Possible Chemical Warfare Nerve Agents

Mil. Med. Sci. Lett. (Voj. Zdrav. Listy) 2013, vol. 82(1), p. 2-24 ISSN 0372-7025 DOI: 10.31482/mmsl.2013.001

REVIEW ARTICLE

BINDING OF QUATERNARY AMMONIUM SALTS TO ACETYLCHOLINE RECEPTORS: POSSIBLE CHEMICAL WARFARE NERVE AGENTS

James C. Ball

1083 Jewell Road, Milan, MI 48160, USA

Received 22 th January 2013. Revised 12 th February 2013. Published 8 th March 2013.

Summary Classical chemical nerve agents are organophosphate based compounds such as Sarin, Soman, Tabun, VX and others. These compounds inhibit acetylcholinesterases in the synapses of nerve junctions. Instead of inactivating acetylcholinesterases, compounds, quaternary amines, can block nerve transmission by binding primarily to the acetylcholine receptor sites. The U.S. Patent Office has published 23 unique patents or invention registrations on the synthesis of compounds that can bind to muscarinic and nicotinic acetylcholine receptors. It is likely that some of these compounds could serve as either polarizing or nonpolarizing nerve agents that bind to the acetylcholine receptor and block binding of acetylcholine. This paper has systematically reviewed a series of bisquaternary amines that could be potentially deadly nerve agents. Analogous tertiary amines, based on the structure of the parent bisquaternary amines, have been proposed as compounds that might be more soluble in membranes making them more bioavailable and toxic. Finally, methods have been discussed that could identify this class of compounds using a bioassay for binding to the acetylcholine receptor. A tool in the identification of these compounds, the Hofmann elimination reaction, has been proposed as a novel method for helping to establish that quaternary or tertiary amines are functional groups of the nerve agent.

Key words: Quaternary ammonium salts; nerve agents; chemical warfare; acetylcholine receptor

INTRODUCTION AND BACKGROUND the skin as well as through inhalation and oral expo - sure. These compounds act on acetylcholinesterases Classical chemical nerve agents are organophos - in the synapses of nerve junctions. The mode of ac - phate based compounds such as Sarin, Soman, tion of these agents involves phosphorylation Tabun, VX and others. These agents have properties of the active site of acetylcholinesterase followed by that make them uniquely suitable as chemical warfare “aging” in some compounds, a chemical reaction in - agents including high toxicity by all routes of expo - volving the spontaneous formation of a stable cova - sure. The hydrophobic nature of these chemicals lent bond in the active site of the enzyme. In a typical makes them susceptible to absorption through nerve conduction scenario, acetylcholine is released from the nerve terminus of the synaptic junction and rapidly diffuses across the synaptic cleft 1083 Jewell Road, Milan, MI 48160, USA to the postjunctional membrane where it binds [email protected] to acetylcholine receptors. When two acetylcholine (734) 429-1280 molecules bind to their respective sites within a re - Ball: Quaternary Ammonium Salts – Possible Chemical Agents ceptor, a conformational change occurs in the recep - receptors and opens the channel for the migration tor proteins allowing the opening of a channel with of cations. Thus, succinylcholine acts to depolarize the influx of Na +/Ca 2+ and efflux of K + [1]. the neuromuscular endplate. Succinylcholine is not The movement of cations through the channel causes hydrolyzed by the usual acetylcholinesterase but is the nerve to become depolarized; the nerve repolar - hydrolyzed by butyrylcholinesterase – a slower izes once the acetylcholine has diffused away from enzyme. The development of nondepolarizing the receptor causing the channel to close. Acetyl - compounds as an adjunct to anesthesia and used as choline is prevented from continually binding to a tool to relax muscles during surgery has resulted the receptor by rapid hydrolysis in a reaction catalyzed in the identification of several bisquaternary by acetylcholinesterase. If this enzyme is inactivated, ammonium compounds such as pancuronium as happens with the binding of the phosphorus-based (Figure 3), vercuronium (Figure 4) and tubocurarine nerve agents, the levels of acetylcholine do not dimin - (Figure 1) [3]. ish, the channel remains open and the nerve remains polarized; nerve conduction ceases to exist because the nerve cannot repolarize. Nicotinic acetylcholine O receptors are cholinergic receptors (responsive O H + to acetylcholine) found in the central nervous system, + N peripheral nervous systems and skeletal muscles. N H H O H OH O O + N O Figure 3. Pancuronium Cation [(2 S,3 S,5 S,8 R,9 S,10 S,13 S,14 S,16 S,17 R)- 17-acetyloxy- O 10,13-dimethyl-2,16-bis(1-methyl-3,4,5,6-tetrahydro-2 H- pyridin-1-yl)-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetra- + OH N decahydro-1 H-cyclopenta[ a]phenanthren-3-yl] acetate H O

Figure 1. d-Tubocurarine 6,6’-dimethoxy-2,2,2’,2’-tetramethyltubocuraran-2,2’- O diium-7’,12’-diol O

+ H N N + OO+ H N N H O OO H Figure 2. Succinylcholine Cation O 2,2'-[(1,4-dioxobutane-1,4-diyl)bis(oxy)]bis( N,N,N - Figure 4. Vercuronium Cation trimethylethanaminium) [(2 S,3 S,5 S,8 R,9 S,10 S,13 S,14 S,16 S,17 S)-17-acetyloxy- 10,13-dimethyl-16-(1-methyl-3,4,5,6-tetrahydro-2 H-pyridin -1-yl)-2-(1-piperidyl)-2,3,4,5,6,7,8,9,11,12,14,15,16,17- Tubocurarine was the first compound identified tetradecahydro-1 H-cyclopenta[a]phenanthren-3-yl] acetate to cause muscle paralysis via binding to the nicotinic acetylcholine receptor. This compound was identified from curare, an extract of a South American plant A key feature of nondepolarizing compounds Pareira, Chondrodendron tomentosum [2]. is that they form a rigid molecular framework. Tubocurarine is a compound with two quaternary Decamethonium (Figure 5), with two quaternary ammonium functional groups (Figure 1) that binds to ammonium centers separated by 10 methylene (-CH 2-) the nicotinic acetylcholine receptor and blocks groups, is sufficiently flexible that both sites for the binding of acetylcholine. The binding of this acetylcholine binding are bound resulting in the compound does not open the membrane channel. nerve becoming depolarized [4, 5]. Two broad Succinylcholine (Figure 2, basically two classes of compounds that bind to acetylcholine acetylcholine molecules joined back to back) binds to receptors are the competitive inhibitors that block both acetylcholine binding sites in the acetylcholine binding of the acetylcholine neurotransmitters, but

