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Design, Synthesis and Evaluation of Orally Available ClioquinolMoracin M Hybrids as Multi-Target-Directed Ligands for Cognitive Improvement in a Rat Model of Neurodegeneration in Alzheimer’s Disease Zhiren Wang, Yali Wang, Bo Wang, Wenrui Li, Ling Huang, and Xingshu Li J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01222 • Publication Date (Web): 16 Oct 2015 Downloaded from http://pubs.acs.org on October 21, 2015

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Design,

Synthesis

and

Evaluation

of

Orally

Available

Clioquinol-Moracin M Hybrids as Multi-Target-Directed Ligands for Cognitive Improvement in a Rat Model of Neurodegeneration in Alzheimer’s Disease Zhiren Wang, Yali Wang, Bo Wang, Wenrui Li, Ling Huang,* and Xingshu Li*

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China

ABSTRACT A novel series of clioquinol-moracin hybrids were designed and synthesized by fusing the pharmacophores of clioquinol and moracin M, and their activities as multi-target-directed ligands against Alzheimer's disease were evaluated. Biological activity results demonstrated that these hybrids possessed significant inhibitory activities against phosphodiesterase 4D (PDE4D) and Aβ aggregation as well as remarkable antioxidant effects and excellent blood-brain barrier permeability. The optimal compound, 18d, exhibited excellent PDE4D inhibitory potency (IC50 = 0.32 µM), significant antioxidant effects, appropriate biometal chelating functions, and interesting properties that modulated self- and metal-induced Aβ aggregation. Two-dimensional NMR studies revealed that 18d had significant interactions with Aβ1–42 at the R5, H6, H14, Q15 and F20 residues. Furthermore, this typical hybrid possessed preeminent neuroprotective effects against inflammation in microglial cells. Most

importantly,

oral

administration

of

18d•HCl

demonstrated

marked

improvements in cognitive and spatial memory in a rat model of Alzheimer’s disease and protected hippocampal neurons from necrosis.

ACS Paragon Plus Environment


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Introduction Alzheimer’s disease (AD) is one of the most dreadful and prevalent neurodegenerative diseases and affects approximately 44 million people worldwide.1 AD has been demonstrated to possess an extremely complex interconnected network of multiple factors and etiological hallmarks, including abnormal β-amyloid (Aβ) deposition and accumulation, low levels of acetylcholine, oxidative stress, neuroinflammation of the central nervous system (CNS) and dyshomeostasis of biometals.2-4 Thus, there has been a major growth in efforts to develop multi-target-directed ligands (MTDLs) for AD networks.5-14 The aberrant accumulation of misfolded Aβ peptides is a major pathological hallmark of AD.15 Aβ peptides can form oligomers, protofibrils, fibrils, and ultimately, insoluble plaques through self-assembly and hydrophobic interactions.16 In particular, the soluble low molecular weight aggregated Aβ species can cause damage to neuronal and

mitochondrial

functions and

lead

to

oxidative

stress

and

neuroinflammation.17 In addition, high levels and miscompartmentalization of metal ions such as Cu2+, Zn2+, Fe2+ and Fe3+ can readily bind to Aβ via three histidine residues, H6, H13 and H14, and facilitate Aβ aggregation.4, 18-21 Meanwhile, under physiological conditions, the interaction between redox-active metal ions and Aβ was demonstrated to generate reactive oxygen species (ROS) via Fenton-like reactions, which led to oxidative stress and, eventually, neuronal death in AD patients.4, 16, 21 Therefore, the modulation of metal–Aβ interactions as well as the metal distribution in the brain has become a promising approach to inhibit Aβ aggregation.16, 18, 21 The


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metal chelating agent, clioquinol (CQ, Fig. 1), and its second generation agent 8-hydroxyquinoline

ionophore,

PBT2

(Fig.

1),

demonstrated a

significant

improvement in cognition and memory by redistributing metal ions and decreasing plasma Aβ levels in Phase II clinical trials.22-27 Recently, the multi-target-directed metal-chelating agents M30, HLA20, tacrine-8-hydroxyquinoline hybrids, L2-b and J2326 were developed and studies demonstrated significant inhibition of Aβ aggregation in vitro or improvements in cognition in vivo in mouse models (Fig. 1).28-35

Figure 1. Several reported multi-target-directed metal-chelating agents, PDE4D inhibitors and Moracin M.

Phosphodiesterase 4D (PDE4D), one of the primary enzymes that hydrolyzes intracellular cyclic adenosine monophosphate (cAMP) to control neuronal stimulation-induced signal transduction, is widely expressed in the hippocampus and cortex.36-38 Gene deletion studies have demonstrated that a lack of PDE4D in mice significantly enhanced long-term hippocampal-dependent memory.39, 40 More recent studies clearly indicate that PDE4D plays a leading role among the PDE subtypes that mediate memory formation.41 Rolipram, a PDE4D inhibitor, has beneficial effects on hippocampal-dependent memory tasks, but PDE4D-deficient mice did not show altered memory with the administration of rolipram, which further demonstrated the



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crucial role of PDE4D in memory consolidation.40 The newly developed PDE4D inhibitor GEBR-7b enhanced spatial and object memory performance in mice and rats (Fig. 1).42-44 In addition, MK-0952 (Fig. 1), another PDE4 inhibitor that enhanced cognition in preclinical studies, prompted clinical trials in patients with mild-to-moderate AD from phase II clinical studies.45,

46

Moracin M is a natural

product that is isolated from the root bark of Morus alba L, and has a broad scope of biological activity such as antioxidant and anti-inflammatory activity, and in particular, the inhibition of PDE4D, which is directly related to AD pathology.47, 48 Considering that both inflammation and biometal dyshomeostasis are important etiological hallmarks of AD, we fused the pharmacophores of CQ and moracin M into one molecule without major structural modifications (Fig. 2). We then evaluated their biological activities with regards to the modulation of Aβ aggregation and blood-brain barrier (PAMPA-BBB) permeability, the inhibition of human PDE4D and the prevention of neuroinflammation. Furthermore, an optimal compound, 18d•HCl, was further assessed in a rat model of AD by conducting behavioral performance and histopathological studies of hippocampal neurons.

Figure 2. Multi-target-directed design strategy by fusing the pharmacophore moieties of CQ and Moracin M.

Results and Discussion


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Chemistry Syntheses of the Wittig reagents (4a-4c) are described in Scheme 1. Compound 2, which was prepared as previously described,49 was reacted with triethyl phosphite to give the key intermediate 3a. Compounds 3b and 3c were obtained by treating 3a with NaClO and I2, respectively. Then, the reaction of compounds 3a-3c with methyl iodide produced the Wittig reagents 4a-4c in good yields. The syntheses of the target compounds 16a-16g, 17a-17d, 17f and 18c-18e are described in Scheme 2. First, the hydroxyl groups of the corresponding salicylic aldehydes were protected by reacting them with chloro(methoxy)methane to give compounds 6a-6g. The subsequent reactions of compounds 6a-6g with Wittig reagents (4a-4c) provided compounds 7a-7g, 8a-8d, 8f and 9c-9e, respectively, which were treated with HCl (37%) in CH3OH to provide compounds 10a-10g, 11a-11d, 11f and 12c-12e. Finally, the target compounds were produced by cyclizing the phenol intermediates with I2, followed by deprotection of the methyl groups with BBr3. The hydrochloride of 18d•HCl was obtained by treating target compound 18d with HCl gas in ethyl acetate. The syntheses of the target compounds 16h and 16i are presented in Scheme 3. The starting material 8-hydroxyquinaldine (3d) was first protected using methyl iodide to generate 4d, followed by condensation with corresponding salicylic aldehydes to produce 7h and 7i. The interesterification of 7h and 7i in the presence of CH3OH and K2CO3 gave the intermediates 10h and 10i, which were treated with I2 to yield 13h and 13i. Finally, the target compounds 16h and 16i were obtained by deprotecting the methyl groups with BBr3.



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Scheme 1. Synthesis of 2, 3a-3c, 4a-4c. Reagents and conditions: a. HCHO, HCl(g), rt, 72 h; b.P(OEt)3, 110 °C, 12 h; c. DMF, KOH, CH3I, rt, 12 h; d. H2O, KOH, NaClO, rt, 2 h; e. H2O, KOH, KI, I2, rt, 6 h.

Scheme 2. Synthesis of 6a-6g, 7a-7g, 8a-8d, 8f, 9c-9e, 10a-10g, 11a-11d, 11f, 12c-12e, 13a-13g, 14a-14d, 14f,

15c-15e and 16a-16g, 17a-17d, 17f, 18c-18e, 18d•HCl. Reagents and conditions: a. CH2Cl2, DIPEA, ClMOM, rt, 5 h; b. DMF, NaH, rt, 4 h; c. CH3OH, HCl (37%), rt, 2 h; d. THF, K2CO3, I2, rt, 16 h; e. anhydrous CH2Cl2, BBr3, -78 °C, 12 h. f. EtOAc, HCl (g), rt, 12 h.


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Scheme 3. Synthesis of 4d, 7h-7i, 10h-10i, 13h-13i, and 16h-16i. Reagents and conditions: a. DMF, K2CO3, CH3I, rt, 12 h; b. acetic anhydride, 150 °C, 12 h; c. CH3OH, K2CO3, rt, 1 h; d. THF, K2CO3, I2, rt, 16 h; e. anhydrous CH2Cl2, BBr3, -78 °C, 12 h.

Inhibition of PDE4D The inhibition of PDE4D2 by the target compounds was determined using a previously described method, with rolipram and moracin M as the reference compounds.50, 51 The results in Table 1 indicated that most of the target compounds exhibited more potent inhibitory activities against PDE4D2 compared to moracin M. Among the three series of compounds 16 (X = H), 17 (X = Cl) and 18 (X = I), 16e (IC50 = 1.21 µM) and 18e (IC50 = 0.23 µM), which bear a phenolic dihydroxyl group on the benzofuran moiety, demonstrated better activities than the other analogs in the series. The removal of the phenolic hydroxyl group on the benzofuran moiety in compounds 16a (IC50 > 10 µM), 16c (> 10 µM) and 18c (0.89 µM) reduced the potency of these compounds, indicating that the phenolic hydroxyl group in the benzofuran moiety was crucial. Compound 16h (8.86 µM) and 16i (2.01 µM), with the benzofuran moiety on the pyridine ring, also exhibited moderate activities. The structure-activity relationship study revealed that the phenolic hydroxyl group on the



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R3 position of the benzofuran moiety was more favorable for the activity than the other positions. Compounds 16d (IC50 = 2.31 µM), 17d (0.96 µM) and 18d (0.32 µM), which bear a phenolic hydroxyl group on the R3 position, exhibited more potent activity than compounds 16b (IC50 > 10 µM), 16c (> 10 µM), 17b (2.08 µM), 17c (1.07 µM) and 18c (0.89 µM), respectively. Compounds 16f (3.67 µM) and 17f (2.92 µM), with a methyl group on the R5 position of the benzofuran moiety, showed lower activity than the parent compounds 16d and 17d, respectively. The presence of a halogen, particularly iodine, on the oxine moiety was demonstrated to be indispensable for the inhibitory activity. Compounds 16d, 17d and 18d, with or without the chlorine and iodine group in the oxine moiety, exhibited IC50 values of 2.31, 0.96 and 0.32 µM, respectively. It is worth noting that compound 16g, which contains a diiodine on the benzofuran moiety, displayed IC50 values of 0.75 µM. Table 1. Inhibition of PDE4D and Aβ Self-aggregation, Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents), Permeability (Pe×10-6 cm s-1) Determined by the PAMPA-BBB Assay for Target Compounds and Predicted Penetration of the CNS.

Aβ1-42 PDE4D2 Comd.

R

self-induced

Pe c

X

ORAC IC50 (µM)a

Pred. (×10-6 cm s-1)d

aggregation (%Inhib.)b

16a

H

H

>10

12.4 ± 1.5

0.5 ± 0.2


4.3 ± 0.4

CNS±

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16b

R1=OH

H

>10

35.1 ± 2.1

3.3 ± 0.1

14.5 ± 0.5

CNS+

16c

R2=OH

H

>10

46.3 ± 5.3

3.5 ± 0.1

15.8 ± 0.7

CNS+

16d

R3=OH

H

2.31 ± 0.32

57.7 ± 7.8

3.9 ± 0.2

15.5 ± 0.1

CNS+

16e

R2=R4=OH

H

1.21 ± 0.07

61.2 ± 6.1

3.1 ± 0.3

2.6 ± 0.1

CNS±

H

3.67 ± 0.43

53.1 ± 3.4

4.4 ± 0.1

16.8 ± 0.7

CNS+

R3=OH, 16f 5

R =Me 16g

R1=R3=I

H

0.75 ± 0.05

13.2 ± 3.2

0.2 ± 0.1

3.6 ± 0.2

CNS±

16h

R=H

-

8.86 ± 0.39

33.8 ± 0.9

4.3 ± 0.3

15.8 ± 0.7

CNS+

16i

R=OH

-

2.01 ± 0.23

48.7 ± 9.8

3.1 ± 0.3

13.3 ± 0.2

CNS+

17a

H

Cl

3.05 ± 0.36

19.2 ± 4.4

0.3 ± 0.1

2.6 ± 0.1

CNS±

17b

R1=OH

Cl

2.08 ± 0.12

48.1 ± 2.6

2.9 ± 0.3

16.4 ± 0.1

CNS+

17c

R2=OH

Cl

1.07 ± 0.08

52.0 ± 4.3

2.4 ± 0.1

17.9 ± 1.5

CNS+

17d

R3=OH

Cl

0.96 ± 0.11

59.1 ± 5.1

2.9 ± 0.3

21.4 ± 2.6

CNS+

Cl

2.92 ± 0.35

51.6 ± 3.9

3.6 ± 0.2

3.5 ± 0.4

CNS±

R3=OH, 17f 5

R =Me 18c

R2=OH

I

0.89 ± 0.09

63.5 ± 8.7

3.5 ± 0.1

16.6 ± 0.9

CNS+

18d

R3=OH

I

0.32 ± 0.02

67.5 ± 1.9

3.6 ± 0.2

19.1 ± 0.8

CNS+

18e

R2=R4=OH

I

0.23 ± 0.01

69.2 ± 8.3

3.1 ± 0.2

0.8 ± 0.1

CNS–

rolipram

-

0.61 ± 0.03

-e

-e

-e

-e

moracin M

-

2.91 ± 0.16

43.1 ± 5.6

2.5 ± 0.2

-e

-e

clioquinol

-

>10

4.7 × 10-6 cm s-1 could cross the blood-brain barrier by passive diffusion. e n.t. = not tested.

Inhibition of Aβ Self-Aggregation The inhibitory activities of the target compounds on Aβ1–42 self-induced aggregation were first determined by a thioflavin T (ThT) fluorescence assay using curcumin and resveratrol as reference compounds (Table 1). None of the tested compounds exhibited significant fluorescence signals under the experimental conditions. Most of the target compounds significantly inhibited Aβ1–42 self-induced aggregation at the tested concentrations. A structure-activity relationship analysis indicated that a relationship between the position of phenolic hydroxyl groups on the benzofuran moieties and PDE4D inhibitory activity was also observed for Aβ1−42 inhibitory activity. Compounds 16d, 17d and 18d, which have a phenolic hydroxyl group at the R3 position, exhibited much higher inhibitory activities (57.7%, 59.1%


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and 67.5%, respectively) than moracin M (43.1%). Comparatively, compounds 16a and 17a, which do not have phenolic hydroxyl groups on the benzofuran moieties, exhibited only 12.4% and 19.2% inhibition, respectively. The types of halogens in the oxine moieties were also important for proper biological activity; compounds 18c-18e, which have iodine on the oxine moieties, exhibited 63.5%, 67.5%, and 69.2% inhibition, respectively, and were more potent than their chloro- or non-substituted counterparts (16c-16e: 46.3%, 57.7% and 61.2%; 17c-17d: 52.0% and 59.1%). 2-D NMR Study of the Direct Interaction between Compound 18d and Aβ in Solution Two-dimensional (2-D) Translational Relaxation Optimized Spectroscopy (TROSY) 1H-15N Heteronuclear Single Quantum Correlation (HSQC) NMR was performed to investigate the direct atomic-level interaction between compound 18d and

15

N-labeled Aβ1-42 (Fig. 3A). When compound 18d was titrated into

15

N-labeled

Aβ1-42, significant chemical shift perturbations (CSP) and broadening of some of the peaks for the Aβ1–42 residues were observed, particularly for Aβ1–42 residues R5, H6, H14, Q15 and F20 (Fig. 3A and 3B). Because these residues are part of and in close contact with the putative metal coordination site of Aβ (H6, H13, and H14), this finding demonstrated that compound 18d was in close proximity to the metal binding residues. Moreover, compound 18d not only possessed a strong targeting ability for Aβ but also may chelated the metal ions surrounded by Aβ (Fig. 3C).19, 20, 28, 31 These results were similar to those of previously reported meta-chelating compounds with bifunctionality in metal chelation and Aβ interaction, which demonstrates the



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significantly ability of this compound to modulate the formation of meta-induced Aβ aggregates and promote disaggregation.28,

29, 31, 52, 53

Substantial chemical shift

perturbations were observed when 5.0 equivalents of compound 18d was added into 15

N-labeled Aβ1-42, which were attributed to a possible change in the conformation of

Aβ1–42 (Fig. 3A). Small chemical shifts were detected for the methionine 35 (M35) residue, demonstrating that compound 18d did not induce Aβ1–42 oxidation.54

Figure 3. 2-D NMR study the interactions of 18d with Aβ1–42 in solution. (A) 2D 1H-15N TROSY-HSQC NMR spectra of 18d-titrated Aβ1–42 (freshly dissolved Aβ1–42 (50 µM) in 5 mM phosphate buffer, 0.5 mM Na2EDTA, and


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0.05 mM NaN3, 7% D2O (v/v), pH 7.5, 950 MHz). (B) Chemical shift perturbations for Aβ1–42 residues in the presence of 18d (Aβ1–42 : 18d = 1 : 2). * Denotes absent or overlapping signals. (C) Residues with the largest changes in chemical shifts mapped onto the structure of Aβ1–42 (PDB 1Z0Q).

