Phenyltetrahydroisoquinoline-pyridinaldoxime conjugates as efficient uncharged reactivators for the dephosphylation of inhibited human acetylcholinesterase

Article abstract of DOI:10.1021/jm3015519

Pyridinium and bis-pyridinium aldoximes are used as antidotes to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus nerve agents. Herein, we described a series of nine nonquaternary phenyltetrahydroisoquinoline- pyridinaldoxime conjugates more efficient than or as efficient as pyridinium oximes to reactivate VX-, tabun- and ethyl paraoxon-inhibited human AChE. This study explores the structure-activity relationships of this new family of reactivators and shows that 1b-d are uncharged hAChE reactivators with a broad spectrum.

Full text of DOI:10.1021/jm3015519

Brief Article
pubs.acs.org/jmc
Phenyltetrahydroisoquinoline−Pyridinaldoxime Conjugates as
Efficient Uncharged Reactivators for the Dephosphylation of
Inhibited Human Acetylcholinesterase
Guillaume Mercey,†,‡,§ Julien Renou,†,‡,§ Tristan Verdelet,†,‡,§ Maria Kliachyna,∥ Rachid Baati,∥
Emilie Gillon,§ Melanie Arboleas,§ Melanie Loiodice,§ Florian Nachon,*,§ Ludovic Jean,†,‡,§
́
́
́
and Pierre-Yves Renard*,†,‡,§
†Universite
‡INSA de Rouen, Avenue de l’Universite,
§CNRS Del
egation Normandie, 14 Rue Alfred Kastler, 14052 Caen Cedex, France
∥Universite
de Strasbourg, Faculte de Pharmacie, CNRS UMR 7199, BP 24, 67401 Illkirch, France
§Cellule Enzymologie, Dep
artement de Toxicologie, Institut de Recherche Biomedicale des Armees, 24, Avenue des Maquis du
Gresivaudan, BP 87, 38702 La Tronche, France
́
̀
de Rouen, COBRA CNRS UMR 6014 and FR 3038, IRCOF, 1 Rue Tesniere, 76821 Mont St. Aignan Cedex, France
́
76800 St. Etienne du Rouvray, France
́
́
́
́
́
́
́
́
S
* Supporting Information
ABSTRACT: Pyridinium and bis-pyridinium aldoximes are used as antidotes to reactivate acetylcholinesterase (AChE) inhibited
by organophosphorus nerve agents. Herein, we described a series of nine nonquaternary phenyltetrahydroisoquinoline−
pyridinaldoxime conjugates more efficient than or as efficient as pyridinium oximes to reactivate VX-, tabun- and ethyl paraoxon-
inhibited human AChE. This study explores the structure−activity relationships of this new family of reactivators and shows that
1b−d are uncharged hAChE reactivators with a broad spectrum.
INTRODUCTION
■
Organophosphorus nerve agents (OPNAs) are used as
insecticides (pest control agents) but also for highly toxic
agents as warfare agents (such as sarin, cyclosarin, tabun,
soman, and VX)1,2 in armed conflicts (e.g., Gulf Wars3) and
terrorist attacks (e.g., subway attack in Tokyo4). The acute
toxic effect of OPNAs is based on the irreversible
phosphylation of the catalytic serine hydroxyl group at the
active site of acetylcholinesterase (AChE, EC 3.1.1.7).5
Irreversible inhibition of the enzyme leads to accumulation of
the neurotransmitter (acetylcholine) in the synaptic cleft, then
to overstimulation of cholinergic receptors, causing seizures,
respiratory arrest, and death. Organophosphorus pesticide
poisoning is also a major health problem worldwide with
over 200 000 deadly intoxications yearly.6 It is of primary
importance to find an efficient treatment for acute and chronic
intoxications by OPNA.
Figure 1. Structure of standard pyridinium oximes and uncharged
AChE reactivators 1b,c.
2 decades,7,8 these reactivators still present serious drawbacks.
