Fluorescent membrane intercalating probes and methods for their use

Abstract

The invention relates to a family of dyes which fluoresce in the UV-VIS, far red and near infrared wavelengths of the spectrum and possess asymmetric lipophilic alkyl chains. The dyes of the invention are soluble in commercially available membrane staining vehicles, are useful as probes for rapidly staining lipophilic structures such as membranes in cells or isolated from cells, and are well-retained therein. Methods of utilizing the dyes to detect stained cells both in vivo and in vitro are also disclosed.

Claims

1. A cyanine dye having a chemical structure selected from the group consisting of: ##STR00020## wherein Z is a biologically compatible counterion.

2. The cyanine dye of claim 1, wherein the counterion is selected from the group consisting of chloride, bromide, iodide, sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate, tetrafluoroborate, nitrate, an anion of an aromatic carboxylic acid and an anion of an aliphatic carboxylic acid.

3. The cyanine dye of claim 2, wherein the counterion is iodide.

4. An in vivo method for detecting abnormal cell shedding rates of mature surface epithelial cells of a warm-blooded animal, comprising the steps of: labeling mature surface epithelial cells at a target site of the animal with the cyanine dye of claim 1, and monitoring the site for the presence or absence of the cyanine dye following said labeling step.

5. The method of claim 4, wherein the counterion is selected from the group consisting of chloride, bromide, iodide, sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate, tetrafluoroborate, nitrate, an anion of an aromatic carboxylic acid and an anion of an aliphatic carboxylic acid.

6. The method of claim 5, wherein the counterion is iodide.

7. The method of claim 4, wherein the site is a mucosal surface.

8. The method of claim 4, wherein the mucosal surface lines the surface of the gastrointestinal tract, the respiratory tract, or the genitourinary tract.

9. The method of claim 7, wherein the site is a mucosal surface of the stomach.

10. The method of claim 7, wherein the site is a mucosal surface of the colon.

11. The method of claim 4, wherein cell shedding is detected by observing changes in the level of the cyanine dye at the site at a pre-selected time following said labeling step.

12. The method of claim 4, further comprising the step of exciting the target site with a light source wherein the light source has a wavelength of from about 600 nm to 1000 nm.

13. The method of 4, wherein the epithelial cells are labeled by direct application of a labeling composition comprising the cyanine dye to the site.

14. The method of claim 13, wherein said labeling composition further comprises a pharmaceutically acceptable vehicle.

15. The method of claim 4, wherein said labeling step comprises applying to mature surface epithelial cells at a target side a labeling composition comprising the cyanine dye.

16. The method of claim 15, wherein said monitoring step comprises observing the rate at which the cyanine dye is lost from the target site.

17. The method of claim 15, wherein said labeling composition further comprises a pharmaceutically acceptable vehicle.

Description

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(1) Reference will now be made in detail to the presently preferred embodiments of the invention. The invention provides compounds that are novel membrane intercalating dyes (also referred to herein as probes) that fluoresce in the UV-VIS, far red and near infrared segments of the spectrum and methods for their use. The invention also provides labeling compositions comprising the probes solubilized in a labeling vehicle. The probes are solubilized in a vehicle compatible with the biological and/or solvent sensitivities.

(2) The compounds contain lipophilic tails (referred to herein in the structures as R and R that in some embodiments are equal in length while in some preferred embodiments, RR.

(3) It is contemplated that the probes of the present invention may be used in conjunction with other labeling techniques and reagents for multiparameter cell tracking and sorting procedures. Usefulness of a dye in combination with others is traditionally determined by two aspects of spectra of light energy interactions: (1) the extent to which a dye molecule is excited by a single illumination wavelength or narrow band of wavelengths and (2) the extent to which each excited dye molecule emits light of wavelength sufficiently different from the other dyes so as to be discernible as a unique color or peak. The first aspect enables the user to illuminate the multiply-stained biological sample with a single wavelength and the second aspect enables the user to observe and record different colors of emission, each of which is associated with a particular cell type or a structure.

(4) There are a number of new detectors under development in which the entire spectrum of light is collected and then the curves characteristic of each probe's emission are devolved from the aggregate signal taken over the entire spectrum. It is also contemplated that the probes of the present invention will be readily detected using such instruments.

(5) The probes of the invention, when used as membrane-intercalating dyes selectively stain cell membranes in vitro, ex vivo and in vivo and do not undesirably affect the nature of the cells. The probes and thus the cells or other lipophilic structures to which they become attached can be readily identified by the fluorescence they emit. In addition, the compounds of the present invention are not cytotoxic when used at appropriate concentrations, are stably retained in cell membranes, and stain cells rapidly. The probes need only be applied to cells for a few minutes to achieve staining intensities 100-1000 times greater than background autofluorescence. Another beneficial aspect of the compounds of the present invention is that they are soluble in isotonic salt free diluents suitable for membrane labeling such as Diluent C (Sigma-Aldrich Corporation). The probes of the present invention are useful generally as agents for cell labeling, cell sorting and cell tracking in vitro and in vivo for both basic (laboratory) and clinical research. For examples of such uses, see U.S. Pat. Nos. 5,385,822; 5,256,532, the disclosures of which are incorporated by reference herein; and U.S. Pat. Nos. 4,859,584; 4,783,401 and 4,762,701. Such probes will be useful, for example, as research reagents for use in existing flow cytometers and confocal imaging systems which have FR and/or NIR capabilities.

(6) The probes of the present invention will 1) bind to cells in sufficient numbers to give a good signal compared to autofluorescence; 2) not be toxic to the cells at that level, and 3) be retained in the cell membrane long enough for tracking and/or sorting of particular subgroups of cells to be completed.

