In 1910 the first deliberately targeted search for a new chemotherapeutic agent came to fruition when Paul Ehrlich introduced Salvarsan for the treatment of syphilis. This thesis presents a detailed review of the history and literature leading up to and following on from Ehrlich’s discovery and thoroughly investigates the chemistry of Salvarsan and related species. A series of arylarsonic acids was prepared and fully characterised by electrospray mass spectrometry, NMR and X-ray crystallography for six examples. A detailed analysis of the hydrogen bonding in crystals of these molecules showed that they adopt several characteristic motifs which govern the packing in the crystals. Two of the examples containing NH2 groups crystallised as zwitterions while one NH2 containing example containing other bulky groups was is its molecular form. Salvarsan (cyclo 3-amino-4-hydroxyphenylarsenic(I)) was prepared by several different methods and analysed in detail using high resolution electrospray mass spectroscopy. This showed that Salvarsan consists of small cyclic species of the type (RAs)n where R is 3-NH2-4-OHC6H3 and n is three or greater. The dominant species in an aqueous solution of Salvarsan were found to be (RAs)3 and (RAs)5 . A detailed analysis is presented of impurities in Salvarsan prepared by different methods and also in a sample of original commercial Salvarsan. Mixtures of (RAs)n and (R’As)n exchange R groups in aqueous solution at room temperature, as shown by ESI-MS. ESI-MS studies are reported for the oxidation product of Salvarsan, RAs(OH)2 (commercially known as Mapharsen) and related As(III) compounds. Oligomers involving As-O-As linkages were found in solution and one tetrameric example (R = 3-NO2-4-OHC6H3) was isolated and structurally characterised. Preliminary ESI-MS studies showed that As(III) species bind to thioredoxin, a possible target for the pharmaceutical activity of Salvarsan and its derivatives

Lloyd, Nicholas

English

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Molecular StructureThe Composition of Ehrlichs Salvarsan:Resolution of a Century-Old Debate**Nicholas C. Lloyd, Hugh W. Morgan,Brian K. Nicholson,* and Ron S. RonimusIn 1910, Ehrlich introduced the compound 3-amino-4-hydroxyphenylarsenic(i) (salvarsan, arsphenamine, or Ehr-lich 606) as a remedy for syphilis, a disease caused by thespirochaete bacterium Treponema pallidum. His methodicalsearch for a specific curative for an identified disease can beregarded as the introduction of targeted chemotherapy.Despite its long history and importance, the actualstructure of salvarsan is still debated.[1, 2] Ehrlich synthesizedthe compound by the reaction of 3-nitro-4-hydroxyphenyl-arsonic acid with dithionite.[3] This simultaneously reducedthe NO2 group to NH2 and AsV to AsI to give a material offormula 3-H2N-4-HOC6H3As (Scheme 1). The product wasisolated as the hydrated hydrochloride salt of formula 3-H2N-4-HOC6H3As·HCl·H2O. This synthesis was not always repro-ducible, and inevitably gave rise to sulfur-containing impur-ities, which may have accounted for the variable toxicity ofdifferent batches of salvarsan.[5] A two-step process thatinvolved initial reduction of the NO2 group by sodiumdithionite followed by reduction of AsV with hypophos-phorous acid was subsequently shown by Christiansen to leadto sulfur-free material (Scheme 1).[4]By analogy with azo compounds, Ehrlich assigned struc-ture 1 to the free base of salvarsan. Although As=As bondsare now known to be found only in sterically crowdedmolecules,[6] the As=As form is repeatedly cited, with text-books and reviews still giving structure 1.