WO1995011217A1

WO1995011217A1 - Isotopically-labeled retinoids, their production and use

Info

Publication number: WO1995011217A1
Application number: PCT/US1994/010768
Authority: WO
Inventors: Marcus F. Boehm
Original assignee: Ligand Pharmaceuticals Incorporated
Priority date: 1993-10-22
Filing date: 1994-09-23
Publication date: 1995-04-27
Also published as: AU7877494A

Abstract

Isotopically-labeled retinoids, methods for their synthesis, and their use in the discovery of Retinoid X Receptor ligands are provided. Use of the isotopically-labeled retinoids in mass spectral metabolism studies are also provided.

Description

Isotopi cal ly-l abel ed reti noids , thei r production and use

Field of the Invention

The present invention relates to the synthesis of isotopically- labeled compounds, and in particular to the synthesis of isotopically-labeled retinoids. The present invention also relates to the use of isotopically-labeled retinoids and related compounds in methods to discover retinoid receptor ligands.

Background of the Invention

The role of retinoids such as all-trans retinoic acid (ATRA), 13- cis retinoic acid (13 -cis RA) and synthetic retinoic acid (RA) analogs in mediating cell growth and differentiation has generated interest in their pharmacological utility for controlling the treatment of dermatological diseases, such as psoriasis and acne, as well as oncological applications such as chemotherapy and chemoprevention. Significant advances in elucidating the molecular basis of retinoid action now offer the potential for designing RA compounds with improved therapeutic indices.

To date, several receptors for retinoic acid have been identified. These receptors are members of a superfamily of intracellular receptors which function as ligand dependent transcription factors. At present, these receptors have been classified into two subfamilies, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The classification of these subfamilies is based primarily on differences in amino acid structure, responsiveness to different naturally occurring and synthetic retinoids, and ability to modulate expression of different target genes. Each RAR and RXR subfamily has three distinct isoforms designated RARα, RARβ and RARγ, and RXRα, RXRβ and RXRγ. The discovery of multiple retinoid receptors raises questions of the functional properties of the distinct subfamilies and their isoforms.

One technique for determining the affinity of retinoic acid and synthetic retinoids to RARs and associated proteins is to employ a competitive ligand binding assay using radio-labelled compounds showing RAR activity. See e^, U.S. Patent No. 5,196,577. See also U.S. Patent Nos. 5,073,361, 5, 149,631. These ligand binding studies require substantial quantities, i.e. greater than 50 milliCuries, of a high specific activity ( > 20 Ci/mmol) radio- labelled compound. In addition, to date, no isotopically-labeled or radio-labelled compounds have been identified that show specific selective activity on RXRs versus activity on RARs.

The entire disclosures of the publications and references referred to above and hereinafter in this specification are incorporated herein by reference.

Summary of the Invention

The present invention provides isotopically-labeled retinoids optionally substituted with deuterium, tritium, carbon 13, carbon 14 or CF3. The invention also provides methods of producing such isotopically-labeled retinoids, as well as methods of identifying retinoid X receptor ligands using such isotopically-labeled retinoids in competitive binding assays and the like, and use of the isotopically-labeled retinoids in mass spectral metabolism studies. These and various other advantages and features of novelty which characterize the invention are pointed out with particularity in the claims annexed hereto and forming a part hereof. However, for a better understanding of the invention, its advantages, and objects obtained by its use, reference should be had to the accompanying drawings and descriptive matter, in which there is illustrated and described preferred embodiments of the invention. Brief Description of the Drawings

The invention may be further illustrated by reference to the accompanying Drawings wherein:

FIG. 1 is a FAB mass spectrum showing a peak at 349 corresponding to 4[l(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid and the corresponding isotopically-labeled 4[1(3-¹³CI_3- 5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid at peak 353, as well as fragments of these compounds at peaks 333 and 337 respectively. Detailed Description of Embodiments of the Invention

