3-Hydroxynaphthalene-2-carboxanilides and their antitrypanosomal activity

Article abstract of DOI:10.1007/s00706-017-2099-1

Abstract: Series of ring-substituted 3-hydroxynaphthalene-2-carboxanilides were screened for their in vitro activity against wild-type S427 (bloodstream form) of Trypanosoma brucei brucei. 3-Hydroxy-N-(3-trifluoromethylphenyl)- and 3-hydroxy-N-(4-trifluoromethylphenyl)naphthalene-2-carboxamides showed the highest biological activity (MIC?=?1.56 and 2.08?μmol/dm³, respectively). Antitrypanosomal activity was correlated with the experimentally determined lipophilicity and acid–base dissociation constants of the compounds as well as with the calculated electronic properties of individual anilide substituents expressed as Hammett’s σ parameters. The substitution in the meta- or para-position of anilide of derivatives with higher lipophilicity by an electron-withdrawing moiety is favourable for higher activity. The optimum thermodynamic pK_a ^T value was found to be ca. 7.5. The structure–activity relationships of all compounds are discussed. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-017-2099-1

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-017-2099-1
ORIGINAL PAPER
3-Hydroxynaphthalene-2-carboxanilides and their antitrypanosomal
activity
Jiri Kos1 Iva Kapustikova1 Carol Clements2 Alexander I. Gray2 Josef Jampilek1
•
•
•
•
Received: 17 October 2017 / Accepted: 13 November 2017
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
Series of ring-substituted 3-hydroxynaphthalene-2-carboxanilides were screened for their in vitro activity against wild-type
S427 (bloodstream form) of Trypanosoma brucei brucei. 3-Hydroxy-N-(3-trifluoromethylphenyl)- and 3-hydroxy-N-(4-
trifluoromethylphenyl)naphthalene-2-carboxamides showed the highest biological activity (MIC = 1.56 and 2.08 lmol/
dm3, respectively). Antitrypanosomal activity was correlated with the experimentally determined lipophilicity and acid–base
dissociation constants of the compounds as well as with the calculated electronic properties of individual anilide substituents
expressed as Hammett’s r parameters. The substitution in the meta- or para-position of anilide of derivatives with higher
lipophilicity by an electron-withdrawing moiety is favourable for higher activity. The optimum thermodynamic pKTa value
was found to be ca. 7.5. The structure–activity relationships of all compounds are discussed.
Graphical abstract
Keywords Carbonyl compounds Á Antitrypanosomal activity Á Bioorganic chemistry Á Structure–activity relationships
Introduction
Sleeping sickness, human African trypanosomiasis, is
caused by protozoan parasites Trypanosoma brucei that
have traditionally been grouped into three subspecies: T. b.
brucei, T. b. gambiense, and T. b. rhodesiense. T. b.
& Carol Clements
c.j.clements@strath.ac.uk
gambiense is found in the West and Central Africa region
and currently accounts for 97% of cases and causes a
& Josef Jampilek
josef.jampilek@gmail.com
chronic infection. T. b. rhodesiense is found in the Eastern
and Southern Africa, represents \ 3% of cases, and causes
1
an acute infection. Currently, the estimated number of
actual cases is 20,000, and the estimated population at risk
is 65 million people. Only five drugs have been used for the
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Comenius University, Bratislava, Slovakia
2
Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, Glasgow, Scotland, UK
123

J. Kos et al.
treatment of sleeping sickness: pentamidine (against T. b.
gambiense) and suramin (against T. b. rhodesiense) are
used for the first stage treatment and melarsoprol (used for
the treatment of both types), eflornithine, and nifurtimox
(against T. b. gambiense) are used for the second stage
treatment. Four from these five drugs approved for the
treatment of sleeping sickness show non-negligible unde-
sirable effects [1, 2]. These facts underline the urgency of
searching for new and inexpensive structure types of drugs
to achieve the effective control of African trypanosomes
[1, 3, 4].
amide. All the compounds were purified by recrystalliza-
tion from ethanol [5].
