Diethyl 2-(phenylcarbamoyl)phenyl phosphorothioates: Synthesis, antimycobacterial activity and cholinesterase inhibition

Article abstract of DOI:10.3390/molecules19067152

A new series of 27 diethyl 2-(phenylcarbamoyl)phenyl phosphorothioates (thiophosphates) was synthesized, characterized by NMR, IR and CHN analyses and evaluated against Mycobacterium tuberculosis H37Rv, Mycobacterium avium and two strains of Mycobacterium kansasii. The best activity against M. tuberculosis was found for O-{4-bromo-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (minimum inhibitory concentration of 4 iM). The highest activity against nontuberculous mycobacteria was exhibited by O-(5-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}- phenyl) O,O-diethyl phosphorothioate with MIC values from 16 iM. Prepared thiophosphates were also evaluated against acetylcholinesterase from electric eel and butyrylcholinesterase from equine serum. Their inhibitory activity was compared to that of the known cholinesterases inhibitors galanthamine and rivastigmine. All tested compounds showed a higher (for AChE inhibition) and comparable (for BChE inhibition) activity to that of rivastigmine, with IC50s within the 8.04 to 20.2 iM range.

Full text of DOI:10.3390/molecules19067152

Molecules 2014, 19, 7152-7168; doi:10.3390/molecules19067152
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Diethyl 2-(Phenylcarbamoyl)phenyl Phosphorothioates:
Synthesis, Antimycobacterial Activity and
Cholinesterase Inhibition
Jarmila Vinšová 1,*, Martin Krátký 1, Markéta Komlóová 1, Echchukattula Dadapeer 1,
Šárka Štěpánková 2, Katarína Vorčáková 2 and Jiřina Stolaříková 3
1
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic;
E-Mails: martin.kratky@faf.cuni.cz (M.K.); marketa.komloova@faf.cuni.cz (M.K.);
dadapeer78@gmail.com (E.D.)
2
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology,
University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic;
E-Mails: sarka.stepankova@upce.cz (Š.Š.); katarina.vorcakova@student.upce.cz (K.V.)
3
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in
Ostrava, Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic; E-Mail: Jirina.Stolarikova@zu.cz
* Author to whom correspondence should be addressed; E-Mail: jarmila.vinsova@faf.cuni.cz;
Tel.: +420-495-067-343; Fax: +420-495-067-166.
Received: 29 April 2014 / in revised form: 26 May 2014/ Accepted: 27 May 2014 /
Published: 30 May 2014
Abstract: A new series of 27 diethyl 2-(phenylcarbamoyl)phenyl phosphorothioates
(thiophosphates) was synthesized, characterized by NMR, IR and CHN analyses and
evaluated against Mycobacterium tuberculosis H37Rv, Mycobacterium avium and two
strains of Mycobacterium kansasii. The best activity against M. tuberculosis was found for
O-{4-bromo-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate
(minimum inhibitory concentration of 4 µM). The highest activity against nontuberculous
mycobacteria was exhibited by O-(5-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}-
phenyl) O,O-diethyl phosphorothioate with MIC values from 16 µM. Prepared
thiophosphates were also evaluated against acetylcholinesterase from electric eel and
butyrylcholinesterase from equine serum. Their inhibitory activity was compared to that of
the known cholinesterases inhibitors galanthamine and rivastigmine. All tested compounds
showed a higher (for AChE inhibition) and comparable (for BChE inhibition) activity to
that of rivastigmine, with IC50s within the 8.04 to 20.2 µM range.

Molecules 2014, 19
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Keywords: antimycobacterial activity; in vitro acetylcholinesterase inhibition;
in vitro butyrylcholinesterase inhibition; salicylanilide; thiophosphates
1. Introduction
Tuberculosis (TB) is an infectious disease caused by intracellular pathogen Mycobacterium
tuberculosis (Mtb). In the past, the occurrence of TB decreased thanks to the progress in medical care
and therapeutic options, but nowadays, affecting more than eight million people worldwide, TB is once
again considered to be a global health problem. In 2012, 1.3 million people died from the disease [1]. The
rise of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) cases and the
co-infection with HIV are the main reasons for its recurrence. Demanding pharmacological treatment
and urgent need for new antimycobacterial agents present serious challenges, stimulating research in
this area.
A series of salicylanilide diethyl phosphates, previously synthesized by our group [2], showed an
increased antimycobacterial activity, a decreased cytotoxicity and improved selectivity indices in
comparison with the parent salicylanilides. Some of the compounds were also found to inhibit
multidrug-resistant Mtb strains with a minimum inhibitory concentration (MIC) of 1 µM. We have
concluded that temporary masking of the free hydroxyl group of salicylanilide compounds may
increase/be beneficial for its possible usage and the formation of this kind of prodrug is an attractive
strategy for an efficient cellular internalization [2].
In previous studies, the synthesis of thiophosphates has been involved in the synthesis of potential
immunotherapeutic oligonucleotide prodrugs [3], thermolytic DNA prodrugs [4] and the synthesis of
thiophosphate esters of 2'-C-methyl ribonucleosides used as inhibitors of RNA-dependent RNA
polymerase of hepatitis C virus [5]. This structure modification served as an inspiration for the
incorporation of sulphur into the phosphate moiety and preparation of thioanalogues with increased
lipophilicity—O,O-diethyl O-[2-(phenylcarbamoyl)phenyl] phosphorothioates (salicylanilide diethyl
thiophosphates).
Compounds with phosphonic, phosphoric or thiophosphoric moieties in their structures are
well-known anticholinesterase agents. They act as irreversible inhibitors of both acetylcholinesterase
(AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) [6]. The role of AChE in the
cholinergic synapsis is the termination of cholinergic impulse by hydrolysis of acetylcholine (ACh), a
neurotransmitter present in central and peripheral nervous system [7]. With the inhibition of AChE, the
level of ACh in the synaptic junction increases, enhancing thus cholinergic transmission. Reversible
inhibitors of AChE are used in the treatment of various diseases, requiring a longer duration of ACh
action on postsynaptic receptors (e.g., myasthenia gravis, Alzheimer’s disease) [8,9]. However, the
irreversible inhibition by organophosphorus compounds can graduate into cholinergic crisis leading to
overstimulation of cholinergic receptors which can be life-threatening. In pathological states
accompanied by AChE depletion, BChE can partially compensate its regulatory function. Lacking the
high specificity for the substrate, BChE is also a very important detoxicating protein, acting as a
scavenger of esteratic exogenous compounds. However, its principal role in the organism still remains

Molecules 2014, 19
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unclear [10]. Organophosphorus irreversible inhibitors express a strong inhibitory activity of both
AChE and BChE, resulting from the formation of covalent bond between the organophosphorus
compound and active site serine of the enzyme [11]. Similarity in thiophosphate moiety led us to
evaluate our salicylanilide thiophosphates also against both of these enzymes. This assumption was
supported by the fact that salicylanilide carbamates were reported as cholinesterases inhibitors [12].
Based on presented facts, we evaluated salicylanilide diethyl thiophosphates as potential
antimycobacterial drugs (salicylanilide derivatives) and anticholinesterase agents (dialkyl aryl
phosphorothioate moiety).
2. Results and Discussion
2.1. Chemistry
The synthetic approach to the preparation of the diethyl (2-phenylcarbamoyl)phenyl thiophosphates
(phosphorothioates) 1 is depicted in Scheme 1. It is similar to the one used earlier for the synthesis of
salicylanilide diethyl phosphates [2]. Starting salicylanilides were routinely obtained by the reaction of
4/5-chloro and 5-bromosalicylic acids with the appropriate anilines mediated by PCl3 in chlorobenzene
using a microwave reactor [13]. Salicylanilides were esterified by O,O-diethyl phosphoro-
chloridothioate in the presence of triethylamine in dichloromethane (DCM). The synthesis, subsequent
isolation and purification gave yields within the 38%–93% range.
Scheme 1. Synthesis of diethyl (2-phenylcarbamoyl)phenyl thiophosphates 1 (R for esters
1 = 4-Cl, 4-Br, 5-Cl; R1 = 3-Cl, 4-Cl, 3,4-diCl, 3-Br, 4-Br, 3-F, 4-F, 3-CF3, 4-CF3.
2.2. In Vitro Antimycobacterial Activity
Antimycobacterial activity was tested in vitro against Mycobacterium tuberculosis strain 331/88
(H37Rv), Mycobacterium avium 330/88, Mycobacterium kansasii 235/80 and Mycobacterium kansasii
6509/96, a strain isolated from a patient. The minimum inhibitory concentration values are presented
in the Table 1.
According to the substitution in the salicylic part of the molecule, the tested compounds could be
divided into three groups: Group 1 includes 4-Br derivatives 1a–i; Group 2 includes 4-Cl derivatives
1j–r; and Group 3 contains 5-Cl derivatives 1s–1zz. It was observed, that halogenation of the position
4 of salicylic part positively contributes to the antimycobacterial activity. This finding correlates with
our previous results [2].

