Design, Syntheses, and in Vitro Evaluation of New Fluorine-18

Sep 18, 2017 - Deposition of aggregates of hyperphosphorylated tau protein is a hallmark of tauopathies like Alzheimer and many other neurodegenerativ...
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Article Cite This: J. Med. Chem. 2017, 60, 8741-8757

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Design, Syntheses, and in Vitro Evaluation of New Fluorine-18 Radiolabeled Tau-Labeling Molecular Probes Luka Rejc,*,†,∇ Lojze Šmid,‡ Vladimir Kepe,§,⊥ Č rtomir Podlipnik,† Amalija Golobič,† Mara Bresjanac,‡ Jorge R. Barrio,§ Andrej Petrič,†,∥ and Janez Košmrlj*,† †

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia Faculty of Medicine, Institute of Pathological Physiology, University of Ljubljana, Zaloška 4, SI-1001 Ljubljana, Slovenia § Department of Molecular and Medical Pharmacology, The David Geffen School of Medicine, University of California, Los Angeles, California 90095, United States ∥ EN-FIST Centre of Excellence, Trg Osvobodilne fronte 13, SI-1000 Ljubljana, Slovenia ‡

S Supporting Information *

ABSTRACT: Deposition of aggregates of hyperphosphorylated tau protein is a hallmark of tauopathies like Alzheimer and many other neurodegenerative diseases. A sensitive and selective method of in vivo detection of tau-aggregate presence and distribution could provide the means of an early diagnosis of tau-associated diseases. Furthermore, the use of selective molecular probes that enable histochemical differentiation of protein aggregates post-mortem would be advantageous for the insight into the properties of tau protein aggregates. We chose to design new molecular probes based on the structure of 2-(1-(6-((2-[18F]fluoroethyl)(methyl)amino)-2-naphthyl)ethylidene)malononitrile to investigate their likelihood of fitting into VQIVYK tau protein binding channel model. In a modular approach, using cross-coupling reactions, we synthesized a series of candidates, radiolabeled them with fluorine-18 radioisotope, and determined their physicochemical and in vitro binding properties. Herein we report the synthesis of a series of molecular probes capable of detection of tau protein deposits in vitro.



INTRODUCTION The tau proteins are important microtubule-associated structural proteins that stabilize the cytoskeleton. Genetic predispositions, pathological stress, and traumatic brain injuries are often the cause of post-translational changes of normal tau protein (Tau), such as hyperphosphorylation and glycosylation.1 These impair biological functions and inactivate Tau from participating in tubulin assembly into microtubules. Its inability to perform primary biological functions promotes formation of insoluble intraneuronal inclusions, known as neurofibrillary tangles (NFTs). The presence of NFTs, along with deposits of amyloid β (Aβ) protein, has been characterized as the pathology hallmark of different neurodegenerative diseases, such as Alzheimer disease (AD), progressive supranuclear palsy (PSP), Huntington disease, Creutzfeldt−Jakob disease, etc.2,3 The role of these deposits in neurodegeneration remains unknown; however the degree of neuronal degeneration in tauopathies was found to correlate well with the deposition of hyperphosphorylated tau protein (pTau) aggregates.4 Furthermore, their presence in the brain was found to occur years before the symptoms, which provides the means for an early diagnosis of neurodegenerative tauopathies.5 The increasing incidence of neurodegenerative tauopathies provides impetus for better sensitive methods of detecting pTau deposits in vivo. © 2017 American Chemical Society

The development of new techniques and the latest research on pTau structures offer new insights into the morphology of Tau deposits; however, the exact atomic structure of different full length tau aggregates from brain samples has not yet been determined.6 A hexapeptide sequence valine−glutamine− isoleucine−valine−tyrosine−lysine (VQIVYK) was found to be the minimal interaction motif that allows tau proteins to selfassemble into PHF in vitro without the presence of any additional compound to promote the assembly.7,8 Such a selfassembly leads to the formation of charged lysine and the lipophilic tyrosine channels.9 This discovery enabled computational analysis of protein−molecular probe interactions through quantum mechanics and molecular docking. By using the proposed VQIVYK model of tau steric zipper, computational methods offer an accessible, affordable, and fast way of screening large number of structures to check their potential affinity to pTau aggregates. The complexity of the protein structure, however, makes computational studies inadequate for drawing the final conclusions about the binding affinity of a molecular probe for detection of tissue targets. Therefore, biological and histochemical tests have to be performed to test these Received: May 24, 2017 Published: September 18, 2017 8741

DOI: 10.1021/acs.jmedchem.7b00764 J. Med. Chem. 2017, 60, 8741−8757

Journal of Medicinal Chemistry

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input. (c) It should hold a reactive site ready for radiolabeling to make it appropriate for the use in radioactive assays and in vivo imaging with PET. (d) The structure has to be sufficiently hydrophilic to be suitable for the intravenous injection and appropriately lipophilic not only to cross the blood−brain barrier (BBB) but also to reach the intracellular targets. The ideal partition coefficient value (log Poct/water) for a successful use in PET imaging is believed to be between 1 and 3, although the compounds with higher log P values have also been successfully used in vivo.22 In this work, compounds 1 (Figure 2A) with a variety of structurally diverse aromatic scaffolds were designed, prepared, and examined to shed light on the structure−physicochemical and binding characteristics. To create the push−pull electron effect found in FDDNP,23 naphthalene, benzene, ethyne, and ethene building blocks have been combined to access new derivatives, having the π-bridge between the electron-donating N-ethyl-N-(2-fluoroethyl) and electron-withdrawing 2-dicyanovinyl groups (Figure 2B). In this work, sensitivity of these molecules for amyloid neuroaggregates was also tested but not other potential tissue targets (e.g., MAO A or MAO B). Herein we report on the synthesis, quantum mechanical (QM) calculations, molecular docking studies, physicochemical properties, and in vitro histochemical evaluations of new molecular probe candidates for a sensitive detection of pTau aggregates.

