.1, 33.8, 28.6, 22.0, 21.6, 21.2, 21.1, 13.5, 6.3.
##STR00128##
[0868] Triethylbenzene receptor 1 (15.0 mg, 0.003 mol), HBTU (W: 2.6 mg, 0.006 mmol) and 5-(aminoacetamido)fluorescein (21.8 mg, 0.048 mmol) were dissolved in DMF (1.25 mL) and H.sub.2O (1.25 mL). The reaction mixture was stirred in the dark, at room temperature for 16 hr then concentrated under reduced pressure. The crude reaction mixture was neutralised to pH 7 with aq. NaOH then concentrated under reduced pressure. Purified by reverse phase flash chromatography on a 12 g SNAP Ultra C18 cartridge elution with 1CV 20% MeOH/H.sub.2O+0.1% formic acid to 65% MeOH/H.sub.2O+0.1% formic acid over 8 CV to 100% H.sub.2O+0.1% formic acid over 0.5CV then 100% H.sub.2O+0.1% formic acid for 3 CV. Product containing fractions were combined, concentrated under reduced pressure, neutralised to pH 7 then lyophilised to give compound 9 as an orange powder. .sup.1H NMR: (400 MHz, D.sub.2O, 298 K): 7.91-7.28 (m, 15H, Ar), 7.25-6.89 (m, 9H, Ar), 6.73-6.32 (m, 12H, Ar), 4.52-4.04 (m, 12H, H.sub.5), 4.02-3.81 (m, 6H, H.sub.22), 2.59-1.61 (m, 156H, H.sub.1+15+16+19+20), 1.21-0.82 (m, 18H, H.sub.1).
Key Intermediates
[0869] ##STR00129##
[0870] 3,4-Diaminobenzoic acid (41.000 g, 0.269 mol) was mixed with Saturated NaHCO.sub.3 (0.40 L) and acetonitrile (0.40 L) to give a brown slurry. Next, solid Fmoc-OSu (99.99 g, 0.296 mol) was added in portions over 5 minutes. The heterogenous suspension was allowed to stir at room temperature for 16 hours and then acidified with 1M HCl.sub.(aq). The solids were collected on a frit and washed with cold diethyl ether (3100 mL), hexane (3100 mL), then MeOH (350 mL) and then dried under vacuum. Brown solid (101 g, 0.269 mol, 100%). This intermediate (10.000 g, 0.027 mol), HOBt (8.181 g, 0.053 mol), and HBTU (20.259 g, 0.053 mol) were dissolved in THF (300) mL) and DIPEA (18.610 mL, 0.107 mol). The heterogenous slurry was stirred at room temperature for 90 minutes after which the solvent was removed in vacuo to afford a viscous oil. The oil was dissolved in EtOAc (80 ml) and was added to a rapidly stirring mixture of water (200 ml) and EtOAc (40 mL). After ca. 2 mins a precipitate formed and diethyl ether (100 mL) was added to the flask. After stirring for 10 minutes, the solids were collected by filtration and washed with water (310 mL) and diethyl ether (210 mL) before drying under vacuum for 16 hours. .sup.1H NMR: (500 MHz, DMSO-d.sub.6) 8.94 (s, 1H), 8.14 (dd, J=17.7, 8.4 Hz, 2H), 8.01-7.93 (m, 1H), 7.90 (d, J=7.6 Hz, 2H), 7.87-7.68 (m, 6H), 7.68-7.61 (m, 1H), 7.54 (dt, J=11.5, 7.5 Hz, 1H), 7.42 (t, J=7.4 Hz, 2H), 7.34 (t, J=7.6 Hz, 2H), 6.89 (d, J=8.6 Hz, 1H), 6.46 (s, 2H), 4.44 (s, 2H), 4.31 (s, 1H), 3.40 (s, 7H), 3.03 (s, 7H), 2.50 (s, 4H).
##STR00130##
[0871] A 100 mL Schlenk flask was charged with compound A1 (2.750 g mg, 2.263 mmol), N,N-diisopropylethylamine (0.585 mL, 3.341 mmol) and anhydrous THF (10 mL). Solid 3-(2-aminoethyl)pentane-1,3,5-N-Boc-triamine* (0.800 g, Moles: 1.911 mmol) was added and the homogenous solution/suspension was left to stir. After 72 hours, TLC (SiO.sub.2, 7:3 EtOAc:DCM) indicated complete consumption of starting materials and formation of the product. The solvent was removed in vacuo and the crude residue was extracted with DCM (210 mL). The filtrate was concentrated to dryness and the residue purified by MPLC (12%.fwdarw.100% DCM in EtOAc). Orange amorphous solid (1.154 g, 1.413 mmol, 74%). .sup.1H NMR: (400 Hz, CDCl.sub.3) 7.00-7.82 (m, 11H, ArH), 6.71 (2H, d, J=8.13 Hz, NH.sub.2), 3.14 (6H, q, J=7 Hz, CH.sub.2NH), 2.04 (app. s, 6H, CCH.sub.2), 1.40 (27H, s, C(CH.sub.3).sub.3). MS: (ESI.sup.+) calculated for C.sub.44H.sub.60N.sub.6O.sub.9Na.sup.+: 839.4314, found [M+Na].sup.+: 839.4318. *Carter, T. S.; Mooibroek, T. J.; Stewart, P. F. N.; Crump, M. P.; Galan, M. C. & Davis, A. P. Angewandte Chemie International Edition, 2016, 55, 9311-9315.
##STR00131##
[0872] NaH (1.05 g, 26.3 mmol, 60% in mineral oil) was added to a Schlenk flask (100 mL) and placed under nitrogen. The mineral oil was removed by washing the solids with 325 mL petroleum ether 60-80 OC. The washed NaH was suspended in anhydrous DMF (10 mL) and vigorously stirred for ca. 10 mins whilst cooling in an ice bath. Solid trifluoroacetamide (4.46 g, 39.46 mmol) was added portion-wise under a nitrogen counter-flow. After five minutes of stirring the mixture was let warm to room temperature. Once the evolution of gas had completely stopped (within 1 hour), solid 1,3,5-tribromomethyl-2,4,6-trimethoxylbenzene* (2.00 g, 4.39 mmol) was added portion-wise under a nitrogen counter-flow and the resulting white suspension was stirred at room temperature. After ca. 18 hours, the suspension was poured into 0.5 M HCl (150 mL) and the light orange precipitate collected on a frit. The solids were washed with water (210 mL) and then dried under vacuum overnight (ca. 10.sup.2 mbar). Off-white solid (2.44 g, 4.491 mol, 102%). The crude product was used without further purification and contained 2-3% DMF (quantified by .sup.1H NMR spectroscopy). .sup.1H NMR: (400 MHz, DMSO-d.sub.6) d. 4.40 (6H, .sup.3J.sub.HH=4.6 Hz, ArCH.sub.2NHC(O)CF.sub.3), s. 3.70 (9H, OCH.sub.3). .sup.13C NMR: (400 MHz, DMSO-d.sub.6) q. 158.3 (.sup.2J.sub.CF=37.1 Hz, C(O)CF.sub.3), 137.3, 132.1 (Ar), q. 116.1 (.sup.1J.sub.CF=287.9 Hz, CF.sub.3), 62.5 (NHCH.sub.2Ar), 32.1 (OCH.sub.3). *Rosien, J.; Seichter, W.; Mazik, M., Organic and Biomolecular Chemistry, 2013. 11(38), 6569-8579.
##STR00132##
[0873] Prepared by an analogous route using 1,3,5-tribromomethyl-2,4,6-trimethylbenzene to Compound 3b. Off-white solid (4.86 g, 0.01 mol 82%). .sup.1H NMR: (500 MHz, CDCl.sub.3/MeOH-d.sub.4) s. 4.47 (6H, ArCH.sub.2NHC(O)CF.sub.3), s. 2.26 (9H, OCH.sub.3). .sup.13C NMR: (125 MHz, CDCl.sub.3/MeOH-d.sub.4) q. 157.6 (.sup.2J.sub.CF=37.2 Hz, C(O)CF.sub.3), 137.8, 131.1 (Ar), q. 115.9 (.sup.1J.sub.CF=287.3 Hz, CF.sub.3), 39.4 ArCH.sub.2NHC(O)CF.sub.3), 16.0 (ArCH.sub.3). MS:(APCI.sup.) 493.9
##STR00133##
[0874] Prepared by an analogous route to Compound 3b using 1,3,5-tris(bromomethyl)benzene (5.07 g, 0.014 mol). The crude product was purified by reverse phase MPLC on a C18 SNAP Ultra 60 g cartridge eluting (10% acetone:water for 1 CV, a gradient of 10-95% acetone:water for 10 CV and 95% acetone:water for 2 CV). White solid (2.16 g, 0.005 mol, 34%). .sup.1H NMR: (400 MHz, MeOH-d.sub.4) 7.07, (s, 3H, ArH), 4.35 (6H, .sup.3J.sub.HH=4.6 Hz, ArCH.sub.2N(O)HCF.sub.3). .sup.13C NMR: (125 MHz, MeOH-d.sub.4) q. 158.1 (.sup.2J.sub.CF=36.9 Hz, C(O)CF.sub.3), 138.1, 132.2 (Ar), q. 114.6 (.sup.1J.sub.CF=286.9 Hz, CF.sub.3), 125.9 (Ar), 42.3, ArCH.sub.2NHC(O)CF.sub.3).
##STR00134##
[0875] NaH (3.809 g, 0.095 mol, 60% in mineral oil) was added to a Schlenk flask (100 mL) and placed under nitrogen. The mineral oil was removed by washing the solids with 325 mL petroleum ether 60-80 C. The washed NaH was suspended in anhydrous DMF (40 mL) and vigorously stirred for ca. 10 mins whilst cooling in an ice bath. Solid trifluoroacetamide (16.147 g, 0.143 mol) was added portion-wise under a nitrogen counter-flow. After five minutes of stirring the mixture was let warm to room temperature. Once the evolution of gas had completely stopped (within 1 hour), solid 1,3,5-tribromomethyl-2,4,6-trimethoxylbenzene* (7.00 g, 0.016 mol) was added portion-wise under a nitrogen counter-flow and the resulting white suspension was stirred at room temperature. After ca. 18 hours, the suspension was poured into 0.5 M HCl (150 mL) and the light orange precipitate collected on a frit. The solids were washed with water (210 mL) and then dried under vacuum overnight (ca. 10.sup.2 mbar). Off-white solid (7.910 g, 0.015 mol 93%). The intermediate acetamide (4.90 g, 0.009 mol) was dissolved in methanol (38.6 mL) and water (38.6 mL). NaOH (1.05 g, 3.150 mol) was added and the reaction mixture was left to stir for ca. 18 hours at 65 C. Solid Boc.sub.2O (7.287 g, 0.033 mol) and triethylamine (2.534 mL, 0.026 mol) were added and the reaction was left to stir for a further 4 hours at ambient temperature. The reaction mixture was diluted with DCM (200 mL) and washed with sat. aq. NaHCO.sub.3 (200 mL), 1 M NaOH (200 mL) and brine (100 mL). The organic phase was concentrated to dryness and the resultant crude product purified by MPLC (0.fwdarw.50% MeOH in DCM). Colourless solid (4.820 g, 0.009 mol, 96%). .sup.1H NMR: (400 MHz, CDCl.sub.3) m.br 4.33 (9H, ArCH.sub.2NHCO.sub.2C(CH.sub.3).sub.3), NH), q. 2.71 (6H, .sup.3J.sub.HH=7.5 Hz, ArCH.sub.2CH.sub.3), s. 1.44 (27H, CO.sub.2C(CH.sub.3).sub.3), t. 1.19 (9H, .sup.3J.sub.HH=7.5 Hz, ArCH.sub.2CH.sub.3). .sup.13C NMR: (125 MHz, CDCl.sub.3) 155.5 (CO.sub.2C(CH.sub.3).sub.3), 143.9, 132.6 (Ar), 79.7 (CO.sub.2C(CH.sub.3).sub.3), 38.9 ArCH.sub.2NHCO.sub.2C(CH.sub.3).sub.3), 28.6 (CO.sub.2C(CH.sub.3).sub.3), 23.0 (ArCH.sub.2CH.sub.3), 16.7 (ArCH.sub.2CH.sub.3). *Rosien, J.; Seichter, W.; Mazik, M., Organic and Biomolecular Chemistry, 2013, 11(38), 6569-6579.
##STR00135##
[0876] Compound 3c (9.123 g, 0.013 mol) was dissolved in MeOH (125 mL), and NaOH (1.689 g, 0.042 mol) at room temp overnight. 0.040 mol) was added followed by BOC anhydride (11.702 g, 0.054 mol) and left to stir overnight. The solvent was removed under vacuum and crude product partitioned between DCM and H.sub.2O. The organic fractions were combined and washed with 0.5M HCl (50 ml), dried with MgSO.sub.4 and concentrated under vacuum to give an off white solid. Purified by MPLC (0.fwdarw.20% EtOAc in petrol). Colourless crystalline solid (5.62 g, 0.011 mol, 83%). .sup.1H NMR: (500 MHz, CDCl.sub.3) d. 4.33 (9H, ArCH.sub.2NHCO.sub.2C(CH.sub.3).sub.3), NH), s. 2.37 (9H, ArCH.sub.3), s. 1.44 (27H, CO.sub.2C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, CDCl.sub.3) 155.7 (CO.sub.2C(CH.sub.3).sub.3), 136.9, 133.5 (Ar), 79.7 (CO.sub.2C(CH.sub.3).sub.3), 40.0 ArCH.sub.2NHCO.sub.2C(CH.sub.3).sub.3), 28.5 (CO.sub.2C(CH.sub.3).sub.3), 16.0 (ArCH.sub.3).
##STR00136##
[0877] Prepared by an analogous route to Compound 4b using Compound 3b (0.919 g, 1.69 mmol) Purified by MPLC (0.fwdarw.40% EtOAc in petroleum ether). Colourless crystalline solid (0.655 g, 1.15 mmol 68%). .sup.1H NMR: (4500 MHz, CDCl.sub.3) d 4.38 (6H, .sup.3J.sub.HH=4.2 Hz ArCH.sub.2NHCO.sub.2C(CH.sub.3).sub.3), s. 3.79 (9H, OCH.sub.3) 1.43 (27H, CO.sub.2C(CH.sub.3).sub.3).
##STR00137##
[0878] Prepared in an analogous fashion to Compound 4b using Compound 3d (2.155 g, 0.005 mol) was dissolved in MeOH (30 mL). Solid NaOH (0.599 g, 0.015 mol). Purified by MPLC (50% EtOAc in DCM) and then recrystalised from DCM/petrol. Colourless crystalline solid (0.88 g, 0.002 mmol 40%). .sup.1H NMR: (400 MHz, CDCl.sub.3) s. 7.08 (1H, ArH) d. 4.24 (6H, .sup.3J.sub.HH=6.6 Hz, ArCH.sub.2N(O)OC(CH.sub.3).sub.3), s. 1.46 (9H, C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, CDCl.sub.3) 155.7 (CO.sub.2C(CH.sub.3).sub.3), 136.8 (Ar), 125.2 (Ar), 78.9 (CO.sub.2C(CH.sub.3).sub.3), 45.2 (ArCH.sub.2NHCO.sub.2C(CH.sub.3).sub.3), 28.2 (CO.sub.2C(CH.sub.3).sub.3).
##STR00138##
[0879] Method A:
[0880] A pre-dried 250 mL round-bottomed flask fitted with a nitrogen inlet and reflux condenser and mounted in a heating block was charged with triphosgene (1.78 g, 6 mmol). The flask was placed under vacuum (ca. 10.sup.2 mbar) for 10 min the re-filled with nitrogen. Anhydrous toluene (150 mL) was added to give a colourless, homogenous solution. To this, a solution of 1,3,5-triaminomethyl-2,4,6-triethylbenzene (0.5 g, 2 mmol) that had been previously dried by one azeotropic distillation of toluene (80 mL), in anhydrous toluene (20 mL) was added via syringe over 10 mins. After the addition was complete the temperature was raised to 125 C. and the reaction allowed to reflux. After 75 min. the flask was allowed to cool to room temperature and the solvent removed in a rotary evaporator. The residue was extracted with toluene (310 mL) and concentrated to dryness to give a pale-yellow oil that crystallised on standing. Pale yellow crystalline solid (350 mg, 1.07 mmol, 54%).
[0881] Method B:
[0882] A pre-dried 200 mL Schlenk flask was charged with a magnetic stirrer and Compound 4a (1.266 g, 2.25 mmol) and then placed under a nitrogen atmosphere. 2-chloropyridine (1.7 mL, 20.21 mmol) and anhydrous DCM (70 mL) were added via syringe to give a colourless, homogenous solution. Triflic anhydride (1.5 mL, 10.11 mmol) was added dropwise at ambient temperature over 2 mins with stirring (400 rpm). The reaction was stirred for 30 mins before a small aliquot of the reaction mixture (ca. 50 mL) was withdrawn and analysed by TLC (SiO.sub.2, 50% Et.sub.2O in petrol), which revealed complete consumption of the starting material (R.sub.f=0.24) and conversion to 5a (R.sub.f=0.5). The solvent was removed on a rotary evaporator to give an off-white solid. The solids were extracted with Et.sub.2O (2 15 mL) and passed through an alumina plug (20 mm20 mm), eluting with a further 20 mL of Et.sub.2O. The colourless filtrate was evaporated to dryness and the residue recrystalised from hexane. Colourless crystalline solid (0.435 g, 1.33 mmol, 59%). .sup.1H NMR: (400 MHz, Toluene-d.sub.6) s. 3.93 (6H, ArCH.sub.2NCO), m. 2.51-2.37 (6H, ArCH.sub.2CH.sub.3), m. 0.96-0.86 (9H, ArCH.sub.2CH.sub.3). .sup.13C NMR: (100 MHz, Toluene-d.sub.6) 143.2, 132.6 (Ar), 124.0 (NCO), 40.4 (ArCH.sub.2NCO), 22.8 (ArCH.sub.2CH.sub.3), 16.0 (ArCH.sub.2CH.sub.3).
##STR00139##
[0883] Prepared by an analogous route to Compound 5a (Method B) using Compound 4c (0.351 g, 0.616 mmol) Colourless crystalline solid (0.144 g, 0.432 mmol 70%). .sup.1H NMR: (400 MHz, Toluene-d.sub.8) d. 3.90 (6H, ArCH.sub.2NCO), s. 3.40 (9H, OCH.sub.3). .sup.13C NMR: (125 MHz, Toluene-d.sub.8) 159.7 (Ar), 121.4 (NCO), 62.9 (ArOCH.sub.3), 36.2 (ArCH.sub.2NCO).
##STR00140##
[0884] Prepared by an analogous route to Compound 5a (Method B) using Compound 4b (0.300 g, 0.985 mmol) Colourless crystalline solid (0.198 g, 0.694 mmol, 71%). .sup.1H NMR: (500 MHz, Toluene-d.sub.8) s. 3.73 (6H, ArCH.sub.2NCO), s. 1.91 (9H, CH.sub.3). .sup.13C NMR: (125 MHz, Toluene-d.sub.8) 135.9, 133.4 (Ar), 124.4 (ArCH.sub.2NCO), 41.2 (ArCH.sub.2NCO), 15.1 (ArCH.sub.3).
##STR00141##
[0885] Prepared by an analogous route to Compound 5a (Method B) using Compound 4d (0.250 g, 0.537 mmol). Colourless oil (0.062 g, 0.255 mmol, 48%). .sup.1H NMR: (500 MHz, Toluene-d.sub.8) s. 6.56 (3H, ArH), s. 3.64 (6H, ArCH.sub.2NCO). .sup.13C NMR: (125 MHz, Toluene-d.sub.8) 138.4 (Ar), 124.2 (Ar, ArCH.sub.2NCO), 45.7 (ArCH.sub.2NCO).
##STR00142##
[0886] A pre-dried round bottom flask was charged with 1,3,5-triaminomethyl-2,4,6-triethylbenzene tristrifluoroacetate (0.535 g, 0.905 mmol). THF (9 mL) was added, followed by CS.sub.2 (1.100 mL, 18.100 mmol) and DCC (0.585 mg, 2.806 mmol). The reaction was stirred for 16 h and then concentrated under vacuum. The resultant residue was triturated with DCM and the filtrate purified by MPLC (5% EtOAc/DCM.fwdarw.40%) to give a white solid (0.168 g, 0.447 mmol, 49%). .sup.1H NMR: (400 MHz, CDCl.sub.3) s. 4.74 (6H, ArCH.sub.2NCS), q. 2.84 (6H, J=7.6 Hz, ArCH.sub.2CH.sub.3), t. 1.26 (9H, J=7.6 Hz, ArCH.sub.2CH.sub.3). .sup.13C NMR: (100 MHz, CDCl.sub.3) 144.2, 132.4 (Ar), 130.1 (NCS), 42.9 (ArCH.sub.2NCS), 23.2 (ArCH.sub.2CH.sub.3), 15.8 (ArCH.sub.2CH.sub.3).
##STR00143##
[0887] A pre-dried round bottom flask was charged with tris(2-aminotheyl)amine (5.000 g, 0.033 mol). THF (500 mL) was added, followed by CS.sub.2 (40.0 mL, 0.660 mol) and DCC (21.258 g, 0.102 mol). The reaction was stirred for 16 h and then filtered. The filtrate was then concentrated under vacuum. The resultant residue was triturated with DCM and the filtrate purified by MPLC (12% EtOAc/DCM.fwdarw.30%) to give a yellow solid (3.340 g, 0.023 mmol, 71%). .sup.1H NMR: (500 MHz, CDCl.sub.3) t. 4.74 (6H, J=6.2 Hz, NCH.sub.2CH.sub.2NCS), t. 2.96 (6H, J=6.2 Hz, NCH.sub.2CH.sub.2NCS). .sup.13C NMR: (125 MHz, CDCl.sub.3) 132.8 (NCS), 54.5 (NCH.sub.2CH.sub.2NCS), 44.4 (NCH.sub.2CH.sub.2NCS).
##STR00144##
[0888] A pre-dried Schlenk tube was charged with compound 5b (18 mg, 0.053 mmol) and compound 84 (333 mg, 0.186 mmol) under a flow of nitrogen. Dry THF (3 mL) and anhydrous pyridine (0.024 mL, 0.053 mmol) was added. A flake of MoO.sub.2Cl.sub.2 was added under a flow of nitrogen. The reaction was stirred for 16 h. The reaction mixture was concentrated to dryness and the crude residue purified by MPLC (2:3 EtOAc:DCM) to give a brown solid (174 mg, 0.030 mmol, 57%). .sup.1H NMR: (500 MHz, CDCl.sub.3) m. 7.77-7.07 (33H, ArH), m. 4.56-4.06 (15H, ArCH.sub.2j, CO.sub.2CH.sub.2CH),), s. 3.66 (9H, ArOCH.sub.3), m. 2.31-1.80z (144H, NHCH.sub.2CH.sub.2C(O)), s. 1.34 (162H, CO.sub.2C(CH.sub.3).sub.3), s. 1.33 (81H, CO.sub.2C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, CDCl.sub.3) 173.1, 173.0 (CONH), 172.7, 172.7 (CO.sub.2C(CH.sub.3).sub.3), 172.6 (ArCONHR), 166.6 (NHC(O)NH), 158.4 (COCH.sub.3), 153.9 (CO.sub.2CH.sub.2CH), 143.8 (CNHFmoc), 143.6 (Fmoc 4), 141.3 (CNHFmoc), 141.2 (Fmoc 4), 127.7 (Fmoc Ar), 127.7, 127.7 (CCO.sub.2NHR), 127.1 (Fmoc Ar), 127.0 (CNHC(O)NH), 125.3, 125.3 (CHCHCNH), 124.5 (Fmoc Ar), 120.0 (Fmoc Ar), 119.9 (CHCNHFmoc), 119.9 (CCH.sub.2NHC(O)NH), 80.5, 80.4 (CO.sub.2C(CH.sub.3).sub.3), 67.3 (CO.sub.2CH.sub.2CH), 60.4 (ArOCH.sub.3), 57.4, 57.4 (C(CH.sub.2CH.sub.2CO.sub.2).sub.3), 57.4 (C(CH.sub.2CH.sub.2CONH).sub.3), 47.2 (CO.sub.2CH.sub.2CH), 39.2 (ArCH.sub.2NHC(O)NH), 32.2 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 31.7 (CH.sub.2CH.sub.2CONH), 29.8 (CH.sub.2CH.sub.2CONH), 29.7 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 28.0 (CO.sub.2C(CH.sub.3).sub.3).
