Dibenzothiazoles as novel amyloid-imaging agents

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Abstract

Novel dibenzothiazole derivatives were synthesized and evaluated as amyloid-imaging agents. In vitro quantitative binding studies using AD brain tissue homogenates showed that the dibenzothiazole derivatives displayed high binding affinities with Ki values in the nanomolar range (6.8–36 nM). These derivatives are relatively lipophilic with partition coefficients (logP oct) in the range of 1.25–3.05. Preliminary structure–activity relationship studies indicated dibenzothiazole derivatives bearing electron-donating groups exhibited higher binding affinities than those bearing electron-withdrawing groups. A lead compound was selected for its high binding affinity and radiolabeled with [125I] through direct radioiodination using sodium [125I] iodide in the presence of Chloramine T. The radioligand (4-[2,6′]dibenzothiazolyl-2′-yl-2-[125I]-phenylamine) displayed moderate lipophilicity (logP oct, 2.70), very good brain uptake (3.71 ± 0.63% ID/g at 2 min after iv injection in mice), and rapid washout from normal brains (0.78% and 0.43% ID/g at 30 and 60 min, respectively). These studies indicated that lipophilic dibenzothiazole derivatives represent a promising pharmacophore for the development of novel amyloid-imaging agents for potential application in Alzheimer’s disease and related neurodegenerative disorders.

Introduction

Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder that is characteristic of amyloid deposition in the forms of senile plaques (SPs) and neurofibrillary tangles (NFTs).1, 2, 3, 4 Both SP and NFT accumulation have been suggested as early and specific events in the pathogenesis of AD.5, 6, 7, 8 Currently postmortem histopathological examination of SP and NFTs in the brain is still the only method for definitive diagnosis. One of the major tasks in AD research is to detect and quantify SP and NFT in living subjects, preferably at early or even pre-symptomatic stages. Toward this goal, nuclear imaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) have been employed. When used in conjunction with trace amount radioligands, PET and SPECT have the capacity to detect and quantify amyloid deposition in vivo. As a prerequisite to using these imaging techniques, amyloid-imaging agents that readily enter the brain and specifically target at SP or NFTs have to be developed.

To date, applications of PET and SPECT for amyloid-imaging have been hampered by the lack of suitable amyloid-imaging probes. Recent efforts have been focused on small-molecule amyloid dyes used widely in AD pathology. Such amyloid dyes include Congo Red (CR), thioflavin T (ThT), and thioflavin S (ThS) (Fig. 1). Because these histological dyes are either positively or negatively charged, they are incapable of penetrating the brain–blood barrier (BBB). This has led to the development of neutral derivatives of the amyloid dyes for potential in vivo imaging studies.

Several types of amyloid-imaging agents have thus been synthesized and evaluated. Systematic modification of CR resulted in a series of bisstyrylbenzene derivatives.9, 10, 11, 12, 13, 14, 15, 16, 17, 18 These bisstyrylbenzene derivatives exhibited high binding affinity and specificity with improved brain uptake. However, no lead compounds have been identified with in vivo pharmacokinetic profiles that meet a series of strict requirements set for in vivo imaging.

Further modification of CR also led to the design and synthesis of a series of stilbene derivatives as amyloid-imaging agents for either PET or SPECT studies.19, 20, 21, 22, 23, 24, 25 Following appropriate radiolabeling, the stilbene derivatives have been evaluated for in vivo and in vitro binding properties to amyloid deposits and pharmacokinetic profiles. Most of these stilbene derivatives readily penetrated the BBB and selectively bound to amyloid deposits at high affinities. These studies have led to the identification of a lead compound, termed [11C]SB-13 ([11C]-4-N-methylamino-4′-hydroxystilbene) (Fig. 2), that can be used for PET amyloid-imaging in human subjects.20 In AD subjects, [11C]SB-13 displayed an accumulate pattern that is considered consistent with the previously reported AD pathology. In contrast, little or no retention of [11C]SB-13 was observed in age-matched control subjects.20

Another amyloid dye that has been extensively studied is thioflavin T (ThT). ThT is a positively charged histological dye for amyloid that cannot penetrate the BBB.26 Elimination of the positive charge has led to the development of a series of benzothiazole and related heterocycles such as 2-aryl-substituted benzothiazole derivatives,17, 27, 28, 29, 30, 31 2-aryl-substituted benzooxazole derivatives,32 2-aryl-substituted benzofuran,33 and imidazo [1,2-α] pyridine derivatives.34, 35 Most of these neutral, lipophilic ThT analogs bind to amyloid fibrils with high affinity and specificity. The in vivo pharmacokinetic profiles of the above heterocyclic compounds have been extensively evaluated as potential amyloid-imaging agents. Compared to neutral CR analogs, lipophilic ThT analogs have even smaller molecular weights and display a higher brain uptake. A lead compound, termed PIB ([11C]-2-(4-(methylamino)phenyl)-6-hydroxybenzothiazole) (Fig. 2), was thus identified for human studies. Extensive clinical PET studies indicated that PIB readily entered the brain and selectively bound to amyloid deposits in AD subjects. PIB accumulation is predominant in the cortical areas known for amyloid deposition in AD subjects. Conversely, PIB showed rapid entry and clearance in all cortical gray matter of healthy control subjects.

As an imaging agent for SPECT, a [123I]-labeled imidazo [1,2-α] pyridine derivative, termed IMPY (6-iodo-2-(4′-dimethylamino-)phenyl-imidazo[1,2]pyridine) (Fig. 2), has been identified and its pharmacological effects have been evaluated in human subjects. Preliminary studies in both AD and normal control subjects demonstrated that IMPY is a safe radiotracer for clinical imaging studies.36 These studies paved the way for the potential use of [123I] IMPY in clinical SPECT imaging of amyloid deposits in human subjects.

