LU101206B1 - PDEdelta Inhibitors - Google Patents

Abstract

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

Description

New LU Application Applicant:University of Luxembourg Our Ref.: LUX16587LU -—Lut01206 PDEdelta Inhibitors

SEE 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 farnesyitransferase 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 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-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 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-Ari2 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_U 101206 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] Thus, there is a need to provide further PDEdelta Inhibitors.

SUMMARY OF THE INVENTION

[007] The present invention relates to a compound according to formula (1) Ri~...R icra,

I O=p—0R,

O So À Ra S Rs

O (1) wherein n is an integer between 1 and 10, preferably 4 to 10; more preferred 6 to 10; most preferably 6 to 9, particularly preferred 6; Ris H, “COR, -SO;Rs, or -CH;Rs, preferably —CORg; Rz is H, “COR, -SO;Rs, or -CH;R;, preferably H: Re is independently selected from substituted or unsubstituted -(Cz-C14)Ar, preferably substituted or unsubstituted -(Cs-C40)Ar; Rs is -(C;-Cs)Alkyl, preferably methyl or ethyl; R4 is —-CO2(C,-C5)Alkyl or -(C,-Cs)Alkyl, preferably —CO,Et or methyl Rs is -(C4-Cg)Alkyl, or substituted or unsubstituted -(C3-C14)Ar, preferably methyl, phenyl, -t-Bu: more preferably methyl; and a solvate, hydrate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.

[008] In a second aspect, the invention is directed to a pharmaceutical composition, comprising said compound.

[009] In a third aspect, the invention further relates to said compound or pharmaceutical composition for use in medicine.

[0010] In a fourth aspect, the invention is directed to said compound or pharmaceutical composition for use in the treatment of cancer.

[0011] It has been found that the compound of the present invention, due to the —(CH2),- group enables high drug efficacy. Further, it has been shown that the presence of the thioester cell penetration group, in formula (I), results in improved cell penetration and thus bioavailability.

BRIEF DESCRIPTION OF THE FIGURES

[0012] Fig. 1 depicts characteristic structural elements of the compounds of the present, based on example compounds A, B, C, D. The proper length of the flexible middle-linker / “spring” probably improves resilience of compounds against Arl2-mediated ejection from PDEdelta resulting in a higher cellular activity (Figs. 3,4).

[0013] Fig. 2: In vitro binding of compounds to PDE6. To a three-fold dilution series of compounds (in 3% DMSO in the starting dilution), a complex of 0.25 uM F-Rheb and 2 uM PDEdelta was added and the reaction mix was incubated for 15 min at RT. Then fluorescence anisotropy was measured using a polarization cube with excitation at 485 (20) nm and emission at 528 (20) nm. Data were fit to log inhibitor vs. response — variable slope (four parameters) equation in GraphPad.

[0014] Fig. 3: Inhibition of K-RasG12V/ PDE3 interaction in HEK293 by compounds. Cells growing under normal conditions were transfected with pmGFP-K-RasG12V and pmCherry- PDEdelta, and 24h later treated with 0.1% DMSO vehicle control or indicated concentrations of compounds for 24 hr. The apparent FRET efficiency was calculated from FLIM data (meant s.e.m., n=2). The numbers in the bars indicate the number of analyzed cells. Statistical significance of differences between control and treated cells were examined using one-way ANOVA complemented with Tukey's tests (n.s., not significant; *** p < 0.001, **** p < 0.0001).

[0015] Fig. 4: K-Ras over H-Ras selectivity of compounds determined by nanoclustering- FRET. K-RasG12V and H-RasG12V nanoclustering-FRET in HEK293 cells. Cells were co- transfected with pmGFP-K-RasG12V and pmCherry-K-RasG12V or pmGFP-H-RasG12V and pmCherry-H-RasG12V, respectively. After 24 h cells were treated with 0.1% DMSO vehicle control or different concentrations of compounds for 24 h. The apparent FRET efficiency was calculated from FLIM data (meant s.e.m., n= 3 for K-RasG12V and n=2 for H-RasG12V). The U101206 numbers in the bars indicate the number of analyzed cells. Statistical significance of differences between control and treated cells were examined using one-way ANOVA complemented with Tukey's tests (n.s., not significant; ** p < 0.01, **** p < 0.0001).

[0016] Fig. 5: Deltaflexins selectively inhibit oncogenic K-Ras driven mammosphere formation. Sphere formation efficiency (SFE) of (A) MDA-MB-231 cells , n 2 4, and (B) Hs 578T cells (D,F), n = 4, cultured in suspension culture for 6 days, followed by a 72h incubation with 10 uM compounds A, B, C, D, or Deltarasin, 0.5 uM FTI277 or 0.1% DMSO control. Error bars denote +SEM. Statistical significance of differences between control and treated cells were examined using one-way ANOVA complemented with Tukey's tests. Statistical significance levels are annotated as ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001.

DETAILED DESCRIPTION OF THE INVENTION HUT01200

[0017] 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

[0018] 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, 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.

[0019] The term "aryl", also abbreviated herein as “Ar”, refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5 to 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl.

[0020] The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 14 carbon atoms, such as 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, or 10 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, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl. The term "cycloalkyl" is also meant to include bicyclic and tricyclic versions thereof. If bicyclic rings are formed it is preferred that the respective rings are connected to each other at two adjacent carbon atoms, however, alternatively the two rings are connected via the same carbon atom, i.e., they form a spiro ring system or they form "bridged" ring systems. Preferred examples of cycloalkyl include C3-Cg-cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, bicyclo[4.1.0jheptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl, and bicyclo[4.2.0joctyl.

[0021] The term “substituted” means, that in the respective underlying group at least oneLU101206 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.

[0022] 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.

[0023] The term "halogen", also abbreviated herein as “Hal” means fluoro, chloro, bromo, or iodo, preferably fluoro.

[0024] "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.

[0025] Diastereomers" are stereoisomers which are non-superimposable and not mirror-images of each other.

[0026] 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.

[0027] "Tautomers" are structural isomers of the same chemical substance that spontaneously interconvert with each other, even when pure.

[0028] 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-LU101206 stoichiometric. A "hydrate" is a solvate wherein the solvent is water.