predictions. Fluorescence microscopy is traditionally used for post-mortem localization of protein deposits.10 The use of molecular probes that can be excited at wavelengths longer than 400 nm and emit light in the near-infrared (NIR) part of the spectra is preferable to minimize wavelength excitation interference with other tissue moieties and stray autofluorescence signals emanating from endogenous fluorescent molecules, hence increasing the signal-noise ratio (S/N).11 On the other hand autoradiography and positron emission tomography (PET) imaging, in cooperation with positronemitter-labeled pTau-specific molecular probes, can be used for in vitro and in vivo studies, respectively. 2-(1-(6-((2-18[F]Fluoroethyl)(methyl)amino)-2-naphthyl)ethylidene)malononitrile ([18F]FDDNP) is a molecular probe successfully used to detect pTau and Aβ in AD and also pTau in predominat tauopathies like those found in PSP patients,12,13 as well as in chronic traumatic encephalopathy cases of retired professional American football players.14 Along with [18F]FDDNP, other tau-molecular probes, including [11C]PBB3,15 [18F]AV-1451,16 [18F]THK535117 and [11C]THK535118 (Figure 1), have been recently developed.19 Most of these



RESULTS AND DISCUSSION Chemistry. Appropriate aniline and acetophenone derivatives were selected as starting substrates due to their suitability for the final transformations into N-ethyl-N-(2-fluoroethyl) and 2-dicyanovinyl end-capped compounds 1 (Figure 2B). The syntheses begun with the preparation of acetylene building blocks 2a, its ethoxyethoxy (EE) protected derivative 2b, and 3 (Scheme 1). While the preparation of 2a was easily achieved by the literature procedure,24 its further transformation into the EE protected derivative 2b failed presumably because of hydrolytic instability of the acetylene moiety under acidic conditions. Instead, compound 2b was obtained by the method similar to that of Cross and Davis24 from 2-(ethyl(phenyl)amino)ethanol. Hydroxyl group protection at the latter, iodination of the phenyl ring, subsequent Sonogashira crosscoupling with 2-methylbut-3-yn-2-ol (mebynol), and partial deprotection with tert-butoxide afforded acetylene 2b. The same Sonogashira cross-coupling−deprotection sequence was used on 2-(4-bromophenyl)-2-methyl-1,3-dioxolane to prepare the phenylacetylene derivative 3. A modular assembly of acetylenes 2a,b and 3 into the precursors of target compounds 1 was easily achieved through just a few of reaction conditions typical for the Sonogashira and Suzuki−Miyaura reactions including some protective/deprotective manipulations as shown in Scheme 2. Briefly, alkynylation of triflates 4 and 6 with 2a gave acetylenes 5 and 7 (Scheme 2A and Scheme 2B). Alkynylation of iodonaphtole 8 with 2b gave compound 9 (Scheme 2C). Compound 9 was activated into triflate 10 and cross-coupled with 3 into 11, which after hydrolysis afforded compound 12. Sonogashira reaction of iodonaphthalene derivative 13 with acetylene 3 gave compound 14, which was upon hydrolysis transformed into 15 (Scheme 2D). Finally, the Suzuki− Miyaura reaction between 4-acetylphenylboronic acid and 2(ethyl(4-iodophenyl)amino)ethanol afforded biphenyl derivative 16 (Scheme 2E), whereas alkynylation of p-bromoaceto-

Figure 1. Selected molecular probes currently in preclinical and clinical trials.

compounds have poor specificity with significant off-target tissue binding in vivo (e.g., [18F]AV-1451), most specifically by binding to MAO A and MAO B with high affinity.20 Clearly, identifying compounds that selectively label pTau over Aβ and provide a significant S/N ratio in PET imaging remains challenging. In this research we envisaged new molecular probe candidates that should be capable of sensitive pTau deposits labeling while offering a bridge between different evaluation methods: in vitro fluorescence microscopy, autoradiography, and in vivo PET imaging. To qualify, the probe’s structure should meet the following criteria: (a) It should enable free electron flow throughout chemical bonds, providing a push− pull electron effect for a desired NIR emission maxima wavelength, large Stokes shift, and substantial quantum yield, prerequisites for the use in fluorescence microscopy. This can be achieved by end-capping the π-conjugated structure with an electron-donating group on one end and with an electronwithdrawing group on the other while keeping the uninterrupted conjugation between these two parts.21 (b) It should be of a rod-like geometry to enable access to the binding site in the tyrosine channel of pTau deposits without a major energy 8742

DOI: 10.1021/acs.jmedchem.7b00764 J. Med. Chem. 2017, 60, 8741−8757

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Figure 2. (A) General representation of elongated FDDNP analogues (oval represents aromatic scaffold). (B) Structures of new biphenyl-, diphenylacetylene-, stilbene-, and naphthalene-based derivatives.

