阅读说明：本技术 作为ep4受体拮抗剂的(r)-4-(1-(1-(4-(三氟甲基)苄基)吡咯烷-2-甲酰胺)环丙基)苯甲酸 ((R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid as EP4 receptor antagonists ) 是由 塞布丽娜·普奇 F·马科维 L·C·罗瓦蒂 于 2019-07-04 设计创作，主要内容包括：以下发明涉及(R)-4-(1-(1-(4-(三氟甲基)苄基)吡咯烷-2-甲酰胺)环丙基苯甲酸(化合物1)或其盐。还描述了用于获得化合物1的有利方法以及包含化合物1的药物组合物。(R)-4-(1-(1-(4-(三氟甲基)苄基)吡咯烷-2-甲酰胺)环丙基苯甲酸(化合物1)或其药学上可接受的盐被描述为用于作为EP4受体拮抗剂在治疗涉及前列腺素E-2(PGE-2)在其发病机理中的活性的病状的中使用。(The following invention relates to (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (compound 1) or a salt thereof advantageous methods for obtaining compound 1 and pharmaceutical compositions comprising compound 1 are also described (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (compound 1) or a pharmaceutically acceptable salt thereof is described for use as an EP4 receptor antagonist in the treatment of conditions involving prostaglandin E 2 (PGE 2 ) In the pathology of activity in its pathogenesis.)

(R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropylbenzoic acid (compound 1), having the following structure:

or a salt thereof.

2. The compound of claim 1, wherein the salt is a sodium salt.

3. The compound of claim 1, wherein the salt is a lithium salt.

4. The compound of claim 1, wherein the salt is a hydrochloride salt.

5. A process for the preparation of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropylbenzoic acid (compound 1), or a salt thereof, according to any one of claims 1 to 4, comprising the steps of:

a) amide formation (intermediate P1) by reaction of (tert-butoxycarbonyl) -D-proline (N-Boc-D-proline) with methyl 4- (1-aminocyclopropyl) benzoate in the presence of a coupling agent

b) Deprotecting the Boc group of intermediate P1 with at least one acid to obtain intermediate P2

c) Alkylating said intermediate P2 with 4-trifluoromethylbenzyl bromide in the presence of a base, in order to obtain the methyl ester derivative (intermediate P3)

And

d) hydrolysis of the derived methyl ester (intermediate P3) in the presence of a strong base, in order to obtain said Compound 1

6. The process of claim 5, wherein the coupling agent of step a) is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

7. The process according to claim 5 or 6, wherein in step b) the at least one acid is 2,2, 2-trifluoroacetic acid.

8. The process of any one of claims 5-7, wherein in step c), the base is cesium carbonate.

9. The process of any one of claims 5-8, wherein in step d), the strong base is sodium hydroxide.

10. A process for the preparation of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropylbenzoic acid (compound 1), or a salt thereof, according to any one of claims 1 to 4, comprising the steps of:

a) alkylation of D-proline with 4-trifluoromethylbenzyl bromide in the presence of a strong base, using an alcohol as solvent, to obtain intermediate P4

b) Amide formation by reaction of said intermediate P4 with methyl 4- (1-aminocyclopropyl) benzoate in the presence of a coupling agent (intermediate P3)

And

c) hydrolyzing said intermediate P3 in the presence of a base to obtain said Compound 1

11. The process of claim 10, wherein in step a), the base is potassium hydroxide and the alcohol is 2-propanol.

12. The process according to any one of claims 10-11, wherein in step b) the coupling agent is preferably 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

13. The process of any one of claims 10-12, wherein in step c) the strong base is sodium hydroxide.

14. A pharmaceutical composition comprising a compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

15. (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (compound 1), or a pharmaceutically acceptable salt thereof, according to any of claims 1-4 for use as a medicament.

16. (R) -4- (1- (1- (4-trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (Compound 1), or a pharmaceutically acceptable salt thereof, according to any of claims 1-4 for use as an EP4 receptor antagonist in therapy involving prostaglandin E₂(PGE₂) In active pathologies in their pathogenesis.

17. (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid, or a pharmaceutically acceptable salt thereof, for use according to claim 16, wherein the condition is selected from the group consisting of rheumatoid arthritis, spondyloarthritis, psoriatic arthritis and ankylosing spondylitis, arthropathies caused by inflammation and acute and chronic pain, osteoarthritic pain, arthritic pain with an immunological etiology and rheumatoid arthritis.

18. (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid, or a pharmaceutically acceptable salt thereof, for use according to claim 16, wherein the condition is a neoplastic disease.

19. (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropylbenzoic acid, or a pharmaceutically acceptable salt thereof, for use according to claim 16, wherein the condition is an eye disease, preferably selected from the group consisting of retinopathy of prematurity (ROP), proliferative diabetic retinopathy and age-related macular degeneration (AMD).

20. (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid, or a pharmaceutically acceptable salt thereof, for use according to claim 16, wherein the condition is endometriosis.

