Alkyl and aryl α-ketophosphonate derivatives as photoactive compounds targeting glutathione S-transferases

Article abstract of DOI:10.1080/10426507.2021.1901703

In this article, photoactive alkyl and aryl α-ketophosphonate derivatives were studied as inhibitors of glutathione S-transferases (GSTs). The α-ketophosphonate monoesters were synthesized by sodium iodide-induced dealkylation of corresponding phosphonate

Full text of DOI:10.1080/10426507.2021.1901703

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gpss20
Alkyl and aryl α-ketophosphonate derivatives as
photoactive compounds targeting glutathione S-
transferases
О. L. Kobzar, Yu. V. Shulha, V. M. Buldenko, G. P. Mrug, M. V. Kolotylo, O. V.
Stanko, P. P. Onysko & А. I. Vovk
To cite this article: О. L. Kobzar, Yu. V. Shulha, V. M. Buldenko, G. P. Mrug, M. V. Kolotylo, O.
V. Stanko, P. P. Onysko & А. I. Vovk (2021): Alkyl and aryl α-ketophosphonate derivatives as
photoactive compounds targeting glutathione S-transferases, Phosphorus, Sulfur, and Silicon and
the Related Elements, DOI: 10.1080/10426507.2021.1901703
To link to this article: https://doi.org/10.1080/10426507.2021.1901703
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
https://doi.org/10.1080/10426507.2021.1901703
Alkyl and aryl a-ketophosphonate derivatives as photoactive compounds
targeting glutathione S-transferases
a
a
a
a
b
b
b
ꢀ
. L. Kobzar , Yu. V. Shulha , V. M. Buldenko , G. P. Mrug , M. V. Kolotylo , O. V. Stanko , P. P. Onysko , and
a
ff. I. Vovk
a
b
V. P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine; Institute of
Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine
ABSTRACT
ARTICLE HISTORY
In this article, photoactive alkyl and aryl a-ketophosphonate derivatives were studied as inhibitors
of glutathione S-transferases (GSTs). The a-ketophosphonate monoesters were synthesized by
sodium iodide-induced dealkylation of corresponding phosphonate diesters. The compounds
being inactive toward the enzyme without UV light were activated by irradiation at 365nm which
caused inhibition of GSTs from equine liver and human placenta with micromolar IC50 values.
Hetaryl a-ketophosphonate monoesters can also be potential candidates for the UV-dependent
enzyme targeting. Inhibitory potency of the phosphonate monoesters was comparable to that of
free phosphonic acid. No effects were observed in case of dialkyl a-ketophosphonates. The light-
induced effects of the inhibitors were found to be related to decrease in maximum velocity but
not to Michaelis constants of the enzyme-catalyzed reaction. The kinetics of a decrease in enzyme
activity was described by pseudo-first order rate constants dependent on inhibitor concentration.
The calculated second order rate constants indicated that the alkyl a-ketophosphonate monoesters
can exhibit higher potency in comparison with that of aryl or hetaryl phosphonate derivatives.
Received 31 December 2020
Accepted 25 February 2021
KEYWORDS
a-ketophosphonate; gluta-
thione S-transferase;
irradiation; inhibitors;
kinetics; molecular docking
GRAPHICAL ABSTRACT
Introduction
Glutathione S-transferases (GSTs; EC 2.5.1.18) belong to
phase II detoxification enzymes catalyzing the nucleophilic con-
jugation of glutathione with different exogenous and endogenous
electrophilic molecules. The superfamily of GSTs is represented
Synthetic and natural phosphonic acids and their derivatives
containing direct carbon–phosphorus bonds are considered
as phosphate bioisosters and may be useful for designing
by one microsomal, one mitochondrial, and seven cytosolic
[
1,2]
bioactive compounds of structurally diverse classes.
The
[12,13]
classes.
Human a-class and p-class GSTs, due to their cata-
structure of a-ketophosphonic acids mimics also a-ketocar-
boxylic acids fragments of some natural mono- and dicar-
boxylic acids. However, despite the advances in the
chemistry and biological activity of phosphonic acid deriva-
lytic activity and regulation signaling pathway, play a role in
[14–16]
many diseases including cancer.
