Design And Preparation Of Flexible Small Molecular Probes Targeting Aβ Protein In Brain Parenchyma And Blood Vessels Of Brain

Senile plaques（SPs）formed by β-amyloid（Aβ）deposited on nerve fibers are an important pathological feature of Alzheimer disease（AD）.Aβ deposited in the cerebral vascular wall especially in the arterial wall of cerebral cortex and small pia mater in 82 % 98 % patients with AD.The deposition of above Aβ occured in the early stage of AD,and its deposition amount was positively correlated with the severity and progress of the disease [1-4].Aβ protein deposited on nerve fibers and Aβ protein deposited on the blood vessel wall（Aβ protein deposited in brain parenchyma）can be used as a drug target for diagnosis of AD.Aβ deposited in the cerebral vascular wall,especially in the cerebral cortex and pia mater,is an important pathological feature of cerebral amyloid angiopathy（CAA）,and the deposition of Aβ in the vessel wall occurs in the early stage of CAA.Aβ protein deposited in the vessel wall can be used as a drug target for diagnosis of CAA.Small molecular probes targeting Aβ protein in the brain parenchyma currently have the following disadvantages:1.Most of the small molecular probes that target Aβ protein in the brain parenchyma have rigid planar structures.These probes have higher lipophilicity and more non-specific binding.2.Most small molecular probes that target Aβ protein in the brain parenchyma cannot distinguish Aβ plaques in different parts of the brain.The development of flexible small molecular probes with less lipotropy targeting Aβ protein in the brain parenchyma can reduce non-specific binding,improve signal-to-noise ratio,and contribute to the early diagnosis of AD.The development of flexible small molecular probes that selectively target Aβ proteins on the vessel wall provides early and specific assessment and quantitative assessment of CAA,and can assist in differential diagnosis of AD and CAA under the help of small molecular probes targeting Aβ protein in the brain parenchyma.This paper mainly explores the following two aspects:（1）Design and preparation of flexible small molecular probes targeting Aβ protein in brain parenchymaMethod: 1.The chemdraw software predicted the Log P values of compounds 1.4,1.5,1.14,1.15,1.16 and 1.17.2.Using p-nitrophenol as starting material,diphenylamino polyethylene glycol（PEG）small molecular compounds 1.4,1.5,1.14,1.15,1.16 and 1.17 which target Aβ protein in brain parenchyma were synthesized by substitution reaction and reduction reaction.The structure and molecular weight of the above materials were evaluated by ¹H NMR and MS.3.The affinity of final compounds 1.4,1.5,1.14,1.15,1.16 and 1.17 to Aβ aggregates was evaluated by competition binding assays.Result: 1.The Log P values for final compounds 1.4,1.5,1.14,1.15,1.16 and 1.17 were: 2.64,4.13,2.33,3.97,2.49 and 3.81,respectively.2.¹H NMR and MS for final compound 1.4:（600 MHz,CDCl₃）δ 6.92 – 6.87（m,2H）,6.85 – 6.82（m,2H）,6.81 – 6.78（m,2H）,6.67 – 6.63（m,2H）,4.23（s,4H）,3.77（s,3H）,3.44（s,2H）;260.1278（m / z）.¹H NMR and MS for final compound 1.5:（600 MHz,CDCl₃）δ 7.56（d,J = 8.6 Hz,2H）,6.79（d,J = 8.6 Hz,2H）,6.73（d,J = 8.6 Hz,2H）,6.65（d,J = 8.6 Hz,2H）,4.24（s,4H）,3.45（s,2H）;356.0143（m / z）.¹H NMR and MS for final compound 1.14:（600 MHz,CDCl₃）δ 6.89 – 6.84（m,2H）,6.84 – 6.80（m,2H）,6.79 – 6.75（m,2H）,6.67 – 6.63（m,2H）,4.09（dt,J = 13.3,4.9 Hz,4H）,3.91 – 3.86（m,4H）,3.76（s,3H）;304.1559（m / z）.¹H NMR and MS for final compound 1.15:（600 MHz,CDCl₃）δ 7.54（d,J = 8.9 Hz,2H）,6.76（d,J = 8.8 Hz,2H）,6.70（d,J = 8.9 Hz,2H）,6.63（d,J = 8.7 Hz,2H）,4.12 – 4.09（m,2H）,4.09 – 4.06（m,2H）,3.92 – 3.89（m,2H）,3.89 – 3.86（m,2H）,3.39（s,2H）;400.0424（m / z）.¹H NMR and MS for final compound 1.16:（600 MHz,CDCl₃）δ 6.85（d,J = 9.1 Hz,2H）,6.81（d,J = 9.1 Hz,2H）,6.75（d,J = 8.7 Hz,2H）,6.64（d,J = 8.7 Hz,2H）,4.06（dt,J = 15.5,4.8 Hz,4H）,3.83（dt,J = 9.6,4.8 Hz,4H）,3.76（s,3H）,3.74（s,4H）;348.1811（m / z）.