Combination drug of EP4 antagonist and immune checkpoint inhibitor for treating tumors

Abstract

The present invention provides a pharmaceutical combination comprising an EP4 antagonist of formula (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid or a pharmaceutically acceptable salt thereof and at least one immune checkpoint inhibitor, preferably the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid. Also described is polymorphic Form A of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate, characterized by a powder XRD spectrum with peaks at values ​​of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, and 15.9 ±0.2 degrees 2θ. Combinations and polymorphic Form A are described for use in the treatment of tumors.

Description

[Technical field]

[0001] The present invention provides a pharmaceutical combination of an EP4 antagonist and an immune checkpoint inhibitor for treating tumors.

[0002] In a preferred embodiment, the present invention relates to polymorphs of the EP4 antagonists of the combination of the present invention. [Background technology]

[0003] Cancer is a major threat to global public health and remains the leading cause of death worldwide. Thus, despite recent therapeutic advances, there is an urgent medical need for the development of more effective therapeutic treatments.

[0004] Immuno-oncology is an innovative research field that aims to harness the patient's immune system to fight cancer. One of the most promising approaches to prevent the suppression of anti-cancer immunity is the blockade of immune checkpoints, molecular pathways that have evolved to prevent T-cell-mediated autoimmunity, but which tumors can also take advantage of. In tumors, the expression of these proteins is deregulated. For this reason, an important line of research has focused on the blockade of inhibitory receptors expressed on T cells, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1), or their corresponding ligands expressed on tumor cells, such as programmed cell death protein 1 (PD-2). Cell Death Ligands The focus has been on immune checkpoint inhibitors (ICIs) that block PD-L1 (Alsaab, HO et al. Front. Pharmacol. 8, 1-15 (2017)).

[0005] A variety of anti-cancer drugs focusing on anti-CTLA-4 and PD-1 / PD-L1 checkpoint inhibitors are currently approved by the FDA. These include monoclonal antibodies against PD-1 and CTLA-4, such as pembrolizumab, nivolumab, durvalumab, tremelimumab and ipilimumab. Cancer immunotherapy by blocking immune checkpoint molecules has demonstrated remarkable clinical efficacy across multiple cancer types, and clinical trials of immune checkpoint immunotherapy have shown good results even in advanced metastatic cancers. (Alsaab,HO et al. Front.Pharmacol.8,1-15(2017)).

[0006] Despite certain successes, not all patients are responsive, presumably because cancers can find other ways to evade immune surveillance. These limitations have driven clinicians to seek new antitumor agents or new treatments that are more effective against immune surveillance. Summary of the Invention

[0007] The inventors have surprisingly found that a combination of an EP4 antagonist and at least one immune checkpoint inhibitor is effective in treating tumors.

[0008] The present invention therefore relates to a pharmaceutical combination comprising an EP4 antagonist of formula (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid or a pharma- ceutical acceptable salt thereof and at least one immune checkpoint inhibitor.

[0009] EP4 antagonists were first described in WO 2013 / 004290 as being included in the general formula. The inventors have surprisingly found that the EP4 antagonist of formula (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid is the only one that can effectively treat tumors when combined with checkpoint inhibitors, as revealed by the experimental part.

[0010] In the present invention, when the definition of "immune checkpoint" is used, it is intended as an auxiliary molecule that can activate cellular pathways in immune cells or cancer cells that promote or inhibit T cell activation.

[0011] In the present invention, when the definition of "immune checkpoint inhibitor" is used, it is intended as a molecule that inhibits the function of an immune checkpoint.

[0012] The inventors have surprisingly found that the sodium salt of the EP4 antagonist (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid is the preferred one for preparing the combination. As is evident from the experimental section, the resulting sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid is an amorphous compound. Surprisingly, the inventors have found a highly stable crystalline form of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate, designated Form A, which allows for the treatment of cancer by itself, preferably when combined with at least one immune checkpoint inhibitor.

[0013] Thus, in another aspect, the present invention relates to polymorphic Form A of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid, characterized by a powder XRD spectrum with peaks at values ​​of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, 15.9 degrees 2θ±0.2°.

[0014] The present invention further relates to polymorphic form A of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid for use as a medicament, preferably for use in the treatment of tumors.

[0015] Thus, the pharmaceutical combination preferably comprises polymorphic Form A of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid and at least one immune checkpoint inhibitor.

[0016] In another aspect, the present invention relates to a pharmaceutical combination comprising an EP4 antagonist selected from (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid or a pharma- ceutical acceptable salt and at least one immune checkpoint inhibitor.

[0017] In another aspect, the present invention relates to a pharmaceutical combination comprising an EP4 antagonist consisting of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate crystalline form A and at least one immune checkpoint inhibitor for use in the treatment of tumors. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 reports the DSC graph of crystalline form A of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate of Example 1. [Diagram 2] FIG. 1 reports the IR spectrum of crystalline form A of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate of Example 1. [Diagram 3] Figure 1 reports the antitumor response of crystal form A of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate of Example 1, and comparative compounds of Example 2 and anti-PD-1 in the CT26 tumor model. Balb / c mice were subcutaneously injected with 1x10^6 CT26 cells. Tumors were measured on day 7, after which mice were randomized and then treated with the indicated treatments. Compound 1 and the compound of Example 2 were orally administered at 30mg / Kg daily. Anti-PD-1 antibodies were injected at 20mg / kg on day 8, 13, 19 and 23 after implantation, and 10mg / kg on days 13, 19 and 23. Tumor volumes were measured twice weekly and are shown as the mean ±SE of 15 mice per group. Arrows indicate anti-PD-1 injection (*p<0.05; **p<0.01 Anova test). [Figure 4] Figure 1 reports the antitumor response of compounds, anti-PD-1 and combination therapy in CT26 tumor model. Balb / c mice were subcutaneously injected with 1x10^6 CT26 cells. Tumors were measured on day 7, after which mice were randomized and then treated with the indicated treatments. Compound 1 and Compound 2 were orally administered at 30mg / kg daily. Anti-PD-1 antibodies were injected at 20mg / kg on day 8, 13, 19 and 22 after implantation, and 10mg / kg on days 13, 19 and 22. Tumor volumes were measured twice weekly and are shown as the mean±SE of 15 mice per group. Arrows indicate anti-PD-1 injection (*p<0.05; **p<0.01 Anova test). [Diagram 5]Figure 1 reports the relative body weight changes throughout treatment with Compound 1, the compound of Example 2, anti-PD-1 antibody and combination therapy. Mouse body weights were measured twice weekly. [Figure 6] FIG. 1 reports the in vitro concentration-dependent reversal of PGE2 effects by Compound 1. [Figure 7] Figure 1 reports the linear relationship between LPS-induced ex vivo TNF-α release: PGE2 inhibition (IC50) and compound 1 treatment dose. Linear regression analysis, R2=0.9268; P-value of slope deviation against 0=0.0086. [Figure 8] Figure 1 reports ex vivo inhibition of TNF-α release in whole blood cultures stimulated by LPS. Inhibitory effect of PGE2 0.03 μM and 0.1 μM after administration of vehicle or compound 1 at doses ranging from 10 mg / kg to 300 mg / kg. Two-way ANOVA analysis; **p<0.001 vs. vehicle; ***p<0.0005 vs. vehicle Dunnett's multiple comparison test. [Figure 9] Figure 1 reports ex vivo inhibition of TNF-α release in whole blood sampled at different times from dosing and stimulated ex vivo with LPS. Inhibitory effect of PGE2 0.03 μM and 0.1 μM after dosing with vehicle or 10 mg / kg compound 1. Two-way ANOVA analysis; ***p<0.0005 vs. vehicle Dunnett's multiple comparison test. [Figure 10] PGE2 IC50 value distribution: Reporting the effect of Compound 1 (10 mg / kg) and Compound 2 (10 mg / kg) after a single oral dose (24 hours after dosing). One-way ANOVA *p<0.001 vs. vehicle Dunnett's multiple comparison test. [Figure 11] PGE2 IC50 value distribution: Reports the effect of compound 1 10 mg / kg after repeated oral administration (qd for 8 days). *P<0.001 vs. vehicle, Mann Withney test. [Figure 12]FIG. 1 reports that compound 1 dose-dependently reverses PGE2-induced inhibition of TNF-α gene expression on human THP-1 cells differentiated into macrophages and stimulated with LPS 10 ng / ml + 0.01 μM PGE2. Results are expressed as the mean percentage of TNF-α expression ± SD of independent experiments performed in triplicate. (LPS lipopolysaccharide, PGE2 prostaglandin E2). [Figure 13] Figure 1 reports that compound 1 inhibited RANKL gene expression on human breast cancer cell line MDA-MB-231 stimulated with 10 μM PGE2. Results are expressed as the mean percentage of RANK-L expression ± SD of independent experiments performed in triplicate. *P<0.05 by one-way ANOVA with Tukey-Kramer multiple comparison test. PGE2 Prostaglandin E2. [Figure 14] FIG. 1 reports that compound 1 dose-dependently reduced Th-17 frequency in human PBMC cells induced toward Th-17 cell differentiation by exposure to IL-2, IL-21, anti-CD3 and CD28 antibodies, and 0.03 μM PGE2 (Th-17 cells were gated as CD4+CCR6+CD45Ro-IL17F+). [Figure 15] FIG. 1 reports that compound 1 reduced Th-3 cell frequency in human PBMC cells induced towards Treg differentiation by exposure to rIL23 and rIL-1β (10 ng / ml) and treated with PGE2 0.03 μM ± 0.1 μM compound 1. Graph reports mean and standard deviation. *p<0.05 One-way Anova. (Th-3 cells were gated as CD3+CD4+FoxP3+CD25low+TGFb+) [Figure 16] FIG. 1 reports that compound 1 reduced iTr35 regulatory cell frequency in human PBMC cells induced towards Treg differentiation by exposure to rIL23 and rIL-1β (10 ng / ml) and treated with PGE2 0.03 μM±0.1 μM compound 1. Graph reports mean and standard deviation. *p<0.05 One-way Anova. (iTr35 cells were gated as CD3+CD4+IL35+). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention relates to a pharmaceutical combination comprising an EP4 antagonist of formula (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid or a pharma- ceutical acceptable salt thereof and at least one immune checkpoint inhibitor.

