Discovery of small molecules for up-regulating the translation of antiamyloidogenic secretase, a disintegrin and metalloproteinase 10 (ADAM10), by binding to the G-quadruplex-forming sequence in the 5' untranslated region (UTR) of its mRNA

Article

pubs.acs.org/jmc

Discovery of Small Molecules for Up-Regulating the Translation of

Antiamyloidogenic Secretase, a Disintegrin and Metalloproteinase

10 (ADAM10), by Binding to the G‑Quadruplex-Forming Sequence in

the 5′ Untranslated Region (UTR) of Its mRNA

Jie Dai,^‡Zhen-Quan Liu,^‡Xiao-Qin Wang, Jing Lin, Pei-Fen Yao, Shi-Liang Huang, Tian-Miao Ou,

Jia-Heng Tan, Ding Li, Lian-Quan Gu, and Zhi-Shu Huang*

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China

S

* Supporting Information

ABSTRACT: Up-regulation of a disintegrin and metalloproteinase 10 (ADAM10) to prevent the formation of β-amyloid (Aβ)

peptides might be a promising strategy to treat Alzheimer’s disease (AD). RNA G-quadruplex motif within the 5′-UTR of the

ADAM10 mRNA is an inhibitory element for ADAM10 translation. Thus, mitigation of the suppressive eﬀect of this motif using

an RNA G-quadruplex-forming G-rich sequence (QGRS) binder might be a new approach for AD therapy. Herein, a series of

new methylquinolinium derivatives were synthesized and screened by surface plasmon resonance (SPR) and the dual-luciferase

reporter assay. Among them, compound 24 showed selective aﬃnity for the QGRS of ADAM10 and could strongly up-regulate

the translation of it. Moreover, treatment with 24 led to a signiﬁcant increase of the secretion of sAPPα, consequently decreasing

the Aβ₄₀in cellular. These results illustrate that the interaction between the RNA QGRS and a small molecule may be a new

molecular strategy to modulate the translation of ADAM10.

Clinical studies have provided evidence that ADAM10 is a

candidate AD susceptibility gene,¹¹and signiﬁcantly reduced

levels of both ADAM10 and sAPPα are present in the platelets

of AD patients.¹³Therefore, up-regulation of ADAM10 and/or

stimulation of ADAM10 activity with the consequence of

reducing the generation of Aβ might be a promising strategy to

treat AD.

The strategies for the modulation of ADAM10 cleavage can

be broadly classiﬁed into four groups: the transcriptional level,

translational level, enzymatic activity, and indirect modulation

(Figure 1). Progress toward development of a direct activator of

α-secretase as a drug treatment for AD has not been

encouraging.¹⁷Transcriptional modulators including acitretin,¹⁴

a vitamin A derivative, as well as GABA-A receptor modulators

including etazolate hydrochloride (EHT-0202)^18,19can pro-

mote sAPPα secretion and are considered to be promising drug

INTRODUCTION

■

Alzheimer’s disease (AD) is a common neurodegenerative

disorder involving a progressive loss of cognitive functions. β-

Amyloid (Aβ) peptide is believed to play a signiﬁcant role in

the neurotoxicity and the development of AD.¹Neurotoxic Aβ

results from the amyloidogenic processing of the amyloid

precursor protein (APP) in which it is sequentially cleaved by

β- and γ-secretase.^1,2Alternatively, a third protease, α-secretase,

can cleave APP within its amyloid domain and liberate a

secreted product, sAPPα, that has neuroprotective properties,^3,4

thereby preventing the production of the neurotoxic Aβ (Figure

1). Moreover, it is well established that the regulated

component of α-secretase can compete with β-secretase and

thereby reduce the generation of neurotoxic Aβ, suggesting that

a pharmacological activation of α-secretase could improve the

therapeutic approach in AD.⁵Some members of a large family

of disintegrin and metalloproteinases (ADAM) have been

shown to exert α-secretase activity.⁶⁻⁹Among these, ADAM10

has been identiﬁed the major member for the α-secretase.¹⁰⁻¹⁶

Received: January 26, 2015

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Figure 1. Schematic representation of the APP processing pathway and strategy to use small molecules to reduce Aβ. APP ectodomain shedding is

mediated by either an α-secretase or the β-secretase. While the APP ectodomain released by α-secretase liberates the neuroprotective sAPPα, β-

secretase/γ-secretase cleavage leads to the release of neurotoxic Aβ peptides. The methods targeting Aβ can be classiﬁed into three groups: the

enhancement of α-secretase activity and the inhibition of β-secretase or γ-secretase cleavage. Among these, regulation of the translational level of α-

secretase (ADAM10) has stimulated the most interest.

Figure 2. Antisense oligonucleotide AS destabilizes the G-quadruplexes of the ADAM10 mRNA to enhance luciferase expression. (A) AS traps RNA

G-quadruplex motifs into double-stranded structures in a concentration-dependent manner. A ﬂuorescently labeled A10-RNA-Wt sequence (Cy3

and Cy5 at its 3′ and 5′ ends, respectively) was titrated with increasing concentrations of the AS deoxyoligonucleotide and analyzed using a native gel

electrophoresis assay. (B) Schematic representation of the plasmids used for the dual-luciferase reporter gene assays. The wild-type QGRS (A10-Wt)

of the ADAM10 5′-UTR or its mutated variants (A10-Mut) and the wild-type full-length 5′-UTR of ADAM10 (5′UTR-Wt) or its QGRS motif-

mutated variants (5′UTR-Mut) were cloned directly in front of the Renilla coding region.²²(C) Eﬀect of antisense deoxyoligonucleotides on the

dual-luciferase assays. The dual-luciferase assay was performed at 24 h post-transfection with AS or random deoxyoligonucleotides. The fold changes

in expression were calculated relative to blank control (control was set as 1). All data are expressed as the mean SD from three independent

experiments. The symbols ∗∗∗ indicate signiﬁcant diﬀerences at P < 0.001 vs control.

candidates for AD.^17,20However, attenuation of AD symptoms

by these drugs has not been demonstrated in patients.¹⁷

Recently, there has been increased interest in the regulation of

ADAM10 expression through mechanisms involving the 5′

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Figure 3. Eﬀect of the antisense oligonucleotide AS on ADAM10 translation. (A) Western blot showing the levels of APP, ADAM10, and β-actin

proteins in HeLa cells transfected with 200 nM AS or random deoxyoligonucleotides. (B) Western blot showing the levels of sAPPα, APP,

ADAM10, and β-actin proteins in HEK-APP cells transfected with 200 nM AS or random deoxyoligonucleotides. All data were normalized to β-

actin, and the fold changes in expression were calculated relative to the blank control (the control was set to 1). All data are expressed as the mean

SD from three independent experiments. The symbols ∗∗ and ∗∗∗ indicate signiﬁcant diﬀerences at P < 0.01 and P < 0.001 vs control.

untranslated region (UTR) of the ADAM10 mRNA.²¹

Importantly, translation of ADAM10 is suppressed by its long

GC-rich 5′-UTR, which contains a G-rich sequence that can

form stable RNA G-quadruplex secondary structures.²²Further

study has demonstrated that the presence of this RNA G-rich

sequence in an overexpression construct of ADAM10 caused a

reduction in the protein levels, while the mRNA levels were not

altered. Mutation of the G-quadruplex motif results in increased

ADAM10 translation and consistently promoted the secretion

of sAPPα.²²These data provide an alternative basis to

hypothesize that the RNA G-quadruplex-forming G-rich

sequence (QGRS) is a new molecular target for therapeutic

eﬀorts to up-regulate ADAM10 expression.²³

An increasing number of reports in the past few years have

focused on the RNA G-quadruplex within the 5′-UTR because

of its critical biological functions especially in translation.²⁴

Down-regulation of translation by RNA G-quadruplex regions

has been reported for several genes including EBNA1,²⁵N-

ras,²⁶Zic-1,²⁷Bcl-2,²⁸and others,²⁹and up-regulation of

translation by this structure has also been reported for

VEGF.³⁰More signiﬁcantly, regulation of translation by

exogenous factors, such as small molecules and antisense

oligonucleotides (ASO) that bind to the RNA G-quadruplex

within the 5′-UTR of mRNAs, has been proven to be

eﬀective.^25,31,32Several examples so far suggest a mechanism

by which small molecules act as RNA G-quadruplex binders to

inhibit translation via stabilization of the 5′-UTR RNA G-

quadruplex structure.³³⁻³⁷For example, Pierre Murat et al.

demonstrated that targeting the EBNA1 mRNA with a small

molecule that stabilized the G-quadruplex structures inhibits

the translation of the EBNA1 mRNA and decreases antigen

presentation.²⁵On the other hand, G-quadruplex binders have

rarely been shown to increase the translation eﬃciency. The

only example is the cationic porphyrin TMPyP4, which has

been reported to destabilize the (CGG)_nintramolecular

quadruplex structure and increase translation eﬃciency of the

5′-UTR mRNA.³⁸These combined data suggest a new strategy

by which exogenous factors such as small molecules may

modify the inhibitory eﬀect of the G-quadruplexes on

translation.

Here, we report the eﬀect of exogenous factors on the

elimination of the inhibitory eﬀect of the G-quadruplexes

located in the 5′-UTR of ADAM10 on the translation of the

mRNA, as well as the discovery of translational up-regulators,

the design, synthesis, and structure−activity relationships

(SARs) of new methylquinolinium derivatives that target the

QGRS in the 5′-UTR of the ADAM10 mRNA, and the in-

depth evaluation of the optimized compound.

RESULTS AND DISCUSSION

■

DNA Antisense Oligonucleotides Targeting RNA

QGRS Increase the Translation Eﬃciency of ADAM10.

Mutation of the G-quadruplex motif in 5′-UTR resulted in

increased ADAM10 translation and consistently promoted the

secretion of sAPPα.²²However, this study provided no

evidence for the ability of exogenous factors to eliminate the

inhibitory eﬀect of the G-quadruplexes on the translation of the

ADAM10 mRNA. Recently, oligonucleotides-based strategies

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Table 1. Binding Data for the Interaction between RNA and Designed Analogs Using the SPR Assay

a

K_Dfor diﬀerent compounds toward the A10-RNA-Wt sequence as determined using SPR. Compounds that did not achieve saturation state at the

highest tested concentration (50 μM) are noted. Compounds noted as >50 μM are considered to have low aﬃnity for the RNA QGRS.

that, using antisense oligonucleotides to modulate RNA G-

quadruplexes folding, aﬀect the translation of speciﬁc mRNAs

were on the rise.^25,32Pierre Murat et al. were the ﬁrst to

demonstrate that destabilization of G-quadruplexes within the

EBNA1 mRNA using antisense oligonucleotides (both the

RNA antisense oligonucleotide and its DNA analog) increases

EBNA1 mRNA translation.²⁵According to this strategy, and

with the goal of verifying the feasibility of designing small-

molecule modulators of QGRS within ADAM10 mRNA to

regulate ADAM10 translation, in an initial study we explored

the eﬀect of DNA antisense oligonucleotides on the elimination

of inhibitory eﬀect of G-quadruplexes in the 5′-UTR on

ADAM10 mRNA translation. First, the results of native gel

electrophoresis assays showed that titration with increasing

concentrations of DNA antisense oligonucleotides (AS) caused

the G-quadruplexes bands (lower bands) to gradually

disappear, indicating the ability of the antisense oligonucleo-

tides to trap the G-quadruplex motifs of ADAM10 into double-

stranded structures (upper bands) (Figure 2A).

of the Renilla luciferase in the psi-CHECK-2 vector (Figure

2B), was used. The G-quadruplex motif mutant variant (A10-

Mut) and psi-CHECK-2 blank vector (A10-Del) were used as

system controls,²²and transfection with no oligonucleotide or a

random deoxyoligonucleotide was used as negative control in

this experiment. The data presented in Figure 2C demonstrate

a 3.4-fold increase in the luciferase expression following

treatment for 24 h with the AS deoxyoligonucleotide in the

cells containing the A10-Wt plasmid. As expected, the eﬀect of

the AS deoxyoligonucleotide on luciferase expression in the

cells containing the mutant or blank plasmids was insigniﬁcant.

