Novel potent bifunctional carboxylesterase inhibitors based on a polyfluoroalkyl-2-imino-1,3-dione scaffold

Article abstract of DOI:10.1016/j.ejmech.2021.113385

An expanded series of alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates (HOPs) 3 was obtained via Cu(OAc)₂-catalyzed azo coupling. All were nanomolar inhibitors of carboxylesterase (CES), while moderate or weak inhibitors of acetylcholinesterase and butyrylcholinesterase. Steady-state kinetics studies showed that HOPs 3 are mixed type inhibitors of the three esterases. Molecular docking studies demonstrated that two functional groups in the structure of HOPs, trifluoromethyl ketone (TFK) and ester groups, bind to the CES active site suggesting subsequent reactions: formation of a tetrahedral adduct, and a slow hydrolysis reaction. The results of molecular modeling allowed us to explain some structure-activity relationships of CES inhibition by HOPs 3: their selectivity toward CES in comparison with cholinesterases and the high selectivity of pentafluoroethyl-substituted HOP 3p to hCES1 compared to hCES2. All compounds were predicted to have good intestinal absorption and blood-brain barrier permeability, low cardiac toxicity, good lipophilicity and aqueous solubility, and reasonable overall drug-likeness. HOPs with a TFK group and electron-donor substituents in the arylhydrazone moiety were potent antioxidants. All compounds possessed low cytotoxicity and low acute toxicity. Overall, a new promising type of bifunctional CES inhibitors has been found that are able to interact with the active site of the enzyme with the participation of two functional groups. The results indicate that HOPs have the potential to be good candidates as human CES inhibitors for biomedicinal applications.

Full text of DOI:10.1016/j.ejmech.2021.113385

European Journal of Medicinal Chemistry 218 (2021) 113385
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Novel potent bifunctional carboxylesterase inhibitors based on a
polyfluoroalkyl-2-imino-1,3-dione scaffold
,
Galina F. Makhaeva a, Sofya V. Lushchekina a b, Natalia P. Boltneva a,
Olga G. Serebryakova a, Nadezhda V. Kovaleva a, Elena V. Rudakova a, Natalia A. Elkina c,
Evgeny V. Shchegolkov c, Yanina V. Burgart c, Tatyana S. Stupina d, Alexey A. Terentiev d,
,
,
Eugene V. Radchenko a e, Vladimir A. Palyulin a e, Victor I. Saloutin c, Sergey O. Bachurin a,
, g, h, *
Rudy J. Richardson f
a Institute of Physiologically Active Compounds Russian Academy of Sciences, Chernogolovka, 142432, Russia
b Emanuel Institute of Biochemical Physics Russian Academy of Sciences, Moscow, 119334, Russia
c Postovsky Institute of Organic Synthesis, Urals Branch of Russian Academy of Sciences, Ekaterinburg, 620990, Russia
d Institute of Problems of Chemical Physics Russian Academy of Sciences, Chernogolovka, 142432, Russia
e Department of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia
f Department of Environmental Health Sciences, University of Michigan, Ann Arbor, MI, 48109, USA
g Department of Neurology, University of Michigan, Ann Arbor, MI, 48109, USA
h Center of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An expanded series of alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates (HOPs) 3 was
obtained via Cu(OAc)2-catalyzed azo coupling. All were nanomolar inhibitors of carboxylesterase (CES),
while moderate or weak inhibitors of acetylcholinesterase and butyrylcholinesterase. Steady-state ki-
netics studies showed that HOPs 3 are mixed type inhibitors of the three esterases. Molecular docking
studies demonstrated that two functional groups in the structure of HOPs, trifluoromethyl ketone (TFK)
and ester groups, bind to the CES active site suggesting subsequent reactions: formation of a tetrahedral
adduct, and a slow hydrolysis reaction. The results of molecular modeling allowed us to explain some
structure-activity relationships of CES inhibition by HOPs 3: their selectivity toward CES in comparison
with cholinesterases and the high selectivity of pentafluoroethyl-substituted HOP 3p to hCES1 compared
to hCES2. All compounds were predicted to have good intestinal absorption and blood-brain barrier
permeability, low cardiac toxicity, good lipophilicity and aqueous solubility, and reasonable overall drug-
likeness. HOPs with a TFK group and electron-donor substituents in the arylhydrazone moiety were
potent antioxidants. All compounds possessed low cytotoxicity and low acute toxicity. Overall, a new
promising type of bifunctional CES inhibitors has been found that are able to interact with the active site
of the enzyme with the participation of two functional groups. The results indicate that HOPs have the
potential to be good candidates as human CES inhibitors for biomedicinal applications.
Received 28 December 2020
Received in revised form
6 March 2021
Accepted 8 March 2021
Available online 15 March 2021
Keywords:
ADMET
Alkyl 2-arylhydrazinylidene-3-oxo-3-
polyfluoroalkylpropionates
Antioxidants
Carboxylesterase
Cholinesterases
Molecular docking
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
hydrolase protein superfamily. CESs, along with CYP450s, are
categorized as Phase I enzymes that metabolize drugs and envi-
In humans and other mammals, carboxylesterases (CESs, EC
3.1.1.1) are important serine esterases that belong to the a/b-
ronmental toxicants [1e3]. They are localized in the lumen of the
endoplasmic reticulum (ER), and they are responsible for the hy-
drolytic biotransformation of a wide range of compounds bearing
ester, thioester, and amide bonds [4e7]. CESs are important cata-
lysts for the bioactivation of numerous prodrugs and bio-
inactivation of antedrugs [8,9]. They can also hydrolyze
endogenous substrates and play important physiological roles in
lipid metabolism and energy homeostasis [10e12].
* Corresponding author. Molecular Simulations Laboratory, Room M6065 SPH-II
2029, University of Michigan, 1415 Washington Heights, Ann Arbor, MI, 48109-
2029, USA.
E-mail address: rjrich@umich.edu (R.J. Richardson).
https://doi.org/10.1016/j.ejmech.2021.113385
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Human CESs (hCESs) can be classified into five major families
(CES1-CES5) according to the homology of their amino acid se-
quences [6]. The major carboxylesterase isozymes involved in hu-
man drug metabolism are CES1 (hCES1, hCES1A, hCE1) and CES2
(hCES2, hCES2A, hiCE2) [1,4,6,13]. These two enzymes share 47%
protein sequence identity, but they exhibit differential tissue dis-
tribution as well as distinct substrate and inhibitor specificities
[5e7].
Currently, the increased interest in CESs is largely caused by two
trends. The first is the extensive use of the ester-containing prodrug
concept for improving the absorption and bioavailability of phar-
macologically active molecules [8,9,14e16]. The second trend is the
ever-increasing amount of information about genetic poly-
morphisms, various diseases, drug interactions, and xenobiotics
that can change CES activity, thus modifying the pharmacokinetics
and efficacy of drugs that are CES substrates [8,17e25]. Moderate to
high inter-individual variability of hCES1 and hCES2 has been re-
ported from 5- to 160-fold for hCES1, and 4- to 34-fold for hCES2
based on protein abundance, 430-fold based on mRNA level, and
127-fold based on hydrolytic activity [9]. Additional interest in CESs
is connected with recently increasing data on their participation in
the metabolism of cholesteryl esters, triacylglycerols, and other
endogenous lipids; thus, CESs can serve as therapeutic targets for
been found among carbamates. They include O-carbamoylated 1-
hexafluoroisopropanols, discovered by our group [42], and carba-
mates WWL113 and WWL229, selectively inhibiting mouse Ces3,
which is a human CES1 ortholog, found by Cravatt’s group [43].
Compounds with a 1,2-dione scaffold (Fig. 1) have been identi-
fied as the most important structures for CES inhibition. These
compounds have Ki values in the nanomolar range and exert potent
and selective inhibitory effects toward hCES1 or hCES2 depending
on the orientation of the dione oxygens [35,39,44,45]. While the
1,2-dione moiety is very beneficial for CES inhibition [39,40], such
compounds do not exhibit untoward inhibitory effects on human
acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase
(BChE, EC 3.1.1.8) [2,33].
TFK (Fig. 1) are some of the most potent CES inhibitors identified
to date with Ki values in the low nanomolar range, and these
compounds mostly demonstrated poor specificity toward hCES1 or
hCES2 [46]. TFK chemotype is very efficient at inhibiting many
enzymes whose catalytic mechanism involves attack by a nucleo-
philic catalytic residue e.g., serine. The extreme inhibitor potency of
this type of compounds is attributed to the polarization of a
carbonyl by the trifluoromethyl group, which greatly increases the
electrophilicity of the carbonyl carbon and hence its susceptibility
to nucleophilic attack [41,47]. The recent QM/MM study demon-
strated that such reaction with AChE occurs almost without energy
barrier [48]. The mechanism of inhibition of esterases by TFK
compounds is associated with formation of so-called transition
state analogs [35,49], as was shown in an X-ray study of AChE in-
hibition by TFK [50], although this type of adduct is more correctly
termed as a “tetrahedral intermediate analog” [51,52].
Recently, our team developed a new polyfluoroalkyl-2-imino-
1,3-dione scaffold that combines in one molecule two chemotypes
e TFK and 1,2-dione e specific for effective CES inhibition [53e56].
Its distinguishing feature is the replacement of one carbonyl func-
tion in the 1,2-dione fragment by a bioisosteric imino group, which
can be chemically modified. We first discovered that alkyl 2-
arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates (Fig. 2,
HOP) showed the ability to inhibit porcine liver CES in the nano-
molar range, with significantly lower activity against two related
enzymes, AChE and BChE [53]. Effective and highly selective CES
inhibitors have also been found among HOP analogs bearing nat-
ural alcohol moieties (HOPN) [56] or an acid residue (HOA) [57] as
well as their cyclic derivatives, 7-hydroxy-7-polyfluoroalkyl-4,7-
dihydroazolo[5,1-c][1,2,4]triazines [58] (Fig. 2). An additional
the treatment of
a variety of human metabolic disorders
[10,11,26e28].
The importance of CES in both human health and drug meta-
bolism has aroused great interest in the discovery of potent CES
modulators, e.g., potent and selective inhibitors of hCES1 and
hCES2. Inhibitors of CESs could be used as co-drugs to improve
pharmacokinetics, efficacy, and safety profiles of clinically
approved drugs for which CESs are involved in their metabolism
and clearance [2,29e32]. Because of the critical role of hCES1 in
metabolizing cholesteryl esters, inhibitors of hCES1 have the po-
tential to treat hypertriglyceridemia, obesity, type 2 diabetes, and
atherosclerosis [33].
The main classes of compounds among which effective and
selective CES inhibitors have been found include bis-arylsulfona-
mides [34], 1,2-diones including benzil and its analogs [35], alkyl-
1,2-diones [36], isatins [37], and 1,2-quinones [35], as well as nat-
ural compounds such as tanshinones [38,39], and
b-lapachones
[40] containing a 1,2-dione moiety (Fig. 1). Trifluoromethyl ketone
(TFK) derivatives [41] are also highly active reversible inhibitors of
CES. Selective CES covalent inhibitors with low acute toxicity have
Fig. 1. CES inhibitors having 1,2-dione and TFK scaffolds.
2

