THIENYL-DIBENZOAZEPINES AND THEIR DERIVATIVES AS DONORS FOR XANTHENE-BASED SHORT-WAVE INFRARED (SWIR) DYES

Abstract

A near infrared dye comprising a counterion and a structure of Formula I

##STR00001##

wherein the at least one of R.sub.1 and R.sub.2 are selected from dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, and thienodihydrodibenzazepine, and X, R, R.sub.3-R.sub.4, R.sub.19, R.sub.20 and R.sub.22-R.sub.29 are as disclosed herein. Materials and compositions comprising the SWIR dye can absorb light at a wavelength of 800 nm to 1400 nm and release the energy in the form of light (fluorescence) or heat (non-radiative). The dyes can also convert the absorbed light to heat and ultrasound waves via the photoacoustic effect. The photoacoustic effect can be used in photoacoustic tomography to image biological materials or processes. Methods for synthesizing the SWIR dyes and materials comprising the same are also disclosed.

Claims

1. A short-wave infrared (SWIR) dye comprising a counterion and a structure of Formula I ##STR00032## wherein X is selected from the group consisting of O, Si and P; R is selected from the group consisting of hydrogen, C(O)OH, a substituted or unsubstituted linear or branched C.sub.1-C.sub.18 alkyl group, a substituted or unsubstituted linear or branched C.sub.2-C.sub.18 alkenyl group, a substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl group, a substituted or unsubstituted linear or branched C.sub.1-C.sub.18 alkoxy group, an ester group represented by the formula C(O)OA.sup.1, wherein A.sup.1 is a substituted or unsubstituted linear or branched C.sub.1-C.sub.18 alkyl group, a substituted or unsubstituted linear or branched C.sub.2-C.sub.18 alkenyl group or a substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl group, an amide group represented by the formula C(O)N(A.sup.2).sub.2, wherein A.sup.2 is selected from the group consisting of a substituted or unsubstituted linear or branched C.sub.1-C.sub.18 alkyl group, a substituted or unsubstituted linear or branched C.sub.2-C.sub.18 alkenyl group, a substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl group, and a substituted or unsubstituted C.sub.6-C.sub.10 aryl group, and an ether group represented by the formula CH.sub.2OA.sup.3 wherein A.sup.3 is selected from the group consisting of a substituted or unsubstituted linear or branched C.sub.1-C.sub.18 alkyl group, a substituted or unsubstituted linear or branched C.sub.2-C.sub.18 alkenyl group, and a substituted or when X is O, then R.sub.3 and R.sub.4 are absent, and when X is Si, then R.sub.3 and R.sub.4 are each singly bonded to the Si atom, and are independently selected from the group consisting of hydrogen, a substituted or unsubstituted C.sub.1-C.sub.18 linear or branched alkyl group, and a substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl group, and when X is P, then R.sub.3 is singly bonded to the P atom and is selected from the group consisting of hydrogen, a substituted or unsubstituted C.sub.1-C.sub.18 linear or branched alkyl group, and a substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl group, and R.sub.4 is singly bonded to the P atom and is a substituted or unsubstituted C.sub.1-C.sub.18 linear or branched alkyl group or a substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl group, or R.sub.4 is doubly bonded to the P atom and is an oxygen; R.sub.19, R.sub.20, R.sub.22, R.sub.23, R.sub.24, R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29 are each independently selected from the group consisting of hydrogen, sulfonate, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, and an alkoxy group having 1 to 20 carbon atoms, or wherein one or more pair(s) of R.sub.22 and R.sub.23, R.sub.24 and R.sub.25, R.sub.25 and R.sub.26, R.sub.26 and R.sub.27, R.sub.27 and R.sub.28, R.sub.28 and R.sub.29, together with the carbons to which they are attached form a saturated or unsaturated 6 membered ring; and R.sub.1 and R.sub.2 are both selected from one of the following Groups A, B and C: Group A R.sub.1 is hydrogen and R.sub.2 is a donor which is substituted or unsubstituted and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine; or Group B both R.sub.1 and R.sub.2 are the same donors, are substituted or unsubstituted, and are selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine; or Group C R.sub.1 and R.sub.2 are different donors and R.sub.1 is a donor which is substituted or unsubstituted and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine, and R.sub.2 is a donor which is substituted or unsubstituted and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, a thienodihydrodibenzazepine, 1-(thiophen-2-yl)piperidine, 1-(thieno[3,2-b]thiophen-2-yl)piperidine, 1-([2,2-bithiophen]-5-yl)piperidine, C.sub.2-C.sub.12 dialkyl amino, indolizine-3-yl, diphenylamino, and julolidinyl.

2. The SWIR dye of claim 1, wherein X is O, and R.sub.3 and R.sub.4 are absent; R is C(O)OH, or R is an ester group, an amide group, or an ether group and A.sup.1, A.sup.2, and A.sup.3 are independently selected from the group consisting of a linear or branched C.sub.1-C.sub.18 alkyl group, a linear or branched C.sub.2-C.sub.18 alkenyl group, a C.sub.3-C.sub.10 cycloalkyl group, and a substituted or unsubstituted C.sub.6-C.sub.10 aryl group that is substituted with 1 to 3 substituents independently selected from a halogen, sulfonate, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, and an alkoxy group having 1 to 20 carbon atoms.

3. The SWIR dye of claim 1, wherein X is O, and R.sub.3 and R.sub.4 are absent; R is C(O)OH or an ester group represented by the formula: C(O)OA.sup.1 wherein A.sup.1 is selected from a linear or branched C.sub.1-C.sub.6 alkyl group, or R is an amide group represented by the formula: C(O)N(A.sup.2).sub.2, wherein A.sup.2 is a C.sub.6-C.sub.10 aryl group which is optionally substituted by an amino group; and R.sub.1 and R.sub.2 are both selected from one of the following Groups A and B: Group A R.sub.1 is hydrogen and R.sub.2 is a donor which is substituted with 0 to 3 substituents and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine; and Group B both R.sub.1 and R.sub.2 are the same donors, are substituted with 0 to 3 substituents, and are selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine; wherein the substituents are independently selected from the group consisting of a halogen, sulfonate, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbons, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbons, and an alkoxy group having 1 to 20 carbon atoms.

