Multivalent butyrylcholinesterase inhibitor discovered by exploiting dynamic combinatorial chemistry

Article abstract of DOI:10.1016/j.bioorg.2021.104656

In this study, we report the generation of a polymer-based dynamic combinatorial library (DCL) incorporating exchangeable side chains using acylhydrazone formation reaction. In combination with tetrameric butyrylcholinesterase (BChE), the most potent binding side chain was identified, and the information obtained was further used for the synthesis of a multivalent BChE inhibitor. In the in vitro biological evaluation, this multivalent inhibitor exhibited not only better inhibitory effect than the commercial reference but also high selectivity on BChE over acetylcholinesterase (AChE).

Full text of DOI:10.1016/j.bioorg.2021.104656

Bioorganic Chemistry 108 (2021) 104656
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Multivalent butyrylcholinesterase inhibitor discovered by exploiting
dynamic combinatorial chemistry
Shuang Zhao, Jintao Xu, Shixin Zhang, Maochun Han, Yao Wu, Yusi Li, Lei Hu*
School of Pharmacy, Jiangsu University, 301 Xuefu Rd., Zhenjiang, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, we report the generation of a polymer-based dynamic combinatorial library (DCL) incorporating
exchangeable side chains using acylhydrazone formation reaction. In combination with tetrameric butyr-
ylcholinesterase (BChE), the most potent binding side chain was identified, and the information obtained was
further used for the synthesis of a multivalent BChE inhibitor. In the in vitro biological evaluation, this multi-
valent inhibitor exhibited not only better inhibitory effect than the commercial reference but also high selectivity
on BChE over acetylcholinesterase (AChE).
Dynamic combinatorial chemistry
Multivalent interaction
Butyrylcholinesterase
Protein inhibition
Macromolecule
1. Introduction
hydrolyzes both acetyl- and butyrylcholine [23,24]. Moreover, while
BChE is found in more tissues, such as blood serum, liver and pancreas,
Dynamic combinatorial chemistry (DCC) falls into the category of
supramolecular chemistry and it utilizes covalent or noncovalent
reversible reactions to generate dynamic combinatorial library (DCL)
from building blocks with complementary functional groups [1–8]. DCL
is fully under thermodynamic control therefore it can respond to the
addition of external (e.g. enzyme, light, metal ion) or internal (e.g. pH,
phase change) stimuli due to its adaptive nature, resulting in the for-
mation of more favorable constituents at the cost of other less suitable
combinations (Fig. 1). DCL, as a whole, keeps shifting from its initial
equilibrium during the interaction with stimuli until an overall optimal
state, being the amplification of the best-fit constituent(s), is eventually
achieved [9–11]. Among its many applications, DCC has been an
outstanding success in chemical biology for identification of novel li-
gands for diverse protein targets [12–14]. DCC integrates compound
library generation and affinity screening in one-pot, offering an efficient
strategy for the investigation of ligand–protein interaction as well as
subsequent drug development [15–17].
than AChE, it exists at much lower concentration in the central and
peripheral nervous systems [25]. Nevertheless, previous studies
revealed that the level of AChE decreases in patient brain while the level
and activity of BChE remain unchanged or even increase during the
progression of Alzheimer’s disease (AD), suggesting that BChE might
play an important role in the late stage of AD [26–28]. Besides, BChE
activity has been identified to associate with diabetes, obesity, hydro-
lysis of hunger hormone Ghrelin and other liver diseases [29–34].
Therefore, developing selective BChE inhibitor is currently becoming
more and more an attractive target for multiple therapeutic interests
despite the challenges mainly due to its structural similarity to AChE as
both enzymes share 65% homologic amino acid sequences [35–37].
