Biomedical Mass Spectrometry 1W6,3, 127 to I36

Mass Spectral Studies on Prostaglandins IV-Prostaglandin

FZa

GYULAHORVATH Research Institute for Pharmaceutical Chemistry, H-I325 Budapest. Pf. 82. Hungary (Received 15 December 1975) Abstract-The mass spectroscopic behaviour of prostaglandin FZ, and its methyl ester has been studied. Mechanisms are suggested for the formation of the prominent ions in the spectra of these compounds. The proposed fragmentation pathways have been confirmed with the aid of low electron voltage spectra, measurements on metastable ion decompositions, high resolution mass measurements and deuterium labelling.

TABLE1. Data of measurements on metastable ion decompositions of compounds 1 and 2

Introduction IN THE previous papers of this series'-3 a mechanistic interpretation of the mass spectral fragmentations of some prostaglandins and their analogues has been attempted. In the present paper studies on the mass spectroscopic behaviour of prostaglandin F,, are reported. The increasing interest in pharmaceutical applications of prostaglandin F,, is also reflected in the large number of communications dealing with the mass spectrometry of its various derivative^.^^^ Therefore, it is surprising that no reports appear to have been published on the mass spectrum of the parent compound itself. As regards methyl prostaglandin F,=, its partial mass spectrum6 and some spectral data' were reported. Nevertheless. a reliable interpretation on the characteristic features of the spectrum of this compound has not yet been reported. This situation prompted the reporting here of mass spectral investigations on prostaglandin F,= (1) and its methyl ester (2). The mechanisms proposed for the fragmentation of 1 and 2 can also be applied to other F-type prostaglandins. H 9 O R =

I

Scheme

Symbol

1

336 350 332

2

289 204

1 1

2

306 263 191

1 1

2

R=H

191

2

165

1

137

1

192

1

2 2

71 264 278 137 153 1-04 221 191 191 194

1

193

1

OH

2 2 1 1

Experimental

2

Prostaglandin F,, (1) was a standard sample of the Upjohn Co. Esterification of 1, yielding compound 2, was performed with diazomethane by the usual procedure. Deuterium labelling was accomplished by direct exchange with CH30D + D,O (1 :2). Mass spectra were taken on a Varian MAT SM-1 instrument under the following operating conditions : resolution, 1250; accelerating voltage, 8 kV; electron energy, _ _ 70 or 12.5 eV; electron current, 300 PA; source temperature, 150 "C ; evaporation temperatures (in "C), 1: 140, 160(12SeV);2: 120, 140(12.5eV). 0 Heyden & Son Ltd. 1976

m,

2

2 R=CH, HO

Compound

1

a

127

w = weak, m

=

2

177

1

196

medium; s

=

Voltage ratio

rn,

1.054 1.051 1.108 1.054 1.100 1.647 1.560 1.089 1.144 1.277 1.665 1.528 1.094 1.834 1.740 1.601 1.168 1.094 1.854 1.684 1.268 1.928 1.526 1.206 1.657 1.094 1.391 1.272 1.258 2.031 1.725 1.295 1.257 1.382 1.455 1.805 1.433 1.652 1.369 1.094 1.876 1.571 1.713 1.347

354 368 368 350 318 336 318 222 350 336 318 292 209 350 332 306 223 209 306 278 209 264 209 165 318 210 99 336 350 278 264 264 278 264 278 350 278 319 264 211 332 278 336 264

Intensiff

strong; vs = very strorlg.

m s W

vs m W

S

s S

m S

m vs s S

m m VS

m m vs m m m S

m vs m m W

W

vs VS

S S W VS

W

S

m W

VS

W

vs

GY. HORVATH

128

For low energy electron ionization, the electron accelerating voltage was set to the ionization potential of oxygen (c. 12.5 eV). Measurements on metastable ion decompositions were performed by scanning the accelerating voltage. The voltage ratios given in Table 1 are those of the voltages necessary to observe daughter ions formed from the appropriate metastable precursor ions vs normal operating voltage (each value was the average of five measurements). High resolution mass measurements were performed at a resolution of 10 OOO (10% valley) using PFK as the reference standard.