(7) The lipophilic nature of the probes of the present invention provides for the efficient incorporation of the probes into various lipid containing or hydrophobic structures, including cell and viral membranes, liposomes, microspheres and the like. When incorporating the probes into cells and virions, a labeling composition comprising the probe and an aqueous labeling vehicle is mixed with the target to be labeled. The labeling composition contains a cyanine dye in a vehicle (diluent) that is safe for application and that provides reproducible cell labeling. Osmolarity regulating agents in which cyanine dyes form stable solutions for at least as long as required for labeling can be used. Acceptable osmolarity regulating agents may be selected from sugars including monosaccharides such as glucose, fructose, sorbose, xylose, ribose, and disaccharides such as sucrose; sugar-alcohols including mannitol, glycerol, inositol, xylitol, and adonitol; amino acids including glycine and arginine; and certain Goods buffers such as N-tris(hydroxymethyl)-methyl-3-aminopropanesulfonic acid. Small amounts of buffering agents may be added to the labeling medium to regulate hydrogen ion concentration (pH) to physiological and/or nontoxic levels. Other conventional agents, such as antibiotics and preservatives, may be also be employed in the vehicle, but only to the extent that they do not create salt concentrations that induce rapid formation of dye micelles or aggregates.

(8) When incorporating the probes into microspheres or liposomes and like materials which can tolerate exposure to non-aqueous labeling vehicles without detrimental effect, a labeling composition comprising the probe and a non aqueous aqueous labeling vehicle is mixed with the target to be labeled. Non aqueous labeling vehicles include polar organic solvents such as ethanol, dimethyl formamide, dimethylsulfoxide, and the like.

(9) Despite their lipophilic nature, we have found that the probes of the present invention are sufficiently soluble in aqueous vehicles to allow efficient and rapid staining of liphophilic structures (membranes and the like) which are detrimentally affected by exposure to polar organic solvents.

(10) Research shows that good solubility in DMF is advantageous for indirectly labeling distant cells in tissue preparations by coating high concentrations of dye on filter nylon falters and inserting them into tissue. See B. Fritzch et al., Brain Res. Bull., 66 249-258 (2005).

(11) The detection step can employ a luminescence microscope or other optical imaging apparatus such as e.g., a fiber optic diagnostic device such as a cystoscope or endoscope and the like, having a filter for absorption of scattered light of the excitation wavelength and for passing the wavelength that corresponds to the fluorescence corresponding to the particular dye label used with the specimen. Preferred methods of observation and analysis include direct visualization with a microscope fitted with a light source and filters appropriate to the excitation and emission wavelengths, and use of a camera attached thereto.

(12) A preferred method of the invention for cell separation and enumeration of live cells appropriately stained with this class of probe reagents, is by use of a flow cytometry apparatus such as a FACSCalibur analyzer or FACSVantage cell sorting instrument. These instruments illuminate a mixed cell population, for example at a given wavelength with an argon laser source of light, and use an emission signal from each cell detected in a moving fluid such as a buffer, to sort each cell as it is flowing past the detector using a variety of bandpass filters for collection of emitted light using techniques well known in the art. The apparatus can count and/or collect cell populations yielding both data and cell fractions for further analysis and use.

(13) In another preferred embodiment, cells are labeled with the probes of the present invention and one or more additional cell markers which also bind to the labeled cells. The probes and the additional cell markers are then detected by flow cytometry. Alternatively, markers may be selected that recognize cells not labeled with the probes of the present invention. Detection of multiple markers in addition to or instead of the probes of the present invention enables identification and/or separation of specific subgroups of cells based on multiple phenotypes. Many additional cell markers such as fluorescent-labeled monoclonal antibodies specific for individual cell surface ligands and the like are known in the art.

(14) Recent advances in technology have made it possible to do fluorescence imaging not only at the microscopic (cellular and subcellular) level but also at the macroscopic (whole tissue or whole body) level. The probes of the present invention provide useful solutions to the problem of spectral pollution or spill, which is frequently encountered when doing multiprobe studies using confocal imaging or other quantitative microscopy techniques. In addition, the NIR probes, when combined with macroscopic imaging methods, provide a useful alternative to radiolabels for monitoring immune cell trafficking, localization and redistribution in intact animals as well as potential for use in photodynamic diagnosis and/or phototherapy.

(15) In another preferred embodiment, the probes of the present invention are administered to a subject for detection of abnormal epithelial cell shedding rates in vivo such as described in U.S. Pat. No. 5,804,389. In this process, the abnormal shedding of epithelial cells in a warm-blooded animal is determined. The process includes the steps of labeling mature surface epithelial cells with the compounds of the present invention at a target site, exposing the cells to light of an excitation wavelength and thereafter monitoring the site for the presence or absence of the label, and observing by the loss of detectable label over a pre-defined period of time. The present invention also provides a method for diagnosing disease states characterized by such abnormal cell shedding rates amongst mature epithelial cells of a warm-blooded animal, comprising labeling mature epithelial cells, determining the shedding rate of the labeled cells and comparing the shedding rate of the labeled cells to the known shedding rate of similarly located healthy epithelial cells.

(16) The probes of the present invention can be utilized to determine abnormal shedding rates on any epithelial surface of the body. Surfaces include those of the stomach, biliary tract, colon, urinary tract, blood vessels, pulmonary tract including the nasal cavity, cornea, esophagus, pancreatic duct, small intestine, and genital organs including the vagina and ovarian duct and the prostate gland. Preferably, the composition in solution form is administered by direct application (e.g. spraying) onto the surface of the epithelial mucosa under direct vision by endoscope, by flooding the surface with a solution, or by orally administering the solution to the subject in the form of a drink.

(17) When in vivo use in humans is contemplated, a solution of any of the compounds of the present invention can be prepared by dissolving an effective amount of the probe in an iso-osmotic, aqueous and preferably salt-free solvent miscible with both water and polar organic solvents. The concentration levels of dyes in compositions for in vivo use according to the present invention will be similar to the concentration levels used in the previously-known in vitro cell staining applications of those dyes. The precise concentration to be administered can be varied and can be readily optimized. The volume of probe composition to be administered will vary depending upon the concentration of the cyanine dye in the composition and upon the size of the target site. The administration volume may vary, for example, from about 1 to 100 ml and can be readily optimized. An administration volume of about 10 ml of the probe composition can be used in many applications. Administration route will vary depending upon the type of cells to be labeled. As indicated above, for staining of epithelial cells may be best achieved by oral administration or direct application. Other modes of administration, such as subcutaneous, intramuscular and intravenous injection and the like are also contemplated.

