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Title:
COMPOUNDS FOR THE DETECTION OF LIGANDROL
Document Type and Number:
WIPO Patent Application WO/2024/046605
Kind Code:
A1
Abstract:
The compounds of formula (IA) or formula (IB) or a salt thereof. The compounds can be used as reference products in the determination of the administration of ligandrol to a subject.

Inventors:
PITSINOS EMMANOUIL (GR)
ANGELIS IOANNIS (GR)
PETROU MICHAEL (CY)
Application Number:
PCT/EP2023/063907
Publication Date:
March 07, 2024
Filing Date:
May 24, 2023
Export Citation:
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Assignee:
NATIONAL CENTER FOR SCIENT RESEARCH DEMOKRITOS (GR)
International Classes:
C07C255/58; C07D207/09; C07D207/26; G01N33/50
Domestic Patent References:
WO2012047617A12012-04-12
WO2009082437A22009-07-02
Other References:
THEVIS MARIO ET AL: "Characterization of a non-approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing : Characterization of LGD-4033 and phase-I metabolites", RAPID COMMUNICATIONS IN MASS SPECTROMETRY, vol. 29, no. 11, 22 April 2015 (2015-04-22), GB, pages 991 - 999, XP093067415, ISSN: 0951-4198, Retrieved from the Internet DOI: 10.1002/rcm.7189
FRAGKAKI ARGYRO G. ET AL: "Human in vivo metabolism study of LGD-4033", DRUG TESTING AND ANALYSIS, vol. 10, no. 11-12, 1 November 2018 (2018-11-01), GB, pages 1635 - 1645, XP093067841, ISSN: 1942-7603, Retrieved from the Internet DOI: 10.1002/dta.2512
M. THEVIS ET AL., RAPID COMMUN. MASS SPECTROM., vol. 29, 2015, pages 991 - 999
A. G. FRAGKAKI ET AL., DRUG TEST. ANAL., vol. 10, 2018, pages 1635 - 1645
Attorney, Agent or Firm:
ROUKOUNAS, Dimitrios (DE)
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Claims:
CLAIMS 1. A compound according to formula (IA) or (IB) or a salt of the compound of formula (IA) or (IB). 2. The compound according to claim 1, wherein the salt is selected from chloride, bromide, iodide, sulfate, nitrate, phosphate, formate, trifluoroacetate, propionate, lactate, citrate, tartrate, succinate, butyrate, maleate, fumarate, lithium, sodium, potassium, calcium, magnesium, barium, ammonium, trimethylammonium, triethylammonium, tributylammonium, or N,N- dimethylethanolammomium. 3. A process for the preparation of the compound of formula (IA), (IA) or a salt thereof, wherein the process comprises a) converting the compound of formula (IIA) to the compound of formula (IIIA) (IIIA) wherein R is a hydroxyl protective group, b) converting the compound of formula (IIIA) to the compound of formula (IVA) (IVA) wherein R is the same as defined above, c) converting the compound of formula (IVA) to the compound of formula (VA) d) converting the compound of formula (VA) to the compound of formula (IA) e) optionally, converting the compound of formula (IA) to a salt thereof. 4. A process for the preparation of the compound of formula (IB), or a salt thereof, wherein the process comprises a) converting the compound of formula (IIB) (IIB) to the compound of formula (IIIB) wherein R is a hydroxyl protective group, b) converting the compound of formula (IIIB) to the compound of formula (IVB) wherein R is the same as defined above, c) converting the compound of formula (IVB) to the compound of formula (VB) (VB) d) converting the compound of formula (VB) to the compound of formula (IB) e) optionally, converting the compound of formula (IB) to a salt thereof. 5. The process according to claim 3 or 4, wherein the protective group is selected from an alkyl ether, a silyl ether, an ester, or a carbonate. 6. The process according to any one of claims 3 to 5, wherein the protective group is selected from 2-(trimethylsilyl)ethoxymethyl (SEM) ether, tert- butyldimethylsilyl (TBDMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS) ether, tert-butyldiphenylsilyl (TBDPS) ether, tris(trimethylsilyl)silyl (sisyl) ether, acetate ester, pivaloate ester, benzoate ester, methyl carbonate, ethyl carbonate, methoxymethyl carbonate. 7. The process according to any one of claims 3 to 6, wherein the protective group is TBDMS. 8. The process according to any one of claims 3 to 7, wherein step b) is carried out with RuO4; iodine and sodium hydrogencarbonate in tetrahydrofuran (THF) or acetonitrile/water or DMSO; [bis(acetoxy)iodo]benzene and tert- butylhydroperoxide in decane/nitromethane; trimethylamine N-oxide and OsO4 in tert-butanol/H2O; potassium permanganate in acetone or potassium permanganate and a phase transfer catalyst in dichloromethane; bis- [(trifluoroacetoxy)iodo]benzene and sodium cyanide in water; iodosobenzene in water; RuO4 and sodium periodate in ethyl acetate/water; CrO3 and 3,5- dimethylpyrazole in dichloromethane; aminoxyl-mediated Shono-type electrochemical oxidation; or gold nanoparticles supported on alumina and O2 in H2O or THF/H2O.

