Design, Synthesis, and Evaluation of Orally Bioavailable Quinoline

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2018, 61, 1871−1894

Design, Synthesis, and Evaluation of Orally Bioavailable Quinoline− Indole Derivatives as Innovative Multitarget-Directed Ligands: Promotion of Cell Proliferation in the Adult Murine Hippocampus for the Treatment of Alzheimer’s Disease Zhiren Wang,†,‡ Jinhui Hu,† Xiaoping Yang,‡ Xing Feng,‡ Xingshu Li,*,† Ling Huang,*,† and Albert S. C. Chan† †

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, School of Medicine, Hunan Normal University, Changsha 410013, China

‡

S Supporting Information *

ABSTRACT: A novel series of quinoline−indole derivatives were synthesized and evaluated as multitarget-directed ligands for the treatment of Alzheimer’s disease (AD). Biological evaluation revealed that the derivatives had multifunctional profiles including antioxidant effects, blood−brain barrier (BBB) penetration, biometal chelation, Aβ aggregation modulation, neurotrophic and neuroprotective properties. Moreover, several representative target derivatives demonstrated hippocampal cell proliferation in living adult mice by intracerebroventricular (icv) injection or oral administration. Further drug-like property analysis demonstrated that the optimized compound, 8d (WI-1758), had liver microsomal metabolic stability, was well tolerated (>2000 mg/kg), and had a rational pharmacokinetic profile, as well as an oral bioavailability of 14.1% and a positive log BB (−0.19) to cross the BBB in vivo. Pharmacodynamics studies demonstrated that chronic oral administration of 8d·HCl substantially ameliorated the cognitive and spatial memory deficits in APP/PS1 AD mice and noticeably reduced overall cerebral β-amyloid deposits.

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INTRODUCTION Alzheimer’s disease (AD), the most prevalent and chronic type of neurodegenerative disease, affects approximately 47 million patients worldwide.1 The pathologic changes of AD are convoluted and multilayered; these changes include the aberrant accumulation of amyloid-β (Aβ) plaques, loss of synapses, neurofibrillary tangles within neurons, oxidative stress, dyshomeostasis of biometals in the brain, etc.2 Presently, the primary therapeutic options for AD treatment are acetylcholinesterase inhibitors (AChEIs), such as donepezil, rivastigmine, and galantamine, as well as the N-methyl-Daspartate (NMDA) receptor (NMDAR) antagonist, memantine, which can improve memory and cognitive function to a certain extent but cannot definitively cure this disease. Due to its complex pathogenesis, the multitarget-directed ligands (MTDLs) approach for AD treatment has been developed in both symptomatic and disease-modifying efficiencies.3−6 Among the numerous neuropathological biomarkers of AD, the extracellular Aβ plaque deposits between neurons and intracellular neurofibrillary tangles within the brains of patient are primary markers.7 The self-assembled and hydrophobic aggregations of Aβ peptides are associated with complex © 2018 American Chemical Society

pathways, soluble oligomers, paranuclei, and protofibrils, and they ultimately generate insoluble filaments.8 The dyshomeostasis of biometals is another biomarker, and through comparison with the age-matched healthy parenchyma within the brains of AD patients, 3- to 5-fold higher concentrations of metal ions in Aβ plaque deposits, such as Cu2+ (0.4 mM, 5.7 times), Zn2+ (1.0 mM, 2.8 times), and Fe2+/3+ (0.9 mM, 2.9 times), were found. These biometals have a comparatively greater affinity toward the N-terminus of the Aβ peptide, especially at residues His6, His13, and His14, which aggravated Aβ aggregation and exacerbated their noxious properties.9 Furthermore, the dyshomeostasis redox active metal ions, including those unbonded and bonded with Aβ peptides, could exacerbate the overproduction of reactive oxygen species (ROS) that jeopardizes biological tissue and finally results in neuronal damage or apoptosis in the central nervous system (CNS) of AD patients.10 Consequently, the use of appropriate metal chelating agents to inhibit the production or accumulation of Aβ plaques and restore biometal homeostasis in the Received: September 23, 2017 Published: February 8, 2018 1871

DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894

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Figure 1. Structures of CQ, multitarget-directed metal-chelating agents, melatonin, melatonin−N-benzylamine hybrids, and several small molecules reported to manipulate neuronal cells in vitro and in vivo.

benzoyl)benzamide (P7C3-S326, Figure 1), which has a preeminent ability to promote adult hippocampal neurogenesis, demonstrated significant efficacies in rodent models of Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), traumatic brain injury (TBI), and age-related cognitive decline.17,18,25−29 Interestingly, the melatonin-N-benzylamine hybrids (Figure 1), which have an indole framework, can promote the effective development of neural stem cells into the neuronal phenotype.6,30 Taking into account neuronal loss, deficits in adult neurogenesis, and the formation of plaques and tangles associated with AD, in this work, we merged the skeleton of indole and CQ to generate a series of quinoline−indole scaffolds to study their ability to modulate adult hippocampal neurogenesis in vivo, biometal dyshomeostasis, and antiamyloid aggregation (Figure 2). Through structure−activity relationship

brains of AD patients has been developed as a potent therapeutic strategy for AD treatment.11 Clioquinol (CQ, Figure 1) and its analogs, with 8-hydroxyquinoline as a crucial scaffold, have a moderate affinity for metal ions and can extract metals from extracellular Aβ aggregates and shift them to copper-carrier proteins in the parenchymal cells without perturbing the overall essential metal biochemistry in the brain, demonstrating safety, noticeably strengthened cognition, and substantially decreased cerebrospinal fluid (CSF) levels of Aβ in recent phase II clinical trials.12,13 The more recently developed preclinical multitarget-directed metal-chelating agents 5-((methyl(prop-2-yn-1-yl)amino)methyl)quinolin-8-ol (M-30), 11-(5-chloro-8-methoxyquinolin-2-yl)undecan-1-ol (J2326), and N1,N1-dimethyl-N4-(pyridin-2-ylmethyl)benzene1,4-diamine (L2-b) revealed noticeable efficacies in multiple accessible murine models of AD (Figure 1).14−16 All adult mammalian species have reservoirs of neuronal stem cells in the hippocampal dentate gyrus (DG), which integrates new neurons into the hippocampus throughout life.17 This adult neurogenesis provides an indispensable mechanism by which lost or damaged neural cells from neurodegeneration can be replaced with functioning normal ones, which facilitates long-term hippocampal-dependent learning, memory, and neuronal plasticity.18,19 Nevertheless, with aging or neurodegeneration in rodents and elderly humans, the rate of regeneration of new neurons from the neural stem cells within the hippocampus decreases exponentially.20 Furthermore, before the new neural precursor cells are fully matured into functional neurons and migrate into the adult hippocampus, which is generally over approximately 30 days for mice, the vast majority of neural precursor cells undergo normal apoptosis during the first week with no explicit reason that has been currently identified.17,18,21 Therefore, the promotion of adult hippocampal neurogenesis with small drug-like molecules was identified as an effective therapeutic strategy to address longterm neurodegeneration, which has significant unmet regenerative medical needs, particularly for AD.19 Allopregnanolone (Figure 1) and the compound (6S,9aS)-N-benzyl-6-(4-hydroxybenzyl)-8-(naphthalen-1-ylmethyl)-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxamide (ICG-001, Figure 1) significantly strengthened hippocampal neurogenesis and markedly reduced the pathological burden in a preclinical in vivo model of AD.22,23 Through enhancing remyelination, benztropine (Figure 1) exhibited a striking mitigation of disease severity in a murine model of relapsing-remitting multiple sclerosis.24 Recently, the compound N-(3-(3,6-dibromo-9Hcarbazol-9-yl)-2-fluoropropyl)-3-methoxyaniline (P7C3A20, Figure 1) and its further analog N-(3-(3,6-dibromo-9Hcarbazol-9-yl)-2-fluoropropyl)-4-(4-(prop-2-yn-1-yloxy)-

Figure 2. Multitarget-directed design strategy by introducing an indole fragment into the CQ framework.

(SAR) studies of antioxidant ability, BBB permeability, modulation of Aβ aggregation, and neuronal cell proliferation activity, the potential compounds with neurotrophic function were further evaluated for their neuroprotective capacities, metabolic stability, acute toxicity, and hippocampal cell proliferation in living adult mice. Ultimately, the pharmacokinetic profile and pharmacodynamics evaluation of the optimal compound was performed.

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RESULTS AND DISCUSSION Chemistry. The synthetic routes of the target compounds (7a−g and 8a−g) are depicted in Scheme 1. The commercially available 8-hydroxyquinaldine or one of its analogs (1a or 2a) was reacted with the corresponding o-nitrobenzaldehyde derivative to produce 3a−g and 4a−g, respectively, followed by reductive cyclization of the o-nitrostyrenes derivatives (3a−g and 4a−g) catalyzed by Pd(OAc)2 under CO, thus leading to the key indole intermediates 5a−g and 6a−g. Finally, the removal of the acetyl group by interesterification in the presence of CH3OH and K2CO3 (for 5a−g) or NaOCH3 (for 6a−g) led to the target compounds 7a−g and 8a−g with good yields. In addition, by treatment of the pure target compounds 1872

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Scheme 1. Synthesis of 7a−g (X = H), 8a−g (X = Cl), 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCla

a Reagents and conditions: (a) acetic anhydride, 150 °C, 12 h; (b) DMF, phen·H2O, Pd(OAc)2, CO, 80 °C, 24 h; (c) CH3OH, K2CO3 (for 5a−g) or NaOCH3 (for 6a−g), rt, 1 h; (d) AcOEt, HCl; (g), rt, 12 h.

Scheme 2. Synthesis of 9a, 9d, and 10da

Reagents and conditions: (a) HCHO, HCl (g), rt, 72 h; (b) P(OEt)3, 110 °C, 12 h; (c) H2O, KOH, NaClO, rt, 2 h; (d) CH2Cl2, DIPEA, ClMOM, rt, 5 h; (e) DMF, NaH, rt, 4 h; (f) DMF, phen·H2O, Pd(OAc)2, CO, 80 °C, 24 h; (g) CH3OH, HCl (37%), rt, 5 h.

a

assay using fluorescein (ORAC-FL) method with a vitamin E analog, Trolox, as the internal standard. Table 1 revealed that all of the target compounds exhibited significantly higher antioxidant efficacies compared with clioquinol (0.5), melatonin (2.4), and clioquinol + melatonin (2.9). The results also showed that the radical scavenging efficacies of the target compounds were principally derived from the indole moiety. An SAR study demonstrated that the phenolic hydroxyl groups on the indole moieties were pivotal for the antioxidant activity. Compounds 7f (5.4) and 8f (4.9), which have phenolic hydroxyl groups at the 5-position of the indole moieties, provided considerably better antioxidant efficacies compared with the analogs that were methylated of the hydroxyl group (7e, 3.5; 8e, 3.4). Notably, compound 9d (6.6), which has an indole moiety on the 5-position of the quinoline, demonstrated the greatest radical scavenging efficacy. The presence of the chlorine on the oxine framework indicated a slight detrimental effect on the antioxidant activity. Compounds 7d, 7f, and 9d, lacking a chlorine atom in the oxine scaffold, exhibited ORAC

7g, 8c, 8d, and 8g with HCl (g) in an ethyl acetate solvent, the corresponding hydrochloride compounds were obtained with a high yield. The synthesis of the target compounds 9a, 9d, and 10d is described in Scheme 2. Compounds 2d and 2e, which were prepared according to a previous method,31 were reacted with chloro(methoxy)methane to give 2f and 2g. The subsequent reactions of 2f and 2g with o-nitrobenzaldehyde or its analogs in the presence of NaH gave 3i, 3j, and 4j, respectively. Finally, target compounds 9a, 9d, and 10d were produced by reductive cyclization of the o-nitrostyrene intermediate (3i, 3j, or 4j) with CO, followed by deprotection of the methoxymethane groups under acidic conditions. Free Oxygen Radical Scavenging Ability. Oxidative stress is one of the earliest events in AD pathogenesis, and oxidative damage is present within the brain of AD patients. Considering that 8-hydroxyquinoline derivatives generally have good metal ion complexing ability, we first determined the antioxidant ability of the quinoline−indole compounds and performed the free oxygen radical absorbance capacity (ORAC) 1873

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Table 1. Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents), Permeability (Pe × 10−6 cm s−1) Determined by the PAMPA-BBB Assay for Target Compounds, Predicted Penetration of the CNS, and Inhibition of Aβ Self-Aggregation

compd

R1

R2

X

ORACa

Pe (×10−6 cm s−1)b

pred

Aβ1−42 self-induced aggregation (% inhib)c

7a 7b 7c 7d 7e 7f 7g 8a 8b 8c 8d (WI-1758) 8e 8f 8g 9a 9d 10d clioquinol melatonin clioquinol + melatonin curcumin resveratrol chlorpromazine hydrocortisone

H F Cl OH OMe OH

H H H H OMe OMe

H F Cl OH OMe OH

H H H H OMe OMe

H H H H H H H Cl Cl Cl Cl Cl Cl Cl H H Cl

4.5 4.2 4.6 5.9 3.5 5.4 4.3 3.1 3.2 3.0 5.0 3.4 4.9 3.2 2.1 6.6 5.3 0.5 2.4 2.9 d d d d

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.0 ± 1.2 10.6 ± 2.3 9.8 ± 1.6 14.8 ± 0.7 5.2 ± 1.3 12.1 ± 2.1 5.9 ± 0.3 6.6 ± 1.4 5.3 ± 0.4 5.6 ± 0.8 10.2 ± 0.1 3.9 ± 1.6 8.4 ± 1.5 4.9 ± 0.2 12.8 ± 1.4 9.1 ± 0.6 6.8 ± 0.4 d d d d d 5.9 ± 0.4 1.1 ± 0.3

CNS+ CNS+ CNS+ CNS+ CNS+ CNS+ CNS+ CNS+ CNS+ CNS+ CNS+ CNS± CNS+ CNS+ CNS+ CNS+ CNS+ d d d d d CNS+ CNS−

6.1 ± 1.3 34.2 ± 3.8 25.1 ± 3.9 51.2 ± 9.1 21.5 ± 7.4 42.3 ± 5.8 19.4 ± 5.0 13.6 ± 4.2 42.9 ± 5.1 35.9 ± 1.9 66.8 ± 8.2 32.8 ± 7.1 50.3 ± 8.3 30.8 ± 5.6 43.1 ± 7.8 57.2 ± 4.8 68.7 ± 7.9 1.9 ± 1.2 19.3 ± 3.4 22.5 ± 5.3 36.7 ± 6.6 42.1 ± 5.8 d d

H OH OH

0.1 0.3 0.2 0.3 0.2 0.1 0.2 0.2 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.2

The mean ± SD of three independent experiments. Data are expressed as μmol of Trolox equivalent/μmol of tested compound. bCompounds were dissolved in DMSO at 5 mg mL−1 and diluted with PBS/EtOH (70:30). Values are expressed as the mean ± SD at least three independent experiments. Compounds with permeabilities Pe > 4.7 × 10−6 cm s−1 could cross the blood−brain barrier by passive diffusion. cThe Aβ1−42/inhibitor ratio was equal to 5/1, with inhibitor at 5 μM and Aβ1−42 at 25 μM. Values are the mean of three independent experiments in triplicate. dNot tested. a

undesirable effect on brain penetration. The chloro-substituted compounds 8d (10.2) and 10d (6.8) exhibited slightly lower BBB permeabilities compared with the hydrogen-substituted counterparts 7d (14.8) and 9d (9.1). Inhibition of Aβ Self-Aggregation. To determine the inhibitory activities of the quinoline−indole derivatives on Aβ1−42 self-induced aggregation, the thioflavin T (ThT) fluorescence assay was first performed with curcumin and resveratrol as reference compounds (Table 1). Among the four series of compounds, 7 (X = H), 8 (X = Cl), 9 (X = H), and 10 (X = Cl), compounds 7d (inhibition, 51.2%) and 8d (inhibition, 66.8%), in which a phenolic hydroxyl group was on the indole moiety at the R1 position, had more potent effects than clioquinol (1.9%), melatonin (19.3%), clioquinol + melatonin (22.5%, and the other analogs. Compounds 7f and 8f, with a methoxy group on the ortho (R2) position of the hydroxyl group, exhibited slightly weaker activities (42.3%, and 50.3%, respectively) than the parent compounds. The methylation of the hydroxyl group or cyclization with the methoxy group seemed unfavorable for the activities, and compounds 7e (21.5%), 8e (32.8%), 7g (19.4%), and 8g (30.8%) showed dramatic decreases in inhibiting Aβ1−42 aggregation, which demonstrated that the phenolic hydroxyl group on the indole moiety was preferable for the inhibitory

values of 5.9, 5.4, and 6.6, respectively, compared with 8d, 8f, and 10d (ORAC values 5.0, 4.9, and 5.3), which have a chlorine in the oxine scaffold. Blood−Brain Barrier Permeability Assay. Penetration of the blood−brain barrier (BBB) is a critical factor for successful CNS drugs. To determine the BBB penetration of the target compounds, we used the parallel artificial membrane permeability (PAMPA) assay, which was initially established by Di et al. and has been successfully applied to a multitude of diverse compounds to predict passive BBB permeation. The results presented in Table 1 indicated that most of the target compounds can significantly permeate the blood−brain barrier by passive diffusion. The SAR study indicated that compounds 7d (Pe = 14.8) and 7f (12.1), which contain a phenolic hydroxyl group on the indole moiety, demonstrated significantly higher BBB permeability compared with the analogs that replaced the phenolic hydroxyl groups with hydrogen (7a, 8.0), halogens (7b, 10.6; 7c, 9.8), dioxoles (7g, 5.9) or the methylation of the phenolic hydroxyl groups (7e, 5.2). In contrast, for compounds with the indole scaffold on the 5position of the quinoline, such as 9a (12.8) and 9d (9.1), the phenolic hydroxyl groups on the indole moiety demonstrated slight unfavorable effects on BBB permeability. Likewise, the effect of chlorine on the oxine moiety displayed a slight 1874

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Figure 3. (A) UV absorbance spectrum of compound 8d (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) UV−vis titration of compound 8d (50 μM) with Cu2+ in buffer (20 mM HEPES, 150 mM NaCl, pH = 7.4) at room temperature. (C) High-resolution mass spectrum of the Cu2+−8d complex in solution.