Free Oxygen Radical Scavenging Ability To evaluate the antioxidant activities of the target compounds, the free oxygen radical Absorbance Capacity (ORAC) assay was performed and trolox and moracin M were used as the reference compounds.55-57 The results presented in table 1 showed that most of the target compounds demonstrated a higher antioxidant ability compared to moracin M and clioquinol. As expected, the pharmacophore for ORAC of the target compounds was primarily rooted in the benzofuran moiety. The structure-activity relationship between the position of phenolic hydroxyl group on benzofuran moiety and the PDE4D inhibitory activity was also observed in the antioxidant activity assay. Compounds possessing a phenolic hydroxyl group on the benzofuran moiety (16b-16f, 16h-16i, 17b-17f, 18c-18e) exhibited significantly higher ORAC values (4.4-2.4) than those lacking the phenolic hydroxyl group (16a, 0.5; 17a, 0.3) or with a replacement of the phenolic hydroxyl with halogen (16g, 0.2). Notably, compounds 16f and 17f, which contain a methyl group on the R5 position of the benzofuran moiety, provided the best ORAC values (4.4 and 3.6, respectively). The presence of a halogen, particularly chlorine, on the oxine moiety was slightly unfavorable for antioxidant activity. Compounds 16d, 17d and 18d, without or with a chlorine or iodine in the oxine moiety, showed ORAC values of 3.9, 2.9 and 3.6, respectively. Blood-Brain Barrier Permeability Assay



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The parallel artificial membrane permeability (PAMPA) assay, which can successfully predict passive BBB permeation, was used to determine the BBB penetration of the target compounds.55-57 From the results presented in Table 1, it could be seen that most of the target compounds exhibited significant BBB permeability. Among them, compounds 16b-16d, 17b-17d and 18c-18d, which bear a single phenolic hydroxyl group on the benzofuran moiety, exhibited much better BBB permeability than compounds without or with di-phenolic hydroxyl groups, such as 16a, 16e, 16g, 17a and 18e, which exhibited questionable BBB permeability. As brain penetration is the first requirement for developing anti-AD drugs, compound 18d was chosen for further evaluation. Neuroprotective

Effects

of

the

Compounds

against

LPS-Stimulated

Inflammation in BV2 Microglial Cells Neuroinflammation plays a crucial role in causing neuronal death and damage, which in turn leads to neurodegenerative diseases such as AD, PD and multiple sclerosis.58 The activation of brain microglial cells in the CNS and the subsequent excess production of inflammatory mediators, such as nitric oxide (NO), may result in uncontrolled neuroinflammation in neurodegenerative diseases.59 Therefore, by inhibiting the production of inflammatory mediators NO in microglial cells, antineuroinflammatory therapy could delay or halt the disease progression prior to irreversible

damage

and

the

occurrence

of

clinical

symptoms.

The

antineuroinflammatory properties of target compound 18d were determined by the Griess assay, which detects the suppression of NO production following LPS-induced


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inflammatory events in BV2 microglia cells using quercetin as a positive control. At first, all of the compounds promoted more than 95% cellular viability at the tested concentrations,

as

determined

by

the

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. As the results in Fig. 4 and Table 2 indicated, compound 18d exhibited significantly higher inhibition (EC50 = 1.5 µM) of NO production in the LPS-stimulated BV2 cells than moracin M (15.1 µM) and clioquinol (> 30 µM). Therefore, this result further demonstrated that the unique structure of compound 18d resulted in improved antineuroinflammatory activities compared to the leading compounds moracin M and clioquinol.

Figure 4. Nitric oxide production inhibiting effect of control (A) and 18d (B).

Table 2. The EC50 Values for Inhibition Effects of NO Production in BV2 Cell.

a

Compound

EC50 (µM) a

Compound

EC50 (µM) a

quercetin

11.0 ± 1.0

clioquinol

> 30

moracin M

15.1 ± 0.8

18d

1.50 ± 0.2

Data are expressed as the mean ± SD at least three independent experiments.

Metal-Chelating Properties of 18d



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Figure 5. (A) UV absosobance spectrum of CQ (50 µM) alone or in the presence of CuSO4 (50 µM), ZnCl2 (50 µM) FeSO4 (50 µM) or FeCl3 (50 µM) in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). (B). Determination of the stoichiometry of complex Cu2+-CQ by Job’s method. (C) UV absosobance spectrum of compound 18d (50 µM)

alone or in the presence of CuSO4 (50 µM), ZnCl2 (50 µM) FeSO4 (50 µM) or FeCl3 (50 µM) in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). (D) UV-vis titration of compound 18d (50 µM) with Cu2+ in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) at room temperature. The concentration of Cu2+ varied from 0 to 50 µM. A breakpoint was observed at 0.5:1 ratio. (E) high-resolution mass spectroscopy of the Cu2+-18d complex in

solution.


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A UV-visible spectroscopy assay was performed to evaluate the ability of the target compounds to chelate biometals such as Cu2+, Zn2+, Fe2+ and Fe3+.28, 55, 57, 60, 61 The results presented in Fig. 5A and 5C show that similar to parent compound CQ (Fig. 5A), new optical bands were detected at 456 and 426 nm after the addition of CuSO4 or ZnCl2 to the solution of compound 18d (Fig. 5C), which demonstrated the production of the corresponding complex via metal chelation. Although there was no obvious optical shift with the addition of FeSO4 or FeCl3, the dramatic increase in absorption indicated a possible interaction between these biometals and the ligand. The results of moracin M indicated that it chelated with Cu2+, but no optical bands shifted in the presence of Zn2+, Fe2+and Fe3+(Fig. S1). Following the absorption at 456 nm, a series of UV-vis spectra were collected of compound 18d titrated with Cu2+, and the isosbestic point demonstrated a 1:2 Cu2+/ligand molar ratio for the unique Cu2+-18d complex shown in Fig. 5D. This result was in accordance with the stoichiometry of the Cu2+-CQ complex (break at a mole fraction of 0.659, indicating 1:2 Cu2+/ligand), which was determined by Job’s method (Fig. 5B). To further confirm the components of the complex, high-resolution mass spectroscopy was performed in solution, and the results in Fig. 5E show the presence of [(18d)2Cu – H]– (calculated: 856.8452, detected: 856.8441) and [(18d)2Cu + Cl]– (calculated: 903.8239, detected: 903.8224), which again indicated a 1:2 Cu2+/ligand molar ratio in the Cu2+-18d complex. Modulation of Self- and Metal-Induced Aβ Aggregation and Disaggregation of Aβ aggregates by Compound 18d



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Figure 6. Experiments of inhibition self-induced Aβ1–42 aggregation and disaggregation of self-induced Aβ1–42 aggregates in the presence of 18d. (a and b) Top: Scheme for the inhibition or disaggregation experiments. Bottom:

The fluorescence intensity of the ThT binding assay, data are expressed as the mean ± SD at least three independent experiments. Statistical significance was analyzed by ANOVA: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (c and d) TEM image analysis of the inhibition of Aβ1–42 aggregation and disaggregation of self-induced Aβ1–42 aggregates. Serial number: (1) fresh Aβ1–42, (2) Aβ1–42 alone, (3) Aβ1–42 + moracin M, (4) Aβ1–42 + CQ, (5) Aβ1–42 + 18d. Experimental conditions: Aβ1–42 (25 µM); compound/Aβ1-42 = 1/1; PBS (50 mM); pH 7.4; 37 °C.

The regulation of self-induced Aβ aggregation and the disassembly of preformed Aβ1-42 aggregates by compound 18d were studied using the ThT assay, and transmission electron microscopy (TEM) was then performed to determine the morphological changes in the Aβ species (Fig. 6). The ThT assay showed that compound 18d inhibited 86.3 ± 4.1% of the self-induced Aβ1-42 aggregation and disassembled 83.9 ± 3.6% of the preformed Aβ1-42 aggregates (Fig. 6a and 6b), which


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were significantly higher than CQ (20.2 ± 7.2% and 13.5 ± 3.2%, respectively, p < 0.001) and moracin M (62.3 ± 5.7% and 60.4 ± 5.3%, respectively, p < 0.05 and 0.01). In accord with the ThT assay, fewer and shorter Aβ1-42 aggregates (Fig. 6c and 6d, sequence 5) were observed after compound 18d was added to the samples compared to moracin M (Fig. 6c and 6d, sequence 3) and CQ (Fig. 6c and 6d, sequence 4). These results strongly indicated 18d could effectively control self-induced Aβ aggregation and disassemble the preformed self-induced Aβ aggregates, which should mainly be derived from its overall framework rather than the individual moieties.

Figure 7. Experiments of inhibition Cu2+-induced Aβ1–42 aggregation and disaggregation of Cu2+-induced Aβ1–42 aggregates in the presence of 18d. (a and b) Top: Scheme for the inhibition or disaggregation experiments. Bottom:

The fluorescence intensity of the ThT binding assay, data are expressed as the mean ± SD at least three independent experiments. Statistical significance was analyzed by ANOVA: ∗ p < 0.01, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (c and d) TEM images analysis of the inhibition of Cu2+-induced Aβ1–42 aggregation and disaggregation of Cu2+-induced Aβ1–42 aggregates. Serial number: (1) fresh Aβ1–42, (2) Aβ1–42 alone, (3) Aβ1–42 + Cu2+, (4) Aβ1–42 + Cu2+ + CQ, (5) Aβ1–42 + Cu2+ + 18d, (6) Aβ1–42 + Cu2+ + moracin M. Experimental conditions: Aβ1–42 (25 µM);



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chelator/Aβ1-42/Cu2+ = 2/1/1; HEPES (20 µM) and NaCl (150 µM); pH 6.6; 37 °C.

In addition, compound 18d also showed remarkable effects on the modulation of Cu2+-induced Aβ aggregation (Fig. 7). Excellent Cu2+-induced Aβ aggregation inhibition (80.4 ± 5.5%) was obtained compared to moracin M and CQ (50.8 ± 4.8% and 64.4 ± 5.0%, respectively, p < 0.01,). Similarly, 81.0 ± 5.6% of the of the preformed Cu(II)-induced aggregates were disaggregated, which was much better than moracin M and CQ (37.3 ± 8.7% and 37.6 ± 5.5%, respectively, p < 0.01). The TEM assay showed that the Cu2+-treated sample of fresh Aβ produced more fibrils than did the non-treated sample (Fig. 7c, sequence 2 and 3). When compound 18d and Cu2+ were incubated with Aβ, fewer Aβ fibrils were detected (Fig. 7c, sequence 5). Fig. 7d shows the TEM assay results for disaggregation in the presence of compound 18d, moracin M and CQ. As indicated, compound 18d also exhibited stronger disaggregation activity on the preformed Cu(II)-induced Aβ fibrils. 18d and CQ possess similar metal binding properties, but a 2D 1H-15N TROSY-HSQC NMR demonstrated that the CQ scaffold had poor interactions with Aβ residues.28 Consequently, the improved effects of 18d on the modulation of Cu(II)-induced Aβ1−42 aggregation could be attributed to the introduction of a benzofuran moiety to CQ, which may have increased the interactions between 18d and the metal coordination residues (especially H6 and H14, Fig. 3) of Aβ1−42. Overall, because of its unique structure, compound 18d could significantly modulate Cu(II)-induced Aβ1−42 aggregation and disassembled the preformed Cu(II)-induced aggregates into smaller, amorphous conformations.


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Metabolic Stability The metabolic stability of compound 18d•HCl in rat liver microsomes was examined and compared with donepezil and CQ using testosterone as a positive control. As shown in Table 3, donepezil, CQ and testosterone had a T1/2 of 73.3, 34.8 and 2.1 min, respectively, in liver microsomes; these values were consistent with previous reports and internal validation data.62,

63

Comparatively, 18d•HCl

demonstrated significant stability after incubation with rat liver microsomes (T1/2 = 293.8 min) under the same conditions. These data suggest that compound 18d•HCl possesses considerably greater metabolic stability in vitro compared with the reference compounds. Table 3. Metabolic Stability of 18d•HCl in Liver Microsomes of SD Rat.

a

compound

k (min-1)

T1/2 (min) a

testosterone b

0.31513 ± 0.04378

2.1 ± 0.3

donepezil c

0.00896 ± 0.00033

73.3 ± 2.6

CQ

0.01988 ± 0.00178

34.8 ± 3.1

18d•HCl

0.00236 ± 0.00008

293.8 ± 7.2

Results are expressed as the mean ± SD of at least three independent experiments performed in triplicate. b, c The

positive control (testosterone) and the compound donepezil exhibited metabolic stability that was consistent with the literature and internal validation data.62, 63

Compound 18d•HCl Demonstrated no Acute Toxicity Twenty KM mice (KM mice, which are common closed colony mice and most widely used in biomedical research in China) were randomly allocated into 4 groups,



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and the test compound 18d•HCl was given in single oral doses of 0, 677, 1333, or 2000 mg/kg. After administration of the compound, mice were monitored continuously for the first 4 h for any abnormal behavior and mortality changes, intermittently for the next 24 h, and occasionally thereafter for 14 days to monitor the onset of any delayed effects. During the experimental period, no acute toxicity such as mortality, significant abnormal changes in water or food consumption or body weight were observed. Furthermore, all mice were sacrificed on the 14th day after drug administration, and no damage to the heart, liver or kidneys was macroscopically detected. Overall, compound 18d•HCl was non-toxic and well tolerated at doses up to 2000 mg/kg. Cognitive and Memory Improvement in a Rat Model of AD

Figure 8. (A) The mean daily body weight profile of each group rats during drug administration period (n = 11).

(B) The escape latency time of each group was counted every day during the period of training trial (mean ± SD, n

= 11). (C) The representative tracks of the rats in the Morris water maze during the training trial period. The

location of the platform and effective region (twofold diameter of the platform) were represented as a blue and


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bright green circle, respectively.

The learning and memory assessments were performed using the Morris water maze by administering compound 18d•HCl to the Aβ-induced AD rat, which suffers from learning and memory dysfunction, with clioquinol and donepezil as the positive control treatments. Fifty-five male Wistar rats (270-300 g) were randomly allocated into 5 groups (n = 11 for each group): sham, model, donepezil (2 mg/kg/day), CQ (30 mg/kg/day) and compound 18d•HCl (30 mg/kg/day).22, 23 The rats for model, CQ, donepezil and compound 18d•HCl groups received intrahippocampal injections of an aggregated Aβ solution, while the sham group of rats were injected with saline. After the surgery, the rats in each group were administered the corresponding drug dosage in a 0.5% CMC-Na solution (4 mL/kg) by intragastric infusion throughout the entire assay (a blank 0.5% CMC-Na solution was used for the sham and model groups). Twenty-six days later, behavioral performance was evaluated using the Morris water maze task, which demanded incremental learning of the location of a fixed, hidden platform throughout the training period (5 consecutive days). The results presented in Fig. 8 showed that the administration of compound 18d•HCl (Fig. 8A) during the drug treatment period (32 consecutive days) did not influence the mean daily body weight profile of the rats, and, in particular, it did not cause any adverse or abnormal events (such as emesis-like or diarrhea behavior) or affect the survival. This demonstrated that long-term administration of compound 18d•HCl is safe at doses of 30 mg/kg/day. During the training trials (Fig. 8B), the mean escape latency and searching



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distance for the rats in each group declined progressively; however, the model rats normally spent more time and required more distance to find the hidden platform, which was located in the center of the first quadrant (Fig. 8B and 8C).

