Synthesis and biological evaluation of new tetramethylpyrazine-based chalcone derivatives as potential anti-Alzheimer agents

Article abstract of DOI:10.1111/cbdd.13355

In the current study, a series of new ligustrazine-based chalcones was synthesized. For insertion of tetramethylpyrazine (TMP, also designated as ligustrazine) in chemical backbone of chalcone, a new ligustrazine-based aldehyde was prepared. New ketones were synthesized for inclusion of quinazolin-4-yl amino and pyrazin-2-yl amino moieties. The newly synthesized compounds were screened for acetylcholinesterase, butyrylcholinesterase, and monoamine oxidases (MAO) inhibitory activities and also for in vitro cytotoxicity on PC12 cells. The effect of these compounds against amyloid β-induced cytotoxicity and aggregation was also investigated. The synthesized compounds effectively inhibited the related enzymes and also exhibited neuroprotective effects. Most of the compounds displayed better inhibitory potencies against Aβ aggregation than reference compounds. Some compounds such as 11e and 16b showed very potent effects on multiple targets exhibiting behavior as multifunctional anti-Alzheimer agents.

Full text of DOI:10.1111/cbdd.13355

DR SYED NASIR ABBAS BUKHARI (Orcid ID : 0000-0001-8125-7972)
Article type : Research Article
Synthesis and biological evaluation of new tetramethylpyrazine based
chalcone derivatives as potential anti-Alzheimer agents
Meng Wanga‡, Hua-Li Qin*a, Jing Lenga, Ameeduzzafarb, Muhammad Wahab Amjadc,
Maria Abdul Ghafoor Rajac, Muhammad Ajaz Hussaind, Syed Nasir Abbas Bukhari*a,b‡
a Department of Pharmaceutical Engineering, School of chemistry, chemical engineering and
life science, Wuhan University of Technology, 205 Luoshi Road, Wuhan,430070,
P.R.China.
b Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Aljouf,
Sakaka2014, Saudi Arabia.
cCollege of Pharmacy, Northern Border University, King Abdullah Road, Rafha, Saudi
Arabia
d Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan
‡These authors contributed equally
* Authors to whom correspondence should be addressed;
Hua-Li Qin: qinhuali@whut.edu.cn; Syed Nasir Abbas Bukhari: snab_hussaini@yahoo.com;
Tel: +86 27 87749300
Fax: +86 27 87749300
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13355
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Abstract
In the current study, a series of new ligustrazine-based chalcones was synthesized. For
insertion of tetramethylpyrazine (TMP, also designated as ligustrazine) in chemical backbone
of chalcone a new ligustrazine-based aldehyde was prepared. New ketones were synthesized
for inclusion of quinazolin-4-yl amino and pyrazin-2-yl amino moieties. The newly
synthesized compounds were screened for acetylcholinesterase, butyrylcholinesterase and
monoamine oxidases (MAO) inhibitory activities and also for in vitro cytotoxicity on PC12
cells. The effect of these compounds against amyloid β-induced cytotoxicity and aggregation
was also investigated. The synthesized compounds effectively inhibited the related enzymes
and also exhibited neuroprotective effects. Most of the compounds displayed better inhibitory
potencies against Aβ aggregation than reference compounds. Some compounds such as 11e
and 16b showed very potent effects on multiple targets exhibiting behavior as multifunctional
anti-Alzheimer agents.
Keywords: Chalcone, Amyloid β, Acetylcholinesterase, Ligustrazine, Monoamine oxidases.
1. INTRODUCTION
Neurodegenerative diseases such as Alzheimer’s (AD) are one of the main causes of
disability and death in aging population [1]. With the quick rise in the aging populations these
diseases are becoming more serious. The main reason behind the development of these
diseases is the neuronal loss [2]. AD has severely affected the quality of life and health in
aging populations. An analysis shows that the occurrence of AD may triple (from 5.1 to 13.8
million) in population aging 65 and more by 2050, if timely efforts are not made regarding its
prevention and cure [3].