Figure 4. Experiments of inhibition self-induced Aβ1−42 aggregation and disaggregation of self-induced Aβ1−42 aggregates in the presence of 8d. (a, 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, d) TEM image analysis of the inhibition of Aβ1−42 aggregation and disaggregation of self-induced Aβ1−42 aggregates: (1) fresh Aβ1−42, (2) Aβ1−42 alone, (3) Aβ1−42 + CQ, (4) Aβ1−42 + melatonin, (5) Aβ1−42 + 8d, (6) Aβ1−42 + 8g. Experimental conditions were the following: Aβ1−42 (25 μM); compound/Aβ1−42 = 1/1; PBS (50 mM); pH 7.4; 37 °C.

activities than compounds 7d, 8d, and 9d, respectively. Compounds 9d (57.2%) and 10d (68.7%), with the indole moiety on the 5-position of the quinoline, had activities

activity. Replacement of the phenolic hydroxyl group with an H, F, or Cl, as in compounds 7a−c (6.1−34.2%), 8a−c (13.6− 42.9%), and 9a (43.1%), also provided much less potent 1875

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Figure 5. Experiments of inhibition Cu2+-induced Aβ1−42 aggregation and disaggregation of Cu2+-induced Aβ1−42 aggregates in the presence of 8d. (a, b) Top: Scheme for the inhibition or disaggregation experiments. Bottom: 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, d) TEM images analysis of the inhibition of Cu2+-induced Aβ1−42 aggregation and disaggregation of Cu2+-induced Aβ1−42 aggregates: (1) fresh Aβ1−42, (2) Aβ1−42 alone, (3) Aβ1−42 + Cu2+, (4) Aβ1−42 + Cu2+ + CQ, (5) Aβ1−42 + Cu2+ + 8d, (6) Aβ1−42 + Cu2+ + melatonin. Experimental conditions were the following: Aβ1−42 (25 μM); chelator/Aβ1−42/Cu2+ = 2/1/1; HEPES (20 μM) and NaCl (150 μM); pH 6.6; 37 °C.

(TEM) and the ThT assay were performed for both the inhibition of Aβ aggregates and disaggregation of Aβ aggregation fibrils in the presence of quinoline−indole derivatives. As indicated by the ThT assay (Figure 4), compounds 8d and 8g provided 71.6−48.6% inhibition of self-induced Aβ1−42 aggregation and 72.7−48.55% disaggregation of the preformed Aβ1−42 aggregate fibrils (Figure 4a and Figure 4b), especially 8d, which were higher than the inhibition values of CQ (19.6% and 13.0%, respectively, p < 0.01 and 0.001) and melatonin (49.1% and 44.3%, respectively, p < 0.05 and 0.01). The morphological assessment of compound 8d by TEM (Figure 4c, sequence 5) showed less dense and thinner fibrils as well as fewer unstructured Aβ1−42 aggregates in the 8dtreated sample compared with the control (Figure 4c, sequence 2), CQ (Figure 4c, sequence 3), or melatonin (Figure 4c, sequence 4). Alternatively, in the disaggregation assay, 8d exhibited narrower and shorter aggregates (Figure 4d, sequence 5) than CQ (Figure 4d, sequence 3) and melatonin (Figure 4d, sequence 4). These results validated that 8d could effectively modulate self-induced Aβ1−42 aggregation by its overall structure rather than its structural components. The abilities of 8d to inhibit Cu2+-induced Aβ aggregation and induce the disaggregation of Aβ aggregation fibrils were investigated (Figure 5). The ThT fluorescence assay demonstrated that 8d had a rate of 85.8% inhibition for Aβ aggregation and 83.3% for disaggregation of Cu2+-associated Aβ aggregation fibrils, which were distinctly better than the values found for CQ (65.4% and 36.9%, respectively, p < 0.01 and 0.01) and melatonin (47.8% and 24.0%, respectively, p < 0.001 and 0.01). These results were further supported by TEM images of Aβ samples, which revealed the smaller-sized nonfibrillar amorphous Aβ species in the presence of Cu2+ and 8d (Figure 5c and Figure 5d, sequence 5). In contrast, larger Aβ aggregates were observed with CQ (Figure 5c and Figure 5d, sequence 4), melatonin (Figure 5c and Figure 5d,

comparable with 7d and 8d. The results also demonstrated that the presence of a chlorine atom on the oxine moiety plays a pivotal role in the inhibitory activity, and compounds 8d (66.8%) and 10d (68.7%) exhibited more potent activities than their hydrogen-substituted counterparts 7d (51.2%) and 9d (57.2%), respectively. Metal-Chelating Property Studies. Considering compound 8d showed excellent antioxidant ability, blood−brain barrier (BBB) permeability, and inhibition of Aβ1−42 selfinduced aggregation and structurally the quinoline−indole derivatives should also have good metal-chelating properties, we chose 8d as typical for studying the metal-chelating ability of the quinoline−indole derivatives.31 The results in Figure 3 demonstrate that substantial new characteristic absorptions were detected at 338, 309, 324, and 309 nm after titrating 8d (314 nm) with CuSO4, ZnCl2, FeSO4, and FeCl3, respectively, which indicated that compound 8d was capable of chelating Cu2+, Zn2+, Fe2+, and Fe3+ and generating corresponding complexes. The stoichiometry of the Cu2+−8d complexes in buffer was initially determined using the inflection point analysis, which measures the absorption at 338 nm by titrating 8d with Cu2+. As shown in Figure 3B, an isoabsorptive point (at 0.5) revealed a unique Cu2+−8d complex via a 1:2 Cu2+/ligand molar ratio. Moreover, high-resolution mass spectrometry was also performed to further probe the explicit components of the chelation complex, and the primary molecular ion peak of [(8d)2Cu]2+ (calculated, 375.0269; detected, 375.0277, Figure 3C) illustrated a molar ratio of 1:2 Cu2+/ligand for the complex. Inhibition of Self- and Metal-Induced Aβ Aggregation and Disaggregation of Aβ Aggregates by Quinoline− Indole Derivatives. To further elucidate the inhibitory effects of quinoline−indole derivatives on self- or metal-induced Aβ aggregation, translating to less toxic or nontoxic off-pathway amorphous Aβ aggregates, transmission electron microscopy 1876

DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894

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Figure 6. (A) Quantification of neuron number using image-based analysis. Statistics were expressed as the percentage relative to control and as the mean ± SD of at least three independent experiments. NGF was tested at a constant concentration of 50 ng/mL. (B) Effects of 8d, 8d + U0126 (ERK inhibitor, 1 μM), and 8d + SB203580 (p38 inhibitor, 1 μM) on neuronal survival measured by MTT assay. Statistical significance was analyzed by ANOVA: (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 by compared with the control group (0 μM quinoline−indole derivatives).

Figure 7. Images from representative experiments showing the quinoline−indole derivatives in undifferentiated PC12 cells: cells were treated with 50 ng/mL NGF or 10 μM melatonin, CQ, 7g, 8c, 8d, or 8g, respectively.

show any neurotoxicity and revealed significant abilities to increase the number of cells. The SAR investigation indicated that replacement of the phenolic hydroxyl group on the indole scaffold of 8d (X = Cl, R1 = OH) with chlorine (8c, X = Cl, R1 = Cl) generated a slightly decreased neurotrophic activity. However, replacement with hydrogen (8a, X = Cl, R1 = H) and fluorine (8b, X = Cl, R1 = F) or the introduction of a methoxy group (8f, X = Cl, R1 = OH, R2 = OMe) resulted in the complete loss of the increased proliferation, and further methylation of the hydroxyl group (8e, X = Cl, R1 = R2 = OMe) displayed increased neurotoxicity. Similar trends were also observed for the compounds containing chlorine on the quinoline moiety, such as compounds 7c (X = H, R1 = Cl) and 7d (X = H, R1 = OH), and when the chlorine was removed from 8c or 8d, they exhibited a rather poor neurotrophic effect. In addition, shifting the indole moiety to the benzene ring of the quinoline, for example, in compounds 9a, 9d, and 10d, led to considerable neurotoxicity compared with the vehicle. Compounds 7g and 8g, which have a dioxole moiety on the indole, demonstrated striking neurotrophic efficacies. The literature has reported that two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and p38, play crucial roles in neurotrophic actions.16,36 Therefore, we used the ERK inhibitor U0126 (CAS ,10951158-2) and the p38 inhibitor SB203580 (CAS, 152121-47-6) to study the effects of neuronal proliferation by quinoline−indole derivatives using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

sequence 6), and the control sample (Figure 5c and Figure 5d, sequence 2). Because CQ exhibited a weaker ability to inhibit Cu2+-induced Aβ aggregation and to disaggregate the wellformed Aβ fibrils and melatonin provided almost no ability for either, these observations suggest that both the indole and 8hydroxyquinoline moieties in 8d could be crucial for redirecting and interacting with metal−Aβ. The above results also demonstrated that substituents on the indole moiety of 8d, especially the hydroxyl group, could influence the regulation of Aβ aggregation. These similar scenarios often appeared in the literature describing the development of specific chemical ligands for antiamyloidogenic activity against metal−Aβ complexes.15,32−34 Taken together, all of the results suggest that 8d has a strong ability to modulate self- and metal-induced Aβ aggregation. Effects of the Quinoline−Indole Derivatives on Neuronal Proliferation of PC12 Cells. Inspired by the melatonin-N-benzylamine hybrids that can promote the development of neural stem cells into the neuronal phenotype, we assessed the efficacies of the target compounds on neuronal proliferation with undifferentiated PC12 neuronal cells according to a well-established method.16,35 After the cells were cultured for 72 h in the presence of the tested compounds (at concentrations ranging from 0.5 μM to 50 μM) or nerve growth factor (NGF, 50 ng/mL, as the positive control), the cells were surveyed under a digital microscope. As the results indicate in Figure 6A, compounds 7g, 8c, 8d, and 8g did not 1877

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Figure 8. Inhibition of t-BuOOH-induced ROS production in the presence of CQ, melatonin, 7g, 8c, 8d, and 8g determined by DCFH-DA. Data are the mean ± SD of at least three independent experiments performed in sextuplicate and expressed as fluorescence unit. Statistical comparisons with control (only t-BOOH) were performed by one-way ANOVA (levels of significance: (∗) p < 0.05; (∗∗) p < 0.01; (∗∗∗) p < 0.001, (ns) p > 0.05).

tetrazolium bromide (MTT, 570 nm) assay. With the addition of the ERK inhibitor U0126 (Figure 6B), the 8d-triggered proliferation of neuronal cells was completely abolished, and the addition of the p38 inhibitor SB203580 significantly decreased the proliferation effect of 8d. These preliminary results suggested that quinoline−indole derivatives might promote neuronal proliferation in a MAPK-dependent mechanism. Additionally, the morphological studies in Figure 7 show that compounds 7g, 8c, 8d, and 8g exhibited neurotrophic effects on PC12 cells, in contrast to the lack of increase in neurons using CQ and melatonin. Neuroprotective Capacity against Intracellular Oxidative Stress. Cellular damage in the central nervous system by oxidative stress is a typical feature in AD, PD, Huntington’s disease (HD), and ALS, especially intracellular oxidative stress, such as mitochondrial ROS, which is closely involved in the most crucial intrinsic pathway leading to apoptosis emanating from the mitochondria.17 To explore the therapeutic potentials for compounds 7g, 8c, 8d, and 8g as close as possible to the physiological conditions of the onset of neurodegenerative disease, an assay to investigate their neuroprotective capacities against intracellular oxidative stress was performed using the human neuroblastoma cell line SH-SY5Y (which is usually used in the determination of ROS) with tert-butyl hydroperoxide (tBuOOH) as the ROS stimulant along with the fluorescence probe dichlorofluorescein diacetate (DCFH-DA), which was used to evaluate the intracellular ROS production. In the trial, all compounds were tested at 1−0.01 μM, a concentration close to their concentrations for promoting neurite outgrowth. As shown in Figure 8, after t-BuOOH was introduced into the cells, the production of intracellular ROS units increased from 37.5 ± 5.2 (vehicle) to 415.5 ± 39.4 (control). In contrast, significantly less ROS production was detected in 7g-, 8c-, 8d-, and 8g-incubated cells. The EC50 values (0.26, 0.82, 0.10, and 0.33 μM, respectively, for 7g, 8c, 8d, and 8g) in Table 2 demonstrated that these compounds have the greatest capacities against intracellular oxidative stress compared with melatonin (EC50 > 1.0 μM) and the parent compound CQ (EC50 > 1.0 μM). Metabolic Stability in Liver Microsomes of SD Rats. Proper liver microsomal stability of the optimal compounds is beneficial for the stability in metabolism. To evaluate this property, the metabolic stabilities of compounds 7g·HCl, 8c· HCl, 8d·HCl, and 8g·HCl in the liver microsomes of SD rats

Table 2. EC50 Values of Neuroprotective Capacity against Intracellular Oxidative Stress in SH-SY5Y Cell compd

EC50 (μM)a

compd

EC50 (μM)a

melatonin 7g 8d

>1.0 0.26 ± 0.07 0.10 ± 0.03

clioquinol 8c 8g

>1.0 0.82 ± 0.12 0.33 ± 0.05

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

a

were examined according the procedure described in a previous study with CQ and donepezil as the reference anti-AD agents and testosterone as a positive control.31 According to the results in Table 3, the hydrochlorides of 8c, 8d, and 8g Table 3. Metabolic Stability of 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCl in Liver Microsomes of SD Rats compd testosterone donepezilb CQ 7g·HCl 8c·HCl 8d·HCl 8g·HCl

b

k (min−1)

T1/2 (min)a

± ± ± ± ± ± ±

2.2 ± 0.4 77.9 ± 5.1 34.4 ± 3.8 58.6 ± 1.8 195.8 ± 23.1 116.8 ± 5.6 389.3 ± 15.5

0.31323 0.00889 0.02012 0.01183 0.00354 0.00593 0.00178

0.04206 0.00041 0.00156 0.00026 0.00038 0.00020 0.00005

Results are expressed as the mean ± SD of at least three independent experiments performed in triplicate. bThe positive control (testosterone) and the compound donepezil exhibited metabolic stability that was consistent with the literature and internal validation data.37,38 a

exhibited T1/2 values in liver microsomes of 195.8, 116.8, and 389.3 min, respectively, which demonstrated better stabilities compared with CQ (T1/2 = 34.4 min) and donepezil (T1/2 = 77.9 min). Compound 7g·HCl displayed a somewhat weaker stability and had a T1/2 of 58.6 min toward liver enzymes, which may be attributed to the lack of chlorine substituent to block the metabolic position of the quinoline scaffold. Overall, these results revealed that the hydrochlorides of 7g, 8c, 8d, and 8g had considerable in vitro metabolic stabilities and thus deserve further detailed studies in vivo.37,38 Acute Toxicity Studies. The acute toxicity profiles of the hydrochlorides of 7g, 8c, 8d, and 8g were evaluated in adult C57BL/6 mice (12 weeks old). Each of the test compounds 1878

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Figure 9. Histological analysis of liver, heart, lung, and brain for the acute toxicity studies of hydrochlorides of 7g, 8c, 8d, and 8g at dosage of 2000 mg/kg in mice; 1000 μm and 100 μm indicate the scale bar of images (HE staining, n = 6). Representative images of HE-stained liver, heart, lung, and brain for each group are shown.

