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    • 专利摘要:
    RODAMINES AND CONJUGATES. The present invention generally relates to new rhodamine dyes which, by conjugation with another molecule, form unique isomeric conjugation products. These new rhodamine dyes contain only a single functional group in the rhodamine molecule for conjugation so that their conjugation products are unique isomeric conjugation products.
    公开号:BR112012025165B1
    申请号:R112012025165-1
    申请日:2011-04-01
    公开日:2021-02-09
    发明作者:Ulf Bremberg；Erik Ringberg；Wei Berts；Anthony de Belder；James S. Strickland
    申请人:Pharmacophotonics, Inc.；
    IPC主号:
    专利说明:

    DESCRIPTION CROSS REFERENCE TO RELATED ORDER
    The present application claims the benefit of U.S. Provisional Patent Application No. 61 / 320,571 filed on April 2, 2010, the contents of which are incorporated herein by reference. RESEARCH OR DEVELOPMENT FINANCED BY THE FEDERAL GOVERNMENT None. FIELD OF THE INVENTION
    The present invention generally relates to new rhodamine dyes which, by conjugation with another molecule, form unique isomeric conjugation products. BACKGROUND OF THE INVENTION
    Rhodamine dyes fluoresce and have been used extensively in research, both as a free dye and as a conjugate for larger molecules, for example, proteins and antibodies (Lee S, McAuliffe DJ, Kodama T, Doukas AG, In vivo transdermal delivery using a shock tube, Shock Waves (2000) 10: 307-307; Janson LW,
    Ragsdale K, Luby-Phelps K, Mechanism and size cutoff for steric exclusion from actin-rich cytoplasmic domains., Biophys J (1996) 71: 1228-1234; Pu R, Robinson KR,
    Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa., J Cell Sci (1998) 111 (Pt 21): 3197-3207; Nishiya T, Kajita E,
    Horinouchi T, Nishimoto A, Miwa S, Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88, FEBS Lett (2007) 581: 3223 3229; Tanner GA, Sandoval RM, Dunn KW, Two-photon in vivo microscopy of sulfonefluorescein secretion in normal and cystic rat kidneys, Am J Physiol Renal Physiol (2004) 286: F152-F160). Structurally, rhodamine is a family of polyurethane dyes related to a xanthene nucleus.

    Xanthene The general structure of rhodamine is as follows:
    Rhodamine amines can be primary amines, secondary amines or tertiary amines. One of the fluorescent rhodamine dyes commonly used is sulforhodamine 101 which contains an element of julolidine structure:
    Sulforrodamine 101 contains bifunctional sulfonyl groups as shown below:
    Sulphonyl rhodamine 101 has been used in neurophysiological experiments that comprise calcium imaging methods as well as an astrocyte counterstain (Nimmerjahn, A., Kirchhoff, F., Kerr, JN, Helmchen, F., Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo, Nature Methods (2004) 1: 31-7).
    A sulfonyl chloride derivative of sulforhododamine 101 is sold by Sigma Aldrich, Inc. (St. Louis, MO) under the trademark Texas Red®. It is used for conjugation with a number of functional groups, especially with primary amines. Texas Red® fluorescence at about 615 nm with an absorption peak at 589 nm. Texas Red® is typically available as a mixture of two monosulfonyl chlorides with the exchangeable SO3 and SO2Cl groups as shown below:

    Other rhodamine derivatives have also been described, such as in the International PCT Application (2009), WO 2009108905 A2 20090903; U.S. Patent Application Publication No. 2004054162 A1 20040318; U.S. Patent No. 5,728,529; U.S. Patent No. 5,686,261; PCT International Application (1997), WO 9700967 A1 19970109; U.K. Patent Application (1995), GB 2283744 A 19950517; and by Kim etl al. (Kim, TG; Castro, J. C; Loudet, A .; Jiao, JG-S .; Hochstrasser, RM; Burgess, K .; Topp, MR, Journal of Physical Chemistry A (2006), 110 (1), 20-27).
    Although several publications show the possibility of using bifunctional rhodamine dyes for conjugation, for example, by the selective reaction of one of the sulfonyl chloride groups in Texas Red® (Titus JA, Haugland R, Sharrow SO, Segal DM, Texas Red, a hydrophilic, redemitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies, J. Immunol. Methods (1982) 50 (2): 193-204), the possibility of double reactivity makes it difficult to establish a reliable process producing only a single isomer product, which is shown in figure 1 in which a bifunctional rhodamine dye with two sulfonyl groups reacting with a primary amine to form two isomeric conjugation products. Two isomeric structures instead of just one in a process offer two major disadvantages: 1) the ratio of isomers changes from batch to batch with impacting product effects, and 2) regulatory requirements (data on toxicity, stability, characterization, etc.). ) will be doubled to cover two substances instead of one.
    The present invention circumvents these difficulties by using new monofunctional derivatives of rhodamine dye with only a single functional group in the rhodamine molecule by conjugation so that their conjugation products are single isomeric conjugation products.
    These and other aspects and attributes of the present invention will be discussed with reference to the following drawings and attached specification.
    BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the conjugation of a bifunctional rhodamine dye with a macromolecule to form two isomeric conjugation products; Figure 2 shows the conjugation of a monofunctional rhodamine dye with a macromolecule to form only a single isomeric conjugation product; Figure 3 shows the formation of the new rhodamine dye of the present invention from 8-hydroxyjulolidine (2 equivalents) and the substituted benzaldehyde (1 equivalent) where R1, R2, R3, R4 and R5 can be an H or any group ; Figure 4 shows the general conjugation reaction under the Ugi reaction conditions between a rhodamine dye with a monofunctional group and a macromolecule; and Figure 5 is an example of conjugation with the Ugi reaction of a monofunctional 2-sulforhododamine dye (2-SHR) containing a single functional primary amino group with carboxymethylated dextran to form a single isomeric conjugation product; Figure 6 is a UV absorption spectrum of Compound 18 sweeping from 200 nm to 800 nm at a scanning speed of 400 nm / minute; Figure 7 is a fluorescence emission scan of Compound 18; Figure 8 is a fluorescence excitation scan of Compound 18; Figure 9 is a 3-dimensional fluorescence scan of Compound 18 where EM is the emission wavelength and EX is the excitation wavelength; Figure 10 is a UV absorption spectrum of the conjugate of Example 26 sweeping from 200 nm to 800 nm at a scanning speed of 400 nm / minute; Figure 11 is a fluorescence emission scan of the conjugate of Example 26; Figure 12 is a fluorescence excitation scan of the conjugate of Example 26; and Figure 13 is a 3-dimensional fluorescence scan of the conjugate of Example 26 where EM is the emission wavelength and EX is the excitation wavelength. DETAILED DESCRIPTION OF THE INVENTION
    While this invention is susceptible of the modalities in several different forms, there are specific modalities that will be described in this in detail with the understanding that the present disclosure is to be considered as an example of the principles of the invention and is not intended to limit the invention to the specific modalities illustrated.
