Novel deoxyvasicinone derivatives as potent multitarget-directed ligands for the treatment of Alzheimer's disease: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2017.09.008

A series of multitarget ligands was designed by introducing several structurally diverse aminoacetamide groups at position 6 of the deoxyvasicinone group, with the aim of obtaining novel multifunctional anti-Alzheimer's disease agents using deoxyvasicinone as the substrate. In vitro studies showed that almost all of the derivatives were potent inhibitors of human recombinant acetylcholinesterase (hAChE) and human serum butyrylcholinesterase (hBChE), with IC₅₀ values in the low nanomolar range, and exhibited moderate to high inhibition of Aβ_1?42 self-aggregation. In particular, compounds 12h, 12n, and 12q showed promising inhibitory activity for hAChE, with IC₅₀ values of 5.31 ± 2.8, 4.09 ± 0.23, and 7.61 ± 0.53 nM, respectively. Compounds 12h and 12q also exhibited the greatest ability to inhibit hBChE, with IC₅₀ values of 4.35 ± 0.32 and 2.35 ± 0.14 nM, respectively. Moreover, enzyme kinetics confirmed that compound 12q caused a mixed type of AChE inhibition, by binding to both the active sites (PAS and CAS) of AChE. Remarkably, compound 12q also demonstrated the highest potential inhibitory activity for Aβ_1?42 self-aggregation (63.9 ± 4.9%, 10 μM), and it was also an excellent metal chelator.

Full text of DOI:10.1016/j.ejmech.2017.09.008

Accepted Manuscript
Novel deoxyvasicinone derivatives as potent multitarget-directed ligands for the
treatment of Alzheimer's disease: Design, synthesis, and biological evaluation
Fang Ma, Hongtao Du
PII:
S0223-5234(17)30690-6
10.1016/j.ejmech.2017.09.008
EJMECH 9723
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 July 2017
Revised Date: 3 September 2017
Accepted Date: 5 September 2017
Please cite this article as: F. Ma, H. Du, Novel deoxyvasicinone derivatives as potent multitarget-
directed ligands for the treatment of Alzheimer's disease: Design, synthesis, and biological evaluation,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.008.
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Novel Deoxyvasicinone Derivatives as Potent Multitarget-Directed Ligands for
the Treatment of Alzheimer’s Disease: Design, Synthesis, and Biological
Evaluation
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Fang Ma b , Hongtao Du a, c, *
a College of Life Sciences, Xinyang Normal University, Xinyang, Henan, China
b School of Geographic Sciences, Xinyang Normal University, Xinyang, Henan, China
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c Shaanxi Key Laboratory of Natural Products & Chemical Biology，School of Chemistry and Pharmacy, Northwest A&F
University, Yangling 712100, Shaanxi Province, P. R. China.
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E-mails: duht@nwsuaf.edu.cn (H. T. Du); mafang_1984@163.com (F.Ma)
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*Corresponding author:
Dr. Hong-Tao Du
1. College of Life Sciences, Xinyang Normal University, Xinyang, Henan 464000, P. R. China
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2. Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Sciences,
Northwest A&F University, Yangling, Shaanxi 712100, P. R. China
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Email: duhongtao8410@163.com
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ABSTRACT
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A series of multitarget ligands was designed by introducing several structurally diverse
aminoacetamide groups at position 6 of the deoxyvasicinone group, with the aim of obtaining novel
multifunctional anti-Alzheimer’s disease agents using deoxyvasicinone as the substrate. In vitro
studies showed that almost all of the derivatives were potent inhibitors of human recombinant
acetylcholinesterase (hAChE) and human serum butyrylcholinesterase (hBChE), with IC50 values in
the low nanomolar range, and exhibited moderate to high inhibition of Aβ1−42 self-aggregation. In
particular, compounds 12h, 12n, and 12q showed promising inhibitory activity for hAChE, with IC50
values of 5.31 ± 2.8, 4.09 ± 0.23, and 7.61 ± 0.53 nM, respectively. Compounds 12h and 12q also
exhibited the greatest ability to inhibit hBChE, with IC50 values of 4.35 ± 0.32 and 2.35 ± 0.14 nM,
respectively. Moreover, enzyme kinetics confirmed that compound 12q caused a mixed type of
AChE inhibition, by binding to both the active sites (PAS and CAS) of AChE. Remarkably,
compound 12q also demonstrated the highest potential inhibitory activity for Aβ1−42 self-aggregation
(63.9 ± 4.9%, 10 µM), and it was also an excellent metal chelator.
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KEYWORDS
Alzheimer’s disease; deoxyvasicinone derivatives; cholinesterase inhibitors; inhibition of Aβ1−42
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self-aggregation; metal chelator
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1. Introduction
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Alzheimer’s disease (AD) is a progressive and degenerative neurodegenerative brain disorder
characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical
regions[1]. AD is the most common form of dementia, affecting approximately 47 million people
worldwide in 2015[2]. With the increase in average life expectancy, the number of affected persons is
projected to exceed 100 million individuals worldwide by 2050[3]. Clinically, the symptoms can
include problems such as loss of memory, language deterioration, mood swings, and loss of bodily
functions, ultimately leading to death[4]. At this time, acetylcholinesterase (AChE) inhibitors (tacrine
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(1), donepezil (2), rivastigmine (3), and galantamine (4), Figure 1) and N-methyl-D-aspartate
antagonist are the only approved drugs that can provide a palliative, therapeutic strategy in mild
forms of AD[5, 6]. Unfortunately, there is no means to cure, or even slow, the progression of the
disease[7]. This alarming situation needs further effort to develop more effective drugs for treatment
of AD.
Although the exact cause of AD is still not fully known, the consensus is that AD is a
multifactorial disease[8]. Low levels of acetylcholine (ACh) in the hippocampus and cortex area of
the brain, deposition of amyloid-β (Aβ) peptide, neurofibrillary tangles (p-Tau), and oxidative stress
are thought to play vital roles in AD pathogenesis[9-12]. Because of the complexity of AD and the
interconnection of molecular events in its progression, the single-target directed drugs that have
reached clinical trials have failed. Therefore, some researchers have invested their efforts in the
design of multitarget-directed ligands (MTDLs) [13-15], which simultaneously interact with two or
more diseased targets, as a better strategy for AD rather than concentrating on reducing just one
target.
