Synthesis and in vivo stability studies of [¹⁸F]-zwitterionic phosphonium aryltrifluoroborate/indomethacin conjugates

Abstract

With the goal of developing new positron emission tomography (PET) probes for imaging inflammation in cancer tumours, we have conjugated zwitterionic phosphonium aryltrifluoroborates as fluoride captors with indomethacin, a known cyclooxygenase 2 inhibitor. The resulting conjugates have been radiolabeled by ¹⁸F–¹⁹F isotopic exchange in aqueous solutions. NMR studies combined with in vivo data show that the nature of the phosphonium substituents plays an important role on the stability of the radiotracers.

---

Introduction

Positron emission tomography, which is used as an important medical diagnostic technique, has stimulated a great deal of synthetic chemistry aimed at the incorporation of the radionuclide ¹⁸F in organic derivatives and biomolecules.^1–3 While traditional approaches have relied on the formation of C–¹⁸F bonds, radiolabelling can also be achieved by conjugation of an organic derivative or biomolecule with a fluorophilic prosthetic group. Given their inherent fluoride ion affinity, Lewis acidic group 13 elements are being considered as fluoride binding sites in a growing numbers of prosthetic groups.^4–10 Inspired by earlier contributions on the use of aryltrifluoroborate prosthetic groups by the group of Perrin,^11–23 we showed that zwitterionic structures which combine the trifluoroborate unit with a proximal cationic group possess superior stability which lessens deactivation of the probe by fluoride anion release.²⁴ Examples of such zwitterions include 1–3 (Fig. 1), which were described by our group in 2012.²⁵ More recently, the less hydrophobic derivatives A²⁶ and B²⁷ have been described (Fig. 1) and, in the case of A, incorporated in a range of peptide based radiotracers.^26–32

Fig. 1 Structures of the known zwitterionic organotrifluoroborates.

As part of our continuing interest in the chemistry of phosphonium-trifluoroborates, we have started to investigate their conjugation with organic derivatives as a mean to generate disease specific radiotracers. With this in mind, we have decided to consider the conjugation of these derivatives with indomethacin (Fig. 2), a nonsteroidal anti-inflammatory drug that selectively binds to the cyclooxygenase 2 (COX-2) enzymes.^33–36 This study was prompted by: (i) the knowledge that COX-2 enzymes are overexpressed at the surface of most cancer cells;^37–39 (ii) the successful use of indomethacin as a targeting agent in both fluorescence and PET imaging agents.^40–45

Fig. 2 Structure of indomethacin.

Results and discussion

Synthesis and characterization of the precursors

To avail ourselves with a broader set of captors and investigate the influence of the phosphorus substituents on the properties of the captors, we decided to synthesize the di-iso-propylphosphine analogue of the carboxylic acid functionalized zwitterionic aryltrifluoroborate 3. This new zwitterionic aryltrifluoroborate 5 was prepared by the deprotonation of the known [ortho-(iPr₂PH)C₆H₄(Bpin)][BF₄]⁴⁶ (4) followed by treatment with 3-iodoproprionic acid at elevated temperature and subsequently KHF₂ at room temperature (Scheme 1). The carboxylic acid derivative 5 was obtained in a 66% overall yield. This new derivative has been fully characterized by multinuclear NMR spectroscopy. The presence of a trifluoroborate moiety has been confirmed by the detection of a ¹¹B NMR signal at 3.1 ppm and a ¹⁹F NMR signal at −134.4 ppm. The ³¹P NMR spectrum shows a singlet at 42.9 ppm corresponding to the phosphonium moiety.

Scheme 1 Scheme showing the synthesis of 5.

The structure of this compound has also been studied by single crystal X-ray diffraction (Fig. 3). The short B(1)–P(1) separation of 3.429(3) Å in 5 which is comparable to that in 3 (3.490(10) Å)²⁵ serves as a reminder of the strong Coulombic stabilization between the oppositely charged phosphonium and trifluoroborate moieties. This stabilization may be complemented by a F(lone pair) → P–C(σ*) donor–acceptor interaction as suggested by the F(1)–P(1) separation of 3.049(3) Å as well as hydrogen bonding interactions between one of the CH group of the iso-propyl substituents and one of the fluorine atoms as shown by the F(1)–C(7) separation of 3.079(3) Å.

Fig. 3 Crystal structure of the 5. Ellipsoids are scaled to the 50% probability level and hydrogen atoms have been omitted for clarity.

The zwitterionic aryltrifluoroborate/indomethacin conjugates were synthesized by reaction of the amine terminated indomethacin derivative 6 with the carboxylic acid derivatives 3 and 5, using EDCI (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), HOBT (1-hydroxybenzotriazole), and DIEA (di-iso-propylmethylamine) as coupling reagents (Scheme 2). The phosphonium trifluoroborate/indomethacin conjugates 7 and 8 were obtained pale yellow powders in a 63 and 57% yield, respectively. These compounds have been characterized by multinuclear NMR spectroscopy. The ³¹P NMR spectrum shows a singlet at 30.6 ppm for 7 and 44.2 ppm for 8 confirming the existence of a phosphonium moiety. In addition, the trifluoroborate group of each compound was detected as a broad singlet by ¹¹B NMR spectroscopy at 2.9 ppm for 7 and 3.0 ppm for 8 as well as ¹⁹F NMR spectroscopy at −133.4 ppm for 7 and −133.9 ppm for 8. The ¹H NMR spectrum of each compound displays two broad triplets at 6.70 and 6.80 ppm for 7 as well as 6.67 and 6.75 ppm for 8 corresponding to the amide signals. The detection of the molecular ion (+Li⁺) by electrospray mass spectrometry at m/z 790.23 for 7 and 722.30 for 8 serves as an additional confirmation for the successful conjugation of the captors with the indomethacin unit. Finally, the lipophilicity of the conjugates 7 and 8 was evaluated by determination of octanol–water partition coefficient (P). The log P values for 7 and 8 were calculated to be 3.60 ± 0.09 and 2.74 ± 0.02, respectively, suggesting the highly lipophilic nature of the conjugate 7.