4. The SWIR dye of claim 1, wherein X is O, and R.sub.3 and R.sub.4 are absent; R is C(O)OH or an ester group represented by the formula: C(O)OA.sup.1 wherein A.sup.1 is selected from a linear or branched C.sub.1-C.sub.6 alkyl group, or R is an amide group represented by the formula: C(O)N(A.sup.2).sub.2, wherein A.sup.2 is a C.sub.6-C.sub.10 aryl group which is optionally substituted by an amino group; and R.sub.1 and R.sub.2 are both selected from Group C: Group C R.sub.1 and R.sub.2 are different donors, R.sub.1 is a donor which is substituted with 0 to 3 substituents and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine, and R.sub.2 is a donor which is substituted with 0 to 3 substituents, and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, a thienodihydrodibenzazepine, 1-(thiophen-2-yl)piperidine, 1-(thieno[3,2-b]thiophen-2-yl)piperidine, 1-([2,2-bithiophen]-5-yl)piperidine, C.sub.2-C.sub.12 dialkyl amino, indolizine-3-yl, diphenylamino, and julolidinyl; wherein the substituents are independently selected from the group consisting of a halogen, sulfonate, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, and an alkoxy group having 1 to 20 carbon atoms.

5. The SWIR dye of claim 1, wherein X is O, and R.sub.3 and R.sub.4 are absent; R is C(O)OH or an ester group represented by the formula: C(O)OA.sup.1 wherein A.sup.1 is selected from a linear or branched C.sub.1-C.sub.6 alkyl group, or R is an amide group represented by the formula: C(O)N(A.sup.2).sub.2, wherein A.sup.2 is a C.sub.6-C.sub.10 aryl group which is optionally substituted by an amino group; and R.sub.1 and R.sub.2 are both selected from Group C: Group C R.sub.1 and R.sub.2 are different donors, R.sub.1 is a donor which is substituted with 0 to 3 substituents and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine and R.sub.2 is a donor which is substituted with 0 to 3 substituents and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, a thienodihydrodibenzazepine, 1-(thiophen-2-yl)piperidine, 1-(thieno[3,2-b]thiophen-2-yl)piperidine, 1-([2,2-bithiophen]-5-yl)piperidine, diethyl amino, and julolidinyl; wherein the substituents are independently selected from the group consisting of halogen, sulfonate, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, and an alkoxy group having 1 to 20 carbon atoms.

6. The SWIR dye of claim 1, wherein the counterion is selected from the group consisting of nitrite, sulfate, phosphate, bicarbonate, trifluoroacetate, pentafluoropropanoate, chloride, bromide, iodide, perchlorate, nitrate, benzenesulfonate, p-toluenesulfonate, methylsulfate, ethylsulfate, propylsulfate, tetrafluoroborate, tetraphenylborate, hexafluorophosphate, benzenesulfinate, acetate, trifluoroacetate, propionacetate, benzoate, oxalate, succinate, malonate, oleate, stearate, citrate, monohydrogen diphosphate, dihydrogen monophosphate, pentachlorostannate, chlorosulfonate, fluorosulfonate, trifluoromethansulfonate, hexafluoroarsenate, hexafluoroantimonate, molybdenate, tungstate, titanate, zirconate ions, and any combination thereof.

7. The SWIR dye of claim 1, wherein the counterion is selected from the group consisting of trifluoroacetate, pentafluoropropanoate, chloride, bromide, iodide, fluorosulfonate, and trifluoromethansulfonate.

8. The SWIR dye of claim 1, wherein R.sub.19, R.sub.20, R.sub.22, R.sub.23, R.sub.24, R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29 are each independently selected from the group consisting of hydrogen, sulfonate, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, an aryl group having 6 to 10 carbon atoms, and a heterocyclic group having 6 to 10 carbon atoms, and wherein 0 or 1 pair of R.sub.22 and R.sub.23, R.sub.24 and R.sub.25, R.sub.25 and R.sub.26, R.sub.26 and R.sub.27, R.sub.27 and R.sub.28, R.sub.28 and R.sub.29, together with the carbons to which they are attached from form a saturated or unsaturated six membered ring.

9. The SWIR dye of claim 1, wherein R.sub.19, R.sub.20, R.sub.22, R.sub.23, R.sub.24, R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, an alkyl group having 1 to 6 carbon atoms, and a phenyl group.

10. The SWIR dye of claim 1, wherein R.sub.19. R.sub.20. R.sub.22. R.sub.23. R.sub.24. R.sub.25. R.sub.26. R.sub.27. R.sub.28. and R.sub.29 arc each hydrogen.

11. The SWIR dye of claim 1, wherein the SWIR dye has one of the following structures: ##STR00033##

12. The SWIR dye of claim 1, wherein the SWIR dye absorbs light having a wavelength of from about 800 nm to about 1400 nm.

13. A composite comprising the SWIR dye of claim 1 in a polymer matrix.

14. The composite according to claim 13, wherein the polymer matrix is solid at room temperature.

15. A composite comprising the SWIR dye of claim 1 encapsulated in a phospholipid-polymer conjugate.

16. The composite according to claim 15, wherein the phospholipid-polymer conjugate is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG).

17. A method for making a SWIR dye, the method comprising steps of: (a) performing a CH arylation reaction by combining a Donor and an Acceptor of Formula II with a catalyst in a solvent to form a reaction mixture, ##STR00034## wherein R.sub.18 and R.sub.21 are independently selected from the group consisting of Cl, Br, I, and OSO.sub.2R.sub.52 wherein R.sub.52 is a hydrogen or C.sub.1-C.sub.4 alkyl; R.sub.19, R.sub.20, R.sub.22, R.sub.23, R.sub.24, R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29 are each independently selected from the group consisting of hydrogen and an alkyl group having 1 to 20 carbons, or wherein one or more pair(s) of R.sub.22 and R.sub.23, R.sub.24 and R.sub.25, R.sub.25 and R.sub.26, R.sub.26 and R.sub.27, R.sub.27 and R.sub.28, R.sub.28 and R.sub.29, together with the carbons to which they are attached from form a saturated or unsaturated six membered ring; and the Donor(s) is substituted or unsubstituted and comprises at least one from Group (X) and one from Group (Y): Group (X) dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, and thienodihydrodibenzazepine; and Group (Y) dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, thienodihydrodibenzazepine, 1-(thiophen-2-yl)piperidine, 1-(thieno[3,2-b]thiophen-2-yl)piperidine, 1-([2,2-bithiophen]-5-yl)piperidine, C.sub.2-C.sub.12 dialkyl amine, indolizine, diphenylamine, and julolidine, to thereby replace the groups at R.sub.18 and R.sub.21 with said Donor(s); (b) a ring opening reaction by transesterification with an alcohol to give the SWIR dye of Formula I ##STR00035## wherein X is O, and R.sub.3 and R.sub.4 are absent; R is selected from C(O)OH or an ester group represented by the formula C(O)OA.sup.1, wherein A.sup.1 is a linear or branched C.sub.1-C.sub.18 alkyl group; are each independently selected from the group consisting of hydrogen and an alkyl group having 1 to 20 carbons, or wherein one or more pair(s) of R.sub.22 and R.sub.23, R.sub.24 and R.sub.25, R.sub.25 and R.sub.26, R.sub.26 and R.sub.27, R.sub.27 and R.sub.28, R.sub.28 and R.sub.29, together with the carbons to which they are attached from form a saturated or unsaturated six membered ring; and R.sub.1 and R.sub.2 are each selected from one of the following Groups B and C: Group B both R.sub.1 and R.sub.2 are the same donors, both are substituted or unsubstituted, and are selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine; and Group C R.sub.1 and R.sub.2 are different donors, R.sub.1 is a donor which is substituted or unsubstituted and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, and a thienodihydrodibenzazepine and R.sub.2 is a donor which is substituted or unsubstituted and is selected from the group consisting of a dibenzazepinyl, a thienyldibenzazepine, a bithienyldibenzazepine, a thienodibenzazepine, a dihydrodibenzazepinyl, a thienyldihydrodibenzazepine, a bithienyldihydrodibenzazepine, a thienodihydrodibenzazepine, 1-(thiophen-2-yl)piperidine, 1-(thieno[3,2-b]thiophen-2-yl)piperidine, 1-([2,2-bithiophen]-5-yl)piperidine, C.sub.2-C.sub.12 dialkyl amino, indolizine-3-yl, diphenylamino, and julolidinyl.

