DOI: 10.1002/asia.201000058

Chromo-Fluorogenic Detection of Nerve-Agent Mimics Using TriggeredCyclization Reactions in Push–Pull Dyes

Ana M. Costero,*[a, b] Margarita Parra,[a, b] Salvador Gil,[a, b] Raffll Gotor,[a, b, d]

Pedro M. E. Mancini,[a, b] Ram�n Mart�nez-M�Çez,*[a, c, d] F�lix Sancen�n,[a, c, d] andSantiago Royo[c, d]

Introduction

The current increase in international concern in relation tocriminal terrorist attacks using chemical warfare (CW)agents has brought about increasing interest in the develop-ment of reliable detection techniques of these lethal chemi-cals. A typical classification of chemical warfare agents in-cludes nerve agents, asphyxiant/blood agents, vesicantagents, choking/pulmonary agents, lachrymatory agents, in-capacitating agents, and cytotoxic proteins.[1,2] Among them,nerve agents are especially dangerous species, and poisoningmay occur through inhalation or consumption of contami-nated liquids or foods. The effects of nerve agents arecaused by their ability to inhibit the action of acetylcholines-terase.[3]

Chemically, nerve gases are highly toxic phosphoric acidesters, which are structurally related to the larger family oforganophosphate compounds. Tabun (GA), sarin (GB), and

Abstract: A family of azo and stilbenederivatives (1–9) are synthesized, andtheir chromo-fluorogenic behavior inthe presence of nerve-agent simulants,diethylchlorophosphate (DCP), diiso-propylfluorophosphate (DFP), and di-ethylcyanophosphate (DCNP) in aceto-nitrile and mixed solution of water/ace-tonitrile (3:1 v/v) buffered at pH 5.6with MES, is investigated. The pre-pared compounds contain 2-(2-N,N-di-methylaminophenyl)ethanol or 2-[(2-N,N-dimethylamino)phenoxy]ethanolreactive groups, which are part of theconjugated p-system of the dyes andare able to give acylation reactionswith phosphonate substrates followedby a rapid intramolecular N-alkylation.The nerve-agent mimic-triggered cycli-

zation reaction transforms a dimethyla-mino group into a quaternary ammoni-um, inducing a change of the electronicproperties of the delocalized systemsthat results in a hypsochromic shift ofthe absorption band of the dyes. Simi-lar reactivity studies are also carriedout with other “non-toxic” organophos-phorus compounds, but no changes inthe UV/Vis spectra were observed. Theemission behaviour of the reagents inacetonitrile and water–acetonitrile 3:1v/v mixtures is also studied in the pres-ence of nerve-agent simulants and

other organophosphorous derivatives.The reactivity between 1–9 and DCP,DCNP, or DFP in buffered water–ace-tonitrile 3:1 v/v solutions under pseudofirst-order kinetic conditions, using anexcess of the corresponding simulant,are studied in order to determine therate constants (k) and the half-lifetimes (t1/2 = ln2/k) for the reaction. Thedetection limits in water/acetonitrile3:1 v/v are also determined for 1–9 andDCP, DCNP, and DFP. Finally, thechromogenic detection of nerve agentsimulants both in solution and in gasphase are tested using silica gel con-taining adsorbed compounds 1, 2, 3, 4,or 5 with fine results.

Keywords: acylation · cyclization ·nerve agents · phosphorus · recep-tors

[a] Prof. Dr. A. M. Costero, Dr. M. Parra, Dr. S. Gil, R. Gotor,Prof. Dr. P. M. E. Mancini, Prof. Dr. R. Mart�nez-M�Çez,Dr. F. Sancen�nInstituto de Reconocimiento Molecular y Desarrollo Tecnol�gico(IDM)Centro mixto Universidad Polit�cnica de Valencia - Universidad deValenciaValencia (Spain)Fax: (+34) 963 543 831E-mail : Ana.Costero@uv.es

[b] Prof. Dr. A. M. Costero, Dr. M. Parra, Dr. S. Gil, R. Gotor,Prof. Dr. P. M. E. ManciniDepartamento de Qu�mica Org�nicaFacultad de Qu�micas, Universidad de ValenciaDoctor Moliner 50, 46100 Burjassot, Valencia (Spain)

[c] Prof. Dr. R. Mart�nez-M�Çez, Dr. F. Sancen�n, S. RoyoDepartamento de Qu�micaUniversidad Polit�cnica de ValenciaCamino de Vera s/n, 46022 Valencia (Spain)

[d] R. Gotor, Prof. Dr. R. Mart�nez-M�Çez, Dr. F. Sancen�n, S. RoyoCIBER de Bioingenier�a, Biomateriales y Nanomedicina (CIBER-BBN)

Chem. Asian J. 2010, 5, 1573 – 1585 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1573

soman (GD) were developed during the Second World War,whereas further research resulted in the discovery of V-nerve agents. The ease of production and extreme toxicityof organophosphorus-containing nerve agents underlines theneed to detect these chemicals. In fact, an intense researcheffort has been directed to develop sensitive and selectivesystems for the detection of these compounds. Currentmethods for nerve-agents monitoring are mainly based onthe use of biosensors,[4] ion-mobility spectroscopy (IMS),[5]

electrochemistry,[6] microcantilevers,[7] photonic crystals,[8]

and optical-fiber arrays.[9] As an alternative to these instru-mental methods, the development of easy-to-use fluorogenicand chromogenic reagents has been gaining interest inrecent years.[10] For instance, paradigms involving PET-basedfluorescent probes,[11] assays using oximate-containing deriv-atives[12] molecular imprinting polymers,[13] nanoparticles,[14]

carbon nanotubes,[15] porous silicon,[16] or displacement-likeprocedures[17] have been recently reported. Most of theseparadigms rely on the changes in fluorescence properties,whereas few examples are related to color modulations. Forinstance, reported chromogenic examples include thesodium perborate-mediated oxidation of the organophos-phorus agent to a peracid, which was able to oxidize certainaromatic amines to give colored derivatives.[18] Also, oxi-mates and hydrazones have been used for the developmentof chromogenic reagents for chemical warfare agents. Whenthese moieties were implemented into an organic scaffoldwith absorption bands in the visible region, the reactionwith the electrophilic phosphorus centers of the simulants,induced significant color changes.[12a, b, d] Finally, gold nano-particles as signalling subunits coupled with an enzymaticassay (based on the inhibition of the enzymatic activity ofthe acetylcholinesterase) have also been reported.[14a, d]

Despite these interesting examples, the development ofselective chromogenic probes for the detection of thesedeadly chemical species in vapors or in aqueous environ-ments is still rare. Following our interest in the developmentof new chromo-fluorogenic chemosensors[19, 20] for targetchemical species, we report herein the design of colorimetricprobes for nerve-agent simulants for their detection in solu-tion or in gas phase. The sensing scheme uses the reactivityof the 2-(2-N,N-dimethylaminophenyl)ethanol or 2-[(2-N,N-dimethylamino)phenoxy]ethanol fragments with phospho-nate substrates and its coupling with a colorimetric eventfollowing a chemodosimeter paradigm. We have already re-ported a preliminary communication of this study recent-ly.[21]

Results and Discussion

Design of the Colorimetric Probes

The colorimetric recognition and signalling of the nerve-agent mimics is based on the use of suitable properties offragment I. This moiety contains a nucleophile, the hydroxylfunctional group, which has been reported to give acylationreactions with phosphonate substrates to form the inter-

mediate II. Furthermore, it is known that II will suffer arapid intramolecular N-alkylation to afford III, a quaternaryammonium salt. Following the scheme of the above-men-tioned paradigm, we envisaged that the use of fragment I asa donor group and its coupling with an acceptor (A) moietyin certain chromophores could be a suitable procedure todevelop colorimetric probes for nerve-agent detection (seeScheme 1). Thus, the overall conversion of the correspond-ing tertiary amine IV to the quaternary ammonium V uponreaction with certain organophosphorus (OP) substrates willinduce a change on the electronic donor properties of thenitrogen atom resulting in a decrease of the push–pull char-acter of the dye and color modulation. Among the differentpossibilities, we have used here as reactive groups 2-2-(N,N-dimethylamino)phenyl)ethanol or 2-[2-(N,N-dimethylami-no)phenoxy]ethanol.

Even though a similar reaction (acylation and intramolec-ular N-alkylation) had been previously used in fluorogenicnerve-agent sensing, this paradigm has been barely used forthe chromogenic signaling of nerve agents.[21]

Synthesis, Characterization, and Spectroscopic Properties

Chromoreagent 1 (see Scheme 2) was prepared by the reac-tion of 2-(2-dimethylaminophenyl)ethanol (11) with the di-azonium salt of 4-nitroaniline using the well-documentedprocedures for the preparation of azo dyes.[22] The startingcompound 11 was synthesized following a previously de-scribed method.[23] First, 2-nitrotoluene was transformedinto 2-(2-nitrophenyl)ethanol. Then, a reductive aminationin the presence of formaldehyde gave rise to 2-(2-dimethyla-minophenyl)ethanol, (11).[24] Chromoreagents 2 and 3 wereprepared by the reaction of ethyl 2-(2-dimethylaminophe-noxy)acetate (13) with the diazonium salts of 4-nitroaniline(for 2) and 4-aminobenzonitrile (for 3). The starting com-

Scheme 1. Colorimetric sensing paradigm. The 2-(2-dimethylaminophen-yl)ethanol group suffers cyclization reaction in the presence of nerve-agent simulants. It acts as a donor system in push–pull chromophores.Upon reaction with the nerve-agent simulants, the donor ability of theamine group is reduced, resulting in a weakening of the push–pull charac-ter of the dye and color modulation.

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FULL PAPERS

pound 13 was prepared from 2-nitrophenol, which was firsttransformed into 2-(N,N-dimethylamino)phenol by reactionwith formaldehyde, followed by catalytic reduction with hy-drogen.