Furthermore, the deoxyoligonucleotide-free control group and

random deoxyoligonucleotide group also demonstrated no

obvious change in luciferase expression. Next, we asked

whether the AS oligonucleotide was capable of improving the

expression level of genomic ADAM10 in a cellular system. To

address this question, we measured the protein levels of

ADAM10 using a Western blotting assay 48 h after transfection

with either the AS deoxyoligonucleotide or a random

deoxyoligonucleotide. The results demonstrated a 2.5-fold

and 2.8-fold increase in ADAM10 expression following

treatment with the AS deoxyoligonucleotide in HeLa and

HEK-APP cells, respectively (Figure 3). Insigniﬁcant changes

were observed in the deoxyoligonucleotide-free and random

control groups. Additionally, RT-PCR was used to assess the

The eﬀects of the AS deoxyoligonucleotide on translation

were subsequently detected using the dual-luciferase reporter

system, which is an appropriate system for evaluating the

capacity of exogenous factors to regulate gene expression. For

these experiments, the reporter plasmid described previously,²²

carrying the ADAM10 G-quadruplex motif (A10-Wt) in front

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Table 2. Binding Data for the Interaction between RNA and the Designed Analogs Using the SPR Assay

a

K_Dfor diﬀerent compounds toward the A10-RNA-Wt sequence as determined using SPR. Compounds that did not achieve saturation state at the

highest tested concentration (50 μM) are noted. Compounds noted as >50 μM are considered to have low aﬃnity for the RNA QGRS..

changes in the mRNA levels on parallel samples. The results

showed no obvious change in the levels of the ADAM10

mRNA (Figure S1 in Supporting Information), indicating that

the AS-induced increase in ADAM10 expression was mainly the

result of the eﬀects of the deoxyoligonucleotide on the

translation process.

Together, our data demonstrated that targeting the QGRS

within 5′-UTR of ADAM10 mRNA with a complementary

antisense oligonucleotide, an exogenous regulator, could

successfully stimulate ADAM10 translation, suggesting that

the strategy to use exogenous factors to eliminate the inhibitory

eﬀect of the G-quadruplexes in the ADAM10 mRNA on its

translation is likely to be feasible.

Initial Screening of RNA QGRS Binders Using SPR.

Indeed, antisense-oligonucleotide-based strategies represent

sequence-speciﬁc modulation for targeting unique RNA G-

quadruplex;^25,32however, their poor pharmacological proper-

ties still represent an issue.²⁹Thus, “druglike” synthetic small

molecules provide an alternative option for targeting RNA G-

quadruplex.^31,37Few previous studies of the use of small

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Table 3. Binding Data for the Interaction between RNA and Analogs 24−27 Using the SPR Assay

a

K_Dfor diﬀerent compounds toward the A10-RNA-Wt sequence as determined using SPR. Compounds that did not achieve saturation state at the

highest tested concentration (50 μM) are noted. Compounds noted as >50 μM are considered to have low aﬃnity for the RNA QGRS.

molecules that can bind to the QGRS domains of RNA to

enhance gene translation have been reported. To identify small

molecules that bind selectively to the QGRS in the 5′-UTR of

the ADAM10 mRNA, we performed a screening experiment

using surface plasmon resonance (SPR) in which biotinylated

wild-type (A10-RNA-Wt) and mutant (A10-RNA-Mut)

QGRSs in ADAM10 mRNA were immobilized on a

neutravidin-coated sensor chip. SPR is a very sensitive method

that measures time-resolved binding events to a surface

immobilized biomolecule and provides information on binding

kinetics and aﬃnity. In an SPR experiment, a higher relative

intensity (RU) indicates a stronger interaction. We tested 7

classes, a total of 52 compounds synthesized by our lab group,

including curcumin derivatives, crytolepine derivatives, berber-

ine derivatives, rutaecarpine derivatives, quinazoline derivatives,

1-methylquinolinium derivatives, and natural products. As

shown in Figure S2A, it is noteworthy that RU values for

three 1-methylquinolinium derivatives (violet bars) for QGRS

(A10-RNA-Wt) in ADAM10 were above 50, suggesting their

potential high aﬃnity for this domain. Furthermore, the

corresponding RU values for the same compounds for the

mutant of QGRS were below 20, indicating that the 1-

methylquinolinium derivatives had low speciﬁc interactions

with A10-RNA-Mut (Figure S2B). These results suggested that

1-methylquinolinium might be a class of promising candidates

for the development of small molecules that could up-regulate

the translation of ADAM10. Encouraged by these results, we

proceeded with the development of novel 1-methylquinolinium

derivatives by medicinal chemistry optimization.

Synthesis of Methylquinolinium Derivatives. A series

of novel 1-methylquinolinium derivatives was developed and

synthesized. It was found that the amine side chain at the 4-

position and the aromatic ring at the 2-position of the quinoline

ring might play important roles in their aﬃnity to the QGRS.

Therefore, the newly synthesized compounds were primarily

designed with changes in these two positions. The synthesis of

the 1-methylquinolinium derivatives (1−28) was performed

following the process shown in Scheme S1 using the method

provided in our previous report.³⁹Taking into consideration

the impact of methylation of the quinoline on the biological

activity, four quinoline derivatives (29−32) were also

synthesized according to the method reported by our group

(Scheme S2). All of the synthetic compounds (1−32) are

shown in Tables 1−4.

SAR Study Using SPR Assay. To search for more potential

RNA QGRS binders, the quinolinium derivatives from the

initial screening study and the newly synthesized analogs were

further analyzed using the SPR assay to determine the

equilibrium dissociation constants (K_D) as indicated by the

analysis of the dynamic process of binding, and the structure/

binding aﬃnity relationship was explored.

First, to investigate the impact of the amine side chain at the

4-position of 1-methylquinolinium moiety on the binding

aﬃnity, two series of analogs that contained a 4-hydroxy-3,5-

dimethoxystyryl group (1−4) or a 2-hydroxy-5-nitrostyryl

group (5−8) at the 2-position of the quinoline were

synthesized. The SPR assay results for the binding of these

compounds to A10-RNA-Wt (Table 1) showed similar SAR

trends, in which the introduction of a morpholine group (2, 6)

or a piperidine group (3, 7) had no signiﬁcant eﬀect on the

aﬃnity of compounds to RNA, but the introduction of a

methylpiperazine group (4, 8) led to an improvement with K_D

values of 16.5 and 16.3 μM, respectively, compared with

compounds 1 and 5 (K_D= 20.5 and 46.3 μM, respectively),

which lacked substitutions at this position. The results indicated

that methylpiperazine motif might be a promising group for the

interaction of compounds with QGRS in ADAM10 mRNA and

should be maintained in future analogs.

To gain a better understanding of the SAR for the aromatic

groups at the 2-position of the quinoline, two series of analogs

that either lacked the 4-amino group (9−16) or contained a 4-

methylpiperazine group (17−24) were designed and synthe-

sized. As shown in Table 2, each series had eight diﬀerent types

of aromatic groups, including substituted phenyl groups,

carbazolyl groups, and indolyl groups, which were linked to

the 2-position of the quinoline moiety by a vinyl linkage. On

the basis of the K_Dvalue results obtained by SPR and shown in

Tables 1 and 2, most of compounds in which the 4-amino

group was absent (1, 5, 9−10, 12, 14−16) showed weaker

binding abilities than those of analogs containing the 4-

methylpiperazine group (4, 8, 17−18, 20, 22−24). These

results further support the signiﬁcance of the methylpiperazine

motif at the 4-position of quinoline. On the other hand, the

presence of an aromatic group (Ar) at the 2-position had a

notable signiﬁcant eﬀect on the binding aﬃnity of the

compounds to the RNA. A signiﬁcant enhancement of binding

aﬃnity of the compounds to the QGRS of the ADAM10

mRNA was observed (K_Dwas less than 10 μM) when aromatic

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Table 4. Binding Data for the Interaction between RNA and Analogs 24 and 28−32 Using the SPR Assay

a

K_Dfor diﬀerent compounds toward the A10-RNA-Wt sequence as determined using SPR. Compounds that did not achieve saturation state at the

highest tested concentration (50 μM) are noted. Compounds noted as >50 μM are considered to have low aﬃnity for the RNA QGRS.

groups as indolyl (15, 23), N-ethylcarbazolyl (24), 4-

morpholinostyryl (22), 4-hydroxylphenyl (20), and 3,4,5-

trimethoxyphenyl (11) groups were present at this position.

The next area for the SAR exploration involved introducing

various lengths of side chains into the methylpiperazine group

at the 4-position of the quinoline. The SPR results (Table 3)

showed that compounds 25−27, which had long side chains at

the methylpiperazine group, had weaker binding aﬃnity than

the corresponding methylpiperazine analog (24), suggesting

that a long side chain at the methylpiperazine group at the 4-

position of the quinoline was unfavorable for the binding

aﬃnity of this class of compounds.

Initial Identiﬁcation of Translational Upregulators of

ADAM10 Using the Dual-Luciferase Reporter Assay.

Having a high binding aﬃnity for the QGRS of the ADAM10

mRNA is only the ﬁrst step in identifying compounds that can

be used as translation regulators. We next aimed to validate the

ability of the QGRS binders to improve translation using the

dual-luciferase reporter assay. Dual-luciferase reporter systems

are widely used to study the simultaneous expression and

measurement of two individual reporter enzymes within a

single system. Most of the analogs with high binding aﬃnity

were tested using this assay. On the basis of MTT results that

all the compounds have no obvious cell toxicity at a

concentration of 2 μM (Table S1), dual-luciferase reporter

assays²²were performed initially using a single concentration (2

μM) of the compounds to quickly establish the relative

activities of the selected analogs. The data presented in Table 5

show that some of the tested compounds (analogs 15, 16, and

22−24) improved the translation eﬃciency by more than 15%.

Notably, treatment with 24 had the most signiﬁcant eﬀect on

enhancing the expression of the Renilla luciferase as indicated

by its activity (38.3%). On the basis of the selective aﬃnity for

the QGRS of the RNA in the SPR experiment and the

enhancement eﬀect on the translation eﬃciency in the dual-

Finally, we explored the signiﬁcance of a methyl moiety at

the 1-position of the quinoline. Four compounds (29−32) that

lacked the 1-methyl group were investigated and compared

with those in which this moiety was retained (16, 22, 24, and

28). The results shown in Table 4 revealed the necessity of the

methyl group at the 1-position of quinoline ring for maintaining

binding aﬃnity for RNA QGRS, as indicated by the fact that

compounds 29−32 were nearly inactive.

None of the above compounds showed any speciﬁc

interaction with the mutant RNA QGRSs (A10-RNA-Mut)

motif in the SPR experiments (data not shown).

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phoresis assays presumably due to its damage of secondary

structure of nucleic acids. These combination results suggest

the association of 24 with the QGRS of the ADAM10 mRNA

in vitro.

Table 5. Change in the Translational Activity for Select

Compounds in the Dual-Luciferase Reporter Assay

a

compd

ratio Renilla/ﬁreﬂy luciferase activity (%)

4

108.0 1.3

98.7 3.7

101.1 2.8

101.9 0.3

92.0 1.1

118.9 2.7

116.3 5.2

91.5 7.3

137.1 0.2

115.8 4.3

138.3 1.6

To further clarify the capacity of 24 to mitigate translational

repression through the QGRS motif, dual-luciferase reporter

assays using the three reporter plasmids described previously

and RT-PCR were applied. For these experiments, after

transfection with the reporter plasmids, the cells were treated

with various concentrations of 24 (0, 0.5, 1, 2.5 μM) for 24 h.

The cells were inspected prior to the luciferase assay, and no

detectable change in the cell number or morphology in any of

the treatment groups was observed. We observed a

concentration-dependent increase in the Renilla luciferase

activity (Figure 5A). As shown, 0.5 μM 24 had a moderate

eﬀect on translation with an increase of 14.1%. Moreover, when

the concentration of 24 was increased to 1 and 2.5 μM, the

translation increased by 46.9% and 87.3%, respectively. In

contrast, the mutant variant and psi-CHECK-2 blank vector

groups showed no obvious eﬀect after treatment with 24

(Figure 5A). The lack of any change in the level of translation

of the deletion mutant and mutant variant can be explained by

assuming that 24 is speciﬁcally interacting with the QGRS in

the ADAM10 mRNA in the cells and signiﬁcantly mitigating

the repressive eﬀect of the G-quadruplex on translation.

Additionally, no obvious eﬀect was observed (Figure S5) after

treatment with an inactive compound 6 (SPR K_D> 50 μM)

also conﬁrmed the eﬀect of 24 was derived from its interaction

with the QGRS in the ADAM10 mRNA. Interestingly, the

results of the RT-PCR assays show very subtle changes in the

mRNA levels for both the wild type and mutants (Figure 5B

and Figure S6). Since some oncogenes also have QGRS in their

5′-UTR, similar dual-luciferase assays on luciferase reporter

plasmid carrying the quadruplex or its mutant sequence of N-

Ras,²⁶Bcl-2,²⁸and VEGF³⁰(Figure S7A) were further

performed to investigate the speciﬁcity of compound 24

among diﬀerent genes. As shown in Figure S7, 24 had no

detectable eﬀect on VEGF even at a concentration of 2.5 μM

and moderate eﬀect on N-Ras and Bcl-2 with an increase of

about 20% (87.3% increase for ADAM10), which indicated the

compound 24 had a certain speciﬁcity (about 3-fold) to the

ADAM10 QGRS.

