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Design, synthesis and evaluation of excessively charged nucleophiles as potential reactivators of aged phosphorylated acetylcholinesterases, and N-bromo imides as universal decontaminants online

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DESIGN, SYNTHESIS AND EVAIUATICIJ OF EXCESSIVEKir CHARGED

NUCIDOPHIIES AS POTENITAL REACITVAK^S OF AGED

FHOSPHORYIATED ACEIYIOiOLINESTERASES , AND N-BRCMD

imCES AS UNIVERSAL EEXX»«1AMINANIS



By
B. RAEHAKRISHNAN



A DISSEREATICW PE^ESQ/TED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FKKEDA

IN PARTIAL RJUTHMENT OF THE RBQUIREMEMIS PC» THE

CEXSREE OF DOCTCR OF FHHiDSOPHY



UNIVERSITY OF FIDRIDA
1989



It) Father and Mother



Gcxxi reasons iniast, of force, give place to better.



- Shake^jeare



ACKNOWIEDGMEITIS

I would like to express my densest gratitude to Dr. Nicholas Bodor
for the invaluable guidance, genercus support, and the unique
opportunity to work directly with him and make this interdisciplinary
program ccroe trtie. I would like to express my sincere appreciation to
Dr. Alan Katritzky for his advice, help, and for having me as the
"adopted son" of his group. In addition, I would like to thank the
members of my graduate ocmraittee. Dr. Hartraut Derendorf, Dr. John
Helling and Dr. Kenneth Wagener, for their advice and enoourageinent.

My special thanks are extended to Dr. Dave Winwood, Dr. Marcus
Brewster and Dr. Gene Brown for their iinnediate guidance and helpful
hints durii^ my work at the Center for Drug Design and Delivery (CDDD)
laboratory at Alachua.

I am grateful to all my colleagues and members of Dr. Bodor's
group and the Center for Dr^jg Design and Delivery at the College of
Pharmacy and members of Dr. Katritzky's group in the chemistry
department. I sincerely thank everyone at the CDDD Labs for their
friendliness and for providing all facilities during the stages of my
"experimental era."

I would also like to express iny de^^est thanks to Laurie Johnson,
Joan Martinago and Julie Drigger for their indisputable part in my work.

iv



I would lite to extend my thcmks persovilly and collectively to all
those in the chemistry and medicinal chemistry d^)art3nents who have
prtjvided their support throughout my stay at the University of Florida.

Finally, I wish to thank all my friends for their moral si^port in
the pursuit of this degree.



TABIE OF GCJTrEinS



Page



ACKNCIWIEI3GMENIS IV

TiTST OF TABLES ^^^

LIST OF FIGUPES ^

KEY TO ABBREVIATICNS ^ii

ABSTRACT ^^

CHAPTERS

I INTRDDUCTiaJ 1

Fharmacology of Acetylcholinesterase and

Organcpho^iiate Poisoning 1

Specific Aiins (Reactivators of Aged AChE) 22

LfrdverseLI Deccartaminant (UD) 33

Specific Ains (N-^Ialoamine Deocntaminants) 45

II RESUnrS AND DISCUSSION 56

Synthesis of ECNRs and Fho^iiate Models 56

Synthesis of N-Brono Ccnpounds and their Derivatives. . 84

Fhysical-ChemiccLL Stuiies of ECNR Catpounds 99

Evaluaticxi of N-Brcmo Oaipounds 149

III EXPERIMENTAL I'^O

Materials I'^O

Methods ^'71

Conpounds ^^2

Fhysical-Qiemical Studies 219

IV SUMMARY AND OCNdJUSIONS 233

REFERENCES ^^^

BIOGRAPHICAL SKETCH 246



VI



LIST OF TABUS



Table ^Se

1-1 Hcilf lives of aging of sane organophosphate

derivatives of acetylcholinesterase 13

2-1 Apparent ionizaticxi caistants of ECNR and reference

ccrpounds ^^^

2-2 Critical micelle ccxioentration of sane ECNR

cind reference carpounds at 35°C 102

2-3 Kinetic data for the hydrolysis of Paraoxon at varicus

ccTJcentraticn of 14 in aqueous buffer pH 9.5 at 35°C. . 106



2-4 Kinetic data for the hydrolysis of octyl methyl 4-nitrcphenyl
phosphate by 14 in aqueous buffer pH 9.3 (0.03M)
at 35°C