The stilbene derivatives 4–9 were synthesized by using aWittig reaction from 2-(4-formyl-(2-dimethylaminophenyl)-ethanol (16) and the corresponding phosphonium salts, orby coupling with 1,4-dimethylpyridinium iodide for chro-moreagent 5. Compound 16 was not commercially available,and its synthesis was performed from 4-nitro-3-methylben-zoic acid (17) by two alternative pathways (see Scheme 3).In the first route, 4-nitro-3-methylbenzoic acid was convert-ed into methyl ester (18), which was condensed with formal-dehyde to afford compound 19. Reductive amination of 19transforms the nitro group into the corresponding dimethy-lamino moiety. The last step was the transformation of themethoxycarbonyl group into the formyl group. This last re-action cannot be carried out in one single step. It was ach-ieved by the reduction of 20 with LiAlH4 and further con-trolled oxidation to the aldehyde 16.

The second pathway followed for the synthesis of 16 start-ed with the conversion of 17 into the corresponding methoxy-methylamide 22. The transformation of 22 into 24 was ach-ieved following the same reaction route for the conversionof 18 to 20. Finally, 24 was reduced with DIBAL to afford16. Even though the first procedure involves one more step,the overall yields were similar for both synthetic routes. Fi-nally, compound 10 was prepared from 5-methoxy-2-nitroto-luene following the same procedure described for the syn-thesis of 11.

1–3 are azo dyes, 4–7 can be classified as donor–acceptorstilbenes, whereas 8 and 9 are described as donor–donor stil-benes. The spectroscopic characteristics of all compoundsare shown in Table 1. 1–9 display a relatively intense absorp-tion band in the 300–400 nm range. Based on their electron-ic properties, compounds 1–9 are chromophores, containinga donor dimethyl amino moiety and a group of variable ac-

ceptor character at the otherend of the molecular frame-work. 1–3 shows adsorptionbands in the visible region,whereas the stilbene derivatives4–9 absorb in the 260–380 nmrange. The influence of the dif-ferent electronic properties ofthe stilbene family (derivatives4–9) on the absorption band ofthe chromophores is evidentfrom the data in Table 1, and itagrees with the expectation thatchanges in the acceptor charac-ter of the appended groups(pyridinium>NO2>CN~an-thraquinone>OCH3) in the p-system will result in a hypso-chromic shift of the absorptionband (labs 5>4>6= 7>8/9).This effect is also observed for

compounds 2 and 3 ; the stronger the acceptor character(NO2>CN), the larger the wavelength of the absorptionband.

Reactivity with Nerve-Agent Simulants

First, the reactivity of the prepared chromoreagents wastested with diethylchlorophosphate (DCP), diisopropylfluor-ophosphate (DFP), and diethylcyanophosphate (DCNP) inacetonitrile (see Scheme 4). Arising from the high toxicityof nerve agents, sarin, soman, and tabun, the related com-pounds DFP, DCP, and DCNP have been typically used asmodels for the design of indicators and sensing systems, asthey have similar reactivity but lack the efficacy of typicalnerve agents. Preliminary studies were carried out with com-pound 1 (1 �10�5 moldm�3), which displays an intense ab-sorption band at 410 nm in acetonitrile, typical of azo-dyederivatives. The addition of DCP, DFP, or DCNP to acetoni-trile solutions of 1 resulted in a clear hypsochromic shift ofthe absorption band and color modulation from yellow tocolorless. The observed results are consistent with the intra-molecular cyclization process shown in Scheme 1 and the re-duction of the donor character of the N,N-dimethylaminomoiety in the chromophore. In this preliminary step, it wasalso confirmed that 1 was not able to react with other orga-nophosphorus derivatives, such as OP1–OP4 (seeScheme 4). A similar reactivity of 1 with DCP, DFP, andDCNP in mixed water–acetonitrile solutions was also found.

Motivated by these favorable features observed for deriv-ative 1, similar studies of reactivity were carried out with de-rivatives 2–9 (1 � 10�5 mol dm�3) in water–acetonitrile (3:1v/v) solutions buffered at pH 5.6 with MES (1�10�1 mol dm�3). Mixtures containing a larger percentage ofwater could not be used in this work because of the poorsolubility of some of the reagents. Furthermore, derivative10 was prepared and used as the model compound. Com-

Scheme 2. Chromoreagents 1–10.

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Chromo-Fluorogenic Detection of Nerve-Agent Mimics

pounds 8 and 9 were separatedin the synthetic process andwere perfectly identified byusing 1H NMR, however, be-cause of their photo-instability,after a short time at room tem-perature, both samples (in solidstate or solution) became iden-tical; that is, a mixture of bothisomers.[25] Therefore, studies inthe presence of the reagents for8 and 9 were carried out in sol-utions containing a mixture ofboth compounds 8/9.

As observed for compound 1,the reagents 2–9 also show hyp-sochromic shifts of the absorp-tion band upon addition of anexcess of the nerve-agent simu-lants DCP, DFP, and DCNP inmixed water–acetonitrile (3:1 v/v) solutions buffered at pH 5.6with MES. For 1–5, a bleachingof the yellow or pale yellow sol-utions could be observed withthe naked eye. Figure 1 shows aphotograph with the colorchanges observed for 3 (1 �10�5 mol dm�3) in water/acetoni-trile (3:1 v/v, MES 1 �10�1 mol dm�3) upon addition ofan excess of nerve-agentmimics DCP, DFP, and DCNP.Moreover, Figure 2 shows theUV/Vis spectrum of chromore-agent 3 (1 �10�5 moldm�3) inwater/acetonitrile (3:1 v/v, MES1 � 10�1 mol dm�3) after the ad-dition of DCP.

Data in Table 1 shows thatthe hypsochromic shift of the

absorption band in the presence of nerve-agent simulantsfor ligands 1–3 (Dl of 125, 114, and 98 nm) was larger thanthat for other prepared reagents (Dl of 19, 53, 44, 5, and<5 nm for 4, 5, 6, 7, and 8/9, respectively). In general, the

Scheme 3. a) HCHO, PhO�, DMSO, 60–70 8C, 1 h; b) H2, Pd-C, 37% HCHO; c) 4-nitroaniline, NaNO2, H+ ;d) HCHO, H2, Pd-C; e) BrCH2COOCH2CH3, Cs2CO3, ACN; f) 4-aminobenzonitrile, NaNO2, H+ ; g) MeOH/H+ , 5 h; h) LiAlH4, Et2O, 30 min; i) MnO2, Et2O, 2.5 h; j) CBr4, Et3N, CH3NH2OCH3Cl, Ph3P, DCM, 2.5 h;k) DIBAL, THF, 45 min.

Table 1. UV absorptions of pure ligands 1–10 (1 � 10�5 mol dm�3) inwater/acetonitrile (3:1 v/v, MES 1� 10�1 mol dm�3) and in the presence ofDCP.

Receptor Receptor+DCPlabs

max [nm] loge [m�1 cm�1] labsmax [nm] loge [m�1 cm�1]

1[a] 407 3.9 282 4.02[c] 428 4.1 314 4.43[b] 412 4.1 314 4.34[c] 366 4.3 347 4.55[c] 378 4.2 325 4.56[b] 309 4.3 265 4.37[c] 310 4.1 305 4.28/9[a,d] 268 4.3 266 4.510[a] 267 4.0 –[e] –

[a] 10 equivalents of DCP were used. [b] 200 equivalents of DCP wereused. [c] 700 equivalents of DCP were used. [d] Arising from the photo-conversion between 8 and 9, the studies were carried out in mixtures ofboth compounds 8/9. [e] Very low shifts of the absorption band (lowerthan 3 nm) were observed in the presence of the DCP simulant. Scheme 4. Chemical structure of different organophosphorus derivatives.

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hypsochromic shifts upon addition of the nerve-agentmimics are in agreement with the expectation that transfor-mation of the donor amine into the corresponding quaterna-ry ammonium in push–pull systems will produce a blue-shiftof the absorption band because of a modulation of thedipole moments of the ground and excited states, and there-fore, a change in the relative energy of HOMO and LUMO.In general, the trend observed in the hypsochromic shiftwhen changing from amine to ammonium for the stilbenederivatives in the presence of simulants agrees with the ac-ceptor strength. This is for instance clear when comparingthe Dl of 53 nm for 4 (having a strong electron-acceptorNO2 group) with the Dl of <5 nm for 8/9, which includes adonor-OCH3 moiety. It was also observed that for a certaindye, the absorption spectra after reaction with the three sim-ulants DCP, DFP, and DCNP, were very similar, corroborat-ing the fact that the same reaction takes place in all cases.The apparent anomalous shift observed for 7 and DCNP(labs

max =265 nm) may be caused by the formation of the cya-nohydrin derivatives as consequence of the reaction of theevolved cyanide with the carbonyl groups in the ligand.

Similar reactivity studies were also carried out with 2–9and other “nontoxic” organophosphorus compounds (seeScheme 4, OP1–OP4), but no changes in the UV/Vis spectrawere observed, indicating that the reactivity with the nerve-agent mimics is selective.

Furthermore, to assess themechanism involved in the ob-served chromogenic response,the corresponding ammoniumsalts (V in Scheme 1) for somecompounds were prepared fol-lowing a different syntheticroute, which involves the reac-tion of the corresponding chro-moreagent with p-toluenesul-fonyl chloride in the presence

of sodium carbonate in acetonitrile. This gave the corre-sponding tosylated derivative, which suffered a spontaneouscyclization that resulted in the formation of the correspond-ing cyclic compounds 25 and 26 when using 1 and 4, respec-tively (Scheme 5). This transformation was easily detected

in the 1H NMR spectra of the final products. For example,in the case of 25, one triplet centered at 4.36 ppm, indicativeof a methylene subunit located near the quaternary ammo-nium, (Figure 3) is shown. Also, a significant long-range1H–13C coupling between this signal and the 13C signals cor-responding to the methyls (54 ppm) and to the aromaticcarbon (149 ppm) attached to the N,N-dimethylaniliniumfragment, points toward the unambiguous formation of thecyclized product. Moreover, UV/Vis data confirmed that the

Figure 1. Color changes observed for 1 (left) and 3 (right) (1 � 10�5 mol dm�3) in water/acetonitrile (3:1 v/v,MES 1� 10�1 mol dm�3) upon addition of nerve-agent mimics DCP, DFP, and DCNP. In each photograph fromleft to right; reactand, reactand +DCP, reactand +DFP, and reactand +DCNP.

