International Journal of Molecular Sciences

Review Novichoks: The Dangerous Fourth Generation of Chemical Weapons

Tanos C. C. Franca 1,2,* , Daniel A. S. Kitagawa 1, Samir F. de A. Cavalcante 3 , Jorge A. V. da Silva 3, Eugenie Nepovimova 4 and Kamil Kuca 5,* 1 Laboratory of Molecular Modeling Applied to the Chemical and Biological Defense (LMCBD), Military Institute of Engineering, Rio de Janeiro 22290-270, RJ, Brazil; [email protected] 2 Center for Basic and Applied Research, Faculty of Informatics and Management, University of Hradec Kralove, Rokitanskeho 62, 50003 Hradec Kralove, Czech Republic 3 Brazilian Army CBRN Defense Institute (IDQBRN), Brazilian Army Technological Center (CTEx), Rio de Janeiro 23020-470, Brazil; [email protected] (S.F.d.A.C.); [email protected] (J.A.V.d.S.) 4 Department of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62, 50003 Hradec Kralove, Czech Republic; [email protected] 5 Philosophical Faculty, University of Hradec Kralove, Rokitanskeho 62, 50003 Hradec Kralove, Czech Republic * Correspondence: [email protected] (T.C.C.F.); [email protected] (K.K.); Tel.: +55-21-2546-7195 (T.C.C.F.); +420-603-289-166 (K.K.)


Received: 24 February 2019; Accepted: 6 March 2019; Published: 11 March 2019


Abstract: “Novichoks” is the name given to the controversial chemical weapons supposedly developed in the former Soviet Union between the 1970s and the 1990s. Designed to be undetectable and untreatable, these chemicals became the most toxic of the nerve agents, being very attractive for both terrorist and chemical warfare purposes. However, very little information is available in the literature, and the Russian government did not acknowledge their development. The intent of this review is to provide the IJMS readers with a general overview on what is known about novichoks today. We briefly tell the story of the secret development of these agents, and discuss their synthesis, toxicity, physical-chemical properties, and possible ways of treatment and neutralization. In addition, we also wish to call the attention of the scientific community to the great risks still represented by nerve agents worldwide, and the need to keep constant investments in the development of antidotes and ways to protect against such deadly compounds.

Keywords: Novichoks; binary weapon; nerve agents; chemical warfare

1. Introduction Despite of all the eradication efforts made by the Chemical Weapons Convention (CWC) (https://www.opcw.org/chemical-weapons-convention/), recent episodes involving nerve agents have proved that this kind of chemical weapon (CW) is still far from controlled. Some examples are the use of sarin several times in the Syrian civil war, and the assassination of Kim Jong-Nam (half-brother of the North Korea dictator Kim Jong-Un) with VX in February of 2017 (in the Kuala Lumpur airport) [1]. Further, a new threat has emerged in this scenario after the recent attempted assassination of the former Russian spy Sergei Skripal and his daughter, Yulia, on UK soil [1]. According to the Organization for Prevention of Chemical Weapons (OPCW) (https://www.opcw.org/), the chemical used in this event was the controversial nerve agent known as novichok, considered the fourth generation of the CWs [2]. This name was given to the weapons created with some compounds of the A-series nerve agents and, supposedly, secretly developed in the former Soviet Union between the 1970s and 1990s.

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Because they were not included in CWC’s list of banned chemicals, the agents of this series were kept unknown from most of the scientific community, and little was done in developing ways of preventing or treating against intoxication with them. Its recent use in the UK presents a great opportunity for warning the scientific community about the risks they represent and urging the need to develop new antidotes as well as ways of detection, protection, and neutralization.

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 11 2. Development of the A-Series Nerve Agents with them. Its recent use in the UK presents a great opportunity for warning the scientific community In his book titled: “State Secrets: An inside chronicle of the Russian chemical weapons program” [3], about the risks they represent and urging the need to develop new antidotes as well as ways of the Russiandetection, dissident protection, exiled and in neutralization. the United States, Vil S. Mirzayanov, denounces the former Soviet Union for having secretly developed novichok agents between the 1970s and 1990s. Mirzayanov is a former scientist2. who Development worked forof the 26 A-Series years at Nerve the State Agents Scientific Research Institute for Organic Chemistry and TechnologyIn in his Moscow, book titled: abbreviated “State Secrets: in RussianAn inside languagechronicle of as the GOSNIIOKhT Russian chemical (G weaponsocHII OXTprogram”in Cyrillic). He claims[3], the to haveRussian witnessed, dissident exiled between in the 1971United and Stat 1973,es, Vil S. Petr Mirzayanov, Kirpichev denounces and his the assistants former Soviet developing a new seriesUnion offor neurotoxichaving secretly agents developed derived novichok from agen thets G-series between andthe 1970s V-series and 1990s. agents, Mirzayanov this was is named a as the A-series.former According scientist who to hisworked testimony, for 26 years the first at the compound State Scientific of this Research series synthesized Institute for andOrganic tested was Chemistry and Technology in Moscow, abbreviated in Russian language as GOSNIIOKhT N-2-diethylaminomethylacetoamidido-methylphosphonofluoridate(ГоcHИИOXT in Cyrillic). He claims to have witnessed, between 1971 (Figure and 1973,1). Essentially,Petr Kirpichev it wasand a sarin derivativehis assistants where the developingO-isopropyl a new group series of was neurotoxic replaced agents by the derived acetoamydin from the G-series radical. and This V-series compound receivedagents, the codenamethis was named A-230 as the and A-series. was According referred toto his as testimony, substance the 84 first in co thempound internal of this reports series of the GOSNIIOKhT.synthesized Agent and A-230 tested presented was N-2-diethylaminomethylacetoamid a toxicity 5–8 times higher thanido-methylphosphonofluoridate the Russian VX (RVX), referred to as substance(Figure 1). 33. Essentially, However, it itwas was a sarin found derivative that it crystallizedwhere the O-isopropyl when stored group at was –10 replaced◦C[3]. Thisby the problem acetoamydin radical. This compound received the codename A-230 and was referred to as substance could be solved by adding N,N-dimethylformamide to the pure agent. After agent A-230, Petr Kirpichev 84 in the internal reports of the GOSNIIOKhT. Agent A-230 presented a toxicity 5–8 times higher than and histhe group Russian synthesized VX (RVX), and referred tested to theas substance derivatives 33. However, A-232, A-234, it was A-242,found that and it A-262crystallized (Figure when1). A-232 and A-234stored (respectively at –10 °C [3]. the This methoxy problem and could ethoxy be solved analogues by adding of A-230)N,N-dimethylformamide presented toxicity to the similar pure to RVX but wereagent. much After more agent volatile A-230, and Petr lessKirpichev stable and in hydrolysis.his group synthesized A-242 and and A-262 tested (the the derivatives guanidine A-232, analogues of A-230 andA-234, A-232, A-242, respectively), and A-262 (Figure were 1). probably A-232 and the A-234 first (respectively solid neurotoxic the methoxy agents and synthesized ethoxy analogues [3]. Duringof A-230) the presented 1980s, A-230 toxicity passed similar allto RVX field but tests, were andmuch in more 1990 volatile it was and approved less stableby in hydrolysis. the Soviet Army A-242 and A-262 (the guanidine analogues of A-230 and A-232, respectively), were probably the first as a new chemical agent that could be used in all types of ammunition. Plans for mass production solid neurotoxic agents synthesized [3]. of this compoundDuring the were 1980s, then A-230 started, passed and all field a new tests, CW and production in 1990 it was factory approved was by designedthe Soviet Army in Pavlodar, Kazakhstanas a new [3]. chemical Funding agent for that this could project, be used however, in all types disappeared of ammunition. with Plans the collapse for mass ofproduction the Soviet of Union, and A-230this wascompound never masswere then produced started, despite and a new the experimentalCW production quantities factory was (a designed few tons) in were Pavlodar, synthesized in the GOSNIIOKhTKazakhstan [3]. Funding branches for of this Volsk project, and however, Volgograd disappeared [3]. Soon with after the collapse A-230, of A-232 the Soviet also Union, passed field and A-230 was never mass produced despite the experimental quantities (a few tons) were tests and was incorporated to the Soviet Army’s arsenal. This agent had the advantages of being synthesized in the GOSNIIOKhT branches of Volsk and Volgograd [3]. Soon after A-230, A-232 also resistantpassed to cold field temperatures, tests and was incorporated and it circumvented to the Soviet the Army’s list of arsenal. chemical This agentsagent had controlled the advantages by the CWC becauseof it being belonged resistant to to the cold series temperatures, of phosphoramidofluoridates. and it circumvented the list Itof was chemical similar agents to controlled the majority by of the organophosphatesthe CWC because (OPs) it belonged used in to agriculturethe series of phosph (pesticidesoramidofluoridates. and herbicides). It was similar OPs to are the not majority included in the schedulesof the organophosphates of CWC because, (OPs) so used far, allin agriculture known OPs (pesticides usedas and chemical herbicides). weapons OPs are not are included phosphonates. As A-232in the and schedules A-234 are of CWC not phosphonates, because, so far, all they known were OPs ideal used toas chemical cheat the weapons CWC inspectorsare phosphonates. [3]. As A-232 and A-234 are not phosphonates, they were ideal to cheat the CWC inspectors [3].

