Rational design and biological evaluation of a new class of thiazolopyridyl tetrahydroacridines as cholinesterase and GSK-3 dual inhibitors for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2020.112751

A key factor in the success of the MTDLs drug discovery approach is the selection of suitable target proteins. Based on the results of our previous research regarding dual-target inhibitors of AChE/GSK-3β and analysis of target proteins, in the current study, 28 hybrids were designed and synthesized. Docking studies allowed us to rationalize the binding mode of the synthesized compounds in both targets. In vitro enzyme inhibition studies identified compound GT15 as a lead molecule with preferential AChE/GSK-3β inhibition (hAChE IC₅₀ = 1.2 ± 0.1 nM; hGSK-3β IC₅₀ = 22.2 ± 1.4 nM). In addition, GT15 showed high kinase selectivity for GSK-3, except for DYRK1, with inhibition rate of 83.69% and 67.94% against DYRK1α and DYRK1β at a concentration of 20 μM. The compound also exhibited good permeability across the blood-brain-barrier and ability to inhibit the phosphorylation of tau protein. Upon oral administration, GT15 exhibited promising cognitive improvement in the scopolamine-induced cognitive deficit mice in the Morris water maze model. These results suggest that AChE and GSK-3 based multitargeted approach have therapeutic potential for Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2020.112751

European Journal of Medicinal Chemistry 207 (2020) 112751
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Rational design and biological evaluation of a new class of
thiazolopyridyl tetrahydroacridines as cholinesterase and GSK-3 dual
inhibitors for Alzheimer’s disease
Xueyang Jiang a, Junting Zhou a, Yang Wang b, Lei Chen f, Yan Duan c, Jianping Huang c,
,
g
,
, *
Chang Liu b, Yao Chen e, Wenyuan Liu b, Haopeng Sun d, Feng Feng a **, Wei Qu a
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, 211198, China
b Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 211198, China
c School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China
d Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, 211198, China
e School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China
f School of Pharmacy, Gannan Medical University, Ganzhou, 341000, China
g Jiangsu Food and Pharmaceutical Science College, No.4 Meicheng Road, Huai’an, 223003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A key factor in the success of the MTDLs drug discovery approach is the selection of suitable target
proteins. Based on the results of our previous research regarding dual-target inhibitors of AChE/GSK-3
and analysis of target proteins, in the current study, 28 hybrids were designed and synthesized. Docking
studies allowed us to rationalize the binding mode of the synthesized compounds in both targets. In vitro
Received 31 May 2020
Received in revised form
6 July 2020
Accepted 9 August 2020
Available online 22 August 2020
b
enzyme inhibition studies identified compound GT15 as a lead molecule with preferential AChE/GSK-3
inhibition (hAChE IC50 ¼ 1.2 0.1 nM; hGSK-3 IC50 ¼ 22.2 1.4 nM). In addition, GT15 showed high
kinase selectivity for GSK-3, except for DYRK1, with inhibition rate of 83.69% and 67.94% against DYRK1
and DYRK1 at a concentration of 20 M. The compound also exhibited good permeability across the
b
b
a
Keywords:
Alzheimer’s disease
b
m
blood-brain-barrier and ability to inhibit the phosphorylation of tau protein. Upon oral administration,
GT15 exhibited promising cognitive improvement in the scopolamine-induced cognitive deficit mice in
the Morris water maze model. These results suggest that AChE and GSK-3 based multitargeted approach
have therapeutic potential for Alzheimer’s disease.
Glycogen synthase kinase3
Acetylcholinesterase
Multi-target-directed ligands
b
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
the Food and Drug Administration (FDA), which include cholines-
terase (ChE) inhibitors and N-methyl-D-aspartate (NMDA) receptor
As
a
common chronic neurodegenerative syndrome, Alz-
noncompetitive antagonists. Nevertheless, all of the approved
drugs provide only temporary symptomatic relief in the mild-to-
moderate phase of the disease and are incapable of stopping its
progression [3].
Despite extensive efforts of academic research groups and
pharmaceutical companies over the past decades, no curative
heimer’s disease (AD) is characterized by severe memory and
cognitive decline and mental and motor disorders [1]. According to
the "World Alzheimer’s Disease Report 2019", this cost is predicted
to surge to US$2 trillion by 2030, while the number of AD patients
worldwide is expected to reach 152 million by 2050 [2]. As a
chronic disease, AD not only exposes the patient’s family to phys-
ical, mental, and economic pressures, but also imposes a heavy
burden on the society. Several drugs for AD have been approved by
therapies have been found so far [4]. In recent years, A
b monoclonal
antibodies and -secretase (BACE1) inhibitors developed by phar-
b
maceutical companies such as Eli Lilly [5], Eisai [6] and Merck [7]
have all ceased in phase III clinical trials, although they can
significantly reduce or even completely eliminate A
patients. The most prominent pathological changes in the brain of
AD patients include brain atrophy, amyloid- (A ) plaque deposi-
tion in extracellular brain parenchyma, neurofibrillary tangles
b in the brain of
* Corresponding author.
b
b
** Corresponding author. Department of Natural Medicinal Chemistry, China
Pharmaceutical University, Nanjing, 211198, China.
E-mail address: popoqzh@126.com (W. Qu).
https://doi.org/10.1016/j.ejmech.2020.112751
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
(NFTs) in neurons, and abnormalities of microtubule-related tau
protein, and loss of neurons and synapses [8]. These factors ulti-
mately lead to progressive loss of cholinergic neurons, memory loss
and cognitive dysfunction [9]. As a consequence of multifactorial
and sporadic nature of the AD pathogenesis, it is difficult to effec-
tively control AD by interfering with single targets [10]. For the
systemic disease of AD, it may be more advantageous to rationally
regulate the neural microenvironment using multi-target drugs.
Multi-target-directed ligands (MTDLs) with a single chemical entity
appear to constitute more promising strategies for the treatment of
complex systemic diseases with pathological mechanisms [11e13].
In recent years, the development of MTDLs has made great progress
for anti-neurodegenerative diseases. In 2019, Lumateperone
developed by Intra-Cellular Therapies was approved by the FDA for
the treatment of adult schizophrenia. Ladostigil, developed by
Avraham Pharmaceuticals, has entered phase II clinical trial for the
treatment of mild cognitive impairment, which can inhibit the
activity of AChE and monoamine oxidase [14].
Fig. 1. The potential neuropathology involved in GSK-3
ways relevant to the development of Alzheimer’s disease.
b and AChE in different path-
Cholinergic projection neurons are primarily distributed in the
basal forebrain and striatum, and emit projection fibers to other
brain regions, participating in exercise, awakening, learning, and
memory functions [15]. The central cholinergic transmitter
acetylcholine (ACh) is essential for maintaining normal learning
and memory in mammals. As one of the clinical symptoms of AD,
cognitive impairment is closely related to the degradation of
cholinergic neurons and the reduction of ACh [16]. The level of
metabolic enzyme acetylcholinesterase (AChE) in the hippocampus
of patients is significantly higher than that of healthy individuals,
which makes the intrasynaptic ACh content insufficient [17].
Therefore, the strategy to restore the patient’s cholinergic level by
inhibiting AChE is an effective way, which has been clinically
verified [18].
Glycogen synthase kinase 3 (GSK-3) is a constitutively active
serine-threonine kinase with two isomers, namely GSK-3
GSK-3 [19]. In the central nervous system (CNS), GSK-3 is mainly
concentrated in neurons and astrocytes, and its expression levels
increases with age [20]. GSK-3 is found hyperactive in the brain of
AD patients and is involved in the regulation of many key cell
biology pathways [21]. As a major tau kinase, GSK-3 involved in
a and
b
b
b
b
the development of the AD tau pathology [22e24]. The transgenic
Drosophila model successfully demonstrated that the aberrant
Fig. 2. The representative tacrine-based AChE/GSK-3 dual inhibitors.
upregulation of GSK-3
toxic tau aggregates [25]. In vitro and in vivo evidence has shown
that GSK-3 activation is involved in A formation and accumula-
tion in the AD brain by modulating the cleavage of amyloid pre-
cursor protein (APP) [26,27]. Transgenic mice overexpressing GSK-
b positively correlates with accumulation of
dual of AChE/GSK-3b inhibitors [40]. However, the unsatisfactory
b
b
targets efficacy and physicochemical properties of this type of hy-
brids have hindered their further development studies. In pre-
sented study, we fully considered the structural characteristics of
3b display reduction in proliferation and maturation of new func-
AChE and GSK-3b, and designed another class of compounds by
tional dentate gyrus neurons and significant memory impairments
[28e31]. Numerous previous studies have demonstrated the pres-
ence of chronic inflammation in the brains of AD patients [32,33].
introducing tetrahydroacridine fragments from different positions
of the thiazolopyridine group (Fig. 3). Herein, we would like to
report their synthesis and in vitro kinase inhibition, selectivity as-
says, and cell-based evaluation.
GSK-3
flammatory response in microglia via multiple pathways [34e36].
GSK-3 inhibitor Tideglusib have entered phase II clinical trial for
the treatment of AD and progressive supranuclear [37,38].
GSK3 and AChE are potential pathological links involved in
b has been identified as an important regulator of the in-
b
2. Results and discussion
b
different aspects and pathways related to the development of
2.1. Molecular design of GSK-3b/AChE dual-target inhibitors
Alzheimer’s disease neuropathology.
Given the above evidence (Fig. 1), AChE and GSK-3
b
appear to be
The two main strategies for developing multi-target drugs are
based on fusion and combination strategies [12]. The hybrid ligand
strategy allows one molecule to display two different pharmaco-
logical activities by linking different active fragments. For the GSK-
promising candidates for the multitarget strategy for the treatment
of AD. However, only two studies describing AChE/GSK-3 dual in-
hibitors have been reported (Fig. 2) [39,40]. K. Oukoloff et al. syn-
thesized a series of eight AChE/GSK-3 dual inhibitors containing
tacrine and isoindolone scaffolds, making use of the copper(I)-
catalysed azide alkyne cycloaddition reaction to link the two moi-
eties [39]. We previous reported the discovery of the first-in-class
3b binding moiety, the ATP competitive inhibitor G1 was selected
(Fig. 3), because of its high affinity and the ability to reduce phos-
phorylated tau in vivo [41]. The previous structure-activity rela-
tionship (SAR) studies indicated that: a) the introduction of a linker

X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
3
Fig. 3. Design strategy of dual GSK-3
b/AChE inhibitors. (A) the co-crystal structure of G1 in the ATP binding domain of GSK-3
b
(PDB id: 4PTC), and the pink dotted line is the solvent
exposed area; (B) Further optimization of hybrids through linking tacrine fragment from the two solvent exposure sites of G1. (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.)
from the amide position of the GSK-3
binding of the compound to GSK-3 , thereby affecting the inhibi-
tory GSK-3 activity; b) to allow the tacrine fragment to protrude
b
inhibitor G1 affects the
respectively. The following acylation with chloroacetyl chloride
afforded 14aꢀ14i, which were further reacted with 9aꢀ9g to give
the desired compounds GT15ꢀGT28 in moderate yield.
b
b
into the AChE, the interior of the narrow catalytic pocket requires a
long enough distance between the tacrine moiety and the pyr-
idothiazole fragment. Based on the previous SAR work of G1, the
optimal attachment position for a linker was identified as the hy-
droxyl moiety of the thiazole ring. We, therefore, retained the
thiazole cyclic carboxamide group and evaluated several different
types of chains of various lengths at the hydroxyl position of the
thiazole ring to obtain the hybrid compounds GT1ꢀGT11 (Fig. 3).
The cyclopropanoyl group of G1 is another solvent exposed area,
which is considered to be the ideal connection site. Therefore, the
ortho-amino group of the pyridine of G1 was connected to tacrine
via an alkyl chain to obtain compounds GT15-GT28. Importantly,
the structure of these compounds is band-shaped, which may be
more suitable for the narrow catalytic cavity of AChE.
2.3. Enzyme inhibitory activity
The main optimization effort is to explore the effect of the
connectionpositionof thepyridinethiazolefragmentontheactivities,
aiming at identifying a new inhibitor with improved GSK-3
tion activity while maintaining acceptable AChE inhibition activity.
Initially, the abilityof the compounds to inhibit both GSK-3 and ChEs
was investigated and compared to SB216763 and tacrine as reference
compounds for the GSK-3 and ChEs inhibition, respectively. The
parent compound G1 was also used for the comparison of the activity.
Furthermore, The GSK-3 inhibitory activity of compounds was
b inhibi-
b
b
b
evaluated by using a Kinase-Glo luminescent assay [42], which
quantifies the decrease in the ATP levels following a kinase reaction.
Compounds displayed low nanomolar activities against GSK-3
b
2.2. Chemical synthesis
(6 nM ꢁ IC50 ꢁ 83 nM), demonstrating their better suitability for
extracting the linking chain from the hydroxyl position of the thiazole
ring in G1. Extending the length of the linker chain can reduce the
effects of the tacrine fragment, thereby increasing the kinase inhibi-
tory activity of the compound (inhibitoryactivity: GT6 < GT7 < GT11).
The ChEs inhibition was examined using the colorimetric Ellman’s
assay (Table 1) [43]. Considering ChEs, the compounds that are
directly linked to triazolo and tacrine fragments (GT1, GT2, GT3)
The synthesis of the target compounds GT1ꢀGT11 was divided
into two parts, i.e., the preparation of the pyridothiazole and acri-
dine building blocks, which were coupled using a click chemistry
approach in the final step. The synthesis of the key pyridothiazole
ester 6 intermediates has been published previously [40]. In the
first instance, a nucleophilic substitution reaction of intermediates
1aꢀ1c with ethanolamine resulted in the formation of tacrine de-
rivatives 2aꢀ2c. The hydroxyl groups of 2aꢀ2c were subsequently
activated to form esters 3aꢀ3c in the presence of methanesulfonyl
chloride (MsCl). Heating 3aꢀ3c with an excess of sodium azide
yielded azide derivatives 4aꢀ4c (Scheme 1). Compound 6 served as
the starting material in the Mitsunobu reaction, which was fol-
lowed by hydrolysis and condensation to afford the acetylenic in-
termediates 8aꢀ8d. In the last step, azides 4aꢀ4c and 5 were
coupled with alkynes 8aꢀ8d using click chemistry to yield com-
pounds GT1ꢀGT11 (Scheme 1).
exhibited low AChE inhibitory activity (IC50 > 1 mM), which may be
affected by the position of the triazolium fragment. The amino acid
residueTrp86islocatedatthebottom ofcatalyticallyactivesite(CAS);
thus, a certain angle between the tacrine scaffold and the linker of the
compoundisrequiredtoform asuitablep-pstackinginteractionwith
the indole group of the Trp86 residue. However, the poor flexibility of
the triazole-linked chain, coupled with the narrower CAS cavity of
AChE, make it challenging for the tacrine fragment to form such an
interaction. Nonetheless, since the cavity of the BChE catalytic region
is relatively large and displays a certain tolerance for rigidity, the
compounds show BChE inhibitory activity. In order to improve the
flexibility, we designed compounds GT4ꢀGT11 by adding two addi-
tional carbon atoms between the triazole and tacrine scaffolds. For
most of the resulting compounds, the AChE inhibitory activity was
improved, with IC50 values ranging from 36 to 269 nM. The effect of
the length of the linking chain on the AChE inhibitory activities of the
compounds is obvious. For example, when the chain length was
increased to nine carbon atoms, the inhibitory activity significantly
improved (GT9, IC50 ¼ 12 nM). We hypothesize that this outcome is
To explore another solvent-exposed area of GSK-3
b for SAR
studies, novel compounds GT12ꢀGT28 have been synthesized here
as shown in Scheme 2. Intermediates 9aꢀ9g were obtained by
nucleophilic substitution of 9-Cl-tetrahydroacridine derivatives
1aꢀ1c with diamines. Compounds GT12ꢀGT14 were obtained by
Mitsunobu reaction and hydrolysis reaction of 10aꢀ10c, followed
by further condensation with 9aꢀ9g. For the synthesis of com-
pounds GT15ꢀGT28, 10aꢀ10c was converted to 13aꢀ13i via Mit-
sunobu reaction, hydrolysis reaction and condensation,

