Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Design, synthesis and biological evaluation of novel carbamates as potential

inhibitors of acetylcholinesterase and butyrylcholinesterase
Jie Wua,b, Marco Pistolozzic, Siyu Liud, Wen Tand,e,
⁎

a
School of Pharmacy, Jinan University, Guangzhou 510632, China
b
YZ Health-tech Inc., Hengqin District, Zhuhai 519000, China
c
School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
d
Institute of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China
e
Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway 47500, Selangor Darul Ehsan, Malaysia

ARTICLE INFO ABSTRACT

Keywords: Rivastigmine, a dual inhibitor of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), has been ap-
Carbamates proved by U.S. Food and Drug Administration to treat Alzheimer’s disease (AD) and Parkinson’s disease (PD)
Acetylcholinesterase dementia. In the current work, a bambuterol derivative lacking one of the carbamoyloxy groups on the benzene
Butyrylcholinesterase ring (BMC-1) and its analogues were synthesized using 1-(3-hydroxyphenyl) ethan-1-one and 1-(4-hydro-
Inhibitor
xyphenyl) ethan-1-one as starting materials. In-vitro cholinesterase assay established that nine compounds were
Bambuterol
more potent to inhibit both electric eel AChE and equine serum BChE than rivastigmine under the same ex-
perimental conditions. Further study confirmed that among the nine carbamates, BMC-3 (IC50(AChE) = 792 nM,
IC50(BChE) = 2.2 nM) and BMC-16 (IC50(AChE) = 266 nM, IC50(BChE) = 10.6 nM) were excellent cholinesterase
inhibitors with potential of permeating through the blood-brain barrier. These carbamates could be used as
potential dual inhibitors of AChE and BChE and to discover novel drugs for the treatment of AD and PD de-
mentia.

1. Introduction By catalyzing the hydrolysis of the neurotransmitter acetylcholine

(ACh), acetylcholinesterase (EC 3.1.1.7; AChE) is the main responsible
Alzheimer's disease (AD) is a type of progressive neurodegenerative for the termination cholinergic signals in the nervous system10–12. Bu-
disorder leading to severe behavioral and psychiatric symptoms and tyrylcholinesterase (EC 3.1.1.8; BChE) is another enzyme capable of
more than 80% of dementia cases in elderly people worldwide are AD hydrolyze ACh and supports AChE in the regulation of ACh levels13–17.
patients1–4. At present, about 24 million people are affected by AD and Cholinesterase inhibitors prolong the duration of action of ACh, thereby
it is estimated that this number will quadruple by 2050. In 2015, the improving the clinical symptoms of dementia caused by the degenera-
overall cost for the management of AD was estimated to be 818 billion tion of cholinergic neurons or impairment of the cholinergic function in
dollars, with an increase of 35.3% compared to 2010 and the figure is the brain18. The fact that both AD and PD dementia are associated with
expected to double by 20305. It was also estimated that if a therapy cortical cholinergic deficits stands as the rationale for the use of cho-
could delay the onset of AD for one year by 2020, there would be 9.2 linesterase (ChE) inhibitors as pharmacological symptomatic therapy to
million fewer AD patients in 20506. Parkinson’s disease (PD) is another treat both AD and PD dementia18,19. Among the ChE inhibitors ap-
neurodegenerative disorder characterized by impaired motor skills, proved by U.S. Food and Drug Administration (FDA), rivastigmine is
shaking, slowness of movement and postural instability. PD typically the only ChE inhibitor that is currently licensed for the treatment of
occurs in 1%–2% of people over the age of 60 years, rising to 3.5% at mild, moderate, and severe AD as well as mild-to-moderate PD de-
age 85–89 years7. More than six million people worldwide are affected mentia18,20,21. Rivastigmine is a pseudo-substrate dual inhibitor of
by PD and this disease is now the world’s second most common neu- AChE and BChE while the other two ChE inhibitors used in clinic for the
rodegenerative disorder8. In addition, PD patients are often also af- treatment of AD (i.e. galantamine and donepezil) are selective re-
fected by AD dementia9. versible inhibitors of AChE22. Since only one dual inhibitor of AChE and

⁎
Corresponding author.
E-mail address: went@gdut.edu.cn (W. Tan).

https://doi.org/10.1016/j.bmc.2020.115324
Received 17 November 2019; Received in revised form 7 January 2020; Accepted 10 January 2020
0968-0896/ © 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Jie Wu, et al., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2020.115324
J. Wu, et al. Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx

Fig. 1. Structures of bambuterol, MONO, terbutaline and BMC-1.*: Chiral center.

Scheme 1. Synthesis of Carbamates. Reagents and conditions: (a) K2CO3, K2CO3·3/2H2O, pyridine, ethyl acetate, 70 °C; (b) CuBr2, ethyl acetate:CHCl3 = 1 : 1, 60 °C;
(c)NaBH4, CH2Cl2, methanol; (d) Isopropanol, 80 °C; (e) HCl in ethanol, 0 °C. *: Chiral center.