[0029] 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, "SN, 18F, 325, CI, and ‘1. Compounds

[0030] The present invention is directed to a compound according to formula (1) R ~ _R aka,

I O=F-OR,

O LA À

O (I) wherein

[0031] n is an integer between 1 and 10. n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably 4 to 10; more preferred 5 to 10; most preferably 6 to 10, particularly preferred 6 to 9, more particularly preferred 6.

[0032] Rı is H, -CORe, -SOzRg, or -CH2Rg, preferably -CORe;

[0033] R; is H, -CORe, -SO;Rs, or -CH:Rg, preferably H;

[0034] Rs may be present in R; and R, and may be a different group if present in R, and Ra.

[0035] Rs is independently selected from substituted or unsubstituted -(Cz-C,4)Ar, preferably substituted or unsubstituted -(Cs-C+0)Ar;

[0036] If Rs is substituted -(Cz-C-4)Ar, , one, two or more substituents may be present. Preferably, at least one, more preferably at least two substituents are present.

[0037] Preferably, the one or more first level substituents of -(C:-C,4)Ar, are independently U101206 selected from COOH, -COO(C,-C;)Alkyl, -NO,, -Hal, -O(C-Cg)Alkyl, -CHals, -(C;-Ce)Alkyl, -(Cs. C44)Cycloalkyl, and -NH,.

[0038] The first level substituents -O(C;-Cs)Alkyl, -(C1-Ce)Alkyl, -(C3.C14)Cycloalkyl, -COO(C:- Ce)Alkyl and —-NHz may themselves be substituted with a second level substituent selected from from -COOH, -COO(C;-Cs)Alkyl, -NO,, -Hal, -O(C;-Ce)Alkyl, -CHalz, -(C,-Cs)Alkyl, -(C3. C14)Cycloalkyl, and -NHz.

[0039] Preferably Re is -(Cs-C44)Ar, more preferably phenyl.

[0040] R; is -(C,-Cs)Alkyl, preferably methyl or ethyl;

[0041] R4 is —-CO2(C,-Ce)Alkyl or -(C,-Cs)Alkyl, preferably —CO,Et or methyl

[0042] Rs is -(C;-Ce)Alkyl, or substituted or unsubstituted -(C;-C44)Ar, preferably methyl, phenyl, -t-Bu; more preferably methyl; and a solvate, hydrate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.

[0043] Preferably, the salt is a pharmaceutically acceptable salt.

[0044] 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.

[0045] In one embodiment, Rs is -(Cs-C44)Ar, preferably phenyl, substituted with at least two substituents, independently selected from -COOH, -COO(C;-Cg)Alkyl, and -NHa.

[0046] In a further embodiment, Re is phenyl, substituted with at least two substituents, wherein one substituent is in para-position, selected from the group consisting of -COOH and -COO(C--

Ce)Alkyl and a second substituent is in the meta-position, selected from the group consisting of_ 101206 NH. Are DO 0000

[0047] In a further embodiment, Rs is NH2 or NH;

[0048] In a preferred embodiment R;ı is —CORe, Re is H, and Re is substituted or unsubstituted -(Cs-C40)Ar.

[0049] In an even more preferred embodiment Ry is “COR, Ra is H “Ore Aero and Re is NH; or NH; preferably 001500 NH, |

[0050] À selection of compounds according to the present invention is listed in the following Table 1. These compounds are designated as Compound A to Compound D. Table 1 3, % R-0 COOMe RO COOMe 0 00 0 O PL

S

HN — 0” “OEt HN = >

O O B A

Js + Js =< U101206 ÿ COOMe 0 COOMe O 0-P-0 O-P-0 OEt 0 a ) > Lö a | Sn

HN HN 0 O

C D Pharmaceutical Composition and Medical Use

[0051] A further aspect of the present invention is directed to a pharmaceutical composition comprising at least one of the inventive compounds.

[0052] “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.

[0053] “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 U101206 mode of administration.

[0054] "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.

[0055] A further embodiment of the invention is directed to the inventive compounds or pharmaceutical composition for use in medicine.

[0056] A further embodiment of the invention is directed to the inventive compounds or pharmaceutical composition for use in the treatment of cancer.

[0057] Preferably, the cancer is selected from K-Ras dependent cancers or from cancers wherein the KRAS gene is mutated.

[0058] 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 breast cancer. xkxkx

[0059] 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. Preparation of Compounds

[0060] N,N,N ,N -tetraisopropyl-1-methoxyphosphanediamine, 1-chloro-N,N-diisopropyl-1- methoxyphosphinamine, 0.45 M 1H-tetrazole solution in CH;CN, 1 M trimethylphosphine solution in toluene, 3-amino-4-(methoxycarbonyl)benzoic acid and 2,6-lutidine were commercial products of Sigma-Aldrich.

[0061] 4-Aminobutanol and 6-aminohexanol were commercial products of TCI. (4- (Aminomethyl)cyclohexyl)methanol was commercial products of Carbosynth. Solvents were purchased from the companies Sigma Aldrich, VWR and Thermo Fisher Scientific. Pyridine,

DMF, MeCN, DCM, EtOAc and 0.45 M 1H4etrazole solution in CH3;CN were dried overLU101206 molecular sieves (3 or 4 A). TEA was dried by refluxing over CaH, and distilled before use

[0062] The preparation of ethyl 4-(acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxobutanoate (26) and S-(4-Hydroxy-3,3-dimethyl-2-oxobutyl)ethanethioate (27), have been described previously

[10]. CO,Me CO2Me CO,Me or | of Hon OH 0 N,

DO COH COzH CANTON acid CO»Me CO:Me COIMe À, NH has HaN SOR A N; OT. CO EI OO CO‚H Lom ! Jd ae az i) HATU, DIPEA, DMF | Scheme 1: Synthesis of precursor A1 and A2

