Biomedical Mass Spectrometry 1975, 2 , 91 to 106

Mass Spectrometry of 1.4-Benzodiazepines S. RENDI~ , L. KLASINC,? V. SUNJI~ , F. KAJFEZ, V. KRAMER~ and P. MILDNER

Compagnia di Ricerca Chemica, Corso San Gottardo 35, CH-6830 Chiasso, Switzerland Institute of Organic Chemistry and Biochemistry, Zagreb, Yugoslavia

(Received 22 January 1975)

Abstract-Mass spectra and fragmentation pathways of some achiral and a number of chiral 1,4-benzodiazepine- 2-ones, and those of their in vitro biotransformation products are reported and discussed. Various derivatives substituted in position 3 are shown to be useful for the determination of fragmentation pathways from low resolu- tion spectra

Introduction

CHIRAL 1 ,4-benzodiazepines1 (1 to 4) possessing a centre of chirality in position 3, were found to be remarkably different in their psychopharmacological activity' and to follow different pathways in biotrans- formation3 While investigating their biotransforma- tion, we prepared compounds 5 to 10 as potential metabolites. Compounds l l a and l lb, 12a and 12b, and 13a and 13b-well resolved by thin-layer chromato- graphy, and characterized by ultraviolet and mass spectrometry-were actually isolated as authentic biotransformation p r o d ~ c t s . ~ Mass spectrometry proved to be the most useful tool for investigating the structures of isolated metabolites. The fragmentation pathways of some 1,4-benzodiazepines studied by high resolution mass spectrometry were reported earlier.6 By using derivatives substituted in position 3, the determination of fragmentation pathways and of fragment ion structures became accessible by the low resolution technique, as described here. The mass spectroscopic data and suggested fragmentation path- ways of the above compounds reported in this paper are intended as a contribution to the mass spectrometry of 1 ,Cbenzodiazepines, a class of compounds including many psychopharmacologic agents in current thera- peutical use.

Experimental

Syntheses of chiral 1,4-benzodiazepine-2-0nes 1 to 4 were carried out according to previously reported procedures.' Methods of synthesis for compounds 5 and 6, starting from compounds 1 and 2, and those for com- pounds 7 and 8, will be published elsewhere. Com- pounds 9 and 10 were prepared as described by Walser et at.5

Metabolites were isolated and identified by thin- layer chromatography, preparative-layer chromato- graphy and circular dichroism, and will be described in detail elsewhere.

t Institute 'R. BoSkoviC', Zagreb, Yugoslavia. 4 Institute J. Stefan', Ljubljana, Yugoslavia.

The 70eV mass spectra were measured on a CEC 21-1 IOC mass spectrometer using direct insertion probes at the following temperatures: 1, 120 "C: 2, 125°C; 3, 130°C; 4, 130°C; 5, 215°C; 6, 125°C; 7, 160°C;8, 130"C;9, 15O0C;1O, 125"C;l la , 160°C; l lb, 140°C; 12a, 145°C; 12b, 160°C; 13a, 140°C; 13b, 160°C; 14, 170°C; 15, 110°C; 16, 140°C; 17, 110 "C.

Results and discussion

The mass spectra of compounds 1 and 2 are presented in Figs. 1 and 2, and their fragmentation pathways are shown in Scheme 1, overleaf. The ions of m/r 242,243 (1) and m/e 256, 257 (2) undergo further fragmentation giving rise to ions with possibly an acridinium struc- ture6 (m/e 214 and m/e 228). The fragmentation path- ways for compounds 1 and 2 were confirmed by measurements of metastable ions in the field free region preceding the electrostatic sector.