4
X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
Scheme 1. Synthesis of hybrids GT1ꢀGT11aaReagents and conditions: (a) ethanolamine, isoamyl alcohol, 125 ꢂC, 24 h, 88e92%; (b) MsCl, TEA, DMAP, dry DCM, r.t., 24 h, 75e80%; (c)
NaN3, DMF, 80 ꢂC, 8 h, 68e88%; (d) alkynols, PPh3, DIAD, THF, r.t., 24 h, 80e88%; (e) 2 N LiOH, MeOH/THF, r.t., 8 h, 75e80%; (f) CH3NH2·HCl, (NH4Cl for 8d) HATU, DIPEA, DMF, r.t.,
24 h, 79e81%; (g) 4aꢀ4c or 5 (5 for GT1ꢀGT3), sodium ascorbate, CuSO4, TEA, n-butanol/H2O, r.t., 24 h, 59e81%.
Scheme 2. Synthesis of hybrids GT12ꢀGT28aaReagents and conditions: (a) diamines, NaI, phenol, 160 ꢂC, 2 h; (b) i: cyclopropanemethanol, DIAD, PPh3, r.t., 24 h; ii: 2 N LiOH, MeOH,
r.t., 7 h; (c) 9aꢀ9g, HATU, DIPEA, DMF, r.t., 10 h; (d) i: alcohols, DIAD, PPh3, r.t., 24 h; ii: 10 N NaOH, MeOH, reflux, 10 h; (e) amines, HATU, DIPEA, DMF, r.t., 10 h; (f) chloroacetyl
chloride, DCM, NaH, DMAP, reflux, 24 h; (g) 9aꢀ9g, K2CO3, MeCN, reflux, 4 h.
due to the long linker being able to expose the pyrithiazole fragment
to peripheral anion site (PAS). Notably, the 6-Chloro-substituted
compound (GT9) display the most optimal AChE inhibitory activities
and highest selectivities. However, the 5-OMe-substituted com-
pounds (GT7, GT8 and GT11) lost nearly all of their AChE inhibitory
activities, which might be due to the steric hindrance created by the
methoxy group preventing tacrine fragment from occupying the CAS
through the cavity of the elongated AChE.
on the thiazole ring led to compound GT15, of which GSK-3b
inhibitory activity is twice that of GT20. SAR showed GT15 turned out
to be the most potent compound, with a formamide group and
cyclopropyl methyl ether group. In addition, compounds
GT15ꢀGT28 have stronger AChE inhibitory activity compared to
compounds GT12ꢀGT14. The alkyl chain extension of compound
GT15 such as butane (GT21), hexane (GT22), and octane (GT23) was
detrimental to the inhibition activity. The replacement of the
cyclopropylmethyl group of GT15 by hydrophobic group such as
methyl (GT24), tert-butyl (GT26), and isopropyl (GT27) was also
detrimental to AChE inhibition activity. Most compounds have better
inhibitory activity on AChE versus BChE, except for GT22 and GT23.
Introducing the tacrine fragment from the carboxamide
(GT12ꢀGT14) position of G1 was detrimental to GSK-3
b inhibition
activity. The ortho-aminopyridine of compound G1 is also located in
the solvent-exposed region (Fig. 3). Numerous structural optimiza-
tions of GSK-3b inhibitors have also focused on this region [44].
Therefore, we synthesized and tested compounds GT15ꢀGT28
2.4. Blood-brain barrier (BBB) Permeation
(Table 2). The extension of the chain length is not conducive to the
inhibition of GSK-3
pound’s ability to inhibit GSK-3
compounds GT15 (n ¼ 1), GT21 (n ¼ 3), and GT22 (n ¼ 5). The
substituents, such as ethyl (GT18) and propyl (GT19) on carboxamide
group on the thiazole ring are not favorable to the inhibition of GSK-
b
. As length of the linker increases, the com-
Due to the tight junction structure of the BBB, the main
approach for drugs to enter the brain is through transcellular pas-
sive diffusion. The parallel artificial membrane permeability assay
(PAMPA) can estimate the passive permeability of compound by
measuring the effective permeability rate (Pe).. To verify the mea-
surement method of permeability, we used 10 commercial drugs,
b
decreases, as can be seen from
3b. Interestingly, the methylation of the carboxamide group position

X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
5
Table 1
Inhibition of the enzymatic activities of hGSK-3
b
, hAChE, and hBChE by compounds (GT1ꢀGT11).
Cpd.
R1
R2
X
IC50 (nM)a
hGSK-3
b
hAChE
hBChE
GT1
GT2
GT3
e
-CH3
-CH3
H
-CH2OCH2-
-CH2CH2eCH2CH2O-
-CH2CH2-
83.9 9.9
51.7 7.5
6.5 0.9
>1000
>1000
>1000
200 37
600 81
>1000
e
e
GT4
GT5
GT6
GT7
GT8
GT9
GT10
GT11
Tac.
6-Cl
H
H
5-OMe
5-OMe
6-Cl
H
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH2-
-CH2-
-CH2CH2-
-CH2-
46.7 8.4
72.0 9.5
64.3 9.6
37.1 5.2
14.6 1.1
26.3 8.1
20.8 2.7
31.0 4.9
>1000
38.7 4.9
269 87
168 31
>1000
>1000
300 52
170 24
37.5 5.4
>1000
>1000
360 79
>1000
-CH2CH2-
>1000
-CH2OCH2eCH2CH2O-
-CH2OCH2eCH2CH2O-
-CH2OCH2eCH2CH2O-
12.1 4.9
44.7 7.5
>1000
171.9 31
>1000
5-OMe
39.6 3.7
>1000
G1
SB216763
17.2 3.0
19.8 2.1
e
e
a
Recombinant human AChE, BChE and GSK-3b were used. Data are the mean of at least three independent determinations.
included high permeability, low permeability and uncertain
permeability. The tested hybrids with a Pe value greater than
4.22 ꢃ 10ꢀ6 cm sꢀ1 are considered to have the ability to pass BBB,
based on established guidelines for predicting BBB penetration
[45].The ability of the selected compounds with both enzyme
(IC50 < 50 nM) to cross the BBB was predicted by PAMPA. The re-
sults (Table 3) indicated that hybrids GT4, GT10, GT15, GT17, GT20
and GT21 may pass the BBB (Pe > 4.22 ꢃ 10ꢀ6 cm sꢀ1). Among
them, GT21 showed the best BBB permeability, with a Pe value of
excellent inhibitory activity against GSK-3b and AChE, as well as
good BBB permeability, GT15 was further evaluated for simulated
docking and molecular dynamics simulation. (Figs. 5 and 6). In the
AChE complex, the tacrine moiety extends to the CAS of AChE,
forming a
p-p stacking interaction with the Trp86 residue; the
pyridhiazole fragment of GT15 extends to the solvent-exposed area
and is located in the PAS of AChE, forming a hydrogen bonding
interaction with the Ser293 and Tyr341. Overall, the in-silico
docking studies reveal reasonable structural design for hybrids. In
order to further investigate the ligand-protein binding process, we
performed a 100 ns molecular dynamics simulation (Fig. 6). The
root-mean-square deviation (RMSD) of the GT15-AChE complex
was shown in Fig. 6A. It was determined that the system reaches
equilibrium at 5 ns. The analysis of binding energy difference
showed that the total energy of the GT15-AChE complex decreases
during the binding process (the value of delta total is ꢀ53.7 kcal/
mol), indicating that the system of the complex is relatively stable.
Residue contribution analysis showed that the Trp86, Trp286 and
Tyr341 were the most important energy contributors. Further en-
ergy decomposition analysis of amino acid residues showed that
van der Waals force and electrostatic force play a major role.
7.1
ꢃ
10ꢀ6 cm sꢀ1
. While GT9, with the Pe values of
4.2 ꢃ 10ꢀ6 cm sꢀ1, may display poor permeability.
2.5. Kinase selectivity profiling of GT15
Since the hybrid contains a competitive ATP fragment (o-ami-
nopyridine), we examined the kinase selectivity profile of the
preferred compound GT15. A panel of 18 related kinases from the
CMGC kinase family was tested at a 0.3 mM concentration of GT15,
and the inhibition rate was reported in Fig. 4. In addition to
showing strong binding affinities with GSK3 family, GT15 also
exhibited a potent inhibitory activity against dual specificity tyro-
Indeed, Trp86 and Trp286 form
fragment and the pyridine group of GT15, respectively (Fig. 5A).
For the GSK-3 complex, the pyridhiazole pharmacophore of the
p-p stacking effect with the tacrine
sine phospho-regulated kinase 1 (DYRK1) (DYRK1
a and DYRK1b
with IC50 of 28.3 nM and 119.7 nM, respectively). For 14 additional
kinases of CMGC kinase family, at least 100-fold selectivity was
b
compound GT15 is well accommodated in the same pocket as the
published ligand, where it binds to the key amino acids, including
Lys85, Tyr134, Val135, and Cys199. The linker and tacrine fragment
of GT15 extends toward the solvent-exposed area, and make fewer
interactions. The molecular dynamics simulation studies demon-
strated in Fig. 6B also reveal that the van der Waals and electrostatic
force of Lys85, Tyr134, Val135, and Glu137 amino acids play key
roles in the protein-ligand interactions.
observed. Many studies have shown that DYRK1
in the premature development of AD by phosphorylating tau and
synuclein and other key substrates [46e48]. DYRK1 could also
phosphorylate presenelin 1 to increase secretase-mediated cleav-
age of APP [49]. Interestingly, DYRK1 has been shown to be
a may participate
a
-
a
a
overexpressed in sporadic AD cases and may act as a priming kinase
by phosphorylates tau protein at Thr212 in vitro [50].
In view of the strong inhibitory activity of GT15 on DYRK1
docking calculation was also studied. GT15 occupies the ATP binding
site of DYRK1 , similar to the binding mode with GSK-3 (Fig. 5D).
The ortho-aminopyridine and formamide groups of GT15 form
hydrogen bond interactions with Leu241, Ser242, and Lys188 of
a, the
2.6. Molecular modeling studies
a
b
To better insight into the potential ligand-protein interactions,
we perform computational molecular docking studies using Dis-
covery Studio and PyMOL 2.3 software. Considering that GT15 has

