Ligand-based design of [18F]OXD-2314 for PET imaging in non-Alzheimer's disease tauopathies

Abstract

Positron emission tomography (PET) imaging of tau aggregation in Alzheimer's disease (AD) is helping to map and quantify the in vivo progression of AD pathology. To date, no high-affinity tau-PET radiopharmaceutical has been optimized for imaging non-AD tauopathies. Here we show the properties of analogues of a first-in-class 4R-tau lead, [¹⁸F]OXD-2115, using ligand-based design. Over 150 analogues of OXD-2115 were synthesized and screened in post-mortem brain tissue for tau affinity against [³H]OXD-2115, and in silico models were used to predict brain uptake. [¹⁸F]OXD-2314 was identified as a selective, high-affinity non-AD tau PET radiotracer with favorable brain uptake, dosimetry, and radiometabolite profiles in rats and non-human primate and is being translated for first-in-human PET studies.

Fig. 1. Structures of tau PET radiotracers divided into structural scaffolds.

A Compounds based on the carbazole scaffold of [¹⁸F]Tauvid™ (a.k.a., [¹⁸F]flortaucipir or [¹⁸F]T807) which also include [¹⁸F]PI-2620. B Janssen’s naphthalene-pyridine linked scaffolds, as well as [¹⁸F]MK-6240 ([¹⁸F]florquinitau) and [¹⁸F]FDDNP. C [¹¹C]PBB3, [¹⁸F]F-PBB3, and [¹⁸F]florzolotau ([¹⁸F]PM-PBB3, [¹⁸F]APN-1607) analogues based on a benzothiazole scaffold. D PET radiotracers based on Oxiant Discovery’s fluoropyridinyl-indole scaffold (OXD analogues) including [¹⁸F]OXD-2115 (a.k.a. CBD-2115) and [¹⁸F]OXD-2314 (this work).

Fig. 2. Synthetic route to OXD-2314 and [¹⁸F]OXD-2314.

Top: chemical precursors 3 or 7 for OXD-2314 and [¹⁸F]OXD-2314, respectively, were synthesized from 2,6-difluoropyridine or 2-chloro-6-nitropyridine according to the route described in the top panel under the following reaction conditions: (a) Hünig’s base, dioxane, 100 °C, 5 h (75% yield); (b) N-bromosuccinimide, acetonitrile, 0 °C, 30 min (96% yield); (c) [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl₂), potassium carbonate (K₂CO₃), dioxane, 90 °C, 1 h (98% yield); (d) tetrabutylammonium fluoride, tetrahydrofuran, 0 °C, 10 min (67% yield). Each step was followed by flash column purification to isolate each intermediate compound. Bottom: Synthesis of OXD-2314 and radiochemical synthesis of [¹⁸F]OXD-2314 from 3 or 7, respectively, under the following reaction conditions: (e) methanol, 150 °C, 45 min under microwave heating (47% yield); (f) cyclotron-generated [¹⁸F]F^-, K₂CO₃, Kryptofix_2.2.2, dimethyl sulfoxide, 160 °C, 20 min; followed by addition of methanol, 130 °C, 20 min. [¹⁸F]OXD-2314 was isolated by semi-preparative high-performance liquid chromatography and formulated in 10% ethanol in sterile saline with radiochemical purities of 95–98%. Toronto: Molar activities of 61.5 ± 26.3 GBq/μmol (n = 4), and radiochemical yields of 8.7 ± 3.7% (not decay-corrected, ndc); Pittsburgh: molar activities of 349 ± 144 GBq/μmol (n = 4) and radiochemical yields of 2.3 ± 1.1% (ndc).

Fig. 3. [³H]OXD-2314 autoradiographic binding in human post-mortem brain tissues of tauopathies.

A Autoradiographic detection of [³H]OXD-2314 binding in post-mortem cortical or hippocampal brain tissue samples from healthy control (HC), Alzheimer’s disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) cases. Top row shows total [³H]OXD-2314 (3 nM) binding and bottom row shows non-specific binding of [³H]OXD-2314 (3 nM) with displacement by unlabeled OXD-2314 (3–10 μM) in adjacent sections. Representative data of 2–3 cases per diagnosis. B High-resolution autoradiography image of [³H]OXD-2314 (3 nM, black) in CBD cortical tissue. [³H]OXD-2314 signal is overlaid with immunohistochemical (AT8) staining to indicate phospho-tau (turquoise) distribution. Scale bar, 20 µm.

Fig. 4. PET imaging of [¹⁸F]OXD-2314 in Sprague-Dawley rats.

A Whole brain time-activity curves of standardized uptake values (SUV) in female (pink, n = 1) and male (blue, n = 1) rat whole brains in baseline PET-CT scans using [¹⁸F]OXD-2314 (0−120 min). B Representative PET summation images of rat brain from baseline PET scans using [¹⁸F]OXD-2314. Top row shows transverse images of a male wild-type rat brain at 0–5, 5–30, and 60–120 min; bottom row shows transverse images of a female wild-type rat brain at the same time points.

Fig. 5. PET imaging of [¹⁸F]OXD-2314 in rhesus macaque monkeys.

A Representative transverse PET summation images (0–11 min and 60–90 min) of macaque brain from baseline PET scan using [¹⁸F]OXD-2314. B Time-activity curves of standardized uptake values (SUV) in macaque (n = 1) whole brain (solid circles,), cortex (triangles,), and cerebellum (rings, () from baseline PET scan using [¹⁸F]OXD-2314 over 0–90 min. C–E Time-activity curves of SUV in macaque (n = 1) from a displacement PET experiment using [¹⁸F]OXD-2314 with unlabeled OXD-2314, (C) whole brain, (D) cortex, and (E) cerebellum. Baseline () and displacement (() PET imaging in macaque, 0–90 min. The displacing dose of unlabeled OXD-2314 (0.5 mg/kg) was administered at 10 min post-injection (indicated by arrows). No significant displacement of [¹⁸F]OXD-2314 could be detected in any brain region, as expected in animals without tau pathology.

Fig. 6. PET imaging and radiometabolite analysis of [¹⁸F]OXD-2314 in baboon.

A Time-activity curves of standardized uptake values (SUV) in male (solids, n = 1) and female (open, n = 1) baboon whole brain (circles), cortex (triangles), and cerebellum (squares) from baseline PET scans using [¹⁸F]OXD-2314 over 0-90 min. B Radiometabolite analysis from male (n = 1) and female (n = 1) baboon venous blood taken during baseline PET scans using [¹⁸F]OXD-2314. The fraction of unchanged [¹⁸F]OXD-2314 in plasma was measured by high-performance liquid chromatography at six-time points between 2 and 90 min post-injection.