Multivalency serves in nature as a fundamental principle for
achieving strong interactions [38]. Inspired by multivalency, numerous
examples, big as the nest stadium and small as velcro, could be found in
our daily life. In biology, multivalent interactions are characterized by
the spontaneous, reversible binding of multiple ligands on one biological
substrate to multiple receptors on another and play key roles in pro-
cesses such as adhesion, recognition and signaling [39–42]. One distinct
character of multivalent interactions between binding partners is the
dramatic gain in affinity, making it a highly efficient tool for the targeted
strengthening among different biological entities [43–46]. In the study,
a polymer-based DCL was designed and generated through reversible
acylhydrazone formation reaction, constructing a dynamic platform
with multiple reaction sites (Fig. 2). This polymer-based DCL was further
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are
two cholinesterase proteins commonly found in mammals [18]. In
human body as well as many other mammals, AChE and BChE mainly
exist as tetrameric glycoproteins with four identical subunits [19–22].
Different from the well-established physiological property of AChE in
regulating cholinergic signaling, the role of BChE has become less
elusive only in the recent decade. BChE, also known as pseudocholin-
esterase, or serum cholinesterase, is a non-specific enzyme that
* Corresponding author.
E-mail address: leihu@ujs.edu.cn (L. Hu).
https://doi.org/10.1016/j.bioorg.2021.104656
Received 7 October 2020; Received in revised form 7 January 2021; Accepted 12 January 2021
Available online 27 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Zhao et al.
Bioorganic Chemistry 108 (2021) 104656
Fig. 1. Conceptual demonstration of DCC.
Fig. 2. Schematic illustration of polymer-based multivalent BChE inhibitor identification via ‘polymer-based’ DCC approach.
subjected to the selection of tetrameric BChE through multivalent
ligand/receptor interactions. The amplification information obtained
from the dynamic system was analyzed and applied for the synthesis of a
specific multivalent BChE inhibitor, which proved to be potent and se-
lective comparing to the tested commercial reference.
was evaporated under vacuum, and the crude product was purified by
silica gel chromatography (hexane:EtOAc = 1:1) to yield product 3e
(200 mg, 23%) as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ: 10.29
(s, 1H), 9.36 (s, 1H), 8.77 (d, J = 4.0 Hz, 2H), 7.85 (d, J = 4.0 Hz, 2H),
7.55 (d, J = 8.0 Hz , 2H), 6.78(d, J = 8.0 Hz , 2H). 13C NMR (100 MHz,
DMSO‑d6) δ: 163.7, 154.5, 150.6, 142.5, 130.5, 122.8, 121.9, 115.5.
2. Materials and methods
2.2.2. Ethyl 2-((7-(dimethylamino)naphthalen-2-yl)oxy)acetate 4a
To a solution of 7-N,N-dimethylamino-2-naphthol (278 mg, 1.48
mmol) and K2CO3 (308 mg, 2.22 mmol) in CH3CN (10 mL) was added
2.1. Materials
Reagents were used as purchased if not specified. Butyr-
ylcholinesterase (C7512) and acetylcholinesterase (C3389) were pur-
chased from Sigma-Aldrich. 1H NMR and 13C NMR data were recorded
on a Bruker Avance 400. Chemical shifts are reported as δ values (ppm),
and J values are given in Hertz (Hz). Thin layer chromatography (TLC)
was performed on precoated G/UV silica plates (0.20 mm, Qingdao-
Haiyang), visualized with UV-detection. Flash column chromatog-
raphy was performed on silica gel 60, 200–300 mesh (Qingdao-
Haiyang). Sephadex G-50 was purchased from Pharmacia Fine Chem-
icals, Sweden. High-resolution mass spectra were analyzed by Micromon
technical corporation, China. Analytical high-performance liquid chro-
matography (HPLC) with revise phase stationary phases was performed
on HP-Agilent 1260 series controller. Solvents for HPLC use were of
spectrometric grade. Gel permeation chromatography was performed on
a Malvern instrument (Herrenberg, Germany) equipped with a refrac-
tive index detector (Viscotek), viscosity detector (Viscotek 270 detector)
ethyl bromoacetate (197 μL, 1.78 mmol) at 0 ℃ dropwise. The reaction
mixture was stirred at 0 ℃ for 1 h and then at r.t. overnight, at which
time the solution was filtered, and the filtrate was concentrated under
vacuum. The crude product was purified by silica gel chromatography
(hexane:EtOAc = 8:1) to yield product 4a (344 mg, 84%) as a white
solid. 1H NMR (400 MHz, DMSO‑d6) δ: 7.65–7.61 (m, 2H), 7.06–7.03 (m,
2H), 6.88–6.83 (m, 2H), 4.84 (s, 2H), 4.20 (q, J = 8.0 Hz 2H), 2.98 (s,
6H), 1.23 (t, J = 8.0 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 169.2,
156.3, 149.3, 136.2, 129.3, 128.6, 122.3, 114.5, 114.2, 106.1, 105.5,
65.0, 61.0, 40.7, 14.5.