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Fig. 2. New CES inhibitors based on the 2-imino-3-polyfluoroalkyl-1,3-dione scaffold.
property of all compounds with the 2-imino-1,3-dione scaffold is
their high radical-scavenging activity [56e58], which we regard as
a positive attribute for the potential medicinal use of these
molecules.
methyl ketones under lithium hydride action to yield 1,3-diketone
reagents [63] (see Scheme 1B).
The use of these techniques was more efficient than previous
ones [64e66], because the yields of targeted arylhydrazones 3 and
5 reached 87%. Moreover, the synthesized compounds did not need
additional purification owing to the absence of side reactions under
the conditions of synthesis.
QM calculations demonstrated that for HOPs 3, the Z-isomer is
more stable than the E-form, in which the polyfluoroacyl oxygen
atom is involved in an intramolecular hydrogen bond (Fig. 3,
Table 1). For example, the energy difference was 3.5 kcal/mol for
compound 3k (Fig. 3). For the Z-isomer, there is also the possibility
of formation of a hydrogen bond between the NH-group of the
arylhydrazone fragment and the oxygen atom of the ester alkoxy
group, but such a conformation was less favorable (5.5 kcal/mol
difference for compound 3k) when the compounds were not bound
to the enzyme active site.
These QM calculation results were in good agreement with our
previous experimental findings [67]. According to 1H, 19F and 13C
NMR spectroscopy and X-ray diffraction analysis, both in the solid
state and in (CD3)2CO or CDCl3 solution, HOPs 3 exist as Z-isomers.
Similar results were obtained in our previous study of HOPsN
bearing higher or natural alcohol moieties [56] (Scheme 2).
We also defined the conformational form of 2-
arylhydrazinylidene-3-oxo esters 3 in aqueous DMSO. According
to 1H and 19F NMR spectroscopy, all esters 3a-s containing a poly-
fluoroalkyl substituent exist only as the Z-isomer in DMSO‑d6. In
contrast to compounds 3a-s, the non-fluorinated analog 3t is a
mixture of Z- and E-isomers in these solutions (Scheme 2, see
Experimental).
The aim of the present study was to elucidate the unique fea-
tures of compounds containing the polyfluoroalkyl-2-imino-1,3-
dione scaffold that give rise to their high activity and selectivity
toward CES compared to cholinesterases. We accomplished this
goal by first synthesizing an expanded series of the most active
HOPs using a more productive synthetic protocol than we had used
previously. Next, we performed a biological evaluation of the
compounds as inhibitors of porcine liver CES along with human
erythrocyte AChE and equine serum BChE accompanied by mo-
lecular docking. Some of the compounds were also studied as in-
hibitors of human CES1 and CES2. Additionally, to assess potential
pharmacokinetic properties of HOPs, we determined their ADMET
profiles computationally and assessed their antioxidant activity by
the ABTS assay as an indicator of their potential hepatoprotective
effect. Finally, we determined the cytotoxicity of the compounds
in vitro with the MTT assay in human FetMSC cells and assessed
their acute toxicity in vivo in mice.
2. Results and discussion
2.1. Chemistry
To synthesize 2-arylhydrazinylidene-3-oxo esters, the
commonly used procedure is azo coupling of 3-oxo esters 1 with
aryldiazonium salts 2, which are obtained from reacting the aryl-
amine with sodium nitrite in aqueous HCl [59]. The usual medium
for azo coupling is a water-miscible organic solvent and water with
sodium acetate or sodium hydroxide for enolate generation [60].
However, the reaction under these conditions can be complicated
by Japp-Klingemann cleavage or formazan formation. Recently, it
has been proposed to use mechanically activated solid-state syn-
thesis of arylhydrazone derivatives via a high-speed ball mill in the
absence of solvent [61]. It has also been reported that Sc(OTf)3
could be used to catalyze transfer diazenylation of 1,3-dicarbonyls
with aryltriazenes through NeN bond cleavage [62]. To increase
the yield of target arylhydrazones, we have carried out azo coupling
in a water-chlorohydrocarbon medium in the presence of tetraal-
kylammonium halide as a phase-transfer catalyst [53].
To synthesize alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluor
oalkylpropionates 3 (HOPs), we decided to apply Cu(OAc)2-cata-
lyzed azo coupling of 3-oxo esters 1a-h with various aryldiazonium
salts 2a-i in a mixture of acetone-water. Using this protocol, we ob-
tainedalarge numberofesters3a-swithhighyields(Scheme 1A). The
same method was used for synthesis of non-fluorinated analog 3t.
Copper (II) acetate was not only a catalyst, but also it served as a re-
agent to bind 3-oxo esters 1 into copper complexes, which were less
prone to undergo cleavage. To obtain trifluoromethyl-containing 2-
tolylhydrazinylidene-1,3-diketones 5a,b, azo coupling of tolyldiazo-
nium salt 2bwas carried out with lithium 1,3-diketonates 4a,b, which
were the intermediates in a Claisen condensation of esters with
2.2. Inhibition studies of CES, AChE and BChE: structure-activity
relationships
The inhibitory potency of our synthesized compounds against
CES and structurally related cholinesterases (i.e., their esterase
profile) was determined using commercially available enzymes:
porcine liver CES, human erythrocyte AChE, and equine serum
BChE (Sigma, USA). AChE from human erythrocytes was used along
with two enzymes of non-human origin, because of their relatively
low cost and the exploratory character of this work. High protein
sequence identities between human and equine BChE (90%) and
human CES1 and porcine liver CES (77%) [68] along with our pre-
vious results [68e70] supported the applicability of this set of en-
zymes for determining esterase profiles of new compounds.
Data characterizing the esterase profile of compounds 3 and 5
are shown in Table 2. All compounds were moderate or weak in-
hibitors of cholinesterases. At the same time, all HOPs 3a-m con-
taining the TFK fragment were highly effective inhibitors of CES
with IC50 values in the nanomolar range. Moreover, the variation of
substituent in the arylhydrazone moiety did not substantially affect
the inhibitory activity of these compounds, with the exception of
compounds 3h,i, where a decrease in activity may be associated
with steric hindrance.
3

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Scheme 1. Synthesis of 2-arylhydrazinylidene-3-oxo esters 3 (A) and 2-arylhydrazinylidene-1,3-diketones 5 (B).
HOPs 3a-m contain two functional groups with a polarized
carbonyl in their structure: a TFK moiety and an ester moiety; both
of them could react with the active site serine and thus affect the
inhibitory potency against serine esterases.
complete disappearance of AChE and BChE inhibition. Upon
replacing the CF3 group (3b) by CH3 (3t), inhibitory activity against
CES also dramatically decreased (from IC50 ¼ 7.41 nM for 3b to
12.1% inhibition at 20
mM for 3t). Thus, the results indicate the
To clarify the role of the TFK group, the inhibitory activity of oxo
esters 3 in which the CF3 group was replaced by CHF2 (3n,o) and
CH3 (3t) was studied. It turned out that replacing CF3 (3b,k) with
CHF2 (3n,o) led correspondingly to about a 400-fold (3b vs 3n)
to100-fold (3k vs 3o) decrease in anti-CES activity, with almost
importance of the TFK moiety, CF3C(O), for potent action of HOPs 3
against the studied esterases, especially CES. The weakening of the
inhibitory activity of esters having CHF2 (3n,o) and CH3 (3t), could
be due to a decrease in
dþ on the carbon atom of the acyl moiety,
which is involved in binding to the active site of CES. Another
4

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Scheme 2. The geometrical isomerism of alkyl 2-arylhydrazinylidene-3-oxo esters 3.
increasing polyfluoroalkyl chain length appeared to be caused by
both electronic and steric factors.
Interestingly, most of the HOP Me esters were 4e9 times less
effective against CES than the corresponding Et esters, except Me
and Et esters 3e,m having a 4-NO2 group in the arylhydrazone
moiety that displayed the same high anti-CES activity (Table 2).
To elucidate the role of the ester moiety in the anti-CES effi-
ciency of HOPs 3, we replaced the ester group with two different
keto groups e MeC(O) and PhC(O) e 1,3-diketones 5a and 5b. The
results provided evidence for the large contribution of the ester
moiety to anti-CES activity: substitution with PhC(O) (5b) reduced
anti-CES activity about 700 times compared to the Et ester 3b and
about 200 times compared to Me ester 3k. Moreover, substitution
with MeC(O) (5a) reduced inhibitor potency by another order of
magnitude (Table 2). Diketones 5a and 5b were also very weak
inhibitors of cholinesterases.
Thus, in order to exhibit maximum anti-CES activity, a molecule
based on the polyfluoroalkyl-2-imino-1,3-dione scaffold should
contain a combination of TFK and an ester group, with the Et esters
preferable.
2.3. Kinetics studies of porcine CES, human AChE, and equine BChE
inhibition
Fig. 3. Energy diagram of the E-isomer and two conformers of the Z-isomer for
compound 3k. Two possible conformations of the Z-isomer have intramolecular
hydrogen bonds with the carbonyl oxygen (Z1) and the oxygen atom of the ester
alkoxy group (Z2).
The compound 3k was selected for a steady-state kinetics study
of inhibition toward porcine CES, human AChE, and equine BChE.
Graphical analysis of steady-state inhibition kinetics data for oxo
ester 3k against the three esterases is shown in Fig. 4. Binding of 3k
to all three enzymes changed both Vmax and Km values. Such alter-
ations are consistent with mixed-type inhibition of all three ester-
Table 1
Energy differences of the two conformers of the Z-isomers relative to E-isomer
energy level for HOPs 3.
Compound
DE, kcal/mol
ases, with Ki ¼ 14.0
Ki ¼ 82.2 5.7 nM (noncompetitive component) for CES. The cor-
responding constants for AChE were Ki ¼ 10100 800 nM and Ki ¼
139000 11000 nM, and for BChE were Ki ¼ 29200 2300 nM and
Ki ¼ 72700 5100 nM.
1.1 nM (competitive component) and
a
Z1-E
Z2-E
a
3a
3b
3c
3d
3e
3f
3g
3h
3i
ꢀ3.7
ꢀ3.6
ꢀ3.6
ꢀ3.5
ꢀ3.7
ꢀ3.6
ꢀ3.7
ꢀ2.5
ꢀ3.8
ꢀ3.8
ꢀ3.5
ꢀ3.7
ꢀ3.9
ꢀ3.1
ꢀ1.5
ꢀ3.0
ꢀ2.3
ꢀ2.4
ꢀ0.1
ꢀ0.8
2.1
2.1
2.1
2.3
ꢀ1.1
2.2
2.1
3.3
2.0
1.7
2.0
1.9
ꢀ1.3
2.5
4.1
2.7
3.0
3.2
5.4
5.0
a
2.4. Inhibition study of human CES1 and CES2. Structure-activity
relationships
All studied HOPs 3 showed high activity and selectivity against
porcine CES compared to cholinesterases. Among these, potent and
selective compounds were studied as inhibitors of the main human
isoenzymes involved in biotransformation of ester-containing
drugs, human CES1 and CES2. Data on the inhibitory activity of
these compounds toward hCES1 and hCES2 are presented in
Table 3.
As can be seen from Table 3, most studied CES inhibitors
showed higher selectivity for hCES1. Analysis of data on inhibition
of human CES isoenzymes revealed that electron donor sub-
stituents in the arylhydrazone moiety, such as Me, OMe (Et esters
3b, 3c, 3d and Me esters 3k, 3l) improved CES1 inhibitory activity
and selectivity, while compounds with electron-withdrawing
substituents NO2, F (Et esters 3e, 3f and Me ester 3m) were
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
decrease in HOP anti-CES activity, although less appreciable,
occurred with increasing the length of the CF3 group to C2F5 (3p),
C3F7 (3q,r) and C4F9 (3s). This decrease of inhibitory activity with
more active against CES2. Oxo esters 3a,j having
a
5