Compounds l l a and l l b were obtained as products of enzymic hydroxylation of compounds 1(S) and 2(S), whereas 12a and 12b have been isolated previously as the biotransformation products of 2(S).3 Fragmentation of the oxygenated compounds l l a and l l b and 12a and 12b, potential metabolites of 1 and 2 [(S) forms], differs from that of their parent compounds, as the former produced intermediate ions m/e 230 (from l l a and l lb) and m/e244 (from 12a and 12b) (Scheme 1). These intermediate ions further fragment to ions m/e 139 (precursors l l a and l lb) and 153, 152 (precursors 12a and 12b) appearing in the mass spectra of oxygen- ated metabolites (Figs. 3 to 6). It must be mentioned that a high relative abundance of ion m/e 139 occurred in the mass spectrum of 7 (Fig. 7), and the same fragment was observed in the mass spectra of acridinone and N - methyl a c r i d i n ~ n e . ~ ? ~ A fragmentation pathway similar to that of acridinone and N-methyl acridinone was, therefore, deduced for compound 7. From these data and by comparison with the mass spectra of compounds 5,6 and 9 (Figs. 10 to 12), it may be concluded that the parent compounds are not oxygenated in positions C-3 or N-4. The actual position of the hydroxyl groups in metabolites cannot be determined from mass spectra

97 0 Heyden & Son Ltd, 1975

98 s. RENDIC, L. KLASINC, v. SUNJIC, F. K A J F E ~ , v. KRAMER AND P. MILDNER

100

ao

., 60 -

I?

LO

2c

100 1 2 9 257

''I, m

m/e FIG. 2. Mass spectrum of compound 2.

R1 = H ; Rz=H (11 m/e 2EL RI =CH3; Rz=H (21 m/e 298

R l = H ; R2=OH ( 1 1 a/bl m/e MO RI = C y , R2=OH (12 a/bl m/e 3M

- co - 28

__c

R, = H , R z = H m/e 2L2

Rl =CHJ, R 2 = H m/e 297 R I = C H ~ , R ~ = H m/e 256

R l = H , R2=OH m/e 299 RI 'H , R 2 = O H m/e 258 R)=CH3;R2=OH m/e 272 Rl=CH3 , R2=OH m/e 313

= H , R 2 = H m/e 283

- CI,-co

H R 1 = ti m/e 230 R l = C% m/e 2LL

m/e 167

m/e 181

- CI,-H,HCN

RI = H m/e 2IL

R l = CH3 m/e 228

SCHEME 1

m/e 151

m/o 165

m / e 139 m/e 153

MASS SPECTROMETRY OF 1 ,.I-BENZODIAZEPINES

1-13

99

Compound R’ R2 R3 R4 R5 R6 R’ Configuration

1 H

3 H

5 H

I H 8 H

2 CH3

4 CH3

6 CH,

9 CH3 10 CH3

l l a a n d l l b H 12a and 12b CH3 13aand 13b H

H - c1 H H H - c1 H H H - CI H H H - c1 H H H 0 CI H H H 0 c1 H H H - c1 OH H H H H H

OH _._ c1 H H -

OCOCH, - CI H H H - CI H OH H CI H OH H - H H OH

-

100-

80.

LO 60 80 100 120 140 160 M ZOO 220 2LO 2M) 280 300 W e

FIG. 3. Mass spectrum of compound l l a .

m/e

FIG. 4. Mass spectrum of compound l lb .

100

100 -

.- - Y [L

80 -

- 60 -

4 0 .

20 -

s. RENDIC, L. KLASINC, v. SUNJIC, F. KAJFEZ, v. KRAMER AND P. MILDNER

272 315

\ \ I

\, \ ,’ ?73 3t3 (Mil \ ,

\\ I \ I

I “,I I 1; I1 I

12a ‘ OH

I 03 139 152 166 181 193 209 221 229 2LL 255 285 297

I 1 1 I I ; i I 1 , I , , I I i I I I I I I I 1 2 6 2

I I 1 1 I 1 , I 1 ; , I I 1 I , ,

I l l

I 55 76 7

/I

11111 ,I l.,d#tl I uull I 11111 I, 11111, 11l11 1 % II2t 11111 1111 I t ,I, 111111111,~ Id., , IL I _,,I,, 1111, I

100 .

80 - - c - - [L 60-

40 .

