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Thermal transformation of cholecalciferol between 100–170°C
23
2115 THERMAL TRANSFORMATION OF CHOLECALCIFEROL BETWEEN lOO-170°C Bohumil Pelt and David H. Marshall M.R.C. Mineral Metabolism Unit, General Infirmary, Leeds LSl 3EX, U.K.
Ret 'd:4/21/T/
ABSTRACT Cholecalciferol is transformed, irreversibly, to pyrocholecalciferol and isopyrocholecalciferol. The rate constant, as a function of temperature, for this transformation from the equilibrium mixture of cholecalciferol and precholecalciferol has been determined for temperatures between lOO-17oOC and is slower than the precholecalciferol-cholecalciferol interconversion. The ratio of rates of production of pyro to isopyro derivatives is 2:l throughout.
INTRODUCTION The formation of pyrocalciferol
(lOa-ergosterol) and isopyrocal-
ciferol (9B-ergosterol) (1) from calciferol (vitamin D2), at 188'C, was first observed by Askew et al. (2).
Busse (3) found that when vitamin
D2 was heated in a sealed tube, with the exclusion of oxygen, it was stable for several hours at 125'C but was completely isomerised within 4 hours at 188'C (4).
Another example of the relative thermal stability
of calciferol is that it could be purified by high vacuum disti llation at temperatures not exceeding 150°C (5).
Recently, precalciferol rather
than calciferol has been proposed as the intermediate in the folrmation of pyro- and isopyrocalciferols
(6-9).
In the present communication, it is shown that the pyro transformation rate is temperature dependant and measurable conversion is observed
Volwne 31, Nwnber I
s
WIIEOXDI
January,
2978
24
TmEOID6
s
at temperatures as low as llO°C, and is not only confined to temperatures greater than 180' (10). EXPERIMENTAL A variety of solvents was chosen to provide a temperature range from 100° to 170°. lo-15 mg of cholecalciferol was dissolved in 2 ml of solvent: dioxan (b.p. lOoOC), toluene AllOoC), n-butyl acetate (125OC), xylene (139OC), dimethylformamide (153 C), diglyme (163OC) and a mixture Air was replaced by nitrogen and the of diglyme and glycol (17OoC). solutions were heated under reflux for between one and 17 hours. The actual measured temperature was 2O-3'C higher than the boiling point of the solvents. The solvent was evaporated under nitrogen below 100°C and the residue applied to an aluminium oxide plate (Merck F254, neutral, 200 x 200 x 0.25 mn) and the plate developed in dichloromethane for The sequence of the four isomers (with the corresponding about 75 min. Rf values) was III (0.23), I (0.32), II (0.42) and IV (0.61), see fig. 1.
cholecekiferd
pyrocholecakiferol
isopyrocholecakiferol
Fig. 1
The individual bands were analysed by HPLC for estimation of purity The absorption wavelengths are: and UV spectrometry for quantitation. precholecalciferol (i max 261nm), cholecalciferol (1 max 265nm), pyrocholecalciferol (lOa+-cholesta-5,7-dien-3B-01, A max 274, 285 and 296nm) and isopyrocholecalciferol (96-cholesta-5,7-dien-38-01, h max 274, 284 The structures of the pyro- and isopyro-compounds were and 295nm) (11). The UV spectra were confirmed by NMR spectroscopy (see appendix). recorded with a Varian-Techtron 635 and HPL chromatographs were obtained The NMR spectra were taken with a Varian HA-100 in with a Varian 8500. carbon tetrachloride, using tetramethylsilane as the internal standard.
S
25
TBEOXDI
RESULTSAND DISCUSSION Pyrocholecalciferols
III and IV were produced at all temperatures The
studied, the conversion increasing with both time atid temperature. ratio of compound III
to compound IV was found to be constant at 2:1,
independent of time and temperature (Fig. 2).
I80-pyrOch0hcrlcihr0l
Fig. 2:
t%)
Composition of pyro and isopyrocalciferols at all times and temperatures.
Under the conditions used to generate the pyro compounds III
IV, we have assumed that the seco sterols (I and II)
and
are in equilibrium.
This assumption is supported by kinetic studies (12) which established that the rate of interconversion of I and II is faster than the rate
S
26
TmEOXDI
of production of pyro-steroids found in the present work by at least an order of magnitude. We did not attempt to measure the amount of
I present at various
times at the reaction temperatures studied for technical reasons. Since, as noted above, the reversible conversion of
I to II is fast
and the equilibrium varies with temperature (12), we recognize that the amounts of
I and II present after removal of the solvent (generally over
a few minutes at temperatures between 50' and 1OO'C) are not the same as the amounts present under the reaction conditions.
