ES2748064A1 - VITAMIN D COMPOUNDS WITH ISOTOPIC MARKING (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Vitamin D compounds with isotopic labeling. The invention is directed to compounds of formula I characterized by being derived from vitamins D2 and D3 with isotopic labeling (13 C,14 C,11 C) in any of its carbons, to procedures for obtaining said compounds and to intermediates useful in their synthesis. These compounds of formula I are useful as internal standards in methods of quantifying vitamin D, its metabolites and/or analogues by mass spectrometry; as tracer ligands of the nuclear vitamin D receptor for its study by nuclear magnetic resonance (NMR), as tools for the study of metabolism and as agents in positron emission tomography (PET) techniques. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Vitamin D compounds with isotopic labeling

Technical sector

The invention is directed to compounds of formula I characterized by being derivatives of vitamins D2 and D3 with isotopic labeling (13C, 14C, 11C) in any of their carbons, to processes for obtaining said compounds and to useful intermediates in their synthesis. These compounds of formula I are useful as internal standards in methods of quantifying vitamin D, its metabolites and / or analogs by mass spectrometry; as tracer ligands of the nuclear receptor of vitamin D for its study by nuclear magnetic resonance (NMR), as tools for the study of metabolism and as agents in positron emission tomography (PET) techniques.

State of the art

The stages of metabolic activation and the functions of vitamin D have been discovered thanks to the use of metabolites and vitamin D analogues that incorporated radioactive tracers.

Metabolites of vitamin D3 with 13C incorporation at positions 7,9 and 19 (see WH Okamura et al., J. Org. Chem. 2002, 67, 1637-1650) are useful as isotopically substituted reference substances of different vitamins. D and metabolites for use in methods of quantifying vitamin D (of its metabolites and / or analogues) in blood plasma and tissues by direct mass spectrometry or by serially coupled (tandem) techniques with gas or liquid chromatography; as tracer ligands of the nuclear vitamin D receptor for their study by NMR and agents in positron emission tomography (PET) techniques.

Description of the Invention

The present invention is directed to compounds with high degrees of incorporation of 13C into the metabolites and analogues of vitamins D3 and D2. Said compounds they can present markings in positions 19, 23, 24, 25, 26, 27 and / or 28. The authors of the present invention have developed a methodology versatile enough to be able to choose the desired position in the vitamin structure mark.

The compounds of the invention have the vitamin's own side chain, without hydroxyl at C25, are compounds with greater complexity than those known and optionally have an epimeric configuration at C1, C3 and / or C20.

In one aspect, the present invention is directed to compounds of formula I

where

A is a single, double or triple bond,

B is a single or double bond,

F is selected from methylene (CH2), 13C-methylene (13CH2), 11C-methylene (11CH2), and -14C-methylene (14CH2),

G is selected from is selected from methylene (CH2), 13C-methylene (13CH2), 11C-methylene (11CH2), and -14C-methylene (14CH2) when N is hydrogen, and is selected from -CH-, -13CH2- , -11CH2-, and -14CH2 when N is not hydrogen, -K is selected from -C-, -13C-, -11C-and -13C-,

M is a methyl, methyl-13C, trideuteromethyl-13C, methyl-11C, methyl-14C, ethyl group N is a hydrogen, methyl, hydroxyl or -OR, where R is selected from the groups methoxymethyl, methoxyethyl, trimethylsilylethoxymethyl, trimethylsilyl , triethylsilyl, t-butyldimethylsilyl, tri- / so-propylsilyl, t-butyldiphenylsilyl, dimethylphenylsilyl, dimethylbenzylsilyl, benzoate, p-nitrobenzoate, pivalate or acetate,

R1, R2, R3 and R4 are independently selected from hydrogen, hydroxyl or -OR, where R is selected from the groups methoxymethyl, methoxyethyl, trimethylsilylethoxymethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, tri- / so-propylsilyl, tbutyldiphenylsilyl, dimethylphenylsilyl, dimethylbenzyl silyl, benzoate, p-nitrobenzoate, pivalate or acetate,

R5 are independently selected from hydrogen, methylene group ( = CH ² ), dideuteromethylene group ( = CD ² ), methylene-13C group ( = 13CH ² ), dideuteromethylene-13C group ( = 13CDi), methylene-11C group ( = 11CH ²⁾ ) or methylene-14C group ( = 14CH ² ),

R6 and R7 are independently selected from hydrogen or -methyl,

R8 are independently selected from hydrogen, hydroxyl, or -OR, where R is selected from the groups methoxymethyl, methoxyethyl, trimethylsilylethoxymethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, tri- / so-propylsilyl, t-butyldiphenylsilyl, dimethylphenylsilyl, dimethylphenylsilyl , p-nitrobenzoate, pivalate or acetate,

In a particular embodiment the invention is directed to compounds of formula I where F is -13CH2- and G can be -13CH2- or -13CH-.

In a particular embodiment the invention is directed to the compounds of formula I where F is -13CH2-, G can be -13CH2- or -13CH- and K is 13C.

In a particular embodiment the invention is directed to the compounds of formula I where F is -13CH2-, G can be -13CH2- or -13CH-, K is 13C and M is 13C-methyl (13CH3).

In a particular embodiment the invention is directed to the compounds of formula I where K is 13C and M is 13C-methyl (13CH3).