[0020] The combination of the present invention may comprise a pharma- ceutically acceptable salt of the EP4 antagonist (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid. The salt may be selected from the group consisting of hydrochloride, sodium salt, potassium salt and lithium salt. Preferably, according to the present invention, the salt of the combination is a sodium salt.

[0021] As is evident from the experimental section, the sodium salt of benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid obtained was an amorphous compound.

[0022] Surprisingly, the inventors have discovered a highly stable crystalline form of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate, designated Form A, for use in combination with at least one immune checkpoint inhibitor.

[0023] Thus, in another aspect, the present invention relates to polymorphic Form A of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid, characterized by a powder XRD spectrum with peaks at values ​​of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, 15.9 degrees 2θ±0.2°.

[0024] The present invention further relates to polymorphic form A of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid for use as a medicament, preferably for use in the treatment of tumors.

[0025] Thus, the pharmaceutical combination preferably comprises polymorphic Form A of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid and at least one immune checkpoint inhibitor.

[0026] In another aspect, the present invention relates to a pharmaceutical combination comprising an EP4 antagonist selected from (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid or a pharma- ceutical acceptable salt and at least one immune checkpoint inhibitor for use as a medicament.

[0027] In another aspect, the present invention relates to a pharmaceutical combination comprising an EP4 antagonist selected from (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid or a pharma- ceutical acceptable salt and at least one immune checkpoint inhibitor for use in the treatment of a tumor.

[0028] In another aspect, the present invention relates to a pharmaceutical combination comprising an EP4 antagonist consisting of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate crystalline form A and at least one immune checkpoint inhibitor for use in the treatment of tumors.

[0029] Therefore, the present invention further relates to a method for treating a tumor, comprising the step of administering to a patient a therapeutically effective amount of a pharmaceutical combination of the present invention.

[0030] Immune checkpoint inhibitors are inhibitors of PD-1 (programmed cell death-1), PD-L1 (programmed cell death-ligand 1), CTLA-4 (cytotoxic T lymphocyte antigen-4), TIM3 (T cell immunoglobulin and mucin-3), OX-40 and its ligand OX40L, LAG-3 (lymphocyte activation gene-3), KIR (killer cell immunoglobulin-like receptor), VISTA (V domain Ig-containing suppressor of T cell activation), IDO1 (indoleamine 2,3-dioxygenase), TIGIT (T cell immunoglobulin and ITIM domain), BTLA (B and T lymphocyte attenuator). These include, but are not limited to, CD276 (cluster of differentiation 276, also known as B7H4), CD27 (cluster of differentiation 27) and its ligands CD27 (cluster of differentiation 27), CD160 (cluster of differentiation 160), and CD39 (cluster of differentiation 39).

[0031] Preferably, the immune checkpoint inhibitor is a neutralizing antibody, anti-PD-1 (e.g., nivolumab (Opdivo), pembrolizumab (Keytruda)), anti-CTLA-4 (e.g., ipilimumab, tremelimumab), anti-TIM-3 antibody (e.g., MBG453) or anti-LAG-3 antibody.

[0032] Prior to administration, the antibody, such as an anti-PD-1 antibody used in the present invention or any of the other immune checkpoint antibodies listed above, will generally be mixed with a pharma- ceutically acceptable substance, such as a saline solution, and may be administered using any suitable method, including, but not limited to, intravenous, intradermal, intraperitoneal, or intrathecal injection.

[0033] A therapeutically "effective amount" refers to the amount of a particular checkpoint inhibitor (e.g., the potency (IC 50 ), Effectiveness (EC 50 It is intended that the amount of EP4 antagonist and at least one checkpoint inhibitor corresponds to an amount that varies depending on factors such as the age, size and weight of the tumor, tumor condition and its severity, the identity of the patient requiring treatment, but can nevertheless be routinely determined by those skilled in the art. Similarly, the duration of treatment and administration period (time between administrations and timing of administration, e.g., before / during / after a meal) of the compounds contained in the combination also varies depending on the identity of the person requiring treatment (e.g., body weight), the specific compound and its characteristics (e.g., pharmaceutical characteristics), tumor and its severity, but can nevertheless be determined by those skilled in the art.

[0034] The EP4 antagonist of the present invention and at least one immune checkpoint inhibitor of the present invention may be administered independently of each other by any suitable route of administration, including both systemic and local administration.

[0035] Systemic administration includes oral administration, parenteral administration, transdermal administration, rectal administration and administration by inhalation.

[0036] The combination of the EP4 antagonist of the present invention and at least one immune checkpoint inhibitor of the present invention may be administered once or according to a dosing regimen in which several doses are administered at various time intervals over a given period of time. Doses may be administered until a desired therapeutic effect is achieved or indefinitely to maintain a desired therapeutic effect. The appropriate dosing regimen of the EP4 antagonist of the present invention and at least one immune checkpoint inhibitor of the present invention depends on the pharmacokinetic properties of such compounds, such as absorption, distribution and half-life, which can be determined by those skilled in the art. The combination of the present invention may also be formulated into a pharmaceutical composition before administration to a patient. The pharmaceutical composition of the present invention is prepared using techniques and methods known to those skilled in the art.

[0037] In a more preferred embodiment, the tumor may include, but is not limited to, colorectal cancer, bladder cancer, adrenal cancer, breast cancer, brain cancer, glioma, glioblastoma, cervical cancer, head and neck cancer, endometrial cancer, lung cancer, ovarian cancer, melanoma, prostate cancer, renal cancer, kidney cancer, liver cancer, thyroid cancer, pancreatic cancer, sarcoma, fibrosarcoma.

[0038] In an even more preferred embodiment, the immune checkpoint inhibitor is an anti-CTLA-4 and / or an anti-PD-1 / PD-L1 checkpoint inhibitor.

[0039] For all treatment methods, in some embodiments, the effective dose of anti-PD-1 antibody used is 0.1 mg / kg to 20 mg / kg of total body weight, with preferred doses being 2 mg / kg or 3 mg / kg.

[0040] It will also be appreciated by those skilled in the art that optimal amounts and intervals of individual administration will be determined by the nature and extent of the condition being treated.

[0041] The invention will now be further detailed with reference to the experimental section.

[0042] Experimental Department Reagents used in the following examples were commercially available from various suppliers and were used without further purification. Solvents were used in anhydrous form. Reactions in an anhydrous environment were carried out under a positive pressure of dry N2.

[0043] Proton nuclear magnetic resonance ( 1 H NMR spectra were recorded on a Bruker Avance 400 MHz instrument. Chemical shifts are reported in ppm (δ) using residual solvent as internal standard. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad signal.

[0044] Mass spectra (MS) were performed on an Ion Trap Thermo LCQ classical spectrometer operating in positive ES (+) and negative ES (-) ionization modes.

[0045] UPLC spectra were performed on a Waters Acquity UPLC-SQD instrument using an Acquity UPLC-BEH C18 column (1.7 μM, 50 × 2.1 mm).

[0046] Flash silica gel chromatography was performed on a Biotage automated flash chromatography system (Isolera system) using Biotage SNAP HP silica cartridges.

[0047] Reverse-phase chromatography was performed on a Biotage automated flash chromatography system (Isolera system) using RediSep Gold C-18Aq cartridges.

[0048] Purification of several basic compounds was carried out using a Phenomenex Strata SCX cartridge (55 μm, 70 A).

[0049] Thin layer chromatography was performed using Merck TLC plates Kieselgel 60F-254 with visualization with UV light, aqueous permanganic acid, and iodine vapor.

[0050] The following abbreviations are used herein: AcOH: acetic acid; DIAD: diisopropyl(E)-diazene-1,2-dicarboxylate; Boc: tertbutyloxycarbonyl; DCM: dichloromethane; TFA: trifluoroacetic acid; DMF: dimethylformamide; THF: tetrahydrofuran; RT: room temperature; AcOEt: ethyl acetate; NaOH: sodium hydroxide; LiOH: lithium hydroxide; DIPEA: N,N-diisopropylethylamine; TEA: triethylamine; NaHCO3: sodium bicarbonate; Na2SO4: sodium sulfate; Cs2CO3: cesium carbonate; NaHMDS: sodium bis(trimethylsilyl)amide; HOBt: 1-hydroxybenzotriazole

[0051] Example 1: Preparation of EP4 antagonists of formula (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid, sodium salt The compounds were obtained according to the following synthetic steps as shown in Scheme 1: [ka] a) conversion of starting material (SM1) to a compound of formula (I) using starting material (SM2) and a suitable coupling agent; b) deprotection of compound (I) in an acidic medium such as TFA in DCM to give compound (II); c) Alkylation of the ring nitrogen with benzyl bromide in the presence of a suitable base gives compound (III); d) hydrolysis of the methyl ester using a suitable inorganic base such as NaOH to give compound (IV); and e) Formation and crystallization of the sodium salt in a suitable solvent.

