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The reaction of vitamin D (calciferol) with tetracyanoethylene. The basis of a possible chemical method to estimate vitamin D
CLINICA
THE
CHXMlCA ACTA
REACTIC)N
ETHYLENE, ESTIMATE
OF VITAMIN
D (CALCIFEROL)
THE BASIS OF A POSSIBLE
WITH
CHEMICAL
TETRACYANOMETHOD
TO
V~TA~~IN D
SUMMARY
The Diets-Alder addition reaction of t~t~acyanoet~yl~ne (TCE) to ergocalciferol and ergocalciferyl acetate has been studied. The characteristics of the reaction and the stability of the adduct of T&E and ergo~alciferyl acetate suggest the possibility of using this reaction in a double~isotope dilution method of estimation for calciferol; this possibility is discussed. The further possibilities of extending such a method to the estimation of ~~drox~Iated calciferol metabolites are discussed.
INTRODUCTION
The reaction between crystalline ergocalciferol (or ergocaleifetyl acetate) and crystalline ~et~acyanoetbyl~ne (TCE) has been studied and is described in this paper. The possibility of using this reaction in a doable-~sotoFe dilution method of estimation of calciferol, particularly in animal tissues, is discussed. &XATERIALS AND METHODS
Crystalline ergocakiferol (I~o~h-Light Co, Ltd.) was used as rec&ved alld t&a~yanoethylen~ (TCE) (Aldrich Chemical Co. Ltd.) was recrystallised from benzene (m.pt. x91-192”). All solvents were redistilled. Ergocalciferyl acetate was prepared in the usual way by the reaction of acetic anhydride in the presence of pyridine. The reactions with TCE were studied by dissolving I mg of ergocalciferol or ergocalciferyl acetate and a 3 x molar excess of TCE in I-Z ml of ethyl acetate or benzene. The reactions were carried out in stoppered test-tubes at 60” or room temperature for up to zq h. AIiquots of the reaction mixtures were chromata~~p~~~d on 300 rnp silica gel thin-layer plates; the absorbent was “Merck” Kieselgel YFa54 which contained a fluorescent indicator. After ~hrorn~t~~aph~T,dark bands could be located ..-----
EVANS
86
under ultra-violet light against the fluorescent background and after carbonising with concentrated sulphuric acid no bands appeared which had not already been seen under ultra-violet light; a yellow band attributable to unchanged TCE did not carbonise. Hence it was thought that viewing under ultra-violet light detected all the products of the reaction in which calciferol participated. The bands detected under ultra-violet light were scraped off and eluted with ethanol. The ultra-violet adsorption spectra of the eluted bands, in ethanol, were measured. Ultra-violet adsorption spectra were measured by a Beckman double-beam recording spectrophotometer and gas chromatography was carried out using a Beckman GC-4 gas chromatograph equipped with dual flame ionisation detectors. RESULTS
When ergocalciferol or ergocalciferyl acetate was reacted with recrystallised TCE in ethyl acetate or benzene at room temperature for I h, no free ergocalciferol, or free ergocalciferyl acetate could be found in the reaction mixture, i.c. none of the located bands absorbed with the maximum at 264-265 nm as expected for calciferol and the acetate. The same result was observed for the longer times and higher temperature. All located bands absorbed with a maximum about ZIO nm. On silica gel, the reaction products of ergocalciferol and ergocalciferyl acetate with TCE had lower migration rates than the corresponding unreacted sterol or steryl acetate. The reaction of TCE with ergocalciferol gave two main products while the reaction with ergocalciferyl acetate gave one main product. The & values on silica gel, with dichloromethane as solvent, are given in Table I.
I?F po
VALUES rnp
OS
K_IESELGELPF25+
layers. !Xwnt:
dichloromethanc.
-.. RF --__”
TCE (DI- OH f TCE) reaction mixture I&-OH (D,--0.11‘ -- TCE) reaction mixture
D2--o:\C
_____-.
0.36; o.rq 0.39
.
