RCM

Letter to the Editor

To the Editor-in-ChiefSir;

Mass spectrometric behaviour ofsome 19-aza and 2-aza squalenederivatives

Obtaining new inhibitors of 2,3-oxi-dosqualene cyclase, a key enzyme inthe biosynthesis of sterols, repre-sents a rational approach for thefuture development of hypocholes-terolemic or antifungal drugs.

The formation of lanosterol inmammals and fungi starts from aninitial protonation of 2,3-oxidosqua-lene by an electrophilic residue ofthe enzyme, giving the C-2 carbon-ium ion intermediate, followed byits cyclization to a series of inter-mediates, the C-10, C-8, C-13 andC-20 carbonium ions or protosterylion. This latter undergoes backbonerearrangements to yield lanosterol.The stereochemistry and the impor-tance of the protosteryl ion in theinteraction with a nucleophilicgroup of the enzyme has become ofrenewed interest in recent years.1–3

With this in mind, various acyclic19-azasqualene derivatives, having a2,3-epoxide and with the nitrogen inthe position corresponding to theC-20 carbonium ion or the proto-steryl ion, were developed.4 Theybehaved as powerful inhibitors of2,3-oxidosqualene cyclase, somederivatives showing an irreversibleaction towards the enzyme of mam-mals. The presence of an epoxidering, together with an acyclic struc-ture is essential for activity, since19-azasqualene derivatives lackingthe 2,3-epoxide function were inac-tive on partially purified oxidosqua-lene cyclase from pig liver. Ourconclusions on the importance of anacyclic structure for specific activityhave been subsequently confirmedby Corey,5 who found that thecorresponding cyclic aza derivative,aminoprotosterol, shows a consider-ably weaker inhibitory activity com-pared with our 2,3-epoxy-19-azasqualenes.

The mass spectrometric behav-iour of some first-generation aza-

squalenes and their analogues, viz.2-azasqualene and 2-azasqualenederivatives, has been previouslystudied by us6 by electron impact(EI) experiments performed usingeither double focusing or ion trapinstruments. In both cases, fragmen-tation pathways well related to thestructure of the neutral species werefound, showing that mass spectrom-etry represents a powerful analyti-cal tool in this field. The presentcommunication deals with massspectrometric studies of 19-aza-18, 19, 22, 23 - tetrahydrosqualene(1), 2,3 - epoxy - 19 -aza -18,19,22,23 - tetrahydrosqualene (3)and 22,23 - epoxy - 2 - aza - 2,3 -dihydrosqualene (5) and theirrespective N-oxides (2, 4 and 6).

Compound 1 [(4E,8E,12E)-N -methyl - N - (4-methylpentyl) -4,9,13,17 - tetramethyl - 4,8,12,16 -octadecatetraenylamine] was syn-thesized as follows: 0.9 mmol ofN-methyl-4-methylpentylamine in10 mL of anhydrous methanol wascooled to 0 °C and NaBH3CN (0.54mmol) added under stirring; C22squalenoid aldehyde (0.45 mmol)dissolved in 2 mL of methanol wasthen added, the mixture brought toroom temperature, and stirred for4 h. The reaction mixture wasextracted with dichloromethane,after addition of brine, dried, andevaporated to dryness in vacuo. Theresulting oil was purified by flashchromatography using light petro-leum + diethylether (first 98:2, then96:4 and finally 94:6 v:v) to give135 mg (72% yield) of 19-aza-

18,19,22,23-tetrahydrosqualene 1,as a colourless oil.

Compound 2 [(4E,8E,12E) - N -methyl - N - (4-methylpentyl)-4,9,13,17 - tetramethyl - 4,8,12,16 -octadecatetraenylamine N-oxide]was synthesized as follows: 0.161mmol of compound 1 was dissolvedin 0.5 mL of methanol and 0.5 mLof 30% H2O2 added. The mixtureimmediately became turbid andfrothy and was left for 12 h understirring, during which time it pro-gressively cleared. Light petroleum(30 mL) was added, the two-phasesystem cooled to 0 °C, vigorouslystirred and MnO2 added in catalyticamounts to decompose unreactedH2O2. When this decompositionwas complete, the suspension wasdried over Na2SO4, filtered andconcentrated in vacuo. The resultingoil was purified by flash chromatog-raphy using acetone + isopropyla-mine (99:1 v:v) to remove traces ofunreacted 1, then methanol to give53 mg (76% yield) of 19-aza-18,19,22,23-tetrahydrosqualene-N-oxide 2 as a colourless oil.