In accordance with a first aspect of the present invention, we have developed isotopically-labeled retinoids of the formulae:

or

wherein, Rι_f R2, R3, and R4 each independently, represent hydrogen or lower alkyl or acyl having 1-4 carbon atoms, R' and R" represent hydrogen, a lower alkyl or acyl having 1-4 carbon atoms, OH, alkoxy having 1-4 carbon atoms, thiol or thio ether, or amino, or R' or R" taken together form an oxo (keto), methano, thioketo, HO-N=, RgO-N-^*-^*, NC-N=, (R7Rs)N-N=, epoxy, cyclopropyl, or cycloalkyl group, and the epoxy, cyclopropyl and cycloalkyl groups can be substituted with lower alkyl having 1-4 carbons or halogen, R5 represents a lower alkyl or acyl having 1-4 carbon atoms, an alkyl ether, halogen, or OH, Re represents alkyl or alkenyl having 1-4 carbon atoms or an alkyl amine, R7 and Rg each independently represent hydrogen or a lower alkyl having 1-6 carbons, Z, Z', Z", and Z'", each independently, represent C, S, O, and N, or a pharmaceutically acceptable salt, but are not O or S if attached by a double bond to another such Z, or if attached by a single bond to another such Z which is N, n = 0-3, and the dashed lines in the structures depict optional double bonds, and wherein any of the hydrogens at positions 1, 4, 6, 7, 11, 12, 14, and 15 can be replaced with deuterium or tritium, and further wherein any of the carbons in the functional groups at R5 can be replaced with ¹ CH3_; ¹³Q_3, CD3, C³H3, (CF)_nCF₃, and ¹ CD₃. As used in this disclosure, pharmaceutically acceptable salts include, but are not limited to: hydrochloric, hydrobromic, hydroiodic, hydrofluoric, sulfuric, citric, maleic, acetic, lactic, nicotinic, succinic, oxalic, phosphoric, malonic, salicylic, phenylacetic, stearic, pyridine, ammonium, piperazine, diethylamine, nicotinamide, formic, urea, sodium, potassium, calcium, magnesium, zinc, lithium, cinnamic, methylamino, methanesulfonic, picric, tartaric, triethylamino, dimethylamino, and tris(hydoxymethyl) aminomethane. Additional pharmaceutically acceptable salts are known to those skilled in the art. Representive isotopically-labeled retinoids of the present invention include, without limitation, 4[l(3-[¹⁴C]H3-5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[l(3-[¹³C]H3-5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[1(3- [ ¹³C]U3-5 , 5 , 8, 8-pentamethyl-5,6, 7, 8-tetrahydro-2-naρhthyl) ethenyl] benzoic acid, 4[l(3-CF₃-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[l(3-CD3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[l(3-C[³H]3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl) ethenyl] benzoic acid, and 4[l(3,5,5,8,8-pentamethyl-6,7-di-[³H]- 5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid. In a second aspect, the present invention also provides methods of discovering retinoid X receptor (RXR) selective ligands. In particular, an isotopically-labeled compound which is capable of binding to a RXR, such as an isotopically-labeled retinoid according to a first aspect of the present invention, can be used in a competition binding assay against the ligand to be tested. By compar- ing the degree to which the isotopically-labeled compound and hgand bind to the RXR relative to the degree of binding of the isotopically-labeled compound to the RXR in the absence of the ligand, the activity of the ligand on the RXR can be determined.

The variations in conducting such competition binding assays are well known to those skilled in the art. For example, one method of conducting such an assay is to first incubate the isotopically-labeled compound in a medium containing an RXR protein until the isotopically-labeled compound is bound to the RXR protein to the point of saturation. Thereafter, the bound RXR protein is washed and incubated with a high concentration (e.g. 200 times) of the ligand to be tested. By comparing the quantity of bound isotopically-labeled compound displaced by the ligand to be tested, the relative selective activity of the ligand on the RXR, if any, can be determined. Alternatively, when the saturation curve of the isotopically-labeled compound on the RXR is already known, then the isotopicially-labelled compound and ligand can be incubated with the RXR at the same time, and the degree of exclusion of the binding of the isotopically-labeled compound to the RXR used to measure the relative selective activity of the hgand on the RXR, if any.

The RXR proteins used in the competition binding assays of the methods of the present invention will generally be recovered from cell lysates of an appropriate cell culture transfected with a recombinant plasmid capable of expressing the RXR proteins. However, any appropriate means of expressing a sufficient quantity of one or more retinoid X receptors to allow for the conducting of the identification methods of the present invention can be employed. Furthermore, any biologically compatible medium in which the competition binding assays of the present invention can function is considered to be within the scope of the present invention.

