Optically Pure Diphenoxy Derivatives as More Flexible Probes for Î²

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Optically Pure Diphenoxy Derivatives as More Flexible Probes for #-Amyloid Plaques Jianhua Jia, Jia Song, Jiapei Dai, Boli Liu, and Mengchao Cui ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00155 • Publication Date (Web): 23 Jun 2016 Downloaded from http://pubs.acs.org on July 8, 2016

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ACS Chemical Neuroscience

Optically Pure Diphenoxy Derivatives as More Flexible Probes for β-Amyloid Plaques Jianhua Jia a, Jia Song a, Jiapei Dai b, Boli Liu a, Mengchao Cui a,*

a

Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing

Normal University, Beijing 100875, P. R. China. b

Wuhan Institute for Neuroscience and Neuroengineering, South-Central University for Nationalities,

Wuhan 430074, P. R. China.

Corresponding Author: *Tel/Fax: +86-10-58808891. E-mail: [email protected] (M. Cui)

1

ACS Paragon Plus Environment


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Abstract The highly rigid and planar scaffold with π-conjugated systems has been widely considered to be indispensable for Aβ binding probes. However, the flexible benzyloxybenzene derivatives [125I]BOB-4 represents an excellent lead candidate for targeting Aβ in AD brains. Based on that, we designed two pairs of more flexible and optically pure diphenoxy derivatives with a chiral center as novel Aβ probes. These compounds possessed high affinity (Ki = 15.8 – 45.0 nM) for Aβ1-42 aggregates, and (R)-enantiomers showed slightly better binding ability than (S)-enantiomers. In addition, the competition binding assay implied the optically pure diphenoxy derivatives with more flexible geometry shared the same binding site as IMPY, a classical rigid and planer Aβ probe. For

125

I-radiolabeled enantiomers,

(S)-[125I]5 and (R)-[125I]5, specific plaques labeling on brain sections of Tg mice and AD patients were observed in in vitro autoradiography, persuasively proved the excellent affinity of the probes. In biodistribution, (S)-[125I]5 and (R)-[125I]5 with relatively low lipophilicity exhibited moderate initial brain uptake (4.37 and 3.72 % ID/g at 2 min) and extremely fast washout from normal mice brain (brain2 min/brain60 min

= 19.0 and 17.7). In summary, the separate enantiomers displayed roughly similar

properties in vitro and in vivo, and (S/R)-[123I]5 may be potential SPECT probes for recognizing Aβ plaques in AD brains.

Key words: Alzheimer’s disease, Aβ plaque, benzyloxybenzene, enantiopure, imaging probe.

2


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Introduction Alzheimer’s disease (AD), known as a common neurodegenerative dementia, results in synaptic failure and neuronal death. This disease starts gradually and then gives rise to cognitive deficits usually involving episodic memory, language and attention1. It is found that one of the main pathological hallmarks of this disease can be attributed to the presence of extracellular senile plaques in the brain and the major constituent of these plaques refers to amyloid-β (Aβ). Despite the fact that there exists no universal agreement regarding the exact role plaques play during the onset of AD, it is widely acknowledged that based on the histopathological studies, extensive Aβ deposition in autopsy is observed.2,

3

Therefore, it is believed that the application of Aβ imaging agents using noninvasive

techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) is of great help in tracking amyloid pathology and achieving early diagnosis of AD. In past decades, reports mainly focused on the variants of Congo red and Thioflavin-T as imaging agents targeting to Aβ plaques. The related imaging agents [18F]AV-45 (florbetapir)4, 5, [18F]GE-067 (flutemetamol)6,

7

and [18F]BAY94-9172(florbetaben)8,

9

gained approval of FDA. Therefore, PET

imaging has been a powerful technique to differentiate AD patients from healthy controls. However, these

18

F-labeled probes are handicapped by complicated synthetic manipulations, special equipments,

high cost and short half-life in clinic. Meanwhile, more SPECT scanners in hospitals urgently need SPECT imaging agents for detection of Aβ.

99m

Tc and

123

I are two common and excellent SPECT

isotopes. The application of 99mTc-labeled Aβ probes was hampered by limited blood-brain barrier (BBB) penetration. Researchers have also developed many

123

I-labeled Aβ probes. [123I]IMPY10, one of

3



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promising SPECT imaging agents derived from Thioflavin-T, has failed in preclinical trials because of the poor signal-to-noise ratio for Aβ labeling. Up to now, no Aβ probes for SPECT imaging received marketing authorization. Recently, our group reported a series of

125

I-labeled flexible benzyloxybenzene derivatives without

π-conjugated systems and rigid planarity structures as Aβ probes. Interestingly, these flexible compounds shared the same binding site with rigid compound IMPY. More important, a promising compound [125I]BOB-4 displayed excellent properties including good affinity for Aβ, effective BBB penetration, rapid clearance ability from normal brain and satisfactory biostability11. Nevertheless, the high lipophilicity of [125I]BOB-4 is still a disadvantage and needs to decrease by structure modifications. Introducing of a hydroxyl group is an easy and efficient method. The affinity would be decline if introduce a hydroxyl on the benzene ring according to the structure-affinity relationships of the benzyloxybenzene structure11. Herein, we introduced a hydroxyl group into the flexible linker between the two benzene rings and a chiral center was generated in this case (Figure 1). So far, [18F]FMAPO, the only one reported Aβ probe, had a chiral center in the side chain of target molecule, however, it was pharmacologically evaluated as racemic mixtures.12 It is known that enantiomers have similar physical and chemical properties, whereas they usually have remarkable differences in pharmacological activities, metabolism and toxicity in vivo. Therefore, it is important and necessary to definite the biological properties of single-enantiomer in the process of drug development. In this paper, we described the design, synthesis, characterization and biological evaluations of

125

I-labeled optically pure diphenoxy

derivatives as SPECT probes for detecting Aβ plaques in AD brains. Compared to benzyloxybenzene derivatives, the enantiopure diphenoxy derivatives is more flexible and tortuous, and the study of 4


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separated enantiomers in vitro and in vivo is more complex and interesting.