BChE is currently available for the therapy of AD and PD dementia, it is inhibition of BMC-3 and BMC-16. Finally, lipophilicity (Log P) and
useful to develop novel dual inhibitors in order to increase the ther- polar surface area (PSA) estimations were performed to demonstrate
apeutic options and improve the management of these conditions. the potential ability of the synthesized analogues in crossing the blood-
Bambuterol, a prodrug of the β2-adrenergic receptor agonist ter- brain barrier.
butaline, has been approved to treat asthma in many countries. BChE is
believed to play a major role in the bioconversion (hydrolysis) of both 2. Results and discussion
bambuterol and its monocarbamate metabolite (MONO) to terbutaline
(Fig. 1)23. During the hydrolytic process, bambuterol acts as a potent 2.1. Chemistry
pseudo-substrate inhibitor of BChE with an IC50 of 3 × 10−9 M whereas
it is a much weaker inhibitor of AChE (IC50 = 3 × 10−5 M). MONO is Previous studies showed that bambuterol is hydrolyzed to terbuta-
also a quite specific pseudo-substrate inhibitor for BChE line by ChEs with a two-step process. The hydrolysis mechanism in each
(IC50 = 5 × 10−8 M) with much weaker inhibitory potency towards step consists of a first stage in which one carbamate group of bambu-
AChE (IC50 = 2 × 10−5 M)24. Based on the structures of selective and terol or MONO is transferred to the active site of the enzyme, resulting
non-selective ChE inhibitors, we anticipated that the hydroxyl group on in its temporary inhibition, and a successive stage in which the enzyme
the benzene ring of MONO could have a key role for its selectivity to- activity is restored by water that removes the carbamyl group from the
wards BChE and wondered what would be the potency and selectivity active site25. Based on the selectivity observed for the hydrolysis of
of a derivative lacking that function (BMC-1, Fig. 1). On this basis, bambuterol and MONO by ChEs, we hypothesized that the presence of a
BMC-1 and fifteen carbamate derivatives based on BMC-1 were de- second substitution on the benzene ring in addition to the carbamate
signed, synthesized and evaluated as ChE inhibitors using electric eel (e.g., a second carbamate for bambuterol or an hydroxyl group for
AChE (elAChE) and equine serum BChE (eqBChE). In addition, the IC50 MONO) could have a key role for the selectivity towards BChE and
of the best inhibitors (BMC-3 and BMC-16) was measured using purified wondered what would be the potency and selectivity of BMC-1, which
human AChE (hAChE) and human plasma as the source of BChE lacks this function. We found that BMC-1 maintained inhibitory activity
(hBChE). A complete kinetic study of AChE and BChE inhibition, in- towards ChEs and could be regarded as a leading compound to find
cluding the measurement of the carbamylation and decarbamylation novel dual inhibitors of AChE and BChE. Therefore we investigated
rate constants, was performed to demonstrate the mechanism of modifications of BMC-1 involving the position of the carbamyl ester and

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Table 1
IC50 values of rivastigmine and carbamates for AChE and BChE obtained after 60 min of preincubation.
Compound elAChE IC50 eqBChE IC50 hAChE IC50 hBChE IC50
mean ± SD (μM) mean ± SD (μM) mean ± SD (nM) mean ± SD (nM)

Rivastigmine 22.7 ± 4.8 0.785 ± 0.056

BMC-1 6.08 ± 0.95 0.383 ± 0.016
BMC-2 4.15 ± 0.22 0.180 ± 0.006
BMC-3 1.87 ± 0.25 0.008 ± 0.001 792 ± 96 2.2 ± 0.1
BMC-4 1.39 ± 0.52 0.057 ± 0.017
BMC-5 67.5 ± 8.2 0.282 ± 0.023
BMC-6 45.4 ± 21.8 0.194 ± 0.010
BMC-7 28.9 ± 6.9 0.381 ± 0.026
BMC-8 70.5 ± 4.2 0.154 ± 0.009
BMC-9 3.74 ± 0.60 0.097 ± 0.007
BMC-10 3.15 ± 0.60 0.179 ± 0.011
BMC-11 > 100 2.19 ± 0.23
BMC-12 7.12 ± 1.39 0.303 ± 0.010
BMC-13 15.0 ± 2.7 5.37 ± 0.56
BMC-14 15.9 ± 5.6 1.57 ± 0.11
BMC-15 3.51 ± 1.75 0.245 ± 0.010
BMC-16 0.655 ± 0.207 0.027 ± 0.004 266 ± 8 10.6 ± 0.9

substitutions of the tert-butylamine group taking into account the with IC50 values of 266 ± 8 nM and 792 ± 96 nM for hAChE re-
structure-activity relationships of the bisdimethylcarbamate derivatives spectively.
of metaproterenol and isoproterenol as well as those of bambuterol
analogues26,27. The selection of groups to replace the tert-butylamine 2.3. Kinetic study of the inhibition of hAChE and hBChE by BMC-3 and
was based on our previous study27. The synthesis of BMC-1 and its BMC-16
analogues is systematically represented in Scheme 1. Differently from
all the other analogues, BMC-1 and BMC-2 were tried to form solid salts As showed in Figs. 2A, B, D and E, the inhibition of hAChE and
with hydrochloric acid. But these two compounds failed to form solid hBChE by BMC-3 and BMC-16 was time-dependent (i.e. the extent of
salts with hydrochloric acid, therefore they could not be isolated using the inhibition increased while increasing the pre-incubation time) and
our previously reported method, and were eventually purified by concentration-dependent (i.e. the extent of the inhibition increased
column chromatography. while increasing the inhibitor concentration). The rates of carbamyla-
tion measured at different inhibitor concentrations (kobs) obtained with
2.2. Measurement of IC50 both hAChE and hBChE were linearly correlated to the concentration of
the inhibitors, with the line passing through the origin (Fig. 2C and F),
The synthesized carbamates were ranked for their inhibitory po- indicating that the Kd of the carbamate-ChE reversible complexes were
tency and compared to rivastigmine using elAChE and eqBChE as model above the concentration range used in these experiments29. The second
ChEs. The measured IC50 values are included in Table 1. A comparative order carbamylation rate constant (kI) of BMC-3 and BMC-16 were
analysis of the IC50 values indicated that all the compounds were more (4.26 ± 0.11) × 104 M−1 min−1 and (9.07 ± 0.23) × 104 M−1
potent inhibitors of eqBChE than that of elAChE. The IC50 values of min−1 for hAChE and (1112 ± 29) × 104 M−1 min−1 and
most of the compounds were lower than 100 μM for elAChE, with the (206 ± 6) × 104 M−1 min−1 for hBChE respectively (Table 2). The kI
exception of BMC-11, while the IC50 values of all the compounds for values of rivastigmine measured in the same conditions used in this
eqBChE were between 8 nM and 6 μM. Changing the position of the N, work (i.e. phosphate buffer 100 mM, pH 8, 37 °C) were reported to be
N’-dimethylcarbamoyl group on the aromatic ring from meta to para (4540 ± 140) M−1 min−1 and (33.3 ± 2.2) × 104 M−1 min−1 for
had different effects on the inhibitory potency depending on the type of hAChE and hBChE, respectively30. Based on these values, the kI of BMC-
derivatization of the amino goup on the side chain. Tert-butyl amine 3 and BMC-16 were about 10-fold and 20-fold higher than that of riv-
and tert-amylamine derivatives (BMC-1, BMC-2, BMC-13 and BMC-14) astigmine for hBChE, respectively, while for AChE the kI of the two
showed a decrease of the inhibitory potency towards both elAChE and compounds were 30-fold and 6-fold higher than that of rivastigmine,
eqBChE whereas piperidine and cyclohexylamine modified derivatives respectively. This indicated that, when present at the same concentra-
(BMC-3, BMC-9, BMC-15 and BMC-16) showed the opposite effect. tion, both BMC-3 and BMC-16 have much faster carbamylation rates
Aromatic amine-modified derivatives (BMC-5, BMC-6, BMC-7 and towards human ChEs than rivastigmine, consistently with the pattern of
BMC-8) were less effective in inhibiting elAChE but more potent in- IC50 values. We have reported that the IC50 and kI of bambuterol for
hibitors of eqBChE than rivastigmine. Nine compounds were more po- hBChE, measured in the same conditions used in this work, are
tent inhibitors of both elAChE and eqBChE than rivastigmine while only (4.29 ± 0.41) nM and (77.7 ± 3.5) × 105 M−1 min−1 respectively27.
BMC-11 was less potent than rivastigmine on both elAChE and eqBChE. BMC-16 was found to be a less potent hBChE inihibitor than bambu-
BMC-16 and BMC-3 were the most potent inhibitors of elAChE and terol; nevertheless its IC50 in the low nanomolar range and its kI value
eqBChE, respectively. Since ChEs activity and susceptibility to in- in the same order of magnitude of bambuterol make this compound a
hibitors have been reported to be different between humans and other very potent BChE inhibitor. Most notably, BMC-3 was about 2-fold
organisms28, we used hAChE and human plasma to confirm the capa- more potent than bambuterol in hBChE inibition with a kI value about
city of BMC-3 and BMC-16 to repress the activity of human ChEs 1.4-fold higher than that of bambuterol.
(Table 1). By comparing the IC50 values of these compounds for hAChE
and hBChE, we found that BMC-3 and BMC-16 were potent inhibitors of 2.4. Determination of the decarbamylation rate constants (k3) by the
both human ChEs. Consistently with the pattern observed for elAChE analysis of the area under the inhibition-time curve
and eqBChE, BMC-3 was a more potent inhibitor of hBChE than BMC-16
with IC50 values of 2.2 ± 0.1 nM and 10.6 ± 0.9 nM, respectively, The decarbamylation rate constant (k3) was determined using a
whereas BMC-16 was a more potent inhibitor of hAChE than BMC-3 recently published data manipulation method that allows to calculate

