WO2022079226A1 - Inhibitors of pdedelta - Google Patents
Inhibitors of pdedelta Download PDFInfo
- Publication number
- WO2022079226A1 WO2022079226A1 PCT/EP2021/078585 EP2021078585W WO2022079226A1 WO 2022079226 A1 WO2022079226 A1 WO 2022079226A1 EP 2021078585 W EP2021078585 W EP 2021078585W WO 2022079226 A1 WO2022079226 A1 WO 2022079226A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compound
- cancer
- group
- mmol
- methyl
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/32—One oxygen, sulfur or nitrogen atom
- C07D239/42—One nitrogen atom
-
- 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
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
Definitions
- the present invention relates to compounds which show activity as PDEdelta/ PDE6D- inhibitors, pharmaceutical compositions comprising these compounds, their use in medicine as well as their use in the treatment of cancer.
- the oncogene Ras is one of the best-established cancer targets without approved inhibitor Ras drug development efforts in the 1990s were thwarted by the failure of farnesyltransferase inhibitors (FTI) in clinical trials ([1] Papke and Der, 2017).
- FTI farnesyltransferase inhibitors
- the highly mutated K-Ras4A/4B and N-Ras, but not H-Ras can be alternatively prenylated by geranylgeranyltransferase I, reinstating Ras plasmamembrane localization and thus activity, even in the presence of FTIs ([2] Lerner et al., 1997).
- PDEdelta also called PDE6D
- PDE6D is a trafficking chaperone of farnesylated proteins, suggesting that its inhibition affects the same clients as inhibition of farnesyltransferase.
- PDEdelta cannot facilitate intracellular diffusion of proteins that are in addition palmitoylated ([4]Chandra et al., 2011; [5] Dharmaiah et al., 2016).
- K-Ras4B (herafter K-Ras) trafficking, but has much less effect on trafficking of dual-pal mitoylated H-Ras ([4] Chandra et al. ,2011 ; [6] Schmick et al., 2014).
- K-Ras In order to relay signaling, K-Ras needs to be localized predominantly to the plasma membrane. This requires vesicular transport of K-Ras to the plasma membrane from the recycling endosome, where it is collected after PDEdelta-assisted diffusion from internal cellular membranes ([4] Chandra et al., 2011 ;[6] Schmick et al., 2014). Unloading of K-Ras from PDEdelta in the perinuclear compartment requires the binding of GTP-Arl2 to PDEdelta, which results in an allosteric conformational change in PDEdelta that effectively releases its cargo ([7] Ismail et al., 2011).
- Deltaflexin-1 and Deltaflexin-2 Another group of competitive PDEdelta inhibitors called Deltaflexin-1 and Deltaflexin-2 has been published by Siddiqui et al. ([19] Siddiqui et al., 2020). However, there is room for improvement of these compounds with respect to their activity in vitro and in cells.
- proteolysis-targeting chimeras Another class of recent inhibitors developed by both the Waldmann and Sheng groups are proteolysis-targeting chimeras (PROTACs). These heterobifunctional compounds bind to PDEdelta also in the prenyl-pocket and instruct the proteasomal degradation through a connected chemical moiety that recruits an E3 ubiquitin ligase complex. ([20] Winzker et al., 2020).
- the present invention relates to a compound according to formula (I) wherein
- RT is selected from the group consisting of unsubstituted piperidinyl and unsubtituted (C C 5 )alkyl, preferably methyl;
- A is selected from the group consisting of
- R 2 is selected from the group consisting of unsubstituted (Ci-C 6 )alkyl, and unsubtituted (C 3 - C 8 )cycloalkyl;
- R 3 is selected from the group consisting of unsubstituted (Ci-C 6 )alkyl, preferably isobutyl;
- R 4 is selected from the group consisting of unsubstituted (C 1 -C 6 )alkyl, preferably (CrC ⁇ alkyl, more preferably methyl;
- X is selected from the group consisting of F, Cl, Br, and I, preferably F; n is an integer between 1 and 10, preferably between 2 and 8, more preferably 6; and a solvate, hydrate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.
- the invention is directed to a pharmaceutical composition, comprising said compound.
- the invention further relates to said compound or pharmaceutical composition for use in medicine.
- the invention is directed to said compound or pharmaceutical composition for use in the treatment of cancer.
- the compounds show a significantly higher activity in cells (Figure 1), while maintaining their on-target activity (Figure 2) and K-Ras4B (hereafter K-Ras) activity ( Figure 3) compared to compounds of the prior art, for example as published in Siddiqui et al. ([19] Siddiqui et al., 2020).
- FIG. 1 Inhibition of 2D cell proliferation.
- FIG. 2 Cellular BRET-data showing on-target activity of inhibitors against the PDEdelta (PDE6D)/ K-Ras interaction.
- HEK 293 EBNA cells were co-transfected with RLuc8- tagged PDE6D and GFP2-tagged KRas4B-G12V (donor: acceptor plasmid ratio 1:20).
- 24 h after transfection cells were treated for 24 h with 0.1% DMSO (control), inventive compounds or control compounds.
- Values represent the mean ⁇ SEM of 3 independent biological repeats. Statistical significance levels were evaluated by comparison to the control condition and are annotated as * p ⁇ 0.05, ** p ⁇ 0.01 and *** p ⁇ 0.001.
- FIG. 3 Cellular K-Ras nanoclustering-BRET data showing on-target activity of inhibitors.
- HEK 293 EBNA cells were co-transfected with RLuc8- and GFP2-tagged KRas4B- G12V (donor : acceptor plasmid ratio 1 : 5).
- 24 h after transfection cells were treated for 24 h with 0.1% DMSO (control) or various concentrations of compounds of the invention and control compounds. Tested concentration ranges are indicated and different ranges have a different color code. Values represent the mean ⁇ SEM of 3 independent biological repeats. Statistical significance levels were evaluated by comparison to the control condition and are annotated as * p ⁇ 0.05, ** p ⁇ 0.01 and *** p ⁇ 0.001.
- FIG. 4 Cellular H-Ras nanoclustering-BRET data to assess K-Ras selectivity.
- HEK 293 EBNA cells were co-transfected with RLuc8- and GFP2-tagged HRas-G12V (donor : acceptor plasmid ratio 1 : 3).
- 24 h after transfection cells were treated for 24 h with 0.1% DMSO (control) or various concentrations of inventive compounds and control compounds. Tested concentration ranges are indicated and different ranges have a different color code. Values represent the mean ⁇ SEM of > 3 independent biological repeats. Statistical significance levels were evaluated by comparison to the control condition and are annotated as * p ⁇ 0.05, ** p ⁇ 0.01 and *** p ⁇ 0.00 .
- alkyl refers to a monoradical of a saturated straight or branched hydrocarbon.
- the alkyl group comprises from 1 to 6 (such as 1 to 6) carbon atoms, i.e., 1 , 2, 3, 4, 5, or 6, carbon atoms (such as 1, 2, 3, 4, 5, 6, carbon atoms), more preferably 1 to 4 carbon atoms.
- Exemplary alkyl groups include methyl, ethyl, propyl, sec-propyl ( alternative name :iso- propyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl- propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n- nonyl, n-decyl, n-undecyl, n-dodecyl, and the like.
- cycloalkyl represents cyclic non-aromatic versions of “alkyl” and "alkenyl” with preferably 3 to 8 carbon atoms, such as 3 to 8 carbon atoms, i.e., 3, 4, 5, 6, 7, or 8 carbon atoms, more preferably 3 to 8 carbon atoms, even more preferably 3 to 7 carbon atoms.
- exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, cyclooctyl.
- cycloalkyl examples include C 3 -C 8 -cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.
- substituted means, that in the respective underlying group at least one hydrogen atom is substituted with another functional group, not selected from hydrogen, as further defined below in each case. In particular this means that all, or only a part of the hydrogen atoms are substituted with another functional group.
- unsubstituted means, that in the respective underyling group none of the hydrogen atoms is further substituted with at least one further functional group.
- halogen also abbreviated herein as “hal” means fluoro, chloro, bromo, or iodo, preferably fluoro.
- “Isomers” are compounds having the same molecular formula but differ in structure ("structural isomers”) or in the geometrical positioning of the functional groups and/or atoms (“stereoisomers”).
- “Enantiomers” are a pair of stereoisomers which are non-superimposable mirror-images of each other.
- a “racemic mixture” or “racemate” contains a pair of enantiomers in equal amounts.
- Diastereomers are stereoisomers which are non-superimposable and not mirror-images of each other.
- the compounds described herein may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), regioisomers, enantiomers or diastereomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated or identified compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures.
- stereoisomers such as double-bond isomers (i.e., geometric isomers), regioisomers, enantiomers or diastereomers.
- Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the person skilled in the art.
- the compounds may also exist in several tautomeric forms including the enol form, the keto form and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated or identified compounds.
- Tautomers are structural isomers of the same chemical substance that spontaneously interconvert with each other, even when pure.
- solvate refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal.
- a solvent such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids)
- a solvent such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like
- isotopically labeled compounds one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons.
- a hydrogen atom may be replaced by a deuterium atom.
- Exemplary isotopes which can be used in the compounds of the present invention include deuterium, 11 C, 13 C, 14 C, 15 N, 18 F, 32 S, 36 CI, and 125 l.
- the present invention is directed to a compound according to formula (I) wherein
- RT is selected from the group consisting of unsubstituted piperidinyl and unsubtituted (C 1 -C 5 )alkyl, preferably methyl.
- the piperidinyl may be a 2, 3 or 4-piperidinyl group, preferably a 4-piperidinyl group. Wherein position 1 corresponds to the position of the nitrogen atom in the 6-membered ring of piperidine.
- A is selected from the group consisting preferably
- R 2 is selected from the group consisting of unsubstituted (Ci-C 6 )alkyl, and unsubtituted (C 3 -C 8 ) cycloalkyl. In one embodiment, R 2 is selected from the group consisting of cyclopropyl, sec-propyl, and ethyl, preferably sec-propyl;
- R 3 is selected from the group consisting of unsubstituted (C 1 -C 6 )alkyl, preferably (C C 4 )alkyl, more preferably isobutyl.
- R 4 is selected from the group consisting of unsubstituted (C 1 -C 6 )alkyl, preferably (C C 3 )alkyl, more preferably methyl;
- X is selected from the group consisting of F, Cl, Br, and I, preferably F; X may be in ortho, meta or para position, preferably in para position.
- n is an integer between 1 and 10, preferably between 2 and 8, more preferably 6.
- the present invention comprises a solvate, hydrate, pharmaceutically acceptable salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.
- the salt is a pharmaceutically acceptable salt.
- “Pharmaceutically acceptable salt” embraces salts with a pharmaceutically acceptable acid or base.
- Pharmaceutically acceptable acids include both inorganic acids, for example hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic, hydroiodic and nitric acid and organic acids, for example citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p- toluenesulphonic acid.
- Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases, for example alkyl amines, arylalkyl amines and heterocyclic amines.
- the preparation of the inventive compounds may be carried out in general steps specified in the following.
- the synthetic steps may be carried out in the order as described below or arranged in a different order where technically possible
- step 1 either step 1a or 1b may be carried out.
- Step 1a In step 1a compound is converted with an amine R 2 NH 2 preferably in the presence of a base in an organic solvent.
- LG may be selected from Cl, Br, I or a sulfonate leaving group, such as mesylate, tosylate, nosylate, triflate, preferably Br.
- R 5 may be selected from (Ci-C 6 )alkyl, preferably methyl and ethyl.
- Any suitable base may be used.
- a weaker base such as K 2 CO 3 , NaCO 3 is used.
- the organic solvent is a polar-aprotic solvent, such as Diethylether, THF, DMF, DMSO, more preferably DMF.
- a polar-aprotic solvent such as Diethylether, THF, DMF, DMSO, more preferably DMF.
- step 1a is carried out at room temperature.
- the reaction time in step 1a is 1 to 5 h, more preferably 2 h.
- Step 1b In step 1b compound n is converted with preferably in the presence of a base in an organic solvent.
- LG may be selected from Cl, Br, I or a sulfonate leaving group, such as mesylate, tosylate, nosylate, triflate, preferably Br.
- R 5 may be selected from (C 1 -C 6 )alkyl, preferably methyl and ethyl.
- Any suitable base may be used.
- a weaker base such as K 2 CO 3 , or NaCO 3 is used.
- the organic solvent is a polar-aprotic solvent, such as Diethylether, THF, DMF, DMSO, more preferably DMF.
- a polar-aprotic solvent such as Diethylether, THF, DMF, DMSO, more preferably DMF.
- step 1a is carried out at room temperature.
- the reaction time in step 1a is 1 to 5 h, more preferably 2 h.
- the organic solvent is a polar protic solvent, such as ethanol.
- any suitable base may be used.
- a weaker base such as K 2 CO 3 , or NaCO 3 is used.
- the reaction temperature is 20 to 100 °C, more preferably 60 to 90 °C, most preferably 70 to 85 °C. o
- Step 2 In step 2 the product obtained in step 1a is converted with the presence of a suitable coupling reagent, a suitable base and optionally a catalyst for creating amid bonds between an amine and an carboxylic acid group in an organic solvent.
- a suitable coupling reagent for creating amid bonds between an amine and an carboxylic acid group in an organic solvent.
- a suitable base for reacting a suitable base with a suitable base with a suitable base with a suitable base and optionally a catalyst for creating amid bonds between an amine and an carboxylic acid group in an organic solvent.
- a catalyst for creating amid bonds are known in the art. If step 1b is carried out, step 2 is not carried out.
- the coupling reagent may be selected for example from 1 -Ethyl-3-(3- dimethylaminopropyl)carbodiimid (EDC) and dicyclohexylcarbodiimid (DCC), 1- [bis(Dimethylamin)methylen]-1/7-1,2,3-triazol[4,5-b]pyridinium-3-oxid-hexafluorophosphat (HATLI), preferably EDC.