20 -

!? 31? 316 2L2 \, /’ \, ,’Id1 ‘\ c’

\ I

CI

12b

OH 55 n 105 139 152 165 I81 l% 209 221 229 2LL 255 262 285 297

I

I I I I

I ! I ! I / I I I I 1 I I I , I I 1

i I I I

111 I ll 11!111 11111 , 1111111 I, I 1 1 l 1 1 I, Ill 11 II_ I I 1111 I II I

m/e

FIG. 6. Mass spectrum of compound 1Zb.

1 1 1 1111 I I1

0 ’ 51 63 77 1CU 128 139 152 166/7 177 19L 205

I j j I j j j i I I ,

, , I I I l l I I I ,

I / I ; I ;

FIG. 7. Mass spectrum of compound 7.

for the following reasons. The relative abundances of ions m/e 139 or 152 are about the same in the mass spectra of metabolites and of their parent compounds but much lower in the mass spectrum of 7, suggesting that hydroxylation has occurred at the 5-phenyl group. This is supported by a relatively low intensity of ion

m/e 77 in the mass spectra of metabolites, as compared with spectra of the parent compounds and with the spectrum of 7. However, because of the absence of an ion corresponding to the structure HOC6H,CN in the mass spectra of metabolites, which would indicate the presence of hydroxyl function in the 5-phenyllo

MASS SPECTROMETRY OF I ,CBENZODIAZEPINES

2 69

, '\, '

235 77 1L9 165 186 195 213 228 2L1 2 %

I I I I I I , I (

' I I I ! I I ( I , I

I I , , I I I I ,

LO

M

dl I LI! Ill 11,lIhl 1111111111 111 I I , I L IIIII 1 1 1 1 loll 111111 1hI1 I8 1111II 1 1 1 I I I I II Ill, Ill 1

101

?70 , (M:I I

I

+

/-=F---- CI aNH2 C E O + 1;: cI a: - C6H5 - C,H,OH

- 93 m/e 154 m/e 126 / - v N H 2 I+

K R = O H m/e 121 m/e 93 R=OH 16 m/e 247 R - H m/e 105 m/e 77 R = H 15 m/e 231

SCHEME 2

substituent, hydroxylation at the phenyl ring cannot be definitely established. In order to determine the site of the hydroxyl group, compound l l a was therefore subjected to acid hydrolysis, which yielded 16 as the product. The mass spectrum of this compound (Fig. 16) was compared to that of 15 (Fig. 15). The fragmenta- tion pathways deduced for these compounds are

14 15,16.17

R , R2

15 H H 1 6 H OH 17 OH 0

presented in Scheme 2. As one might expect, both 15 and 16 undergo fragmentation in closely similar ways, a

feature which can assist in recognizing the site of oxygenation. The molecular ion m/e 247 in the spectrum of 16 differs by 16 mass units from that in the spectrum of 15 (m/e231), thus suggesting the presence of an hydroxyl group in 16. The mass spectrum of 16 shows high relative abundances of ions mle 121 and mle 93, whereas in the mass spectra of compounds 15 and 17 (obtained as an acid hydrolysis product of 7) highly abundant ions mle 77 and m/e 105 appear. From these data it is inferred that the respective parent compounds are hydroxylated in the 5-phenyl ring

The mass spectrum of 14, isolated from an incubation mixture of 2 [(S) and (R) form^]^.^ is shown in Fig. 8 : ions resulting from the loss of H - and CH,. (m/e 254), H - and CO (m/e 241), C1. (mle 235) and NCO. (mle 228) can be clearly seen in this spectrum.