Scheme 1. Synthesis of Acetylene Building Blocks 2a,b and 3a

Reagents and conditions: (a) ethyl vinyl ether, p-TsOH, rt, Ar, 24 h; (b) I2, pyridine/dioxane, 0 °C, Ar, 1 h; (c) 2-methylbut-3-yn-2-ol, Pd(OAc)2, PPh3, CuI, DIPA, Ar, 70 °C, 0.5−14 h; (d) then t-BuOK, toluene, reflux, 20−30 min.

a

Compounds 1e−g, which were found to exhibit the best physicochemical and biological properties (vide infra), were also prepared as fluorine-18 (18F) radiolabeled analogues [18F] 1e, [18F]1f, [18F]1g. To this end, the reaction sequence from Scheme 3A was altered accordingly; the initial introduction of the 2-dicyanovinyl group in derivatives 16−18 by Knoevenagel condensation was followed by the primary hydroxyl group activation via tosylate (19−21) and radiofluorination with fluorine-18 by using [18F]KF/Kryptofix 222 (Scheme 3B). All new compounds under this investigation were fully characterized by 1D and 2D NMR spectroscopic techniques (1H, 13C, 19F, 1H−13C gs-HSQC and 1H−13C gs-HMBC),

phenone with 2a gave tolan 17 (Scheme 2F). Catalytic reduction of tolan 17 provided a mixture of E- and Z-stilbene derivative 18, which was cleanly transformed into E-18 upon treatment with formic acid at elevated temperature (Scheme 2F). Finally, by use of an established two-step reaction sequence, fluorination of the primary alcohol group with diethylaminosulfur trifluoride (DAST), and subsequent Knoevenagel condensation of acetyl functionality with malononitrile, the above precursors 5, 7, 12, 15−18 were readily transformed into the target derivatives 1a−g (Scheme 3A). 8743

DOI: 10.1021/acs.jmedchem.7b00764 J. Med. Chem. 2017, 60, 8741−8757

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Scheme 2. Synthesis of Precursors of Target Compounds 1a

Reagents and conditions: (a) Pd(OAc)2, PPh3, CuI, DIPA, Ar, 70 °C, 0.5−14 h; (b) Tf2O, Ar, −15 °C, 1−3 h; (c) acetone, p-TsOH, rt, 1.5−3 h; (d) 2-(ethyl(4-iodophenyl)amino)ethanol, Pd(PPh3)4, NaHCO3, DME, Ar, 90 °C, 2−4 h; (e) Pd(OAc)2, KOH, DMF, Ar, 145 °C, 20 min; (f) HCOOH, dioxane, 80 °C, 4 h. a

(d3 = 1.366 Å for CAr−N and d4 = 1.486 Å for CAr−Csp2) compared with those reported in the literature (1.371 and 1.488 Å average values, respectively). In contrast, the triple carbon−carbon bond is longer (d5 = 1.192 Å) than usually observed (1.189 Å), suggesting a lower triple bond character between the acetylene carbon atoms. Similar results were seen in the cases of 1a, 1d, 1e, 1f, and 1g (see Figure S1 in the Supporting Information). As also shown in Figure 3, the entire π-extended electron scaffold is nearly planar with only slight out-of-planarity of the benzene ring at the 2-dicyanovinyl terminal. Quantum Mechanical (QM) Calculations and Molecular Docking Studies. QM calculations and molecular docking studies showed the favorable geometry of the designed structures to fit inside the narrow channels running along the model of VQIVYK binding channel. A possibility of binding to hexapeptide sequence lysine−leucine−valine−phenylalanine− phenylalanine−alanine (KLVFFA), which presents an inter-

elemental analysis, high-resolution mass spectrometry, and IR. UV/vis and fluorescence spectra and single crystal diffraction analyses were performed for compounds 1a−g. Single Crystal X-ray Diffraction Analysis. We succeeded in growing crystals suitable for X-ray crystallography from compounds 1a, 1b, 1d, 1e, 1f, and 1g, and the results corroborated the initial hypothesis on the extended electron πdelocalization between the electron-donating N-ethyl-N-(2fluoroethyl) and electron-withdrawing 2-dicyanovinyl groups attached to the aromatic scaffold. For example, in the case of 1b (Figure 3), the compound with the highest calculated affinity to VQIVYK channel model (vide infra), the bond length between benzene and naphthalene ring (d1 = 1.479 Å) and the distance between acetylene carbon atom and naphthalene ring (d2 = 1.429 Å) are shorter compared to the reported average values (1.487 and 1.434 Å, respectively).25 Also in agreement are shorter bond lengths between the electron-donating and electron-withdrawing groups attached to the aromatic scaffold 8744

DOI: 10.1021/acs.jmedchem.7b00764 J. Med. Chem. 2017, 60, 8741−8757

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Scheme 3. Synthesis of Compounds 1 and Their Radiolabeled Analoguesa

