GB1592170A - 1a-hydroxycholecalciferol-25-hydroxy esters and their use in radioimmunoassay - Google Patents

1a-hydroxycholecalciferol-25-hydroxy esters and their use in radioimmunoassay Download PDF

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GB1592170A
GB1592170A GB538/77A GB53877A GB1592170A GB 1592170 A GB1592170 A GB 1592170A GB 538/77 A GB538/77 A GB 538/77A GB 53877 A GB53877 A GB 53877A GB 1592170 A GB1592170 A GB 1592170A
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dihydroxycholecalciferol
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Priority to NL7800224A priority patent/NL7800224A/en
Priority to DK7878A priority patent/DK7878A/en
Priority to DE19782800782 priority patent/DE2800782A1/en
Priority to FR7800414A priority patent/FR2376863A1/en
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Description

(54) 1a-HYDROXYCHOLECALCIFEROL-25- HYDROXY ESTERS AND THEIR USE IN RADIOIMMUNOASSAY (71) We, F. HOFFMAN-LA ROCHE & CO. AKTIEN GESELLSCHAFT, a Swiss Company of 124--284 Grenzacherstrasse, Basle, Switzerland, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to the determination of la,25dihydroxycholecalciferol and of optical enantiomers and racemates thereof. More particularly, the invention is concerned with a radioimmunoassay and a reagent for the determination of 1 a,25-dihydroxycholecalciferol and of optical enantiomers and racemates thereof, with novel antigens and antibodies useful in said process and with a process for the preparation of said antigens and antibodies. The invention is also concerned with novel haptens useful in the preparation of said antigens and with a process for the preparation of said haptens.
Recent advances in the metabolism of vitamin D3 (cholecalciferol) have shown that it is hydroxylated in the liver and that the kidney subsequently produces a metabolite, namely la,25-dihydroxycholecalciferol, which has high biological activity. This knowledge has stimulated the need for assays of this metabolite which are suitable for use in clincial medicine. Hitherto, assays for the aforementioned metabolite have used competitive protein binding techniques [Brumbaugh et al, (1974) Biochemistry 13 4091]. These assays, although clinically useful in obtaining aetiological and therapeutic information, are not entirely satisfactory for routine clinical practice since, inter alia, they are laborious to perform and require large volumes of blood.
As compared with assays hitherto used for 1a,25-dihydroxycholecalciferol, the radioimmunoassay provided by the present invention is a relatively simple and rapid procedure. In addition, the present radioimmunoassay requires substantially smaller volumes of biological fluids than hitherto used.
In one aspect of the present invention there is provided a novel hapten useful in preparing the aforementioned antigens. This novel hapten is a compound of the general formula
wherein n is a number from 2 to 10, or an optical enantiomer or racemate thereof.
In a preferred embodiment, n in the compound of formula 1 is 2 or 3. In a particularly preferred embodiment n is 2, i.e. the compound is la- hydroxycholecalciferol-25-hydroxy hemisuccinate.
The process provided by the present invention for the preparation of the compounds of formula I and the optical enantiomers and racemates thereof comprises reacting a compound of the general formula
wherein R represents an alkyl group, R' represents a iower alkyl group and n has the significance given earlier, or an optical enantiomer or racemate thereof with an alkali metal hydroxide in an aqueous lower alkanol.
In the foregoing process, the secondary acyl groups of a compound of formula II are selectively cleaved by means of an alkali metal hydroxide (e.g. sodium, potassium or lithium hydroxide) dissolved in a suitable lower alkanol (e.g. methanol, ethanol or propanol) at a temperature of from about 0 C to 800 C.
Lithium hydroxide is the preferred alkali metal hydroxide and ethanol is the preferred lower alkanol. A hydrolysis temperature of about 25"C is also preferred.
The novel antigens aforesaid, which form another aspect of the present invention, comprise a compound of formula I hereinbefore or an optical enantiomer or racemate thereof covalently bonded, through the carboxyl group to an immunogenic carrier material.
As used in this specification, the term "immunogenic carrier material" includes those materials which have the property of independently eliciting an immunogenic response in a host animal and which can be covalently coupled to a compound of formula I or an optical enantiomer or racemate thereof. Suitable immunogenic carrier materials for the purposes of the present invention include, for example, proteins, natural or synthetic polymeric compounds e.g. polypeptides (e.g. polylysine or copolymers of other amino acids), and polysaccharides.
Particularly preferred immunogenic carrier materials are proteins and polypeptides, especially proteins.
When a protein is used as the immunogenic carrier material, the identity thereof is not critical. Examples of suitable proteins include mammalian serum proteins e.g. human gamm globulin, human serum albumin, bovine serum albumin, methylated bovine serum albumin, rabbit serum albumin and bovine gamma globulin. Bovine serum albumin is preferred. Other suitable proteins will be readily apparent. It is generally preferred, but is not absolutely necessary, that there is used a protein which is foregin to the animal host in which the resulting antigen will be used.
The process provided by the present invention for the manufacture of the aforementioned novel antigens comprises covalently coupling a compound of formula I or an optical enantiomer or racemate thereof through the carboxyl group to an immunogenic carrier material.
The covalent coupling of a compound of formula I or an optical enantiomer thereof to an immunogenic carrier material can be carried out according to methods known per se for the formation of amide or ester bonds.
Thus, for example, in one type of method a compound of formula I or an optical enantiomer or racemate thereof can be converted into an isolatable activated derivative prior to the coupling to the immunogenic carrier material. An example of a suitable isolatable activated derivative is the N-hydroxysuccinimide ester.