8

9

13

14

15

16

17

22

23

24

a

2 μM compounds were used in dual-luciferase reporter assays, and

the values represent the averages and standard deviations of three

independent measurements relative to the no-treatment control.

luciferase reporter assays, we decided to pursue compound 24

as the most potent compound in further studies.

Compound 24 Had a High Aﬃnity for RNA QGRS and

a High Capacity To Enhance Translation Activity of a

Reporter Gene. To further conﬁrm the binding ability of

compound 24 to the QGRS of the ADAM10 mRNA,

microscale thermophoresis (MST) and native gel electro-

phoresis assays were performed. First, the MST technology,

which is a contact-free optical and thermodynamic method, is

complementary to SPR and it measures the equilibrium

dissociation constants (K_d) of compound/RNA interactions

in solution at the microliter scale. Consistently, both results of

SPR and MST showed high aﬃnity of 24 to QGRS in

ADAM10 mRNA with K_Dvalue of 6.1 μM and K_dvalue of 4.9

μM, respectively (Figure S3). In contrast, 24 bound more

weakly to the RNA mutant sequence of ADAM10 mRNA, K_D

value of 0.7 M (Figure S3A). Additionally, as shown in Figure

4, increasing the concentration of 24 led to a progressive

Given that the G-quadruplex motif is an inhibitory element

within the entire context of the ADAM10-5′-UTR,^21,22to

further demonstrate the eﬀect of 24 on ADAM10 translation,

we performed additional dual-luciferase reporter assays using a

reporter plasmid that carries the entire 5′-UTR (5′UTR-Wt) or

its QGRS mutant variant (5′UTR-Mut) in front of the Renilla

luciferase in the psi-CHECK-2 vector (Figure 2B). The QGRS

mutant variant (5′UTR-Mut) was used as system control. As

shown in Figure 5C, the relative luciferase activity for the

5′UTR-Wt plasmid increased approximately 9.7% in the

presence of 0.5 μM 24, 22.5% in the presence of 1 μM 24,

and 44.8% when the cells were treated with 2.5 μM 24. The

relative luciferase activity of the mutant variant group showed

no obvious change after interaction with 24 (Figure 5C). The

RT-PCR results also showed no change in the levels of the

luciferase mRNA (Figure 5D and Figure S6) for any of the

tested samples, indicating that the gain in activity occurred at

the translational level in the cells. These results conﬁrmed that

compound 24 could eliminate the translational suppression of

the ADAM10 QGRS motif.

Figure 4. Structures of compound 24 and the binding of 24 to

ADAM10 RNA G-quadruplex. Migration of ADAM10 RNA G-

quadruplex in the presence of 0, 0.5, 1, 5, 10-fold excess concentrations

of 24 in a native 16% polyacrylamide gel.

decrease in the quantity of the RNA G-quadruplex bands

(lower bands) and an increase in the RNA−ligand complexes

(upper bands) in native gel electrophoresis assays. Consistently,

the results of CD spectrum showed that titrating increasing

concentrations of compound 24 resulted in a little decrease of

the quadruplex secondary structure in the CD signal at 263 nm

(Figure S4), reﬂecting the migration in native gel electro-

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Figure 5. Reporter gene assay results reﬂect the eﬀect of 24 on the ADAM10 QGRS motif in dual-luciferase assay. (A) Histogram showing the

percentage of activity of the translation of the Renilla gene as a function of the concentration of 24. (B) Quantiﬁcation of the mRNA level of the

dual-luciferase reporter gene is shown in Supporting Information Figure S6. (C) Histogram showing the percentage of activity of the translation of

the Renilla gene as a function of the concentration of 24. The Renilla luciferase activity was normalized to the ﬁreﬂy luciferase activity, and the value

for the blank sample in each group was set to 100%. (D) Quantiﬁcation of the mRNA level of the dual-luciferase reporter gene is shown in

Supporting Information Figure S6. For all data, the Renilla luciferase activity was normalized to the ﬁreﬂy luciferase activity, and the value for the

blank sample in each group was set to 100%. The symbol ∗ indicates a signiﬁcant diﬀerence at P < 0.05 vs the ligand-free control (the ligand-free

control was set to 1). All data are expressed as the mean SD of three independent experiments performed in triplicate.

Compound 24 Eﬃciently Enhanced ADAM10 Trans-

lation and sAPPα Secretion in a Cellular Model. To

directly assess the impact of 24 on ADAM10 translation, a

further evaluation of the ability of 24 to modulate the

translation of the ADAM10 gene in cells was conducted. The

protein and mRNA levels of ADAM10 in HEK-APP cells were

measured after culturing with 0, 0.5, 0.75, 1, 1.5 μM 24 for 48 h.

The results of the Western blotting and RT-PCR assays showed

a concentration-dependent increase in the ADAM10 protein

levels (20.2%, 55.1%, 76.8%, and 92.9% for treatment with 0.5,

0.75, 1, and 1.5 μM 24, respectively) (Figure 6A), while only

small changes in the mRNA levels of ADAM10 were detected

in the 24-treated cell samples (Figure 6B). It was also found

that 24 (it is intrinsically ﬂuorescent) accumulated in the

cytoplasm (Figure S8), strongly suggesting that 24 was cell

permeable. Moreover, a 3.3-fold increase in the levels of

secreted sAPPα, a soluble form of APP cleaved by α-secretase,

was detected in the supernatants of the cells that were cultured

with 1.5 μM 24 for 48 h (Figure 6A). It is noticeable that for

treatment with the ASO the level of sAPPα is lower compared

to treatment with compound 24, to explore the possible causes

of this discrepancy, we further detected the impact of 24 on the

levels of the APP protein as well as on the α-secretase activity of

ADAM10 in vitro. As shown in Figure 6, no obvious impact of

24 on the levels of the APP protein was detected (Figure 6A),

but a light increase of the α-secretase activity of ADAM10 was

observed (Figure 6C). Thus, the increase of sAPPα in cells

might be a combinational result of the increase of ADAM10

protein as well as the enhancement of the α-secretase activity of

ADAM10 induced by treatment with compound 24. The

experiment in which Hela cells were cultured with various

concentrations of 24 also produced consistent results (Figure

S9).

Compound 24 Led to a Decrease in Aβ₄₀Production

in HEK-APP Cells. The hallmark of Alzheimer′s disease is the

signiﬁcantly elevated Aβ concentrations in cells. We therefore

next measured the ability of 24 to reduce the cellular

production of Aβ. Because Aβ₄₀constitutes more than 90%

of the secretion of Aβ, we measured the change in Aβ₄₀

production. HEK-APP cells, which overexpress APP, were

treated with increasing concentrations of 24 for 48 h, and the

Aβ₄₀production of the cells was quantitatively determined

using ELISA assays. As shown in Figure 6D, as expected, 24

was able to inhibit the production of Aβ₄₀in a dose-dependent

manner. The production of Aβ₄₀decreased 7.7% in the

presence of 0.75 μM 24, 20.7% in the presence of 1 μM 24, and

40.8% when the cells were treated with 1.5 μM 24.

CONCLUSIONS

■

In this study, 1-methylquinolinium derivatives were identiﬁed

with favorable predicted ADAM10 mRNA QGRS binding

properties. On the basis of these results, a series of 1-

methylquinolinium derivatives optimized at the 2- and 4-

positions was designed and synthesized to provide valuable

insights into the structure−activity relationships of ADAM10

mRNA QGRS binders as well as to screen for translational up-

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Figure 6. Eﬀect of 24 on HEK-APP cells. HEK-APP cells were treated with various concentrations of 24 for 48 h. (A) Eﬀect of 24 on the protein

levels of APP, ADAM10, sAPPα, and β-actin. The results were normalized to β-actin. (B) Eﬀect of 24 on the levels of ADAM10 and β-actin mRNA.

The results were normalized to β-actin. (C) Eﬀect of 24 on the α-secretase activity of ADAM10. The ADAM10 protein was treated with various

concentrations of 24 for 20 min, and the α-secretase activity of ADAM10 was detected using a ﬂuorogenic peptide substrate. (D) Detection of the

eﬀects of 24 on the secretion of Aβ₄₀using an ELISA kit (Invitrogen). All data are expressed as the mean SD from three independent experiments.

The fold changes in expression were calculated relative to the blank control (the blank control was set to 1). The symbols ∗ and ∗∗∗ indicated

signiﬁcant diﬀerences at P < 0.05 and P < 0.001 vs control.

regulators of ADAM10. The analysis of the in vitro binding

properties of the synthesized derivatives by SPR indicated the

following aspects of the SAR: (1) the methyl group at the 1-

position of quinoline was necessary for the binding to the RNA

QGRS sequence, (2) introduction of a methylpiperazine group

at the 4-position may enhance the aﬃnity of the compounds for

the QGRS, and (3) the type of aromatic groups (Ar) at the 2-

position had signiﬁcant eﬀects on the binding ability of

compounds for the target. In particular, several potent

ADAM10 mRNA QGRS binders were identiﬁed with K_D

values in the micromolar range. Further dual-luciferase reporter

assays allowed us to identify one carbazolmethylquinolinium

analog, compound 24, as a novel ADAM10 mRNA QGRS

binder and translational enhancer. The selected compound 24

was then tested for its ability to enhance ADAM10 translation

and sAPPα secretion as well as to decrease Aβ₄₀production by

the cells. Our results indicated that using ADAM10 mRNA

QGRS binders, such as 24, is a feasible strategy to up-regulate

ADAM10 and thus reduce the formation of Aβ.

Because translation of ADAM10 is suppressed by the G-

quadruplexes within its long GC-rich 5′-UTR,²²in principle

there are at least two mechanisms by which 24 targeting of

ADAM10 mRNA QGRS could modulate the mRNA trans-

lation: (1) by destabilizing the RNA G-quadruplex structure

and thus stimulating translation^40,41or (2) by interfering with a

biologically essential RNA G-quadruplex/protein binding

event.^29,42Given that 24 led to a little decrease of the RNA

quadruplex secondary structure and had no obvious eﬀect on

decreasing the thermodynamic stability of the RNA G-

quadruplex in ADAM10 mRNA (data not shown), 24 may

interfere with a biologically essential RNA G-quadruplex/

protein binding event, including interference with an RNA−

translation inhibitor protein or recruitment of a translation

initiation protein.

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8.19 (m, 1H), 8.13 (d, 1H, J = 8.0), 7.99−7.95 (m, 1H), 4.45 (s, 3H),

3.10 (s, 3H). ¹³C NMR (100 Hz, DMSO-d₆): δ 161.1, 145.3, 135.0,

128.9, 125.1, 119.0, 39.9, 23, 3.

Notably, most of the G-quadruplex (G4) motifs in the 5′-

UTRs of mRNA have been found to repress translation by

approximately 50%, suggesting that functional proteins exist to

maintain the equilibrium of the folded state in which the G-

quadruplex is formed and translation is prevented. Recently, the

ﬁrst systematic approach to identifying proteins that bind to G-

quadruplex elements in 5′-UTRs of mRNAs using pull-down

assays and MALDI-TOF MS was published.⁴³The interaction

between the cellular proteins and the G-quadruplexes located in

two mRNAs (MMP16 and ARPC2) was investigated.

Furthermore, a selective mechanism for the translational

control associated with the G4 motifs in the 5′-UTRs has

been elucidated. In another study, elf4A was demonstrated to

be required for the translation of mRNAs containing G-

quadruplexes in their 5′-UTRs.⁴⁴

General Procedure A: Preparation of Compounds 6a and

6b. A suspension of compound 2a (1.77 g, 10 mmol), alicyclic amine

(20 mmol), and p-toluenesulfonic acid (0.60 g, 3.2 mmol) was stirred

under reﬂux for 10 h. After completion of the reaction, the reaction

mixture was cooled to room temperature, poured into ice−water (100

mL), and then aqueous NaOH was added to make the solution basic.

The mixture was extracted with three 50 mL portions of CH₂Cl₂. The

combined organic phase was washed with 40 mL of water, dried over

magnesium sulfate, and concentrated under reduced pressure. The

crude product was puriﬁed by using ﬂash column chromatography to

give 6a and 6b.