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Aβ plays an important role among the multiple factors in AD[16]. Neurofibrillary tangles and
aggregated Aβ peptide deposition in senile plaques have been identified as the main pathological
hallmarks[17-19]. Thus, drugs that are expected to reduce Aβ production, prevent Aβ aggregation,
and promote Aβ clearance are promising approaches for the treatment of AD[20].
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AD is ascribed to reduced levels of ACh, which is an important neurotransmitter involved in
memory and learning in the brain[21]. AChE inhibitors (AChEIs) can increase the amount of ACh in
the synaptic cleft. Recent studies have also shown that AChEIs possibly affect the expression and/or
the metabolic processing of amyloid precursor protein, which may influence Aβ generation[22, 23].
Therefore AChEIs remain the preferred target for the treatment of AD[24, 25]. Recently, some
articles have reported that butyrylcholinesterase (BChE), another cholinesterase present in the brain,
has the similar biological function as AChE for hydrolysis of ACh in a healthy human brain[26].
Moreover, BChE inhibitors (BChEIs) not only improve the cognitive performance of aged rats
without the classic adverse effects associated with AChE inhibition but they also inhibit amyloid
fibril formation[27]. Furthermore, inhibitors of both of these cholinesterase enzymes have shown
significant clinical utility in conferring cognitive improvements. Thus, developing MTDLs with dual
inhibitors may have benefits for the treatment of AD[28, 29].
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Biometals, including Cu(II), Zn(II), and Fe(II, III), have also been found to promote aggregation
of Aβ[30, 31]. In addition, the interaction of Aβ with Cu(II) is involved in the production of reactive
oxygen species and oxidative stress[32], which implies that modulation of these biometals in the
brain may be a potential therapeutic strategy for the treatment of AD[33, 34].
Deoxyvasicinone (5) (Figure 2), consisting of a quinazolinone moiety conjugated with a
pyrrolidine, is a naturally occurring alkaloid with antibacterial[35], antiinflammatory[36], and
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antiproliferative activities[37]. Structure−activity relationships (SARs) of the quinazolinone ring
system examined in various studies suggest that position 6 is suitable for substituents[38-41].
Recently, deoxyvasicinone and its derivatives have been considered as cholinesterase inhibitors,
because the structure of deoxyvasicinone is similar to that of tacrine (1). Deoxyvasicinone exhibits
moderate inhibitory activity towards AChE (from Electric Eel) and BChE (from equine serum), with
IC50 values of 82.5 and 25.1 µM, respectively[39]. Compounds 6 and 7 demonstrate dramatic
inhibitory activities towards AChE and BChE, with IC50 values of 69.2 nM and 1.95 µM,
respectively[40, 41]. In addition, there are several reports that suggest that carbamate-containing
compounds (e.g., rivastigmine (3) and compounds 7 and 8) can be used for BChE-inhibiting
activity[41,42]. We assume that the effects of the aminoacetamide structure on BChE-inhibitory
activity is similar to that of carbamate. Furthermore, the aminoacetamide structure also has metal ion
complexing action. On the basis of the forgoing, we introduced different aminoacetamide groups into
the 6 position of deoxyvasicinone, to obtain a novel series of derivatives that are expected to be
AChEIs and BChEIs, inhibitors of self-induced Aβ aggregation, and biometal chelators. For
investigation into the SARs of this class of compounds, several hydrophobic substituents (aliphatic
and aromatic moieties) were added to the aminoacetamide groups (Figure 3).
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2. Results and discussion
2.1. Chemistry
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The synthetic route to compounds 12a−12s is depicted in Scheme 1. The starting material
deoxyvasicinone (5) was synthesized as previously described[35]. A nitration reaction of compound
deoxyvasicinone with H2SO4−HNO3 provided the nitration product, which was reduced by
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Na2S·9H2O in the presence of sodium hydroxide as a base, to afford compound 10. The nucleophilic
substitution reaction of compound 10 with different bromohydrocarbons using sodium hydride as a
base with a catalytic amount of potassium iodide in acetonitrile as a solvent yielded the target
compounds 11a−11s. Finally, compounds 12a−12s were obtained after the deprotection of the Boc
group in the presence of CF3COOH and dry dichloromethane.
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2.2. In Vitro AChE and BChE Inhibition Assays
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The inhibitory activities of the synthesized compounds 12a−12s against human recombinant
AChE (hAChE) and human serum BChE (hBChE) were evaluated by the method of Ellman et al.,
where tacrine (1) and deoxyvasicinone (5) were used as reference compounds. IC50 values and
selectivity ratios are listed in Table 1.
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It is evident that the synthesized derivatives were potent inhibitors of hAChE, with IC50 values in
the low nanomolar range. Furthermore, all compounds showed much stronger inhibitory activity than
the parent compound (deoxyvasicinone (5), IC50 = 62.5 µM). Encouragingly, 14 of the 19 derivatives
displayed higher inhibitory activity against hAChE than that of the positive control (tacrine (1), IC50
= 76.5 nM). Notably, compounds 12h, 12n, and 12q exhibited very potent AChE inhibitory activity,
with IC50 values of 5.31, 4.09, and 7.61 nM, respectively. In addition, several SARs were observed.
The inhibitory potency of these derivatives (except for compounds 12f and 12g) against hAChE was
enhanced with increasing length of the carbon chain (12a vs. 12b vs. 12c vs. 12d vs. 12e vs. 12h, 12i
vs. 12j, and 12m vs. 12n). These findings are in accordance with the results from Decker’s
group[40]. On lengthening the tether chain from 2 to 16 carbon units, the inhibitory activity against
hAChE increased by between 112-fold and 11,770-fold (12a vs. 12h). The inhibitory activity also
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increased with the degree of unsaturation: for example, compounds 12i and 12l, with an unsaturated
group, exhibited higher AChE inhibitory activity (IC50 = 66.6 and 46.4 nM, respectively) than the
derivative with a saturated group (12b, IC50 = 557 nM). When aromatic groups were added to the
aminoacetamide moiety, hAChE inhibitory activity increased in the presence of electron-donating
substituents in the o-position or when the conjugated system was enlarged (12m vs. 12o vs. 12p vs.
12r vs. 12s). These results imply that extending the conjugated system could improve the
combination of derivatives and hAChE.