Results and discussion The mass spectra of compounds 1 and 2 are shown in Figs. 1 and 2, respectively. It should be noted that 1 and 2 are fairly stable thermally: their spectra do not change upon increasing the evaporation temperature even by 50 "C above the lowest applicable value. Data of keasurements on metastable ion decompositions are listed in Table 1 ;data of high resolution mass measurements are given in Table 2.

The fragmentation pattern of 1 and 2 can be rationalized by applying the principles outlined in the previous papers of this series.'*2 However, these compounds undergo a range of processes affording prominent ions, which have no analogues in the spectra of A- or E-type prostaglandins. These special features will be discussed here in detail, while argument is limited in the case of fragmentation mechanisms which resemble closely those of the corresponding processes in prostaglandins El and E,. As regards these latter processes, it should be emphasized that the mass spectroscopic data obtained on the relevant ions of 1 and 2 are in accordance with the mechanisms suggested previously.' The loss of water from the molecular ion of 1 and 2 is suggested to proceed analogously to the same process in prostaglandins E.' As shown in Scheme 1, this involves the interaction of the C-11 and C-15 hydroxyl groups. Accordingly, this process corresponds almost exactly to the elimination of D,O in the spectra of the 0-deuteriated derivative of both 1 and 2. An intense metastable peak could be observed for the transition [MI: -+a in both compounds (Table 1). The loss of water from ion a gives rise to abundant ions at inle 318 and m/e 332 in the spectra of 1 and 2,

TABLE 2. Data of high resolution mass measurements on ions of compounds 1 and 2 Mass decimals Scheme

Symbol

Compound

mle

Measured

Calculated

2

350 289 283 265 279 204" 292 2 74 191" 192 165" 165 137

0.24357 0.21805 0.15304 0.14555 0.15941 0.1 1464 0.20208 0.19299 0.14412 0.14952 0.12788 0.09131 0.13384

0.24571 0.21676 0.15455 0.14399 0.15964 0.1 1503 0.20385 0.19328 0.14359 0.15143 0.12794 0.09156 0.13303 0.08099 0.08608 0.20893 0.21228 0.22793 0.09 156 0.14085 0.07043 0.06260 0.05478 0.05478 0.18780 0.15416 0.17998 0.13068 0.12286 0.16433 0.14633

1 1

1

2 1 1 1

1 1

2 2 1 2 1 1 1 2 1 1 1 1 1 1

1 2 1 2 1

2 1

99"

0.08079

71" 264 265" 279" 153 150 81 80 79 67 204 1 21 191 194 193" 177 196

0.08632 0.20923 0.2 1 184 0.2296 1 0.09051 0.14004 0.07094 0.06229 0.05427 0.05465 0.18742 0.15295 0.18032 0.13075 0.12277 0.16367 0.14613

Elemental composition

14 (ppm)

6.1 4.5 5.3 C1SH2305 5.9 ' S H 2 lo, 0.8 C16H2304 1.9 C13H1602 6.1 C18H2803 2.6 C18H2.5O2 2.8 ' 3H'91 9.9 C13H200 0.4 CllHl7O 1.5 C10H1302 5.9 '10H17 2.0 C6HllO 3.4 ( 3 1 1 1.1 ' 7H2802 1.7 C16'3CH2802 6.0 cl 7 1 3 C H 3 ~ 0 2 6.9 'gH2'31 5.4 CllHlS 6.3 C6H9 3.9 6.5 C,H 7 1.9 CSH7 1.9 C15H24 5.5 C14H2lO2 1.8 C14H23 0.4 C12Hl802 0.5 C12H1702 3.7 C13H21 '2 4'43Hl

C19H2902

1

1

1

C12H2oO2

1.O

'This is the major component of the doublet at this mie value. (The minor components of some doublets have been omitted as they were of negligible abundance or corresponded to an isotopic peak.)

PROSTAGLANDIN F,,

120

e 264

50

100 O / .

40

30 I

I

$? Y

20 191

10 b

& 300

50

100

200

I50

250 rn / e

*

I

f

191

137

I

274

100

I50

200

m/e

FIG.1. (a) 70 eV mass spectrum of prostaglandin F,, (1 : [MI?