[0052] Example 1a) Synthesis of starting material 1 (SM1) The starting material SM1, reported in Scheme 1 above, was obtained by the following steps, reported in Scheme 2 below: [ka] a) (R)-1-(tert-butoxycarbonyl)-4-oxopiperidine-2-carboxylic acid (R) (R) (SEQ ID NO:1) was prepared using MeI and a suitable base such as Cs2CO3. Tetrah (prepared according to Edron (1997) 15671-15680) to give the methyl ester (IX). b) Wittig reaction using methyltriphenylphosphonium bromide in toluene to obtain compound (X) c) Deprotection of the piperidine nitrogen using a suitable acidic reagent such as HCl in MeOH followed by protection using Cbz chloride in DCM gives compound (XI). d) Cyclopropane formation using an organometallic reagent such as diethylzinc in THF, diiodomethane and TFA to give compound (XII). e) Deprotection of the piperidine nitrogen using a reducing agent such as hydrogen in MeOH and palladium on charcoal followed by protection using (BOC)2O gives compound (XIII). f) Hydrolysis of the methyl ester using a suitable inorganic base such as LiOH in THF / MeOH gives (SM1). 。

[0053] - Synthesis of intermediate (IX) 1-(tert-butyl) 2-methyl (R)-4-oxopiperidine-1,2-dicarboxylate Referring to Scheme 2, intermediate (IX) was prepared.

[0054] In a round-bottom flask, (R)-1-(tert-butoxycarbonyl)-4-oxopiperidine-2-carboxylic acid (10 g; 0.041 mol) was dissolved in DMF (25 ml) and cooled to 3° C. Cesium carbonate (0.6 eq.) was added, followed by dropwise addition of methyl iodide (1.1 eq.) and after 2 h at RT, the mixture was diluted with water (250 ml) and extracted with AcOEt (3×150 ml). The combined organic layers were washed with water (150 ml×3) followed by brine (150 ml), dried over sodium sulfate, filtered and concentrated at 40° C. to give the title compound (9 g; 85%) as a light brown solid. 1H NMR(400MHz,CHLOROFORM-d)δ=5.28-4.75(m,1H),4.13-4.03(m,1H),3.76(s,3H),3.72-3.55(m,1H),2.92-2.70(m,2H),2.53(br s,2H),1.50(br s,9H)ESI+m / z 258[M+H] +

[0055] - Synthesis of intermediate (X) 1-(tert-butyl) 2-methyl (R)-4-methylenepiperidine-1,2-dicarboxylate Referring to Scheme 2, intermediate (X) was prepared.

[0056] Methyltriphenylphosphonium bromide (1.1 eq.) was dissolved in dry toluene (400 ml) and cooled to 3° C., then NaHMDS solution (1.05 eq.) was added dropwise slowly. After 1 h at 3° C. under nitrogen atmosphere, intermediate (IX) (9 g, 0.035 mol) in dry toluene (200 ml) was added and stirred for 1 h. After completion, the reaction was quenched with ice / water (800 ml), the two layers were separated and the organic layer was washed with water (350 ml) followed by brine (350 ml), dried over sodium sulfate and concentrated. The residue was purified by column chromatography using silica gel eluting with hexane / AcOEt 95-5 to 60-40 to give the title compound as a pale yellow solid (8 g; 90%). 1H NMR(400MHz,CHLOROFORM-d)δ=5.12-4.76(m,3H),4.27-3.97(m,1H),3.73(s,3H),3.23-2.92(m,1H),2.86-2.69(m,1H),2.57-2.38(m,1H),2.21(br s,2H),1.49(br s,9H).ESI+m / z 256[M+H] +

[0057] - Synthesis of intermediate (XI) 1-benzyl 2-methyl (R)-4-methylenepiperidine-1,2-dicarboxylate Referring to Scheme 2, intermediate (XI) was prepared.

[0058] Intermediate (X) (8 g; 0.031 mol) was dissolved in anhydrous methanol (150 ml) and cooled to 0° C., then 300 ml of 3M methanolic HCl solution was added slowly. After 2 h at RT, the solvent was evaporated to dryness, the residue was dissolved in DCM (250 ml) and cooled to 0° C., then triethylamine (2.5 eq.) and benzyl chloroformate (1.2 eq.) were added after 1 h at RT. After completion, the reaction was quenched with ice-cold water; the two layers were separated and the organic layer was washed with water followed by a brine solution (250 ml). The organic layer was dried over sodium sulfate and concentrated below 40° C. to give a residue which was purified by column chromatography using silica gel eluting with hexane / AcOEt 95-5 to 60-40 to give the title compound as a white solid (6.7 g; 74%). 1H NMR(400MHz,CHLOROFORM-d)δ=7.45-7.30(m,5H),5.24-4.93(m,3H),4.83(s,2H),4.31-4.13(m,1H),3 .84-3.62(m,3H),3.26-3.03(m,1H),2.86-2.74(m,1H),2.55-2.43(m,1H),2.34-2.17(m,2H).ESI+m / z 290[M+H] +

[0059] - Synthesis of intermediate (XII) 6-benzyl 5-methyl (R)-6-azaspiro[2.5]octane-5,6-dicarboxylate Referring to Scheme 2, intermediate (XII) was prepared.

[0060] DCM (150ml) was cooled to 0°C, then diethylzinc solution in THF (2.3eq) was added slowly and stirred for 30 minutes. Trifluoroacetic acid (2.0eq) was added slowly at 0°C, stirred for 60 minutes, then diiodomethane (4.0eq) was added and stirred for 60 minutes at 0°C. Intermediate (XI) (6g; 0.02mol) in dry dichloromethane (50ml) was added slowly at 0°C, then left stirring at 25°C for 20 hours. The reaction mass was quenched with 10% sodium bicarbonate solution (400ml). The solid precipitate was filtered off, the layers were separated from the filtrate and the organic layer was washed with water (250ml x 2) followed by brine solution (250ml). The organic layer was dried over sodium sulfate and concentrated below 40° C. to give a residue which was purified by column chromatography using silica gel eluted with hexane / AcOEt 95-5 to 60-40 to give the title compound as a white solid (4.72 g; 75%). 1H NMR(400MHz,CHLOROFORM-d)dppm 0.03-0.47(m,78H)0.76-0.92(m,1H)1.26-1.48(m,1H)1.49-1.70(m,2 H)1.75(s,1H)1.88-2.04(m,1H)2.19(s,1H)2.35-2.41(m,1H)3.17-3. 39(m,1H)3.61-3.83(m,3H)4.14(m,J=11.74Hz,1H)4.96(m,J=4.89Hz,1H)5.12-5.25(m,2H)7.15-7.28(m,1H)7.37(m,J=9.29Hz,5H).ESI+m / z 304[M+H] +

[0061] - Synthesis of intermediate (XIII) 6-(tert-butyl) 5-methyl (R)-6-azaspiro[2.5]octane-5,6-dicarboxylate Referring to Scheme 2, intermediate (XIII) was prepared.

[0062] Intermediate (XII) (4.5g, 0.015mol) was dissolved in methanol (200ml) and Pd / C 10% (400mg) was added and the suspension was then hydrogenated at 3bar for 2h. After completion, the reaction was filtered through a bed of celite and washed with methanol (200ml). The solution was concentrated to 150ml and cooled to 20°C and then boc anhydride (1.2eq) was added slowly and stirred at 25°C for 16h. After completion of the reaction, the solvent was evaporated and the residue was purified by column chromatography using silica gel eluting with hexane / AcOEt 95-5 to 60-40 to give the title compound as a white solid (3.68g; 92%). 1H NMR(400MHz,CHLOROFORM-d)δ=5.03-4.71(m,1H),4.21-3.90(m,1H),3.74(s,3H),3.35-3.04(m,1H),2.27-2. 13(m,1H),2.02-1.83(m,J=4.6,13.1,13.1Hz,1H),1.56-1.38(m,10H),0.95-0.72(m,1H),0.43-0.20(m,4H). ESI+m / z 270[M+H] +

[0063] - Synthesis of starting material 1 (SM1) (R)-6-(tert-butoxycarbonyl)-6-azaspiro[2.5]octane-5-carboxylic acid Referring to Scheme 2, the starting material (SM1) was obtained.

[0064] Intermediate (XIII) (3.5 g; 0.013 mol) was dissolved in THF (100 ml) and methanol (100 ml), cooled to 0° C., and then lithium hydroxide solution (3.0 equivalents in 50 ml water) was added dropwise.

[0065] The mixture was stirred at RT for 8 h. The reaction was cooled to 10° C., quenched with acetic acid to pH 5 (50 ml) and concentrated by distilling off MeOH and THF. The concentrated mass was diluted with ice water and extracted with ethyl acetate (300 ml×2). The combined organic layers were washed with water (200 ml) followed by brine (200 ml). The organic layers were dried over sodium sulfate and concentrated below 50° C. Petroleum ether (300 ml) was added to the residue; the solid was filtered off and dried in vacuum at 40° C. for 24 h. Yield 3 g (92%) 1H NMR(400MHz,CHLOROFORM-d)δ=5.11-4.77(m,1H),4.18-3.88(m,1H),3.32-3 .09(m,1H),2.32-2.14(m,1H),2.03-1.88(m,1H),1.67-1.55(m,1H),1.50(br s,9H),0.97-0.75(m,1H),0.52-0.26(m,4H).ESI+m / z 256[M+H] +

[0066] Example 1b) Synthesis of starting material 2 (SM2) Starting material 2 (SM2) was prepared according to known literature procedures (WO2008104055, Example 1, Step 2).