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It is of note that, in MTDLs, strong AChE inhibitory activity is just one part of the rationale
behind the development of such synthetic molecules. Evidence suggests that BChE is primarily
expressed and secreted by glial cells, and its level remains constant or increases in advanced AD,
while AChE concentration in certain brain regions decreases[27]. Inhibition of central BChE activity
has also been researched as a potential therapeutic approach to ameliorate the cholinergic deficit in
moderate forms of AD[29,43]. These reports show that balanced inhibition of both cholinesterases
could be conducive to the treatment of AD[28]. Thus, the test compounds were also evaluated for
hBChE inhibitory activity, and the results are summarized in Table 1.
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As planned in the compound design, the synthesized compounds exhibited potent hBChE
inhibitory activity (IC50 values ranging from 1.186 µM to 2.35 nM), much higher than that of the
parent compound (deoxyvasicinone (5), IC50 = 45.1 µM). Two derivative compounds (12h and 12q)
displayed better inhibitory activity against hBChE (IC50 value of 4.35 and 2.35 nM, respectively)
than the positive control (tacrine (1), IC50 = 10.8 nM). Remarkably, compounds 12h and 12q also
showed potent hAChE inhibitory activity (IC50 = 5.31 and 7.61 nM, respectively). These results
indicate that the two candidate drugs could be effective in treating mild to moderate as well as severe
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forms of AD when AChE takes over the role played by BChE. In addition, when a saturated alkyl or
an aromatic group was incorporated in the aminoacetamide moiety, the SARs toward hBChE
revealed similar trends to those observed for hAChE. However, with an unsaturated alkyl in the
aminoacetamide group, a slightly different trend was observed: for example, compound 12i, which
has an allyl group, showed higher inhibitory activity against hBChE (IC50 = 16.6 nM) than the
derivative with a but-1-en-4-yl group (12j, IC50 = 81.5 nM). These results show that deoxyvasicinone
derivatives featuring aminoacetamide groups exhibit higher inhibitory activity against hAChE and
hBChE than compounds with carbamate groups.
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2.3. Kinetic Evaluation of Compound 12q on hAChE
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To assess the AChE inhibition mechanism of deoxyvasicinone derivatives, the potent compound
12q was chosen for an enzyme kinetic study. Lineweaver−Burk reciprocal plots were constructed by
plotting the double reciprocal of the increasing inhibitor and substrate concentrations (Figure 4). The
plots reveal both increasing slopes (decreased Vmax) and intercepts (higher Km) at increasing
concentrations of compound 12q. This pattern indicated a mixed-type inhibition. The results suggest
that compound 12q could bind simultaneously to the catalytic active site (CAS) as well as to the
peripheral anionic site (PAS) of hAChE. This is a desirable effect for treatment of AD because Aβ
aggregation is catalyzed by the PAS of AChE. The inhibitor dissociation constants Ki for the
enzyme−inhibitor and K′i for the enzyme−substrate−inhibitor were estimated to be 9.85 and 15.81
nM, respectively (Supporting Information, Table S1 and Figure S1).
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2.4. Inhibition of Aβ1−42 Self-aggregation Assay
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Aβ1−42 aggregation is deemed to play a crucial role in the pathogenesis of AD[44]. Thus, the
inhibition of Aβ1−42 self-aggregation has been investigated as an attractive therapeutic strategy to
more efficiently combat AD[45]. In this work, inhibitory activity of the deoxyvasicinone derivatives
against Aβ1−42 self-aggregation was evaluated using the thioflavin T fluorescence assay[46], with
deoxyvasicinone and curcumin as reference compounds. The results (Table 1) show that the
derivatives exhibited moderate to good potencies, with inhibition in the range 6.46−63.9% at 10 µM.
It is interesting that most of substituted alkyl groups in the aminoacetamide moiety generally
enhanced inhibition activity with increasing length of the carbon chain (with the exception of
compound 16). Compounds 12m−12s, with a substituted benzyl group in the aminoacetamide moiety,
generally gave better results for inhibition activity (25.0−63.9%): compound 12q, with a
3,5-difluorobenzyl group in the aminoacetamide moiety, was the most potent inhibitor of Aβ1−42
aggregation (63.9%). Compound 12b, with a propyl group in the aminoacetamide moiety, exhibited
inhibition activity of 7.38%. On the other hand, compounds 12i and 12l, with an allyl and propargyl
group in the aminoacetamide moiety, respectively, led to 37.9% and 52.3% inhibition, respectively.
These results imply that increased unsaturation of substituents in the aminoacetamide group could
improve inhibitory activity.
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2.5. Metal-Chelating Properties of Compound 12q
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The ability of compound 12q to bind biometals such as Cu(II), Fe(II), Fe(III), and Zn(II) was
investigated by ultraviolet–visible (UV−vis) and fluorescence spectrometry[47, 48], and the results
are shown in Figure 5. Cu(II), Fe(II), and Fe(III) cause an impressive decrease in the intrinsic
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fluorescence of compound 12q (Figure 5A), and this effect increases in a dose-dependent manner
with concentration. However, the fluorescence intensity of compound 12q did not differ substantially
in the absence or presence of Zn(II) (Supporting Information, Figure S2). In addition, after CuSO4
was added to a solution of compound 12q, the maximum absorption wavelength shifted from 282 to
291 nm, indicating the formation of a 12q−Cu(II) complex (Figure 5B). The maximum absorption at
282 nm decreased with the addition of FeSO4 and FeCl3, which suggests that Fe(II) and Fe(III)
possibly interacted with compound 12q. However, when ZnCl2 was added, there was no significant
change in the UV spectrum, which is in accordance with the fluorescence spectrum.
To determine the binding stoichiometry of compound 12q with Cu(II), the UV spectra were used
to measure the absorbance of the complex of compound 12q and Cu(II) at different concentrations at
451 nm. As depicted in Figure 5C, when the absorbance changes at 451 nm were plotted, two straight
lines intersected at a mole fraction of 0.86, indicating a 1:1 stoichiometry for the complex
12q−Cu(II).