To help ensure an accurate measurement of the amount of isotopically-labeled compound that can bind to a particular RXR, it is preferred that the specific binding of the isotopically-labeled compound be determined by titrating the bound isotopically-labeled compound against an excess quantity of the same compound in a non-labeled form. For example, the isotopically-labeled compound is first incubated with a given RXR to the point of saturated binding. An appropriate binding curve is then generated which shows total binding, both specific binding to the RXR pocket, and nonspecific binding to other associated structures (e.g. lipids). Thereafter, the RXR bound with the isotopically-labeled compound is incubated with a large excess of the non-labeled version of the isotopically-labeled compound (e.g. 200 times or greater concentration). Any isotopically-labeled compound that remains bound, as shown by an appropriate binding curve, represents nonspecifically bound isotopically-labeled compound. By subtracting the nonspecifically bound protein from the total bound protein, a binding curve can be generated which shows the total specific binding of the isotopically-labeled compound to the RXR. This in turn provides the reference useful for determining the concentration of a tested ligand that displaces the isotopically-labeled compound from an RXR in the method of the present invention. In a preferred aspect of the method of the present invention, the isotopically-labeled compound will show specific, selective activity (e.g. binding) on retinoid X receptors (RXRs) versus retinoic acid receptors (RARs). In such an instance, the isotopically-labeled compound can be used to identify compounds that activate RXRs. For example, such an isotopically-labeled compound will prove useful for identifying ligands that activate one or more of the RXRs.

Another use of the isotopically-labeled compounds of the present invention is in metabolism studies using a mass spectrometer to locate metabolites of both the isotopically-labeled and non-labeled retinoids. For example, a 1 : 1 mixture of a ¹³CD₃ isotopically-labeled compound (such as 4[l(3-¹³CH₃-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid) and a non-labeled ¹²CH3 (naturally occurring) compound (such as 4[l(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid) is administered to an in vitro (e.g., an appropriate cell line) or an in vivo (e.g., an animal model, such as laboratory mice) system, followed by extraction of metaboUtes from the system (e.g. whole cell lysates or animal bodily fluids). The metabolites may now be identified by their characteristic mass spectral profiles. In particular, such metabolites will exhibit a pair of spectral peaks four mass units apart, which will be vizualized in a 1 : 1 ratio. A reproduction of such a FAB mass spectral profile is given in FIG. 1, which shows a FAB mass spectrograph showing a peak at 349 corresponding to 4[1(3,5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid and the corresponding isotopically-labeled 4[ 1 (3-¹³CH3-5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl) ethenyl] benzoic acid at peak 353, as well as fragments of these compounds at peaks 333 and 337 respectively. The retinoid compounds that are isotopically-labeled according to the present invention can be made by a variety of organic synthesis techniques well known to those skilled in the art. Methodologies of generating such retinoids are disclosed in Assignee's pending patent application Serial No. 08/052,051, filed April 21, 1993, the disclosure of which is herein incorporated by reference. More specifically, the non-labeled retinoid starting materials can be generated according to the following scheme.

V

Compounds of structure I when R₅ = halo, OH, amino or thio are prepared by standard Friedel-Crafts reaction conditions combining the appropriate substituted benzene with 2,5-dichloro-2,5-dimethyl hexane in the presence of aluminum chloride. Condensation of I with mono-methyl terephthalate II was carried out by addition of PC1₅ to I and II in CH₂C1 followed by addition of A1C1₃ at room temperature.

The resulting methyl ester HI was treated with methyltriphosphonium bromide-sodium amide in THF, which afforded methano compound IV.

The carboxylic acid V was formed by adding KOH to methano compound IV in MeOH, followed by acidification. In resulting compound V, R₅ = Br, R_x to R₄ = CH₃, and Z = C.

Using these non-labeled retinoid starting materials, the isotopically-labeled retinoids of the present invention can be made by replacing various hydrogen substituents at the 1, 4, 6, 7, 11, 12, 14 and 15 positions with deuterium or tritium, or by replacing the functional groups at R₅ with compounds such as ¹⁴CH_3; ¹³CH₃, CD₃, C³H₃, (CF)_nCF₃, and ¹³CD₃. Further, the replacement of such functional groups, through the use of reagents such as labeled methyl iodides, including [¹⁴C]H₃I, C[³H]₃I, ¹³CH₃I, ¹³CD₃I, CD3I, and CF3I, are well known to those skilled in the art, and can be accomplished using the following scheme.

