Pharmaceutical combination of EP4 antagonist and immune checkpoint inhibitor for treatment of tumors

Abstract

The present application relates to a pharmaceutical combination of an EP4 antagonist and an immune checkpoint inhibitor for the treatment of tumors. The present invention provides a pharmaceutical combination comprising an EP4 antagonist of the 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. Also described is a polymorphic form A of a sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl) benzyl)-6-azaspiro [2.5] octane-5-formylamino) cyclopropyl) benzoic acid characterized by a powder XRD spectrum having a peak at a value of an angle of 2 [theta] + / -0.2 DEG of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, 15.9. The combination and polymorphic Form A are described for use in the treatment of tumors.

Description

[0001] This application is a divisional application of an application with an application date of April 8, 2020, application number 202080036803.6, and invention name “Drug combination of EP4 antagonists and immune checkpoint inhibitors for treating tumors”. Field of the Invention

[0002] The present invention provides a drug combination of an EP4 antagonist and an immune checkpoint inhibitor for treating tumors.

[0003] In a preferred aspect, the present invention relates to polymorphic forms of the EP4 antagonists of the combination of the present invention. Background of the Invention

[0005] Cancer is a major threat to global public health and remains the leading cause of death worldwide. Therefore, despite recent advances in therapy, there is an urgent medical need to develop more effective therapeutic treatments.

[0006] Immuno-oncology is an innovative research field that aims to use the patient's immune system to fight cancer. One of the most promising methods to prevent the suppression of anti-cancer immunity is to block immune checkpoints, that is, to prevent T cell-mediated autoimmunity but tumors can also take advantage of its molecular pathways (molecular pathways). In tumors, the expression of these proteins is unrestricted. For this reason, an important research path has been focused on immune checkpoint inhibitors (ICIs) to 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)).

[0007] Different anticancer drugs focusing on anti-CTLA-4 and PD-1 / PD-L1 checkpoint inhibitors are now approved by the FDA. These include monoclonal antibodies for PD-1 and CTLA-4, such as pembrolizumab, nivolumab, durvalumab, tremelimumab and ipilimumab. Cancer immunotherapy by blocking immune checkpoint molecules has demonstrated significant clinical efficacy in a variety of cancer types. In addition, clinical trials of immune checkpoint immunotherapy have shown good results even for advanced metastatic cancer. (Alsaab, HO et al. Front. Pharmacol. 8, 1-15 (2017)).

[0008] Despite the undoubted success, not all patients respond, possibly because cancer can find other ways to evade immune surveillance. These limitations have pushed clinicians toward new antitumor agents or new therapies that are more effective against immune surveillance. SUMMARY OF THE INVENTION

[0010] The present inventors surprisingly found that the combination of an EP4 antagonist and at least one immune checkpoint inhibitor is effective in the treatment of tumors.

[0011] Therefore, 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.

[0012] The EP4 antagonist was first described in WO2013 / 004290 contained in the general formula. The inventors 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 EP4 antagonist that can effectively treat tumors when combined with a checkpoint inhibitor, as will be apparent from the experimental part.

[0013] When the definition of "immune checkpoint" is used in the present invention, it is intended as an accessory molecule capable of activating cellular pathways that promote or inhibit T-cell activation in immune cells or cancer cells.

[0014] When the definition of "immune checkpoint inhibitor" is used in the present invention, it is intended as a molecule that inhibits the function of an immune checkpoint.

[0015] The inventors were surprised to find that the sodium salt of the EP4 antagonist (R) -4- (1- (6- (4- (trifluoromethyl) benzyl) -6- azaspiro [2.5] octane -5- formamido) cyclopropyl) benzoic acid is preferred for the preparation of the combination. As will be clear from the experimental section, the obtained sodium salt of (R) -4- (1- (6- (4- (trifluoromethyl) benzyl) -6- azaspiro [2.5] octane -5- formamido) cyclopropyl) benzoic acid is an amorphous compound. Surprisingly, the inventors have found a very stable crystalline form of sodium (R) -4- (1- (6- (4- (trifluoromethyl) benzyl) -6- azaspiro [2.5] octane -5- formamido) cyclopropyl) benzoate, which is named Form A, allowing cancer to be treated alone, preferably when combined with at least one immune checkpoint inhibitor.

[0016] 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 having peaks at values ​​of 2θ±0.2° angle of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, 15.9.

[0017] The present invention also 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.

[0018] Therefore, 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.

[0019] 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.

[0020] In another aspect, the present invention relates to a pharmaceutical combination comprising an EP4 antagonist consisting of crystalline form A of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate and at least one immune checkpoint inhibitor for use in the treatment of tumors.

[0021] Description of the drawings

[0022] Figure 1 The DSC chart 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 is reported.

[0023] Figure 2 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 is reported.

[0024] Figure 3 The anti-tumor response of the crystalline form A of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate of Example 1 and the comparative compound of Example 2 and anti-PD-1 in the CT26 tumor model is reported. Balb / c mice were injected subcutaneously with 1×10^6 CT26 cells. After measuring the tumor on day 7, the mice were randomized and then treated with the specified therapy. Compound 1 and the compound of Example 2 were orally administered 30 mg / Kg per day. On the 8th day after transplantation, anti-PD-1 antibody was injected with 20 mg / kg, and on the 13th, 19th and 23rd days after transplantation, anti-PD-1 antibody was injected with 10 mg / kg. Tumor volume was measured twice a week and is shown as the mean ± SE of 15 mice per group. Arrows indicate anti-PD-1 injections (*p<0.05; **p<0.01Anova test).

[0025] Figure 4 The anti-tumor response of compounds, anti-PD-1 and combination therapy in the CT26 tumor model is reported. Balb / c mice were injected subcutaneously with 1×10^6 CT26 cells. After measuring the tumor on the 7th day, the mice were randomized and then treated with the specified therapy. Compound 1 and compound 2 were orally administered 30 mg / Kg every day. On the 8th day after transplantation, anti-PD-1 antibody was injected with 20 mg / kg, and on the 13th, 19th and 22nd days after transplantation, anti-PD-1 antibody was injected with 10 mg / kg. Tumor volume was measured twice a week and shown as the mean ± SE of 15 mice per group. Arrows indicate anti-PD-1 injections (*p<0.05; **p<0.01Anova test).

[0026] Figure 5 Relative body weight changes throughout treatment with Compound 1, the compound of Example 2, anti-PD-1 antibody, and combination therapy are reported. Mouse body weights were measured twice a week.

[0027] Figure 6 reported a concentration-dependent reversal of the effects of PGE2 by compound 1 in vitro.

[0028] Figure 7 reported LPS-induced TNF-α release in vitro: under PGE2 inhibition (IC 50 ) and the treatment dose of compound 1. Linear regression analysis, R2 = 0.9268; slope deviation to zero (slope deviation to zero) P value = 0.0086.

[0029] Figure 8 Inhibition of TNF-α release in vitro in whole blood cultures stimulated by LPS is reported. Inhibition 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 relative to vehicle; ***p<0.0005 relative to vehicle, Dunnett's multiple comparison test.

[0030] Fig. 9 Ex vivo inhibition of TNF-α release in whole blood sampled at various times from dosing and stimulated ex vivo by LPS is reported. Inhibitory effects of PGE2 0.03 μM and 0.1 μM following vehicle or 10 mg / kg Compound 1 administration. Two-way ANOVA analysis; ***p<0.0005 vs. vehicle, Dunnett's multiple comparison test.

[0031] Fig.10 PGE2IC reported 50 Distribution of values: Effects of Compound 1 (10 mg / kg) and Compound 2 (10 mg / kg) after single oral administration (24 hours after dosing). One-way ANOVA *p<0.001 vs. vehicle, Dunnett's multiple comparison test.

[0032] Fig.11 PGE2IC reported 50 Distribution of values: Effects of Compound 1 10 mg / kg after repeated oral administration (qd for 8 days). *P < 0.001 vs. vehicle, Mann Withney test.