##STR00145##
[0889] A pre-dried Schlenk tube was charged with compound 84 (441 mg, 0.245 mmol) under a flow of nitrogen and a solution of compound 5c (20 mg, 0.070 mmol) in THF (0.5 mL) added. Dry THF (3.5 mL) and anhydrous pyridine (0.006 mL, 0.070 mmol) was added. The reaction was stirred for 16 h at 50 C. The reaction mixture was concentrated to dryness and the crude residue purified by reverse phase MPLC on a 120 g SNAP Ultra C18 cartridge elution (70-95% acetone/H.sub.2O) to give a white solid (197 mg, 0.035 mmol, 50%). .sup.1H NMR: (400 MHz, methanol-d.sub.4) m. 7.86-7.03 (33H, ArH), m. 4.48-4.04 (15H, ArCH.sub.2, CO.sub.2CH.sub.2CH),), s. 2.37 (9H, ArCH.sub.3), m. 2.31-1.86z (144H, NHCH.sub.2CH.sub.2C(O)), s. 1.40 (243H, CO.sub.2C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, methanol-d.sub.4) 175.6, 175.5 (CONH), 174.4 (CO.sub.2C(CH.sub.3).sub.3), 174.4 (ArCONHR), 157.1 (NHC(O)NH), 153.0 (CO.sub.2CH.sub.2CH), 145.0 (CNHFmoc), 142.6 (Fmoc 4), 134.4 (CCH.sub.3), 131.2 (Fmoc Ar), 128.9 (CCO.sub.2NHR), 128.2 (CNHC(O)NH), 126.3 (CHCHCNH), 121.4 (Fmoc Ar), 121.1 (CHCNHFmoc), 121.1 (CCH.sub.2NHC(O)NH), 81.6 (CO.sub.2C(CH.sub.3).sub.3), 59.4, 58.8 (C(CH.sub.2CH.sub.2CO.sub.2).sub.3), 58.7 (C(CH.sub.2CH.sub.2CONH).sub.3), 40.1 (ArCH.sub.2NHC(O)NH), 30.7 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 30.5 (CH.sub.2CH.sub.2CONH), 28.5 (CH.sub.2CH.sub.2CONH, CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 28.4 (CO.sub.2C(CH.sub.3).sub.3), 16.2 (ArCH.sub.3).
##STR00146##
[0890] A pre-dried Schlenk tube was charged with compound 84 (441 mg, 0.245 mmol) under a flow of nitrogen and a solution of compound 5d (212 mg, 0.049 mmol) in THF (0.5 mL) was added. Dry THF (2.5 mL) and anhydrous pyridine (0.004 mL, 0.049 mmol) was added. The reaction was stirred for 32 h. The reaction mixture was concentrated to dryness and the crude residue purified by reverse phase MPLC on a 120 g SNAP Ultra C18 cartridge elution (70-95% acetone/H.sub.2O) to give a white solid (110 mg, 0.020 mmol, 40%). .sup.1H NMR: (500 MHz, methanol-d.sub.4) (m. 8.03-7.06 (33H, ArH), m. 4.55-4.17 (15H, ArCH.sub.2, CO.sub.2CH.sub.2CH), m. 2.27-1.85 (144H, NHCH.sub.2CH.sub.2C(O)), s. 1.41 (243H, CO.sub.2C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, methanol-d.sub.4) 175.5 (CONH), 174.4 (CO.sub.2C(CH.sub.3).sub.3), 174.4 (ArCONHR), 157.3 (NHC(O)NH), 153.9 (CO.sub.2CH.sub.2CH), 143.8 (CNHFMoc), 143.6 (Fmoc 4), 141.1 (Fmoc 4), 129.0 (CCO.sub.2NHR), 127.7 (Fmoc Ar), 126.3 (CHCHCNH), 124.4 (Fmoc Ar), 122.0 (CHCCH.sub.2NHC(O)NH), 120.9 (CHCNHFmoc), 120.0 (Fmoc Ar), 81.6 (CO.sub.2C(CH.sub.3).sub.3), 58.7 (C(CH.sub.2CH.sub.2CO.sub.2).sub.3, C(CH.sub.2CH.sub.2CONH).sub.3), 40.7 (CO.sub.2CH.sub.2CH), 40.1 (ArCH.sub.2NHC(O)NH), 30.7 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 30.5 (CH.sub.2CH.sub.2CONH), 28.5 (CH.sub.2CH.sub.2CONH, CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 28.5 (CO.sub.2C(CH.sub.3).sub.3).
Compound 6h-1 and 6h-2
[0891] ##STR00147## ##STR00148##
[0892] A Schlenk flasked was charged with a stirrer bar, compound 84 (0.933 g, 0.519 mmol) and compound 5a (0.100 g, 0.305 mmol) were dissolved in anhydrous THF (6 mL), pyridine (0.147 mL, 1.833 mmol) was added and the mixture was then heated to 50 C. for 5 hours. Compound 11 (0.228 g, 0.397 mmol) in anhydrous THF (1 mL) was added in one portion and the reaction stirred for a further 12 hours. The reaction mixture was transferred to a RBF washing the Schlenk with CH.sub.2Cl.sub.2 before concentrating under vacuum. The crude residue obtained was then purified by reverse phase flash chromatography on a 120 g SNAP Ultra C18 cartridge elution (1CV 85% acetone/H.sub.2O, 10 CV 85-95% acetone/H.sub.2O, 2CV 95% acetone) gave resolved peaks (fr17-29) 6h-1 (511 mg, 37%) and (fr8-15) 6h-2 (458 mg, 46%).
Compound 6h-1
[0893] .sup.1H NMR: (400 MHz, (CD.sub.3OD): 8.02-7.46 (19H, br. m, ArH), 7.46-7.11 (14H, br. m, ArH), 4.60-4.30 (12H, br. m, NHCH.sub.2Ph and FmocH), 4.19 (3H, br. s, NHCH.sub.2Ph and FmocH), 3.71-3.55 (14H, m, PEG CH.sub.2), 3.33 (2H, m, PEG CH.sub.2), 2.85 (6H, br. s, CH.sub.2), 2.35-1.86 (96H, m, dendrimer CH.sub.2), 1.42 (162H, s, CH.sub.3), 1.23 (9H, br. s, CH.sub.3); HRMS: (ESI.sup.+) calculated for C.sub.244H.sub.352N.sub.21O.sub.57Na.sub.3.sup.2+: 1520.8407, found [M+3Na].sup.3+: 1520.8395.
Compound 6h-2
[0894] .sup.1H NMR: (400 MHz, (CD.sub.3OD): 8.01-7.51 (19H, br. m, ArH), 7.43-7.17 (14H, br. m, ArH), 4.54-4.25 (12H, br. m, NHCH.sub.2Ph and FmocH), 4.15 (3H, br. s, NHCH.sub.2Ph and FmocH), 3.69-3.47 (28H, m, PEG CH.sub.2), 3.26 (4H, m, PEG CH.sub.2), 2.82 (6H, br. s, CH.sub.2), 2.33-1.80 (48H, m, dendrimer CH.sub.2), 1.42 (81H, s, CH.sub.3), 1.17 (9H, br. s, CH.sub.3);
##STR00149##
[0895] Into a dry Schlenk equipped with a stirrer bar, compound 5a (55.0 mg, 0.168 mmol) and compound 2 (480 mg, 0.588 mmol) were weighed. Anhydrous THF (3.3 mL) was added. The reaction mixture was stirred at 50 degrees for 20 hours, then transferred to a round bottom flask and concentrated to dryness. The crude product was purified by MPLC, on a 60 g C18 SNAP ULTRA cartridge, elution (3CV 70% acetone/water, 10CV 70-95% acetone/water, 3CV 95% acetone) produced a resolved peak (fr18-22). White solid 273 mg, 59%. .sup.1H NMR: (400 MHz, CD.sub.3OD) 7.05-7.85 (33H, m, ArH), 4.35 (3H, s, Flu-CH), 4.26 (6H, s, Flu-CHCH.sub.2O), 3.01 (18H, m, H16), 2.71 (6H, m, ArCH.sub.2CH.sub.3), 1.93 (18H, m, H15), 1.30 (81H, s, .sup.tBu), 1.06 (9H, t, J=7.00 Hz, ArCH.sub.2CH.sub.3). .sup.13C NMR: (101 MHz, CD.sub.3OD) 15.59 (9C, ArCH.sub.2CH.sub.3), 22.23 (3C, ArCH.sub.2CH.sub.3), 27.31 (27C, .sup.tBu), 34.89 (9C, CCH.sub.2CH.sub.2NH), 35.21 (9C, CCH.sub.2CH.sub.2NH), peak not observed (1C, Flu-CHCH2), 51.32 (1C, C(CH.sub.2CH.sub.2NHBoc).sub.3), peak not observed (9C, .sup.tBu), peak not observed (1C, Flu-CH), 119.60-127.47 (60C, Ar), 141.28 (9C, Boc CO), 143.64 (1C, Fmoc CO), 156.89 (1C, ArCO).
##STR00150##
[0896] To a stirred solution of 6b (0.170 g, 0.030 mmol) was in DCM (5 mL) at 0 C., was added distilled DBU (0.006 mL, 0.040 mmol). The reaction was stirred at 0 C. for 2 h before concentrating under vacuum. The crude residue obtained was then purified by reverse phase flash chromatography on a 120 g SNAP Ultra C18 cartridge elution (1CV 80% acetone/H.sub.2O, 10 CV 80-95% acetone/H.sub.2O, 2CV 95% acetone) to give an off-white solid (0.133 g, 0.026 mmol, 89%). .sup.1H NMR: (500 MHz, methanol-d.sub.4) m. 7.30-7.27 (3H, CHCNH (Ar)), m. 7.22-7.15 (6H, CHCHCNH, CHCNH.sub.2 (Ar)), s. 4.54 (6H, ArCH.sub.2NH), s. 3.89 (9H, ArOCH.sub.3), m. 2.25-1.91 (144H, NHCH.sub.2CH.sub.2C(O)), s. 1.43 (243H, CO.sub.2C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, methanol-d.sub.4) 175.5, 175.4 (CONH), 174.4, 174.3 (CO.sub.2C(CH.sub.3).sub.3), 170.6, 170.0 (ArCONHR), 160.2 (NHC(O)NH), 157.9 (COCH.sub.3), 141.4, 140.7 (CNH.sub.2), 134.7, 132.7 (CCO.sub.2NHR), 129.9, 125.6 (CNHC(O)NH), 124.4, 123.6, 120.7, 118.8 (CHCHCNH), 117.2, 116.6 (CHCNH.sub.2), 115.5 (CCH.sub.2NHC(O)NH), 81.6 (CO.sub.2C(CH.sub.3).sub.3), 59.3 (ArOCH.sub.3), 58.7, 58.6 (C(CH.sub.2CH.sub.2CO.sub.2).sub.3), 54.8 (C(CH.sub.2CH.sub.2CONH).sub.3), 35.0, 35.0 (ArCH.sub.2NHC(O)NH), 32.7, 32.5 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 32.2, 32.2 (CH.sub.2CH.sub.2CONH), 30.7 (CH.sub.2CH.sub.2CONH), 30.4, (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 28.5, 28.4 (CO.sub.2C(CH.sub.3).sub.3).
##STR00151##
[0897] Prepared in a manner analogous to 7b from compound 6c (0.097 g, 0.017 mmol). Purified by reverse phase flash chromatography on a 120 g SNAP Ultra C18 cartridge elution (1CV 80% acetone/H.sub.2O, 10 CV 80-95% acetone/H.sub.2O, 2CV 95% acetone) to give a white solid (0.072 g, 0.014 mmol, 85%). .sup.1H NMR: (500 MHz, methanol-d.sub.4) m. 7.42-7.30 (3H, CHCNH (Ar)) m. 7.31-7.29 CHCNH.sub.2(Ar)), m. 7.23-7.19 (3H, CHCHCNH (Ar)), m. 4.54-4.48 (6H, ArCH.sub.2NH), s. 2.49 (6H, ArCH.sub.3), s. (3H, ArCH.sub.3), m. 2.28-1.90 (144H, NHCH.sub.2CH.sub.2C(O)), s. 1.44 (243H, CO.sub.2C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, methanol-d.sub.4) 175.5 (CONH), 174.4, 174.3 (CO.sub.2C(CH.sub.3).sub.3), 170.1 (ArCONHR), 158.1 (NHC(O)NH), 141.3 (CNH.sub.2), 135.4 (CCO.sub.2NHR), 134.7 (CNHC(O)NH), 130.0 (CCH.sub.3), 124.4, 118.9 (CHCHCNH), 117.4 (CHCNH.sub.2), 111.4 (CCH.sub.2NHC(O)NH), (C(CH.sub.2CH.sub.2CONH).sub.3), 81.6 (CO.sub.2C(CH.sub.3).sub.3), 58.7 (C(CH.sub.2CH.sub.2CO.sub.2).sub.3, C(CH.sub.2CH.sub.2CONH).sub.3), 40.3 (ArCH.sub.2NHC(O)NH), 32.5 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 32.2 (CH.sub.2CH.sub.2CONH), 30.7 (CH.sub.2CH.sub.2CONH), 30.5, (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 28.4 (CO.sub.2C(CH.sub.3).sub.3), 16.3 (ArCH.sub.3).
##STR00152##
[0898] Prepared in a manner analogous to 7b from compound 6d (0.110 g, 0.020 mmol). Purified by reverse phase flash chromatography on a 120 g SNAP Ultra C18 cartridge elution (1CV 80% acetone/H.sub.2O, 10 CV 80-95% acetone/H.sub.2O, 2CV 95% acetone) to give a white solid (0.022 g, 0.004 mmol, 21%). .sup.1H NMR: (500 MHz, methanol-d.sub.4) m. 7.43-7.13 (12H, Ar), m. 4.50-4.29 (6H, ArCH.sub.2NH), m. 2.34-1.86 (144H, NHCH.sub.2CH.sub.2C(O)), s. 1.43 (243H, CO.sub.2C(CH.sub.3).sub.3). .sup.13C NMR: (HSQC) (125 MHz, methanol-d.sub.4) 175.5 (CONH), 174.4 (CO.sub.2C(CH.sub.3).sub.3), 174.4 (ArCONHR), 157.3 (NHC(O)NH), 141.2 (CNH.sub.2), 135.4 (CCO.sub.2NHR), 134.7 (CNHC(O)NH), 126.3 (CHCHCNH), 124.4, (CHCHCNH), 122.0 (CHCCH.sub.2NHC(O)NH), 118.9 (CHCHCNH), 115.6 (CHCNH.sub.2), 81.6 (CO.sub.2C(CH.sub.3).sub.3), 58.7 (C(CH.sub.2CH.sub.2CO.sub.2).sub.3, C(CH.sub.2CH.sub.2CONH).sub.3), 44.4 (ArCH.sub.2NHC(O)NH), 35.1 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 34.6 (CH.sub.2CH.sub.2CONH), 30.4 (CH.sub.2CH.sub.2CONH), 30.2, (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 28.0 (CO.sub.2C(CH.sub.3).sub.3).
##STR00153##
[0899] Compound 13 (231 mg, 0.30 mmol) was dried by azeotrope with toluene, then under an inert N.sub.2 atmosphere was dissolved in anhydrous dichloromethane (1.5 mL). Pyridine (41 uL, 0.51 mmol) was added, followed by a solution of compound 5a (8 mg, 0.086 mmol) in anhydrous dichloromethane (0.5 mL) and the reaction mixture was stirred at 34 C. until complete by TLC. The solvent was removed under vacuum and the crude product purified by flash column chromatography (SiO.sub.2, EtOAC:CH.sub.2Cl.sub.2 1:4 to 1:2 then MeOH:CH.sub.2Cl.sub.2 5:95) to afford the FMOC-protected intermediate. HRMS: (ESI.sup.+) calculated for C.sub.150H.sub.192N.sub.12Na.sub.3O.sub.30.sup.3+ 983.7859, found [M+3Na].sup.3+: 983.7844. Under an inert N.sub.2 atmosphere, the FMOC-protected intermediate (140 mg, 0.053 mmol) was dissolved in anhydrous dichloromethane (9 mL) and cooled to 0 C. DBU (50 L, 0.34 mmol) was added dropwise and the reaction mixture warmed to room temperature and stirred for 1 hour. The solvent was removed under vacuum and the crude product purified by flash column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2 then 7.5% MeOH:CH.sub.2Cl.sub.2) to afford compound 7e (95 mg, 0.048 mmol, 91%). .sup.1H NMR: (400 MHz, CD.sub.3OD, 298 K): 7.40 (s, 3H, NH), 7.35 (d, J=8.2 Hz, 3H, H.sub.11), 7.21 (d, J=2.0 Hz, 3H, H.sub.8), 7.10 (dd, J=8.2, 2.0 Hz, 3H, H.sub.10), 4.52 (s, 6H, H.sub.5), 2.94-2.87 (m, 6H, H.sub.2), 2.30-2.25 (m, 18H, H.sub.16), 2.12-2.06 (m, 18H, His), 1.45 (s, 81H, H.sub.19), 1.26 (t, J=7.4 Hz, 9H, H.sub.1); HRMS: (ESI.sup.+) calculated for C.sub.105H.sub.164N.sub.12O.sub.24.sup.2+ 989.1002, found [M+2H].sup.2+: 989.1004.
##STR00154##
[0900] Prepared in an analogous fashion to compound 2 by treating compound A1 (3.308 g, 5.443 mmol), (3.308 g, Moles: 5.443 mmol) was added and dissolved in S1 (Vol: 22.680 mL). R3 (Vol: 1.270 mL, Moles: 7.258 mmol) was added dropwise followed by R2 (Vol: 1.000 mL, Moles: 4.536 mmol) and the reaction was left to stir until complete by TLC. TLC 70% EtOAc/CH.sub.2Cl.sub.2 showed the reaction to be complete at 24 h (visualised by UV/weak ninhydrin stain). The reaction mixture was transferred to a RBFwashing with CH.sub.2Cl.sub.2 and concentrated under vacuum to yield a brown residue. The crude product was purified by flash column chromatography eluting with 50% to 100% EtOAc/CH.sub.2Cl.sub.2. Colourless amorphous solid (2.50 g, 4.35 mmol, 80%). .sup.1H NMR: (400 MHz, CDCl.sub.3) 7.74 (d, J=7.5 Hz, 2H, ArH), 7.60 (d, J=2.0 Hz, 2H, ArH), 7.45 (s, 1H, ArH), 7.38 (t, J=7.5 Hz, 2H, ArH), 7.26 (d, J=9.6 Hz, 2H, ArH), 7.05 (s, 1H, ArH), 6.84 (s, 1H ArH), 6.65 (d, J=8.4 Hz, 1H C(O)NHCH.sub.2), 4.49 (s, 1H, Flu-CH.sub.2), 4.18 (s, 2H, Flu-OCH.sub.2), 3.68-3.45 (m, 14H, OCH.sub.2), 3.27 (t, J=5.0 Hz, 2H C(O)NHCH.sub.2), 1.34 (d, J=6.7 Hz, 2H, N.sub.3CH.sub.2).
##STR00155##
[0901] Prepared in an analogous fashion to compound 2 by treating compound A1 (2.000 g, 3.291 mmol) in 16.5 mL DCM and DIPEA (0.917 mL, 5.266 mmol) with propargyl amine (0.422 mL, Moles: 6.583 mmol) for 72 hours. Purified by column chromatography using ethyl acetate as the eluent. White solid (1.36 g, 3.290 mmol, 99%). .sup.1H NMR: (400 MHz, Methanol-d.sub.4) 7.95 (d, J=7.5 Hz, 2H, ArH), 7.89-7.80 (m, 3H, ArH), 7.61 (d, J=8.4 Hz, 1H, ArH), 7.52 (t, J=7.4 Hz, 2H, ArH), 7.44 (s, 2H), 6.87 (d, J=8.4 Hz, 1H, ArH), 4.45 (s, 2H), 4.40 (s, 1H, Flu-CH.sub.2), 4.17 (d, J=2.5 Hz, 2H, Flu-OCH.sub.2), 2.87 (t, J=2.4 Hz, 1H, CH.sub.2CCH), 2.67 (p, J=1.9 Hz, 2H, CH.sub.2CCH).
##STR00156##
[0902] A 50 mL RBF was charged with compound A1 (500 mg, 1.017 mmol), di-tert-butyl-4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate* (560 mg, 1.35 mmol) (560 mg, 1.35 mmol) and anhydrous toluene (10 mL). The slurry was evaporated to dryness and the residue re-dissolved in anhydrous pyridine (5 mL) and DCM (3 mL). The mixture was stirred at 50 C. for 16 hours. The solvent was removed to give a viscous brown oil, which was partitioned between EtOAc and 1M aq HCl. The organic phase was washed with water then brine. The combined organic fractions were concentrated and then absorbed onto silica gel and purified by flash chromatography (EtOAc:DCM (20-50%). Pink powder (467 mg, 0.612 mmol, 60%). .sup.1H NMR: (400 MHz, CDCl.sub.3) 7.79 (d, J=7.6 Hz, 2H, ArH), 7.64 (d, J=2.1 Hz, 1H, ArH), 7.43 (t, J=7.5 Hz, 2H, ArH), 7.34 (s, 3H, ArH), 6.78 (d, J=9.0 Hz, 1H, NH), 6.60 (s, 1H, ArH), 6.30 (s, 1H, ArH), 4.56 (s, 2H, Flu-CH.sub.2O), 4.28 (s, 1H, Flu-CH.sub.2), 4.08 (s, 2H), 2.30 (dd, J=8.8, 6.7 Hz, 6H, CH.sub.2C(O)), 2.16-2.04 (m, 6H, CCH.sub.2), 1.44 (s, 24H, C(CH.sub.3).sub.3). *Newkome, George R.; Weis, Claus D. Organic Preparations and Procedures International, 1996, vol. 28, #4 p. 495-498
##STR00157##
[0903] A Schlenk flash was charged with (3S,4S)-pyrrolidine-3,4-idol (100.0 mg, 0.970 mmol), di-tert-butyl 4-(3-(tert-butoxy)-3-oxopropyl)-4-isocyanatoheptanedioate (385.4 mg, 0.873 mmol) and anhydrous DMF under N.sub.2 to give orange solution. The solution was left to stir for 16 hours the poured into water (10 mL) and extracted with EtOAc (10 mL). The organic layer was separated, dried and concentrated to dryness. Off white solid. (430 mg, 0.79 mmol, 81%). .sup.1H NMR: (400 MHz, CDCl.sub.3) 4.65 (s, 1H, C(O)NH), 4.15-4.06 (m, 2H, CHOH), 3.95 (s, 2H, OH), 3.52 (dd, J=10.9, 4.2 Hz, 2H, NCH.sub.2), 3.21 (d, J=10.8 Hz, 2H, NCH.sub.2), 2.17 (dd, J=9.0, 6.7 Hz, 6H, CH.sub.2C(O)), 2.02-1.82 (m, 6H, CCH.sub.2), 1.37 (s, 27H, C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, CDCl.sub.3) 176.8 (s, NC(O)N), 168.1 (s, CCO.sub.2C), 79.7 (s, CO.sub.2C(CH.sub.3).sub.3), 75.7 (s, COH), 51.5 (s, CH.sub.2N), 30.8 (s, CH.sub.2C(O)), 29.8 (s, CCH.sub.2), 28.1 (s, CO.sub.2C(CH.sub.3).sub.3).
##STR00158##
[0904] A Schlenk flash was charged with Compound 14 (400.0 mg, 0.734 mmol) anhydrous DCM and TEA (0.409 mL, 2.93 mmol) and neat methylsulfonyl chloride (0.125 mL, 1.62 mmol) was added dropwise. The orange solution was allowed to stir overnight to give turbid orange solution. TLC (SiO.sub.2, 100% EtOAc) indicated complete consumption of the starting material to a new product (Rf=0.4). Water was added (10 mL) and the organic layer separated, dried with MgSO.sub.4 and concentrated to dryness. Orange crystalline solid (428 mg, 0.611 mmol, 83%). .sup.1H NMR: (400 MHz, CDCl.sub.3) 5.42 (s, 1H, C(O)NH), 5.2 (d, J=4.5, 2H, CHOS(O.sub.2)O), 3.77 (dd, J=12.2, 4.5 Hz, 2H, NCH.sub.2), 3.65 (d, J=12.2 Hz, 2H, NCH.sub.2), 3.11, (s, 1H, CH.sub.3S(O).sub.2O), 2.24 (t, J=8.0 Hz, 6H, CH.sub.2C(O)), 1.97 (m, 6H, CCH.sub.2), 1.42 (s, 27H, C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, CDCl.sub.3) 176.9 (s, NC(O)N), 168.3 (s, CCO.sub.2C), 79.8 (s, CO.sub.2C(CH.sub.3).sub.3), 79.0 (s, CH.sub.3S(O).sub.2OC), 49.0 (CH.sub.2N), 38.6 (s, CH.sub.3S(O).sub.2O), 30.5 (s, CH.sub.2C(O)), 30.2 (s, CCH.sub.2), 28.4 (s, CO.sub.2C(CH.sub.3).sub.3).
##STR00159##
[0905] Compound 15 (428.0 mg, 0.611 mmol), dissolved in dry DMF and solid NaN.sub.3 (119.2 mg, 1.833 mmol, 3.000 eq) added in one portion. The orange suspension was heated to 100 C. for 16 h to give a dark brown solution. Partitioned between EtOAc and water, washed with 2 10 mL 5% LiCl(aq), brine (110 mL), dried over MgSO.sub.4 then concentrated to dryness with ca. 1 g silica gel. This silica absorbed crude product was loaded onto a frit on top of 1 cm2 cm plug of fresh silica and eluted with 50% EtOAc in petrol (ca. 20 mL). The colourless filtrate was evaporated to dryness. Colouress crystalline solid (256 mg, 0.43 mmol, 74%). .sup.1H NMR: (400 MHz, CDCl.sub.3) 5.21 (s, 1H, C(O)NH), 3.92 (m, 2H, CHN.sub.3), 3.60 (dd, J=10.9, 5.7 Hz, 2H, NCH.sub.2), 3.30 (ds, J=10.9, 3.3 Hz, 2H, NCH.sub.2), 2.19 (t, J=7.5 Hz, 6H, CH.sub.2C(O)), 1.92 (m, 6H, CCH.sub.2), 1.37 (s, 27H, C(CH.sub.3).sub.3). .sup.13C NMR: (125 MHz, CDCl.sub.3) 176.8 (s, NC(O)N), 169.3 (s, CCO.sub.2C), 79.8 (s, CO.sub.2C(CH.sub.3).sub.3), 64.0 (s, CN.sub.3), 48.5 (s, CH.sub.2N), 30.3 (s, CH.sub.2C(O)), 29.9 (s, CCH.sub.2), 28.3 (s, CO.sub.2C(CH.sub.3).sub.3).