In a particular aspect, the invention is directed to the following compounds of formula I:

19.23.24.25.26.27- 13C6-1a-hydroxyvitamin D3 (1a),

19.23.24.25.26.27- 13C6-1a, 25-dihydroxyvitamin D3 (1b),

23.24.25.26.27- 13C5-vitamin D3 (1c),

23.24.25.26.27- 13C5-25-hydroxyvitamin D3 (1d),

23.24.25.26.27- 13C5-1a-hydroxyvitamin D3 (1e),

23.24.25.26.27- 13C5-1a, 25-dihydroxyvitamin D3 (1f),

23.24.25.26.27- 13C5-3-epi-1a, 25-dihydroxyvitamin D3 (1g),

19.23.24.25.26.27- 13C6-1a, 25-dihydroxyvitamin D2 (2a),

23.24.25.26.27- 13C5-vitamin D2 (2b),

23.24.25.26.27- 13C5-25-hydroxyvitamin D2 (2c),

23.24.25.26.27- 13C5-1a-hydroxyvitamin D2 (2d),

23.24.25.26.27- 13C5-1a, 25-dihydroxyvitamin D2 (2e),

23,24,25,26,27-13C5-3-epi-1a, 25-dihydroxyvitamin D2 (2f).

Synthesis of the compounds of formula I

Another aspect of the invention relates to a process for the preparation of the compounds of formula I, which comprises a coupling of compounds II and III in the presence of a catalyst,

where A, B, F, G, K, M, N, R1, R2, R3, R4, R6, R7 and R8 are selected as in formula I,

R9 are independently selected from methylene (= CH ² ), dideuteromethylene (= CD2), 13C-methylene- (13CH ² ), 13C-dideuteromethylene (= 13CD ² ), 11C-methylene (= 11CH ² ) or

14C-methylene (= 14CH ² ),

X is indium dichloride (-InCh), zinc bromide (-ZnBr) or dialkoxyboro [-B (OR) 2].

In a particular aspect, the catalyst is preferably selected from tetra-k / striphenylphosphine palladium (0), palladium (II) dichloride bis-triphenylphosphine, palladium (II) dichloride bis- (diphenylphosphine) ferrocene, palladium (II) acetate , palladium (II) dichloride bis-acetonitrile, tris (dibenzylidene ketone) dipalladium (0), nickel (II) acetylacetonate, or bis (1,5-dicyclooctadienyl) nickel (0).

In another aspect of the invention it is directed to an alternative process for the preparation of the compounds of formula I comprising a Wittig-Horner reaction between compounds IV and V in the presence of a base,

seleccion fórmula I. where

I select formula I.

The high degree of isotopic incorporation of the compounds of formula I that give it high versatility when preparing compounds derived from vitamin D with isotopic labeling in certain positions, can only be obtained from a compound of formula II and IV with the same degree of isotopic incorporation. According to this, the intermediates. Accordingly, the intermediates of formula V are key since each and every one of its functional groups is selected to allow isotopic incorporation into the vitamin D derivatives of the present invention, and allows to obtain the compounds of formula II and IV effectively and in a few stages.

In a particular embodiment, the invention relates to the preparation of the compounds of formula II described above from the compounds of Va or Vb, which comprises

(a) catalytic hydrogenation,

(b) lack of protection, and

(c) oxidation,

where F, G, K, M, N and R8 are selected as in formula I.

where R9 is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl or tert-butyldimethylsilyl, acetate, benzoate, para-methoxybenzoate, pivalate.

where F, G, K, M, N and R8 are selected as in formula I.

In a particular embodiment, the complete reduction of the double bond and the triple bond is carried out in the presence of a metal catalyst in a hydrogen atmosphere or by hydrogen transfer. In a particular embodiment the metal catalyst is selected from palladium on carbon, platinum on carbon, rhodium on carbon, nickel-Raney and Wilkinson's catalyst.

A person skilled in the art can select different conditions for the deprotection of step b), where a hydroxyl is obtained from the -OR9 group. These conditions are selected depending on the value of R9, for example among the conditions described in Wuts, PGM, Greene, TW; "Protective Groups in Organic Synthesis”, 4th Ed., John Wiley & Sons, Inc. 2007, New Jersey, pages 24-222. For example, in a case where R9 is trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl or tert-butyldimethylsilyl, deprotection can be carried out by reaction with tetrabutylammonium fluoride, with aqueous solution of hydrofluoric acid. with pyridinium fluoride.

A person skilled in the art can select different conditions to carry out the oxidation of step c) as described in R.C. Larock. Comprehensive Organic Transformations. John Wiley & Sons, Inc. 2nd. edition, 1999, New York, pages 1234 1248. For example, oxidation can be carried out using a chromium derivative, such as pyridinium dichromate, pyridinium chlorochromate, chromium trioxide, chromic acid; Dess-Martin periodinan or tetrapropylammonium perruthenate.

In a particular embodiment, the invention relates to the preparation of the compounds of formula II described above from the Vc or Vd compounds, which comprises

(a) partial hydrogenation of the triple bond to a cis alkene,

(b) formation of a good leaving group (independently selected from a carbamate group [-OC (O) NH-], a dialkylphosphate group [-OP (O) (OR) 2] or an ester group [-OC ( O) R],

(c) double-bond assisted bimolecular nucleophilic substitution,

(d) lack of protection, and

(c) oxidation,

where F, G, K, M, N and R8 are selected as in formula I.

where R9 is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl or tert-butyldimethylsilyl, acetate, benzoate, para-methoxybenzoate, pivalate.

In a particular embodiment, the partial reduction of the triple bond is carried out in the presence of a metal catalyst poisoned in a hydrogen atmosphere or by hydrogen transfer. In a particular embodiment the metal catalyst is select from Lindlar catalyst, palladium on carbon, and Wilkinson's catalyst.

A person skilled in the art can select different conditions for the formation of the carbamate from step b). In a particular embodiment, phenyl isocyanate in solution in pyridine is used.

A person skilled in the art knows the methodology for carrying out a double-bond-assisted nucleophilic substitution on the carbamate with stereochemistry without the use of cuprates (see A. Mouriño et al., J. Org. Chem. 1986, 51, 1264 1269).

