Evaluation of polyanionic cyclodextrins as high affinity binding scaffolds for fentanyl

Abstract

Cyclodextrins (CDs) have been previously shown to display modest equilibrium binding affinities (K_a ~ 100-200 M^-1) for the synthetic opioid analgesic fentanyl. In this work, we describe the synthesis of new CDs possessing extended thioalkylcarboxyl or thioalkylhydroxyl moieties and assess their binding affinity towards fentanyl hydrochloride. The optimal CD studied displays a remarkable affinity for the opioid of K_a = 66,500 M^-1, the largest value reported for such an inclusion complex to date. One dimensional ¹H Nuclear Magnetic Resonance (NMR) as well as Rotational Frame Overhauser Spectroscopy (2D-ROESY) experiments supported by molecular dynamics (MD) simulations suggest an unexpected binding behavior, with fentanyl able to bind the CD interior in one of two distinct orientations. Binding energies derived from the MD simulations work correlate strongly with NMR-derived affinities highlighting its utility as a predictive tool for CD candidate optimization. The performance of these host molecules portends their utility as platforms for medical countermeasures for opioid exposure, as biosensors, and in other forensic science applications.

Figure 1

(a) Chemical structures of fentanyl and morphine, the potency of the opioid relative to morphine (~ 100x) is provided along with its systemic circulation half-life (t_1/2); (b) structures of two of the most common antidotes used to treat fentanyl poisoning, naloxone and naltrexone along with their systemic circulation half-lives; (c) structures of fentanyl analogs encountered in several overdose cases in the US (acetylfentanyl) as well as others used as incapacitating agents (carfentanil and remifentanil), their potencies relative to morphine are provided; (d) structure of β-cyclodextrin showing the seven glucose units linked in an α1,4-fashion giving rise to a conical structure (frustrum) open at both ends with a hydrophobic interior and a hydrophilic exterior; (e) representation of a hypothetical inclusion complex formed between β-CD and fentanyl.

Figure 2

Basic structural scheme of a βCD with its upper rim, C6-hydroxyl moiety replaced with a generalized R group. There are two types of moieties investigated in this work, the anionic ones comprising CDs featuring the 2-mercaptoacetyl (SBX-1, 1), 3-mercaptopropionoyl (SBX, 2) and 4-mercaptobutanoyl (SBX + 1, 3) groups. The second type of moieties involve ones bearing neutral moieties in the upper rim featuring the 2-mercaptoethanol (SBN-1, 4), 3-mercaptopropanol (SBN, 5), and 4-mercaptobutanol (SBN + 1, 6). In addition to these βCD-based targets, SAX (Sualphadex, 7) and SGX (Sugammadex, 8) were also synthesized and evaluated separately, however, both did not show any significant binding to fentanyl.

Figure 3

NMR binding affinities (simple 1:1 model) versus enthalpic energies determined from simulation for the dominant conformer of fentanyl bound to hosts 1–6 and to α- and β-cyclodextrin. Note the affinity reported here for SBX + 1 is the dominant conformation from the more complex two-state 1:1 model (67 mM^-1) Neutral CD scaffolds given as circles; all others as filled squares. The solid line is a linear fit to filled data points considering measurement uncertainties. The dashed line has slope RT (0.602 kcal mol^-1 at 303 K).

Figure 4

MD results for the host:guest complex with two orientations: (a) fentanyl (carbon atoms in cyan) aligned “down” with amide half near primary rim of SBX (carbon atoms in green), (b) fentanyl in alternate, “up” position within SBX, c) binding energies for all three SBX complexes.

Figure 5

Partial ROESY spectrum of SBX + 1:fentanyl HCl complex. Correlations between fentanyl aromatic protons from the phenethyl ring and SBX + 1 methylenes are highlighted by the dashed box. Full spectrum is given in Fig. 3 of Supporting Information.