Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/545—Heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
  • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
  • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
  • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
  • C07D235/14—Radicals substituted by nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

• TITLE BIFUNCTIONAL COMPOUNDS OF THE PROTAC TYPE TARGETING PXR, THEIR PREPARATION METHOD AND THEIR USE
• the present invention relates to the treatment of cancer and more particularly cancers overexpressing the PXR nuclear receptor, such as colorectal cancer.
• Colorectal cancer is the third most common cancer and is the third leading cause of cancer death.
• Current treatments include surgery, radiation therapy, and chemotherapy, sometimes in combination with targeted therapies that demonstrate minor improvement.
• the efficacy of these treatments is seriously compromised by the frequent appearance of resistance which leads to relapse in patients after stopping treatment (50% of patients).
• cancer cell subpopulations, cancer stem cells (CSCs) have been shown to be involved in tumor initiation, metastatic development and drug resistance, thus leading to tumor recurrence. .
• NR112 nuclear receptor PXR
• shRNA RNA
• Inhibition of the PXR nuclear receptor (NR112) therefore makes it possible to sensitize cancer stem cells to current treatments.
• PXR antagonists identified to date are, however, either non-specific and/or toxic at the concentrations necessary for PXR inactivation, or not yet validated for clinical use.
• PROTACs (“Proteolysis Targeting Chimera”) are bifunctional molecules that simultaneously bind a target protein and an E3-ubiquitin ligase. This causes poly-ubiquitination of the target protein which is thus degraded into small peptides and amino acids by the proteasome complex.
• the PROTAC approach is therefore a chemical protein knock-down strategy
• the present invention relates to the bifunctional compounds corresponding to the general formula (I):
• L(PXR) is a ligand capable of binding to the PXR nuclear receptor
• L(E3 ligase) represents a ligand of the E3 ubiquitin ligase
• Linker represents a group that makes it possible to covalently bind L(PXR) to
• the ubiquitin-proteasome (UPP) pathway is an essential cellular pathway that regulates key regulatory proteins and degrades misfolded or abnormal proteins. UPP is central to several cellular processes. If it is faulty or unbalanced, it leads to the pathogenesis of various diseases.
• the covalent attachment of ubiquitin to specific protein substrates is achieved by the action of ubiquitin ligases E3. These ligases include more than 500 different proteins and are classified into several classes defined by the structural element of their functional activity E3.
• the E3 ligase ligand which is a functional modality of the present compounds, binds to an E3 ubiquitin ligase.
• the ligase catalyzes the covalent attachment of ubiquitin to the target protein, which in turn induces the degradation of the target protein by native proteasomes.
• the compounds of the present invention are designed in a manner that exploits native cellular degradative processes but where the degradative action is directed to undesirable target proteins that are implicated in disease etiology.
• the PROTACs according to the invention act as degradation enzymes with a super-stoichiometric action capacity.
• L(PXR) which is a functional modality of the present compounds which binds to PXR.
• the targeting ligand is an analog of the ligands of
• L(PXR) can be chosen from the groups of formula
• the compounds according to the invention can correspond to the following formula (1-1): where Linker, L(E3 ligase) are as defined above or below, or a pharmaceutically acceptable salt.
• the E3 ligase ligand binds to cereblon.
• L(E3 ligase) can in particular be chosen from: - the groups of formula (NIA): and
• X is NH
• X' is -C(O)- or -CH 2 -;
• Y represents H or a C1-C6 alkyl group; represents the group's attachment to Linker.
• the compounds according to the invention may in particular correspond to the formula (I-) or (1-3): where Linker, L(PXR), L(E3 ligase), X, X′, Y are as defined above or below; or a pharmaceutically acceptable salt.
• Linker provides covalent attachment of the targeting ligand with the E3 ligase ligand.