Selective Promotion of Cell Proliferation in the Hippocampus of Living Adult Mice. To further determine the efficacies of the target quinoline−indole compounds on cell proliferation in the hippocampus of living adult mice, an unbiased in vivo screening technique was performed.29 Each compound (hydrochlorides of 7g, 8c, 8d, and 8g, at a concentration of 10 μM) was administered by intracerebroventricular (icv) injection at a constant rate (0.5 μL/h) over 7 days into the left lateral ventricle of living adult wild type C57BL/6 mice (12 weeks old, n = 6 for each compound) via subcutaneously implanted osmotic minipumps.28 According to the previous studies, the attainable intracerebroventricular concentrations of the compounds were in a low-micromolar to mid-nanomolar concentration range.17 During the infusion of the compound, animals received a daily intraperitoneal (ip) injection with the thymidine analog bromodeoxyuridine (BrdU, 50 mg kg−1 day−1) to mark new cells and the survival of proliferating hippocampal neural precursor cells. Apart from the icv injection method, the administration of hydrochlorides of 7g, 8c, 8d, and 8g (po group, 30 mg kg−1 day−1) by oral gavage

was given in single doses of 0, 677, 1333, or 2000 mg/kg by intragastric infusion (n = 6 for each dosage, three male and three female). After the administration of the compounds, any abnormal behavior and mortality changes were observed continuously during the first 4 h, intermittently for the following 24 h, and occasionally thereafter for 14 consecutive days for any delayed effects. During the observation period, no acute toxicity phenomena, including mortality, significant abnormal behaviors, drastically altered consumption of water or food, and marked weight loss or gain, were observed. Additionally, no macroscopic abnormities of the stomach, intestines, heart, lung, liver, spleen, kidneys, and other organs were detected after all of the mice were sacrificed on the 14th day. Furthermore, no significant histological abnormal changes were detected in the liver, heart, lung, and brain of the mice at the maximum tested dosage 2000 mg/kg in the histopathological studies by hematoxylin and eosin (HE) staining (Figure 9). Overall, these results revealed that 7g·HCl, 8c·HCl, 8d· HCl, and 8g·HCl were well tolerated and exhibited no acute toxicity at a dose up to 2000 mg/kg. 1879

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Figure 10. Administration of quinoline−indole compounds by icv indicated selective BrdU incorporation in the hippocampus of living adult mice, including the dentate gyrus (DG), cornus ammonis (CA) 1, CA2, and CA3 regions, rather than cortex and striatum (immunohistochemical staining, ×40, n = 6).

Figure 11. Immunohistochemically visualized BrdU incorporation in the new hippocampal neurons of living adult C57BL/6 mice (immunohistochemical staining, ×200, n = 6). The compounds 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCl were administered icv at the indicated concentration (0.5 μL/h) for 7 days, during which mice were dosed ip daily with BrdU (50 mg kg−1 day−1) to label new hippocampal neurons. CA1, CA2, CA3, and DG indicate the cornus ammonis (CA) 1, CA2, CA3, and dentate gyrus (DG) regions of the hippocampus, respectively. Representative 200× magnification images of an immunohistochemically stained hippocampus for each group were shown.

staining of BrdU cells in the brains of mice illustrated that the quinoline−indole compounds could specifically incorporate BrdU in the hippocampal regions, including the DG, cornus ammonis (CA) 1, CA2, and CA3 regions, rather than the regions near the hippocampus, such as the cortex and striatum (Figure 10). This valid evidence further demonstrated that the target compounds had a preeminent ability to promote cell proliferation in the hippocampus of living adult mice rather than universally throughout the entire brain. This characteristic is very important for the treatment of AD, as all adult mammals form new neurons within the hippocampus, which is indispensable for learning, memory, and neuronal plasticity. The cell proliferation efficacies displayed in Figures 11 and 12 demonstrated a substantial difference in the number of BrdU+ cells in the subgranular zone (SGZ) of DG regions

to evaluate the efficacy of cell proliferation in the hippocampus of adult C57BL/6 mice was also performed (n = 6 for each group). To ensure a low baseline level of cell proliferation, mice were housed individually without access to running wheels 7 days prior to the assay, as social interaction and voluntary exercise stimulate hippocampal neurogenesis. After 1 week of the administration of the compounds (including the icv and po groups), all animals were sacrificed and transcardially perfused. BrdU immunohistochemistry was used to quantify cell proliferation by counting the number of BrdU+ cells in the brain hemisphere opposite the side of infusion to avoid complications arising from the surgical implantation of the pump. The number of BrdU+ cells was normalized against the volume of the hippocampus to obtain consistent data across six animals.26 It is worth noting that the immunohistochemical 1880

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surviving hippocampal neurons in the DG regions of the hippocampus were 43.6% (p < 0.001), 28.5% (p < 0.01), 41.3% (p < 0.001), and 31.2% (p < 0.01), respectively, which revealed strikingly greater results than for the vehicle (7.8%) and CQ (6.2%) groups. Melatonin displayed moderate effects (27.4%) on the hippocampal neurons, which was consistent with previously reported results.30 Consequently, the results indicated that the quinoline−indole compounds could promote cell proliferation from the reservoirs of neuronal stem cells in the subgranular zone (SGZ) of the hippocampal DG. Furthermore, the icv injection of the hydrochlorides of 7g, 8c, 8d, and 8g noticeably induced the cell proliferation of neural precursor cells in the CA1, CA2, and CA3 regions of the hippocampus (7g, 61.9%, 56.1%, and 49.3%, p < 0.001; 8c, 15.3%, 18.1%, and 20.3%, p < 0.01; 8d, 50.1%, 39.6%, and 36.5%, p < 0.001; 8g, 49.8%, 38.0%, and 37.6%, p < 0.001), which demonstrated significantly greater effectiveness compared with the vehicle (3.2%, 2.4%, and 1.4%), CQ (2.7%, 2.1%, and 1.5%), or melatonin (2.7%, 2.1%, and 1.2%) groups (Figures 11 and 12). These results further suggested that compounds 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCl can induce cell proliferation in the hippocampus of living adult mice. Alternatively, after oral gavage administration of compounds at the dose of 30 mg kg−1 day−1 for 7 days, the new hippocampal neurons in the DG, CA1, CA2, and CA3 regions of the hippocampus for quinoline−indole-compound treated groups (7g, 7.6%, 8.0%, 7.5%, and 3.9%; 8c, 4.4%, 4.8%, 5.1%, and 4.7%; 8d, 30.6%, 21.1%, 24.3%, and 23.9%, p < 0.01; 8g, 17.0%, 14.6%, 16.3%, and 17.1%, p < 0.05) were higher than the vehicle (2.2%, 2.3%, 2.4%, and 1.5%), CQ (2.0%, 2.4%, 2.3%, and 1.4%), or melatonin (9.4%, 3.2%, 1.6%, and 1.5%)

Figure 12. Efficacy of 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCl in the living adult mouse model of cell proliferation within the hippocampus. The compounds were administered icv at the indicated concentration (0.5 μL/h) for 7 days, during which time the mice were dosed ip daily with BrdU (50 mg kg−1 day−1) to label new hippocampal neurons. CA1, CA2, CA3, and DG indicate the cornus ammonis (CA) 1, CA2, CA3, and dentate gyrus (DG) regions of the hippocampus, respectively. The results were expressed as the mean ± SD (n = 6), and statistical significance was analyzed by two-way ANOVA: (ns) p > 0.05, (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 versus the vehicle, CQ, and melatonin groups.

between the quinoline−indole compound-treated mice, CQ- or melatonin-treated mice, and the vehicle group. After the living mice were treated with the hydrochlorides of 7g, 8c, 8d, and 8g over the 7 day period, the immunohistochemical results indicated that the rates of BrdU-incorporated new and

Figure 13. Immunohistochemically visualized BrdU incorporation in the new hippocampal neurons of living adult C57BL/6 mice (immunohistochemical staining, ×200, n = 6). The compounds were administered orally at the indicated dose (30 mg kg−1 day−1) for 7 days, during which time mice were dosed ip daily with BrdU (50 mg kg−1 day−1) to label new hippocampal neurons. CA1, CA2, CA3, and DG indicate the cornus ammonis (CA) 1, CA2, CA3, and dentate gyrus (DG) regions of the hippocampus, respectively. Representative 200× magnification images of an immunohistochemically stained hippocampus for each group were shown. 1881

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HCl had T1/2 = 3.58 h (T1/2 > 3.0 h), Tmax = 0.5 h (Tmax < 1.0 h), Cmax = 1001 μg/L, and a rational oral bioavailability of F = 14.1%. Most importantly, the LC−MS/MS analysis of plasma and brain levels of 8d with the oral dose of 30 mg/kg revealed corresponding compound concentrations of 902 ng/g in the plasma and 579 ng/g in brain tissue after oral dosing for 1 h, which showed a log BB value of −0.19, which was substantially higher than the marginal value that determines whether a compound will be able to penetrate the BBB (log BB > −1). Overall, these favorable drug-like pharmacokinetic properties in vivo facilitated the pharmacodynamics evaluation of 8d·HCl in a murine model of AD. Cognitive and Memory Strengthening in APP/PS1 AD Mice by Long-Term Oral Uptake of 8d·HCl and 8g·HCl. To evaluate the learning and memory efficacies of 8d·HCl and 8g·HCl in vivo, the Morris water maze (MWM) test, using double transgenic APP/PS1 mice (six months old), a wellestablished animal model of AD that exhibits early senile plaques in the brain and cognitive dysfunction, was performed (oral administration at 30 mg kg−1 day−1; clioquinol, 30 mg kg−1 day−1 as the positive control) and wild type (WT) C57BL/6 mice were used as a negative control according to pharmacokinetic properties and previous studied dosage.13 The corresponding drug dosage was dissolved in a 0.5% carboxymethylcellulose sodium (CMC-Na) solution by intragastric infusion for a total period of 80 days, and a blank 0.5% CMC-Na solution was used as a placebo for the WT and vehicle group mice. The results illustrated in Figure 15 indicated that during the entire administration period, 8d· HCl and 8g·HCl did not trigger any significant abnormalities in body weight compared with the vehicle- and CQ-treated mice, and additionally, no detrimental or abnormal events, including diarrhea, emesis-like behavior, or an increased mortality were observed. During the final 5 days of treatment with the compounds, the Morris water maze (MWM) test of training trials was performed. The results indicated that the vehicle mice had adverse effects regarding spatial learning, and they spent more time and required more paths to locate the hidden escape platform in the pool compared with the wild type mice (Figure 15A2 and Figure 15B). Conversely, the 8d·HCl- and 8g·HCltreated groups, especially the 8d·HCl group (p < 0.01), had shortened mean escape latency and path searching in the APP/ PS1 mice compared with the vehicle- or CQ-treated groups. This result revealed that the long-term oral intake of 8d·HCl significantly strengthened the spatial learning and memory capability in the APP/PS1 AD mice. Twenty-four hours after the last training, the long-term memory retention was assessed by performing the spatial probe trial. The platform in the pool was removed, and the original platform location and its 2-fold diameter location were set as

group (Figures 13 and 14). The reduced efficacies of compounds 7g·HCl and 8c·HCl by oral administration in

Figure 14. Efficacy of 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCl in the adult mouse model of neurogenesis. The compounds 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCl were administered orally at the indicated dose (30 mg kg−1 day−1) for 7 days, during which time mice were dosed ip daily with BrdU (50 mg kg−1 day−1) to label new hippocampal neurons. CA1, CA2, CA3, and DG indicate the cornus ammonis (CA) 1, CA2, CA3, and dentate gyrus (DG) regions of the hippocampus, respectively. The results were expressed as the mean ± SD (n = 6), and statistical significance was analyzed by two-way ANOVA: (ns) p > 0.05, (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 versus the vehicle, CQ, and melatonin groups.

contrast with icv injection may be attributed their poor BBB permeabilities (see Table 1) and undesirable metabolic stabilities (see Table 2). Taken together, compound 8d·HCl exhibited a significant ability to selectively promote cell proliferation in the hippocampus by both icv injection and oral administration, meeting the requirements of acceptable physicochemical properties for an oral drug in developing successful CNS drugs and drug candidates.39,40 Therefore, compound 8d·HCl was chosen for further pharmacokinetics investigation and pharmacodynamics evaluation. Pharmacokinetic Profile and log BB Values for the BBB Penetration of Compound 8d·HCl in SD Rats. To evaluate the pharmacokinetic properties of 8d·HCl, a preliminary pharmacokinetics analysis in SD rats was performed. The results in Table 4 demonstrated that 8d·HCl had C0 = 4926 μg/L, AUC(0−∞) = 2124 h·μg/L, a moderate clearance CL = 2.356 L h−1 kg−1 (0.6 L h−1 kg−1 < CL < 3.0 L h−1 kg−1), and a temperate volume of distribution Vz = 2.491 L/kg (1 L/kg < Vz < 10 L/kg) for the intravenous administration (iv, dose = 5 mg/kg) mode. In addition, after single oral gavage administrations (po, dose = 30 mg/kg), 8d· Table 4. Pharmacokinetic Profile of 8d·HCl in SD Rats iva

T1/2 (h)

C0 (μg/L)

8d·HCl pob

0.93 ± 0.21 T1/2 (h)

4926 ± 266 Tmax (h)

8d·HCl 3.58 ± 1.13 0.5 ± 0.0 poc (1 h after dosing) 8d·HCl

AUC(0−t) (h·μg/L) 2109 ± 14 Cmax (μg/L)

AUC(0−∞) (h·μg/L)

VZ (mL/kg)

CL (mL h−1 kg−1)

MRT(0−t) (h)

2124 ± 23 2491 ± 1724 2356 ± 24 0.38 ± 0.06 AUC(0−t) (h·μg/L) AUC(0−∞) (h·μg/L) MRT(0−t) (h)

1001 ± 128 1502 ± 49 plasma (ng/g) 902 ± 136

1791 ± 323 brain (ng/g) 579 ± 89

MRT(0−∞) (h·μg/L)

VSS (mL/kg)

0.41 ± 0.07 954 ± 163 MRT(0−∞) (h·μg/L) F (%)

2.53 ± 0.74 4.88 ± 2.84 log BB (brain, log BB > −1)

14.1

−0.19 ± 0.06

a

iv = intravenous administration, dose = 5 mg/kg, n = 3. bpo = oral administration, dose = 30 mg/kg, n = 3. c1 h after oral administration, dose = 30 mg/kg, n = 3. 1882

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Figure 15. (A1) Mean daily body weight profile of each group mice (including APP/PS1 AD mice and WT mice) during 80 consecutive days of oral drug administration period (WT = wild type mice, n = 10). (A2) The escape latency time of each group was counted every day during the period of training trial (mean ± SD, n = 10). Statistical significance was analyzed by two-way ANOVA: (ns) p > 0.05, (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 versus the vehicle, 8d·HCl, and 8g·HCl. (B) Representative tracks of the mice in the Morris water maze during the training trial period. The location of the platform and effective region (2-fold diameter of the platform) were represented as a blue and bright green circle, respectively.

Figure 16. (A) Effect of long-term memory retention on the spatial probe trial (with the platform removed from the pool) in the Morris water maze test after 80 consecutive days of oral drug administration treatment in APP/PS1 AD mice. The results were expressed as the mean ± SD (n = 10). Statistical significance was analyzed by two-way ANOVA: (ns) p > 0.05, (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 versus the vehicle and compounds 8d·HCl and 8g·HCl. (1) Average swimming speed for mice in each group. (2) Number of virtual platform (the original platform location) crossings. (3) Number of effective region (2-fold diameter of the original platform location) crossings. (4) Time spent in the virtual platform. (5) Time spent in the effective region. (6) Time spent in the virtual platform quadrant (correct quadrant). (7) Swimming path length in the virtual platform. (8) Swimming path length in the effective region (2-fold diameter of the platform). (9) Swimming path length in the virtual platform quadrant (correct quadrant). (B) Representative tracks of the mice in the Morris water maze during the spatial probe trial period. The location of the original platform and the effective region (2-fold diameter of the platform) were represented as a blue and bright green circle, respectively.