---------Figure 1 ----------

Results and discussion Chemistry The synthesis of the desired optically pure diphenoxy derivatives and their precursors is outlined in Scheme 1. Based on an alternative base-catalyzed SN2 reaction (t-BuOK/DMF), the hydroxy group of 4-methoxyphenol

or

4-nitrophnol

was

coupled

with

optically

pure

(R)-3-(-)-

or

(S)-3-(+)-chloropropane-1,2-diol to provide dihydroxy group compounds (R)-1, (S)-1, (R)-2 and (S)-2 in 47.5 - 90.7% yields. Tosylation of the hydroxyl groups of (R)-1, (S)-1, (R)-2 and (S)-2 produced monotosylate and ditosylate compounds, and the monotosylate compounds (S)-3, (R)-3, (S)-4 and (R)-4 were isolated in yields of 18.8 - 28.1%. 4-Iodophenol replaced the monotosylate group using base-catalyzed reaction (K2CO3/DMF) to afford (S)-5, (R)-5, (S)-6 and (R)-6 in yields of 20.4 - 36.3%. The nitro-compounds (S)-6 and (R)-6 were reduced with SnCl2.2H2O/HCl/EtOH to give amino compounds (S)-7 and (R)-7 efficiently. Conversion of (S)-7 and (R)-7 to the dimethylamino derivatives (S)-8 and (R)-8 was achieved by a common dimethylation method with (HCHO)n/NaBH3CN/HAc in yields of 44.5% and 41.7%, respectively. The tributyltin precursors (S)-9 and (R)-9 for 125I-labeling were produced using iodine to tributyltin exchange reaction catalyzed by (PPh3)4Pd from the corresponding 5



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iodo-compounds (S)-5 and (R)-5 in yields of 13.5% and 18.1%, respectively. A pair of single crystals of (R/S)-5 were obtained by slow evaporation from petroleum ether/ethyl acetate system at room temperature, the chiral structure was confirmed via the X-ray analysis as displayed in Figure 2, and the relative crystallographic data are shown in Table 1. The values of Flack parameter, near 0 and a small standard uncertainty (-0.030(15) and -0.024(19)), given the absolute configuration of (S)-5 and (R)-513. From the crystal structures of (S)-5 and (R)-5, we can find that they have non-planar structure and the dihedral angles between the two phenyl rings (pink and blue) are 54.486° and 54.489°, respectively. In addition, the opticity of (S)-5 and (R)-5 was confirmed by polarimeter, and the optical rotations were + 4.4° and - 4.4° in methanol, respectively.

---------Scheme 1 Figure 2 Table 1 ----------

In vitro binding assay using Aβ1-42 aggregates According to conventional methods14, we carried out an inhibition binding assay using [125I]IMPY as a competitive radio-ligand to quantitatively evaluate the binding affinity of these optically pure diphenoxy derivatives for Aβ1-42 aggregates (Table 2 and Figure S2). The compounds with more flexible geometry also inhibited the binding of [125I]IMPY in a dose-dependent manner, implying they have the 6


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same binding site as IMPY. Ligands (S)-5, (R)-5, (S)-8 and (R)-8 exhibited good affinity for Aβ1-42 aggregates with Ki values of 34.0 ± 2.7 nM, 25.0 ± 1.3 nM, 45.0 ± 13.0 nM and 15.8 ± 11.5 nM, respectively, which were equivalent to the value of IMPY (Ki = 25.3 ± 2.6 nM) in the identical assay conditions. Compared with methoxy- substituted benzyloxybenzene derivative, BOB-4 (Ki = 24.3 ± 6.8 nM)11, methoxy- substituted diphenoxy enantiomers have comparable affinity for Aβ1-42 aggregates, which implied the more flexible structure and introduction of a hydroxyl group did not affect the binding affinity. The Ki values suggest that (R)-enantiomers have better binding abilities than (S)-enantiomers, and the substituent group methoxy- and N,N-dimethylamino- group have little influence on the binding affinity. The excellent affinities imply that these ligands can conveniently label Aβ plaques in AD brains. Compared to the N,N-dimethylamino- substituent ligands (S)-8 and (R)-8, the methoxy- substituent ligands (S)-5 and (R)-5 possess easier synthetic route and more stable property, therefore, they were labeled with 125I and subsequently bioevaluated in detail.

