Polyfluorinated salicylic acid derivatives as analogs of known drugs: Synthesis, molecular docking and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2016.10.014

We have developed the convenient methods for synthesis of polyfluorosalicylic acids and their derivatives. For the first time the biological properties of polyfluorosalicylates were investigated in vitro (permeability through the biological membranes, COX-1 inhibitory action) and in vivo (anti-inflammatory, analgesic activities, acute toxicity). Molecular docking of polyfluorinated salicylates confirmed in vitro and in vivo experiments.

Full text of DOI:10.1016/j.bmc.2016.10.014

Accepted Manuscript
Polyfluorinated salicylic acid derivatives as analogs of known drugs: synthesis,
molecular docking and biological evaluation
E.V. Shchegol’kov, I.V. Shchur, Ya.V. Burgart, V.I. Saloutin, A.N. Trefilova,
G.A. Ljushina, S.Yu. Solodnikov, L.N. Markova, V.V. Maslova, O.P. Krasnykh,
S.S. Borisevich, S.L. Khursan
PII:
S0968-0896(16)30941-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.014
BMC 13341
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 September 2016
7 October 2016
10 October 2016
Please cite this article as: Shchegol’kov, E.V., Shchur, I.V., Burgart, Ya.V., Saloutin, V.I., Trefilova, A.N., Ljushina,
G.A., Solodnikov, S.Yu., Markova, L.N., Maslova, V.V., Krasnykh, O.P., Borisevich, S.S., Khursan, S.L.,
Polyfluorinated salicylic acid derivatives as analogs of known drugs: synthesis, molecular docking and biological
evaluation, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.10.014
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Polyfluorinated salicylic acid derivatives as analogs of known drugs:
synthesis, molecular docking and biological evaluation
E.V. Shchegol’kova*, I.V. Shchura, Ya.V. Burgarta, V.I. Saloutina,
A.N. Trefilovab, G.A. Ljushinab , S.Yu. Solodnikov b, L.N. Markovab, V.V. Maslovab,
O.P. Krasnykhb, S.S. Borisevichc, S.L. Khursanc
aPostovsky Institute of Organic Synthesis of Ural Branch of Russian Academy of Science, S. Kovalevskoy Str., 22,
Ekaterinburg 620990, Russia.
bPerm National Research Polytechnic University, Komsomolsky Av., 29, Perm 614990, Russia.
cUfa Institute of Chemistry of Russian Academy of Science, Octyabrya Av., 71, Ufa 450078, Russia.
Abstract. We have developed the convenient methods for synthesis of polyfluorosalicylic acids and their
derivatives. For the first time the biological properties of polyfluorosalicylates were investigated in vitro
(permeability through the biological membranes, COX-1 inhibitory action) and in vivo (anti-inflammatory, analgesic
activities, acute toxicity). Molecular docking of polyfluorinated salicylates confirmed in vitro and in vivo
experiments.
Keywords: Polyfluoronated salicylates, Nucleophilic substitution, ortho-Methoxylation, Molecular docking,
Anti-inflammatory and analgesic activities, COX-1 inhibition, Toxicity, Permeability.
1. Introduction
Salicylic acid (SA), its derivatives (salicylates), and particularly acetylsalicylic acid (aspirin,
ASA), are among the most successful drug classes ever: it has been serving on the drug market
since the end of 19th century and is still broadly used current days to treat a variety of pain,
fever, inflammation and other diseases.
The successful chemical modifications of initially quite toxic and ineffective SA allowed
obtaining a number of salicylates such as sodium salicylate, methyl salicylate, trolamine
salicylate, salsalate, ASA etc. Due to triad of anti-inflammatory, antipyretic and analgesic
effects, salicylates take place in the registry of clinically used non-steroidal anti-inflammatory
drugs (NSAIDs).1 Another equally important property of ASA is antiplatelet action and it is
successfully used in the treatment of cardiovascular diseases.2 Recent studies show that ASA
reduces blood sugar levels,3 has anti-cancer effect,4 and reduces the activity of the brain during
migraine attacks5 and so on.
However, the salicylate drugs retain some strong side effects. The main side effects of
salicylates including ASA are gastrointestinal ulcers, stomach bleeding, and ringing in the ears.
* Corresponding author. Tel./fax: +7-343-3745954; e-mail: schegolkov@ios.uran.ru
1

In children and adolescents, aspirin is not recommended for flu-like symptoms or viral illnesses,
because of the risk of Reye's syndrome.6
However, in experts’ opinion, the chemistry of SA and its derivatives is still an important
area for new drug research.7 It is connected with the development of new salicylates with lower
side effects, a high degree of activity, and the appearance of new types of biological action.
In addition to medicine, the SA and its derivatives have a great value in organic synthesis as
important reagents for various open-chain7 and heterocyclic molecules, i.e. such as coumarins,8
cumarones,9 1,2-benzisoxazoles,10 1H-indazoles,11 benzopyrans12 and isoflavanones13 and others.
Progress in this area we may see in polyfluoro-containing derivatives synthesis as analogs of
known salicylate drugs and in investigation of their biological properties. The fluorine atom has
being effectively and successfully used in medicinal chemistry for modulating the
physiochemical and biological properties of lead compounds in drug discovery programs. Due to
high electronegativity of fluorine atom, the fluorinated compounds are capable of forming strong
intermolecular fluorine-hydrogen like nucleotide-DNA bonds, which is important for the
directed drug synthesis.14,15 In fact, about 20 per cent of used pharmaceutical drugs have at least
a one fluorine atom in structure, and it is aromatic fluorine.16 Salicylate derivatives are also not
deprived of attention. For example, fluorinated drug diflunisal has anti-inflammatory activity in
two times more than ASA, and less irritating to the mucous membranes of the gastrointestinal
tract.17
The minimal information of biological effects of polyfluorosalicylic acids derivatives
suggests the future development in this direction. A number of polyfluorosalicylic amides is
patented as anticoagulants,18 as agents against obesity and diabetes,19 as inhibitors of platelet
aggregation and P2Y12 receptors.20 Recently, we have observed the high tuberculostatic activity
of polyfluorosalicylic acids. It has been found that minimum inhibitory concentration of the
compounds is from 0.7 to 6.5 µg/mL depending on the structure.21
2. Discussion
2.1. Chemistry
2.1.1. Synthesis of polyfluorosalicylic acids
Although polyfluorosalicylic acids has been known, but they were remaining hardly
accessible compounds until our investigations. It was connected with some obstacles in their
synthesis owing to presence of fluorine atoms, which are capable to substitution. It makes
ineffective of method for non-fluorinated salicylic acid synthesis.22 The described methods for
polyfluorosalicylic acid synthesis include the oxidation of exotic fluorooxoheterocylces,23 the
carboxylation of rather expensive partial fluorinated phenols,24 which demand complex
2

