Organic Mass Spectrometry, 1973, Vol. 7, pp. 737 to 752. Heyden & Son Limited. Printed in Northern Ireland

HETEROCYCLIC STUDIES-XXX:"

APPLICATION OF MASS SPECTROMETRY TO THE STUDY OF COVALENT HYDRATION

JIM CLARK and A. E. CUNLIFFE Department of Chemistry and Applied Chemistry, University of Salford,

Salford, M5 4WT, Lancashire, England.

(Received 9 June 1972; accepted (revised) 4 December 1972)

Abstract-Comparison of the mass spectra of 3 ,Chydrated 4-trifluoromethylpteridine derivatives with those of corresponding anhydrous compounds shows that this technique can be used to deduce the position of covalent hydration in heterocyclic compounds.

It is less easy to demonstrate the presence of added water molecules in 5,6,7,8-dihydrated deriva- tives of ethyl pteridine-4-carboxylates, but spectra run at low ionising voltages give the required informa tion.

Two hydrates (3,4-hydrates of 4-ethoxycarbonyl- and 4-trifluoroniethyl-pteridine-2(lH)-thione) which are very much more thermally stable than usual are also discussed.

Metastable peaks and accurate mass measurements support many of the postulated fragmentation pathways.

MANY electron deficient nitrogen-heterocyclic compounds have been shown to undergo reversible addition of water molecules across polarised C=N linkages.2 This phenomenon is known as covalent hydration and is typified by the example of pteridine (I; X = H) which, in aqueous solution is in equilibrium with its 3,4- hydrate (11; X = H).3 Although many of the hydrates are very unstable and can only be studied in solution some stable isolable adducts have been made (see for example Refs. 4 to 6).

Techniques which have been used to determine the position and/or extent of hydration include U.V. spectroscopy, ionisation constants, chemical oxidation2 and n.m.r. spectro~copy.~ It is now shown that mass spectrometry can be used to determine the position of hydration of compounds which have sufficient thermal sta- bility.

4-Trifluoromethylpteridine (I; X = CF,) and many of its derivatives form 3,4-hydrates (11; X = CF,) which have been isolated and thoroughly characterised by existing methods.6 Mass spectra of ten of the anhydrous pteridines were published recently* and some of these are now compared with spectra of corresponding hydrates.

The 3,4-hydrate of 4-trifluoromethylpteridine-3,4-dihydro-4-hydroxy-4-trifluoro- methylpteridine (11; X = CF,)-is substantially dehydrated on sublimation ,6 so it is not surprising that the spectrum obtained from the hydrate (Fig. 1) contains all the peaks? observed in that of the anhydrous compound.* There are several new peaks including the base peak C,H,N,O (m/e 149), however, which are clearly derived from the hydrate, although the latter does not show a molecular ion at 70 eV. The initial loss of CF, from the molecular ion (nz/e 218) of (R1 = R2 = H) would only be strongly favoured if the OH group was at position 4 so that the resonance stabilised ion (m/e 149) ( a ; R1 = R2 = H) (base peak) is formed. (cf. fragmentation of tertiary

* For Part XXIX, see Ref. 1 . -t The peak at m/e 181 (Fig. 1) was incorrectly placed at m/e 180 in Fig. l a of Ref. 8.

737

738 J. CLARK and A. E. CUNLIFFE

Z q ( o m \\ I "

d rz

Heterocyclic studies-XXX 73 9

alcoholsg"). The position of hydration is therefore established. I o n a subsequently fragmented mainly by successive losses of CO and two molecules of HCN to give peaks at m/e 121 (C,H,N,) (ion b; R1 = R2 = H), 94 and 67, respectively.

This fragmentation pathway was confirmed by the spectra of the hydrates of the 7-methyl- (111; R2 = Me, R1 = H) and 6,7-diniethyl (111; R1 = R2 = Me) deriva- tives (Table l), which gave corresponding ions a at in/e 163 and 177 (base peak in

TABLE 1 . IMPORTANT PEAKS& IN THE MASS SPECTRA OF SOME HYDRATED AND ANHYDROUS PTERIDINE

DERIVATIVES

3,4-Dil1ydvo-4-hydroxy-7-metl~yl-4-fri~uorome~hylpteridine.-232 (l), 214 (46), 187 (21), 164 (11), 163 (loo), 146 (21), 119 (lo), 118 (17), 108 (19), 67 (ll), 59 (14), 57 (17), 56 (11).