2.2.3. Ethyl 2-(3-(dimethylamino)phenoxy)acetate 4b
Procedure for compound 4a was followed. Compound 4b was ob-
tained as a yellow solid (43%). 1H NMR (400 MHz, CDCl3) δ: 7.14 (t, J =
8.4 Hz, 1H), 6.40 (dd, J = 8.4, 2.0 Hz, 1H), 6.34 (t, J = 2.4 Hz, 1H), 6.23
(dd, J = 7.6, 2.0 Hz, 1H), 4.61 (s, 2H), 4.27 (q, J = 7.2 Hz, 2H), 2.92 (s,
6H), 1.30 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.2,
158.9, 152.0, 129.7, 106.6, 101.5, 100.0, 65.5, 61.2, 40.5, 14.2.
and Viscotek A-Columns [set 1: A3000 (6
μ
m, 300 mm × 8 mm) +
A2000 (8 m, 300 mm × 8 mm) and set 2: 2 × A6000M (13
μ
μ
m, 300 mm
× 8 mm)]. Millipore water was used as solvent with 8.5 g/L NaNO3 and
0.2 g/L NaN3. The flow rate was 0.5 mL/min, and calibration was con-
ducted with poly(ethylene glycol) standards. Absorbance in the IC50
evaluation was measured on a Bio-Rad iMark ELISA microtiter plate
reader.
2.2.4. Ethyl 2-(pyridin-3-yloxy)acetate 4c
Procedure for compound 4a was followed. Compound 4c was ob-
tained as a white solid (29%). 1H NMR (400 MHz, CDCl3) δ: 8.79 (dd, J
= 4.0, 1.6 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H), 7.46 (dd, J = 9.2, 2.8 Hz,
1H), 7.36 (dd, J = 8.4, 4.4 Hz, 1H), 7.02 (d, J = 2.8 Hz, 1H), 4.75 (s, 2H),
4.30 (q, J = 7.2 Hz, 2H), 1.31(t, J = 6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ: 168.5, 155.9, 148.4, 144.6, 135.0, 131.1, 129.0, 122.1, 121.5,
106.6, 65.6, 61.5, 14.1.
2.2. Synthesis
2.2.1. N-(4-hydroxyphenyl)pyridine-4-carboxamide 3e
4-aminophenol (443 mg, 4.06 mmol), hydroxybenzoic acid (500 mg,
4.06 mmol) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (934 mg, 4.87 mmol) were dissolved in acetone (25 mL).
The mixture was refluxed for 20 h under N2 atmosphere. The solution
2.2.5. Ethyl 2-(quinolin-6-yloxy)acetate 4d
Procedure for compound 4a was followed. Compound 4d was ob-
tained as a white solid (77%). 1H NMR (400 MHz, CDCl3) δ: 8.34–8.32
2

S. Zhao et al.
Bioorganic Chemistry 108 (2021) 104656
(m, 1H), 8.26 (dd, J = 4.4, 2.0 Hz, 1H), 7.25–7.18 (m, 2H), 4.57 (s, 2H),
4.26 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ: 168.2, 154.1, 143.1, 138.0, 123.8, 121.6, 65.5, 61.6, 14.1.