Compound 8, lacking a substituent in position 7, gives rise to the molecular ion rnle250 (Fig. 9) which generally follows a fragmentation pathway similar to that outlined for 1 (Scheme 1). In biotransformation experiments with 8 two metabolites, 13a and 13b, analogous to l l a and l lb, were isolated. The U.V. and mass spectra were very similar to those of l l a and Ilb, suggesting that 8 is also hydroxylated in the 5-phenyl

102

loo -

80 - - €

d 60- -

s. RENDIC, L. KLASINC, v. ~ U N J I ~ , F. KAJFEZ, v. KRAMER AND P. MILDNER

1 1 1 1 1 1 I

IL I I,. 11111 ,,,I I u Ill,, ,1111, I! I, ,111 ,,I,, 111, ,,I. 11 I ,I,,, I I 0 LO 60 80 100 120 YO 160 180 200 220 Z O 26U

m/e

FIG. 9. Mass spectrum of compound 8.

I

group, and follows a fragmentation pathway similar to that of lla and llb. The mass spectra of 13a and 13b show ions m/e 196, m/e 167 and m/e 139 in analogy to ions of the same m/e ratios in the mass spectra of lla and llb.

Figures 10 and 11 show the mass spectra of 5 and 6. Loss of H. and CO leads to the formation of ions m/e 271 (5) and 285 (6), and after loss of 0, ions m/e 255 and m/e 269, presumably with quinazolinium structures, are formed (Scheme 3). The molecular ions of compounds

20

t i 5 C I

Ph

21L 229 2LO 265 271 283

I l l ' I I ~ 1 1 i / I

2 5 5

I

m/e

FIG. 10. Mass spectrum of compound 5.

100 .

a0 -

X 5 601

jnj 204 I I

LO 60 80 W 120 ILO 160

I I I I Ill I

180 200 220 240 260 280 300 320 m/e

FIG. 1 1 . Mass spectrum of compound 6.

MASS SPECTROMETRY OF 1,4-BENZODIAZEPINES 103

R = H (51 m/e 300 \ m/e 299 R-CH, ( 6 ) m/e 314 [- OH] m/e 313

m/e 277

m/o 285 m/e 255

m/e 269

R-H m/e 283 R-CH, m/e 297

SCHEME 3

5 and 6 apparently lose C1, whereby ions mle 265 and mle279 are formed, as seen in the mass spectra of 5 and 6. These spectra also show ions mle283 and 297, formed by loss of HO radical from molecular ions. The ion m/e 297 undergoes further fragmentation as des- cribed for 1 and 2. Additionally, ions m/e 105 with a benzoyl structure are present in mass spectra of both 5 and 6. The benzoyl ion is probably formed by a mechan- ism described by Sadee and 0 thers~3~ in which an oxazirane ring system acts as an intermediate.

The most abundant ion mle 271 in the mass spectrum of 9 is formed by loss of CH,CO from the molecular ion (Fig. 12). The relatively abundant ion corresponding to [CH,CO]' (m/e 43) in the mass spectrum of 9 confirms the proposed fragmentation pathways (Scheme 4). The pathway starting with elimination of H,O from the molecular ion 9 leads to the formation of ion mle296

which, after loss of CH,CN and CO, yields the ion m/e 228, possibly with an acridinium structure.

In the mass spectrum of 10 (Fig. 13), after the loss of ketene from the molecular ion, a fragmentation similar to that of 9 is observed. The base peak is again ion m/e 271. The fragmentation of 10 is presented in Scheme 5. The fragmentation pathways proposed in Schemes 4 and 5 were confirmed by measurements of metastable ions.

Figure 14 shows the mass spectrum of 4. The most abundant ion is formed by loss of the substituent from position 3. Loss of C,H7. radical leaves an ion m/e 283. The relatively abundant ion corresponding to the tropylium ion (m/e 91) can be seen in the mass spectrum of 4 (Fig. 14). The structure of ion mle 283 seems to be the same as that of the ion formed by loss of hydrogen from 1,4-benzodiazepines unsubstituted in position C-3.6 The molecular ion of compounds 3 and 4 also

100 - ~ 3 ; ; C I ' -N

77 IOL 110 151/2 16L 13 177 193 221 228 213 256

I

1 I I

I

I. ... I... .,I1 I , .,.,.I I: 1 4 1 1 . 11111 I. .., .Jilt. I ..I. JIL ,.I1

LO 60 80 100 1M 1LO 160 180 200 220 2LO 260 280 300 320 m/e

FIG. 12. Mass spectrum of compound 9.