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3. Conclusion
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In summary, all 19 deoxyvasicinone derivatives exhibited high hAChE and hBChE inhibitory
activity at nanomolar concentrations. In particular, inhibitory activities against hAChE (IC50 = 7.61
nM) and hBChE (IC50 = 2.35 nM) of one derivative (12q) increased 8729- and 19191-fold in
comparison with the parent compound. Remarkably, compound 12q also demonstrated the highest
potential inhibitory activity for Aβ1−42 self-aggregation (63.9 ± 4.9%, 10 µM), and it was also an
excellent metal chelator. Thus, compound 12q is a promising multifunctional candidate for the
treatment of AD. Further investigations of AD therapeutic candidates are in progress, and results will
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be presented later. Meanwhile these beneficial effects of the derivatives highlight deoxyvasicinone as
a lead molecule and aminoacetamide structure as a rewarding group to be developed in the search for
multi-target drugs for the treatment of AD.
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4. Experimental part
4.1. General Remarks.
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All reagents were commercial grade, and were used without further purification unless
otherwise indicated. Silica gel (100–200 mesh) for column chromatography and silica GF254 for
thin-layer chromatography were obtained from the Qingdao Marine Chemical Company (China).
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Melting points were measured using an XT-4 melting-point apparatus, and were uncorrected. H
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nuclear magnetic resonance (NMR) (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a
Bruker Avance using CDCl3 or deuterated dimethyl sulfoxide (DMSO-d6) as the solvent and
tetramethylsilane as the internal standard. Chemical shifts are reported in parts per million (ppm).
Multiplicities are reported as follows: singlet (s), doublet (d), triplet (t), doublet of doublet (dd),
doublet of doublet of doublet (ddd) and multiplet (m). Electron ionization mass spectroscopy
(ESI-MS) was undertaken with a Thermo Fisher spectrometry instrument.
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4.2. Chemistry
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4.2.1. Synthesis of deoxyvasicinone (5)
The deoxyvasicinone (5) was synthesized according to a previously described method
(Supporting Information,Scheme S1)[35]. White solid, yield: 54 %. Mp: 108−109 °C. 1H NMR (500
MHz, CDCl3) δ 8.29 (dd, J = 8.5, 1.3 Hz, 1H), 7.73 (ddd, J = 8.5, 7.6, 1.5 Hz, 1H), 7.65 (d, J = 7.8
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Hz, 1H), 7.49–7.41 (m, 1H), 4.22 (t, J =7.3 Hz 2H), 3.19 (t, J = 8.0 Hz, 2H), 2.35–2.24 (m, 2H). 13C
NMR (126 MHz, CDCl3) δ 170.6, 169.0, 158.7, 143.7, 136.4, 136.0, 135.8, 130.1, 56.1, 42.1, 29.1.
ESIMS calcd for C11H10N2O [M+H]+,187.09; Found, 187.13.
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4.2.2. Synthesis of compounds 9 and 10
A cold solution (25 mL, −10 °C) of H2SO4−HNO3 (2:3, v/v) was added 0.931 g (5 mmol) of
deoxyvasicinone (5) with stirring, and the reaction mixture was kept at −10 °C for 3 h. Upon
completion of the reaction, the solution was poured into ice water, basified with 20% aq. NaOH at
pH 9, and extracted with ethyl acetate. The combined extracts were washed with water, brine, dried
over anhydrous Na2SO4. Evaporation of the solvent under reduced pressure gave a nitration product.
Afterwards, a solution of nitration product in EtOH (50 mL) was added slowly to a suspension of
sodium sulfide hydrate (2.420 g, 10 mmol) and NaOH (0.802 g, 20 mmol) in distilled water (80 mL).
The mixture was heated to reflux for 4 h. After cooling, the solution was concentrated under reduced
pressure. The compound 9 (0.792 g, 79 % yield) was isolated as a yellow solid by a column
chromatography on elution with CH2Cl2/MeOH (10:1, v/v)
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A suspension of the compound 9 (0.509 g, 2.5 mmol) in dichloromethane (30 mL) was added
slowly to a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.566g, 3
mmol) and 2-((tert-butoxycarbonyl)amino)acetic acid (0.524 g, 3 mmol) in dichloromethane (40 mL).
The mixture was refluxed for 6 h and concentrated in vacuum. The reaction solution was washed
with water, brine, dried over anhydrous Na2SO4, and concentrated in vacuum to give the crude
product (10) as a yellow solid.
4.2.3. General procedure for the preparation of compounds 11a−11s and 12a−12s
A mixture of compound 10 (127 mg, 0.5 mmol), KI (17 mg, 0.1 mmol), NaH (18 mg, 0.75 mmol),
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and the alkyl bromide (1 mmol) in acetonitrile (50 mL) was refluxed. When the compound 10
disappeared (as detected by TLC), the solvents were removed under reduced pressure. Distilled water
(50 mL) was then added, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic
solvent phase was washed by water (15 mL × 2), brine (15 mL × 2), dried over anhydrous Na2SO4,
and evaporated under vacuum. The compounds 11a−11s were purified by a flash chromatography on
silica gel. CF3COOH (1 mL, 14 mmol) was then added dropwise to a solution of compound 11a−11s
in dry CH2Cl2 (50 mL). The solution was refluxed for 7 h. After cooling, the solvent was
concentrated under reduced pressure to give a yellow oil, which was purified by a flash
chromatography on silica gel using CH2Cl2/MeOH as the elution system to obtain the compound
12a−12s.
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2-(ethylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide
(12a).
White solid, yield: 53 %. Mp: 198−199 °C. 1H NMR (500 MHz, DMSO-d6) δ 9.80 (s, 1H), 8.41 (d, J
= 2.5 Hz, 1H), 7.97 (dd, J = 8.8, 2.5 Hz, 1H), 7.52 (d, J = 8.1, 1H), 4.22 (s, 1H), 4.10 (t, J =7.3 Hz,
2H), 3.17 (s, 2H), 3.10 (t, J = 7.9 Hz, 2H), 2.66 (m, 2H), 2.26−2.21 (m, 2H), 1.13 (t, J = 6.2 Hz, 3H).
13C NMR (126 MHz, CDCl3) δ 172.3, 166.4, 160.3, 145.6, 136.5, 126.9, 126.2, 120.8, 115.2, 57.9,
48.6, 47.6, 32.3, 19.5, 13.1. ESIMS calcd for C15H18N4O2 [M+H]+,286.33; Found, 286.34.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-2-(propylamino)acetamide
(12b).