• Linker represents a C1 -C20 alkylene group, optionally interrupted or optionally ending at one and/or the other of the two ends, by one of the groups -0-, -S-, -N (R') -, -C(O)-, -C(0)0-, - OC(O) -, -0C(0)0 -, - C(NOR')-, -C(0 )N(R')-, -C(0)N(R')C(0)-, -C(0)N(R')C(0)N(R')-, -N(R' )C(0)-, -N(R' )C(0)-, -N(R' )C(0)-, -N(R' )C(0)-, -N(R' )C(0)-, -N(R' )C(0)-, -N(R' )C(0)
• Linker can be chosen from C4-C20 alkylene groups, optionally interrupted by and/or ending with one or more groups chosen from -NH-, -O-, -C(O) -; piperidinyl, piperazinylene.
• Linker can be represented among the groups of formula
• Li and L 2 identical or different, independently represent an alkylene group of 1 to 12 carbon atoms optionally interrupted or ending in a heterocyclene of 3 to 12 members and comprising 1, 2 or 3 heteroatoms chosen from N,
• Li is bound is linked to L(E3 ligase);
• Z represents H or a C1-C6 alkyl group.
• Li is a C7-alkylene group
• L 2 is a (C2 to C8)-alkylene group optionally interrupted by a piperidinyl group.
• the compounds according to the invention may correspond to the following formula (V): in which L 2 represents a linear C2-C8 alkylene group optionally interrupted by a piperidinyl group, and L(E3 ligase) is as defined above or below.
• Alkyl means an aliphatic hydrocarbon group which may be linear or branched having about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups have 1 to about 12 carbon atoms in the chain, especially 1 to 6 carbon atoms. Branched means that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a straight alkyl chain. “Lower alkyl” means from about 1 to about 4 carbon atoms in the chain which may be straight or branched.
• the alkyl may be substituted with one or more "alkyl group substituents", which may be the same or different and include halo, cycloalkyl, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy, alkoxycarbonyl, aralkoxycarbonyl, heteroaralkoxycarbonyl or Y 1 Y 2 NCO-, wherein Y 1 and Y 2 are, independently, hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl or optionally substituted heteroaralkyl, or Y 1 and Y 2 , taken together in conjunction with N through which Y 1 and Y 2 are linked, form a 4 to 7 membered heterocyclyl.
• alkyl group substituents may be the same or different and include halo, cycloalkyl, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy, alkoxycarbonyl,
• alkyl groups include methyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n-propyl, 17-propyl, n-butyl, t-butyl, n-pentyl, 3- pentyl, methoxyethyl, carboxymethyl, methoxycarbonylethyl, benzyloxycarbonylmethyl, pyridylmethyloxycarbonylmethyl.
• Alkylene designates a bivalent alkyl group as defined above.
• Preferred alkylene groups are lower alkylene groups having 1 to about 6 carbon atoms.
• Typical examples of alkylene groups include methylene and ethylene.
• Cycloalkyl means a mono- or multi-cyclic non-aromatic ring system of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. Preferred ring sizes of the rings of the ring system include about 5 to about 6 ring atoms, optionally substituted with one or more substituents.
• Exemplary monocyclic cycloalkyls include cyclopentyl, cyclohexyl, cycloheptyl, and the like.
• Exemplary multicyclic cycloalkyls include 1-decalin, norbornyl, adamant-(1 or 2-)yl, and the like.
• Cycloalkylene means a saturated, divalent cycloalkyl group as defined above, such as in particular cyclohexylene.
• Heterocyclyl means a non-aromatic saturated monocyclic or multicyclic ring system of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms, wherein one or more of the carbon atoms in the system ring is/are hetero element(s) different from carbon, for example nitrogen, oxygen or sulphur.
• Preferred ring sizes of the rings of the ring system include about 5 to about 6 ring atoms.
• the designation of aza, oxa or thia as a prefix before heterocyclyl defines that at least one nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom.
• the heterocyclyl may be optionally substituted with one or more substituents, which may be the same or different, and are as defined herein.
• the nitrogen atom of a heterocyclyl may be a basic nitrogen atom.
• the nitrogen or sulfur atom of the heterocyclyl can also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.
• Exemplary monocyclic heterocyclyl rings include piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
• heterocyclene denotes a bivalent heterocyclyl radical as defined above.