    It will be understood that the chemical structures that are used to define compounds of the present invention are each representations of one of the possible resonance structures that each given structure can be represented by. Furthermore, it will be understood that by definition, resonance structures are merely a graphical representation used by those skilled in the art to represent electron displacement, and that what the present reveals is in no way limited to showing a particular resonance structure for a given structure.
    It will also be understood that the chemical structures that are used to define compounds of the present invention also include their structures in their respective salt forms.
    The present invention generally relates to new rhodamine dyes which, after conjugation with another molecule, to form unique isomeric conjugation products.
    Although several publications show the possibility of using bifunctional rhodamine dyes for conjugation, for example, by the selective reaction of one of the sulfonyl chloride groups in Texas Red® (Titus JA, Haugland R, Sharrow SO, Segal DM, Texas Red, a hydrophilic, redemitting fluorophore for use with fluorescent in dual parameter flow microfluorometric and fluorescentence microscopic studies, J. Immunol. Methods (1982) 50 (2): 193- 204), the possibility of double reactivity makes it difficult to establish a reliable process producing only a single isomer product. As shown in figure 1, a bifunctional rhodamine dye with two sulfonyl groups reacts with a primary amine to form two isomeric conjugation products. Two isomeric structures instead of just one in a process offer two major disadvantages: 1) the ratio of isomers changes from batch to batch with impacting product effects, and 2) regulatory requirements (data on toxicity, stability, characterization, etc.). ) will be duplicated to cover two substances instead of one.
    The present invention circumvents these difficulties by using new monofunctional derivatives of rhodamine dye with only a single functional group in the rhodamine molecule for conjugation so that their conjugation products are unique isometric conjugation products as illustrated in figure 2. Figure 2 illustrates an example of the present invention in which a rhodamine derivative with a single sulfonyl group reacts with a primary amine to form only a single isometric conjugation product. What is meant by a "functional group" is that the group is suitable for conjugation. The functional group suitable for conjugation is reacted to another molecule, such as a macromolecule, to form a conjugate through a covalent bond. A rhodamine derivative containing only one "functional group" is known as monofunctionalized or a monofunctional derivative (such as rhodamine monosulfonyl in Figure 2) that differentiates from rhodamine derivatives containing more than one "functional group" such as sulfonyl rhodamine 101 or Texas Red ®. Examples of functional groups suitable for conjugation include but are not limited to amines, isocyanates, isothiocyanates, thiols, carboxylic acids and the like. "Functionalized" in this means that the rhodamine derivative was derived to contain a "functional group". An example is "amino-functionalized" meaning that the functional group contains the reactive amino group.
    In one embodiment, the new rhodamine dyes of the present invention have a general structure of:
    wherein R1, R2, R3, R4 and R5 can be an H or any group. However, between R1, R2, R3, R4 and R5, only one of these groups can have a "functional group" so that the rhodamine dye has only a single "functional group" capable of conjugation to another molecule, such as a macromolecule , to form a single isomeric conjugation product. This general structure can be formed by reacting 8-hydroxyjulolidine (2 equivalents) with the substituted benzaldehyde (1 equivalent) as shown in figure 3. 8-hydroxyjulolidine and the substituted benzaldehyde can be mixed with 60% aqueous sulfuric acid (11 , 1 mL / mmol of benzaldehyde) and stirred at 150 ° C for 24 h under an atmosphere of air. The reaction mixture can be added to the ice (28 g / mmol benzaldehyde), after which 60% NaOH can be carefully added at pH6-7 to precipitate the crude product. The crude product can be extracted between dichloromethane (DCM) and water. The organic phase can be separated, and washed with brine. The organic solvent can be removed and the final product dried by evaporation with ethanol and toluene 5 times to provide the crude product. Detailed methods for preparing the specific examples of rhodamine dyes of the present invention are described in the Examples below.
    Some examples of general structures of monofunctional rhodamine derivatives suitable to form unique isomeric conjugation products are shown below with the general formula of 2-sulforhodhodamine:
    or the general formula of 4-carboxyrodamine:
    or the general formula of 3-carboxyrodamine:
    or the general formula of 4-arylhodamine:
    where for all 4 general formulas above, Ar is an aryl group, and R1 and / or R2 form a spacer with a unique functional group in each R1 or R2 suitable for conjugation to another molecule, where the spacer can be, but it is not limited to, hydrogen, alkyl, aryl, amide, alkyl sulfonamide, alkyl ether, alkyl amide and the like, or mixtures thereof. The alkyl groups mentioned above preferably have a carbon chain length of 1 to 20. R1 and R2 can also be connected to form a cyclic structure, such as, but not limited to, the structure as shown below:
    or Although the examples shown above show that the sulfo group is in position 2, the carboxy group is in position 3 or 4 and the aryl group is in position 4, it should be noted that these and other groups containing "functional" group may be in position 2, 3 or 4. The examples below (Examples 4-24) illustrate the synthesis of some of the members belonging to the groups shown above. However, the synthesis of other compounds related to the sulfo, carboxy, aryl or any other group positioned in any desired position in the new rhodamine dye of the present invention should be obvious to those skilled in the art with the illustrations of the Examples.
    In a preferred embodiment, the new rhodamine derivatives are in the form of a salt, such as, but not limited to, trifluoroacetate, chloride, hydrochloride, hydrobromide, iodhydrate, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate , lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, format, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate and p-toluenesulfonate. In another preferred embodiment, the salt is trifluoroacetate or chloride. In yet another preferred embodiment, the salt is a pharmaceutically acceptable salt.
    One of the applications for these new monofunctional rhodamine derivatives is their ability to conjugate with another molecule, such as a macromolecule. The molecule, such as a macromolecule, when conjugated to the rhodamine dye can be easily detected and / or quantified. Generally macromolecules used in this include, but are not limited to, polymers, proteins (such as antibodies), dextrans, celluloses, carbohydrates, lipids, nucleic acids (such as DNA and RNA) and the like. The conjugation of rhodamine dyes with macromolecules and their applications is well known to those skilled in the art and has been described in detail in the specific literature, as described by Titus et al. (Titus JA, Haugland R, Sharrow SO, Segal DM, Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies, J. Immunol. Methods 50 (1982) 2: 193 204 ) and by Haugland et al. in U.S. Patent No. 5,798,276. The rhodamine conjugate with macromolecules such as antibodies are readily commercially available as the human IgG antibody conjugated to rhodamine from Abcam (Cambridge, MA) and various rhodamine dye conjugated proteins from Sigma Aldrich (St. Louis, MO). Any synthetic methodology that creates a covalent bond between the functional group of the dye and the macromolecule can be used for conjugation. The general conjugation of the reaction between the rhodamine dye and the macromolecule under the Ugi reaction conditions is illustrated in figure 4, exemplified by the conjugation of the amino-functionalized sulfonamide dye (Compound 1 shown in Example 6) for carboxymethylated dextran with the Ugi reaction, as shown in figure 5.
