Synthesis of O-aryl-diarylphosphinates and study of their interaction with neurotoxic esterase and acetylcholinesterase

Full text of DOI:10.1023/A:1010471710580

Pharmaceutical Chemistry Journal
Vol. 35, No. 4, 2001
SYNTHESIS OF O-ARYL-DIARYLPHOSPHINATES AND STUDY
OF THEIR INTERACTION WITH NEUROTOXIC ESTERASE
AND ACETYLCHOLINESTERASE
1
1
1
1
G. F. Makhaeva, S. B. Fomicheva, L. V. Zhuravleva, A. N. Yarkevich,
A. V. Kharitonov, and V. V. Malygin1
1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 35, No. 4, pp. 14 – 16, April, 2001.
Original article submitted December 7, 2000.
Many organophosphorus compounds (OPCs), including
those used as drugs for the treatment of Alzheimer’s disease
and glaucoma [1, 2], exhibit significant acute toxicity related
to the inhibition of acetylcholinesterase (ACE) activity in
neuronal synapses and, in addition, lead to delayed distal
neuropathy manifested by a long axon degeneracy in the spi-
nal cord and peripheral nerves. The latter phenomenon is re-
ferred to as the delayed neurotoxicity of organophosphorus
compounds (DNOPC) [3 – 6]. A target for the axonotoxic
OPCs is one of the serine hydrolases of the nerve tissue,
which is called neurotoxic esterase (NTE) [3 – 5, 7, 8]. In
contrast to the acute toxicity of OPCs, related to the ACE in-
hibition, the mechanism of DNOPC initiation includes two
stages: (i) inhibition of the catalytic activity of NTE by
phosphorylation of the enzyme followed by (ii) extremely
rapid dealkylation of a phosphoryl group covalently bound to
serine. The latter stage is equivalent to “aging” of the
phosphorylated enzyme, leading to the appearance of a mod-
ified protein that is believed to initiate the axon degeneration
process [3 – 5, 7, 8].
Thus, the search for new agents for the prophylaxis of
DNOPC must be directed toward the synthesis of compounds
which, first, effectively inhibit NTE while not promoting ag-
ing of the phosphorylated enzyme and, second, do not (or
very slightly) inhibit the ACE activity.
In the search for nontoxic neuroprotectors effective
against the compounds inducing DNOPC, we have synthe-
sized a series of O-aryl-diarylphosphinates with the general
formula Ar P(O)OC H X, where Ar = phenyl (Ph) or
2
6
4
para-tolyl ( p-Tol), and studied in vitro the kinetics of inter-
action of these compounds with NTE and ACE from hen
brain (these compounds are standard test objects for DNOPC
investigations [3 – 5]. Aryl esters of diphenylphosphinic acid
(Ia – Ic) and di( p-tolyl)phosphinic acid (IIa – IId) (Table 1)
were synthesized by interaction of Ph P(O)Cl and
2
( p-Tol) P(O)Cl chloroanhydrides with p-substituted phenols
2
in the presence of organic bases.
EXPERIMENTAL CHEMICAL PART
Compounds acylating the enzyme but, for certain rea-
sons, excluding the possibility of the aging stage following
do not induce DNOPC. Moreover, the results of experiments
in vivo showed that such agents produce a protective effect
when introduced prior to neurotoxic OPC [3, 4, 9 – 11].
These compounds include carbamates, phosphinates, and
sulfonylfluorides [3, 4. 9 – 11]. For example, the interaction
of phosphinates with NTE leads to the formation of a
phosphorylated NTE possessing certain structural features
3
1
The P NMR spectra of the synthesized compounds
were measured on a Bruker CXP spectrometer with a work-
ing frequency of 81 MHz using chloroform as the solvent
and 85 % H PO as the external standard. The melting points
3
4
were determined on a Boetius PHMK-05 heating stage. The
yields, physicochemical characteristics, and parameters of
3
1
the P NMR spectra are listed in Table 1. The data of ele-
mental analyses (C, H, N) agreed with the results of analyti-
cal calculations according to the empirical formulas.
(
the absence of P–O–C and P–N–C bonds) making detach-
Diphenylphosphinic acid aryl esters (Ia – Ic). To a so-
lution of 2.37 g (10 mmole) of Ph P(O)Cl [12] in 20 ml of
ment of an alkyl (or aryl) radical impossible. Therefore, this
phosphorylated enzyme does not exhibit aging. On the other
hand, phosphinates are potentially capable of effectively in-
hibiting ACE and, hence, may possess a high acute toxicity.
2
anhydrous benzene were added 1.40 ml (10 mmole) of
freshly distilled triethylamine and then, dropwise with stir-
ring, a solution of 10 mmole of the corresponding phenol in
5 – 15 ml of anhydrous benzene. The reaction mixture was
heated for 30 min with weak boiling of the solvent. Then the
1
Institute of Physiologically Active Substances, Russian Academy of Sci-
ences, Chernogolovka, Moscow Region, Russia.
190
0
091-150X/01/3504-0190$25.00 © 2001 Plenum Publishing Corporation