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The precise cause of AD has not been elucidated till date. The main pathological
characteristic of AD is the accumulation of amyloid β (Aβ) in brain, according to amyloid
hypothesis. The neurodegeneration and neuronal cell death occurs owing to the induction of
microglial activation by Aβ [4]. One of the important methods in AD treatment is increasing
the acetylcholine level in brain using acetylcholinesterase inhibitors [5]. Cholinesterase
inhibitors (ChE) have been found to sustain mental function in around 50% of patients for 1
year [6]. The hydrolysis of acetylcholine is done by two enzymes such as
butyrylcholinesterase (BChE) and acetylcholinesterase (AChE). AChE is considered as the
‘cholinergic target’ in AD treatment and is considered as the principle enzyme involved in
central nervous system (CNS). So, several studies have focused AChE in AD treatment
owing to its pivotal involvement in cholinergic neurotransmission. Both AChE and BChE
have been found to regulate cellular propagation and neurite growth during the development
of CNS in various species, including rodents [7], and BChE has also been found to have
substantial role in normal CNS functions [7-9].
MAO inhibitors (MAOIs) are used in neurology for the treatment of Alzheimer’s and
Parkinson’s diseases while in psychiatry for depressive disorders treatment. The dynamics of
regular monoamine transmitters including dopamine, serotonin or noradrenaline are altered
by the accumulation of exogenous and endogenous substrates (which are themselves
triggered by MAO inhibition). MAO-A and MAO-B are the two isoforms of MAO. MAO
enzymes have different tissue distribution, inhibitor specificity and substrate preference and
are involved in the oxidative deamination of xenobiotic and endogenous amines [10].
Noradrenaline, adrenaline and serotonin are mainly metabolized by MAO-A [11] while
MAO-B primarily metabolizes benzylamine and b-phenylethylamine [12]. Other amines
including dopamine and tyramine are common substrates for both MAO-A and MAO-B [13].
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A drug possessing single target mechanism of action may not prove to be effective in the
treatment of diseases involving multiple pathways including AD. To address this issue, the
synergistic delivery of two or more drugs, may provide useful effects and possibly result in
the modification of disease. Still, some negative aspects are associated with this strategy
which includes pharmacokinetic interferences and poor patient compliance. Instead, to
interact with various systems and/or targets involved in the onset or progression of disease a
single therapeutic agent can be designed on purpose. The disease would be attacked from
multiple sides using this multi-target approach, therefore significantly augmenting the
chances of disease modification [14, 15].
To date, there is no approved causal AD treatment. The antibody and chemical drug
development pipelines appear to meet the bottleneck. Several researchers are striving to
discover a disease-modifying drug by putting emphasis on natural- and natural-derived anti-
AD agents. In complete modulation of multi-target diseases, conventional Chinese medicine
offers some benefits as it focuses on the overall regulation of body’s pathophysiological
condition. For the treatment of cerebrovascular and cardiovascular diseases including stroke,
hypertension and atherosclerosis, a rhizome of Ligusticum chuanxiong Hort.(Ligusticum
wallichii Franch) has long been used in conventional oriental medicine [16, 17]. A potent
constituent of the conventional Chinese medicine Chuanxiong is tetramethylpyrazine (TMP)
also called as ligustrazine. In neurodegenerative conditions including ischemic stroke, AD
and Parkinson’s disease (PD), TMP has exhibited promising therapeutic effects [18-21].
Moreover it has also exhibited numerous activities such as enhancement of mitochondrial
biosynthesis, protection against kainite-induced excitotoxicity, blockade of calcium channel
and prevention of oxidative stress-induced neuronal death which are all related to AD
pathogenesis [22, 23]. Though the precise mechanisms of TMP-mediated neuroprotection are
not entirely explained, nonetheless it has been found to protect antioxidant enzyme, increase
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transcription of redox proteins, scavenge free radicals and maintain mitochondrial function
[21, 22]. Moreover, its blood-brain barrier (BBB)-penetrating ability is worth mentioning
[24]. After intravenous administration, TMP was found to efficiently penetrate BBB, yielding
a higher brain/blood ratio at 10-120 min [25].