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Table 2
Esterase profiles and ABTS·þ-scavenging activity of HOPs 3 and diketones 5.
No.
Compound
Inhibitory activity IC50 SEM (nM) or inhibition % at 20 mМ
.
ABTS$þ-scavenging
R
R1
R2
Porcine CES
Human AChE
Equine BChE
TEACa
IC50, mM
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF2H
CF2H
C2F5
C3F7
C3F7
C4F9
Me
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OMe
OMe
OMe
OMe
OEt
OMe
OEt
OEt
OMe
OEt
OEt
Me
H
6.14 0.55
7.41 0.58
7.27 0.58
13.1 1.0
4.95 0.39
7.16 0.57
5.14 0.41
41.6 3.3
47.1 3.2
55.5 4.4
27.8 2.5
46.7 3.2
7.65 0.68
3230 280
1570 120
164 11
3220 280
7320 580
30400 2100
4620 410
759 61
2670 210
1360 110
481 38
3790 260
20100 1600
28300 2200
30300 2400
20900 1800
18.2 1.4%
5.8 1.1%
11.5 1.9%
23.6 2.1%
7.1 1.3%
17.2 1.5%
6.8 1.2%
11.0 1.9%
9.1 1.7%
n.a.
8420 720
8340 660
38100 2900
24.3 1.9%
8060 640
11300 900
2020 140
0.80 0.04
0.95 0.05
0.85 0.04
1.0 0.04
0.04 0.01
0.60 0.04
0.53 0.03
0.04 0.01
0.1 0.01
0.78 0.04
0.98 0.03
0.97 0.05
0.03 0.01
0.37 0.02
0.38 0.02
0.36 0.02
0.30 0.02
0.40 0.03
0.26 0.02
0.38 0.03
1.33 0.2
1.7 0.18
n.d.
26.7 1.2
21.4 0.9
25.4 1.1
19.3 0.5
n.d.
35.3 1.4
36.3 1.5
n.d.
193 2.6
26.6 1.3
21.2 0.9
22.5 1.1
n.d.
53.3 2.1
52.6 2.5
55.2 2.1
62.3 1.8
50.3 2.1
66.6 2.5
52.4 1.8
15.6 0.9
12.8 0.5
n.d.
4-Me
3-Me
4-OMe
2-NO2
4-F
4-Br
2-CO2Et
4-CO2Et
H
122
7
19600 1600
14500 1100
33000 2300
49600 3900
45100 4100
12.4 1.9%
2.3 0.5%
31100 2100
42100 3700
21.2 1.9%
4.0 1.1%
n.a.
13.4 2.3%
19.4 1.6%
n.a.
n.d.
n.d.
4-Me
4-OMe
2-NO2
4-Me
4-Me
4-Me
4-Me
4-OMe
4-Me
4-Me
4-Me
4-Me
3q
3r
3s
3t
5a
5b
102
103
8
9
89.3 4.2
12.1 1.8%
56600 5100
5540 440
1800 110
n.d.
CF3
CF3
Ph
BNPP
Trolox
Ascorbic acid
Catechol
n.d.
n.d.
n.d.
1.0
0.98 0.03
1.2 0.04
20.4 0.6
21.6 1.2
16.4 0.9
n.d.
n.d.
n.d.
Data are expressed as mean SEM, n ¼ 3.
Esterase profiles: n.a. ¼ not active at 20 M.
Values expressed as % correspond to % inhibition at 20
m
m
M.
Values without units of measurement correspond to IC50 values in nM.
n.d. ¼ not determined.
a
TEAC (Trolox equivalent antioxidant capacity) was determined from the ratio of the slopes of the concentration-response curves test compound/Trolox.
phenylhydrazone substituent (R2 ¼ H) displayed the same degree
of inhibition of human CES1 and CES2.
toward the catalytic histidine or away from it (Fig. 5A and B). In
both cases, the position of the TFK-group in the active site was such
that the carbonyl oxygen atom was in the oxyanion hole and the
carbonyl carbon atom was facing the catalytic serine. Such a posi-
tion would favor a subsequent covalent interaction with the for-
mation of a hemiketal. Moreover, the position of the TFK group
shown in Fig. 5B also had an additional hydrogen bond between the
NH-group of Gly142 in the oxyanion hole and the oxygen atom of
the ester alkoxy group.
When the ester group was in the active site, the poses again
differed by a 180ꢁ flip of the TFK-arylhydrazone fragment, while the
position of the ester group itself remained the same (Fig. 5C and D).
Both configurations suggest the possibility of subsequent hydro-
lysis of this group, followed by reinhibition of the enzyme by the
product [57].
For compounds with longer fluorinated substituents, the
replacement of CF3 with C2F5 (3p) and C3F7 (3r) led to decreasing
inhibitory activity against both human CES isoenzymes (compare
IC50 3p and 3b; 3r and 3l), but at the same time, selectivity toward
hCES1 was maintained at a similar level as that of the CF3 de-
rivatives. It may reasonably be assumed that hCES1 selectivity is
determined by the presence of the electron-donating Me or MeO
substituent in the arylhydrazone moiety.
2.5. Molecular modeling
To gain insight into the structural aspects for the high efficiency
and selectivity of HOPs toward CES, we performed molecular
docking studies of HOPs interactions with CES, AChE, and BChE. X-
ray crystal structures of the human enzymes were used.
Molecular docking of the Z-isomers of HOPs 3a-m into the CES
active site revealed two major clusters of binding poses suggesting
further reaction: one cluster with the TFK group in the active site
(Fig. 5A and B) and the other with the ester group in the active site
(Fig. 5C and D). For each of the two clusters, there were two possible
orientations of the ligands with respect to the catalytic residues.
Thus, there were four major binding positions for compounds in the
CES catalytic site.
Estimates of docked free energies for all four types of binding
poses were very close, within 0.5 kcal/mol, which is less than the
AutoDock standard deviation of 2.5 kcal/mol [71].
Such binding tothe CES active site is associated with the loss of the
inherent planarity of aromatic system found in the free compounds
and, in the case of binding of the ester group in the active site, there is
impairment of intramolecular hydrogen bonding. However, these
destabilizations of the ligands are compensated by their specific in-
teractions with the enzyme, and subsequent reactions would explain
the high inhibitory activity of the compounds toward CES.
Molecular docking also showed strong additional binding of
compounds 3e and 3m in the CES active site, consisting of binding
For the TFK group in the active site, the entire molecule could be
flipped by 180ꢁ, such that the eCF3 group was directed either
6

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Fig. 4. Steady state inhibition of (A) porcine liver CES, (B) AChE from human erythrocytes, (C) BChE from equine serum by oxo ester 3k. Lineweaver-Burk double-reciprocal plots of
initial velocity and substrate concentrations in the presence of inhibitor (three concentrations) and without inhibitor are presented. [S], mM e concentration of substrates; V e
initial velocity rate; A e absorbance.
Table 3
Inhibitory activity of HOPs 3 against hCES1 and hCES2.
No.
Compound
IC50 (nM) SEM
Selectivity Ratio
R
R1
R2
hCES1
hCES2
IC50(hCES2)/IC50(hCES1)
3a
3b
3c
3d
3e
3f
3g
3j
3k
3l
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
C2F5
C3F7
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OMe
OMe
OMe
OMe
OEt
H
6.10 0.48
2.92 0.23
5.17 0.47
4.29 0.34
24.12 2.11
22.31 1.72
15.32 1.14
11.54 0.93
16.33 1.31
21.31 1.42
102.34 8.4
47.42 4.21
111.28 8.33
4.33 0.38
0.70
24.7
4.5
16.4
0.17
0.44
0.71
4.1
11.8
15.9
0.17
33.9
11.1
4-Me
3-Me
4-OMe
2-NO2
4-F
72.31 5.12
23.44 1.61
70.51 6.33
4.04 0.28
9.84 0.78
4-Br
H
10.81 0.83
47.74 3.82
193.34 15.18
339.25 30.32
16.92 1.34
610.24 54.12
230.09 18.21
4-Me
4-OMe
2-NO2
4-Me
4-OMe
3m
3p
3r
OMe
7

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Fig. 5. Binding poses of compounds (with compound 3k shown as an example) to hCES1: A and B d two different orientations of the TFK group in the active site; C and D d ester
group in the active site with different orientations of the remainder of the molecule, the TFK-arylhydrazone fragment. E ꢀ compound 3m pose with the nitro group in the active site.
Yellow dashes show hydrogen bonds, whereas red dashes indicate directions of potential subsequent nucleophilic attack. Carbon atoms of enzyme residues are colored green;
ligand carbons are colored light blue; fluorine atoms are colored cyan. Nitrogen ¼ blue; oxygen ¼ red. When hydrogens are included, they are colored white.
spacious CES active site could allow reorientation of the ligand to
one of the reactive positions.
Increasing the length of the fluorinated substituent made the
reactive position of the polyfluoroalkyl keto group in the CES active
site less favorable due to steric conflicts with surrounding residues.
As shown in Fig. 6 for compound 3s (RF ¼ C4F9), the carbonyl atom
is displaced from a reactive position in the active site. These results
explain why RF ¼ CF3 is optimal for maximum anti-CES inhibitor
activity, whereas compounds with longer polyfluoroalkyl groups
were 10e20 times less potent (Table 2).
Differences in binding modes of HOPs 3 to CES, AChE, and BChE
active sites determine the reasons for their selectivity. The large
CES1 active site (~1300 Å3) is lined predominately by hydrophobic
amino acids with aliphatic chains, allowing the entry of numerous
structurally diverse substrates [32].
Fig. 6. Binding pose of compound 3s (RF ¼ C4F9) in the CES with the polyfluoroalkyl
Due to differences in their active site shape and size compared
keto group in the active site. Protein residue carbons ¼ green; ligand carbons ¼ light
blue; nitrogens ¼ blue; oxygens ¼ red; fluorines ¼ cyan; when hydrogens are depicted,
they are colored white. Yellow dashed lines ¼ hydrogen bonds.
to CES, the cholinesterases exhibited less diversity in their inhibitor
binding poses than was seen with CES. Moreover, whereas AChE
had binding poses of HOP 3k in both the gorge and active site, poses
in the active site did not suggest the possibility of a subsequent
covalent reaction (Fig. 7A). BChE had only one potentially reactive
orientation of 3k with the TFK group in the active site. A specific
of the NO2 group to the oxyanion hole (Fig. 5E). Such a binding pose
does not suggest further covalent interactions; however, the
8