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J. Wu, et al. Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx

Fig. 2. Time- and concentration-dependent inhibition of hAChE and hBChE after pre-incubation with various concentrations of BMC-3 (A, hAChE; D, hBChE) and
BMC-16 (B, hAChE; E, hBChE). Linear correlations of the kobs values obtained for BMC-3 and BMC-16 versus their respective concentrations (C, hAChE; F, hBChE).

k3 from the same data set used to determine kI29. This method is based in Table 2, the k3 values obtained after incubation of BMC-3 and BMC-
on the analysis of the area under the inhibition-time curves (AUIC) from 16 with hAChE were 2-fold higher than those obtained with hBChE. In
which k3 is obtained as the slope of a secondary plot by simple linear particular, the k3 values of hAChE and hBChE conjugates obtained with
regression. BMC-3 were (34.5 ± 2.4) × 10−3 min−1 and (18.9 ± 1.3) × 10−3
In principle, upon reaction with BMC-3 and BMC-16, hAChE and min−1, respectively and those obtained with BMC-16 were
hBChE were expected to form N, N′-dimethylcarbamoyl conjugates, (33.8 ± 2.1) × 10−3 min−1 and (14.8 ± 1.3) × 10−3 min−1, re-
similarly to bambuterol and MONO. The previously reported k3 values spectively, consistently with the previous results. These data confirmed
of the N, N′-dimethylcarbamoyl conjugates of human ChEs obtained that BMC-3 and BMC-16 formed N, N’-dimethylcarbamoyl conjugates
after incubation with bambuterol, MONO and their enantiomers in- with human ChEs.
dicated that the k3 of the hAChE conjugate is roughly double as that of
the hBChE conjugate. In particular, the measured k3 values ranged from 2.5. In-silico chemical analysis of Log P and PSA
(21.7 ± 0.8) to (34 ± 2) × 10−3 min−1 for hAChE and from
(10.2 ± 0.6) to (15.7 ± 0.6) × 10−3 min−1 for hBChE29. As shown The ability of carbamates to penetrate the blood-brain barrier (BBB)
was estimated by calculating their Log P and PSA using computational
tools (Table 3). The acid group was ignored for salts. In order to easily
Table 2
cross the BBB, compounds should have log P = 2–5 and PSA ≤ 70
Summary of the kinetic parameters measured for the inhibition of hAChE and
Å231,32. Ten of the carbamates synthesized in this work had log
hBChE by BMC-3 and BMC-16.
P = 2–5, while all the compounds had PSA values in the range between
Compound and enzyme kI (×104 M−1 min−1) k3 (×10−3 min−1) 42 and 50 Å2. BMC-3 and BMC-16 had the same Log P and PSA values
(2.54 and 49.62 Å2 respectively), indicating that they are likely to cross
BMC-3-hAChE 4.26 ± 0.11 34.5 ± 2.4
BMC-3-hBChE 1112 ± 29 18.9 ± 1.3 the BBB.
BMC-16-hAChE 9.07 ± 0.23 33.8 ± 2.1
BMC-16-hBChE 206 ± 6 14.8 ± 1.3 3. Conclusion
Rivastigmine-hAChE 0.454 ± 0.014a 0.3 ± 0.01a
Rivastigmine-hBChE 33.3 ± 2.2a 2.3a
We designed and synthesized 16 carbamates and determined their
Data are expressed as mean ± S.D. (n = 3). ability to inhibit AChE and BChE. Nine of the carbamates were found to
a
Values obtained from literature30. be more potent elAChE and eqBChE inhibitors than rivastigmine under

