2-Hydroxy-N-Phenylbenzamides and their esters inhibit acetylcholinesterase and Butyrylcholinesterase

Article abstract of DOI:10.3390/biom9110698

The development of novel inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) represents a viable approach to alleviate Alzheimer’s disease. Thirty-six halogenated 2-hydroxy-N-phenylbenzamides (salicylanilides) with various substitution patterns and their esters with phosphorus-based acids were synthesized in yields of 72% to 92% and characterized. They were evaluated for in vitro inhibition of AChE from electric eel and BuChE from equine serum using modified Ellman’s spectrophotometric method. The benzamides exhibited a moderate inhibition of AChE with IC₅₀ values in a narrow concentration range from 33.1 to 85.8 μM. IC₅₀ values for BuChE were higher (53.5–228.4 μM). The majority of derivatives inhibit AChE more efficiently than BuChE and are comparable or superior to rivastigmine—an established cholinesterases inhibitor used in the treatment of Alzheimer’s disease. Phosphorus-based esters especially improved the activity against BuChE with 5-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl diethyl phosphite 5c superiority (IC₅₀ = 2.4 μM). This derivative was also the most selective inhibitor of BuChE. It caused a mixed inhibition of both cholinesterases and acted as a pseudo-irreversible inhibitor. Several structure-activity relationships were identified, e.g., favouring esters and benzamides obtained from 5-halogenosalicylic acids and polyhalogenated anilines. Both 2-hydroxy-N-phenylbenzamides and esters share convenient physicochemical properties for blood-brain-barrier penetration and thus central nervous system delivery.

Full text of DOI:10.3390/biom9110698

biomolecules
Communication
2-Hydroxy-N-phenylbenzamides and Their Esters
Inhibit Acetylcholinesterase
and Butyrylcholinesterase
Martin Krátký 1,
*
, Šárka Šteˇpánková 2 , Neto-Honorius Houngbedji 1, Rudolf Vosátka 1,
Katarína Vorcˇáková 2 and Jarmila Vinšová 1
1
Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles
University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic;
houngben@faf.cuni.cz (N.-H.H.); vosatkar@faf.cuni.cz (R.V.); vinsova@faf.cuni.cz (J.V.)
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of
2
Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic; Sarka.Stepankova@upce.cz (Š.Š.);
katarina.vorcakova@upce.cz (K.V.)
*
Correspondence: martin.kratky@faf.cuni.cz; Tel.: +420-495067343; Fax: +420-495067166
Received: 16 September 2019; Accepted: 1 November 2019; Published: 5 November 2019
Abstract:
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
The development of novel inhibitors of acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) represents a viable approach to alleviate Alzheimer’s disease. Thirty-six
halogenated 2-hydroxy-N-phenylbenzamides (salicylanilides) with various substitution patterns and
their esters with phosphorus-based acids were synthesized in yields of 72% to 92% and characterized.
They were evaluated for in vitro inhibition of AChE from electric eel and BuChE from equine
serum using modified Ellman’s spectrophotometric method. The benzamides exhibited a moderate
inhibition of AChE with IC50 values in a narrow concentration range from 33.1 to 85.8
µM. IC50 values
for BuChE were higher (53.5–228.4 M). The majority of derivatives inhibit AChE more efficiently than
µ
BuChE and are comparable or superior to rivastigmine—an established cholinesterases inhibitor used
in the treatment of Alzheimer’s disease. Phosphorus-based esters especially improved the activity
against BuChE with 5-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl diethyl phosphite 5c
superiority (IC50 = 2.4 µM). This derivative was also the most selective inhibitor of BuChE. It caused
a mixed inhibition of both cholinesterases and acted as a pseudo-irreversible inhibitor. Several
structure-activity relationships were identified, e.g., favouring esters and benzamides obtained from
5-halogenosalicylic acids and polyhalogenated anilines. Both 2-hydroxy-N-phenylbenzamides and
esters share convenient physicochemical properties for blood-brain-barrier penetration and thus
central nervous system delivery.
Keywords: acetylcholinesterase; benzamides; butyrylcholinesterase; enzyme inhibition; esters;
in vitro inhibition; phosphorus derivatives; salicylanilides
1. Introduction
Alzheimer’s disease (AD) is a chronic neurodegenerative disease and the most common cause of
dementia, with heavy social and economic costs and problems, which gradually worsen over a number
of years [
1,
2]. Fifty million people worldwide are living with dementia and the World Alzheimer report
2018 estimates an incensement to more than 152 million cases by 2050 [
3
]. The disease is associated
with a loss of cholinergic neurons in the brain and decreased levels of neurotransmitter acetylcholine
(ACh). Enzymes called cholinesterases terminate its action. The brain contains two major forms of
cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Both enzymes may
have roles in the aetiology and progression of AD beyond regulation of synaptic ACh levels. They differ
Biomolecules 2019, 9, 698; doi:10.3390/biom9110698
www.mdpi.com/journal/biomolecules

Biomolecules 2019, 9, 698
2 of 16
genetically, structurally and in their kinetics. The enzyme acetylcholinesterase (a serine hydrolase
found at neuromuscular junctions and cholinergic brain synapses) is involved in the termination of
impulse transmission by rapid hydrolysis of ACh in numerous cholinergic pathways in the central
and peripheral nervous systems. Butyrylcholinesterase serves as a co-regulator of AChE activity [
While the overall amount of AChE in the brain decreases with the progression of AD, the enzymatic
activity of BuChE remains unchanged or even increases and can compensate the loss of AChE [ ].
The presence of AChE in the patients’ brains is associated with -amyloid plaques and neurofibrillary
tangles (NFT), which are the hallmarks of the AD pathogenesis. Increased AChE levels have been
discovered around amyloid plaques and NFT [ ]. Analogously, overexpression of BuChE is involved
in the formation of -amyloid protein and senile plaques. Therefore, inhibiting BuChE activity could
4].
5
β
6
β
prove beneficial as a hypothetically disease-modifying approach to AD; BuChE is considered as an
adjunctive therapeutic target in later stages of AD [7].
Based on these facts, the main medicines used in the treatment of AD are AChE inhibitors.
In addition to potent enzyme inhibition, these molecules must cross the blood-brain barrier (BBB) to be
sufficiently available in the central nervous system (CNS). According to the mode of action, AChE
inhibitors are irreversible or reversible. Reversible inhibitors, competitive or non-competitive (such
as the current AD drugs donepezil and galantamine) and pseudo-irreversible inhibitor rivastigmine,
a unique dual inhibitor of AChE and BuChE [8], have therapeutic applications, while toxic effects are
normally associated with irreversible AChE inhibitors such as organophosphate pesticides and nerve
warfare agents.
Moreover, inhibitors of cholinesterases can be used in the therapy of other diseases than AD,
such as other dementias, sleep behaviour disorder, myasthenia gravis, glaucoma, postoperative ileus,
bladder distention and intoxication of anticholinergic agents [9–11]. Cholinergic hypothesis was the
first comprehensive paradigm to explain the memory deficit and cognitive impairment in AD. After
that, the loss of cholinergic neurons has been associated with other neurodegenerative disorders.
Later, the cholinergic hypothesis in dementia has lost some interest, in particular because of the
limited clinical efficacy of ACh augmentation therapy and the rising highlight of the role of A
β
and tau protein pathology. Recently, the cholinergic hypothesis has been “revisited” due to many
reasons [12]. In addition to this approach, other hypotheses have been formulated, e.g., amyloid
cascade, hyperphosphorylation of tau protein, oxidative stress, metal ions hypothesis, or inflammatory
hypothesis [13].
Current treatment cannot stop the disease from progressing; it can only temporarily improve
symptoms or slow down the progress at best. Thus, a worldwide effort is needed to find better ways
to treat the Alzheimer’s disease, delay its onset, and prevent it from developing. Despite multiple
features of AD pathogenesis and a wide range of proposed potential targets, no better therapeutic
interventions than inhibitors of cholinesterases and NMDA-receptor antagonist memantine have been
introduced. AChE inhibition may decrease A
efficacy of cholinesterases inhibitors, AChE is one of the most investigated targets for AD [
β
plaques and NFT formation. Despite moderate clinical
]. Thus,
6
AChE- and BuChE-oriented inhibitors-related research can still be considered useful. In particular,
these dual inhibitors modulating also other pathogenesis pathway(s) are up-to-date.
2-Hydroxy-N-phenylbenzamides (salicylanilides or 2-hydroxybenzanilides) are weakly acidic
phenolic compounds. Depending on their substitution, they can be divided into different
groups, including halogenated (mono-, di- and polyhalogenated compounds) and nonhalogenated
derivatives, O-esters including carbamates with antibacterial, mainly antimycobacterial and
antifungal activity [14–18]. Several halogenated salicylanilides are widely used as veterinary
drugs against helminths and ectoparasites [19]. Commercially available closantel, niclosamide,
oxyclozanide, rafoxanide and resorantel are also potent against Gram-positive bacteria such as
Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes [20]. Niclosamide and
N-(3,5-dichlorophenyl)-5-chloro-2-hydroxybenzamide exhibited anti-filamentation and anti-biofilm
activity against fungus Candida albicans, and multidrug-resistant yeast Candida auris [21].