18. The method for making the SWIR dye of claim 17, wherein the compound of Formula II is: ##STR00036##

19. A composition comprising the SWIR dye of claim 1 and a pharmaceutically acceptable carrier or a solid polymer matrix.

20. A method for imaging a biological sample, the method comprising steps of: (a) contacting the biological sample with an effective amount of the composition of claim 19; (b) exposing the biological sample and the composition to SWIR radiation; and (c) observing photoacoustic resonance or fluorescence in the biological sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0105] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0106] FIG. 1 shows the normalized absorption spectra of a dibenzazepine donor conjugated to thiophene (SCR-1), thienothiophene (SCR-2), or bithiophene (SCR-3) in CH.sub.2Cl.sub.2.

[0107] FIG. 2 shows the normalized emission spectra of SCR-1, SCR-2, and SCR-3 in CH.sub.2Cl.sub.2.

[0108] FIG. 3A shows the normalized absorbance spectra of a ratiometric nanoparticle for nitric oxide (rNP-NO) before addition of NO (100 ?m).

[0109] FIG. 3B shows the normalized absorbance spectra of rNP-NO after addition of NO (100 ?m).

[0110] FIG. 3C shows the normalized ratiometric turn-on (?.sub.SCR-NO/?.sub.IR-1061) of rNP-NO after addition of NO (100 ?M) and its vehicle control.

[0111] FIG. 3D shows the ratiometric turn-on (?.sub.SCR-NO/?.sub.IR-1061) of rNP-NO after treatment with various biological analytes for 1 hour. Concentrations were at 100 ?M, except for cysteine (Cys) (200 ?M), glutathione (GSH) (5 mM), and hydroxyl radical (1 ?M).

[0112] FIG. 3E shows photoacoustic (PA) images of rNP-NO (0.1, 0.2, or 0.3 g/mL) embedded in a tissue-mimicking phantom that was 3 cm thick. The mages were compiled from multiple experiments recorded using the same imaging conditions.

[0113] FIG. 3F shows quantified data from the data shown in FIG. 3E. In FIG. 3F, n=3 for all experiments. Error bars=standard error of mean (SEM). Statistical analysis was performed using a two-tailed t-test (?=0.05, ** P<0.05).

[0114] FIG. 4 shows a proposed reaction mechanism between NO (via N.sub.2O.sub.3) and SCR-NO in image a). In image a), SCR-NO and the free carboxylate are PA-active; however, after hydrolysis to the corresponding carboxylic acid product, spontaneous spiro-lactonization yields the closed PA-inactive product. Image b) of FIG. 4 shows the chemical structure of the NO-Responsive Probe (SCR-NO), PA-inactive turnover product form of SCR-NO, and the SWIR Reference Dye (IR-1061). Image c) of FIG. 4 shows a cartoon schematic showing the production of two SWIR signals before reaction with NO and one SWIR signal (from the reference IR-1061) after reaction with NO.

[0115] FIG. 5A shows normalized absorption spectra of SCR-NO in CH.sub.2Cl.sub.2 and NP-SCR-NO in water.

[0116] FIG. 5B shows normalized absorption spectra of IR-1061 in CH.sub.2Cl.sub.2 and NP-IR-1061 in water.

[0117] FIG. 5C shows the normalized absorption spectra of SCR-NO in CH.sub.2Cl.sub.2, IR-1061 in CH.sub.2Cl

[0118] FIG. 6A shows representative cross-sectional shortwave-infrared meso-patterned imaging (SWIR) photoacoustic (PA) images of the liver from mice treated with saline (control, n=3) and representative cross-sectional shortwave-infrared meso-patterned imaging (SWIR) photoacoustic (PA) images of the liver from mice treated with acetaminophen (APAP) (treatment group, n=4).

[0119] FIG. 6B shows a cartoon schematic identifying the liver in cross-sectional view.

[0120] FIG. 6C shows quantified ratiometric data from FIGS. 6A and 6B. The scale bar represents 5 mm. The statistical analysis was performed using a two-tailed t-test (?=0.05, * P<0.05).

DETAILED DESCRIPTION

[0121] Small molecule organic dyes may be attractive for clinical applications because of their tendency to metabolize in the cells and their potential for low toxicity. Disclosed herein is a method for making SWIR organic dyes by combining donor and acceptor groups. In one aspect, the choice of a good donoracceptor pair can significantly lower the optical bandgap of a dye due to the promotion of charge transfer events.

[0122] The novel SWIR dyes can be used in biological imaging such as in vivo photoacoustic or fluorescence imaging of tumor angiogenesis monitoring, blood oxygenation mapping, functional brain imaging, skin melanoma detection, methemoglobin measuring, etc. In one aspect, the disclosure provides new materials that absorb light in the SWIR region where biological tissues are most transparent. In a further aspect, the compositions allow for direct, real-time laser imaging of biological samples at a faster, more affordable rate than an MRI, while also potentially allowing real time analysis during surgery.

[0123] The xanthene-based dyes of the disclosure have outstanding photophysical properties and stimuli responses. For instance, the new SWIR xanthene-based PA imaging agents SCR-1, SCR-2 and SCR-3 have absorption maxima at 840, 950 and 1040, respectively.

[0124] These three dyes were made using the donor-acceptor-donor (D-A-D) design. These dyes were based on two factors: (i) a good overlap of the thiophene donor and xanthene acceptor to lower the bandgap of the dye due to charge transfer events, and (ii) an amino group connected to the thiophene to increase donor strength in the push-pull mechanism of xanthene-based dyes.