Table 1
Characterization of APG and APG5b.
Mna (Da)
Mwb (Da)
PDIc
NMRd (Da)
APG
8470
9550
1.128
1.175
7684
9744
2.2.6. Ethyl 2-(4-(isonicotinamido)phenoxy)acetate 4e
APG5b
12,000
14,100
Procedure for compound 4a was followed. Compound 4e was ob-
tained as a white solid. (116 mg, 42%) 1H NMR (400 MHz, DMSO‑d6) δ:
10.41 (s, 1H), 8.78 (d, J = 5.2 Hz, 2H), 7.86 (d, J = 5.2 Hz, 2H), 7.68 (d,
J = 8.4 Hz, 2H), 6.96 (d, J = 8.8 Hz , 2H), 4.77 (s, 2H), 4.18 (q, J = 7.2
Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ:
169.2, 164.0, 154.6, 150.7, 142.4, 132.7, 122.4, 121.9, 115.0, 65.3,
61.0, 14.5.
a
Mn, Number-average molecular weight.
Mw, Weight-average molecular weight.
b
c
d
PDI, Polymer dispersity index is defined as Mw/Mn.
Molecular weight calculated through NMR integration.
155.3, 148.7, 146.8, 131.9, 126.1, 122.5, 115.3, 66.9, 48.6. HRMS (ESI-
TOF): ([Mꢀ I], C15H17N4O3; cal.: 301.12952, found: 301.12944).
2.2.7. 7-(2-hydrazinyl-2-oxoethoxy)-N,N,N-trimethylnaphthalen-2-
aminium iodide 5a
2.2.12. N-(4-phenoxyacetohydrazide)pyridine-4-carboxamide 5f
To a solution of compound 4e (95 mg, 0.317 mmol) in methanol (3
To a solution of compound 4a (324 mg, 1.18 mmol) in CH3CN was
added methyl iodide (1.84 mL, 29.6 mmol). The reaction mixture was
stirred at r.t. overnight, at which time the solvent was removed under
vacuum. The residue was dissolved in water and filtered. The solvent
was removed under vacuum to yield crude methylated product (374
mg). The crude product (20 mg, 0.048 mmol) was dissolved in methanol
mL) was added hydrazine hydrate (153 μL, 1.583 mmol). The reaction
mixture was refluxed for 3 h, then the solvent was removed under
vacuum. The residue was washed with DCM (10 mL × 3) and dried to
1
afford product 5f (58 mg, 64%) as a white solid. H NMR (400 MHz,
DMSO‑d6) δ: 10.41 (s, 1H), 9.36 (s, 1H), 8.78 (d, J = 8.0 Hz, 2H), 7.86 (d,
J = 4.0 Hz 2H), 7.68 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 8.0 Hz , 2H), 4.49
(s, 2H), 4.35 (s, 2H). 13C NMR (100 MHz, DMSO‑d6) δ: 167.1, 164.0,
154.8, 150.7, 142.4, 132.6, 122.4, 121.9, 115.1, 66.9. HRMS (ESI-TOF):
([M+H], C14H15N4O3; cal.: 287.11387, found: 287.11371).
(3 mL), and hydrazine hydrate (22 μL, o.48 mmol) was added to this
solution. The reaction mixture was refluxed for 3 h, at which time the
solvent was removed under vacuum. The solid was washed with
dichloromethane (10 mL × 3) and dried to yield product 5a (17 mg,
88%) as a white solid. 1H NMR (400 MHz, D2O) δ: 8.13 (d, J = 4.0 Hz,
1H), 7.99 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 12.0 Hz, 1H), 7.69–7.66 (m,
1H), 7.31–7.25 (m, 2H), 4.65 (s, 2H), 3.63 (s, 9H). 13C NMR (100 MHz,
D2O) δ: 169.3, 156.4, 144.4, 133.5, 130.6, 129.6, 128.7, 120.7, 117.6,
115.0, 108.1, 65.9, 57.0; HRMS (ESI-TOF): ([Mꢀ I], C15H20N3O2; cal.:
274.1550, found: 274.1549).