Figure 2. Absorption spectra of chromoreagent 3 (1 � 10�5 mol dm�3) inwater/acetonitrile (3:1 v/v, MES 1� 10�1 mol dm�3) and 3 after the addi-tion of an excess of DCP.

Scheme 5. Chemical structure of cyclized derivatives 25 and 26.

Figure 3. 1H NMR (aliphatic zone) of ligand 1 and its correspondingcyclic compound 25.

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Chromo-Fluorogenic Detection of Nerve-Agent Mimics

products obtained by tosylation displayed the same spectro-scopic characteristics as the products obtained from the re-action of the corresponding chromoreagent with the nerve-agent simulants.

The UV/Vis spectrum of compounds 1–9 are character-ized by the presence of intense absorption bands centered inthe 300–430 nm range. The emission behavior of reagents 1–9 in acetonitrile and water–acetonitrile (3:1 v/v) mixtures(1 �10�5 moldm�3) was also studied (see Table 2 for data in

acetonitrile). Chromogenic reagents 1–3 are poorly fluores-cent, and no remarkable emission modulations were foundin the presence of nerve-gas simulants. Therefore, the stud-ies detailed below were only performed with the stilbene-de-rivatives 4–9. The photophysics of donor–acceptor stilbenes(similar to 4–7) and donor–donor stilbenes (similar to 8 and9) have been widely studied in the literature. In general, thespectroscopic behavior of such dyes is often determined byan intramolecular charge-transfer process upon optical exci-tation and the presence of different emitting states involvingplanar geometry and double-bond twist- and single-bondtwist states.[26] As expected for this class of dye compounds,4–9 show characteristics broad and structureless emissionbands with a large Stokes shift. The addition of OP1–OP4to 4–9 resulted in negligible changes in the emission-intensi-ty profiles of the reagents. In contrast, the addition of thenerve-agent simulants DCP, DFP, and DCNP, inducedchanges in the fluorescence behavior (see Table 2 for datawith DCP). The changes in fluorescence intensity upon reac-tion with DCP, comprise fluorescence enhancement (for 4,5, and 7) and fluorescence quenching (for 6 and 8/9). Thefluorescence-quenching factors are relatively low for 6 andlarge for 8/9, whereas fluorescence-enhancement factorsrange from small (for 4) to medium (for 5 and 7). In allcases, the presence of the simulant DCP induced the disap-pearance of the fluorescence band of the reactand, and thegrowth of a new emission band that is either red- or blue-shifted depending on the receptor used.

Similar emission studies were carried out for receptors 4–9 (1� 10�5 mol dm�3) in water–acetonitrile (3:1 v/v, MES 1 �10�1 mol dm�3) solutions. In this medium, 4, 5, and 7 werenot fluorescent, whereas 6 and mixtures of 8/9 were stillemissive. The addition of OP1–OP4 on 6 or 8/9 resulted innegligible changes in the fluorescence. Furthermore, thefluorescence behavior of 6 showed no significant changes in

the presence of nerve-agent simulants. In contrast, the addi-tion of DCP, DFP, and DCNP to 8/9 resulted in an enhance-ment of the emission. This result is displayed in Figure 4,which shows the emission spectra (lexc =280 nm) of 8/9 and8/9 in the presence of 4 equivalents of DCP in water/aceto-nitrile (3:1 v/v); a remarkable 3.5-fold enhancement of theemission intensity was observed.

The design using the formation of azo and stilbene dyes,shown in Scheme 2, in relation to the sensing response ob-served for compounds 1–9 is evident when one comparesthe performance of these derivatives with the model com-pound 10, for which no remarkable changes in the UV/Vis(Dl of <5 nm) nor in the emission spectra were observedupon the addition of the nerve-agent simulants.

To ascertain that the chromogenic and fluorogenic re-sponse observed was caused by a nucleophilic addition–in-tramolecular cyclization mechanism and not by a protona-tion of the nitrogen atom of the N,N-dimethylanilinemoiety, control experiments were carried out. Thus, the ad-dition of HCl at typical simulant concentrations to water–acetonitrile (3:1 v/v) solutions (MES, pH 5.6) of reagents 1–9 induced negligible changes in the UV/Vis and fluorescenceprofiles of the products. This experiment disables the possi-bility that the response observed was solely caused by thepartial hydrolysis of DCP.

Kinetic and Detection-Limit Studies

To achieve a better understanding of the reaction, the reac-tivity between 1–9 and DCP, DCNP, or DFP in bufferedwater–acetonitrile (3:1 v/v) solutions under pseudo first-order kinetic conditions, using an excess of the correspond-ing simulant, was studied. By monitoring the changes in theabsorbance intensity and by plotting ln ACHTUNGTRENNUNG[(A0-A)/A] versustime (in which, A0 is the final absorbance and A is the ab-sorbance at a given time), allowed us the determination ofthe rate constants (k) and the half-life times (t1/2 = ln2/k) forthe reaction of the nerve-gas simulants with reagents 1–9.

Table 2. Fluorescence properties of reagents 4–10 (1 � 10�5 mol dm�3) inacetonitrile and after the reaction with 100 equiv of DCP.

Reagent Reagent+DCP Enhancementlabs

max [nm] lemmax [nm] lem

max [nm] Ireagent+DCP/Ireagent

4 381 580 576 1.255 404 627 415 5.406 270 570 590 0.687 317 453 509 3.338/9[a] 304 430 390 0.03

[a] Arising from the photoconversion between 8 and 9, the studies werecarried out in mixtures of both compounds 8/9.

Figure 4. Emission spectra (lexc =280 nm) of 8/9 and 8/9 (10�5 mol dm�3)in the presence of DCP (4 eq.) in water/acetonitrile (3:1 v/v, MES 1�10�1 mol dm�3).

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Figure 5 shows the absorbance changes at the UV/Vis bandmaximum for dyes 1 and 4 with DCP and DFP, respectively.In all cases, the reaction is relatively quick, and completeUV/Vis changes are observed after a few seconds. It hasbeen suggested, in fluorogenic systems using similar cycliza-tion paradigms, that the reaction of the alcohol with thephosphate derivatives to form the phosphate-ester inter-mediate is slow relative to the intramolecular cyclization.However, for a given nerve-agent mimic, the rate constantsare quite different depending on the reactand used, suggest-ing that cyclization could also possibly play some role in theoverall rate constant of the reaction process.

Table 3 also includes the detection limits observed for thedetection of these chemicals determined from changes inthe UV/Vis spectra (for 1–6) and from changes in the emis-sion (for 8/9) in aqueous water–acetonitrile (3:1 v/v) mix-tures. The lower detection limits (DLs) have been observedfor the chromogenic reactand 1 (DLs of 1 � 10�4 mol dm�3

for DCP and DCNP) and especially for the fluorogenic re-actand 8/9 (DL of 1 � 10�5 mol dm�3 for DCP).

Sensing in Mixed Aqueous Environments and in Gas Phase

Motivated by the favorable chromogenic and fluorogenicsensing features shown by the compounds studied, we tooka step forward towards the potential use of these ligands forin-situ sensing and rapid-screening applications. With thisidea in mind, we simply adsorbed compound 1, 2, 3, 4, or 5on silica gel (resulting in modified yellow/orange solids con-taining the probes), and tested their chromogenic ability tonerve-agent simulants both in solution and in gas phase.Thus, in the first test, the colored silica support containingthe probe was prepared by adsorption of a (1 �10�5 mol dm�3) solution of the corresponding probe in water/acetonitrile (3:1 v/v) buffered at pH 5.6 (MES 1 �10�1 mol dm�3). These samples were put in contact with anacetonitrile solution containing DCP (CDCP =1.0 �10�3 mol dm�3), and a very rapid bleaching was observedover a few seconds. An additional assay was related to thepossible detection of vapors of the simulants. For this pur-pose, an ambient vapor containing a low amount of DCP(5 ppm) was prepared. At the same time, the orange/yellow-colored silica was placed on a column, and the air contain-ing the simulant was passed through. As the silica comes incontact with the air sample, it becomes colorless (see

Figure 5. Absorption changes at 410 nm upon reaction of 1 with DCP (left) and at 360 nm and upon reaction of 4 with DFP (right). The inset in bothcases shows the first-order kinetic plot.

Table 3. Detection limits and kinetic data.

Chromoreagent 1 2 3 4

Simulant DFP DCP DCNP DFP DCP DCNP DFP DCP DCNP DFP DCP DCNPDetection limit � 103ACHTUNGTRENNUNG[mol dm�3]

1.0 0.1 0.1 10 4.5 8.1 7 1.8 4.1 10 7.0 6.0

k [s�1] 0.20 0.13 0.061 0.38 0.22 5.11 0.86 0.52 1.35 0.57 0.28 173.2t1/2 [s] 3.5 5.3 11.4 1.8 3.1 0.1 0.8 1.3 0.5 1.2 2.5 0.004

Chromoreagent 5 6 7 8/9

Simulant DFP DCP DCNP DFP DCP DCNP DFP DCP DCNP DFP DCP DCNPDetection limit � 103ACHTUNGTRENNUNG[mol dm�3]

8.0 7.0 7.0 1.0 2.0 1.0 10 7.0 11 0.04 0.01 0.04

k [s�1] 0.86 0.15 0.46 693.1 14.4 346.5 0.69 0.23 1.64 31.5 18.2 77t1/2 [s] 0.80 4.5 1.5 <0.001 0.048 0.002 1 3 0.42 0.022 0.038 0.005

Chem. Asian J. 2010, 5, 1573 – 1585 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemasianj.org 1579

Chromo-Fluorogenic Detection of Nerve-Agent Mimics

Figure 6 for ligand 1). Similar bleaching responses were ob-served when the simulants DFP and DCNP were used,whereas the modified silica remained yellow-orange in the

presence of vapors of the OP1–OP4 derivatives. This simpletest suggests that this or similar chromogenic systems basedon the reactivity of group I in Scheme 1 might prove usefulfor the development of easy-to-use chromogenic alarmassays, and studies relating to real applications are beingperformed in our laboratory.