##STR00160##
[0906] Compound 15a (200.0 mg, 0.336 mmol) was dissolved in EtOH (1.000 mL) and mixed with 10% Pd/C (25.0 mg, 0.235 mmol). The reaction flask was purged nitrogen and then a hydrogen. The resulting mixture was stirred for 20 hours under a hydrogen atmosphere (supplied from a balloon). The crude reaction mixture was filtered through Celite washing and concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO.sub.2, MeCN:CH.sub.2Cl.sub.2 1:4 then MeOH:CH.sub.2Cl.sub.2 1:9) to afford compound 16 (93 mg, 0.171 mmol, 51%). .sup.1H NMR: (400 MHz, CDCl.sub.3, 298 K): 4.59 (s, 1H, NH), 3.63 (dd, J=10.1, 5.3 Hz, 2H, H.sub.8), 3.08 (q, J=5.7, 5.0 Hz, 2H, H.sub.9), 2.94 (dd, J=10.3, 5.7 Hz, 2H, H.sub.8), 2.17 (t, J=7.8 Hz, 6H, H.sub.4), 1.95-1.85 (m, 6H, H.sub.5), 1.38 (s, 27H, H.sub.1). .sup.13C NMR: (100 MHz, CDCl.sub.3, 298 K): 173.18 (C.sub.3), 155.63 (C.sub.7), 80.57 (C.sub.2), 58.37 (C.sub.9), 56.74 (C.sub.6), 52.03 (C.sub.8), 30.53 (C.sub.5), 29.91 (C.sub.4), 28.13 (C.sub.1); HRMS: (ESI.sup.+) calculated for C.sub.27H.sub.50N.sub.4NaO.sub.7.sup.+ 565.3572, found [M+Na].sup.+: 565.3547.
Compound 2001,3,5-tris(bromomethyl)-2-bromo-4, 6-dimethylbenzene
[0907] ##STR00161##
[0908] 2-bromo-4,6-dimethylbenzene (5.000 g, 0.027 mol, 1.000 eq), paraformaldehyde (12.736 g, 0.424 mol, 15.700 eq) and AcOH/HBr 33% (70 mL) were added to a dry 200 mL round bottom flask. The mixture was stirred while ZnBr.sub.2 (15.211 g, 0.068 mol, 2.500 eq) was slowly added and the mixture was heated to 90 C. After 24 hours, an additional portion paraformaldehyde (12.736 g, 0.424 mol, 15.700 eq) and 2.7 g ZnBr.sub.2 (12.736 g, 0.424 mol, 15.700 eq) was added. The yellow solution was heated an additional. 140 hours. The reaction mixture was then cooled to room temperature and the colourless crystals isolated by filtration, washing with AcOH (310 mL) and then water until the pH of the filtrate was neutral. Dried under vacuum for 2 day to afford colourless crystals (7.50 g, 0.016 mol, 60%).
[0909] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.78 (s, 4H, CH.sub.2), 4.54 (s, 2H, CH.sub.2), 2.54 (s, 6H, CH.sub.3).
Compound 2011,3,5-tris(azidomethyl)-2-bromo-4, 6-dimethylbenzene
[0910] ##STR00162##
[0911] 1,3,5-tris(bromomethyl)-2-bromo-4,6-dimethylbenzene (1.326 g, 2.859 mmol) was dissolved in dry DMF (30.000 mL) under nitrogen and stirred while NaN.sub.3 (1.115 g, 17.154 mmol). The reaction mixture was heated to 40 C. and stirred overnight. The reaction was cooled and poured into water (100 mL) and ppt extracted with EtOAc (350 mL). The combined organic fractions were washed with 5% LiCl (220 mL) and then brine, dried (MgSO.sub.4) and concentrated to dryness behind a blast shield to afford a colourless oil the crystallised on standing. (1.00 g, 2.856 mmol, 99%).
[0912] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.72 (s, 4H, CH.sub.2), 4.55 (s, 2H, CH.sub.2), 2.50 (s, 6H, CH.sub.3).
Compound 2021,3,5-tris(aminomethyl)-2-bromo-4, 6-dimethylbenzene trihydrochloride
[0913] ##STR00163##
[0914] 1,3,5-tris(azidomethyl)-2-bromo-4,6-dimethylbenzene (0.600 g, 1.713 mmol, 1.000 eq) and triphenylphopshine (3.011 g, 11.48 mmol, 6.7) were dissolved in THF (12 mL) and water (0.375 mL, 20.78 mmol, 12.13 eq) added. Stirred at 40 C. overnight. Solvent removed completely and treated with 0.5 M HCl. (10 mL). Extracted with EtOAc (210 mL) and the water layered reserved and concentrated to near dryness the added to rapidly stirred acetone (20 mL). The white precipitate was collected on a frit and washed with acetone (10 mL) and the dried under vacuum. Colourless solid (0.65 g, 1.71 mmol, 99%).
[0915] .sup.1H NMR (400 MHz, D.sub.2O) 4.54 (s, 4H, CH.sub.2), 4.38 (s, 2H, CH.sub.2), 2.53 (s, 6H, CH.sub.3).
Compound 203di-tert-butyl ((2-bromo-5-(((tert-butoxycarbonyl)amino)methyl)-4,6-dimethyl-1,3-phenylene)bis(methylene))dicarbamate
[0916] ##STR00164##
[0917] 1,3,5-tris(aminomethyl)-2-bromo-4,6-dimethylbenzene trihydrochloride (0.68 g, 1.78 mmol), BOC.sub.2O (2.33 g, 10.7 mmol) and triethylamine (1.5 mL, 10.7 mmol) was dissolved in MeOH (70 mL) and stirred at room temperature overnight. The solvent was removed and the residue portioned between EtOAc (50 mL) and 0.5 M citric acid. The aqueous layer was extracted with EtOAc (220 mL) and the combined organic fractions combined, dried (MgSO.sub.4) and concentrated to dryness to yield a colourless oil which crystallised upon standing (0.80 g, 1.4 mmol, 78%)
[0918] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.77 (br. s, 2H, NH), 4.55 (d, J=5.7 Hz, 4H, CH.sub.2), 4.36 (br. s, 3H, NH and CH.sub.2), 2.48 (s, 6H, CH.sub.3), 1.44 (s, 9H, CH.sub.3);
Compound 2041-bromo-2,4,6-tris(isocyanatomethyl)-3,5-dimethylbenzene
[0919] ##STR00165##
[0920] Prepared by an analogous route to Compound 103 using Compound 202 (0.20 g, 0.35 mmol) to give Compound 204 as a light pink crystalline solid (94 mg, 0.27 mmol, 77%).
[0921] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.70 (s, 4H, CH.sub.2), 4.49 (s, 2H, CH.sub.2), 2.52 (s, 6H, CH.sub.3);
[0922] .sup.13C NMR (101 MHz, CDCl.sub.3) 138.4 (C), 135.1 (C), 134.4 (C), 129.1 (C), 124.4 (C), 45.6 (CH.sub.2), 41.3 (CH.sub.2), 16.7 (CH.sub.3);
[0923] HRMS (ESI.sup.+) calculated for C.sub.14H.sub.13BrN.sub.3O.sub.3.sup.+: requires 350.0135, found [M+H].sup.+: 350.0139.
##STR00166##
[0924] A Schlenk was dried under vacuum by heat-gun and allowed to cool to RT. Compound 84 (628.4 mg, 0.350 mmol, 3.5 eq) was added to the flask and dissolved in anhydrous toluene (5 mL). The solvent was removed on the line to azeotropically dry the reagent and the resulting residue left to dry for 30 mins prior to use. The solid was re-dissolved in anhydrous toluene (4 mL) and a solution of 1-bromo-2,4,6-tris(isocyanatomethyl)-3,5-dimethylbenzene (Compound 204, 35.0 mg, 0.100 mmol, 1.0 eq) in anhydrous toluene (1 mL) was added to the flask. Pyridine (0.048 mL, 0.600 mmol, 6.0 eq) was added and the reaction heated to 34 C. for 24 h. (N.B. the reaction became a vibrant orange colour overtime). TLC (5% MeOH/CH.sub.2Cl.sub.2, UV and Seebach visualisation) indicated that the reaction was complete. The reaction was concentrated under vacuum and purified by reverse phase flash chromatography (on a SNAP Ultra C18 120 g cartridge, 70:30 Acetone:Water to 100:0 over 12 CV). Fraction 6-11 contained excess 3-G2MM Linker which was recovered and fraction 15-18 contained Compound 205 as an off-white foam (348 mg, 61%).
[0925] .sup.1H NMR (400 MHz, CD.sub.3OD) 8.09-7.49 (br. m, 19H, ArH), 7.51-7.10 (br. m, 14H, ArH), 4.74-4.54 (br. m, 4H, benzylic CH.sub.2 and FmocH), 4.51-4.05 (br. s, 11H, benzylic CH.sub.2 and FmocH), 2.50 (br. s, 6H, methyl CH.sub.3), 2.31-1.81 (m, 144H, dendrimer CH.sub.2), 1.41 (s, 243H, dendrimer CH.sub.3);
[0926] HRMS (Nanospray ESI.sup.+) calculated for C.sub.308H.sub.466BrN.sub.21O.sub.75.sup.4+: requires 1435.8134, found [M+4H].sup.4+: 1435.8147.
##STR00167##
[0927] Compound 205 (327.0 mg, 0.057 mmol, 1.000 eq) was dissolved in anhydrous CH.sub.2Cl.sub.2 (2.9 mL) and cooled to 0 C. DBU (0.051 mL, 0.342 mmol, 6.0 eq) was added dropwise and the reaction stirred for 2 h. The reaction mixture was then concentrated under vacuum and purified by reverse phase flash chromatography (on a SNAP Ultra C18 60 g cartridge, 70:30 Acetone:Water to 100:0 over 12 CV) to give the Compound 206 as a light pink solid (0.278 g, 97%).
[0928] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.40 (dd, J=8.3, 3.5 Hz, 3H, ArH), 7.30 (d, J=2.1 Hz, 3H, ArH), 7.21 (dd, J=8.3, 2.1 Hz, 3H, ArH), 4.73 (br. s, 4H, benzylic CH.sub.2), 4.52 (br. s, 2H, benzylic CH.sub.2), 2.60 (br. s, 6H, CH.sub.3), 2.31-2.14 (m, 72H, dendrimer CH.sub.2), 2.13-2.04 (m, 18H, dendrimer CH.sub.2), 2.00-1.88 (m, 54H, dendrimer CH.sub.2), 1.44 (s, 243H, dendrimer CH.sub.3);
[0929] .sup.13C NMR (126 MHz, CD.sub.3OD) 175.5 (C), 174.4 (C), 170.1 (C), 158.1 (C), 158.0 (C), 141.4 (C), 140.2 (C), 137.1 (C), 136.1 (C), 132.8 (C), 130.7 (C), 130.0 (C), 129.9 (C), 124.4 (CH), 118.9 (CH), 117.3 (CH), 81.7 (C), 59.4 (C), 58.7 (C), 32.5 (CH.sub.2), 32.2 (CH.sub.2), 30.7 (CH.sub.2), 30.5 (CH.sub.2), 28.5 (CH.sub.3), 17.1 (CH.sub.3);
[0930] HRMS (Nanospray ESI.sup.+) calculated for C.sub.263H.sub.435BrN.sub.21O.sub.69.sup.3+: requires 1691.0137, found [M+3H].sup.3+: 1691.0132.
##STR00168##
[0931] Dissolved Compound 206 (270.0 mg, 0.053 mmol, 1.0 eq) in anhydrous pyridine (22.3 mL and heated the reaction mixture to 40 C. in a dry syn, external temperature of heat probe set to 40 C. in a separate pear-shaped flask TEB NCO (Compound 103, 20.9 mg, 0.064 mmol, 1.2 eq) was dissolved in anhydrous CH.sub.22 (2.3 mL). The TEB NCO solution was added by syringe pump at 0.85 mL/h. Once the addition was complete the reaction was left overnight at 40 C. The reaction mixture was concentrated under vacuum on a liquid nitrogen cold finger rotary evaporator to dryness. The resulting foam was co-evaporated with toluene twice and the resulting foam purified by reverse phase chromatography (loading in MeCN and eluting on a SNAP Ultra C18 60 g cartridge starting at Acetone:Water 70:30 to 100:0 over 12 CV). Fraction 3-6 contained Compound 207 as a colourless foam (196 mg, 68%).
[0932] .sup.1H NMR (500 MHz, CD.sub.3OD) 8.20-7.83 (m, 4H, ArH), 7.73-7.56 (m, 5H, ArH), 7.44 (s, 5H, NH), 4.55-4.31 (m, 12H, benzylic CH.sub.2), 2.96-2.74 (m, 6H, ethyl CH.sub.2), 2.55 (s, 6H, methyl CH.sub.3a), 2.36-1.80 (m, 144H, dendrimer CH.sub.2), 1.44 (s, 243H, dendrimer CH.sub.3), 1.25-1.16 (m, 9H, ethyl CH.sub.3);
[0933] .sup.13C NMR (126 MHz, CD.sub.3OD) 175.5, 175.4, 174.3, 169.5, 169.3, 158.3, 158.0, 157.9, 157.5, 144.4, 144.4, 139.7, 136.9, 136.2, 135.2, 134.3, 134.1, 132.7, 132.0, 131.4, 130.7, 129.8, 125.9, 125.8, 125.1, 124.5, 122.6, 81.6, 59.5, 59.4, 58.8, 58.7, 43.5, 40.1, 38.9, 32.4, 32.2, 32.2, 30.7, 30.7, 30.4, 28.5, 23.6, 17.1, 16.9, 16.7;
[0934] HRMS (Nanospray ES was) calculated for vacuum C.sub.281H.sub.455BrN.sub.24O.sub.72Na.sup.3+: liquid nit: require 1808.0615, found [M+2H+Na].sup.3+: 1808.0601.
##STR00169##
[0935] Dissolved Compound 207 (196 mg, 0.036 mmol, 1.0 eq) in CH.sub.2Cl.sub.2 (9.0 mL) and TFA (2.4 mL) was added. The reaction was left overnight at RT and added dropwise to 300 mL of H.sub.2O to precipitate the acid. This suspension was centrifuged in 50 mL batches and then washed and sonicated with H.sub.2O. The isolated solid was then dried under high vacuum to give Compound 208 as a colourless solid (108 mg, 77%).
[0936] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 12.04 (s, 27H, COOH), 8.23-7.66 (m, 6H, ArH), 7.59-7.41 (m, 3H, ArH), 7.41-7.16 (m, 6H, NH), 6.67-6.34 (m, 6H, NH), 4.45-4.20 (m, 12H, benzylic CH.sub.2), 2.81 (s, 6H, ethyl CH.sub.2), 2.47 (s, 6H, methyl CH.sub.3), 2.28-1.75 (m, 144H, dendrimer CH.sub.2), 1.20-1.08 (m, 9H, ethyl CH.sub.3);
[0937] .sup.13C NMR (126 MHz, DMSO-d.sub.6) 174.4, 172.4, 165.7, 158.5, 158.3, 156.0, 155.6, 154.8, 150.6, 142.9, 138.0, 135.4, 135.1, 134.7, 133.9, 133.5, 133.1, 130.2, 129.7, 129.1, 122.9, 78.7, 57.4, 56.4, 30.8, 30.4, 29.0, 28.1 22.4, 16.7, 16.2.
##STR00170##
[0938] Prepared in an analogous fashion to Compound 2 by treating HBTU activated linker (Compound A1, 14.3 g, 11.8 mmol, 1.0 eq) in CH.sub.2Cl.sub.2 (16.5 mL) and DIPEA (2.7 mL, 21 mmol, 1.8 eq) with propargyl amine (1.6 mL, 25 mmol, 2.1 eq) for 72 hours. Purified by column chromatography using ethyl acetate as the eluent to give Compound 209 as colourless solid (2.24 g, 5.4 mmol, 46%).
[0939] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.87-7.60 (m, 5H, ArH), 7.57-7.45 (m, 1H, ArH), 7.45-7.23 (m, 4H, ArH), 6.79 (d, J=8.4 Hz, 1H, ArH), 4.45 (br. s, 2H, Fmoc CH.sub.2), 4.27 (br. s, 1H, Fmoc CH), 4.11 (d, J=2.5 Hz, 2H, alkyne CH.sub.2), 2.56 (p, J=2.5 Hz, 1H, alkyne CH).
[0940] HRMS (ESI.sup.+) calculated for C.sub.25H.sub.21N.sub.3O.sub.3Na.sup.+: requires 434.1475, found [M+Na].sup.+: 434.1479.
Compounds 210a and 210b
[0941] ##STR00171## ##STR00172##
[0942] A Schlenk flasked was charged with a stirrer bar, Compound 84 (0.933 g, 0.519 mmol, 1.7 eq) and Compound 5a (0.100 g, 0.305 mmol, 1.0 eq) and dissolved in anhydrous THF (6 mL) and pyridine (0.145 mL, 1.833 mmol, 6.0 eq) then heated to 50 C. for 5 h. Compound 209 (0.189 g, 0.458 mmol, 1.5 eq) was added in one portion and the reaction stirred for a further 12 h. The reaction mixture was transferred to an RBF washing the Schlenk with CH.sub.2Cl.sub.2 before concentrating under vacuum. The crude residue obtained was then purified by reverse phase flash chromatography (loaded with MeCN) on a 120 g SNAP Ultra C18 cartridge elution (1CV 85% acetone/H.sub.2O, 10 CV 85-95% acetone/H.sub.2O, 2CV 95% acetone) to give (fr18-24) identified as Compound 210b a colourless solid (312 mg, 35%), (fr37-47) identified as Compound 210a a colourless solid (524 mg, 40%) and (fr57-60) identified as Compound 108 as colourless solid (354 mg, 20%).
[0943] .sup.1H NMR Compound 210a (400 MHz, CDCl.sub.3) 8.06-7.54 (m, 19H, ArH), 7.48-7.11 (14H, m, ArH), 4.60-4.28 (m, 13H, benzylic CH.sub.2 and FmocH), 4.23-4.09 (m, 4H, benzylic CH.sub.2, FmocH and alkyne CH.sub.2), 2.84 (br. s, 6H, ethyl CH.sub.2), 2.61 (t, J=2.5 Hz, 1H, alkyne CH), 2.31-1.87 (m, 96H, dendrimer CH.sub.2), 1.43 (s, 162H, dendrimer CH.sub.3), 1.20 (br. s, 9H, ethyl CH.sub.3);
[0944] HRMS Compound 210a (Nanospray ESI.sup.+) calculated for C.sub.239H.sub.342N.sub.18O.sub.54Na.sub.2.sup.2+: requires 2188.2210, found [M+2Na].sup.2+: 2188.2224.
[0945] .sup.1H NMR Compound 210b (400 MHz, CDCl.sub.3) 8.01-7.51 (m, 19H, ArH), 7.47-7.07 (m, 14H, ArH), 4.61-4.27 (m, 13H, benzylic CH.sub.2 and FmocH), 4.21-4.08 (m, 4H, benzylic CH.sub.2, FmocH and alkyne CH.sub.2), 2.84 (br. s, 6H, ethyl CH.sub.2), 2.61 (t, J=2.5 Hz, 2H, alkyne CH), 2.34-1.82 (m, 48H, dendrimer CH.sub.2), 1.44 (s, 81H, dendrimer CH.sub.3), 1.19 (br. s, 9H, ethyl CH.sub.3);
[0946] HRMS Compound 210b (Nanospray ESI.sup.+) calculated for C.sub.166H.sub.213N.sub.15O.sub.33Na.sub.2.sup.2+: requires 1495.7634, found [M+2Na].sup.2+: 1495.7628.
##STR00173##
[0947] A RBF charged with Compound 210a (0.524 g, 0.121 mmol, 1.0 eq) dissolved in anhydrous CH.sub.2Cl.sub.2 (6 mL) under nitrogen was cooled to 0 C. DBU (0.108 mL, 0.726 mmol, 6.0 eq) was added dropwise and the reaction mixture was left for 2 h at 0 C. The reaction mixture was then concentrated under vacuum and purified by normal phase flash chromatography on a SNAP KP-Sil 50 g cartridge (eluting with CH.sub.2Cl.sub.2:MeOH 100:0 to 90:10 over 12 CV). This material was then purified by reverse phase chromatography (loaded with MeCN) on a SNAP Ultra C18 60 g cartridge (eluting with Acetone:Water 75:25 to 100:0 over 12 CV) to give Compound 211 as a colourless solid (0.200 g, 0.055 mmol, 45%).
[0948] .sup.1H NMR (500 MHz, CD.sub.3OD) 7.69-7.54 (m, 1H, ArH) 7.46-7.32 (m, 3H, ArH), 7.33-7.26 (m, 2H, ArH), 7.23-7.19 (m, 2H, ArH), 7.17-7.12 (m, 1H, ArH), 4.54-4.43 (m, 6H, benzylic CH.sub.2) 4.11 (d, J=2.5, 2H, alkyne CH.sub.2), 2.88 (m, 6H, ethyl CH.sub.2), 2.58 (t, J=2.5, 1H, alkyne CH), 2.32-2.03 (m, 60H, dendrimer CH.sub.2), 2.01-1.84 (m, 36H, dendrimer CH.sub.2), 1.43 (s, 162H, dendrimer CH.sub.3), 1.25 (m, 9H, ethyl CH.sub.3);
[0949] HRMS (Nanospray ESI.sup.+) calculated for C.sub.194H.sub.315N.sub.18O.sub.48.sup.3+: requires 1222.4271, found [M+3H].sup.3+: 1222.4281.
##STR00174##
[0950] A RBF charged with Compound 210b (0.312 g, 0.106 mmol, 1.0 eq) dissolved in anhydrous CH.sub.2Cl.sub.2 (5.3 mL) under nitrogen was cooled to 0 C. DBU (0.095 mL, 0.635 mmol, 6.0 eq) was added dropwise and the reaction mixture was left for 2 h at 0 C., The reaction mixture was then concentrated under vacuum and purified by normal phase flash chromatography on a SNAP KP-Sil 50 g cartridge eluting (CH.sub.2Cl.sub.2:MeOH 100:0 to 90:10 over 12 CV). This material was then purified by reverse phase chromatography (loaded with MeCN) on a SNAP Ultra 018 60 g cartridge (eluting with Acetone:Water 75:25 to 100:0 over 12 CV) to give Compound 212 as a colourless solid (0.130 g, 0.057 mmol, 54%).
[0951] .sup.1H NMR (500 MHz, CD.sub.3OD) 7.71-7.50 (m, 1H, ArH) 7.46-7.32 (m, 3H, ArH), 7.31-7.22 (m, 2H, ArH), 7.22-7.16 (m, 1H, ArH), 7.16-7.06 (m, 2H, ArH), 4.61-4.34 (m, 6H, benzylic CH.sub.2) 4.11 (br. s, 4H, alkyne CH.sub.2), 2.88 (q, J=7.5, 6H, ethyl CH.sub.2), 2.58 (t, J=2.5, 2H, alkyne CH), 2.33-2.04 (m, 30H, dendrimer CH.sub.2), 2.02-1.86 (m, 18H, dendrimer CH.sub.2), 1.44 (s, 81H, dendrimer CH.sub.3), 1.24 (t, J=7.5, 9H, ethyl CH.sub.3);
[0952] HRMS (Nanospray ESI.sup.+) calculated for C.sub.121H.sub.185N.sub.15O.sub.27.sup.2+: requires 1140.6798, found [M+2H].sup.2+: 1140.6805.
##STR00175##
[0953] Dissolved Compound 211 (200.0 mg, 0.055 mmol, 1.0 eq) in anhydrous pyridine (22.9 mL) and heated the reaction to 40 C. in a dry syn, external temperature of heat probe set to 40 C. In a separate pear-shaped flask Compound 5a (21.6 mg, 0.066 mmol, 1.2 eq) was dissolved in anhydrous CH.sub.2Cl.sub.2 (2.3 mL). The Compound 5a solution was added by syringe pump at 0.85 mL/h. Once the addition was complete the reaction was left overnight at 40 C. The reaction mixture was concentrated under vacuum on a liquid nitrogen cold finger rotary evaporator to dryness. The resulting foam was co-evaporated with toluene twice and the resulting foam purified by reverse phase chromatography (loading in MeCN and eluting on a SNAP Ultra C18 60 g cartridge starting at Acetone:Water 70:30 to 100:0 over 12 CV). Fraction 5-9 contained Compound 213 as a colourless foam (115 mg, 53%).