instrumentation, and nucleophilic substitution of available polyfluorobenzoic acids by sodium
hydroxide25 or alkali metals alkoxides26 which proceeds non-selectively with preferable para-
product formation.
However, we have found that the selective ortho-substitution of fluorine atom in
pentafluorobenzoic acid 1a proceeded under the action of magnesium methoxide followed by
acid hydrolysis of intermediate in situ allowed us to obtain tetrafluorosalicylic acid 4a (yield 42
%).27 Then, we have improved ortho-methoxylation of acid 1a-d,21 which allowed us to increase
the yields of target polyfluorosalicylic acids from high (4a, 65%) toward quantitative (4b-d, 93-
95%) (Scheme 1). As a result, we have developed a convenient and efficient method for
polyfluorosalicylic acid synthesis via the selective ortho-methoxylation of commercially
available polyfluorobenzoic acids.
Scheme 1
We have applied our new strategy to 3,5,6-trifluorosalicylic acid 4e synthesis (Scheme 2). In
this case, the successful synthesis of acid 4e was complicated by a choice of conditions for the
selective ortho-mono-methoxylation due to probable ortho-disubstituted by product 5e formation
(Scheme 2). The reaction of 1 eq. of acid 1e and 2.5 eq. of magnesium methoxide did not lead to
conversion of initial reagents in comparison with previous results for acid 1a (Scheme 1). The
increasing of Mg(OMe)2 quantity (5 eq.) and refluxing time (8 h) allowed us to yield a mixture
with good ratio of acids 1e, 2e, 5e (Table 1). Obviously, it is connected with poor solubility of
intermediate magnesium salt of acid 1e in toluene. The best conditions for full conversion of acid
1e with more selective formation of 2-methoxy-3,5,6-trifluorobenzoic acid 2e were refluxing in
diglyme with ratio of 1 eq. of acid 1e and 3.5 eq. of magnesium methoxide. The control of
19
reaction was performed by F NMR spectroscopy. Then, we treated the reaction mixture with
phosphorus pentachloride followed by fractional distillation and separation of the resulting
3

chloride of 2-methoxy-3,5,6-tetrafluorobenzoic acid 3e (Scheme 2). Hydrolysis of
chloroanhydride 3e in 48% hydrobromic acid led to 3,5,6-trifluorosalicylic acid 4e.
Scheme 2
Table 1.Optimization of orto-monomethoxylation of acid 1e.
Solvent
Mg(OMe)2, eq
Ratio of mixture, % in accordance with 19F NMR
spectroscopy in СDCl3
Compound 1e
Compound 2e
Compound 5e
diglyme
toluene
3.5
2.5
5
0
15
8
81
70
77
10
19
15
15
0
2.5
90
2.1.2. Modifications of polyfluorosalicylic acids
Further, we have carried out the synthesis of polyfluorinated analogues of the known drugs in
salicylic series based on modification of polufluorosalicylic acids 4a-e by carboxyl and hydroxyl
groups.
So, the treatment of acids 4a,b with sodium hydroxide gave to water-soluble sodium
salicylates 6a,b (Scheme 3).
4

Scheme 3
Ethyl 2-hydroxy-3,4,5,6-tetrafluorobenzoate 7a had been described previously.24a,28
However, the authors had some obstacles during the synthesis and isolation ester 7a due to
incomplete conversion of the initial acid 4a and the yield did not exceed 75 %. We managed to
increase the yields of ethyl tetrafluoro- and trifluorobenzoates 7a,b up to 85-88% (Scheme 3) by
using of 100-fold excess of ethanol in reactions with polyfluorosalicylic acids 4a,b. There was
the full conversion of parent acids 4a,b in accordance with thin-layer chromatography.
Use of the same quantity of methanol in this reaction allows to give methyl esters 8a,b
(analogs of methyl salicylate) with yields 87-90% (Scheme 3). Carrying out the reaction under
microwave irradiation can significantly reduce the synthesis time from 15 to 2 h and to increase
the yield up to 95%.
Polyfluorinated analogs of aspirin 9a-e were synthesized by acylation of polyfluorosalicylic
acids 4a-e under heating in acetic anhydride for 30 min (Scheme 3).
The development of convenient methods for synthesis of polyfluorosalicylic acids and
derivatives allowed us to investigate of their biological properties.
5

2.2.Biological activity
After oral administration, the amount of drug that reaches systematic circulation depends on
many factors such as active substance physicochemical properties and multiple parameters
characterizing its interaction on the molecular level with cell and tissue medium, cell membrane
barriers, guards (e.g. efflux pumps, transporters, metabolizing enzymes, etc.) and so on. We have
evaluated two characteristics - lipophilicity of synthesized compounds (calculated СlogP values)
and permeability through the lipid membranes (in vitro data for selected set) - which are of
special importance for the first steps of drug’s journey in vivo.
2.2.1. Calculation of lipophilicity
Lipophilicity represented as СlogPs was calculated using ChemBioDraw Ultra 13.0.2.302
(Table 3). The СlogP values depend on the number of fluorine atoms as well as on their
positioning in the benzene ring and overall these values are belonging in the numerical interval
1.0 – 2.6 which is not warning in terms of bioavailability of compounds. It is interesting that
alkylation or acetylation of phenolic OH group (compounds 2a,b and 9a-e correspondingly)
leads to reduction of lipophilicity while alkylation of COOH functionality (compounds 8a and
8b) slightly improves this property. The ClogP value for ASA is similar to one for its
tetrafluorinated derivative 9a.
87,9
128
64
22,1
32
18,6
16,4
16
5,1
8
4,7
pH 5
3,0
4
pH 6.2
pH 7.4
1,8
2
1
0,5
0.5
0.25
0.125
0.0625
0,2
0,1
0,1
0,1
0,1
ketoprofene ranitidine
ASA
4a
6a
Figure 1. Permeability of salicylates, Pe (10-6 cm/s), in logarithmic scale
2.2.2. In vitro bioavailability evaluation
Permeation of drug through the biological membranes is the crucial factor defining the
processes of drug absorption and distribution. The ability of synthesized compounds to passively
6