9. The process according to any one of claims 3 to 8, wherein step b) is carried out with RuO4, generated in situ from RuCl3 or RuO2 and aqueous NaIO4. 10. The process according to any one of claims 3 to 9, wherein step c) is carried out with tetrabutylammonium fluoride (TBAF), cesium fluoride, or hydrogenfluoride-pyridine. 11. The process according to any one of claims 3 to 10, wherein step d) is carried out with LiOH, NaOH, KOH, or Ba(OH)2. 12. The process according to any one of claims 3 to 11, wherein steps c) and d) are carried out in a single step. 13. Use of a compound according to claim 1 or 2 in the determination of the administration of ligandrol to a subject, by analysis of a sample obtained from a subject. 14. Use according to claim 13, wherein the subject is a mammal, preferably a human. 15. Use according to claim 13 or 14, wherein the sample is a urine sample or a blood sample.

Description:
COMPOUNDS FOR THE DETECTION OF LIGANDROL FIELD OF THE INVENTION The present invention relates to the detection of the nonsteroidal selective androgen receptor modulator ligandrol in a biological sample. BACKGROUND OF THE INVENTION Ligandrol, also known as LGD-4033, has the chemical name 4-((R)-2-((R)-2,2,2- trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl )benzonitrile and the following structure: Ligandrol Ligandrol is a nonsteroidal selective androgen receptor modulator (SARM) which was disclosed in international patent application WO2009082437. SARMs are a class of compounds which act upon the androgen receptor. They exhibit some of the desired effects of androgens and have been studied as potential treatments for a number of diseases including cancer, osteoporosis, sexual dysfunction and muscle wasting. However, due to their anabolic effects, they have also become popular substances of abuse in competitive sport. Ligandrol belongs to the list of prohibited compounds of the World Anti-Doping Agency. Several studies related to the determination of the illicit use of ligandrol by humans and on horses have been reported in the literature and have led to the identification of related metabolites in urine samples. M. Thevis, et al. in Rapid Commun. Mass Spectrom., 2015, 29, 991–999 have proposed a compound having the following structure as a metabolite of ligandrol, which can be used as a reference compound in the detection of the use of ligandrol, for example, for doping control purposes. In the above chemical formula, R is a hydroxyl group. However, the above compound is expected to be very labile, due to the presence of the N-bridged bis-hemiaminal moiety, and actually the chemical synthesis of the above compound has not been reported to date. Furthermore, A. G. Fragkaki, et al. in Drug Test. Anal., 2018, 10, 1635–1645 have proposed that an epimer of the above compound (at the carbon atom which is bound to the hydroxyl and trifluoromethyl groups), arising either from the in vivo epimerization of LGD-4033 or from metabolism of the LGD-4033 epimer that could be present in the supplement used for doping, might be more desirable as a reference compound in the detection of the use of ligandrol. SUMMARY OF INVENTION The present invention provides compounds which are metabolites of SARM ligandrol and/or the corresponding epimeric alcohol and can be used as references in the determination of the administration of ligandrol to a subject. The present invention also provides a process for the preparation of the compounds. Furthermore, the present invention provides the use of the compounds in the determination of the administration of ligandrol to a subject. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows: A) the full scan mass spectrum, and B) the product ion mass spectrum of the deprotonated molecular ion [M−H] with m/z 369 at a collision energy of 25 eV of (4R,5R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-6,6,6-t rifluoro-5- hydroxyhexanoic acid (a compound of the present invention). Figure 2 shows the extracted ion chromatogram of the deprotonated molecular ion [M−H] with m/z 369 of (4R,5R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-6,6,6- trifluoro-5-hydroxyhexanoic acid (a compound of the present invention). Figure 3 shows the LC-HRMS analysis of: A) (4R,5R)-4-((4-cyano-3- (trifluoromethyl)phenyl)amino)-6,6,6-trifluoro-5-hydroxyhexa noic acid (a compound of the present invention), and B) a LGD-4033 positive, urine-derived sample. Figure 4 shows the LC-HRMS analysis of a mixture of (4R,5R)-4-((4-cyano-3- (trifluoromethyl)phenyl)amino)-6,6,6-trifluoro-5-hydroxyhexa noic acid (a compound of the present invention) and a LGD-4033 positive, urine-derived sample. DETAILED DESCRIPTION OF THE INVENTION The present invention provides the compound of formula (IA), or the compound of formula (IB), (IB) or a salt of the compound of formula (IA) or (IB). The compounds of the present invention possess the spectroscopic and chromatographic characteristics of long-term metabolites of LGD-4033 (ligandrol). Thus, the presence of a compound with identical spectroscopic and chromatographic characteristics with a compound of the present invention in a sample obtained from a subject indicates that ligandrol has been administered to the subject. Therefore, the compounds of the present invention can be used as reference products in the determination of the administration of ligandrol to a subject. Such determination may be carried out, for example, for doping control purposes. The subject may be a human, or a non-human animal, such as a horse, a dog, or any other domesticated or non-domesticated animal. Preferably, the subject is a mammal. More preferably, the subject is a human. The determination of the administration of ligandrol is carried out in a sample obtained from the subject. The sample may be, for example, a urine sample, a blood sample, a serum sample, a saliva sample, a tear sample, a tissue sample, such as hair or nails, a swab obtained from the oral cavity, nose or throat, and/or other body secretion. Preferably, the sample is a urine sample or a blood sample. The analysis of a sample can be performed, for example, for doping control purposes. The analysis can be carried out using methods well known in the art. For example, the analysis can be carried out using gas chromatography, or liquid chromatography, such as high-performance liquid chromatography (HPLC), typically combined with a suitable analyzer, such as a mass spectrometer. The chromatographic and/or spectroscopic characteristics obtained from the sample are then compared to the corresponding characteristics obtained from reference products to determine the presence of certain compounds in the sample. Since the compounds of the present invention are metabolites of ligandrol, they can be used as reference products in the analysis of the sample. The presence of a compound of the present invention in the sample indicates that ligandrol has been administered to a subject. The compounds of the present invention can also be used in the preparation of biosensors or lab-on-a-chip sensors following processes well known in the art. For example, the compounds of the present invention can be used in the preparation of antibodies or molecularly imprinted polymers (MIPs). Typically, in MIPs a recognition site within a polymer matrix is formed around a target template, commonly through non-covalent interactions, which is then removed to leave an “imprint”. MIPs or antibodies can be deposited on chips or similar surfaces, such as Surface Plasma Resonance biosensor chips or photonic immunosensors. Such chips can then be used in devices for the detection of a target molecule. Thus, the compounds of the present invention can be used as target molecules in a biosensor or lab-on-a-chip sensor for the determination of the administration of ligandrol to a subject. The compounds of formula (IA) and formula (IB) contain an amino group and can form the corresponding acid addition salts of an inorganic or organic acid. Examples of acid addition salts include chloride, bromide, iodide, sulfate, nitrate, phosphate, formate and