3 Ball: Quaternary Ammonium Salts – Possible Chemical Agents do not activate the ion channel and compounds that The development of new agents, which do not bind tightly to both sites in the acetylcholine receptor depend on the inactivation of acetylcholinesterase, causing depolarization of the nerve. Depolarization would make the use of these three countermeasures of the nerve allows initial transmission of the nerve mute. New agents would also significantly delay impulse to the muscle resulting in twitching the forensic identification of the agent and its origin. of the muscles (fasciculations) followed by paralysis An example of potential new nerve agents comes of the affected muscles. from an examination of the U.S. Patent Office which has published 23 unique patents or invention Phosphorus-based nerve agents were first registrations on the synthesis of compounds that can synthesized by German chemists prior to and during block nerve transmission by binding to muscarinic World War II and were, fortunately, never used and nicotinic acetylcholine receptors [8-32]. It is in combat [6]. There has been little research likely that some of these compounds could serve as published on the development of new nerve agents either polarizing or nonpolarizing nerve agents that and it is difficult to determine the extent to which any bind to the acetylcholine receptor and block binding country has developed new and novel nerve agents. of acetylcholine. There are a number of motivations why countries would pursue such research. For example, some The primary purpose of this paper is to report on of the phosphorous-based nerve agents can be an investigation of the open literature for clues to counteracted three ways: possible new classes of chemical weapons, or in this case, a new class of nerve agents. This paper will 1) through the use of agents that reactivate endeavor to review some of the physical properties acetylcholinesterase (2-PAM, 2-pyridine aldoxime (e.g. solubility, hydrophobicity) and potential methyl chloride; Figure 6); toxicities of a new class of nerve agents and their potential for use in chemical warfare. There are 2) by protection of the enzyme from attack several objectives of this study: (pyridostigmine; Figure 7) [4]; 1) to understand the structure, properties and 3) or by replacement of inactivated possible modes of action of a “second class” of nerve acetylcholinesterase by the intravenous injection agents that does not involve the inhibition of (i.v.) of human butyrylcholinesterase [7]. acetylcholinesterase, a mechanism common to the phosphorous based nerve agents – the original class of nerve agents, + + N N 2) review structural characteristics of these compounds that appear to be important Figure 5. Decamethonium Cation in the structure-toxicity relationship, Trimethyl-(10-trimethylammoniodecyl)ammonium 3) review characteristics of quaternary ammonium compounds that optimize their binding

+ to the nicotinic or muscarinic acetylcholine receptors, N OH N 4) discuss the possibility that more potent nerve agents may be derived from structurally similar Figure 6. 2-PAM, 2-pyridine aldoxime compounds with properties that would enhance their 2-[(hydroxyimino)methyl]-1-methylpyridin-1-ium lipid solubility and their absorption via inhalation, ingestion or dermal exposure,

5) discuss 3-quinuclidinyl benzilate as a classical + N example of a tertiary amine that is also an O incapacitating agent developed and stock-piled for ON potential use in a conflict,

Figure 7. Pyridostigmine 6) discuss possible chemical and biochemical 3-[(dimethylcarbamoyl)oxy]-1-methylpyridinium methods that could be used to detect and identify

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nerve agents discussed in this report, including General Synthetic Method the following: biochemical methods to detect this class of nerve agents, utilization of the Hoffman A common synthetic and purification scheme is elimination reaction of quaternary amines for apparent when the syntheses in the all the patents detection and identification, and analytical chemistry are reviewed and compared. The synthesis and methods to identify these compounds, and purification of several examples for each patent are presented in adequate detail in the patents. 7) to discuss recent results showing similar The chemistry used to synthesize these compounds studies on other published bisquaternary amines. is straight forward and typically involves the mixing of an alkyl bromide with a tertiary amine in the presence of a suitable solvent and allowing the components to combine in a simple U.S. Patents Issued to the U.S. Army - Chemical nucleophilic displacement reaction. After a suitable Agents time, the volume of the reaction mixture is concentrated, treated with decolorizing charcoal, In the 1970s and 80s, 23 unique U.S. patents (two filtered and allowed to precipitate or crystallize. patents were duplicates) described the synthesis and The compound maybe induced to precipitate or rudimentary toxicity (intravenous injection, LD 50 crystallize from solution using a combination measurements) of chemical nerve agents based on of methods including the addition of another less mono and bisquaternary ammonium functional polar solvent and lower temperatures. groups (Table 1). The patents were assigned to The combination of solvents/temperature used in the U.S. government represented by the United the purification of these compounds represents States Army [8-32]. It is not clear why these the “art” of the synthesis and varies among patents were released as they are potentially deadly the different patents but is similar within one set nerve agents. They were originally submitted to the of compounds represented by one patent. One Patent Office in the 1960s, but were not published should note that sophisticated column until ~20 years later. Perhaps these were released in chromatography (e.g. preparative HPLC or preparation for the ratification of the Conventional normal phase silica gel column chromatography) Weapons Treaty that prohibits the development of is not required to achieve products of reasonable new chemical weapons [34]. One may argue that purity. Reasonable purity, in this case, is defined the medicinal research was in the process of as the ability to obtain an adequate C, H, N identifying nerve blocking agents for muscle elemental analysis, which means that relaxants and anesthetic uses. It made sense to lay the elemental analyses are with 0.4% of claim to the hundreds of compounds already the theoretical values [35]. The rather simple investigated that may have some therapeutic chemistry involved in the syntheses of benefit. The problem with this argument, however, these compounds allows the syntheses to be carried is that none of the patents claim any potential out in an enclosed environment (e.g. a glove box) therapeutic benefits; they all cite the potential for minimizing the exposure of the chemist to these the compounds to be chemical warfare agents. lethal compounds. Regardless of the reasoning behind the submission of these patents, they reveal a potentially new class of nerve agents. Each of these patents represents a number of different compounds due to Toxicology a combination of substituents and anions selected in the synthesis of the compounds, presumably to These patents give no information on the pharma- study their structure-activity relationships. cokinetics of these compounds. There is no The number of compounds represented by these information on the absorption, distribution, patents could easily be in the hundreds. For most of these patents, one, two, or three examples of the synthesis of representative compounds are + + N presented. In some cases, the key precursor to N the compounds is also shown. There is sufficient detail discussed in these patents that a skilled chemist Figure 8. Hexamethonium Cation could easily synthesize and purify these compounds. N,N,N,N',N',N' -hexamethylhexane-1,6-diaminium

5 Ball: Quaternary Ammonium Salts – Possible Chemical Agents metabolism or elimination via inhalation, oral or (Figure 8), a doubly charged bisquaternary dermal exposure. Based on the charged nature ammonium salt, via the inhalation route of exposure of these compounds it seems likely that these [37]. Exposure by inhalation to a hexamethonium compounds would have a difficult time crossing aerosol resulted in a steady decline in lung function a membrane via dermal contact or though absorption resulting in the death of the subject one month after in the GI tract to ultimately enter systemic circulation initial exposure even after hospitalization and where they could be transported to muscarinic and/or respiratory support. Thus, it seems possible that nicotinic acetylcholine receptors. However, there is these agents could be toxic via the inhalation route one anecdotal report of poisoning by hexamethonium of exposure using an aerosol formulation.

Table 1. Patented potential chemical warfare agents - quaternary ammonium salts

Toxicity a Substituents i.v. injection Compounds Compound (μg/kg) Ref. Studied LD 50 LD 50 Compound 1 Compound 2 Rabbits Mice

1 R, R = CH 3, C 2H5, Compound 1 n-C 3H7, i-C 3H7, N - N n-C 4H9, i-C 4H9, 2 X 1 R = CH 3 2.7 μg/kg 7 μg/kg t-C 4H9, n-C 5H11 , R, R = CH 3 1 OOO O R = C 2H5 R O O R + + n-C 6H13 n = 10 [8] N N – – n = 8 1 1 n = 2-12 X = Br – – Compound 2 R R – X = Br N n N X = halides, − – – HC 2O4 , ClO 4 , NO 3 , – – (C 6H6)4B , HSO 4 2.7 μg/kg 10 μg/kg

Compound 1

1 N - N R, R = CH 3, C 2H5, 2 X R = CH 3 R = CH 3 n-C 3H7, i-C 3H7, 1 1 4 μg/kg 11 μg/kg R = C 2H5 R = C 2H5 O O O O n-C 4H9, C 6H5CH 2 R R + + n = 8 n= 6 [9] N N n = 3-14 – – – – 1 1 – X = Br X = Br Compound 2 N R R N X = monovalent, n polyvalent anion 4 μg/kg 6 μg/kg