Conclusions

In summary, a family of new reagents for the chromo-fluoro-genic detection of nerve-agent simulants has been prepared.These chromoreagents display an intramolecular cyclizationreaction coupled with a color change upon interaction withcertain nerve-agent simulants. The sensing paradigm relieson the use of 2-(2-(N,N-dimethylamino)phenyl)ethanol or 2-(2-(N,N-dimethylamino)phenoxy)ethanol reactive sites thatare also part of the conjugated p-system in azo and stilbene-dye scaffolds. Furthermore, the synthesis is easy and the ap-proach is highly modular, bearing in mind that a number ofacceptor groups could be anchored to the I donor moieties.In all cases, the reaction of the reactants with the nerve-agent mimics is quick, and complete UV/Vis changes are ob-served after a few seconds. These reagents react only withDCP, DFP, and DCNP that show close chemical structuresto sarin, soman, and tabun, whereas they remain silent inthe presence of other organophosphorus derivatives, such as,OP-1–4. Finally, the fact that the probe retains its signalingabilities upon adsorption on silica and displays color modu-lations to nerve-agent simulants in both vapors and mixedaqueous solution, are additional issues of interest.

Experimental Section

General Procedures and Materials

THF was distilled from Na/benzophenone under Ar prior to use. Allother reagents were commercially available, and were used without pu-rification. Silica gel 60 F254 (Merck) plates were used for TLC. 1H and13C NMR spectra were recorded with the deuterated solvent as the lockand residual solvent as the internal reference. High-resolution mass spec-

tra were recorded in the positive ion mode on a VG-AutoSpec mass spec-trometer. UV/Vis spectra were recorded using a 1 cm path length quartzcuvette. All measurements were carried out at 293 K (thermostated).Fluorescence spectra were carried out in a Varian Cary Eclipse fluorime-ter.

2-(2-N,N-dimethylaminophenyl)ethanol (11): 2-Nitrotoluene (9.2 g,67.2 mmol), sodium phenoxide (0.06 g, 0.52 mmol), and para-formalde-hyde (0.9 g, 25 mmol, 95%) in DMSO (20 mL) were heated under stir-ring at 60–70 8C for 1 h. The cold reaction mixture was poured into water(20 mL) and extracted with ethyl ether. The combined organic phaseswere washed (with aq. NaCl), dried (using Mg SO4), evaporated, and theresidue was distilled (Kugelrohr, 160 8C, 0.2 mmHg) to give 2-(2-nitro-phenyl)ethanol (4.3 g, 37%). 2-(2-Nitrophenyl)ethanol (3.30 g,19.7 mmol), formaldehyde (4.83 mL, 32.3 mmol, 37 %), ethanol (200 mL,99%), and Pd/C (480 mg, 10%) were placed under an H2 atmosphereuntil the uptake of hydrogen ceased. After filtration through celite, thesolvent was evaporated, and the residue was dissolved in chloroform, andextracted with an aqueous solution of HCl (1 n, 2� 50 mL). The aqueousphase was basified with a saturated solution of Na2CO3 and extractedwith chloroform. After drying (using MgSO4), the solvent was evaporatedto give 2-(2-(N,N-dimethylamino)phenyl)ethanol (2.85 g, 84%) as a lightyellow oil. IR (neat): n =3367, 3059, 3020, 2937, 2860, 2826, 2785, 1597,1492, 1451, 1046, 945, 768, 749 cm�1; 1H NMR (400 MHz, CDCl3): d=

7.3–7.0 (m, 4 H), 5.3 (br. s, 1H), 3.85 (t, J =11.6 Hz, 2 H), 3.01 (t, J=

11.6 Hz, 2 H), 2.70 ppm (s, 6H); 13C{1H} NMR (100 MHz, CDCl3): d=

152.1, 136.0, 131.1, 127.6, 124.9, 120.0, 64.2, 44.9, 36.0 ppm; HRMS (EI):m/z (%) calcd for C10H15NO: 165.1154 [M]+ ; found: 165.1153.

2-((E)-5-(2-(4-nitrophenyl)diazenyl)-2-dimethylaminophenyl)ethanol (1):4-Nitroaniline (0.836 g, 6.05 mmol), concentrated sulfuric acid (1.25 mL,23 mmol), and water (8 mL) were stirred and slightly heated until the 4-nitroaniline was completely dissolved, and then placed in an ice bath (0–5 8C) for 10 min before a solution of NaNO2 (0.418 g, 6.05 mmol) inwater (4 mL) was added dropwise. After stirring for an additional10 mins at 0–5 8C, a solution of 11 (1.0 g, 6.05 mmol), concentrated HCl(1.3 mL, 15.1 mmol), and water (4 mL) was added dropwise during15 min. The resulting orange solution was stirred for 15 min in an icebath, and 15 min at room temperature, then was neutralized withNa2CO3 and extracted with CH2Cl2. The dried (using MgSO4) organicphases were evaporated to give a dark oil that was dissolved in ethyl ace-tate. Upon addition of hexane, a red dark precipitate appeared, identifiedas 1 (1.2 g, 63 %). M.p.: 98–100 8C; IR (neat): n=2948, 2873, 1591, 1517,1345, 1099, 1039, 858, 827 cm�1; 1H NMR (400 MHz, CDCl3): d =8.36 (d,J =9.0 Hz, 2H), 7.98 (d, J=9.0 Hz, 2 H), 7.85 (d, J =8.1 Hz, 1H), 7.81 (s,1H), 7.26 (d, J= 8.1 Hz, 1H), 3.95 (t, J =5.8 Hz, 2 H), 3.10 (t, J =5.8 Hz,2H), 2.83 ppm (s, 6H); 13C{1H} NMR (100 MHz, CDCl3): d =156.7, 155.9,148.8, 148.4, 135.4, 125.4, 124.7, 123.9, 123.2, 119.9, 63.8, 44.6, 35.8 ppm;HRMS (EI): m/z (%) calcd for C16H18N4O3: 314.1379 [M]+ ; found:314.1371.

2-(N,N-dimethylamino)phenol (12): 2-Nitrophenol (2.68 g, 19.3 mmol),formaldehyde (3.54 mL, 42.46 mmol, 36 %), and Pd/C (480 mg, 10%)were dissolved in absolute ethanol (43 mL), and placed under an H2 at-mosphere until uptake of hydrogen ceased. After filtration throughcelite, the solvent was evaporated, and the product was purified by subli-mation at 155 8C to give 2-(N,N-dimethylamino)phenol (1.81 g, 69%) as awhite solid. 1H NMR (300 MHz, CDCl3): d= 7.08 (dd, J =7.8 Hz, 1.5 Hz,1H), 6.96 (dt, J= 7.7 Hz, 1.5 Hz, 1 H), 6.84 (dd, J =8.07 Hz, 1.49 Hz, 1H),6.77 (dt, J =7.55 Hz, 1.55 Hz, 1H), 6.56 (s, 1H), 2.62 ppm (s, 6H);13C{1H} NMR (75 MHz, CDCl3): d=151.9, 141.0, 126.4, 121.1, 120.5,114.6, 45.5 ppm.

2-(2-Dimethylaminophenoxy)acetate (13): A mixture of 2-(N,N-dimethy-lamino)phenol (12) (1.81 g, 13.2 mmol), ethyl bromoacetate (1.50 mL,13.2 mmol, 98%), and caesium carbonate (12.9 g, 39.6 mmol) in acetoni-trile (100 mL) was refluxed overnight. The volatile materials were re-moved by concentration on a rotary evaporator, and the residue was par-titioned between water (100 mL) and EtOAc (100 mL). The organiclayer was separated, and the aqueous layer was extracted with EtOAc(2 � 100 mL). The combined organic extracts were dried over anhydrousmagnesium sulfate, filtered, and concentrated to give the title compound

Figure 6. Left: a glass tube with silica gel containing adsorbed 1. Right:silica gel containing 1 after passing through the tube (1000 mL) of aircontaining DCP (5 ppm).

1580 www.chemasianj.org � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Asian J. 2010, 5, 1573 – 1585

FULL PAPERSA. M. Costero, R. Martinez-M�Çez et al.

(2.54 g, 86 %) as a brown oil. 1H NMR (300 MHz, CDCl3): d=6.96–6.76(m, 3H), 6.74–6.64 (m, 1H), 4.62 (s, 1 H), 4.17 (q, J= 7.1 Hz, 2 H), 2.76 (s,6H), 1.21 ppm (t, J =7.1 Hz, 3H); 13C{1H} NMR (75 MHz, CDCl3): d=

169.6, 150.8, 143.3, 122.6, 122.5, 118.9, 113.5, 65.9, 61.6, 43.6, 14.5 ppm.