White solid, yield: 53 %. Mp: 292−294 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.44 (d,
J = 2.5 Hz, 1H), 7.95 (dd, J = 8.8, 2.5 Hz, 1H), 7.56 (dd, J = 8.6, 1H), 4.25 (s, 1H), 4.10 (t, J =7.3 Hz,
2H), 3.20 (s, 2H), 3.13 (t, J = 7.9 Hz, 2H), 2.69 (t, J = 7.1 Hz, 2H), 2.29−2.24 (m, 2H), 1.40−1.32 (m,
2H), 0.96 (t, J = 6.8 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.9, 164.4, 161.2, 143.2, 137.7,
135.0, 129.5, 122.5, 114.8, 55.8, 55.0, 43.9, 33.5, 29.1, 23.2, 14.3. ESIMS calcd for C16H20N4O2
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[M+H]+, 301.36; Found, 301.36.
2-(butylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide
(12c).
3
White solid, yield: 58 %. Mp: 157−158 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.46 (d,
J = 2.4 Hz, 1H), 7.95 (dd, J = 8.8, 2.5 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 4.21−3.96 (m, 3H), 3.27 (s,
2H), 3.09 (t, J = 7.9 Hz, 2H), 2.71 (t, J = 7.1 Hz, 2H), 2.29−2.24 (m, 2H), 1.43−1.30 (m, 4H), 0.88 (t,
J = 6.8 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.5, 162.5, 159.6, 147.3, 139.5, 127.8, 127.0,
122.9, 114.9, 53.6, 52.9, 46.8, 36.4, 29.5, 25.4, 19.5, 15.6. ESIMS calcd for C17H22N4O2 [M+H]+,
315.18; Found, 315.20.
4
5
6
7
8
9
2-(hexylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide
(12d).
10
11
12
13
14
15
16
17
18
19
20
21
22
White solid, yield: 44 %. Mp: 211−213 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.46 (d,
J = 2.4 Hz, 1H), 7.93 (dd, J = 8.8, 2.5 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 4.21−3.96 (m, 3H), 3.12 (s,
2H), 3.11 (t, J = 7.9 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 2.26−2.21 (m, 2H), 1.38 (m, 2H), 1.29−1.28
(m, 6H), 0.81 (t, J = 6.8 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.5, 160.3, 159.6, 145.6, 136.9,
127.0, 126.2, 120.9, 114.9, 53.1, 52.5, 46.8, 32.2, 29.5, 28.9, 28.2, 27.6, 19.5, 12.3. ESIMS calcd for
C19H26N4O2 [M+H]+, 343.21; Found, 315.24.
2-(octylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide(12e). White
solid, yield: 47 %. Mp: 145−146 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.46 (d, J = 2.4
Hz, 1H), 7.93 (dd, J = 8.8, 2.5 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 4.21−3.82 (m, 3H), 3.13 (s, 2H),
3.10 (t, J = 7.9 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 2.23−2.18 (m, 2H), 1.38 (m, 2H), 1.29 (m, 2H),
13
1.28−1.26 (m, 8H), 0.80 (t, J = 6.8 Hz, 3H). C NMR (126 MHz, CDCl3) δ 168.5, 168.0, 161.3,
142.6, 139.8, 123.7, 122.3, 121.2, 118.9, 53.1, 53.0, 48.9, 34.5, 31.9, 29.9, 29.7, 29.3 (2C), 27.2, 22.8,
14.2. ESIMS calcd for C21H30N4O2 [M+H]+, 371.24; Found, 3371.34.
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2-(decylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide(12f). White
solid, yield: 45 %. Mp: 189−190 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.46 (d, J = 2.4
Hz, 1H), 7.93 (dd, J = 8.8, 2.5 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 4.21−3.82 (m, 3H), 3.12 (s, 2H),
3.11 (t, J = 7.9 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 2.23−2.18 (m, 2H), 1.38 (m, 2H), 1.29 (m, 2H),
3
4
13
5
1.28−1.26 (m, 12H), 0.78 (t, J = 6.8 Hz, 3H). C NMR (126 MHz, CDCl3) δ 169.5, 166.0, 164.3,
6
142.5, 138.8, 123.7, 122.6, 121.6, 118.7, 54.2, 54.0, 48.6, 34.9, 32.4, 31.7, 29.4, 29.7, 29.7, 29.7 (2C),
27.2, 22.3, 14.1. ESIMS calcd for C23H34N4O2 [M+H]+, 399.28; Found, 399.35.
7
8
2-(dodecylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide(12g).
White solid, yield: 35 %. Mp: 223−225 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.23 (s, 1H), 8.17 (d,
J = 2.4 Hz, 1H), 7.88 (dd, J = 8.8, 2.5 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 3.87 (t, J =7.3 Hz, 2H), 3.83
(s, 1H), 3.12 (s, 2H), 3.11 (t, J = 7.9 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 2.23−2.18 (m, 2H), 1.38 (m,
2H), 1.29 (m, 2H), 1.28 (m, 2H), 1.27−1.26 (m, 14H), 0.86 (t, J = 6.8 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 167.5, 165.0, 164.3, 142.6, 138.3, 123.4, 122.4, 121.8, 118.2, 53.1, 53.0, 48.7, 34.1, 31.4,
31.4, 29.7 (5C), 29.6 (2C), 27.1, 22.7, 14.4. ESIMS calcd for C25H38N4O2 [M+H]+, 427.60; Found,
427.68.
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11
12
13
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15
16
17
18
19
20
21
22
2-(hexadecylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide(12h).
White solid, yield: 49 %. Mp: 280−281 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.23 (s, 1H), 8.47 (d,
J = 2.4 Hz, 1H), 7.94 (dd, J = 8.8, 2.5 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 4.21 (s, 1H), 4.13 (t, J =7.3
Hz, 2H), 3.13 (s, 2H), 3.11 (t, J = 7.9 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 2.23−2.18 (m, 2H), 1.38 (m,
2H), 1.29 (m, 2H), 1.28 (m, 2H), 1.27−1.26 (m, 22H), 0.86 (t, J = 6.8 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 168.4, 166.0, 164.5, 142.5, 138.6, 123.5, 122.5, 121.6, 118.4, 54.2, 54.1, 48.6, 34.7, 31.7,
31.3, 29.8 (9C), 29.4 (2C), 27.1, 22.7, 14.4. ESIMS calcd for C29H46N4O2 [M+H]+, 483.37; Found,
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483.44.