Figure 9. (A) Effect of memory retention on the spatial probe trial (with the platform removed from the pool) in

the water maze test. The results were expressed as the means ± SD (n = 11). Statistical significance was analyzed by two-way ANOVA: ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus the model and compound 18d•HCl groups. (1) Number of virtual platform (the original platform location) crossings. (2) The average swimming speed

for the rats. (3) The swimming path length in the virtual platform test. (4) The time spent in the virtual platform

quadrant. (5) The swimming path length in the effective region (two-fold diameter of the platform). (6) The time

spent in the effective region. (B) The representative tracks of the rats in Morris water maze during the spatial probe

trial period. The location of the platform and the effective region (twofold diameter of the platform) were

represented as a blue and bright green circle, respectively.

Twenty-four hours after the last training trial, memory retention was assessed by


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performing a spatial probe trial, with the platform removed from the pool and the two-fold diameter of the platform was set as the effective region. (The statistical significance of each was analyzed by ANOVA versus the model and compound 18d•HCl groups.) The results in Fig. 9 indicated that the sham group had significantly higher numbers of virtual platform (the original platform location) crossings (5.0 ± 0.8, p < 0.001) compared to the model group rats (2.1 ± 0.9), which strongly suggested that intrahippocampal injection of Aβ led to a spatial memory deficiency in the rats (Fig. 9A1). The numbers of virtual platform crossings for the rats that were administered donepezil (3.9 ± 1.1, p < 0.01), CQ (4.0 ± 0.9, p < 0.01) and compound 18d•HCl (6.0 ± 1.7, p < 0.001) were remarkably improved compared to the model group. Interestingly, the rats treated with compound 18d•HCl (6.0 ± 1.7) demonstrated a more favorable amelioration of the cognitive and memory functions compared to the donepezil (3.9 ± 1.1, p < 0.01) and CQ (4.0 ± 0.9, p < 0.01) groups (Fig. 9A1). Meanwhile, the average swimming speed for each group of rats was virtually equivalent, which further demonstrated that the long-term uptake of compound 18d•HCl did not impair the animals’ motility and exploratory activities (Fig. 9A2, p > 0.05, sham = 20.3 ± 2.9, model = 21.0 ± 3.1, donepezil = 21.9 ± 4.1, CQ = 22.9 ± 2.0 and compound 18d•HCl = 22.2 ± 4.0 cm/s). On one hand, the swimming path length in the virtual platform location of the compound 18d•HCl (33.4 ± 18.1, p < 0.001, p < 0.05 and p < 0.01) and sham (25.7 ± 8.0, p < 0.01) groups were longer than the model (10.8 ± 5.4), donepezil (25.0 ± 11.5) and CQ (19.9 ± 2.1) groups (Fig. 9A3). On the other hand (Fig. 9A4), the rats treated with compound



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18d•HCl (1.9 ± 1.0, p < 0.05) spent more time in the virtual platform location than the model rats (0.9 ± 0.6). These results further revealed that administration of 18d•HCl led to a substantial improvement of the conventional reference spatial memory and cognitive abilities compared to the CQ-treated, donepezil-treated or untreated littermates. Similarly, the sham group (102.6 ± 6.7, p < 0.05), as well as the compound 18d•HCl group (121.3 ± 45.4, p < 0.001 and p < 0.05), demonstrated an obviously longer swimming path length in the effective region than the model (72.1 ± 6.0) and donepezil (92.6 ± 17.2) groups (Fig. 9A5). The time spent in the effective region for the sham (5.7 ± 1.2, p < 0.01) and compound 18d•HCl (7.1 ± 2.7, p < 0.001, p < 0.01 and p < 0.05) groups were significantly difference from the model (3.3 ± 1.1), donepezil (5.0 ± 0.9) and CQ (5.1 ± 0.9) groups (Fig. 9A6), which further suggested that the compound 18d•HCl-treated rats, unlike the rats in the model, donepezil or CQ groups, formed a more clear and definite spatial preference for the correct quadrant of the platform (Fig. 9B). Taken together, these overall behavioral performance observations and results demonstrated that compound 18d•HCl can markedly improve spatial memory and cognition compared to CQ and donepezil at the tested dosage. Histopathological Studies in the Hippocampus


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Figure 10. Histopathological changes in rat hippocampal neurons (HE staining × 200, n = 11). CA1, CA2, CA3

and DG indicate the CA1, CA2, CA3 and Dentate Gyrus (DG) regions of the hippocampus. Representative 200

×magnification images of HE-stained hippocampus for each group are shown. The rats in the sham and compound

18d•HCl groups did not demonstrate histopathological abnormalities or neuronal loss; the hippocampal neurons

were clear and intact, and the cells were properly aligned. In the model group, the neurons were loosely arranged,

with a condensed cytoplasm, an irregular shape and a pyknotic nucleus. In the CQ and donepezil groups, the

damage was less than the model group, but greater than the sham and compound 18d•HCl groups.

The histopathological changes of the hippocampal neurons were evaluated by hematoxylin and eosin (HE) staining, which demonstrated significant neuronal abnormalities in the hippocampus of the rats in the model group (Fig. 10). The pyramidal neurons in the CA1, CA2, CA3 and dentate gyrus (DG) regions of the hippocampus were disintegrated, with remarkable nuclear pyknosis, neuronal



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shrinkage, an irregular shape and more darkly stained cells, which indicated a necrotic morphology. In contrast, the hippocampal neurons from the sham group were clear and intact, and the cells were properly aligned. In the compound 18d•HCl-treated group, no significant neuronal abnormalities were detected in the hippocampus; the pyramidal cells in each region were arranged neatly and tightly, the nucleolus was visible, the boundary between the cytoplasm and nucleus was clear, and no cell loss was found, which strongly indicated that compound 18d•HCl significantly protected the hippocampal neurons from damage and death. Additionally, the cells in the donepezil and CQ groups possessed a better morphology than the model group, but not as good as the sham and compound 18d•HCl groups. The denatured cell index (DCI = the number of denatured cells/the number of total cells) in each region of the hippocampus demonstrated that the oral intake of compound 18d•HCl significantly reduced the ratio of dead neurons in the CA1 (11.2% ± 5.7, p < 0.001), CA2 (10.3% ± 5.6, p < 0.001), CA3 (6.9% ± 2.1, p < 0.001) and DG (12.2% ± 6.5, p < 0.001) regions compared to the model (87.5% ± 9.5, 70.3% ± 13.6, 86.8% ± 8.1 and 80.1% ± 10.8, respectively), donepezil (46.8% ± 8.5, 51.3% ± 10.0, 45.6% ± 5.3 and 55.6% ± 10.9, respectively) and CQ (47.8% ± 5.3, 36.3% ± 5.8, 33.6% ± 8.5 and 62.0% ± 18.5, respectively) groups (Fig. 11). In general, the compound 18d•HCl treatment significantly reduced the number of apoptotic neurons in the hippocampus compared to the donepezil and CQ treatments at the dosage used here.


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Figure 11. The denatured cell index (DCI = the number of denatured cells/the number of total cells) of the rat

hippocampal neurons (HE staining, n = 11). CA1, CA2, CA3 and DG indicate the CA1, CA2, CA3 and Dentate

Gyrus (DG) regions of the hippocampus. The results were expressed as the means ± SD (n = 11). Statistical significance was analyzed by two-way ANOVA: ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus the model and compound 18d•HCl groups.

CONCLUSION In summary, this study involved the design, synthesis and biological evaluation of a novel series of multi-target-directed ligands against AD by fusing the pharmacophores of clioquinol and moracin M. Most of the target compounds displayed excellent inhibition of Aβ aggregation and PDE4D activity as well as remarkable BBB penetration and antioxidant effects. The optimal candidate compound, 18d, directly interacted with crucial residues of Aβ in solution, as shown by 2D NMR, and modulated metal-induced Aβ aggregation, and distinctively disaggregated self- or metal-induced Aβ aggregates. This unique hybrid demonstrated significant effects in protecting neuronal cells against LPS-stimulated inflammation. Most importantly, an oral intake of 30 mg/kg/day of compound 18d•HCl by AD



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model rats was non-toxic, significantly improved their spatial memory, and protected hippocampal neurons against damage. All of these results highlight compound 18d’s potential as a promising lead in the development of orally active therapies for AD. EXPERIMENTAL SECTION Chemistry The NMR spectra were recorder using TMS as the internal standard on a Bruker AvanceIII spectrometer at 400.132(1H NMR) and 100.614(13C NMR) MHz. MS spectra were generated on an Agilent LC−MS 6120 instrument with an ESI and APCI mass selective detector. The melting points were determined using an SRS-Opti Melt automated melting point instrument. The reactions were followed by thin-layer chromatography (TLC) on glass-packed precoated silica gel plates and visualized in an iodine chamber or with a UV lamp. Flash column chromatography was performed using silica gel (200–300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The high-resolution mass spectra (HRMS) were obtained using a Shimadzu LCMS-IT-TOF mass spectrometer. The purity (≥95%) of the samples was determined by HPLC, conducted on a Shimadzu LC-20AT series system, TC-C18 column (4.6×250 mm, 5 µm), eluted with CH3CN/H2O = 90/10, at a flow rate of 0.5 mL/min. Compound 2 was synthesised according to the literature.49 Diethyl ((8-hydroxyquinolin-5-yl)methyl)phosphonate (3a)64

To a solution of triethyl phosphite (18.86 mL, 3.0 equiv) was added 1.0 equiv of compound 2 in one portion. The mixture was stirred at 110 °C for 12 h. After the


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completion of the reaction, the residue was directly purified by flash chromatography on silica gel with ethyl acetate as the elution solvent to afford the pure product. Pale yellow solid, 78% yield. Rf = 0.32 (CH2Cl2/CH3OH = 20/1). 1H NMR (400 MHz, CDCl3) δ 8.79 (dd, J = 4.2, 1.3 Hz, 1H), 8.45 (dd, J = 8.6, 1.4 Hz, 1H), 7.49 (dd, J = 8.6, 4.2 Hz, 1H), 7.40 (dd, J = 7.9, 3.8 Hz, 1H), 7.13 (dd, J = 7.9, 0.9 Hz, 1H), 4.03 – 3.88 (m, 4H), 3.50 (d, J = 21.3 Hz, 2H), 1.18 (t, J = 7.1 Hz, 6H). LC/MS (ESI): 296.1 [M+H]+.

Diethyl ((7-chloro-8-hydroxyquinolin-5-yl)methyl)phosphonate (3b)

5.0 g of 3a suspended in 30 mL H2O and 1.0 equiv (17 mL) of 1 N KOH solution, 15.3 mL (7.5 % excess) of NaClO solution was added over about 30min with vigorously stirred, the reaction was stirred at room temperature and monitored by TLC. After the completion of the reaction, the solution was adjusted to pH = 6.0 by 10 % hydrochloric acid, filtrated to get the product without further purification. White solid, 84% yield. Rf = 0.33 (CH2Cl2/CH3OH = 20/1).1H NMR (400 MHz, CDCl3) δ 8.81 (dd, J = 4.1, 1.0 Hz, 1H), 8.44 (dd, J = 8.6, 1.2 Hz, 1H), 7.51 (dd, J = 8.6, 4.2 Hz, 1H), 7.45 (d, J = 3.8 Hz, 1H), 4.05 – 3.93 (m, 4H), 3.50 (s, 1H), 3.44 (s, 1H), 1.21 (t, J = 7.1 Hz, 6H). LC/MS (ESI): 330.1 [M+H]+.

Diethyl ((8-hydroxy-7-iodoquinolin-5-yl)methyl)phosphonate (3c)

To a solution of 30 mL H2O containing 3.0 equiv of KOH (8.55 g), 5.0 g of 3a was dissolved at room temperature. A mixture of iodine (1.0 equiv) in the potassium



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iodide (2.0 equiv) was prepared and added to the above solution. The mixture was stirred for 2 h and then hydrochloric acid (10 %) was added to lead the formation of precipitation. The mixture was extracted by CH2Cl2, washed with brine, dried over anhydrous Na2SO4, evaporated the solvent and purified by flash chromatography on silica gel with ethyl acetate as the elution solvent. Brown solid. 65% yield. Rf = 0.32 (CH2Cl2/CH3OH = 20/1). 1H NMR (400 MHz, CDCl3) δ 8.77 (dd, J = 4.2, 1.2 Hz, 1H), 8.43 (dd, J = 8.6, 1.3 Hz, 1H), 7.74 (d, J = 3.9 Hz, 1H), 7.53 (dd, J = 8.6, 4.2 Hz, 1H), 4.10 – 3.90 (m, 4H), 3.47 (s, 1H), 3.42 (s, 1H), 1.21 (t, J = 7.1 Hz, 6H). LC/MS (ESI): 422.0 [M+H]+.

General Procedures for the Synthesis of 4a-4c

The soluotion of 1.0 equiv of 3 (3.0 g) and 1.5 equiv of KOH in 10 mL DMF was stirred at room temperature for 30 min, 1.1 equiv of iodomethane was added and the reaction was further stirred for 12 h. Water was added to the reaction solution, the product was extracted by ethyl acetate, washed with brine, dried over anhydrous Na2SO4, evaporated the solvent and purified by silica gel column chromatography to afford the product.

Diethyl ((8-methoxyquinolin-5-yl)methyl)phosphonate (4a) Yellow oil. 87% yield. Rf = 0.40 (CH2Cl2/CH3OH = 20/1). 1H NMR (400 MHz, CDCl3) δ 8.96 (dd, J = 4.1, 1.4 Hz, 1H), 8.43 (dd, J = 8.6, 1.5 Hz, 1H), 7.50 (dd, J = 8.6, 4.1 Hz, 1H), 7.46 (dd, J = 8.0, 3.9 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 4.09 (s, 3H),


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3.94 (dddd, J = 17.2, 8.4, 7.2, 3.0 Hz, 4H), 3.56 (s, 1H), 3.51 (s, 1H), 1.16 (t, J = 7.1 Hz, 6H). LC/MS (ESI): 310.1 [M+H]+. Diethyl ((7-chloro-8-methoxyquinolin-5-yl)methyl)phosphonate (4b) Yellow oil. 79% yield. Rf = 0.44 (CH2Cl2/CH3OH=20/1). 1H NMR (400 MHz, CDCl3) δ 8.97 (dd, J = 4.1, 1.4 Hz, 1H), 8.43 (dd, J = 8.6, 1.5 Hz, 1H), 7.51 (d, J = 3.8 Hz, 1H), 7.48 (dd, J = 8.6, 4.2 Hz, 1H), 4.17 (s, 3H), 4.07 – 3.95 (m, 4H), 3.54 (s, 1H), 3.49 (s, 1H), 1.22 (t, J = 7.1 Hz, 6H). LC/MS (ESI): 344.1 [M+H]+. Diethyl ((7-iodo-8-methoxyquinolin-5-yl)methyl)phosphonate (4c) Yellow oil. 75% yield. Rf = 0.42 (CH2Cl2/CH3OH = 20/1). 1H NMR (400 MHz, CDCl3) δ 8.92 (dd, J = 4.0, 1.6 Hz, 1H), 8.43 (dd, J = 8.6, 1.5 Hz, 1H), 7.83 (d, J = 3.9 Hz, 1H), 7.50 (dd, J = 8.6, 4.1 Hz, 1H), 4.14 (s, 3H), 4.06 – 3.96 (m, 4H), 3.52 (s, 1H), 3.47 (s, 1H), 1.22 (t, J = 7.1 Hz, 6H). LC/MS (ESI): 436.0 [M+H]+. 8-Methoxyquinoline (4d)65 To a solution of 10 g 2-methylquinolin-8-ol (1.0 equiv) and 13 g K2CO3 (1.5 equiv) in 10 mL DMF, 4.3 mL iodomethane (1.1 equiv)was added. The reaction was stirred at ambient temperature for 12 h and monitored by TLC. 150 mL of water was added, and then extracted by ethyl acetate, washed with brine, dried over Na2SO4, purified by flash chromatography on silica gel as a whiter solid. 89% yield. Rf = 0.29 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 8.4 Hz, 1H), 7.43 – 7.28 (m, 3H), 7.03 (d, J = 7.4 Hz, 1H), 4.08 (s, 3H), 2.80 (s, 3H). LC/MS (ESI):160.1 [M+H]+. General Procedures for the Synthesis of 6a-6g