Reagents and conditions: (a) DAST, rt, 1−2 h; (b) CH2(CN)2, Py, 80 °C, 3−23 h; (c) p-Ts2O, Py, rt, Ar, 15 min−24 h; (d) [18F]KF/Kryptofix 222, 90 °C, 15−20 min. a

Table 1. Energy Contributions of Compound 1b to the Energy of Binding inside the VQIVYK (pTau) and KLVFFA Models (Aβ Model) protein model

Glide XP (kcal/mol)

VQIVYK KLVFFA

−14.5 −12.34

lipophilicity H-bond −10.38 −9.61

−0.7 0

low MW

electro

0 0

−0.09 −0.06

factor in the binding event. Accordingly, results showed preferential binding to the more lipophilic tyrosine channel of the VQIVYK binding model, as observed for the parent compound FDDNP. Moreover, the geometries of the structures, when docked into the proposed tyrosine channel, comply well with the X-ray crystal structures of the synthesized molecules (see Figure 3B). This indicates that no significant energy input is required for the binding site to accommodate the designed structures, assuming the crystal structure takes the lowest possible energy. Absorption and Emission Spectra. To inspect the appropriateness for the use in fluorescence microscopy, absorption (λab), and emission maxima (λem) were determined for the solutions of the synthesized molecular probes in solvents of different polarity: hexane, dichloromethane, acetonitrile, methanol, and water. Observed absorption maxima in dichloromethane, the solvent found to mimic the environment in the amyloid aggregate’s binding sites the best, were above or close to 400 nm (Table 2). This made the synthesized derivatives applicable for the use in fluorescence microscopy without significant interference of autofluorescence. Furthermore, biphenyl, diphenylacetylene, and stilbene derivatives exhibited an increased Stokes shifts as compared to FDDNP, its analogue 1,1-dicyano-2-((6-dimethylamino)naphthalene-2-yl)propene (DDNP), and other naphthalene derivatives. By using an appropriate excitation, this promised a further differentiation of the signal that is emitted from the bound probe and the emission filters, thus increasing the S/N ratio. Large differences in intensities and wavelengths of the emission maxima with increasing solvent polarity, especially in biphenyl and diphenylacetylene derivatives 1e and 1f, suggest a

Figure 3. (A) Ball and stick presentation of molecule 1b as determined by single crystal X-ray diffraction analysis with selected bond distances and the plane passing through the naphthalene ring (translucent green plane). Relative to this, in the crystal lattice, the benzene ring at the 2dicyanovinyl terminal end (left-hand side) is out-of-plane by approximately 34°. (B) Superposition of the calculated structure (turquoise) and the single crystal X-ray diffraction structure (brown) of 1b, located inside the tyrosine channel of VQIVYK binding model.

action motif in Aβ plaques, was also examined. The calculated energies of binding of derivatives 1a−g range from −10.74 to −14.85 kcal/mol in the VQIVYK model (−10.44 kcal/mol for FDDNP) and from −8.96 to −13.44 kcal/mol in the KLVFFA model (−7.7 kcal/mol for FDDNP), suggesting higher affinity of these compounds to the selected targets compared to the parent compound (see Table S2 in the Supporting Information). The higher predicted affinity to VQIVYK and KLVFFA model of naphthalene-based analogues 1a−d is believed to be due to a substantially higher lipophilic contribution to the overall energy, as depicted for the derivative with the highest calculated binding affinity, 1b (Table 1), making lipophilicity of the compounds the most important 8745

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Table 2. Spectroscopic Data (Recorded in Dichloromethane), QM Calculations, and Binding Quantification of Molecular Probe Candidatesa DDNP λab (nm) λem (nm) ε × 10−4 (L mol−1 cm−1) Φb dipole moment (Debye)c KD (nmol/L) log P e

441 563 2.4 0.028 11.03/11.13d

FDDNP 428 549 2.3

74.7 3.612

1b

1c

407 654 2.8 0.496 11.07

1a

381 507 3.2

383 497 5.5

5.416

7.211

1d 403 2.6 11.42/12.73d

12.57 6.844

5.04

1e

1f

1g

414 615 2.2 0.117 11.88 3.06 4.623

411 654 2.3 0.207 9.93 288.6 4.256

437 669 1.6 0.272 10.44 >0.64f 5.386

a

For the full table of physicochemical and binding properties see Table S3 in the Supporting Information. bQuantum yields were calculated relative to the reported quantum yield of DDNP (see section 3.1.1. in the Suporting Information). cEnergies for the lowest energy mode and dipole moments were calculated by Schrödinger’s Glide. dDDNP and 1d crystallize in two different crystals forms. ePartition coefficient values were calculated using www.molinspiration.com software. fDue to solubility issues, only a partial inhibition was observed.