6
X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
Table 2
Inhibition of the enzymatic activities of hGSK-3
b
, hAChE, and hBChE by compounds (GT12ꢀGT28).
Cpd.
R1
R2
R3
R4
n
IC50 (nM)
hGSK-3
b
hAChE
hBChE
GT12
GT13
e
e
e
e
e
e
e
103.6 5.5
311.6 9.4
215.9 16.3
e
e
80.7 1.5
295.2 17.3
30.7 2.8
GT14
GT15
-CH3
e
e
e
e
92.8 21.5
22.0 1.4
393.6 15.4
1.2 0.1
173.4 10.9
149.8 12.6
H
-CH3
1
GT16
GT17
GT18
GT19
GT20
GT21
GT22
GT23
e
e
e
e
e
e
e
e
6-Cl
-CH3
-CH3
- Et
1
1
1
1
1
3
5
7
62.6 1.5
39.3 1.8
114.4 17.8
239.3 76.2
42.6 3.5
41.6 6.8
62.0 11.1
62.2 4.9
40.5 0.4
30.7 2.9
45.2 3.0
4.9 0.7
612.9 48.7
>1000
7-OMe
H
H
H
H
H
H
512.4 49.9
89.5 7.1
189.8 14.3
48.3 2.4
8.2 0.7
-Pr
-H
0.8 0.2
-CH3
-CH3
-CH3
23.1 1.8
21.9 1.6
15.2 1.3
3.2 0.2
GT24
GT25
e
e
H
H
-OMe
-CH3
-CH3
1
1
41.1 9.3
130.4 3.5
97.4 6.7
155.3 5.8
262.4 20.9
276.7 14.0
GT26
GT27
e
e
H
H
-CH3
-CH3
1
1
165.3 8.8
71.6 1.1
102.9 1.9
235.6 16.2
330.1 7.1
237.4 27.9
GT28
e
H
-CH3
1
132.4 47.3
49.2 2.8
619.4 49.6
a
Recombinant human AChE, BChE and GSK-3b were used. Data are the mean of at least three independent determinations.
Table 3
Permeability (Pe) of commercial drugs and the selected hybirds in the PAMPA-BBB assay.
a
b
Commercial drugs
Bibliography
Pe (10ꢀ6 cm sꢀ1
)
Cpd.
Pe (10ꢀ6 cm sꢀ1
)
Prediction
Dopamine
Ofloxacin
Lomefloxacin
Piroxacin
Clonidine
Chlorpromazine
Progesterone
Desipramine
Verapamil
0.2
0.8
1.1
2.5
5.3
6.5
9.3
12.0
16.0
17.0
0.3 0.1
1.5 0.5
0.9 0.4
3.9 1.1
6.5 1.5
7.1 0.9
10.5 1.8
14.8 3.3
16.5 1.7
18.1 1.9
GT4
GT9
GT10
GT15
GT17
GT20
GT21
-
4.7
4.2
4.6
6.9
5.7
4.9
7.1
-
CNSþ
CNS
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
-
-
-
-
-
-
-
Testosterone
a
The Pe value of 10 commercial drugs in PAMPA-BBB model.
Data shown are the means of two replicates (n ¼ 2).
b
DYRK1a, respectively. The tacrine fragment is located in the solvent-
phosphorylation, GSK-3
b
phosphorylates the Ser396, Ser199, and
exposed area and forms a hydrogen bond with Leu164 (Fig. 5C).
Ser413 sites of the tau protein. The cellular GSK-3b activity of GT15
was confirmed by evaluating its effect on the phosphorylation of
tau protein in neuroblastoma N2a cells overexpressing human tau
(Fig. 7). The level of phosphorylation of tau protein was clearly
2.7. Effect of hybrid on tau phosphorylation
Microtubule-associated protein tau is essential in maintaining
the stability of microtubules. As a key kinase for tau protein
decreased for the Ser396 epitope at 6 h incubation with 5
GT15, a 49% reduction compared to the untreated cells. As the
mM of

X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
7
concentration of GT15 was increased to 15
mM, the level of phos-
phorylated tau protein was reduced to 17%. The level of total tau
protein showed no differences in the electrophoretic profile be-
tween different doses of GT15 and untreated cells.
2.8. Inhibition of LPS-induced inflammatory responses
Due to the brain high utilization rate of oxygen, it is susceptible
to oxidative damage, especially the elderly. Reactive oxygen species
(ROS)-induced oxidative stress may be an important mediator of
damaged cell structure in microglia-mediated pathogenesis [51,52].
GSK-3b inhibitors can improve the clinical symptoms of central
inflammatory diseases, such as experimental autoimmune
encephalomyelitis [53]. To evaluate the preliminary anti-
inflammatory effects, we tested the effect of hybrids on the level
of ROS in mouse neuroglia BV2 cells. As the result shown in Fig. 8,
hybrids were determined to significantly prevent the increase of
ROS caused by LPS without affecting the morphology of the BV2
cells, which indicated that the compounds have the ability to
reduce oxidative stress.
2.9. Behavioral studies
Given the balanced in vitro activity profile, hybrids GT10 and
GT15 were further tested in vivo to ascertain whether they could
ameliorate the cholinergic activity (the scopolamine-induced
Fig. 4. Protein kinase selectivity of GT15 in a kinses interaction assay (ChemPartner).
GT15 was screened at a 300 nM concentration on an 18-kinase panel. The inhibition
rate is reported as the mean (n ꢄ 2).
Fig. 5. Preferred compound GT15 was docked with hGSK-3
binding mode of GT15 with AChE; (B) the action site and specific binding mode of GT15; (C) specific binding mode of GT15 with DYRK1
DYRK1 (green). The pyridinethiazole fragment of GT15 almost completely overlap, occupying the ATP binding site of kinase. (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.)
b
(PDB id: 4PTC), hAChE (PDB id: 4EY7), and hDYRK1
a
(PDB id: 5AIK), respectively. (A) the site of action and specific
a
; (D) the overlay of GSK-3 (blue) and
b
a

8
X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
Fig. 6. (A) The molecular dynamics simulation of GT15 with AChE; (B) The molecular dynamics simulation of GT15 with GSK-3b.
B), which indicates that the scopolamine modeling was successful.
Compared with the model group, the average time and distance to
the platform of the tacrine group decreased significantly, indicating
that tacrine has a function of significantly improving memory and
cognition in mice. The cognitive impairment of the mice caused by
scopolamine was further improved by compounds GT15 and GT10.
By comparing the swimming speed of the five groups of mice
(Fig. 9C), it was found that there was no significant difference be-
tween the groups, indicating that compounds GT15 and GT10
would not negatively affect the mobility of the mice. The repre-
sentative trajectory of each group of mice is shown in Fig. 9D.
Compared with the control group, the trajectory of the model group
of mice was more chaotic and longer. Furthermore, compared with
the model group, the trajectory of the compounds GT15 and GT10
groups were considerably simplified. These observations suggested
that the cognitive impairment of the mice recovered and com-
pounds GT15 and GT10 improved the memory and cognitive the
function of the investigated mice. Overall, the obtained results
indicated that GT15 and GT10 greatly improved the cognitive
impairment of the examined mice. GT15 displayed better proper-
ties than tacrine.
Fig. 7. Effect of hybrid GT15 on tau phosphorylation at Ser396 in N2a cells over-
expressing human tau. Protein expression was detected by western blots analysis, and
was quantified using ImageJ analysis software.
cognitive impairment in ICR mice) using a Morris water maze test
[54]. Scopolamine is an anticholinergic agent used to induce
memory impairment in animal models [55,56]. The cognitive def-
icits caused by scopolamine in the test subjects were comparable to
those caused by the cholinergic deficit characteristics of AD [57].
Memory training and learning were conducted for five days, and a
probe trial was conducted on the sixth day. The results are evalu-
ated based on swimming speed, travel distance (the distance
required to find the platform) and escape latency (time required to
find the platform). As it can be seen, compared to the control group,
the average escape latency and traveled distance of the
scopolamine-induced mice was significantly prolonged (Fig. 9A and
3. Conclusions
So far, attempts to target only one of the pathogenic mecha-
nisms of AD have been disappointing. The multi-target strategy is
considered to be a promising method to solve complex and multi-
Fig. 8. Detection of the amount of ROS produced in BV2 cells using fluorescence microscopy analysis. The green fluorescence intensity represents the amount of intracellular ROS
produced. To avoid errors in the exposure time to test fluorescence brightness, the exposure time was locked at 25 ms. (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)

X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
9
Fig. 9. Effects of the oral administration of GT15 (15 mg/kg), GT10 (15 mg/kg), and tacrine (15 mg/kg) on scopolamine-induced cognitive impairment in ICR mice. (A) The latency to
the platform. (B) The path to the platform. (C) The swimming speeds. (D) Representative tracks of the mice in the Morris water maze during the trial training period. Data are
presented as the mean SEM (n ¼ 8; **p < 0.01; ***p < 0.001 vs. scopolamine group).
factor diseases, including cancer and neurodegenerative diseases.
The challenge of obtaining multi-target compounds is to transform
these low affinity binders into potent leads, while maintaining the
affinity of the compounds to the original target. Based on the
pathological features of AD and our previous work, we believe that
selective detector. The purity of compounds GT1ꢀGT28 (higher
than 95%) was determined by HPLC, conducted on a Shimadzu LC-
20AT series system with the use of a Hedera ODS-2 column
(4.6 mm ꢃ 250 mm, 5
mm), eluted with a 50:50 methanol/distilled
water mixture [0.1% formic acid], at a flow rate of 1.0 mL/min.
AChE and GSK-3b play a key role in the development of AD. In the
present study, we synthesized a series of 28 ChEs/GSK-3 dual-target
inhibitors by connecting tetrahydroacridine scaffolds from three
different sites of pyridothiazole core. One of these new hybrids
(GT15) has shown balanced in vitro activities against AChE and
GSK-3b, with potency in the low nanomolar range. In addition,
GT15 has shown an attractive BBB permeability profile, as well as
anti-neuroinflammation in cells, with no sign of toxicity. It is worth
4.1.1. General procedure for the preparation of compounds 8aꢀ8d
To a solution of 7aꢀ7c (1 mmol) in 10 mL of DMF, DIPEA (1.6 mL,
10 mmol) and HATU (570 mg, 1.5 mmol) were added. After reaction
at room temperature for 2 h under argon, NH4Cl or methylamine
hydrochloride (10 mmol) was added, and the mixture was stirred at
room temperature for 24 h under the protection of argon. The re-
action was complete by TLC. The solvent was evaporated under a
vacuum to give the crude product which was purified by column
chromatography on silica gel (DCM/MeOH ¼ 10/1, v/v) to obtain the
desired product8aꢀ8d as a yellow solid.
noting that GT15 has an IC50 value of 28.3 nM for DYRK1
a. DYRK1a
are responsible for both AD major neurotoxic processes, the for-
mation of A
AD. DYRK1
b
a
plaques and NFTs in the premature development of
phosphorylates amyloid precursor protein at the
Thr668 residue and tau protein at Thr212, which leads to the for-
mation of A plaques and NFTs, respectively. Considering that
DYRK1 and GSK-3 play a similar role in the development of AD
and the similarity of their protein structures, the development of
dual inhibitors is a very meaningful exploration. Further, kinases
4.1.1.1. 4-(but-3-yn-1-yloxy)-2-(2-(cyclopropanecarboxamido)pyr-
idin-4-yl)-N-methylthiazole-5-carboxamide (8a). Synthesized as
per the general procedure described above. Using compound 7a
(357 mg) and methylamine hydrochloride (670 mg) to get com-
pound 8a as a yellow solid (292 mg, 79%).1H NMR (300 MHz,
b
a
b
such as DYRK1a and GSK-3b are involved in the pathogenesis of AD,
CDCl3):
d 11.09 (s, 1H, -CONH-), 8.64 (m, 1H, AreH), 8.45 (d, 1H,
and AChE is related to the symptoms of AD patients, so simulta-
neous inhibition of the three main pathogenic processes may
produce a higher efficacy. The potential triple action of MTDLs
J ¼ 1.8Hz, AreH), 7.54 (d, J ¼ 6.2 Hz, 1H, AreH), 4.47 (t, 1H,
J ¼ 6.3 Hz, eOCH2-), 2.90 (s, 1H, eC^CH), 2.68 (t, 1H, J ¼ 6.3 Hz,
eCH2CH2-), 2.03 (m, 1H, -COCH-), 0.84 (m, 4H, -(CH2)2-); 13C NMR
based on AChE, GSK-3
b and DYRK1a deserves to be further
(75 MHz, CDCl3):
d 181.3, 159.4, 155.1, 150.8, 148.7, 147.3, 146.9,
explored in the field of anti-AD.
145.8, 120.4, 115.9, 80.7, 68.9, 65.4, 28.9, 23.5, 17.8, 9.4; ESI/MS (m/
z): 369.1 ([M ꢀ H]-).
4. Experimental procedures
4.1.1.2. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-N-methyl-4-
(pent-4-yn-1-yloxy)thiazole-5-carboxamide (8b). Synthesized as
per the general procedure described above. Using compound 7b
(371 mg) and methylamine hydrochloride (670 mg) to get com-
pound 8b as a yellow solid (311 mg, 81%).1H NMR (300 MHz,
4.1. Chemistry
Unless otherwise noted, all solvents and starting materials used
were commercially available without further purification. 1H and
13C NMR spectra were acquired using Bruker spectrometer (300
and 400 MHz). Standard abbreviations indicating spin multiplic-
ities are given as follows: s (singlet), d (doublet), t (triplet), br s
(broad singlet), or m (multiplet). The mass spectra data were ob-
tained on an Agilent LCꢀMS 6120 instrument with an ESI mass
DMSO‑d6):
d 11.05 (s, 1H, -CONH-), 8.65 (m, 1H, eCONHCH3), 8.45
(m, 1H, AreH), 7.58 (d, 1H, J ¼ 1.8 Hz, AreH), 7.50 (m, 1H, AreH),
4.59 (t, 1H, J ¼ 6.3 Hz, eOCH2-), 2.85 (s, 1H, eC^CH), 2.83 (d, 3H,
J ¼ 6.2 Hz, -NHCH3), 2.35 (m, 2H, eCH2-), 2.05 (m, 2H, eCH2), 0.85
(m, 2H, -(CH2)2-); 13C NMR (75 MHz, DMSO‑d6):
d 179.4,171.6, 151.0,