4-(N-Methylpiperazin-1-yl)-2-methylquinoline (6a). Com-

pound 2a was reacted with N-methylpiperazine according to general

1

procedure A to aﬀord 6a as pale yellow liquid in 75% yield. H NMR

Some reports have characterized small molecules that

modulate the DNA G-quadruplex/protein binding event.^45,46

However, there have been fewer developments in the ﬁeld of

functional RNA G-quadruplex ligands. Recently, Zamiri et al.

showed the cationic porphyrin TMPyP4 bound to the

_r(GGGGCC)₈RNA of the C9orf 72 gene and blocked

interaction with RNA-binding proteins.⁴⁷On the basis of the

previous research that identiﬁed several proteins, including

ASF/SF2 and hnRNPA, that selectively bind to the

_r(GGGGCC)₈RNA in vivo,^48,49the authors found that

TMPyP4 disrupted the binding of ASF/SF2 and hnRNPA1

(400 MHz, CDCl₃): δ 7.98 (t, J = 8.0 Hz, 2H), 7.62 (t, J = 8.0 Hz,

1H), 7.42 (t, J = 8.0 Hz, 1H), 6.74 (s, 1H), 3.27 (s, 4H), 2.73 (s, 4H),

2.69 (s, 3H), 2.43 (s, 3H).

4-(Piperidin-1-yl)-2-methylquinoline (6b). Compound 2a was

reacted with piperidine according to general procedure A to aﬀord 6b

as a white solid in 64% yield. Mp 41.2−42.6 °C. ¹H NMR (400 MHz,

CDCl₃): δ 7.95 (d, J = 8.0 Hz, 2H), 7.59 (t, J = 8.0 Hz, 1H), 7.40 (t, J

= 8.0 Hz, 1H), 6.71 (s, 1H), 3.15 (s, 4H), 2.66 (s, 3H), 1.84 (s, 4H),

1.68(s, 2H). ¹H NMR (400 MHz, CDCl₃): δ 7.95 (d, J = 8.0 Hz, 2H),

7.59 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 6.71 (s, 1H), 3.15 (s,

4H), 2.66 (s, 3H), 1.84 (s, 4H), 1.68 (s, 2H).

General Procedure B: Preparation of (2-Aromatic vinyl)-1-N-

methylquinolinium Derivatives 1−28. A suspension of 3a and 3b

(0.65 g, 0.2 mmol), aromatic aldehydes (0.2 mmol, 1.0 equiv amount),

1.5 equiv amount secondary amine, and 15 mL of ethanol was heated

in a ﬂask or sealed tube for 3 h at 80 °C. A precipitate was formed

during the process of the reaction. The reaction mixture was allowed

to cool to room temperature. The ﬂask or sealed tube was then placed

in an ice bath, and the solution was ﬁltered and washed thoroughly

with anhydrous ethanol to aﬀord the (2-aromatic vinyl)-1-N-

methylquinolinium derivatives.

(E)-2-(4-Hydroxy-3,5-dimethoxystyryl)-1-methylquinolin-1-

ium Iodide (1). Following general procedure B for (2-aromatic

vinyl)-1-N-methylquinolinium derivatives, the product was obtained as

a green-yellow solid. Mp > 200 °C.¹H NMR (400 Hz, DMSO-d₆): δ

9.42 (s, 1H), 8.99 (d, 1H, J = 8.0), 8.54 (d, 2H, J = 8.0), 8.32 (d, 1H, J

= 8.0), 8.18 (d, 1H, J = 16.0), 8.15 (d, 1H, J = 8.0), 7.93 (t, 1H, J =

8.0), 7.76 (d, 1H, J = 16.0), 7.32 (s, 2H), 4.56 (s, 3H), 3.90 (s, 6H).

¹³C NMR (100 MHz, DMSO-d₆): δ 156.4, 148.5, 148.2, 143.3, 140.1,

to (GGGGCC)₈, suggesting that this may be a viable avenue

r

for the development of therapeutic treatments. Thus, further

studies are needed to conﬁrm the speculation that compound

24 can enhance the translation of ADAM10 via binding to the

RNA G-quadruplexes and interfering with a biologically

essential RNA G4/protein binding event.

Our study provided new insight into the design and

discovery of an RNA QGRS binder and proposed a new

molecular strategy by which interactions of these molecules

with RNA QGRS may modulate translation of ADAM10.

EXPERIMENTAL SECTION

■

General Methods. ¹H and ¹³C NMR spectra were recorded using

TMS as the internal standard in DMSO-d₆or CDCl₃with a Bruker

BioSpin GmbH spectrometer at 400 and 100 MHz, respectively. The

chemical shifts are reported in parts per million (ppm) relative to

139.2, 134.6, 129.9, 128.6, 127.4, 125.4, 120.6, 119.2, 115.7, 107.6,

56.3, 18.5. Purity: 99.8% by HPLC. ESI-HRMS (C₂₀H₂₀NO₃): calcd,

322.1438 [M]⁺; found, 322.1446.

1

residual CHCl₃(δ = 7.26, H; δ = 77.0, ¹³C) and DMSO (δ = 2.50,

¹H; δ = 39.5, ¹³C) in the corresponding deuterated solvents. The mass

spectra (MS) were recorded on a Shimadzu LCMS-2010A instrument

with an ESI or ACPI mass-selective detector, and high-resolution mass

spectra (HRMS) were obtained using a Shimadzu LCMS-IT-TOF

instrument. The melting points (Mp) were determined using an SRS-

OptiMelt automated melting point instrument without correction.

Flash column chromatography was performed with silica gel (200−300

mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The

purities of the synthesized compounds were conﬁrmed to be higher

than 95% by analytical HPLC performed using a dual pump Shimadzu

LC-20AB system equipped with an Ultimate XB-C18 column and

eluted with methanol/water (80%) containing 0.1% TFA at a ﬂow rate

of 0.2 mL min⁻¹. Aniline, ethyl acetoacetate, polyphosphoric acid,

POCl_3,ethanol, n-butanol, 1,4-dioxane, THF, and all the amines were

commercially available.

(E)-2-(4-Hydroxy-3,5-dimethoxystyryl)-1-methyl-4-morpho-

linoquinolin-1-ium Iodide (2). Following general procedure B for

(2-aromatic vinyl)-1-N-methylquinolinium derivatives, the product

was obtained as a green-yellow solid. Mp > 200 °C. ¹H NMR (400 Hz,

DMSO-d₆): δ 9.15 (s, 1H), 8.32 (d, 1H, J = 8.0), 8.20 (d, 1H, J = 8.0),

8.04 (t, 1H, J = 8.0), 7.85 (d, 1H, J = 16.0), 7.75 (t, 1H, J = 8.0), 7.59

(d, 1H, J = 16.0), 7.49(s, 1H), 7.20 (s, 2H), 4.27 (s, 3H), 3.91 (s, 4H),

3.88 (s, 6H), 3.77 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆): δ 159.3,

154.5, 148.1, 144.5, 140.8, 138.9, 133.7, 126.5, 126.3, 125.6, 119.7,

119.3, 116.6, 106.8, 105.0, 65.8, 56.3, 52.0, 38.3. Purity: 99.0% by

HPLC. ESI-HRMS (C₂₄H₂₇N₂O₄): calcd, 407.1965 [M]⁺; found,

407.1945.

(E)-2-(4-Hydroxy-3,5-dimethoxystyryl)-1-methyl-4-(piperi-

din-1-yl)quinolin-1-ium Iodide (3). Following general procedure B

for (2-aromatic vinyl)-1-N-methylquinolinium derivatives, the product

was obtained as a green-yellow solid. Mp > 200 °C. ¹H NMR (400 Hz,

DMSO-d₆): δ 8.17 (d, 1H, J = 8.0), 8.03 (d, 1H, J = 8.0), 7.94 (t, 1H, J

= 8.0), 7.80 (d, 1 H, J = 16.0), 7.65 (t, 1H, J = 8.0), 7.35 (s, 1H), 7.31

(d, 1H, J = 16.0), 7.15 (s, 2H), 4.14 (s, 3H), 3.83 (s, 6H), 3.62 (s, 4H),

1.82 (s, 4H), 1.74 (s, 2H),1.53 (s, 1H). ¹³C NMR (100 MHz, DMSO-

d₆): δ 158.7, 153.8, 148.6, 144.0, 140.8, 133.2, 126.2, 125.5, 123.7,

Synthesis of Intermediates. The intermediates 1a, 2a, 3a, and 3b

were prepared following the process shown in Scheme S1 using the

method by our previous report.³⁹

1-N-Methyl-2-methylquinolinium Iodode (4a). The prepara-

tion was according to the synthetic method of intermediates 3a and

3b. ¹H NMR (400 Hz, DMSO-d₆): δ 9.11 (d, 1H, J = 8.0), 8.60−8.57

(dd, 1H, J = 4.0, J = 8.0), 8.41−8.38(dd, 1H, J = 4.0, J = 4.0), 8.23−

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119.7, 118.8, 113.8, 108.6, 107.2, 104.4, 56.2, 44.5, 37.8, 25.4, 23.5.

Purity: 98.8% by HPLC. ESI-HRMS (C₂₅H₂₉N₂O₃): calcd, 405.2173

[M]⁺; found, 405.2141.

(E)-2-(4-Fluoro-3-methoxystyryl)-1-methylquinolin-1-ium

(10). Following general procedure B for (2-aromatic vinyl)-1-N-

methylquinolinium derivatives, the product was obtained as a green-

yellow solid. Mp > 200 °C. ¹H NMR (400 Hz, DMSO-d₆): δ 9.12 (d,

1H, J = 8.0), 8.59 (t, 2H, J = 8.0), 8.39 (d, 1H, J = 4.0), 8.22−8.18 (m,

2H), 8.00−7.92 (m, 2H), 7.81 ( d, 1H, J = 8.0), 7.57 (t, 1H, J = 4.0),

7.41−7.37 (m, 1H), 4.62 (s, 3H), 3.99 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 156.1, 152.1, 147.6, 146.0, 144.3, 139.2, 135.0, 132.0,

130.0, 129.1, 127.8, 123.3, 121.2, 119.4, 119.3, 116.6, 116.4, 113.6,

72.6, 56.5, 40.2. Purity: 98.4% by HPLC. ESI-HRMS (C₁₉H₁₇NOF):

calcd, 294.1289 [M]⁺; found, 294.1285.

(E)-2-(4-Hydroxy-3,5-dimethoxystyryl)-1-methyl-4-(4-meth-

ylpiperazin-1-yl)quinolin-1-ium Iodide (4). Following general

procedure B for (2-aromatic vinyl)-1-N-methylquinolinium derivatives,

1

the product was obtained as a green-yellow solid. Mp > 200 °C. H

NMR (400 Hz, DMSO-d₆): δ 8.30 (d, 1H, J = 8.0), 8.14 (d, 1H, J =

7.2), 8.03 (s, 1H), 7.89 (d, 1H, J = 16.0), 7.75 (s, 1H), 7.58 (d, 1H, J =

16.0), 7.50 (s, 1H), 7.23 (s, 2H), 4.26 (s, 3H), 3.87 (s, 6H), 3.80 (s,

4H), 2.75 (s, 4H), 2.39 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

159.2, 154.4, 148.1, 144.4, 140.7, 138.8, 133.7, 126.5, 126.3, 125.6,

119.7, 119.3, 116.6, 106.8, 105.1, 56.4, 54.0, 51.2, 45.1, 38.3. Purity:

99.7% by HPLC. ESI-HRMS (C₂₅H₃₀N₃O₃): calcd, 420.2282 [M]⁺;

found, 420.2274.

(E)-2-(2-Hydroxy-5-nitrostyryl)-1-methylquinolin-1-ium Io-

dide (5). Following general procedure B for (2-aromatic vinyl)-1-N-

methylquinolinium derivatives, the product was obtained as a green-

yellow solid. Mp > 200 °C. ¹H NMR (400 Hz, DMSO-d₆): δ

8.82−8.80 (m, 2H), 8.49 (d, 1H, J = 12.0), 8.36 (s, 2H), 8.22 (d, 2H, J

= 8.0), 8.06 (t, 1H, J = 8.0), 7.84−7.79 (m, 2H), 6.26 (d, 1H, J = 12.0),

4.36 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 179.1, 157.8, 148.5,

141.2, 138.9, 133.6, 131.9, 130.2, 129.4, 127.4, 126.9, 126.4, 123.0,

121.4, 119.7, 118.2, 113.5, 38.2. Purity: 98.8% by HPLC. ESI-HRMS

(C₁₈H₁₅N₂O₃): calcd, 307.1077 [M]⁺; found, 307.1092.