In addition, amyloid-imaging agents have also been derived from other histological dyes such as acridine orange,37, 38 fluorene,39 and DDNP.40, 41, 42 In fact, the first PET amyloid-imaging studies in human subjects were carried out with a F-18-labeled DDNP analog termed [18F] FDDNP ([18F]-2-(1-(2-(N- (2-fluoroethyl) -N-methylamino) naphthalene-6-yl) ethylidene) malononitrile)43 (Fig. 2). Clinical studies suggested that [18F]FDDNP’s retention in amyloid deposit regions may be due to selective binding to both SPs and NFTs in the brain.

In this work, we report a series of dibenzothiazole derivatives as amyloid-imaging agents. The dibenzothiazole pharmacophore is seen in several histological dyes such as primuline44 and thioflavin S (ThS).45, 46, 47 Primuline and ThS have been commonly used as viability stains of starch in phytoplankton and a fluorescent stain for amyloid, respectively. However, Primuline and ThS exist as a mixture of several components. The major component of these dyes contains two conjugated benzothiazole units.48 We thus designed and synthesized a series of dibenzothiazole derivatives. Compared with primuline and ThS, these dibenzothiazole derivatives are lipophilic and readily enter the brain, making it possible for potential in vivo amyloid-imaging agents. In vitro evaluations suggested that these dibenzothiazole derivatives bound to amyloid deposits in AD brain homogenates with high affinities. In vivo brain permeability studies of selected compounds displayed high initial brain uptake. These studies thus expand the current portfolio of amyloid-imaging agents for potential clinical applications.

Section snippets

Chemistry, synthesis, and radiolabeling

The synthesis of dibenzothiazole derivatives is described in Scheme 1 starting from commercially available p-aminobenzothiazole (1). As shown in Scheme 1, 2-aminobenzothiazole-6-carboxylic acid (2) was first prepared from 1 based on previously reported procedures.49 Basic hydrolysis of 2 followed by neutralizing in HCl and ZnCl2 yielded the Zinc salt of 4-amino-3-mercaptobenzoic acid (3), which was coupled immediately with p-nitrobenzoyl chloride to give

Conclusion

In summary, we have developed a new type of amyloid-imaging agent based on the dibenzothiazole pharmacophore. These derivatives displayed high binding affinities for AD brain homogenates. When labelled with 125I, [125I] 18 readily enters the brain at early time intervals followed by rapid washout from the normal mouse brain, indicating low non-specific binding. Further studies are under way to systematically evaluate these novel amyloid-imaging agents for potential in vivo studies. Once fully

General remarks

All chemicals were purchased from Sigma-Aldrich and used without further purification. 1H NMR spectra were obtained at 300 MHz on Bruker DPX-300 (QNP probe) NMR spectrometers using 5 mm NMR tubes (Wilmad 528-PP) in CDCl3 or DMSO-d6 (Aldrich or Cambridge Isotopes) solutions at room temperature. Chemical shifts are reported as δ values relative to internal TMS. HR-ESIMS were acquired under the electron spray ionization (ESI) condition. The radioactivities of 125I and 3H were calculated by the

Acknowledgments

We thank Dr. Anil Gulati (University of Illinois at Chicago) for assistance in the in vitro binding assays. This work was supported in part by grants from the Alzheimer’s Association, the Institute for the Study of Aging (Y.W.), and the National Institute on Aging (AG22048, Y.M.). This work was also supported by National Natural Science Foundation of P.R. China (30470496, C. Wu) and Natural Science Foundation of Jiangsu Province, P.R. China (BK2004-423, C. Wu).

References and notes (53)

  • J.Q. Trojanowski et al.

    Neurobiol. Aging

    (1997)
  • W.E. Klunk et al.

    Neurobiol. Aging

    (1995)
  • C.D. Link et al.

    Neurobiol. Aging

    (2001)
  • K. Ishii et al.

    Neurosci. Lett.

    (2002)
  • Z.P. Zhuang et al.

    Nucl. Med. Biol.

    (2005)
  • M. Ono et al.

    Nucl. Med. Biol.

    (2003)
  • M. Ono et al.

    Nucl. Med. Biol.

    (2005)
  • W. Zhang et al.

    Nucl. Med. Biol.

    (2005)
  • C.A. Mathis et al.

    Bioorg. Med. Chem. Lett.

    (2002)
  • Z.P. Zhuang et al.

    Nucl. Med. Biol.

    (2001)
  • M. Ono et al.

    Nucl. Med. Biol.

    (2002)
  • T. Suemoto et al.

    Neurosci. Res.

    (2004)
  • C.W. Lee et al.

    Nucl. Med. Biol.

    (2003)
  • E.D. Agdeppa et al.

    Neuroscience

    (2003)
  • K. Shoghi-Jadid et al.

    Am. J. Geriatr. Psychiatry

    (2002)
  • G.T. Westermark et al.

    Methods Enzymol.

    (1999)
  • C. Wu et al.

    Curr. Top. Dev. Biol.

    (2005)
  • A. Nordberg

    Lancet Neurol.

    (2004)
  • J.A. Hardy et al.

    Science

    (1992)
  • J. Hardy et al.

    Science

    (2002)
  • H. Braak et al.

    J. Neural. Transm. Suppl.

    (1998)
  • D.J. Selkoe

    Ann. N.Y. Acad. Sci.

    (2000)
  • J. Naslund et al.

    JAMA

    (2000)
  • D.J. Selkoe

    JAMA

    (2000)
  • M. Manczak et al.

    Hum. Mol. Genet.

    (2006)
  • S.D. Styren et al.

    J. Histochem. Cytochem.

    (2000)
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