Lately, our research group reported the synthesis of α, β-unsaturated carbonyl-based
compounds as multipotent ChEIs for the treatment of AD and we discovered that it is
probable to discover agents which show anticholinesterase potential in addition to other
activity associated with the neurodegenerative perspective of AD [26, 27]. Extending our
research, we have synthesized new ligustrazine-based α, β-unsaturated carbonyl-based
compounds (chalcones) with pyrazin-2-yl amino and quinazolin-4-yl amino moieties. The
pharmacological assessment of these compounds includes AChE, BuChE and MAO
inhibition, effects on self-induced Aβ aggregation and amyloid β-induced cytotoxicity in
PC12 cells.
2. METHODS AND MATERIALS
2.1.
Synthesis of ligustrazine based chalcone derivatives
Previously available literature was followed to synthesize compounds (3a-e) for the
subsequent ketones synthesis (5a-e). Likewise, 2a-e and its chlorinated derivatives (3a-e)
were synthesized [29] (Scheme 1). Using Boekelheide reaction, 2-hydroxymethyl-3,5,6-
trimethylpyrazine (9) was first synthesized by taking ligustrazine as the starting material (8).
The 2-iodoxybenzoic acid (IBX) oxidation of (9) yielded an aldehyde group-containing
intermediate (10) that was used for synthesis of all new compounds [30] (Scheme 3).
In ethanol (10 mL), a mixture of 4-aminoacetophenone (2.75 mmol) and 3a-e or 6a-c (2.5
mmol) was heated for 8 h under reflux. The reaction yielded a solid which was then filtered
and recrystallized from ethanol to obtain new quinazolin-4-yl amino and pyrazin-2-yl amino
ketones 5a-e and 7a-c respectively (Scheme 2).
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Our recent report described the synthesis of α, β-unsaturated carbonyl-based chalcone
derivatives using direct coupling method (Scheme 3) (Table 1) [31]. For the synthesis of 17
new compounds, base-catalyzed Claisen-Schmidt condensation was used to react various
ketones with ligustrazine-based aromatic aldehyde (10) at feed ratio 1:1. The synthesis path
for new chalcones is shown in scheme 3. An aromatic aldehyde (10 mmol, 1 equivalant) and
a ketone (10 mmol, 1 equivalent) were mixed and dissolved in 15 mL ethanol followed by
stirring for few min at 5°C. A 40% NaOH solution (in ethanol) was added drop wise to this
solution and allowed to stir for 1-24 h at 27 °C. The change in the color of reaction mixture
and the appearance of precipitate indicated product formation. The progress of the reaction
was monitored using TLC while acidified ice was used to end the reaction upon completion.
Either column chromatography or recrystallization was used to isolate compounds. The
characterization data of most active compounds are provided in supplementary materials.
2.2.
Pharmacology
To investigate the cytotoxicity or neuroprotective effects of synthetic compounds on PC12
cells, MTT assay was carried out. The AChE and BuChE inhibitory activity of compounds
was investigated using a modified Ellman’s test [32]. Using specific substrate serotonin or
benzylamine, the compounds’ activity towards MAO-A and MAO-B selectivity was
determined. Bovine brain mitochondria were isolated using Basford method [33]. MAO-A
and MAO-B activities were determined using a fluorimetric technique described by
Matsumoto and co-workers [34].Using a previously reported method, the protein
concentration was measured [35]. The results of MAO-A and MAO-B as IC50 are presented
in Table 2. The negative control used in the study was propylene glycol. To study the
inhibition of Aβ1-42 aggregation, a thioflavin T (ThT)-binding assay (previously described by
Bartolini and colleagues) with minor alterations was used [36]. All pharmacological
evaluation methods are explained in detail (Supplementary Materials).