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To a mixture of 2.0 g of salicylic aldehyde or acetophenone derivative (1.0 equiv) and 9 mL of diisopropylethylamine (4.0 equiv) in 10 mL DCM, 1.5 equiv of chloromethyl methyl ether was dropwise added. After stirred 5 h, 100 mL of saturated NaHCO3 solution was added, then extracted by DCM, washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography. 2-(Methoxymethoxy)benzaldehyde (6a)66 Yellow oil. 86% yield. Rf = 0.51 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 10.51 (d, J = 0.7 Hz, 1H), 7.84 (dd, J = 7.7, 1.8 Hz, 1H), 7.53 (ddd, J = 8.5, 7.3, 1.8 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 5.31 (s, 2H), 3.53 (s, 3H). LC/MS (ESI): 167.1 [M+H]+. 3-Methoxy-2-(methoxymethoxy)benzaldehyde (6b)67 Yellow oil. 92% yield. Rf = 0.50 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 10.48 (s, 1H), 7.49 – 7.39 (m, 1H), 7.21 – 7.08 (m, 2H), 5.23 (d, J = 1.1 Hz, 2H), 3.89 (s, 3H), 3.57 (s, 3H). LC/MS (ESI): 197.1 [M+H]+. 4-Methoxy-2-(methoxymethoxy)benzaldehyde (6c)68 Yellow oil. 84% yield. Rf = 0.57 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 10.33 (d, J = 0.7 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 6.71 (d, J = 2.3 Hz, 1H), 6.61 (ddd, J = 8.7, 2.3, 0.6 Hz, 1H), 5.29 (s, 2H), 3.86 (s, 3H), 3.53 (s, 3H). LC/MS (ESI): 197.1 [M+H]+. 5-Methoxy-2-(methoxymethoxy)benzaldehyde (6d)69


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Yellow oil. 81% yield. Rf = 0.53 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 10.47 (s, 1H), 7.33 (d, J = 3.1 Hz, 1H), 7.18 (d, J = 9.1 Hz, 1H), 7.11 (dd, J = 9.1, 3.1 Hz, 1H), 5.24 (s, 2H), 3.81 (s, 3H), 3.52 (s, 3H). LC/MS (ESI): 197.1 [M+H]+. 2,4-Dimethoxy-6-(methoxymethoxy)benzaldehyde (6e) Yellow oil. 90% yield. Rf = 0.49 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 10.37 (s, 1H), 6.33 (d, J = 2.1 Hz, 1H), 6.14 (d, J = 2.1 Hz, 1H), 5.25 (s, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.51 (s, 3H). LC/MS (ESI): 227.1 [M+H]+. 1-(5-Methoxy-2-(methoxymethoxy)phenyl)ethanone (6f)70 Yellow oil. 87% yield. Rf = 0.52 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 3.2 Hz, 1H), 7.13 (d, J = 9.0 Hz, 1H), 7.00 (dd, J = 9.0, 3.2 Hz, 1H), 5.21 (s, 2H), 3.80 (s, 3H), 3.51 (s, 3H), 2.64 (s, 3H). LC/MS (ESI): 211.1 [M+H]+. 3,5-Diiodo-2-(methoxymethoxy)benzaldehyde (6g) Yellow oil. 72% yield. Rf = 0.39 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1H), 8.35 (d, J = 2.1 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 5.14 (s, 2H), 3.61 (s, 3H). LC/MS (ESI): 418.8 [M+H]+. General Procedures for the Synthesis of 7a-7g, 7m-7n, 8a-8d, 8f and 9c-9e To a solution of 4 (1.0 equiv) in 10 mL anhydrous DMF, 3.0 equiv of sodium hydride (80 %) was added in portion at an ice bath. The mixture was further stirred for 30 min, a solution of the corresponding aldehyde or ketone (1.0 equiv) in 2 mL DMF was added in dropwise and monitored by TLC. Water was added to the solution, extracted



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by DCM, washed with brine, dried over anhydrous Na2SO4, evaporated the solvent under reduced pressure, purified by silica gel column chromatography. (E)-8-Methoxy-5-(2-(methoxymethoxy)styryl)quinoline (7a) Yellow oil. 67% yield. Rf = 0.31 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.96 (dd, J = 4.0, 1.4 Hz, 1H), 8.54 (dd, J = 8.6, 1.2 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.70 (t, J = 12.7 Hz, 2H), 7.48 (dd, J = 8.7, 4.2 Hz, 1H), 7.44 (d, J = 16.1 Hz, 1H), 7.26 (dd, J = 15.5, 1.4 Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 7.08 (dd, J = 14.8, 7.7 Hz, 2H), 5.27 (s, 2H), 4.12 (s, 3H), 3.52 (s, 3H). LC/MS (ESI): 322.1 [M+H]+. (E)-8-Methoxy-5-(3-methoxy-2-(methoxymethoxy)styryl)quinoline (7b) Yellow oil. 71% yield. Rf = 0.36 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.89 (dd, J = 4.1, 1.6 Hz, 1H), 8.47 (dd, J = 8.6, 1.6 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 16.2 Hz, 1H), 7.47 – 7.35 (m, 2H), 7.25 (dd, J = 7.9, 1.1 Hz, 1H), 7.10 – 6.98 (m, 2H), 6.80 (dd, J = 8.1, 1.2 Hz, 1H), 5.09 (s, 2H), 4.05 (s, 3H), 3.81 (s, 3H), 3.49 (s, 3H). LC/MS (ESI): 352.1 [M+H]+. (E)-8-Methoxy-5-(4-methoxy-2-(methoxymethoxy)styryl)quinoline (7c) Yellow oil. 76% yield. Rf = 0.30 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.96 (dd, J = 4.1, 1.6 Hz, 1H), 8.54 (dd, J = 8.6, 1.6 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 6.3 Hz, 1H), 7.59 (s, 1H), 7.48 (dd, J = 8.6, 4.1 Hz, 1H), 7.35 (d, J = 16.1 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 6.76 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 8.6, 2.4 Hz, 1H), 5.26 (s, 2H), 4.12 (s, 3H), 3.84 (s, 3H), 3.52 (s, 3H). LC/MS (ESI): 352.1 [M+H]+. (E)-8-Methoxy-5-(5-methoxy-2-(methoxymethoxy)styryl)quinoline (7d)


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Yellow oil. 69% yield. Rf = 0.32 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.96 (dd, J = 4.1, 1.6 Hz, 1H), 8.53 (dd, J = 8.6, 1.5 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 16.2 Hz, 1H), 7.49 (dd, J = 8.6, 4.1 Hz, 1H), 7.41 (d, J = 16.1 Hz, 1H), 7.22 (d, J = 3.0 Hz, 1H), 7.11 (d, J = 1.7 Hz, 1H), 7.09 (s, 1H), 6.81 (dd, J = 8.9, 3.0 Hz, 1H), 5.19 (s, 2H), 4.13 (s, 3H), 3.85 (s, 3H), 3.52 (s, 3H). LC/MS (ESI): 352.1 [M+H]+. (E)-5-(2,4-Dimethoxy-6-(methoxymethoxy)styryl)-8-methoxyquinoline (7e) Yellow oil. 62% yield. Rf = 0.30 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.94 (dd, J = 4.1, 1.6 Hz, 1H), 8.53 (dd, J = 8.6, 1.6 Hz, 1H), 8.05 (d, J = 16.3 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.46 (dd, J = 8.6, 4.1 Hz, 1H), 7.32 (d, J = 16.3 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 6.26 (d, J = 2.3 Hz, 1H), 5.27 (s, 2H), 4.11 (s, 3H), 3.91 (s, 3H), 3.84 (s, 3H), 3.53 (s, 3H). LC/MS (ESI): 382.1 [M+H]+. (E)-8-Methoxy-5-(2-(5-methoxy-2-(methoxymethoxy)phenyl)prop-1-en-1-yl)quin oline (7f) Yellow oil. 65% yield. Rf = 0.41 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.89 (dd, J = 4.1, 1.5 Hz, 1H), 8.44 (dd, J = 8.5, 1.5 Hz, 1H), 7.42 (dd, J = 8.5, 4.1 Hz, 1H), 6.98 (dd, J = 15.3, 8.5 Hz, 2H), 6.91 (s, 1H), 6.75 (d, J = 8.1 Hz, 1H), 6.63 (dd, J = 9.0, 3.1 Hz, 1H), 6.47 (d, J = 3.1 Hz, 1H), 4.82 (s, 2H), 3.99 (s, 3H), 3.56 (s, 3H), 3.27 (s, 3H), 2.31 (d, J = 1.1 Hz, 3H). LC/MS (ESI): 366.2 [M+H]+. (E)-5-(3,5-Diiodo-2-(methoxymethoxy)styryl)-8-methoxyquinoline (7g)



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Yellow oil. 59% yield. Rf = 0.34 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 4.0, 1.4 Hz, 1H), 8.54 (dd, J = 8.6, 1.3 Hz, 1H), 8.03 (d, J = 1.9 Hz, 1H), 7.96 (d, J = 1.9 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 2.6 Hz, 1H), 7.52 (dd, J = 8.6, 4.0 Hz, 1H), 7.29 (d, J = 6.3 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 5.08 (s, 2H), 4.13 (s, 3H), 3.63 (s, 3H). LC/MS (ESI): 574.0 [M+H]+. General Procedures for the Synthesis of 7h-7i 1.0 g quinoline (1.0 equiv) and benzaldehyde (1.0 equiv) was dissolved in 20 mL acetic anhydride, and the mixture was stirred at 150 °C for 10 h. After cooled to room temperature, the mixture was filtrated off and the precipitation was then washed with water and dried under vacuum to get the product. (E)-5-Methoxy-2-(2-(8-methoxyquinolin-2-yl)vinyl)phenyl acetate (7h) Yellow solid. 87% yield. Rf = 0.43 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 16.5 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.39 (d, J = 7.3 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 6.85 (dd, J = 8.8, 2.3 Hz, 1H), 6.67 (d, J = 2.4 Hz, 1H), 4.10 (s, 3H), 3.82 (s, 3H), 2.42 (s, 3H). LC/MS (ESI): 350.1 [M+H]+. (E)-3,5-Dimethoxy-2-(2-(8-methoxyquinolin-2-yl)vinyl)phenyl acetate (7i) Yellow solid. 84% yield. Rf = 0.41 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.05 (dd, J = 8.1, 2.5 Hz, 1H), 7.78 – 7.65 (m, 2H), 7.61 (dd, J = 16.5, 2.4 Hz, 1H), 7.43 – 7.28 (m, 2H), 7.02 (d, J = 4.4 Hz, 1H), 6.41 (s, 1H), 6.30 (s, 1H), 4.08 (s, 3H), 3.91 (s, 3H), 3.81 (s, 3H), 2.40 (s, 3H). LC/MS (ESI): 380.1 [M+H]+. (E)-7-Chloro-8-methoxy-5-(2-(methoxymethoxy)styryl)quinoline (8a)


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Yellow oil. 62% yield. Rf = 0.28 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 4.1, 1.5 Hz, 1H), 8.52 (dd, J = 8.6, 1.5 Hz, 1H), 7.82 (s, 1H), 7.68 (dd, J = 8.7, 7.4 Hz, 2H), 7.53 – 7.43 (m, 2H), 7.31 – 7.27 (m, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.07 (t, J = 7.3 Hz, 1H), 5.29 (s, 2H), 4.19 (s, 3H), 3.53 (s, 3H). LC/MS (ESI): 356.0 [M+H]+ (E)-7-Chloro-8-methoxy-5-(3-methoxy-2-(methoxymethoxy)styryl)quinoline (8b) Yellow oil. 68% yield. Rf = 0.30 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 4.1, 1.6 Hz, 1H), 8.53 (dd, J = 8.6, 1.6 Hz, 1H), 7.81 (s, 1H), 7.73 – 7.66 (m, 1H), 7.52 (d, J = 16.1 Hz, 1H), 7.46 (dd, J = 8.6, 4.1 Hz, 1H), 7.30 (dd, J = 7.9, 1.0 Hz, 1H), 7.13 (t, J = 8.0 Hz, 1H), 6.91 (dd, J = 8.1, 1.2 Hz, 1H), 5.17 (s, 2H), 4.19 (s, 3H), 3.89 (s, 3H), 3.58 (s, 3H). LC/MS (ESI): 386.1 [M+H]+. (E)-7-Chloro-8-methoxy-5-(4-methoxy-2-(methoxymethoxy)styryl)quinoline (8c) Yellow oil. 67% yield. Rf = 0.34 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 4.1, 1.6 Hz, 1H), 8.51 (dd, J = 8.6, 1.5 Hz, 1H), 7.79 (s, 1H), 7.58 (dd, J = 12.3, 7.9 Hz, 2H), 7.45 (dd, J = 8.6, 4.1 Hz, 1H), 7.39 (d, J = 16.0 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 8.6, 2.4 Hz, 1H), 5.28 (s, 2H), 4.18 (s, 3H), 3.85 (s, 3H), 3.53 (s, 3H). LC/MS (ESI): 386.1 [M+H]+. (E)-7-Chloro-8-methoxy-5-(5-methoxy-2-(methoxymethoxy)styryl)quinoline (8d) Yellow oil. 69% yield. Rf = 0.33 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.96 (dd, J = 4.1, 1.6 Hz, 1H), 8.53 (dd, J = 8.6, 1.5 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 16.2 Hz, 1H), 7.49 (dd, J = 8.6, 4.1 Hz, 1H), 7.41 (d, J = 16.1 Hz, 1H), 7.22 (d, J = 3.0 Hz, 1H), 7.11 (d, J = 1.7 Hz, 1H), 7.09 (s, 1H), 6.81 (dd,



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J = 8.9, 3.0 Hz, 1H), 5.19 (s, 2H), 4.13 (s, 3H), 3.85 (s, 3H), 3.52 (s, 3H). LC/MS (ESI): 386.1 [M+H]+. (E)-7-Chloro-8-methoxy-5-(2-(5-methoxy-2-(methoxymethoxy)phenyl)prop-1-en1-yl)quinoline (8f) Yellow oil. 62% yield. Rf = 0.36 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.91 (dd, J = 4.1, 1.5 Hz, 1H), 8.41 (dd, J = 8.5, 1.6 Hz, 1H), 7.38 (dd, J = 8.5, 4.1 Hz, 1H), 7.11 (s, 1H), 6.99 (d, J = 9.0 Hz, 1H), 6.85 (s, 1H), 6.65 (dd, J = 9.0, 3.1 Hz, 1H), 6.43 (d, J = 3.1 Hz, 1H), 4.92 (s, 2H), 4.09 (s, 3H), 3.58 (s, 3H), 3.31 (s, 3H), 2.30 (d, J = 1.3 Hz, 3H). LC/MS (ESI): 400.1 [M+H]+. (E)-7-Iodo-8-methoxy-5-(4-methoxy-2-(methoxymethoxy)styryl)quinoline (9c) Yellow oil. 78% yield. Rf = 0.32 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.95 (dd, J = 4.1, 1.5 Hz, 1H), 8.50 (dd, J = 8.6, 1.4 Hz, 1H), 8.12 (s, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.53 (d, J = 16.1 Hz, 1H), 7.47 (dd, J = 8.6, 4.1 Hz, 1H), 7.36 (d, J = 16.1 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 8.6, 2.4 Hz, 1H), 5.28 (s, 2H), 4.16 (s, 3H), 3.85 (s, 3H), 3.53 (s, 3H). LC/MS (ESI): 478.0 [M+H]+. (E)-7-Iodo-8-methoxy-5-(5-methoxy-2-(methoxymethoxy)styryl)quinoline (9d) Yellow oil. 65% yield. Rf = 0.30 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.96 (dd, J = 4.1, 1.6 Hz, 1H), 8.50 (dd, J = 8.6, 1.6 Hz, 1H), 8.14 (s, 1H), 7.62 (d, J = 16.1 Hz, 1H), 7.48 (dd, J = 8.6, 4.1 Hz, 1H), 7.42 (d, J = 16.1 Hz, 1H), 7.19 (d, J = 3.0 Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H), 6.84 (dd, J = 9.0, 3.0 Hz, 1H), 5.21 (s, 2H), 4.18 (s, 3H), 3.85 (s, 3H), 3.53 (s, 3H). LC/MS (ESI):478.0 [M+H]+.


