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

Abstract

To provide an effective and novel antitumor agent.SOLUTION: The present invention provides a pharmaceutical combination comprising the 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. A polymorphic form A of 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 the angle 2θ±0.2° of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, 15.9 is also described. The combination and the polymorphic form A are described for use in the treatment of tumors.SELECTED DRAWING: None

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 a 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 field of research aimed at harnessing a patient's immune system to fight cancer. One of the most promising approaches to prevent suppression of anti-cancer immunity is the blockade of immune checkpoints, molecular pathways that evolved to prevent T cell-mediated autoimmunity, but tumors can also take advantage of them. In tumors, the expression of these proteins is deregulated. For this reason, a significant body of research has focused on immune checkpoint inhibitors (ICIs) that block 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 ligand-1 (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 based on immune checkpoint blockade has demonstrated remarkable clinical efficacy across multiple cancer types, and clinical trials of immune checkpoint immunotherapy have shown favorable results even in advanced metastatic cancers (Alsaab, HO et al., Front. Pharmacol. 8, 1–15 (2017)).

[0006] Despite significant success, not all patients respond, likely because cancers can find other ways to escape immune surveillance. These limitations have driven clinicians to seek new antitumor agents or novel 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] Accordingly, 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 pharmaceutically 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 will become clear from the experimental section.

[0010] In the present invention, when the definition of "immune checkpoint" is used, it is intended as an accessory 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 present 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 a preferred compound for preparing the combination. As can be seen 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 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, which, by itself, allows for the treatment of cancer, 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 ±0.2° 2θ.

[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 pharmaceutically acceptable salt thereof 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 treating tumors. [Brief explanation 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. [Figure 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. [Figure 3] Figure 1 reports the anti-tumor response of crystalline form A of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate sodium of Example 1, and the comparative compound of Example 2 and anti-PD-1 in a 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 30 mg / kg daily. Anti-PD-1 antibody was injected at 20 mg / kg on day 8, and at 10 mg / kg on days 13, 19, and 23 after implantation. Tumor volumes were measured twice weekly and are shown as the mean ± SE for 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 responses of compounds, anti-PD-1, and combination therapy 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 Compound 2 were orally administered daily at 30 mg / kg. Anti-PD-1 antibody was injected at 20 mg / kg on day 8 and at 10 mg / kg on days 13, 19, and 22 after implantation. Tumor volumes were measured twice weekly and are shown as the mean ± SE for 15 mice per group. Arrows indicate anti-PD-1 injection (*p<0.05; **p<0.01 Anova test). [Figure 5]Figure 1 reports the relative body weight changes during treatment with Compound 1, the compound of Example 2, an anti-PD-1 antibody, and combination therapy. Mouse body weights were measured twice a week. [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 treatment dose of Compound 1. Linear regression analysis, R2=0.9268; P-value of slope deviation relative to 0=0.0086. [Figure 8] Figure 1 reports the ex vivo inhibition of TNF-α release in whole blood cultures stimulated with 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 the ex vivo inhibition of TNF-α release in whole blood sampled at different times from administration and stimulated ex vivo with LPS. Inhibitory effect of PGE2 0.03 μM and 0.1 μM after administration of 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: Reports the effect of Compound 1 (10 mg / kg) and Compound 2 (10 mg / kg) after a single oral dose (24 hours after administration). One-way ANOVA *p<0.001 vs. vehicle Dunnett's multiple comparison test. [Figure 11] PGE2 IC50 value distribution: Reporting 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]This figure 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 the 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] This figure reports that compound 1 dose-dependently reduced Th-17 frequencies 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 frequencies 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. Graphs report mean and standard deviation. *p<0.05 One-Way Anova. (Th-3 cells were gated as CD3+CD4+FoxP3+CD25low+TGFb+) [Figure 16] This figure reports that Compound 1 reduced the frequency of iTr35 regulatory cells in human PBMC cells induced toward Treg differentiation by exposure to rIL23 and rIL-1β (10 ng / ml) and treated with PGE2 0.03 μM ± 0.1 μM Compound 1. Graphs report mean and standard deviation. *p<0.05 One-Way Anova. (iTr35 cells were gated as CD3+CD4+IL35+). DETAILED DESCRIPTION OF THE INVENTION

[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 pharmaceutically acceptable salt thereof and at least one immune checkpoint inhibitor.