• Heteroaryl means an aromatic monocyclic or multicyclic ring system of about 5 to about 14 carbon atoms, preferably about 5 to about 10 carbon atoms, wherein one or more of the carbon atoms in the ring system is/are heteroelement(s) other than carbon, for example nitrogen, oxygen or sulphur. Preferred ring sizes of the rings of the ring system include about 5 to about 6 ring atoms. "Heteroaryl” can also be substituted with one or more substituents. The designation of aza, oxa or thia as a prefix before heteroaryl defines that at least one nitrogen, oxygen or sulfur atom is present respectively as a ring atom.
• a nitrogen atom of a heteroaryl can be a basic nitrogen atom and can also be optionally oxidized to the corresponding N-oxide.
• exemplary heteroaryl and substituted heteroaryl groups include pyrazinyl, thienyl, isothiazolyl, oxazolyl, pyrazolyl, furazanyl, pyrrolyl, 1,2,4-thiadiazolyl, pyridazinyl, quinoxalinyl, phthalazinyl, l imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl, benzofurazanyl, azaindolyl, benzimidazolyl, benzothienyl, thienopyridyl, thienopyrimidinyl, pyrrolopyridyl, imidazopyridyl, benzoazaindole, 1,2,4-triazinyl, benzthiazolyl, furanyl
• Heteroarylene denotes a bivalent heteroaryl radical as defined above. “Substituents” denotes one or more identical or different groups chosen from halogen, cyano, cycloalkyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, aroylamino, carboxy, alkoxycarbonyl, aralkoxycarbonyl, heteroaralkoxycarbonyl.
• the compounds of the present invention may be in the form of a free acid or a free base, or a pharmaceutically acceptable salt.
• pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its purified form with an organic or inorganic acid and isolating the salt thus formed.
• acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate salts.
• Acid addition salts can also be prepared by separately reacting the purified compound in its acid form with an organic or inorganic base and isolating the salt thus formed.
• Acid addition salts include amine and metal salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum salts. Sodium and potassium salts are preferred.
• Suitable basic inorganic addition salts are prepared from metallic bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide .
• Suitable basic amine addition salts are prepared from amines which have sufficient alkalinity to form a stable salt, and preferably include amines which are often used in medicinal chemistry due to their low toxicity and acceptability.
• ammonia ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)- aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino
• stereoisomer may have at least one chiral center and therefore may be in the form of a stereoisomer, which as used herein encompasses all isomers of individual compounds which differ only in orientation. of their atoms in space.
• stereoisomer includes mirror image isomers (enantiomers that include the (R-) or (S-) configurations of compounds), mixtures of mirror image isomers (physical mixtures of enantiomers and racemates or racemic mixtures ) of geometric compounds (cis/trans or E/Z, R/S isomers) of compounds and isomers of compounds with more than one chiral center that are not mirror images of each other (diastereoi somers).
• the chiral centers of compounds can undergo epimerization in vivo; thus, for these compounds, administration of the compound in its (R ⁇ ) form is considered equivalent to administration of the compound in its (S-) form. Accordingly, the compounds of the present invention can be made and used as individual isomers and substantially free of other isomers, or as a mixture of various isomers, eg, racemic mixtures of stereoisomers.
• the following compounds are suitable for binding Cereblon and PXR:
• the compounds according to the invention can be chosen from the compounds corresponding to one of the following formulas:
• the present invention also relates to the process for the preparation of a compound according to the invention.
• the compounds of general formula (I) can be prepared by applying or adapting any method known per se to and/or within the reach of those skilled in the art, in particular those described by Larock in Comprehensive Organic Transformations, VCH Pub., 1989 , or by application or adaptation of the methods described in the examples which follow.
• said method comprises the coupling of a compound of formula (B) and a compound of formula (C):
• the term "complementary reactive functions" designates two functions capable of reacting together to form a function providing a covalent bond between T and T'.
• T and T' are such that T has a terminal function of the amine type and T' has a terminal function of the carboxylic acid type.