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J. Wu, et al. Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx

Table 3 aliquot of 60 mL of water was added to the mixture and stirred for 2 h at
70 °C. The reaction mixture was cooled down to room temperature and
In-silico analysis of the carbamates. subjected to phase separation. The organic phase was washed with
water and diluted sulfuric acid (2% H2SO4), dried over MgSO4 and
filtered. The filtrate was concentrated to give product B, which was
Compound R1 R2 M MolLog P MolPSA
used in the next step without further purification.
BMC-1 OOCN(CH3)2 H 1.49 48.99 Å2 CuBr2 (2 equivalents) were mixed with B (6 g) in ethyl acetate
(50 mL) and chloroform (50 mL) and stirred at 60 °C and monitored by
BMC-2 OOCN(CH3)2 H 2.00 49.46 Å2 TLC. The mixture was strained to remove the solid residues and the
filtrate was washed with water five times, dried over MgSO4 and fil-
BMC-3 OOCN(CH3)2 H 2.54 49.62 Å2
tered again. The filtrate was finally concentrated and crystallized using
ethyl acetate and petroleum ether to give compound C.
BMC-4 OOCN(CH3)2 H 2.18 49.99 Å2
Compound C (4 g) was mixed with 3 equivalents of NaBH4 and the
BMC-5 OOCN(CH3)2 H 2.33 48.57 Å2 mixture was dissolved in 80 mL of dichloromethane at room tempera-
ture. Twenty mL of methanol were added to the suspension under ice
BMC-6 OOCN(CH3)2 H 2.60 48.57 Å2 bath and stirred for 10 min. Then the mixture was stirred at 40 °C and
monitored by TLC. Sixty mL of NH4Cl solution were added to the re-
BMC-7 OOCN(CH3)2 H 3.12 48.57 Å2
action mixture and stirred for 2 h at room temperature to form distinct
aqueous and organic layers. The resulting solution was separated and
BMC-8 OOCN(CH3)2 H 2.88 48.57 Å2 the water phase was further extracted twice with dichloromethane. The
combined organic phase was dried over MgSO4, filtered and con-
BMC-9 OOCN(CH3)2 H 1.94 42.07 Å2 centrated to produce D. The product D was used in the next step
without further purification.
BMC-10 OOCN(CH3)2 H 1.58 42.44 Å2
D (0.5 g) was mixed with amines in 20 mL of isopropanol and was
BMC-11 OOCN(CH3)2 H 0.63 49.98 Å2 stirred at 80 °C and monitored by TLC. After cooling down to room
temperature, the resulting solution was concentrated to generate the
BMC-12 OOCN(CH3)2 H 2.43 48.91 Å2
crude product. The product was dissolved in 30 mL of dichloromethane
BMC-13 H OOCN(CH3)2 1.49 48.99 Å2 and washed three times with water. Then the organic phase was dried
BMC-14 H OOCN(CH3)2 2.00 49.46 Å2
over MgSO4, filtered and concentrated to give the yellow oil E. The
crude product E was purified by flash column chromatography using
BMC-15 H OOCN(CH3)2 1.94 42.07 Å2 ethyl acetate and petroleum ether or dichloromethane and methanol.

BMC-16 H OOCN(CH3)2 2.54 49.62 Å2 4.2.2. Method 2: Preparation of compounds as hydrochloride salts (BMC-1
and BMC-2)
The crude product E was dissolved in ethanol and the pH of the
*: Chiral center.
solution was adjusted to 1 using hydrochloric acid in ethanol. The
mixture was stirred continuously at 0 °C for 4 h. Then the fraction was
the same experimental conditions. Among these nine compounds, BMC-
purified by flash column chromatography using dichloromethane and
3 and BMC-16 displayed excellent inhibition of hAChE and hBChE with
methanol.
potential ability to permeate the BBB. These data showed that BMC-3
and BMC-16 may be useful lead compounds for the discovery of novel
4.2.3. 3-(2-(tert-butylamino)-1-hydroxyethyl)phenyl dimethylcarbamate
AD and PD therapeutical agents.
hydrochloride (BMC-1)
Yield:49%; 1H NMR (400 MHz, DMSO) δ = 7.39 (t, J = 7.9 Hz, 1H,
4. Experimental section Harom), 7.28 (d, J = 7.7 Hz, 1H, Harom), 7.18 (d, J = 1.7 Hz, 1H, Harom),
7.03–7.09 (m, 1H, Harom), 5.00 (d, J = 8.5 Hz, 1H, CH), 2.86–3.08 (m,
4.1. Chemistry 8H, CH2 and CON(CH3)2), 1.31 (s, 9H, C(CH3)3) ppm. 13C NMR
(101 MHz, DMSO) δ = 154.43, 151.82, 143.92, 129.62, 123.24,
1
H NMR spectra were acquired with a Bruker AvanceIII 400 MHz 121.74, 119.87, 68.92, 56.63, 49.04, 36.76, 36.57, 25.57 ppm. TOF MS
spectrometers. 13C NMR was performed with a Bruker AvanceIII ES(+)(m/z): calculated for C15H25N2O3 ([M + H]+) 281.1860; found
101 MHz spectrometers. All NMR analyses were carried out at room 281.1856.
temperature. Chemical shifts were reported as parts per million (ppm)
relative to TMS, used as an internal standard for both 1H NMR and 13C 4.2.4. 3-(1-hydroxy-2-(tert-pentylamino)ethyl)phenyl dimethylcarbamate
NMR. Mass spectra were recorded on a Q-TOF mass spectrometer hydrochloride (BMC-2)
(Bruker) equipped with an ESI source. TLC was performed on silica Yield: 26%; 1H NMR (400 MHz, DMSO) δ = 7.39 (t, J = 7.9 Hz, 1H,
F254 and detection was performed by UV light at 254 nm. Anhydrous Harom), 7.27 (d, J = 7.7 Hz, 1H, Harom), 7.18 (d, J = 1.8 Hz, 1H, Harom),
solvents were used in all experiments, unless otherwise specified. 7.03–7.09 (m, 1H, Harom), 6.25 (s, 1H, OH), 5.00 (d, J = 8.9 Hz, 1H,
CH), 2.84–3.11 (m, 8H, CH2 and CON(CH3)2), 1.67 (q, J = 7.4 Hz, 2H,
4.2. General procedures for the preparation of carbamates CH2CH3), 1.25 (s, 6H, C(CH3)2), 0.88 (t, J = 7.5 Hz, 3H, CH2CH3) ppm;
13
C NMR (101 MHz, DMSO) δ = 154.45, 151.83, 143.94, 129.59,
4.2.1. Method 1: Preparation of compounds without salt formation (BMC-3 123.24, 121.69, 119.86, 68.98, 59.51, 49.04, 36.76, 36.57, 30.42,
to BMC-16) 22.70, 22.61, 8.37 ppm; TOF MS ES(+)(m/z): calculated for
A mixture of compound A (88 mmol), K2CO3·3/2H2O (69 mmol), C16H27N2O3 ([M + H]+) 295.2016; found 295.2011.
K2CO3 (19 mmol) and pyridine (0.5 mL) in ethyl acetate (60 mL) were
stirred and heated to 70 °C. A solution of dimethylcarbamic chloride 4.2.5. 3-(2-(cyclohexylamino)-1-hydroxyethyl)phenyl dimethylcarbamate
(40 mL, 132 mmol) in ethyl acetate was added dropwise to this mixture (BMC-3)
and the suspension was stirred at 70 °C and monitored by TLC. An Yield: 21%; 1H NMR (400 MHz, DMSO) δ = 7.26–7.34 (m, 1H,