(4E,8E,12E) - N - methyl - N -(4-methylpentyl) - 16,17 - epoxy -4,9,13,17 - tetramethyl - 4,8,12 -octadecatrienylamine (3), (4E,8E,12E) - N - methyl - N - (4-methyl-pentyl) - 16,17 - epoxy - 4,9,13,17 -tetramethyl - 4,8,12 - octadeca-trienylamine N - oxide (4),(4E,8E,12E,16E) - 20,21 - epoxy -N,N - dimethyl - 4,8,13,17,21 - pen-tamethyl - 4,8,12,16 - docosatetrae-nylamine (5) and (4E,8E,12E,16E) -20,21 - epoxy - N,N - dimethyl -4,8,13,17,21 - pentamethyl - 4,8,12,16-docosatetraenylamine N-oxide(6) were synthesized, purified andcharacterized as reported in theliterature.4,7

Because of the presence, in com-pounds 2, 3, and 4–6, of N-oxideand/or epoxide moieties, wethought it of interest for their char-acterization to use, in parallel toelectron ionization, a softer ion-ization technique, viz fast-atombombardment (FAB).8 Both EI andFAB measurements were per-formed on a double focusing,reverse geometry VG ZAB 2Finstrument9 (VG Analytical, Man-chester, UK). EI mass spectra wereobtained with an electron energy of70 eV (200 µA) at a source tem-perature of 180 °C. FAB8 massspectra were obtained by bombard-ing glycerol solutions of 1–6 with8 keV xenon atoms. Metastable ionstudies were performed by mass-

LETTER TO THE EDITOR 321

CCC 0951–4198/97/030321–04 $17.50 © 1997 by John Wiley & Sons, Ltd.

Compound 1 2 3 4Ionic Species FAB MIKE FAB MIKE FAB MIKE FAB MIKE

m/z m/z m/z m/z m/z m/z m/z m/z(R.A.%) (R.A.%) (R.A.%) (R.A.%) (R.A.%) (R.A.%) (R.A.%) (R.A.%)

[M+H]+ 416 / 432 / 432 / 448 /(31) (91) (100) (92)

[M+H–O]+ / / 416 416 / / 432 /(15) (100) (17)

[M+H–H2O]+ / / / / / 414 / 430(100) (100)

[M+H–C5H11]+.

345 345 / / / 361 / /Cleavage 1 (9) (40) (30)[M+H–(C5H11, O]+

./ / / 345 / / / 361

Cleavage 1–O (5) (9)Cleavage 2 129 129 / / / 129 / /

(100) (100) (70)Cleavage 2–O / / 129 129 / / 129 129

(100) (20) (100) (19)Cleavage 3 211 211 227 / / / 227 227

(15) (27) (9) (9) (3)Cleavage 3–O / / 211 211 / / 211 211

(12) (9) (10) (7)Cleavage 4 279 279 295 295 / 279 / 295

(6) (25) (2) (6) (46) (3)Cleavage 4–O / / / 279 / / / 279

(3) (2)Cleavage 5 / / / / / 196 / /

(47)Cleavage 6 / / / / / 290 / /

(21)[M+H–C3H7]+

./ / / / / 389 / /

(34)[M+H–C4H9]+

./ / / / / / / 391

(16)

Table 1. FAB mass spectra and MIKE spectra of the [M+H]+ ions of compounds 1–4

analysed ion kinetic energy (MIKE)spectrometry.10

The EI and FAB mass spectra ofcompound 1 are reported in Fig. 1.Under EI conditions (Fig. 1(a)), aquite abundant molecular ion isdetected at m/z 415, together withfragment ions at m/z 346, 344, 278,210 and 128. The ionic species atm/z 344 and 128 correspond tosymmetrical cleavages â to the Natom, leading to quaternary ammo-nium cations. The ions at m/z 346,278 and 210 originate from cleav-ages â to the double bonds withC5H9, C10H17 and C15H25 lossesrespectively. These decompositionroutes are in agreement with ourprevious findings:6 in fact for aza-squalene derivatives the cleavagesin the position â to the N atom andto double bonds are the mostfavoured processes under EIconditions.

Electron ionization is ineffective

for the structural characterizationof compounds 2–6. In all cases,molecular ions are of negligibleabundance and most of the total ioncurrent is due to ionic species of lowmass, possibly due to molecular ionsoriginating by pyrolysis processesaccompanying the process of sam-ple vaporization.

On the contrary, FAB leads to theproduction of abundant protonatedmolecules, but the low energy depo-sition arising from the enthalpy ofprotonation is only able to activatedecomposition reaction in loweryield (see as an example, Fig. 1(b)).For this reason, as well as to dis-criminate product ions from thechemical background due to thematrix, the employment of metast-able ion investigation on the proto-nated molecules becomes ofinterest.