[0033] Fig.12Reported that compound 1 dose-dependently reversed the PGE2-induced inhibition of TNF-α gene expression of human THP-1 cells differentiated into macrophages and stimulated with LPS 10 ng / ml plus 0.01 μM PGE2. Results are expressed as the average TNF-α expression percentage ± SD of independent experiments performed in triplicate. (LPS lipopolysaccharide, PGE2 prostaglandin 2).

[0034] Fig.13 Compound 1 inhibits RANKL gene expression in the human breast cancer cell line MDA-MB-231 stimulated with 10 μM PGE2. Results are expressed as mean RANK-L expression percentage ± SD from independent experiments performed in triplicate. *P<0.05 by one-way ANOVA with Tukey-Kramer multiple comparison test. PGE2 Prostaglandin 2.

[0035] Fig.14 Compound 1 dose-dependently reduced Th-17 frequency in human PBMC cells that were induced to differentiate toward Th-17 cells by exposure to IL-2, 11-21, anti-CD3 and CD28 antibodies, and 0.03 μM PGE2. (Th-17 cells were gated as CD4+CCR6+CD45Ro-IL17F+).

[0036] Fig.15 It is reported that compound 1 reduces the frequency of Th-3 cells in human PBMC cells, which were induced to differentiate towards Treg by exposure to rIL23 and rIL-1β (10ng / ml) and treated with PGE2 0.03μM±0.1μM compound 1. In the figure, the mean and standard deviation are reported. *p<0.05, one-way Anova. (Th-3 cells were gated as CD3+CD4+FoxP3+CD25low+TGFb+)

[0037] Fig.16 It is reported that compound 1 reduces the frequency of iTr35 regulatory cells in human PBMC cells, which are induced to differentiate towards Treg by exposure to rIL23 and rIL-1β (10ng / ml) and treated with PGE2 0.03μM±0.1μM compound 1. In the figure, the mean and standard deviation are reported. *p<0.05, one-way Anova. (iTr35 cells are gated as CD3+CD4+IL35+). DETAILED DESCRIPTION OF THE INVENTION

[0039] 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.

[0040] 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.

[0041] As will be clear from the experimental part, the sodium salt of (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoic acid obtained is an amorphous compound.

[0042] Surprisingly, the present inventors have discovered a very 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.

[0043] 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 having peaks at values ​​of 2θ±0.2° angle of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, 15.9.

[0044] The present invention also 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.

[0045] Therefore, 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.

[0046] In another aspect, the present invention relates to a pharmaceutical combination for use as a medicament, 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.

[0047] In another aspect, the present invention relates to a pharmaceutical combination for use in the treatment of tumors, 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.

[0048] In another aspect, the present invention relates to a pharmaceutical combination for use in the treatment of tumors, comprising an EP4 antagonist consisting of crystalline form A of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate and at least one immune checkpoint inhibitor.

[0049] Therefore, the present invention also relates to a method for treating tumors, which comprises administering a therapeutically effective amount of the pharmaceutical combination of the present invention to a patient.

[0050] Immune checkpoint inhibitors include, but are not limited to, PD-1 (programmed death-1), PD-L1 (programmed 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 inhibitor of T cell activation), IDO1 (indoleamine 2,3-dioxygenase), TIGIT (T cell immunoglobulin and ITIM domain), BTLA (B and T lymphocyte attenuator), A2AR (adenosine receptor A2), SIGLEC7 (sialic acid-binding immunoglobulin-type lectin 7), GITR (glucocorticoid-induced TNFR family-related gene), ICOS (Inducible T-cell co-stimulator). 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).

[0051] 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.

[0052] Antibodies, such as anti-PD-1 for use in the present invention, or antibodies against any of the other immune checkpoints listed above, will typically be mixed in a pharmaceutically acceptable substance such as a saline solution prior to administration, and may be administered using any suitable method, including but not limited to intravenous, intradermal, intraperitoneal, or intrathecal injection.

[0053] A therapeutically "effective amount" is intended to be an amount of an EP4 antagonist and at least one checkpoint inhibitor, which amount will correspond to a specific amount that will depend on, for example, the potency (IC) of the specific checkpoint inhibitor. 50 ), efficacy (EC 50 ) and biological half-life), tumor condition and its severity, the identity of the patient in need of treatment (e.g., age, size and weight), but can still be routinely determined by those skilled in the art. Similarly, the duration of treatment and the administration period (the period between doses and the timing of the dose, such as before / with / after meals) of the compounds included in the combination will vary depending on the identity of the person in need of treatment (e.g., weight), the specific compound and its properties (e.g., drug properties), the tumor and its severity, however, these can be determined by those skilled in the art.

[0054] 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.

[0055] Systemic administration includes oral administration, parenteral administration, transdermal administration, rectal administration and inhalation administration.

[0056] 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 multiple doses are administered at different time intervals within a given time period. The dose can be administered until the desired therapeutic effect is achieved, or it can be administered indefinitely to maintain the desired therapeutic effect. The suitable dosing regimen for 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 a technician. The combination of the present invention can also be formulated into a pharmaceutical composition before being administered to a patient. The pharmaceutical composition of the present invention is prepared using techniques and methods known to those skilled in the art.

[0057] In a more preferred aspect, the tumors may include, but are 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, fibrosarcoma.

[0058] In an even more preferred aspect, the immune checkpoint inhibitor is an anti-CTLA-4 and / or anti-PD-1 / PD-L1 checkpoint inhibitor.

[0059] For all treatment methods, in some embodiments, the effective dose of the anti-PD-1 antibody used is 0.1 mg / kg to 20 mg / kg of total body weight, with a preferred dose being 2 mg / kg or 3 mg / kg.

[0060] Those skilled in the art will also recognize that the optimal amount and spacing of individual dosages will be determined by the nature and extent of the condition being treated.

[0061] The present invention will now be described in further detail with reference to the Experimental Section.

[0062] Experimental Section

[0063] The reagents used in the following examples are commercially available from various suppliers and were used without further purification. Solvents were used in dry form. Reactions in an anhydrous environment were carried out under dry N 2 under positive pressure.

[0064] Proton NMR ( 1H NMR) spectra were recorded on a Bruker Avance 400 MHz instrument. Chemical shifts are reported in ppm (δ) using the residual solvent line as an internal standard. Splitting patterns are designated as: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad signal.

[0065] Mass spectra (MS) were run on an ion trap Thermo LCQ Classic spectrometer operated in positive ES (+) and negative ES (-) ionization modes.

[0066] 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).

[0067] Flash silica gel chromatography was performed on a Biotage automated flash chromatography system (Isolera systems) using Biotage SNAP HP silica gel columns.

[0068] Reverse phase chromatography was performed on a Biotage automated flash chromatography system (Isolera Systems) using a RediSep Gold C-18Aq column.

[0069] Purification of some basic compounds was performed using a Phenomenex Strata SCX column (55 μm, 70 A).

[0070] Thin layer chromatography was performed using Merck TLC plates Kieselgel 60F-254 with visualization using UV light, aqueous permanganate solution, iodine vapor.

[0071] The following abbreviations are used herein: AcOH: acetic acid; DIAD: diisopropyl (E)-diazene-1,2-dicarboxylate; Boc: tert-butoxycarbonyl; 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; NaHCO 3 :Sodium bicarbonate; Na 2 SO 4 :Sodium sulfate; Cs 2 CO 3 : cesium carbonate; NaHMDS: sodium bis(trimethylsilyl)amide; HOBt: 1-hydroxybenzotriazole

[0072] Embodiment 1:

[0073] Formula (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl) Preparation of EP4 antagonist of sodium benzoate

[0074] The compound was obtained by following the synthetic steps in Scheme 1 below:

[0075]

[0076] a) converting the starting material (SM1) into a compound of formula (I) using the starting material (SM2) and a suitable coupling agent;

[0077] b) deprotecting compound (I) in an acidic medium such as DCM containing TFA to obtain compound (II);

[0078] c) alkylating the nitrogen on the ring with benzyl bromide in the presence of a suitable base to obtain compound (III);

[0079] d) hydrolyzing the methyl ester using a suitable inorganic base such as NaOH to obtain compound (IV); and

[0080] e) Formation and crystallization of the sodium salt in a suitable solvent.