The structures of the investigated compounds are men-
tioned in Table 1 together with the physicochemical
parameters: electronic properties of individual anilide
substituents expressed as Hammett’s r constants predicted
using sw. ACD/Percepta ver. 2012; lipophilicity values of
the anilides expressed as logarithm of capacity factor
log k experimentally determined by RP-HPLC [5]; and
acid–base dissociation constants expressed as pKTa experi-
mentally determined using capillary electrophoresis.
Lipophilicity and acid–base dissociation constants are
fundamental and most important physicochemical param-
eters that influence the bioavailability of drugs, i.e., their
transport through biological membranes to their active site
[22, 23].
3-Hydroxynaphthalene-2-carboxanilides, cyclic ana-
logues of salicylanilides, were recently prepared and pub-
lished as compounds with noteworthy biological activities
[5, 6] based on the presence of an amide group [3, 7, 8] and
a spatially close hydroxy moiety [6, 9–14] in the naph-
thalene scaffold. Salicylanilides are known as multitarget
agents with a broad spectrum of biological activities
[15, 16]. The properties of the amide moiety can be easily
modified by various substitutions [17]. The amide moiety
also occurs in many antiprotozoan drugs [2–4, 18–20]. Due
to the formation of hydrogen bonds between the CONH
group of amides and target proteins, changes in protein
conformation may occur, which results in killing proto-
zoans [21].
The determination of acid–base dissociation constant
(pKa) using capillary zone electrophoresis is based on the
measurement of the effective electrophoretic mobility of
the studied compound as a function of separation elec-
trolyte pHs. This relationship is then fitted by a sigmoidal
curve, where pKa is the value of pH in the sigmoid
inflection point. Then, pKa value obtained in buffer solu-
tions is recalculated to thermodynamic pKTa , which corre-
spond to water medium with zero ionic strength [23–25].
The pKa and pKTa values of individual compounds are given
in Table 1.
Thus, in the context of the above-mentioned facts, the
recently prepared series of ring-substituted 3-hydroxy-
naphthalene-2-carboxanilides [5] were additional tested on
their in vitro antitrypanosomal activity against T. b. brucei.
In vitro antitrypanosomal activity
3-Hydroxynaphthalene-2-carboxanilides showed the high-
est potency against wild-type S427 (bloodstream form) of
T. b. brucei compared to other positional isomers, e.g.,
2-hydroxynaphthalene-1-carboxanilides or 1-hydroxy-
naphthalene-2-carboxanilides [26]. The potency of the
compounds was expressed as the minimum inhibitory
concentration (MIC). Activity was primarily associated
with meta- and para-substituted derivatives. 3-Hydroxy-N-
Results and discussion
Chemistry
All the studied compounds were prepared according to
Scheme 1 by means of microwave-assisted synthesis in one
step. Briefly, the carboxyl group was activated with phos-
phorus trichloride, and then, aminolysis of acyl chloride by
ring-substituted aniline in dry chlorobenzene yielded a final
(3-trifluoromethylphenyl)-(7b), 3-hydroxy-N-(4-trifluo-
romethylphenyl)-(7c), 3-hydroxy-N-(3-nitrophenyl)-(8b),
Scheme 1
a) PCl3, anhydrous chlorobenzene, MW: 1st phase: 10 min, 100 °C, 100 W; 2nd phase: 15 min, 120°C,
500 W; 3rd phase: 20 min, 130 °C, 500 W
123

3-Hydroxynaphthalene-2-carboxanilides and their antitrypanosomal activity
Table 1 Structure of 3-hydroxynaphthalene-2-carboxanilides 1–8c,
experimentally determined values of lipophilicity log k [5], predicted
values of electronic Hammett’s r parameters of substituents R,
experimentally determined values of pKa expressed as mean SD
(n = 3 experiments), coefficients of determination (R2), calculated
thermodynamic acid–base dissociation constants pKTa and in vitro
antitrypanosomal activity against T. b. brucei (MIC/lmol/dm3) in
comparison with suramin (SUR) standard
Comp.