Figure 4. In vitro fluorescence microscopy using naphthalene 1a (U-MNV filter, 40× magnification), biphenyl 1e (U-MWIB filter, 1e−pTau, 40× magnification, and 1e−Aβ, 20× magnification), diphenylacetylene 1f (U-MWIB filter, 1f−pTau, 20× magnification, and 1f−Aβ, 40× magnification), and stilbene 1g (U-MWIB filter, 1g−pTau, 20× magnification, and 1g−Aβ, 40× magnification) derivatives. Binding to NFT of aggregates of pTau and dense core as well as diffused plaques of Aβ were observed in all cases. Autofluorescence emanating from lipofuscine can be easily resolved from the rest of the signal, appearing as yellow spheres. Significant difference in emission wavelengths emanating from pTau and Aβ aggregates was observed in cases of 1e and 1f, suggesting a more hydrophobic environment at the pTau binding site.

allow for the naked eye discrimination between green-to-yellow plaques and red-to-orange tangles. It not only provides with an easier differentiation between the plaques and the tangles but also reflects a significant difference in the local environment inside the binding pocket of the two protein aggregates. The emission differences between the light emanating from NFTs and Aβ correspond well with the difference in the emission maxima of 1f in dichloromethane and acetonitrile, suggesting higher lipophilicity at the binding site of pTau than Aβ deposits. While molecular probes 1e−1g and 1a were adequately soluble in aqueous ethanol, the poor solubility of 1b−d, even in aqueous DMSO, prevented their use in biological assays. Radiometric Competitive Binding Assay. To exclude molecular probes with a high binding affinity to Aβ, a competitive binding assay was carried out. Competitive binding assays of analogues 1e−g with radioactively labeled probe [18F]FDDNP were performed using the nonradiolabeled FDDNP as a point of reference. The use of more lipophilic analogues 1a−d was hampered by low solubility in water or water−ethanol solutions. In line with the fluorescence microscopy results, biphenyl and stilbene-based derivatives 1e and 1g, respectively, showed a higher affinity to Aβ fibrils as

large effect of the environment on the energy of the ground and excited state of the two compounds. Fluorescence and Confocal Microscopy. Alzheimer disease brain tissue was treated, incubated, and inspected as described in the Experimental Section. In general, 40× magnification was used to distinguish between the labeled deposits; however, in some cases the labeled proteins were clearly visible even under 20× magnification. The inspection of the tissue incubated with biphenyl analogue 1e and naphthalene-based analogue 1a revealed an apparent better visualization of the Aβ plaques than of NFTs. In contrast, when the tissue was incubated with diphenylacetylene (1f) and stilbene analogues (1g), the signal emanating from NFTs was much more intense than the one from Aβ, observing the fibrils under 20× magnification, while 40× magnification was needed to observe the plaques. While binding assay with stilbene 1g resulted in a red color signal emanating from pTau fibrils and Aβ plaques, a difference in emission maxima was observed between the two types of aggregates stained with 1e and 1f (Figure 4). A closer examination by confocal microscope revealed a 15 nm difference in emission wavelength maxima (580 nm in Aβ plaques compared to 595 nm in NFTs) of 1f (Figure 5). This gap is large enough in this emission region to 8746

DOI: 10.1021/acs.jmedchem.7b00764 J. Med. Chem. 2017, 60, 8741−8757

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showed lower affinity to the Aβ fibrils compared to FDDNP (288.6 and 74.7 nmol/L, respectively, under the experimental conditions used (1% ethanol in PBS) (Table S3 in the Supporting Information)). A nearly 100% inhibition of [18F]FDDNP by the analogue 1e or 1f indicated their accommodation by the same binding site as FDDNP. Insufficient solubility of 1g under the experimental conditions prevented meaningful inhibition measurements at concentrations above 10 nmol/L. Autoradiography. Brain sections of patient with advanced PSP, a known taupathy, where pTau is expressed mainly in the cortex, were incubated with [18F]1e, [18F]1f, and [18F]1g to show their affinity to the deposits at low concentrations. Large difference in the signal intensities between pTau rich gray matter and myelin rich white matter of the PSP brain slices was observed (Figure 7). Contrarily, no significant difference between the signals emanating from gray and white matter was observed in the control tissue (Figure 7). A high contrast observed in PSP tissue compared to the control tissue is comparable with the results obtained with positive control [18F]FDDNP (Figure 7G) and suggests high affinity of [18F]1e, [18F]1f, and [18F]1g to the pTau deposits in the gray matter. While the signal might also arise due to the binding to Aβ plaques, when present in the cortex of late stage cases of PSP, the affinity of these probes to Aβ was proven to be low by the radiometric competitive binding assay, suggesting that the majority of the observed signal is due to a high potency toward pTau deposits.



Figure 5. Confocal microscopy of 1f labeled pTau (A) and Aβ (B) aggregates associated with Alzheimer disease. Peak fluorophore emission wavelength (C) in dense core senile plaques was 580 nm and differed from that of neurofibrillary tangles (595 nm). Markedly different lipofuscin autofluorescence spectrum (with peak emission at 566 nm) and low tissue background fluorescence contributed to good pathology visualization. Compound 1f clearly labeled pTau aggregates in neurofibrillary tangles (A, NFT) and dystrophic neurites (B, DN). Senile plaques (with dense core and diffuse β amyloid aggregates, B, SP) were also labeled by this fluorophore. Scale bar size is 20 nm.