First, these reactivators are permanently charged cationic
compounds that poorly cross the blood−brain barrier (BBB)
and cannot readily reactivate cholinesterases in the central
nervous system (CNS).9 Moreover, there is no single broad-
spectrum oxime suitable for the antidotal treatment of any
OPNA. For instance, HI-6 rapidly reactivates VX-AChE in vitro
but is inefficient onto tabun-AChE.10
The current treatment of poisoning by OPNA is a
combination of an antimuscarinic agent (e.g., atropine),
AChE reactivator such as one of the standard pyridinium
oximes (pralidoxime or 2-PAM, trimedoxime or TMB-4,
obidoxime, HI-6, and HLo-7)7 (Figure 1), and anticonvulsant
̈
drug (e.g., diazepam).2 2-PAM is the oxime currently used in
the military forces of most countries. The high nucleophilicity
of oximes allows the displacement of the phosphoryl group
from the catalytic serine and thus induces the recovery of the
enzyme catalytic activity.
During the past couple of years, several research groups have
reported new efforts toward the development of original
reactivators aimed at tackling the above cited drawbacks.11 de
Since the discovery of monopyridinium and bispyridinium
oximes7 as reactivators for inhibited AChE and despite
hundreds of oximes synthesized and evaluated during the past
Received: October 23, 2012
Published: November 14, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Preparation of Phenyltetrahydroisoquinoline−Pyridinaldoxime Reactivators 1a,d−k
a
Conditions and reagents: (a) 7a−d, Pd(PPh3)4, CuI, NEt3, THF, rt, 15 h; (b) H2 (1 atm), Pd(OH)2/C, EtOAc, 15 h, rt; (c) (1) TBSCl, imidazole,
DMF, rt, 2 h; (2) DIBAL-H, CH2Cl2, −78 °C, 10 min; (3) TBAF, THF, 0 °C, 30 min; (d) NH2OH·HCl, NaOAc, EtOH, rt, 1 h.
Table 1. Reactivation Rate Constant (kr, min−1), Dissociation Constant (KD, μM), and Bimolecular Reactivation Rate Constant
(kr2, mM−1·min−1) for Reactivation of VX-, Tabun-, and Ethyl Paraoxon-Inhibited Human AChE
VX-hAChE
KD
tabun-hAChE
ethyl paraoxon-hAChE
reactivator
kr
kr2
kr
KD
kr2
kr
KD
kr2
a
a
a
a
e
a
a
2-PAM
0.06 0.01
0.60 0.05
215 75
54 12
0.28
11
0.01 0.0005
0.040 0.006
706 76
0.01
0.16
0.19
0.17 0.007
187.3 19
0.9
e
e
e
obidoxime
250 110
106 15
1.22 0.01
65 17
19
3
a
a
a
HLo-7
0.49
7.8
63
0.020 0.0007
0.63 0.04
210 31
̈
b
b
b
e
e
HI-6
TMB-4
1a
0.44 0.15
50 26
9
⌀
⌀
⌀
0.16 0.004
478 11
0.3
e
e
e
0.085 0.005
0.0034 0.0002
0.045 0.002
0.015 0.001
0.021 0.0015
145 25
0.6
0.5
1.7
2.7
1.5
1.5
0.97 0.01
62 19
16
3
0.45 0.11
0.68 0.06
0.35 0.05
0.29 0.03
117 30
3.8
20
7
2
0.6 0.07
1.17 0.09
0.4 0.041
208 26
1b
33
6
4
27
4
60
31
6
9
19
13
11
1c
2
58
5.6 1.4
1d
38
nd
nd
3
7.6
14
2
0.168 0.004
14.5 0.1
c
c
c
c
1e
nd
3
0.006 0.0003
4
1
c
c
d
d
1f
nd
0.8
nd (kobs at 100 μM = 0.004 min−1
)
)
nd (kobs at 100 μM = 0.056 min−1
)
d
1g
0.31 0.09
0.28 0.03
0.037 0.001
0.10 0.02
350 140
0.9
2.4
0.5
1.3
nd (kobs at 100 μM = 0.003 min−1
0.095 0.005
38.7 3.5
39.9 3.5
2.5
6.8
1h
113
79
7
0.08 0.006
75
51
9
3
1
0.27 0.01
d
1i
1
0.028 0.0008
0.5
nd (kobs at 100 μM = 0.08 min−1
)
)
d
d
1j
77 26
nd (kobs at 100 μM = 0.002 min−1
)
)
nd (kobs at 100 μM = 0.05 min−1
c
c
c
d
d
1k
nd
nd
0.2
nd (kobs at 100 μM = 0.002 min−1
nd (kobs at 100 μM = 0.018 min−1
)
a
b
c
Data from ref 20. No reactivation up to 5 mM HI-6. If [reactivator] ≪ KD, then there is a linear dependence between kobs and [reactivator]: kobs
=
d
(kr/KD)[reactivator]. In this case, kr and KD cannot be determined, but kr2 = kr/KD is the slope of the line. Not determined if kobs at 100 μM is
e
<0.005 min−1 for tabun-hAChE or <0.1 min−1 for paraoxon-hAChE. Data from ref 21.
