Dual-Acting Cholinesterase-Human Cannabinoid Receptor 2 Ligands Show Pronounced Neuroprotection in Vitro and Overadditive and Disease-Modifying Neuroprotective Effects in Vivo

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Dual-Acting Cholinesterase−Human Cannabinoid Receptor 2

Ligands Show Pronounced Neuroprotection in Vitro and

Overadditive and Disease-Modifying Neuroprotective Eﬀects in Vivo

Matthias Scheiner,^†Dominik Dolles,^†Sandra Gunesch,^†Matthias Hoﬀmann,^†Massimo Nabissi,^‡

Oliviero Marinelli,^‡Marina Naldi,^§Manuela Bartolini,^§Sabrina Petralla,^∥Eleonora Poeta,^∥

Barbara Monti,^∥Christina Falkeis,^⊥Michael Vieth,^⊥Harald Hubner,^#Peter Gmeiner,^#

̈

Rangan Maitra,^∇Tangui Maurice,^○and Michael Decker*^,†

^†Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, Julius Maximilian University of Wu

Am Hubland, 97074 Wurzburg, Germany

^‡School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy

^§Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy

^∥Department of Pharmacy and Biotechnology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy

^⊥Pathology, Clinical Center Bayreuth, Preuschwitzer Straße 101, 95445 Bayreuth, Germany

^#Medicinal Chemistry, Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nu

19, 91052 Erlangen, Germany

^∇Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, North Carolina 27709, United States

^○MMDN, University of Montpellier, INSERM, EPHE, UMR-S1198, 34095 Montpellier, France

̈

rzburg,

̈

rnberg, Schuhstraße

S

* Supporting Information

ABSTRACT: We have designed and synthesized a series of 14 hybrid

molecules out of the cholinesterase (ChE) inhibitor tacrine and a

benzimidazole-based human cannabinoid receptor subtype 2 (hCB₂R)

agonist and investigated them in vitro and in vivo. The compounds

are potent ChE inhibitors, and for the most promising hybrids, the

mechanism of human acetylcholinesterase (hAChE) inhibition as well

as their ability to interfere with AChE-induced aggregation of β-

amyloid (Aβ), and Aβ self-aggregation was assessed. All hybrids were

evaluated for aﬃnity and selectivity for hCB₁R and hCB₂R. To ensure

that the hybrids retained their agonist character, the expression of

cAMP-regulated genes was quantiﬁed, and potency and eﬃcacy were

determined. Additionally, the eﬀects of the hybrids on microglia activation and neuroprotection on HT-22 cells were

investigated. The most promising in vitro hybrids showed pronounced neuroprotection in an Alzheimer’s mouse model at low

dosage (0.1 mg/kg, i.p.), lacking hepatotoxicity even at high dose (3 mg/kg, i.p.).

remain unknown. However, a variety of biochemical changes

during the pathological process take place. AD hallmarks are the

formation of amyloid-beta (Aβ) plaques in the extracellular

brain parenchyma,³the abnormal hyperphosphorylation of the

microtubule-associated tau protein, and the formation of

neuroﬁbrillary tangles (NFTs) within neurons.⁴Both the Aβ

aggregates and the NTFs lead to a progressive loss of cholinergic

neurons in the brain, memory deﬁcits, and cognitive

dysfunction.⁵⁻⁷Aβ itself has been shown to exert cytotoxic

eﬀects on cultured neurons,⁸induce oxidative stress, and modify

ionic homeostasis.^9,10Furthermore, in the post-mortem analysis

of AD brain, an increased expression of inﬂammatory mediators

INTRODUCTION

■

About 50 million people worldwide had to live with dementia in

2018.¹In about 30 years, an increase of cases up to 152 million is

expected. The most common form of dementia is Alzheimer’s

disease (AD). People aged 65 and over are considered at risk and

are most likely to be aﬀected.¹Unfortunately, pharmacotherapy

is very limited. Currently, only four drugs are approved for the

treatment of AD. Three of them (donepezil, rivastigmine, and

galantamine) are cholinesterase (ChE) inhibitors, while the

fourth (memantine) is an N-methyl-^D-aspartate (NMDA)

receptor noncompetitive antagonist. All approved drugs are

only symptomatically eﬀective in the moderate stages of the

disease and reduce but do not stop the progression and do not

aﬀect the cause of the disease.²The exact molecular

mechanisms, as well as the causes for an AD outbreak still

Received: April 30, 2019

A

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

has been observed.^11,12The increase seems to be partly triggered

by microglia activation. Microglia cells are activated by various

stimuli, including misfolded Aβ, its precursor protein (APP),

and misfolded tau.^13,14In the early stage of AD, stimulation leads

to a neuroprotective M2 microglia phenotype and can,

therefore, promote Aβ clearance via microglia’s scavenger

receptors (SRs), which hinder AD progression.^15,16However,

it has been shown that there is a switch from the neuroprotective

M2 to a more classically activated M1 phenotype at the later

stage of AD. The M1 phenotype generally mediates defense

from pathogens and tumor cells, and it is characterized by the

production of proinﬂammatory cytokines, such as TNF-α, IL1β,

or reactive oxygen species (ROS), which are associated with the

loss of neurons. Therefore, this switch can lead to an increased

Aβ production, a decreased Aβ clearance, and, ultimately, to

neuronal damage and progression of AD.¹⁶

The human endocannabinoid system has been intensively

investigated, and two cannabinoid receptor (hCBR) subtypes

have been discovered. The hCBR subtype 1 (hCB₁R) is the most

abundant metabotropic receptor in the brain, and it is involved

in a variety of physiological processes.¹⁷⁻¹⁹The psychoactive

eﬀect of tetrahydrocannabinol (THC), the main psychoactive

constituent of cannabis, is due to hCB₁R agonism.²⁰The hCB₂R

is mainly expressed in peripheral immune cells and has been

originally described as the peripheral CBR,²¹but later, it was

found also in the central nervous system (CNS) where it is

expressed on microglia and astrocytes.^22,23While the expression

of the hCB₁R remains unchanged, the hCB₂R is abundantly and

selectively expressed in neuritic plaque-associated astrocytes and

microglia.^24,25In several in vitro and in vivo models of acute and

chronic neurodegenerative disorders, the activation of the

hCB₂R was shown to exert beneﬁcial eﬀects.²⁶hCB₂R agonists

can suppress microglia activation and the production of

neurotoxic factors such as ROS, nitric oxide (NO), and

proinﬂammatory mediators (TNF-α and cytokines).²⁷⁻²⁹

Administration of an hCB₂R agonist to rats, previously and

intracerebrally treated with Aβ₄₀, the forty-amino acid long

isoform of Aβ, promoted Aβ clearance, decreased secretion of

proinﬂammatory mediators, and, ultimately, led to an increased

synaptic plasticity, cognition, and memory.³⁰Furthermore, in

vivo studies with transgenic Tg2576 mice overexpressing

hAPP_Sweshowed a lower Aβ production, reduced reactive

microglia cells, and improved cognition performance upon

treatment with an hCB₂R agonist.³¹Additionally, an hCB₂R

knockout experiment with APP transgenic mice supports the

potential of cannabinoid therapies targeting hCB₂R to reduce

Aβ, as the knockout results in an increased Aβ pathology and an

alternation in tau processing.³²

observed since BChE can take over the hydrolysis of ACh.³⁹

Results from further studies were in accordance with the role of

the BChE in AD brains and showed a positive correlation

between selective BChE inhibition and improved cognitive

performance and memory.⁴⁰⁻⁴⁴

There is even more diversity in the pathological processes and

reasons for AD beyond the ones herein described. Because of

this multifactorial and sporadic nature of AD, the classical

approach “one target−one disease” reaches its limits or even

makes it ineﬀective to develop an eﬀective drug.⁴⁵Conversely, a

more promising approach seems to be the development of a

multitarget drug capable of simultaneously acting at diﬀerent

targets.⁴⁶To obtain such multitarget drugs, there are two

common strategies: the hybrid and the “merged ligand”.⁴⁷In the

merged ligand strategy, two pharmacophores are fused into a

single small molecule. The hybrid strategy foresees the

combination of two molecules with two diﬀerent pharmacolog-

ically active moieties by using a linker to form a larger

molecule.^45,47Through the merged ligand strategy, we have

recently achieved low-molecular-weight hCB₂R ligands en-

dowed with hBChE inhibitory activity, which proved to exert

neuroprotection even in vivo.⁴⁸The design and improvement of

such multi-target directed ligands (MTDLs) are challenging

since minor chemical alternations can improve aﬃnity at one

target and diminish activity at the second one. Here, we describe

hybrids out of similar benzimidazole units with the established

tacrine unit to yield higher aﬃnity at both targets.

Design. Since AChE is a well-established target for the

treatment of AD at the early and moderate stages, while BChE is

a promising target for the moderate-to-advanced forms of AD,

an inhibitor that is able to tackle both ChEs should be eﬀective

for a longer time span along AD progression. Such dual

inhibitors have been described in the literature, among which

tacrine 1.⁴⁹Tacrine was the ﬁrst approved drug for the treatment

of AD but was withdrawn from the market because of its dose-

dependent hepatotoxicity.^50,51Due to its low molecular weight

and excellent ChE inhibitory activity, tacrine 1 is an ideal starting

point for the development of multitarget drugs (for recent

review articles, see refs 52 and 53).

In developing tacrine hybrids, it is important to keep the

potential hepatotoxic eﬀect in mind. Linking or coupling with

hepatoprotective compounds can reduce or even eliminate such

a side eﬀect.⁵⁴⁻⁵⁶

In addition to the inhibition of the ChEs, the activation of the

hCB₂R, as previously mentioned, represents a promising

strategy in the treatment of AD. An example of a selective

hCB₂R agonist is the benzimidazole-based derivative 2

described by AstraZeneca (see Figure 1)⁵⁷and further

investigated by us.^48,58−60

Acetylcholinesterase (AChE)-inhibiting drugs for sympto-

matic cognition improvement in AD patients are related to the

oldest theory of AD pathophysiology, the cholinergic hypoth-

esis.²The theory describes the loss of cholinergic neurons and

reduction of the neurotransmitter acetylcholine (ACh).³³

Inhibition of the metabolizing enzyme AChE raises ACh levels,

leading to an improved cognitive performance. However, since

AChE level decreases with the progression of AD,³⁴⁻³⁶AChE

inhibition seems to be only eﬀective at early stages of disease. On

the other hand, the levels of the less speciﬁc butyrylcholinester-

ase (BChE) remain unaltered or increase with AD pro-

gression.³⁵⁻³⁸BChE can hydrolyze ACh and, thereby,

compensate the reduction of AChE activity. An experiment

with AChE knockout mice supported this hypothesis. Indeed,

no cholinergic hyperactivation in the absence of AChE was

RESULTS AND DISCUSSION

■

Following the hybrid concept, coupling of structures 1 and 2

into a single molecule through a linker should yield a molecule

with inhibitory properties toward ChEs and aﬃnity for the

hCB₂R. Deﬁning a suitable connecting position for the linker

chain is a crucial task because an unsuitable connection between

the two pharmacophore units can easily lead to a complete loss

of activity or can reverse the behavior of the drug at the target,

especially with regard to the hCB₂R (e.g., turn an agonist into an

antagonist).⁶⁰Linkage at the primary amine moiety at the

position 9 of 1 is chemically easy to access, and substituted

derivatives still show pronounced inhibitory properties toward

ChEs.⁶¹

B

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

The nitro moiety was then reduced with tin(II) chloride

dihydrate to obtain the anilinic amines 18a−f, which were

directly used for the next step. 2-(4-Ethoxyphenyl)acetic acid

was activated with HBTU and added to amines 18a−f. In the

last step, the amides 19a−f were cyclized in acetic acid to yield

compounds 3a−e and 6 (Scheme 2).

4-Fluoro-3-nitrobenzoic acid 15 served as the starting

material for derivatives 4a−e, 5, 7, and 8. In the ﬁrst step, the

ethyl ester was formed as a protection group, and the ﬂuorine

atom was substituted with 3-methylbutan-1-amine to yield

substituted ester 20 in a good yield. In the next step, the nitro

moiety was reduced with hydrogen over Pd/C to yield amine 21,

which was then directly reacted with HBTU-activated 2-(4-

ethoxyphenyl)acetic acid to form amide 22. Benzimidazole 23

was formed by stirring 22 in acetic acid under reﬂux. The ester

was hydrolyzed under basic conditions. In the last step, acid 24

was activated with HBTU and reacted with the respective

amines 12a−g and 14 to yield hybrids 4a−e, 5, 7, and 8 (Scheme

3).

Figure 1. Tacrine 1 and selective hCB₂R agonist 2.⁵⁸

Taking advantage of the previous work on the structure−

activity relationships (SARs) of the hCB₂R ligand 2, two

possible attachment positions for a linker were identiﬁed, that is,

the amide moiety and the N¹of the imidazole.⁵⁸Connection of

structures 1 and 2 through these two connection points lead to

two sets of hybrid compounds: 3a−e and 4a−e (see Figure 2).

Modiﬁcation of the tertiary amide group of the parent

molecule 2 into a secondary amide group in derivatives 4a−e

might aﬀect aﬃnity and activity at the target. Hence, to

investigate a possible eﬀect of the number of substituents at this

position, we designed a tertiary amide hybrid (hybrid 5). After

optimizing the spacer length, we designed hybrids with a

functionalized linker. Therefore, we introduced a 2-poly-

(ethylene glycol) (PEG) linker for higher rigidity and polarity

(hybrids 6 and 7). In addition, cystamine was introduced as a

linker at the amide position to archive neuroprotective and

putative hepatoprotective properties (8, see Figure 3).⁶²

Synthesis. Based on these design strategies, tacrine-amine

derivatives 12a−g and 14 were synthesized and coupled to the

benzimidazole core. Anthranilic acid 9 and cyclohexanone 10

were heated up with an excess of phosphoroxylchloride to yield

the tetrahydroacridine derivative 11.⁶³By substitution with the

corresponding diamine in excess, the tacrine-amine derivatives

12a−g were obtained (Scheme 1).

PHARMACOLOGICAL PROFILE

■

Inhibition of Cholinesterases. All synthesized compounds

were assayed for their ability to inhibit human butyrylcholines-

terase (hBChE) and human acetylcholinesterase (hAChE)

using the colorimetric Ellman’s assay (Table 1).⁶⁴Tacrine 1 was

selected as the reference compound and showed an

pIC₅₀(hBChE) = 7.9 and pIC₅₀(hAChE) = 6.8. Previous studies

showed no signiﬁcant ChE inhibitory activity of the hCB₂R

agonist 2 at 10 μM.⁵⁸According to our expectations, all

synthesized hybrids acted as ChE inhibitors. Compared to 1,

hybrids 3a, 3b, 3e, and 4b showed an increased inhibitory

potency toward hBChE, while 3a and 4b, in addition, also

showed a higher inhibitory activity toward hAChE. The higher

inhibitory potency might be due to interaction with the PAS of

the AChE (see Inhibition and Mode of Action toward hAChE),

which was consequently followed up (cf. Figure 5). For a better

overview of the selectivity, we correlated pIC₅₀values of hAChE

and hBChE in Figure 4.

For the synthesis of derivative 14, the primary amine of

tacrine-amine derivative 12c was acetylated to form amide 13 in

quantitative yield. In the next step, the amide was reduced to the

secondary amine 14 in the presence of lithium aluminum

hydride (Scheme 1).

Synthesis of derivatives 3a−e and 6 started with 4-ﬂuoro-3-

nitrobenzoic acid 15. To form the diethylamide 16, oxalyl

chloride and a catalytic amount of DMF were used, and then

diethylamine was added. In the next step, the ﬂuorine atom was

substituted with the respective amine derivatives 12a−f to form

compounds 17a−f, the reaction gave good to quantitative yield.

Inhibition Constants and Mode of Action toward

hAChE. To get further insights into the inhibition of hAChE,

the mechanism of inhibition was investigated for one hybrid of

each set with a pronounced inhibition of AChE. Speciﬁcally,

hybrid 3e of set 1 and 4b of set 2 were selected. Additionally,

cystamine hybrid 8 was investigated.

For the three selected compounds, the mechanism of

inhibition was investigated by monitoring the catalytic rate at

several substrate and inhibitor concentrations (see Experimental

Section for details). Lineweaver−Burk reciprocal plots showed

increasing slope (decreasing V_max) and increasing x intercept

(increasing K_mvalues) at increasing inhibitor concentration (see

Figure 5). This trend indicates a mixed-type inhibition, and thus,

the inhibitor could bind simultaneously with the CAS and the

PAS of hAChE. The inhibitor inhibition constant K_iand the

inhibition constant for the enzyme−substrate−inhibitor com-

plex (K_i′) were estimated for each hybrid and are reported in

Figure 5.

Inhibition of Aβ Aggregation. Compounds able to bind

the AChE’s PAS may interfere with the AChE-induced Aβ

oligomerization and ﬁbrillization.^65,66Hence, given the ability of

3e, 4b, and 8 to contact AChE’s PAS, the inhibitory activity on

AChE-induced Aβ₄₀aggregation was investigated by a thioﬂavin

T (ThT)-based ﬂuorometric assay⁶⁵and compared with that of

the anti-AD drug 1 (see Figure 6). Results showed that

Figure 2. Designed hybrids 3a−e and 4a−e used to investigate the

optimal spacer length and the attachment point.

C

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 3. Designed hybrids 5−8.

a

Scheme 1. Synthesis of Tacrine-Amine Intermediates 12a−g and 14

a

Reagents and conditions: (a) POCl₃, reﬂux; (b) respective diamine, hexanol, reﬂux; (c) Ac₂O, r.t.; (d) LiAlH₄, THF, reﬂux.

a

Scheme 2. Synthesis of Hybrids 3a−e and 6

a

Reagents and conditions: (a) HNEt₂, cat. DMF, (COCl)₂, CH₂Cl₂at 0 °C → r.t.; (b) respective amines 12a−f, THF, NEt₃, r.t.; (c) SnCl₂·2H₂O,

EtOH, reﬂux; (d) 2-(4-ethoxyphenyl)acetic acid, HBTU, NEt₃, DMF, r.t.; (e) AcOH, reﬂux.

compounds 3e and 4b were able to signiﬁcantly reduce the

proaggregating action of hAChE, while 1 was inactive, in

agreement with a previous study.⁶⁵Interestingly, percent

inhibition values archived for 3e and 4b are close to activity

values previously obtained for the well-known multifunctional

compound bis(7)tacrine (68.0% inhibition in the same assay

conditions).⁶⁷In the same experimental conditions, the

cystamine derivate 8 was not able to signiﬁcantly inhibit the

AChE-induced Aβ₄₀aggregation.

Although 1 is not able to inhibit amyloid self-aggregation at a

signiﬁcant extent, several studies have shown that tacrine homo-

and heterodimers may be endowed with such a beneﬁcial

property.⁵²Hence, the three selected hybrids were also assayed

for their ability to inhibit Aβ₄₂self-aggregation by a similar ThT

D

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 3. Synthesis of Hybrids 4a−e, 5, 7, and 8^a

a

Reagents and conditions: (a) (I) cat. H₂SO₄(97%), EtOH, reﬂux; (II) NEt₃3-methylbutan-1-amine, r.t.; (b) cat. Pd/C (10%), MeOH, H₂atm, 8

bar, r.t.; (c) 2-(4-ethoxyphenyl)acetic acid, HBTU, NEt₃, DMF, r.t.; (d) AcOH, reﬂux; (e) LiOH, H₂O, reﬂux; (f) respective amines 12a−g, 14,

HBTU, NEt₃, DMF, r.t.

ﬂuorescence assay.⁶⁸Quite interestingly, compounds 3e and 8

strongly inhibited Aβ₄₂aggregation when assayed at a 1:1 ratio

with Aβ₄₂, while, in the same conditions, inhibition by 4b was

quite weak (see Figure 6) since each compound is representative

of a diﬀerent subset of hybrids, SARs cannot be derived.

However, it is clear that the spatial arrangement of

pharmacophore fragments and the nature of the spacer chain

strongly aﬀect the inhibitory ability toward the spontaneous

aggregation of the 42-amino acid isoform of Aβ.

The three hybrids showed diﬀerent inhibition proﬁles toward

the two aggregation phenomena. While hybrid 8 was able to

signiﬁcantly interfere with Aβ self-aggregation but not with the

chaperonic action of hAChE toward Aβ, hybrid 4b was able to

signiﬁcantly interfere with the chaperonic action of hAChE

toward Aβ but only weakly with Aβ self-aggregation, and, ﬁnally,

hybrid 3e able to signiﬁcantly inhibit both aggregation

phenomena. Because of the strong inhibitory potency toward

Aβ self-aggregation exerted by 3e, it cannot be excluded that this

activity can also synergistically contribute to the good inhibitory

potency recorded in the AChE-induced amyloid aggregation

assay. This might also explain the high inhibition value recorded

in this assay, notwithstanding a K′_ivalue in the submicromolar

range.

Radioligand Binding Assays at hCB₁R and hCB₂R. All

synthesized compounds were tested for aﬃnity to hCB₁R and

hCB₂R using radioligand binding assays. Brieﬂy, hCBRs were

isolated from cell lines stably expressing receptors (CHO cells

for hCB₁R or HEK cells in the case of hCB₂R). [3H]CP 55,940

was used as radioligand. As a positive control, the hCB₂R

selective ligand 2 was used. In our tests, we measured a

K_i(hCB₂R) = 37 nM and selectivity over hCB₁R [24%

replacement (hCB₁R) of radioligand at 10 μM)]. All

compounds showed aﬃnity to hCB₂R within single- to two-

digit micromolar range. In the case of compounds 3a−e, the

aﬃnity toward hCB₂R increased with the length of the spacer,

from K_i= 38.5 μM (3a) to K_i= 4.5 μM (3e). Part of this eﬀect

might be due to an increasing lipophilicity, which is a favorable

feature for high-aﬃnity hCB₂R ligands.⁷¹Compounds 4a−e all

show a single-digit micromolar aﬃnity toward hCB₂R.

Experimental data did not support the correlation between

aﬃnity and chain length. The tertiary amide derivative 5, which

is the equivalent of derivative 4c, showed the same aﬃnity

toward hCB₂R but a higher aﬃnity toward hCB₁R, resulting in

low selectivity. Hence, the number of substituents at the amide

group inﬂuences selectivity. Hybrids 6 and 7 with a 2-PEG-

linker, which confers a higher hydrophilicity and rigidity,

showed a weaker aﬃnity toward hCB₂R compared to the

compounds with the longest alkylene linkers (3e and 4e). The

disulﬁde bond in hybrid 8 has no signiﬁcant eﬀect on aﬃnity

toward hCB₂R as the equivalent derivative 4e showed almost the

same aﬃnity (Table 1). Overall, the linkage of the two molecules

has been associated with a loss of aﬃnity at the hCB₂R compared

to the parent molecule 2, but the ligands still retain moderate

aﬃnity and good selectivity at hCB₂R. Since parent molecule 2

shares a high structural similarity with the known μ-opioid

receptor (MOR) ligand etonitazene,⁴⁸we investigated potential

aﬃnity and found a signiﬁcant aﬃnity of hybrids 3a, 4a, and 4e

(see Supplementary Table 1). In our previous work, we were

able to show that an MOR “design out” approach is possible, if

this is required with regard to the side eﬀect proﬁle.⁴⁸

Eﬃcacy at hCB₂R. The hCB₂R is coupled through G_i/0

protein, so adenylate cyclase is inhibited in the case of activation.

This results in a lower level of intracellular cAMP in the case of

agonist binding or to an increased cAMP level in the case of an

inverse agonist binding.⁷²The cAMP increase by forskolin

(FSK) is further enhanced by an antagonist/reverse agonist

binding, while the binding of an agonist reverts the eﬀect.⁷³This

makes the intracellular detection of cAMP levels by immuno-

assays a common method for the characterization of the eﬃcacy

of hCB₂R ligands. Going down the signaling pathway mediated

by a conformational change of the receptor, the resulting

diﬀerence in the cellular cAMP level changes the expression of

cAMP-regulated genes (the reporter). The transcription of these

genes is regulated by the transcription factor cAMP response

element-binding protein (CREB). Binding of CREB to the

cAMP responding element (CRE) is upstreaming gene

expression.⁷⁴A high cAMP level, induced by an antagonist

binding, leads to a high expression of cAMP-regulated genes,

while an agonist leads to the opposite eﬀect. By quantifying the

expression of these genes, it is possible to investigate the

behavior of the ligand at the receptor. An example of a cAMP-

regulated gene is the macrophage migration inhibitory factor

(MIF) with a CRE in the proximal promoter region.⁷⁵A further

example is the signal transducer and activator of transcription

E

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 1. Results of the in Vitro Evaluation of the Inhibitory Eﬀect of AChE/BChE and Radioligand Binding Studies at hCB₁R/

a

hCB₂R

a

b

n.a. activity lower than 10% at the highest tested concentration (10 μM); n.d., not determined. Values are the means of at least three independent

c

d

determinations; hBChE from human serum. Values are the means of at least three independent determinations; recombinant hAChE. Selectivity

index (SI) toward hBChE = IC₅₀(hAChE)/IC₅₀(hBChE). Screened on membranes of HEK cells stably expressing hCB₂R using 10 μM of the

compound; values are mean values from at least two independent experiments, each performed in triplicate. Screened on membranes of CHO cells

e

f

stably expressing hCB₁R using 10 μM of the compound; values are mean values from at least two independent experiments, each performed in

triplicate.