10
X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
149.7,148.1,147.0,144.9,142.5,119.7,117.9, 81.9, 70.8, 61.7, 31.8, 26.7,
purity. 1H NMR (300 MHz, CDCl3):
d 8.73 (br s, 1H, -CONH-), 8.59 (s,
15.4, 13.8, 8.6; ESI/MS (m/z): 383.1 ([M- H]-).
1H, -CONH-), 8.31 (d, 1H, J ¼ 1.8 Hz, AreH), 8.09 (t, J ¼ 8.2 Hz, 1H,
AreH), 7.73 (m, 1H, AreH), 7.69 (t, J ¼ 8.2 Hz, 1H, AreH), 7.45 (m,
2H, AreH), 7.22 (m,1H, AreH), 7.11 (m,1H, AreH), 4.76 (t, J ¼ 6.2 Hz,
2H, eOCH2-), 3.23 (t, J ¼ 6.2 Hz, 2H, Ar-CH2-), 3.15 (t, J ¼ 6.2 Hz, 2H,
Ar-CH2-), 3.04 (d, J ¼ 3.2 Hz, 3H, -NHCH3), 2.51 (m, 2H, eCH2-), 2.07
(m, 1H, -CH-), 1.98 (m, 2H, eCH2-), 1.82 (m, 2H, eCH2-),1.02 (m, 2H,
4.1.1.3. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-4-(hex-5-yn-
1-yloxy)-N-methylthiazole-5-carboxamide (8c). Synthesized as per
the general procedure described above. Using compound 7c
(385 mg) and methylamine hydrochloride (670 mg) to get com-
pound 8c as a yellow solid (318 mg, 80%).1H NMR (300 MHz,
eCH2-), 0.87 (m, 2H, eCH2-); 13C NMR (75 MHz, CDCl3):
d 173.5,
DMSO‑d6):
d
11.07 (s, 1H, -CONH-), 8.73 (m, 1H, eCONHCH3), 8.68
162.9, 160.5, 160.4, 160.2, 153.5, 149.6, 147.1, 146.7, 141.0, 139.3,130.1,
128.7, 127.8, 125.2, 123.3, 122.0, 115.4, 110.7, 109.1, 71.0, 46.2, 33.7,
28.7, 26.8, 24.6, 22.4, 22.0, 21.9, 14.7, 8.3 ( ꢃ 2); HRMS (m/z): calcd
for C32H33N8O3S [M þ H]þ, 609.2391; found, 609.402.
(m, 1H, AreH), 7.52 (d, 1H, J ¼ 7.9 Hz, AreH), 8.43 (d, 1H, J ¼ 1.8 Hz,
AreH), 4.59 (m, 2H, eOCH2-), 3.97 (s, 2H, -CH2C^CH-), 3.71 (m, 2H,
eCH2-), 2.85 (s, 1H, eC^CH), 2.89 (d, 3H, J ¼ 6.2 Hz, -NHCH3), 2.02
(m, 2H, eCH2), 0.86 (m, 2H, -(CH2)2-); 13C NMR (75 M Hz,
DMSO‑d6):
d
181.4,169.7,153.8,150.1,148.4,147.0,145.9,144.1,120.7,
4.1.2.3. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-4-(2-(1-
(1,2,3,4-tetrahydroacridin-9-yl)-1H-1,2,3-triazol-4-yl)ethoxy)thia-
zole-5-carboxamide (GT3). Synthesized as per the general proced-
ure described above. Using compound 8d (383 mg) and
intermediate compound 5 to get compound GT3. The product GT3
was isolated as a yellow oil by column chromatography (DCM/
MeOH ¼ 40/1, v/v) with a yield of 68%, mp 235e237 ꢂC, 96.9% HPLC
117.6, 79.4, 77.6, 68.0, 65.9, 59.4, 26.7, 18.0, 7.9; ESI/MS (m/z): 399.1
([M- H]-).
4.1.1.4. 4-(but-3-yn-1-yloxy)-2-(2-(cyclopropanecarboxamido)pyr-
idin-4-yl)-N-methylthiazole-5-carboxamide (8d). Synthesized as
per the general procedure described above. Using compound 7c
(357 mg) and NH4Cl (540 mg) to get compound 8d as a yellow solid
(326 mg, 85%).
purity. 1H NMR (300 MHz, DMSO‑d6):
d 11.01 (br s, 1H, -CONH-),
8.64 (m, 1H, AreH), 8.41 (br s, 2H, -CONH-), 8.05 (d, 1H, J ¼ 6.3 Hz,
AreH), 7.85 (m, 1H, AreH), 7.75 (t, J ¼ 8.2 Hz, 1H, AreH), 7.56 (d,
J ¼ 3.1 Hz, 1H, AreH), 7.50 (t, J ¼ 6.3 Hz, 1H, AreH), 7.08 (m, 1H,
AreH), 7.03 (d, J ¼ 8.2 Hz, 1H, AreH), 4.66 (t, J ¼ 6.2 Hz, 2H, eOCH2-
), 3.12 (t, J ¼ 6.2 Hz, 2H, Ar-CH2-), 3.02 (t, J ¼ 6.2 Hz, 2H, Ar-CH2-),
2.49 (m, 2H, Ar-CH2-), 2.32 (m, 2H, eCH2-), 2.00 (m, 1H, -CH-), 1.89
(m, 2H, eCH2-), 1.72 (m, 2H, eCH2-), 0.82 (m, 2H, -(CH2)2-); 13C
4.1.2. General procedure for the preparation of compounds
GT1ꢀGT11
Compound 8aꢀ8d (1 mmol) and intermediate compound
4aꢀ4c or 5 (5 for GT1ꢀGT4) (1 mmol) were dissolved in 20 mL n-
butanol/H2O (3:1 v/v). Et3N (2 mmol, 2 eq) and catalytic amounts of
sodium ascorbate and copper sulfate were added under argon. The
reaction was stirred at room temperature for 24 h. The reaction was
completed by TLC. The solvent was washed with water (2 ꢃ 10 mL)
and saturated aqueous sodium chloride (10 mL), dried over sodium
sulfate, and filtered. The solvent was evaporated under a vacuum to
give the crude product which was purified by column chromatog-
raphy on silica gel (DCM/MeOH ¼ 30/1, v/v) to obtain the desired
product GT1ꢀGT11 as a yellow oil.
NMR (75 MHz, DMSO‑d6):
d 173.5, 163.2, 161.6, 160.8, 160.3, 153.6,
149.7, 147.0, 146.7, 141.0, 139.3, 130.1, 128.7, 127.8, 125.2,123.4, 122.0,
115.5, 110.9, 109.1, 70.9, 33.7, 28.7, 24.6, 22.4, 22.0, 21.9, 19.1, 14.7, 8.3
( ꢃ 2); HRMS (m/z): calcd for C31H31N8O3S [M þ H]þ, 595.2234;
found, 595.2227.
4.1.2.4. 4-(2-(1-(2-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)
e t h y l ) - 1 H - 1, 2 , 3 - t r i a z o l - 4 - y l ) e t h o x y ) - 2 - ( 2 - ( c y c l o -
propanecarboxamido)pyridin-4-yl)-N-methylthiazole-5-carboxamide
(GT4). Synthesized as per the general procedure described above.
Using compound 8b (383 mg) and intermediate compound 4b
(302 mg) to get compound GT4. The product GT4 was isolated as a
yellow solid by column chromatography (DCM/MeOH ¼ 40/1, v/v)
with a yield of 81%, mp 185e187 ꢂC, 98.9% HPLC purity. 1H NMR
4.1.2.1. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-N-methyl-4-
(2-((1-(1,2,3,4-tetrahydroacridin-9-yl)-1H-1,2,3-triazol-4-yl)
methoxy)ethoxy) thiazole-5-carboxamide (GT1). Synthesized as per
the general procedure described above. Using compound 8a
(369 mg) and intermediate compound 5 to get compound GT1. The
product GT1 was isolated as a yellow oil by column chromatog-
(300 MHz, DMSO‑d6):
d 11.03 (br s, 1H, -CONH-), 8.63 (br s, 1H,
raphy (DCM/MeOH
¼
40/1, v/v) with
a
yield of 80%, mp
8.75
-CONH-), 8.43 (d, J ¼ 3.1 Hz, 1H, AreH), 7.93 (d, J ¼ 3.1 Hz, 1H,
AreH), 7.86 (m, 2H, AreH), 7.62 (m, 1H, AreH), 7.54 (d, J ¼ 6.1 Hz,
1H, AreH), 7.25 (d, J ¼ 6.3 Hz, 1H, AreH), 5.81 (m 1H, eCH2NH-),
4.56 (t, J ¼ 6.2 Hz, 2H, eOCH2-), 4.54 (m, 2H, -NHCH2-), 3.90 (m, 2H,
Ar-CH2-), 3.10 (t, J ¼ 6.2 Hz, 2H, - Ar-CH2-), 2.79 (m, 2H, eCH2-), 2.78
(d, J ¼ 3.2 Hz, 3H, -NHCH3), 2.53 (m, 2H, eCH2-), 2.04 (m, 1H, -CH-),
1.73 (m, 4H, -(CH2)2-), 0.85 (m, 4H, -(CH2)2-); 13C NMR (75 MHz,
200e202 ꢂC, 98.3% HPLC purity. 1H NMR (300 MHz, CDCl3):
d
(br s, 1H, -CONH-), 8.71 (m, 1H, -CONH-), 8.30 (d, 1H, J ¼ 1.8Hz,
AreH), 8.07 (t, J ¼ 8.2Hz, 1H, AreH), 7.84 (m, 1H, AreH), 7.70 (t,
J ¼ 8.2Hz, 1H, AreH), 7.44 (m, 2H, AreH), 7.26 (m, 1H, AreH), 7.08
(m, 1H, AreH), 4.94 (s, 2H, eCH2-), 4.73 (m, 2H, -OCH2-), 4.06 (m,
2H, -OCH2-), 3.23 (t, J ¼ 6.2Hz, 2H, eCH2-), 2.97 (d, J ¼ 3.2Hz, 3H,
-NHCH3), 2.58 (m, 2H, eCH2-), 2.03 (m, 2H, eCH2-), 1.98 (m, 1H,
-CH-),1.84 (m, 2H, eCH2-),1.11 (m, 2H, eCH2-), 0.91 (m, 2H, eCH2-);
DMSO‑d6):
d 173.6, 169.6, 162.9, 160.3, 160.1, 153.6, 149.7, 143.9,
141.1, 141.0, 133.7, 126.4, 125.7, 124.3, 123.8, 118.4, 116.7, 115.6, 115.5,
110.8, 109.8, 70.5, 50.4, 48.1, 29.5, 26.5, 25.4, 25.2, 22.7, 22.3, 14.8,
8.4 ( ꢃ 2); HRMS (m/z): calcd for C33H35ClN9O3S [M þ H]þ,
672.2267; found, 672.2268; calcd for C33H34ClN9O3SNa [M þ Na]þ,
694.2086; found, 694.2088.
13C NMR (75 MHz, CDCl3):
d 172.7, 162.8, 161.1, 160.1, 152.6, 148.5,
146.9, 145.3, 144.9, 141.8, 138.8, 129.7, 128.7, 127.7, 127.5, 124.9,
123.2, 121.3, 115.6, 111.7, 109.8, 70.2, 68.7, 64.3, 33.8, 26.4, 24.8, 22.5,
22.0, 15.8, 8.6 ( ꢃ 2); HRMS (m/z): calcd for C32H33N8O4S [M þ H]þ,
625.2340; found, 625.2333.
4.1.2.5. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-N-methyl-4-
(2-(1-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-
triazol-4-yl)ethoxy)thiazole-5-carboxamide (GT5). Synthesized as
per the general procedure described above. Using compound 8b
(383 mg) and intermediate compound 4a (267 mg) to get com-
pound GT5. The product GT5 was isolated as a yellow solid by
column chromatography (DCM/MeOH ¼ 40/1, v/v) with a yield of
69%, mp 200e202 ꢂC, 96.1% HPLC purity. 1H NMR (300 MHz,
4.1.2.2. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-N-methyl-4-
(2-(1-(1,2,3,4-tetrahydroacridin-9-yl)-1H-1,2,3-triazol-4-yl)ethoxy)
thiazole-5-carboxamide (GT2). Synthesized as per the general pro-
cedure described above. Using compound 8c (398 mg) and inter-
mediate compound 5 to get compound GT2. The product GT2 was
isolated as
a yellow oil by column chromatography (DCM/
MeOH ¼ 25/1, v/v) with a yield of 59%, mp 203e205 ꢂC, 96.4% HPLC