R, R’ = CH 3, C 2H5, Compound 1 n-C 3H7, C 2H5OH 1 1 R, R’ = CH 3, - R , R ’= H, CH 3, R, R’ = CH 3 N 2 X 1 1 C2H5OH 4.5 μg/kg 9 μg/kg 1 2 C2H5 R , R ’= H R R R 1 1 N + + 2 2 2 2 R , R ’= H R , R ’ = H, OH, CH 3, R , R ’ = H 2 2 [10] O N (CH 2)10 N OH 1 2 R , R ’ = H C2H5, n-C 3H7 n = 1 O R´ R ´ n R ´ n = 1 Compound 2 n = 1-8 X– = Br – X– = Br – X– = monovalent, polyvalent anion 5.4 μg/kg 14 μg/kg

Compound 1

1 2 3 R , R , R = CH 3, R = CH 3 R = CH 3 + C2H5, n-C 3H7, 1 1 6 μg/kg 13 μg/kg N R = CH 3 R = CH 3 R i-C 3H7, n-C 4H9 2 2 + 1 R = CH 3 R = CH 3 [11] (CH 2)n N R n = 5-16 O O - 2 – n = 10 n = 8 X = monovalent, – – – – Compound 2 2 X R X = (C 6H6)4B X = (C 6H6)4B N polyvalent anion 4.6 μg/kg 14 μg/kg

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Toxicity a Substituents i.v. injection Compounds Compound (μg/kg) Ref. Studied LD 50 LD 50 Compound 1 Compound 2 Rabbits Mice

1 R, R = CH 3, C 2H5, Compound 1 n-C 3H7, i-C 3H7,

n-C 4H9, i-C 4H9, 1 1 R, R = CH 3 R, R = CH 3 ZZn-C 5H11 , n-C 6H13 R O O R n = 10 n = 8 5 μg/kg 14 μg/kg + + n = 2-12 – – – – N N X = Br X = Br O 1 1 O – R R X = monovalent, [12] - n Z = H Z = H N 2 X N polyvalent anion O O dimethylcarbamoxy dimethylcarbamoxy Compound 2 Z = H, CH 3, C 2H5 group in o-position group in o-position dimethylcarbamoxy group in o, m, p 6 μg/kg 18 μg/kg positions

Compound 1 1 R, R = CH 3, C 2H5, N - N 2 X n-C 3H7, i-C 3H7, R = CH 3 R = CH 3 1 1 5 μg/kg 7 μg/kg O O O O n-C 4H9 R = C 2H7 R = n-C 3H7 R R [13] + + n = 3-14 n = 6 n = 8 N N – – – – – Compound 2 1 1 X = monovalent, X = Br X = Br R R n polyvalent anion 10 μg/kg 45 μg/kg

1 R, R = C 2H5, Compound 1 n-C 3H7, n-C 4H9 1 Z = C 2H5, n-C 3H7, 1 R, R = CH 3 - R, R = n-C 4H9 5 μg/kg 28 μg/kg N 2 X n-C 4H9 Z = C 6H11 N R Z = n-C 4H9 + + When Z = n-C 8H17 , – – (cyclohexane) [14] O N (CH 2)10 N Z X = Br – – 1 n-C 12 H25 , C 6H11 then X = Br O R 1 Compound 2 R, R = CH 3, C 2H5 X– = monovalent, polyvalent anion 10 μg/kg 28 μg/kg

Compound 1 1 R, R = CH 3, C 2H5, 1 - n-C 3H7, n-C 4H9 R, R = CH 3 1 N 2 X R, R = CH 3 5.6 μg/kg 10 μg/kg R n =1-8 n = 1 N + + – – – n = 3 [15] O N (CH 2)10 N (CH 2)n N X = halides, X = Br – – 1 − – – X = C 6H6)4B Compound 2 O R HC 2O4 , ClO 4 , NO 3 , – – (C 6H6)4B , HSO 4 5.6 μg/kg 11 μg/kg

1 R, R = CH 3, C 2H5, Compound 1 n-C 3H7, i-C 3H7, - 1 N 2 X N n-C 4H9, i-C 4H9, R, R = CH 3 – – b R 5 11 6 13 6 6 4 N + + n-C H , n-C H X = (C H ) B ND [16] – O N (CH 2)10 N OH 1 X = monovalent, O R polyvalent anion 5,8 μg/kg 22 μg/kg

Compound 1 R = H, OH, CH 3, - N 2 X CHNOH in the o, m 7 μg/kg 13 μg/kg N and p positions R = H R = p-CHNOH + + – – – – – [17] N (CH ) N X = monovalent, X = Br X = Br O 2 10 Compound 2 O R polyvalent anion

5.8 μg/kg 18 μg/kg

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Toxicity a Substituents i.v. injection Compounds Compound (μg/kg) Ref. Studied LD 50 LD 50 Compound 1 Compound 2 Rabbits Mice

NRR 1R2 =

+ Compound 1 N

+ N

OH + 1 2 1 2 5.9 μg/kg 11 μg/kg - N NRR R = NRR R = N 2 X + O N + R N N + + 1 + N H3C [18] O N (CH 2)10 N R OH 2 H3C – – – – O R + X = Br X = Br N H3C Compound 2

+ N H3C OH X– = monovalent, polyvalent anion 6 μg/kg 10 μg/kg

R, R’ = CH 3, C 2H5 Compound 1 1 1 2 2 R , R ’, R , R ’ = H, R, R’ = CH 3 - 1 1 R, R’ = CH 3 N CH 3 R , R ’ = H 1 1 2 2 2 X 1 2 R , R ’, R , R ’ = H 6.3 μg/kg 13 μg/kg N RRR Z = CH 3, C 2H5, [19, + + 2 2 O N (CH 2)10 N O n-C 3H7, n-C 4H9, R , R ’ = H, CH 3 20] 1 2 Z = n-C 3H7 Compound 2 O R´ R ´ R ´ Z n-C 5H11 Z = CH 3 X– = Br – O X– = monovalent, X– = Br – polyvalent anion 6 μg/kg 13 μg/kg

Compound 1 c

N - N 2 X – >32 μg/kg >20 μg/kg O O O O X = monovalent n = 1 n = 6 – – – – [21] + + n = 1-9 X = Br X = Br N N Compound 2 c N n N 6.3 μg/kg 2.6 μg/kg

Compound 1 1 2 3 R , R , R = CH 3,

- C2H5, n-C 3H7, 1 2 1 2 2 X R, R , R = CH 3 R, R , R = CH 3 7 μg/kg 22 μg/kg i-C 3H7, n-C 4H9 N R n =10 n = 8 [22] + + 1 n = 5-16 – – – – O N (CH 2)n N R – X = Br X = Br 2 X = monovalent, Compound 2 O R polyvalent anion 7 μg/kg 14 μg/kg

1 R,R = CH 3, C 2H5, 1 n-C 3H7, i-C 3H7, R, R = CH 3 N - O Compound 2 X n-C 4H9 CH3 O O O ON R CH3 CH3 + + ON ND [23] N N 1 CH3 N In p– positi–on N R 8 O Dimethyl carbamoxy X = Br O group in o, m. or p 8 μg/kg 18 μg/kg X– = monovalent, polyvalent anion

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Toxicity a Substituents i.v. injection Compounds Compound (μg/kg) Ref. Studied LD 50 LD 50 Compound 1 Compound 2 Rabbits Mice

– – – X = monovalent, X = C 6H6)4B polyvalent anion Compound

- ND [24] N 4 X N N N + + + + O N (CH 2)10 N (CH 2)10 N (CH 2)10 N O 8 μg/kg 32 μg/kg O O

1 R, R = CH 3 Z =

N Compound 1

3-dimethylcarbamoxyphenyl

2-dimethylcarbamoxybenzyl

N N

3-dimethylcarbamoxy- a-picolinyl 1 R, R = CH 3 Z = 28 μg/kg 18 μg/kg

When Z = O O O Z = R R O N N + + O N N a Z N - N Z 3-dimethylcarbamoxy- -picolinyl 1 1 2 X 3-dimethylcarbamoxy- a-picolinyl N [25] R R R,R 1 combine to 3-dimethylcarbamoxyphenyl R,R 1 combine to form form – – X = Br + N + H C N 3 Compound 2 H3C pyrrolidinio pyrrolidinio – – + X = Br N H3C 3-pyrrolinio