[0020] The combination of the present invention may comprise a pharmaceutically 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 clear from the experimental section, the sodium salt of benzyl-6-azaspiro[2.5]octane-5-carboxamidocyclopropylbenzoic acid obtained was an amorphous compound.

[0022] Surprisingly, the inventors have discovered a highly stable crystalline form of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate sodium, 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 ±0.2° 2θ.

[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 pharmaceutically acceptable salt thereof 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 pharmaceutically acceptable salt thereof and at least one immune checkpoint inhibitor for use in treating 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 treating tumors.

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

[0030] Immune checkpoint inhibitors include 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), and 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 ligand 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, e.g., anti-PD-1 or any of the other immune checkpoint antibodies listed above used in the present invention, is generally mixed with a pharmaceutically acceptable substance, such as 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 potency (IC) of a particular checkpoint inhibitor (e.g., 50 ), efficacy (EC 50 It is contemplated that the amounts of the EP4 antagonist and at least one checkpoint inhibitor will vary depending on factors such as the tumor size and biological half-life, tumor condition and its severity, and the characteristics of the patient requiring treatment (e.g., age, size, and weight), but can nevertheless be routinely determined by those skilled in the art. Similarly, the duration of treatment and the administration period (the period between administrations and the timing of administration, e.g., before / during / after meals) of the compounds contained in the combination will also vary depending on the characteristics of the person requiring treatment (e.g., body weight), the specific compound and its characteristics (e.g., pharmaceutical properties), the tumor and its severity, but can nevertheless be determined by those skilled in the art.

[0034] The EP4 antagonist of the present invention and the 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 can be administered once or according to a dosing regimen in which several doses are administered at various time intervals over a given period. Doses can be administered until the desired therapeutic effect is achieved or indefinitely to maintain the 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 can 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 more preferred embodiments, 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, kidney cancer, renal cancer, liver cancer, thyroid cancer, pancreatic cancer, sarcoma, and fibrosarcoma.

[0038] In an even more preferred embodiment, the immune checkpoint inhibitor is an anti-CTLA-4 and / or 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 the optimal amount and interval of individual administrations 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 The 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] Reversed-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: terbutyloxycarbonyl; 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 antagonist 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 to give 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 (prepared according to Thetrahedron (1997) 15671-15680) is converted to the methyl ester (IX) using MeI and a suitable base such as Cs2CO3. 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 hydrogen in MeOH and a reducing agent such as palladium on carbon 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 equivalents) was added, followed by dropwise addition of methyl iodide (1.1 equivalents), 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 equiv.) was dissolved in anhydrous toluene (400 ml) and cooled to 3°C. NaHMDS solution (1.05 equiv.) was then slowly added dropwise. After 1 h at 3°C ​​under a nitrogen atmosphere, intermediate (IX) (9 g, 0.035 mol) in anhydrous toluene (200 ml) was added and stirred for 1 h. Upon 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, followed by the slow addition of 300 ml of 3M methanolic HCl solution. After 2 h at RT, the solvent was evaporated to dryness, and the residue was dissolved in DCM (250 ml) and cooled to 0°C. Triethylamine (2.5 equiv.) and benzyl chloroformate (1.2 equiv.) were added after 1 h at RT. Upon completion, the reaction was quenched with ice-cold water; the two layers were separated, and the organic layer was washed with water followed by brine solution (250 ml). The organic layer was dried over sodium sulfate and concentrated below 40°C to give a residue that 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 (150 ml) was cooled to 0° C., then diethylzinc solution (2.3 eq.) in THF was slowly added and stirred for 30 minutes. Trifluoroacetic acid (2.0 eq.) was slowly added at 0° C. and stirred for 60 minutes, then diiodomethane (4.0 eq.) was added and stirred for 60 minutes at 0° C. Intermediate (XI) (6 g; 0.02 mol) in dry dichloromethane (50 ml) was slowly added at 0° C. and then left to stir at 25° C. for 20 hours. The reaction mass was quenched with 10% sodium bicarbonate solution (400 ml). The solid precipitate was filtered off, the layers were separated from the filtrate, and the organic layer was washed with water (250 ml×2) followed by 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 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)d ppm 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) (4.5 g, 0.015 mol) was dissolved in methanol (200 ml), Pd / C 10% (400 mg) was added, and the suspension was then hydrogenated at 3 bar for 2 hours. After completion, the reaction was filtered through a bed of celite and washed with methanol (200 ml). The solution was concentrated to 150 ml, cooled to 20°C, and then boric anhydride (1.2 equiv.) was added slowly and stirred at 25°C for 16 hours. 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 (3.68 g; 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 hours. 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 x 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 under vacuum at 40°C for 24 hours. 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 a known literature procedure (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 equivalents) and EDC-HCl (1.1 equivalents) were added, and the mixture was stirred at 20 °C for 30 minutes. Starting material 2 (1.02 equivalents) was added, followed by TEA (1.2 equivalents); the reaction was left stirring at 30 °C for 6 hours 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), and 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, and the suspension was stirred at 20 °C for 17 hours. The white solid was then 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 equivalents) was added, and the solution was stirred at 20°C for 5 hours. The reaction mixture was evaporated 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. tert-butyl methyl ether (200 ml) was added, and the solvent was evaporated again. 300 ml of tert-butyl methyl ether was added, and the suspension was stirred at 20°C for 17 hours. The white solid was then filtered and washed with cold tert-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 CsCO (2 equivalents) and 4-(trifluoromethyl)benzyl bromide (1.2 equivalents dissolved in 75 ml of THF) were added. The reaction mixture was stirred at 25° C. for 8 hours, and then 350 ml of DCM and 350 ml of 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 the solvent was evaporated again. 150 ml of n-heptane was added, and the suspension was stirred at 20° C. for 4 hours, 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 acid form, was obtained.