[0067] Example 1c) Synthesis of intermediate (I) tert-butyl (R)-5-((1-(4-(methoxycarbonyl)phenyl)cyclopropyl)carbamoyl)-6-azaspiro[2.5]octane-6-carboxylate Referring to Scheme 1, intermediate (I) was obtained.

[0068] 10 g (0.039 mmol) of starting material 1 was dissolved in DCM (200 ml), HOBt hydrate (1.1 eq.) and EDC-HCl (1.1 eq.) were added and the mixture was stirred at 20° C. for 30 min. Starting material 2 (1.02 eq.) was added, followed by TEA (1.2 eq.); the reaction was left stirring at 30° C. for 6 h and then quenched with water (100 ml). The organic phase was washed with 5% sodium bicarbonate solution (100 ml), 1 M citric acid solution (200 ml), water (200 ml). DCM was evaporated, t-butyl methyl ether (200 ml) was added and the solvent was evaporated again. 400 ml of t-butyl methyl ether was added, the suspension was stirred at 20° C. for 17 h, then the white solid was filtered and washed with cold t-butyl methyl ether. The product was dried under vacuum at 50° C. Yield: 14.7g (88%) 1H NMR(400MHz,CHLOROFORM-d)δ=8.01-7.93(m,2H),7.28(s,2H),6.74(s,1H),4.83(br s,1H),4.21(br s,1H),3.92(s,3H),3.10-2.87(m,1H),2.10-1.99(m,1H),1.97-1.85(m,1H),1.84-1.75(m,1H),1.52(s,9H),1.40(br s,4H),0.88-0.81(m,1H),0.63-0.45(m,1H),0.45-0.29(m,2H),0.28-0.18(m,1H). ESI+m / z 429[M+H] +

[0069] Example 1d): Synthesis of intermediate (II) methyl (R)-4-(1-(6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate Referring to Scheme 1, intermediate (II) was obtained.

[0070] Intermediate (I) (14 g; 0.032 mmol) was dissolved in DCM (150 ml), TFA (10 eq.) was added and the solution was stirred at 20° C. for 5 h. The reaction mixture was distilled under vacuum, DCM (100 ml) was added and saturated sodium bicarbonate solution was added slowly at 15-25° C. (300 ml, significant foaming). The organic phase was washed with water (200 ml) and evaporated under reduced pressure. t-Butyl methyl ether (200 ml) was added and then the solvent was evaporated again. 300 ml of t-Butyl methyl ether was added and the suspension was stirred at 20° C. for 17 h, then the white solid was filtered and washed with cold t-Butyl methyl ether. The product was dried under vacuum at 50° C. Yield 9.65 g (90%) 1H NMR(400MHz,CHLOROFORM-d)δ=7.96(d,J=8.3Hz,2H),7.61(br s,1H),7.26(d,J=8.3Hz,2H),3.91(s,3H),3.46-3.39(m,1H),3.13-3.04(m,1H),2.92-2.82 (m,1H),1.87-1.70(m,3H),1.41-1.28(m,5H),1.01-0.93(m,1H),0.46-0.24(m,4H).ESI+m / z 329[M+H] +

[0071] Example 1e) Synthesis of intermediate (III) methyl (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate Referring to Scheme 1, intermediate (III) was obtained.

[0072] Intermediate (II) (9.5 g, 0.029 mmol) was dissolved in dry THF (150 ml) and then Cs2CO3 (2 eq.) and 4-(trifluoromethyl)benzyl bromide (1.2 eq. dissolved in 75 ml THF) were added. The reaction mixture was stirred at 25° C. for 8 h and then 350 ml DCM and 350 ml water were added. The organic phase was washed with water (150 ml) and brine (250 ml) and evaporated under reduced pressure. n-heptane (1500 ml) was added and then the solvent was evaporated again. 150 ml n-heptane was added and the suspension was stirred at 20° C. for 4 h and then the white solid was filtered and washed with n-heptane. The product was dried under vacuum at 50° C. Yield 12.95 g (92%) 1H NMR(400MHz,CHLOROFORM-d)δ=7.96-7.89(m,2H),7.66-7.61(m,2H),7.47-7.40(m,3H),7.27- 7.21(m,J=8.8Hz,2H),3.90(s,3H),3.84(d,J=14.7Hz,1H),3.35(d,J=14.7Hz,1H),3.05(dd,J= 3.9,10.3Hz,1H),2.93-2.86(m,1H),2.31-2.22(m,1H),2.07-1.98(m,1H),1.88-1.78(m,1H), 1.45-1.27(m,4H),1.17-1.08(m,1H),1.01-0.94(m,1H),0.50-0.36(m,3H),0.33-0.26(m,1H). ESI+m / z 487[M+H] +

[0073] Example 1f): Synthesis of intermediate (IV) (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid Referring to Scheme 1, intermediate (IV), an EP4 antagonist in its acid form, was obtained.

[0074] Intermediate (III) (12 g, 0.024 mmol) was dissolved in THF (70 ml) and then NaOH 2N (4 eq.) was added. The reaction was left stirring at 5° C. for 5 h, then water (200 ml) was added and the organics were evaporated. 200 ml of dichloromethane was added and the pH of the mixture was adjusted to 4.5-5.0 with acetic acid; the organic phase was washed with water and brine (200 ml×3) and evaporated to give the title compound (9.9 g; 85%). 1 H NMR(400MHz,DMSO-d6)δ ppm 12.72(1H,s),8.73(1H,s),7.78(2H,d,J=8.6Hz),7.69(2H,d,J=8.3Hz),7.64(2H,d,J=8.3Hz),7.20(2H,d,J=8.6Hz),3.79(2H,d,J=13.9Hz ),3.28(1H,d,J=13.9Hz),2.93(1H,dd,J=3.1;10.3Hz),2.73(1H,m),2.08(2H,m),1.78(1H,m),1.10-1.31(5H,m),0.87(1H,m),0.32(4H,m). ESI+m / z 473[M+H] + .

[0075] The EP4 antagonist thus obtained was analyzed and found to be a zwitterion as an amorphous solid.

[0076] Example 1g Preparation of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid The only crystalline form that could be obtained from the zwitterion of Example 1f) was a solvate, which readily gave rise to an amorphous form upon thermal stress (e.g., heating to reduce the solvent content to ICH levels) or mechanical stress.

[0077] Therefore, the hydrochloride salt was prepared by adding intermediate (IV) HCl in ethyl ether.

[0078] The hydrochloride salt of intermediate (IV) was investigated and an amorphous form and two crystalline forms (anhydrous A and solvate B) were identified from the screen. Anhydrous form A was further investigated. Intermediate (IV) hydrochloride form A showed a tendency to convert to an amorphous form under mechanical or thermal stress. The chemical stability was comparable to the amorphous zwitterion of example 1f).

[0079] After solubilizing intermediate (IV) in a solvent, the sodium salt was prepared by adding NaOH in EtOH. Various solvents were tested (n-propanol, n-butanol; iPrOH), but n-propanol was selected due to its higher yield and easy drying. Two crystalline forms, designated Forms A and D, were obtained: Form A was obtained directly from n-propanol crystallization, while Form D was obtained only by hydration of Form A. During physical stability testing, Form D was found to be susceptible to conversion to an amorphous form by mechanical stress (milling) and during drying.

[0080] Conversely, sodium salt form A was found to be more stable to amorphization by thermal stress, but amorphization was observed upon mechanical stress at high energy. Form A was the most thermodynamically stable anhydrous form, and form D was the most stable hydrated form. The other forms were less stable or metastable. These forms as well as the amorphous form were easily converted to the stable form A by solid slurry from acetone, diethyl ether, isopropyl acetate, THF, heptane.

[0081] The synthesis protocol for (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate sodium (Form A) (also referred to as compound 1) was established as reported below.

[0082] Intermediate (IV) (50 g, 0.1 mol) was dissolved in n-propanol (400 ml) and then a chilled solution of NaOH (1.02 equiv.) in EtOH (70 ml) was added. The suspension was stirred at 20-24° C. for 18-20 h, then filtered, washed with pre-chilled n-propanol (100 ml) and dried at 70° C. for 24 h. Yield 43 g white solid (87%) 1 H NMR(400MHz,DMSO-d6)δ ppm 8.68(1H,s),7.73(2H,d,J=8.3Hz),7.69(2H,d,J=8.3Hz),7.64(2H,d,J=8.3Hz),7.02(2H,d,J=8.3Hz),3.81(2H,d,J=13.9Hz),3.26(1H ,d,J=13.9Hz),2.92(1H,dd,J=3.1;10.5Hz),2.72(1H,m),2.07(2H,m),1.78(1H,m),1.16(4H,m),1.09(1H,m),0.85(1H,m),0.30(4H,m). ESI+m / z 473[M+H] +

[0083] Solubility, dissolution and bioavailability studies were carried out on the zwitterion of Example 1f), both the salt form and the all sodium form. The salts, especially both forms of the sodium salt, showed improved dissolution properties compared to the amorphous form. PK studies in both rats and dogs showed no significant differences between the various forms in terms of exposure and bioavailability, however sodium salt form A appears to be the form of choice for development given its stability and pharmaceutical manufacturability properties.