[1, 2] Various sugges-tions for the true structure of salvarsan have been proposed,including large polycyclic molecules[7] and polymers,[8] butlack strong supporting data.We now report the first definitive evidence for thecomposition of salvarsan based on electrospray ionizationmass spectrometric data. Salvarsan was synthesized by using amodification of the two-step synthesis first described byChristiansen.[4] The compound was isolated as the hydrochlo-ride, giving consistent yields of a pale-yellow material thatwas analyzed as the monohydrate, corresponding exactly toEhrlichs original reports.[9] We also isolated the mixedH2PO2/H2PO3 salt form (31P NMR spectroscopic data) bydirect precipitation from the reaction mixture.1H and 13C NMR spectroscopic data were recorded forsalvarsan, but were not particularly useful for structuralassignment; ESI MS proved much more practical for thispurpose. A typical spectrum is shown in Figure 1. Althoughthere are many peaks, they can be readily assigned. Threeclear series can be identified (Table 1). The first of these is themajor one and consists of [(RAs)n+H]+ peaks (R= 3-H2N-4-HOC6H3) for n= 3–8. Peaks corresponding to n= 3 and n= 5are clearly dominant in this series. A lesser series comprisesions generated by loss of R from (RAs)n, that is, [Rn1Asn]+for n= 4–8. There are also minor peaks, which can be assignedto species [(RAs)n+O+H]+ arising from mild oxidation,presumably through the insertion of an oxygen atom into theAsn ring. There is a significant peak ([R2As]+) which connotesa fragmentation product of the cyclic polyarsines (see below).Peaks corresponding to [RAs(OH)2+H]+ and [RAs(OH)]+arise from complete oxidative cleavage of As–As bonds.The small peak at m/z 459 is a doubly charged ion (asdetermined by the spacing in the high-resolution isotopeScheme 1. Two routes to salvarsan (1). The initial route of Ehrlich andBertheim involves simultaneous reduction of the nitro group and AsV(top arrow).[3] Christiansen’s two-step process of sequential reductionsis shown below. [4][*] N. C. Lloyd, Prof. H. W. Morgan, Prof. B. K. Nicholson,Dr. R. S. RonimusDepartments of Chemistry and Biological SciencesUniversity of WaikatoPrivate Bag 3105, Hamilton (New Zealand)Fax: (+64)7-838-4219E-mail: b.nicholson@waikato.ac.nz[**] We thank the Marsden Fund, administered by the Royal Society ofNew Zealand, for financial support.AngewandteChemie941Angew. Chem. Int. Ed. 2005, 44, 941 –944 DOI: 10.1002/anie.200461471  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheimpattern) and can be attributed to [(RAs)5+ 2H]2+. Similarly,there is a small overlapping contribution to the [(RAs)3+H]+peak at m/z 550 from doubly protonated [(RAs)6+ 2H]2+ions. There were no features in any of the mass spectra of thetype expected for higher-molecular-mass polymeric com-pounds.All the evidence indicates that salvarsan in solutionconsists of cyclic species (RAs)n, with n= 3 (2) and n= 5 (3) asthe preferred sizes. We are confident that the ESI MS dataprovide an accurate profile of the components of salvarsan insolution, and that the species observed are not artifactsgenerated in the mass spectrometer, for the followingreasons:1) In many other areas it has been demonstrated thatESI MS provides an accurate guide to solution speciation,as the chemical ionization process is mild and does notlead to significant fragmentation.