Synthesis, Characterization, and Potential Usefulness in Liver Function Assessment of Novel Bile Acid Derivatives with Near-Infrared Fluorescence (NIRBAD)

Conventional serum markers often fail to accurately detect cholestasis accompanying many liver diseases. Although elevation in serum bile acid (BA) levels sensitively reflects impaired hepatobiliary function, other factors altering BA pool size and enterohepatic circulation can affect these levels. To develop fluorescent probes for extracorporeal noninvasive hepatobiliary function assessment by real-time monitoring methods, 1,3-dipolar cycloaddition reactions were used to conjugate near-infrared (NIR) fluorochromes with azide-functionalized BA derivatives (BAD). The resulting compounds (NIRBADs) were chromatographically (FC and PTLC) purified (>95%) and characterized by fluorimetry, 1H NMR, and HRMS using ESI ionization coupled to quadrupole TOF mass analysis. Transport studies using CHO cells stably expressing the BA carrier NTCP were performed by flow cytometry. Extracorporeal fluorescence was detected in anesthetized rats by high-resolution imaging analysis. Three NIRBADs were synthesized by conjugating alkynocyanine 718 with cholic acid (CA) at the COOH group via an ester (NIRBAD-1) or amide (NIRBAD-3) spacer, or at the 3α-position by a triazole link (NIRBAD-2). NIRBADs were efficiently taken up by cells expressing NTCP, which was inhibited by taurocholic acid (TCA). Following i.v. administration of NIRBAD-3 to rats, liver uptake and consequent release of NIR fluorescence could be extracorporeally monitored. This transient organ-specific handling contrasted with the absence of release to the intestine of alkynocyanine 718 and the lack of hepatotropism observed with other probes, such as indocyanine green. NIRBAD-3 administration did not alter serum biomarkers of hepatic and renal toxicity. NIRBADs can serve as probes to evaluate hepatobiliary function by noninvasive extracorporeal methods.


■ INTRODUCTION
Bile acids (BAs) are steroids synthesized by the liver from cholesterol.These compounds are characterized by their marked organotropism toward tissues of the so-called enter-ohepatic circuit.BA vectorial properties are due to the presence in hepatocytes and intestinal epithelial cells (mainly in the ileum) of transmembrane proteins accounting for highly effective BA uptake from sinusoidal blood and the intestinal lumen, respectively.Although Na + -independent transporters, belonging to the OATP family, are involved in BA uptake by hepatocytes, this process mainly occurs via Na + -dependent transporters encoded by a member of the family 10 of the solute carrier (SLC) superfamily of genes, namely, the hepatic Na +taurocholate cotransporting polypeptide (NTCP, SLC10A1).The intestinal apical sodium-dependent BA transporter (ASBT, SLC10A2) plays a similar role in the ileum. 1 Owing to the high affinity and specificity of both transporters for BA as substrates, these compounds have been used as shuttles for molecules with pharmacological properties targeted toward tissues within this circuit, such as chlorambucil, 2 nucleosides, 3 nitrogenous bases, 4 polyamines, 5 or cisplatin, 6,7 thus increasing their bioavailability in the liver and intestine while reducing adverse side effects. 8oreover, in the assessment of liver function within clinical settings, diverse methodologies leveraging labeled BA derivatives (BADs) have been explored (for a recent review, see ref 9).These include 18 F-labeled BA derivatives as positron emitter tomography (PET) tracers to study hepatic transporters. 10It is noteworthy that currently used approaches for the diagnosis of several hepatobiliary disorders (e.g., focal lesions, tumors, and cholestasis) also include cholescintigraphy using 99m Tc-labeled iminodiacetic acid derivatives and magnetic resonance imaging (MRI) using hepatocyte-specific contrast agents, such as gadoxetate.However, all probes mentioned lack enterohepatic vectoriality and consequently lack tissue specificity. 11o overcome this limitation, a wide variety of fluorescent BADs have been synthesized.This includes several amido fluorescein (amF) derivatives, such as cholyl-amF, cholyl-glycyl-amF (CGamF), chenodeoxycholyl-glycyl-amF, and ursodeoxycholyl-glycyl-amF, which have been used with in vitro and in vivo models to study BA transport, drug−BA interactions, and cytosol-nucleus traffic. 12,13They have facilitated the exploration of BA organotropism, and the usefulness of drug targeting of BAD vectorized toward cells expressing BA transporters. 6,14esides, cholyl-L-lysyl-fluorescein (CLF) has been used in vivo in combination with intravital imaging performed through confocal microscopy in anesthetized animals for assessing liver function. 15Dansylated cholic acid (CA) derivatives have proven valuable for determining BA amphipathic characteristics and aggregation behavior, as well as their binding to proteins and their handling by the liver. 16ll the fluorescent BADs mentioned above emit in the visible range of the spectrum, which limits their usefulness.The present study aimed to synthesize novel compounds (NIRBADs) labeled with tags emitting near-infrared fluorescence (NIR, 780−2500 nm) with greater tissue penetration and enhanced liver organotropism due to their conjugation with BADs.The aim of synthesizing these novel compounds was to enhance their usefulness by adding the advantage of enabling their visualization from outside the body without the need for invasive procedures.