[0084] Characterization of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate sodium (Form A), also known as compound 1 solid state NMR Solid-state NMR measurements were performed on a Bruker AVANCE II 400 instrument operating at 400.23 MHz and 100.65 MHz for 1H and 13C, respectively. 13 C CPMAS spectra were recorded at room temperature at a spinning speed of 12 kHz. A cylindrical 4 mm outer diameter zirconia rotor with a sample volume of 80 μL was used. The CPMAS experiments used a ramp cross-polarization pulse sequence with a contact time of 3 ms, a 1H 90° pulse of 4.0 μs, a recycle delay of 5-10 s, and a transient response of 2000-4000. A two-pulse phase-modulated decoupling scheme was used with a frequency field of 75 KHz. 13 C chemical shift (ppm) δ=177.1;144.8;143.1;136.9;129.3;124.9;71.4;65.5;61.3;57.3;51.2;39.0;35.1;23.2;18.0;14.9;12.6

[0085] XRPD XRPD measurements were performed on a PANalytical X'pert Pro X-ray powder diffractometer with Bragg-Brentano geometry and equipped with: a) Detector X'Celerator b) Multisampler c) Spinner d) Ceramic X-ray tube Cu LFF (λ1=1.54051A; λ2=1.54430A) Range: 4~40°2θ [Table 1]

[0086] DSC Thermograms were obtained using a Perkin-Elmer DSC 8000 with a scan rate of 10° C. / min from 30° C. to 300° C. The thermograms are reported in FIG. Peaks detected: T onset = 252.64 °C, ΔH = 56.11 J / g

[0087] IR The IR spectrum was measured in the ATR (attenuated total reflection) mode from 4000 to 650 cm -1 The IR spectrum was recorded on a Perkin Elmer Spectrum 100 FT / IR instrument between 1000 and 2000 rpm. The IR spectrum is reported in Figure 2. The main absorption frequencies are reported in the table below: [Table 2]

[0088] Stability of Form A At various humidity levels and temperatures ( The stability of the sodium salt of R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid, form A (also called compound 1), was investigated. The results are reported in the following table: [Table 3]

[0089] Example 2: Preparation of (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido)cyclopropyl)benzoic acid, sodium salt (also called compound 2) Example 1 EP 4 The antagonist may be any of the compounds disclosed in WO 2013 / 004290.4 The antagonist was compared with (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido)cyclopropyl)benzoic acid.

[0090] The compounds were obtained according to the following synthetic steps as shown in Scheme 3: [ka] a) converting (R)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid into a compound of formula (V) using starting material (SM2) and a suitable coupling agent; b) deprotection of compound (V) in an acidic medium such as TFA in DCM to give compound (VI); c) Alkylation of the ring nitrogen with benzyl bromide in the presence of a suitable base gives compound (VII); d) hydrolysis of the methyl ester using a suitable inorganic base such as LiOH to give compound (VIII); and e) Formation of the sodium salt using NaOH in an appropriate solvent mixture

[0091] Example 2a: Synthesis of intermediate (V) tert-butyl (R)-2-((1-(4-(methoxycarbonyl)phenyl)cyclopropyl)carbamoyl)piperidine-1-carboxylate Boc-D-pipecolic acid (500 mg, 2.181 mmol) was dissolved in DCM (13 ml). N-Hydroxybenzotriazole hydrate (2.62 mmol) and EDCI (3.05 mmol) were added and the reaction mixture was left stirring for 40 min. SM2 (2.268 mmol) was added followed by TEA (2.94 mmol). The reaction mixture was left stirring at room temperature for 15 h and then water (20 ml) was added. The phases were separated and the aqueous layer was extracted with DCM (2×15 mL). The combined organic layers were evaporated and loaded onto a SNAP Ultra-HP Sphere-Si (10 g) column eluting with cyclohexane / AcOEt 100% to 70 / 30. Yield 810 mg (92%), pale yellow foam. 1H NMR(400MHz,CHLOROFORM-d)δ=8.00-7.93(m,J=8.3Hz,2H),7.27-7.22(m,J=8.3Hz,2H),6.76(br s,1H),4.85-4.67(m,1H),4.12(br s,1H),3.92(s,3H),2.97-2.66(m,1H),2.29(br s,1H),1.72-1.59(m,3H),1.54-1.22(m,15H). ESI+m / z 403[M+H] +

[0092] Example 2b: Synthesis of intermediate (VI) methyl (R)-4-(1-(piperidine-2-carboxamido)cyclopropyl)benzoate According to Scheme 3, intermediate (VI) was obtained.

[0093] Intermediate (V) (800 mg, 1.988 mmol) was dissolved in DCM (8 mL). TFA (19.88 mmol) was added and the reaction mixture was left stirring at room temperature for 4 h. The solvent was evaporated and the residue was loaded onto a SPE-SCX (5 g) cartridge eluting with MeOH and NH3 1M in MeOH. The ammoniacal fraction was evaporated to give the title compound (590 mg; 98%). 1H NMR(400MHz,CHLOROFORM-d)δ=8.00-7.92(m,J=8.8Hz,2H),7.58(br s,1H),7.28-7.24(m,2H),3.91(s,3H),3.33-3.27(m,1H),3.10-3.03(m,1H),2.80-2.69(m,1H),2.20(br s,1H),2.06-1.97(m,1H),1.84-1.74(m,1H),1.65-1.58(m,1H),1.54-1.31(m,7H).ESI+m / z 303[M+H] +

[0094] Example 2c: Synthesis of intermediate (VII) methyl (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido)cyclopropyl)benzoate Intermediate (VI) (585 mg, 1.9 mmol) was dissolved in THF (12 ml). The mixture was stirred until the material was dissolved. Cesium carbonate (3.87 mmol) was added followed by 4-(trifluoromethyl)benzyl bromide (2.42 mmol) and the mixture was stirred for 24 h. THF was evaporated and the residue was dissolved in a mixture of DCM / NaHCO3 saturated solution (50 ml). The phases were separated and the aqueous layer was extracted with DCM (2 x 15 ml). The combined organic layers were evaporated and the residue was loaded onto a SNAP Ultra-HP Sphere-Si (10 g) column eluting with cyclohexane / ethyl acetate 100% to 70 / 30. Yield 850 mg (95%) 1H NMR(400MHz,DMSO-d6)δ=8.71(s,1H),7.84-7.78(m,2H),7.72-7.66(m,2H),7.63-7.58(m,2H),7.26-7.20(m,J=8.8Hz,2H),3.82(s,3H),3. 73(d,J=14.2Hz,1H),3.25(d,J=14.2Hz,1H),2.87-2.80(m,1H),2.79-2.70(m,J=11.7Hz,1H),2.01-1.93(m,1H),1.86-1.78(m,1H),1.70(br d,J=10.3Hz,2H),1.57-1.38(m,2H),1.35-1.23(m,3H),1.21-1.11(m,2H).ESI+m / z 461[M+H] +

[0095] Example 2d: Synthesis of intermediate (VIII) (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido)cyclopropyl)benzoic acid Lithium hydroxide monohydrate (3.32 mmol) was added to a solution of intermediate (VII) (850 mg, 1.846 mmol) in water / dioxane. The reaction mixture was left stirring at room temperature for 5 h, then 1 ml of AcOH was added, dioxane was evaporated and the residue was loaded onto a Biotage C18 10 g SPE-column eluted with water (2 VC) and MeOH (3 VC). Yield 94% (772 mg, white solid). 1H NMR(400MHz,DMSO-d6)δ=12.72(br s,1H),8.69(s,1H),7.83-7.76(m,2H),7.72-7.66(m,2H),7.64-7.59(m,2H) ,7.24-7.18(m,2H),3.74(d,J=14.2Hz,1H),3.25(d,J=14.2Hz,1H),2.88-2. 79(m,1H),2.79-2.72(m,1H),2.01-1.93(m,1H),1.88-1.77(m,1H),1.76-1. 63(m,2H),1.57-1.38(m,2H),1.37-1.21(m,3H),1.21-1.09(m,2H).ESI+m / z 447[M+H] +

[0096] Example 2e: Synthesis of (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido)cyclopropyl)benzoic acid, sodium salt Intermediate (VIII) (40 g, 89.7 mmol) was dissolved in water / dioxane and then NaOH (98 mmol) was added. After stirring for 1 h, the THF was evaporated. The residue was loaded onto a Biotage C18 150 g SPE-column (8 injections) eluted with water (4 VC) to MeOH (gradient 4 CV). The fractions containing the desired product were evaporated and the resulting solid was dried under vacuum at 60° C. for 3 days. Yield 98% (41 g, white solid). 1H NMR(400MHz,DMSO-d6)δ=8.60(s,1H),7.75-7.65(m,J=7.8,7.8Hz,4H),7.65-7.57 (m,2H),7.05-6.96(m,2H),3.75(d,J=14.2Hz,1H),3.24(d,J=13.7Hz,1H),2.84-2 .78(m,1H),2.78-2.71(m,J=11.7Hz,1H),2.01-1.91(m,1H),1.86-1.76(m,1H),1. 74-1.63(m,2H),1.56-1.36(m,2H),1.34-1.20(m,1H),1.19-1.00(m,4H).ESI+m / z 447[M+H] +

[0097] Example 3: Efficacy of compound 1 ((R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid sodium salt form A) and compound 2 in combination with anti-mouse pd-1 antibody in a syngeneic model of mouse colorectal cancer. Materials and Methods Male Balb / c mice, 4-6 weeks of age, were housed five per cage in a temperature- and humidity-controlled room with a 12 h / 12 ​​h light / dark cycle. Animals had free access to food and water throughout the experiment. Balb / c mice were randomly divided into six groups (n=15 in each group): vehicle, compound 1, compound 2, anti-PD-1, compound 1 + anti-PD-1, and compound 2 + anti-PD-1 groups.