- EDC Ethyl-3-(3- dimethylaminopropyl)carbodiimid
- DCC dicyclohexylcarbodiimid
- HTLI 1- [bis(Dimethylamin)methylen]-1/7-1,2,3-triazol[4,5-b]pyridinium-3-oxid-hexafluorophosphat
- the catalyst may be selected for example from 4- dimethylaminopyridine (DMAP) and hydroxybenzotriazole (HOBt), preferably HOBt.
- the base is preferably selected from an organic amine base with low nucleophilicity, for example triethylamin, and diisopropylethylamin (DIPEA).
- the solvent is preferably an aprotic organic solvent, for example dichloromethane, and DMF, more preferably DMF.
- the reaction is carried out at room temperature.
- Step 3 The ester group in the product of step 1b and step 2 is hydrolized to the corresponding carboxyl group.
- Suitable conditions for this reaction type are generally known to the person skilled in the art.
- the reaction is carried out in the presence of an inorganic base, water and an organic solvent.
- the inorganic base may be selected from LiOH, and NaOH, preferably LiOH.
- the organic solvent may be selected from, THF, ethanol, methanol, preferably THF.
- Step 4 The product of step 3 is reacted with R ⁇ under conditions as described for step 2.
- step 4 if is piperidinyl, the nitrogen atom in piperidinyl is protected with a suitable protecting group, preferably Boc.
- a suitable protecting group preferably Boc.
- HATLI is used as coupling reagent.
- DI PEA is used as base.
- DMF is used as solvent.
- RT is piperidinyl
- the nitrogen atom in piperidinyl is protected with a suitable protecting group, preferably Boc.
- Reductive amination may be carried out in the presence of NaCNBH 3 , an organic acid such as acetic acid an in an polar-protic solvent such as an alcohol like methanol or ethanol.
- Step 5 Remaining protecting groups such as Boc introduced in step 4 may be removed under conditions known in the art. Boc may be for example be removed under acidic conditions such as in the presence of an inorganic acid like hydrochlorid acid preferably in dioxane.
- a further aspect of the present invention is directed to a pharmaceutical composition comprising at least one of the inventive compounds.
- “Pharmaceutical composition” refers to one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
- Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
- Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered orally.
- Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions.
- Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
- the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
- the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
- Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
- Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
- the formulation should suit the mode of administration.
- Therapeutically effective amount is an amount of the inventive compounds or a combination of two or more such compounds, which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition.
- a therapeutically effective amount can also be an amount which is prophylactically effective. The amount which is therapeutically effective will depend upon the patient's size and gender, the condition to be treated, the severity of the condition and the result sought. For a given patient, a therapeutically effective amount can be determined by methods known to those of skill in the art.
- a further embodiment of the invention is directed to the inventive compounds or pharmaceutical composition for use in medicine.
- a further embodiment of the invention is directed to the inventive compounds or pharmaceutical composition for use in the treatment of cancer.
- the cancer is selected from K-Ras dependent cancers or from cancers wherein the KRAS gene is mutated.
- the cancer is selected from glioma, breast cancer, colorectal cancer, pancreatic cancer, stomach cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, particularly preferred pancreatic cancer.
- Step-1 Synthesis of compound (lnt.-8) (Ethyl 7-(cyclopentylamino)heptanoate)
- Step-2 Synthesis of compound (lnt.-10)
- Ethyl 7-(cyclopentylamino)heptanoate (0.750 g, 4.6 mmol, 1.0 eq) in DMF (10 ml) was added EDC.HCI (2.0 g,10.7 mmol, 2.0eq), DIPEA (2.7 ml, 16.0 mmol, 3.0 eq), HOBT(0.144 g,1.07 mmol, 0.2 eq) and 4-Fluorobenzoic acid (1.5 g, 6.42 mmol, 1.2eq) at RT. The reaction mixture was stirred at RT for 16 h.
- Step-3 Synthesis of compound (lnt.-11)
- Step-4 Synthesis of compound (lnt.-13)
- Step-5 Synthesis of compound (A-342a)
- Step-6a Synthesis of compound (lnt.-12) tert-butyl 4-((((2-(methylamino)pyrimidin-4-yl)methyl)amino)methyl)piperidine- 1 - carboxylate
- Step-7 Synthesis of compound (A-442a)
- Step-6b Synthesis of compound (lnt-12’)
- Step-1 Synthesis of compound (lnt.-8)
- Step-2 Synthesis of compound (lnt.-10)
- Ethyl 7-(isopropylamino)heptanoate (0.650 g, 4.6 mmol, 1.0 eq) in DMF was added EDC.HCI (1.7g, 9.2 mmol, 2.0 eq), DIPEA (2.4ml, 13.9 mmol, 3.0 eq), HOBT (0.125 g, 0.92 mmol, 0.2 eq) and 4-fluorobenzoic acid (0.990 g, 4.6 mmol, 1.0 eq). The mixture was stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml).
- Step-3 Synthesis of compound (lnt.-11)
- Step-4 Synthesis of compound (lnt.-13)
- Step-5 Synthesis of compound (A-342b)
- Step-7 Synthesis of compound (A-442b)
- Step-1 Synthesis of compound (lnt.-8)
- Ethyl 7-(ethylamino)heptanoate (1.50 g, 7.46 mmol, 1.0 eq) in DMF(15 ml) was added EDC.HCI (1.46 g, 10.44 mmol, 2.0 eq), DIPEA (2.26 g, 22.38 mmol, 3.0 eq), HOBT (0.20 g,1.49 mmol, 0.2 eq) and 4-Fluorobenzoic acid (1.46 g, 10.44 mmol, 1.4 eq). The mixture was stirred at RT for 16 h . After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml).
- Step-3 Synthesis of compound (lnt.-11)
- Step-4 Synthesis of compound (lnt.-13)
- Step-1 Synthesis of compound (lnt.-3)
- Step-2 Synthesis of compound (lnt.-4)
- Step-3 Synthesis of compound (I nt. -5)
- Step-4 Synthesis of compound (I nt. -6)
- K-Ras4B (hereafter K-Ras) is a major driver of cell proliferation.
- uncontrolled proliferation typically depends on the former.
- BRET-biosensors Two types have been contructed to measure directly K-Ras displacement from PDEdelta (RLuc8-PDE6D/ GFP2-K-RasG12V) or the functional organization of K-Ras in nanoscale signaling complexes of the plasma membrane, called nanocluster (RLuc8-K-RasG12V/ GFP2- K-RasG12V). Nanoclustering-BRET decreases with the inhibition of any process upstream of nanoclustering, including improper posttranslational modification of Ras, trafficking defects and disrupted plasma membrane anchorage. BRET-biosensors were expressed in HEK293 EBNA cells to assess in cellulo on- target activity.
- All expression constructs were produced by multi-site Gateway cloning technology as described (Wall et al., 2014). Briefly, three entry clones with compatible LR recombination sites, encoding the CMV promoter, RLuc8 or GFP2 tag and a gene of interest with stop codon, either K-Ras4B- G12V, H-Ras-G12V or PDE6D, were obtained from Addgene. The three clones were inserted into a destination vector, pDest-305 or pDest-312, using Gateway LR Clonase II enzyme mix (#11791020, Thermo Fisher Scientific). The reaction mix was transformed into ccdB sensitive E.coli strain DH10B (#EC0113, Thermo Fisher Scientific) and positive clones were selected in the presence of ampicillin.
- HEK293 EBNA (HEK) cells were a gift of Prof. Florian M. Wurm, EPFL, Lausanne, Switzerland, and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza Pharma).
- DMEM Dulbecco’s modified Eagle’s medium
- MDA-MB- 231 ATCC HTB-26
- RPMI Roswell Park Memorial Institute medium
- MIA PaCa-2 ATCC CRM-CRL-1420
- All media were supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Lonza Pharma) (complete medium).
- Cells were grown at 37 °C in a water-saturated, 5% CO 2 atmosphere and sub-cultured twice a week.
- MDA-MB-231 and MIA PaCa-2 cells were plated in complete medium into 96-well cell culture plates (#655 180, Greiner bio-one, Merck KGaA) at a density of 1000 cells/ 100 .L and allowed to attach for 24 h. Test compounds were then added at indicated concentrations and DMSO (0.1% v/v) was used as a vehicle control. After 72 h incubation in the presence of the compounds, the cell viability was assessed using the alamarBlue reagent (#DAL1100, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, alamarBlue reagent was added to each well of the plate (10% final volume) and incubated for 4 h at 37 °C.
- the fluorescence intensity was read at the excitation wavelength of 530 ⁇ 10 nm and emission wavelength of 590 ⁇ 10 nm using a CLARIOstar plate reader (BMG LABTECH GmbH).
- the obtained raw fluorescence intensity data were normalized to vehicle control (100% viability) and plotted against the compound concentration.
- DSS3 Drug sensitivity score analysis
- DSS drug sensitivity score
- the output file provides several drug sensitivity measures including IC 50 and AUC.
- DSS3 value DSS 2 X2 X1 n cc
- DSS2 is given by the equation
- DSS 2 and DSS1 is given by the equation
- DSS3 was employed as it takes drug responses over a wider concentration range into account, as compared to drugs that show increased response only at the higher end of the concentration range.
- AUC was determined as exact solution.
- a 10% minimal activity threshold (t) was set.
- the maximum (Cmax) and minimum (Cmin) concentrations used for screening of the inhibitors, with Cmax x2 and x1 concentration with minimal activity t.
- the parameter a is the value of the top asymptote, which can be different from 100% inhibition as obtained with100 pM benzethonium chloride (#53751 , Merck KGaA) treatment.
- HEK293 EBNA cells were plated in 1 mL complete DMEM into 12-well cell culture plates (#665 180, Greiner bio-one, Merck KGaA) at a density of 150,000 to 200,000 cells/ mL and allowed to attach for 24 h. Then, RLuc8-tagged donor and GFP2-tagged acceptor constructs were transfected into cells using the jetPRIME transfection reagent (Polyplustransfection SA) following the manufacturer’s instructions. Each well was transfected with about 1 pg of plasmid DNA using 3 pL of jetPRIME reagent.
- the concentration of donor plasmid 50 ng was kept constant and the concentration of acceptor plasmid was increased from 0 to 1000 ng.
- the empty pcDNA3.1 plasmid was used to top-up the total DNA load per transfection. 24 h after transfection, cells were treated with compounds or vehicle control (DMSO 0.1% v/v in complete medium) at specified concentrations for 24 h.
- the fluorescence intensity (RFU) of GFP2 was measured with excitation at 405 ⁇ 10 nm and emission 515 ⁇ 10 nm ; it is proportional to the acceptor concentration [Acceptor], BRET readings were taken well by well by adding 10 pL of 100 pM coelenterazine 400a (DeepBlueC, #C-320, Gold Biotechnology) RLuc8 substrate to each well (final concentration of 10 pM) using the injector present in the plate reader. Luminescence emission intensities were simultaneously recorded at 410 ⁇ 40 nm (RLU, proportional to [Donor]) and 515 ⁇ 15 nm (BRET-signal).
- the raw BRET ratio was calculated as the BRET signal measured at 515 nm divided by emission signal measured at 410 nm (RLU).
- the BRET ratio was obtained by subtracting the raw BRET ratio by a background BRET signal measured for cells expressing only the donor.
- the BRET ratio was calculated using the formula
- the BRET ratio was plotted against the ratio of acceptor to donor plasmid amounts (A/D plasmid ratio) that were transfected or the relative expression.
- the A/D plasmid ratio at which the BRET ratio changes most linearly with the relative expression was determined for each BRET sensor and then used for compound profiling.
- GraphPad Prism version 8.00 for Windows, GraphPad Software
- the GDI-like solubilizing factor PDEb sustains the spatial organization and signalling of Ras family proteins. Nat Cell Biol 14, 148-158.
- KRas localizes to the plasma membrane by spatial cycles of solubilization, trapping and vesicular transport. Cell 157, 459-471.
- Rapalogs can promote cancer cell sternness in vitro in a Galectin-1 and H-ras-dependent manner.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Plural Heterocyclic Compounds (AREA)
Abstract
The present invention relates to compounds which show activity as PDEdelta/PDE6D Inhibitors and pharmaceutical compositions comprising these compounds, their use in medicine as well as their use in the treatment of cancer.
Description
Inhibitors of PDEdelta
The work leading to this invention was supported by the Luxembourg Fonds National de Recherche (FNR) under Grant Nr. Grant Nr. PF18/13246472/econoRAS2.
TECHNICAL FIELD OF THE INVENTION
[001] The present invention relates to compounds which show activity as PDEdelta/ PDE6D- inhibitors, pharmaceutical compositions comprising these compounds, their use in medicine as well as their use in the treatment of cancer.
BACKGROUND ART
[002] The oncogene Ras is one of the best-established cancer targets without approved inhibitor Ras drug development efforts in the 1990s were thwarted by the failure of farnesyltransferase inhibitors (FTI) in clinical trials ([1] Papke and Der, 2017). At that time, it was disregarded that the highly mutated K-Ras4A/4B and N-Ras, but not H-Ras, can be alternatively prenylated by geranylgeranyltransferase I, reinstating Ras plasmamembrane localization and thus activity, even in the presence of FTIs ([2] Lerner et al., 1997).
[003] Ras drug development has recently gained track again, with direct and indirect targeting approaches ([3] Spiegel et al., 2014). The indirect target PDEdelta, also called PDE6D, is a trafficking chaperone of farnesylated proteins, suggesting that its inhibition affects the same clients as inhibition of farnesyltransferase. However, PDEdelta cannot facilitate intracellular diffusion of proteins that are in addition palmitoylated ([4]Chandra et al., 2011; [5] Dharmaiah et al., 2016). Thus PDEdelta inhibition selectively affects K-Ras4B (herafter K-Ras) trafficking, but has much less effect on trafficking of dual-pal mitoylated H-Ras ([4] Chandra et al. ,2011 ; [6] Schmick et al., 2014).
