LU502224B1 - Inhibitors of PDE6D - Google Patents

Abstract

The present invention relates to compounds which show activity as PDE6D Inhibitors and pharmaceutical compositions comprising these compounds, their use in medicine as well as their use in the treatment of cancer.

Description

New Patent Application

Applicant: UNIVERSITE DU LUXEMBOURG

Our Ref.: LUX17597LU

LU502224

Inhibitors of PDE6D

The work leading to this invention was supported by the Luxembourg Fonds National de

Recherche (FNR) under Grant Nr. Grant Nr. PoC20/15269106-inhibitPDE-RASv2.

TECHNICAL FIELD OF THE INVENTION

[001] The present invention relates to compounds which show activity as PDE©D/ PDEdelta- 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 |, reinstating Ras plasma membrane 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 PDE6D, also called PDEdelta, is a trafficking chaperone of farnesylated proteins, suggesting that its inhibition affects the same clients as inhibition of farnesyltransferase. However, PDE6D cannot facilitate intracellular diffusion of proteins that are in addition palmitoylated ([4] Chandra et al., 2011; [5] Dharmaiah et al, 2016). Thus, PDE6©D inhibition selectively affects K-Ras4B (herafter K-Ras) trafficking but has much less effect on trafficking of dual-palmitoylated 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 PDE6D-assisted diffusion from internal cellular membranes ([4] Chandra et al., 2011;[6] Schmick et al., 2014). Unloading of K-Ras from PDE6D in the perinuclear compartment requires the binding of GTP-Arl2 to PDE6D, which results in an allosteric conformational change in PDE6D that effectively releases its cargo ([7] Ismail et al., 2011). Unfortunately, this ejection mechanism also applies to the first two generations of PDEGD inhibitors Deltarasin ([8] Zimmermann et al., 2013) and Deltazinone ([9] Papke et al., 2016;

WO2015189433A1).

[005] Only the last generation of PDE6D 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 also bound with nanomolar affinity, but again had only micromolar cellular activity ([18] Jiang et al., 2017).

[007] Another group of competitive PDEGD 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. 1

[008] Another class of recent inhibitors developed by both the Waldmann and Sheng gro RE 502224 are proteolysis-targeting chimeras (PROTACs). These heterobifunctional compounds bind t©

PDEGD 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 PDEGD Inhibitors.

SUMMARY OF THE INVENTION

[0010] The present invention relates to a compound according to formula (I)

O

Ans A n J Ri

B

(D wherein

R, is selected from the group consisting of unsubtituted (C--Cs)alkyl, preferably methyl; 0

OT

- LA RP

À is selected from the group consisting of X ;

La

B is selected from the group consisting of , ", E;

Ra is selected from the group consisting of unsubstituted (C--Cs)alkyl, and unsubtituted (Cs-

Cs)cycloalkyl;

X is selected from the group consisting of F, CI, Br, and |, preferably F;

Rs x Rs

Re D

J is selected from the group consisting of Rs ;

Rs, Ra, Rs, Rs are independently selected from the group consisting of hydrogen, -NH; and -

NOz;

D is selected from the group consisting of -COOH, -COO(C4-Cs)alkyl, -C(O)(C--Cs)alkyl, -

C(O)(Ca-Cs)alkenyl and halogen, preferably —-COOH, -COOCHz, and Br,

E is -COO(Cy-Cs)alkyl; nis 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

[0011] In a second aspect, the invention is directed to a pharmaceutical composition, 502224 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 low nanomolar affinity (Table 2), cellular on-target activity (Figure 1) and improved K-Ras4B (hereafter K-Ras) activity (Figure 2), but also H-Ras activity (Figure3). Activity of some compounds against UNC119A, which is related structurally to

PDE6D, was found (Figure 4). As compared to Deltazinone, pERK and pS6 levels, indicative of inhibition of the MAPK- and mTORC1-pathways, were stronger reduced by some compounds (Figures 5,6). In agreement with this, activity in cells was higher than of previous compounds (Figure 7a) with improved selectivity for PDE6D-dependent and KRAS-mutant cancer cell lines (Figure 7b).

BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1: Cellular PDE6D / K-Ras BRET data showing on-target activity of compounds of the present invention. HEK293 EBNA cells were co-transfected with RLuc8- tagged PDE6D and GFP2-tagged K-Ras4B-G12V (donor : acceptor plasmid ratio 1 : 20). 24 h after transfection, cells were treated for 24 h with 0.1 % DMSO (control) or various concentrations of compounds and three reference PDEGD inhibitors (Deltarasin, Deltazinone1,

Deltasonamide1). Tested concentrations are in coded on the bottom in grey scale, with look-up- table on the top. Values represent the mean + SEM of 3-4 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.

[0016] Figure 2: Cellular K-Ras nanoclustering-BRET data to assess K-Ras selectivity of compounds of the present invention. HEK293 EBNA cells were co-transfected with RLuc8- and GFP2-tagged K-Ras4B-G12V (donor : acceptor plasmid ratio 1 : 5). 24 h after transfection, cells were treated for 24 h with 0.1% DMSO (control) or indicated concentrations of compounds, including a farnesylation inhibitor (FTI-277) as control and the reference PDEGD inhibitors (Deltarasin, Deltazinone1, Deltasonamide1). Tested concentrations are in coded on the bottom in grey scale, with look-up-table on the top. Concentrations for FTI-277 are to the left. Values represent the mean + SEM of 3-4 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 H-Ras nanoclustering-BRET data to assess H-Ras effect of compounds of the present invention. HEK293 EBNA cells were co-transfected with RLuc8- and GFP2-tagged H-Ras-G12V (donor : acceptor plasmid ratio 1 : 3). 24 h after transfection, cells were treated for 24 h with 0.1% DMSO (control) or indicated concentrations of compounds, including a farnesylation inhibitor (FTI-277) as control and the reference PDEGD inhibitors (Deltarasin, Deltazinone1, Deltasonamide1). Tested concentrations are in coded on the bottom in grey scale, with look-up-table on the top. Concentrations for FTI-277 are to the left. Values 3 represent the mean + SEM of 3-4 independent biological repeats. Statistical significance levels) 502224 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 UNC119A/ Src BRET data to evaluate off-target activity against

UNC119A of selected compounds of the present invention. HEK293 EBNA cells were co- transfected with RLuc8-tagged UNC119A and GFP2-tagged Src. 24 h after transfection, cells were treated with 0.1 % DMSO (control) or 5 uM inhibitor for 24 h. (a) Treatment with reference

PDE6D inhibitors Deltarasin, Deltazinone1, Deltasonamide1 or UNC119A inhibitor Squarunkin-

A. (b) Treatment with selected compounds of the present invention DA1_B, DA1_B2 and

DA1_B4 or as a positive control UNC119A inhibitor Squarunkin-A. Independent biological repeats n=3 for all conditions.