110



2-5 Hydrolysis of Paraoxoi by hydroocamic acid 15 (non-micellar

analogue 14) in pH 9.5 (0.03M) at 35°C 112

2-6 Kinetic data of the reaction of oodme ECNRs with
ParaoxDn and octyl methyl FWPP at 35°C
in pH 9.4 (0.03M) 1^4

2-7 Effect of CIAB oi the rate of reactiai of 14 with Paraoxon

at pH 9.3 ^^"^

2-8 The reactivity of hydroxamic acid 14 with Paraoxcai at
various pH buffers (0.03M) at 35°C 119

2-9 Kinetic data for the reactioi of bis 4-nitrcphenyl

pho^i^te (32) by hydroxamic acid 14 in aqueous buffer

pH 9.3 (0.03M) at 35°C 134

2-10 Kinetic data for the biphasic reaction of 14 with bis
2,4-dinitrcphenyl pho^ihate (33) in aquecus
buffer pH 9.4, 8.0 and 7.0 at 35°C 128

2-11 Kinetic data for the hydrolysis of dodecyl, octyl, tutyl and
benzyl 4-nitrophenyl pho^jhates by 14 i^ aquecus buffer
pH 9.5 at 35°C 132



Vll



2-12 Corpariscai of BCNR reactivity with other r^»rted systems

aginst ethyl 4-nitrophenyl pho^iiate Q9) 137

2-13 Kinetic data for the hydrolysis of ethyl 4-nitrcphenyl

pho^iiate (29) by hydroxamic acid 2A in aquecus buffer pH
9.5 at 35°C 138

2-14 Kirjetic data for the hydrolysis of 4-nitrc|iienyl

phenylFhosphonate by JA at 35°C in pH 9.5 (0.03M) 143

2-15 Equilibrium ccnstants of the reversible reactioi between N-
brxxiinating agent and suocinimide to form free
oxazolidinone and N-branosuocinimide (NBS) 152

2-16 Stability of NEMDS (47) , NEMDSuocn (49) , tetrabraro dimethyl
glycouril ( 54 fb)) and NBO (43) (reference) in varicxis
buffer soluticxi and in solid form 156

2-17 Stability of tetrabrono dimethylglyccuril 54(b) in

hydroocyprcpyl ^-cyclodextrin (HPCD) formulation 158

2-18 An estimation of release of active branine ^secies (OBr )
in solution of 3-*»rcino oxazolidinone system as a
function of the solution pH 162

2-19 Hydrtalysis of Paraoxcn by NEMDS (47) as a functirai of pH at

30°C 1^^

2-20 Hydrolysis of Paraoxai by NEMDSuccn (49) as a functicn of pH

at 30''C l^''

