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Facile synthesis of spin labelled steroids by esterification
Journul o/ Steroid B,orhmmisrrj Vol. 16. pp. 133 to 134. 1982 Printed in Great Britain. All rlghls reserved
C022-4731/82iOlOl33-02503.00/O Copyright 0 1982 Pcrgamon Press Ltd
SHORT COMMUNICATION FACILE
SYNTHESIS OF SPIN LABELLED BY ESTERIFICATION
STEROIDS
T. L. K~RLEY and H. B. HABALL* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221, U.S.A. (Received 2 June 1981) SUMMARY Hydroxy steroids have been spin-labelled by esterification at various positions around the steroid ring nucleus. This has been made possible by means of an amino pyridine catalyst in conjunction with dicyclohexylcarbodiimide as a coupling agent. The desired products are synthesized under very mild and simple reaction conditions and are easily purified. The yields are higher than those obtained by other synthesis methods.
interest in studying the type and extent of steroid binding to both cell receptors and serum proteins has been evident for many years. One of the most common methods used in these studies has been equilibrium dialysis. However, the technique is time consuming and imprecise, and alternative methods are being used with greater frequency. A technique which has come into its own in this field is the use of spin-labelled analogues of steroids to study the thermodynamics of steroid-protein interactions [ 1,2]. This method has none of the disadvantages of equilib~um dialysis, assuming that the proper analogues are either comrnercially available or easily synthesized. For this reason, it is important to improve the present methods of synthesizing spin-labelled compounds for use in studying biochemical systems. EXPERIMENTAL
Materials Steroids and dicyclohexyIcar~diimide (DCC) were obtained from Sigma. 2,2,5,5-Tetramethyl-3-pyrrolin-l-
oxyl-3-carboxylic acid and 4-p~ro~idinopyridine were obtained from Eastman Chemicals and Fluka Ag respectively. All solvents were reagent grade or better. (B) General synthesis and purification procedure A solution of 2,2,5,5-tetramethyl-3-pyrrolin-I-oxyl-3-carboxyiic acid (1) (1.41 mmol), DCC (3.1 mmol), the steroid (1.49 mrnol) and 4-pyrrolidinopyridine (0.23 mmol) in 40 ml of dichloromethane was stirred for 24 h at room temperature. The white precipitate (N,N-dicycfohexylurea) was filtered off and the filtrate extracted with distilled water (3 x 50ml), 5% acetic acid (3 x 501111)and distilled water (3 x 501111).After drying the crude product with MgS04 (_ 1 g), the solvent was evaporated in vacua and purification on silica gel (using benzene:ethyI acetate 1: l)‘yielded pure product. In the case of the spin-labelled 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) the chromatography solvent system was chloroform-ether (9: 1).
(A)
* Author t,o whom correspondence
should be addressed.
Table 1. Summary of spectra-physical Steroid esterified Product structure number (Fig. 1) Yield of purified product Melting point (“C) u.v. ~ln.,(.nll (Abs ETOH) emax i.r. (in CHClJ C==O stretch Elemental analysis % C theoretical ;< C found 7; H theoretical % H found “/bN theoretical “/, N found
Testosterone
RESULTS
The pure compounds obtained were characterized and identified by Uv, IR and ESR spectroscopy as well as mass spectral and elemental analysis. The four steroids esterified chemical properties of the product analogues
Corticosterone
2
3
897; 212-213 237 19,220 1714 1670
58”/;, 197-198 237 19,600 1730
73.97 73.7s 8.87 8.63 3.08 3.00
70.28 70.18 8.26 8.30 2.73 2.73 133
Deoxycorticosterone
4 47% 129-131 236 19,450 1720.1660
72.55 72.45 8.52 8.31 2.82 2.67
Estrone
5
517; 176-177 214 27.200 1730
74.28 74.42 7.86 8.04 3.21 3.16
134
Short communication
0
by this method were: (a) 17-B-hydroxy-4-androsten-3-one (testosterone), (b) 1 l/3,21-dihydroxy-4-pregnene-3,20 dione (corticosterone), (c) 21-hydroxy-4-pregnene-3,20 dione (deoxycorticosterone), (d) 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone). In the case of corticosterone, the 21 hydroxy position was selectively esterified (as shown by examination of mass spectral fragmentation patterns). Table 1 is a summary. of the spectra-physical chemical properties of the spinlabelled analogues. All products were ESR active. The resulting product structures have been labelled 2-5 respectively in Fig. 1.
