A convenient and efficient approach to polyfluorosalicylic acids and their tuberculostatic activity

Article abstract of DOI:10.1016/j.bmcl.2016.03.107

We have developed the practical method for polyfluorosalicylic acids synthesis via nucleophilic ortho-mono-substitution of fluorine atom with magnesium methoxide. We have managed to increase the yield of targeted polyfluorosalicylic acids from good to quantitative. We have studied the tuberculostatic activity of polyfluorosalicylic acids. It has been found that minimum inhibitory concentration (MIC) of compounds is from 0.7 to 6.5 μg/mL depending on the structure.

Full text of DOI:10.1016/j.bmcl.2016.03.107

Accepted Manuscript
A convenient and efficient approach to polyfluorosalicylic acids and their tu-
berculostatic activity
Evgeny V. Shchegol’kov, Irina V. Shchur, Yanina V. Burgart, Victor I. Saloutin,
Sergey Yu. Solodnikov, Olga P. Krasnykh, Marionella A. Kravchenko
PII:
S0960-894X(16)30339-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.107
BMCL 23750
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 January 2016
29 March 2016
30 March 2016
Please cite this article as: Shchegol’kov, E.V., Shchur, I.V., Burgart, Y.V., Saloutin, V.I., Solodnikov, S.Y.,
Krasnykh, O.P., Kravchenko, M.A., A convenient and efficient approach to polyfluorosalicylic acids and their
tuberculostatic activity, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.03.107
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1
A convenient and efficient approach to polyfluorosalicylic acids and
their tuberculostatic activity
Evgeny V. Shchegol’kova,, Irina V. Shchura, Yanina V. Burgarta, Victor I. Saloutina,
Sergey Yu. Solodnikovb, Olga P. Krasnykhb, and Marionella A. Kravchenkoc
aPostovsky Institute of Organic Synthesis of Ural Branch of Russian Academy of Science, S. Kovalevsloy Str., 22, Ekaterinburg 620990, Russia
bPerm National Research Polytechnic University, Komsomolsky Av., 29, Perm 614990, Russia
cUral Research Institute of Phthisiopulmonology, 22 Parts’ezda, 50, Ekaterinburg 620039, Russia
Abstract. We have developed the practical method for polyfluorosalicylic acids synthesis via nucleophilic
ortho-mono-substitution of fluorine atom with magnesium methoxide. We have managed to increase the yield of
targeted polyfluorosalicylic acids from good to quantitative. We have studied the tuberculostatic activity of
polyfluorosalicylic acids. It has been found that minimum inhibitory concertation (MIC) of compounds is from 0.7
to 6.5 g/mL depending on the structure.
Keywords: Polyfluorosalicylic acid, Nucleophilic substitution, Anti-mycobacterial activity, Tuberculosis,
Toxicity
Tuberculosis (TB) still remains a serious disease in the world. In accordance with Global
Tuberculosis Report for 2015 (World Health Organization)1 9.6 million people fell ill with TB
and 1.5 million people died of TB. 27% from died people were HIV-infected persons as a result
of the high risk of developing TB among them. Besides, the 480000 cases of multi-drug resistant
(MDR) TB were recorded and 10 % of them were extensively drug-resistant TB.
One of the widely used drugs for TB treatment is para-aminosalicylic acid (PAS), which is
applied in MDR therapy. In one hand, PAS is the metabolic precursor that incorporated into the
folate pathway by dihydropteroate synthase – dihydrofolate synthase, generating a toxic
dihydrofolate analog that subsequently inhibits dihydrofolate reductase activity.2 On the other
hand, PAS reveals inhibitory effect on mycobactin biosynthesis. PAS is structural analog of
salicylic acid, which responsible for the biosynthesis of mycobactin and carboxymycobactin
participating in the cell membrane construction.3
We envisioned the progress in anti-TB treatment by use of fluorinated salicylic acids
structural analogs as possible inhibitors of mycobactin biosynthesis. The fluorine atom has been
effectively and successfully used in medicinal chemistry for modulating the physiochemical and
biological properties of lead compounds in drug discovery programs. High electronegativity of
fluorine atom combined with extremely low polarization make these organic compounds quite
unique.4 In fact, about 20 per cent of used pharmaceutical drugs have at least one fluorine atom
in structure, and it is an aromatic fluorine.5
 Corresponding author. Tel./fax: +7-343-3745954; e-mail: schegolkov@ios.uran.ru

