DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12/22/2025 has been entered.
Status of Claims
Claims 1, 5 and 13 have been amended. Claims 1, 5, 7, 9, 10, 13 and 17-20 are pending and are examined herein on the merits for patentability.
Response to Arguments
Applicant’s arguments have been fully considered. Any rejection not reiterated herein has been withdrawn as being overcome by claim amendment. New grounds for rejection are set forth, necessitated by claim amendment.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1, 9, 10 and 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Cui et al. (J. Photochemistry and Photobiology A: Chem., 2007, 186, p. 85-92) in view of Taniguchi et al. (Med. Chem. Commun., 2019, 10, 1121).
Cui teaches the synthesis and spectral properties and photostability of novel boron–dipyrromethene dyes. Generally, the BODIPY fluorophores present advantageous photo-spectral properties, such as high extinction coefficients and high fluorescence quantum yields, which facilitate their applications in DNA sequencing and bio-analysis. However, various substituents at BODIPY framework may lead to large difference on their spectral properties, especially on the quantum yields.
Two series of novel boron–dipyrromethene (BODIPY) dyes containing 8-phenyl groups have been synthesized and their spectral properties have been studied (page 85).
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The terminal COOH in 2e may be activated in the form of succinimide ester and used to label bio-molecules directly.
In summary, we have synthesized two series of BODIPY dyes (1 and 2) and researched their spectral properties. Dyes 2 with four methyl groups show much higher fluorescence quantum yields and extinction coefficients than dyes 1. The X-ray structure analysis of the crystals of 1c and 2c is used to reveal that blocking the rotation of 8-phenyl moiety by 1- and 7-methyl groups will suppress the intramolecular vibronic relaxation and internal conversion. The “push–pull” electronic effect caused by methyl groups at 3- and 5-position of BODIPY is another positive factor for the high quantum yields of 2. The photostability of dyes 1 are higher than that of dyes 2, and the electron withdrawing p-substituents at phenyl moiety of the dyes are beneficial to increasing the photostability. The BODIPY dyes with better photostability present comparatively lower quantum yields in our research (page 91).
Compound 1b is within the scope of the instant claims such that R1-R6, R21, R22, R24 and R25 are hydrogen, R23 is a carboxy group and Q1 is a halogen.
Compound 2b is within the scope of the instant claims such that R1, R2, R5 and R6 are Formula A (bond-alkyl), R3 and R4 are hydrogen, R21, R22, R24 and R25 are hydrogen, i.e. a substituent, R23 is a carboxy group and Q1 is a halogen.
Cui does not specifically teach wherein Q2 is an alkyl group.
Taniguchi teaches the photophysical properties and application in live cell imaging of B,B-fluoro-perfluoroalkyl BODIPYs. The photophysical properties of newly identified B,B-fluoro-perfluoroalkyl BODIPYs (2 and 3), which possess a fluoro group and a trifluoromethyl or pentafluoroethyl group at the boron center, were investigated. B,B-Fluoro-perfluoroalkyl BODIPYs 2 and 3 exhibited better photophysical/chemical properties than B,Bdifluoro-BODIPY 1, as follows: (1) higher photostability both in methanol solvent and in a live cell environment, (2) higher stability against acid degradation, and (3) improved fluorescence signal-to-noise ratios in a cell system. These favorable properties of B,B-fluoro-perfluoroalkyl BODIPYs are likely due to the highly electron-withdrawing nature of the perfluoroalkyl groups on the boron atom, which reduces the reactivity to 1 O2 and strengthens the complexation of the dipyrromethene ligands to the boron atom. Thus, B,Bfluoro perfluoroalkyl BODIPYs may be useful functional molecules for various applications.