+ N H3C piperidinio

+ N O 10 μg/kg 22 μg/kg H3C morpholinio X– = monovalent, polyvalent anion

O O n = 3-16 - Compound N O 2 X O N X– = halides, n= 8 − – – – HC 2O4 , ClO 4 , X = Br ND [26] – – + + NO 3 , (C 6H6)4B , N N – HSO 4 16 μg/kg 6 μg/kg n

1 - R, R = CH 3, C 2H5, N Compound 2 X n-C 3H7, i-C 3H7, 1 R, R = CH 3 O O O n-C 4H9 R n = 1 ND [27] + + (CH 2)n n = 1-9 – – N N – X = Br 1 OH X = monovalent, R 17 μg/kg 32 μg/kg N 8 polyvalent anion

9 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Toxicity a Substituents i.v. injection Compounds Compound (μg/kg) Ref. Studied LD 50 LD 50 Compound 1 Compound 2 Rabbits Mice

1 2 3 R , R , R = CH 3, O C2H5, n-C 3H7, Compound - i-C 3H7, n-C 4H9 1 2 3 N O 2 X R , R , R = CH 3 n = 5-16 – n = 10 ND [28] X = halides, – – + R − – X = (C 6H6)4B N + 1 HC 2O4 , ClO 4 , – – (CH 2)n N R NO 3 , (C 6H6)4B , 25 μg/kg ND 2 – R HSO 4

N - Compound X – O O X = monovalent, – – X = Br ND [29] + polyvalent anion N Br 80 μg/kg 45 μg/kg N 8

LD 50 MED 50 m =2-6 (i.v., mice) (i.v., mice) - O n = 6-16 2 X – m = 2 + N X = halides, [30, N + − – n = 10 ND HO HC 2O4 , ClO 4 , – - 31] (CH 2)n N (CH 2)m O – – X = Br NO 3 , (C 6H6)4B , 560 μg/kg 56 μg/kg – HSO 4

N - 2 X n = 4-14 – O O X = halides, n = 8 d − – – – ND NR NR [32] + + HC 2O4 , ClO 4 , X = Br – – N N NO 3 , HSO 4 N n

a LD 50 – Lethal dose producing 50% mortality usually by intravenous injection (i.v.); MED 50 – minimum effective dose for 50% of the population. b ND = Not determined c The lowest and highest toxicities were selected as examples for this compound as a range of toxicities were reported. d NR = Not reported

Structure-Toxicity Relationships animals to die. There was no discussion of morbidity effects of these compounds at sub lethal doses. The mode of action of these compounds is Additional studies that examine clinical effects at sub unknown and was not discussed in the patents. lethal doses might help inform the mode of action The toxicity data in Table 1 are limited to and structure-toxicity relationships more accurately. the intravenous injection (i.v.) of the compound and Even with this limited toxicity data set, one can noting the concentration of the compound required observe common characteristics of those compounds to kill 50 % of the animals (LD 50 in rabbits or mice). that were the most toxic in rabbits. The data in Table There are no data showing how long animals were 1 (and subsequent tables) are ranked by their relative observed, their symptoms, nor how long it took toxicity in rabbits because rabbits are the more

10 Ball: Quaternary Ammonium Salts – Possible Chemical Agents sensitive of the two animal models (rabbits and mice) methylene (or the equivalent) units (see Table 2 for studied. There are no data to suggest which animal a specific change in toxicity as a function of model might be more representative of human increasing methylene units) [3]. One additional exposure, especially since humans would likely be structural similarity among the more potent exposed via inhalation or skin contact, not by compounds is the requirement for an aromatic ring an intravenous injection. Three structural features are with two substituents in the ortho position. readily apparent from an inspection of Tables 1 and The aromatic ring can be a picolinic quaternary amine 2. One obvious and well known feature of these or a benzylic quaternary amine with the quaternary compounds are the requirement for two quaternary amine ortho to a dimethylcarbamoxy functional amines for optimal lethality. This is no surprise given group (Figure 9). These common structural the structure of well known toxic compounds such as characteristics are found in the first 16 patents shown decamethonium (Figure 5), succinylcholine (Figure in Table 1. The dimethylcarbamoxy functional group 2), tubocurarine (Figure 1), and pancuronium is similar to the well known pryidostigmine (Figure (Figure 3). The compounds shown in Table 1 7), a compound used to protect acetylcholinesterase appear to be more similar to decamethonium and by forming a temporary enzyme-carbamate that succinylcholine compared to the curare derived subsequently hydrolyzes to yield an active compounds (tubocurarine and pancuronium). acetylcholinesterase [36]. It is not known if these The flexibility of these compounds in binding to compounds participate in the transfer of a carbamate the receptors is thought to be a key structural residue to an appropriate nucleophile in the receptor feature possibly causing depolarization of or if they participate in hydrogen bonding within the endplate of the neuromuscular junctions [3]. the receptor. A second aromatic ring (pyridinyl or These compounds tend to bind tightly to their phenyl) near the second quaternary amine is not receptor keeping the endplate from repolarizing and required for enhanced lethality as suggested by blocking nerve transmission. The optimal distance structures that do not have this feature and have between the two charges appears to be 8-10 simple alkyl substituents.

Table 2. LD 50 toxicity of bis (dimethyl)carbamoxypyridine derivatives with spacing of 1-9 methylene groups.

i.v LD 50 Compound [21] n, X – (µg/kg) Rabbits Mice 1, Br – >20,000 >32,000 2, Br – 5,600 3,200 3, Br – 56 63 N - N 2 X 4, Br – 17.6 17.8

O O O O – 5, Br 5.6 13 + + N N 6, Br – 2.6 6.3 N N n 7, Br – 2.7 11 8, Br – 4.2 10 8, I – 5 10 9, B r– 5 9

A key structural feature of any quaternary nicotinic receptors is about 5.9 Å while the optimal ammonium compound that binds to muscarinic or distance between charges for muscarinic receptors is nicotinic receptor sites is the relative spacing between 4.3-4.4 Å. Although molecular models have not the positively charged ammonium group and made and the optimal geometries have not been an electron-donating functional group (e.g. carbonyl estimated for the compounds shown in Tables 1-3, it oxygen) that forms a hydrogen bond to the receptor appears that many of the compounds described by [38]. The optimal distance for compounds binding to these patents will have the appropriate geometry

11 Ball: Quaternary Ammonium Salts – Possible Chemical Agents and/or flexibility for either the muscarinic or nicotinic binding sites, depending on the number OH O of spacing groups and their rigidity. N O

O N - O Figure 10. Figure 10. 3-Quinuclidinyl benzilate 1 X R 1-azabicyclo[2.2.2]oct-3-yl 2-hydroxy-2,2-diphenylacetate + N N (CH 2)n R Table 1 potentially have the optimal geometries for Dimethylcarbamoxy functional group ortho binding to nicotinic or muscarinic acetylcholine to the picolinic quaternary ammonium group receptors but have poor lipid solubility characteristics. It is possible that these compounds could be modified O N to enhance their lipid solubility and their overall - absorption while maintaining a structure that binds to O X 1 acetylcholine receptors. One such set of compounds R + would be the analogous tertiary amines instead of N (CH 2)n the positively charged, quaternary amines (Table 3). R It is well known that at physiological pH (~pH 7.4) Dimethylcarbamoxy functional group ortho a large fraction of the tertiary amines in Table 3 would to the benzylic quaternary ammonium group be positively charged. These compounds would have the potential to be as toxic as the parent quaternary Figure 9. Picolinic or benzylic ammonium carbamate amino compound because a significant fraction of structures common to agents in Table 1. the tertiary amines would be positively charged. Yet these compounds might have enhanced lipid solubility because a fraction of the tertiary amines Tertiary Amine Derivatives – Possible would not be charged and, in this form, would be Lipophilic Analogs better able to cross membranes, including potentially the blood-brain barrier membrane. The idea that The bisquaternary ammonium structure of these tertiary amines can have profound toxic nerve effects compounds plays an important role in binding of the is illustrated by the well known incapacitating agent compounds to acetylcholine receptors. However, these 3-quinuclidinyl benzilate (Figure 10) and other positively charged amines also make the compounds compounds [39 -41]. The extrapolation of quaternary more water soluble which inhibits their movement amines to tertiary amines is a logical extension of across membranes. Thus, the absorption of the evaluation of quaternary amines and if one the compounds via inhalation, ingestion or through assumes quaternary amines are potential chemical dermal exposure is confounded by these positively weapon threats, one should also make a similar charged amines. Some of the compounds shown in assumption for the analogous tertiary amines.