Alternatively, it is possible to use methods which do not require the isolation of an activated derivative of a compound of formula I or of an optical enantiomer or racemate thereof. Such methods include the use of EEDQ (N ethoxycarbonyl - 2 - ethoxy - 1,2 - dihydroquinoline) as the coupling agent or the use of the mixed anhydride method.
Other coupling methods may also be used. Thus, for example, the coupling of a compound of formula I or an optical enantiomer or racemate thereof to the immunogenic carrier material may be carried out in the presence of a carbodiimide, for example I - ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide.
In a preferred embodiment, the covalent coupling of a compound of formula I or an optical enantiomer or racemate thereof to an immunogenic carrier material is carried out according to the mixed anhydride method. In this method, a compound of formula I or an optical enantiomer or racemate thereof is dissolved in an inert water-miscible organic solvent (e.g. a cyclic ether e.g. dioxane), the solution is neutralised with a tri(lower alkyl)amine e.g. tri(n-butyl)-amine and is then treated with a (lower alkyl)-haloformate e.g. isobutylchloroformate. The resulting solution is then added, at a temperature of from about 0 C to 8"C, to a solution of the immunogenic carrier material in water or in a 1:1 mixture of water and an inert water-miscible organic solvent (e.g. a cyclic ether e.g. dioxane), the latter solution being made basic by the addition of an alkali metal hydroxide solution e.g. sodium hydroxide solution. The coupling is then allowed to proceed for a period of 30 minutes to overnight. Following dialysis, the desired antigen can be obtained by acidification and subsequent centrifugation of the precipitate formed.
The novel antigens provided by the present invention may be used to induce the formation of antibodies to la,25-dihydroxycholecalciferol or an optical enantiomer or racemate thereof in host animals by injecting the antigen into such a host, preferably using a conventional adjuvant material. Suitable host animals for this purpose include mammals e.g. rabbits, horses, goats, guinea pigs, rats, cows and sheep. Improved titres can be obtained by repeated injections over a period of time. The resulting antisera will complex with la,25-dihydroxycholecalciferol or an optical enantiomer or racemate thereof or an antigen prepared therefrom as described hereinbefore.
The antibodies provided by another aspect of the present invention are useful as reagents for the determination of the concentration of 1 ,25- dihydroxycholecalciferol or an optical enantiomer or racemate thereof in biological fluids, preferably plasma. Consequently they may be incorporated into a test kit for the determination of la,25-dihydroxycholecaliferol.
As mentioned earlier, the present invention provides in a further aspect a method for the radioimmunoassay of 1a,25-dihydroxycholecalciferol or an optical enantiomer or racemate thereof in a sample, which method comprises mixing said sample with a known amount of radiolabelled 1 ,25-dihydroxycholecalciferol or an optical enantiomer or racemate thereof and an antibody which will complex with said la,25-dihydroxychblecalciferol or optical enantiomer or racemate thereof and with said radiolabelled la,25-dihydroxycholecalciferol or optical enantiomer or racemate thereof, said antibody being obtained by the use of an antigen consisting of a compound of formula I hereinbefore or an optical enatiomer or racemate thereof covalently bonded through the carboxyl group to an immunogenic carrier material, measuring the degree of binding of said radiolabelled la,25- dihydroxycholecalciferol or optical enantiomer or racemate thereof and determining the amount of la,25-dihydroxycholecalciferol or optical enantiomer or racemate thereof in said sample by comparing said degree of binding to a standard curve obtained by mixing known amounts of said la,25- dihydroxycholecalciferol or optical enantiomer or racemate thereof with fixed amounts of radiolabelled 1 la,25-dihydroxycholecalciferol or optical enantiomer or racemate thereof and said antibody and determining the degree of binding for each known amount of said la,25-dihydroxycholecalciferol or optical enantiomer or racemate thereof.
Suitably, the radiolabelled la,25-dihydroxycholecalciferol or optical enantiomer or racemate thereof which is used in the aforementioned radioimmunoassay is la,25-dihydroxycholecalciferol or an optical enantiomer or racemate thereof which is radiolabelled with tritium (3H) carbon 14 (14C) or iodine 125 ('25I).
The compounds of formula II and optical enantiomers and racemates thereof which are required for the preparation of the novel haptens aforesaid can be prepared as described hereinafter.
In the formulae hereinafter, the various substituents are shown as joined to the nucleus by one of two notations; namely a solid line (-) indicating a substituent which is in the p-orientation (i.e. above the plane of the molecule) or a dotted line (- - -) indicating a substituent which is in the cr-orientation (i.e. below the plane of the molecule). The formulae have all been drawn to show the compounds in their absolute stereochemical configurations. Since the starting materials are derived from naturally occurring materials, the final products exist in the single absolute configuration depicted herein. However, the procedures described hereinafter are intended to apply as well to the synthesis of compounds of the racemic series. Thus, the synthesis may proceed from racemic starting materials in order to prepare racemic products. Optically active products can then be prepared by resolution of the racemic products used in the preparation thereof, as hereinafter described, by standard and well-known resolution techniques.
As used in this specification, the term "alkyl" denotes a straight-chain or branched-chain saturated hydrocarbon group containing from 1 to 18 carbon atoms. The term "alkanol" denotes an alcohol derived by combination of an alkyl group and a hydroxyl group. The term "lower" refers to the numerical range of from I to 8.
In the first step for the preparation of a compound of formula 11, a Ia acyloxy - 25 - hydroxy - 7 - dehydrocholesterol - 3 - acylate of the general formula
wherein R represents a lower alkyl group, the preparation of which is described hereinafter, is converted into an acyl ester of the general formula
wherein R, R' and n have the significance given earlier.
