25-OH VITAMIN D DERIVATIVES FOR DETERMINING VITAMIN D METABOLITES
20170115313 ยท 2017-04-27
Assignee
Inventors
Cpc classification
C07C2602/24
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to new vitamin D compounds which are bonded to a labelling group at the C3 stereocentre by means of a linker. The present invention further relates to a method for producing these vitamin D compounds and to the use of an intermediate for producing these compounds. The present invention also relates to a method for quantitatively determining vitamin D using a vitamin D compound according to the invention as a tracer. Furthermore, the present invention relates to a reagent for determining vitamin D, containing a compound according to the invention, and to the use thereof for determining vitamin D.
Claims
1. Vitamin D compound having a structure according to formula (I), ##STR00011## wherein R is a C.sub.2-C.sub.10 alkyl or alkenyl group, which is optionally substituted by a hydroxy group, L is a linker, and W is a labelling group that is not a vitamin D compound.
2. Compound according to claim 1, characterised in that the linker has a chain length of from 6 to 110 atoms, selected from C atoms and optionally heteroatoms such as N, O and/or S, the chain length preferably being 8 to 80, 12 to 60 or 30 to 50 atoms.
3. Compound according to either claim 1, characterised in that L is represented by a structure according to formula (II):
Q.sub.1CH.sub.2.sub.nYCH.sub.2.sub.2.sub.mQ.sub.2(II) wherein Q2 is: C(O)NR.sup.1., C(O)O., C(S)NR.sup.1., C(S)O., C(O)S., C(S)S., O., S., NR.sup.1., CH.sub.2O., CH.sub.2S. or CH.sub.2NR.sup.1., Q1 is: C(O)., NR.sup.2C(O)., NR.sup.2C(S)., OC(O)., C(O)NR.sup.2., C(S)NR.sup.2., C(S)., OC(S)., SC(O)., SC(S)., O., S. or NR.sup.2., Y is: O or CH.sub.2, R.sup.1 and R.sup.2, independently of each other, are H or C.sub.1-C.sub.6 alkyl, and m is 1-30, and n is 1-12, Q1 being bonded to the group W and Q2 being bonded to the parent substance of the vitamin D compound according to formula (I), and in each case (.) representing the binding site of Q2 to the parent substance of the vitamin D compound or of Q1 to the group W.
4. Compound according to claim 1, characterised in that W is biotinyl, a fluorescent group or a chemiluminescent group.
5. Compound according to claim 3, characterised in that Y is O.
6. Compound according to claim 3, characterised in that Q2 is: C(O)NR.sup.1., Q1 is: C(O)., NR.sup.2C(O)., OC(O)., C(O)NR.sup.2., O., S. or NR.sup.2., Y is: O, R.sup.1 and R.sup.2, independently of each other, are H or C.sub.1-C.sub.6 alkyl and in particular H, m is 1-30, and n is 1-12.
7. Compound according to claim 1, characterised in that R is a vitamin D side chain, preferably having a hydroxy group such as a structure according to formula (III) ##STR00012## or according to formula (IV) ##STR00013##
8. Compound according to claim 1, having a structure according to formula (Ib) ##STR00014## or formula (Ic) ##STR00015##
9. Method for producing a compound according to claim 1, comprising the steps of: (a) nucleophilically substituting, at the hydroxy group of the C3 atom, a compound having a structure according to formula (V) ##STR00016## wherein R is defined as in claim 1, wherein the hydroxy group is replaced by a group X with inversion of the configuration at the C3 stereocentre, in order to obtain a compound having a structure according to formula (VI), ##STR00017## R is defined as in claim 1, and X is an optionally protected hydroxy group, thiol group or primary or secondary amino group, and (b) coupling a labelling group W which is not a vitamin D compound to the group X of the compound having a structure according to formula (VI) via a linker, in order to obtain a compound having a structure according to formula (I).
10. In-vitro method for quantitatively determining vitamin D in a sample, using a compound according to claim 1 as a tracer.