O O O O O O hexamethylene diacrylate

O O O O O + + AgO, H O, heat N O O N 2 O

O O O cistracurium O O N O laudanosine

Figure 11. Example of the Hofmann elimination reaction

12 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Forensic Identification potential compounds and the complexity of the mass spectra, it is unlikely that GC/MS or GC/LC/MS will There are many reasons for wanting to identify be able to identify these compounds. It makes sense nerve agents after an attack (or preferably before an then, to develop a method that first identified attack occurs) including the development of antidotes the biological target of the compound and then tailor (if feasible), general management/treatment of the the chemical analysis for final identification of symptoms and the identification of the perpetrators. the compound. In the case of agents that bind to It is likely that people exposed to sub lethal doses of the acetylcholine receptor, it would be prudent to these nerve agents will require a different treatment develop a very sensitive and selective assay for these protocol than used for active site inhibitors of compounds so that even a small sample would reveal acetylcholinesterase that is typically found using the primary mode of action of the compound. For organophosphate nerve agents. example, a bioassay using isolated acetylcholine receptors (e.g. the isolation of nicotinic acetylcholine receptors from Torpedo californica ) [42] and a radioactively labeled compound that binds to Bioassay for Acetylcholine Receptor Binding the acetylcholine receptor is feasible, sensitive and selective for this class of compounds. Thus, a small It is evident that there are a very large number of amount of sample containing one the compounds in compounds (Tables 1-3 and Table 5) that have this paper would compete for the binding site on the potential to be used as chemical weapons; it will the acetylcholine receptor releasing a radioactively likely be every difficult to narrow down the structure labeled compound that could be detected using of an agent to a specific compound. There are several conventional methods. Other strategies are also possible approaches to identifying compounds of possible, but the development of such assays is the type presented in this paper. In a typical scenario, beyond the scope of this study. Once the assay only small amounts of sample, perhaps bound to suggests that one of the compounds in this paper is clothing or swabs taken from the scene of attack, will being used, one can employ other tools to help be available for analysis. Given the large number of identify the compounds.

Table 3. Potential tertiary amines

Substituents in original quaternary amine proposed Original Quaternary Amine Proposed Tertiary Amine (if possible) Ref. for the analogous tertiary amine compound

N - N N N 2 X R = CH 3, C 2H5, n-C 3H7, i-C 3H7, n-C 4H9, i-C 4H9, OOO O OOO O [8] R O O R R O O R + + n-C 5H11 , n-C 6H13 N N N N 1 1 n = 2-12 R R N n N N n N

N - N N N 2 X R = CH 3, C 2H5, n-C 3H7, O O O O O O O O i-C 3H7, n-C 4H9, C 6H5CH 2 [9] R R R R + + N N N N n = 3-14 1 1 R R N n N N n N

R’ = CH 3, C 2H5, n-C 3H7, - C2H5OH N 2 X 1 2 N 1 2 1 1 R R R R R R R , R ’= H, CH 3, C 2H5 N + + N 2 2 [10] O N (CH 2)10 N OH O N (CH 2)10 N OH R , R ’ = H, OH, CH 3, 1 2 1 2 O R´ R ´ n R ´ O R ´ n R ´ C2H5, n-C 3H7 n = 1-8

13 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Substituents in original quaternary amine proposed Original Quaternary Amine Proposed Tertiary Amine (if possible) Ref. for the analogous tertiary amine compound

+ N R A simple analogous compound is not a + 1 NA [11] (CH 2)n N R possible due to heterocyclic amino group O O - 2 2 X R N

1 R, R = CH 3, C 2H5, n-C 3H7,

ZZ ZZ i-C 3H7, n-C 4H9, i-C 4H9, R O O R O O + + R R n-C 5H11 , n-C 6H13 N N N N O 1 1 O O O n = 2-12 [12] R R - n N n N N 2 X N Z = H, CH 3, C 2H5 O O O O dimethylcarbamoxy group in O,M,P positions

N - N N N 2 X R = CH 3, C 2H5, n-C 3H7, O O O O O O O O R R R R i-C 3H7, n-C 4H9 [13] + + N N N N n = 3-14 1 1 R R n n

- R = C 2H5, n-C 3H7, n-C 4H9 N 2 X N Z = C 2H5, n-C 3H7, n-C 4H9 R R N + + N When Z = n-C 8H17 , [14] O N (CH 2)10 N Z N (CH ) N Z 1 O 2 10 n-C 12 H25 , C 6H11 O R O then R = CH 3, C 2H5

- N 2 X N R = CH 3, C 2H5, n-C 3H7, R R N + + N i-C 3H7, n-C 4H9 [15] O N (CH 2)10 N (CH 2)n N O N (CH 2)10 N (CH 2)n N 1 n =1-8 O R O

- N 2 X N N N R = CH 3, C 2H5, n-C 3H7, R R N + + N i-C 3H7, n-C 4H9, i-C 4H9, [16] O N (CH 2)10 N OH O N (CH 2)10 N OH 1 n-C 5H11 , n-C 6H13 O R O

- N 2 X Simple analogous compounds are not N + + NA [17] O N (CH 2)10 N possible due to heterocyclic amino group O R

14 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Substituents in original quaternary amine proposed Original Quaternary Amine Proposed Tertiary Amine (if possible) Ref. for the analogous tertiary amine compound

When NRR 1R2 =

+ N

+ N - N 2 X OH Simple analogous compounds are not + R N N + + 1 possible due to heterocyclic [18] O N (CH 2)10 N R 2 amino groups O O R + N H3C

+ N H3C

+ N H3C OH

- R = CH 3, C 2H5 N - 2 X 1 2 N 2 X 1 1 2 2 1 2 R , R ’, R , R ’ = H, CH 3 RRR N RRR N + [19, + Z = CH 3, C 2H5, n-C 3H7, O N (CH 2)10 N O O N (CH 2)10 N O 1 2 1 2 20] O R´ R ´ R ´ Z O R ´ R ´ Z n-C 4H9, n-C 5H11 O O

N - N N N 2 X O O O O O O O O n = 1-9 [21] + + N N N N N n N N n N

- 1 2 X R, R , = CH 3, C 2H5, n-C 3H7, N R R i-C 3H7, n-C 4H9 + + 1 N 1 [22] O N (CH 2)n N R N (CH ) N R n = 5-16 2 O 2 n O R O

R = CH 3, C 2H5, n-C 3H7, N - N 2 X i-C 3H7, n-C 4H9 O O O O O R R + + CH3 [23] N N N N ON 1 N N N R N CH3 8 O 8 O O O Dimethyl carbamoxy group in o, m. or p

- N 4 X N N N N N N N + + + NA [24] + O N (CH 2)10 N (CH 2)10 N (CH 2)10 N O O N (CH ) N (CH 2)10 N (CH 2)10 N O 2 10 O O O O