The esterification is carried out using an acyl halide of the general formula XCO(CH2)nCOOR' (V) wherein R' and n have the significance given earlier and X represents a chlorine or bromine atom, dissolved in a halocarbon solvent (e.g. dichloromethane) in the presence of an acid-acceptor (e.g. a heteroaromatic amine e.g. pyridine, picoline or collidine) at a temperature of from about -20"C to about 25 C.
In the next step of the sequence, a compound of formula IV dissolved in a suitable inert organic solvent is first irradiated by means of a low pressure mercury lamp equipped with a cooling finger fabricated from, for example, Vycor (trade mark) glass or quartz, for a period of time necessary to bring about 300/ to 50% conversion of the compound of formula IV, and then by means of a high pressure lamp equipped with a uranium glass filter or a Corning (trade mark) CS 52 Filter, in the presence of a photosensitiser, both irradiations being carried out under an inert atmosphere (e.g. nitrogen, helium, argon) at a temperature of about -400C to about +25"C. The preferred irradiation temperature is -5"C.
Suitable inert organic solvents for the irradiation include mixtures of saturated aliphatic hydrocarbons (e.g. pentane, hexane, isooctane) and ethereal solvents (e.g. monoglyme, diglyme, tetrahydrofuran, tetrahydropyran). A mixture of n-hexane and tetrahydrofuran is the preferred solvent.
Suitable sources of irradiation energy include Pen Ray (trade mark) low pressure mercury lamps and high pressure lamps (e.g. mercury, xenon-mercury and thallium-mercury lamps). A 450W Hanovia (trade mark) high pressure mercury lamp is the preferred source of energy for the second irradiation.
Upon completion of the irradiation, the solvents are removed by evaporation and the residue is separated into a pure compound of the general formula
wherein R, R' and n have the significance given earlier, and pure unchanged diene of formula IV on a high pressure liquid chromatograph using a solid absorbent column and an inert organic eluent. Suitable inert organic eluents for the separation include mixtures of hydrocarbons (e.g. n-hexane, isooctane, benzene, toluene) and esters of aliphatic and aromatic carboxylic acids (e.g. ethyl acetate, ethyl benzoate). Suitable solid absorbents include Porasil (trade mark), Corasil, Biosil, Zorbax, Sorbax-Sil, Sil-x. A Waters Associates Chromatograph Model 202 using a Porasil A column (244 cmx0.95 cm) and mixtures of'n-hexane and ethyl acetate as the eluent is the prefered high pressure liquid chromatographic system.
As suitable photosensitisers, there may be mentioned aromatic hydrocarbons (e.g. anthracene, phenanthrene) and aromatic ketones (e.g. fluorenone).
Phenanthrene is the preferred photosensitiser.
By first irradiating a previtamin of formula IV with a low pressure mercury lamp and then with a high pressure lamp in the presence of a photosensitiser, the formation of the major by-product, namely a tachysterol of the general formula
wherein R, R' and n have the significance given earlier, is markedly diminished or possibly eliminated [see S. C. Eyley and D. H. Williams, J. C. S. Chem. Comm:, 858 (1975)]. The use of the two step irradiation technique in conjunction with the recyclisation of unchanged diene of formula IV renders the present process for the preparation of a previtamin of formula VI more efficient than previously disclosed processes such as those described by, for example, H. DeLuca, et al., Tetrahedron Letters, 4147 (1972) and R. H. Hesse, etal., United States Patent Specification No.
3,901,928.
In the third step of the synthesis, a previtamin of formula VI is isomerised to a compound of formula II hereinbefore without elimination of the tertiary acyl group. The isomerisation is carried out by heating a previtamin of formula VI in an inert organic solvent (e.g. benzene, toluene) under an inert atmosphere (e.g. argon, nitrogen, helium). Benzene is the preferred solvent. While the isomerisatin temperature is not narrowly critical, it is preferred to carry out the isomerisation at about the reflux temperature of the solvent.
A Ia - acyloxy - 25 - hydroxy - 7 - dehydrocholesterol - 3 - acylate of formula III hereinbefore is prepared by acylating la,25 - dihydroxycholesterol with an alkanoic anhydride (e.g. acetic anhydride, propionic anhydride, butanoic anhydride and the like) in the presence of an acid acceptor (e.g. pyridine, lutidine, s-collidine) and an acylation accelerator (e.g. N,N - dimethyl - 4 - aminopyridine) to give a la - acyloxy - 25 - hydroxy - 7 - dehydrocholesterol - 3 - acylate, which is allylically halogenated with a suitable halogenating agent (e.g. 1,3 - dibromo 5,5 - dimethylhydantoin, N-chlorosuccinimide, N-bromosuccinimide, Nchloroacetamide, N-bromoacetamide to give a mixture of a 7a- and a 7p - halo la - acyloxy - 25 - hydroxycholesterol - 3 - acylate. The mixture of 7 - halo Ia - acyloxy - 25 - hydroxycholesterol - 3 - acylates is dehydrohalogenated by means of a heteroaromatic base (e.g. pyridine, lutidine, s-collidine) to yield a mixture of a 1a - acyloxy - 25 - hydroxy - 7 - dehydrocholesterol - 3 - acylate and a 1a - acyloxy - 25 - hydroxy - 4,6 - cholestadiene - 3 - acylate which is saponified by means of an alkali hydroxide (e.g. sodium hydroxide, potassium hydroxide, lithium hydroxide) in an anhydrous alkanol (e.g. methanol, ethanol, propanol) to yield a mixture of difficultly separable 1 a,25 - dihydroxy - 7 dehydrocholesterol and la,25 - dihydroxy - 4,6 - cholestadien - 3 - ol. Selective dehydration of the 4,6 - cholestadien - 3 - ol component of the mixture by means of a strong acid (e.g. sulphuric acid, p-toluenesulphonic acid, methanesulphonic acid) affords a mixture of lea,25 - dihydroxy - 2,4,6 - cholestatriene and lea,25 dihydroxy - 7 - dehydrocholesterol, from which la,25 - dihydroxy - 7 dehydrocholesterol can be readily separated in pure form by well-known chromatographic techniques. Acylation of pure la,25 - dihydroxy - 7 dehydrocholesterol with an alkanoic anhydride (e.g. acetic anhydride, propionic anhydride, butanoic anhydride) gives the requisite Ia - acyloxy - 25 - hydroxy 7 - dehydrocholesterol - 3 - acylate of formula III.