trifluoroacetate. The compounds of formula (IA) and formula (IB) contain also a carboxylic group and can form the corresponding base addition salts of an inorganic or organic base. Examples of base addition salts include lithium, sodium, potassium, calcium, magnesium, barium, ammonium, trimethylammonium, triethylammonium, tributylammonium, N,N-dimethylethanolammomium and the like. The acid or base addition salts can be produced by methods well known in the art. The present invention also provides a process for the preparation of the compound of formula (IA) (IA) or formula (IB). or a salt of the compound of formula (IA) or (IB), wherein the process comprises a) converting the compound of formula (IIA) or the compound of formula (IIB) (IIB) to the compound of formula (IIIA) or to the compound of formula (IIIB), respectively, wherein R is a hydroxyl protective group, b) converting the compound of formula (IIIA) or the compound of formula (IIIB) to the compound of formula (IVA) or to the compound of formula (IVB), respectively, (IVB) wherein R is the same as defined above, c) converting the compound of formula (IVA) or the compound of formula (IVB) to the compound of formula (VA) or to the compound of formula (VB), respectively d) converting the compound of formula (VA) or the compound of formula (VB) to the compound of formula (IA) or to the compound of formula (IB), respectively e) optionally, converting the compound of formula (IA) or the compound of formula (IB) to a salt thereof. The starting material of the process of the present invention [ i.e., compound of formula (IIA or compound of formula IIB)] is ligandrol (LGD-4033) or the corresponding epimeric alcohol. Ligandrol or the corresponding epimeric alcohol can be prepared by following methods well known in the art. For example, they can be prepared by following the method disclosed in WO2009082437 and depicted in Scheme 1 below. According to this method, a mixture of D-prolinol (1), 4-fluoro-2- trifluoromethylbenzonitrile (2), and triethylamine is stirred overnight in tetrahydrofuran at 60°C. Standard work-up of the reaction mixture provides R-4-(2- hydroxylmethylpyrrolidinyl)-2-trifluoromethyl- benzonitrile (3). The intermediate alcohol is oxidized by sulfur trioxide pyridine complex to give R-4-(2-formyl- pyrrolidinyl)-2-trifluoromethyl- benzonitrile (4). The aldehyde intermediate is treated with trimethyl(trifluoromethyl)-silane to provide a mixture of two diastereoisomers. HPLC separation generates pure ligandrol and the corresponding epimeric alcohol. Scheme 1 Step a) in the process of the present invention involves the protection of the hydroxyl group of ligandrol or the corresponding epimeric alcohol. The protective group can be any group suitable for the protection of an alcohol that is stable under the subsequent oxidation conditions but labile under the subsequent deprotection and/or hydrolysis conditions. Examples of protective groups which can be used in the process of the present invention include alkyl ethers [such as 2-(trimethylsilyl)ethoxymethyl (SEM)], silyl ethers [such as tert-butyldimethylsilyl (TBDMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), tris(trimethylsilyl)silyl (sisyl)], esters (such as acetate, pivaloate, benzoate), and carbonates (such as methyl, ethyl, methoxymethyl). The alcohol protection can be carried out following reactions well known in the art. Preferably, the protective group is TBDMS and can be employed by reacting, for example, ligandrol or the corresponding epimeric alcohol with TBDMS chloride and imidazole in dimethylformamide (DMF). Step b) involves the oxidation of the pyrrolidine ring of the compound of formula (IIIA) or the oxidation of the pyrrolidine ring of the compound of formula (IIIB). Such oxidation can be carried out following methods well known in the art, such as RuO4; iodine and sodium hydrogencarbonate in tetrahydrofuran or acetonitrile/water or DMSO; [bis(acetoxy)iodo]benzene and tert-butylhydroperoxide in decane/nitromethane; trimethylamine N-oxide and OsO4 in