Technical Field

The present invention describes novel derivatives of D-proline as potent and selective antagonists of the EP4 receptor, the compound (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropylbenzoic acid of the formula (Compound 1)

And pharmaceutically acceptable salts thereof, processes for their preparation, pharmaceutical compositions containing them, and their use as medicaments for the treatment of diseases in which prostaglandin E is involved in the pathogenesis₂(PGE₂) Plays a fundamental role through its interaction with the EP4 receptor subtype.

Field of the invention

The EP4 receptor is a type of 7-transmembrane receptor, the activation of which is usually associated with increased intracellular levels of cyclic adenosine monophosphate (cAMP).

Prostaglandin E₂(PGE₂) The EP4 receptor, activated by the activation, functions as a cytokine amplification system such as interleukin-6 (IL-6) and induces differentiation and expansion of pro-inflammatory T helper lymphocytes (Th) (Yokoyama et al, pharmacol. rev.2013; 65: 1010-52).

WO2013/004290 describes cyclic amino derivatives as EP4 receptor antagonists. In particular, in example 7, 4- (1- (6- (4- (trifluoromethyl) benzyl) -6-azaspiro [2.5] is described]Octyl-5-carboxamide) cyclopropyl) benzoic acid (known as E7). E7, enantiomer R abbreviated CR6086, shows high affinity and selectivity for the human EP4 receptor (K)_I16.6nM) in stimulated cAMP PGE₂Act as pure antagonists in the production of (c). In an experimental model of E7 cell culture, E7 inhibition was taken as a classRole of important cytokines as mediators of rheumatoid arthritis: for example, it decreases the expression of IL-6 and Vascular Endothelial Growth Factor (VEGF) in macrophages, decreases the release of IL-23 in dendritic cells, decreases the release of IL-17 in Th-17 lymphocytes. Furthermore, in the rat and mouse model of collagen arthritis (CIA), CR6086 has been shown to improve all parameters of induced arthritis: histopathology, inflammation, pain (Caselli et al, Arthritis Research)&Therapy(2018)20:39)。

However, CR6086 is obtained by a complex chemical synthesis as shown in scheme 1 below:

analyzing this synthesis described in WO2013/004290 and WO2011/006960, the complexity of the preparation of the synthetic intermediate CR6086, i.e. the following (R) -6- (tert-butoxycarbonyl) -6-azaspiro [2.5] octane-5-carboxylic acid, is clearly shown:

as indicated in scheme 1 above, the synthesis consists of 8 steps in sequence: after starting from the commercially available piperidin-4-one, the amino group is protected with benzyl chloroformate. Under standard conditions, a wittig reaction occurs to convert the ketone to a double bond. The double bond was converted to cyclopropyl using diethylzinc and the protecting group on the amine was removed by hydrogenolysis. The amine was reprotected with tert-butyl carbonate anhydride and the obtained intermediate was carbonated to give the acid as a racemic mixture. Resolution of the racemate occurs by selective precipitation of a single enantiomer as a salt using (R) -N-benzyl-1-phenylethane-1-amine. Finally, the intermediate (R) -6- (tert-butoxycarbonyl) -6-azaspiro [2.5] octane-5-carboxylic acid was obtained by acidification of the racemic salt solution with HCl.

The need is therefore felt for new similar inhibitors of the EP4 receptor, but whose synthesis is less complex.

It is therefore a first object of the present invention to provide a novel selective antagonist of the EP4 receptor, for the pharmacological treatment of diseases requiring antagonists of the above mentioned receptors, which is at least equivalent to CR6086, but at the same time is available using a synthetic route of much lower complexity.

Summary of The Invention

The above indicated object was obtained by means of the original derivative of D-proline, i.e. (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (compound 1) having the following structure:

or a salt thereof.

Thus, the present invention relates to compound 1, said compound 1 being a strong antagonist of the EP4 receptor.

Furthermore, as will be apparent from the description below, the present inventors have prepared compound 1 by two synthetic procedures that overcome the disadvantages of the prior art processes for preparing CR 6086.

Thus, in a further aspect, the present invention relates to a process for the preparation of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropylbenzoic acid (compound 1), or a salt thereof, comprising the steps of:

a) amide formation (intermediate P1) by reaction of (tert-butoxycarbonyl) -D-proline (N-Boc-D-proline) with methyl 4- (1-aminocyclopropyl) benzoate in the presence of a coupling agent

b) Deprotection of the Boc group of intermediate P1 with at least one acid to give intermediate P2

c) Alkylating intermediate P2 with 4-trifluoromethylbenzyl bromide in the presence of a base, so as to obtain the methyl ester derivative (intermediate P3);

and

d) hydrolysis of the derived methyl ester (intermediate P3) in the presence of a strong base to obtain Compound 1

The process of the present invention illustrated above is extremely advantageous both due to the simplicity of its operating steps and also due to the use of N-Boc-D-proline in step a), the commercial cost of which is at least 20 times lower than the commercial cost of the intermediate of the prior art, i.e. (R) -6- (tert-butoxycarbonyl) -6-azaspiro [2.5] octane-5-carboxylic acid, used for the synthesis of CR 6086.