0.73 0.79
~.._
The reaction mixtures were also gas chromato~aphed on a xo ft. x 2 mm i.d. column of I:$ OV-17 on Gas Chrom Q at 240’. No free ergocalciferol or ergocalciferyl acetate could be found in the reaction mixtures. Fig. I shows the gas chromatography, under the conditions set out above, of the one-hour reaction mixture of ergocalciferyl acetate and TCE. It can be seen that there is one main peak and no ergocalciferyl acetate in the reaction mixture. Since this new peak has a long retention time compared to the peaks of pyro- and isopyro-ergocalciferyl acetate, and since pure TCE gave no response at the detector under the same conditions, it is assumed that this new peak is an adduct of ergocalciferyl acetate and TCE formed in a D&-Alder addition reaction. The true absorption spectra of the compounds eluted from the sihca gel were, at first, uncertain because the extraction of Kieselgel PF254 alone, with the same
POSSIBLE
CHEMICAL
90 (b)
70
METHOD
TO ESTIMATE
(a)
VITAMIN
D
l
i 260
300
260
Wave~~h
50
240
220
2
fnm)
(b)
d ti Li $
E max (206 n m) = 9.700
I
30
300
260
260
240
220
2
Wavekngth (nm) Fig. I. Gas chromatography of the ergocalciferyl acetate-TCE adduct. x(a.). I ,~g ergocaiciferyl acetate; r(b), one-hour reaction mixture of ergocalciferyl acetate and TCE (corresponding to approximately 0.5 @g of ergocalciferyl acetate). Peak I = pyroergocalciferyl-OAc; peak 2 = isopyroergocalciferyl-OAc; peak 3 = ergocalciferyl-OAc-TCE adduct. Column: IO ft. /: 2 mm i.cl. I 96 OV--17 on silanised Gas Chrom Q (IOO-rzo mesh). Carrier gas: nitrogen at 60 ml/min; +Ittenuation: z x 10~ (1~40 amps f.s.d.). Temperatures: inlet 250°, column z.@, detector line zoo”, detector Beckman LX-4 with dual flame ionisation detectors. KP *; instrument: Fig. z. Wtra-vi&et absorption spectra. (a) of an ethanof extract of Kieselgel PFz.54 : (b) of a crystaliine sample of the adduct of ergocalciferyf acetate and TCE.
solvent (ethanol) gave a solution which absorbed also about 2x0 nm (Fig. za). However, a crystalline sample of the adduct of ergocalciferyl acetate and TCE was prepared and a solution of this in ethanol (I mg/zo ml) absorbed with a maximum at 206 nm (Fig. zb) and molar extinction coeffkient = 97oo. The crystalline adduct was prepared as follows: 50 mg of ergocalciferyl acetate was reacted with 50 mg of TCE overnight at room temperature, in benzene. The reaction mixture was evaporated to dryness and redissolved in dichloromethane. The unreacted TCE was removed by chromatography on a column of silica gel using dichloromethane as solvent. The eluate, containing the adduct, was evaporated to dryness and rccrystaliised from ethanol. EIemental analysis gave C 75,1:/, H 7.8%, N 9.5% (expected figures were C‘ 76,3~iOP H &roj,, N 9.9%), The gas chromatography of this crystalline compound gave a peak which had the same retention time as the peak, shown in Fig. I, obtained from the one-hour reaction mixture. The adduct, ergocalciferyl acetate and ergocalciferol were spotted in varying CEit2.ChiWt.
AGtCZ,
38
(1972)
85-89
amounts (5-50 ,I.& on silicagel plates. They were left in air at room temperature for various times, from I h to overnight (16 h). The plates were then developed in dichloromethane, dried, sprayed with concentrated sulphuric acid and heated at IIOO for ~-IO min. The criterion for the oxidation of the samples by air and, hence, the production of more polar products, was the appearance of streaking extending from the origin up the plate to the sample spot; this could be seen after spraying and heating. These experiments showed the adduct was not affected by the action of air under conditions where ergocalciferol and ergocalciferyl acetate were extensively attacked. Even after overnight exposure to air, when ergocalciferol and ergocalciferyl acetate showed extensive streaking, the adduct showed no streaking.