Scheme 2 Scheme showing the synthesis of the conjugates 7 and 8.

Stability of the precursors in aqueous solution

As shown in various studies,^24,25,47,48 aryltrifluoroborates undergo hydrolysis in aqueous media to produce the corresponding boronic acids according to a first order rate reaction (ν = k_obs[ArBF₃]).^47,49 To probe the possible influence of the phosphorus substituents, we first investigated the hydrolysis of the bis(iso-propyl) derivative 5 by ¹⁹F NMR spectroscopy. As previously done for 1, 2, and 3,²⁵ 5 was dissolved in D₂O–CD₃CN (8/2 vol) at pH 7.5 ([phosphate buffer] = 500 mM, [ArBF₃] = 20 mM with ArBF₃ = zwitterionic trifluoroborate) and monitored periodically. Based on the amount of ArBF₃ in solution as determined by ¹⁹F NMR, we found that the first order hydrolysis rate constant of 5 (k_obs = 5.2 × 10⁻⁵ min⁻¹) is slightly higher than those of 2 (k_obs = 3.9 × 10⁻⁵ min⁻¹) and 3 (k_obs = 1.5 × 10⁻⁵ min⁻¹) and noticeably higher than that of 1 (k_obs = 3.4 × 10⁻⁶ min⁻¹). These experiments confirm that the presence of iso-propyl substituents decreases the kinetic stability of the complexes. A comparison between the hydrolysis rate constants of 2 and 5 also show that the pendant carboxylic acid functionality plays a labilizing role.

Next, we decided to examine the stability of our new indomethacin conjugates compounds 7 and 8 in aqueous media. Unfortunately, 7 and 8 did not dissolve in D₂O–CD₃CN (8/2 vol) phosphate buffer solution. To address this solubility problem, we decided to use the neutral surfactant triton X-100. We found that both conjugates can be dissolved in 10% w/v triton X-100 in H₂O/DMSO (7/3 vol) at pH = 7.5 ([phosphate buffer] = 500 mM, [ArBF₃] = 20 mM with ArBF₃ = zwitterionic trifluoroborate). In order to get a reference point in this new medium, we first evaluated the stability 3 and 5 and obtained hydrolysis rate constants (k_obs) of 4.4 × 10⁻⁶ min⁻¹ and 1.2 × 10⁻⁵ min⁻¹, respectively. These hydrolysis rate constants are at least three times lower than those measured in D₂O–CD₃CN (8/2 vol), which reflects the lower water content of the new H₂O/DMSO (7/3 vol) medium. Using these new conditions, we observed that the hydrolysis reaction of the indomethacin conjugates is extremely slow. In the case of the diphenylphosphonium derivative 7, we did not see any evidence of hydrolysis even after 7 days. In the case of the di-iso-propylphosphonium derivative 8, we did notice a trace amount of free fluoride at the same period of time. By expanding this experiment to a longer timescale, we have been able to calculate k_obs = 4.0 × 10⁻⁷ for 8. These results are consistent with the previous studies on the stability of the ortho-phosphonium aryltrifluoroborates which show that the rate of hydrolysis of the diphenylphosphonium derivative 1 is slower than that of the di-iso-propylphosphonium derivative 2, respectively.^24,25 These results also show that the targeting group, in this case indomethacin, may have a drastic effect on the hydrolytic stability of the trifluoroborate.

Radiosynthesis and in vivo imaging

Using the conditions that we developed for the radiofluorination of zwitterionic aryltrifluoroborates,²⁵ we decided to radiolabel the novel indomethacin conjugates by ¹⁸F–¹⁹F isotopic exchange. This radiosynthetic approach, which is gaining interest, has also been used for the radiofluorination of main group molecules, for example, BF₄⁻,⁵⁰ organoboranes^51–53 and fluorosilane derivatives.^54,55 In a typical experiment, 7 (0.28 μmol) was dissolved in DMSO (20 μL) and mixed with a solution of irradiated [¹⁸O] water (100 μL, pH 2) containing 45 mCi of ¹⁸F at 75 °C for 10 minutes (Scheme 3). The same approach was applied to the radiofluorination of conjugate 8.

Scheme 3 Scheme showing the radiolabeling of 7 and 8 by isotopic exchange in aqueous solution.

As shown in Fig. 4, [^18/19F]–7 and [^18/19F]–8 are cleanly obtained in 95.1% and 93.5% radiochemical yield (RCY) based on the integration of the TLC trace. The identity of [^18/19F]–7 and [^18/19F]–8 were confirmed by comparing the radio TLC trace with the R_f value of the non-labelled analogue. The labelled compounds were purified by HPLC. The decay corrected specific activities of [^18/19F]–7 and [^18/19F]–8 were calculated to be 53.3 mCi μmol⁻¹ and 49.7 mCi μmol⁻¹, respectively, based on the integration of HPLC peak.

Fig. 4 Crude radio-signal TLC profiles obtained in the radiofluorination of [^18/19F]–7 (A) and [^18/19F]–8 (B).

After HPLC purification, we first evaluated the in vitro stability of these probes in phosphate buffer solution (PBS). As shown in Fig. 5(A) and (C), both [^18/19F]–7 and [^18/19F]–8 showed >98.5% radiochemical purity right after reformulation in 1× PBS. However, after 2 hours incubation, the radiochemical purity of [^18/19F]–7 stayed as high as 96.5% (Fig. 5(B)), while the radiochemical purity of [^18/19F]–8 dropped to 72.1% (Fig. 5(D)). This difference clearly indicated that [^18/19F]–8 is less stable than [^18/19F]–7 and undergoes slow defluorination in physiological environments. The lower stability of [^18/19F]–8 is consistent with the results obtained for 8 in H₂O/DMSO (7/3 vol) in the presence of the neutral surfactant triton X-100 (vide supra). The latter conditions appear to slow down the rate of hydrolysis, an effect that we assign to the presence of the surfactant and the relatively large amount of DMSO which is used as an organic co-solvent.