2
It should be noted that polyfluorosalicylic derivatives remain hardly accessible and therefore
the little known class of compounds until recently. However, based on the scarce information on
their biological effects one can suggest the future development in this direction. So, a number of
polyfluorosalicylic amides is patented as anticoagulant agents,6 polyfluorosalicylic N-
alkohenalkylsulfone amides can be used as agents against obesity and diabetes.7 Polyfluorinated
N-{[4-oxo-6-fluoro-7-(cyclohexylamine)-1-cyclopentyl-1,4-dihydroquinolin-3-yl]methyl}-2-
hydroxybenzamides are claimed as inhibitors of platelet aggregation and P2Y12 receptors.8
The use of Kolbe-Schmitt9 reaction for the preparation of hardly available polyfluorosalicylic
acids cannot be applied because of possible nucleophilic substitution of fluorine in fluorinated
phenols.
There are a few known methods for polyfluorosalicylic acid synthesis but all of them have no
preparative value due to their disadvantages. So, 2,3,4,5-tetrafluorosalicylic acid was obtained
with yield of 85% from 2,3,4,5-tetrafluorophenol in the presence of n-butyllithium at -75 °C
under CO2 atmosphere.10,11 The following variations of the reaction conditions and organolithium
reagents allowed the regioisomeric polyfluorosalicylic acids to be obtained with yields of 61-
93%.12 In spite of high yields of target acids, the method of fluoro-containing phenol
carboxylation has some significant disadvantages such as the use of hardly accessible and rather
expensive polyfluorophenols as well as complex instrumentation for low-temperature synthesis
process.
3,5-Difluorosalicylic
acid
can
be
obtained
from
2,4-difluorophenol
and
hemathylenetetramine in trifluoroacetic acid via Duff reaction with subsequent oxidation of
intermediate aldehyde.13
Tetrafluorosalicylic acid was obtained by oxidation of 3-formyl- or 2-methyl-3-
ethoxycarbonyl-5,6,7,8-tetrafluorochromones with yield of 30%.14 The yield up to 64% was
achieved by continuous extraction for 15 h with diethyl ether.15 Obvious disadvantages of this
method are difficulty of target acid isolation accompanied by the low or moderate yields. In
addition, the preparation of starting polyfluorochromones is required and it is a particular non-
trivial task.
The direct fluorination of salicylic acid with elemental fluorine in acetonitrile or hydrogen
fluoride led to a mixture of mono 5-fluorosalicylic and 3,5-difluorosalicylic acids in ratio 2…7 to
1 correspondingly and was accompanied by incomplete conversion of initial reagents.16
The synthesis of polyfluorosalicylic acid by nucleophilic ortho-substitution of fluorine atom
in polyfluorobenzoic acids with hydroxyl groups under heating with NaOH in 1,3-dimethyl-2-
imidazoline has been described.17 However, this way is characterized by the low selectivity
process as a result of the regioisomeric products mixture formation. The use of alkali metals

3
alkoxides in reactions with pentafluorobenzoic acid leads to selective formation of para-
substituted products.18
Previously, we have described the selective ortho-substitution of fluorine atom in
pentafluorobenzoic acid 1a under the action of magnesium methoxide.19 To obtain 2-methoxy-
3,4,5,6-tetrafluorobenzoic acid 2a the most suitable condition was heating the acid 1a with
magnesium methoxide in diglyme at 100 ºC for 2 h. The further temperature and the reaction
time increase led to formation of disubstituted by product - 2,6-dimethoxy-3,4,5-trifluorobenzoic
acid 3, which when being heated at 130 ºC for 4 h became the main product.19 Subsequent
hydrolysis of acid 2a in situ resulted in tetrafluorosalicylic acid 4a formation. However, one
significant disadvantage of this quite convenient and simple method is low yield of the desired
compound 4a (42%).
In this research, the method of polyfluorosalicylic acid synthesis by selective nucleophilic
ortho-substitution of fluorine in polyfluorobenzoic acid under magnesium methoxide action has
been improved.
For this purpose, we attempted to carry out ortho-mono-methoxylation of pentafluorobenzoic
acid 1a using various ratios of magnesium methoxide in weak polar solvents (Table 1).The
choice of solvents can be explained by the need of fluorine nucleofugicity decreasing to prevent
para-substituted isomers formation. At the same time, the use of weak polar solvent brings about
the requirement of carrying out the process at high temperatures. Besides, to easy isolation and
purification of target product, it is preferable for solvent to have not good miscibility with water
unlike diglyme. In our view, toluene satisfies this set of requirements well because it has a
sufficiently high boiling point (110 ºC), weak polarity ( 2.4) and low miscibility with water
(0.52 g/L).20 In addition, this solvent can be easily regenerated for reuse in the reaction of
methoxylation.
In this regard, we have focused on the use of toluene as a solvent for ortho-mono-
methoxylation of acid 1a. The ratio of magnesium methoxide to acid 1a was ranged from 1 to 12
molar equivalents. The optimization results are presented in Table 1.
The optimal conditions for ortho-mono-substitution of fluorine in pentafluorobenzoic acid 1a
with more selective formation of 2-methoxy-3,4,5,6-tetrafluorobenzoic acid 2a were refluxing in
toluene with ratio of 1 eq. of acid 1a and 2.5 eq. of magnesium methoxide (Scheme 1). However,
in these conditions it was not possible to obtain the product 2a in pure form as a result of
incomplete conversion of parent acid 1a. All attempts to separate compounds 1a and 2a were
failed because of their co-crystallization since acids 1a and 2a have similar nature. Efforts to
achieve a full conversion of pentafluorobenzoic acid 1a by adding of more magnesium
methoxide equivalents (from 3 to 5) were unsuccessful. In this case, the yield of compound 2a is