(STAT-3) gene, which expression is also under CRE promoter

control.⁷⁶Both genes are highly expressed in multiple myeloma

cells.^77,78Compounds 3e and 4c were evaluated, as previously

described, at a concentration of 50 μM.⁴⁸In general, before

examining the eﬃcacy of the compounds, an MTT assay was

carried out on U266 myeloma cells to evaluate a nontoxic

concentration of the compounds (see Supplementary Figure 1).

The eﬃcacy of the compounds was investigated by the

quantiﬁcation of the cAMP-regulated MIF and STAT-3 genes

by a qRT-PCR methodology. AM630 was measured as a

F

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 6. AChE-mediated and self-induced Aβ aggregation. Footnote a

denotes inhibition of Aβ₄₂self-aggregation investigated by the

thioﬂavin T ﬂuorescence assay. Assays were carried out in the presence

of 50 μM inhibitor and 50 μM Aβ₄₂([I] = [Aβ₄₂]). Values are expressed

Figure 4. Plot of pIC₅₀values toward hBChE against pIC₅₀values

toward hAChE of all hybrids.

as means

SEM of two independent experiments, and each was

performed in duplicate. Footnote b denotes % inhibition of hAChE-

induced Aβ₄₀aggregation at [I] = 100 μM. The Aβ₄₀/hAChE ratio was

reference antagonist/reverse agonist. Cells were treated with

compound 2 as a reference agonist, 3e and 4c alone, and in

combination with AM630. Compared to the vehicle, com-

pounds 2, 3e, and 4c show a decreased expression of both genes.

Also, in combination experiments, all three compounds show a

decreased level of both genes compared to the levels of only the

AM630-treated cells (see Figure 7 and Supplementary Figure 2).

Furthermore, compounds 3e and 4c were characterized using

a functional ﬂuorescent hCB₂-activated G_αq16-coupled intra-

cellular calcium mobilization assay in CHO-K1 cells over-

expressing the receptor.^79,80Brieﬂy, CHO-K1 cells were

equal to 100:1. Values are expressed as means

SEM of two

independent experiments, and each was performed in duplicate.

engineered to stably co-express hCB1 or hCB2 and G_αq16

.

Activation of hCB₁/CB₂by an agonist then leads to generation

of inositol phosphatase 3 (IP3) and activation of IP3 receptors,

which, in turn, mobilizes intracellular calcium. Compounds 3e

and 4c were identiﬁed as agonists of hCB₂R with an EC₅₀of 911

42 nM for compound 4c and an E_max= 39%. Compound 3e

shows an EC₅₀of 3.05 0.2 μM and an E_max= 51%. Therefore,

Figure 5. Kinetic study on the mechanism of hAChE inhibition by 3e, 4b, and 8. Lineweaver−Burk reciprocal plots of hAChE initial velocity at

increasing substrate (acetylthiocholine, ACth) concentration in the absence of the inhibitor (control) and the presence of increasing concentrations of

the inhibitor are shown. Values are the means of at least three independent determinations.

G

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

the expression of iNOS, TREM2, and TGFβ2 were tested

through Western blot analysis.

Nitrite production due to iNOS induction and IL1β release is

the marker of M1, neurotoxic activated microglia, and induced

by the LPS treatment, while the phagocytic proteins TREM2

and TGFβ2 are both makers of M2, neuroprotective microglia.

As shown in Figure 9, compound 3e strongly reduces in a dose-

dependent way IL1β release and nitrite accumulation, as well as

iNOS expression induced by LPS-mediated activation of

microglial cells, with no parallel change in TREM2 and

TGFβ2 expression, thus indicating an immunomodulatory

eﬀect of the compound, which is similar to compounds 2 and 4a.

Neuroprotective Properties on HT-22 Cells. For the

biological evaluation of compound 8 regarding its eﬀects on

murine hippocampal neurons (HT-22), we performed a

glutamate assay as previously described.^81,82This neuronal cell

line is derived from murine hippocampal tissue and is glutamate

sensitive.⁸³HT-22 cells lack ionotropic glutamate receptors.

The addition of extracellular glutamate at high concentrations

can be used to introduce intracellular ROS accumulation by

blocking the cystine/glutamate antiporter, resulting in gluta-

thione depletion. This neuronal oxidative stress results in cell

injuries and eventually cell death.⁸⁴⁻⁸⁶Before performing the

neuroprotectivity assays, potential neurotoxicity of both

compounds 4e and 8 were evaluated in an MTT assay (see

Figure 10A). Compound 8 showed concentration-dependent

neuroprotectivity starting at 1 μM (see Figure 10C). Treatment

with 5 μM compound 8 leads to neuroprotection comparable to

the positive control quercetin at 25 μM. Compound 4e shows a

similar neuroprotective behavior but decreases viability at 10

μM due to the neurotoxic eﬀect at this dose (see Figure 10B).

The use of cystamine as a linker yielded a hybrid with lower

neurotoxicity compared to the sulfur-free analogue 4e with a

neuroprotective behavior at a comparable low concentration.

Furthermore, compounds 3a, 3d, 3e, 4a, and 4b were also

investigated toward their neurotoxicity and neuroprotective

behavior. In summary, compounds 3a, 3d, and 3e show no

neurotoxicity at the doses tested (max. 10 μM), while 4a is toxic

above 5 μM, and 4b shows ﬁrst toxicity eﬀects at 5 μM.

Compounds 4b and 4a show neuroprotective behavior only at 5

μM, while other compounds tested showed low neuroprotective

behavior (3e) or none (3a and 3d) (see Supplementary Figure

3).

Figure 7. Test compounds (2, 3e, and 4c) reduce MIF and STAT-3

gene expression. U266 cells were treated with hybrid compounds (50

μM), FSK (10 μM), or AM630 (25 μM) for 2 h. In a combined

experiment (AM630 plus test compounds), U266 cells were

preincubated with AM630 for 30 min. Before adding the test

compounds, MIF and STAT-3 mRNA levels were determined by

qRT-PCR. GAPDH was used for normalization. Data are expressed as

relative fold with respect to vehicle-treated cells used as the control.

Values are means of at least two independent experiments, and each was

performed in duplicate. Data are expressed as means SD *p < 0.01 vs

untreated; # vs AM630.

both compounds are partial agonists at hCB₂R. Both

compounds were devoid of activity at hCB₁R. (see Figure 8).

Compounds 3e and 4c are therefore partial agonists at the

hCB₂R as conﬁrmed in two diﬀerent assays. Since in our

previous work, we also found agonist behavior for structurally

related derivatives and because of the high structural similarity of

all the hybrids described, we assume that all hybrids are

agonists.⁴⁸

Eﬀects on Microglia Activation. To test a possible

immunomodulatory eﬀect, that is, the shift from the M1

neurotoxic to the M2 neuroprotective phenotype, of com-

pounds 2, 3e, and 4a, the murine microglial cell line N9 was

exposed to 100 ng/mL lipopolysaccharide (LPS), which induces

an M1 activation state, in the presence or absence of increasing

concentrations (1, 2.5, and 5 μM) of the test compounds. After

24 h of treatment, microglial conditioned media were collected,

partly used for nitrite measurement, and partly concentrated for

Western blot analysis. In parallel, microglial cells were collected,

and the protein content was determined. Accumulation of nitrite

in microglial conditioned media was measured by a colorimetric

assay based on the Griess reaction, while the release of IL1β and

In Vivo Studies. The most promising compounds were

tested regarding their procognitive eﬀects in an in vivo AD

mouse model. To induce AD such as neuroinﬂammation and

Figure 8. Calcium mobilization assay in CHO-K1 cells overexpressing hCB₁or hCB₂and G_αq16.Compounds were tested in each cell type as described

under Experimental Section. Agonism was noted in cells expressing hCB₂(left) but not in cells expressing hCB₁(right). The synthetic full agonist CP

55,040 served as a comparator and positive control. No signiﬁcant signal was noted in vehicle control cells.

H

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 9. Eﬀect of compounds 2, 3e, and 4a on microglial activation in N9 cells previously activated by LPS (100 ng/mL). IL1β release and expression

of iNOS, TREM2, and TGFβ2 were tested through Western blot analysis after 24 h of treatment with LPS in the presence of increasing concentrations

(1, 2.5, and 5 μM) of compounds (A) 2, (B) 3e, and (C) 4a, respectively, and (G−R) quantiﬁed through densitometry. NO release was evaluated

through the Griess reaction in media conditioned for 24 h by microglial cells treated by LPS in the presence of compounds (D) 2, (E) 3e, and (F) 4a,

showing an increase in NO release in media conditioned by LPS-treated cells that are reduced by the co-treatment with the compounds, with the

strongest eﬀect of compound 2. Both (G−I) IL1β release and (J−L) iNOS expression, which are markers of M1 neurotoxic microglia, strongly increase

in LPS-treated cells but signiﬁcantly decrease in cells co-treated with compounds (G, J) 2, (H, K) 3e, and (I, L) 4a in a dose-dependent way, while the

expression of the M2 microglial markers (M−O) TREM2 and (P−R) TGFβ2 is not reduced by co-treatment with the compounds. All quantitative

data are presented as means SEM from at least three independent experiments. Statistical signiﬁcance between diﬀerent treatments was calculated by

using one-way analysis of variance (ANOVA) followed by post hoc comparison through Bonferroni’s test. *p < 0.05; **p < 0.01 compare to control; ^#p

< 0.05; ^##p < 0.01 compare to LPS.

cognitive deﬁcits, oligomerized Aβ₂₅₋₃₅peptide was intra-

cerebroventricularly injected into the mouse brain.^44,87,88

Since the addressed targets are located in the brain, hybrids

must be able to penetrate the BBB. We ﬁrst tested compounds,

with the shortest spacer lengths, 3a and 4a with a low molecular

weight. After the ﬁrst positive results, we also tested compounds

with a more pronounced in vitro proﬁle.

Compounds were injected once daily (o.d.) and intra-

peritoneally (i.p.) from day 1, 20 min after the peptide injection,

until day 7. Compounds were solubilized in pure DMSO at a

concentration of 2 mg/mL and then diluted in water. Final

DMSO concentration in the vehicle solution remained,

however, high (60%), and innocuity was controlled by a daily

observation on the mice behavior and weight gain control. None

of the treatment aﬀected signiﬁcantly the weight gain during the

I

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 10. Compounds 4e and 8 were studied on neuronal HT-22 cells for (A) neurotoxicity eﬀects and (B, C) neuroprotection against glutamate-

induced oxidative stress at 1−25 μM. Results of the modiﬁed MTT test are presented as means SD of three independent experiments, each

performed in sextuplicate, and refer to untreated control cells, which were set as 100% values. Statistical analysis was achieved by applying one-way

ANOVA followed by Dunnett’s multiple comparison post-test. Levels of signiﬁcance: *p < 0.005; **p < 0.01; ***p < 0.001. Treated cells were

compared to (A) untreated cells and (B) cells treated with glutamate only.

Figure 11. Eﬀect of the compounds on Aß₂₅₋₃₅-induced learning impairments in mice: spontaneous alternation performance (upper panels) and

passive avoidance response (lower panels). Mice received Aβ₂₅₋₃₅(9 nmol, i.c.v.) or vehicle solution (3 μL, i.c.v.) on day 1 and then compound (a, b)

3a, (c, d) 3d, (e, f) 3e, (g, h) 4a, or (i, j) 8 in the 0.1−3 mg/kg, i.p. dose range, o.d. between day 1 to 7. Mice were then tested for (a, c, e, g, and i)

spontaneous alternation on day 8 and (b, d, f, h, and j) passive avoidance on days 9 and 10. Data show means SEM (upper panels) or median and

interquartile range (lower panels). ANOVA: F_(4,79)= 3.05, p < 0.05, n = 12−18 in (a); F_(4,62)= 3.77, p > 0.01, n = 11−15 in (c); F_(4,64)= 3.85, p < 0.01, n

= 10−15 in (e); F_(4,67)= 3.14, p < 0.05, n = 11−17 in (g); F_(4,50)= 5.40, p < 0.01, n = 8−12 in (i). Kruskal−Wallis ANOVA: H = 9.87, p < 0.05, n = 12−

15 in (b); H = 17.4, p < 0.01, n = 11−14 in (d); H = 21.9, p < 0.001, n = 12−13 in (f); H = 13.9, p < 0.01, n = 12−17 in (h); H = 11.8, p < 0.05, n = 12−15

in (j). Post hoc: *p < 0.05, **p < 0.01, and ***p < 0.001 vs (V + V)-treated group; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 vs (V + Aß₂₅₋₃₅)-treated

group; Dunnett’s (upper panels) or Dunn’s test (lower panels).

week of treatment. Animals lost up to 1 g after the peptide

injection but then regained regularly between 0.2 and 0.4 g daily

(see Supplementary Figure 4).

Behavioral examination was performed between day 8 and 10

according to the procedure schematized in Supplementary

J

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 2. Results of the Liver Histology Performed with High Dose (3 mg/kg)-Treated Mice

microvesicular steatosis

necrosis

treatment

V/V

Aß/V

Aß/3a (3 mg/kg)

Aß/3d (3 mg/kg)

Aß/4a (3 mg/kg)

Aß/8 (3 mg/kg)

positive/specimen

% of specimen

reported area %

positive/specimen

% of specimen

reported area %

6/10

7/10

8/12

6/6

2/9

8/11

60

58

66

100

22

72

30−40

10−30

5−30

3−45

10−30

20−40

5/10

11/12

8/12

5/6

6/9

8/11

50

91

66

83

66

72

2−30

1−30

2−50

5−15

5−10

5−50

Figure 5. All animals were sacriﬁced on day 11 (see

Supplementary Figure 5).

mm slices. At least two-thirds of the parenchyma was embedded,

including all macroscopically visible abnormalities. The material

underwent standardized dehydration and paraﬃn embedding as

well as hematoxylin−eosin staining after well-established

routine protocols. All slides were assessed by the same

pathologist, completely blinded to the treatment protocols.

The assessment included all typical liver changes seen with drug

toxicity: Necrosis was reported focal, segmental, diﬀuse, or zonal

in percentages and divided between fresh and long-standing

necrosis. Steatosis was reported as macro- or microvesicular,

given in percentages, as diﬀuse or zonal (lobular zones I−III).

Fibrosis was assessed according to stages I to IV after Batts and

Ludwig.⁹⁰Inﬂammation is reported semiquantitatively in a four-

tiered system 0−3 and divided into portal and/or lobular.

Cholestasis was also reported in a four-tiered system 0−3

speciﬁed to intrahepatocytic or canalicular. Cell ballooning was

also stated in percentage. After the assessment, the ﬁndings were

realigned with the treating groups. Microvesicular steatosis was

present in all groups, including controls (6/10) showing the

highest ratio in group 3d (6/6) and the lowest in group 4a (2/

9). Necrosis was diﬀerentiated into fresh necrosis and older

necrosis. The fresh necrosis in these specimen was completed

without reaction, only hours old. It was seen in all groups,

including controls. The highest ratio was observed in the Aß/V

group (11/12) and group 3d (5/6) with nearly all specimen

aﬀected. Older necrosis was reported in two specimen only, both

in group Aß/V. Other ﬁndings, such as discrete lobular

inﬂammation, were seen only in a single specimen, not related

to treatment groups. The results suggest drug-independent

reasons for steatosis and fresh necrosis because they were also

found in the control groups in a high percentage. One possible

cause of the observed liver damage could be the high level of

DMSO used for compound injection.⁹¹No hepatotoxic relation

to compound treatment could be established. Further experi-

ments for long-time toxicity must be performed.

Spontaneous alternation performance, which is an index of

spatial working memory, was tested on day 8 in the Y-maze test.

Long-term memory response was measured on day 10, in a step-

through type passive avoidance task, with retention assessed for

24 h after training. Results conﬁrmed that Aβ₂₅₋₃₅induced

signiﬁcant learning impairments in the behavioral tests.

Moreover, all compounds tested showed signiﬁcant attenuations

of Aβ₂₅₋₃₅-induced learning impairments in both the short-term

and the long-term memory responses (see Figure 11). Eﬀective

doses were slightly lower in the spontaneous alternation test (see

Figure 11, upper panels) than in the passive avoidance test (see

Figure 11, lower panels) and appeared often bell-shaped (see

Figure 11d,f,i,j). Compound 3a signiﬁcantly prevented Aβ₂₅₋₃₅

-

induced learning impairments at doses of 1 mg/kg and higher

(see Figure 11b, 1). All other compounds, namely, 3d, 3e, 4a,

and 8, signiﬁcantly prevented the learning deﬁcits at the lower

dose of 0.3 mg/kg (see Figure 11c−j). Notably, compounds 3e,

4a, and 8 completely prevented Aβ₂₅₋₃₅impairments in both

tests at the most eﬀective doses (resp., 0.3, 1, and 0.3 mg/kg),

outlining the high eﬃciency of the compounds. It should be

noted, at this point, that the hybrids show a signiﬁcantly higher

eﬃcacy compared to the parent molecules 1 and 2, which were

also investigated in vivo in our previous work.^48,87It is possible

that MOR activity of the compounds may marginally contribute

to the neuroprotective eﬀect observed here. However, the only

indirect measure we get from our experiments was that no

signiﬁcant change in pain sensitivity to footshock was measured,

even at the highest drug dose during the passive avoidance

training session. Shock sensitivity was 1.1 0.3 for the group

treated with compound 3a, 3 mg/kg, and 0.9

0.3 for

compound 4a, 1 mg/kg, versus 1.4 0.3 for Veh-treated

animals, suggesting that pain response was not aﬀected by the

compound treatments. Nevertheless, MORs have been shown to

be involved in memory formation, particularly, in the context of

memory formation associated with opiate drug abuse, by

regulating GABAergic inputs and facilitating neuronal excitatory

activity through the hippocampal CA1 area.⁸⁹Therefore, the

agonist (or antagonist) activity of the compounds at MORs and

synergy of MOR and CB₂R activities on memory and

neuroprotection isat least in principlepossible. Indeed,

the compounds tested exhibit a remarkably high in vivo eﬃcacy,

with sub-mg/kg active doses as compared with their low

micromolar aﬃnities in vitro on CB₂Rs or MORs. Their precise

PK/PD proﬁles and any possible synergy between pharmaco-

logical targets should be examined to conﬁrm that the

compounds exhibit a very unique and interesting pharmaco-

logical activity.

CONCLUSIONS

■

In the current study, sets of hybrid compounds combining

tacrine 1 as ChE inhibitor and the hCB₂R agonist 2 were

designed and synthesized using diﬀerent molecular attachment

points in the benzimidazole core, diﬀerent spacer lengths, and

structures. The newly synthesized compounds were ﬁrst

evaluated in a series of experiments to conﬁrm activity on AD-

relevant targets, namely, AChE, BChE, and hCB₂R. The hybrid

compounds showed higher inhibition of ChEs compared to 1.

To follow this up, a kinetic study was performed. The study

showed a mixed-type inhibition by compounds tested and

suggests binding to a second site in ChE leading to higher

inhibition. While 1 has no eﬀect on Aβ aggregation when

assayed at 1/1 ratio, all hybrids tested had an eﬀect. Above all, 3e

showed a pronounced inhibition of self- and AChE-induced Aβ

aggregation. Radioligand binding studies on hCB₂R showed a

Liver Histology. Livers of mice treated with the high dose (3

mg/kg) were dissected out on day 12, after the behavioral

experiments, post-ﬁxed in buﬀered formalin 4%, and cut into 2

K

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

coupling constant in hertz (s⁻¹). The abbreviations of the indicated

signal diversities were s = singlet, d = doublet, t = triplet, q = quartet, and

m = multiplet.

lower aﬃnity from the nanomolar range of 2 to the single-digit

micromolar range for the hybrids. Hybrids 3e and 4c,

representative for the two diﬀerent sets, were investigated by a

cAMP-regulated gene expression experiment, which showed

they maintained their agonist behavior at the hCB₂R. To

investigate the eﬀectiveness, microglial activity was investigated.

Therefore, diﬀerent M1 and M2 markers were quantiﬁed.

Despite the signiﬁcantly lower aﬃnity for the hCB₂R compared

to 2, the compounds showed an immunomodulatory eﬀect

similar to the parent molecule 2. The concept of incorporating a

disulﬁde into the linker to introduce neuroprotection was

investigated in a HT22 cell assay. Both tested compounds, 8 and

the sulfur-free analogue 3e, showed neuroprotection against

glutamate-induced oxidative stress. Due to the promising in

vitro proﬁle, several compounds were tested in vivo and showed

pronounced eﬀects on short- and long-term memory, proving

their ability to cross the BBB. Keeping the hepatotoxicity of 1 in

mind, liver histology of high dose-treated animals (3 mg/kg) was

carried out (Table 2). In ﬁrst-line experiments, the tested

hybrids showed no hepatotoxicity eﬀect, but this must be further

investigated. The high in vivo eﬃcacy of the compounds should

be emphasized, which is signiﬁcantly higher in particular for the

compounds 3e and 8 (0.1 mg/kg), than for the parent molecules

1 and 2, which were investigated in our previous work.^48,87Due

to the signiﬁcantly lower dosage compared to 1, a lower liver-

damaging side eﬀect of the novel hybrids in a potential clinical

application might be assumed. We consider these data also of

interest for other drug discovery eﬀorts with regard to tacrine-

containing experimental therapeutics as well as hybrid molecules

with higher molar mass aiming at CNS activity.

Liquid Chromatography/Mass Spectrometry (LC/MS). Measure-

ments for veriﬁcation and purity of the compounds were performed by

LC/MS (from Shimadzu), comprising a DGU-20A3R controller, pump

LC-20AB, degasser DGU-20A, and SPD-20A UV/Vis detector. ESI

ionization was accomplished by an LCMS-2020 single quadrupol mass

spectrometer. As a stationary phase, for analytical purpose, a Synergi 4U

fusion-RP 80 Å (150 × 4.6 mm) column and for preparative purpose, a

Synergi 4U fusion-RP 80 Å (250 × 10.0 mm) were used. As a mobile

phase, a gradient of MeOH/water (both containing 0.1% formic acid)

(phase A/phase B) was used. The compounds were dissolved in MeOH

and ﬁltered through syringe ﬁlters.

The parameters for the methods are given as follows: ﬂow rate, 1.0

mL/min; UV detection, 254 nm; scan range, 100−800 m/z; gradient:

A, H₂O (0.1% CF₃COOH) and B, MeOH (0.1% CF₃COOH); 0−8

min 5% → 90% B, 8−13 min 90% B, 13−14 min 90% → 10% B, and

14−18 min 10% → 5% B.

Compounds were only used for biological evaluation if the purity was

≥95%.

General Procedures. General Procedure I for Nucleophilic

Substitution of Aryl Chloride by a Diamine (for Compounds 12a−

g). The chloride compound 11 was dissolved in hexanol, and the

corresponding diamine was added. The mixture was stirred for 24 h

under reﬂux conditions under an argon atmosphere. The solvent was

evaporated under high vacuum using an evaporator. The residue was

dissolved in CH₂Cl₂and extracted with water. The organic layer was

dried over anhydrous MgSO₄, and the solvent was removed in vacuo.

General Procedure II for Aromatic Substitution of Fluoride by

Amines (for Compounds 17a−f and 20). The respective ﬂuoride

compound and the respective amine were dissolved in THF, and then

NEt₃was added. The mixture was stirred at room temperature

overnight. The solvent was then removed in vacuo, and the residue was

dissolved in CH₂Cl₂and washed with water. The organic layers were

combined and dried over anhydrous MgSO₄, and the solvent was

removed in vacuo.

General Procedure III for Reduction of an Aromatic Nitro Moiety

with Stannous Chloride Dihydrate (for Compounds 18a−f). The

respective nitro compound was dissolved in ethanol, and SnCl₂·2H₂O

was added. The mixture was stirred under reﬂux conditions under an

argon atmosphere for 5 h. Then, the mixture was basiﬁed with NH_3(aq)

(25%), and the precipitate was ﬁltered oﬀ by suction. The ﬁltrate was

concentrated in vacuo. The residue was dissolved in CH₂Cl₂and

washed with 1M NaOH. The organic layers were combined and dried

over anhydrous MgSO₄, and the solvent was removed in vacuo. The

product was directly used in the next reaction, without further

puriﬁcation, and just characterized by mass spectrometry.