(18) According to the preferred method of the invention, the cyanine dye label is detected by exposing the site of labeled cells to excitation light and observing and/or measuring the intensity of the fluorescence.

(19) The probes of this invention can be synthesized, as set forth in detail in the examples below.

(20) The UV-Vis, FR and NIR probes of the present invention can be detected by commercial cytometry instrumentation with the ability to excite fluors which absorb in the 400-700 nm range and emit in the UV-VIS range from 400-600 nm, FR from 600-700 nm and NIR >700 nm range (e.g, BD FACSVantage, FACSCalibur, Beckman Coulter Altra, and Cytomation MoFlo flow cytometer/sorters, and BioRad 1024ES or 1024MP confocal imaging systems). The advent of small benchtop cytometers (e.g. volume capillary cytometer in development by Biometric Imaging/BD which incorporate inexpensive diode lasers and diode detection systems will provide additional detection systems to quantify NIR fluorescence.

(21) This near infrared/far red wavelength also is advantageous in that the background fluorescence in this region normally is low in biological systems and high sensitivity can be achieved.

(22) A preferred labeling composition of the present invention comprises a cyanine dye of the formula:
[A-CR.sub.1CR.sub.2YCR.sub.3CR.sub.4B].sup.+Z.sup.

(23) wherein Y

(24) is selected from the group consisting of

(25) CR.sub.1, CR.sub.1CR.sub.2CR.sub.3, CR.sub.5, CR.sub.6CR.sub.7CR.sub.8, CR.sub.9CR.sub.10CR.sub.11CR.sub.12CR.sub.13, and

(26) ##STR00001##

(27) wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is independently H, halogen or an alkyl group having 1-4 carbons;

(28) X is selected from the group consisting of H, halogen, O-alkyl, O-aryl, S-alkyl and S-aryl;

(29) Z is a biologically compatible counterion;

(30) A- is a structure selected from the group consisting of

(31) ##STR00002##

(32) and B is selected from the group of structures consisting of

(33) ##STR00003##

(34) wherein D and E are each independently O, S or CR.sub.14R.sub.15, where R.sub.14 and R.sub.15, which may be the same or different, are independently alkyl groups having 1-6 carbons. Alternatively, R.sub.14R.sub.15 taken in combination complete a 5- or 6-membered saturated ring. Preferably X equals Y (yielding a symmetrical cyanine). In some preferred embodiments, both D and E are CR.sub.14R.sub.15, where CR.sub.14R.sub.15 are methyl or ethyl, more typically methyl.

(35) Each of R.sub.16-R.sub.47 is independently H, halogen, phenyl (optionally substituted with halogen, cyano, and nitro), phenylsulfonate, NO.sub.2 or an alkyl group of 1-4 carbons.

(36) R and R are each independently linear or branched hydrocarbons, saturated or containing unsaturated double bonds having 7-36 carbons, with the proviso that one of R and R must be at least 3 carbons.

(37) Z is a counterion that is typically an anion that balances the intrinsic positive charge of the cyanine dye and is present in such a number and with such a total charge as to make the overall molecule electrically neutral. As used herein, a substance that is biologically compatible is not toxic as used, and does not have a substantially deleterious effect on biomolecules. Such compounds also must not undesirably affect cell viability in the concentrations required for labeling. Accordingly, pharmaceutically acceptable forms of the cyanine dye other than the iodide salt may be employed in some instances, including other pharmaceutically acceptable salts. Examples of useful counterions for dyes having a net positive charge include, but are not limited to, chloride, bromide, iodide, sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate, tetrafluoroborate, nitrate and anions of aromatic or aliphatic carboxylic acids. Selection of an appropriate anion, however will be limited by the particular anions' affect on solubility, since it is known that the anion associated with the various lipophilic molecules can effect the solubility of the compound.

(38) In a preferred embodiment, the counter ion is iodide. In certain instances, such as for in vivo administration of the probes, it may be preferable to substitute the iodide counterion with a chloride, since some individuals are allergic to iodine.

(39) In one embodiment, the cyanine dye has a chemical structure selected from the group consisting of:

(40) ##STR00004##

(41) The present invention is further described in the following examples which are provided for illustrative purposes only and are not to be construed as limiting. Standard techniques well known in the art or the techniques specifically described below are utilized.

Example 1

Synthesis of Unsubstituted FR Cyanine Dyes F279A, F280A, F283A, F284A, F285A, F299A, F300A with Approximately 665 nm Emissions

(42) These membrane probes with the above-referenced emissions were prepared using the same generic synthetic scheme and reaction conditions as described below using the appropriate alkyl iodide or alkyl 4-chlorobenzene sulfonate ester or unsaturated alkyl 4-chlorobenzene sulfonate ester to provide the required hydrocarbon tail length.

(43) ##STR00005##

(44) Preparation of 1-tetradecyl-2,3,3-trimethylindolinium Iodide (compound 5) was carried out according to the following protocol. Tetradecyl-1-(4-chlorobenzenesulfonate): To a stirred solution of tetradecanol (26.3 g, 0.123 mol, Aldrich Chemical Co., Milwaukee, Wis.) and 4-chlorobenzenesulphonyl chloride (28.48 g, 0.123 mol, Aldrich) in dichloromethane (500 ml) at room temperature is added triethylamine (28 ml, Aldrich) in dichloromethane (200 ml) dropwise. The resulting solution is stirred for 48 hours. The reaction mixture is then washed with water (3400 ml) and the dichloromethane layer dried over sodium sulfate and concentrated. The crude solid obtained was recrystallized from methanol to provide pure tetradecyl-1-(4-chlorobenzenesulphonate) (29.9 g, 62%) as a white solid. 200 MHz proton NMR (CDCl.sub.3): 0.88 (t, Jm 7.0 Hz, 3H), 1.10-1.80 (m, 24H), 4.05 (t, J=7.0 Hz, 2H), 7.50-7.60 (m, 2H), 7.80-7.90 (m, 2H).