In a preferred embodiment the requisite compound of formula Il, wherein n is 2 or 3, is prepared by a sequence comprising the steps of converting a i cr - acyloxy 25 - hydroxy - 7 - dehydrocholesterol - 3 - acylate into a Ia - acyloxy - 7 dehydrocholesterol - 3 - acylate - 25 - hydroxy alkylsuccinate or alkylglutarate, irradiating said la - acyloxy - 7 - dehydrocholesterol - 3 - acylate - 25 - hydroxy alkylsuccinate or alkylglutarate to a lce - acyloxyprecholecalciferol - 3 - acylate 25 - hydroxy alkylsuccinate or alkylglutarate and isomerising said la - acyloxyprecholecaliciferol - 3 - acylate - 25 - hydroxy alkylsuccinate or alkylglutarate to a la - acyloxycholecalciferol - 3 - acylate - 25 - hydroxy alkylsuccinate or alkylglutarate.
The compounds of formulae I, II, IV and VI hereinbefore and optical enantiomers and racemates thereof are novel and it will be appreciated that they and their preparation also form part of the present invention.
The following Examples illustrate the present invention: Example I (a) la,25-Dihydroxy-7-dehydrocholesterol A solution of 1.04 g (2.5 mmol) of 1a,25-dihydroxycholesterol, 561 mg (5.5 mmol) of acetic anhydride, 8 ml of anhydrous pyridine and 450 mg of N,N - dimethyl - 4 aminopyridine was stirred under an atmosphere of nitrogen for 5 hours. 50 ml of ethyl acetate were then added to the mixture. The solution was washed with five 20 ml portions l-N-hydrochloric acid, two 20 ml portions of 10% sodium bicarbonate solution, water and saturated sodium chloride solution and then dried over anhydrous sodium sulphate. The mixture was filtered and the filtrate evaporated to give 1.31 g of la - acetoxy - 25 - hydroxycholesterol - 3 - acetate.
The foregoing residue (1.25 g, 2.5 mmol) was dissolved without further purification in two 5 ml portions of hexane and 0.40 g (1.4 mmol) of 1,3 - dibromo 5,5 - dimethylhydantoin and 1.25 g of sodium bicarbonate were added. The mixture was heated under reflux for 20 minutes and then evaporated under reduced pressure. The residue was triturated with 10 ml of xylene. The insoluble material was collected and the filter cake was washed with xylene.
The filtrate was added dropwise over a period of 5 minutes to a boiling solution of 600 mg (5.0 mmol) of freshly distilled s-collidine and 10 ml of xylene and the resulting mixture was heated under reflux for 90 minutes. The mixture was then allowed to cool to room temperature and benzene was added. The solution was washed with two 10 ml portions of l-N hydrochloric acid, one 10 ml portion of saturated bicarbonate solution and one 10 ml portion of water and subsequently dried over anhydrous sodium sulphate. The drying agent was collected and the filtrate evaporated to give 1.58 g of a mixture of Ia - acetoxy - 25 - hydroxy - 7 dehydrocholesterol - 3 - acetate and la - acetoxy - 25 - hydroxy - 4,6 cholestadien - 3 - acetate.
A solution of 1.19 g (2.5 mmol) of the mixture obtained according to the preceding paragraph, 20 ml of 50 /n methanolic potassium hydroxide solution and 15 ml of anhydrous dioxane was stirred at room temperature for 17 hours. 100 ml of water were added to the mixture and the solution was extracted with three 50 ml portions of 5% ethanol/chloroform. The layers were separated and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate, filtered and evaporated to give 1.19 g of a mixture of it,25 dihydroxy - 7 - dehydrocholesterol and I a,3p,25 - trihydroxy - 4,6 cholestadiene.
A solution of 1.19 g of the mixture obtained according to the preceding paragraph, 100 mg of p-toluenesulphonic acid and 25 ml of anhydrous dioxane was heated at 700C under a blanket of nitrogen for 1 hour and then allowed to coolto room temperature. 100 ml of water were added to the mixture and the solution was extracted with 75 ml of 50% ethanol/chloroform and then with three 50 ml portions of chloroform. The layers were separated and the organic phase was washed with 10% sodium bicarbonate solution, dried over anhydrous sodium sulphate, filtered and evaporated. The residue was placed on a column (0.95 cm 30 cm) of 125 g of silica gel and the column was eluted with 300 ml of 5% methanol/chloroform to give 630 mg (590/, yield based on la,25-dihydroxycholesterol) of lcr,25 - dihydroxy - 7 dehydrocholesterol after removal of the impurity in the initial 660 ml of eluent.