[004] In order to relay signaling, K-Ras needs to be localized predominantly to the plasma membrane. This requires vesicular transport of K-Ras to the plasma membrane from the recycling endosome, where it is collected after PDEdelta-assisted diffusion from internal cellular membranes ([4] Chandra et al., 2011 ;[6] Schmick et al., 2014). Unloading of K-Ras from PDEdelta in the perinuclear compartment requires the binding of GTP-Arl2 to PDEdelta, which results in an allosteric conformational change in PDEdelta that effectively releases its cargo ([7] Ismail et al., 2011). Unfortunately, this ejection mechanism also applies to the first two generations of PDEdelta inhibitors Deltarasin ([8] Zimmermann et al., 2013) and Deltazinone ([9] Papke et al., 2016; WO2015189433A1).
[005] Only the last generation of PDEdelta inhibitors, the Deltasonamides, could largely withstand Arl2-mediated ejection, as they were highly optimized for sub-nanomolar affinity. However, these compounds had a low partitioning coefficient, suggesting low cell penetration ([13] Martin-Gago et al., 2017).
[006] Similarly, inhibitors developed by the Sheng group (Shanghai, China) also bound with nanomolar affinity, but again had only micromolar cellular activity ([18] Jiang et al., 2017).
[007] Another group of competitive PDEdelta inhibitors called Deltaflexin-1 and Deltaflexin-2 has been published by Siddiqui et al. ([19] Siddiqui et al., 2020). However, there is room for improvement of these compounds with respect to their activity in vitro and in cells.
[008] Another class of recent inhibitors developed by both the Waldmann and Sheng groups are proteolysis-targeting chimeras (PROTACs). These heterobifunctional compounds bind to
PDEdelta also in the prenyl-pocket and instruct the proteasomal degradation through a connected chemical moiety that recruits an E3 ubiquitin ligase complex. ([20] Winzker et al., 2020).
[009] Thus, there is a need to provide further PDEdelta Inhibitors.
SUMMARY OF THE INVENTION
RT is selected from the group consisting of unsubstituted piperidinyl and unsubtituted (C C5)alkyl, preferably methyl;
R2 is selected from the group consisting of unsubstituted (Ci-C6)alkyl, and unsubtituted (C3- C8)cycloalkyl;
R3 is selected from the group consisting of unsubstituted (Ci-C6)alkyl, preferably isobutyl;
R4 is selected from the group consisting of unsubstituted (C1-C6)alkyl, preferably (CrC^alkyl, more preferably methyl;
X is selected from the group consisting of F, Cl, Br, and I, preferably F; n is an integer between 1 and 10, preferably between 2 and 8, more preferably 6; and a solvate, hydrate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.
[0011] In a second aspect, the invention is directed to a pharmaceutical composition, comprising said compound.
[0012] In a third aspect, the invention further relates to said compound or pharmaceutical composition for use in medicine.
[0013] In a fourth aspect, the invention is directed to said compound or pharmaceutical composition for use in the treatment of cancer.
[0014] . The compounds show a significantly higher activity in cells (Figure 1), while maintaining their on-target activity (Figure 2) and K-Ras4B (hereafter K-Ras) activity (Figure 3) compared to compounds of the prior art, for example as published in Siddiqui et al. ([19] Siddiqui et al., 2020).
BRIEF DESCRIPTION OF THE FIGURES
[0015] Figure 1 : Inhibition of 2D cell proliferation. KRAS-mutant cancer cell lines were treated with PDE6D inhibitors Deltaflexin-1, Deltaflexin-2 (concentration range 0.39 - 100 pM, n = 3), Inventive compounds A342a, A342b, A342c, A372, A442a, A442b, A442c and A472 (0.3 - 80 pM, n > 3), Deltarasin (0.078 - 20 pM, n = 5), or Deltazinone (0.156 - 40 pM, n=5). Activity is compared to reference compounds FTI-277 (0.3 - 80 pM, n = 2), AMG-510 (0.0039 - 1 pM, n = 3) and ARS-1620 (0.078 - 20 pM, n = 3). Cell viability was measured by alamarBlue assay after 72 h of drug treatment. Dose response data were analyzed using the Breeze-platform to obtain the DSS3 score, which is a normalized area under the curve measure that takes different tested concentration ranges into account. Values represent mean ± SEM of n independent biological repeats.
[0016] Figure 2: Cellular BRET-data showing on-target activity of inhibitors against the PDEdelta (PDE6D)/ K-Ras interaction. HEK 293 EBNA cells were co-transfected with RLuc8- tagged PDE6D and GFP2-tagged KRas4B-G12V (donor: acceptor plasmid ratio 1:20). 24 h after transfection, cells were treated for 24 h with 0.1% DMSO (control), inventive compounds or control compounds. Values represent the mean ± SEM of 3 independent biological repeats. Statistical significance levels were evaluated by comparison to the control condition and are annotated as * p < 0.05, ** p < 0.01 and *** p < 0.001.
[0017] Figure 3: Cellular K-Ras nanoclustering-BRET data showing on-target activity of inhibitors. HEK 293 EBNA cells were co-transfected with RLuc8- and GFP2-tagged KRas4B- G12V (donor : acceptor plasmid ratio 1 : 5). 24 h after transfection, cells were treated for 24 h with 0.1% DMSO (control) or various concentrations of compounds of the invention and control compounds. Tested concentration ranges are indicated and different ranges have a different color code. Values represent the mean ± SEM of 3 independent biological repeats. Statistical significance levels were evaluated by comparison to the control condition and are annotated as * p < 0.05, ** p < 0.01 and *** p < 0.001.
[0018] Figure 4. Cellular H-Ras nanoclustering-BRET data to assess K-Ras selectivity. HEK 293 EBNA cells were co-transfected with RLuc8- and GFP2-tagged HRas-G12V (donor : acceptor plasmid ratio 1 : 3). 24 h after transfection, cells were treated for 24 h with 0.1% DMSO (control) or various concentrations of inventive compounds and control compounds. Tested concentration ranges are indicated and different ranges have a different color code. Values represent the mean ± SEM of > 3 independent biological repeats. Statistical significance levels were evaluated by comparison to the control condition and are annotated as * p < 0.05, ** p < 0.01 and *** p < 0.00 .
DETAILED DESCRIPTION OF THE INVENTION
[0019] The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the Figures and reflected in the claims.
Definitions
[0020] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 6 (such as 1 to 6) carbon atoms, i.e., 1 , 2, 3, 4, 5, or 6, carbon atoms (such as 1, 2, 3, 4, 5, 6, carbon atoms), more preferably 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, sec-propyl ( alternative name :iso- propyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl- propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n- nonyl, n-decyl, n-undecyl, n-dodecyl, and the like.
[0021] The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 8 carbon atoms, such as 3 to 8 carbon atoms, i.e., 3, 4, 5, 6, 7, or 8 carbon atoms, more preferably 3 to 8 carbon atoms, even more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, cyclooctyl. Preferred examples of cycloalkyl include C3-C8-cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.
[0022] The term “substituted” means, that in the respective underlying group at least one hydrogen atom is substituted with another functional group, not selected from hydrogen, as further defined below in each case. In particular this means that all, or only a part of the hydrogen atoms are substituted with another functional group.
[0023] The term “unsubstituted” means, that in the respective underyling group none of the hydrogen atoms is further substituted with at least one further functional group.
[0024] The term "halogen", also abbreviated herein as “hal” means fluoro, chloro, bromo, or iodo, preferably fluoro.
[0025] "Isomers" are compounds having the same molecular formula but differ in structure ("structural isomers") or in the geometrical positioning of the functional groups and/or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers which are non-superimposable mirror-images of each other. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts.
[0026] Diastereomers" are stereoisomers which are non-superimposable and not mirror-images of each other.
[0027] The compounds described herein may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), regioisomers, enantiomers or diastereomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated or identified compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the person skilled in the art. The compounds may also exist in several tautomeric
forms including the enol form, the keto form and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated or identified compounds.
[0028] "Tautomers" are structural isomers of the same chemical substance that spontaneously interconvert with each other, even when pure.
[0029] The term "solvate" as used herein refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal. The amount of solvent contained in the addition complex may be stoichiometric or non- stoichiometric. A "hydrate" is a solvate wherein the solvent is water.
[0030] In isotopically labeled compounds one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons. For example, a hydrogen atom may be replaced by a deuterium atom. Exemplary isotopes which can be used in the compounds of the present invention include deuterium, 11C, 13C, 14C, 15N, 18F, 32S, 36CI, and 125l.
Compounds
[0032] RT is selected from the group consisting of unsubstituted piperidinyl and unsubtituted (C1-C5)alkyl, preferably methyl.
[0033] The piperidinyl may be a 2, 3 or 4-piperidinyl group, preferably a 4-piperidinyl group. Wherein position 1 corresponds to the position of the nitrogen atom in the 6-membered ring of piperidine.
[0034] In one preferred embodiment,
is unsubstituted (C1-C5)alkyl, more preferably preferably methyl.
[0036] R2 is selected from the group consisting of unsubstituted (Ci-C6)alkyl, and unsubtituted (C3-C8) cycloalkyl. In one embodiment, R2 is selected from the group consisting of cyclopropyl, sec-propyl, and ethyl, preferably sec-propyl;
[0037] R3 is selected from the group consisting of unsubstituted (C1-C6)alkyl, preferably (C C4)alkyl, more preferably isobutyl.
[0038] R4 is selected from the group consisting of unsubstituted (C1-C6)alkyl, preferably (C C3)alkyl, more preferably methyl;
[0039] X is selected from the group consisting of F, Cl, Br, and I, preferably F; X may be in ortho, meta or para position, preferably in para position.
[0040] n is an integer between 1 and 10, preferably between 2 and 8, more preferably 6.
[0041] Further, the present invention comprises a solvate, hydrate, pharmaceutically acceptable salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.
[0042] Preferably, the salt is a pharmaceutically acceptable salt.
[0043] “Pharmaceutically acceptable salt” embraces salts with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids, for example hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic, hydroiodic and nitric acid and organic acids, for example citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p- toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases, for example alkyl amines, arylalkyl amines and heterocyclic amines.
[0044] A selection of compounds according to the present invention is listed in the following Table 1.
Preparation of Compounds
[0045] The preparation of the inventive compounds may be carried out in general steps specified in the following. The synthetic steps may be carried out in the order as described below or arranged in a different order where technically possible
[0046] In step 1 either step 1a or 1b may be carried out. o
[0047] Step 1a: In step 1a compound
is converted with an amine R2NH2 preferably in the presence of a base in an organic solvent.
LG may be selected from Cl, Br, I or a sulfonate leaving group, such as mesylate, tosylate, nosylate, triflate, preferably Br.
R5 may be selected from (Ci-C6)alkyl, preferably methyl and ethyl.
Any suitable base may be used. Preferably a weaker base such as K2CO3, NaCO3 is used.
Preferably, the organic solvent is a polar-aprotic solvent, such as Diethylether, THF, DMF, DMSO, more preferably DMF.
Preferably, step 1a is carried out at room temperature.
Preferably, the reaction time in step 1a is 1 to 5 h, more preferably 2 h.
L ) 'R5 "Q
[0048] Step 1b: In step 1b compound n is converted with preferably in the presence of a base in an organic solvent.
LG may be selected from Cl, Br, I or a sulfonate leaving group, such as mesylate, tosylate, nosylate, triflate, preferably Br.
R5 may be selected from (C1-C6)alkyl, preferably methyl and ethyl.
Any suitable base may be used. Preferably a weaker base such as K2CO3, or NaCO3 is used.
Preferably, the organic solvent is a polar-aprotic solvent, such as Diethylether, THF, DMF, DMSO, more preferably DMF.
Preferably, step 1a is carried out at room temperature.
Preferably, the reaction time in step 1a is 1 to 5 h, more preferably 2 h.
H N'N =S R3 >
[0049] Wherein may be obtained by reaction of R3-CH2-NH-NH2 with (Isothiocyanatomethyl)benzene in an organic solvent in the presence of a base.
Preferably, the organic solvent is a polar protic solvent, such as ethanol.
Any suitable base may be used. Preferably a weaker base such as K2CO3, or NaCO3 is used. Preferably, the reaction temperature is 20 to 100 °C, more preferably 60 to 90 °C, most preferably 70 to 85 °C. o
[0050] Step 2: In step 2 the product obtained in step 1a is converted with
the presence of a suitable coupling reagent, a suitable base and optionally a catalyst for creating amid bonds between an amine and an carboxylic acid group in an organic solvent. A variety of reaction conditions for creating amid bonds are known in the art. If step 1b is carried out, step 2 is not carried out. The coupling reagent may be selected for example from 1 -Ethyl-3-(3- dimethylaminopropyl)carbodiimid (EDC) and dicyclohexylcarbodiimid (DCC), 1- [bis(Dimethylamin)methylen]-1/7-1,2,3-triazol[4,5-b]pyridinium-3-oxid-hexafluorophosphat (HATLI), preferably EDC. The catalyst may be selected for example from 4- dimethylaminopyridine (DMAP) and hydroxybenzotriazole (HOBt), preferably HOBt. The base is preferably selected from an organic amine base with low nucleophilicity, for example triethylamin, and diisopropylethylamin (DIPEA). The solvent is preferably an aprotic organic solvent, for example dichloromethane, and DMF, more preferably DMF. Preferably, the reaction is carried out at room temperature.
[0051] Step 3: The ester group in the product of step 1b and step 2 is hydrolized to the corresponding carboxyl group. Suitable conditions for this reaction type are generally known to
the person skilled in the art. Usually, the reaction is carried out in the presence of an inorganic base, water and an organic solvent. The inorganic base may be selected from LiOH, and NaOH, preferably LiOH. The organic solvent may be selected from, THF, ethanol, methanol, preferably THF.