• T represents a group of formula (T-B):
• T' represents a group
• TC in which Li and L 2 are as defined above.
• Said coupling can advantageously be carried out in the presence of a peptide coupling agent such as BOP (benzotriazol-l -yloxytris (dimethylamino) phosphonium hexafluorophosphate, typically in the presence of an organic base such as Hünig base (N, /V- diisopropylethylamine (DIPEA or DIEA).
• BOP benzotriazol-l -yloxytris (dimethylamino) phosphonium hexafluorophosphate
• organic base such as Hünig base (N, /V- diisopropylethylamine (DIPEA or DIEA).
• compound (B) corresponds to formula (A):
• the compound (C) corresponds to the formula (C-1): ligase)
• said method may also comprise the step consisting in isolating the product of formula (I) obtained.
• the product can, if desired, be further purified by various techniques, such as recrystallization, reprecipitation or the various chromatography techniques, including column chromatography or preparative thin layer chromatography.
• compounds useful according to the present invention may contain asymmetric centers. These asymmetric centers may be independently in the R or S configuration. It will be apparent to those skilled in the art that certain compounds which are useful according to the invention may also exhibit geometric isomerism. It is to be understood that the present invention includes individual geometric isomers and stereoisomers and mixtures thereof, including racemic mixtures, of compounds of formula (I) above.
• This type of isomers can be separated from their mixtures, by the application or adaptation of known methods, for example chromatography techniques or recrystallization techniques, or they are prepared separately from the appropriate isomers of their intermediates.
• the basic products or reagents used are commercially available and/or can be prepared by the application or adaptation of known methods, for example methods as described in the Reference Examples or their obvious chemical equivalents.
• the process according to the invention can implement the intermediate of formula (A) which is new.
• the present invention therefore also relates to the compound of formula (A):
• the compound of formula (A) can be prepared by coupling the following compounds:
• This coupling can typically be achieved by applying or adapting the procedure described in example 1.
• the compounds of formula (I) are capable of inducing the targeted proteolysis of PXR.
• the compounds of formula (I) are therefore useful in the treatment and/or prevention of cancers, in particular cancers overexpressing PXR.
• a subject of the present invention is therefore also pharmaceutical compositions comprising a compound according to the invention with a pharmaceutically acceptable excipient.
• said composition contains an effective amount of the compound according to the invention.
• the present invention also relates to a compound of general formula (I) for the treatment and/or prevention of cancers, in particular cancers overexpressing PXR.
• Cancers overexpressing PXR include colorectal cancer, and cancers of the pancreas, liver and breast.
• the compounds according to the invention can be used in combination with an anti-cancer agent.
• anticancer agents can be chosen in particular from 5-Fluorouracil (5-FU), Irinotecan (CPT11), Oxaliplatin, Cisplatin, Tamoxifen, Paclitaxel, Doxorubicin, Vonblastine, Cyclophosphamide (CPA), Isophosphamide (IFO).
• said composition is administered to a patient in need thereof.
• Said patient is in particular a patient resistant to the aforementioned anti-cancer agents.
• the type of formulation of the pharmaceutical compositions of the invention depends on the mode of administration, which may include enteral (e.g. oral), parenteral (e.g. subcutaneous (sc), intravenous (iv), intramuscular (im) injection. and intrasternal, or infusion techniques, intravenous or intravenous), arterial, intramedullary, intrathecal, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical mucosa, nasal, buccal, sublingual, intratracheal instillation, bronchial instillation and/or inhalation .
• enteral e.g. oral
• parenteral e.g. subcutaneous (sc), intravenous (iv), intramuscular (im) injection. and intrasternal, or infusion techniques, intravenous or intravenous
• compositions are formulated for oral or intravenous administration (eg, systemic intravenous injection).
• pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle suitable for administering compounds of the present invention to mammals.
• Suitable carriers can include, for example, liquids (both aqueous and non-aqueous and combinations thereof), solids, encapsulating materials, gases and combinations thereof (e.g. semi-solids) , which function to carry or transport the compound from one organ or body part to another organ or body part.