[0063] Methyl 2-azido-4-((4-hydroxybutyl)carbamoyl)benzoate (A1). 3-Amino-4- (methoxycarbonyl)benzoic acid (5.0 g, 25.6 mmol) was dissolved in a mixture of H,O (35.7 ml) and concentrated HCI (43 ml) at 0 °C and a solution of sodium nitrite (2.00 g, 29.4 mmol) was added. The mixture was stirred for 20 min. and sodium azide (5.13 g, 78.9 mmol) was added drop wise upon 1 h. The mixture was allowed to warm up and stirred overnight at room temperature. The reaction mixture was diluted in water and extracted with ethyl acetate. The organic phase was separated, dried over Na,SQ,, filtrated and evaporated to dryness to obtain 50 g (88%) of the crude azide.To a solution of the crude azide (3-azido-4- (methoxycarbonyl)benzoic acid) (5.00 g, 22.6 mmol) and HATU (12.9 g, 33.6 mmol) in DMF (50 ml), DIPEA (11.8 ml, 67.8 mmol) was added. After 5 min, a solution of 4-aminobuthanol (3.02 g,

33.9 mmol) in DMF (10 ml) was added and the reaction solution was stirred at 60 °C for 1h. The solvent was evaporated under reduced pressure and the residue was purified by silica gel chromatography eluting with a mixture of DCM and MeOH (98:1, vA). Compound A1 was obtained as a white solid in 48% yield (3.2 g). 'H NMR (600 MHz, CD;CN): à = 7.83 (d, 1H, J =

8.1 Hz, Ar), 7.68 (d, 1H, J = 1.5 Hz, Ar), 7.62 — 7.59 (m, 1H , Ar), 7.39 (br s, NH), 3.88 (s, 3H,

OCHj3), 3.56 (m, 2H, CH2OH), 3.39 (m, 2H, NCH, 2.76 (m, 1H, OH), 1.69 — 1.63 (m, 2H, CH,),LU101206

1.60 — 1.55 (m, 2H, CH,). °C NMR (151 MHz, CD;CN): à = 165.2 (OCO), 165.1 (NCO), 139.8,

139.2, 131.4, 125.0, 123.0 and 119.2 (Ar), 61.3 (CH,OH), 52.1 (OCH;), 39.6 (NCH,), 29.9 and

25.8 (2 x CHz). HRMS (ESI) m/z: [M+Na]" calcd for C13H16N,04Na* 315,1064; found 315,1063.

[0064] Methyl 2-azido-4-((6-hydroxyhexyl)carbamoyl)benzoate (A2). To a solution of 3- azido-4-(methoxycarbonyl)benzoic acid (5.00 g, 22.6 mmol) and HATU (12.9 g, 33.6 mmol) in DMF (50 mi), DIPEA (11.0 mi, 67.8 mmol) was added. After 5 min, a solution of 6-aminohexanol (3.97 g, 33.9 mmol) in DMF (10 ml) was added and the reaction solution was stirred at 60 °C for 1h. The solvent was evaporated under reduced pressure and the residue was purified by silica gel chromatography eluting with a mixture of DCM and MeOH (98:1, vA). Compound A2 was obtained as a white solid in 51% yield (3.0 g). 'H NMR (600 MHz, CDsCN): 6 = 7.86 (d, 1H, J =

7.9 Hz, Ar), 7.70 (s, 1H, Ar), 7.61 (m, 1H, Ar), 7.23 (br s, NH), 3.90 (s, 3H, OCH3), 3.51 (m, 2H, CH,0H), 3.38 (m, 2H, NCH), 2.76 (s, unknown), 2.51 (m, 1H, OH), 2.19 (s, unknown), 1.66 —

1.58 (m, 2H, CH), 1.54 — 1.48 (m, 2H, CH), 1.43 — 1.37 (m, 4H, 2 x CH,). °C NMR (151 MHz, CD+4CN): à = 165.3 (OCO), 165.1 (NCO), 139.8, 139.3, 131.3, 125.1, 123.1, and 119.2 (Ar), 61.5 (CH2OH), 52.1 (OCHz), 39.6 (NCH), 37.9 (unknown), 32.5, 29.1, 26.5, 25.3 (4 x CH,). HRMS (ESI) m/z: [M+Na]’ calcd for C+5sHz0N,0,Na* 343,1377; found 343,1374.

Ns AH LU101206 0 \ HN—(CH,)5

FN 0 9 Sn ort Pr Sog MeO 9 Ho PrN MeO _ 9 A2 T “SAC + === PEN J SA; —" 26 0 Na G 0 Nj 0 AA «JOE | N : > OMe ~~ “Ft wl) gre gp Bs AN, 0 Fo TT = “(che À / SA > “(CHyle Jo sac" J (CHals 0 9 © 0 i TetH, MeCN | ii) TetH, MeCN iii} 1, THF, H,O, 2,6-lutidine iv) PMes in toluere, THF, H,O OH i SR Oo N AC 214 9 0 LC >< et MeO, 3 MeO =O DOE *P-NiPr, “OE pd PrN MeO, ? A1 Ho r=, i TOT gap ee G SA eee PON g Sac 26 Q Nz 0 9 Ng oO Wo u que x >OEt ä Meo ® N OMe Sa v. € >; =. „Or m — > N „oO pp — O0 — 7 _ UY (CH: J sac SAN (CHa © Jo SAc 0 0 Scheme 2: Synthesis of Compound A and C

[0065] Methyl 4-((6-(((4-(acetylthio)-2-(ethoxycarbonyl)-2-methyl-3- oxobutoxy)(methoxy)phosphoryl)oxy)hexyl)carbamoyl)-2-aminobenzoate (A). Ethyl 4- (acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxobutanoate (26) (0.17 g, 0.67 mmol) was dissolved in CH3CN (3.0 ml) under nitrogen. N,N,N',N'-tetraisopropyl-1-methoxyphosphanediamine (0.23 mi, 0.79 mmol) and 0.45 M 1H-tetrazole solution in CH;CN (1.49 mi, 0.67 mmol) were added and the mixture was stirred at room temperature for 30 minutes. The course of the phosphitylation was followed by *'P NMR spectroscopy (126 MHz, CD3CN, the product: à =

149.4 and 149.2 ppm). Methyl 2-azido-4-((6-hydroxyhexyl)carbamoyl)benzoate (A2) (0.30 g,

0.93 mmol) together with 0.45 M solution of 1H-tetrazole in CH3CN (4.15 mi, 1.86 mmol) was then added to the reaction mixture under nitrogen (*'P NMR: à = 139.9 ppm for the phosphite triester). The mixture was stirred for 15 min and the phosphite ester was oxidized with I, (0.20 g,

