Kinetic assessment of N-methyl-2-methoxypyridinium species as phosphonate anion methylating agents

Article abstract of DOI:10.1021/ol400054m

Organophosphate nerve agents and pesticides are potent inhibitors of acetylcholinesterase (AChE). Although oxime nucleophiles can reactivate the AChE-phosphyl adduct, the adduct undergoes a reaction called aging. No compounds have been described that reactivate the aged-AChE adduct. A family of 2-methoxypyridinium species which reverse aging in a model system is presented. A kinetic study of this system, which includes an SAR analysis, demonstrates that the reaction is highly tunable based on the ring substituents.

Full text of DOI:10.1021/ol400054m

ORGANIC
LETTERS
2013
Vol. 15, No. 5
1084–1087
Kinetic Assessment of
N‑Methyl-2-methoxypyridinium
Species as Phosphonate Anion
Methylating Agents
Joseph J. Topczewski and Daniel M. Quinn*
Department of Chemistry, The University of Iowa, Iowa City, Iowa 52242, United States
daniel-quinn@uiowa.edu
Received January 14, 2013
ABSTRACT
Organophosphate nerve agents and pesticides are potent inhibitors of acetylcholinesterase (AChE). Although oxime nucleophiles can reactivate
the AChE-phosphyl adduct, the adduct undergoes a reaction called aging. No compounds have been described that reactivate the aged-AChE
adduct. A family of 2-methoxypyridinium species which reverse aging in a model system is presented. A kinetic study of this system, which
includes an SAR analysis, demonstrates that the reaction is highly tunable based on the ring substituents.
Organophosphorus (OP) agents have been used as
both pesticides and chemical warfare agents for most of
the past century.1,2 Pesticides based on phosphate or thio-
phosphate agents (e.g., parathion, diazinon, or malathion)
are or have been used in domestic and commercial agri-
culture to protect crops from a variety of destructive
species. Despite numerous efforts to ban or limit the
use of OP pesticides, their continued use results in over
200,000 fatalities and over 1 million instances of morbidity
annually due to OP exposure. Phosphonate nerve agents,
including sarin, soman, VX, and tabun, are extremely toxic
and are considered a serious threat to national security due
to their potential use in terrorist actions.1
These nerve agents elicit their acute toxicity by inhibiting
the cholinergic system which results in an overstimula-
tion of muscles due to the buildup of acetylcholine in
the neuromuscular junction. The ultimate result is muscle
fasciculation and subsequent cardiopulmonary arrest.1,2
Specifically, OPs inhibit acetylcholinesterase (AChE) by
covalently bonding to the serine residue in the catalytic
triad of the active site (Figure 1). The well documented and
Figure 1. AChE inhibition by organophosphonates.
tremendous affinity of OP agents for the active cite of
AChE is a result of an OP’s structural similarity to the
tetrahedral transition state of the cognate hydrolysis reac-
tion of acetylcholine.3
There are several potential treatments for both acute
and chronic organophosphate poisoning. Most include
a nucleophilic oxime to reactivate the enzyme along with
atropine as an acetylcholine receptor agonist and diaz-
epam to treat seizures that arise from inhibition of AChE
in the CNS.1,2 If administered shortly after exposure, the
oxime (i.e., 2-PAM) is able to displace the bound OP and
liberate the serine residue (Figure 1).4 If administration of
(1) Mercey, G.; Verdelet, T.; Renou, J.; Kliachyna, M.; Baati, R.;
Nachon, F.; Jean, L.; Renard, P.-Y. Acc. Chem. Res. 2012, 45, 756.
(2) Jokanovic, M.; Prostran, M. Curr. Med. Chem. 2009, 16, 2177.
(3) Quinn, D. M. Chem. Rev. 1987, 87, 955.
r
10.1021/ol400054m
Published on Web 02/14/2013
2013 American Chemical Society

the oxime is delayed a process called aging occurs where
the enzyme bound phosphonate experiences a solvolytic
loss of an alkyl group (Figure 1: R = isopropyl for sarin,
pinacoyl for soman).1,5 The aged adduct is a phosphonate
ester monoanion, which is intrinsically less reactive as
an electrophile than the neutral phosphonate ester of
the initial phosphonyl-AChE adduct. The aged adduct is
further stabilized by several key interactions with the
AChE active site and is refractory to oxime reactivation.5
No compounds have yet been discovered that are able
to recover AChE activity once aging has occurred, and
therefore there are no antidotes against aged AChE-OP
adducts.1,4
We hypothesize that realkylation of the aged adduct
could lead to a reactivatable AChE adduct and have
initiated studies to demonstrate this. Because the aged
complex has long been thought of as a dead enzyme, we
term this concept “resurrection” of the aged adduct
(Figure 1). Presented here is the description of a family
of N-methyl-2-methoxypyridinium species which are
effective at “resurrecting” a model phosphonate system.