3,4-Dil~vdro-4-liydroxy-6,7-dimethy1-4-tr~~uoromethylpreridine.-246 (l), 228 (53), 187 (46), 177 (loo), 160 (24), 146 (18), 119 (12), 100 (14), 91 (24), 77 (14), 69 (15), 53 (15), 52 (ll), 42 (68).

Ethyl 7-nzethylpteridin-4-carboxylate.-218 (12), 174 (26), 147 (ll), 146 (LOO), 145 (ll), 119 (41), 118 (37), 91 (22).

Ethyr 6,7-dimethylpreridin-4-carboxylafe.-232 (15), 188 (19), 161 (14), 160 (loo), 159 (ll), 146 (ll), 133 (42), 132 (28), 119 (14), 91 (33), 53 (26), 44 (18), 43 (lo), 42 (16), 41 (11).

Ethyl 2-chlovopteridin-4-carboxylate.-240 (6), 238 (14), 196 (1 l), 194 (32), 168 (33), 167 (14), 166 (loo), 165 (14), 131 (25), 105 (26), 104 (52), 77 (16), 53 (lo), 52 (36).

Ethyl 5,6,7,8-tetrahydro-6,7-dihydroxy-7-methylpteridin-4-carboxylate (a) at 70 eV.-254 (l), 21 8 (19), 174 (26), 173 (12), 147 (14), 146 (loo), 145 (14), 119 (39), 118 (39), 91 (22). (b) at 11 eV.-254 (18). 220 (27), 219 (45), 218 (49, 182 (27), 175 (15), 174 (loo), 147 (lo), 146 (18). Ethyl 5,6,7,8-tetrahydro-6,7-dihydroxy-6,7-diinethylpteridin-4-carboxylate-(a) at 70 eV.-268 (I), 232 (14), 188 (20), 161 (12), 160 (loo), 159 (lo), 133 (42), 132 (22), 91 (27), 53 (22), 42 (12). (b) at 11 eV.-269 (lo), 268 (36), 234 (37), 233 (72), 232 (loo), 189 (ll), 188 (81), 182 (36), 161 (17), 160 (36).

3,4- Dihydro-4-hyduoxy-4-ethoxycavbonylpteridin-2(1H)-thione (at 250", 70 eV; cf. Fig. 7).-254 (6), 236 (36), 192 (13), 182 (ll), 181 (81), 180 (15), 165 (27), 164 (loo), 163 (90), 137 (20), 136 (48), 122 (]I), 109 (18), 105 (18), 104 (45), 94 (151, 93 (161, 79 (11), 77 (191, 66 (101, 53 (151, 52 (451, 44 (12), 40 (13).

R. mle > 40; relative intensity > 10% (all molecular ions quoted).

each case), and b at m/e 135 and 149, respectively. The methyl derivatives did give molecular ions but they were of only about 1 % relative abundance.

Similar comparison of the spectrum (Fig. 2) of the 2-methoxy compound (IV) with that of its anhydrous analogue* revealed significant new peaks at m/e 248 (3 o/,) [MI+. and 179 (100%) with compositions of C,H,F,N,O, and C,H,N40,. The position of the base peak at m/e 179 [M - CF,]+ again revealed that the hydroxy group was on the same carbon atom as the CF, group so that the stabilised ion (c) was formed.

Another series of pteridine derivatives which form relatively stable hydrates consists of 4-ethoxycarbonylpteridine (V; X = R1 = R2 = H) and its derivatives. Unlike 4-trifluoromethylpteridines, however, these usually form 5,6,7,8-dihydrates

J. CLARK and A. E. CUNLIFFE

Heterocyclic studies--XXX 741

(Vl)>35 Spectra of the simple anhydrous compounds (V) have not been published, so these are described before the hydrates are considered.