Fig. 5 Radio HPLC profiles of [^18/19F]–7 (A) and [^18/19F]–8 (C) right after reformulation in 1× PBS and radio HPLC profiles of [^18/19F]–7 (B) and [^18/19F]–8 (D) after incubating in 1× PBS for 2 hours.

Next, we endeavoured to evaluate the in vivo stability these probes in a murine model. We injected 0.1 mCi of [^18/19F]–7 and [^18/19F]–8 into female nude mice. At 1 hour post-injection, static microPET scans were obtained. The resulting sagittal images for [^18/19F]–7 and [^18/19F]–8 are shown in Fig. 6 (above). At 1 hour post injection, [^18/19F]–7 showed a clear localization in the liver because of its lipophilic property while [^18/19F]–8 was more quickly cleared through the urinary track as indicated by the appearance of a signal in the bladder of the animal. In addition, we also observed that the [^18/19F]–8 undergoes a high degree of defluorination in vivo as confirmed by a distinct skeletal signal especially obvious in the sagittal images at 1 hour post-injection, which correlated well with the in vitro stability test. Bio-distribution analysis shows that the use of [^18/19F]–8 leads to a bone uptake of 3.4 %ID per g at 1 hour post-injection (Fig. 6 (below)). These values, which are significantly higher than those measured for [^18/19F]–7 (0.29 %ID per g at 1 hour post-injection), can be correlated to the higher hydrolysis rate constants measured for the di-iso-propyl derivatives compared to their diphenyl analogues.

Fig. 6 (Above) Decay-corrected whole-body microPET Sagittal images of nude mice from a static scan at 1 hour after injection of [^18/19F]–7 and [^18/19F]–8. The images are corrected based on the injected activity. The signal intensity is reported in % of the injected dose per gram (%ID per g). (Below) Bio-distribution for [^18/19F]–7 and [^18/19F]–8 in nude mice at 1 hour post injection time point.

Conclusions

This report establishes that phosphonium aryltrifluoroborates can be conjugated with molecules of biological interest such as indomethacin using simple synthetic approaches. The resulting conjugates are amenable to late stage radiofluorination via simple isotopic ¹⁸F–¹⁹F exchange in aqueous solution. This approach affords PET probes which have been tested in vivo in a murine model. One of the key findings of these animal studies is the influence of the captor structure which largely governs the bio-distribution and the stability of the probe.

Experimental

General consideration

tert-Butyl(2-aminoethyl) carbamate,⁵⁶ ortho-(Ph₂(CH₂CH₂COOH)P)C₆H₄(BF₃),²⁵ and [ortho-(iPr₂PH)C₆H₄(Bpin)][BF₄]⁴⁶ were synthesized according to published procedures. Indomethacin was purchased from Enzo life sciences; 1,2-diaminoethane was purchased from Mallinckrodt; hydroxylbenzotriazole (HOBT) was purchased from Advanced ChemTech; tert-butylcarbamate, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDCI), N,N-diisopropylethylamine, and trifluoroacetic acid were purchased from Alfar Aesar. All chemicals were used without further purification. CH₂Cl₂ was dried by passing through an alumina column. Electrospray mass spectra were acquired from a MDS Sciex API QStar Pulsar. NMR spectra were recorded on a Varian Unity Inova 400 NMR and an Inova 500B spectrometer at ambient temperature. Chemical shifts are given in ppm, and are referenced to residual ¹H and ¹³C solvent signals as well as external H₃PO₄ (³¹P NMR) and BF₃–Et₂O (¹¹B NMR and ¹⁹F NMR).

Synthesis of (2-((2-carboxyethyl)diisopropylphosphonio) phenyl) trifluoroborate (5). To a stirred solution of the [ortho-(iPr₂PH)C₆H₄(Bpin)][BF₄] (4) (0.84 g, 2.07 mmol) in CH₃CN (15 mL) was added NaNH₂ (0.08 g, 2.07 mmol) at room temperature. The mixture was stirred for 1 h. The mixture was then filtered through celite to remove a sodium salt. The solvent was removed in vacuo affording a pale yellow powder. Without further purification, the powder was dissolved in toluene (15 mL) and 3-iodoproprionic acid (0.415 g, 2.07 mmol) was added to the solution. The reaction mixture was heated to 90 °C for 18 h. After 18 h, toluene was removed under vacuum yielding a pale yellow solid. The solid was then dissolved in methanol (6 mL) and treated with a solution of KHF₂ (0.485 g, 6.21 mmol) in water (6 mL). The resulting solution was sonicated for 15 minutes and stirred for 1 h. The mixture was extracted with dichloromethane (3 × 15 mL) and the organic layer was dried with MgSO₄. After filtration, the solution was concentrated to 1 mL and treated with Et₂O (15 mL), leading to the precipitation of 5 as a pale yellow solid (0.456 g, 66.2% yield). ¹H NMR (399.5 MHz, CD₃CN): δ 1.25 (dd, 6H, isopropyl-CH₃, ³J_H-P = 16.78 Hz, ³J_H-H = 7.59 Hz), 1.37 (dd, 6H, isopropyl-CH₃, ³J_H-P = 16.78 Hz, ³J_H-H = 7.19 Hz), 2.62 (m, 2H, –PCH₂CH₂COOH), 2.90 (m, 2H, –PCH₂CH₂COOH), 3.37 (m, 2H, isopropyl-CH), 7.43 (m, 1H, phenyl-CH), 7.52–7.64 (m, 2H, phenyl-CH), 7.97 (dd, 1H, J_H-H = 7.19, 4.39 Hz). ¹³C NMR (100.5 MHz, CD₃CN): δ 11.39 (d, J_C-P = 3.52 Hz), 11.89 (d, J_C-P = 3.02 Hz), 16.35 (d, J_C-P = 3.12 Hz), 17.25 (d, J_C-P = 3.12 Hz), 23.69 (d, J_C-P = 3.72 Hz), 24.14 (d, J_C-P = 3.72 Hz), 27.58, 127.04 (d, J_C-P = 12.26 Hz), 132.53, 132.76 (d, J_C-P = 10.75 Hz), 136.19 (d, J_C-P = 3.82 Hz), 136.35 (d, J_C-P = 3.82 Hz). ¹¹B NMR (128.2 MHz, CD₃CN): δ 3.05 (q, J_B-F = 47.56 Hz). ¹⁹F NMR (375.9 MHz, CD₃CN): δ −134.36. ³¹P NMR (161.7 MHz, CD₃CN): δ 42.92. Mass (ESI⁻): calcd for C₁₅H₂₂BF₃O₂P (M − H)⁻, 333.14; found 332.83.