A person skilled in the art can select different conditions for the deprotection of step b), where a hydroxyl is obtained from the -OR9 group. These conditions are selected depending on the value of R9, for example among the conditions described in Wuts, P. G. M., Greene, T. W .; "Protective Groups in Organic Synthesis", 4th Ed., John Wiley & Sons, Inc. 2007, New Jersey, pages 24-222. For example, in a case where R9 is an ester, deprotection can be carried out by the reaction with lithium aluminum hydride, with aluminum (III) hydride, with alkaline and alkaline earth hydroxides in THF / H2O.

A person skilled in the art can select different conditions to carry out the oxidation of step c) as described in R.C. Larock. Comprehensive Organic Transformations. John Wiley & Sons, Inc. 2nd. edition, 1999, New York, pages 1234 1248. For example, oxidation can be carried out using a chromium derivative, such as pyridinium dichromate, pyridinium chlorochromate, chromium trioxide, chromic acid; Dess-Martin periodinan or tetrapropylammonium perruthenate.

In a particular embodiment, the invention relates to the preparation of the compounds of formula Va or Vb described above from the compounds of Via or VIb, which comprises

(a) formation of tosylate or iodide,

(b) bimolecular nucleophilic substitution with the protected 2-methyl-3-butin-2-ol anions incorporating isotopic labels at positions 23,24,25,26 and 27 (positions F; G, K, M); 23,24 and 25 (positions F, G, K); 25,26 and 27 (positions K, M); or 23 and 24 (positions F; G).

(c) lack of protection, and

(d) oxidation,

where F, G, K, M, N and R8 are selected as in formula I.

where R9 is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl or tert-butyldimethylsilyl, acetate, benzoate, para-methoxybenzoate, pivalate.

where R9 is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl or tert-butyldimethylsilyl, acetate, benzoate, para-methoxybenzoate, pivalate.

The preparation form of the compounds of formula Vía and VIb are described in patent ES2396524 (Versatile and functionalized intermediates in the synthesis of vitamin D and new derivatives of vitamin D).

In a particular embodiment, the invention relates to the preparation of the compounds of formula Vc or Vc described above from the compounds of VIc or VId, comprising

(a) nucleophilic addition with the protected 2-methyl-3-butin-2-ol anions incorporating isotopic labels at positions 23,24,25,26 and 27 (positions F; G, K, M); 23,24 and 25 (positions F, G, K); 25,26 and 27 (positions K, M); or 23 and 24 (positions F; G),

(b) partial hydrogenation of the triple bond to a cis alkene,

(c) formation of a good leaving group (independently selected from a carbamate group [-OC (O) NH-], a dialkylphosphate group [-OP (O) (OR) 2] or an ester group [-OC ( O) R],

(d) double-bond assisted bimolecular nucleophilic substitution,

(e) lack of protection, and

(f) oxidation,

where R9 is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl or tert-butyldimethylsilyl, acetate, benzoate, para-methoxybenzoate, pivalate.

In a particular embodiment, the partial reduction of the triple bond is carried out in the presence of a metal catalyst poisoned in a hydrogen atmosphere or by hydrogen transfer. In a particular embodiment, the metal catalyst is selected from Lindlar catalyst, palladium on carbon, and Wilkinson's catalyst.

A person skilled in the art can select different conditions for the formation of the carbamate from step b). In a particular embodiment, phenyl isocyanate in solution in pyridine is used.

A person skilled in the art knows the methodology for carrying out a double-bond-assisted nucleophilic substitution on the carbamate with stereochemistry without the use of cuprates (see A. Mouriño et al., J. Org. Chem. 1986, 51, 1264 1269).

A person skilled in the art can select different conditions for the deprotection of step b), where a hydroxyl is obtained from the -OR9 group. These conditions are selected depending on the value of R9, for example among the conditions described in Wuts, P. G. M., Greene, T. W .; "Protective Groups in Organic Synthesis", 4th Ed., John Wiley & Sons, Inc. 2007, New Jersey, pages 24-222. For example, in a case where R9 is an ester, deprotection can be carried out by the reaction with lithium aluminum hydride, with aluminum (III) hydride, with alkaline and alkaline earth hydroxides in THF / H2O.

A person skilled in the art can select different conditions to carry out the oxidation of step c) as described in R.C. Larock. Comprehensive Organic Transformations. John Wiley & Sons, Inc. 2nd. edition, 1999, New York, pages 1234 1248. For example, oxidation can be carried out using a chromium derivative, such as pyridinium dichromate, pyridinium chlorochromate, chromium trioxide, chromic acid; Dess-Martin periodinan or tetrapropylammonium perruthenate.

A person skilled in the art can prepare the compounds of formula VIc and VIc from compounds Via and VIb (patent ES2396524, Versatile and functionalized intermediates in the synthesis of vitamin D and new derivatives of vitamin D).

Compounds of formula VII, VIII and III, which incorporate isotopic labeling at C19,

where

* means isotopic labeling at position C19 (13C, 11C or 14C-methylene ( = 14CH ² ), E is a SiR3, SiR2R ', where R and R' are alkyl or aryl radicals, or a methoxymethyl group. It is known how to prepare these compounds from compound IX (described by BM Trost et al., J. Am. Chem.Soc. 1982, 114, 9836 9845).

A person skilled in the art is capable of synthesizing protected 2-methyl-3-butin-2-ol that incorporate isotopic labels at positions 23,24,25,26 and 27 (formula I, positions F; G, K, M) ; 23,24 and 25 (formula I, positions F; G, K); 25,26 and 27 (formula I, positions K, M); or 23 and 24 (formula I, positions F; G) from the following commercial compounds: 13C3-ethyl propyolate, 13C3-acetone and 13C2-trimethylsilylacetylene.