Molecules 2014, 19
Table 1. Antimycobacterial activity of salicylanilide diethyl thiophosphates 1.
7155
MIC [µM]
M. kansasii
Mtb 331/88
M. avium
M. kansasii
6509/96
14 d
500
C
code
R
R1
(H37Rv)
330/88
235/80
14 d
500
500
250
125
250
250
500
62.5
62.5
250
62.5
250
500
500
500
500
500
125
125
32
logP
14 d 21 d 14 d
21 d
>500
500
7 d
125
125
62.5
125
125
62.5
125
32
21 d
500
500
500
125
250
500
>500
125
125
500
62.5
500
>500
500
>500
>500
>500
250
125
32
7 d
125
125
62.5
125
62.5
62.5
125
32
21 d
1a
1b
1c
1d
1e
1f
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
3-Cl
4-Cl
5.73
5.73
8
16
4
16
16
4
500
500
500
125
250
250
500
125
125
500
500
125
125
250
250
250
125
125
500
125
250
500
500
500
500
500
250
125
62.5
62.5
250
62.5
250
500
125
32
500
3,4-di-Cl 6.25
500
125
3-Br
4-Br
3-F
6.00
6.00
5.35
5.35
6.09
6.09
5.45
5.45
16
8
16
16
16
32
8
125
125
250
250
8
500
250
1g
1h
1i
4-F
32
8
>500
125
250
3-CF3
4-CF3
3-Cl
4-Cl
62.5
62.5
500
8
8
125
32
32
1j
32
32
8
62.5 >500 >500
250
32
250
62.5
125
125
250
250
125
125
62.5
32
1k
1l
32
8
500
500
125
3,4-di-Cl 5.97
>500 >500
125
125
250
250
250
125
62.5
32
250
1m
1n
1o
1p
1q
1r
1s
3-Br
4-Br
3-F
5.73
5.73
5.07
5.07
5.82
5.82
5.45
5.45
16
32
32
16
8
16
32
32
16
8
500
500
500
500
500
500
125
125
500
500
500
500
>500
500
125
125
500
500
500
4-F
250
3-CF3
4-CF3
3-Cl
4-Cl
500
8
8
125
16
32
16
32
32
16
32
32
8
32
32
62.5
62.5
62.5
125
1t
32
32
1u
1v
1w
1x
1y
1z
1zz
3,4-di-Cl 5.97
16 >500 >500
32
32
32
32
3-Br
4-Br
3-F
5.73
5.73
5.07
5.07
5.85
5.82
32
32
32
32
500
250
250
125
500
250
250
250
500
62.5
32
125
32
250
32
32
32
32
62.5
125
32
125
250
125
16
250
500
250
16
32
4-F
62.5
32
62.5
62.5
16
250
3-CF3
4-CF3
62.5 500
125
16
1
62.5
16
32
INH
PAS
0.5–1
>250 >250 >250 >250 >250
32 125 125 1000 >1000
2–4
32
4
4–8
500
62.5 62.5
125
INH = isoniazid; PAS = para-aminosalicylic acid. The best MIC values for each strain are given in bold.

Molecules 2014, 19
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Concerning the aniline part of the molecule, the choice of substituents was motivated by our
experience with structure-activity relationships observed in the diethyl phosphate series. To summarize
the obtained results, there is an obvious contribution of 3- or 4-trifluoromethyl and mainly 3,4-dichloro
substitution to the potency against Mtb. 3,4-Dichloro derivatives (1c, 1l, 1u) showed improved potency
when compared to either 3-Cl or 4-Cl monosubstituted ones. The most efficient compound was O-{4-
bromo-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate 1c with MIC of 4 µM.
Five of the compounds, 3-trifluoromethyl (1h, 1q), 4-trifluoromethyl (1i, 1r) and 3,4-dichloro (1l)
derivatives also showed significant MIC of 8 µM. Even though the structure-activity relationships are
in correlation with salicylanilide diethyl phosphates [2], diethyl thiophosphate compounds expressed
only moderate antimycobacterial activity against Mtb and atypical mycobacteria. The introduction of
sulphur into the phosphate group did not meet our expectations about increased antimycobacterial
activity, not only against Mtb, but also against M. avium and M. kansasii, which were inhibited even at
higher concentrations (≥ 16 µM) than Mtb. The best activity against these last mentioned strains was
described for 1zz. M. avium showed the lowest susceptibility to salicylanilide phosphates with MIC
values ≥ 62.5 µM.
None of the diethyl thiophosphates 1 exhibited the activity similar to isoniazid (INH) against
M. tuberculosis and one strain of M. kansasii (6509/96). On the other hand, ten salicylanilide
derivatives (1d, 1e, 1h, 1i, 1s, 1t, 1w–1y, and 1zz) were significantly more active against INH-resistant
M. avium (with all MIC values ≤ 250 μM) and fourteen molecules (1d, 1e, 1h, 1i, 1k, 1r–1x, 1z, and
1zz) share MICs for M. kansasii 235/80 lower (≤ 250 μM) than INH. All diethyl thiophosphates 1
outstripped the activity of para-aminosalicylic acid (PAS), a second-line oral antimycobacterial drug
sharing a structural similarity, against Mtb and none of them exhibited markedly lower growth
inhibition of M. kansasii 235/80. The inverse relationship was discovered for M. avium—only O-(5-
chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl) O,O-diethyl phosphorothioate 1zz showed an
equal in vitro potency.
2.3. In Vitro AChE a BChE Inhibition
The ability of the tested compounds to inhibit AChE from electric eel (Electrophorus electricus L.)
and BChE from equine serum was screened in vitro using modified Ellman’s method for the evaluation
of cholinesterase activity. The effectiveness of the inhibitors is expressed as IC50, representing the
concentration of an inhibitor required for 50% inhibition of the enzyme. The activities of the tested
compounds were compared with standards rivastigmine and galanthamine (Table 2). These two
standards were chosen due to their different structures. Rivastigmine is an acylating pseudo-reversible
carbamate inhibitor of cholinesterases that inhibits AChE as well as BChE, while galanthamine is a
non-acylating competitive reversible cholinesterase inhibitor. Furthermore, galanthamine acts as an
allosteric ligand at nicotinic acetylcholine receptors. The choice of these drugs with different
mechanism of action can provide relevant results. All tested compounds expressed a good inhibitory
activity with IC50s in low micromolar values. These values are summarized in Table 2.
It is possible to conclude that most of the tested compounds inhibit AChE somewhat better than
BChE. Derivatives of Group 2 (R = 4-Cl) and Group 3 (R = 5-Cl) are more effective inhibitors of

Molecules 2014, 19
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AChE than derivatives of Group 1 (R = 4-Br). For Groups 1 and 3, the relationship is inverse when
compared to anti-TB activity.
Table 2. The IC50 values of thiophosphates 1 for AChE and BChE inhibition.
Code
1a
1b
1c
IC50 for AChE [µM]
12.60 ± 0.05
12.9 ± 0.1
IC50 for BChE [µM]
13.90 ± 0.05
13.5 ± 0.9
14.0 ± 0.1
18.8 ± 0.7
1d
1e
11.30 ± 0.05
10.7 ± 0
20.0 ± 0.2
18.9 ± 0.2
1f
12.70 ± 0.05
12.40 ± 0.05
13.0 ± 0.02
11.20 ± 0.02
9.97 ± 0.24
9.78 ± 0.11
9.33 ± 0.11
14.1 ± 0.2
12.2 ± 0.0
1g
1h
1i
12.6 ± 0.3
20.0 ± 0.5
20.2 ± 1.6
1j
14.80 ± 0.15
12.70 ± 0.10
17.4 ± 0.5
1k
1l
1m
1n
1o
1p
15.1 ± 1.3
9.32 ± 0.06
9.94 ± 0.20
13.6 ± 1.1
15.1 ± 0.1
11.8 ± 0.6
14.9 ± 0.8
1q
12.1 ± 0.2
19.9 ± 0.2
1r
9.70 ± 0.18
9.79 ± 0.20
10.8 ± 0.3
9.56 ± 0.21
9.98 ± 0.43
10.12 ± 0.08
8.04 ± 0.27
11.6 ± 0.3
11.4 ± 0.3
9.16 ± 0.17
501 ± 3.08
4 ± 0.13
15.6 ± 0.7
12.5 ± 0.1
13.80 ± 0.05
14.7 ± 0.2
14.9 ± 1.0
16.4 ± 0.2
8.68 ± 0.54
8.67 ± 0.49
15.0 ± 0
1s
1t
1u
1v
1w
1x
1y
1z
1zz
18.3 ± 1.0
19.95 ± 0.20
7.96 ± 0.59
Rivastigmine
Galanthamine
AChE and BChE inhibition is expressed as mean ± SD (n = 3 experiments). The best values for each enzyme
are given in bold. The values of IC50 of rivastigmine and galanthamine were published previously [14].
As in the case of the inhibition of BChE, there is no observed effect of aniline substitution by
chlorine or bromine on the power of inhibition. Both 3- and 4-fluorine represent the most convenient
substitution patterns for the aniline ring for BChE (1f, 1g, 1o, 1p, and especially 1x and 1y). However,
IC50s of all derivatives are within a narrow concentration range (8–20 µM) without any sharp
differences. We presume that this is a consequence of their mechanism of action—irreversible
phosphorylation of enzyme active site; from this point of view, salicylanilide core serves as a carrier of
phosphorylating agents. In general, the IC50 values of all diethyl thiophosphates 1 were within a
narrow concentration range without any sharp differences: 8.04–14.1 μM for AChE and 8.67–20.2 μM