5.
& d 0
Y 0
0
3
.
Fig. 1. Structures of the spin-labelled compounds.
1. DlSCUSSlON
0
0 2.
-c
II
-
7$
/ ,lli”” N
0.
0
Ho+
3. 0
4.
0
do+
The methods presently used in the synthesis of spinlabelled steroids by esterification leave much to be desired. The synthesis of esters by prior formation of the acid chloride has been accomplished with varying degrees of success [l, 3,4], and mostly low yields. Dodd and Lewis[4] have had limited success using N,N’-thionyldiimidazole as a coupling agent. However, no high yield, general method which is mild enough to be used safely with spin labels has been found. Hassner and Alexanian have reported [5] that certain carboxylic acids can be esterified using DCC and an aminopyridine catalyst. The adaptation of their procedure reported here has proved to be very useful, and the technique can undoubtedly be applied to many other similar steroids and biomolecules. In summary, the technique has the following advantages: (1) It is a simple, one step reaction. (2) The reaction conditions are very mild. (3) There are few side products. (4) The yields are good. Acknowledgements-This work was supported grant HD 13207 and NSF grant GP 8490.
by NIH
REFERENCES
1. Basset M., Defaye G. and Chambaz E. M.: Interaction of purified human transcortin with spin-labelled steroids. FEBS Lett. 60 (1975) 364368. 2. Halsall H. B., Kirley T. L. and Sprague E. D.: Preparation and binding of spin-labelled steroids to orosomucoid. Fed. Proc. 39 (1980) 1906. 3. Rozantzev E. G.: Free Nitroxyl Radicals. Plenum Press, New York (1970) p. 209. 4. Dodd J. R. and Lewis A. J.: Spin-label synthesis using N,N’-thionyldiimidazole coupling reagent. J. them. Sot. them. Comm. (1975) 52&521. 5. Hassner A. and Alexanian V.: Direct room temperature esterification of carboxylic acids. Tetrahedron Lett. 46 (1978) 4475-4478.
C022-4731/82iOlOl33-02503.00/O Copyright 0 1982 Pcrgamon Press Ltd
SHORT COMMUNICATION FACILE
SYNTHESIS OF SPIN LABELLED BY ESTERIFICATION
STEROIDS
T. L. K~RLEY and H. B. HABALL* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221, U.S.A. (Received 2 June 1981) SUMMARY Hydroxy steroids have been spin-labelled by esterification at various positions around the steroid ring nucleus. This has been made possible by means of an amino pyridine catalyst in conjunction with dicyclohexylcarbodiimide as a coupling agent. The desired products are synthesized under very mild and simple reaction conditions and are easily purified. The yields are higher than those obtained by other synthesis methods.
interest in studying the type and extent of steroid binding to both cell receptors and serum proteins has been evident for many years. One of the most common methods used in these studies has been equilibrium dialysis. However, the technique is time consuming and imprecise, and alternative methods are being used with greater frequency. A technique which has come into its own in this field is the use of spin-labelled analogues of steroids to study the thermodynamics of steroid-protein interactions [ 1,2]. This method has none of the disadvantages of equilib~um dialysis, assuming that the proper analogues are either comrnercially available or easily synthesized. For this reason, it is important to improve the present methods of synthesizing spin-labelled compounds for use in studying biochemical systems. EXPERIMENTAL
Materials Steroids and dicyclohexyIcar~diimide (DCC) were obtained from Sigma. 2,2,5,5-Tetramethyl-3-pyrrolin-l-
oxyl-3-carboxylic acid and 4-p~ro~idinopyridine were obtained from Eastman Chemicals and Fluka Ag respectively. All solvents were reagent grade or better. (B) General synthesis and purification procedure A solution of 2,2,5,5-tetramethyl-3-pyrrolin-I-oxyl-3-carboxyiic acid (1) (1.41 mmol), DCC (3.1 mmol), the steroid (1.49 mrnol) and 4-pyrrolidinopyridine (0.23 mmol) in 40 ml of dichloromethane was stirred for 24 h at room temperature. The white precipitate (N,N-dicycfohexylurea) was filtered off and the filtrate extracted with distilled water (3 x 50ml), 5% acetic acid (3 x 501111)and distilled water (3 x 501111).After drying the crude product with MgS04 (_ 1 g), the solvent was evaporated in vacua and purification on silica gel (using benzene:ethyI acetate 1: l)‘yielded pure product. In the case of the spin-labelled 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) the chromatography solvent system was chloroform-ether (9: 1).