O O O P N O P N O P N F N F N F N

A-230 A-232 A-234

O O P N O P N F N F N N N

A-242 A-262

Figure 1. Structures reported by Mirzayanov for the A-series agents developed in the State Scientific ResearchFigure Institute 1. Structures for Organic reported Chemistry by Mirzayanov and Technologyfor the A-series (GOSNIIOKhT) agents developed by in Petrthe State Kirpichev Scientific [3 ]. Research Institute for Organic Chemistry and Technology (GOSNIIOKhT) by Petr Kirpichev [3].

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3. Binary Weapons In chemical warfare, a binary weapon is defined as a CW where the toxic agent is produced during the flying time of the ammunition (rocket, missile, or grenade) towards the target [4]. The purpose of this kind of CW is to reduce the risks in the production, storage, transport, and even destruction of the toxic agent. This is possible because the starting materials are non-toxic (or less toxic) precursors, which are mixed to produce the chemical agent only after the ammunition is launched. For this task to be feasible the reaction has to be controlled to avoid overheating or explosion during the flight. Also, it should preferably run without solvent and has to be completed, with a high yield, in a matter of seconds. Usually the body of a binary projectile contains two separate canisters, one behind the other, each containing one starting material. The force of launching causes the breaking of the canisters, which mixes the starting materials and triggers the reaction to produce the chemical agent. The idea of designing this kind of ammunition originates from World War II [4], when military chemists developed, but never used, a binary bomb to deliver the blood agent arsine (AsH3). The blister agent N-(2-chloroethyl)-N-nitrosocarbamate (code named KB-16) was also considered as a potential binary weapon in the 1940s, but never fully developed. The first nerve agents developed as binary weapons were GB-2, GD-2, and VX-2, the binary versions of sarin, soman, and VX, respectively. They were all developed by the USA during the Cold War [5–7] and were considered the third generation of CWs [2]. GB-2 and GD-2 can be produced from the reaction between methylphosphonic difluoride (DF) and isopropyl or pinacolyl alcohol in an isopropylamine (IPA) solution (Scheme1). VX-2 is produced from the reaction of O-Ethyl O-2-diisopropylaminoethyl methylphosphonite (QL) with elemental sulfur to produceInt. J. Mol. the Sci. intermediate 2019, 20, x FOR (1) PEER that REVIEW quickly rearranges to VX (Scheme1). 4 of 11

O IPA O OH + P F P O + HF F F Isopropyl DF GB alcohol

O IPA O OH + P F P O + HF F F Pinacolyl DF GD alcohol

N N N O o O 30 - 50 oC S 130 C P + 1/8S P S 8 P O O 10 min O O QL XV (1)

Scheme 1. Production reactions of sarin (GB), soman (GD), and XV in binary weapons [5–7]. Scheme 1. Production reactions of sarin (GB), soman (GD), and XV in binary weapons [5–7]. In response to the USA binary program, the Soviet Union started the FOLIANT program in the 1980s [8,9], with the goal of also developing this kind of CW. According to Mirzayanov [3], under this program, researchers from the GOSNIIOKhT only successfullyN weaponized A-232 and O substance 33 as binary weapons, both in 1990. However, otherO sources in the literature mention P CN + N P S +HCN that A-234 was also weaponized,HS as novichok 7 [2,10,11]. Binary A-232, weaponized as novichok O 5, is reported as the product of the reaction between the methyl phosphorocyanidofluoridateO (PCF) PCN DEAT RVX

O O N O P CN + N O P N + HCN F HN F PCF NNDA A-232

Scheme 2. Production reactions of A-232 and substance 33 in novichoks # and 5 [3].

4. Other Possible Chemicals in the A-Series Due to the high level of secrecy surrounding the FOLIANT program, very little is known about the real structures of the A-series nerve agents, and many speculative structures have been reported in the literature [3,11–13]. There is some consensus that all compounds of the A-series should be derivatives of one of the three scaffolds reported by Chai et al. [11], as shown in Figure 2. However, there is much speculation about the possibilities for the groups R1, R2, and R3. Different from Mirzayanov [3], Hoenig [12] speculates that compounds A-230, A-232, and A-234 are fluorophosphonates, as shown in Figure 3, where the N atom is not directly bound to the P atom, and

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O IPA O OH + P F P O + HF F F Isopropyl DF GB alcohol

O IPA O OH + P F P O + HF F F Int. J. Mol. Sci. 2019Pinacolyl, 20, 1222 DF GD 4 of 10 alcohol and N,N-diethyl-2-iminopropan-1-amine (NNDA) (Scheme2). Binary substance 33, weaponized as novichok # [10], is produced from the reaction between 2-methylpropyl methylphosphonocyanidate N N (PCN) and 2-(diethylamino)ethanethiol (DEAT) (Scheme2N). Despite reporting the precursors, O o O 30 - 50 oC S 130 C neither MirzayanovP nor+ other1/8S literature sources give any reactional details of theseP binaryS weapons. 8 P O MirzayanovO also reported that, ironically, the former President of the10 USSR, min MikhailO Gorbachev, who O won the Nobel Peace Prize in 1990, signed the secret documents on 23 September 1989 that authorized QL XV the implementation of the project to transform A-232 into(1) a binary weapon. Almost immediately after, the USA and USSR governments signed the Wyoming Memorandum of Understanding regarding CW disarmament [3]. Scheme 1. Production reactions of sarin (GB), soman (GD), and XV in binary weapons [5–7].