The compounds of formula I are isotopically labeled compounds derived from vitamins D2 and D3, useful as internal standards in quantitative methods of vitamin D, its metabolites and / or analogues by mass spectrometry, as tracers of the nuclear receptor receptor for vitamin D for its study by NMR, as tools for the study of metabolism and and agents in positron emission tomography (PET) techniques.

Detailed description of the invention

The compounds of formula I are prepared by a convergent synthesis through either of the two procedures shown in Scheme 1.

The compounds of formula I are prepared by coupling between a compound of formula II and a compound of formula III, in the presence of a catalyst, wherein the coupling is carried out in the presence of a catalyst preferably selected from tetra-to / s -triphenylphosphine palladium (0), palladium (II) dichloride bistriphenylphosphine, palladium (II) dichloride bis- (diphenylphosphin) ferrocene, palladium (II) acetate, palladium (II) dichloride bis-acetonitrile, tris (dibenzylidene acetone) (0), nickel (II) acetylacetonate, or bis (1,5-dicyclooctadienyl) nickel (0), with a compound of formula III. If necessary, the deprotection of the protecting groups is carried out, particularly with tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature, with hydrofluoric acid or with acidic ion exchange resin AG W50 X4 in deoxygenated methanol.

In particular, this coupling can be carried out by reacting the anion of the compound of formula V, which is prepared by reacting the compound of formula V with n-butyllithium, lithium di- / so-propylamide, tert-butoxide sodium, potassium hexamethyldisilazanide or sodium hexamethyldisilazanide at -78 ° C in anhydrous tetrahydrofuran, with compounds of formula IV. If necessary, it is possible to carry out the deprotection of the protecting groups. This deprotection is preferably carried out with tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature and with AG W50 X4 acidic ion exchange resin in deoxygenated methanol.

Particular embodiment of the invention

The following examples and comments are provided to enable those of skill in the art to have a clearer understanding and to practice the invention. They should not be considered as limiting the scope of the invention, but illustrative and representative thereof.

The compounds of formula II can be prepared in a particular way by a sequence of reactions from the already known compound of formula 3 (Scheme 2). The compounds of formula 4 are obtained nucleophilic substitution with the different anions of the protected 2-methyl-3-butin-2-ol that incorporate isotopic labels at positions 23,24,25,26 and 27; 23,24 and 25; 25,26 and 27 or 23 and 24 (see MR Uskokovic et al, J. Org. Chem. 1986, 51, 3098-3108).

The compounds of formula 5 are prepared by hydrogenation of the triple bond by treatment with hydrogen in the presence of Pd / C (10%) in anhydrous methanol.

Scheme 2

8a, 13C5-23,24,25,26,27 7a, 13C5-23,24,25,26,27 6a, 13C5-23,24,25,26,27 8b, 13C3-23,24,25 7b, 13C3-23,24,25 6b, 13C3-23,24,25 8c, 13C3-25,26,27 7c, 13C3-25,26,27 6c, 13C3-25,26,27 8d, 13C2-23,24 7d, 13C2-23,24 6d, 13C2-23,24

The compounds of formula 6 are prepared by reaction with tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature.

The compounds of formula 7 are obtained by oxidation (pyridinium dichromate, Dess-Martin periodinan, TEMPO) in anhydrous dichloromethane.

Compounds of formula 8 are prepared by a Wittig reaction with bromomethyl triphenylphosphonium ylide, followed by a Miyaura reaction (palladium catalyzed Br / B exchange), or by the Sato method (see F. Sato et al, J. Org. Chem.

2003, 68, 9767-9772).

In particular, the compounds of formula 12 are obtained from compounds 5 (Scheme 3). The compounds of formula 9 are prepared by a selective deprotection reaction of methoxymethyl ether.

The compounds of formula 10 are prepared by a deoxygenation reaction of the hydroxyl group by formation of a xanthate and subsequent reaction with hydride of tributyltin or by dehydration followed by catalytic hydrogenation (see A. Mouriño et al, Chem. Eur. J. 2018, 24, 3314-3320).

The compounds of formula 11 are prepared by reaction with tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature, with hydrofluoric acid or with acidic ion exchange resin AG W50 X4 in deoxygenated methanol.

The compounds of formula 12 are obtained by oxidation with pyridinium dichromate in anhydrous dichloromethane.

Scheme 3

13d, 13C2-23,24 12d, 13C2-23,24 12d, 13C2-23,24

Alternatively the compounds of formula I can be prepared in a particular way by a sequence of reactions from the already known compound of formula 14 (Scheme 4).

The compounds of formula 15 are prepared by stereoselective addition with the different protected 2-methyl-3-butin-2-ol anions incorporating isotopic labels at positions 23,24,25,26 and 27 and 25,26 and 27.

The compounds of formula 16 are obtained by partial hydrogenation of the triple bond by treatment with hydrogen and the Lindlar catalyst (10% Pd on BaSO4, quinoline) in anhydrous methanol, and subsequent reaction of the allyl alcohols prepared with phenyl isocyanate in anhydrous pyridine .

The compounds of formula 17 are prepared by substitution reaction with methyl cuprates.

The compounds of formula 18 are obtained by reaction with lithium aluminum hydride in anhydrous tetrahydrofuran, followed by oxidation of the resulting alcohols with pyridinium dichromate in anhydrous dichloromethane.

Compounds of formula 19 are prepared by a Wittig reaction with bromomethyl triphenylphosphonium ylide, followed by a Miyaura reaction (palladium catalyzed Br / B exchange), or by the Sato method (see F. Sato et al, J. Org. Chem.

2003, 68, 9767-9772).

In particular, the compounds of formula 23 are obtained from compounds 17 (Scheme 5). The compounds of formula 20 are prepared by a selective deprotection reaction of methoxymethyl ether.