(45) 2,3,3-trimethyl-(3H)-indoleine (4) (6.36 g, 0.04 mol, Aldrich) and tetradecyl-1-(4-chlorobenzenesulfonate) (15.52 g, 0.04 mol) are heated together with stirring at 130-140 C. for 3 h. The reaction mixture is then cooled to room temperature and dissolved in ethanol (200 ml). A saturated solution of potassium iodide (200 ml) is added and this mixture is stirred for 30 mins. 1.5 L of distilled water is then added and after a further 15 minutes stirring, the solid precipitate is collected, washed with water and dried under vacuum. The crude material is recrystallized from ethyl acetate to provide pure intermediate compound 5 (11.9 g, 62%). 300 MHz proton NMR (CDCl.sub.3): 0.88 (t, J=7 Hz, 3H), 1.25-1.50 (m), 1.67 (s, 6H), 1.93 (m, 2H), 3.13 (s, 3H), 4.70 (t, J=7.70 Hz, 2H), 7.60-7.66 (m, 4H).

Preparation of 1-docosanyl-2,3,3-trimethylindolinium Iodide (6)

(46) Docosanyl-1-(4-chlorobenzenesulfonate): To a stirred solution of docosanol (47.8 g, 0.15 mol) and 4-chlorobenzenesulphonyl chloride (34.01 g, 0.16 ml, Aldrich) in dichloromethane (500 ml) at room temperature is added triethylamine (33.5 ml, Aldrich) in dichloromethane (200 ml) dropwise. The resulting solution is stirred for 48 h. The reaction mixture is then washed with water (3400 ml), the organic layer dried over sodium sulfate and concentrated to 400 ml to initiate crystallization of the product. After cooling and aging the precipitate is collected by filtration and dried under vacuum to provide pure docosanyl-1-(4-chlorobenzenesulphonate) (48.9 g, 73%) as a white solid. 300 MHz proton nmr (CDCl.sub.3): 0.88 (t, J=7.0 Hz, 3H), 1. 1 5-1.40 (m), 1.55-1.75 (m, 2H), 4.02 (t, J=7.0 Hz, 2H), 7.48-7.60 (m, 2H), 7.78-7.90 (m, 2H).

(47) 2,3,3-trimethyl-(3H)-indoleine (6.3 g, 0.04 mol, Aldrich) and docosanyl-1-(4-chicrobenzenesulfonate) (20.0 g, 0.04 mol) are heated together with stirring at 130-140 C. for 3 h. The reaction mixture is then cooled to room temperature and the waxy solid dissolved in ethanol (250 ml). A saturated solution of potassium iodide (200 ml) is added and this mixture stirred for 30 mins 1.0 L of distilled water is then added and after a further 15 minutes stirring, the solid precipitate is collected, washed with water and dried under vacuum. The crude material is recrystallized from dichloromethane/hexane to provide pure intermediate compound 6 (14.5 g, 61%). 200 MHz proton NMR (CDCl.sub.3): 0.87 (t, J=7.0 Hz, 3H), 1.15-1.50 (m), 1.67 (s, 6H), 1.85-2.00 (m, 2H), 3.10 (s, 3H), 4.70 (t, J=7.7 Hz, 2H), 7.55-7.70 (m, 4H).

Preparation of 1-docosanyl-2-[(4-N-phen-N-acetyl-amino)-1,3-butadienyl]-3,3-dimethylindolinilim iodide (7)

(48) A solution of (6) (2-38 g, 4 mmol) and malonaldehyde bisphenylimine hydrochloride (1.10 g, 4.4 mmol, TCI America, Portland, Oreg.) in acetic anhydride (30 ml) is heated at 100-110 C. for 1 h, cooled to room temperature and filtered. The filtrate is then diluted with 300 ml of water and placed in a refrigerator at 0-5 C. for h. The resulting precipitate is collected and dried under vacuum to provide (7) (0.65 g, 21%). 300 MHz proton NMR (CDCl.sub.3): 0.88 (t, J=6.5 Hz, 3H), 1.20-1.40 (m), 1.70 (m), 1.80 (s, 6H), 2.24 (s, 3H), 4.36 (t, J-z 7.5 Hz, 2H), 5.79 (t, Jm 12.8 Hz, 1H), 6.71 (d, J=15.0 Hz, 1H), 7.25-7.70 (m, 10H).

(49) Intermediate compound 7 (0.544 g, 0.71 mmol) and intermediate 5 (0.343 g, 0.71 mmol) are heated together in refluxing dichloromethane (10 ml) containing 10 drops of triethylamine for 3 h. The reaction is monitored by TLC (5% methanol in dichloromethane). The reaction mixture is then concentrated by rotary evaporation and the residue crystallized from methanol at 20 C. overnight. The solid is collected and purified further by silica gel flash column chromatography eluting with 5-7.5% methanol in dichloromethane. Pure fractions are combined and concentrated to provide compound 8 (210 mgs, 30%). Purity >97% by HPLC. 300 MHz proton NMR (CDCl.sub.3): 0.88 (t, J=6.3 Hz, 6H), 1.25-1.55 (m), 1.66 (m), 1.81 (s, 16H), 4.04 (t, J=7.4 HZ, 4H), 6.29 (d, J=13.6 Hz, 2H), 6.78 (t, J=12.4 Hz, 1H), 7.05 (d, J=7.8 Hz, h.), 7.20-7.70 (m, 2H), 7.33-7.38 (m, 4H), 8.32 (t, J=13.0 Hz, 2H). Electrospray mass spectroscopy. M=860. UV/VIS (ethanol): ma,x=648 nm, c=189,270M-1 cm-1. Fluorescence (ethanol): excitation max.=653 nm, emission max.=672 nm.