---------Table 2 ----------

Radiochemistry To obtain the desired (S)-[125I]5 and (R)-[125I]5, an iododestannylation reaction was proceeded starting from the corresponding tributyltin precursors in high radiochemical yields of 84.0% and 81.6%, respectively. The specific activity of the 125I-labeled final products was anticipated to be 2200 Ci/mmol , 7



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similar to that of [125I]NaI. Sample purification was conducted using HPLC to afford the high radiochemical purity of

125

I-radiotracers (> 95%). The retention time of these radiotracers was close to

that of corresponding non-radioactive compounds in co-injection and co-elution HPLC analysis, which successfully verified their chemical identities (Figure S1 and Table S1). In vitro autoradiography In vitro autoradiography, being able to directly characterize the good binding affinity for Aβ plaques, was carried out on brain sections from transgenic (Tg) mice and AD patients with (S)-[125I]5 and (R)-[125I]5. As shown in Figure 3A and Figure 3B, clusters of radioactive spots were observed in the cortical and cerebellar regions of Tg mice brain, and the distribution and location were line with the fluorescent dots stained with a fluorescent Aβ probe DANIR 3b15. Likewise, as displayed in Figure 4A and 4B, many radioactive spots, which accumulated on brain sections of different AD patients, concluding Aβ plaques and cerebrovascular amyloids, coincided precisely with the fluorescent staining results. Conversely, no significant accumulation of radioactivity was observed on brain sections of wild-type mice and healthy human (Figure 3C, 3D, 4I and 4K). These results explicitly validated the specific binding affinity of (S)-5 and (R)-5 in the competition binding assay, and proved the feasibility and practicality of (S)-[125I]5 and (R)-[125I]5 as Aβ probes at tracer dose in vitro.

---------Figure 3 Figure 4 ---------8


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In vivo biodistribution The ability of BBB penetration and washout properties of tracers are considered as two important factors for imaging agents, therefore, biodistribution studies were performed in normal ICR mice. The partition coefficients of these two radiolabeled ligands were firstly determined to figure out their ability to penetrate BBB. The log D values of (S)-[125I]5 and (R)-[125I]5 were 3.26 ± 0.12 and 2.97 ± 0.04, respectively. Compared with that of [125I]BOB-4 (4.00 ± 0.08)11, the lipophilicity of the enantiomers was decreased, which is anticipated to be more beneficial to reduction of nonspecific and increase of washout from normal brain. In biodistribution studies, as shown in Figure 5 and Table S2, (S)-[125I]5 and (R)-[125I]5 penetrated BBB with medium initial brain uptake levels of 4.37 ± 0.70% ID/g and 3.72 ± 0.31% ID/g at 2 min post-injection and rapidly dropped to 0.23 ± 0.04% ID/g and 0.21 ± 0.12% ID/g at 60 min post-injection, displaying the high washout rate of 19.0 and 17.7 (brain2 min/brain60 min). The liver and small intestine is significant for drug metabolism, specially, a maximum tracer concentration in the liver was found to gradually decrease and a low accumulation of radio-activity in the small intestine kept drastically increasing with time. In addition, low levels of radioactivity in thyroids were observed, which indicated that the two radiolabeled ligands were stable in vivo. From the values of initial brain uptake and washout rate, the pharmacokinetics of the enantiomers (S)-[125I]5 and (R)-[125I]5 had no significant differences in vivo, and (S)-enantiomer showed slight superiority. Compared with reported [125I]BOB-4, which displayed moderate brain uptake (6.18% ID/g at 2 min) and rapid clearance rate with 16.3 (brain2 11 min/brain60 min) ,

(S)-[125I]5 and (R)-[125I]5 had a slight advantage in the aspect of washout rate but

inferior in terms of BBB penetration. However, compared to those reported 9


125

I-labeled tracers with


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rigid structure, like Crystamine G derivatives16, Thioflavin-T derivatives17-21, stilbene derivatives22 and plant pigment derivatives23-26, the superiority of (S)-[125I]5 and (R)-[125I]5 is more significant on the washout rate from normal mice brain. These results suggested (R/S)-5, when labeled with [123I], are suitable for detection of Aβ plaques in AD brains as SPECT imaging agents. ---------Figure 5 ----------

In conclusion, we synthesized more flexible and optically pure diphenoxy derivatives targeting Aβ plaques of AD patients by modifying benzyloxybenzene scaffold, a novel flexible target molecule for Aβ. In in vitro binding assay, two pairs of enantiomers displayed good affinity for Aβ1-42 aggregates, especially for the (R)-enantiomers, their affinity was comparable or superior to IMPY under the same conditions. The more flexible and twisty compounds could well compete with rigid IMPY, which completely change the previous cognition about planar Aβ probes. The high affinity of (S)-[125I]5 and (R)-[125I]5 were verified powerfully by the excellent labeling Aβ plaques on brain sections of Tg mice and AD patients in in vitro autoradiography studies. In biodistribution, optically pure enantiomers (S)-[125I]5 and (R)-[125I]5 exhibited moderate ability of BBB penetration (4.37 and 3.72 %ID/g at 2 min) and very fast clearance from normal mice brain (brain2 min/brain60 min = 19.0 and 17.7), which implied the separate enantiomers had roughly similar properties in vivo. The preliminary results implied the more flexible and optically pure (R/S)-[125I]5 would be potential imaging agents for Aβ plaques by SPECT when labeled with 123I. In addition, the research of more flexible and chiral Aβ probes gives a new sight 10


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of understanding the complexity and diversity of Aβ protein, and offers a successful example to develop versatilely flexible Aβ imaging agents.