(b) I a-Acetoxy-25-hydroxy-7-dehydrocholesterol-3-acetate A solution of 1.62 g (3.88 mmol) of lea,25 - dihydroxy - 7 dehydrocholesterol, 10 ml of dry pyridine and 10 ml of acetic anhydride was stirred at room temperature for 28 hours under a nitrogen atmosphere. The mixture was evaporated in vacuo and the residue dissolved in 150 ml of ethyl acetate. The solution was washed successively with three 50 ml portions of 2-N hydrochloric acid, three 50 ml portions of water, 50 ml of 2-N potassium bicarbonate solution and three 50 ml portions of water. The layers were separated and the brganic layer was dried over anhydrous sodium sulphate, filtered and the filtrate evaporated in vacuo to give 1.90 g (98 /n) of Icr - acetoxy - 25 - hydroxy - 7 dehydrocholesterol - 3 - acetate in the form of a thick oil.
(c) 1 a-Acetoxy-7-dehydrocholesterol-3-acetate 25-hydroxy methylsuccinate A solution of 2.30 g (4.59 mmol) of la - acetoxy - 25 - hydroxy - 7 dehydrocholesterol - 3 - acetate in 50 ml of dry dichloromethane was cooled to 0 C and treated dropwise with a solution of 7.50 g (49.8 mmol) of 3carbomethoxypropionyl chloride dissolved in 25 ml of dichloromethane. A solution of 1.69 ml (20.9 mmol) of pyridine dissolved in 5 ml of dry dichloromethane was then added over a period of 1 hour under mtrogen)and the resulting mixture was stirred at OOC for 26 hours. 20 ml of ice-water were then added to the mixture. The aqueous phase was separated and extracted with dichloromethane. The combined organic phases were washed successively with 2-N hydrochloric acid, water, 2-N potassium bicarbonate solution and brine, dried over anhydrous magnesium sulphate and evaporated to dryness. The residue (2.90 g) was chromatographed on 275 g of silica gel (Merck silica gel 60). Elution with hexane/ethyl acetate (3:1) yielded 1.80 g (64 /n) of Ia - acetoxy - 7 - dehydrocholesterol - 3 - acetate 25hydroxy methylsuccinate as a thick oil.
(d) I a-Acetoxyprecholecalciferol-3-acetate-25-hydroxy methylsuccinate A solution of 390 mg (0.634 mmol) of 1α-acetoxy-7-dehydrocholesterol-3- acetate 25-hydroxy methylsuccinate, 360- ml of n-hexane and 40 ml of dry tetrahydrofuran was cooled at -50C and irradiated for 30 minutes under argon with a low pressure mercury lamp (Pen Ray 5W). 50 mg of phenanthrene were added to the irradiation mixture and the solution was irradiated for a further 15 minutes using a high pressure mercury lamp (Hanovia, 450W) and a uranium glass filter.
The solvent was evaporated in vacuo at room temperature and the residue purified with a Waters Associates Chromatograph (Model 202) using a 244 cmx0.95 cm Porasil A column and hexane/ethyl acetate (5:1) as eluent to give 196 mg of unreacted starting material and 93 mg of Ia - acetoxyprecholecalciferol - 3 acetate - 25 - hydroxy methylsuccinate.
(e) 1α-Acetoxycholecalciferol-3-acetate-25-hydroxy methylsuccinate A solution of 595 me (0.968 mmol) of 1α - acetoxyprecholecalciferol - 3 acetate - 25 - hydroxy methylsuccinate in 65 ml of dry benzene, saturated with argon, was heated under reflux and under an argon atmosphere for 2 hours. The mixture was then allowed to cool. The solvent was evaporated in vacuo and the residue separated with a liquid chromatograph (Waters Associates Model 202) using a 244 cmx0.95 cm Porasil A column and n-hexane/ethyl acetate (5:1) for the elution into 210 mg of unchanged starting material and 227 mg of 1α acetoxycholecalciferol - 3 - acetate - 25 - hydroxy methylsuccinate as a thick oil.
(f) la-Hydroxycholecalciferol-25-hydroxy hemisuccinate A solution of 227 mg (0.369 mmol) of la - acetoxycholecalciferol - 3 acetate - 25 - hydroxy methylsuccinate, 30 ml of absolute ethanol and 4 ml of a 5% solution of lithium hydroxide monohydrate in water was stirred at room temperature under argon for 24 hours. The hydrogen form of a cation exchange resin (BioRad AG 50W-X4, 4 g) was added to the mixture. The suspension was stirred for 5 minutes and then filtered. The filtrate was evaporated in vacuo at room temperature. The residue was dissolved in 20 ml of methyl formate and the solution evaporated in vacuo at room temperature to give 189 mg (99 /" yield) of Ia hydroxycholecalciferol - 25 - hydroxy hemisuccinate as a white foam of melting rabbit and different doses of conjugate were used as indicated in the following Table: TABLE Number and type of rabbits used for each dose regimen
Dose of conjugate per rabbit Type of rabbit 200,ug 400 mug Male White Half Lop 2 3 Female White Half Lop l 5 Male Inbred "Antiserüm-producing" ' strain 3 For administration to each rabbit, the appropriate dose of conjugate was dissolved in 1 ml of normal saline and mixed with 1 ml of Freund's complete adjuvant (Calbiochem (trade mark) modification). The resulting 2 ml solution was injected, in 30-40 divided doses, intradermally into the shaved skin on the lateral aspect of the back of the rabbit. All rabbits were bled from ear veins prior to immunisation. All rabbits were bled from ear veins three weeks after the initial injections and subsequently at one to two weekly intervals. The rabbits receiving 200 tLg doses of conjugate were boosted by the intramuscular injection of 200 ssg of conjugate 54 days after the initial injections. The blood collected from the rabbits was left to clot and the serurn was separated and subsequently stored in aliquots at -20 C.