Biomolecules 2019, 9, 698
3 of 16
We have found salicylanilide O-substituted derivatives with significant activity against AChE and
BuChE. Diethyl phosphates act as pseudo-irreversible cholinesterases inhibitors [22], N-phenethyl [23
]
and N,N-diphenyl carbamates [24] produced clearly favourable BuChE inhibition when compared
to other analogues. In addition, salicylanilide diethyl thiophosphates were active, especially against
AChE [25]—but there is no study dealing with the inhibition property of parent benzamides with free
phenolic group. That is why we initiated this systematic study dealing with the halogenated
salicylanilides as potential inhibitors of both cholinesterases and their phosphorus analogues.
The halogenation ensures sufficient lipophilicity, which should contribute to the passive targeting of
CNS. The current study should answer whether parent salicylanilides are able to inhibit AChE and
BuChE, how selective and if the intrinsic activity of this scaffold could participate in the inhibition
previously described for salicylanilide carbamates and (thio)phosphates [22–25]. The second goal
is focused on the influence of esterification by phosphorus-based acids on enzyme inhibition and
selectivity. The compounds with low IC50 values and dual inhibition of AChE and BuChE with optimal
physicochemical properties to penetrate BBB were aimed at in this study.
2. Materials and Methods
2.1. Chemistry
2.1.1. General Methods
All the reagents and solvents were purchased from Sigma-Aldrich (Darmstadt, Germany) or
Penta Chemicals (Prague, Czech Republic) and they were used as received. The reactions and the
purity of the products were monitored by thin-layer chromatography using a mixture with a ratio
of toluene to ethyl acetate of 4:1 or 9:1 (v/v) as the eluent. Plates were coated with 0.2 mm Merck
60 F254 silica gel (Merck Millipore, Darmstadt, Germany) and were visualized by UV irradiation
(254 nm). The melting points were determined on a Büchi Melting Point B-540 apparatus (BÜCHI,
Flawil, Switzerland) using open capillaries. The reported values are uncorrected. Infrared spectra were
recorded on a FT-IR spectrometer using ATR-Ge method (Nicolet 6700 FT-IR, Thermo Fisher Scientific,
Waltham, MA, USA) in the range 650–4000 cm−1. The NMR spectra were measured in DMSO-d6,
CDCl3 or acetone-d6 at ambient temperature using a Varian V NMR S500 instrument (500 MHz for 1H
and 126 MHz for 13C; Varian Comp. Palo Alto, CA, USA). The chemical shifts
δ are given in ppm with
respect to tetramethylsilane as an internal standard. The coupling constants (J) are reported in Hz.
Elemental analysis (C, H, N) was performed on an automatic microanalyser CHNS-O CE instrument
(FISONS EA 1110, Milano, Italy). Flash chromatography was performed on a CombiFlash Rf 200
automated chromatograph (Teledyne Isco, Lincoln, NE, USA) using columns filled with Kieselgel 60,
0.040–0.063 mm (Merck, Germany) and a detection wavelength of 280 nm.
2.1.2. Synthesis of 2-Hydroxy-N-phenylbenzamides 1–4
The microwave-assisted synthesis of salicylanilides 1–4 was described previously by our group
(e.g., [26]). Briefly, appropriate salicylic acid (1 mmol) was suspended in 10 mL of chlorobenzene, then
substituted aniline (1 mmol) was added, followed by phosphorus trichloride (0.5 mmol). The reaction
mixture was irradiated in microwave reactor (530 W, 25 min; MicroSYNTH Milestone Ethos 1600
(Milestone, Bergamo, Italy)). The reaction mixture was filtered while hot and was then let to stand at
+4 ◦C for 12 h. The resulted precipitate was filtered off and crystallized from ethanol/water to obtain
pure benzamides.
Some of the compounds
we adopted the salicylanilides from two works: Kr
3f 3k, and 4k) and Paraskevopoulos et al. [15] (derivatives 2l
(4a, 4b, 4f, 4h–4j) were prepared as synthetic precursors of salicylanilide diethyl phosphates [17].
1
–
4
were synthesized and reported previously by our group. For this study,
tk et al. [26] (compounds 2a 2c 2f 2k 3a 3c
3l 4c 4g, and 4l). Bromosalicylanilides
á
ý
1,
–
,
–
,
– ,
–
,
,
,