[0074] Intermediate (III) (12 g, 0.024 mmol) was dissolved in THF (70 ml) and then NaOH 2N (4 equivalents) was added. The reaction was left stirring at 5°C for 5 hours, 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 x 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 screening identified an amorphous form and two crystalline forms (anhydrous A and solvate B). 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. Chemical stability was comparable to that of 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 chosen due to its higher yield and ease of 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 due to thermal stress, but amorphization was observed due to high-energy mechanical stress. 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 stable Form A by solid slurry from acetone, diethyl ether, isopropyl acetate, THF, and heptane.

[0081] A synthetic 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 cooled solution of NaOH (1.02 eq.) 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 conducted on the zwitterion of Example 1f), both the salt form and all sodium forms. 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 with respect to exposure and bioavailability, although 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 referred to 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 experiment used a ramp cross-polarization pulse sequence with a 3 ms contact time, a 4.0 μs 1H 90° pulse, a 5-10 s recycle delay, and a 2000-4000 transient response. A two-pulse phase-modulated decoupling scheme was used with a 75 kHz frequency field. 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 an X-ray powder diffractometer PANalytical X'pert Pro 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 acquired 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. Detected peaks: 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 1500 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 The stability of Form A of the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid (also referred to as Compound 1) was investigated at various humidity values ​​and temperatures. 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 known as compound 2) The EP4 antagonist of Example 1 was compared to another EP4 antagonist disclosed in WO 2013 / 004290, namely (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 a suitable 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 hours. The solvent was evaporated and the residue was loaded onto an SPE-SCX (5 g) cartridge eluted with MeOH and NH3 1 M in MeOH. Evaporation of the ammoniacal fractions gave 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 hours, then 1 ml of AcOH was added, the dioxane was evaporated and the residue was loaded onto a Biotage C18 10 g SPE-column eluting 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) eluting 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: Effect 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 mouse colorectal cancer model. Materials and Methods Male Balb / c mice, 4-6 weeks old, were housed five per cage in a temperature- and humidity-controlled room with a 12-hour / 12-hour 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 per group): vehicle, compound 1, compound 2, anti-PD-1, compound 1 + anti-PD-1, and compound 2 + anti-PD-1.

[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% CO incubator. Cells were subcultured twice a week until the required number of cells for inoculation was obtained.

[0099] On the day of transplantation (day 0), logarithmically growing cells were harvested and diluted in PBS, at 10 in 0.2 ml of PBS. ^6 Cells were inoculated subcutaneously into the flank region 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 once daily at 30 mg / kg repeatedly from day 7 to day 23, 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 day 8 after transplantation and at a dose of 10 mg / kg on days 13, 19, and 22 after transplantation.