Methods General information All reagents for chemical synthesis were purchased without further purification. [125I]NaI was purchased from PerkinElmer. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were obtained on a Bruker Avance III NMR spectrometer in CDCl3 solutions at room temperature. Mass spectra were acquired with GCT CA127 Micronass UK instrument and Bruker microOTOF-QII instrument. X-ray crystallographic data of compounds (S)-5 and (R)-5 were collected on a Bruker Smart APEX II diffractometer (Bruker Co., Germany) and deposited at the Cambridge Crystallographic Data Centre as supplementary publication ((R)-5, No. CCDC 1447649; (S)-5, No. CCDC 1447638). HPLC analysis was performed on a Shimadzu SCL-20 AVP equipped with a Bioscan Flow Count 3200 NaI/PMT γ-radiation scintillation detector and a SPD-20A UV detector, λ = 254 nm. A Venusil MP C18 reverse phase column (Bonna-Agela Technologies, 5 µm, 4.6 × 250 mm) was used for separations (acetonitrile:water = 65:35) and purity determinations (acetonitrile:water = 70:30) with a binary gradient system at a 1.0 mL/min flow rate. Fluorescent observation was carried out on the Axio Observer Z1 inverted fluorescence microscope (Zeiss, Germany) equipped with an AF488 filter set. Post-mortem brain sections from autopsy-confirmed AD subjects (91-year-old, male, temporal lobe; 64-year-old, female, temporal lobe) and a control subject (79-year-old, male, temporal lobe) were obtained from the Chinese Brain Bank Centre. Tg mice (C57BL6, APPswe/PSEN1, 12-month-old, female) and wild-type mice (C57BL6, 11



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12-month-old, female) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. Normal ICR mice (5 weeks, male) in vivo biodistribution were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd. All experiments on mice were performed in accordance with the guidelines approved by the animal care committee of Beijing Normal University. (R)-3-(4-methoxyphenoxy)propane-1,2-diol ((R)-1) To a solution of 4-methoxyphenol (6.21 g, 50.0 mmol) in DMF (20 mL) was added potassium tert-butoxide (8.42 g, 75.0 mmol). The resulting suspension was stirred at 150 °C, 1 h later, (R)-3-(-)-chloropropane-1,2-diol (11.05 mg, 100.0 mmol) was added into the mixture and stirred at 120 °C for additional 48 h. Then the paste reaction mixture was diluted by methanol (20 mL) and filtered to remove the produced salt in the process of reaction. The filtrate was evaporated in vacuum at 110 °C to remove solvent, the obtained residue was again dissolved in a little ethyl acetate and it was purified by silica gel chromatography (petroleum ether/AcOEt = 2/1 – 1/3). Brown crystal, 5.66 g. Yield, 57.1%. M.p. 61.3 – 62.5 °C. 1H NMR (400 MHz, CDCl3) δ 6.88 – 6.81 (m, 4H), 4.11 – 4.05 (m, 1H), 4.03 – 3.95 (m, 2H), 3.85 – 3.81 (m, 1H), 3.77 (s, 3H), 3.76 – 3.72 (m, 1H), 2.35 (s, 1H), 2.32 (s, 1H). 13

C NMR (100 MHz, CDCl3) δ 154.27, 152.76, 115.72, 114.85, 70.79, 69.97, 63.86, 55.84. HRMS

(ES+): m/z calcd for [C10H14O4 + Na]+ 221.0784; found 221.0779. (S)-3-(4-methoxyphenoxy)propane-1,2-diol ((S)-1) The same procedure described above for the synthesis of (R)-1 was employed to give (S)-1 (column chromatography condition: petroleum ether/AcOEt = 2/1 – 1/3). Brown crystal, 9.42 g. Yield, 47.5%. M.p. 61.8 – 63.4 °C. 1H NMR (400 MHz, CDCl3) δ 6.88 – 6.81 (m, 4H), 4.11 – 4.06 (m, 1H), 4.03 – 12


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3.95 (m, 2H), 3.85 – 3.81 (m, 1H), 3.77 (s, 3H), 3.76 – 3.72 (m, 1H), 2.46 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 154.26, 152.76, 115.72, 114.84, 70.78, 69.96, 63.85, 55.84. HRMS (ES+): m/z calcd for [C10H14O4 + Na]+ 221.0784; found 221.0776. (R)-3-(4-nitrophenoxy)propane-1,2-diol ((R)-2) The same procedure described above for the synthesis of (R)-1 was employed to give (R)-2 (column chromatography condition: petroleum ether/AcOEt = 3/1 – 1/5). Pale yellow waxy solid, 10.75 g. Yield, 64.3%. M.p. 56.3 – 57.9 °C. 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 9.3 Hz, 2H), 6.99 (d, J = 9.3 Hz, 2H), 4.18 – 4.09 (m, 3H), 3.87 (dd, J = 11.4, 3.5 Hz, 1H), 3.77 (dd, J = 11.4, 5.1 Hz, 1H), 2.55 (s, 2H). 13