Koning et al. described uncharged reactivators, though their
ability to reactivate inhibited human AChE remains modest
with no efficiency for tabun-AChE.12 Kalisiak et al. described
amidine oximes with enhanced ability to cross the BBB, whose
second order reactivation rates constants (efficiency rates) are
greater than that of monoisonitrosoacetone (MINA).13 Yet
binding to phosphylated AChE but sufficiently low for binding
to the uninhibited enzyme.
Our preliminaries results encouraged us (1) to synthesize
and evaluate a series of nine analogues of 1b,c to explore the
effect of structural modifications on the reactivation efficiency,
namely, the linker length, attachment position of the linker
onto the pyridyl ring, insertion of a heteroatom into the linker,
and (2) to complete the biological evaluation with a
representative pesticide, ethyl paraoxon.
́
they remain less efficient than pralidoxime. Radic et al. reported
a large library of uncharged oximes, whose promising lead is a
2-hydroxyiminoacetamide with an 8-fold improved efficiency
for VX-AChE compared to pralidoxime but with 3- and 4-fold
lower efficiency for paraoxon and tabun, respectively.14 In the
same period, our group described the synthesis and evaluation
of two promising nonquaternary reactivators 1b,c (Figure 1).15
They display an in vitro ability to reactivate VX- and tabun-
AChE at least equivalent to that of the best bispyridinium
oximes, i.e., 1−2 order of magnitudes superior to 2-PAM.
These reactivators are composed of 3-hydroxy-2-pyridinealdox-
ime16 linked to a peripheral site ligand of AChE, a
phenyltetrahydroisoquinoline moiety.17 Phenyl tetrahydroiso-
quinoline provides a balanced affinity sufficiently high for
CHEMISTRY
■
The syntheses of the different reactivators are shown in Scheme
1. First, Sonogashira coupling reaction between alkynes 2−6
with bromopyridines 7a−d15,18 afforded the desired 8−16.
Then reduction of alkyne and deprotection of phenol function
furnished 17−25. For the introduction of the required aldehyde
function, the best yields were obtained through the sequence
comprising the temporary protection of the phenol group as a
TBS ether, the reduction of methyl ester into the
corresponding aldehyde by using DIBAL-H, and a subsequent
deprotection, which gave the desired products 26−34. Finally, a
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Journal of Medicinal Chemistry
Brief Article
treatment of the latter with hydroxylamine afforded the
analogues 1a,d−k, whose linker is attached to position C6,
C5, or C4 of the pyridine ring. Oxime 1i bearing an additional
methyl on position 6 of the pyridine should allow us to study
the interaction with residues in the AChE catalytic site and the
subsequent effect on reactivation rates. Besides, the insertion of
one oxygen into the linker has been evaluated (1e in Scheme 1)
for potential H-bonds between the linker and tyrosines lining
AChE active site gorge.19
oximes have IC50 > 100 μM, indicating that the affinity toward
hAChE is lower than toward OPNA-inhibited AChE and that
these reactivators will not significantly inhibit hAChE once it is
reactivated.
CONCLUSION
■
We have described a series of nine analogues 1a,d−k as hAChE
reactivators to study the influence of some structural
parameters on the reactivation of VX-, tabun-, and ethyl
paraoxon-inhibited hAChE. The best global reactivation
efficiency has been obtained with a linker length of four or
five carbon atoms attached on position 6 of the pyridine ring,
i.e., for 1b and 1c. Their derivatives show a lower ability to
reactivate VX-inhibited hAChE, yet superior to that of 2-PAM.