Synthesis of tert-butyl (2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)ethyl)carbamate. To a stirred solution of indomethacin (0.870 g, 2.43 mmol) in DMF (25 mL) was added HOBt (0.558 g, 3.65 mmol) and EDCI (0.512 g, 2.67 mmol) at 0 °C. The solution was stirred at 0 °C for 15 min. After 15 min, tert-butyl (2-aminoethyl) carbamate (1.170 g, 7.32 mmol) and DIEA (1.30 mL, 7.36 mmol) were added to the mixture. The reaction mixture was stirred at room temperature for 16 h. Removal of the solvent in vacuo afforded a residue, to which 30 mL water was added before extraction with EtOAc (3 × 25 mL). The organic layer was dried over MgSO₄, concentrated in vacuo, and precipitated from hexane affording the t-butyl carbamate product as a yellow powder (1.260 g, 93.8% yield). ¹H NMR (299.9 MHz, DMSO-d₆): δ 1.37 (s, 9H, CH₃), 2.22 (s, 3H, CH₃), 3.00 (m, 2H, –CH₂–), 3.09 (m, 2H, –CH₂–), 3.50 (s, 2H, CH₂CO), 3.77 (s, 3H, OCH₃), 6.72 (dd, J_H-H = 9.00, 2.34 Hz, 1H, indolyl H-6), 6.79 (t, J_H-H = 4.80 Hz, 1H, amide-H), 6.97 (d, J_H-H = 9.00 Hz, 1H, indolyl H-7), 7.10 (d, J_H-H = 2.40 Hz, indolyl H-4), 7.65 (d, J_H-H = 8.10 Hz, 2H, p-chlorobenzoyl H-3, H-5), 7.71 (d, J_H-H = 8.40 Hz, 2H, p-chlorobenzoyl H-2, H-6), 8.04 (t, J_H-H = 5.40 Hz, 1H, amide-H). ¹³C NMR (125.58 MHz, DMSO-d₆): δ 13.81, 28.62, 31.58, 55.83, 78.10, 102.29, 111.71, 114.64, 115.05, 129.46, 130.71, 131.32, 131.57, 134.69, 135.57, 137.96, 155.98, 156.04, 168.27, 169.95. Mass (ESI⁺): calcd for C₂₆H₃₀ClN₃O₅Na (M + Na)⁺, 522.98; found 522.16.

Synthesis of 2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)ethanaminium trifluoroacetate (6). tert-Butyl(2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)ethyl)carbamate (1.260 g, 2.521 mmol) was dissolved in 50% (vol) solution of trifluorocarboxylic acid (TFA) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was then concentrated in vacuo to a volume of 2 mL. Addition of hexanes (20 mL) led to the precipitation of 6 as a light brown solid (1.256 g, 96.9% yield). ¹H NMR (299.9 MHz, DMSO-d₆): δ 2.22 (s, 3H, CH₃), 2.86 (m, 2H, CH₂C), 3.29 (m, 2H, CCH₂), 3.53 (s, 2H, CH₂CO), 3.75 (s, 3H, OCH₃), 6.71 (dd, J_H-H = 9.00, 2.55 Hz, 1H, indolyl H-6), 6.94 (d, J_H-H = 9.30 Hz, 1H, indolyl H-7), 7.08 (d, J_H-H = 2.43 Hz, indolyl H-4), 7.64 (d, J_H-H = 8.70 Hz, 2H, p-chlorobenzoyl H-3, H-5), 7.69 (d, J_H-H = 8.70 Hz, 2H, p-chlorobenzoyl H-2, H-6), 7.82 (s, br, 3H, NH₃⁺), 8.23 (m, 1H, NHCOCH₂). ¹³C NMR (125.58 MHz, DMSO-d₆): δ 13.36 31.07 36.66 38.66 55.42, 101.93, 111.21, 113.90, 114.57, 129.06, 130.31, 130.33, 131.16, 134.21, 135.34, 137.65, 155.59, 158.47 (q, J_C-F = 36.42 Hz) 167.87, 170.38. ¹⁹F NMR (469.92 MHz, DMSO-d₆): δ −74.32. Mass (ESI⁺): calcd for C₂₁H₂₃ClN₃O₃ (M + H)⁺, 400.50; found 400.12.