2

50 mg

la. R = "CH₃

lb. R = H

la

A non-labeled retinoid, such as 4[l(3-bromo-5,5,8,8-tetramethyl- 5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid 2 is treated with 2.2 equivalents of an organo-metalo base, such as nBuLi, at -78 °C followed by addition of labeled methyl iodide in the presence of DMBU. The labeled methyl iodide includes [¹⁴C]H₃I, C[³H]₃I, ¹³CH₃I, ¹³CD₃I, CD3I, and CF3I. After addition of the labeled methyl iodide (such as ¹⁴QE_3l, shown below), the reaction is acidified and purified by crystallization or HPLC to give the isotopically-labeled material, such as 4[l(3-[¹⁴C]H3-5,5,8,8-tetramethyl-5,6,7,8- tetrahydro-2-naphthyl) ethenyl] benzoic acid la.

Alternatively, high specific activity isotopically-labeled retinoids, labeled with deuterium or tritium, such as [ H]-4[l(3,5,5,8,8-pentamethyl-6,7- ditritio-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, can be synthesized by the following route. Olefin 7 is reduced with tritium gas under metallo catalysis to give tritium labeled tetrahydronaphthalene 8, using the methodology of Rhee et al., Synthesis of a New Class of Retinoid. 3H-labeled TTNPB, with a High Specific Activity. 8 J. Labeled Compounds and Radiophram., 843-849 (1985), the disclosure of which is herein incorporated by reference. Aluminum chloride catalyzed Friedel Crafts condensation of 8 with monomethyl terephthallic acid chloride yields the ketone 9. Ketone 9 is then treated with the sodium amide anion of methyl triphenyl phosphonium bromide to give olefin 10. Hydrolysis of ester 10 in refluxing methanolic KOH followed by HC1 acidification yields isotopically-labeled retinoid [³H]-4[l(3,5,5,8,8-pentamethyl- 6,7-ditritio-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid 11, with a specific activity of >50 Ci/mmol.

AICI ₃, CH ₂CI₂

10

11 The invention will be further illustrated by reference to the following non-limiting Examples.

EXAMPLE 1 4rif3-|^'14C1H^-5.5.8,8-pentamethyl-5.6.7.8-tetrahvdro-2-naphthyl. ethenyll benzoic acid (la):

To 50 mg (0.12 mmol) of 4[l(3-bromo-5,5,8,8-tetramethyl- 5,6,7, 8-tetrahydro-2-naphthyl) ethenyl] benzoic acid 2 in 2 mL of dry tetrahydrofuran (THF) at -78 °C under dry nitrogen gas was added 130 mL (0.3 mmol) of a 2.3 M nBuLi solution. The reaction instantly became red and was stirred for 20 m. To this mixture was added 1 mL of a 10% DMPU solution in THF and the reaction stirred for an additional 10 m. The red reaction mixture was transferred by canula to ¹ CH3l (1 mCi, 60 mCi/mmol) at -78 °C and stirred for an additional 1 hr. The reaction was quenched with 1 mL of saturated aqueous NH4CI followed by addition of 2 mL of aqueous 10% HC1 solution. The organic products were extracted with ether (3X), the ether dried (MgSO₄), filtered and concentrated to give a mixture of labeled 4[l(3-[¹⁴C]H3-5,5,8,8- pentam_thyl-5,6,7,8-tetr_hydro-2-naphthyl) ethenyl] benzoic acid la and 4[l(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid lb which were separated by HPLC (85:15:0.5 MeOH.H₂O:AcOH, ODS column) to give 529 mCi of 4[l(3-[¹⁴C]H₃-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl) ethenyl] benzoic acid (la) at specific activity of 55 mCi/mmol (radiochemical yield is 53 %). TLC (20% methanol-80% chloroform) R_f = 0.28; UN λ_MeOH= 264 nm, e = 16,400, ^H-ΝMR (CDCI3) δl.28 (s, (CH₃)₂), 1.31 (s, (CH₃)₂),1.70 (s, (CH₂)₂)_ 1-95 (s, CH₃), 5.35 (s, =CH₂), 5.83 (s, =CH₂), 7.08 (s, CH), 7J3 (s, CH), 7.38 (d, J = 8.1 Hz, 2 CH), 8.03 (d, J = 8.1 Hz, 2 CH).