(50) Probe with this emission wavelength was selected for synthesis because it is red shifted by >100 nm compared with most other visible fluorescent probes used for cell labeling and there-fore has minimum spectral overlap with them. Such probe could therefore be ideal for use in flow cytometry and confocal microscopy when multiprobe labeling is required. Varying alkyl chain lengths can be incorporated as needed to adjust strength/stability of membrane association.

Example 2

Synthesis of FR Probe F288A

(51) This compound is prepared as shown in Scheme 1 above using similar types of reactions and conditions but using 5-tert-buty-2,3,3-trimethyl(3H)indolenine as the starting material.

Example 3

Synthesis of FR Probe F289A

(52) This compound is prepared as shown in Scheme 1 above using similar types of reactions and conditions but using 5-bromo-2,3,3-trimethyl(3H)indolenine as the starting material.

Example 4

Synthesis of NIR Probe F277A

(53) This compound is prepared as shown in Scheme 2 below, using similar types of reactions and conditions to those already described above.

Example 5

Synthesis of NIR Probe F301A

(54) This compound is prepared as shown in Scheme 2 below, using similar types of reactions and conditions to those already describe above but using 2,3,3-trimethyl(3H)indolenine as the starting material.

(55) ##STR00006##

Example 6

Synthesis of NIR Probes F291A

(56) This compound is prepared as shown in Scheme 3 below, using similar types of reactions and conditions to those already describe above but using 2,3,3-trimethyl(3H)indolenine as the starting material and displacing the chloro functionality on the bridging cyclohexene ring with a thiophenyl group upon reaction with sodium benzthiolate in dimethylformamide at room temperature.

(57) ##STR00007##

(58) Intermediate 17 was prepared in 28% yield from cyclohexanone according to techniques known in the art. The optimal conditions for coupling of 17 with 2 mole equivalents of 10 is reflux in ethanol containing sodium acetate. The resulting compound is treated with potassium iodide followed by purification using silica gel chromatography.

Example 7

Synthesis of Probe F295B

(59) This compound is prepared as shown in Scheme 1 above using similar types of reactions and conditions but using 6-phenyl-2,3,3-trimethyl(3H)indolenine as the starting material for one side of the headgroup and 2,3,3-trimethyl(3H)indolenine for the other side.

Example 8

Synthesis of Probe F296B

(60) This compound is prepared as shown in Scheme 1 above using similar types of reactions and conditions but using 6-phenyl-2,3,3-trimethyl(3H)indolenine as the starting material for both sides of the headgroup.

Example 9

Synthesis of Probe F297B

(61) F297B was prepared from F295B, upon treatment with 20% fuming sulfuric acid in a solution of concentrated sulfuric acid. Yields were 65%.

Example 10

Synthesis of Probe F298B

(62) F298B was prepared from F296B upon treatment with 20% fuming sulfuric acid in a solution of concentrated sulfuric acid. Yields were 79% respectively.

Example 11

Synthesis of Probe F305B

(63) F305B was synthesized as shown below.

(64) ##STR00008##

(65) Reaction of 2-methylthio-benzthiaxole with tetradecyl-4-chlorobenzene sulphonate ester at 120 C. for 2 h provides salt (1) and reaction of 2-methylbenzthiazole with docosanyl-4-chlorobenzene sulphonate ester at 120 C. for 12 h followed by treatment with saturated potassium iodide provides salt (2). Coupling of (1) and (2) with DBU in DMF at 60-70 C. for 3-4 h then provides F305B which is purified by recrystallization and chromatography.

Example 12

Synthesis of Probe F306B

(66) Step 1:

(67) ##STR00009##

(68) In a 100 ml round bottom flask was placed 7.25 g (0.044 mol) of 2-Amino-4-tert-butylphenol, (Aldrich Cat #193282) with 6.0 g (0.055 mol) of Methyl acetimidate hydrochloride (Aldrich Cat #254940) with 50 ml of MeOH. This was refluxed for 4 hours. Then the MeOH was removed under vacuum. This was separated between 50 ml of ether and 50 ml of water. The ether was separated, dried, and removed under vacuum. Then 50 ml of hexanes was added to the resulting solid. Remaining 2-Amino-4-tert-butylphenol was insoluble in hexanes and was removed by filtration. The remaining oil was chromatographed using 2% MeOH/CH.sub.2Cl.sub.2 to provide 5-tert-butyl-2-methylbenzoxazole (Yield=2 g, 24%)

(69) Step 2:

(70) ##STR00010##

(71) In a 100 ml round bottom flask was placed 2 g (0.0106 mol) of SM1 and 4.7 g (0.0106 mol) of SM2 under argon. This was heated at 150 C. for 4 hours. Then the oil bath was allowed to cool to 120 C. and 4.71 g (0.0318 mol) of SM3 and 20 ml of anhydrous. pyridine was added under argon and refluxed for 2 hours. After cooling to room temperature the reaction mixture was poured into 100 ml of a 5% KI solution and placed in 0-5 C. refrigerator overnight. The residue that remained was washed with 2100 ml of water and chromatographed using 5% MeOH/CH.sub.2Cl.sub.2 to provide PTIR306 (Yield=840 mg, 7%).

Example 13

Synthesis of Probe F304B

(72) This compound was made in an analogous fashion to PTIR306 but using hexadecyl-4-chlorobenzene sulphonate ester as SM2 instead of octadecyl-4-chlorobenzene sulphonate ester.