2.2.13. Acylydrazone polymer APG5b
To a solution of polymer APG (50 mg, 0.0065 mmol) in methanol (2
mL) with anhydrous MgSO4 (10 mg) was added compound 5b (23 mg,
0.065 mmol). The reaction mixture was refluxed for 2 h, then the so-
lution was filtered. The filtrate was concentrated under vacuum, and the
crude product was purified by Sephadex G-50 column using water as
eluent to yield polymer APG5b as a white solid (42 mg, 58%) (charac-
terization see Table 1).
2.2.8. 3-(2-hydrazinyl-2-oxoethoxy)-N,N,N-trimethylbenzenaminium
iodide 5b
Procedure for compound 5a was followed. Compound 5b was ob-
tained as a yellow solid (55%). 1H NMR (400 MHz, D2O) δ: 7.58 (t, J =
8.4 Hz, 1H), 7.46 (dd, J = 8.4, 2.4 Hz, 1H), 7.41 (t, J = 2.4 Hz, 1H), 7.17
(dd, J = 8.0, 2.0 Hz, 1H), 4.74 (s, 2H), 3.62 (s, 9H); 13C NMR (100 MHz,
DMSO‑d6) δ: 166.5, 159.0, 148.6, 131.3, 116.3, 113.2, 108.6, 67.1, 56.9;
2.3. General procedure of the formation, templating and analysis of DCL
Acylhydrazides (1
APG (1 L, 50 mM of aldehyde group concentration in 0.1 M, pH 6.2 PBS
buffer), aniline (5 L, 1 M in DMSO) were added to PBS buffer (993 L,
μL, 50 mM each dissolved in DMSO), polymer
μ
μ
μ
HRMS (ESI-TOF): ([Mꢀ I],
C11H18N3O2; calc.: 224.1394, found:
0.1 M, pH 6.2) in a screw-cap vial, which was assembled onto a rotary
mixer at r.t.. 10 h later, pH of the DCL was raised to 8 by addition of
aqueous NaOH solution. Polymer was separated by using ultrafiltration
(MW cut-off 3500 Da at 10,000 rpm for 10 min). Equilibrium of the DCL
was determined by HPLC analysis to verify the free acylhydrazide
concentrations.
224.1389).
2.2.9. 6-(2-hydrazinyl-2-oxoethoxy)-1-methylquinolin-1-ium iodide 5c
Procedure for compound 5a was followed. Compound 5c was ob-
tained as a white solid (64%). 1H NMR (400 MHz, D2O) δ: 9.02 (d, J =
5.6 Hz, 1H), 8.95 (d, J = 8.4 Hz, 1H), 8.35 (d, J = 9.6 Hz, 1H), 7.96–7.91
(m, 2H), 7.66 (d, J = 2.8 Hz, 1H), 4.89 (s, 2H), 4.61 (s, 3H); 13C NMR
(100 MHz, D2O) δ: 168.8, 157.3, 147.0, 146.0, 135.05, 131.4, 127.7,
122.1, 120.3, 109.1, 66.4, 45.4; HRMS (ESI-TOF): ([Mꢀ I], C12H14N3O2;
calc.: 232.1081, found: 232.7087).
Butyrylcholinesterase (0.25 eq, 9 mg) was subsequently added to the
equilibrated DCL mixture, which was rotated for 20 h at r.t.. pH of the
mixture was raised to 8 by addition of aqueous NaOH solution to quench
any further exchange, and CH3CN was added to denature the enzyme.
