Dimethylamino-fluorenes: ligands for detecting β-amyloid plaques in the brain

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Abstract

Formation of β-amyloid (Aβ) plaques in the brain is a major contributing factor in the pathogenesis of Alzheimer’s disease (AD). Detection of Aβ plaques in the brain will be potentially useful in early diagnosis and monitoring the progression of the disease. A series of novel Aβ aggregate-specific ligands based on fluorenes, which are simple and rigid tricyclic molecules, are synthesized and characterized. Starting with 2- or 3-aminofluorenes, 1a-1f, the amino group was converted to the N,N-dimethylamino group (2a-2f) in excellent yield. It was found that 7-iodo-2-N,N-dimethylaminofluorene (2f) showed an extremely high binding affinity to preformed Aβ40 aggregates (Ki = 0.9 nM). In vitro autoradiography study using brain sections obtained from transgenic mice (Tg2576) with [125I]2f showed exquisitely high specific binding to Aβ plaques. The same section also displayed an equivalent labeling when stained by Thioflavin-S, a commonly used fluorescent dye for Aβ plaques. When [125I]2f was injected intravenously into normal mice, it exhibited an excellent brain uptake. Taken together the data suggest that [125I]2f may be useful as an in vivo imaging agent to detect Aβ plaques in the brain.

Introduction

Alzheimer’s disease (AD) is a neurodegenerative disease of the brain characterized by dementia, cognitive impairment and memory loss. Formation of β-amyloid (Aβ) plaques in the brain is a major contributing factor in the pathogenesis of Alzheimer’s disease (AD) [5], [16]. Detection of Aβ plaques in the brain will be potentially useful in early diagnosis and monitoring the progression of the disease. Potential imaging agents including, analogs of Chrysamine G, Congo Red [3], [7], [21], styrylbenzenes [X-34 [19], ISB [13], BSB [18] and IMSB [23]], benzothiazoles [6-OH-BTA-1 [9], [14], [20], TZDM [23], IBOX [22]], IMPY [12] and FDDNP [1], [17], have been reported.

In the search for novel ligands which bind to Aβ plaques, a series of fluorenes was investigated. It was previously reported from our group that benzothiazoles are not the only structures which showed selective binding towards Aβ plaques. Relatively simple molecules, such as stilbenes, showed highly effective binding to the same amyloid plaques [10]. These stilbene-based probes contain a simple two-phenyl ring. One of the aromatic rings contains an electron-donating group, p-Me2N-, -OMe or -OH, which appears to be essential for the binding affinity. There is a bulk tolerance for the second aromatic ring, on which radiolabel, 99mTc, 123I or 18F, can be readily attached without detrimental effects on binding affinity to Aβ aggregates [10]. On a closer inspection of the stilbene structure it is apparent that the aromatic rings and the vinyl group are conjugated and form a rigid system. One end of the molecule contains an electron donating group attached to an aromatic ring, which is required to provide the binding affinity. It appears that only one end of the aromatic ring plays a critical role in exerting the high binding affinity. Adding extra halogens on the other phenyl ring of the stilbenes increases the potency on binding to Aβ aggregates [10]. To investigate the potential reduction of molecular size, which is essential for penetrating intact blood brain barrier, we have explored a series of probes based on a highly rigid tri-cyclic fluorene, using the assumption that only one side of the fluorene ring system may be critical for binding to Aβ aggregates, while the other portion of the molecule may be subjected to modification. Reported herein are preliminary results of the structure-activity relationship of fluorene derivatives as ligands for binding to Aβ aggregates.