Donors

[0125] The xanthene dyes can be in a donor-acceptor (D-A) design or a donor-acceptor-donor (D-A-D) design. For the D-A-D design, the donors can be symmetrical or unsymmetrical. For symmetrical dyes, R.sub.1 and R.sub.2 are identical donors. For unsymmetrical dyes, R.sub.1 and R.sub.2 are not identical.

[0126] Although the structures described and drawn herein show the cationic charge on a single atom, the donors can be in resonance with one another such that the cationic charge can move from the donor at R.sub.1 to the donor at R.sub.2. R.sub.1 and R.sub.2 can be selected from one of the following Groups A, B and C:

Group A

[0127] R.sub.1 is hydrogen and R.sub.2 is a donor which is selected from a substituted or unsubstituted dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, and thienodihydrodibenzazepine;

Group B (Symmetrical Dyes)

[0128] Both R.sub.1 and R.sub.2 are the same donors, are substituted or unsubstituted and are selected from dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, and thienodihydrodibenzazepine; and

Group C (Unsymmetrical Dyes)

[0129] R.sub.2 is a different donor from R.sub.1, and R.sub.1 is a donor which is selected from a substituted or unsubstituted dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, and thienodihydrodibenzazepine, and R.sub.2 is a donor which is substituted or unsubstituted, and is selected from dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, thienodihydrodibenzazepine, 1-(thiophen-2-yl)piperidine, 1-(thieno[3,2-b]thiophen-2-yl)piperidine, 1-([2,2-bithiophen]-5-yl)piperidine, a C.sub.2-C.sub.12 dialkyl amine, indolizine derivative, diphenylamine, and julolidine.

[0130] In Group B, the dyes are symmetrical, such as XanthCR-880. In Group C, the dyes are unsymmetrical. For instance, the donor at R.sub.1 could be thienyldibenzazepine and at R.sub.2 could be bithienyldibenzazepine; or both R.sub.1 and R.sub.2 can be thienyldibenzazepine but one is substituted and the other is not.

[0131] The preferred structures of the donors are shown below.

1. Dibenzazepine

[0132] ##STR00009##

wherein R.sub.34, R.sub.35, R.sub.36, R.sub.37, R.sub.38, R.sub.39, R.sub.40, R.sub.41, R.sub.42, and R.sub.43 are independently selected from hydrogen, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, an aryl group having 6 to 10 carbon atoms, and a heterocyclic group having 3 to 16 carbon atoms. Preferably the dibenzazepine has 3 or fewer substituents, more preferably 1 substituent and most preferably no substituents. These dibenzazepine donors are derived from the following ring structure:

##STR00010##

[0133] The disclosure also includes dihydrogenated dibenzazepines as optional donors, wherein the above structures containing the R.sub.34, R.sub.35, R.sub.36, R.sub.37, R.sub.38, R.sub.39, R.sub.40, R.sub.41, R.sub.42, and R.sub.43 ligands are modified such that the carbons bonded to the R.sub.38 and R.sub.39 ligands each have an additional hydrogen attached thereto and the double bond between the carbons bonded to the R.sub.38 and R.sub.39 ligands is a single bond. These dihydrogenated dibenzazepine donors are sometimes referred to herein as dihydrodibenzazepinyl groups and are derived from a 10,11-dihydro-5H-dibenzo[b,f]azepine ring.

##STR00011##

2. Thienyldibenzazepine

[0134] ##STR00012##

wherein R.sub.34, R.sub.35, R.sub.36, R.sub.37, R.sub.38, R.sub.39, R.sub.40, R.sub.41, R.sub.42, and R.sub.43 are as described above. R.sub.44 and R.sub.45 are each independently selected from hydrogen, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, and an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms. Preferably, the thienyldibenzazepine has 3 or fewer substituents, more preferably 1 substituent and most preferably no substituents. These thienyldibenzazepine donors are derived from the following ring structure:

##STR00013##

[0135] The disclosure also includes dihydrogenated thienyldibenzazepines as optional donors, wherein the above-structures containing the R.sub.34-R.sub.45 ligands are modified such that the carbons bonded to the R.sub.38 and R.sub.39 ligands each have an additional hydrogen attached thereto and the double bond between the carbons bonded to the R.sub.38 and R.sub.39 ligands is, instead, a single bond. These dihydrogenated thienyldibenzazepine donors are sometimes referred to herein as thienyldihydrodibenzazepine groups and are derived from the following ring structure:

##STR00014##

3. Bithienyldibenzazepine

[0136] ##STR00015##

wherein R.sub.34, R.sub.35, R.sub.36, R.sub.37, R.sub.38, R.sub.39, R.sub.40, R.sub.41, R.sub.42, and R.sub.43 are as described above. R.sub.46, R.sub.47, R.sub.48 and R.sub.49 are each independently selected from hydrogen, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, and an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms. Preferably, the bithienyldibenzazepine has 3 or fewer substituents, more preferably 1 substituent and most preferably no substituents. These bithienyldibenzazepine donors are derived from the following ring structure:

##STR00016##

[0137] The disclosure also includes dihydrogenated bithienyldibenzazepines as optional donors, wherein the above-structures containing the R.sub.34-R.sub.43 and R.sub.46-R.sub.49 ligands are modified such that the carbons bonded to the R.sub.38 and R.sub.39 ligands each have an additional hydrogen attached thereto and the double bond between the carbons bonded to R.sub.38 and R.sub.39 ligands is instead a single bond. These dihydrogenated bithienyldibenzazepine donors are sometimes referred to herein as bithienyldihydrodibenzazepine groups and are derived from the following ring structure:

##STR00017##

Thienodibenzazepine

[0138] ##STR00018##

wherein R.sub.34, R.sub.35, R.sub.36, R.sub.37, R.sub.38, R.sub.39, R.sub.40, R.sub.41, R.sub.42, and R.sub.43 are as described above. R.sub.50 and R.sub.51 are each independently selected from hydrogen, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbons, an alkoxy group having 1 to 20 carbon atoms, and an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms. Preferably, the thienodibenzazepine has 3 or fewer substituents, more preferably 1 substituent and most preferably no substituents. These thienodibenzazepine donors are derived from the following ring structure:

##STR00019##

[0139] The disclosure also includes dihydrogenated thienodibenzazepines as optional donors, wherein the above-structures containing the R.sub.34-R.sub.43 and R.sub.50-R.sub.51 ligands are modified such that the carbons bonded to the R.sub.38 and R.sub.39 ligands each have an additional hydrogen attached thereto and the double bond between the carbons bonded to R.sub.38 and R.sub.39 ligands is instead a single bond. These dihydrogenated thienodibenzazepine donors are sometimes referred to herein as thienodihydrodibenzazepine groups and are derived from the following ring structure:

##STR00020##

1-(thiophen-2-yl)piperidine

[0140] ##STR00021##

wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are each independently selected from hydrogen, sulfonate, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbons, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, and an alkylether group having 2-20 carbon atoms and 1 to 5 oxygen atoms. Preferably, the 1-(thiophen-2-yl)piperidine has 3 or fewer substituents, more preferably 1 substituent and most preferably no substituents, in other words, most preferably all of R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are hydrogen.