The compounds of formula 21 are prepared by formation of a xanthate and subsequent reaction with tributyltin hydride or by dehydration followed by catalytic hydrogenation (see A. Mouriño et al, Chem. Eur. J. 2018, 24, 3314-3320). The compounds of formula 22 are prepared by reaction with tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature.

The compounds of formula 23 are obtained by oxidation (pyridinium dichromate, Dess-Martin periodinan, TEMPO) in anhydrous dichloromethane.

As shown in Scheme 1, the compounds of formula III corresponding to rings A are prepared by a sequence of reactions from the compound of formula 24, which is already known (Scheme 6). The compound of formula 25 is obtained by epoxidation of the double bond with mete-chloroperbenzoic acid, followed by oxidative breakdown of the epoxide with periodic acid.

The compound of formula 26 is obtained by reaction with the isotope-substituted organolytic (13C, 11C, 14C or 13C-2H) obtained by metalation of the corresponding 13C-methyl (13CH3I) iodides, 13C-trideuteromethyl (13CD3I), of 11C-methyl (11CH3I), or 14C-methyl (14CH3I) in ethyl ether at low temperature.

The compound of formula 27 is prepared by oxidation (pyridinium dichromate, Dess-Martin periodinan, TEMPO) in anhydrous dichloromethane.

The compound of formula 28 is prepared by treatment with a base (lithium di- / sopropylamide, lithium hexamethyldisilazanide, potassium tert-butoxide) in anhydrous THF followed by entrapment of the enolate with W- (5-chloro-2-pyridyl) triflimide.

Scheme 6

Example 1

19,23,24,25,26,27-13C6-1a-Hydroxyvitamin D3 ( 1a )

An aqueous solution of K3PO4 (3 mL, 2M) and PdCl2 (PPh3) 2 (3mg, 0.00039mmol) were added over a solution of 13a (43mg, 0.0102mmol) and 28 (58mg, 0.11mmol) in THF ( 4 mL). The mixture was vigorously stirred. The reaction was stopped by the addition of water. The mixture was extracted with MTBE. The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography. The residue was dissolved in THF (6 mL). A solution of TBAF in THF (0.30 mL) was added. The mixture was stirred for 48h. Saturated NH4CL solution was added. The mixture was extracted with Et2O. The combined organic phase was dried, filtered, and concentrated. The residue was purified by HPLC Phenomenex Luna 5 ^ silica, 20% PrOH / hexanes, rt = 13.5 min to give 1a [28 mg, 69% (2 steps), white foam]. 1H NMR (500 MHz, CDCh) 56.34 (d, J = 11.2 Hz, 1H, H-6), 5.99 (d, J = 11.3 Hz, 1H, H-7), 5.27 (d, J = 158.9 Hz, 1H , H-19), 4.98 (d, J = 160.7 Hz, 1H, H-19), 3.90 (tt, J = 6.8, 3.5 Hz, 1H, H-3), 1.02 (dd, J = 7.4, 3.6 Hz , 6H, Me-26, Me-27), 0.90 (d, J = 6.5 Hz, 3H, Me-21), 0.51 (s, 3H, Me-18).

Example 2

19,23,24,25,26,27-13Ce-1a, 25-Dihydroxyvitamin D3 (1b)

Following the procedure described in Example 1, an aqueous solution of K3PO4 (2 mL, 2M), PdCl2 (PPh3) 2 (2mg, 0.00026mmol) 9a (22mg, 0.052mmol) and 28 (30mg, 0.058mmol) they provided 1b [17 mg, 78% (2 steps), white foam]. 1H NMR (500 MHz, CDCh) 56.35 (d, J = 11.2 Hz, 1H, H-6), 5.98 (d, J = 11.3 Hz, 1H, H-7), 5.29 (d, J = 158.9 Hz, 1H , H-19), 4.97 (d, J = 160.7 Hz, 1H, H-19), 4.40 (dt, J = 7.6, 3.6 Hz, 1H, H-1), 4.20 (tt, J = 6.8, 3.5 Hz , 1H, H-3), 1.24 (dd, J = 7.4, 3.6 Hz, 6H, Me-26, Me-27), 0.91 (d, J = 6.5 Hz, 3H, Me-21), 0.52 (s, 3H, Me-18).

Example 3

23,24,25,26,27-13C5-Vitamin D3 (1c)

To a solution of 1.23 mmol of phosphine oxide 29 in 10 mL of anhydrous tetrahydrofuran cooled to -78 ° C, 1.23 mmol of 1.5M n-butyllithium in hexane are added dropwise under argon atmosphere. After stirring for 30 minutes, a solution of 0.15 mmol of ketone 12a in 6 mL of anhydrous tetrahydrofuran is added dropwise over a period of 30 minutes. The reaction mixture is then stirred at -78 ° C for 2 hours. The reaction is stopped by the addition of an aqueous sodium chloride solution and the resulting mixture is allowed to reach room temperature. Isolation of the title compound can be done by extraction with ethyl acetate and purification by flash chromatography on silica gel. The removal of the silicon protecting group can be carried out by treating a solution of the silylated title compound in 2 mL of deoxygenated and anhydrous methanol, with 500 mg of DOWEX AG 50W-X4 resin under argon atmosphere and stirring in the dark at temperature. environment for 24 hours. This reaction mixture is stopped by addition of an aqueous sodium chloride solution and the title compound is isolated by extraction with ethyl acetate and purified by flash column chromatography on silica gel to give 1c (89% yield) . 1H NMR (250 MHz, CDCh) 56.22 (d, J = 11 Hz, 1H, H-6), 5.99 (d, J = 11 Hz, 1H, H-7), 5.21 (br s, 1H, H-19 ), 4.98 (br s, 1H, H-19), 4.39 (d, J = 3.6 Hz, 1H, H-1), 4.18 (t, J = 3.5 Hz, 1H, H-3), 1.03 (dd, J = 7.4, 3.6 Hz, 6H, Me-26, Me-27), 0.90 (d, J = 6.5 Hz, 3H, Me-21), 0.51 (s, 3H, Me-18).