Example 4 Radioimmunoassay Materials: 3H- I a,25-Dihydroxycholecalciferol 1 a,25-Dihydroxycholecalciferol Antiserum in PO4 buffer NaH2PO4 Na2HPO4 sodium barbitone Sodium acetate Hydrochloric acid Methanol Dextran T 40 Charcoal Norit OL (trade mark) Triton (trade mark) X100 Scintillation Grade Biofluor Scintillation System or Toluene/Triton X100/PPO/POPOP Buffers: 0.05-M phosphate buffer; pH 7.5 (a) 0.05-M NaH2PO42H2O 7.8 g/l (b) 0.05-M Na2HPO4 7.098 g/l (a) and (b) are mixed in the ratio 160 ml:640 ml.
B arbitone/acetate/hydrochloric acid buffer; pH 7.9 sodium barbitone 2.943 g sodium acetate 1.943 g sodium chloride 15.3 g Distilled water 1900 ml 0.I-NHCl 57 ml Dextran coated charcoal: 5% charcoal and 0.25% dextran T 40 in barbitone acetate buffer. The mixture was made up 30 minutes before use and was stirred continuously and kept at 4 C.
In assay tubes: 0.5 ml PO4 buffer containing antiserum; 20 ,ul methanol; 1500 c.p.m. 2H-la,25-dihydroxycholecalciferol < 4800 d.p.m. (disintegrations per minute) # 90 pg (picograms); Standard solutions of I a,25-dihydroxycholecalciferol (0, 250 pg, 500 pg, I ng, 2 ng, 4 ng and 8 ng per tube) in methanol; 10 yI Triton X100 1:36 in PO4 buffer; 250 ,ul Dextran coated charoal.
Procedure: 1. An aliquot of serum obtained according to Example 3 was thawed from its storage temperature (-20 C) and diluted in PO4 buffer.
2. Standard solutions of unlabelled la,25-dihydroxycholecalciferol (0, 250 pg, 500 pg, 1 ng, 2 ng, 4 ng and 8 ng per tube) in methanol were added to different assay tubes.
3. The aforementioned tritiated la,25-dihydroxycholecalciferol was added to these assay tubes. The radiolabelled la,25-dihydroxycholecalciferol was dissolved in 100 M1 of methanol for each tube.
4. The contents of the assay tubes were dried down under oxygen-free nitrogen.
5. The residue in each assay tube was re-dissolved in 20 ,ul of methanol.
6. 0.5 ml of antiserum in PO4 buffer was added to each assay tube.
7. The assay tubes were vortex mixed.
8. The assay tubes were incubated at 250C for I hour.
9. The assay tubes were placed on ice for 10 minutes.
10. 10 ,ul of Triton X100 diluted 1:36 in PO4 buffer were added to each assay tube. The tubes were vortex mixed and left to stand for 10 minutes.
11. 250 1 of Dextran coated charcoal were added to each of the assay tubes.
(Plain Dextran in buffer was added to total tubes). The tubes were vortex mixed and subjected to centrifugation for 15 minutes at 2000 revolutions per minute.
12. The supernatant of each assay tube was sampled; 0.5 ml aliquots being placed in counting vials. 4.5 ml of Biofluor scintillation fluid were added, mixing was carried out and a count was taken for radioactivity.
13. The counts per minute registered with the different concentrations of unlabelled la,25-dihydroxycholecalciferol were used to establish a standard curve.
The above procedure was repeated with serum samples of human origin in lieu of the standard solutions of unlabelled 1 la,25-dihydroxycholecalciferol. Prior to the assay, these serum samples were extracted with chloroform/methanol (2:1), dried down, re-dissolved in 7.5 , ether in hexane and chromatographed on silicic acid.
The amount of la,25-dihydroxycholecalciferol in the unknown specimens was determined by comparison with the standard curve.

Claims (53)

WHAT WE CLAIM IS:
1. Process for the preparation of a vitamin derivative of the formula
wherein n is a number from 2 to 10; or an optical enantiometer or racemate thereof, which comprises reacting a compound of the formula
wherein R is alkyl, R' is lower alkyl and n is a number from 2 to 10; or an optical enantiomer or racemate thereof, with an alkali metal hydroxide in an aqueous lower alkanol.
2. Process according to claim 1, wherein n is 2 or 3.
3. Process according to claim-l or claim 2, wherein R is methyl, R' is methyl and n is 2.
4. Process according to any one of claims 1 to 3, wherein the alkali metal hydroxide is lithium hydroxide.
5. Process according to any one of claims I to 4, wherein the lower alkanol is ethanol.
6. Process according to any one of claims 1 to 5, wherein the temperature is about 25"C.
7. Process for the preparation of a vitamin derivative of the formula
wherein R is lower alkyl, R' is lower alkyl and n is a number from 2 to 10; or an optical enantiomer or racemate thereof, which comprises heating a compound of the formula
wherein R, R' and n are as above; in an inert solvent.
8. Process according to claim 7, wherein n is 2 or 3.
9. Process according to claim 7 or claim 8, wherein R is methyl, R' is methyl and n is 2.
10. Process according to any one of claims 7 to 9, wherein the inert solvent is an inert organic solvent.
Il. Process according to claim 10, wherein the inert organic solvent is benzene.
12. Process for the preparation of a previtamin dervative of the formula
wherein R is lower alkyl, R' is lower alkyl and n is a number from 2 to 10; or an optical enantiomer or racemate thereof, which comprises (a) irradiating a starting compound of the formula
wherein R, R' and n are as above; in an inert solvent with a low pressure lamp, adding a photosensitiser and irradiating with a high pressure lamp using a filter to form a mixture of previtamin and unreacted starting compound; (b) separating the mixture to yield pure previtamin; and (c) recycling unreacted starting compound to yield additional quantities of pure previtamin.