=

250

b

1

300

354); (b) 12.5 eV mass spectrum of compound 1

I

350

GY. HORVATH

A

(a)

e 278 100 %

50 2

40 -

99 30-

-s

7

c-(

137

81

20 -

332

I 19 I

I

1

177

221 I

I

I

D 350

c b 297 306314 288 1

50

°

I

~

100

150

200

Ill1

I

[MI?

I

d

300

250

3 50

m /e

e 100%

2 ( 12.5 e V )

2

I9 I

I

i

209

0

1

I

71 I I I

I

100

I50

I 200

250

300

350

m /e

FIG.2. (a) 70 eV mass spectrum of methyl prostaglandin F,, (2: [MI?= 368): (b) 12.5 eV mass spectrum of compound 2.

PROSTAGLANDIN Fjz

131

-. OH

, /

H- rearrangement I

I

OH

il-I

H - rearrangement \

SCHEME 1

respectively. The fact that ion m/e 332 contains no deuterium atoms in the spectrum of 0-deuteriated 2 also confirms the mechanisms proposed for the formation of ion a. To a lesser extent a third molecule of water can also be ejected from the molecular ion of 1 and 2, giving the ions m/e 300 and m/e 314. In accordance with the structure proposed for ion a2, the ion [a - H,O]t can lose a C H O radical, affording the ion m/e 289 in the case of 1 (Tables 1 and 2). In the case of 2 the loss of .OCH, radical occurs from the ions a and [a - H,O]+, giving ions m/e 319 and m/e 301, respectively. As expected,' these two ions have reduced relative abundances in the low eV spectrum of 2. The fragmentations depicted in Scheme 2 closely

resemble the respective processes of prostaglandins El and E,, which have been discussed in detail previously.2 Due to the presence of the C-9 hydroxyl group in 1 and 2 instead of the C-9 0x0 group in prostaglandins E, however, an additional loss of water occurs from ions d and h. It should be noted that pathway M I + c+ d is much less preferred here than in prostaglandins El and E,. Ion d is only about 10% of the corresponding peaks in the spectra of 1 and 2 (cf Table 2). Measurement on metastable ion decompositions leading to ion m/e 204 in the case of 1 showed that the respective neutral parts can also be eliminated in a reversed sequence (or even synchronously in the first field free region).

OH

OH

t m/e 229-

H-rearrangements 01

-

h

I m/e

- HO ,

204

2 m / e 218

-O=CHC,H,, SCHEME

2

- ROH

g

GY. HORVATH

132

H '0 J'-

\

\

OH

- C H2= CHOH b

f

- '(CH2),COOR 0 02

-'C6 H ,,COOR OH

- CH,=CHOH

H-reorrongement

m / e 137( I )

m / e 165

SCHEME 3

The mechanisms proposed for the primary fragmentations of ion a, shown in Scheme 3 are based on arguments similar to those described for the corresponding processes in methyl prostaglandin E, .2 Although the hydrogen of the hydroxyl group might be involved equally in the hydrogen rearrangement leading to the elimination of H,C=CHOH from ion a,, the spectrum of O-deuteriated 2 showed that structure b can be ascribed to the ions formed with greater probability since only about 20% of the deuterium content of ion a is lost with that neutral part. The preference of this pathway may be due to the extended conjugation in ion b. The loss of water from ion b yields ions mle 274 and m/e 288 in the spectra of 1 and 2, respectively. The formation of ion m/e 191(2) from ion b is accompanied by a metastable peak (Table 1): otherwise this process is almost 'invisible' since the ion has the same elemental composition as the ion m/e 191(1). Nevertheless, the higher than expected deuterium content of the ions at m/e 191 also indicates the presence of ion m/e 191(2). 'Metastable defocusing' performed at peaks mle 191 and 192 in the case of deuteriated 1 afforded further evidence on ion m/e 191(2) as a metastable transition from ion b has only been observed at mle 192. The ion m/e 209 contains one deuterium atom in the spectra of both