In a further aspect, the present invention relates to another process for the preparation of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (compound 1), or a salt thereof, comprising the steps of:

a) alkylation of D-proline with 4-trifluoromethylbenzyl bromide in the presence of a strong base, using an alcohol as solvent, to obtain intermediate P4

b) Amide formation by reaction of intermediate P4 with methyl 4- (1-aminocyclopropyl) benzoate in the presence of a coupling agent (intermediate P3)

And

c) hydrolysis of intermediate P3 in the presence of a strong base to obtain Compound 1

From a synthetic point of view, this process for the preparation of compound 1 or a pharmaceutically acceptable salt thereof proves to be more advantageous, providing a further reduction of the synthetic steps thereof, in this case starting from the direct alkylation of D-proline with 4- (trifluoromethyl) benzyl bromide, thus avoiding the protection and subsequent deprotection with N-Boc-D-proline.

In further aspects, the invention relates to compound 1, or a pharmaceutically acceptable salt thereof, for use as a medicament, as well as to pharmaceutical compositions comprising compound 1, or a pharmaceutically acceptable salt thereof, of the invention and at least one pharmaceutically acceptable excipient.

In yet another aspect, the invention relates to compound 1 or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease in the pathogenesis of which prostaglandin E is involved₂(PGE₂) Plays a fundamental role through its interaction with the EP4 receptor subtype.

Accordingly, the present invention relates to compound 1 or a pharmaceutically acceptable salt thereof for use as a selective EP4 antagonist for the treatment of diseases in which prostaglandin E is involved in the pathogenesis₂(PGE₂) Plays a basic role.

Thus, compound 1 of the present invention has been shown to inhibit prostaglandin E mediated by the EP4 receptor₂(PGE₂) Activity of (2).

Thus, according to the present invention, compound 1 or a pharmaceutically acceptable salt thereof is a selective antagonist of the EP4 receptor for use in the treatment of a condition (arthritis) selected from the group consisting of rheumatoid arthritis, spondyloarthritis (such as, for example, psoriatic arthritis and ankylosing spondylitis), arthropathies caused by inflammation, and acute and chronic pain such as osteoarthritis pain or arthritic pain with an immunological etiology such as rheumatoid arthritis.

In another aspect, the present invention relates to compound 1, or a pharmaceutically acceptable salt thereof, for use as an antagonist of EP4 in neoplastic diseases. In this case, the inventors hypothesize that compound 1 may act in two different, alternative, and concomitant ways: 1) restoring an immune response against cancer cells, and 2) inhibiting angiogenesis that nourishes tumor tissue.

In yet another aspect, the present invention is directed to compound 1, or a pharmaceutically acceptable salt thereof, as an antagonist of the EP4 receptor in the treatment of ocular diseases. Among these eye diseases, mention may be made of retinopathy of prematurity (ROP), proliferative diabetic retinopathy and age-related macular degeneration (AMD), which have ocular neovascularization as a common pathological basis, which is largely associated with the activation of the EP4 receptor, and which are the main cause of blindness in developed countries.

In a further aspect, the present invention relates to compound 1, or a pharmaceutically acceptable salt thereof, as an antagonist of the EP4 receptor in the treatment of endometriosis. In this case, the compound is extremely advantageous, since endometriosis is a serious chronic disease that leads to infertility and chronic pelvic pain in 10% -20% of women of childbearing age.

The present invention will now be described in conjunction with the following detailed description and the annexed drawings.

Brief Description of Drawings

The characteristics and advantages of the invention will become apparent from the following detailed description, from the embodiments provided by way of illustrative and non-limiting examples, and from the accompanying drawings, in which:

FIG. 1 shows PGE of example 9₂Reversal of the effect of LPS-induced TNF α release (1 hour sample after treatment);

FIG. 2 shows the CIA edema assessment of example 10; and

fig. 3 shows the CIA clinical evaluation of example 10.

Detailed Description

Accordingly, the present invention relates to (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (compound 1) having the structure:

or a salt thereof.

Thus, the present invention relates to compound 1, said compound 1 being a strong antagonist of the EP4 receptor.