Biomolecules 2019, 9, 698
4 of 16
The identity of the known compounds was established using NMR and IR spectroscopy by the
comparison with previously reported data. Additionally, their purity was checked by melting points
measurement and elemental analysis.
◦
5-Chloro-N-(3,5-dichlorophenyl)-2-hydroxybenzamide (2d). White solid; yield 82%; mp 246–248 C
1
(247–249 ◦C [27]). IR: 3324 (N-H), 1630 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.54 (1H, s, OH),
10.54 (1H, s, NH), 8.09 (2H, d, J = 2.3 Hz, H20, H6’), 7.85 (1H, d, J = 2.7 Hz, H6), 7.45 (1H, dd, J = 8.8,
2.7 Hz, H4), 7.37 (1H, t, J = 2.3 Hz, H40), 7.01 (1H, d, J = 8.8 Hz, H3). 13C NMR (126 MHz, DMSO):
δ
165.2, 156.7, 139.8, 133.3, 130.6, 128.7, 124.1, 123.0, 120.3, 120.2, 119.2. Anal. C 49.41, H 2.31, N 4.51%,
calculated for C13H8Cl3NO2 (316.56), C 49.32, H 2.55, N 4.42%.
5-Chloro-N-(3,4,5-trichlorophenyl)-2-hydroxybenzamide (2e). White solid; yield 79%; mp 286–288.5 ◦C
1
(287–290 ◦C [27]). IR: 3323 (N-H), 1628 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.53 (1H, s, OH),
10.63 (1H, s, NH), 8.04 (2H, s, H20, H60), 7.83 (1H, d, J = 2.7 Hz, H6), 7.45 (1H, dd, J = 8.6, 2.5 Hz, H4),
7.05 (1H, d, J = 8.8 Hz, H3). 13C NMR (126 MHz, DMSO):
δ
165.8, 157.4, 149.8, 139.0, 134.6, 132.2, 129.4,
123.0, 120.7, 119.2, 113.5. Anal. C 44.34, H 1.94, N 4.00%, calculated for C13H7Cl4NO2 (351.00), C 44.48,
H 2.01, N 3.99%.
◦
4-Chloro-N-(3,5-dichlorophenyl)-2-hydroxybenzamide (3d). White solid; yield 80%; mp 257–259 C
1
(255–256 ◦C [28]). IR: 3310 (N-H), 1605 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.70 (1H, s, OH),
10.49 (1H, s, NH), 7.84–7.78 (3H, m, H6, H20, H60), 7.31 (1H, t, J = 1.9 Hz, H40), 7.05–6.99 (2H, m, H3,
H5). 13C NMR (126 MHz, DMSO):
166.2, 159.0, 140.9, 137.7, 133.7, 131.4, 122.3, 120.1, 118.8, 118.2,
δ
116.4. Anal. C 49.60, H 2.41, N 4.70%, calculated for C13H8Cl3NO2 (316.56), C 49.32, H 2.55, N 4.42%.
◦
4-Chloro-N-(3,4,5-trichlorophenyl)-2-hydroxybenzamide (3e). White solid; yield 76%; mp 260–262 C
1
(261–262 ◦C [29]). IR (ATR): 3320 (N-H), 1609 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.75 (1H, s,
OH), 10.55 (1H, s, NH), 8.03 (2H, s, H20, H60), 7.83 (1H, d, J = 8.5 Hz, H6), 7.06 (1H, d, J = 2.1 Hz, H3),
7.02 (1H, dd, J = 8.5, 2.1 Hz, H5). 13C NMR (126 MHz, DMSO):
δ
165.6, 158.3, 138.6, 136.5, 133.0, 131.4,
120.6, 119.6, 119.2, 116.9, 114.0. Anal. C 44.40, H 1.97, N 4.10%, calculated for C13H7Cl4NO2 (351.00), C
44.48, H 2.01, N 3.99%.
◦
5-Bromo-N-(3,5-dichlorophenyl)-2-hydroxybenzamide (4d). White solid; yield 81%; mp 244–245.5 C
1
(243–245 ◦C [28]). IR: 3327 (N-H), 1628 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.47 (1H, s, OH),
10.54 (1H, s, NH), 7.93 (1H, d, J = 2.5 Hz, H6), 7.81 (2H, d, J = 1.9 Hz, H20, H60), 7.56 (1H, dd, J = 8.8,
2.6 Hz, H4), 7.33 (1H, t, J = 1.9 Hz, H40), 6.95 (1H, d, J = 8.8 Hz, H3). 13C NMR (126 MHz, DMSO):
δ
165.2, 156.8, 140.8, 136.1, 134.2, 131.6, 123.5, 121.1, 119.6, 118.4, 110.4. Anal. C 43.40, H 2.27, N 3.75%,
calculated for C13H8BrCl2NO2 (361.02), C 43.25, H 2.23, N 3.88%.
5-Bromo-N-(3,4,5-trichlorophenyl)-2-hydroxybenzamide (4e). White solid; yield 78%; mp 297–298.5 ◦C
1
(297–299 ◦C [28]). IR: 3322 (N-H), 1634 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.46 (1H, s, OH),
10.57 (1H, s, NH), 8.01 (2H, s, H20, H60), 7.91 (1H, d, J = 2.6 Hz, H6), 7.56 (1H, dd, J = 8.8, 2.6 Hz, H4),
6.95 (1H, d, J = 8.8 Hz, H3). 13C NMR (126 MHz, DMSO):
δ
165.2, 156.7, 138.6, 136.2, 133.5, 131.7, 124.4,
121.0, 120.6, 119.6, 110.4. Anal. C 39.41, H 1.91, N 3.55%, calculated for C13H7BrCl3NO2 (395.46), C
39.48, H 1.78, N 3.54%.
2.1.3. Synthesis of Phosphorus-Based Derivatives 5
The syntheses of diethyl phosphate 5a and thiophosphate 5b were reported previously by our
group [17,25]. It is based on the reaction of appropriate salicylanilide with a mild excess of diethyl
chlorophosphate or diethyl chlorothiophosphate in the presence of a tertiary base.
Diethyl phosphite 5c was obtained by the following procedure: 1 mmol of the salicylanilide 3k
was suspended in 10 mL of dichloromethane (DCM) and treated by triethylamine (1.5 of equivalents).

Biomolecules 2019, 9, 698
5 of 16
After a complete dissolution, diethyl chlorophosphite was added in one portion (1.5 of equivalents).
The reaction mixture was stirred for 24 h at room temperature. Then, it was evaporated to provide a
viscous liquid. Ethyl acetate was added, and the insoluble portion was filtered off. The filtrate was
washed sequentially by sodium carbonate solution (10%), hydrochloric acid (0.1 M) and saturated
brine. The organic phase was dried using sodium sulphate and evaporated to provide a viscous oily
liquid, which was purified using flash chromatography (mobile phase composition was with a ratio of
ethyl acetate to hexane of 1:3 (v/v)).
Cyclic analogues 5d and 5e were synthesized by modified procedure adopted from [30]. 1 mmol of
the salicylanilide 3k was suspended in 10 mL of dichloromethane (DCM) and treated by triethylamine (3
of equivalents). After a complete dissolution, phenylphosphonic dichloride (or phenylphosphonothioic
dichloride) was added in one portion (1.5 of equivalents). The reaction mixture was heated for 4 h
and then stirred at room temperature. After 20 h, it was evaporated to dryness. The resulting crystals
were suspended in ethyl acetate. The insoluble portion was filtered off and the filtrate was washed
sequentially by sodium carbonate solution (10%), hydrochloric acid (0.1 M) and saturated brine. The
organic phase was dried using sodium sulphate and the crystallization was initiated by the addition
of n-hexane. The reaction mixture was stored for 24 h at +4 ◦C. Then, the resulting precipitate was
filtered off and the crystals were recrystallized from ethyl acetate/hexane mixture.
5-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl diethyl phosphite (5c). Brownish viscous oil; yield
28%. IR: 3310 (N-H), 1684 (C=O), 1601, 1323, 1292, 1165, 1118, 1066, 1033, 964, 845, 766, 689 cm−1
.
1H-NMR (500 MHz, acetone-d6):
1.1 Hz, H3), 7.76–7.72 (2H, m, H20, H60), 7.52–7.51 (1H, m, H6), 7.44 (1H, ddd, J = 8.3, 2.0, 0.9 Hz, H4),
4.28-4.20 (4H, m, CH2), 1.29 (6H, td, J = 7.1 Hz, J = 1.1 Hz, CH3). 13C NMR (126 MHz, CDCl3):
162.0,
δ
9.99 (1H, s, NH), 8.06-8.02 (2H, m, H30, H50), 7.80 (1H, dd, J = 8.3,
δ
147.5 (d, J = 7.1 Hz), 141.3, 138.1 (d, J = 2.1 Hz), 132.9 (d, J = 1.0 Hz), 126.2 (d, J = 1.5 Hz), 126.0 (d,
J = 1.7 Hz), 126.2 (q, J = 3.8 Hz), 126.2 (q, J = 32.6 Hz), 124.1 (q, J = 270.0 Hz), 121.7 (d, J = 3.0 Hz), 119.6,
64.1 (d, J = 6.0 Hz), 14.0 (d, J = 6.8 Hz). Anal. C 49.68, H 4.29, N 3.09%, calculated for C18H18ClF3NO4P
(435.76), C 49.61, H 4.16, N 3.21%.
(RS)-7-Chloro-2-phenyl-3-[4-(trifluoromethyl)phenyl]-3-hydrobenzo[e][1,3,2]oxazaphosphinin-4-one 2-oxide
(
5d). White solid; yield 76%; mp 185–186 ◦C. IR: 3337, 1656 (C=O), 1575, 1564, 1330, 1219, 1181, 1118,
1
1070, 715 cm−1. H-NMR (500 MHz, CDCl3):
δ
8.16 (1H, d, J = 8.5 Hz, H5), 7.77–7.72 (2H, m, H30,
H50), 7.66–7.56 (3H, m, H20, H60, H400), 7.50-7.44 (2H, m, H300, H500), 7.39-7.28 (4H, m, H6, H8, H200,
H600). 13C NMR (126 MHz, CDCl3):
δ
161.1 (d, J = 4.3 Hz), 151.3 (d, J = 7.9 Hz), 141.9, 136.6, 134.4 (d,
J = 3.2 Hz), 132.3 (d, J = 11.2 Hz), 131.5, 131.0 (q, J = 32.7 Hz), 130.2 (d, J = 2.4 Hz), 129.0 (d, J = 16.1 Hz),
126.5 (q, J = 3.9 Hz), 125.9, 125.5 (d, J = 182.6 Hz), 123.5 (q, J = 272.6 Hz), 119.4 (d, J = 9.2 Hz), 115.8 (d,
J = 2.0 Hz). Anal. C 54.65, H 2.69, N 3.29%, calculated for C20H12ClF3NO3P (437.74), C 54.88, H 2.76,
N 3.20%.
(RS)-7-Chloro-2-phenyl-3-[4-(trifluoromethyl)phenyl]-3-hydrobenzo[e][1,3,2]oxazaphosphinin-4-one
2-sulfide (5e). Yellow solid; yield 52%; mp 136.5–139 ◦C. IR: 3076, 1675 (C=O), 1610, 1417, 1330, 1315,
1299, 1130, 1069, 949, 750, 692 cm−1 1H-NMR (500 MHz, CDCl3):
. δ 8.13 (1H, d, J = 8.4 Hz, H5),
7.94-7.88 (2H, m, H30, H50), 7.64–7.59 (1H, m, H400), 7.53 (2H, d, J = 8.3 Hz, H20, H60), 7.50–7.44 (2H, m,
H300, H500), 7.34 (1H, td, J = 8.5, 1.3 Hz, H6), 7.30–7.24 (3H, m, H8, H200, H600). 13C NMR (126 MHz,
CDCl3):
δ 161.3 (d, J = 3.7 Hz), 151.5 (d, J = 8.7 Hz), 141.7, 134.4 (d, J = 3.3 Hz), 132.2 (d, J = 12.9 Hz),
131.4, 130.6 (d, J = 2.4 Hz), 130.8 (q, J = 32.6 Hz), 129.6 (d, J = 139.3 Hz), 128.7 (d, J = 15.8 Hz), 126.1 (q,
J = 3.8 Hz), 125.8, 123.5 (q, J = 272.5 Hz), 120.9, 119.9 (d, J = 8.4 Hz), 116.6 (d, J = 3.3 Hz). Anal. C 53.11,
H 2.77, N 2.94%, calculated for C20H12ClF3NO2PS (453.80), C 52.94, H 2.67, N 3.09%.