[0954] .sup.1H NMR (500 MHz, CD.sub.3OD) 8.07-7.94 (m, 6H, ArH), 7.68 (d, J=8.4 Hz, 2H, ArH), 7.63 (d, J=8.4 Hz, 1H, ArH), 7.46 (s, 1H, NH), 4.59-4.34 (m, 12H, benzylic CH.sub.2), 4.17 (d, J=2.5 Hz, 2H, alkyne CH.sub.2), 3.00-2.85 (m, 6H, ethyl CH.sub.2), 2.85-2.74 (m, 6H, ethyl CH.sub.2), 2.63 (t, J=2.5 Hz, 1H, alkyne CH), 2.36-2.09 (m, 60H, dendrimer CH.sub.2), 1.97 (t, J=8.2 Hz, 36H, dendrimer CH.sub.2), 1.45 (s, 162H, dendrimer CH.sub.3), 1.26-1.18 (m, 18H, dendrimer CH.sub.3);
[0955] .sup.13C NMR (126 MHz, CD.sub.3OD) 175.5, 175.5, 174.4, 169.3, 169.0, 158.4, 158.3, 157.2, 157.2, 144.5, 144.5, 144.4, 137.1, 136.9, 134.3, 134.2, 134.0, 133.8, 131.2, 130.1, 130.1, 129.4, 126.1, 125.9, 125.5, 125.3, 122.7, 122.3, 81.6, 80.8, 72.2, 59.4, 58.8, 58.7, 38.9, 38.8, 38.8, 38.7, 32.4, 32.2, 30.7, 30.4, 28.4, 23.6, 16.8, 16.7, 16.6;
[0956] HRMS (Nanospray ESI.sup.+) calculated for C.sub.212H.sub.336N.sub.21O.sub.51.sup.3+: requires 1331.4802, found [M+3H].sup.3: 1331.4784.
##STR00176##
[0957] Dissolved Compound 213 (111 mg, 0.028 mmol, 1.0 eq) in CH.sub.2Cl.sub.2 (7.0 mL) and TFA (1.9 mL) was added. The reaction was left overnight at RT and added dropwise to 300 mL of H.sub.2O to precipitate the acid. This suspension was centrifuged in 50 mL batches and then washed and sonicated with H.sub.2O. The isolated solid was then dried under high vacuum to give Compound 214 as a colourless solid (36 mg, 43%).
[0958] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 12.02 (s, 27H, COOH), 8.73 (s, 1H, NH), 8.19-7.67 (m, 6H, ArH), 7.64-7.12 (m, 9H, ArH and NH), 6.48 (s, 3H, NH), 6.39 (s, 3H, NH), 4.52-4.15 (m, 12H, benzylic CH.sub.2), 4.11-3.93 (m, 2H, alkyne CH.sub.2), 3.34 (s, 6H, NH), 3.09 (s, 1H, alkyne CH), 2.82 (s, 6H, ethyl CH.sub.2), 2.66 (s, 6H, ethyl CH.sub.2), 2.28-1.62 (m, 144H, dendrimer CH.sub.2), 1.26-1.01 (m, 18H, ethyl CH.sub.3);
[0959] .sup.13C NMR (126 MHz, DMSO-d.sub.6) 174.9, 172.9, 166.0, 165.9, 156.4, 156.1, 155.0, 142.8, 142.7, 135.5, 135.4, 133.9, 133.8, 133.4, 133.3, 129.2, 128.7, 128.4, 127.5, 124.6, 123.9, 123.6, 123.1, 119.9, 119.3, 82.1, 73.1, 57.9, 56.8, 37.6, 37.2, 31.2, 30.8, 29.5, 28.9, 28.6, 22.8, 22.5, 16.8, 16.7.
##STR00177##
[0960] Dissolved Compound 212 (130.0 mg, 0.057 mmol, 1.0 eq) in anhydrous pyridine (23.8 mL) and heated the reaction to 40 C. in a dry syn, external temperature of heat probe set to 40 C. In a separate pear-shaped flask. Compound 5a (22.0 mg, 0.068 mmol, 1.2 eq) was dissolved in anhydrous CH.sub.2Cl.sub.2 (2.4 mL). The Compound 5a solution was added by syringe pump at 0.85 mL/h. Once the addition was complete the reaction was left overnight at 40 C. The reaction mixture was concentrated under vacuum on a liquid nitrogen cold finger rotary evaporator to dryness. The resulting foam was co-evaporated with toluene twice and the resulting foam purified by reverse phase chromatography (loading in MeCN and eluting on a SNAP Ultra C18 60 g cartridge starting at Acetone:Water 70:30 to 100:0 over 12 CV). Fraction 3-4 contained Compound 215 as a colourless foam (33 mg, 23%).
[0961] .sup.1H NMR (500 MHz, CD.sub.3OD) 8.03 (d, J=2.1 Hz, 2H, ArH), 7.99-7.92 (m, 4H, ArH), 7.66 (dd, J=8.6, 2.1 Hz, 1H, ArH), 7.61 (dd, J=8.6, 2.1 Hz, 2H, ArH), 7.45 (s, 1H, NH), 4.56-4.36 (m, 12H, benzylic CH.sub.2), 4.15 (d, J=2.5 Hz, 4H, alkyne CH.sub.2), 2.97-2.84 (m, 6H, ethyl CH.sub.2), 2.84-2.72 (m, 6H, ethyl CH.sub.2), 2.61 (t, J=2.5 Hz, 2H, alkyne CH), 2.32-2.09 (m, 30H, dendrimer CH.sub.2), 2.01-1.88 (m, 18H, dendrimer CH.sub.2), 1.43 (s, 81H, dendrimer CH.sub.3), 1.28-1.13 (m, 18H, ethyl CH.sub.3);
[0962] .sup.13C NMR (126 MHz, CD.sub.3OD) 175.6, 175.5, 174.4, 169.4, 169.1, 158.4, 158.3, 157.3, 157.2, 144.5, 144.5, 144.5, 137.2, 136.9, 134.3, 134.2, 133.9, 133.8, 131.1, 130.1, 130.1, 129.3, 126.1, 126.0, 125.5, 125.3, 122.7, 122.2, 81.7, 80.8, 72.1, 59.5, 58.8, 58.7, 38.9, 38.8, 38.7, 38.7, 32.4, 32.2, 32.2, 30.7, 30.4, 30.0, 28.4, 23.6, 16.7, 16.6, 16.5;
[0963] HRMS (Nanospray ESI.sup.+) calculated for C.sub.139H.sub.206N.sub.18O.sub.30.sup.2+: requires 1304.2589, found [M+2H].sup.2+: 1304.2606.
##STR00178##
[0964] Dissolved Compound 215 (30 mg, 0.012 mmol, 1.0 eq) in CH.sub.2Cl.sub.2 (3.0 m:) and TFA (0.81 mL) was added. The reaction was left overnight at RT and added dropwise to 300 mL of H.sub.2O to precipitate the acid. This suspension was centrifuged in 50 mL batches and then washed and sonicated with H.sub.2O. The isolated solid was then dried under high vacuum to give Compound 216 as a colourless solid (17 mg, 67%).
[0965] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 12.03 (s, 27H, COOH), 8.75 (t, J=5.7 Hz, 1H, NH), 8.21-7.93 (m, 6H, ArH), 7.91-7.73 (m, 4H, NH), 7.60-7.42 (m, 3H, ArH), 7.38-7.21 (m, 6H, ArH and NH), 6.46 (s, 3H, NH), 6.37 (s, 3H, NH), 4.52-4.21 (m, 12H, benzylic CH.sub.2), 4.03 (d, J=2.5 Hz, 2H, alkyne CH.sub.2), 3.10 (t, J=2.5 Hz, 1H, alkyne CH), 2.83 (s, 6H, ethyl CH.sub.2), 2.69 (m, 6H, ethyl CH.sub.2), 2.18-1.72 (m, 144H, dendrimer CH.sub.2), 1.19-1.09 (m, 18H, ethyl CH.sub.2);
[0966] .sup.13C NMR (126 MHz, DMSO-d.sub.6) 174.9, 172.9, 166.0, 165.9, 156.3, 156.1, 155.0, 149.3, 142.8, 142.8, 137.6, 135.5, 135.4, 133.9, 133.8, 133., 130.6, 129.2, 128.7, 128.3, 127.5, 124.7, 124.6, 123.9, 123.7, 123.1, 120.4, 119.9, 119.3, 82.1, 73.1, 57.8, 56.8, 37.5, 37.3, 31.2, 30.8, 29.5, 28.9, 28.58, 22.8, 22.5, 16.8, 16.7.
##STR00179##
[0967] N-Boc-L-serine (5.00 g, 24.4 mmol, 1.0 eq) was dissolved in anhydrous DMF (50 mL), cooled to 0 C. and NaH (2.05 g, 51.1 mmol, 2.1 eq) (60% dispersion in mineral oil) was added. After stirring for 30 min at 0 C., 3-bromopropyne (2.52 mL, 26.7 mmol) (80% solution in toluene) was added dropwise. After stirring at 0 C. for 30 minutes the ice bath was removed and stirring continued overnight at ambient temperature. The solution was dark brown in colour after this time. Aqueous sulfate buffer (16 g Na.sub.2SO.sub.4, 2 mL H.sub.2SO.sub.4 made up to 150 mL with H.sub.2O) was added to the flask gradually to avoid any exotherm generated upon quenching. A precipitate formed and then slowly dissolved to give an orange solution. Brine (150 mL) was added and the reaction mixture extracted with EtOAc (3150 mL). The combined organics were washed with H20 (3150 mL), dried over MgSO4, filtered and the resulting filtrate concentrated under vacuum. Some DMF was transferred over during this step. Before the organic had completely concentrated silica gel was added and then concentrated to dryness. The silica was then washed with CH.sub.2Cl.sub.2 (500 mL) before eluting the desired compound with 10% MeCN/CH.sub.2Cl.sub.2 (500 mL) or until fractions did not contain the product (PMA stain, 10% MeCN/CH.sub.2Cl.sub.2, streaky black spot 0.3-0.4 R.sub.f). The resulting filtrate was concentrated under vacuum to give the desired compound as a yellow gum after drying under high vacuum (4.9 g, 83%). (S)-2-[(tert-butoxycarbonyl)amino]-3-(prop-2-yn-1-yloxy)propanoic acid (2.1 g, 8.6 mmol, 1.0 eq) was dissolved in CH.sub.2Cl.sub.2 (33.6 mL) and TFA (33.1 mL) was added. The reaction was stirred at RT until complete by TLC. The reaction mixture was concentrated under vacuum and azeotrope with toluene to give the TFA salt of O-(prop-2-yn-1-yl)serine which was taken directly onto the next step. Dissolved O-(prop-2-yn-1-yl)serine (2.22 g, 8.63 mmol, 1.0 eq) in acetone (14.4 mL) and H.sub.2O (14.4 mL). Sodium carbonate (2.75 g, 25.9 mmol, 3.0 eq) and Fmoc-OSu (3.06 g, 9.07 mmol, 1.05 eq) was added and the reaction left to stir overnight. The reaction mixture was acidified to pH 3 with HCl (3 M) and extracted with EtOAc (3100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and the resulting filtrate concentrated under vacuum. The crude residue was purified by column chromatography (eluting with 2.5% MeOH in CH.sub.2Cl.sub.2) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(prop-2-yn-1-yl)serine as a white solid (2.83 g, 90%). Second generation dendritic amine (Compound 82, 600 mg, 0.417 mmol, 1.0 eq) was dissolved in THF (3.6 mL). N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(prop-2-yn-1-yl)serine (183 mg, 0.500 mmol, 1.2 eq), COMU (0.21 g, 0.50 mmol, 1.2 eq), K-Oxyma (90 mg, 0.50 mmol, 1.2 eq) and DIPEA (0.22 mL, 1.25 mmol, 3.0 eq) were then added to the solution. The reaction was left to stir overnight at RT. The reaction was concentrated under vacuum to remove THF. The residue was redissolved in EtOAc (40 mL) and then washed sequentially with KHSO.sub.4 (100 mL), sat. NaHCO.sub.3 (100 mL) and brine (100 mL). The organic phase was dried over MgSO.sub.4, filtered and concentrated under vacuum to give a crude yellow oil. Purification by reverse phase flash chromatography on a SNAP Ultra C18 120 g cartridge (eluting with Acetone:Water 78:22 for 3CV then 91:9 for 4CV) gave the Fmoc-protected intermediate (fr10-13) as an off white solid (602 mg, 81%). To a RBF dried under vacuum by heat-gun was added Fmoc-protected intermediate (1.2 g, 0.67 mmol, 1.0 eq). Anhydrous CH.sub.2Cl.sub.2 (33.6 mL) was added and the flask cooled to 0 C. DBU (0.20 mL, 1.3 mmol, 2.0 eq) was added dropwise and the reaction stirred until complete by TLC (5% MeOH/CH.sub.2Cl.sub.2, stain with ninhydrin to visualise). The reaction flask was warmed to RT after 2 h. The reaction was concentrated under vacuum and the crude residue purified by reverse phase chromatography on a SNAP Ultra C18 120 g cartridge (eluting with Acetone:Water 75:25 to 100:0 over 12 CV). Fr5-18 contained Compound 217 as a yellow foam (0.95 g, 91%).
[0968] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.58 (br. s, 1H, NH), 4.30-4.15 (m, 2H, alkyne CH.sub.2), 3.72 (dd, J=9.2, 5.4 Hz, 1H, serine CHH), 3.63 (dd, J=9.2, 5.4 Hz, 1H, serine CHH), 3.48 (t, J=5.4 Hz, 1H, serine CH), 2.90 (t, J=2.4 Hz, 1H, alkyne CH), 2.29-1.90 (m, 48H, dendrimer CH.sub.2), 1.45 (s, 81H, dendrimer CH.sub.3);
[0969] HRMS (ESI.sup.+) calculated for C.sub.82H.sub.142N.sub.5O.sub.23Na.sup.2+: requires 793.9991, found [M+H+Na].sup.2+: 793.9974.
##STR00180##
[0970] A RBF was dried under vacuum using a heat-gun. Once cool, Compound 217 (0.45 g, 0.29 mmol, 1.0 eq) and HBTU activated linker (0.21 g, 0.35 mmol, 1.2 eq) were added and suspended in anhydrous THF (1.44 mL). DIPEA (0.08 mL, 0.46 mmol, 1.6 eq) was then added and the reaction stirred overnight at RT. The reaction was heterogenous initially and became a homogeneous (dark brown) solution after 16 h at RT. The reaction was concentrated under vacuum and purified by reverse phase flash chromatography on a SNAP Ultra C18 120 g cartridge (eluting with Acetone:Water 66:34 for 3CV then 88:12 for 5CV). Fr7-11 contained the Compound 218 as a yellow solid (441 mg, 80%).
[0971] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.80-7.42 (m, 6H, ArH), 7.41-7.07 (m, 4H, ArH), 6.72 (d, J=8.5 Hz, 1H, ArH), 4.47 (d, J=5.3 Hz, 1H, serine CH), 4.39 (br. s, 2H, Fmoc CH.sub.2), 4.19 (br. s, 1H, Fmoc CH), 4.17-4.07 (t, J=2.4 Hz, 2H, alkyne CH.sub.2), 3.83 (dd, J=9.6, 5.3 Hz, 1H, serine CHH), 3.75 (dd, J=9.6, 5.3 Hz, 1H, serine CHH), 2.81 (t, J=2.4 Hz, 1H, alkyne CH), 2.17-1.73 (m, 48H, dendrimer CH.sub.2), 1.32 (s, 81H, dendrimer CH.sub.3);
[0972] HRMS (ESI.sup.+) calculated for C.sub.104H.sub.157N.sub.7O.sub.26Na.sub.2.sup.2+: requires 983.5497, found [M+2Na].sup.2+: 983.5496.
##STR00181##
[0973] Compound 218 (0.64 mg, 0.33 mmol, 3.5 eq) was concentrated into a pear-shaped flask and dried through azeotropic distillation with anhydrous toluene (5 mL) followed by drying under high vacuum for 30 min. The resulting residue was dissolved in anhydrous CH.sub.2Cl.sub.2 (4.7 mL), Compound 5a (31 mg, 0.095 mmol, 1.0 eq) and pyridine (0.046 mL, 0.57 mmol, 6.0 eq) were added and the reaction heated to 34 C. for 12 h. The reaction mixture was concentrated under vacuum and the resulting residue purified by flash column chromatography on a SNAP Ultra C18 120 g cartridge (eluting with Acetone:Water 70:30 to 95:5 over 12CV). Fr1-8 recovered Compound 218 and fr11-14 contained Compound 219 as an off-white solid (476 mg, 83%).
[0974] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.93-7.36 (m, 19H, ArH), 7.34-7.05 (m, 14H, ArH), 4.49 (t, J=5.0 Hz, 3H, serine CH), 4.38 (br. s, 6H, benzylic CH.sub.2), 4.27 (br. s, 6H, Fmoc CH.sub.2), 4.15 (t, J=2.2 Hz, 6H, alkyne CH.sub.2), 4.07 (s, 3H, Fmoc CH), 3.84 (dd, J=9.6, 5.0 Hz, 3H, serine CHH), 3.76 (dd, J=9.6, 5.0 Hz, 3H, serine CHH), 2.81 (t, J=2.2 Hz, 1H, alkyne CH), 2.74 (br. s, 6H, ethyl CH.sub.2), 2.22-1.71 (m, 144H, dendrimer CH.sub.2), 1.32 (s, 243H, dendrimer CH.sub.3), 1.09 (t, J=7.4 Hz, 9H, ethyl CH.sub.3).
[0975] HRMS (Nanospray ESI) calculated for C.sub.330H.sub.496N.sub.24O.sub.81.sup.4+: requires 1523.6382, found [M+4H].sup.4+: 1523.6409.
##STR00182##
[0976] A RBF charged with Compound 219 (0.451 g, 0.074 mmol, 1.0 eq) dissolved in anhydrous CH.sub.2Cl.sub.2 (3.7 mL) under nitrogen and cooled to 0 C. DBU (0.066 mL, 0.444 mmol, 6.0 eq) was added dropwise and the reaction mixture was left for 2 h at 0 C. The reaction mixture was then concentrated under vacuum and purified by reverse phase flash chromatography on a SNAP Ultra C18 60 g cartridge (eluting with Acetone:Water 70:30 to 100:0 over 12 CV). Fr3-7 contained Compound 220 as a colourless solid (0.279 g, 69%).
[0977] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.37 (d, J=8.3 Hz, 3H, ArH), 7.24 (d, J=2.1 Hz, 3H, ArH), 7.15 (dd, J=8.2, 2.1 Hz, 3H, ArH), 4.48 (t, J=5.3 Hz, 3H, serine CH), 4.42 (br. s, 6H, benzylic CH.sub.2), 4.25-4.10 (m, 6H, alkyne CH.sub.2), 3.84 (dd, J=9.6, 5.3 Hz, 3H, serine CHH), 3.76 (dd, J=9.6, 5.3 Hz, 3H, serine CHH), 2.83 (t, J=2.4 Hz, 3H, alkyne CH), 2.80 (q, J=6.1 Hz, 6H, ethyl CH.sub.2), 2.20-1.73 (m, 144H, dendrimer CH.sub.2), 1.34 (s, 243H, dendrimer CH.sub.3), 1.22-1.12 (m, 9H, ethyl CH.sub.2).
[0978] .sup.13C NMR (126 MHz, CD.sub.3OD) 175.4, 174.4, 171.6, 170.4, 157.8, 145.1, 141.4, 133.9, 131.4, 130.5, 124.4, 119.0, 117.4, 81.7, 80.4, 77.0, 70.4, 59.4, 59.4, 58.7, 56.2, 39.3, 32.2, 32.1, 30.7, 30.5, 28.5, 23.9, 17.0;
[0979] HRMS (Nanospray ESI.sup.+) calculated for C.sub.285H.sub.466N.sub.24O.sub.75.sup.4+: requires 1357.0870, found [M+4H].sup.4+: 1357.0824.
##STR00183##
[0980] Compound 220 (265.0 mg, 0.049 mmol, 1.0 eq), DMAP (18 mg, 0.147 mmol, 3.0 eq) and n-octyl glucoside (29 mg, 0.098 mmol, 2.0 eq) were weighed into a round bottom flask and anhydrous toluene was added. This was removed in situ under high vacuum. Procedure repeated, and the resulting foam allowed to dry for 30 min. The reagents were then dissolved in anhydrous CH.sub.2Cl.sub.2 (98 mL) and heated to 34 C. In a separate dried RBF, Compound 5a (19.2 mg, 0.059 mmol, 1.2 eq) was weighed into a flask and dissolved in anhydrous CH.sub.2Cl.sub.2 (9.8 mL).
[0981] This solution was then syringe pumped into the reaction mixture at 1 mL/hr. After completion, the reaction was left for a further 24 h at 34 C. The reaction mixture was cooled and concentrated under vacuum. Purification by reverse phase flash chromatography on a SNAP Ultra C18 120 g column (eluting with Acetone:Water 70:30 to 100:0 over 12 CV). Fr1-8 were taken and purified by prep HPLC (C18 20150 mm, 5 m, 20 mL/min, Acetone:Water 70:30 to 100:0 over 30 min). Analysis at this stage was difficult because of the presence of n-octyl glucose (110 mg, 39%). Mass spec confirmed material obtained after prep HPLC contained Compound 221.
[0982] HRMS (Nanospray ESI.sup.+) calculated for C.sub.303H.sub.487N.sub.27O.sub.78.sup.4+: requires 1438.6254, found [M+4H].sup.4+: 1438.6232.
##STR00184##
[0983] Compound 221 (110 mg, 0.019 mmol, 1.0 eq) was dissolved in CH.sub.2Cl.sub.2 (4.8 mL) and TFA (1.3 mL) was added. The reaction was left overnight at RT and then concentrated under vacuum. Purification by reverse phase flash chromatography on a SNAP Ultra C18 30 g column (eluting with MeOH:Water+0.1% formic acid 10:90 to 100:0 over 12 CV). Fr30-32 were taken and purified by prep HPLC (C18 20150 mm, 5 m, 20 mL/min, MeOH:Water+0.1% formic acid 10:90 to 100:0 over 30 min) gave Compound 222 as a colourless solid (30 mg, 37%).
[0984] .sup.1H NMR (400 MHz, CD.sub.3OD) 8.46 (s, 3H, NH), 8.17 (s, 3H, ArH), 8.02 (d, J=8.5 Hz, 3H, ArH), 7.62 (d, J=8.5 Hz, 3H, ArH), 4.65 (t, J=5.4 Hz, 3H, serine CH), 4.53-4.33 (m, 12H, benzylic CH.sub.2), 4.37-4.23 (m, 6H, alkyne CH.sub.2), 4.10-3.84 (m, 6H, serine CH.sub.2), 2.98 (t, J=2.4 Hz, 3H, alkyne CH), 2.76 (s, 6H, ethyl CH.sub.2), 2.69 (s, 6H, ethyl CH.sub.2), 2.48-1.68 (s, 144H, dendrimer CH.sub.2), 1.41-1.00 (m, 18H, ethyl CH.sub.3);
[0985] .sup.13C NMR (126 MHz, CD.sub.3OD) 182.8, 175.2, 175.0, 170.6, 170.0, 160.8, 157.4, 156.8, 144.2, 132.3, 131.9, 128.3, 127.8, 124.4, 68.7, 58.3, 58.2, 54.7, 37.6, 31.6, 30.8, 30.6, 22.6, 22.3, 15.4.
##STR00185##
[0986] A pre-dried Schlenk tube was charged with Compound 84 (441 mg, 0.245 mmol) under a flow of nitrogen and a solution of Compound 5c (20 mg, 0.070 mmol) in THF (0.5 mL) added. Dry THF (3.5 mL) and anhydrous pyridine (0.006 mL, 0.070 mmol) was added. The reaction was stirred for 16 h at 50 C. The reaction mixture was concentrated to dryness and the crude residue purified by reverse phase MPLC on a 120 g SNAP Ultra C18 cartridge elution (70-95% acetone/H.sub.2O) to give a Compound 223 white solid (197 mg, 0.035 mmol, 50%).
[0987] HRMS (Nanospray ESI) calculated for C.sub.309H.sub.469N.sub.21O.sub.75.sup.4+: requires 1419.3407, found [M+4H].sup.4+: 1419.3391.
##STR00186##
[0988] Prepared in a manner analogous to Compound 7b from Compound 223 (0.097 g, 0.017 mmol). Purified by reverse phase flash chromatography on a 120 g SNAP Ultra C18 cartridge elution (1CV 80% acetone/H.sub.2O, 10 CV 80-95% acetone/H.sub.2O, 2CV 95% acetone) to give a white solid (0.072 g, 0.014 mmol, 85%).
[0989] .sup.1H NMR (500 MHz, methanol-d.sub.4) m. 7.42-7.30 (3H, CHCNH (Ar)) m. 7.31-7.29 CHCNH.sub.2 (Ar)), m. 7.23-7.19 (3H, CHCHCNH (Ar)), m. 4.54-4.48 (6H, ArCH.sub.2NH), s. 2.49 (6H, ArCH.sub.3), s. (3H, ArCH.sub.3), m. 2.28-1.90 (144H, NHCH.sub.2CH.sub.2C(O)), s. 1.44 (243H, CO.sub.2C(CH.sub.3).sub.3).
[0990] .sup.13C NMR (125 MHz, methanol-d.sub.4) 175.5 (CONH), 174.4, 174.3 (CO.sub.2C(CH.sub.3).sub.3), 170.1 (ArCONHR), 158.1 (NHC(O)NH), 141.3 (CNH.sub.2), 135.4 (CCO.sub.2NHR), 134.7 (CNHC(O)NH), 130.0 (CCH.sub.3), 124.4, 118.9 (CHCHCNH), 117.4 (CHCNH.sub.2), 111.4 (CCH.sub.2NHC(O)NH), (C(CH.sub.2CH.sub.2CONH).sub.3), 81.6 (CO.sub.2C(CH.sub.3).sub.3), 58.7, (C(CH.sub.2CH.sub.2CO.sub.2).sub.3), 40.3 (ArCH.sub.2NHC(O)NH), 32.5 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 32.2 (CH.sub.2CH.sub.2CONH), 30.7 (CH.sub.2CH.sub.2CONH), 30.5, (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 28.4 (CO.sub.2C(CH.sub.3).sub.3), 16.3 (ArCH.sub.3).