C NMR (100 MHz, CDCl3) δ 163.73, 141.83, 126.04, 114.69, 70.33, 69.79, 63.51. HRMS (ES+): m/z

calcd for [C9H11NO5 + Na]+ 236.0529; found 236.0552. (S)-3-(4-nitrophenoxy)propane-1,2-diol ((S)-2) The same procedure described above for the synthesis of (R)-1 was employed to give (S)-2 (column chromatography condition: petroleum ether/AcOEt = 3/1 – 1/5). Pale yellow waxy solid, 15.47 g. Yield, 90.7%. M.p. 62.4 – 63.8 °C. 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 9.3 Hz, 2H), 6.99 (d, J = 9.3 Hz, 2H), 4.22 – 4.10 (m, 3H), 3.88 (dd, J = 11.4, 3.6 Hz, 1H), 3.78 (dd, J = 11.4, 5.1 Hz, 1H), 2.53 (s, 2H). 13

C NMR (100 MHz, CDCl3) δ 163.64, 141.93, 126.08, 114.68, 70.31, 69.81, 63.49. HRMS (ES+): m/z

calcd for [C9H11NO5 + Na]+ 236.0529; found 236.0543. (S)-2-hydroxy-3-(4-methoxyphenoxy)propyl 4-methylbenzenesulfonate ((S)-3) To a solution of (R)-1 (4.97 g, 25.0 mmol) and 4-methylbenzenesulfonyl chloride (5.73 g, 30.0 mmol) in anhydrous CH2Cl2 (20 mL) was added triethylamine (5 mL) under ice bath. The resulting mixture was stirred at room temperature for 4 h, then 30 mL of water was added to the mixture and it 13



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was extracted by CH2Cl2 (3 × 20 mL). The combined organic layers were dried over MgSO4, filtered, and the solvent was removed under vacuum. The residue was purified by silica gel chromatography (petroleum ether/AcOEt = 5/1 – 1/1). Pale yellow oil, 2.48 g. Yield, 28.1%. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 6.85 – 6.73 (m, 4H), 4.24 – 4.14 (m, 3H), 3.93 (d, J = 4.8 Hz, 2H), 3.77 (s, 3H), 2.43 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 154.38, 152.40, 145.27, 132.57, 130.10, 128.09, 115.68, 114.79, 70.59, 68.63, 68.13, 55.84, 21.75. HRMS (ES+): m/z calcd for [C17H20O6S + Na]+ 375.0872; found 375.0872. (R)-2-hydroxy-3-(4-methoxyphenoxy)propyl 4-methylbenzenesulfonate ((R)-3) The same procedure described above for the synthesis of (S)-3 was employed to give (R)-3 (column chromatography condition: petroleum ether/AcOEt = 5/1 – 1/1). Colorless oil, 2.30 g. Yield, 18.8%. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 6.88 – 6.72 (m, 4H), 4.23 – 4.13 (m, 3H), 3.93 (d, J = 4.7 Hz, 2H), 3.77 (s, 3H), 2.55 (s, 1H), 2.43 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 154.36, 152.40, 145.28, 132.54, 130.10, 128.09, 115.67, 114.79, 70.62, 68.62, 68.12, 55.84, 21.76. HRMS (ES+): m/z calcd for [C17H20O6S + Na]+ 375.0872; found 375.0874. (S)-2-hydroxy-3-(4-nitrophenoxy)propyl 4-methylbenzenesulfonate ((S)-4) The same procedure described above for the synthesis of (S)-3 was employed to give (S)-4 (column chromatography condition: petroleum ether/AcOEt = 5/1 – 1/1). White solid, 0.86 g. Yield, 23.3%. M.p. 92.2 – 92.8 °C. 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 9.3 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 9.3 Hz, 2H), 4.30 – 4.19 (m, 3H), 4.10 (d, J = 5.0 Hz, 2H), 2.44 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.22, 145.53, 142.03, 132.38, 130.16, 128.06, 125.98, 114.71, 70.17, 68.61, 67.86, 21.76. HRMS (ES+): m/z calcd for [C16H17NO7S + Na]+ 390.0618; found 390.0639. 14


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(R)-2-hydroxy-3-(4-nitrophenoxy)propyl 4-methylbenzenesulfonate ((R)-4) The same procedure described above for the synthesis of (S)-3 was employed to give (R)-4 (column chromatography condition: petroleum ether/AcOEt = 5/1 – 1/1). Light yellow solid, 2.81 g. Yield, 24.1%. M.p. 62.2 – 62.8 °C. 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 9.3 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 9.3 Hz, 2H), 4.30 – 4.19 (m, 3H), 4.10 (d, J = 5.0 Hz, 2H), 2.44 (s, 3H).

13

C NMR (100 MHz, CDCl3) δ 163.23, 145.56, 141.99, 132.33, 130.16, 128.05, 125.98, 114.71,