■ RESULTS AND DISCUSSION
Molecules with NIR fluorescence are helpful tools in clinical practice.For instance, indocyanine green (ICG) has been used to carry out extracorporeal detection of lymph nodes and vessels in cancer and other diseases with an ischemic profile, to study the liver function in patients with hepatic tumors before surgical removal and during laparoscopic cholecystectomy for the identification of biliary anatomy. 17However, ICG, like other available NIR probes, has the limitation of lacking tissueselective characteristics.In contrast, the NIR probes synthesized here are cholephilic compounds, i.e., efficiently taken up by hepatocytes and secreted into bile.Triazoles have proven helpful in generating a broad range of molecules with biological activity. 18,19These compounds are synthesized by the copper-catalyzed Huisgen 1,3-dipolar cycloaddition, the most popular example of the click reaction, which involves alkynes and azides. 20Using 1,2,3-triazole moieties as linkers offers advantages due to their stability under typical physiological conditions and their ability to form hydrogen bonds.Additionally, the capacity of 1,2,3-triazoles to mimic the topological and electronic features of amide bonds makes them well suited for designing peptidomimetics with enhanced medicinal properties. 18,21revious studies have established the click strategy's efficacy in producing BA derivatives by binding them to active molecules with diverse residues. 18An example has been BA binding to peptides to obtain novel compounds in supramolecular chemistry. 22Additionally, significant strides have been made in the field of antimicrobial development through the binding of BA to beta-lactams, resulting in the synthesis of innovative antibiotic agents. 23Furthermore, clickable conjugates of BAs and nucleosides have been synthesized and assayed in vitro as anticancer and antituberculosis agents. 19In the present study, we leveraged this background to synthesize a new family of compounds (NIRBADs) (Figure 1) targeted to the enter-ohepatic circuit through a BA moiety and capable of emitting NIR fluorescence due to the linked fluorochrome, alkynocyanine 718.This compound was selected for this purpose due to two characteristics: (i) near-infrared emission wavelength (Ex664/Em718 nm) and (ii) a terminal triple bond, which eliminated the need for structural modifications prior to carrying out the envisioned 1,3-dipolar cycloaddition reaction with BAazide derivatives.
For the synthesis of NIRBAD-2 (Figure 3), functionalization of the hydroxyl group at the 3α position of the CA steroid core was performed.First, the carboxylic acid of CA was protected by Fischer esterification using thionyl chloride in methanol, obtaining the corresponding methyl ester (7).Next, due to the basic character of the hydroxide anion making it a poor  leaving group, the tosylation of the hydroxyl group at position 3 was carried out to obtain the tosyl derivative of CA (8).Then, an S N 2 nucleophilic substitution reaction with inversion of the configuration of the tosylate group by iodine was performed, using KI and DMF as the solvent (9), resulting in the inversion of C3 configuration from R to S. Finally, to preserve the original hydroxyl configuration R, a subsequent S N 2 substitution of the iodine was performed using sodium azide in DMF, resulting in the (3R) isomer (10) due to the new inversion of the configuration.The next step was the deprotection of the carboxylic acid by a basic hydrolysis reaction with LiOH in methanol (11).Finally, the 1,3-dipolar cycloaddition reaction between the azide derived (11) from CA and alkynocyanine 718 was performed.Once the crude reaction product was obtained, it was purified by preparative thin layer chromatography (PTLC) to obtain the purified NIRBAD-2 compound (12).