Regarding the reactivation of tabun-inhibited hAChE, i.e., the
conjugate that is the most difficult to reactivate, three new
compounds reported in this study, 1d, 1e, and 1h, are more
efficient than TMB-4, while 1a and 1i are as efficient as TMB-4.
Moreover, this study shows that 1b−d are as efficient as
obidoxime and TMB-4 toward paraoxon-inhibited hAChE.21
Given their general efficiency and broad spectrum, 1b−d will be
evaluated in vivo in priority as nonquaternary hAChE
reactivators.
RESULTS AND DISCUSSION
■
The apparent dissociation constant of the reactivator/phosphyl-
AChE complex (KD), the maximal reactivation rate (kr), and
the bimolecular rate constant (kr2 = kr/KD) have been measured
to evaluate the potential of oximes 1a−k for in vitro
reactivation of VX-, tabun-, and ethyl paraoxon-hAChE,
(Table 1 and SI for details). 1b and 1c are the most efficient
compound for every OPNA tested and show that the general
optimal linker length is 4/5 methylenes with an attachment
point at position 6 onto the pyridine, i.e., in position meta
relative to the oxime function. Substitution at position 5 is the
most detrimental. Note that the relative linker/oxime meta
position, the most favorable for the reactivation reaction, is
harmful to the nucleophilicity of bispyridinium aldoxime.
Indeed, the conjugation of the oxime with the pyridium
nitrogen is lost, resulting in a higher oxime pKa.
EXPERIMENTAL SECTION
■
The synthesis of 1d is described below. The general chemistry,
experimental information, and syntheses of all other compounds are
supplied in the Supporting Information. Purity of all final compounds
as determined by HPLC analysis is ≥95%.
For VX-hAChE, most of the other derivatives are
significantly more efficient than 2-PAM, 1d being only
marginally less efficient than HI-6 or obidoxime. For tabun-
hAChE, 5 out of 9 analogues are at least as efficient as TMB-4,
one of the best tabun-hAChE reactivators to date:20 1d and 1e
are 2.5-fold more efficient, 1h is 2-fold more efficient, while 1a
and 1i are about as efficient as TMB-4.
OP pesticide poisoning is also a major health problem
worldwide; therefore, these encouraging results for the
reactivation of VX- and tabun-hAChE prompted us to evaluate
this series of reactivators for the reactivation of human AChE
inhibited by the widespread diethylphosphoryl pesticides, e.g.,
ethyl paraoxon. As shown in Table 1, the efficiency toward
ethyl paraoxon-hAChE is correlated to that toward VX-hAChE.
Oxime 1b is as efficient as obidoxime for in vitro reactivation of
paraoxon-hAChE, while 1c,d are slightly less efficient (1.5 and
1.7 less efficient, respectively). Yet they remain among the best
reactivators described to date for this family of pesticides, which
further enlightens the broad spectrum of this new family of
reactivators.
Methyl 3-(Benzyloxy)-6-(6-(6,7-dimethoxy-1-phenyl-3,4-di-
hydroisoquinolin-2(1H)-yl)hex-1-ynyl)picolinate (9). To a
Schlenk tube were added 5 (500 mg, 1.4 mmol), 7a (460 mg, 1
equiv), CuI (30 mg, 0.1 equiv), Pd(PPh3)4 (90 mg, 0.05 equiv), and
degassed THF (5 mL) and NEt3 (2.5 mL). The resulting mixture was
stirred at room temperature for 15 h under argon atmosphere. After
concentration under reduced pressure, the residue was purified by flash
chromatography on silica gel (cyclohexane/EtOAc 3:2) to give 9 (772
mg, 93%) as a light brown solid. Rf = 0.3 (cyclohexane/EtOAc 1:1).
1H NMR (300 MHz, CDCl3) δ (ppm) 1.44−1.67 (m, 4H), 2.25−2.36
(m, 3H), 2.47−2.60 (m, 2H), 2.76 (dt, J = 4.4, 15.4 Hz, 1H), 2.91−
3.04 (m, 1H), 3.15 (dt, J = 5.2, 11.5 Hz, 1H), 3.59 (s, 3H), 3.85 (s,
3H), 3.95 (s, 3H), 4.47 (s, 1H), 5.20 (s, 2H), 6.17 (s, 1H), 6.60 (s,
1H), 7.21−7.29 (m, 6H), 7.30−7.45 (m, 6H). 13C NMR (75 MHz,
CDCl3) δ (ppm) 19.1, 25.9, 26.0, 26.9, 28.2, 46.8, 52.7, 53.4, 55.7,
55.8, 68.1, 70.8, 79.3, 90.5, 110.7, 111.6, 121.8, 126.9, 127.1, 128.1,
128.2, 128.7, 129.6, 130.0, 130.2, 135.5, 135.6, 140.0, 144.2, 147.0,
147.3, 152.8, 164.9.