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The fluoro-perfluoroalkyl BODIPYs 2 and 3 exhibited higher photostability and chemical stability than the corresponding BF2-BODIPY 1. Interestingly, when 2 and 3 were applied to a live cell system, the fluorescence signal-to-noise ratios were markedly improved compared with 1. In addition, the higher photostability of 2 and 3 compared with that of 1 was retained in the live cell environment. Mechanistic studies suggest that the strongly electron-withdrawing perfluoroalkyl groups render BODIPYs 2 and 3 more electron-deficient, which reduces the reactivity to 1 O2 and stabilizes the B–N bonds. The B,B-fluoroperfluoroalkyl BODIPYs complement previous BODIPYs whose photostability is improved by other structural modifications. In addition, the B-perfluoroalkyl modification was accessible to the dipyrromethene containing an alkyne functionality at the meso position,11 demonstrating potential applicability of the new BODIPY for the bioconjugation (page 1123).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify a compound 1b or 2b taught by Cui to have at least one alkyl or substituted alkyl group according to one of variables Q1 or Q2 of the instant claims when the teaching of Cui is taken in view of Tanaguchi. Each of Cui and Tanaguchi are directed to fluorescent BODIPY dyes, including for conjugation to a biomolecule. While Cui teaches halide at the stated position, one would have been motivated to provide a halide and alkyl halide according to B,B-fluoro perfluoroalkyl BODIPYs, with a reasonable expectation of success, because Tanaguchi teaches that doing so allows for high photostability which was retained in the live cell environment and that the strongly electron-withdrawing perfluoroalkyl groups render BODIPYs more electron-deficient, which reduces the reactivity to oxygen and stabilizes the B–N bonds. Regarding claim 18, it would have been further obvious to provide links between the dye and a biomolecule because Cui teaches that a dye having a terminal COOH may be activated in the form of succinimide ester and used to label bio-molecules directly.
Claim(s) 1, 9, 10 and 17-20 are rejected under 35 U.S.C. 103 as being unpatentable over Cui et al. (J. Photochemistry and Photobiology A: Chem., 2007, 186, p. 85-92) in view of Taniguchi et al. (Med. Chem. Commun., 2019, 10, 1121), in further view of Schirner et al. (CA 2385593).
The rejection over Cui in view of Taniguchi is applied as above.
With regard to claims 19-20, Cui and Taniguchi do not specifically recite wherein a biomolecule is a protein and that an amide bond is formed between N-hydroxysuccinimide ester in a compound with an amino group in a biological substance.
Schirner teaches antibody dye conjugates which are suited for binding to structures of newly formed vessels and to their use for interoperatively depicting pathological angiogenesis (abstract). Dyes include bodipy and derivatives (page 8). Dyes in the antibody-dye conjugates from the above-mentioned classes, which have one or more carboxyl groups, which are coupled to amino groups of antibodies or antibody fragments after chemical activation.
Dyes with carboxyl groups are activated first by conversion into a reactive ester (e. g., N-hydroxysuccinimide ester) according to methods that are known in the art (page 15). See also Example 1 directed to conjugation of an antibody with an N-hydroxysuccinimide ester activated dye.
It would have been obvious to one of ordinary skill in the art at the time of the invention to provide a protein as a biomolecule which is conjugated to a compound taught by Cui and Taniguchi when the teachings of Cui and Taniguchi are taken in view of Schirner. One would have been motivated to do because Cui teaches that a bodipy dye having a terminal COOH may be activated in the form of succinimide ester and used to label bio-molecules directly, and Schirner teaches that a protein/antibody conjugated to a dye, including bodipy derivative, via NHS-activated carboxylic acid coupled to an amino group is useful for interoperatively depicting pathological angiogenesis.
Claim(s) 1, 5, 7, 9, 10, 13 and 17-20 are rejected under 35 U.S.C. 103 as being unpatentable over Cui et al. (J. Photochemistry and Photobiology A: Chem., 2007, 186, p. 85-92) in view of Taniguchi et al. (Med. Chem. Commun., 2019, 10, 1121), in further view of RU 2680090 and Kanazawa et al. (EP 3531132).
The rejection over Cui and Taniguchi is applied as above.
With regard to claims 5, 7 and 13, Cui and Taniguchi do not teach a sulfo group or formula A at positions R3 and R4, or alkyl at positions R21 and R22.