HN R,
N^N
HN,
[0052] Step 4: The product of step 3 is reacted with R< under conditions as described for step 2. Wherein in step 4, if is piperidinyl, the nitrogen atom in piperidinyl is protected with a suitable protecting group, preferably Boc. In step 4, preferably HATLI is used as coupling reagent. Preferably, DI PEA is used as base. Preferably DMF is used as solvent.
NH2-CH2- I . Wherein, if RT is piperidinyl, the nitrogen atom in piperidinyl is protected with a suitable protecting group, preferably Boc. Reductive amination may be carried out in the presence of NaCNBH3, an organic acid such as acetic acid an in an polar-protic solvent such as an alcohol like methanol or ethanol.
Step 5: Remaining protecting groups such as Boc introduced in step 4 may be removed under conditions known in the art. Boc may be for example be removed under acidic conditions such as in the presence of an inorganic acid like hydrochlorid acid preferably in dioxane.
Pharmaceutical Composition and Medical Use
[0053] A further aspect of the present invention is directed to a pharmaceutical composition comprising at least one of the inventive compounds.
[0054] “Pharmaceutical composition" refers to one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
[0055] “Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[0056] "Therapeutically effective amount" is an amount of the inventive compounds or a combination of two or more such compounds, which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition. A therapeutically effective amount can also be an amount which is prophylactically effective. The amount which is therapeutically effective will depend upon the patient's size and gender, the condition to be treated, the severity of the condition and the result sought. For a given patient, a therapeutically effective amount can be determined by methods known to those of skill in the art.
[0057] A further embodiment of the invention is directed to the inventive compounds or pharmaceutical composition for use in medicine.
[0058] A further embodiment of the invention is directed to the inventive compounds or pharmaceutical composition for use in the treatment of cancer.
[0059] Preferably, the cancer is selected from K-Ras dependent cancers or from cancers wherein the KRAS gene is mutated.
[0060] Most preferably, the cancer is selected from glioma, breast cancer, colorectal cancer, pancreatic cancer, stomach cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, particularly preferred pancreatic cancer.
[0061] A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.
EXAMPLES OF THE INVENTION
1. Synthesis of Compounds
Synthesis of N-cyclopentyl-4-fluoro-N-(7-(((2-(methylamino)pyrimidin-4- yl)methyl)(piperidin-4-ylmethyl)amino)-7-oxoheptyl)benzamide (A-342a)
Scheme 1 : Synthesis of N-cyclopentyl-4-fluoro-N-(7-(((2-(methylamino)pyrimidin-4-yl)methyl)(piperidin-4- ylmethyl)amino)-7-oxoheptyl)benzamide (A-342a)
[0062] Step-1: Synthesis of compound (lnt.-8) (Ethyl 7-(cyclopentylamino)heptanoate)
To a solution of cyclopentanamine (1.0 g, 11.7mmol, 1.0eq) in DMF (10 ml) was added K2CO3 (3.24g, 22.5mmol,2.0eq) and resulting mixture was stirred at RT 1 h. After 1 h Ethyl 7- bromoheptanoate (3.31g, 14.10 mmol, 1.2 eq) was added dropwise at RT and resulting mixture was stirred at RT for 3 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by coloumn chromatography on silica gel (60-120 mesh size) using Dichloromethane:Methanol (2-5%) as eluent. The desire product isolated as solid (1.5 g, 52.9%). LCMS: 100%, ESI-MS 242.2 m/z [M+H]+. 1H NMR (400 MHz, Chloroform-d) 5 4.16-4.10(q, 2H), 3.49 (s, 1 H), 3.31-3.24(m, 1 H), 2.81-2.77(t, 2H), 2.31- 2.27 (t, 2H), 2.03-2.00 (m, 2H), 1.83-1.70 (m ,6H), 1.65-1.57 (m ,4H), 1.37-1.36(m ,4H), 1.30- 1.24 (m ,3H).
[0063] Step-2: Synthesis of compound (lnt.-10)
(Ethyl 7-(N- cyclopentylfluorobenzamido)heptanoate)
To a solution of Ethyl 7-(cyclopentylamino)heptanoate (lnt.-8) (0.750 g, 4.6 mmol, 1.0 eq) in DMF (10 ml) was added EDC.HCI (2.0 g,10.7 mmol, 2.0eq), DIPEA (2.7 ml, 16.0 mmol, 3.0 eq), HOBT(0.144 g,1.07 mmol, 0.2 eq) and 4-Fluorobenzoic acid (1.5 g, 6.42 mmol, 1.2eq) at RT. The reaction mixture was stirred at RT for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by coloumn chromatography on silica gel (60-120 mesh size) using Dichloromethane: Methanol (3-5%) as eluent. The desire product isolated as solid (1.4 g, 61.98%). LCMS: 100%, ESI-MS 364.36 m/z
SUBSTITUTE SHEET (RULE 26)
[M+H]+. 1H NMR (400 MHz, DMS0-d6) 6 7.41-7.68(t, 2H), 7.28-7.24 (t, 2H), 4.08-4.03(m, 2H), 3.19(s, 2H), 2.27 (s, 2H), 1.64-1.41 (m, 12H), 1.25-1.17 (m ,8H).
[0064] Step-3: Synthesis of compound (lnt.-11)
(7-(N-cyclopentyl-4-fluorobenzamido)heptanoic acid)
To a solution of Ethyl 7-(N-cyclopentyl-4-fluorobenzamido)heptanoate (lnt.-10) (1.4 g, 3.8 mmol, 1.0 eq) in THF:H2O (1 :1) (14 mL), LiOH (0.320 g,7.7 mmol, 2.0 eq) was added at RT. The mixure stirred at RT for 3 h. After completion of reaction, reaction mixture was diluted in water (50 ml), and extracted with ethyl acetate (1 x50ml). The aqeuous layer was acidify with 2N HCI untill pH~2, It was extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was directly used in next step without further purification. (1.0 g, 90%) LCMS: 100%, ESI-MS 336.34 m/z [M+H]+.
[0065] Step-4: Synthesis of compound (lnt.-13)
Tert-butyl 4-((7-(N-cyclopentyl-4-fluorobenzamido)-N-((2-(methylamino)pyrimidin-4- yl)methyl)heptanamido)methyl)piperidine-1 -carboxylate
To a solution of 7-(N-cyclopentyl-4-fluorobenzamido)heptanoic acid (lnt.-11) (0.10 g, 0.28 mmol, 1.0 eq) in DMF (1 ml) was added HATU (0.168 g, 0.4 mmol, 1.5 eq), DIPEA (0.14 ml, 0.8 mmol, 3.0 eq) followed by addition of tert-butyl 4-((((2-(methylamino)pyrimidin-4- yl)methyl)amino)methyl)piperidine-1-carboxylate (lnt.-12) (0.10 g, 0.28 mmol, 1.0 eq) at RT. The reaction mixuture stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x50 ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was directly used in next step. (0.230g, crude) LCMS: 47.70%, ESI-MS 653.7 m/z [M+H]+.
[0066] Step-5: Synthesis of compound (A-342a)
N-cyclopentyl-4-fluoro-N-(7-(((2-(methylamino)pyrimidin-4-yl)methyl)(piperidin-4- ylmethyl)amino)-7-oxoheptyl)benzamide
To a solution of tert-butyl 4-((7-(N-cyclopentyl-4-fluorobenzamido)-N-((2- (methylamino)pyrimidin-4-yl)methyl)heptanamido)methyl)piperidine-1 -carboxylate (lnt.-13)
(0.230 g, 0.3 mmol, 1.0 eq) in DCM (10 ml) was added 4N HCI in dioxane (2.5 ml) drop wise at RT. Reaction was stirred at RT for 2 h. After completion of reaction the reaction mixture was evaporated under vacuum and it was then purified by Prep.HPLC purification using 5 mm ABC + 0.1% NH3 in water: Acetonitrile as eluent. The desired product was isolated as white solid (35 mg, 20%). LCMS: 100%, ESI-MS 553.7 m/z [M+H]+. 1H NMR (400 MHz, DMSO-d6-High temp.350.5 K) 5 8.22-8.17 (d, 1 H), 7.39 (dd, J = 8.4, 5.5 Hz, 2H), 7.23 (t, J = 8.7 Hz, 2H),6.76- 6.66 (d, 1 H), 6.396 (d,1H), 4.38 (s, 2H), 4.04 (m,1 H), 3.21 (s, 4H), 3.08 (s, 4H), 2.93 (s, 2H), 2.83 (d, J = 4.9 Hz, 3H), 2.40 (d, J = 12.4 Hz, 2H), 2.28 (s, 1H), 1.75 - 1.65 (m, 6H), 1.50 (d, J = 29.2, 7H) 1.26 (d, J = 15.7 Hz, 4H), 1.05 (s, 2H).
[0067] Step-6: Synthesis of compound (lnt-15)
2-(methylamino)pyrimidine-4-carbaldehyde
4-(dimethoxymethyl)-N-methylpyrimidin-2-amine (2.0 g, 11.05 mmol, 1.0 eq) was taken in 3N HCI (20 ml). Reaction mixture was stirred at 50°C for 18 h. The progressed reaction was monitered by TLC. After completion of reaction, reaction mixture was neutralised by NaHCO3 solution untill pH- 8. The product was extracted with ethylacetate (2x200 ml). The combined SUBSTITUTE SHEET (RULE 26)
organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was directly used in next step without further purification. The desired product was isolated as yellow gum (1.25 g-crude).
[0068] Step-6a: Synthesis of compound (lnt.-12) tert-butyl 4-((((2-(methylamino)pyrimidin-4-yl)methyl)amino)methyl)piperidine- 1 - carboxylate
To a solution of 2-(methylamino)pyrimidine-4-carbaldehyde (lnt.-15) (1.0 g, 7.26 mmol, 1.0 eq) in Methanol (10 ml) was added Acetic acid (0.05 g, 0.729 mmol, 0.1 eq) and tert-butyl 4- (aminomethyl)piperidine-l -carboxylate (1.75g, 8.17 mmol, 1.5 eq) at RT. Reaction mixture was stirred at RT for 3-4 h. Sodium cyanoborohydride (0.68 g, 10.93 mmol, 1.5 eq) was added in reaction mixture at RT portion wise. After completion of reaction, the reaction mixture was quenched in ice water (100 ml), the resulting mixture was extracted with ethyl acetate (3 x 100 ml). The combined organic layer was washed with brine (200 ml), It was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by coloumn chromatography on silica gel (60-120 mesh size) using Dichloromethane:Methanol (2-5%) as eluent. The desire product isolated as semi-solid (0.65 g, 65.10%). LCMS: 91.2%, ESI-MS 336.36 m/z [M+H]+.
Synthesis of N-cyclopentyl-N-(7-(ethyl((2-(methylamino)pyrimidin-4-yl)methyl)amino)-7- oxoheptyl)-4-fluorobenzamide(A-442a)
Scheme 2: Synthesis of N-cyclopentyl-N-(7-(ethyl((2-(methylamino)pyrimidin-4-yl)methyl)amino)-7- oxoheptyl)-4-fluorobenzamide(A-442a)
[0069] Step-7: Synthesis of compound (A-442a)
N-cyclopentyl-N-(7-(ethyl((2-(methylamino)pyrimidin-4-yl)methyl)amino)-7-oxoheptyl)-4- fluorobenzamide
To a solution of 7-(N-cyclopentyl-4-fluorobenzamido)heptanoic acid (0.15 g, 0.44 mmol, 1.0 eq) in DMF (1 ml) was added HATLI (0.168 g, 0.4 mmol, 1.5 eq), DIPEA (0.14 ml, 0.8 mmol, 3.0 eq) followed by addition of 4-((ethylamino)methyl)-N-methylpyrimidin-2-amine (lnt.-12’) (0.11 g, 0.67 mmol, 1.5 eq) at RT. The reaction mixuture stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, and extracted with ethylacetate
SUBSTITUTE SHEET (RULE 26)
(3x50 ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was purified by Prep.HPLC purification using 5 mm ABC + 0.1% NH3 in waterAcetonitrile as eluent. The desired product was isolated as white solid (10 mg, 10%). LCMS: 100%, ESI-MS 484.28 m/z [M+HJ+. 1H NMR (400 MHz, DMSO-d6-High temp.350.5 K) 5 8.19 (s, 1 H), 7.39 (dd, J = 8.4, 5.5 Hz, 2H), 7.23 (t, J = 8.9 Hz, 2H), 6.70 (s, 1 H), 6.37 (s, 1 H), 4.37 (s, 2H), 4.04-4.02 (m,1H), 3.29 (s,2H), 3.21 (s, 2H), 2.83 (d, J = 4.9 Hz, 3H), 2.35-2.34 (m, 2H), 2.27 (s, 1 H), 1.85 - 1.60 (m, 6H), 1.60 - 1.39 (m, 6H), 1.28-1.13 (m, 4H), 1.09 (d, J = 30.8 Hz, 2H).
[0070] Step-6b: Synthesis of compound (lnt-12’)
4-((ethylamino)methyl)-N-methylpyrimidin-2-amine
To a solution of 2-(methylamino)pyrimidine-4-carbaldehyde (lnt.-15) (1.0 g, 7.26 mmol, 1.0 eq) in Methanol (10 ml) was added Acetic acid (0.05 g, 0.729 mmol, 0.1 eq) and Ethylamine hydrochloride (0.36 g, 8.19 mmol, 1.5 eq) at RT. Reaction mixture was stirred at RT for 3-4 h. Sodium cyanoborohydride (0.68 g, 10.93 mmol, 1.5 eq) was added in reaction mixture at RT portion wise. After completion of reaction, the reaction mixture was quenched in ice water (100 ml), the resulting mixture was extracted with ethyl acetate (3 x 100 ml). The combined organic layer was washed with brine (200 ml), it was then dried over Na2SO4 and concentrated under vacuum. The crude product was purified by coloumn chromatography on silica gel (60-120 mesh size) using Dichloromethane:Methanol (2-5%) as eluent. The desire product isolated as semi-solid (0.75 g, 68%). LCMS: 72.26%, ESI-MS 167.0 m/z [M+H]+.