tert-butanol/H2O; potassium permanganate in acetone or potassium permanganate and a phase transfer catalyst in dichloromethane; bis-[(trifluoroacetoxy)iodo]benzene and sodium cyanide in water; iodosobenzene in water; RuO 4 and sodium periodate in ethyl acetate/water; CrO 3 and 3,5-dimethylpyrazole in dichloromethane; aminoxyl- mediated Shono-type electrochemical oxidation; gold nanoparticles supported on alumina and O 2 in H 2 O or THF/H 2 O. Preferably, the oxidation is carried out with the use of RuO 4 , generated in situ from RuCl 3 or RuO 2 and aqueous NaIO 4 . Such reaction can be carried out under two-phase conditions, for example in EtOAc/H 2 O, or single- phase conditions, for example in tert-BuOH/H 2 O. Step c) involves the deprotection of the alcohol group of the compound of formula (IVA) or the deprotection of the alcohol group of the compound of formula (IVB). The deprotection can be carried out following methods well known in the art. For example, when the protective group is SEM or a silyl ether, the deprotection can be carried out with tetrabutylammonium fluoride (TBAF), cesium fluoride, or hydrogenfluoride- pyridine. When the protective group is TBDMS, the deprotection is preferably carried out with tetrabutylammonium fluoride (TBAF). Step d) involves the hydrolytic opening of the lactam ring of the compound of formula (VA) or the hydrolytic opening of the lactam ring of the compound of formula (VB). The hydrolysis can be carried out following methods well known in the art, such as LiOH or NaOH or KOH or Ba(OH)2 in water or THF/H2O. Preferably, the hydrolysis is carried out with LiOH, for example, in a mixture of tetrahydrofuran (THF), methanol and water. Step e) involves the conversion of the compound of formula (IA) to a salt or the conversion of the compound of formula (IB) to a salt, when the corresponding salt is the desired product. Such a conversion can be carried out following methods well known in the art. For example, the compound of formula (IA) or the compound of formula (IB) can react with an acid, for the formation of the corresponding acid addition salt, or a base, for the formation of the corresponding base addition salt, in the presence of a solvent, such as water, ethanol, acetone and the like. According to an embodiment, steps c) and d) of the method of the present invention are carried out in one step, which reduces the duration and the cost of the method. For example, when the protective group is an ester or a carbonate, the concomitant deprotection of the alcohol group and the hydrolytic opening of the lactam ring of the compound of formula (IVA) or of the lactam ring of the compound of formula (IVB) can be carried out with LiOH or NaOH or KOH or Ba(OH) 2 in water or THF/H 2 O. EXAMPLES EXAMPLE 1 This example discloses the synthesis of the compound of formula (IA) from ligandrol [i.e., the compound of formula (IIA)]. The compound of formula (IB) can be synthesized by the same process when the compound of formula (IIA) is substituted by epi-ligandrol [i.e., the compound of formula (IIB)]. General methods All reactions were carried out under a dry argon atmosphere with anhydrous solvents (freshly distilled over the appropriate desiccant or dried over 3 Å molecular sieves) under anhydrous conditions, unless otherwise noted. All reactions were magnetically stirred with Teflon stir bars, and temperatures were measured externally. Reactions requiring anhydrous conditions were carried out in oven dried (120 °C, 24 h) or flame dried (vacuum < 0.5 Torr) glassware. All reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Yields refer to chromatographically and spectroscopically homogeneous materials, unless otherwise noted. All reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F254). UV light was used for visualization and an acidified ethanolic solution of p-anisaldehyde or an acidified aqueous solution of ceric ammonium molybdate and heat were used as developing agents. E. Merck silica gel (60 Å, particle size 0.040–0.063 mm) or Acros Organics silica gel (60 Å, particle size 0.035–0.070 mm) were used for flash column chromatography. Optical rotations were recorded using a Perkin-Elmer 241 polarimeter at the sodium D line (589 nm) using a 10 cm path-length cell in the solvent and concentration indicated. Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance DRX 500 MHz or Bruker Avance III 250 MHz instrument and were calibrated using as internal reference the residual nondeuterated solvent for 1 H-NMR and the deuterated solvent for 13 C-NMR, respectively (e.g., CDCl 3 : δ H = 7.26 ppm, δ C = 77.16 ppm; CD 3 OD: δ H = 3.31 ppm, δ C = 49.00 ppm). Multiplicities are designated as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint.) or multiplet (m). Broad or obscured peaks are indicated as “br” or “obs”, respectively. To facilitate NMR spectra comparisons, the same LGD-4033 skeleton numbering has been used for all compounds when assigning signals. The skeleton numbering is reproduced in the formula of LGD-4033 below. High resolution mass spectra (HRMS) were acquired on a LC qExactive plus HRMS (Thermo Scientific, Bremen, Germany) instrument. 4-((R)-2-((R)-1-((tert-butyldimethylsilyl)oxy)-2,2,2-trifluo roethyl)pyrrolidin-1-yl)- 2-(trifluoromethyl)-benzonitrile: LGD-4033 (32.6 mg, 96.4 μmol) was placed in a 5 mL pear-shaped flask and imidazole (195 mg, 2.9 mmol), TBDMSCl (241.1 mg, 1.6 mmol), and DMF (0.15 mL) were added sequentially under an argon atmosphere. The mixture was stirred at ambient temperature for 48 h. Volatiles were removed under reduced pressure and the residue was dissolved in dichloromethane (2 mL). Silica gel (0.5 g) was added, volatiles were removed under reduced pressure, the residue was loaded on top of a chromatography column (silica gel), and eluted with n- hexane/dichloromethane 75:25 to provide TBS-protected LGD-4033 as amorphous white solid (41.4 mg, 91.5 μmol, 95% yield). R = 0.71 (silica gel, CH Cl ); [α]D f 2 2 25 = +20 (c = 0.97, CH Cl ); 1 2 2 H NMR (250 MHz, CDCl3): δ = 7.57 (d, J = 8.8 Hz, 1 H, H-6), 7.02 (br s, 1 H, H-3), 6.79 (dd, J = 8.8, 2.7 Hz, 1 H, H-5), 4.20–4.14 (m, 1 H, H-8), 3.81 (dq, J = 8.8, 6.0 Hz, 1 H, H-12), 3.55–3.48 (m, 1 H, H-11), 3.30–3.19 (m, 1 H, H-11′), 2.23– 1.98 (m, 4 H, H-9 & H-10), 0.66 (s, 9 H, (CH3)3CSi), 0.05 & −0.23 (2 s, 6 H, CH3SiCH3) ppm; 13C NMR (62.5 MHz, CDCl3): δ = 151.1 (s), 135.6 (s, C-6), 133.8 (q, J = 32 Hz), 124.9 (q, J = 284 Hz), 122.8 (q, J = 274 Hz), 117.4 (s), 115.0 (s, C-5), 111.3 (s, C-3), 95.4 (s), 73.4 (q, J = 28 Hz, C-12), 58.6 (s, C-8), 49.0 (s, C-11), 29.4 (s, C-10), 25.3 (s, CH3)3CSi), 23.0 (s, C-9), 17.8 (s, (CH3)3CSi), −4.7, −5.5 (2 s, CH3SiCH3) ppm; HRMS (ESI +): calculated for C20H27F6N2OSi [M+H]: 453.1791, found 453.1780; HRMS (ESI −): m/z calculated for C21H27F6N2O3Si [M+HCOO]: 497.1690, found 497.1705. 4-((R)-2-((R)-1-((tert-butyldimethylsilyl)oxy)-2,2,2-trifluo roethyl)-5- oxopyrrolidin-1-yl)-2-(trifluoro-methyl)benzonitrile: A 5 mL round bottom flask equipped with an efficient magnetic stirring bar was charged with RuCl 3 (5.4 mg, 26 μmol) and 10% w/v aqueous NaIO 4 solution (0.25 mL, 115 μmol) was added. To the stirred black solution that ensued was added dropwise a solution of 4-((R)-2-((R)-1- ((tert-butyldimethylsilyl)oxy)-2,2,2-trifluoroethyl)pyrrolid in-1-yl)-2-(trifluoromethyl)- benzonitrile (18.5 mg, 40.9 μmol) in ethyl acetate (0.9 mL), the flask was sealed, and the mixture was vigorously stirred at ambient temperature for 3 h. Water (1 mL) and ethyl acetate (3 mL) was added and the organic phase was separated. The aqueous layer was extracted with ethyl acetate (2 × 3 mL). To the combined organic layers was added isopropanol (0.5 mL), the mixture was stirred for 1 h, and the black precipitate formed was removed by filtration through a short pad of Celite. The filtrate was washed with brine (2 × 3 mL), dried over Na 2 SO 4 , and concentrated. Flash column chromatography (silica gel, n-hexane/EtOAc 9:1 to 8:2) gave the title compound as light brown oil (13.7 mg, 29.4 μmol, 72% yield). Rf = 0.52 (silica gel, hexane/EtOAc 1:1); [α]D 25 = +3.3 (c = 0.95, CHCl 