Stability of corticosteroids under anaerobic conditions

Vol. 2 - 1 9 8 o Pharmaceutisch Weekblad Scientific Edition 59

LISBOA, B. P. ([964)J. Chromatog. t 3, 391. MASON, H. L. (1938)Proc. Staff Meetings Mayo Clhl. I3,

235. SYKES, P. (1965) A guidebook to mechanism in organic

chemistry. Second Edition, Longmans London, 172.

WENDLER, N. L. (I967) Molecular Rearrangements. Part Two (P. MAYO, Ed.) Interscience, New York, [o68.

Received July i979 . Accepted for publication November [979-

Stability o f corticosteroids under anaerobic conditions

IV. D-homosteroid 1

D. DEKKER 2

A B S T R A C T

One of the identified decomposition products formed during the anaerobic decomposition of prednisolone is a D-homosteroid.

The chromatographic properties, the isolation and the structure elucidation of the D-homosteroid are given. Also a mechanism leading to this compound is postulated. (Pharm. Weekblad Sci. Ed. 2, 59-64)

I N T R O D U C T I O N

In part I of this series (DEKKER I979) I7-deoxyprednisolone and in part II (DEKKER I980) 17-deoxy-2 i-dehydroprednisolone were described as decomposition products of prednisolone under anaerobic conditions. In both products the dihydroxy-acetone side chain has been changed. In part 1IX (DEKKER and BUnS I980 ) the I7-ketosteroid was described, in this product the side chain has disappeared.

In the D-homosteroid (Fig. I B,C) a part of the side chain of prednisolone (Fig. I A) is lost, while the rest, on rearrangement, results in the formation of a six membered D-ring. In this publication the isolation of this decomposition product from the decomposition mixture is described. Chromatographic properties are given, to identify this compound in a decomposition mixture. The structure elucidation is given. A decomposit ion mechanism of prednisolone leading to the D-homosteroid is postulated.

M A T E R I A L S A N D M E T H O D S

Chemicals The chemicals used were of European Pharmacopoeia quality unless mentioned otherwise.

Chromatography

A. Thin-layer chromatography I. Prednisolone and the decomposition products in gen- eral. See part I. The destructive method of visualisation was: spraying with a sulphuric acid-ethanol (20+80) mixture, heating at [2o~ for about [o min, and viewing under daylight or ov light of 365 nm. 11. The D-homosteroid. The same solvent and plate as in part I were used. Detection: a. Zimmermann's reagent; heavy spraying with a freshly

H.'•-OH 20C? B

C

Fig. i. A: Prednisolone; B: I7a-hydroxy-[7a-hydrogen- [7-keto-D-homosteroid; C: I7a-keto-[7-hydroxy-I 7-

hydrogen- D-homosteroid

Part nl, see DEKKER, D., and D. J. BULLS (I980) Pharm. Weekblad Sci. Ed. 2, 54-59- -" Department of Analytical Pharmacy, Faculty of Pharmacy, State University of Utrecht, Catharijnesingel 60, 35 [ [ GH Utrecht (The Netherlands).

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prepared mixture of equal parts of a 2.0% (w/v) ethanolic solution of m-dinitrobenzenc and 1.2 N ethanolic potassium hydroxide, and warming for 2 rain at 80~ repcating this action twice and finally warming for 5-io rain.

b. Spraying with a fi'eshly prepared mixture of 1 part of volume of o.2% (w/v) tetrazolium blue in methanol and 3 parts of volume 12",, (w/v) sodium hydroxide in methanol, and after drying at room temperature re- peating this action twice.

B. High pressure liqutd chromatography L For analytical purposes, sec part L IL For isolation purposes, see part L

Mas.s spectrometry See part i

Decomposition condittons See part i .

Isolation of the D-homosteroid

A. Microgram quant#ie.~. See part I .

B. Milligram quantities. From fractions obtained with the preparative liquid chromatograph milligram quantities of the D-homosteroid were obtained with the anah, tical liquid chromatograph. For this isolation the mobile phase was methanol (analytical grade)-water (5o + 5o, w/wl. The flow rate was 1.o ml/min: the sensitwity was o.32 AUVS for the isolation of o.4 nag D-homosteroid; the detection was performed at 313 nm.

