16. Narayanan N, Patonay G. A New Method for the Synthesis of Heptamethine Cyanine Dyes: Synthesis of New Near-Infrared Fluorescent Labels. 1995;60:2391-5 doi:10.1021/jo00113a018
17. Strekowski L, Lipowska M, Patonay G. Substitution reactions of a nucleofugal group in heptamethine cyanine dyes. Synthesis of an isothiocyanato derivative for labeling of proteins with a near-infrared chromophore. 1992;57:4578-80 doi:10.1021/jo00043a009
Among the cyanine dyes evaluated, compound 3 (IR783), the polymethine cyanine-based dye with the indolenine nucleus, a chlorinated cyclohexenyl center and a sulfonate group, was found to be the best candidate for NIR fluorescence tumor imaging in the series of NIRFs 1-5, 7-10, Cypate (6), ICG and IR820 probed for their absorbance/fluorescence properties. Although, NIRF 3 was the best both in terms of the spectral properties and tumor affinity, it would not be feasible to use it in the original form for our purposes. It needed further functionalization before conjugation to HPPH (3-(1'-hexyloxyethyl)pyropheophorbide-a), a highly effective photosensitizer undergoing Phase II human clinical trials . In addition, cyanine dyes 5, 7-10 were synthesized from their parent IR820 and 3 (IR783) by replacing the central chlorinated cyclohexenyl group with 3-mercaptobenzoic acid (to yield 7 and 10), 4-mercaptobenzoic acid (to yield 5) and 4-aminophenol (to yield 8 and 9). Also, among the functionalized NIRFs (5, 7-10), the best structural substitute in terms of tumor uptake was found to be 4-aminothiophenol. At the same time, the photophysical data showed that the substitution with 4-aminothiophenol caused quenching of the fluorescence in substituted NIRFs 8 and 9. Based on the results reported in this study, further studies were performed: the NIRFs 5, 7, 8, 9 and 10 were conjugated with HPPH in the mono and di-forms. See the succeeding paper (Part-2, pages 703 - 718). NIRFs 5 and 7 were used to assess whether the position of the dye with respect to HPPH within the conjugate made a difference in PDT response.
Near-infrared (NIR) absorbing fluorescent probes with pH-dependent optical properties offer many advantages over visible dyes, such as low background signal from tissues or other biological samples. Due to their excellent photophysical properties and ease of synthetic modifications, NIR heptamethine cyanine dyes have been increasingly preferred over other fluorescent probes for pH sensing applications. Two major classes of these pH-sensitive dyes include norcyanine dyes with a pH responsive indolium nitrogen atom and ketocyanine dyes with keto-enol isomerization site at the meso-position of heptamethine dye (–). Recently, we reported barbituric acid-incorporated cyanine dyes to generate pH-dependent NIR dyes for biological applications (). Although these dyes show good pH-dependent optical properties, their pKa values were too low for conventional biological applications (pKa~3.5).
Syntheses of pH-dependent pyrazole-substituted NIR cyanine dyes 1-3 are shown in . Meso-pyrazole substituted dyes were obtained from commercially available chloro-substituted dye, IR-820, using modified published procedure (). Under standard Suzuki-coupling conditions, the presence of common base required for these reactions promote decomposition of base-labile heptamethine cyanine dyes or the formation of by-products. These complications were circumvented by conducting the experiment in water/DMF mixture, which served as both a base and reaction solvent. The reaction mixture was purified by column chromatography on reversed phase (RP) silica gel and the precipitate formed after lyophilization of the combined fractions was recrystallized from MeOH/diethyl ether. The UV-Vis and LC-MS data are consistent with the expected features for dyes 1-3.