Molecules 2014, 19
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for BChE. O-{5-Chloro-2-[(3-fluorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1x) was
the most effective AChE inhibitor (IC50 = 8.04 ± 0.27 μM), and the second best one for inhibition of
butyrylcholinesterase (IC50 = 8.68 ± 0.54 μM). A bit more effective BChE inhibitor is O-{5-chloro-2-[(4-
fluorophenyl)-carbamoyl]phenyl} O,O-diethyl phosphorothioate (1y) (IC50 = 8.67 ± 0.49 μM). Then again
the least effective AChE inhibitor is O-{2-[(3-bromophenyl)carbamoyl]-4-chlorophenyl} O,O-diethyl
phosphorothioate (1m, IC50 = 14.1 ± 0.20 μM) and the least effective BChE inhibitor was O-(4-bromo-2-
{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl) O,O-diethyl phosphorothioate (1i, IC50 = 20.2 ± 1.6 µM).
Salicylanilide diethyl thiophosphates provide significantly lower IC50 values for AChE than
salicylanilide N-alkyl carbamates [12].
3. Experimental Section
3.1. General Information
Solvents and reagents were purchased from Sigma-Aldrich (Darmstadt, Germany) and Penta
Chemicals (Prague, Czech Republic) and were used as received. Reactions were monitored by
thin-layer chromatography (TLC) using commercially available coated plates (Merck Kieselgel 60
F254 silica) with UV light (256 nm) visualization. The melting points were determined on a Büchi
Melting Point B-540 apparatus (Bűchi Labortechnik AG, Flawil, Switzerland) using open capillaries
and were uncorrected. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA) in the range of 400–4000 cm−1 using the ATR technique. The NMR
spectra were recorded at ambient temperature on a Varian Mercury (500 MHz for 1H, 125 MHz for 13C
and 202 MHz for 31P; Varian Comp. Palo Alto, CA, USA) using deuterated chloroform (CDCl3)
solutions of the samples. Proton spectra are referenced to TMS as the internal standard; the carbon
shifts are given against the central line of the solvent signal (CDCl3 at δ = 77.1 ppm). The coupling
constants (J) are reported in Hz. Elementary analysis was performed on CE Instruments EA-1110
CHN analyser (CE Instruments, Wigan, UK). The calculated logP values (ClogP), which are the
logarithms of the partition coefficients for octan-1-ol/water, were determined using the program CS
ChemOffice Ultra version 12.0 (CambridgeSoft, Cambridge, MA, USA).
3.2. Chemistry
General Procedure for the Synthesis of Diethyl (2-phenylcarbamoyl)thiophosphates
Appropriate salicylanilide (2.0 mmol) was suspended at 20 °C in dichloromethane (10 mL) and then
1.5 equivalents of triethylamine (0.418 mL; 3.0 mmol) were added under vigorous stirring. After
5 min, 1.2 equivalents of O,O-diethyl phosphorochloridothioate (0.347 mL) was added to the mixture.
The mixture was stirred at the room temperature for 2 h; the reaction was monitored by TLC using a
4:1 toluene/ethyl acetate mixture as eluent. After this time, the solution was added to a
chromatography column using chloroform or chloroform/hexane 9:1 as eluent. The solvent was
removed under reduced pressure to obtain the product, which was recrystallized from acetone-hexane,
if necessary.

Molecules 2014, 19
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O-{4-Bromo-2-[(3-chlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1a). Yield: 86%,
white solid; m.p. 55–56 °C; IR: 1678 (amide I), 1593 (ν CCaromatic), 1529 (amide II), 1478 (ν CCaromatic
)
cm−1; 1H-NMR: δ 9.06 (1H, bs, NH), 8.15 (1H, dd, J = 2.5 Hz, J = 1.0 Hz, H3), 7.86 (1H, t, J = 2.0 Hz,
H2'), 7.61–7.57 (2H, m, H5, H6'), 7.30–7.26 (2H, m, H6, H5'), 7.14–7.11 (1H, m, H4'), 4.28–4.16 (4H,
13
m, CHAHB), 1.32 (6H, dt, J = 7.0 Hz, 0.9 Hz, CH3); C-NMR: δ 161.4, 146.7 (1C, d, J = 8.1 Hz),
139.0, 135.4 (1C, d, J = 1.5 Hz), 134.6, 134.5, 130.0, 128.5 (1C, d, J = 6.1 Hz), 124.6, 122.9 (1C, d,
J = 3.0 Hz), 120.1, 119.1 (1C, d, J = 2.0 Hz), 118.0, 66.0 (2C, d, J = 5.6 Hz, CHAHB), 15.8 (2C, d,
31
J = 7.4 Hz, CH3); P-NMR: δ 62.6. Anal. Calcd. for C17H18BrClNO4PS (478.72): C, 42.65; H, 3.79;
N, 2.93. Found: C, 42.45; H, 4.00; N, 2.96.
O-{4-Bromo-2-[(4-chlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1b). Yield: 89%,
white solid; m.p. 75–77 °C; IR: 1666 (amide I), 1592 (ν CCaromatic), 1530 (amide II), 1492, 1478 (ν
CCaromatic) cm−1; 1H-NMR: δ 9.05 (1H, bs, NH), 8.14 (1H, dd, J = 2.5 Hz, 1.0 Hz, H3), 7.72–7.69 (2H,
m, H2', H6'), 7.58 (1H, dd, J = 8.5 Hz, 2.5 Hz, H5), 7.34–7.31 (2H, m, H3', H5'), 7.27 (1H, dd,
J = 8.5 Hz, 1.0 Hz, H6), 4.28–4.15 (4H, m, CHAHB), 1.31 (6H, dt, J = 7.0 Hz, 0.7 Hz, CH3); 13C-NMR:
δ 161.4, 146.6 (1C, d, J = 8.5 Hz), 136.4, 135.3 (1C, d, J = 1.4 Hz), 134.5, 129.6, 129.0, 128.7 (1C, d,
J = 6.3 Hz), 122.9 (1C, d, J = 2.8 Hz), 121.3, 119.0 (1C, d, J = 2.0 Hz), 66.0 (2C, d, J = 5.5 Hz,
CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.6. Anal. Calcd for C17H18BrClNO4PS (478.72):
C, 42.65; H, 3.79; N, 2.93. Found: C, 42.76; H, 3.70; N, 2.99.
O-{4-Bromo-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1c). Yield:
69%, white solid; m.p. 80–81 °C; IR: 1678 (amide I), 1585 (ν CCaromatic), 1525 (amide II), 1476 (ν
1
CCaromatic) cm−1; H-NMR: δ 9.13 (1H, bs, NH), 8.15 (1H, dd, J = 2.5 Hz, 1.0 Hz, H3), 8.00 (1H, d,
J = 2.5 Hz, H2'), 7.61–7.57 (2H, m, H5, H6'), 7.41 (1H, d, J = 8.5 Hz, H5'), 7.28 (1H, dd, J = 8.5 Hz,
13
1.0 Hz, H6), 4.29–4.16 (4H, m, CHAHB), 1.32 (6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); C-NMR: δ 161.4,
146.7 (1C, d, J = 8.4 Hz), 137.3, 135.6 (1C, d, J = 1.5 Hz), 134.6, 132.8, 130.5, 128.1 (1C, d, J = 6.1 Hz),
127.8, 122.9 (1C, d, J = 2.9 Hz), 121.7, 119.3, 119.1 (1C, d, J = 1.9 Hz), 66.1 (2C, d, J = 5.5 Hz,
CHAHB), 15.8 (2C, d, J = 7.3 Hz, CH3); 31P-NMR: δ 62.5. Anal. Calcd for C17H17BrCl2NO4PS
(513.17): C, 39.79; H, 3.34; N, 2.73. Found: C, 39.88; H, 3.46; N, 2.85.
O-{4-Bromo-2-[(3-bromophenyl)carbamoyl]phenyl} diethyl phosphate (1d). Yield: 63%, white solid;
m.p. 67–68 °C (acetone-hexane); IR: 1677 (amide I), 1591 (ν CCaromatic), 1530 (amide II), 1476 (ν
1
CCaromatic) cm−1; H-NMR: δ 9.05 (1H, bs, NH), 8.15 (1H, dd, J = 2.5 Hz, 1.0 Hz, H3), 8.00 (1H, t,
J = 1.8 Hz, H2'), 7.67–7.64 (1H, m, H6'), 7.59 (1H, dd, J = 8.5 Hz, 2.5 Hz, H5), 7.30–7.21 (3H, m, H6,
13
H4', H5'), 4.29–4.16 (4H, m, CHAHB), 1.32 (6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); C-NMR: δ 161.4,
146.7 (1C, d, J = 8.3 Hz), 139.1, 135.4 (1C, d, J = 1.5 Hz), 134.6, 130.3, 128.5 (1C, d, J = 6.3 Hz),
127.6, 122.9, 122.9 (1C, d, J = 2.9 Hz), 122.6, 119.1 (1C, d, J = 1.9 Hz), 118.5, 66.0 (2C, d, J = 5.5 Hz,
CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.6. Anal. Calcd for C17H18Br2NO4PS (523.18):
C, 39.03; H, 3.47; N, 2.68. Found: C, 39.14; H, 3.34; N, 2.55.
O-{4-Bromo-2-[(4-bromophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1e). Yield: 69%,
white solid; m.p. 82–83 °C (acetone-hexane); IR: 1666 (amide I), 1589 (ν CCaromatic), 1527 (amide II),
1
1488, 1441 (ν CCaromatic) cm−1; H-NMR: δ 9.05 (1H, bs, NH), 8.14 (1H, dd, J = 2.5 Hz, 1.0 Hz, H3),