Biomolecules 2019, 9, 698
6 of 16
2.1.4. Determination of Physicochemical Parameters
The molecular weights, logP values and topological polar surface areas (tPSA) were calculated
using the CS ChemOffice Ultra program (version 18.0, CambridgeSoft, Cambridge, MA, USA).
2.2. Determination of Cholinesterases Inhibition
The IC50 values were determined using the spectrophotometric Ellman’s method modified
according to Zdražilová et al. [31], which is a simple, rapid and direct method to determine the
SH and -S-S- group content in proteins. This method is widely used for the screening of the
efficiency of cholinesterases inhibitors. The enzymatic activity is measured indirectly by quantifying
the concentration of the 5-thio-2-nitrobenzoic acid ion formed in the reaction between the thiol
reagent 5,50-dithio-bis(2-nitrobenzoic acid) and thiocholine, a product of substrate hydrolysis (i.e.,
acetylthiocholine; ATCH) by cholinesterases [32].
The enzyme activity in final reaction mixture (2000
of acetylthiocholine (or butyrylthiocholine, BTCH) 40
5,50-dithio-bis(2-nitrobenzoic acid) 0.1 mM for all reactions. The tested compounds were
dissolved in DMSO and then diluted in demineralized water to the concentration of 1 M. For all
µL) was 0.2 U/mL, concentration
µM
and concentration of
µ
tested compounds and standard (rivastigmine), five different concentrations of inhibitor in final
reaction mixture were typically used. All measurements were carried in triplicate and the average
values of reaction rate (v0-uninhibited reaction, vi-inhibited reaction) were used for construction of the
dependence v0/vi vs. concentration of inhibitor. From obtained equation of regression curve, the value
of IC50 was calculated (based on the definition of IC50).
The determination of mechanism of enzyme inhibition was described in detail previously [22].
The inhibitor 5c was evaluated at three different concentrations for each enzyme.
For the determination of the type of inhibition Lineweaver-Burk plot [33] was used and the
measuring procedure was similar to that for the determination of IC50 (modified Ellman’s method was
used). The enzyme activity in the final reaction mixture (2000
µL) was 0.2 U/mL, a concentration of
ATCH or BTCH 20–80
µ
M and a concentration of 5,50-dithio-bis(2-nitrobenzoic acid) 0.1 mM. For each
of the substrate concentrations, four different concentrations of inhibitor were chosen. The dependence
absorbance vs. time was observed and the reaction rate was calculated. All measurements were
carried in duplicate and the average values of reaction rate were used for the construction of the
Lineweaver-Burk plot. From obtained equations of regression curves, the values of KM (Michaelis
constant) and Vm (maximum velocity) were calculated and the type of inhibition was evaluated.
Acetylcholinesterase was obtained from electric eel (Electrophorus electricus L.) and
butyrylcholinesterase was from an equine serum. Rivastigmine was used as a reference drug.
All of the enzymes and rivastigmine were purchased from Sigma-Aldrich (Prague, Czech Republic).
3. Results and Discussion
3.1. Chemistry
2-Hydroxy-N-phenylbenzamides 1–4l were synthesized from substituted salicylic acids and
anilines using phosphorus trichloride (PCl3) under microwave (MW) irradiation (Scheme 1). When
compared to analogous reaction performed under refluxing for 6 h, this MW-mediated synthesis offers
shorter reaction time and higher yields (72–92%) [26]. The majority of these benzamides have been
reported in our previous studies [15,17,26]; for details, see Section 2.1.2.
The selected benzamide 3k was esterified using corresponding chlorides of phosphorus-based
acids. It was converted by triethylamine (Et3N; 1.5 of equivalents for 5a
and 5e) in situ into triethylammonium salts in dichloromethane (DCM) and then a mild excess (1.5 of
equivalents) of chlorides were added [17 25]. After 24 h of stirring, the final esters (Scheme 2) were
–5c, 3 of equivalents for 5d
,
5
obtained in yields of 28% to 93%. The preparation of the cyclic analogues 5d and 5e involved also
initial refluxing for 4 h.

Biomolecules 2019, 9, 698
7 of 16
Scheme 1. Synthesis of salicylanilides 1-4
(R1 = H, 5-Cl, 4-Cl, 5-Br; R2 = H, 3-F, 3-Cl, 3-Br, 3-CF3, 4-F,
4-Cl, 4-Br, 4-CF3, 3,4-diCl, 3,5-diCl, 3,4,5-triCl, 3,5-bis-CF3; Ph-Cl: chlorobenzene).
Scheme 2. Synthesis of the esters 5 (X = O, S, or is missing; Y = O, S; DCM: dichloromethane).
The compounds were characterised by melting points, IR, and NMR spectra. Their purity was
checked by thin-layer chromatography and elemental analysis.
The structures of all the compounds are reported in Table 1.
3.2. In Vitro Inhibition of Acetylcholinesterase and Butyrylcholinesterase
Thirty-six benzamides
(EeAChE) and BuChE from equine serum (EqBuChE) using Ellman’s spectrophotometric method
modified according to Zdražilov et al. [31]. Their efficacy is expressed as IC50, i.e., the concentration
1–4l were evaluated as potential inhibitors of AChE from electric eel
á
required for 50% inhibition of the enzymatic activity. Based on inhibition of both cholinesterases,
we calculated selectivity indexes (SI) as the ratio of IC50 value for AChE/IC50 value for BuChE to
express the selectivity quantitatively (Table 1). The results were compared with those obtained for
rivastigmine, an established carbamate drug used in the therapy of Alzheimer’s and Parkinson’s
dementias. Rivastigmine is a dual acylating pseudo-irreversible inhibitor of both AChE and BuChE.
Focusing on inhibition of AChE, IC50 values were in a close range 33.13 (4d)–85.75 (4b
) µM.
These results are fully comparable to those obtained for phenolic carbamate drug rivastigmine
(56.10
(49.71
µ
M). Interestingly, unsubstituted salicylanilide
M) than a majority of salicylanilides, i.e., the halogenation of salicylanilide moiety is not
1 produced a comparatively better inhibition
µ
essential for inhibition of AChE. However, the bromination of salicylic acid in combination with a
polyhalogenated aniline improved the activity ( vs. 4d 4e 4j, and 4k) and provided the three
most active compounds in this study, 5-bromo-2-hydroxy-N-(3,5-dichlorophenyl)benzamide 4d
(33.13 M), followed by 5-bromo-2-hydroxy-N-(3,4,5-trichlorophenyl)benzamide 4e (42.08 M) and
5-bromo-2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide 4j (42.67 µM).
1
,
,
µ
µ