RU 2680090 teaches salts of the compound of formula I with alkali metals, replacing hydrogen atoms in both sulfo groups, where R means the N-oxysuccinimidyl group. Also proposed a method of obtaining salts and their use. Salts of the compounds of formula I are fluorescent dyes with better characteristics than their analogues – water solubility, quantum yield, and extinction coefficient – and can be used as fluorescent labels of protein molecules (abstract).
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General formula I is taught, page 3:
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,
Including where R is H, Hal, alkyl, aryl, cycloalkyl, alkylaryl, acyl and sulfo group.
See compound IIIa, page 5.
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.
Alkali metal salts of the compounds of formula III are proposed for sulfo groups, alkali metals preferably Na and K.
The objective of the invention is to obtain 2- (4,4-difluoro-1,3,5,7-tetramethyl-2,6-disulfo-4-boro-3a, 4a-diaza-s-indacen-8-yl)-benzoic acid (compound IIIa), as well as its derivatives with N-oxysuccimidyl groups, characterized by the possibility of using for fluorescent labeling of proteins (fluorescein channel, 490-520 nm), characterized by solubility in water, higher quantum yield, higher extinction coefficient, compared to fluorescein and with known dyes of the formula (I), for example BODIPY FL, used for the fluorescein channel.
Kanazawa teaches compounds represented by Formula (1) that can be used by being bound to the first binding substance such as an antibody as a fluorescent label, and the compound represented by Formula (1) is useful as a fluorescent labeling agent (pages 2-3). A fluorescently labeled antibody bound through an amide bond is taught (page 4).
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Compounds in Example 1 include:
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It would have been obvious to one of ordinary skill in the art to provide a sulfonated variable including a sulfo group or Formula A at position R3 and R4 of a bodipy derivative corresponding to the instant claims when the teaching of Cui and Taniguchi are taken in view of RU 2680090 and Kanazawa. Each of Cui, Tanaguchi, RU 2680090 and Kanazawa are directed to fluorescent BODIPY dyes, including for conjugation to a biomolecule. One would have been motivated to do so, with a reasonable expectation of success because RU 2680090 teaches that doing a sulfo group allows for solubilization of the dye. One would have had a reasonable expectation of success in doing so because RU 2680090 teaches a sulfonation method by sulfonation with chlorosulfonic acid (see methods) and teaches a carboxyl group as one of the active groups capable of forming a chemical bond with the ligand (claim 2).
Regarding claim 5, Kanazawa further teaches a sulfonated derivative corresponding to variables R3 and R4 as increasing solubility.
Regarding variables R21 and R22 of the instant claims, it would have been further obvious to provide an alkyl moiety because Kanazawa teaches formula Ar-1 having variables R121 and R122 equivalent to the stated positions which each independently represent a halogen atom, an alkyl group, or an alkoxy group, provided that R121 and R122 each may have a hydrophilic part or a capture part capable of forming a covalent bond with a biological molecule, n represents an integer of 0 to 4, and in a case where n is 2 or more, a plurality of R122's may be the same or different from each other (page 3-4).
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Accordingly, one of ordinary skill in the art would have found it obvious to select alkyl as a suitable substituent to represent R121 or R122 from among the limited number of variables set forth at position R21 and R22 of the instant claims.
Regarding claims 18-20, it would have been further obvious to provide links between the dye and a biomolecule because Kanazawa teaches that a NHS activated carboxylic acid is used to label an antibody via an amide bond.
Claim(s) 1, 5, 9, 10 and 17-20 are rejected under 35 U.S.C. 103 as being unpatentable over Kanazawa et al. (EP 3531132) in view of Taniguchi et al. (Med. Chem. Commun., 2019, 10, 1121).
Kanazawa teaches compounds represented by Formula (1) that can be used by being bound to the first binding substance such as an antibody as a fluorescent label, and the compound represented by Formula (1) is useful as a fluorescent labeling agent (pages 2-3). A fluorescently labeled antibody bound through an amide bond is taught (page 4).