    To illustrate the invention, a number of structures suitable for conjugation have been synthesized (Compounds 1-19) which are shown in Examples 6-24 below.
    Experimental details of additional examples of conjugations are shown with Examples 25-28 below, using dyes from the synthesized examples (Compound 18, Compound 3, Compound 15 and Compound 16) conjugating to carboxymethylated dextran (CM-dextran).
    While the present invention is described in connection with what is presently considered to be the most practical and preferred modalities, it should be appreciated that the invention is not limited to the described modalities, and is intended to cover various modifications and equivalent provisions included within the spirit and scope of the claims. Modifications and variations in the present invention can be made without departing from the new aspects of the invention as defined in the claims. The appended claims must be interpreted broadly and in a manner consistent with the spirit and scope of this invention. EXAMPLES
    Solvents and reagents are used as received from Labscan (Gliwice, Poland) and Sigma Aldrich (St. Louis, MO), respectively. An Agilent Technologies Liquid Chromatography Mass Spectroscopy (LCMS) system (Santa Clara, CA) is used, which consists of a G1379B degasser, a G1312A binary pump, a G1329A auto-injector, a G1316A column oven, a G1365B detector UV-Vis (used to detect maximum absorbance) and a 6110 Quadrupole MS detector. High Performance Liquid Chromatography (HPLC) purities are measured with an ACE-C8 column (50 x 4.6 mm) maintained at 35 ° C and eluted with 10-97% acetonitrile in 0.1% trifluoral acetic acid at (TFA) during a 3-minute gradient.
    The compounds in the Examples below are named based on Marvin Sketch 5.2.6, using IUPAC Preferred nomenclature definitions for naming structures. However, other nomenclature systems can be employed to name these compounds. Example 1: Preparation of Intermediate 1 The following compound is prepared:

    The reaction mixture of 8-hydroxyjulolidine (1.1 g, 5.8 mmol) and sodium 2-formylbenzene-1-sulfonate (0.6 g, 2.9 mmol) in 60% aqueous H2S04 (10 mL) is stirred at 150 ° C under an air atmosphere for 2 hours, after which time the starting materials were completely converted to the expected product. The reaction pH is adjusted to about 7 with 60% aqueous NaOH, in which procedure the expected product is precipitated. The precipitation is filtered and washed with toluene (3 x 50 ml) and dried in vacuo. The crude product is dissolved in hot ethanol (EtOH), and filtered. Insoluble solids are discarded and the filtrate is evaporated in vacuo with toluene (3 x 50 mL), and 1.1 g of the title molecule are obtained with 90% HPLC purity and 73% yield. Example 2: Preparation of Intermediate 2 The following compound is prepared:
    Intermediate 2 8-Hydroxyijulolidine (1.4 g, 7.2 mmols) and 4-formyl-benzoic acid (500 mg, 3.6 mmols) are mixed with 60% aqueous sulfuric acid (40 ml) and stirred at 150 ° C for 24 hours under an air atmosphere. The reaction mixture is added to ice (100 g), after which 60% NaOH is carefully added at pH 6-7, precipitating the crude product. The crude product is extracted between dichloromethane (DCM) and water. The organic phase is separated and washed with brine. The organic solvent is removed and the final products are dried by evaporation with toluene and EtOH 5 times to obtain 1.1 g of product (61% yield). Purity as determined by HPLC is 100%. MS (ESI) [M +] = 491. Example 3: Preparation of intermediate 3 The following compound is prepared:
    Intermediate 3 8-Hydroxyijulolidine (1.4 g, 7.2 mmols) and 3-formyl-benzoic acid (500 mg, 3.6 mmols) are mixed with 60% aqueous sulfuric acid (40 ml) and stirred at 150 ° C for 24 hours under an air atmosphere. The reaction mixture is added to ice (100 g), after which 60% NaOH is added carefully at pH 6-7, precipitating the crude product. The crude product is extracted between DCM and water. The organic phase is separated and washed with brine. The organic solvent is removed and the final products are dried by evaporation with toluene and EtOH 5 times to obtain 1.7 g of acidic product (94% yield). Purity as determined by HPLC is 95%. MS (ESI) [M +] = 491. Example 4: Preparation of Intermediate 4 The following compound is prepared:
    Intermediate 4 8-Hydroxyijulolidine (1.4 g, 7.2 mmol) and 4-bromobenzaldehyde (670 mg, 3.6 mmol) are mixed with 60% aqueous sulfuric acid (40 mL) and stirred at 150 ° C for 24 hours under air atmosphere. The reaction mixture is added to ice (100 g), after which 60% NaOH is added carefully at pH 6-7, precipitating the crude product and drying under vacuum overnight to 1.12 g of a black solid. The crude product is dissolved in 90/10 CHCl3 / methanol (5 ml) and applied to a silica column (35 x 100 mm) and eluted with 10-18% methanol in CHCl3. The pure fractions were combined and the solvent was evaporated under reduced pressure to 448 mg of product. Example 5: Preparation of Intermediate 5 The following compound is prepared:
    Intermediate 5 Intermediate 4 prepared as described in Example 4 (236 mg, 0.45 mmol) and tert-butyl-N - {[4 - (dihydroxyboranyl) phenyl] methyl} carbamate (225 mg, 0.90 mmol) are transferred to a 50 mL flask with ethanol and 2M aqueous K2C03 solution (672 μL, 1.34 mmol) added. The reaction mixture is rapidly degassed and [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II). DCM (18 mg, 0.022 mmol)) is added, degassing again and stirring under nitrogen at room temperature. After 50 minutes of stirring, add more [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II). DCM (13 mg, 0.016 mmol) is added and the mixture is heated to 65 ° C for 1 hour. This is followed by evaporation and dissolution in DCM, the addition of 4 g of silica and evaporation and chromatography through a 12 x 2.5 cm silica column, packed with DCM, eluted with 5 - 20% methanol (MeOH) in DCM and evaporation of the pure fraction gave 236 mg of a sticky golden brown solid with 90% pure HPLC. Example 6: Preparation of a 2-sulforhododamine trifluoroacetate (Compound 1) The following compound is prepared:
    Compound 1 Compound 1 is 16- {2 - [(6-aminohexyl) -sulfamol] - phenyl} -3-oxa-9À5,23-diazaheptacyclo [17.7.1.15'9.02'17.04'15. 023'27.013'28] octacosa- 1 (27), 2 (17), 4.9 (28), 13,15,18-heptaen- 9-ilium; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared as follows. Intermediate 1 (0.66 mmol, 350 mg) is dissolved in DCM (10 mL) and a drop of dimethylformamide (DMF). Oxalyl chloride (3.98 mmols, 500 mg) is added and the reaction mixture is stirred at room temperature for one hour. A gas evolution is immediately noticed. The solvent is evaporated, mixed with toluene (10 ml) and re-evaporated, the residue dissolved in DCM (12 ml), cooled in an ice bath, and divided into two equal portions. One portion is carefully (less than 5 minutes) added to an ice cold solution of hexamethylenediamine (1.06 mmol, 120 mg) in DCM (5 mL) and triethylamine (0.40 mmol, 40 mg) in DCM (5 mL). The dark bluish solutions immediately change to dark red. The reaction is completed within 30 minutes. A portion of the crude mixture is purified on preparative HPLC, ACE-C8 column with a gradient of methanol in 0.1% TFA in water to obtain 58 mg (24%) of product as a sparkling dark blue copper glass. Purity as determined by HPLC is 100%. MS (ESI) [M +] = 625. Maximum absorbance is 586 nm. Example 7: Preparation of a 2-sulforhododamine trifluoroacetate (Compound 2) The following compound is prepared:
    Compound 2 Compound 2 is 16- {2- [3- (aminomethyl) pyrrolidine-1-sulfonyl] phenyl} -3-oxa-9 5,23-diazaeptacyclo [17.7.1.15'9.02'17.04,15.023'27.013'28 ] octacous-1 (27), 2 (17), 4, 9 (28), 13,15,18-heptaen-9-yl; 2,2,2-trifluoroacetate according to the nomenclature system used and is prepared as follows. Intermediate 1 (0.66 mmol, 350 mg) is dissolved in DCM (10 mL) and a drop of DMF. Oxalyl chloride (3.98 mmols, 500 mg) is added and the reaction mixture is stirred at room temperature for one hour. A gas evolution is immediately noticed. The solvent is evaporated, mixed with toluene (10 ml) and re-evaporated, the residue dissolved in DCM (12 ml), cooled in an ice bath, divided into two equal portions. One portion is carefully (less than 5 minutes) added to an ice-cooled solution of tert-butyl N- (pyrrolidin-3-ylmethyl) carbamate (1.06 mmol, 90 mg) in DCM (5 mL) and triethylamine (0 , 40 mmol, 40 mg) in DCM (5 ml). The dark bluish solutions immediately change to dark red. The reaction is completed within 30 minutes. TFA (1 ml in ca of 2 ml of DCM) is added, completing the deprotection in one hour. A portion of the crude product is purified on preparative HPLC, ACE-C8 column with a gradient of methanol in 0.1% TFA in water to obtain 95 mg (39%) as a sparkling dark blue copper glass. Purity as determined by HPLC is 100%. MS (ESI) [M +] = 605. Maximum absorbance is 590 nm. Example 8: Preparation of a 4-carboxyrodamine trifluoroacetate (Compound 3) The following compound is prepared:
    Compound 3 Compound 3 is 16- {4 - [(2-aminoethyl) carbamol] phenyl} - 3-oxa-9À5,23-diazaheptacyclo [17.7.1.15,9.02'17.04'15.023'27.013'28] octacous- 1 (27 ), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ileum; 2,2,2- trifluoroacetate according to the nomenclature system we use and is prepared as follows. Intermediate 2 (150 mg, 0.31 mmol) is dissolved in DMF-CH3CN (1 - 4.7 ml). Triethylamine (94 mg, 0.93 mmol) and solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorin-2, 4,6-trioxide in ethyl acetate ( 592 μL, 0.93 mmol) are added and the mixture was stirred at room temperature for 15 minutes. The 1/6 part is removed and added to 1,2-diaminoethane (19 mg, 0.31 mmol) and the reaction mixture was stirred at room temperature for 1.5 hours. Purification is performed on preparative HPLC, ACE-C8 column with a gradient of methanol in 0.1% TFA in water to obtain 24 mg (72%) as a dark blue glass. Purity as determined by HPLC is 93%. MS (ESI) [M +] = 533. Maximum absorbance is 586. Example 9: Preparation of a 4-carboxyrodamine trifluoroacetate (Compound 4) The following compound is prepared:
    Compound 4 Compound 4 is 16- {4 - [(3-aminopropyl) carbamol] phenyl} -3-oxa-9À5,23-diazaheptacycle [17.7.1.15'9.02'17.04,15.023'27.013'28] octacous- 1 (27 ), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ile; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure used to prepare Compound 3, but with 1,3-diaminopropane replacing 1,2-diaminoethane (23 mg, 0.31 mmol) to obtain 25 mg (73% yield) of the product. Purity as determined by HPLC is 100%. MS (ESI) [M +] = 547. Max absorbance is 584 nm. Example 10: Preparation of a 4-carboxyrodamine trifluoroaceate (Compound 5) The following compound is prepared:
    Compound 5 Compound 5 is 16- {4 - [(2,2-dimethoxyethyl) carbamol] phenyl} -3-oxa-9À5,23-diazaeptacycle [17.7.1.15'9.02'17.04'15.023>27.013'28] octacous- 1 (27), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ile; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure used to prepare Compound 3, but with 2,2-dimethoxyethane-1-amine replacing 1,2-diaminoethane (33 mg, 0.31 mmol) to obtain 26 mg (73% yield) of the product. Purity as determined by HPLC is 100%. MS (ESI) [M +] = 578. Max absorbance is 584 nm. Example 11: Preparation of a 4-carboxyrodamine trifluoroaceate (Compound 6) The following compound is prepared:
    Compound 6 Compound 6 is 16- {4 - [(6-aminohexyl) carbamol] phenyl} -3-oxa-9À5,23-diazaheptacyclo [17.7.1.15'9.02'17.04'15.023'27.013,28] octacous- 1 (27 ), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ile; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure used to prepare Compound 3, but with 1,6-diaminohexane replacing 1,2-diaminoethane (36 mg, 0.31 mmol) to obtain 32 mg (88% yield) of product. Purity as determined by HPLC is 100%. MS (ESI) [M +] = 589. Max absorbance is 586 nm. Example 12: Preparation of a 4-carboxyrodamine trifluoroaceate (Compound 7) The following compound is prepared:
    Compound 7 is 16- [{- 4- (aminomethyl) phenyl] methyl} carbamol) phenyl] -3-oxa-9À5,23-diazaheptacyclo [17.7.1.15'9.02,17.04'15.023'27.013'28] octacosa - l (27), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ile; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure used to prepare Compound 3, but with [4 - (aminomethyl) phenyl] methanamine replacing 1,2-diaminoethane (42 mg, 0.31 mmol) to obtain 36 mg (96% yield) of the product. Purity as determined by HPLC is 99%. MS (ESI) [M +] = 609. Maximum absorbance is 590 nm. Example 13: Preparation of a 4-carboxyrodamine trifluoroaceate (Compound 8) The following compound is prepared:
    Compound 8 Compound 8 is 16- {4 - [(4-oxopiperidin-1-yl) carbonyl] phenyl} -3-oxa-9À5,23-diazaheptacyclo [17.7.1.15'9.02'17.04'15.023 '7.013'28] octacous - 1 (27), 2 (17), 4.9 (28), 13,15,18-heptaen-9-yl; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure as that used for Compound 3, but with piperidin-4-one replacing 1,2-diaminoethane (31 mg, 0, 31 mmol) to obtain 28 mg (79% yield) of the product. Purity as determined by HPLC is 82%. MS (ESI) [M +] = 572. Max. is 588 nm. Example 14: Preparation of a 3-carboxyrodamine trifluoroaceate (Compound 9) The following compound is prepared:
    Compound 9 Compound 9 is 16- {3 - [(2-aminoethyl) carbamol] phenyl} - 3-oxa-9À5,23-diazaheptacyclo [17.7.1.15,9.02'17.04'15.023'27.013'28] octacous- 1 (27 ), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ile; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure as the one used for Compound 3, but with intermediate 3 replacing 1,2-diaminoethane as acid and 1,2-diaminoethane (19 mg, 0.31 mmol) as amine to obtain 20 mg of product (60% yield) . Purity as determined by HPLC is 100%. MS (ESI) [M +] = 533. Max absorbance is 586 nm. Example 15: Preparation of a 3-carboxyrodamine trifluoroaceate (Compound 10) The following compound is prepared:
    Compound 10 Compound 10 is 16- {3 - [(3-aminopropyl) carbamol] phenyl} -3-oxa-9À5,23-diazaheptacycle 17.7.1.15'9.02'17.04'15.023'27.013'28] octacous- 1 (27) , 2 (17), 4,9 (28), 13,15,18-heptaen-9-ilii; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure as the used for the preparation of Compound 9, but with 1,3-diaminopropane replacing 1,2-diaminoethane (23 mg, 0.31 mmol) to obtain 19 mg (56% yield) of the product. Purity as determined by HPLC is 100%. MS (ESI) (M +] = 547. Max. Absorbance is 584 nm Example 16: Preparation of a 3-carboxyrodamine trifluoroaceate (Compound 11) The compound as follows is prepared:
    Compound 11 Compound 11 is 16- {3 - [(6-aminohexyl) carbamol] phenyl} -3-oxa-9À5,23-diazaheptacycle [17.7.1.15,9.02'17.04'15.023'27.013'28] octacous-1 (27 ), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ileum; 2,2,2- trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure as that used for the preparation of Compound 9, but with 1,6-diaminohexane replacing 1,2-diaminoethane (36 mg, 0.31 mmol) to obtain 27 mg (74% yield) of the product. Purity as determined by HPLC is 100%. MS (ESI) [M +] = 589. Max. is 584 nm. Example 17: Preparation of a 3-carboxyrodamine trifluoroaceate (Compound 12) The following compound is prepared:
    Compound 12 Compound 12 is 16- [3 - ({[4- (aminomethyl) phenyl] methyl} carbamol) phenyl] -3-oxa-9 5,23-diazaheptacyclo [17.7.1.15'9.02,17.04,15.023'27.013 ' 28] octacosa- l (27), 2 (17), 4.9 (28), 13,15,18-heptaen-9-yl; 2,2,2- trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure as used to prepare Compound 9, but with [4 - (aminomethyl) phenyl] methanamine replacing 1,2-diaminoethane (42 mg, 0.31 mmol) to obtain 32 mg of product (86% yield). Purity as determined by HPLC is 100%. MS (ESI) [M +] = 609. Max absorbance is 587 nm. Example 18: Preparation of a 3-carboxyrodamine trifluoroaceate (Compound 13)
    The following compound is prepared: Compound 13 Compound 13 is 16- {3 - [(4-oxopiperidin-1-yl) carbonyl] phenyl} -3-oxa-9À5,23-diazaeptacycle [17.7.1.15'9.02'17.04 '15.023'27.013'28] octacosa- 1 (27), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ile; 2,2,2-trifluoroacetate according to the nomenclature that we use and is prepared using the same procedure as that used for the preparation of Compound 9, but with piperidin-4-one replacing 1,2-diaminoethane (31 mg, 0.31 mmol) to obtain 34 mg (yield of 96%) of the product. Purity as determined by HPLC is 80%. MS (ESI) [M +] = 572. Max absorbance is 584 nm. Example 19: Preparation of a 3-carboxyrodamine trifluoroaceate (Compound 14) The following compound is prepared:
    Compound 14 Compound 14 is 16- {3 - [(2,2-dimethoxyethyl) carbamol] phenyl} -3-oxa-9 5,23-diazaheptacyclo [17.7.1.15'9.02'17.04'15.023'27.013'28] octacosa- 1 (27), 2 (17), 4, 9 (28), 13,15,18-heptaen-9-ile; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared using the same procedure as that used for the preparation of Compound 9, but with 2,2-dimethoxyethane-1-amine replacing 1,2-diaminoethane (33 mg, 0.31 mmol) to obtain 29 mg of the product (81% yield) . Purity as determined by HPLC is 98%. MS (ESI) [M +] = 578. Max absorbance is 588 nm. Example 20: Preparation of a 4-carboxyrodamine trifluoroaceate (Compound 15) The following compound is prepared:
    Compound 15 Compound 15 is 16- {4- [4- (aminomethyl) phenyl] phenyl} -3-oxa-9À5,23-diazaheptacyclo [17.7.1.15'9.02'17.04'15.023'27.013'28] octacous-1 (27 ), 2 (17), 4, 9 (28), 13,15,18-heptaen-9-ile; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared as follows. Intermediate 5 (236 mg, 0.35 mmol) is dissolved in 4 ml of DCM / TFA (3/1). After 30 minutes, the solvent is blown with air. The crude product is purified on an ACE-C8 elution column (150 x 30 mm), with 20 - 100% methanol in 0.1% TFA. Pure fractions are evaporated to 115 mg (% yield) of a dark blue / red solid with a green tint. Purity as determined by HPLC is 98%. MS (ESI) [M +] = 552. Max absorbance is 578 nm.