11. In-vitro method according to claim 10, comprising the steps of: a) contacting a vitamin D-containing sample with the tracer, b) quantitatively determining the tracer under conditions in which the detected quantity of tracer allows a conclusion to be drawn as to the overall concentration of vitamin D in the sample.
12. Method according to either claim 10, characterised in that the tracer is determined by an immunological method, preferably by a competitive immunological method.
13. Method according to claim 10, characterised in that an antibody which specifically recognises the side chain R and preferably specifically recognises a vitamin D3 or vitamin D2 side chain having a hydroxy group at C25 is used to determine the tracer.
14. Method according to claim 10, characterised in that the tracer is added to the sample in a quantity to set a final concentration of from 0.2 to 100 ng/ml of this compound.
15. Method according to claim 10, characterised in that the sample to be tested is a biological fluid which is preferably selected from blood, serum, plasma or milk.
16. Method according to claim 10, characterised in that the vitamin D to be determined comprises a hydroxy group at C25 and is selected from the group comprising 25-hydroxy vitamin D3, 25-hydroxy vitamin D2, 1,25-dihydroxy vitamin D3, 1,25-dihydroxy vitamin D2 and mixtures thereof, the vitamin D preferably being 25-hydroxy vitamin D3 and/or 25-hydroxy vitamin D2.
17. Reagent for determining vitamin D, comprising a compound according to claim 1.
18. Reagent according to claim 17, further comprising a reagent for releasing bound vitamin D and optionally diluting agents, carriers, solvents, adjuvants, stabilisers, preservatives and/or dispersants.
19. Reagent according to claim 17, characterised in that it is present as a dry or wet reagent.
20. Use of a compound according to claim 1 for determining vitamin D.
21. Use of a compound having a structure according to formula (VI) ##STR00018## wherein R is a C.sub.2-C.sub.10 alkyl or alkenyl group, which is optionally substituted by a hydroxy group, and X is NHR.sup.3, OH or SH, wherein R.sup.3 is H or C.sub.1-C.sub.6 alkyl, for producing a Vitamin D compound according to claim 1.
Description
LEGEND TO THE FIGURES
[0086]
[0087]
[0088]
[0089]
EXAMPLE 1
Synthesis of 3-[-biotinamide--dodeca(ethylene glycol)-amide]-25-hydroxy-deoxyvitamin D3 (FIG. 1)
[0090] .sup.1H-NMR (at 300 MHz) spectra were recorded on a Bruker AC300 (Bruker, Karlsruhe, Bruker) using the solvent CDCl.sub.3. The evaluation was carried out in ppm with tetramethylsilanes as the internal standard. The mass spectra were determined on a Varian MAT 311 spectrometer (70 eV, EI). F.256 silica gel plates (Merck Darmstadt, Germany) were used for the analytical thin-layer chromatography. Chromatographic purification was carried out in silica gel columns, 230-400 mesh ASTM. All the reagents were obtained from Sigma Aldrich.
1.1: Synthesis of 3-phthalimide-25-hydroxy-deoxyvitamin D3
[0091] A solution of 25-hydroxy-vitamin D3 (10.0 mg, 25 mol), phthalimide (4.5 mg, 31.25 mol), triphenylphosphine (8.2 mg, 31.25 mol) and diethylazodicarboxylate (5.4 mg, 31.25 mol) in tetrahydrofuran (2.0 ml) was stirred at room temperature for 24 hours. After hexane (2.0 ml) was added, the mixture was filtered through a silica gel filter. After the solvent was removed, the solids were resuspended in 5 ml 10% Na.sub.2CO.sub.3 and were extracted twice using 10 ml diethyl ether each time. The combined ether phases were dried over MgSO.sub.4 and concentrated under vacuum to form a colourless oil. The product was purified by silica gel column chromatography. 3-phthalimide-25-hydroxy-deoxyvitamin D3 (
1.2: Synthesis of 3-amino-25-hydroxy-deoxyvitamin D3
[0092] 3-Phthalimide-25-hydroxy-deoxyvitamin D3 (2), (7.2 mg, 13.5 mmol) was dissolved in 0.5 ml 8M methylamine in ethanol in a nitrogen atmosphere. The mixture was first stirred for 2 hours at room temperature and then for 16 hours at 4 C. The solvent was evaporated at negative pressure and the residue was resuspended in 5 ml 10% Na.sub.2CO.sub.3 and was extracted three times using 10 ml diethyl ether each time. The combined ether phases were dried over MgSO.sub.4 and concentrated under vacuum to form a colourless oil. The product was purified by silica gel column chromatography. 3-Amino-25-hydroxy-deoxyvitamin D3 was isolated as a colourless oil (4), (5.2 mg). NMR: 0.57 (s, HC(18), 3H), 0.85 (d, J=6.4 Hz, HC(21), 3H), 1.21 (s, HC(26 and 27), 6H), AB quartet: 4.83 (d, J=2.4 Hz, HC(19)E, 1H) and 5.00 (d, J=1.8 Hz, HC(19)Z, 1H), AB quartet: 5.97 (d, J=11.0 Hz, HC(7), 1H) and 6.27 d, J=11.0 Hz, HC(6), 1H), MS: m/e (100) 400.37 (M.sup.+).