1

!'I"

,______j , ~ _ _ j b ~ " V A 2t~ lS

I ' i o I o s o

MINUTE8

) -12 _~

8 <:

Fig. 2. The HPLC chromatogram of i op l of the filtrate of the decomposition mixture. Column: p-Bondapak (Wa- ters Assoc.), temperature 25~ flow rate l.O ml/min, mobile phase: methanol-water (50 + 5 o, w/w), to this mixture I% (v/w) acetic acid was added, wavelength 254 nm,

sensitivity o.64 AUFS

RESULTS AND D I S C U S S I O N

i . Chromatographic properties

A. Analvtical. The partition chromatogram of the filtrate of the decomposit ion mixture obtained with high pressure liquid chromatography is illustrated in Figure z. Peak 4 is the D-homosteroid. The two peaks under I are the two proposed configurations of I7-deoxyprednisolone (part l, DEKKER 1979), peak 3 is the 17-ketosteroid (part lit, DEKKER and auus I98o), peak P is prednisolone.

A thin-layer chromatogram of the chloroform extract of the acidified filtrate of the decomposit ion mixture and of the D-homosteroid and prednisolone is illustrated in Figure 3. Spot i is the I7-deoxyprednisolone (part I, DEKKER I979), spot 3 is the I7-ketosteroid (part Ill, DEKKER and 8uus ]98o). A thin-layer chromatogram of the decomposit ion mixture shows the D-homosteroid as a blue coloured spot with tetrazolium blue spray reagent. Also

FRONT

3 0 0 0

1 0

8 o

O A B C

Fig. 3. Thin-layer chromatogram of: A. The chloroform extract of the acidified filtrate of the decomposition mixture; B. The D-homosteroid; C. Prednisolone. Precoated silicagel plates (60 F254 Merck, 20 • 20 cm, thickness 0.25 mm), solvent chloroform-methanol (90 + to); detection: spraying with a sulphuric acid-ethanol (20 + 80) mixture, heating at t2o~ for about Io min, and viewing under

daylight or uv light of 365 nm

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prednisolone and I7-deoxyprednisolone appear as blue coloured spots with the tetrazolium blue reagent. The specificity of the tetrazolium blue reaction will be discussed under Structure elucidation. The chromatographic properties as illustrated in Figure 2 and 3 can be used to identify prednisolone, the D-homosteroid and other decomposit ion products in a decomposit ion mixture.

B. Prepara'tive. In Figure 4, a thin-layer chromatogram is given of the fractions I6 to 3o obtained with the preparative high pressure liquid chromatograph. The dot ted spots are the D- homosteroid. This compound is present in the fractions 20 up to 29. With partition chromatography using the analytical liquid chromatograph, the D-homosteroid could be isolated from these fractions. In Figure 5 the chromatogram illustrates this isolation from fraction 23; of peak A, only the part between the arrows is isolated. In this way in one run 0.4 mg of the D-homosteroid is obtained. The isolated compound gives one spot on a thin- layer chromatogram based on adsorption chromatography and also one peak appears with partition chromatography using the high pressure liquid chromatograph.

FRONT

16 17 18 19 20 21 22 23 ";'4 25 26 27 28 29 30

Fig. 4. The thin-layer chromatogram of the fractions x6 up to 3o obtained with the preparative high pressure liquid chromatograph. The same plate, solvent and detection

were used as in Figure 3

I 0.032 A

I I I I 15 10 w 0

MINUTES

Fig. 5. The HPLC isolation of the D-homosteroid. A = the D-homosteroid. Column:/t-Bondapak (Waters Assoc.), temperature 25~ flow rate i.o ml/min, mobile phase: methanol-water (50 + 50, w/w), wavelength 313 nm,

sensitivity 0.32 AUFS

It. Structure elucidation

A. Thin-layer chromatography. A thin-layer chromatogram of the decomposit ion mixture (Fig. 3), shows the D-homosteroid as a blue coloured spot with tetrazolium blue. With prednisolone and I7-deoxyprednisolone (part I) the blue colour appears after a single spraying. For the D-homosteroid it is necessary to spray three times to get a positive reaction. Consequently this compound reacts slower with tetrazolium blue. The tetrazolium blue reaction is based on a reduction of tetrazolium blue to the blue coloured mono- and diformazans, by oxidation of the a-ketol side chain of corticosteroids (GOROG and aORVM'H I978). The a-ketol side chain is absent (see Mass spectrometry) in the D-homosteroid, but the positive tetrazolium blue reaction shows the possible presence of an a-ketol group.