    Example 21: Preparation of a 4-carboxyrodamine trifluoroaceate (Compound 16) The following compound is prepared:
    Compound 16 Compound 16 is 16- [4- (2-amino-4-methylphenyl) phenyl] - 3-oxa-9 5,23-diazaheptacyclo [17.7.1.15'9.02'17.04'15.023'27.013'28] octacous- 1 (27), 2 (17), 4, 9 (28), 13,15,18-heptaen-9-yl; 2,2,2-trifluoroacetate according to the nomenclature system we use and is prepared as follows. Intermediate 4 (152 mg, 0.29 mmol) and 5-methyl-2- (4,4,5,5-tetramethyl) - (1,2,2-dioxaborolan-2-yl) -phenylamine (135 mg, 0 , 58 mmol) are dissolved in ethanol (10 mL) and treated with 2M K2C03 (434 μL, 0.87 mmol) and [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II) DCM (11 mg, 0.015 mmol ). The reaction mixture is heated to 65 ° C for 1 hour. The crude product is filtered through celite and purified on an ACE-C8 column (150x30 mm) eluting with 60 - 100% methanol in 0.1% TFA, evaporating pure fractions to 80 mg (50% yield) of solid dark blue / red with a green tinge. Purity as determined by HPLC is 90%. MS (ESI) [M +] = 552. Maximum absorbance is 580 nm. Example 22: Preparation of a 4-carboxyrodamine trifluoroaceate (Compound 17) The following compound is prepared:
    Compound 17 Compound 17 is 16- [4- (4-acetylphenyl) phenyl] -3-oxa- 9 5,23-diazaheptacyclo [17.7.1.15'9.02'17.04'15.023'27.013'28] octacous- 1 (27), 2 (17), 4.9 (28), 13,15,18-heptaen-9-ileum; 2,2,2-trifluoroacetate according to the nomenclature system used and is prepared as follows. Intermediate 4 (152 mg, 0.29 mmol) and phenyl 4-ethanone boronic acid (95 mg, 0.58 mmol) are dissolved in ethanol (10 mL) and treated with 2M K2C03 (434 μL, 0.87 mmol) and [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II). DCM (11 mg, 0.015 mmol). The reaction mixture is heated to 65 ° C for 1 hour. The crude product is filtered through celite and purified on an ACE-C8 column (150x30 mm), eluting with 60 - 100% methanol in 0.1% TFA, evaporating the pure fractions at 54 mg (33% yield) a dark blue / red solid with a green tinge. Purity as determined by HPLC is 92%. MS (ESI) [M +] = 565. Max absorbance is 580 nm. Example 23: Preparation, purification and crystallization of a 2-sulforhododamine dichloride (Compound 18) The following compound is prepared:
    Compound 18 Compound 18 is 16- {2 - [(6-aminohexyl) sulfamol] phenyl} -3-oxa-9À5,23-diazaheptacyclo [17.7.1.15'9.02'17.04'15.023'27.013'28] octacous- 1 (27 ), 2 (17), 4.9 (28), 13, 15.18-heptaen-9-ileum; dichloride according to the nomenclature system we use and is prepared as follows.
    Synthetic step 1: A mixture of 8-Hydroxyjulolidine (200 g) and sodium 2-formylbenzene-1-sulfonate (110 g) is added to 1.8 L of 60% aqueous H2S04 preheated to 150 ° C and stirred for 4 hours. After the reaction has ended monitored by LC-MS, the reaction mixture is cooled to 0 ° C. 60% sodium hydroxide (aqueous) is added slowly, until the pH value of the reaction mixture is 2 (product is precipitate). Celite (800 g) is added to the reaction mixture with the precipitated crude product and the reaction mixture is filtered. The resulting solid with celite is washed with toluene (3 x 500 ml) and added to anhydrous ethanol (4 L) and heated with stirring at 60 ° C for 0.5 hours. The reaction mixture is then filtered. The filtrate is concentrated in vacuo and then coevaporated with toluene, to get rid of traces of water and to obtain the crude Rhodonic sulfonic acid intermediate (270g) as a dark blue solid. Synthetic step 2: Sulfonic acid intermediate Crude rhodamine from Step 1 (100 g, 0.19 mol) is dissolved in a solvent mixture of dichloromethane (500 mL) and DMF (13.9 g). The reaction solution is cooled to 0 ° C and oxalyl chloride (48.1 g, 0.379 mol) is added dropwise. The reaction mixture is stirred for an additional 2 hours at 0 ° C. The reaction mixture is then concentrated in vacuo and for the resulting residue four iterations of adding toluene (100 ml) and evaporation are carried out. The raw Rhodamine sulfonic acid chloride intermediate is used directly in the next step, after drying under reduced pressure for 6 hours.
    Synthetic step 3: a solution of hexane-1,6-diamine (176 g, 1.52 mol) in dichloromethane (800 ml) is cooled to 0 ° C. A solution of all the raw Rhodamine sulfonic acid chloride intermediate obtained in Step 2, in dichloromethane (500 ml) is added dropwise with continued cooling to 0 ° C. The reaction mixture is stirred for an additional period of 4 hours at 0 ° C. After checking with layer chromatography fine (TLC) the reaction is complete, the reaction mixture is quenched by filtration through celite paper, and evaporating the filtrate in vacuo four times with additions of toluene (150 mL) each time. The resulting solid is dried under reduced pressure. Purification: Purification by chromatography using silica gel is carried out using a solvent mixture of dichloromethane and MeOH (gradient from 15: 1 to 1: 1), saturated with HCl gas. The second chromatography on silica gel produces 12.5 g of 2-sulphorodamine dichloride (Compound 18) as a dark green solid (purity> 98% by HPLC). The total yield for all 3 stages is 11.0%. Crystallization: 2.5 g of 2-sulforhododamine dichloride (Compound 18) with a purity of approximately 88% is dissolved in ethanol (approximately 50 mL) and ethyl acetate (approximately 150 mL) is added, which precipitates a sticky solid . The supernatant is treated with silica gel (5 g) and filtered. The silica is strained with 1 M HCl (200 ml), the resulting purple solution is then used to dissolve the sticky solid which is precipitated with ethyl acetate in an earlier step. The solution is heated to a boil and treated with a hot saline solution (400 mL, saturated aqueous NaCl at room temperature). The solution is allowed to cool to room temperature and little precipitation is observed. The pH is adjusted from -0.2 to +0.3 by careful addition of solid NaHC03. The mixture is heated to boiling, an aliquot of the cooled solution is analyzed for pH 0.0. Solid NaHCO3 is added to the hot solution until a cooled sample shows pH 0.1. The suspension is left to cool to room temperature, after two days the supernatant is analyzed to a pH of 0.0. Solid NaHCO3 is carefully added to the mixture, with no further precipitation being observed (use a red lamp!). The solid is separated by centrifugation and the supernatant discarded (pH 0.0). The solid is suspended in the same volume of 20% aqueous NaCl as used in the previous precipitation and centrifuged, discarding the supernatant (pH 0.5). The solid is resuspended in an equal