R
log k [5]
ra
pKa SD
R2
pKTa
MIC/lmol/dm3
1
H
0.6310
0.6916
0.6971
0.5951
0.6936
0.8831
0.8753
0.7303
0.8296
0.7317
0.9509
1.0796
1.0687
0.9715
1.1536
1.1459
0.8762
1.2053
1.2835
0.8710
0.8143
0.9175
-
0
7.551 0.012
7.802 0.034
7.344 0.034
7.743 0.041
7.587 0.028
7.484 0.012
7.646 0.040
7.422 0.017
7.455 0.010
7.506 0.017
7.455 0.016
7.638 0.036
7.563 0.030
7.638 0.041
7.592 0.032
7.614 0.052
7.638 0.022
7.445 0.000
7.937 0.176
7.633 0.036
7.529 0.051
7.509 0.054
-
0.9994
0.9930
0.9936
0.9962
0.9947
0.9994
0.9867
0.9980
0.9992
0.9987
0.9994
0.9979
0.9977
0.9972
0.9983
0.9933
0.9986
0.9976
0.9735
0.9921
0.9822
0.9789
-
7.594
7.846
7.387
7.787
7.630
7.527
7.689
7.465
7.499
7.550
7.499
7.682
7.606
7.681
7.636
7.658
7.681
7.489
7.981
7.677
7.573
7.553
-
NA
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
SUR
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
- 0.28
0.12
- 0.27
- 0.17
- 0.07
- 0.17
0.06
0.34
0.06
0.22
0.37
0.23
0.22
0.39
0.23
0.51
0.43
0.51
0.77
0.71
0.78
-
12.50
5.21
6.25
NA
NA
NA
NA
3-F
NA
4-F
NA
2-Cl
25.00
12.50
16.67
25.00
5.21
5.73
25.00
1.56
2.08
25.00
2.34
1.69
0.13
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CF3
3-CF3
4-CF3
2-NO2
3-NO2
4-NO2
-
NA no activity
aCalculated using the ACD/Percepta ver. 2012 (Advanced Chemistry Development, Inc., Toronto, ON, Canada)
and 3-hydroxy-N-(4-nitrophenyl)naphthalene-2-carboxami-
des (8c) demonstrated the highest activity within the series
with MIC = 1.56, 2.08, 2.34, and 1.69 lmol/dm3, respec-
tively. Unfortunately, as was published recently by Kos
et al. [5], both nitro derivatives 8b and 8c showed signifi-
cant cytotoxic effect against human monocytic leukemia
THP-1 cells [LD50 = 2.5 0.9 lmol/dm3 (8b) and
LD50 \ 0.37 lmol/dm3 (8c)]. For example, LD50 of
oxaliplatin is 1.7 0.6 lmol/dm3 and camptothecin LD50
is 0.20 0.07 lmol/dm3 [5, 27]. The toxicity of nitro
derivatives correlates with the position of the substituents
on the aniline moiety, where the toxicity increases with the
nitro group shift from the ortho- to para-position. The
highest dose of other effective compounds in the medium
(30 lmol/dm3) did not lead to significant lethal effect on
THP-1 cells, i.e., LD50 [ 30 lmol/dm3 [5].
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
3-CF
3
4-CF
3
4-NO2
3-NO2
3-Br
3-OCH3
4-Br
4-OCH3
3-Cl
4-Cl
2-OCH3
2-NO2
0.8
3 2-Cl
2-Br
1.0
2-CF
0.5
0.6
0.7
0.9
log k
1.1
1.2
1.3
ortho
meta
para
Fig. 1 Dependence of antitrypanosomal activity expressed as
log[1/(MIC/mol/dm3)] of investigated compounds on lipophilicity
expressed as log k
The dependence of antitrypanosomal activity on
lipophilicity, expressed as log k, is shown in Fig. 1. There
are three separate trends in the chart. The activity decreases
with increasing lipophilicity for the least effective ortho-
substituted derivatives. In general, for meta- and para-
substituted compounds, it can be stated that the activity
increases with increasing lipophilicity; however, for
methoxy, nitro, and trifluoromethyl derivatives, i.e.,
123