deuteriated 1 and 2. This labelling atom is lost upon formation of ion m/e 191(1). Metastable transitions leading to ion mle 191(1) indicate that via this pathway the elimination of the neutral parts from ion a, can also proceed in a reversed sequence or even both of them can be ejected in the field free region (cf. Table 1). When corrected for the isotopic contribution of the m/e 191 ion, the ion m/e 192 occurs in a relative abundance of c. 3.5:,, in both the 70 and 12.5eV spectra of 1 while in those of 2 it is 5.40,; and 2.39,. respectively. Since the transition [ a - H,O]t -+ 192 is accompanied by an intense metastable peak (see Table l), it can be assumed that the m/e 192 ion is an analogue of ion mje 191(1), but the hydrogen atom lost together with the C-9 hydroxyl group had been rearranged from the C-1-C-7 sidechain in ion a,. (The original numbering of prostaglandin F,, is used for the fragment ions throughout this paper.) The mechanism of this process may be a counterpart of the McLafferty rearrangement in methyl prostaglandin E,.' The above observations shed some light on the mechanisms of the loss of water from ion a as well. Ion mle 165 in Scheme 3 can represent the prevalent portion of the ions at that mje value in the spectra of 1 and 2 on the basis of the following evidence. High

PROSTAGLANDIN F,,

resolution data (Table 2) showed that about 90% of this peak corresponds to the elemental composition C,,H,,O. An intense metastable peak has been observed for the transition 209- 165. As shown in Table 1, a peak of medium intensity formed by the loss of C,H,,COOR from the metastable ion b (or from an isobaric ion ; vide supra) also occurs, indicating the possibility of a reversed sequence of eliminations from ion a, along this pathway. (The metastable transition 278-+ 165 in Table 1 is assumed to belong to the minor, C,oH,,02 component of this peak.) In the spectrum of deuteriated 2 the d,-content of the ions at mle 165 is only about 20%, showing that the deuterium atom in the m/e 209 atom is lost predominantly upon formation of the m/e 165 ion. It can be assumed that the extended conjugation in ion mle 165 makes its formation energetically favourable. Data on metastable ion decompositions indicate that part of the singlet peak (cf. Table 2) at mle 137 in the spectra of 1 and 2 arises from ions mle 165 and m/e 209, respectively. The mechanism proposed for the former process yielding ion m / e 137(1)is depicted in Scheme 3. It involves a [ 1,2] hydrogen rearrangement’ followed by the loss of carbon monoxide. 137 might proceed by rearrangeThe process 209 ment of the H atom from the hydroxyl group of the mle 209 ion, followed by the loss of O=CHCH,CH=O, yielding the mle 137(2)ion (vide infra). The considerable increase in the relative d,-content of ions m / e 137 in the 12.5 eV spectrum of deuteriated 1 confirms these assumptions, since it is obviously due to the reduced rate of formation of the m/e 137(1)ion, the do component (cf. Scheme 3; the abundance of the m/e 165 ion in this spectrum is only a quarter of the value in the corresponding 70 eV spectrum). The ions mje 141, 140 and 109, prominent in the case of methyl prostaglandin E,,’ also appear in the spectra of 2, but in lower abundances. As suggested earlier,’ the abundant ion at m / e 99 is formed in a double hydrogen rearrangement. In accordance with its proposed structure + O r C - C 5 H , (cf. Table 2) this ion can lose CO, yielding ion mle 71, as shown by an intense metastable peak observed for this transition. While the ions discussed so far are formed in pathways more or less analogous to those occurring in prostaglandins E, the fragmentation processes depicted in Scheme 4 are more specific for 1,2 and other F-type prostaglandins. These processes include the formation and the further fragmentations of ion e which gives rise to the base peak in the spectra of 1 and 2. It must be mentioned that the loss of the 90 mass units, resulting in ion e, was assigned formerly to the elimination of H,O and C,H12 from the molecular ion of prostaglandins F7 and this view was advanced again in connection with compound 1.” High resolution data (Table 2) show, however, that actually this pathway is not to be found in the fragmentation of 1 and 2. -+