0.79 mmol) in a mixture of THF (3.0 ml), HO (1.5 ml) and 2,6-lutidine (0.75 ml) by stirring overnight at room temperature. The solvent was evaporated and the residue was dissolved in DCM and washed with 5% aq NaHSO; (100 ml). The organic phase was separated, dried over

Na,SO, and evaporated to dryness. To reduce N3 to NH, group, the crude product was| J101206 dissolved in a mixture of THF (6.0 ml) and H,O (1.2 ml). 1 M trimethylphosphine solution (0.76 ml) in toluene was added and the mixture was stirred for 15 min. The residue was dissolved in DCM and washed with H,O and brine. The organic phase was dried over Na,SO, and evaporated to dryness. The crude product was purified by RP-HPLC (Phenomenex 250 X 10, Synenergi 4um Fusion-RP 80A, flow rate 3 ml/min) by using isocratic elution with 46% CH,CN in HO and then a gradient elution from 46% to 80% CH4CN in H,O over 5 min. The product fractions were combined and lyophilized to yield 46 mg (11%) of the product as a viscous oil. 'H NMR (500 MHz, CDCks): à = 7.89 (d, 1H, J = 8.0 Hz, Ar), 7.17 (d, 1H, J = 1.5 Hz, Ar), 6.94 (m, 1H, Ar), 6.43 (br m, NH,), 4.48-4.37 (m, 2H, POCH,C,), 4.27 (q, 2H, J = 7.5 Hz, OCH,CHjs),

4.07 (m, 2H,POCH,), 4.04-3.93 (m, 2H, and SCH,), 3.90 (s, 3H, OCH3), 3.77 (2 x d, 3H, J= 11.0 Hz, POCHz), 3.45 (m, 2H, NCH), 2.37 (2 x s, 3H, AcS), 1.71 (quintet, J = 6.5 Hz, 2H, CH),

1.64 (quintet, J = 7.0 Hz, 2H, CH,), 1.58 (s, 3H, CH;), 1.47-1.43 (m, 4H, CH,CH,), 1.30 (t, 3H, J = 7.5 Hz, OCH,CH;). "°C NMR (126 MHz, CDCl;): à = 198.8 (CO), 193.7 (SCO), 169.6 and

169.5 (COOEt), 168.0 (COOMe), 167.0 (NCO), 150.5, 139.8, 131.7, 115.6, 113.7 and 112.5 (Ar), 69.1 and 69.0 (POCH,), 67.9 and 67.9 (POCH,), 62.4 (OCH;CHs), 60.2 and 60.1 (quaternary C), 54.5 and 54.4 (POCHsz), 51.7 (OCH), 39.7 (NCH), 36.7 (CH,S), 30.1 (CH-CO),

30.0, 29.9 and 29.3 (CHz), 26.1 (CH), 24.7 (CH,), 17.6 (CH3), 14.0 (OCH2CHs). *'P NMR (202 MHz, CDsCN): 6 = -0.38 ppm. HRMS (ESI) m/z. [M+Na]' calcd for CyH3gN,O:/PSNa*

641.1904; found 641.1897.

[0066] Methyl 4-((4-(((4-(acetylthio)-2-(ethoxycarbonyl)-2-methyl-3- oxobutoxy)(methoxy)phosphoryl)oxy)butyl)carbamoyl)-2-aminobenzoate (C). Ethyl 4- (acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxobutanoate (26) (0.18 g, 0.73 mmol) was dissolved in CH:CN (3.0 ml) under nitrogen. N,N,N',N'-tetraisopropyl-1-methoxyphosphanediamine (0.24 mi, 0.84 mmol) and 0.45 M 1H-tetrazole solution in CH3CN (1.61 ml, 0.73 mmol) were added. The mixture was stirred at room temperature for 30 minutes upon which the course of the phosphitylation was followed by *'P NMR spectroscopy (126 MHz, CD;CN: à = 149.4 and 149.2 ppm for the product). Methyl 2-azido-4-((4-hydroxybutyl)carbamoyl)benzoate (A1) (0.28 g, 0.96 mmol) was added in 0.45 M 1H-tetrazole solution in CH;CN (4.27 mi, 1.92 mmol) under nitrogen (*'P NMR: à = 139.9 ppm for the phosphite triester). After stirring 5 min, the phosphite ester was oxidized with |, (0.21 g, 0.84 mmol) in a mixture of THF (3.0 ml), H,O (1.5 ml) and 2,6-lutidine (0.75 ml) by stirring at room temperature overnight. The solvent was evaporated to dryness and the residue was dissolved in DCM and washed with 5% aq NaHSO; (100 mi). The organic phase was dried over Na,SO, evaporated to dryness. To reduce N3 to NH, group, the crude product was dissolved in a mixture of THF (6.0 ml) and H,O (1.2 ml). 1 M trimethylphosphine solution (0.70 ml) in toluene was added and the mixture was stirred 15 min. The residue was U101206 dissolved in DCM and washed with H,O and brine. The organic phase was dried over Na,SO, and evaporated to dryness. The crude product was purified by RP-HPLC (Thermo Scientific 250 X 10 Hypersil ODS 5um, flow rate 3 ml/min) by using isocratic elution with 35% CH3CN in H,O to yield C as a viscous oil (60 mg, 18%). 'H NMR (500 MHz, CDCIs): à = 7.91 (d, 1H, J = 8.4 Hz, Ar), 7.20 (d, 1H, J = 1.2 Hz, Ar), 7.00 (d, 1H, J = 8.4 Hz, Ar), 6.60 (br s, NH), 4.50 — 4.37 (m, 2H, POCH,C,), 4.27 (quartet, 2H, J = 7.2 Hz, OCH,CHj3), 4.13 (m, 2H, POCH,), 4.00 (m, 2H, SCH), 3.91 (s, 3H, OCH), 3.78 (d, 3H, J = 11.2 and 1.0 Hz, POCH;), 3.50 (m, 2H, NCH2), 2.36 (s, 3H, AcS), 1.83-1.73 (m, 4H, CH2CH2), 1.58 (d, 3H, J = 2.4 Hz, CH3C,), 1.31 (t, 3H, J = 7.1 Hz, OCH:CH;). *C NMR (126 MHz, CDCIs): § = 198.9 (CO), 193.9 (SCO), 169.6 (COOEt),

168.0 (COOMe), 166.9 (NCO), 150.0, 139.6, 131.7, 115.9, 114.3 and 112.9 (Ar), 69.2 and 69.1 (POCHzC;), 68.0 and 67.9 (POCHy), 62.4 (OCH,CHj3), 60.2, 60.1, 60.0 and 60.0 (quaternary C),

54.6 and 54.5 (POCHsz), 51.8 (OCHj3), 39.5 (NCH2), 36.8 and 36.7 (CH,S), 30.1 (CH;COS),

27.6 and 25.5 (CHzCHz), 17.6 (CH3Ca), 14.0 (CH:CH20). *'P NMR (202 MHz, CDCl3): & = -0.46 and -0.50 ppm. HRMS (ESI) m/z. [M+Na]' calcd for C,4H35N2O4+PSNa* 613.1591; found

613.1579.