2-(allylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide(12i). Yellow
1
3
solid, yield: 58 %. Mp: 182−183 °C. H NMR (500 MHz, CDCl3) δ 9.47 (s, 1H), 8.35 (dd, J = 8.8,
4
2.5 Hz, 1H), 8.10 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 5.91 (ddt, J = 16.8, 10.2, 6.5 Hz, 2H),
5.30 (d, J = 8.8 Hz, 1H), 4.23 (t, J =7.3 Hz, 2H), 3.25 (m, 4H), 3.19 (t, J = 7.9 Hz, 2H), 2.26−2.21 (m,
3H). 13C NMR (126 MHz, CDCl3) δ 169.7, 160.7, 158.4, 145.6, 136.1, 134.0, 127.8, 126.4, 120.8,
119.2, 115.1, 58.1, 57.4, 46.5, 32.4, 19.6. ESIMS calcd for C16H18N4O2 [M+H]+, 299.15; Found,
299.19.
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6
7
8
9
2-(but-3-en-1-ylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide(12j
). Yellow solid, yield: 57 %. Mp: 210−212 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.50
(d, J = 2.4 Hz, 1H), 8.06 (dd, J = 8.8, 2.5 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 5.91 (ddt, J = 16.8, 10.2,
6.5 Hz, 2H), 5.30 (d, J = 8.8 Hz, 1H), 4.23 (t, J =7.3 Hz, 2H), 4.07 (dd, J = 15.1, 7.8 Hz, 2H), 3.55
(m, 2H), 3.27 (t, J =2.2 Hz, 2H), 3.08 (t, J = 7.9 Hz, 2H), 2.35−1.98 (m, 3H). 13C NMR (126 MHz,
DMSO-d6) δ 168.7, 160.3, 159.5, 145.5, 136.9, 135.3, 127.6, 126.7, 120.8, 119.2, 115.2, 59.4, 56.7,
46.8, 42.7, 32.1, 19.5. ESIMS calcd for C17H20N4O2 [M+H]+, 313.37; Found, 313.42.
2-((3-methylbut-2-en-1-yl)amio)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)aceta
mide (12k). Yellow solid, yield: 61 %. Mp: 184−185 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.85 (s,
1H), 8.47 (d, J = 2.4 Hz, 1H), 7.89 (dd, J = 8.8, 2.5 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 5.53 (t, J = 7.6
Hz, 1H), 4.18 (d, J = 7.6 Hz, 2H), 4.14 (s, 1H), 4.08 (t, J =7.3 Hz, 2H), 3.28 (t, J =2.3 Hz, 2H), 3.08
(t, J = 7.9 Hz, 2H), 2.24−2.16 (m, 2H), 1.85 (s, 3H), 1.75 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
166.9, 163.2, 160.2, 146.1, 136.6, 135.8, 128.1, 126.5, 120.9, 118.8, 115.7, 58.1, 56.8, 46.8, 32.2,
26.6, 19.5, 18.9. ESIMS calcd for C18H22N4O2 [M+H]+, 327.18; Found, 3327.22.
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N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-2-(prop-2-yn-1-ylamino)acetamide
1
(12l). Yellow solid, yield: 58 %. Mp: 183−184 °C. H NMR (500 MHz, DMSO-d6) δ 10.16 (s, 1H),
3
8.50 (d, J = 2.4 Hz, 1H), 7.98 (dd, J = 8.8, 2.4 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 4.12 (s, 1H), 4.08 (t,
J =7.3 Hz, 2H), 3.55(m, 4H), 3.28 (t, J = 7.9 Hz, 2H), 2.57 (s, 1H), 2.29−2.21 (m, 2H). 13C NMR
(126 MHz, DMSO-d6) δ 168.7, 160.3, 159.5, 145.5, 137.0, 131.1, 122.6, 120.8, 115.2, 79.4, 76.6,
56.7, 46.8, 42.7, 32.2, 19.5. ESIMS calcd for C16H16N4O2 [M+H]+, 297.14; Found, 297.17.
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5
6
7
2-(benzylamino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide
(12m).
8
Yellow solid, yield: 68 %. Mp: 188−189 °C. 1H NMR (500 MHz, CDCl3) δ 9.28 (s, 1H), 8.24 (dd, J
= 8.8, 2.5 Hz, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.42−7.40 (m, 3H), 7.34−7.29
(m, 2H), 4.57 (s, 1H), 4.25 (t, J =7.3 Hz, 2H), 3.79 (s, 4H), 3.33 (t, J = 7.9 Hz, 2H), 2.31−2.20 (m,
2H). 13C NMR (126 MHz, CDCl3) δ 169.3, 160.7, 158.4, 145.7, 136.7, 136.0, 129.1 (2C), 127.9 (2C),
126.3, 123.4, 122.6, 120.8, 115.0, 59.8, 58.0, 46.6, 32.4, 19.6. ESIMS calcd for C20H20N4O2 [M+H]+,
349.17; Found, 349.23.
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N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-2-((3-phenylpropyl)amino)acetamide
1
(12n). White solid, yield: 59 %. Mp: 208−210 °C. H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H),
8.46 (d, J = 2.2 Hz, 1H), 7.95 (dd, J = 8.8, 2.1 Hz, 1H),7.93 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 8.0 Hz,
2H), 7.63 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 4.63 (s, 1H), 4.25 (t, J =7.3 Hz, 2H), 3.85 (s,
2H), 3.76 (t, J = 7.8 Hz, 2H), 3.38 (t, J = 7.8 Hz, 2H), 3.21 (t, J = 7.9 Hz, 2H), 2.76−2.63 (m, 2H),
2.52−2.43 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 166.5, 160.3, 158.2, 146.9, 136.9, 136.3,
129.8 (2C), 127.8 (2C), 126.2, 123.2, 122.4, 118.6, 115.5, 46.8, 43.2, 35.5, 32.2, 27.2, 23.9, 19.5.
ESIMS calcd for C22H24N4O2 [M+H]+, 377.20; Found, 377.25.