[0141] The wavy line is globally used herein to refer to the location of the bond between the donor and the xanthene core structure.

1-(Thieno[3,2-b]thiophen-2-yl)piperidine

[0142] ##STR00022##

wherein R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are as described above. R.sub.12 and R.sub.13 are each independently selected from hydrogen, sulfonate, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, and an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms. Preferably, the 1-(thieno[3,2-b]thiophen-2-yl)piperidine has 3 or fewer substituents, more preferably 1 substituent and most preferably no substituents.

1-([2,2-Bithiophen]-5-yl)piperidine

[0143] ##STR00023##

wherein R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are as described above. R.sub.14, R.sub.15, R.sub.16, and R.sub.17 are each independently selected from hydrogen, sulfonate, halogen, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, and an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms. Preferably the 1-([2,2-bithiophen]-5-yl)piperidine has 3 or fewer substituents, more preferably 1 substituent and most preferably no substituents.

C.SUB.2.-C.SUB.12 .Dialkyl Amine

[0144] A C.sub.2-C.sub.12 dialkyl amine which is bonded to the xanthene core at the amine nitrogen, and is optionally substituted with 1 to 3 substituents selected from an alkenyl group having 2 to 10 carbon atoms, an alkynyl group having 2 to 10 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, an aryl group having 6 to 10 carbon atoms, and a heterocyclic group having 3 to 16 carbon atoms. Preferably, the C.sub.2-C.sub.12 dialkyl amine has 1 substituent and most preferably no substituents.

Indolizin-3-yl

[0145] Indolizin-3-yl which is bonded to the xanthene core at the 3-position and is substituted or unsubstituted. Preferably, the indolizine-3-yl donor is substituted with 1 to 3 substituents which are independently selected from halogen, sulfonate, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an aryl group having 6 to 10 carbon atoms, a heterocyclic group having 3 to 16 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, and an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms. Preferably, the indolizine-3-yl donor has 3 or fewer substituents; even more preferably the indolizine-3-yl donor is substituted at the 1- and 2-positions with an alkyl group having 1 to 5 carbons and an aryl group having 6 to 10 carbon atoms; and even more preferably, the indolizine-3-yl donor is substituted at the 1-position with an alkyl group having 1 to 4 carbon atoms and at the 2-position with an aryl group having 6 to 8 carbon atoms; and most preferably, the indolizine donor is 1-methyl-2-phenylindolizin-3-yl.

Diphenylamine

[0146] The diphenylamine preferably has the following cationic structure (the corresponding nonionic structure is not shown):

##STR00024##

wherein R.sub.30, R.sub.31, R.sub.32, and R.sub.33 are independently selected from hydrogen, an alkyl group having 1 to 20 carbons, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, and an aryl group having 6 to 10 carbon atoms, at least one combination of (R.sub.30 and R.sub.31) and (R.sub.32 and R.sub.33) together with N forming a substituted or unsubstituted pyrrolidine ring, a substituted or unsubstituted piperidine ring, a substituted or unsubstituted morpholine ring, a substituted or unsubstituted tetrahydropyridine ring or a substituted or unsubstituted cyclohexylamine ring.

Julolidine

[0147] Julolidine preferably has the following cationic structure (the corresponding nonionic structure is not shown):

##STR00025##

Julolidine is optionally substituted with 1 to 3 substituents independently selected from a halogen, sulfonate, hydroxy, amino, nitro, cyano, carboxy, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, an alkylether group having 2 to 20 carbon atoms and 1 to 5 oxygen atoms, an aryl group having 6 to 10 carbon atoms, and a heterocyclic group having 3 to 16 carbon atoms. Preferably, the julolidine donor is not substituted or has one substituent.

[0148] The wavy line is refers to the location of the bond between the donor and the xanthene core structure.

Pharmaceutically Acceptable Carriers and Biocompatibility

[0149] In various aspects, the dyes of the present disclosure can be given to a patient in a biocompatible composition in an amount effective to allow for PA or Fl analysis of a particular tissue, organ or system. As used herein, biocompatible refers to a material or composition that does not cause harm to living tissue. In one aspect, the SWIR dyes disclosed herein are biocompatible. Herein, the term biocompatible is used interchangeably with pharmaceutical or pharmaceutically acceptable. As used herein, pharmaceutically acceptable carriers means one or more of a pharmaceutically acceptable diluents, preservatives, antioxidants, solubilizers, emulsifiers, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, and adjuvants.

[0150] In another aspect, the present disclosure relates to pharmaceutical compositions suitable for parenteral administration, such as intravenous administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the SWIR dyes are being administered for imaging.

[0151] In various aspects, the present disclosure also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and an effective amount of the SWIR dye compounds of the present disclosure for bioimaging, a product of the method of the disclosure, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof. In a further aspect, the compounds of the present disclosure, a product of the method of the disclosure, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof, or any subgroup or combination thereof may be formulated into various pharmaceutical forms for administration purposes.

[0152] Pharmaceutical compositions of the present disclosure suitable for parenteral administration can include sterile aqueous or oleaginous solutions, suspensions, or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some aspects, the final injectable form is sterile and must be effectively fluid for use in a syringe. The pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0153] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, such as a HEPES Buffered Saline or similar solutions.

[0154] As used herein, nontoxic refers to a material or composition that does not kill cells or organisms. In a further aspect, the SWIR dyes disclosed herein are nontoxic.

Absorbance and Fluorescence of SWIR Dyes

[0155] In terms of brightness, luminosity is dependent on the extinction coefficient (molar absorptivity) of the fluorophores or their ability to absorb light, and the quantum efficiency or effectiveness at transforming absorbed light into emitted luminescence. The SWIR dyes themselves are not very fluorescent, but they are sufficiently fluorescent for brightness imaging. For instance, when the SWIR dye binds to proteins, the protein becomes more easily detectable.