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J. Wu, et al. Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx

Harom), 7.18 (d, J = 7.7 Hz, 1H, Harom), 7.08 (s, 1H, Harom), 6.92–7.00 Harom), 5.80 (t, J = 5.4 Hz, 1H, NH), 5.58 (d, J = 4.3 Hz, 1H, OH),
(m, 1H, Harom), 4.60 (dd, J = 8.6, 3.7 Hz, 1H, CH), 2.97 (d, 4.69–4.77 (m, 1H, CH), 4.01 (s, 1H, C^CH), 3.18–3.27 (m, 1H, CH2),
J = 52.9 Hz, 6H, CON(CH3)2), 2.66 (ddd, J = 20.5, 11.8, 6.3 Hz, 2H, 3.07–3.14 (m, 1H, CH2), 2.99 (d, J = 53.5 Hz, 6H, CON(CH3)2) ppm;
13
CH2), 2.42 (tt, J = 10.0, 3.5 Hz, 1H, CHring), 1.53–1.83 (m, 4H, C NMR (101 MHz, DMSO) δ = 154.53, 151.72, 149.16, 146.13,
CH2ring), 0.90–1.27 (m, 6H, CH2ring) ppm; 13C NMR (101 MHz, DMSO) 129.62, 129.22, 123.24, 122.53, 120.92, 119.80, 119.57, 115.43,
δ = 154.54, 151.66, 146.61, 129.14, 123.05, 120.70, 119.66, 71.61, 113.53, 84.96, 79.66, 70.78, 51.37, 36.75, 36.57 ppm; TOF MS ES(+)
56.25, 54.85, 36.74, 36.55, 33.40, 33.18, 26.26, 24.84 ppm; TOF MS ES (m/z): calculated for C19H21N2O3 ([M + H]+) 325.1547; found
(+)(m/z): calculated for C17H27N2O3 ([M + H]+) 307.2016; found 325.1530.
307.2008.
4.2.11. 3-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenyl dimethylcarbamate
4.2.6. 3-(2-(cyclopentylamino)-1-hydroxyethyl)phenyl dimethylcarbamate (BMC-9)
(BMC-4) Yield: 24%; 1H NMR (400 MHz, CDCl3) δ = 7.31 (t, J = 7.8 Hz, 1H,
Yield: 69%; 1H NMR (400 MHz, DMSO) δ = 7.31 (t, J = 7.8 Hz, 1H, Harom), 7.14–7.21 (m, 2H, Harom), 7.01 (dd, J = 8.0, 1.5 Hz, 1H, Harom),
Harom), 7.17 (d, J = 7.7 Hz, 1H, Harom), 7.07 (s, 1H, Harom), 6.96 (dd, 4.74 (dd, J = 10.6, 3.4 Hz, 1H, CH), 3.05 (d, J = 34.4 Hz, 6H, CON
J = 7.9, 1.8 Hz, 1H, Harom), 4.61 (dd, J = 8.6, 3.8 Hz, 1H, CH), (CH3)2), 2.70 (s, 2H, piperidine), 2.53 (dd, J = 12.6, 3.5 Hz, 1H, CH2),
2.89–3.07 (m, 7H, CHring and CON(CH3)2), 2.61 (ddd, J = 20.5, 11.8, 2.42 (dd, J = 12.3, 10.8 Hz, 3H, CH2 and piperidine), 1.62 (tt,
6.3 Hz, 2H, CH2), 1.70 (ddd, J = 19.2, 12.4, 6.1 Hz, 2H, CH2ring), J = 13.9, 7.0 Hz, 4H, piperidine),1.41–1.52 (m, 2H, piperidine) ppm;
13
1.55–1.64 (m, 2H, CH2ring), 1.44 (ddd, J = 13.9, 7.4, 3.4 Hz, 2H, C NMR (101 MHz, CDCl3) δ = 154.92, 151.65, 144.00, 129.08,
CH2ring), 1.22–1.33 (m, 2H, CH2ring) ppm; 13C NMR (101 MHz, DMSO) 122.53, 120.68, 119.12, 68.24, 66.64, 54.44, 36.67, 36.44, 25.93,
δ = 154.55, 151.66, 146.66, 129.14, 123.04, 120.68, 119.65, 71.57, 24.11 ppm; TOF MS ES(+)(m/z): calculated for C16H25N2O3
59.39, 56.61, 36.74, 36.56, 33.01, 32.92, 24.03 ppm; TOF MS ES(+) ([M + H]+) 293.1860; found 293.1848.
(m/z): calculated for C16H25N2O3 ([M + H]+) 293.1860; found
293.1851. 4.2.12. 3-(1-hydroxy-2-(pyrrolidin-1-yl)ethyl)phenyl dimethylcarbamate
(BMC-10)
4.2.7. 3-(1-hydroxy-2-(phenylamino)ethyl)phenyl dimethylcarbamate Yield: 45%; 1H NMR (400 MHz, DMSO) δ = 7.30 (t, J = 7.8 Hz, 1H,
(BMC-5) Harom), 7.18 (d, J = 7.6 Hz, 1H, Harom), 7.07 (s, 1H, Harom), 6.96 (dd,
Yield: 44%; 1H NMR (400 MHz, DMSO) δ = 7.34 (t, J = 7.8 Hz, 1H, J = 7.9, 1.4 Hz, 1H, Harom), 4.65 (dd, J = 7.6, 5.1 Hz, 1H, CH), 2.97 (d,
Harom), 7.24 (d, J = 7.7 Hz, 1H, Harom), 7.15 (s, 1H, Harom), 7.08 (dd, J = 53.2 Hz, 6H, CON(CH3)2), 2.51–2.65 (m, 5H, CH2 and pyrrolidine),
J = 8.2, 7.5 Hz, 2H, Harom), 6.98–7.03 (m, 1H, Harom), 6.63 (d, 1.90 (s, 1H, pyrrolidine), 1.66 (s, 4H, pyrrolidine) ppm; 13C NMR
J = 7.8 Hz, 2H, Harom), 6.54 (t, J = 7.2 Hz, 1H, Harom), 5.54 (s, 2H, NH (101 MHz, DMSO) δ = 154.54, 151.63, 146.76, 129.09, 123.25,
and OH) , 4.69–4.78 (m, 1H, CH), 3.07–3.27 (m, 2H, CH2), 2.98 (d, 120.73, 119.79, 71.20, 64.45, 54.43, 36.74, 36.56, 23.61 ppm; TOF MS