CONCLUSIONS Highly efficient modular approach has been designed and realized to access a small library of novel molecular probes in just a few simple synthetic steps. The target compounds were designed to have a rod-like geometry and extended πconjugated scaffold that was end-capped with electron-donating N-ethyl-N-(2-fluoroethyl) and electron-withdrawing 2-dicyanovinyl groups. The π-conjugated scaffold comprised various combinations of naphthalene, benzene, ethyne, and ethene building blocks. The desired geometry and π-conjugation were confirmed by single crystal X-ray structure analysis, making the synthesized compounds prime candidates for a successful binding to pTau deposits. A possibility of a favorable interaction with pTau aggregate was additionally confirmed by molecular docking calculations of the designed probes into the VQIVYK channel model. In addition to this, optical characteristics of all compounds under investigation meet the requirements of the standard fluorescence and confocal microscopes. The solubility in aqueous ethanol prioritized the use of biphenyl (1e), diphenylacetylene (1f), and stilbene (1g) derivatives. While the three compounds clearly bound both pTau NFTs and Aβ plaques in fluorescence microscopy, diphenylacelylene 1f shows a higher sensitivity toward pTau, compared to Aβ aggregates. In further radiometric competitive binding assay the affinity to synthetic Aβ fibrils was 100- and 500-fold lower for the diphenylacetylene 1f compared to biphenyl 1e and stilbene 1g, respectively. These results are compliant with the observed data from fluorescence microscopy. Additionally, fluorescence and confocal microscopy of biphenyl and diphenylacetylene analogues revealed their capability of differentiating the environment inside the binding pockets of the Aβ and pTau aggregates based on the emitted wavelength maxima. This feature permits, using a single ligand, the differential visualization of pTau and amyloid aggregates.

compared to FDDNP under the experimental conditions (Figure 6). On the other hand, the diphenylacetylene 1f

Figure 6. Dose−response curves for competitive binding assay of molecular probe candidates 1e (R2 = 0.9684), 1f (R2 = 0.9551), 1g (R2 = 0.9927), and FDDNP (R2 = 0.9727) to amyloid β fibrils. 8747

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Figure 7. Autoradiography with [18F]1e (A = PSP, B = control), [18F]1g (C = PSP, D = control), [18F]1f (E = PSP, F = control), [18F]FDDNP (G = PSP, H = control). The control tissue used was thicker (150 μm) than the PSP tissue (40 μm), resulting in a higher retention of 18F-marker; hence the background white matter appears darker. aryl halide), tetrakis(triphenylphosphine)palladium (0.01 equiv) and aqueous sodium carbonate were added. The reaction mixture was stirred under argon atmosphere at 75 °C for 15 min. Then a solution of 4-acetylphenylboronic acid (1.2 equiv) in absolute ethanol (1 mL/ mmol of 4-acetylphenylboronic acid) was added. The reaction mixture was stirred for 4 h at 75 °C. The reaction mixture was diluted with dichloromethane, the solution was washed with water, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was purified by radial chromatography on silica gel, using eluent as indicated below. General Procedure for Synthesis of Triflates (Procedure C). A round-bottom flask was purged with argon, charged with a solution of a naphthol (1 equiv) in anhydrous pyridine, and cooled to −15 °C on an ice/methanol bath. Trifluoromethanesulfonic anhydride (2 equiv) was added dropwise to the solution and the reaction mixture stirred under argon at −15 °C for the time indicated below (1−2.5 h). The reaction mixture was diluted with dichloromethane, the solution was washed with water, and the organic layer was dried over anhydrous sodium sulfate and filtered. The solvent was removed under reduced pressure and the crude product was purified by chromatography on silica gel by using eluent as indicated below. General Procedure for Fluorination (Procedure D). To a solution of hydroxyl precursor (1 equiv) in anhydrous dichloromethane, a solution of DAST (2 equiv) in 2 mL of anhydrous dichloromethane was added dropwise. The reaction mixture was stirred at room temperature under argon for the time indicated below (1−2 h). The mixture was poured on ice, the resulting slurry was neutralized with sodium hydrogen carbonate, and the product was extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The crude product was purified by chromatography on silica gel or crystallization, using solvent as indicated below. General Procedure for Knoevelagel Condensation (Procedure E). To a solution of ketone (1 equiv) in pyridine, malononitrile (10−20 equiv) was added. The reaction mixture was stirred at 80 °C for the time indicated below (3−23 h). The resulting mixture was diluted with dichloromethane and washed with water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by chromatography (silica gel, eluted with ethyl acetate/petroleum ether) and further crystallized to obtain crystals suitable for X-ray analysis. General Procedure for Synthesis of a Tosylate (Procedure F). A round-bottom flask was purged with argon and charged with a solution of hydroxyl precursor (1 equiv) in freshly distilled pyridine. Upon the addition of an excess of p-toluenesulfonic anhydride (3−7 equiv) the solution was stirred under argon at room temperature for the time indicated below (0.5−2 h). The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was