N O O P CN + N P S +HCN HS O O PCN DEAT RVX

O O N O P CN + N O P N + HCN F HN F PCF NNDA A-232

Scheme 2. Production reactions of A-232 and substance 33 in novichoks # and 5 [3]. Scheme 2. Production reactions of A-232 and substance 33 in novichoks # and 5 [3]. 4. Other Possible Chemicals in the A-Series 4. Other Possible Chemicals in the A-Series Due to the high level of secrecy surrounding the FOLIANT program, very little is known about the Due to the high level of secrecy surrounding the FOLIANT program, very little is known about real structures of the A-series nerve agents, and many speculative structures have been reported in the the real structures of the A-series nerve agents, and many speculative structures have been reported literaturein the [3 literature,11–13]. There[3,11–13]. is some There consensus is some consensu that alls compounds that all compounds of the A-series of the A-series should beshould derivatives be of onederivatives of the three of one scaffolds of the three reported scaffolds by reported Chai et by al. Chai [11], et as al. shown [11], as inshown Figure in Figure2. However, 2. However, there is muchthere speculation is much about speculation the possibilities about the forpossibilities the groups for R the1,R groups2, and RR31., DifferentR2, and R from3. Different Mirzayanov from [3], HoenigMirzayanov [12] speculates [3], Hoenig that compounds [12] speculates A-230, A-232,that compounds and A-234 areA-230, fluorophosphonates, A-232, and A-234 as shownare in FigureInt. J. Mol.fluorophosphonates,3, Sci. where 2019, the 20, x N FOR atom asPEER shown is REVIEW not in directly Figure bound3, where to the the N Patom atom, is not and directly they containbound to several the P atom, halogens and 5 of in 11 the structure. Similar versions of these compounds were also reported by Ellison [14] and Patocka [13] (Figurethey contain4). several halogens in the structure. Similar versions of these compounds were also reportedIt is veryby Ellison difficult [14] to and confirm Patocka if the [13] structures (Figure reported4). in Figure2 are the authentic scaffolds of the A-seriesIt is nervevery difficult agents. Thisto confirm is due notif the only structures to the high reported level ofin secrecyFigure surrounding2 are the authentic these compounds,scaffolds of butthe alsoA-series to the nerve possibility agents. of This many is ofdue these not compoundsonly to the beinghigh fakelevel structuresof secrecy intentionally surrounding leaked these bycompounds, counter intelligence but also services.to the possibility Currently, theof CWCmany recognizesof these thecompoundsO-alkyl alkylphosphonamidatesbeing fake structures (Figureintentionally2) as possible leaked A-seriesby counter derivatives, intelligence and classifiesservices. themCurrently, as schedule the CWC 2.B.04. recognizes However, the there O-alkyl is no detailedalkylphosphonamidates information about (Figure this series 2) as of possible OP compounds. A-seriesComparing derivatives, to and existing classifies Schedule them I nerveas schedule agents listed2.B.04. in However, the CWC, there these is compounds no detailed can information be regarded about as cholinesterase this series of OP inhibitors. compounds. Therefore, Comparing exposure to toexisting them maySchedule lead to I the nerve SLUDGEM agents (Salivation,listed in the Lachrymation, CWC, these Urination, compounds Defecation, can be Gastrointestinal, regarded as Emesis,cholinesterase and Miosis) inhibitors. Syndrome Therefore, [15]. exposure to them may lead to the SLUDGEM (Salivation, Lachrymation, Urination, Defecation, Gastrointestinal, Emesis, and Miosis) Syndrome [15]. O O O R3 R3 R1 P N R1O P N R1O P O NR R2 R2 R2 3

Figure 2. PossiblePossible scaffolds for the A-series nerve agen agentsts already reported in the literature [[11].11].

Cl Cl Cl O O O O P O F O P O F O P O F F N F N F N Cl Cl Cl A-230 A-232 A-234

Figure 3. Possible A-series nerve agents, as reported by Hoenig [12].

Cl O O O O O P O Cl O P O Cl O P O F O P O F N N F F F N N N Cl Cl Cl Cl Cl

NC

O Cl O O O O P O F O P O F O P O F O P O F F N Cl N F N N F F Cl F Cl

Cl Cl O O O O O P O F O P O F O P O F O P O F N N F F F F N F N Cl NO 2 Cl Cl F

Figure 4. Possible structures of A-series nerve agents,as reported by Ellison [14] and Patocka [13].

5. Synthesis of A-series Nerve Agents Discussion about the A-series nerve agents must be done carefully. Those compounds have not been confirmed yet, and most of the information available today is speculative or comes from questionable sources. One relevant aspect of the proposed synthetic route is related to the idea behind

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theyInt. J. containMol. Sci. 2019 several, 20, x FORhalogens PEER REVIEW in the structure. Similar versions of these compounds were 5 ofalso 11 reported by Ellison [14] and Patocka [13] (Figure 4). theyIt containis very difficultseveral tohalogens confirm in if thethe structuresstructure. Similarreported versions in Figure of 2 theseare the compounds authentic scaffoldswere also of thereported A-series by Ellisonnerve agents.[14] and This Patocka is due [13] not(Figure only 4). to the high level of secrecy surrounding these compounds,It is very but difficult also toto confirmthe possibility if the structures of ma nyreported of these in Figure compounds 2 are the being authentic fake scaffolds structures of intentionallythe A-series leakednerve agents.by counter This intelligenceis due not services.only to theCurrently, high level the of CWC secrecy recognizes surrounding the O these-alkyl alkylphosphonamidatescompounds, but also to(Figure the possibility2) as possible of A-seriesmany of derivatives, these compounds and classifies being themfake asstructures schedule 2.B.04.intentionally However, leaked there by is counterno detailed intelligence information services. about Currently, this series theof OP CWC compounds. recognizes Comparing the O-alkyl to existingalkylphosphonamidates Schedule I nerve (Figure agents 2) aslisted possible in theA-series CWC, derivatives, these compounds and classifies can them be regardedas schedule as cholinesterase2.B.04. However, inhibitors. there is noTherefore, detailed informationexposure to about them this may series lead of toOP the compounds. SLUDGEM Comparing (Salivation, to existing Schedule I nerve agents listed in the CWC, these compounds can be regarded as Lachrymation, Urination, Defecation, Gastrointestinal, Emesis, and Miosis) Syndrome [15]. cholinesterase inhibitors. Therefore, exposure to them may lead to the SLUDGEM (Salivation, O R O O Lachrymation, Urination, Defecation,3 Gastrointestinal,R3 Emesis, and Miosis) Syndrome [15]. R P N R O P N R O P O 1 O 1 O 1 O R3 R3 NR R2 R2 R2 3 R1 P N R1O P N R1O P O R R R NR3 Figure 2. Possible scaffolds2 for the A-series nerve2 agents already reported2 in the literature [11]. Int. J. Mol. Sci. 2019, 20, 1222 5 of 10 Figure 2. Possible scaffolds for the A-series nerve agents already reported in the literature [11]. Cl Cl Cl O O O Cl Cl O P O F Cl O P O F O O PO O F O F N N F N O P O F O FP O F O P O F Cl Cl Cl F N F N F N A-230 Cl A-232 Cl A-234 Cl A-230 A-232 A-234 Figure 3. Possible A-series nerve agents, as reported by Hoenig [12]. Figure 3. Possible A-series nerve agents, as reported by Hoenig [12]. Figure 3. Possible A-series nerve agents, as reported by Hoenig [12]. Cl O O O O O P O Cl O P O Cl Cl O O O PO O F O OP O F N N OFP O Cl O FP O Cl F N N N Cl Cl O P O F Cl O P O F N N Cl Cl F F F N N N Cl Cl Cl Cl Cl

NC

O Cl NC O O O O POO F Cl O OP O F O PO O F O P O F N O N OFP O F N ClO P O F F O FP O F O FP ON F F N Cl N Cl F N F N Cl F F Cl F Cl