##STR00187##
[0991] Dissolved Compound 224 (200 mg, 0.040 mmol, 1.0 eq) in anhydrous pyridine (16.8 mL) and heated the reaction to 40 C. in a dry syn, external temperature of heat probe set to 40 C. In a separate pear-shaped flask Compound 5c (13.7 mg, 0.048 mmol, 1.2 eq) was dissolved in anhydrous CH.sub.2Cl.sub.2 (1.7 mL). The Compound 5c solution was added by syringe pump at 0.85 mL/h. Once the addition was complete the reaction was left overnight at 40 C. The reaction mixture was concentrated under vacuum on a liquid nitrogen cold finger rotary evaporator to dryness. The resulting foam was co-evaporated with toluene twice and the resulting foam purified by reverse phase chromatography (loading in MeCN and eluting on a SNAP Ultra C18 60 g cartridge starting at Acetone:Water 70:30 to 100:0 over 12 CV). Fraction 4-6 contained Compound 225 as a colourless foam (127 mg, 60%).
[0992] .sup.1H NMR (500 MHz, CD.sub.3OD) 8.07 (s, 2H, NH), 7.95 (s, 3H, ArH), 7.89 (br. s, 3H, ArH), 7.66 (d, J=8.6 Hz, 3H, ArH), 7.44 (s, 6H, NH), 4.51 (s, 6H, benzylic CH.sub.2), 4.45 (s, 1H, benzylic CH.sub.2), 2.49 (s, 9H, methyl CH.sub.3), 2.43 (s, 9H, methyl CH.sub.3), 2.32-1.91 (m, 144H, dendrimer CH.sub.2), 1.43 (s, 243H, dendrimer CH.sub.3).
[0993] .sup.13C NMR (126 MHz, CD.sub.3OD) 175.5, 174.4, 169.4, 158.6, 157.6, 137.5, 137.4, 135.1, 134.7, 131.4, 129.9, 126.0, 122.6, 81.6, 59.5, 58.8, 40.2, 40.0, 32.5, 32.2, 30.7, 30.5, 28.5, 16.7, 16.3.
[0994] HRMS (Nanospray ESI.sup.+) calculated for C.sub.279H.sub.450N.sub.24O.sub.72Na.sub.4.sup.4+: requires 1345.7981, found [M+4Na].sup.4+: 1345.7994.
##STR00188##
[0995] Dissolved Compound 225 (120 mg, 0.023 mmol, 1.0 eq) in CH.sub.2Cl.sub.2 (5.8 mL) and TFA (1.5 mL) was added. The reaction was left overnight at RT and added dropwise to 300 mL of H.sub.2O to precipitate the acid. This suspension was centrifuged in 50 mL batches and then washed and sonicated with H.sub.2O. The isolated solid was then dried under high vacuum to give Compound 226 as a colourless solid (80 mg, 91%).
[0996] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 12.04 (s, 27H, COOH), 8.00 (s, 3H, ArH), 7.93 (d, J=8.5 Hz, 3H, ArH), 7.78 (s, 3H, NH), 7.73 (s, 3H, NH), 7.54 (d, J=8.5 Hz, 3H, ArH), 7.41 (s, 3H, NH), 7.27 (s, 9H, NH), 6.54 (s, 3H, NH), 6.44 (s, 3H, NH), 4.36 (s, 12H, benzylic CH.sub.2), 2.42 (s, 9H, methyl CH.sub.3), 2.35 (s, 9H, methyl CH.sub.3), 2.25-1.71 (m, 144H, dendrimer CH.sub.2).
[0997] .sup.13C NMR (126 MHz, DMSO-d.sub.6) 174.9, 172.8, 166.0, 156.6, 155.2, 135.9, 129.4, 128.8, 124.8, 123.7, 119.7, 67.5, 57.9, 56.8, 31.2, 30.8, 29.5, 28.5, 16.3.
##STR00189##
[0998] A Schlenk flask equipped with a magnetic stirrer was charged with Compound 108 (200 mg, 0.04 mmol, 1.0 eq), DMAP (14.5 mg, 0.12 mmol, 3.0 eq) and n-octyl glucoside (23.2 mg, 0.08 mmol, 2.0 eq) dissolved in anhydrous CH.sub.2Cl.sub.2 (40 mL) and then warmed to 34 C. A solution of 1,3,5-triisocyanatobenzene (12.5 mg, 0.051 mmol) in toluene (ca. 85% purity) was added to the flask and the reaction was left for 16 h. The solvent was removed under vacuum and the crude product was purified by reverse phase MPLC on a C18 SNAP Ultra 60 g cartridge (eluting with Acetone:Water 70:30 to 100:0 over 12CV) giving Compound 227 as a white solid (67 mg, 0.012 mmol, 32%).
[0999] .sup.1H NMR (500 MHz, CD.sub.3OD) 8.11 (d, J=8.6 Hz, 3H, ArH), 7.76-7.69 (m, 6H, ArH), 7.14 (s, 3H, ArH), 4.43 (s, 6H, benzylic CH.sub.2), 4.29 (s, 6H, benzylic CH.sub.2), 2.80-2.70 (m, 6H, ethyl CH.sub.2), 2.31-1.86 (m, 144H, dendrimer CH.sub.2), 1.43 (s, 243H, dendrimer CH.sub.3), 1.21 (t, J=7.4 Hz, 9H, ethyl CH.sub.3).
[1000] HRMS (ESI.sup.+) calculated for C.sub.279H.sub.450N.sub.24O.sub.72Na.sub.4.sup.4+: requires 1345.7981, found [M+4Na].sup.4+: 1345.7985.
##STR00190##
[1001] A 50 mL flask, equipped with a magnetic stirrer and a tapped gas adapter, was charged with Compound 13 (89 mg, 0.045 mmol). Compound 13 was placed under vacuum for 5 min, then placed under nitrogen. The Compound 13 was dissolved in dry pyridine (20 mL, 0.002 M), and the solution heated to 40 C. A solution of Compound 103 (16 mg, 0.050 mmol) in dry dichloromethane (1.0 mL, 0.050 M) was added at a rate of 0.1 mL/h. The reaction stirred for a further 12 h at 40 C. before being concentrated under vacuum. The residue was purified by reverse phase flash chromatography on a 60 g SNAP Ultra C18 cartridge elution (1 CV 60% acetone/water, 10 CV 60-100% acetone/water, 6 CV 100% acetone).
[1002] .sup.1H NMR (500 MHz, CD.sub.3OD) 7.99 (d, J=8.5, 3H, ArH), 7.95 (d, J=2.1, 3H, ArH), 7.62 (s, 3H, RNHCO), 7.53 (dd, J=8.6, 2.1, 3H, ArH), 4.48 (s, 6H, BnH), 4.41 (s, 6H, BnH), 2.85 (q, J=7.5, 6H, CH.sub.2CH.sub.3), 2.75 (q, J=7.5, 6H, CH.sub.2CH.sub.3), 2.28 (t, J=8.0, 18H, CCH.sub.2CH.sub.2), 2.10 (t, J=8.0, 18H, CCH.sub.2CH.sub.2), 1.45 (s, 81H, .sup.tBuH), 1.21 (t, J=7.4, 18H, CH.sub.2CH.sub.3).
[1003] .sup.13C NMR (126 MHz, CD.sub.3OD) 208.96 (C), 173.18 (C), 168.38 (C), 156.81 (C), 155.83 (C), 143.16 (C), 143.10 (C), 134.95 (C), 132.69 (C), 132.24 (C), 129.85 (C), 127.95 (C), 123.89 (CH), 123.46 (CH), 120.73 (CH), 80.34 (C), 58.34(C), 37.39 (CH.sub.2), 29.42 (CH.sub.2), 29.19 (CH.sub.2), 26.98 (CH.sub.3), 22.25 (CH.sub.2), 15.25 (CH.sub.3), 15.22 (CH.sub.3).
[1004] HRMS (Nanospray) calculated for C.sub.123H.sub.185N.sub.150O.sub.27: requires 1152.6793, found [M+2H].sup.2+: 1152.6798.
##STR00191##
[1005] A 10 mL flask, equipped with a magnetic stirrer, was charged with Compound 228 (27 mg, 0.012 mmol). Compound 228 was dissolved in dichloromethane (2.9 mL, 0.004 M) before trifluoroacetic acid was added. The reaction stirred for 12 h at rt. The reaction mixture was dripped into rapidly stirring water (150 mL) which resulted in the formation of a white precipitate. The precipitate was centrifuged out and dissolved in acetone. After being transferred to a round bottomed flask, the acetone was removed under vacuum, and the white solid azeotroped with toluene.
[1006] .sup.1H NMR (600 MHz, D.sub.2O) 7.86 (s, 3H, ArH), 7.76 (d, J=8.5, 3H, ArH), 7.52 (d, J=8.5, 3H, ArH), 4.44 (s, 12H, BnH), 2.84-2.64 (m, 12H, CH.sub.2CH.sub.3), 2.29-2.16 (m, 18H, CCH.sub.2CH.sub.2), 2.15-2.01 (m, 18H, CCH.sub.2CH.sub.2), 1.24-1.10 (m, 18H, CH.sub.2CH.sub.3).
[1007] .sup.13C NMR (126 MHz, DMSO-d.sub.8) 174.50 (C), 166.09 (C), 155.49 (C), 154.56 (C), 142.30 (C), 134.21 (C), 132.81 (C), 128.34 (C), 128.20 (C), 123.05 (CH), 122.59 (CH), 119.33 (CH), 57.10 (C), 37.04 (CH.sub.2), 29.06 (CH.sub.2), 28.22 (CH.sub.2), 21.04 (CH.sub.2), 16.34 (CH.sub.3).
##STR00192##
[1008] Compound 11 (1.384 g, 2.409 mmol) and Compound 5a (0.200 g, 0.611 mmol) were placed in a round bottomed flask under nitrogen and dissolved in anhydrous DMF (21 mL). To this solution was added dry pyridine (0.147 mL, 1.833 mmol) then heated to 30 C. for 100 hours. The solvent was evaporated under vacuum to give a gum which was purified by column chromatography by pre-adsorbing onto silica gel (20 g) by dissolving the crude in a mixture of dichloromethane and methanol and removing the solvent under vacuum to give a free-flowing powder. This pre-adsorbed material was loaded into an empty cartridge and put in line with a 100 g SNAP HP Sil cartridge and eluting with a gradient of dichloromethane with increasing concentration of methanol gave recovered Compound 11 (367 mg), and Compound 230 (1.171 g, 93%).
[1009] .sup.1H NMR: (400 MHz, (CD.sub.3OD): m.br. 7.95-7.20 (33H, ArH), s. 4.40 (6H, ArCH.sub.2NH), m. 4.30-4.10 (9H, FmocH), m. 3.71-3.60 (42H, CH.sub.2CH.sub.2O, and OCH.sub.2CH.sub.2NH.sub.2), m. 3.33 (6H, OCH.sub.2CH.sub.2N.sub.3), m.br. 2.79 (6H, ArCH.sub.2CH.sub.3), s.br. 1.19 (9H, ArCH.sub.2CH.sub.3).
##STR00193##
[1010] To a stirred suspension of Compound 230 (1.171 g, 0.571 mmol) in DCM (5 mL) at room temperature was added distilled DBU (0.426 mL, 2.854 mmol). After 10 minutes the reaction mixture became a clear solution and was stirred for a total of 2 hours before loading directly onto a flash chromatography column 50 g SNAP KP Sil cartridge that was equilibriated with dichloromethane and then eluting with a gradient of dichloromethane with increasing concentration of methanol to give the desired product contaminated with the fluorenyl by-product. Partial evaporation of the product-containing fractions gave a thick white slurry where the solid was separated by centrifuge. The wet solid was resuspended in methanol and centrifuged again and the solid dried to give Compound 231 as an off-white solid (0.344 g, 43%).
[1011] .sup.1H NMR (400 MHz, CDCl.sub.3/methanol-d.sub.4) m. 7.12-6.85 (9H, ArH), s. 4.32 (6H, ArCH.sub.2NH), m. 3.50-3.40 (42H, CH.sub.2CH.sub.2O, and OCH.sub.2CH.sub.2N.sub.3), t. 3.20 (6H, OCH.sub.2CH.sub.2N.sub.3), m.br. 2.66 (6H, CH.sub.2), s.br. 1.09 (9H, CH.sub.3).
##STR00194##
[1012] Dissolved Compound 231 (226 mg, 163 mmol) in dry DMSO (1 mL) and diluted with dry pyridine (70 mL). Stirred under nitrogen at 40 C. and added a solution of Compound 5a (60 mg, 183 mmol) in dry DCM (2 mL) by syringe pump over 6 hours, then left stirring at 40 C. for a further 60 hours. Evaporation of the solvent on a rotary evaporator gave an orange coloured gum which was dissolved in methanol containing a little water. This was loaded onto a Biotage 120 g reverse phase column and eluted with a water/methanol gradient. The compound which eluted at about 80% methanol was collected, evaporated, redissolved in methanol, concentrated by boiling off the solvent and the hot solution (3 mL) was left to cool overnight to give the product (Compound 232) as clumps of white crystals (83 mg, 30%).
[1013] .sup.1H NMR (400 MHz, D.sub.2O) s. 8.09 (3H, ArH), d. 7.98 (3H, ArH), d. 7.58 (3H, ArH), m. 4.41-4.45 (12H, ArCH.sub.2NH), m. 3.5-3.7 (42H, OCH.sub.2CH.sub.2), m. 2.7-2.9 (12H, ArCH.sub.2CH.sub.3), m. 1.20 (18H, ArCH.sub.2CH.sub.3).
[1014] HRMS: (ESI.sup.+) calculated for C.sub.81H.sub.116N.sub.24O.sub.18.sup.2+[M+2H].sup.2+ 856.4449, found: 856.4470.
##STR00195##
[1015] Compound 232 (22 mg, 0.013 mmol) was dissolved in a mixture of warm methanol (2 mL) and water (0.1 mL) and to this was added triphenylphosphine (26 mg, 0.099 mmol) under an atmosphere of nitrogen. The reaction mixture was then heated for 16 hours at 60 C. before allowing to cool to room temperature. The cloudy mixture was diluted with more methanol and water, followed by 40 L of 1 M aqueous hydrochloric (until it was acidic). Added more water then extracted it a couple of times with DCM to remove the triphenylphosphine based compounds. The slightly cloudy aqueous layer was passed through a bond-elut (500 mg) several times until the eluant emerged clear and tic showed that no product was passing through. Then eluted with water 6 mL, then 24 mL of 25% MeOH in water, then 44 mL of 50%, 24 mL of 75% MeOH in water. Tlc showed that the product had eluted in the 25% to 75% methanol fractions which were combined and evaporated before being redissolved in water and freeze-dried to give Compound 233 (19 mg, 89%) as an off-white solid.
[1016] .sup.1H NMR (400 MHz, D.sub.2O) s. 7.88 (3H, ArH), d. 7.71 (3H, ArH), d. 7.43 (3H, ArH), s.br. 4.20 (12H, ArCH.sub.2NH), m. 3.4-3.6 (42H, OCH.sub.2CH.sub.2, and OCH.sub.2CH.sub.2NH.sub.2), t. 3.00 (6H, OCH.sub.2CH.sub.2NH.sub.2), m. 2.48 (12H, ArCH.sub.2CH.sub.3), m. 0.96 (18H, ArCH.sub.2CH.sub.3).
[1017] HRMS: (MALDI.sup.+) calculated for C.sub.81H.sub.120N.sub.18O.sub.18Na.sup.+[M+Na].sup.+ 1655.8920, found: 1655.8932.
Hexa-Carboxylate Macrocycle (Compound H8Receptor 11)
[1018] ##STR00196## ##STR00197##
N-(4, 5-dimethyl-2-nitrophenyl) acetamide (Compound H2)
[1019] ##STR00198##
[1020] 4,5-dimethyl-2-nitroaniline (Compound H1, 2 g, 12 mmol) was suspended in glacial acetic acid (24 mL) and heated to 90 C. Acetic anhydride (1.2 mL, 13 mmol) was added and the mixture stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature and poured into water (300 mL). The yellow precipitate was filtered, washed with water and recrystallized from ethanol to yield Compound H2 (2.4 g, 11.6 mmol, 97%) as a yellow crystalline solid.
[1021] .sup.1H NMR: (400 MHz, (CDCl.sub.3): 2.25 (s, 6H, C(9, 10)H.sub.3), 2.32 (s, 3H, C(1)H.sub.3), 7.93 (s, 1H, C(8)H), 8.51 (s, 1H, C(5)H), 10.26 (br s, 1H, NH).
[1022] .sup.13C NMR: (100 MHz, (CDCl.sub.3): 19.1 (C10), 20.5 (C9), 25.6 (C1), 122.6 (C8), 125.9 (C5), 132.3 (C7), 132.7 (C6), 134.1 (C3), 146.8 (C4), 168.9 (C(2)O); v.sub.max 3341, 2987, 2901, 1708, 1695, 1576, 1323, 1151, 759 cm.sup.1.
[1023] HRMS: (ESI.sup.+) Found [M+Na].sup.+: 231.0745.
4-amino-5-nitrophthalic acid (Compound H3)
[1024] ##STR00199##
[1025] Under an inert N.sub.2 atmosphere, Compound H2 (2 g, 9.6 mmol) and KMnO.sub.4 (6 g, 37.9 mmol)) was suspended in water (50 mL) and stirred at reflux for 3 days. Additional KMnO.sub.4 (3 g, 19 mmol) was added halfway through the reaction time. The resultant brown precipitate was filtered hot and washed with water. The yellow filtrate was acidified to pH 3 with 1M HCl, extracted with EtOAc (3100 mL), washed with brine (100 mL) and dried (MgSO.sub.4). The solvent was removed under vacuum to yield Compound H3 (0.77 g, 2.88 mmol, 60%) as a yellow orange solid.
[1026] .sup.1H NMR: (400 MHz, (CDCl.sub.3): 7.25 (s, 1H, C(6)H), 7.63 (br s, 2H, NH.sub.2), 8.70 (s, 1H, C(3)H), 11.50 (br s, 2H, C(7, 8)O.sub.2H).
[1027] .sup.13C NMR: (100 MHz, (CDCl.sub.3): 117.7 (C4), 118.7 (C6), 129.3 (C3), 132.6 (C5), 141.5 (C2), 146.9 (C1), 167.8 (C7), 169.0 (C8); v.sub.max 3486, 3364, 2972, 2901, 1712, 1681, 1626, 1502, 1252, 1057, 882 cm.sup.1.
[1028] LRMS: (EI) Found [M].sup.+: 226.1.
dimethyl 4-amino-5-nitrophthalate (Compound H4)
[1029] ##STR00200##
[1030] Compound H3 (0.8 g, 3.5 mmol) was dissolved in MeOH (30 mL) and concentrated H.sub.2SO.sub.4 (0.5 mL) added. The reaction mixture was stirred at reflux for 3 hours and then the solvent was removed under vacuum. The residue was dissolved in EtOAc (60 mL), washed with 5% NaHCO.sub.3 (60 mL), brine (60 mL) and dried (MgSO.sub.4). The solvent was removed under vacuum and the crude solid purified by flash column chromatography (100% CH.sub.2Cl.sub.2) to yield Compound H4 (0.72 g, 2.8 mmol, 80%) as an orange solid.
[1031] .sup.1H NMR: (400 MHz, (CDCl.sub.3): 3.88 (s, 3H, C(10)H.sub.3), 3.92 (s, 3H, C(9)H.sub.3), 6.91 (s, 1H, C(6)H), 7.26 (br s, 2H, NH.sub.2), 8.74 (s, 1H, C(3)H).
[1032] .sup.13C NMR: (100 MHz, (CDCl.sub.3): 52.5 (C9), 53.1 (C10), 116.9 (C4), 118.3 (C6), 129.7 (C3), 131.2 (C5), 140.7 (C2), 146.6 (C1), 164.7 (C7), 168.0 (C8); v.sub.max 3486, 3342, 2987, 2901, 1736, 1697, 1621, 1502, 1435, 1339, 1250, 1027, 762 cm.sup.1.
[1033] LRMS: (ESI) Found [M+Na].sup.+: 277.1.
dimethyl 4-amino-5-nitrophthalate (Compound H5)
[1034] ##STR00201##
[1035] Under an inert N.sub.2 atmosphere, a solution of Compound H4 (0.1 g, 0.39 mmol) in MeOH (10 mL) was added to Pd/C (10 mg). The reaction vessel was then purged with hydrogen (1 atm) and the reaction mixture stirred at room temperature for 1 hour. After which, the reaction mixture was filtered through celite and washed with CH.sub.2Cl.sub.2, and the filtrate concentrated under vacuum. The crude product was then purified by flash column chromatography (5% MeOH:CH.sub.2Cl.sub.2) to afford Compound H5 (81 mg, 0.36 mmol, 93%) as light brown solid.
[1036] .sup.1H NMR: (400 MHz, (CDCl.sub.3): 3.84 (s, 6H, C(5)H.sub.3), 7.02 (s, 2H, C(2)H).
[1037] .sup.13C NMR: (100 MHz, (CDCl.sub.3): 52.4 (C5), 116.6 (C2), 124.1 (C3), 1136.6 (C1), 168.4 (C4).
[1038] HRMS: (ESI.sup.+) Found [M+Na].sup.+: 247.0685.
Hexa-Ester Amino Half Receptor (Compound H6)
[1039] ##STR00202##
[1040] Under an inert N.sub.2 atmosphere, Compound H5 (80 mg, 0.36 mmol) was dissolved in dry pyridine (10 mL) and heated to 40 C. A solution of TEB isocyanate (Compound 103, 20 mg, 0.06 mmol) in dry CH.sub.2Cl.sub.2 (2 mL) was added over 1 hour and the reaction stirred at 40 C. for 16 hours. The reaction mixture was concentrated under vacuum and residual pyridine co-evaporated with toluene (330 mL). The crude product was then suspended in CH.sub.2Cl.sub.2, filtered and air dried to afford Compound H6 (49 mg, 0.049 mmol, 82%) as a light brown solid.
[1041] .sup.1H NMR: (400 MHz, ((CD.sub.3).sub.2SO): 1.19 (t, J=7.2 Hz, 9H, C(1)H.sub.3), 2.79 (br q, 6H, C(2)H.sub.2), 3.72, 3.73 (s, 29H, C(14, 16)H.sub.3), 4.36 (s, 6H, C(5)H.sub.2), 5.87 (br s, 6H, NH.sub.2), 6.51 (br t, 3H, NHC(5)), 6.84 (s, 3H, C(11)H), 8.13 (s, 3H, C(8)H), 8.60 (s, 3H, NH).
[1042] .sup.13C NMR: (100 MHz, ((CD.sub.3).sub.2SO): 16.9 (C1), 22.8 (C2), 37.7 (C5), 52.2, 52.5 (C14 and C16), 114.0 (C11), 117.3 (C9), 121.7 (C8), 126.9 (C7), 128.6 (C10), 133.2 (C4), 142.2 (C12), 143.4 (C4), 155.5 (C6), 167.3, 169.2 (C9).
[1043] HRMS: (ESI.sup.+) Found [M+H].sup.+: 1000.4041.
Hexa-Ester Hexa-Urea Macrocycle (Compound H7)
[1044] ##STR00203##
[1045] Method A:
[1046] Under an inert N.sub.2 atmosphere, Compound H5 (40 mg, 0.18 mmol) was dissolved in dry pyridine (100 mL) and heated to 40 C. A solution of TEB isocyanate (Compound 103, 38 mg, 0.12 mmol) in dry CH.sub.2Cl.sub.2 (2 mL) was added over 1 hour and the reaction stirred at 40 C. for 16 hours. The reaction mixture was concentrated under vacuum and residual pyridine co-evaporated with toluene (330 mL). The crude product was then purified by reverse phase HPLC (100% water.fwdarw.100% acetonitrile) to afford Compound H7 (9.5 mg, 0.007 mmol, 12%) as a white solid.
[1047] Method B:
[1048] Under an inert N.sub.2 atmosphere, Compound H6 (20 mg, 0.02 mmol) was dissolved in dry pyridine (20 mL) and heated to 40 C. A solution of TEB isocyanate (Compound 103, 7.8 mg, 0.024 mmol) in dry CH.sub.2Cl.sub.2 (2 mL) was added and the reaction stirred at 40 C. for 16 hours. The reaction mixture was concentrated under vacuum and residual pyridine co-evaporated with toluene (330 mL). The crude product was then purified by reverse phase HPLC (100% water.fwdarw.100% acetonitrile) to afford Compound H7 (8 mg, 0.006 mmol, 31%) as a white solid.
[1049] .sup.1H NMR: (400 MHz, (CD.sub.3OD): 1.22 (t, J=7.4 Hz, 18H, C(1)H.sub.3), 2.79 (q, J=7.4 Hz, 12H, C(2)H.sub.2), 3.86 (s, 18H, C(11)H.sub.3), 4.42 (s, 12H, C(5)H.sub.2), 8.34 (s, 6H, C(8)H).
[1050] .sup.13C NMR: (100 MHz, (CD.sub.3OD): 15.3 (C1), 22.4 (C2), 37.5 (C5), 51.5 (C11), 121.6 (C8), 126.3 (C7), 131.6 (C3), 131.9 (C9), 143.2 (C4), 155.6 (C6), 168.1 (C10).
[1051] HRMS: (ESI.sup.+) Found [M+H].sup.+: 1327.5635.