2-21 Hydrolysis of Paraoxon as the function of the concentratioi of

NEMDS (47) in pH 11.17 at 35°C 168



Vlll



UST OF FIGURES

Figure ^^

1-1 Organc|3ho^iTDrus anticholinesterase agents 2

1-2 Nornal functioning of acetylcholinesterase 5

1-3 Inhibition of acetylcholinesterase by an

organopho^jhorus derivative 7

1-4 Reactivation of inhibited AChE by a ccawentioncil

codme (2-PAM) 1°

1-5 Aging of the poiscxied (pho^Dhorylated) AChE 12

1-6 Diester anion - 2-PAM ccnplex 21

1-7 General structure of hydroxamic acid ECNRs 24

1-8 Retrosynthetic scheme for hydroxaitu.c acid ECNRs 25

1-9 Genercil structure of oxime ECNRs 26

1-10 Retix3synthetic scheme for oocime ECNRs 27

1-11 Genercil stnicture of aged enzyme models and

organopho^ixjrus substrates 28

1-12 Retrosynthetic scheme for the pho^iiate substrates 30

1-13 Reaction of hypochlorite anicxi with Sonan 39

1-14 Reaction of mustard agent with hypobroracus acid



40



1-15 Retrosynthetic scheme for the derivatives of

4^ydro(xy-4-methyl-2-axazolidinaTes 48

1-16 Retrosynthetic scheme for dimethylglyccuril systems 51

2-1 General synthetic scheme for hydroxamic acid ECNRs 57



IX



2-2 Direct synthesis of N-(^-diethylaminoethyl)H>4Denzyl

hydraxyleanine ^

2-3 Ihe ^H NMR ^)ec±r\im (300 MHz) of 65 in CDCI3 64

2-4 Genercil synthetic scheaaes for oxime ECNR ccrrpcunds 67

2-5 Proposed pathway of decariDoxylaticn reactions of the

l-cartx3xyirethylpyridimLiin-2-aldaxinie 68

2-6 Alternative synthetic schfeme to pyridinium ECNR: Via

quatemization with branoesters of aminoalcohols 70

2-7 Preparation of brooaacylesters of quaternary aminoalcchols. . 72

2-8 The % NMR spectrum (300 MHz) of IglfeL in EMSCMy

trace up '*

2-9 Ihe ^H NMR ^3ectr\m (300 MHz) of 3-8 (b) in CMSO-d^

trace Up '^

2-10 Ihe ^H NMR spectrum (300 MHz) of 19(b) in EMSO-d^

trace Kf '°

2-11 General synthetic sequence for esters of 4-nitrcphenyl

pho^iioric acids

2-12 Diester hydrogen pho^iiate synthesis

2-13 The ^H NMR ^)ectrum (90 MHz) of n-tutyl 4-nitrcphenyl

hydrogen pho^shate in CDCI3 ^^

2-14 Synthetic scheme of (4^rano-4-inethyl-2-oxazolidinOTie-4-yl)

methyl sulfate ^^

2-15 Broninaticn of (4-inethyl-2-oxazolidinone-4-yl) methyl sulfate 89

2-16 Synthetic scheme for polar derivatives of 4-hydroxymethyl-4-
roBthyl-2-oxazolidincaie

2-17 The ^'c chemical shift assignment of _ _

4- (/3-diethylamiiioethoxymethyl-4-methyl-2-oxazolidinone

(50) and model ccnpcunds ^^

2-18 The "c NMR spectrum (74.5 MHz, EMSO-d^) of 4-^ydroxymethyl-
4-jnBthyl-2-axazolidincr>e (44)

2-19 The "c NMR ^jectnm (74.45 MHz, CDCI3)

4-(^-dimethylaminoethaxymethyl-4-methyl-2-oxazolidinone

(50) ^^



2-20 Synthetic schearae to dimethylglyccuril (54) ard attertpted

nonosubstituticxi reactioTS ^"7

2-21 Ihe stnjcture of ECNR ocnpcunds 107

2-22 HvdrtJlvsis of Paraoxon by hydroxamic acid 14 in pH 9.3 at

35°C 108

2-23 Hydrolysis of octyl methyl 4-nitriophenyl pho^iiate by

hydroxamic acid H in pH 9.4 at 35°C 108

2-24 Hartley model of micelle 109

2-25 Relative reactivity of oxime BCNPs against Paraoxcxi 115

2-26 Hydrolytic rate of Paraooorai by hydroxamic acid 14 vmder

nan first-order rate conditions 121

2-27 First-order rate constants for the hydrolysis of bis

4-nitrophenyl pho^ihate (IS) at various amount 14

in pH 9.5 at 35°C 125

2-28 Hydrolysis of dodedecyl 4-nitrcfiienyl phosphate (26) by

2A iii aqueous buffer pH 9.5 at 35°C 133

2-29 Hydrolysis of ethyl 4-nitrophenyl phosphate (29) by 14 139

2-30 Relative reactivity of BCNR against aged enzyme

models 14°

2-31 Hydrolysis of ethyl 4-nitrophenyl phosphate by 14 under

non first-order rate ccnditions 141

2-32 Hydrolysis of dodedecyl 4-nitrc|iienyl phosphate by 14

under non first-order rate conditions 141

2-33 Hydrolysis of phenyl 4-nitrophenylphosphonate by hydroxamic

acid 14 in pH 9. 6 at 35''C 144

2-34 A molecular model r^resentation of the mode of action of 14

with organophosphorus monoanicxi 147

2-35 Hydrolysis of Paraoxon at various ccncentratioi of NEMOS (47)

in pH 11.2 at 35''C 1^



XI



KEY TO ABEREVIATIONS

CMC: criticcil micelle cxmoentratiai

Ci3^: Hexadecyltrimethylaiiinonium brcmide (cso-surfactant)