[0019] Figure 5: Inhibition of MAPK signalling in MIA PaCa-2 cells by selected compounds of the present invention. (a) Western blot analysis of ERK phosphorylation in

MIA PaCa-2 cells treated with 0.1% DMSO vehicle control either without (-EGF) or with 200 ng/mL EGF treatment for 10 min (+EGF). Values represent the mean + SEM of n=6 independent biological repeats; statistical significance **** p < 0.0001. (b) Western blot analysis of ERK phosphorylation in MIA PaCa-2 cells treated for 4 h with 0.1% DMSO vehicle control or with 2.5, 5, 10, or 20 uM of the control compound Deltazinone1 or selected compounds of the present invention and stimulated with 200 ng/ML EGF for 10 min. Values represent the mean + SEM of n=2 independent biological repeats. Statistical significance levels were evaluated by comparison to the control condition (DMSO + EGF) and are annotated as * p < 0.05.

[0020] Figure 6: Inhibition of mTORC1 signalling in MIA PaCa-2 cells by selected compounds of the present invention. (a) Western blot analysis of S6 phosphorylation in MIA

PaCa-2 cells treated with 0.1% DMSO vehicle control either without (-EGF) or with 200 ng/mL

EGF treatment for 10 min (+EGF). Values represent the mean + SEM of n=6 independent biological repeats; statistical significance **** p < 0.0001. (b) Western blot analysis of S6 phosphorylation in MIA PaCa-2 cells treated for 4 h with 0.1% DMSO vehicle control or with 2.5, 5, 10, or 20 uM of the control compound Deltazinone1 or selected compounds of the present invention and stimulated with 200 ng/ML EGF for 10 min. Values represent the mean + SEM of nz2 independent biological repeats. Statistical significance levels were evaluated by comparison to the control condition (DMSO + EGF) and are annotated as * p < 0.05.

[0021] Figure 7: Inhibition of 2D cell proliferation by selected compounds of the present invention. (a) PDE6D-dependent and KRAS-mutant, KRAS-mutant, HRAS-mutant, or BRAF- mutant cancer cell lines were treated with selected compounds of the present invention, or reference PDE6D inhibitors (Deltarasin, Deltazinone1 and Deltasonimide1). Additional control compounds: farnesylation inhibitor (FTI-277), KRAS-G12C inhibitors (AMG-510, ARS-1620),

BRAF inhibitor (Vemurafenib) or MEK inhibitor (Trametinib). Cell viability was measured by alamarBlue assay after 72 h of drug treatment. Dose response data were analyzed to obtain the

DSS3 score, which is a normalized area under the curve measure that takes different tested concentration ranges into account. Higher DSS3 score relates to a higher activity of the compound. The heatmap was generated using the DSS3 scores. KRAS-G12C mutant cell lines are labeled with an asterisk. (b) The average of DSS3 scores obtained from PDE6D-dependent 4 and KRAS-mutant cell lines (first set) were plotted against the average of DSS3 scores oe Lp50222 4 from HRAS-mutant cell lines (second set) per compound. Values represent the mean + SEM o n=3-5 independent biological repeats. The numbers above the bars indicate the ratio between the averages of the first and second set of scores obtained.

[0022] Figure 8: Inhibition of microtumor formation in CAM assay. MDA-MB-231 cells were allowed to form microtumors on the frertized chicken egg CAM. Tumors were treated daily for 5 days with either 0.1% DMSO vehicle control, 10 uM of Deltazinone1 or DA1_B4. The weight of the tumors was then determined. Values represent the mean + SEM of n=3 independent biological repeats. The numbers above the scatter dot plots indicate the number of analyzed

CAMs. Statistical significance levels were evaluated by comparison to the control condition and are annotated as * p < 0.05.

DETAILED DESCRIPTION OF THE INVENTION LUS02224

[0023] 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

[0024] 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.

[0025] 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 C;-Cs-cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.

[0026] 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.

[0027] 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.

[0028] The term "halogen", also abbreviated herein as “hal” means fluoro, chloro, bromo, or iodo, preferably fluoro.

[0029] 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.

[0030] "Tautomers" are structural isomers of the same chemical substance that spontaneously interconvert with each other, even when pure.

[0031] 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, 6 n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two Plisoz224 more of these liquids), wherein the addition complex exists in the form of a crystal or mixe 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.

[0032] 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, "'C, °C, ™C, "°N, "°F, Ss, CI, and ‘|.

Compounds

[0033] The present invention is directed to a compound according to formula (I)

O

Ans A n J Ri

B

(D wherein

[0034] R, is selected from the group consisting of unsubtituted (C--Cs)alkyl, preferably methyl. 0 0 > NE NE

JR R

[0035] A is X ? - preferably F 2

DO

[0036] B is selected from the group consisting of E, and

[0037] R; is selected from the group consisting of unsubstituted (C4-Cg)alkyl, and unsubtituted (C5-Cs)cycloalkyl. In one embodiment, Ra is selected from the group consisting of cyclopropyl, sec-propyl, and ethyl, preferably sec-propyl;

[0038] X is selected from the group consisting of F, CI, Br, and |, preferably F; X may be in ortho, meta or para position, preferably in para position.

Rs x Rs

Re D

[0039] J is selected from the group consisting of Rs ;

[0040] Rs, R4, Rs, Re are independently selected from the group consisting of hydrogen, -NH, and -NO;;

[0041] D is selected from the group consisting of -COOH, -COO(C+-Ce)alkyl, -C(O)(C+-Cs)alkyl, -C(O)(C»-Cs)alkenyl and halogen, preferably -COOH, -COOCH>, and Br; 7

[0042] E is -COO(C+-Cg)alkyl. LU502224

[0043] n is an integer between 1 and 10, preferably between 2 and 8, more preferably 6:

[0044] Further, the present invention comprises a solvate, hydrate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.

[0045] Preferably, the salt is a pharmaceutically acceptable salt.

[0046] “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.

[0047] A selection of compounds according to the present invention is listed in the following

Table 1.