J = 53.5 Hz, 6H, CON(CH3)2) ppm; 13C NMR (101 MHz, DMSO) ES(+)(m/z): calculated for C15H23N2O3 ([M + H]+) 279.1703; found
δ = 154.54, 151.72, 149.07, 146.28, 129.35, 129.22, 123.20, 120.87, 279.1696.
119.77, 116.27, 112.76, 70.78, 51.72, 36.76, 36.58 ppm; TOF MS ES
(+)(m/z): calculated for C17H21N2O3 ([M + H]+) 301.1547; found 4.2.13. 3-(1-hydroxy-2-morpholinoethyl)phenyl dimethylcarbamate
301.1537. (BMC-11)
Yield: 72%; 1H NMR (400 MHz, CDCl3) δ = 7.32 (t, J = 7.8 Hz, 1H,
4.2.8. 3-(2-((4-fluorophenyl)amino)-1-hydroxyethyl)phenyl Harom), 7.15–7.21 (m, 2H, Harom), 7.03(dd, J = 8.0, 1.6 Hz, 1H, Harom),
dimethylcarbamate (BMC-6) 4.77 (dd, J = 10.5, 3.4 Hz, 1H, CH), 3.68–3.83 (m, 4H, morpholine),
Yield: 38%; 1H NMR (400 MHz, DMSO) δ = 7.33 (t, J = 7.8 Hz, 1H, 3.05 (d, J = 34.8 Hz, 6H, (CH3)2NCO), 2.68–2.79 (m, 2H, CH2), 2.53
Harom), 7.23 (d, J = 7.7 Hz, 1H, Harom), 7.14 (d, J = 1.8 Hz, 1H, Harom), (ddd, J = 41.0, 14.8, 8.0 Hz, 4H, morpholine) ppm; 13C NMR
6.97–7.02 (m, 1H, Harom), 6.86–6.94 (m, 2H, Harom), 6.59–6.66 (m, 2H, (101 MHz, CDCl3) δ = 154.87, 151.71, 143.47, 129.17, 122.49,
Harom), 5.54 (s, 1H, NH), 5.49 (s, 1H, OH), 4.72 (s, 1H, CH), 2.86–3.25 120.85, 119.15, 68.19, 66.94, 66.46, 53.45, 36.68, 36.44 ppm; TOF MS
(m, 8H, CH2 and CON(CH3)2) ppm; 13C NMR (101 MHz, DMSO) ES(+)(m/z): calculated for C15H23N2O4 ([M + H]+) 295.1652; found
δ = 155.93, 154.54, 153.63, 151.71, 146.25, 145.83, 129.21, 123.21, 295.1639.
120.89, 119.79, 115.76, 115.54, 113.53, 113.45, 70.80, 52.23, 36.75,
36.57 ppm; TOF MS ES(+)(m/z): calculated for C17H20FN2O3 4.2.14. 3-(1-hydroxy-2-((2-phenylpropan-2-yl)amino)ethyl)phenyl
([M + H]+) 319.1452; found 319.1441. dimethylcarbamate (BMC-12)
Yield: 50%; 1H NMR (400 MHz, DMSO) δ = 7.38–7.44 (m, 2H,
4.2.9. 3-(1-hydroxy-2-((4-iodophenyl)amino)ethyl)phenyl Harom), 7.24–7.32 (m, 3H, Harom), 7.16 (t, J = 7.3 Hz, 1H, Harom), 7.09
dimethylcarbamate (BMC-7) (d, J = 7.7 Hz, 1H, Harom), 6.99 (s, 1H, Harom), 6.94 (dd, J = 7.9,
Yield: 30%; 1H NMR (400 MHz, DMSO) δ = 7.33 (t, J = 6.2 Hz, 3H, 2.2 Hz, 1H, Harom), 5.31 (s, 1H, OH), 4.54 (t, J = 6.0 Hz, 1H, CH), 2.96
Harom), 7.22 (d, J = 7.5 Hz, 1H, Harom) , 7.14 (s, 1H, Harom), 6.99 (d, (d, J = 51.3 Hz, 6H, CON(CH3)2), 2.29–2.39 (m, 2H, CH2), 1.33–1.40
J = 8.0 Hz, 1H, Harom), 6.50 (d, J = 8.4 Hz, 2H, Harom), 5.84 (s, 1H, (m, 6H, C(CH3)2) ppm; 13C NMR (101 MHz, DMSO) δ = 154.53,
NH), 5.57 (s, 1H, OH), 4.71 (s, 1H, CH), 2.88–3.22 (m, 8H, CH2 and 151.58, 148.58, 146.67, 129.05, 128.41, 126.30, 126.19, 123.11,
CON(CH3)2) ppm; 13C NMR (101 MHz, DMSO) δ = 154.41, 151.83, 120.65, 119.66, 72.61, 55.63, 51.59, 36.74, 36.55, 30.05, 29.86 ppm;
148.17, 143.82, 137.44, 129.36, 124.21, 120.91, 120.39, 115.91, TOF MS ES(+)(m/z): calculated for C20H27N2O3 ([M + H]+)
76.86, 66.25, 59.41, 36.74, 36.55 ppm; TOF MS ES(+)(m/z): calculated 343.2016; found 343.2010.
for C17H20IN2O3 ([M + H]+) 427.0513; found 427.0492.
4.2.15. 4-(2-(tert-butylamino)-1-hydroxyethyl)phenyl dimethylcarbamate
4.2.10. 3-(2-((3-ethynylphenyl)amino)-1-hydroxyethyl)phenyl (BMC-13)
dimethylcarbamate (BMC-8) Yield: 25%; 1H NMR (400 MHz, CDCl3) δ = 7.29 (d, J = 8.4 Hz, 2H,
Yield: 37%; 1H NMR (400 MHz, DMSO) δ = 7.34 (t, J = 7.8 Hz, 1H, Harom), 7.07 (d, J = 8.4 Hz, 2H, Harom), 3.89 (dd, J = 8.9, 4.9 Hz, 1H,
Harom), 7.24 (d, J = 7.6 Hz, 1H, Harom), 7.15 (s, 1H, Harom), 7.07 (t, CH), 3.53 (dd, J = 10.6, 4.9 Hz, 1H, CH2), 3.31 (d, J = 9.5 Hz, 1H,
J = 7.8 Hz, 1H, Harom), 7.00 (dd, J = 7.9, 1.3 Hz, 1H, Harom), 6.73 (s, CH2), 3.05 (d, J = 34.3 Hz, 6H, CON(CH3)2), 2.58 (s, 2H, NH and OH),
1H, Harom), 6.69 (d, J = 8.2 Hz, 1H, Harom), 6.64 (d, J = 7.4 Hz, 1H, 1.05 (s, 9H, C(CH3)3) ppm; 13C NMR (101 MHz, CDCl3) δ = 154.85,