DOD: n-dodecyl grtxp

But: rHxityl grcup

Et: ethyl grxx^)

FNPP: p-nitrcphenylphosphate moiety

FNP Phospn: p-nitrcphenylphosphaiate moiety

2,4-CNPP: 2,4-dinitrcphenylpho^±iate moiety

HA: imidcizolium hydroxamic acid

B2HA: benzyl protected imidazolixim hydroxamic acid

r.t: room teirperature

t^^: half life, based on log of activity vs time

K^ : e^^parent bimolecular rate coTstant

jr . psaxio first-order rate constant

f^i end point absorption

A : absorptiai at time t

r: correlation coefficient

accn: reaction rate acceleration based on buffer hydrolysis

Exct: excitation

Uniss: emission

X: wavelength

log: log^o

xii



M: mole per liter

Bis: disubstitxited

mono: monosubstituted

min: minutes

sec: seocnds

nm: nancmeter

UV: xiltraviolet

/il: microliter

BCNR: excessively charged nucleophilic reagent



Xlll



Abstract of Dissertatioi Presented to the Graduate School
of the University of Florida in Particd Fulfillment of the
Requirements for the Degree of Doctor of Riiloscphy

EESIGN, SYNTHESIS AND EVAIUATICN OF EXCESSIVELSf CHARGED

NUdEOFHILES AS POTENITAL REAC ITVA IORS OF AGED

PH0SHO?YIATED ACEIYLCHOUNESTERASES , AND

N-EKMD IMIEES AS UNIVERSAL CEXXWTAMINANIS

By

B. Radhakrishnan

Deceanber 1989

Chairman: Alan R. KatritzJcy
Oochciirman: Nicholas Bodor
Major D^artment: Chemistry

Acetylcholinesterase (AChE) enzyme has a major physiological
function in humans in ensuring that najrotransmission is of finite
duration by cleavii^ neurotransmitter acetylcholine at the nerve
j\jncticns. Ihe organophosphorus nerve agents inhibit this process by
irreversibly phosphonylating or phosphorylating the enzyme, which
results in elevated levels of acetylcholine, causing convulsion,
nusculcu: peiralysis auid death.

Inhibited AChE can be reactivated by coTventicxTal nuclecphilic
antidotes such as 2-PAM and ObicJoxiine. However, the inhibited enzyme
"ages" fran a reactivatable phosphate triester state to a
nonreactivatable pho^iiate diester anicxi or an analog thereof, vMch is
extremely inactive towarc3s any nucleophiles due to anionic repulsion.
So far, no antidotes have been shown to reactivate the aged enzyme.



XIV



A series of molticaticaiic nuclecphiles, neonely, excessively
charged nuclecphilic reagents (ECNR) , sane having surface activity, have
been designed, synthesized and investigated for the hypothesized mode of
action: (a) neutralization of repulsive charge and (b) sinultanecus
nuclecphilic attack against the aged enzyme models such as pho^jhate
diester and pho^iTonate mcnoanions.

Ihe ECNR carrying hydroxamate anion shewed hydrolytic cleavage of
the nonoanions with 40-to 2,600-fold rate enhancement at pH 9.5. Ihe
reaction appears not to be influenced by micellar action but takes place
by the hypothesized mode. The ECNR carrying aximate aniens were less
effective against the aged enzyme models. Both types of ECUR shewed
rapid acceleration of hydrolysis of organcphosphate triesters. Seme of
the ECNR should have in vivo applications.