Methyl 6-(6-(6,7-Dimethoxy-1-phenyl-3,4-dihydroisoquino-
lin-2(1H)-yl)hexyl)-3-hydroxypicolinate (18). To a solution of 9
(245 mg, 0.5 mmol) in degassed EtOAc (10 mL) was added
Pearlman’s catalyst (143 mg, 0.2 equiv, 20% Pd, moisture 50%). The
solution was bubbled with H2, and the reaction was stirred at room
temperature under H2 atmosphere (1 atm) for 15 h. The mixture was
filtered through Celite and concentrated under reduced pressure to
furnish 18 (213 mg, 85%) as a light brown solid. The product was
used in the next step without further purification. 1H NMR (300 MHz,
CDCl3) δ (ppm) 1.12−1.29 (m, 4H), 1.36−1.51 (m, 2H), 1.52−1.68
(m, 2H), 2.18−2.34 (m, 1H), 2.39−2.59 (m, 2H), 2.64−2.79 (m, 3H),
2.85−3.00 (m, 1H), 3.04−3.16 (m, 1H), 3.58 (s, 3H), 3.83 (s, 3H),
4.02 (s, 3H), 4.46 (s, 1H), 6.15 (s, 1H), 6.58 (s, 1H), 7.14−7.30 (m,
7H), 10.55 (br s, 1H). 13C NMR (75 MHz, CDCl3) δ (ppm) 26.6,
27.1, 28.1, 29.2, 30.1, 37.7, 46.7, 53.2, 54.1, 55.7, 55.8, 67.8, 110.7,
111.6, 126.6, 126.9, 127.0, 128.0, 128.4, 128.7, 129.2, 129.6, 130.1,
144.3, 147.0, 154.3, 157.2, 170.2.
Ideally, reactivators must not be strong inhibitors of hAChE
at their practical reactivation concentration. We measured the
IC50 of eight reactivators for hAChE (Table 2). All of these
Table 2. Inhibitory Activity (IC50) of Uncharged
Reactivators toward Native Human AChE
reactivator
IC50 (μM)
1a
1b
1c
1d
1f
260
>100
>100
>400
>200
320
1h
1j
6-(6-(6,7-Dimethoxy-1-phenyl-3,4-dihydroisoquinolin-
2(1H)-yl)hexyl)-3-hydroxypicolinaldehyde (27). To a solution of
18 (195 mg, 0.39 mmol) in dry DMF (10 mL) were successively
300
1k
>500
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added imidazole (80 mg, 3 equiv) and TBDMSCl (128 mg, 2.2 equiv).
The mixture was stirred at room temperature for 2 h under argon
atmosphere. EtOAc (30 mL) was added, and the organic layer was
washed with water (thrice), dried over MgSO4, and concentrated
under reduced pressure. To a solution of the resulting residue in dry
CH2Cl2 (10 mL) was added dropwise DIBAL-H (780 μL, 1 M in
CH2Cl2, 2 equiv) at −78 °C. Then the reaction mixture was stirred at
this temperature for 10 min. The reaction was quenched with MeOH
(780 μL), and the mixture was allowed to warm at room temperature.
The organic layer was washed with an aqueous solution of NaOH
(1M), dried over MgSO4, and concentrated under reduced pressure.