Cl Cl O O Cl Cl O O O O POO F O P O F O OP O F O OP O F N O P ON FF O FP O F F O PF ON F O PF O N F N Cl N FNO F F 2 F N Cl F N Cl Cl F NO 2 Cl Cl F FigureFigure 4. Possible 4. Possible structures structures of of A-series A-series nerve nerve agents,agents,a ass reported reported by by Ellison Ellison [14] [ 14and] and Patocka Patocka [13]. [13]. Figure 4. Possible structures of A-series nerve agents,as reported by Ellison [14] and Patocka [13]. 5. Synthesis of A-series Nerve Agents 5. Synthesis of A-series Nerve Agents 5. Synthesis of A-series Nerve Agents DiscussionDiscussion about about the the A-series A-series nervenerve agents agents must must be bedone done carefully. carefully. Those Thosecompounds compounds have not have not beenbeen confirmedconfirmedDiscussion yet,about and and the mostA-series most of of nervethe the informatio information agents mustn available be available done carefully.today today is Thosespeculative is speculative compounds or comes or have comes from not from questionablequestionablebeen confirmed sources. sources. yet, One Oneand relevant most of aspect aspectthe informatio of the of theproposedn proposedavailable synthetic today synthetic ro uteis speculative is routerelated is to or relatedthe comes idea behind tofrom the idea questionable sources. One relevant aspect of the proposed synthetic route is related to the idea behind behind the development of these agents. As they have been planned to be prepared from chemicals not prohibited by the CWC, precursors should not be under the scrutiny of the OPCW at that time. Therefore, they could be disguised as other compounds, with lower toxicity and with fair use as pesticides. The other issue is that they may be weaponized as binary agents, being delivered on-site, and handled with safety. According to Mirzayanov [3], A-230 can be synthesized following a synthetic route similar to the one used to obtain sarin and soman, where an amydine (instead of an alcohol) is condensed with DF (see Scheme3). If O-methyl phosphonyldifluoride or O-methyl phosphonylfluoro cyanide is used instead of DF, A-232 can be obtained, while A-234 comes from the reaction with O-ethyl phosphonyldifluoride or O-ethyl phosphonylfluoro cyanide. A-242 and A-262 on this turn, can be obtained if the amydine is replaced by a 1,1,3,3-tetraethylguanidine (TEG) in the reactions to produce A-230 and A-232, respectively (see Scheme3). As they are fluorophosphonates, A-232, A-234, and A-262 can be easily synthesized from the non-scheduled compounds O-ethyl phosphonyldifluoride and O-methyl phosphonyldifluoride and, therefore, circumvent the CWC. For analytical purposes, and to contribute to the OPCW Central Analytical Database (OCAD), in the development of ways of detection for the A series, Hosseini et al. [16] have reported a microsynthesis of a series of O-alkyl N-[bis(dimethylamino)methylidene]-P-methylphosphonamidates. They replaced TEG by 1,1,3,3-tetramethylguanidine (TMG), and extended the synthetic route of A-242 through an additional reaction of the final product with the appropriate alcohol in the presence of NaH (see Scheme4). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 11

the development of these agents. As they have been planned to be prepared from chemicals not prohibited by the CWC, precursors should not be under the scrutiny of the OPCW at that time. Therefore, they could be disguised as other compounds, with lower toxicity and with fair use as pesticides. The other issue is that they may be weaponized as binary agents, being delivered on-site, and handled with safety. According to Mirzayanov [3], A-230 can be synthesized following a synthetic route similar to the one used to obtain sarin and soman, where an amydine (instead of an alcohol) is condensed with DF (see Scheme 3). If O-methyl phosphonyldifluoride or O-methyl phosphonylfluoro cyanide is used instead of DF, A-232 can be obtained, while A-234 comes from the reaction with O-ethyl phosphonyldifluoride or O-ethyl phosphonylfluoro cyanide. A-242 and A-262 on this turn, can be obtained if the amydine is replaced by a 1,1,3,3-tetraethylguanidine (TEG) in the reactions to produce A-230 and A-232, respectively (see Scheme 3). As they are fluorophosphonates, A-232, A-234, and A- 262 can be easily synthesized from the non-scheduled compounds O-ethyl phosphonyldifluoride and O-methyl phosphonyldifluoride and, therefore, circumvent the CWC. For analytical purposes, and to contribute to the OPCW Central Analytical Database (OCAD), in the development of ways of detection for the A series, Hosseini et al. [16] have reported a microsynthesis of a series of O-alkyl N-[bis(dimethylamino)methylidene]-P- methylphosphonamidates. They replaced TEG by 1,1,3,3-tetramethylguanidine (TMG), and extended Int. J. Mol. Sci. 2019, 20, 1222 6 of 10 the synthetic route of A-242 through an additional reaction of the final product with the appropriate alcohol in the presence of NaH (see Scheme 4).

O O N P X + N P N + HX F HN F NNDA A-230

O O N + R1O P X N R1O P N + HX F HN F

NNDA A-232 (R1 = CH3)

A-234 (R1 = CH2CH3)

O O N P N P X + HN F N + HX N F N

TEG A-242

O O N P N O P X + HN F N + HX N F N

TEG A-262

Scheme 3. Possible synthesis routes for the A-series based on Mirzayanov’s report [2]. The leaving groupScheme X is either 3. Possible fluorine synthesis (F) or cyanide routes for (CN). the A-series based on Mirzayanov’s report [2]. The leaving Int. J. Mol.group Sci. X 2019 is either, 20, x fluoriFOR PEERne (F) REVIEW or cyanide (CN). 7 of 11 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 11 O O O N P N TEA, DCM OP N ROH, NaH O + N + HF OP F HN N F N P ORN TEA, DCM P N ROH, NaH N F + N N N + HF P F NH F N OR F N N N TMG TMG Scheme 4. Microsynthesis of O-Alkyl N-[bis(dimethylamino)methylidene]-P-methylphosphonamidates, as reportedScheme by Hosseini4. Microsynthesis et al. [16]. of O-Alkyl N-[bis(dimethylamino)methylidene]-P- Schememethylphosphonamidates, 4. Microsynthesis as reported ofby HosseiniO-Alkyl et al. [16].N-[bis(dimethylamino)methylidene]- P- Differentmethylphosphonamidates, from Mirzayanov as [ 3reported], Hoenig by Hosseini [12] hypothesized et al. [16]. that their alleged A-series compounds Different from Mirzayanov [3], Hoenig [12] hypothesized that their alleged A-series compounds can be synthesizedDifferent from following Mirzayanov the two-step[3], Hoenig route [12] hypo shownthesized in Scheme that their5. alle Accordingged A-series to him, compounds starting can be synthesized following the two-step route shown in Scheme 5. According to him, starting from fromcan a di-substituted be synthesized fluorophosphite following the two-step (2), the route nitroso show formn in Scheme of O-chlorodihaloaldoxime 5. According to him, starting (3) (which from is aa di-substituteddi-substituted fluorophosphitefluorophosphite (2),(2), thethe nitrosonitroso formform ofof OO-chlorodihaloaldoxime-chlorodihaloaldoxime (3) (3) (which (which is is structurally related to phosgene) acts as a nucleophile, affording a dihalophosphite intermediate (4). structurallystructurally relatedrelated toto phosgene)phosgene) acts acts as as a a nucleo nucleophile,phile, affording affording a a dihalophosphite dihalophosphite intermediate intermediate (4). (4). Heating leads to a rearrangement to the fluorophosphate derivatives (5), which he claims to be the Heating leadsleads toto aa rearrangementrearrangement toto thethe fluorophosphfluorophosphateate derivatives derivatives (5), (5), which which he he claims claims to to be be the the scaffold of the A-series. Some authors speculate that the novichoks are in fact the intermediates (4) or scaffoldscaffold ofof thethe A-series.A-series. SomeSome authors authors speculate speculate tha thatt the the novichoks novichoks are are in in fact fact the the intermediates intermediates (4) (4) or or precursorsprecursors of derivatives ofof derivativesderivatives (5) (5) [(5)12 [12,17].,[12,17].17].