13. Process according to claim 12, wherein n is 2 or 3.
14. Process according to claim 12 or claim 13, wherein R is methyl, R' is methyl and n is 2.
15. Process according to any one of claims 12 to 14, wherein the inert solvent is an inert organic solvent.
16. Process according to claim 15, wherein the inert organic solvent is n hexane-tetrahydrofuran.
17. Process according to any one of claims 12 to 16, wherein the filter is a glass filter.
18. Process according to claim 17, wherein the glass filter is a uranium glass filter.
19. Process according to any one of claims 12 to 18, wherein the photosensitiser is an organic photosensitiser.
20. Process according to claim 19, wherein the organic photosensitiser is phenanthrene.
21. Process for the manufacture of an antigen comprising covalently coupling a compound of the formula
wherein n is a number from 2 to 10; or an optical enantiomer or racemate thereof through the carboxyl group to an immunogenic carrier material.
22. Process according to claim 21, wherein n in the compound of formula I is 2 or 3.
23. Process according to claim 21 or claim 22, wherein the compound of formula I is la - hydroxycholecalciferol - 25 - hydroxy hemisuccinate.
24. Process according to any one of claims 21 to 23, wherein the immunogenic carrier material is a protein.
25. Process according to claim 24, wherein the protein is bovine serum albumin.
26. Process for the manufacture of an antibody to la,25dihydroxycholecalciferol, wherein a non-human host animal is innoculated with an antigen comprising a compound of the formula
wherein n is a number from 2 to 10; or an optical enantiomer or racemate thereof convalently bonded through the carboxyl group to an immunogenic carrier material.
27. Antigen comprising a compound of the formula
wherein n is a number from 2 to 10; or an optical enantiomer or racemate thereof covalently bonded through the carboxyl group to an immunogenic carrier material.
28. Antigen according to claim 27, wherein n in the compound of formula I is 2 or 3.
29. Antigen according to claim 27 or claim 28, wherein the compound of formula I is Ia - hydroxycholecalciferol - 25 - hydroxy hemisuccinate.
30. Antigen according to any one of claims 27 to 29, wherein the immunogenic carrier material is a protein.
31. Antigen according to claim 30, wherein the protein is bovine serum albumin.
32. Antibody to la,25-dihydroxycholecalciferol obtained by innoculating a non-human host animal with an antigen comprising a compound of the formula
wherein n is a number from 2 to 10; or an optical enantiomer or racemate thereof covalently bonded through the carboxyl group to an immunogenic carrier material.
33. Antibody according to claim 32, wherein n in the compound of formula I is 2 or 3.
34. Antibody according to claim 32 or claim 33, wherein the compound of formula I is la - hydroxycholecalciferol - 25 - hydroxy hemisuccinate.
35. Antibody according to any one of claims 32 to 34, wherein the immunogenic carrier material is a protein.
36. Antibody according to claim 35, wherein the protein is bovine serum albumin.
37. A compound of the formula
wherein n is a number from 2 to 10; or an optical enantiomer or racemate thereof.
38. A compound according to claim 37, wherein n is 2 or 3.
39. A compound according to claim 37 or claim 38, which is la - hydroxycholecalciferol - 25 - hydroxy hemisuccinate.
40. A compound of the formula
wherein R is lower alkyl, R' is lower alkyl and n is a number from 2 to 10; or an optical enantiomer or racemate thereof.
41. A compound according to claim 40, wherein n is 2 or 3.
42. A compound according to claim 41, which is la - acetoxycholecalciferol 3 - acetate - 25 - hydroxy methylsuccinate.
43. A compound of the formula
wherein R is lower alkyl, R' is lower alkyl and n is a number from 2 to 10; or an optical enantiomer or racemate thereof.
44. A compound according to claim 43, wherein n is 2 or 3.
45. A compound according to claim 44, which is l Ia - acetoxy - 7 - dehydrocholesterol - 3 - acetate - 25 - hydroxy methylsuccinate.
46. A compound of the formula
wherein R is lower alkyl, R' is lower alkyl and n is a number from 2 to 10; or an optical enantiomer or racemate thereof.
47. A compound according to claim 46, wherein n is 2 or 3.
48. A compound according to claim 47, which is la - acetoxyprecholecalciferol - 3 - acetate - 25 - hydroxy - methylsuccinate.
49. Method for the radioimmunoassay of lea,25 - dihydroxycholecalciferol or an optical enantiomer or racemate thereof in a sample, which method comprises mixing said sample with a known amount of radiolabelled lea,25 dihydroxycholecalciferol or an optical enantiomer or racemate thereof and an antibody which will complex with said lcr,25 - dihydroxycholecalciferol or optical enantiomer or racemate thereof and with said radiolabelled I a,25 - dihydroxycholecalciferol or optical enantiomer or racemate thereof, said antibody being obtained by the use of an antigen consisting of a compound of the formula
wherein n is a number from 2 to 10; or an optical enantiomer or racemate thereof covalently bonded through the carboxyl group to an immunogenic carrier material, measuring the degree of binding of said radiolabelled lea,25 dihydroxycholecalciferol or optical enantiomer or racemate thereof and determining the amount of la,25 - dihydroxycholecalciferol or optical enantiomer or racemate thereof in said sample by comparing said degree of binding to a standard curve obtained by mixing known amounts of said 1&alpha;25 - dihydroxycholecalciferol or optical enantiomer or racemate thereof with fixed amounts of radiolabelled la,25 - dihydroxycholecalciferol or optical enantiomer or racemate thereof and said antibody and determining the degree of binding for each known amount of said lea,25 - dihydroxycholecalciferol or optical enantiomer or racemate thereof.