OH LU101206 a HN (Cha) O nr - 246 OS © MeO. Nip wd a Le PEN 7 MeO SC A2 DD iPraN Oy | N Ho Isa man Pin” © J "BAG me 27 ON hi Ns 3 7 _ LS OMe , ~~ TB MeO” <Q N Que Na MeO Cl H ope DN iv N._ OP OL SO SCHE SA J (CH J SAC Tr (Cols 6 © © 0 i} TetH, MeCN it) TetH, MeCN iii) I». THF H,0. 2 6-lutidine iv) PMe, in toluene, THF, HO

SE OH a HN- ch + a {CHa MeO, Ni MeO Nm © vs —NiPr, Se Xx PLN MeO X A1 HO — T sac - +. mms cc PON © J sae ee ML Tn re eS 27 Oo Na O ON | “ À 5 Mo OMe WT vo > à H Que x iv P la 1 N 0P-0 " WALA O0 sa SC T SCH, © Sac Ô (CH2k © 0 © Scheme 3: Synthesis of compound B and D

[0067] Methy! 4-((6-(((4-(acetylthio)-2,2-dimethyl-3- oxobutoxy)(methoxy)phosphoryl)oxy)hexyl)carbamoy!)-2-aminobenzoate (B). S-(4- hydroxy-3,3-dimethyl-2-oxobutyl)ethanethioate (27) (0.13 g, 0.70 mmol) was dissolved in CH3CN (3.0 ml) under nitrogen. N,N,N ,N-tetraisopropyl-1-methoxyphosphanediamine (0.22 mi,

0.78 mmol) and 0.45 M 1H-tetrazole solution in CH;CN (1.56 mi, 0.70 mmol) were added and the mixture was stirred at room temperature for 30 minutes. The course of the phosphitylation was followed by *'P NMR spectroscopy (126 MHz, CDzCN: à = 148.5 ppm for the product). Methyl 2-azido-4-((6-hydroxyhexyl)carbamoyl)benzoate (A2) (0.30 g, 0.94 mmol) was added together with a 0.45 M 1H-tetrazole solution in CH3CN (4.16 ml, 1.87 mmol) under nitrogen (SP NMR: à = 139.5 ppm for the phosphite triester). After stirring 15 min, the phosphite ester was oxidized with I» (0.20 g, 0.78 mmol) in a mixture of THF (3.0 ml), H,O (1.5 ml) and 2,6-lutidine (0.75 ml) by stirring at room temperature overnight. The solvent was evaporated to dryness and the residue was dissolved in DCM and washed with 5% aq NaHSO; (100 ml). The organic phase was dried over Na:SO, and evaporated © dryness. To reduce N; to NH, group, the crudel U101206 product was dissolved in a mixture of THF (4.0 ml) and H,O (0.8 ml). 1 M trimethylphosphine solution (0.46 ml) in toluene was added and the mixture was stirred 15 min. The residue was dissolved in DCM and washed with H,O and brine. The organic phase was dried over Na,SO, and evaporated to dryness. The crude product was purified by RP-HPLC (Phenomenex 250 X 10, Synenergi 4um Fusion-RP 80A, flow rate 3 ml/min) by using isocratic elution with 46% CH3CN in HO and then gradient elution from 46% to 80% CH3CN over 5 min. Compound B was obtained as a viscous oil (22 mg, 6%). 'H NMR (500 MHz, CDCIs): 5 = 7.85 (d, 1H, J = 8.0 Hz, Ar), 7.17 (d, 1H, J = 1.5 Hz, Ar), 7.04 (br s, NH), 6.93 (dd, 1H, J = 8.0, and 1.5 Hz, Ar), 6.17 (br s, NH), 4.07 (d, 2H, J = 5.0 Hz, POCH,C,), 4.04-4.00 (m, 2H, POCH; and SCH), 3.86 (s, 3H, OCHj3), 3.71 (d, 3H, J = 11.0 Hz, POCH;), 3.44 (m, 2H, NCH,), 2.35 (s, 3H, AcS), 1.68 (quintet, J = 7.0 Hz, 2H, CH), 1.59 (quintet, J = 7.0 Hz, 2H, CH2), 1.45-1.40 (m, 4H, CHzCH2),

1.24 (s, 6H, 2 x CHz). °C NMR (126 MHz, CDCIs): 6 = 205.6 (CO), 194.4 (SCO), 167.9 (COOMe), 166.4 (NCO), 151.0, 140.3, 131.2, 115.4, 113.4 and 111.6 (Ar), 72.3 and 72.2 (POCH,), 67.7 and 67.6 (POCH;z), 54.0 and 53.9 (POCHz), 51.3 (OCH), 48.5 (quaternary C),