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3. RESULTS AND DISCUSSION
3.1. Chemistry
Equimolar amounts of appropriate substituted benzaldehydes and acetophenones were
reacted using Claisen-Schmidt condensation in 1 mmol, 50% ethanolic NaOH solution to
yield ten novel chalcones (scheme-3)[37]. To get vital α,β-unsaturated carbonyl compounds
(ligustrazine-based chalcones), analytical grade precursor ketones 13a-e, 15a-b and 17a-b
were purchased from Sigma-Aldrich as starting materials and were used for reaction with
ligustrazine-based aldehyde (10) without further processing. A selective nucleophilic
aromatic substitution (SNAr) of 4-chlorine atom on substituted 4-chloro-quinazolines (3a-e)
and 4-chloro-pyrazine (6a-c) with 4-aminoacetophenone (4) yielded 5a-e and 7a-c (Scheme
2). Analytical grade 4-chloro-pyrazine (6a-c) was purchased from Sigma-Aldrich and was
used for reaction with 4-aminoacetophenone (4) without further processing. The substituted
quinazolin-4(3H)-one (2a-e) and its chlorinated derivatives (3a-e) were synthesized by
referring to the 1iterature [38] (Scheme 1).
For all new chalcones, same ligustrazine-based aldehyde (10) was used in combination with
different types of ketones as stated previously. For the synthesis of this most important
compound, starting from ligustrazine (8), Boekelheide reaction was used to first synthesize 2-
hydroxymethyl-3,5,6-trimethylpyrazine (9). An aldehyde group-containing intermediate (10)
was produced after IBX oxidation of (9) (Scheme 3)[30].
3.2.
PC12 cells viability
To study the effect of newly synthesized ligustrazine-based chalcones on cell viability, MTT
assay was performed using PC12 cells. The cells were treated with various concentrations
(0.01-100 μM) of new chalcones bearing pyrazin-2-yl amino and quinazolin-4-yl amino
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moieties for 24 h and the compounds were discovered to be nontoxic to cells at all tested
concentrations.
3.3.
Neuroprotective effects of synthetic compounds
In AD pathogenesis, Aβ produced from amyloidogenic proteolytic process of amyloid
precursor protein (APP) has been proposed to be the main player. At an early stage of AD
cascade of events, the accumulation of Aβ takes place in cellular dysfunction through
neuroinflammation [39]. One of the critical strategies in the prevention of AD might be the
suppression of Aβ-induced neuroinflammatory toxicity. MTT assay was carried out to
explore the compounds’ effect on Aβ-induced PC12 cell toxicity. The Aβ-treated cells
showed considerably lower viability up to 45% in contrast to control. The cells were
protected against Aβ-induced cell death by up to 77% when pre-treated with the compounds
(100 μM). Against Aβ-induced cell toxicity, compounds such as 11c, 11d, 11e and 12a were
discovered to be most protective . In contrast to selegiline (positive control), the protective
effect of compounds was significantly better, with 11e showing the best results. The novel
ligustrazine-based chalcone derivatives with quinazolin-4-yl amino substitution at position 4
of ketone ring (compounds 11c-e) showed greatest protective action. In quinazoline moiety,
substitution pattern on the phenyl ring of neighboring pyrimidine played an important role in
exhibiting protective effects as compound 11a which did not bear any substitution exhibited
weak effect in comparison with others possessing various groups. The protective effect of
compound 11b was increased after the inclusion of chlorine at R2, still it was found to be
weaker in comparison to 11c, 11d and 11e that bear F at position R1 and Br at position R3.
With trimethoxy substitutions at R2, R3 and R4, 11e showed maximum protection (77%). In
comparison to chalcones with substituted quinazolin-4-yl amino moiety, a derivative 12a
with pyrazin-2-yl amino substitution at position 4 on ketone ring exhibited weaker activity. In
compounds 12b and 12c, methyl substitution on pyrazin-2-yl amino moiety was found to
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decrease the activity. A protection of 55-69 % was shown by all other chalcones (Table 2).