X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
11
DMSO‑d6):
d
9.02 (br s,1H, -CONH-), 8.69 (br s,1H, -CONH-), 8.29 (d,
to get compound GT8. The product GT8 was isolated as a yellow
solid by column chromatography (DCM/MeOH ¼ 40/1, v/v) with a
yield of 78%, mp 213e215 ꢂC, 98.0% HPLC purity. 1H NMR (300 MHz,
1H, J ¼ 1.6 Hz, AreH), 7.95 (d, J ¼ 6.1 Hz, 1H, AreH), 7.85 (d,
J ¼ 6.1 Hz, 1H, AreH), 7.74 (m, 1H, AreH), 7.63 (s, 1H, AreH), 7.51 (t,
J ¼ 6.1 Hz, 1H, AreH), 7.39 (d, J ¼ 3.2 Hz, 1H, AreH), 7.31 (t,
J ¼ 6.2 Hz, 1H, AreH), 5.30 (m 1H, eCH2NH-), 4.72 (t, J ¼ 6.2 Hz, 2H,
eOCH2-), 4.67 (m, 2H, -NHCH2-), 4.10 (m, 2H, Ar-CH2-), 3.22 (t,
J ¼ 6.2 Hz, 2H, - Ar-CH2-), 3.06 (m, 2H, -CH2-), 3.01 (d, J ¼ 3.2 Hz, 3H,
-NHCH3), 2.62 (m, 2H, -CH2-), 1.83 (m, 4H, -(CH2)2-), 1.70 (m, 1H,
-CH-), 1.10 (m, 2H, eCH2-), 0.91 (m, 2H, eCH2-); 13C NMR (75 MHz,
CDCl3): d 9.03 (br s,1H, -CONH-), 8.95 (br s,1H, -CONH-), 8.60 (s,1H,
AreH), 8.28 (d, J ¼ 3.1 Hz, 1H, AreH), 7.73 (d, J ¼ 6.1 Hz, 1H, AreH),
7.71 (d, J ¼ 3.1 Hz, 1H, AreH), 7.39 (t, J ¼ 3.1 Hz, 1H, AreH), 7.18 (m,
1H, AreH), 7.12 (t, J ¼ 3.2 Hz, 1H, AreH), 6.94 (d, J ¼ 6.1 Hz, 1H,
AreH), 4.74 (m, 2H, eOCH2-), 4.55 (t, J ¼ 6.1 Hz, 2H, -NHCH2-), 4.21
(m, 2H, Ar-CH2-), 3.89 (s, 3H, eOCH3), 3.49 (m, 2H, Ar-CH2-), 2.99 (d,
J ¼ 3.2 Hz, 3H, -NHCH3), 2.88 (t, J ¼ 6.2 Hz, 2H, - Ar-CH2-), 2.60 (m,
2H, -CH2-), 2.20 (t, J ¼ 6.2 Hz, 2H, Ar-CH2-),1.82 (m, 2H, eCH2-),1.80
(m, 1H, -CH-), 1.26 (m, 2H, eCH2-), 1.10 (m, 2H, eCH2-), 0.92 (m, 2H,
DMSO‑d6):
d 172.7, 162.7, 161.0, 160.0, 158.7, 152.4, 151.0, 148.6,
144.4, 141.7, 134.2, 129.6, 126.1, 124.6, 123.0, 122.5, 119.1, 116.0, 115.6,
111.2, 109.6, 69.8, 50.3, 47.9, 29.7, 26.4, 25.8, 24.7, 22.5, 22.0,15.9, 8.6
( ꢃ 2); HRMS (m/z): calcd for C33H36N9O3S [M þ H]þ, 638.2656;
found, 638.2646.
eCH2-); 13C NMR (75 MHz, CDCl3):
d 173.5,162.8,160.4,160.3,156.6,
153.5,149.7,146.2,140.9,123.1,122.5,120.1,115.4,110.6,109.0,102.5,
70.7, 56.1, 50.0, 49.0, 47.3, 28.6, 26.7, 25.4, 22.5, 21.7, 14.7, 8.4 ( ꢃ 2);
HRMS (m/z): calcd for C35H40N9O4S [M þ H]þ, 682.2918; found,
682.2935.
4.1.2.6. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-N-methyl-4-
(3-(1-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-
triazol-4-yl)propoxy)thiazole-5-carboxamide (GT6).
Synthesized as per the general procedure described above. Using
compound 8b (383 mg) and intermediate compound 4a (267 mg)
to get compound GT6. The product GT6 was isolated as a yellow
solid by column chromatography (DCM/MeOH ¼ 40/1, v/v) with a
yield of 73%, mp 207e209 ꢂC, 97.2% HPLC purity. 1H NMR (300 MHz,
4.1.2.9. 4-(2-((1-(2-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)
ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)
-2-(2-(cyclo-
propanecarboxamido)pyridin-4-yl)-N-methylthiazole-5-carboxamide
(GT9). Synthesized as per the general procedure described above.
Using compound 8a (369 mg) and intermediate compound 4b
(302 mg) to get compound GT9. The product GT9 was isolated as a
yellow solid by column chromatography (DCM/MeOH ¼ 40/1, v/v)
with a yield of 59%, mp 185e187 ꢂC, 96.4% HPLC purity. 1H NMR
CDCl3):
d 8.82 (br s, 1H, -CONH-), 8.64 (br s, 1H, -CONH-), 8.30 (m,
1H, AreH), 8.03 (d, J ¼ 6.1Hz, 1H, AreH), 7.99 (d, J ¼ 6.1 Hz, 1H,
AreH), 7.65 (s, 1H, AreH), 7.48 (t, J ¼ 6.1 Hz, 1H, AreH), 7.40 (d,
J ¼ 3.2 Hz, 1H, AreH), 7.31 (t, J ¼ 6.2 Hz, 1H, AreH), 7.14 (d,
J ¼ 1.8 Hz, 1H, AreH), 6.16 (m 1H, eCH2NH-), 4.77 (m, 2H, eOCH2-),
4.58 (t, J ¼ 6.1 Hz, 2H, -NHCH2-), 4.26 (m, 2H, Ar-CH2-), 3.09 (t,
J ¼ 6.2 Hz, 2H, - Ar-CH2-), 3.00 (d, J ¼ 3.2 Hz, 3H, -NHCH3), 2.90 (t,
J ¼ 6.2 Hz, 2H, Ar-CH2-), 2.63 (m, 2H, -CH2-), 2.23 (m, 2H, -CH2-),
1.84 (m, 4H, -(CH2)2-), 1.71 (m, 1H, -CH-), 0.92 (m, 2H, eCH2-), 0.86
(300 MHz, CDCl3): d 8.88 (br s, 1H, -CONH-), 8.60 (br s, 1H, -CONH-),
8.30 (s, 1H, AreH), 8.01 (d, J ¼ 3.1 Hz,1H, AreH), 7.99 (m, 1H, AreH),
7.93 (m, 1H, AreH), 7.39 (m, 1H, AreH), 7.23 (m, 1H, AreH), 7.18 (m,
1H, AreH), 6.50 (m 1H, eCH2NH-), 4.88 (m, 2H, eOCH2-), 4.65 (m,
2H, -NHCH2-), 4.34 (m, 2H, eOCH2-), 3.88 (s, 2H, eOCH2-), 3.15 (m,
2H, Ar-CH2-), 3.10 (d, J ¼ 3.2 Hz, 3H, -NHCH3), 2.93 (m, 2H, - Ar-CH2-
), 2.63 (m, 2H, -CH2-), 1.85 (m, 4H, -(CH2)2-), 1.72 (m, 1H, -CH-), 1.12
(m, 2H, eCH2-), 0.93 (m, 2H, eCH2-); 13C NMR (75 M Hz, CDCl3):
(m, 2H, eCH2-); 13C NMR (75 MHz, CDCl3):
d 172.8, 162.8, 161.2,
160.3,154.7, 152.9,152.5,148.7,147.0,141.8,141.7, 130.6,124.9, 123.8,
123.2, 122.6, 117.9, 115.7, 114.5, 110.9, 109.7, 70.4, 50.0, 47.9, 29.7,
28.7, 26.5, 24.5, 22.3, 22.0, 21.4, 15.9, 8.6 ( ꢃ 2); HRMS (m/z): calcd
for C34H38N9O3S [M þ H]þ, 652.2813; found, 652.2827.
d
173.5, 163.2, 161.5, 160.5, 154.8, 153.6, 151.4, 149.8, 140.9, 140.1,
135.0, 131.9, 131.8, 129.8, 129.2, 129.1, 121.1, 115.5, 110.9, 109.1, 58.9,
55.3, 53.7, 51.3, 46.5, 42.0, 19.1, 18.4, 17.1, 14.7, 12.6, 8.3 ( ꢃ 2); HRMS
(m/z): calcd for
702.2356.
C34H37ClN9O4S [M
þ
H]þ, 702.2372; found,
4.1.2.7. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-4-(2-(1-(2-
((7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-
triazol-4-yl)ethoxy)-N-methylthiazole-5-carboxamide
(GT7).
4.1.2.10. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-N-methyl-4-
(2-((1-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-
Synthesized as per the general procedure described above. Using
compound 8a (369 mg) and intermediate compound 4c (297 mg) to
get compound GT7. The product GT7 was isolated as a yellow solid
by column chromatography (DCM/MeOH ¼ 40/1, v/v) with a yield
of 81%, mp 210e212 ꢂC, 98.9% HPLC purity. 1H NMR (300 MHz,
triazol-4-yl)methoxy)ethoxy)thiazole-5-carboxamide
(GT10).
Synthesized as per the general procedure described above. Using
compound 8a (369 mg) and intermediate compound 4a (267 mg)
to get compound GT10. The product GT10 was isolated as a yellow
solid by column chromatography (DCM/MeOH ¼ 40/1, v/v) with a
yield of 62%, mp 179e181 ꢂC, 98.8% HPLC purity. 1H NMR (300 MHz,
DMSO‑d6):
d 8.88 (br s, 1H, -CONH-), 8.63 (br s, 1H, -CONH-), 8.29
(m, 1H, AreH), 7.89 (m, 1H, AreH), 7.72 (d, J ¼ 6.1 Hz, 1H, AreH),
7.65 (d, J ¼ 3.1 Hz, 1H, AreH), 7.38 (d, J ¼ 3.1 Hz, 1H, AreH), 7.20 (m,
1H, AreH), 6.93 (d, J ¼ 6.3 Hz, 1H, AreH), 5.82 (m 1H, eCH2NH-),
4.79 (m, 2H, -NHCH2-), 4.68 (t, J ¼ 6.2 Hz, 2H, eOCH2-), 4.24 (m, 2H,
Ar-CH2-), 3.88 (s, 3H, eOCH3), 3.32 (t, J ¼ 6.2 Hz, 2H, - Ar-CH2-), 3.08
(m, 2H, eCH2-), 3.01 (d, J ¼ 3.2 Hz, 3H, -NHCH3), 2.63 (m, 2H, eCH2-
), 1.83 (m, 4H, -(CH2)2-), 1.46 (m, 1H, -CH-), 0.95 (m, 2H, eCH2-),
CDCl3): d 8.94 (br s,1H, -CONH-), 8.58 (br s,1H, -CONH-), 8.28 (s,1H,
AreH), 8.09 (m, 1H, AreH), 8.06 (m, 1H, AreH), 8.04 (m, 1H, AreH),
7.42 (d, J ¼ 6.2 Hz, 1H, AreH), 7.37 (d, J ¼ 6.2 Hz, 1H, AreH), 7.23 (m,
1H, AreH), 7.15 (m, 1H, AreH), 6.81 (m 1H, eCH2NH-), 4.92 (m, 2H,
eOCH2-), 4.70 (m, 2H, -OCH2-), 4.63 (s, 2H, eOCH2-), 4.35 (m, 2H,
Ar-CH2-), 3.85 (m, 3H, -NHCH3), 3.09 (m, 2H, - Ar-CH2-), 2.93 (m,
2H, - Ar-CH2-), 2.60 (m, 2H, -CH2-), 1.82 (m, 4H, -(CH2)2-), 1.79 (m,
1H, -CH-), 0.92 (m, 2H, eCH2-), 0.88 (m, 2H, eCH2-); 13C NMR
0.89 (m, 2H, eCH2-); 13C NMR (75 MHz, DMSO‑d6):
d 172.7, 166.1,
162.6, 160.9, 159.9, 156.6, 152.9, 152.4, 151.4, 148.6, 144.4, 141.7,
125.1, 124.6, 123.5, 122.2, 119.2, 115.6, 111.1, 109.6, 101.6, 69.8, 55.9,
50.0, 47.4, 29.7, 26.4, 25.8, 24.9, 22.4, 21.5, 15.8, 8.6 ( ꢃ 2); HRMS (m/
z): calcd for C34H38N9O4S [M þ H]þ, 668.2762; found, 668.779.
(75 MHz, CDCl3):
d 172.9, 162.7, 161.1, 161.0, 159.9, 154.2, 152.5,
148.6, 144.7, 141.5, 131.3, 125.0, 124.6, 123.7, 116.8, 115.5, 115.4, 111.4,
111.3, 109.7, 70.2, 68.2, 64.1, 50.6, 49.8, 47.8, 45.8, 29.6, 26.4, 24.4,
22.0, 15.7, 8.7 ( ꢃ 2); HRMS (m/z): calcd for C34H38N9O4S [M þ H]þ,
668.2762; found, 668.2749.
4.1.2.8. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-4-(3-(1-(2-
((7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-
triazol-4-yl)propoxy)-N-methylthiazole-5-carboxamide
Synthesized as per the general procedure described above. Using
compound 8b (383 mg) and intermediate compound 4c (297 mg)
(GT8).
4.1.2.11. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-4-(2-((1-(2-
((7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)ethoxy)-N-methylthiazole-5-carboxamide