Table 1 a à / N J N

So No HO” Yo

F F

DA1_B DA1_B1 0 À, ot

N A

NH,

QO. ng 0 Dé

F F

DA1_B2 DA1_B4 8 r ( LU502224 à à

N N

NO,

O Dé NO, O D

HO” 50 Ho "0

F F

DA1_B6 DA1_B5

À, oh

NO on y z ~o Yo - ON

Y Br Y

F

F

DA1_B7 . . - A442b_intermediate 9

Preparation of Compounds

[0048] 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 0 “NP oR

[0049] Step 1: In step 1 compound n is converted with an amine RaNHz preferably in the presence of a base in an organic solvent.

LG may be selected from CI, Br, | or a sulfonate leaving group, such as mesylate, tosylate, nosylate, triflate, preferably Br.

R; may be selected from the group consisting of unsubstituted (Cy-Cs)alkyl, and unsubtituted (C5-Cs)cycloalkyl. In one embodiment, R7 is selected from the group consisting of cyclopropyl, sec-propyl, and ethyl, preferably sec-propyl

R; may be selected from (C--Cs)alkyl, preferably methyl and ethyl.

Any suitable base may be used. Preferably a weaker base such as K,CO+, NaCOs is used.

Preferably, the organic solvent is a polar-aprotic solvent, such as Diethylether, THF, DMF,

DMSO, more preferably DMF.

Preferably, step 1 is carried out at room temperature.

Preferably, the reaction time in step 1 is 1 to 5 h, more preferably 2 h.

O

[0050] Step 2: In step 2 the product obtained in step 1 is converted with ad in 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]-1H-1,2, 3-triazol[4,5-b]pyridinium-3-oxid-hexafluorophosphat (HATU), preferably EDC. The catalyst may be selected for example from 4- dimethylaminopyridine (DMAP) and hydroxybenzotriazole (HOB), 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 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.

PG y 9

[0052] Step 4: The product of step 3 is reacted with Ri under conditions as described for step 2. PG represents a protecting group for amines known to the person skilled in the art 502224 preferably Boc. In step 4, preferably HATU is used as coupling reagent. Preferably, DIPEA is used as base. Preferably DMF is used as solvent.

PG

N

PG

"

J

[0053] Wherein Ri may be prepared by reductive amination of Soand a respective amine

NH.-CH--R;. Reductive amination may be carried out in the presence of NaCNBHs,, an organic acid such as acetic acid an in an polar-protic solvent such as an alcohol like methanol or ethanol.

[0054] 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.

Ra O

O. NA yg,

X

NA NH

[0055] Step 6: The compound X obtained in steps 4 and 5 may be further

G Z

NX

= reacted with -YC(O)(C4-Ce)alkyl, -YC(O)(Cz-Cs)alkenyl, or X in the presence of a base, such as an amine base e.g. triethylamin, diisopropylethylamine, K,CO;. The solvent may be an aprotic-unpolar or aprotic polar solvent, such as dichloromethane, or dimethylsulfoxide.

Wherein Y is a halogen, preferably chloro. Z is -C(O)O(Cy-Cs)alkyl, or halogen. G is selected from -NO, and not present.

Re 9

O Ng, a C

N

= Ne TL

[0056] Step 7: In step 7, compound Fz , wherein Z is -C(O)O(C--

Cs)alkyl may be hydrolyzed to Z is -COOH under conditions as described in step 3.

Re 9

Oo Ng, a C

N

= Ne TL 4

[0057] Step 8: In step 8, G in ar may be optionally reduced to -NHz.

General conditions for reduction of aromatic nitro groups to amino groups are known in the art.

Step 8 may be carried out using for example Pd/C as catalyst and hydrogen as reduction agent. 11

Pharmaceutical Composition and Medical Use LU502224

[0058] À further aspect of the present invention is directed to a pharmaceutical composition comprising at least one of the inventive compounds.

[0059] “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.

[0060] “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.

[0061] "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.

[0062] A further embodiment of the invention is directed to the inventive compounds or pharmaceutical composition for use in medicine.

[0063] A further embodiment of the invention is directed to the inventive compounds or pharmaceutical composition for use in the treatment of cancer.

[0064] Preferably, the cancer is selected from K-Ras dependent cancers or from cancers wherein the KRAS gene is mutated.

[0065] 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. *kkk 12

[0067] A better understanding of the present invention and of its advantages will be had FO 11502224 the following examples, offered for illustrative purposes only. The examples are not intended © limit the scope of the present invention in any way.

EXAMPLES OF THE INVENTION

Synthesis of Methyl 4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)benzoate (A442b_Intermediate)

Oa OMe

N = OMe F © SOL 2N Ethylamine 0 Np LI © © A

HN - lose 5 TEM NII

F A442b_Int-4 F Int-1 step-2 A

A442b_Intermediate

Scheme 1: Synthesis of Methyl 4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)benzoate (A442b_Intermediate)

[0068] N-(7-(ethylamino)-7-oxoheptyl)-4-fluoro-N-isopropylbenzamide (Int-1): To a solution of ethyl 7-(4-fluoro-N-isopropylbenzamido)heptanoate (A442b_Int-4) (2 g, 5.93 mmol) in THF (2 mL) was added Ethylamine (2M in THF, 20 mL) followed by TBD (1.64 g, 11.86 mmol) at 0 °C and the reaction mixture was stirred for 16 h at room temperature (monitored by TLC). The reaction mixture quenched with water (5 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic extracts were dried over sodium sulphate, evaporated and purified by column chromatography (1:1 ethyl acetate in hexane) to get N-(7-(ethylamino)-7-oxoheptyl)-4-fluoro-N- isopropylbenzamide (Int-1) (1.8 g 90% yield). LCMS: 337.7 [M+H]".