Cl R R1 O Cl 2 R1 Cl Cl R O O F R1 O ClP F Δ 2 R1 O P F + O Cl R OO P O F N FCl R OP FO Δ 1 OP F + O 2 F R O P FO N F R2 N Cl R O O 1 O 2 F F N Cl R2 Cl (5) (2) (3) (4) Cl (2) (3) (4) Cl (5) Scheme 5. Synthetic route proposed by Hoenig [12] for the synthesis of the A-series nerve agents. Scheme 5. Synthetic route proposed by Hoenig [12] for the synthesis of the A-series nerve agents. Scheme 5. Synthetic route proposed by Hoenig [12] for the synthesis of the A-series nerve agents. 6. Physico-Chemical Properties of A-series Nerve Agents 6. Physico-Chemical Properties of A-series Nerve Agents Mirzayanov [3] reported that, like the other nerve agents, A-230, A-232, and A-234 are liquids, whileMirzayanov A-242 and [3]A-262 reported are the that, first like nerve the agentsother nerve known agents, to be inA-230, the solid A-232, state and at A-234 room aretemperature. liquids, whileHowever, A-242 as and there A-262 is virtually are the firstno information nerve agents in knownthe literature to be in about the solid this series state atof roomwarfare temperature. agents, their However,physico-chemical as there is properties virtually noare information mostly estimated in the literature [2,3,7]. Table about 1 thisreviews series some of warfare properties agents, collected their physico-chemicalfrom the literature properties (many estimated) are mostly andestimated lists the [2,3,7]. Log TablePs, calculated 1 reviews using some the properties chemicalize collected server from(https://chemicalize.com) the literature (many estimated)for tabun, andsarin, lists soman, the Log VX, Ps, and calculated some of usingthe structures the chemicalize of the A-seriesserver (https://chemicalize.com)reported by Mirzayanov for[3], tabun,Hoenig sarin, [12], andsoman, Elliso VX,n [14]. and The some low of vapor the structures pressures, of similar the A-series to tabun, reportedreported by for Mirzayanov A-230, A-232, [3], Hoenig and A-234 [12], and [3], Elliso suggestn [14]. that The they low vaporare moderately pressures, similarpersistent to tabun, in the reportedenvironment, for A-230, while A-232,the estimated and A-234 values [3], of suggestLog P suggest that they a considerable are moderately lipophilicity, persistent which in the will environment,increase the absorptionwhile the estimatedby the body. values According of Log toP suggestEllison [14],a considerable these comp lipophilicity,ounds can be which hydrolyzed will increaseto produce the absorptionHF, HCl, HCN, by the and body. highly According toxic oximes. to Ellison [14], these compounds can be hydrolyzed to produce HF, HCl, HCN, and highly toxic oximes. Table 1. Physical-chemical properties of some nerve agents [2,3,7,17]. Table 1. Physical-chemical properties of some nerve agents [2,3,7,17]. Melting Point Boiling Point Vapor Solubility at 25 Nerve Agent Melting Point Boiling Point Vapor Solubility at 25 Log P Nerve Agent (°C) (°C) Pressure (Pa) °C (g/L) Log P (°C) (°C) Pressure (Pa) °C (g/L) Tabun −50 240 0.057 9.8 − Tabun −50 240 0.057 9.8 − Sarin −57 147 2.9 Miscible − Sarin −57 147 2.9 Miscible − Soman −42 167–200 0.4 2.1 − Soman −42 167–200 0.4 2.1 − VX Below −51 298 0.0007 Miscible <2.4 °C − VX Below −51 298 0.0007 Miscible <2.4 °C − A−230 5.56 259.92 2.13 4.826 2.14 A−230 5.56 259.92 2.13 4.826 2.14 A−232 5.65 266.59 1.48 1.775 2.55 A−232 5.65 266.59 1.48 1.775 2.55

Int. J. Mol. Sci. 2019, 20, 1222 7 of 10

6. Physico-Chemical Properties of A-series Nerve Agents Mirzayanov [3] reported that, like the other nerve agents, A-230, A-232, and A-234 are liquids, while A-242 and A-262 are the first nerve agents known to be in the solid state at room temperature. However, as there is virtually no information in the literature about this series of warfare agents, their physico-chemical properties are mostly estimated [2,3,7]. Table1 reviews some properties collected from the literature (many estimated) and lists the Log Ps, calculated using the chemicalize server (https://chemicalize.com) for tabun, sarin, soman, VX, and some of the structures of the A-series reported by Mirzayanov [3], Hoenig [12], and Ellison [14]. The low vapor pressures, similar to tabun, reported for A-230, A-232, and A-234 [3], suggest that they are moderately persistent in the environment, while the estimated values of Log P suggest a considerable lipophilicity, which will increase the absorption by the body. According to Ellison [14], these compounds can be hydrolyzed to produce HF, HCl, HCN, and highly toxic oximes.

