Fluorinated ΔF508-CFTR Correctors and Potentiators for PET Imaging

Abstract

¹⁹F-modified bithiazole correctors and phenylglycine potentiators of the ΔF508-CFTR chloride channnels were synthesized and their function assayed in cells expressing human ΔF508-CFTR and a halide-sensitive fluorescent protein. Fluorine was incorporated into each scaffold using prosthetic groups for future biodistribution imaging studies using positron emission tomography (PET). The ΔF508-CFTR corrector and potentiator potencies of the fluorinated analogs were comparable to or better than those of the original compounds.

Cystic fibrosis (CF) is the most prevalent lethal hereditary disease among Caucasians, affecting approximately one in 2,500 individuals.¹ The average life expectancy in CF is about 40 years. The principal cause of mortality in CF is deterioration of lung function caused by chronic lung infection.^2,3 Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, the most common being a deletion of phenylalanine at position 508 (ΔF508).^4,5 This mutation causes CFTR protein misfolding, resulting in retention and rapid degradation in the endoplasmic reticulum.^6–9

Compounds have been synthesized that rescue defective ΔF508-CFTR protein processing (correctors) and defective chloride channel gating (potentiators).^10–12 Bithiazole 1 (corr-4a) and indolylacetamidophenylacetamide 2 (PG-01) are the benchmark corrector and potentiator studied here, respectively (Figure 1). Structure-activity relationship studies based on corr-4a revealed that substitution of a 2-chloro-5-(N,N-dimethyl-amino)phenyl moiety for the 5-chloro-2-methoxyphenyl group improved water solubility (3; Figure 1)¹³ and that locking the bithiazole system into an s-cis conformation with a 7-membered ring (3; Figure 1) improved corrector efficacy.¹¹

Figure 1.

ΔF508-CFTR correctors (1/3/4) and potentiator (2).

We recently reported the synthesis of a fluorescently-labeled bithiazole corrector 4 (Figure 1), which enabled the evaluation of its biodistribution in mice by fluorescence imaging.¹⁴ Though this approach produced information about the in vivo handling of a functional CF corrector, the inclusion of a fluorophore label resulted in poor compound metabolic stability, limiting the time over which the non-modified compounds could be studied. Also, the limited penetration of light into tissues precluded non-invasive whole-body imaging.

To address these issues, we report here a first step to apply positron emission tomography (PET) imaging to non-invasively visualize the biodistribution of a bithiazole corrector and a phenylglycine potentiator.^15–17 Analogs suitable for PET imaging have the advantage of minimally modifying the original compound structure (replacing a H with an ¹⁸F),¹⁸ which increases the likelihood of maintaining activity while enabling in vivo monitoring of uptake and biodistribution. Indeed, imaging modalities can be relevant at all stages of CF drug development, with potential applications in animal models, tissue preparations, and human in vivo studies. Relevant imaging could enlighten at the basic mechanistic level leading to a better understanding CF pathophysiology, at the level of assessing the potential of new correctors or potentiators, at the effectiveness of treatment level, and/or at the level of measuring function to better drug delivery.¹⁹

PET produces images through the detection of positron emitting radioisotopes. The most commonly used radionucleotide is fluorine-18 due to its relatively long half-life (t_1/2 = 110 min). Other elements, such as carbon (¹¹C t_1/2 = 20.4 min), nitrogen (¹³N t_1/2 = 9.97 min), and oxygen (¹⁵O t_1/2 = 2.04 min), have much shorter half-lives. To address the need for new disease-specific probes, prosthetic groups¹⁸ have been developed for the rapid incorporation of radioisotopes into the specific tracers. A variety of amine-reactive prosthetic groups have been developed, the most commonly being [¹⁸F]N-succinimidlyl-4-fluorobenzoate (5; Figure 2).²⁰ Other ¹⁸F-acylating agents include the carboxylate reactive [¹⁸F]4-fluoroaniline prosthetic group (6; Figure 2).²¹ A strategy that has received considerable attention is the use of click chemistry to incorporate small molecular weight ¹⁸F-labeled alkynes such as [¹⁸F]5-fluoroalkyne (7; Figure 2) into azide substituted peptides.²² Copper(I)-catalyzed 1,2,3-triazole formation is fast, chemoselective, and performed under mild conditions.²²

Figure 2.

Potential ¹⁸F-prosthetics.

As a first step in enabling CF-relevant PET studies, we report here the design, synthesis, and ΔF508-CFTR activity of two ¹⁹F-labeled correctors (8 and 9; Figure 3) and two ¹⁹F-labeled potentiators (10 and 11; Figure 3). Since ¹⁸F has a 110 min half-life, late-stage introduction of the fluorinated moiety is essential and, consequently, targets 8–11 were designed with the aforementioned ¹⁸F-prosthetic groups in mind.