[0069] Methyl 4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)benzoate (A442b_Intermediate): To a solution of

N-(7-(ethylamino)-7-oxoheptyl)-4-fluoro-N-isopropylbenzamide (Int-1) (0.5 g, 1.49 mmol) in

DMF (5 mL) was added NaH (118 mg, 2.98 mmol) at 0 °C. The reaction mixture was stirred for minutes at 0 °C and methyl 4-(bromomethyl)benzoate (682 mg, 2.98 mmol) was added. The reaction mixture stirred at room temperature for 16 h (monitored b TLC) and quenched by water (20 mL), extracted with ethyl acetate (20 mL x 3). The combined organic extracts were dried over sodium sulphate, evaporated and purified by column chromatography eluting with 50% ethyl acetate in hexane to get methyl 4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)benzoate (A442b_Intermediate) (80 mg, 12% yield) as a colorless sticky material. LCMS: 485.4 [M+H]*, HPLC: 100%, 'H NMR (400MHz, CDCIs) 5 = 8.03-8.00 (m, 2H), 7.40 — 7.30 (m, 4H), 7.14 — 7.09 (m, 2H), 4.67 — 4.61 (m, 2H), 3.96 (m, 4H), 3.50 (q, J= 7.2 Hz, 1H), 3.33 (m, 4H), 2.44 (bs, 1H), 2.31 (bs, 1H), 1.70 (m, 2H), 1.47 — 1.38 (m, 4H), 1.29 — 1.13 (m, 10H). 13

Synthesis of Methyl 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoate (DA1_B4)

CHO “NH, fe ° © 9 LA

NaBH(OAc) 3, AcOH + 0 nas A oo“ ronan OX zz

Boc Step-1 N F Step-2 N 137076-22-3 oo A442b_Int-11 int2 Boc”

O

0 0 + y Oo

HCI Dioxane mon o— © nr 0°Cr 16h x A OY Cs DMSO 5 os

Step-3 HCl HN 80 °C, 16 h

Int-3 Step F DA1_B4

Scheme 2: Synthesis of Methyl 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoate (DA1_B4)

[0070] tert-butyl 4-((ethylamino)methyl)piperidine-1-carboxylate (1): To a solution of tert- butyl 4-formylpiperidine-1-carboxylate (18 g, 84.5 mmol) in THF (200 mL) was added ethylamine (42.25 mL, 84.5 mmol) at 0 °C followed by drop of acetic acid (cat.) and the reaction mixture was stirred for 1 h at room temperature (monitored by TLC). The reaction mixture cooled to 0 °C and NaBH(OAc); (17.91 g, 84.5 mmol) was added and stirred at room temperature for 16 h. The reaction mixture quenched with Sat. NaHCO; and organic solvent is removed under reduced pressure. The aqueous layer was washed with ethyl acetate once to remove non polar impurities (discarded). The aqueous layer is further extracted with 5% MeOH :

DCM (200 mL x 3). The combined layers of MeOH: DCM extracts were dried over sodium sulphate, evaporated to dryness to get pure tert-butyl 4-((ethylamino)methyl)piperidine-1- carboxylate (15 g, 73% yield) as a colorless oil. LCMS: 243.7 [M+H]".

[0071] tert-butyl 4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidine-1-carboxylate (2): To a stirred solution of 7-(4-fluoro-N-isopropylbenzamido)heptanoic acid (A442b_Int-5) (3 g, 9.7 mmol) in dry THF (30 mL) was added HATU (5.5 g, 14.5 mmol) at 0 °C and DIPEA (8.5 mL, 48.0 mmol). The reaction mixture was stirred at room temperature for 1 h and tert-butyl 4 ((ethylamino)methyl)piperidine-1-carboxylate (4.2 g, 17.4 mmol) was added. The reaction mixture was stirred at room temperature for 16 h (monitored by TLC) and quenched with water (20 mL). The aqueous layer is extracted with ethyl acetate (50 mL x 3) and combined organic layer was evaporated to dryness to get tert-butyl 4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidine-1-carboxylate (2) (5.1 g, quantitative yield) as colorless sticky material. LCMS: 534.6 [M+H]"

[0072] Methyl 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoate (DA1_B4): To a stirred solution of tert-butyl 4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidine-1-carboxylate (2) (10 g, 23.0 mmol) in dry

DCM (20 mL) was added 4M HCI in dioxane (30 mL) at 0 °C and reaction mixture was stirred 14 for 4 h (monitored by TLC). The solvent was removed under reduced pressure and resulting HE 502224 salt (10 g) was used for next step without further purification.

To a solution of above crude (10 g) in DMSO (100 mL) was added K,CO; (15.8 g, 115.2 mmol) (pH~9) followed by methyl 4-fluorobenzoate (7.09 g, 46.0 mmol). The reaction mixture was heated to 80 °C for 16 h (Monitored by TLC). Quenched with cold water and extracted with ethyl acetate (50 mL x 3). The combined organic extracts were dried, evaporated and purified by flash column chromatography eluting with 15% MeOH: DCM to product. The obtained product was further purified using 0.1% formic acid in water: acetonitrile to get methyl 4-(4-((N-ethyl-7- (4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoate (DA1_B4) (3.3 g, 31% yield over two step) as a white solid. LCMS: 568.5 [M+H]*, HPLC: 99.95%, 'H NMR (400MHz, DMSO-d6) à = 7.76 (d, J = 6 Hz, 2H), 7.40 (m, 2H), 7.29-7.23 (m, 2H), 6.97 (m, 2H), 3.98 - 3.89 (m, 2H), 3.76 (s, 3H), 3.73 (m, 1H), 3.16 (m, 3H), 2.82 (t, J = 12 Hz, 2H), 2.33 (m, 3H), 1.85 (s, 1H), 1.67-1.58 (m, 6H), 1.42-1.31 (m, 4H), 1.23-0.98 (m, 13H).

Synthesis of compounds Methyl 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2-nitrobenzoate (DA1_B), (4-((N- ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2- nitrobenzoic acid (DA1_B6), 2-amino-4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoic acid (DA1_B1)

NOz NO; O

F LC

0 0 a Ç _ YY oe

DS 151504813 oO CCD) LiOHH2O

HCI HN 80°C, 16 h Step-2

Step-1

DA1_B4-Int-3 F DA1_B

NO2 Q NH, O

Dé Oo NZ OH

O SOC) Hz, Pd/C (10%) o SSP N

N MeOH, rt, 4h TO 3 [ Step-3 4 (

F DA1_B6 F DA1_B1

Scheme 3: Synthesis of compounds Methyl 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2-nitrobenzoate = (DA1_B), (4-((N-ethyl-7-(4- fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2-nitrobenzoic acid (DA1_B6), 2-amino- 4-(4-((N-ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoic acid (DA1_B1)

[0073] 4-(4-((N-ethyI-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1- yl)-2-nitrobenzoate (DA1_B): To a solution N-(7-(ethyl(piperidin-4-ylmethyl)amino)-7- oxoheptyl)-4-fluoro-N-isopropylbenzamide hydrochloride (DA1_B4-Int-3) (300 mg, 0.69 mmol) in

DMSO (3 mL) was added K,CO; (191 mg, 1.38 mmol) (pH~9) followed by methyl 4-fluoro-2- nitrobenzoate (165 mg, 0.82 mmol). The reaction mixture was heated to 80 °C for 16 h (Monitored by TLC). Quenched with cold water and extracted with ethyl acetate (10 mL x 3).