(E)-1-Methyl-2-(3,4,5-trimethoxystyryl)quinolin-1-ium Io-

dide (11). Following general procedure for (2-aromatic vinyl)-1-N-

methylquinolinium derivatives, the product was obtained as a green-

yellow solid. Mp > 200 °C. ¹H NMR (400 Hz, DMSO-d₆): δ 9.08 (d,

1H, J = 8.0), 8.58 (d, 1H, J = 12.0), 8.55 (d, 1H, J = 12.0), 8.36 (d, 1H,

J = 12.0), 8.21 (d, 1H, J = 8.0), 8.17 (d, 1H, J = 16.0), 7.79 (t, 1H, J =

8.0), 7.79 (d, 1H, J = 16.0), 7.33 (s, 2H), 4.60 (s, 3H), 3.91 (s, 6H),

3.77 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.9, 156.1, 143.7,

140.7, 133.5, 131.4, 130.9, 126.8, 126.3, 126.1, 122.1, 121.7, 119.8,

119.1, 116.8, 104.6, 99.6, 65.9, 52.0, 37.8. Purity: 99.8% by HPLC. ESI-

HRMS (C₂₁H₂₂NO₃): calcd, 336.1594 [M]⁺; found, 336.1594.

(E)-2-(4-Hydroxystyryl)-1-methylquinolin-1-ium Iodide (12).

Following general procedure B for (2-aromatic vinyl)-1-N-methyl-

quinolinium derivatives, the product was obtained as a green-yellow

1

(E)-2-(2-Hydroxy-5-nitrostyryl)-1-methyl-4-morpholinoqui-

nolin-1-ium Iodide (6). Following general procedure B for (2-

aromatic vinyl)-1-N-methylquinolinium derivatives, the product was

solid. Mp > 200 °C. H NMR (400 Hz, DMSO-d₆): δ 10.37 (s, 1H),

8.98 (d, 1H, J = 8.0), 8.56 (d, 1H, J = 8.0), 8.52 (d, 1H, J = 16.0), 8.32

(d, 1H, J = 8.0), 8.21 (d, 1H, J = 16.0), 8.16 (t, 1H, J = 8.0), 7.92 (t,

1H, J = 8.0), 7.88 (d, 2H, J = 8.0), 7.71 (d, 1H, J = 16.0), 6.93 (d, 2H, J

= 8.0), 4.53 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.1, 156.2,

147.6, 143.1, 138.9, 134.5, 131.7, 129.8, 128.5, 127.2, 126.1, 120.6,

119.0, 116.0, 115.1, 39.8. Purity: 99.8% by HPLC. ESI-HRMS

(C₁₈H₁₆NO): calcd, 262.1226 [M]⁺; found, 262.1236.

1

obtained as a green-yellow solid. Mp > 200 °C. H NMR (400 Hz,

DMSO-d₆): δ 8.65 (d, 1H, J = 12.0), 8.34 (d, 1H, J = 4.0), 8.23 (d, 1H,

J = 8.0), 8.15 (d, 1H, J = 8.0), 7.99 (t, 1H, J = 8.0), 7.92 (d, 1H, J =

16.0), 7.80 (dd, 1H, J = 4.0, J = 8.0), 7.70 (t, 1H, J = 8.0), 7.51 (s, 1H),

6.26 (d, 1H, J = 8.0), 4.16 (s, 3H), 3.91 (t, 4H, J = 4.0), 3.69 (t, 4H, J =

4.0). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.9, 156.1, 143.7, 140.7,

133.5, 131.4, 130.9, 126.8, 126.3, 126.1, 122.1, 121.7 119.8, 119.1,

116.8, 104.6, 99.6, 65.9, 52.0, 37.8. Purity: 97.7% by HPLC. ESI-

HRMS (C₂₂H₂₂N₃O₄): calcd, 392.1605 [M]⁺; found, 392.1592.

(E)-2-(2-Hydroxy-5-nitrostyryl)-1-methyl-4-(piperidin-1-yl)-

quinolin-1-ium Iodide (7). Following general procedure B for (2-

aromatic vinyl)-1-N-methylquinolinium derivatives, the product was

(E)-2-(3-Methoxy-4-morpholinostyryl)-1-methylquinolin-1-

ium Iodide (13). Following general procedure B for (2-aromatic

vinyl)-1-N-methylquinolinium derivatives, the product was obtained as

1

a green-yellow solid. Mp > 200 °C. H NMR (400 Hz, DMSO-d₆): δ

9.00 (d, 1H, J = 8.0), 8.55 (t, 2H, J = 12.0), 8.33 (d, 1H, J = 8.0), 8.23

(d, 1H, J = 16.0), 8.16 (t, 1H, J = 6.0), 7.93 (t, 1H, J = 8.0), 7.79 (d,

1H, J = 16.0), 7.59 (s, 1H), 7.52 (d, 1H, J = 8.0), 7.00 (d, 1H, J = 8.0),

4.56 (s, 3H), 3.94 (s, 3H), 3.75 (s, 4H), 3.14 (s, 4H). ¹³C NMR (100

MHz, DMSO-d₆): δ 156.3, 151.6, 147.7, 144.5, 143.3, 139.2, 134.6,

129.9, 128.7, 128.7, 127.4, 124.7, 120.8, 119.2, 117.5, 116.2, 111.5,

66.1, 55.9, 50.0, 39.4. Purity: 98.6% by HPLC. ESI-HRMS

(C₂₃H₂₅N₂O₂: calcd, 361.1911 [M]⁺; found, 361.1893.

(E)-1-Methyl-2-(4-morpholinostyryl)quinolin-1-ium Iodide

(14). Following general procedure B for (2-aromatic vinyl)-1-N-

methylquinolinium derivatives, the product was obtained as a green-

yellow solid. Mp > 200 °C. ¹H NMR (400 Hz, DMSO-d₆): δ 8.90 (d,

1H, J = 8.0), 8.53 (t, 1H, J = 8.0), 8.47 (t, 1H, J = 8.0), 8.29−8.25 (m,

2H), 8.22 (d, 1H, J = 4.0), 8.14−8.09 (m, 2H), 7.89 (d, 1H, J = 16.0),

7.67 (d, 1H, J = 4.0), 7.07 (d, 2H, J = 8.0), 4.49 (s, 3H), 3.76 (d, 4H),

3.36 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆): δ 156.3, 153.2, 148.1,

142.6, 139.2, 134.3, 131.5, 129.8, 128.3, 127.1, 124.7, 120.4, 119.0,

113.8, 113.8, 65.8, 46.7, 39.1. Purity: 98.9% by HPLC. ESI-HRMS

(C₂₂H₂₃N₂O): calcd, 331.1805 [M]⁺; found, 331.1809.

(E)-2-(2-(1H-Indol-2-yl)vinyl)-1-methylquinolin-1-ium Iodide

(15). Following general procedure B for (2-aromatic vinyl)-1-N-

methylquinolinium derivatives, the product was obtained as a green-

yellow solid. Mp > 200 °C. ¹H NMR (400 Hz, DMSO-d₆): δ 12.29 (s,

1H), 8.80 (d, 1H, J = 8.0), 8.64 (d, 1H, J = 16.0), 8.59 (d, 1H, J = 8.0),

8.40 (t, 2H, J = 12.0), 8.22 (t, 2H, J = 8.0), 8.07 (t, 1H, J = 8.0), 7.84

(d, 1H, J = 8.0), 7.53 (t, 2H, J = 8.0), 7.30 (s, 2H), 4.46 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 156.6, 142.8, 141.6, 139.1, 137.5,

134.5, 134.0, 129.7, 127.8, 126.6, 125.2, 123.4, 121.9, 120.3, 119.8,

118.7, 114.5, 112.8, 111.4, 38.9. Purity: 98.8% by HPLC. ESI-HRMS

(C₂₀H₁₇N₂): calcd, 285.1386 [M]⁺; found, 285.1391.

1

obtained as a green-yellow solid. Mp > 200 °C. H NMR (400 Hz,

DMSO-d₆): δ 8.66 (d, 1H, J = 12.0), 8.30 (d, 1H, J = 4.0), 8.18 (d, 1H,

J = 12.0), 8.06 (d, 1H, J = 8.0), 7.96 (t, 1H, J = 8.0), 7.87 (d, 1H, J =

16.0), 7.80 (m, 1H), 6.69 (t, 1H, J = 8.0), 7.44 (s, 1H), 6.16 (d, 1H, J =

8.0), 4.13 (s, 3H), 3.65 (t, 4H, J = 4.0), 1.83 (s, 4H), 1.75 (s, 2H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 179.3, 158.9, 156.1, 144.2, 140.5,

132.9, 131.2, 129.1, 126.4, 125.9, 125.3, 122.4, 121.6, 119.8, 118.5,

115.1, 103.7, 52.4, 37.1, 25.1, 23.2. Purity: 95.2% by HPLC. ESI-

HRMS (C₂₃H₂₄N₃O₃): calcd, 390.1812 [M]⁺; found, 390.1815.

(E)-2-(2-Hydroxy-5-nitrostyryl)-1-methyl-4-(4-methylpipera-

zin-1-yl)quinolin-1-ium Iodide (8). Following general procedure B

for (2-aromatic vinyl)-1-N-methylquinolinium derivatives, the product

was obtained as a green-yellow solid. Mp > 200 °C. ¹H NMR (400 Hz,

DMSO-d₆): δ 8.58 (s, 1H), 8.29 (d, 1H, J = 12.0), 8.17 (s, 1H), 8.14

(d, 1H, J = 12.0), 8.09 (d, 1H, J = 8.0), 8.06 (t, 1H, J = 8.0), 7.88 (d,

1H, J = 16.0), 7.75(t, 1H, J = 8.0), 7.50 (s, 1H), 6.92 (d, 1H, J = 8.0),

4.20 (s, 3H), 3.85 (s, 4H), 2.83 (s, 4H), 2. 54 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆) δ 159.2, 154.7, 140.7, 139.2, 136.8, 133.9, 127.5,

126.9, 126.6, 126.4, 122.3, 120.9, 119.7, 119.2, 118.4, 105.4, 99.6, 53.6,

50.7, 44.6, 38.2. Purity: 95.4% by HPLC. ESI-HRMS (C₂₃H₂₅N₄O₃):

calcd, 405.1921 [M]⁺; found, 405.1908.

(E)-1-Methyl-2-styrylquinolin-1-ium Iodide (9). Following

general procedure B for (2-aromatic vinyl)-1-N-methylquinolinium

derivatives, the product was obtained as a green-yellow solid. Mp >

200 °C. ¹H NMR (400 Hz, DMSO-d₆): 9.13 (d, 1H, J = 8.0), 8.61 (t,

2H, J = 8.0), 8.40 (t, 1H, J = 8.0), 8.25−8.20 (m, 2H), 8.02−7.96 (m,

4H), 7.55 (s, 3H), 4.60 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

156.2, 146.7, 144.3, 139.2, 135.0, 134.8, 131.3, 130.1, 129.1, 127.9,

121.3, 119.5, 119.4, 48.6. Purity: 98.8% by HPLC. ESI-HRMS

(C₁₈H₁₆N): calcd, 246.1277 [M]⁺; found, 246.1286.

(E)-2-(2-(9-Ethyl-9H-carbazol-2-yl)vinyl)-1-methyl-quinolin-

1-ium Iodide (16). Following general procedure B for (2-aromatic

vinyl)-1-N-methylquinolinium derivatives, the product was obtained as

L

DOI: 10.1021/acs.jmedchem.5b00139

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Journal of Medicinal Chemistry

Article

1

a green-yellow solid. Mp > 200 °C. H NMR (400 Hz, DMSO-d₆): δ

(E )-1 -M e thyl-4 -(4- methylpip era zin- 1- yl) -2- (4-

morpholinostyryl)quinolin -1-ium Iodide (22). Following general

procedure B for (2-aromatic vinyl)-1-N-methylquinolinium derivatives,

8.95 (d, 1H, J = 8.0), 8.87 (s, 1H), 8.61 (d, 1H, J = 8.0), 8.49 (t, 2H, J

= 8.0), 8.30−8.24(m, 2H), 8.13 (t, 2H, J = 8.0), 7.96−7.88 (m, 2H),

7.78 (dd, 2H, J = 8.0, J = 12.0), 7.54 (t, 1H, J = 8.0), 7.33 (t, 1H, J =

8.0), 4.58 (s, 3H), 4.50 (d, 2H, J = 8.0), 1.36 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆): δ 156.1, 149.0, 142.8, 141.5, 140.1, 138.9, 134.3,

129.7, 128.3, 127.9, 127.0, 126.6, 126.0, 122.7, 122.4, 122.1, 120.6,

120.4, 119.8, 118.8, 115.2, 109.8, 109.7, 39.6, 37.3, 13.8. Purity: 99.5%

by HPLC. ESI-HRMS (C₂₆H₂₃N₂): calcd, 363.1856 [M]⁺; found,

363.1866.