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Compounds in Example 1 include:
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The Exemplary compounds are within the scope of the instant claims such that R1 and R2 are formula A including bond-alkyl, R5 and R6 are formula A including alkylene-aryl, R3 and R4 are formula A including aryl-formula (1-3) and comprising a sulfo group, R21, R22, R24 and R25 are hydrogen, R23 is a carboxy group and Q1 is a halogen.
Kanazawa does not specifically teach wherein Q2 is an alkyl group.
Taniguchi teaches the photophysical properties and application in live cell imaging of B,B-fluoro-perfluoroalkyl BODIPYs. The photophysical properties of newly identified B,B-fluoro-perfluoroalkyl BODIPYs (2 and 3), which possess a fluoro group and a trifluoromethyl or pentafluoroethyl group at the boron center, were investigated. B,B-Fluoro-perfluoroalkyl BODIPYs 2 and 3 exhibited better photophysical/chemical properties than B,Bdifluoro-BODIPY 1, as follows: (1) higher photostability both in methanol solvent and in a live cell environment, (2) higher stability against acid degradation, and (3) improved fluorescence signal-to-noise ratios in a cell system. These favorable properties of B,B-fluoro-perfluoroalkyl BODIPYs are likely due to the highly electron-withdrawing nature of the perfluoroalkyl groups on the boron atom, which reduces the reactivity to 1 O2 and strengthens the complexation of the dipyrromethene ligands to the boron atom. Thus, B,Bfluoro perfluoroalkyl BODIPYs may be useful functional molecules for various applications.
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The fluoro-perfluoroalkyl BODIPYs 2 and 3 exhibited higher photostability and chemical stability than the corresponding BF2-BODIPY 1. Interestingly, when 2 and 3 were applied to a live cell system, the fluorescence signal-to-noise ratios were markedly improved compared with 1. In addition, the higher photostability of 2 and 3 compared with that of 1 was retained in the live cell environment. Mechanistic studies suggest that the strongly electron-withdrawing perfluoroalkyl groups render BODIPYs 2 and 3 more electron-deficient, which reduces the reactivity to 1 O2 and stabilizes the B–N bonds. The B,B-fluoroperfluoroalkyl BODIPYs complement previous BODIPYs whose photostability is improved by other structural modifications. In addition, the B-perfluoroalkyl modification was accessible to the dipyrromethene containing an alkyne functionality at the meso position,11 demonstrating potential applicability of the new BODIPY for the bioconjugation (page 1123).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the Example 1 compounds taught by Kanazawa to have at least one alkyl or substituted alkyl group according to one of variables Q1 or Q2 of the instant claims when the teaching of Kanazawa is taken in view of Tanaguchi. Each of Kanazawa and Tanaguchi are directed to fluorescent BODIPY dyes, including for conjugation to a biomolecule. While Kanazawa teaches halide at the stated position, one would have been motivated to provide a halide and alkyl halide according to B,B-fluoro perfluoroalkyl BODIPYs, with a reasonable expectation of success, because Tanaguchi teaches that doing so allows for high photostability which was retained in the live cell environment and that the strongly electron-withdrawing perfluoroalkyl groups render BODIPYs more electron-deficient, which reduces the reactivity to oxygen and stabilizes the B–N bonds. Regarding claims 18-20, it would have been further obvious to provide links between the dye and a biomolecule because Kanazawa teaches that a NHS activated carboxylic acid is used to label an antibody via an amide bond.
Regarding variables R21 and R22 of the instant claims, it is noted that Kanazawa teaches formula Ar-1 having variables R121 and R122 equivalent to the stated positions which each independently represent a halogen atom, an alkyl group, or an alkoxy group, provided that R121 and R122 each may have a hydrophilic part or a capture part capable of forming a covalent bond with a biological molecule, n represents an integer of 0 to 4, and in a case where n is 2 or more, a plurality of R122's may be the same or different from each other.
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Accordingly, one of ordinary skill in the art would have found it obvious to select alkyl, halo or alkoxy as a suitable substituent to represent R121 or R122 from among the limited number of variables set forth at the stated position.
Conclusion
No claims are allowed at this time.
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/LHS/
/Michael G. Hartley/Supervisory Patent Examiner, Art Unit 1618