[0098] CT26 cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM; ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich) at 37°C in a 5% CO2 incubator. Cells were subcultured twice a week until the required cell number for inoculation was obtained.

[0099] On the day of transplantation (day 0), logarithmic-phase cells were harvested and diluted in PBS at 10 in 0.2 ml of PBS. ^6 Cells were inoculated subcutaneously into the flank area of ​​each mouse using a 26G syringe.

[0100] Seven days after inoculation, mice were randomized into six treatment groups of 15 animals each receiving vehicle, compound 1, compound 2, anti-PD-1, and combination therapy.

[0101] Compound 1 and compound 2 were orally administered at 30 mg / kg once a day repeatedly from the 7th to 23rd days, and anti-mouse PD-1 antibody was intraperitoneally administered to mice in the anti-mouse PD-1 antibody monotherapy group and the combination therapy group at a dose of 20 mg / kg on the 8th day after transplantation and at a dose of 10 mg / kg on the 13th, 19th, and 22nd days after transplantation.

[0102] Using digital calipers, measure tumor size (mm 3 Tumor length along the major and minor axes was measured and calculated using the formula: volume = [(length) 2 Tumor volume was calculated using [×width] / 2.

[0103] result To investigate the effect of the combination therapy of the compounds of Compound 1 and Example 2 with anti-mouse PD-1 antibody, a mouse colorectal cancer cell line allograft model was used. CT26 cancer cell tumors grown subcutaneously in mice were treated with the compounds of Example 1 or 2 as monotherapy, or with the compounds of Example 1 or 2 in combination with anti-mouse PD-1 antibody for 3 weeks.

[0104] The graph in Figure 3 reports the weekly measured tumor volumes plotted against the number of days after tumor cell inoculation. It can be seen that monotherapy with compound 1 had a mild inhibitory effect on cancer growth starting from the second week of treatment, while the compound of Example 2 showed only a slight impairment of tumor growth. According to data reported in the literature, anti-mouse PD-1 therapy delayed cancer growth, even if the inhibition of cancer growth was only partial. (Shindo, Y. et al. Anticancer Res. 35, 129-136 (2015)). In contrast, as reported in Figure 4, the antitumor efficacy was significantly enhanced by the combined treatment of compound 1 with anti-mouse PD-1 antibody.

[0105] Furthermore, assessment of the animals' body weight throughout the treatment indicated that the combination treatment did not affect the overall health of the animals (Figure 5).

[0106] conclusion The results reported above demonstrated that Compound 1 and Compound 2 inhibited tumor growth when given as monotherapy. Furthermore, Compound 1 significantly enhanced the antitumor effect of PD-1 antibodies when used in combination.

[0107] Example 4 Effects of two EP4 antagonists on specific binding of 3H-PGE2 to human recombinant EP4 receptor subtypes method Cell membranes were prepared from the HEK293 cell line (human embryonic kidney 293) stably overexpressing the human recombinant EP4 receptor. Cells were grown adherently in DMEM with Glutamax I containing 10% FBS at 37°C with 5% CO2. For membrane preparation, 150 cm 2Culture medium was aspirated from the flask. The cell monolayer was washed with 10 ml of hypotonic lysis buffer (TRIS 5 mM + EDTA 5 mM - pH 7.4) and then the cells were detached and lysed by mechanical scraping in the same buffer. The lysate was centrifuged at 40000 x g for 22 min at 4 °C. The pellet was stored at -80 °C until use.

[0108] [3H]-Prostaglandin E2 ([3H]-PGE2) binding assays were performed in 10 mM MES-KOH buffer pH 6 containing 10 mM MgCl2 and 1 mM CaCl2. Ten micrograms of protein from membrane fractions were incubated with 1 nM [3H]-PGE2 in a total volume of 0.1 ml. To determine total or non-specific binding, 1% DMSO or 1 μM PGE2 was added to the reaction mixture, respectively. Specific binding accounted for more than 85% of total binding. In competition curves, the diluent was replaced by the test compound (8 concentrations spanning at least two orders of magnitude; duplicate points). In an independent set of experiments, competition curves of the same compounds were performed in the presence of 0.5% bovine serum albumin. Incubation was carried out for 90 min at room temperature in 96 multiwell plates before separation of bound and free radioligand by rapid filtration through glass fiber filters (Unifilter GFB 96, PerkinElmer Inc) presoaked in 0.3% polyethyleneimine. Filters were washed with ice-cold buffer pH 7.4 (50 mM HEPES, NaCl 500 mM, BSA 0.1%) and dried for 30 min at 30°C, then 0.1 ml of MICROSCINT-20 (PerkinElmer Inc.) was added. After at least 1 h of stabilization, residual [3H]-PGE2 binding was determined by solid scintillation counter (TopCount, PerkinElmer Inc). Competition curve results were analyzed using IC 50 The K values ​​were expressed as K and the corresponding K was calculated according to the Chang-Prousoff equation. Finally, the K values ​​were converted to pK (negative logarithm of K).

[0109] result Both compounds under investigation exhibited nanomolar affinity for the human recombinant EP4 receptor. However, in the presence of protein (i.e., BSA), compound 2 exhibited a 6-fold lower affinity (7.5 and 6.7 in the absence or presence of BSA, respectively) as indicated by the lower pKi values ​​reported in the table below for compound 1. [Table 4]

[0110] Affinity (pKi) of Compound 1 and Compound 2 for human recombinant EP4 receptor in the absence or presence of 0.5% BSA.

[0111] conclusion The results obtained suggest that the compound of Example 2 has a higher binding capacity to proteins (e.g., BSA) in vitro when compared to compound 1, thus affecting its interaction with the EP4R under test.

[0112] Example 5: In vitro effects of compound 1 on TNFα release induced by lipopolysaccharide (LPS) and regulated by prostaglandin E2 (rat whole blood cultures) Materials and Methods Male Wistar rats (250-300 g bw, Charles River, Italy) were housed six per cage in a temperature-controlled room with a 12 h / 12 ​​h light / dark cycle set to maintain temperature within the range of 20 °C ± 2 °C and relative humidity within the range of 55% ± 10. Throughout the study, animals had free access to standard laboratory chow (Teklad Rodent Chow 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. Care and handling of the animals followed local government guidelines and European Community regulations. Approval for the experimental procedures was granted by the Italian Ministry of Health.

[0113] On the day of the experiment, animals were anesthetized with 2.5–3% isoflurane in O2 in an induction chamber and blood was collected from the abdominal aorta. Whole blood samples were collected in tubes containing 0.1–0.2 U / ml heparin. Aliquots (0.5 ml) were dispensed into a series of tubes and after addition of 0.1 μg / ml lipopolysaccharide (Escherichia coli serotype 055:B5; LPS; Sigma Aldrich) (control sample) or a mixture of 0.1 μg / ml LPS + 0.1 μM PGE2 (Cayman Chemical), samples were incubated for 4 h at 37 °C in a 5% CO2 incubator. The stimulator concentration chosen was the lowest concentration from previous titration assays that resulted in cell activation. Otherwise, it is possible that modest changes in cytokine production could be masked. Similarly, the time of stimulation was optimal and as short as possible, since immune cell function could be affected if immune cells are removed from the animal. Four hours after stimulation, cytokine release was near maximal and plateaued after 18–24 hours.

[0114] In samples representing basal cytokine release, LPS was replaced with the same volume of sterile PBS. LPS and PGE2 were prepared as stock solutions in sterile PBS + BSA 0.2%. At the end of the incubation, 10 mM EDTA was added and samples were centrifuged at 4°C to obtain plasma, which was then stored in aliquots at -80°C until ELISA assay for cytokine determination. The level of TNF-α release in rat whole blood cultures was assessed using a rat TNF-α ELISA kit (Diaclone, France). TNF-α levels in samples were expressed as concentration (pg / ml) compared to a reference standard curve.

[0115] result A concentration-dependent reversal of the effect of PGE2 on TNF-α release by compound 1 in vitro was demonstrated. The IC curves of compound 1 (0.1–30 μM) versus fixed PGE2 concentrations that determined approximately 80% of the inhibition of TNF-α release were shown in Figure 6. 50 This made it possible to calculate a value of 2.3 μM.

[0116] conclusion The inhibitory effect of PGE2 on LPS-induced TNF-α production was useful to demonstrate in vitro the activity of the EP4 antagonist compound 1. The results obtained in these experiments highlight that compound 1 reverses the PGE2 reduction of TNF-α release in LPS-stimulated blood cells.