The properties of these
enzymes form an interest in new inhibitors for designing anti-
[17]
cancer drugs and therapeutics against drug resistance.
[
3,4]
tives,
phosphonates.
only a few studies deal with bioactivity of a-keto-
The glutathione analogs and their conjugates, as well as
[
5–8]
In the recent years, peptides containing
derivatives of aliphatic, aromatic, and heterocyclic com-
a-ketophosphonate group have been described as light-
switched inhibitors of PTP1B, MptpA, and STAT5b. As
phosphotyrosine mimetics, the enzymatically stable benzoyl
phosphonate derivatives were activated by irradiation with
UV light forming triplet intermediate which provided modi-
[13,18–20]
pounds were described as inhibitors of GSTs.
The
inhibitory effects on activity of GSTs were observed also in
[21,22]
the presence of phosphonic acid derivatives.
[
9–11]
fication of protein structures.
CONTACT ff. I. Vovk
vovk@bpci.kiev.ua
V. P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of
Ukraine, Murmanska, 1, 02094 Kyiv, Ukraine.
ß 2021 Taylor & Francis Group, LLC

2
О. L. KOBZAR ET AL.
In this article, the light-induced inhibition of cytosolic GSTs during UV irradiation at 365 nm. The GSTs from
GSTs of a- and p-class by some alkyl, aryl, and hetaryl equine liver and human placenta, as well as human recom-
a-ketophosphonate derivatives was studied.
binant GSTA1-1 and GSTP1-1 were used in the experiments
in vitro. Phosphonic acid diesters 1b–d and phosphonic acid
3
were also chosen to compare their properties with corre-
Results and discussion
sponding a-ketophosphonate monoesters. All the monoester
and diester compounds as well as free acid 3 in concentra-
tion of 25 mM showed no inhibitory activity against GSTs
without UV irradiation of reaction mixture. Compounds 2b
and 3 in concentration of 250 mM demonstrated low inhib-
ition effects (7%–12% in case of GSTs from equine liver and
human placenta).
Synthesis of a-ketophosphonate derivatives
a-Ketophosphonates presented by phosphonic diesters 1a–f,
monoesters 2a–f, or phosphonic acid 3 were obtained according
to the reaction shown in Scheme 1. The syntheses of compounds
1
a, 1e, and 1f as well as 2a, 2b, 2e, and 2f have been described
[23]
previously.
The sodium iodide dealkylation of the diesters 1
After a series of experiments, a dose-dependent influence
of the photoactive compounds on GSTs was represented as
IC50 (50% inhibitory concentration). Figure 1 demonstrates
decrease in activity of GSTs from equine liver and human
placenta upon 5 min UV irradiation of solution containing
compound 2b. Shapes of the dose-dependent curves empha-
size the differences in the inhibition of the two GSTs by
compound 2b (apparent Hill coefficients were of 0.83 and
to the monoesters 2 was found to depend on chemical nature of
Rꞌ substituent. So, the dealkylation of dimethyl ester 1c
occurred at room temperature within several hours whereas con-
version of its diisopropyl analog 1d to 2d was not achieved even
after boiling of reaction mixture in acetone for 2 days. The struc-
ture of obtained phosphonic diesters 1a–f, monoesters 2a–f, and
1
31
13
phosphonic acid 3 were confirmed by H, P, and C NMR
spectroscopy. The compounds exhibit similar spectral character-
istics of the O ¼ C–POR fragment. Phosphorus chemical shifts
were observed in a very close region (dP ꢀ3.4 to 1.5 ppm)
whereas the PC ¼ O carbon signals for ketophosphonic mono-
1
.25, respectively).
The IC50 values for a-ketophosphonate derivatives as
light-induced inhibitors of GSTs from equine liver and
human placenta are shown in Table 1. The observed inhibi-
tory effects on recombinant human GSTA1-1 and GSTP1-1
13
esters 2 in the C NMR spectra were shifted to low field as com-
pared to diesters 1 (Dd 8.5–11.3 ppm).