70.24, 68.58, 67.80, 21.75. HRMS (ES+): m/z calcd for [C16H17NO7S + Na]+ 390.0618; found 390.0635. (S)-1-(4-iodophenoxy)-3-(4-methoxyphenoxy)propan-2-ol ((S)-5) To a solution of (S)-3 (0.73 g, 2.1 mmol) and 4-iodophenol (0.46 g, 2.1 mmol) in anhydrous DMF (5 mL) was added potassium carbonate (0.35 g, 2.5 mmol). The mixture was stirred at 90 °C for 5 h. After reaction, DMF was removed in vacuum at 110 °C. The crude product was extracted by CH2Cl2 (3 × 20 mL), and purified by silica gel chromatography (petroleum ether/AcOEt = 10/1). White powder, 0.24 g. Yield, 28.0%. M.p. 112.9 – 113.8 °C. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 9.0 Hz, 2H), 6.89 – 6.82 (m, 4H), 6.72 (d, J = 9.0 Hz, 2H), 4.40 – 4.32 (m, 1H), 4.15 – 4.05 (m, 4H), 3.77 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 158.55, 154.41, 152.69, 138.46, 117.17, 115.79, 114.92, 83.58, 69.65, 69.15, 68.92, 55.91. HRMS (ES+): m/z calcd for [C16H17O4I + H]+ 401.0250; found 401.0254. (R)-1-(4-iodophenoxy)-3-(4-methoxyphenoxy)propan-2-ol ((R)-5) The same procedure described above for the synthesis of (S)-5 was employed to give (R)-5 (column chromatography condition: petroleum ether/AcOEt = 10/1). White powder, 0.30 g. Yield, 36.3%. M.p. 115.1 – 115.7 °C. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.9 Hz, 2H), 6.89 – 6.81 (m, 4H), 6.71 (d, J = 8.9 Hz, 2H), 4.38 – 4.32 (m, 1H), 4.15 – 4.05 (m, 4H), 3.77 (s, 3H), 1.99 (s, 1H). 13C NMR (100 MHz, 15



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CDCl3) δ 158.54, 154.42, 152.69, 138.46, 117.16, 115.79, 114.92, 83.57, 69.64, 69.14, 68.92, 55.91. HRMS (ES+): m/z calcd for [C16H17O4I + H]+ 401.0250; found 401.0254. (S)-1-(4-iodophenoxy)-3-(4-nitrophenoxy)propan-2-ol ((S)-6) The same procedure described above for the synthesis of (S)-5 was employed to give (S)-6 (column chromatography condition: petroleum ether/AcOEt = 4/1). White powder, 0.49 g. Yield, 20.4%. M.p. 106.7 – 108.5 °C. 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 9.3 Hz, 2H), 7.58 (d, J = 8.9 Hz, 2H), 7.01 (d, J = 9.3 Hz, 2H), 6.72 (d, J = 8.9 Hz, 2H), 4.46 – 4.41 (m, 1H), 4.26 – 4.21 (m, 2H), 4.17 – 4.11 (m, 2H), 2.54 (s, 1H).

13

C NMR (100 MHz, CDCl3) δ 163.51, 158.31, 142.02, 138.52, 126.09, 117.08,

114.74, 83.82, 69.57, 68.78, 68.61. HRMS (ES+): m/z calcd for [C15H14INO5 + Na]+ 437.9809; found 437.9806. (R)-1-(4-iodophenoxy)-3-(4-nitrophenoxy)propan-2-ol ((R)-6) The same procedure described above for the synthesis of (S)-5 was employed to give (R)-6 (column chromatography condition: petroleum ether/AcOEt = 4/1). White powder, 0.63 g. Yield, 21.8%. M.p. 105.9 – 107.2 °C. 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 9.3 Hz, 2H), 7.58 (d, J = 9.0 Hz, 2H), 7.01 (d, J = 9.3 Hz, 2H), 6.72 (d, J = 9.0 Hz, 2H), 4.46 – 4.40 (m, 1H), 4.28 – 4.21 (m, 2H), 4.17 – 4.10 (m, 2H).

13

C NMR (100 MHz, CDCl3) δ 163.44, 158.29, 142.13, 138.55, 126.10, 117.06, 114.72, 83.84,

69.53, 68.75, 68.61. HRMS (ES+): m/z calcd for [C15H14INO5 + Na]+ 437.9809; found 437.9810. (S)-1-(4-aminophenoxy)-3-(4-iodophenoxy)propan-2-ol ((S)-7) To a solution of (S)-6 (0.49 g, 1.1 mmol) and SnCl2.2H2O (1.07 g, 4.4 mmol) in EtOH (20 mL) was added concentrated HCl (5 mL). The resulting mixture was stirred at 110 °C for 4 h. After reaction, EtOH was removed in vacuum and the residue was poured into ice, then the pH was adjusted to 7 by 16


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adding ammonia. The mixture was extracted by CH2Cl2 (3 × 20 mL) to afford crude product, which was pure enough for next steps. Yellow solid, 0.81 g. Yield, 100.0%. M.p. 84.8 – 85.7 °C. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 8.5 Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.5 Hz, 2H), 4.36 – 4.29 (m, 1H), 4.12 – 4.01 (m, 4H), 3.43 (s, 2H).

13

C NMR (100 MHz, CDCl3) δ

158.57, 151.94, 140.14, 138.43, 117.17, 116.92, 115.96, 83.51, 69.72, 69.21, 68.87. HRMS (ES+): m/z calcd for [C15H16INO3 + H]+ 386.0248; found 386.0236. (R)-1-(4-aminophenoxy)-3-(4-iodophenoxy)propan-2-ol ((R)-7) The same procedure described above for the synthesis of (S)-7 was employed to give (R)-7. Yellow solid, 0.56 g. Yield, 100.0%. M.p. 89.9 – 91.4 °C. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 9.0 Hz, 2H), 6.76 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 8.9 Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 4.36 – 4.30 (m, 1H), 4.13 – 4.02 (m, 4H), 3.12 (s, 2H).