Scheme 3: Synthesis of 4-fluoro-N-isopropyl-N-(7-(((2-(methylamino)pyrimidin-4-yl)methyl)(piperidin-4- ylmethyl)amino)-7-oxoheptyl)benzamide (A-342b)
[0071] Step-1: Synthesis of compound (lnt.-8)
Ethyl 7-(isopropylamino)heptanoate
To a solution of Propan-2-amine (1.0 g, 11.7mmol, 1.0 eq) in DMF was added K2CO3 (3.2 g, 22.5 mmol, 2.0 eq) and resulting mixture was stirred at RT for 1 h. After 1h Ethyl 7- bromoheptanoate (1.86g, 7.85 mmol, 0.668 eq) was added dropwise and resulting mixture was stirred at room temperature for 2.5 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml). The combined organic layer was dried over
SUBSTITUTE SHEET (RULE 26)
Na2SO4 and concentrated under vacuum. The crude product was purified by coloumn chromatography on silica gel (60-120 mesh size) using Dichloromethane:Methanol (2-5%) as eluent. The desire product isolated as solid (1.2 g, 32.9%). LCMS: 100%, ESI-MS 216.44 m/z
[M+HJ+.
[0072] Step-2: Synthesis of compound (lnt.-10)
Ethyl 7-(4-fluoro-N-isopropylbenzamido)heptanoate
To a solution of Ethyl 7-(isopropylamino)heptanoate (lnt.-8) (0.650 g, 4.6 mmol, 1.0 eq) in DMF was added EDC.HCI (1.7g, 9.2 mmol, 2.0 eq), DIPEA (2.4ml, 13.9 mmol, 3.0 eq), HOBT (0.125 g, 0.92 mmol, 0.2 eq) and 4-fluorobenzoic acid (0.990 g, 4.6 mmol, 1.0 eq). The mixture was stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by coloumn chromatography on silica gel (60-120 mesh size) using Dichloromethane: Methanol (2-5%) as eluent. The desire product isolated as solid (1.15 g, 73.39%). LCMS: 99%, ESI-MS 338.36 m/z [M+HJ+.
[0073] Step-3: Synthesis of compound (lnt.-11)
7-(4-fluoro-N-isopropylbenzamido)heptanoic acid
To a solution of Ethyl 7-(4-fluoro-N-isopropylbenzamido)heptanoate (lnt.-10) (1.15 g, 3.4 mmol, 1.0 eq) in THF:H2O (1:1) (12 ml), LiOH (0.286 g,6.8 mmol, 2.0 eq) was added. The mixure stirred at RT for 3 h. After completion of reaction, reaction mixture was diluted in water (50 ml), and extracted with ethyl acetate (1x50ml). The aqeuous layer was acidify with 2N HCI untill pH- 2, It was then extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was directly used in next step without further purification. (0.85g, 90%) LCMS: 100%, ESI-MS 310.30 m/z [M+H]+.
[0074] Step-4: Synthesis of compound (lnt.-13)
Tert-butyl 4-((7-(4-fluoro-N-isopropylbenzamido)-N-((2-(methylamino)pyrimidin-4- yl)methyl)heptanamido)methyl)piperidine-1 -carboxylate
To a solution of 7-(4-fluoro-N-isopropylbenzamido)heptanoic acid (0.10 g, 0.32 mmol, 1.0 eq) in DMF (1 ml) was added HATU (0.181 g, 0.2 mmol, 1.5 eq), DIPEA (0.14 ml, 0.8 mmol, 3.0 eq) followed by addition of tert-butyl 4-((((2-(methylamino)pyrimidin-4- yl)methyl)amino)methyl)piperidine-1 -carboxylate (lnt.-12) (0.108 g, 0.32 mmol, 1.0 eq) at RT. The reaction mixuture stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x50 ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was directly used in next step. (0.310 g-crude) LCMS: 73.63%, ESI-MS 627.67 m/z [M+H]+.
[0075] Step-5: Synthesis of compound (A-342b)
4-Fluoro-N-isopropyl-N-(7-(((2-(methylamino)pyrimidin-4-yl)methyl)(piperidin-4- ylmethyl)amino)-7-oxoheptyl)benzamide
To a solution of tert-butyl tert-butyl 4-((7-(4-fluoro-N-isopropylbenzamido)-N-((2-
(methylamino)pyrimidin-4-yl)methyl)heptanamido)methyl)piperidine-1 -carboxylate (0.310g, 0.14
SUBSTITUTE SHEET (RULE 26)
mmol, 1.0 eq) (lnt.-13) (0.230 g, 0.3 mmol, 1.0 eq) in DCM (10 ml) was added 4N HCI in dioxane (2.5 ml) drop wise at RT. Reaction was stirred at RT for 2 h. After completion of reaction the reaction mixture was evaporated under vacuum and it was the purified by Prep.HPLC purification using 5 mm ABC + 0.1% NH3 in waterAcetonitrile as eluent. The desired product was isolated as white solid (25 mg, 25%). LCMS: 100%, ESI-MS 527.68 m/z [M+HJ+. 1H NMR (400 MHz, DMSO-d6-High temp.350.5 K) 5 8.23-8.17(d, 1 H),7.38 (dd, J = 8.3, 5.4 Hz, 2H), 7.23 (dd, J = 10.0, 7.4 Hz, 2H), 6.77-6.66 (d, 1 H),6.40 (s, 1 H), 4.39 (s, 2H), 4.01 (s, 1 H), 3.21 (s, 4H), 3.08 (s, 4H), 2.93 (s, 2H), 2.82 (d, J = 4.8 Hz, 3H), 2.40 (d, J = 13.6 Hz, 2H), 2.28 (s, 1 H), 1.70-1.69 (m,1 H), 1.53 (s, 5H), 1.26 (d, J = 19.9 Hz, 3H), 1.17 (d, J = 6.7 Hz, 6H), 1.05 (s, 2H).
Synthesis of N-(7-(ethyl((2-(methylamino)pyrimidin-4-yl)methyl)amino)-7-oxoheptyl)-4- fluoro-N-isopropylbenzamide (A-442b)
Scheme 4: Synthesis of N-(7-(ethyl((2-(methylamino)pyrimidin-4-yl)methyl)amino)-7-oxoheptyl)-4-fluoro- N-isopropylbenzamide (A-442b)
[0076] Step-7: Synthesis of compound (A-442b)
N-(7-(ethyl((2-(methylamino)pyrimidin-4-yl)methyl)amino)-7-oxoheptyl)-4-fluoro-N- isopropylbenzamide
To a solution of 7-(4-fluoro-N-isopropylbenzamido)heptanoic acid (0.100 g, 0.32 mmol, 1.0 eq) in DMF (1 ml) was added HATLI (0.168 g, 0.4 mmol, 1.5 eq), DIPEA (0.14 ml, 0.8 mmol, 3.0 eq) followed by addition of 4-((ethylamino)methyl)-N-methylpyrimidin-2-amine (lnt.-12’) (0.11 g, 0.67 mmol, 1.5 eq) at RT. The reaction mixuture stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x50 ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was purified by Prep.HPLC purification using 5 mm ABC + 0.1 % NH3 in waterAcetonitrile as eluent. The desired product was isolated as white solid (10 mg, 11 %). LCMS: 100%, ESI-MS 458.57 m/z [M+HJ+. 1 H NMR (400 MHz, DMSO-d6-High temp.350.5 K) 5 8.19 (s, 1 H), 7.38 (dd, J = 8.4, 5.5 Hz, 2H), 7.23 (t, J = 8.7 Hz, 2H), 6.70 (s, 1 H), 6.37 (s, 1 H), 4.37 (s, 2H), 4.01 (s, 1 H), 3.39 (s, 2H), 3.20 (s, 2H), 2.83 (d, J = 4.9 Hz, 3H), 2.46-2.34 (m, 2H), 2.27 (s, 1 H), 1.54 (s, 4H), 1.28 (s, 4H), 1.17 (d, J = 6.7 Hz, 6H), 1.05-0.96 (m, 2H).
SUBSTITUTE SHEET (RULE 26)
Synthesis of compounds A342c and A442c
Synthesis of N-ethyl-4-fluoro-N-(7-(((2-(methylamino)pyrimidin-4-yl)methyl)(piperidin-4- ylmethyl)amino)-7-oxoheptyl)benzamide (A-342c)
Scheme 5: Synthesis of N-ethyl-4-fluoro-N-(7-(((2-(methylamino)pyrimidin-4-yl)methyl)(piperidin-4- ylmethyl)amino)-7-oxoheptyl)benzamide (A-342c)
[0077] Step-1: Synthesis of compound (lnt.-8)
Ethyl 7-(ethylamino)heptanoate
To a solution of Ethanamine (3.0 g, 66 mmol, 1.0 eq) in DMF (20 ml) was added K2CO3 (18.2 g, 132 mmol, 2.0eq) and resulting mixture was stirred at RT 1 h. After 1 h Ethyl 7- bromoheptanoate (7.2 g, 4.44 mmol, 0.668 eq) was added dropwise and resulting mixture was stirred at RT for 2.5 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by coloumn chromatography on silica gel (60-120 mesh size) using Dichloromethane:Methanol (5-7%) as eluent. The desire product isolated as solid (3.5 g, 35%).
1H NMR (400 MHz, DMSO-d6) 5 4.07-4.02 (m, 2H), 2.54-2.39 (m, 4H), 2.32-2.35 (m, 2H), 1.53- 1.50 (m, 2H), 1.38-1.35 (m, 2H), 1.272 (S, 4H), 1.20-1.16 (m, 3H), 1.017-0.982 (m, 3H).
SUBSTITUTE SHEET (RULE 26)
[0078] Step-2: Synthesis of compound (lnt.-10)
Ethyl 7-(N-ethyl-4-fluorobenzamido)heptanoate
To a solution of Ethyl 7-(ethylamino)heptanoate (1.50 g, 7.46 mmol, 1.0 eq) in DMF(15 ml) was added EDC.HCI (1.46 g, 10.44 mmol, 2.0 eq), DIPEA (2.26 g, 22.38 mmol, 3.0 eq), HOBT (0.20 g,1.49 mmol, 0.2 eq) and 4-Fluorobenzoic acid (1.46 g, 10.44 mmol, 1.4 eq). The mixture was stirred at RT for 16 h . After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by coloumn chromatography on silica gel (60-120 mesh size) using Dichloromethane:Methanol (2-5%) as eluent. The desire product isolated as solid (1.5 g, 62.00%). LCMS: 95%, ESI -MS 324.34 m/z [M+HJ+.
[0079] Step-3: Synthesis of compound (lnt.-11)
7-(N-ethyl-4-fluorobenzamido)heptanoic acid
To a solution of Ethyl 7-(N-ethyl-4-fluorobenzamido)heptanoate (lnt.-10) (1.5 g, 4.62 mmol, 1.0 eq) in THF:H2O (1 :1) (20 ml), LiOH (0.45 g, 9.2 mmol, 2.0 eq) was added. The mixure stirred at RT for 3 h. After completion of reaction, reaction mixture diluted in water (50 ml), extracted with ethyl acetate (1x50ml). The aqeuous layer was acidify with 2N HCI untill pH- 2, It was then extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was directly used in next step without further purification. (1.2 g, 87%). LCMS: 100%, ESI-MS 296.34 m/z [M+H]+.
[0080] Step-4: Synthesis of compound (lnt.-13)
Tert-butyl 4-((7-(N-ethyl-4-fluorobenzamido)-N-((2-(methylamino)pyrimidin-4- yl)methyl)heptanamido)methyl)piperidine-1-carboxylate
To a solution of 7-(N-ethyl-4-fluorobenzamido)heptanoic acid (0.200 g, 0.6 mmol, 1.0 eq) in DMF (5ml) was added HATU (0.386 g, I.Ommol, 1.5 eq), DI PEA (0.14 ml, 0.8 mmol, 3.0 eq) followed by addition of tert-butyl 4-((((2-(methylamino)pyrimidin-4- yl)methyl)amino)methyl)piperidine-1 -carboxylate (0.340 g, 1.01mmol, 1.5 eq). The reaction mixuture stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x50 ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was directly used in next step. (0.22 g, crude)
LCMS: 84.20%, ESI-MS 613.39 m/z [M+H]+.