2-((2-methylbenzyl)amino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide
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(12o). Yellow solid, yield: 69 %. Mp: 223−225 °C. 1H NMR (500 MHz, CDCl3) δ 8.89 (s, 1H), 8.05
(dd, J = 8.8, 2.3 Hz, 1H), 7.92 (d, J = 2.1 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.37 (d, J = 7.4 Hz, 2H),
7.22 (dd, J = 13.2, 5.8 Hz, 2H), 4.60 (s, 1H), 4.24 (t, J =7.3 Hz, 2H), 3.80 (s, 2H), 3.34 (s, 2H), 3.19
(t, J = 7.9 Hz, 2H), 2.36−2.27 (m, 2H), 2.03 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 169.4, 160.6,
158.3, 145.5, 136.9, 135.9, 135.4, 130.8, 130.3, 128.0, 127.6, 126.3, 126.2, 120.7, 114.8, 58.8, 58.5,
46.5, 32.4, 19.6, 19.3. ESIMS calcd for C21H22N4O2 [M+H]+, 363.18; Found, 363.22.
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4
5
6
7
2-((2-fluorobenzyl)amino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide
1
8
(12p). White solid, yield: 70 %. Mp: 109−111 °C. H NMR (500 MHz, CDCl3) δ 9.55 (s, 1H), 8.22
9
(d, J = 2.2 Hz, 1H), 8.18 (dd, J = 8.8, 2.4 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 7.4 Hz, 2H),
7.13−7.06 (m, 2H), 4.56 (s, 1H), 4.28−4.22 (m, 2H), 3.85 (s, 2H), 3.34 (s, 2H), 3.21 (t, J = 7.9 Hz,
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11
12
13
14
15
16
17
18
19
20
21
22
1
2H), 2.37−2.29 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 169.1, 161.7 (d, JCF = 245.5 Hz), 158.3,
145.5, 136.2, 134.9, 131.7 (d, 3JCF = 4.0 Hz), 129.9 (d, 3JCF = 8.7 Hz), 127.6, 126.3, 124.4, 124.3 (d,
2JCF = 19.0 Hz), 120.8, 115.8 (d, 2JCF = 12.5 Hz), 115.2, 58.1, 53.9, 46.5, 32.4, 19.6. ESIMS calcd for
C20H19FN4O2 [M+H]+, 367.16; Found, 367.19.
2-((3,5-difluorobenzyl)amino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetami
de (12q). White solid, yield: 64 %. Mp: 266−268 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.02 (s, 1H),
8.46 (d, J = 2.5 Hz, 1H), 7.93 (dd, J = 8.8, 2.5 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.21 (d, J = 1.9 Hz,
2H), 7.10 (tt, J = 9.3, 2.2 Hz, 1H), 4.48 (s, 1H), 4.09 (t, J = 7.4 Hz, 2H), 3.86 (s, 2H), 3.39 (s, 2H),
3.09 (t, J = 7.9 Hz, 2H), 2.25 – 2.15 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 169.5, 162.9 (d, 1JCF
3
= 245.9, 2C), 160.3, 159.5, 145.6, 144.2 (t, JCF = 8.8 Hz), 136.9, 127.5, 126.7, 120.8, 115.4, 112.0
(dd, 2JCF = 19.6 Hz, 3JCF = 5.4 Hz, 2C), 103.0 (t, 2JCF = 25.8 Hz), 57.6, 57.1, 46.8, 32.2, 19.5. ESIMS
calcd for C20H18F2N4O2 [M+H]+, 385.15; Found, 385.20.
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2-((2-nitrobenzyl)amino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide
1
(12r). White solid, yield: 73 %. Mp: 178−180 °C. H NMR (500 MHz, DMSO-d6) δ 10.16 (s, 1H),
3
8.49 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.95 (dd, J = 8.8, 2.1 Hz, 1H), 7.83−7.60 (m, 3H),
7.58 (d, J = 8.8 Hz, 1H), 4.61 (s, 1H), 4.08 (t, J =7.2 Hz, 2H), 3.79 (s, 2H), 3.48 (s, 2H), 3.158 (t, J =
7.8 Hz, 2H), 2.56−2.48 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 168.5, 166.2, 161.3, 146.6, 146.2,
139.9, 128.5, 127.7, 123.8, 122.6, 121.8, 119.8, 117.4, 115.0, 112.4, 53.7, 52.1, 51.3, 36.2, 19.9.
ESIMS calcd for C20H19N5O4 [M+H]+, 394.15; Found, 394.21.
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5
6
7
8
2-((2-cyanobenzyl)amino)-N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)acetamide
1
9
(12s). White solid, yield: 71 %. Mp: 115−117 °C. H NMR (500 MHz, DMSO-d6) δ 10.23 (s, 1H),
10
11
12
13
14
8.32 (d, J = 2.7 Hz, 1H), 8.17(d, J = 8.0 Hz, 1H), 7.95 (dd, J = 8.8, 2.1 Hz, 1H), 7.81−7.67 (m, 3H),
7.58 (d, J = 8.8 Hz, 1H), 4.32 (s, 1H), 4.16 (t, J =7.2 Hz, 2H), 3.93 (s, 2H), 3.56 (s, 2H), 3.08 (t, J =
7.8 Hz, 2H), 2.24−2.16 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 170.3, 160.3, 159.5, 146.4, 145.5,
137.0, 132.6, 132.5, 129.5, 127.6, 127.4, 126.4, 120.8, 119.4, 115.0, 110.1, 62.7, 52.3, 46.7, 32.1,
19.5. ESIMS calcd for C21H19N5O2 [M+H]+, 373.15; Found, 373.19.
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4.3. hAChE and hBChE Inhibition Assays
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17
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21
AChE and BChE inhibitory activities for the tested compounds were obtained using the method
of Ellman et al[47]. hAChE, hBChE, 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent; DTNB),
acetylthiocholine iodide (ATCI), and butyrylthiocholine iodide (BTCI) were purchased from
Sigma-Aldrich. At least five different concentrations (10–4–10–9 M) of each test compound were used
to determine the enzyme inhibition activity. In summary, the procedure was as follows: 50 µL of
hAChE (0.02 unit/mL) or hBChE (0.02 unit/mL) and 10 µL of the compounds was incubated at
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37 °C for 6 min; next, 30 µL of 0.01 M substrate (ATCI or BTCI solution) was added, and the
solution further incubated at 37 °C for 12 min; and, finally, 150 µL of 0.01 M DTNB was added, and
the activity measured at a wavelength of 415 nm using an Evolution 300 PC UV-Vis
Spectrophotometer. The IC50 value (the concentration of the compound required for a 50% reduction
in cholinesterase activity) was calculated using Origin 8.0 software. The results are expressed as the
mean ± SEM of at least four experiments performed in triplicate.