Synthesis of O-Aryl-diarylphosphinates
191
3
1
precipitated triethylamine hydrochloride was sep- TABLE 1. Yields, Physicochemical Characteristics, and Parameters of the
NMR Spectra of the Synthesized Ar P(O)C H -p-X Compounds
P
arated by filtration. The organic solvent was
washed with water, dried over Na SO , and evap-
2
6
4
3
1
P NMR
spectrum
(CHCl ):
2
4
Com-
pound
Empirical
formula
orated in a rotor evaporator. The residue was
recrystallized from a benzene – hexane mixture
and dried in vacuum.
Ar
X
Yield, %
M.p., °C
3
d, ppm
*
Ia
Ph
Ph
H
89
134.5 – 135.0
147 – 149
120 – 122
93.5 – 94.5
136 – 137
109 – 110
107 – 108
C
C
C
C
C
C
C
18
18
19
20
20
20
21
H
H
H
H
H
H
H
15
O
2
P
30.92
33.22
30.30
32.20
34.36
32.66
31.80
Di(p-tolyl)phosphinic acid aryl esters
Ib
Ic
NO
2
89
91
79
84*
75
72
14NO
4
P
P
(
IIa – IId). To a suspension of 1.48 g (6 mmole)
Ph
CH
H
3
17
19
O
O
2
2
P
of di(p-tolyl)phosphinic acid [13] in 15 ml of an-
hydrous benzene was added by portions with stir-
ring in a flow of argon 1.37 g (6.6 mmole) of
comminuted phosphorus pentachloride. The reac-
tion mixture wad heated for 30 min with weak
boiling of the solvent. Then the solvent and
IIa
p-Tol
p-Tol
p-Tol
p-Tol
P
IIb
IIc
IId
NO
2
18NO
4
Br
2
18BrO P
CH
3
21 2
O P
Note: * Cf. [20].
POCl were distilled off in vacuum. The oily resi-
3
due was dissolved in 20 ml of anhydrous ben-
zene. To this solution were added 0.48 ml
(
6 mmole) of freshly distilled pyridine and then, dropwise
also determined by spectrophotometry on a Gilford Model
250 instrument as described by Ellman et al. [18] using ace-
tylcholine iodide (Sigma, USA) as the substrate. The kinetics
of NTE and ACE inhibition was studied by incubating a
sample of the corresponding enzyme with a compound tested
with stirring and cooling to 10°C, a solution of 6 mmole of
the corresponding phenol in 5 – 15 ml of anhydrous benzene.
The reaction mixture was allowed to stand for 12 h, after
which the precipitated pyridine hydrochloride was separated
by filtration. The organic solvent was washed with water,
dried over Na SO , and evaporated in a rotor evaporator. The
(in the presence of 5 vol.% DMSO) for various times under
the conditions [I ] > [E ] , after which the residual activity
0
0
2
4
was determined as described above [19]. All tests were car-
ried out in duplicate.
residue was recrystallized from a benzene – heptane mixture
and dried in vacuum.
The pseudomonomolecular enzyme inhibition rate con-
stants (k¢) were determined from the relationship
EXPERIMENTAL BIOLOGICAL PART
log V = log V + k¢t/2.3,
t
0
The source of NTE was the previously developed stable
lyophilized preparation from hen brain [14] with a specific
activity of 50 nmole of phenylvalerate per min per mg pro-
tein. This NTE preparation was obtained by rapid freezing
and lyophilization of a P + P membrane fraction of hen
where V is the rate of enzymatic hydrolysis of the substrate
in the absence of an inhibitor and V is the analogous value
0
t
determined upon the enzyme reaction with inhibitor for a
2
3
time t.
brain treated with paraoxon (40 mM, 40 min) [14, 15]. ACE
was obtained either from the same membrane fraction of hen
brain not treated with paraoxon [15] or from a commercial
human erythrocyte ACE preparation (Institute of Vaccines
and Sera, Perm) with a specific activity of 4 U/mg protein.
The NTE activity was determined by spectrophotometry
on a Gilford Model 250 instrument (England) using a differ-
ential Johnson technique [16] with small modifications de-
scribed in [14, 17]. The enzyme was prepared by suspending
the initial protein in a buffer (50 mM Tris-HCl, 0.2 mM
EDTA, pH 8.0) in a Potter homogenizer with a Teflon pestle.
The activity with respect to phenylvalerate (substrate) was
determined 0.5 – 1 h after preparation, using a 1 ml sample
TABLE 2. Inhibiting Activity of the Synthesized O-Aryl-diaryl-
phosphinates with Respect to Neurotoxic Esterase (NTE) and Ace-
tylcholinesterase (ACE)
Ar
O
P
Ar
O
X
Com-
pound
– 1
(NTE), M min
– 1
– 1
i
k (ACE), M min
– 1
Ar
X
k
i
with
(
a
microbiuret). The reaction of phenylvalerate with the en-
protein concentration of 0.1 – 0.14 mg/ml
1
4
2
2
3
3
1
Ia
Ph
Ph
H
(6.12 ± 0.71) ´ 10
(1.02 ± 0.08) ´ 10
(2.27 ± 0.16) ´ 10
(1.18 ± 0.08) ´ 10
(3.22 ± 0.17) ´ 10
(1.70 ± 0.06) ´ 10
(8.17 ± 0.76) ´ 10
No inhibition
1
< 10
Ib
NO
2
zyme was terminated 20 min after the beginning by adding a
sodium dodecylsulfate solution in a buffer containing
Ic
Ph
CH
H
3
No inhibition
No inhibition
IIa
IIb
IIc
IId
p-Tol
p-Tol
p-Tol
p-Tol
4
-aminoantipyrine. The amount of phenol was evaluated by
1
NO
Br
2
< 10
the intensity of the red color (measured at 510 nm,
e = 13,900) developed upon treating the reaction mixture
with an aqueous K Fe(CN) solution. The ACE activity was
No inhibition
No inhibition
CH
3
3
6

1
92
G. F. Makhaeva et al.
The bimolecular inhibition rate constants (k ) were deter-
mined from the relationship
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5
6
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7
3
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This study was supported by the Russian Foundation for
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1
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1
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