4
decreased owing to disubstituted product 3 formation via nucleophilic substitution of both ortho-
fluoine atoms in acid 1a (see Table 1).
For effective isolation and purification of acid 2a we suggested to treat the reaction mixture
with thionylchloride or phosphorus pentachloride followed by fractional distillation and
separation of the resulting chlorides of 2-methoxy-3,4,5,6-tetrafluorobenzoic acid 5a and
pentafluorobenzoic acid 5b (Scheme 1). Hydrolysis of chloroanhydride 5a in 48% hydrobromic
acid leads to tetrafluorosalicylic acid 4a. So, we managed to increase the total yield of
tetrafluorosalicylic acid 4a by this optimization from 42% to 65% in comparison with the
previous results19 and return ~10-12% of the initial acid 1a by hydrolysis of the
pentafluorobenzoyl chloride 5b. The analytically pure sample of 2-methoxy-3,4,5,6-
tetrafluorobenzoic acid 2a was obtained by hydrolysis of chloroanhydride 5a with 20% NaOH in
water.
Table 1.Optimization of orto-monomethoxylation of acid 1a.
Solvent
diglyme
toluene
Mg(OMe)2, eq
Ratio of mixture A, % in accordance with 19F NMR
spectroscopy in СDCl3
Compound 1a
Compound 2a
Compound 3
12
12
5
0
25
15
35
63
76
65
46
75
85
63
25
10
8
0
2
3
12
14
26
51
2.5
2
1
3

5
Scheme 1
We carried out X-Ray analysis for tetrafluorosalicylic acid 4a for the first time (Fig. 1,
CCDC 1447869). It is found that the effective intramolecular hydrogen bond is realized in
molecule 4a between hydrogen atom of hydroxyl group and oxygen atom of carbonyl moiety
H1…O1 1.71(4) Å. The same bond in non-fluorinated analogue is characterized by a somewhat
larger value of length 1.85(4) Å.21 Hence we can assume that the hydroxyl group in
tetrafluorosalicylic acid 4a is more acidic in comparison with a similar group in non-fluorinated
salicylic acid. In general, the acidic properties of tetrafluorosalicylic acid 4a should be higher. It
confirms the difference in calculated pKa values of compound 4a (1.53±0.28)22 and salicylic
acid (3.01±0.10).22 The enhancement of acidic nature of substance 4a can be caused by influence
of electron-withdrawing fluorine atoms. There are planar chain dimers through intermolecular
hydrogen bonds in crystal of the molecule 4a [dimers: O1…H2*, H2…O1* 1.81(3) Å, 1.81(2)
Å, chains: O2…H1** 2.44(2) Å (Fig. 1)] similarly, the non-fluorinated analogue.21
Figure 1. ORTEP view of molecule 4a.
The developed approach has been applied for selective ortho-mono-methoxylation of 2,3,4,5-
tetrafluoro- 1b, 2,3,4-trifluoro- 1c and 2,3,5-trifluoro- 1d benzoic acids which have the only
fluorine atom in the ortho-position as compared to pentafluorobenzoic acid 1a. It is significantly
simplifies the method for corresponding 3,4,5-trifluoro-, 3,4-difluoro- and 4,5-difluorosalicylic
acid synthesis. So, acids 1b-d interact with 4 eq. of magnesium methoxide, which is necessary to
full conversion of initial reagents 1b-d, by refluxing in toluene (or diglyme) to give 2-methoxy-
3,4,5-trifluoro- 2b, 2-methoxy-3,4-difluoro 2c, and 2-methoxy-4,5-difluoro- 2d benzoic acids as
single products. The subsequent hydrolysis of acids 2b-d in 48% hydrobromic acid leads to
3,4,5-trifluoro- 4b, 3,4-diflluoro- 4c, and 4,5-difluoro- 4d salicylic acids (Scheme 2) with nearly
quantitative yields.