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(E)-5-(2,4-Dimethoxy-6-(methoxymethoxy)styryl)-7-iodo-8-methoxyquinoline (9e) Yellow oil. 73% yield. Rf = 0.31 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.93 (dd, J = 4.1, 1.6 Hz, 1H), 8.49 (dd, J = 8.6, 1.6 Hz, 1H), 8.10 (s, 1H), 7.97 (d, J = 16.2 Hz, 1H), 7.45 (dd, J = 8.6, 4.1 Hz, 1H), 7.32 (d, J = 16.2 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 6.25 (d, J = 2.2 Hz, 1H), 5.28 (s, 2H), 4.16 (s, 3H), 3.92 (s, 3H), 3.84 (s, 3H), 3.53 (s, 3H). LC/MS (ESI): 508.0 [M+H]+. General Procedures for the Synthesis of 10a-10g, 11a-11d, 11f and 12c-12e To a solution containing 0.5 g of compound 7, 8, or 9 in 10 mL of CH3OH, 2 mL 37% hydrochloric acid was added. After the reaction was stirred at room temperature for 2 h, 200 mL of saturated NaHCO3 solution was added, the mixture was filtrated off and the precipitation was then washed with pure water and dried under vacuum to get the product without any further purification. (E)-2-(2-(8-Methoxyquinolin-5-yl)vinyl)phenol (10a) Yellow solid. 88% yield. Rf = 0.21 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, DMSO) δ 8.88 (dd, J = 4.0, 1.5 Hz, 1H), 8.75 (dd, J = 8.7, 1.5 Hz, 1H), 7.86 (dd, J = 16.2, 12.3 Hz, 2H), 7.77 (dd, J = 7.8, 1.2 Hz, 1H), 7.60 (dd, J = 8.6, 4.1 Hz, 1H), 7.39 (d, J = 16.2 Hz, 1H), 7.25 (d, J = 8.3 Hz, 1H), 7.17 – 7.06 (m, 1H), 6.94 – 6.79 (m, 2H), 3.99 (s, 3H). LC/MS (ESI): 278.1 [M+H]+. (E)-2-Methoxy-6-(2-(8-methoxyquinolin-5-yl)vinyl)phenol (10b) Yellow solid. 91% yield. Rf = 0.23 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, DMSO) δ 8.88 (d, J = 3.8 Hz, 1H), 8.74 (d, J = 8.5 Hz, 1H), 7.86 (dd, J = 11.9, 9.1 Hz,



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2H), 7.60 (dd, J = 8.6, 4.0 Hz, 1H), 7.46 – 7.37 (m, 2H), 7.25 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 6.83 (t, J = 7.9 Hz, 1H), 4.00 (s, 3H), 3.83 (s, 3H). LC/MS (ESI): 308.1 [M+H]+. (E)-5-Methoxy-2-(2-(8-methoxyquinolin-5-yl)vinyl)phenol (10c) Yellow solid. 81% yield. Rf = 0.22 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, DMSO) δ 8.86 (dd, J = 4.0, 1.4 Hz, 1H), 8.72 (dd, J = 8.7, 1.4 Hz, 1H), 7.77 (dd, J = 14.7, 12.3 Hz, 2H), 7.66 (d, J = 8.5 Hz, 1H), 7.58 (dd, J = 8.6, 4.1 Hz, 1H), 7.32 (d, J = 16.1 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 6.46 (d, J = 2.5 Hz, 1H), 6.42 (dd, J = 8.2, 2.5 Hz, 1H), 3.98 (s, 3H), 3.72 (s, 3H). LC/MS (ESI): 308.1 [M+H]+. (E)-4-Methoxy-2-(2-(8-methoxyquinolin-5-yl)vinyl)phenol (10d) Yellow solid. 83% yield. Rf = 0.20 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, DMSO) δ 8.88 (dd, J = 4.0, 1.6 Hz, 1H), 8.79 (dd, J = 8.7, 1.5 Hz, 1H), 7.90 (d, J = 16.2 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.61 (dd, J = 8.6, 4.1 Hz, 1H), 7.38 (d, J = 16.2 Hz, 1H), 7.34 (d, J = 2.9 Hz, 1H), 7.25 (d, J = 8.3 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 6.73 (dd, J = 8.8, 3.0 Hz, 1H), 3.99 (s, 3H), 3.77 (s, 3H). LC/MS (ESI): 308.1 [M+H]+. (E)-3,5-Dimethoxy-2-(2-(8-methoxyquinolin-5-yl)vinyl)phenol (10e) Yellow solid. 78% yield. Rf = 0.22 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, CDCl3) δ 8.95 (dd, J = 4.1, 1.6 Hz, 1H), 8.49 (dd, J = 8.6, 1.6 Hz, 1H), 7.76 (dd, J = 16.0, 12.4 Hz, 2H), 7.47 (dd, J = 8.6, 4.1 Hz, 1H), 7.20 (d, J = 16.4 Hz, 1H), 7.08 (d, J = 8.2 Hz, 1H), 6.20 (d, J = 2.3 Hz, 1H), 6.16 (d, J = 2.3 Hz, 1H), 4.11 (s, 3H), 3.88 (s, 3H), 3.83 (s, 3H). LC/MS (ESI): 338.1 [M+H]+.


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(E)-4-Methoxy-2-(1-(8-methoxyquinolin-5-yl)prop-1-en-2-yl)phenol (10f) Yellow solid. 85% yield. Rf = 0.21 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, DMSO) δ 8.78 (dd, J = 4.1, 1.6 Hz, 1H), 8.46 (dd, J = 8.5, 1.6 Hz, 1H), 7.50 (dd, J = 8.5, 4.1 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.88 (s, 1H), 6.64 (t, J = 5.1 Hz, 1H), 6.53 (dd, J = 8.8, 3.1 Hz, 1H), 6.24 (d, J = 3.1 Hz, 1H), 3.85 (s, 3H), 3.40 (s, 3H), 2.21 (d, J = 1.4 Hz, 3H). LC/MS (ESI): 322.1 [M+H]+. (E)-2,4-Diiodo-6-(2-(8-methoxyquinolin-5-yl)vinyl)phenol (10g) Yellow solid. 87% yield. Rf = 0.19 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, DMSO) δ 8.94 – 8.85 (m, 2H), 8.18 (d, J = 1.8 Hz, 1H), 7.95 (s, 1H), 7.92 (dd, J = 4.3, 2.3 Hz, 2H), 7.61 (dd, J = 8.1, 4.6 Hz, 1H), 7.38 (d, J = 16.0 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 4.00 (s, 3H). LC/MS (ESI): 530.0 [M+H]+. General Procedures for the Synthesis of 10h-10i Compound 7h or 7i (1.0 g, 1.0 equiv ) and 1.0 equiv of K2CO3 in 10 mL CH3OH was stirred at room temperature, after the completion of the reaction, 200 mL water was added, filtrated, the precipitation was then washed with pure water and dried under reduced pressure to get the the product. (E)-5-Methoxy-2-(2-(8-methoxyquinolin-2-yl)vinyl)phenol (10h) Yellow solid. 92% yield. Rf = 0.31 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 8.7 Hz, 1H), 7.87 (d, J = 16.4 Hz, 1H), 7.68 (dd, J = 12.6, 7.8 Hz, 2H), 7.41 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 7.4 Hz, 1H), 7.32 (dd, J = 8.2, 1.4 Hz, 1H), 7.01 (dd, J = 7.4, 1.3 Hz, 1H), 6.40 (dd, J = 8.6, 2.5 Hz, 1H), 6.29 (d, J = 2.4 Hz, 1H), 3.99 (s, 3H), 3.57 (s, 3H). LC/MS (ESI): 308.1 [M+H]+.



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(E)-3,5-Dimethoxy-2-(2-(8-methoxyquinolin-2-yl)vinyl)phenol (10i) Yellow solid. 87% yield. Rf = 0.29 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 8.7 Hz, 1H), 7.96 (dd, J = 36.4, 16.9 Hz, 2H), 7.83 (d, J = 8.7 Hz, 1H), 7.41 – 7.37 (m, 1H), 7.35 (dd, J = 8.2, 1.7 Hz, 1H), 7.04 (dd, J = 7.2, 1.6 Hz, 1H), 6.13 – 5.94 (m, 2H), 4.02 (s, 3H), 3.85 (s, 3H), 3.60 (s, 3H). LC/MS (ESI): 338.1 [M+H]+. (E)-2-(2-(7-Chloro-8-methoxyquinolin-5-yl)vinyl)phenol (11a) Yellow solid. 81% yield. Rf = 0.29 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, DMSO) δ 9.00 (dd, J = 4.0, 1.1 Hz, 1H), 8.82 (dd, J = 8.3, 0.9 Hz, 1H), 8.02 – 7.85 (m, 2H), 7.80 (dd, J = 9.8, 3.1 Hz, 1H), 7.65 (dd, J = 8.6, 4.1 Hz, 1H), 7.49 (dd, J = 15.9, 5.5 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 6.90 (dt, J = 14.9, 6.3 Hz, 2H), 4.10 (s, 3H). LC/MS (ESI): 312.1 [M+H]+. (E)-2-(2-(7-Chloro-8-methoxyquinolin-5-yl)vinyl)-6-methoxyphenol (11b) Yellow solid. 83% yield. Rf = 0.27 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, DMSO) δ 9.03 (dd, J = 4.3, 1.4 Hz, 1H), 8.92 (dd, J = 8.7, 1.4 Hz, 1H), 7.95 (s, 1H), 7.91 (d, J = 16.2 Hz, 1H), 7.71 (dd, J = 8.6, 4.3 Hz, 1H), 7.53 (d, J = 16.2 Hz, 1H), 7.43 (dd, J = 7.9, 1.1 Hz, 1H), 6.95 (dd, J = 8.0, 1.2 Hz, 1H), 6.84 (t, J = 7.9 Hz, 1H), 4.10 (s, 3H), 3.84 (s, 3H). LC/MS (ESI): 342.1 [M+H]+. (E)-2-(2-(7-Chloro-8-methoxyquinolin-5-yl)vinyl)-5-methoxyphenol (11c) Yellow solid. 76% yield. Rf = 0.28 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, DMSO) δ 8.98 (dd, J = 4.1, 1.5 Hz, 1H), 8.80 (dd, J = 8.7, 1.5 Hz, 1H), 7.86 (s, 1H), 7.79 (d, J = 16.2 Hz, 1H), 7.72 (d, J = 9.3 Hz, 1H), 7.63 (dd, J = 8.6, 4.1 Hz, 1H),


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7.41 (d, J = 16.1 Hz, 1H), 6.49 (dd, J = 5.6, 2.4 Hz, 2H), 4.08 (s, 3H), 3.75 (s, 3H). LC/MS (ESI): 342.1 [M+H]+. (E)-2-(2-(7-Chloro-8-methoxyquinolin-5-yl)vinyl)-4-methoxyphenol (11d) Yellow solid. 83% yield. Rf = 0.26 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, DMSO) δ 8.99 (d, J = 4.0 Hz, 1H), 8.85 (d, J = 8.7 Hz, 1H), 7.97 – 7.86 (m, 2H), 7.64 (dd, J = 8.6, 4.1 Hz, 1H), 7.46 (d, J = 16.1 Hz, 1H), 7.36 (d, J = 2.8 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 6.76 (dd, J = 8.8, 2.9 Hz, 1H), 4.08 (s, 3H), 3.76 (s, 3H). LC/MS (ESI): 342.1 [M+H]+. (E)-2-(1-(7-Chloro-8-methoxyquinolin-5-yl)prop-1-en-2-yl)-4-methoxyphenol (11f) Yellow solid. 87% yield. Rf = 0.25 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, DMSO) δ 8.92 (dd, J = 4.1, 1.5 Hz, 1H), 8.55 (dd, J = 8.6, 1.6 Hz, 1H), 7.56 (dd, J = 8.6, 4.2 Hz, 1H), 7.12 (s, 1H), 6.94 (s, 1H), 6.68 (d, J = 8.8 Hz, 1H), 6.59 (dd, J = 8.8, 3.1 Hz, 1H), 6.29 (d, J = 3.1 Hz, 1H), 4.00 (s, 3H), 3.44 (s, 3H), 2.24 (d, J = 1.3 Hz, 3H). LC/MS (ESI): 356.1 [M+H]+. (E)-2-(2-(7-Iodo-8-methoxyquinolin-5-yl)vinyl)-5-methoxyphenol (12c) Yellow solid. 77% yield. Rf = 0.23 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, DMSO) δ 8.95 (dd, J = 4.1, 1.5 Hz, 1H), 8.80 (dd, J = 8.7, 1.5 Hz, 1H), 8.14 (s, 1H), 7.73 (dd, J = 12.7, 8.8 Hz, 2H), 7.65 (dd, J = 8.6, 4.1 Hz, 1H), 7.37 (d, J = 16.1 Hz, 1H), 6.55 – 6.42 (m, 2H), 4.05 (s, 3H), 3.75 (s, 3H). LC/MS (ESI): 434.0 [M+H]+. (E)-2-(2-(7-Iodo-8-methoxyquinolin-5-yl)vinyl)-4-methoxyphenol (12d)



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Yellow solid. 86% yield. Rf = 0.23 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, DMSO) δ 8.95 (dd, J = 4.1, 1.5 Hz, 1H), 8.80 (dd, J = 8.7, 1.5 Hz, 1H), 8.14 (s, 1H), 7.73 (dd, J = 12.7, 8.8 Hz, 2H), 7.65 (dd, J = 8.6, 4.1 Hz, 1H), 7.37 (d, J = 16.1 Hz, 1H), 6.55 – 6.42 (m, 2H), 4.05 (s, 3H), 3.75 (s, 3H). LC/MS (ESI): 434.0 [M+H]+. (E)-2-(2-(7-Iodo-8-methoxyquinolin-5-yl)vinyl)-3,5-dimethoxyphenol (12e) Yellow solid. 82% yield. Rf = 0.25 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.94 (d, J = 3.4 Hz, 1H), 8.45 (d, J = 8.6 Hz, 1H), 7.99 (s, 1H), 7.71 (d, J = 16.2 Hz, 1H), 7.46 (dd, J = 8.2, 3.9 Hz, 1H), 7.18 (d, J = 16.2 Hz, 1H), 6.22 (s, 1H), 6.12 (s, 1H), 4.12 (s, 3H), 3.86 (s, 3H), 3.81 (s, 3H). LC/MS (ESI): 464.0 [M+H]+. General Procedures for the Synthesis of 13a-13i, 14a-14d, 14f, and 15c-15e The solution of phenols compound (0.5 g, 1.0 equiv ) and 5.0 equiv of K2CO3 in 20 mL THF was stirred at room temperature for 30 min, then, 5.0 equiv of iodine was added in one portion and the reaction was monitored by TLC. 200 mL saturate Na2S2O3 solution was added and the mixture was stirred for another 30 min. The product was extracted by CH2Cl2, washed with brine and dried over anhydrous Na2SO4, evaporated the solvent under vacuum, purified by silica gel column chromatography. 5-(Benzofuran-2-yl)-8-methoxyquinoline (13a)71 White solid. 77% yield. Rf = 0.26 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.00 (d, J = 2.7 Hz, 1H), 8.81 (dd, J = 8.6, 1.2 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 7.2 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.52 (dd, J = 8.6, 4.1 Hz, 1H),


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7.31 (ddd, J = 15.4, 11.1, 3.9 Hz, 2H), 7.14 (d, J = 8.2 Hz, 1H), 6.97 (s, 1H), 4.16 (s, 3H). LC/MS (ESI): 276.1 [M+H]+. 8-Methoxy-5-(7-methoxybenzofuran-2-yl)quinoline (13b) White solid. 83% yield. Rf = 0.23 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 4.1, 1.6 Hz, 1H), 8.79 (dd, J = 8.7, 1.6 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.53 (dd, J = 8.6, 4.1 Hz, 1H), 7.24 (d, J = 1.3 Hz, 1H), 7.23 – 7.19 (m, 1H), 7.14 (d, J = 8.2 Hz, 1H), 6.96 (s, 1H), 6.86 (dd, J = 7.6, 1.2 Hz, 1H), 4.16 (s, 3H), 4.06 (s, 3H). LC/MS (ESI): 306.1 [M+H]+. 8-Methoxy-5-(6-methoxybenzofuran-2-yl)quinoline (13c) White solid. 67% yield. Rf = 0.22 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 4.1, 1.6 Hz, 1H), 8.80 (dd, J = 8.6, 1.6 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.54 – 7.47 (m, 2H), 7.15 – 7.10 (m, 2H), 6.93 (dd, J = 8.5, 2.2 Hz, 1H), 6.89 (d, J = 0.7 Hz, 1H), 4.15 (s, 3H), 3.89 (s, 3H). LC/MS (ESI): 306.1 [M+H]+. 8-Methoxy-5-(5-methoxybenzofuran-2-yl)quinoline (13d) White solid. 82% yield. Rf = 0.24 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 4.1, 1.6 Hz, 1H), 8.81 (dd, J = 8.6, 1.6 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.52 (dd, J = 8.6, 4.1 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 7.11 (d, J = 2.5 Hz, 1H), 6.94 (dd, J = 8.9, 2.6 Hz, 1H), 6.91 (d, J = 0.7 Hz, 1H), 4.15 (s, 3H), 3.88 (s, 3H). LC/MS (ESI): 306.1 [M+H]+. 5-(4,6-Dimethoxybenzofuran-2-yl)-8-methoxyquinoline (13e) White solid. 82% yield. Rf = 0.21 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 4.1, 1.6 Hz, 1H), 8.81 (dd, J = 8.6, 1.6 Hz, 1H), 7.84 (d, J =