2

50 mg

la. R = »<CH₃ la lb. R = H

EXAMPLE 2

4f 1 (3 -CF 5.5.8.8-tetramethyl-5.6.7. -tetrahvdro-2-naphthyl . ethenyl] benzoic acid (4V

50 mg (0J2 mmol) of 4[l(3-bromo-5,5,8,8-tetramethyl-5,6,7,8- te_.ahydro-2-napb.thyl) ethenyl] benzoic acid 2 in 2 mL of dry tetrahydrofuran (THF) at -78 °C under dry nitrogen gas was added 130 mL (0.3 mmol) of a 2.3 M nBuLi solution. The reaction instantly became red and was stirred for 20 m. To this mixture was added 1 mL of a 10% DMPU solution in THF and the reaction stirred for an additional 10 m. Into this solution was bubbled CF3I gas until the solution turned yellow. The reaction was quenched with 1 mL of saturated aqueous NH4CI followed by addition of 2 mL of aqueous 10% HC1 solution. The organic products were extracted with ether (3X), the ether dried(MgSO4), filtered and concentrated to give crude 4[l(3-CF3-5, 5,8,8- tetramethyl-5,6,7,8-tetrahydro-2- naphthyl) ethenyl] benzoic acid (4) which was purified by crystallization from hexane to give 13 mg of 4[l(3-CF3-5,5,8,8- tetramethyl-5,6,7,8-tetrahydro-2-naρhthyl) ethenyl] benzoic acid (4). TLC (20% methanol-80% chloroform) Rf = 0.32; -^H-NMR (CDCI3) δl.28 (s, (CH₃)₂), 1.30 (s, (CH₃)2)J-70 (s, (CH₂)₂), 5.37 (s, =CH₂), 5.92 (s, =CH₂), 7.20 (s, CH), 7.26 (s, CH), 7.37 (d, j = 8J Hz, 2 CH), 8.04 (d, J = 8J Hz, 2 CH).

EXAMPLE 3

4[l('3-¹³CH -5.5.8.8-Dentamethyl-5.6,7.8-tetrahvdro-2-naphthyl. ethenyl] benzoic acid (5):

To 50 mg (0J2 mmol) of 4[l(3-bromo-5,5,8,8-tetramethyl- 5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid 2 in 2 mL of dry tetrahydrofuran (THF) at -78 °C under dry nitrogen gas was added 130 mL (0.3 mmol) of a 2.3 M nBuLi solution. The reaction instantly became red and was stirred for 20 m. To this mixture was added 1 mL of a 10% DMPU solution in THF and the reaction stirred for an additional 10 m. To this solution was added 23 mL ( 0.36 mmol) of ¹³CH3l. The reaction was quenched with 1 mL of saturated aqueous NH4CI followed by addition of 2 mL of aqueous 10% HCl solution. The organic products were extracted with ether (3X), the ether dried(MgSO4), filtered and concentrated to give crude 4[l(3-¹³CH3-5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid (5) which was purified by crystallization from hexane to give 25 mg of 4[l(3-¹³CH3-5, 5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid (5). TLC (20% methanol-80% chloroform) Rf = 0.30; -^H-NMR (CDCI3) δ 1.28 (s, (CH₃)2). 1-30 (s, (CH₃)₂),l-70 (s, (CH₂) ), 1-94 (d, J -^*-^* 126 Hz, 1³CH₃), 5.34 (s, =CH₂), 5.83 (s, =CH₂), 7.11 (d, J = 4 Hz), CH), 7.13 (s, CH), 7.37 (d, J = 8.1 Hz, 2 CH), 8.01 (d, J = 8.1 Hz, 2 CH).