Section snippets

Chemistry

Synthesis of N,N-dimethylamino derivatives of fluorene was successfully achieved by a reductive methylation reaction shown in Scheme 1.Starting with 2- or 3-aminofluorenes, 1a-1f, the amino group was converted to the N,N-dimethylamino group (2a-2f) in excellent yield (>90%) using paraformaldehyde in the presence of sodium cyanoborohydride as a reducing agent [4]. A same reaction was applied in the methylation of 9-fluorenones (Scheme 2) by which the amino-9-fluorenones were converted to

Biological studies

Using in vitro binding assay it was demonstrated that substituted fluorenes competed with [125I]TZDM binding to Aβ40 aggregates showing excellent binding affinities (Scheme 1, Scheme 2, Scheme 3). When fluorenes with un-methylated amino groups, 1a-1f, were tested, they displayed no binding affinity (Ki > 1,000 nM) (Scheme 1). However, the 7-bromo-2-amino derivative, 1d, showed a high binding affinity (Ki = 56 ± 2 nM). When the aminofluorenes were transformed to the N,N-dimethylamino derivatives

Experimental

All reagents used in the synthesis were commercial products and were used without further purification unless otherwise indicated. Anhydrous Na2SO4 was used as a drying agent. Flash column chromatography was performed on 230-400 mesh silica gel. 1H NMR spectra were obtained on NMR spectrometers (Bruker DPX 200 and AMX 500). Chemical shifts are reported as δ values with respect to residual protons in CDCl3 unless otherwise mentioned. Coupling constants are reported in Hz.

Preparation of radioiodinated ligands

[125I]TZDM was prepared according to the method described previously [23]. The desired [125I]2f was prepared using iododestannylation reactions with tributyltin precursors, 12 [11]. Hydrogen peroxide (50 μL, 3% w/v) was added to a mixture of 50 μL of the corresponding tributyltin precursor (1μg/μL EtOH), 50 μL of 1N HCl and [125I]NaI (1-5 mCi) in a sealed vial. The reaction was allowed to proceed for 10 min at room temperature and terminated by addition of 100 μL of sat. NaHSO3. The reaction

Partition coefficient determination

Partition coefficients were measured by mixing [125I]2f with 3 g each of 1-octanol and buffer (0.1 M phosphate, pH 7.4) in a test tube. The test tube was vortexed for 3 min at room temperature, followed by centrifugation for 5 min. Two weighed samples (0.5 g each) from the 1-octanol and buffer layers were assayed for radioactivity content in a well counter. The partition coefficient was determined by calculating the ratio of cpm/g of 1-octanol to that of buffer. Samples from the 1-octanol layer

Binding assays using aggregated Aβ40 peptide in solution

The solid form of peptide Aβ40 was purchased from Bachem (King of Prussia, PA). Aggregation of peptide was carried out by gently dissolving the peptide (0.5 mg/mL) in a buffer solution (pH 7.4) containing 10 mM sodium phosphate and 1 mM EDTA. The solutions were incubated at 37°C for 36-42 h with gentle, constant shaking. Binding studies were carried out in 12 × 75 mm borosilicate glass tubes according to the procedure described [8] with some modifications. For inhibition studies, 1mL of the

In vitro autoradiography and fluorescent staining of brain sections of Tg 2576 (APPSW) mouse

The frozen brain of a 16-month-old Tg2576 mouse was kindly provided by Dr. David Holtzman (Washington University). After equilibrium to −20°C, consecutive 20 μm coronal sections were cut on a cryostat (Hacker Instruments, Fairfield, NJ), thaw-mounted on Fisher Superfrost Plus slides and stored at −70°C until use. The sections were thawed, then labeled with 0.3 nM [125I]2f at room temperature for 1 h and washed 2 x 3 min with saturated Li2CO3 in 40% EtOH, 2 min in 40% EtOH and 30 sec in H2O.

In vivo biodistribution in normal mice

While under ether anesthesia, 0.15 mL of a 0.1% bovine serum albumin solution containing [125I]2f (5-10 μCi) was injected directly into the tail vein of male ICR mice (2-3 month-old, average weight 20-30 g). The mice were sacrificed by cardiac excision at various time points post injection. The organs of interest were removed and weighed, and the radioactivity was counted with an automatic gamma counter (Packard 5000). The percentage dose per organ was calculated by a comparison of the tissue

Acknowledgements

This work was supported by grants awarded from the National Institutes of Health (NS 18509, H.F.K.), Institute for the Study of Aging (M-P.K); brain samples of Tg2576 mice were kindly provided by Dr. David Holtzman, Washington University. Theracor Pharmaceuticals Inc. has reached an agreement with the University of Pennsylvania to develop compounds related to this work as diagnostic imaging agents for Alzheimer’s disease.

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