133

The mechanism proposed for the formation of ion e is based upon the following observations. As the COOR group is evidently present in ion e, its elemental composition (C,,H280, in the case of 1) indicates an attached hydrocarbon portion with three double bond equivalents and at the same time the loss of the elements C,H,O, from the molecular ion upon formation of this ion. The only metastable transition to ion e commences from ion [M - H,O]+ and hence the remaining C,H,O, atoms must be ejected from that ion as a single unit. In the spectrum of 0-deuteriated 2 one of the labelling atoms is retained in ion e. All these observations are consistent with the assumption that ion e is formed from ion a2 by a hydrogen rearrangement from the C-9 hydroxyl group, followed by the elimination of a neutral malonaldehyde molecule. The migrating hydrogen atom seems to originate exclusively from this O H group, since the deuterium content of ion e (do < 18%, d , > 82%) is almost identical to that of ion a in the spectrum of 0-deuteriated 2. The fact that the formation of ion e from ion a,, although a rearrangement process, is accompanied only by a metastable peak of medium intensity, led to the assumption that this process should require not only a low activation energy but also possess a relatively large frequency factor. Consequently the rearrangement should proceed through a 6-membered ring transition state,” and the fragmentation process may be formulated as in Scheme 4. The deuterium contents of the daughter ions of ion e in the case of deuteriated 1 and 2 are in accordance with this assumption. Stereochemical considerations suggest that the 8, 12 double bond in ion e should have a trans configuration at the moment of its formation, while the 14, 15 double bond is trans already in ion a’. It is interesting to note the analogy that prostaglandin endoperoxides prostaglandin G, and prostaglandin H, are transformed enzymatically into a heptadecatrienoic acid derivative via the elimination of malonaldehyde.” It is also worth mentioning that the formation of ion e confirms the assumption that the cyclopentane ring has been opened in ion a2 and in the corresponding ions of prostaglandins E.’ A similar structure has been ascribed to one of the enzymatic transformation products of PGGz and P G H ~ It seems reasonable to assume that in statu nascendi the electron deficiency occurs in ground-state ion e on the middle 8,12 double bond in the form of a oneelectron n-bond, as every significant fragmentation process starting from ion e can be explained by using this structural model. The process affording ion mle 137(2) is depicted as being a direct cleavage in Scheme 4, since the relative abundance of this ion decreased considerably upon lowering the electron energy.’ This effect is more pronounced in the case of 2. As the ratio of the m / e 137(1)and m / e 137(2) ions cannot be determined, the extent of deuterium retention

GY. HORVATH

134

m / e 13712'1

/

d--Y

A

-'C,h,,COOR

I \

\

'

I

H

=c,

\Y

I

m / e 191131

I

COOR

-+

I

-C+,=Ci?

CH,=C;:

-Cti=CH

-(CH,j,-COOd+'

rn

H- reorrongernent -CH,=CH-Y

e

Ck;

=cr

1+' -CH=CH-CH=CH-C,H,,

150 SCHEME

in ion m/e 137(2)of O-deuteriated 2 could not be calculated exactly. Nevertheless, calculations based on complete retention of the deuterium content of ion e gave the result that 62S{ of the relevant ions would correspond to structure mie 137(2) in the spectrum of deuteriated 2. Thus, although a partial loss of deuterium label upon formation of ion m/e I37(2) could not be excluded, it can be concluded that the greater part of the deuterium content of ion e is retained in this process. The cleavage at the opposite side of the 8, 12 double bond in ion e results in ions of low abundance in the spectra of 1 and 2. Data in respect of measurements on the respective ion of 1 (mje 153) are given in Tables 1 and 2. The processes leading to ions 171 and m/e 150 (Scheme 4), have been observed also in the case of various polyunsaturated fatty acid esters' related to arachidonic acid, the biosynthetic precursor of prostaglandin F2=. The low abundance of these ions in the spectra of 1 and 2. when compared with that of the analogous ions in the spectra of all-cis polyunsaturates, is assumed to be

4

due to the fact that the hydrogen rearrangements depicted can only proceed at the cis configuration of the middle double bond. These rearrangements were assumed 1 3 b to involve the specific migration of one of the hydrogen atoms from the CHI-units separating the double bonds in those esters. Accordingly. the considerably lower deuterium content of ion in/e 150 (d, 30:;) compared with ion e in the spectrum of deuteriated 2. is in keeping with the depicted structure of ion e bearing the rearranged hydrogen atom at C-13 position, from where it can be eliminated partially upon formation of the mie 150 ion. The abundant ions in the lower mass region of the spectra of 1 and 2 (at m l e 81. 67, etc.) occur commonly in the mass spectra of the above-mentioned polyunsaturates. A discussion on their formation is beyond the scope of this paper; nevertheless, data of mass measurements on some of these ions have been included in Table 2. Ion e can lose CH,=C(OH)-OR in a hydrogen rearrangement to the COOR grouping. as indicated by 'metastable' and mass measurement data of 1 (see Tables 1 and 2). In the case of 1 this ion appears as the