Table 1. Physical-chemical properties of some nerve agents [2,3,7,17]. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 11 Int. J. Mol. Sci. 2019, 20, x FOR PEERMelting REVIEW Point Boiling Point Vapor Pressure Solubility at 25 ◦C 8 of 11 Int. J. Mol.Nerve Sci. 2019 Agent, 20, x FOR PEER REVIEW◦ ◦ 8 ofLog 11 P Int. J. Mol. Sci. 2019, 20, x FOR PEER (REVIEWC) ( C) (Pa) (g/L) 8 of 11 Int. J. Mol. Sci. 2019A−234, 20, x FOR PEER REVIEW 3.06 264.11 1.7 0.6512 8 of 112.97 Int. J. Mol. Sci.TabunA 2019−234, 20 , x FOR PEER −REVIEW50 3.06 240264.11 0.0571.7 9.80.6512 8 of 2.97 11− Int. J. Mol. Sci.A Sarin2019−234, 20 , x FOR PEER −REVIEW57 3.06 147264.11 2.91.7 Miscible0.6512 8 of 2.97 11− Int. J. Mol. Sci.A 2019−A234−242, 20 , x FOR PEER REVIEW 3.0621.46 264.11284.85 1.70.579 0.65121000 8 of 2.97 110.45 SomanA−242234 −4221.463.06 167–200284.85264.11 0.40.5791.7 2.10.65121000 0.452.97− AVX−242234 Below −5121.463.06 298284.85264.11 0.00070.5791.7 Miscible <2.40.65121000◦C 2.970.45− A−O234 3.06 264.11 1.7 0.6512 2.97 A−−242234 21.463.06 284.85264.11 0.5791.7 0.65121000 0.452.97 OAOAP−242230O Cl 5.5621.46 259.92284.85 2.130.579 4.8261000 2.140.45 O AOPA−−O242232 Cl 5.6521.46−12.33 266.59284.85218.93 1.480.57918.5 1.7751000 16.74 2.550.451.76 OA−F242N −21.4612.33 218.93284.85 0.57918.5 16.741000 1.760.45 O AFP−O234N ClCl 3.06 264.11 1.7 0.6512 2.97 O POA−O242 Cl −21.4612.33 218.93284.85 0.57918.5 16.741000 1.760.45 OF N Cl −12.33 218.93 18.5 16.74 1.76 O AOP−O242N Cl 21.46 284.85 0.579 1000 0.45 O FP O Cl −12.33 218.93 18.5 16.74 1.76 OF O N Cl O P ON Cl −12.33 218.93 18.5 16.74 1.76 O OOFP PO O Cl F −12.33 218.93 18.5 16.74 1.76 OF N O P O ClF −12.33−12.33−35.05 218.93218.93191.37 18.518.573.8 16.74 16.74 37.64 1.761.761.47 OF F N N Cl −35.05 191.37 73.8 37.64 1.47 O P ON ClF O PFO O F Cl −35.05 191.37 73.8 37.64 1.47 OF N Cl O OP ON F −35.05 191.37 73.8 37.64 1.47 O FP O ClF −35.05 191.37 73.8 37.64 1.47 OF O N Cl O P ON F −35.05 191.37 73.8 37.64 1.47 O OFPO PO O ClF F −35.05−35.05 191.37191.37 73.873.8 37.64 37.64 1.471.47 FO N Cl −35.05−58.28 191.37162.02 73.8 304 37.64 84.02 1.471.18 O OFP FON N ClF O P ON F −58.28 162.02 304 84.02 1.18 O PFO O ClF F −58.28 162.02 304 84.02 1.18 OF N F −58.28 162.02 304 84.02 1.18 O OFP ON F O OP O F −58.28 162.02 304 84.02 1.18 O POF ON F −58.28−58.28 162.02162.02 304 304 84.02 84.02 1.181.18 OO PF O N FF −58.28 162.02 304 84.02 1.18 OO P ON F O P FO N F FF −58.28 162.02 304 84.02 1.18 O FF N F 48.34 256.81 1.53 9.940 1.34 O FP ON FF NO 48.34 256.81 1.53 9.940 1.34 O PO O F F 2 OF N FFNO 48.34 256.81 1.53 9.940 1.34 O P ON FF 2 OF F NO 2 48.34 256.81 1.53 9.940 1.34 O P ON F F O POF O FF NO 2 48.3448.34 256.81256.81 1.531.53 9.940 9.940 1.341.34 F NO F O P O F F NO 2 48.34 256.81 1.53 9.940 1.34 F ON FNO 48.34 256.81 1.53 9.940 1.34 ON PF OF 2Cl F O NO 2 48.34 256.81 1.53 9.940 1.34 O P OF N Cl −1.27 237.51 7.04 5.374 2.12 O FF NO 2 −1.27 237.51 7.04 5.374 2.12 O FP ON ClCl O PO OF Cl −1.27 237.51 7.04 5.374 2.12 OF N Cl −1.27−1.27 237.51237.51 7.047.04 5.374 5.374 2.122.12 O OP ON Cl O FP O Cl −1.27 237.51 7.04 5.374 2.12 OF O N Cl O P ON Cl −1.27 237.51 7.04 5.374 2.12 OOF P O Cl F −1.27 237.51 7.04 5.374 2.12 O FP ON Cl O OP O ClF −1.27−23.2 237.51211.21 7.0427.4 5.374 12.15 2.121.83 OF F N N Cl −23.2 211.21 27.4 12.15 1.83 O FP ON F Cl O PO O FCl −23.2−23.2 211.21211.21 27.427.4 12.15 12.15 1.831.83 OF N Cl −23.2 211.21 27.4 12.15 1.83 O OFP ON ClF O P OO F −23.2 211.21 27.4 12.15 1.83 O POF ON ClF −23.2 211.21 27.4 12.15 1.83 OF N Cl −23.2 211.21 27.4 12.15 1.83 O OFPO PONO F F O P O ClF −23.2−46.48 211.21183.14 27.4 110 12.15 27.29 1.831.54 OF F N NCl −46.48 183.14 110 27.29 1.54 O P ON ClF −46.48 183.14 110 27.29 1.54 O PFO O F F −46.48 183.14 110 27.29 1.54 OF N F O OP ON F −46.48 183.14 110 27.29 1.54 O FP O F −46.48 183.14 110 27.29 1.54 OOF N F O P ON F −46.48 183.14 110 27.29 1.54 OOO PFP OO FF −46.48 183.14 110 27.29 1.54 O F N F −46.48 183.14 110 27.29 1.54 O OP FFO NNF FF 58.8558.85 273.49273.49 0.5090.509 3.1573.157 1.701.70 O P ON FF F NO 58.85 273.49 0.509 3.157 1.70 O PFO O F 2 OF N FFNO 58.85 273.49 0.509 3.157 1.70 O OP ON FF 2 58.85 273.49 0.509 3.157 1.70 O FP O F F NO 2 OF N FNO 58.85 273.49 0.509 3.157 1.70 O P ON FF F 2 O FP O F F NO 58.85 273.49 0.509 3.157 1.70 F N FNO 2 58.85 273.49 0.509 3.157 1.70 7. Toxicity ofN A-seriesF F 2 Nerve Agents58.85 273.49 0.509 3.157 1.70 F F NO 2 7. Toxicity of A-seriesNO Nerve Agents 7. Toxicity of A-seriesF 2 Nerve Agents 7. ToxicityAccording of A-seriesF to Mirzayanov Nerve Agents [3] and some other literature sources [6,14], A-230 was found to be 5– 7. ToxicityAccording of A-series to Mirzayanov Nerve Agents [3] and some other literature sources [6,14], A-230 was found to be 5– 7. 8Toxicity timesAccording more of A-series toxicto Mirzayanov than Nerve RVX, Agents [3] which and someis similar other in literature toxicity to sources VX, while [6,14], A-232 A-230 presented was found a toxicity to be 5– 10 7.8 timesToxicityAccording more of toxicA-series to Mirzayanov than Nerve RVX, Agentswhich [3] and is somesimilar other in to literaturexicity to sourcesVX, while [6,14], A-232 A-230 presented was found a toxicity to be 5–10 87. times ToxicityAccording highermore of toxicA-series than to Mirzayanovthan soman. Nerve RVX, He Agentswhich also[3] and reported is somesimilar otherthat in A-242to literaturexicity and to VX,sourcesA-262 while are [6,14], “ultra-highly”A-232 A-230 presented was toxic found a toxicity derivatives to be 105– 8times timesAccording higher more thantoxic to soman. Mirzayanovthan RVX, He alsowhich [3] reportedand is somesimilar that other in A-242 to literaturexicity and to A-262 VX,sources while are [6,14],“ultra-highly” A-232 A-230 presented was toxic found a derivatives toxicity to be 105– times8 oftimes A-230According higher more and than toxic A-232, to soman.Mirzayanov than but RVX, didHe alsonot which [3] define reportedand is some similar this that termother in A-242 neitto literaturexicityher and compared to A-262 sourcesVX, whileare it [6,14],“ultra-highly” to A-232 the A-230 toxicity presented was oftoxic foundany a derivatives toxicityother to be agent. 5–10 timesof8 times A-230According higher more and than A-232,toxic to soman.Mirzayanov than but didRVX, He not also which [3]define reportedand is this somesimilar term that other in neitA-242 to literatureherxicity and compared to A-262 VX,sources whileare it to [6,14],“ultra-highly” the A-232 toxicity A-230 presented wasof toxic any found aother derivatives toxicity to agent. be 105– 8oftimes Consideringtimes A-230 higher more and A-232,thantoxic this soman. than information,but didRVX, He not alsowhich define we reported isestimated this similar term that in neitA-242the toxicityher values and compared to A-262 ofVX, LCt while are it50 to “ultra-highly”and the A-232 toxicityLD presented50 for of toxictheseany aother derivativestoxicitycompounds agent. 10 ofConsideringtimes8 times A-230 higher more and A-232,than thistoxic information,soman. than but didRVX, He not also which definewe reported estimated is this similar term that thein neitA-242 to valuesherxicity and compared to ofA-262 VX, LCt whileare50 it toand “ultra-highly” the A-232 LD toxicity50 presentedfor ofthese toxicany aothercompounds derivatives toxicity agent. 