Hexa-Carboxylate Hexa-Urea Macrocycle (Compound H8Receptor 11)
[1052] ##STR00204##
[1053] Compound H7 (8 mg, 0.006 mmol) was dissolved in MeOH (4 mL) and then NaOH (5 M, 1 mL) added dropwise. The solution was stirred at 40 C. for 1 hour and the reaction diluted with water (5 mL). The MeOH was removed under vacuum and the aqueous solution neutralised to pH 7.4 with acidic ion exchange resin, filtered and freeze dried to afford Compound H8 (7.8 mg, 0.0056 mmol, 93%) as a white solid.
[1054] .sup.1H NMR: (600 MHz, D.sub.2O): 1.19 (t, J=7.4 Hz, 18H, C(1)H.sub.3), 2.75 (br q, 12H, C(2)H.sub.2), 4.47 (s, 12H, C(5)H.sub.2), 7.73 (s, 6H, C(8)H).
[1055] .sup.13C NMR: (100 MHz, D.sub.2O): 15.4 (C1), 22.5 (C2), 37.6 (C5), 124.6 (C8), 128.3 (C7), 131.8 (C3), 134.9 (C9), 143.3 (C4), 157.4 (C6), 176.4 (C10).
##STR00205##
[1056] Compound 15 (2.000 g, 3.363 mmol) was dissolved in THF (145.0 mL) and mixed with triphenylphosphine (0.838 g, 3.195 mmol). The reaction was stirred at room temperature for 24 h. Water (75 mL) was added and the reaction heated at 50 C. for 3 h. After cooling the reaction mixture was diluted with water and extracted with EtOAc (3100 mL). Combined organic layers were concentrated in vacuo and the crude residue purified by reverse phase MPLC on a C18 SNAP Ultra 120 g cartridge eluting (40% acetone:water to 60% acetone:water) to give Compound 234 as a white solid (1.510 g, 2.655 mmol, 79%).
[1057] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.57 (s, 1H, NH), 3.86 (dt, J=5.8, 4.0 Hz, 1H, NCH.sub.2), 3.73 (dd, J=11.2, 5.8 Hz, 1H, NCH.sub.2), 3.59 (dd, J=10.6, 6.1 Hz, 1H, NCH.sub.2), 3.41-3.32 (m, 2H, CHN.sub.3, CHNH.sub.2), 3.13 (dd, J=10.6, 4.1 Hz, 1H NCH.sub.2), 2.22 (m, 6H, CH.sub.2C(O)), 1.95 (m, 6H, CCH.sub.2), 1.45 (s, 27H, C(CH.sub.3).sub.3).
[1058] .sup.13C NMR (100 MHz, CD.sub.3OD) 174.9 (s, CCO.sub.2C), 157.8 (s, NC(O)N), 81.7 (s, CO.sub.2C(CH.sub.3).sub.3), 67.2 (s, CNH.sub.2), 58.3 (CN.sub.3), 56.8 (s, CH.sub.2N), 52.5 (CH.sub.2N), 31.2 (s, CH.sub.2C(O)), 30.8 (s, CCH.sub.2), 28.4 (s, CO.sub.2C(CH.sub.3).sub.3).
##STR00206##
[1059] Compound 234 (67 mg, 0.118 mmol) and Compound 103 (11 mg, 0.034 mmol) were dissolved in DCM (1 mL) and stirred together for 18 h. The solvent was removed in vacuo and the residue purified by reverse phase MPLC on a C18 SNAP Ultra 120 g cartridge eluting 70% acetone:water to 100% acetone:water to give Compound 235 as a white solid (60 mg, 0.030 mmol, 89%).
[1060] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.39 (s, 6H, ArCH.sub.2NH), 4.24 (dt, J=6.7, 4.7 Hz, 3H, CHNH), 4.05 (dt, J=6.0, 4.5 Hz, 3H, NCH.sub.2), 3.68 (dd, J=10.8, 6.7 Hz, 3H, NCH.sub.2), 3.61 (dd, J=11.3, 6.0 Hz, 3H, NCH.sub.2), 3.32 (m, 3H, CHN.sub.3), 3.17 (dd, J=10.8, 4.6 Hz, 3H NCH.sub.2), 2.78 (q, J=7.4 Hz, 6H, ArCH.sub.2CH.sub.3), 2.21 (dd, J=9.3, 6.6 Hz, 18H, CH.sub.2C(O)), 1.94 (dd, J=9.3, 6.6 Hz, 18H, CCH.sub.2), 1.44 (s, 81H, C(CH.sub.3).sub.3), 1.18 (t, J=7.4 Hz, 9H, ArCH.sub.2CH.sub.3).
[1061] .sup.13C NMR (100 MHz, CD.sub.3OD) 174.9 (s, CCO.sub.2C), 157.8, 152.4 (s, NC(O)N), 137.5, 133.7 (Ar), 81.8 (s, CO.sub.2C(CH.sub.3).sub.3), 69.1 (s, CHNH), 61.5 (CN.sub.3), 58.5 (s, CH.sub.2N), 51.0 (CH.sub.2N), 39.3 (ArCH.sub.2NH), 31.2 (s, CH.sub.2C(O)), 30.9 (s, CCH.sub.2), 28.4 (s, CO.sub.2C(CH.sub.3).sub.3), 22.2 (s, ArCH.sub.2CH.sub.3), 15.6 (s, ArCH.sub.2CH.sub.3).
##STR00207##
[1062] To a solution of Compound 235 (100 mg, 0.049 mmol) in MeOH (15 mL) was added a slurry of Pd/C (30 mg) in DCM. The reaction was placed under a hydrogen atmosphere and left to stir overnight. Filtration through Celite and concentration of the filtrate gave a white solid (91 mg, 0.047 mmol, 96%).
[1063] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.46-4.33 (m, 6H, ArCH.sub.2NH), 4.25 (d, J=6.9 Hz, 3H, CHNH), 3.88-3.74 (m, 6H, NCH.sub.2), 3.60 (d, J=6.4 Hz, 3H, NCH.sub.2), 3.33-3.27 (m, 3H, CHNH.sub.2), 3.20 (dd, J=10.4, 7.0z Hz, 3H, NCH.sub.2), 2.78 (q, J=8.2 Hz, 6H, ArCH.sub.2CH.sub.3), 2.30-2.14 (m, 18H, CH.sub.2C(O)), 2.04-1.85 (m, 18H, CCH.sub.2), 1.44 (s, 81H, C(CH.sub.3).sub.3), 1.19 (t, J=7.4 Hz, 9H, ArCH.sub.2CH.sub.3).
[1064] .sup.13C NMR (100 MHz, CD.sub.3OD) 174.8 (s, CCO.sub.2C), 157.9, 151.5 (s, NC(O)N), 137.2, 132.0 (Ar), 81.7 (s, CO.sub.2C(CH.sub.3).sub.3), 68.1 (s, CHNH), 67.5 (CNH.sub.2), 58.2 (s, CH.sub.2N), 54.8 (CH.sub.2N), 43.6 (ArCH.sub.2NH), 31.2 (s, CH.sub.2C(O)), 30.8 (s, CCH.sub.2), 28.4 (s, CO.sub.2C(CH.sub.3).sub.3), 22.3 (s, ArCH.sub.2CH.sub.3), 16.9 (s, ArCH.sub.2CH.sub.3).
##STR00208##
[1065] A mixture of Compound 236 (110 mg, 0.056 mmol), n-octyl glucoside (33 mg, 0.112 mmol) and DMAP (21 mg, 0.168 mmol) were azeotroped to dryness with toluene in a two-necked flask and then placed under N.sub.2. The residue was then dissolved in DCM (110 mL) and cooled to 0 C. A solution of TEB NCO (Compound 103, 18 mg, 0.056 mmol) in DCM (20 mL) was added. The reaction mixture was heated to 35 C. for 16 h. Solvent was removed under reduced pressure, and the crude product purified by reverse phase MPLC on a C18 SNAP Ultra 60 g cartridge eluting 70% acetone:water to 100% acetone:water (46 mg, 0.020 mmol, 36%).
[1066] .sup.1H NMR (500 MHz, CD.sub.3OD) 4.79-4.33 (azeotroped, 6H, ArCH.sub.2NH), 4.31-4.10 (m, 6H, ArCH.sub.2NH), 3.97-3.80 (m, 6H, CHNH), 3.73-3.37 (m, 9H, NCH.sub.2) 2.96-2.56 (m, 9H, NCH.sub.2, ArCH.sub.2CH.sub.3), 2.35-2.20 (m, 24H, ArCH.sub.2CH.sub.3, CH.sub.2C(O)), 2.10-1.87 (m, 18H, CCH.sub.2), 1.48-1.40 (m, 81H, C(CH.sub.3).sub.3), 1.24-1.10 (m, 18H, ArCH.sub.2CH.sub.3).
[1067] .sup.13C NMR (125 MHz, (CD.sub.3).sub.2SO) 172.5 (s, CCO.sub.2C), 157.6, 155.4 (s, NC(O)N), 141.6, 133.9 (Ar), 79.7 (s, CO.sub.2C(CH.sub.3).sub.3), 56.2 (NHC(CH.sub.2).sub.3), 52.7 (s, CHNH), 50.2 (s, CH.sub.2N), 36.6 (ArCH.sub.2NH), 29.4 (s, CH.sub.2C(O)), 29.3 (s, CCH.sub.2), 27.8 (s, CO.sub.2C(CH.sub.3).sub.3), 22.0 (s, ArCH.sub.2CH.sub.3), 16.3 (s, ArCH.sub.2CH.sub.3).
[1068] MS: (ESI.sup.+) calculated for C.sub.117H.sub.195N.sub.18O.sub.27.sup.2+: 1141.7180, found [M+2H].sup.2+: 1141.7196
##STR00209##
[1069] Compound 237 (8 mg, 3.504 mol) dissolved in TFA (1.3 mL), and heated to 30 C. with stirring for 20 hours. The reaction mixture was allowed to cool, whereupon pentane (25 mL) was added. The resultant suspension was centrifuged, and the supernatant removed. The residual oil was dissolved in 0.2 M aq. NaHCO.sub.3 solution (3.5 mL) and this solution was desalted by 20 mL column of G-25 sephadex. The resultant solution was freeze dried to give Compound 238 as a white solid (6.5 mg, 3.290 mmol, 94%).
[1070] .sup.1H NMR (500 MHz, D.sub.2O) 4.66-4.50 (m, 3H, ArCH.sub.2NH), 4.27-4.03 (m, 9H, ArCH.sub.2NH), 4.02-3.82 (m, 3H, CHNH), 3.79-3.34 (m, 12H, CHNH, NCH.sub.2), 3.09-2.75 (m, 6H, ArCH.sub.2CH.sub.3), 2.75-2.27 (m, 9H, NCH.sub.2, ArCH.sub.2CH.sub.3), 2.26-2.13 (m, 18H, CH.sub.2C(O)), 2.03-1.89 (m, 18H, CCH2), 1.23-1.09 (m, 18H, ArCH.sub.2CH.sub.3).
[1071] .sup.13C NMR (125 MHz, D.sub.2O) 183.4 (s, CCO.sub.2), 160.2, 159.7, 159.3 (NHC(O)NH), 157.6 (s, NHC(O)N), 144.9, 144.2, 144.1 (Ar), 58.1 (NHC(CH.sub.2).sub.3), 58.0, 57.9 (s, CHNH), 49.1, 48.4, 47.7 (s, CH.sub.2N), 39.8, 38.8, 37.6 (ArCH.sub.2NH), 32.2, 32.1 (s, CH.sub.2C(O)), 31.7 (s, CCH.sub.2), 23.5, 22.7, 22.1 (s, ArCH.sub.2CH.sub.3), 16.1, 15.9, 15.5 (s, ArCH.sub.2CH.sub.3).
[1072] MS: (ESI.sup.+) calculated for C.sub.117H.sub.195N.sub.18O.sub.27.sup.2+: 1141.7180, found [M+2H].sup.2+: 1141.7196
##STR00210##
[1073] A 1 L flask, with a side-arm gas adaptor, equipped with a magnetic stirrer was dried under vacuum using a heat-gun. The flask was cooled to RT and charged with G2 amine (Compound 82, 2.50 g, 1.74 mmol) under flowing nitrogen. THF (100 mL) and Et.sub.3N (0.25 mL, 1.80 mmol) were added, and the flask cooled to 0 C. with an ice bath. A solution of triphosgene (0.26 g, 1.00 mmol) in THF (25 mL) was added dropwise over the course of 20 min. After 3 h, the solvent was removed under vacuum and the resultant residue taken up in chloroform (50 mL) and washed with water (50 mL). The organic layer was dried (MgSO.sub.4) and concentrated under vacuum to give Compound 239 as a white foam (2.50 g, 1.71 mmol, 98%).
[1074] .sup.1H NMR (400 MHz, toluene-d.sub.8) 2.26 (m, 18H, CH.sub.2), 2.07 (m, 30H, CH.sub.2), 1.40 (s, 81H, CH.sub.3).
[1075] .sup.13C NMR (100 MHz, toluene-d.sub.8) 173.3 (CCO.sub.2C), 172.4 (CONH), 123.7 (NCO), 80.4 (C(CH.sub.3).sub.3), 58.2 (NHC(CH.sub.2).sub.3), 53.7 (C(NCO)), 30.7 (CH.sub.2), 30.5 (CH.sub.2), 28.7 (CH.sub.2), 28.5 (CH.sub.3).
##STR00211##
[1076] A Schlenk flash was charged with (3S,4S)-pyrrolidine-3,4-diol (185.0 mg, 1.790 mmol), G2 NCO (2.500 g, 2.000 mmol) and anhydrous DMF (100 mL) under N.sub.2. The solution was left to stir for 16 hours, then poured into 5% aq. LiCl (700 mL) and extracted with EtOAc (300 mL). The organic layer was separated, dried and concentrated to a gummy solid (2.760 g, 1.759 mmol, 88%).
[1077] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.24 (s, 3H, C(O)NH), 6.13 (2H, s, OH), 4.19 (s, 1H, NC(O)NH), 3.63 (dd, J=11.5, 3.7 Hz, 2H, CHOH), 3.54-3.37 (m, 4H, NCH.sub.2), 2.17 (dd, J=10.3, 6.4 Hz, 24H, CH.sub.2C(O)), 1.92 (dd, J=9.9, 6.4 Hz, 24H, CCH.sub.2), 1.41 (s, 81H, C(CH.sub.3).sub.3).
[1078] .sup.13C NMR (100 MHz, CDCl.sub.3) 173.4 (C(O)NH), 172.9 (CCO.sub.2C), 162.6 (s, NC(O)N), 80.8 (s, CO.sub.2C(CH.sub.3).sub.3), 80.7 (s, COH), 57.4 (s, CH.sub.2N), 30.1 (s, CH.sub.2C(O)), 30.0 (s, CH.sub.2C(O)NH), 29.9 (s, CCH.sub.2), 29.9 (CCH.sub.2), 28.2 (s, CO.sub.2C(CH.sub.3).sub.3).
##STR00212##
[1079] Compound 240 (1.851 g 1.180 mmol) was dissolved in DCM (13.9 mL), and triethylamine (0.66 mL, 4.719 mmol) was added. The reaction was cooled to 0 C. and mesyl chloride (0.20 mL, 2.595 mmol) was added dropwise. The reaction was stirred at room temperature for 16 h, before being washed with 5% aq. KHSO.sub.4, sat. aq. NaHCO.sub.3 and brine. The organic layer was concentrated in vacuo to give Compound 241 as a white foam (1.350 g, 0.783 mmol, 66%).
[1080] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.95 (s, 1H, NC(O)NH), 6.17 (s, 3H, C(O)NH), 5.32-5.13 (m, 2H, CHOSO.sub.2), 3.94-3.56 (m, 4H, NCH.sub.2), 3.13 (s, J=1.6 Hz, 2H, SO.sub.2CH.sub.3), 2.31-2.11 (m, 24H, CH.sub.2C(O)), 2.04-1.86 (m, 24H, CCH.sub.2), 1.41 (s, 81H, C(CH.sub.3).sub.3).
[1081] .sup.13C NMR (100 MHz, CDCl.sub.3) 173.3 (C(O)NH), 172.7 (CCO.sub.2C), 156.3 (s, NC(O)N), 80.6 (s, CO.sub.2C(CH.sub.3).sub.3), 80.1 (s, COSO.sub.2), 57.4 (s, CH.sub.2N), 38.6 (SO.sub.2CH.sub.3), 29.9 (s, CH.sub.2C(O)), 29.9 (s, CH.sub.2C(O)NH), 29.8 (s, CCH.sub.2), 29.8 (CCH.sub.2), 28.1 (s, CO.sub.2C(CH.sub.3).sub.3).
##STR00213##
[1082] NaN.sub.3 (0.131 g, 2.017 mmol) was added to a solution of Compound 241 (1.160 g, 0.672 mmol) in DMF (3.4 mL) at 0 C. The reaction was then heated at 100 C. for 16 h. The reaction was cooled and diluted with EtOAc (30 mL) and washed with water (30 mL), 5% aq. LiCl (230 mL) and brine (30 mL). The organic layer was concentrated in vacuo and the residue purified by reverse phase MPLC on a C18 SNAP Ultra 60 g cartridge eluting (70% acetone:water to 100% acetone:water) to give Compound 242 as a white solid (788 mg, 0.487 mmol, 72%).
[1083] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.29-4.18 (m, 2H, CHN.sub.3), 3.84-3.70 (m, 2H, NCH.sub.2), 3.55-3.44 (m, 2H, NCH.sub.2), 2.32-2.19 (m, 24H, CH.sub.2C(O)), 2.06-1.98 (m, 24H, CCH.sub.2), 1.52 (s, 81H, C(CH.sub.3).sub.3)
[1084] .sup.13C NMR (100 MHz, CD.sub.3OD) 175.8 (C(O)NH), 174.4 (CCO.sub.2C), 158.1 (s, NC(O)N), 81.7 (s, CO.sub.2C(CH.sub.3).sub.3), 65.1 (s, CH.sub.2N.sub.3), 62.2 (NC(O)NHC(CH.sub.2).sub.3), 61.5 (NHC(CH.sub.2).sub.3), 58z.8 (CH.sub.2N), 30.7 (s, CH.sub.2C(O)), 30.7 (s, CH.sub.2C(O)NH), 30.5 (s, CCH.sub.2), 30.5 (CCH.sub.2), 28.4 (s, CO.sub.2C(CH.sub.3).sub.3).
##STR00214##
[1085] Compound 241 (300 mg, 0.185 mmol) was dissolved in THF (8.0 mL). Triphenylphosphine (46 mg, 0.176 mmol) was added and the reaction stirred for 18 h. Water (4.0 mL) was added and the reaction heated at 50 C. for 6 h. The reaction mixture was concentrated in vacuo and the residue purified by reverse phase MPLC on a C18 SNAP Ultra 60 g cartridge eluting (75% acetone:water to 100% acetone:water) to give a Compound 243 as a white solid (187 mg, 0.117 mmol, 66%).
[1086] .sup.1H NMR (400 MHz, CD.sub.3OD) 3.92-3.72 (m, J=5.8, 4.0 Hz, 3H, NCH.sub.2), 3.61 (dd, J=10.7, 6.0 Hz, 1H, CHN.sub.3), 3.41-3.32 (m, 1H, CHNH.sub.2), 3.24-3.12 (m, 1H NCH.sub.2), 2.23-2.16 (m, 24H, CH.sub.2C(O)), 1.99-1.91 (m, 24H, CCH.sub.2), 1.45 (s, 81H, C(CH.sub.3).sub.3).
[1087] .sup.13C NMR (100 MHz, CDCl.sub.3) 173.3 (C(O)NH), 172.7 (CCO.sub.2C), 158.2 (s, NC(O)N), 81.6 (s, CO.sub.2C(CH.sub.3).sub.3), 58.7 (CNH.sub.2), 58.6 (s, CH.sub.2N), 58.4 (CN.sub.3), 33.1 (s, CH.sub.2C(O)), 32.3 (s, CH.sub.2C(O)NH), 30.7 (s, CCH.sub.2), 30.5 (CCH.sub.2), 28.4 (s, CO.sub.2C(CH.sub.3).sub.3).
##STR00215##
[1088] Compound 7b (80 mg, 0.016 mmol) was azeotroped with toluene to dryness in reaction flask and then redissolved in pyridine (8.0 mL). This was heated to 40 C. and a solution of Compound 5a (6.2 mg, 0.019 mmol) in DCM (0.7 mL) was added by syringe pump over 3 h. The reaction was then cooled to room temperature and stirred for a further 16 h. The reaction was concentrated under vacuum and the crude residue obtained was then purified by reverse phase flash chromatography on a 30 g SNAP Ultra C18 cartridge elution (1CV 80% acetone/H.sub.2O, 10 CV 80-95% acetone/H.sub.2O, 2CV 100% acetone) to give Compound 244 as a white solid (55 mg, 0.010 mmol, 64%).
[1089] .sup.1H NMR (500 MHz, methanol-d.sub.4) .sup.zd. 8.12 (3H, J=2.1 Hz, Ar), dd. 7.56 (3H, J=8.4, 2.1 Hz, Ar), d. 7.41 (3H, J=8.4 Hz), s. 4.58 (6H, ArCH.sub.2NH), s. 4.45 (6H, ArCH.sub.2NH), s. 3.85 (9H, ArOCH.sub.3), m. 2.82-2.72 (6H, ArCH.sub.2CH.sub.3), m. 2.35-1.89 (144H, NHCH.sub.2CH.sub.2C(O)), s. 1.43 (243H, CO.sub.2C(CH.sub.3).sub.3), t. 1.20 (9H, J=7.3 Hz, ArCH.sub.2CH.sub.3).
[1090] .sup.13C NMR (125 MHz, methanol-d.sub.4) 175.5 (CONH), 174.4 (CO.sub.2C(CH.sub.3).sub.3), 171.1 (ArCONHR), 81.6 (CO.sub.2C(CH.sub.3).sub.3), 59.3 (ArOCH.sub.3), 58.7 (C(CH.sub.2CH.sub.2CO.sub.2).sub.3), 54.6 (C(CH.sub.2CH.sub.2CONH).sub.3), 44.2, 43.7 (ArCH.sub.2NHC(O)NH), 32.2 (CH.sub.2CH.sub.2CONH, CH.sub.2CH.sub.2CONH), 30.7 (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 30.5, (CH.sub.2CH.sub.2CO.sub.2C(CH.sub.3).sub.3), 28.5 (CO.sub.2C(CH.sub.3).sub.3), 16.9 (ArCH.sub.2CH.sub.3).
[1091] MS: (ESI) calculated for C.sub.282H.sub.456N.sub.24O.sub.75.sup.3+: 1795.0979, found [M+3H].sup.3+: 1795.0938.
##STR00216##
[1092] Compound 244 (8.5 mg, 0.002 mmol) was dissolved in DCM (0.5 mL) and formic acid (0.5 mL, 13.3 mmol) was added. After 24 h the reaction mixture was added dropwise into stirring water (20 mL) and the white precipitate collected by centrifuging. The supernatant was decanted off, and the white solid was neutralised using 10 mM NaOH solution to pH 7. The resulting solution was desalted by 10 mL column of G-25 sephadex. The resultant solution was freeze dried to give a white solid (1 mg, 0.32 mol, 16%).
[1093] .sup.1H NMR (500 MHz, CD.sub.2Cl.sub.2/formic acid-d.sub.2, 1:1) .sup.1H NMR (500 MHz, methanol-d.sub.4) s. 8.18 (3H, Ar), s. 7.50 (6H, Ar), s. 4.48 (6H, ArCH.sub.2NH), s. 4.41 (6H, ArCH.sub.2NH), s. 3.83 (9H, ArOCH.sub.3), m. 2.69-2.60 (6H, ArCH.sub.2CH.sub.3), m. 2.45-1.81 (144H, NHCH.sub.2CH.sub.2C(O)), m. 1.16-1.08 (9H, ArCH.sub.2CH.sub.3).
##STR00217##
[1094] Prepared in a manner analogous to Compound 244 from Compounds 7a (202.0 mg, 0.040 mmol) and 5c (11.4 mg, 0.040 mmol). Purified by reverse phase flash chromatography on a 120 g SNAP Ultra C18 cartridge elution (1CV 80% acetone/H.sub.2O, 10 CV 80-97% acetone/H.sub.2O, 4CV 97% acetone) to give Compound 246 as a white solid (108.0 mg, 50.6%).
[1095] .sup.1H NMR (500 MHz, methanol-d.sub.4) m. 8.08-7.58 (9H, Ar), m. 4.63-4.30 (12H, ArCH.sub.2NH), m. 2.95-2.60 (9H ArCH.sub.3), m. 2.32-1.84 (150H, NHCH.sub.2CH.sub.2C(O), ArCH.sub.2CH.sub.3), s. 1.43 (243H, CO.sub.2C(CH.sub.3).sub.3), s. 1.29 (9H, ArCH.sub.2CH.sub.3).
##STR00218##
[1096] Compound 246 (108 mg, 0.02 mmol) was dissolved in anhydrous DCM (Vol: 20 mL) and TFA (4.8 mL, 60.0 mmol)_at RT. The resulting off yellow solution was stirred for 12 h at room temperature. Volatiles were removed under vacuum to give a yellow solid. The solid which was purified by reverse phase MPLC on a C18 SNAP Ultra 60 g cartridge by loading the sample in 1:1 MeOH/H.sub.2O+0.1% formic acid). The resulting white solid was neutralised using 100 mM NaOH solution to pH 7 and the resulting solution concentrated to dryness under vacuum. White crystalline solid (30 mg, 0.008 mmol, 40%).