Light-induced inhibition of GSTs by a-ketophosphonate
derivatives
The a-ketophosphonate monoesters 2a–f were found to be
photoactive compounds exhibiting inhibitory effects against
Figure 1. Dose-dependent curves of UV light-induced inhibition of GST from
Scheme 1. Synthesis of a-ketophosphonate derivatives.
equine liver (
᭺
) and GST from human placenta (
w) by a-ketophosphonate 2b.
Table 1. IC50 values of a-ketophosphonate derivatives as photoactive inhibitors of GSTs.
IC50 (lM)
Compound
GST from equine liver
GST from human placenta
GSTA1-1
GSTP1-1
1
1
1
2
2
2
2
2
2
3
b
c
>100
>100
>100
>100
ND
ND
ND
ND
ND
ND
d
a
b
c
d
e
f
65.6 ± 10.5
6.6 ± 1.5
6.8 ± 1.3
7.6 ± 2.1
>100
10.9 ± 3.3
10.8 ± 1.7
3.4 ± 0.5
5.5 ± 0.7
17.0 ± 3.9
5.6 ± 0.3
6.7 ± 1.0
8.3 ± 1.3
59.9 ± 17.3
19.6 ± 0.6
25.2 ± 7.3
4.0 ± 0.9
4.3 ± 0.6
65.3 ± 21.5
49.9 ± 14.0
>100
ND
35.6 ± 4.5
16.2 ± 4.1
91.2 ± 9.3
ND
ND
ND
>100
38.6 ± 11.5
ND
>100
9.1 ± 1.1
ND
Ethacrynic acid
IC50 values represent the mean of 2–3 assays ± standard deviation.
ND
not determined.

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
Figure 2. UV light-induced inhibition of GSTs from equine liver (A, B) and human placenta (C, D) by 4 and 6 mM a-ketophosphonate 2b, respectively.
Figure 3. Pseudo-first-order kinetics (A, C) and relationship between kobs and concentration of a-ketophosphonate 2b (B, D) as UV light-induced inhibitor of GSTs
from equine liver (A, B) and human placenta (C, D).
turned out to be lower (reaction mixture contained 10 lM of a-ketophosphonate monoesters. The alkyl a-ketophospho-
dithiothreitol). Phosphonic acid diesters 1b–d displayed very nate monoesters 2a and 2b with IC50 values of 5.6 of
low or no activity at concentration <100 mM, while inhibi- 6.8 lM, respectively, were more potent inhibitors as com-
tory potency of phosphonic acid 3 was comparable to that pared to aryl and hetaryl derivatives 2e and 2f. The low

4
О. L. KOBZAR ET AL.
activity of a-ketophosphonate 2d can be attributed to steric time. As seen from Figure 3, there were no significant devia-
hindrance of tert-butyl group. Ethacrynic acid as a reference tions from linear plots of kobs versus inhibitor concentration
compound, non-sensitive to UV light, showed IC50 value of used in the experiments. This is consistent with a low affin-
5
.5 and 4.3 mM for GST from equine liver and human pla- ity of the inhibitors to the enzyme which was evidenced also
centa, respectively.
in experiments without UV-irradiation. The calculated
It has been reported previously that benzoyl phosphonate second-order rate constants showing the efficiency of
derivatives activated by UV light can cause modification of
enzyme transformation to its inactivated form were depend-
ent on the inhibitor structure. The alkyl a-ketophosphonate
derivatives 2a and 2d were 2–10 times more effective than
a-ketophosphonates 2f and 2e, exhibiting some selectivity to
GST from human placenta (Table 2). These observations, as
well as analysis of activities of other mono- and diesters of
a-ketophosphonic acids such as 1b–d and 2d can be related
to a different mode of binding of activated inhibitor to the
enzyme undergoing a rapid irreversible modification.
protein structure with formation of a covalent cross-linking
[
9,10]
product or inactive oxidized form of the enzyme.