These experiments show that TCE reacts with ergocalciferyl acetate to form one main compound within I h at room temperature. Thus, at least at the milligram level of ergocalciferyl acetate, a good yield of the adduct is formed under conditions where ergocalciferyl acetate is not breaking down quickly and the adduct seems stable enough to survive an extensive purification process. Also, this adduct is quite stable to attack and breakdown by air; certainly it is more stable in this respect than ergocalciferyl acetate and ergocalciferol. These characteristics suggest that a possibility exists of using this reaction in a double-isotope dilution method to estimate calciferol in tissues. The sensitivity of the method is a question of the specific activities of tritiated talciferol and %arbon-TCE used, but it should be possible to cover the physiolo~cal range of calciferol in serum and maybe other tissues. There has been little work on the chemical estimation of vitamin D (calciferol) in animal tissues compared to the work on pharmaceutical preparations and fish liver oils; this is because the amounts of calciferol in animal tissues are fairly low (with the notable exception of the body oils of some fish). Of the methods that have been applied to animal tissuesI-5, all have involved pre-treatment of the whole animal with calciferol or, in the case of serum or plasma, known amounts of crystalline calciferol have been added. Thus, at present, the chemical methods of estimation of calciferol do not cover the normal range of physiological concentrations in animal tissues. This possible method may meet this need. The reaction of ergocalciferyl acetate with TCE is thought to be a Diels-Alder addition reaction as occurs in the reaction of ergocalciferyl acetate with maleic anhydride’l and para-benzoquinone 18, the reactive part of the calciferol molecule being the cisoid diene system extending over carbon atoms 6,5, IO and 19. This diene system is maintained in the h~7droxycholecalciferols found as metabolites: 2Qydroxycholecalciferol6 and doubly-hydroxylated cholecalciferols7s8. Thus, TCE would probably form an adduct with all the known metabolites of calciferol which display biological activity. For serum, it seems that the biological (antiricketic) activity is accounted for by calciferol and 25-hydroxycalciferols. TCE would probably react with both acetates of these compounds and the difference in hydroxylation may give a separation of the free hydroxyl compounds (after hydrolysis of the acetate group) in an adsorption chromatographic system. It should be noted, however, that in such a method as outlined above, the sum of the amounts of the compound with the ergosterol side-chain (e.g. ergocalciferol) and the compound with the cholesterol side-chain (s-g. cholecalci-
POSSIBLE
CHEMICAL METHOD TO ESTIMATE
VITAMIN
D
89
ferol) would be measured. In general, some form of partition chromato~aphy (e.g. gas c~omato~aphy) is needed to separate a compound carrying the ergosterol sidechain from the similar compound carrying the cholesterol side-chain. Thus it has been seen that gas chromatography can separate isovitamin D, from isovitamin D, (ref. 3), pyrocalciferol, from pyrocalciferol, (ref. 5), and isotachysterol, from isotachysterol, (ref. IO). In the present case it is not yet known if the chole- and ergo- forms of the adduct can be separated by gas chromatography. This is not necessary, however, if the total amount of calciferol needs to be estimated.
The financial support of the Wellcome Trust is gratefully acknowledged. The advice and encouragement of the late Professor Paul Fourman is gratefully acknowledged. I thank Dr. Peter Brook of the Organic Chemistry Department, University of Leeds, for helpful discussions on the chemistry of vitamin D. REFERENCES I E. KOIXER z 3 4 5
A~TDD. R. ASHBY, Bin&em. J. 57 (1954) xii. P. NAIR, C. BUCA~, S. DE LEOK AND D. A. TURNER, An&. Ckewz., 37 (1~65) 631. I<. MURRAY, EI. C. DAY AND E. KQDICEK, Bioc&m. J., 98 (1966) 293. J. DE VRUB, F. J. MULDER ANU K. J. KEUNING, J. ~~~t~~~nQ~~g~, 15 (196~) 189. R. EVANS, Ph. D. Tkesis. University ofleeds, 1971. I?. f>E Luca, Amer. f. Cli~z. N&v., 22 (1969) 312. SU~A, H. F. DE LUCA, H. K. SCHNOES, G. PONCHON, ‘1’. TANAKA AND M. F. HORLICK, Biochemistry, 9 (1970) 2917. D. E. M. L.~WSON, D. R. FRASER, E. KODI~TZK, H. R. &IORRIS AND D. R. WILLIAMS, N&m, 230 (rg71) 228. E. B. MAWER, G. A. LUMB, R. ScehEFmz AND S. W. STAXBURY, Cl&. .%., 40 (1971) 39. A. J. SHEPPARD, D. E. LA CROIX AND A. R. PROSSER, J. Assoc. O&c. Asal. Chem., gr(Ig68) 834. A. WINDAIJS AND W. THIELE. Ann. Chmie, 521 (1936) 160. M. LOKA-TAMAYO, J. L. LEEN AND C. ESTADA, d. Ovg. Chem., 17 (1952) 812.