[0081] Example 1a) Synthesis of Starting Material 1 (SM 1)

[0082] The starting material SM1 reported in Scheme 1 above was obtained by the following steps reported in Scheme 2 below:

[0083]

[0084] a) Using MeI and a suitable base such as Cs 2 CO 3 Conversion of (R)-1-(tert-butoxycarbonyl)-4-oxopiperidine-2-carboxylic acid (prepared according to Tetrahedron (1997) 15671-15680) to the methyl ester (IX)

[0085] b) Wittig reaction using methyltriphenylphosphonium bromide in toluene to obtain compound (X)

[0086] c) Deprotection of the piperidine nitrogen using a suitable acidic reagent such as HCl in MeOH and subsequent protection using Cbz chloride in DCM to afford compound (XI)

[0087] d) Formation of cyclopropane using an organometallic reagent such as diethylzinc in THF, diiodomethane and TFA to obtain compound (XII)

[0088] e) Deprotection of the piperidine nitrogen using a reducing agent such as hydrogen and palladium on carbon in MeOH and subsequent reaction with (BOC) 2 O is protected to obtain compound (XIII)

[0089] f) hydrolysis of the methyl ester using a suitable inorganic base such as LiOH in THF / MeOH to obtain (SM 1)

[0090] -Synthesis of Intermediate (IX) 1-(tert-butyl) 2-methyl (R)-4-oxopiperidine-1,2-dicarboxylate

[0091] Referring to Scheme 2, intermediate (IX) was prepared.

[0092] 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): after 2 h at RT, the mixture was diluted with water (250 ml) and extracted with AcOEt (3 x 150 ml). The combined organic layers were washed with water (150 ml x 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.

[0093] 1H NMR (400 MHz, 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] +

[0094] -Synthesis of Intermediate (X) 1-(tert-butyl) 2-methyl (R)-4-methylenepiperidin-1,2-dicarboxylate

[0095] Referring to Scheme 2, intermediate (X) was prepared.

[0096] Methyltriphenylphosphonium bromide (1.1 equivalents) is dissolved in dry toluene (400ml), cooled to 3°C, then slowly dripped NaHMDS solution (1.05 equivalents). After 1 hour under 3°C nitrogen atmosphere, the intermediate (IX) (9g, 0.035mol) added in dry toluene (200ml) and stirring continued for 1 hour. After completion, the reaction was quenched with ice / water (800ml), and the two layers were separated, and the organic layer was washed with water (350ml), then washed with brine (350ml), dried over sodium sulfate and concentrated. The residue was purified by column chromatography using silica gel eluted with hexane / AcOEt 95-5 to 60-40, to provide the title compound (8g, 90%) as a light yellow solid.

[0097] 1H NMR (400 MHz, 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] +

[0098] -Synthesis of Intermediate (XI) 1-Benzyl 2-methyl (R)-4-methylenepiperidin-1,2-dicarboxylate

[0099] Referring to Scheme 2, intermediate (XI) was prepared.

[0100] Intermediate (X) (8 g; 0.031 mol) was dissolved in dry methanol (150 ml), cooled to 0°C, then 300 ml of 3M methanolic HCl solution was slowly added. After 2 hours at RT, the solvent was evaporated to dryness, the residue was dissolved in DCM (250 ml), cooled to 0°C, then triethylamine (2.5 eq.) and benzyl chloroformate (1.2 eq.) were added after 1 hour 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 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 afford the title compound (6.7 g; 74%) as a white solid.

[0101] 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] +

[0102] -Synthesis of Intermediate (XII) 6-Benzyl 5-methyl (R)-6-azaspiro[2.5]octane-5,6-dicarboxylate

[0103] Referring to Scheme 2, intermediate (XII) was prepared.

[0104] DCM (150ml) was cooled to 0°C, then diethylzinc solution (2.3 eq) in THF was slowly added and stirring continued for 30 minutes. Trifluoroacetic acid (2.0 eq) was slowly added at 0°C and stirring continued for 60 minutes, then diiodomethane (4.0 eq) was added and stirring continued for 60 minutes at 0°C. Intermediate (XI) (6 g; 0.02 mol) in dry dichloromethane (50ml) was slowly added at 0°C, then stirring was maintained 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×2) and then with 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 eluting with hexanes / AcOEt 95-5 to 60-40 to afford the title compound as a white solid (4.72 g; 75%).

[0105] 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,2H)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] +

[0106] -Synthesis of Intermediate (XIII) 6-(tert-butyl) 5-methyl (R)-6-azaspiro[2.5]octane-5,6-dicarboxylate

[0107] Referring to Scheme 2, intermediate (XIII) was prepared.

[0108] Intermediate (XII) (4.5g, 0.015mol) is dissolved in methanol (200ml), Pd / C 10% (400mg) is added, and then the suspension is hydrogenated at 3 bar for 2 hours. After completion, the reaction is filtered through a diatomaceous earth bed and washed with methanol (200ml). The solution is concentrated to 150ml, cooled to 20°C, then boc-anhydride (1.2 equivalents) is slowly added, and stirring at 25.5°C continues for 16 hours. After the reaction is complete, the solvent is evaporated, and then the residue is purified by column chromatography using silica gel eluted with hexane / AcOEt 95-5 to 60-40, to provide the title compound (3.68g; 92%) as a white solid.

[0109] 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).

[0110] ESI+m / z 270[M+H] +

[0111] - Starting Material 1 (SM 1) Synthesis of (R)-6-(tert-butoxycarbonyl)-6-azaspiro[2.5]octane-5-carboxylic acid

[0112] Referring to Scheme 2, the starting material (SM1) was obtained.

[0113] 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 eq. in 50 ml of water) was added dropwise.

[0114] The mixture was stirred at RT for 8 hours. The reaction was cooled to 10°C, quenched with acetic acid to pH 5 (50ml), and concentrated by distilling out MeOH and THF. The concentrated material was diluted with ice-water and extracted with ethyl acetate (300ml×2). The combined organic layers were washed with water (200ml) and then with brine (200ml). The organic layer was dried over sodium sulfate and concentrated below 50°C. Petroleum ether (300ml) was added to the residue; the solid was filtered off and dried under vacuum at 40°C for 24 hours. Yield 3g (92%)

[0115] 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] +

[0116] Example 1b) Synthesis of Starting Material 2 (SM2)

[0117] The starting material 2 (SM 2) was prepared according to known literature procedures (WO2008104055, Example 1, Step 2).

[0118] Example 1c) Intermediate (I) (R)-5-((1-(4-(methoxycarbonyl)phenyl)cyclopropyl)carbamoyl)-6- Synthesis of tert-butyl azaspiro[2.5]octane-6-carboxylate

[0119] Referring to Scheme 1, intermediate (I) was obtained.

[0120] 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 minutes. Starting material 2 (1.02 eq) was added, followed by TEA (1.2 eq); the reaction was kept stirring at 30°C for 6 hours, 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, tert-butyl methyl ether (200 ml) was added, and the solvent was evaporated again. 400 ml of tert-butyl methyl ether was added, 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 at 50°C under vacuum. Yield 14.7 g (88%)

[0121] 1H NMR (400MHz, chloroform-d) δ = 8.01-7.93 (m, 2H), 7.28 (s, 2H), 6.74 (s, 1H), 4.83 (brs, 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).

[0122] ESI+m / z 429[M+H] +

[0123] Example 1d): Intermediate (II) (R)-4-(1-(6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzene Synthesis of Methyl Formate

[0124] Referring to Scheme 1, intermediate (II) was obtained.

[0125] 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 hours. The reaction mixture was distilled under vacuum, DCM (100 ml) was added, and saturated sodium bicarbonate solution (300 ml, significant foaming) was slowly added at 15° C.-25° C. 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, the suspension was stirred at 20° C. for 17 hours, then the white solid was filtered and washed with cold tert-butyl methyl ether. The product was dried at 50° C. under vacuum. Yield 9.65 g (90%)

[0126] 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] +

[0127] Example 1e): Intermediate (III) (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane Synthesis of Methyl (5-((2-( ...

[0128] Referring to Scheme 1, intermediate (III) was obtained.