General Procedure IV for Amide Formation with HBTU-Activated

Ester (for Compounds 19a−f and 22). The respective acid was

dissolved in DMF then NEt₃, and HBTU was added. This mixture was

added to a solution of the respective amine in DMF. The mixture was

stirred overnight at room temperature, and the solvent was then

removed in vacuo. The residue was dissolved in CH₂Cl₂and washed

with a saturated NaHCO₃solution. The organic layers were combined

and dried over anhydrous MgSO₄, and the solvent was removed in

vacuo.

General Procedure V for Ring Closure to Benzimidazole (for

Compounds 3a−e, 6, and 23). The respective amide was dissolved in

glacial acetic acid. The mixture was stirred depending on the reaction

progress for 2−6 h under reﬂux conditions and was then concentrated

in vacuo. The residue was basiﬁed with NH_3(aq)(25%) and extracted

with CH₂Cl₂. The organic layers were combined, dried over anhydrous

MgSO₄, and concentrated in vacuo.

EXPERIMENTAL SECTION

■

Chemistry. General Information. Reagents and Solvents. All

reagents were used without further puriﬁcation and bought from

common commercial suppliers. For anhydrous reaction conditions,

THF was dried prior to use by reﬂuxing over sodium slices with

benzophenone as an indicator under an argon atmosphere.

Thin-Layer Chromatography (TLC). To monitor reaction progress,

thin-layer chromatography (precoated plates with silica gel, from

Machery-Nagel: ALUGRAM Xtra SIL G/UV₂₅₄) was carried out. The

spots were visualized by UV light (254 and 366 nm), by staining in the

iodine chamber, orby using spray reagents (Ehrlich’s reagent: primary

amines turn red). The eluent systems are indicated in volume (v) ratios

of the respective solvents (v/v).

Preparative Thin-Layer Chromatography. Silica gel (Merck KGaA:

Silica gel 60 GF₂₅₄) was mixed with water. Glass plates were coated

evenly with the mixture and dried at room temperature. The compound

was dissolved in CH₂Cl₂and applied to the starting line in small drops.

The plate was then transferred to a preparative TLC chamber with the

mobile phase. After successful separation, the plate was dried, and the

product was detected under UV light (254 and 366 nm), scraped oﬀ,

and then extracted with MeOH.

Column Chromatography. Silica gel (particle size, 0.063−0.2 mm;

Merck) was used for column chromatography. The eluent systems are

indicated in volume (v) ratios of the respective solvents (v/v). Column

chromatography was also performed using an Interchim Puri Flash 430

(Ultra Performance Flash Puriﬁcation) instrument (Montluc

France) connected to an Interchim Flash ELSD. Used columns were

Silica 25 g (30 μm) (Interchim, Montlucon, France).

̧on,

̧

Nuclear Magnetic Resonance Spectroscopy (NMR). Measurements

Synthesis. 2-(4-Ethoxybenzyl)-N,N-diethyl-1-(2-((1,2,3,4-tetrahy-

droacridin-9-yl)amino)ethyl)-1H-benzo[d]imidazole-5-carboxa-

mide (3a). The reaction was carried out according to general procedure

V using 3-(2-(4-ethoxyphenyl)acetamido)-N,N-diethyl-4-((2-

((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)amino)benzamide 19a

(258 mg, 0.43 mmol). The crude product was puriﬁed by column

chromatography (20:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)), and

of NMR spectra were performed on a Bruker Advance 400 (¹H NMR:

1

400 MHz; ¹³C NMR: 101 MHz). H and ¹³C NMR spectra were

calibrated with the hydrogen signal of the respective solvent as the

internal standard. In this work, CDCl₃, CD₃OD, and D₂O were used as

solvents (¹H: CDCl₃, 7.26 ppm; ¹H: CD₃OD, 3.31 ppm; ¹H: D₂O, 4.79

ppm; ¹³C: CDCl₃, 77.16 ppm; ¹³C: CD₃OD, 49.0 ppm). J is the

L

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

product 3a (210 mg, 0.36 mmol, 84%) was obtained as a yellow oil. ¹H

NMR (400 MHz, CDCl₃): δ 7.86 (d, J = 8.2 Hz, 1H), 7.70 (s, 1H), 7.57

(d, J = 8.3 Hz, 1H), 7.51−7.45 (m, 1H), 7.24−7.15 (m, 2H), 7.11 (d, J

= 8.3 Hz, 1H), 6.90 (d, J = 8.6 Hz, 2H), 6.66−6.61 (m, 2H), 4.13 (t, J =

6.6 Hz, 2H), 4.04 (s, 2H), 3.88 (s, 1H), 3.82 (q, J = 7.0 Hz, 2H), 3.56−

3.23 (m, 6H), 2.96 (t, J = 6.4 Hz, 2H), 2.31 (t, J = 6.3 Hz, 2H), 1.83−

1.75 (m, 2H), 1.74−1.66 (m, 2H), 1.27 (t, J = 7.0 Hz, 3H), 1.15 (s, 6H)

ppm. ¹³C NMR (101 MHz, CDCl₃): δ 171.7, 158.8, 158.2, 154.9, 149.2,

147.2, 142.2, 135.9, 131.6, 129.4, 128.9, 128.7, 127.7, 124.6, 122.0,

121.7, 120.6, 118.0, 117.9, 114.9, 109.5, 63.5, 47.3, 44.5, 34.0, 33.8,

24.8, 22.9, 22.7, 14.9, 14.2 ppm. ESI: m/z calcd for C₃₆H₄₂N₅O₂[M +

H]⁺, 576.33; found, 576.25; retention time, 7.90 min; HPLC purity,

97%.

2-(4-Ethoxybenzyl)-N,N-diethyl-1-(3-((1,2,3,4-tetrahydroacridin-

9-yl)amino)propyl)-1H-benzo[d]imidazole-5-carboxamide (3b).

The reaction was carried out according to general procedure V using

3-(2-(4-ethoxyphenyl)acetamido)-N,N-diethyl-4-((3-((1,2,3,4-tetra-

hydroacridin-9-yl)amino)propyl)amino)benzamide 19b (104 mg, 0.17

mmol). The crude product was puriﬁed by preparative thin-layer

chromatography (10:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)), and

product 3b (23 mg, 39.0 μmol, 23%) was obtained as a yellow oil. ¹H

NMR (400 MHz, CDCl₃): δ 8.16 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 8.0

Hz, 1H), 7.74 (s, 1H), 7.56−7.49 (m, 2H), 7.27−7.22 (m, 2H),7.15 (d,

J = 7.9 Hz, 2H), 6.62 (d, J = 8.3 Hz, 2H), 4.43 (s, 2H), 4.00−3.92 (m,

2H), 3.77 (q, J = 6.9 Hz, 2H), 3.57−3.48 (m, 2H), 3.40−3.13 (m, 4H),

3.08−3.03 (m, 2H), 2.59−2.51 (m, 2H), 2.20−2.12 (m, 2H), 1.75−

1.71 (m, 4H), 1.29−1.17 (m, 8H) ppm. ¹³C NMR (101 MHz, CDCl₃):

δ 171.0, 158.4, 156.7, 154.2, 151.0, 145.9, 142.3, 134.2, 132.2, 129.8,

128.4, 128.1, 127.5, 124.5, 122.4, 121.8, 119.7, 117.7, 115.9, 115.0,

111.7, 63.5, 46.0, 42.8, 33.9, 32.0, 29.8, 24.8, 22.4, 22.1, 14.9, 14.2 ppm.

ESI: m/z calcd for C₃₇H₄₅N₅O₂[M + 2H]²⁺, 295.68; found, 295.60;

retention time, 7.82 min; HPLC purity, 98%.

2-(4-Ethoxybenzyl)-N,N-diethyl-1-(4-((1,2,3,4-tetrahydroacridin-

9-yl)amino)butyl)-1H-benzo[d]imidazole-5-carboxamide (3c). The

reaction was carried out according to general procedure V using 3-(2-

(4-ethoxyphenyl)acetamido)-N,N-diethyl-4-((4-((1,2,3,4-tetrahydroa-

cridin-9-yl)amino)butyl)amino)benzamide 19c (166 mg, 0.27 mmol).

The crude product was puriﬁed by column chromatography (10:1:0.1

CH₂Cl₂/MeOH/NH_3(aq)(25%)), and product 3c (46 mg, 76.2 μmol,

28%) was obtained as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 7.90

(d, J = 8.3 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.75 (s, 1H), 7.55 (t, J =

11.1, 4.0 Hz, 1H), 7.34 (t, J = 7.3 Hz, 1H), 7.29−7.26 (m, 1H), 7.18 (d,

J = 8.3 Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.76 (d, J = 8.6 Hz, 2H), 4.20

(s, 2H), 3.97 (t, J = 7.1 Hz, 2H), 3.87 (q, J = 7.0 Hz, 2H), 3.64−3.36 (m,

4H), 3.30 (t, J = 6.7 Hz, 2H), 3.05 (t, J = 5.8 Hz, 2H), 2.62 (t, J = 5.7 Hz,

2H), 1.92−1.83 (m, 4H), 1.61−1.49 (m, 4H), 1.30 (t, J = 7.0 Hz, 3H),

1.26−1.19 (m, 6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 171.8, 158.7,

157.2, 154.5, 150.3, 147.5, 142.2, 135.9, 131.3, 129.5, 128.9, 128.5,

127.9, 124.1, 122.5, 121.5, 120.6, 117.8, 116.9, 115.9, 109.5, 63.5, 48.7,

43.9, 33.9, 29.8, 28.9, 27.0, 25.0, 23.1, 22.9, 14.8, 14.2 ppm. ESI: m/z

calcd for C₃₈H₄₇N₅O₂[M + 2H]²⁺, 302.69; found, 302.65; retention

time, 7.92 min; HPLC purity, 98%.

21.3, 14.8, 14.1 ppm. ESI: m/z calcd for C₃₉H₄₉N₅O₂[M + 2H]²⁺,

309.70; found, 309.65; retention time, 8.07 min; HPLC purity, 99%.

2-(4-Ethoxybenzyl)-N,N-diethyl-1-(6-((1,2,3,4-tetrahydroacridin-

9-yl)amino)hexyl)-1H-benzo[d]imidazole-5-carboxamide (3e). The

reaction was carried out according to general procedure V using 3-(2-

(4-ethoxyphenyl)acetamido)-N,N-diethyl-4-((6-((1,2,3,4-tetrahydroa-

cridin-9-yl)amino)hexyl)amino)benzamide 19e (142 mg, 0.22 mmol).

The crude product was puriﬁed by preparative thin-layer chromatog-

raphy (10:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)), and product 3e (70

mg, 0.11 mmol, 50%) was obtained as a yellow oil. ¹H NMR (400 MHz,

CDCl₃): δ 7.96−7.90 (m, 2H), 7.75 (s, 1H), 7.55 (t, J = 8.1, Hz, 1H),

7.33 (t, J = 9.2 Hz 1H), 7.30−7.27 (m, 1H), 7.25−7.22 (m, 1H), 7.11

(d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 4.23 (s, 2H), 3.98−3.90

(m, 4H), 3.61−3.33 (m, 6H), 3.09−3.03 (m, 2H), 2.70−2.64 (m, 2H),

1.92−1.86 (m, 4H), 1.59−1.47 (m, 4H), 1.33 (t, J = 7.0 Hz, 3H), 1.30−

1.13 (m, 10H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 171.9, 158.2,

154.5, 151.1, 146.9, 142.19, 136.0, 131.2, 129.6, 128.7, 128.3, 128.1,

125.4, 123.9, 122.9, 121.5, 120.1, 117.7, 155.8, 115.0, 109.6, 63.6, 49.3,

44.1, 33.9, 33.7, 31.6, 29.5, 26.8, 26.7, 24.9, 23.1, 22.7, 14.9, 14.3 ppm.

ESI: m/z calcd for C₄₀H₅₁N₅O₂[M + 2H]²⁺, 316.70; found, 316.90;

retention time, 8.27 min; HPLC purity, 95%.

2-(4-Ethoxybenzyl)-1-isopentyl-N-(2-((1,2,3,4-tetrahydroacridin-

9-yl)amino)ethyl)-1H-benzo[d]imidazole-5-carboxamide (4a). The

reaction was carried out according to general procedure V using 2-(4-

ethoxybenzyl)-1-isopentyl-1H-benzo[d]imidazole-5-carboxylic acid 24

(271 mg, 0.74 mmol), N¹-(1,2,3,4-tetrahydroacridin-9-yl)ethane-1,2-

diamine 12a (179 mg, 0.74 mmol), HBTU (281 mg, 0.74 mmol), and

NEt₃(145 μL, 1.05 mmol). The crude product was puriﬁed by

preparative thin-layer chromatography (10:1:0.1 CH₂Cl₂/MeOH/

NH_3(aq)(25%)), and product 4a (35.0 mg, 59.3 μmol, 8%) was

obtained as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 8.24 (s, 1H),

8.13 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 8.6 Hz,

1H), 7.44 (t, J = 7.7 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.30−7.24 (m,

2H), 7.06 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 4.21 (s, 2H),

4.04−3.99 (m, 4H), 3.92−3.83 (m, 4H), 2.83−2.79 (m, 2H), 2.69−

2.62 (m, 2H), 1.85−1.71 (m, J = 25.6, 5.6 Hz, 4H), 1.63−1.52 (m, 1H),

1.44−1.39 (m, 2H), 1.34 (t, J = 7.0 Hz, 3H), 0.90 (d, J = 6.6 Hz, 6H)

ppm. ¹³C NMR (101 MHz, CDCl₃): δ 168.8, 158.2, 155.3, 155.0, 152.3,

147.8, 142.1, 138.0, 130.6, 129.7, 128.5, 127.5, 125.5, 124.7, 122.1,

120.0, 118.7, 117.1, 115.1, 114.9, 109.8, 63.6, 44.7, 42.5, 40.6, 38.3,

33.7, 31.5, 29.8, 26.3, 24.2, 22.5, 21.3, 14.9 ppm. ESI: m/z calcd for

C₃₇H₄₅N₅O₂[M + 2H]²⁺: 295.68; found: 295.75; retention time: 8.96

min; HPLC purity, 96%.

2-(4-Ethoxybenzyl)-1-isopentyl-N-(3-((1,2,3,4-tetrahydroacridin-

9-yl)amino)propyl)-1H-benzo[d]imidazole-5-carboxamide (4b).

The reaction was carried out according to general procedure V using

2-(4-ethoxybenzyl)-1-isopentyl-1H-benzo[d]imidazole-5-carboxylic

acid 24 (370 mg, 1.01 mmol), N¹-(1,2,3,4-tetrahydroacridin-9-

yl)propane-1,3-diamine 12b (200 mg, 0.78 mmol), HBTU (326 mg,

1.33 mmol), and NEt₃(184 μL, 1.33 mmol). The crude product was

puriﬁed by column chromatography (20:1:0.1 CH₂Cl₂/MeOH/

NH_3(aq)(25%)), and product 4b (67.0 mg, 0.11 mmol, 14%) was

obtained as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 8.37 (s, 1H),

8.24 (d, J = 8.7 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 8.5 Hz,

1H), 7.54 (t, J = 7.7 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.25 (d, J = 10.0

Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 4.19 (s, 2H),

3.97−3.88 (m, 6H), 3.71−3.65 (m, 2H), 3.14−3.09 (m, 2H), 2.75 (t, J

= 5.8 Hz, 2H), 2.07−2.00 (m, 2H), 1.85−1.74 (m, 4H), 1.57−1.48 (m,

1H), 1.35 (t, J = 7.0 Hz, 3H), 1.33−1.24 (m, 2H), 0.87 (d, J = 6.6 Hz,

6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 169.4, 158.2, 155.5, 155.0,

152.0, 148.3, 142.3, 137.7, 131.6 129.6, 128.1, 127.8, 125.0, 124.2,

122.2, 119.1, 117.8, 116.6, 115.6, 114.9, 109.4, 63.6, 44.3, 42.8, 38.2,

36.2, 36.7, 33.8, 31.2, 29.3, 26.2, 24.6, 22.5, 21.1, 14.9 ppm. ESI: m/z

calcd for C₃₈H₄₇N₅O₂[M + 2H]²⁺, 302.69; found, 302.75; retention

time, 8.82 min; HPLC purity, 98%.

2-(4-Ethoxybenzyl)-N,N-diethyl-1-(5-((1,2,3,4-tetrahydroacridin-

9-yl)amino)pentyl)-1H-benzo[d]imidazole-5-carboxamide (3d).

The reaction was carried out according to general procedure V using

3-(2-(4-ethoxyphenyl)acetamido)-N,N-diethyl-4-((5-((1,2,3,4-tetra-

hydroacridin-9-yl)amino)pentyl)amino)benzamide 19d (346 mg, 0.47

mmol). The crude product was puriﬁed by column chromatography

(10:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)), and product 3d (22 mg,

1

35.6 μmol, 51%) was obtained as a yellow oil. H NMR (400 MHz,

CDCl₃): δ 8.05−7.98 (m, 2H), 7.72 (s, 1H), 7.56 (t, J = 7.6 Hz, 1H),

7.33 (t, J = 7.7 Hz, 1H), 7.25−7.19 (m, 2H), 7.11 (d, J = 8.5 Hz, 2H),

6.78 (d, J = 8.5 Hz, 2H), 4.22 (s, 2H), 3.98 (t, J = 7.2 Hz, 2H), 3.92 (q, J

= 7.0 Hz, 2H), 3.61 (t, J = 7.1 Hz, 2H), 3.56−3.32 (m, 4H), 3.06−3.01

(m, 2H), 2.55−2.49 (m, 2H), 1.83−1.75 (m, 4H), 1.67−1.52 (m, 4H),

1.32 (t, J = 7.0 Hz, 3H), 1.28−1.13 (m, 8H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 172.0, 158.2, 154.7, 154.7, 153.4, 142.1, 141.1, 135.9, 131.1,

131.1, 129.6, 128.0, 124.7, 123.9, 122.7, 121.4, 117.6, 117.3, 115.0,

112.4, 109.7, 63.6, 48.1, 44.0, 33.8, 31.1, 26.7, 29.2, 24.5, 24.2, 22.3,

2-(4-Ethoxybenzyl)-1-isopentyl-N-(4-((1,2,3,4-tetrahydroacridin-

9-yl)amino)butyl)-1H-benzo[d]imidazole-5-carboxamide (4c). The

reaction was carried out according to general procedure V using 2-(4-

ethoxybenzyl)-1-isopentyl-1H-benzo[d]imidazole-5-carboxylic acid 24

(286 mg, 0.78 mmol), N¹-(1,2,3,4-tetrahydroacridin-9-yl)butane-1,4-

M

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

diamine 12c (161 mg, 0.60 mmol), HBTU (250 mg, 0.66 mmol), and

NEt₃(141 μL, 1.02 mmol). The crude product was puriﬁed by

preparative thin-layer chromatography (10:1:0.1 CH₂Cl₂/MeOH/

NH_3(aq)(25%)), and product 4c (38.0 mg, 0.62 mmol, 10%) was

obtained as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 8.26 (s, 1H),

8.18 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.5, 1H),

7.51 (t, J = 7.6, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H),

7.06 (d, J = 8.6 Hz, 2H), 6.72 (d, J = 8.4 Hz, 2H), 4.18 (s, 2H), 3.96−

3.88 (m, 6H), 3.56−3.49 (m, 2H), 3.03 (t, J = 5.9 Hz, 2H), 2.60 (t, J =

5.7 Hz, 2H), 1.96−1.88 (m, 2H), 1.81−1.70 (m, 6H), 1.56−1.48 (m,

1H), 1.34 (t, J = 7.0 Hz, 3H), 1.31−1.23 (m, 2H), 0.86 (d, J = 6.6 Hz,

6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 168.4, 158.2, 155.8, 154.9,

153.2, 142.9, 142.1, 137.5, 132.2, 129.6, 128.6, 127.7, 125.1, 124.7,

122.1, 120.5, 118.6, 116.5, 116.0, 114.9, 109.4, 63.6, 47.9, 42.8, 39.3,

38.3, 33.7, 29.8, 28.6, 28.1, 26.8, 26.2, 23.9, 22.4, 20.8, 14.9 ppm. ESI:

m/z calcd for C₃₉H₄₉N₅O₂[M + 2H]²⁺, 309.70; found, 309.70;

retention time, 8.77 min; HPLC purity, 98%.

2-(4-Ethoxybenzyl)-1-isopentyl-N-(5-((1,2,3,4-tetrahydroacridin-

9-yl)amino)pentyl)-1H-benzo[d]imidazole-5-carboxamide (4d).

The reaction was carried out according to general procedure V using

2-(4-ethoxybenzyl)-1-isopentyl-1H-benzo[d]imidazole-5-carboxylic

acid 24 (286 mg, 0.78 mmol), N¹-(1,2,3,4-tetrahydroacridin-9-

yl)pentane-1,5-diamine 12d (170 mg, 0.60 mmol), HBTU (250 mg,

0.66 mmol), and NEt₃(141 μL, 1.02 mmol). The crude product was

puriﬁed by column chromatography (20:1:0.1 CH₂Cl₂/MeOH/

NH_3(aq)(25%)), and product 4d (57.0 mg, 0.32 mmol, 15%) was

obtained as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 8.15 (d, J = 8.7

Hz, 1H), 8.04 (s, 1H), 7.85 (d, J = 8.5, 1H), 7.82−7.77 (m, 1H), 7.60 (t,

J = 7.3 Hz, 1H), 7.40−7.34 (m, 2H), 7.08 (d, J = 8.1 Hz, 2H), 6.67 (d, J

= 7.0 Hz, 2H), 4.28 (s, 2H), 4.10−4.04 (m, 2H), 3.91−3.84 (m, 4H),

3.49−3.45 (m, 2H), 2.89−2.85 (m, 2H), 2.43−2.38 (m, 2H), 1.90−

1.85 (m, 2H), 1.65−1.58 (m, 2H), 1.60−1.56 (m, 4H), 1.46−1.43 (m,

1H), 1.32 (t, J = 7.0 Hz, 4H), 1.26−1.25 (m, 5H), 0.94 (d, J = 6.8 Hz,

6H) ppm.¹³C NMR (101 MHz, CDCl₃): δ 169.5, 158.2, 155.5, 154.2,

153.6, 143.3, 142.3, 137.4, 130.7, 129.8, 128.5, 127.4, 125.2, 124.2,

122.5, 119.7, 118.0, 117.3, 115.8, 114.9, 109.9, 63.6, 47.5, 43.0, 39.2,

38.3, 33.1, 29.8, 29.0, 28.2, 27.9, 26.8, 26.3, 23.6, 22.5, 20.4, 14.9 ppm.

ESI: m/z calcd for C₄₀H₅₁N₅O₂[M + 2H]²⁺, 316.70; found, 316.75;

retention time, 8.82 min; HPLC purity, 98%.

2-(4-Ethoxybenzyl)-1-isopentyl-N-(6-((1,2,3,4-tetrahydroacridin-

9-yl)amino)hexyl)-1H-benzo[d]imidazole-5-carboxamide (4e). The

reaction was carried out according to general procedure V using 2-(4-

ethoxybenzyl)-1-isopentyl-1H-benzo[d]imidazole-5-carboxylic acid 24

(114 mg, 0.31 mmol), N¹-(1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-

diamine 12e (92.2 mg, 0.31 mmol), HBTU (118 mg, 0.31 mmol), and

NEt₃(65.1 μL, 0.47 mmol). The crude product was puriﬁed by column

chromatography (20:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)), and

product 4e (40.0 mg, 61.9 μmol, 20%) was obtained as a yellow oil.