Methods for Making SWIR Dyes

[0156] In an aspect, the disclosure relates to a method for making SWIR dyes, the method comprising: [0157] (a) performing a CH arylation reaction by combining a Donor and an Acceptor of Formula II with a catalyst in a solvent to form a reaction mixture,

##STR00026##

wherein R.sub.18 and R.sub.21 are individually selected from Cl, Br, I and OSO.sub.2R.sub.52 and R.sub.52 is a hydrogen or lower alkyl; [0158] wherein R.sub.19, R.sub.20, R.sub.22, R.sub.23, R.sub.24, R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29 are each independently selected from hydrogen or an alkyl group having 1 to 20 carbon atoms, or wherein one or more pair(s) of R.sub.22 and R.sub.23, R.sub.24 and R.sub.25, R.sub.25 and R.sub.26, R.sub.26 and R.sub.27, R.sub.27 and R.sub.28, R.sub.28 and R.sub.29, together with the carbons they are attached form a saturated or unsaturated 6 membered ring; and [0159] wherein the Donor(s) is substituted or unsubstituted and comprises: [0160] group (X) selected from dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, and thienodihydrodibenzazepine; and [0161] group (Y) selected from dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, thienodihydrodibenzazepine, 1-(thiophen-2-yl)piperidine, 1-(thieno[3,2-b]thiophen-2-yl)piperidine, 1-([2,2-bithiophen]-5-yl)piperidine, C.sub.2-C.sub.12 dialkyl amine, indolizine, diphenylamine, and julolidine, [0162] to thereby replace the groups at R.sub.18 and R.sub.21 with said Donor(s); [0163] (b) a ring opening reaction by transesterification with an alcohol to give the SWIR dye of formula I

##STR00027## [0164] wherein X is O, R.sub.3 and R.sub.4 are absent; [0165] R is selected from C(O)OH or an ester group represented by the formula C(O)OA.sup.1, wherein A.sup.1 is a linear or branched C.sub.1-C.sub.18 alkyl group; [0166] R.sub.19, R.sub.20, R.sub.22, R.sub.23, R.sub.24, R.sub.25, R.sub.26, R.sub.27, R.sub.28, and R.sub.29 are as described above; and [0167] R.sub.1 and R.sub.2 are independently selected from one of the following Groups B and C:

Group B

[0168] Both R.sub.1 and R.sub.2 are the same donors and both are substituted or unsubstituted, and are selected from dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, and thienodihydrodibenzazepine; and

Group C

[0169] R.sub.1 and R.sub.2 are different donors and R.sub.1 is a donor which is substituted or unsubstituted and is selected from dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, and thienodihydrodibenzazepine and R.sub.2 is a donor which is substituted or unsubstituted and is selected from dibenzazepinyl, thienyldibenzazepine, bithienyldibenzazepine, thienodibenzazepine, dihydrodibenzazepinyl, thienyldihydrodibenzazepine, bithienyldihydrodibenzazepine, thienodihydrodibenzazepine, 1-(thiophen-2-yl)piperidine, 1-(thieno[3,2-b]thiophen-2-yl)piperidine, 1-([2,2-bithiophen]-5-yl)piperidine, C.sub.2-C.sub.12 dialkyl amino, indolizine-3-yl, diphenylamino, and julolidinyl. In Group C, the dyes are unsymmetrical. For instance, the donor at R.sub.1 could be thienyldibenzazepine and at R.sub.2 could be thienodibenzazepine; or both R.sub.1 and R.sub.2 can be thienyldibenzazepine but one is substituted and the other is not.

[0170] Preferably, the compound formed in step (a) has the following structure:

##STR00028##

[0171] In one aspect, the solvent in step (a) is selected from N-methyl-2-pyrrolidone (NMP), N,N-dimethylacetamide (DMA), dimethylformamide (DMF), toluene, tetrahydrofuran (THF), dioxane, and any combination thereof.

[0172] In one aspect, in step (a), the reaction mixture is heated at a temperature from about 80? C. to about 150? C., or at about 80? C., 85? C., 90? C., 95? C., 100? C., 105? C., 110? C., 115? C., 120? C., 125? C., 130? C., 135? C., 140? C., 145? C., or about 150? C., or a combination of any of the foregoing values, or a temperature within a range encompassing any of the foregoing values. In another aspect, the reaction mixture can be heated for from about 6 hours to about 30 hours, or for about 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or about 24 hours, or a combination of any of the foregoing values, or for a time within a range encompassing any of the foregoing values. In one aspect, step (a) can be conducted in an inert atmosphere such as, for example, nitrogen.

[0173] In one aspect, step (a) further includes admixing a catalyst with the compound of Formula II and the Donor. The catalyst can be bis(triphenylphosphine)palladium(II) dichloride (PdCl.sub.2(PPh.sub.3).sub.2), Palladium(II)acetate (Pd(OAc).sub.2), tris(dibenzylideneacetone)dipalladium(0) (Pd(dba).sub.3)CHCl.sub.3, or any combination thereof. In a further aspect, from about 0.01 to about 0.1 moles of catalyst can be used per mole of compound of Formula II. Further in this aspect, about 0.01, 0.02, 0.03, 0.04 or about 0.05 moles of catalyst can be used, or a combination of any of the foregoing values, or an amount within a range encompassing any of the foregoing values.

[0174] In another aspect, step (a) further includes admixing a base with the compound of Formula II and the Donor. In still another aspect, the base can be potassium acetate (KOAc), sodium acetate (NaOAc), Cs.sub.2CO.sub.3, KO.sup.tBu, NaO.sup.tBu, K.sub.2CO.sub.3, Na.sub.2CO.sub.3, or any combination thereof. In one aspect, from about 2.0 moles to about 6.0 moles of base can be used per mole of compound of Formula II. Further in this aspect, about 2.0, 2.7, 3.0, 3.2, or about 3.3 moles to about 6.0, 5.5, 5.0, or about 4.5 moles of base can be used, or a combination of any of the foregoing values, or a number of moles within a range encompassing any of the foregoing values.

[0175] In still another aspect, step (a) further includes admixing a ligand with the compound of Formula II and the Donor compound. In a further aspect, the ligand can be triphenylphosphine (PPh.sub.3), Dicyclohexyl[2,4,6-tris(propan-2-yl)[1,1-biphenyl]-2-yl]phosphane (Xphos), 2,2-bis(diphenylphosphino)-1-1-binaphthyl (BINAP), (.sup.tBu).sub.2PMeHBF.sub.4, or any combination thereof. In an aspect, from about 1 to about 4 moles of ligand can be used per mole of catalyst, or about 1.5 to about 2.5, or about 2 moles of ligand per mole of catalyst can be used, or a combination of any of the foregoing values, or a number of moles within a range encompassing any of the foregoing values.

[0176] In an aspect, in step (b), the alcohol used in the transesterification reaction is also a solvent or cosolvent. The alcohol may be selected from methanol, ethanol, propanol, isopropanol, and butyl alcohol. In an aspect, the reaction mixture is heated to reflux. In another aspect, the reaction mixture can be heated for from about 6 hours to about 24 hours, or for about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 hours, or a combination of any of the foregoing values, or for a time within a range encompassing any of the foregoing values. In one aspect, step (b) can be conducted in an inert atmosphere such as, for example, nitrogen.