J. Kos et al.
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4-NO2
3-CF
3
4-NO2
3-NO2
4-CF
3
3-CF
3
4-CF
3
3-NO2
3-OCH3
3-Br
3-Br
4-Br
4-OCH3
2-OCH3
4-Br
3-OCH3
4-OCH3
2-OCH3
3-Cl
2-Br
2-CF3
3-Cl
4-Cl
2-Cl
4-Cl
2Cl
2-NO2
2-Br
2-CF
2-NO2
3
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
-0.4
-0.2
0.0
0.2
0.4
0.6
para
0.8
1.0
pKaT
meta
σ
ortho
para
ortho
meta
Fig. 3 Dependence of antitrypanosomal activity expressed as
log[1/(MIC/mol/dm3)] of investigated compounds on acid–base
properties of C(3) phenolic moiety expressed as thermodynamic pKaT
values. Excluded compounds are marked by an empty symbol
Fig. 2 Dependence of antitrypanosomal activity expressed as
log[1/(MIC/mol/dm3)] of the tested compounds on Hammett r
constants of individual substituents R. Excluded compounds are
marked by an empty symbol
evaluation. For poorly active ortho-substituted derivatives, it
can be stated that the activity increases with the increasing
pKTa value, i.e., with the decrease of the acidity and ioniz-
ability of phenolic hydroxyl. For effective meta- and para-
substituted derivatives, a bilinear trend with the optimal pKaT
value between 7.49 compound 7b (3-CF3) and 7.55 8b (4-
NO2) can be observed in Fig. 3.
substituents with an ability to form hydrogen bonds and
other weak intermolecular interactions, the dependence of
the activity on log k seems to be as quasi-parabolic, while
for chlorinated, brominated (and also triflouromethyl sub-
stituted) compounds, the efficiency increases with
increasing lipophilicity. Thus, it seems that log k about 1.2
can be considered as a lipophilicity optimum. It should be
noted that compounds 5b (R = 3-Cl) and 5c (R = 4-Cl)
showed worse solubility in the testing medium than the
corresponding bromo (compounds 6b, 6c) and tri-
flouromethyl (compounds 7b, 7c) substituted derivatives.
From the distribution of the individual substituents in
Fig. 1, it is evident that the antitrypanosomal activity is
also dependent on the electron properties of individual
substituents R that are expressed as Hammett r constants.
This dependence is illustrated in Fig. 2. In general, less
active ortho-substituted isomers show again a clear trend,
where the activity decreases with the electron-withdrawing
character of the substituent R. When the least effective
compounds 5b (3-Cl) and 5c (4-Cl) from meta- and para-
substituted derivatives are excluded, the opposite trend can
be observed for meta- and para-derivatives; activity
increases with electron-withdrawing properties.
Conclusion
A recently synthesised series of 22 ring-substituted 3-hydrox-
ynaphthalene-2-carboxanilides were screened for their in vitro
activity against wild-type S427 (bloodstream form) of Try-
panosoma b. brucei. 3-Hydroxy-N-(3-trifluoromethylphenyl)-
(7b) and 3-hydroxy-N-(4-trifluoromethylphenyl)naphthalene-
2-carboxamides (7c) showed the highest biological activity
(MIC = 1.56 and 2.08 lmol/dm3, respectively). It can be
stated that higher potency is connected with the meta-andpara-
positions of anilide substituents, while ortho-substituted
derivatives showed much less efficiency, and their activity is
not significantly influenced by neither lipophilicity, electron
properties nor acid–base properties. On the other hand, the
antitrypanosomal activity of the meta- and para-substituted
3-hydroxynaphthalene-2-carboxanilides is positively affected
by increasing lipophilicity and electron-withdrawing proper-
ties of anilide substituents. The optimum pKTa value seems to be
7.5.