6
Scheme 2
This method of tri- and difluorosalicylic acids 4b-d synthesis is an excellent alternative to
carboxylation of tri- and difluorophenols.12 Our approach is distinguished by the ease of process,
high yield of the desired acids 4b-d (total yield over 90 %) and the absence of expensive initial
reagents polyfluorophenols.
Synthesized polyfluorosalicylic acids 4a-d were screened for activity against Mycobacterium
tuberculosis H37Rv, avium, terrae, and MDR strains. An efficiency of compounds 4a-d were
compared to that of known drugs izoniazid, ofloxacin (Table 2) and PAS (MIC for strain H37Rv
is 0.3-1 g/mL and M. avium - 32–128 g/mL).23 It is noted that salicylic acid in small doses
activates the process of mycobactin synthesis,3 whereas a suppression growth of M. tuberculosis
occurs in high concentrations (MIC > 250 g/mL).24 We have found that the presence of fluorine
atoms in aromatic ring of salicylic acids has the significant influence on activity in relation to
Mycobacterium. The MIC reduces to 0.7…6.5 g/mL depending on the structure of compounds
4a-d (Table 2). Moreover, acids 4a,c,d have the considerable activity against M. avium, M.
terrae and MDR strains.
The toxicity of polyfluorosalicylic acids 4a-c is comparable to that of isoniazid (Table 2).
However, decreasing the fluorine content in the molecule reduces a toxicity. So, 2-hydroxy-4,5-
difluorobenzoic acid 4d combines a high activity with lower LD50 (Table 2). Moreover, the
fluorinated compounds have a great potential for further chemical transformations toward
increasing of their efficiency and reducing of toxicity.
In summary, we have developed the convenient and efficient approach to
polyfluorosalicylic acids 4a-d synthesis via nucleophilic ortho-fluorine atom substitution. The
MIC values 0.7 g/mL of polyfluorosalicylic acids 4a,d for all strains allow us to conclude about
perceptivity of research area concerning chemistry of polyfluorosalicylic derivatives.

7
Table 2. In vitro anti-tuberculosis activity of compounds 4a-d and their acute in vivo toxicity in
mice
Compound
Anti-mycobacterial activity against different strains of
LD50, mg/kg
Mycobacterium tuberculosis (MIC in g/mL)
H37Rv
0.7
M. avium
M. terrae
0.7
MDR
0.7
0.7
n.d.a
100
>100
>150
>300
5450d
100d
4a
4b
6.5
n.d.
1.5
n.d.
1.5
1.5
1.5
4c
0.7
0.7
0.1
0.7
0.7
4d
Ofloxacin
Izoniazid
PAS
0.1
0.1
0.1
n.a.b
0.1
0.3-1c
0.1
0.1
32-128 (n.a.)c
n.d.
n.d.
4000d
a n.d. – not detected, b n.a. – non-active, c Ref 23, d DrugBank
Acknowledgements
This work was financially supported by the Ural Branch of the Russian Academy of Sciences
(Grant No. 15-21-3-5), the Russian Foundation for Basic Research [Grant No. 14-03-31541 for
E.V. Shchegol’kov (biological and physico-chemical investigations)] and Russian Scientific
Foundation [Grant No. 16-13-10255 for V.I. Saloutin (synthetic part)]. The authors are grateful
for Dr. Yu.S. Kudyakova and A.N. Trefilova for assistance in manuscript preparation.
Supplementary material
Supplementary data (CIF file, experimental procedures, characterization of new compounds
and biological assays protocols) associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl….
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10
A convenient and efficient approach to polyfluorosalicylic acids
and their tuberculostatic activity
Evgeny V. Shchegol’kova,, Irina V. Shchura, Yanina V. Burgarta,
Victor I. Saloutina, Sergey Yu. Solodnikovb, Olga P. Krasnykhb, and Marionella A. Kravchenkoc
aPostovsky Institute of Organic Synthesis of Ural Branch of Russian Academy of Science, S. Kovalevsloy Str., 22,
Ekaterinburg 620990, Russia; bPerm National Research Polytechnic University, Komsomolsky Av., 29, Perm 614990,
Russia; cUral Research Institute of Phthisiopulmonology, 22 Parts’ezda, 50, Ekaterinburg 620039, Russia