[0129] Intermediate (II) (9.5 g, 0.029 mmol) was dissolved in dry THF (150 ml), and then Cs 2 CO 3 (2 eq.) and 4-(trifluoromethyl)benzyl bromide (1.2 eq., dissolved in 75 ml of THF). The reaction mixture was stirred at 25°C for 8 hours, 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, the suspension was stirred at 20°C for 4 hours, then the white solid was filtered and washed with n-heptane. The product was dried at 50°C under vacuum. Yield 12.95 g (92%)

[0130] 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).

[0131] ESI+m / z 487[M+H] +

[0132] Example 1f): Intermediate (IV) (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane- Synthesis of 5-(5-formylamino)cyclopropyl)benzoic acid

[0133] Referring to Scheme 1, intermediate (IV), an EP4 antagonist in the acid form, is obtained.

[0134] Intermediate (III) (12 g, 0.024 mmol) was dissolved in THF (70 ml), then NaOH 2N (4 equiv.) was added. The reaction was kept stirring at 5 °C for 5 hours, then water (200 ml) was added and the organic matter was 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 obtain the title compound (9.9 g; 85%).

[0135] 1 H NMR(400MHz,DMSO-d6)δppm 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).

[0136] ESI+m / z 473[M+H] + .

[0137] The EP4 antagonist thus obtained was analyzed and found to be a zwitterion as an amorphous solid.

[0138] Example 1g): (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamide Preparation of sodium salt of 1-(2-(4-(4-cyclopropyl)benzoic acid)

[0139] The only crystalline form obtainable from the zwitterion of Example 1f) is a solvate which readily yields an amorphous form under thermal stress (eg heating to reduce the solvent content to ICH levels) or mechanical stress.

[0140] Thus, the hydrochloride salt was prepared by adding HCl in ether to intermediate (IV).

[0141] The hydrochloride salt of intermediate (IV) was studied, and an amorphous form and two crystalline forms (anhydrous A and solvate B) were identified from the screening. Anhydrous form A was further studied. Intermediate (IV) hydrochloride salt 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).

[0142] Sodium salt is prepared by adding NaOH in EtOH after dissolving intermediate (IV) in solvent. Different solvents (n-propyl alcohol, n-butyl alcohol; iPrOH) were tested, but n-propyl alcohol was selected for higher yield and easy drying. Two crystalline forms referred to as Form A and Form D were obtained: Form A was obtained directly from n-propyl alcohol crystals, and Form D was obtained only by the hydration of Form A. During the physical stability test, it was found that Form D was easily converted into an amorphous form by mechanical stress (milling) and during drying.

[0143] In contrast, sodium salt Form A was found to be more stable to amorphization by thermal stress, although amorphization by mechanical stress was observed at high energy. Form A is the most thermodynamically stable anhydrous form, and Form D is the most stable hydrated form. Other forms are less stable or metastable. These forms, as well as the amorphous form, are easily converted to stable Form A by solid slurries from acetone, ether, isopropyl acetate, THF, heptane.

[0144] As reported below, a synthetic protocol for sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate (Form A), also referred to as Compound 1, was established.

[0145] 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° C.-24° C. for 18-20 hours, then filtered, washed with pre-cooled n-propanol (100 ml), and dried at 70° C. for 24 hours. 43 g of a white solid (87%) was obtained.

[0146] 1 H NMR(400MHz,DMSO-d6)δppm 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).

[0147] ESI+m / z 473[M+H] +

[0148] Solubility, dissolution and bioavailability studies were performed on both the zwitterion, salt form and all sodium forms of Example 1f). The salts, especially the two forms of the sodium salt, showed improved dissolution properties compared to the amorphous form. PK experiments in both rats and dogs showed no significant differences between the various forms in terms of exposure and bioavailability, but from the perspective of stability and pharmaceutical manufacturability property, sodium salt form A appears to be the form of choice for development.

[0149] Characterization of sodium (R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido)cyclopropyl)benzoate (Form A) (also known as Compound 1)

[0150] Solid-state NMR

[0151] Solid-state NMR measurements were performed on a Bruker AVANCE II 400 instrument operating at 400.23 MHz for 1H and 100.65 MHz for 13C. The NMR spectra were recorded at room temperature with a spinning speed of 12 kHz. 13 C CPMAS spectroscopy. A cylindrical 4 mm od zirconia rotor was used, and a sample volume of 80 μL. For the CPMAS experiment, a ramp cross polarization pulse sequence with a contact time of 3 ms, a 1H 90° pulse of 4.0 μs, a cycle delay of 5s-10s, and a transient of 2000-4000 was used. A dual pulse phase modulation decoupling scheme was used with a frequency field of 75 KHz.

[0152] 13 C chemical shift (ppm)

[0153] 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

[0155] XRPD

[0156] XRPD measurements were performed on an X-ray powder diffractometer PANalytical X'pert Pro with Bragg-Brentano geometry and equipped with:

[0157] a) X'Celerator detector

[0158] b) Multi-purpose sampler

[0159] c) Rotator

[0160] d) Ceramic X-ray tube Cu LFF (λ 1 =1.54051A;λ 2 =1.54430A)

[0161] Range 4°-40°2θ

[0162] Characteristic peak (°2θ) 4.3±0.2°2θ 5.0±0.2°2θ 5.8±0.2°2θ 6.4±0.2°2θ 7.1±0.2°2θ 8.3±0.2°2θ 8.7±0.2°2θ 12.8±0.2°2θ 15.3±0.2°2θ 15.9±0.2°2θ

[0163] DSC

[0164] Thermograms were obtained using a Perkin-Elmer DSC8000 with a scan rate of 10°C / min from 30°C to 300°C. Figure 1 Report in.

[0165] Detected peaks: Tstart = 252.64°C, ΔH = 56.11 J / g

[0166] IR

[0167] IR spectra were recorded at 4000 cm-1 on a Perkin Elmer Spectroscopy 100 FT / IR instrument using the ATR (attenuated total reflectance) mode. -1 and 650cm -1 IR spectrum Figure 2 The main absorption frequencies are reported in the following table:

[0168] <![CDATA[with (cm- 1 )]]> 3301 2996 2938-2821 1659 1590 1540 1411

[0169] Stability of Form A

[0170] 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) at different humidity values ​​and temperatures was studied. The results are reported in the following table:

[0171]

[0172] RH = relative humidity

[0173] Embodiment 2:

[0174] (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidin-2-carboxamido)cyclopropyl)benzoic acid sodium salt (also known as Preparation of Compound 2

[0175] The EP4 antagonist of Example 1 was compared with another EP4 antagonist disclosed in WO2013 / 004290, namely (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido)cyclopropyl)benzoic acid.

[0176] The compound was obtained by following the synthetic steps in Scheme 3 below:

[0177]

[0178] a) converting (R)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid into a compound of formula (V) using the starting material (SM2) and a suitable coupling agent;

[0179] b) deprotecting compound (V) in an acidic medium such as DCM containing TFA to obtain compound (VI);

[0180] c) alkylating the nitrogen on the ring with benzyl bromide in the presence of a suitable base to obtain compound (VII);

[0181] d) hydrolyzing the methyl ester using a suitable inorganic base such as LiOH to obtain compound (VIII); and

[0182] e) Formation of the sodium salt using NaOH in a suitable solvent mixture.

[0183] - Example 2a: Intermediate (V) (R)-2-((1-(4-(methoxycarbonyl)phenyl)cyclopropyl)carbamoyl)piperidin Synthesis of tert-Butylpyridine-1-carboxylate

[0184] Boc-D-piperidin (500mg, 2.181mmol) is dissolved in DCM (13ml).Add N-hydroxybenzotriazole hydrate (2.62mmol) and EDCI (3.05mmol), and the reaction mixture is kept stirring for 40min.Add SM2 (2.268mmol), then add TEA (2.94mmol).The reaction mixture is kept stirring for 15h at room temperature, then water (20ml) is added.Separate each phase, and the water layer is extracted with DCM (2×15mL).The organic layer merged is evaporated and loaded on SNAP Ultra-HP Sphere-Si (10g) post, which is eluted with cyclohexane / AcOEt 100% to 70 / 30.Yield 810mg (92%), light yellow foam.