In a further aspect, the present invention relates to a process for the preparation of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (compound 1), or a salt thereof, comprising the steps of:

a) amide formation (intermediate P1) by reaction of (tert-butoxycarbonyl) -D-proline (N-Boc-D-proline) with methyl 4- (1-aminocyclopropyl) benzoate in the presence of a coupling agent

b) Deprotection of the Boc group of intermediate P1 with at least one acid to give intermediate P2

c) Alkylation of intermediate P2 with 4-trifluoromethylbenzyl bromide in the presence of a base to obtain the methyl ester derivative (intermediate P3)

And

d) hydrolysis of the derived methyl ester (intermediate P3) in the presence of a strong base to obtain Compound 1

Preferably, in step a), methyl 4- (1-aminocyclopropyl) benzoate is prepared as described in WO2013/004290, and the coupling agent is preferably 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

In particular, step a) provides the formation of an amide between D-N-Boc proline and methyl 4- (1-aminocyclopropyl) benzoate in the presence of HOBt, EDCl and TEA for 24 hours; sequentially with NaHCO₃Saturated solution of (3), NH₄A saturated solution of Cl and finally washing the organic phase with water; the solvent was evaporated and the product was purified by chromatography on silica gel eluting with a cyclohexane/ethyl acetate gradient (100: 0; 25/75).

In step b), the at least one acid is preferably 2,2, 2-trifluoroacetic acid.

In particular, step b) provides deprotection of the Boc group with trifluoroacetic acid and subsequent release of the salt on an ion exchange resin.

In step c), the base is preferably cesium carbonate.

In particular, step c) comprises adding to Cs₂CO₃Is alkylated with 4- (trifluoromethyl) benzyl bromide for 24 hours; subsequently washing with 4.5% NaCl solution, dehydrating and evaporating the solvent; the residue dissolved in DCM was purified by precipitation using n-heptane as co-solvent; the filtrate was then purified by flash chromatography on silica gel eluting with a DCM/ethyl acetate gradient (from 100/0 to 85/15).

In step d), the strong base is preferably sodium hydroxide. In step d), the strong base is preferably sodium hydroxide. Step d) preferably takes place in a water-miscible organic solvent mixture, more preferably the water-miscible organic solvent is Tetrahydrofuran (THF). In particular, step d) provides for hydrolysis of the methyl ester in the presence of NaOH, followed by purification by reverse phase chromatography eluting with water only and then methanol, to give compound 1 in the form of the sodium salt.

As indicated, the process of the invention described above results extremely advantageous not only due to the simplicity of its operating steps but also due to the truly low-cost reagents used.

In a further aspect, the present invention relates to another process for the preparation of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropanebenzoic acid (compound 1), or a salt thereof, comprising the steps of:

a) alkylation of D-proline with 4-trifluoromethylbenzyl bromide in the presence of a strong base, using an alcohol as solvent, to obtain intermediate P4

b) Amide formation by reaction of intermediate P4 with methyl 4- (1-aminocyclopropyl) benzoate in the presence of a coupling agent (intermediate P3)

And

c) hydrolysis of intermediate P3 in the presence of a strong base to obtain Compound 1

In step a), the base is preferably potassium hydroxide and the alcohol is 2-propanol.

In particular, step a) consists of: alkylating D-proline with 4-trifluoromethylbenzyl bromide in the presence of a strong base using an alcohol, preferably 2-propanol, as solvent; the product was recovered by treatment with aqueous HCl and subsequent filtration, and purified by treatment with acetone and tert-butyl ether as co-solvents.

In step b), methyl 4- (1-aminocyclopropyl) benzoate is preferably prepared as described in WO2013/004290, and the coupling agent is preferably 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

In particular, step b) provides 4- (trifluoromethyl) benzyl prepared according to the previous step a) in the presence of hydroxybenzotriazole hydrate, TEA, EDCl dissolved in DCM solutionAmide formation between yl) -D-proline and methyl 4- (1-aminocyclopropyl) benzoate hydrochloride; with NaHCO₃The organic phase is washed with water and finally the organic phase is washed with water, dehydrated and the solvent evaporated, the compound (R) -methyl 4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoate thus formed is purified and recovered by treatment with methyl tert-butyl ether and precipitation.

In step c), the strong base is preferably sodium hydroxide. Step c) preferably takes place in a water-miscible organic solvent mixture, more preferably the water-miscible organic solvent is Tetrahydrofuran (THF).

In step c), in particular, the hydrolysis of the methyl ester thus obtained and the recovery of compound 1 according to what is described in point d) of the first process, according to what is described in point b) previously, take place continuously.

As indicated above, the process of preparation of this compound proved to be more advantageous from a synthetic point of view, providing a further reduction of its synthetic pathway (passage), in this case starting from the direct alkylation of D-proline with 4- (trifluoromethyl) benzyl bromide, thus avoiding the protection and subsequent deprotection with N-Boc-D-proline.

In an advantageous aspect, compound 1 is in the form of a salt, preferably in the form of a pharmaceutically acceptable salt, more preferably as the hydrochloride or lithium or sodium salt.

In a further advantageous aspect, compound 1 is in the form of a zwitterion.

In another aspect, the invention relates to compound 1, or a pharmaceutically acceptable salt thereof, for use as a medicament, and a pharmaceutical composition comprising compound 1, or a pharmaceutically acceptable salt thereof, of the invention and at least one pharmaceutically acceptable excipient.

In yet another aspect, the invention relates to compound 1 or a pharmaceutically acceptable salt thereof for use in the treatment of diseases in which prostaglandin E is implicated in the pathogenesis₂(PGE₂) Plays a fundamental role through its interaction with the EP4 receptor subtype.