(A)
* Author t,o whom correspondence
should be addressed.
Table 1. Summary of spectra-physical Steroid esterified Product structure number (Fig. 1) Yield of purified product Melting point (“C) u.v. ~ln.,(.nll (Abs ETOH) emax i.r. (in CHClJ C==O stretch Elemental analysis % C theoretical ;< C found 7; H theoretical % H found “/bN theoretical “/, N found
Testosterone
RESULTS
The pure compounds obtained were characterized and identified by Uv, IR and ESR spectroscopy as well as mass spectral and elemental analysis. The four steroids esterified chemical properties of the product analogues
Corticosterone
2
3
897; 212-213 237 19,220 1714 1670
58”/;, 197-198 237 19,600 1730
73.97 73.7s 8.87 8.63 3.08 3.00
70.28 70.18 8.26 8.30 2.73 2.73 133
Deoxycorticosterone
4 47% 129-131 236 19,450 1720.1660
72.55 72.45 8.52 8.31 2.82 2.67
Estrone
5
517; 176-177 214 27.200 1730
74.28 74.42 7.86 8.04 3.21 3.16
134
Short communication
0
by this method were: (a) 17-B-hydroxy-4-androsten-3-one (testosterone), (b) 1 l/3,21-dihydroxy-4-pregnene-3,20 dione (corticosterone), (c) 21-hydroxy-4-pregnene-3,20 dione (deoxycorticosterone), (d) 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone). In the case of corticosterone, the 21 hydroxy position was selectively esterified (as shown by examination of mass spectral fragmentation patterns). Table 1 is a summary. of the spectra-physical chemical properties of the spinlabelled analogues. All products were ESR active. The resulting product structures have been labelled 2-5 respectively in Fig. 1.
5.
& d 0
Y 0
0
3
.
Fig. 1. Structures of the spin-labelled compounds.
1. DlSCUSSlON
0
0 2.
-c
II
-
7$
/ ,lli”” N
0.
0
Ho+
3. 0
4.
0
do+
The methods presently used in the synthesis of spinlabelled steroids by esterification leave much to be desired. The synthesis of esters by prior formation of the acid chloride has been accomplished with varying degrees of success [l, 3,4], and mostly low yields. Dodd and Lewis[4] have had limited success using N,N’-thionyldiimidazole as a coupling agent. However, no high yield, general method which is mild enough to be used safely with spin labels has been found. Hassner and Alexanian have reported [5] that certain carboxylic acids can be esterified using DCC and an aminopyridine catalyst. The adaptation of their procedure reported here has proved to be very useful, and the technique can undoubtedly be applied to many other similar steroids and biomolecules. In summary, the technique has the following advantages: (1) It is a simple, one step reaction. (2) The reaction conditions are very mild. (3) There are few side products. (4) The yields are good. Acknowledgements-This work was supported grant HD 13207 and NSF grant GP 8490.
by NIH
REFERENCES
1. Basset M., Defaye G. and Chambaz E. M.: Interaction of purified human transcortin with spin-labelled steroids. FEBS Lett. 60 (1975) 364368. 2. Halsall H. B., Kirley T. L. and Sprague E. D.: Preparation and binding of spin-labelled steroids to orosomucoid. Fed. Proc. 39 (1980) 1906. 3. Rozantzev E. G.: Free Nitroxyl Radicals. Plenum Press, New York (1970) p. 209. 4. Dodd J. R. and Lewis A. J.: Spin-label synthesis using N,N’-thionyldiimidazole coupling reagent. J. them. Sot. them. Comm. (1975) 52&521. 5. Hassner A. and Alexanian V.: Direct room temperature esterification of carboxylic acids. Tetrahedron Lett. 46 (1978) 4475-4478.