39.2 (NCH), 36.1 (CH.S), 29.8 and 29.7 (CH), 29.4 (CH;CO), 29.0 (CH), 26.0 (CH,), 24.7 (CH), 20.7 (CHz). *'P NMR (202 MHz, CDsCl): 6 = -0.43 ppm. HRMS (ESI) m/z: [M+Na]" calcd for C24H37N20,PSNa' 583.1850; found 583.1825. The synthesis of B was repeated by the company of Piramal Enterprises Ltd with a similar strategy, but with slightly different reagent ratios. S-(4-Hydroxy-3,3-dimethyl-2-oxobutyl)ethanethioate (27) (1.0 g, 5.26 mmol) was dissolved in CHCN (100 ml) under nitrogen. N,N,N ,N-tetraisopropyl-1- methoxyphosphanediamine (1.52 g, 1.66 mi, 5.79 mmol) and 1H-tetrazole (0.37 g, 5.26 mmol) were added and the mixture was stirred at room temperature for 30 minutes. Methyl 2-azido-4- ((6-hydroxyhexyl)carbamoyl)benzoate (A2) (0.84 g, 2.63 mmol) was added together with a 0.45 M 1H-tetrazole solution in CH3CN (11.7 mi, 5.26 mmol) under nitrogen. After stirring 15 min, the phosphite ester was oxidized with I, (3.0 g, 6.3 mmol) in a mixture of THF (11.4 mi), H,O (5.6 ml) and 2,6-lutidine (6.5 ml) by stirring at room temperature for 1 h. LCMS analysis showed 33% product conversion. The solvent was evaporated and the residue was dissolved in DCM and washed with 5% aq NaHSO; (100 ml). The organic phase was separated, dried over Na,SO, and evaporated to dryness. The crude product was passed through a silica column eluting with a 7:3 (vW) mixture of hexane and ethylacetate and used in a next step without further purification. To reduce N3 to NH; group, the crude product (1.0 g, 1.70 mmol) was dissolved in a mixture of THF (10.0 ml) and H,O (6.0 ml). Trimethylphosphine (0.16 g, 2.04 mmol) (1 M solution in toluene) was added and the mixture was stirred 15 min. The residue was dissolved in DCM and washed with H,O and brine. The organic phase was dried with Na,SO, and evaporated to dryness. The crude product was purified by RP-HPLC by using isocratic elution with 46% CH3CN in HO. Compound B was obtained as a viscous oil (0.4 g, 42 %). The synthesis was repeated to yield compound B 27 g. 'H NMR (500 MHz, CD3CN): à = 7.85 (d; y101206 1H, J= 8.2 Hz, Ar), 7.17 (d, 1H, J = 1.6 Hz, Ar), 7.06 (br s, NH), 6.93 (dd, 1H, J = 8.2, and 1.6 Hz, Ar), 6.17 (br s, NH), 4.07 (d, 2H, J = 4.7 Hz, POCH,C,), 4.04-4.00 (m, 4H, POCH, and SCH), 3.86 (s, 3H, OCHz), 3.71 (d, 3H, J= 11.1 Hz, POCH;), 3.34 (m, 2H, NCH), 2.35 (s, 3H, AcS), 1.68 (quintet, J = 6.8 Hz, 2H, CH2), 1.59 (quintet, J = 7.0 Hz, 2H, CH,), 1.44-1.39 (m, 4H, CH2CH2), 1.24 (s, 6H, 2 x CHz). ®C NMR (126 MHz, CDCl): § = 205.6 (COC,), 194.4 (SCO),

167.9 (COOMe), 166.4 (NCO), 151.0, 140.3, 131.2, 115.4, 113.4 and 111.6 (Ar), 72.4 and 72.3 (POCHC,), 67.7 and 67.6 (POCH), 54.0 and 53.9 (POCHs), 51.3 (OCH,), 48.5 (quaternary C),

39.3 (NCH), 36.1 (CH,S), 29.9 and 29.8 (CHz), 29.4 (CH:CO), 29.0 (CH,), 26.0 (CHz), 24.8 (CHa), 20.7 (CHz). *'P NMR (202 MHz, CDsCl): à = -0.43 ppm. HRMS (ESI) m/z: [M+Na]* calcd for C24Hz3N20g9PS* 561.2030; found 561.2032.

[0068] 4-((4-(((4-(acetylthio)-2,2-dimethyl-3- oxobutoxy)(methoxy)phosphoryl)oxy)butyl)carbamoyl)-2-aminobenzoate (D). S-(4- hydroxy-3,3-dimethyl-2-oxobutyl)ethanethioate (27) (0.136 g, 0.71 mmol) was dissolved in CH;CN (3.0 mi) under nitrogen. N,N,N',N-tetraisopropyl-1-methoxyphosphanediamine (0.24 ml,

0.83 mmol) and 0.45 M 1H-tetrazole solution in CH3CN (1.59 ml, 0.72 mmol) were added and the mixture was stirred at room temperature for 30 minutes. The course of the phosphitylation was followed by *'P NMR spectroscopy (126 MHz, CD;CN: à = 149.2 ppm for the product). Methyl 2-azido-4-((4-hydroxybutyl)carbamoyl)benzoate (A1) (0.29 g, 0.94 mmol) was added together with 0.45 M 1H-tetrazole solution in CH;CN (4.17 ml, 1.88 mmol) under nitrogen (*'P NMR: à = 139.9 ppm for the phosphite triester). After 10 min stirring, the phosphite ester was oxidized with I, (0.21 g, 0.83 mmol) in a mixture of THF (3.0 ml), H,O (1.5 ml) and 2,6-lutidine (0.75 ml) by stirring at room temperature overnight. The solvent was evaporated and the residue was dissolved in DCM and washed with 5% aq NaHSO; (100 ml). The organic phase was dried over Na,SO, and evaporated to dryness. To reduce N; to NH, group, the crude product was dissolved in a mixture of THF (6.0 ml) and H,O (1.2 mi). 1 M trimethylphosphine solution (0.70 ml) in toluene was added and the mixture was stirred for 15 min. The residue was dissolved in DCM and washed with H,O and brine. The organic phase was separated, dried over Na:SO, and evaporated to dryness. The crude product was purified by RP-HPLC (Thermo Scientific 250 X 10 Hypersil ODS 5um, flow rate 3 ml/min) by using isocratic elution with 34% CHzCN in H,O to yield D as a viscous oil (23 mg, 6%). 'H NMR (500 MHz, CDCI;): à = 7.90 (d, 1H, J=8.5 Hz, Ar), 7.17 (d, 1H, J = 1.5 Hz, Ar), 6.98 (dd, 1H, J = 8.5, and 1.5 Hz, Ar), 6.63 (br s, NH), 5.92 (br s, NH,), 4.14-4.07 (m, 4H, POCHzC,, POCH2), 3.99 (s, 2H, SCH), 3.90 (s, 3H, OCHz), 3.78 (d, 3H, J= 11.0 Hz, POCHj3), 3.55-3.47 (m, 2H, NCH,), 2.35 (s, 3H, AcS), 1.87-1.73 (m, 4H, CH,CH,), 1.30 and 1.29 (2 x s, 6H, 2 x CHz). *C NMR (126 MHz, CDCIs): § = 205.7 (CO), 194.5 (SCO), 168.0 (COOMe), 167.0 (NCO), 150.5, 139.6, 131.7, 115.7, 113.8 and