Synthesis of (2-((3-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)ethyl)amino)-3-oxopropyl) diphenylphosphonio)phenyl)trifluoroborate (7). To a stirred solution of ortho-(Ph₂(CH₂CH₂COOH)P)C₆H₄(BF₃) (3) (0.280 g, 0.53 mmol) in DMF (25 mL) was added HOBt (0.122 g, 0.80 mmol) and EDCI (0.112 g, 0.58 mmol) at 0 °C. In a separate vial, DIEA (0.28 mL, 1.60 mmol) was added to a solution of 6 (0.409 g, 0.80 mmol) in DMF (5 mL). The solution of 6 was subsequently mixed with a solution of ortho-(Ph₂(CH₂CH₂COOH)P)C₆H₄(BF₃) (3) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Removal of the solvent in vacuo afforded a residue, to which 30 mL of water was added and extracted with EtOAc (3 × 25 mL). The organic layers were combined and washed with 2 M HCl (3 × 30 mL), saturated NaHCO₃ (3 × 30 mL), and brine (1 × 30 mL), respectively. Finally, the organic layer was dried over MgSO₄, concentrated in vacuo, and mixed with hexane leading to the precipitation of 7 as a yellow powder (0.261 g, 62.83% yield). ¹H NMR (499.4 MHz, CD₃CN): δ 2.27 (s, 3H, CH₃), 2.46 (m, 2H, CH̲₂CH₂P), 3.17 (s, br, 4H, NHCH̲₂CH̲₂NH), 3.56 (m, 2H, CH₂CH̲₂P), 3.56 (s, 2H, CH₂), 3.79 (s, 3H, OCH₃), 6.62 (dd, 1H, J_H-H = 8.99, 2.35 Hz, indolyl H-6), 6.68 (s, br, 1H, amide-H), 6.78 (s, br, 1H, amide-H), 6.96 (d, 1H, J_H-H = 8.49 Hz, indolyl H-7), 7.02 (d, 1H, J_H-H = 1.70 Hz, indolyl H-4), 7.16 (dd, 1H, phenyl-CH̲, ³J_H-P = 13.98 Hz, ³J_H-H = 6.49 Hz), 7.36 (m, 1H, phenyl-CH̲), 7.53–7.81 (m, 11H, phenyl-CH̲), 7.99 (m, 1H, phenyl-CH̲). ¹³C NMR (125.59 MHz, CD₃CN): δ 12.98, 29.44, 31.56, 39.19, 39.57, 55.43, 101.43, 111.57, 113.82, 115.13, 122.39, 123.08, 127.06, 127.17, 129.12 (d, J_C-P = 2.26 Hz), 129.16, 129.48, 129.59, 129.68, 131.05, 131.20, 131.35, 132.95, 133.02, 133.22 (d, J_C-P = 3.77 Hz), 133.42, 133.49, 133.83 (d, J_C-P = 3.01 Hz), 134.72, 135.17, 135.28, 135.41, 135.54, 136.31, 138.51, 156.17, 168.49, 170.34, 170.38, 170.51. ¹¹B NMR (128.2 MHz, CD₃CN): δ 2.88. ¹⁹F NMR (375.9 MHz, CD₃CN): δ −133.4. ³¹P NMR (161.7 MHz, CD₃CN): δ 30.6. Mass (ESI⁺): calcd for C₄₂H₃₉ClN₃O₄PBF₃Li (M + Li)⁺, 790.25; found 790.23.

Synthesis of (2-((3-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)ethyl)amino)-3-oxopropyl) diisopropylphosphonio)phenyl)trifluoroborate (8). To a stirred solution of ortho-(iPr₂(CH₂CH₂COOH)P)C₆H₄(BF₃) (5) (0.288 g, 0.86 mmol) in DMF (25 mL) was added HOBt (0.198 g, 1.29 mmol) and EDCI (0.182 g, 0.95 mmol) at 0 °C. In the separated vial, DIEA (0.45 mL, 2.58 mmol) was added to a solution of 6 (0.575 g, 1.12 mmol) in DMF (5 mL). The solution of 6 was then combined with a solution of ortho-(iPr₂(CH₂CH₂COOH)P)C₆H₄(BF₃) (5) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Removal of solvent in vacuo afforded a residue, to which 30 mL of water was added before extraction with EtOAc (3 × 25 mL). The organic layers were combined and washed with 2 M HCl (3 × 30 mL), saturated NaHCO₃ (3 × 30 mL), and brine (1 × 30 mL), respectively. Finally, the organic layer was dried over MgSO₄, concentrated in vacuo, and mixed with hexane leading to the precipitation of 8 as a yellow powder (0.351 g, 57.03% yield). ¹H NMR (499.4 MHz, CD₃CN): δ 1.14 (dd, 6H, isopropyl-CH₃, ³J_H-P = 16.48 Hz, ³J_H-H = 7.49 Hz), 1.31 (dd, 6H, isopropyl-CH₃, ³J_H-P = 16.48 Hz, ³J_H-H = 7.49 Hz), 2.25 (s, 3H, CH₃), 2.35 (m, 2H, CH̲₂CH₂P), 2.76 (m, 2H, CH₂CH̲₂P), 3.18 (m, 4H, NHCH̲₂CH̲₂NH), 3.32 (m, 2H, isopropyl-CH), 3.53 (s, 2H, CH₂), 3.76 (s, 3H, OCH₃), 6.62 (s, br, 1H, amide-H), 6.64 (dd, 1H, J_H-H = 8.90, 2.40 Hz, indolyl H-6), 6.70 (s, br, 1H, amide-H), 6.94 (d, 1H, J_H-H = 9.00 Hz, indolyl H-7), 6.97 (d, 1H, J_H-H = 2.40 Hz, indolyl H-4), 7.37 (m, 1H), 7.46–7.58 (m, 4H), 7.68 (m, 2H), 7.93 (m, 1H). ¹³C NMR (125.59 MHz, CD₃CN): δ 13.01, 16.39 (d, ²J_C-P = 2.14 Hz), 17.25 (d, ²J_C-P = 2.14 Hz), 23.73 (d, ¹J_C-P = 4.40 Hz), 24.08 (d, ¹J_C-P = 4.40 Hz), 28.93 (d, ¹J_C-P = 4.02 Hz), 31.57, 39.33, 39.54, 55.43, 101.41, 111.61, 113.77, 115.19, 126.85, 126.93, 129.14, 129.18, 131.06, 131.18, 131.37, 132.34 (d, J_C-P = 3.39 Hz), 132.60, 132.68, 134.69, 136.11 (d, J_C-P = 3.89 Hz), 136.24 (d, J_C-P = 3.52 Hz), 136.34, 138.55, 156.22, 168.49, 170.39, 170.49, 170.60. ¹¹B NMR (128.2 MHz, CD₃CN): δ 3.00. ¹⁹F NMR (375.9 MHz, CD₃CN): δ −133.9. ³¹P NMR (161.7 MHz, CD₃CN): δ 44.2. Mass (ESI⁺): calcd for C₃₆H₄₃ClN₃O₄PBF₃Li (M + Li)⁺, 722.91; found 722.30.