Ethyl 2-[(E)-5-(2-(4-nitrophenyl)diazenyl)-2-(dimethylamino)phenoxy]-acetate (14): 4-Nitroaniline (0.182 g, 1.29 mmol), concentrated sulfuricacid (0.5 mL, 9.2 mmol), and water (10 mL) were stirred and slightlyheated until the 4-nitroaniline was completely dissolved, then, the mix-ture was placed in an ice bath (0–5 8C) for 10 min before a solution ofNaNO2 (0.089 g, 1.29 mmol) in water (5 mL) was added dropwise. Afterstirring for an additional 10 min at 0–5 8C, a solution of the ethyl 2-(2-di-methylaminophenoxy)acetate (13) (0.287 g, 1.29 mmol) in ethanol wasadded dropwise during 30 min. The resulting orange solution was stirredfor 30 min in an ice bath, and 30 min at room temperature, and was neu-tralized with potassium acetate. The precipitate was filtered, and theaqueous phase was extracted with CH2Cl2. The precipitated and extractedproduct was purified by column chromatography with silica gel usinghexane/ethyl acetate (9/1, v/v) as solvent. The final product (0.28 g, 58%)was obtained as a dark red powder. 1H NMR (300 MHz, CDCl3): d=8.28(d, J =8.91 Hz, 2H), 7.88 (d, J =8.91 Hz, 2H), 7.61 (dd, J =8.5 Hz,2.1 Hz, 1H), 7.33 (d, J =2.1 Hz, 1H), 6.93 (s, 1H), 4.70 (s, 2H), 4.23 (q,J =6.7 Hz, 2 H), 2.99 (s, 3 H), 1.26 ppm (t, J=6.7 Hz, 3H); 13C{1H} NMR(75 MHz, CDCl3): d=168.8, 156.6, 150.0, 148.31, 148.0, 147.4, 147.3,125.1, 124.1, 123.3, 117.3, 103.9, 65.9, 61.9, 43.3, 14.6 ppm.

2-[(E)-5-(2-(4-nitrophenyl)diazenyl)-2-dimethylaminophenoxy]ethanol(2): To a solution of ester 14 (0.05 g, 0.1344 mmol) in dry THF (20 mL),LiAlH4 (0.27 mmol) was slowly added in portions at 0 8C. The reactionmixture was stirred at 0 8C for an additional 1 h. Then, the reaction waswarmed to room temperature, stirred for 1 h more, and then, water(1 mL) was carefully added. The mixture was stirred for 2 h, the precipi-tate was filtered off, and washed with THF (40 mL). The combined or-ganic solutions were dried with MgSO4, filtered, and evaporated. Thefinal product was purified by column chromatography with aluminumoxide using dichloromethane as solvent to give the title compound(0.042 g, 95%) as a dark red powder. 1H NMR (300 MHz, CDCl3): d=

8.41 (d, J =9.13 Hz, 2H), 8.04 (d, J =9.13 Hz, 2H), 7.69 (dd, J =8.5 Hz,2.2 Hz, 1 H), 7.58 (d, J =2.1 Hz, 1 H), 7.10 (d, J=8.5 Hz, 1H), 4.21 (t, J =

5.12 Hz, 2 H), 3.99 (t, J= 5.12 Hz, 2H), 3.02 ppm (s, 6H); 13C{1H} NMR(75 MHz, CDCl3): d=156.5, 151.5, 148.6, 147.2, 140.0, 125.1, 123.5, 123.0,118.0, 106.2, 77.8, 77.6, 77.4, 77.0, 72.1, 61.4, 43.9 ppm; HRMS (TOF):m/z (%) calcd for C16H19N4O4: 331.1406 [M+1]+ ; found: 331.1396.

Ethyl 2-[(E)-5-(2-(4-cyanophenyl)diazenyl)-2-(dimethylamino)phenoxy]-ACHTUNGTRENNUNGacetate (15): 4-Aminobenzonitrile (0.230 g, 1.91 mmol, 98 %), concentrat-ed sulfuric acid (0.5 mL, 9.2 mmol), and water (10 mL) were stirred andslightly heated until the 4-aminobenzonitrile was completely dissolved.The mixture was placed in an ice bath (0–5 8C) for 10 min, and then, a so-lution of NaNO2 (0.132 g, 1.91 mmol) in water (5 mL) was added drop-wise. After stirring for an additional 10 min at 0–5 8C, a solution of ethyl2-(2-dimethylaminophenoxy)acetate (13) (0.425 g, 1.91 mmol) in ethanol(20 mL) was added dropwise during 30 min. The resulting red solutionwas stirred for 30 min in an ice bath, and another 30 min at room temper-ature. Then, the resulting solution was neutralized with potassium ace-tate. The mixture was extracted with CH2Cl2. The product was purifiedby column chromatography with silica gel using hexane/ethyl acetate (8/2, v/v) as eluent. The final pure product (0.19 g, 27%) was isolated as anorange powder. 1H NMR (300 MHz, CDCl3): d=7.81 (d, J =8.78 Hz,2H), 7.68 (d, J =8.78 Hz, 2H), 7.57 (dd, J= 8.5 Hz, 2.1 Hz, 1 H), 7.30 (d,J =2.1 Hz, 1H), 6.88 (d, J=8.5 Hz, 1 H), 4.66 (t, J =6.9 Hz, 2H), 4.21 (q,J =7.1 Hz, 2 H), 2.95 (s, 6 H), 1.23 ppm (t, J=7.1 Hz, 3H); 13C{1H} NMR(75 MHz, CDCl3): d=168.8, 155.3, 150.0, 147.2, 146.9, 133.5, 123.7, 123.3,119.2, 117.2, 113.1, 104.0, 65.9, 61.8, 43.2, 14.6 ppm.

2-[(E)-5-(2-(4-cyanophenyl)diazenyl)-2-dimethylaminophenoxy]ethanol(3): To a solution of ester 15 (0.064 g, 0.182 mmol) in dry THF (20 mL),LiAlH4 (2 m) in THF (155 mL, 0.31 mmol) was slowly added at 0 8C. Thereaction was then stirred at 0 8C for 1 h, warmed to room temperature,stirred for another hour, and then water (1 mL) was carefully added. Themixture was stirred for 2 h, and the precipitate was filtered off, andwashed with THF (40 mL). The combined organic solutions were dried

with MgSO4, filtered, and evaporated. The product was purified bycolumn chromatography with aluminum oxide using dichloromethane assolvent to give the final compound (0.044 g, 79 %) as an orange powder.1H NMR (300 MHz, CDCl3): d=8.41 (d, J= 8.7 Hz, 2H), 8.04 (d, J=

8.7 Hz, 2H), 7.69 (dd, J =8.5 Hz, 2.2 Hz, 1H), 7.58 (d, J =2.1 Hz, 1H),7.10 (d, J=8.5 Hz, 1 H), 4.21 (t, J =4.42 Hz, 2 H), 3.99 (t, J =4.42 Hz,2H), 3.02 ppm (s, 6 H); 13C{1H} NMR (75 MHz, CDCl3): d= 155.3, 151.6,148.0, 147.6, 133.5, 123.4, 123.1, 119.1, 117.7, 113.4, 106.1, 72.0, 61.5,43.5 ppm; HRMS (EI): m/z (%) calcd for C17H19N4O2: 311.1508 [M+1]+ ;found: 311.1511.

Methyl 3-methyl-4-nitrobenzoate (18): Sulfuric acid (0.3 mL) was addedto a solution of 3-methyl-4-nitrobenzoic acid (5 g, 27.6 mmol) in metha-nol (12 mL). The mixture was refluxed for 5 h, and then the methanolwas evaporated. The reaction mixture was poured into water (25 mL)and extracted with ethyl ether. The combined organic phases werewashed with NaHCO3 and aqueous solution of NaCl. After drying (usingMgSO4), the solvent was evaporated to give methyl 3-methyl-4-nitroben-zoate (18) (4.8 g, 87 %) as a pale yellow solid. 1H NMR (400 MHz,CDCl3): d =8.03–7.97 (m, 3H), 3.97 (s, 3H), 2.63 ppm (s, 3H);13C{1H} NMR (100 MHz, CDCl3): d =165.3, 151.9, 134.0, 133.7, 133.5,128.0, 124.6, 52.7, 20.0 ppm.

Methyl 3-(2-hydroxyethyl)-4-nitrobenzoate (19): Methyl 3-methyl-4-nitro-benzoate (18) (4.0 g, 30.3 mmol), sodium phenoxide (0.026 g, 0.22 mmol),and para-formaldehyde (0.42 g, 12 mmol, 95 %) in DMSO (10 mL) wereheated under stirring at 60–70 8C for 1 h. The cold reaction mixture waspoured into water (20 mL) and extracted with ethyl ether. The combinedorganic phases were washed (with aq. NaCl), dried (using MgSO4),evaporated, and the residue was purified by flash chromatography(EtOAc/hexane, 1:2) to afford methyl 3-(2-hydroxyethyl)-4-nitrobenzoate(19) (0.9 g, 24%) and the recovered starting material (3.02 g, 75%).1H NMR (400 MHz, CDCl3): d=8.10 (s, 1H), 8.01 (d, J =8.5 Hz, 1H),7.90 (d, J= 8.5 Hz, 1H), 3.85 (s, 3H), 3.84 (t, J =6.4 Hz, 2H), 3.17 ppm (t,J =6.4 Hz, 2H); 13C{1H} NMR (100 MHz, CDCl3): d=165.3, 152.5, 134.0,133.9, 133.6, 128.6, 124.7, 62.3, 52.8, 35.7 ppm.

Methyl 4-dimethylamino-3-(2-hydroxyethyl)benzoate (20): Methyl 3-(2-hydroxyethyl)-4-nitrobenzoate (19) (1.0 g, 4.4 mmol), formaldehyde(1.1 mL, 7.4 mmol, 37 %), ethanol (54 mL, 99%), and Pd/C (110 mg,10%) were placed under an H2 atmosphere until the uptake of hydrogenceased. After filtration through celite, the solvent was evaporated, andthe residue was dissolved in chloroform, and was extracted with aqueoussolution of HCl (1 n, 2� 50 mL). The aqueous phase was basified with asaturated solution of Na2CO3 and was extracted with chloroform. Afterwashing (with aq. NaCl), and drying (using MgSO4), the solvent wasevaporated to give methyl 4-dimethylamino-3-(2-hydroxyethyl)benzoate(20) (0.95 g, 89 %) as a light yellow oil. 1H NMR (400 MHz, CDCl3): d=

7.79 (d, J= 8.4 Hz, 1 H), 7.77 (s, 1H), 7.01 (d, J= 8.4 Hz, 1H), 4.10 (br s,1H), 3.80 (s, 3 H), 3.79 (t, J= 5.9 Hz, 2H), 2.94 (t, J =5.9 Hz, 2H),2.66 ppm (s, 6 H).