15 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Substituents in original quaternary amine proposed Original Quaternary Amine Proposed Tertiary Amine (if possible) Ref. for the analogous tertiary amine compound

R = CH 3 Z = O

3-dimethylcarbamoxyphenyl

2-dimethylcarbamoxybenzyl

N N

3-dimethylcarbamoxy- a-picolinyl When Z = O

O O O O N N R R R R [25] + + 3-dimethylcarbamoxy- a-picolinyl Z N - N Z Z N N Z 1 1 R 2 X R Simple analogous compounds are not possible due to heterocyclic amino groups when R,R 1 combine to form

+ N H3C pyrrolidinio

+ N H3C 3-pyrrolinio

+ N H3C piperidinio

+ N O H3C morpholinio

O O - N O 2 X O N A simple analogous compound is not NA [26] possible due to heterocyclic amino groups + + N N n

N - N 2 X R = CH 3, C 2H5, n-C 3H7, O O O O O O i-C 3H7, n-C 4H9 R R [27] + + (CH 2)n (CH 2)n n = 1-9 N N 1 OH N N OH R N 8 N 8

O - N O 2 X A simple analogous compound is not NA [28] + R possible due to heterocyclic amino group N + 1 (CH 2)n N R 2 R

16 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Substituents in original quaternary amine proposed Original Quaternary Amine Proposed Tertiary Amine (if possible) Ref. for the analogous tertiary amine compound

N - N X O O O O None [29] + N Br N Br N 8 N 8

- O 2 X A simple analogous compound is not [30, + N NA N + possible due to heterocyclic amino group 31] HO (CH 2)n N (CH 2)m O

N - N 2 X O O O O n = 4-14 [32] + + N N N N N N n n a NA = Not applicable

Hofmann Elimination Reaction in the acetylcholine receptor assay, this would suggest that quaternary amines were the primary One novel tool for aiding in the identification of class of nerve agents present in the sample. It is also quaternary and tertiary amines is through possible that one of the tertiary amines (Table 3) is the identification of products from the Hofmann the nerve agent responsible. Exhaustive methylation elimination reaction. As discussed previously, of the tertiary amine with methyl iodide followed the compounds in Table 1 are all quaternary amines by the Hoffman elimination reaction would help and many are susceptible to the Hofmann elimination provide evidence that tertiary amines were the class reaction. The Hofmann elimination reaction of of nerve agents being used. The use of the Hofmann cisatracurium (See Figure 11 for an example of the elimination reaction also has potential forensic Hofmann elimination reaction [43]) proceeds benefits in that many of the alkenes, dienes and spontaneously at physiological pH (pH 7.4) and amines shown in Table 4 would be identifiable in vivo. The reaction proceeds by elimination of the using conventional GC/MS with or without quaternary ammonium group yielding a double derivatization. A conclusive identification of the bond from the carbon containing the fewest nerve agent responsible may not be possible substituents (e.g. CH 3 > CH 2 > CHR). Hofmann because of the wide range of compounds, the elimination products, in this example, would likely expected complex pattern of the mass spectra and yield hexamethylene diacrylate with physical the very small amounts of sample available for properties more amenable to analysis by analyses. However, a conclusive identification may conventional methods such as GC/MS. Some of the not be required for many of the more simple Hofmann elimination products of those treatment/preventative objectives once the compound compounds listed in Table 1 are shown in Table 4. has been detected as a chemical that binds strongly If a sample from a suspected nerve agent attack was to acetylcholine receptors. On the other hand, it may subjected to conditions of the Hofmann elimination be prudent to begin developing a mass spectral reaction and the sample was no longer positive library of all of the compounds discussed in this

17 Ball: Quaternary Ammonium Salts – Possible Chemical Agents paper. The Chemical Weapons Convention treaty would have developed a bisquaternary amine or a allows the synthesis of small amounts (less than tertiary amine different than those in Tables 1-3, 100 g) of agents that could be used for this 5; a library of mass spectra of known chemicals purpose. There is also the possibility, however, that that bind to acetylcholine receptors would not be a foreign government, doing their own research, helpful in this situation.

Table 4. Possible Hofmann elimination products of quaternary ammonium salts a

Compound Tertiary amine Alkene Substituents Ref.

1 N - N R, R = CH 3, C 2H5, 2 X n-C 3H7, i-C 3H7, OOO O Products complex; loss R O O R Alkenes n-C 4H9, i-C 4H9, [8] + + of small chain alkenes N N n-C 5H11 , n-C 6H13 1 1 R R N n N n = 2-12

N - N N 1 2 X R, R = CH 3, C 2H5, n-C 3H7, i-C 3H7, O O O O O O Dienes R R n-C 4H9, C 6H5CH 2 [9] + + R n-Alkenes N N n = 3-14 1 1 N R R 1 N n N N R

N R, R’ = CH 3, C 2H5, N n-C 3H7, C 2H5OH - 1 1 N 2 X 1 2 O N R , R ’= H, CH 3, R R R [10, N + + O C2H5 2 2 33] O N (CH 2)10 N OH 1 2 1 1,9-Decadiene R , R ’ = H, OH, 2 R R R O R´ R ´ n R ´ b.p. 169 °C CH 3, C 2H5, n-C 3H7 N OH 1 2 n-Alkenes n = 1-8 R´ R ´ n R ´

1 2 3 + R , R , R = CH 3, N N R Dienes C2H5, n-C 3H7, + 1 [11] (CH 2)n N R O O R n-Alkenes i-C 3H7, n-C 4H9 O O - 2 1 2 X R NR n = 5-16 2 N N R

1 R, R = CH 3, C 2H5, n-C 3H7, i-C 3H7, ZZ R O O R n-C 4H9, i-C 4H9, + + N N Products complex; loss n-C 5H11 , n-C 6H13 O 1 1 O Alkenes [12] R R - n of small chain alkenes n = 2-12 N 2 X N O O Z = H, CH 3, C 2H5 dimethylcarbamoxy group in o, m, p positions

N - N N 1 2 X R, R = CH 3, C 2H5, O O O O O O Dienes n-C 3H7, i-C 3H7, R R [13] + + R n-Alkenes n-C 4H9 N N N 1 1 n = 3-14 R R 1 n R

18 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Compound Tertiary amine Alkene Substituents Ref.

1 R, R = C 2H5, N - n-C 3H7, n-C 4H9 N N 2 X Z = C 2H5, n-C 3H7, R O N N + + n-C 4H9 [14] O O N (CH 2)10 N Z 1,9-Decadiene 1 When Z = n-C 8H17 , O R R b.p. 169 °C N Z n-C 12 H25 , C 6H11 then 1 n-Alkenes R R, R 1 = CH 3, C 2H5

N 1 - N R, R = CH 3, C 2H5, N 2 X O N R n-C 3H7, i-C 3H7, N + + O [15] n-C 4H9 O N (CH 2)10 N (CH 2)n N 1,9-Decadiene 1 R O R b.p. 169 °C n =1-8 N (CH ) N 1 2 n n-Alkene nitriles R

N N 1 - O N R, R = CH 3, C 2H5, N 2 X N O n-C 3H7, i-C 3H7, N R [16] + + 1,9-Decadiene n-C 4H9, i-C 4H9, O N (CH 2)10 N OH N 1 R n-C 5H11 , n-C 6H13 O R b.p. 169 °C N OH n-Alkenes 1 R

N - N N 2 X R = H, OH, CH 3, O N N + + O CHNOH in the o, m [17] O N (CH 2)10 N 1,9-Decadiene and p positions R O N b.p. 169 °C R

N N O N NRR 1R2 = O R 1 N R 2 R + N N + - N N 2 X N R OH N + 1 + OH + [18] O N (CH 2)10 N R N 2 1,9-Decadiene O R N b.p. 169 °C O O + N H3C H3C N + N H3C H3C N + N H3C H C N 3 OH OH

N R, R’ = CH 3, C 2H5 - N 1 1 2 2 N R , R ’, R , R ’ = H, 2 X 1 2 O N RRR CH 3 N + + O 1 2 [19,20] O N (CH 2)10 N O 1,9-Decadiene Z = CH 3, C 2H5, 1 2 R R R O Z R´ R ´ R ´ N O b.p. 169 °C n-C 3H7, O 1 2 R´ R ´ R ´ Z n-Alkenes n-C 4H9, n-C 5H11 O

19 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Compound Tertiary amine Alkene Substituents Ref.