P. T, E. J. 6 H. 7 T. 8 g xo II 12
CEin. Chim. Acta, 38 (1972)
85-89
THE
CHXMlCA ACTA
REACTIC)N
ETHYLENE, ESTIMATE
OF VITAMIN
D (CALCIFEROL)
THE BASIS OF A POSSIBLE
WITH
CHEMICAL
TETRACYANOMETHOD
TO
V~TA~~IN D
SUMMARY
The Diets-Alder addition reaction of t~t~acyanoet~yl~ne (TCE) to ergocalciferol and ergocalciferyl acetate has been studied. The characteristics of the reaction and the stability of the adduct of T&E and ergo~alciferyl acetate suggest the possibility of using this reaction in a double~isotope dilution method of estimation for calciferol; this possibility is discussed. The further possibilities of extending such a method to the estimation of ~~drox~Iated calciferol metabolites are discussed.
INTRODUCTION
The reaction between crystalline ergocalciferol (or ergocaleifetyl acetate) and crystalline ~et~acyanoetbyl~ne (TCE) has been studied and is described in this paper. The possibility of using this reaction in a doable-~sotoFe dilution method of estimation of calciferol, particularly in animal tissues, is discussed. &XATERIALS AND METHODS
Crystalline ergocakiferol (I~o~h-Light Co, Ltd.) was used as rec&ved alld t&a~yanoethylen~ (TCE) (Aldrich Chemical Co. Ltd.) was recrystallised from benzene (m.pt. x91-192”). All solvents were redistilled. Ergocalciferyl acetate was prepared in the usual way by the reaction of acetic anhydride in the presence of pyridine. The reactions with TCE were studied by dissolving I mg of ergocalciferol or ergocalciferyl acetate and a 3 x molar excess of TCE in I-Z ml of ethyl acetate or benzene. The reactions were carried out in stoppered test-tubes at 60” or room temperature for up to zq h. AIiquots of the reaction mixtures were chromata~~p~~~d on 300 rnp silica gel thin-layer plates; the absorbent was “Merck” Kieselgel YFa54 which contained a fluorescent indicator. After ~hrorn~t~~aph~T,dark bands could be located ..-----
EVANS
86
under ultra-violet light against the fluorescent background and after carbonising with concentrated sulphuric acid no bands appeared which had not already been seen under ultra-violet light; a yellow band attributable to unchanged TCE did not carbonise. Hence it was thought that viewing under ultra-violet light detected all the products of the reaction in which calciferol participated. The bands detected under ultra-violet light were scraped off and eluted with ethanol. The ultra-violet adsorption spectra of the eluted bands, in ethanol, were measured. Ultra-violet adsorption spectra were measured by a Beckman double-beam recording spectrophotometer and gas chromatography was carried out using a Beckman GC-4 gas chromatograph equipped with dual flame ionisation detectors. RESULTS
When ergocalciferol or ergocalciferyl acetate was reacted with recrystallised TCE in ethyl acetate or benzene at room temperature for I h, no free ergocalciferol, or free ergocalciferyl acetate could be found in the reaction mixture, i.c. none of the located bands absorbed with the maximum at 264-265 nm as expected for calciferol and the acetate. The same result was observed for the longer times and higher temperature. All located bands absorbed with a maximum about ZIO nm. On silica gel, the reaction products of ergocalciferol and ergocalciferyl acetate with TCE had lower migration rates than the corresponding unreacted sterol or steryl acetate. The reaction of TCE with ergocalciferol gave two main products while the reaction with ergocalciferyl acetate gave one main product. The & values on silica gel, with dichloromethane as solvent, are given in Table I.
I?F po
VALUES rnp
OS
K_IESELGELPF25+
layers. !Xwnt:
dichloromethanc.
-.. RF --__”
TCE (DI- OH f TCE) reaction mixture I&-OH (D,--0.11‘ -- TCE) reaction mixture
D2--o:\C
_____-.
0.36; o.rq 0.39
.