Accordingly, the present inventionRelates to the use of compound 1 or a pharmaceutically acceptable salt thereof as a selective EP4 antagonist for the treatment of diseases in which prostaglandin E is involved in the pathogenesis₂(PGE₂) Plays a basic role.

Thus, according to the present invention, compound 1 or a pharmaceutically acceptable salt thereof is a selective antagonist of the EP4 receptor for use in the treatment of a condition selected from the group consisting of rheumatoid arthritis, spondyloarthritis (such as, for example, psoriatic arthritis and ankylosing spondylitis), arthropathies caused by inflammation, and acute and chronic pain such as osteoarthritis pain or arthritic pain with an immunological etiology such as rheumatoid arthritis.

In another aspect, the present invention relates to compound 1, or a pharmaceutically acceptable salt thereof, for use as an antagonist of EP4 in neoplastic diseases. In this case, the inventors hypothesize that compound 1 may act in two different, alternative, and concomitant ways: 1) restoring an immune response against cancer cells, and 2) inhibiting angiogenesis that nourishes tumor tissue.

In yet another aspect, the present invention is directed to compound 1, or a pharmaceutically acceptable salt thereof, as an antagonist of the EP4 receptor in the treatment of ocular diseases. Among these eye diseases, mention may be made of retinopathy of prematurity (ROP), proliferative diabetic retinopathy and age-related macular degeneration (AMD), which have ocular neovascularization as a common pathological basis, which is largely associated with the activation of the EP4 receptor, and which are the main cause of blindness in developed countries.

In a further aspect, the present invention relates to compound 1, or a pharmaceutically acceptable salt thereof, as an antagonist of the EP4 receptor in the treatment of endometriosis. In this case, the compound is extremely advantageous, since endometriosis is a serious chronic disease that leads to infertility and chronic pelvic pain in 10% -20% of women of childbearing age.

Further examples of embodiments of the present invention are given below by way of non-limiting examples.

Examples

Reagents used in the following examples were purchased from various suppliers and used without further purification. The solvent is used in anhydrous form. Reaction in an anhydrous Environment at N₂Under positive pressure.

Proton nuclear magnetic resonance spectrum (¹H NMR) was recorded on an instrument Bruker Avance 400 MHz. Chemical shifts are reported in ppm (δ) using the residual solvent line as an internal standard. The diversity of the signal (sign) is specified as: s, singlet; d, double peak; t, triplet; q, quartet; m, multiplet; br, spread signal.

UPLC-Massa spectroscopy was performed on an instrument Waters acquisition UPLC-SQD using an acquisition UPLC-BEH C18 column (1.7. mu.M, 50X 2.1 mm).

Flash chromatography on silica gel was performed on a Biotage automatic flash chromatography system (Sp1 and Isolera systems) using SNAP HP Biotage silica cartridges. Reverse phase chromatography was performed on a Biotage (Isolera) automated flash chromatography system using a RediSep Gold C-18Aq column.

The SPE-SCX cartridge is an ion exchange column for solid phase extraction.

Optical rotation (rotatory power) was measured with an Autopol V polarimeter (Rudolph Sci.).

The following abbreviations are used herein:

NH₄cl: ammonium chloride; NaHCO 2₃: sodium bicarbonate; cs₂CO₃: cesium carbonate; NaOH: sodium hydroxide; KOH: potassium hydroxide; TEA: triethylamine; NH (NH)₃: ammonia; HCl: hydrochloric acid; EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide; na (Na)₂SO₄: sodium sulfate; DCM: dichloromethane; EtOH: ethanol; MeOH: methanol; IsoH: 2-propanol; THF: tetrahydrofuran; t.a.: and (4) room temperature.

Example 1:

preparation of Compound 1 by the first Process of the invention

Preparation 1: (R) -2- ((1- (4- (methoxycarbonyl) phenyl) cyclopropyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester Preparation of butyl ester (intermediate P1).

N-hydroxybenzotriazole hydrate (25.6g,167mmol) and EDC (40.1g,209mmol) were added to a solution of (tert-butoxycarbonyl) -D-proline (30g,139mmol) in DCM (550ml) and the mixture was stirred at room temperature for 1 hour. Thereafter, methyl 4- (1-aminocyclopropyl) benzoate hydrochloride (33.0g,145mmol) and TEA (26.2ml,188mmol) were added. The reaction was stirred for 24 hours. When the reaction was complete, water (350mL) was added and the two phases were stirred for about 10 minutes. The organic phase is separated from the aqueous phase and successively treated with NaHCO₃Saturated solution (300ml), NH₄A saturated solution of Cl (300mL) was washed and finally with 300mL of water.

Na for organic phase₂SO₄Dried and concentrated to give a pale yellow solid which was purified by flash chromatography on silica gel with a cyclohexane/ethyl acetate gradient (100: 0; 25/75) to afford the desired compound (49.5 g; yield 88%). (intermediate P1).