101
I
3
I
6
I
ho&
I
l2
1
I
”
‘*
Fig. 3: Decrease in I+11 as a function of time and temperature.
Nonetheless, conversion
Fig. 4: Conversion rate plotted against l/T
the data are consistent
with a first
order
constant irreversible
to III and IV from the equilibrium mixture of I and II.
s
27
TIIEOXDI
Apparent rate constants at each temperature can be calculated from the rate of decrease of the mixture of I and II with time, expressing the amount of I and II
present as a percentage of the total:
I t II = 100
exp. [-K(T)t] w here K(T) is the rate constant for the production of I II + IV at temperature T and t is time in hours.
The sum of I plus II
a function of time and temperature is shown in Fig. 3.
as
The linearity
Of these plots indicates that the reaction is both first order and irreversi ble.
The plot of the logarithm of the rate constants against the
reciprocal of temperature (T in OK) yields a straight line in accord with the Arrhenuis equation (Fig. 4). From the slope of this line the equation for the rate Constant for the production of III
plus IV can be written:
log10 KolB = 13.58 - 7
The rate constants for the individual isomers are therefore given loglo Ka = 13.40 - 6052
T
for the 10a isomer (III)
and 'Og10 KB = 13"'
- 6052
T
for the gB isomer (IV)
since they are in the ratio of 2:l. The data for these calculations is based on the percentage of I and II in the recovered mixture of compounds I to IV.
This recovery
of compounds I-IV in the final mixture was commonly 85-958 but at the higher temperatures this decreased to about 65%.
28
SL
TDEOID6
The conversion of cholecalciferol to the pyro compounds is believed to take place through the intermediate precholecalciferol
(I)
(6-8).
The slow irreversible transformation of precholecalciferol is probably influenced by the geometry of two energy-rich conformers which lead to the pyro- compounds in disrotatory cyclization
(9).
between the position of equilibrium between I and II
The difference under the reaction
conditions and that presumably re-established during isolation could be appreciable.
Calculations based on the equations of Hanewald et al.
(12) suggest values between 1:3.1 at 50' (the lowest evaporation temperature) and 1.8:1 at 170' (the highest experimental temperature). Therefore, use of the sum of I and II constant was necessary.
to calculate an apparent rate
To calculate a true rate constant for the
pyro transformation, it would have been necessary to measure the actual amount of I present under the conditions of the reaction.
APPENDIX NMR spectrum of compound III confirmed that the 38-OH group is axial, due to a narrow multiplet of the 3a-H. Similarly the 36-OH group in compound IV is equatorial due to a broad multiplet of the 3a-H. Compound (III) (CC14) 0.84 (l8-H~,s), 1.07(19-H3,s), 3.95 (3~11, narrow m), 5.36 (6,7-H2, q, J=6Hz). Compound (IV) (CC14) 0.85(18-H3,s), 1.24(19-H3,s), 3.40(3a-H, broad m.), 5.42(6,7-H2, q, J=6Hz).
S
TDEOXD6
29
REFERENCES 1. 2.
i: 5. 6. 7. 8.
1:: 11. 12.
Castells, J., Jones, E.R.H., Meakins, G.D. and Williams, R.W.J., JCtjEMF ;OC. 1159(1959). Bourdillon, R.B., Bruce, H.M., Callow, R.K., Philpot, J.S.L: and'iebster, T.A ., PROC. ROY. SOC. B., 109, 488 (1932). Busse, P., Z. PHYS. CHEM. 214, 211 (1933). Fieser, L.F. and Fieser, ASTEROIDS, Reinhold, N. York, p.137 (1959). Fieser, L.F., and Fieser, M. STEROIDS, Reinhold, N. York, p.126 (1959). Verloop, A., Koevoet, A.L. and Havinga, E., REC. TRAV. CHIM. _’ 76 689 (1957). Havinga, E. and Schlatmann, J.L.M.A., Tetrahedron, 16, 146 (1961). Sanders, G.M., Pot, J. and Havinga, E. PROGRESS INTHE CHEMISTRY OF ORGANIC NATURAL PRODUCTS, Ed. L. Zechmeister, Springer Verlag, 1969, vol. 27, p.151. Pfoertner, K., HELV. CHIM. ACTA., 55, 937 (1972). Kirk, D.N. and Hartshorn, M.P., STE’ITOIDREACTION MECHANISMS, Elsevier, 1968, p.416. Shaw, W.H.C., Jefferies, J.P. and Holt, T.E., ANALYST, 82, 2 (1957). Hanewald, K.H., Rappoldt, M.P. and Roborgh, J.R., REC. '!'RAV.CHIM. 80, 1003 (1961). -
2115 THERMAL TRANSFORMATION OF CHOLECALCIFEROL BETWEEN lOO-170°C Bohumil Pelt and David H. Marshall M.R.C. Mineral Metabolism Unit, General Infirmary, Leeds LSl 3EX, U.K.