EXAMPLE 4

4fl⁽3-¹³CD2-5.5.8.8-pentamethyl-5.6.7.8-tetrahvdro-2-naρhthyl. ethenyll benzoic acid (6):

To 50 mg (0.12 mmol) of 4[l(3-bromo-5,5,8,8-tetramethyl- 5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid 2 in 2 mL of dry tetrahydrofuran (THF) at -78 °C under dry nitrogen gas was added 130 mL (0.3 mmol) of a 2.3 M nBuLi solution. The reaction instantly became red and was stirred for 20 m. To this mixture was added 1 mL of a 10% DMPU solution in THF and the reaction stirred for an additional 10 m. To this solution was added 23 mL ( 0.36 mmol) of ¹³CD3l. The reaction was quenched with 1 mL of saturated aqueous NH4CI followed by addition of 2 mL of aqueous 10% HCl solution. The organic products were extracted with ether (3X), the ether dried(MgSO4), filtered and concentrated to give crude 4[l(3-¹³CH3-5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid (6) which was purified by crystallization from hexane to give 20 mg of 4[l(3-¹³CD3-5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid (6). TLC (20% methanol-80% chloroform) Rf = 0.30; Η-NMR (CDCI3) δl.28 (s, (CH₃)₂), 1-30 (s, (CH₃)₂)J.70 (s, (CH₂)₂), 5.34 (s, =CH₂), 5.83 (s, =CH₂), 7J0 (d, J = 4 Hz), CH), 7.12 (s, CH), 7.36 (d, J = 8.1 Hz, 2 CH), 8.01 (d, J ^***- 8.1 Hz, 2 CH). MS (FAB) 353

EXAMPLE 5

For binding studies, retinoids receptors are used employing a baculovirus expression system. The methods concerning growth, purification, and assays of recombinant viruses follows the protocol outlined by Summers, M.D., and Smith, G.E., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agric. Exp. Stat. Bull, No. 155 (1987). The recombinant plasmids are cotransfected into SF21 cells with wild-type AcNPN DNA, See U.S. Patent No. 5,071,773, and the recombinant viruses are plaque purified. For the mock (control) extracts, wild-type AcNPV-infected cells are used.

For ligand binding assays, the baculovirus-infected cells are disrupted by Dounce homogenization (Kontes Co., Vineland, NJ) in 10 nM Tris (pH 7.6), 5 mM dithiothreitol (DTT), 2 mM EDTA 0.5% CHAPS, and 1 mM phenylmethyisulfonyl fluoride. The KCI concentration is adjusted to 0.4 M after cell lysis. The cell lysates are centrifuged for 1 hr at 4°C, 100,000 x g, and the supernatant is recovered as a high-salt, whole cell extract. For the saturation binding analysis, cell extracts (50 microgram protein) are incubated at 0°C for 2.0 hr with a isotopically-labeled retinoid (e.g., 4[l(3-¹³CH₃-5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid) in the presence or absence of 200-fold excess unlabelled ligand. Specific ligand binding to receptor is determined by a hydroxyapatite binding assay according to the protocol of Wecksler, W.R., and Norman, A.W., A Hydroxylapatite Batch Assay for the Quantitation of 12.25-Dihydroxyvitamin D^-Receptor Complexes. 92 Anal. Biochem., 314-323 (1979).

To characterize the binding of a desired retinoid to baculo virus- derived RXRs, saturation binding analysis with isotopically-labeled compounds of the invention is performed. Total specific and nonspecific binding are determined. Nonspecific binding is determined by adding a low concentration of the isotopically -labled retinoid (e.g., 1 nM of 4[l(3-¹ CH3-5,5,8,8-pen_a__ethyl- 5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid) with an excess concentration (e.g., 200 nM) of non-labeled retinoid (e.g., 4[1(3,5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid), effectively competing off the specifically bound isotopically-labeled retinoid with the non- labeled retinoid. Any remaining non-bound isotopically-labeled retinoid is due to nonspecific binding. Specific binding is determined by subtracting the values for nonspecifically bound isotopically-labeled retinoid from total bound isotopically- labeled retinoid.

While in accordance with the patent statutes, description of the preferred embodiments and processing conditions have been provided, the scope of the invention is not to be limited thereto or thereby. Various modifications and alterations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.

Consequently, for an understanding of the scope of the present invention, reference is made to the following claims.