112.5 (Ar), 72.9 and 72.8 (POCH,), 67.8 and 67.7 (POCH,), 54.5 and 54.4 (POCHs), 51.7LU101206 (OCH), 48.8 and 48.7 (quaternary C), 39.5 (NCH,), 36.5 (CH,S), 30.2 (CH3CO), 27.7, 27.6 and

25.5 (CH,CH,), 21.5 and 21.5 (CHz). *'P NMR (202 MHz, CDCIs): & = -0.35 ppm. HRMS (ESI) m/z. [M+Na]" calcd for Cz2Hz3N,O,PSNa* 555.1537; found 555.1520.

2. Biological Testing Example 2.1: Fluorescence Polarization Assay to assess direct competition of inventive compounds with a PDEJ substrate

[0069] Using a previously published fluorescence polarization assay, which detects displacement of a fluorescently labelled, farnesylated peptide derived from the PDE5 client Rheb (F-Rheb) from PDEJ, the binding of the compounds A and B to PDE3 (Ismail et al., 2011

[7]) has been analysed in vitro.

[0070] The binding of compounds to PDE6D was assessed in a fluorescence polarization (FP) assay using fluorescein-tagged farnesylated Rheb (F-Rheb) peptide (Blazevits, Sci report 2016

[14]) as the probe. The F-Rheb peptide was a gift from Eyad K. Fansa (Dortmund, Germany) and PDE6D was produced in-house, adapted as described (Ismail et al., 2011 [7]). FP assays were performed on a black low volume, round-bottom, non-binding surface 384-well plate (Corning, #4514) in an assay buffer composed of 30 mM Tris, 150 mM NaCl and 3 mM dithiothreitol. To a three-fold dilution of compounds in assay buffer, complex of 0.25 uM F-Rheb peptide and 2 uM PDE6D was added and the reaction mix was incubated for 15 min at RT. Then the FP was recorded on Synergy H1 hybrid plate reader (Biotek) equipped with a polarization cube with excitation wavelength of 485 + 10 nm and emission wavelength of 528 + nm. The FP data were plotted against the logarithmic concentration of compounds and fit into log inhibitor vs response variable slope (four parameters) equation in GraphPad and the ICso was determined.

[0071] The results are listed in Table 2.

Table 2. Affinity of compounds cE

Example 2.2: The inventive compounds. suppress K-Ras/PDEdelta interaction and y101206 selectively K-Ras membrane organization

[0072] Next, on-target activity in cells has been tested, using a FRET-assay which directly detects the interaction between K-RasG12V and PDEdelta. A FRET-reporter consisting of mGFP-K-RasG12V and mCherry-PDEdelta was transiently expressed in HEK293 (hereafter HEK) cells and loss of FRET after incubation of cells with compounds was monitored using Fluorescence Lifetime Imaging Microscopy (FLIM).

[0073] Cell Culture: Human Embryonic Kidney (HEK) 293-EBNA, Hs 578T cell lines were maintained in Dulbecco's modified Eagle’s medium (DMEM, Cat. No. D6171, Sigma-Aldrich, Helsinki, Finland), supplemented with 10% Fetal Bovine Serum (Cat. No. S1810, Biowest, Nuaille, France) and 2 mM L-glutamine (Cat. No. G7513, Sigma-Aldrich). MDA-MB-231 cells were cultured in Roswell Park Memorial Institute medium (RPMI, Cat. No. R5886, Sigma- Aldrich), containing 10% FBS and 2 mM L-glutamine. All cells were incubated at 37 °C, with 5% CO,, in a humidified cell incubator.

[0074] DNA constructs: Plasmids pmGFP/mCherry-H-rasG12V and pmGFP/mCherry-K- rasG12V were previously described (Posada et al., 2017b [12], Najumudeen et al., 2016 [16]). Plasmids for mCit-Rheb and mCherry-PDEd have been previously described (Chandra et al., 2011 [17)).

[0075] Fluorescence Lifetime Imaging Microscopy (FLIM)-FRET: HEK293 EBNA cells were seeded in 12-well plates onto sterile cover slips. The next day, cells were transfected using FuGENE HD (Cat. No. E2311, Promega Biotech AB, Nacka, Sweden) or jetPRIME (Cat. No. 114-75, Polyplus, Illkrich-Graffenstaden, France) transfection reagent with a total of 800 ng of plasmids. For donor samples, cells were transfected with mGFP or mCitrine-tagged plasmid. In FRET pairs donor and acceptor were added at a 1:3 ratio. Acceptors were cherry tag plasmids. After 24 h of transfection, cells were treated with either 0.1% DMSO control or various concentrations of test compound or Deltarasin (Cat. No. 9001536, Cayman Chemical, Tallinn, Estonia), or FTI (Cat. No. Sc-215058, Santa Cruz Biotechnology, Dallas, USA) for 24 h and fixed in 4% PFA for 12 minutes before mounting with Mowiol 4-88 (Cat. No. 81381, Sigma— Aldrich). The donor fluorescence lifetime was measured using a fluorescence microscope (Zeiss AXIO Observer D1) with a fluorescence lifetime imaging attachment (Lambert Instruments, Groningen, The Netherlands), as previously described in Guzman et al., 2016 [15] and Najumudeen et al., 2016 [16].

[0076] In agreement with the in vitro data, compound B performed better than A in disrupting K- RasG12V/ PDEdelta binding. Compounds C and D, which are identical to compounds A and B,

respectively, except for their shorter, tetramethy middle moiety (Figure 1), performed worse; 101206 Their activity relation furthermore showed that the 2,2-dimethyl-substitution in the protecting group is preferable, in particular in the cellular context.