104

I I 1 1 1 II I I I I /II I I Ill I , l"Il" I1 " Ill

S. RENDIk, L. KLASINC, V. SUNJIC, F. KAJFEF, V. KRAMER AND P. MILDNER

Ill I l j I I ,

m/e 220 H

SCHEME 4

100

80

60 - - n"

LO

20

W e

FIG. 13. Mass spectrum of compound 10.

77 91 1 1

o 4 103 131 15112 165 177 193 205 221 227 2L3 271

I

I I

317

I I I

I

Lo 60 80 100 I20 1LO 160 180 200 220 2LO 260 280 30(1 320

FIG. 14. Mass spectrum of compound 4.

3L6

- 310 360

m iQ.

I

3 80

MASS SPECTROMETRY OF I ,CBENZODIAZEPINES

c!i.-c=o -CH,CO

314 m/e 271

105

0 (10) M:

297 m/e 3 m/e 256

SCHEME 5

undergoes fragmentation by loss of CO which leads to the formation of ion m/e346 (4). The subsequent loss of C,H,* results in formation of the ion mje 255, which probably possesses a quinazolinium structure such as ions commonly encountered in the fragmentation of 1,4- benzodiazepines unsubstituted at C-36 (Scheme 6). The ions m/e 316 and m/e 317 appearing in the mass spectra of 4 (Fig. 14) could have been formed only after loss of the N,-C, moiety from the molecular ion, although this type of fragmentation was never observed

The metastable measurements indicated a formation of the above ion (m/e 316). The same fragmentation pathway was also deduced from the mass spectrum Of 3. A fragment containing C-2, C-3 and the benzyl sub- stituent appears as an abundant ion (m/e 131) in the mass spectra of compounds 3 and 4. This fragment could have been formed by loss of the C,-C, moiety from the molecular ion. Further fragmentation of ions mje243, 255 and 283 proceeds as described for com- pounds 1 and 2 and for 1 A-benzodiazepines unsubstitu-

in 1,4-benzodiazepine-2-ones unsubstituted at C-3.6 ;ed at C-3.6

2LO

106 s. RENDIC, L. KLASINC, v. SUNJIC, F. K A J F E ~ , V. KRAMER.AND P. MILDNER

R R R

R-H (3lM+rn/e 360

R-CH, (4lM?rn/e 37L

rn/e 269

m/e 283

SCHEME 6

0 m/e 243

120 ILO 160 180 200 220 2 ~ 0 LO 60 80 loo

FIG. 16. Mass spectrum of compound 16.

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2. SunjiC, V.; Kuftinec, J. ; Kajfei, F. Areneimittel-Forsrh. In

3. RendiC, S.; Sunjit, V.; Kajfei, F.; Klasinc, L.; Mildner, P.

4. SunjiC, V.; Kajfef, F.; Kolbah, D.; Hofman, H . ; Stromar, M.

5. Walser, A.; Silverman, G.; Blount, J . ; Freyer. Jan I.; Sternbach,

J . Heterocyclic. Chem. 1973, 10, 581.

press.

Chimia (Aarau) 1974, 28, 232.

Tetrahedron Letters 1973, 34, 3209.

L. H. J . Org. Chem. 1971, 36, 1465.

7. Sadee, W.; Wan der Kleijn, E. J. Pharm. Sci. 1971, 60, 135. 8. Bowie, J . H.; Cooks, R. G . ; Prager, R. H.; Thredgold. H. M .

Australiun J . Chem. 1967, 20, 1179. 9. Porter, Q. N.; Baldas, J . In Mass Spectrometry of Heterocyfic

Compounds. Weisberger, A.; Taylor, E. C . ; editors. Wiley: New York, 1971, p. 420.

10. Schwartz, M. A,; Vane, F. M . ; Postma, E. Biorhem. Pharmacol. 1968, 17,965.