Claims

What is claimed is:

1. A isotopically-labeled retinoid of the formulae:

or

wherein, Ri_, R_2, R3, and R4 each independently, represent hydrogen or lower alkyl or acyl having 1-4 carbon atoms, R' and R" represent hydrogen, a lower alkyl or acyl having 1-4 carbon atoms, OH, alkoxy having 1-4 carbon atoms, thiol or thio ether, or amino, or R' or R" taken together form an oxo (keto), methano, thioketo, HO-N= R6θ-N=, NC-N= (R₇R₈)N-N=, epoxy, cyclopropyl, or cycloalkyl group, and the epoxy, cyclopropyl and cycloalkyl groups can be substituted with lower alkyl having 1-4 carbons or halogen, R₅ represents a lower alkyl or acyl having 1-4 carbon atoms, an alkyl ether, halogen, or OH, B_β represents alkyl or alkenyl having 1-4 carbon atoms or an alkyl amine, R₇ and Rg each independently represent hydrogen or a lower alkyl having 1-6 carbons, Z, Z', Z", and Z'", each independently, represent C, S, O, and N, or a pharmaceutically acceptable salt, but are not O or S if attached by a double bond to another such Z, or if attached by a single bond to another such Z which is N, n = 0-3, and the dashed lines in the structures depict optional double bonds, and wherein any of the hydrogens at positions 1, 4, 6, 7, 11, 12, 14, and 15 can be replaced with deuterium or tritium, and further wherein any of the functional groups at R₅ can be replaced with ¹⁴CH₃, ¹³CH₃, CD₃, C³H3, (CF)_nCF₃, and ¹³CD₃.

2. The isotopically-labeled retinoid of claim 1, wherein the hydrogens at position 6 and 7 are replaced with deuterium or tritium.

3. The isotopically-labeled retinoid of claim 1, wherein R5 comprises ¹⁴CH₃, ¹³CH₃, CD3, C³H₃, (CF)_nCF₃, or ¹³CD₃.

4. The isotopically-labeled retinoid of claim 1, selected from the group consisting of 4[l(3-[¹⁴C]H3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro- 2-naphthyl) ethenyl] benzoic acid, 4[l(3-[¹³C]H3-5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[l(3-[¹³C]D3-5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[1(3-CF3- 5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[1(3- CD3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[l(3-C[³H]3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, and 4[l(3,5,5,8,8-pentamethyl-6,7-di-[³H]-5,6,7,8-tetrahydro-2- naphthyl) ethenyl] benzoic acid.

5. 4[l(3,5,5,8,8-pentamethyl-6,7-di-[³H]-5,6,7,8-tetrahydro- 2-naphthyl) ethenyl] benzoic acid.

6. 4[l(3-[¹ C]H₃-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl) ethenyl] benzoic acid.

7. 4[l(3-[¹³C]D₃-5,5,8,8-pentamethyl-5,6,7,

8-tetrahydro-2- naphthyl) ethenyl] benzoic acid. A method for producing a isotopically-labeled retinoid comprising: (a) providing a retinoid of the formulae:

or

wherein, Ri₍ R2_, R3, and R4 each independently, represent hydrogen or lower alkyl or acyl having 1-4 carbon atoms, R' and R" represent hydrogen, a lower alkyl or acyl having 1-4 carbon atoms, OH, alkoxy having 1-4 carbon atoms, thiol or thio ether, or amino, or R' or R" taken together form an oxo (keto), methano, thioketo, HO-N= __6θ-N=, NC-N=, (R₇Rs)N-N=, epoxy, cyclopropyl, or cycloalkyl group, and the epoxy, cyclopropyl and cycloalkyl groups can be substituted with lower alkyl having 1-4 carbons or halogen, R₅ represents a lower alkyl or acyl having 1-4 carbon atoms, an alkyl ether, halogen, or OH, R_ό represents alkyl or alkenyl having 1-4 carbon atoms or an alkyl amine, R₇ and Rg each independently represent hydrogen or a lower alkyl having 1-6 carbons, Z, Z\ Z", and Z'", each independently, represent C, S, O, and N, or a pharmaceutically acceptable salt, but are not O or S if attached by a double bond to another such Z, or if attached by a single bond to another such Z which is N, n = 0-3, and the dashed lines in the structures depict optional double bonds; and (b) replacing any of the hydrogens at positions 1, 4, 6, 7, 11,

12, 14, and 15 with deuterium or tritium, or any of the functional groups at R5 with ¹ CH_3, ¹³CH₃, CD₃, C³H₃, (CF)_nCF₃, or ¹³CD₃.