3 ); 1H NMR (500 MHz, CDCl 3 ): δ = 8.12 (dd, J = 8.7, 2.3 Hz, 1 H, H-5), 8.03 (d, J = 2.3 Hz, 1 H, H-3), 7.81 (d, J = 8.6 Hz, 1 H, H-6), 4.66 (t, J = 7.2 Hz, 1 H, H-8), 4.07 (quint., J = 6.3 Hz, 1 H, H-12), 2.76 (ddd, J = 17.8, 11.4, 9.4, 1 H, H-10), 2.60 (ddd, J = 17.9, 9.7, 1.9 Hz, 1 H, H-10′), 2.45–2.29 (m, 2 H, H-9 & H-9′), 0.75 (s, 9 H, (CH 3 ) 3 CSi), −0.08 & −0.04 (2 s, 6 H, CH 3 SiCH 3 ) ppm; 13C NMR (125 MHz, CDCl 3 ): δ = 174.8 (s, C-11), 143.0 (s, C-4), 135.6 (s, C-6), 133.8 (q, J = 32.8 Hz, C-2), 124.5 (q, J = 284.4 Hz, C-14), 123.8 (s, C-5), 122.3 (q, J = 274.1 Hz, C-16), 118.6 (q, J = 5.0 Hz, C-3), 115.5 (s, C-15), 105.1 (br d, J = 2.3 Hz, C-1), 70.7 (q, J = 29.3 Hz, C-12), 58.7 (s, C-8), 30.7 (s, C-10), 25.3 (s, CH 3 ) 3 CSi), 21.3 (s, C-9), 17.9 (s, (CH 3 ) 3 CSi), −5.0 & −5.3 (2 s, CH 3 SiCH 3 ) ppm; HRMS (ESI −): m/z calculated for C 21 H 25 F 6 N 2 O 4 Si [M+HCOO]: 511.1482, found 511.1488. 4-((R)-2-oxo-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)pyrrolidi n-1-yl)-2- (trifluoromethyl)benzonitrile: A solution of 4-((R)-2-((R)-1-((tert- butyldimethylsilyl)oxy)-2,2,2-trifluoroethyl)-5-oxopyrrolidi n-1-yl)-2-(trifluoro- methyl)benzonitrile (12.4 mg, 26.6 μmol) in THF (0.5 mL) was treated at ambient temperature and under an atmosphere of argon with 1.0 M solution of TBAF in THF (0.04 mL, 40 μmol). After 0.5 h, half saturated aqueous NH4Cl solution (0.5 mL) was added and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, and concentrated. Flash column chromatography (silica gel, CH2Cl2/EtOAc 9:1) gave the title compound as colorless solid (8.8 mg, 25.0 μmol, 94% yield). R f = 0.26 (silica gel, CH 2 Cl 2 /EtOAc 8:2); [α]D 2 5 = −3.2 (c = 0.99, MeOH); 1H NMR (500 MHz, CD3OD): δ = 8.19 (s, 1 H, H- 3), 7.97–7.93 (m, 2 H, H-5 & H-6), 4.92 (ddt, J = 6.8, 5.2, 1.7 Hz, 1 H, H-8), 4.19 (qd, J = 7.5, 5.2 Hz, 1 H, H-12), 2.89–2.79 (m, 1 H, H-10), 2.54–2.45 (m, 2 H, H-10′ & H- 9), 2.20 (td, J = 10.4, 2.2 Hz, 1 H, H-9′) ppm; 13C NMR (125 MHz, CD3OD): δ = 177.7 (s, C-11), 145.0 (s, C-4), 136.6 (s, C-6), 133.8 (q, J = 32.6 Hz, C-2), 127.6 (s, C-5), 126.1 (q, J = 282.9 Hz, C-14), 123.9 (q, J = 273.0 Hz, C-16), 122.6 (q, J = 5.1 Hz, C- 3), 116.5 (s, C-15), 106.2 (br d, J = 2.0 Hz, C-1), 72.3 (q, J = 29.5 Hz, C-12), 60.1 (s, C-8), 31.8 (s, C-10), 23.4 (s, C-9) ppm; HRMS (ESI −): m/z calculated for C15H11F6N2O4 [M+HCOO]: 397.0618, found 397.0624. (4R,5R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-6,6,6-t rifluoro-5- hydroxyhexanoic acid: To a stirred solution of 4-((R)-2-oxo-5-((R)-2,2,2-trifluoro-1- hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitril e (7.5 mg, 21.3 μmol) in THF/MeOH/H 2 O 8:1:4 (0.4 mL) was added at 0 °C LiOH·H 2 O (11 mg, 262 μmol). The mixture was allowed to reach ambient temperature and it was then stirred at 30 °C for 4 h. The reaction was quenched at 0 °C with the dropwise addition of AcOH/H 2 O 1:1 (1 mL). The mixture was diluted with EtOAc (5 mL) and washed with half saturated brine (2 × 5 mL). The aqueous washings were extracted with EtOAc (3 × 5 mL) and the combined organic layers were dried over Na 2 SO 4 , and concentrated under reduced pressure. Benzene (3 × 5 mL) was added to the residue and volatiles were removed under reduced pressure. The light-yellow solid thus obtained was dissolved in 2% MeOH in dichloromethane (2 mL), silica gel (0.1 g) was added, volatiles were removed under reduced pressure, and the residue was loaded on top of a chromatography column (silica gel, CH 2 Cl 2 /EtOAc 8:2). Elution with CH 2 Cl 2 /EtOAc 8:2 to CH 2 Cl 2 /EtOAc/AcOH 80:20:1 provided the title compound as colorless glass (7.1 mg, 19.2 μmol, 90% yield). R f = 0.34 (silica gel, CH 2 Cl 2 /EtOAc/AcOH 80:20:1); [α]D25 = +12 (c = 0.14, MeOH); 1H NMR (500 MHz, CD 3 OD): δ = 7.58 (d, J = 8.7 Hz, 1 H, H- 6), 7.08 (d, J = 2.5 Hz, 1 H, H-3), 6.89 (dd, J = 8.7, 2.5 Hz, 1 H, H-5), 4.10–4.04 (m, 2 H, H-8 & H-12), 2.40 (t, J = 7.2 Hz, 2 H, H-10 & H-10′), 1.99 (q, J = 7.2 Hz, 2 H, H- 9 & H-9′) ppm; 13C NMR (125 MHz, CD 3 OD): δ = 176.8 (s, C-11), 153.4 (s, C-4), 137.4 (s, C-6), 134.9 (q, J = 31.6 Hz, C-2), 126.4 (q, J = 282.9 Hz, C-14), 124.3 (q, J = 272.9 Hz, C-16), 118.3 (s, C-15), 114.8 (br s, C-5), 111.5 (br s, C-3), 94.5 (br d, J = 2.4 Hz, C-1), 71.3 (q, J = 29.7 Hz, C-12), 52.0 (s, C-8), 30.9 (s, C-10), 28.7 (s, C-9) ppm; HRMS (ESI −): m/z calculated for C 14 H 11 F 6 N 2 O 3 [M−H]: 369.0668, found 369.0671. The full scan mass spectrum of (4R,5R)-4-((4-cyano-3- (trifluoromethyl)phenyl)amino)-6,6,6-trifluoro-5-hydroxyhexa noic acid [compound of formula (IA)] is shown in Figure 1A and the product ion mass spectrum of the deprotonated molecular ion [M-H] of the same compound with m/z 369 at a collision energy of 25 eV is shown in Figure 1B. The extracted ion chromatogram of the deprotonated molecular ion [M−H] with m/z 369 is shown in Figure 2. EXAMPLE 2 This example shows the results of chromatographic analysis of a urine sample carried out on a sample obtained from a human subject who was confirmed that had used ligandrol in the weeks preceding the analysis compared to the chromatographic analysis of (4R,5R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-6,6,6-t rifluoro-5- hydroxyhexanoic acid [compound of formula (IA)]. The samples were analysed by liquid chromatography–high resolution mass spectrometry (LC–HRMS). LC-HRMS Analysis A Dionex UHPLC system (Thermo Scientific, Bremen, Germany) was used for the chromatographic separation. The system consisted of a vacuum degasser, a high- pressure binary pump, an autosampler with a temperature-controlled sample tray set at 7 °C and a column oven set at 30 °C. Chromatographic separation was performed at 30 °C using a Zorbax Eclipse Plus C18 column (100 × 2.1 mm i.d., 1.8 µm particle size; Agilent Technologies). The mobile phase consisted of 5 mM ammonium formate in 0.02% formic acid (solvent A) and a mixture of acetonitrile/water (90:10 v/v) containing 5 mM ammonium formate and 0.01% formic acid (solvent B). A gradient elution program was employed at a constant flow rate of 0.2 mL min −1 with solvent B starting at 5% for 3 min, increasing to 30% in 4 min, increasing to 90% in 11 min and then, set back to 5% in 11.5 min. Post-run equilibrium time was 3.5 min. The injection volume was 5 mL. The mass spectrometer was a QExactive plus benchtop Orbitrap-based mass spectrometer (ThermoScientific, Bremen, Germany) operated in the negative polarity mode and equipped with a heated electro-spray ionization (HESI) source. Source parameters were: sheath gas (nitrogen) flow rate, auxiliary gas (nitrogen) flow rate and sweep gas flow rate: 40, 10 and 1 arbitrary units respectively, capillary temperature: 300 °C, heater temperature: 30 °C, spray voltage: +4.0 kV (positive polarity). The instrument operated in FS mode from m/z 100–1000 at 17,500 resolving power and duty cycle of 100 ms and in MS/MS mode from m/z 100–1000 at 17,500 resolving power and duty cycle of 62 ms (product ion mode). The automatic gain control (AGC) was set to 106. The mass calibration of the Orbitrap instrument was evaluated in both positive and negative modes daily and external calibration was performed prior to use following the manufacturer′s calibration protocol. Figure 3A shows the chromatogram obtained from the LC-HRMS analysis of (4R,5R)- 4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-6,6,6-trifluoro -5-hydroxyhexanoic acid [compound of formula (IA)]. Figure 3B shows the corresponding chromatogram obtained from the LC-HRMS analysis obtained from the urine sample of the subject. Furthermore, Figure 4 shows the corresponding chromatogram obtained from the co- injection of (4R,5R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-6,6,6-t rifluoro-5- hydroxyhexanoic acid [compound of formula (IA)] with a LGD-4033 positive, urine- derived, sample. It is clear that the three chromatograms are identical. This means that (4R,5R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-6,6,6-t rifluoro-5- hydroxyhexanoic acid [compound of formula (IA)] can be used as a reference product in the determination of the administration of ligandrol.