N - N N 2 X O O O O O O Dienes n = 1-9 [21] + + N N N N n N N

1 2 3 - N R , R , R = CH 3, 2 X O N Dienes C2H5, n-C 3H7, N R + + 1 O [22] N (CH ) N R n-Alkenes i-C 3H7, n-C 4H9 O 2 n R 2 1 n = 5-16 O R N R 2 R

N 1 R,R = CH 3, C 2H5, N - n-C 3H7, i-C 3H7, 2 X O O n-C 4H9 O O N R Dimethyl carbamoxy + + N [23] N N 1,9-Decadiene group in o, m. or p 1 N N R R b.p. 169 °C O 8 O N 1 N CH R n-Alkenes 3 O O ON O CH3

- N 4 X N N 1,9-Decadiene N N + + + + N NA [24] N (CH ) N (CH ) N O b.p. 169 °C O N (CH 2)10 2 10 2 10 O N O O O N 1-dimethylamino-9-decene

Products complex if reaction ocurrs at all; when R, R 1 = CH 3 and Z = O

3-dimethylcarbamoxyphenyl

2-dimethylcarbamoxybenzyl Hofmann O elimination N N products complex – a O O 3-dimethylcarbamoxy- -picolinyl ring-opening of R R heterocyclic amines + + Products complex when Z = ND [25] Z N - N Z O is likely when 1 2 X 1 R R N N Z = O 3-dimethylcarbamoxy- a-picolinyl N N and R,R 1 combine to form 3-dimethylcarbamoxy- a-picolinyl + N H3C pyrrolidinio

+ N H3C 3-pyrrolinio

+ N H3C piperidinio

+ N O H3C morpholinio

20 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

Compound Tertiary amine Alkene Substituents Ref.

O O O - N O 2 X O N N O Dienes n = 4-16 [26] + + N N N n

N 1 N - R, R = CH 3, C 2H5, 2 X O O 1,9-Decadiene n-C 3H7, i-C 3H7, O O O N R b.p. 169 °C n-C 4H9 [27] + + (CH 2)n N N N 1 OH n-Alkene n = 2-9 R N 8 R O carboxylic acids N (CH ) 1 2 n n-Alkenes R OH

O O - N O 1 2 3 N O 2 X R , R , R = CH 3, Dienes C2H5, n-C 3H7, [28] N + R n-Alkenes i-C 3H7, n-C 4H9 N + 1 n = 5-16 (CH 2)n N R R 2 1 R NR2 R

N - X N Alkene bromide O O O O likely to react with NA [29] + N Br N AgO N 8 N

- N 2 X O HO Dienes + m =2-6 N + N n-Alkene [30, 31] O HO (CH 2)n N (CH 2)m O n = 6-16 N dimethylcarbamates N (CH 2)n O

N - N 2 X O O O O Dienes n = 4-14 [32] + + N N N N n N a Representative amines possible from the Hofmann eliminate reaction, not a comprehensive list as some of the amines can be quite complex due to elimination of alkenes from substituents bound to the tertiary amine. ND = Not determined NA = Not applicable

Other Studies therapeutic potential to cause muscle relaxation while attempting to minimize the toxicity of the compounds. An update on other quaternary amines similar to It is apparent from inspection that the compounds those in Table 1 is illustrated in Table 5 [44]. Two synthesized are remarkably similar to those in the oxopyrimidine structures were studied for their U.S. Patents. The synthesis of these compounds

21 Ball: Quaternary Ammonium Salts – Possible Chemical Agents basically follows the methods outlined in the U.S. They measured the relative toxicity of their Patents; the synthetic methods are extremely simple compounds using intraperitoneal (i.p.) injections with high yields and essentially no purification compared to intravenous injections (i.v.) in the U.S. required. The study also reported chemical Patents. The toxicity of these oxopyrimidines were characterization using infrared and 1H nuclear much less toxic (mg/kg; on the order of 100-1000 magnetic resonance spectra; they were able to verify times less toxic) than the compounds in the U.S the structure of their products. This paper reported on Patents (ug/kg). This maybe due to a lower inherent biological effects not studied in the U.S. Patents. For toxicity or could reflect the route of exposure since example, they measured the binding constant of these i.p. injections would require these compounds compounds to nicotinic acetylcholine receptors and to cross membranes to enter systemic circulation compared their results with well known antagonists where they could reach neuromuscular junctions. of these receptors (tubocurarine, hexamethonium and The authors also tested to see if these compounds decamethonium). One set of oxopyrimidines binds inhibited acetylcholinesterase; there was no almost as effectively as tubocurarine when inhibition of this enzyme as expected based on the spacing between the positively charged the hypothesized mode of action of these ammonium groups is about 10 methylene groups. compounds.

Table 5. Structure and biological effects of oxopyrimidinyl bisquaternary amines (Zobov et al. 2004).

c Myorelaxant Substituent LD 50 , i.p. a b Activity Compound pK B pI 50 Mice d MW ED 50 n m (mg/kg) (g/mole) (mg/kg) 3 6 694.5 5.2 3.0 45 15.0 4 6 722.6 4.7 3.4 20 6.5 5 6 750.7 <4.0 4.0 20 4.0 - O 2 Br O 6 6 778.7 5.8 4.1 15 3.0 N N + + O N (CH 2)n N (CH 2)m N (CH 2)n N O 7 6 806.8 5.6 4.1 15 2.2 3 10 750.7 5.8 3.2 5.0 2.0 4 10 778.7 5.8 4.3 4.5 1.5 6 10 834.9 6.2 4.9 5.0 1.7 7 10 862.9 5.9 4.8 7.0 2.2 - 2 Br 2 6 636.5 ND 3.0 7.8 1.6 + + O (CH 2)n N (CH 2)m N (CH 2)n O 3 6 664.5 6.4 4.4 7.0 2.0 N N 2 10 692.6 8.0 4.1 3.1 0.8 H2N NH2 N N 3 10 720.6 8.1 4.2 4.0 1.2

- 2 Br + + N N NA NA 362.2 ND <3.0 120 25.0

Hexamethonium dibromide

OH O + N O O NA NA 624.8 8.9 <3.0 0.42 0.2 + OH N H O d-Tubocurarine chloride - 2 Br + + N N NA NA 418.3 ND <3.0 4.0 0.6 Decamethonium bromide a pK b = negative log of the dissociation constant for binding of the compound to nicotinic acetylcholine receptors from frog abdominal muscles. b pI 50 = negative log of the concentration of the compound causing 50% inhibition of human erythrocyte acetylcholinesterase. c LD 50 = dose of the compound causing mortality in 50% of the mice tested. d ED 50 = effective concentration of the compound that causes myorelaxation (inability to perform standard tretbahn test for 30 minutes) in 50% of the mice tested. ND = not determined.