These results show that a few new ligustrazine-based chalcones might reduce cell damage
brought about by Aβ-induced cytotoxicity. The death of cells caused by Aβ was found to be
considerably reduced in a dose-dependent manner when pretreated with new compounds.
The neuroprotective effects of ligustrazine-based derivatives have been previously reported
with thorough mechanistic studies. Chen et al. reported the synthesis of a similar series of
tetramethylpyrazine derivatives and investigated their activity in prevention against oxidative
stress-induced neuronal damage and scavenging free radicals in vitro. They discovered some
compounds to be potential neuroprotective agents in neurological diseases treatment [40]. Xu
and coworkers also synthesized a derivative of TMP that showed protective effect in
excitotoxicity-challenged cerebellar granule neurons model and exhibited pro-differential
effect, which rendered this compound to be a multi-target drug candidate for AD therapy
principally ischemic stroke [41].
3.4.
Acetylcholinesterase and butyrylcholinesterase inhibition activity
For the care of AD patients, AChE inhibitors treatment is only one feature of the package.
AChE inhibitors are the only drugs presently licensed for the treatment of AD, which act via
acetylcholinesterase (AChE) inhibition. To evaluate the inhibitory effect of compounds on
AChE and BChE, an assay based on Ellman’s method was carried out [28]. The compounds
were found to be moderate to strong AChE inhibitors (Table 2). In comparison to the
reference compounds tacrine (IC50=0.035 µM) and donepezil (IC50=0.055 µM), the
compound 16b exhibited better AChE inhibitory activity (IC50 = 0.025 µM). Similarly, 16b
was also found to inhibit BuChE (IC50=7.2 µM) similar to positive control donepezil (IC50
=7.1 µM). In comparison to AChE, the compounds were discovered to be less potent against
BChE. For BChE, other chalcones exhibited IC50 in range of 20 to 78 μM.
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The structure activity relationship (SAR) study revealed that the strong activity of
compounds 16a and 16b was due to the presence of 1-Pyridin-3-yl-ethanone as a ketone
moiety in chalcone back bone. In comparison to those bearing pyridine, chalcones 18a and
18b bearing pyrazine in ketone moiety were discovered to be less active. A detailed analysis
also revealed that there was an evident effect of substitution pattern as mono methoxy
substituted 16a was found to be more potent than 16b with dimethoxy substitution.
In comparison to chalcones with substituted quinazolin-4-yl amino moiety, those bearing
pyrazin-2-yl amino substitution at position 4 on ketone ring also showed less activity. An
exciting behavior of substitution pattern on aromatic ring of quinazoline part was observed in
new chalcones bearing quinazolin-4-yl amino moiety (11a-e). Those bearing substitutions
were found to be more potent in comparison to those without any substitution (11a). The best
results were seen in case of Trimethoxy substitution (11e). Zhang et al reported the protective
effect of tetramethylpyrazine on memory and learning in D-galactose-lesioned mice [42]. In
this model group, lowered N-methyl-D-aspartate (NMDA) receptor binding sites in
hippocampus, decreased M-cholinergic receptor binding sites in the cortex and AChE
activity, lowered superoxide dismutase (SOD) activity, elevated malondialdehyde (MDA)
content and deficit of memory and learning were observed, in comparison to normal control.
TMP can protect NMDA receptor activity, improve central cholinergic system function,
strengthen antioxidative function and therefore improve the memory and learning in D-gal-
lesioned mice [42].
3.5.