Biomolecules 2019, 9, 698
8 of 16
Table 1. Structures of 2-hydroxybenzamides and IC50 for acetylcholinesterase and butyrylcholinesterase.
Selectivity
AChE/BuChE
Code
R1
R2
IC50 (EeAChE) [µM]
IC50 (EqBuChE) [µM]
1
H
H
3-Cl
4-Cl
3,4-diCl
3,5-diCl
3,4,5-triCl
3-Br
4-Br
3-F
4-F
49.71 ± 3.73
59.50 ± 3.80
76.53 ± 2.17
60.79 ± 6.88
46.21 ± 0.63
51.06 ± 0.49
50.03 ± 0.15
58.25 ± 4.87
58.65 ± 3.98
48.27 ± 8.90
54.41 ± 0.25
58.47 ± 2.80
50.18 ± 0.88
58.08 ± 1.20
56.19 ± 3.23
46.26 ± 0.56
50.15 ± 0.26
57.78 ± 4.05
60.03 ± 0.42
58.32 ± 1.57
60.79 ± 1.74
62.85 ± 1.53
68.28 ± 3.78
60.29 ± 2.39
54.72 ± 2.05
62.04 ± 6.48
85.75 ± 6.10
60.83 ± 3.65
33.13 ± 0.47
42.08 ± 2.41
69.72 ± 9.97
70.30 ± 2.85
54.44 ± 0.73
61.22 ± 1.08
42.67 ± 0.18
44.79 ± 3.44
48.46 ± 1.07
56.10 ± 1.41
205.73 ± 12.15
228.42 ± 11.29
169.60 ± 3.09
186.47 ± 15.69
111.59 ± 7.98
102.72 ± 0.97
141.04 ± 1.13
118.74 ± 6.65
154.40 ± 8.38
132.99 ± 15.81
120.30 ± 3.02
154.65 ± 3.58
64.44 ± 1.34
134.29 ± 0.24
152.00 ± 3.13
100.27 ± 1.15
141.10 ± 2.80
132.36 ± 4.31
167.80 ± 2.06
173.99 ± 4.02
145.66 ± 4.07
151.76 ± 7.58
163.18 ± 11.68
199.53 ± 2.02
150.50 ± 0.87
134.00 ± 1.63
130.61 ± 3.99
122.60 ± 7.20
135.92 ± 0.14
140.07 ± 6.20
145.78 ± 3.53
112.16 ± 1.79
119.24 ± 2.53
136.57 ± 1.47
116.91 ± 2.23
134.81 ± 0.86
53.46 ± 3.20
38.40 ± 1.97
0.24
0.26
0.45
0.33
0.41
0.50
0.35
0.49
0.38
0.36
0.45
0.38
0.78
0.43
0.37
0.46
0.36
0.44
0.36
0.34
0.42
0.41
0.42
0.30
0.36
0.46
0.66
0.50
0.24
0.30
0.48
0.63
0.46
0.45
0.36
0.33
0.91
1.46
2a
2b
2c
2d
2e
2f
2g
2h
2i
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
2j
2k
2l
3-CF3
4-CF3
3,5-bis-CF3
3-Cl
3a
3b
3c
3d
3e
3f
3g
3h
3i
4-Cl
3,4-diCl
3,5-diCl
3,4,5-triCl
3-Br
4-Br
3-F
4-F
3j
3k
3l
3-CF3
4-CF3
3,5-bis-CF3
3-Cl
4a
4b
4c
4d
4e
4f
4g
4h
4i
4-Cl
3,4-diCl
3,5-diCl
3,4,5-triCl
3-Br
4-Br
3-F
4-F
4j
4k
4l
3-CF3
4-CF3
3,5-bis-CF3
Rivastigmine
AChE and BuChE inhibition are expressed as the mean
values for each enzyme are shown in bold.
±
SD (n = three experiments). The two or three lowest IC50
Additionally, several general structure-activity relationships were identified:
1.
2.
3.
4.
Slightly better inhibition is associated with derivatives of 5-substituted salicylic acid (series 2
and 4).
The substitution of the position 3 at the aniline ring led to improved activity when compared to
the position 4 (majority of the series 3 and the pair 2h and 2i are partial exceptions).
The order of preferred aniline substituents in the case of monosubstitution is as follows: CF3 > F
> Br > Cl (series 4); Cl > Br > F > CF3 (series 3); F > Br > CF3 > Cl (series 2).
The di- and tri-substitution of aniline (both by chlorine c–e and trifluoromethyl group l)
resulted predominantly in significantly more potent inhibition of AChE when compared to
monosubstituted analogues.
5.
Regarding polychlorinated anilides, the derivatives of 3,5-dichloroaniline
3,4-dichloroanilides and also to 3,4,5-trichloroanilides ; the additional substitution of the
3,5-dichloro compounds by 4-chlorine (i.e., providing trichloro structures ) is detrimental but
it is still superior to 3,4-dichloroanilides c. Series 3 is a partial exception.
d are superior to
c
e
d
e

Biomolecules 2019, 9, 698
9 of 16
In contrast to AChE, somewhat different results were obtained for BuChE. This enzyme
was inhibited at higher concentrations and in a broader concentration range starting from
53.46
(38.40
of the salicylanilide
µ
M (4l) up to 228.42
M). The halogenation of the salicylanilide core is essential and it improves IC50 values
Two amides share significantly low IC50 values: 5-bromo- and
µM (2a); all the compounds are less potent than rivastigmine
µ
1.
5-chloro-2-hydroxy-N-[3,5-bis(trifluoromethyl)phenyl]benzamides 2l and 4l (64.44 and 53.46 µM,
respectively). The following SAR were revealed:
1.
2.
The most potent inhibition was exhibited by the derivatives of 5-bromosalicylic acid (series
followed by 5-chlorosalicylic acid (2).
The order of preferred atoms on monosubstituted aniline ring is as follows: Br > CF3 > F > Cl
(series ); Cl > F > Br > CF3 (series ); CF3 > F > Br > Cl (series ; i.e., the identical results as
4)
2
3
4
obtained for AChE). There was no sharp difference in the activity of 3- and 4-substituted anilines.
The presence of di- and tri-substituted anilines increases the BuChE inhibition predominantly,
especially for trifluoromethyl derivatives.
3.
4.
Regarding polychlorinated anilides within the series
are superior to 3,5-dichloroanilides and also to 3,4,5-trichloroanilides
SAR was found in the series 2.
3
and
4, the derivatives of 3,4-dichloroaniline
c
d
e. However, the reverse
Regarding selectivity to both cholinesterases, it can be distinguished based on the values of SI.
SI lower than 1 means selectivity for AChE. On the other hand, compounds with SI higher than 1
tends to the selectivity for BuChE. The salicylanilides share an obvious tendency to inhibit AChE
1–4
more efficiently (SI within the range of 0.24 to 0.91; Table 1). Only the bis-trifluoromethyl derivative 4l
exhibited almost identical inhibition of both enzymes.
To investigate the influence of phosphorus-based substituents, a known scaffold for the inhibition
of cholinesterases [8,22,25], of phenolic group on inhibition, we prepared several derivatives of
4-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide 3k, i.e., the salicylanilide with a moderate
activity against AChE and low activity against BuChE. Thus, if the presence of phosphorus-based
moiety was beneficial, a drop in IC50 values would be more significant.
Previously, we synthesized diethyl phosphate 5a [17,22] and diethyl thiophosphate 5b [25]. Both
derivatives share an enhanced inhibition of BuChE (up to 20.3 times) and moreover, thiophosphate 5b
was 6.6 times more effective AChE inhibitor (Table 2). Then, we prepared analogous diethyl phosphite
5c. Regarding BuChE, this modification was the most successful one with IC50 value of 2.37 µM (84.2
times more potent than the parent 3k) and highly selective for BuChE (SI 28.0), but still remained the
dual inhibitor of both cholinesterases. The removal of oxygen from phosphate 5a to phosphite 5c
improved the activity especially against BuChE (4.2 times). Then, we prepared two cyclic analogues,
7-chloro-2-phenyl-3-[4-(trifluoromethyl)phenyl]-3-hydrobenzo[e][1,3,2]oxazaphosphinin-4-one 2-oxide
(
5d)/2-sulfide (5e). The first derivative showed an enhanced inhibitory potency against both enzymes
when compared to parent salicylanilide and, regarding AChE, also when compared to the majority of
the compounds involved in this study (IC50 48.13 M). However, the isosteric replacement of oxygen
µ
by sulphur (5d→5e) resulted in a substantial lower activity comparable to parent amide 3k; it contrasts
with the pair 5a and 5b, where this switch led to a sharply stronger AChE inhibition. The activity of
[1,3,2]oxazaphosphinine-4-ones 5d and 5e suggests that the amidic hydrogen is not essential for the
existence of biological activity, but its presence contributes to more potent enzyme inhibition.
Four phosphorus-based esters (5a
them against AChE (5b 5d). Interestingly, the type of phosphorus functional group can modulate the
AChE/BuChE selectivity. The presence of only oxygen(s) leads to preferential inhibition of BuChE (SI
of 1.93–28.0; 5a 5c and 5d) while the incorporation of sulphur increased the relative affinity to AChE
(SI 0.37 and 0.50 for 5e and 5b, respectively).
–5d) were superior to rivastigmine against BuChE and two of
,
,