MS:(ES/+)m/z:389[MH^-]

¹H NMR (400MHz, chloroform-d) δ ppm 7.94(2H, d, J ═ 8.31Hz),7.73(1H, br.s.),7.23(2H, d, J ═ 7.83Hz),4.29(1H, br.s.),3.91(3H, s),3.45(2H, br.s.),1.77-2.53(4H, m),1.47(9H, br.s.), 1.29-1.42(4H, m).

Preparation 2: preparation of methyl (R) -4- (1- (pyrrolidine-2-carboxamide) cyclopropyl) benzoate (intermediate P2)

Tert-butyl (R) -2- ((1- (4- (methoxycarbonyl) phenyl) cyclopropyl) carbamoyl) pyrrolidine-1-carboxylate (49.5g,122mmol) was dissolved in DCM (400mL) and the mixture was cooled to 0 ℃, then 2,2, 2-trifluoroacetic acid (80mL,1038mmol) was added. The reaction was allowed to reach room temperature and then stirred for 3 hours. The solvent was evaporated and the crude residue loaded onto an SPE-SCX cartridge (150g), eluting first with MeOH only, and then with 1M NH in MeOH₃And (4) eluting. The ammonia-containing fractions were evaporated to obtain the desired residual solid (30.9g, yield 88%). (intermediate P2).

MS:(ES/+)m/z:289

¹H NMR(400MHz,DMSO-d6)δppm8.62(1H,s),7.80-7.91(2H,m),7.17-7.27(2H,m),3.83(3H,s),3.49-3.58(1H,m),2.77-2.92(3H,m),1.91-2.03(1H,m),1.56-1.74(3H,m),1.17-1.34(4H,m)。

Preparation 3: (R) -4- (1- (1- (4-trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid methyl ester Preparation of (intermediate P3)

Methyl (R) -4- (1- (pyrrolidine-2-carboxamide) cyclopropyl) benzoate (29.6g,103mmol) was dissolved in THF (500 ml). Sequential addition of Cs₂CO₃(57.2g,176mmol) and 4- (trifluoromethyl) benzyl bromide (16.82ml,109mmol), and the mixture was stirred at room temperature for 24 hours. THF was evaporated and the residue partitioned between DCM (250ml) and water (250ml) and the resulting mixture stirred vigorously for about 10 minutes. The organic phase was separated from the aqueous phase and washed twice with 4.5% NaCl solution (250ml) over Na₂SO₄Dried and concentrated to obtain a white solid (46.5g), to which DCM (100mL) was added and heated to about 40 ℃ until complete dissolution. At this time, the solution was brought to room temperature, and then n-heptane (500mL) was added to obtain a white precipitate, which was then filtered and dried at 50 ℃ for 24 hours to obtain the desired compound (50 g; yield: 89%). (intermediate P3).

MS:(ES/+)m/z:447

¹H NMR(400MHz,DMSO-d6)δppm 8.47(1H,s),7.77-7.85(2H,m),7.66-7.71(2H,m),7.58-7.65(2H,m),7.14(2H,d,J＝8.31Hz),3.78-3.90(4H,m),3.66(1H,d,J＝13.20Hz),3.11(1H,dd,J＝9.05,4.65Hz),2.98-3.05(1H,m),2.34-2.42(1H,m),2.05-2.20(1H,m),1.70-1.87(3H,m),1.15-1.32(2H,m),0.98-1.15(2H,m)。

Preparation 4: (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-formamide) cyclopropyl) benzoic acid (formula) Preparation of the sodium salt of Compound 1).

Methyl (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoate (40.95g,92mmol) (intermediate P3) was dissolved in a mixture of THF (450ml) and water (250ml) and NaOH (4.40g,110mmol) was added. The whitish suspension was stirred at room temperature for 24 hours. The organic solvent was evaporated and the residue was loaded onto a RediSep Gold C-18Aq column, eluting first with water only (2 column volumes) and finally with MeOH (3 column volumes). The MeOH containing fractions were concentrated and the resulting residual solid was dried under vacuum at 60 ℃ to give 35.5g of the sodium salt of compound 1 (84% yield).

C₂₃H₂₂F₃N₂NaO₃

MS:(ES/+)m/z:433[MH^-]

¹H NMR(400MHz,DMSO-d6)δppm 8.38(1H,s),7.65-7.77(4H,m),7.56-7.64(2H,m),6.87-7.01(2H,m),3.86(1H,d,J＝13.20Hz),3.64(1H,d,J＝13.69Hz),3.09(1H,dd,J＝9.05,4.65Hz),2.94-3.04(1H,m),2.29-2.41(1H,m),2.03-2.19(1H,m),1.69-1.86(3H,m)。

Specific rotation power (specific rotation power): (α) D20 at H₂O1% ═ 30.2 °.