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8.2 Hz, 1H), 7.50 (dd, J = 8.6, 4.1 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.98 (s, 1H), 6.81 – 6.68 (m, 1H), 6.38 (d, J = 1.8 Hz, 1H), 4.15 (s, 3H), 3.95 (s, 3H), 3.89 (s, 3H). LC/MS (ESI): 336.1 [M+H]+. 8-Methoxy-5-(5-methoxy-3-methylbenzofuran-2-yl)quinoline (13f) White solid. 65% yield. Rf = 0.25 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 4.1, 1.7 Hz, 1H), 8.32 (dd, J = 8.6, 1.7 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.46 (dd, J = 8.6, 4.1 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 7.04 (d, J = 2.5 Hz, 1H), 6.95 (dd, J = 8.8, 2.6 Hz, 1H), 4.16 (s, 3H), 3.91 (s, 3H), 2.24 (s, 3H). LC/MS (ESI): 320.1 [M+H]+. 5-(5,7-Diiodobenzofuran-2-yl)-8-methoxyquinoline (13g) White solid. 56% yield. Rf = 0.20 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 9.01 (dd, J = 4.1, 1.6 Hz, 1H), 8.82 (dd, J = 8.7, 1.6 Hz, 1H), 7.96 (d, J = 1.5 Hz, 1H), 7.92 (t, J = 4.6 Hz, 2H), 7.57 (dd, J = 8.7, 4.1 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 7.01 (s, 1H), 4.17 (s, 3H). LC/MS (ESI): 527.9 [M+H]+. 8-Methoxy-2-(6-methoxybenzofuran-2-yl)quinoline (13h) White solid. 58% yield. Rf = 0.29 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.6 Hz, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.54 (dd, J = 9.0, 4.7 Hz, 2H), 7.48 – 7.41 (m, 1H), 7.38 (dd, J = 8.2, 1.3 Hz, 1H), 7.14 (d, J = 1.9 Hz, 1H), 7.07 (dd, J = 7.6, 1.2 Hz, 1H), 6.91 (dd, J = 8.6, 2.3 Hz, 1H), 4.12 (s, 3H), 3.89 (s, 3H). LC/MS (ESI): 306.1 [M+H]+. 2-(4,6-Dimethoxybenzofuran-2-yl)-8-methoxyquinoline (13i)


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White solid. 76% yield. Rf = 0.27 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 8.6 Hz, 1H), 7.60 (s, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.37 (dd, J = 8.1, 1.1 Hz, 1H), 7.06 (d, J = 7.7 Hz, 1H), 6.77 (d, J = 0.9 Hz, 1H), 6.34 (d, J = 1.8 Hz, 1H), 4.11 (s, 3H), 3.94 (s, 3H), 3.88 (s, 3H). LC/MS (ESI): 336.1 [M+H]+. 5-(Benzofuran-2-yl)-7-chloro-8-methoxyquinoline (14a) White solid. 69% yield. Rf = 0.31 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.03 (dd, J = 4.1, 1.6 Hz, 1H), 8.82 (dd, J = 8.7, 1.6 Hz, 1H), 7.96 (s, 1H), 7.68 (dd, J = 7.6, 0.6 Hz, 1H), 7.63 – 7.55 (m, 1H), 7.51 (dd, J = 8.7, 4.1 Hz, 1H), 7.42 – 7.34 (m, 1H), 7.31 (td, J = 7.5, 1.0 Hz, 1H), 7.06 (d, J = 0.7 Hz, 1H), 4.25 (s, 3H). LC/MS (ESI): 310.1 [M+H]+. 7-Chloro-8-methoxy-5-(7-methoxybenzofuran-2-yl)quinoline (14b) White solid. 74% yield. Rf = 0.30 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.02 (dd, J = 4.1, 1.6 Hz, 1H), 8.81 (dd, J = 8.7, 1.6 Hz, 1H), 7.98 (s, 1H), 7.51 (dd, J = 8.6, 4.1 Hz, 1H), 7.26 – 7.20 (m, 2H), 7.04 (s, 1H), 6.89 (dd, J = 7.4, 1.4 Hz, 1H), 4.24 (s, 3H), 4.06 (s, 3H). LC/MS (ESI): 340.1 [M+H]+. 7-Chloro-8-methoxy-5-(6-methoxybenzofuran-2-yl)quinoline (14c) White solid. 79% yield. Rf = 0.32 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.01 (dd, J = 4.1, 1.6 Hz, 1H), 8.82 (dd, J = 8.7, 1.6 Hz, 1H), 7.92 (s, 1H), 7.56 – 7.44 (m, 2H), 7.13 (d, J = 1.9 Hz, 1H), 6.99 (d, J = 0.7 Hz, 1H), 6.95 (dd, J = 8.6, 2.2 Hz, 1H), 4.23 (s, 3H), 3.90 (s, 3H). LC/MS (ESI): 340.1 [M+H]+. 7-Chloro-8-methoxy-5-(5-methoxybenzofuran-2-yl)quinoline (14d)



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White solid. 77% yield. Rf = 0.33 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.02 (dd, J = 4.1, 1.5 Hz, 1H), 8.82 (dd, J = 8.7, 1.6 Hz, 1H), 7.94 (s, 1H), 7.55 – 7.43 (m, 2H), 7.12 (d, J = 2.5 Hz, 1H), 7.04 – 6.90 (m, 2H), 4.24 (s, 3H), 3.89 (s, 3H). LC/MS (ESI): 340.1 [M+H]+. 7-Chloro-8-methoxy-5-(5-methoxy-3-methylbenzofuran-2-yl)quinoline (14f) White solid. 62% yield. Rf = 0.34 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.01 (dd, J = 4.1, 1.7 Hz, 1H), 8.34 (dd, J = 8.6, 1.7 Hz, 1H), 7.70 (s, 1H), 7.45 (dd, J = 8.6, 4.1 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 6.98 (dd, J = 8.9, 2.6 Hz, 1H), 4.26 (s, 3H), 3.92 (s, 3H), 2.27 (s, 3H). LC/MS (ESI): 354.0 [M+H]+. 7-Iodo-8-methoxy-5-(6-methoxybenzofuran-2-yl)quinoline (15c) White solid. 74% yield. Rf = 0.33 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 4.1, 1.6 Hz, 1H), 8.80 (dd, J = 8.6, 1.6 Hz, 1H), 8.25 (s, 1H), 7.52 (dd, J = 8.6, 3.9 Hz, 2H), 7.13 (d, J = 1.9 Hz, 1H), 6.97 (d, J = 0.7 Hz, 1H), 6.94 (dd, J = 8.6, 2.2 Hz, 1H), 4.22 (s, 3H), 3.90 (s, 3H). LC/MS (ESI): 432.0 [M+H]+. 7-Iodo-8-methoxy-5-(5-methoxybenzofuran-2-yl)quinoline (15d) White solid. 72% yield. Rf = 0.30 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 4.1, 1.6 Hz, 1H), 8.81 (dd, J = 8.7, 1.6 Hz, 1H), 8.27 (s, 1H), 7.52 (dd, J = 8.6, 4.1 Hz, 1H), 7.48 (d, J = 8.9 Hz, 1H), 7.11 (d, J = 2.6 Hz, 1H), 7.05 – 6.90 (m, 2H), 4.22 (s, 3H), 3.89 (s, 3H). LC/MS (ESI): 432.0 [M+H]+. 5-(4,6-Dimethoxybenzofuran-2-yl)-7-iodo-8-methoxyquinoline (15e)


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White solid. 75% yield. Rf = 0.32 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 4.1, 1.6 Hz, 1H), 8.81 (dd, J = 8.6, 1.6 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.50 (dd, J = 8.6, 4.1 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.98 (s, 1H), 6.81 – 6.68 (m, 1H), 6.38 (d, J = 1.8 Hz, 1H), 4.15 (s, 3H), 3.95 (s, 3H), 3.89 (s, 3H). LC/MS (ESI): 462.0 [M+H]+. General Procedures for the Synthesis of 16a-16i, 17a-17d, 17f and 18c-18e

To a solution of compounds 13, 14 or 15 (200 mg, 1.0 equiv) in anhytrous CH2Cl2, BBr3 (6.0 equiv, 9.0 equiv to compound 16e, 16l and 18e) was added in dropwise at -78 °C. The resulting solution was slowly warmed to room temperature and stirred overnight. saturate NaHCO3 solution (100 mL) was added slowly, the mixture was filtrated off and the precipitation was then washed with pure water, the crude product was purified by flash chromatography on silica gel with ethyl acetate as the elution solvent.

5-(Benzofuran-2-yl)quinolin-8-ol (16a)71 Yellow solid. 63% yield. Rf = 0.31 (CH2Cl2/CH3OH = 10/1), mp 139.0-139.5 °C. 1H NMR (400 MHz, DMSO) δ 8.96 (dd, J = 4.1, 1.5 Hz, 1H), 8.87 (dd, J = 8.7, 1.5 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.74 – 7.65 (m, 3H), 7.40 – 7.27 (m, 3H), 7.23 (d, J = 8.1 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 154.54, 154.45, 154.16, 148.40, 138.34, 133.77, 128.75, 125.89, 124.23, 123.09, 122.75, 120.95, 117.71, 111.02, 104.51; FT-IR 3329, 3059, 1576, 1507, 1474, 1411, 1260, 1218, 1188, 963, 784, 735, 651;



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HRMS (ESI) m/z [M-H]- for C17H11NO2 pred. 260.0717, meas. 260.0713; HPLC purity: 98.59%, retention time: 12.503 min. 5-(7-Hydroxybenzofuran-2-yl)quinolin-8-ol (16b) Yellow solid. 59% yield. Rf = 0.26 (CH2Cl2/CH3OH = 10/1), mp 179.1-180.6 °C. 1H NMR (400 MHz, DMSO) δ 8.95 (dd, J = 4.0, 1.4 Hz, 1H), 8.89 (dd, J = 8.7, 1.4 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.71 (dd, J = 8.7, 4.1 Hz, 1H), 7.26 – 7.20 (m, 2H), 7.13 (dd, J = 7.6, 1.0 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.79 (dd, J = 7.5, 0.9 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 154.34, 154.23, 148.37, 143.15, 142.40, 138.32, 133.91, 130.58, 128.68, 125.89, 123.79, 122.71, 117.89, 111.49, 111.01, 110.55, 104.89; FT-IR 3392, 3310, 3051, 1617, 1579, 1508, 1473, 1449, 1415, 1305, 1280, 1226, 1187, 967, 782, 720, 606; HRMS (ESI) m/z [M-H]- for C17H11NO3 pred. 276.0666, meas. 276.0657; HPLC purity: 98.01%, retention time: 6.559 min. 5-(6-Hydroxybenzofuran-2-yl)quinolin-8-ol (16c) Yellow solid. 67% yield. Rf = 0.24 (CH2Cl2/CH3OH = 10/1), mp 199.7-200.6 °C. 1H NMR (400 MHz, CDCl3) δ 8.84 (dd, J = 5.6, 1.5 Hz, 1H), 8.83 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.54 (dd, J = 8.3, 4.5 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.24 (s, 1H), 7.07 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 0.8 Hz, 1H), 6.83 (dd, J = 8.4, 2.2 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 157.40, 156.80, 154.97, 154.93, 149.34, 139.92, 135.62, 129.21, 127.67, 123.35, 122.96, 122.04, 120.63, 113.33, 111.58, 105.50, 98.56; FT-IR 3439, 3201, 1623, 1579, 1508, 1489, 1472, 1415, 1276, 1208, 1143, 1110, 972, 814, 784, 713, 621; HRMS (ESI) m/z [M-H]- for C17H11NO3 pred. 276.0666, meas. 276.0662; HPLC purity: 97.87%, retention time: 10.323 min.


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5-(5-Hydroxybenzofuran-2-yl)quinolin-8-ol (16d) Yellow solid. 73% yield. Rf = 0.22 (CH2Cl2/CH3OH = 10/1), mp 194.5-195.3 °C. 1H NMR (400 MHz, MeOD) δ 8.76 (t, J = 6.3 Hz, 2H), 7.74 (d, J = 8.1 Hz, 1H), 7.48 (dd, J = 8.6, 4.2 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 2.3 Hz, 1H), 6.83 (s, 1H), 6.70 (dd, J = 8.8, 2.3 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 157.12, 155.33, 154.57, 150.87, 149.41, 139.93, 135.60, 131.24, 129.65, 127.77, 123.46, 120.41, 113.99, 112.09, 111.55, 106.50, 105.50; FT-IR 3198, 1619, 1602, 1578, 1506, 1463, 1415, 1367, 1285, 1190, 972, 948, 780, 654; HRMS (ESI) m/z [M-H]- for C17H11NO3 pred. 276.0666, meas. 276.0653; HPLC purity: 99.50%, retention time: 9.907 min. 2-(8-Hydroxyquinolin-5-yl)benzofuran-4,6-diol (16e) Yellow solid. 59% yield. Rf = 0.26 (CH2Cl2/CH3OH = 5/1), mp 149.6-150.1 °C. 1H NMR (400 MHz, MeOD) δ 8.71 (d, J = 8.9 Hz, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.43 (dd, J = 7.9, 3.9 Hz, 1H), 7.04 (d, J = 7.9 Hz, 1H), 6.85 (s, 1H), 6.39 (s, 1H), 6.12 (s, 1H). 13C NMR (101 MHz, MeOD) δ 158.60, 157.72, 154.74, 153.07, 152.30, 149.34, 139.96, 135.62, 128.93, 127.63, 123.29, 120.81, 112.61, 111.60, 102.88, 98.79, 90.71; FT-IR 3240, 2922, 2851, 1616, 1580, 1506, 1467, 1414, 1284, 1243, 1133, 991, 813, 785, 720, 637; HRMS (ESI) m/z [M-H]- for C17H11NO4 pred. 292.0615, meas. 292.0606; HPLC purity: 95.59%, retention time: 7.370 min. 5-(5-Hydroxy-3-methylbenzofuran-2-yl)quinolin-8-ol (16f) Yellow solid. 64% yield. Rf = 0.27 (CH2Cl2/CH3OH = 10/1), mp 232.6-233.4 °C. 1H NMR (400 MHz, DMSO) δ 8.93 (dd, J = 4.1, 1.5 Hz, 1H), 8.29 (dd, J = 8.6, 1.5 Hz,



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1H), 7.66 – 7.60 (m, 2H), 7.39 (d, J = 8.7 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 2.3 Hz, 1H), 6.79 (dd, J = 8.7, 2.4 Hz, 1H), 2.15 (s, 3H). 13C NMR (101 MHz, DMSO) δ 154.29, 153.10, 150.55, 148.41, 147.83, 138.35, 134.22, 130.68, 130.14, 127.21, 122.48, 117.79, 112.89, 112.52, 111.12, 110.77, 103.94, 8.79; FT-IR 3232, 2940, 2913, 1603, 1578, 1510, 1455, 1421, 1283, 1175, 1089, 951, 790, 716, 613; HRMS (ESI) m/z [M-H]- for C18H13NO3 pred. 290.0811, meas. 290.0823; HPLC purity: 99.73%, retention time: 6.477 min. 5-(5,7-Diiodobenzofuran-2-yl)quinolin-8-ol (16g) Yellow solid. 58% yield. Rf = 0.21 (CH2Cl2/CH3OH = 10/1), mp 208.1-209.2 °C. 1H NMR (400 MHz, DMSO) δ 8.97 (d, J = 3.3 Hz, 1H), 8.87 (d, J = 8.5 Hz, 1H), 8.06 (s, 1H), 7.97 (dd, J = 8.7, 4.5 Hz, 2H), 7.73 (dd, J = 8.6, 4.0 Hz, 1H), 7.41 (s, 1H), 7.26 (d, J = 8.1 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 155.80, 155.02, 154.28, 148.51, 139.11, 138.23, 133.50, 131.06, 129.37, 129.29, 125.80, 122.95, 116.48, 111.16, 104.41, 88.03, 77.65; FT-IR 3303, 3062, 2923, 1574, 1559, 1506, 1469, 1415, 1394, 1278, 1236, 1189, 1147, 1072, 975, 931, 841, 785, 718, 684; HRMS (ESI) m/z [M-H]- for C17H9NO2I2 pred. 511.8650, meas. 511.8646; HPLC purity: 99.48%, retention time: 6.486 min. 2-(6-Hydroxybenzofuran-2-yl)quinolin-8-ol (16h) Yellow solid. 66% yield. Rf = 0.29 (CH2Cl2/CH3OH = 10/1), mp 315.4-317.1 °C. 1H NMR (400 MHz, DMSO) δ 8.41 (d, J = 8.7 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.96 (d, J = 0.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.49 – 7.37 (m, 2H), 7.14 (dd, J = 6.6, 2.3 Hz, 1H), 7.06 (d, J = 0.8 Hz, 1H), 6.84 (dd, J = 8.4, 2.1 Hz, 1H). 13C NMR (101 MHz,