A phenolic moiety in position 3 of the naphthalene scaf-
fold of the discussed compounds is important for their bio-
logical activity [5, 9]. As mentioned above, acid–base
properties also play an important role in biological activity of
many drugs; therefore, the pKa values of this phenolic moiety
were determined by capillary electrophoresis. Figure 3 illus-
trates the dependence of antitrypanosomal activity on acid–
base properties expressed as thermodynamic pKTa values. It
seems that pKTa values of the phenolic moiety in the C(3)
position are important for effective meta- and para-substi-
tuted derivatives. Compound 7c (4-CF3) showed limited
solubility in the electrophoretic system (SD was higher than
for other compounds); therefore, it was excluded from the
Experimental
Synthesis
The detailed synthetic pathway and full analytical charac-
terization (m.p., HPLC purity, IR, NMR, HR-MS) of
123

3-Hydroxynaphthalene-2-carboxanilides and their antitrypanosomal activity
discussed compounds 1–8a are provided in Kos et al. [5].
All the studied compounds are presented in Table 1.
of each analyte and the migration time of methanol as
electroosmotic flow marker according to the equation:
ꢀ
ꢁ
1
1
LdLt
U
leff
¼
À
;
ð1Þ
Acid–base dissociation constant determination
tmig tEOF
where Lt is the total length of capillary (m), Ld is the
effective length (m), U is the applied voltage (V), tmig is the
migration time of analyte (s), and tEOF is the migration time
of electroosmotic flow marker(s) [24, 29, 30].
All chemicals and solvents were of analytical-reagent
grade. MES, TAPS, CAPS, TES, and methanol were pur-
chased from Sigma-Aldrich (Germany). Sodium hydroxide
was from Fluka (Switzerland), and calibration buffers were
from WTW (Weilheim in Oberbayern, Germany). Experi-
ments were carried out using an Agilent 3D CE system
(Agilent Technologies, Waldbronn, Germany) equipped
with a diode array detector. Results were enregistered by a
system Agilent ChemStation and processed in the program
SigmaPlot v. 10.0 (San Rafael, CA, USA). The pH values
of used background electrolytes were measured with a pH
meter WTW InoLab pH Level 2 equipped with a combined
electrode WTW SenTix 41Ò (Germany). Electrophoretic
measurements were performed in a fused-silica capillary of
33.0 cm total length, 24.5 cm effective length (from
injection to detector), and 50 lm internal diameter. Before
first using and when background electrolyte was changed,
capillary was rinsed with 0.1 mol/dm3 NaOH for 15 min,
then deionised water for 15 min, and background elec-
trolyte for 15 min. Between consecutive runs, capillary
was rinsed for 30 s with water and 30 s with background
electrolyte. The sample was injected by pressure of
30–50 mbar for 3–5 s. A constant voltage of 15 kV was
applied at 25 °C. Detection was at wavelength 200 and
254 nm.
The dependence of experimentally obtained effective
mobilities on background electrolyte pH was fitted by the
sigmoid curve, which is described by the following equa-
tion for monoprotic weak acid:
À
lA
leff
¼
;
ð2Þ
1 þ 10ðpK ÀpHÞ
a
À
where lA is the ionic mobility of fully deprotonated acid.
This relationship was fitted by a sigmoidal mobility curve,
where pKa is the value of pH in the sigmoid inflection point
[24, 25].