Example 14

Synthesis of Probe F308B

(73) Step 1:

(74) ##STR00011##

(75) In a 25 ml round bottom flask was placed 2 g (0.00365 mol) SM1 with 15 ml H.sub.2SO.sub.4. This was stirred in an ice bath for 15 minutes then 0.3 g (0.00365 mol) NaNO.sub.3 was added all at once. After stirring for 30 minutes in an ice bath the reaction mixture was stirred at room temperature for 3 hours. This was then poured over 50 grams of ice. NaCl was then added with stirring until saturated. A brown solid separated. Most of the waster water was decanted. The solid was dissolved in CHCl.sub.3 and washed twice with 50 ml of H.sub.2O. The CHCl.sub.3 was dried with sodium sulfate and removed under vacuum. The residue was chromatographed on silica gel eluting with 10% MeOH/90% CH.sub.2Cl.sub.2 to provide the nitro product (Yield=1.12 g, 77%)

(76) Step 2:

(77) ##STR00012##

(78) In a 50 ml round bottom flask was placed 1 g (0.0017 mol) of SM1 and 0.46 g (0.0017 mol) of SM2 with 25 ml of Ac.sub.2O. This was refluxed in a preheated oil bath at 150 C. for 1 hour. This was then poured into 100 ml of 10% KI and placed in the refrigerator overnight. The resulting solid was used in the next step without purification

(79) Step 3:

(80) ##STR00013##

(81) In a 100 ml round bottom flask was placed 1.38 g (0.0018 mol) of SM1 and 0.72 g (0.0018 mol) of SM2 with 50 ml of CH.sub.2Cl.sub.2 and the mixture refluxed for 1 hour. This was then placed on 75 g of reverse phase silica flushing with 10% H.sub.2O/90% MeOH and 0.1% TFA until all the brown color was off. The column was then eluted with 100% MeOH/0.1% TFA to furnish pure product (Yield=0.29 g, 16%)

Example 15

Synthesis of Probe F317B

(82) Step 1:

(83) ##STR00014##

(84) In a 100 ml round bottom flask was placed 5 g (0.0068 mol) of SM1, 1.75 g (0.0068 mol) of SM2 and 60 ml of Ac.sub.2O. This was heated at 150 C. for 1 hour in a preheated oil bath. This was then poured into 300 ml of a 10% KI solution and then placed in the refrigerator overnight. The solid was collected, dissolved in CH.sub.2Cl.sub.2, dried with sodium sulfate and removed under vacuum. Product was used as is in step 3.

(85) Step 2:

(86) ##STR00015##

(87) In a 50 ml round bottom flask was placed 2.86 g (20 mmol) of lepidine and 4.04 g (22 mmol) of 1-iodobutane. The reaction was then heated to reflux overnight. The solution was then taken up in 40 ml of CH.sub.2Cl.sub.2 and washed with 40 ml of 5% Na.sub.2CO.sub.3 and then 240 ml of H.sub.2O. The organic layer was dried over sodium sulfate, filtered and concentrated under vacuum to provide the product. (Yield=6 g, 92%).

(88) Step 3:

(89) ##STR00016##

(90) In a 100 ml round bottom flask was placed 2.4 g (0.003 mol) of SM1, 0.98 g (0.003 mol) of SM2, 0.45 g (0.003 mol) of DBU with 50 ml of EtOH. The reaction mixture was then refluxed for 1 hour with stirring. After this time heating was stopped and the mixture stirred overnight. The EtOH was then removed under vacuum and the resulting material was placed onto 50 g of reverse phase silica. The column was eluted with 20% H.sub.2O/80% MeOH+0.1% TFA initially and then with 100% MeOH+0.1% TFA. Pure fractions were collected and the concentrated. The resulting material was dissolved in CH.sub.2Cl.sub.2, dried over sodium sulfate, filtered, and concentrated under vacuum to provide the product (Yield=156.6 mg, 6%).

Example 16

Synthesis of Probe F319B

(91) Step 1:

(92) ##STR00017##

(93) In a 100 ml round bottom flask was placed 4.86 g (0.88 mmol) of N-tetradecyl-2,3,3-trimethylindolinium 4-chlorobenzenesulfonate salt (A) and 50 ml of H.sub.2SO.sub.4. This solution was stirred in an ice bath for 15 minutes then 0.75 g (0.88 mmol) of sodium nitrite was added. After 30 minutes of stirring in an ice bath the reaction mixture was stirred at room temperature for 3 hours. It was then poured over 100 g of ice and once the ice melted, sodium chloride was added with stirring until a saturated salt solution was obtained. A brown solid separated and most of the water was decanted. The resulting solid was dissolved in 200 ml CHCl.sub.3 and washed twice with 100 ml of H.sub.2O. The CHCl.sub.3 was dried over sodium sulfate and concentrated under vacuum. The residue was chromatographed on silica gel eluting with 10% MeOH/CH.sub.2Cl.sub.2 to provide 1.7 g of product (B). Yield=48%

(94) Step 2:

(95) ##STR00018##

(96) In a 100 ml round bottom flask was placed 50 ml of Ac.sub.2O with 2 g (3.36 mmol) of N-docosanyl-2,3,3-trimethylindolinium iodide (C) and 1.05 g (3.7 mmol) of glutaconaldehydedianil hydrochloride. This mixture was refluxed for 2 hours at 150 C. in a preheated oil bath. It was then allowed to cool to room temperature and poured into 200 ml of a 10% KI solution. The resulting solid was filtered off. The solid was dissolved in CH.sub.2Cl.sub.2 and the residual water removed by separation. The organic phase was concentrated and the resulting 2.64 g of material (D) used without any further purification.

(97) Step 3:

(98) ##STR00019##

(99) In a 50 ml round bottom flask was placed 25 ml of CHCl.sub.3 with 0.792 g (1.0 mmol) of (D) and 0.4 g (1.0 mmol) of (B). This was refluxed for 1 hour, then allowed to cool. The solvent was removed under vacuum. The residue was placed on a regular phase silica gel column and the column flushed with 1 L of 30% EtOAc/Hexanes, followed by 100% CH.sub.2Cl.sub.2, and then finally with 10% MeOH/CH.sub.2Cl.sub.2 to remove the product. The resulting material was further purified using 75 g of C18 reverse phase silica and eluting with 10% H.sub.2O/MeOH/0.1% TFA followed by 100% MeOH/0.1% TFA. 90 mg of (E) was obtained (8.5% yield).