diffuse through the membranes was assessed in vitro using filter-immobilized artificial
membranes in Parallel Artificial Membrane Permeability Assay (PAMPA)29 for ASA, 4a, 6a, 9a
and 9b at pH 5.0, 6.2 and 7.4. Drugs ketoprofen and ranitidine were used as reference samples
with correspondingly high and low permeability.
Tetrafluorosalicylic acid 4a and its sodium salt 6a (i.e. corresponding anion) effectively
and better than ASA pass through the membrane at рН 5.0, however, they almost do not pass at
рН 7.4, which is a known characteristic of acidic compounds ionized in base medium (Fig. 1).
ASA is observed in a donor plate at both pH 5.0 and pH 7.4.
Compounds 9a and 9b are not found after incubation in both donor and acceptor plates.
Instead, the UV absorbance of the products of hydrolysis, i.e. corresponding fluorinated SAs 4a
and 4b, are observed in the donor plate at all used pHs (see Suppl. section). The data imply that
hydrolysis of acetylated derivatives starts quite promptly, in the first few minutes of the PAMPA
experiment. This raises the question: why do both tetrafluorosalicylic acid 4a and its sodium salt
6a pass through the membrane at pH 5.0 quite decently, however the acetylated derivatives 9a
and 9b – if suggest that they are just hydrolyzed – at all used values of pH do not show any
detectable presence in the acceptor plate neither themselves nor products of their hydrolysis? H1
NMR spectra of aqueous solutions of the compounds 9a and 9b at pH 7.4 do not indicate the
occurrence of any hydrolysis. It has been suggested that deacetylation of 9a and 9b in the
PAMPA experiment may be affected by the presence of some specific components in the model
biological fluids used in the assay and composition of which is not disclosed by manufacturer.
2.2.3. COX-1 inhibition
The basic mechanism underlying most pharmacological effects of ASA is an inhibition of
pro-inflammatory eicosanoid biosynthesis and particularly, the first step of arachidonic acid
(ACD) transformation catalyzed by prostaglandin endoperoxide H synthase - bifunctional
enzyme which works for two sequential reactions as cyclooxygenase (COX) and peroxidase
correspondingly. ASA irreversibly acetylates the serine residue in COX active site blocking
activity of constitutive COX-1 and impairing the activity of isoform COX-2 which in most
tissues is induced during the inflammation. A simplified and initially dominated view on the
COX-1 and -2 inhibitors where inhibition of COX-1 (constitutive isoform) was accounted for
most side effects of drugs, while their anti-inflammatory and analgesic activities attributed
mainly to COX-2 acetylation, was gradually significantly deflated. Selective inhibition of one of
these isoforms can lead to both desirable and not desirable side effects which is the case for ASA
as well.30-32
7

As a first step in mechanism of action study, the ability of the synthesized fluorinated SA
derivatives to inhibit ovine COX-1 was evaluated. COX Fluorescent Inhibitor Screening Assay
Kit (Cayman Chemical) was used and two concentrations, 2µM and 200µM, were tested (Table
2, Suppl. materials section for additional information).
Table 2. Inhibition of COX-1 enzyme by selected synthesized compounds
Compound
Inhibition of
COX-1, %
(C=2 µM)a
31
Inhibition of COX-1, %
(C=200 µM)a
ASA
2a
36
64
48
<10
not tested
2b
4a
34
59
47
35
39
31
48
18
6a
8a
9b
aThe mean values are a result of three determinations
It was reported that ASA is a moderate inhibitor of COX-1 with IC50 near 0.3 µM.33 The
synthesized compounds have very similar to ASA activity toward COX-1 in our experiment. It is
interesting that methylation of both functionalities in salicylic acid - phenolic OH and carboxylic
group – leads to the active towards COX-1 compounds: methyl-2-hydroxytetrafluorobenzoate 8a
and 2-methoxytetrafluorobenzoic acid 2a retain the potency close in value to one of the parent 2-
hydroxytetrafluorobenzoic acid 4a.
2.2.4. Anti-inflammatory activity in vivo
The carrageenan induced hind paw edema model in rats was used for a primary evaluation
of the anti-inflammatory activity of fluorinated derivatives SA.34 The measurements of paw
volume were conducted at the 1st, 3rd and 5th hours after carrageenane injection. Pre-treatment
of rats with synthesized compound at doses 25 or 50 mg/kg leads to a various effect at different
time points (Table 3). For most compounds the response is not developed in the first hour (data
are not presented), however prominently appears when measured on the 3rd and / or 5th hour.
Analysis of the anti-inflammatory effect revealed that tetrafluorinated and trifluorinated SAs
4a, b and ASAs 9a, b are active at both time points (3rd and 5th hours) at 25 and/or 50mg/kg.
The exception represents tetrafluorosalicylic acid 4a which causes moderate inhibition of
inflammation at the dose 25mg/kg on 5th hour only. However, at the dose of 50 mg/kg it is
active at both time points. All but one (9c) of tested difluorosubstituted SAs and ASAs are not
active. Functionalization of carboxyl moiety in fluorinated SAs leads to the loss of the activity
8