10 timesConsideringofreported A-230 higher and in thanA-232,Tablethis soman.information, 2. but Mirzayanov did He not also definewe reported[3] estimated did this not term that report the A-242neit values herth eand comparedtoxicity ofA-262 LCt of are50 itA-234, andto “ultra-highly” the LD but toxicity50 considering for ofthese toxic any compounds other derivativesits structural agent. Consideringreportedoftimes A-230 higher andin Table A-232,thanthis 2.information,soman. Mirzayanovbut did He not also definewe [3] reported estimateddid this not term thatreport the neitA-242 thvalueshere toxicityand compared ofA-262 LCtof A-234, 50are it andto “ultra-highly” the butLD toxicity considering50 for ofthese toxicany its compoundsother derivatives structural agent. ofreportedConsideringsimilarity A-230 andin Tableto A-232,this A-232, 2.information, Mirzayanovbut we did assumed not definewe [3] that didestimated this notit would term report the neit have thvalueshere toxicity similar compared of LCtoftoxicity. A-234, 50it toand the Basedbut LD toxicity considering50 onfor these ofthese any considerations, its othercompounds structural agent. reportedsimilarityConsideringof A-230 andin to Table A-232,A-232,this 2.information, Mirzayanovwebut assumeddid not definewe [3] that didestimated itthis notwould term report the haveneit thvaluesher esimilar toxicity compared of toxicity. LCtof A-234,50 it andto Based the but LD toxicity considering 50on for these ofthese any considerations, its compoundsother structural agent. Consideringsimilarityreportedwe can intell to Table A-232,thatthis 2.A-230,information, Mirzayanovwe assumed A-232, weand [3]that estimateddid A-234 it notwould arereport the thehave thvaluesmost esimilar toxicity toxic of toxicity. LCtnerveof A-234,50 and agents Based butLD consideringknown.50on for these these Speculating considerations, itscompounds structural about similaritywereportedConsidering can tell in to thatTable A-232,this A-230, 2.information, Mirzayanovwe A-232,assumed and we [3]that A-234 didestimated it notwould are report the thehave most thvalues esimilar toxicity toxic of nervetoxicity. LCtof A-234,50 agents and Based but LD known. considering50on for these Speculatingthese considerations, itscompounds structural about reportedwesimilarityA-242 can telland in to thatTable A-262A-232, A-230, 2. toxicity, Mirzayanovwe A-232,assumed if Mirzayanovand [3] that A-234 did it notwould are [3] report themeant have most the thatsimilar toxicity toxic “ultra-highly nervetoxicity. of A-234, agents Based toxic”but known. considering on is thesemore Speculating considerations,toxic its structuralthan about A-230 weA-242similarityreported can and tell in to A-262 thatTable A-232, A-230, toxicity,2. Mirzayanovwe A-232,assumed if Mirzayanov and [3] that A-234 did it notwould [3]are reportmeant the have most th that esimilar toxicitytoxic “ultra-highly nervetoxicity. of A-234, agents toxic”Based but known. consideringison more these Speculating toxic considerations, its than structural A-230about similarityA-242weand can A-232,and tell to A-262 that A-232,these A-230, toxicity, compounds we A-232,assumed if Mirzayanov and would that A-234 itinstead would [3] are meant the be have themost that mostsimilar toxic“ultra-highly toxic. nervetoxicity. The agents fact toxic”Based that known. ison they more these areSpeculating toxic considerations, solids, than however, A-230about A-242andwesimilarity can A-232, and tell toA-262 thesethat A-232, A-230, compoundstoxicity, we A-232,assumed if Mirzayanov would and that A-234 instead it would[3] are meantbe the thehave most thatmost similar toxic“ultra-highly toxic. nervetoxicity. The agentsfact toxic”Based that known. they ison more these are Speculating toxicsolids, considerations, than however, A-230about weandA-242renders can A-232, and tell them A-262thesethat A-230,almost compoundstoxicity, A-232,useless if Mirzayanov would and for A-234 weaponization instead [3]are meantbe the the most mostthatbu ttoxic “ultra-highly cantoxic. nervemake The agentsfactthem toxic” that very known. they is moreattractive are Speculating solids,toxic forthan however, terrorism aboutA-230 andrendersA-242we canA-232, and tellthem A-262thesethat almost A-230, compoundstoxicity, useless A-232, if Mirzayanov would forand weaponization A-234 instead [3] are meantbe the the mostbu mostthatt cantoxic “ultra-highly toxic. make nerve The them factagents toxic” thatvery known. they isattractive more are Speculating solids,toxic for than however,terrorism A-230about A-242rendersandpurposes. A-232, and them A-262 these As almost guanidine compoundstoxicity, useless ifderivatives, Mirzayanov wouldfor weaponization instead it is[3] very meantbe the likely bu thatmostt thatcan “ultra-highly toxic. makethey The have them fact toxic”good thatvery solubility they isattractive more are toxicsolids, in for water than terrorismhowever, and,A-230 for renderspurposes.andA-242 A-232, and them As A-262these guanidinealmost compoundstoxicity, useless derivatives, if Mirzayanov wouldfor weaponization insteadit is [3]very meantbe likely the bu mostthat thatt can “ultra-highly toxic.they make haveThe them fact good toxic” thatvery solubility they isattractive more are intoxicsolids, water for than terrorismhowever, and, A-230 for andpurposes.rendersexample, A-232, them As theseare guanidine almostperfect compounds uselessto derivatives,contaminate would for weaponization insteadit the is very water be likely the supply. bu most thatt can toxic. they make haveThe them fact good thatvery solubility they attractive are insolids, water for however,terrorism and, for purposes.example,rendersand A-232, them are As these perfectguanidine almost compounds to useless contaminate derivatives, wouldfor weaponization theinsteadit is water very be likelysupply. the bu most thatt can toxic.they make haveThe them fact good thatvery solubility they attractive are in solids, water for terrorismhowever, and, for rendersexample,purposes.No them are Asantidote perfect guanidinealmost is to useless yetcontaminate derivatives, reported for weaponization the forit is waterthe very neurotoxic supply.likely bu tthat cans theyof make the have A-series,them good very solubility but attractive it is inbelieved waterfor terrorism and, that for the example,purposes.rendersNo them antidoteare As perfect guanidinealmost is toyet useless contaminate derivatives,reported for weaponizationfor the it theis water very neurotoxic supply.likely bu thatt scan of they makethe haveA-series, them good very butsolubility attractiveit is believed in water for terrorism thatand, thefor purposes.example,commercialNo antidote are As perfect guanidineoximes, is toyet like contaminate derivatives,reported 2-PAM and for theit HI-6, isthe water very neurotoxic should likelysupply. work thats of againstthey the have A-series, intoxication good butsolubility withit is thesebelieved in water agents thatand, [9]. thefor The commercialexample,purposes.No antidote are As oximes, perfectguanidine is liketoyet contaminate 2-PAMderivatives,reported and for theHI-6, it theis water very should neurotoxic supply.likely work thats againstof they the have intoxicationA-series, good butsolubility with it isthese believed in agentswater that and,[9]. The thefor example,commercialfirstNo reports antidoteare oximes, perfect on experiments is liketoyet contaminate 2-PAMreported in and this for theHI-6, direction the water should neurotoxic supply.are work eagerly s againstof anticipated.the intoxicationA-series, but with it isthese believed agents that [9]. Thethe commercialfirstexample, reportsNo antidote are oximes,on perfect experiments is like toyet contaminate 2-PAMreported in this and fordirection HI-6,the the water should neurotoxic are supply. eagerly works against ofanticipated. the intoxicationA-series, but with it isthese believed agents that [9]. Thethe firstcommercial reportsNo antidote oximes,on experiments is likeyet 2-PAMreported in this and fordirection HI-6, the shouldneurotoxic are eagerly works against ofanticipated. the intoxicationA-series, but with it is these believed agents that [9]. Thethe firstcommercial reportsNo antidote oximes,on experiments is likeyet 2-PAMreported in this and fordirection HI-6, the should neurotoxic are eagerly works against ofanticipated. the intoxicationA-series, but with it isthese believed agents that [9]. Thethe commercialfirst reports oximes,on experiments like 2-PAM in this and direction HI-6, should are eagerly work against anticipated. intoxication with these agents [9]. The firstcommercial reports oximes,on experiments like 2-PAM in this and direction HI-6, should are eagerly work against anticipated. intoxication with these agents [9]. The first reports on experiments in this direction are eagerly anticipated.