([MI '' of 111)

H

1''

H

(V) .(VI)

Comparison of the spectrum of 4-ethoxycarbonylpteridine (V; X = R1 = R2 = H) with those of its 7-methyl (V; X = R1 = H, R2 = Me) and 6,7-dimethyl derivatives (V; X=H, R1 = R2 = Me), shows that all peaks above m/e 100 are shifted upwards by 14 and 28 mass units, respectively (Fig. 3 and Table 1). Clearly it is the pyrimidine

742 J. CLARK and A. E. CUNLIPFE

I I I I I I I I # I - 0 0 !2 P (Y

0 m 0

0 -

0 ‘0 cy

0 P N

0 N N

0 0 (Y

0 z

0 2

u .. E

0 2

0 N - 0 2

54

0 ‘0

0 P

Heterocyclic studies-XXX 743

ring which fragments preferentially and this is preceded by breakdown of the ester group according to Scheme 1. Successive losses of CH,CHO and CO are not common

1''

l+

C7H6Na (R1 == H, R* = Me) (S 1 C)6H5N3 (R1= H, R2 =.Me)

1-H*

C,H,N3 (R* = H, R2 =Me)

-* - R A W 1 N~c-c~c-~~_=c---R~

(4 C ~ H ~ N Z (R1 = Me)

SCHEME 1

in ethyl esters of aromatic but in this case they could yield ions (e ; R1 = R2 = H), (e ; R1 = R2 = Me) and (e; R1 = H, R2 = Me), which correspond to molecular ions of pteridine, its 6,7-dimethyl and its 7-methyl derivative, respectively. Pteridine fragments by two successive losses of HCN and 7-methyl pteridine by loss of HCN then MeCN,1O however, whereas the present ions of similar compositions both lose mainly HCN, H* and HCN or H., HCN and HCN. This suggests that the present ions have different distributions of excitation energy when compared with ions derived by direct ionisation of pteridine or methylpteridines. Breakdown of ions h chiefly by loss of R2CN is consistent with the fact that the 7-methyl derivative loses mainly HCN at this stage while the 6,7-dimethyl compound loses MeCN. The 2-chloro compound (V; R1 = R2 = H, X = Cl) broke down in exactly the same way (Table 1) with ions corresponding to e , f, g and h being linked by losses of C1- and ClCN instead of Ha and HCN.

7

744 J. CLARK and A. E. CUNLIFFE

5 i?

* - 0- N

Heterocyclic studies-XXX 745

Spectra of the dihydrates of 4-ethoxycarbonylpteridine and its 7-methyl and 6,7-dimethyl derivatives (VII; R1 = R2 = H) (VII; R1 = H; R2 = Me) and (VII; R1 = R2 = Me), respectively, when run with an ionising voltage of 70 eV (Fig. 4 and Table l), were almost identical with those (Fig. 3 and Table 1) of corre- sponding anhydrous compounds (V) except that each showed an additional peak of very low relative abundance (about 1 %) corresponding to the molecular ion of the dihydrate. This close similarity is partly due to the fact that the hydrates are largely thermally dehydrated on sublimation4 and partly to electron-impact induced dehydra- tion. When the spectra were run at 11 eV (nominal) (Fig. 4 and Table l), however, they showed molecular ions and other ions clearly attributable to the hydrates and of use for structure elucidation. Firstly, it was clear from the molecular ions that two molecules of water were added to the heteroaromatic system and secondly the common peak m/e 182, which appeared in the spectra of all three compounds and which was assigned structure j , showed that the 6- and 7-substituents were lost with the oxygen atoms (probably as R1-CO-CO-R2). Peaks at m/e [M - 34]+. indicated loss of two .OH radicals to give ions k , which lost two He atoms successively to give [M - 35]+ and [M - 36]f- ions. The latter (I) corresponded to molecular ions of the anhydrous compounds. The 11 eV spectra show appreciable [M + 11 peaks and the presence of [M + 1 - 34]+ ions indicates that the [M + 11 ions also lose the elements of hydrogen peroxide (Fig. 4 and Table 1).