• a carrier is "acceptable” in the sense that it is physiologically inert and compatible with the other components of the formulation and is non-toxic to the subject or patient. Depending on the type of formulation,
• the compounds of formula I can be formulated into solid compositions (eg powders, tablets, dispersible granules, capsules, cachets and suppositories), liquid compositions (eg solutions in which the compound is dissolved, suspensions in which the particles of the compound are dispersed, emulsions and solutions containing liposomes, micelles or nanoparticles, syrups and elixirs); semi-solid compositions (for example, gels, suspensions and creams); and gases (eg propellants for aerosol compositions).
• solid compositions eg powders, tablets, dispersible granules, capsules, cachets and suppositories
• liquid compositions eg solutions in which the compound is dissolved, suspensions in which the particles of the compound are dispersed, emulsions and solutions containing liposomes, micelles or nanoparticles, syrups and elixirs
• semi-solid compositions for example, gels, suspensions and creams
• excipients which are suitable for solid administration are derivatives of cellulose or microcrystalline cellulose, alkaline-earth carbonates, magnesium phosphate, starches, modified starches, lactose for the forms solid.
• water, aqueous solutions, saline, isotonic solutions are the most conveniently used vehicles.
• the dosage may vary within significant limits depending on the therapeutic indication and the route of administration, as well as the age and weight of the subject.
• Figure 1 depicts the PXR affinity of pre-PROTAC JMV6944 measured by RT-FRET.
• FIG 2 illustrates the activation of PXR by the pre-PROTAC JMV6944 and the resulting PROTACs measured using a luciferase reporter gene placed under the control of the CYP3A4 promoter, the target gene of PXR.
• Figures 3A and 3B represent the induction of a PXR target gene (i.e. CYP3A4) by pre-PROTAC JMV6944 and subsequent PROTACs measured by RT-qPCR.
• Figures 4A and 4B illustrate and represent the effect of PROTACs JMV7048 and JMV7965 on the induction of CYP34 by Western blot.
• FIGs 5A and 5B illustrate the effects of PROTACs on cell viability in different cell lines (LS174T, FIT29) and primary culture (CRC1) from colon cancer.
• Figures 6A-E represent the effects of PROTACs on the degradation of the PXR protein in LS174T cells, measured by Western blot.
• FIG 7 Figures 7A and 7B respectively represent the effects of PROTACs on the degradation of the PXR protein in FIEPG2 (7A) and ASPC1 (7B) cells, measured by Western blot.
• FIGs 8A-B illustrate the importance of the proteasome pathway in the effects of PROTACs on PXR protein degradation measured by Western blot.
• Figures 9A-C represent the effect of JMV7048 on the degradation of the PXR protein in vivo, on xenografts of LS174T cells in SCID mice, respectively.
• Figures 10A-D represent respectively the effects of PROTACs on the cancer stem cell population: inhibition of ALDFI activity (10A), inhibition of their self-renewal capacity (10B) and sensitization to chemotherapy (10C and 10D)
• FIG 11 Figure 11 illustrates the mode of interaction of JMV6944 with the LBD of hPXR.
• 11 A Whole structure of the complex. The F112 activating helix is indicated. The arrow symbolizes the extension of the PROTACs synthesized thereafter.
• 11 B Zoom on the JMV6944 exit pathway and superposition with the structure of the hPXR-LBD/SR12813 complex. The end of the H2' helix, residues 206 to 209, rearranges in the presence of the ligand.
• 11C Interactions of JMV6944 with hPXR binding pocket residues and representation of ligand electron density (omit type difference map).
• the following examples illustrate the invention, without however limiting it.
• the starting products used are known products or products prepared according to known procedures.