Inhibition of MAO-A and MAO-B
The fact that expression of MAO-B in neuronal tissue rises around 4-fold with age
(particularly in glial cells) has sparked in developing the inhibitors of MAO-B for the
treatment of neurodegenerative diseases. Most of MAO-B inhibitors act irreversibly by
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making a N(5)-flavocyanine adduct with the enzyme resulting in probable immunogenic
effects together with long-term inhibition of enzyme. They bear the essential structural
characteristics for selective binding to human MAO-B as they contain two aromatic rings
attached via a three-carbon α,β-unsaturated carbonyl system [43].
To study the MAO-inhibitory potential of ligustrazine derivatives, an assay was carried out.
As an alternative to MAO-B inhibition alone, dual MAO-A and MAO-B inhibition may give
better effects as depressive symptoms are shown by AD patients [44]. So, compounds’
inhibitory activity against both isoforms was studied. Some compounds were found to
effectively inhibit both MAO-A and MAO-B at micromolar concentration however the new
derivatives inhibited MAO-B stronger than MAO-A (Table 2).
An IC50 in the range of 11.4 to 57.8 μM was exhibited by the derivatives for MAO-A
inhibition. None of the compounds was found to be as active as positive control tracine
(0.050 µM). The best activity was shown by 16b (11.4 µM). Instead, strong MAO-B
inhibition was shown by new ligustrazine derivatives ranging from 2.6 to 18.7 µM. When
compared with positive control donepezil (15.2µM), most of the compounds exhibited
superior activity.
The SAR of new derivatives was presented by their MAO-B inhibitory activities.
Ligustrazine derivatives with substituted quinazolin-4-yl amino moiety (11a-e) exhibited
potent effects and similar to AChE inhibition, an exciting behavior of the substitution pattern
on aromatic ring of quinazoline part was seen. The compound 11e was found to be most
active with trimethoxy substitution on aromatic ring of quinazoline moiety. 14e bearing
methoxy substitution at R5 was found to be most active among ligustrazine derivatives (14a-
e). Weak inhibition was shown by other related compounds with methoxy substitution at
various positions. Kelekci and coworkers reported the synthesis of a new series of pyrazoline
derivatives starting from a quinazolinone ring and studied them for MAO-A and -B
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inhibitory, anxiogenic and antidepressant activities in vivo and in vitro. The starting
compound bearing quinazolinone ring inhibited MAO-A reversibly and competitively while
quinazolinone hydrazine inhibited MAO-B irreversibly and competitively [45].
3.6.
Self-induced Aβ1–42 aggregation inhibitory activities
It is now possible to target two or more pathways by using newly designed compounds
courtesy multi-target drug discovery (MTDD). The realism of this strategy has been
emphasized by the effective clinical transition of numerous compounds [46]. Apart from the
analysis of MAO and ChEs inhibition, the self-induced Aβ1–42 aggregation inhibitory
activity of compounds was assessed using thioflavin T-based fluorometric assay [47].
An oral medication (donepezil) which is being used in the treatment of alzheimer's
disease was used as a reference compound in this study. As compared to donepezil (14.86 %
at 10 µM), ligustrazine derivatives exhibited moderate to strong potencies (16.5 to 62.7 % at
10 µM concentration) (Table 3). At a concentration of 10 µM, 11a and 11b exhibited
strongest activities (62.7% and 59.2%). The ligustrazine derivatives with substituted
quinazolin-4-yl amino moiety (11a-e) potently inhibited the aggregation of Aβ except 11e
which contained tri methoxy substitution however it was found to be a strong AChE inhibitor
and also exhibited protection against Aβ-induced cell toxicity (Table 3). Ligustrazine
derivative 11a substituted with quinazolin-4-yl amino was discovered to be the most potent
inhibitor of aggregation and it did not bear any substitution on aromatic ring of substituted
quinazoline moiety. Halogen substitutions at various positions on aromatic ring of this
quinazoline part displayed noticeable reduction in the inhibitory activities however it was
contrary to their effects on AChE.