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

[0103] result To investigate the effect of combination therapy of Compound 1 and Compound 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 for 3 weeks with Compound 1 or 2 as monotherapy, or with Compound 1 or 2 in combination with anti-mouse PD-1 antibody.

[0104] The graph in Figure 3 reports the weekly measured tumor volume 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 tumor growth, starting from the second week of treatment, while Compound 2 showed only a slight impairment of tumor growth. According to data reported in the literature, anti-PD-1 therapy delayed cancer growth, even if the inhibition was only partial (Shindo, Y. et al., Anticancer Res. 35, 129-136 (2015)). In contrast, as reported in Figure 4, combined treatment with Compound 1 and an anti-PD-1 antibody significantly enhanced antitumor efficacy.

[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 showed that Compound 1 and Compound 2 inhibit tumor growth when given as monotherapy. Furthermore, Compound 1 significantly enhances the antitumor effect of PD-1 antibodies when used in combination.

[0107] Example 4 Effects of two EP4 antagonists on the 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 containing Glutamax I containing 10% FBS at 37°C in 5% CO2. For membrane preparation, cells were seeded on a 150cm plate. 2The culture 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 40,000 × g for 22 minutes at 4 °C. The pellet was stored at -80 °C until use.

[0108] [H]-prostaglandin E2 ([H]-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 [H]-PGE2 in a total volume of 0.1 ml. To determine total or nonspecific binding, 1% DMSO or 1 μM PGE2 was added to the reaction mixture, respectively. Specific binding accounted for >85% of total binding. For competition curves, the diluent was replaced with test compound (eight concentrations spanning at least two orders of magnitude; duplicate points). In an independent series of experiments, competition curves of the same compound were performed in the presence of 0.5% bovine serum albumin. Incubation was carried out for 90 min at room temperature in a 96-multiwell plate 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, 500 mM NaCl, 0.1% BSA) and dried at 30°C for 30 min, followed by addition of 0.1 ml of MICROSCINT-20 (PerkinElmer Inc.). After stabilization for at least 1 h, residual [3H]-PGE2 binding was determined by solid-state 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, as indicated by the lower pKi values ​​reported in the table below for Compound 1 (7.5 and 6.7 in the absence or presence of BSA, respectively). [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 a temperature within a range of 20 °C ± 2 °C and a relative humidity within a range of 55% ± 10%. Throughout the study, animals had free access to standard laboratory chow (Teklad Rodent Diet 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. Animal care and handling 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 and 0.1 μM PGE2 (Cayman Chemical), the samples were incubated for 4 hours at 37°C in a 5% CO2 incubator. The selected stimulator concentration was the lowest concentration from previous titration assays that resulted in cell activation. Otherwise, modest changes in cytokine production may be hidden. Similarly, the duration of stimulation was optimally kept as short as possible, as immune cell function may be affected if immune cells are removed from the animal. Cytokine release was near maximal 4 hours after stimulation and plateaued after 18-24 hours.

[0114] In samples representing basal cytokine release, LPS was replaced with an equal volume of sterile PBS. LPS and PGE2 were prepared as stock solutions in sterile PBS + 0.2% BSA. 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 for compound 1 (0.1–30 μM) against fixed PGE2 concentrations, which determined approximately 80% of the inhibition of TNF-α release, were shown in Figure 6. 50 This allowed a value of 2.3 μM to be calculated.

[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 a temperature within a range of 20 °C ± 2 °C and a relative humidity within a range of 55% ± 10%. Throughout the study, animals had free access to standard laboratory chow (Teklad Rodent Diet 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. Animal care and handling followed local government guidelines and European Community regulations. Approval for the experimental procedures was granted by the Italian Ministry of Health.

[0118] The 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 administration.