2-(2-Dimethylamino-5-(hydroxymethyl)phenyl)ethanol (21): Methyl 4-di-methylamino-3-(2-hydroxyethyl)benzoate (20) (0.41 g, 1.84 mmol) inethyl ether (2 mL) was added dropwise to a solution of lithium aluminumhydride (0.07 g, 1.8 mmol) in ethyl ether (5 mL). The mixture was stirredfor 30 min, and then aqueous solution of ethyl ether and water wasadded. The solution was extracted with ethyl acetate. The combined or-ganic phases were washed (with aq. NaCl), dried (using MgSO4), evapo-rated, and the residue was purified by flash chromatography (EtOAc/hexane, 1:2) to give 2-(2-dimethylamino-5-(hydroxymethyl)phenyl)etha-nol (21) (0.34 g, 81 %). 1H NMR (400 MHz, CDCl3): d=7.11–7.05 (m,3H), 4.49 (s, 2H), 3.71 (t, J= 5.5 Hz, 2H), 2.88 (t, J= 5.5 Hz, 2H),2.58 ppm (s, 6 H); 13C{1H} NMR (100 MHz, CDCl3): d=151.4, 137.9,136.2, 130.2, 129.9, 126.3, 120.1, 64.5, 64.2, 45.0, 36.1 ppm.

N-Methoxy-N,3-dimethyl-4-nitrobenzamide (22): 3-Methyl-4-nitrobenzoicacid (17) (9.05 g, 50 mmol), tetrabromomethane (16.6 g, 50 mmol), trie-thylamine (7.0 mL, 50 mmol), and N,O-dimethylhydroxylamine hydro-chloride (5.63 g, 55 mmol), were stirred in dry dichloromethane (75 mL)under argon atmosphere. Then, a solution of triphenylphosphine (50 mL,1m) in dichloromethane was added within 5 min, and the reaction was al-lowed to run for 2.5 h. Hexane was added, and the precipitate was fil-

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Chromo-Fluorogenic Detection of Nerve-Agent Mimics

tered. The organic solvents were washed once with Na2CO3 (10 %) andNaHCO3 (10 %). Aqueous layers were extracted, and the combined or-ganic layers were dried (with MgSO4), evaporated, and purified by flashchromatography (EtOAc/hexane, 15:85) to give N-methoxy-N,3-dimeth-yl-4-nitrobenzamide (22) (6.93 g, 62%). 1H NMR (300 MHz, CDCl3): d=

7.89 (d, J =7.2 Hz, 2 H), 7.54 (s, 1H), 7.52 (d, J= 7.2 Hz, 1 H), 3,47 (s,3H), 3,30 (s, 3H), 2,53 ppm (s, 3H); 13C{1H} NMR (75 MHz, CDCl3): d=

168.2, 150.3, 138.8, 133.8, 132.9, 126.9, 124.7, 61.7, 33.6, 20.7 ppm; HRMS(EI): m/z (%) calcd for C10H12N2O4: 224,0797 [M]+ ; found: 224,0805.

3-(2-Hydroxyethyl)-N-methoxy-N-methyl-4-nitrobenzamide (23): N-me-thoxy-N,3-dimethyl-4-nitrobenzamide (22) (2.44 g, 10.9 mmol), sodiumphenoxide (0.010 g, 0.09 mmol), and para-formaldehyde (0.13 g, 4 mmol,95%) in DMSO (30 mL) were heated under stirring at 60–70 8C for 1 h.The cold reaction mixture was poured into water (30 mL) and extractedwith ethyl acetate. The combined organic phases were washed (with aq.NaCl), dried (using MgSO4), evaporated, and the residue was purified byflash chromatography (EtOAc:hexane, 70:30 to 80:20) to afford 3-(2-hy-droxyethyl)-N-methoxy-N-methyl-4-nitrobenzamide (23) (1.05 g, 41%) asa yellow oil. 1H NMR (300 MHz, CDCl3): d =7.85 (d, J=7.2, 2 H), 7.65(s, 1H), 7.59 (d, J =7.20 Hz, 1H), 3.88 (t, J =6.8 Hz, 2H), 3.49 (s, 3H),3.29 (s, 3H), 3.10 (t, J=7.2 Hz, 2 H), 1.81 ppm (bs, 1H); 13C{1H} NMR(75 MHz, CDCl3): d=168.2, 151.0, 138.6, 134.2, 133.0, 127.6, 124.8, 62.8,61.8, 36.3, 33.7 ppm; HRMS (EI): m/z (%) calcd for C11H14N2O5:254.0910 [M]+ ; found: 254.0903.

4-Dimethylamino-3-(2-hydroxyethyl)-N-methoxy-N-methylbenzamide(24): 3-(2-Hydroxyethyl)-N-methoxy-N-methyl-4-nitrobenzamide (23)(2.19 g, 8.61 mmol), formaldehyde (2.2 mL, 15.1 mmol, 37 %), ethanol(220 mL, 99 %), and Pd/C (200 mg, 10%) were placed under an H2 at-mosphere (pressure of 3.5 bar) until the uptake of hydrogen ceased.After filtration through celite, the solvent was evaporated, and the resi-due was dissolved again in ethyl acetate, dried (using MgSO4), andevaporated. Flash chromatography purification (EtOAc/hexane, 90:10)afforded 4-dimethylamino-3-(2-hydroxyethyl)-N-methoxy-N-methylben-zamide (24) (1.75 g, 80%). 1H NMR (400 MHz, CDCl3): d= 7.51 (dd, J=

8.3 and 2.1 Hz, 1H), 7.46 (d, J=2.0 Hz, 1 H), 7.09 (d, J =8.3 Hz, 1H),3.79 (t, J= 5.8 Hz, 2 H), 3.51 (d, J=3.0 Hz, 3H), 3.28 (s, 3H), 2.95 (t, J =

5.7 Hz, 2H), 2.67 ppm (s, 6H); 13C{1H} NMR (100 MHz, CDCl3): d=

169.5, 154.6, 135.3, 131.4, 130.0, 127.9, 119.3, 64.3, 61.0, 44.7, 36.0,33.9 ppm; HRMS (EI): m/z (%) calcd for C13H20N2O3: 252.1476 [M]+ ;found: 252.1474.

4-Dimethylamino-3-(2-hydroxyethyl)benzaldehyde (16): 2-(2-Dimethyla-mino-5-(hydroxymethyl)phenyl)ethanol (21) (0.25 g, 1.28 mmol), manga-nese dioxide (1.28 g, 14.7 mmol), previously activated in ethyl ether(10 mL), was stirred at room temperature for 2.5 h. The mixture was fil-tered and washed with chloroform and ethanol. The organic layers werecombined and evaporated to afford 4-dimethylamino-3-(2-hydroxyethyl)-benzaldehyde (16) (0.22 g, 75%) as yellow oil. In an alternative proce-dure, 4-dimethylamino-3-(2-hydroxyethyl)-N-methoxy-N-methylbenza-mide (24) (253.2 mg, 1 mmol) in dry THF (11 mL) was stirred underargon atmosphere at �78 8C. Then, a solution of DIBAL (3 mL, 1 m) wasadded dropwise, and the mixture was allowed to react during 45 min. Re-action is quenched with acetone (1 mL) and after 15 min, solution waspoured over water (2.5 mL). Mixture was extracted with EtOAc, and thecombined organic layers were dried (using MgSO4), and evaporated.Flash chromatography purification (EtOAc/hexane, 90:10) afforded 4-di-methylamino-3-(2-hydroxyethyl)benzaldehyde (16) (0.130 g, 67%) asyellow oil. 1H NMR (300 MHz, CDCl3): d=9.81 (s, 1 H), 7.62 (m, 3H),7.11 (d, J=9.2 Hz, 1 H), 3.81 (t, J =6.6 Hz, 2H), 3.42 (bs, 1H), 2.96 (t, J=

7.0 Hz, 2H), 2.72 ppm (s, 6H); 13C{1H} NMR (75 MHz, CDCl3): d=193.2,160.5, 136.5, 134.0, 133.8, 131.9, 121.4, 65.5, 46.3, 37.5 ppm; HRMS (EI):m/z (%) calcd for C11H16NO2: 194.1181 [M+1]+ ; found: 194.1178.

(E)-2-(2-dimethylamino-5-(4-nitrostyryl)phenyl)ethanol (4): (4-Nitroben-zyl)triphenylphosphonium bromide (149.5 mg, 0.31 mmol) in dry THF(1 mL) was stirred at 0 8C under argon atmosphere, and then, a solutionof BuLi (0.20 mL, 0.3 mmol, 1.6m,) was added dropwise. After 15 min, asolution of 4-dimethylamino-3-(2-hydroxyethyl)benzaldehyde (16)(0.25 mL, 0.25 mmol, 1 m) in dry THF was added at room temperature.24 h later, the reaction mixture was poured over a saturated solution of

NaHSO3. The aqueous layer was extracted with EtOAc, and the organiclayers were dried (using MgSO4), and evaporated. The reddish oil waspurified by flash chromatography (EtOAc/hexane, 30:70) to afford (E)-2-(2-dimethylamino-5-(4-nitrostyryl)phenyl)ethanol (4) (53 mg, 68%) as ared solid. 1H NMR (300 MHz, CDCl3): d=8.13 (dt, J=8.8 and 1.9 Hz,2H), 7.53 (dt, J =8.8 and 1.9 Hz, 2 H), 7.34 (dd, J =8.3 and 2.1 Hz, 1H),7.28 (d, J =2.1 Hz, 1H), 7.14 (d, J =16.3 Hz, H-8, 2 H), 7.12 (d, J= 8.0, H-11, 1H), 6.98 (d, J= 16.3 Hz, H-7, 1H), 3.82 (t, J =6.0 Hz, 2 H), 2.96 (t,J =5.6 Hz, 2H), 2.67 ppm (s, 6H); 13C{1H} NMR (75 MHz, CDCl3): d=

145.6, 143.0, 135.3, 131.7, 131.6, 128.7, 125.7, 125.5, 124.5, 123.1, 119.3,63.3, 43.8, 35.2 ppm; HRMS (EI): m/z (%) calcd for C11H15NO2: 312.1470[M]+ ; found: 312.1474.