The combined organic extracts were dried, evaporated and purified by flash column chromatography eluting with ethyl acetate in hexane (7:3) to get methyl 4-(4-((N-ethyl-7-(4-

fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2-nitrobenzoate (DA1_B) (190 502224 mg, 38.36% yield) as a colorless sticky material. LCMS: 613.5 [M+H]*, HPLC: 100%, 'H NMR (400MHz, DMSO-d6) 6 = 7.73 (d, J = 8.8 Hz, 1H), 7.40 (m, 2H), 7.31-7.23 (m, 3H), 7.12 (m, 1H), 4.06 (m, 2H), 3.74 (m, 4H), 3.29 (m, 2H), 3.16 (m, 4H), 2.92 (t, J = 12 Hz, 2H), 2.33 (m, 3H), 1.89 (s, 1H), 1.66-1.58 (m, 5H), 1.31 (m, 3H), 1.23-0.97 (m, 12H).

[0074] 4-(4-((N-ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1- yl)-2-nitrobenzoicacid (DA1_B6):

Methyl 4-(4-((N-ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2- nitrobenzoate (DA1_B) (280 mg, 0.4 mmol) in THF:MeOH:Water (9 mL, 1:1:1) and LIOH-H20 (96 mg, 2.2 mmol) at 0 °C was added. The reaction mixture was stirred at room temperature for 16 h (monitored by TLC) and the solvent was removed under reduced pressure. The residue was dissolved in water (1 mL) and acidified to pH~ 3 at 0 °C. The aqueous layer is extracted with ethyl acetate (5 mL x 3) and combined organic layer was evaporated to dryness. The obtained semisolid was further purified by RP-prep-HPLC using 0.1% formic acid in water and acetonitrile to get pure 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2-nitrobenzoic acid (DA1_B6) (150 mg, 55% yield) as a white solid. LCMS: 599.5 [M+H]*, HPLC: 100%, 'H NMR (400MHz, DMSO-d6) 0=774(d, J = 8.8 Hz, 1H), 7.40 (m, 2H), 7.33-7.24 (m, 3H), 7.10 (m, 1H), 4.05 (m, 2H), 3.78 (s, 1H), 3.18 (m, 4H), 2.91 (t, J = 12.4 Hz, 2H), 2.34 (m, 2H), 1.90 (bs, 1H), 1.67-1.58 (m, 6H), 1.33 (m, 3H), 1.25-1.02 (m, 14H). (Note: Acid proton not observed in '"H NMR).

[0075] 2-amino-4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoic acid (DA1_B1):

To a stirred solution of 4-(4-((N-ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2- nitrobenzoic acid (DA1_B6) (120 mg, 0.02 mmol) in MeOH (3 mL), 10% Pd/C (50 mg, 50% moisture) was added. The reaction mixture was stirred at room temperature for 4 h (monitored by TLC) and catalyst was filtered out by using celite bed. The filtrate was evaporated under reduced pressure and residue was purified by RP-prep-HPLC using 0.1% formic acid in water and acetonitrile to get 2-amino-4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoic acid (DA1_B1) (60 mg, 53% yield) as a white solid. LCMS: 569.5 [M+H]*, HPLC: 100%, 'H NMR (400MHz, DMSO-d6) à = 7.51 (dd, J = 8.8, 2.4 Hz, 1H), 7.40 (m, 2H), 7.29-7.23 (m, 2H), 6.18 (m, 2H), 4.85 (bs, 1H), 4.42 (m, 1H), 3.83 (m, 2H), 3.17 (m, 5H), 2.75 (t, J = 10 Hz, 2H), 2.34 (m, 2H), 1.83 (s, 1H), 1.67- 1.57 (m, 6H), 1.33 (bs, 3H), 1.25-1.01 (m, 14H). (Note: Acid proton not observed in 'H NMR). 16

Synthesis of 4-(4-((N-ethyl-7-(4-fluoro-N- oo LU502224 isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-3-nitrobenzoic acid (DA1_B5) and 3-amino-4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoate (DA1_B2)

ON

F— SP oO oO oO O o— O,N _ ON 000 so © RR Yr LT o yn " co Si oc ron & 0e : BA

HCI HN step-1 [ [

DA1_B4-Int-3 4 Int-1 È DA1_B5 9 oO

Da NO ~~ HN o © SSP N Hz, PU/C (10%) ON oO cd RO ‘

Step-2 [

F Int-1 F DA1_B2

Scheme 4: Synthesis of 4-(4-((N-ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin- 1-yl)-3-nitrobenzoic acid (DA1_B5) and 3-amino-4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoate (DA1_B2)

[0076] Methyl 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-2-nitrobenzoate (Int-1) and 4-(4- ((N-ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yi)-3- nitrobenzoic acid (DA1_B5): To a solution of N-(7-(ethyl(piperidin-4-ylmethyl)amino)-7- oxoheptyl)-4-fluoro-N-isopropylbenzamide hydrochloride (DA1_B4-Int-3) (300 mg, 0.69 mmol) in

DMSO (3 mL) was added K,CO; (191 mg, 1.38 mmol) (pH~9) followed by methyl 4-fluoro-3- nitrobenzoate (165 mg, 0.82 mmol). The reaction mixture was heated to 80 °C for 16 h (Monitored by TLC). Quenched with cold water and extracted with ethyl acetate (10 mL x 3).

The combined organic extracts were dried, evaporated and purified by flash column chromatography eluting with ethyl acetate in hexane (7:3) to get methyl 4-(4-((N-ethyl-7-(4- fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-3-nitrobenzoate (Int-1) (100 mg, 26.17% yield) as a colorless sticky material and its corresponding hydrolyzed product acid 4-(4-((N-ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-3- nitrobenzoic acid (DA1_B5) which was further purified by RP-prep-HPLC using 0.1% formic acid in water and acetonitrile to get pure 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-3-nitrobenzoic acid (DA1_B5) (10 mg, 3% yield) as a colorless sticky material. Int-1: LCMS: 613.5 [M+H]*, DA1_B5: LCMS: 599.4 [M+H]*, HPLC: 99.72%, 'H NMR (400MHz, DMSO-d6) à = 8.28 (s, 1H), 8.00 (d, J = 8 Hz, 3sH), 7.35 (d, J = 8.4 Hz, 3H), 3.84 (s, 6H), 3.02 (t, J = 11.2 Hz, 5H), 2.24 (d, J = 5.2 Hz, 6H), 1.82 (d,

J = 10.4 Hz, 5H), 1.69 (s, 3H), 1.22 (m, 6H), 0.96 (s, 4H).