[0117] Example 6 Ex vivo TNF-α release induced by LPS: PGE2 inhibition (IC 50 ) and Compound 1 treatment dose Materials and Methods Male Wistar rats (250-300 g bw, Charles River, Italy) were housed six per cage in a temperature-controlled room with a 12 h / 12 ​​h light / dark cycle set to maintain temperature within the range of 20 °C ± 2 °C and relative humidity within the range of 55% ± 10. Throughout the study, animals had free access to standard laboratory chow (Teklad Rodent Chow 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. Care and handling of the animals followed local government guidelines and European Community regulations. Approval for the experimental procedures was granted by the Italian Ministry of Health.

[0118] Modulation of the effect of PGE2 (Cayman Chemical) on TNF-α release ex vivo was assessed using whole blood culture samples from animals orally treated with vehicle or various doses of Compound 1 and collected 1 hour after dosing.

[0119] In detail, on the day of the experiment, animals were anesthetized with 2.5-3% isoflurane in O2 in an induction chamber and blood (average 7 ml / rat) was collected from the abdominal aorta. Whole blood samples were collected in tubes containing 0.1-0.2 U / ml heparin. Aliquots (0.5 ml) were dispensed into a series of tubes and after addition of LPS (control samples) or a mixture of 0.1 μg / ml LPS + various concentrations of PGE2, the samples were incubated for 24 h at 37 °C in a 5% CO2 incubator. In samples representing basal release of cytokines, LPS was replaced with the same volume of sterile PBS. LPS and PGE2 were prepared as 100x stock solutions in sterile PBS + BSA 0.2%. At the end of the incubation, 10 mM EDTA was added, samples were centrifuged at 1500 g for 10 min at 4 °C and plasma was removed and stored in aliquots at -80 °C until ELISA assay for cytokine determination. To analyze the levels of TNF-α release in rat whole blood cultures, a rat TNF-α ELISA kit (Diaclone, France) was used. TNF-α levels in samples were expressed as concentration (pg / ml) compared to a reference standard curve.

[0120] For each animal, the % inhibition by various PGE2 concentrations relative to the control sample and the corresponding IC 50 (or median inhibitory concentration ranging from 0 to 100% inhibition) were calculated by linear regression analysis. Additionally, either the mean TNF-α level and the mean % inhibition by PGE2 were calculated for each treatment group. To determine the statistically significant effect of each dose treatment relative to the vehicle group, a two-way ANOVA was performed considering each point on the PGE2 inhibition curve.

[0121] Linear regression analysis of compound 1 dose and PGE2 IC obtained 50 To calculate the relationship between the mean IC 50 Values ​​were plotted as a function of Compound 1 dose administered.

[0122] result To demonstrate the modulatory activity of compound 1 with respect to the known PGE2 inhibitory effect on TNF-α release, an ex vivo model based on LPS-stimulated whole blood cultures was developed. In particular, the agonist IC in the presence of antagonists was 50 The ex vivo effect of compound 1 administered orally at five different doses ranging from 10 mg / kg to 300 mg / kg was evaluated against the PGE2 inhibition curve to calculate the shift in TNF-α. TNF-α production induced by LPS 0.1 μg / ml was measured after 24 h of ex vivo stimulation. Blood was collected at 1 h and then treated with compound 1 t max The low doses used (10-30 mg / kg), chosen as representative of, were in accordance with their previously determined pharmacological activity in a rat model of rheumatoid arthritis.

[0123] PGE2 inhibited cytokine release, and the presence of Compound 1 in the blood, which interacts with EP4R, determined a statistically significant reversal of this effect. 50 The IC value was calculated and a 3-fold to up to 9-fold increase in this value was observed as a result of treatment with Compound 1. 50 The linear relationship between the values ​​is shown in FIG.

[0124] Consistent with the targeted binding of compound 1 to rat EP4R, the analysis of ex vivo release of TNF-α induced by LPS, inhibited by PGE2, and modulated by compound 1 was focused on low nanomolar concentrations of PGE2, as this is representative of the concentration range typically observed in the tumor microenvironment. When the inhibitory effect on TNF-α release determined by PGE2 was plotted against the compound 1 administered dose, an overall dose-dependent reversal was observed for both PGE2 concentrations. The results are reported in Figure 8.

[0125] Furthermore, as can be observed in the graph of Figure 9, although a slight time-dependent decrease in the effect of Compound 1 could be observed, the time course of the blood sampling analysis (1-3-24 hours) highlighted the prolonged binding of EP4R by the minimally effective dose of Compound 1 at 10 mg / kg, as the reversal of PGE2 TNF-α release inhibition was still evident 24 hours after Compound 1 administration.

[0126] conclusion The inhibitory effect of PGE2 on TNF-α production induced by LPS was useful to demonstrate the activity of the EP4 antagonist Compound 1. Overall, these results strongly demonstrate the ability of Compound 1 to reverse PGE2-induced inhibition of TNF-α release in blood cells. Indeed, in ex vivo experiments using blood samples obtained from animals orally treated with vehicle (control) or Compound 1 and collected 1 hour after administration, a statistically significant reversal of the expected effect of PGE2 was determined by the presence of Compound 1. Moreover, the reversal of PGE2 TNF-α release inhibition was still evident 24 hours after Compound 1 administration.

[0127] Example 7 Ex vivo effects of single treatments of Compound 1 and Compound 2 on LPS-induced and PGE2-inhibited TNFα release 24 hours after administration The modulatory effect of PGE2 on TNF-α release ex vivo was assessed using whole blood culture samples from Wistar Hansen rats (275-300 g bw) orally treated with vehicle, Compound 1 or the compound of Example 2, both dosed at 10 mg / kg. Blood samples were collected 24 hours after dosing.

[0128] Animals were housed six per cage in a temperature-controlled room with a 12 h / 12 ​​h light / dark cycle set to maintain temperature within the range of 20 °C ± 2 °C and relative humidity within the range of 55% ± 10. Throughout the study, animals had free access to standard laboratory chow (Teklad Rodent Chow 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. Animal care and handling were in accordance with local government guidelines and European Community regulations. Approval for the experimental procedures was granted by the Italian Ministry of Health.

[0129] In detail, on the day of the experiment, animals were anesthetized with 2.5-3% isoflurane in O2 in an induction chamber and blood was collected from the abdominal aorta. Samples were collected in tubes containing 0.1-0.2 U / ml heparin and aliquots (0.5 ml) were dispensed into a series of tubes, which were incubated for 4 h at 37 °C in a 5% CO2 incubator after the addition of LPS 0.1 μg / ml (control sample) or a mixture of LPS + PGE2 at various concentrations. In samples representing basal release of cytokines, LPS was replaced with the same volume of sterile PBS. LPS and PGE2 were prepared as stock solutions in sterile PBS + BSA 0.2%. At the end of the incubation, 10 mM EDTA was added, samples were centrifuged at 1500 g for 10 min at 4 °C and plasma was removed and stored in aliquots at -80 °C until ELISA assay (Diaclone France) for cytokine determination.

[0130] result PGE2 IC obtained in control (vehicle) or treatment groups 50 The values ​​were analyzed. The presence of Compound 1 in the blood 24 hours after administration determined a statistically significant reversal of PGE2 inhibition of TNFα release induced by LPS versus the vehicle-treated group ( FIG. 10 ), and indeed the PGE2 IC 50 A statistically significant nine-fold shift in IC values ​​was observed. In contrast, in the presence of Compound 2, IC 50Only modest, statistically non-significant shifts in values ​​were observed. Neither compound interfered with cytokine release by itself.

[0131] conclusion The inhibitory effect of PGE2 on TNF-α production induced by LPS was useful to demonstrate the activity of EP4 antagonists. Compound 1 was able to reverse the PGE2 inhibitory effect on TNF-α release 24 hours after a single oral dose. In contrast, compound Example 2 only slightly reduced PGE2 inhibition without statistical significance.

[0132] Example 8 Ex vivo effects of repeated administration of compound 1 on lipopolysaccharide-induced and prostaglandin E2-regulated TNF-α release Materials and Methods Male Wistar rats (250-300 g bw; Charles River, Italy) were housed six per cage in a temperature-controlled room with a 12 h / 12 ​​h light / dark cycle set to maintain temperature within the range of 20 °C ± 2 °C and relative humidity within the range of 55% ± 10. Throughout the study, animals had free access to standard laboratory chow (Teklad Rodent Chow 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. Animal care and handling were in accordance with local government guidelines and European Community regulations. Approval for the experimental procedures was granted by the Italian Ministry of Health.

[0133] The modulation of the effect of PGE2 on TNF-α release ex vivo was assessed using whole blood culture samples from animals orally treated with vehicle or 10 mg / kg compound 1 qd for 8 days and collected 24 hours after the last treatment.

[0134] In detail, on the day of the experiment, animals were anesthetized with 2.5-3% isoflurane in O2 in an induction chamber and blood was collected from the abdominal aorta. Whole blood samples were collected in tubes containing 0.1-0.2 U / ml heparin. Aliquots (0.4 ml) were dispensed into a series of tubes pre-warmed at 37°C for 10 min, and after addition of LPS (control) or a mixture of LPS + various concentrations of PGE2, samples were incubated for 4 h at 37°C in a 5% CO2 incubator. In samples representing basal release of cytokines, LPS was replaced with the same volume of sterile PBS. LPS and PGE2 were prepared as 100x stock solutions in sterile PBS + BSA 0.2%. At the end of the incubation, 10 mM EDTA was added, samples were centrifuged at 1500 g for 10 min at 4°C, and plasma was removed and stored in aliquots at -80°C until ELISA assay for cytokine determination.

[0135] The level of TNF-α release in rat whole blood cultures was assessed using a rat TNF-α ELISA kit (Diaclone, France).