volume, centrifuged and the supernatant discarded (pH 0.55). The second supernatant is not colorless and the washing procedure is interrupted. The solid is dried in a vacuum oven to 1.8 g of golden green material, 96% purity with the HPLC Syntagon method. The above crystallization process can be used with a 2-sulforhodamine acetate salt (such as the trifluoroacetate salt as in Compound 1) instead of the dichloride salt of Compound 18. Figure 6 is a UV absorption spectrum of Compound 18 scanning from 200 nm to 800 nm, at a scanning speed of 400 nm / minute. The maximum UV absorption is at 586 nm. FIG. 7 is a fluorescence emission scan and FIG. 8 is a fluorescence excitation scan of Compound 18. FIG. 9 is a 3-dimensional fluorescence analysis of Compound 18 where EM is the emission wavelength and EX is the excitation wavelength. Excitation (max), which is the maximum absorbance wavelength is 566 nm and emission (max), which is the wavelength with maximum emission intensity, is 618 nm. Example 24: Readjustment of compound 18 to form an isomer (Compound 19) The following compound is prepared:
    Compound 19 Compound 18 can be readjusted to form a Compound 19 isomer, which can be used as its parent compound Compound 18 and the other new rodamine dye of the present invention in the formation of conjugates with other molecules, such as macromolecules for form unique isomeric conjugation products. Compound 19 is 16- [N- (6-azaniumylhexyl) benzenesulfonamido] -3-oxa- 9X5,23-diazaheptacyclo [17.7.1.15,9.02'17.04'15.023,27.013'28] octacous- 1 (27), 2 ( 17), 4.9 (28), 13,15,18-heptaen-9-ileum; dichloride according to the nomenclature system used and is prepared as follows. Compound 18 as the dichloride salt (1.16 g, 1.6 mmol) is dissolved in methanol (30 ml). Aqueous sodium hydroxide (1.0 M, 16 mL) is added dropwise to the 2-SHR solution at room temperature. The resulting colorless solution is stirred for 30 min at room temperature and then evaporated with ethanol (10 ml) and toluene (10 ml) to a colorless solid. The solid dissolved in methanol (20 ml) and aqueous hydrochloric acid (1.0 M, 16 ml) are added, which reform the deep red color. The mixture is evaporated and the solid residue purified with silica chromatography, eluting with methanol (10% -12.5%) in chloroform containing 0.1% concentrated hydrochloric acid. The selected pure fractions are evaporated with ethanol and toluene, drying under high vacuum overnight results in 120 mg of 2-SHR-iso (11% yield) as a metallic green solid. Purity as determined by HPLC is 97.8%. MS (ESI) [M +] = 625. Max absorbance is 586 nm. Similarly, compound 1 or any other salt of 2-sulphorodamine Compound 18 to form the corresponding isomeric product with the corresponding salt. Example 25: Synthesis of conjugate with Compound 15 and carboxymethylated dextrans (CM-dextran)
    To a solution of Compound 15 (Mw: 665.77 g / mol, n: 0.038 mmol, m: 25 mg) dissolved in dimethyl sulfoxide (DMSO) (3 ml) is added cold acetaldehyde from a stock solution (118 μT) with agitation. After 15 minutes, a solution of CM-dextran 150 (300 mg) in distilled water (3 ml) is added with rapid stirring, followed by cyclohexyl-isonitrile (Mw: 109.1 g / mol, n: 0.21 mmol, m: 22.9 mg, δ: 0.878g ml, V: 26.5 μT). The pH is adjusted to 5.9 with a few drops of 1M aqueous HCl. The reaction mixture is left under stirring for four hours.
    After four hours, the pH increased to 6.02, Ethanolamine (Mw: 61.08 g mol, n: 3.34 mmols, m: 0.204 g, δ: 1.02, V: 200 μT) is added and the reaction is left for 90 minutes with stirring.
    Ninety minutes after the addition of ethanolamine, the pH increased to 11.2. After the addition of saturated sodium chloride (0.5 ml), the reaction mixture is slowly poured into ethanol (96%, 50 ml) with rapid stirring where after the blue solid precipitates it is allowed to settle overnight. The supernatant is decanted and the residue is filtered through a glass filter funnel (p3). The precipitate is washed with Ethanol (3x10 ml) and filtered. The product is re-precipitated until it is free of unreacted dye. It is vacuum dried at 60 ° C for 15 hours. The yield is 225 mg.
    The excitation (max) is 587 nm; Emission (max) is 608. Example 26: Synthesis of Compound 18 with CM-dextran A in a solution of Compound 18 (chloride salt, Mw: 697.7 g / mol, n: 0.136 mmol, m: 95, 4 mg) dissolved in DMSO (14 mL) cold acetaldehyde from a stock solution (391 μl) is added with stirring. After 15 minutes a solution of CM-dextran 150 (1.8 g) in distilled water (19.2 mL) is added with rapid stirring, followed by cyclohexyl-isonitrile (Mw: 109.1 g / mol, n: 1, 4 mmol, m: 152.6 mg, δ: 0.878g / mL, V: 172 μl). The pH is adjusted to 5 with a few drops of 1M aqueous HCl. The reaction mixture is allowed to stir overnight.
    Ethanolamine (Mw: 61.08 g / mol, n: 6.64 mmol, m: 0.40 g, δ: 1.02, V: 400 μl) is added and the reaction is left for 60 minutes with stirring.
    Ninety minutes after the addition of ethanolamine, the pH increased to 11.2. After the addition of saturated sodium chloride (0.5 ml), the reaction mixture is slowly poured into ethanol (96%, 50 ml) with rapid stirring whereupon the precipitated blue solid is left to settle overnight.
    The supernatant is decanted and the residue is filtered through a glass filter funnel (p3). The precipitate is washed with ethanol (3 x 10 ml) and filtered. The product is precipitated until it is free of the unreacted dye. It is vacuum dried at 60 ° C for 15 hours. The yield is 1.3 g. Figure 10 is a UV absorption spectrum of the scan of the conjugate from 200 nm to 800 nm at a scan speed of 400 nm / minute. The maximum UV absorption is at 589.5 nm. Figure 11 is a fluorescence emission scan and Figure 12 is a fluorescence excitation scan of the conjugate. Figure 13 is a three-dimensional fluorescence scan of the conjugate where EM is the emission wavelength and EX is the excitation wavelength.
    The excitation (max) is 589 nm; The emission (max) is 608 nm. Example 27: Synthesis of Compound 3 with CM-dextran To a solution of compound 3 (TFA salt, Mw: 646.7 g / mol, n: 0.037 mmol, m: 24 mg) dissolved in DMSO (3 mL) cold acetaldehyde from a stock solution (118 μl) is added with stirring. After 15 minutes, a solution of CM-dextran 150 (300 mg) in distilled water (3 mL) is added with rapid stirring, followed by cyclohexyl-isonitrile (Mw: 109.1 g / mol, n: 0.21 mmol, m: 22.9 mg, δ: 0.878g / mL, V: 26.5 μl). The pH is adjusted to 5 with a few drops of 1M aqueous HCl. The reaction mixture is left under stirring for 4 hours.