Crystallographic measurements

Single crystals of 5 were obtained by slow evaporation of a solution of 5 in CH₂Cl₂: toluene (4 : 1 (v/v)) mixture. The crystallographic measurement of 5 was performed using a Bruker APEX-II CCD area detector diffractometer, with graphite-monochromated Mo-K_α radiation (λ = 0.71069 Å). A specimen of suitable size and quality was selected and mounted onto a nylon loop. The semi-empirical method SADABS was applied for absorption correction. The structure was solved by direct methods, and refined by full-matrix least-square method against F² with the anisotropic temperature parameters for all non-hydrogen atoms. All H atoms were geometrically placed and refined using the riding model approximation. Data reduction and further calculations were performed using the Bruker SAINT+ and SHELXTL NT program packages.

Kinetic studies

A sample of 5 (5 mg) was dissolved in 0.2 mL of CD₃CN and 0.8 mL D₂O (pH 7.5, phosphate buffer, 500 mM). The ¹⁹F NMR spectrum of 5 was collected periodically. The decomposition of the aryltrifluoroborates were monitored by integration of the decreasing aryltrifluoroborate signal in conjunction with the increasing signal corresponding to free F⁻. In addition, a sample of 3, 5, 7, and 8 was also dissolved in 0.3 mL DMSO-d₆ and 0.7 mL of 10% w/v triton X-100 in D₂O phosphate buffer (pH 7.5, 500 mM) for the stability test. The rate of aryltrifluoroborate hydrolysis was monitored by ¹⁹F NMR spectroscopy. All spectra were processed using the VNMRJ Version 2.2 NMR software. The rate constant, k_obs, was calculated using a well-established NMR method reported in the literature.⁴⁷ This method is based on the fact that the concentration in ArBF₃ species is proportional to the ¹⁹F NMR integration of ArBF₃ signal divided by the sum of the integration of ArBF₃ signal and the free fluoride signal. For convenience, the value of the ArBF₃ integration is arbitrarily set at 100 and the free fluoride integration determined.

Octanol–water partition coefficient

To determine the lipophilicity of the compounds, a sample of 7 and 8 (5 mg) were dissolved in 0.5 mL of octanol and 0.5 mL PBS (1×). After vigorous mixing for 10 min, the mixture was separated by 5 min centrifuge (5000 rpm). Then aliquots (n = 3) of the octanol layer and PBS layer were collected and loaded onto Nanodrop UV-Vis Spectrophotometer to measure the UV absorbance at 250 nm. The log P values reported as an average of 3 independent measurements plus the standard deviation.

Radiochemistry experiments

All chemicals from commercial were of analytic grade and used without further purification. Analytical reversed-phase high-performance liquid chromatography (HPLC) was accomplished on a Waters 515 chromatography system. A Waters 2487 dual λ absorbance detector and model 2200 scaler ratemeter radiation detector were added to the HPLC. HPLC was performed on a phenomenex Luna 5μ C18 column with a flow of 1 mL min⁻¹. The mobile phase was programmed to change from 95% solvent A and 5% solvent B (0–2 min) to 5% solvent A and 95% solvent B at 22 min, where solvent A is 0.1% TFA in water and solvent B is 0.1% TFA in acetonitrile.

Radiochemistry

The radiolabelling reactions were carried out using the following protocol. Compound 7 and 8 (0.28 μmol) were dissolved in 20 μL DMSO. 45 mCi of [¹⁸F]-fluoride in 100 μL [¹⁸O] water was added. pH was adjusted to 2 by adding 6 N HCl. The reaction mixture was incubated at 75 °C for 10 min. Then the reaction mixture was loaded onto the Radio-TLC with a mobile phase of 95% acetonitrile in water to measure the percentage of the reaction conversion. The labeled compounds were confirmed by comparison of the R_f value with the starting non-radioactive compounds.

In vitro stability test

The HPLC purified fresh [^18/19F]–7 and [^18/19F]–8 were re-injected into HPLC for radio profile standard. Then the probes were added with 0.1 N NaOH to adjust pH to 7. Then 10× PBS was added to each probes to reconstruct the solution to 1× PBS. After 2 hours incubation, a fraction of each probe (50 μCi) was injected into HPLC. The radio purity was calculated based on the integration of the product peak and other minor peaks.

MicroPET imaging

MicroPET imaging were acquired at 1 h post injection. For PET image acquiring, each female nude mice was injected with 0.1 mCi of [^18/19F]–7 and [^18/19F]–8 in 1× PBS pH 7.5 (200 μL) via the tail vein. At 1 hour post injection, the mice were anesthetized using isoflurane (2% in oxygen), then placed into imaging chambers equipped with a heated coil to maintain body temperature and gas anesthesia as well. The static microPET acquisition were then achieved and reconstructed for analysis.

Bio-distribution analysis

The mice were sacrificed at 1 hour post injection time and different organs were collected and weighed. Gamma counting was carried out by PerkinElmer 2480 Automatic Gamma Counter. All animal related experiments were performed in compliance with the relevant laws and institutional guidelines approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee (IACUC).

Acknowledgements

This work was supported by the Cancer Prevention Research Institute of Texas (RP130604), the National Institute of Biomedical Imaging and Bioengineering (1R01EB014354-01A1), the National Cancer Institute (P30-CA016086-35-37), and the Biomedical Research Imaging Center, University of North Carolina at Chapel Hill. K. C. gratefully acknowledges the Development and Promotion of Science and Technology (DPST) project under Royal Thai Government for the financial support.