22 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

SUMMARY 8. Sommer, H. Z.; Wicks, G. E. Jr.; Owens, O. O. Chemical agents. U.S. Patent 4,246,416, 1981 . This paper has systematically reviewed a number 9. Sommer, H. Z.; Owens, O. O. Chemical agents. of bisquaternary amines that could be potentially U.S. Patent 4,686,293, 1987 . deadly nerve agents. Presumably, these agents would 10. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. function by blocking the effect of acetylcholine by U.S. Patent 4,241,212, 1980 . binding to acetylcholine receptors and stopping nerve 11. Sommer, H. Z.; Wicks, G. E., Jr. Unsymmetrical transmission. Analogous tertiary amines, based on pyrrolino benzyl quaternary compounds. U.S. the structure of the parent bisquaternary amines, have Patent 4,240,965, 1980 . been proposed as compounds that might be more 12. Sommer, H. Z.; Wicks, G. E., Jr.; Owens, O. O. soluble in membranes making them more Ketobenzylcarbamates. U.S. Patent 4,677,222, 1987 . bioavailable and toxic. Methods have been discussed 13. Sommer, H. Z.; Owens, O. O. Chemical agents. that could identify this class of compounds using U.S. Invention Registration H443, 1988 . a bioassay for binding to the acetylcholine receptor. 14. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. A tool in the identification of these compunds, U.S. Patent 4,672,123, 1987 . the Hofmann elimination reaction, has been proposed 15. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. as a novel method for helping to establish that U.S. Patent 4,672,124, 1987 . quaternary or tertiary amines are functional groups 16. Sommer, H. Z.; Wicks, G. E., Jr. Picolyl of the nerve agent. Finally, other studies unsymmetrical bis-quaternary carbamates. U.S. on the development of similar compounds were Patent 4,246,415, 1981 . presented. 17. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. U.S. Patent 4,241,210, 1980 . 18. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. U.S. Patent 4,672,119, 1987 . REFERENCES 19. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. U.S. Patents 4,672,122, 1987 . 1. Conti-Tronconi, B. M.; Raftery, M. A., Nicotinic 20. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. acetylcholine receptor contains multiple binding U.S. Patent 4,672,069, 1987 . sites: evidence from binding α- dendrotoxin. Proc. 21. Sommer, H. Z.; Krenzer, J.; Owens, O. O.; Miller, Natl. Acad. Sci. 1986 , 83, 6646-6650. J. L. Chemical agents. U.S. Patent 4,677,204, 2. Sobell, H. M.; Sakore, T. D.; Tavale, S. S.; 1987 . Canepa, F. G.; Pauling, P.; Petcher, T. J., 22. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. Stereochemistry of a curare alkaloid: O,O', N- U.S. Patent 4,241,218, 1980 . trimethyl-d-tubocurarine. Proc. Nat. Acad. Sci. 23. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. 1972 , 69, 2212-2215. U.S. Patent 4,241,211, 1980 . 3. Raghavendra, T., Neuromuscular blocking drugs: 24. Sommer, H. Z.; Wicks, G. E., Jr.; Witten, B. discovery and development. J. Royal Soc. Med. Chemical agents. U.S. Patent 4,672,120, 1987 . 2002 , 95, 363-367. 25. Sommer, H. Z.; Chemical agents. U.S. Patent 4. Kuca, K.; Hrabinova, M.; Soukup, O.; Tobin, G.; 4,692,530, 1987 . Karasova, J.; Pohanka, M. Pralidoxime-the gold 26. Sommer, H. Z.; Owens, O. O.; Miller, J. L. standard of acetylcholinesterase reactivators - Isoquinilinium chemical agents. U.S. Patent reactivation in vitro efficacy. Bratisl. Lek. Listy. 4,673,745, 1987 . 2010 , 111, 502-504. 27. Sommer, H. Z.; Wicks, G. E., Jr. Unsymmetrical 5. Lee, C.; Jones, T., Molecular conformation- bis-quaternary amino acids. U.S. Patent activity relationship of decamethonium 4,246,418, 1981 . congeners. Br. J. Anaesth. 2002 , 88, 692-699. 28. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agents. 6. Paxman, J.; Harris, R. A., Higher Form of Killing: U.S. Patent 4,241,209, 1980 . The Secret History of Chemical and Biological 29. Sommer, H. Z.; Wicks, G. E., Jr. Chemical agent. Warfare. Hill and Wang, New York 1982, pp. 53-67. U.S. Patent 4,677,205, 1987 . 7. Mumford, H. E.; Price, M., Lenz, D. E.; Cerasoli, 30. Sommer, H. Z.; Miller, J. L. Haloalkyl- D. M. Post-exposure therapy with human carbamoxyalkyl derivatives. U.S. Patent butyrylcholinesterase following percutaneous 3,956,365, 1976 . VX challenge in guinea-pigs. Clin. Toxicol. 2011 , 31. Sommer, H. Z.; Miller, J. L. Hydroxyquinuclidine 49, 287-97. derivatives. U.S. Patent 3,919,241, 1975 .

23 Ball: Quaternary Ammonium Salts – Possible Chemical Agents

32. Sommer, H. Z.; Miller, J. L. Chemical agents. 40. 3-Quinuclidnyl benzilate; Expert Meeting: U.S. Patent 4,675,411, 1987 . Incapacitating Chemical Agents: Implications for 33. Physical properties of 1,9-decadiene International Law, Montreux, Switzerland, March http://www.sigmaaldrich.com/catalog/ProductDe 2010 87 pp tail.do?D7=0&N5=Product%20No.%7CBRAND http://www.icrc.org/eng/assets/files/publications/i _KEY&N4=347841%7CAldrich&N25=0&QS= crc-002-4051.pdf ON&F=SPEC 41. Kawai, H.; Carlson, B. J.; Okita, D. K.; Raftery, 34. Library of Congress, Thomas, Chemical Weapons M. A. Eserine and other tertiary amine Convention, Treaty Number 103-21, ratified interactions with Torpedo acetylcholine receptor April 12, 1997. postsynaptic membrane vesicles. Biochemistry. 35. ACS, American Chemical Society, January 2012, 1999 , 38, 134-141. The Journal of Organic Chemistry Guidelines for 42. Baenziger, J. E.; Ryan, S. E.; Goodreid, M. M.; Authors Vuong, N. Q.; Sturgeon, R. M.; daCosta, C. J. B. http://pubs.acs.org/paragonplus/submission/jocea Lipid composition alter drug action at the h/joceah_authguide.pdf Nicotinic Acetylcholine Receptor. Mol. 36. Geoghegan, J.; Tong, J. L. Chemical warfare Pharmacol. 2008 , 73, 880-890. agents. Contin. Educ. Anaesth. Crit. Care Pain. 43. Welch, R. M.; Brown, A.; Ravitch, J.; Dahl, R. The 2006 , 6, 230-234. in vitro degradation of cisatracurium, the R, cis-R'- 37. Savulescu, J.; Spriggs, M. Current controversy: isomer of atracurium, in human and rat plasma. The hexamethonium asthma study and the death Clin. Pharmacol. Ther. 1995 , 58, 132-142. of a normal volunteer in research. J. Med. Ethics. 44. Zobov, V. V.; Aslyamova, A. A.; Berezinsky, L. 2002 , 28, 3-4. A.; Reznik, V. S.; Akamsin, V. D.; 38. Beers, W. H.; Reich, E. Structure and activity of Galyametdinova, I. V.; Nafikova, A. A. Synthesis acetylcholine. Nature. 1970 , 228(5), 917-922. and biological activity of α, ω- 39. Zhang Z.; Zheng G.; Pivavarchyk M.; Deaciuc A. bis(ammonio)alkalis containing oxopyrimidinyl G.; Dwoskin L. P.; Crooks P.A. Novel bis, tris-, radicals. Pharm. Chem. J. 2004 , 38, 540-544. and tetrakis-tertiary amino analogs as antagonists at neuronal nicotinic receptors that mediate nicotine-evoked dopamine release. Bioorg. Med. Chem. Lett. 2011 , 21, 88-91.