Enzyme and polymer were separated from the reaction mixture by ul-
trafiltration (MW cut-off 3500 Da at 10,000 rpm for 10 min). The final
acylhydrazide distribution was analyzed by HPLC analysis, and the re-
sults were compared with that of in equilibrium.
2.2.10. 3-(2-hydrazinyl-2-oxoethoxy)-1-methylpyridin-1-ium iodide 5d
Procedure for compound 5a was followed. Compound 5d was ob-
1
tained as a red solid (94%). H NMR (400 MHz, D2O) δ: 8.62 (s, 1H),
8.47 (d, J = 6.0 Hz, 1H), 8.13 (dd, J = 9.2, 2.4 Hz, 1H), 7.917 (dd, J =
8.8, 6.0 Hz, 1H), 4.89 (s, 2H), 4.37 (s, 3H); 13C NMR (100 MHz, D2O) δ:
167.9, 156.5, 138.7, 133.8, 130.7, 128.6, 67.1, 48.5; HRMS (ESI-TOF):
([Mꢀ I], C8H12N3O2 ; calc.: 182.0924, found: 182.0918).
2.4. HPLC condition
Column, a tandem column system with one Agilent Zorbax C8 (3.5
μ
m, 250 mm × 4.6 mm) and one Agilent InfinityLab Poroshell 120 C18
(4
stituents; flow rate, 0.5 mL/min; wavelength, 273 nm; injection volume,
20 L; gradient, NH4OAc (0.1 M)/MeOH at 85% for 25 min followed by
85–25% over 5 min.
μm, 250 mm × 4.0 mm) was applied to efficiently separate all con-
2.2.11. 4-((4-(2-hydrazinyl-2-oxoethoxy)phenyl)carbamoyl)-1-
methylpyridin-1-ium iodide 5e
μ
Procedure for compound 5a was followed. Compound 5e was ob-
tained as a yellow solid (134 mg, 88%) 1H NMR (400 MHz, DMSO‑d6) δ:
10.82 (s, 1H), 9.36 (s, 1H), 9.20 (d, J = 8.0 Hz, 2H), 8.52 (d, J = 8.0 Hz,
2H), 7.69 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H), 4.50 (s, 2H), 4.43
(s, 2H), 3.38 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 167.0, 160.9,
3

S. Zhao et al.
Bioorganic Chemistry 108 (2021) 104656
Scheme 1. Synthesis of acylhydrazide derivatives.
2.5. General kinetic studies and determination of Km
the same time. After an additional 24 h incubation, each well was added
with 20 L of 5 mg/mL MTT stock solution. Then the cells were further
incubated for 4 h. Subsequently, staining agent was replaced with 100
μ
BChE activity assays were carried out by Ellman’s method with slight
modification [47]. To a solution of 50 mM phosphate buffer (pH 7.4)
μL of DMSO. The plates were placed on a table oscillator for 20 min, and
was added DTNB (250
μM) with different concentration of BTCh to make
the absorbance was measured at 570 nm. Results were expressed at half
maximal inhibitory concentration (IC50), which was the dose of sample
leading to a 50% loss of cell viability.
a total volume of 2 mL. BChE (0.5 U in 1 mL buffer) was added to initiate
the hydrolysis process. The formation of 5-thio-2-nitrobenzoate (TNB)
was recorded by UV–Vis spectroscopy at 412 nm using a time-drive
analysis method over the first 1 min. Blank reactions were performed
using buffer solution. Km was obtained using non-linear regression
analysis (GraphPad).