Molecules 2014, 19
7160
7.67–7.64 (2H, m, H2', H6'), 7.58 (1H, dd, J = 8.5 Hz, J = 2.5 Hz, H5), 7.49–7.45 (2H, m, H3', H5'),
7.27 (1H, dd, J = 8.5 Hz, 1.3 Hz, H6), 4.27–4.15 (4H, m, CHAHB), 1.31 (6H, dt, J = 7.0 Hz, 1.0 Hz,
13
CH3); C-NMR: δ 161.4, 146.6 (1C, d, J = 8.4 Hz), 136.9, 135.3 (1C, d, J = 1.4 Hz), 134.5, 132.0,
128.7 (1C, d, J = 6.0 Hz), 122.9 (1C, d, J = 2.9 Hz), 121.6, 119.1 (1C, d, J = 1.9 Hz), 117.2, 66.0 (2C,
d, J = 5.5 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.5. Anal. Calcd for
C17H18Br2NO4PS (523.18): C, 39.03; H, 3.47; N, 2.68. Found: C, 39.18; H, 3.34; N, 2.71.
O-{4-Bromo-2-[(3-fluorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1f). Yield: 73%,
oily liquid; IR: 1680 (amide I), 1606 (ν CCaromatic), 1540 (amide II), 1492, 1473 (ν CCaromatic) cm−1;
1H-NMR: δ 9.09 (1H, bs, NH), 8.15 (1H, d, J = 2.5 Hz, H3), 7.70 (1H, d, J = 11.0 Hz, H6'), 7.58 (1H,
dd, J = 8.5 Hz, 2.5 Hz, H5), 7.41–7.25 (2H, m, H6, H2', H5'), 6.85 (1H, td, J = 8.5 Hz, 1.8 Hz, H4'),
13
4.26–4.17 (4H, m, CHAHB), 1.31 (6H, t, J = 7.0 Hz, CH3); C-NMR: δ 162.9 (1C, d, J = 243.3 Hz,
C3'), 161.5, 146.6 (1C, d, J = 8.4 Hz), 139.3 (1C, d, J = 10.8 Hz), 135.3 (1C, d, J = 1.5 Hz), 134.5,
130.0 (1C, d, J = 9.3 Hz), 128.6 (1C, d, J = 6.1 Hz), 122.9 (1C, d, J = 3.0 Hz), 119.0 (1C, d, J = 2.0 Hz),
115.3 (1C, d, J = 2.9 Hz), 111.3 (1C, d, J = 21.4 Hz), 107.5 (1C, d, J = 26.5 Hz), 66.0 (2C, d, J = 5.5 Hz,
CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.6. Anal. Calcd for C17H18BrFNO4PS (462.27):
C, 44.17; H, 3.92; N, 3.03. Found: C, 44.03; H, 4.05; N, 3.20.
O-{4-Bromo-2-[(4-fluorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1g). Yield: 49%,
oily liquid; IR: 1664 (amide I), 1614 (ν CCaromatic), 1541 (amide II), 1509, 1470 (ν CCaromatic) cm−1;
1H-NMR: δ 9.00 (1H, bs, NH), 8.14 (1H, dd, J = 2.5 Hz, 1.0 Hz, H3), 7.73–7.69 (2H, m, H2', H6'), 7.57
(1H, dd, J = 8.7 Hz, 2.5 Hz, H5), 7.28 (1H, dd, J = 8.5 Hz, 1.0 Hz, H6), 7.08–7.03 (2H, m, H3', H5'),
13
4.27–4.16 (4H, m, CHAHB), 1.30 (6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); C-NMR: δ 161.3, 159.5 (1C, d,
J 242.6 Hz, C4'), 146.6 (1C, d, J = 8.5 Hz), 135.2 (1C, d, J = 1.5 Hz), 134.5, 133.9 (1C, d,
J = 2.9 Hz, C1'), 128.80 (1C, d, J = 6.1 Hz), 122.9 (1C, d, J = 3.0 Hz), 121.7 (2C, d, J = 7.8 Hz, C2',
C6'), 119.0 (1C, d, J = 2.0 Hz), 115.6 (2C, d, J = 22.4 Hz, C3', C5'), 65.9 (2C, d, J = 5.6 Hz, CHAHB),
15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.6. Anal. Calcd for C17H18BrFNO4PS (462.27): C, 44.17;
H, 3.92; N, 3.03. Found: C, 44.30; H, 3.99; N, 2.87.
O-(4-Bromo-2-{[3-(trifluoromethyl)phenyl]carbamoyl}phenyl) O,O-diethyl phosphorothioate (1h).
Yield: 38%, oily liquid; IR: 1677 (amide I), 1606 (ν CCaromatic), 1537 (amide II), 1492, 1472 (ν
1
CCaromatic) cm−1; H-NMR: δ 9.21 (1H, bs, NH), 8.19 (1H, dd, J = 2.8 Hz, 1.1 Hz, H3), 8.04 (1H, s,
H2'), 7.97 (1H, d, J = 8.2 Hz, H6'), 7.60 (1H, dd, J = 8.7 Hz, 2.6 Hz, H5), 7.49 (1H, t, J = 8.0 Hz, H5'),
7.41 (1H, d, J = 7.8 Hz, H4'),7.31 (1H, dd, J = 8.7 Hz, 1.3 Hz, H6), 4.29–4.17 (4H, m, CHAHB), 1.31
(6H, t, J = 7.1 Hz, CH3); 13C-NMR: δ 161.55, 146.76 (1C, d, J = 8.2 Hz), 138.39, 135.51 (1C, d,
J = 1.8 Hz), 134.64 (1C, d, J = 1.0 Hz), 131.39 (1C, q, J = 32.5 Hz, C3'), 129.57, 128.27 (1C, d,
J = 6.2 Hz), 123.83 (1C, q, J = 272.5 Hz, CF3), 123.12, 122.89 (1C, d, J = 2.9 Hz), 121.12 (1C, q,
J = 3.9 Hz, C2'), 119.09 (1C, d, J = 2.1 Hz), 116.78 (1C, q, J = 3.9 Hz, C4'), 66.05 (2C, d, J = 5.6 Hz,
CHAHB), 15.76 (2C, d, J = 7.3 Hz, CH3); 31P-NMR: δ 62.7. Anal. Calcd for C18H18BrF3NO4PS
(512.28): C, 42.20; H, 3.54; N, 2.73. Found: C, 42.41; H, 3.59; N, 2.64.
O-(4-Bromo-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl) O,O-diethyl phosphorothioate (1i).
Yield: 61%, white solid; m.p. 83–84 °C (acetone-hexane); IR: 1683 (amide I), 1604 (ν CCaromatic), 1540