The synthesis of these compounds provides insight into their distinct properties arising from structural variations.Although NIRBAD-1 and NIRBAD-3 have undergone functionalization on their side chains, this has been achieved through chemically different linkers, i.e., an ester and an amide, respectively.Consequently, their biodistribution is anticipated to exhibit varying profiles due to the change of a hydrogen bond acceptor to a donor.Moreover, it is worth noting that esters and amides undergo metabolism at significantly different rates, further contributing to the potential dissimilarities. 24In contrast, NIRBAD-2 has been functionalized at the hydroxyl in the C3 position, maintaining the side chain in a carboxylate form.This distinction is also expected to influence its biodistribution.Furthermore, the stereochemistry of the chiral centers in the original BA, CA, has been preserved to retain its native structure.This characteristic ensures that the synthesized BA derivative is still efficiently recognized as a substrate by BA transporters.
The analysis of the fluorescent properties of these compounds revealed that the excitation and emission wavelengths differed from those of the initial fluorescent probe (Figure 4A).Differences among NIRBADs were also found (Figure 4B− D).Moreover, the relative quantum yields of NIRBAD-1 and NIRBAD-3 were similar but lower than that of NIRBAD-2 (Figure S13).This diversity may likely be attributed to the fact that, while the wavelengths stem from the pi-electron delocalization within the indole-alkene conjugate system, slight modifications may arise from the altered chemical environment induced by the presence of triazoles.The lower signal found for ester NIRBAD-1 as compared to that of the amide NIRBAD-3 was not due to bleaching or hydrolysis, as their photostability (Figure S14) and chemical stability (Figure S15) were similarly preserved when kept in solution at 37 °C in the dark for up to 60 min, i.e., longer than the incubation time during uptake experiments (Figure 5).
To assess the hepatocyte-targeting properties of NIRBADs, flow cytometry studies were carried out using CHO cells that either did not express any human BA transporter or stably express NTCP.The uptake of NIRBADs in the absence and  presence of TCA revealed that NTCP can transport NIRBADs in a TCA-inhibitable manner (Figure 5).The order of efficacy for this transport was NIRBAD-3 ≫ NIRBAD-2 > NIRBAD-1.Based on these results, further in vivo assays were carried out using NIRBAD-3 as a proof of concept.
Upon i.v.injection of NIRBAD-3 or alkynocyanine 718, the fluorescence emitted from the upper abdominal region was extracorporeally recorded.In the case of NIRBAD-3, an efficient liver load, followed by migration of the fluorescence toward the intestine, was observed (Figure 6).The hepatic elimination was achieved at approximately 60 min.Determinations carried out in blood samples collected 120 min after NIRBAD-3 administration revealed no change in serum biomarkers of hepatic and renal toxicity (Table 1).
In contrast to NIRBAD-3, the fluorescence due to alkynocyanine 718 persisted in the liver with no transfer to the intestinal area.We also administered ICG for comparison purposes, whose fluorescence was dispersedly distributed in the abdominal region without showing any hepatic selectivity.Moreover, the fluorescence remained in this location throughout the 60 min experimental period (Figure 6).
In conclusion, novel BA derivatives have been synthesized.They are selectively taken up by the liver and efficiently transferred to the intestine by biliary secretion, as expected for a cholephilic substance.This organotropic characteristic, together with their ability to emit NIR fluorescence, permits extracorporeal detection for monitoring their liver handling.The usefulness of these probes in noninvasively assessing liver function in health and diseases using experimental models deserves further investigation.
General Chemical Procedures.Solvents were purified by standard procedures and distilled before use.Reagents and starting materials obtained from commercial suppliers were used without further purification.Melting points are given in °C. 1 H NMR spectra were recorded on a Bruker Avance spectrophotometer at 400, 200, and 100 MHz, as appropriate. 1H NMR chemical shifts are reported in ppm with tetramethylsilane (TMS) as the internal standard or using the residual solvent 1 H resonance as a reference.The coupling constants, J, are reported in Hertz (Hz).Data for 1 H NMR are reported as follows: chemical shift (in ppm), number of hydrogen atoms, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br s = broad singlet).Splitting patterns that could not be clearly distinguished are denoted as multiplets (m).Highresolution mass spectral analyses (HRMS) were performed using ESI ionization and a quadrupole TOF mass analyzer.All compounds were routinely checked by TLC using precoated silica gel 60 F254, aluminum foil, and the spots were detected under UV light at 254 and 365 nm or were revealed spraying with 10% phosphomolybdic acid in ethanol.Flash chromatography (FC) was performed on 70−200 mesh silica gel using a different composition of the mobile phase according to the polarity of the compound.