[10] Ions present insolution are transferred directly into the source. This iswhy ESI is so much more informative than EI, FAB, orMALDI in speciation studies.2) The overall ESI MS profile does not change significantlywith varying skimmer cone voltage (20–100 V), withvarying pH, or with the different electrospray desolvationmechanisms of the two different instruments used. If thesmaller rings were fragmentation products of largermolecules, then the distribution of peaks would changemarkedly under the different conditions. Furthermore,when fragmentation of the specific rings was deliberatelyinduced in MS/MS experiments, the observed processesinvolved elimination of R, R2As, and R3As fragments,with no sign at all of conversion from larger into smallerrings.Whereas the ESI MS results are believed to give a truequalitative reflection of the composition of salvarsan, quanti-tative allocation to different ring sizes is less certain, as the ionsignal reflects ease of chemical ionization as well as relativeabundance.[10] However, each molecule in the series is chemi-cally ionized (protonated) on the same type of NH2 group.Therefore, a moderate correlation between signal intensityand the amount present should exist. Certainly it appears that(RAs)3 and (RAs)5 are the predominant molecules in themixture, with significant (RAs)4 and (RAs)6 present, and onlyminor representation from larger ring sizes.It is pertinent to ask why elucidation of the structure ofsalvarsan has proved so difficult, as it has been establishedthat other organoarsenic(i) compounds such as (PhAs)n and(MeAs)n can form rings (n= 5 and 6 for (PhAs)n, and n= 5together with a polymeric ladder form for (MeAs)n).[1] This isa result of its resilient physical features—the material is amixture of compounds; it does not crystallize in a formsuitable for X-ray crystallographic analysis; it is not volatile,so it cannot be examined by traditional mass spectrometricmethods; it is readily oxidized in solution (particularly atneutral or higher pH values[11]) ; and it has strong hydrogenbonding functional groups, which will complicate cryoscopicmass measurements (salvarsan forms gels in aqueous solu-tions at critical concentrations and pH values[4,12]).We have attempted to separate salvarsan into its constit-uent ring species with HPLC experiments. However, theseFigure 1. The ESI mass spectrum of salvarsan in H2O. Ions derivedfrom the different cycloarsines, (RAs)n, are indicated as values of n.Assignment of other ions is given in Table 1.Table 1: ESI mass spectral data for an aqueous solution of salvarsan.m/z Irel [%] Assignment[a]1465 2 [(RAs)8+H]+1356 2 [(RAs)8R]+1282 4 [(RAs)7+H]+1173 2 [(RAs)7R]+1115 3 [(RAs)6O+H]+1099 18 [(RAs)6+H]+990 3 [(RAs)6R]+932 5 [(RAs)5O+H]+916 90 [(RAs)5+H]+807 10 [(RAs)5R]+749 5 [(RAs)4O+H]+733 43 [(RAs)4+H]+624 10 [(RAs)4R]+550 100 [(RAs)3+H]+458.5 30 [(RAs)6+2H]2+291 61 [R2As]+218 50 [RAs(OH)2+H]+200 25 [RAs(OH)]+[a] R=3-NH2-4-HOC6H3-; see Figure 1Communications942  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org Angew. Chem. Int. Ed. 2005, 44, 941 –944have so far been unsuccessful, presumably because of the highreactivity and hydrogen bonding properties of the compound.The (RAs)n rings formed by salvarsan are of interest,especially the trimer, as an As3 ring does not seem to havebeen confirmed before, although examples of both (RP)3 and(RSb)3 are known.[13] It is striking that the trimer andpentamer appear to be the main forms when R= 4-hydroxy-3-aminophenyl, whereas the hexamer and pentamer are theonly characterized examples for R= phenyl.[1, 7] (We note thatWest and co-workers found a peak corresponding to[(PhAs)3]+ in the EI mass spectrum of (PhAs)n, but concludedit to be a fragmentation ion rather than a true component ofthe mixture.[14])It has been accepted that salvarsan in the clinicallyadministered form differs from the active form in vivo.Oxidation is needed to activate the compound.