Molecules 2014, 19
7161
1
(amide II), 1472 (ν CCaromatic) cm−1; H-NMR: δ 9.23 (1H, bs, NH), 8.17 (1H, dd, J = 2.5 Hz, 1.3 Hz,
H3), 7.89 (2H, d, J = 8.5 Hz, H3', H5'), 7.62 (2H, d, J = 8.5 Hz, H2', H6'), 7.60 (1H, dd, J = 9.0 Hz,
2.5 Hz, H5), 7.23 (1H, dd, J = 9.0 Hz, 1.3 Hz, H6), 4.29–4.16 (4H, m, CHAHB), 1.31 (6H, dt, J = 7.0 Hz,
13
1.0 Hz, CH3); C-NMR: δ 161.6, 146.7 (1C, d, J = 8.5 Hz), 140.9 (1C, d, J = 1.1 Hz), 135.5 (1C, d,
J = 1.6 Hz), 134.6, 128.4 (1C, d, J = 6.1 Hz), 126.3 (2C, q, J = 3.8 Hz, C3', C5'), 126.3 (1C, q,
J = 32.8 Hz, C4'), 124.0 (1C, q, J = 270.1 Hz, CF3), 122.9 (1C, d, J = 2.9 Hz), 119.7, 119.1 (1C, d,
31
J = 2.0 Hz), 66.0 (2C, d, J =5.6 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); P-NMR: δ 62.5. Anal.
Calcd for C18H18BrF3NO4PS (512.28): C, 42.20; H, 3.54; N, 2.73. Found: C, 42.39; H, 3.38; N, 2.91.
O-{4-Chloro-2-[(3-chlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1j). Yield: 75%,
white solid; m.p. 52–54 °C; IR: 1677, 1658 (amide I), 1597 (ν CCaromatic), 1540 (amide II), 1476, 1428
(ν CCaromatic) cm−1; 1H-NMR: δ 9.07 (1H, bs, NH), 8.00 (1H, dd, J = 2.8 Hz, 1.3 Hz, H3), 7.87 (1H, t,
J = 2.0 Hz, H2'), 7.59 (1H, dd, J = 8.3 Hz, 1.3 Hz, H6'), 7.43 (1H, dd, J = 8.8 Hz, 2.8 Hz, H5), 7.35
(1H, dd, J = 8.5 Hz, 1.5 Hz, H6), 7.28 (1H, t, J = 8.0 Hz, H5'), 7.14–7.11 (1H, m, H4'), 4.28–4.16 (4H,
m, CHAHB), 1.32 (6H, dt, J = 7.0 Hz, 0.9 Hz, CH3); 13C-NMR: δ 161.5, 146.1 (1C, d, J = 8.4 Hz), 139.0,
134.6, 132.4 (1C, d, J = 1.8 Hz), 131.6, 131.6 (1C, d, J = 1.9 Hz), 130.0, 128.2 (1C, d, J = 6.1 Hz),
124.6, 122.6 (1C, d, J = 3.0 Hz), 120.1, 118.0, 66.0 (2C, d, J = 5.6 Hz, CHAHB), 15.8 (2C, d, J = 7.5 Hz,
CH3); 31P-NMR: δ 62.7. Anal. Calcd for C17H18Cl2NO4PS (434.27): C, 47.02; H, 4.18; N, 3.23. Found:
C, 47.15; H, 4.08; N, 3.32.
O-{4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1k). Yield: 62%,
white solid; m.p. 53–54 °C; IR: 1666 (amide I), 1594 (ν CCaromatic), 1536 (amide II), 1492 (ν CCaromatic
)
cm−1; 1H-NMR: δ 9.06 (1H, bs, NH), 7.99 (1H, d, J = 2.5 Hz, H3), 7.71 (2H, d, J = 8.5 Hz, H2', H6'),
7.43 (1H, dd, J = 8.8 Hz, 2.5 Hz, H5), 7.35–7.29 (3H, m, H6, H3', H5'), 4.28–4.15 (4H, m, CHAHB),
1.30 (6H, t, J = 7.0 Hz, CH3); 13C-NMR: δ 161.5, 146.1 (1C, d, J = 8.6 Hz), 136.4, 132.3, 131.5, 129.5,
129.0, 128.4 (1C, d, J = 6.3 Hz), 122.6 (1C, d, J = 2.6 Hz), 121.3, 66.0 (2C, d, J = 5.5 Hz, CHAHB),
15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.6. Anal. Calcd for C17H18Cl2NO4PS (434.27): C, 47.02;
H, 4.18; N, 3.23. Found: C, 47.12; H, 4.30; N, 3.17.
O-{4-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1l). Yield:
87%, white solid; m.p. 90–91 °C (acetone-hexane); IR: 1677 (amide I), 1586 (ν CCaromatic), 1524
1
(amide II), 1477 (ν CCaromatic) cm−1; H-NMR: δ 9.14 (1H, bs, NH), 8.01–7.99 (2H, m, H3, H2'), 7.59
(1H, dd, J = 9.0 Hz, 2.5 Hz, H6'), 7.44 (1H, dd, J = 8.8 Hz, 2.8 Hz, H5), 7.41 (1H, dd, J = 9.0 Hz, 0.8
Hz, H5'), 7.34 (1H, dd, J = 8.8 Hz, 1.0 Hz, H6), 4.29–4.17 (4H, m, CHAHB), 1.32 (6H, tt, J = 7.2 Hz,
13
0.9 Hz, CH3); C-NMR: δ 161.5, 146.1 (1C, d, J = 8.5 Hz), 137.3, 132.8, 132.6 (1C, d, J = 1.8 Hz),
131.7, 131.6 (1C, d, J = 2.0 Hz), 130.5, 127.9 (1C, d, J = 6.3 Hz), 127. 8, 122.6 (1C, d, J = 3.0 Hz),
121.7, 119.3, 66.1 (2C, d, J = 5.5 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.6. Anal.
Calcd for C17H17Cl3NO4PS (468.72): C, 43.56; H, 3.66; N, 2.99. Found: C, 43.44; H, 3.57; N, 3.16.
O-{2-[(3-Bromophenyl)carbamoyl]-4-chlorophenyl} O,O-diethyl phosphorothioate (1m). Yield: 76%,
white solid; m.p. 57–58 °C (acetone-hexane); IR: 1677 (amide I), 1592 (ν CCaromatic), 1530 (amide II),
1
1476 (ν CCaromatic) cm−1; H-NMR: δ 9.06 (1H, bs, NH), 8.01–7.99 (2H, m, H3, H2'), 7.66 (1H, ddd,
J = 8.0 Hz, 2.0 Hz, 1.0 Hz, H6'), 7.44 (1H, dd, J = 8.8 Hz, 2.8 Hz, H5), 7.35 (1H, dd, J = 8.8 Hz, 1.3