13

C NMR (100 MHz, CDCl3) δ 158.56, 151.81, 140.43, 138.44,

117.15, 116.72, 115.97, 83.49, 69.68, 69.16, 68.90. HRMS (ES+): m/z calcd for [C15H16INO3 + H]+ 386.0248; found 386.0239. (S)-1-(4-(dimethylamino)phenoxy)-3-(4-iodophenoxy)propan-2-ol ((S)-8) To a solution of (S)-7 (0.81 g, 2.1 mmol) and paraformaldehyde (0.63 g, 21.0 mmol) in acetic acid (10 mL) in ice bath was added NaBH3CN (0.66 g, 10.0 mmol) in batches. The reaction mixture was stirred at room temperature for 12 h and the residue was poured into ice, then the pH was adjusted to 7 by adding ammonia. The resulting mixture was extracted by CH2Cl2 (3 × 40 mL) and purified by silica gel column chromatography to give final product (petroleum ether/AcOEt = 4/1). White solid, 0.39 g. Yield, 44.5%. M.p. 101.6 – 102.2 °C. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 6.78 (s, 2H), 6.71 (d, J = 9.0 Hz, 2H), 4.37 – 4.32 (m, 1H), 4.14 – 4.05 (m, 4H), 2.89 (s, 17



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6H), 2.58 (s, 1H).

13

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C NMR (100 MHz, CDCl3) δ 158.58, 150.80, 146.34, 138.44, 117.16, 115.81,

114.89, 83.48, 69.68, 69.16, 68.95, 41.82. HRMS (AP+): m/z calcd for [C17H20NO3I+H]+ 414.0566; found 414.0565. (R)-1-(4-(dimethylamino)phenoxy)-3-(4-iodophenoxy)propan-2-ol ((R)-8) The same procedure described above for the synthesis of (S)-8 was employed to give (R)-8 (column chromatography condition: petroleum ether/AcOEt = 4/1). White solid, 0.26 mg. Yield, 41.7%. M.p. 107.8 – 108.6 °C. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.9 Hz, 2H), 6.87 (d, J = 8.9 Hz, 2H), 6.78 (s, 2H), 6.71 (d, J = 8.9 Hz, 2H), 4.39 – 4.31 (m, 1H), 4.14 – 4.05 (m, 4H), 2.89 (s, 6H), 2.59 (s, 1H). 13

C NMR (100 MHz, CDCl3) δ 158.59, 150.78, 146.37, 138.44, 117.16, 115.82, 114.87, 83.48, 69.69,

69.17, 68.95, 41.83. HRMS (AP+): m/z calcd for [C17H20NO3I+H]+ 414.0566; found 414.0564. (S)-1-(4-methoxyphenoxy)-3-(4-(tributylstannyl)phenoxy)propan-2-ol ((S)-9) To a solution of (S)-5 (205.3 mg, 0.5 mmol) and (Bu3Sn)2 (892.7 mg, 1.5 mmol) in toluene (10 mL) was added (Ph3P)4Pd (58.9 mg, 0.05 mmol) and 3 mL of Et3N. The mixture was stirred at 110 °C for 6 h. Toluene was evaporated under vacuum and the residue was purified by silica gel chromatography (petroleum ether/AcOEt = 10/1). Colorless oil, 72.5 mg. Yield, 13.5%. 1H NMR (400 MHz, CDCl3) δ 7.43 – 7.28 (m, 2H), 7.00 – 6.92 (m, 2H), 6.89 – 6.82 (m, 4H), 4.40 – 4.33 (m, 1H), 4.19 – 4.06 (m, 4H), 3.77 (s, 3H), 1.68 – 1.61 (m, 2H), 1.53 – 1.47 (m, 4H), 1.39 – 1.25 (m, 8H), 1.04 – 1.00 (m, 4H), 0.88 (t, J = 7.3 Hz, 9H). 13C NMR (100 MHz, CDCl3) δ 158.72, 154.37, 152.77, 137.70, 133.03, 115.75, 114.86, 114.67, 69.69, 69.02, 68.60, 55.86, 29.22, 27.49, 13.80, 9.74. HRMS (ES+): m/z calcd for [C28H44O4Sn + H]+ 565.2340; found 565.2317. (R)-1-(4-methoxyphenoxy)-3-(4-(tributylstannyl)phenoxy)propan-2-ol ((R)-9) 18


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The same procedure described above for the synthesis of (S)-9 was employed to give (R)-9 (column chromatography condition: petroleum ether/AcOEt = 10/1). Colorless oil, 63.8 mg. Yield, 18.1%. 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 6.89 – 6.82 (m, 4H), 4.40 – 4.33 (m, 1H), 4.18 – 4.06 (m, 4H), 3.77 (s, 3H), 2.58 (d, J = 5.1 Hz, 1H), 1.68 – 1.59 (m, 2H), 1.55 – 1.49 (m, 4H), 1.42 – 1.28 (m, 8H), 1.04 – 1.00 (m, 4H), 0.88 (t, J = 7.3 Hz, 9H). 13C NMR (100 MHz, CDCl3) δ 158.74, 154.37, 152.79, 137.71, 133.02, 115.76, 114.87, 114.69, 69.71, 69.03, 68.63, 55.86, 29.23, 27.50, 13.81, 9.75. HRMS (ES+): m/z calcd for [C28H44O4Sn + H]+ 565.2340; found 565.2350. Radiolabeling The (S)-[125I]5 and (R)-[125I]5 were prepared from the corresponding tributyltin precursors according to the reported procedures previously.24 The rude product was purified by radio-HPLC under the following conditions: Venusil MP C18 column (Bonna-Agela Technologies, 4.6 mm × 250 mm), CH3CN/H2O = 65/35, 1 mL/min, UV = 254 nm. The