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Fig. 7. Example of binding of HOP 3 to cholinesterases: (A) compound 3k in the active site of AChE; (B) 3k in the active site of BChE. Nitrogens ¼ blue; oxygens ¼ red; AChE residue
carbons ¼ light cyan, BChE residue carbons ¼ light pink; ligand carbons ¼ light blue; fluorines ¼ cyan; when hydrogens are depicted, they are colored white.
Fig. 8. All binding poses obtained by molecular docking of compound 3p to CES1 (A) and CES2 (B). Ligand poses are colored according to estimated binding affinity increasing from
white to red. Protein residue colors: carbon ¼ green; nitrogen ¼ blue; oxygen ¼ red; hydrogen ¼ white.
feature of this binding was a hydrogen bond between the arylhy-
drazone fragment NH-group of 3k and Ser198 (Fig. 7B). However, a
hydrogen bond of this fragment with the active site Glu197 could
prevent a chemical reaction, and unlike CES, the BChE active site
did not allow reorientation of the 3k molecule to a more reactive
position. In addition, such binding was associated with a more
pronounced loss of planarity in the conjugated arylhydrazone
group than was the case with CES, which makes a reactive position
less favorable. These observations serve to explain the reduced
efficiency of HOP 3 against cholinesterases compared to CES.
Molecular modeling also allowed us to see differences in the
binding of pentafluoroethyl-substituted HOP 3p that correlated
with the highest selectivity between human CES isoenzymes:
IC50CES2/IC50CES1 ¼ 33.9 (Table 3). In docking compound 3p to
CES1, active site poses were most prevalent (Fig. 8A), while docking
3p to the CES2 homology model resulted in much more diverse
binding poses mostly outside of the active site (Fig. 8B).
2.6. Antioxidant activity
CES and cytochromes P450 are enzymes of phase I metabolism
of xenobiotics [72,73] and have close localization in hepatocytes. In
this regard, a potentially useful additional property of CES in-
hibitors could be their antioxidant action [56], which can protect
the liver from damage caused by highly reactive metabolites arising
from the biotransformation of drugs under the action of the cyto-
chrome P450 microsomal enzyme system [74,75].
The antioxidant activity of compounds 3 and 5 was evaluated in
the ABTS radical cation (ABTSꢂþ) scavenging assay [76], using Trolox
as the reference antioxidant. As shown in Table 1, the ABTSꢂþ
-
scavenging activity of the tested HOPs
3 containing a tri-
fluoromethyl group depended on the substituent in the arylhy-
drazone moiety. Compounds 3b-d,k,l with electron-donor Me or
MeO substituents had a high antiradical activity comparable to that
of Trolox (TEAC range, 0.85e1.0), ascorbic acid, and catechol, and
9

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
similar to that of earlier studied analogs e HOPN containing various
fragments of higher or natural alcohols [56]. The oxo esters 3a,j
having a non-substituted phenylhydrazone moiety also revealed
Although the limited solubility of the compounds has prevented
the exact cytotoxicity and acute toxicity measurements, it should
be stressed that their inhibitory concentrations are at least several
orders of magnitude lower, ensuring extremely wide safety
margins.
good radical-scavenging action (TEAC 0.78e0.8). The ABTSꢂþ
-
scavenging activity decreased for compounds with electron-
withdrawing substituents 3f (p-F, TEAC 0.6), 3g (p-Br,
¼
TEAC ¼ 0.53), with the lowest values for compounds 3i (p-CO2Et,
TEAC ¼ 0.1), 3e, 3h and 3m (o-NO2, o-CO2Et, TEAC ¼ 0.03e0.04).
Replacement of the trifluoromethyl group (3b, TEAC ¼ 0.95)
with CHF2 (3n, TEAC ¼ 0.37) and CH3 (3t, TEAC ¼ 0.38) as well as
elongation of the polyfluoroalkyl group (3p e C2F5, 3q e C3F7, 3s e
C4F9) decreased radical-scavenging activity (TEAC ¼ 0.36, 0.30,
0.26, respectively). Similar data were obtained for Me esters: 3k
(CF3, TEAC ¼ 0.98) vs 3o (CHF2, TEAC ¼ 0.38) and 3r (C3F7,
TEAC ¼ 0.4). Thus, the presence of a trifluoromethyl group in the
oxo esters 3 along with an electron-donor substituent in the aryl-
hydrazone moiety were the optimal conditions for high radical-
scavenging activity.
2.8. Predicted ADMET and physicochemical profiles
The results of the computational estimation of a number of
ADMET properties for HOPs 3 are shown in Table 5. As can be seen,
all the compounds had predicted high values for intestinal ab-
sorption, enabling their oral administration. The majority of the
compounds was predicted to have good blood-brain barrier
permeability (brain concentration is about 70e165% of the plasma
concentration), ensuring very efficient action on the CNS, although
the permeability of compounds 3e and 3m may be more moderate
(brain concentration about 40% of the plasma concentration). Both
parameters of the cardiac toxicity risk (pKi and pIC50) for all
analyzed compounds (3.8e5.6 log units) were in the lower part of
their possible range (3e9 log units). The predicted lipophilicities
and aqueous solubilities of the compounds were also good,
although some improvement would be desirable for a few com-
pounds. Finally, the integral quantitative estimates of drug-likeness
(QED) values for most of the compounds were close to 0.4. Thus, the
predicted ADMET properties of compounds 3 were quite acceptable
for potential lead compounds at the early drug development stages.
The most active scavengers of free radicals among the studied
compounds were 2-arylhydrazylidene-1,3-diketones 5a,b in which
the ester fragment was replaced with a ketone. Furthermore, a
diketone with a benzoyl moiety 5b (TEAC ¼ 1.7) was more effective
than the acyl-containing analog 5a (TEAC ¼ 1.33).
The calculated HOMO/LUMO gap values (DFT (B3LYP)/
6‑31þþG** method) for compounds 3h (3.89 eV), 3k (3.77 eV) and
5b (3.60 eV) agreed with increasing antioxidant activity in this
series (TEAC values 3h < 3k < 5b, Table 2).
2.9. PAINS analysis
2.7. Cytotoxicity studies and acute toxicity evaluation
The Pan Assay INterference compoundS (PAINS) filter check for
the compounds listed in Table 5 identified only one alert for the
potentially reactive imino-carbonyl moieties (“imine_one_A(321)”).
As discussed above, the reaction of the inhibitor carbonyl groups
with the catalytic serine hydroxyl indeed play a important role in
the proposed mechanism of action of the arylhydrazone derivatives
containing TFK and ester groups. Their specific activity was
confirmed by their selectivity to CES over AChE and BChE and by
the structure-activity relationships identified in the experiment
and explained by molecular modeling, as well as by kinetic studies,
experimental concentration-response curves, and IC50 measure-
ments. ABTS-scavenging activity of the compounds was confirmed
by their experimentally determined structure-activity relation-
ships, which agreed with the calculated HOMO-LUMO gaps,
Cell viability was assessed using the MTT assay. All studied HOPs
3a-c,e-g,o were found to have relatively low potency against hu-
man FetMSC cells in the MTT assay, with IC50 values exceeding
500 mM (Table 4). Higher concentrations were not used because of
the limited solubility of compounds.
The acute toxicity of some of the most active compounds 3a-
c,e,g,m was estimated in CD-1 mice. The tested compounds in 2%
(w/v) starch mucilage solution were injected intraperitoneally.
Animals were observed for lethality during 14 days. The obtained
preliminary data (Table 4) allowed us to conclude that LD50 values
for these compounds were expected to exceed 300 mg/kg, because
after administering this dose none of the animals died. Increasing
the dose of 3a to 600 mg/kg and 3b to 900 mg/kg did not lead to the
death of any animals in the experiment.
experimental
concentration-response
curves
for
TEAC
Table 4
Cytotoxicity of HOPs 3 and their acute toxicity in mice.
Compound
Cytotoxicity,
IC50
Dose, mg/kg
Viability after 14 days
(number of animals in the experiment/number of survivors)
,
m
M
No.
R
R1
R2
3a
3b
3c
3e
3f
3g
3m
3o
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CHF2
OEt
OEt
OEt
OEt
OEt
OEt
OMe
OMe
H
>500
>500
>500
>500
>500
>500
>500
>500
600
900
300
300
n.d.
300
300
n.d.
3/3
3/3
3/3
3/3
n.d.
3/3
3/3
n.d.
4-Me
3-Me
2-NO2
4-F
4-Br
2-NO2
4-Me
n.d. ¼ not determined.
10

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
Table 5
Predicted ADMET and physicochemical profiles of HOPs 3.
Compound
LogBB
HIA, %
hERG, pKi
hERG, pIC50
LogPow
pS
QED
No.
R
R1
R2
3a
3b
3c
3d
3e
3f
3g
3h
3i
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OMe
OMe
OMe
OMe
OEt
OMe
OEt
OEt
OMe
OEt
OEt
H
ꢀ0.06
ꢀ0.06
ꢀ0.06
ꢀ0.06
ꢀ0.41
ꢀ0.06
ꢀ0.06
ꢀ0.08
ꢀ0.14
ꢀ0.06
ꢀ0.06
ꢀ0.06
ꢀ0.41
ꢀ0.06
ꢀ0.06
ꢀ0.06
ꢀ0.06
ꢀ0.06
ꢀ0.06
ꢀ0.06
100
100
100
100
96
100
100
100
100
93
93
93
90
100
93
4.41
4.68
4.45
4.68
3.95
4.68
4.68
4.48
4.81
4.27
4.53
4.53
3.82
4.68
4.53
4.68
4.68
4.53
4.68
4.68
4.57
4.71
4.67
4.66
4.08
4.88
4.67
4.28
4.67
4.51
4.65
4.60
4.02
4.57
4.51
4.83
4.98
4.87
5.09
4.49
3.95
4.47
4.45
4.12
3.83
4.26
4.86
4.28
4.33
3.47
4.07
3.64
3.55
3.93
3.58
5.22
5.76
5.38
6.17
2.71
4.88
5.62
5.61
5.32
4.92
5.60
6.26
5.41
5.54
4.49
5.14
4.94
4.67
4.27
4.04
6.36
6.01
5.47
6.59
3.85
0.39
0.39
0.39
0.38
0.28
0.30
0.38
0.35
0.35
0.39
0.40
0.39
0.29
0.38
0.38
0.28
0.25
0.25
0.21
0.37
4-Me
3-Me
4-OMe
2-NO2
4-F
4-Br
2-CO2Et
4-CO2Et
H
3j
3k
3l
4-Me
4-OMe
2-NO2
4-Me
4-Me
4-Me
4-Me
4-OMe
4-Me
4-Me
3m
3n
3o
3p
3q
3r
3s
3t
CF3
CF2H
CF2H
C2F5
C3F7
C3F7
C4F9
Me
100
100
93
100
100
Note: LogBB e blood-brain barrier permeability, HIA e human intestinal absorption [%], hERG pKi e hERG potassium channel affinity [elog(M)], hERG pIC50 e hERG potassium
channel inhibitory activity [elog(M)], LogPow e octanol-water partition coefficient, pS e aqueous solubility [elog(M)], QED e quantitative estimate of drug-likeness.
determination, and IC50 measurements. Thus, we have demon-
strated in several different experimental assays and computational
approaches that the compounds flagged by the PAINS screen were
genuinely active.
product. Mechanisms of these reactions have been described using
extensive QM/MM calculations (unpublished results; manuscript in
preparation).
The results of molecular modeling allowed us to explain some
structure-activity relationships of CES inhibition by HOPs 3, their
selectivity toward CES in comparison with cholinesterases, and the
higher selectivity of pentafluoroethyl-substituted HOP 3p to hCES1
compared to hCES2.
Computational ADMET studies revealed that all HOPs 3 were
predicted to have good intestinal absorption and blood-brain bar-
rier permeability, low cardiac toxicity risks, good lipophilicity and
aqueous solubility, and reasonable overall drug-likeness, i.e. they
should have good or acceptable properties for potential lead com-
pounds at the early drug development stages.
3. Conclusions
In summary, an expanded series of HOPs 3 using the more
productive synthetic protocol via Cu(OAc)2-catalyzed azo coupling
was obtained while 2-arylhydrazinylidene-1,3-diketones 5 were
prepared from lithium 1,3-diketonates as the starting reagents.
According to physical structural studies and QM calculations, 2-
arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates
3 exist
as Z-isomers with intramolecular hydrogen bond between the NH-
group and the carbonyl oxygen atom of the ester group.
All of the fully fluorinated HOPs 3, in contrast to their CHF2 and
CH3 derivatives and 1,3-diketone analogs 5, were potent and highly
selective inhibitors of porcine liver CES and moderate or weak in-
hibitors of cholinesterases. Among the studied CES inhibitors,
selectivity to both human CES1 and CES2 were found, while the
selectivity to CES1 was more pronounced. It should be stressed that
their inhibitory concentrations are at least several orders of
magnitude lower than toxic concentrations, ensuring extremely
wide safety margins.
Steady-state kinetics studies showed that HOPs 3 are mixed
type inhibitors of all three esterases.
To exhibit maximum anti-CES activity, a molecule based on the
polyfluoroalkyl-2-imino-1,3-dione scaffold should contain a com-
bination of a TFK group and an ester group.
In addition, HOPs 3, which have a TFK group and electron-donor
substituents in the arylhydrazone moiety, were found to be potent
antioxidants. Accordingly, we anticipate that the radical-
scavenging action of the developed selective inhibitors of CES en-
zymes would be an additional useful feature that could protect the
liver from damage induced by highly reactive metabolites formed
during the P450-mediated biotransformation of drugs. All tested
compounds 3 possessed low cytotoxicity and low acute toxicity.
Thus, using kinetic studies and molecular modeling, we have
shown that the high anti-CES activity of HOPs based on a poly-
fluoroalkyl-2-imino-1,3-dione scaffold is due to the presence of two
functional groups, TFK and an ester, capable of interacting with the
active site of the enzyme. Overall, the results indicate that HOPs 3
have the potential to be good candidates as human CES inhibitors
for biomedicinal applications.
Molecular docking studies demonstrated the possibility of both
functional groups in the structure of HOP e TFK and ester groups e
binding to the CES active site. This suggests the possibility of sub-
sequent reactions according to two mechanisms: (1) formation of a
tetrahedral adduct; and (2) a slow hydrolysis reaction. The latter
could be followed by reinhibition of the enzyme by the hydrolysis
4. Experimental section
4.1. Chemistry
All solvents, chemicals, and reagents were obtained
11