Notes and references

1. K. Chansaenpak, B. Vabre and F. P. Gabbaï, Chem. Soc. Rev., 2016, 45, 954–971.
2. P. W. Miller, N. J. Long, R. Vilar and A. D. Gee, Angew. Chem., Int. Ed., 2008, 47, 8998–9033.
3. L. Cai, S. Lu and V. W. Pike, Eur. J. Org. Chem., 2008, 2853–2873.
4. B. P. Burke, G. S. Clemente and S. J. Archibald, Contrast Media Mol. Imaging, 2015, 10, 96–110.
5. S. Liu, D. Li, Z. Zhang, G. K. Surya Prakash, P. S. Conti and Z. Li, Chem. Commun., 2014, 50, 7371–7373.
6. S. Liu, D. Li, H. Shan, F. P. Gabbaï, Z. Li and P. S. Conti, Nucl. Med. Biol., 2014, 41, 120–126.
7. E. J. Keliher, J. A. Klubnick, T. Reiner, R. Mazitschek and R. Weissleder, ChemMedChem, 2014, 9, 1368–1373.
8. S. Liu, T.-P. Lin, D. Li, L. Leamer, H. Shan, Z. Li, F. P. Gabbaï and P. S. Conti, Theranostics, 2013, 3, 181–189.
9. J. A. Hendricks, E. J. Keliher, D. Wan, S. A. Hilderbrand, R. Weissleder and R. Mazitschek, Angew. Chem., Int. Ed., 2012, 51, 4603–4606.
10. Z. Li, T.-P. Lin, S. Liu, C.-W. Huang, T. W. Hudnall, F. P. Gabbaï and P. S. Conti, Chem. Commun., 2011, 47, 9324–9326.
11. R. Ting, M. J. Adam, T. J. Ruth and D. M. Perrin, J. Am. Chem. Soc., 2005, 127, 13094–13095.
12. C. W. Harwig, R. Ting, M. J. Adam, T. J. Ruth and D. M. Perrin, Tetrahedron Lett., 2008, 49, 3152–3156.
13. R. Ting, C. Harwig, U. auf dem Keller, S. McCormick, P. Austin, C. M. Overall, M. J. Adam, T. J. Ruth and D. M. Perrin, J. Am. Chem. Soc., 2008, 130, 12045–12055.
14. R. Ting, J. Lo, M. J. Adam, T. J. Ruth and D. M. Perrin, J. Fluorine Chem., 2008, 129, 349–358.
15. Y. Li, A. Asadi and D. M. Perrin, J. Fluorine Chem., 2009, 130, 377–382.
16. U. auf dem Keller, C. L. Bellac, Y. Li, Y. Lou, P. F. Lange, R. Ting, C. Harwig, R. Kappelhoff, S. Dedhar, M. J. Adam, T. J. Ruth, F. Bénard, D. M. Perrin and C. M. Overall, Cancer Res., 2010, 70, 7562–7569.
17. Y. Li, R. Ting, C. W. Harwig, D. K. U. auf, C. L. Bellac, P. F. Lange, J. A. H. Inkster, P. Schaffer, M. J. Adam, T. J. Ruth, C. M. Overall and D. M. Perrin, Med. Chem. Commun., 2011, 2, 942–949.
18. Z. Liu, Y. Li, J. Lozada, J. Pan, K.-S. Lin, P. Schaffer and D. M. Perrin, J. Labelled Compd. Radiopharm., 2012, 55, 491–496.
19. Y. Li, J. Guo, S. Tang, L. Lang, X. Chen and D. M. Perrin, Am. J. Nucl. Med. Mol. Imaging, 2013, 3, 44–56.
20. Y. Li, Z. Liu, C. W. Harwig, M. Pourghiasian, J. Lau, K.-S. Lin, P. Schaffer, F. Benard and D. M. Perrin, Am. J. Nucl. Med. Mol. Imaging, 2013, 3, 57–70.
21. Z. Liu, Y. Li, J. Lozada, P. Schaffer, M. J. Adam, T. J. Ruth and D. M. Perrin, Angew. Chem., Int. Ed., 2013, 52, 2303–2307.
22. Z. Liu, Y. Li, J. Lozada, M. Q. Wong, J. Greene, K.-S. Lin, D. Yapp and D. M. Perrin, Nucl. Med. Biol., 2013, 40, 841–849.
23. Z. Liu, N. Hundal-Jabal, M. Wong, D. Yapp, K. S. Lin, F. Benard and D. M. Perrin, Med. Chem. Commun., 2014, 5, 171–179.
24. C. R. Wade, H. Zhao and F. P. Gabbaï, Chem. Commun., 2010, 6380–6381.
25. Z. Li, K. Chansaenpak, S. Liu, C. R. Wade, H. Zhao, P. S. Conti and F. P. Gabbaï, Med. Chem. Commun., 2012, 3, 1305–1308.
26. Z. Liu, M. Pourghiasian, M. A. Radtke, J. Lau, J. Pan, G. M. Dias, D. Yapp, K.-S. Lin, F. Bénard and D. M. Perrin, Angew. Chem., Int. Ed., 2014, 53, 11876–11880.
27. K. Chansaenpak, M. Wang, Z. Wu, R. Zaman, Z. Li and F. P. Gabbai, Chem. Commun., 2015, 51, 12439–12442.
28. M. Pourghiasian, Z. Liu, J. Pan, Z. Zhang, N. Colpo, K.-S. Lin, D. M. Perrin and F. Bénard, Bioorg. Med. Chem., 2015, 23, 1500–1506.
29. Z. Liu, D. Chao, Y. Li, R. Ting, J. Oh and D. M. Perrin, Chem.–Eur. J., 2015, 21, 3924–3928.
30. Z. Liu, G. Amouroux, Z. Zhang, J. Pan, N. Hundal-Jabal, N. Colpo, J. Lau, D. M. Perrin, F. Bénard and K.-S. Lin, Mol. Pharm., 2015, 12, 974–982.
31. Z. Liu, M. A. Radtke, M. Q. Wong, K.-S. Lin, D. T. Yapp and D. M. Perrin, Bioconjugate Chem., 2014, 25, 1951–1962.