0.73 0.79
~.._
The reaction mixtures were also gas chromato~aphed on a xo ft. x 2 mm i.d. column of I:$ OV-17 on Gas Chrom Q at 240’. No free ergocalciferol or ergocalciferyl acetate could be found in the reaction mixtures. Fig. I shows the gas chromatography, under the conditions set out above, of the one-hour reaction mixture of ergocalciferyl acetate and TCE. It can be seen that there is one main peak and no ergocalciferyl acetate in the reaction mixture. Since this new peak has a long retention time compared to the peaks of pyro- and isopyro-ergocalciferyl acetate, and since pure TCE gave no response at the detector under the same conditions, it is assumed that this new peak is an adduct of ergocalciferyl acetate and TCE formed in a D&-Alder addition reaction. The true absorption spectra of the compounds eluted from the sihca gel were, at first, uncertain because the extraction of Kieselgel PF254 alone, with the same
POSSIBLE
CHEMICAL
90 (b)
70
METHOD
TO ESTIMATE
(a)
VITAMIN
D
l
i 260
300
260
Wave~~h
50
240
220
2
fnm)
(b)
d ti Li $
E max (206 n m) = 9.700
I
30
300
260
260
240
220
2
Wavekngth (nm) Fig. I. Gas chromatography of the ergocalciferyl acetate-TCE adduct. x(a.). I ,~g ergocaiciferyl acetate; r(b), one-hour reaction mixture of ergocalciferyl acetate and TCE (corresponding to approximately 0.5 @g of ergocalciferyl acetate). Peak I = pyroergocalciferyl-OAc; peak 2 = isopyroergocalciferyl-OAc; peak 3 = ergocalciferyl-OAc-TCE adduct. Column: IO ft. /: 2 mm i.cl. I 96 OV--17 on silanised Gas Chrom Q (IOO-rzo mesh). Carrier gas: nitrogen at 60 ml/min; +Ittenuation: z x 10~ (1~40 amps f.s.d.). Temperatures: inlet 250°, column z.@, detector line zoo”, detector Beckman LX-4 with dual flame ionisation detectors. KP *; instrument: Fig. z. Wtra-vi&et absorption spectra. (a) of an ethanof extract of Kieselgel PFz.54 : (b) of a crystaliine sample of the adduct of ergocalciferyf acetate and TCE.
solvent (ethanol) gave a solution which absorbed also about 2x0 nm (Fig. za). However, a crystalline sample of the adduct of ergocalciferyl acetate and TCE was prepared and a solution of this in ethanol (I mg/zo ml) absorbed with a maximum at 206 nm (Fig. zb) and molar extinction coeffkient = 97oo. The crystalline adduct was prepared as follows: 50 mg of ergocalciferyl acetate was reacted with 50 mg of TCE overnight at room temperature, in benzene. The reaction mixture was evaporated to dryness and redissolved in dichloromethane. The unreacted TCE was removed by chromatography on a column of silica gel using dichloromethane as solvent. The eluate, containing the adduct, was evaporated to dryness and rccrystaliised from ethanol. EIemental analysis gave C 75,1:/, H 7.8%, N 9.5% (expected figures were C‘ 76,3~iOP H &roj,, N 9.9%), The gas chromatography of this crystalline compound gave a peak which had the same retention time as the peak, shown in Fig. I, obtained from the one-hour reaction mixture. The adduct, ergocalciferyl acetate and ergocalciferol were spotted in varying CEit2.ChiWt.
AGtCZ,
38
(1972)
85-89
amounts (5-50 ,I.& on silicagel plates. They were left in air at room temperature for various times, from I h to overnight (16 h). The plates were then developed in dichloromethane, dried, sprayed with concentrated sulphuric acid and heated at IIOO for ~-IO min. The criterion for the oxidation of the samples by air and, hence, the production of more polar products, was the appearance of streaking extending from the origin up the plate to the sample spot; this could be seen after spraying and heating. These experiments showed the adduct was not affected by the action of air under conditions where ergocalciferol and ergocalciferyl acetate were extensively attacked. Even after overnight exposure to air, when ergocalciferol and ergocalciferyl acetate showed extensive streaking, the adduct showed no streaking.