6
J. Wu, et al. Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx

150.60, 140.33, 127.64, 121.84, 66.76, 58.27, 51.71, 36.69, 36.42, (0.125 U/mL) or diluted plasma (1 : 150) in phosphate buffer (sodium
30.26 ppm; TOF MS ES(+)(m/z): calculated for C15H25N2O3 phosphate buffer 100 mM, pH 8) were pre-incubated at 37 °C with
([M + H]+) 281.1860; found 281.1846. 25 μL solutions of each inhibitor at different concentrations. At pre-
determined time, the reactions were terminated by adding 25 μL solu-
4.2.16. 4-(1-hydroxy-2-(tert-pentylamino)ethyl)phenyl dimethylcarbamate tion containing the substrate (2.5 mM) and DTNB (3.4 mM) in 100 mM
(BMC-14) sodium phosphate buffer, pH 8. The enzyme activity (vi) was de-
Yield: 62%; 1H NMR (400 MHz, CDCl3) δ = 7.37 (d, J = 8.4 Hz, 2H, termined by measuring absorbance at 412 nm for 5 min at 37 °C. A
Harom), 7.08 (d, J = 8.5 Hz, 2H, Harom), 4.70 (dd, J = 8.8, 3.2 Hz, 1H, sample incubated with blank in place of the inhibitor was analyzed in
CH), 3.37 (s, 2H, NH and OH), 3.05 (d, J = 36.0 Hz, 6H, CON(CH3)2), parallel to measure the maximum enzyme activity (v0). A blank sample
2.88 (dd, J = 11.9, 3.4 Hz, 1H, CH2), 2.58 (dd, J = 11.8, 9.0 Hz, 1H, containing phosphate buffer (pH 8) in place of the enzyme was mea-
CH2), 1.44 (q, J = 7.4 Hz, 2H, CH2CH3), 1.08 (s, 6H, C(CH3)2), 0.86 (t, sured for each inhibitor concentration to account for the spontaneous
J = 7.5 Hz, 3H, CH2CH3) ppm; 13C NMR (101 MHz, CDCl3) δ = 154.97, conversion of the substrate into product and the values obtained were
150.79, 139.43, 126.64, 121.62, 71.32, 53.58, 49.76, 36.70, 36.45, subtracted from each measured activity.
33.19, 26.21, 26.14, 8.24 ppm; TOF MS ES(+)(m/z): calculated for
C16H27N2O3 ([M + H]+) 295.2016; found 295.2002. 4.3.2. Measurement of IC50 with ChEs or human plasma
This approach was based on the general method for measuring
4.2.17. 4-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenyl dimethylcarbamate cholinesterase activity. The residual ChE activity was measured after
(BMC-15) 60 min of pre-incubation of the enzyme with seven different con-
Yield: 75%; 1H NMR (400 MHz, CDCl3) δ = 7.35(d, J = 8.4 Hz, 2H, centrations of the synthesized compounds. Each concentration was
Harom), 7.08 (d, J = 8.5 Hz, 2H, Harom), 4.73 (dd, J = 10.6, 3.4 Hz, 1H, analyzed in triplicate.
CH), 3.05 (d, J = 35.6 Hz, 6H, CON(CH3)2), 2.70 (s, 2H, CH2),
2.34–2.51 (m, 4H, piperidine), 1.61 (dt, J = 17.5, 5.9 Hz, piperidine), 4.3.3. Kinetic analysis of the inhibition of hAChE and hBChE by BMC-3
1.43–1.51 (m, 2H, piperidine) ppm; 13C NMR (101 MHz, CDCl3) and BMC-16: Determination of kI
δ = 154.97, 150.77, 139.20, 129.73, 126.69, 121.56, 121.38, 69.77, The kinetic constants describing the interaction of BMC-3 and BMC-
68.22, 66.87, 59.99, 54.46, 36.69, 36.44, 26.39, 26.03, 24.37, 16 with hAChE and human plasma were determined as previously de-
24.19 ppm; TOF MS ES(+)(m/z): calculated for C16H25N2O3 scribed25,27. The enzyme and plasma were pre-incubated with BMC-3
([M + H]+)293.1860; found 293.1842. and BMC-16 for predetermined time intervals (1 min, 2 min, 4 min,
8 min, 15 min, 30 min, 45 min and 60 min) and ChE activities were
4.2.18. 4-(2-(cyclohexylamino)-1-hydroxyethyl)phenyl measured as described in paragraph 4.3.1. The concentrations of BMC-3
dimethylcarbamate (BMC-16) used were 5 × 10−6 M, 2 × 10−6 M, 1 × 10−6 M and 5 × 10−7 M for
Yield: 41%; 1H NMR (400 MHz, DMSO) δ = 7.33 (d, J = 8.3 Hz, the experiments with hAChE and 2.5 × 10−8 M, 1 × 10−8 M,
2H, Harom), 7.03 (d, J = 8.3 Hz, 2H, Harom), 4.57 (dd, J = 8.2, 3.7 Hz, 5 × 10−9 M and 2.5 × 10−9 M for the experiments with human
1H, CH), 2.96 (d, J = 53.1 Hz, 6H, CON(CH3)2), 2.64 (ddd, J = 20.2, plasma. The concentrations of BMC-16 used were 2.5 × 10−6 M,
11.7, 6.4 Hz, 2H, CH2), 2.38 (dd, J = 11.7, 8.4 Hz, 1H, CHring), 1.79 (t, 1 × 10−6 M, 5 × 10−7 M and 3 × 10−7M for the experiments with
J = 12.6 Hz, 2H, CH2ring), 1.59 (dd, J = 42.3, 10.2 Hz, 3H, CH2ring), hAChE and 1 × 10−7 M, 5 × 10−8 M, 2.5 × 10−8 M and 1.5 × 10−8
1.07–1.27 (m, 3H, CH2ring), 0.99 (dd, J = 22.1, 11.3 Hz, 2H, CH2ring) M for the experiments with human plasma.
ppm; 13C NMR (101 MHz, DMSO) δ = 154.60, 150.54, 141.86, 127.10, The values of kI for BMC-3 and BMC-16 towards hAChE and hBChE
121.77, 71.71, 56.28, 55.13, 36.74, 36.55, 33.55, 33.39, 26.31, were calculated using the following equations25,27.
24.87 ppm; TOF MS ES(+)(m/z): calculated for C17H27N2O3 vi
([M + H]+) 307.2016; found 307.2001. Rt =
v0 (1)