¹H NMR (400 MHz, CDCl₃): δ 8.11 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H),

7.78−7.75 (m, 2H), 7.55−7.50 (m, 1H), 7.37−7.32 (m, 1H), 7.29−

7.26 (m, 1H), 7.08 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 4.21 (s,

2H), 4.02−3.86 (m, 6H), 3.64 (t, J = 6.6 Hz, 2H), 3.47−3.40 (m, 2H),

2.62 (t, J = 5.7 Hz, 2H), 1.87−1.80 (m, 4H), 1.75−1.68 (m, 2H), 1.65−

1.59 (m, 2H), 1.56−1.51 (m, 1H), 1.42−1.32 (m, 9H), 0.88 (d, J = 6.6

Hz, 6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 168.3, 158.2, 155.2,

154.7, 153.4, 143.3, 142.2, 137.5, 130.5, 129.6, 128.9, 127.7, 124.4,

124.0, 122.0, 118,1, 118.0, 117,0, 115,1, 114,9, 109.6, 63.6, 48.9, 42.8,

42.5, 39.9, 38.2, 36.6, 33.8, 31.3, 31.1, 29.5, 26.4, 26.2, 24.1, 22.4, 21.8,

14.9 ppm. ESI: m/z calcd for C₄₁H₅₃N₅O₂[M + 2H]²⁺, 323.71; found,

323.80; retention time, 8.94 min; HPLC purity, 95%.

product 5 (21 mg, 32.5 μmol, 60%) was obtained as a brown oil. H

NMR (400 MHz, CDCl₃): δ 7.89 (d, J = 8.1 Hz, 1H), 7.75−7.73 (s,

1H), 7.56−7.51 (m, 1H), 7.36−7.30 (m, 1H), 7.27−7.23 (m, 3H), 7.14

(d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 4.24 (s, 2H), 4.02−3.95

(m, 4H), 3.58−3.42 (m, 4H), 3.08−3.03 (m, 2H), 2.77−2.69 (m, 2H),

1.94−1.88 (m, 4H), 1.83−1.65 (m, 6H), 1.59−1.51 (m, 1H), 1.41−

1.35 (m, 6H), 1.29−1.23 (m, 2H), 0.90 (d, J = 6.6 Hz, 6H) ppm. ¹³C

NMR (101 MHz, CDCl₃): δ 168.4, 158.2, 155.4, 154.8, 153.7, 142.2,

142.2, 137.5, 130.5, 129.6, 128.9, 127.9, 125.0, 124.0, 121.3, 119.9,

117.8, 116.9, 115.2, 115.0, 109.7, 63.6, 52.4, 48.0, 44.1, 42.8, 41.1, 38.3,

33.9, 30.5, 28.3, 26.3, 24.2, 22.5, 22.3, 21.2, 14.9, 14.2. ppm. ESI: m/z

calcd for C₄₁H₅₂N₅O₂[M + 1H]¹⁺, 646.41; found, 646.25; retention

time 8.94 min; HPLC purity, 99%.

2-(4-Ethoxybenzyl)-N,N-diethyl-1-(2-(2-(2-((1,2,3,4-tetrahydroa-

cridin-9-yl)amino)ethoxy)ethoxy)ethyl)-1H-benzo[d]imidazole-5-

carboxamide (6). The reaction was carried out according to general

procedure V using 3-(2-(4-ethoxyphenyl)acetamido)-N,N-diethyl-4-

((2-(2-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethoxy)ethoxy)-

ethyl)amino)benzamide 19f (398 mg, 0.58 mmol). The crude product

was puriﬁed by column chromatography (CH₂Cl₂/MeOH/NH_3(aq)

(25%) 16:1:0.1), and product 6 (220 mg, 33.1 mmol, 57%) was

obtained as a brown oil. ¹H NMR (400 MHz, CDCl₃): δ 7.95−7.89 (m,

2H), 7.68 (s, 1H), 7.50 (t, J = 6.9 Hz, 1H), 7.31−7.27 (m, 1H), 7.22 (d,

J = 8.3, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H), 4.25 (s,

2H), 4.13 (t, J = 5.4 Hz, 2H), 3.90 (q, J = 7.0 Hz, 2H), 3.58 (t, J = 5.1

Hz, 2H), 3.51 (t, J = 5.4 Hz, 2H), 3.46−3.41 (m, 4H), 3.54−3.32 (m,

4H), 3.39−3.35 (m, 2H), 3.02 (t, J = 6.0 Hz, 2H), 2.62 (t, J = 5.9 Hz,

2H), 1.86−1.75 (m, 4H), 1.31 (t, J = 7.0 Hz, 3H), 1.21−1.06 (m, 6H)

ppm. ¹³C NMR (101 MHz, CDCl₃): δ 171.7, 158.0, 157.1, 155.3, 151.6,

145.5, 142.0, 135.1, 131.1, 129.5, 129.1, 127.9, 126.9, 124.1, 123.0,

122.2, 119.7, 117.4, 116.4, 114.8, 109.8, 70.8, 70.3, 69.4, 63.4, 48.2,

47.9, 44.1, 33.5, 32.6, 24.4, 22.7, 22.3, 14.8, 14.1 ppm. ESI: m/z calcd for

C₄₀H₅₁N₅O₄[M + 2H]²⁺, 332.70; found, 332.70; retention time, 7.89

min; HPLC purity, 99%.

2-(4-Ethoxybenzyl)-1-isopentyl-N-(2-(2-(2-((1,2,3,4-tetrahydroa-

cridin-9-yl)amino)ethoxy)ethoxy)ethyl)-1H-benzo[d]imidazole-5-

carboxamide (7). The reaction was carried out according to general

procedure V using 2-(4-ethoxybenzyl)-1-isopentyl-1H-benzo[d]-

imidazole-5-carboxylic acid 24 (40 mg, 0.11 mmol), N-(2-(2-(2-

aminoethoxy)ethoxy)ethyl)-1,2,3,4-tetrahydroacridin-9-amine 12f

(36.2 mg, 0.11 mmol), HBTU (41.7 mg, 0.11 mmol), and NEt₃

(23.6 μL, 0.17 mmol). The crude product was puriﬁed by column

chromatography (16:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)), and

product 7 (33.0 mg 48.7 μmol, 44%) was obtained as a brown oil. ¹H

NMR (400 MHz, CDCl₃): δ 8.08 (s, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.94

(d, J = 8.4 Hz, 1H), 7.72 (d, J = 8.5, 1H), 7.56−7.52 (m, 1H), 7.32 (t, J =

8.0, 1H), 7.15 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 6.76 (d, J =

8.7 Hz, 2H), 4.17 (s, 2H), 3.95−3.90 (m, 4H), 3.85−3.81 (m, 2H),

3.72−3.67 (m, 10H), 3.02−2.98 (m, 2H), 2.58 (t, J = 7.8 Hz, 2H),

1.85−1.78 (m, 4H), 1.56−1.49 (m, 1H), 1.35 (t, J = 7.0 Hz, 3H), 1.30−

1.27 (m, 2H), 0.86 (d, J = 6.6 Hz, 6H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 168.0, 158.2, 155.2, 154.95, 153.3, 143.0, 142.1, 137.5,

130.5, 129.7, 128.5, 127.7, 124.6, 123.7, 122.1, 118.4, 117.9, 117.0,

114.7, 114.9, 109.5, 70.4, 70.3, 70.0, 69.9, 63.6, 48.1, 42.8, 39.8, 38.2,

33.8, 31.2, 29.8, 26.2, 23.9, 22.4, 21.8, 14.9 ppm. ESI: m/z calcd for

C₄₁H₅₃N₅O₄[M + 2H]²⁺, 339.80; found, 339.70; retention time, 8.75

min; HPLC purity, 98%.

2-(4-Ethoxybenzyl)-1-isopentyl-N-(2-((2-((1,2,3,4-tetrahydroacri-

din-9-yl)amino)ethyl)disulfaneyl)ethyl)-1H-benzo[d]imidazole-5-

carboxamide (8). The reaction was carried out according to general

procedure V using 2-(4-ethoxybenzyl)-1-isopentyl-1H-benzo[d]-

imidazole-5-carboxylic acid 24 (48 mg, 0.13 mmol), N-(2-((2-

aminoethyl)disulfaneyl)ethyl)-1,2,3,4-tetrahydroacridin-9-amine (35

mg, 0.11 mmol), HBTU (49 mg, 0.13 mmol), and NEt₃(26 μL, 0.19

mmol). The crude product was puriﬁed by preparative thin-layer

chromatography (18:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)), and

2-(4-Ethoxybenzyl)-N-ethyl-1-isopentyl-N-(4-((1,2,3,4-tetrahy-

droacridin-9-yl)amino)butyl)-1H-benzo[d]imidazole-5-carboxa-

mide (5). The reaction was carried out according to general procedure

V using 2-(4-ethoxybenzyl)-1-isopentyl-1H-benzo[d]imidazole-5-car-

boxylic acid 24 (23.7 mg, 64.6 μmol), N¹-ethyl-N⁴-(1,2,3,4-

tetrahydroacridin-9-yl)butane-1,4-diamine 14 (16.0 mg, 53.8 μmol),

HBTU (24.5 mg, 64.6 μmol), and NEt₃(14.9 μL, 0.11 mmol). The

solvent was removed in vacuo, and the residue was dissolved in CH₂Cl₂

and washed with NaHCO_3(aq). The crude product was puriﬁed by

preparative TLC (20:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)). The

1

product 8 (35 mg, 51.4 μmol, 47%) was obtained as a yellow oil. H

NMR (400 MHz, CDCl₃): δ = 8.15 (d, J = 1.3 Hz, 1H), 8.00−7.96 (m,

1H), 7.84 (dd, J = 8.5, 0.8 Hz, 1H), 7.77 (dd, J = 8.5, 1.6 Hz, 1H), 7.53−

7.50 (m, 1H), 7.35−7.32 (m, 1H), 7.29−7.25 (m, 1H), 7.13−7.08 (m,

N

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2H), 6.86 (t, J = 5.8 Hz, 1H), 6.80−6.76 (m, 2H), 4.23 (s, 2H), 4.01−

3.92 (m, 4H), 3.89−3.82 (m, 2H), 3.75 (q, J = 6.3 Hz, 2H), 3.04−2.96

(m, 2H), 2.95−2.86 (m, 4H), 2.71 (s, 2H), 1.86 (t, J = 2.8 Hz, 4H),

1.62−1.50 (m, 1H), 1.42−1.33 (m, 5H), 0.88 (d, J = 5.3 Hz, 6H) ppm.

¹³C NMR (101 MHz, CDCl₃): δ 168.3, 158.2, 157.4, 155.3, 151.2,

145.8, 142.3, 137.7, 129.6, 129.4, 128.4, 127.7, 127.1, 124.4, 123.1,

122.1, 119.9, 118.2, 116.6, 115.0, 109.6, 63.6, 46.9, 42.9, 39.0, 38.9,

38.2, 37.9, 33.8, 33.0, 26.3, 24.8, 22.8, 22.5, 14.9 ppm. ESI: m/z calcd for

C₃₉H₄₉N₅O₂S₂[M + 2H]²⁺, 341.66; found, 341.70; retention time, 9.38

min; HPLC purity, 96%.

3.02−2.98 (m, 2H), 2.96−2.89 (m, 2H), 2.66−2.60 (m, 4H), 1.62−

1.39 (m, 6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 158.5, 150.6,

146.6, 128.6, 128.2, 123.5, 122.7, 120.3, 115.9, 49.3, 41.9, 33.2, 32.8,

31.7, 27.4, 24.8, 23.0, 22.6 ppm. ESI: m/z calcd for C₁₈H₂₆N₃[M + H]⁺,

284.21; found, 284.15; retention time, 4.66 min.

N¹-(1,2,3,4-Tetrahydroacridin-9-yl)hexane-1,6-diamine (12e).

The reaction was carried out according to general procedure I using

9-chloro-1,2,3,4-tetrahydroacridine 11 (500 mg, 2.30 mmol) and

hexane-1,6-diamine (1.50 mL, 11.5 mmol). The product 12e was

obtained as a brown oil (343 mg, 1.15 mmol, 50%).¹H NMR (400

MHz, CDCl₃): δ 7.94 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.53

(t, J = 6.8 Hz, 1H), 7.32 (t, J = 6.8 Hz, 1H), 3.47 (t, J = 7.2 Hz, 2H), 3.04

(t, J = 6.5 Hz, 2H), 2.72−2.65 (m, 4H), 1.92−1.88 (m, 4H), 1.69−1.60

(m, 2H), 1.46−1.35 (m, 6H) ppm.¹³C NMR (101 MHz, CDCl₃): δ

158.8, 151.0, 147.74, 129.0, 128.8, 124.1, 123.3, 120.6, 116.2, 49.9, 42.5,

34.4, 34.0, 32.2, 27.3, 27.1, 25.2, 23.4, 23.2 ppm. ESI: m/z calcd for

C₁₉H₂₈N₃[M + 2H]²⁺, 149.62; found, 149.65; retention time, 5.38 min.

N-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-1,2,3,4-tetrahydroacridin-

9-amine (12f). The reaction was carried out according to general

procedure I using 9-chloro-1,2,3,4-tetrahydroacridine 11 (600 mg, 2.76

mg) and 2,2′-(ethane-1,2-diylbis(oxy))bis(ethan-1-amine) (2.00 mL,

13.8 mmol). The product 12f (864 mg, 2.62 mmol, 95%) was obtained

as a brown oil. ¹H NMR (400 MHz, CDCl₃): δ = 7.95 (d, J = 8.5 Hz,

1H), 7.89 (d, J = 8.5 Hz, 1H), 7.58−7.59 (m, 1H), 7.37−75.30 (m,

1H), 3.67−3.56 (m, 8H), 3.50 (t, J = 5.2 Hz, 2H), 3.05 (t, J = 6.0 Hz,

2H), 2.88−2.84 (m, 2H), 2.76 (t, J = 5.7 Hz, 2H), 1.94−1.86 (m, 4H)

ppm. ¹³C NMR (101 MHz, CDCl₃): δ 158.7, 150.4, 147.5, 128.8, 128.2,

123.8, 122.7, 120.8, 117.5, 73.6, 70.5, 70.3, 70.3, 48.6, 41.8, 24.7, 23.1,

22.9, 22.6 ppm. ESI: m/z calcd for C₁₉H₂₈N₃O₂[M + H]⁺, 330.22;

found, 330.05; retention time, 4.60 min.

9-Chloro-1,2,3,4-tetrahydroacridine (11). 2-Aminobenzoic acid 9

(1.00 g, 7.29 mmol) and cyclohexanone 10 (883 μL, 8.53 mmol) were

combined in a ﬂask and cooled down to 0 °C. Then, POCl₃(8.00 mL)

was slowly added. Afterward, the mixture was stirred for 3 h under reﬂux

conditions and then concentrated to a slurry in vacuo. The slurry was

dissolved in ethyl acetate and neutralized with aqueous sodium

hydroxide. The mixture was extracted with ethyl acetate. The organic

layers were combined and dried over anhydrous MgSO₄, and the

solvent was removed in vacuo. The crude product was puriﬁed by

recrystallization from acetone. The product 11 (988 mg, 4.54 mmol,

62%) was obtained as a brown solid. mp = 66 °C. ¹H NMR (400 MHz,

CDCl₃): δ 8.17 (d, J = 8.4, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.69−7.63 (m,

1H), 7.56−7.50 (m, 1H), 3.13 (t, J = 5.6 Hz, 2H), 3.03 (t, J = 6.4 Hz,

2H), 1.97−1.93 (m, 4H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 159.4,

146.2, 141.8, 129.4, 128.9, 128.3, 126.6, 125.4, 123.7, 33.9, 27.5, 22.6,

21.5 ppm. ESI: m/z calcd for C₁₃H₁₃ClN [M + H]⁺, 218.07; found,

218.00; retention time, 9.97 min.

N¹-(1,2,3,4-Tetrahydroacridin-9-yl)ethane-1,2-diamine (12a).

The reaction was carried out according to general procedure I using

9-chloro-1,2,3,4-tetrahydroacridine 11 (800 mg, 3.67 mmol) and

ethane-1,2-diamine (1.23 mL, 18.35 mmol). The product 12a (557 mg,

N-(2-((2-Aminoethyl)disulfaneyl)ethyl)-1,2,3,4-tetrahydroacri-

din-9-amine (12g). 2,2′-Dithiobis(ethylamine)dihydrochloride (608

mg, 2.70 mmol) was suspended in hexanol, and NEt₃(1.49 mL, 10.8

mmol) was added. The reaction vessel was closed and stirred for 30 min

at 160 °C. Then, 9-chloro-1,2,3,4-tetrahydroacridine 11 (117 mg, 0.54

mg) was added, and the mixture was stirred for further 12 h at 160 °C.

The solvent was removed under high vacuum at 60 °C. The crude

product was puriﬁed using RP ﬂash chromatography to yield 12g (80

1

2.31 mmol, 63%) was obtained as a brown oil. H NMR (400 MHz,

CDCl₃): δ 7.94 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.48 (t, J =

10.0, 5.1 Hz, 1H), 7.26 (t, 1H), 4.75 (s, 1H), 3.43−3.36 (m, 2H), 3.03−

2.97 (m, 2H), 2.89−2.81 (m, 2H), 2.72−2.65 (m, 2H), 1.86−1.80 (m,

4H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 157.5, 149.9, 146.4, 127.7,

127.2, 122.6, 121.8, 119.5, 115.5, 50.0, 41.4, 33.0, 23.8, 22.0, 21.8 ppm.

ESI: m/z calcd for C₁₅H₂₀N₃[M + H]⁺, 242.17; found, 242.10;

retention time, 4.09 min.

1

mg, 0.33 mmol, 44%) as a pale yellow oil. H NMR (400 MHz,

N¹-(1,2,3,4-Tetrahydroacridin-9-yl)propane-1,3-diamine (12b).

The reaction was carried out according to general procedure I using

9-chloro-1,2,3,4-tetrahydroacridine 11 (600 mg, 2.76 mmol) and

propane-1,3-diamine (1.15 mL, 13.8 mmol). The product 12b (530

mg, 1.97 mmol, 80%) was obtained as a brown oil (417 mg, 1.63 mmol,

59%). ¹H NMR (400 MHz, CDCl₃): δ 7.87 (m, 1H), 7.83−7.79 (m,

1H), 7.46−7.39 (m, 1H), 7.24−7.19 (1 H), 3.48 (t, J = 6.7 Hz, 2H),

3.01 (t, J = 6.1 Hz, 2H), 2.89 (t, J = 6.2 Hz, 2H), 2.60 (t, J = 5.9 Hz, 2H),

1.85−1.76 (m, 4H), 1.85−1.76 (m, 2H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 157.3, 149.9, 146.3, 127.6, 127.2, 122.6, 121.9, 119.1, 114.8,

47.0, 39.4, 33.1, 26.4, 24.1, 22.1, 21.8 ppm. ESI: m/z calcd for C₁₆H₂₂N₃

[M + H]⁺, 256.18; found, 256.10; retention time, 4.26 min.

MeOD): δ 8.44 (d, J = 8.7 Hz, 1H), 7.88−7.86 (m, 2H), 7.65−7.61 (m,

1H), 4.33 (t, J = 6.6 Hz, 2H), 3.34−3.29 (m, 2H), 3.24 (t, J = 6.6 Hz,

2H), 3.08 (t, J = 5.3 Hz, 2H), 3.03 (t, J = 6.9 Hz, 2H), 2.79 (t, J = 5.7 Hz,

2H), 2.02−1.94 (m, 4H) ppm. ¹³C NMR (101 MHz, MeOD): δ =

156.8, 151.0, 138.2, 132.8, 125.3, 124.9, 118.9, 115.9, 112.3, 46.29, 38.0,

36.7, 33.7, 28.1, 23.9, 21.6, 20.4 ppm. ESI: m/z calcd for C₁₇H₂₄N₃S₂

[M + H]⁺, 334.14; found, 334.00; retention time, 5.31 min.

N-(4-((1,2,3,4-Tetrahydroacridin-9-yl)amino)butyl)acetamide

(13). N¹-(1,2,3,4-Tetrahydroacridin-9-yl)butane-1,4-diamine 12c

(51.0 mg, 0.19 mmol) was dissolved in CH₂Cl₂, and acetic acid

anhydride (359 μL, 3.80 mmol) was added at room temperature. After

stirring for 5 min, the mixture was basiﬁed by washing with a saturated

NaHCO₃solution. The organic layer was dried over anhydrous MgSO₄

and concentrated in vacuo. The product 13 (59.2 mg, 0.19 mmol,

quant.) was obtained as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃):

δ 8.13−8.10 (m, 1H), 7.95−7.91 (m, 1H), 7.53−7.48 (m, 1H), 7.34−

7.30 (m, 1H), 3.79 (t, J = 7.1 Hz, 2H), 3.26 (q, J = 6.5 Hz, 2H), 2.99 (t, J

= 6.0 Hz, 2H), 2.55 (t, J = 5.7 Hz, 2H), 1.84−1.75 (m, 6H), 1.66−1.59

(m, 2H), 1.23 (s, 3H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 171.2,

155.11 151.7, 139.3, 131.6, 124.7, 124.3, 120.8, 116.2, 111.3, 47.8, 38.7,

28.5, 28.1, 26.5, 24.1, 23.1, 22.0, 20.8 ppm. ESI: m/z calcd for

C₁₉H₂₆N₃O [M + H]⁺, 312.21; found, 312.05; retention time, 6.58 min.

N¹-Ethyl-N⁴-(1,2,3,4-tetrahydroacridin-9-yl)butane-1,4-diamine

(14). N-(4-((1,2,3,4-Tetrahydroacridin-9-yl)amino)butyl)acetamide

13 (218 mg, 0.70 mmol) was dissolved in dry THF. LiAlH₄(133 mg,

3.50 mmol) was added, the mixture was stirred for 48 h under reﬂux

conditions, and then NH_3(aq)(25%) was carefully added to the mixture

on ice. The mixture was concentrated in vacuo, dissolved in ethyl

acetate, and washed with a mixture of water/NH_3(aq)(25%) (1:1). The

combined organic layers were dried over anhydrous MgSO₄and

concentrated in vacuo. The crude product was puriﬁed by column

N¹-(1,2,3,4-Tetrahydroacridin-9-yl)butane-1,4-diamine (12c).

The reaction was carried out according to general procedure I using

9-chloro-1,2,3,4-tetrahydroacridine 11 (535 mg, 2.45 mmol) and

butane-1,4-diamine (1.08 g, 12.3 mmol). The product 12c (530 mg,

1

1.97 mmol, 80%) was obtained as a brown oil. H NMR (400 MHz,

CDCl₃): δ 7.99−7.92 (m, 2H), 7.57−7.52 (m, 1H), 7.36−7.31 (m,

1H), 3.53 (t, J = 7.1 Hz, 2H), 3.07 (t, J = 6.3 Hz, 2H), 2.76 (t, J = 6.9 Hz,

2H), 2.71 (t, J = 6.2 Hz, 2H), 1.93−1.88 (m, 4H), 1.76−1.68 (m, 2H),

1.61−1.52 (m, 2H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 157.9, 151.1,

146.7, 128.6, 128.1, 123.7, 122.9, 119.9, 115.6, 49.29, 41.72, 33.56,

30.76, 29.06, 24.82, 22.98, 22.61 ppm. ESI: m/z calcd for C₁₇H₂₄N₃[M

+ H]⁺, 270.20; found, 270.10; retention time, 4.26 min.

N¹-(1,2,3,4-Tetrahydroacridin-9-yl)pentane-1,5-diamine (12d).

The reaction was carried out according to general procedure I using

9-chloro-1,2,3,4-tetrahydroacridine 11 (600 mg, 2.76 mmol) and

pentane-1,5-diamine (1.62 mL, 13.8 mmol). The product 12d (427 mg,

1

1.51 mmol, 55%) was obtained as a brown oil. H NMR (400 MHz,

CDCl₃): δ 8.09 (d, J = 8.4 Hz, 1H), 7.91−7.86 (m, 1H), 7.62−7.56 (m,

1H), 7.30−7.24 (m, 1H), 3.41 (t, J = 7.0 Hz, 2H), 3.07−3.03 (m, 2H),

O

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

chromatography (4:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)). The

product 14 (144 mg, 0.48 mmol, 69%) was obtained as a yellow oil.

¹H NMR (400 MHz, CDCl₃): δ 7.98−7.90 (m, 2H), 7.56−7.51 (m,

1H), 7.35−7.29 (m, 1H), 3.52 (t, J = 6.8 Hz, 2H), 3.07−3.03 (m, 2H),

2.72−2.65 (m, 6H), 1.92−1.86 (m, 4H), 1.76−1.61(m, 4H), 1.14 (t, J =

7.2 Hz, 3H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 157.8, 151.2,

146.6, 128.7, 127.9, 123.8, 123.0, 119.9, 115.6, 49.2, 49.0, 44.0, 33.5,

29.3, 27.1, 24.8, 23.0, 22.6, 14.7 ppm. ESI: m/z calcd for C₁₉H₂₈N₃[M +

2H]²⁺, 149.62; found, 149.65; retention time, 4.77 min.

= 7.1 Hz, 6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 169.4, 154.4,

151.0, 146.6, 145.8, 135.4, 131.0, 128.9, 128.1, 125.8, 124.2, 124.1,

122.8, 120.0, 116.1, 113.9, 53.6, 48.9, 42.9, 33.5, 29.3, 26.5, 25.0, 23.0,

22.7, 13.8 ppm. ESI: m/z calcd for C₂₈H₃₆N₅O₃[M + H]⁺, 490.28;

found, 490.15; retention time, 8.04 min.