Example 2.3: K-Ras selectivity

[0077] A previously developed additional cellular FRET-assay has been employed, which is able to detect the functional membrane anchorage of Ras proteins in an isoform-specific manner and with higher sensitivity than confocal imaging (Posada et al., 2017a; [11] 2017b

[12]). By coexpressing FRET-pairs of Ras proteins, one can observe FRET due to nanoscale clustering (nanoclustering-FRET) of Ras oligomers, including dimers, on the membrane (Prakash et al., 2017 [13}).

[0078] The reference PDEdelta-inhibitor, Deltarasin, reduced FRET of both Ras isoforms (Figure 4). This indicates off-target effects of Deltarasin that may underlie its relatively high general toxicity (Papke et al., 2016, [9]).

[0079] The preference of a 6-membered over a 4-membered carbon chain as the middle moiety was supported by comparing the activities of compounds A and B, with their tetramethyl- counterparts, C and D, respectively. Furthermore A and B displayed a clear, dose-dependent reduction of the K-RasG12V FRET, while no effect on H-RasG12V FRET was observed, demonstrating a K-Ras selectivity.

[0080] These results demonstrate that A and B are as potent in cells, as in vitro and act on target in cells to selectively downregulated K-RasG12V, but not H-RasG12V activity.

Example 2.4: Deltaflexins selectively inhibit oncogenic K-Ras driven tumorosphere formation

[0081] The effect of compounds A,B,C, D, Deltarasin and control compounds on tumorosphere (specifically mammosphere) formation of the K-RasG13D-mutated and -dependent MDA-MB- 231 and H-RasG12D-mutated and -dependent Hs578T breast cancer cell lines has been tested.

[0082] Mammosphere Assay: Mammosphere formation assays were performed in 96-well suspension culture plates (Cat. No. 655185, Cellstar, Greiner Bio-One, Frickenhausen, Germany). 1500 cells/ per well were seeded in 50 yl DMEM or RPMI medium containing 1X B27 (Cat. No. 17504044, Gibco, Thermo Fisher Scientific), 25 ng/ml EGF (Cat. No. E9644, Sigma-Aldrich) and 25 ng/ml FGF (Cat. No. RP-8628, Thermo Fisher Scientific). Cells were cultured for 6 days and then treated with test compounds or DMSO control (0.1%, v/v) for an 401206 additional 3 days or described previously Najumudeen et al., 2016 [16].

[0083] Compounds A and B show good acitivity against MDA-MB-231 on tumorosphere formation, but very low to no activity against the tumorosphere formation of Hs578T, while deltarasin showed a high activity against the on tumorosphere formation of both cell lines. Compounds C and D were not active any more against any of the cell lines in this assay (Figure 5).

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Claims (13)

1. A compound according to formula (I) Rı .R 1 N 2 0 ACHg)_

I O=P-OR3

O

LA À

O (1) wherein n is an integer between 1 and 10, preferably 4 to 10; more preferred 5 to 10; most preferably 6 to 10, particularly preferred 6 to 9, more particularly preferred 6: Riis H, —CORg, -SO2Rg, or -CH2Rg, preferably -CORe; Rz is H, -CORe, -SO2Rg, or -CHzRg, preferably H; Re is substituted or unsubstituted -(Cz-C44)Ar, preferably substituted or unsubstituted -(Cs- Cio)Ar, Rs is -(C4-Cg)Alkyl, preferably methyl or ethyl; R4 is —CO2(C4-Cs)Alkyl or -(C4-Cs)Alkyl, preferably —CO,Et or methyl Rs is -(C,-Cs)Alkyl, preferably methyl, phenyl, -t-Bu: and a solvate, hydrate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, and isotopically enriched forms thereof.

2. The compound according to claim 1, wherein I) Re is -(Cs-C40)Ar, preferably phenyl, substituted with at least one substituent, selected from — COOH, -COO(C1-Cg)Alkyl, -NO,, -Hal, -O(C;-Cg)Alkyl, -CHals, -(C1-Cg)Alkyl, -(C3.C1o)Cycloalkyl, , and -NH,, or I) Re is -(Cs-C1o)Ar, preferably phenyl, substituted with at least two substituents, independently selected from —COOH, -COO(C+-Ce)Alkyl, -NOz, -Hal, -O(C,-Cg)Alkyl, -CHal,, -(C4-Cg)Alkyl, -(C3. C10)Cycloalkyl, and -NHz, or

| 27 Ill) Re is -(Cs-C1o)Ar, preferably phenyl, substituted with at least two substituents, independently LU101206 selected from ~COOH, -COO(C+;-Ce)Alkyl, and -NH,, or IV) Re is phenyl, substituted with at least two substituents, wherein one substituent is in para- position, selected from the group consisting of -COOH and -COO(C--C;)Alkyl and a second substituent is in the meta-position, selected from the group consisting of -NH-.

3. The compound according to claim 1, wherein Re is phenyl substituted with at least one substituent which is located in para position.

4. The compound according to any one of claims 1 to 3, wherein R4 is -CO2(C1-Cg)Alkyl, preferably —-CO,Me or —CO,Et, more preferably —CO,Et.

5. The compound according to any one of claims 1 to 3, wherein R4 is -(C4-Cg)Alkyl, preferably methyl or ethyl, more preferably methyl.

6. The compound according to any one of claims 1 to 5, wherein Ore Aeron Rs is NH2 or NH; .

7. The compound according to any one of claims 1 to 6, wherein R, is -CORe, Rzis H, and Re is substituted or unsubstituted -(Cs-C40)Ar.

8. A compound selected from Ys » 0 Q 9 COOMe PO COOMe 0 0-P-0 0 do 7 Ss HN 0 OEt HN O , = 0 ,

Ms ° LU101206 o 0 Q COOMe R-O COOMe o-P-0 a db D a NH, Das / NH, s

HN HN O , ana © 0 .

9. The compound according to any one of claim 1 or 8, for use in medicine.

10. The compound according to any one of the claims 1 to 8, for use in the treatment of cancer.

11. The compound for use according to claim 10, wherein the cancer is selected from K-Ras dependent cancers, preferably from cancers wherein the K-Ras gene is mutated.

12. The compound for use according to claims 10 or 11, wherein the cancer is selected from glioma, breast cancer, colorectal cancer, pancreatic cancer, stomach cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, preferably breast cancer.

13. A pharmaceutical composition, comprising the compound according to any one of the claims 1 to 8.