Biomolecules 2019, 9, 698
10 of 16
Table 2. Phosphorus-bases inhibitors of AChE and BuChE.
Selectivity
AChE/BuChE
Code
X
IC50 (EeAChE) [µM]
IC50 (EqBuChE) [µM]
5a [22]
5b [25]
5c
5d
5e
O
S
−
O
S
86.3 ± 4.9
9.16 ± 0.17
66.37 ± 1.14
48.13 ± 1.70
63.48 ± 1.41
60.29 ± 2.39
56.10 ± 1.41
9.84 ± 0.06
18.3 ± 1.0
8.77
0.50
28.00
1.93
0.37
0.30
1.46
2.37 ± 0.01
25.00 ± 0.30
170.10 ± 13.58
199.53 ± 2.02
38.40 ± 1.97
3k
Rivastigmine
AChE and BuChE inhibition are expressed as the mean
each enzyme is shown in bold.
±
SD (n = three experiments). The lowest IC50 value for
We have demonstrated unequivocally that both salicylanilides and their phosphorus-based
analogues act as dual AChE-BuChE inhibitors. Based on anti-AD drug rivastigmine, it was
shown that this dual inhibition might be beneficial for the therapy of dementia [34]. The parent
2-hydroxybenzamides inhibit AChE preferentially and their modification did not improve the activity
predominantly. On the other hand, the esterification of phenolic hydroxyl led to a significantly
enhanced inhibition of BuChE. Regarding the structure of phosphorus part of the molecules, it is
possible to modulate selectivity to each cholinesterase.
Mechanism and Type of Inhibition
Previously, we identified salicylanilide diethyl phosphates as pseudo-irreversible inhibitors of
both cholinesterases [22]. That is why we also investigated the mechanism of inhibition of the most
potent BuChE inhibitor 5c.
Based on the changes of enzyme activity in the presence of tested inhibitor during prolonged
incubation, it is possible to distinguish reversible, pseudo-irreversible and irreversible inhibition [35,36].
In the case of reversible inhibition, the inhibitor is bound to the enzyme for a short period of time,
the activity of the enzyme goes down immediately and, after dissociation of complex enzyme-inhibitor,
the enzyme activity is restored. During pseudo-irreversible inhibition, the inhibitor is bound to the
enzyme molecule covalently, but the bond is slowly broken down and the enzyme activity returns
to the initial state. For irreversible inhibition, covalent and permanent bond between enzyme and
inhibitor is typical and therefore the enzyme becomes inactivated. Irreversible inhibitor decreases
enzyme activity successively and the dependence log% A vs. time in the presence of inhibitor is
linear [37,38].
The procedure of measurement and data evaluation were in detail described previously in the
reference [22]. From the dependence log% A vs. time the mechanism of action of tested inhibitor can
be deduced. Obtained results assert the pseudo-irreversible inhibition of both cholinesterase enzymes
(Figure 1 for AChE, Figure 2 for BuChE).
The enzyme inhibitors may bind to the active site or at some other sites. Based on this fact, they can
be divided into four categories: competitive, non-competitive, uncompetitive or mixed. Competitive
inhibitors compete with the substrate for the same binding site on the enzyme, the so-called active
site. Non-competitive, uncompetitive and mixed inhibitors do not bind at the active site but at some
other [39].

Biomolecules 2019, 9, 698
11 of 16
2.5
2
10 μM
30 μM
50 μM
1.5
1
0.5
0
0
50
100
150
200
250
t (min)
Figure 1. The dependence log% A vs. time. Enzyme: acetylcholinesterase, derivative 5c, concentration
of inhibitor: 10 µM (rhomb), 30 µM (square), 90 µM (triangle).
2.5
3 μM
6 μM
9 μM
2
1.5
1
0.5
0
0
50
100
150
200
250
t (min)
Figure 2. The dependence log% A vs. time. Enzyme: butyrylcholinesterase, derivative 5c, concentration
of inhibitor: 3 µM (rhomb), 6 µM (square), 9 µM (triangle).
The type of inhibition could be distinguished based on the Lineweaver-Burk plot [33] and the
comparison of the kinetic parameters KM and Vm of uninhibited and inhibited reactions. The diagnostic
criterion for competitive inhibition is that Vm is unaffected by inhibitor and KM for inhibited reaction
is increased. All lines in Lineweaver-Burk plot share a common y-intercept. For non-competitive
inhibitor KM is unchanged, but Vm of inhibited reaction is decreased. All lines in Lineweaver-Burk
plot share a common x-intercept. Uncompetitive inhibitor decreases KM and Vm, but KM/Vm remains
the same as for the uninhibited reaction. The pattern obtained in Lineweaver-Burk plot is a set of
parallel lines. For mixed inhibition KM, Vm and KM/Vm are altered. The lines in Lineweaver-Burk plot
intercept in the quadrant II or III [33].
From obtained results of KM and Vm and Lineweaver-Burk plots presented in Figure 3 (AChE) and
Figure 4 (BuChE), it is possible to conclude, that the inhibitor 5c acts via mixed inhibitory mechanism.

Biomolecules 2019, 9, 698
12 of 16
Figure 3. Lineweaver-Burk plot for AChE inhibition (ATCH = acetylthiocholine).
Figure 4. Lineweaver-Burk plot for BuChE inhibition (BTCH = butyrylthiocholine).
3.3. Prediction of Physicochemical Parameters, Drug-Likeness and CNS Delivery
We determined physicochemical parameters that are important for the prediction of drug-likeness
and also CNS availability (Table 3). Lipinski’s rule of five defines four physicochemical parameters
that are associated with a favorable profile for oral administration. It postulates that the molecular
weight (MW) should not be higher than 500, logP over five, number of hydrogen bond donors and
acceptors should be of
for MW and the number of hydrogen bond donors and acceptors. One salicylanilide (4l) and four
phosphoric derivatives (5a 5b 5d, and 5e) share slightly higher logP values. However, the escalated
≤
5 and
≤
10, respectively [40]. All of the derivatives 1–5 meet the criteria
,
,
lipophilicity may help to overcome bioavailability-related problems of the parent compounds
which is known substantially low for salicylanilides, despite generally meeting Lipinski’s rule [14
1–4,
,
41].
Increased lipophilicity should also improve crossing biological barriers including BBB by a non-specific
passive diffusion.