N,N-Diethyl-3-nitro-4-((5-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)pentyl)amino)benzamide (17d). The reaction was carried out

according to general procedure II using N,N-diethyl-4-ﬂuoro-3-

nitrobenzamide 16 (221 mg, 0.92 mmol), 12d (237 mg, 0.84 mmol),

and NEt₃(173 μL, 1.26 mmol). The crude product was puriﬁed by ﬂash

chromatography (gradient CH₂Cl₂, MeOH), and the product 17d (160

mg, 0.32 mmol, 38%) was obtained as a brown oil. ¹H NMR (400 MHz,

CDCl₃): δ 8.21 (d, J = 2.0 Hz, 1H), 8.13−8.05 (m, 3H), 7.56−7.51 (m,

1H), 7.50−7.46 (m, 1H), 7.35−7.30 (m, 1H), 6.81 (d, J = 8.9 Hz, 1H),

3.72 (t, J = 7.2 Hz, 2H), 3.45−3.35 (m, 3H), 3.33−3.27 (m, 2H), 3.12−

3.07 (m, 2H), 2.67−2.63 (m, 2H), 1.87−1.79 (m, 6H), 1.77−1.70 (m,

2H), 1.58−1.50 (m, 2H), 1.17 (t, J = 7.1 Hz, 6H) ppm. ¹³C NMR (101

MHz, CDCl₃): δ 169.4, 154.6, 153.3, 145.8, 142.7, 135.2, 130.7, 130.4,

125.7, 124.5, 124.3, 123.8, 123.7, 118.0, 114.0, 113.5, 53.1, 48.5, 42.9,

31.1, 30.0, 28.6, 24.6, 24.3, 22.5, 21.7, 13.7 ppm. ESI: m/z calcd for

C₂₉H₃₈N₅O₃[M + H]⁺, 504.30; found, 504.15; retention time, 8.36

min.

N,N-Diethyl-3-nitro-4-((6-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)hexyl)amino)benzamide (17e). The reaction was carried out

according to general procedure II using N,N-diethyl-4-ﬂuoro-3-

nitrobenzamide 16 (341 mg, 1.42 mmol), 12e (458 mg, 1.56 mmol),

and NEt₃(297 μL, 2.13 mmol). The product 17e (230 mg, 0.44 mmol,

31%) was obtained as a brown oil and used in the next reaction step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃): δ 8.55 (d, J

= 8.4 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.18−8.13 (m, 2H), 7.73−7.67

(m, 1H), 7.57−7.52 (m, 1H), 7.48−7.42 (m, 1H), 6.86 (d, J = 8.9 Hz,

1H), 3.94 (t, J = 7.0 Hz, 2H), 3.51−3.37 (m, 4H), 3.36−3.29 (m, 4H),

2.60 (t, J = 6.2 Hz, 2H), 1.91−1.83 (m, 4H), 1.69−1.58 (m, 4H), 1.57−

1.53 (m, 4H), 1.21 (t, J = 7.1 Hz, 6H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 168.5, 155.45, 152.0, 146.0, 142.04 135.4, 129.52, 129.2,

125.4, 124.9, 123.7, 123.2, 121.6, 118.7, 114.0, 113.6, 53.1, 48.2, 43.0,

31.3, 31.2, 28.8, 26.8, 26.6, 26.3, 23.8, 20.8, 13.8 ppm. ESI: m/z calcd for

C₃₀H₄₀N₅O₃[M + H]⁺, 518.31; found, 518.40; retention time, 8.55

min.

N,N-Diethyl-4-ﬂuoro-3-nitrobenzamide (16). 4-Fluoro-3-nitro-

benzoic acid 15 (1.00 g, 5.4 mmol) was dissolved in CH₂Cl₂and

cooled down to 0 °C. A catalytic amount of N,N-dimethylformamide

was added, and oxalylchloride (2.31 mL, 27.01 mmol) was added

dropwise. The mixture was stirred for 10 min at 0 °C, allowed to come

to room temperature, and then stirred again for 1 h. The mixture was

then evaporated, and the residue was dissolved in CH₂Cl₂and cooled

down to 0 °C. Then, a mixture of HNEt₂(621 μL, 1.1 mmol) and NEt₃

(2.24 mL, 16.2 mmol) in CH₂Cl₂was slowly added to the cold mixture.

After stirring for 3 h at room temperature, the mixture was washed with

water. The combined organic layers were dried over anhydrous MgSO₄

and concentrated in vacuo. The product 16 (918 mg, 3.82 mmol, 71%)

was obtained as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 8.11 (d, J =

7.0 Hz, 1H), 7.66−7.70 (m, 1 H), 7.35 (d, J = 10.4 Hz, 1H), 3.65−3.18

(m, 4H), 1.30−1.11 (m, 6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ

167.5, 156.8, 154.1, 136.9, 133.8, 124.4, 118.8, 43.4, 14.0 ppm. ESI: m/z

calcd for C₁₁H₁₄FN₂O₃[M + H]⁺, 241.10; found, 241.10.

N,N-Diethyl-3-nitro-4-((2-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)ethyl)amino)benzamide (17a). The reaction was carried out

according to general procedure II using N,N-diethyl-4-ﬂuoro-3-

nitrobenzamide 16 (351 mg, 1.46 mmol), 12a (388 mg, 1.61 mmol),

and NEt₃(304 μL, 2.19 mmol). The crude product was puriﬁed by

column chromatography (20:1:0.1 CH₂Cl₂/MeOH/NH_3(aq)(25%)).

The product 17a (475 mg, 1.03 mmol, 71%) was obtained as a yellow

oil. ¹H NMR (400 MHz, CDCl₃): δ 8.27 (t, J = 5.2 Hz, 1H), 8.24 (d, J =

1.8 Hz, 1H), 7.91 (t, J = 8.5 Hz, 2H), 7.53 (t, J = 7.6 Hz, 1H), 7.46 (d, J

= 8.8 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 4.27 (s,

1H), 3.77−3.70 (m, 2H), 3.55−3.48 (m, 2H), 3.46−3.32 (m, 4H), 3.04

(t, J = 6.0 Hz, 2H), 2.71 (t, J = 5.8 Hz, 2H), 1.91−1.81 (m, 4H), 1.18 (t,

J = 7.0 Hz, 6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 169.2, 158.9,

149.5, 147.3, 145.5, 135.1, 131.3, 128.9, 128.6, 125.7, 124.5, 124.4,

122.2, 120.8, 118.2, 113.8, 47.4, 43.6, 34.0, 25.0, 22.9, 22.7 ppm. ESI:

m/z calcd for C₂₆H₃₃N₅O₃[M + H]⁺, 462.57; found, 462.15; retention

time, 7.718 min.

N,N-Diethyl-3-nitro-4-((3-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)propyl)amino)benzamide (17b). The reaction was carried out

according to general procedure II using N,N-diethyl-4-ﬂuoro-3-

nitrobenzamide 16 (173 mg, 0.72 mmol), 12b (165 mg, 0.65 mmol),

and NEt₃(136 μL, 0.98 mmol). The crude product was puriﬁed by ﬂash

chromatography (gradient CH₂Cl₂, MeOH), and the product 17b (110

mg, 0.23 mmol, 36%) was obtained as a brown oil. ¹H NMR (400 MHz,

CDCl₃): δ 8.23 (d, J = 2.0 Hz, 1H), 8.16 (t, J = 5.1 Hz, 1H), 7.89 (d, J =

8.4, 2H), 7.54−7.50 (m, 1H), 7.49−7.45 (m, 1H), 7.33−7.27 (m, 1H),

6.74 (d, J = 8.9 Hz, 1H), 3.60 (t, J = 6.9 Hz, 2H), 3.49−3.30 (m, 6H),

3.03 (t, J = 5.3 Hz, 2H), 2.68 (t, J = 5.5 Hz, 2H), 2.09−1.98 (m, 2H),

1.90−1.81 (m, 4H), 1.18 (t, J = 7.1 Hz, 6H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ = 169.9, 154.6, 151.6, 145.9, 143.1, 135.8, 130.3, 129.1,

128.6, 124.2, 124.8, 123.5, 122.6, 119.0, 115.5, 113.8, 52.9, 42.6, 41.6,

34.7, 26.9, 24.5, 22.4, 22.0, 13.3 ppm. ESI: m/z calcd for C₂₇H₃₄N₅O₃

[M + H]⁺, 476.26; found, 476.15; retention time, 7.83 min.

N,N-Diethyl-3-nitro-4-((4-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)butyl)amino)benzamide (17c). The reaction was carried out

according to general procedure II using N,N-diethyl-4-ﬂuoro-3-

nitrobenzamide 16 (430 mg, 1.79 mmol), 12c (439 mg, 1.63 mmol),

and NEt₃(340 μL, 2.45 mmol). The crude product was puriﬁed by ﬂash

chromatography (gradient CH₂Cl₂, MeOH), and the product 17c (212

mg, 0.43 mmol, 26%) was obtained as a brown oil. ¹H NMR (400 MHz,

CDCl₃): δ 8.26 (d, J = 2.0 Hz, 1H), 8.16 (t, J = 4.9 Hz, 1H), 7.96−7.92

(m, 2H), 7.58−7.51 (m, 2H), 7.37−7.32 (m, 1H), 6.81 (d, J = 8.9 Hz,

1H), 3.57 (t, J = 6.2 Hz, 2H), 3.45−3.33 (m, 6H), 3.09−3.05 (m, 2H),

2.72−2.67 (m, 2H), 1.92−1.86 (m, 4H), 1.85−1.79 (m, 4H), 1.20 (t, J

N,N-Diethyl-3-nitro-4-((2-(2-(2-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)ethoxy)ethoxy)ethyl)amino)benzamide (17f). The reaction

was carried out according to general procedure II using N,N-diethyl-4-

ﬂuoro-3-nitrobenzamide 16 (475 mg, 1.98 mmol), 12f (718 mg, 2.18

mmol), and NEt₃(410 μL, 2.97 mmol). The crude product was puriﬁed

by ﬂash chromatography (gradient CH₂Cl₂/MeOH), and product 17f

(575 mg, 1.05 mmol, 53%) was obtained as a brown oil and used in the

1

next reaction step without further puriﬁcation. H NMR (400 MHz,

CDCl₃): δ 8.41−8.35 (m, 2H), 8.22 (d, J = 2.0 Hz, 1H), 8.11−8.07 (m,

1H), 7.66−7.60 (m, 1H), 7.56−7.52 (m, 1H), 7.42−7.37 (m, 1H), 6.86

(d, J = 8.9 Hz, 1H), 4.01−3.95 (m, 2H), 3.83−3.73 (m, 4H), 3.75−3.70

(m, 4H), 3.52−3.48 (m, 2H), 3.47−3.35 (m, 4H), 3.27−3.22 (m, 2H),

2.68−2.63 (m, 2H), 1.90−1.83 (m, 4H), 1.20 (t, J = 7.1 Hz, 6H) ppm.

¹³C NMR (101 MHz, CDCl₃): δ 169.3, 154.30, 151.4, 145.8, 143.1,

135.4, 131.1, 125.7, 125.0, 124.3, 124.2, 123.6, 121.3, 117.6, 116.0,

114.1, 70.9, 70.5, 70.0, 69.0, 52.7, 48.1, 42.8, 30.1, 23.9, 22.4, 21.4, 13.7

ppm. ESI: m/z calcd for C₃₀H₄₀N₅O₅[M + H]⁺, 550.30; found, 550.25;

retention time, 8.05 min.

3-Amino-N,N-diethyl-4-((2-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)ethyl)amino)benzamide (18a). The reaction was carried out

according to general procedure III using N,N-diethyl-3-nitro-4-((2-

((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)amino)benzamide 17a

(475 mg, 1.03 mmol) and SnCl₂·2H₂O (1.44 g, 6.39 mmol). The

product 18a (325 mg, 0.75 mmol, 73%) was obtained as a colorless oil

and directly used for the next reaction without puriﬁcation. ESI: m/z

calcd for C₂₆H₃₅N₅O [M + H]⁺, 432.28; found, 432.30; retention time,

7.534 min.

3-Amino-N,N-diethyl-4-((3-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)propyl)amino)benzamide (18b). The reaction was carried out

according to general procedure III using N,N-diethyl-3-nitro-4-((3-

((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)amino)benzamide 17b

P

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(110 mg, 0.23 mmol) and SnCl₂·2H₂O (323 mg, 1.43 mmol). The

product 18b (89.1 mg, 0.20 mmol, 87%) was obtained as a colorless oil

and directly used for the next reaction without puriﬁcation. ESI: m/z

calcd for C₂₇H₃₆N₅O [M + H]⁺, 223.65; found, 223.65; retention time,

7.84 min.

3-Amino-N,N-diethyl-4-((4-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)butyl)amino)benzamide (18c). The reaction was carried out

according to general procedure III using N,N-diethyl-3-nitro-4-((4-

((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)amino)benzamide 17c

(212 mg, 0.43 mmol) and SnCl₂·2H₂O (602 mg, 2.67 mmol). The

product 18c (154 mg, 0.34 mmol, 79%) was obtained as a colorless oil

and directly used for the next reaction without puriﬁcation. ESI: m/z

calcd for C₂₈H₃₈N₅O [M + 2H]²⁺, 230.66; found, 230.60; retention

time, 7.17 min.

3-Amino-N,N-diethyl-4-((5-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)pentyl)amino)benzamide (18d). The reaction was carried out

according to general procedure III using N,N-diethyl-3-nitro-4-((5-

((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)amino)benzamide 17d

(236 mg, 0.47 mmol) and SnCl₂·2H₂O (657 mg, 2.91 mmol). The

product 18d (222 mg, 0.47 mmol, quant.) was obtained as a colorless

oil and directly used for the next reaction without puriﬁcation. ESI: m/z

calcd for C₂₉H₄₀N₅O [M + 2H]²⁺, 237.67; found, 237.65; retention

time, 7.73 min.

3-Amino-N,N-diethyl-4-((6-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)hexyl)amino)benzamide (18e). The reaction was carried out

according to general procedure III using N,N-diethyl-3-nitro-4-((6-

((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)amino)benzamide 17e

(230 mg, 0.44 mmol) and SnCl₂·2H₂O (616 mg, 2.73 mmol). The

product 18e (140 mg, 0.29 mmol, 66%) was obtained as a colorless oil

and directly used for the next reaction without puriﬁcation. ESI: m/z

calcd for C₃₀H₄₂N₅O [M + 2H]²⁺, 244.68; found, 244.65; retention

time, 7.93 min.

3-Amino-N,N-diethyl-4-((2-(2-(2-((1,2,3,4-tetrahydroacridin-9-

yl)amino)ethoxy)ethoxy)ethyl)amino)benzamide (18f). The reac-

tion was carried out according to general procedure III using N,N-

diethyl-3-nitro-4-((2-(2-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)-

ethoxy)ethoxy)ethyl)amino)benzamide 17f (575 mg, 1.05 mmol) and

SnCl₂·2H₂O (1.47 g, 8.51 mmol). The product 18f (290 mg, 0.56

mmol, 53%) was obtained as a colorless oil and directly used for the

next reaction without puriﬁcation. ESI: m/z calcd for C₃₀H₄₂N₅O₃[M +

2H]²⁺, 260.67; found, 260.60; retention time, 7.50 min.

3-(2-(4-Ethoxyphenyl)acetamido)-N,N-diethyl-4-((2-((1,2,3,4-tet-

rahydroacridin-9-yl)amino)ethyl)amino)benzamide (19a). The re-

action was carried out according to general procedure IV using 3-

amino-N,N-diethyl-4-((2-((1,2,3,4-tetrahydroacridin-9-yl)amino)-

ethyl)amino)benzamide 18a (325 mg, 0.75 mmol), 2-(4-

ethoxyphenyl)acetic acid (30 mg, 0.83 mmol), HBTU (313 mg, 0.83

mmol), and triethylamine (156 μL, 1.13 mmol). The product 19a (258

mg, 0.43 mmol, 57%) was obtained as an orange/brown oil. ¹H NMR

(400 MHz, CDCl₃): δ 8.72 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.89 (d, J =

8.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.23 (d, J

= 8.5 Hz, 2H), 7.15 (d, J = 1.4 Hz, 1H), 7.00 (dd, J = 8.3, 1.4 Hz, 1H),

6.74 (d, J = 8.5 Hz, 2H), 6.46 (d, J = 8.4 Hz, 1H), 4.73 (s, 1H), 4.64 (t, J

= 5.3 Hz, 1H), 3.85 (q, J = 7.0 Hz, 2H), 3.66−3.59 (m, 4H), 3.39 (s,

4H), 3.26−3.18 (m, 2H), 3.01 (t, J = 6.1 Hz, 2H), 2.63 (t, J = 6.0 Hz,

2H), 1.89−1.75 (m, 4H), 1.32 (t, J = 7.0 Hz, 3H), 1.15 (s, 6H) ppm.

¹³C NMR (101 MHz, CDCl₃): δ 171.7, 171.1, 158.1, 157.9, 150.9,

7.59−7.55 (m, 1H), 7.47−7.44 (m, 1H), 7.29−7.28 (m, 1H), 7.24 (d, J

= 8.4 Hz, 2H), 7.05−7.02 (m, 1H), 6.76−6.74 (m, 2H), 6.56−6.52 (m,

1H), 3.92−3.83 (m, 4H), 3.73 (s, 2H), 3.42−3.36 (m, 4H), 3.22−3.15

(m, 2H), 2.73−2.68 (m, 2H), 2.43−2.36 (m, 2H), 2.03−1.97 (m, 2H),

1.71−1.65 (m, 4H), 1.32 (t, J = 7.0 Hz, 3H), 1.20−1.09 (m, 6H) ppm.

¹³C NMR (101 MHz, CDCl₃): δ 172.1, 171.9, 162.7, 157.2, 151.9,

146.6, 143.5, 130.6, 129.4, 127.3, 126.7, 125.8, 124.7, 124.6, 123.0,

121.1, 119.5, 118.5, 115.5, 114.8, 111.0, 63.5, 46.1, 42.8, 42.5, 39.4,

29.8, 29.5, 24.8, 22.4, 22.1, 14.9, 13.7 ppm. ESI: m/z calcd for

C₃₇H₄₇N₅O₃[M + 2H]²⁺, 304.69; found, 304.70; retention time, 8.23

min.

3-(2-(4-Ethoxyphenyl)acetamido)-N,N-diethyl-4-((4-((1,2,3,4-tet-

rahydroacridin-9-yl)amino)butyl)amino)benzamide (19c). The re-

action was carried out according to general procedure IV using 3-

amino-N,N-diethyl-4-((4-((1,2,3,4-tetrahydroacridin-9-yl)amino)-

butyl)amino)benzamide 18c (139 mg, 0.30 mmol), 2-(4-

ethoxyphenyl)acetic acid (121 mg, 0.33 mmol), HBTU (125 mg,

0.33 mmol), and triethylamine (62.3 μL, 0.45 mmol). The product 19c

(166 mg, 0.27 mmol, 90%) was obtained as a brown oil. ¹H NMR (400

MHz, CDCl₃): δ 7.97 (s, 1H), 7.79−7.77 (m, 1H), 7.53−7.50 (m, 1H),

7.34−7.31 (m, 1H), 7.24 (d, J = 8.6 Hz, 2H), 7.17−7.14 (m, 1H),

7.06−7.02 (m, 1H), 6.81 (d, J = 8.6 Hz, 2H), 6.50−6.46 (m, 1H),

4.03−3.91 (m, 4H), 3.64 (s, 2H), 3.42−3.31 (m, 4H), 3.05−3.00 (m,

2H), 2.94−2.90 (m, 2H), 2.63−2.57 (m, 2H), 1.87−1.80 (m, 4H),

1.73−1.56 (m, 4H), 1.33 (t, J = 7.0 Hz, 3H), 1.19−1.11 (m, 6H) ppm.

¹³C NMR (101 MHz, CDCl₃): δ 171.7, 171.4, 162.1, 158.4, 152.4,

144.7, 143.8, 130.5, 129.8, 127.2, 126.9, 125.9, 124.9, 124.3, 123.6,

121.9, 119.5, 118.9, 115.7, 115.1, 111.1, 63.6, 48.9, 47.2, 44.4, 43.1,

32.2, 28.7, 26.3, 24.7, 22.7, 22.2, 14.9, 13.6 ppm. ESI: m/z calcd for

C₃₈H₄₉N₅O₃[M + 2H]²⁺, 311.69; found, 311.70; retention time, 8.39

min.

3-(2-(4-Ethoxyphenyl)acetamido)-N,N-diethyl-4-((5-((1,2,3,4-tet-

rahydroacridin-9-yl)amino)pentyl)amino)benzamide (19d). The

reaction was carried out according to general procedure IV using 3-

amino-N,N-diethyl-4-((5-((1,2,3,4-tetrahydroacridin-9-yl)amino)-

pentyl)amino)benzamide 19d (222 mg, 0.47 mmol), 2-(4-

ethoxyphenyl)acetic acid (191 mg, 0.52 mmol), HBTU (197 mg,

0.52 mmol), and triethylamine (98.4 μL, 0.71 mmol). The product 19d

1

(299 mg, 0.47 mmol, quant.) was obtained as a brown oil. H NMR

(400 MHz, CDCl₃): δ 8.11 (d, J = 8.7 Hz, 1H), 7.59−7.56 (m, 2H),

7.43−7.36 (m, 2H), 7.27−7.25 (m, 2H), 6.92−6.87 (m, 1H), 6.79 (d, J

= 8.6 Hz, 2H), 6.33 (d, J = 8.5 Hz, 1H), 3.95 (q, J = 14.0, 7.0 Hz, 2H),

3.86−3.82 (m, 2H), 3.69 (s, 2H), 3.46−3.37 (m, 4H), 3.00−2.95 (m,

2H), 2.69−2.64 (m, 2H), 2.50−2.45 (m, 2H), 1.80−1.72 (m, 6H),

1.69−1.63 (m, 2H), 1.37−1.33 (m, 5H), 1.30−1.21 (m, 6H) ppm. ¹³C

NMR (101 MHz, CDCl₃): δ 171.8, 171.2, 161.4, 157.6, 153.1, 146.7,

143.7, 130.5, 129.5, 127.3, 126.7, 125.8. 124.8, 124.5, 123.5, 122.2,

119.6, 118.8, 115.7, 114.9, 111.3, 63.6, 48.6, 47.4, 45.1, 43.2, 30.2, 29.9,

28.4, 28.2, 23.56, 22.2, 22.1, 14.9, 13.7 ppm. ESI: m/z calcd for

C₃₉H₅₁N₅O₃[M + 2H]²⁺, 318.70; found, 318.65; retention time, 8.75

min.

3-(2-(4-Ethoxyphenyl)acetamido)-N,N-diethyl-4-((6-((1,2,3,4-tet-

rahydroacridin-9-yl)amino)hexyl)amino)benzamide (19e). The re-

action was carried out according to general procedure IV using 3-

amino-N,N-diethyl-4-((6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-

hexyl)amino)benzamide 18e (140 mg, 0.29 mmol), 2-(4-

ethoxyphenyl)acetic acid (52.3 mg, 0.29 mmol), HBTU (133 mg,

0.35 mmol), and triethylamine (61.0 μL, 0.44 mmol). The product 19e

(142 mg, 0.22 mmol, 76%) was obtained as a brown oil. ¹H NMR (400

MHz, CDCl₃): δ 8.56 (s, 1H), 8.15 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 8.4

Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.25 (d, J =

8.6 Hz 2H), 7.04−7.00 (m, 1H), 6.75 (d, J = 8.6 Hz, 2H), 6.48−6.44

(m, 1H), 3.92 (q, J = 7.0 Hz, 2H), 3.81 (m, 2H), 3.71 (s, 2H), 3.49−

3.32 (m, 4H), 2.96−2.91 (m, 2H), 2.88−2.83 (m, 2H), 2.54−2.49 (m,

2H), 1.80−1.68 (m, 6H), 1.54−1.47 (m, 2H), 1.37−1.31 (m, 4H), 1.25

(t, J = 7.3 Hz, 3H), 1.14−1.09 (m, 6H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 172.0, 170.8, 162.7, 158.1, 156.0, 150.5, 144.07, 130.4,

129.4, 127.6, 127.0, 125.8, 125.1, 124.9, 123.8, 122.6, 119.9, 119.0,

115.7, 114.7, 111.3, 63.5, 48.1, 46.3, 45.2, 43.2, 31.5, 30.3, 28.6, 28.3,

146.5, 143.2, 130.2, 128.7, 127.8, 127.2, 125.3, 125.2, 124.7, 124.0,

123.8, 122.9, 120.2, 116.9, 114.7, 111.1, 63.4, 47.6, 44.3, 42.9, 33.5,

24.8, 22.9 , 22.6, 14.8 ppm. ESI: m/z calcd for C₃₆H₄₅N₅O₃[M + H]⁺,

594.78; found, 594.25; retention time, 8.101 min.