An analysis of the kinetics of these reactions demonstrates
the highly tunable reactivity of N-methyl-2-methoxypyr-
idinium species as alkylating agents for the methyl methane-
phosphonate monoanion.
that utilized N-methyl-2-methoxypyridinium as a catalyst.7
The methyl transfer reactivity of these species and their
structural similarity to the oxime AChE reactivator 2-PAM
lead to the consideration of them as aged AChE-OP
resurrecting agents.
The synthesis of N-methyl-2-methoxypyridinium species
was conducted by exposure of various commercial pyri-
dines to trimethoxonium tetrafluoroborate (Scheme 1).
This resulted in a family of nine pyridinium tetrafluorobo-
rates with varied substituents. Other work demonstrated
the utility of 1H NMR in measuring constants associated
with this family of compounds.8 Previous reports have
1
evaluated the H NMR chemical shift of the pyridinium
N-methyl as a measure of electron density.8a In an expan-
sion of this, Figure 2 shows a multiple linear correlation of
the methoxy methyl shift against the SwainꢀLupton field
and resonance parameters9,10 of the ring substituents. The
correlation shows that the chemical shift of the methoxy
moiety, and hence its electron density, is markedly depen-
dent on both field and resonance effects of substituents.
Further correlations with reactivity are discussed below.
Scheme 1. Synthesis
Our studies began with a literature search of alkylating
agents that are known to react with phosphate or phos-
phonate anions. Although several alkylating agents or
reactive intermediates are capable of phosphonate anion
alkylation, few provide practical solutions to the aging
problem due to their aqueous instability, poor selectivity,
or toxicity (i.e., MeOTf is not suitable).6 This led to the
consideration of less traditional methyl transfer reagents.
Particularlyappealing wereclassic reportsthatdescribe the
equilibration of 2-methoxypyridine to N-methylpyridone
The effectiveness of these compounds as methyl transfer
agents was determined by using sodium methyl methane-
phosphonate as an analogue of the aged AChE-OP adduct.
Model reactions were conducted by mixing equimolar
concentrations of phosphonate salt with the pyridinium
species (27 mM in d6-DMSO), and the reaction was mon-
itored by 1H NMR spectroscopy (see header of Table 1).
All reactants and products in the header of Table 1
could be simultaneously monitored. Moreover, a broad
(4) The synthesis of improved oximes is an ongoing field of research:
(a) Kalisiak, J.; Ralph, E. C.; Zhang, J.; Cashman, J. R. J. Med. Chem.
2011, 54, 3319. (b) Kalisiak, J.; Ralph, E. C.; Cashman, J. R. J. Med.
Chem. 2012, 55, 465. (c) Mercey, G.; Verdelt, T.; Saint-Andre, G.;
Gillon, E.; Wagner, A.; Baati, R.; Jean, L.; Nachon, F.; Renard, P.-Y.
Chem. Commun. 2011, 47, 5295. (d) Sit, R. K.; Radic, Z.; Gerardi, V.;
Zhang, L.; Garcia, E.; Katalinic, M.; Amitai, G.; Kovarik, Z.; Fokin,
V. V.; Sharpless, K. B.; Taylor, P. J. Biol. Chem 2011, 286, 19422.
(5) Characterization of aged OP-AChE adducts: (a) Millard, C. B.;
Kryger, G.; Ordentlich, A.; Greenblatt, H. M.; Harel, M.; Raves, M. L.;
Segall, Y.; Barak, D.; Shafferman, A.; Silman, I.; Sussman, J. L.
Biochemistry 1999, 38, 7032. (b) Sanson, B.; Nachon, F.; Colletier, J.;
Froment, M.; Toker, L.; Greenblatt, H. M.; Sussman, J. L.; Ashani, Y.;
Masson, P.; Silman, I.; Weik, M. J. Med. Chem. 2009, 52, 7593. (c)
Carletti, E.; Colletier, J.; Dupeux, F.; Trovaslet, M.; Masson, P.;
Nachon, F. J. Med. Chem. 2010, 53, 4002. (d) Masson, P.; Nachon,
F.; Lockridge, O. Chem.-Biol. Interact. 2010, 187, 157.