1.3: Synthesis of 3-[-biotinamide--dodeca(ethylene glycol)-amide]-25-hydroxy-deoxyvitamin D3
[0093] 3-Amino-25-hydroxy-deoxyvitamin D3 (3), (5.2 mg, 12.9 mol) triethylamine (0.02 L), NHS-PEG.sub.12-biotin (9.4 mg, 10.0 mol, Thermo Scientific, Prod. No. 21312) were dissolved in 0.2 ml dimethylformamide and stirred at room temperature overnight. The solvent was evaporated at negative pressure and the residue was purified by silica gel column chromatography. Here, 5.9 mg 3-[-biotinamide-w-dodeca(ethylene glycol)-amide]-25-hydroxy-deoxyvitamin D3 (4) was obtained in the form of a colourless wax. NMR: 0.58 (s, HC(18), 3H), 0.85 (d, J=6.4 Hz, HC(21), 3H), 1.21 (s, HC(26 and 27), 6H), 3.62 (m, HC(ethylene glycol), 50H), AB quartet: 4.84 (d, J=2.4 Hz, HC(19)E, 1H) and 5.06 (d, J=1.9 Hz, HC(19)Z, 1H), AB quartet: 6.02 (d, J=11.0 Hz, HC(7), 1H) and 6.26 (d, J=11.0 Hz, HC(6), 1H), MS: m/e (100) 1247.80 (M+Na.sup.+).
EXAMPLE 2
Preparation of a Vitamin D Release Reagent
[0094] Sodium toluenesulfonate was dissolved in water and adjusted with stock solutions of 1 M sodium citrate and 1 M iron(III) chloride to a final concentration of 1 M sodium toluenesulfonate, 100 mM sodium citrate and 50 mM iron(III) chloride.
EXAMPLE 3
Binding of a 25-OHD Antibody to a Solid Phase
[0095] An antibody against 25-OH-vitamin D3 or 25-OH-vitamin D2 was diluted in Tris-buffered saline pH 8.0 to a concentration of 1 g/ml. The wells of a microtitre plate (Maxisorb, Nunc) were coated with 100 l of the diluted antibody, dried at 37 C. and stored at 4 C. until the test was carried out.