[0077] Methyl 3-amino-4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoate (DA1_B2): To a stirred solution of methyl 4-(4-((N-ethyl-7-(4-fluoro-N- isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)-3-nitrobenzoate (Int-1) (100 mg, 0.167 mmol) in MeOH (3 mL), 10% Pd/C (40 mg, 50% moisture) was added. The reaction mixture was stirred at room temperature for 4 h (monitored by TLC) and catalyst was filtered out by using celite bed. The filtrate was evaporated under reduced pressure and residue was purified by RP- 17 prep-HPLC using 0.1% formic acid in water and acetonitrile to get Methyl 3-amino-4-(4-((NT 502224 ethyl-7-(4-fluoro-N-isopropylbenzamido)heptanamido)methyl)piperidin-1-yl)benzoate (DA1_B2) (50 mg, 52% yield) as a colorless sticky material. LCMS: 583.5 [M+H]*, HPLC: 97.82%, 'H NMR (400MHz, DMSO-d6) 5 = 7.45 (m, 2H), 7.32-7.18 (m, 4H), 6.93 (m, 1H), 4.92 (m, 2H), 3.78 (s, 3H), 3.34 - 3.15 (m, 10H), 2.34 (m, 2H), 1.83 (s, 1H), 1.70-1.55 (m, 5H), 1.41 - 1.34 (m, 6H), 1.25-1.01 (m, 10H). (Note: Acid proton not observed in '"H NMR).

Synthesis of N-(7-(((1-(4-bromo-2-nitrophenyl)piperidin-4-yl)methyl)(ethyl)amino)-7- oxoheptyl)-4-fluoro-N-isopropylbenzamide (DA1_B7)

NO, ° 5 ol Ss ~~ Dos

TO 364-73-8 0 LOSC N

PY K,CO,, DMSO OO

F OY rt, 0.5 h 3 [

HCI HN Stop-1

DA1_B4-Int-3 F DA1_B7

Scheme 5: Synthesis of N-(7-(((1-(4-bromo-2-nitrophenyl)piperidin-4-yl)methyl) (ethyl)amino)-7- oxoheptyl)-4-fluoro-N-isopropylbenzamide (DA1_B7)

[0078] N-(7-(((1-(4-bromo-2-nitrophenyl)piperidin-4-yl)methyl)(ethyl)amino)-7-oxoheptyl)- 4-fluoro-N-isopropylbenzamide (DA1_B7): To a solution N-(7-(ethyl(piperidin-4- ylmethyl)amino)-7-oxoheptyl)-4-fluoro-N-isopropylbenzamide hydrochloride (DA1_B4-Int-3) (500 mg, 1.5 mmol) in DMSO (5 mL) was added K,CO; (794 mg, 5.7 mmol) (pH~9) followed by 4- bromo-1-fluoro-2-nitrobenzene (502 mg, 2.3 mmol). The reaction mixture was stirred at room temperature for 0.5 h (Monitored by TLC). Quenched with cold water and extracted with ethyl acetate (10 mL x 3). The combined organic extracts were dried, evaporated and purified by RP- prep-HPLC using 0.1% formic acid in water and acetonitrile to get N-(7-(((1-(4-bromo-2- nitrophenyl)piperidin-4-yl) methyl) (ethyl)amino)-7-oxoheptyl)-4-fluoro-N-isopropylbenzamide (DA1_B7) (290 mg, 43% yield) as a yellow colored sticky material. LCMS: 635.2 [M+H]*, HPLC: 99.16%, "H NMR (400MHz, DMSO-d6) 5 = 8.02 (m, 1H), 7.72 (m, 1H), 7.41 (m, 2H), 7.28 (t, J = 8.4 Hz, 3H), 3.80 (m, 1H), 3.38 (m, 2H), 3.20 — 3.15 (m, 6H), 2.94 — 2.73 (m, 2H), 2.34 (m, 2H), 1.76 (s, 1H), 1.76-1.55 (m, 6H), 1.32-1.19 (m, 6H), 1.12-0.88 (m, 9H). 2. Biological Testing

[0079] Compounds of the present invention have low nanomolar affinities

The compounds of the present invention (Table 1) were tested in a fluorescence polarization assay, using the same method that was previously employed to determine the affinity of reference compounds from the Waldmann group (Deltarasin, Deltazinone1, Deltasonamide1).

Fluorescein-labelled Atorvastatin (F-Ator), which binds to the prenyl-pocket of PDE©D, was used as a probe. Using this assay, published affinity data of the reference compounds were essentially reproduced (Table 2). The dissociation constants of compounds ranged mostly in the low-nanomolar regime, with DA1_B having even sub-nanomolar affinity. 18

[0080] Compounds of the present invention show improved activity against PDE6D (N, 502224

BRET-experiments

Compounds of the present invention were tested in cellular BRET-experiments. The BRET- biosensor RLuc8-PDE6D/ GFP2-K-Ras4B-G12V was expressed in HEK293ebna cells and the

BRET response was determined. Several compounds, such as DA1_B2 and DA1_B4 showed a strong, dose-depedent reduction in BRET signal, consistent with a displacement of the K-

Ras4B-G12V construct from PDE6D. Other compounds had less activity, suggesting poor in cell activity, e.g. due to problems with cell-penetration or degradation (Figure 1).

[0081] Compounds of the present invention disrupt Ras membrane organisation

Loss of Ras nanoclustering-dependent BRET indicates disruption of functional membrane organisation of Ras, such as by inhibition of its lipid modification, trafficking or nanoclustering itself. By employing a K-RasG12V-specific assay (Figure 2) with H-RasG12V specific assay (Figure 3), the activity of compounds against either of these Ras isoforms can be determined.

Compounds displayed various degrees of K-Ras and H-Ras directed activities, with the most relevant being DA1_B2 having a slight K-Ras selectivity (Figures 2, 3). The H-Ras directed activity has also been observed with Deltazinone1, and may suggest that with higher potency also H-Ras membrane trafficking is affected, as has been described before ([4] Chandra et al.,2011; [6] Schmick et al., 2014).