Example 17

Absorbance/Emission Spectra

(100) Referring now to Tables 1 and 2, there are shown the absorbance and emission maxima of some example compounds of the present invention. Absorption and Fluorescence spectra were run using 0.25 M solutions in ethanol on a Jasco, Inc. UV-530 UV/VIS spectrophotometer and FP 750 spectrofluorometer, respectively. Spectral properties are summarized in Table 1 and Table 2. Approximate extinction coefficients were estimated from the absorbance scans by dividing the maximum absorbance by the nominal concentration (25 M).

(101) TABLE-US-00001 TABLE 1 Extinction Coefficient Flourescence Fluorescence Compound # Abs Max (M.sup.1cm.sup.1) Ex Max Em Max F277A 788 nm 205,400 790 nm 814 nm F279A 646 nm 229,500 649 nm 668 nm F280A 647 nm 185,700 650 nm 688 nm F283A 646 nm 200,700 650 nm 666 nm F284A 646 nm 193,700 650 nm 665 nm F285A 646 nm 192,900 650 nm 650 nm F288A 658 nm 215,350 658 nm 679 nm F289A 655 nm 223,500 656 nm 674 nm F291A 789 nm 209,100 798 nm 815 nm F301A 750 nm 199,300 750 nm 771.5 nm

(102) TABLE-US-00002 TABLE 2 Extinction Coefficient Flourescence Fluorescence Compound # Abs Max (M.sup.1cm.sup.1) Ex Max Em Max F295B 650 nm 259,000 652 nm 669 nm F296B 654 nm 256,000 654 nm 670 nm F297B 651 nm 239,000 652 nm 670 nm F298B 655 nm 241,000 655 nm 672 nm F299B 647 nm 178,000 650 nm 664 nm F300B 657 nm 197,000 650 nm 664 nm F305B 425 nm 81,200 420 nm 461 nm F304B 493 nm 160,000 494 nm 508 nm F306B 492 nm 135,000 493 nm 508 nm F308B 649 nm 134,610 493 nm 671 nm F317B 718 nm 149,000 652 nm 771.5 nm F319B 743 nm 112,500 776 nm

Example 18

Chemical Stability of FR/NIR Probes

(103) Chemical stability of example compounds of the present invention when stored at room temperature as a solid or as a 1 mM solution in ethanol were determined by HPLC using the same conditions as above. Integration of the peaks detected for example compounds of the present invention at 260 nm was used to estimate chemical stability. Certain compounds tested displayed <5% change in HPLC purity when held as a solid at room temperature for approximately two months and when held as a 1 mM liquid solution in ethanol at room temperature for approximately one month.

Example 19

Photostability of FR/NIR Probes

(104) The photostability of example compounds of the present invention when stored in solution for 24 hours under ambient fluorescent lighting is represented in Table 3. 1 mM solutions of the indicated probes in >60% ethanol were placed under normal fluorescent room lighting. Aliquots were taken from each sample shortly after preparation and the absorbance measured at the absorbance maximum for example compounds. The absorbance of each sample was again measured after 24 hours at room temperature under ambient fluorescent light. Certain compounds tested displayed <20% photobleaching.

Example 20

Solubility of FR/NIR Probes

(105) Effects of the various combinations of asymmetrical and symmetrical side chains and methine bridges on the solubility of the compounds of the present invention were examined. Solubility in a commercially available probe diluent. Diluent C, was also tested. Triplicate 1:50 dilutions of stock 1 mM probe solutions were made in either ethanol or test diluent. After 30 minutes at room temperature solutions were centrifuged at 10,000g for 10 minutes to remove any microagregates and 100 ul aliquots were transferred to 2.0 ml of ethanol for absorbance determinations. Results are expressed as % of absorbance obtained from a control diluted in 100% ethanol as shown in Table 3. Based on the teachings of Leung et al. and on solubility specifications found on Sigma Certificates of Analysis for the commercial diluent (diluent supernatant absorbance at 30 minutes at least 60% of ethanol supernatant absorbance after 30 minutes), a preliminary specification at least 60% of the ethanol control was set as a surrogate for solubility sufficient to achieve cell labeling with S/N ratios greater than 100 and to minimize potential problems with brightness or reproducibility of labeling. Certain example compounds were soluble after 30 minutes in Diluent C.

(106) TABLE-US-00003 TABLE 3 Dye Type Solubility FR: 600-700 nm >60% of EtOH Compound # NIR: 700-900 nm Control @ 30 min F276A NIR 84% F277A NIR 95% F283A FR 82% F288A FR 88% F289A FR 70% F290A NIR 67% F291A NIR 92%

(107) Diluent C is a commercially available isotonic salt-free vehicle (Sigma-Aldrich Chemical Company) that is commonly used. This example demonstrates that the modified asymmetrical probes of the present invention are soluble in commercially available membrane probe diluents. In addition, it shows that useful levels of cell labeling can be obtained even when solubilities in iso-osmotic diluents are less than those taught by Horan et al.

Example 21

Characterization of Violet Probe Effects on Cell Viability, Cell Growth, and S/N Ratios

(108) U937 cells were stained at a concentration of 10.sup.7 cells/ml with increasing concentrations of probe by rapidly admixing 2 cells suspended in Diluent C with 2 probe in Diluent C. After 5 minutes at room temperature, the staining was stopped by the addition of cell culture medium. Cells were then washed thoroughly and cultured under standard conditions for 22-24 hours. Cell numbers were compared to those in a control culture treated with diluent alone.

