Sterically shielded heptamethine cyanine dyes
The near-infrared window of fluorescent heptamethine cyanine dyes greatly facilitates biological imaging because there is deep penetration of the light and negligible background fluorescence. But dye instability, aggregation, and poor pharmacokinetics are current drawbacks that limit performance and the scope of possible applications. All these limitations are simultaneously overcome with a new molecular design strategy that produces a charge balanced and sterically shielded fluorochrome. The key design feature is a meso-Aryl group that simultaneously projects two shielding arms directly over each face of a linear heptamethine polyene. Cell and mouse imaging experiments compared a shielded heptamethine cyanine dye (and several peptide and antibody bioconjugates) to benchmark heptamethine dyes and found that the shielded systems possess an unsurpassed combination of photophysical, physiochemical and biodistribution properties that greatly enhance bioimaging performance.
1 . A compound of Formula I:
or a salt thereof;
wherein
G 1 and G 2 are each independently-(C 1 -C 4 )alkyl-(Z)—R W ;
each Z is independently 5- or 6-membered heterocycle;
each R W is independently —X 1 (C 1 -C 4 )alkyl-(O(C 2 -C 4 )alkyl) m -O(C 1 -C 4 )alkyl, or H;
each X 1 is independently absent, O, S, or NH;
each m is independently 0-500;
G 3 is —(C═O)R A wherein R A is OR B , NR B R C , H, or halo;
R B and R C are each independently H, N-succinimidyl, drug, or biologic;
J1 and J2 are each independently SO 3 H or CO 2 H;
Q 1 and Q 2 are each independently —(C 2 -C 6 )alkyl-X 2 ;
each X 2 is independently N + (R D ) 3 , N(R D ) 2 , CO 2 H, SO 3 H, or —O(C 1 -C 6 )alkyl;
each R D is independently-(C 1 -C 6 )alkyl;
R 1 , R 2 , R 3 and R 4 are each independently —(C 1 -C 4 )alkyl or H;
R 5 and R 6 are each independently H or —(C 1 -C 4 )alkyl; or
R 5 and R 5 taken together form a 5- or 6-membered carbocycle; and
x and y are each independently 0-3;
wherein the moieties (C 2 -C 4 )alkyl or (C 2 -C 6 )alkyl are optionally branched, and charged moieties present in the compound are counterbalanced with counterions.
2 . The compound of claim 1 wherein the compound is s775z:
3 . The compound of claim 1 wherein Z is imidazole, triazole, or tetrazole.
4 . The compound of claim 1 wherein G 1 and G 2 are
5 . The compound of claim 1 wherein G 3 is CO 2 H.
6 . The compound of claim 1 wherein G 3 is —(C═O)NHR C .
7 . The compound of claim 6 wherein R C is an arginylglycylaspartic acid or peptide, or immunoglobulin.
8 . The compound of claim 1 wherein J 1 and J 2 are SO 3 H.
9 . The compound of claim 1 wherein Q 1 and Q 2 are —(CH 2 ) 3 N + (CH 3 ) 3 .
10 . The compound of claim 1 wherein R 1 , R 2 , R 3 and R 4 are methyl.
11 . The compound of claim 1 wherein R W is —CH 2 (OCH 2 CH 2 ) 3 —O(C 1 -C 4 )alkyl.
12 . The compound of claim 1 wherein R 5 and R 6 are H.
13 . The compound of claim 1 wherein x and y are 1.
14 . The compound of claim 1 wherein the compound is represented by Formula II, III, or IV:
wherein R W is —(C 1 -C 4 )alkyl-(O(C 2 -C 4 )alkyl) m -O(C 1 -C 4 )alkyl, and m is 1-6.
15 . A method for imaging a targeted biological organism comprising:
a) conjugating a compound of claim 1 to a drug or biologic to form a bioconjugate wherein the bioconjugate has selective affinity for a targeted biological organism or biomolecule;
b) contacting the bioconjugate and the biological organism or biomolecule;
c) exciting the bioconjugate a suitable wavelength for inducing a fluorescence signal or photoacoustic signal; and
d) imaging the excited bioconjugate fluorescence signal or photoacoustic signal emanating from within the biological organism;
wherein the targeted biological organism is thereby imaged.
16 . The method of claim 15 wherein the bioconjugate is a bioconjugate of the compound s775z and an arginylglycylaspartic acid or immunoglobulin.
17 . The method of claim 15 wherein the biological organism is cancer tissue.
18 . A compound of Formula V:
or a salt thereof,
wherein
G 4 is pyridyl, pyridiniumyl, or
wherein L 1 and L 2 are each independently a substituted 2-indolyl group or substituted 2-indolium group, wherein the 2-indolyl or 2-indolium is optionally fused to a benzo ring;
R A is OR B , NR B R C , H, or halo;
R B and R C are each independently H, N-succinimidyl, —(C 1 -C 6 )alkyl, drug, or biologic;
Z 1 and Z 2 are each independently halo, N 3 , or a triazole substituted with polyethylene glycol wherein each triazole is optionally alkyl quaternized; and
n is 0-3;
wherein charged moieties when present in the compound are counterbalanced with counterions.
19 . The compound of claim 18 wherein the compound is:
20 . A method for synthesizing a sterically shielded heptamethine cyanine compound of Formula VI:
or a salt thereof;
wherein
L 1 and L 2 are each independently a substituted 2-indolyl or substituted 2-indolinyl group, wherein the 2-indolyl or 2-indolinyl is optionally fused to a benzo ring;
R A is OH, OCH 3 , OC(CH 3 ) 3 , or halo;
Z 1 and Z 2 are each independently a triazole substituted with polyethylene glycol wherein each triazole is optionally alkyl quaternized; and
n is 0-3;
wherein charged moieties present in the compound are counterbalanced with counterions; the method comprising:
a) contacting a pyridinium compound of Formula VIIa and an indolium compound of Formula VIIb under suitable Zincke reaction conditions to form the cyanine compound:
wherein
R A , Z 1 , Z 2 and n are as defined above;
X − and Y − are each independently a counterion;
J 2 is SO 3 H, CO 2 H, phenyl-SO 3 H, polyethylene glycol, or H;
J 4 is H; or
J 4 and J 2 taken together form a fused benzo ring; or
J 4 , R 3 and R 4 taken together form a fused benzo ring;
Q 2 is —(C 2 -C 6 )alkyl-X 2 or polyethylene glycol;
X 2 is N + (R D ) 3 , N(R D ) 2 , CO 2 H, SO 3 H, or —O(C 1 -C 6 )alkyl;
each R D is independently —(C 1 -C 6 )alkyl; and
R 3 and R 4 are each independently —(C 1 -C 4 )alkyl or H, when R 3 and R 4 do not form a fused benzo ring together with J 4 ;
wherein each fused benzo ring when present is substituted optionally with one or more SO 3 H; and
b) quenching the reaction;
wherein the sterically shielded heptamethine cyanine compound is thereby formed.