[0082] Compounds of the present invention disrupt binding of Src to UNC119A

UNC119A is a structurally highly related to PDE6D and a trafficking chaperone of myristoylated proteins, such as Src. We constructed a BRET-biosensor UNC119A-Rluc8/ Src-GFP2, which shows reduced BRET upon displacement of the Src-construct by myristoylation-pocket inhibitors of UNC119A. The compound Squarunkin A was identified by others as a UNC119A inhibitor and we observed a strong reduction of the BRET-signal with its treatment (Figure 4) ([22] Garivet et al). As compared to Deltazinone1, the three top compounds in our invention,

DA1_B, DA1_B2 and DA1_B4 also reduced the BRET-signal, suggesting some off-target activity against UNC119A. The same was observed with the subnanomolar inhibitor

Deltasonamide1, suggesting that low nanomolar affinity of inhibitors for PDE©D may be accompanied by a significant off-target activity against related UNC119A.

[0083] Compounds of the present invention reduce phosphorylation of ERK and S6.

Active Ras stimulates a number of effector pathways, including the MAPK- and Akt/mTORC1- pathways. Phosphorylation of the kinase ERK and the ribosomal protein S6, respectively, are a result of the activation of these pathways. We tested the activity of top compounds as compared to Deltazinone1 and found that DA1_B4, DA1_B and DA1_B2 significantly reduced phosphorylation levels of ERK and S6 at concentrations between 5 and 20 pM (Figures 5,6).

This is in line with the loss of Ras membrane organisation (Figures 2, 3), which inactivates Ras.

[0084] Compounds of the present invention reduce cancer cell proliferation and microtumor formation.

We tested the activity of top compounds DA1_B, DA1_B4 and DA1_B2 as compared to a set of reference inhibitors, including Deltazinone1, against proliferation of a panel of nine cancer cell lines (Figure 7a). As annotated in the figure, cancer cell lines were classified as either PDE6D- dependent and KRAS-mutant, KRAS-mutant, HRAS-mutant or as a control BRAF-mutant. As expected, none of our compounds had a strong effect on the BRAF-mutant cancer cell line.

When comparing the selectivity of compounds for PDE6D-dependent and KRAS-mutant cells as compared to HRAS-mutant cells, DA1_B showed the highest selectivity, followed closely by

DA1_B2 (Figure 7b). We tested the anti-tumorigenic activity of DA1_B4 as compared to 19

Deltazinone1 using the CAM assay, where cancer cells are cultured on top of chick embryos INıso2224 fertilized eggs. DA1_B4 was of comparable potency to Deltazinone (Figure 8).

Table 2: Affinities of reference compounds Deltarasin, Deltazinone1, Deltasonamide1 and compounds of the present invention as determined in fluorescence polarization measurements using FITC-labelled Atorvastatin as a probe.

[0085] 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 Il enzyme mix (#11791020, Thermo Fisher Scientific). The reaction mix was transformed into ccdB sensitive

E.coli strain DH10B (#£C0113, Thermo Fisher Scientific) and positive clones were selected in the presence of ampicillin.

[0086] Cell culture

HEK293 EBNA 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). NCI-H358 (ATCC CRL-5807), MDA-MB-231 (ATCC HTB-26) and IGR-39 (DSMZ ACC 239) were maintained in Roswell Park Memorial Institute medium (RPMI). PANC-1 (ATCC CRL-1469), MIA

PaCa-2 (ATCC CRM-CRL-1420), Hs-578T (DSMZ ACC 781) and T24 (DSMZ ACC-376) were maintained in DMEM. SW-620 (ATCC CCL-227) and SW-480 (DSMZ ACC-313) were maintained in Leibovitz's L-15 Medium (Lonza Pharma). 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, atmosphere and sub-cultured twice a week.

[0087] 2D cell viability assay

MIA PaCa-2, PANC-1, SW-620, SW-480, MDA-MB-231, NCI-H358, Hs-578T, T24 and IGR-39 cells were plated in complete medium into 96-well cell culture plates (#655 180, Greiner bio- one, Merck KGaA) at a density of 2500 cells/ 100 uL 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. Concentration range was between 0.156 - 40 uM for DA1_B4, DA1_B, DA1_B2,

FTI-277, deltazinone1 and deltasonamide1, between 0.078 - 20 uM for deltarasin and ARS- 1620, and 0.0039 - 1 uM for AMG-510, vemurafenib and trametinib. After 72 h incubation in the presence of the compounds, the cell viability was assessed using the alamarBlue "229° 502224 (#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.

[0088] Drug sensitivity score analysis (DSS3)

To quantitatively profile the drug sensitivity with a more robust parameter than the ICs, or ECsg 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 (hites.//breeze fimm f/) and analyzed on the site (Potdar et al, 2020).

The output file provides several drug sensitivity measures including ICs, and AUC. We plotted the DSS3 value, which was calculated as DSS; = DSS, i where DSS2 is given by the equation DSS, = on 4 . AUC-t(x,— x and DSS1 is given by the equation DSS; = és

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 with 100 uM benzethonium chloride (#53751,

Merck KGaA) treatment.

[0089] BRET assay

BRET assays were essentially performed as described ([23] Okutachi S 2021). 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 (Polyplus-transfection SA) following the manufacturer's instructions. Each well was transfected with about 1 pg of plasmid DNA using 3 pL of jetPRIME reagent.

For BRET experiments, the concentration of donor plasmid (50 ng) was kept constant and the concentration of acceptor plasmid was increased from O to 1000 ng (titration experiments) or kept at the indicated plasmid ratio. 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 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 21 fluorescence intensity followed by BRET readings were carried out on a CLARIOstar (BMG 502224

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; this value is proportional to the acceptor concentration and plotted as [Acceptor]. BRET readings were taken well by well by adding 10 pL of 100 coelenterazine 400a (DeepBlueC, #C-320, Gold Biotechnology) RLuc8 substrate to each well (final concentration of 10 uM) using the injector present in the plate reader. Luminescence emission intensities were simultaneously recorded at 410 + 40 nm (RLU, proportional to donor concentration and plotted as [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

BRET ratio = Aem 515 NM(Donor + Acceptor) _ Aem 515 NM(Donor only) dem 410 NM(Donor+Acceptor) dem 410 NM(Donor 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 RFU/ RLU.

For BRET donor saturation titration experiments, the BRET ratio was plotted against [Acceptor]/[DBonor]. 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.

[0090] 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 and **** p < 0.0001.