[0081] Step-5: Synthesis of compound (A-342c)
N-ethyl-4-fluoro-N-(7-(((2-(methylamino)pyrimidin-4-yl)methyl)(piperidin-4- ylmethyl)amino)-7-oxoheptyl)benzamide
To a solution of tert-butyl 4-((7-(N-ethyl-4-fluorobenzamido)-N-((2-(methylamino)pyrimidin-4- yl)methyl)heptanamido)methyl)piperidine-1-carboxylate (lnt.-13) (0.22 g, 0.30 mmol, 1.0 eq) in DCM (10 ml) was added 4N HCI in dioxane (2.5 ml) drop wise at RT. Reaction was stirred at RT for 2 h. After completion of reaction the reaction mixture was evaporated under vacuum and it was the purified by Prep.HPLC purification using 5 mm ABC + 0.1% NH3 in waterAcetonitrile as eluent. The desired product was isolated as white solid (40 mg, 25%). LCMS: 100%, ESI-MS 513.18 m/z [M+H]+. 1H NMR (400 MHz, DMSO-d6-High temp.350.5 K) 5 8.22 (s, 1 H), 7.40 (t, J = 6.9 Hz, 2H), 7.23 (t, J = 8.8 Hz, 2H), 6.37 (s, 1H), 6.39 (s, 1H), 4.38 (s, 2H), 3.32 (m, 4H), 3.22 (d, J = 7.1 Hz, 2H), 3.078 (s, 4H), 2.94 (d, J = 12.1 Hz, 2H), 2.82 (d, J = 4.8 Hz, 3H), 2.45 (d, J =
12.9 Hz, 2H), 2.27 (s, 1 H), 1.70 (m, 1 H), 1.53 (s, 5H), 1.23 (s, 3H), 1.10 (t, J = 7.1 Hz, 3H), 1.08 (m, 1 H)
Synthesis of N-ethyl-N-(7-(ethyl((2-(methylamino)pyrimidin-4-yl)methyl)amino)-7- oxoheptyl)-4-fluorobenzamide(A-442c)
Scheme: Step-7: Synthesis of compound (A-442c)
[0082] To a solution of 7-(N-ethyl-4-fluorobenzamido)heptanoic acid (0.100 g, 0.32 mmol, 1.0 eq) in DMF (1 ml) was added HATLI (0.168 g, 0.4 mmol, 1.5 eq), DIPEA (0.14 ml, 0.8 mmol, 3.0 eq) followed by addition of 4-((ethylamino)methyl)-N-methylpyrimidin-2-amine (lnt.-12’) (0.11 g, 0.67 mmol, 1.5 eq) at RT. The reaction mixuture stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x50 ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was purified by Prep. HPLC purification using 5 mm ABC + 0.1% NH3 in waterAcetonitrile as eluent. The desired product was isolated as white solid (10 mg, 11%). LCMS: 100%, ESI-MS 444.02 m/z [M+HJ+. 1 H NMR (400 MHz, DMSO-d6-High temp.350.5 K) 5 8.20 (s, 1 H), 7.40 (t, J = 6.9 Hz, 2H), 7.23 (t, J = 8.7 Hz, 2H), 6.69 (s, 1 H), 6.37 (s, 1 H), 4.37 (s, 2H), 3.39-3.30 (m, 5H), 2.83 (d, J = 4.9 Hz, 3H), 2.27 (s, 1 H), 1.54 (s, 4H), 1.28 (s, 7H), 1.11 (t, J = 7.0 Hz, 5H).
Synthesis of 7-((4-benzyl-5-isobutyl-4H-1 ,2,4-triazol-3-yl)thio)-N-((2-
Scheme 6: Synthesis of 7-((4-benzyl-5-isobutyl-4H-1 ,2,4-triazol-3-yl)thio)-N-((2-(methylamino)pyrimidin- 4-yl)methyl)-N-(piperidin-4-ylmethyl)heptanamide (A-372)
[0083] Step-1: Synthesis of compound (lnt.-3)
4-Benzyl-5-isobutyl-2,4-dihydro-3H-1,2,4-triazole-3-thione
Isopentylhydrazine (0.600 g, 5.1 mmol, 1.0 eq) and (Isothiocyanatomethyl)benzene (0.770 g, 5.1 mmol, 1.0 eq) were taken in ethanol (10 ml) and refluxed for 2 h. The reaction mixture was cooled to RT and added K2CO3 (0.650 g, 5.1 mmol, 1.0 eq) in one portion. Reaction mixture was then refluxed for another 1 h. After completion of reaction, rection mixture was poured into water and neutralized by 1 N HCI untill pH - 7. The resulting solid was filtered and wash with water (50 ml). The desire product was isolated as solid (0.735 g, 50%). LCMS: 100%, ESI-MS 248.1 m/z [M+H]+ 1H NMR (400 MHz, DMSO-d6) 5, 7.31-7.39 (m,3H), 7.27 (d, J =4Hz ,2H), 5.30 (s, 2H), 2.369 (d, J =7.2 Hz, 2H), 1.80-2.02 (m,1 H), 0.753 (d, J =6.4 Hz, 6H).
[0084] Step-2: Synthesis of compound (lnt.-4)
Ethyl 7-((4-benzyl-5-isobutyl-4H-1,2,4-triazol-3-yl)thio)heptanoate
To a solution of 4-Benzyl-5-isobutyl-2,4-dihydro-3H-1 ,2,4-triazole-3-thione (0.300 g, 1.20mmol, 1.0 eq) and Ethyl 7-bromoheptanoate (0.287g,1.20mmol,1.0eq) in DMF (10ml) was added K2CO3 (0.225 g, 1.20 mmol, 1.0 eq) at RT. The reaction mixture was stirred at 50 °C for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum to give crude product. The desire product isolated as solid (0.40 g, 84.00%). LCMS: 97.20%, ESI-MS 404.4 m/z [M+H]+. 1H NMR (400 MHz, DMSO-D6) 5 7.303-7.359 (m, 3H), 7.223 (d, J = 6.8Hz, 2H) , 5.303(s,2H) , 4.145 (s, 2H), 4.036 (d, J =6.8 Hz ,2H), 2.337(s, 2H), 2.241 (t, J =6.8 Hz, 2H), 1.763 (s,4H), 1.507(s, 2H), 1.288 (s, 3H), 1.157 (t, J =6.4 Hz, 3H) ,0.798 (d, J =6.0 Hz, 6H).
SUBSTITUTE SHEET (RULE 26)
[0085] Step-3: Synthesis of compound (I nt. -5)
7-((4-Benzyl-5-isobutyl-4H-1,2,4-triazol-3-yl)thio)heptanoic acid
To a solution of Ethyl 7-((4-benzyl-5-isobutyl-4H-1 ,2,4-triazol-3-yl)thio)heptanoate (lnt.-4) (0.400 g, 0.99 mmol, 1.0 eq) in THF:H2O (1 :1) (16ml), LiOH (0.25 g, 1.8 mmol, 2.0 eq) was added. The mixure stirred at RT for 3 h. After completion of reaction, reaction mixture diluted with water (50 ml), extracted with ethyl acetate (1 x50ml). The aqueous layer was acidify with 2N HCI untill pH- 2, It was then extracted with ethylacetate (3x100ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was directly used in next step without further purification. (0.350 g, 94%). LCMS: 88%, ESI-MS 375.5.
[0086] Step-4: Synthesis of compound (I nt. -6)
Tert-butyl4-((7-((4-benzyl-5-isobutyl-4H-1,2,4-tnazol-3-yl)thio)-N-((2-
(methylamino)pyrimidin-4-yl)methyl)heptanamido)methyl)piperidine-1 -carboxylate
To a solution of 7-((4-benzyl-5-isobutyl-4H-1 ,2,4-triazol-3-yl)thio)heptanoic acid
(0.200 g, 0.53 mmol, 1.0 eq) in DMF was added HATLI (0.300g, 0.798 mmol, 1.5 eq), DIPEA (0.51ml, 0.16 mmol, 3.0 eq) followed by addition of tert-butyl 4-((((2-(methylamino)pyrimidin-4- yl)methyl)amino)methyl)pipe,ridine-1-carboxylate (0.214 g, 0.639 mmol, 1.2 eq). The reaction mixuture stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x50 ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was directly used in next step. (0.190g, 48%) LCMS: 100%, ESI-MS 694.0 m/z [M+H]+.
[0087] Step-5: Synthesis of compound (A-372)
7-((4-benzyl-5-isobutyl-4H-1,2,4-triazol-3-yl)thio)-N-((2-(methylamino)pyrimidin-4- yl)methyl)-N-(piperidin-4-ylmethyl)heptanamide
To a solution of tert-butyl 4-((7-((4-benzyl-5-isobutyl-4H-1 ,2,4-triazol-3-yl)thio)-N-((2- (methylamino)pyrimidin-4-yl)methyl)heptanamido)methyl)piperidine-1 -carboxylate (0.190g, 0.14 mmol, 1.0 eq) in DCM (10 ml) was added 4N HCI in dioxane (2.5 ml) drop wise at RT. Reaction was stirred at RT for 2 h. After completion of reaction the reaction mixture was evaporated under vacuum and it was then purified by Prep. HPLC purification using 5 mm ABC + 0.1 % NH3 in waterAcetonitrile as eluent. The desired product was isolated as white solid (10 mg, 22%). LCMS: 100% ESI-MS 593.2 m/z [M+H]+.
1H NMR (400 MHz, DMSO-d6-High temp.350.5 K) 68.22-8.13(d,1H), 7.41 - 7.27 (m, 3H), 7.07 (d, J = 7.4 Hz, 2H), 6.77 (s, 1 H), 6.37 (d, J = 19.5 Hz, 1 H), 5.18 (s, 2H), 4.39 (s, 2H), 3.22 (d, J = 7.0 Hz, 2H), 3.08-3.04 (s, 4H), 2.93 (s, 2H), 2.82 (d, J = 4.8 Hz, 3H), 2.41 (s, 2H), 2.38 (s, 2H), 2.28 (s, 1 H), 1.99 (dt, J = 13.4, 6.7 Hz, 1 H), 1.57 (d, 8H), 1.51 (s,4H) 1.05 (s, 2H), 0.90 (d, J = 6.6 Hz, 6H).
Synthesis of 7-((4-benzyl-5-isobutyl-4H-1,2,4-triazol-3-yl)thio)-N-ethyl-N-((2- (methylamino)pyrimidin-4-yl)methyl)heptanamide (A-472)
Scheme 7: Synthesis of 7-((4-benzyl-5-isobutyl-4H-1 ,2,4-triazol-3-yl)thio)-N-ethyl-N-((2- (methylamino)pyrimidin-4-yl)methyl)heptanamide (A-472)
[0088] Step-5: Synthesis of compound (A-472)
7-((4-benzyl-5-isobutyl-4H-1,2,4-triazol-3-yl)thio)-N-ethyl-N-((2-(methylamino)pyrimidin-4- yl)methyl)heptanamide
To a solution of 7-((4-benzyl-5-isobutyl-4H-1,2,4-triazol-3-yl)thio)heptanoic acid
(0.100 g, 0.266 mmol, 1.0 eq) in DMF (5 ml) was added HATLI (0.151 g, 0.399mmol, 1.5 eq), DIPEA (0.14 ml, 0.8 mmol, 3.0 eq) followed by addition of 4-((ethylamino)methyl)-N- methylpyrimidin-2-amine (lnt.-12’) (0.053 g, 0.319 mmol, 1.2 eq). The reaction mixuture stirred at room temperature for 16 h. After completion of reaction, rection mixture was poured into water, extracted with ethylacetate (3x50 ml). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting crude product was purified by Prep.HPLC purification using 5 mm ABC + 0.1% NH3 in water: Acetonitrile as eluent. The desired product was isolated as white solid (10 mg, 8%). LCMS: 100%, ESI-MS 524.0 m/z [M+HJ+.
1H NMR (400 MHz, DMSO-d6-High temp.350.5 K) 8.19 (d, 1 H), 7.43 - 7.26 (m, 3H), 7.07 (d, J = 7.4 Hz, 2H), 6.76-6.69(d, 1 H), 6.36 (d,1 H), 5.18 (s, 2H), 4.37 (s, 2H), 3.39 (s, 2H), 2.83 (d, J = 4.9 Hz, 3H), 2.34 (s,2H), 2.28(m, 1H), 2.02-1.97(m, 1H) 1.58 (d, J = 34.5 Hz, 4H), 1.28 (s, 6H), 1.12 (s, 4H), 0.90 (d, J = 6.7 Hz, 6H).
2. Biological Testing
[0089] Inhibition of 2D cell proliferation by the compounds of the present invention
K-Ras4B (hereafter K-Ras) is a major driver of cell proliferation. In cancer cell lines expressing constitutively active mutant K-Ras, uncontrolled proliferation typically depends on the former.
[0090] The anti-proliferative effect of the novel compounds as listed in Table 1 on KRAS mutant MDA-MB-231 (KRAS-G13D) breast cancer and MIA-PaCa-2 (KRAS-G12C) pancreatic cancer cell lines at various concentrations has been tested. Obtained IC50 values are given in Table 2. Dose response data were converted into the DSS3 summary score, which assessed the normalized area under the curve with the consideration of different tested concentration ranges (Figure 1). For comparison, the effect of Deltaflexin-1 and -2 compounds has been reassessed ([19] Siddiqui et al., 2020). While several compounds according to the present invention (A- 342a, A-342c, A-442a, and A-442c ) exhibited comparable activities to Deltaflexin-2, at least in one of the cell lines, two compounds showed the same activity in MDA-MB-231 and a significantly higher activity against MIA-PaCa-2 cells (A442b and A472). Of note, the activity of
these two compounds in the latter cell line reached the same or higher level of PDEdelta inhibitors Deltarasin and Deltazinone or of the potent farnesyltransferase inhibitor FTI-277. More impressively, however, these latter two compounds were as potent as AMG-510 the currently leading first direct K-Ras inhibitor that is on fast-track FDA approval after successful clinical trial results ([21] Hong et al., 2020).
[0091] Several new compounds display on-target activity in cellular BRET-assays
[0092] Further, it has been analysed whether the observed anti-proliferative activity correlates with on-target effects in cells. Two types of BRET-biosensors have been contructed to measure directly K-Ras displacement from PDEdelta (RLuc8-PDE6D/ GFP2-K-RasG12V) or the functional organization of K-Ras in nanoscale signaling complexes of the plasma membrane, called nanocluster (RLuc8-K-RasG12V/ GFP2- K-RasG12V). Nanoclustering-BRET decreases with the inhibition of any process upstream of nanoclustering, including improper posttranslational modification of Ras, trafficking defects and disrupted plasma membrane anchorage. BRET-biosensors were expressed in HEK293 EBNA cells to assess in cellulo on- target activity.
[0093] As expected, control compounds Deltarasin and Deltazinone robustly inhibited the BRET signal of RLuc8-PDE6D/ GFP2-K-RasG12V (Figure 2). In agreement with previous FRET data, Deltaflexin-1 and -2 also lowered the BRET-signal, albeit with lower potency than the former compounds. Importantly, several new compounds, including A342a, A442a and in particular A442b and A472 were more potent than Deltaflexin-1 and -2. Of note, A342c did not show an effect, suggesting that there could be off-target issues with the A3xx scaffold.