3-(2-(4-Ethoxyphenyl)acetamido)-N,N-diethyl-4-((3-((1,2,3,4-tet-

rahydroacridin-9-yl)amino)propyl)amino)benzamide (19b). The

reaction was carried out according to general procedure IV using 3-

amino-N,N-diethyl-4-((3-((1,2,3,4-tetrahydroacridin-9-yl)amino)-

propyl)amino)benzamide 18b (154 mg, 0.34 mmol), 2-(4-

ethoxyphenyl)acetic acid (136 mg, 0.37 mmol), HBTU (140 mg,

0.37 mmol), and triethylamine (70.6 μL, 0.51 mmol). The product 19b

1

(104 mg, 0.17 mmol, 50%) was obtained as an orange/brown oil. H

NMR (400 MHz, CDCl₃): δ 8.07 (s, 1H), 8.02 (d, J = 9.5 Hz, 1H),

Q

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

26.2, 25.9, 21.8, 20.7, 14.9, 13.7 ppm. ESI: m/z calcd for C₄₀H₅₃N₅O₃

[M + 2H]²⁺, 325.70; found: 325.75; retention time: 8.76 min.

3-(2-(4-Ethoxyphenyl)acetamido)-N,N-diethyl-4-((2-(2-(2-

((1,2,3,4-tetrahydroacridin-9-yl)amino)ethoxy)ethoxy)ethyl)-

amino)benzamide (19f). The reaction was carried out according to

general procedure IV using 3-amino-N,N-diethyl-4-((2-(2-(2-

((1,2,3,4-tetrahydroacridin-9-yl)amino)ethoxy)ethoxy)ethyl)amino)-

benzamide 18f (290 mg, 0.56 mmol), 2-(4-ethoxyphenyl)acetic acid

(122 mg, 0.62 mmol), HBTU (235 mg, 0.62 mmol), and triethylamine

(116 μL, 0.84 mmol). The product 19f (398 mg, 0.58 mmol, quant.)

was obtained as a brown oil.¹H NMR (400 MHz, CDCl₃): δ 8.03−7.96

(m, 2H), 7.87 (d, J = 8.3 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.31 (t, J =

7.2 Hz 1H), 7.20 (d, J = 8.6 Hz, 2H), 7.07−7.03 (m, J = 8.4, 1.9 Hz,

1H), 6.81 (d, J = 8.6 Hz, 2H), 6.56−6.52 (m, 1H), 3.99−3.92 (m, 2H),

3.71−3.67 (m, 2H), 3.63−3.56 (m, 10H), 3.42−3.31 (m, 4H), 3.17 (t, J

= 5.3 Hz, 2H), 2.99−2.94 (m, 2H), 2.67−2.62 (m, 2H), 1.84−1.76 (m,

4H), 1.36 (t, J = 7.2 Hz, 3H), 1.14−1.09 (m, 6H) ppm. ¹³C NMR (101

MHz, CDCl₃): δ 171.5, 170.9, 158.3, 156.6, 152.1, 144.9, 143.4, 130.5,

129.5, 127.0, 126.4, 125.7, 124.6, 124.3, 123.5, 123.3, 119.5, 118.7,

115.9, 115.0, 111.6, 70.4, 70.3, 70.2, 69.6, 63.5, 53.5, 48.3, 45.8, 43.5,

29.8, 24.4, 22.8, 22.3, 14.9, 13.7 ppm. ESI: m/z calcd for C₄₀H₅₁N₅O₅

[M + 2H]²⁺, 341.70; found, 341.70; retention time, 8.34 min.

Ethyl 4-(Isopentylamino)-3-nitrobenzoate (20). 4-Fluoro-3-nitro-

benzoic acid 15 (1.00 g, 5.40 mmol) was dissolved in ethanol, and a cat.

amount (few drops) of H₂SO₄(95−97%) was added. After stirring

overnight under reﬂux conditions, the mixture was basiﬁed with

triethylamine, and 3-methylbutan-1-amine (691 μL, 5.94 mmol) was

added. After stirring under reﬂux conditions overnight, the mixture was

concentrated in vacuo. The residue was dissolved in CH₂Cl₂and

washed with 1 M HCl_(aq)and NaHCO₃solution. The organic layers

were combined, dried over anhydrous MgSO₄, and concentrated in

vacuo. The product 20 (1.46 g, 5.22 mmol, 97%) was obtained as a

yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 8.88−8.85 (m, 1H), 8.31 (s,

1H), 8.07−8.03 (m, 1H), 6.88−6.83 (m, 1H), 4.35 (q, J = 7.1 Hz, 2H),

3.39−3.33 (m, 2H), 1.83−1.72 (m, 1H), 1.65 (q, J = 7.1 Hz, 2H), 1.38

(t, J = 7.1 Hz, 3H), 0.99 (d, J = 6.7 Hz, 6H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 165.3, 147.9, 136.5, 131.3, 129.6, 117.5, 113.6, 61.1, 41.6,

37.8, 26.1, 22.6, 14.5 ppm. ESI: m/z calcd for C₁₄H₂₁N₂O₄[M + H]⁺,

281.15; found, 281.00; retention time, 11.85 min.

Ethyl 3-Amino-4-(isopentylamino)benzoate (21). Ethyl 4-(iso-

pentylamino)-3-nitrobenzoate 20 (1.46 g, 5.22 mmol) was dissolved in

MeOH and a cat. amount of Pd/C (10%) was added. The mixture was

stirred under a hydrogen atmosphere (8 bar) at room temperature for 3

h. The clear mixture was ﬁltered oﬀ by suction over Celite. The

combined organic layers were dried over anhydrous MgSO₄and

concentrated in vacuo, and product 36 (1.31 g, 5.22 mmol, quant.) was

obtained as a colorless oil. The product was directly used for the next

reaction step without further puriﬁcation. ESI: m/z calcd for

C₁₄H₂₃N₂O₂[M + H]⁺, 251.18; found, 251.05; retention time, 10.34

min.

Ethyl 3-(2-(4-Ethoxyphenyl)acetamido)-4-(isopentylamino)-

benzoate (22). The reaction was carried out according to general

procedure V using 3 ethyl 3-amino-4-(isopentylamino)benzoate 21

(1.31 g, 5.22 mmol), 2-(4-ethoxyphenyl)acetic acid (1.03 g, 5.74

mmol), HBTU (2.18 g, 5.74 mmol), and NEt₃(1.08 mL, 7.83 mmol).

The product 22 (2.15 g, 5.22 mmol, quant.) was obtained as an orange/

brown oil. ¹H NMR (400 MHz, CDCl₃): δ 7.86−7.82 (m, 1H), 7.73−

7.71 (m, 1H), 7.32−7.29 (m, 2H), 6.97−6.94 (m, 2H), 6.65 (d, J = 8.7

Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.74 (s, 2H),

3.14−3.08 (m, 2H), 1.70−1.65 (m, 1H), 1.50−1.40 (m, 5H), 1.37 (t, J

= 7.1 Hz, 3H), 0.96 (d, J = 6.6 Hz, 6H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ 170.8, 158.6, 147.2, 130.5, 130.2, 130.1, 127.8, 126.5, 121.5,

118.4, 115.4, 114.6, 63.7, 60.5, 41.8, 38.8, 38.3, 26.1, 22.7, 14.9, 14.6

ppm. ESI: m/z calcd for C₂₄H₃₃N₂O₄[M + H]⁺, 413.24; found, 413.10;

retention time, 11.04 min.

NH_3(aq)(25%)), and product 23 (1.67 g, 4.23 mmol, 81%) was

obtained as a purple oil. ¹H NMR (400 MHz, CDCl₃): δ 8.44 (s, 1H),

7.95−7.91 (m, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 8.8 Hz, 2H),

6.78 (d, J = 8.8 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.20 (s, 2H), 3.96−

3.89 (m, 4H), 1.56−1.46 (m, 1H), 1.39−1.28 (m, 8H), 0.85 (d, J = 6.8

Hz, 6H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 167.2, 158.1, 155.1,

142.2, 138.5, 129.5, 127.7, 124.4, 123.8, 121.7, 114.9, 108.9, 63.4, 60.7,

42.7, 38.0, 33.8, 26.1, 22.3, 14.7, 14.4 ppm. ESI: m/z calcd for

C₂₄H₃₁N₂O₃[M + H]⁺, 395.23; found, 395.15; retention time, 11.11

min.

2-(4-Ethoxybenzyl)-1-isopentyl-1H-benzo[d]imidazole-5-carbox-

ylic Acid (24). Ethyl 2-(4-ethoxybenzyl)-1-isopentyl-1H-benzo[d]-

imidazole-5-carboxylate 23 (250 mg, 0.63 mmol) was dissolved in

THF, and lithium hydroxide (150 mg, 0.63 mmol) in water was added.

After 48 h of harsh stirring under reﬂux conditions, the mixture was

concentrated in vacuo, and the residue was acidiﬁed with 2 M HCl_(aq)

.

Then, CH₂Cl₂was added, and the organic layer was washed with water.

The organic layers were combined, dried over anhydrous MgSO₄, and

concentrated in vacuo. Product 24 (231 mg, 0.63 mmol, quant.) was

obtained as a purple oil. ¹H NMR (400 MHz, D₂O): δ 8.10−8.07 (m,

1H), 7.80−7.75 (m, J = 9.9 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H), 7.06 (d, J

= 8.5 Hz, 2H), 6.78 (d, J = 8.3 Hz, 2H), 4.10 (s, 2H), 3.94−3.88 (m,

2H), 3.86−3.80 (m, 2H), 1.36−1.27 (m, 1H), 1.21 (t, J = 6.9 Hz, 3H),

1.08−1.01 (m, 2H), 0.66 (d, J = 6.6 Hz, 6H) ppm. ¹³C NMR (101

MHz, DMSO): δ 167.6, 166.2, 158.4, 135.7, 132.8, 130.6, 128.0, 126.5,

125.3, 125.2, 117.2, 115.5, 63.7, 43.8, 37.1, 31.1, 25.8, 22.2, 14.8 ppm.

ESI: m/z calcd for C₂₂H₂₇N₂O₃[M + H]⁺, 367.20; found, 367.15;

retention time, 10.13 min.

Pharmacology. Inhibition of hBChE. BChE (E.C. 3.1.1.8, from

humans) was kindly provided by Oksana Lockridge from the University

of Nebraska Medical Center. 5,5′-Dithiobis(2-nitrobenzoic acid) and

ATC iodide were obtained from Fluka Analytical, and tacrine

hydrochloride was purchased from Sigma-Aldrich. Inhibitory activities

were evaluated using Ellman’s method.⁶⁴The stock solutions of the test

compounds were prepared in ethanol (33.3 mM) and diluted to the

desired concentrations. For the testing, 50 μL of 5,5′-dithiobis(2-

nitrobenzoic acid) and 50 μL of enzyme were added to 1.5 mL of the

buﬀer. After 50 μL of the test compound was added, the mixture was

incubated for 4.5 min. Afterward, 10 μL of acetylthiocholine iodide was

added, and the mixture was allowed to incubate for further 2.5 min.

Enzyme activity was then observed via UV (λ = 412 nm). IC₅₀values

were determined graphically from inhibition curves using Pad Prism 5.0

software. Experiments were carried out three times independently.

Inhibition of hAChE and hBChE. The hAChE inhibitory activity was

evaluated spectrophotometrically at 37 °C by Ellman’s method.⁶⁴

AChE stock solution was prepared by dissolving recombinant hAChE

lyophilized powder (Sigma, Italy) in 0.1 M phosphate buﬀer (pH = 8.0)

containing Triton X-100 0.1%. The stock solution of BChE from

human serum (Sigma, Italy) was prepared by dissolving the lyophilized

powder in an aqueous solution of gelatin 0.1%. Stock solutions of

inhibitors (1 or 2 mM) were prepared in methanol. The assay solution

consisted of a 0.1 M phosphate buﬀer at pH 8.0, with the addition of

340 μM 5,5′-dithiobis(2-nitrobenzoic acid), 0.02 unit/mL hAChE or

hBChE, and 550 μM substrate (acetylthiocholine iodide or

butyrylthiocholine iodide for AchE or BchE, respectively). Fifty

microliter aliquots of increasing concentration of the tested compound

were added to the assay solution and preincubated for 20 min at 37 °C

with the enzyme. Upon addition of the substrate, the increase in

absorbance at 412 nm was monitored for 3 min. Assays were carried out

with a blank containing all components except the enzyme to account

for the nonenzymatic reaction. The reaction rates were compared, and

the percent inhibition was calculated. IC₅₀values were determined

graphically from inhibition curves using GraphPad Prism 4.03 software.

Experiments were carried out three times independently.

Investigation of Inhibitory Constants and Mode of Inhibition

toward hAChE. To assess the mechanism of action and inhibitory

constant reciprocal plots of 1/V versus 1/[S] were constructed using

relatively low concentrations of substrate (ACTh, 0.111−0.554 mM).

The evaluation of the enzyme activity was carried out by Ellman’s

method.⁶⁴The plots were assessed by a weighted least square analysis

Ethyl 2-(4-Ethoxybenzyl)-1-isopentyl-1H-benzo[d]imidazole-5-

carboxylate (23). The reaction was carried out according to general

procedure V using ethyl 3-(2-(4-ethoxyphenyl)acetamido)-4-

(isopentylamino)benzoate 22 (2.15 g, 5.22 mmol). The crude product

was puriﬁed by column chromatography (20:1:0.1 CH₂Cl₂/MeOH/

R

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

that assumed the variance of V to be a constant percentage of V for the

entire data set. The mechanism of inhibition was assessed by comparing

the overlaid Lineweaver−Burk plots with the theoretical trends for

competitive, mixed-type, and noncompetitive inhibition. To determine

the inhibition constant K_i, slopes of reciprocal plots were replotted

against the concentration of the tested inhibitor (3e, range 0−100 nM;

4b, range 0−7.5 nM; 8, range 0−25.0 nM), and K_iwas determined as

the intersect on the negative x axis. The K′_i(dissociation constant for

the enzyme−substrate−inhibitor complex) value was determined by

plotting the apparent 1/V_maxversus inhibitor concentration.⁹²Data

analysis was performed using GraphPad Prism 4.03 software.

Experiments were carried out three times independently.

(Genova, Italy). Cells were cultured in an RPMI 1640 medium (Lonza,

Milan, IT) supplemented with 10% fetal bovine serum (FBS), 2 mM ^L-

glutamine, 100 IU/mL penicillin, 100 μg/mL ampicillin/streptomycin,

1 mM sodium pyruvate and grown at 37 °C with 5% CO₂and 95%

humidity.

Cell Thawing. Cells were removed from liquid nitrogen stocks and

warmed up in a 37 °C water bath. Then, the cells were resuspended in

10 mL of DMEM (high glucose) and 8−10% FCS. The suspension was

centrifuged for 5 min at 1000 rpm, and the supernatant was discarded.

The pellet was resuspended in 10 mL of DMEM (high glucose) with 2.5

μL (25 μg/mL) of zeocin and 8−10% FCS. The suspension was

transferred into a tissue culture ﬂask and then placed in an incubator

(37 °C, 5% CO₂, humid). The medium was changed on the following

day.

Inhibition of AChE-Induced Aβ₄₀Aggregation.⁶⁵Aβ₄₀, supplied as

triﬂuoroacetate salt, was purchased from Bachem AG (Switzerland).

Aβ₄₀(2 mg mL⁻¹) was dissolved in 1,1,1,3,3,3-hexaﬂuoro-2-propanol

(HFIP), lyophilized, and redissolved in DMSO to achieve a 2.3 mM

stock solution. Stock solutions of tested inhibitors were prepared in

methanol (2.0 mM) and diluted in the assay buﬀer. Aliquots of Aβ₄₀

peptide (2 μL) were incubated for 24 h at room temperature in 0.215 M

sodium phosphate buﬀer (pH 8.0) at a ﬁnal concentration of 230 μM.

For co-incubation experiment aliquots (16 μL) of hAChE solution

(ﬁnal concentration, 2.30 μM; Aβ/AChE molar ratio, 100:1) and

hAChE in the presence of 2 μL of the tested inhibitor (ﬁnal inhibitor

concentration, 100 μM) in 0.215 M sodium phosphate buﬀer solution

(pH 8.0) were added. Blanks containing Aβ₄₀alone, hAChE alone, and

Aβ₄₀plus tested inhibitors in 0.215 M sodium phosphate buﬀer (pH

8.0) were also prepared. The ﬁnal volume of each sample was 20 μL. To

quantify amyloid ﬁbril formation, the thioﬂavin T ﬂuorescence method

was then applied.⁹³The ﬂuorescence intensities related to ﬁbril

formation were monitored for 300 s at λ_em= 490 nm (λ_exc= 446 nm).

Fluorescence intensities of samples without and with an inhibitor were

compared, and percent inhibition was calculated.⁶⁵The experiment was

carried out two times independently, and each was performed in

duplicate.

Inhibition of Aβ₄₂Self-Aggregation. As reported in a previously

published protocol,⁶⁸HFIP-pretreated Aβ₄₂samples (Bachem AG,

Switzerland) were solubilized with a CH₃CN/0.3 mM Na₂CO₃/250

mM NaOH (48.4:48.4:3.2) mixture to obtain a 500 μM stock solution.

Experiments were performed by diluting (ﬁnal Aβ concentration, 50

μM) and incubating the peptide in 10 mM phosphate buﬀer (pH = 8.0)

containing 10 mM NaCl, at 30 °C for 24 h with and without an inhibitor

(50 μM, Aβ/inhibitor = 1:1). Blanks containing the tested inhibitors

were also prepared. Each assay was run in duplicate. To quantify

amyloid ﬁbril formation, the thioﬂavin T ﬂuorescence method was

used.⁹³After incubation, samples were diluted to a ﬁnal volume of 2.0

mL with 50 mM glycine−NaOH buﬀer (pH 8.5) containing 1.5 μM

thioﬂavin T. A 300 s time scan of the ﬂuorescence intensity was carried

out (λ_exc= 446 nm; λ_em= 490 nm, FP-6200 ﬂuorometer, Jasco Europe),

and values at the plateau were averaged after subtracting the

background ﬂuorescence of 1.5 μM thioﬂavin T solution. The

ﬂuorescence intensities obtained in the absence and in the presence

of tested inhibitors were compared, and the percent inhibition due to

the presence of the inhibitor was calculated by the following formula:

100 − (IF_i/IF_o× 100), where IF_iand IF_oare the ﬂuorescence intensities

obtained for Aβ₄₂in the presence and in the absence of inhibitor,

respectively. The experiment was carried out two times independently,

and each was performed in duplicate.

Cell Passaging. The passaging rate was determined by examining the

cells under the light microscope. The medium was discarded, and cells

were washed gently by adding 5 mL of PBS. The PBS was discarded,

and 3 mL of Trypsin/EDTA was added. The ﬂask was placed in the

incubator for 5 min. After incubation, 7 mL of DMEM (high glucose)

with 8−10% FCS was added. The cells were washed down and

transferred into a falcon tube. The tube was centrifuged for 5 min at

1000 rpm. The supernatant was discarded, and 5−10 mL of DMEM

(high glucose) with zeocin (25 μg/mL) and 8−10% FCS was added.

The cell suspension was divided into aliquots according to the

passaging rate of the cells. Cells were passaged twice a week.

Binding Studies. Membrane Preparation. The cells were passaged

according to their passaging rate (1:8) and plated on petri dishes 3 days

before preparation. The preparation itself was carried out on ice. The

medium was dispensed, and the cells were washed with 5 mL of PBS.

Then, 3.5 mL of preparation buﬀer (50 mM Tris, 1 mM MgCl₂·6H₂O, 1

mM EDTA, pH 7.4) were added. The cells were scrubbed from the

surface, and the cell suspensions were combined. The combined

suspension was treated two times for 10 s with an ultraturrax. The cell

lysate was then centrifuged for 10 min at 3200 rpm at 4 °C. The

supernatant was transferred to ultracentrifugation tubes and

centrifugated for 50 min at 37,000 rpm at 4 °C. The supernatant was

discarded, and the pellet was resuspended in binding buﬀer (50 mM

Tris, 5 mM MgCl₂·6 H₂O, 2.5 mM EDTA, pH 7.4). The protein

concentration of the pellet was determined using the Bradford assay

(according to vendor’s description). The suspension was passaged, and

the aliquots were shock-frosted in liquid nitrogen and stored at −80 °C

until further use.

Radioligand Binding Assay. SR-144,528 (inverse agonist for

hCB₂R) was purchased from Santa Cruz Biotechnology Inc. Unlabeled

CP 55,940 (agonist for hCB₂R and hCB₁R) was obtained from Sigma-

Aldrich Life Science. Radioactive labeled [³H]CP 55,940 was acquired

by Hartmann Analytic GmbH. Rimonabant (inverse agonist for

hCB₁R) was obtained by an in-house synthesis. Saturation assays

were carried out similar to Murkherjee et al.⁹⁴to determine the K_Dvalue

of the membrane samples. Saturation assays were carried out using

eight concentrations of [³H]CP 55,940, ranging from 0.088 to 4.4 nM.

Reactions were started by adding 8 μg membrane per well of a 96 well

Multiscreen ﬁlter plate (Millipore) containing the radioligand in assay

buﬀer (50 mM Tris-HCl, pH 7.4; 5 mM MgCl₂·6 H₂O; 2.5 mM EDTA;

2 mg/mL BSA). After incubating for 3 h at RT, the reaction was

stopped by vacuum ﬁltration, and each well was washed four times with

100 μL of cold binding buﬀer (50 mM Tris-HCl, pH 7.4; 5 mM MgCl₂·

6 H₂O; 2.5 mM EDTA). The ﬁlter plate was dried at 40 °C. The activity

was measured in a Microbeta Trilux counter (Wallac) using an IRGA

Safe plus-scintillation cocktail (PerkinElmer). Competition assays were

performed with 5−11 concentrations of replacing ligands (0.1 nM−0.4

mM) and 0.44 nM [³H]CP 55,940. Nonspeciﬁc binding was

determined using 10 μM 2 for hCB₂R and 10 μM rimonabant for

hCB₁R.

Radioligand Binding Studies on hCB₂R and hCB₁R and Eﬃcacy.

HEK hCB₂R Cell Line. Human embryonic kidney cells (HEK) stably

expressing the hCB₂R were grown in Dulbecco’s modiﬁed Eagle’s

medium containing high glucose supplemented with 8% fetal calf serum

and 25 μg/mL zeocin in a 37 °C incubator in the presence of 5% CO₂.

Cells were passaged twice a week.

CHO hCB₁R Cell Line. Chinese hamster ovary cells (CHO) stably

expressing the hCB₁R were grown in Ham’s F-12 Nutrient Mix

supplemented with 8% fetal calf serum and 400 μg/mL geneticin in a 37

°C incubator in the presence of 5% CO₂. Cells were passaged twice a

week.

Statistical Analysis. To determine the IC₅₀values, statistical

evaluations and sigmoidal dose−response curve ﬁttings were performed

using GraphPad Prism 5 software applying nonlinear regression and

one site ﬁt logIC₅₀as curve ﬁtting functions. K_ivalues were determined

according to the Cheng−Prusoﬀ equation when the displacement of

U266 Cell Line. The U266 cell line was purchased from ATCC (LGC

Standards, Milan, IT). Cell authentication was performed by IST

S

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

[³H]CP 55,940 was higher than 60% at 100 μM test compound

concentration

Eﬀects on Microglia. Cell Cultures. Mouse N9 microglial cells

were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM)

supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1%

penicillin/streptomycin, and 2 mM glutamine (all cell cultures’ reagents

were from Aurogene Srl, Rome, Italy). At conﬂuence, after a short wash

with sterile PBS, microglia were trypsinized for 5 min at 37 ° C, and

trypsin was inactivated with a complete DMEM medium. Detached

cells were then collected, centrifuged for 5 min at 300g, and

resuspended to be counted. For experiments, microglial cells were

plated at the density of 2.5 × 10⁵in a 35 mm Ø dish and exposed to 100

ng/mL lipopolysaccharide (LPS), in the presence or absence of

increasing concentrations of the compound to be tested. After 24 h of

treatment, microglial conditioned media were collected and partly used

for nitrite measurement, partly ﬁltered through 0.22 μm ﬁlters,

concentrated using Microcon YM-3 (Millipore, Billerica, MA), and

resuspended in 12 μL of 4× loading buﬀer (0.2 M Tris-HCl, pH 6.8; 8%

sodium dodecyl sulfate; 40% glycerol; 0.4% bromophenol blue, and 0.4

M dithiothreitol; Sigma-Aldrich) for Western blot analysis. In parallel,

microglial cells were collected in 2× loading buﬀer (LB; 50 μL per dish)

for Western blot analysis.