12
X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
(GT11). Synthesized as per the general procedure described above.
Using compound 8a (369 mg) and intermediate compound 4c
(297 mg) to get compound GT11. The product GT11 was isolated as a
yellow solid by column chromatography (DCM/MeOH ¼ 40/1, v/v)
with a yield of 71%, mp 190e192 ꢂC, 97.5% HPLC purity. 1H NMR
160.4,156.4,153.8,149.8,141.0,140.1,135.1,133.2,129.3,125.4,121.2,
119.5, 115.8, 115.5, 111.7, 110.4, 109.2, 76.1, 54.1, 42.3, 30.4, 28.4, 24.9,
21.8, 20.7, 18.5, 17.2, 12.9, 10.8, 3.7 ( ꢃ 2); HRMS (m/z): calcd for
C
34H38N6O3S [M þ H]þ, 611.2804; found, 611.2800.
(300 MHz, CDCl3):
d
11.05 (br s, 1H, -CONH-), 8.61 (br s, 1H, -CONH-
4.1.3.3. 2-(2-acetamidopyridin-4-yl)-4-(cyclopropylmethoxy)-N-(3-
((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)thiazole-5-
carboxamide (GT14). Yellow solid, 68% yield, mp 235e237 ꢂC, 96.9%
), 8.43 (s, 1H, AreH), 8.05 (m, 1H, AreH), 7.83 (d, J ¼ 6.2Hz, 1H,
AreH), 7.57 (m, 1H, AreH), 7.53 (m, 1H, AreH), 7.45 (m, 2H, AreH),
5.76 (m 1H, eCH2NH-), 4.73 (m, 2H, eOCH2-), 4.52 (m, 2H, -NHCH2-
), 4.22 (m, 2H, eOCH2-), 3.91 (s, 3H, eOCH3), 3.74 (m, 2H, Ar-CH2-),
3.40 (m, 2H, -Ar-CH2-), 2.93 (m, 2H, -Ar-CH2-), 2.76 (d, J ¼ 3.2 Hz,
3H, -NHCH3), 2.61 (m, 2H, -CH2-), 2.05 (m, 1H, -CH-), 1.75 (m, 4H,
HPLC purity.1H NMR (300 MHz, DMSO‑d6):
d 10.76 (s, 1H, -CONH-),
8.75 (s, 1H, AreH), 8.54 (d, J ¼ 8.2 Hz, 1H, AreH), 8.47 (d, J ¼ 8.2 Hz,
1H, AreH), 8.36e8.39 (m, 1H, -CONHCH2-), 8.25 (s, 1H, AreH),
7.81e7.84 (m, 1H, AreH), 7.75 (d, J ¼ 8.3 Hz, 1H, AreH), 7.70 (s, 1H,
AreH) 7.53e7.56 (m, 1H, AreH), 4.33 (d, J ¼ 8.4 Hz, 2H, eOCH2-),
3.97 (d, J ¼ 8.2 Hz, 2H, -NHCH2-), 3.62 (m, 2H, eNCH2-), 3.14e3.16
(m, 2H, -CONHCH2-), 2.94 (t, J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.67 (t,
J ¼ 7.8 Hz, 2H, -NHCH2-), 2.14 (s, 3H, eCOCH3), 1.81 (m, 4H,
eCH(CH2) 2-), 1.27 (m, 1H, eCH2CH-), 0.53e0.55 (m, 2H,
-CHCH2CH2-), 0.41 (m, 2H, -CHCH2CH2-); 13C NMR (75 MHz,
-(CH2)2-), 0.85 (m, 4H, -(CH2)2-); 13C NMR (75 MHz, CDCl3):
d 173.5,
162.9, 160.3, 160.2, 157.0, 153.5, 151.3, 149.7, 144.0, 140.9, 125.0,
124.8, 124.0, 122.3, 118.7, 115.4, 113.4, 111.3, 111.1, 109.0, 103.4, 70.6,
67.7, 63.5, 56.4, 55.3, 49.9, 47.1, 45.8, 26.6, 22.1, 20.8, 14.7, 8.3 ( ꢃ 2);
HRMS (m/z): calcd for C35H40N9O5S [M þ H]þ, 698.2868; found,
698.2858.
DMSO‑d6):
d 170.3, 163.2, 160.8, 160.4, 156.4, 153.7, 151.6, 150.8,
4.1.3. General procedure for the preparation of compounds
GT12ꢀGT14
149.9,141.0,140.1,138.3,133.2,129.3,125.6,121.2,119.5,112.9,109.2,
76.2, 54.1, 42.3, 30.4, 28.4,19.3,18.6, 17.2, 13.0, 10.8, 3.8 ( ꢃ 2); HRMS
(m/z): calcd for C31H34N6O3S [M þ H]þ, 571.2491; found, 571.2493.
To a solution of 11aꢀ11c (0.5 mmol) in 10 mL of DMF, DIPEA
(0.8 mL, 5 mmol) and HATU (285 mg, 0.75 mmol) were added. After
reaction at room temperature for 1 h under argon, amine com-
pounds 9aꢀ9g (0.75 mmol) was added, and the mixture was stirred
at room temperature for 24 h under the protection of argon. The
product GT12ꢀGT14 was isolated as a yellow solid by column
chromatography (DCM/MeOH ¼ 20/1, v/v).
4.1.3.4. 4-(cyclopropylmethoxy)-N-methyl-2-(2-(2-((2-((1,2,3,4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT15). Yellow solid, 81% yield, mp
185e187 ꢂC, 98.9% HPLC purity. 1H NMR (400 MHz, DMSO‑d6):
d
10.45 (s, 1H, -CONH-), 8.61 (d, J ¼ 6.1 Hz, 1H, AreH), 8.42 (d,
J ¼ 5.2 Hz, 1H, AreH), 8.23 (d, J ¼ 8.5 Hz, 1H, AreH), 7.62 (d,
J ¼ 3.3 Hz, 1H, AreH), 7.56e7.61 (m, 2H, AreH), 7.39 (t, J ¼ 7.7 Hz,
1H, AreH), 4.37 (d, J ¼ 8.3 Hz, 2H, -OCH2CH-), 3.67 (t, J ¼ 6.0 Hz, 2H,
eCOCH2-), 3.44 (s, 2H, -CH2-), 2.89 (m, 2H, -NHCH2-), 2.88 (m, 2H,
Ar-CH2-), 2.84 (d, J ¼ 6.3 Hz, 3H, -NHCH3), 2.73 (t, J ¼ 7.8 Hz, 2H, Ar-
CH2-), 1.79e1.81 (m, 4H, -(CH2)2-), 1.35e1.41 (m, 1H, eCH2CH-),
0.55e0.60 (m, 2H, -CH2CH2CH-), 0.41e0.45 (m, 2H, -CH2CH2CH-);
4.1.3.1. 2-(2-(cyclopropanecarboxamido)pyridin-4-yl)-4-(cyclo-
propylmethoxy)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)
thiazole-5-carboxamide (GT12). Yellow solid, 80% yield, mp
200e202 ꢂC, 98.3% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
d
11.05 (s, 1H, -CONH-), 8.75 (s, 1H, AreH), 8.61 (d, J ¼ 8.1 Hz, 1H,
AreH), 8.52 (d, J ¼ 8.1 Hz, 1H, AreH), 8.44e8.46 (m, 1H, -CONHCH3-
), 7.80 (d, J ¼ 7.8 Hz, 1H, AreH), 7.76 (d, J ¼ 7.8 Hz, 1H, AreH),
7.69e7.72 (m, 1H, AreH), 7.50e7.53 (m, 1H, AreH), 4.31e4.33 (d,
J ¼ 8.4 Hz, 2H, eOCH2-), 3.94 (d, J ¼ 8.2 Hz, 2H, -NHCH2-), 3.18 (d,
J ¼ 8.2 Hz, 2H, -CONHCH2-), 2.92 (t, J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.64 (t,
J ¼ 7.8 Hz, 2H, -NHCH2-), 2.03 (t, J ¼ 6.3 Hz, 2H, -CH2CH2CH2-), 2.03
(m, 1H, -COCH-), 1.80 (m, 4H, eCH(CH2) 2-), 1.29 (m, 1H, eCH2CH-),
0.85e0.86 (m, 2H, -CHCH2CH2-), 0.85 (m, 2H, -CHCH2CH2-), 0.52
(m, 2H, -CHCH2CH2-), 0.39 (m, 2H, -CHCH2CH2-); 13C NMR (75 MHz,
13C NMR (100 MHz, DMSO‑d6,
d: 171.7, 162.3, 160.4, 160.3, 155.5,
152.6, 152.3, 149.6, 144.2, 140.8, 129.6, 125.4, 124.0, 123.9, 118.7,
115.6, 114.4, 110.5, 108.5, 75.8, 52.2, 49.1, 47.6, 31.8, 26.5, 24.5, 22.4,
21.8, 10.5, 3.5 ( ꢃ 2); HRMS (m/z): calcd for C31H35N7O3S [M þ H]þ,
586.2600; found, 586.2587.
4.1.3.5. 2-(2-(2-((2-((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)
ethyl)amino)acetamido)pyridin-4-yl)-4-(cyclopropylmethoxy)-N-
methylthiazole-5-carboxamide (GT16). Yellow solid, 69% yield, mp
200e202 ꢂC, 96.1% HPLC purity. 1H NMR (300 MHz, DMSO‑d6)
DMSO‑d6):
d 171.7, 160.6, 160.6, 156.9, 152.8, 151.8, 149.9, 145.6,
141.2, 129.2, 127.0, 123.9, 119,8, 115.9, 115.3, 110.9, 108.7, 76.1, 52.8,
49.4, 48.3, 32.9, 30.9, 29.4, 29.1, 26.7, 25.3, 23.0, 22.5, 10.8 ( ꢃ 2), 3.8
( ꢃ 2); HRMS (m/z): calcd for C33H36N6O3S [M þ H]þ, 597.2648;
found, 597.2640.
d
10.43 (s, 1H, -CONH-), 8.64 (s, 1H, AreH), 8.43 (d, J ¼ 8.3 Hz, 1H,
AreH), 8.17 (d, J ¼ 8.3 Hz, 1H, AreH), 7.70 (s, 1H, AreH), 7.60 (m, 1H,
-CONH-), 7.59 (d, J ¼ 7.9 Hz,1H, AreH), 7.30 (d, J ¼ 7.9 Hz,1H, AreH),
4.39 (d, J ¼ 7.8 Hz, 2H, eOCH2-), 3.54 (d, J ¼ 8.1 Hz, 2H, eCOCH2-),
3.42 (m, 2H, -NHCH2-), 2.85 (t, J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.81 (d,
J ¼ 6.3 Hz, 3H, -NHCH3), 2.73 (t, J ¼ 7.9 Hz, 2H, Ar-CH2-), 2.51 (m, 2H,
-NHCH2-), 1.80 (m, 4H, -(CH2)2-), 1.37e1.41 (m, 1H, eCH2CH-),
0.57e0.60 (m, 2H, -CH2CH2CH-), 0.43e0.45 (m, 2H, -CH2CH2CH-);
4.1.3.2. 2-(2-(cyclobutanecarboxamido)pyridin-4-yl)-4-(cyclo-
propylmethoxy)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)
thiazole-5-carboxamide (GT13). Yellow solid, 59% yield, mp
203e205 ꢂC, 96.4% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
d
10.58 (s, 1H, -CONH-), 8.76 (s, 1H, AreH), 8.67 (d, J ¼ 8.0 Hz, 1H,
13C NMR (75 MHz, DMSO‑d6):
d 172.0, 162.6, 160.5, 159.6, 152.9,
AreH), 8.52 (d, J ¼ 8.0 Hz, 1H, AreH), 8.43e8.45 (m, 1H, -CONHCH3-
), 7.80e7.81 (d, J ¼ 8.1 Hz, 1H, AreH), 7.75 (d, J ¼ 8.1 Hz, 1H, AreH),
7.51e7.53 (m, 1H, AreH), 7.49e7.50 (m, 1H, AreH), 4.34 (d,
J ¼ 8.4 Hz, 2H, eOCH2-), 3.95 (d, J ¼ 8.2 Hz, 2H, -NHCH2-), 3.14e3.16
(m, 2H, -CONHCH2-), 3.12e3.13 (m, 1H, eCH2CHCH2-), 2.90 (t,
J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.66 (t, J ¼ 7.8 Hz, 2H, -NHCH2-), 2.24 (t,
J ¼ 7.8 Hz, 2H, -NHCH2-), 2.10e2.13 (m, 2H, -CHCH2CH2-), 2.04e2.06
(m, 2H, -CHCH2CH2-), 2.02e2.04 (m, J ¼ 6.3 Hz, 2H, -CH2CH2CH2-),
2.02e2.04 (m, 1H, -COCH-), 1.80 (m, 4H, eCH(CH2) 2-), 1.26 (m, 1H,
eCH2CH-), 0.53e0.55 (m, 2H, -CHCH2CH2-), 0.40e0.41 (m, 2H,
151.1, 149.9, 148.0, 141.9, 134.1, 127.0, 126.0, 123.8, 118.8, 116.3, 115.8,
108.8, 106.3, 76.1, 52.6, 49.7, 48.2, 33.9, 26.8, 25.1, 23.0, 22.7, 19.1,
10.8, 3.8 ( ꢃ 2); HRMS (m/z): calcd for C31H34ClN7O3S [M þ H]þ,
620.2211; found, 620.2190.
4.1.3.6. 4-(cyclopropylmethoxy)-2-(2-(2-((2-((7-methoxy-1,2,3,4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)-
N-methylthiazole-5-carboxamide(GT17). Yellow solid, 73% yield, mp
207e209 ꢂC, 97.2% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
d
10.48 (s, 1H, -CONH-), 8.63 (s, 1H, AreH), 8.43 (d, J ¼ 8.1 Hz, 1H,
-CHCH2CH2-); 13C NMR (75 MHz, DMSO‑d6):
d 174.7, 163.2, 160.8,
AreH), 7.67 (d, J ¼ 8.1 Hz, 1H, AreH), 7.58 (d, J ¼ 8.2 Hz, 2H, AreH),