Example 4

23,24,25,26,27-13C5-25-Hydroxyvitamin D3 ( 1d )

Following the procedure described in Example 3, 2.45 mmol of phosphine oxide 29 in 15 mL of tetrahydrofuran, 2.45 mmol of 1.5M n-butyllithium, 0.61 mmol of ketone 7a in 8 mL of tetrahydrofuran provided 1d (90% yield , Two steps). 1H NMR (250 MHz, CDCh) 56.32 (d, J = 11 Hz, 1H, H-6), 5.98 (d, J = 11 Hz, 1H, H-7), 5.27 (br s, 1H, H-19 ), 4.98 (br s, 1H, H-19), 3.91 (tt, J = 6.8, 3.5 Hz, 1H, H-3), 1.21 (d, J = 7.4 Hz, 6H, Me-26, Me-27 ), 0.92 (d, J = 6.5 Hz, 3H, Me-21), 0.52 (s, 3H, Me-18). Example 5

23,24,25,26,27-13C5-1a-Hydroxyvitamin D3 (1e)

Following the procedure described in Example 3, 0.31 mmol of phosphine oxide 30 in 8 mL of tetrahydrofuran, 0.30 mmol of 1.5M n-butyllithium, 0.20 mmol of ketone 12a in 6 mL of tetrahydrofuran provided 1e (86% yield , Two steps).

1H NMR (250 MHz, CDCh) 56.32 (d, J = 11 Hz, 1H, H-6), 5.98 (d, J = 11 Hz, 1H, H-7), 5.24 (br s, 1H, H-19 ), 4.96 (br s, 1H, H-19), 3.91 (tt, J = 6.8, 3.5 Hz, 1H, H-3), 1.02 (d, J = 7.4 Hz, 6H, Me-26, Me-27 ), 0.90 (d, J = 6.5 Hz, 3H, Me-21), 0.51 (s, 3H, Me-18).

Example 6

23,24,25,26,27-13C5-1a, 25-Dihydroxyvitamin D3 (1f)

Following the procedure described in Example 3, 1.2 mmol of phosphine oxide 30 in 15 mL of tetrahydrofuran, 1.15 mmol of 1.5M n-butyllithium, 0.85 mmol of ketone 7a in 12 mL of tetrahydrofuran provided 1f (90% yield two steps) .1H NMR (250 MHz, CDCh) 56.32 (d, J = 11 Hz, 1H, H-6), 5.98 (d, J = 11 Hz, 1H, H-7), 5.27 (br s, 1H, H-19), 4.98 (br s, 1H, H-19), 4.37 (d, J = 3.6 Hz, 1H, H-1), 4.20 (t, J = 3.5 Hz, 1H, H-3) , 1.22 (d, J = 7.4 Hz, 6H, Me-26, Me-27), 0.93 (d, J = 6.5 Hz, 3H, Me-21), 0.53 (s, 3H, Me-18).

Example 7

23,24,25,26,27-13C5-3-epi-1a, 25-Dihydroxyvitamin D3 (1g)

Following the procedure described in Example 3, 1.22 mmol of phosphine oxide 31 in 20 mL of tetrahydrofuran, 1.20 mmol of n-butyl lithium 1.5 M, 0.81 mmol of ketone 7a in 15 mL of tetrahydrofuran provided 1j (88% yield two steps) .1H NMR (500 MHz, CDCh) 56.43 (d, J = 11 Hz, 1H, H-6), 6.02 (d, J = 11 Hz, 1H, H-7), 5.29 (br s, 1H, H-19), 5.00 (br s, 1H, H-19), 4.31 (d, J = 4.5 Hz, 1H, H-1), 4.05 (m, 1H, H-3), 1.21 (d, J = 7.4 Hz, 6H, Me-26, Me-27), 0.94 (d, J = 6.5 Hz, 3H, Me-21), 0.54 (s, 3H, Me-18).

Example 8

19,23,24,25,26,27-13Ce-1a, 25-Dihydroxyvitamin D2 (2a)

Following the procedure described in Example 1, an aqueous solution of K3PO4 (3 mL, 2M) and PdCh (PPh3) 2 (3 mg, 0.00039 mmol), a solution of 19a (34 mg, 0.078 mmol) and 28 (45 mg 0.088 mmol) in THF (4 mL) provided 2a [26 mg, 79% (2 steps), white foam]. 1H NMR (500 MHz, CDCh) 56.34 (d, J = 11.2 Hz, 1H, H-6), 6.01 (d, J = 11.3 Hz, 1H, H-7), 5.35 (m, 2H, H-22, H-23), 5.28 (d, J = 158.9 Hz, 1H, H-19), 4.96 (d, J = 160.7 Hz, 1H, H-19), 4.37 (dt, J = 7.6, 3.6 Hz, 1H, H-1), 4.18 (tt, J = 6.8, 3.5 Hz, 1H, H-3), 1.17 (s, 3H, Me-26 or Me-27), 1.13 (s, 3H, Me-27 or Me- 26), 1.03 (d, J = 6.5 Hz, 3H, Me-21), 0.98 (dd, J = 6.2, 2.2Hz, 6H, Me-26, Me-27), 0.55 (s, 3H, Me-18 ).