Molecules 2014, 19
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Hz, H6), 7.28 (1H, ddd, J = 8.0 Hz, 2.0 Hz, 1.0 Hz, H4'), 7.22 (1H, t, J = 8.0 Hz, H5'), 4.28–4.16 (4H,
13
m, CHAHB), 1.32 (6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); C-NMR: δ 161.5, 146.1 (1C, d, J = 8.3 Hz),
139.1, 132.4 (1C, d, J = 1.5 Hz), 131.6, 131.6 (1C, d, J = 1.8 Hz), 130.3, 128.2 (1C, d, J = 6.1 Hz),
127.6, 122.9, 122.6 (2C, d, J = 3.0 Hz), 122.6, 118.5, 66.0 (2C, d, J = 5.5 Hz, CHAHB), 15.8 (2C, d,
J = 7.4 Hz, CH3); 31P-NMR: δ 62.7. Anal. Calcd for C17H18BrClNO4PS (478.72): C, 42.65; H, 3.79; N,
2.93. Found: C, 42.79; H, 3.65; N, 2.82.
O-{2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl} O,O-diethyl phosphorothioate (1n). Yield: 81%,
white solid; m.p. 60–62 °C; IR: 1677, 1660 (amide I), 1600, 1572 (ν CCaromatic), 1538 (amide II), 1489
1
(ν CCaromatic) cm−1; H-NMR: δ 9.06 (1H, bs, NH), 7.99 (1H, dd, J = 2.7 Hz, 1.2 Hz, H3), 7.68–7.63
(2H, m, H2', H6'), 7.49–7.45 (2H, m, H3', H5'), 7.43 (1H, dd, J = 8.8 Hz, 2.7 Hz, H5), 7.33 (1H, dd,
J = 8.8 Hz, 1.3 Hz, H6), 4.27–4.15 (4H, m, CHAHB), 1.31 (6H, dt, J = 7.0 Hz, 0.8 Hz, CH3); 13C-NMR:
δ 161.8, 146.1 (1C, d, J = 8.4 Hz), 137.0, 132.3 (1C, d, J = 1.8 Hz), 132.0, 131.6 (1C, d, J = 2.0 Hz),
128.4 (1C, d, J = 6.3 Hz), 122.6 (1C, d, J = 2.9 Hz), 121.6, 117.2, 66.0 (2C, d, J = 5.5 Hz, CHAHB),
15.8 (2C, d, J = 7.5 Hz, CH3); 31P-NMR: δ 62.7. Anal. Calcd for C17H18BrClNO4PS (478.72): C,
42.65; H, 3.79; N, 2.93. Found: C, 42.49; H, 4.01; N, 3.10.
O-{4-Chloro-2-[(3-fluorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1o). Yield: 65%,
white solid; m.p. 46–48 °C; IR: 1659 (amide I), 1612 (ν CCaromatic), 1548 (amide II), 1473, 1446 (ν
1
CCaromatic) cm−1; H-NMR: δ 9.10 (1H, bs, NH), 8.00 (1H, dd, J = 2.5 Hz, 1.0 Hz, H3), 7.70 (1H, dt,
J = 11.0 Hz, 2.2 Hz, H6'), 7.43 (1H, dd, J = 8.8 Hz, 2.5 Hz, H5), 7.35 (1H, dd, J = 8.8 Hz, 1.0 Hz, H6),
7.36–7.27 (2H, m, H2', H5'), 6.85 (1H, ddt, J = 8.0 Hz, 2.5 Hz, J = 0.9 Hz, H4'), 4.28–4.16 (4H, m,
CHAHB), 1.31 (6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); 13C-NMR: δ 162.9 (1C, d, J = 243.5 Hz, C3'), 161.6,
146.1 (1C, d, J = 8.4 Hz), 139.3 (1C, d, J = 10.9 Hz), 132.3 (1C, d, J = 1.6 Hz), 131.6 (1C, d,
J = 1.3 Hz), 131.5, 130.0 (1C, d, J = 9.3 Hz), 128.3 (1C, d, J = 6.1 Hz), 122.6 (1C, d, J = 2.9 Hz),
115.3 (1C, d, J = 3.0 Hz, C6'), 111.3 (1C, d, J = 21.3 Hz), 107.5 (1C, d, J = 26.4 Hz), 66.0 (2C, d,
J = 5.5 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.7. Anal. Calcd for C17H18ClFNO4PS
(417.82): C, 48.87; H, 4.34; N, 3.35. Found: C, 48.75; H, 4.23; N, 3.52.
O-{4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1p). Yield: 68%,
liquid; IR: 1670 (amide I), 1615 (ν CCaromatic), 1541 (amide II), 1509, 1473 (ν CCaromatic) cm−1;
1H-NMR: δ 9.02 (1H, bs, NH), 7.99 (1H, dd, J = 2.8 Hz, 1.2 Hz, H3), 7.75–7.69 (2H, m, H2', H6'),
7.42 (1H, dd, J = 8.8 Hz, 2.8 Hz, H5), 7.34 (1H, dd, J = 8.8 Hz, 1.2 Hz, H6), 7.09–7.03 (2H, m, H3',
13
H5'), 4.27–4.14 (4H, m, CHAHB), 1.30 (6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); C-NMR: δ 161.4, 159.5
(1C, d, J = 242.6 Hz, C4'), 146.1 (1C, d, J = 8.5 Hz), 133.9 (1C, d, J = 2.8 Hz, C1'), 132.2 (1C, d,
J = 1.5 Hz), 131.5, 128.5 (1C, d, J = 6.1 Hz), 122.6 (1C, d, J = 2.9 Hz), 121.7 (2C, d, J = 7.8 Hz, C2',
C6'), 115.6 (2C, d, J = 22.3 Hz, C3', C5'), 65.9 (2C, d, J = 5.6 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz,
31
CH3); P-NMR: δ 62.7. Anal. Calcd. for C17H18ClFNO4PS (417.82): C, 48.87; H, 4.34; N, 3.35. Found:
C, 48.98; H, 4.50; N, 3.19.
O-(4-Chloro-2-{[3-(trifluoromethyl)phenyl]carbamoyl}phenyl) O,O-diethyl phosphorothioate (1q).
Yield: 67%, white solid; m.p. 49–51 °C; IR: 1678 (amide I), 1606 (ν CCaromatic), 1564 (amide II), 1450
(ν CCaromatic) cm−1; 1H-NMR: δ 9.22 (1H, bs, NH), 8.06–8.02 (2H, m, H3, H2'), 7.98 (1H, d, J = 8.0 Hz,

Molecules 2014, 19
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H6'), 7.49 (1H, t, J = 8.0 Hz, H5'), 7.45 (1H, dd, J = 8.8 Hz, 2.8 Hz, H5), 7.40 (1H, d, J = 8.0 Hz, H4'),
7.37 (1H, dd, J = 8.8 Hz, 1.3 Hz, H6), 4.29–4.16 (4H, m, CHAHB), 1.31 (6H, t, J = 7.0 Hz, CH3);
13C-NMR: δ 161.7, 146.2 (1C, d, J = 8.4 Hz), 138.4, 132.5 (1C, d, J = 1.6 Hz), 131.7 (1C, d, J = 1.1 Hz),
131.6 (1C, d, J = 1.9 Hz), 131.4 (1C, q, J = 32.3 Hz, C3'), 129.6, 128.0 (1C, d, J = 6.1 Hz), 123.8 (1C,
q, J = 270.9 Hz, CF3), 123.1 (1C, d, J = 0.9 Hz), 122.6 (1C, d, J = 2.9 Hz), 121.1 (1C, q, J = 3.8 Hz,
C2'), 116.8 (1C, q, J = 3.9 Hz, C4'), 66.0 (2C, d, J = 5.5 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3);
31P-NMR: δ 62.7. Anal. Calcd for C18H18ClF3NO4PS (467.83): C, 46.21; H, 3.88; N, 2.99. Found: C,
45.98; H, 4.04; N, 3.19.
O-(4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl) O,O-diethyl phosphorothioate (1r).
Yield: 93%, white solid; m.p. 81–82 °C; IR: 1684 (amide I), 1603 (ν CCaromatic), 1540 (amide II), 1475
(ν CCaromatic) cm−1; 1H-NMR: δ 9.24 (1H, bs, NH), 8.02 (1H, dd, J = 2.8 Hz, 1.2 Hz, H3), 7.89 (2H, d,
J = 8.5 Hz, H3', H5'), 7.62 (2H, d, J = 8.5 Hz, H2', H6'), 7.45 (1H, dd, J = 8.8 Hz, 2.8 Hz, H5), 7.35
(1H, dd, J = 8.8 Hz, 1.3 Hz, H6), 4.28–4.16 (4H, m, CHAHB), 1.31 (6H, td, J = 7.0 Hz, 1.0 Hz, CH3);
13C-NMR: δ 161.8, 146.1 (1C, d, J = 8.7 Hz), 140.9, 132.6 (1C, d, J = 1.8 Hz), 131.7 (1C, d, J = 1.0 Hz),
131.6 (1C, d, J = 2.6 Hz), 128.1 (1C, d, J = 6.3 Hz), 126.3 (1C, q, J = 32.6 Hz, C4'), 126.2 (2C, q,
J = 3.9 Hz, C3', C5'), 124.0 (1C, q, J = 270.0 Hz, CF3), 122.6 (1C, d, J = 3.0 Hz), 119.7, 66.0 (2C, d,
J = 5.6 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.6. Anal. Calcd for
C18H18ClF3NO4PS (467.83): C, 46.21; H, 3.88; N, 2.99. Found: C, 46.32; H, 3.93; N, 3.20.
O-{5-Chloro-2-[(3-chlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1s). Yield: 79%,
semisolid; IR: 1660 (amide I), 1593 (ν CCaromatic), 1532 (amide II), 1482 (ν CCaromatic) cm−1; 1H-NMR:
δ 9.06 (1H, bs, NH), 7.98 (1H, d, J = 8.5 Hz, H3), 7.87 (1H, t, J = 2.0 Hz, H2'), 7.58 (1H, dd, J = 8.3 Hz,
1.8 Hz, H6'), 7.40 (1H, t, J = 1.5 Hz, H6), 7.32 (1H, td, J = 8.5 Hz, 1.0 Hz, H4), 7.26 (1H, t, J = 8.0 Hz,
13
H5'), 7.12–7.08 (1H, m, H4'), 4.28–4.17 (4H, m, CHAHB), 1.32 (6H, t, J = 7.0 Hz, CH3); C-NMR: δ
161.9, 147.9 (1C, d, J = 8.5 Hz), 139.1, 138.0 (1C, d, J = 1.9 Hz), 134.6, 132.9, 129.9, 126.3 (1C, d,
J = 1.1 Hz), 125.2 (1C, d, J = 6.0 Hz), 124.5, 121.5 (1C, d, J = 3.0 Hz), 120.1, 118.0, 66.1 (2C, d,
J = 5.4 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.4. Anal. Calcd for C17H18Cl2NO4PS
(434.27): C, 47.02; H, 4.18; N, 3.23. Found: C, 46.91; H, 4.29; N, 3.34.
O-{5-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1t). Yield: 91%,
white solid; m.p. 67–68 °C (acetone-hexane); IR: 1658 (amide I), 1604, 1546 (ν CCaromatic), 1546
(amide II), 1492 (ν CCaromatic) cm−1; 1H-NMR: δ 9.07 (1H, bs, NH), 7.99 (1H, d, J = 8.5 Hz, H3), 7.74–7.69
(2H, m, H2', H6'), 7.40 (1H, d, J = 1.8 Hz, H6), 7.34–7.29 (3H, m, H4, H3', H5'), 4.29–4.17 (4H, m,
13
CHAHB), 1.32 (6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); C-NMR: δ 161.9, 147.9 (1C, d, J = 8.8 Hz), 137.9
(1C, d, J = 1.6 Hz), 136.6, 132.8, 129.4, 129.0, 126.3, 125.4 (1C, d, J = 6.1 Hz), 121.5 (1C, d, J = 2.6 Hz),
121.2, 66.1 (2C, d, J = 5.5 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.3. Anal. Calcd
for C17H18Cl2NO4PS (434.27): C, 47.02; H, 4.18; N, 3.23. Found: C, 47.20; H, 4.02; N, 3.10.
O-{5-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1u). Yield:
91%, white solid; m.p. 70–71 °C; IR: 1650 (amide I), 1599, 1587 (ν CCaromatic), 1533 (amide II), 1472
(ν CCaromatic) cm−1; 1H-NMR: δ 9.14 (1H, bs, NH), 8.02 (1H, d, J = 2.5 Hz, H2'), 8.00 (1H, dd, J = 8.5 Hz,
1.0 Hz, H3), 7.58 (1H, dd, J = 8.8 Hz, 2.3 Hz, H6'), 7.42–7.39 (2H, m, H6, H5'), 7.32 (1H, td, J = 8.5 Hz,