(4a vs 8a, 4b vs 6b) with exception the data on 3rd hour for 8b which expresses moderate anti-
inflammatory activity at 25 mg/kg. Contrary, the methylation of phenolic OH group leads to
active derivatives: 4a vs 2a and 4b vs 2b. The O-acetylation leads to similarly active compounds
in case of 4a (vs 9a) and 4c (vs 9c). However, it causes complete loss of the anti-inflammatory
activity in case of trifluorinated SA 4b (vs 9b) and appearance of some mild activity for only one
of the tested disubstituted derivatives (4c vs 9c).
Overall data is indicating that an acetylated (as in ASA) or free (as in SA) 2-OH group is not
strictly required for the anti-inflammatory activity of fluorinated SA derivatives thus it can be
substituted for OMe, for example. This implies that different other types of O-substitution can be
done during the leads optimization.
Anti-inflammatory activity of several synthesized compounds is comparable to that of ASA.
Since the carrageenan induced model is based on the only parameter (an inflamed paw volume),
other test systems will be useful for re-evaluating the most active compounds. Additional test
results are also required for drawing the meaningful conclusion regarding the structure-activity
relationship.
2.2.5. Analgesic activity in vivo
The analgesic activity of the synthesized compounds was evaluated in “hot plate” test in
rats38 at the dose of 25 mg/kg. All of the tested compounds (Table 3) demonstrated an analgesic
activity comparable to one of acetylsalicylic acid or significantly higher. Two compounds, 4a
and 4b, are more active than diclofenac at the same dose. The only three not active compounds
among the tested in this group (4c, 8a and 9d) do not represent an uniform group from the point
of view of their structures since they are different in terms of numbers and positions of fluorine
atoms as well as in terms of functional groups. Two of them (4c, 8a) also did not inhibit the
carrageenan induced inflammation and 9d was not tested for anti-inflammatory activity.
The overall in vivo data does not reveal any clear correlation between anti-inflammatory
and analgesic action of tested compounds, leading to the hypothesis that antinociceptive
properties of fluorine SA and ASA derivatives and their anti-inflammatory activity may result
from modulating the function of distinct biological targets or the combinations of these targets
which are not completely overlapping for the two types of activity. Additionally,
pharmacokinetic properties should be taken later into the consideration for drawing the
conclusion regarding the structure –activity relationship.
9

2.2.6. Acute toxicity
The acute toxicity of compounds was estimated in CD-1 mice. The preliminary data
(Table 3) allows us to conclude that LC50 values for most of the synthesized fluorinated salicylic
acids derivatives are expected to belong in the range 100 – 300 mg/kg. Overall acute toxicity of
the fluorinated derivatives seems to be higher than ASA toxicity, especially on the molar basis.
Wherein, difluorosubstituted derivatives are slightly less toxic than tri- and tetrafluorosubstituted
ones.
10

Table 3. ClogP values and biological activity of fluorinated salicylates in vivo
Compound ClogP
Anti-inflammatory activity:
swelling inhibition,%
Analgesic
activity:
Acute toxicity:
dose, mg/kg
latent period
prolongation,%
(viability, %)
after 3 h
53.8b, **
after 5 h
46.9b, *
1.80
100.8
73,2
n/t
2a
1.81
40.0c, **
30.0c, *
50 (100), 150 (80),
300 (0) §§
2b
n/a b
33.1 b, **
143.2
75 (66), 300 (0) §
2.33
4a
64.0 c , ****
56.5c, ****
2.47
46.7 c, ***
n/a b
46.9 c, ****
n/a b
178.1
n/a
300 (0) §
4b
2.38
100(66) 150(66),
300(33) §
4с
b
2.58
n/a
n/a b
60.6
100 (100), 150 (66),
4d
300(33) §
b, **
2.18
47.0
41.7 b, *
n/aс
60.3
66.8
n/a
150 (66) §
150(66) §
4e
0.85
n/aс
6b
-2.26a
2.35
n/a c
n/ac
50 (100), 150 (90),
300 (0) §§
8a
2.58
40,3c, *
42.9 b, *
n/a c
n/t
100(100), 150 (100),
8b
300 (0) §§
1.48
56,8 b, *
83.2
71.2
50 (100), 150 (80),
300 (0) .§§
9a
65.6 c, **** 45.1 c , **
9b
1.71
n/a c
n/a c
50 (100), 150 (70),
300 (0) §§.
1.09
1.29
1.08
1.48
n/ab
n/t
35.3b, *
n/t
98.8
300(33) §
9с
9d
150 (100) §
n/t
n/a
37.3b, **
30.6-56.2b
32.7b, **
29.4 - 44.9b
86.5
9e
57.9 17.0b, d
ASA
100 (100), 150
(100), 300 (33) §.
179.6 (109.1-295.5),
mice, ip35
74, mice, ip36
64.4 9.8 b, e 54.4 3.1b, e
DIF
-
56.0 - 102.3b, f
(diclofenac)
a
for an anion; b at the dose of 25 mg/kg; c at the dose of 50 mg/kg; n/t – not tested; n/a – not active;
median for 6 different independent experiments standard deviation; median for 4 different independent
d
e
experiments standard deviation; f- interval of values obtained in 6 different experiments.
* p<0,05; ** p<0,01; ***p<0,001; ****p<0,0001.
§
set of experiments with 3 mice per group; §§ set of experiments with 10 mice per group
11

2.3. Molecular docking
We have performed the docking process of polyfluorinated salicylates (2a-9e) into the
tyrosine sites of COX-1 chosen as the basic targets (PDB code: 1DIY) and have compared the
calculated docking scores (∆Gbind) and ligand efficiencies of these derivatives with the values
characterizing the native ACD ligand and well-known COX inhibitors, DIF and ASA. According
to the calculation results the values of binding energy (∆Gbind) of 2a-9e are comparable with that
of the native ligand and COX-1 inhibitors (Table 4).
Table 4. Results of docking of all compounds to the active site of COX-1.
Ligand
|LE|a
RMSD
Ligand
|LE|
RMSD
∆Gbind
,
∆Gbind,
kcal/mol
-11.56
-7.32
-9.73
-7.39
-9.70
-9.22
-6.95
-7.39
-8.61
-7.71
-7.54
kcal/mol
-10.51
-8.81
ACD
SA
2a
0.52
0.73
0.65
0.53
0.63
0.71
0.54
0.62
0.66
0.55
0.58
0.02
0.15
0.07
0.06
0.07
0.08
0.15
0.09
0.06
0.07
0.06
DIF
ASA
7a
0.55
0.68
0.49
0.56
0.58
0.65
0.60
0.47
0.48
0.43
0.54
0.08
0.07
0.07
0.08
0.06
0.09
0.06
0.07
0.07
0.08
0.06
-7.81
-8.38
2b
4a
7b
8a
-8.74
-9.07
4b
4c
8b
9a
-10.24
-7.58
4d
4e
9b
9c
-7.66
-6.49
6a
9d
9e
-8.14
6b
a – ligand efficiency (LE = ∆Gbind/Nheavy atoms);
All compounds show interactions with surrounding amino acids: Val349, Leu352, Ile523,
Ser353, Ala527, Leu531, Arg120, Val116, Tyr355, Phe518, Leu359, Pro528, Ile345 and Ser530
(Fig. 2). More pictures are shown in Suppl. Section. The hydrogen bridges were identified
between the oxygen atoms of the ligands with Arg120 and Tyr355. The hydrophobic interactions
(yellow field is shown in the Fig. 2) of the inhibitors were recognized within the active site
residues. According to results of calculation the lead-compounds 2a, 4a, 4b and 9a are
determined. Docking results confirmed in vitro and in vivo tests.
12