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Figure 17. Oral drug administration of 8d·HCl and 8g·HCl reduces the total brain Aβ plaque burden in the APP/PS1 transgenic mice after treatment at the dosage of 30 mg kg−1 day−1 for 80 consecutive days (n = 10). Representative images of coronal sections from WT (wild type mice), vehicle, CQ-, 8d·HCl-, and 8g·HCl-treated mice after immunohistochemical staining for Aβ with β-amyloid (mOC64) antibody. Scale bar equals 1000 and 400 μm, respectively.

behavioral performance observations and outcomes demonstrated that the spatial memory impairment and cognitive dysfunction in vehicle-treated APP/PS1 mice were significantly ameliorated by the long-term oral administration of 8d·HCl compared with CQ at the comparative testing dosage. Finally, all of the mice were sacrificed after the completion of the assays, and no expanded brain tissue or carcinogenicity in the subcutaneous layer, stomach, intestines, heart, lung, liver, spleen, kidneys, and other organs or tissues was detected with 8d·HCl and 8g·HCl administration, which again suggested that 8d·HCl and 8g·HCl do not universally promote cell proliferation. In summary, these convincing results demonstrated that the long-term oral uptake of compounds 8d·HCl and 8g·HCl was well-tolerated and safe at doses of 30 mg kg−1 day−1. Reduction of Amyloid Burden in APP/PS1 Transgenic Mice by Chronic Oral Administration of 8d·HCl and 8g· HCl. The efficacy of 8d·HCl and 8g·HCl long-term oral treatment in strengthening learning and memory encouraged us to further investigate whether there was a correlation in the reduction of Aβ plaque deposition and quinoline−indole derivatives in APP/PS1 mice. The potential association of 8d· HCl and 8g·HCl with amyloid pathology was performed by Aβ plaque immunostaining in the brain tissue of APP/PS1 AD mice. The results revealed that 8d·HCl and 8g·HCl, especially 8d·HCl, treatment showed significant reductions in the amounts and area occupied by Aβ deposits in the coronal sections of the brain, including the cortex and hippocampus (Figure 17). Quantification analysis of the immunoreactivity revealed that 8d·HCl treatment demonstrated up to a 54% (p < 0.001, 8d·HCl vs vehicle) reduction in plaque burden compared with vehicle littermate AD mice (Figure 18), which was substantially mitigated compared to CQ (35%, p < 0.05, 8d·HCl vs CQ) treated AD mice. In addition, a 26% decrease in the brain Aβ burden was detected after chronic treatment with 8g·HCl (p < 0.05, 8g·HCl vs vehicle). In brief, these results suggest that chronic oral treatment with 8d·HCl and 8g· HCl markedly reduced the overall Aβ plaque deposition in APP/PS1 mice.

the virtual platform and effective region, respectively. In the trial, each mouse was permitted only one chance to swim freely for 90 s to search for the virtual platform. The results in Figure 16A1 indicated that the swimming velocities in the quadrants for groups treated with 8d·HCl, 8g·HCl, CQ, and vehicle were approximately equivalent, which suggests that the animals’ locomotion and exploratory activities were not jeopardized by the long-term administration of compounds 8d·HCl and 8g· HCl (Figure 16A1, p > 0.2, WT = 16.9 ± 4.5, vehicle = 17.8 ± 2.5, CQ = 18.7 ± 2.9, 8d·HCl = 18.9 ± 3.2, and 8g·HCl = 19.0 ± 1.9 cm/s). This effect further convincingly demonstrated the safety and nontoxic nature of 8d·HCl and 8g·HCl by long-term oral administration. The significantly higher numbers crossing the virtual platform (the original platform location, Figure 16A2) and effective region (2-fold diameter of the original platform location, Figure 16A3) in WT (4.8 ± 3.1, p < 0.001 and 5.7 ± 2.0, p < 0.001), 8d·HCl (4.5 ± 1.7, p < 0.001 and 4.9 ± 0.7, p < 0.001), and 8g·HCl (3.3 ± 2.4, p < 0.05 and 4.4 ± 0.5, p < 0.01) mice contrasted with their vehicle-treated (1.7 ± 1.1 and 2.1 ± 1.1) and CQ-treated (2.7 ± 1.6, p < 0.05 and 3.3 ± 1.9, p > 0.05) littermates and revealed the significantly better cognitive and memory function for 8d·HCl- and 8g·HCltreated mice. The duration spent close to the platform location, including the time in the virtual platform (Figure 16A4), effective region (Figure 16A5), and correct quadrant (Figure 16A6), for the 8d·HCl (3.2 ± 0.8, p < 0.05; 18.0 ± 8.3, p < 0.05; and 27.4 ± 5.1, p < 0.01) and 8g·HCl (2.6 ± 0.8, p > 0.05; 16.7 ± 8.0, p > 0.05; and 24.0 ± 4.1, p > 0.05) groups, especially the 8d·HCl group, was noticeably longer than for the vehicle (1.9 ± 0.2, 9.0 ± 3.8, and 20.6 ± 4.4) and CQ (2.1 ± 0.7, p > 0.05; 15.1 ± 5.3, p > 0.05; and 24.2 ± 3.9, p > 0.05) groups. These treatment outcomes further demonstrated that the oral uptake of 8d·HCl led to a considerable strengthening of the spatial memory retention and conventional cognitive capabilities compared with vehicle- or CQ-treated AD mice. Meanwhile, the swimming path lengths in the virtual platform (Figure 16A7), effective region (Figure 16A8), and correct quadrant (Figure 16A9) for the 8d·HCl (22.1 ± 7.3, p < 0.001; 71.3 ± 17.1, p < 0.001; and 533.3 ± 71.1, p < 0.001) and 8g· HCl (15.0 ± 7.7, p < 0.05; 65.5 ± 16.9, p < 0.001; and 473.5 ± 27.2, p < 0.05) groups were remarkably longer than for the vehicle (9.1 ± 2.8, 44.2 ± 4.9, and 295.3 ± 46.9) and CQ (15.1 ± 5.3, p < 0.05; 45.8 ± 13.9, p > 0.05; and 416.4 ± 80.9, p < 0.001) groups, indicating that a more distinct specific spatial reference was formed in the memory vault for mice treated with compounds 8d·HCl and 8g·HCl. Taken together, these overall

■

CONCLUSION In summary, this work presented the design, synthesis, and biological evaluation of a novel class of quinoline−indole derivatives as MTDLs against AD by merging the skeleton of indole and CQ. SAR studies revealed that most of the target derivatives had excellent free oxygen radical scavenging ability, 1884

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Journal of Medicinal Chemistry

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Hz, 1H), 7.57 (t, J = 7.4 Hz, 1H), 5.32 (s, 1H), 4.90 (s, 1H). LC/MS (ESI): 194.0 [M + H]+. Diethyl ((8-Hydroxyquinolin-5-yl)methyl)phosphonate (2d).31 To a solution of triethyl phosphite (18.86 mL, 3.0 equiv) was added 1.0 equiv of compound 2c (7.0 g) in one portion. The mixture was stirred at 110 °C for 12 h. After the 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 (2e). Compound 2d (5.0 g) was suspended in 30 mL of H2O containing 17 mL of 1 N KOH solution. 15.3 mL of NaClO solution (7.5% excess) was added in 30 min with vigorously stirring, and then the mixture was stirred at room temperature. After the completion of the reaction (monitored by TLC), the solution was adjusted to pH 6.0 by adding 10% hydrochloric acid and then filtered to get the solid 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]+. General Procedures for the Synthesis of 2f,g. To a mixture of 2d or 2e (4.0 g, 1.0 equiv) and diisopropylethylamine (4.0 equiv) in 20 mL of DCM was added 1.5 equiv of chloromethyl methyl ether dropwise, and the mixture was stirred at room temperature. After the completion of the reaction, the solvent was evaporated under vacuum, and the crude product was purified by silica-column chromatography. Diethyl ((8-(Methoxymethoxy)quinolin-5-yl)methyl)phosphonate (2f). Yellow oil. 72% yield. Rf = 0.32 (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), 7.48 (dd, J = 8.6, 4.1 Hz, 1H), 7.42 (dd, J = 8.1, 3.6 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 5.50 (s, 2H), 4.03−3.89 (m, 4H), 3.57 (s, 3H), 3.55 (s, 1H), 3.49 (s, 1H), 1.17 (t, J = 7.1 Hz, 6H). LC/MS (ESI): 340.1 [M + H]+. Diethyl ((7-Chloro-8-(methoxymethoxy)quinolin-5-yl)methyl)phosphonate (2g). Yellow oil. 75% yield. Rf = 0.36 (CH2Cl2/CH3OH = 20/1). 1H NMR (400 MHz, CDCl3) δ 8.92 (dd, J = 4.0, 1.3 Hz, 1H), 8.42 (dd, J = 8.6, 1.5 Hz, 1H), 7.50 (d, J = 3.8 Hz, 1H), 7.46 (dd, J = 8.6, 4.1 Hz, 1H), 5.62 (s, 2H), 4.05−3.95 (m, 4H), 3.72 (s, 3H), 3.53 (s, 1H), 3.48 (s, 1H), 1.21 (t, J = 7.1 Hz, 6H). LC/MS (ESI): 374.0 [M + H]+. General Procedures for the Synthesis of 3a−g and 4a−g. Quinoline (1.0 equiv, 1.0 g) and benzaldehyde (1.0 equiv) were dissolved in 20 mL of acetic anhydride, and the mixture was stirred at 150 °C for about 8 h. After cooled to room temperature, the mixture was filtered, washed with water and dried under vacuum to get the product. (E)-2-(2-Nitrostyryl)quinolin-8-yl Acetate (3a). Yellow solid. 82% yield. Rf = 0.29 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 16.0 Hz, 1H), 8.18 (d, J = 8.6 Hz, 1H), 8.01 (dd, J = 8.2, 1.1 Hz, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.70 (dd, J = 8.1, 1.3 Hz, 1H), 7.64 (dd, J = 10.2, 4.5 Hz, 2H), 7.49 (dt, J = 7.1, 5.5 Hz, 2H), 7.44 (dd, J = 7.5, 1.3 Hz, 1H), 7.33 (d, J = 16.0 Hz, 1H), 2.57 (s, 3H). LC/MS (ESI): 335.1 [M + H]+. (E)-2-(5-Fluoro-2-nitrostyryl)quinolin-8-yl Acetate (3b). Yellow solid. 75% yield. Rf = 0.31 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.28 (dd, J = 15.9, 1.0 Hz, 1H), 8.19 (d, J = 8.5 Hz, 1H), 8.10 (dd, J = 9.1, 5.1 Hz, 1H), 7.71 (dd, J = 8.1, 1.3 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.55 (dd, J = 9.7, 2.3 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.45 (dd, J = 7.5, 1.4 Hz, 1H), 7.31 (d, J = 16.0 Hz, 1H), 7.18−7.12 (m, 1H), 2.57 (s, 3H). LC/MS (ESI): 353.1 [M + H]+. (E)-2-(5-Chloro-2-nitrostyryl)quinolin-8-yl Acetate (3c). Yellow solid. 89% yield. Rf = 0.33 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 16.0 Hz, 1H), 8.15 (d, J = 8.5 Hz,

Figure 18. Percentage of plaque burden in the overall half coronal hemisphere of mice was analyzed and quantified by the area of Aβ immunoreactivity expressed as a percentage of full area using ImageJ software. The results were expressed as the mean ± SD (n = 10). Statistical significance was analyzed by two-way ANOVA: (ns) p > 0.05, (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 versus the vehicle and compounds 8d·HCl and 8g·HCl.

BBB permeability by passive diffusion, inhibition of Aβ selfaggregation, and stimulation of neuronal cell proliferation. The optimal compound, 8d·HCl (WI-1758), had a metal-chelating ability and an eminent neuroprotective capacity via intracellular antioxidant effects (EC50 = 0.1 μM), providing 71.6% and 85.8% inhibition of self- or Cu2+-induced Aβ aggregation as well as 72.7% and 83.3% disaggregation of preformed self- or Cu2+associated Aβ1−42 aggregate fibrils. Moreover, 8d·HCl had reasonable pharmacokinetic properties, with substantial metabolic stability (T1/2 = 116.8 min in liver microsomes and T1/2 = 3.58 h in vivo), oral bioavailability (F = 14.1%), salient log BB values (log BB = −0.19) for penetration of the CNS in SD rats, and no acute toxicity up to 2000 mg/kg. Further biological property analysis demonstrated that this optimal compound markedly and selectively promoted adult hippocampal cell proliferation in living adult C57BL/6 mice by icv injection with osmotic minipumps or oral administration at a dosage of 30 mg kg−1 day−1. Most importantly, pharmacodynamics studies by the Morris water maze test demonstrated that long-term (80 consecutive days) oral intake of compound 8d·HCl was not toxic and remarkably ameliorated cognitive and spatial memory deficits in the double transgenic APP/PS1 AD mice at a dosage of 30 mg kg−1 day−1. Altogether, these convincing biochemical results strongly highlight 8d·HCl as a reliable and potent drug candidate for treating AD and warrant further investigation of its mechanisms of action.

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EXPERIMENTAL SECTION

Chemistry. The NMR spectra were recorded using TMS as the internal standard on a Bruker Avance III 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 SRSOpti 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 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 mm × 250 mm, 5 μm), eluted with CH3CN/H2O = 90/10, at a flow rate of 0.5 mL/min. 5-(Chloromethyl)quinolin-8-ol Hydrochloride (2c·HCl). Compound 2c·HCl was synthesized according to the literature.41 Pale yellow solid, 98% yield. Rf = 0.42 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, DMSO) δ 9.32 (t, J = 7.7 Hz, 1H), 9.14 (dd, J = 19.3, 4.9 Hz, 1H), 8.16 (ddd, J = 36.0, 8.6, 5.3 Hz, 1H), 7.80 (dd, J = 75.6, 8.0 1885

DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894

Journal of Medicinal Chemistry

Article

(E)-5,7-Dichloro-2-(5-chloro-2-nitrostyryl)quinolin-8-yl Acetate (4c). Yellow solid. 79% yield. Rf = 0.27 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 8.7 Hz, 1H), 8.27 (d, J = 15.9 Hz, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 2.2 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.64 (s, 1H), 7.44 (dd, J = 8.7, 2.2 Hz, 1H), 7.26 (d, J = 15.9 Hz, 1H), 2.61 (s, 3H). LC/MS (ESI): 437.0 [M + H]+. (E)-2-(5-Acetoxy-2-nitrostyryl)-5,7-dichloroquinolin-8-yl Acetate (4d). Yellow solid. 92% yield. Rf = 0.39 (petroleum/EtOAc = 3/ 1). 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 8.8 Hz, 1H), 8.33 (d, J = 15.9 Hz, 1H), 8.10 (d, J = 8.9 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.64 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 7.27 (d, J = 9.7 Hz, 1H), 7.23 (d, J = 2.6 Hz, 1H), 2.60 (s, 3H), 2.38 (s, 3H). LC/MS (ESI): 461.0 [M + H]+. (E)-5,7-Dichloro-2-(4,5-dimethoxy-2-nitrostyryl)quinolin-8yl Acetate (4e). Yellow solid. 87% yield. Rf = 0.33 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 8.8 Hz, 1H), 8.42 (d, J = 16.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.20 (t, J = 8.0 Hz, 2H), 4.07 (s, 3H), 4.00 (s, 3H), 2.60 (s, 3H). LC/MS (ESI): 463.0 [M + H]+. (E)-2-(5-Acetoxy-4-methoxy-2-nitrostyryl)-5,7-dichloroquinolin-8-yl Acetate (4f). Yellow solid. 68% yield. Rf = 0.38 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 8.7 Hz, 1H), 8.30 (d, J = 15.9 Hz, 1H), 7.84−7.60 (m, 3H), 7.55 (s, 1H), 7.26 (s, 1H), 7.19 (d, J = 15.9 Hz, 1H), 3.96 (s, 3H), 2.61 (s, 3H), 2.39 (s, 3H). LC/MS (ESI): 491.0 [M + H]+. (E)-5,7-Dichloro-2-(2-(6-nitrobenzo[d][1,3]dioxol-5-yl)vinyl)quinolin-8-yl Acetate (4g). Yellow solid. 72% yield. Rf = 0.31 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 8.7 Hz, 1H), 8.31 (d, J = 16.0 Hz, 1H), 7.71−7.57 (m, 2H), 7.53 (s, 1H), 7.26−6.93 (m, 2H), 6.16 (s, 2H), 2.60 (s, 3H). LC/MS (ESI): 447.0 [M + H]+. (E)-7-Chloro-8-(methoxymethoxy)-5-(5-(methoxymethoxy)2-nitrostyryl)quinoline (4j). Yellow solid. 70% yield. Rf = 0.31 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.95 (dd, J = 4.1, 1.6 Hz, 1H), 8.50 (dd, J = 8.6, 1.6 Hz, 1H), 8.12 (d, J = 9.1 Hz, 1H), 7.83 (s, 1H), 7.69 (d, J = 15.8 Hz, 1H), 7.51 (d, J = 16.7 Hz, 1H), 7.47 (dd, J = 8.7, 4.2 Hz, 1H), 7.35 (d, J = 2.6 Hz, 1H), 7.10 (dd, J = 9.1, 2.6 Hz, 1H), 5.67 (s, 2H), 5.32 (s, 2H), 3.74 (s, 3H), 3.54 (s, 3H). LC/MS (ESI): 431.1 [M + H]+. General Procedures for the Synthesis of 5a−g, 5i,j, 6a−g, and 6j. An autoclave was charged with Pd(OAc)2 (0.05 equiv), 1,10phenathroline·H2O (0.1 equiv), and 5 mL of DMF. After the mixture was stirred at room temperature for 30 min, 1.0 g of nitro compound (1.0 equiv) was added and the vessel was purged six times with CO and then pressurized to 40 bar with this gas. After the mixture was stirred at 80 °C for overnight, 100 mL of water was added, then, filtered, washed with pure water, and dried under vacuum to afford the product. 2-(1H-Indol-2-yl)quinolin-8-yl Acetate (5a). Yellow solid. 88% yield. Rf = 0.23 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 9.46 (s, 1H), 8.19 (d, J = 8.6 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.71 (dd, J = 7.6, 2.0 Hz, 2H), 7.57−7.40 (m, 3H), 7.31−7.27 (m, 1H), 7.20 (s, 1H), 7.16 (t, J = 7.5 Hz, 1H), 2.62 (s, 3H). LC/MS (ESI): 303.3 [M + H]+. 2-(5-Fluoro-1H-indol-2-yl)quinolin-8-yl Acetate (5b). Yellow solid. 65% yield. Rf = 0.34 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 9.44 (s, 1H), 8.18 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.58−7.40 (m, 2H), 7.32 (dt, J = 9.8, 5.1 Hz, 2H), 7.11 (s, 1H), 7.01 (td, J = 9.1, 2.3 Hz, 1H), 2.59 (s, 3H). LC/MS (ESI): 321.1 [M + H]+. 2-(5-Chloro-1H-indol-2-yl)quinolin-8-yl Acetate (5c). Yellow solid. 74% yield. Rf = 0.58 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 9.46 (s, 1H), 8.19 (d, J = 8.7 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.70 (dd, J = 7.9, 1.5 Hz, 1H), 7.64 (d, J = 1.9 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.45 (dd, J = 7.5, 1.6 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.21 (dd, J = 8.7, 2.0 Hz, 1H), 7.09 (d, J = 1.2 Hz, 1H), 2.58 (s, 3H). LC/MS (ESI): 337.1 [M + H]+. 2-(5-Acetoxy-1H-indol-2-yl)quinolin-8-yl Acetate (5d). Yellow solid. 83% yield. Rf = 0.21 (petroleum/EtOAc = 2/1). 1H NMR (400