[0185] 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).

[0186] ESI+m / z 403[M+H] +

[0187] - Example 2b): Synthesis of Intermediate (VI) (R)-4-(1-(piperidine-2-carboxamido)cyclopropyl)benzoic acid methyl ester become

[0188] According to Scheme 3, intermediate (VI) is obtained.

[0189] Intermediate (V) (800 mg, 1.988 mmol) was dissolved in DCM (8 mL). TFA (19.88 mmol) was added and the reaction mixture was kept stirring at room temperature for 4 hours. The solvent was evaporated and the residue was loaded on a SPE-SCX (5 g) column which was washed with MeOH and NH 3 Elution was 1 M. The ammonia fractions were evaporated to obtain the title compound (590 mg; 98%).

[0190] 1H NMR (400MHz, chloroform-d) δ = 8.00-7.92 (m, J = 8.8Hz, 2H), 7.58 (brs, 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(brs,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 / z303[M+H] +

[0191] - Example 2c): Intermediate (VII) (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido) Synthesis of Methyl Cyclopropylbenzoate

[0192] 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 extracted into DCM / NaHCO3 The mixture of saturated solutions (50 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 the residue was loaded onto a SNAP Ultra-HPSphere-Si (10 g) column eluted with cyclohexane / ethyl acetate 100% to 70 / 30. 850 mg (95%) was obtained.

[0193] 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] +

[0194] - Example 2d): Intermediate (VIII) (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido) Synthesis of cyclopropyl)benzoic acid

[0195] 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 kept stirring at room temperature for 5 h, then 1 ml of AcOH was added, the dioxane was evaporated, and the residue was loaded onto a Biotage C18 10 g SPE-column, which was eluted with water (2 VC) and MeOH (3 VC). Yield 94% (772 mg, white solid).

[0196] 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,2 H),7.24-7.18(m,2H),3.74(d,J=14.2Hz,1H),3.25(d,J=14.2Hz,1H),2.8 8-2.79(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] +

[0197] - Example 2e): (R)-4-(1-(1-(4-(trifluoromethyl)benzyl)piperidine-2-carboxamido)cyclopropyl)benzyl Synthesis of sodium salt of acid

[0198] Intermediate (VIII) (40 g, 89.7 mmol) was dissolved in water / dioxane and NaOH (98 mmol) was then added. After stirring for 1 hour, THF was evaporated. The residue was loaded onto a Biotage C18 150 g SPE-post (8 injections), which was eluted with water (4 VC) to MeOH (gradient 4 CV). The fractions containing the desired product were evaporated and the obtained solid was dried at 60 ° C under vacuum for 3 days. Yield 98% (41 g, white solid).

[0199] 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] +

[0200] Embodiment 3:

[0201] Compound 1 ((R)-4-(1-(6-(4-(trifluoromethyl)benzyl)-6-azaspiro[2.5]octane-5-carboxamido) Combination therapy of sodium salt of cyclopropyl)benzoic acid (Form A) and compound 2 with anti-mouse PD-1 antibody in the colorectal cancer of mice Role in syngeneic models of cancer.

[0202] Materials and methods

[0203] Male Balb / c mice aged 4-6 weeks were housed in a temperature and humidity controlled room with a 12h / 12h light / dark cycle in 5 per cage. Animals had free access to food and water throughout the experiment. Balb / c mice were randomly divided into 6 groups (n=15 in each group): vehicle group, compound 1 group, compound 2 group, anti-PD-1 group, compound 1+anti-PD-1 group, compound 2+anti-PD-1 group.

[0204] CT26 cells were purchased from ATCC and cultured in a 5% CO 2 The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich) in a 37° C. incubator. The cells were subcultured twice a week until the desired cell number for seeding was obtained.

[0205] On the day of transplantation (day 0), cells in logarithmic growth phase were harvested, diluted in PBS, and 10 μl of PBS in 0.2 ml was added using a 26G syringe. ^6 The cells were inoculated subcutaneously into the flank region of each mouse.

[0206] On day 7 after inoculation, mice were randomized into 6 treatment groups with 15 animals in each group, receiving vehicle, compound 1, compound 2, anti-PD-1, and combination therapy, respectively.

[0207] Compound 1 and Compound 2 were repeatedly orally administered from day 7 to day 23 at 30 mg / kg once a day, while anti-mouse PD-1 antibody was intraperitoneally administered to mice in the anti-mouse PD-1 antibody-monotherapy group and mice in 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.

[0208] Tumor size (mm) was assessed twice weekly using digital calipers. 3 Tumor volume was calculated by measuring tumor length along the major and minor axes and using the following formula: Volume = [(length) 2 × width] / 2.

[0209] result

[0210] To investigate the effects of combination therapy with Compound 1 and the compound of Example 2 and anti-mouse PD-1 antibody, an allogeneic transplant model of mouse colorectal cancer cell lines was used. CT26 cancer cell tumors grown subcutaneously in mice were treated with monotherapy with the compound of Example 1 or Example 2 or with the compound of Example 1 or Example 2 in combination with anti-mouse PD-1 antibody for a period of 3 weeks.

[0211] Figure 3 The graph reports the weekly measured tumor volume plotted against the days after tumor cell inoculation. It can be seen that starting from the second week of treatment, monotherapy with compound 1 has a mild inhibitory effect on cancer growth, while the compound of Example 2 only shows a slight weakening of tumor growth. Anti-mouse PD-1 therapy slows cancer growth, although according to data reported in the literature, the inhibition of cancer growth is only partial. (Shindo, Y. et al. Anticancer Res. 35, 129-136 (2015)). In contrast, as Figure 4 As reported in , the anti-tumor efficacy was significantly enhanced by the combination therapy of compound 1 and anti-mouse PD-1 antibody.

[0212] Furthermore, evaluation of the animals' body weight throughout the treatment period showed that the combination therapy did not affect the animals' general health ( Figure 5 ).

[0213] in conclusion

[0214] The results reported above show that when given as monotherapy, Compound 1 and Compound 2 inhibited tumor growth. In addition, when used in combination, Compound 1 significantly enhanced the anti-tumor effect of PD-1 antibody.

[0215] Example 4

[0216] Effects of two EP4 antagonists on the specific binding of 3H-PGE2 to human recombinant EP4 receptor subtypes

[0217] method

[0218] Cell membranes were prepared from the HEK293 cell line (human embryonic kidney 293) stably overexpressing the human recombinant EP4 receptor. Cells were cultured in DMEM with Glutamax I containing 10% FBS at 37°C and 5% CO. 2 For membrane preparation, cells were seeded from a 150 cm 2The medium was aspirated from the flask. The cell monolayer was washed with 10 ml of hypotonic lysis buffer (TRIS 5mM+EDTA 5mM-pH 7.4), and then the cells were separated and lysed with the same buffer and mechanically scraped. The lysate was centrifuged at 40000×g for 22 min at 4°C. The pellet was stored at -80°C until use.

[0219] [3H]-Prostaglandin E2 ([3H]-PGE2) binding assay was performed in a 10 mM MgCl 2 and 1 mM CaCl 2 10mM MES-KOH buffer pH 6 was carried out.10 micrograms of protein from the membrane part was incubated with 1nM [3H]-PGE2 in a total volume of 0.1ml. In order to determine total binding or non-specific binding, 1% DMSO or 1μM PGE2 was added to the reaction mixture respectively. Specific binding represents total binding>85%. In the competition curve, the diluent was replaced by the test compound (8 concentrations spanning at least 2 orders of magnitude; duplicate points). In an independent experimental series, the competition curve of the same compound was carried out in the presence of bovine serum albumin 0.5%. At room temperature, incubation continued for 90 minutes in 96 multiwell plates, then the radioligand bound and free radioligand were separated by rapid filtration on a glass fiber filter (Unifilter GFB96, PerkinElmer Inc) pre-soaked in 0.3% polyethyleneimine. The filter was washed with ice-cold buffer pH 7.4 (50 mM HEPES, NaCl 500 mM, BSA 0.1%), dried at 30°C for 30 minutes, and then 0.1 ml of MICROSCINT-20 (PerkinElmer Inc) was added. After at least 1 hour of stabilization, residual [3H]-PGE2 binding was determined by a solid scintillation counter (TopCount, PerkinElmer Inc). The results of the competition curve are expressed as IC 50 And the corresponding Ki is calculated according to the Chang-Prousoff equation. Finally, the Ki value is converted to pKi (negative logarithm of Ki).