• Step 3 (9FI-fluoren-9-yl)methyl N-[7-(5-amino-1-benzyl-1H-1,3-benzodiazol-2-yl)heptyllcarbamate
• Step 4 N-[2-(7-aminoheptyl)-1-benzyl-1H-1.3-benzodiazol-5-yl1-2.4.6-trimethylbenzene-1-sulfonamide
• a solution containing (9H-fluoren-9-yl)methyl N-[7-(5-amino-1-benzyl-1H-1,3-benzodiazol-2-yl)heptyl]carbamate 0.386 g, 0.69 mmol
• a pyridine / DCM (1/1) mixture 5 ml / 5 ml
• 2-mesitylenesulfonyl chloride (0.166 g, 0.75 mmol
• the reaction medium containing 4-Fluorophthalic anhydride (2.43 g, 14.63 mmol) and 3-aminopiperidine-2,6-dione (2.38 g, 14.63 mmol) and sodium acetate (2.4 g, 29.26 mmol) in the acetic acid (50 ml) is heated at 100° C. for 24 hours. After cooling to room temperature add water (150ml) to the reaction mixture, drain the mixture and wash with ether several times.
• the 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione compound (500 mg, 1.81 mmol) is dissolved in NMP (7 ml) at ambient temperature.
• DIEA (0.89 ml, 5.43 mmol)
• t-butyl 1 -piperazinecarboxylate (370.9 mg, 1.99 mmol) are added and the mixture is stirred at 140° C. for 24 hours.
• the solution is diluted in water (100 ml). Extract with ethyl acetate twice and the organic phase is washed with saturated NaCl and dried over magnesium sulphate.
• Step 3 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione
• 6-Bromohexanoic acid 152 mg, 0.73 mmol
• DIEA 0.193 ml, 1.16 mmol
• the solution is concentrated under reduced pressure.
• the oil obtained is purified by preparative HPLC. After lyophilization, a yellow powder is obtained with a mass of 90 mg (65% yield).
• Step 1 7- ⁇ 4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]piperazin-1-yljheptanoic acid
• Step 1 8- ⁇ 4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]piperazin-1-yljoctanoic acid
• Step 1 tert-butyl 4-( ⁇ 4-[2-(2,6-dioxopiperidin-3-yl),3-dioxo-2,3-dihydro-1H-isoindol-5-yl]piperazin-1- yl ⁇ methyl)piperidine-1-carboxylate
• Step 2 2-(2,6-dioxopiperidin-3-yl)-5- ⁇ 4-[(piperidin-4-yl)methyl]piperazin-1-yl ⁇ -2,3-dihydro-1H-isoindole- 1,3-dione
• the tert-butyl compound 4-( ⁇ 4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]piperazin-1 -yl ⁇ methyl)piperidine-1-carboxylate (100 mg, 0.19 mmol) is dissolved in DCM (50ml). TFA (5ml) is added drop by drop to the reaction medium and stirred at room temperature for 5 hours. The solution is concentrated under reduced pressure. The oil obtained (75mg, yield 92%) is used as is in step 3.
• Step 3 tert-butyl 2-[4-( ⁇ 4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl ]piperazin-1 -yl ⁇ methyl)piperidin-1 -yljacetate
• Step 4 2-[4-( ⁇ 4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]piperazin acid -1 -yl ⁇ methyl)piperidin-1 -yljacetic
• Step 1 tert-butyl 4- ⁇ 4-[(2,6-dioxopiperidin-3-yl)carbamoyl]phenyl ⁇ piperazine-1-carboxylate To a solution containing 4-[4-(tert-butoxycarbonyl)piperazino]benzoic acid
• Step 3 7-(4- ⁇ 4-[(2,6-dioxopiperidin-3-yl)carbamoyl]phenyl ⁇ piperazin-1 - yl)heptanoic acid
• N-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)benzamide compound 50 mg, 0.15 mmol
• DMF 5 ml
• 7-bromoheptanoic acid 66 mg, 0.31 mmol
• DIEA 0.078 ml, 0.47 mmol
• the solution is concentrated under reduced pressure.
• the oil obtained is purified by preparative HPLC. After freeze-drying, a yellow powder is obtained with a mass of 38 mg (55% yield).