X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
13
7.49 (s,1H, AreH), 7.22e7.25 (m, J ¼ 8.2 Hz,1H, -CONH-), 5.64 (s,1H,
AreH), 4.38 (d, J ¼ 8.4 Hz, 2H, eOCH2-), 3.87 (s, 3H, eOCH3), 3.54 (d,
J ¼ 8.2 Hz, 2H, eCOCH2-), 3.45 (m, 2H, -NHCH2-), 2.85 (t, J ¼ 7.8 Hz,
2H, Ar-CH2-), 2.84 (d, J ¼ 6.3 Hz, 3H, -NHCH3), 2.77 (t, J ¼ 7.9 Hz, 2H,
Ar-CH2-), 2.76 (m, 2H, Ar-CH2-), 1.79 (m, 4H, -(CH2)2-), 1.39 (m, 1H,
eCH2CH-), 0.57e0.59 (m, 2H, -CH2CH2CH-), 0.43e0.44 (m, 2H,
138.6, 133.6, 125.7, 125.4, 120.0, 120.0, 116.3, 113.5, 109.7, 107.6, 76.2,
52.0, 49.4, 46.9, 36.3, 29.5, 24.4, 22.0, 20.9, 10.8, 3.7; HRMS (m/z):
calcd for C30H33N7O3S [M þ H]þ, 572.2444; found, 572.2411.
4.1.3.10. 4-(cyclopropylmethoxy)-N-methyl-2-(2-(2-((4-((1,2,3,4-
tetrahydroacridin-9-yl)amino)butyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT21). Yellow solid, 62% yield, mp
179e181 ꢂC, 98.8% HPLC purity.1H NMR (300 MHz, DMSO‑d6):
-CH2CH2CH-); 13C NMR (75 MHz, DMSO‑d6):
d 171.5, 162.1, 160.1,
155.5, 154.6, 152.5, 150.3, 149.4, 141.2, 140.7, 128.5, 120.5, 116.3,
115.3, 110.2, 108.4, 101.8, 75.6, 55.4, 52.2, 49.2, 47.3, 32.4, 27.1, 26.3,
24.9, 22.6, 22.2, 20.9, 10.3, 3.3 ( ꢃ 2); HRMS (m/z): calcd for
d
8.63 (s, 1H, AreH), 8.41 (d, J ¼ 8.2 Hz,1H, AreH), 8.17 (d, J ¼ 8.2 Hz,
1H, AreH), 7.72 (d, J ¼ 7.9 Hz, 1H, AreH), 7.58 (d, J ¼ 7.9 Hz, 1H,
AreH), 7.55 (s, 1H, AreH), 7.37 (m, 1H, AreH), 4.38 (d, J ¼ 7.8 Hz, 2H,
eOCH2-), 3.60 (d, J ¼ 8.2 Hz, 2H, eCOCH2-), 3.37 (m, 2H, -NHCH2-),
2.90 (t, J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.84 (d, J ¼ 6.3 Hz, 3H, -NHCH3),
2.69 (t, J ¼ 7.9 Hz, 2H, Ar-CH2-), 2.55e2.49 (m, 2H, -NHCH2-), 1.79
(m, 4H, -(CH2)2-), 1.64e1.66 (m, 2H, -CH2CH2-), 1.49e1.51 (m, 2H,
-CH2CH2-), 1.38e1.41 (m, 1H, eCH2CH-), 0.57e0.59 (m, 2H,
-CH2CH2CH-), 0.43e0.44 (m, 2H, -CH2CH2CH-); 13C NMR (75 MHz,
C
32H37N7O4S [M þ H]þ, 616.2706; found, 616.2691.
4.1.3.7. 4-(cyclopropylmethoxy)-N-ethyl-2-(2-(2-((2-((1,2,3,4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT18). Yellow solid, 81% yield, mp
210e212 ꢂC, 98.9% HPLC purity.1H NMR (300 MHz, DMSO‑d6):
d
10.48 (s, 1H, -CONH-), 8.63 (s, 1H, AreH), 8.43 (d, J ¼ 7.9 Hz, 1H,
AreH), 8.27 (d, J ¼ 7.9 Hz, 1H, AreH), 7.77 (d, J ¼ 8.2 Hz, 1H, AreH),
7.66 (d, J ¼ 8.2 Hz, 1H, AreH), 7.60e7.61 (m, 1H, AreH), 7.42e7.45
(m, J ¼ 8.2 Hz, 1H, -CONH-), 7.40 (m, 1H, AreH), 4.38e4.41 (d,
J ¼ 8.4 Hz, 2H, eOCH2-), 3.71 (d, J ¼ 8.2 Hz, 2H, eCOCH2-), 3.45 (m,
2H, -NHCH2-), 3.34e3.38 (m, 2H, -NHCH2-), 2.90 (t, J ¼ 7.8 Hz, 2H,
Ar-CH2-), 2.89e2.91 (m, J ¼ 6.3 Hz, 2H, -CONHCH2-), 2.74 (t,
J ¼ 7.9 Hz, 2H, Ar-CH2-), 1.81 (m, 4H, -(CH2)2-), 1.38e1.39 (m, 1H,
eCH2CH-), 1.14 (t, J ¼ 6.3 Hz, 3H, -CH2CH3), 0.58e0.60 (m, 2H,
-CH2CH2CH-), 0.44e0.45 (m, 2H, -CH2CH2CH-); 13C NMR (75 MHz,
DMSO‑d6): d 172.8, 171.7, 162.6, 160.7, 160.6, 156.7, 152.0, 150.1,
150.0, 146.1, 145.3, 141.2, 129.5, 129.4, 124.1, 124.0, 119.6, 115.2, 108.7,
76.1, 52.7, 49.1, 48.2, 32.7, 29.5, 28.6, 26.8, 25.3, 22.9, 22.4, 10.8, 3.8
( ꢃ 2); HRMS (m/z): calcd for C33H39N7O3S [M þ H]þ, 614.2910;
found, 614.2911.
4.1.3.11. 4-(cyclopropylmethoxy)-N-methyl-2-(2-(2-((6-((1,2,3,4-
tetrahydroacridin-9-yl)amino)hexyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT22). Yellow solid, 71% yield, mp
190e192 ꢂC, 97.5% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
DMSO‑d6):
d 172.0, 162.7, 160.7, 159.9, 152.9, 149.9, 147.2, 141.2,
130.4, 124.7, 124.6, 124.5, 115.9, 114.3, 111.1, 109.0, 108.9, 76.2, 52.6,
49.3, 47.9, 34.5, 31.6, 30.3, 29.5, 24.7, 22.6, 22.0,15.4,10.8, 3.7; HRMS
(m/z): calcd for C32H37N7O3S [M þ H]þ, 600.2757; found, 600.2749.
d
8.62 (s, 1H, AreH), 8.41 (d, J ¼ 8.1 Hz, 1H, AreH), 8.15 (d, J ¼ 8.1 Hz,
1H, AreH), 7.75 (d, J ¼ 7.8 Hz, 1H, AreH), 7.58 (d, J ¼ 7.8 Hz, 1H,
AreH), 7.54e7.57 (m, 1H, AreH), 7.34e7.36 (m, 1H, AreH), 5.93 (m,
1H, AreH), 4.37 (d, J ¼ 8.3 Hz, 2H, eOCH2-), 3.60 (m, 2H, -NHCH2-),
3.38 (d, J ¼ 8.2 Hz, 2H, eCOCH2-), 2.90 (t, J ¼ 7.8 Hz, 2H, Ar-CH2-),
2.84 (d, J ¼ 6.3 Hz, 3H, -NHCH3), 2.67 (t, J ¼ 7.9 Hz, 2H, Ar-CH2-),
2.55 (m, 2H, -NHCH2-), 1.78 (m, 4H, -(CH2)2-), 1.58 (m, 2H,
-CH2CH2CH2-), 1.40e1.41 (m, 2H, -CH2CH2CH2-), 1.38e1.39 (m, 1H,
eCH2CH-), 1.29 (m, 2H, -CH2CH2CH2-), 1.20e1.24 (m, 2H,
-CH2CH2CH2-), 0.56e0.59 (m, 2H, -CH2CH2CH-), 0.42e0.44 (m, 2H,
4.1.3.8. 4-(cyclopropylmethoxy)-N-propyl-2-(2-(2-((2-((1,2,3,4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT19). Yellow solid, 78% yield, mp
213e215 ꢂC, 98.0% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
d
10.46 (s, 1H, -CONH-), 8.61 (s, 1H, AreH), 8.42 (d, J ¼ 8.2 Hz, 1H,
AreH), 8.28 (d, J ¼ 8.2 Hz, 1H, AreH), 7.75 (d, J ¼ 8.2 Hz, 1H, AreH),
7.66 (d, J ¼ 8.2 Hz, 1H, AreH), 7.58e7.62 (m, 1H, AreH), 7.58 (m,
J ¼ 8.2 Hz,1H, -CONH-), 7.40e7.45 (m, 1H, AreH), 4.39 (d, J ¼ 8.4 Hz,
2H, eOCH2-), 3.73 (d, J ¼ 8.2 Hz, 2H, eCOCH2-), 3.46 (m, 2H,
-NHCH2-), 3.25e3.29 (m, 2H, -NHCH2-), 2.90 (t, J ¼ 7.8 Hz, 2H, Ar-
CH2-), 2.89 (m, 2H, -CONHCH2-), 2.73 (t, J ¼ 7.9 Hz, 2H, Ar-CH2-),
1.81 (m, 4H, -(CH2)2-), 1.51e1.58 (m, 2H, -CH2CH2-), 1.29e1.36 (m,
1H, eCH2CH-), 0.90e0.94 (t, J ¼ 6.3 Hz, 3H, -CH2CH3), 0.58e0.60
(m, 2H, -CH2CH2CH-), 0.43e0.44 (m, 2H, -CH2CH2CH-); 13C NMR
-CH2CH2CH-); 13C NMR (75 MHz, DMSO‑d6):
d 171.5, 162.6, 160.7,
160.6, 156.8, 156.7, 152.0, 150.0, 145.3, 141.2, 129.4, 127.1, 126.6,
124.0, 124.0, 119.6, 115.9, 115.1, 108.7, 76.1, 52.7, 49.3, 48.2, 32.7, 30.9,
29.6, 29.5, 26.8, 26.6, 25.3, 22.9, 22.4, 10.8, 3.8 ( ꢃ 2); HRMS (m/z):
calcd for C35H44N7O3S [M þ H]þ, 642.3226; found, 642.3221.
4.1.3.12. 4-(cyclopropylmethoxy)-N-methyl-2-(2-(2-((8-((1,2,3,4-
tetrahydroacridin-9-yl)amino)octyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT23). Yellow solid, 80% yield, mp
200e202 ꢂC, 98.3% HPLC purity.1H NMR (300 MHz, DMSO‑d6):
(75 MHz, DMSO‑d6):
d 172.0, 162.8, 160.7, 160.0, 154.8, 153.4, 152.9,
149.9,143.3,141.2, 130.5,124.6, 124.5, 121.9, 118.4, 115.9, 114.2,110.9,
108.8, 76.2, 52.5, 49.3, 47.9, 31.4, 29.5, 24.7, 22.9, 22.6, 21.9, 11.7, 10.8,
3.7 ( ꢃ 2); HRMS (m/z): calcd for C33H39N7O3S [M þ H]þ, 614.2913;
found, 614.2910.
d
8.64 (s,1H, AreH), 8.42 (d, J ¼ 8.0 Hz,1H, AreH), 8.14 (d, J ¼ 8.0 Hz,
1H, AreH), 7.72 (d, J ¼ 7.9 Hz, 1H, AreH), 7.57 (d, J ¼ 7.9 Hz, 1H,
AreH), 7.54 (M, 1H, AreH), 7.33e7.38 (m, 1H, AreH), 4.37 (d,
J ¼ 8.4 Hz, 2H, eOCH2-), 3.60 (d, J ¼ 8.2 Hz, 2H, eCOCH2-), 3.38 (m,
2H, -NHCH2-), 2.90 (t, J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.85 (d, J ¼ 6.3 Hz,
3H, -NHCH3), 2.67 (t, J ¼ 7.9 Hz, 2H, Ar-CH2-), 2.55 (m, 2H, -NHCH2-
), 1.79 (m, 4H, -(CH2)2-), 1.55 (m, 4H, -CH2CH2CH2-), 1.38e1.40 (m,
1H, eCH2CH-), 1.33 (m, 4H, -CH2CH2CH2-), 1.22 (m, 4H,
-CH2CH2CH2-), 0.57e0.59 (m, 2H, -CH2CH2CH-), 0.43e0.44 (m, 2H,
4.1.3.9. 4-(cyclopropylmethoxy)-2-(2-(2-((2-((1,2,3,4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT20). Yellow solid, 59% yield, mp
185e187 ꢂC, 96.4% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
d
10.23 (s, 1H, -CONH-), 8.55 (d, J ¼ 8.2 Hz, 1H, AreH), 8.42 (d,
J ¼ 8.2 Hz, 1H, AreH), 8.18 (s, 1H, AreH), 7.91e7.95 (m, 1H, AreH),
7.79 (m, 1H, AreH), 7.53 (s, 2H, eCONH2), 7.41 (m, 1H, AreH), 7.07
(m, 1H, AreH), 4.38 (d, J ¼ 8.4 Hz, 2H, eOCH2-), 3.97 (d, J ¼ 8.2 Hz,
2H, eCOCH2-), 3.51e7.57 (m, 2H, -NHCH2-), 3.17 (m, 1H,
eCH2NHCH2-), 2.96 (m, J ¼ 6.8 Hz, 2H, -NHCH2CH2-), 2.89 (m,
J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.73 (m, J ¼ 7.8 Hz, 2H, Ar-CH2-), 1.81 (m,
4H, -(CH2)2-), 1.34e1.38 (m, 1H, eCH2CH-), 0.59e0.61 (m, 2H,
-CH2CH2CH-), 0.43 (m, 2H, -CH2CH2CH-); 13C NMR (75 MHz,
-CH2CH2CH-); 13C NMR (75 MHz, DMSO‑d6):
d 171.6, 162.6, 162.6,
160.5,156.8,152.7,151.8,149.9,147.8,147.3,141.2,129.2,126.9,123.9,
123.8, 115.9, 115.9, 115.3, 108.7, 76.1, 52.8, 49.4, 49.4, 48.2, 32.9, 30.9,
30.8, 29.2, 26.9, 26.7, 25.3, 22.9, 10.8, 3.8 ( ꢃ 2); HRMS (m/z): calcd
for C35H44N7O3S [M þ H]þ, 670.3539; found, 670.3536.
4 .1. 3 .13 . 4 - m e t h o x y- N - m e t hyl - 2 - ( 2- ( 2 - ( ( 2 - ( ( 1, 2 , 3 , 4 -
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT24). Yellow solid, 59% yield, mp
DMSO‑d6): d 170.3,163.1,162.8,161.7,161.0,156.8,156.2,154.6,141.2,