Decontamination by hydrolytic cleavage using positive halogen
sources is one effective method to decCTitaminate nerve gas and nustard
agents in field or under various environmentcil ooTditions. Seme stable,
water solx±>le, "soft" braninating N-trano 2-oxazolidiiicaie systems, as
well as an N-brcno glyoouril system with increased molar positive
brcmine, were designed, synthesized and investigated for use as
universal decontaminants. Ihe water-soluble 2-oxazolidinane system
shewed high stability and activity against both mustard and nerve gas
(arganoFhosphate) agent nodels. The glyccuril system is exceptionally
stable as a solid.



XV



All synthetic strategies ani methods to target molecailes and the
strcjctural characterizatican of products and side products as well as
physical-chemical stuiies and kinetic processes are discussed.



XVI



CHAPTER I
INIRDDUCnON



This thesis will examine the design, synthetic methods, and
evaluation strategies that are relevant to sucx:essful develcpnent of
potential reactivators (antidotes) of organopho^Dhate blocked "aged"
acetylcholinesterases (AChE) and a new class of decontaminants of
organophosphates .

Pharmacoloav of Acetylcholinesterase and Orcr anophosphate Poisoning
Ihe organc^osphate anticholinesterase agents (nerve agents) are
hi^ily toxic derivatives of phosphonic and phosphoric acid.
Representatives of this class of oaipounds have widespread use in
agriculture (e.g., parathion, malathion) , pharmacology and medicine
(e.g., DFP, paraoxon), ard are of corK:em to the military because of
their application as chemical weapons in war (e.g., tabun [GA] , sarin
[VD] and [VX] ) . Ihe structures of these ccrpounds and rat 11^ values
are given in Figure 1-1.

All of these organophosphates elicit their prijonary effects by
phosphorylating or phosphonylating the serine hydroxyl group at the
active site of the enzyme acetylcholinesterase, causing essentially
irreversible inhibition of the enzyme. Differences in their ultimate
pharmacological effects and their lethality (toxicity) are derived from
their relative ability to penetrate into the central nervous system
(CNS), their rate of reaction with AChE, and their selectivity for



2

reaction with the enzyme at specific loci, and their behavior (e.g.,
aging) once attached at the active site on the enzyme.

i - C3H7O \ 9
(CH3)2N p \/

CaHsO^'^N CH3 F

Tabun (GD) Sarin (GB)

LD50 0.20 0.14



CH3
t-Bu-C-0. .0 C2H5O. -O

H ^P



r\/ "'\p/



P-L ^SCH2CH2-N(i-C3H7)2

CH, ^ ^"^3



■"3

Soman (GD)
LD50 0.10



vx

0.02



-x/ _ -3H,0 O



Eto'^ ^O-^ VnOs i-C3H70



Paraoxon
LD50 0.50



DFP

3.00



LD5o(rat) mg/kg, Route sc
Figure 1-1: Organcphosphorus antidiolinesterase agents.



3

Nn-rmal Function and Inhibition of AC3iE

Acetylcholine is found as a metabolite in hi^ concentrations in
nervous tissue and in nctor nerve tracts, as an active neurotransmitter
substance. When a nerve cell enervates a inascle fibre, the nerve-cell
endirg at the muscle junction forms a morphologiccaiy distinct and
functicxially discrete contact called a synapse. Ihis is a gap
(typically of about 500 A) between the presynaptic nerve-ending membrane
and postsynaptic membrane of the muscle-cell membrane. This interaction
transmits a depolarization signal to the muscle membrane in a manner as
yet inperfectly understood.

The physiological role of acetylcholinesterase is to ensure the
nerve inpulse is of finite duration by Icwering the acetylcholine
concentiBtion in the cleft via hydrolysis. This enzymatic cleavage will
lower the ooncentraticxi of free acetylcholine in the synaptic cleft, and
the resultirg perturbaticai of equilibrium will induce dissociation of
the acetylcholine-acetylcholine receptor ccnplex, turning off the signal
to the muscle cell. Active acetylcholinesterase is crucial for normal
neuronascular function, and inhibition of this enzyme produces tetanic
shock, with eventual muscle paralysis. In severe cases asphyxiation can
result. Not surprisingly, this enzyme is a target for nerve agents and
insecticides such as Soman and diisoprcpylphosphofluoridate (DFP) .