[0119] Specifically, 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 sample) or a mixture of 0.1 μg / ml LPS and various concentrations of PGE2, the samples were incubated at 37°C in a 5% CO2 incubator for 24 hours. For samples representing basal cytokine release, LPS was replaced with the same volume of sterile PBS. LPS and PGE2 were prepared as 100x stock solutions in sterile PBS + 0.2% BSA. At the end of the incubation, 10 mM EDTA was added, and the samples were centrifuged at 1500 g for 10 minutes at 4°C. Plasma was removed and stored in aliquots at -80°C until ELISA assay for cytokine determination. To analyze the level 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 The median inhibitory concentration (or half-maximal inhibitory concentration ranging from 0 to 100% inhibition) was calculated by linear regression analysis. Additionally, either the mean TNF-α level or the mean % inhibition by PGE2 was 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] Compound 1 dose and PGE2 IC obtained by linear regression analysis 50 The mean IC for each group was calculated to calculate the relationship between 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 an antagonist was developed. 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 production induced by 0.1 μg / ml LPS after 24 hours of ex vivo stimulation. Blood was collected at 1 hour and then compared with that of compound 1. max The low doses (10–30 mg / kg) used, chosen as representative of the RA, were in accordance with its 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 values ​​were calculated and a 3-fold to up to 9-fold increase in this value was observed as a result of treatment with Compound 1. Compound 1 dose and PGE2 IC 50 The linear relationship between the values ​​is shown in Figure 7.

[0124] Consistent with target binding of Compound 1 to rat EP4R, analysis of ex vivo TNF-α release induced by LPS, inhibited by PGE2, and regulated 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 PGE2-determined TNF-α release was plotted against Compound 1 dose, a global 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 in Figure 9, although a slight time-dependent decrease in the effect of Compound 1 can 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, 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 LPS-induced TNF-α production was useful in demonstrating 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 in the presence of Compound 1. Furthermore, 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, all administered at 10 mg / kg. Blood samples were collected 24 hours after administration.

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

[0129] Specifically, 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. Aliquots (0.5 ml) were dispensed into a series of tubes. After addition of LPS 0.1 μg / ml (control sample) or a mixture of LPS and various concentrations of PGE2, these were incubated for 4 h at 37°C in a 5% CO2 incubator. For 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 and 0.2% BSA. At the end of the incubation, 10 mM EDTA was added, and the samples were centrifuged at 1500 g for 10 min at 4°C. 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 presence of Compound 1 in the blood 24 hours after administration determined a statistically significant reversal of PGE2 inhibition of LPS-induced TNFα release relative to the vehicle-treated group (FIG. 10), and indeed, the PGE2 IC 50 A statistically significant 9-fold shift in IC values ​​was observed. In contrast, in the presence of Compound 2, IC values ​​were significantly higher than those in the vehicle-treated group. 50Only modest, statistically insignificant shifts in values ​​were observed. Both compounds did not interfere with cytokine release by themselves.

[0131] conclusion The inhibitory effect of PGE2 on LPS-induced TNF-α production was useful in demonstrating 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 administration. In contrast, compound 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 a temperature within a range of 20 °C ± 2 °C and a relative humidity within a range of 55% ± 10%. Throughout the study, animals had free access to standard laboratory chow (Teklad Rodent Diet 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. Animal care and handling followed 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 treated orally with vehicle or 10 mg / kg Compound 1 qd for 8 days and collected 24 hours after the last treatment.

[0134] Specifically, 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 preheated at 37°C for 10 min. After addition of LPS (control) or a mixture of LPS and various concentrations of PGE2, the samples were incubated for 4 h at 37°C in a 5% CO2 incubator. For samples representing basal cytokine release, LPS was replaced with the same volume of sterile PBS. LPS and PGE2 were prepared as 100x stock solutions in sterile PBS + 0.2% BSA. At the end of the incubation, 10 mM EDTA was added, and the samples were centrifuged at 1500 g for 10 min at 4°C. 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] TNF-α levels in the samples were expressed as concentration (pg / ml) relative to a reference standard curve. For each animal, the % inhibition by various PGE2 concentrations relative to the control sample and the corresponding IC 50 The mean IC50 (or half-maximal inhibitory concentration ranging from 0 to 100% inhibition) was calculated by linear regression analysis. Additionally, 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 IC50 for each group was calculated. 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 assessed 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, exceeding the IC 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 Figure 11, the IC50 Mean IC values ​​for Compound 1 treatment groups 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 manufacturer's instructions. 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 hours. 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 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 using normalization to 18S amplification values ​​was performed according to Thermo Fisher Scientific's specific instructions for relative gene expression quantification. All individual data are the result of at least three separate 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 hours.

[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 ± 10 μM Compound 1 for 24 hours. 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 using 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 separate analyses for each sample.