2a
9a
4a
4b
Figure 2. Location of some polyfluorinated salicylates in the active tyrosine site of COX-1: the
hydrogen interactions are shown yellow dash; pose 9a: field of hydrophobic interactions are
shown yellow; the hydrogen bond acceptors – blue color and the hydrogen bond donors – rose
color.
3.
Conclusion
In summary, we have developed the convenient practical method for polyfluorosalicylic
acids synthesis, which allowed us to carry out their chemical modifications and the primary
biological investigations. Polyfluorosalicylates were evaluated in vitro (permeability through the
biological membranes, COX-1 inhibitory action) and in vivo (anti-inflammatory, analgesic
activities, acute toxicity) experiments and in silico by molecular docking. We have revealed the
leader-compounds 2a, 4a, b, 8b, 9a in a number of polyfluorinated salicylates. The obtained
results showed promising studies on further chemical modification polyflurosalicylates for the
development of new drugs.
13

4. Expiremental
4.1.Chemical part
Melting points were measured in the open capillaries on “Stuart SMP30” melting point
apparatus and uncorrected. The IR spectra were recorded on “Perkin Elmer Spectrum One FT-
IR” by diffuse reflection accessory (DRA) and “Thermo Nicolet 6700 FT-IR” spectrometers at
1
4000-400 cm-1 using the “Frustrated total internal reflection” method (FTIR). The H NMR
spectra were registered on “Bruker Avance DRX-400” 400 MHz or “Bruker Avance DRX-500”
500 MHz spectrometers relative to SiMe4. The 19F NMR spectra were obtained on “Bruker
Avance DRX-400” or “Bruker Avance DRX-500” spectrometers (19F, 376 or 470 MHz) using
C6F6 as an internal standard. The chemical shifts were converted from C6F6 to CCl3F. The
microanalyses (C, H, N) were carried out on “Perkin Elmer PE 2400” series II elemental
analyzer. The microwave syntheses were performed in sealed vessels (35 ml) with polymer caps
in microwave apparatus “CEM Discover & Explorer” with a power output ranging from 0 to 300
W. The temperature was monitored via an IR sensor located directly below the vessel. The vessel
contents were stirred by means of an adjustable speed electromagnet located below the
microwave cavity and a Teflon-coated magnetic stir bar inside the vessel. Temperature and
power profiles were recorded using computer controlled software. Acids 1a-d are commercially
available via P&M Invest® and Alfa Aesar®. Acid 1e have been obtained from compound 1a by
the known method.37
All physico-chemical characteristic of compounds 2a-d, 3a, 4a-d are represented in our
previous publication.21
ortho-Monomethoxylation of acids 1e. The magnesium shavings (8.4 g, 350 mmol) and a
few of iodine crystals were placed into two-necked flask with condenser and thermometer,
heated to fully disappear iodine. After cooling an absolute methanol (60 ml) and absolute
diglyme (200 ml) were added. A mixture was refluxed to fully dissolve of magnesium shavings.
Methanol was removed from the reaction mass. After cooling acid 1e (19.4 g, 100 mmol) was
added. The reaction mixture was refluxed for 2 hrs. Then, diglyme was removed in vacuo. The
residue decomposed with 10% HCl (200 ml) and precipitate filtered off. Recrystallization from
hexane gives a mixture of acids 2e and 5e in a ratio of 81 : 19.
2-Methoxy-3,5,6-trifluorobenzoyl chloride (3e). PCl5 (10 g, 0.05 mol) was added carefully
to a mixture of acid 2e and 5e (10 g, 0.05 mol) after methoxylation. The resulting liquid mass
o
was heated at 70 C for 1 h. The fractional distillation in vacuo gave chloride 3e. Yield 77%,
from clear to light-yellow liquid, bp 95-100 °С/15 Torr. IR (FTIR): ν 1785 (COCl), 1620, 1525,
1495 (С=С), 1280-1200 (С–F) cm-1. 1Н NMR (CDCl3): δ 4.03 (d, 3H, OMe, J 2.1 Hz), 7.15(ddd,
14