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DMSO) δ 157.14, 156.41, 152.18, 152.08, 145.94, 138.13, 136.78, 127.71, 122.44, 120.42, 117.93, 113.51, 112.38, 108.26, 97.65; FT-IR 3406, 3230, 2261, 1604, 1560, 1491, 1442, 1375, 1292, 1194, 1163, 1119, 835, 747, 636; HRMS (ESI) m/z [M-H]for C17H11NO3 pred. 276.0666, meas. 276.0654; HPLC purity: 96.27%, retention time: 6.714 min. 2-(8-Hydroxyquinolin-2-yl)benzofuran-4,6-diol (16i) Yellow solid. 75% yield. Rf = 0.31 (CH2Cl2/CH3OH = 5/1), mp 148.9-150.0 °C. 1H NMR (400 MHz, DMSO) δ 8.37 (d, J = 8.7 Hz, 1H), 8.00 (t, J = 4.2 Hz, 2H), 7.42 (q, J = 8.0 Hz, 2H), 7.15 (dd, J = 6.6, 1.4 Hz, 1H), 6.58 (s, 1H), 6.32 (d, J = 1.3 Hz, 1H). 13

C NMR (101 MHz, DMSO) δ 157.80, 157.44, 152.75, 152.01, 151.55, 146.66,

138.17, 136.88, 127.49, 127.13, 117.72, 117.55, 111.54, 110.89, 105.00, 98.07, 89.40; FT-IR 3236, 1638, 1601, 1562, 1509, 1453, 1321, 1235, 1193, 1145, 1087, 991, 939, 828, 737, 721, 621; HRMS (ESI) m/z [M-H]- for C17H11NO4 pred. 292.0615, meas. 292.0605; HPLC purity: 95.74%, retention time: 5.401 min. 5-(Benzofuran-2-yl)-7-chloroquinolin-8-ol (17a) Yellow solid. 70% yield. Rf = 0.28 (CH2Cl2/CH3OH = 20/1), mp 169.0-170.1 °C. 1H NMR (400 MHz, DMSO) δ 9.05 – 8.99 (m, 1H), 8.91 (dd, J = 8.7, 1.0 Hz, 1H), 8.03 (s, 1H), 7.79 – 7.66 (m, 3H), 7.49 – 7.28 (m, 3H). 13C NMR (101 MHz, DMSO) δ 154.26, 152.92, 150.22, 149.24, 138.53, 134.50, 128.61, 128.55, 124.66, 124.51, 123.26, 122.97, 121.19, 118.37, 115.77, 111.16, 105.61; FT-IR 3394, 2923, 1576, 1503, 1450, 1409, 1287, 1217, 975, 880, 812, 787, 724, 656; HRMS (ESI) m/z



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[M-H]- for C17H10NO2Cl pred. 294.0327, meas. 294.0314; HPLC purity: 99.60%, retention time: 15.637 min. 7-Chloro-5-(7-hydroxybenzofuran-2-yl)quinolin-8-ol(17b) Yellow solid. 57% yield. Rf = 0.24 (CH2Cl2/CH3OH = 20/1), mp 220.0-221.1 °C. 1H NMR (400 MHz, MeOD) δ 9.06 – 8.57 (m, 2H), 7.88 (s, 1H), 7.52 (d, J = 4.2 Hz, 1H), 7.22 – 6.81 (m, 3H), 6.69 (d, J = 7.5 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 152.59, 150.24, 149.35, 145.46, 143.20, 138.73, 134.35, 130.39, 128.45, 124.46, 123.98, 122.95, 118.48, 115.61, 111.68, 110.86, 105.94; FT-IR 3532, 3048, 2611, 1607, 1578, 1503, 1404, 1409, 1286, 1221, 1201, 972, 883, 813, 788, 720, 656; HRMS (ESI) m/z [M-H]- for C17H10NO3Cl pred. 310.0276, meas. 310.0263; HPLC purity: 95.59%, retention time: 7.366 min. 7-Chloro-5-(6-hydroxybenzofuran-2-yl)quinolin-8-ol (17c) Yellow solid. 62% yield. Rf = 0.23 (CH2Cl2/CH3OH = 20/1), mp 211.0-211.8 °C. 1H NMR (400 MHz, DMSO) δ 9.00 (dd, J = 4.1, 1.4 Hz, 1H), 8.88 (dd, J = 8.7, 1.4 Hz, 1H), 7.95 (s, 1H), 7.73 (dd, J = 8.7, 4.1 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.04 (d, J = 1.5 Hz, 1H), 6.82 (dd, J = 8.4, 2.1 Hz, 1H).13C NMR (101 MHz, DMSO) δ 155.81, 155.46, 151.17, 149.80, 149.12, 138.74, 134.19, 127.68, 124.21, 122.66, 121.26, 120.61, 118.69, 115.60, 112.59, 105.45, 97.53; FT-IR 3400, 3041, 1626, 1579, 1500, 1490, 1409, 1293, 1199, 1140, 1112, 976, 881, 813, 787, 725, 656; HRMS (ESI) m/z [M-H]- for C17H10NO3Cl pred. 310.0276, meas. 310.0262; HPLC purity: 97.70%, retention time: 13.463 min. 7-Chloro-5-(5-hydroxybenzofuran-2-yl)quinolin-8-ol (17d)


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Yellow solid. 73% yield. Rf = 0.22 (CH2Cl2/CH3OH = 20/1), mp 221.4-222.0 °C. 1H NMR (400 MHz, DMSO) δ 8.99 (dd, J = 4.0, 1.3 Hz, 1H), 8.88 (dd, J = 8.7, 1.3 Hz, 1H), 7.96 (s, 1H), 7.72 (dd, J = 8.7, 4.1 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.25 (s, 1H), 7.00 (d, J = 2.4 Hz, 1H), 6.79 (dd, J = 8.8, 2.4 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 153.47, 153.38, 150.20, 149.25, 148.52, 138.71, 134.25, 129.31, 128.24, 124.38, 122.84, 118.53, 115.60, 113.37, 111.35, 105.47, 105.34; FT-IR 3531, 3168, 1604, 1500, 1456, 1409, 1240, 1197, 881, 784, 655; HRMS (ESI) m/z [M-H]- for C17H10NO3Cl pred. 310.0276, meas. 310.0261; HPLC purity: 99.21%, retention time: 6.994 min. 7-Chloro-5-(5-hydroxy-3-methylbenzofuran-2-yl)quinolin-8-ol (17f) Yellow solid. 53% yield. Rf = 0.25 (CH2Cl2/CH3OH = 20/1), mp 223.4-224.6 °C. 1H NMR (400 MHz, DMSO) δ 8.99 (dd, J = 4.1, 1.4 Hz, 1H), 8.32 (dd, J = 8.6, 1.4 Hz, 1H), 7.75 (s, 1H), 7.67 (dd, J = 8.6, 4.2 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.82 (dd, J = 8.8, 2.4 Hz, 1H), 2.17 (s, 3H). 13C NMR (101 MHz, DMSO) δ 153.73, 150.58, 149.71, 149.34, 148.42, 138.81, 135.66, 130.98, 130.72, 126.42, 123.27, 119.04, 116.10, 114.06, 113.82, 111.79, 104.55, 9.28; FT-IR 3194, 2918, 1610, 1580, 1503, 1459, 1412, 1291, 1277, 1197, 1094, 955, 881, 790, 721, 657; HRMS (ESI) m/z [M-H]- for C18H12NO3Cl pred. 324.0433, meas. 324.0426; HPLC purity: 96.86%, retention time: 7.267 min. 5-(6-Hydroxybenzofuran-2-yl)-7-iodoquinolin-8-ol (18c) Yellow solid. 67% yield. Rf = 0.24 (CH2Cl2/CH3OH = 20/1), mp 175.4-176.5 °C. 1H NMR (400 MHz, DMSO) δ 8.95 (d, J = 3.6 Hz, 1H), 8.84 (d, J = 8.6 Hz, 1H), 8.19 (s,



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1H), 7.73 (dd, J = 8.6, 4.0 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.23 (s, 1H), 7.03 (s, 1H), 6.80 (dd, J = 8.4, 1.8 Hz, 1H).13C NMR (101 MHz, DMSO) δ 155.75, 155.43, 154.22, 150.96, 148.93, 137.25, 135.38, 134.28, 125.23, 123.00, 121.23, 120.64, 119.73, 112.54, 105.32, 97.51, 79.33; FT-IR 3280, 2923, 1623, 1601, 1573, 1491, 1442, 1402, 1372, 1293, 1139, 1112, 975, 815, 786, 721, 651; HRMS (ESI) m/z [M-H]- for C17H10NO3I pred. 401.9633, meas. 401.9616; HPLC purity: 97.21%, retention time: 8.145 min. 5-(5-Hydroxybenzofuran-2-yl)-7-iodoquinolin-8-ol (18d) Yellow solid. 75% yield. Rf = 0.25 (CH2Cl2/CH3OH = 20/1), mp 159.7-160.2 °C. 1H NMR (400 MHz, DMSO) δ 8.97 (d, J = 3.0 Hz, 1H), 8.87 (d, J = 8.5 Hz, 1H), 8.23 (s, 1H), 7.75 (dd, J = 8.6, 4.0 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.24 (s, 1H), 7.01 (d, J = 2.3 Hz, 1H), 6.80 (dd, J = 8.8, 2.3 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 154.62, 153.44, 153.19, 149.01, 148.49, 137.22, 135.93, 134.30, 129.36, 125.37, 123.11, 119.56, 119.53, 113.28, 111.32, 105.30, 79.32; FT-IR 3298, 1597, 1494, 1451, 1399, 1285, 1193, 976, 847, 814, 786, 718, 650; HRMS (ESI) m/z [M-H]- for C17H10NO3I pred. 401.9633, meas. 401.9622; HPLC purity: 96.75%, retention time: 8.039 min. 5-(5-Hydroxybenzofuran-2-yl)-7-iodoquinolin-8-ol hydrochloride (18d•HCl) Compound 18d (5.0 g, HPLC purity > 96%) was dissolved in 50 mL EtOAc, HCl gas was bubbled into the solution at room temperature for 12 h. The mixture was filtrated off and the orange precipitation was then washed with 40 mL EtOAc, dried under reduced pressure as a orange solid which was used in the vivo assay and metabolic stability study. Orange solid. 97% yield. Rf = 0.25 (CH2Cl2/CH3OH = 20/1), mp


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216.3-217.0 °C. 1H NMR (400 MHz, DMSO) δ 9.00 (d, J = 4.0 Hz, 1H), 8.95 (d, J = 8.6 Hz, 1H), 8.26 (s, 1H), 7.81 (dd, J = 8.6, 4.2 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.26 (s, 1H), 7.05 (d, J = 1.5 Hz, 1H), 6.83 (dd, J = 8.7, 1.7 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 154.41, 154.02, 153.43, 149.04, 136.74, 136.15, 129.80, 125.99, 123.68, 120.27, 113.93, 111.86, 106.07, 105.86, 81.26. HRMS (ESI) m/z [M+H]+ for C17H10NO3I pred. 403.9778, meas. 403.9779; HPLC purity: 98.96%, retention time: 9.761 min (eluted with CH3CN/H2O = 70/30, 0.5 mL/min). 2-(8-Hydroxy-7-iodoquinolin-5-yl)benzofuran-4,6-diol (18e) Yellow solid. 77% yield. Rf = 0.28 (CH2Cl2/CH3OH = 10/1), mp 182.4-183.9 °C. 1H NMR (400 MHz, DMSO) δ 8.95 (dd, J = 4.1, 1.4 Hz, 1H), 8.81 (dd, J = 8.7, 1.4 Hz, 1H), 8.15 (s, 1H), 7.74 (dd, J = 8.7, 4.1 Hz, 1H), 7.18 (s, 1H), 6.51 (d, J = 0.7 Hz, 1H), 6.24 (d, J = 1.7 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 156.74, 156.63, 153.94, 151.12, 148.95, 148.86, 137.28, 135.06, 134.26, 125.18, 122.99, 119.96, 110.51, 103.02, 97.83, 89.30, 79.34; FT-IR 3235, 1608, 1496, 1454, 1401, 1335, 1281, 1200, 1157, 1118, 1062, 988, 809, 651; HRMS (ESI) m/z [M-H]- for C17H10NO4I pred. 417.9582, meas. 417.9568; HPLC purity: 99.59%, retention time: 7.599 min.

Biological assays Human PDE4D2 Inhibitory Screening Assays The expression and purification of the human PDE4D2 (catalytic domain, residues 86–413) protein was performed according to a previously described method.50, 51 The inhibition of enzymatic activities by the synthesized compounds were performed



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using 3H-cAMP (18000-22000 cpm/assay) as the substrate, and the reactions were conducted in buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM DTT, pH 7.5) at room temperature (25 °C) for 15 min. Then, 0.2 M ZnSO4 and 0.2 M Ba(OH)2 were added to terminate the reaction. The reacted product 3H-AMP was precipitated, while the unreacted 3H-cAMP remained in the supernatant and was used to measure the radioactivity in 1.8 mL of Ultima Gold liquid scintillation cocktails (PerkinElmer) using a liquid scintillation analyzer (TRI-CARB 2900TR). The IC50 values were calculated by non-linear regression of at least three independent experiments. The IC50 values of the reference compound, rolipram, were measured before other assays were performed. ThT assay72 Aβ1–42 (Millipore, counter ion: NaOH) was dissolved in ammonium hydroxide (1% v/v) to give a stock solution (2000 µM), which was aliquoted into small samples and stored at -80 °C. For the experiment of self-mediated Aβ1–42 aggregation inhibition, the Aβ stock solution was diluted with 50 mM phosphate buffer (pH 7.4) to 50 µM before use. A mixture of the peptide (10 µL, 25 µM, final concentration) with or without the tested compound (10 µL) was incubated at 37 °C for 48 h. Blanks using 50 mM phosphate buffer (pH 7.4) instead of Aβ with or without inhibitors were also carried out. The sample was diluted to a final volume of 200 µL with 50 mM glycine–NaOH buffer (pH 8.0) containing thioflavin T (5 µM). Then the fluorescence intensities were recorded five minutes later (excitation, 450 nm; emission, 485 nm). The percent


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inhibition of aggregation was calculated by the expression (1-IFi/IFc) × 100%，in which IFi and IFc are the fluorescence intensities obtained for Aβ in the presence and absence of inhibitors after subtracting the background, respectively. For the experiment of copper-mediated Aβ1–42 aggregation inhibition, the Aβ stock solution was diluted in 20 µM HEPES (pH 6.6) with 150 µM NaCl. The mixture of the peptide (10 µL, 25 µM, final concentration) with or without copper (10 µL, 25 µM, final concentration) and the tested compound (10 µL, 50 µM, final concentration) was incubated at 37 °C for 24 h. Then 20 µL of the sample was diluted to a final volume of 200 µL with 50 mM glycine–NaOH buffer (pH 8.0) containing thioflavin T (5 µM). The detection method was the same as that of self-mediated Aβ1–42 aggregation experiment. For the experiment of self-induced Aβ fibrils disaggregation, the Aβ stock solution was diluted with 10 mM phosphate buffer (pH 7.4). The peptide (15µL, 50 µM) was incubated at 37 °C for 24 h. The tested compound (15µL, 50 µM) was then added and incubated at 37 °C for another 24 h. Then 20 µL of the sample was diluted to a final volume of 200 µL with 50 mM glycine–NaOH buffer (pH 8.0) containing thioflavin T (5 µM). The detection method was the same as above. For the experiment of copper-induced Aβ fibrils disaggregation, the Aβ stock solution was diluted in 20 µM HEPES (pH 6.6) with 150 µM NaCl. The mixture of the peptide (10 µL, 25 µM, final concentration) with copper (10 µL, 25 µM, final concentration) was incubated at 37 oC for 24 h. The tested compound (10µL, 50 µM, final concentration) was then added and incubated at 37 °C for another 24 h.50 Then



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20 µL of the sample was diluted to a final volume of 200 µL with 50 mM glycine–NaOH buffer (pH 8.0) containing thioflavin T (5 µM). The detection method was the same as above. 2D NMR Spectroscopy The interaction of monomeric Aβ1–42 with compound 18d was determined by Two-dimensional (2-D) Translational Relaxation Optimized Spectroscopy (TROSY) 1