1

the product was obtained as a green-yellow solid. Mp > 200 °C. H

NMR (400 Hz, DMSO-d₆): δ 8.27 (d, 1H, J = 8.0), 8.12 (d, 1H, J =

8.0), 8.01−7.95 (t, 1H, J = 8.0), 7.88 (d, 1H, J = 12.0), 7.79 (d, 2H, J =

12.0), 7.72 (t, 1H, J = 8.0), 7.48 (t, 2H, J = 8.0),7.05 (d, 2H, J = 8.0),

4.22 (s, 3H), 3.75 (s, 8H), 3.32 (s, 4H), 2.63 (s, 4H), 2.31 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 159.0, 154.4, 152.4, 143.7, 140.7,

133.5, 130.3, 126.3, 126.0, 125.1, 119.6, 119.1, 115.0, 113.9, 104.7,

65.8, 54.2, 51.5, 47.0, 45.5, 38.1. Purity: 96.6% by HPLC. ESI-HRMS

(C₂₇H₃₃N₄O): calcd, 429.2649 [M]⁺; found, 429.2649.

(E)-1-Methyl-4-(4-methylpiperazin-1-yl)-2-styrylquinolin-1-

ium Iodide (17). Following general procedure B for (2-aromatic

vinyl)-1-N-methylquinolinium derivatives, the product was obtained as

(E)-2-(2-(1H-Indol-2-yl)vinyl)-1-methyl-4-(4-methylpipera-

zin-1-yl)quinolin 1-ium Iodide (23). Following general procedure B

for (2-aromatic vinyl)-1-N-methylquinolinium derivatives, the product

was obtained as a green-yellow solid. Mp > 200 °C. ¹H NMR (400 Hz,

DMSO-d₆): δ 12.01 (s, 1H), 8.27 (t, 2H, J = 8.0), 8.17 (s, 2H), 8.12

(d, 1H, J = 8.0), 8.00 (t,1H, J = 8.0), 7.72 (t, 1H, J = 8.0), 7.53 (s, 2H),

7.41 (d, 1H, J = 16.0), 7.28 (d, 2H, J = 4.0), 4.25 (s, 3H), 3.72(s, 4H),

2.65 (s, 4H), 2.32 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 158.8,

155.2, 140.7, 138.8, 137.4, 133.4, 132.5, 126.3, 126.0, 125.0, 122.9,

121.2, 120.2, 119.8, 119.1, 113.6, 112.6, 104.3, 54.2, 51.5, 48.5, 45.5,

37.9. Purity: 97.8% by HPLC. ESI-HRMS (C₂₅H₂₇N₄): calcd,

383.2230 [M]⁺; found, 383.2212.

1

a green-yellow solid. Mp > 200 °C. H NMR (400 Hz, DMSO-d₆): δ

8.32−8.28 (m, 1H), 8.18−8.15 (m, 1H), 8.04 (t, 1H, J = 12.0), 7.92−

7.85 (m, 3H), 7.76−7.71 (m, 2H), 7.99 (d, 1H, J = 8.0), 7.50 (s, 4H),

4.24 (s, 3H), 3.84 (s, 4H), 2.65 (s, 4H), 2.32 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆) δ 159.5, 153.7, 142.6, 140.8, 135.1, 133.8, 132.6,

130.3, 129.2, 128.9, 128.4, 126.7, 126.2, 120.2, 119.5, 119.2, 105.2,

54.2, 51.6, 45.4, 38.3. Purity: 96.1% by HPLC. ESI-HRMS

(C₂₃H₂₆N₃): calcd, 344.2121 [M]⁺; found, 344.2115.

(E)-2-(4-Fluoro-3-methoxystyryl)-1-methyl-4-(4-methylpi-

perazin-1-yl) quinolin-1-ium Iodide (18). Following general

procedure B for (2-aromatic vinyl)-1-N-methylquinolinium derivatives,

(E)-2-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)-1-methyl-4-(4-meth-

yl piperazin-1-yl)quinolin-1-ium Iodide (24). Following general

procedure B for (2-aromatic vinyl)-1-N-methylquinolinium derivatives,

1

the product was obtained as a green-yellow solid. Mp > 200 °C. H

NMR (400 Hz, DMSO-d₆): δ 8.30 (d, 1H, J = 8.0), 8.15 (d, 1H, J =

8.0), 8.04 (t, 1H, J = 8.0), 7.83 (d,1H, J = 20.0), 7.78 (d, 1H, J = 16.0),

7.74 (s, 1H), 7.70−7.68 (m, 1H),7.46 (s, 2H), 7.34 (t, 1H, J = 12.0),

4.24 (s, 3H), 3.95 (s, 3H), 3.82 (s, 4H), 2.61 (s, 4H), 2.29(s, 2H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 159.4, 153.9, 153.6, 151.4, 147.5,

147.3, 141.8, 140.7, 133.8, 132.2, 126.6, 126.2, 122.1, 120.0, 119.4,

119.2, 116.3, 116.1, 113.3, 105.3, 56.5, 54.3, 51.7, 45.4, 38.5. Purity:

95.3% by HPLC. ESI-HRMS (C₂₄H₂₇N₃OF): calcd, 392.2133 [M]⁺;

found, 392.2114.

1

the product was obtained as a green-yellow solid. Mp > 200 °C. H

NMR (400 Hz, DMSO-d₆): δ 8.73 (s, 1H), 8.31 (d, 1H, J = 12.0), 8.23

(d, 1H, J = 8.0), 8.16 (d, 1H, J = 8.0), 8.13 (s, 1H), 8.07−8.01 (m, 2H,

J = 8.0, J = 8.0), 7.75 (t, 3H, J = 8.0), 7.69 (d, 1H, J = 8.0), 7.57−7.51

(m, 2H, J = 8.0, J = 8.0), 7.30 (t, 1H, J = 8.0), 4.53−4.48 (m, 2H, J =

4.0, J = 8.0), 4.29 (s, 3H), 3.78 (s, 4H), 2.65 (s, 4H), 2.32 (s, 3H), 1.36

(t, 3H, J = 8.0). ¹³C NMR (100 MHz, DMSO-d₆) δ 159.1, 154.2,

144.8, 140.8, 140.6, 140.0, 133.4, 126.7, 126.3, 126.2, 126.1, 126.0,

122.5, 122.1, 121.7, 120.5, 119.6, 119.0, 116.1, 109.6, 109.5, 104.8,

54.2, 51.6, 45.5, 38.2, 37.2, 13.8. Purity: 98.6% by HPLC. ESI-HRMS

(C₃₁H₃₃N₄): calcd, 461.2700 [M]⁺; found, 461.2703.

(E)-1-Methyl-4-(4-methylpiperazin-1-yl)-2-(3,4,5-

trimethoxystyryl)quinolin-1-ium Iodide (19). Following general

procedure B for (2-aromatic vinyl)-1-N-methylquinolinium derivatives,

1

the product was obtained as a green-yellow solid. Mp > 200 °C. H

(E)-2-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)-4-(4-(2-

hydroxyethyl)piperazin-1-yl)-1-methylquinolin-1-ium Iodide

(25). Following general procedure B for (2-aromatic vinyl)-1-N-

methylquinolinium derivatives, the product was obtained as a green-

yellow solid. Mp > 200 °C. ¹H NMR (400 Hz, DMSO-d₆): δ 8.73 (s,

1H), 8.31 (d, 1H, J = 8.0), 8.23 (d, 1H, J = 8.0), 8.13 (t, 2H, J = 8.0),

8.07−8.00 (m, 2H), 7.76−7.72 (m, 3H), 7.68 (d, 1H, J = 8.0), 7.56−

7.51(m, 2H), 7.30 (t, 1H, J = 8.0), 4.51(t, 2H, J = 4.0), 4.29 (s, 3H),

3.80 (s, 4H), 3.60 (t, 2H, J = 8.0, J = 4.0), 2.76 (s, 4H), 2.55 (t, 2H, J =

8.0), 1.36 (t, 3H, J = 8.0). ¹³C NMR (100 MHz, DMSO-d₆): δ 159.0,

154.2, 144.7, 140.8, 140.6, 140.1, 133.5, 126.7, 126.4, 126.3, 126.2,

126.0, 122.6, 122.2, 121.7, 120.6, 119.6, 119.5, 119.0, 116.1, 109.6,

109.5, 104.7, 59.8, 58.5, 52.8, 51.7, 38.2 37.2, 13.8. Purity: 96.7% by

HPLC. ESI-HRMS (C₃₂H₃₅N₄O): calcd, 491.2805 [M]⁺; found,

491.2778.

(E)-4-(4-(2-(Dimethylamino)ethyl)piperazin-1-yl)-2-(2-(9-

ethyl-9H-carbazol-3 -yl)vinyl)-1-methylquinolin-1-ium Iodide

(26). Following general procedure B for (2-aromatic vinyl)-1-N-

methylquinolinium derivatives, the product was obtained as a green-

yellow solid. Mp > 200 °C. ¹H NMR (400 Hz, DMSO-d₆): δ 8.75 (s,

1H), 8.32 (d, 1H, J = 8.0), 8.23 (d, 1H, J = 8.0), 8.16 (t, 2H, J = 8.0),

8.07 (d, 1H, J = 12.0), 8. 01 (t, 1H, J = 8.0), 7.81−7.73 (m, 3H), 7.70

(d, 1H, J = 8.0),7.59 (s, 1H), 7.57−7.52 (m, 1H, J = 4.0, J = 8.0), 7.31

(t, 1H, J = 8.0), 4.55−4.50 (m, 2H, J = 8.0, J = 8.0), 4.31 (s, 3H), 3.82

(s, 4H), 3.32 (s, 2H), 3.15 (t, 2H, J = 4.0), 2.83 (s, 6H), 2.73 (s, 4H),

1.88 (s, 2H), 1.36 (t, 3H, J = 8.0). ¹³C NMR (100 MHz, DMSO-d₆): δ

159.1, 154.4, 144.9, 141.0, 140.8, 140.1, 133.6, 126.7, 126.4, 126.2,

126.1, 122.6, 122.2, 121.7, 120.8, 120.5, 120.1, 119.7, 119.6, 119.2,

116.3, 109.9, 109.7, 109.6, 109.6, 104.9, 55.5, 54.2, 52.2, 51.6, 42.4,

38.2, 37.2, 20.9, 13.8. Purity: 99.7% by HPLC. ESI-HRMS

(C₃₄H₄₀N₅): calcd, 518.3278 [M]⁺; found, 518.3160.

NMR (400 Hz, CDCl₃): δ 8.00−7.88 (m, 4H), 7.71 (d, 1H, J = 16.0),

7.64−7.61 (m, 1H), 7.41 (s, 1H), 7.21 (s, 2H), 4.37 (s, 3H), 3.93 (m,

4H), 3.91 (s, 6H), 3.75 (s, 3H), 2.76 (m, 4H), 2.41 (s, 3H). ¹³C NMR

(100 Hz, CDCl₃): δ 159.7, 155.1, 153.1, 144.8, 141.2, 140.0, 134.3,

130.2, 126.7, 126.1, 118.6, 118.4, 106.7, 106.1, 60.8, 57.4, 54.7, 52.4,

45.8, 40.4. Purity: 99.4% by HPLC. ESI-HRMS (C₂₆H₃₂N₃O₃): calcd,

434.2438 [M]⁺; found, 434.2411.

(E)-2-(4-Hydroxystyryl)-1-methyl-4-(4-methylpiperazin-1-yl)-

quinolin-1-ium Iodide (20). Following general procedure B for (2-

aromatic vinyl)-1-N-methylquinolinium derivatives, the product was

1

obtained as a green-yellow solid. Mp > 200 °C. H NMR (400 Hz,

DMSO-d₆): δ 8.29 (d, 1H, J = 8.0), 8.14 (d, 1H, J = 8.0), 8.02 (t, 1H, J

= 8.0), 7.85 ( d, 1H, J = 16.0), 7.75 (m, 3H), 7.49 (m, 2H), 6.90 (d,

2H, J = 8.0), 4.22 (s, 3H), 3.79 (s, 4H), 2.68 (s, 4H), 2.34 (s, 3H). ¹³C

NMR (100 Hz, DMSO-d₆): δ 160.0, 159.1, 154.3, 143.6, 140.6, 133.6,

130.7, 126.4, 126.3, 126.2, 119.6, 119.1, 116.0, 115.8, 105.0, 54.0, 51.3,

45.1, 42.8. Purity: 97.2% by HPLC. ESI-HRMS (C₂₃H₂₆N₃O): calcd,

360.2070 [M]⁺; found, 360.2083.