The dihydrate of pteridine-4-carboxylic acid, which exists as the zwitterion (VIII) in solutionll and probably in the solid state, behaved very much like the correspond- ing ester (VII; R1 = R2 = H) except that even at 11 eVno molecular ion was observed. Nevertheless, the peak at m/e 178 due to ion m was clearly derived from the dihydrate and its abundance relative to the mje 176 peak (ion n) increased sharply at low ionising voltages (Fig. 5). M/e 132, corresponding to ionised pteridine, was a large peak under all conditions and much of this probably arises from pteridine produced by thermal decarboxylation and dehydration as well as by loss of CO, from ion n. The peak at m/e 134 in the 11 eV spectrum indicated that CO, loss also occurs from ion m to give the dihydropteridine ion (0). The base peak, m/e 44 was due to [CO,]'. ions.

Two compounds which have not been considered so far are the 2-thiones of the 4-trifluoromethyl (X; X = CF,) and 4-ethoxycarbonyl (X; X = C0,Et) series. These hydrates were known to exhibit a higher order of chemical and thermal stabil- ity5*12 than the other hydrates and this was attributed to urea type resonance stabilisa- tion. The situation was repeated in the molecular ions of the compounds which were much more stable (Figs. 6 and 7) than those of other hydrates and again resonance stabilisation can be postulated (Scheme 2).

The 4-trifluoromethyl derivative (X; X = CF,), ([MI+. m/e 250) lost a CF, radical to give the m/e 181 ion ( p ) whose structure is analogous to those from other trifluoro- methylpteridine hydrates. A minor fragmentation pathway (p -+ q -+ r ) proceeds by successive losses of HNCS and CO (Scheme 2). This breakdown resembles that of thiouracils.~J* A more important pathway ( p --f t 3 u -+ u) which involves successive losses of NH,, CO and HCN is readily rationalised (Scheme 2) if ionp first rearranges to another stabilised ion (s). It was shown that the loss of 17 mass units from m/e 181 -+ 164 involved NH, rather than OH when the N,N,O-trideuterio derivative lost 20 mass units at this stage.

2 e a

d c;;

Heterocyclic studies-= 747

( j ) m/e 182

B l+.

(VIII)

I -2H. t-

H (0) iy/e 134

748 J. CLARK and A. E. CUNLIFFE

Heterocyclic studies-XXX 749

750 J. CLARK and A. E. CUNLIFFE

HO+ ( p ) m/e 181 CCHsNr OS

8 -HNCS I H-N

*I-..

""x>

+o=c (s) m/e 181

-NI13 I S S = N

0-c ( t ) m/e 164 CBHzN30S

i -co I S==C=N x )

(u) m/e 136 C5HzN3S

*l-HCN

.S=C=N-EC-$ECH (0) m/e 109 C4HNtS

SCHEME 2

A spectrum of the corresponding ester (X; X = C0,Et) run quickly at 135" (Fig. 7) was almost identical from m/e 181 downwards with that of the trifluoromethyl compound (Fig. 6). It was clear that the loss of *CO,Et and CF,, respectively from these compounds gave identical ions (p ) which broke down in each case according to Scheme 2. If the ester was left in the heated source or the spectrum was run at a higher temperature e.g. 250", however, certain peaks, notably mle 236, 192, 164 and 163 (Table 1) increased in intensity relative to the molecular ion m/e 254. These peaks were clearly attributable to the anhydrous compound (XI), which was pro- duced by thermal dehydration and which broke down according to Scheme 3. In the case of the trifluoromethyl derivative the m/e 164 peak had been exclusively due to C,H,N,OS ions ( t ) but in the case of the ester its composition gradually changed

Heterocyclic studies-XXX 751

COzEt

(XI, [M] +’) m/e 236 CsHJ%OiS

CHO

(w) m/e 192

+co

-HNCS I ( u ) m/e 136

C ~ H ~ N B S

NGC ntle 104

SCHEME 3

from predominantly C,H,N,OS ions (1 ) to predominantly C,H,N,S ions (x) as the compound was kept in the source or the temperature was raised. It was clear that the ester hydrate, although still much more stable than most hydrates studied, was rather more prone to thermal dehydration than its trifluoromethyl analogue.