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
commercially and used without purification. Melting points were
measured in open capillaries on a Stuart SMP30 melting point
apparatus and were uncorrected. The 1H NMR spectra were regis-
tered on a Bruker DRX-400 (400 MHz) or Bruker AvanceIII 500
(500 MHz) spectrometers relative to SiMe4. The 19F NMR spectra
were obtained on a Bruker DRX-400 (376 MHz) or Bruker AvanceIII
500 (470 MHz) spectrometers using C6F6 as an internal standard.
The microanalyses (C, H, N) were carried out on a PerkinElmer PE
2400 series II elemental analyzer.
General Procedures for synthesis of compounds 3. To 60 ml of a
solution of amine (40 mmol) in 1 M hydrochloric acid, a solution of
sodium nitrite (2.76 g, 40 mmol) in 40 ml of water at 0 ꢁC was added
dropwise. Then, in another container, solutions of copper (II) ace-
tate (20 mmol) in water (20 ml) and 3-oxo ester 1 (40 mmol) in
acetone (20 ml) were mixed. To the resulting suspension, a solution
of the aryldiazonium salt 2 was slowly added dropwise at 10 ꢁC. The
resulting product 3 was extracted by chloroform (2 ꢃ 20 ml), and
the chloroform layer was separated and dried with anhydrous so-
dium sulfate. The chloroform was removed in vacuo, and compound
3 was recrystallized from ethanol.
OCH2Me), 4.5 (q, 3JH,H 7.1 Hz, 2H, OCH2Me), 7.34, 7.73, 8.07, 8.30 (all
m, 4H, C6H4), 14.71 (s, 1H, NH). 19F NMR (376 MHz, CDCl3):
d 90.96
(s, CF3). Elemental analysis calculated (%) for С12Н10F3N3О5 (M
333.06): C 43.25, H 3.02, F 17.10, N 12.61. Found: C 43.15, H 3.07, F
17.35, N 12.80.
Ethyl (2Z)-4,4,4-trifluoro-2-[2-(4-fluorophenyl)hydrazinylidene]-
3-oxobutanoate (3f) was obtained according to the general pro-
cedure from 3-oxo ester 1a (7.36 g, 40 mmol) and 4-
fluorobenzenediazonium chloride prepared from 4-fluoroaniline
(4.44 g, 40 mmol) as an orange powder (10.02 g, 82% yield), mp
79e80 ꢁC (eluent e CH2Cl2). 1H NMR (400 MHz, CDCl3):
d 1.42 (t,
3
3JH,H 7.1 Hz, 3H, OCH2Me), 4.41 (q, JH,H 7.1 Hz, 2H, OCH2Me),
7.12e7.17, 7.38e7.42 (both m, 4H, C6H4), 13.52 (s, 1H, NH). 19F NMR
(376 MHz, CDCl3):
d 47.08 (m, 1F, FAr), 91.28 (s, 3F, CF3). Elemental
analysis calculated (%) for C12H10F4N2O3 (M 306.22): C 47.07, H 3.29,
N 9.15. Found: C 47.06, H 3.03, N 9.33.
Ethyl (2Z)-4,4,4-trifluoro-2-[2-(4-bromophenyl)hydrazinylidene]-
3-oxobutanoate (3g) was obtained according to the general pro-
cedure from 3-oxo ester 1a (7.36 g, 40 mmol) and 4-
bromobenzenediazonium chloride prepared from 4-bromoaniline
(6.88 g, 40 mmol) as a yellow powder (11.75 g, 80% yield), mp
Ethyl (2Z)-4,4,4-trifluoro-3-oxo-2-(2-phenylhydrazinylidene)buta-
noate (3a) was obtained according to the general procedure from 3-
oxo ester 1a (7.36 g, 40 mmol) and benzenediazonium chloride pre-
pared from aniline (3.75 g, 40 mmol) as a yellow powder (9.56 g, 83%
109e110 ꢁC (eluent e CH2Cl2). 1H NMR (400 MHz, CDCl3):
d 1.42 (t,
3
3JH,H 7.1 Hz, 3H, OCH2Me), 4.41 (q, JH,H 7.1 Hz, 2H, OCH2Me), 7.29,
3
7.56 (both d, JH,H 8.8 Hz, 4H, C6H4), 13.44 (s, 1H, NH). 19F NMR
yield), mp 90e91 ꢁC [53,77]. 1H NMR (400 MHz, DMSO‑d6):
d
1.32 (t,
(376 MHz, CDCl3): d 91.19 (s, CF3). Elemental analysis calculated (%)
for C12H10BrF3N2O3 (M 367.12): C 39.26, H 2.75, N 7.63. Found: C
39.35, H 2.72, N 7.41.
3
3JH,H 7.1 Hz, 3H, OCH2Me), 4.36 (q, JH,H 7.1 Hz, 2H, OCH2Me),
7.25e7.28, 7.46e7.50, 7.56e7.58 (all m, 4H, C6H4),12.95 (s,1H, NH).19
NMR (470 MHz, DMSO‑d6): 93.35 (s, CF3).
F
d
Ethyl 2-[(2Z)-2-(1-ethoxy-4,4,4-trifluoro-1,3-dioxobutan-2-ylidene)
hydrazinyl]benzoate (3h) was obtained according to the general pro-
cedure from 3-oxo ester 1a (7.36 g, 40 mmol) and 2-(ethoxycarbonyl)
benzenediazonium chloride prepared from 2-ethoxycarbonylaniline
(6.60 g, 40 mmol) as a yellow powder (12.38 g, 86% yield), mp
Ethyl (2Z)-4,4,4-trifluoro-2-[2-(4-methylphenyl)hydrazinylidene]-
3-oxobutanoate (3b) was obtained according to the general pro-
cedure from 3-oxo ester 1a (7.36 g, 40 mmol) and 4-
methylbenzenediazonium chloride prepared from 4-toluidine
(4.28 g, 40 mmol) as a yellow powder (13.74 g, 87% yield), mp
104e105 ꢁC (EtOH). 1H NMR (500 MHz, CDCl3):
d 1.44 (m, 6H,
75e76 ꢁC [53]. 1H NMR (400 MHz, DMSO‑d6):
d
1.28 (t, 3JH,H 7.1 Hz,
2OCH2Me), 4.49 (m, 4H, 2OCH2Me), 7.24e7.27, 7.63e7.66, 7.96e7.97,
3
3H, OCH2Me), 2.28 (s, 3H, C6H4Me), 4.30 (q, JH,H 7.1 Hz, 2H,
8.09e8.11 (all m, 4H, C6H4), 14.88 (s, 1H, NH). 19F NMR (470 MHz,
OCH2Me), 7.18e7.36 (m, 4H, C6H4), 11.65 (s, 1H, NH). 19F NMR
CDCl3):
1H NMR (500 MHz, DMSO‑d6):
(m, 4H, 2OCH2Me), 7.35e7.39, 7.80e7.83, 8.05e8.07 (all m, 4H,
C6H4), 14.59 (s, 1H, NH). 19F NMR (470 MHz, DMSO‑d6):
93.19 (s,
d 91.31 (s, CF3).
(376 MHz, DMSO‑d6):
d
93.5 (s, CF3).
d 1.36 (m, 6H, 2OCH2Me), 4.40
Ethyl (2Z)-4,4,4-trifluoro-2-[2-(3-methylphenyl)hydrazinylidene]-
3-oxobutanoate (3c) was obtained according to the general pro-
cedure from 3-oxo ester 1a (7.36 g, 40 mmol) and 3-
methylbenzenediazonium chloride prepared from m-toluidine
(4.28 g, 40 mmol) as a yellow powder (12.95 g, 82% yield), mp
d
CF3). Elemental analysis calculated (%) for C15H15F3N2O5 (M
360.29): C 50.01, H 4.20, N 7.78. Found: C 50.04, H 4.07, N 7.74.
Ethyl 4-[(2Z)-2-(1-ethoxy-4,4,4-trifluoro-1,3-dioxobutan-2-ylidene)
hydrazinyl]benzoate (3i) was obtained according to the general pro-
cedure from 3-oxo ester 1a (7.36 g, 40 mmol) and 4-(ethoxycarbonyl)
benzenediazonium chloride prepared from 4-ethoxycarbonylaniline
(6.60 g, 40 mmol) as a yellow powder (11.81 g, 82% yield), mp
61e62 ꢁC (eluent e CHCl3). 1H NMR (400 MHz, CDCl3):
d 1.42 (t,
3
3JH,H 7.1 Hz, 3H, OCH2Me), 2.40 (s, 3H, C6H4eMe), 4.41 (q, JH,H
7.1 Hz, 2H, OCH2Me), 7.06e7.08, 7.23e7.24, 7.30e7.32 (all m, 4H,
C6H4), 13.46 (s, 1H, NH). 19F NMR (470 MHz, CDCl3):
d 91.31 (s, CF3).
Elemental analysis calculated (%) for C13H13F3N2O3 (M 302.25): C
51.66, H 4.34, N 9.27. Found: C 51.73, H 4.39, N 9.14.
106e107 ꢁC(EtOH).1HNMR(500MHz,CDCl3): 1.41,1.43(botht,3JH,H
d
7.1 Hz, 6H, 2OCH2Me), 4.39, 4.43 (both q, 3JH,H 7.1 Hz, 4H, 2OCH2Me),
7.45, 8.13 (both d, 3JH,H 8.8 Hz, 4H, C6H4), 13.43 (s, 1H, NH). 19F NMR
Ethyl (2Z)-4,4,4-trifluoro-2-[2-(4-methoxyphenyl)hydrazinylidene]-
3-oxobutanoate (3d) wasobtained according tothe generalprocedure
from 3-oxo ester 1a (7.36 g, 40 mmol) and 4-methoxyben
zenediazonium chloride prepared from 4-anisidine (4.92 g,
40 mmol) as a yellow powder (13.48 g, 82% yield), mp 130e131 ꢁC
(470MHz,CDCl3):
15H15F3N2O5 (M 360.29): C 50.01, H 4.20, N 7.78. Found: C 50.12, H
4.21, N 7.60.
d91.09(s, CF3). Elementalanalysiscalculated(%)for
C
Methyl (2Z)-4,4,4-trifluoro-3-oxo-2-(2-phenylhydrazinylidene)buta
noate (3j) was obtained according to the general procedure from 3-
oxo ester 1b (7.36 g, 40 mmol) and benzenediazonium chloride pre-
pared from aniline (3.75 g, 40 mmol) as orange crystals (8.76 g, 80%
[66].1HNMR(400MHz,DMSO‑d6): 1.32(t, 3JH,H 7.1Hz,3H, OCH2Me),
d
3.78 (s, 3H, C6H4eOMe), 4.34 (q, 3JH,H 7.1 Hz, 2H, OCH2Me), 7.06, 7.54
(both d, 3JH,H 9.0 Hz, 4H, C6H4), 13.10 (s, 1H, NH). 19F NMR (376 MHz,
DMSO‑d6):
d
93.52 (s, CF3).
yield), mp 84e85 ꢁC (MeOH).1H NMR (400 MHz, CDCl3):
d
3.96 (s, 3H,
OMe), 7.27e7.29, 7.44e7.45 (both m, 5H, Ph), 13.49 (s, 1H, NH). 19
NMR (376 MHz, CDCl3): 91.31 (d, J 0.7 Hz, CF3). Elemental analysis
Ethyl (2Z)-4,4,4-trifluoro-2-[2-(2-nitrophenyl)hydrazinylidene]-3-
oxobutanoate (3e) was obtained according to the general proced-
ure from 3-oxo ester 1a (7.36 g, 40 mmol) and 2-
nitrobenzenediazonium chloride prepared from 2-nitroaniline
(5.52 g, 40 mmol) as a yellow powder (10.78 g, 81% yield), mp
F
d
calculated (%) for C11H9F3N2O3 (M 274.20): C 48.18, H 3.31, N 10.22.
Found: C 48.24, H 3.15, N 10.20.
Methyl (2Z)-4,4,4-trifluoro-2-[2-(4-methylphenyl)hydrazinylidene]-
3-oxobutanoate (3k) was obtained according tothe generalprocedure
99e100 ꢁC [53]. 1H NMR (400 MHz, CDCl3): 1.45 (t, 3JH,H 7.1 Hz, 3H,
d
12