Ret 'd:4/21/T/
ABSTRACT Cholecalciferol is transformed, irreversibly, to pyrocholecalciferol and isopyrocholecalciferol. The rate constant, as a function of temperature, for this transformation from the equilibrium mixture of cholecalciferol and precholecalciferol has been determined for temperatures between lOO-17oOC and is slower than the precholecalciferol-cholecalciferol interconversion. The ratio of rates of production of pyro to isopyro derivatives is 2:l throughout.
INTRODUCTION The formation of pyrocalciferol
(lOa-ergosterol) and isopyrocal-
ciferol (9B-ergosterol) (1) from calciferol (vitamin D2), at 188'C, was first observed by Askew et al. (2).
Busse (3) found that when vitamin
D2 was heated in a sealed tube, with the exclusion of oxygen, it was stable for several hours at 125'C but was completely isomerised within 4 hours at 188'C (4).
Another example of the relative thermal stability
of calciferol is that it could be purified by high vacuum disti llation at temperatures not exceeding 150°C (5).
Recently, precalciferol rather
than calciferol has been proposed as the intermediate in the folrmation of pyro- and isopyrocalciferols
(6-9).
In the present communication, it is shown that the pyro transformation rate is temperature dependant and measurable conversion is observed
Volwne 31, Nwnber I
s
WIIEOXDI
January,
2978
24
TmEOID6
s
at temperatures as low as llO°C, and is not only confined to temperatures greater than 180' (10). EXPERIMENTAL A variety of solvents was chosen to provide a temperature range from 100° to 170°. lo-15 mg of cholecalciferol was dissolved in 2 ml of solvent: dioxan (b.p. lOoOC), toluene AllOoC), n-butyl acetate (125OC), xylene (139OC), dimethylformamide (153 C), diglyme (163OC) and a mixture Air was replaced by nitrogen and the of diglyme and glycol (17OoC). solutions were heated under reflux for between one and 17 hours. The actual measured temperature was 2O-3'C higher than the boiling point of the solvents. The solvent was evaporated under nitrogen below 100°C and the residue applied to an aluminium oxide plate (Merck F254, neutral, 200 x 200 x 0.25 mn) and the plate developed in dichloromethane for The sequence of the four isomers (with the corresponding about 75 min. Rf values) was III (0.23), I (0.32), II (0.42) and IV (0.61), see fig. 1.
cholecekiferd
pyrocholecakiferol
isopyrocholecakiferol
Fig. 1
The individual bands were analysed by HPLC for estimation of purity The absorption wavelengths are: and UV spectrometry for quantitation. precholecalciferol (i max 261nm), cholecalciferol (1 max 265nm), pyrocholecalciferol (lOa+-cholesta-5,7-dien-3B-01, A max 274, 285 and 296nm) and isopyrocholecalciferol (96-cholesta-5,7-dien-38-01, h max 274, 284 The structures of the pyro- and isopyro-compounds were and 295nm) (11). The UV spectra were confirmed by NMR spectroscopy (see appendix). recorded with a Varian-Techtron 635 and HPL chromatographs were obtained The NMR spectra were taken with a Varian HA-100 in with a Varian 8500. carbon tetrachloride, using tetramethylsilane as the internal standard.
S
25
TBEOXDI
RESULTSAND DISCUSSION Pyrocholecalciferols
III and IV were produced at all temperatures The
studied, the conversion increasing with both time atid temperature. ratio of compound III
to compound IV was found to be constant at 2:1,
independent of time and temperature (Fig. 2).
I80-pyrOch0hcrlcihr0l
Fig. 2:
t%)
Composition of pyro and isopyrocalciferols at all times and temperatures.
Under the conditions used to generate the pyro compounds III
IV, we have assumed that the seco sterols (I and II)
and
are in equilibrium.
This assumption is supported by kinetic studies (12) which established that the rate of interconversion of I and II is faster than the rate
S
26
TmEOXDI
of production of pyro-steroids found in the present work by at least an order of magnitude. We did not attempt to measure the amount of
I present at various
times at the reaction temperatures studied for technical reasons. Since, as noted above, the reversible conversion of
I to II is fast
and the equilibrium varies with temperature (12), we recognize that the amounts of
I and II present after removal of the solvent (generally over
a few minutes at temperatures between 50' and 1OO'C) are not the same as the amounts present under the reaction conditions.