[0177] As noted above, the donors can be symmetrical or unsymmetrical in a D-A-D designed molecule. For symmetrical dyes, such as XanthCR-880, the donor is combined with the acceptor using twice the moles of the donor when compared to the moles of acceptor. On the other hand, an SWIR dye having an unsymmetrical D1-A-D2 design could be prepared by essentially the same synthesis except that some of the DI reactants are replaced with D2 reactants, such that: the total moles (D1+D2) is twice the moles of A. In addition, SWIR dyes having R.sub.1?H could be made by starting with an acceptor having only one leaving group and using equimolar amounts of acceptor and donor.

[0178] Also disclosed are SWIR dyes produced by the disclosed methods.

Compositions, Methods, and Devices Using the SWIR Dyes

[0179] In one aspect, disclosed herein is a composition including an SWIR dye disclosed herein and which optionally includes a carrier. In a further aspect, the carrier can be a pharmaceutically acceptable carrier. In still another aspect, the compositions can be biocompatible and/or nontoxic.

[0180] Also disclosed herein are methods for imaging a biological sample. In one aspect, the method includes the steps of (a) contacting the biological sample with a disclosed composition; (b) exposing the biological sample and the composition to SWIR radiation; and (c) observing PA emission in the biological sample. In a further aspect, the biological sample includes an organelle, a cell, a tissue, an organ, or any combination thereof.

[0181] Other potential applications include composites comprising the dyes in a polymer matrix for commodity items such as eyeglasses, night vision glasses or smart glasses, sensors, laser, and optoelectronic materials for electrical devices.

[0182] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.

EXAMPLES

[0183] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in 20? C.-22? C. or is at ambient temperature, and pressure is at or near atmospheric.

[0184] All chemicals and solvents were purchased from commercial suppliers and used without further purification unless other-wise specified. Tetrahydrofuran (THF), isopropyl alcohol (IPA), and hydrogen peroxide (H2O2, 30% w/v) were purchased from Macron Fine Chemicals. IR-1061, acetaminophen (APAP), potassium superoxide, tert-butyl hydroperoxide, and tetrakis(acetonitrile)copper(I) hexafluorophosphate were purchased from Sigma-Aldrich. mDSPE-PEG MW 5,000 was purchased from Laysan Bio. Sodium chloride, 50K centrifugal filters, and Mohr's salt were purchased from MilliporeSigma. DEA-NONOate was purchased from Cayman Chemical. 1? phosphate saline buffer (Corning) was purchased from Thermo-Fisher Scientific. 25 mM HEPES and L-cysteine were purchased from Oakwood Chemical. Glutathione (reduced) was purchased from Acros Organics Chemicals. Isopentyl nitrite was purchased from Alfa Aesar Chemicals. Thieno[3,2-b]thiophene, 2-bromothiophene, 5-bromo-2,2-bithiophene, and 5H-dibenzo[b,f]azepine were purchased from Sigma Aldrich or Fisher Scientific and used directly without further purification.

[0185] Thiophene (SCR-1), thienothiophene (SCR-2), and bithiophene (SCR-3) were incorporated into xanthene dye structures. The synthesis of the dyes began with the CN cross-coupling of the dibenzazepine core with the corresponding bromothiophene derivatives (see Schemes 1-3 below), followed by a direct diarylation reaction with 3,6-dibromofluoran. From here, the dyes were trapped into the opened form by converting them to the corresponding ethyl ester, giving rise to SCR-1, SCR-2, and SCR-3. It is noteworthy that the synthesis of the dyes occurs in three steps from the bromothiophene derivatives.

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[0186] An assessment of the photophysical properties of SCR-1, SCR-2, and SCR-3 in dichloromethane (DCM) revealed the absorption and emission bands of all three dyes extended well into the NIR to SWIR region, thus fulfilling the primary design objective (FIGS. 1 and 2, Table 1). The quantum yield (QY) of the dyes decreased as the absorption wavelength increased, which is common to these dyes. However, it is worth noting that the Stokes shift also increased with the conjugation length.

TABLE-US-00001 TABLE 1 Photophysical properties of SCR-1, SCR-2, and SCR-3 ?.sub.abs ?.sub.em QY Stokes ? Dyes (nm) (nm) (%) Shift (M.sup.?1cm.sup.?1) ? 10.sup.5 SCR-1 840 950 0.31 110 1.20 SCR-2 950 1100 0.036 150 1.24 SCR-3 1040 1260 0.016 220 0.80

Nitrous Oxide Nanosensor

[0187] An aspect of the invention is NO-responsive molecules, an example of which is SCR-NO that used the SCR scaffold.

[0188] The NO reactive species, SCR-NO (see the first PA-Active species in FIG. 4, image a)), was formed using an SCR dye with an NO-responsive unit (i.e., o-phenylenediamine trigger), thus locking the molecule in its open PA-active form. The reaction with NO (via the active species N.sub.2O.sub.3) would then generate an acyl triazole intermediate that can undergo spontaneous rate-limiting hydrolysis to yield the free dye. After this cleavage event, the dye could immediately close to the PAinactive lactone form (see FIG. 4). Having developed SCR-NO, a second SWIR-absorbing dye that does not respond to NO to serve as an internal reference was used. The internal reference, SWIR Reference Dye (IR-1061) was chosen because it had NO-stability and exhibited minimal spectral overlap with SCR-NO to enable ratiometric calibration.

[0189] To ensure the relative ratio of the probe and reference remained constant, a nanoparticle (NP) system that demonstrates minimal leaching of the encapsulated dye components was used. Moreover, the NP would ideally display excellent biocompatibility, display sufficient permeability to allow NO to diffuse into the core to access the encapsulated probe, and exhibit intrinsic liver targeting. The SCR-NO and IR-1061 were encapsulated using DSPE-PEG (a common phospholipids-polymer conjugate) to give NP-SCR-NO and NP-IR-1061, respectively. Both sets of NPs showed the predicted absorbance properties, where the spectra in water were similar to the un-encapsulated parent molecules in organic solvent (FIGS. 5A-5C). However, when the components were co-encapsulated (rNP-NO, ratiometric NP for NO), there was observed a favorable ?100 nm red-shift of the SCR-NO ?.sub.abs into the SWIR region (FIG. 3A). The corresponding change for IR-1061 was minimal (1 nm).

[0190] Next, the baseline stability of the NPs was tested. Each solution was found to be stable, as no change in the absorbance intensity was observed compared to the initial time point over several days. However, when rNP-NO was treated with NO, the signal corresponding to the probe decreased while the signal from the reference remained unchanged (FIG. 3B). The ratiometric turn-on, defined as ratio of ?.sub.SCR-NO/?.sub.IR-1061, changed from 1.00?0.02 to 0.79?0.06 after reaction with NO (FIG. 3C). Together, the results indicate the chosen 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) matrix imparts sufficient NP stability and is permeable to NO.