Example 2:

preparation of Compound 1 by the second Process of the invention

Preparation 1: preparation of (4- (trifluoromethyl) benzyl) -D-proline (intermediate P4)

50g (0.43 mol) of D-proline was added to a solution of 73g (1.3 mol) of potassium hydroxide dissolved in 500ml of 2-propanol and then 74ml of 4- (trifluoromethyl) benzyl bromide (0.477 mol) dissolved in 300ml of 2-propanol was added in portions; the solution was reacted at 50 ℃ for 12 hours with stirring. The solution was then cooled to 0 ℃ and the pH was adjusted to about 4 ± 1 with aqueous 32% HCl. The precipitated solid formed was filtered, washed with a small amount of 2-propanol, dissolved in acetone, filtered hot, and reprecipitated by adding methyl tert-butyl ether to obtain the desired compound after filtration and drying (99 g; 83% yield). (intermediate P4).

MS:(ES/+)m/z:274

¹H NMR(400MHz,DMSO-d6)δppm 11.66(vbs),7.69(2H,d),7.59(2H,d),4.07(1H,d),3.68(1H,d),3.25-3.32(1H,m),2.93(1H,ddd),2.39-2.48(1H,m),2.10(1H,dq),1.67-1.92(3H,m)。

Preparation 2: (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid methyl ester Preparation of (intermediate P3)

To a solution of 26g (95.4mmol) of 4- (trifluoromethyl) benzyl) -D-proline and N-hydroxybenzotriazole hydrate (17.6g,114mmol) dissolved in 600ml of DCM were added 80ml of TEA (570mmol) and 20g (105mmol) of EDCl under a stream of nitrogen. The reaction mixture was allowed to stir for about 6 hours, then 24g (105mmol) of methyl 4- (1-aminocyclopropyl) benzoate hydrochloride was added and then the reaction was continued for another 24 hours at room temperature with stirring all the time. When the reaction was complete, water (500mL) was added and the two phases were stirred for about 10 minutes. The organic phase is separated from the aqueous phase and successively treated with NaHCO₃Was washed with 400mL of water and finally with 400mL of water. Na for organic phase₂SO₄Dried and concentrated to obtain a cream colored solid. The solid was suspended in 1 liter of methyl t-butyl ether, heated to boiling, hot filtered, and the clear solution thus obtained was concentrated to dryness under vacuum to obtain a white solid, which was then dried at 50 ℃ for 24 hours to obtain the desired compound (32.5 g; yield: 75% (. sup.: intermediate P3).

MS:(ES/+)m/z:447

¹H NMR (400MHz, chloroform-d) Δ ppm7.80-8.01(3H, m),7.62(2H, d),7.41(2H, d),7.12-7.24(2H, m),3.86(4H, s),3.66(1H, d),3.27(1H, dd),3.07-3.18(1H, m),2.46(1H, td),2.21-2.38(1H, m),1.70-2.03(3H, m),1.05-1.44(4H, m).

Preparation 3: (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-formamide) cyclopropyl) benzoic acid (formula) Preparation of the sodium salt of Compound 1).

Methyl (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoate (40.95g,92mmol) (intermediate P3) was dissolved in a mixture of THF (450ml) and water (250ml) and NaOH (4.40g,110mmol) was added. The whitish suspension was stirred at room temperature for 24 hours. The organic solvent was evaporated and the residue was loaded onto a RediSep Gold C-18Aq column, eluting first with water only (2 column volumes) and finally with MeOH (3 column volumes). The MeOH containing fractions were concentrated and the resulting residual solid was dried under vacuum at 60 ℃ to obtain 35.5g of the sodium salt of compound 1 (84% yield).

C₂₃H₂₂F₃N₂NaO₃

MS:(ES/+)m/z:433[MH^-]

¹H NMR(400MHz，DMSO-d6)δppm 8.38(1H，s)，7.65-7.77(4H，m)，7.56-7.64(2H，m)，6.87-7.01(2H，m)，3.86(1H，d，J＝13.20Hz)，3.64(1H，d，J＝13.69Hz)，3.09(1H，dd，J＝9.05，4.65Hz)，2.94-3.04(1H，m)，2.29-2.41(1H，m)，2.03-2.19(1H，m)，1.69-1.86(3H，m)。

Specific optical rotation: (α) D20 at H₂O1% ═ 30.2 °.

Example 3

Preparation of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid (compound 1 zwitterion).

1.7ml (29.4mmol) of acetic acid were added to 80ml of H₂A solution of 6.1g (13.4mmol) of the sodium salt of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid in O and the solution was extracted twice with 80ml of DCM. The combined organic phases are washed with H₂O washed, and then Na₂SO₄Dried and concentrated under vacuum to give a light cream-colored solid with an amorphous appearance (5.7 g; yield 96%).