32. Z. Liu, D. M. Perrin, M. Pourghiasian, F. Benard, J. Pan and K.-S. Lin, J. Nucl. Med., 2014, 55, 1499–1505.
33. L. H. Rome and W. E. M. Lands, Proc. Natl. Acad. Sci. U. S. A., 1975, 72, 4863–4865.
34. R. J. Kulmacz and W. E. M. Lands, J. Biol. Chem., 1985, 260, 2572–2578.
35. A. S. Kalgutkar, B. C. Crews, S. W. Rowlinson, A. B. Marnett, K. R. Kozak, R. P. Remmel and L. J. Marnett, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 925–930.
36. A. S. Kalgutkar, A. B. Marnett, B. C. Crews, R. P. Remmel and L. J. Marnett, J. Med. Chem., 2000, 43, 2860–2870.
37. R. A. Gupta and R. N. DuBois, J. Natl. Cancer Inst., 2002, 94, 406–407.
38. T. J. Maier, K. Schilling, R. Schmidt, G. Geisslinger and S. Grosch, Biochem. Pharmacol., 2004, 67, 1469–1478.
39. W. C. Black, C. Bayly, M. Belley, C. C. Chan, S. Charleson, D. Denis, J. Y. Gauthier, R. Gordon, D. Guay, S. Kargman, C. K. Lau, Y. Leblanc, J. Mancini, M. Ouellet, D. Percival, P. Roy, K. Skorey, P. Tagari, P. Vickers, E. Wong, L. Xu and P. Prasit, Bioorg. Med. Chem. Lett., 1996, 6, 725–730.
40. M. J. Uddin, B. C. Crews, K. Ghebreselasie and L. J. Marnett, Bioconjugate Chem., 2013, 24, 712–723.
41. L. J. Marnett, J. Org. Chem., 2012, 77, 5224–5238.
42. M. J. Uddin, B. C. Crews, K. Ghebreselasie, I. Huda, P. J. Kingsley, M. S. Ansari, M. N. Tantawy, J. Reese and L. J. Marnett, Cancer Prev. Res., 2011, 4, 1536–1545.
43. M. J. Uddin, B. C. Crews, A. L. Blobaum, P. J. Kingsley, D. L. Gorden, J. O. McIntyre, L. M. Matrisian, K. Subbaramaiah, A. J. Dannenberg, D. W. Piston and L. J. Marnett, Cancer Res., 2010, 70, 3618–3627.
44. E. F. J. de Vries, A. van Waarde, A. R. Buursma and W. Vaalburg, J. Nucl. Med., 2003, 44, 1700–1706.
45. T. J. McCarthy, A. U. Sheriff, M. J. Graneto, J. J. Talley and M. J. Welch, J. Nucl. Med., 2002, 43, 117–124.
46. A. L. Gott, W. E. Piers, J. L. Dutton, R. McDonald and M. Parvez, Organometallics, 2011, 30, 4236–4249.
47. R. Ting, C. W. Harwig, J. Lo, Y. Li, M. J. Adam, T. J. Ruth and D. M. Perrin, J. Org. Chem., 2008, 73, 4662–4670.
48. J. Bernard, R. Malacea-Kabbara, G. S. Clemente, B. P. Burke, M.-J. Eymin, S. J. Archibald and S. Jugé, J. Org. Chem., 2015, 80, 4289–4298.
49. A. J. J. Lennox and G. C. Lloyd-Jones, J. Am. Chem. Soc., 2012, 134, 7431–7441.
50. M. Jauregui-Osoro, K. Sunassee, A. J. Weeks, D. J. Berry, R. L. Paul, M. Cleij, J. Banga, M. J. O'Doherty, P. K. Marsden, S. E. M. Clarke, J. R. Ballinger, I. Szanda, S. Y. Cheng and P. J. Blower, Eur. J. Nucl. Med. Mol. Imaging, 2010, 37, 2108–2116.
51. Z. Liu, N. Hundal-Jabal, M. Wong, D. Yapp, K. S. Lin, F. Benard and D. M. Perrin, MedChemComm, 2014, 5, 171–179.
52. Z. B. Liu, M. Pourghiasian, M. A. Radtke, J. Lau, J. H. Pan, G. M. Dias, D. Yapp, K. S. Lin, F. Benard and D. M. Perrin, Angew. Chem., Int. Ed., 2014, 53, 11876–11880.
53. Z. B. Liu, M. A. Radtke, M. Q. Wong, K. S. Lin, D. T. Yapp and D. M. Perrin, Bioconjugate Chem., 2014, 25, 1951–1962.
54. R. Schirrmacher, G. Bradtmoller, E. Schirrmacher, O. Thews, J. Tillmanns, T. Siessmeier, H. G. Buchholz, P. Bartenstein, B. Waengler, C. M. Niemeyer and K. Jurkschat, Angew. Chem., Int. Ed., 2006, 45, 6047–6050.
55. E. Schirrmacher, B. Wangler, M. Cypryk, G. Bradtmoller, M. Schafer, M. Eisenhut, K. Jurkschat and R. Schirrmacher, Bioconjugate Chem., 2007, 18, 2085–2089.
56. T. Schröder, K. Schmitz, N. Niemeier, T. S. Balaban, H. F. Krug, U. Schepers and S. Bräse, Bioconjugate Chem., 2007, 18, 342–354.

---

Footnotes

† Electronic supplementary information (ESI) available: Experimental, characterization and imaging data. CCDC 1058977. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra26323a

‡ Contributed equally to the work.

§ Jointly conceived the study.

---