[1097] .sup.1H NMR (500 MHz, methanol-d.sub.4) m. 8.37-7.81 (9H, Ar), m. 4.57-4.22 (12H, ArCH.sub.2NH), m. 2.85-1.66 (159H, ArCH.sub.3, NHCH.sub.2CH.sub.2C(O), ArCH.sub.2CH.sub.3), t. 1.25 (9H, J=9.2 Hz, ArCH.sub.2CH.sub.3).
2,3:4,5-bis-O-(1-methylethylidene)-1-O-2-propynyl-L-arabinitol (Compound 248)
[1098] ##STR00219##
[1099] To a suspension of the sodium hydride (500 mg of 60% by weight in oil, 12.9 mmol) in THF (20 mL) was added 2,3:4,5-bis-O-(1-methylethylidene)-L-arabinitol (2.00 g, 8.61 mmol). The suspension was heated at 50 C. for 30 minutes then cooled in an ice-bath before adding the propargyl bromide (2.56 g, 17.2 mmol). After stirring at 0 C. for 30 mins, the reaction mixture was warmed to room temp and stirred for another hour before working up by carefully adding water then evaporating the organic solvents away. The residue was dissolved in a mixture of DCM and aqueous citric acid, took organic layer and dried over sodium sulfate and evaporated to give an orange oil 2.38 g. Silica gel chromatography eluting with DCM to 10% diethyl ether in DCM gradient gave 1.47 g of a yellow oil. This material was subjected to another silica gel column eluting with 15% EtOAc in Petrol to give on evaporation 2,3:4,5-bis-O-(1-methylethylidene)-1-O-2-propynyl-L-arabinitol (1.26 g, 54%) as a colourless oil.
[1100] .sup.1H NMR (400 MHz, CDCl.sub.3) m. 4.23 (2H, OCH.sub.2CCH), m. 4.15-4.02 (3H), dd. 3.95 (1H), dd. 3.82 (1H), t. 3.71 (1H), dd. 3.63 (1H), t. 2.42 (1H, OCH.sub.2CCH), s. 1.405 (3H), s. 1.40 (3H), s. 1.37 (3H), s. 1.33 (3H).
[1101] .sup.13C NMR (400 MHz, CDCl.sub.3) 109.97, 109.78, 79.60, 79.60, 77.84, 77.24, 74.76, 70.43, 67.77, 58.81, 27.16, 27.12, 26.85, 25.36.
1-O-2-propynyl-L-arabinitol (Compound 249)
[1102] ##STR00220##
[1103] 2,3:4,5-bis-O-(1-methylethylidene)-1-O-2-propynyl-L-arabinitol (1.26 g, 4.66 mmol) was dissolved in a mixture of TFA (4 mL) and water (2 mL). After 3 hours the solvent was evaporated and the residue dissolved in methanol with heating. After evaporating and redissolving several times in methanol, the residue was dissolved in the minimum amount of hot methanol and allowed to crystallise. The crystals were filtered and washed with a little cold methanol to give 1-O-2-propynyl-L-arabinitol (135 mg, 15%) as fine white crystals.
[1104] .sup.1H NMR (400 MHz, D.sub.2O) m. 4.22 (2H, OCH.sub.2CCH), m. 4.03 (1H), dd. 3.79 (1H), m. 3.73-3.59 (4H), m. 3.52 (1H), t. 2.86 (1H, OCH.sub.2CCH).
[1105] .sup.13C NMR (400 MHz, D.sub.2O) 79.55, 76.07, 71.58, 70.97, 70.87, 68.51, 63.03, 58.24.
##STR00221##
[1106] Compound 233 (25 mg, 0.015 mmol), sodium ascorbate (11.6 mg, 0.058 mmol) and Compound 249 (16.7 mg, 0.088 mmol) were dissolved in degassed THF (5 ml) and water (2 mL). To this was added a solution of copper sulfate (10.9 mg, 0.044 mmol) in water (0.5 mL), whereupon the blue copper colour rapidly turned to brown then faded over a few seconds to give a colourless solution. After 1 minute the solution started to turn cloudy then over an hour an orange solid had formed. The reaction mixture was evaporated to dryness and triturated in a mixture of DCM and methanol before loading the supernatant onto a normal phase column eluting with an increasing gradient (0 to 50%) methanol in DCM, however the solubility of the compound gave poor recovery of impure material. This impure material was purified by reverse phase chromatography eluting with a water-methanol gradient, and freeze-dried to give Compound 250 (7 mg, 20%) as a white solid.
[1107] HRMS: (nanospray.sup.+) calculated for C.sub.105H.sub.158N.sub.24O.sub.33.sup.2+ [M+2H].sup.2+ 1142.0726, found: 1142.0708.
Monocyclic Receptor Synthesis
[1108] ##STR00222##
[1109] Tetra-hydroxy anthracene (33) was prepared according to literature procedure as described in J. Org. Chem., 1989, 54, 1018.
Tetra-tert-butyl-2,2,2,2-((9, 10-dimethylanthracene-2, 3, 6, 7-tetrayl)tetrakis(oxy))tetraacetate (34)
[1110] ##STR00223##
[1111] Under an inert N.sub.2 atmosphere, tetra-hydroxy anthracene 33 (2.35 g, 8.7 mmol) was dissolved in anhydrous THF (500 mL). K.sub.2CO.sub.3 (4.9 g, 35.2 mmol) and tert-butyl bromoacetate (7 mL, 47.4 mmol) were added and the reaction mixture stirred under reflux for 16 hours. The mixture was cooled to room temperature and the solvent removed under vacuum. The crude residue was then dissolved in CH.sub.2Cl.sub.2 (500 mL) and washed with water (150 mL), brine (200 mL) and dried (MgSO.sub.4). The solvent was removed under vacuum and the crude residue purified by flash column chromatography (1% MeOH:CH.sub.2Cl.sub.2) to yield 34 (3.8 g, 5.2 mmol, 60%) as a yellow solid. .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.49 (s, 36H, 3C(1)H.sub.3), 2.85 (s, 6H, 2C(9)H.sub.3), 4.76 (s, 8H, C(4)H.sub.2), 7.38 (s, 4H, 4C(6)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 14.6 (C(9)H.sub.3), 28.1 (C(1)H.sub.3), 66.6 (C(4)H.sub.2), 82.3 (C(2)(CH.sub.3).sub.3), 105.7 (C(6)H), 124.3 (C8), 126.2 (C7), 147.2 (C5), 167.8 (C(3)O); V.sub.max 2987, 2901, 1750, 1453, 1369, 1145, 1066 cm.sup.1; HRMS: (ESI.sup.+) Found [M+Na].sup.+: 749.3520.
tetra-tert-butyl-2,2,2,2-((9,10-bis(bromomethyl)anthracene-2,3,6,7-tetrayl)tetrakis(oxy)) tetraacetate (35)
[1112] ##STR00224##
[1113] Under an inert N.sub.2 atmosphere, 34 (3 g, 4.1 mmol) was dissolved in anhydrous CH.sub.2Cl.sub.2 (500 mL). NBS (1.84 g, 10.3 mmol) and ABCN (50 mg, 5 mol %) were added, and the mixture stirred under reflux for 1.5 hours. The reaction mixture was then cooled to room temperature and diluted with CH.sub.2Cl.sub.2 (300 mL). The solution was washed with NaOH (300 mL, 1M), water (300 mL) and the solvent removed under vacuum to yield 35 (3.5 g, 4.0 mmol, 98%) as an orange solid. .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.52 (s, 36H, 3C(1)H.sub.3), 4.81 (s, 8H, C(4)H.sub.2), 5.22 (s, 4H, 2C(9)H.sub.2), 7.38 (s, 4H, 4C(6)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 28.1 (C(1)H.sub.3), 29.7 (C(9)H.sub.3), 66.4 (C(4)H.sub.2), 82.6 (C(2)(CH.sub.3).sub.3), 104.2 (C(6)H), 126.2 (C8), 126.4 (C7), 148.7 (C5), 167.4 (C(3)O); V.sub.max 2987, 2933, 1706, 1488, 1362, 1228, 1183, 1066 cm.sup.1; HRMS: (ESI.sup.+) Found [M+Na].sup.+: 905.1709, 907.1692.
tetra-tert-butyl-2, 2,2,2-((9,10-bis(azidomethyl)anthracene-2,3,6,7-tetrayl)tetrakis(oxy)) tetraacetate (36)
[1114] ##STR00225##
[1115] Under an inert N.sub.2 atmosphere, 35 (3.5 g, 4.0 mmol) was dissolved in anhydrous MeCN (300 mL). NaN.sub.3 (1 g, 15.9 mmol) was added and the reaction stirred under reflux for 3 hours. The reaction mixture was cooled to room temperature and the solvent removed under vacuum. The crude product was dissolved in CH.sub.2Cl.sub.2 (400 mL), washed with water (3100 mL) and the solvent removed under vacuum to yield 36 (3.2 g, 3.9 mmol, 98%) as an orange solid. .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.52 (s, 36H, 3C(1)H.sub.3), 4.78 (s, 8H, C(4)H.sub.2), 5.08 (s, 4H, 2C(9)H.sub.2), 7.40 (s, 4H, 4C(6)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 28.1 (C(1)H.sub.3), 46.9 (C(9)H.sub.3), 66.4 (C(4)H.sub.2), 82.6 (C(2)(CH.sub.3).sub.3), 104.6 (C(6)H), 124.2 (C8), 126.8 (C7), 148.7 (C5), 167.5 (C(3)O); v.sub.max 2988, 2931, 2091, 1736, 1498, 1364, 1227, 1186, 1062 cm.sup.1; HRMS: (ESI.sup.+) Found [M+Na].sup.+: 831.3530.
tetra-tert-butyl-2,2,2,2-((9, 1O-bis(aminomethyl)anthracene-2,3,6, 7-tetrayl)tetrakis(oxy)) tetraacetate (37)
[1116] ##STR00226##
[1117] Under an inert N.sub.2 atmosphere, 36 (100 mg, 0.12 mmol) was dissolved in anhydrous degassed THF (8 mL). PMe.sub.3 was added (2.5 mL, 2.5 mmol, 1M in THF) and the mixture stirred at room temperature for 3 hours. Degassed water (2 mL) was added and the reaction mixture stirred for 1 hour. The solvent was then evaporated under a flow of nitrogen and the crude residue dissolved in THF/H.sub.2O (5:1, 3 mL). The solvent was then removed by freeze-drying to yield 37 (90 mg, 0.12 mmol, 96%) as a pale brown solid. .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.50 (s, 36H, 3C(1)H.sub.3), 4.58 (s, 4H, 2C(9)H.sub.2), 4.77 (s, 8H, C(4)H.sub.2), 7.49 (s, 4H, 4C(6)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 28.1 (C(1)H.sub.3), 38.9 (C(9)H.sub.2), 66.5 (C(4)H.sub.2), 82.4 (C(2)(CH.sub.3).sub.3), 105.0 (C(6)H), 125.8 (C8), 126.2 (C7), 148.0 (C5), 167.6 (C(3)O);); v.sub.max 2982, 2926, 1729, 1497, 1358, 1222, 1144, 1069 cm.sup.1; HRMS: (MALDI.sup.+) Found [M+H].sup.+: 757.3909.
##STR00227## ##STR00228##
tetra-tert-butyl-2,2,2,2-((9,10-bis(isocyanatomethyl)anthracene-2,3, 6, 7-tetrayl)tetrakis(oxy)) tetraacetate (38)
[1118] ##STR00229##
[1119] Under an inert N.sub.2 atmosphere, a flask was charged with 37 (30 mg, 0.04 mmol) and NaHCO.sub.3 (12 mg, 0.14 mmol). CH.sub.2Cl.sub.2 (1 mL) and H.sub.2O (1 mL) were added, the mixture cooled to 0 C. and rapidly stirred. Triphosgene (9.4 mg, 0.016 mmol) was added and the reaction mixture stirred at room temperature for 30 minutes. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (10 mL) and the organic layer separated, dried (MgSO.sub.4) and the solvent removed under vacuum to afford 38 (29 mg, 0.036 mmol, 91%) as an orange solid. .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.51 (s, 36H, 3C(1)H.sub.3),), 4.78 (s, 8H, C(4)H.sub.2), 5.07 (s, 4H, 2C(9)H.sub.2), 7.35 (s, 4H, 4C(6)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 28.0 (C(1)H.sub.3), 39.83 (C(9)H.sub.2), 66.6 (C(4)H.sub.2), 82.7 (C(2)(CH.sub.3).sub.3), 104.4 (C(6)H), 125.5 (C8), 126.0 (C7), 148.7 (C5), 167.3 (C(3)O); V.sub.max 2979, 2934, 2251, 1734, 1493, 1367, 1225, 1144, 1064 cm.sup.1; HRMS: (ESI) Found [M+Na].sup.+: 831.3319. tert-butyl protected half receptor (39)
##STR00230##
[1120] Under an inert N.sub.2 atmosphere, 1,2-phenylene diamine (0.3 g, 2.70 mmol) was dissolved in dry degassed CH.sub.2Cl.sub.2 (120 mL). A solution of 38 (55 mg, 0.068 mmol) in dry degassed CH.sub.2Cl.sub.2 (50 mL) was added dropwise over 10 minutes and then stirred at room temperature for 30 minutes. The solvent was removed under vacuum and the crude solid purified by flash column chromatography (80:20 EtOAc:hexane.fwdarw.5:95 MeOH:CH.sub.2Cl.sub.2.fwdarw.10:90 MeOH:CH.sub.2Cl.sub.2) to yield 39 (53 mg, 0.052 mmol, 77%) as an orange brown solid. .sup.1H NMR: (400 MHz, ((CD.sub.3).sub.2SO): 1.47 (s, 36H, 3C(1)H.sub.3),), 4.59 (s, 4H, N(19)H.sub.2), 4.87 (s, 8H, C(4)H.sub.2), 5.12 (s, 4H, 2C(9)H.sub.2), 6.50-6.57 (m, 4H, C(13)H and N(18)H), 6.66-6.70 (m, 2H, C(15)H), 6.77 (t, J=7.6 Hz, 2H, C(14)H), 7.42-7.48 (m, 4H, C(12)H and N(18)H), 7.66 (s, 4H, 4C(6)H); .sup.13C NMR: (100 MHz, ((CD.sub.3).sub.2SO): 28.2 (C(1)H.sub.3), 36.7 (C(9)H.sub.2), 66.8 (C(4)H.sub.2), 82.9 (C(2)(CH.sub.3).sub.3), 105.2 (C(6)H), 114.5 (C15), 118.9 (C13), 122.8 (C11), 125.2, 125.5 (C12 and C14), 125.8 (C8), 126.2 (C7), 148.3 (C5), 149.5 (C16), 154.2 (C10), 167.5 (C(3)O); V.sub.max 3315, 2973, 2901, 1733, 1622, 1494, 1393, 1225, 1146, 1057, 742 cm.sup.1; HRMS: (ESI.sup.+) Found [M+Na].sup.+: 1047.4689. tert-butyl protected tetra-urea macrocycle (40)
##STR00231##
[1121] Under an inert N.sub.2 atmosphere, 38 (40 mg, 0.049 mmol) was dissolved in dry degassed CH.sub.2Cl.sub.2 (600 mL). To this was added a solution of 39 (50 mg, 0.049 mmol) in dry degassed pyridine (60 mL) dropwise over 20 minutes. The reaction was stirred at room temperature for 16 hours and then the solvent removed under reduced pressure. The crude residue was suspended in HPLC grade water and freeze-dried to afford a fine crude solid. The product was then purified by reverse phase HPLC and freeze dried to afford 40 (50 mg, 0.027 mmol, 56%) as an off white solid. .sup.1H NMR: (400 MHz, ((CD.sub.3).sub.2CO): 1.49 (s, 36H, 3C(1)H.sub.3),), 4.78 (m, 16H, C(4)H.sub.2), 5.12 (m, 8H, C(9)H.sub.2), 6.95 (s, 4H, C(13)H), 7.65 (s, 8H, C(6)H), 8.07 (s, 4H, C(12)H); .sup.13C NMR: (100 MHz, ((CD.sub.3).sub.2CO): 27.3 (C(1)H.sub.3), 36.0 (C(9)H.sub.2), 66.0 (C(4)H.sub.2), 81.6 (C(2)(CH.sub.3).sub.3), 105.3 (C(6)H), 126.3 (C8), 126.9 (C7), 127.4 (C12), 132.2 (C13), 135.6 (C11), 147.8 (C5), 155.5 (C10), 167.9 (C(3)O); HRMS: (ESI.sup.+) Found [M+Na].sup.+: 1856.8145, [M+2Na].sup.2+: 939.9019.
##STR00232## ##STR00233## ##STR00234## ##STR00235##
9,10-bis(isocyanatomethyl)anthracene (55)
[1122] ##STR00236##
[1123] Under an inert N.sub.2 atmosphere, a flask was charged with anthracene-9,10-diyldimethanamine An-NH.sub.2* (20 mg, 0.085 mmol) and NaHCO.sub.3 (26 mg, excess). CH.sub.2Cl.sub.2 (1 mL) and H.sub.2O (1 mL) were added, the mixture cooled to 0 C. and rapidly stirred. Triphosgene (20 mg, 0.068 mmol) was added and the reaction mixture stirred at room temperature for 30 minutes. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (10 mL) and the organic layer separated, dried (MgSO.sub.4) and the solvent removed under vacuum to afford 55 (22 mg, 0.076 mmol, 91%) as a yellow solid. .sup.1H NMR: (400 MHz, (CDCl.sub.3): 5.38 (s, 4H, C(5)H.sub.2), 7.66 (dd, J=6.9, 3.2 Hz, 4H, C(1)H), 8.35 (dd, J=6.9, 3.2 Hz, 4H, C(2)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 39.0 (C(5)H.sub.2), 124.1 (C(1)H), 126.7 (C(8)H), 127.3 (C4), 129.3 (C3), 167.3; V.sub.max 2921, 2234, 1620, 1491, 1448, 1324, 1185, 858, 751 cm.sup.1; HRMS: (ESI.sup.+) Found [M+Na].sup.+: 311.0785. * prepared according to the synthetic procedures described in literature procedure as described in Org. Biomol. Chem., 2005, 3, 48.
Diamino Tert-Butyl Protected Anthracene Half Receptor (88)
[1124] ##STR00237##
[1125] Under an inert N.sub.2 atmosphere, 84 (350 mg, 0.195 mmol) was dissolved in anhydrous dichloromethane (10 mL). 55 (25 mg, 0.098 mmol) was added and the reaction heated to reflux for 2 days. The reaction was cooled to room temperature and the solvent removed under vacuum. The crude residue was purified by reverse phase HPLC to afford the Fmoc protected product 86 (254 mg, 0.66 mmol, 67%) as a white solid. Conversion to 86 was confirmed by limited NMR studies* and high resolution mass spectrometry (ESI.sup.+): m/z calculated for [M+2Na].sup.2+1963.1077, found 1963.1067. Under an inert N.sub.2 atmosphere, 86 was dissolved in anhydrous dichloromethane (10 mL) and cooled to 0 C. DBU (45 L, 0.28 mmol) was added dropwise and the reaction mixture warmed to room temperature and stirred for 2 hours. The solvent was removed under vacuum and the crude product purified by flash column chromatography (5% MeOH:CH.sub.2Cl.sub.2) to afford 88 (215 mg, 0.063 mmol, 95%) as an off-white solid. .sup.1H NMR: (400 MHz, (CD.sub.3OD): 1.43 (s, 162H, C(23)H.sub.3), 1.93 (m, 36H, C(20)H.sub.2), 2.08 (m, 12H, C(15)H.sub.2), 2.18 (m, 48H, C(19, 16)H.sub.2), 5.36 (s, 4H, C(5)H.sub.2), 7.18 (dd, J=2.1, 8.3 Hz, 2H, C(9)H), 7.26 (d, J=2.1 Hz, 2H, C(11)H), 7.41 (d, J=8.3 Hz, 2H, C(8)H), 7.41 (s, 6H, NH), 7.60 (dd, J=3.3, 6.9 Hz, 4H, C(1)H), 7.89 (s, 2H, NH), 8.47 (dd, J=3.3, 6.9 Hz, 4H, C(2)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 27.1 (C23), 29.1 (C20), 29.3 (C21), 31.0 (C15), 31.1 (C16), 57.4 (C18), 58.0 (C14), 80.2 (C22), 115.9 (C11), 117.4 (C9) 123.1 (C8), 124.6 (C2), 125.9 (C1), 128.5 (C10), 130.1 (C4), 130.5 (C3), 131.4 (C7), 140.0 (C12), 156.7 (C6), 168.6 (C13), 173.0 (C21), 174.1 (C17); HRMS: (ESI.sup.+). Found [M+H+Na].sup.2+: 1730.5507. * Limited NMR studies were only possible due to believed slow conformational exchange of 86 resulting in very broad signals of low intensity.
Tert-butyl protected G2 anthracene tetra urea macrocycle (89)
[1126] ##STR00238##
[1127] Under an inert N.sub.2 atmosphere, 55 (5.3 mg, 0.018 mmol) was dissolved in anhydrous degassed dichloromethane (600 mL) and heated to reflux. 88 (63 mg, 0.018 mmol) in anhydrous degassed dichloromethane (50 mL) was added over 30 mins and stirred at reflux for 4 days. The solvent was then removed under vacuum and the crude product purified by reverse phase HPLC and then freeze dried to afford 89 (25 mg, 6.7 mol, 37%) as a white solid. .sup.1H NMR: (400 MHz, (CD.sub.3OD): 1.45 (s, 162H, C(23)H.sub.3), 1.99 (m, 36H, C(20)H.sub.2), 2.17 (m, 12H, C(15)H.sub.2), 2.24 (m, 48H, C(19)H.sub.2), 2.31 (m, 12H, C(16)H.sub.2), 5.39 (s, 8H, C(5)H.sub.2), 7.31, 7.44 (br s, 4H, C(1)H), 7.49 (s, 6H, NH), 7.70 (dd, J=2.1, 8.5 Hz, 2H, C(9)H), 7.89 (d, J=8.5 Hz, 2H, C(8)H), 7.98 (s, 2H, C(11)H), 8.39 (br s, 8H, C(2)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 27.0 (C23), 29.1 (C20), 29.3 (C21), 30.8 (C15), 31.1 (C16), 57.3 (C18), 58.1 (C14), 80.3 (C22), 121.6 (C9), 121.77 (C12) 124.4 (C2), 124.7 (C11), 125.8, 125.9 (C1), 130.0 (C10), 130.4 (C3), 130.6 (C3), 131.4 (C7, 12), 156.0, 156.8 (C6), 168.1 (C13), 173.1 (C21), 174.1, 174.2 (C17); HRMS: (ESI.sup.+) Found [M+3Na].sup.3+: 1264.3835, [M+4Na].sup.4+: 954.0378.
Fmoc Protected Methyl Ester G2 Linker (94)
[1128] ##STR00239##
[1129] 84 (215 mg, 0.12 mmol) was dissolved in dichloromethane (5 mL) and TFA (5 mL) added dropwise. The solution was stirred at room temperature for 16 hours and the TFA and solvent evaporated under a flow of N.sub.2. The residue was redissolved in methanol (5 mL) and trimethyl orthoformate (5 mL). HCl (5% v/v, 0.5 mL) was added and the mixture stirred for 24 hours. The solvent was then removed under vacuum and the crude product purified by flash column chromatography (5% MeOH:CH.sub.2Cl.sub.2) to afford 94 (147 mg, 0.10 mmol, 87%). .sup.1H NMR: (400 MHz, (CDCl.sub.3): 1.99 (m, 18H, C(23)H.sub.2), 2.108 (m, 6H, C(18)H.sub.2), 2.25 (m, 24H, C(22, 19)H.sub.2), 3.60 (s, 27H, C(25)H.sub.3), 4.23 (m, 1H, C(7)H), 4.48 (m, 2H, C(8)H.sub.2), 6.31 (s, 3H, NH), 6.72 (d, J=8.4 Hz, 1H, C(13)H), 7.26 (m, 3H, C(4, 12)H), 7.38 (t, J=7.4 Hz, 2H, C(3)H), 7.63 (m, 2H, C(5)H), 7.75 (d, J=7.9 Hz, 3H, C(2, 15)H), 8.12 (s, 1H, NH); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 28.3 (C22), 29.6 (C23), 31.8 (C19), 32.2 (C18), 47.2 (C7), 51.8 (C25), 57.3 (C21), 58.2 (C17), 67.1 (C8), 116.4 (C12), 120.0 (C2), 122.6 (C10), 124.4 (C14), 125.1 (C4), 125.2 (C15), 126.6 (C13), 127.7 (C5), 127.8 (C3), 141.3 (C1), 143.6 (C11), 143.8 (C6), 155.0 (C9), 166.6 (C16), 173.3 (C20), 173.8 (C24); V.sub.max 3330, 2976, 2961, 1727, 1658, 1531, 1452, 1367, 1249, 1150, 846 cm.sup.1; HRMS: (ESI.sup.+) Found [M+2Na].sup.2+: 731.3279, [M+Na].sup.+: 1439.6353.