1H), 7.97 (d, J = 8.7 Hz, 1H), 7.83 (d, J = 2.1 Hz, 1H), 7.67 (dd, J = 8.0, 1.3 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.44 (dd, J = 7.5, 1.4 Hz, 1H), 7.41 (dd, J = 8.7, 2.2 Hz, 1H), 7.29 (d, J = 15.9 Hz, 1H), 2.57 (s, 3H). LC/MS (ESI): 369.0 [M + H]+. (E)-2-(5-Acetoxy-2-nitrostyryl)quinolin-8-yl Acetate (3d). Yellow solid. 81% yield. Rf = 0.36 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 16.0 Hz, 1H), 8.16 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 8.9 Hz, 1H), 7.68 (dd, J = 8.0, 1.1 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.44 (dd, J = 7.4, 1.2 Hz, 1H), 7.28 (d, J = 16.0 Hz, 1H), 7.22 (dd, J = 8.9, 2.4 Hz, 1H), 2.57 (s, 3H), 2.37 (s, 3H). LC/MS (ESI): 393.1 [M + H]+. (E)-2-(4,5-Dimethoxy-2-nitrostyryl)quinolin-8-yl Acetate (3e). Yellow solid. 92% yield. Rf = 0.26 (petroleum/EtOAc = 3/1). 1 H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 8.8 Hz, 1H), 8.42 (d, J = 16.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.20 (t, J = 8.0 Hz, 2H), 4.07 (s, 3H), 4.00 (s, 3H), 2.60 (s, 3H). LC/MS (ESI): 395.1 [M + H]+. (E)-2-(5-Acetoxy-4-methoxy-2-nitrostyryl)quinolin-8-yl Acetate (3f). Yellow solid. 72% yield. Rf = 0.25 (petroleum/EtOAc = 2/ 1). 1H NMR (400 MHz, CDCl3) δ 8.26 (dd, J = 15.9, 3.9 Hz, 1H), 8.17 (dd, J = 8.4, 4.2 Hz, 1H), 7.70 (dd, J = 7.1, 3.5 Hz, 1H), 7.67− 7.60 (m, 2H), 7.58 (d, J = 4.0 Hz, 1H), 7.54−7.47 (m, 1H), 7.47−7.41 (m, 1H), 7.23 (dd, J = 16.1, 4.2 Hz, 1H), 3.96 (s, 3H), 2.58 (s, 3H), 2.39 (s, 3H). LC/MS (ESI): 423.1 [M + H]+. (E)-2-(2-(6-Nitrobenzo[d][1,3]dioxol-5-yl)vinyl)quinolin-8-yl Acetate (3g). Yellow solid. 89% yield. Rf = 0.32 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 15.8 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 7.66 (dd, J = 20.5, 8.1 Hz, 2H), 7.55−7.42 (m, 3H), 7.25−7.15 (m, 2H), 6.15 (s, 2H), 2.56 (s, 3H). LC/MS (ESI): 379.1 [M + H]+. General Procedures for the Synthesis of 3i,j and 4j. To a solution of 2f or 2g (1.0 equiv) in 10 mL of anhydrous DMF, 3.0 equiv of sodium hydride (80%) was added in portions at ice bath. The mixture was stirred for 30 min, a solution of the corresponding aldehyde (1.0 equiv) in 2 mL of DMF was added in dropwise, and the reaction was monitored by TLC. After the completion of the reaction, water was added and the product was extracted by DCM, washed with brine, dried over anhydrous Na2SO4, evaporated the solvent under reduced pressure to afford the crude product which was purified by silica-column chromatography. (E)-8-(Methoxymethoxy)-5-(2-nitrostyryl)quinoline (3i). Yellow solid. 70% yield. Rf = 0.32 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 9.00 (dd, J = 4.1, 1.6 Hz, 1H), 8.54 (dd, J = 8.6, 1.6 Hz, 1H), 8.01 (dd, J = 8.2, 1.1 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.70−7.62 (m, 2H), 7.58 (d, J = 15.8 Hz, 1H), 7.51 (dd, J = 8.6, 4.1 Hz, 1H), 7.49−7.41 (m, 2H), 5.55 (s, 2H), 3.60 (s, 3H). LC/MS (ESI): 337.1 [M + H]+. (E)-8-(Methoxymethoxy)-5-(5-(methoxymethoxy)-2nitrostyryl)quinoline (3j). Yellow solid. 91% yield. Rf = 0.28 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 9.00 (dd, J = 4.1, 1.5 Hz, 1H), 8.54 (dd, J = 8.6, 1.5 Hz, 1H), 8.10 (d, J = 9.1 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 15.8 Hz, 1H), 7.60 (d, J = 15.8 Hz, 1H), 7.51 (dd, J = 8.6, 4.1 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.38 (d, J = 2.6 Hz, 1H), 7.12−7.02 (m, 1H), 5.55 (s, 2H), 5.32 (s, 2H), 3.60 (s, 3H), 3.54 (s, 3H). LC/MS (ESI): 397.1 [M + H]+ (E)-5,7-Dichloro-2-(2-nitrostyryl)quinolin-8-yl Acetate (4a). Yellow solid. 91% yield. Rf = 0.28 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 8.7 Hz, 1H), 8.30 (d, J = 15.9 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.76−7.58 (m, 3H), 7.50 (t, J = 7.6 Hz, 1H), 7.29 (d, J = 17.1 Hz, 1H), 2.60 (s, 3H). LC/MS (ESI): 403.0 [M + H]+. (E)-5,7-Dichloro-2-(5-fluoro-2-nitrostyryl)quinolin-8-yl Acetate (4b). Yellow solid. 87% yield. Rf = 0.23 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J = 8.7 Hz, 1H), 8.33 (dd, J = 15.9, 0.9 Hz, 1H), 8.11 (dd, J = 9.1, 5.1 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.65 (s, 1H), 7.54−7.50 (m, 1H), 7.27 (d, J = 15.9 Hz, 1H), 7.20−7.14 (m, 1H), 2.60 (s, 3H). LC/MS (ESI): 421.0 [M + H]+. 1886

DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894

Journal of Medicinal Chemistry

Article

MHz, CDCl3) δ 9.45 (s, 1H), 8.15 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.68 (dd, J = 7.7, 1.8 Hz, 1H), 7.50−7.42 (m, 2H), 7.36 (dd, J = 5.5, 3.3 Hz, 2H), 7.12 (d, J = 1.3 Hz, 1H), 6.97 (dd, J = 8.8, 2.2 Hz, 1H), 2.58 (s, 3H), 2.33 (s, 3H). LC/MS (ESI): 361.1 [M + H]+. 2-(5,6-Dimethoxy-1H-indol-2-yl)quinolin-8-yl Acetate (5e). Yellow solid. 61% yield. Rf = 0.35 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 9.48 (s, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.66 (dd, J = 6.7, 2.8 Hz, 1H), 7.54−7.29 (m, 2H), 7.06 (s, 1H), 7.05 (d, J = 1.8 Hz, 1H), 6.75 (s, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 2.55 (s, 3H). LC/MS (ESI): 363.1 [M + H]+. 2-(5-Acetoxy-6-methoxy-1H-indol-2-yl)quinolin-8-yl Acetate (5f). Yellow solid. 79% yield. Rf = 0.22 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, DMSO) δ 8.41 (d, J = 8.7 Hz, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.85 (d, J = 7.3 Hz, 1H), 7.54 (q, J = 7.4 Hz, 2H), 7.32 (s, 2H), 7.20 (s, 1H), 3.84 (s, 3H), 2.60 (s, 3H), 2.28 (s, 3H). LC/MS (ESI): 391.2 [M + H]+. 2-(8H-[1,3]Dioxolo[4,5-g]indol-7-yl)quinolin-8-yl Acetate (5g). Yellow solid. 81% yield. Rf = 0.25 (petroleum/EtOAc = 2/1). 1 H NMR (400 MHz, CDCl3) δ 9.34 (s, 1H), 8.10 (d, J = 8.7 Hz, 1H), 7.80 (dd, J = 8.7, 4.7 Hz, 1H), 7.65 (dd, J = 6.7, 2.8 Hz, 1H), 7.44− 7.42 (m, 1H), 7.28 (dd, J = 8.3, 1.0 Hz, 1H), 7.05−6.99 (m, 2H), 6.86 (d, J = 14.5 Hz, 1H), 5.97 (s, 2H), 2.57 (s, 3H). LC/MS (ESI): 347.1 [M + H]+. 5-(1H-Indol-2-yl)-8-(methoxymethoxy)quinoline (5i). White solid. 91% yield. Rf = 0.26 (petroleum/EtOAc = 1/1). 1H NMR (400 MHz, CDCl3) δ 8.97 (dd, J = 4.1, 1.7 Hz, 1H), 8.63 (dd, J = 8.6, 1.7 Hz, 1H), 8.47 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.48−7.39 (m, 3H), 7.27−7.22 (m, 1H), 7.18 (td, J = 7.6, 1.0 Hz, 1H), 6.71 (dd, J = 2.0, 0.8 Hz, 1H), 5.53 (s, 2H), 3.59 (s, 3H). LC/MS (ESI): 305.1 [M + H]+. 8-(Methoxymethoxy)-5-(5-(methoxymethoxy)-1H-indol-2yl)quinoline (5j). White solid. 93% yield. Rf = 0.22 (petroleum/ EtOAc = 1/1). 1H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 4.1, 1.7 Hz, 1H), 8.64 (dd, J = 8.6, 1.7 Hz, 1H), 8.30 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.45 (dd, J = 8.4, 3.9 Hz, 1H), 7.36 (t, J = 5.6 Hz, 2H), 7.00 (dd, J = 8.7, 2.4 Hz, 1H), 6.64 (d, J = 1.3 Hz, 1H), 5.55 (s, 2H), 5.24 (s, 2H), 3.60 (s, 3H), 3.55 (s, 3H). LC/MS (ESI): 365.1 [M + H]+. 5,7-Dichloro-2-(1H-indol-2-yl)quinolin-8-yl Acetate (6a). Yellow solid. 88% yield. Rf = 0.52 (petroleum/EtOAc = 10/1). 1H NMR (400 MHz, CDCl3) δ 9.36 (s, 1H), 8.46 (d, J = 8.9 Hz, 1H), 7.95 (d, J = 8.9 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.56 (s, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.29 (d, J = 7.1 Hz, 1H), 7.20 (d, J = 1.2 Hz, 1H), 7.17−7.08 (m, 1H), 2.64 (s, 3H). LC/MS (ESI): 371.0 [M + H]+. 5,7-Dichloro-2-(5-fluoro-1H-indol-2-yl)quinolin-8-yl Acetate (6b). Yellow solid. 67% yield. Rf = 0.22 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.33 (s, 1H), 8.48 (d, J = 8.9 Hz, 1H), 7.93 (d, J = 8.9 Hz, 1H), 7.58 (s, 1H), 7.35 (dd, J = 9.0, 4.4 Hz, 1H), 7.31 (dd, J = 9.3, 2.4 Hz, 1H), 7.14 (d, J = 1.4 Hz, 1H), 7.04 (td, J = 9.0, 2.5 Hz, 1H), 2.63 (s, 3H). LC/MS (ESI): 389.0 [M + H]+. 5,7-Dichloro-2-(5-chloro-1H-indol-2-yl)quinolin-8-yl Acetate (6c). Yellow solid. 80% yield. Rf = 0.26 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.38 (s, 1H), 8.46 (d, J = 8.9 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.64 (s, 1H), 7.54 (s, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.22 (dd, J = 8.7, 1.3 Hz, 1H), 7.09 (s, 1H), 2.64 (s, 3H). LC/MS (ESI): 405.0 [M + H]+. 2-(5-Acetoxy-1H-indol-2-yl)-5,7-dichloroquinolin-8-yl Acetate (6d). Yellow solid. 87% yield. Rf = 0.34 (petroleum/EtOAc = 2/1). 1H NMR (400 MHz, CDCl3) δ 9.33 (s, 1H), 8.43 (d, J = 8.9 Hz, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.51 (s, 1H), 7.36 (dd, J = 5.5, 3.2 Hz, 2H), 7.10 (d, J = 1.5 Hz, 1H), 6.99 (dd, J = 8.8, 2.1 Hz, 1H), 2.64 (s, 3H), 2.35 (s, 3H). LC/MS (ESI): 429.0 [M + H]+. 5,7-Dichloro-2-(5,6-dimethoxy-1H-indol-2-yl)quinolin-8-yl Acetate (6e). Yellow solid. 82% yield. Rf = 0.27 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.31 (d, J = 8.9 Hz, 1H), 7.76 (d, J = 8.9 Hz, 1H), 7.40 (s, 1H), 7.05 (s, 1H), 6.97 (d, J = 1.5 Hz, 1H), 6.82 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 2.68 (s, 3H). LC/MS (ESI): 431.0 [M + H]+. 2-(8-Acetoxy-5,7-dichloroquinolin-2-yl)-6-methoxy-1Hindol-5-yl Acetate (6f). Yellow solid. 85% yield. Rf = 0.26 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, DMSO) δ 8.54