[0145] result To evaluate whether compound 1 can 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 ± 10 μM of Compound 1 for 24 hours, 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 counteracts 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 the 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 Th-17 cells by stimulation with IL-12 and IL-2 (both 5 ng / ml) combined with 1.5 ng / ml of anti-CD3 and anti-CD28 antibodies, and Compound 1 at concentrations of 0.03 μM PGE2 ± 0.01-0.03-0.1-0.3 μM 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 counted using 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 the PBMCs using a human naive CD4+ T cell isolation kit (Miltenyi Biotech). 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 30 nM PGE2 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 counted using 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 shown 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 highlight the effectiveness of compound 1 in reducing PGE2-induced ex vivo Treg differentiation. 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 correlate with poor prognosis. (Duran-Aniotz, C. et al. Cancer Immunol. Immunother. 62, 761-772 (2013)). Furthermore, a similar inhibition of PGE2-induced differentiation was observed for another subpopulation of Treg cells, namely iTR35 cells, as reported in Figure 16.

[0156] iTr35 cells are characterized by the production and release of the potent immunosuppressant IL-35. Furthermore, 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 aimed at restoring immune responses against cancer cells. [Section 1] Compounded medicines, including: EP4 antagonists of the formula: (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid, or pharmaceutically acceptable salts thereof, and At least one immune checkpoint inhibitor. [Section 2] Item 1. The combination pharmaceutical according to Item 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sodium salt, potassium salt and lithium salt. [Section 3] Item 3. The combination pharmaceutical according to Item 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. [Section 4] 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, and 15.9 degrees 2θ ± 0.2°. [Section 5] Polymorphic form A according to item 4 for use as a pharmaceutical. [Section 6] Polymorphic form A according to paragraph 4 for use in the treatment of tumors. [Section 7] Item 5. A combination pharmaceutical comprising 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 according to Item 4 and at least one immune checkpoint inhibitor. [Section 8] The at least one immune checkpoint inhibitor is selected from the group consisting 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), and IL-1 (IL-1). Item 5. The combination pharmaceutical according to any one of Items 1 to 4, wherein the target antigen is selected from the group consisting of: A2AR (adenosine receptor A2), SIGLEC7 (sialic acid-binding immunoglobulin lectin 7), GITR (glucocorticoid-inducible TNFR family related gene), ICOS (inducible T cell costimulator), NOX-2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), arginase I, CD276 (cluster of differentiation 276, also known as B7H4), CD27 (cluster of differentiation 27) and its ligand CD27 (cluster of differentiation 27), CD160 (cluster of differentiation 160), and CD39 (cluster of differentiation 39). [Section 9] Item 9. The combination pharmaceutical of Item 8, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of 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. [Section 10] 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 pharmaceutically acceptable salt thereof, and at least one immune checkpoint inhibitor, for use in treating a tumor. [Section 11] A combination pharmaceutical composition comprising an EP4 antagonist consisting of polymorphic form A of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate according to item 4, and at least one immune checkpoint inhibitor, for use in treating a tumor.

Claims

1. A combination pharmaceutical comprising 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 having peaks at angles 2θ±0.2° of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, and 15.9, and at least one immune checkpoint inhibitor.

2. The at least one immune checkpoint inhibitor is selected from the group consisting 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, 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), and IL-1 (IL-1).

2. The combination pharmaceutical of claim 1, wherein the target antigen is selected from the group consisting of: IL-16 (antigen), A2AR (adenosine receptor A2), SIGLEC7 (sialic acid-binding immunoglobulin lectin 7), GITR (glucocorticoid-inducible TNFR family related gene), ICOS (inducible T cell costimulator), NOX-2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), arginase I, CD276 (cluster of differentiation 276, also known as B7H4), CD27 (cluster of differentiation 27), CD160 (cluster of differentiation 160), and CD39 (cluster of differentiation 39).

3. 3. The combination pharmaceutical of claim 2, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of anti-PD-1 (nivolumab (Opdivo), pembrolizumab (Keytruda)), anti-CTLA-4 (ipilimumab, tremelimumab), anti-TIM-3 antibody (MBG453), or anti-LAG-3 antibody.

4. A pharmaceutical combination comprising an EP4 antagonist consisting of polymorphic Form A of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate, characterized by a powder XRD spectrum having peaks at angle 2θ±0.2° values ​​of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, and 15.9, and at least one immune checkpoint inhibitor, for use in treating a tumor.

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