Diamino Methyl Ester Protected Anthracene Half Receptor (97)
[1130] ##STR00240##
[1131] Under an inert N.sub.2 atmosphere, 94 (160 mg, 0.11 mmol) was dissolved in anhydrous dichloromethane (5 mL). 55 (15 mg, 0.054 mmol) was added and the reaction heated to reflux for 4 days. The reaction was cooled to room temperature and the solvent removed under vacuum. The crude residue was purified by flash column chromatography (5% MeOH:CH.sub.2Cl.sub.2) to afford the Fmoc protected product 97a (190 mg, 0.061 mmol, 56%) as a white solid. Conversion to 97a was confirmed by limited NMR studies* and high resolution mass spectrometry (ESI.sup.+): m/z calculated for [M+2Na].sup.2+ 1584.1834, found 1584.1849. Under an inert N.sub.2 atmosphere, 97a was dissolved in anhydrous dichloromethane (10 mL) and cooled to 0 C. DBU (50 L, 0.30 mmol) was added dropwise and the reaction mixture warmed to room temperature and stirred for 4 hours. The solvent was removed under vacuum and the crude product purified by flash column chromatography (4% MeOH:CH.sub.2Cl.sub.2) to afford 97 (151 mg, 0.057 mmol, 93%) as an off white solid. .sup.1H NMR: (400 MHz, (CD.sub.3OD): 1.94 (m, 36H, C(20)H.sub.2), 2.07 (m, 12H, C(15)H.sub.2), 2.24 (m, 48H, C(19, 16)H.sub.2), 3.60 (s, 54H, C(22)H.sub.3), 4.94 (s, 4H, C(5)H.sub.2), 7.05 (d, J=8.3 Hz, 2H, C(9)H), 7.15 (m, 4H, C(11, 8)H), 7.37 (s, 6H, NH), 7.48 (dd, J=3.3, 6.9 Hz, 4H, C(1)H), 7.85 (s, 2H, NH), 8.21 (dd, J=3.3, 6.9 Hz, 4H, C(2)H); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 28.5 (C20), 29.3 (C21), 31.0 (C15), 31.1 (C16), 51.8 (C22), 57.3 (C18), 58.1 (C14), 116.7 (C11), 117.5 (C9) 123.0 (C8), 124.4 (C2), 125.8 (C1), 128.6 (C10), 130.0 (C4), 130.5 (C3), 131.4 (C7), 140.1 (C12), 155.5 (C6), 166.7 (C13), 173.3 (C17), 173.9 (C21); HRMS: (ESI.sup.+) Found [M+2Na].sup.2+: 1361.1096, Found [M+3Na].sup.3+: 915.7415. * Limited NMR studies were only possible due to believed slow conformational exchange of 97a resulting in very broad signals of low intensity.
Methyl Ester Protected G2 Anthracene Tetra Urea Macrocycle (98)
[1132] ##STR00241##
[1133] Under an inert N.sub.2 atmosphere, 55 (7.9 mg, 0.027 mmol) was dissolved in anhydrous degassed dichloromethane (600 mL) and heated to reflux. 97 (73 mg, 0.027 mmol) in anhydrous degassed dichloromethane (50 mL) was added over 30 mins and stirred at reflux for days. The solvent was then removed under vacuum and the crude product purified by reverse phase HPLC and then freeze dried to afford 98 (21 mg, 7.0 mol, 26%) as a pale yellow solid. .sup.1H NMR: (400 MHz, (CD.sub.3).sub.2SO): 1.89 (m, 36H, C(20)H.sub.2), 1.95 (m, 12H, C(15)H.sub.2),), 2.12 (m, 12H, C(16)H.sub.2), 2.22 (m, 36H, C(19)H.sub.2), 5.25 (s, 8H, C(5)H.sub.2), 7.32 (s, 6H, NH), 7.42 (br s, 4H, C(1)H), 7.52 (m, 6H, C(1, 9)H), 7.74 (s, 2H, NH), 7.87 (d, J=8.5 Hz, 2H, C(8)H), 8.07 (s, 2H, C(11)H), 8.37 (br s, 8H, C(2)H); 13C NMR: (100 MHz, (CD.sub.3).sub.2SO): 28.3 (C20), 29.2 (C21), 29.5, 29.8 (C15), 30.7, 31.0 (C16), 35.7 (C5), 51.8 (C22), 56.8 (C18), 57.9 (C14), 120.0 (C8), 122.8 (C9), 123.3 (C11), 125.4 (C2), 125.7 (C12), 126.4, 126.5 (C1), 129.0, 129.5 (C3), 129.9 (C4), 135.6 (C7) 155.1, 155.7 (C6), 166.0 (C13), 172.9 (C17), 173.7 (C21); HRMS: (ESI.sup.+) Found [M+2Na].sup.2+: 1506.1621, [M+3Na].sup.3+: 1011.7687.
Deprotected Anthracene Tetra Urea Macrocycle (90)
[1134] ##STR00242##
[1135] Protected receptor 89 (30 mg, 0.008 mmol) was dissolved in dichloromethane (HPLC grade, 2.7 ml) and cooled to 0 C. Trifluoroacetic acid (TFA) (0.3 mL) was added dropwise and the reaction warmed to room temperature and stirred for 4 hours. The solvent was then removed under a flow of nitrogen, then the residue was co-evaporated with toluene (310 mL) to remove residual TFA, suspended in water and freeze dried. The product was then purified by preparative HPLC (Waters CSH C18 5 m 19250 mm) eluting with 100% Water (buffered with 0.1% TFA).fwdarw.100% methanol over 40 minutes. The solvent was removed under vacuum, the residue co-evaporated with toluene (310 mL), the product suspended in water and freeze dried. The solid was then suspended in water, neutralised to pH 7.4 with NaOH (aq), filtered and then freeze dried to afford 90 as a white solid (21 mg, 0.0068 mmol, 85%). .sup.1H NMR: (400 MHz, 75 C., D.sub.2O): b 2.40-2.50 (m, 36H, C(19)H.sub.2), 2.58-2.71 (m, 48H, C(20)H.sub.2, C(15)H.sub.2), 2.80-2.90 (m, 12H, C(16)H.sub.2), 5.63, 5.81 (br s, 4H, C(5)H.sub.2), 7.43-7.50, 7.98-8.06 (br m, 4H, C(1)H), 8.25 (br s, 2H, C(11)H), 8.27 (d, 2H, C(9)H), 8.39 (d, 2H, C(8)H), 7.63 (d, J=8.3 Hz, 3H, C(10)H), 8.58-8.65, 8.82-8.89 (br m, 4H, C(2)H).
Scheme for Tetra-Methoxy Anthracene Isocyanate (95)
[1136] ##STR00243##
2,3,6,7-Tetramethoxy-9, 10-bis(bromomethyl)anthracene, (92)
[1137] ##STR00244##
[1138] Under an inert N.sub.2 atmosphere, 91 (2 g, 6.1 mmol), NBS (4 g, 22.6 mmol) and ABCN (73 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (150 mL), and the mixture stirred at reflux for 4 hours. The mixture was then cooled to 0 C. and filtered. The solid was then dried under high vacuum to afford 92 (2.1 g, 4.3 mmol, 71%) as a bright yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 4.11 (s, 12H, C(1)H), 5.35 (s, 4H, C(6)H), 7.40 (s, 4H, C(3)H), .sup.13C NMR (100 MHz, CDCl.sub.3) 28.7 (C6), 56.0 (C1), 101.8 (C3), 125.7 (C5), 125.9 (C4), 150.1 (C2).
2,3,6,7-Tetramethoxy-9,10-bis(azidoomethyl)anthracene, (93)
[1139] ##STR00245##
[1140] Under an inert N.sub.2 atmosphere, 92 (2.7 g, 5.58 mmol) and NaN.sub.3 were suspended in anhydrous MeCN (70 mL). The mixture was stirred at reflux for 16 hours and then cooled to room temperature and the solvent was evaporated in vacuo. The remaining residue was then suspended in water (200 mL) and filtered. The solid was washed with ethanol (3100 mL) and dried under high vacuum to afford 93 (1.6 g, 3.9 mmol, 70%) as a brown solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 4.10 (s, 12H, C(1)H), 5.19 (s, 4H, C(6)H), 7.42 (s, 4H, C(3)H), .sup.13C NMR (100 MHz, CDCl.sub.3) 47.7 (C6), 56.1 (C1), 101.8 (C3), 123.8 (C5), 126.9 (C4), 150.2 (C2); HRMS: (ESI.sup.+). Found [M+Na].sup.+ 431.1448.
2,3,6,7-Tetramethoxy-9, 10-bis(aminomethyl)anthracene, (94)
[1141] ##STR00246##
[1142] Under an inert N.sub.2 atmosphere, azide 93 (1.6 g, 3.90 mmol) and PPh.sub.3 (8 g, 31.4 mmol) were suspended in degassed THF (80 mL). Degassed water (4 mL) was added and the reaction heated to 60 C. for 16 hours. The reaction was cooled to room temperature and the solvent removed under vacuum. The crude residue was suspended in toluene (200 mL), filtered, washed with toluene (2100 mL) and dried under high vacuum to afford 94 (1.05 g, 2.96 mmol, 76%) as a pale brown solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 4.08 (s, 12H, C(1)H), 4.69 (s, 4H, C(6)H), 7.49 (s, 4H, C(3)H), .sup.13C NMR (100 MHz, CDCl.sub.3) 39.5 (C6), 56.0 (C1), 101.9 (C3), 125.3 (C5), 125.5 (C4), 149.7 (C2); HRMS: (ESI.sup.+) Found [M+Na].sup.+ 379.1626.
9,10-bis(isocyanatomethyl)-2,3,6, 7-tetramethoxyanthracene (95)
[1143] ##STR00247##
[1144] Under an inert N.sub.2 atmosphere, a flask was charged with triphosgene (324 mg, 1.1 mmol) and anhydrous toluene (15 mL) was added A suspension of 94 (200 mg, 0.55 mmol) in anhydrous toluene (5 mL) was added dropwise and the reaction mixture stirred at reflux for 2 hours. The reaction mixture was cooled and the solvent removed under high vacuum. The crude solid was resuspended in dichloromethane (50 mL) and filtered. The filtrate was collected and the solvent removed under vacuum to afford 95 (121 mg, 0.30 mmol, 54%) as a brown solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 4.11 (s, 12H, C(1)H), 5.21 (s, 4H, C(6)H), 7.37 (s, 4H, C(3)H), V.sub.max 2934, 2832, 2255, 1498, 1435, 1245, 1204, 1169, 1028 cm.sup.1; HRMS: (ESI.sup.+) Found [M+Na].sup.+ 431.1218.
Scheme for Octa-Methoxy Anthracene Bis Urea Receptor
[1145] ##STR00248## ##STR00249##
Diamino Tert-Butyl Protected Methoxy-Anthracene Half Receptor (96)
[1146] ##STR00250##
[1147] Under an inert N.sub.2 atmosphere, 84 (507 mg, 0.283 mmol) and 94 (50 mg, 0.13 mmol) were dissolved in anhydrous dichloromethane (8 mL). Pyridine (60 L, 0.74 mmol) was added and the reaction heated to reflux for 16 hours. The reaction was cooled to room temperature and the solvent removed under vacuum. The crude residue was purified by reverse phase HPLC to afford the Fmoc protected product 95 (336 mg, 0.84 mmol, 65%) as a white solid. Conversion to 95 was confirmed by limited NMR studies* and high resolution mass spectrometry (ESI.sup.+): m/z calculated for [M+2Na].sup.2+2015.6171, found 2015.6176. Under an inert N.sub.2 atmosphere, 95 (100 mg, 0.028 mmol) was dissolved in anhydrous dichloromethane (10 mL) and cooled to 0 C. DBU (50 L, 0.31 mmol) was added and the reaction mixture warmed to room temperature and stirred for 1 hour. The solvent was removed under vacuum and the crude product purified by flash column chromatography (6% MeOH:CH.sub.2Cl.sub.2) to afford 96 (91 mg, 0.026 mmol, 92%) as an off white solid. .sup.1H NMR: (400 MHz, (CD.sub.3OD): 1.42 (s, 162H, C(23)H.sub.3), 1.84-2.0 (m, 36H, C(20)H.sub.2), 2.02-2.14 (m, 12H, C(15)H.sub.2), 2.13-2.31 (m, 48H, C(19, 16)H.sub.2), 3.95 (br s, 12H, C(24)H.sub.3), 4.58 (br s, 4H, C(5)H.sub.2), 7.13 (d, J=8.3 Hz, 2H, C(9)H), 7.23 (s, 2H, C(11)H), 7.31 (d, J=8.3 Hz, 2H, C(8)H), 7.38 (br s, 4H, C(2)H), 7.41 (s, 6H, NH), 7.89 (s, 2H, NH); .sup.13C NMR: (100 MHz, (CDCl.sub.3): 527.1 (C23), 29.1 (C20), 29.3 (C21), 30.8 (C15), 31.1 (C16), 54.9 (C24), 57.4 (C18), 58.1 (C14), 80.3 (C22), 101.9 (C2), 115.8 (C11), 117.3 (C9) 123.3 (C8), 126.1 (C4), 126.1 (C3), 128.2 (C10), 131.6 (7), 140.3 (C12), 149.5 (C1), 157.0 (C6), 168.5 (C13), 173.0 (C21), 174.1 (C17); HRMS: (ESI.sup.+) Found [M+2Na].sup.2+: 1208.3682. * Limited NMR studies were only possible due to believed slow conformational exchange of 95 resulting in very broad signals of low intensity.
Tert-Butyl Protected Octa-Methoxy Anthracene Tetra Urea Macrocycle (97)
[1148] ##STR00251##
[1149] Under an inert N.sub.2 atmosphere, 96 (25 mg, 0.007 mmol) and DMAP (1.7 mg, 0.014 mmol) were dissolved in anhydrous degassed dichloromethane (12 mL) and heated to reflux. 94 (2.7 mg, 0.007 mmol) in anhydrous degassed dichloromethane (2 mL) was added and the reaction stirred at reflux for 2 days. The solvent was then removed under vacuum and the crude product purified by reverse phase HPLC and then freeze dried to afford 89 as a white solid.
Binding Studies
[1150] Isothermal titration calorimetry (ITC) and .sup.1H NMR were used to determine the binding affinities between the compounds of the present invention (e.g. receptor compound 1 and receptor compound 90) and a number of saccharides (e.g. glucose, mannose and cellobiose), together with other small molecules (e.g. uracil and uric acid). ITC and .sup.1H NMR titrations were performed according to the general procedure described hereinabove and the ITC traces, .sup.1H NMR spectra and binding affinities are summarised in
.SUP.1.H NMR Titrations
[1151] .sup.1H-NMR titrations were performed on a Varian VNMR cryogenically cooled S600 spectrometer. Solutions of saccharides in D.sub.2O (99.9%), containing receptor at a known concentration to be used in the experiment, were prepared and allowed to equilibrate overnight before use if necessary. Aliquots were then added to an NMR tube containing a known concentration of receptor solution (typically 50 M-250 M). The receptor concentration was therefore held constant while the carbohydrate concentration was increased. The sample tube was shaken after each addition, centrifuged and .sup.1H-NMR spectra were acquired at 298 K.
[1152] If the receptor bound saccharide slower than the NMR sample rate (slow exchange), the K.sub.a was determined by analysing the NMR integral of a peak assigned to the Host-Guest complex. The variable X was defined as the integral of an isolated resonance of the complex (typically in the aromatic region) divided by the integral of all the related resonances (typically the whole aromatic region). As X is proportional to fraction of host in the bound state, the change in X could be plotted as a function of the guest concentration to give a curve which could be fitted to a 1:1 binding model to yield the association constant K.sub.a. Mathematically, the fitting process is essentially identical to that employed for binding with fast exchange, except that the integral of a peak due to the complex replaces the chemical shift of a peak due to bound+unbound receptor. The calculation was performed using a non-linear least squares curve-fitting programme implemented within Excel. The programme yields binding constants K.sub.a and limiting X (X.sub.lim) as output. K.sub.a values are listed in Table 4 below. An estimated error for Ka was obtained from individual data points by assuming the determined K.sub.a and X.sub.lim. These errors are reported in Table 4 and are typically well below 5%.
TABLE-US-00001 TABLE 1 Relative integrations of -H1 and -H2 during NMR titration. Glucose concentration Relative integration -D-glucose (M) (H1:H2) (%) 57 .sup.a 114 .sup.a 170 1:2.08 67% 225 1:2.01 66% 280 1:1.85 64% 334 1:2.04 67% 387 1:1.88 65% 440 1:1.84 64% 492 1:1.82 64% 544 1:1.82 64% .sup.avalues for integration not obtained due to low intensity of and broadness of signals.
TABLE-US-00002 TABLE 2 Relative integrations of -H1 and -H2 over time from pure -D- glucose, with and without Receptor 1 (0.2 mM) present. Relative integration (H1:H2) D-glucose D-glucose (5 mM) and Time (min) (5 mM) only receptor X (0.2 mM) 0 1:0.01 1:0.06 10 1:0.06 1:0.11 30 1:0.19 1:0.22 60 1:0.30 70 1:0.34 90 1:0.38 100 1:0.50 120 1:0.59 1:0.52 150 1:0.70 1:0.66 180 1:0.86 210 1:0.93
TABLE-US-00003 TABLE 3 Calculated values for K.sub.a when titrating cellobiose (250 mM) against receptor 1 (0.11 mM). The integrals of the peak at 8.02 ppm (denoted with *, see FIG. 31) were made relative to integral of the same peak (8.02 ppm) when all receptor is assumed to be saturated with guest (i.e. the final addition in the titration, row denoted with yellow). These relative integrals are then used to determine the amount of Host-Guest [HG]. This value for [HG] along with calculated values for free host [H] .sub.free and free guest [G].sub.free can be used to calculate the K.sub.a at each point in the titration. An average of the values obtained (denoted in blue) was then used as the overall K.sub.a (31.1 M.sup.1) along with the associated standard deviation and error. Not all K.sub.a values calculated were included in the averaged K.sub.a value. The earlier integrations are unreliable due to the very small intensity of the peak at 8.02 ppm. The later integrations were also deemed unreliable due to large deviations in baseline of the spectra due to the large excess of guest present. The selected values for K.sub.a used for the average calculation and the averaged K.sub.a itself corroborate with the results obtained from ITC. Integral of peak* vs Volume integral of Guest [Host- of peak* added [Host].sub.free/ [Guest].sub.free/ [Host].sub.total/ [Guest].sub.total/ Guest]/ Integral when K.sub.a/ (L) M mM M mM M (peak 8.02 ppm*) [H].sub.free = 0. M.sup.1 0 110 0 110 0 0 0 4 110 20 110 20 0 0.00005 0.0011 0.55 4 99 39 110 39 11 0.00465 0.102 29.0 4 93 58 110 58 17 0.007 0.154 31.2 8 86 96 110 96 24 0.01 0.220 29.4 8 75 132 110 132 35 0.0143 0.315 34.9 8 47 167 110 167 63 0.0262 0.577 81.5 20 38 251 110 251 72 0.0296 0.650 74.0 20 8 329 110 329 102 0.0421 0.927 386 40 0 470 110 470 110 0.0455 1 Average 31.1 K.sub.a/M.sup.1 Std Dev 2.66 (Error) (9%)
Isothermal Titration MicroCalorimetry (ITC) Titrations
[1153] Isothermal Titration MicroCalorimetry (ITC) experiments were performed on a MicroCal iTC200 microcalorimeter and/or a MicroCali VP-ITC. ITC experiments were carried out at 298 K. Saccharide solutions were prepared in HPLC-grade water with 10 mM phosphate buffer solution (pH 7.4) and allowed to equilibrate overnight if necessary. The sample cell was charged with a known concentration of receptor solution in HPLC-grade water with 10 mM phosphate buffer solution at pH 7.4 (typically 50 M-200 M). Then, aliquots (typically 1.0 L) of carbohydrate solution were added and the evolution of heat was followed as a function of time. Heats of dilution were measured by injecting the same carbohydrate solution into HPLC-grade water with 10 mM phosphate buffer solution at pH 7.4, using identical conditions. For every addition, the heat of dilution was subtracted from the heat of binding using a MicroCal software programme implemented in ORIGIN 7.0. This gave an XY matrix of heat vs. total guest concentration. This matrix was then imported into a specially written Excel programme to fit the data to a 1:1 binding model to give a K.sub.a. G can be derived from K.sub.a and thus S can be derived from H and G using common thermodynamic equations. The fitting procedure also yields errors in K.sub.a as in the case of NMR described above. This method consistently produced more accurate fits than fitting the data to an S-curve, as in the MicroCal software (S-curves are typically not observed for binding constants below 104-105 M.sup.1). Although fits produced using the supplied MicroCal software were generally similar to those calculated using the Excel programme, they also consistently overestimated the K.sub.a by approximately 10%. It was found that better corroboration of the ITC data with the equivalent NMR data was achieved using the Excel programme. ITC outputs for heat of dilution of substrates, binding events between substrates and receptor 1, and analysis curves are included in the Figures. An overview of the binding data, including thermodynamic quantities and errors is given in Table 4 below.
TABLE-US-00004 TABLE 4 binding affinities of various substrates for receptor 1. Determined by NMR Determined by ITC (kJ mol.sup.1) Substrate (medium) K.sub.a (M.sup.1) K.sub.a (M.sup.1) G H TS D-Glucose 18,026 1.04% 18,600 14.3% 24.4 7.8 16.6 D-Glucose pH 6 (PBS) 17,300 3.8%.sup. 24.2 2.6 21.6 D-Glucose pH 7 (PBS) 17,800 5.5%.sup. 24.3 2.2 22.0 D-Glucose pH 8 (PBS) 18,300 1.8%.sup. 24.3 2.6 21.8 D-Glucose (human serum) 2477 5.7% 19.4 4.1 15.3 D-Glucose (DMEM cell culture) 5637 2.1% 21.4 5.2 16.2 D-Glucose (DMEM salt control) 5164 5.9% 21.2 5.0 16.2 D-Glucose (Leibovitz's L-15 5214 8.6% 21.2 4.2 17.0 cell culture) Methyl -D-Glucoside 7522 5.5% 7886 16.4% 22.2 3.2 21.2 Myo-inositol 7328 7.4% 7563 4.2% 22.1 22.1 2.4 D-Glucuronic Acid n.d..sup.a 5348 3.5% 21.3 27.8 6.5 D-Xylose n.d..sup.a 5804 3%.sup. 21.5 8.0 13.5 2-Deoxy-D-Glucose n.d..sup.a 725 5.7% 16.3 2.9 13.4 D-Galactose 132 10% 182 2.3% 12.9 8.8 4.2 D-Mannose 140 1.3% 143 1.1% 12.3 11.8 0.6 D-Ribose 267 3.8% 216 1.9% 13.3 23.0 9.7 D-Fructose 51 5.5% 60 2.7% 10.6 20.0 9.5 D-Cellobiose 31 9% 30 15.9% 8.5 9.2 0.7 Mannitol 0 Gluconate.sup.b 0 0 Methyl -D-Glucoside 0 0 N-Acetyl-D-glucosamine 0 D-Maltose 0 L-Fucose 0 Uracil (PBS) 0 Uric Acid (PBS) 0 Cytosine 0 Adenosine 0 Paracetamol 0 Ascorbic Acid 0 L-Phenylalanine 0 L-Tryptophan 0 Affinities (K.sub.d) were measured in D.sub.2O (NMR) or H.sub.2O (ITC) containing phosphate buffer (10 mM, pH = 7.4) at T = 298 K. N.d. = not determined due to broadening of NMR signals on addition of substrate All solutions at pH 7.4 in 10 mM Phosphate buffer solution unless otherwise stated. Human blood Serum and cell culture media were dialysed at 10k MWCO and then buffered with 10 mM phosphate buffer solution at pH 7.4. DMEM Salt control composition: ferric nitrate (0.2 M), calcium chloride (1.8 mM), magnesium sulfate (0.81 mM), potassium chloride (5.3 mM), sodium bicarbonate (44 mM), sodium chloride (110 mM) and sodium phosphate monobasic (0.9 mM). PBS = phosphate buffered saline at pH 7.4, composition: sodium chloride (137 mM), potassium chloride (2.7 mM), disodium phosphate (10 mM), monopotassium phosphate (1.8 mM). .sup.aK.sub.a not determined due to intermediate exchange rate on NMR timescale resulting in complex broad spectra, evidence of binding was indicated regardless. .sup.bPrepared by dissolution of D-glucono--lactone in 10 mM phosphate buffer, pH 7.4. After 4 h .sup.1H NMR indicated that the lactone had hydrolysed to give the acyclic gluconate.
TABLE-US-00005 TABLE 5 Summary of binding results for anthracene receptor 90. Determined by NMR Determined by ITC (kJ mol.sup.1) Substrate K.sub.a (M.sup.1) K.sub.a (M.sup.1) G H TS D-Glucose 5 3.6% D-Cellobiose 46 0.9% 38 6.5% 9.0 5.7 3.3 D-cellotriose 950 0.3% 955 1.2% 17.0 16.5 0.6 D-cellotetraose n.d. D-cellopentaose n.d. D-maltose 15 11.8% D-Maltotriose 20 3.3% Uric Acid 0
TABLE-US-00006 TABLE 6 Measured binding affinities displayed by Receptors 2 to 14 towards D-glucose Binding affinity for D-glucose (K.sub.a (M.sup.1)) Determined Determined Determined Receptor by ITC by .sup.1H-NMR by CD 2 13 4 3 5760 269 4 .sup.6490 72.6 5 10400 132 6 7 6886 190 8 4210 73 9 2554 96 10 481 57 11 2000 1819 152 12 0* 13 1310 33 14 14926 1566 *No measurable binding using a 7.1 mM L- or D-glucose solution and a 0.4 mM solution of Receptor 12.
[1154] While specific embodiments of the invention have been described for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.