[0091] Fluorescence Polarization

Fluorescence polarization (FP) assays were performed as described ([23] Okutachi 2021). The

ICso of compounds were determined in a binding/ displacement assay using fluorescein-labelled

Atorvasatin (F-Ator) as the probe as described before by others ([24] Zimmermann 2013) and in house purified human PDEGD. The F-Ator was used at 5 nM concentration with 5 nM of PDEGD.

FP assays were carried out in black low volume round bottom 384-well plates (cat. no. 4514,

Corning) with a reaction volume of 20 uL. Compounds were three-fold diluted in assay buffer (DPBS, without Ca**/Mg”*, with 0.05% CHAPS as reported). The FP signals were recorded on the Clariostar (BMG labtech) plate reader with excitation at 482 + 8 nm and emission at 530 + nm at 25 °C. The blank corrected milli Polarization value (mP or P x 1000) calculated from

MARS (BMG labtech) program was plotted against the log concentration of inhibitor. The data were fitted into log inhibitor vs response — 4 parametric equation of Prism (GraphPad) to obtain the ICso values. The ICs, values were converted into Ky using the modified Cheng-Prusoff 22 equation, Ku = = ‚where Ka is the dissociation constant between PDE6D and inhibitor, [L] j$;502024

K the ligand or probe concentration used in the assay and Kg is the dissociation constant between the PDE6D and the ligand or fluorescent probe. The reported Ky value of FITC-Atorvastatin to

PDEGD was 7.1 + 4 nM ([24] Zimmermann 2013). Note that the concentration of PDE6D is not part of the equation.

[0092] Western Blot Analysis

Following a 16 h serum starvation, MIA PaCa-2 cells were treated with compounds at 37°C for 4 h and then stimulated with 200 ng/mL EGF (Merck) at 37°C for 10 min. /n situ cell lysis was performed in ice-cold lysis buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCl, 0.1% SDS, 5 mM

EDTA, 1% Nonidet P-40, 1% Triton X-100, 1% sodium-deoxycholate, 1 mM Na;VO,, 10 mM

NaF, 100 mM leupeptin and 100 mM E64D) containing a cocktail of protease inhibitors and a cocktail of phosphatase inhibitors (both from Roche Diagnostics GmbH). Lysate clarification was done by centrifugation at 13200 rpm for 15 min at 4°C and total protein concentration was determined by Bradford assay. Proteins (50 ug per lane) were resolved by SDS-PAGE in a 10% homemade polyacrylamide gel under reducing conditions, and transferred to a nitrocellulose membrane (Bio-Rad) by semi-dry transfer. Membranes were saturated in phosphate-buffered saline (PBS) containing 2% bovine serum albumin and 0.2% Tween for 1 h at room temperature, then incubated with primary antibodies overnight at 4°C, either in a combination mouse anti-phosphoERK (Cell Signalling Technologies, CST9106) and rabbit anti-ERK (CST9102) or in a combination rabbit anti-phosphoS6 (CST4858) and mouse anti-S6 (CST2317) or with the mouse anti-actin antibody alone (Sigma A5441). Incubation with secondary antibodies IRDye 680 RD donkey anti-mouse and IRDye 800 CW goat anti-rabbit (both from Thermo Fisher Scientific) was performed for 1 h at room temperature. Each antibody incubation was followed by at least three wash steps in PBS supplemented with 0.2% Tween.

Signal intensities were quantified using the Odyssey Infrared Image System (LI-COR

Biosciences). The ratio between the intensities obtained for phosphorylated protein versus total protein was calculated and then normalized to the sum of all the ratios calculated for one blot to make blots comparable by accounting for technical day-to-day variability. For representative purposes, data were scaled to the controls present on each blot and are represented as the mean +/- SEM of n independent biological repeats.

[0093] Chick Chorioallantoic Membrane (CAM) Assay

Fertilized chicken eggs were incubated at 37°C in a 60% humidified egg hatcher incubator (MG200/300, Fiem). After 8 days, 2 x 10° MDA-MB-231 cells were resuspended in a 1:1 mix of

RPMI without FBS and Matrigel (Corning), and then deposited within sterilized 7 mm diameter plastic rings cut from PCR tubes (Greiner) on the surface of a chicken embryo CAM. After 24 h, the growing tumors were treated with a volume identical to the deposited cell suspension of 0.1% DMSO vehicle control or test compounds diluted in RPMI without FBS. Treatment was performed daily and after 5 days of treatment the microtumors were harvested and the tumor weight was determined. 23

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Claims (11)

1. A compound according to formula (I) O Ans A n J Ri B (D wherein R, is selected from the group consisting of unsubstituted piperidinyl and unsubstituted (C-- Cs)alkyl, preferably methyl; 0 OT Ais XE Te “O0 B is selected from the group consisting of S, E Ra is selected from the group consisting of unsubstituted -(C,-Cs)alkyl, and unsubstituted (Cz- Cs)cycloalkyl; X is selected from the group consisting of F, CI, Br, and |, preferably F; Rs x Rs Re D J is selected from the group consisting of Rs ; Rs, Ra, Rs, Rs are independently selected from the group consisting of hydrogen, -NH; and - NOz; D is selected from the group consisting of -COOH, -COO(C4-Cs)alkyl, -C(O)(C--Cs)alkyl, - C(O)(Ca-Cs)alkenyl and halogen, preferably —-COOH, -COOCH; and Br; E is -COO(Cy-Cs)alkyl; nis 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 26

; I 4: LU502224 i) X is in para position and/or il) X is F and/or iii) Rz is selected from the group consisting of cyclopropyl, sec-propyl, and ethyl, preferably sec-propyl and/or iv) R, is unsubstituted (C--Cs)alkyl, preferably methyl.

3. The compound of claim 1 or 2, wherein 0 oY Ais F Re

4. The compound of any one of claims 1 to 3, wherein the compound is selected from the group consisting of 0 C Oo. A, Oo. À, © N N NH, O ng NO, 0. hg NH, O. D >07 0 HO No >07 0 F 1 F 1 F 1 N N N NO,

O. D O. D NO, O. D So No Ho So Ho So F 1 F 1 F 1 27

( LU502224 ON X g ‚NO, >97 No - T Br C T F , and F .

5. The compound of any one of claims 1 to 3, wherein the compound is selected from the group consisting of ° N N N ; „NH, 8 NO, O D O hg NO, oO nd O D É OF , F and F :

6. The compound of any one of claims 1 to 3, wherein the compound is selected from the group consisting of oe 0 A, © N ; NH, © ON O dr F ,and F .

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. 28

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. 29