125

I-labeled enantiomers were confirmed by

co-injection and co-elution with the corresponding cold iodo-compounds on HPLC under the following conditions: Venusil MP C18 column (Bonna-Agela Technologies, 4.6 mm × 250 mm), CH3CN/H2O = 70/30, 1 mL/min, UV = 254 nm. Biological evaluations Biological evaluations (in vitro inhibition binding assay, in vitro autoradiography, partition coefficient determination and biodistribution) were all performed using previously reported methods24. Author information Corresponding author E-mail: [email protected]. Tel/Fax: +86-10-58808891. 19



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Notes The authors declare no competing financial interests. Acknowledgement This work was funded by the National Natural Science Foundation of China (No. 21571022) and the Research Fund for the Doctoral Program of Higher Education of China (No. 20130003120012). Supplementary data In vitro and in vivo evaluations are showed including two figures and two tables as described in the manuscript, as well as spectrograms of all synthesized compounds. This material is available free of charge on the ACS Publications website at DOI: XXX.

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Figure 1. Classic Aβ probes with rigid and planar structure previously reported, and the design of optically pure diphenoxy derivatives as more flexible Aβ probes.

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Figure 2. Crystal structure of compound (S)-5 and (R)-5 with the thermal ellipsoids drawn at the 30% probability level (The dihedral angle between pink and blue plant reflect the angle between two benzene rings).

26


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Figure 3. In vitro autoradiography of (S)-[125I]5 and (R)-[125I]5 on mice brain sections. (A and B) Tg, C57BL6, APPswe/PSEN1, 11-month-old, female; (C and D) wild-type, C57BL6, 11-month-old, female. The presence and location of Aβ plaques were precisely confirmed by the results of fluorescence staining using DANIR 3b.

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Figure 4. In vitro autoradiography of (S)-[125I]5 and (R)-[125I]5 on human brain sections. (A and C) AD case 1: 91-year-old, male, temporal lobe; (E and G) AD case 2: 64-year-old, female, temporal lobe; (I and K) Healthy case: 79-year-old, male, temporal lobe. (B, D, F, H, J and L) Corresponding fluorescence staining using DANIR 3b on the same brain sections.

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Figure 5. Brain uptake (A) and thyroid uptake (B) of (S)-[125I]5 and (R)-[125I]5 in normal mice (ICR, 5 weeks, male).

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Scheme 1. Reagents and conditions: a. t-BuOK, DMF, (R)-3-(-)- or (S)-3-(+)-chloropropane-1,2-diol, 120 °C, 48 h; b. TsCl, Et3N, CH2Cl2, r.t., 4 h; c. 4-iodophenol, K2CO3, DMF, 90 °C, 5 h; d. SnCl2.2H2O, HCl, EtOH, 110 °C, 4 h; e. (CH2O)n, NaBH3CN, HAc, r.t., 8 h; f. (Bu3Sn)2, (PPh3)4Pd, Et3N, toluene, 110 °C, 6 h; g. (1)[125I]NaI, HCl (1M), H2O2 (3%), r.t., 15 min; (2) NaHCO3.

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Table 1. Summary of the X-ray crystallographic data for compounds (S)-5 and (R)-5. Compound

(S)-5

(R)-5

Empirical formula

C16H17IO4

C16H17IO4

Formula weight (g/mol)

400.20

400.20

Tempreture (K)

103.6

102.1

Crystal system

monoclinic

monoclinic

Space group

P 1 21 1

P 1 21 1

Cell volume (Å3)

1514.06(7)

1519.13(12)

a (Å)

7.20433(19)

7.2126(3)

b (Å)

11.9715(4)

11.9794(5)

c (Å)

17.7871(4)

17.8131(9)

α (deg)

90

90

β (deg)

99.266(2)

99.239(5)

γ (deg)

90

90

Z

4

4

Calculated density (mg/m3)

1.756

1.750

Absorption coefficient (mm-1)

2.128

2.121

Final R indices [I > 2σ (I)]

R1 = 0.0272(5718)

R1 = 0.0307(5728)

wR2 = 0.0524(5930)

wR2 = 0.0902(5944)

-0.030(15)

-0.024(19)

Flack

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Table 2. Inhibition constants of optically pure diphenoxy derivatives for the binding of [125I]IMPY to Aβ1-42 aggregates. a

a

Compound

Ki (nM)

(S)-5

34.0 ± 2.7

(R)-5

25.0 ± 1.3

(S)-8

45.0 ± 13.0

(R)-8

15.8 ± 11.5

IMPY

25.3 ± 2.6

The Ki values were determined in three independent experiments (n = 3).

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For Table of Contents Use Only Title: Optically Pure Diphenoxy Derivatives as More Flexible Probes for β-Amyloid Plaques Authors: Jianhua Jia a, Jia Song a, Jiapei Dai b, Boli Liu a, Mengchao Cui a,*

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Optically Pure Diphenoxy Derivatives as More Flexible Probes for Î²

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