Finally, the synthesis described herein allows an easy access to radiolabeled molecular probes. Three radiotracers [18F]1e, [18F]1f, [18F]1g were prepared and successfully used in autoradiography, suggesting their possible applicability for in vivo PET imaging. The new fluorine-18 radiolabeled diphenylacetylene-based molecular probe [18F]1f exhibits preferable physicochemical properties and shows high sensitivity for binding to phosphorylated pTau aggregates in vitro. Indeed, toxicity and pharmacokinetic studies are always needed for human administration, but due to the very high specific activity of the fluorine-18 compounds (>74 GBq/ μmol), toxicity is rarely an issue due to the extremely low mass injected per human dose (99%) were determined by HPLC (reverse layer Waters Symmetry C18 analytical HPLC column (5 μm, 4.6 mm × 150 mm), acetonitrile/water = 3:2 used as an eluent, flow rate 1.5 mL/min, detection by γ detection and by UV absorption at 254 and 366 nm). 2-(1-(4′-(Ethyl(2-[18F]fluoroethyl)amino)[1,1′-biphenyl]-4-yl)ethylidene)malononitrile ([18F]1e). Radiofluorination of compound 19 was carried out according to procedure G to obtain pure [18F]1e (220.5 MBq; 1.5% radiochemical yield; AM = 100−200 GBq/ μmol). 2-(1-(4-((4-(Ethyl(2-[18F]¯uoroethyl)amino)phenyl)ethynyl)phenyl)ethylidene)malononitrile ([18F]1f). Radiofluorination of compound 20 was carried out according to procedure G to obtain pure [18F]1f (4.48 GBq; 30% radiochemical yield; AM = 100−200 GBq/μmol). (E)-2-(1-(4-(4-(Ethyl(2-[18F]¯uoroethyl)amino)styryl)phenyl)ethylidene)malononitrile ([18F]1g). Radiofluorination of compound 21 was carried out according to procedure G to obtain pure [18F]1g (4.47 GBq; 15% radiochemical yield; AM = 100−200 GBq/ μmol). QM and Molecular Docking Calculations. Structural parameters, energies, and dipole moments for compounds of our interest were calculated using B3LYP DFT method along with valence-split basis set 6-31G** using Schrödinger’s Jaguar version 8.8. Glide XP, also provided by Schrödinger, was used for molecular docking. Full Glide docking protocol is composed of ligand preparation (LigPrep), protein preparation (Protein Preparation Wizard), molecular docking (GlideXP), and analysis of the result by XP visualizer. Maestro GUI was used for setting, running, and controlling QM and molecular docking computations. Optical Properties. Solubility permitting, solutions at four different concentrations were prepared in hexane, dichloromethane, acetonitrile, methanol, and water. The solute concentrations were 1− 10 μmol/L. To avoid reabsorption effects, the concentrations were adjusted so the absorbance maxima did not exceed 0.1.29 Absorption spectra were recorded using standard 10 mm cuvette using a Varian Cary 50 probe UV−visible spectrophotometer. Emission spectra were recorded using a Varian Cary Eclipse fluorescence spectrophotometer equipped with a xenon light source and Hamamatsu R928 photomultiplier tube. Typical spectral response curve of the photomultiplier was used for correction of emission curves. To calculate quantum yields, integrals of the corrected emission intensities were plotted against the absorbance at the excitation wavelength. Gradients of the obtained linear curves were calculated by Origin 8 software, provided by the Faculty of Chemistry and Chemical Technology, University of Ljubljana. Quantum yields were calculated relative to DDNP, as described previously.30 Fluorescence and Confocal Microscopy. Formalin fixed paraffin embedded 20 μm thick AD brain sections (Department of Pathology at Faculty of Medicine, University of Ljubljana) were deparaffinated, defatted, and rehydrated in baths of gradually