[0220] result

[0221] Both compounds under study showed nanomolar affinity for the human recombinant EP4 receptor. However, in the presence of protein (i.e., BSA), compound 2 showed one-sixth the affinity, as indicated by the lower pKi values ​​reported in the table below relative to compound 1 (7.5 and 6.7, respectively, in the absence or presence of BSA).

[0222]

[0223] Affinity (pKi) of Compound 1 and Compound 2 for human recombinant EP4 receptor in the absence or presence of 0.5% BSA.

[0224] in conclusion

[0225] The results obtained show that the compound of Example 2 has a higher binding potential for proteins (eg BSA) in vitro if compared to compound 1, thereby influencing its interaction with the EP4R under investigation.

[0226] Embodiment 5:

[0227] Effect of Compound 1 on TNFα release induced by lipopolysaccharide (LPS) and regulated by prostaglandin E2 in vitro (large Mouse whole blood culture)

[0228] Materials and methods

[0229] Male Wistar Han rats (250 g-300 g, bw, Charles River, Italy) were housed 6 per cage in a temperature-controlled room set to maintain the temperature within the range of 20 ° C ± 2 ° C and the relative humidity within the range of 55% ± 10, and with a 12-h / 12-h light / dark cycle. Throughout the study, animals had free access to standard laboratory food (Teklad rodent diet 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.

[0230] On the day of the experiment, the induction chamber was filled with O 2 Animals were anesthetized with 2.5%-3% isoflurane in 4% paraformaldehyde (PFA) and blood was drawn from the abdominal aorta. Whole blood samples were collected in tubes containing 0.1U / ml-0.2U / ml heparin. Aliquots (0.5ml) were distributed in a series of tubes and after adding 0.1μg / ml lipopolysaccharide (Escherichia coli serotype 055:B5; LPS; Sigma Aldrich) (control sample) or mixture 0.1μg / ml LPS+0.1μM PGE2 (Cayman Chemical), the samples were incubated at 37°C in 5% CO 2Incubation in the incubator lasts for 4 hours. The selected stimulator concentration is the lowest concentration that causes cell activation from the previous titration determination; otherwise, it is possible that moderate changes in cytokine production can be masked. Similarly, the time of stimulation is optimal and as short as possible, because once the immune cells are taken out of the animal, their function may be potentially affected. After 4 hours of stimulation, cytokine release almost reaches the maximum and is in a plateau after 18-24 hours.

[0231] In the sample representing the basic release of cytokines, LPS is replaced by the sterile PBS of the same volume.LPS and PGE2 are prepared in sterile PBS+BSA 0.2% as stock solution.When incubation ends, 10mM EDTA is added, and the sample is centrifuged at 4 ℃ to obtain blood plasma, which is subsequently stored at -80 ℃ with aliquots until the ELISA assay determined for cytokines.The level of TNF-α release in rat whole blood cultures is evaluated using rat TNFα ELISA kit (Diaclone, France).Compared with reference standard curve, the TNF-α level in the sample is expressed as concentration (pg / ml).

[0232] result

[0233] A concentration-dependent reversal of the effect of PGE2 on TNF-α release in vitro by compound 1 was demonstrated. Curves of compound 1 (0.1 μM-30 μM) against fixed PGE2 concentrations determined an inhibition of TNF-α release of approximately 80%, allowing calculation of the IC 50 Value 2.3μM, such as Figure 6 as shown in .

[0234] in conclusion

[0235] The inhibitory effect of PGE2 on TNF-α production induced by LPS can be used 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 in TNF-α release in blood cells stimulated with LPS.

[0236] Example 6

[0237] In vitro TNF-α release induced by LPS: PGE2 inhibition (IC 50 ) and the linear relationship between the treatment dose of compound 1 Tie

[0238] Materials and methods

[0239] Male Wistar Han rats (250 g-300 g, bw, Charles River, Italy) were housed 6 per cage in a temperature-controlled room set to maintain the temperature within the range of 20 ° C ± 2 ° C and the relative humidity within the range of 55% ± 10, and with a 12-h / 12-h light / dark cycle. Throughout the study, animals had free access to standard laboratory food (Teklad rodent diet 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.

[0240] Modulation of the effects of PGE2 (Cayman Chemical) released ex vivo by TNF-α was assessed using whole blood culture samples from animals treated orally with different doses of vehicle or Compound 1 and collected 1 hour after dosing.

[0241] In detail, on the day of the experiment, the air was inhaled in the chamber with O 2 Animals were anesthetized with 2.5%-3% isoflurane in 4% paraformaldehyde (PFA) and blood was drawn from the abdominal aorta (average 7 ml / rat). Whole blood samples were collected in a tube containing 0.1U / ml-0.2U / ml heparin. Aliquots (0.5 ml) were distributed in a series of tubes, and after adding LPS (control sample) or a mixture of 0.1 μg / ml LPS+ PGE2 of different concentrations, the samples were incubated at 37°C in 5% CO 2 Incubator, incubate for 24 hours.In the sample representing the basic release of cytokines, LPS is replaced by the sterile PBS of the same volume.LPS and PGE2 are prepared as 100 × stock solutions in sterile PBS+BSA0.2%.At the end of incubation, 10mM EDTA is added, and the sample is centrifuged at 4 ℃ with 1500g for 10 minutes, and plasma is taken out and stored at-80 ℃ with aliquots until the ELISA assay determined for cytokines.In order to analyze the level of TNF-α release in rat whole blood cultures, rat TNFα ELISA kit (Diaclone, France) was used.Compared with the reference standard curve, the TNF-α level in the sample is expressed as concentration (pg / ml).

[0242] For each animal, linear regression analysis was used to calculate the inhibition percentage of different PGE2 concentrations relative to the control sample and the corresponding IC 50(or half maximum inhibition concentration within the range of 0%-100%). In addition, in each treatment group, the average % inhibition of average TNF-α level and PGE2 was calculated. Two-way ANOVA was performed to determine the statistically significant effect of each dose treatment relative to the vehicle group and considering each point of the PGE2 inhibition curve.

[0243] The average IC of each group 50 The values ​​were plotted as a function of the administered Compound 1 dose in order to calculate the PGE2IC between the Compound 1 dose and the resulting 50 The relationship between.

[0244] result

[0245] In order to demonstrate the modulatory activity of compound 1 relative to the known inhibitory effect of PGE2 on TNF-α release, an in vitro model based on whole blood cultures stimulated with LPS was used. In particular, the in vitro effect of compound 1 administered orally at five different doses ranging from 10 mg / kg up to 300 mg / kg was evaluated against the PGE2 inhibition curve in order to calculate the agonist IC in the presence of the antagonist. 50 After 24 hours of in vitro stimulation, TNF-α production induced by LPS 0.1 μg / ml was measured. The blood sampling at 1 hour was selected as the compound 1t 最大 The low doses used (10 mg / kg-30 mg / kg) were representative of , and were based on their pharmacological activity as previously measured in a rat model of rheumatoid arthritis.

[0246] PGE2 inhibits cytokine release, and the presence of compound 1, which interacts with EP4R, in the blood determined a statistically significant reversal of this effect. The PGE2 IC for vehicle was calculated 50 values, and it was observed that the values ​​increased by 3-fold to as high as 9-fold as a result of treatment with compound 1. 50 The linear relationship between the values ​​is Figure 7 Displayed in.

[0247] Consistent with the target engagement of Compound 1 with rat EP4R, analysis of ex vivo release of TNF-α induced by LPS, inhibited by PGE2, and modulated by Compound 1 focused on low nanomolar concentrations of PGE2, as these represent the concentration range typically observed in the tumor microenvironment. When the inhibition of TNF-α release determined by PGE2 was plotted relative to the dose of Compound 1 administered, an overall dose-dependent reversal was observed for both PGE2 concentrations. Figure 8 Report in.