AOiE is loosely associated with postsynaptic membrane. As a
catalytic entity, AQiE is remar)cably efficient, with a turn over number
(^t) °^ 25,000 sec'^ and will cleave a substrate molecule once every 40
usee. The active site of AChE as depicted in Figure 1-2, is considered
to consist basically of two subsites: an anionic site, possibly



4

r^resented by a glutamate ion, and an esteratic site, located about 2.5
A frtxn the anionic site which has been shown to incorporate a serine
iroiety ard may contain a histidine residue (pK, 7.2) and a tyrosine
residue (pK^ 9.3).' A hydrophcbic area at the active site is also
indicated.

In normal functioning, acetylcholine, after acting at a
cholinergic receptor, ccnplexes reversibly with the active site of the
enzyme. In a fast step, the acetyl group is transferred from the
acetylcholine molecule to a serine hydroxyl group, forming acetylated
enzyme and releasing choline. This is followed by a rate limiting
hydrolysis of the acetate ester group, presumably acid catalyzed by the
imidazolium ion and possibly also aided by nucleophilic action of the
tyrosine hydrxsxyl, releasing acetate anion and providing reactivated
enzyme available to hydrolyze another molecule of acetylcholine. The
released choline is transported back into the nerve ending for
reconversion to acetylcholine and storage.

It is recognized that, although the active site has been fairly
well elucidated, acetylcholinesterase is an enzyme of highly complex
structure that has by no means been fully characterized. The enzyme
appears normally to consist of several submits, and enzyme obtained
from different species and different tissues (e.g., erythrocyte vs.
brain) can show significant guantitative differences in sensitivity to
organophosphorus agents and ability to be reactivated after inhibition.




Hist Ser

ESTERATIC SITE



Tyr



ENZYME SURFACE



Glu
ANIONIC SITE



Step 1
Enzyme — OH

Step 2



O

II +

CH3C-OCH2CH2N(CH3)3

Acetylcholine



Complex



Enzyme-0-CCH3 +



HOCH2CH2N(CH3)3 (Choline)



Step 3



H,0



Enzyme-OH +



Rate-limiting



CH



I



3^ - 0



H,0^



Figure 1-2: Normal functioning of acetylcholinesterase.



6

ThB orgcincFhosphorvis anticholinestxases react rapidly with the
enzyme; the nerve agents react particularly rapidly. A lethal dose of
Sonan, for exairple, will have corpletely reacted within minutes of
administration to an animal. A hypothetical formilaticai of the process
is deleted in Figure 1-3. The more potent organcphosphorus agents
react so r^idly with enzyme (in seconds), initicil kinetic studies
indicated the reaction to be a sinple bimolecular process without
intervention of a binding caiplex of the reversible Michaelis^fenton
type. Since the reaction is stoichionetric, one molecule of inhibitor
abolishing the functioning of one enzyme active site, the extreme
toxicity of the organcphosphorus agents can be understood vdien one
considers that acetylcholinesterase is present in the body in only small

amounts.

Ihe organc^iiosphorus esters act by acylating the serine hydroxyl
group at the active site of AChE, causing essentially irreversible
inhibition of the enzyme with consequent elevatiwi of acetylcholine
levels. Acetylcholine accumulates in the peripheral and central nervous
system (CNS) . Ultimately, at sufficiently hi^ level there is
d^ression of the respiratory center in the brain abetted by peripheral
neuronuscular blodcage, causing respiratory paralysis and death.
Generally more lipophilic, organcphosphorus esters show high
neurotoxicity as the result r^id penetration to the CNS system.
However, the acute toxicity effects of the organcphosphonos
anticholinesterase agents are elicited by the elevated acetylcholine
levels resulting frcm inhibition of the enzyme.



In sharp contrast to the rc^id hydrolysis of the acetylated
enzyme, hydrolysis of phosphorylated or phosphonylated enzyme is
extremely slow, with a half-life typically of the order of hours to
days.' Thus the enzyme is effectively inactivated and prevented frcm
carrying out its norroeil function of hydrolyzing acetylcholine.