[0191] Beyond NO treatment, the rNP-NO were subjected to a panel of biologically relevant analytes found in the liver (FIG. 3D). For example, glutathione is present at concentrations in the mM range where it functions as an antioxidant to help the body remove toxins. When rNP-NO was treated with GSH at a concentration of 5 mM, no significant change in the ?.sub.SCR-NO/?.sub.IR-1061 was observed. The same results were found when cysteine, another thiol containing amino acid, was examined at 100 ?M. Next, rNP-NO was incubated with various reactive oxygen species (ROS) including hydrogen peroxide, peroxynitrite, tert-butyl hydrogen peroxide, superoxide, and hydroxyl radical. No evidence of probe decomposition was observed. This is an unexpected advantage, since many dye platforms are prone to oxidative decomposition, especially in the presence of highly reactive molecules like hydroxyl radical. Lastly, the stability of rNP-NO against iron and copper was evaluated, which can generate reactive oxygen species (ROS) via Fenton and Fenton-like chemistry, respectively. The ?.sub.SCR-NO/?.sub.IR-1061 ratio was again unaffected. Together, these results demonstrate that besides reactivity with NO, rNP-NO would be stable to conditions found in the liver when administered to live animals.

In Vitro Assessment of PA Properties

[0192] One determinant of a strong PA signal is the magnitude of the extinction coefficient (EC) of a molecule. This often supersedes the quantum yield (QY) since the former of these two terms dominates the PA brightness value (EC?(1?QY)). With a calculated value of 8.28?10.sup.4 M.sup.?1cm.sup.?1 for SCR-1, it was anticipated that strong ultrasound waves will be produced upon irradiation of rNP-NO. To test this, dense tissue-mimicking phantoms comprised of milk and agar were formulated. Individual samples containing each of the NPs were inserted to perform PA imaging. In the case of NP-SCR-NO, a PA signal was observed only when excited at the probe wavelength. Similarly, a PA signal originating from IR-1061 irradiation was the only ultrasound source from NP-IR-1061. However, PA signals were recorded for both the probe and reference when rNP-NO was subjected to the same in vitro test. As can be seen in FIGS. 3E and 3F, there was a dose-dependent increase in the PA intensity as the NP concentration increases; however, the ratio of ?.sub.SCR-NO/?.sub.IR-1061 remained unchanged.

Evaluation of rNP-NO in a Murine Model of Drug-Induced Liver Injury

[0193] For in vivo testing of rNP-NO in live mice, an advanced MSOT (multispectral optoacoustic (photoacoustic) tomography) system was employed to track rNP-NO. PA imaging was performed using the MSOT because: 1) it can be used to visualize the entire animal (including the liver) and present the processed PA images in an easy-to-interpret cross-sectional view; 2) signals from the probe (SCR-NO) and reference dye (IR-1061) can be readily isolated from each other, as well as from interfering endogenous PA-active pigments via spectral unmixing; and 3) it can rapidly switch between the two SWIR wavelengths to facilitate ratiometric calibration. It is noteworthy that the ratiometric feature of rNP-NO is superior to systems where monitoring occurs at a single wavelength. First, a cohort of BALB/c mice (female 6-8-weeks old) were treated with a solution of rNP-NO (3-4 mg/mL) via systemic administration (intraperitoneal (i.p.) injection). The biodistribution profile was determined by tracking rNP-NO circulation at two wavelengths. Within 15 minutes, rNP-NO had predominately localized to the liver.

[0194] Next, the utility of the nanosensor in a drug-induced liver injury (DILI) model was established. Drug-induced liver injury (DILI) is responsible for 60% of acute liver failure cases in the United States and is notoriously difficult to diagnose. Visualizing molecular level changes in the liver during DILI allows for early intervention and is an important step toward deciphering the mechanistic underpinnings of the disease. For example, NO is believed to contribute to the progression of DILI due to the upregulation of inducible nitric oxide synthases in hepatocytes. Moreover, it is one of the earliest indicators of liver failure as the immune response is rapidly and aggressively activated. By injecting BALB/c mice with acetaminophen (APAP), a drug known to cause DILI when given at high doses, it was reasoned that reliable monitoring of the overproduction of NO could be obtained using rNP-NO and SWIR PA imaging. Mice selected to generate the DILI model were intraperitoneal injection (i.p.) injected with a solution (200 ?L) of APAP at a dose of 300 mg/kg. In contrast, mice belonging to the control group were injected with the same volume of 0.9% saline. Then 16 hours were allowed to lapse thereby giving the mice time to succumb to DILI before rNP-NO was administered for SWIR PA imaging. Interestingly, the SWIR PA signal from the reference dye was localized to the liver for both groups and the overall signal intensity (color coded in green) was nearly identical (FIG. 6A, shown below). However, the SWIR PA signal from the probe could only be seen in the liver of the control animals and was clearly absent in the mice treated with APAP (FIG. 6B). Specifically, the (?.sub.SCR-NO/?.sub.IR-1061).sub.Final/(?.sub.SCR-NO/?.sub.IR-1061).sub.Init ratio for the control group was determined to be 2.13?1.15, whereas the corresponding ratio from the APAP treated cohort decreased to 0.56?0.31 (FIG. 6C). This result indicates that NO production was indeed upregulated in response to DILI and our probe exhibited sufficient sensitivity to detect this change.

CONCLUSION

[0195] CH activation chemistry was employed to develop a panel of synthetically accessible SWIR dyes and to highlight their utility for in vivo activity-base sensing by developing a SWIR PA nanosensor. By incorporating a dibenzazepine donor conjugated to thiophene (SCR-1), thienothiophene (SCR-2), and bithiophene (SCR-3) moiety, the absorbance band of the resulting xanthene dyes bathochromically shifted into the SWIR region. Capping of the pendant carboxylate group of SCR-1 with an NO-responsive unit resulted in SCR-NO, which remained open prior to encountering NO. This probe was able to stay on or turn off to design rNP-NO. When encapsulated, the SCR-NO dye resulted in a bathochromic shift to the SWIR region. Co-encapsulation of SCR-NO with IR-1061 as the internal reference dye allowed for a large dynamic range and reliable ratiometric confirmation of NO sensing. This is highlighted in the series of in vitro studies described above as well as in an in vivo DILI model, where rNP-NO was able to differentiate between mice primed for liver damage using APAP versus a saline control. The use of SWIR to monitor the probe and internal reference assures that the PA signals are originating from a deep region of the liver. Beyond activity-based sensing, the SCR panel of SWIR dyes can be readily transformed into contrast agents by capping carboxylate with an alcohol to yield stable ester products. These SWIR molecules are useful in cancer treatment (e.g., SWIR PA surgical guidance).

[0196] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0197] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0198] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0199] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0200] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is noted that the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0201] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

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