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13
1.0 Hz, H4), 4.31–4.18 (4H, m, CHAHB), 1.34 (6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); C-NMR: δ 161.9,
148.0 (1C, d, J = 8.8 Hz), 138.2 (1C, d, J = 1.5 Hz), 137.5, 132.9, 132.8, 130.5, 127.6, 126.3, 124.9 (1C, d,
J = 6.0 Hz), 121.7, 121.5 (1C, d, J = 2.6 Hz), 119.3, 66.2 (2C, d, J = 5.5 Hz, CHAHB), 15.8 (2C, d,
31
J = 7.3 Hz, CH3); P-NMR: δ 62.3. Anal. Calcd for C17H17Cl3NO4PS (468.72): C, 43.56; H, 3.66; N,
2.99. Found: C, 43.69; H, 3.90; N, 3.18.
O-{2-[(3-Bromophenyl)carbamoyl]-5-chlorophenyl} O,O-diethyl phosphorothioate (1v). Yield: 49%,
colour liquid; IR: 1681 (amide I), 1589 (ν CCaromatic), 1532 (amide II), 1479, 1420 (ν CCaromatic) cm−1;
1H-NMR: δ 9.07 (1H, bs, NH), 8.05–8.96 (2H, m, H3, H2΄), 7.65 (1H, d, J = 7.5 Hz, H6'), 7.41 (1H, s,
H6), 7.34–7.18 (3H, m, H4, H4', H5'), 4.33–4.16 (4H, m, CHAHB), 1.33 (6H, t, J = 7.0 Hz, CH3);
13C-NMR: δ 161.9, 147.9 (1C, d, J = 8.5 Hz), 139.2, 138.0 (1C, d, J = 1.9 Hz), 132.9, 130.2, 127.4,
126.3, 125.2 (1C, d, J = 6.0 Hz), 122.9, 122.6, 121.5 (1C, d, J = 2.4 Hz), 118.5, 66.1 (2C, d, J = 5.4 Hz,
CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.4. Anal. Calcd for C17H18BrClNO4PS
(478.72): C, 42.65; H, 3.79; N, 2.93. Found: C, 42.54; H, 4.00; N, 3.01.
O-{2-[(4-Bromophenyl)carbamoyl]-5-chlorophenyl} O,O-diethyl phosphorothioate (1w). Yield: 93%,
white solid; m.p. 80–82 °C; IR: 1681 (amide I), 1649, 1600, 1589 (ν CCaromatic), 1529 (amide II), 1488
1
(ν CCaromatic) cm−1; H-NMR: δ 9.06 (1H, bs, NH), 7.99 (1H, d, J = 8.5 Hz, H3), 7.68–7.64 (2H, m,
H2', H6'), 7.49–7.44 (2H, m, H3', H5'), 7.40 (1H, t, J = 1.8 Hz, H6), 7.31 (1H, td, J = 8.5 Hz, 1.0 Hz,
H4), 4.29–4.17 (4H, m, CHAHB), 1.33 (6H, tt, J = 7.1 Hz, 0.8 Hz, CH3); 13C-NMR: δ 161.9, 147.9 (1C,
d, J = 8.8 Hz), 138.0 (1C, d, J = 1.4 Hz), 137.1, 132.9, 131.9, 126.3 (1C, d, J = 1.4 Hz), 125.4 (1C, d,
J = 6.1 Hz), 121.6, 121.5 (1C, d, J = 2.8 Hz), 117.1, 66.1 (2C, d, J = 5.4 Hz, CHAHB), 15.8 (2C, d,
J = 7.4 Hz, CH3); 31P-NMR: δ 62.3. Anal. Calcd for C17H18BrClNO4PS (478.72): C, 42.65; H, 3.79; N,
2.93. Found: C, 42.77; H, 3.84; N, 2.78.
O-{5-Chloro-2-[(3-fluorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1x). Yield: 48%,
oily liquid; IR: 1662 (amide I), 1599 (ν CCaromatic), 1541 (amide II), 1491 (ν CCaromatic) cm−1; 1H-NMR:
δ 9.11 (1H, bs, NH), 7.99 (1H, dd, J = 8.5 Hz, 1.0 Hz, H3), 7.71 (1H, dt, J = 11.0 Hz, 2.0 Hz, H6'),
7.42–7.26 (4H, m, H4, H6, H2', H5'), 6.84 (1H, td, J = 8.5 Hz, 1.8 Hz, H4'), 4.30–4.18 (4H, m, CHAHB),
1.32 (6H, dt, J = 7.0 Hz, 0.9 Hz, CH3); 13C-NMR: δ 162.9 (1C, d, J = 243.3 Hz, C3'), 162.0, 147.9 (1C,
d, J = 8.6 Hz), 139.4 (1C, d, J = 10.9 Hz), 138.0 (1C, d, J = 1.9 Hz), 132.9, 130.0 (1C, d, J = 9.4 Hz), 126.3
(1C, d, J = 1.4 Hz), 125.3 (1C, d, J = 6.1 Hz), 121.5 (1C, d, J = 3.0 Hz), 115.3 (1C, d, J = 3.0 Hz, C6'),
111.2 (1C, d, J = 21.3 Hz), 107.5 (1C, d, J = 26.4 Hz), 66.1 (2C, d, J = 5.5 Hz, CHAHB), 15.8 (2C, d,
J = 7.3 Hz, CH3); 31P-NMR: δ 62.4. Anal. Calcd for C17H18ClFNO4PS (417.82): C, C, 48.87; H, 4.34;
N, 3.35. Found: C, 49.02; H, 4.50; N, 3.24.
O-{5-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl} O,O-diethyl phosphorothioate (1y). Yield: 83%,
white solid; m.p. 41–42 °C; IR: 1655 (amide I), 1640, 1616 (ν CCaromatic), 1560 (amide II), 1507, 1484
1
(ν CCaromatic) cm−1; H-NMR: δ 9.02 (1H, bs, NH), 7.99 (1H, dd, J = 8.8 Hz, 1.0 Hz, H3), 7.75–7.69
(2H, m, H2', H6'), 7.40 (1H, t, J = 1.8 Hz, H6), 7.31 (1H, ddd, J = 8.8 Hz, 2.0 Hz, 1.0 Hz, H4), 7.08–7.02
13
(2H, m, H3', H5'), 4.29–4.17 (4H, m, CHAHB), 1.32 (6H, dt, J = 7.0 Hz, 0.9 Hz, CH3); C-NMR: δ
161.8, 159.4 (1C, d, J = 242.5 Hz, C4'), 147.9 (1C, d, J = 8.9 Hz), 137.8 (1C, d, J = 1.9 Hz), 134.0 (1C,
d, J = 2.8 Hz, C1'), 132.8, 126.2 (1C, d, J = 1.4 Hz), 125.5 (1C, d, J = 6.1 Hz), 121.7 (2C, d, J = 7.8 Hz,