[0136] The TNF-α levels in the samples were expressed as concentration (pg / ml) relative to the reference standard curve. For each animal, the % inhibition by various PGE2 concentrations relative to the control sample and the corresponding IC 50 (or median inhibitory concentration ranging from 0 to 100% inhibition) was calculated by linear regression analysis. In addition, either the mean TNF-α level or the mean % inhibition by PGE2 was calculated for each treatment group. PGE2 inhibition curves were compared graphically, and the mean IC 50 values ​​were calculated.

[0137] result The effect of Compound 1 (10 mg / kg) administered orally qd for 8 days on the inhibition of PGE2 was evaluated 24 hours after the last treatment. The presence of Compound 1 in the blood confirmed a statistically significant reversal of the inhibitory effect of PGE2, in fact, IC50 of the vehicle-only treated control group. 50 Values ​​are IC of the 10 mg / kg compound 1 treatment group. 50 In particular, as shown in FIG. 11, the IC50 Mean IC value for compound 1 treatment group 50 A statistically significant five-fold increase in values ​​was observed.

[0138] conclusion The results of this study demonstrate that repeated dose treatment with Compound 1 can modulate PGE2-induced inhibition of TNF-α release in rat blood cells, even 24 hours after the last dose.

[0139] Example 9 TNF-α expression in in vitro cultures of human macrophages Materials and Methods The human monocytic cell line THP-1 obtained from ATCC was grown according to the instructions provided. THP-1 cells were differentiated into macrophages with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich) for 4 days. Macrophages were then stimulated with 10 ng / ml lipopolysaccharide (LPS) (Sigma Aldrich) and 0.01 μM PGE2 for 3 h. Total RNA was purified using an ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT-PCR analysis was performed using the Applied Biosystems 7500 Fast Real-Time PCR System with specific TaqMan assays (number Hs00174128_m1; Thermo Fisher Scientific) and the 18S Pre-Developed TaqMan® Assay (Thermo Fisher Scientific) as an endogenous control. Data analysis with normalization to 18S amplification values ​​was performed according to Thermo Fisher Scientific's specific instructions for relative quantification of gene expression. All individual data are the result of at least three different analyses for each sample.

[0140] result To evaluate the potential of compound 1 to counteract PGE2-induced inhibition of TNF-α gene expression in human macrophages, THP-1 cells were differentiated into macrophages and then treated with LPS 10 ng / ml + PGE2 10 nM ± 0.01 μM to 10 μM concentrations of compound 1 for 3 h.

[0141] As shown in the graph in Figure 12, reporting the percentage of TNF-α gene expression compared to LPS+PGE2 stimulated human macrophages (set at 100%), compound 1 increased TNF-α gene expression levels, achieving a 2- to 3-fold increase over cells treated with PGE2+LPS at concentrations as low as 0.1 μM.

[0142] conclusion These data provide evidence that compound 1 modulates TNF-α expression levels and counteracts PGE2-induced inhibition of TNF-α expression in human immune cells such as macrophages.

[0143] Example 10: Effect of Compound 1 on RANK-L expression in cancer cell lines Materials and Methods Human breast adenocarcinoma MDA-MB-231 cells were obtained from ATCC and grown according to the instructions provided.

[0144] Cells were treated with 10 μM PGE2 ± Compound 1 10 μM for 24 h. Total RNA was purified using an ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA) and reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT-PCR analysis was performed using an Applied Biosystems 7500 Fast Real-Time PCR System with a specific TaqMan assay (Hs00243522_m1; Thermo Fisher Scientific) and an 18S Pre-Developed TaqMan® assay (Thermo Fisher Scientific) as an endogenous control. Data analysis with normalization to 18S amplification values ​​was performed according to Thermo Fisher Scientific's specific instructions for gene expression relative quantification. All individual data are the result of at least three different analyses for each sample.

[0145] result To evaluate whether compound 1 could reverse the increase in RANK-L gene expression induced by PGE2 on cancer cells, a human breast cancer cell-based model was used.

[0146] MDA-MB-231 cells were treated with 10 μM PGE2 ± Compound 1 10 μM for 24 h and the level of RANKL gene expression was evaluated by quantitative RT-PCR.

[0147] The results are reported in Figure 13 and show the average percentage of RANK-L expression compared to cells stimulated with 10 μM PGE2, which was set at 100%. Compound 1 significantly reduced the level of RANK-L gene expression.

[0148] conclusion The data obtained provide evidence that compound 1, which opposes the effects of PGE2, reduces RANK-L gene expression in human cancer cells.

[0149] Example 11: Ex vivo reduction of human Th-17 cell differentiation Materials and Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers using density gradient centrifugation, and CD4+ naive T cells were enriched from PBMCs using a human naive CD4+ T cell isolation kit (Miltenyi Biotech). Isolated CD4+ T cells were maintained in RPMI medium and supplemented with IL-12 and IL-2 (both 5 ng / ml) in combination with 1.5 ng / ml of antibodies anti-CD3 and anti-CD28, and 0.03 μM PGE2 ± 0.01-0.03-0.1-0.3. μM The cells were differentiated into Th-17 cells by stimulation with Compound 1 at a concentration of 100 mM for 48 hours.

[0150] At the end of the incubation period, cells were stained with fluorescently conjugated antibodies specific for CD4, CCR6, CD45, CD25 and IL-17 (all from BD Bioscience). Finally, the number of Th-17 cells was determined by flow cytometry and events were measured by a fluorescence-activated cell sorter (FACS; BD Bioscience) and analyzed with dedicated software.

[0151] result The results obtained are reported in Figure 14, where the frequency of Th-17F+ cells is plotted against the concentration of compound 1. Compound 1 induced a significant reduction in the number of naive Th-17 cells in a dose-dependent manner, reaching a plateau of maximum effect at concentrations above 0.1 μM.

[0152] conclusion The data obtained provide evidence that Compound 1 negatively regulates Th-17 cell differentiation.

[0153] Example 12: Ex vivo human T regulatory cell differentiation Materials and Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers using density gradient centrifugation, and CD4+ naive T cells were enriched from PBMCs using a human naive CD4+ T cell isolation kit (Miltenyi Biotech). The isolated CD4+ T cells were maintained in RPMI medium and differentiated into a Treg phenotype using two interleukins, rIL23 and rIL-1β, both at a concentration of 10 ng / ml, and co-stimulated with PGE2 at a concentration of 30 nM for a 144-hour incubation period.

[0154] At the end of the incubation period, cells were stained with fluorescently conjugated antibodies specific for CD3, CD4, FoxP3, CD25, IL35 and CRTH2 (all from BD Bioscience). Finally, the number of Treg cells was determined by flow cytometry and events were measured by a fluorescence-activated cell sorter (FACS; BD Bioscience) and analyzed with dedicated software.

[0155] result Regulatory T cells (Tregs), a subset of CD4+ T cells, have been found to play a key role in maintaining a suppressive tumor microenvironment and thus contribute to cancer progression (Shindo, Y. et al. Anticancer Res. 35, 129-136 (2015)). PGE2 is a well-known inducer of FoxP3 cell differentiation (Zhang, L. et al. Cell Biol. Int. 38, 639-646 (2014)). The data reported here highlighted the efficacy of compound 1 in reducing the ex vivo differentiation of Tregs induced by PGE2. Indeed, 0.03 μM PGE2 significantly promoted the differentiation of FoxP3 positive cells, whereas 0.1 μM compound 1 significantly reduced the differentiation of Th-3 cells, as reported in Figure 15. Th-3 cells are a specific subset of T regulatory cells whose activation and expansion in cancer correlates with the worst prognosis. (Duran-Aniotz, C. et al. Cancer Immunol. Immunother. 62, 761-772 (2013)). Moreover, a similar inhibition of PGE2-induced differentiation was observed for another subpopulation of Treg cells, namely iTR35 cells, as reported in FIG. 16.

[0156] iTr35 cells are characterized by the production and release of the potent immunosuppressant IL-35. Moreover, iTr35 cells are commonly found at high levels in breast and colorectal cancers, where they are involved in tumor immune tolerance through the suppression of effector T cell proliferation (Hao, S. et al. Carcinogenesis 39, 1488-1496 (2018); Ma, Y. et al. Oncotarget 7, 73003-73015 (2016)).

[0157] conclusion The data obtained provide evidence that compound 1 negatively regulates T regulatory cell differentiation, and therefore the results presented strongly support the application of this compound in immuno-oncology therapy aiming at restoring the immune response against cancer cells.

Claims

1. 1. A combination medicament for use in the treatment of tumors, comprising: EP4 antagonists of the formula: 【Chemistry 1】 (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid, or and pharma- ceutically acceptable salts thereof. at least one immune checkpoint inhibitor, proviso that said at least one immune checkpoint inhibitor is an anti-PD-1 antibody.

2. 2. The pharmaceutical formulation for use according to claim 1, wherein the pharma- ceutically acceptable salt is selected from the group consisting of hydrochloride, sodium salt, potassium salt and lithium salt.

3. 3. The combination pharmaceutical for use according to claim 1 or 2, wherein the EP4 antagonist is the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid.

4. A pharmaceutical combination comprising an EP4 antagonist selected from (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid or a pharma- ceutical acceptable salt thereof, and at least one immune checkpoint inhibitor, wherein the at least one immune checkpoint inhibitor is an anti-PD-1 antibody, for use in treating a tumor.

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