(E)-4-(4-dimethylamino-3-(2-hydroxyethyl)styryl)-1-methylpyridiniumiodide (5): 4-dimethylamino-3-(2-hydroxyethyl)benzaldehyde (16)(30 mg, 0.16 mmol) and 1,4-dimethylpyridinium iodide (43 mg,0.19 mmol) in methanol (20 mL) were stirred until complete dissolution,and then, fresh distilled triethylamine (0.25 mL, 0.19 mmol) was added.The mixture was refluxed for 24 h, and then the reaction mixture was al-lowed to cool. Toluene (2 mL) was added, and the methanol was evapo-rated. The residue was diluted again with dichloromethane and dried(with MgSO4.). After evaporating the solvents, Al2O3 flash chromatogra-phy (MeOH/EtOAc, 20:80 to 40:60) afforded (E)-4-(4-dimethylamino-3-(2-hydroxyethyl)styryl)-1-methylpyridinium iodide (5) (mg, 58 %) asorange-ish solid. 1H NMR (400 MHz, [D4]MeOH): d= 8.73 (d, J =6.6 Hz,1H), 8.66 (d, J =6.8 Hz, 2H), 8.11 (d, J =6.9 Hz, 2 H), 7.91 (d, J =6.4 Hz,1H), 7.88 (d, J =16.3 Hz, 1H), 7.64 (d, J =2.1 Hz, 1H), 7.58 (dd, J =8.4and 2.2 Hz, 1H), 7.31 (d, J=16.2 Hz, 1 H), 7.19 (d, J =8.4 Hz, 1H), 4.29(s, 3 H), 3.85 (t, J =7.1 Hz, 2 H), 3.00 (t, J=7.0 Hz, 2H), 2.76 ppm (s,6H); 13C{1H} NMR (100 MHz, [D4]MeOH): d =157.1, 145.8, 143.1, 135.1,131.9, 131.2, 129.6, 128.9, 124.6, 121.8, 120.8, 63.3, 47.6, 45.0, 35.7,21.9 ppm; HRMS (EI): m/z (%) calcd for C11H15NO2: 283.1815 [M]+ ;found: 283.1810.

(E)-4-(4-dimethylamino-3-(2-hydroxyethyl)styryl)benzonitrile (6): 1-(Bro-momethyl)-4-cyanobenzene (1.96 g, 10 mmol) was dissolved in dry tolu-ene (25 mL), then, triphenylphosphine (2.62 g, 10 mmol) was added. Themixture was refluxed for 15 h, and was cooled. A white precipitate wasfiltered, washed with toluene, and crystallized with ethanol to afford (4-cyanobenzyl)triphenylphosphonium bromide[27] (3.92 g, 85%). (4-Cyano-benzyl)triphenylphosphonium bromide (329 mg, 0.72 mmol) in dry THF(21 mL) was stirred at 0 8C under argon atmosphere, and then, a solutionof BuLi (0.47 mL, 0.75 mmol, 1.6 m) was added dropwise. After 30 min, 4-dimethylamino-3-(2-hydroxyethyl)benzaldehyde (16) (111 mg,0.57 mmol) in dry THF (0.6 mL) was added at �78 8C, and the reactionwas then allowed to slowly reach room temperature. 18 h later, the reac-tion mixture was poured over a solution of NaHCO3 (10 %). The aqueouslayer was extracted with EtOAc, and the organic layers were dried (usingMgSO4), and evaporated. The fluorescent oil was purified by flash chro-matography (EtOAc:hexane, 40:60) to afford 4-(4-dimethylamino-3-(2-hydroxyethyl)styryl)benzonitrile (119 mg, 71 %) as a yellow solid, as adiastereoisomeric mixture (E/Z, 1:0.7).

(E)-4-(4-dimethylamino-3-(2-hydroxyethyl)styryl)benzonitrile (6):1H NMR (300 MHz, CDCl3): d=7.46 (d, J= 8.6 Hz, 2H), 7.29 (d, J=

8.3 Hz, 2 H), 6.98 (bs, 2H), 6.93 (bs, 1H), 6.59 (d, J=12.2 Hz, 1 H), 6.45(d, J=12.2 Hz, 1 H), 3.70 (t, J =5.4 Hz, 2H), 2.82 (t, J =5.7 Hz, 2H),2.64 ppm (s, 6 H); 13C{1H} NMR (100 MHz, CDCl3): d=141.2, 134.8,131.5, 131.4, 131.4, 131.0, 130.9, 128.5, 127.1, 125.8, 125.4, 119.3, 118.9,117.9, 109.5, 63.2, 44.1, 34.9 ppm; HRMS (EI): m/z (%) calcd forC19H20N2O: 292.1570 [M]+ ; found: 292.1576.

(E)-2-(4-dimethylamino-3-(2-hydroxyethyl)styryl)anthracene-9,10-dione(7): 2-(Bromomethyl)anthracene-9,10-dione (301 mg, 1 mmol) was dis-solved in dry toluene (2.5 mL), then, triphenylphosphine (262 mg,1 mmol) was added. The mixture was refluxed for 24 h, and then, themixture was cooled. A white precipitate was filtered, washed with tolu-ene, and crystallized with ethanol to afford ((9,10-dioxo-9,10-dihydroan-thracen-2-yl)methyl)triphenylphosphonium bromide[28] (459 mg, 82%).

((9,10-Dioxo-9,10-dihydroanthracen-2-yl)methyl)triphenylphosphoniumbromide (177 mg, 0,31 mmol) in dry THF (10 mL) was stirred at �78 8Cunder argon atmosphere, and then, a solution of BuLi (0.20 mL,

1582 www.chemasianj.org � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Asian J. 2010, 5, 1573 – 1585

FULL PAPERSA. M. Costero, R. Martinez-M�Çez et al.

0.33 mmol, 1.6m) was added dropwise. After 30 min, a solution of 4-di-methylamino-3-(2-hydroxyethyl)benzaldehyde (16) (0.25 mL, 0.25 mmol,1m) in dry THF was added at �78 8C, and then the reaction was allowedto slowly reach room temperature. 24 h later, the reaction mixture waspoured over a saturated solution of NaHSO3. The aqueous layer was ex-tracted with EtOAc, and the organic layers were dried (with MgSO4),and evaporated. The fluorescent oil was purified by flash chromatogra-phy (EtOAc/hexane, 30:70) to afford (E)-2-(4-dimethylamino-3-(2-hy-droxyethyl)styryl)anthracene-9,10-dione (7) (40 mg, 40%) as an oil.1H NMR (400 MHz, CDCl3): d =8.40 (d, J =1.6 Hz, 1 H), 8.34–8.30 (m,2H), 8.28 (d, J =8.1 Hz, 1H), 7.86 (dd, J=8.1 and 1.8 Hz, 1 H), 7.82–7.78(m, 2H), 7.44 (dd, J =8.3 and 2.1 Hz, 1H), 7.38 (d, J=2.0 Hz, 1H), 7.33(d, J =16.3 Hz, 1H), 7.21 (d, J =8.2 Hz, 1H), 7.14 (d, J =16.3 Hz, 1H),3.93–3.87 (m, 2H), 3.04 (t, J=5.6 Hz, 2 H), 2.74 ppm (s, 6H);13C{1H} NMR (100 MHz, CDCl3): d =183.3, 182.6, 168.9, 166.7, 152.8,151.5, 151.5, 143.4, 136.4, 134.2, 133.9, 133.9, 133.7, 133.6, 132.9, 132.4,132.0, 131.4, 129.8, 128.0, 127.2, 127.2, 126.5, 125.9, 124.6, 120.4, 64.4,44.9, 36,4 ppm; HRMS (EI): m/z (%) calcd for C26H23NO3: 397.1674[M]+ ; found: 397.1680.

2-(2-dimethylamino-5-(4-methoxystyryl)phenyl)ethanol (8,9): Phosphorustribromide (0.31 mL, 3.3 mmol) was added dropwise to a solution of (4-methoxyphenyl)methanol (1.39 g, 10 mmol) in dry dichloromethane(15 mL) at 0 8C, under argon atmosphere. 2 h later, the reaction mixturewas poured over a solution of NaHCO3 (15 mL, 1 m) and was extracted(with dichloromethane). The organic layers were dried (using MgSO4)and evaporated to afford the oily residue (1.81 g, 90%). The bromide(1.70 g, 8.5 mmol) was redissolved in dry toluene (22 mL), and then, tri-phenylphosphine (2.21 mg, 8.5 mmol) was added. The mixture was re-fluxed for 15 h, and was cooled. A white precipitate was filtered, washedwith toluene, and crystallized with ethanol/hexane to afford (4-methoxy-benzyl)triphenylphosphonium bromide[29] (3.56 g, 91 %). (4-Methoxyben-zyl)triphenylphosphonium bromide (88 mg, 0.19 mmol) in dry THF(4.5 mL) was stirred at �78 8C under argon atmosphere. Then, a solutionof BuLi (0.11 mL, 0.17 mmol, 1.6 m) was added dropwise. After 30 min, asolution of 4-dimethylamino-3-(2-hydroxyethyl)benzaldehyde (16)(0.15 mL, 0.15 mmol, 1 m) in dry THF was added at room temperature.16 h later, the reaction mixture was poured over a saturated solution ofNaHSO3. The aqueous layer was extracted with EtOAc, and the organiclayers were dried (using MgSO4), and evaporated. The oil was purifiedby flash chromatography (EtOAc/hexane, 30:70) to afford a mixture ofisomers, (E)-2-(2-dimethylamino-5-(4-methoxystyryl)phenyl)ethanol (8)(29 mg, 63%) and (Z)-2-(2-dimethylamino-5-(4-methoxystyryl)phenyl)e-thanol (9) (9 mg, 20%).