19
1Н, HAr, J 10.6, 9.8, 7.4 Hz). F NMR (CDCl3): δ -142.71 (ddd, 1F, FAr, J 22.1, 13.8, 7.4 Hz), -
139.42 (dd, 1F, FAr, J 22.1, 9.7 Hz), -130.33 (ddd, 1F, FAr, J 13.1, 11.0, 1.8 Hz). Anal. calcd. for
C8H4ClF3O2. С, 42.79; Н, 1.80. Found: С, 42.60; Н, 1.85.
2-Hydroxy-3,5,6-trifluorobenzoic acid (4e). A mixture of chloroanhydride 3e (15 mmol)
o
and 48% HBr (9 ml) was heated at 70 C for 2 hrs and cooled. The precipitation was filtered off
andcrystallized from appropriate water and toluene. Yield 95%, colorless crystals, mp 165–167
ºС. (lit.24c mp 166-168 ºС).
Synthesis of sodium salts of polyfluorosalicylic acid (6a,b). A mixture of acid 4a,b (2
mmol) and NaOH (2 mmol) in ethanol (5 ml) was mixed for 2 hrs at 20 ºС. Removal of solvent
gave the salts 6a,b.
Sodium 2-Hydroxy-3,4,5,6-tetrafluorobenzoate (6a). Yield 93%, white powder, mp >300
1
ºС. IR (DRA): ν 3075 (OH), 1665 (COO-), 1620, 1550, 1500 (С=С), 1270-1210 (С–F) cm-1. Н
NMR (DMSO-d6): δ 18.53 (br.s, 1H, OH). 19F NMR (DMSO-d6): δ -168.35 (m, 1F, FAr), -
162.90 (m, 1F, FAr), -158.36 (m, 1F, FAr), -144.14 (d, 1F, FAr, J 16.1 Hz). Anal. calcd. for
C7HF4NaO3. С, 36.23; Н, 0.43; F, 32.75. Found: С, 36.01; Н, 0.41; F, 32.93.
Sodium 2-Hydroxy-3,4,5-trifluorobenzoate (6b). Yield 94 %, white powder, mp >300 ºС.
1
IR (DRA): ν 3081 (OH), 1660 (COO-), 1610, 1530, 1490 (С=С), 1283-1208 (С–F) cm-1. Н
19
NMR (DMSO-d6): δ 7.36 (ddd, 1Н, HAr, J 11.0, 9.2, 2.4 Hz), 17.17 (br.s, 1H, OH). F NMR
(DMSO-d6): δ -160.87 (d, 1F, FAr, J 19.5 Hz), -157.32 (m, 1F, FAr), -154.04 (m, 1F, FAr). Anal.
calcd. for C7H2F3NaO3. С, 39.27; Н, 0.94, F, 26.62. Found: С, 39.60; Н, 0.67; F, 26.92.
Esterification of polyfluorosalicylic acids 4a,b. Method A. A mixture of acid 4a,b (5
mmol), corresponding alcohol (25 ml) and sulfuric acid (4 ml) was refluxed for 15 hrs, cooled
and poured into water (50 ml). The precipitation was filtered off and dried. Method B. A mixture
of 4a (0.3 g, 1.5 mmol), methanol (8 ml) or ethanol (10 ml) and sulfuric acid (1 ml) in sealed
vessel was irradiated for 2 hrs with 30 or 50 W to a temperature of 70 or 90 °C, cooled and
poured into water (15 ml). The precipitation was filtered off and dried to obtain esters 7a,b, 8a,b.
Ethyl-2-hydroxy-3,4,5,6-tetrafluorobenzoate (7a). Yield 85% (method A) and 93%
(method B), colorless crystals, mp 43-44 ºС. (lit.24a mp 45-46 ºС). IR (DRA): ν 2991 (OH), 1673
1
(CO2Et), 1645, 1524 (С=С), 1271-1158 (С–F) cm-1. Н NMR (CDCl3): δ 1.44 (t, 3Н, Me, J 7.2
Hz), 4.49 (q, 2Н, CH2, J 7.2 Hz), 11.38 (br.s, 1Н, OH). 19F NMR (CDCl3): δ -170.72 (m, 1F,
FAr), -163.70 (m, 1F, FAr), -148.04 (m, 1F, FAr), -135.74 (m, 1F, FAr). Anal. calcd. for C9H6F4O3.
С, 45.39; Н 2.54. Found: С, 45.31; Н, 2.53. C9H6F4O3.
15

Ethyl-2-hydroxy-3,4,5-trifluorobenzoate (7b). Yield 88%, colorless crystals, mp 65-66 ºС.
1
IR (DRA): ν 3084 (OH), 1684 (CO2Et), 1629, 1530 (С=С), 1282-1159 (С–F) cm-1. Н NMR
(CDCl3): δ 1.43 (t, 3Н, Me, J 7.2 Hz), 4.44 (q, 2Н, CH2, J 7.2 Hz), 7.48 (ddd, 1Н, HAr, J 10.3,
19
8.0, 2.5 Hz), 10.89 (d, 1H, OH, J 1.2 Hz). F NMR (CDCl3): δ -156.66 (dd, 1F, FAr, J 18.6, 2.5
Hz), -149.15 (m, 1F, FAr), -146.85 (m, 1F, FAr). Anal. calcd. for C9H7F3O3. С, 49.10; Н, 3.20.
Found: C, 49.13; Н, 3.18.
Methyl-2-hydroxy-3,4,5,6-tetrafluorobenzoate (8a). Yield 87% (method A) and 95%
(method B), colorless crystals, mp 61-62 ºС. IR (FTIR): ν 3074 (OH), 1679 (CO2Me), 1647,
1
1525 (С=С), 1297-1150 (С–F) cm-1. H NMR (CDCl3): δ 4.03 (s, 3Н, Me), 11.30 (s, 1Н, OH).
19F NMR (CDCl3): δ -170.45 (m, 1F, FAr), -163.45 (m, 1F, FAr), -147.63 (m, 1F, FAr), -135.84
(m, 1F, FAr). Anal. calcd. for C8H4F4O3. С, 42.87; Н, 1.80. Found: С, 42.43; Н, 1.74.
Methyl-2-hydroxy-3,4,5-trifluorobenzoate (8b). Yield 90%, colorless crystals, mp 60-61
1
ºС. IR (FTIR): ν 3088 (OH), 1683 (CO2Me), 1636, 1525 (С=С), 1281-1207 (С–F) cm-1. Н
NMR (CDCl3): δ 3.98 (s, 3Н, Me), 7.47 (ddd, 1Н, HAr, J 10.2, 8.2, 2.3 Hz), 10.79 (s, 1H, OH).
19F NMR (CDCl3): δ -156.48 (dd, 1F, FAr, J 18.7, 2.3 Hz), -148.85 (m, 1F, FAr), -146.63 (m, 1F,
FAr). Anal. calcd. for C8H5F3O3. С, 46.62; Н, 2.45. Found: С, 45.34; Н, 2.47.
Acylation of polyfluorosalicylic acid (4a-e). A mixture of acid 4a-e (5 mmol), acetic
anhydride (2.4 ml, 25 mmol) and two drops of sulfuric acid was refluxed for 30-40 min, cooled,
poured into water, and extracted with ether. Removal of solvent gave crude, which washed with
water and dried to obtain acids 9a-e.
2-Acetyloxy-3,4,5,6-tetrafluorobenzoic acid (9a). Yield 65% (from toluene:hexane – 1:1),
light-yellow crystals, mp 104–105 ºС. (lit.23b mp 104-106 ºС). IR (FTIR): ν 2948 (OH), 1790
1
(OAc), 1706 (CO2H), 1646, 1558 (С=С), 1253-1176 (С–F) cm-1. Н NMR (CDCl3): δ 2.37 (s,
19
3Н, Me). F NMR (CDCl3): δ -157.82 (t, 1F, FAr, J 20.4 Hz), -151.58 (dd, 1F, FAr, J 20.4, 9.6
Hz), -148.43 (td, 1F, FAr, J 20.4, 5.2 Hz), -136.61 (ddd, 1F, FAr, J 21.6, 9.4, 5.3 Hz). Anal. calcd.
for C9H4F4O4. С, 42.88; Н, 1.60. Found: С, 42.85; Н, 1.55.
2-Acetyloxy-3,4,5-trifluorobenzoic acid (9b). Yield 75 %, colorless crystals, mp 118–119
ºС. IR (FTIR): ν 2860 (OH), 1787 (OAc), 1694 (CO2H), 1618, 1526 (C=C), 1281-1178 (C–F)
1
cm-1. Н NMR (CDCl3): δ 2.39 (s, 3H, Me), 7.77 (ddd, 1Н, HAr, J 10.2, 8.0, 2.2 Hz), 10.56 (br.s,
1H, OH). 19F NMR (CDCl3): δ -147.92 (m, 1F, FAr), -145.89 (ddd, 1F, FAr, J 19.2, 5.0, 2.3 Hz), -
135.85 (ddd, 1F, FAr, J 20.5, 10.7, 5.0 Hz). Anal. calcd. for C9H5F3O4. С, 46.17; Н, 2.15. Found:
С, 45.99; Н, 2.10.
16