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C2', C6'), 121.5 (1C, d, J = 3.0 Hz), 115.6 (2C, d, J = 21.2 Hz, C3', C5'), 66.0 (2C, d, J = 5.6 Hz, CHAHB),
15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.4. Anal. Calcd for C17H18ClFNO4PS (417.82): C, 48.87;
H, 4.34; N, 3.35. Found: C, 48.66; H, 4.30; N, 3.18.
O-(5-Chloro-2-{[3-(trifluoromethyl)phenyl]carbamoyl}phenyl) O,O-diethyl phosphorothioate (1z).
Yield: 78%, oily liquid; IR: 1681 (amide I), 1598 (ν CCaromatic), 1552 (amide II), 1493, 1446 (ν
1
CCaromatic) cm−1; H-NMR: δ 9.22 (1H, bs, NH), 8.07 (1H, s, H2'), 8.01 (1H, dd, J = 8.8 Hz, 1.0 Hz,
H3), 7.96 (1H, d, J = 8.0 Hz, H6'), 7.48 (1H, t, J = 7.8 Hz, H5'), 7.43 (1H, t, J = 1.5 Hz, H6), 7.39 (1H,
d, J = 8.0 Hz, H4'), 7.32 (1H, ddd, J = 8.5 Hz, 1.8 Hz, 1.0 Hz, H4), 4.30–4.18 (4H, m, CHAHB), 1.32
13
(6H, dt, J = 7.0 Hz, 1.0 Hz, CH3); C-NMR: δ 162.0, 148.0 (1C, d, J = 8.6 Hz), 138.5, 138.2 (1C, d,
J = 2.0 Hz), 132.9, 131.3 (1C, q, J = 32.3 Hz, C3'), 129.5, 126.3 (1C, d, J = 1.4 Hz), 125.0 (1C, d,
J = 6.1 Hz), 123.8 (1C, q, J = 270.9 Hz, CF3), 123.1 (1C, d, J = 0.9 Hz), 121.5 (1C, d, J = 3.1 Hz),
121.0 (1C, q, J = 3.8 Hz, C2'), 116.7 (1C, q, J = 4.0 Hz, C4'), 66.1 (2C, d, J = 5.5 Hz, CHAHB), 15.7
(2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.5. Anal. Calcd for C18H18ClF3NO4PS (467.83): C, 46.21; H,
3.88; N, 2.99. Found: C, 46.37; H, 3.75; N, 3.13.
O-(5-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl) O,O-diethyl phosphorothioate (1zz).
Yield: 78%, white solid; m.p. 61–62 °C (acetone-hexane); IR: 1668 (amide I), 1594 (ν CCaromatic), 1542
1
(amide II), 1474 (ν CCaromatic) cm−1; H-NMR: δ 9.25 (1H, bs, NH), 8.01 (1H, dd, J = 8.7 Hz, 0.9 Hz,
H3), 7.89 (2H, d, J = 8.4 Hz, H3', H5'), 7.62 (2H, d, J = 8.4 Hz, H2', H6'), 7.42 (1H, t, J = 1.8 Hz, H6),
7.31 (1H, ddd, J = 8.6 Hz, 2.0 Hz, 1.0 Hz, H4), 4.33–4.15 (4H, m, CHAHB), 1.32 (6H, dt, J = 7.0 Hz,
J = 0.9 Hz, CH3); 13C-NMR: δ 162.1, 148.0 (1C, d, J = 8.9 Hz), 141.0, 138.2 (1C, d, J = 2.0 Hz), 132.9
(1C, d, J = 1.0 Hz), 126.3 (1C, d, J = 1.5 Hz), 125.1 (1C, d, J = 6.1 Hz), 126.2 (2C, q, J = 3.8 Hz, C3',
C5'), 126.2 (1C, q, J = 32.5 Hz, C4'), 124.1 (1C, q, J = 269.9 Hz, CF3), 121.5 (1C, d, J = 3.0 Hz),
119.7, 66.1 (2C, d, J = 5.6 Hz, CHAHB), 15.8 (2C, d, J = 7.4 Hz, CH3); 31P-NMR: δ 62.3. Anal. Calcd
for C18H18ClF3NO4PS (467.83): C, 46.21; H, 3.88; N, 2.99. Found: C, 46.06; H, 3.71; N, 2.82.
3.3. In Vitro Antimycobacterial Activity
The in vitro antimycobacterial activity of the synthesized compounds was determined against
Mycobacterium tuberculosis My 331/88 (H37Rv; dilution of strain 10−3), M. avium My 330/88 (dilution
of strain 10−5), M. kansasii My 235/80 (dilution of strain 10−4) and M. kansasii 6509/96 (dilution of
strain 10−4). All of the strains were obtained from the Czech National Collection of Type Cultures
(CNCTC, Brno, Czech Republic) with the exception of M. kansasii 6509/96, which was clinically
isolated. The antimycobacterial activity of the compounds was determined in a Šula’s semisynthetic
medium (SEVAC, Prague, Czech Republic) via the micromethod for the determination of the
minimum inhibitory concentration (MIC) at 37 °C after 14 and 21 days and after 7, 14 and 21 days for
M. kansasii [15]. The tested compounds and para-aminosalicylic acid (PAS) were added to the
medium in DMSO solutions, and INH was used as a standard in a sterile water solution. The
concentrations of the tested compounds were used as following: 500, 250, 125, 62.5, 32, 16, 8, 4, 2, 1
and 0.5 µM. The same concentrations within the range from 0.5 to 250 µM were used for INH and
PAS; these compounds were selected as comparative drugs.

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3.4. Determination of IC50s for Cholinesterases
The IC50 values were determined using the spectrophotometric Ellman’s method, which is a simple,
rapid and direct method to determine the SH and -S-S- group content in proteins [16]. This method is
widely used for cholinesterase activity evaluation and screening of the efficiency of cholinesterase
inhibitors. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-
nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-
nitrobenzoic acid (DTNB) and thiocholine, a product of substrate hydrolysis by cholinesterase (i.e.,
acetylthiocholine, ATCh) [17]. All the tested compounds were dissolved in dimethyl sulfoxide
(concentration 0.01 M) and then diluted in demineralised water (concentration 0.001 M and 0.0001 M).
The Ellman’s method is slightly modified according to Zdrazilova et al. [14]. Acetylcholinesterase was
obtained from electric eel (Electrophorus electricus L.) and butyrylcholinesterase from equine serum.
4. Conclusions
A series of 27 new salicylanilide derivatives containing diethyl thiophosphate moieties was
synthesized with the goal of improving the antimycobacterial activity of salicylanilide diethyl
phosphates as potential salicylanilide prodrugs presented earlier. However, the expected improvement
was not achieved. Derivatives of 5-bromosalicylic acid, 3,4-dichloroaniline, 3- and 4-(trifluoro-
methyl)aniline exhibited superior antimycobacterial activity. The thiophosphate moiety also inspired
us to evaluate this series against acetylcholinesterase and butyrylcholinesterase. All the discussed
compounds expressed a significant inhibition of both AChE and BChE. Their activities were compared
to those of the known acetylcholinesterase inhibitors galanthamine and rivastigmine. All tested
compounds showed higher or comparable activity to that of rivastigmine and slightly lower than that of
galanthamine. IC50 values for salicylanilide diethyl thiophosphates were found to be similar for all of
the compounds and also for both AChE and BChE. We demonstrated unequivocally that salicylanilide
diethyl thiophosphates share dual biological activity as inhibitors of both cholinesterases and various
mycobacteria. Obviously, there is no correlation between both activities. It is a result of different
expected mechanisms of action and the active part of the molecule: the salicylanilide scaffold confers
antimicrobial activity preferentially, but the cholinesterase inhibition arises from the presence of the
thiophosphate moiety.
Acknowledgments
The work was financially supported by the Research project IGA NT 13346 (2012).
This publication is a result of the project implementation: “Support of establishment,
development, and mobility of quality research teams at the Charles University”, project number
CZ.1.07/2.3.00/30.0022, supported by The Education for Competitiveness Operational Programme
(ECOP) and co-financed by the European Social Fund and the state budget of the Czech Republic.
The publication is co-financed by the European Social Fund and the state budget of the Czech
Republic. Project No. CZ.1.07/2.3.00/30.0061.
The publication is co-financed by the European Social Fund and the state budget of the Czech
Republic Project No. CZ.1.07/2.3.00/20.0235, the title of the project: TEAB.

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This work was supported also by Faculty of Chemical Technology, University of Pardubice and
project SGSFChT_2014005.
The language assistance of J. Urbanová, M.A., is greatly appreciated.
Author Contributions
This study was performed under the guidance of Prof. Jarmila Vinšová. Echchukattula Dadapeer
and Martin Krátký synthesized compounds and M. Krátký characterized all presented compounds.
Markéta Komlóová dealt with theoretical part of cholinesterases inhibition. Šárka Štěpánková and
Katarína Vorčáková did the enzymes inhibition studies and Jiřina Stolaříková made antimycobacterial
measurement. J. Vinšová, M. Krátký, M. Komlóová and Š. Štěpánková wrote the whole article, made
discussion and conclusions.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1a–1zz are available from the authors.
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