first reports on experiments in this direction are eagerly anticipated.

Int. J. Mol. Sci. 2019, 20, 1222 8 of 10

7. Toxicity of A-series Nerve Agents According to Mirzayanov [3] and some other literature sources [6,14], A-230 was found to be 5–8 times more toxic than RVX, which is similar in toxicity to VX, while A-232 presented a toxicity 10 times higher than soman. He also reported that A-242 and A-262 are “ultra-highly” toxic derivatives of A-230 and A-232, but did not define this term neither compared it to the toxicity of any other agent. Considering this information, we estimated the values of LCt50 and LD50 for these compounds reported in Table2. Mirzayanov [ 3] did not report the toxicity of A-234, but considering its structural similarity to A-232, we assumed that it would have similar toxicity. Based on these considerations, we can tell that A-230, A-232, and A-234 are the most toxic nerve agents known. Speculating about A-242 and A-262 toxicity, if Mirzayanov [3] meant that “ultra-highly toxic” is more toxic than A-230 and A-232, these compounds would instead be the most toxic. The fact that they are solids, however, renders them almost useless for weaponization but can make them very attractive for terrorism purposes. As guanidine derivatives, it is very likely that they have good solubility in water and, for example, are perfect to contaminate the water supply.

Table 2. Toxicological data of some nerve agents [6,14].

3 Neurotoxic Compounds LCt50 for Humans (mg-min/m ) LD50 (g/70 kg man) GA 400 1 GB 55 1.7 GD 70 0.35 VX 15 0.006–0.01 A-230 1.9–3 * 7.5 × 10−4–0.002 * A-232 7 * 0.035 * A-234 7 * 0.035 * * Estimated values.

No antidote is yet reported for the neurotoxics of the A-series, but it is believed that the commercial oximes, like 2-PAM and HI-6, should work against intoxication with these agents [9]. The first reports on experiments in this direction are eagerly anticipated.

8. Protection, Treatment, and Decontamination of A-Series Agents There is no information available yet on protection and decontamination against the A-series agents. However, it is believed that no improvement will be needed in the protective wear currently in use to face these agents. In regards to decontamination, it is believed that, based on their structural similarities to other nerve agents, the A-series compounds can be destroyed by basic solutions with more than 10% by weight of NaOH or NaCO3, or undiluted household bleach. It is also speculated that reactive oximes, such as potassium 2,3-butanedione monoximate and basic peroxides, should rapidly detoxify these compounds. It has been reported that the compounds of the A-series should act in the same way as the nerve agents of the G-series and V-series, by inhibiting the enzyme acetylcholinesterase (AChE) and triggering cholinergic syndrome [17–19]. Therefore, preferential prophylaxis would involve the administration of a cocktail containing: (1) an anticholinergic, to reduce the concentration of ACh; (2) an anticonvulsant, to combat the effects of the cholinergic syndrome; and (3) an antidote (usually an oxime) to reactivate AChE. However, it is speculated that the oximes currently in use could be non-effective for these compounds because the intermediate formed during the interaction of these compounds with AChE would be hard to reactivate [17]. This points to bioscavengers as an alternative option for the treatment of intoxication by the A-series agents [17].

9. Final Remarks The A-series of nerve agents represents today a matter of concern in the field of CWs. There are many uncertainties on this issue that still need to be clarified. An urgent, complete, and Int. J. Mol. Sci. 2019, 20, 1222 9 of 10 undoubtful elucidation of their structures and properties is essential at this moment for a better understanding of the real risks posed by these compounds and for the development of effective protection. The knowledge of the correct structures will make it possible for theoretical and experimental investigations towards the discovery of the most appropriate antidotes. Also, its synthesis will allow the evaluation of its physical-chemical properties and toxicology, and it will allow the development of biological and analytical protocols for the identification of its misuse. We believe that this review will bring light to this issue, and it will stimulate researchers to start investigations on this matter, accelerating the process of mastering all aspects related to the A-series nerve agents.

Author Contributions: T.C.C.F. and K.K. conceived and designed the paper; D.A.S.K. contributed to Sections5 and6 ; S.F.d.A.C. contributed to Sections4 and5; J.A.V.d.S. contributed to Section3 and constructed the graphical abstracts; E.N. contributed to Section8 and revised the paper; K.K. revised the paper, and T.C.C.F. wrote and finalized the paper. Funding: This research was funded by Brazilian financial agencies CNPq, grant number [306156/2015-6] and FAPERJ, grant number [E-02/202.961/2017]. This work was also supported by the OPCW Research Project Support Program (L/ICA/ICB/201062/15) and the excellence project FIM. Also supported by the Long-term development project UHK. Acknowledgments: The authors wish to thank the Military Institute of Engineering and Faculty of Informatics and Management, University of Hradec, for the infrastructure. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

AChE Acetylcholinesterase CW Chemical Weapons CWC Chemical Weapons Convention DEAT 2-(diethylamino)ethanethiol DF Methylphosphonic difluoride GA Tabun GB Sarin GB-2 Binary GB GD Soman GD-2 Binary GD GOSNIIOKhT Institute for Organic Chemistry and Technology in Moscow IJMS International Journal of Molecular Sciences IPA Isopropylamine LCt50 Median Lethal Concentration LD50 Median Lethal Dose OCAD OPCW Central Analytical Database OP Organophosphate OPCW Organization for Prevention of Chemical Weapons PCF Methyl phosphorocyanidofluoridate PCN 2-methylpropyl methylphosphonocyanidate QL O-Ethyl O-2-diisopropylaminoethyl methylphosphonite NNDA N,N-diethyl-2-iminopropan-1-amine RVX Russian VX SLUDGEM Salivation, Lachrymation, Urination, Defecation, Gastrointestinal, Emesis and Miosis TEG 1,1,3,3 tetraethylguanidine TMG 1,1,3,3 tetramethylguanidine UK United Kingdom USA United States of America USSR Union of Soviet Socialist Republics VX-2 Binary VX Int. J. Mol. Sci. 2019, 20, 1222 10 of 10

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