-

PROSTAGLANDIN F,,

.

minor component of the doublet at m/e 204 (cf. ion inle 204 in Scheme 2). As regards the mechanisms proposed for the further fragmentations of ion e some introductory remarks are appropriate. The observation that the relative abundalices of ions m/e 177, i or 1 did not decrease markedly in the low eV spectra. of 1 and 2 4 e s p i t e the fact that their formation from the molecular ion involves at least three consecutive fragmentation steps-indicated that these ions cannot be formed from ion e in a ‘direct cleavage’ process of high activation energy.* Moreover, the formation of ion m/e 177 would formally require a vinylic fission, while that of ion I the formal cleavage of a double bond. Consequently, it had to be assumed that these fragmentations should be preceded by bond rearrangements in ion e, namely by cyclizations resulting in the formation of an (isolated) radical site. It has been shown’ that bond cleavages can proceed at a rapid rate in the neighbourhood of such sites, i.e. with low activation energy, or [1,2] hydrogen rearrangements can occur. The nature of these cyclizations is postulated on the basis of the following considerations. It is assumed that ring closures take place in ion e via the interaction of the middle oneelectron n-bond with one of the double bonds. Similar cyclizations are known in the solution chemistry of various homoallyl cations or radicals14 as well as in the photochemistry of l,4-dienes.’ Ab initio molecular orbital calculations showed that isolated homoallyl cations collapse without an energy barrier to cyclopropylcarbinyl cations,16 which are energetically more stable than the corresponding cyclobutyl cations, especially when alkyl-substituted at the carbinyl centre.” The cyclopropyl ring has a stabilizing effect on an adjacent cationic centre greater than that of the phenyl ring.I8 The stability of pentadienyl cations, as e.g. ions m/e 177 and k, is well known in carbonium ion c h e m i ~ t r y , ’ ~ and the cycloreversion of cyclobutene to butadiene runs ~ m o o t h l y . ’To ~ conclude, the comparison of the results cited above with the fragmentation pathways depicted in Scheme 4 showed that the latter are probable processes which may lead to quite stable ions. On the other hand these mechanisms are in accordance with the aforementioned observations too. The formation of ions i and m/e 191(3) from ion e is assumed to proceed through an intermediate state in which a cyclopropyl ring has been formed, as shown in Scheme 4. Ion i is more abundant in the spectra of both 1 and 2 than ion m/e 191(3) which gives only about 15 :d of the corresponding peak of 1 (cf. Table 2). The pathway e-+ 191(3) is attested by an intense metastable peak in the case of both 1 and 2 (Table 1). As expected, ion i is of similar deuterium content to ion e in the spectrum of deuteriated 2. In addition to cleaving the adjacent bond, the radical site in the above intermediate structure can also initiate a [ 1,2] hydrogen rearrangement leading to the formation of ion 1 as depicted in Scheme 4. In accordance with the proposed position of the rearranged