In Table 1 the FAB mass spectraand the MIKE spectra of the corre-

sponding [M + H]+ ions of com-pounds 1–4 are reported. For all thecompounds, abundant protonatedmolecular ions are detected andfragment ions due to cleavages â tothe N atom are present, togetherwith those originating by cleavagesâ to the double bonds. The massesof these fragments clearly indicatethat the protonation reaction occurson the nitrogen atom, which repre-sents the more basic site of themolecules. For the N-oxide deriva-tives, compounds 2 and 4, loss ofoxygen is detected in the FAB massspectra, but only for 2 is it present inthe MIKE spectrum, while for 4 theloss of water, typical of an epoxygroup, is a more favoured decom-position process.

In Table 2 the FAB mass spectraand the MIKE spectra of the corre-sponding [M + H]+ ions of com-pounds 5 and 6 are reported. Forboth compounds, the protonated

322 LETTER TO THE EDITOR

Compound 5 6Ionic Species FAB MIKE FAB MIKE

m/z m/z m/z m/z(R.A.%) (R.A.%) (R.A.%) (R.A.%)

[M+H]+ 430 / 446 /(100) (100)

[M+H–O]+ / / 430 /(5)

[M+H–H2O]+ / 412 / 428(100) (100)

[M+H–C3H9N]+ / 371 / 387Cleavage 1 (10) (16)[M+H–C5H9O]+ / / / 361Cleavage 2 (5)[M+H–C4H11N]+ / 359 / /Cleavage 3 (21)[M+H–C10H17O]+ / 277 / 293Cleavage 4 (19) (4)

[C14H27N]+ / 209 / 209(19) (4)

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Table 2. FAB mass spectra and MIKE spectra of the[M+H]+ ions of compounds 5 and 6

Figure 1. (a) EI mass spectrum of compound 1 and (b) FAB mass spectrum of compound 1.

LETTER TO THE EDITOR 323

molecular ions are responsible forthe base peak of FAB spectra. Noother significant fragment ions areobserved for compound 5, whereasthe loss of oxygen gives rise to anionic species at m/z 430 for com-pound 6. In the MIKE spectra of[M + H]+ ions, the loss of water,leading to ions at m/z 412 for 5 and428 for 6, is the most favoureddecomposition channel, owing tothe presence of the epoxy group.Contrary to what is observed forcompounds 1–4, losses of neutralfragments containing the N atomare observed, leading to ionic spe-cies with charge retention on theepoxy-containing residue.

Acknowledgements

This work was supported byMURST (40% and 60%) and CNRgrants.

Maurizio Ceruti1 andRoberta Seraglia2*

1Dipartimento di Scienza e Tecno-logia del Farmaco, Via Pietro

Giuria 9, I-10125 Torino, Italy.2CNR, Area della Ricerca, Corso

Stati Uniti 4, I-35100 Padova, Italy.

* Correspondence to: R. SeragliaContract grant sponsor: MURST, ItalyContract grant sponsor: CNR, Italy

REFERENCES

1. L. Cattel, M. Ceruti, G. Balliano, F.Viola, G. Grosa, F. Rocco and P. Brusa,Lipids 30, 235 (1995).

2. L. Cattel and M. Ceruti, in Biochemistryand Function of Sterols, E. J. Parish andW. D. Nes (Eds), American Oil Chem-ists’ Society, Ch.1 (1997).

3. I. Abe, M. Rohmer and G. D. Prestwich,Chem. Rev. 93, 2189 (1993).

4. M. Ceruti, F. Rocco, F. Viola, G. Balliano,G. Grosa, F. Dosio and L. Cattel, Eur. J.Med. Chem. 28, 675 (1993).

5. E. J. Corey, D. C. Daley and H. Cheng,Tetrahedron Lett. 37, 3287 (1996).

6. M. Ceruti, R. Seraglia and P. Traldi,Rapid Commun. Mass Spectrom. 8, 59(1994).

7. F. Viola, M. Ceruti, G. Balliano, O.Caputo and L. Cattel, Il Farmaco 45, 965(1990).

8. M. Barber, R. S. Bordoli, R. D. Sedgwickand A. N. Tyler, J. Chem. Soc., Chem.Commun. 7, 325 (1981).

9. R. P. Morgan, J. H. Beynon, R. H.Bateman and B. N. Green, Int. J. MassSpectrom. Ion Phys. 28, 171 (1978).

10. R. G. Cooks, J. H. Beynon, R. M.Caprioli and R. G. Lester, MetastableIons, Elsevier, Amsterdam (1973).

Received 5 December 1996Accepted 3 January 1997

324 LETTER TO THE EDITOR