B. The oxidation with tetrazolium blue. When the tetrazolium blue reaction is performed in a solution ( i o / zg D-homosteroid in o.I ml reagent), the oxidation product can be-isolated with high pressure liquid chromatography. In Figure 6 is illustrated the partition chromato-

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,

J

I0~ IA

25 29 15 10 6 0

MINUTES

i i 2S 20

L

i IS

1 tO

MINUTES

I O.OlA

i i 6 o

Fig. 6. The HPLC chromatogram of 8pl of the tetrazolium blue reaction mixture. I: after 35 seconds reaction time; 2: after 6 minutes reaction time. A = the D-homosteroid; B = the oxidation product of the D-homosteroid; C = assumed m o n o - and diformazans. Column: p-Bondapak (Waters Assoc.), temperature 25~ flow rate 2.0 ml /min , mobile phase: meth-

anol-water (50 + 5o, w/w), wave leng th 254 nm, sensi t ivi ty o.o8 AUFS

gram of the reaction mixture after 35 seconds (z) and after 6 min (2) reaction time. Peak A is the D-homosteroid, peak B is the oxidation product and C is assumed to be the mono- and diformazans. After 25 min the oxidation is completed.

In Figure 7 is illustrated the cz-mass spectrum of this oxidation product. The parent peak of the oxidation product is 328 (cz 329). The parent peak of the D-homosteroid is 33 ~ (cI 33z) (Fig. 8). The o -mass spectrum of the D-homosteroid shows the presence of two hydroxyl groups in the molecule, because the parent minus z • H 2 0 (m/z 313) and the parent minus 2 • H 2 0 (m/z 295) are present. In the oxidation product only one hydroxyl group appears to be present, which is shown by the peak m/z 3 z L due to the loss of z x H 2 0 from the parent. Consequently, one hydroxyl group less is present in the oxidation product. When

C I M E I ~ V ~ E ~ q P . s ~ 9 4 6 8

15

,d, t~ " 14 .... ~ ~ .... ~e6 "

Fig. 7. The o-mass spectrum of the oxidation product of

the D-homosteroid

this is combined with the fact that the parent peak of the oxidation product is two mass units less than the parent peak of the D-homosteroid, it can be concluded that a hydroxyl group in the D-homosteroid has been oxidised

El I00 121/122 >220 X

2 4 0

" 1 - - ~ :"" : , i. . .,I. h, ~.,L~,

4 "'": " z4 " "

RMP.= 00065368

2O

tO0

20

, T " 3 ~ . . . .

1 0 0 21 Z2

i S

CI METHRNE RPIP. : 00005608 ~'345 X 5

15

l~ ' '14 .... 24 .... 24 .... 34

3 1 3

l T 2 9 5

. I , , , I . , t 1 I

Fig, 8. The m- and cI-mass spectra of the D-homosteroid

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Vol. 2 - 1980 Pharmaceutisch Weekblad Scientific Edition 63

H OH

A o

C

B

Fig. 9. The oxidation of the D-homosteroid with tetrazolium blue. A. 17a-hydroxy-17a-hydrogen-i7-keto-D- homosteroid; B. I7a-keto-17-hydroxy-17-hydrogen-D-

homosteroid; C. The I7a-I7-diketo-D-homosteroid

to a ketone group. The oxidation was achieved with tetrazolium blue and consequently an a-ketol group of the D-homosteroid has been transformed to a diketone group in the oxidation product. In Figure 9 this oxidation of the D-homosteroid with an a-ke to l group (A, B) to a D-homostero id with a diketone group is illustrated (C). The cx-mass spectrum of the oxidation product shows an intense peak at m/z x87. This peak is due to cleavage e, as illustrated in Figure Io and discussed in part I (DEKKER I979). The peak at m/z I25 is due to the remaining D-ring fragment.

C. Mass spectrometry. The EI- and cI-mass spectra of the D-homosteroid are given in Figure 8. Fragmentations leading to the same fragments for both prednisolone and the D- homosteroid, do appear as peaks at m/z I2I- L22; m/z I35; m/z I6I; m/z I73; m/z I87; m/z 225 and m/z 240. These fragmentations have

Fig. Io. An important fragmentation of the I7a-I7-diketo-D-homosteroid

been discussed in part I (DEKK~R I979) and show that the isolated D-homosteroid can only be different from prednisolone at CI6 or CI7 or C2o or C2I. In the D-homosteroid two hydroxyl groups are present, as described under Structure e lucidat ion (B). In the mass spectra of prednisolone (see part I, DEt<KEl~ I979) peak m/z 265 can be derived from the parent peak minus 2 x H20 and minus the side chain. In the mass spectra of I7-deoxyprednisolone peak m/z 267 (see part I, Dr~:KER I979) can be derived from the parent peak minus I x HzO and minus the side chain. Because no hydroxyl group is present at CI7 in I7-deoxyprednisolone, this fragment has a difference of two mass units to the corresponding fragment of prednisolone (an H atom at CI7 and one at CI6). In the mass spectra of the D-homosteroid peaks at m/z 265 and m/z 267 are not present (Fig. 8). Thus CI7 has been changed. Prednisolone has a parent peak of 360 (cI 36I) and the D- homosteroid 330 (cI 33 I). The difference is 30 mass units, thus COH2 has disappeared. Conse- quently it may be possible that the C2I group attached to C2o in prednisolone (Fig. I IA) has been substituted by an H atom, resulting in the I7-hydroxy-2o-aldehyde (Fig. I IB). But peak m/z 265 is absent, thus the remaining part of the side chain (the C2o group) is not attached to CI 7 together with a hydroxyl group at CI7 or CI6. If the hydroxyl group is attached to C2o, a peak at 267 should be present (as in I7-deoxyprednisolone) and the compound should have acidic properties. Peak m/z 267 is absent and the compound is neutral, thus this possibility can be excluded. Finally, it can be concluded that D-homoannula t ion of the