To
evaluate the mechanism of action of the photoactive a-keto-
phosphonate derivatives against GSTs, a preliminary study
with dithiothreitol was performed. It was established that
DTT does not influence the enzyme activity after UV irradi-
ation without inhibitor, is able to prevent inactivation in the
reaction mixture with enzyme and inhibitor during UV
irradiation, and does not restore the enzyme activity after
UV-induced inhibition by a-ketophosphonate. These results
are consistent with the mechanism involving irreversible oxi-
dation of cysteine residues to sulfinic acid and sulfonic acid
forms. Another possible mechanism assumes irreversible
modification of tyrosine side chain residues which are
located at the enzyme active site.
Molecular docking
The blind molecular docking on examples of compounds 2a
[
24]
and 2b was carried out by Autodock Vina software
for
identification of possible binding sites for the a-ketophosph-
onates. Given that GSTs are catalytically active in dimeric
The dependence of enzyme velocity from substrate con-
centration with and without inhibitor were obtained and
presented as Lineweaver–Burk plots for each of two sub-
[
12,25]
forms,
the molecular surfaces of homodimeric struc-
tures of GSTA1-1 and GSTP1-1 (PDB codes 6ATO and
[
26]
strates of GSTs from equine liver and human placenta 6GSS,
respectively) were used for the calculations. The
(Figure 2). The decreasing in maximum velocity (Vmax
)
obtained results showed that a-ketophosphonate derivatives
without changing in the Michaelis–Menten constant (Km), may occupy the pocket between C- and N-terminal domains
which is similar to a noncompetitive type of inhibition, sup- of monomeric structure of GSTA1-1. It is known that
ports the irreversible inactivation mechanism.
deregulation of interdomain contacts can lead to disruption
Under the experimental conditions, the inactivation of of the catalytic function of the enzyme.
[
27]
GSTs was described by pseudo-first-order kinetics. The
Two binding sites for the a-ketophosphonate derivatives
observed first-order rate constants (kobs) were estimated for were predicted on the surface of GSTP1-1. One of them was
a-ketophosphonate derivatives 2a, 2b, 2f, 2e, and 3 from the located at the bottom of V-shape cleft, which is interdimer
slopes of a semi-logarithmic plot of inhibition percent versus cavity of GST and considered as non-substrate ligand-bind-
[
28–30]
ing site.
In this region (Figure 4), amino acid residues
Table 2. The second-order rate constants of GSTs inhibition by photoactive
a-ketophosphonate compounds.
Asn66, Arg70, Asp94, and Glu97 of chain A as well as the
same amino acid residues of chain B surrounded a-keto-
phosphonate 2b. The phosphonate fragment of compound
–
1
–1
k
2
(mM min )
Compound
GST from equine liver GST from human placenta
2
b may form salt bridges with two side chain residues of
2
2
2
2
3
a
b
f
0.013
0.024
0.007
0.011
0.026
0.028
0.043
0.004
0.010
0.040
Arg70 belonging to subunits A and B. The distance between
carbonyl group of the a-ketophosphonate derivative and two
interdomain Cys101, which are involved in regulation of
e
[31]
GSTP1-1 activity, was about 7–8 Å.
Figure 4. Possible binding modes of a-ketophosphonate derivative 2b with GSTP1-1.

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
5
Another binding site was located in the active center (H- Dimethyl (4-(tert-butyl)benzoyl)phosphonate (1c)
site) of GSTP1-1 (Figure 4). In this case, the monomethyl
Trimethyl phosphite (0.63 g, 5.1 mmol) was added to 1 g
5.1 mmol) of 4-(tert-butyl)benzoyl chloride. The mixture
was stirred overnight and distilled in vacuum. Yield 1.24 g
pivaloyl phosphonate (2b) formed hydrogen bonds with
Arg13, Tyr108, and Gly205. Amino acid residues Tyr7,
Phe8, and Val10 surrounded the tert-butyl group of the
compound, while methyl substituent of phosphonate frag-
ment was directed to Ile104. The carbonyl group of a-keto-
phosphonate derivative 2b occupied the region between
Tyr7 and Tyr108, which are considered as potential nucleo-
(
ꢂ
1
(
91%), b.p. 139–141 ꢁ (0.1 mm). H NMR (400 MHz,
3
CDCl ): d ¼ 1.31 (s, 9H, Me C), 3.89 (d, J
¼ 12.0 Hz,
3
3
HP
6
ꢃ, MeO), 7.50 (d, J ¼ 8.0 Hz, 2ꢃ, 3,5-H ), 8.16 (d,
Ar
J ¼ 8.0 Hz, 2ꢃ, 2,6-H ). 31ꢄ NMR (202.3 MHz, CDCl ):
Ar
3
3
d ¼ 1.4, septet,
J
¼ 12.0 Hz. 13ꢁ NMR (75.8 MHz,
2
[
32–34]
PH
philes for covalent inhibitors.