Western Blotting. Concentrated microglial conditioned media and

cell samples were brieﬂy sonicated and loaded into 12% sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad).

After electrophoresis and transfer onto nitrocellulose membranes

(GE Healthcare, Milano, Italy), membranes were blocked for 1 h in

blocking solution PBS−0.1% Tween-20 (Sigma-Aldrich), 4% nonfat

dry milk (Bio-Rad) and incubated overnight at 4 °C with primary

antibodies in PBS−0.1% Tween-20. Primary antibodies used were

rabbit anti-iNOS, rabbit anti-IL1β, rabbit anti-TREM2, and rabbit anti-

TGFβ2, and mouse anti-GAPDH (all 1:1000 dilution except for anti-

GAPDH used 1:20,000 dilution, all from Santa Cruz Biotechnology).

Membranes were then incubated with speciﬁc secondary antibodies

conjugated to horseradish peroxidase (goat anti-rabbit and goat anti-

mouse, both at 1:2000 dilution and from Santa Cruz) for 90 min at

room temperature in PBS−0.1% Tween-20. Labeled proteins were

visualized by using the Clarity Western ECL substrate (Bio-Rad) and

detected using Bio-Rad Image Lab software with a ChemiDoc MP

imaging system (Bio-Rad).

Nitrite Assay. Accumulation of nitrite in microglial conditioned

media was measured by a colorimetric assay based on the Griess

reaction. A nitrate standard curve was performed with NaNO₂at known

concentrations. Sulfanilamide (5 mM; Sigma-Aldrich) was added to the

culture medium and the standard curve. Sulfanilamide reacts with

nitrite under acidic conditions to form a diazonium cation, which

subsequently couples to N-1-naphthyl-ethylenediamine dihydrochlor-

ide (NEDA, 40 mM; Sigma-Aldrich) to produce a colored azo dye.

After 15 min of incubation at room temperature in the dark, absorbance

was read at 540 nm with a multiplate spectrophotometric reader (Bio-

Rad Laboratories Srl, Segrate, Milano, Italy).

Statistical Analysis. All quantitative data are presented as means

SE from at least three independent experiments. Statistical signiﬁcance

between diﬀerent treatments was calculated using GraphPad Prism 6

software applying one-way analysis of variance (ANOVA) followed by

post hoc comparison through Bonferroni’s test. A value of p < 0.05 was

considered statistically signiﬁcant.

IC₅₀

K_i=

[L*]

1 +

K_D

with [L*] as radioligand concentration (0.44 nM), and the K_ivalue was

calculated for at least two individual experiments. K_Dvalues and

standard errors were determined for hCB₂R K_D(hCB₂R) = 4.16 3.04

and for CB₁R K_D(hCB₁R) = 2.24 1.15.

hCB₂R Eﬃcacy. MTT Assay. The U266 cell line (4 × 104 cells/mL)

was plated on 96-well plates to a ﬁnal volume of 100 μL/ well. After

incubating for 1 day, compounds or vehicles were added at diﬀerent

concentrations. Six replicates were used for each treatment. At the

indicated time point, cell viability was assessed by adding 0.8 mg/mL 3-

[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)

(Sigma-Aldrich) to the medium. After 3 h, the plates were centrifuged,

the supernatant was discarded, and the pellet was solubilized with 100

μL/well DMSO. The absorbance of the samples against a background

control (medium alone) was measured at 570 nm using an ELISA

reader microliter plate (BioTek Instruments, Winooski, VT, USA). For

some experiments, 1 h of preincubation with Forskolin or AM630 was

performed. Each sample was evaluated in six wells and in two

independent experiments.

cAMP Assay. U266 cells (1 × 10⁶/mL ) were plated in 24-well plates

and treated with the appropriate compounds for 2 h. After treatment,

the cells were processed for the detection of cAMP levels, using the

cAMP assay kit (Enzo Life Sciences, Farmingdale, NY, USA) in

accordance to the manufacturer’s protocol. U266 cells were treated

with Forskolin (10 μM), AM630 (25 μM), or compound (50 μM) for 2

h. For combination treatments, U266 cells were preincubated with

AM630 (25 μM) for 30 min before adding the compounds. The

concentration of cAMP was calculated by measuring the absorbance at

450 nm with an ELISA reader. Each compound was evaluated in

duplicate and in two independent assays. cAMP levels were stated as

pmol/mg protein.

RNA Extraction and qRT-PCR. Total RNA from treated and vehicle

U266 cells was extracted using the RNeasy Mini Kit (Qiagen) at the

appropriate time points, and cDNA was synthesized using the High-

Capacity cDNA Archive Kit (Applied Biosystems, Foster City, PA)

according to the manufacturer’s instructions. Quantitative real-time

polymerase chain reactions (qRT-PCR) for MIF, STAT-3, and

GAPDH were performed using the iQ5 multicolor real-time PCR

detection system (Bio-Rad, Hercules, CA). The PCR reaction was

performed with SYBR Green qPCR mastermix (Qiagen) using 500 ng

of cDNA for the reaction, following the ampliﬁcation protocol indicated

by the manufacturer’s instruction. All samples were tested in triplicates

in the same plate, GAPDH levels were used to normalize mRNA

contents, and target gene levels were calculated by the 2^−ΔΔCTmethod.

cDNA from Forskolin-treated cells was used as CREB-induced MIF

and STAT-3 as a positive control. cDNA from MD-48-treated cells was

used for CB₂agonist MIF and STAT-3 gene expression control. cDNA

from AM630-treated cells was used for antagonist/inverse agonist CB₂

MIF and STAT-3 expression control. Each sample was evaluated in

triplicate and in three diﬀerent experiments.

Neuroprotection on HT-22 Cells. Cell Culture. HT-22 cells were

grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM, Sigma-

Aldrich, Munich, Germany) supplemented with 10% (v/v) heat-

inactivated fetal calf serum (FCS) and 1% (v/v) penicillin/

streptomycin. Cells were passaged every 2 days and incubated at 37

°C with 5% CO₂in a humidiﬁed incubator. Compounds were dissolved

in DMSO, (Sigma-Aldrich, Munich, Germany) and diluted with

medium. Generally, 80% conﬂuent cells were seeded with 5000 cells per

well into sterile 96-well plates and were incubated for 24 h.

Neurotoxicity. For the neurotoxicity assay, the previous medium was

discarded, and diﬀerent concentrations of the compound were added to

the wells. DMSO (0.5%) in DMEM served as control. Cells were

incubated for 24 h. After incubation, an MTT assay was performed.

Neuroprotection. For neuroprotection, 5 mM glutamate (mono-

sodium-^L-glutamate, Sigma-Aldrich, Munich, Germany) was co-

Statistical Analysis. The statistical signiﬁcance for MTT and qRT-

PCR assays was determined using the analysis of variance (ANOVA)

test. The calculation of IC₅₀was performed by a nonlinear ﬁt of log-dose

versus response, using GraphPad Prism 5.01 software. cAMP

concentration was calculated utilizing a four-parameter logistic (4PL)

curve ﬁtting program.

Calcium Mobilization Assay. Compounds were tested using a

ﬂuorescence-based assay. Brieﬂy, CHO-K1 cells were engineered to

either co-express hCB₁R and G_αq16or hCB₂R and G_αq16. Activation of

the receptor therefore leads to mobilization of intracellular calcium.

Cells were seeded out in 96-well plates and incubated overnight. The

next day, cells were loaded with the ﬂuorescent dye calcein-4 AM.

Calcium ﬂux was monitored using an automated plate reader

(FlexStation, Molecular Devices). Statistical analysis was performed

using GraphPad Prism 5 software.

T

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

incubated with diﬀerent concentrations of respective compounds for 24

h. Quercetin (25 μM, Sigma-Aldrich, Munich, Germany) served as a

positive control. After 24 h of incubation, an MTT assay was performed.

MTT Assay. Cell viability was determined using the 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT,

Sigma-Aldrich, Munich, Germany) assay. MTT solution (4 mg/mL

in PBS) was diluted 1:10 with medium and added to the wells after

removal of the previous medium. Cells were incubated for 3 h when the

supernatant was removed and lysis buﬀer (10% SDS) was applied. The

next day, absorbance at 560 nm was determined with a multiwell plate

photometer (Tecan, SpectraMax 250).

or number of arm entries <10) were discarded from the calculation. In

this study, 11 animals were discarded accordingly (3.2% attrition).

Passive Avoidance Test. On days 9 and 10, a passive avoidance test

was performed. The apparatus is a two-compartment (15 × 20 × 15 cm

high) box with one compartment illuminated with white polyvinyl

chloride walls and the other darkened with black polyvinyl chloride

walls and a grid ﬂoor. A guillotine door separates each compartment. A

60 W lamp positioned 40 cm above the apparatus lights up the white

compartment during the experiment. Scrambled foot shocks (0.3 mA

for 3 s) were delivered to the grid ﬂoor using a shock generator

scrambler (Lafayette Instruments, Lafayette, USA). The guillotine door

was initially closed during the training session. During the training

session, on day 9, each mouse was placed into the white compartment.

After 5 s, the door was raised. When the mouse entered the darkened

compartment and placed all its paws on the grid ﬂoor, the door was

closed, and the foot shock was delivered for 3 s. The step-through

latency, that is, the latency spent to enter the darkened compartment,

and the number of vocalizations was recorded. The retention test was

carried out 24 h after training, on day 10. Each mouse was placed again

into the white compartment. After 5 s, the door was raised. The step-

through and escape latencies (corresponding to the re-exit from the

darkened compartment) were recorded up to 300 s. Animals that show

all latencies during the training and retention session lower than 10 s are

considered as failing to respond to the procedure and were discarded

from the calculations. In this study, nine animals were discarded

accordingly (2.6% attrition).

Sacriﬁce and Brain and Liver Sampling. On day 11, all animals

were sacriﬁced, their brains dissected out to isolate the hippocampus

and cortex. Samples were frozen in liquid nitrogen and stored at −80 °C

awaiting further analysis. For treated with the high dose (3 mg/kg) of

compounds, the livers were also dissected out, post-ﬁxed in formalin,

and kept at 4 °C.

Statistical Analyses. All values, except passive avoidance latencies,

were expressed as means SEM. Statistical analyses were performed on

the diﬀerent conditions using one-way ANOVA (F value), followed by

Dunnett’s post hoc multiple comparison test. Passive avoidance

latencies do not follow a Gaussian distribution since upper cutoﬀ

times are set. They were therefore analyzed using a Kruskal−Wallis

nonparametric ANOVA (H value), followed by Dunn’s multiple

comparison test. p < 0.05 was considered as statistically signiﬁcant.

Statistical Analysis. Results are presented as percentage to untreated

control cells. Data are expressed as means

SD of three diﬀerent

independent experiments, and each was performed in sextuplicate.

Analysis was accomplished using GraphPad Prism 5 software applying

one-way ANOVA followed by Dunnett’s multiple comparison post-test.

Levels of signiﬁcance: *p < 0.05; **p < 0.01; ***p < 0.001.

In Vivo Studies. Aim of the Study. To test ﬁve compounds 3a, 3d,

3e, 4a, and 6 as protectant drugs in the in vivo mouse model of

Alzheimer’s disease induced by intracerebroventricular (i.c.v.) injection

of oligomerized Aβ₂₅₋₃₅peptide. Each compound was injected

intraperitoneally (i.p.) o.d. between day 1 and 7, as summarized in

Supplementary Figure 4. The peptide was injected on day 1, and

behavioral examination was performed between days 8 and 10. All

animals were then sacriﬁced on day 11 and their brain stored at −80 °C

awaiting further biochemical analyses. In addition, the livers of high

dose-treated animals (3 mg/kg) were removed and ﬁxed in form-

aldehyde solution.

Animals. Male Swiss mice, 6 weeks old and weighing 30−35 g, from

Janvier (Saint-Berthevin, France), were kept for housing, and

experiments took place within the animal facility building of the

University of Montpellier (CECEMA, Oﬃce of Veterinary Services

agreement #B-34-172-23). Animals were housed in groups with access

to food and water ad libitum, except during behavioral experiments.

They were kept in a temperature and humidity-controlled animal

facility on a 12 h/12 h light/dark cycle (lights oﬀ at 07:00 p.m.). All

animal procedures were conducted in strict adherence to the European

Union directive of September 22, 2010 (2010/63/UE) and authorized

(ﬁle #1485-15034) by the National Ethic Committee (Paris, France).

Drug Preparation. Compounds were weighed and solubilized in

pure DMSO, and a stock solution at 2 mg/mL was prepared in DMSO/

ddH₂O. The percentage of DMSO in ddH₂O for 3 mg/kg dose was

30%. Stock solution in DMSO was stored for 1 week at +4 °C, and the

injection solutions were made fresh daily. Vehicle solutions used for

control groups were DMSO 30% in H₂O.

Amyloid Peptide Preparation and Injection. Mice were anes-

thetized with isoﬂurane 2.5% and were injected i.c.v. with Aβ₂₅₋₃₅

peptide (9 nmol/mouse) or vehicle solution (distilled water), in a ﬁnal

volume of 3 μL/mouse, according to the previously described

method.^87,95−99Homogeneous oligomeric preparation of Aβ₂₅₋₃₅

peptide was performed by incubation for 4 days at 37 °C according

to Maurice et al.⁸⁷Since it is well established that vehicle solution

similar as a control scrambled Aβ₂₅₋₃₅peptide failed to induce toxicity

and therefore aﬀect learning abilities, vehicle-treated animals served as

controls.^87,95−100

Spontaneous Alternation Performances. On day 8, all animals

were tested for spontaneous alternation performance in the Y maze, an

index of spatial working memory. The Y maze is made of gray polyvinyl

chloride. Each arm is 40 cm long, 13 cm high, 3 cm wide at the bottom,

10 cm wide at the top, and converging at an equal angle. Each mouse

will be placed at the end of one arm and allowed to move freely through

the maze during an 8 min session. The series of arm entries, including

possible returns into the same arm, was checked visually. An alternation

was deﬁned as entries into all three arms on consecutive occasions. The

number of maximum alternations is, therefore, the total number of arm

entries minus two, and the percentage of alternation was calculated as

(actual alternations/maximum alternations) × 100. Parameters

included the percentage of alternation (memory index) and the total

number of arm entries (exploration index).^{87,95,96,98,99}Animals that

shown an extreme behavior (alternation percentage of <20% or >90%

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.9b00623.

RP-HPLC chromatograms of the lead compounds (PDF)

Molecular formula stings (CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: michael.decker@uni-wuerzburg.de. Tel.: +49-931-31-

89676.

■

ORCID

Peter Gmeiner: 0000-0002-4127-197X

Rangan Maitra: 0000-0001-6663-6800

Michael Decker: 0000-0002-6773-6245

Author Contributions

Synthesis of the target compounds was performed by M.S. and

D.D. Inhibitory, kinetic studies on hAChE and Aß aggregation

were performed by M.O., M.N., and M.B. Inhibitory potency on

hBChE and hAChE was performed by S.G. and M.H.,

radioligand binding studies on CBRs by M.S., and CB₂R eﬃcacy

by cAMP-regulated gene expression by M.N. The calcium

mobilization assay on CBRs was performed by R.M. and

U

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

neuroprotection and neurotoxicity experiments by S.G. and

M.S. Microglia eﬀects were investigated by S.P., E.P., and B.M.,

and in vivo experiments were performed by M.S. and T.M. and

liver histology by C.F. and M.V. Finally, MOR binding studies

were performed by H.H. and P.G. All authors contributed to

writing the manuscript and read and approved the ﬁnal version.

REFERENCES

■

(1) Patterson, C. World Alzheimer report 2018; ADI: London, 2018;

pp 1−48.

(2) Zemek, F.; Drtinova, L.; Nepovimova, E.; Sepsova, V.; Korabecny,

J.; Klimes, J.; Kuca, K. Outcomes of Alzheimer’s disease therapy with

acetylcholinesterase inhibitors and memantine. Expert Opin. Drug Saf.

2014, 13, 759−774.

(3) Glenner, G. G.; Wong, C. W. Alzheimer’s disease: initial report of

the purification and characterization of a novel cerebrovascular amyloid

protein. Biochem. Biophys. Res. Commun. 1984, 120, 885−890.

(4) Grundke-Iqbal, I.; Iqbal, K.; Tung, Y. C.; Quinlan, M.; Wisniewski,

H. M.; Binder, L. I. Abnormal phosphorylation of the microtubule-

associated protein τ (tau) in Alzheimer cytoskeletal pathology. Proc.

Natl. Acad. Sci. U. S. A. 1986, 83, 4913−4917.

(5) Ferrer, I. Defining Alzheimer as a common age-related

neurodegenerative process not inevitably leading to dementia. Prog.

Neurobiol. 2012, 97, 38−51.

(6) Tsai, J.; Grutzendler, J.; Duff, K.; Gan, W.-B. Fibrillar amyloid

deposition leads to local synaptic abnormalities and breakage of

neuronal branches. Nat. Neurosci. 2004, 7, 1181.

Funding

M.D. acknowledges the German Research Council (Deutsche

Forschungsgemeinschaft (DFG) DFG DE 1546/6-3 and M.D.

and T.M. also acknowledge support from Campus France (PHC

Procope) and the German Academic Exchange Service (DAAD)

with funds of the Federal Ministry of Education and Research

(BMBF). The Italian Ministry of Education, Universities and

Research (MIUR) is acknowledged for ﬁnancial support. M.H.

acknowledges the German Academic Scholarship Foundation

(“Studienstiftung des deutschen Volkes”) for a Ph.D. fellowship

and the Elite Network of Bavaria (International Doctoral

program “Receptor Dynamics”) for support.

́

(7) Gomez-Isla, T.; Hollister, R.; West, H.; Mui, S.; Growdon, J. H.;

Petersen, R. C.; Parisi, J. E.; Hyman, B. T. Neuronal loss correlates with

but exceeds neurofibrillary tangles in Alzheimer’s disease. Ann. Neurol.

1997, 41, 17−24.

Notes

The authors declare no competing ﬁnancial interest.

(8) Loo, D. T.; Copani, A.; Pike, C. J.; Whittemore, E. R.;

Walencewicz, A. J.; Cotman, C. W. Apoptosis is induced by beta-

amyloid in cultured central nervous system neurons. Proc. Natl. Acad.

Sci. U.S.A. 1993, 90, 7951−7955.

ACKNOWLEDGMENTS

■

We thank O. Lockridge (University of Nebraska Medical

Center) for providing human BChE. We thank S. Kachler and

Professor K.-N. Klotz (Institute of Pharmacology and

(9) Zhu, X.; Raina, A. K.; Lee, H. G.; Casadesus, G.; Smith, M. A.;

Perry, G. Oxidative stress signalling in Alzheimer’s disease. Brain Res.

2004, 1000, 32−39.

Toxicology, University of Wurzburg) for technical support

̈

(10) LaFerla, F. M. Calcium dyshomeostasis and intracellular

signalling in Alzheimer’s disease. Nat. Rev. Neurosci. 2002, 3, 862−872.

(11) Akiyama, H.; Barger, S.; Barnum, S.; Bradt, B.; Bauer, J.; Cole, G.

M.; Cooper, N. R.; Eikelenboom, P.; Emmerling, M.; Fiebich, B. L.;

Finch, C. E.; Frautschy, S.; Griffin, W. S. T.; Hampel, H.; Hull, M.;

Landreth, G.; Lue, L.-F.; Mrak, R.; Mackenzie, I. R.; McGeer, P. L.;

O’Banion, M. B.; Pachter, J.; Pasinetti, G.; Plata-Salaman, C.; Rogers, J.;

Rydel, R.; Shen, Y.; Streit, W.; Strohmeyer, R.; Tooyoma, I.; Van

Muiswinkel, F. L.; Veerhuis, R.; Walker, D.; Webster, S.; Wegrzyniak,

B.; Wenk, G.; Wyss-Coray, T. Inflammation and Alzheimer’s disease.

Neurobiol. Aging 2000, 21, 383−421.

(12) Wyss-Coray, T.; Mucke, L. Inflammation in neurodegenerative

diseasea double-edged sword. Neuron 2002, 35, 419−432.

(13) Barger, S. W.; Harmon, A. D. Microglial activation by Alzheimer

amyloid precursor protein and modulation by apolipoprotein E. Nature

1997, 388, 878−881.

and workspace for radioligand binding studies. Mrs. Nicola

Arveda’s technical assistance is acknowledged for the assays

involving hAChE.

ABBREVIATIONS

■

Aβ, amyloid-beta; ACh, acetylcholine; AChE, acetylcholinester-

ase; ACth, acetylthiocholine; AD, Alzheimer’s disease; ANOVA,

analysis of variance; APP, amyloid precursor protein; BBB,

blood−brain-barrier; BChE, butyrylcholinesterase; cAMP,

cyclic adenosine monophosphate; CAS, catalytic active site;

ChE, cholinesterase; CHO, Chinese hamster ovary; CNS,

central nervous system; Cpd, compound; CRE, cAMP response

element; CREB, cAMP response element-binding protein;

DMSO, dimethyl sulfoxide; FSK, forskolin; GAPDH, glycerin-

aldehyde-3-phosphat-dehydrogenase; GTP_ϒS, guanosine 5′[ϒ-

thio]triphosphate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-

tetramethyluronium hexaﬂuorophosphate; hCB_1/2R, human

cannabinoid receptor 1/2; HEK, human embryotic kidney;

i.c.v., intracerebroventricular; Il1β, interleukin 1 beta; iNOS,

inducible nitric oxide synthase; i.p.., intraperitoneal; LPS,

lipopolysaccharide; MIF, macrophage migration inhibitory

factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-

lium bromide; NMDA, N-methyl-^D-aspartate;; MOR, μ-opioid

receptor; PAS, peripheral anionic site; PEG, polyethylene glycol;

ROS, reactive oxygen species; (q)RT/PCR, (quantitative)

reverse transcription polymerase chain reaction; SAR, struc-

ture−activity relationship; SD, standard deviation; SR, scav-

enger receptor; STAT-3, signal transducer and activator of

transcription; ST-PA, step-through passive avoidance; TGFβ2,

transforming growth factor beta-2; THC, tetrahydrocannabinol;

THF, tetrahydrofuran; TNF, tumor necrosis factor; TREM2,

triggering receptor expressed on myeloid cells 2; V or VEI,

vehicle; YMT, Y-maze test

(14) Meda, L.; Cassatella, M. A.; Szendrei, G. I.; Otvos, L., Jr.; Baron,

P.; Villalba, M.; Ferrari, D.; Rossi, F. Activation of microglial cells by β-

amyloid protein and interferon-γ. Nature 1995, 374, 647−650.

(15) Yang, C.-N.; Shiao, Y.-J.; Shie, F.-S.; Guo, B.-S.; Chen, P.-H.;

Cho, C.-Y.; Chen, Y.-J.; Huang, F.-L.; Tsay, H.-J. Mechanism mediating

̈

oligomeric Aβ clearance by naive primary microglia. Neurobiol. Dis.

2011, 42, 221−230.

(16) Wang, W.-Y.; Tan, M.-S.; Yu, J.-T.; Tan, L. Role of pro-

inflammatory cytokines released from microglia in Alzheimer’s disease.

Ann. Transl. Med. 2015, 3, 136.

(17) Katona, I.; Freund, T. F. Multiple functions of endocannabinoid

signaling in the brain. Annu. Rev. Neurosci. 2012, 35, 529−558.

(18) Mechoulam, R.; Parker, L. A. The endocannabinoid system and

the brain. Annu. Rev. Psychol. 2013, 64, 21−47.

(19) Lutz, B.; Marsicano, G.; Maldonado, R.; Hillard, C. J. The

endocannabinoid system in guarding against fear, anxiety and stress.

Nat. Rev. Neurosci. 2015, 16, 705−718.

(20) Ameri, A. The effects of cannabinoids on the brain. Prog.

Neurobiol. 1999, 58, 315−348.

(21) Munro, S.; Thomas, K. L.; Abu-Shaar, M. Molecular character-

ization of a peripheral receptor for cannabinoids. Nature 1993, 365,

61−65.

V

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(22) Van Sickle, M. D.; Duncan, M.; Kingsley, P. J.; Mouihate, A.;

Urbani, P.; Mackie, K.; Stella, N.; Makriyannis, A.; Piomelli, D.;

Davison, J. S.; Marnett, L. J.; Di Marzo, V.; Pittman, Q. J.; Patel, K. D.;

Sharkey, K. A. Identification and functional characterization of

brainstem cannabinoid CB2 receptors. Science 2005, 310, 329−332.

(40) Nordberg, A.; Ballard, C.; Bullock, R.; Darreh-Shori, T.;

Somogyi, M. A review of butyrylcholinesterase as a therapeutic target

in the treatment of Alzheimer’s disease. Prim. Care Companion CNS

Disord. 2013, 15, 1−30.

