Synthesis, Novel Crystal Structure, and β-Amyloid Binding Property of Re(I) (tricarbonyl)+ EHIDA Analogue

A neutral compound Re(CO)3(L) (L: 2-((2-(2,6-diethylphenylamino)-2-oxoethyl)(2-ethoxy-2-oxoethyl)amino)acetic acid, an IDA analogue) has been synthesized and evaluated for in vitro imaging probes of β-amyloid (Aβ) aggregates. Results of X-ray measurement of Re(CO)3(L) demonstrated that the coordination mode of Re(CO)3(L) was different from that of classical Re/Tc(I) (tricarbonyl)-IDA analogues; the structure of Re(CO)3(L) was confirmed by means of infrared spectrum, HPLC-UV, TOF MS, and X-ray measurements (Cambridge Crystallographic Data Centre number is 732731): monoclinic P21/c, a = 15.6636 (12) Å, b = 10.9360 (8) Å, c = 27.756 (2) Å, α = 90.000 (0)°, β = 90.783 (5)°, γ = 90.000 (0)°, and Z = 8. The binding affinity for β-amyloid plaques was assessed by in vitro binding assay using preformed synthetic Aβ (1–40) aggregates. The neutral compound Re(CO)3(L) showed binding affinity to Aβ aggregates at micromolar level by fluorescence spectroscopy, and this work will encourage for further exploration of imaging agents labeled by 99mTc(CO)3 + center as probes for β-amyloid plaques in vivo.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, cognitive impairment, and memory loss, and so far the only definitive confirmation of AD is universally accepted by histopathologic examination of extracellular amyloid plaques comprised of amyloid-beta (Aβ) aggregates and intracellular numerous neurofibrillary tangles in the postmortem brain; furthermore, formation of Aβ aggregates in the brain became the hallmark feature on early pathogenesis of AD before another alternative hypothesis emerged [1]. Several compounds have been selected to be radiolabeled and used in the noninvasive detection of Aβ aggregates by imaging techniques of positron emission tomography (PET) and single photon emission-computed tomography (SPECT); in addition, these representative Aβdetectors were derivatives of congo red, thioflavin, stilbene, and DDNP (6-dialakylamino-2-naphthylidene), such as 11 C-PIB, 125 I-TZDM, 11 C-SB-13, and 18 F-FDDNP [2]. However, the progress of SPECT imaging agents labeled by techenetium-99m center fell behind, because of their low rates of penetration through the blood-brain barrier (BBB) [3]. Iminodiacetic acid (IDA) derivatives EHIDA labeled by 99m Tc(CO) 3 has been studied on physicochemical properties and biological evaluation [4,5], but the structure of technetium(I)-tricarbonyl EHIDA has not been identified. In this paper, we reported the synthesis of Re(CO) 3 (L) (L is esterified EHIDA), and the novel crystal structure of this complex was determined by X-ray measurement: the coordination mode of Re(CO) 3 (L) was different from that of classical Re/Tc(I) (tricarbonyl)-IDA derivatives. The neutral compound Re(CO) 3 (L) showed binding affinity to Aβ aggregates in vitro at micromolar level by fluorescence spectroscopy.  Aggregates. Aβ (1∼40) peptide was purchased from Shang Hai "Supermed" Trade Limited Company. Re(CO) 5 Br, Thioflavin T, Phosphate Buffer Solution (PBS, pH = 7.4), and EDTA were purchased from Sigma-Aldrich Chemical Company. Other chemicals were all purchased from Beijing Chemical Reagents Company. EHIDA was synthesized according to the previous method [6].

Syntheses and X-Ray
Measurement. EHIDA (2, 2 -(2-(2,6-diethylphenylamino)-2-oxoethylazanediyl)diacetic acid) was synthesized according to the previous method [6], and the structure of EHIDA has been confirmed by using melting point mensuration, IR, 1 H-NMR, 13 C-NMR, and Mass Spectrometry (data did not be shown). 200 μmol Re(CO) 5 Br reacted with the equimolar EHIDA in the refluxing mixture of 50 mL ethanol and 0.1 mL H 2 SO 4 (4 mol/L) for 48 hours, then the reaction solution was washed by saturated NaHCO 3 solution and the target product was extracted by ethyl acetate, and after evaporating the ethyl acetate, the yellowish solid Table 2: Selected bond distances (Å) and bond angles ( • ) for the neutral complex: Re(CO) 3 (L).