In addition, the Tyr7
CDCl ): d ¼ 30.8 (Me C), 35.3 (CMe ), 54.0 (d,
J
CP
¼
3
3
3
can be responsible for stabilization of glutathione molecule
6
8.0 Hz, MeO), 125.8 (3,5-C ), 129.8 (2,6-C ), 133.0 (d,
Ar Ar
[
35]
in thiolate anion form.
The distances between carbonyl
2
1
JCP ¼ 6.3 Hz, 1-C ), 159.0 (4-CAr), 197.5 (d, JCP
Ar
¼
oxygen of a-ketophosphonate 2b and oxygen atoms of Tyr7
and Tyr108 are 3.8 and 2.8 Å, respectively.
1
74.0 Hz, C ¼ O). Anal. Calcd. for C H O P: C, 57.77; H,
13
19 4
7
.09; P, 11.46. Found: C, 58.02; H, 7.21; P, 11.31%.
Conclusions
Diisopropyl (4-(tert-butyl)benzoyl)phosphonate (1d)
Our finding showed that low molecular weight alkyl and
aryl a-ketophosphonate derivatives can be of biological
interest as photoactive agents with inhibitory activity against
GSTs. During UV-irradiation, the a-ketophosphonate mono-
esters 2a, 2b, 2c, 2e, 2f provided time-dependent decrease in
activity of GSTs from equine liver and human placenta with
IC50 values in the micromolar range. The kinetics of inhib-
ition was described by pseudo-first order rate constants as
well as second-order rate constants showing the efficiency of
transformation of the enzyme to the inactivated form. In
case of GST from human placenta, the alkyl a-ketophospho-
nates 2a and 2b were ꢁ2–10 times more effective than aryl
and hetaryl derivatives 2f and 2e. The data obtained as well
as molecular docking results can be important for further
designing photoactive inhibitors of GSTs.
A solution of triisopropyl phosphite (0.01 mol) in ether
(
10 mL) was added to cold solution of 4-(tert-butyl)benzoyl
ꢂ
chloride (0.01 mol) in ether (15 mL) and stirred at 0 C for
1
5 min. Then, the reaction mixture was left at r.t. for 2 h.
The solvent was evaporated in vacuum to about 2 mL and
residue was left overnight. Crystals of compound 1d precipi-
tated from the solution. Yield 0.98 g (30%), m.p. 86–88 C.
ꢂ
1
H NMR (500 MHz, CDCl ): d ¼ 1.33 (s, 9H, Me C), 1.37
3
3
3
3
(d, J
¼ 6.0 Hz, 6ꢃ, Me C), 1.38 (d, J
¼ 6.0 Hz, 6ꢃ,
HH
2
HH
Me C), 4.8–4.9 (m, 2H, OCH), 7.51 (d, J ¼ 8.2 Hz, 2ꢃ, 3,5-
2
H ), 8.21 (d, J ¼ 8.2 Hz, 2ꢃ, 2,6-H ). 13ꢁ NMR (126 MHz,
Ar
Ar
3
3
CDCl ): d ¼ 23.8 (d, J ¼ 5.0 Hz, Me C), 24.1 (d, J
¼
¼
3
CP
2
CP
2
4
.0 Hz, Me C), 31.0 (Me C), 35.3 (CMe ), 73.0 (d, J
2 3 3
CP
7
.0 Hz, CHO), 125.7 (3,5-C ), 129.9 (2,6-C ), 133.2 (d,
Ar
Ar
2
1
JCP ¼ 64.2 Hz, 1-C ), 158.6 (4-C ), 198.8 (d, JCP
¼
Ar
Ar
1
75.7 Hz, C ¼ O). 31ꢄ NMR (81 MHz, CDCl3): d ¼ 0.4, t,
Materials and methods
3
J
¼ 8.0 Hz. Anal. Calcd. for C H O P: C, 62.56; H,
17 27 4
PH
8
.34; P, 9.49. Found: C, 62.81; H, 8.51; P, 9.32%.