(e.g., Soman)




Hist Ser

ESTERATIC SITE



, modified Sfil side chain



X' +H*



INHIBITED ENZYME



Glu
ANIONIC SITE



Figure. 1-3: Inhibition of acetylcholinesterase by an organcphosphorus
derivative.



8

Reac±ivators (Antidcftes) of AQiE

Despite considerable effort taken in design of antidotes
(particularly by the military ever sinse the develcpnent of Tabun in
Germany in 1937) no fully satisfactory therapeutic or prophylactic
agents have yet been devised.^ One of the most effective reactivation
nuclec?iiiles yet found is pyridine-aldoxime methiodide, or 2-PAM. It
illustrates rational drtjg design based on a knowledge of enzyme
mechanism. Ihe military has considerable interest in prcphylactic as
well as ther^jeutic agents, the former intended for protection of
personnel in the event that nerve agent attack appears imminent.

Design of antidotes has focused on essentially two ways to counter
the toxicity of organcphosphorus derivatives, namely (1) blocking the
cholinergic effects of the elevated acetylcholine levels, and (2)
reducing those levels. So far no single ocnpound acting in either of
these ways has proved ccnpletely satisfactory. Efforts to achieve
reduction of the elevated levels of acetylcholine induced by the
organcphosphortis nerve agents have largely focused on the develcpnent of
nuclec^iiilic agents that can reactivate inhibited AQiE by cleaving the
bond attaching the phosphoryl group to the serine hydroxyl at the active

site.

A large number of nuclecphilic agents were pr^jared and tested in
the early 1950s; two groups, hydroxamic acids and oximes, emerged as
pronisii^ reactivators. With respect to hydroxamic acid, the effective
nucleophile is hydroxamate anion, at physiological pH. However, the
oximes aj^ieared to offer more premise than hydroxamic acids. Ihe
prototype agent for this purpose is an oxime derivative, 2-



9

pyridinealdoxitoe methiodide (1, 2-PAM) or methyl methylsulfonate (P2S) ,
designed seme years ago by Wilson et al. Since then structure-
activity relationship among the huge number of analogues of 2-EAM that
have been pr^ared have been stiriied, (Saidoxine (2.,ToxogcxTin) has been
found to be scinewhat more effective in reactivating AChE in animals.




CH,



^=CH— (/ N-CHjOCHfN /)■^CH^^



2-PAM (1) Obidoxijne (2)

A hypothetical picture of the way in vAiich 2-EAM may reactivate
inhibited enzyme is presented in Figure 1-4. The pyridinium ion
coiplexes with carboxylate anion at the anicxiic site, andioring the
oximino anian in position to di^laoe the pho^iTonos ester groip fran
the electrostatic site. The tyrosine and the histamine residues
possibly assist in the di^lacement. It shculd be noted that
reactivation with 2-EAM requires the anti-isaner of 2-PAM; the anti-syn
interconversion is a very facile process that may occur in the oxinate
form at the enzyme level. ^



10



,^==^






H



Hist Ser Tyr

ESTERATIC SITE




\



/'



Hist
ESTERATIC SITE



Tyr



INHIBITED ENZYME



Glu
ANIONIC SITE



.^^^



^.



7— °\ /^,y

I. ^t^=CH N +

R I

CHr,



Q^^



Glu
ANIONIC SITE



■P OH

I.
R



,^^^



N +

I

CH,



Figure 1-4: Reactivation of inhibited AOiE by a conventional codme
(2-PAM) .



11

2-PAM chloride, in conjunction with atropine, has generally been
adc^jted in the tftiited States for treatment of poisoning by
organcphosphonis esters, v^iereas Cfcidoxime is preferred in sane European
countries and the methasulfonate salt of 2-PAM (P2S) in others.
However, a major dravADack of all available nuclecphilic reactivators is
that they cannot reactivate aaed enzvme,
Aging of Inhibited Acetylcholinesterase

It is genercilly acc^jted that cifter an organcphosphate reacts with


1 3 4 5 6 7 8 9 10 11 12 13 14

Online LibraryB RadhakrishnanDesign, synthesis and evaluation of excessively charged nucleophiles as potential reactivators of aged phosphorylated acetylcholinesterases, and N-bromo imides as universal decontaminants → online text (page 1 of 14)