135

hydrogen atom in ion e. ion I almost completely retained (do < 300,/,, d , > 70::) the deuterium content of ion e in the case of deuteriated 2. The interaction between the one-electron n-bond and one of the double bonds in ion e can also result in the formation of an intermediate state containing a cyclobutyl ring. The ions n7/e 177 and k are suggested to be formed via such intermediates. Ion k is of lower abundance than the m!e 177 ion and this difference increases in the low eV spectra of 1 and 2. In the case of deuteriated 2 the deuterium content of ion e is almost completely retained in the m/e 177 ion (do < 28 %, d, > 72 %). As depicted in Scheme 4, the formation of intermediate states containing 3- and 4-membered rings, respectively, are competitive processes. These reactions presumably pass over transition states of similar energy, as shown by the nearly equal total abundances of ions i and m/e 191(3) and ions inle 177 and k , respectively. If this is so, the ratio for cyclopropyl vs cyclobutyl ringcontaining intermediates formed in the reaction of a given double bond is dependent upon their relative stabilities. Stereochemical considerations show that the ‘cyclobutyl intermediate’ formed as shown in Scheme 4 from a cis double bond will contain the substituents on the ring in the trans configuration, while in case of a trans double bond the cis configuration will be obtained. The lower stability of this latter intermediate might be the reason for the large difference between the relative abundances of ions i and k, which consequently suggests a trails configuration of the 14,15 double bond in ion e. The fact that the ions corresponding to i and k are of equal abundance in the mass spectra of methyl 6.9,12-cis-octadecatrienoateand its homologues’ 3b cofroborates this assumption. The above rationalization also indirectly supports the mechanisms proposed for the fragmentations of ion e involved. Direct evidence for cyclization upon the fragmentation of ion e is furnished by the formation of ion j shown in Scheme 5. Ion j can be observed separately only in the spectra of compound 1, since it coincides with the isotopic peak of ion m/e 209 and with another ion at m/e 210 (precursor of ion ntle 192; vide supra) in the spectra of 2. An intense metastable peak attests the formation of ion j from ion e (Table 1). According to mass measurements ion j of 1 has the elemental composition C , , H 2 0 0 2 . These facts indicate that in the process leading to ion j the C,H, unit is eliminated from the central part of ion e while the chains in ion e have interlocked. The pathway proposed for this process is depicted in Scheme 5. In accordance with this mechanism and with the proposed position of the rearranged hydrogen atom in ion e, in the spectrum of 0-deuteriated 1 one of the deuterium atoms present in ion e (do 4 %,d , 17 ”/, d , 79 %) is lost upon formation of ion j (do < 3076, d , > 700,/,). In conclusion, this paper has attempted to contribute to a better understanding of the mass spectroscopic

136

GY. HORVATH

t

X=(CH,),COOR, or

X=C,H,,

Y'=C,H,

, Y'=(CH,),COOR

SCHEME 5

behaviour of prostaglandin Fz,, a natural product of great biological importance. Some of the fragmentation reactions of compounds l and 2 show analogy to those of E-type prostaglandins, but most of the abundant ions of 1 and 2 are formed in pathways characteristic only for prostaglandins F. The majority of these latter fragmentation processes start with the most abundant ion in the spectra of 1 and 2, respectively. A specific hydrogen rearrangement process followed by the loss of malonaldehyde has been suggested for the formation of this ion (ion e in Scheme 4) from ion u 2 , which arises from the molecular ion by the elimination of water as shown in Scheme 1. The site to which this hydrogen atom has rearranged in ion e could be identified by interpretation of the mass spectral data obtained on the decomposition processes of this ion, as being the C-13 carbon atom of the prostaglandin skeleton. The mechanisms proposed for the fragmentations of ion e involve cyclizations which are supported by analogy to carbonium ion chemistry and by indirect and direct mass spectroscopic evidence. The fragmentation pattern of natural prostaglandins. outlined in the papers of this series, has been applied to various synthetic analogues of prostaglandins in this laboratory, resulting in a direct and reliable determination of their structures. Work is in progress to investigate C-deuteriated analogues of natural prostaglandins in order to check their proposed fragmentation mechanisms.

ACKNOWLEDGEMENTS Prostaglandin FZzwas the gift of Dr J. E. Pike (The Upjohn Co.. Kalamazoo. Michigan, U.S.A.). Special thanks are due to Dr G. Ambrus for preparing compound 2. The technical assistance of Miss M. Feher is pratefully acknowledged.

REFERENCES I . Horvath. Gy. Biomed. Mass Specrrom. 1975. 2. 190. 2. Horvath. Gy. Biomed. Mass Spectrom. 1976. 3. 4. 3. Horvath. Gy.: Ambrus. G . Presented at the 3rd International Symposium on Mass Spectrometry in Biochemistry and Medicine. Alghero. Italy. 1 6 - 1 8 June 1975. To be published in

Adcances in Mass Spectrometry in Biochemistry and Medicine, Vol. 11. Frigerio, A , ; editor. Spectrum Publications: New

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