[0248] Furthermore, the time course of blood sampling analysis (1 h-3 h-24 h) highlights persistent engagement of EP4R by the minimal effective dose of 10 mg / kg Compound 1, because, as Fig. 9 As can be observed in the figure, although a slight time-dependent decrease in the effect of Compound 1 can be observed, the reversal of the inhibition of PGE2TNF-α release is still evident 24 hours after Compound 1 administration.

[0249] in conclusion

[0250] PGE2 can be used to prove the activity of EP4 antagonist compound 1 relative to the inhibitory effect produced by LPS-induced TNF-α.In general, these results have proved that compound 1 reverses the ability of the inhibition induced by PGE2 of TNF-α release in hemocytes.In fact, in the isolated experiment that blood sample is derived from the animal of oral treatment with vehicle (control) or compound 1 and collected 1 hour after administration, the existence of compound 1 determines the statistically significant reversal of the expected effect of PGE2.In addition, 24 hours after compound 1 was used, the reversal of PGE2TNF-α release inhibition also remains obvious.

[0251] Example 7

[0252] Compound 1 and Compound 2 monotherapy had an effect on TNF-α induced by LPS and inhibited by PGE2 at 24 hours after administration. In vitro effects of α release

[0253] Modulation of the effects of PGE2 released ex vivo by TNF-α was assessed using whole blood culture samples from Wistar Han rats (275 g-300 g bw) treated orally with vehicle, Compound 1, both administered at 10 mg / kg, or the compound of Example 2. Blood samples were collected 24 hours after dosing.

[0254] Animals were housed in a temperature-controlled room 6 per cage, which was set to maintain the temperature within the range of 20°C ± 2°C and the relative humidity within the range of 55% ± 10, and with a 12-h / 12-h light / dark cycle. Throughout the study, animals had free access to standard laboratory food (Teklad rodentdiet 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. The care and handling of animals was in accordance with the guidelines of the local government and the regulations of the European Community. Approval for the experimental procedures was granted by the Italian Ministry of Health.

[0255] In detail, on the day of the experiment, the air was inhaled in the chamber with O 2Animals were anesthetized with 2.5%-3% isoflurane in 4% paraformaldehyde and blood was drawn from the abdominal aorta. Samples were collected in tubes containing 0.1U / ml-0.2U / ml heparin, aliquots (0.5ml) were distributed in a series of tubes, and after adding LPS 0.1μg / ml (control sample) or mixture LPS + different concentrations of PGE2, they were incubated at 37°C in 5% CO 2 Incubate for 4 hours.In the sample representing the basic release of cytokines, LPS is replaced by the sterile PBS of the same volume.LPS and PGE2 are prepared as stock solutions in sterile PBS+BSA0.2%.At the end of incubation, 10mM EDTA is added, and the sample is centrifuged at 4 ℃ for 10 minutes at 1500g, and plasma is taken out and stored at -80 ℃ with aliquots until the ELISA assay (Diaclone France) determined for cytokines.

[0256] result

[0257] Analysis of PGE2IC obtained in the 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 ), in fact, it was observed that PGE2IC 50 In contrast, in the presence of Compound 2, a 9-fold increase in IC 50 The two compounds themselves did not interfere with cytokine release.

[0258] in conclusion

[0259] PGE2 can be used to prove the activity of EP4 antagonists relative to the inhibitory effect produced by TNF-α induced by LPS.Compound 1 can reverse the PGE2 inhibitory effect released by TNF-α in 24 hours after single oral administration.On the contrary, the compound of embodiment 2 only slightly reduces PGE2 inhibition, without statistical significance.

[0260] Example 8

[0261] In vitro effects of repeated administration of compound 1 on lipopolysaccharide-induced and prostaglandin E2-regulated TNF-α release

[0262] Materials and methods

[0263] Male Wistar Han rats (250 g-300 g, bw; Charles River, Italy) were housed 6 per cage in a temperature-controlled room set to maintain the temperature within the range of 20 ° C ± 2 ° C and the relative humidity within the range of 55% ± 10, and with a 12-h / 12-h light / dark cycle. Throughout the study, animals had free access to standard laboratory food (Teklad rodent diet 2018, Harlan Laboratories, S. Pietro al Natisone, UD, Italy) and drinking water. The care and handling of animals was in accordance with the guidelines of the local government and the regulations of the European Community. Approval for the experimental procedures was granted by the Italian Ministry of Health.

[0264] Modulation of the effects of PGE2 on TNF-α release ex vivo was assessed using whole blood culture samples from animals treated orally qd with vehicle or 10 mg / kg of Compound 1 for 8 days and collected 24 hours after the last treatment.

[0265] In detail, on the day of the experiment, the air was inhaled in the chamber with O 2 Animals were anesthetized with 2.5%-3% isoflurane in 4% paraformaldehyde (PFA) and blood was drawn from the abdominal aorta. Whole blood samples were collected in tubes containing 0.1U / ml-0.2U / ml heparin. Aliquots (0.4ml) were distributed in a series of tubes preheated at 37°C for 10 minutes, and after adding LPS (control) or mixture LPS+ PGE2 of different concentrations, the samples were incubated at 37°C in 5% CO 2 Incubate for 4 hours in an incubator. In samples representing the basal release of cytokines, LPS was replaced by the same volume of sterile PBS. LPS and PGE2 were prepared as 100× stock solutions in sterile PBS+BSA0.2%. At the end of incubation, 10 mM EDTA was added, the samples were centrifuged at 1500 g for 10 minutes at 4°C, plasma was removed and stored at -80°C in aliquots until the ELISA assay for cytokine determination was performed.

[0266] The level of TNF-α release in rat whole blood cultures was assessed using a rat TNFα ELISA kit (Diaclone, France).

[0267] The TNF-α levels in the samples were expressed as concentrations (pg / ml) compared to the reference standard curve. For each animal, the percentage inhibition of different PGE2 concentrations relative to the control sample and the corresponding IC 50(or half maximal inhibitory concentration in the range of 0%-100% inhibition). In addition, in each treatment group, the mean TNF-α level or the mean % inhibition of PGE2 was calculated. The PGE2 inhibition curves were compared graphically and the mean IC 50 value.

[0268] result

[0269] 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, the IC of the control group treated with vehicle alone was 50 The value was significantly lower than the IC value of the 10 mg / kg compound 1 treatment group. 50 In particular, the average IC 50 IC values ​​relative to the control 50 A 5-fold and statistically significant increase in Fig.11 as shown in .

[0270] in conclusion

[0271] The results of this study demonstrate that repeated dose treatment with Compound 1 can modulate the PGE2-induced inhibition of TNF-α release in rat blood cells even 24 hours after the last dose.

[0272] Example 9

[0273] TNF-α expression on in vitro cultures of human macrophages

[0274] Materials and methods

[0275] The human monocytic cell line THP-1 obtained from ATCC was grown according to the instructions provided. THP-1 cells were differentiated into macrophages for 4 days with 100nM phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich). Macrophages were then stimulated with lipopolysaccharide (LPS) (Sigma Aldrich) 10ng / ml and PGE2 0.01 μM for 3 hours. Total RNA was purified using ABIPrism 6100Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA), and reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Applied Biosystems 7500 fast real-time PCR system, specific TaqMan assay (numbering Hs00174128_m1; Thermo Fisher Scientific) and 18S pre-developed as endogenous control RT-PCR analysis was performed using the RT-PCR assay (Thermo Fisher Scientific). Data analysis, in which 18S amplification values ​​were normalized, was performed according to the specific instructions of Thermo Fisher Scientific for relative quantification of gene expression. All individual data are the results of at least three different analyses per sample.

[0276] result

[0277] To evaluate the potential of Compound 1 in counteracting PGE2-induced inhibition of TNF-α gene expression in human macrophages, THP-1 cells were differentiated into macrophages and subsequently treated with LPS 10 ng / ml plus PGE2 Compound 1 in concentrations ranging from 10 nM ± 0.01 μM to 10 μM for 3 hours.