4.3. Measurement of ChE activity Rt = R 0 ·exp ( kobs a· t ) + R (2)

kobs = kI ·[I ] (3)

A modified Ellman’s assay was performed to determine the ChE
inhibition ability of the compounds25,27,33. All the experiments were where vi is the enzyme activity measured after pre-incubation with
conducted in 96-well plates and the readings were obtained using a inhibitors for a determined time period, while v0 is the enzyme activity
multimode microplate reader (Berthold Technologies) or an EnSpire measured after pre-incubation with blank for the same given time of
Multimode 2300 plate reader (Perkin Elmer, MA). elAChE (from elec- pre-incubation and represents the maximum enzyme activity. Rt, R0 and
tric eel), eqBChE (from equine serum), hAChE (from human ery- R∞ are the residual fractional activities at time t, 0 and infinite, re-
throcytes), S-acetylthiocholine iodide (ATCh), S-butyrylthiocholine io- spectively. The pseudo-first order rate constants (kobs) were obtained by
dide (BTCh) and 5-5′-dithiobis (2-nitrobenzoic) acid (DTNB) were fitting the residual activity measured after different pre-incubation
purchased from Sigma-Aldrich (Shanghai, PRC). Blood from apparently times to a one-phase exponential decay equation (Eq. (2)). The values of
healthy individuals (males) was collected and prepared as previously kI were obtained as the slope of the linear correlation of kobs versus
described26. Briefly, blood was collected in a heparinized tube and concentration, according to Eq. (3).
centrifuged at 1000g for 20 min at 25 °C to separate plasma from the
erythrocytes. Plasma served as the source of hBChE and was stored at 4.3.4. Determination of the decarbamylation rate constants by the analysis
–80 °C until analysis. The experiment was performed in accordance to of the area under the inhibition-time curve
the declaration of Helsinki. Stock solutions of substrates (ATCh or The data were analyzed according to the following equation29.
BTCh) were made fresh in water. DTNB (Ellman’s reagent) stock solu-
AUCcE [t 1, t 2] 1 cE (t 2) cE (t 1) k
tion was freshly prepared in sodium phosphate buffer (100 mM, pH 7, = · + obs
AUCE [t1, t 2] k3 AUCE [t1, t 2] k3 (4)
0.15% NaHCO3 w/v). Stock solutions of the test compounds were
prepared in a mixture of acetonitrile and water (v : v = 1 : 9). All the According to Michaelis-Menten equation, the activity measured in
measurements were performed at pH 8. saturating conditions is proportional to the concentration of active
enzyme, therefore the fractional concentration of active enzyme (E)
4.3.1. Modified ellman's assay/ChE assay could be considered equal to the measured residual activity (Rt) and the
200 μL of purified elAChE (0.04 U/mL), eqBChE (0.1 U/mL), hAChE concentration of carbamylated enzyme (cE) could be calculated as

7
J. Wu, et al. Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx

cE = 1 − E. The plots of E and cE versus time (Fig. 2A, B, D and E) were bromophenol derivatives with cyclopropyl moiety: Ring opening of cyclopropane
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