9. The method of claim 8, wherein the R5 functional group is replaced with a substituent selected from the group consisting of ¹⁴CH_3, ¹³CH₃, CD₃, C³H₃, (CF)_nCF₃, and ¹³CD₃.

10. The method of claim 8, wherein the isotopically-labeled retinoid is selected from the group consisting of 4[l(3-[¹⁴C]H₃-5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[1(3- [¹³C]H3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[l(3-[¹³C]D3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[l(3-CF3-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[l(3-CD3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl) ethenyl] benzoic acid, 4[l(3-C[³H]₃-5,5,8,8-ρentamethyl-5,6,7,8- tetrahydro-2-naphthyl) ethenyl] benzoic acid, and 4[l(3,5,5,8,8-pentamethyl-6,7- di-[³H]-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid.

11. A method for identifying retinoid X receptor ligands comprising:

(a) incubating a isotopically-labeled compound capable of binding to a retinoid X receptor in a medium containing the retinoid X receptor and a hgand, wherein the isotopically-labeled compound is of the formulae:

or

wherein, Rι_; R2_, R3, and R4 each independently, represent hydrogen or lower alkyl or acyl having 1-4 carbon atoms, R' and R" represent hydrogen, a lower alkyl or acyl having 1-4 carbon atoms, OH, alkoxy having 1-4 carbon atoms, thiol or thio ether, or a ino, or R' or R" taken together form an oxo (keto), methano, thioketo, HO-N=, R^O-N----, NC-N= (R₇R8)N-N=, epoxy, cyclopropyl, or cycloalkyl group, and the epoxy, cyclopropyl and cycloalkyl groups can be substituted with lower alkyl having 1-4 carbons or halogen, R5 represents a lower alkyl or acyl having 1-4 carbon atoms, an alkyl ether, halogen, or OH, Rό represents alkyl or alkenyl having 1-4 carbon atoms or an alkyl amine, R7 and Rg each independently represent hydrogen or a lower alkyl having 1-6 carbons, Z, Z', Z", and Z'", each independently, represent C, S, O, and N, or a pharmaceutically acceptable salt, but are not O or S if attached by a double bond to another such Z, or if attached by a single bond to another such Z which is N, n = 0-3, and the dashed lines in the structures depict optional double bonds, and wherein any of the hydrogens at positions 1, 4, 6, 7, 11, 12, 14, and 15 can be replaced with deuterium or tritium, and further wherein any of the functional groups at R5 can be replaced with ¹⁴CH₃, ¹³CH₃, CD3, C³H , (CF)_nCF₃, or ¹³CD₃; and

(b) comparing the degree to which the ligand binds to the retinoid X receptor in competition with the isotopically-labeled compound relative to the degree to which the isotopically-labeled compound binds to the retinoid X receptor in the absence of the ligand.

12. The method of claim 11 , wherein the isotopically-labeled compound is incubated in the medium with the retinoid X receptor such that the binding of the isotopically-labeled compound to the retinoid X receptor is saturated prior to the addition of the ligand to the medium.

13. The method of claim 11 , wherein the isotopically-labeled compound is selected from the group consisting of 4[l(3-[¹⁴C]H3-5, 5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, 4[1(3- [¹³C]D3-5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid, and 4[ 1 (3 , 5 , 5 , 8, 8-pentamethyl-6, 7-di-[³H]-5, 6, 7, 8-tetrahydro-2-naphthyl) ethenyl] benzoic acid.

14. The method of claim 11, wherein the isotopically-labeled compound is used to identify a retinoid X receptor selective ligand.

15. A method of performing mass spectral metabolism studies comprising,

(a) administering substantially equal quantities of a compound of claim 1 and a non-labeled version of the compound to an in vitro or in vivo system,

(b) extracting metabolites of the compounds from the system, and (b) performing a mass spectral analysis of the metabolites.

16. The method of claim 15, further comprising, separating the metaboUtes recovered from the system.