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4. CONCLUSION
Chalcones and associated α, β-unsaturated carbonyl-based compounds are intermediates in
the synthesis of numerous heterocyclic compounds which have exhibited therapeutic and
diagnostic activities in the treatment of Alzheimer’s disease. A series of new ligustrazine-
based chalcones was synthesized with inclusion of new moieties in ketone domain of
molecule as quinazolin-4-yl amino and pyrazin-2-yl amino moieties and were evaluated as
multifunctional anti-Alzheimer agents. The Aβ-induced death was decreased by 77% when
the PC12 cells were pretreated with synthetic compounds (100 μM). In comparison to
reference compounds, most derivatives showed improved inhibitory potencies against Aβ
aggregation. Compounds including 11e and 16b exhibited potent effects on multiple targets
exhibiting potential as multifunctional anti-Alzheimer agents. On the basis of these findings it
can be stated that the selected compounds might prove to be promising drug candidates in the
treatment of neurodegenerative disorders. So, the BBB penetrating property, toxicity profile
and pharmacokinetics of these compounds need additional assessment.
ACKNOWLEDGMENTS
HQ and SNAB are grateful to Wuhan University of technology for financial support.
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Figure legends
Scheme 1: Synthesis of (2a-e) and its chlorinated derivatives (3a-e) (i) HCONH2 (ii) POCl3.
Scheme 2: Synthesis of new quinazolin-4-yl amino and pyrazin-2-yl amino ketones (5a-e)
and (7a-c), (iii) Reflux, EtOH, ∆.
Scheme 3: Synthesis scheme of ligustrazine aldehyde (10) and new chalcone derivatives.
Reagents and conditions: (i-a) 30% H2O2, acetic acid, 70 °C, 8 h.; (i-b) acetic anhydride,
reflux, 2 h; (i-c) 20% NaOH; (ii) IBX, DMSO, room temperature, 0.5 h; (iii) NaOH, EtOH,
Room temperature.
Conflict of interest
The authors declare no conflicts of interest in this work.
Supplementary data
Supplementary data associated with this article can be found in the online version.
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R
O
R
Cl
N
R
O
N
1
1
1
R
R
2
R
2
2
ii
i
OCH3
NH2
N
NH
R
R
3
R
3
3
R
R
4
R
4
4
(3a-e)
(1a-e)
(2a-e)
Scheme 1: Synthesis of (2a-e) and its chlorinated derivatives (3a-e) (i) HCONH2 (ii) POCl3
O
R
Cl
N
1
R
2
R HN
1
N
iii
R
2
N
R
3
O
(5a-e)
O
R
4
R
N
3
+
(3a-e)
Cl
R
4
H2N
iii
R
(4)
2
N
HN
N
R
2
R
1
N
(6a-c)
(7a-c)
N
R
1
Scheme 2: Synthesis of new quinazolin-4-yl amino and pyrazin-2-yl amino ketones (5a-e)
and (7a-c), (iii) Reflux, EtOH, ∆.
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O
N
N
(5a-e)
R HN
1
N
N
(11a-e)
R
2
N
N
O
R
N
3
(8)
N
N
R
Ligustrazine (TMP)
4
(7a-c)
i
HN
(12a-c)
R
2
N
OH
R
O
N
1
R
1
N
(9)
R
2
R
O
R
1
R
R
R
N
N
3
5
ii
2
R
O
4
iii
(13a-e)
N
R
3
5(14a-e)
H
R
O
R
1
R
4
R
2
N
(10)
R
O
1
R
N
N
2
R
N
3
5
(15a-c)
R
N
R
4(16a-b)
3
O
R
N
2
O
R
N
N
N
N
2
R
N
R
3
5
(17a-b)
R
R
3
5(18a-b)
Scheme 3: Synthesis scheme of ligustrazine aldehyde (10) and new chalcone derivatives.
Reagents and conditions: (i-a) 30% H2O2, acetic acid, 70 °C, 8 h.; (i-b) acetic anhydride,
reflux, 2 h; (i-c) 20% NaOH; (ii) IBX, DMSO, room temperature, 0.5 h; (iii) NaOH, EtOH,
Room temperature.