(109) Maximum Tolerated Concentration (MTC) of F305B was found to be 20 micromol/L for cultured U937 cells stained with commercially available reference dye (PKH26) or with F305B at each dye's MTC showed comparable growth for when monitored daily for 4 days (approximately 4 cell doublings). Cells labeled with both dyes were also monitored daily for changes in mean fluorescence intensity (MFI), using a FACSAria flow cytometer with 488 nm excitation for PKH26 and 405 nm excitation for F305B. The correlation between increasing cell count (cell growth) and decreasing fluorescence intensity (dye dilution) was also found to be comparable for F305B and PKH26, indicating that F305B, like PKH26, has excellent membrane retention properties and is suitable for cell proliferation monitoring using the dye dilution method. As expected, F305B had a lower signal:noise ratio at T0 than PKH26, due to higher levels of cellular autofluorescence in the ultraviolet and violet range. However, initial MFI's were high enough to detect at least 4 and possibly 5 or 6 cell doublings before the fluorescence intensity distribution of labeled cells began to overlap substantially with that of unstained cells. Color overlap of F305B into filter windows used for detection of 488 nm or 633 nm excited dyes was minimal. Since 405 nm excited dyes are less commonly available than 488 nm and 633 nm excited dyes of this nature were therefore deemed useful, despite their somewhat lesser signal:noise ratio, for providing increased flexibility in choice of spectral window to be used for monitoring membrane labeled cells and their proliferation.

(110) Maximum Tolerated Concentration (MTC) of F308B was found to be 15 micromol/L for cultured U937 cells (a continuous myelomonocytic cell line) labeled in Diluent C according to the method described above U937 cells stained with commercially available reference dye (PKH26) or with F308B at each dye's MTC showed comparable growth for when monitored daily for 4 days (approximately 4 cell doublings). Cells labeled with both dyes were also monitored daily for changes in mean fluorescence intensity (MFI), using a FACSCalibur flow cytometer with 488 nm excitation for PKH26 and 633 nm excitation for F308B. The correlation between increasing cell count (cell growth) and decreasing fluorescence intensity (dye dilution) was also found to be comparable for F308B and PKH26, indicating that F308B, like PKH26, has excellent membrane retention properties and is suitable for cell proliferation monitoring using the dye dilution method. As expected, F308B had a greater signal:noise ratio at T0 than PKH26 due to lower levels of cellular autofluorescence in the far red. Although the cells were followed for only 4 doublings, the dye dilution curve suggested that at least 7-8 and possibly up to 9 cell doublings could be monitored using F308B before the fluorescence intensity distribution of labeled cells began to overlap substantially with that of unstained cells. Color overlap of F308B was minimal in filter windows used for detection of fluorescein or phycoerythin and modest but easily correctable in the filter window used to detect Texas Red or PE-Cy5 tandem conjugate dyes.

(111) Maximum Tolerated Concentration (MTC) of F317B was found to be 20 micromol/L for cultured U937 cells (a continuous myelomonocytic cell line) labeled in Diluent C according to the method described above to a final cell concentration of 110.sup.7 cells/mL. U937 cells stained with commercially available reference dye (PKH26) or with F317B at each dye's MTC showed comparable growth for when monitored daily for 4 days (approximately 4 cell doublings). Cells labeled with both dyes were also monitored daily for changes in mean fluorescence intensity (MFI), using a FACSCalibur flow cytometer with 488 nm excitation for PKH26 and 633 nm excitation for F317B. The correlation between increasing cell count (cell growth) and decreasing fluorescence intensity (dye dilution) was also found to be comparable for F317B and PKH26, indicating that F317B, like PKH26, has excellent membrane retention properties and is suitable for cell proliferation monitoring using the dye dilution method. However, due to the fact that the excitation maximum for F317B is not as well matched to the 633 nm laser as that of F308B, signal:noise ratio was substantially lower for F317B than for F308B and at most 4 cell doublings could be monitored before the fluorescence intensity distribution of labeled cells began to overlap substantially with that of unstained cells. Substantial color overlap was seen for F317B in all filter windows on the FACSCalibur, further indicating that F308B was a better dye for proliferation monitoring at far red wavelengths.

(112) Maximum Tolerated Concentration (MTC) of F319B was found to be 20 micromol/L for cultured U937 cells (a continuous myelomonocytic cell line) when cells were labeled at a final cell concentration of 110.sup.7 cells/mL in Diluent C according to the method described. U937 cells stained with comparable concentrations of the commercially available reference dye (PKH26, 12 micromol/L) or with F319B (12.5 micromol/L) and monitored daily for the next 6 days showed comparable growth. Cells labeled either with PKH26 or with F319B were also monitored daily for changes in mean fluorescence intensity (MFI), using a FACSAria flow cytometer with 488 nm excitation for PKH26 and 785 nm excitation for F319B. The correlation between increasing cell count (cell growth) and decreasing fluorescence intensity (dye dilution) was also found to be comparable for F319B and PKH26, indicating that F319B, like PKH26, has excellent membrane retention properties and is suitable for cell proliferation monitoring using the dye dilution method. F319B had a substantially greater signal:noise ratio at T0 than PKH26 (approx 5), due in part to lower levels of cellular autofluorescence in the near infrared and in part to the more optimal match between laser line and excitation maximum for this dye than for PKH26. Although the cells could only be followed for 4-5 doublings when labeled with PKH26, at least 6 doublings could be monitored using F319B before the fluorescence intensity distribution of labeled cells began to overlap substantially with that of unstained cells. Color overlap of F319B was minimal in filter windows used for detection of fluorescein, phycoerthyrin (PE), allophycocyanin (APC), and PE-Texas Red, PE-Cy5 or PerCP-Cy5.5 tandem conjugate dyes. Modest but easily correctable overlap was seen in the filter window used to detect APC-Cy7 tandem conjugate dyes, and more significant but still correctable overlap was seen in the filter window used to detect Alexa 700 dye.

(113) The above descriptions and drawings are only illustrative of a preferred embodiment which achieves the objects, features, and advantages of the present invention, and it is not intended that the present invention be limited thereto. Any modifications of the present invention which come within the spirit and scope of the following claims is considered part of the present invention.