H-15N Heteronuclear Single Quantum Correlation (HSQC) NMR measurements by

titrating a 50 µM solution of

15

compound 18d in DMSO-d6.54,

N-labeled Aβ1-42 with a 25 mM stock solution of 73

The concentration of DMSO-d6 used for each

sample was less than 1% v/v (final concentration), so that the influence of DMSO-d6 on the spectrum of Aβ was minor.14, 74 Compound 18d was well solubilized (with no precipitation at 4 °C) in the buffer at the concentration used for the experiment. The 15

N-labeled Aβ1-42 was purchased from rPeptide (Bogart, GA, U. S. A.) and stored at

-80 °C. One sample (1.0 mg) was predissolved in aqueous NaOH solution (1 mM, pH = 10) in a 1:1 ratio (mg:mL) with sonication for 1 min at 4 °C. The basic pH = 10 solution of the

15

N-labeled Aβ1-42 peptides was diluted with a 4 °C potassium

phosphate buffer solution (5 mM, pH 7.5) that contained 0.50 mM ethylenediamine tetraacetic acid (Na2EDTA), and 0.05 mM NaN3, 7% D2O (v/v), verified to be pH 7.5 before the start of each titration. Each spectrum was obtained with 9.5 kHz, a 2.7 kHz spectral width, 2048 and 256 complex data points in the 1H and

15

N dimensions,

respectively, 96 scans per free induction decay, and a 1.0 s relaxation delay on a Bruker Avance 950 MHz spectrometer at 4 °C. The 2D data were processed using


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TOPSPIN 3.2 (from Bruker) and analyzed with Sparky version 3.113. The 1H-15N HSQC peaks were assigned by comparing them with previously published values.20, 54, 73, 75, 76

The normalized chemical shift perturbations were calculated from equation

1.52

∆δ =

(∆δ ) ( .(∆δ ))

(eq 1)

TEM assay77 For the metal-free experiment, Aβ stock solution was diluted with a 10 mM phosphate buffer (pH = 7.4); for the copper-induced experiment, Aβ stock solution was diluted with 20 µM HEPES (pH = 6.6) and 150 µM NaCl. The sample preparation was same as that for the ThT assay. Aliquots (10 µL) of the samples were placed on a carbon-coated copper/rhodium grid for 2 min. Each grid was stained with uranyl acetate (1%, 5 µL) for 2 min. After draining off the excess staining solution, the specimen was transferred for imaging in a transmission electron microscope (JEOL JEM-1400). All compounds are solubilized in the buffer which was used for the experiment. Oxygen radical absorbance capacity (ORAC-FL) assay78, 79 The tested compound and fluorescein (FL) stock solution were diluted with 75 mM phosphate buffer (pH 7.4) to 10 µM (or 20 µM) and 0.117 µM, respectively. The solution of (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was diluted with the same buffer to 100, 80, 60, 50, 40, 20, and 10 µM. The solution of 2,2'-azobis-(amidinopropane)dihydrochloride (AAPH) was prepared by dissolving 108.4 mg AAPH in 10 mL 75 mM phosphate buffer (pH 7.4) to a final concentration



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of 40 mM. The mixture of the tested compound (20 µL) and FL (120 µL; 70 nM, final concentration) was pre-incubated for 10 min at 37ºC, and then 60 µL of the AAPH solution was added. The fluorescence was recorded every minute for 120 min (excitation, 485 nm; emission, 520 nm). A blank using phosphate buffer instead of the tested compound was also carried out. All reaction mixtures were prepared triple and at least three independent runs were performed for each sample. The Antioxidant curves (fluorescence versus time) were normalized to the curve of the blank. The area under the fluorescence decay curve (AUC) was calculated as following equation:

AUC = 1 + (fi/f0)

Where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i. The net AUC was calculated by the expression: AUCsample – AUCblank. Regression equations between net AUC and Trolox concentrations were calculated. ORAC-FL value for each sample were calculated by using the standard curve which means the ORAC-FL value of tested compound expressed as Trolox equivalents. Inhibiting NO Production of LPS-Stimulated BV2 Microglial Cells10 The BV2 microglial cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. The BV2 microglial cells were plated in 96-well microplates at a density of 4 × 105 cells/mL (100 µL/well). After the cells had attached, the medium was removed and the BV2 microglial cells were incubated with the compounds and LPS (purchased from Sigma, diluted with medium to a final


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concentration 100 nM) for 24 h. The same volume of Griess assay agent was added into the 96-well plates and mixed with the culture medium. The mixture solution was incubated at room temperature for 10 min, and the absorbance at 540 nm was measured in a multifunctional microplate reader. The percent of inhibition of NO production was calculated by the formula: (FL – F0 – FC)/(FL – F0) × 100, where FC = absorbance of the neurons treated with the tested compound and LPS, FL = absorbance of the neurons treated with LPS, and F0 = absorbance of the normal neurons. The IC50 values of the tested compounds were calculated by linear regression plots from at least three independent experiments. Blood-Brain Barrier Permeation Assay The blood-brain barrier penetration of compounds was evaluated using the parallel artificial membrane permeation assay (PAMPA) described by Di et al.80-82 Commercial drugs were purchased from Sigma and Alfa Aesar. Porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm) and acceptor microplate were both from Millipore. The 96-well UV plate (COSTAR®) was from Corning Incorporated. The acceptor 96-well microplate was filled with 300 µL PBS/EtOH (7:3), and the filter membrane was impregnated with 4 µL PBL in dodecane (20 mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to a final concentration of 100 µg/mL. Then, 200 µL of the solution was added to the donor wells. The acceptor filter plate was carefully placed on the donor plate to form a sandwich, which was left undisturbed for 10 h at 25°C. After incubation, the donor



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plate was carefully removed, and the concentration of compounds in the acceptor wells was determined using the UV plate reader (Flexstation® 3). Every sample was analyzed at five wavelengths in four wells and in at least three independent runs. Pe was calculated by the following expression: Pe = -Vd×Va/[(Vd+Va)A×t] × ln(1-drugacceptor/drugequilibrium) where Vd is the volume of donor well; Va, volume in acceptor well; A, filter area; t, permeation time; drugacceptor, the absorbance obtained in the acceptor well; drugequilibrium, the theoretical equilibrium absorbance. The results are given as the mean ± standard deviation. In the experiment, 13 quality control standards (Table S1, see Supporting Information) of known BBB permeability were included to validate the analysis set. A plot of the experimental data versus literature values gave a strong linear correlation, Pe (exp.) = 1.4574Pe (lit.) - 1.0773 (R2 = 0.9427) (Figure S2). From this equation and the limit established by Di et al. ( Pe (lit.) = 4.0× 10-6 cm/s) for blood-brain barrier permeation, we concluded that compounds with a permeability greater than 4.7 × 10-6 cm/s could cross the blood-brain barrier (Table S2). Metal-chelating study The chelating studies were performed with a UV-Vis spectrophotometer. The absorption spectra of each compound (50 µM, final concentration) alone or in the presence of CuSO4, FeSO4, FeCl3 or ZnCl2 (50 µM, final concentration) for 30 min in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were recorded at room temperature. For the stoichiometry of the compound-Cu2+ complex, a fixed amount of 18d (50 µM) was mixed with growing amounts of copper ion (0-50 µM), and the difference UV-vis


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spectra were examined to investigate the ratio of ligand/metal in the complex. Metabolic Stability Rat liver microsomes were prepared according to previously described methods.83 Compounds 18d•HCl, CQ, donepezil and testosterone were dissolved in CH3CN (for 18d•HCl, CH3CN : H2O = 60/40) as a 10 mM stock solution and incubated with rat liver microsomes (1 mg of protein per mL, final concentration) at a final concentration of 100 µM in a final volume of 0.5 mL buffer solution (100.0 mM PBS; 3.0 mM MgCl2; 1.3 mM β-NADPNa2; 3.3 mM glucose 6-phosphate; 0.4 units/mL glucose 6-phosphate dehydrogenase; pH = 7.4). The samples were incubated for various time intervals (0, 5, 10, 30, 60, 90, 120, 150, 180 min) at 37 °C in a water bath. The incubations were terminated at different time points by adding 0.5 mL of ice-cold CH3CN.

A

parallel

incubation

was

performed

in

the

absence

of

a

NADPH-regenerating system and using microsomes as the negative control, and these reactions were terminated after the corresponding incubation times. After centrifugation with 12500 rpm at 4 °C for 10 min, the supernatants were directly analyzed by a HPLC-UV system (Agilent HPLC 1200 instrument, Fig. S3 and S4). Three independent experiments were performed in triplicate (Fig. S5). Acute toxicity The procedure to test for acute toxicity study followed similar protocols from our previous studies.56, 57 A total of 20 KM mice (KM mice, which are common closed colony mice and most widely used in biomedical research in China; male, 22 days, 18-20 g) purchased from the laboratory animal center of Sun Yat-sen University



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(Guangzhou, China) were used to evaluate the acute toxicity of compound 18d•HCl. Mice were maintained with a 12-h light/dark cycle (light from 07:00 to 19:00) at 20-22 °C and 60-70% relative humidity. Sterile food and water were provided according to institutional guidelines. Prior to each experiment, mice were fasted overnight and allowed free access to water. Compound 18d•HCl was suspended 0.5% carboxymethyl cellulose sodium (CMC-Na) salt solution (677, 1333, and 2000 mg/kg) and given via oral administration according to the divided experimental groups. After administration of the compounds, the mice were observed continuously for the first 4 h for any abnormal behavior and mortality changes, intermittently for the next 24 h, and occasionally thereafter for 14 days for the onset of any delayed effects. All animals were sacrificed on the 14th day after drug administration and were macroscopically examined for possible damage to the heart, liver, and kidneys. Cognitive and Memory Improvement in a Rat Model of AD Chemicals and reagents. Aβ1-42 (Sigma) was dissolved in sterilized normal saline (5 µg/µL) and incubated at 37 °C for 7 days to produce the neurotoxic form (aggregated form). CQ, donepezil and compound 18d•HCl were dissolved in a 0.5% carboxymethyl cellulose sodium (CMC-Na) salt solution. Animals. Fifty-five male Wistar rats (270-300 g), purchased from the laboratory animal center of Sun Yat-sen University (Guangzhou, China), were randomly allocated into 5 groups (n = 11 for each group): sham, model, donepezil (2 mg/kg/day), CQ (30 mg/kg/day) and compound 18d•HCl (30 mg/kg/day). The rats were maintained on a 12 h light/dark cycle (light from 07:00 to 19:00) at 23 °C and


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60-70% relative humidity, with free access to sterile food and water. All experiments were strictly executed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Ethics Committee of Sun Yat-sen University. Intrahippocampal Injection of Aβ. Stereotaxic surgery was performed under chloral hydrate (7%, intraperitoneal injection) anesthesia (350 mg/kg body weight). The solution of the aggregated form Aβ1-42 (2 µL, 5 µg/µL) was laterally injected into the hippocampal fissure using a microsyringe (10 µL-gauge). The stereotaxic coordinates were determined as AP = – 4.2 mm; L= 3.0 mm from the bregma, H= + 4.2 mm from the dura. The injection lasted 2 min (1 µL/min) and the syringe was left in place for 8 min after the infusion was complete. The sham rats were injected with 2 µL of saline using the same procedure. The animals were maintained after suture until they recuperated from the anesthesia. After the surgery, the animals were taken to their individual cages and the rats in each group were administered the corresponding drug dosage in a 0.5% CMC-Na solution (4 mL/kg) by intragastric infusion throughout the entire assay (using a blank 0.5% CMC-Na solution for the sham and model groups). Morris Water Maze Test. Twenty-six days later, behavioral performance was evaluated using the Morris water maze task. (The water maze apparatus consisted of a circular pool, 121 cm in diameter and 62 cm high, filled to a depth of 32 cm with fresh, opaque water (kept at 25 °C, the water was refilled each day before the trails), which was made opaque by the addition of non-toxic white TiO2 at a concentration of



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0.25 g/L.) The task demanded incremental learning of the location of a fixed, hidden platform (10 cm in diameter and 30 cm high, submerged 2.0 cm under the water level) throughout the training period. In the single training assay, each rat underwent reference memory training (four trials per day and a 30 min intertrial interval) with a fixed hidden-platform located in the center of first quadrant (northeast) of the pool for 5 consecutive days. The rats were placed in the water facing the wall of the pool from one of the four designated starting locations (west, north, south and east), each of the starting locations was used once in the four trials and the order of the starting locations was varied between the 5 consecutive training days. If the rat did not find the platform within 90 s, the rat was guided onto the platform and the escape latency was recorded as 90 s. For each trial, the rat was allowed 10 s on the platform, and the escape latency and trajectories were recorded using a computerized-digitizing video-tracking system (Chromo track, Meng Tai Instruments, ChengDu, China). All of experiments were performed between 9:00 and 15:00 at 2 h after each drug infusion, and the room temperature was maintained at 25 °C. The extra-maze visual cues remained in fixed positions in the room where the Morris water maze apparatus was located; olfactory auditory cues were minimized by maintaining a fresh and quiet environment. To assess memory retention, a spatial probe trial was performed 24 h after the last training trial, with the platform removed from the pool and the two-fold diameter of the platform was set as the effective region. Each rat was allowed to swim freely in the pool for 90 s (only one trial) from the southwest, a new starting location, which was farthest from the virtual platform location. The number of times the


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swimming rat crossed the virtual platform location (former platform location), the average swimming speed, and the time spent and the swimming path length in the virtual platform and the effective region were recorded by the computerized-digitizing video-tracking system and to analyze the level of memory retention. Hematoxylin and Eosin (HE) Staining and Histopathological Studies in the Hippocampus After the Morris water maze tasks were completed, all of the rats in each group were anesthetized with 7% chloral hydrate (350 mg/kg) by intraperitoneal injection and the heart was exposed. Normal saline (300 mL, 4 °C) was perfused into the aorta through a left ventricular catheter for 2 min, and, subsequently, 4% paraformaldehyde (200 mL, 4 °C) was perfused until the tail and limbs were rigid and pale. The brain was carefully removed, post-fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin. Coronal sections (4 µm) were cut through the entire hippocampus at a site 4.0 mm behind the bregma for HE staining. The paraffin sections were conventionally dewaxed and rehydrated, followed by hematoxylin staining for 5 min and eosin staining for 1 min. Then, the sections were dehydrated in a graded series of alcohol, made transparent with xylene, and sealed. The hippocampal histopathological abnormalities were investigated under a light microscope. The denatured cell index (DCI = the number of denatured cells/the number of total cells) in the hippocampal region of each section was examined in a blinded manner by 2 pathologists, and the average number was used as the final result.



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Page 72 of 90

Statistical Analysis All data were expressed as mean ± standard deviation, results were subjected to Student’s t test or one-way analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.05 was accepted to indicate the significance.

Supporting Information Supporting Information Available: The HPLC data and FT-IR spectra of the target compounds, the method for PAMPA, meta-chelating data and in vitro metabolic stability. Corresponding author *For

L.H.:

phone,

+086-20-3994-3051;

[email protected].

For

X.L.:

fax, phone,

+086-20-3994-3051; +086-20-3994-3050;

e-mail, fax,

+086-20-3994-3050; e-mail, [email protected]. Acknowledgments We thank the National Natural Science Foundation of China (No. 21302235, 20972198), Guangdong Natural Science Foundation (2014A030313124) and the Fundamental Research Funds for the Central Universities (15ykpy04) for financial support of this study. All NMR experiments for Aβ were performed at the Beijing NMR Center and the NMR facility of the National Center for Protein Sciences at Peking University. ABBREVIATIONS AD, Alzheimer's disease; Aβ, amyloid-β; BBB, blood-brain barrier; cAMP, cyclic


Page 73 of 90

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


adenosine monophosphate; CMC-Na, carboxymethyl cellulose sodium; CNS, central nervous system; CQ, clioquinol; CSP, chemical shift perturbations; DG, dentate gyrus; DCI, denatured cell index; HSQC, Heteronuclear Single Quantum Correlation; HE, hematoxylin

and

eosin;

lipopolysaccharide

;

HRMS, MTDLs,

high-resolution

mass

multi-target-directed

spectra; ligands;

LPS, MTT,

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NO, nitric oxide; ORAC, oxygen radical Absorbance Capacity; PAMPA, parallel artificial membrane permeability; PDE4D, Phosphodiesterase 4D; PBL, Porcine brain lipid; ROS, reactive oxygen species; ThT, thioflavin T; TROSY, Translational Relaxation Optimized Spectroscopy;

TEM,

transmission

electron

microscopy;

TLC,

thin-layer

chromatography.

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