(41) Hartmann, J.; Kiewert, C.; Duysen, E. G.; Lockridge, O.; Greig,

N. H.; Klein, J. Excessive hippocampal acetylcholine levels in

acetylcholinesterase-deficient mice are moderated by butyrylcholines-

terase activity. J. Neurochem. 2007, 100, 1421−1429.

(42) Furukawa-Hibi, Y.; Alkam, T.; Nitta, A.; Matsuyama, A.;

Mizoguchi, H.; Suzuki, K.; Moussaoui, S.; Yu, Q. S.; Greig, N. H.;

Nagai, T.; Yamada, K. Butyrylcholinesterase inhibitors ameliorate

cognitive dysfunction induced by amyloid-β peptide in mice. Behav.

Brain Res. 2011, 225, 222−229.

́

(23) Nunez, E.; Benito, C.; Pazos, M. R.; Barbachano, A.; Fajardo, O.;

̃

́

Gonzalez, S.; Tolon, R. M.; Romero, J. Cannabinoid CB2 receptors are

expressed by perivascular microglial cells in the human brain: an

immunohistochemical study. Synapse 2004, 53, 208−213.

(24) Grunblatt, E.; Zander, N.; Bartl, J.; Jie, L.; Monoranu, C. M.;

̈

Arzberger, T.; Ravid, R.; Roggendorf, W.; Gerlach, M.; Riederer, P.

Comparison analysis of gene expression patterns between sporadic

Alzheimer’s and Parkinson’s disease. J. Alzheimer’s Dis. 2007, 12, 291−

311.

(43) Greig, N. H.; Utsuki, T.; Ingram, D. K.; Wang, Y.; Pepeu, G.;

Scali, C.; Yu, Q. S.; Mamczarz, J.; Holloway, H. W.; Giordano, T.; Chen,

D.; Furukawa, K.; Sambamurti, K.; Brossi, A.; Lahiri, D. K. Selective

butyrylcholinesterase inhibition elevates brain acetylcholine, augments

learning and lowers Alzheimer β-amyloid peptide in rodent. Proc. Natl.

Acad. Sci. 2005, 102, 17213−17218.

́

́ ́

(25) Benito, C.; Nunez, E.; Tolon, R. M.; Carrier, E. J.; Rabano, A.;

̃

Hillard, C. J.; Romero, J. Cannabinoid CB2 receptors and fatty acid

amide hydrolase are selectively overexpressed in neuritic plaque-

associated glia in Alzheimer’s disease brains. J. Neurosci. 2003, 23,

11136−11141.

́

(26) Bisogno, T.; Oddi, S.; Piccoli, A.; Fazio, D.; Maccarrone, M.

Type-2 cannabinoid receptors in neurodegeneration. Pharmacol. Res.

2016, 111, 721−730.

(27) Ehrhart, J.; Obregon, D.; Mori, T.; Hou, H.; Sun, N.; Bai, Y.;

Klein, T.; Fernandez, F.; Tan, J.; Shytle, R. D. Stimulation of

cannabinoid receptor 2 (CB 2) suppresses microglial activation. J.

Neuroinflammation 2005, 2, 29.

(44) Maurice, T.; Strehaiano, M.; Simeon, N.; Bertrand, C.;

Chatonnet, A. Learning performances and vulnerability to amyloid

toxicity in the butyrylcholinesterase knockout mouse. Behav. Brain Res.

2016, 296, 351−360.

(45) Morphy, R.; Rankovic, Z. Designed multiple ligands. An

emerging drug discovery paradigm. J. Med. Chem. 2005, 48, 6523−

6543.

(28) Molina-Holgado, F.; Molina-Holgado, E.; Guaza, C.; Rothwell,

N. J. Role of CB1 and CB2 receptors in the inhibitory effects of

cannabinoids on lipopolysaccharide-induced nitric oxide release in

astrocyte cultures. J. Neurosci. Res. 2002, 67, 829−836.

(29) Sheng, W. S.; Hu, S.; Min, X.; Cabral, G. A.; Lokensgard, J. R.;

Peterson, P. K. Synthetic cannabinoid WIN55, 212-2 inhibits

generation of inflammatory mediators by IL-1β-stimulated human

astrocytes. Glia 2005, 49, 211−219.

(30) Wu, J.; Bie, B.; Yang, H.; Xu, J. J.; Brown, D. L.; Naguib, M.

Activation of the CB2 receptor system reverses amyloid-induced

memory deficiency. Neurobiol. Aging 2013, 34, 791−804.

(31) Martín-Moreno, A. M.; Brera, B.; Spuch, C.; Carro, E.; García-

García, L.; Delgado, M.; Pozo, M. A.; Innamorato, N. G.; Cuadrado, A.;

de Ceballos, M. L. Prolonged oral cannabinoid administration prevents

neuroinflammation, lowers β-amyloid levels and improves cognitive

performance in Tg APP 2576 mice. J. Neuroinflammation 2012, 9, 1−

15.

(32) Koppel, J.; Vingtdeux, V.; Marambaud, P.; d’Abramo, C.;

Jimenez, H.; Stauber, M.; Friedman, R.; Davies, P. CB2 receptor

deficiency increases amyloid pathology and alters tau processing in a

transgenic mouse model of Alzheimer’s disease. Mol. Med. 2013, 19,

29−36.

(33) Davies, P.; Maloney, A. J. F. Selective loss of central cholinergic

neurons in Alzheimer’s disease. Lancet 1976, 308, 1403.

̈

(34) Arendt, T.; Bruckner, M. K.; Lange, M.; Bigl, V. Changes in

(46) Proschak, E.; Stark, H.; Merk, D. Polypharmacology by design: a

medicinal chemist’s perspective on multitargeting compounds. J. Med.

Chem. 2019, 62, 420−444.

(47) Dolles, D.; Decker, M. Dual-acting compounds acting as receptor

ligands and enzyme inhibitors. In Design of hybrid molecules for drug

development, Elsevier: Amsterdam, 2017; pp 137−165.

̈

(48) Dolles, D.; Hoffmann, M.; Gunesch, S.; Marinelli, O.; Moller, J.;

Santoni, G.; Chatonnet, A.; Lohse, M. J.; Wittmann, H.-J.; Strasser, A.;

Nabissi, M.; Maurice, T.; Decker, M. Structure−activity relationships

and computational investigations into the development of potent and

balanced dual-acting butyrylcholinesterase inhibitors and human

cannabinoid receptor 2 ligands with pro-cognitive in vivo profiles. J.

Med. Chem. 2018, 61, 1646−1663.

(49) Freeman, S. E.; Dawson, R. M. Tacrine: a pharmacological

review. Prog. Neurobiol. 1991, 36, 257−277.

(50) Summers, W. K.; Majovski, L. V.; Marsh, G. M.; Tachiki, K.;

Kling, A. Oral tetrahydroaminoacridine in long-term treatment of senile

dementia, Alzheimer type. N. Engl. J. Med. 1986, 315, 1241−1245.

(51) Watkins, P. B.; Zimmerman, H. J.; Knapp, M. J.; Gracon, S. I.;

Lewis, K. W. Hepatotoxic effects of tacrine administration in patients

with Alzheimer’s disease. JAMA 1994, 271, 992−998.

(52) Milelli, A.; De Simone, A.; Ticchi, N.; H. Chen, H.; Betari, N.;

Andrisano, V.; Tumiatti, V. Tacrine-based multifunctional agents in

Alzheimer’s disease: an old story in continuous development. Curr.

Med. Chem 2017, 24, 3522−3546.

acetylcholinesterase and butyrylcholinesterase in Alzheimer’s disease

resemble embryonic developmenta study of molecular forms.

Neurochem. Int. 1992, 21, 381−396.

(35) Greig, N. H.; Utsuki, T.; Yu, Q. S.; Zhu, X.; Holloway, H. W.;

Perry, T.; Lee, B.; Ingram, D. K.; Lahiri, D. K. A new therapeutic target

in Alzheimer’s disease treatment: attention to butyrylcholinesterase.

Curr. Med. Res. Opin. 2001, 17, 159−165.

(36) Giacobini, E. Cholinergic function and Alzheimer’s disease. Int. J.

Psychiatry 2003, 18, S1−S5.

(37) Grossberg, G. T. Cholinesterase inhibitors for the treatment of

Alzheimer’s disease: getting on and staying on. Curr. Ther. Res. 2003,

64, 216−235.

(38) Darvesh, S.; Hopkins, D. A.; Geula, C. Neurobiology of

butyrylcholinesterase. Nat. Rev. Neurosci. 2003, 4, 131−138.

(39) Mesulam, M. M.; Guillozet, A.; Shaw, P.; Levey, A.; Duysen, E.;

Lockridge, O. Acetylcholinesterase knockouts establish central

cholinergic pathways and can use butyrylcholinesterase to hydrolyze

acetylcholine. Neuroscience 2002, 110, 627−639.

(53) Lin, H.; Li, Q.; Gu, K.; Zhu, J.; Jiang, X.; Chen, Y.; Sun, H.

Therapeutic agents in Alzheimer’s disease through a multi-target

directed ligands strategy: Recent progress based on tacrine core. Curr.

Top. Med. Chem. 2017, 17, 3000−3016.

(54) Chen, X.; Zenger, K.; Lupp, A.; Kling, B.; Heilmann, J.; Fleck, C.;

Kraus, B.; Decker, M. Tacrine-silibinin codrug shows neuro-and

hepatoprotective effects in vitro and pro-cognitive and hepatoprotec-

tive effects in vivo. J. Med. Chem. 2012, 55, 5231−5242.

(55) McEneny-King, A.; Osman, W.; Edginton, A. N.; Rao, P. P. N.

Cytochrome P450 binding studies of novel tacrine derivatives:

predicting the risk of hepatotoxicity. Bioorg. Med. Chem. Lett. 2017,

27, 2443−2449.

(56) Zenger, K.; Chen, X.; Decker, M.; Kraus, B. In-vitro stability and

metabolism of a tacrine−silibinin codrug. J. Pharm. Pharmacol. 2013,

65, 1765−1772.

́

(57) Page, D.; Balaux, E.; Boisvert, L.; Liu, Z.; Milburn, C.; Tremblay,

M.; Wei, Z.; Woo, S.; Luo, X.; Cheng, Y.-X.; Yang, H.; Srivastava, S.;

Zhou, F.; Brown, W.; Tomaszewski, M.; Walpole, C.; Hodzic, L.; St-

W

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Onge, S.; Godbout, C.; Salois, D.; Payza, K. Novel benzimidazole

derivatives as selective CB₂agonists. Bioorg. Med. Chem. Lett. 2008, 18,

3695−3700.

(73) Ross, R. A.; Brockie, H. C.; Stevenson, L. A.; Murphy, V. L.;

Templeton, F.; Makriyannis, A.; Pertwee, R. G. Agonist-inverse agonist

characterization at CB1 and CB2 cannabinoid receptors of L759633,

L759656 and AM630. Br. J. Pharmacol. 1999, 126, 665−672.

(74) Williams, C. cAMP detection methods in HTS: selecting the best

from the rest. Nat. Rev. Drug Discov. 2004, 3, 125−135.

(75) Waeber, G.; Thompson, N.; Chautard, T.; Steinmann, M.;

Nicod, P.; Pralong, F. P.; Calandra, T.; Gaillard, R. C. Transcriptional

activation of the macrophage migration-inhibitory factor gene by the

corticotropin-releasing factor is mediated by the cyclic adenosine 3′, 5′-

monophosphate responsive element-binding protein CREB in pituitary

cells. Mol. Endocrinol. 1998, 12, 698−705.

(76) Kato, K.; Nomoto, M.; Izumi, H.; Ise, T.; Nakano, S.; Niho, Y.;

Kohno, K. Structure and functional analysis of the human STAT3 gene

promoter: alteration of chromatin structure as a possible mechanism for

the upregulation in cisplatin-resistant cells. Biochim. Biophys. Acta Gene

Struct. Expr. 2000, 1493, 91−100.

(77) Alas, S.; Bonavida, B. Inhibition of constitutive STAT3 activity

sensitizes resistant non-Hodgkin’s lymphoma and multiple myeloma to

chemotherapeutic drug-mediated apoptosis. Clin. Cancer. Res. 2003, 9,

316−326.

(58) Dolles, D.; Nimczick, M.; Scheiner, M.; Ramler, J.; Stadtmuller,

̈

P.; Sawatzky, E.; Drakopoulos, A.; Sotriffer, C.; Wittmann, H.-J.;

Strasser, A.; Decker, M. Aminobenzimidazoles and structural isomers as

templates for dual-acting butyrylcholinesterase inhibitors and hCB2R

ligands to combat neurodegenerative disorders. ChemMedChem 2016,

11, 1270−1283.

(59) Dolles, D.; Strasser, A.; Wittmann, H. J.; Marinelli, O.; Nabissi,

M.; Pertwee, R. G.; Decker, M. The first photochromic affinity switch

for the human cannabinoid receptor 2. Adv. Ther. 2018, 1, 1700032.

(60) Nimczick, M.; Pemp, D.; Darras, F. H.; Chen, X.; Heilmann, J.;

Decker, M. Synthesis and biological evaluation of bivalent cannabinoid

receptor ligands based on hCB2R selective benzimidazoles reveal

unexpected intrinsic properties. Bioorg. Med. Chem. 2014, 22, 3938−

3946.

(61) Mishra, P.; Kumar, A.; Panda, G. Anti-cholinesterase hybrids as

multi-target-directed ligands against Alzheimer’s disease (1998-2018).

Bioorg. Med. Chem. 2019, 27, 895−930.

(62) Minarini, A.; Milelli, A.; Tumiatti, V.; Rosini, M.; Simoni, E.;

Bolognesi, M. L.; Andrisano, V.; Bartolini, M.; Motori, E.; Angeloni, C.;

Hrelia, S. Cystamine-tacrine dimer: a new multi-target-directed ligand

as potential therapeutic agent for Alzheimer’s disease treatment.

Neuropharmacology 2012, 62, 997−1003.

(78) Zheng, Y.; Wang, Q.; Li, T.; Qian, J.; Lu, Y.; Li, Y.; Bi, E.; Reu, F.;

Qin, Y.; Drazba, J.; Hsi, E.; Yang, J.; Cai, Z.; Yi, Q. Role of myeloma-

derived MIF in myeloma cell adhesion to bone marrow and

chemotherapy response. J. Natl. Cancer Inst. 2016, 108, djw131.

(79) Fulp, A.; Zhang, Y.; Bortoff, K.; Seltzman, H.; Snyder, R.; Wiethe,

R.; Amato, G.; Maitra, R. Pyrazole antagonists of the CB1 receptor with

reduced brain penetration. Bioorg. Med. Chem. 2016, 24, 1063−1070.

(80) Seltzman, H. H.; Shiner, C.; Hirt, E. E.; Gilliam, A. F.; Thomas, B.

F.; Maitra, R.; Snyder, R.; Black, S. L.; Patel, P. R.; Mulpuri, Y.;

Spigelman, I. Peripherally selective cannabinoid 1 receptor (CB1R)

agonists for the treatment of neuropathic pain. J. Med. Chem. 2016, 59,

7525−7543.

(63) Hu, M.-K.; Wu, L.-J.; Hsiao, G.; Yen, M.-H. Homodimeric

tacrine congeners as acetylcholinesterase inhibitors. J. Med. Chem.

2002, 45, 2277−2282.

(64) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Featherstone, R.

M. A new and rapid colorimetric determination of acetylcholinesterase

activity. Biochem. Pharmacol. 1961, 7, 88−95.

(65) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. β-Amyloid

aggregation induced by human acetylcholinesterase: inhibition studies.

Biochem. Pharmacol. 2003, 65, 407−416.

̈

(81) Schramm, S.; Huang, G.; Gunesch, S.; Lang, F.; Roa, J.; Hogger,

(66) Castro, A.; Martinez, A. Targeting beta-amyloid pathogenesis

through acetylcholinesterase inhibitors. Curr. Pharm. Des. 2006, 12,

4377−4387.

́

P.; Sabate, R.; Maher, P.; Decker, M. Regioselective synthesis of 7-O-

esters of the flavonolignan silibinin and SARs lead to compounds with

overadditive neuroprotective effects. Eur. J. Med. Chem. 2018, 146, 93−

107.

(67) Zha, X.; Lamba, D.; Zhang, L.; Lou, Y.; Xu, C.; Kang, D.; Chen,

L.; Xu, Y.; Zhang, L.; De Simone, A.; Samez, S.; Persaresi, A.; Stojan, J.;

Lopez, M. G.; Egea, J.; Andrisano, V.; Bartolini, M. Novel tacrine−

benzofuran hybrids as potent multitarget-directed ligands for the

treatment of Alzheimer’s disease: design, synthesis, biological

evaluation, and X-ray crystallography. J. Med. Chem. 2016, 59, 114−

131.

(82) Schramm, S.; Gunesch, S.; Lang, F.; Saedtler, M.; Meinel, L.;

̈

Hogger, P.; Decker, M. Investigations into neuroprotectivity, stability,

and water solubility of 7-O-cinnamoylsilibinin, its hemisuccinate and

dehydro derivatives. Arch. Pharm. 2018, 351, 1800206.

(83) Davis, J. B.; Maher, P. Protein kinase C activation inhibits

glutamate-induced cytotoxicity in a neuronal cell line. Brain Res. 1994,

652, 169−173.

(68) Bartolini, M.; Bertucci, C.; Bolognesi, M. L.; Cavalli, A.;

Melchiorre, C.; Andrisano, V. Insight into the kinetic of amyloid β (1−

42) peptide self-aggregation: elucidation of inhibitors’ mechanism of

action. ChemBioChem 2007, 8, 2152−2161.

(84) Murphy, T. H.; Miyamoto, M.; Sastre, A.; Schnaar, R. L.; Coyle, J.

T. Glutamate toxicity in a neuronal cell line involves inhibition of

cystine transport leading to oxidative stress. Neuron 1989, 2, 1547−

1558.

(69) Lange, J. H. M.; van Stuivenberg, H. H.; Coolen, H. K. A. C.;

Adolfs, T. J. P.; McCreary, A. C.; Keizer, H. G.; Wals, H. C.; Veerman,

W.; Borst, A. J. M.; de Looff, W.; Verveer, P. C.; Kruse, C. G.

Bioisosteric replacements of the pyrazole moiety of rimonabant:

synthesis, biological properties, and molecular modeling investigations

of thiazoles, triazoles, and imidazoles as potent and selective CB1

cannabinoid receptor antagonists. J. Med. Chem. 2005, 48, 1823−1838.

(70) Yao, B. B.; Hsieh, G. C.; Frost, J. M.; Fan, Y.; Garrison, T. R.;

Daza, A. V.; Grayson, G. K.; Zhu, C. Z.; Pai, M.; Chandran, P.; Salyers,

A. K.; Wensink, E. J.; Honore, P.; Sullivan, J. P.; Dart, M. J.; Meyer, M.

D. In vitro and in vivo characterization of A-796260: a selective

cannabinoid CB2 receptor agonist exhibiting analgesic activity in

rodent pain models. Br. J. Pharmacol. 2008, 153, 390−401.

(71) Nimczick, M.; Decker, M. New approaches in the design and

development of cannabinoid receptor ligands: multifunctional and

bivalent compounds. ChemMedChem 2015, 10, 773−786.

(72) Howlett, A. C.; Barth, F.; Bonner, T. I.; Cabral, G.; Casellas, P.;

Devane, W. A.; Felder, C. C.; Herkenham, M.; Mackie, K.; Martin, B.

R.; Mechoulam, R.; Pertwee, R. G. International Union of

Pharmacology. XXVII. Classification of cannabinoid receptors.

Pharmacol. Rev. 2002, 54, 161−202.

(85) Tan, S.; Wood, M.; Maher, P. Oxidative stress induces a form of

programmed cell death with characteristics of both apoptosis and

necrosis in neuronal cells. J. Neurochem. 1998, 71, 95−105.

(86) Tan, S.; Schubert, D.; Maher, P. Oxytosis: a novel form of

programmed cell death. Curr. Top. Med. Chem. 2001, 1, 497−506.

(87) Maurice, T.; Lockhart, B. P.; Privat, A. Amnesia induced in mice

by centrally administered β-amyloid peptides involves cholinergic

dysfunction. Brain Res. 1996, 706, 181−193.

̈

(88) Lahmy, V.; Meunier, J.; Malmstrom, S.; Naert, G.; Givalois, L.;

Kim, S. H.; Villard, V.; Vamvakides, A.; Maurice, T. Blockade of Tau

hyperphosphorylation and Aβ 1−42 generation by the amino-

tetrahydrofuran derivative ANAVEX2-73, a mixed muscarinic and σ 1

receptor agonist, in a nontransgenic mouse model of Alzheimer’s

disease. Neuropsychopharmacology 2013, 38, 1706−1723.

(89) McQuiston, A. R.; Saggau, P. Mu-opioid receptors facilitate the

propagation of excitatory activity in rat hippocampal area CA1 by

disinhibition of all anatomical layers. J. Neurophysiol. 2003, 90, 1936−

1948.

(90) Batts, K. P.; Ludwig, J. An update on terminology and reporting.

Am. J. Surg. Pathol. 1995, 19, 1409−1417.

X

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(91) Mathew, T.; Karunanithy, R.; Yee, M.; Natarajan, P.

Hepatotoxicity of dimethylformamide and dimethylsulfoxide at and

above the levels used in some aflatoxin studies. Lab. Invest. 1980, 42,

257−262.

(92) Silverman, R. B. The organic chemistry of enzyme-catalyzed

reactions. Academic Press: London, 2002.

(93) Naiki, H.; Higuchi, K.; Nakakuki, K.; Takeda, T. Kinetic analysis

of amyloid fibril polymerization in vitro. Lab. Invest. 1991, 65, 104−110.

(94) Mukherjee, S.; Adams, M.; Whiteaker, K.; Daza, A.; Kage, K.;

Cassar, S.; Meyer, M.; Yao, B. B. Species comparison and

pharmacological characterization of rat and human CB₂cannabinoid

receptors. Eur. J. Pharmacol. 2004, 505, 1−9.

(95) Maurice, T.; Su, T.-P.; Privat, A. Sigma1 (σ1) receptor agonists

and neurosteroids attenuate β25−35-amyloid peptide-induced amnesia

in mice through a common mechanism. Neuroscience 1998, 83, 413−

428.

(96) Meunier, J.; Ieni, J.; Maurice, T. The anti-amnesic and

neuroprotective effects of donepezil against amyloid beta25-35

peptide-induced toxicity in mice involve an interaction with the

sigma1 receptor. Br. J. Pharmacol. 2006, 149, 998−1012.

(97) Meunier, J.; Villard, V.; Givalois, L.; Maurice, T. The γ-secretase

inhibitor 2-[(1R)-1-[(4-chlorophenyl) sulfonyl](2, 5-difluorophenyl)

amino] ethyl-5-fluorobenzenebutanoic acid (BMS-299897) alleviates

Aβ1−42 seeding and short-term memory deficits in the Aβ25−35

mouse model of Alzheimer’s disease. Eur. J. Pharmacol. 2013, 698,

193−199.

(98) Villard, V.; Espallergues, J.; Keller, E.; Alkam, T.; Nitta, A.;

Yamada, K.; Nabeshima, T.; Vamvakides, A.; Maurice, T. Antiamnesic

and neuroprotective effects of the aminotetrahydrofuran derivative

ANAVEX1-41 against amyloid β 25−35-induced toxicity in mice.

Neuropsychopharmacology 2009, 34, 1552−1566.

(99) Villard, V.; Espallergues, J.; Keller, E.; Vamvakides, A.; Maurice,

T. Anti-amnesic and neuroprotective potentials of the mixed

muscarinic receptor/sigma₁(σ₁) ligand ANAVEX2-73, a novel

aminotetrahydrofuran derivative. J. Psychopharmacol. 2011, 25,

1101−1117.

(100) Zussy, C.; Brureau, A.; Delair, B.; Marchal, S.; Keller, E.; Ixart,

G.; Naert, G.; Meunier, J.; Chevallier, N.; Maurice, T.; Givalois, L.

Time-course and regional analyses of the physiopathological changes

induced after cerebral injection of an amyloid β fragment in rats. Am. J.

Pathol. 2011, 179, 315−334.

Y

DOI: 10.1021/acs.jmedchem.9b00623

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Dual-Acting Cholinesterase-Human Cannabinoid Receptor 2 Ligands Show Pronounced Neuroprotection in Vitro and Overadditive and Disease-Modifying Neuroprotective Effects in Vivo

DOI: 10.1021/acs.jmedchem.9b00623

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.9b00623

Full text of DOI:10.1021/acs.jmedchem.9b00623

Products guided by the article

R&D Labs maybe for 474018-94-5

Relevant to this article

Hot Product