Figure 3.

Fluorine-containing corrector and potentiator analogs.

Targeting fluorobenzoyl analogs ¹⁹F-8 and ¹⁹F-9 (Scheme 1), the first synthetic step was the condensation of 3-chloropentane-2,4-dione with thiourea in refluxing ethanol over 12 h to afford aminothiazole 12 in 95% yield.¹¹ The acetyl group of 12 was subsequently α-brominated with bromine in acetic acid to produce 13 in 86% yield. Aminothiazole building block 15 was obtained in 79% yield by the condensation of 13 with 1-(2-chloro-5-dimethylaminophenyl)thiourea (14) in refluxing ethanol. The final step in the synthesis of ¹⁹F-bithiazole 8 proved to be more challenging than expected as numerous attempts to couple it with fluorobenzoic acid by amide-coupling condensation with HATU, EDC, or CDI proved unsuccessful. Eventually, bithiazole 15 was acylated with 4-fluorobenzoyl chloride (prepared by treatment of 4-fluorobenzoic acid with oxalyl chloride) in the presence of triethylamine at room temperature (10 min) to afford ¹⁹F-8.

Scheme 1.

Synthesis of fluorinated corrector analogs.^a

^aReagents: (a) EtOH, reflux; (b) Br₂, AcOH; (c) 1-(2-chloro-5-dimethylaminophenyl)thiourea (14), EtOH, reflux; (d) i. 4-fluorobenzoic acid, CH₂Cl₂, oxalyl chloride, ii. 4-fluorobenzoyl chloride (16), CH₂Cl₂, TEA; (e) 4-fluorobenzoic acid, CDI, DMF, 100 °C; (f) PyrH⁺Br₃⁻, 33% HBr in AcOH; (g) 1-(5-chloro-2-methoxyphenyl)thiourea (19), EtOH, reflux.

Given these difficulties, a new strategy was developed for the synthesis of fluorinated bithiazole 9. Work began here with the CDI-mediated coupling of aminothiazole 12 with 4-fluorobenzoic acid. Thiazole 17 was markedly easier to purify than 8. Bromination of 17 proved significantly more difficult than 12, requiring the use of a stronger acidic medium (HBr vs. AcOH) with pyridinium tribromide to afford 18 (91% yield). The final reaction involved condensation of 18 with 1-(5-chloro-2-methoxyphenyl)thiourea (19), delivering ¹⁹F-9 in 76% yield.

Yield and purification difficulties were due to the poor solubility of aminobithiazole intermediate 15 as well as the corresponding fluorinated bithazoles 8 and 9. Because [¹⁸F]N-succinimidlyl-4-fluorobenzoate (¹⁸F-SFB) would be used in picomolar quantities for the final coupling to aminobithiazole intermediates, the reaction solution would be quite dilute and solubility is not expected to be a significant issue.

The ΔF508-CFTR corrector activity of ¹⁹F-8 and ¹⁹F-9 was assayed using a cell-based fluorescence assay in Fischer rat thyroid (FRT) cells co-expressing human ΔF508-CFTR and the halide-sensitive fluorescent protein YFP-H148Q/I152L.¹¹ The concentration-dependence data show slightly greater potency of ¹⁹F-8 and ¹⁹F-9, compared to benchmark corrector corr-4a, as shown in Figure 4. EC₅₀ values (in μM) were: 8 = 1.4; 9 = 1.4; corr-4a = 2.3].

Figure 4.

Concentration-dependence of ΔF508-CFTR corrector activity of ¹⁹F-8 and ¹⁹F-9 compared to corr-4a.

Synthesis of fluoroaniline potentiator ¹⁹F-10 (Scheme 2) began with an EDC-mediated coupling of 4-fluoroaniline to Boc-N-methyl-L-phenylglycine (Scheme 2).^10,23 Boc-protected 21, obtained in 61% yield, was then deprotected by treatment with TFA and subsequent EDC-activated coupling with 3-indole acetic acid and DMAP gave ¹⁹F-10 in 72% yield over two steps.

Scheme 2.

Synthesis of fluorinated potentiator ¹⁹F-10 and azido intermediate 23.^a

^aReagents: (a) CuI, NaN₃, L-proline, NaOH, DMSO, 60 °C; (b) EDC, HOBt, DMF, DCM, 0 °C to rt; (c) i. TFA, DCM, ii. EDC, DMAP, DCM, DMF, 0 °C to rt.