(6) Phosphponate or phosphate anion alkylation with bioincompat-
ible reagents: (a) Moriarty, R. M; Condeiu, C.; Tao, A.; Prakash, O.
Tetrahedron Lett. 1997, 38, 2401. (b) Di Raddo, P.; Chan, T.-H. J. Chem.
Soc., Chem. Commun. 1983, 16. (c) Schole, J.; Schole, C.; Eikemeyer, J.
Tetrahedron 1994, 50, 1125. (d) Andre, V.; Lahrache, H.; Robin, S.;
Rousseau, G. Tetrahedron 2007, 63, 10066. (e) Pitsch, S.; Pombo-Villar,
E.; Eschenmoser, A. Helv. Chim. Acta 1994, 77, 2251. (f) Jankowski, S.;
Kovacs, J.; Huben, K.; Blaszczyk, M.; Glowka, M.; Keglevich, G.
Heteroat. Chem. 2006, 17, 369. (g) Ayukawa, H.; Ohuchi, S.; Ishikawa,
M.; Hata, T. Chem. Lett. 1995, 81. (h) Chavez, M. R.; Zhao, P.; Kovacs,
Z.; Sherry, A. D. Lett. Org. Chem. 2004, 1, 194.
1
resonance in the H NMR spectra centered at 3.4 ppm
was interpreted as low levels of water, which gives rise
to a competing hydrolysis reaction of the N-methyl-2-
methoxypyridinium reactants to generate methanol.
Indeed, methanol was detected in product spectra through
its methyl resonance at 3.2 ppm. Consequently, the overall
kinetics of the reaction system can be described by the
(8) (a) Huang, S.; Wong, J. C. S.; Leung, A. K. C.; Chan, Y. M.;
Wong, L.; Fernendez, M. R.; Miller, A. K.; Wu, W. Tetrahedron Lett.
2009, 50, 5018. (b) Wong, F. M.; Capule, C. C.; Chen, D. X.; Gronert, S.;
Wu, W. Org. Lett. 2008, 10, 2757. (c) Titsky, G. D.; Mitchenko, E. S.;
Dereza, L. I. Ukrainskii Khimicheskii Zhurnal 1993, 59, 2077. (d)
Tummatorn, J.; Albiniak, P. A.; Dudley, B. D. J. Org. Chem. 2007,
72, 8962.
(9) (a) Swain, C. G.; Lupton, E. C. J. Am. Chem. Soc. 1968, 90, 4328.
(b) Swain, C. G.; Unger, S. H.; Rosenquist, N. R.; Swain, M. S. J. Am.
Chem. Soc. 1983, 105, 492. (c) Ewing, D. F. Correlation of NMR
chemical shifts with Hammett sigma values and analogous parameters.
In Correlation Analysis in Chemistry-Recent Advances; Chapman, N. B.,
Shorter, J., Eds.; Plenum Press: New York, 1978; pp 357ꢀ392. (d) Hamer,
G. K.; Peat, I. R.; Reynolds, W. F. Can. J. Chem. 1973, 51, 897.
(10) Carpenter, B. K. Determination of Organic Reaction Mechanisms;
John Wiley & Sons: New York, 1984; pp 76ꢀ82.
(7) (a) Beak, P.; Bonham, J.; Lee, J. T., Jr. J. Am. Chem. Soc. 1968, 90,
1569. (b) Beak, P.; Bonham, J. Chem. Commun. 1966, 631. (c) Beak, P.;
Lee, J. T., Jr. J. Org. Chem. 1969, 34, 2125.
Org. Lett., Vol. 15, No. 5, 2013
1085

d[Q]/dt. For each of the nine methyl transfer reactions,
these four differential equations were numerically inte-
grated by fourth-order RungeꢀKutta integration10 (see
Supporting Information (SI) for details), which provided
least-squares estimates of the rate constants kMe for all nine
N-methyl-2-methoxypyridinium reactants and khyd values
for all but the compound for which R = 5-F (see Table 1
for rate constants). Nearly 3 orders of magnitude distin-
guish the values of the fastest and slowest rate constants of
methyl transfer among the nine compounds evaluated.