50. Method according to claim 49, wherein the radio-labelled 1&alpha;25 - dihydroxycholecalciferol is 3H - 1&alpha;,25 - dihydroxycholecalciferol.
51. Reagent for the determination of 1&alpha;,25 - dihydroxycholecalciferol comprising an antibody according to claims 32 to 36.
52. Test kit for the determination of lea,25 - dihydroxycholecalciferol containing an antibody according to claims 32 to 36.
53. An antibody according to any one of claims 32 to 36, for use in the determination of lea,25 - dihydroxycholecalciferol.
GB538/77A 1977-01-07 1977-01-07 1a-hydroxycholecalciferol-25-hydroxy esters and their use in radioimmunoassay Expired GB1592170A (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
GB538/77A GB1592170A (en) 1977-01-07 1977-01-07 1a-hydroxycholecalciferol-25-hydroxy esters and their use in radioimmunoassay
JP31478A JPS5387344A (en) 1977-01-07 1978-01-06 11alpha * 255dihidroxy cholecalciferol and its optical enantiomer and racemic body
IT19080/78A IT1092713B (en) 1977-01-07 1978-01-06 DETERMINATION OF 1 ALPHA, 25 OF HYDROXY COLECALCIFEROL
NL7800224A NL7800224A (en) 1977-01-07 1978-01-06 THE DETERMINATION OF 1 ALPHA.25-DIHYDROXYCHOLECALCIFEROL.
DK7878A DK7878A (en) 1977-01-07 1978-01-06 PROCEDURE AND REAGENT FOR RADIO IMMUNOLOGICAL DETERMINATION
DE19782800782 DE2800782A1 (en) 1977-01-07 1978-01-09 DETERMINATION OF 1ALPHA, 25-DIHYDROXYCHOLECALCIFEROL
FR7800414A FR2376863A1 (en) 1977-01-07 1978-01-09 DETERMINATION OF 1A, 25-DIHYDROXYCHOLECALCIFEROL
DK125480A DK125480A (en) 1977-01-07 1980-03-21 METHOD OF PRODUCING VITAMIN DERIVATIVES

Applications Claiming Priority (1)

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GB538/77A GB1592170A (en) 1977-01-07 1977-01-07 1a-hydroxycholecalciferol-25-hydroxy esters and their use in radioimmunoassay

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DE (1) DE2800782A1 (en)
DK (1) DK7878A (en)
FR (1) FR2376863A1 (en)
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IT (1) IT1092713B (en)
NL (1) NL7800224A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3048698A1 (en) * 1980-12-23 1982-07-15 Wisconsin Alumni Research Foundation, 53707 Madison, Wis. 1-Alpha-hydroxy-vitamin=D prepn. - by allylic oxidn. of 5,6-trans-vitamin=D followed by irradiation
EP0130509A1 (en) * 1983-07-01 1985-01-09 F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft Process for the preparation of previtamin D and photosensitizers
CN108205064A (en) * 2018-03-28 2018-06-26 北京丹大生物技术有限公司 A kind of 3 detection method of 25(OH)VD and kit
CN115103591A (en) * 2019-12-27 2022-09-23 度勒科特公司 Crystalline and liquid crystalline 25-hydroxy-cholest-5-en-3-sodium sulfate and process for preparing same

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3267108D1 (en) * 1982-04-20 1985-12-05 Teijin Ltd Novel vitamin d3 derivatives, processes for preparation thereof, and antigens comprising said derivatives to be used for preparation of antibodies for immunochemical assay and antibodies prepared therefrom
US5206229A (en) * 1988-04-21 1993-04-27 Leo Pharmaceutical Products Ltd Vitamin d analogues
FR2631025B1 (en) * 1988-05-04 1991-04-12 Ire Medgenix Sa NOVEL VITAMIN D3 DERIVATIVES AND APPLICATION TO DETERMINATIONS OF VITAMIN D3 METABOLITES

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3048698A1 (en) * 1980-12-23 1982-07-15 Wisconsin Alumni Research Foundation, 53707 Madison, Wis. 1-Alpha-hydroxy-vitamin=D prepn. - by allylic oxidn. of 5,6-trans-vitamin=D followed by irradiation
EP0130509A1 (en) * 1983-07-01 1985-01-09 F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft Process for the preparation of previtamin D and photosensitizers
US4582641A (en) * 1983-07-01 1986-04-15 Hoffmann-La Roche Inc. Compounds of o-(6-hydroxy-3-oxo-3H-thio(seleno)xanthen-9-yl)benzene sulfonic(benzoic) acid useful as photosensitizers
CN108205064A (en) * 2018-03-28 2018-06-26 北京丹大生物技术有限公司 A kind of 3 detection method of 25(OH)VD and kit
CN108205064B (en) * 2018-03-28 2020-08-21 北京丹大生物技术有限公司 25OHD3 detection reagent, kit and detection method thereof
CN115103591A (en) * 2019-12-27 2022-09-23 度勒科特公司 Crystalline and liquid crystalline 25-hydroxy-cholest-5-en-3-sodium sulfate and process for preparing same

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IT1092713B (en) 1985-07-12
DK7878A (en) 1978-07-08
JPS5387344A (en) 1978-08-01
FR2376863B1 (en) 1980-08-01
DE2800782A1 (en) 1978-07-13
NL7800224A (en) 1978-07-11
FR2376863A1 (en) 1978-08-04
IT7819080A0 (en) 1978-01-06

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