¹H NMR and MS for final compound 1.17:（600 MHz,CDCl₃）δ 7.53（d,J = 8.6 Hz,2H）,6.75（d,J = 8.7 Hz,2H）,6.69（d,J = 8.6 Hz,2H）,6.62（d,J = 8.7 Hz,2H）,4.09 – 4.07（m,2H）,4.06 – 4.03（m,2H）,3.86 – 3.83（m,2H）,3.83 – 3.80（m,2H）,3.74（s,4H）,3.42（s,2H）;444.0684（m / z）.3.The affinity of final compounds 1.4,1.5,1.14,1.15,1.16 and 1.17 for Aβ aggregates in vitro was: 0.27 ± 0.70 μM,0.22 ± 0.11 μM,3.60 ± 2.67 μM,0.98 ± 0.30 μM,1.31 ± 0.89 μM and 1.06 ± 0.33 μM.Conclusion: 1.The Log P values for final compounds 1.4,1.5,1.14,1.15,1.16 and 1.17 were consistent with the desired requirements for small molecular probes targeting Aβ in brain parenchymal.2.Final compounds 1.4,1.5,1.14,1.15,1.16 and 1.17 were successfully synthesized and characterized by ¹H NMR and MS.3.Final compounds 1.4,1.5,1.14,1.15,1.16 and 1.17 had weak affinity for Aβ aggregates in vitro and still require further structural optimization.（2）Design and preparation of flexible small molecular probes for targeting Aβ protein in the cerebrovascular wallMethod: 1.2.1 reacted with the amino compounds 1.14,1.16 or 1.17 through addition and dehydration condensation reaction to form Schiff base derivatives 2.2,2.4 or 2.5.The Schiff base derivatives 2.2,2.4 and 2.5 were subjected to a reduction reaction to produce final product,namely benzyldiamine derivatives 2.6,2.8 and 2.9.The benzyl bromide compound 2.12 was synthesized by hydrolysis reaction,reduction reaction,and substitution reaction using methyl 2-fluoroterephthalate as a raw material.The benzyl bromide compound 2.12 and the amino compound 1.15 were subjected to substitution reaction to form final products,namely benzyl diamine derivative 2.7.The labeled precursor 2.15 was synthesized by hydrolysis,reduction and oxidation reaction using methyl 2-nitroterephthalate as raw material.The compound 2.16 synthesized by hydrolysis,reduction and substitution reaction was subjected to a substitution reaction with an amino compound 1.15 to form a labeled precursor 2.17.¹H NMR and MS evaluation of final products 2.6,2.7,2.8 and 2.9 structure and molecular weight.2.The affinity of final compounds 2.6,2.7,2.8 and 2.9 to Aβ aggregates was evaluated by competition binding assays.3.Labeled precursor 2.15 and ¹⁸F^-synthesized [¹⁸F]2.1,[¹⁸F]2.1 and amino compound 1.16 synthesized [¹⁸F]2.8 by reductive amination reaction.Or labeled precursor 2.17 with ¹⁸F^- synthesize [¹⁸F] 2.8 via SN2 reaction.The mixed solution after the reaction was separated and identified by high performance liquid chromatography（HPLC）.Result: 1.¹H NMR and MS for final compound 2.6:（600 MHz,CDCl₃）δ 7.33（t,J = 7.8 Hz,1H）,7.08（t,J = 7.7 Hz,2H）,6.86（d,J = 8.8 Hz,4H）,6.84 – 6.75（m,8H）,6.67 – 6.50（m,4H）,4.32（s,2H）,4.26（s,2H）,4.09（dt,J = 14.4,4.9 Hz,8H）,3.88（q,J = 6.7,6.2 Hz,8H）,3.76（s,6H）;727.3403（m / z）.¹H NMR and MS for final compound 2.7:（600 MHz,CDCl₃）δ 7.56 – 7.50（m,4H）,7.32（t,J = 7.7 Hz,1H）,7.10 – 7.05（m,2H）,6.81 – 6.75（m,4H）,6.71 – 6.66（m,4H）,6.57（dd,J = 24.7,8.8 Hz,4H）,4.32（s,2H）,4.26（s,2H）,4.09（dt,J = 22.7,4.7 Hz,8H）,3.88（dt,J = 19.3,4.8 Hz,8H）;919.1112（m / z）.¹H NMR and MS for final compound 2.8:（600 MHz,CDCl₃）δ 7.36（s,1H）,7.08（s,2H）,6.87 – 6.83（m,4H）,6.83 – 6.77（m,8H）,6.61（s,4H）,4.31（s,2H）,4.25（s,2H）,4.06（dt,J = 14.0,4.9 Hz,8H）,3.83（dt,J =8.8,5.0 Hz,8H）,3.75（s,6H）,3.74（s,8H）;815.4001（m / z）.¹H NMR and MS for final compound 2.9:（600 MHz,CDCl₃）δ 7.55 – 7.50（m,4H）,7.35（s,1H）,7.08（s,2H）,6.79（s,4H）,6.71 – 6.67（m,4H）,6.59（s,4H）,4.32（s,2H）,4.26（s,2H）,4.06（dt,J = 18.1,4.6 Hz,8H）,3.83（dt,J = 18.3,4.9 Hz,8H）,3.73（s,8H）;1007.1628（m / z）.2.The affinity of final compounds 2.6,2.7,2.8 and 2.9 for Aβ aggregates in vitro was: 1.86 ± 0.37 n M,2.15 ± 0.64 n M,1.61 ± 0.38 n M and 1.53 ± 0.20 n M,respectively.3.Retention time of stabilized fluorine compounds 2.1,2.4 and 2.8 by HPLC analysis was 5.355 min,27.582 min and 13.217 min.After the labeling reaction of the labeled precursor 2.15,the mixed solution was subjected to HPLC separation,and the peak retention time of the radiation signal was 2.549 min,5.466 min and 10.828 min.After the labeling reaction of the labeled precursor 2.17,the mixed solution was subjected to HPLC separation,and the peak retention time of the radiation signal was 5.130 min and 6.936 min.[¹⁸F]2.8 radiolabeled yield prepared by labeled the precursor 2.15 or 2.17: 0%.Conclusion: 1.The final products 2.6,2.7,2.8 and 2.9 were successfully synthesized and characterized by ¹H NMR and MS.2.The final products 2.6,2.7,2.8 and 2.9 had higher affinity for Aβ aggregates in vitro.3.The preparation conditions for [¹⁸F]2.8 are still to be explored.