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
3
from 3-oxo ester 1b (7.36 g, 40 mmol) and 4-methylbenzene
diazonium chloride prepared from 4-toluidine (4.28 g, 40 mmol) as
orange crystals (9.91 g, 86% yield), mp 111e112 ꢁC (eluent e CHCl3).1H
3.78, 3.85 (both s, 6H, C6H4eOMe, OMe), 7.06, 7.51 (both d, JH,H
8.4 Hz, 4H, C6H4), 13.03 (s, 1H, NH). 19F NMR (376 MHz, DMSO‑d6):
38.48 (m, 2F, CF2), 50.99 (m, 2F, CF2), 82.81 (m, 3F, CF3). Elemental
d
NMR (500 MHz, CDCl3):
7.24, 7.33 (both d, 3JH,H 8.4 Hz, 4H, C6H4), 13.52 (s, 1H, NH). 19F NMR
(470MHz,CDCl3): 91.37(s, CF3). Elemental analysiscalculated(%)for
12H11F3N2O3 (M 288.23): C 50.01, H 3.85, N 9.72. Found: C 49.85, H
d
2.38 (s, 3H, C6H4eMe), 3.94 (s, 3H, OMe),
analysis calculated (%) for C15H13F7N2O3 (M 402.27): C 44.79, H
3.26, N 6.96. Found: C 44.69, H 3.15, N 6.78.
d
Methyl
(2Z)-4,4,5,5,6,6,6-heptafluoro-2-[2-(4-methoxyphenyl)
C
hydrazinylidene]-3-oxohexanoate (3r) was obtained according to the
general procedure from ester 1g (10.80 g, 40 mmol) and 4-
methoxybenzenediazonium chloride prepared from 4-anisidine
(4.92 g, 40 mmol) as a yellow powder (13.25 g, 82% yield), mp
3.77, N 9.66.
Methyl
(2Z)-4,4,4-trifluoro-2-[2-(4-methoxyphenyl)hydrazinyli-
dene]-3-oxobutanoate (3l) was obtained according to the general
procedure from 3-oxo ester 1b (7.36 g, 40 mmol) and 4-
methoxybenzenediazonium chloride prepared from 4-anisidine
(4.92 g, 40 mmol) as an orange powder (10.58 g, 87% yield), mp
142e144 ꢁC [66].
3
95e96 ꢁC [67] 1H NMR (500 MHz, DMSO‑d6): 1.31 (t, JH,H 7.1 Hz,
3H, OCH2Me), 2.31 (s, 3H, C6H4eMe), 4.34 (q, 3JH,H 7.1 Hz 2H, OCH2),
2
7.05 (t, JH,F 53.7 Hz, 1H, HCF2), 7.24e7.26, 7.48e7.50 (all m 4H,
C6H4), 12.71 (s, 1H, NH). 19F NMR (470 MHz, DMSO‑d6):
2JH,F 53.7 Hz, HCF2).
d 35.25 (d,
Methyl (2Z)-4,4,4-trifluoro-2-[2-(2-nitrophenyl)hydrazinylidene]-
3-oxobutanoate (3m) was obtained according to the general pro-
cedure from 3-oxo ester 1b (7.36 g, 40 mmol) and 2-
nitrobenzenediazonium chloride prepared from 2-nitroaniline
(5.52 g, 40 mmol) as an orange powder (10.59 g, 83% yield), mp
Ethyl (2Z)-4,4,5,5,6,6,7,7,7-nonafluoro-2-[2-(4-methylphenyl)hydra
zinylidene]-3-oxoheptanoate (3s) was obtained according to the gen-
eral procedure from ester 1h (13.36 g, 40 mmol) and 4-
methylbenzenediazonium chloride prepared from 4-toluidine
(4.28 g, 40 mmol) as a yellow powder (14.30 g, 82% yield), mp
123e124 ꢁC (MeOH). 1H NMR (500 MHz, CDCl3):
d
4.02 (s, 3H, OMe),
7.33e7.37, 7.76e7.79, 8.06e8.08, 8.30e8.32 (all m, 4H, C6H4), 14.77
(s, 1H, NH). 19F NMR (470 MHz, CDCl3):
90.99 (d, J 0.6 Hz, CF3).
43e45 ꢁC [78].1H NMR (400 MHz, DMSO‑d6): 1.31 (t, 3JH,H 7.1 Hz, 3H,
d
d
OCH2Me), 2.31 (s, 3H, C6H4eMe), 4.35 (q, 3JH,H 7.1 Hz, 2H, OCH2Me),
7.28, 7.44 (both d, 3JH,H 8.4 Hz, 4H, C6H4), 12.92 (s, 1H, NH). 19F NMR
Elemental analysis calculated (%) for C11H8F3N3O5 (M 319.20): C
41.39, H 2.53, N 13.16. Found: C 41.43, H 2.64, N 13.19.
(376 MHz, DMSO‑d6): d 37.66 (m, 2F, CF2), 41.88 (m, 2F, CF2), 51.33 (m,
Ethyl (2Z)-4,4-difluoro-2-[2-(4-methylphenyl)hydrazinylidene]-3-
oxobutanoate (3n) was obtained according to the general proced-
ure from 3-oxo ester 1c (6.64 g, 40 mmol) and 4-
methylbenzenediazonium chloride prepared from 4-toluidine
(4.28 g, 40 mmol) as yellow crystals (9.42 g, 83% yield), mp
2F, CF2), 82.20 (m, 3F, CF3).
Ethyl (2E)-2-[2-(4-methylphenyl)hydrazinylidene]-3-oxobutanoate
(3t) was obtained according to the general procedure from ester 1i
(5.20 g, 40 mmol) and 4-methylbenzenediazonium chloride pre-
pared from 4-toluidine (4.28 g, 40 mmol) as an orange powder
96e97 ꢁC [67]. 1H NMR (500 MHz, DMSO‑d6): 1.31 (t, JH,H 7.1 Hz,
(8.13 g, 82% yield), mp 80e81 ꢁC [77]. 1H NMR (400 MHz, DMSO‑d6):
3
3H, OCH2Me), 2.31 (s, 3H, C6H4eMe), 4.34 (q, 3JH,H 7.1 Hz, 2H, OCH2),
Z-isomer (70%):
d
1.29 (t, JH,H 7.1 Hz, 3H, OCH2Me), 2.28 (s, 3H,
3
2
7.05 (t, JH,F 53.7 Hz, 1H, HCF2), 7.24e7.26, 7.48e7.50 (all m 4H,
C6H4eMe), 2.37 (s, 3H, Me), 4.30 (q, 3JH,H 7.1 Hz, 2H, OCH2Me), 7.19,
C6H4), 12.71 (s, 1H, NH). 19F NMR (470 MHz, DMSO‑d6):
2JH,F 53.7 Hz, HCF2).
d 35.25 (d,
7.34 (both d, 3JH,H 8.4 Hz, 4H, C6H4),11.65 (s,1H, NH), E-isomer (70%):
d
1.29 (t, 3JH,H 7.1 Hz, 3H, OCH2Me), 2.30 (s, 3H, C6H4eMe), 2.37 (s, 3H,
Methyl (2Z)-4,4-difluoro-2-[2-(4-methylphenyl)hydrazinylidene]-
3-oxobutanoate (3o) was obtained according to the general pro-
cedure from 3-oxo ester 1d (6.08 g, 40 mmol) and 4-
methylbenzenediazonium chloride prepared from 4-toluidine
(4.28 g, 40 mmol) as an orange powder (8.64 g, 80% yield), mp
102e103 ꢁC [67]. 1H, 19F NMR (DMSO‑d6) described in Ref. [67].
Ethyl (2Z)-4,4,5,5,5-pentafluoro-2-[2-(4-methylphenyl)hydraziny-
lidene]-3-oxopentanoate (3p) was obtained according to the general
procedure from 3-oxo ester 1e (9.36 g, 40 mmol) and 4-
methylbenzenediazonium chloride prepared from 4-toluidine
(4.28 g, 40 mmol) as a yellow powder (11.97 g, 85% yield), mp
Me), 4.25 (q, 3JH,H 7.1 Hz, 2H, OCH2Me), 7.23, 7.41 (both d, 3JH,H 8.4 Hz,
4H, C6H4), 14.37 (s, 1H, NH).
General Procedures for synthesis of compounds 5. To a solution of
amine (40 mmol) in a solution of 1 M hydrochloric acid (60 ml) was
added dropwise a solution of sodium nitrite (2.76 g, 40 mmol) in
40 ml of water at 0 ꢁC. Then, in another container, solutions of
sodium acetate (40 mmol) in water (20 ml) and lithium salt 4
(40 mmol) in ethanol (20 ml) were mixed. To the resulting sus-
pension was slowly dripped solution of the aryldiazonium salt 2 at
10 ꢁC. The resulting product 5 was extracted by chloroform
(2 ꢃ 20 ml), dried with sodium sulfate. The chloroform was
removed in vacuum. The compound 5 was recrystallized from
ethanol.
(3E)-1,1,1-Trifluoro-3-[2-(4-methylphenyl)hydrazinylidene]
pentane-2,4-dione (5a) was obtained according to the general pro-
cedure from lithium salt 4a (6.40 g, 40 mmol) and 4-methylben
zenediazonium chloride prepared from 4-toluidine (4.28 g,
40 mmol) as a yellow powder (8.70 g, 80% yield), mp 95e96 ꢁC. 1H
53e54 ꢁC (eluent e CHCl3). 1H NMR (400 MHz, CDCl3):
d 1.42 (t,
3
3JH,H 7.1 Hz, 3H, OCH2Me), 2.38 (s, 3H, C6H4eMe), 4.40 (q, JH,H
7.1 Hz, 2H, OCH2Me), 7.24, 7.32 (both d, 3JH,H 8.4 Hz, 4H, C6H4), 13.56
(s, 1H, NH). 19F NMR (376 MHz, CDCl3):
d
47.08 (m, 2F, CF2), 80.76
1.32 (t, 3JH,H 7.1 Hz, 3H,
(m, 3F, CF3). 1H NMR (400 MHz, DMSO‑d6):
d
OCH2Me), 2.32 (s, 3H, C6H4eMe), 4.34 (q, 3JH,H 7.1 Hz, 2H, OCH2Me),
7.29, 7.45 (both d, 3JH,H 8.4 Hz, 4H, C6H4), 12.96 (s, 1H, NH). 19F NMR
(376 MHz, DMSO‑d6):
Elemental analysis calculated (%) for C14H13F5N2O3 (M 352.26): C
47.74, H 3.72, N 7.95. Found: C 47.83, H 3.65, N 8.01.
d
48.49 (m, 2F, CF2), 82.14 (m, 3F, CF3).
NMR (400 MHz, CDCl3):
d
2.38 (s, 3H, C6H4eMe), 2.63 (s, 3H, Me), 7.25,
7.38 (both d, JH,H 8.5 Hz, 4H, C6H4), 15.25 (s, 1H, NH). 19F NMR
(376 MHz, CDCl3):
91.60 (s, 3F, CF3). 1H NMR (400 MHz, DMSO‑d6):
2.33 (s, 3H, C6H4eMe), 2.53 (s, 3H, Me), 7.31, 7.54 (bothd, 3JH,H 8.5 Hz,
4H, C6H4),14.66 (s,1H, NH). 19F NMR (376 MHz, DMSO‑d6):
93.70 (s,
3
d
Ethyl (2Z)-4,4,5,5,6,6,6-heptafluoro-2-[2-(4-methylphenyl)hydra-
zinylidene]-3- oxohexanoate (3q) was obtained according to the
general procedure from 3-oxo ester 1f (11.36 g, 40 mmol) and 4-
methylbenzenediazonium chloride prepared from 4-toluidine
(4.28 g, 40 mmol) as a yellow powder (13.98 g, 87% yield), mp
d
d
CF3). Elemental analysis calculated (%) for C12H11F3N2O2 (M 272.23): C
52.95, H 4.07, N 10.29. Found: C 52.74, H 4.17, N 10.22.
(2E)-4,4,4-Trifluoro-2-[2-(4-methylphenyl)hydrazinylidene]-1-
phenylbutane-1,3-dione (5b) was obtained according to the general
procedure from lithium salt 4b (8.88 g, 40 mmol) and 4-
methylbenzenediazonium chloride prepared from 4-toluidine
(4.28 g, 40 mmol) as a yellow powder (8.70 g, 80% yield), mp
38e39 ꢁC. (eluent e CHCl3). 1H NMR (400 MHz, CDCl3):
d 1.42 (t,
3JH,H 7.1 Hz, 3H, OCH2Me), 2.37 (s, 3H, C6H4eMe), 4.40 (q, JH,H
3
7.1 Hz, 2H, OCH2Me), 7.24, 7.30 (both d, 3JH,H 8.4 Hz, 4H, C6H4), 13.56
(s, 1H, NH). 19F NMR (376 MHz, CDCl3):
d
37.49 (m, 2F, CF2), 49.51
(m, 2F, CF2), 81.37 (m, 3F, CF3). 1H NMR (400 MHz, DMSO‑d6):
d),
133e134 ꢁC [79] 1H NMR (400 MHz, DMSO‑d6):
d 2.30 (s, 3H,
13