A B

Fig. I I. A. The dihydroxy-acetone side chain of prednisolone; B. The I7-hydroxy-2o-aldehyde side chain

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Ht~.---OH L,

2

~ ' ~ .~176 H~s.OH

3

OH ~ ,.OH

/ ~

A B

Fig. I2. Decomposition mechanism of prednisolone to the D-homosteroid

I7 -hydroxy-2I -a ldehyde (Fig. I IB) gives the D-homos te ro id (Fig. IB), resulting in the same parent peaks.

The D-homos te ro id is likely to be present as I7a -hydroxy- I7a -hydrogen- I7 -ke to (Fig. IB) and as 17a-keto- 17-hydroxy- 17-hydrogen (Fig. IC). This can be der ived from the fact that a ke to /enol equil ibrium is possible be tween C i7a and CI7. Details about these two configurations or conformations are under investigation.

uI. D e c o m p o s i t i o n m e c h a n i s m

In Eq. I (Fig. 12) is illustrated the proposed first step in the decomposi t ion mechanism of prednisolone leading to the D-hon'msteroid. In this step a hydroxyl at tack at C2I leads to a

disappearance of the C2I group, resulting in the formation of the I7-hydroxy-20-a ldehyde compound. In the second step (Eq. 2, Fig. I2) is proposed a r ea r rangemen t of the obta ined I7-hydroxy-20-a ldehyde compound by a D- homoannulation mechanism to the I7a-hydroxy- I7a -hydrogen- I7 -ke to -D-homos te ro id . In the D-homoannu la t ion mechanism the hydroxyl group at CI7 is changed to a ketone group, the bond between CI 3 and CI7 is broken and a new bond is fo rmed be tween C2o and CI 3. The a ldehyde group at C20 is then changed to a hydroxyl group. Consequent ly C20 is enclosed in a six m e m b e r e d ring in which case its no- tation is changed to CI7a (Fig. I and Fig. 12). The mechanism of the D-homoannu la t ion of I7-hydroxy-2o-keto-21-methyl steroids is well known (WENDLER 1967). This mechanism is proposed to be the same for the D- homoannula t ion of the 17-hydroxy-2o-aldehyde compound (Eq. 2 Fig. I2). In Eq. 3 (Fig. 12) is illustrated the possibility of the D-homos te ro id to be present as I7a -hydroxy- I7a -hydrogen- I7-keto (A) and as I7a -ke to - I7 -hydroxy- t7 -hydrogen (B), via the enol (C). It is possible that the D-homos te ro id is also present in the enol form. Finally it is clear that the hydrogen atom and the hydroxyl group can be present in a or fl orientation.

A c k n o w l e d g e m e n t s

I thank Prof. Dr. A. W. M. INDEMANS for the discussions and critical reading of the manu- script, Mrs. I. D. M. WAGEMAKER-ENGELS, Dr. J. G. v. M. LEFERINK and Dr. J. RENEMA for the mass spectrometr ic advice and assistance, and Mr. o. A. G. J. VAN DER HOUWEN for the chromatographic suggestions.

REFERENCES DEKKER, O. (I979)Pharrn. Weekblad Sci. Ed. t, 112. DEKKER, O. (I980) Pharm. Weekblad Sci. Ed. 2, I4. OEKKER, O., and D. J. BOUS (I980) Pharm. Weekblad Sci.

Ed. 2, 54. G6ROG, S., and a. HORVs (I978) Analyst to3, 346. WENDLER, N. L. (I967) Molecular Rearrangements, Part

Two (v. MAYO, Ed. )Interscience, New York, I t 14- t 12 i.

Received August 1979. Accepted for publication January I98o.

57 2 Pharmaceutisch Weekblad t t5- t98o

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Stability of corticosteroids under anaerobic conditions