    Ethanolamine (Mw: 61.08 g / mol, n: 6.64 mmol, m: 0.20 g, δ: 1.02, V: 200 μl.) Is added and the reaction is left for 60 minutes with stirring. The reaction product, after the addition of saturated sodium chloride (0.5 ml), is slowly poured into ethanol (96%, 50 ml) with rapid stirring with which the precipitated blue solid is left to settle overnight.
    The supernatant is decanted and the residue is filtered through a glass filter funnel (p3). The precipitate is washed with ethanol (3x10 ml) and filtered. The product is precipitated until it is free of the unreacted dye. It is vacuum dried at 60 ° C for 15 hours. The yield is 339 mg. The excitation (max) is 588 nm; The emission (max) is 609 nm. Example 28: Synthesis of Compound 16 with CM-dextran To a solution of compound 16 (TFA salt, Mw: 665.7 g / mol, n: 0.038 mmol, m: 25 mg) dissolved in DMSO (3 mL) cold acetaldehyde from a stock solution (118 μE) is added with stirring. After 15 minutes, a solution of CM-dextran 150 (300 mg) in distilled water (3 mL) is added with rapid stirring, followed by cyclohexyl-isonitrile (Mw: 109.1 g / mol, n: 0.21 mmol, m: 22.9 mg, δ: 0.878g / mL, V: 26.5 μl). The pH is adjusted to 5 with a few drops of 1M aqueous HCl. The reaction mixture is left under stirring for 4 hours.
    Ethanolamine (Mw: 61.08 g / mol, n: 6.64 mmols, m: 0.20 g, δ: 1.02, V: 200 μl) is added and the reaction is left for 60 minutes with stirring. The reaction product, after adding saturated sodium chloride (0.5 ml), is slowly poured into ethanol (96%, 50 ml) with rapid stirring as the precipitated blue solid is left to settle overnight. The supernatant is decanted and the residue is filtered through a glass filter funnel (p3). The precipitate is washed with ethanol (3x10 ml) and filtered. The product is precipitated until it is free of the unreacted dye. It is vacuum dried at 60 ° C for 15 hours. The yield is 167 mg. The excitation (max) is 585 nm; The emission (max) is 606 nm. REFERENCES 1. Lee S, McAuliffe DJ, Kodama T, Doukas AG, In vivo transdermal delivery using a shock tube, Shock Waves (2000) 10: 307-307 2. Janson LW, Ragsdale K, Luby-Phelps K, Mechanism and size cutoff for steric exclusion from actin-rich cytoplasmic domains., Biophys J (1996) 71: 1228-1234 3. Pu R, Robinson KR, Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa., J Cell Sci (1998) 111 (Pt 21): 3197-3207 4. Nishiya T, Kajita E, Horinouchi T, Nishimoto A, Miwa S, Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88, FEBS Lett (2007) 581: 3223-3229 5. Tanner GA, Sandoval RM, Dunn KW, Two-photon in vivo microscopy of sulfonefluorescein secretion in normal and cystic rat kidneys, Am J Physiol Renal Physiol (2004) 286: F152-F160 6. Titus JA, Haugland R, Sharrow SO, Segal DM, Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluo rometric and fluorescence microscopic studies, J. Immunol. Methods (1982) 50 (2): 193-204 7. Nimmerjahn, A., Kirchhoff, F., Kerr, JN, Helmchen, F., Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo, Nature Methods ( 2004) 1: 31-7 8. Kim, TG; Castro, J. C; Loudet, A .; Jiao, J. 5 G.-S .; Hochstrasser, R. M .; Burgess, K .; Topp, M. R., Journal of Physical Chemistry A (2006), 110 (1), 20-27
    权利要求:
    Claims (19)
    [0001]
    1. Composition characterized by comprising a rhodamine dye or a salt thereof with the general structure of:
    [0002]
    2. Composition according to claim 1, characterized by the fact that the rhodamine dye is capable of conjugation with carboxymethylated dextran.
    [0003]
    3. Composition according to claim 1, characterized by the fact that the rhodamine dye is capable of conjugation with a macromolecule.
    [0004]
    4. Composition according to claim 3, characterized by the fact that the macromolecule is selected from the group consisting of polymers, proteins, polysaccharides, derivatives of polysaccharides, lipids and nucleic acids.
    [0005]
    5. Composition according to claim 4, characterized in that the protein is an antibody.
    [0006]
    6. Composition according to claim 4, characterized in that the nucleic acid is a DNA or an RNA.
    [0007]
    7. Composition according to claim 1, characterized in that the salt is selected from the group consisting of trifluoroacetate, chloride, hydrochloride, hydrobromide, iodhydrate, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicitinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartarate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate and p-toluenesulfonate.
    [0008]
    8. Composition according to claim 1, characterized in that the salt is trifluoroacetate or chloride.
    [0009]
    Composition according to claim 1, characterized in that the salt is a pharmaceutically acceptable salt.
    [0010]
    10. Composition, according to the fact that Ri, R2, R4 and R5 are H and R3 are:
    [0011]
    11. Composition, according to claim 10, characterized by the fact that it undergoes a readjustment to form an isomer having the structure of:
    [0012]
    12. Composition according to claim 11, characterized in that the rhodamine dye is a salt.
    [0013]
    13. Composition according to claim 12, characterized in that the salt is dichloride.
    [0014]
    14. Composition according to claim 11, characterized in that the rhodamine dye is able to form a conjugate with a macromolecule by means of the functional amino group to form a unique isomeric conjugation product.
    [0015]
    15. Composition according to claim 4, characterized in that the polysaccharide is a dextran.
    [0016]
    16. Composition according to claim 1, characterized by the fact that R1, R2, R4 and R5 are H and R3 are:
    [0017]
    17. Composition, according to claim 1, characterized by the fact that R1, R2, R4 and R5 are H and R3 are:
    [0018]
    18. Composition according to claim 17, 10 characterized in that the other molecule is carboxymethylated dextran.
    [0019]
    19. Composition, according to claim 1, characterized by the fact that R1, R2, R4 and R5 are H and R3 are:
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    法律状态:
    2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2018-07-17| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|
    2019-07-09| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2020-06-16| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-09-24| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2021-01-12| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-02-09| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/04/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    2022-01-25| B21F| Lapse acc. art. 78, item iv - on non-payment of the annual fees in time|Free format text: REFERENTE A 11A ANUIDADE. |
    优先权:
    申请号 | 申请日 | 专利标题
    US32057110P| true| 2010-04-02|2010-04-02|
    US61/320,571|2010-04-02|
    PCT/US2011/030999|WO2011123820A2|2010-04-02|2011-04-01|Novel rhodamine dyes and conjugates|
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