Ligand
Re(CO) 3 (L) Re 1 -C CO 1.896 (7)    of Aβ (1∼40) aggregates was added to 500 μL PBS solution of Re(CO) 3 (L) (the concentrations were 19, 20, 21, 22, 23, 24 μM, resp.), and the mixed solution was incubated for 1 minute at room temperature before measuring the fluorescence intensity (slit-width was 2 nm). Excitation wavelength was 320 nm, and a 350 ∼ 600 nm scan range was performed. All the data were performed in triplicate. The linear relationship between the concentrations (Re(CO) 3 (L) only and Re(CO) 3 (L) incubated with Aβ (1∼40) aggregates) and the integration of corresponding fluorescence intensity was analysed by Microcal Origin 6.0. Results were shown in Figure 6.

Comparison of HPLC Analysis between Re(CO) 3 (L) and
99m Tc(CO) 3 (EHIDA). The retention time of Re(CO) 3 (L) (7.2 minutes) was not similar to that of 99m Tc(CO) 3 (EHIDA) (18.0 minutes) [5] (shown in Figure 2), which is speculated due to the change of lipophilicity of the ligand from EHIDA to esterification form. Esterified EHIDA (L) was more lipophilic than EHIDA; so the retention times of Re(CO) 3 3 (L) and EHIDA. The UV-visible absorption spectra of the complexes are illustrated in Figure 3. Upon EHIDA, the visible intraligand (π-π * ) bands at 240 nm and 265 nm were due to the fact that these two bands are of mainly bpy-localized in nature. In comparison, the additional visible MLCT (d-π * ) band of Re(CO) 3 (L) at 280 nm was clearly associated with the coordinational -C=O group on comparison with the UV spectra of the analogue Re(I)(tricarbonyl) + complexes [10] without the -CH 2 -C=O group.

Description of the Structure of Re(CO) 3 (L):
The neutral crystal of Re(CO) 3 . Selected distances and bond angles of the neutral crystal of Re(CO) 3 (L) are listed in Table 2, and the molecular views are presented in Figure 4. From the ORTEP drawing of the Re(CO) 3 (L), it can be known that there were two different structures of Re(CO) 3 (L) in one crystal cell.
The IDA derivatives often coordinate with Re/Tc(I) (tricarbonyl) + centers via classic coordination manner, which is that each oxygen atom of the two carboxyl groups and one N atom combine to the empty orbit in the rhenium or technetium [11], oppositely, our research of the crystal structure of Re(CO) 3 (L) demonstrated that oxygen atom of carbonyl has more stronger coordination ability than the carboxyl group, so that an oxygen atom in the carbonyl group, an oxygen atom in the carboxyl group, and the N atom coordinated with the Re (CO) 3 + core in the molecular of Re(CO) 3 (L), and as a result, 2-((2-(2,6-diethylphenylamino)-2-oxoethyl)(2-ethoxy-2oxoethyl)amino)acetic acid (L or Esterified EHIDA) reacted with Re(CO) 5 Br resulting in neutral but not negative Re(CO) 3 (L) ( Figure 5). to one another in PBS solution, the enhance of fluorescence intensity was mainly because of the breakage of the system of conjugated bonds being inhabited by the microsurrounding of β-amyloid aggregates when Aβ (1∼40) aggregates was added [12]; as a result, an increased MLCT of Re(CO) 3 (L) then occurred. However, the dissociation constant of Thioflavin T equaled 2 μM [8], lower than that of Re(CO) 3 (L), because of there is no obvious enhance of fluorescence intensity when the concentration of Re(CO) 3 (L) was little than 19 μM. Generally, probes which bind characteristically to β-amyloid fribils have more than one conjugationg system and electrondonating groups such as Me 2 N-, MeNH-, MeO-, and HOin their molecular skeletons [13], opposite to the structure of Re(CO) 3

Studies on Binding Aβ
where Y is the fluorescence intensity; X is the concentrations, from 19 to 24 μM; R is the linear correlation coefficient; SD is the standard error. When binding to β-amyloid aggregates, there was also a great linear relationship between the concentrations of Re(CO) 3 where Y is the fluorescence intensity; X is the concentrations of Re(CO) 3 (L), from 19 to 24 μM; R is the linear correlation coefficient; SD is the standard error.

Conclusion
In conclusion, the synthesis and X-ray measurement of Re(CO) 3 (L) was successfully completed, providing a novel crystal structure which was not similar to that of classical Re/Tc(I)(tricarbonyl) + IDA derivatives. Evaluation of its binding affinity to Aβ (1∼40) aggregates by the fluorescence method demonstrated that the binding characteristic was at micromolar level, which suggested that the structural modification should be achieved for future exploration of 99m Tc(CO) 3 + core labeled EHIDA derivatives as imaging agents for Aβ (1∼40) plaques in vivo, and this work will encourage for further exploration of probes for β-amyloid plaques.