3. Results and discussion
3.1. Design and synthesis of initial binding blocks
Acylhydrazone formation reaction was selected in current study to
generate the DCL. This type of reversible reaction has been very often
applied to DCC-based medicinal chemistry investigations as acylhy-
drazone products offer balanced kinetic and thermodynamic properties
[48,49]. An aldehyde-functionalized linear poly(glycidol) (APG), which
was reported in our previous works [11,16] was chosen as the reaction
platform. Density of the aldehyde group was optimized to incorporate as
many reaction sites while maintaining its water solubility. Accordingly,
six acylhydrazides were designed as the complementary binding blocks
of polymer APG for DCL generation (Scheme 1). Although different in
structure, acylhydrazides 5a-5e were all functionalized with quaternary
ammonium salt groups, which in principle would specifically interact
with certain amino acid residues situated in the active site gorge of BChE
2.6. Determination of inhibition constants
Ki and
αKi were determined using the Ellman method by adding
enzyme into BTCh and inhibitor solution. The concentration of BTCh
was varied from 10 μM to 240 μM, while the concentration of inhibitor
was fixed to 0 nM, 33 nM, 100 nM. Tacrine was used as the reference
compound. The inhibition constants were calculated using non-linear
regression analysis in a mixed binding model (GraphPad).
2.7. Cytotoxicity evaluation
The cytotoxicity of polymer APG, inhibitor APG5b and acylhy-
drazide 5b against a series of human cancer cell lines was measured
using MTT method with slight modification. Doxorubicin was used as
reference in this bioassay. Specifically, cells were seeded at a density
through cation-π-binding. A neutral acylhydrazide 5f was also designed
as its close analog was reported to be an effective BChE inhibitor [50].
Specifically, the synthetic route began with the substitution of phenolic
compound 3 to ethyl 2-bromoacetate, obtaining intermediate 4. Com-
pounds 3a-3d were commercially available while compound 3e was
synthesized through amidation between starting materials 1 and 2. After
methylation and hydrazine substitution, intermediates 4a-4e were
converted to the corresponding target molecules 5a-5e, and the neutral
about 3000 cells/well on a 96-well plate in 100 μL complete medium
containing Dulbecco’s modified Eagle’s medium (DMEM), 5% fetal
bovine serum (FBS), 50 unit/mL penicillin and 50 μg/mL streptomycin.
After incubation in 5% CO2 for 12 h at 37 ℃, the medium was removed.
Complete medium containing the test material was then added to the
well while control experiment without any test sample was conducted at
4

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Bioorganic Chemistry 108 (2021) 104656
Scheme 2. Generation of dynamic combinatorial library.
Fig. 3. HPLC chromatographic analyses of free acylhydrazides in DCL: (a) at equilibrium; (b) after BChE intervention.
acylhydrazone 5f was obtained by skipping the methylation step from
intermediate 4e.
more a suitable tool for analysis. Instead, HPLC was applied to monitor
the process of the dynamic system by analyzing the composition of free
acylhydrazides, which were obtained through ultrafiltration, in the re-
action mixture. Therefore, distribution of acylhydrazone side chains on
the polymer was inversely proportional to that of free acylhydrazines.
Under the optimal HPLC conditions, all six acylhydrazides could be
detected (Fig. 3a), and the equilibrium was established within 10 h.
3.2. Generation and equilibrium determination of DCL
Subsequently, the polymer-based DCL was generated by mixing
equal molar amount of six acylhydrazone derivatives and aldehyde
functionality (0.1 eq. of polymer APG) in pH 6.2 PBS buffer using aniline
as acylhydrazone formation catalyst (Scheme 2). As concentration of
constituents in the DCL was considerably low, NMR spectroscopy was no
Scheme 3. Synthesis of multivalent BChE inhibitor APG5b.
5

S. Zhao et al.
Bioorganic Chemistry 108 (2021) 104656
Fig. 4. Inhibition constant of (a) APG5b on BChE; (b) monomer 5b on BChE; (c) Tacrine on BChE; (d) APG5b on AChE; (e) monomer 5b on AChE (●) 0 nM, 33 nM
(■), 100 nM (▴).