Cell Culture.The Chinese ovary hamster cell line, CHO-K1 (ATCC CCL-61), was purchased from the American Type Culture Collection (LGC Standards, Barcelona, Spain) and maintained in DMEM high-glucose medium (Merck, Madrid, Spain) supplemented with 50 g/L of L-proline (Merck), GlutaMAX solution (Fisher Scientific), 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin−streptomycin− amphotericin B (Fisher Scientific).Cells were cultured at 37 °C Bioconjugate Chemistry in a 5% CO 2 atmosphere with 80% relative humidity.To ensure the absence of mycoplasma contamination in the culture, periodic PCR tests were conducted using the mycoplasma Gel Form Kit (Biotools B&M Laboratories, Madrid, Spain).Monoclonal cells stably expressing the ORF of NTCP were obtained by transduction with recombinant lentiviral vectors (pWPI) added to target cells at an appropriate multiplicity of infection (MOI = 25) in the presence of hexadimethrine bromide (Polybrene, Merck) as described elsewhere. 31The clone with the highest capacity to carry out the uptake of CGamF, a fluorescent NTCP substrate, 32 was used in further studies.Flow Cytometry Determinations.Both wild-type (WT) CHO cells (transduced with pWPI empty vectors), used here as a control (Mock), and cells stably expressing NTCP (CHO-NTCP) were cultured according to previously established methods. 33To assess drug uptake, experiments were carried out using three to five different cultures for each data point.Subconfluent cultures were resuspended in uptake medium (96 mM NaCl, 0.8 mM MgSO 4 , 5.3 mM KCl, 1.1 mM KH 2 PO 4 , 1.8 mM CaCl 2 , 11 mM D-glucose, and 10 mM HEPES/Tris, pH 7.4).The cells were then incubated in the presence of 10 μM NIRBADs with or without 100 μM TCA at 37 °C for 15 min.Uptake was stopped by rinsing the culture dishes with 0.9 mL of ice-cold buffer, and the intracellular fluorescence was measured with a flow cytometer (FACSCalibur, Becton Dickinson, Madrid).
In Vivo Assays.Male Wistar rats weighing 220−250 g were obtained from the Animal House, University of Salamanca, Spain.They were housed under controlled environmental conditions of temperature (20 °C) and light (12 h/12 h light/ dark cycle).They had free access to water and standard rodent chow (Panlab, Madrid, Spain).All experimental methods adhered to ethical guidelines and regulations, with protocols approved by the University of Salamanca Ethical Committee for Laboratory Animals, after confirming that they complied with ethical approval from the Spain Ministry of Health and followed European guidelines for the care and use of laboratory animals, in accordance with the NIH Guide for the Care and Use of Laboratory Animals.All experiments were conducted under pentobarbital anesthesia (50 mg/kg b.wt., i.p., Nembutal N.R.; Abbot, Madrid), which was also used for euthanizing the animals at the end of the experiments.
Statistical Analysis.Post hoc analyses, such as paired or unpaired Student t tests, were applied appropriately to calculate the statistical significance of differences among groups.Differences were considered significant when p < 0.05.Microsoft Excel (version 15.32) and GraphPad (Prism5) were used for these purposes.
■ ASSOCIATED CONTENT * sı Supporting Information

Figure 5 .
Figure 5. NTCP-mediated NIRBAD uptake.CHO cells transfected with empty vectors (Mock) or lentiviral particles to induce stable expression of NTCP were incubated for 15 min at 37 °C with 10 μM NIRBAD-1, NIRBAD-2, or NIRBAD-3 in the absence (white bars) or in the presence of 100 μM taurocholic acid (TCA) (black bars).Uptake was determined by flow cytometry.The detected fluorescence expressed as arbitrary units of fluorescence (AUF) per cell and 15 min were normalized in each experiment by considering 100% the value of AUF in Mock cells incubated in the absence of TCA (Control).Results are mean ± SEM from six different measurements carried out in three separate cultures.*p < 0.05; comparing with uptake by Mock cells.†p < 0.05; comparing with uptake in the absence of TCA (Control) in each experimental group by paired t-test.

Figure 6 .
Figure 6.Time course of extracorporeal fluorescence detected in the abdominal area after intravenous administration of 1 μmol NIRBAD-3, alkynocyanine 718, or indocyanine green to anesthetized Wistar rats.These compounds were first dissolved in DMSO and then diluted with saline to inject 1 mL (<12% DMSO).At these doses, neither DMSO nor NIRBAD-3 caused acute liver/renal toxicity.