These experiments show that TCE reacts with ergocalciferyl acetate to form one main compound within I h at room temperature. Thus, at least at the milligram level of ergocalciferyl acetate, a good yield of the adduct is formed under conditions where ergocalciferyl acetate is not breaking down quickly and the adduct seems stable enough to survive an extensive purification process. Also, this adduct is quite stable to attack and breakdown by air; certainly it is more stable in this respect than ergocalciferyl acetate and ergocalciferol. These characteristics suggest that a possibility exists of using this reaction in a double-isotope dilution method to estimate calciferol in tissues. The sensitivity of the method is a question of the specific activities of tritiated talciferol and %arbon-TCE used, but it should be possible to cover the physiolo~cal range of calciferol in serum and maybe other tissues. There has been little work on the chemical estimation of vitamin D (calciferol) in animal tissues compared to the work on pharmaceutical preparations and fish liver oils; this is because the amounts of calciferol in animal tissues are fairly low (with the notable exception of the body oils of some fish). Of the methods that have been applied to animal tissuesI-5, all have involved pre-treatment of the whole animal with calciferol or, in the case of serum or plasma, known amounts of crystalline calciferol have been added. Thus, at present, the chemical methods of estimation of calciferol do not cover the normal range of physiological concentrations in animal tissues. This possible method may meet this need. The reaction of ergocalciferyl acetate with TCE is thought to be a Diels-Alder addition reaction as occurs in the reaction of ergocalciferyl acetate with maleic anhydride’l and para-benzoquinone 18, the reactive part of the calciferol molecule being the cisoid diene system extending over carbon atoms 6,5, IO and 19. This diene system is maintained in the h~7droxycholecalciferols found as metabolites: 2Qydroxycholecalciferol6 and doubly-hydroxylated cholecalciferols7s8. Thus, TCE would probably form an adduct with all the known metabolites of calciferol which display biological activity. For serum, it seems that the biological (antiricketic) activity is accounted for by calciferol and 25-hydroxycalciferols. TCE would probably react with both acetates of these compounds and the difference in hydroxylation may give a separation of the free hydroxyl compounds (after hydrolysis of the acetate group) in an adsorption chromatographic system. It should be noted, however, that in such a method as outlined above, the sum of the amounts of the compound with the ergosterol side-chain (e.g. ergocalciferol) and the compound with the cholesterol side-chain (s-g. cholecalci-
POSSIBLE
CHEMICAL METHOD TO ESTIMATE
VITAMIN
D
89
ferol) would be measured. In general, some form of partition chromato~aphy (e.g. gas c~omato~aphy) is needed to separate a compound carrying the ergosterol sidechain from the similar compound carrying the cholesterol side-chain. Thus it has been seen that gas chromatography can separate isovitamin D, from isovitamin D, (ref. 3), pyrocalciferol, from pyrocalciferol, (ref. 5), and isotachysterol, from isotachysterol, (ref. IO). In the present case it is not yet known if the chole- and ergo- forms of the adduct can be separated by gas chromatography. This is not necessary, however, if the total amount of calciferol needs to be estimated.
The financial support of the Wellcome Trust is gratefully acknowledged. The advice and encouragement of the late Professor Paul Fourman is gratefully acknowledged. I thank Dr. Peter Brook of the Organic Chemistry Department, University of Leeds, for helpful discussions on the chemistry of vitamin D. REFERENCES I E. KOIXER z 3 4 5
A~TDD. R. ASHBY, Bin&em. J. 57 (1954) xii. P. NAIR, C. BUCA~, S. DE LEOK AND D. A. TURNER, An&. Ckewz., 37 (1~65) 631. I<. MURRAY, EI. C. DAY AND E. KQDICEK, Bioc&m. J., 98 (1966) 293. J. DE VRUB, F. J. MULDER ANU K. J. KEUNING, J. ~~~t~~~nQ~~g~, 15 (196~) 189. R. EVANS, Ph. D. Tkesis. University ofleeds, 1971. I?. f>E Luca, Amer. f. Cli~z. N&v., 22 (1969) 312. SU~A, H. F. DE LUCA, H. K. SCHNOES, G. PONCHON, ‘1’. TANAKA AND M. F. HORLICK, Biochemistry, 9 (1970) 2917. D. E. M. L.~WSON, D. R. FRASER, E. KODI~TZK, H. R. &IORRIS AND D. R. WILLIAMS, N&m, 230 (rg71) 228. E. B. MAWER, G. A. LUMB, R. ScehEFmz AND S. W. STAXBURY, Cl&. .%., 40 (1971) 39. A. J. SHEPPARD, D. E. LA CROIX AND A. R. PROSSER, J. Assoc. O&c. Asal. Chem., gr(Ig68) 834. A. WINDAIJS AND W. THIELE. Ann. Chmie, 521 (1936) 160. M. LOKA-TAMAYO, J. L. LEEN AND C. ESTADA, d. Ovg. Chem., 17 (1952) 812.
P. T, E. J. 6 H. 7 T. 8 g xo II 12
CEin. Chim. Acta, 38 (1972)
85-89