2-Acetyloxy-4,5-difluorobenzoic acid (9c). Yield 78 % (from benzene:hexane – 1:1),
colorless crystals, mp 148–150 ºС. IR (FTIR): ν 2865 (OH), 1783 (OAc), 1683 (CO2H), 1623,
1
1520 (C=C), 1280-1183 (C–F) cm-1. Н NMR (CDCl3): δ 2.40 (s, 3H, Me), 7.17 (td, 1Н, HAr, J
9.0, 7.0 Hz), 7.90 (ddd, 1Н, HAr, J 9.0, 5.6, 2.3 Hz), OH signal is not observed. 19F NMR
(CDCl3): δ -151.15 (ddd, 1F, FAr, J 20.7, 7.0, 2.3 Hz), -126.47 (ddd, 1F, FAr, J 20.7, 9.0, 5.6 Hz).
Anal. calcd. for C9H6F2O4. С, 50.01; Н, 2.80. Found: С, 49.96; Н, 2.86.
2-Acetyloxy-3,4-difluorobenzoic acid (9d). Yield 72 %, colorless crystals, mp 133–135 ºС.
IR (FTIR): ν 2855 (OH), 1777 (OAc), 1700 (CO2H), 1620, 1515 (C=C), 1285-1170 (C–F) cm-1.
1Н NMR (DMSO-d6): δ 2.25 (s, 3H, Me), 7.52 (dd, 1Н, HAr, J 10.9, 7.0, Hz), 7.93 (dd, 1Н, HAr,
19
J 10.9, 9.0, Hz), 13.51 (br.s, 1H, OH). F NMR (DMSO-d6): δ -140.64 (ddd, 1F, FAr, J 23.0,
11.0, 7.0 Hz), -129.82 (ddd, 1F, FAr, J 23.0, 10.9, 9.0 Hz). Anal. calcd. for C9H6F2O4. С,
50.01.17; Н, 2.80. Found: С, 50.18; Н, 2.75.
2-Acetyloxy-3,5,6-trifluorobenzoic acid (9e). Yield 70 %, dark-yellow crystals, mp 112–
114 ºС. IR (FTIR): ν 2850 (OH), 1795 (OAc), 1705 (CO2H), 1630, 1535 (C=C), 1290-1183 (C–
F) cm-1. 1Н NMR (DMSO-d6): δ 2.31 (s, 3H, Me), 7.99 (td, 1Н, HAr, J 10.3, 7.4 Hz), 14.39 (br.s,
1H, OH). 19F NMR (DMSO-d6): δ -141.97 (ddd, 1F, FAr, J 21.6, 14.2, 7.4 Hz), -135.66 (ddd, 1F,
FAr, J 23.4, 10.8, 2.3 Hz), -129.08 (ddd, 1F, FAr, J 12.4, 9.9, 2.3 Hz). Anal. calcd. for C9H5F3O4.
С, 46.17; Н, 2.15. Found: С, 46.29; Н, 2.11.
4.2. Biological part
4.2.1. In vitro experiments
Parallel Artificial Membrane Permeability Assay (PAMPA)
PAMPA was performed according to the protocol for GIT test described in Instruction
Manual for PAMPA Explorer (Pion, USA). Stock solutions of all compounds and standard drugs
were prepared by dissolving in 1 ml of DMSO to gain 10 mM concentration. 5 µl of each stock
solution were diluted in Prisma HT buffer according to the protocol and pH-map (pH=7.4, 6.2,
5.0). The diluted solutions of all tested compounds were transferred into the UV sensitive plate
(reference plate) to read the signals using Tecan Infinite M1000 PRO (Fisher Scientific, USA).
Into donor compartment of Sandwich Stirwell Plate equipped with magnetic stirrers the diluted
solutions were transferred column by column. GIT-0 lipid solution (P/N 110669) was gently
dispensed by 5 µl on each membrane back side. The Acceptor Sink Buffer (ASB) was dispensed
into all wells of acceptor compartment. The acceptor plate was placed at the top of the donor
plate, the whole sandwich was covered by lid and placed into Gut-Box with a wet sponge to
17