14
X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
203e205 ꢂC, 96.4% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
10.49 (s, 1H, -CONH-), 8.63 (s, 1H, AreH), 8.44 (d, J ¼ 8.2 Hz, 1H,
4.1. 3.17. 4-Isobutoxy-N-methyl-2-(2-(2-((2-((1, 2, 3, 4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT28). Yellow solid, 73% yield, mp
207e209 ꢂC, 97.2% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
d
AreH), 8.29 (d, J ¼ 8.2 Hz, 1H, AreH), 7.82 (d, J ¼ 8.0 Hz, 1H, AreH),
7.67 (d, J ¼ 8.0 Hz, 1H, AreH), 7.62 (m, 1H, AreH), 7.42 (m, 1H,
AreH), 4.16 (s, 3H, eOCH3), 3.73 (d, J ¼ 7.9 Hz, 2H, eCOCH2-), 3.48
(d, J ¼ 8.0 Hz, 2H, -NHCH2-), 3.17 (d, J ¼ 8.2 Hz, 2H, -NHCH2-), 2.92
(t, J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.81 (d, J ¼ 6.3 Hz, 3H, -NHCH3), 2.74 (t,
J ¼ 7.9 Hz, 2H, Ar-CH2-), 1.81 (m, 4H, Ar-CH2(CH2)2-); 13C NMR
d
10.48 (s, 1H, -CONH-), 8.63 (s, 1H, AreH), 8.45 (d, J ¼ 7.8 Hz, 1H,
AreH), 8.25 (d, J ¼ 7.8 Hz, 1H, AreH), 7.78 (d, J ¼ 8.2 Hz, 1H, AreH),
7.61 (s, 1H, AreH), 7.46 (m, 1H, -CONHCH3), 7.41 (m, 2H, AreH), 4.33
(d, 1H, J ¼ 8.4 Hz, 2H, eOCH2-), 3.68 (d, J ¼ 8.2 Hz, 2H, eCOCH2-),
3.45 (d, J ¼ 8.2 Hz, 2H, -NHCH2-), 2.92 (t, J ¼ 7.8 Hz, 2H, Ar-CH2-),
2.91 (t, J ¼ 7.8 Hz, 2H, -NHCH2-), 2. 86 (d, J ¼ 6.4 Hz, 3H, -NHCH3),
2.74 (t, J ¼ 7.9 Hz, 2H, Ar-CH2-), 2.17e2.21 (m, 1H, -CH2CH(CH3)2),
1.81 (m, 4H, eCH(CH2) 2-), 0.99e1.01 (m, 6H, eCH(CH3)2); 13C NMR
(75 MHz, DMSO‑d6):
d 171.9, 162.6, 161.0, 160.4, 157.9, 150.0, 141.2,
130.7, 129.9, 124.8, 124.7, 124.6, 117.1, 115.9, 114.9, 111.2, 110.6, 108.9,
101.7, 58.7, 49.2, 47.8, 31.6, 30.3, 26.8, 22.6, 14.4 ( ꢃ 2); HRMS (m/z):
calcd for C28H32N7O3S [M þ H]þ, 546.2287; found, 546.2280.
(75 MHz, DMSO‑d6):
d 172.0, 162.7, 160.8, 160.6, 153.0, 150.0, 141.2,
4.1.3.14. 4-(cyclobutylmethoxy)-N-methyl-2-(2-(2-((2-((1,2,3,4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT25). Yellow solid, 68% yield, mp
235e237 ꢂC, 96.9% HPLC purity.1H NMR (300 MHz, DMSO‑d6):
130.4, 130.1, 125.5, 124.6,124.6, 124.4, 124.4, 118.9,115.9, 114.6, 111.2,
110.6, 108.9, 77.6, 52.5, 49.3, 47.9, 31.6, 30.3, 29.5, 28.2, 26.8, 24.8,
19.4; HRMS (m/z): calcd for C31H38N7O3S [M þ H]þ, 588.2757;
found, 588.2745.
d
10.46 (s, 1H, -CONH-), 8.62 (s, 1H, AreH), 8.44 (d, J ¼ 8.2 Hz, 1H,
AreH), 8.28 (d, J ¼ 8.2 Hz, 1H, AreH), 7.76 (d, J ¼ 8.1 Hz, 1H, AreH),
7.64 (d, J ¼ 8.1 Hz, 1H, AreH), 7.61 (m, 1H, -CONHCH3), 7.43e7.44
(m, 2H, AreH), 6.24 (m, 1H, AreH), 4.53 (d, J ¼ 8.4 Hz, 2H, eOCH2-),
3.73 (d, J ¼ 8.2 Hz, 2H, eCOCH2-), 3.45 (d, J ¼ 8.2 Hz, 2H, -NHCH2-),
3.27 (m, 1H, - CH2NHCH2-), 2.92 (t, J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.91 (d,
J ¼ 7.8 Hz, 2H, -NHCH2-), 2.84 (d, J ¼ 6.3 Hz, 3H, -NHCH3), 2.74 (t,
J ¼ 7.9 Hz, 2H, Ar-CH2-), 2.70 (m, 1H, eCH2CH-), 2.08 (m, 2H,
4.2. Biological evaluation
4.2.1. Cholinesterase inhibition
The compound’s cholinesterase inhibitory ability is tested by
Ellman analysis by Ellman assay [43]. Briefly, 40
buffer (pH 8.0) was first added to the 96-well plate, and then add
different concentrations of the tested compounds (10 L), enzyme
(AChE/BChE (2.5 U/mL), 10
L), and 50-dithiobis (2-nitrobenzoic
acid) (10 mM, 20 L) at room temperature for 1 min. After the
plate was incubated for 1 min, 20 L of acetylthiocholine iodide or
mL of phosphate
m
-CHCH2CH2-), 1.88 (m, 4H, eCH(CH2) 2-), 1.82 (m, 4H, -(CH2)2-); 13
C
m
NMR (75 MHz, DMSO‑d6):
d
172.0, 162.7, 160.8, 160.6, 157.7, 153.0,
m
150.0, 141.2, 131.0, 130.5, 124.8, 124.6, 124.5, 118.6, 115.9, 114.5, 110.9,
108.9, 75.3, 52.5, 51.9, 49.2, 48.2, 41.4, 24.5, 29.5, 26.8, 24.8, 22.6,
22.0, 18.5 ( ꢃ 2); HRMS (m/z): calcd for C32H38N7O3S [M þ H]þ,
600.2757; found, 600.2760.
m
butyrylthiocholine iodide (75 mM) was added to the reaction sys-
tem. The plate was placed at 37 ꢂC for 10 min, and then the
absorbance was recorded at 415 nm using a microplate reader. Each
experiment was independently repeated at least three times. 10
of enzyme was replaced by water as 0% of enzyme activity, and
10 L of compound was replaced by water as 100% of enzyme ac-
tivity. The IC50 of the compound is calculated based on the inhibi-
tory ability of the compound at different concentrations by using
GraphPad Prism 8.0 software.
mL
4.1.3.15. N-methyl-4-(neopentyloxy)-2-(2-(2-((2-((1,2,3,4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT26). Yellow solid, 81% yield, mp
185e187 ꢂC, 98.9% HPLC purity. 1H NMR (300 MHz, DMSO‑d6):
m
d
10.51 (s, 1H, -CONH-), 8.60 (s, 1H, AreH), 8.44 (d, J ¼ 8.4 Hz, 1H,
AreH), 8.33 (d, J ¼ 8.4 Hz, 1H, AreH), 7.83 (d, J ¼ 8.2 Hz, 1H, AreH),
7.71 (d, J ¼ 8.2 Hz, 1H, AreH), 7.47 (m, 1H, -CONHCH3), 7.30 (m, 2H,
AreH), 6.69 (m, 1H, AreH), 4.28 (d, J ¼ 7.7 Hz, 2H, eOCH2-), 3.71 (d,
J ¼ 8.2 Hz, 2H, eCOCH2-), 3.48 (d, J ¼ 8.2 Hz, 2H, -NHCH2-), 2.94 (t,
J ¼ 7.8 Hz, 2H, Ar-CH2-), 2.91 (t, J ¼ 7.8 Hz, 2H, -NHCH2-), 2.87 (d,
J ¼ 6.3 Hz, 3H, -NHCH3), 2.69 (t, J ¼ 7.9 Hz, 2H, Ar-CH2-),1.82 (m, 4H,
eCH(CH2)2-), 1.04 (m, 9H, eC(CH3)3); 13C NMR (75 MHz, DMSO‑d6):
4.2.2. Inhibition of GSK-3
Human recombinant GSK-3
substrate peptide, cat. 12e533) were obtained from Millipore.
b
b
(cat. 14e306) and GSM (GSK3
Kinase-Glo Luminescent Kinase Assay was purchased from Prom-
ega. The GSK-3
Kinase-Glo assays method developed by Baki et al. [42]. Briefly,
10 L of tested compound at different concentrations and 10
GSK-3 (20 ng) were added to the black 96-well plate, and then
10 L of GSM substrate (25 M) and 10 L of ATP (0.5 M) were
b inhibitory activity of compound was tested using
d
171.2, 162.7, 161.1, 160.7, 160.6,157.7, 152.9,150.0,141.1, 131.6, 131.4,
m
mL
125.2, 124.9, 124.7, 118.9, 116.0, 109.3, 108.9, 81.0, 52.0, 48.1, 41.3,
b
32.4, 30.3, 29.5, 26.8 ( ꢃ 3), 24.7, 22.4, 21.6; HRMS (m/z): calcd for
m
m
m
m
C
32H40N7O3S [M þ H]þ, 602.2913; found, 602.2883.
added, respectively. After the plate was incubated at 30 ꢂC for
30 min, Kinase-Glo reagent was added to the reaction system. The
value of luminescence was recorded within 5 min using Molecular
Devices ID5 multimode reader.
4.1.3.16. 4-Isopropoxy-N-methyl-2-(2-(2-((2-((1,2,3,4-
tetrahydroacridin-9-yl)amino)ethyl)amino)acetamido)pyridin-4-yl)
thiazole-5-carboxamide (GT27). Yellow solid, 69% yield, mp
200e202 ꢂC, 96.1%HPLCpurity.1H NMR (300 MHz, DMSO‑d6):
d
10.50
4.2.3. In vitro BBB permeation assay
(s,1H, -CONH-), 8.64 (s,1H, AreH), 8.44 (d, J ¼ 8.2 Hz,1H, AreH), 8.24
(d, J ¼ 8.2 Hz, 1H, AreH), 7.78 (d, J ¼ 7.8 Hz, 1H, AreH), 7.61 (d,
J ¼ 7.8Hz,1H, AreH), 7.57e7.59 (m,1H, -CONHCH3), 7.40e7.44 (m, 2H,
AreH), 5.26e5.34 (m, 1H, -OCH(CH3)2), 3.67 (d, J ¼ 8.2 Hz, 2H,
eCOCH2-), 3.47 (d, J ¼ 8.2 Hz, 2H, -NHCH2-), 2.91e2.92 (t, J ¼ 7.8 Hz,
2H, Ar-CH2-), 2.86e2.87(t, J ¼ 7.8Hz, 2H, -NHCH2-), 2.75(d, J ¼ 6.5 Hz,
3H, -NHCH3), 2.53 (t, J ¼ 7.9 Hz, 2H, Ar-CH2-),1.81 (m, 4H, eCH(CH2) 2-
Commercial drugs (Dopamine, Ofloxacin, Lomefloxacin, Pirox-
acin, Clonidine, Chlorpromazine, Progesterone, Desipramine,
Verapamil, and Testosterone) of known BBB permeability were
obtained from Sigma-Aldrich. Porcine brain lipid (PBL) was pur-
chased from Avanti Polar Lipids. The UV plate (COSTAR) was ob-
tained from Corning Inc. The donor microplates (PVDF membrane,
pore size 0.45
from Millipore. The BBB permeability of the tested compounds was
evaluated using PAMPA developed by Di et al. [45]. Briefly, 200 L of
PBS/EtOH (7:3) was added to the 96-well microwell receptor plate,
and the filter was impregnated with 4 L of dodecane containing
PBL (20 mg/mL). 200 L of tested compound (100 g/mL) was
mm) and the acceptor microplates were obtained
),1.41e1.45 (m, 6H, eCH(CH3)2);13CNMR(75MHz,DMSO‑d6):
d172.0,
162.8, 160.6, 160.1, 153.0, 150.0, 141.2, 130.1, 129.7, 125.2, 124.5, 124.3,
124.1, 118.8, 115.9, 114.5, 111.2, 109.0, 108.9, 75.5, 52.6, 49.4, 47.9, 31.8,
26.8, 24.8, 22.7, 22.4 ( ꢃ 2), 22,1; HRMS (m/z): calcd for C30H36N7O3S
[M þ H]þ, 574.2600; found, 574.2593.
m
m
m
m

X. Jiang et al. / European Journal of Medicinal Chemistry 207 (2020) 112751
15
added to the donor well and the donor plate was placed on the
acceptor plate. After the plate was left at room temperature for
20 h, the liquid (100 L) in the acceptor plate was transferred to the
UV plate. The concentrations of the compounds were determined
based on measuring the OD value of the UV plate at different
wavelengths using a microplate reader.
Acknowledgements
m
We gratefully thank the support from the grants (Nos.
81973207and 81573281) of National Natural Science Foundation of
China. X.Y. Jiang especially wishes to thank for the invaluable
spiritual support received from his wife L.Q. Kang over the past
months.
4.2.4. Western blot analysis in N2a-tau cell
Mouse neuroblastoma N2a-tau cells were seeded at
Appendix A. Supplementary data
6.0 ꢃ 105 cells/well in a 6-well plate. N2a cells were pre-treated
with GT15 (5, 10, and 15 mM) for 1 h, and then treated with oka-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112751.
daid acid (40 nM) for 5 h. Briefly, the cells were lysed with RIPA
lysate containing 1% protease inhibitor and 1% phosphatase inhib-
itor. The total protein content was determined using the BCA pro-
References
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secondary antibody. The membrane was again washed with Tween
20-TBS and incubated with ECL chemiluminescent reagent. Lumi-
nescence was captured in the Tanon 5200 multi-functional auto-
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