Biomolecules 2019, 9, 698
13 of 16
Table 3. Lipinski’s rule of five parameters and PSA values of the derivatives 1-5.
H-bond
Donors
H-bond
Acceptors
Number of
Violations
tPSA
[Å2]
Code
R1
R2
MW
LogP
1
H
H
3-Cl
4-Cl
3,4-diCl
3,5-diCl
3,4,5-triCl
3-Br
4-Br
3-F
4-F
3-CF3
4-CF3
3,5-bis-CF3 383.67
3-Cl
4-Cl
3,4-diCl
3,5-diCl
3,4,5-triCl
3-Br
4-Br
3-F
4-F
3-CF3
4-CF3
3,5-bis-CF3 383.67
3-Cl
4-Cl
3,4-diCl
3,5-diCl
3,4,5-triCl
3-Br
4-Br
3-F
4-F
3-CF3
4-CF3
213.24
282.12
282.12
316.56
316.56
351.00
326.57
326.57
265.67
265.67
315.68
315.68
2.45
3.57
3.57
4.12
4.12
4.68
3.84
3.84
3.17
3.17
3.93
3.93
4.85
3.57
3.57
4.12
4.12
4.68
3.84
3.84
3.17
3.17
3.93
3.93
4.85
3.84
3.84
4.4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
0
0
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
6
5
5
4
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
0
1
1
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
49.33
73.86
56.79
56.79
46.61
29.54
2a
2b
2c
2d
2e
2f
2g
2h
2i
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
-
2j
2k
2l
3a
3b
3c
3d
3e
3f
3g
3h
3i
282.12
282.12
316.56
316.56
351.00
326.57
326.57
265.67
265.67
315.68
315.68
3j
3k
3l
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
5d
5e
326.57
326.57
361.02
361.02
395.46
371.03
371.03
310.12
310.12
360.13
360.13
4.4
4.95
4.11
4.11
3.44
3.44
4.2
4.2
3,5-bis-CF3 428.13
5.12
5.08
5.82
4.70
5.64
6.37
-
-
-
-
-
451.76
467.82
435.76
437.74
453.80
-
-
-
-
Polar surface area (PSA) was described as another crucial factor for drugs targeting CNS. PSA is
calculated as a sum of polar atoms surfaces (i.e., nitrogens, oxygens and their attached hydrogens) [42].
A PSA value of less than 76 Å2 should indicate an increased probability of BBB permeability [43].
Table 3 reports topological polar surface areas (tPSA) calculated using ChemOffice software. All the
investigated derivatives exhibited a sufficiently low tPSA values.
Ghose et al. [43] analysed a range of CNS and non-CNS drugs and, in addition to tPSA,
they provided additional parameters for designing CNS drugs including: (i) ideally one or two
nitrogen atom(s), (ii) fewer than seven (two to four) linear chains outside of rings, (iii) fewer than three
(zero or one are optimal) polar hydrogens. Obviously, our compounds meet these guidelines.
4. Conclusions
We evaluated 36 lipophilic halogenated 2-hydroxy-N-phenylbenzamides
1–4 prepared using
PCl3 under MW conditions in high yields. The selected salicylanilide was esterified by chlorides of

Biomolecules 2019, 9, 698
14 of 16
phosphorus-based acids in the presence of a tertiary base. These derivatives were screened in vitro for
their activity against acetylcholinesterase and butyrylcholinesterase.
All salicylanilides exhibited moderate inhibition of both cholinesterases in micromolar
concentration range starting from 33.1 µM with a clear tendency to affect AChE at lower concentrations
and in a closer range of IC50. Some of them were superior to phenolic carbamate rivastigmine against
this enzyme. Structure-activity relationships were also identified. Introduction of a phosphorus-based
acid fragment (monovalent or bivalent) improved inhibitory potency for BuChE significantly uniformly.
Importantly, depending of type of phosphorus containing moiety, it is possible to modulate the
selectivity to cholinesterases. For the most active BuChE inhibitor, type and mechanism of inhibition
were determined experimentally. Based on in silico prediction/calculation, the investigated compounds
meet the criteria for drug-likeness and various parameters for BBB permeability/CNS availability.
Parent 2-hydroxy-N-phenylbenzamides (salicylanilides) with free phenolic group can be
considered as convenient compounds (with an intrinsic activity especially against AChE and good
physicochemical properties for CNS delivery) for conjugation with another cholinesterase inhibiting
scaffolds to obtain drugs that are more active.
Author Contributions: Conceptualization, M.K. and J.V.; methodology, M.K. and Š.Š.; investigation, M.K.,
N.-H.H., R.V., Š.Š. and K.V.; writing—original draft preparation, M.K. and Š.Š.; writing—review and editing, K.V.
and J.V.; supervision, M.K. and J.V.
Funding: This research was funded by the Czech Science Foundation, grant number 17-27514Y, by the Czech
Ministry of Education, Youth and Sports, grant number SVV 260 401, and by the project EFSA-CDN [grant No.
CZ.02.1.01/0.0/0.0/16_019/0000841] co-funded by ERDF. The authors wish to acknowledge financial support from
the University of Pardubice, Faculty of Chemical Technology.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Bondi, M.W.; Edmonds, E.C.; Salmon, D.P. Alzheimer’s Disease: Past, Present, and Future. J. Int. Neuropsychol.
Soc. 2017, 23, 818–831. [CrossRef] [PubMed]
Karlawish, J.; Jack, C.R.; Rocca, W.A.; Snyder, H.M.; Carrillo, M.C. Alzheimer’s disease: The next
frontier-Special Report 2017. Alzheimers Dement. 2017, 13, 374–380. [CrossRef] [PubMed]
World Alzheimer Report 2018. Available online: https://www.alz.co.uk/research/world-report-2018 (accessed
on 20 June 2019).
Sawatzky, E.; Wehle, S.; Kling, B.; Wendrich, J.; Bringmann, G.; Sotriffer, C.A.; Heilmann, J.; Decker, M.
Discovery of Highly Selective and Nanomolar Carbamate-Based Butyrylcholinesterase Inhibitors by Rational
Investigation into Their Inhibition Mode. J. Med. Chem. 2016, 59, 2067–2082. [CrossRef] [PubMed]
Darvesh, S. Butyrylcholinesterase as a diagnostic and therapeutic target for Alzheimer’s disease.
Curr. Alzheimer Res. 2016, 13, 1173–1177. [CrossRef]
5.
6.
7.
8.
9.
García-Ayllón, M.-S.; Small, D.H.; Avila, J.; Saez-Valero, J. Revisiting the Role of Acetylcholinesterase in
Alzheimer’s Disease: Cross-Talk with P-tau and β-Amyloid. Front. Molec. Neurosci. 2011, 4, 22. [CrossRef]
Li, Q.; Yang, H.Y.; Chen, Y.; Sun, H.P. Recent progress in the identification of selective butyrylcholinesterase
inhibitors for Alzheimer’s disease. Eur. J. Med. Chem. 2017, 132, 294–309. [CrossRef]
Mehta, M.; Adem, A.; Sabbagh, M. New Acetylcholinesterase Inhibitors for Alzheimer’s Disease. Int. J.
Alzheimers Dis. 2012, 2012, 728983. [CrossRef]
Colovic, M.B.; Krstic, D.Z.; Lazarevic-Pasti, T.D.; Bondzic, A.M.; Vasic, V.M. Acetylcholinesterase Inhibitors:
Pharmacology and Toxicology. Curr. Neuropharmacol. 2013, 11, 315–335. [CrossRef]
10. Horáková, E.; Drabina, P.; Brož, B.; Šteˇpánková, Š.; Vorcˇáková, K.; Královec, K.; Havelek, R.; Sedlák, M.
Synthesis, characterization and in vitro evaluation of substituted N-(2-phenylcyclopropyl)carbamates as
acetyl- and butyrylcholinesterase inhibitors. J. Enzyme Inhib. Med. Chem. 2016, 31, 173–179. [CrossRef]
11. Carrarini, C.; Russo, M.; Dono, F.; Di Pietro, M.; Rispoli, M.G.; Di Stefano, V.; Ferri, L.; Barbone, F.; Vitale, M.;
Thomas, A.; et al. A Stage-Based Approach to Therapy in Parkinson’s Disease. Biomolecules 2019, 9, 388.
[CrossRef]