Although the results from the last two compounds suggest that hydrates which are chemically and thermally stable may be readily studied by mass spectrometry without interference from dehydration, this is not necessarily so. For example the published spectrumlo of ‘6-hydroxypteridine’ (XII) which exists as 7,8-dihydro-7-hydroxypteri-

J? H

din-6-(5H)-one (XIII) in the solid state and in solution,15 and which could not be dehydrated thermally,16 shows no trace of peaks attributed to the hydrated form. Thus further work on the factors which influence the relative ease of thermal and electron-impact-induced dehydrations is required before mass spectrometry can give predictable results in the study of covalent hydrates.

752 J. CLARK and A. E. CUNLIFFE

EXPERIMENTAL Compounds were synthesised by published methods; 3,4-dihydro-4-hydroxy-4-trifluoromethyl-

pteridine (11; X = CF,) and its 7-methyl and 6,7-dimethyl derivatives$ 3,4-dihydro-4-hydroxy-2- methoxy-4-trifluoromethylpteridine (1V) ;I2 ethyl 5,6,7,8-tetrahydro-6,7-dihydroxypteridin-4-carboxy- late and its 7-methyl and 6,7-dimethyl derivatives (most stable isorner~);~ ethyl pteridin-4-carboxylate and its 6- and 7-methyl derivative^;'^ ethyl 2-chloropteridin-4-carboxylate and ethyl 3,4-dihydro-4- hydroxy-4-ethoxycarbonylpteridin-2(lH)-thione (X; X = COaEt);5 5,6,7,8-tetrahydro-6,7-dihy- droxypteridin-4-carboxylic acid (VII1)II and 3,4-dihydro-4-hydroxy-4-trifluoromethylpteridin-2( 1H)- thione (X; X = CF,>.IZ

M.p.3 and other physical data were identical with those quoted in the original papers. Mass spectra were measured on an AEI MS-12 spectrometer with an ionising voltage of 70 eV

except where a voltage of 11 eV (nominal) is specified. Samples were introduced on a direct insertion probe into the source which was maintained at 150 to 160" except for compounds VIII (140") and X; (X = C0,Et) (135" and 250"). Accurate mass measurements were made on an AEI MS-902s spectrometer usually at a resolving power of 10,000. Compositions of ions determined in this way are indicated in reaction schemes or in the text.

Acknowledgement-We thank Mrs Ruth Maynard for accurate mass measurements and for preparing the line diagrams.

R E F E R E N C E S 1. J. Clark, Org. Muss Spectroni. 7, 225 (1973). 2. A. Albert and W. L. F. Armarego, Advan. Heterocyclic Chem. 4, 1 (1965). 3. D. D. Perrin, J . Chem. SOC. 645 (1962). 4. J. Clark, J. Cheni. Soc. ( C ) 313 (1968). 5. J. Clark and W . Pendergast, J. Chem. SOC. (C) 1124 (1968). 6. J. Clark and W. Pendergast, J . Chem. SOC. ( C ) 1751 (1969). 7. T. J. Batterham, J . Chem. Soc. (C) 999 (1966). 8. J. Clark and F. S . Yates, Org. Muss Spectrom. 5, 1419 (1971). 9(a) H. Budzikiewicz, C. Djerassi and D. H. Williams, Mass Spectrometry of Organic Compounds,

Holden-Day Inc, San Francisco, 1967, p. 97; (b) H. Budzikiewicz, C. Dierassi and D. H. Williams, Mass Spectrometry of Organic- Compounds, HoIden-Day Inc, San Francisco, 1967, p. 197.

10. 11. 12. 13. 14. 15. 16. 17.

T. Goto, A. Tatematsu and S . Matsuura, J . Org. Chem. 30, 1844 (1965). J. Clark, W. Pendergast, F. S. Yates and A. E. Cunliffe, J. Chem. SOC. (C) 375 (1971). J. Clask and F. S. Yates, J. Chem. Soc. (C) 2278 (1971). R. W. Reiser, Org. Muss Spectrom. 2,467 (1969). J. Clark, Z . Munawar and A. W . Timms, J . Chem. SOC. Perkin II 233 (1972). D. J. Brown and S. F. Mason, J. Chem. Soc. 3443 (1956). A. Albert, J . Chem. SOC. 2690 (1955). J. Clark, J . Chem. Sac. (C) 1543 (1967).