maintain a high relative humidity in order to minimize evaporation. The assembly was allowed
to incubate for 2 h and at unstirred water layer (UWL) 40 µm.
After 2 hours, the sandwich was removed from the Gut-Box and disassembled and both
acceptor and donor compartments were transferred to the UV sensitive plates and spectra were
read. The results were processed by using PAMPA Explorer Software.
COX-1 fluorescent inhibitor Screening Assay
The ability of ASA derivatives to inhibit ovine COX-1 was determined using COX
Fluorescent Inhibitor Screening Assay Kit (catalogue no 700100, Cayman Chemical). The
method used for evaluation of inhibitory activity is based on the reaction between PGG2 and
fluorometric substrate ADHP (10-acetyl-3,7-dihydroxyphenoxazine) that results in highly
fluorescent compound production (resorufin). The intensity of fluorescence is proportional to the
amount of resorufin and proportional to the amount of PGG2 which is released by the action of
the enzyme. (https://www.caymanchem.com/pdfs/700100.pdf )
4.2.2. In vivo experiments
Laboratory animals (Wistar rats and CD-mice) were obtained from the Animal Unit
“Pushino” at the M.M. Shemyakin and Yu.A. Ovchinnikov from Institute of Bioorganic
Chemistry RAS (Russia). The animals were housed at natural light cycle and otherwise in a
controlled environment, in propylene cages (Bioskape), on standard bedding (Rehofix MK 2000,
J.Rettenmaier&Söhne, Germany), supplied with feed for conventional laboratory rodents (Chara,
“Assortiment-Agro”, Russia) and water ad libitum. Animal care and all the procedures were
performed according to the Federal Law №61-FZ38 and guidelines for pre-clinical study of
medicinal products.34b
The carrageenan-induced paw edema model.
Anti-inflammatory activity was studied in Wistar or Sprague-Dawley rats of both genders
(6 animals per group) by using the carrageenan-induced paw edema model.34 Tested compounds
(25-50 mg/kg in 2% starch mucilage solution) were injected intraperitoneally 30 min prior to
injection of 0.1 ml of 1% carrageenan (λ-carrageenan, type IV; Sigma Aldrich) solution in the
plantar surface of the right hind paw. The animals in control group were treated only with 2%
starch mucilage solution. ASA (25 or 50 mg/kg in 2% starch mucilage solution) was used as a
reference drug. Paw volumes were measured oncometrically using water plethysmometer (TSE
Volume Meter, Germany) before administration of carrageenan, at 1, 3 and 5 h after
administration of phlogogen. The anti-inflammatory activity was evaluated by percentage of
increment of volume of the inflamed paw after carrageenan treatment to volume of the paw
before carrageenan treatment.
18

The Hot plate test
The hot plate test was conducted according to established guidelines34 on Hot plate 60200
series (TSE-systems, Germany). Rats were administered with substances suspension 25 mg/kg in
1% starch mucilage intraperitoneally. Baseline controls were animals that received vehicle only.
ASA was used as a reference substance. Animals were tested 1 h after administration. They were
placed on an electrically heated plate at 50 °С in a plexiglas cylindric restrainer (19 cm diameter
× 30 cm). The nociceptive response time was measured by observing movements consisting of
jumping, licking, or shaking their hind paws. Maximal cutoff time was 30 s regardless of
response. Data are expressed as a percentage change in withdrawal response time from baseline.
Acute toxicity evaluation
The toxicity evaluation was performed on CD-1 mice. The procedure was based on OECD
recommendations39 and guidelines for pre-clinical study of medicinal products.34b
For general estimating of the toxicity range of synthesized compounds, ten animals per group
(one dose) were used for several compounds. Later reduction of the number of used animals was
done and three mice per one dose were applied for the rest of samples. The tested compounds in
2% starch mucilage solution were injected intraperitoneally. Animals were observed during 14
days, the number of deaths was counted and % of viability calculated.
Statistical analysis
The data were analyzed by a statistical software package GraphPad Prism 6 using the
«Multiple tests» approach. The values were considered significantly different at p < 0.05.
4.3. Docking protocol
Ligands and protein preparation
The tyrosine active site of cyclooxygenase-1 with ACD (native ligand) was chosen as biological
target. The crystallographic structure of enzyme (PDB ID: 1DIY) was downloaded from the
RCSB PDB database40 and prepared with the aid of Protein Preparation Wizard by Schrodinger
Suite.41 All water molecules and all low-molecular-weight compounds except for the native
ligand were removed and missing hydrogen atoms were added in the active site. The geometric
parameters of the receptor were optimized using the OPLS-2005 force field algorithm.
The structures of polyfluorinated salicylates were integrated into a common database. All
ionized states and molecular conformations possible at pH 7.0 0.2 were calculated by default
DFT-methods.42 Their geometric parameters were optimized in the presence of water.
19

Docking studies
The docking was performed under the following conditions: a flexible ligand and protein,
extra prediction accuracy, the grid-box size of 15Å with the native ligand being located at the
center, the possibility of rotation in the ARG120, TYR355, TYR385 and SER530 amino acid
residues that define the tyrosine site. The ACD redocking into the COX-1 active sites correctly
reproduced the mode of enzyme and ligand binding determined by X-ray crystallography. The
root-mean-square deviation (RMSD) for ACD was 0.336 Å. Technical details of docking were
shown in Suppl. Section.
Acknowledgements
This work was supported by the Russian Scientific Foundation (Grant No. 16-13-10255).
The authors express gratitude to Mrs Elvira Pozjumko for helpful assistance at PAMPA
experiment. All calculations were carried out on a cluster in the regional center for shared
computer equipment at the Ufa Institute of Chemistry RAS.
Supplementary materials
Supplementary data associated with this article can be found, in the online version, at http://
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Polyfluorinated salicylic acid derivatives as analogs of
known drugs: synthesis, molecular docking and
biological evaluation
E.V. Shchegol’kova, I.V. Shchura, Ya.V. Burgarta, V.I.
Saloutina, A.N. Trefilovab, G.A. Ljushinab , S.Yu.
Solodnikovb, L.N. Markovab, V.V. Maslovab, O.P.
Krasnykhb, S.S. Borisevichc, S.L. Khursanc
aPostovsky Institute of Organic Synthesis UB RAS, S.
Kovalevskoy Str., 22, Ekaterinburg 620990, Russia.
bPerm National Research Polytechnic University,
Komsomolsky Av., 29, Perm 614990, Russia.
cUfa Institute of Chemistry of RAS, Octyabrya Av.,
71, Ufa 450078, Russia.
24