(E)-2-(3-Methoxy-4-morpholinostyryl)-1-methyl-4-(4- meth-

ylpiperazin-1-yl)quinolin-1-ium Iodide (21). Following general

procedure B for (2-aromatic vinyl)-1-N-methylquinolinium derivatives,

1

the product was obtained as a green-yellow solid. Mp > 200 °C. H

NMR (400 Hz, DMSO-d₆): δ 8.28 (d, 1H, J = 12.0), 8.13 (d, 1H, J =

8.0), 8.02 (t, 1H, J = 8.0), 7.88 (t, 1H, J = 16.0), 7.73 (t, 1H, J = 8.0),

7.59 (d, 1H, J = 16.0), 7.48 (d, 2H, J = 4.0), 7.42 (d, 1H, J = 8.0), 6.95

(d, 1H, J = 8.0), 4.23 (s, 3H), 3.91 (s, 3H), 3.77 (s, 4H), 3.73 (s, 4H),

3.07 (s, 4H), 2.63 (s, 4H), 2.30 (s, 3H). ¹³C NMR (100 MHz, DMSO-

d₆) δ 159.3, 154.2, 151.7, 143.5, 140.8, 136.6, 133.7, 129.2, 126.5,

126.2, 123.2, 119.7, 119.2, 117.5, 117.3, 111.2, 105.0, 66.2, 55.8, 54.2,

51.5, 50.2, 45.4, 38.2. Purity: 99.0% by HPLC. ESI-HRMS

(C₂₈H₃₅N₄O₂): calcd, 459.2755 [M]⁺; found, 459.2746.

(E)-4-(4-(3-Dimethylamino)propyl)piperazin-1-yl)-2-(2-(9-

ethyl-9H-carbazol-3-yl) vinyl)-1-methylquinolin-1-ium Iodide

M

DOI: 10.1021/acs.jmedchem.5b00139

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Journal of Medicinal Chemistry

Article

(27). Following general procedure B for (2-aromatic vinyl)-1-N-

8.06 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 16 Hz,

1H), 7.81 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 7.50−7.38 (m,

5H), 7.28 (d, J = 8.0 Hz, 1H), 7.18 (s, 1H), 4.40−4.37 (m, 2H), 3.35

(s, 4H), 2.77 (s, 4H), 2.46 (s, 3H), 1.46 (t, J = 8.0 Hz, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 157.0, 157.0, 149.7, 140.4, 140.3, 135.1, 129.7,

129.2, 127.8, 126.7, 126.0, 125.1, 124.7, 123.5, 123.4, 123.0, 122.7,

120.5, 119.8, 119.2, 108.8, 108.7, 106.8, 55.2, 52.1, 46.2, 37.7, 13.9.

Purity: 99.9% by HPLC. ESI-HRMS(C₃₀H₃₀N₄): calcd, 447.2543 [M

+ H]⁺; found, 447.2521.

methylquinolinium derivatives, the product was obtained as a green-

1

yellow solid. Mp > 200 °C. H NMR (400 Hz, DMSO-d₆): δ 8.75 (s,

1H), 8.34 (d, 1H, J = 12.0), 8.23 (d, 1H, J = 8.0), 8.16 (t, 2H, J = 8.0),

7.76 (t, 3H, J = 8.0), 7.70 (d, 1H, J = 8.0), 7.60 (s, 1H), 7.54 (t, 1H, J =

8.0),7.31 (t, 1H, J = 8.0), 4.55−4.49 (m, 2H, J = 8.0, J = 8.0), 4.31 (s,

3H), 3.84 (s, 4H), 3.31 (s, 4H), 2.86 (s, 6H), 2.80 (s, 4H), 1.36 (t, 3H,

J = 8.0). ¹³C NMR (100 MHz, DMSO-d₆): δ 159.1, 154.4, 144.9,

141.0, 140.8, 140.1, 133.6, 126.7, 126.4, 126.2, 126.1, 122.6, 122.2,

121.7, 120.8, 120.5, 120.1, 119.7, 119.6, 119.2, 116.3, 109.9, 109.7,

109.6, 109.6, 104.9, 55.5, 54.2, 52.2, 51.6, 42.4, 38.2, 37.24, 20.9, 13.8.

Purity: 98.3% by HPLC. ESI-HRMS (C₃₅H₄₂N₅): calcd, 532.3435

[M]⁺; found, 532.3314.

(E)-4-(Piperid-1-yl)-2-[(9-ethyl-9H-carbazol-3-yl)ethenyl]-

quinoline (32). Compound 6b was reacted with 9-ethyl-9H-carbazol-

3-aldehyde following general procedure C to give the desired product

1

32 as an orange-yellow solid in 65% yield. Mp 185.9−187.4 °C. H

(E)-2-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)-1-methyl-4-(piperi-

din-1-yl)quinolin-1-ium Iodide (28). Following general procedure

B for (2-aromatic vinyl)-1-N-methylquinolinium derivatives, the

product was obtained as a green-yellow solid. Mp > 200 °C. ¹H

NMR (400 Hz, DMSO-d₆): δ 8.72 (s, 1H), 8.29 (d, 1H, J = 12.0), 8.23

(d, 1H, J = 8.0), 8.13 (s, 1H), 8.12−8.02 (m, 4H), 7.77−7.67 (m, 3H),

7.52 (t, 2H, J = 12.0, J = 4.0), 7.30 (t, 1H, J = 8.0), 4.50 (s, 2H), 4.27

(s, 3H), 3.76 (s, 4H), 1.85 (s, 4H), 1.79 (s, 2H), 1.36 (t, 3H, J = 8.0).

¹³C NMR (100 MHz, DMSO-d₆): δ 159.5, 154.2, 144.4, 140.9, 140.8,

140.1, 133.5, 126.6, 126.5, 126.4, 126.2, 125.9, 122.6, 122.2, 121.6,

120.5, 119.7, 119.6, 119.1, 116.4, 109.7, 109.6, 104.4, 52.8, 38.0, 37.2,

25.4, 23.5, 13.7. Purity: 99.1% by HPLC. ESI-HRMS (C₃₁H₃₂N₃):

calcd, 446.2591 [M]⁺; found, 446.2555.

NMR (400 MHz, CDCl₃): δ 8.37 (d, J = 6.0 Hz, 1H), 8.12 (t, J = 8.0

Hz, 1H), 8.06 (s, 1H), 7.97 (t, J = 8.0 Hz, 1H), 7.88−7.76 (m, 2H),

7.64−7.60 (m, 1H), 7.49−7.41 (m, 5H), 7.27−7.24 (m, 1H), 7.16 (d, J

= 8.0 Hz, 1H), 4.40−4.35 (m, 2H), 3.26 (s, 4H), 1.88 (s, 4H), 1.73 (s,

2H), 1.48−1.40 (m, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 158.3,

156.9, 140.4, 140.3, 129.3, 128.9, 127.8, 127.5, 125.9, 125.1, 124.5,

123.8, 123.4, 123.0, 122.9, 121.8, 121.4, 119.8, 119.2, 108.8, 108.7,

108.0, 106.3, 53.7, 37.7, 26.2, 24.5, 13.9. Purity: 98.9% by HPLC. ESI-

HRMS(C₃₀H₂₉N₃): calcd, 432.2434 [M + H]⁺; found, 432.2421.

Oligonucleotides. The RNA oligonucleotides used were the

following: A10-RNA-Wt, 5′-UGGGGGACGGGUAGGGGCGGGAG-

G UAGGG G-3 ′; A10-RNA-Mut, 5′-UA GAA GACG A-

AUAGAAGCGAAAAGUAGAAG-3′. The 5′-end biotinylated A10-

RNA-Wt and 5′-end biotinylated A10-RNA-Mut sequences were

purchased from Integrated DNA Technologies, Inc. The A10-RNA-Wt

sequence, 5′-end labeled with Cy3 and 3′-end labeled with Cy5, was

also purchased from Integrated DNA Technologies, Inc. The DNA

oligonucleotide used was the DNA antisense oligonucleotide (AS), 5′-

CCCCTACCTCCCGCCCCTACCCGTCCCCCA-3′. The AS DNA

sequence was purchased from Life Technologies, Inc.

Surface Plasmon Resonance (SPR). SPR was performed on a

ProteOn XPR36 protein interaction system (Bio-Rad Laboratories,

Hercules, CA) using a neutravidin-coated GLH sensor chip. The 5′-

end biotinylated RNAs (A10-RNA-Wt or A10-RNA-Mut) were

immobilized on the sensor chip in degassed SPR running buﬀer (50

mM Tris-HCl, pH 7.5, 100 mM KCl). For the initial screening

experiment, the ligands were diluted with running buﬀer to the same

concentration (10 μM). In the RNA binding experiments, solutions of

ligands 1−32 were freshly prepared in running buﬀer by serial

dilutions from stock solution. For both experiments, the ligand was

injected at a ﬂow rate of 20 μL/min during the association phase,

which was followed by a disassociation phase. The GLH sensor chip

was regenerated using a brief injection of 50 mM NaOH between

consecutive measurements. The equilibrium dissociation constants

(K_D) were determined with ProteOnmanager software using the

equilibrium method.

Native Gel Electrophoresis Assay. The A10-RNA-Wt sequence

labeled with Cy3 and Cy5 was folded in the presence of 100 mM KCl,

10 mM Tris-HCl, and 0.1 mM EDTA (pH 7.5) by heating for 5 min at

95 °C followed by cooling to room temperature over a 90 min period.

After cooling, the RNA samples (2 μM) were incubated with various

concentrations of the DNA antisense oligonucleotide (AS) or

compound 24 for 15 and 5 min, respectively, at room temperature

in the dark. The samples were then loaded on a native 16% gel and

electrophoresed at 4 °C before being exposed to a gel image system

(Tanon-4500 sf).

cDNA Constructs. Plasmids A10-Wt, A10-Mut, 5′UTR-Wt, and

5′UTR-Mut were constructed as previously described.²¹Brieﬂy, the

ADAM10 G-quadruplex motif, the 5′-UTR of ADAM10, and their

QGRS motif-mutated variants (Figure 2B) inserted in front of the

Renilla luciferase of the psiCHECK-2 vector (Promega) were generated

by PCR using the unique Nhe I restriction site upstream from the

Renilla luciferase start codon. All cDNAs were veriﬁed by sequencing.

Cell Culture. HeLa cells were cultured in Dulbecco’s modiﬁed

Eagle medium (DMEM) supplemented with 10% fetal bovine serum

and 2 mM glutamine. The HEK-APP cell line was cultured in DMEM

(E)-2-[(9-Ethyl-9H-carbazol-3-yl)ethenyl]quinoline (29). A

suspension of 2-methylquinoline (1.0 mmol), 9-ethyl-9H-carbazol-3-

aldehyde (1.05 mmol), and acetic anhydride (15 mL) was heated at

125 °C for 24 h. After completion of the reaction, the reaction mixture

was allowed to cool to room temperature and then poured into ice−

water (40 mL), and then aqueous NaOH was added to make the

solution basic. The solids formed were ﬁltered oﬀ and washed with

water, and the crude product was puriﬁed by using ﬂash column

chromatography with CH₂Cl₂/petroleum ether (3:2) elution to give

desired product 29 as a pale yellow solid in 67% yield. Mp 183.5−

1

184.9 °C. H NMR (400 MHz, CDCl₃): δ 8.37 (d, J = 4.0 Hz, 1H),

8.12 (d, J = 4.0 Hz, 3H), 7.95−7.86 (m, 1H), 7.81−7.71 (m, 4H),

7.49−7.41 (m, 5H), 7.26 (t, J = 4.0 Hz, 1H), 4.44−4.31 (m, 2H),

1.48−1.44 (m, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 156.7, 148.3,

140.4, 140.4, 136.2, 135.6, 129.6, 129.1, 127.7, 127.5, 127.2, 126.3,

126.0, 125.8, 125.2, 123.4, 123.0, 120.5, 119.9, 119.3, 119.1, 108.8,

108.7, 37.7, 13.9. Purity: 99.1% by HPLC. ESI-HRMS (C₂₅H₂₀N₂):

cacld, 349.1699 [M + H]⁺; found, 349.1709.