NMR spectra were recorded with a Bruker Avance DRX 500
1
31
spectrometer with operating frequency 500 ( H), 202 ( P),
1
3
1
26 MHz ( C), a Varian Unity Plus 400 instrument with
operating frequency 400.4 MHz ( H), and a Gemini 200
Varian spectrometer with operating frequency 80.95 MHz
P). Chemical shifts are reported relative to internal TMS
H, C) and external 85%-H PO ( P) standards.
1
Sodium methyl (4-(tert-butyl)benzoyl)phosphonate (2c)
3
1
1
Anhydrous sodium iodide (0.31 g, 2.04 mmol) was added
with stirring to a solution of dimethyl (4-(tert-butyl)ben-
zoyl)phosphonate (1c) in anhydrous acetone (10 mL). After
(
(
13
31
3
4
1
5 min the precipitation of solid was observed. The mixture
Synthesis
Dimethyl pivaloylphosphonate (1b)
was stirred overnight. The solid was isolated by filtration
and washed with acetone (2 ꢃ 1 mL). Yield 0.47 g (91%),
ꢂ
1
white crystals, m. p. > 300 C. H NMR (400 MHz, D O):
2
3
Trimethyl phosphite (2.06 g, 17 mmol) was added dropwise d ¼ 1.20 (s, 9H, Me C), 3.54 (d, J ¼ 12.0 Hz, 3ꢃ, MeO),
3
HP
with stirring to 2.0 g (17 mmol) of pivaloyl chloride and 7.53 (d, J ¼ 8.0 Hz, 2ꢃ, 3,5-H ), 8.02 (d, J ¼ 8.0 Hz, 2ꢃ,
Ar
1
3
obtained mixture was left overnight. Then, the removal of 2,6-H ). ꢁ NMR (75.8 MHz, DMSO-d ): d ¼ 31.0 (Me C),
Ar
6
3
2
solvent and vacuum distillation afforded 2.71 g (84%) of a 34.9 (CMe ), 52.2 (d, J ¼ 6.2 Hz, MeO), 125.0 (3,5-C ),
3
CP
Ar
2
crystalline residue of the dimethyl pivaloyl phosphonate; b.p. 129.3 (2,6-C ), 135.2 (d, J ¼ 51.2 Hz, 1-C ), 155.7 (4-
Ar
CP
Ar
ꢂ
ꢂ
[36]
1
1
5
2–54 ꢁ (0.1 mm) (lit. b.p. 78 ꢁ (2 mm) ) H NMR (500 C ), 209.7 (d, JCP ¼ 166.0 Hz, C ¼ O). 31ꢄ NMR
Ar
3
3
ꢂHz, CDCl ): d ¼ 1.26 (s, 9ꢃ, (ꢁꢃ ) ꢁ), 3.83 (d, J
¼
(202.3 MHz, D O): d ¼ 1.5, q, J ¼ 12.0 Hz. Anal. Calcd.
3
3 3
ꢃꢄ
2
PH
31
8
Hz, 6ꢃ, ꢀꢁꢃ ) ppm. ꢄ NMR (202 ꢂHz, CDCl ): for C H NaO P: C, 51.80; H, 5.80; P, 11.13. Found: C,
3 3 12 16 4
d¼ ꢀ0.6 ppm.
52.01; H, 5.93; P, 10.95%.

6
О. L. KOBZAR ET AL.