101
I
3
I
6
I
ho&
I
l2
1
I
”
‘*
Fig. 3: Decrease in I+11 as a function of time and temperature.
Nonetheless, conversion
Fig. 4: Conversion rate plotted against l/T
the data are consistent
with a first
order
constant irreversible
to III and IV from the equilibrium mixture of I and II.
s
27
TIIEOXDI
Apparent rate constants at each temperature can be calculated from the rate of decrease of the mixture of I and II with time, expressing the amount of I and II
present as a percentage of the total:
I t II = 100
exp. [-K(T)t] w here K(T) is the rate constant for the production of I II + IV at temperature T and t is time in hours.
The sum of I plus II
a function of time and temperature is shown in Fig. 3.
as
The linearity
Of these plots indicates that the reaction is both first order and irreversi ble.
The plot of the logarithm of the rate constants against the
reciprocal of temperature (T in OK) yields a straight line in accord with the Arrhenuis equation (Fig. 4). From the slope of this line the equation for the rate Constant for the production of III
plus IV can be written:
log10 KolB = 13.58 - 7
The rate constants for the individual isomers are therefore given loglo Ka = 13.40 - 6052
T
for the 10a isomer (III)
and 'Og10 KB = 13"'
- 6052
T
for the gB isomer (IV)
since they are in the ratio of 2:l. The data for these calculations is based on the percentage of I and II in the recovered mixture of compounds I to IV.
This recovery
of compounds I-IV in the final mixture was commonly 85-958 but at the higher temperatures this decreased to about 65%.
28
SL
TDEOID6
The conversion of cholecalciferol to the pyro compounds is believed to take place through the intermediate precholecalciferol
(I)
(6-8).
The slow irreversible transformation of precholecalciferol is probably influenced by the geometry of two energy-rich conformers which lead to the pyro- compounds in disrotatory cyclization
(9).
between the position of equilibrium between I and II
The difference under the reaction
conditions and that presumably re-established during isolation could be appreciable.
Calculations based on the equations of Hanewald et al.
(12) suggest values between 1:3.1 at 50' (the lowest evaporation temperature) and 1.8:1 at 170' (the highest experimental temperature). Therefore, use of the sum of I and II constant was necessary.
to calculate an apparent rate
To calculate a true rate constant for the
pyro transformation, it would have been necessary to measure the actual amount of I present under the conditions of the reaction.
APPENDIX NMR spectrum of compound III confirmed that the 38-OH group is axial, due to a narrow multiplet of the 3a-H. Similarly the 36-OH group in compound IV is equatorial due to a broad multiplet of the 3a-H. Compound (III) (CC14) 0.84 (l8-H~,s), 1.07(19-H3,s), 3.95 (3~11, narrow m), 5.36 (6,7-H2, q, J=6Hz). Compound (IV) (CC14) 0.85(18-H3,s), 1.24(19-H3,s), 3.40(3a-H, broad m.), 5.42(6,7-H2, q, J=6Hz).
S
TDEOXD6
29
REFERENCES 1. 2.
i: 5. 6. 7. 8.
1:: 11. 12.
Castells, J., Jones, E.R.H., Meakins, G.D. and Williams, R.W.J., JCtjEMF ;OC. 1159(1959). Bourdillon, R.B., Bruce, H.M., Callow, R.K., Philpot, J.S.L: and'iebster, T.A ., PROC. ROY. SOC. B., 109, 488 (1932). Busse, P., Z. PHYS. CHEM. 214, 211 (1933). Fieser, L.F. and Fieser, ASTEROIDS, Reinhold, N. York, p.137 (1959). Fieser, L.F., and Fieser, M. STEROIDS, Reinhold, N. York, p.126 (1959). Verloop, A., Koevoet, A.L. and Havinga, E., REC. TRAV. CHIM. _’ 76 689 (1957). Havinga, E. and Schlatmann, J.L.M.A., Tetrahedron, 16, 146 (1961). Sanders, G.M., Pot, J. and Havinga, E. PROGRESS INTHE CHEMISTRY OF ORGANIC NATURAL PRODUCTS, Ed. L. Zechmeister, Springer Verlag, 1969, vol. 27, p.151. Pfoertner, K., HELV. CHIM. ACTA., 55, 937 (1972). Kirk, D.N. and Hartshorn, M.P., STE’ITOIDREACTION MECHANISMS, Elsevier, 1968, p.416. Shaw, W.H.C., Jefferies, J.P. and Holt, T.E., ANALYST, 82, 2 (1957). Hanewald, K.H., Rappoldt, M.P. and Roborgh, J.R., REC. '!'RAV.CHIM. 80, 1003 (1961). -