Interestingly, an excellent linear free energy correlation,
plotted in Figure 3, is noted between methyl transfer rate
constants and the methoxy methyl chemical shifts that
are plotted in the multiple linear correlation of Figure 2.
This correlation and that in Figure 2 suggest in turn that
there will be a good correlation between log(kMeR) and
the SwainꢀLupton parameters, and this is indeed the case
(see SI for plot); the corresponding parameters are field =
1.4 ( 0.2, res = 1.0 ( 0.2, r2 = 0.93, n = 8. These data
indicate that kMe is markedly sensitive to both the field
and resonance effects of substituents, as one would expect
if the transition state for methyl transfer from N-methyl-2-
methoxypyridinium species is relatively late and the
leaving group strongly resembles the N-methylpyridone
product. Moreover, the correlation between log(kMe) and
the methoxy methyl chemical shift indicates that addi-
tional methyl donor substrates can be readily screened by
simple 1H NMR spectroscopy.
Figure 2. Multiple linear correlation of OCH3 chemical shift.
Parameters of the fit are field = 0.25 ( 0.02, res = 0.11 ( 0.03,
r2 = 0.93, n = 8. See text for elaboration.
Table 1. Methylation of Model Systema
H
entry R =
kMe
khyd
kMe/kMe
1a
1b 3-F
1c 5-F
H
(4.6 ( 0.1) ꢁ 10ꢀ5
(3.1 ( 0.1) ꢁ 10ꢀ2
(1.6 ( 0.2) ꢁ 10ꢀ4
(2.8 ( 0.2) ꢁ 10ꢀ6
(1.1 ( 0.1) ꢁ 10ꢀ3
NDb
1
670
3.5
60
1d 5-CF3 (2.83 ( 0.03) ꢁ 10ꢀ3 (6.0 ( 0.1) ꢁ 10ꢀ4
1e
1f
6-CF3 (4.63 ( 0.08) ꢁ 10ꢀ3 (8.7 ( 0.2) ꢁ 10ꢀ4
100
120
50
5-NO2 (5.6 ( 0.1) ꢁ 10ꢀ3
(2.2 ( 0.1) ꢁ 10ꢀ2
1g 4-CN (2.47 ( 0.06) ꢁ 10ꢀ3 (3.3 ( 0.2) ꢁ 10ꢀ4
1h 5-CN (5.13 ( 0.07) ꢁ 10ꢀ3 (3.9 ( 0.1) ꢁ 10ꢀ3
110
125
1i
6-CN (5.8 ( 0.2) ꢁ 10ꢀ3
(2.1 ( 0.1) ꢁ 10ꢀ3
a Reactions were conducted in d6-DMSO at 25.0 ( 0.5 °C and were
monitored by 1H NMR spectroscopy at 500 MHz. b ND = not
determined.
following component differential equations:
ꢀd[B]=dt ¼ d[Q]=dt ¼ kMe[A][B]
ð1Þ
Figure 3. Linear free-energy correlation between methyl transfer
rate constants and methoxy methyl chemical shifts. Parameters of
the fitare slope =6.9( 0.4, intercept = ꢀ29(2, r2 = 0.98, n = 8.
ꢀd[A]=dt ¼ d[P]=dt ¼ kMe[A][B] þ khyd[H2O][A] ð2Þ
In these equations, A and B are the methoxypyridinium
and phosphonate monoanion reactants, respectively; P
and Q are the pyridone and neutral phosphonate products,
respectively; and kMe and khyd are the respective second-
order rate constants for the methyl transfer and hydrolysis
reactions. Since time-dependent changes in the concentra-
tionsofall fourspecies A, B, P, and Q can be measured, one
can write from eqs 1 and 2 four independent differential
equations, one each for ꢀd[A]/dt, d[P]/dt, ꢀd[B]/dt, and
In a practical sense, the fastest compound (R = 3-F) was
capableof40% methyltransfertothe methylmethanepho-
sphonate monoanion in less than 10 min. This is within the
expected time frame for the treatment of OP exposure. The
marked sensitivity of the methyl transfer rate constant to
the substituent provides a temporal tuneability that will
likely be important in the eventual design of resurrection
agents for aged OP-AChE adducts.