Sodium isopropyl (4-(tert-butyl)benzoyl)phosphonate (2d)
Molecular modeling
The mixture of 0.2 g diisopropyl (4-(tert-butyl)benzoyl)- The PDB crystals of GSTA1-1 and GSTP1-1 (PDB codes
[
26]
phosphonate (1d) and 0.11 g of sodium iodide in anhydrous 6ATO and 6GSS,
respectively) were downloaded from
[
38]
acetone (3 mL) was refluxed for 48 h. The precipitated solid server RCSB PDB (https://www.rcsb.org).
was separated by filtration and washed with acetone docking was performed by Autodock Vina.
Molecular
Before the
[
24]
(
3 ꢃ 6 mL). Yield 80 mg (40%), white crystals, m. p. > calculations all ligands, water molecules, and amino acid
ꢂ
1
3
3
5
7
2
¼
00 C. H NMR (300 MHz, DMSO-d ): d ¼ 1.09 (d, J
¼
conformers were removed from the PDB files. The struc-
6
HH
.9 Hz, 6ꢃ, Me C), 1.27 (s, 9H, Me C), 4.2 (m, 1H, OCH), tures of compounds were drawn using MarvinSketch
2
3
.44 (d, J ¼ 8.0 Hz, 2ꢃ, 3,5-H ), 8.15 (d, J ¼ 8.0 Hz, 2ꢃ, (MarvinSketch 5.2.4, 2009, ChemAxon, http://www.chem-
Ar
1
3
3
,6-H ). ꢁ NMR (75.8 MHz, DMSO-d ): d ¼ 24.4 (d, J
axon.com) and optimized with AM1 semi-empirical quan-
Ar
6
CP
2
[39]
3.9 Hz, Me C), 30.9 (Me C), 34.7 (CMe ), 66.7 (d, J
CP
¼
tum mechanical method in program MOPAC2016.
The
2
3
3
5
.6 Hz, CHO), 124.6 (3,5-C ), 129.2 (2,6-C ), 135.4 (d, files for the molecular docking were prepared by program
Ar
Ar
2
1
[40]
JCP ¼ 51.2 Hz, 1-C ), 155.1 (4-C ), 209.7 (d, JCP
¼
AutodockTools (version 1.5.6).
The program Discovery
Ar
Ar
1
65 Hz, C ¼ O). 31ꢄ NMR (81 MHz, DMSO-d ): d ¼ 0.8, q, Studio 3.5 (Accelrys Software Inc., San Diego, CA, USA)
6
3
J
¼ 10.0 Hz. Anal. Calcd. for C H NaO P: C, 54.90; H, was used for the analysis of the predicted binding models.
PH
14 20
4
6
.58; P, 10.11. Found: C, 55.13; H, 6.29; P, 9.92%.
Acknowledgments
Pivaloylphosphonic acid (3)
This work was supported by the National Academy of Science of
Ukraine under Grants of the NAS of Ukraine to research laboratories/
groups of young scientists of the NAS of Ukraine in 2020, Project 21/
Bromotrimethylsilane (0.79 g, 5.2 mmol) was added with stir-
ring to dimethyl pivaloyl phosphonate (1b) (0.5 g, 2.6 mmol).
After 3 h, the mixture was evaporated and 5 mL MeOH was
added to the residue, the mixture was stirred for 30 min and
02-2020.
ꢂ
1
References
evaporated. Yield 0.355 g (83%), m.p. 52–54 ꢁ. H NMR
13
(
400 MHz, D O): d ¼ 0.97 (s, 9H); ꢁ NMR (75.8 MHz,
2
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2
DMSO-d ): d ¼ 25.3 (Me C), 45.3 (d, JCP ¼ 50.0 Hz,
6
3
1
31
CMe ), 219.4 (d,
J
CP
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ꢄ NMR
3
(
3
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5
11 4
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[
[
1
7
ml of a mixture consisted of 50 mM Tris-HCl buffer (pH
.5), 50 mM NaCl, 1 mM DTT, 5 mM EDTA and 50 vol.%
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