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
C6H4eMe), 7.24e7.26, 7.37e7.40, 7.56e7.60, 7.70e7.74, 7.80e7.82 (all
m, 9H, C6H4, C6H5), 12.52 (s, 1H, NH). 19F NMR (376 MHz, DMSO‑d6):
increasing concentrations of a test compound and 5e6 decreasing
concentrations of the substrates. Test compounds were incubated
with the enzymes at 25 ꢁC for 10 min, followed by the addition of
substrates. A parallel control was made to assay of the rate of hy-
drolysis of the same concentrations of substrates in solutions with
no inhibitor. Kinetic parameters of substrate hydrolysis were
determined. Measurements were performed in a FLUOStar Optima
microplate reader (LabTech, Ortenberg, Germany). Each experi-
ment was performed in triplicate.
d
93.49 (s, CF3).
4.2. Inhibition in vitro of porcine liver CES, human CES1 and CES2,
human AChE, and equine BChE
The following enzymes were purchased from Sigma-Aldrich
(Saint Louis, MO, USA): porcine liver carboxylesterase (CES, EC
3.1.1.1), human recombinant carboxylesterases CES1 (E0287, Car-
boxylesterase 1 isoform b human recombinant, expressed in
baculovirus-infected BTI insect cells) and CES2 (E4749, Carbox-
ylesterase 2 human recombinant, expressed in mouse NSO cells,
ꢄ95% (SDS-PAGE)), human erythrocyte acetylcholinesterase (AChE,
EC 3.1.1.7) and equine serum butyrylcholinesterase (BChE, EC
3.1.1.8).
Substrates for CES, AChE, and BChE were 4-nitrophenyl acetate
(4-NPA), acetylthiocholine iodide (ATCh), and butyrylthiocholine
iodide (BTCh), respectively, and the colorimetric reagent for the
AChE and BChE assays was 5,50-dithio-bis-(2-nitrobenzoic acid)
(DTNB) (Sigma-Aldrich).
All the kinetic experiments were performed under standard
conditions, according to the protocol of IPAC RAS for a reversible
inhibitors study.
CES (porcine liver) activity was assessed colorimetrically in
0.1 M K/Na phosphate buffer pH 8.0, 25 ꢁC, by measuring the
absorbance of 4-nitrophenol at 405 nm [80]. Final enzyme and
substrate (4-NPA) concentrations were 0.02 unit/mL and 1 mM,
respectively. Reagent blanks included all constituents except
enzyme.
4.4. ABTS radical cation scavenging assay
The antioxidant activity of compounds 3 and 5 was evaluated by
the ABTS radical cation (ABTSꢂþ) scavenging assay by the ability of
the compounds to decolorize the ABTSꢂþ solution [76] with some
modifications [56,58]. Trolox was used as the antioxidant standard,
and the well-known antioxidants ascorbic acid and catechol were
used as the comparison compounds. All tested compounds and
standards were dissolved in DMSO.
ABTS (2,2ʹ-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid))
was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo,
Japan). Potassium persulfate (di-potassium peroxydisulfate), Tro-
lox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid),
ascorbic acid, catechol, and HPLC-grade ethanol were obtained
from Sigma-Aldrich Chemical Co. Aqueous solutions were prepared
using deionized water.
The solution of ABTSꢂþ was produced by incubation of ABTS
with potassium persulfate in deionized water for 12e16 h at room
temperature in the dark. Radical scavenging capacity of the com-
pounds was analyzed by mixing 10
m
l of compound with 240
ml of
The activity of hCES1 and hCES2 were determined spectropho-
tometrically by the release of 4-nitrophenol at 405 nm [80]. The
assay solution consisted of 0.1 M K/Na phosphate buffer pH 7.4,
25 ꢁC as well as 1 mM 4-nitrophenyl acetate and 0.02 unit/mL of
hCES1 or 0.01 unit/mL of hCES2. Assays were carried out with a
blank containing all components except CES1 and CES2,
respectively.
ABTSꢂþ working solution in ethanol (100
mM final concentration),
and after 1 h of incubation the decrease in absorbance was
measured spectrophotometrically at 734 nm using a Bio-Rad xMark
microplate UV/VIS spectrophotometer (Bio-Rad, Hercules, CA,
USA). Ethanol blanks were run in each assay. Values were obtained
from three replicates of each sample and three independent
experiments.
Ellman’s colorimetric assay was used to measure AChE and BChE
activity in 0.1 M K/Na phosphate buffer at pH 7.5, 25 ꢁC [81]. Final
concentrations of reactants were 0.33 mM DTNB, 0.02 unit/mL of
AChE or BChE and 1 mM of substrate (ATCh or BTCh, respectively).
Reagent blanks consisted of reaction mixtures without enzyme to
assess non-enzymatic hydrolysis of substrates.
Test compounds were dissolved in DMSO. Reaction mixtures
contained a final DMSO concentration of 2% (v/v). This concentra-
tion of DMSO on its own did not significantly affect the activity of
any of the enzymes in the study (data not shown). Enzyme inhi-
Antioxidant activity was reported as Trolox equivalent antioxi-
dant capacity (TEAC values), consisting of the ratio between the
slopes obtained from the linear correlation for concentrations of
the tested compounds and Trolox with absorbance of ABTS radical.
For the most active compounds, we also determined IC50 values
(compound concentration required for 50% reduction of the ABTS
radical).
4.5. Cytotoxicity studies
bition was first assessed at a single concentration of 20
m
M for each
Human fetal mesenchymal stem cells (FetMSC), non-
immortalized fibroblast-like cells obtained from bone marrow of
5- to 6-week-old fetuses [82], were maintained in F12/DMEM
compound after a 10 min incubation at 25 ꢁC in three separate
experiments. The most active compounds were then selected for
determination of the IC50 (inhibitor concentration resulting in 50%
inhibition of control enzyme activity). Compounds (eight concen-
trations ranging between 10ꢀ12 and 10ꢀ4 M were selected to ach-
ieve 20e80% inhibition) were incubated with each enzyme for
10 min at 25 ꢁC. Substrate was then added and residual enzyme
activity relative to an inhibitor-free control was measured using a
FLUOStar Optima microplate reader (LabTech, Ortenberg,
Germany).
medium supplemented with 10% (v/v) FBS,
L-glutamine (3 mM), 50
IU/ml penicillin and 50 g/ml streptomycin. The FetMSC cell line is
m
a certified culture available from the Russian collection of verte-
brate cell cultures (RCVCC) of the Institute of Cytology, Russian
Academy of Sciences. The cell line was purchased from RCVCC in
2014. Mycoplasma contamination was tested quarterly with DAPI
staining. A cell-line authentication procedure was not set; hence,
cells were not used for more than 10e15 passages.
To investigate the cytotoxicity of compounds in FetMSC cells, the
MTT cell viability assay was used [83]. Cells were plated in 96-well
plates (6 ꢃ 103 cells/well). At 24 h after plating, the incubation
medium was replaced with fresh medium containing test com-
4.3. Porcine liver CES, human AChE, and equine BChE inhibition
kinetics: inhibition constants at steady state
To elucidate the inhibition mechanisms for active compounds,
porcine liver CES, human erythrocyte AChE, and equine serum
BChE residual activities were determined in the presence of 3
pounds at concentrations of 100, 250 and 500 mM. Control cells
were treated with 0.1% (v/v) DMSO. After 72 h exposure, 0.5 mg/ml
of MTT was added to each well and cells were incubated for 4 h,
14