Then TBAF (430 μL, 1 M in THF, 1.1 equiv) was added at 0 °C to
the residue in dry THF (20 mL), and the mixture was stirred for 30
min at this temperature. After concentration under reduced pressure, a
purification by flash chromatography on silica gel (cyclohexane/EtOAc
1:1) afforded the desired compound 27 (59 mg, 32%) as a pale yellow
oil. Rf = 0.3 (cyclohexane/EtOAc 1:1). 1H NMR (300 MHz, CDCl3) δ
(ppm) 1.20−1.28 (m, 4H), 1.42−1.51 (m, 2H), 1.63−1.67 (m, 2H),
2.25−2.31 (m, 1H), 2.47−2.56 (m, 2H), 2.69−2.79 (m, 3H), 2.90−
2.96 (m, 1H), 3.13 (dt, J = 5.0, 11.4 Hz, 1H), 3.59 (s, 3H), 3.85 (s,
3H), 4.47 (s, 1H), 6.16 (s, 1H), 6.60 (s, 1H), 7.20−7.28 (m, 7H),
10.02 (s, 1H), 10.63 (br s, 1H). 13C NMR (75 MHz, CDCl3) δ (ppm)
26.7, 27.1, 28.2, 29.1, 29.8, 37.3, 46.8, 54.0, 55.7, 55.8, 67.9, 110.7,
111.6, 126.3, 126.9, 127.0, 128.1, 129.6, 129.8, 130.2, 135.7, 144.3,
147.0, 147.3, 155.2, 157.0, 198.8.
6-(6-(6,7-Dimethoxy-1-phenyl-3,4-dihydroisoquinolin-
2(1H)-yl)hexyl)-3-hydroxypicolinaldehyde Oxime (1d). To a
solution of 27 (45 mg, 95 μmol) in absolute EtOH (5 mL) were added
successively HONH2·HCl (9.9 mg, 1.5 equiv) and NaOAc (11.7 mg,
1.5 equiv). The mixture was stirred at room temperature for 30 min
under argon atmosphere. After concentration under reduced pressure,
the residue was purified by flash chromatography on silica gel
(cyclohexane/EtOAc 1:1) to give 1d (40 mg, 86%) as a white solid. Rf
= 0.3 (cyclohexane/EtOAc 1:1). 1H NMR (300 MHz, CDCl3) δ
(ppm) 1.20−1.30 (m, 4H), 1.49−1.60 (m, 4H), 2.29−2.38 (m, 1H),
2.46−2.55 (m, 1H), 2.60−2.69 (m, 3H), 2.82 (dt, J = 4.8, 16.2 Hz,
1H), 2.95−3.05 (m, 1H), 3.17 (dt, J = 4.8, 11.1 Hz, 1H), 3.59 (s, 3H),
3.84 (s, 3H), 4.59 (s, 1H), 6.15 (s, 1H), 6.61 (s, 1H), 6.96 (d, J = 8.4
Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.19−7.28 (m, 5H), 8.37 (s, 1H),
10.00 (s, 1H), 10.48 (br s, 1H). 13C NMR (75 MHz, CDCl3) δ (ppm)
26.1, 27.2, 27.5, 29.0, 30.0, 37.0, 46.5, 54.3, 55.7, 55.8, 67.7, 110.8,
111.5, 123.7, 124.7, 126.4, 127.4, 128.2, 129.4, 129.9, 135.0, 142.6,
147.1, 147.6, 152.5, 153.0, 153.4. MS (ESI+) m/z (%): 490 [M + H]+.
HRMS (ESI+): m/z calcd for C29H36N3O4 490.2700; found 490.2706.
HPLC: tR = 23.2 min (purity, 95%).
DETOXNEURO and ANR_09_BLAN_0192 ReAChE pro-
grams), Defense Threat Reduction Agency (DTRA) (Contract
HDTRA1-11-C-0047), and the Region Haute Normandie
́
(Crunch program). We thank Dr. Anthony Romieu (Universite
de Rouen, France) for MS analyses.
́
ABBREVIATIONS USED
■
OPNA, organophosphorus nerve agent; OP, organophospho-
rus; hAChE, human acetylcholinesterase; BBB, blood−brain
barrier; MINA, monoisonitrosoacetone; CNS, central nervous
system
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■
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Corresponding Author
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For P.-Y.R.: phone, +33 235522476; e-mail, pierre-yves.
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ACKNOWLEDGMENTS
This work was supported by Direction Gen
(through Ph.D. fellowship to T.V., Postdoctoral Fellowship
REI-DGA 2009-34-0023 to G.M., and Contract PDH-2-NRBC-
4-C-403 to F.N.), Institut Universitaire de France, Agence
Nationale pour la Recherche (through ANR_06_BLAN_0163
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