[15] This wasearlier assumed to give rise to what was originally formulatedas the oxide (RAs=O), but is undoubtedly RAs(OH)2 or acondensation product thereof.[12] In essence, salvarsan (RAs)nappears to serve as a slow-release source of RAs(OH)2. Thisoxidation process was confirmed by our ESI MS results. Peaksat m/z 218 and 200 can be assigned to [RAs(OH)2+H]+ and[RAs(OH)]+, respectively (Figure 1). As the sample wasexposed to air, these signals increased in intensity at theexpense of those from the various [(RAs)n+H]+ species, untilafter a few hours they completely dominated the spectrum.In conclusion, we have demonstrated for the first time thatsalvarsan consists of small rings (RAs)n, particularly 2 and 3,rather than the commonly written form 1. Although thisassignment is based mainly on ESI mass spectrometry of themixture because the properties of the compound have so farprecluded separation into components, we are confident thatthis work has provided an accurate description of thedominant structures of salvarsan.Experimental SectionESI mass spectra were recorded on VG Platform and Finnegan LCQmass spectrometers. For the former, the compounds were dissolved inthe appropriate solvent and injected through a Rheodyne injectorequipped with a 10-mL sample loop. A flow rate of 0.02 mLmin1 anda source temperature of 60 8Cwas used. Nitrogen was used as both thenebulizing and drying gas. For the LCQ instrument, the sample wasdirectly injected at 5 mLmin1 with a syringe pump. The capillarytemperature was set at 100 8C. Nitrogen was used as the drying gas,and argon for collisionally induced fragmentation. NMR spectro-scopic data were recorded with a Bruker Avance 300 spectrometer. 3-Nitro-4-hydroxyphenylarsonic acid (roxarsone) was purchased fromAldrich.Preparation of 3-amino-4-hydroxyphenylarsonic acid (adaptedfrom the method of Fargher[16]): 3-Nitro-4-hydroxyphenylarsonic acid(13.1 g, 0.05 mol) was dissolved in a solution of aqueous NaOH (1m,100 mL) and cooled to 0 8C in an ice/salt bath. Sodium dithionite(Na2S2O4) (30.25 g) was added in one portion with vigorous stirring.The solution effervesced. As soon as the color changed from orangeto pale yellow, concentrated aqueous HCl (12 mL) was added. Thismixture was held at < 0 8C until all frothing ceased and the productprecipitated from solution. The precipitate was filtered and washedtwice with ice-cold water to give crude 3-amino-4-hydroxyphenyl-arsonic acid (6.50 g, 56%) as a cream-colored solid. This was driedunder vacuum for 24 h.Purification of 3-amino-4-hydroxyphenylarsonic acid (adaptedfrom the method of Christiansen[4]): The crude product (6.0 g)obtained above was dissolved in dilute aqueous HCl (H2O (25 mL),concd HCl (2 mL)), and the solution was stirred with decolorizingcharcoal for 15 min. The mixture was filtered, and a solution ofsodium acetate (25%) was added to the filtrate until the solution wasno longer acidic to Congo Red. The solution was cooled to 4 8C for20 min. The resulting precipitate was collected by filtration and driedunder vacuum to yield 3-amino-4-hydroxyphenylarsonic acid (4.7 g,78%) as an off-white microcrystalline solid. 1H NMR ([D6]DMSO):d= 6.97, 6.81 ppm; 13C NMR: d= 114.5, 115.0, 119.0, 122.6, 137.8,148.3 ppm; ESI MS (H2O): positive ion m/z 234 [M+H]+, 449[2MH2O+H]+, 664 [3M2H2O+H]+; negative ion m/z 232[MH] , 465 [2MH] , 698 [3MH] . Elemental analysis calcdfor C6H8AsNO4: C 30.90, H 3.43, N 6.01%; found: C 30.76, H 3.41,N 6.32%.Preparation of salvarsan (3-amino-4-hydroxyphenylarsenic(i))(adapted from the method of Christiansen[4]): All solvents andsolutions were degassed and the reaction was carried out undernitrogen. 