New Synthetic Approaches to Small Molecules for Imaging

Division Of Basic Sciences - Nci

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Abstract

Fluorophores in the NIR range enables in vivo optical imaging due to the significant tissue penetration of light in this range. Despite a central role in modern biology and medicine, the compounds employed in NIR fluorescence techniques have changed little in recent decades. Using molecular design concepts borrowed from related fields (e.g. medicinal chemistry and modern organic synthesis), we seek to develop new agents with improved utility for cancer-related imaging and microscopy. The long-term goal is to identify readily synthesized, stable, and bright fluorophores with optimal properties for biomedical imaging. The heptamethine cyanine class of near-IR fluorophores are used for many applications, with extensive recent progress in the context of fluorescence-guided surgery. A number of years ago, we developed a new rearrangement reaction that enables the synthesis of previously inaccessible variants. Compared to existing agents, the compounds we have prepared exhibit improved optical properties and significantly greater chemical stability to biological nucleophiles. Through an extensive optimization campaign, we have developed molecules that are exceptionally resistant to aggregation following labeling on both targeting antibodies and nanoparticles. These molecules exhibit reduced liver uptake and improved in vivo signal when compared to existing agents. In related work, we have shown that small changes in the polar functional groups appended to these fluorophores can have a dramatic impact on biodistribution and tumor accumulation when used without targeting motifs. Over the past year, we have continued to develop agents for use in labeled sensitive abdominal features such as bile duct anatomy and ureter structures. These agents have been tested in several settings, and we are carrying out additional preclinical studies. Motivated by the need for a general synthetic strategy, we came to recognize that these efforts would benefit from further flexibility with respect to cyanine chromophore substitution. Recent studies by Klan and coworkers described a pyridine-derivatization approach using 2,4-dinitrophenyl-pyridinum (Zincke) salts. These studies clearly demonstrate the significant strategic benefits of a pyridine-based cyanine synthesis. However, in seeking to apply this method, we found the requirement of intervening aniline substitution and the use of potentially problematic dinitrophenyl-pyridinium salts were significant impediments, particularly for more polar compounds. We therefore screened several other pyridinium salts, and found that pyridinium-benzoxazole (PyBox) reagents are exceptional for cyanine synthesis. These PyBox intermediates undergo direct transformation to indocyanines without aniline-containing intermediates with short reaction times (typically 15 minutes) using only sodium acetate as an additive. Most notably, the reaction mixtures are exceptionally easy to purify on up to gram scales, often requiring only trituration. In initial studies, we have assembled 35 chemically diverse C4' and C3' substituted cyanines, which have enabled the studies below. Historically, cyanine dyes have been used most successfully for applications involving extracellular targets (e.g., as mAb labels) - something we have found in our work. This is because cyanines are intrinsically charged and, in the case of hydrophobic cell-permeable variants, can only be effectively targeted to the lysosome (with amine-modification) or to the mitochondria (with cationic hydrophobic variants). This effect is so robust that the attachment of targeting ligands can only rarely overcome this bias. The ability to apply NIR probes effectively to intracellular targets would have numerous applications. For FGS, many solid-tumor associated targets are intracellular, and therefore can only be addressed with cell-permeable small molecule conjugates. We hypothesize the cyanines capable of polarity-dependent cyclization would enable new approaches to imaging intracellular targets and fluorogenic probe discovery. In initial efforts, we found that conventional 6-membered ring-containing cyanines substituted at the C4' position with a 2-carboxy aryl ring (prepared through Suzuki coupling) do not undergo efficient spirocyclization, likely due to steric effects. The PyBox chemistry described above allowed us to characterize the reactivity of various nucleophiles at the C4' position in the absence of ring substitution. As shown in Figure 5, we have found the C4'-carboxy species undergo efficient, solvent-dependent cyclization to butanolide derivatives (which were fully assigned by NMR using the dramatic solvent dependance of the closed state in d6-DMSO vs. the open state in CD3OD). This reactivity can be tuned by the introduction of 1,1,1-trifluoro-ethyl substituents on the indolenine ring, which promote the ring-closed state. In closer analogy to rhodamine derivatives, we also found that the C4''-2-carboxy aryl derivatives also undergo polarity-dependent spirocyclization, with similar effects following introduction of a 1,1,1-trifluoro-ethyl substituent. To further tune the ring-open/ring-closed equilibria (particularly to further stabilize the closed state), we will generate additional derivatives, including recently reported sulfonamide derivatives. Notably, our results to date are the first example of polarity-dependent intramolecular cyclization chemistry on the cyanine scaffold. With access to these molecules, we will test the utility of these agents for several intracellular applications with an emphasis on targets of interest for FGS applications. Fluorogenic probes in the near-infrared (NIR) region could provide stimuli-dependent information in living organisms. We recently developed the first class of NIR-responsive fluorogenic probes, which are based on the broadly used heptamethine cyanine scaffold. These compounds were created by modification of heptamethine norcyanines with stimuli-responsive carbamate linkers. These cyanine carbamates (CyBams) exhibit exceptional turn-ON ratios (170x) due to dual requirements for NIR emission, carbamate cleavage through 1,6-elimination and chromophore protonation. We have applied these several settings, including the usef a gamma-glutamate substituted CyBam which was applied to imaging gamma-glutamyl transpeptidase (GGT) activity in a metastatic model of ovarian cancer. By optimizing the cellular uptake and retention of these probes, we have been able to create mAb-targeted variants, which are being applied to study the properties of mAb linkers as described below in detail. Wavelengths between 1000 to 2000 nm, referred to as the shortwave-infrared (SWIR) or NIR-II range, can enable high-resolution in vivo imaging at depths not possible with conventional NIR wavelengths. There is a significant need for the type of bioconjugatable probes that have proven invaluable for multicolor imaging in the visible and NIR range. Enabled by a rational design process, we generated persulfonated nonamethine indocyanine dyes with absorbance/emission maxima beyond 800 nm. These compounds are ideally suited for targeted multiplexed imaging in combination with existing cyanines and have extended the range of wavelengths available for targeted multicolor in vivo imaging. In recent studies, we have developed responsive (i.e. turn-ON probes) in this region that respond to local changes in pH. We anticipate probe in this class will be useful to measuring the internalization of various targeting agents.