[0094] A similar activity profile was seen with the K-Ras nanoclustering-BRET biosensor (Figure 3). As expected, the farnesyltransferase inhibitor FTI-277 has little effect, in agreement with the alternative prenylation of K-Ras. However, the HMG-CoA inhibitor mevastatin robustly reduced the BRET-signal, typically indicative of non-prenylated, fully soluble K-Ras. When compounds were tested against the analogous H-Ras nanoclustering-BRET biosensor (Figure 4), most of them remained with little effect, except for A472, which showed a similar effect as Deltazinone, but not as much as Deltarasin.
Table 2. Effect of compounds on 2D cell viability
IC50 + SEM ( iM), n > 3
Compounds MIA PaCa-2 MDA-MB-231
Deltaflexin-1 66 ± 8 49 ± 14
Deltaflexin-2 98 ± 53 44 ± 34
A342a 23 ± 8 121 ± 36
A342b NA NA
A342c 86 ± 5 45 ± 25
A372 93 ± 9 NA
A442a 21 ± 6 62 ± 15
A442b 11 ± 5 86 ± 5
A442c 46 ± 15 162 ± 4
A472 4.3 ± 0.8 10.2 ± 0.1
Deltarasin 1.4 ± 0.3 1.2 ± 0.2
Deltazinone 6 ± 1 9 ± 2
FTI-277 (n=2) 27 ± 2 42 ± 13
AMG-510 0.02 ± 0.01 NA
ARS-1620 2 ± 1 35 ± 8
[0095] Expression constructs
All expression constructs were produced by multi-site Gateway cloning technology as described (Wall et al., 2014). Briefly, three entry clones with compatible LR recombination sites, encoding the CMV promoter, RLuc8 or GFP2 tag and a gene of interest with stop codon, either K-Ras4B- G12V, H-Ras-G12V or PDE6D, were obtained from Addgene. The three clones were inserted into a destination vector, pDest-305 or pDest-312, using Gateway LR Clonase II enzyme mix (#11791020, Thermo Fisher Scientific). The reaction mix was transformed into ccdB sensitive E.coli strain DH10B (#EC0113, Thermo Fisher Scientific) and positive clones were selected in the presence of ampicillin.
[0096] Cell culture
SUBSTITUTE SHEET (RULE 26)
HEK293 EBNA (HEK) cells were a gift of Prof. Florian M. Wurm, EPFL, Lausanne, Switzerland, and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza Pharma). MDA-MB- 231 (ATCC HTB-26) were maintained in Roswell Park Memorial Institute medium (RPMI) and MIA PaCa-2 (ATCC CRM-CRL-1420) in DMEM. All media were supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Lonza Pharma) (complete medium). Cells were grown at 37 °C in a water-saturated, 5% CO2 atmosphere and sub-cultured twice a week.
[0097] 2D cell viability assay
MDA-MB-231 and MIA PaCa-2 cells were plated in complete medium into 96-well cell culture plates (#655 180, Greiner bio-one, Merck KGaA) at a density of 1000 cells/ 100 .L and allowed to attach for 24 h. Test compounds were then added at indicated concentrations and DMSO (0.1% v/v) was used as a vehicle control. After 72 h incubation in the presence of the compounds, the cell viability was assessed using the alamarBlue reagent (#DAL1100, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, alamarBlue reagent was added to each well of the plate (10% final volume) and incubated for 4 h at 37 °C. Then, the fluorescence intensity was read at the excitation wavelength of 530 ± 10 nm and emission wavelength of 590 ± 10 nm using a CLARIOstar plate reader (BMG LABTECH GmbH). The obtained raw fluorescence intensity data were normalized to vehicle control (100% viability) and plotted against the compound concentration.
[0098] Drug sensitivity score analysis (DSS3)
To quantitatively profile the drug sensitivity with a more robust parameter than the IC50 or EC50 values, the drug sensitivity score (DSS) analysis was employed. DSS values are essentially normalized area under the curve (AUC) measures of dose-response inhibition data (Yadav et al., 2014). Drug response Excel data files containing raw fluorescence intensity measurements were prepared according to the example file obtained from the DSS pipeline website (https://breeze.fimm.fi/) and analyzed on the site (Potdar et al., 2020).
The output file provides several drug sensitivity measures including IC50 and AUC. We plotted the DSS3 value, which was calculated as DSS3 = DSS2 X2 X1
n cc where DSS2 is given by the equation DSS2 =
and DSS1 is given by the equation
[0099] DSS3 was employed as it takes drug responses over a wider concentration range into account, as compared to drugs that show increased response only at the higher end of the concentration range. After logistic fitting of the dose-response inhibition data, the AUC was determined as exact solution. A 10% minimal activity threshold (t) was set. The maximum (Cmax) and minimum (Cmin) concentrations used for screening of the inhibitors, with Cmax = x2 and x1 concentration with minimal activity t. The parameter a is the value of the top asymptote, which can be different from 100% inhibition as obtained with100 pM benzethonium chloride (#53751 , Merck KGaA) treatment.
SUBSTITUTE SHEET (RULE 26)
[00100] BRET assay
BRET assays were essentially performed as described in the literature (Bery et al., 2018; Lavoie et al., 2013). HEK293 EBNA cells were plated in 1 mL complete DMEM into 12-well cell culture plates (#665 180, Greiner bio-one, Merck KGaA) at a density of 150,000 to 200,000 cells/ mL and allowed to attach for 24 h. Then, RLuc8-tagged donor and GFP2-tagged acceptor constructs were transfected into cells using the jetPRIME transfection reagent (Polyplustransfection SA) following the manufacturer’s instructions. Each well was transfected with about 1 pg of plasmid DNA using 3 pL of jetPRIME reagent.
[00101] For BRET donor saturation titration experiments, the concentration of donor plasmid (50 ng) was kept constant and the concentration of acceptor plasmid was increased from 0 to 1000 ng. The empty pcDNA3.1 plasmid was used to top-up the total DNA load per transfection. 24 h after transfection, cells were treated with compounds or vehicle control (DMSO 0.1% v/v in complete medium) at specified concentrations for 24 h.
[00102] The cells from one well of a 12-well plate were collected, washed, and re-plated in PBS (#14190-094, Gibco, Thermo Fisher Scientific) on flat-bottom, white 96-well plates (#236108, Nunc, Thermo Fisher Scientific) as four technical repeats containing 90 pL of cell suspension per well. Then fluorescence intensity followed by BRET readings were carried out on a CLARIOstar (BMG LABTECH GmbH) plate reader at 25 °C. The fluorescence intensity (RFU) of GFP2 was measured with excitation at 405 ± 10 nm and emission 515 ± 10 nm ; it is proportional to the acceptor concentration [Acceptor], BRET readings were taken well by well by adding 10 pL of 100 pM coelenterazine 400a (DeepBlueC, #C-320, Gold Biotechnology) RLuc8 substrate to each well (final concentration of 10 pM) using the injector present in the plate reader. Luminescence emission intensities were simultaneously recorded at 410 ± 40 nm (RLU, proportional to [Donor]) and 515 ± 15 nm (BRET-signal).
[00103] The raw BRET ratio was calculated as the BRET signal measured at 515 nm divided by emission signal measured at 410 nm (RLU). The BRET ratio was obtained by subtracting the raw BRET ratio by a background BRET signal measured for cells expressing only the donor.
The BRET ratio was calculated using the formula
'Icrn 515 i l-(Donor+Acceptor) .GTH 5157l77l(£)onor only)
BRET ratio = - r— - - - -
AC771410 l'irn(j)Onor+Acceptor) em 410 TT-TTT- (jjonor only) with Donor+Acceptor denoting cells transfected with a BRET pair and Donor only being cells expressing only the donor. The relative expression of acceptor relative to donor ([Acceptor]/[Donor]) was determined as,
[00104] For BRET donor saturation titration experiments, the BRET ratio was plotted against the ratio of acceptor to donor plasmid amounts (A/D plasmid ratio) that were transfected or the relative expression. The A/D plasmid ratio at which the BRET ratio changes most linearly
with the relative expression was determined for each BRET sensor and then used for compound profiling.
[00105] Statistical Analysis
For statistical analysis, the GraphPad Prism (version 8.00 for Windows, GraphPad Software) was used. The sample size n represents the number of independent biological repeats and is indicated in the respective figure legends. All graphs show mean values +/- SEM across all technical and biological repeats. Unless otherwise stated, we employed one-way ANOVA with Tukey’s multiple comparison test or student’s t-test to determine statistical differences to control samples. A p value of < 0.05 is considered statistically significant. Statistical significance levels are annotated in the plots as * = p < 0.05; ** = p < 0.01 ; *** = p <0.001.
REFERENCES
1. Papke, B., and Der, C.J. (2017). Drugging RAS: Know the enemy. Science 335, 1158- 1163
2. Lerner, E.C., Zhang, T.T., Knowles, D. B., Qian, Y. Hamilton, A.D., and Sebti, S.M. (1997). Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidometrics and requires both of a farnesyltransferase and a geranylgeranyl transferase I inhibitor in human tumor cell lines. Oncogene 15, 1283-1288.
3. Spiegel, J., Cromm, P.M., Zimmermann, G., Grossmann, T.N., and Waldmann, H. (2014). Small-molecule modulation of Ras signaling. Nat Chem Biol 10, 613-622.
4. Chandra, A., Grecco, H.E., Pisupati, V., Perera, D., Cassidy, L., Skoulidis, F., Ismail,
5.A., Hedberg, C., Hanzal-Bayer, M., Venkitaraman, A.R., et al. (2011). The GDI-like solubilizing factor PDEb sustains the spatial organization and signalling of Ras family proteins. Nat Cell Biol 14, 148-158.
5. Dharmaiah, S., Bindu, L., Tran, T.H., Gillette, WK., Frank, P.H., Ghirlando, R., Nissley,
D.V., Esposito, D., McCormick, F, Stephen, A.G., et al. (2016). Structural basis of recognition of farnesylated and methylated KRAS4b by PDEb. Proceedings of the National Academy of Sciences 113, E6766-E6775.
6. Schmick, M., Vartak, N., Papke, B., Kovacevic, M., Truxius, D.C., Rossmannek, L., and Bastiaens, P.I.H. (2014). KRas localizes to the plasma membrane by spatial cycles of solubilization, trapping and vesicular transport. Cell 157, 459-471.
7. Ismail, S.A., Chen, Y.-X., Rusinova, A., Chandra, A., Bierbaum, M., Gremer, L., Triola, G., Waldmann, H., Bastiaens, P.I.H., and Wittinghofer, A. (2011). Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo. Nat Chem Biol 7, 942-949.
8. Zimmermann, G., Papke, B., Ismail, S., Vartak, N., Chandra, A., Hoffmann, M., Hahn, S.A., Triola, G., Wittinghofer, A., Bastiaens, P.I.H., et al. (2013). Small molecule inhibition of the KRAS-PDEb interaction impairs oncogenic KRAS signalling. Nature 497, 638-642.
9. Papke, B., Murarka, S., Vogel, H.A., Martin-Gago, P, Kovacevic, M., Truxius, D.C., Fansa,
E.K., Ismail, S., Zimmermann, G., Heinelt, K., et al. (2016). Identification of pyrazolopyridazinones as PDEb inhibitors. Nat Commun 7, 11360.
10. Kiuru, E., Ahmed, Z., Ldnnberg, H., Beigelman, L., Ora, M. J. Org. Chem. 2013, 78, 950-959.
11. Posada, I.M.D., Lectez, B., Sharma, M., Oetken-Lindholm, C., Yetukuri, L., Zhou, Y, Aittokallio, T., and Abankwa, D. (2017a). Rapalogs can promote cancer cell sternness in vitro in a Galectin-1 and H-ras-dependent manner. Oncotarget 8, 44550-44566.
12. Posada, I.M.D., Lectez, B., Siddiqui, F.A., Oetken-Lindholm, C., Sharma, M., and Abankwa, D. (2017b). Opposite feedback from mTORCI to H-ras and K-ras4B downstream of
SREBP1. Sci. Rep. 7, 8944.
13. Prakash, P, Sayyed-Ahmad, A., Cho, K.-J., Dolino, D.M., Chen, W., Li, H., Grant, B.J., Hancock, J.F., and Gorfe, A.A. (2017). Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers. Sci. Rep. 7, 40109.
14. Blazevits O, Mideksa YG, Solman M, Ligabue A, Ariotti N, Nakhaeizadeh H, Fansa EK, Papageorgiou AC, Wittinghofer A, Ahmadian MR, Abankwa D. Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering. Sci Rep. 2016 Apr 18;6:24165. doi: 10.1038/srep24165. PubMed PMID: 27087647; PubMed Central PMCID: PMC4834570.
15. Guzman, C., Oetken-Lindholm, C., and Abankwa, D. (2016). Automated High-Throughput Fluorescence Lifetime Imaging Microscopy to Detect Protein-Protein Interactions. J. Lab. Autom.
16. Najumudeen, A.K., Jaiswal, A., Lectez, B., Oetken-, C., Lacey, E., Aittokallio, T., Abankwa, D., Oetken-Lindholm, C., Guzman, C., Siljamaki, E., et al. (2016). Cancer stem cell drugs target K-ras signaling in a. Oncogene 28-54.
17. Chandra, A., Grecco, H.E., Pisupati, V., Perera, D., Cassidy, L., Skoulidis, F, Ismail, S.A., Hedberg, C., Hanzal-Bayer, M., Venkitaraman, A.R., et al. (2011). The GDI-like solubilizing factor PDEb sustains the spatial organization and signalling of Ras family proteins. Nat. Cell Biol. 14, 148-158.