(d, J = 9.0 Hz, 1H), 8.29 (d, J = 9.0 Hz, 1H), 7.99 (s, 1H), 7.42 (s, 1H), 7.35 (s, 1H), 7.18 (s, 1H), 3.84 (s, 3H), 2.67 (s, 3H), 2.28 (s, 3H). LC/MS (ESI): 459.0 [M + H]+. 2-(5H-[1,3]Dioxolo[4,5-f ]indol-6-yl)-5,7-dichloroquinolin-8yl Acetate (6g). Yellow solid. 71% yield. Rf = 0.24 (petroleum/EtOAc = 5/1). 1H NMR (400 MHz, CDCl3) δ 9.24 (s, 1H), 8.41 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H), 7.07 (s, 1H), 7.01 (s, 1H), 6.86 (s, 1H), 5.99 (s, 2H), 2.62 (s, 3H). LC/MS (ESI): 415.0 [M + H]+. 7-Chloro-8-(methoxymethoxy)-5-(5-(methoxymethoxy)-1Hindol-2-yl)quinoline (6j). White solid. 92% yield. Rf = 0.37 (petroleum/EtOAc = 3/1). 1H NMR (400 MHz, CDCl3) δ 8.91 (dd, J = 4.1, 1.7 Hz, 1H), 8.59 (dd, J = 8.6, 1.7 Hz, 1H), 8.39 (s, 1H), 7.66 (s, 1H), 7.40 (dd, J = 8.6, 4.1 Hz, 1H), 7.37 (d, J = 3.0 Hz, 1H), 7.36 (d, J = 3.4 Hz, 1H), 7.02 (dd, J = 8.8, 2.3 Hz, 1H), 6.66 (d, J = 1.3 Hz, 1H), 5.68 (s, 2H), 5.24 (s, 2H), 3.75 (s, 3H), 3.55 (s, 3H). LC/ MS (ESI): 399.1 [M + H]+. General Procedures for the Synthesis of 7a−g. Compounds 5a−g (1.0 g, 1.0 equiv) and K2CO3 (1.0 equiv) in 15 mL of CH3OH were stirred at room temperature. After the completion of the reaction, 100 mL of water was added, filtered, washed with pure water, and dried under vacuum to get the crude product, which was purified by flash chromatography on silica gel and further recrystallized from CH3OH. 2-(1H-Indol-2-yl)quinolin-8-ol (7a). Yellow solid. 83% yield. Rf = 0.35 (petroleum/EtOAc = 1/1), mp 241.5−242.3 °C. 1H NMR (400 MHz, CDCl3) δ 9.54 (s, 1H), 8.13 (d, J = 8.7 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.47 (dd, J = 8.2, 0.8 Hz, 1H), 7.43−7.36 (m, 1H), 7.34−7.25 (m, 2H), 7.22−7.16 (m, 2H), 7.16− 7.09 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 151.76, 148.17, 137.48, 137.06, 136.73, 136.23, 128.99, 127.56, 127.28, 124.04, 121.54, 120.48, 119.16, 118.12, 111.43, 110.92, 103.18. FT-IR 3397, 3369, 3046, 1598, 1566, 1505, 1441, 1413, 1338, 1308, 1244, 1142, 1084, 831, 784, 753, 720, 661, 471; HRMS (ESI) m/z [M − H]− for C17H12N2O pred 259.0877, meas 259.0866. HPLC purity: 98.43%. Retention time: 8.960 min. 2-(5-Fluoro-1H-indol-2-yl)quinolin-8-ol (7b). Yellow solid. 91% yield. Rf = 0.39 (petroleum/EtOAc = 1/1), mp 257.2−257.8 °C. 1H NMR (400 MHz, DMSO) δ 8.36 (d, J = 8.6 Hz, 1H), 8.13 (d, J = 8.6 Hz, 1H), 7.53 (dd, J = 8.7, 4.4 Hz, 1H), 7.49−7.38 (m, 3H), 7.36 (s, 1H), 7.16 (d, J = 7.1 Hz, 1H), 7.10 (td, J = 9.3, 2.5 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 158.80 (s), 156.49 (s), 153.47 (s), 147.83 (s), 139.31 (s), 137.76 (s), 137.32 (s), 134.24 (s), 129.19 (d, J = 10.5 Hz), 127.95 (s), 119.21 (s), 117.95 (s), 112.99 (d, J = 9.7 Hz), 112.04 (d, J = 26.3 Hz), 111.52 (s), 105.97 (d, J = 23.1 Hz), 102.56 (d, J = 4.8 Hz). FT-IR 3390, 3364, 3051, 1628, 1599, 1567, 1549, 1507, 1494, 1442, 1418, 1315, 1286, 1243, 1201, 1129, 1110, 953, 832, 782, 752, 717, 657, 605, 566, 484, 462; HRMS (ESI) m/z [M + H]+ for C17H11FN2O pred 279.0928, meas 279.0929. HPLC purity: 95.24%. Retention time: 7.403 min. 2-(5-Chloro-1H-indol-2-yl)quinolin-8-ol (7c). Yellow solid. 86% yield. Rf = 0.41 (petroleum/EtOAc = 1/1), mp 305.5−306.3 °C. 1H NMR (400 MHz, DMSO) δ 8.37 (d, J = 8.6 Hz, 1H), 8.14 (d, J = 8.6 Hz, 1H), 7.70 (s, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.48−7.39 (m, 2H), 7.36 (s, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 7.1 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 153.40, 147.70, 139.06, 137.71, 137.35, 135.90, 130.13, 128.01, 127.97, 124.61, 123.64, 120.66, 119.23, 118.02, 113.48, 111.52, 102.09. FT-IR 3374, 3328, 2923, 1599, 1569, 1547, 1506, 1440, 1471, 1412, 1318, 1245, 1225, 1183, 1142, 1083, 1058, 915, 832, 778, 748, 716, 689, 655, 605, 571, 533, 476; HRMS (ESI) m/z [M + H]+ for C17H11ClN2O pred 295.0633, meas 295.0630. HPLC purity: 97.89%. Retention time: 7.495 min. 2-(5-Hydroxy-1H-indol-2-yl)quinolin-8-ol (7d). Yellow solid. 87% yield. Rf = 0.31 (petroleum/EtOAc = 1/1), mp 282.7−283.1 °C. 1 H NMR (400 MHz, DMSO) δ 8.31 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.43−7.35 (m, 2H), 7.30 (d, J = 8.7 Hz, 1H), 7.17 (s, 1H), 7.11 (dd, J = 7.0, 1.6 Hz, 1H), 6.94 (d, J = 2.0 Hz, 1H), 6.77 (dd, J = 8.7, 2.2 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 152.61 (s), 150.84 (s), 147.82 (s), 137.18 (d, J = 6.1 Hz), 137.07 (s), 136.49 (s), 131.75 (d, J = 14.8 Hz), 129.21 (d, J = 4.4 Hz), 127.13 (s), 126.95 (s), 1887

DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894

Journal of Medicinal Chemistry

Article

General Procedures for the Synthesis of 8a−g. Compounds 6a−g (1.0 g, 1.0 equiv) and NaOCH3 (6.0 equiv) in 15 mL of CH3OH were stirred at room temperature. After the completion of the reaction, 100 mL of water was added, and then the mixture was filtered, washed with pure water, and dried under vacuum to get the crude product, which was purified by flash chromatography on silica gel and further recrystallized from CH3OH. 5,7-Dichloro-2-(1H-indol-2-yl)quinolin-8-ol (8a). Yellow solid. 93% yield. Rf = 0.36 (petroleum/EtOAc = 1/1), mp 265.0−265.9 °C. 1 H NMR (400 MHz, DMSO) δ 12.10 (s, 1H), 8.36 (d, J = 8.7 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 7.67 (d, J = 7.1 Hz, 2H), 7.54 (d, J = 8.1 Hz, 1H), 7.38 (s, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 149.70 (d, J = 2.6 Hz), 149.10 (s), 138.43 (s), 137.71 (d, J = 15.6 Hz), 136.53 (d, J = 13.8 Hz), 133.72 (s), 128.89 (d, J = 4.4 Hz), 127.39 (s), 124.32 (s), 123.75 (s), 121.95 (s), 120.32 (s), 119.62 (s), 115.58 (s), 112.05 (d, J = 5.6 Hz), 104.00 (d, J = 3.5 Hz). FT-IR 3524, 3401, 3319, 3059, 1657, 1617, 1596, 1545, 1493, 1435, 1415, 1392, 1340, 1306, 1250, 1218, 1182, 1146, 982, 948, 890, 858, 829, 785, 746, 725, 625, 560, 487, 441; HRMS (ESI) m/z [M + H]+ for C17H10Cl2N2O pred 329.0242, meas 329.0243. HPLC purity: 98.28%. Retention time: 7.370 min. 5,7-Dichloro-2-(5-fluoro-1H-indol-2-yl)quinolin-8-ol (8b). Yellow solid. 84% yield. Rf = 0.31 (petroleum/EtOAc = 1/1), mp 278.7−279.4 °C. 1H NMR (400 MHz, DMSO) δ 8.48 (d, J = 8.9 Hz, 1H), 8.29 (d, J = 8.9 Hz, 1H), 7.76 (s, 1H), 7.52 (dd, J = 8.9, 4.5 Hz, 1H), 7.45−7.42 (m, 2H), 7.11 (td, J = 9.2, 2.5 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 157.68 (d, J = 232.9 Hz), 149.47 (s), 149.15 (s), 138.43 (s), 138.27 (d, J = 14.5 Hz), 134.45 (d, J = 15.6 Hz), 134.00 (s), 129.00 (dd, J = 10.6, 3.8 Hz), 127.65 (s), 123.92 (s), 120.43 (s), 119.67 (s), 115.66 (s), 113.21 (dd, J = 9.1, 5.5 Hz), 112.70 (d, J = 26.4 Hz), 106.15 (d, J = 23.3 Hz), 103.95 (d, J = 3.9 Hz). FT-IR 3521, 3292, 3069, 1614, 1595, 1545, 1490, 1435, 1417, 1343, 1306, 1254, 1223, 1199, 1146, 1111, 985, 952, 850, 829, 780, 722, 826, 594, 557, 496, 484, 440; HRMS (ESI) m/z [M + H]+ for C17H9Cl2FN2O pred 347.0149, meas 347.0152. HPLC purity: 96.21%. Retention time: 7.366 min. 5,7-Dichloro-2-(5-chloro-1H-indol-2-yl)quinolin-8-ol (8c). Yellow solid. 77% yield. Rf = 0.37 (petroleum/EtOAc = 1/1), mp 293.1−294.0 °C. 1H NMR (400 MHz, DMSO) δ 12.26 (s, 1H), 8.47 (d, J = 0.9 Hz, 1H), 8.28 (d, J = 2.7 Hz, 1H), 7.73 (d, J = 13.7 Hz, 2H), 7.53 (d, J = 7.6 Hz, 1H), 7.43 (s, 1H), 7.24 (d, J = 7.9 Hz, 1H). 13 C NMR (101 MHz, DMSO) δ 163.11, 150.21, 146.34, 144.68, 143.43, 132.61, 131.94, 127.65, 124.99, 121.34, 119.50, 119.36, 118.70, 116.98, 115.73, 105.10, 99.85. FT-IR 3594, 3516, 3438, 3318, 3074, 1717, 1618, 1595, 1559, 1542, 1493, 1471, 1435, 1410, 1390, 1341, 1305, 1255, 1218, 1178, 1146, 1060, 983, 948, 918, 864, 851, 831, 792, 746, 721, 690, 629, 557, 490, 440; HRMS (ESI) m/z [M + H]+ for C17H9Cl3N2O pred 362.9853, meas 362.9839. HPLC purity: 99.37%. Retention time: 9.760 min. 5,7-Dichloro-2-(5-chloro-1H-indol-2-yl)quinolin-8-ol Hydrochloride (8c·HCl). Yellow solid. 90% yield. Rf = 0.37 (petroleum/ EtOAc = 1/1), mp 304.1−304.9 °C. 1H NMR (400 MHz, DMSO) δ 8.08 (d, J = 8.7 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.33 (s, 1H), 7.32− 7.19 (m, 2H), 6.91 (s, 1H), 6.67 (d, J = 8.4 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 164.02, 151.05, 147.26, 146.50, 143.25, 136.24, 131.01, 129.57, 125.01, 120.43, 119.89, 119.62, 118.09, 117.06, 116.07, 105.01, 100.29. FT-IR 3593, 3512, 3439, 3317, 3073, 1718, 1617, 1596, 1559, 1542, 1493, 1472, 1434, 1412, 1391, 1342, 1306, 1257, 1219, 1178, 1145, 1061, 984, 949, 918, 865, 852, 832, 791, 747, 722, 691, 629, 558, 491, 443; HRMS (ESI) m/z [M + H]+ for C17H9Cl3N2O pred 362.9853, meas 362.9836. HPLC purity: 99.91%. Retention time: 15.996 min. 5,7-Dichloro-2-(5-hydroxy-1H-indol-2-yl)quinolin-8-ol (8d). Yellow solid. 91% yield. Rf = 0.36 (petroleum/EtOAc = 1/1), mp 276.4−277.6 °C. 1H NMR (400 MHz, DMSO) δ 8.31 (d, J = 8.7 Hz, 8H), 8.07 (d, J = 8.7 Hz, 8H), 7.43−7.35 (m, 16H), 7.30 (d, J = 8.7 Hz, 8H), 7.17 (s, 8H), 7.11 (dd, J = 7.0, 1.6 Hz, 8H), 6.94 (d, J = 2.0 Hz, 8H), 6.77 (dd, J = 8.7, 2.2 Hz, 8H). 13C NMR (101 MHz, DMSO) δ 152.61 (s), 150.84 (s), 147.82 (s), 137.18 (d, J = 6.1 Hz), 137.07 (s), 136.49 (s), 131.75 (d, J = 14.8 Hz), 129.21 (d, J = 4.4 Hz), 127.13 (s),

118.58 (s), 117.43 (s), 113.97 (d, J = 9.7 Hz), 111.76 (d, J = 5.1 Hz), 110.75 (d, J = 5.3 Hz), 104.33 (d, J = 7.8 Hz), 101.29 (d, J = 4.6 Hz). FT-IR 3480, 3399, 3357, 1708, 1632, 1598, 1566, 1548, 1504, 1442, 1420, 1338, 1314, 1242, 1210, 1135, 1081, 949, 834, 783, 753, 719, 665, 618, 467; HRMS (ESI) m/z [M − H]− for C17H12N2O2 pred 275.0826, meas 275.0820. HPLC purity: 97.30%. Retention time: 4.914 min. 2-(5,6-Dimethoxy-1H-indol-2-yl)quinolin-8-ol (7e). Yellow solid. 83% yield. Rf = 0.32 (petroleum/EtOAc = 1/1), mp 320.1− 320.7 °C.1H NMR (400 MHz, DMSO) δ 11.89 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H), 8.00 (d, J = 8.7 Hz, 1H), 7.47−7.32 (m, 2H), 7.21−7.13 (m, 2H), 7.11 (s, 1H), 7.00 (s, 1H), 3.88 (s, 3H), 3.81 (s, 3H). 13C NMR (101 MHz, DMSO) δ 153.17, 148.70, 148.45, 145.54, 137.79, 136.90, 135.99, 132.57, 127.44, 127.20, 121.87, 118.87, 117.94, 111.31, 103.21, 102.78, 94.78, 56.20, 55.98. FT-IR 3426, 3381, 3005, 2933, 1626, 1598, 1566, 1544, 1509, 1483, 1452, 1440, 1406, 1390, 1347, 1317, 1287, 1245, 1211, 1133, 1006, 887, 849, 833, 796, 743, 720, 651, 600, 572, 476, 457; HRMS (ESI) m/z [M + H]+ for C19H16N2O3 pred 321.1234, meas 321.1224. HPLC purity: 99.23%. Retention time: 7.600 min. 2-(5-Hydroxy-6-methoxy-1H-indol-2-yl)quinolin-8-ol (7f). Yellow solid. 81% yield. Rf = 0.28 (petroleum/EtOAc = 1/1), mp 302.2−302.9 °C. 1H NMR (400 MHz, DMSO) δ 11.75 (s, 1H), 9.64 (s, 1H), 8.10 (d, J = 8.7 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.70 (d, J = 3.0 Hz, 1H), 7.36−7.31 (m, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.07 (d, J = 7.2 Hz, 1H), 7.03 (s, 1H), 6.63 (s, 1H), 3.99 (s, 3H). 13C NMR (101 MHz, DMSO) δ 153.12, 148.66, 148.18, 139.59, 137.82, 136.71, 135.33, 131.78, 127.33, 126.94, 123.24, 118.95, 117.89, 115.15, 111.15, 103.22, 93.52, 56.38. FT-IR 3476, 3413, 3353, 3018, 2947, 1662, 1587, 1558, 1531, 1511, 1479, 1447, 1436, 1418, 1386, 1351, 1309, 1290, 1268, 1209, 1142, 1062, 879, 852, 841, 789, 745, 728, 649, 606, 574, 469, 458; HRMS (ESI) m/z [M + H]+ for C18H12N2O3 pred 307.1077, meas 307.1063. HPLC purity: 95.80%. Retention time: 7.385 min. 2-(8H-[1,3]Dioxolo[4,5-g]indol-7-yl)quinolin-8-ol (7g). Yellow solid. 76% yield. Rf = 0.36 (petroleum/EtOAc = 1/1), mp 273.2− 273.9 °C. 1H NMR (400 MHz, DMSO) δ 8.28 (d, J = 8.7 Hz, 1H), 8.01 (d, J = 8.6 Hz, 1H), 7.37 (q, J = 8.3 Hz, 2H), 7.22 (s, 1H), 7.10 (d, J = 6.6 Hz, 2H), 6.99 (s, 1H), 6.00 (s, 2H). 13C NMR (101 MHz, DMSO) δ 152.61 (d, J = 14.3 Hz), 147.78 (s), 145.76 (s), 142.78 (s), 137.25 (d, J = 6.3 Hz), 136.46 (s), 135.73 (d, J = 13.7 Hz), 132.54 (d, J = 14.9 Hz), 126.96 (s), 126.72 (s), 122.50 (d, J = 4.2 Hz), 118.30 (s), 117.46 (d, J = 2.3 Hz), 110.77 (d, J = 5.6 Hz), 102.74 (d, J = 3.5 Hz), 100.44 (s), 99.12 (s), 91.81 (d, J = 5.2 Hz). FT-IR 3357, 2893, 1598, 1564, 1543, 1504, 1468, 1416, 1350, 1315, 1284, 1239, 1211, 1086, 1036, 948, 829, 782, 751, 717, 663, 451; HRMS (ESI) m/z [M + H]+ for C18H14N2O3 pred 305.0921, meas 305.0911. HPLC purity: 98.66%. Retention time: 10.282 min. General Procedures for the Synthesis of 7g·HCl, 8c·HCl, 8d· HCl, and 8g·HCl. Compound 7g, 8c, 8d, or 8g (1.0 g, HPLC purity >95%) was dissolved in 30 mL of EtOAc, and then HCl gas was bubbled into the solution at room temperature for 12 h. The mixture was filtered and the precipitation was washed with 40 mL of EtOAc, dried under reduced pressure to obtain an orange solid which was used in the in vivo assay, pharmacokinetics, and metabolic stability study. 2-(8H-[1,3]Dioxolo[4,5-g]indol-7-yl)quinolin-8-ol Hydrochloride (7g·HCl). Yellow solid. 93% yield. Rf = 0.36 (petroleum/ EtOAc = 1/1), mp 299.2−299.9 °C. 1H NMR (400 MHz, DMSO) δ 12.11 (s, 1H), 8.34 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 8.7 Hz, 1H), 7.44− 7.34 (m, 2H), 7.29 (d, J = 1.8 Hz, 1H), 7.15 (dd, J = 7.0, 1.8 Hz, 1H), 7.09 (s, 1H), 7.00 (s, 1H), 6.01 (s, 2H). 13C NMR (101 MHz, DMSO) δ 152.29 (s), 147.72 (s), 146.80 (s), 143.54 (s), 138.17 (s), 136.10 (d, J = 5.1 Hz), 134.72 (d, J = 7.8 Hz), 133.72 (d, J = 17.8 Hz), 127.52 (d, J = 10.3 Hz), 122.88 (dd, J = 8.7, 4.0 Hz), 119.15 (s), 118.13 (s), 112.28 (s), 105.00 (d, J = 7.0 Hz), 101.10 (s), 99.57 (d, J = 2.5 Hz), 92.37 (d, J = 5.5 Hz). FT-IR 3356, 2892, 1599, 1568, 1547, 1504, 1469, 1416, 1355, 1316, 1285, 1241, 1213, 1087, 1036, 949, 829, 783, 750, 718, 662, 453; HRMS (ESI) m/z [M + H]+ for C18H14N2O3 pred 305.0921, meas 305.0904. HPLC purity: 99.88%. Retention time: 15.780 min. 1888

DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894

Journal of Medicinal Chemistry

Article

126.95 (s), 118.58 (s), 117.43 (s), 113.97 (d, J = 9.7 Hz), 111.76 (d, J = 5.1 Hz), 110.75 (d, J = 5.3 Hz), 104.33 (d, J = 7.8 Hz), 101.29 (d, J = 4.6 Hz). FT-IR 3547, 3464, 3302, 3213, 2777, 1711, 1594, 1542, 1493, 1434, 1396, 1305, 1260, 1226, 1199, 1147, 1083, 983, 951, 854, 835, 783, 721, 628, 493, 426; HRMS (ESI) m/z [M − H]− for C17H10N2O2Cl2 pred 343.0047, meas 343.0034. HPLC purity: 99.35%. Retention time: 8.807 min. 5,7-Dichloro-2-(5-hydroxy-1H-indol-2-yl)quinolin-8-ol Hydrochloride (8d·HCl). Yellow solid. 96% yield. Rf = 0.36 (petroleum/EtOAc = 1/1), mp 312.7−313.3 °C. 1H NMR (400 MHz, DMSO) δ 8.43 (d, J = 8.9 Hz, 1H), 8.22 (d, J = 8.9 Hz, 1H), 7.72 (s, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.27 (s, 1H), 6.97 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.7, 2.2 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 151.61 (s), 150.00 (d, J = 3.6 Hz), 148.99 (s), 138.49 (s), 136.68 (dd, J = 14.2, 7.2 Hz), 133.71 (s), 132.61 (d, J = 15.5 Hz), 129.58 (dd, J = 8.7, 4.3 Hz), 127.24 (s), 123.67 (s), 120.40 (s), 119.70 (s), 115.52 (s), 115.29 (s), 112.58 (d, J = 5.6 Hz), 104.92 (s), 103.24 (d, J = 3.8 Hz). FT-IR 3546, 3466, 3306, 3218, 2776, 1714, 1597, 1543, 1496, 1438, 1397, 1305, 1260, 1228, 1199, 1149, 1085, 983, 958, 857, 836, 787, 726, 628, 495, 428; HRMS (ESI) m/z [M + H]+ for C17H10Cl2N2O2 pred 345.0192, meas 345.0179. HPLC purity: 99.58%. Retention time: 26.961 min. 5,7-Dichloro-2-(5,6-dimethoxy-1H-indol-2-yl)quinolin-8-ol (8e). Yellow solid. 75% yield. Rf = 0.34 (petroleum/EtOAc = 1/1), mp 245.8−246.4 °C. 1H NMR (400 MHz, DMSO) δ 12.03 (s, 1H), 8.33 (d, J = 8.6 Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H), 7.65 (s, 1H), 7.22 (s, 1H), 7.09 (s, 1H), 6.94 (s, 1H), 3.85 (s, 3H), 3.80 (s, 3H). 13C NMR (101 MHz, DMSO) δ 149.77, 149.09, 145.67, 138.91, 133.42, 132.96, 132.80, 127.04, 126.90, 123.49, 121.77, 120.00, 115.50, 104.07, 103.85, 103.12, 94.72, 56.18, 55.95. FT-IR 3583, 3524, 3313, 3246, 3075, 2998, 2934, 2834, 1595, 1541, 1484, 1462, 1436, 1412, 1350, 1294, 1250, 1210, 1141, 1082, 1008, 984, 947, 852, 812, 751, 714, 622, 516, 485, 438; HRMS (ESI) m/z [M + H]+ for C19H14Cl2N2O3 pred 389.0454, meas 389.0450. HPLC purity: 99.45%. Retention time: 7.580 min. 5,7-Dichloro-2-(5-hydroxy-6-methoxy-1H-indol-2-yl)quinolin-8-ol (8f). Yellow solid. 69% yield. Rf = 0.26 (petroleum/ EtOAc = 1/1), mp 310.9−311.3 °C. 1H NMR (400 MHz, DMSO) δ 11.78 (s, 1H), 10.42 (s, 1H), 8.22 (d, J = 9.0 Hz, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.78 (s, 1H), 7.64 (s, 1H), 7.04 (s, 1H), 6.73 (d, J = 0.9 Hz, 1H), 4.00 (s, 3H). 13C NMR (101 MHz, DMSO) δ 150.21 (d, J = 1.6 Hz), 148.80 (d, J = 2.6 Hz), 148.69 (d, J = 4.1 Hz), 139.82 (d, J = 12.8 Hz), 138.60 (d, J = 7.6 Hz), 134.40 (s), 133.30 (s), 132.23 (s), 126.69 (s), 123.30 (s), 123.21 (s), 120.34 (s), 119.74 (d, J = 5.7 Hz), 115.36 (d, J = 3.0 Hz), 114.96 (d, J = 6.8 Hz), 104.81 (s), 93.45 (s), 56.37 (s). FT-IR 3589, 3546, 3332, 3265, 3056, 2989, 2941, 2840, 1558, 1516, 1449, 1426, 1406, 1392, 1346, 1289, 1248, 1209, 1135, 1078, 1003, 979, 951, 869, 822, 753, 712, 626, 511, 483, 434; HRMS (ESI) m/z [M + H]+ for C18H11Cl2N2O3 pred 375.0298 meas 375.0292. HPLC purity: 95.18%. Retention time: 5.112 min. 2-(5H-[1,3]Dioxolo[4,5-f ]indol-6-yl)-5,7-dichloroquinolin-8ol (8g). Yellow solid. 79% yield. Rf = 0.35 (petroleum/EtOAc = 1/1), mp 292.9−293.3 °C. 1H NMR (400 MHz, DMSO) δ 8.29 (d, J = 8.7 Hz, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.58 (s, 1H), 7.17 (s, 1H), 7.04 (s, 1H), 6.91 (s, 1H), 5.98 (s, 2H). 13C NMR (101 MHz, DMSO) δ 148.99, 146.36, 143.31, 140.10, 135.97, 135.82, 133.42, 133.34, 133.27, 127.22, 123.89, 122.92, 119.67, 115.71, 103.99, 100.97, 99.52, 92.46. FT-IR 3307, 3111, 3074, 2889, 1595, 1541, 1499, 1471, 1440, 1416, 1349, 1309, 1284, 1242, 1210, 1185, 1151, 1040, 983, 947, 844, 826, 783, 745, 722, 683, 624, 560, 501, 463; HRMS (ESI) m/z [M + H]+ for C18H10Cl2N2O3 pred 373.0141, meas 373.0137. HPLC purity: 99.22%. Retention time: 12.052 min. 2-(5H-[1,3]Dioxolo[4,5-f ]indol-6-yl)-5,7-dichloroquinolin-8ol Hydrochloride (8g·HCl). Yellow solid. 92% yield. Rf = 0.35 (petroleum/EtOAc = 1/1), mp 321.9−322.3 °C. 1H NMR (400 MHz, DMSO) δ 8.39 (d, J = 8.9 Hz, 1H), 8.17 (d, J = 8.9 Hz, 1H), 7.69 (s, 1H), 7.31 (s, 1H), 7.10 (s, 1H), 7.01 (s, 1H), 6.02 (s, 2H). 13C NMR (101 MHz, DMSO) δ 149.79 (d, J = 3.3 Hz), 148.88 (s), 146.72 (s), 143.46 (s), 138.51 (s), 135.22 (dd, J = 14.5, 7.2 Hz), 133.49 (dd, J = 11.2, 4.5 Hz), 126.87 (s), 123.39 (s), 122.84 (dd, J = 8.5, 4.0 Hz),

120.04 (s), 119.68 (s), 115.45 (s), 104.64 (d, J = 3.1 Hz), 101.07 (s), 99.63 (s), 92.34 (d, J = 5.6 Hz). FT-IR 3316, 3112, 3078, 2891, 1596, 1543, 1499, 1473, 1442, 1417, 1349, 1309, 1286, 1245, 1213, 1186, 1154, 1041, 984, 949, 846, 828, 786, 747, 726, 685, 628, 561, 503, 467; HRMS (ESI) m/z [M + H]+ for C18H10Cl2N2O3 pred 373.0141, meas 373.0132. HPLC purity: 99.96%. Retention time: 17.382 min. General Procedures for the Synthesis of 9a, 9d, and 10d. To a solution of 9a, 9d, or 10d (1.0 g, 1.0 equiv) in 10 mL of CH3OH, 2 mL of 37% hydrochloric acid was added. After being stirred at room temperature for 2 h, the reaction mixture was filtered and the residue was dissolved in 200 mL of saturated NaHCO3 solution. Then, it was filtered, washed with pure water, and dried under vacuum to get the crude product which was further purified by recrystallization from AcOEt. 5-(1H-Indol-2-yl)quinolin-8-ol (9a). Yellow solid. 81% yield. Rf = 0.26 (CH2Cl2/CH3OH = 10/1), mp 194.8−195.1 °C. 1H NMR (400 MHz, CDCl3) δ 8.83 (dd, J = 4.1, 1.5 Hz, 1H), 8.68 (dd, J = 8.6, 1.5 Hz, 1H), 8.26 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.49 (dd, J = 8.6, 4.2 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.26− 7.22 (m, 2H), 7.22−7.14 (m, 1H), 6.70 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 152.38, 148.03, 138.36, 136.40, 135.47, 134.59, 128.99, 128.32, 126.90, 122.31, 122.26, 121.84, 120.53, 120.32, 110.86, 109.56, 103.35. FT-IR 3342, 3054, 2506, 1880, 1580, 1500, 1469, 1454, 1413, 1366, 1343, 1297, 1271, 1226, 1190, 1158, 1094, 924, 903, 840, 790, 742, 694, 654, 642, 566, 517, 492, 435; HRMS (ESI) m/z [M − H]− for C17H12N2O pred 259.0877, meas 259.0868. HPLC purity: 95.83%. Retention time: 5.862 min. 5-(5-Hydroxy-1H-indol-2-yl)quinolin-8-ol (9d). Yellow solid. 85% yield. Rf = 0.22 (CH2Cl2/CH3OH = 10/1), mp 235.7−236.3 °C. 1 H NMR (400 MHz, DMSO) δ 8.91 (dd, J = 4.0, 1.4 Hz, 1H), 8.69 (dd, J = 8.6, 1.4 Hz, 1H), 7.73−7.57 (m, 2H), 7.21 (t, J = 8.6 Hz, 2H), 6.92 (d, J = 2.1 Hz, 1H), 6.66 (dd, J = 8.6, 2.2 Hz, 1H), 6.47 (s, 1H). 13 C NMR (101 MHz, DMSO) δ 153.45 (s), 151.22 (d, J = 11.8 Hz), 148.67 (s), 138.91 (s), 136.56 (d, J = 14.6 Hz), 134.74 (s), 131.63 (d, J = 15.0 Hz), 129.71 (d, J = 3.5 Hz), 128.66 (s), 127.02 (s), 122.74 (s), 122.05 (d, J = 5.4 Hz), 112.00 (d, J = 4.5 Hz), 111.33 (s), 104.11 (d, J = 6.1 Hz), 101.60 (d, J = 4.2 Hz). FT-IR 3404, 3335, 2922, 1702, 1624, 1581, 1504, 1465, 1453, 1413, 1365, 1270, 1223, 1179, 1097, 1072, 953, 837, 785, 714, 647, 592, 566, 485, 459; HRMS (ESI) m/z [M + H]+ for C17H12N2O2 pred 277.0972, meas 277.0971. HPLC purity: 99.25%. Retention time: 14.006 min. 7-Chloro-5-(5-hydroxy-1H-indol-2-yl)quinolin-8-ol (10d). Yellow solid. 90% yield. Rf = 0.29 (CH2Cl2/CH3OH = 20/1), mp 325.0−326.1 °C. 1H NMR (400 MHz, DMSO) δ 8.56 (d, J = 8.4 Hz, 2H), 7.55 (s, 1H), 7.34 (dd, J = 8.5, 4.1 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 2.1 Hz, 1H), 6.56 (dd, J = 8.5, 2.2 Hz, 1H), 6.28 (s, 1H). 13C NMR (101 MHz, DMSO) δ 163.64, 151.06, 146.03, 145.22, 138.25, 134.26, 131.29, 131.15, 130.08, 127.59, 121.12, 116.76, 111.55, 110.86, 108.57, 103.81, 99.63. FT-IR 3404, 3211, 1623, 1592, 1551, 1521, 1500, 1449, 1377, 1355, 1306, 1251, 1196, 1165, 1101, 871, 801, 785, 732, 684, 649, 611, 590, 501, 460; HRMS (ESI) m/z [M + H]+ for C17H11N2O2Cl pred 311.0582, meas 311.0594. HPLC purity: 95.22%. Retention time: 9.986 min. Oxygen Radical Absorbance Capacity (ORAC-FL) Assay.42,43 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-2,5,7,8tetramethylchroman-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 of AAPH in 10 mL of 75 mM phosphate buffer (pH 7.4) to a final concentration of 40 mM. The mixture of the tested compound (20 μL) and FL (120 μL; 70 nM, final concentration) was preincubated for 10 min at 37 °C, 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 in triplicate, and at least three independent runs were performed for each sample. The antioxidant curves (fluorescence versus time) were normalized to the curve of the 1889

DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894

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blank. The area under the fluorescence decay curve (AUC) was calculated as following equation:

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 (15 μL, 50 μM) was incubated at 37 °C for 24 h. The tested compound (15 μL, 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 °C for 24 h. The tested compound (10 μL, 50 μM, final concentration) 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. TEM Assay. For the metal-free experiment, Aβ stock solution was diluted with a 10 mM phosphate buffer (pH = 7.4); for the copperinduced experiment, Aβ stock solution was diluted with 20 μM HEPES (pH = 6.6) and 150 μM NaCl. The sample preparation was the 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. Metal-Chelating Study. The chelating studies were performed with a UV−vis spectrophotometer. The absorption spectra of compound 8d (50 μM, final concentration) alone or in the presence of CuSO4, FeSO4, FeCl3, or ZnCl2 (50 μM, final concentration) in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were incubated for 30 min and then recorded at room temperature, respectively. For the stoichiometry of the compound−Cu2+ complex, a fixed amount of 8d (50 μM) was mixed with growing amounts of copper ion (0−100 μM), and the difference UV−vis spectra were examined to investigate the ratio of ligand/metal in the complex. Neuronal Cell Proliferation Assay.16,35 The undifferentiated rat neuronal cells PC12 (purchased from cell bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in F12K medium supplemented with 10% horse serum and 5% fetal bovine serum. The cells were placed into the wells of 6-well plates at 1 × 105 cells/well (used for morphological studies) and 96-well plates at 5 × 104 cells/mL (used for neuron numbers quantification) held in an incubator at 37 °C under a 5% CO2 atmosphere. After 24 h, the medium was replaced with fresh medium, and the undifferentiated neuronal cells were treated with vehicle (0.1% (v/v) DMSO), NGF (50 ng/mL, Peprotech, London, U.K.), or 0, 0.5, 1, 5, 10, 20, 40, or 50 μM quinoline−indole derivative for 3 days. At least six fields were randomly selected and photographed per well at 40× magnification (morphological studies) and 4× magnification (neuron numbers studies) using an EVOS FL microscope (EVOS FL Auto) equipped with a digital camera. Neurite length and neuron numbers were analyzed using Image-ProPlus based on morphological results. For the possible mechanism studies of mitogenic effect, neuronal survival was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 570 nm) assay and cells that were only treated with vehicle were set as control (100%). At least three independent experiments were performed. Anti-Intracellular Oxidative Stress in SH-SY5Y Cells. SH-SY5Y cells were seeded at 1 × 104 cells/well in 96-well plates. After 24 h, the medium was removed and replaced with test compounds at 37 °C and kept for another 24 h. The cells were washed with PBS and incubated with 5 μM DCFH-DA (a fluorescent probe) in PBS at 37 °C in 5% CO2 for 30 min. After removal of DCFH-DA and further washing, the cells were exposed to 0.1 mM t-BuOOH (tert-butyl hydroperoxide, a compound used to induce oxidative stress) in PBS for 30 min. At the

i = 120

AUC = 1 +

∑

(fi /f0 )

i=1

where f 0 is the initial fluorescence reading at 0 min and f i is the fluorescence reading at a specific time. The net AUC was calculated by the following expression: AUCsample − AUCblank. Regression equations between net AUC and Trolox concentrations were calculated. ORACFL value for each sample were calculated by using the standard curve which means the ORAC-FL value of tested compound expressed as Trolox equivalents. 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.44−46 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 of PBS/EtOH (7:3), and the filter membrane was impregnated with 4 μL of 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 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)At]} × 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 in 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). ThT Assay.47 Aβ1−42 (Millipore, counterion: 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 5 min later (excitation, 450 nm; emission, 485 nm). The percent 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 with a solution of 20 μM HEPES (pH 6.6) and 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 1890

DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894

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end of the incubation, the fluorescence of the cells from each well was measured (λ excitation = 485 nm, λ emission = 535 nm) with a multifunctional microplate reader (Molecular Devices, FlexStation 3). The antioxidant activity was expressed as percentage of control cells and calculated by the formula [(Ft − Fnt)/(Ft′ − Fnt)] × 100, where Ft = absorbance of treated neurons with tested compound, Ft′ = absorbance of treated neurons without tested compound, and Fnt = absorbance of neurons not treated with t-BuOOH. Metabolic Stability. The rat liver microsomes were prepared according to the previously described methods.31,48 Compounds 7g· HCl, 8c·HCl, 8d·HCl, 8g·HCl, CQ, donepezil, and testosterone were dissolved in CH3CN as 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 of buffer solution (PBS, 100.0 mM; MgCl2, 3.0 mM; 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 incubation was terminated at different time points by adding 0.5 mL of ice-cold CH3CN. A parallel incubation was performed in the absence of NADPH-regenerating system and microsomes as the negative control, and the reactions were terminated after the corresponding time incubation. After centrifugation with 12 500 rpm at 4 °C for 10 min, the supernatant was directly analyzed by the HPLC−UV systems (Agilent HPLC 1200 instrument). Three independent experiments were performed in triplicate. Acute Toxicity and Histological Studies. The procedures for acute toxicity study followed were the similar protocols in our previous studies.31,49 C57BL/6 mice (half male and half female, 12 weeks old, 25−30 g) purchased from the laboratory animal center of Sun Yat-sen University (Guangzhou, China) were used to evaluate the acute toxicity of compounds 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCl. Mice were maintained with a 12 h light/dark cycle (light from 07:00 to 19:00) at 20−22 °C with a 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. Compounds 7g·HCl, 8c·HCl, 8d·HCl, and 8g·HCl were in suspension in 0.5% carboxymethylcellulose sodium (CMC-Na) salt solution (677, 1333, and 2000 mg/kg) and given via oral administration according to the divided experimental groups. After the 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, lung, and brain. The organs were carefully collected, postfixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin. For the brain, coronal sections (4 μm) were cut through the entire hippocampus; for other organs, the sections (4 μm) were cut through the maximum transverse sections and used 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 histopathological abnormalities were investigated under EVOS FL microscope (EVOS FL Auto) equipped with a digital camera. Hippocampal Cell Proliferation In Living Adult C57BL/6 Mice.17,25,29 Adult (12 weeks old) wild type C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China) for in vivo study. In order to ensure a low baseline level of neurogenesis, mice were individually housed without access to running wheels beginning 7 days prior to pump implantation and throughout the entire procedure, as both social activity and voluntary exercise could stimulated hippocampal neurogenesis. 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. For the icv method group, each compound was dissolved at 10 μM (containing 0.05% DMSO) in an artificial cerebrospinal fluid (aCSF:

NaCl (128 mM), KCl (2.5 mM), CaCl2 (0.95 mM), MgCl2 (1.9 mM)) and then infused icv into the left lateral ventricle of six adult (12 weeks old) wild type C57BL/6 mice by means of surgically implanted Alzet osmotic minipumps which delivered the solution into animals at a constant rate of 0.5 μL/h for 7 days, respectively. Surgical implantation of Alzet osmotic minipumps (Alzet 1007D; Cupertino, CA) was conducted as follows. Animals were anesthetized with intraperitoneally administered sodium pentobarbital (dosage, 0.15 mL/10g; concentration, 0.3% in normal saline), and the back of the neck and upper back area were then shaved and swabbed with 70% ethanol and betadine. A small midline incision was made in the skin between the scapulae to allow use of a hemostat to create a small pocket by spreading the subcutaneous connective tissue apart. The pump was inserted into the pocket with the flow moderator pointing away from the incision, and the incision was closed with surgical thread. Surgery to implant the cannula into the lateral ventricle followed immediately, while animals were still anesthetized. The top portion of the head was shaved and the animal was then placed in a stereotactic holder. The head was swabbed with 70% alcohol followed by betadine, and an incision was made in the scalp to expose the surface of the skull in the area of bregma. The skull was cleaned of fascial tissue and a burr hole was made in the skull such that the guide cannula could be lowered into the burr hole to a depth H = +3 mm deep to the pial surface, AP = −0.3 mm anteroposterior relative to bregma, and L = −1.3 mm lateral to midline. The guide cannula was secured to the skull with dental cement, and the scalp incision was closed by suture thread. Animals were then placed in a clean warm cage on a heating pad until mobile. For the oral administration group, the mice in each group were administered the corresponding drug dosage in a 0.5% CMC-Na solution (0.02 mL/g) by intragastric infusion for 7 days (using a blank 0.5% CMC-Na solution as placebo for vehicle groups). All mice had free access to food and water, and bromodeoxyuridine (BrdU, Sigma-Aldrich) was injected intraperitoneally at 50 mg kg−1 day−1 for 6 days during pump infusion in order to detect agents that either promote proliferation or augment survival of proliferating neural stem cells in the hippocampus. The injection site was alternated between right and left sides. Animals were monitored daily for general health and weighed every day. Accumulated weight loss of >20% relative to presurgical weight was considered a sign of sickness and any such animals were euthanized for humane treatment. Twenty-four hours after the final BrdU administration, mice were sacrificed by transcardial perfusion with 4% paraformaldehyde at pH 7.4, and their brains were processed for immunohistochemical detection of incorporated BrdU in the hippocampus. Dissected brains were immersed in 4% paraformaldehyde overnight at 4 °C, then embedded in paraffin before being sectioned with a Leica SM2000R sliding microtome coronally into 30 μm thick free-floating sections. Unmasking of BrdU antigen was achieved through incubating tissue sections for 2 h in 50% formamide/2× SSC at 65 °C, followed by 5 min wash in 2× SSC and subsequent incubation for 30 min in 2 M HCl at 37 °C. Sections were processed for immunohistochemical staining with mouse monoclonal anti-BrdU (1:100, Sigma-Aldrich). Images were analyzed with a motorized research microscope, and the number of BrdU+ cells in the entire hippocampus in the contralateral hemisphere (opposite side of surgically implanted pump) was quantified by counting BrdU+ cells within hippocampus in every fifth section throughout the entire hippocampus and then normalizing for hippocampus volume. Pharmacokinetics Analysis and log BB Values Studies. Pharmacokinetic properties of 8d·HCl were analyzed by Medicilon Companies, Shanghai, China. Male SD rats with body weight of 220− 250 g were purchased from Shanghai SIPPR-BK LAB Animal Ltd., Shanghai, China. Compound 8d·HCl was dissolved in 5% DMSO, 10% Solutol, and 85% saline for intravenous administration (iv, 1 mg/ mL) and oral administration (po, 3 mg/mL). A single dosage of 5 mg/ kg for iv (three SD rats) and 30 mg/kg for po (three SD rats) of the formulated compounds were administrated, with the blood samples taken at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after the compound administration. The concentration of the corresponding compound in blood was analyzed by LC−MS/MS (Shimadzu 1891

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liquid chromatographic system and API4000 mass spectrometer, Applied Biosystems, Ontario, Canada). For the log BB values studies, a single dosage of 30 mg/kg for po (three SD rats) of the formulated compounds was administrated, 1 h after the oral administration of compound 8d·HCl, the three SD rats were sacrificed, the plasma and brains were collected from the three animals, and the concentration of the corresponding compound in blood and brain was analyzed by LCMS/MS. Cognitive and Memory Improvement in APP/PS1 AD Mice by Long-Term Oral Uptake of Compounds.31 Animals. Sixmonth-old double transgenic (APP/PS1) AD mice and C57BL/6 littermates mice (wild type, WT) were purchased from the Beijing HFK Bioscience Co., Ltd. (Beijing, P. R. China), the APP/PS1 mice were randomly allocated into four groups (n = 10 for each group): vehicle, CQ (30 mg kg−1 day−1), 8d·HCl (30 mg kg−1 day−1), and 8g· HCl (30 mg kg−1 day−1). The mice were maintained on a 12 h light/ dark cycle (light from 07:00 to 19:00) at 23 °C and 60−70% relative humidity, with ad libitum 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. The animals were taken to their individual cages, and the mice in each group were administered the corresponding drug dosage in a 0.5% CMC-Na solution (0.02 mL/g) by intragastric infusion throughout the entire assay (using a blank 0.5% CMC-Na solution as placebo for vehicle and WT groups). Morris Water Maze Test. Seventy-four days later, behavioral performance was evaluated using the Morris water maze task. (The water maze apparatus consisted of a circular pool, 65 cm in diameter and 40 cm high, filled to a depth of 17 cm with fresh water (kept at 25 °C, the fresh water was refilled each day before the trails), which was made opaque by the addition of nontoxic white TiO2 at a concentration of 0.25 g/L.) The task demanded incremental learning of the location of a fixed, hidden platform (6.5 cm in diameter and 16 cm high, submerged 1.0 cm under the opaque water level) throughout the training period. In the single training assay, each mouse underwent reference memory training (four trials per day and a 30 min intertrial interval) with a fixed hidden-platform located in the center of third quadrant (southwest) of the pool for 5 consecutive days. The mouse was 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 mouse did not find the platform within 90 s, the mouse was guided onto the platform and the escape latency was recorded as 90 s. For each trial, the mouse was allowed 15 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 2-fold diameter of the original platform location was set as the effective region. Each mouse was allowed to swim freely in the pool for 90 s (only one trial) from the northeast, a new starting location, which was farthest from the virtual platform location. The number of times the swimming mouse crossed the virtual platform location (original platform location), the average swimming speed, the swimming path length as well as time spent the in the virtual platform, the effective region, and the correct quadrant were recorded by the computerized digitizing video-tracking system to analyze the level of memory retention. Aβ Plaque Immunohistochemistry in the Brain of APP/PS1 AD Mice. After the Morris water maze experiment, animals were deeply anesthetized with an intraperitoneal injection of and transcardially perfused with 4% paraformaldehyde. The brain was carefully

removed, postfixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin. Three coronal sections (30 μm in thickness and 120 μm interval) were cut through the medial hippocampus for immunohistochemical staining. The sections were pretreated with 3% H2O2 for 25 min to block endogenous peroxidase activity, then the sections washed with distilled water and 0.1 M Tris-buffered saline (TBS), pH 7.4. Sections were immunostained using the following antibodies: incubated at 4 °C overnight with the primary amyloid-β monoclonal antibody (mOC64; 1:300; Abcam, U.K.), subsequently rinsed and incubated with biotinylated secondary antibody (Dako) for 50 min, finally with the chromogen diaminobenzidine. After staining, the sections were dehydrated and observed under EVOS FL microscope (EVOS FL Auto) equipped with a digital camera. The Aβ plaque deposition in brain was calculated as the percentage of the Aβ deposition area with respect to the overall area in three representative images for each mouse by using the ImageJ software. Statistical Analysis. All data were expressed as the mean ± standard deviation. Results were subjected to Student’s t test or oneway analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.05 was accepted to indicate the significance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01417. HPLC data of the target compounds and the method for PAMPA (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*X.L.: phone, +086-20-3994-3050; fax, +086-20-3994-3050; email, [email protected]. *L.H.: phone, +086-20-3994-3051; fax, +086-20-3994-3051; email, [email protected]. ORCID

Zhiren Wang: 0000-0002-2016-0517 Xingshu Li: 0000-0002-7433-739X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Grants 21772241 and 21302235), Guangdong Natural Science Foundation (Grant 2014A030313124), Guangdong High-Level Personnel of Special Support ProgramYoung Top-Notch Talent Project (Grant 2015TQ01R244), Guangzhou Pearl River New Star Fund Science and Technology Planning Project (Grant 201610010111), and The Fundamental Research Funds for the Central Universities (Grant 15ykpy04) for financial support of this study.

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ABBREVIATIONS USED Aβ, amyloid-β; AD, Alzheimer’s disease; ADME, absorption, distribution, metabolism, and excretion; aCSF, artificial cerebral spinal fluid; ALS, amyotrophic lateral sclerosis; BBB, blood− brain barrier; BrdU, bromodeoxyuridine; CA, cornus ammonis; CMC-Na, carboxymethylcellulose sodium; CNS, central nervous system; CQ, clioquinol; CSF, cerebrospinal fluid; DCFH-DA, dichlorofluorescein diacetate; DG, dentate gyrus; ERK, extracellular signal-regulated kinase; HD, Huntington’s disease; HE, hematoxylin and eosin; HRMS, high-resolution mass spectra; icv, intracerebroventricular; ip, intraperitoneal; 1892

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MAPK, mitogen-activated protein kinase; MTDL, multitargetdirected ligand; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; MWM, Morris water maze; NMDAR, N-methyl-D-aspartate (NMDA) receptor; ORAC, oxygen radical absorbance capacity; PAMPA, parallel artificial membrane permeability; PBS, phosphate buffered saline; PD, Parkinson’s disease; PO, oral administration; ROS, reactive oxygen species; SAR, structure−activity relationship; SGZ, subgranular zone; TBI, traumatic brain injury; t-BuOOH, tertbutyl hydroperoxide; TEM, transmission electron microscopy; ThT, thioflavin T; TLC, thin-layer chromatography; WT, wild type

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Journal of Medicinal Chemistry

Article

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DOI: 10.1021/acs.jmedchem.7b01417 J. Med. Chem. 2018, 61, 1871−1894