7
4.4. Kinetic Study of AChE Inhibition Assay
8
9
The mechanism of AChE inhibition by compound 12q was determined by using Ellman’s method
[49]. Relatively low concentrations of the substrate (0.1−0.5 mM) were reacted with AChE in the
absence or presence of different concentrations of compound 12q (1−10 nM). The Vmax and Km
values for Michaelis−Menten kinetics were obtained by a weighted least squares analysis from the
substrate–velocity curves using GraphPad Prism 5. In addition, the inhibitor constant (Ki) was
calculated by linear regression from the Lineweaver−Burk plot versus the inhibitor concentration,
and the Ki′ values were determined by plotting the apparent 1/vmax versus inhibitor concentration.
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15
4.5. Aβ1−42 Self-aggregation Inhibition Assay
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The thioflavin T fluorescence method was used to quantify amyloid fibril formation[46].
Thioflavin T, Aβ1−42, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were acquired from
Sigma-Aldrich. HFIP-pretreated Aβ1−42 samples were dissolved in a 50 mM phosphate buffer (pH
7.4), to obtain a stable stock solution (Aβ concentration of 500 µM). The peptide was incubated in 10
mM phosphate buffer (pH 8.0) at 30 °C for 24 h (final Aβ concentration of 50 µM) with or without
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the tested compounds at 10 µM (Aβ : inhibitor = 5:1). After incubation, the samples were diluted to a
final volume of 200 µL with 50 mM glycine−NaOH buffer (pH 8.5) containing 1.5 µM thioflavin T.
Next, a 300 s time scan of the fluorescence intensity was carried out (λexc = 446 nm, λem = 490 nm),
and the plateau values averaged after subtracting the background fluorescence of the thioflavin T
solution. The percentage inhibition was calculated by the following formula:
inhibition (%) = (1− IFi/IFo) × 100
where IFi and IFo are the fluorescence intensities obtained from Aβ1−42 in the presence and absence of
inhibitor, respectively.
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4.6. Metal-Chelating Assay
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Compound 12q was investigated as a metal chelator using UV−vis and fluorescence
spectrophotometers in 20 % (v/v) ethanol/buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). A fixed
amount of compound 12q (50 µM) was mixed with increasing amounts of Cu(II), Fe(II), Fe(III), and
Zn(II) (0−1000 µM) for 30 min, and the fluorescence intensity (λexc = 285 nm) examined. In addition,
the UV absorption spectra of compound 12q (50 µM, final concentration) alone or in the presence of
CuSO4, FeSO4, FeCl3, or ZnCl2 (50 µM) were recorded at room temperature. To obtain the
stoichiometry of the compound–Cu2+ complex, a fixed amount of compound 12q (50 µM) was mixed
with increasing amounts of Cu2+ (0−100 µM), and the difference in the UV spectra assessed to give
the ligand:metal ratio in the complex.
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Acknowledgements
This work was supported by grants from the Program for Natural Science Foundation of China
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(Grant No. 21602178).
Conflicts of interest
The authors declare no conflict of interest about this article.
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Legend of Scheme and Figures
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Scheme 1. Synthesis of Deoxyvasicinone Derivatives.
Figure 1. Structures of AChE inhibitors 1−4 used for the management of AD.
Figure 2. Deoxyvasicinone and its derivatives.
Figure 3. Design strategy for deoxyvasicinone derivatives.
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Figure 4. Kinetic study on the mechanism of hAChE inhibition by compound 12q. Overlaid
Lineweaver−Burk reciprocal plots of AChE initial velocity at increasing substrate
concentration (0.1−0.5 mM) in the absence and in the presence (1.0−10.0 nM) of
compound 12q are shown. Lines were derived from a weighted least-squares analysis of
the data points.
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Figure 5. (A) Fluorescence (λexc = 285 nm) and (B) UV spectra of compound 12q (50 µM) alone and
in the presence of CuSO4, FeSO4, FeCl3 or ZnCl2 (50 µM) in 20 % (v/v) ethanol/buffer
(20 mM HEPES, 150 mM NaCl, pH 7.4). (C) Determination of the stoichiometry of
complex 12q−Cu(II) by molar ratio method.
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Table 1. hAChE and hBChE Inhibitory Activities (IC50), Selectivity Ratios, and Inhibition of Aβ1-42
Self-Aggregation of Compounds 1, 5, and 12a−12s.
2
3
O
H
R
N
N
N
H
O
N
hAChE
IC50 (nM) ±
SEMa
inhibition of Aβ1−42
self-aggregation (% ±
SEM)c
hBChE
IC50 (nM) ± SEMa
selectivity
ratiob
Comp.
R
12a
12b
12c
12d
12e
12f
12g
12h
12i
557 ± 31
310 ± 27
1001 ± 66
1186 ± 70
107 ± 12
0.556
0.261
0.198
2.41
6.46 ± 0.25
7.38 ± 0.53
10.5 ± 0.82
22.9 ± 1.3
17.6 ± 0.9
22.1 ± 1.5
26.0 ± 1.7
39.6 ± 2.2
37.9 ± 2.5
42.2 ± 3.1
21.2 ± 1.9
25.3 ± 1.3
10.6 ± 0.4
24.1 ± 2.1
36.7 ± 1.9
5.31 ± 2.8
66.6 ± 5.5
13.3 ± 0.8
10.5 ± 0.6
45.7 ± 3.8
37.2 ± 2.5
26.8 ± 2.0
4.35 ± 0.32
16.6 ± 0.8
81.5 ± 5.9
0.232
0.648
1.37
1.22
4.01
12j
0.163
12k
12l
25.9 ± 1.5
46.4 ± 2.6
231 ± 19
16.3 ± 1.3
12.4 ± 0.8
145 ± 12
1.59
3.74
1.59
31.0 ± 2.4
52.3 ± 3.8
30.5 ± 2.5
12m
12n
4.09 ± 0.23
20.7 ± 1.5
0.20
43.3 ± 5.1
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