18. Jiang Y et al., (2017). Structural Biology-Inspired Discovery of Novel KRAS-PDEb Inhibitors. J. Med. Chem. 60: 9400-9406.
19. Siddiqui FA et al., (2020). PDE6D Inhibitors with a New Design Principle Selectively Block K-RAS Activity. ACS Omega. 5: 832-842.
20. Winzker M et al., (2020). Development of a PDEb-Targeting PROTACs that Impair Lipid Metabolism. Angew.
21. Hong, D.S., Fakih, M.G., Strickler, J.H., Desai, J., Durm, G.A., Shapiro, G.I., Falchook, G.S., Price, T.J., Sacher, A., Denlinger, C.S., et al. (2020). KRAS(G12C) Inhibition with Sotorasib in Advanced Solid Tumors. N Engl J Med 383, 1207-1217.
Claims
RT is selected from the group consisting of unsubstituted piperidinyl and unsubstituted (C C5)alkyl, preferably methyl;
R2 is selected from the group consisting of unsubstituted (C1-C6)alkyl, and unsubstituted (C3- C8)cycloalkyl;
R3 is selected from the group consisting of unsubstituted (C1-C6)alkyl, preferably isobutyl;
R4 is selected from the group consisting of unsubstituted (C1-C6)alkyl, preferably (C C^alkyl, more preferably methyl;
X is selected from the group consisting of F, Cl, Br, and I, preferably F; n is an integer between 1 and 10, preferably between 2 and 8, more preferably 6; and a solvate, hydrate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.
2. The compound of claim 1 , wherein i) X is in para position and/or ii) X is F and/or iii) R2 is selected from the group consisting of cyclopropyl, sec-propyl, and ethyl, preferably sec-propyl and/or iv) R3 is isobutyl and/or
v) R4 is methyl and/or vi) RT is unsubstituted (Ci-C5)alkyl, preferably methyl.
7. The compound according to any one of claim 1 to 6, for use in medicine.
8. The compound according to any one of the claims 1 to 7, for use in the treatment of cancer.
9. The compound for use according to claim 8, wherein the cancer is selected from K-Ras dependent cancers, preferably from cancers wherein the K-Ras gene is mutated.
10. The compound for use according to claims 8 or 9, wherein the cancer is selected from glioma, breast cancer, colorectal cancer, pancreatic cancer, stomach cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, preferably pancreatic cancer.
11. A pharmaceutical composition, comprising the compound according to any one of the claims 1 to 10 and at least one pharmaceutically acceptable carrier.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
LU102135A LU102135B1 (en) | 2020-10-16 | 2020-10-16 | Inhibitors of PDEdelta |
LULU102135 | 2020-10-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022079226A1 true WO2022079226A1 (en) | 2022-04-21 |
Family
ID=73834576
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2021/078585 WO2022079226A1 (en) | 2020-10-16 | 2021-10-15 | Inhibitors of pdedelta |
Country Status (2)
Country | Link |
---|---|
LU (1) | LU102135B1 (en) |
WO (1) | WO2022079226A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
LU502224B1 (en) * | 2022-06-08 | 2023-12-11 | Univ Luxembourg | Inhibitors of PDE6D |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015189433A1 (en) | 2014-06-13 | 2015-12-17 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Pyridazinones for the treatment of cancer |
-
2020
- 2020-10-16 LU LU102135A patent/LU102135B1/en active IP Right Grant
-
2021
- 2021-10-15 WO PCT/EP2021/078585 patent/WO2022079226A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015189433A1 (en) | 2014-06-13 | 2015-12-17 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Pyridazinones for the treatment of cancer |
Non-Patent Citations (26)
Title |
---|
AITTOKALLIO, T.ABANKWA, D.: "Rapalogs can promote cancer cell sternness in vitro in a Galectin-1 and H-ras-dependent manner", ONCOTARGET, vol. 8, 2017, pages 44550 - 44566 |
BJ?RN PAPKE ET AL: "Identification of pyrazolopyridazinones as PDE[delta] inhibitors", NATURE COMMUNICATIONS, vol. 7, 20 April 2016 (2016-04-20), pages 11360, XP055348695, DOI: 10.1038/ncomms11360 * |
CHANDRA, A., GRECCO, H.E., PISUPATI, V., PERERA, D., CASSIDY, L., SKOULIDIS, F., ISMAIL,S.A.,C.,HANZAL-BAYER,M.,VENKITARMAN,A.R.: "The GDI-like solubilizing factor POPE sustains the spatial organization and signalling of Ras family proteins. ", NAT CELL BIOL, vol. 14, 2011, pages 148 - 158 |
CHENG JUNFEI ET AL: "Discovery of Novel PDE[delta] Degraders for the Treatment of KRAS Mutant Colorectal Cancer", vol. 63, no. 14, 23 July 2020 (2020-07-23), US, pages 7892 - 7905, XP055798054, ISSN: 0022-2623, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/acs.jmedchem.0c00929> DOI: 10.1021/acs.jmedchem.0c00929 * |
ESPOSITO, D.MCCORMICK, F.STEPHEN, A.G. ET AL.: "Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 113, 2016, pages E6766 - E6775 |
GUNTHER ZIMMERMANN ET AL: "Small molecule inhibition of the KRAS-PDE[delta] interaction impairs oncogenic KRAS signalling", NATURE, vol. 497, no. 7451, 22 May 2013 (2013-05-22), London, pages 638 - 642, XP055335434, ISSN: 0028-0836, DOI: 10.1038/nature12205 * |
GUZMAN, C.OETKEN-LINDHOLM, C.ABANKWA, D.: "Automated High-Throughput Fluorescence Lifetime Imaging Microscopy to Detect Protein-Protein Interactions", J. LAB., 2016 |
HANCOCK, J.F.GORFE, A.A.: "Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers", SCI. REP., vol. 7, 2017, pages 40109 |
HEDBERG, C.HANZAL-BAYER, M.VENKITARAMAN, A.R. ET AL.: "The GDI-like solubilizing factor POPE sustains the spatial organization and signalling of Ras family proteins", NAT. CELL BIOL., vol. 14, 2011, pages 148 - 158 |
ISMAIL, S.A.CHEN, Y.-X.RUSINOVA, A.CHANDRA, A.BIERBAUM, M.GREMER, L.TRIOLA, G.WALDMANN, H.BASTIAENS, P.I.H.WITTINGHOFER, A.: "Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo", NAT CHEM BIOL, vol. 7, 2011, pages 942 - 949 |
JIANG Y ET AL.: "Structural Biology-Inspired Discovery of Novel KRAS-POPE Inhibitors", J. MED. CHEM., vol. 60, 2017, pages 9400 - 9406, XP055490026, DOI: 10.1021/acs.jmedchem.7b01243 |
JOCHEN SPIEGEL ET AL: "Small-molecule modulation of Ras signaling", NATURE CHEMICAL BIOLOGY, vol. 10, no. 8, 15 June 2014 (2014-06-15), New York, pages 613 - 622, XP055231387, ISSN: 1552-4450, DOI: 10.1038/nchembio.1560 * |
KIURU, E.AHMED, Z.LONNBERG, H.BEIGELMAN, L.ORA, M., J. ORG. CHEM., vol. 78, 2013, pages 950 - 959 |
LERNER, E.C., ZHANG, T.T., KNOWLES, D.B., QIAN, Y. HAMILTON, A.D., AND SEBTI, S.M.: "Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidometrics and requires both of a farnesyltransferase and a geranylgeranyl transferase I inhibitor in human tumor cell lines", ONCOGENE, vol. 15, 1997, pages 1283 - 1288 |
NAJUMUDEEN, A.K.JAISWAL, A.LECTEZ, B.OETKEN-, C.LACEY, E.AITTOKALLIO, T.ABANKWA, D.OETKEN-LINDHOLM, C.GUZMAN, C.SILJAMAKI, E. ET A: "Cancer stem cell drugs target K-ras signaling in a", ONCOGENE, 2016, pages 28 - 54 |
PAPAGEORGIOU ACWITTINGHOFERAAHMADIAN MRABANKWA D: "Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering", SCI REP., vol. 6, 18 April 2016 (2016-04-18), pages 24165 |
PAPKE, B.DER, C.J.: "Drugging RAS: Know the enemy", SCIENCE, vol. 335, pages 1158 - 1163 |
PAPKE, B.MURARKA, S.VOGEL, H.A.MARTIN-GAGO, P.KOVACEVIC, M.TRUXIUS, D.C.FANSA, E.K.ISMAIL, S.ZIMMERMANN, G.HEINELT, K. ET AL.: "Identification of pyrazolopyridazinones as POPE inhibitors", NAT COMMUN, vol. 7, 2016, pages 11360, XP055348695, DOI: 10.1038/ncomms11360 |
POSADA, I.M.D.LECTEZ, B.SIDDIQUI, F.A.OETKEN-LINDHOLM, C.SHARMA, M.ABANKWA, D.: "Opposite feedback from mTORC1 to H-ras and K-ras4B downstream of SREBP1", SCI. REP., vol. 7, 2017, pages 8944 |
PRICE, T.J.SACHER, A.DENLINGER, C.S. ET AL.: "KRAS(G12C) Inhibition with Sotorasib in Advanced Solid Tumors", N ENGL J MED, vol. 383, 2020, pages 1207 - 1217 |
SCHMICK, M.VARTAK, N.PAPKE, B.KOVACEVIC, M.TRUXIUS, D.C.ROSSMANNEK, L.BASTIAENS, PI.H.: "KRas localizes to the plasma membrane by spatial cycles of solubilization, trapping and vesicular transport", CELL, vol. 157, 2014, pages 459 - 471, XP028847313, DOI: 10.1016/j.cell.2014.02.051 |
SIDDIQUI FA ET AL.: "PDE6D Inhibitors with a New Design Principle Selectively Block K-RAS Activity", ACS OMEGA, vol. 5, 2020, pages 832 - 842 |
SPIEGEL, J., CROMM, P.M., ZIMMERMANN, G., GROSSMANN, T.N., AND WALDMANN, H.: "Small-molecule modulation of Ras signaling", NAT CHEM BIOL, vol. 10, 2014, pages 613 - 622, XP055231387, DOI: 10.1038/nchembio.1560 |
WINZKER M ET AL.: "Development of a PDEO-Targeting PROTACs that Impair Lipid Metabolism", ANGEW, 2020 |
YAN JIANG ET AL: "Structural Biology-Inspired Discovery of Novel KRAS-PDE[delta] Inhibitors", JOURNAL OF MEDICINAL CHEMISTRY, vol. 60, no. 22, 22 November 2017 (2017-11-22), US, pages 9400 - 9406, XP055490026, ISSN: 0022-2623, DOI: 10.1021/acs.jmedchem.7b01243 * |
ZIMMERMANN, G.PAPKE, B.ISMAIL, S.VARTAK, N.CHANDRA, A.HOFFMANN, M.HAHN, S.A.TRIOLA, G.WITTINGHOFER, A.BASTIAENS, P.I.H. ET AL.: "Small molecule inhibition of the KRAS-POPE interaction impairs oncogenic KRAS signalling", NATURE, vol. 497, 2013, pages 638 - 642, XP055335434, DOI: 10.1038/nature12205 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
LU502224B1 (en) * | 2022-06-08 | 2023-12-11 | Univ Luxembourg | Inhibitors of PDE6D |
Also Published As
Publication number | Publication date |
---|---|
LU102135B1 (en) | 2022-04-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9840464B2 (en) | IAP binding compounds | |
AU2021200421B2 (en) | Novel piperazine and piperidine derivatives, their synthesis and use thereof in inhibiting VDAC oligomerization, apoptosis and mitochondria dysfunction | |
RU2267488C2 (en) | Acridine derivatives, method for their preparing and pharmaceutical composition based on thereof | |
WO2017007612A1 (en) | Methods to induce targeted protein degradation through bifunctional molecules | |
EP3392261B1 (en) | Steroidal compound, composition containing the same and use thereof | |
KR20220079866A (en) | therapeutic conjugate | |
LU102135B1 (en) | Inhibitors of PDEdelta | |
EP4342899A1 (en) | Salt form and crystal form of heterocyclic substituted purinone derivative | |
Cai et al. | Design and synthesis of proteolysis-targeting chimeras (PROTACs) as degraders of glutathione peroxidase 4 | |
US10787423B2 (en) | Piperazine and piperidine derivatives, their synthesis and use thereof in inhibiting VDAC oligomerization, apoptosis and mitochondria dysfunction | |
Wuest et al. | Fluorine-and rhenium-containing geldanamycin derivatives as leads for the development of molecular probes for imaging Hsp90 | |
LU502224B1 (en) | Inhibitors of PDE6D | |
Jiangzhou et al. | Discovery of proqodine A derivatives with antitumor activity targeting NAD (P) H: quinone oxidoreductase 1 and nicotinamide phosphoribosyltransferase | |
LU101206B1 (en) | PDEdelta Inhibitors | |
US20220220120A1 (en) | Crystal form of wee1 inhibitor compound and use thereof | |
US20240254145A1 (en) | Salt form and crystal form of heterocyclic substituted purinone derivative | |
WO2024026262A1 (en) | Substituted pyrazolyl-pyridinyl compounds as ligand directed degraders of irak3 | |
WO2024145315A1 (en) | Targeted protein degraders of indoleamine 2,3-dioxygenase 1 (ido1) | |
CN114716436A (en) | KRAS G12C mutation inhibitor and application thereof | |
CN114957219A (en) | Degradation agent for targeted degradation of Gli1 protein and preparation method and application thereof | |
KR20210091865A (en) | N-phenylbenzothiazol-2-amine Compounds Having Vascular Tube Formation Inhibition Effect and Pharmaceutical Composition Comprising the Same | |
Du et al. | Discovery of Novel Egfr-Protacs Overcoming Various Types of Acquired Drugs Resistant |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21801837 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 21801837 Country of ref document: EP Kind code of ref document: A1 |