As the measurements were carried out in the system of
buffer solutions with the ionic strength of 0.01 mol/dm3,
the obtained values of pKa had to be converted to the
thermodynamic dissociation constants (pKaT), which cor-
respond to aquatic environment of the zero ionic strength:
pKaT ¼ pKa À log cA
where the following formula was used for the calculation
;
ð3Þ
À
of the activity coefficients:
pffiffi
0:5084z2
I
pffiffi
log c ¼ À
:
ð4Þ
Stock solutions of tested substances were prepared in
methanol (0.5 mg/cm3). Working solution was prepared by
diluting stock solution of analytes in methanol to the final
concentration of 0.1 mg/cm3. Measurements were per-
formed in the system of seven or eight buffer solutions with
constant ionic strength 0.01 mol/dm3 within pH range
5.50–11.20. The online program Buffer Calculator was
employed to calculate the composition of the series of
background electrolytes with various pH values [28]. The
pH range of background electrolyte solutions was chosen to
guarantee the practically full deprotonation (A-) of ana-
lytes at the highest pH and the absence of their ionisation
(HA) at the lowest pH. An increase of the absolute value of
effective mobility with increasing pH was observed. At the
highest pH, the mobility of analyte is maximal, which
indicates the predominance of anionic species. As pH
decreases, a new species with zero electric charge is
formed by protonation of analyte, and its effective mobility
goes to zero. Measurement of each compound in each of
background electrolytes was repeated three times.
1 þ 0:3281a I
3
˚
For the ionic strength of 0.01 mol/dm and a = 5 A,
log c has the value of - 0.0437 [29, 30]. The pKa and pKaT
values of individual compounds are given in Table 1.
In vitro antitrypanosomal activity
The biological activity of the prepared compounds against
African trypanosome was performed using a modified
alamarBlue assay according to Raz et al. [31]. Biological
assays were performed in 96-well microtiter plates using
the alamarBlue reduction indicator. Resazurin (blue),
which is reduced to light pink fluorescence resorufin in the
presence of live parasite, is the active ingredient. Try-
panosoma brucei brucei S427 (a bloodstream form) was
cultivated at an optimal density of 3 9 104 trypanosome/cm3
in HMI-9 medium (Invitrogen, USA) supplemented with
10% fetal calf serum at 37 °C and 5% CO2. The tested
compounds were dissolved in DMSO, the concentration of
which did not exceed 1% in the total sample. The final
concentration of the discussed compounds in the range of
10–0.08 lmol/dm3 was obtained from a 200 lmol/dm3
The effective electrophoretic mobilities leff (m2/V/s) of
analytes were calculated from the observed migration times
123

J. Kos et al.
13. Gonec T, Pospisilova S, Holanova L, Stranik J, Cernikova A,
Pudelkova V, Kos J, Oravec M, Kollar P, Cizek A, Jampilek J
(2016) Molecules 21:1189
14. Kos J, Nevin E, Soral M, Kushkevych I, Gonec T, Bobal P,
Kollar P, Coffey A, O’Mahony J, Liptaj T, Kralova K, Jampilek J
(2015) Bioorg Med Chem 23:2035
15. Kratky M, Vinsova J (2011) Curr Pharm Des 17:3494
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Pharm Sci 77:197
17. Pattabiraman VR, Bode JW (2011) Nature 480:471
18. Ferrins L, Gazdik M, Rahmani R, Varghese S, Sykes ML, Jones
AJ, Avery VM, White KL, Ryan E, Charman SA, Kaiser M,
stock solution by repeated dilution with the medium.
Prior to the addition of alamarBlue (20 mm3/well,
0.49 mmol/dm3 in 19 PBS, pH 7.4), the plates were
incubated at 37 °C, 5% CO2 for 48 h. Then, an additional
24 h of incubation followed. Fluorescence values were
measured using a Wallac Victor 2 microplate reader (Per-
kin Elmer, USA) (excitation 560 nm, 590 nm emission)
giving control values in %. MIC values were obtained for
compounds showing less than 20% control values against
T. brucei brucei. The experiment was performed in dupli-
cate three times according to the previously described
studies [32, 33]. Suramin sulfate (Sigma) was used as a
standard. The results are summarized in Table 1.
¨
Bergstrom CA, Baell JB (2014) J Med Chem 57:6393
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Acknowledgements This study was supported by Grants FaF UK/13/
2016 and FaF UK/4/2017 and by SANOFI-AVENTIS Pharma Slovakia.
20. Masand VH, El-Sayed NNE, Mahajan DT, Rastija V (2017)
Environ Res 28:165
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