3-Amino-4-hydroxyphenylarsonic acid (2.3 g, 0.01 mol) wasdissolved in a solution of hypophosphorous acid (14 mL, 50%) inwater (73 mL). An aqueous solution of KI (1 mL, 3%) was added,and the solution was heated gradually to 55 8C and held between 55and 608C for 90 min. The mixture was cooled to 10 8C and pouredwith vigorous stirring into a cold mixture of HCl/H2O (1:1, 164 mL).Salvarsan precipitated as a pale yellow gelatinous solid, which wascollected and dried under vacuum as the hydrochloride. Elementalanalysis calcd for C6H6AsNO·HCl·H2O: C 30.37, H 3.80, N 5.91%;found: C 30.66, H 3.70, N 5.62%. Alternatively, ethanol was added tothe cooled reaction mixture after the heating step with vigorousstirring, until a precipitate formed. The solid was collected and driedas above, and was characterized as a mixed hypophosphite/phosphitesalt of 3-amino-4-hydroxyphenylarsenic(i). 31P NMR (D2O): d=9.7 ppm (t, 1JH-P= 536 Hz) H2PO2 , d= 4.1 ppm (d, 1JH-P= 648 Hz)HPO32 ( 5:1). Elemental analysis calcd for C6H6AsNO·H2-PO2·H2O: C 26.97, H 4.12, N 5.24%; found: C 27.50, H 3.92,N 5.14%.Received: July 29, 2004Revised: October 8, 2004Published online: December 29, 2004.Keywords: arsenic · arsphenamine · mass spectrometry ·salvarsan · structure elucidation[1] O. M. N. Dhubhghaill, P. J. Sadler, Struct. Bonding (Berlin) 1991,78, 129.[2] A. Lykknes, L. Kvittingen, J. Chem. Educ. 2003, 80, 497; A. S.Levanson, J. Chem. Educ. 1977, 54, 98.[3] P. Ehrlich, A. Bertheim, Ber. Dtsch. Chem. Ges. 1912, 45, 756.See also W. G. Christiansen, J. Am. Chem. Soc. 1921, 43, 2202;for a review, see: S. Riethmiller, Bull. Hist. Chem. 1999, 24, 28.[4] W. G. Christiansen, J. Am. Chem. Soc. 1920, 42, 2402.[5] W. G. Christiansen, J. Am. Chem. Soc. 1922, 44, 847; H. King, J.Chem. Soc. 1921, 119, 1107; H. King, J. Chem. Soc. 1921, 119,1415; R. G. Fargher, F. L. Pyman, J. Chem. Soc. 1920, 117, 370.[6] B. Twamley, C. D. Sofield, M. M. Olmstead, P. P. Power, J. Am.Chem. Soc. 1999, 121, 3357.[7] L. R. Smith, J. L. Mills, J. Organomet. Chem. 1975, 84, 1; T.Klapotke, Biol. Met. 1988, 69; C. Elschenbroich, A. Salzer,Organometallics: a Concise Introduction, 2nd ed. , VCH, Wein-heim, 1992, p.158.[8] M. Ya. Kraft, E. B. Agracheva,Dokl. Akad. Nauk Az. SSR 1955,100, 279 (CAN 50:8273); M. Ya. Kraft, I. A. Bashchuk, Dokl.Akad. Nauk Az. SSR 1949, 65, 509 (CAN 45:16367).AngewandteChemie943Angew. Chem. Int. Ed. 2005, 44, 941 –944 www.angewandte.org  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim[9] P. Ehrlich, A. Bertheim, US Patent 986148, 1911, (CAN5:10870).[10] R. B. Cole, Electrospray Ionization Mass Spectrometry, Wiley,New York, 1997; W. Henderson, B. K. Nicholson, L. J. McCaf-frey, Polyhedron 1998, 17, 4291; R. Colton, A. DAgostino, J. C.Traeger, Mass Spectrom. Rev. 1995, 14, 79; N. B. Cech, C. G.Enke, Mass Spectrom. Rev. 2001, 20, 362.[11] C. Voegtlin, H. W. Smith, J. Pharmacol. Exp. Ther. 1920, 16, 199;R. P. Hogan, H. Eagle, J. Pharmacol. Exp. Ther. 1944, 80, 93.[12] G. O. Doak, H. G. Steinman, H. Eagle, J. Am. Chem. Soc. 1941,63, 99; P. V. Ioannou, Appl. Organomet. Chem. 2000, 14, 261.[13] C. Frenzel, E. Hey-Hawkins, Phosphorus Sulfur Silicon Relat.Elem. 1998, 143, 1; H. J. Breunig, R. Rosler, E. Lork, Organo-metallics 1998, 17, 5594.[14] P. S. Elmes, S. Middleton, B. O. West, Aust. J. Chem. 1970, 23,1559.[15] C. Voegtlin, Physiol. Rev. 1925, 5, 63; H. Eagle, G. O. Doak,Pharm. Rev. 1951, 107.[16] R. G. Fargher, J. Chem. Soc. 1919, 115, 982.Communications944  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org Angew. Chem. Int. Ed. 2005, 44, 941 –944

A Reinvestigation of Salvarsan and Related Arsenic Chemistry.

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