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Org. Lett., Vol. 15, No. 5, 2013

The hydrolytic stability of the N-methyl-2-methoxypyr-
idinium species is an issue of considerable significance,
since antidotes against aged OP-AChE adducts must
survive the aqueous media of the bloodstream to reach
their target. Consequently, initial rates of hydrolysis were
obtained by observing pseudo-first-order hydrolysis in
D2O by 1H NMR spectroscopy (Table 2). Since the initial
rate is Vi = khyd[A], the corresponding first-order rate
constant is calculated as khyd = Vi/[A]. No hydrolysis
was observed for compounds R = H, 5-F, and 6-CF3 over
one week in D2O. The rate constants of hydrolysis for all
compounds were significantly slower than those of methyl
transfer in DMSO, typically by 2 orders of magnitude.
The 5-NO2 compound showed the lowest selectivity for
methyl transfer over hydrolysis. Another and perhaps
more germane measure of hydrolytic stability versus
methyl transfer activity was gauged by measuring rate
constants of hydrolysis in phosphate buffer. Table 2 shows
that the rate constants of hydrolysis in buffer at near-
physiological pH are significantly faster than those in pure
D2O but are still markedly slower than methyl transfer in
DMSO. The only compound that showed methyl transfer
to the phosphate buffer was that with R = 3-F, which
showed slower kinetics of methyl transfer in aqueous
media than hydrolysis. The kinetic selectivity of methyl
transfer from pyridinium compounds in DMSO, as a model
of the AChE active site, versus aqueous hydrolysis suggests
that blood serum lifetimes of select pyridinium compounds
will be sufficient to reach the aged OP-AChE targets at
neuromuscular junctions. On the other hand, the kinetic
selectivity of methyl transfer in DMSO versus that to
phosphate buffer components in aqueous media suggests
that unselective alkylation of other biological phosphorus
molecules (e.g., DNA, RNA, or ATP) will be minimal.
In conclusion, a family of N-methyl-2-methoxypyridi-
nium species has been shown to be capable of methylating
Table 2. Rate Constants of Hydrolysisa
entry
R =
khyd in D2O
khyd in bufferb
1a
1b
1c
1d
1e
1f
H
NDc
NDc
3-F
(2.3 ( 0.2) ꢁ 10ꢀ5
(7.1 ( 0.3) ꢁ 10ꢀ5
5-F
NDc
NDc
5-CF3
6-CF3
5-NO2
4-CN
5-CN
6-CN
(1.10 ( 0.05) ꢁ 10ꢀ6
NDc
(7.0 ( 0.5) ꢁ 10ꢀ5
(1.20 ( 0.06) ꢁ 10ꢀ4
(2.40 ( 0.07) ꢁ 10ꢀ3
(1.60 ( 0.04) ꢁ 10ꢀ4
(8.3 ( 0.2) ꢁ 10ꢀ4
(4.10 ( 0.04) ꢁ 10ꢀ4
(6.4 ( 0.4) ꢁ 10ꢀ5
(1.40 ( 0.03) ꢁ 10ꢀ6
(1.40 ( 0.05) ꢁ 10ꢀ5
(4.1 ( 0.3) ꢁ 10ꢀ6
1g
1h
1i
a Rate constants given in units of minꢀ1 were determined by 1H NMR
spectroscopy. b 50 mM phosphate buffer pH = 7.2. c Not determined, the
reaction was too slow to observe hydrolysis.
a weakly basic phosphonate anion, which is a model for
an aged AChE-OP adduct. This reaction is highly tunable
based on substituent dependent electronic factors, and
gratifyingly high rates of methyl transfer have been
observed.11 On the other hand, these compounds are
relatively stable in aqueous solution. The resurrection
activity of these compounds versus aged AChE-OP ad-
ducts is being explored and will be reported in due course.
Acknowledgment. Mr. Alexander Lodge (U-Iowa) is
acknowledged for the preparation of sodium methyl
methanephosphonate. Supported by the CounterACT
Program, National Institutes of Health Office of the
Director, and the National Institute of Neurological
Disorders and Stroke, Grant No. 5 R21 NS076430.
(11) An SN2 reaction is depicted in the abstract. The absence of
observable intermediates in these reactions would support this. However,
a reviewer suggested an alternative mechanism, which is shown below.
Supporting Information Available. Experimental pre-
paration, 1H and 13C NMR for all compounds, and
expanded kinetic analysis. This material is available free
of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 5, 2013
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