Example 9

23,24,25,26,27-13C5-Vitamin D2 (2b)

Following the procedure described in Example 3, 0.22 mmol of phosphine oxide 29 in 5 mL of tetrahydrofuran, 0.21 mmol of n-butyl lithium 1.5 M, 0.21 mmol of ketone 23a in 5 mL of tetrahydrofuran provided 2b (89% yield , Two steps). 1H NMR (250 MHz, CDCh) 56.30 (d, J = 11 Hz, 1H, H-6), 5.96 (d, J = 11 Hz, 1H, H-7), 5.35 (m, 2H, H-22, H-23), 5.21 (br s, 1H, H-19), 4.97 (br s, 1H, H-19), 3.91 (tt, J = 6.8, 3.5 Hz, 1H, H-3), 0.96 (dd , J = 7.4, 2.4 Hz, 6H, Me-26, Me-27), 0.92 (d, J = 6.5 Hz, 3H, Me-28), 0.89 (d, J = 6.5 Hz, 3H, Me-21) , 0.52 (s, 3H, Me-18).

Example 10

23,24,25,26,27-13C5-25-Hydroxyvitamin D2 ( 2c )

Following the procedure described in Example 3, 1.2 mmol of phosphine oxide 29 in 15 mL of tetrahydrofuran, 1.15 mmol of n-butyl lithium 1.5 M, 0.85 mmol of ketone 18a in 15 mL of tetrahydrofuran provided 2c (89% yield , Two steps). 1H NMR (250 MHz, CDCh) 56.30 (d, J = 11 Hz, 1H, H-6), 5.96 (d, J = 11 Hz, 1H, H-7), 5.35 (m, 2H, H-22, H-23), 5.21 (br s, 1H, H-19), 4.97 (br s, 1H, H-19), 3.91 (tt, J = 6.8, 3.5 Hz, 1H, H-3), 0.96 (dd , J = 7.4, 2.4 Hz, 6H, Me-26, Me-27), 0.92 (d, J = 6.5 Hz, 3H, Me-28), 0.89 (d, J = 6.5 Hz, 3H, Me-21) , 0.52 (s, 3H, Me-18).

Example 11

23,24,25,26,27-13C5-1a-Hydroxyvitamin D2 (2d)

Following the procedure described in Example 3, 1.1 mmol of phosphine oxide 30 in 15 mL of tetrahydrofuran, 1.05 mmol of n-butyl lithium 1.5 M, 0.75 mmol of ketone 23a in 10 mL of tetrahydrofuran provided 2d (92% yield , Two steps). 1H NMR (250 MHz, CDCh) 56.33 (d, J = 11 Hz, 1H, H-6), 5.98 (d, J = 11 Hz, 1H, H-7), 5.35 (m, 2H, H-22, H-23), 5.21 (br s, 1H, H-19), 4.97 (br s, 1H, H-19), 4.34 (dt, J = 7.6, 3.6 Hz, 1H, H-1), 3.89 (tt , J = 6.8, 3.5 Hz, 1H, H-3), 0.96 (dd, J = 7.4, 2.4 Hz, 6H, Me 26, Me-27), 0.92 (d, J = 6.5 Hz, 3H, Me-28), 0.89 (d, J = 6.5 Hz, 3H, Me-21), 0.52 (s, 3H, Me-18) .

Example 12

23,24,25,26,27-13C5-1a, 25-Dihydroxyvitamin D2 (2e)

Following the procedure described in Example 3, 1.1 mmol of phosphine oxide 30 in 15 mL of tetrahydrofuran, 1.05 mmol of n-butyl lithium 1.5 M, 0.75 mmol of ketone 18a in 10 mL of tetrahydrofuran provided 2e (93% yield , Two steps). 1H NMR (250 MHz, CDCh) 56.30 (d, J = 11 Hz, 1H, H-6), 5.96 (d, J = 11 Hz, 1H, H-7), 5.35 (m, 2H, H-22, H-23), 5.21 (br s, 1H, H-19), 4.97 (br s, 1H, H-19), 4.34 (dt, J = 7.6, 3.6 Hz, 1H, H-1), 3.91 (tt , J = 6.8, 3.5 Hz, 1H, H-3), 0.96 (dd, J = 7.4, 2.4 Hz, 6H, Me-26, Me-27), 0.92 (d, J = 6.5 Hz, 3H, Me- 28), 0.89 (d, J = 6.5 Hz, 3H, Me-21), 0.52 (s, 3H, Me-18).

Example 13

23,24,25,26,27-13C5-3-epi-1a, 25-Dihydroxyvitamin D2 (2f)

Following the procedure described in Example 3, 1.02 mmol of phosphine oxide 31 in 12 mL of tetrahydrofuran, 0.96 mmol of 1.5M n-butyllithium, 0.71 mmol of the Ketone 7a in 12 mL tetrahydrofuran provided 2j (86% yield, two steps) .1H NMR (250 MHz, CDCl3) 56.43 (d, J = 11 Hz, 1H, H-6), 6.02 (d, J = 11 Hz, 1H, H-7), 5.35 (m, 2H, H-22, H-23), 5.21 (br s, 1H, H-19), 5.00 (br s, 1H, H-19), 4.31 ( d, J = 4.5 Hz, 1H, H-1), 4.05 (m, 1H, H-3), 1.21 (d, J = 7.4 Hz, 6H, Me-26, Me-27), 0.94 (d, J = 6.5 Hz, 3H, Me-21), 0.54 (s, 3H, Me-18).