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
then culture medium was aspirated and MTT formazan was dis-
solved in 100% DMSO. MTT staining intensity was read at 570 nm.
The MTT staining of control cells was taken as 100%.
256 runs, 25 ꢃ 106 evaluations, 27 ꢃ 104 generations, and a pop-
ulation size of 3000.
Figures for molecular structures were prepared with PyMOL
(www.pymol.org).
4.6. Acute toxicity evaluation
4.8. Prediction of ADMET and physicochemical profiles
The toxicity evaluation was performed on CD-1 mice. Laboratory
animals (CD-mice) were obtained from the Animal Unit “Pushino”
at the M.M. Shemyakin and Yu. A. Ovchinnikov Institute of Bio-
Human intestinal absorption (HIA) [96], blood-brain barrier
permeability (LogBB) [97], and hERG-mediated cardiac toxicity risk
(channel affinity pKi and inhibitory activity pIC50) [98] were esti-
mated using the integrated online service for ADMET properties
prediction ADMET Prediction Service [99] (http://qsar.chem.msu.
ru/admet/Accessed Dec 01, 2020). It implements the predictive
QSAR models based on accurate and representative training sets,
fragmental descriptors, and artificial neural networks. The lip-
ophilicity (LogPow) and aqueous solubility (pS) were estimated by
the ALogPS 3.0 neural network model implemented in the OCHEM
platform [100]. The quantitative estimate of drug-likeness (QED)
values [101] were calculated by RDKit version 2020.03.4 software
[102].
organic Chemistry RAS (Russia). Animals were housed in
a
controlled environment with a natural light cycle in polypropylene
cages (Bioskape), on standard bedding (Rehofix MK 2000, J.
€
Rettenmaier & Sohne, Germany), supplied with feed for conven-
tional laboratory rodents (Chara, “Assortiment-Agro”, Russia) and
water ad libitum. Animal care and all the procedures were per-
ꢁ
formed by professional staff according to the Federal Law N 61-FZ
[84] and guidelines for pre-clinical study of medicinal products
[85]. The procedure for toxicity evaluation was based on OECD
recommendations [86] and guidelines for pre-clinical study of
medicinal products [85]. For general estimation of the toxicity
range of synthesized compounds, three animals per group (one
dose) were used for several compounds. The tested compounds in
2% (w/v) starch mucilage solution were injected intraperitoneally.
Animals were observed during 14 days, the number of deaths was
counted, and percent viability calculated.
4.9. PAINS analysis
The Pan Assay INterference compoundS (PAINS) alerts were
checked using RDKit version 2020.03.4 software [102]. A descrip-
tion of the experimental evidence for specific activity of the re-
ported compounds with potential PAINS liability is presented in the
following Results and Experimental sections: inhibition in vitro of
porcine liver CES, human CES1 and CES2, human AChE, and equine
BChE; porcine liver CES, human AChE and equine BChE inhibition
kinetics: inhibition constants at steady state; and ABTS radical
cation scavenging assay as a measure of antioxidant activity.
Computational evidence is presented in the sections describing
results and methods of quantum mechanical calculations and
molecular docking.
4.7. Molecular modeling studies
4.7.1. Quantum mechanical calculations
Quantum mechanical optimizations of geometries of all
considered compounds (Z-, E-isomers and major conformers) were
performed with the GAMESS US [87] package at B3LYP/6-31G* and
B3LYP/6e311þþG** (for frontier orbitals calculations) levels of
theory using an implicit PCM solvent model.
4.7.2. Molecular docking
An X-ray crystal structure of human CES was used for molecular
modeling studies due to its high quality. Previous studies showed
that the available X-ray crystal structure of the porcine enzyme had
certain problems impairing its use for molecular modeling [56],
while results with the homology model of the porcine enzyme
were very close to those obtained with the X-ray crystal structure of
the human protein.
The protein structure of hCES1 for molecular docking was pre-
pared using the A-chain of the X-ray crystal structure PDB ID 2H7C
[88] (hCES; 2.00 Å resolution); the missing Glu99 side chain was
reconstructed manually. The homology model of hCES2 was pre-
pared as described previously [56]. For cholinesterases, X-ray
crystal structures of human AChE (PDB ID 4EY4 and 4EY7 [89]) and
human BChE (PDB ID 1P0I [90]) were used. The BChE structure was
prepared as described previously [91]. For the other two structures,
all crystallographic waters and co-crystallized molecules were
removed. Hydrogen atoms were added with Reduce [92] software,
which protonates histidine rings with regard to the surrounding
hydrogen-bonding network. The MD-optimized homology model
of CES2 was described recently [56].
4.10. Statistical analyses
Results are presented as mean SEM (n ꢄ 3 independent ex-
periments) calculated using GraphPad Prism version 6.05 for
Windows, GraphPad Software (La Jolla, CA, USA). Plots, linear re-
gressions, and values of IC50, rate, and equilibrium constants were
determined using Origin 6.1 for Windows, OriginLab (North-
ampton, MA, USA).
Declaration of competing interest
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.
Acknowledgments
This work was supported by RFBR (project 20-03-00312) (syn-
thesis and characterization of compounds) and the IPAC RAS State
Targets Project No. 0090-2019-0005 (biochemical studies).
Analytical studies were carried out using equipment of the Center
for Joint Use “Spectroscopy and Analysis of Organic Compounds” at
the Postovsky Institute of Organic Synthesis of UB RAS; biochemical
studies were carried out using equipment of the “Center for Col-
lective Use of IPAC RAS”. The authors thank Dr. O.P. Krasnykh (Perm
National Research Polytechnic University, Russia) for determination
of acute toxicity. Computer modeling was carried out using
equipment from the shared research facilities of the HPC
computing resources at Lomonosov Moscow State University [103].
Partial atomic charges on ligand atoms were assigned from QM
€
data according to the Lowdin [93] scheme, corresponding to a
recent comparison of different atomic charge distribution schemes
[94].
Molecular docking was performed with AutoDock 4.2.6 software
[71]. The grid box for docking included the entire active site gorge
of CES and AChE (22.5 Å ꢃ 22.5 Å ꢃ 22.5 Å grid box dimensions),
and BChE (15 Å ꢃ 20.25 Å ꢃ 18 Å) with a grid spacing of 0.375 Å. The
main Lamarckian Genetic Algorithm (LGA) [95] parameters were
15

G.F. Makhaeva, S.V. Lushchekina, N.P. Boltneva et al.
European Journal of Medicinal Chemistry 218 (2021) 113385
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s13238-017-0437-z.
We thank acknowledge also the Joint Supercomputer Center of the
Russian Academy of Sciences for provision of computational time.
Support for RJR’s contributions was provided in part by a research
grant from the Alternatives Research and Development Foundation
(ARDF) and an Mcubed grant from the University of Michigan. The
funding sources had no role in the study design; collection, analysis
and interpretation of data; writing of the manuscript; or the deci-
sion to submit the article for publication.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113385.
Abbreviations
CES
mammalian carboxylesterase
human carboxylesterase 1
human carboxylesterase 2
acetylcholinesterase
butyrylcholinesterase
trifluoromethyl ketone
hCES1
hCES2
AChE
BChE
TFK
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tion of prodrugs, Curr. Drug Metabol. 11 (2010) 793e805, https://doi.org/
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in contemporary drug design and development, Nat. Rev. Drug Discov. 17
(2018) 559e587, https://doi.org/10.1038/nrd.2018.46.
TEAC
ROS
Trolox equivalent antioxidant capacity
radical oxygen species
RNS
radical nitrogen species
4-NPA
ATCh
BTCh
DTNB
DMSO
ADMET
CNS
4-nitrophenyl acetate
acetylthiocholine iodide
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morphism, Biomol Ther 17 (2009) 335e347, https://doi.org/10.4062/
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butyrylthiocholine iodide
5,50-dithio-bis-(2-nitrobenzoic acid)
Dimethyl Sulfoxide
absorption, distribution, metabolism, excretion, toxicity
central nervous system
Metabol.
Toxicol.
6
(2010)
261e271,
https://doi.org/10.1517/
17425250903483215.
[19] S.D. Varfolomeev, S.V. Lushchekina, A.V. Nemukhin, A.M. Kulakova, E.D. Kots,
G.F. Makhaeva, H. Delacour, O. Lockridge, P. Masson, Molecular poly-
morphism of human enzymes as the basis of individual sensitivity to drugs.
Supercomputer-assisted modeling as a tool for analysis of structural changes
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https://doi.org/10.1007/s11172-016-1487-8.
LogBB
HIA
blood-brain barrier permeability
human intestinal absorption [%]
hERG pKi hERG potassium channel affinity [elog(M)]
[20] H.B. Rasmussen, D. Bjerre, K. Linnet, G. Jurgens, K. Dalhoff, H. Stefansson,
T. Hankemeier, R. Kaddurah-Daouk, O. Taboureau, S. Brunak, T. Houmann,
P. Jeppesen, A.K. Pagsberg, K. Plessen, J. Dyrborg, P.R. Hansen, P.E. Hansen,
T. Hughes, T. Werge, I. Consortium, Individualization of treatments with
drugs metabolized by CES1: combining genetics and metabolomics, Phar-
macogenomics 16 (2015) 649e665, https://doi.org/10.2217/pgs.15.7.
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M. Paile-Hyvarinen, M.K. Itkonen, M.T. Holmberg, V. Karja, V.T. Mannisto,
P.J. Neuvonen, J. Pihlajamaki, J.T. Backman, M. Niemi, Effects of genetic
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hERG pIC50 hERG potassium channel inhibitory activity [elog(M)]
LogPow
pS
octanol-water partition coefficient
aqueous solubility [elog(M)]
QED
ABTS
Trolox
quantitative estimate of drug-likeness
2,2ʹ-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic
acid
QM/MM quantum mechanics/molecular mechanics
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide;
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variation in carboxylesterase drug metabolism, Expet Opin. Drug Metabol.
FetMSC cells human fetal mesenchymal stem cells
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