EXAMPLE 4
Competitive Binding Analysis
[0096] Defined concentrations of 25-OHD3 were added to vitamin D-free serum matrix (Seracon, Seracare Inc.). In each case 80 l of the concentration series were introduced into a microtitre plate well pre-loaded with 3-[-biotinamide--dodeca(ethylene glycol)-amide]-25-hydroxy-deoxyvitamin D3 (25OHD-biotin tracer reagent) from example 1 as a tracer and incubated for 5 minutes at RT (20-27 C.) for resolubilisation of the tracer reagent. 80 l of the release reagent from example 2, which dissociates vitamin D in the sample from the complex with DBP, were then added to the sample batch and mixed. 100 l of the samples diluted with vitamin D release reagent were transferred into wells coated with 25OHD antibody and incubated for 30 minutes at RT. The 25OHD contained in the sample now competes with the 25OHD biotin tracer for binding sites on the 25OHD antibody. The sample batch was then removed and the wells were rinsed three times with 200 l of wash buffer each time. The antibody-bound biotin-25OHD tracer was detected with peroxidase-labelled streptavidin and a tetramethylbenzidine (TMB) colour reaction. After the substrate reaction had been stopped by addition of 100 l of 50 mM phosphoric acid, the optical density was measured at 450 nm. Here, a specific competition of the biotin-25OHD tracer was demonstrated, as shown in
EXAMPLE 5
Determination of 25-Hydroxy Vitamin D in Serum or Plasma in the Automated Alegria 25-OH Vitamin D3/D2 Test System
[0097] In order to study the correlation between HPLC+LC-MS/MS-based 25OHD3/D2 determination methods and the automated Alegria 25-OH vitamin D3/D2 test system (ORG270, Orgentec Diagnostika GmbH), serum or plasma samples, the 25OHD3/D2 concentration of which was characterised by HPLC+LC-MS/MS-based methods (6PLUS1 Multilevel Serum Calibrator Set 25-OH-Vitamin D3/D2, Chromsystems GmbH), and dilutions thereof were stored in aliquots at 20 C. until the determination.
[0098] For the determination, in each case 80 l of the samples were placed in well A of the Alegria test strip. The 8 wells of a test strip contained the vitamin D release reagent from example 2, streptavidin-HRP conjugate, TMB substrate, a 25OHD calibrator solution and 25-OHD antibody-coated wells for in each case one sample determination. Test processing took place automatically in the Alegria machine. Inside a test strip, the sample and the calibrator, internal to the test strip, were in each case mixed with 25OHD-biotin tracer and vitamin D release reagent, transferred to the 25OHD antibody-coated wells and washed after being incubated for 30 minutes. The antibody-bound biotin-25OHD tracer was detected with peroxidase-labelled streptavidin and a tetramethylbenzidine (TMB) colour reaction by measuring the optical density at 650 nm.
[0099] As is shown in
EXAMPLE 6
Determination of the Serum Stability of 25-OH Vitamin D Derivatives
[0100] For comparative testing of the serum stability of biotin 25-OH vitamin D derivatives, the test compounds were incorporated in a vitamin D-free serum matrix (Seracon, Seracare Inc.). In a first batch (VD1), the 25-OH vitamin D derivative 3-[-biotinamido--dodeca(ethylene glycol)-amido]-25-hydroxy-deoxyvitamin D.sub.3 that is in accordance with example 1 and is present at the C3 atom in the non-natural (R) configuration was set to a concentration of 3 ng/ml in a vitamin D-free serum matrix. In a second batch (VD2), the 25-OH vitamin D derivative 3-3[-biotinamido--dodeca(ethylene glycol)-amido]amidopropyl-ether-25-hydroxy-vitamin D.sub.3 that is in accordance with US20080317764 and is present at the C3 atom in the natural (S) configuration was set to a concentration of 3 ng/ml in a vitamin D-free serum matrix.
[0101] Aliquots of 1 ml each of the batches VD1 and VD2 were stored at 20 C. and were incubated for 24 hours at 37 C. The samples were then equilibrated for 1 hour at room temperature (22 C.). 500 l of each of the reference samples stored at 20 C. and of each of the samples incubated for 24 hours at 37 C. were added to and mixed with 500 l release reagent according to example 2 in each case. 100 l of the samples diluted with vitamin D release reagent were transferred into 25OHD antibody-coated wells according to example 3 and were incubated at room temperature for 30 minutes, so that the biotin-25-OH vitamin D derivatives present in the samples bound to the 25OHD antibody.
[0102] The sample batches were then removed and the wells were rinsed three times with 200 l of wash buffer each time. The antibody-bound biotin-25-OH vitamin D derivatives were detected with peroxidase-labelled streptavidin and a tetramethylbenzidine (TMB) colour reaction. After the substrate reaction had been stopped by addition of 100 l of 50 mM phosphoric acid, the optical density was measured at 450 nm.
[0103] This demonstrated, as shown in