E Isomer (8): 1H NMR (400 MHz, CDCl3): d =7.36 (d, J =8.7 Hz, 2H),7.28 (dd, J =8.3 Hz, 2.1 Hz, 1 H), 7.21 (d, J=2.0 Hz, 1H), 7.10 (d, J=

8.3 Hz, 1H), 6.92 (d, J= 16.3 Hz, 1 H), 6.86 (d, 1 H), 6.82 (d, J =8.8 Hz,2H), 3.81 (t, J=5.4 Hz, 2H), 3.75 (s, 3H), 2.96 (t, 2 H), 2.67 ppm (s, 6H);13C{1H} NMR (101 MHz, CDCl3): d =158.2, 129.1, 129.1, 128.0, 126.8,126.6, 124.8, 124.5, 119.2, 113.1, 112.6, 95.3, 63.4, 54.4, 44.1, 35.4 ppm;HRMS (EI): m/z (%) calcd for C19H23NO2: 297.1729 [M]+ ; found:297.1728.

Z Isomer (9): 1H NMR (400 MHz, CDCl3): d =7.21 (d, J =8.6 Hz, 2H),7.13 (dd, J=8.2 and 2.0 Hz, 1H), 7.06 (d, J =2.0 Hz, 1H), 7.04 (d, J=

8.3 Hz, 1H), 6.77 (d, J= 8.8 Hz, 2 H), 6.49 (d, J =12.2 Hz, 1H), 6.41 (d,J =12.2 Hz, 1H), 3.79 (s, 3 H), 3.77 (t, J=5.5 Hz, 2 H), 2.91–2.84 (m, 2H),2.70 ppm (s, 6 H); 13C{1H} NMR (100 MHz, CDCl3): d=158.7, 150.7,135.8, 134.3, 131.7, 130.2, 130.1, 129.7, 129.4, 128.1, 119.7, 114.1, 113.6,64.5, 55.2, 45.0, 36.1 ppm; HRMS (EI): m/z (%) calcd for C19H23NO2:297.1729 [M]+ ; found: 297.1727.

2-(2-Dimethylamino-5-methoxyphenyl)ethanol (10): 4-Methoxy-2-methyl-1-nitrobenzene (2.99 g, 17.9 mmol), sodium phenoxide (0.030 g,0.26 mmol), and para-formaldehyde (0.24 g, 8 mmol, 95 %) in DMSO(10 mL) were heated under stirring at 60–70 8C for 1 h. The cold reactionmixture was poured into water (10 mL) and extracted with hexane. Thecombined organic phases were washed (with aq. NaCl), dried (usingMgSO4), and evaporated to afford an orange oil, 2-(5-methoxy-2-nitro-phenyl)ethanol (0.77 g, 39 %), which was used without further purifica-tion. 1H NMR (300 MHz, CDCl3): d =7.90 (d, J= 9.3 Hz, 1 H), 6.75 ACHTUNGTRENNUNG(s,

1H), 6.67 (d, J =9.3 Hz, 1 H), 3.79 (t, J =6.6 Hz, 2 H), 3.76 (s, 3H),3.04 ppm (t, J= 6.6 Hz, 2H); 13C{1H} NMR (75 MHz, CDCl3): d=163.4,142.6, 137.6, 128.1, 118.4, 112.9, 62.7, 56.2, 37.4 ppm.

2-(5-Methoxy-2-nitrophenyl)ethanol (1.05 g, 5.33 mmol), formaldehyde(1.5 mL, 18.5 mmol, 37%), absolute ethanol (70 mL), and Pd/C (100 mg,10%) were placed under an H2 atmosphere until the uptake of hydrogenceased. After filtration through celite, the solvent was evaporated, andthe residue was dissolved in chloroform, and extracted with an aqueoussolution of HCl (1 n, 2� 50 mL). The aqueous phase was basified with sa-turated Na2CO3 and extracted with chloroform. After washing (with aq.NaCl) and drying (using MgSO4), the solvent was evaporated and waspurified by flash chromatography (EtOAc/hexane, 50:50) to give 2-(2-di-methylamino-5-methoxyphenyl)ethanol (10) (0.84 g, 43 %) as a lightyellow oil. 1H NMR (300 MHz, CDCl3): d=7.14 (d, J= 8.7 Hz, 1H), 6.73(m, 2 H), 3.85 (t, J =5.5 Hz, 2H), 3.78 (s, 3H), 2.95 (t, J= 5.5 Hz, 2H),2.64 ppm (s, 6H); 13C{1H} NMR (75 MHz, CDCl3): d= 156.8, 145.2, 138.0,121.1, 116.8, 112.7, 64.2, 55.3, 45.3, 36.5 ppm.

Cyclic ammonium salt from 1 (25): To a stirred mixture of 1 (63 mg,0.2 mmol) and K2CO3 (56 mg, 0.4 mmol) in acetonitrile (1 mL), tosylchloride (36 mg, 0.2 mmol) in acetonitrile (0.5 mL) was slowly added.After stirring at room temperature for 6 h, toluene (5 mL) was added.The formed precipitate was filtered to give 25 (as its tosylate salt)(46 mg, 0.1 mmol) as a white solid. M.p.: >350 8C; IR (neat): n =3082,2961, 1628, 1398, 1381, 1127, 1007, 831, 701, 664 cm�1; 1H NMR(400 MHz, CD3OD): d=8.46 (d, J =8.8 Hz, 2H), 8.15 (m, 4 H), 8.02 (d,J =8.7 Hz, 1H), 7.72 (d, J=7.8 Hz, 2 H), 7.25 (d, J =7.8 Hz, 2H), 4.36 (t,J =7.2 Hz, 2 H), 3.65 (s, 6 H), 3.59 ppm (t, J=7.2 Hz, 2H); 13C{1H} NMR(100 MHz, CDCl3): d 155.2, 153.8, 149.0, 142.1, 140.4, 128.4, 125.6, 124.9,124.6, 123.6, 120.3, 118.2, 68.5, 54.0, 26.6, 19.9 ppm; HRMS (EI): m/z (%)calcd for C16H17N4O2: 297.1352 [M]+ ; found: 297.1355.

Cyclic ammonium salt from 4 (26): To a stirred mixture of 4 (31 mg,0.1 mmol) and triethylamine (14 mL, 0.1 mmol) in acetonitrile (1 mL),tosyl chloride (19 mg, 0.1 mmol) in acetonitrile (0.5 mL) was slowlyadded. After stirring at room temperature for 6 h, toluene (5 mL) wasadded. The formed precipitate was filtered to give 26 (as its tosylatesalt). 1H NMR (300 MHz, CD3OD): d=7.96 (d, J =8.7 Hz, 2H), 7.51 (m,4H), 7.43 (d, J =8.7 Hz, 1H), 7.16 (d, J =8.1 Hz, 2 H), 6.96 (d, J =8.1 Hz,2H), 3.99 (t, J =7.2 Hz, 2 H), 3.30 (s, 6H), 3.22 (t, J =7.2 Hz, 2H),2.09 ppm (s, 3 H); HRMS (EI): m/z (%) calcd for C18H19N2O2: 295.1441[M]+ ; found: 295.1439.

General Procedure for Detection-limit Determinations

To the corresponding receptor (1 � 10�5 mol dm�3) in water/acetonitrile(3:1 v/v, 3 dm3) buffered with MES (1 � 10�1 mol dm�3), increasingamounts of the corresponding simulant solutions were added. UV spectrawere recorded in 1 cm path length quartz cells at 20 8C (thermostated).Representation of absorbance at the appropriate wavelength versus con-centration of the simulant allows the calculation of the detection limit.

Detection in Solid-phase Experiments

Reagents 1, 2, 3, 4, or 5 (25 mL, 1� 10�5 mol dm�3) in water/acetonitrile(3:1 v/v, MES 1� 10�1 mol dm�3) were adsorbed on silica gel. The corre-sponding solid material was put in contact with a freshly prepared solu-tion of DCP (1.0 � 10�3 mol dm�3) in acetonitrile. A clear decolorationwas observed. Similar bleaching responses were observed when the simu-lants DFP and DCNP were used, whereas the modified silica remainedyellow-orange in the presence of vapors of the OP1–OP4 derivatives.Moreover, as a control experiment, the same materials were treated witha solution of pure acetonitrile without simulant. No color changes werefound.

Detection of DCP Vapors

Following a similar procedure as above, reagents 1, 2, 3, 4, or 5 (25 mL,1� 10�5 mol dm�3) in water/acetonitrile (3:1 v/v, MES 1� 10�1 mol dm�3)were adsorbed on silica gel. Air, containing a DCP (5 ppm), was pre-pared. The orange/yellow-colored silica, containing the corresponding re-agent, was placed on a column, and the air, containing the simulant, waspassed through. As the silica comes in contact with the air sample, it be-

Chem. Asian J. 2010, 5, 1573 – 1585 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemasianj.org 1583

Chromo-Fluorogenic Detection of Nerve-Agent Mimics

comes colorless. Similar bleaching responses were observed when thesimulants DFP and DCNP (5 ppm) were used, whereas the modifiedsilica remained yellow-orange in the presence of vapors of the OP1–OP4derivatives.

Acknowledgements

We thank the Spanish Government (projects MAT2009-14564-C04) andthe Generalitat Valencia (project PROMETEO/2009/016 andACOMP07/080) for support. S.R. is grateful to the Generalitat Valenci-ana for the fellowship. SCSIE (Universidad de Valencia) is gratefully ac-knowledged for all the equipment employed.

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Received: January 21, 2010Revised: March 9, 2010

Published online: May 28, 2010

Chem. Asian J. 2010, 5, 1573 – 1585 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemasianj.org 1585

Chromo-Fluorogenic Detection of Nerve-Agent Mimics