decreasing concentrations of ethanol solution in water. Where stated, the hydrated tissue was preincubated in a water solution of potassium permanganate (1.6 mol/L) for 20 min to suppress tissue autofluorescence (this step was omitted in confocal microscopy). The tissue sections were incubated for 15 min with 1−10 μmol/L solution of a molecular probe in 1% ethanol in water or DMSO where stated. After rinsing with water, the sections were briefly washed in 50%, 70%, or 90% aqueous ethanol. The tissue sections were examined under the fluorescence microscope31 using U-MNV (excitation filter, 400−410 nm; emission filter, 455 nm), U-MNBV (excitation filter, 420−440 nm; emission filter, 475 nm), and/or U-MWIB filter (excitation filter, 460−495 nm; emission filter, 510 nm) at 20× and/or 40× magnification. Confocal microscopy was performed using Leica TCS SP5 MP confocal microscope with excitation wavelength of 458 nm. Immunohistochemistry with AT8 antibodies was used to confirm the presence of hyperphosphorylated tau aggregates in the tissue. Radiometric Competitive Binding Assay. Aβ fibrils were prepared as described previously.32 In brief, 1 mg of Aβ (1−40) protein was dissolved in phosphate buffered saline (PBS; 1 mL, pH 7.4). Protein solutions were stirred at 37 °C for 4 days, resulting in a turbid solution containing visible white particles. The suspension was centrifuged at 28 000 rpm at 4 °C for 15 min, and supernatant was removed. The presence of the fibrils in the resulting white residue was confirmed with Congo red as described previously.32 Fibrils were suspended in 4 mL of PBS to give 0.25 mg/mL suspension and used immediately after formation. [18F]FDDNP was synthesized according to a previously described procedure.12 Fresh solutions of competitors FDDNP, 1e, 1f, and 1g in 1% ethanol in PBS (pH 7.4) were prepared for each assay. Aβ fibrils (10 μg) were incubated with [18F]FDDNP (0.148 MBq) and various competitor concentrations (0.2 pmol−4 nmol) in 0.88% ethanol in PBS (4 mL) for 1 h. The resulting suspensions were vacuum filtered through APFF glass fiber prefilters (0.7 μm particle retention; Millipore, Bedford, MA, USA) using 12sample manifold (Millipore) modified with stainless steel support screens (Millipore) and glass sample chambers. Each filter was washed twice with 3 mL of 0.88% ethanol in PBS (pH 7.4) and dried by suction for 10 min. The radioactivity retained by glass fiber filters was measured by γ counter (Packard Cobra II Auto-Gamma γ counter) and decay-corrected to a common reference time. To determine 100% specific binding, fibrils were incubated with [18F]FDDNP without any competitor. The radioactivity uptake by the glass fiber prefilters was determined by vacuum filtration of the solution of [18F]FDDNP in 0.88% ethanol in PBS. Each assay was performed in triplicate. The results were analyzed, and KD values calculated based on a dose− response curve fit by Origin 8 software.33 Autoradiography. Formalin fixed, 40 μm thick mid-brain PSP tissue was defatted in a bath of xylenes for 1 h and then rehydrated in aqueous ethanol solutions by gradually decreasing the content of ethanol. The tissue was incubated with 10 mL of 0.384 MBq/mL solution of radiotracer ([18F]1e, [18F]1f, [18F]1g, [18F]FDDNP; molar activity AM = 100−200 GBq/μmol) in 1% aqueous ethanol for 30 min. The excess of the incubation solution was removed and the slides were washed in Milli-Q water (18 MΩ) while shaking at 80 rpm for 30 s. The tissue washing was performed in 90% aqueous tert-butanol solution twice for 3 min (for [18F]1e, [18F]1f, and [18F]FDDNP) or 6 min (for [18F]1g) while shaking at 80 rpm, followed by 30 s wash in Milli-Q water (18 MΩ) while shaking at 80 rpm. Formalin fixed, 150 μm thick tissue sections with no protein deposits observed was used as a control tissue. The tissue was treated, incubated with [18F]1e, [18F]1f, [18F]1g, and [18F]FDDNP, and washed as described for PSP tissue sections. The tissue washing was performed in 90% aqueous tert-butanol solution twice for 8 min. The slides were air-dried and exposed to an autoradiography film (FUJIFILM imaging plate, BAS-IP MS 2025) for 45 min, then read by FUJIFILM BAS 5000 image reader. 8755

DOI: 10.1021/acs.jmedchem.7b00764 J. Med. Chem. 2017, 60, 8741−8757

Journal of Medicinal Chemistry





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REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00764. X-ray data collection, molecular docking and QM calculations, physicochemical and biological properties, and NMR spectra (PDF) SMILES molecular formula strings (CSV)



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AUTHOR INFORMATION

Corresponding Authors

*L.R.: e-mail, [email protected]. *J.K.: e-mail, [email protected]. ORCID

Janez Košmrlj: 0000-0002-3533-0419 Present Addresses ⊥

V.K.: Cleveland Clinic, Cleveland, OH 44195. L.R.: CIC biomaGune, Paseo Miramon 182, 20014 San Sebastian, Spain. ∇

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors acknowledge the financial support from the Slovenian Research Agency (Research Core Funding Grant P10230, Young Researcher Grant to L.R., Project J1-8147, and Grant P3-0171 to L.Š. and M.B.). This work was partially supported within the infrastructures of the Centre for Research Infrastructure at the Faculty of Chemistry and Chemical Technology of the University of Ljubljana, and the EN-FIST Centre of Excellence, Trg Osvobodilne fronte 13, SI-1000 Ljubljana, Slovenia. The suggestions and insight of Dr. Kendall Houk and Dr. Hung Pham, Department of Chemistry and Biochemistry, UCLA, with docking studies and the invaluable help of personnel at the UCLA Biomedical Cyclotron are also acknowledged.



ABBREVIATIONS USED AD, Alzheimer disease; AM, molar activity; Aβ, amyloid β; BBB, blood−brain barrier; DAST, diethylaminosulfur trifluoride; DDNP, 1,1-dicyano-2-((6-dimethylamino)naphthalene-2-yl)propene; DIPA, diisopropylamine; DMSO, dimethylsulfoxide; EE, ethoxyethoxy; FDDNP, 2-(1-(6-((2-fluoroethyl)(methyl)amino)-2-naphthyl)ethylidene)malononitrile; HRMS, high resolution mass spectrometry; KLVFFA, lysine−leucine−valine− phenylalanine−phenylalanine−alanine; NFT, neururofibrilary tangle; NIR, near-infrared; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; PET, positron emission tomography; PHF, paired helical filament; ppm, parts per million; PSP, progressive supranuclear palsy; pTau, hyperphosphorylated tau protein; QM, quantum mechanical; S/N, signal-to-noise; Tau, normal tau protein; THF, tetrahydrofuran; TLC, thin layer chromatograpy; TMS, tetramethylsilane; VQIVYK, valine−glutamine−isoleucine−valine−tyrosine−lysine 8756

DOI: 10.1021/acs.jmedchem.7b00764 J. Med. Chem. 2017, 60, 8741−8757

Journal of Medicinal Chemistry

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DOI: 10.1021/acs.jmedchem.7b00764 J. Med. Chem. 2017, 60, 8741−8757