General Procedure C: Preparation of Compounds 30−32. A

suspension of compound 6a or 6b (1.0 mmol), various aromatic

aldehydes (1.05 mmol), and acetic anhydride (15 mL) was heated at

150 °C for 36 h. After completion of the reaction, the reaction mixture

was allowed to cool to room temperature and poured into ice−water

(40 mL), and then aqueous NaOH was added to make the solution

basic. The solids formed were ﬁltered and washed with water, and the

crude product was puriﬁed by using ﬂash column chromatography

with CH₂Cl₂/petroleum ether (1:1) or EtOAc/methanol (200:3)

elution to give the desired products.

(E)-4-(4-Methylpiperazin-1-yl)-2-(4-(morpholin-1-yl)styryl)-

quinoline (30). Compound 6a was reacted with 4-morpholinyl-

benzaldehyde following general procedure C to give the desired

1

product 30 as an orange solid in 66% yield. Mp 206.6−208.9 °C. H

NMR (400 MHz, CDCl₃): δ 7.99 (d, J = 8.9 Hz, 2H), 7.65−7.45 (m,

4H), 7.44−7.34 (m, 1H), 7.21 (d, J = 12.0 Hz, 1H), 7.14−7.00 (m,

1H), 6.92 (s, 2H), 3.87 (s, 4H), 3.25 (s, 8H), 2.74 (s, 4H), 2.51−2.33

(m, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 157.0, 156.8, 151.3, 149.6,

133.8, 129.6, 129.2, 128.4, 128.1, 126.5, 124.7, 123.4, 122.6, 115.2,

106.8, 66.8, 55.2, 52.1, 48.6, 46.1. Purity: 98.6% by HPLC. ESI-HRMS

(C₂₆H₃₀N₄O): cacld, 415.2492 [M + H]⁺; found, 415.2474.

(E)-4-(4-Methylpiperazin-1-yl)-2-[(9-ethyl-9H-carbazol-3-yl)-

ethenyl]quinoline (31). Compound 6a was reacted with 9-ethyl-9H-

carbazol-3-aldehyde according to general procedure C to give the

desired product 31 as a yellow solid in 66% yield. Mp 188.2−191.3 °C.

¹H NMR (400 MHz, CDCl₃): δ 8.38 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H),

N

DOI: 10.1021/acs.jmedchem.5b00139

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

supplemented with 10% fetal bovine serum, 2 mM glutamine, and 0.2

mg/mL G418.

5′-TCTTTGTGGGCCACGACTGGGG-3′, antisense, 5′-

GGCAGCGAACTCCTCAGGCTCC-3′. Fireﬂy luciferase: sense, 5′-

GCCGTGGCCAAGCGCTTTCATC-3′, antisense, 5′-

CTCCCAGGGTCTTGCCGGTGTC-3′. ADAM10: sense, 5′-

TTTGATGATGGCGTACTTGG-3′, antisense, 5′-AGTTTGTC-

CCCAGATGTTGC-3′. APP: sense, 5′-CACCACAGAG-

TCTGTGGAAGA-3′, antisense, 5′-AGGTGTCTGAGATACTTGT-

3′. For each corresponding RNA sample used in the dual-luciferase

reporter assay, the Renilla luciferase RNA expression was normalized

to ﬁreﬂy luciferase as described.²¹The ADAM10 and APP levels were

normalized to β-actin levels where indicated. The quantiﬁcation was

performed by densitometry using Quantity ONE software (Bio Rad).

Measurement of ADAM10 Activity. α-Secretase (ADAM10)

activity was determined using recombinant human ADAM10 protein

(R&D) and a ﬂuorogenic α-secretase substrate (MERCK). Before

performance of the activity assay, the ADAM10 protein was incubated

with various concentrations of 24 (0, 0.25, 0.5, 1, 2, 4 μM) for 20 min.

A total of 50 μL of sample (total protein 50 ng) was added to each

well followed by 50 μL of 2× reaction buﬀer (50 μM Tris, 4 μM

ZnCl₂, 0.01% (w/v) Brij-35, pH 9.0) and 20 μM α-secretase substrate.

The samples were then incubated in the dark at 37 °C for 1 h. The

ﬂuorescence was read at excitation and emission wavelengths of 340

and 490 nm, respectively, using a monochromator-based multimode

microplate reader (M1000, Tecan). α-Secretase activity is proportional

to the ﬂuorimetric reaction and is expressed as nmol/mg protein per

minute.

Dual Luciferase Reporter Assay. HeLa cells were transfected

with 0.8 μg of A10-Wt, A10-Mut, or A10-Del plasmid using

LipofectAMINE 2000 (Invitrogen) as a transfecting reagent. Trans-

fections were performed by addition of the vector (250 ng/well). The

culture medium was replaced 12 h after transfection, and the cells were

transiently transfected with either the AS or a random deoxyoligonu-

cleotide, each at a ﬁnal concentration of 200 nM. After treatment for

24 h, cell lysates were prepared, and the luciferase activity was

measured with the dual-luciferase reporter assay system (Promega)

according to the manufacturer’s instructions. In experiments for 11

selected compounds, after transfection with A10-Wt, A10-Mut, or

A10-Del plasmid as above, the cells were transiently incubated with a

single concentration of selected compounds (2 μM). After treatment

for 24 h, the luciferase activity was measured with the dual-luciferase

reporter assay system. In further experiments for the most potent

compound, 24, after transfection with 0.8 μg of A10-Wt, A10-Mut,

A10-Del, 5′UTR-Wt, or 5′UTR-Mut plasmid for 12 h, the HeLa cells

were incubated with 0, 0.5, 1, or 2.5 μM 24 for 24 h, and the dual-

luciferase reporter assays were performed. The quantiﬁcation was

performed using a multimode reader (Molecular Devices). The Renilla

luciferase activity was normalized to the ﬁreﬂy luciferase activity. All of

the above samples were also prepared for RT-PCR assay.

Protein Analysis. HEK-APP cells were plated in 10 cm dishes, and

48 h after treatment with 0, 0.5, 0.75, 1, or 1.5 μM 24, cell lysates were

prepared using lysis buﬀer containing 50 mM Tris-HCl, pH 7.6, 150

mM NaCl, 2 mM EDTA, 1% Triton X-100, and protease inhibitors

(Sigma) and analyzed for ADAM10, APP, and β-actin protein levels by

immunoblotting. The cell lysates were collected, transferred to

Eppendorf tubes, and centrifuged at 15 000g for 2 min to collect the

cell debris. For analysis of APP processing, equal amounts of the

conditioned cell culture medium were collected and analyzed for

sAPPα and Aβ₄₀protein level by immunoblotting and ELISA,

respectively. All of the above samples were also prepared for analysis

by RT-PCR.

Western Blotting. Protein samples denatured in SDS sample

buﬀer (125 mmol/L Tris-HCl, pH 7.0, 50% (v/v) glycerol, 2% (w/v)

SDS, 5% (v/v) mercaptoethanol, and 0.01% (w/v) bromophenol blue)

were subjected to SDS−PAGE and blotted onto polyvinylidene

diﬂuoride (Millipore) membranes. The blotted membranes were

blocked with 5% (w/v) skim milk in Tris-buﬀered saline containing

0.1% (v/v) Tween 20 for 1 h and then incubated with primary

antibodies for 16 h at 4 °C. After three washes in Tris-buﬀered saline

containing 0.1% (v/v) Tween 20, the membranes were incubated with

anti-mouse or anti-rabbit peroxidase-linked secondary antibody

(1:2000; Santa Cruz) for 2 h. The immunoreactive signals were

detected using a ChemiDoc XRS imaging system (Bio Rad) and

quantiﬁed with Image Lab 3.0 software (Bio Rad). Key proteins were

examined by Western blotting using speciﬁc antibodies including anti-

ADAM10 (1:1000; Abcam), anti-APP (1:1000; Convance), anti-

sAPPα (1:1000; Convance), and anti-β-actin (1:1000; Cell Signaling).

Immunolabeled bands were quantiﬁed by densitometry using Quantity

ONE software (Bio Rad), and representative blots are shown. The

ADAM10 and APP levels were normalized to the β-actin levels where

indicated.

ASSOCIATED CONTENT

■

S

* Supporting Information

1

Additional experiment results, H and ¹³C NMR spectra, and

LC/MS and HPLC assay data for compounds. This material is

available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: 8620-39943056. Fax: 8620-39943056. E-mail:

ceshzs@mail.sysu.edu.cn.

■

Author Contributions

^‡J.D. and Z.-Q.L. contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the Natural Science Foundation of China (Grants

81273433, 91213302, and 81330077), Specialized Research

Fund for the Doctoral Program of Higher Education of China

(Grant 20110171110051), and Guangdong Provincial Key

Laboratory of Construction Foundation (Grant

2011A060901014) for ﬁnancial support of this study.

ABBREVIATIONS USED

■

ADAM10, a disintegrin and metalloprotease 10; Aβ, β-amyloid;

AD, Alzheimer’s disease; QGRS, G-quadruplex forming G-rich

sequence; UTR, untranslated region; SPR, surface plasmon

resonance; HEK-APP, HEK 293 cell line stably overexpressing

Swedish-mutated amyloid precursor protein; APP, amyloid

precursor protein; ADAM, a family of disintegrin and

metalloproteinases; PKC, protein kinase C; GABA-A, γ-

aminobutyric acid type A; PDE4, phosphodiesterase 4; MTT,

methylthiazolyltetrazolium; MST, microscale thermophoresis;

sAPPα, human soluble amyloid precursor protein α; AS, DNA

antisense oligonucleotide; RT-PCR, reverse transcription PCR;

RU, relative intensity; SAR, struture−activity relationship; p-

TSA, p-toluenesulfonic acid; FMRP, fragile-X mental retarda-

tion protein; hnRNP, heterogeneous nuclear ribonucleopro-

Enzyme-Linked Immunosorbent Assay (ELISA). HEK-APP cell

culture media were collected to quantify the secreted Aβ₄₀peptide by

sandwich immunoassay using an ELISA kit (Invitrogen) according to

the manufacturer’s instructions.

Reverse-Transcription PCR (RT-PCR). Total RNA was isolated

from HeLa cells or HEK-APP cells samples using RNAiso Plus

(Takara) according to the manufacturer’s protocol. After the ﬁrst

strand cDNA was synthesized using Oligo (dT), the cDNA was used

for ampliﬁcation of speciﬁc target genes by PCR. β-Actin was used as

the RNA loading control. The PCR products were separated on 1.5%

(w/v) agarose gels and analyzed using the AlphaImager EC Imaging

System (Alpha Innotech). The PCR primer sequences were as follows.

β-Actin: sense, 5′-CTGAATCTGCACCAAGCATGA-3′, antisense,

5′-TAAAACGCAGCTCAGTAACAGTCC-3′. Renilla luciferase: sense,

O

DOI: 10.1021/acs.jmedchem.5b00139

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

essential for Notch signalling but not for alpha-secretase activity in

fibroblasts. Hum. Mol. Genet. 2002, 11, 2615−24.

tein; ASF/SF2, alternative splicing factor/splicing factor 2; MS,

mass spectra; HRMS, high-resolution mass spectra; Mp,

melting point; EDTA, ethylenediaminetetraacetic acid;

DMEM, Dulbecco’s modiﬁed Eagle medium; FBS, fetal bovine

serum; SDS, sodium dodecyl sulfate; ELISA, enzyme-linked

immunosorbent assay

(16) Jorissen, E.; Prox, J.; Bernreuther, C.; Weber, S.; Schwanbeck,

R.; Serneels, L.; Snellinx, A.; Craessaerts, K.; Thathiah, A.; Tesseur, I.;

Bartsch, U.; Weskamp, G.; Blobel, C. P.; Glatzel, M.; De Strooper, B.;

Saftig, P. The disintegrin/metalloproteinase ADAM10 is essential for

the establishment of the brain cortex. J. Neurosci. 2010, 30, 4833−44.

(17) De Strooper, B.; Vassar, R.; Golde, T. The secretases: enzymes

with therapeutic potential in Alzheimer disease. Nat. Rev. Neurol. 2010,

6, 99−107.

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DOI: 10.1021/acs.jmedchem.5b00139

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of small molecules for up-regulating the translation of antiamyloidogenic secretase, a disintegrin and metalloproteinase 10 (ADAM10), by binding to the G-quadruplex-forming sequence in the 5' untranslated region (UTR) of its mRNA

DOI: 10.1021/acs.jmedchem.5b00139

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