Biomolecules 2019, 9, 698
15 of 16
12. Bohnen, N.I.; Grothe, M.J.; Ray, N.J.; Müller, M.L.T.M.; Teipel, S.J. Recent Advances in Cholinergic Imaging
and Cognitive Decline—Revisiting the Cholinergic Hypothesis of Dementia. Curr. Geri. Rep. 2018, 7, 1–11.
[CrossRef] [PubMed]
13. Liu, Z.; Zhang, A.; Sun, H.; Han, Y.; Kong, L.; Wang, X. Two decades of new drug discovery and development
for Alzheimer’s disease. RSC Adv. 2017, 7, 6046–6058. [CrossRef]
14. Kr
Curr. Pharm. Des. 2011, 17, 3494–3505. [CrossRef] [PubMed]
15. Paraskevopoulos, G.; Monteiro, S.; Vos tka, R.; Kr tk , M.; Navr
Vinšov , J. Novel salicylanilides from 4,5-dihalogenated salicylic acids: Synthesis, antimicrobial activity and
cytotoxicity. Bioorg. Med. Chem. 2017, 25, 1524–1532. [CrossRef] [PubMed]
16. Kr tk , M.; Vinšov , J. Salicylanilide N-monosubstituted carbamates: Synthesis and in vitro antimicrobial
activity. Bioorg. Med. Chem. 2016, 24, 1322–1330. [CrossRef] [PubMed]
17. Vinšov , J.; Kozic, J.; Kr tk , M.; Stolarˇ kov , J.; Mand kov , J.; Trejtnar, F.; Buchta, V. Salicylanilide diethyl
átký, M.; Vinšová, J. Salicylanilide ester prodrugs as potential antimicrobial agents–a review.
á
á
ý
átilová, L.; Trejtnar, F.; Stolarˇíková, J.;
á
á
ý
á
á
á
ý
í
á
í
á
phosphates: Synthesis, antimicrobial activity and cytotoxicity. Bioorg. Med. Chem. 2014, 22, 728–737.
[CrossRef] [PubMed]
18. Krátký, M.; Vinšová, J.; Stolarˇíková, J. Antimycobacterial Assessment of Salicylanilide Benzoates including
Multidrug-Resistant Tuberculosis Strains. Molecules 2012, 17, 12812–12820. [CrossRef]
19. Swan, G.E. The pharmacology of halogenated salicylanilides and their anthelmintic use in animals. J. S. Afr.
Vet. Assoc. 1999, 70, 61–70. [CrossRef]
20. Rajamuthiah, R.; Fuchs, B.B.; Conery, A.L.; Kim, W.; Jayamani, E.; Kwon, B.; Ausubel, F.M.; Mylonakis, E.
Repurposing salicylanilide anthelmintic drugs to combat drug resistant Staphylococcus aureus. PLoS ONE
2015, 10, e0124595. [CrossRef]
21. Garcia, C.; Burgain, A.; Chaillot, J.; Pic, E.; Khemiri, I.; Sellam, A. A phenotypic small-molecule screen
identifies halogenated salicylanilides as inhibitors of fungal morphogenesis, biofilm formation and host cell
invasion. Sci. Rep. 2018, 8, 11559. [CrossRef]
22. Krátký, M.; Šteˇpánková, Š.; Vorcˇáková, K.; Vinšová, J. Salicylanilide Diethyl Phosphates as Cholinesterases
Inhibitors. Bioorg. Med. Chem. 2015, 58, 48–52. [CrossRef] [PubMed]
23. Krátký, M.; Vorcˇáková, K.; Vinšová, J.; Šteˇpánková, Š. Investigation of salicylanilide and 4-chlorophenol-based
N-monosubstituted carbamates as potential inhibitors of acetyl- and butyrylcholinesterase. Bioorg. Chem.
2018, 80, 668–673. [CrossRef] [PubMed]
24. Krátký, M.; Šteˇpánková, Š.; Vorcˇáková, K.; Švarcová, M.; Vinšová, J. Novel Cholinesterases Inhibitors Based
on O-Aromatic N,N-Disubstituted Carbamates and Thiocarbamates. Molecules 2016, 21, 191. [CrossRef]
[PubMed]
25. Vinšová, J.; Krátký, M.; Komlóová, M.; Dadapeer, E.; Šteˇpánková, Š.; Vorcˇáková, K.; Stolarˇíková, J. Diethyl
2-(Phenylcarbamoyl)phenyl Phosphorothioates: Synthesis, Antimycobacterial Activity and Cholinesterase
Inhibition. Molecules 2014, 19, 7152–7168. [CrossRef]
26. Krátký, M.; Vinšová, J. Antifungal Activity of Salicylanilides and Their Esters with 4-(Trifluoromethyl)benzoic
Acid. Molecules 2012, 17, 9426–9442. [CrossRef]
27. Lee, I.Y.; Gruber, T.D.; Samuels, A.; Yun, M.; Nam, B.; Kang, M.; Crowley, K.; Winterroth, B.; Boshoff, H.I.;
Barry, C.E. Structure-activity relationships of antitubercular salicylanilides consistent with disruption of the
proton gradient via proton shuttling. Bioorg. Med. Chem. 2013, 21, 114–126. [CrossRef]
28. Institute of Medicinal Molecular Design, Inc.; Muto, S.; Itai, A. Inhibitors against the Production and Release
of Inflammatory Cytokines. Patent No. EP1512396, A1, 9 March 2005.
29. Geigy, A.G.; Bindler, J.; Model, E. Poly Halo-Salicylanilides. Patent No. US2703332 (A), 1 March 1955.
30. Charles University, Faculty of Pharmacy in Hradec Králové; Vinšová, J.; Krátký, M.; Paraskevopoulos, G.
Substituted derivative of oxyphosphorus acids, its use and pharmaceutical preparation containing it. Patent
Application No. WO 2016095878 A1, 23 June 2016.
31. Zdrazilova, P.; Stepankova, S.; Komers, K.; Ventura, K.; Cegan, A. Half-inhibition concentrations of new
cholinesterase inhibitors. Z. Naturforsch. C 2004, 59, 293–296. [CrossRef]
32. Sinko, G.; Calic, M.; Bosak, A.; Kovarik, Z. Limitation of the Ellman method: Cholinesterase activity
measurement in the presence of oximes. Anal. Biochem. 2007, 370, 223–227. [CrossRef]
33. Lineweaver, H.; Burk, D. The Determination of Enzyme Dissociation Constants. J. Am. Chem. Soc. 1934, 56,
658–666. [CrossRef]

Biomolecules 2019, 9, 698
16 of 16
34. Kandiah, N.; Pai, M.C.; Senanarong, V.; Looi, I.; Ampil, E.; Park, K.W.; Karanam, A.K.; Christopher, S.
Rivastigmine: The advantages of dual inhibition of acetylcholinesterase and butyrylcholinesterase and its
role in subcortical vascular dementia and Parkinson’s disease dementia. Clin. Interv. Aging 2017, 12, 697–707.
[CrossRef]
35. Szedlacsek, S.E.; Duggleby, R.G. [6] Kinetics of slow and tight-binding inhibitors. Methods Enzymol. 1995
,
249, 144–180.
36. Singh, J.; Petter, R.C.; Bailli, T.A.; Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 2011
10, 307–317. [CrossRef]
,
37. Jann, M.W.; Shirley, K.L.; Small, G.W. Clinical Pharmacokinetics and Pharmacodynamics of Cholinesterase
Inhibitors. Clin. Pharmacokinet. 2002, 41, 719–739. [CrossRef]
38. Bartolini, M.; Cavrini, V.; Andrisano, V. Characterization of reversible and pseudo-irreversible
acetylcholinesterase inhibitors by means of an immobilized enzyme reactor. J. Chromatogr.
1144, 102–110. [CrossRef]
A 2007,
39. Biochemistry. Fourth edition. Available online: http://gtu.ge/Agro-Lib/Reginald%20H.%20Garrett,
%20Charles%20M.%20Grisham%20-%20Biochemistry%20(4th%20ed.)%20-%202010.pdf (accessed on
20 June 2019).
40. Lipinski, C.A. Lead- and drug-like compounds: The rule-of-five revolution. Drug. Discov. Today Technol.
2004, 1, 337–341. [CrossRef]
41. Zhang, X.; Zhang, Y.; Zhang, T.; Zhang, J.; Wu, B. Significantly enhanced bioavailability of niclosamide
through submicron lipid emulsions with or without PEG-lipid: A comparative study. J. Microencapsul. 2015
32, 496–502. [CrossRef]
,
42. Hitchcock, S.A.; Pennington, L.D. Structure-Brain Exposure Relationships. J. Med. Chem. 2006, 49, 7559–7583.
[CrossRef]
43. Ghose, A.K.; Herbertz, T.; Hudkins, R.L.; Dorsey, B.D.; Mallamo, J.P. Knowledge-Based, Central Nervous
System (CNS) Lead Selection and Lead Optimization for CNS Drug Discovery. ACS Chem. Neurosci. 2012, 3,
50–68. [CrossRef]
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).