[0278] like Fig.12 As shown in the figure, which reports the percentage of TNF-α gene expression compared to LPS+PGE2-stimulated human macrophages (set to 100%), compound 1 increased TNF-α gene expression levels, achieving a 2-fold to 3-fold increase relative to cells treated with PGE2+LPS at concentrations as low as 0.1 μM.

[0279] in conclusion

[0280] These data provide evidence that Compound 1 modulates TNF-α expression levels and antagonizes PGE2-induced inhibition of TNF-α expression in human immune cells such as macrophages.

[0281] Embodiment 10:

[0282] Effect of compound 1 on RANK-L expression in cancer cell lines

[0283] Materials and methods

[0284] Human breast adenocarcinoma MDA-MB-231 cells were obtained from ATCC and grown according to the instructions provided.

[0285] Cells were treated with 10 μm PGE2 ± compound 1 10 μm for 24 hours. Total RNA was purified using ABIPrism 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA) and reverse transcribed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Applied Biosystems 7500 fast real-time PCR system, specific TaqMan assay (Hs00243522_m1; Thermo Fisher Scientific) and 18S pre-developed as an endogenous control were used. RT-PCR analysis was performed using the RT-PCR assay (Thermo Fisher Scientific). Data analysis, in which 18S amplification values ​​were normalized, was performed according to the specific instructions of Thermo Fisher Scientific for relative quantification of gene expression. All individual data are the results of at least three different analyses per sample.

[0286] result

[0287] To evaluate whether compound 1 could reverse the increase in RANK-L gene expression on cancer cells induced by PGE2, a model based on human breast cancer cells was used.

[0288] MDA-MB-231 cells were treated with 10 μM PGE2 ± Compound 1 10 μM for 24 hours and the level of RANKL gene expression was evaluated by quantitative RT-PCR.

[0289] The results are Fig.13 The results are reported in , where the mean percentage of RANK-L expression is shown compared to cells stimulated with 10 μM PGE2 set as 100%. Compound 1 significantly reduced the level of RANK-L gene expression.

[0290] in conclusion

[0291] The data obtained provide evidence that compound 1, which antagonizes the effects of PGE2, reduces RANK-L gene expression in human cancer cells.

[0292] Embodiment 11:

[0293] Reduced human Th-17 cell differentiation in vitro

[0294] Materials and methods

[0295] Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers using density gradient centrifugation, and CD4+ naive T cells were enriched from PBMC using a human naive CD4+ T cell isolation kit (Miltenyi Biotech). The isolated CD4+ T cells were maintained in RPMI culture medium and differentiated into Th-17 cells by the following stimulation for 48 hours: both 5ng / ml of IL-12 and IL-2 with 1.5ng / ml of antibodies anti-CD3 and anti-CD28 and 0.03μM PGE2±compound 1 in a concentration of 0.01μM-0.03μM-0.1μM-0.3μM.

[0296] At the end of the incubation time, cells were stained with fluorescent 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 using dedicated software.

[0297] result

[0298] The results obtained in Fig.14 , where the frequency of Th-17F+ cells was plotted against the concentration of compound 1. Compound 1 dose-dependently induced a significant reduction in the number of naive Th-17 cells, reaching a plateau of maximal effect at concentrations above 0.1 μM.

[0299] in conclusion

[0300] The data obtained provide evidence that compound 1 negatively regulates Th-17 cell differentiation.

[0301] Embodiment 12:

[0302] Ex vivo human regulatory T cell differentiation

[0303] Materials and methods

[0304] Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers using density gradient centrifugation, and CD4+ naive T cells were enriched from PBMC using a human naive CD4+ T cell separation kit (Miltenyi Biotech). The isolated CD4+ T cells were maintained in RPMI culture medium, and in an incubation period of 144 hours, two interleukins, i.e. rIL23 and rIL-1β, both at a concentration of 10 ng / ml were used to differentiate to the Treg phenotype, and co-stimulated with 30 nM concentration of PGE2.

[0305] At the end of incubation, cells were stained with fluorescent 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 fluorescence activated cell sorter (FACS; BD Bioscience) and analyzed using dedicated software.

[0306] result

[0307] Regulatory T cells (Treg), a subset of CD4+T cells, have been found to play a key role in maintaining a suppressive tumor microenvironment and thus promote 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 herein emphasize the effectiveness of compound 1 in reducing the in vitro differentiation of Treg induced by PGE2. In fact, while 0.03 μM PGE2 significantly promoted the differentiation of FoxP3-positive cells, 0.1 μM compound 1 significantly reduced Th-3 cell differentiation, such as Fig.15 Th-3 cells are a specific subset of regulatory T cells, whose activation and expansion in cancer are associated with the worst prognosis. (Durán-Aniotz, C. et al. Cancer Immunol. Immunother. 62, 761-772 (2013)). In addition, similar inhibition of PGE2-induced differentiation was observed for another subset of Treg cells, iTR35 cells, such as Fig.16 reported in .

[0308] iTr35 cells are characterized by the production and release of the potent immunosuppressive factor IL-35. In addition, iTr35 cells are often found at high levels in breast cancer and colorectal cancer, where they participate in tumor immune tolerance via inhibition of effector T cell proliferation (Hao, S. et al. Carcinogenesis 39, 1488-1496 (2018); Ma, Y. et al. Oncotarget 7, 73003-73015 (2016)).

[0309] in conclusion

[0310] The data obtained provide evidence that compound 1 negatively regulates regulatory T cell differentiation, and the results shown therefore strongly support the use of this compound in immuno-oncology therapy, aiming to restore the immune response against cancer cells.

Claims

1. A drug 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, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of: PD-L1 (programmed 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 (T cell activated V domain containing Ig inhibitor) , IDO1 (indoleamine 2,3-dioxygenase), TIGIT (T cell immunoglobulin and ITIM domain), BTLA (B and T lymphocyte attenuator), A2AR (adenosine receptor A2), SIGLEC7 (sialic acid-binding immunoglobulin-type lectin 7), GITR (glucocorticoid-induced TNFR family-related gene), ICOS (inducible T-cell co-stimulator), 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).

2. The pharmaceutical combination according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sodium salt, potassium salt and lithium salt.

3. The pharmaceutical combination according to claim 1 or claim 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. 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, Features Powder XRD spectrum having peaks at 2θ±0.2° angle values ​​of 4.3, 5.0, 5.8, 6.4, 7.1, 8.3, 8.7, 12.8, 15.3, 15.

9.

5. Use of polymorphic form A according to claim 4 for the preparation of a medicament for treating a tumor, wherein the tumor is selected from the group consisting of 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.

6. A drug combination comprising a 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 claim 4 and at least one immune checkpoint inhibitor, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of: PD-L1 (programmed 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 (T cell activated V domain Ig inhibitor The proteins involved in the study are TNFR family members (TNFR family members), SGLT2 (SGLT2), TNFR family members (TNFR family members), TNFR family members (TNFR family members), EGFR (EGFR family members), TNFR family members (TNFR family members), EGFR (EGFR family members), TNFR family members (TNFR family members), EGFR (EGFR family members), EGFR family members (TNFR ...

7. The drug combination according to claim 1 or 6, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of a neutralizing antibody anti-CTLA-4 (e.g., ipilimumab, tremelimumab), an anti-TIM-3 antibody (e.g., MBG453), or an anti-LAG-3 antibody.

8. A use of a drug 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 for the preparation of a medicament for treating a tumor, wherein the tumor is selected from the group consisting of 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.

9. A use of a drug combination comprising an EP4 antagonist consisting of 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 claim 4 and at least one immune checkpoint inhibitor for the preparation of a medicament for treating a tumor, wherein the tumor is selected from the group consisting of 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.

10. A use of a drug 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 for the preparation of a medicament for treating a tumor, wherein the at least one immune checkpoint inhibitor is anti-PD-1 (programmed death-1), and the tumor is selected from the group consisting of: 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.

11. A drug combination comprising an EP4 antagonist consisting of 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 claim 4 and at least one immune checkpoint inhibitor for the preparation of a medicament for treating a tumor, wherein the at least one immune checkpoint inhibitor is anti-PD-1 (programmed death-1), and the tumor is selected from the group consisting of 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.

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