Claims (1)

1. A compound of formula I:

where A is a single, double or triple bond, B is a single or double bond, F is selected from methylene (CH2), 13C-methylene (13CH2), 11C-methylene (11CH2), and -14C-methylene (14CH2), G is selected from is selected from methylene (CH2), 13C-methylene (13CH2), 11C-methylene (11CH2), and -14C-methylene (14CH2) when N is hydrogen, and is selected from -CH-, -13CH2- , -11CH2-, and -14CH2 when N is not hydrogen, -K is selected from -C-, -13C-, -11C-and -13C-, M is a methyl, methyl-13C, trideuteromethyl-13C, methyl-11C, methyl-14C, ethyl group N is a hydrogen, methyl, hydroxyl or -OR, where R is selected from the groups methoxymethyl, methoxyethyl, trimethylsilylethoxymethyl, trimethylsilyl , triethylsilyl, t-butyldimethylsilyl, tri- / so-propylsilyl, t-butyldiphenylsilyl, dimethylphenylsilyl, dimethylbenzylsilyl, benzoate, p-nitrobenzoate, pivalate or acetate, R1, R2, R3 and R4 are independently selected from hydrogen, hydroxyl or -OR, where R is selected from the groups methoxymethyl, methoxyethyl, trimethylsilylethoxymethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, dimethyl diphenylsilyl, dimethyl diphenylsilyl , dimethylbenzyl silyl, benzoate, p- nitrobenzoate, pivalate or acetate, R5 are independently selected from hydrogen, methylene group ( = CH ² ), dideuteromethylene group ( = CD ² ), methylene-13C group ( = 13CH ² ), dideuteromethylene-13C group ( = 13CD2), methylene-11C group ( = 11CH ²⁾ ) or methylene-14C group ( = 14CH ² ), R6 and R7 are independently selected from hydrogen or -methyl, R8 are independently selected from hydrogen, hydroxyl, or -OR, where R is selected from methoxymethyl, methoxyethyl, trimethylsilylethoxymethyl groups, trimethylsilyl, triethylsilyl, f-butyldimethylsilyl, tri- / so-propylsilyl, f-butyldiphenylsilyl, dimethylphenylsilyl, dimethylbenzyl silyl, benzoate, p-nitrobenzoate, pivalate or acetate. 2. A compound of formula I, selected from 19,23,24,25,26,27-13C6-1a-hydroxyvitamin D3 (1a), 19.23.24.25.26.27- 13C6-1a, 25-dihydroxyvitamin D3 (1b), 23.24.25.26.27- 13C5-vitamin D3 (1c), 23.24.25.26.27- 13C5-25-hydroxyvitamin D3 (1d), 23.24.25.26.27- 13C5-1a-hydroxyvitamin D3 (1e), 23,24,25,26,27-13C5-1a, 25-dihydroxyvitamin D3 (1f), 23.24.25.26.27- 13C5-3-epi-1a, 25-dihydroxyvitamin D3 (1g), 19.23.24.25.26.27- 13C6-1a, 25-dihydroxyvitamin D2 (2a), 23.24.25.26.27- 13C5-vitamin D2 (2b), 23.24.25.26.27- 13C5-25-hydroxyvitamin D2 (2c), 23,24,25,26,27-13C5-1a-hydroxyvitamin D2 (2d), 23.24.25.26.27- 13C5-1a, 25-dihydroxyvitamin D2 (2e), 23.24.25.26.27- 13C5-3-epi-1a, 25-dihydroxyvitamin D2 (2f). 3. A process for the preparation of the compounds of formula I,

wherein A, B, F, G, K, M, N, R1, R2, R3, R4, R5, R6, R7 and R8 are selected as in claim 1, comprising a coupling of compounds II and III in the presence of a catalyst,

where A, B, F, G, K, M, N, R1, R2, R3, R4, R6, R7 and R8 are selected as in claim 1, R9 are independently selected from methylene ( = CH ² ), dideuteromethylene (= CD2), 13C-methylene- ( 13CH ² ), 13C-dideuteromethylene ( = 13CD ² ), 11C-methylene ( = 11CH ² ) or 14C-methylene ( = 14CH ² ), X is indium dichloride (-InCh), zinc bromide (-ZnBr) or dialkoxyboro [-B (OR) 2], and The process according to claim 3, wherein the catalyst used is preferably selected from tetra-triphenylphosphine palladium (0), palladium (II) dichloride bistriphenylphosphine, palladium (II) dichloride bis- (diiphenylphosphine) ferrocene, palladium acetate (II), palladium (II) dichloride bis-acetonitrile, tris (dibenzylidenacetone) dipalladium (0), nickel (II) acetylacetonate or bis (1,5-dicyclooctadienyl) nickel (0). 5. A process for the preparation of the compounds of formula I,

wherein A, B, M, N, R1, R2, R3, R4, R5, R6, R7 and R8 are selected as in claim 1, comprising a Wittig-Horner reaction between compounds IV and V in the presence of a base,

wherein A, B, M, N, R1, R2, R3, R4, R5, R6, R7 and R8 are selected as in claim. 6. A process according to claim 5, characterized in that the base used is preferably selected from n-butyllithium, potassium f-butoxide, sodium hydride or sodium amide. 7. The compounds of formula II,

wherein R6, R7 and R8 are selected as in claim 1, X is indium dichloride (-InCh), zinc bromide (-ZnBr) or dialkoxyboro [-B (OR) 2] 8. The compounds of formula IV,

wherein R6, R7 and R8 are selected as in claim 1, 9. The compounds of formula III,

wherein R1, R2, R3, R4 and R5 are selected as in claim 1. 10. Use of the compounds of formula I, according to claim 1, in methods of quantification of vitamin D, its metabolites and / or analogs by mass spectrometry. 11. Use of the compounds of formula I, according to claim 1, in methods of identification and quantification of vitamin D, its metabolites and / or analogues by nuclear magnetic resonance. 12. Use of the compounds of formula I, according to claim 1, in positron emission tomography techniques as a tracer.

s Category of documents cited X: of particular relevance O: referring to unwritten disclosure Y: of particular relevance combined with other / s of the P: published between the priority date and the filing date same category of the application A: reflects the state of the art E: previous document, but published after the date application submission This report has been made 0 for all claims D for claims no: Date of completion of the Examiner report Page 30.04.2019 G. Esteban García 1/3