• the 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione compound (250mg, 0.90 mmol) is dissolved in anhydrous DMF (5ml) and the reaction medium is stirred and brought to 0. °C. Add the NaH in portions and leave to stir for 20 minutes. Add the iodomethane and stir for 2 hours. Stop the reaction with a solution of NH4Cl. Extract with ethyl acetate and wash twice with NaCl sat the organic phase. Dry over MgS04, filter and concentrate under reduced pressure.
• Step 2 tert-butyl 4-[2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1 H-isoindol-5-yl]piperazine- 1-carboxylate
• Step 3 2 (1-methyl-2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)-2,3-dihydro-1H-isoindole-
• Step 4 6- ⁇ 4-[2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]piperazin acid -1-yl ⁇ hexanoic
• the human PXR receptor ligand-binding domain (hPXR-LBD, residues 130-434), was produced as a recombinant protein in E. coli BL21-DE3 bacteria.
• the protein was purified on an affinity column then by size exclusion chromatography. After concentration, hPXR-LBD was crystallized in the presence of the JMV6944 ligand.
• the structure of the hPXR-LBD/JMV6944 complex was determined by X-ray crystallography by the molecular replacement method, then reconstructed and refined on the basis of the electron density (diffraction data collected at the ESRF synchrotron, Grenoble). The structure is shown in Figure 11 .
• the entire structure of the complex shows the binding mode of JMV6944.
• the originality of JMV6944 lies in the position of the extension added to the parent molecule JMV6845 and its way out of the protein domain.
• the extension grafted onto the agonist JMV6845 does not extend towards the H12 helix but points in the opposite direction between the helices H2', H6, H7 and the S1 strand to finally reach the outer surface of the LBD.
• JMV6944 also establishes hydrogen bonds with I ⁇ 407 and S247, as well as hydrophobic interactions with L411 and F428 on the one hand, and the residues of the “p-trap” region. on the other hand (F288, W299, Y306).
• the binding affinity between the JMV6944 molecule (prePROTAC) and the ligand binding domain (LBD) of PXR was quantified by FRET using the LanthaScreen TR-FRET PXR Competitive binding assay Kit (Invitrogen). The molecules were incubated for 1 hour 30 minutes at ambient temperature with the LBD of PXR in the presence of a fluorescent reference ligand. The displacement of the fluorescent ligand caused by the prePROTAC or the PXR ligand SR12813 was measured by reading the emissions at 520nm and 495nM after excitation at 337 nM on a PHERA-Star device (BMG LABTECH). The results are illustrated in Figure 1 which shows that the JMV6944 molecule is a PXR ligand with an affinity of 18.38 nM.
• LS174T cells stably transfected with an expression vector encoding the PXR protein, a Luciferase reporter gene placed under the control of the CYP3A4 promoter (target gene of PXR) and an expression cassette encoding the GFP protein placed under CMV promoter control for signal normalization.
• the cells were treated for 48 h with 5 mM of the molecules JMV6944 (prePROTACs), the PROTACs JMV7048 and JMV7605, as well as rifampicin (5 mM, PXR ligand).
• the transcriptional activity of PXR is measured by the ratio of the luciferase/GFP signals measured on a PHERA-Star device (BMG LABTECH).
• Figure 2 shows that only prePROTAC JMV6944 and rifampicin are able to activate PXR transcriptional activity.
• the LS174T cells were treated for 48 hours with 5 mM of the molecules JMV6944 (prePROTACs), JMV7048, JMV7505, or JMV5159 (inactive equivalent of JMV7048 following the addition of a methyl group to the CNBR ubiquitin ligase ligand) in the presence or in the absence of rifampicin (ligand of PXR) at 5 mM final.
• JMV6944 prePROTACs
• JMV7048 JMV7505, or JMV5159
• rifampicin ligand of PXR
• FIG. 5A illustrates the absence of toxicity of PROTACs JMV7048, JMV7505 and JMV7605 on the LS174T line.
• FIG. 5B it is seen that PROTAC JMV7048 does not affect the viability of HT29 cells or CRC1 protoculture (from a patient with colon cancer.
• the effect of PROTACs on the level of expression of the PXR protein was studied by Western-blot.