Synthesis of 4-(3-fluoropropyl)-1H-1,2,3-triazole analog 11 began with the synthesis of 4-azidoaniline via a CuI-catalyzed, proline-promoted coupling reaction²⁴ followed by an EDC-mediated condensation of 20 (Scheme 2) with Boc-N-methyl-L-phenylglycine to afford 22 (20, 74%; and 22, 97%, respectively). Subsequent Boc removal (TFA) followed by treatment with EDC-activated 3-indole acetic acid and DMAP afforded azido phenylglycine intermediate 23 (90% yield).

The synthesis of 5-fluoroalkyne for the triazole-forming cycloaddition with azido-phenylglycine intermediate 23 proved to be unsuccessful. Initially, we focused on utilizing diethylaminosulfur trifluoride (DAST) for the conversion of the alcohol moiety of 4-pentyne-1-ol into an alkyl fluoride.²⁵ Unfortunately, no fluorinated alkyne was isolated. Das et al. reported that ionic liquids can be used in an improved purification procedure of the dehydroxy-fluorination reaction with DAST; again, no desired product was isolated.²⁶ Likewise, a modified procedure employing tosylate 24 (synthesized by the treatment of 4-pentyn-1ol with p-toluenesulfonyl chloride and triethylamine²⁷) and nucleophilic fluoride [KF plus 18-Crown-6 or Bu₄N⁺F⁻(tBuOH)₄²⁸] was unsuccessful.

With this as a backdrop, tosylate 24 was clicked to azido intermediate 23 in a Huisgen copper-catalyzed 1,3-dipolar cycloaddition to afford 25 in 85% yield (Scheme 3). Finally, potentiator analog ¹⁹F-11 was obtained by treatment of 25 with Bu₄N⁺F⁻(tBuOH)₄ (26) in 31% yield.

Scheme 3.

Synthesis of fluorinated potentiator ¹⁹F-11.

^aReagents: (a) TsCl, TEA, DCM, 0 °C to rt; (b) Na ascorbate, CuSO₄, 23, DCM, tBuOH, H₂O; (c) i. Bu₄N⁺F⁻ H₂O, tBuOH, hexane, 90 °C, 30 min forms Bu₄N⁺F⁻(tBuOH)₄ (26), ii. 25, 26, ACN, 70 °C, 1 h.

The ΔF508-CFTR potentiator activity of ¹⁹F-10 and ¹⁹F-11 was assayed in FRT cells co-expressing ΔF508-CFTR and YFP-H148Q/I152L after rescue of ΔF508-CFTR by 24 h culture at 27 °C.^11,12 Fluorinated phenylglycine analogs had EC₅₀ of 0.09 μM (¹⁹F-10) and 1.1 μM (¹⁹F-11), comparable to that of 0.3 μM reported previously for PG-01,¹⁰ and much better than that of 7.0 μM for benchmark potentiator genistein (Figure 5).

Figure 5.

Concentration-dependence of ΔF508-CFTR potentiator activity of ¹⁹F-10 and ¹⁹F-11 compared to genistein.

The retained corrector activity of ¹⁹F-8 and ¹⁹F-9 and potentiator activity of ¹⁹F-10 is likely the consequence of the enhanced binding interactions of fluorine.²⁹ Indeed, fluorine is found in approximately 5–15% of drugs that have come to market over the past 50 years.²⁹ Fluorine has also been shown to improve metabolic stability and generally enhance physicochemical properties,²⁹ features which would be useful in future in vivo studies.

In conclusion, two active fluorinated corrector analogs and two active fluorinated potentiator analogs were synthesized and the introduction of a fluorine atom was found to improved potency. Both fluorinated correctors (¹⁹F-8 and ¹⁹F-9) and a fluorinated potentiator (¹⁹F-10) showed improved or comparable activity to the original compounds. These active, fluorinated analogs are potentially useful for non-invasive PET imaging of in vivo compound uptake and biodistribution. Given the short half-life of ¹⁹F (t_1/2 = 110 min), the next step en route to PET studies with these compounds will be to develop synthetic routes to ¹⁹F-8, ¹⁹F-9, and ¹⁹F-10 where the ¹⁹F moiety is introduced in the last synthetic step. Current efforts are focused on developing viable and high-yielding routes to ¹⁹F-8 from N²-(2-chloro-5-(dimethylamino)phenyl)-4′-methyl-[4,5′-bithiazole]-2,2′-diamine (15) + 7 and ¹⁹F-10 from (S)-2-(2-(1H-indol-3-yl)-N-methylacet-amido)-2-phenylacetic acid + 6.