3.3. Identification of the amplified side chain
3.5. Measurement of the Michaelis constant and inhibition constant
With the equilibrium property determined, BChE was added to the
equilibrated system. After stirred for 20 h at room temperature, pH of
the mixture was adjusted to 8 by addition of NaOH to freeze further
exchange, and CH3CN was added to denature the protein. Free acylhy-
drazides were separated from the polymer and protein by ultrafiltration.
According to the HPLC analysis (Fig. 3b), concentration of acylhy-
drazide 5b decreased dramatically, while that of acylhydrazide 5f
sharply increased. The concentration of the rest four hydrazides (5a, 5c,
5e, 5d) only decreased slightly compared to acylhydrazide 5b. The
change in free acylhydrazide concentration indicated that acylhydrazide
5b was preferentially functionalized on the polymer at cost of other less
favored constituents through multivalent specific binding to BChE.
Slightly modified Ellman’s method was applied to determine the
Michaelis constant (Km) of BChE and inhibition constant (Ki) of various
substrates. Km of BChE was determined to be 105 μM, and results indi-
cated that the multivalent inhibitor APG5b displayed a mixed inhibition
pattern, with a competitive inhibition constant of 59 nM (Ki) and a
noncompetitive inhibition constant of 82 nM (αKi) (Fig. 4a). As a com-
parison, acylhydrazide 5b exhibited much lower effective inhibition
(Fig. 4b) with Ki = 2.8 M and Ki = 2.1 M. Tacrine, a commercial
BChE inhibitor, was used as reference. Compared to APG5b, Tacrine
showed a very similar inhibition with Ki of 62 nM and Ki of 102 nM,
μ
α
μ
α
respectively (Fig. 4c). A possible explanation for the good inhibition
potency of polymer APG5b was that the polymer and enzyme could
form a cross-linked network instead of a 1:1 binding complex, leading to
the significant increase in binding affinity. In order to test the selectivity
of inhibitor APG5b, we also evaluated its inhibitory effect on AChE.
Results showed that multivalent inhibitor APG5b displayed a Ki of 924
3.4. Synthesis of multivalent BChE inhibitor
After identification of amplified constituent, a multivalent BChE in-
hibitor containing only acylhydrazide 5b functionalized side chain was
synthesized and purified for the following biological evaluation (Scheme
3).
nM and a
hibitor APG5b on BChE over AChE. In addition, Ki of monomer 5b on
AChE was determined to be 5.2 M (Fig. 4e), which was not significantly
different from that of on BChE. However, the inhibition difference of
αKi of 2.2 μM (Fig. 4d), indicating a good selectivity of in-
μ
6

S. Zhao et al.
Bioorganic Chemistry 108 (2021) 104656
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MCF-7 (
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A549 (
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H1229 (
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AC16 (μM)
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´
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5b
69.74 ± 2.18
182.48 ± 6.57
7.92 ± 2.31
73.29 ± 5.72
188.27 ± 5.82
10.17 ± 1.84
99.45 ± 6.42
202.19 ± 6.77
18.63 ± 2.15
78.83 ± 5.37
179.93 ± 4.28
3.56 ± 1.14
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In summary, we have demonstrated the generation of a polymer-
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Declaration of Competing Interest
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Interact. 187 (2010) 23–26.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[25] B. Li, J.A. Stribley, A. Ticu, W. Xie, L.M. Schopfer, P. Hammond, S. Brimijoin, S.
H. Hinrichs, O. Lockridge, Abundant tissue butyrylcholinesterase and its possible
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Acknowledgments
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S. Alpan, H.S. Günes¸, E. Erciyas, 1H-benzimidazole derivatives as
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This work was supported by the Natural Science Foundation of
Jiangsu Province (BK20180853), China Postdoc Science Foundation
(2019M651717) and the Senior Talent Program of Jiangsu University
(17JDG003).
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Appendix A. Supplementary material
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H. Hinrichs, O. Lockridge, Abundant Tissue butyrylcholinesterase and its possible
function in the acetylcholinesterase knockout mouse, J. Neurochem. 75 (3) (2000)
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Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104656.
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