• the LS174T cells were transfected in the absence or presence of 50 nM of an siRNA targeting PXR (siPXR: NR112 Silencer, Thermofischer) or treated with PROTACs. After lysis of the cells (RIPA+antiproteases), the proteins were purified and assayed before being deposited (9C ⁇ g) on a 10% SDS-PAGE gel.
• Figures 6D and 6E illustrate the effect of JMV7048 on the level of expression of PXR according to the treatment time (maximum effect reached after 3 hours of treatment) and the concentration used (decrease dependent on the dose, with a maximum effect observed from 500 nM).
• FIGS. 7A and 7B illustrate the effect of PROTACs JMV7048 and JMV7965 on the level of expression of PXR in hepatic (7A) or pancreatic (7B) cancer cells
• LS174T cells were treated for 24 hours with JMV7048 in the presence or absence of CNBR ubiquitin ligase (MLN4924) or proteasome inhibitor (Bortezomib).
• Figures 8A and 8B confirm the importance of the proteasome pathway on the decrease in the level of expression of the PXR protein induced by PROTAC JMV7048: the decrease in the level of expression of PXR induced by JMV7048 is reversed by inhibitors ubiquitin ligase CRBN (MLN4924, Figure 8A) or by a 26S proteasome inhibitor (Bortezomib, Bz; Figure 8B), whereas the JMV7048 mutant (i.e. JMV7159, not allowing CNBR recruitment) does not cause any decrease in PXR expression level.
• the JMV7048 mutant i.e. JMV7159, not allowing CNBR recruitment
• PROTACs The effects of PROTACs on the survival and self-renewal of colon cancer stem cells were studied in vitro on the HT29 line or cancer cells isolated from patients (CRC1).
• the cells were treated with or without 5mM PROTACs for 48 hours before being analyzed: Aldefluor labeling, enzymatic activity preferentially present in cancer stem cells (Figure 10A); formation of tumorspheres under aseric and non-adherent conditions, (FIG. 10B), and finally resistance to chemotherapies (FIG. 10C and 10D).
• FIG. 10A shows that the PROTACs JMV7048, JMV 7505, JMV7506 and JMV7965 significantly reduce the percentage of ALDH-positive cells after dissociation of CRC1 cells and labeling with AldefluorTM (STEMCELL Technologies) compared to untreated cells.
• FIG. 10B shows that the JMV7048 and JMV7965 molecules significantly reduce the number of HT29 cells capable of surviving anoikis () and of inducing the formation of T umorphers (Sphere Forming Cells). Tumorphers of more than 50 mM in diameter were counted 10 days after treatment and culturing 200 cells per well (previously treated with poly2Hema in order to prevent any cell adhesion) in 100 m ⁇ medium devoid of calf serum.
• FIGS. 10A-D show that a treatment at 5 mM for 2 days with the PROTACs JMV7048 and JMV7965 significantly decreases the survival and the chemoresistance of stem cells in colon cancer cell lines.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Medicinal Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Pharmacology & Pharmacy (AREA)
• Life Sciences & Earth Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Engineering & Computer Science (AREA)
• Public Health (AREA)
• Epidemiology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicinal Preparation (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=76284058

Family Applications (1)

Country Status (7)

Families Citing this family (3)

Family Cites Families (1)

• 2021
  • 2021-05-19 FR FR2105217A patent/FR3122989B1/fr active Active
• 2022
  • 2022-05-18 JP JP2023572137A patent/JP2024519536A/ja active Pending
  • 2022-05-18 US US18/562,251 patent/US20240252658A1/en active Pending
  • 2022-05-18 EP EP22733859.7A patent/EP4340887A1/de active Pending
  • 2022-05-18 WO PCT/EP2022/063422 patent/WO2022243365A1/fr not_active Ceased
  • 2022-05-18 CN CN202280035417.4A patent/CN117295523A/zh active Pending
  • 2022-05-18 CA CA3218875A patent/CA3218875A1/fr active Pending

Also Published As

Similar Documents

Legal Events