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Table 1: New synthetic compounds.
Compound
5a
R1
R2
R3
R4
R5
-
-
-
-
-
Cl
-
5b
-
-
-
-
Br
5c
-
-
-
F
5d
-
-
-
-
OCH3
OCH3
OCH3
-
5e
-
7a
-
-
-
-
-
CH3
-
7b
-
-
-
-
CH3
7c
-
-
-
11a
11b
11c
11d
11e
12a
12b
12c
14a
14b
14c
14d
14e
16a
16b
18a
18b
-
-
-
-
-
Cl
-
-
-
-
-
Br
-
-
-
F
-
-
-
-
OCH3
OCH3
OCH3
-
-
-
-
-
-
-
CH3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CH3
-
-
-
CH3
-
-
-
-
CH3
CH3
-
CH3
CH3
CH3
-
OCH3
-
-
-
-
-
-
-
-
OCH3
-
-
OCH3
-
-
OCH3
OCH3
-
-
-
-
-
-
CH3
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Table 2: Effects of synthetic compounds on AChE, BChE and MAO.
AChE
IC50 (µM)
12.5±1.0
8.2±1.4
BuChE
IC50 (µM)
54.2±3.9
47.4±2.8
42.4±2.5
37.6±3.9
22.4±2.3
65.6±2.9
48.6±3.5
59.6±5.2
77.9±4.3
42.4±2.5
37.6±3.9
67.4±4.8
69.6±2.9
7.2±1.9
MAO-A
MAO-B
Compounds
IC50 (µM) IC50 (µM)
40.4±2.9
56.9±2.4
57.8±3.5
52.4±2.8
47.4±1.9
>100
17.5±1.8
8.4±2.0
9.9±1.8
2.9±1.0
2.6±0.5
18.5±3.7
12.4±1.5
14.9±4.6
13.7±2.9
11.5±0.9
11.2±1.5
7.5±1.9
4.2±1.2
9.4±1.2
8.9±1.7
18.7±4.2
15.9±3.7
15.2±2.1
11a
11b
11c
11d
11e
12a
12b
12c
14a
14b
14c
14d
14e
16a
16b
18a
7.5±1.6
2.4±0.4
0.10±0.02
19.2±2.5
14.5±3.9
10.1±3.1
18.7±1.9
7.5±1.6
>100
>100
46.5±1.4
42.5±2.4
39.6±2.7
>100
2.4±0.4
4.9±1.2
1.4±1.0
>100
0.062±0.02
0.025±0.01
6.5±2.1
17.6±2.5
11.4±2.1
37.5±2.3
31.9±1.8
788±51
2.7±1.4
27.8±1.9
20.3±1.3
7.1±0.97
5.7±1.3
18b
0.055±0.09
Donepezil
0.035±0.04 0.0057±0.004 0.050±0.06 0.17±0.5
Tacrine
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Table 3: Inhibition of self-induced Aβ1–42 aggregation and protective effects of
synthetic compounds against Aβ1-42-induced cell damage. Data is expressed as means
± standard deviation (SD) of at least three independent experiments.
% Inhibition
at
% Protection
at
Compound
10 µM
62.7±1.2
100 µM
69±5
11a
11b
11c
11d
11e
12a
12b
12c
14a
14b
14c
14d
14e
16a
16b
18a
59.2±2.9
50.5±4.6
36.9±4.5
26.7±1.4
38.8±2.9
32.6±5.7
27.1±2.9
52.8±2.5
47.3±2.9
47.0±1.2
29.8±2.5
21.4±2.7
19.8±1.9
16.5±3.4
29.2±3.2
21.9±4.4
68±2
74±4
75±6
77±4
73±2
69±4
66±3
54±3
56±4
57±2
55±5
57±2
67±4
68±3
52±6
56±5
-
18b
Donepezil
14.86±2.5
7.5±2.0
-
-
Tacrine
Selegiline
55±4
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