C₂₃H₂₃F₃N₂O₃。

MS：(ES/+)m/z：433

¹H NMR(400MHz，DMSO-d6)δppm 12.68(1H，br.s.)，8.45(1H，s)，7.76-7.83(2H，m)，7.65-7.72(2H，m)，7.58-7.65(2H，m)，7.08-7.17(2H，m)，3.86(1H，d，J＝13.69Hz)，3.66(1H，d，J＝13.20Hz)，3.12(1H，dd，J＝9.29，4.40Hz)，2.97-3.06(1H，m)，2.34-2.43(1H，m)，2.05-2.19(1H，m)，1.71-1.86(3H，m)，1.15-1.30(2H，m)，0.97-1.14(2H，m)

Specific optical rotation: (α) D20, 1% in methanol 28.9 °.

Example 4

Preparation of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid (Compound 1 HCl).

HCl 1N (30mL, 30mmol) was added to a solution of 7g (16.2mmol) of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid (compound 1 "zwitterion") dissolved in 300mL of butan-2-one. The reaction mixture was stirred for 12 hours, and the white precipitate formed in suspension was filtered and dried under vacuum at 60 ℃ to obtain 5.8g of compound 1 hydrochloride (yield 85%).

C₂₃H₂₃F₃N₂O₃*HCl。

MS:(ES/+)m/z:433

¹H NMR (400MHz, methanol-d 4) δ ppm7.89-7.97(2H, m),7.70-7.83(4H, m),7.18(2H, d, J ═ 8.31Hz),4.45-4.60(2H, m),4.28(1H, dd, J ═ 9.05,6.60Hz),3.68-3.79(1H, m),3.39-3.49(1H, m),2.61-2.75(1H, m),2.22-2.35(1H, m),2.01-2.15(2H, m),1.19-1.38(2H, m),1.13(1H, ddd, J ═ 10.51,6.85,5.14Hz),0.89-1.00(1H, m)

Specific optical rotation: (α) D20 at H₂O1% ═ 18.3 °.

Example 5

Preparation of lithium salt of (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid.

1.4g (33.1mmol) of lithium hydroxide monohydrate were added dissolved in 50% H₂A solution of 8g (18mmol) of methyl (R) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoate in a mixture of O-dioxane. The solution was allowed to stir for 6 hours and then the dioxane was evaporated under vacuum; the residue was loaded onto a Biotage C-18 column, washed first with water only (2 column volumes)Strip and finally elute with MeOH (3 column volumes). The MeOH containing fractions were concentrated and the resulting solid was dried under vacuum at 60 ℃ to obtain 6.9g of the lithium salt of compound 1 (yield 88%).

C₂₃H₂₂F₃N₂LiO₃

MS:(ES/+)m/z:433

¹H NMR (400MHz, methanol-d 4) δ ppm 7.88-7.83(m,2H),7.66-7.62(m,2H),7.60-7.55(m,2H),7.16-7.10(m,2H),3.87-3.72(m,2H),3.18(dd, J ═ 4.4,9.8Hz,2H),2.55-2.44(m,1H),2.32-2.20(m,1H),1.89(br s,3H),1.35-1.25(m,1H),1.22-1.15(m,1H),1.11-1.04(m,1H),0.99-0.90(m, 1H).

Example 6 reference Compound, i.e.Sodium salt of (S) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid (Compound 2)Preparation of。

Compound 2 was prepared according to the first process of the invention and therefore the whole process including amounts and reagents described in example 1 was followed, but this time starting from (tert-butoxycarbonyl) -L-proline rather than (tert-butoxycarbonyl) -D-proline.

C₂₃H₂₂F₃N₂NaO₃。

MS:(ES/+)m/z:433

¹H NMR(400MHz,DMSO-d₆)δ＝8.38(s,1H),7.75-7.66(m,4H),7.64-7.56(m,2H),6.96(d,J＝8.8Hz,2H),3.86(d,J＝13.2Hz,1H),3.63(d,J＝13.2Hz,1H),3.13-3.07(m,1H),3.03-2.96(m,1H),2.40-2.31(m,1H),2.19-2.05(m,1H),1.87-1.69(m,3H),1.21-1.06(m,2H),1.05-0.91(m,2H)

Specific optical rotation: (α) D20 at H₂O1% — 28.5 °.

Pharmacological evaluation

Example 7

Studies of binding to the human EP4 receptor.

The experiments were carried out according to the procedure described by Abramovitz et al (Biochemical and Biophysica Acta; 1483,285-293, 2000).

Preparing tissues:

cell membranes carrying the human EP4 receptor stably expressed by HEK293 cells (human embryonic kidney 293) were prepared as described below. After removal of the medium (DMEM, supplemented with Glutamax I and containing 10% foetal calf serum and 10. mu.g/ml blasticidin), washing of the cell monolayer was carried out with 10ml of hypotonic lysis buffer (TRIS-Cl 5mM + EDTA5mM-pH 7.4). Then separated from the growth vessel (150cm2 flask) at 37 ℃ with 5% CO₂The growing cells were adhered in the above mentioned medium and lysed by mechanical action by addition of 20ml of fresh lysis buffer. The lysate was stirred for 30 seconds and centrifuged at 40000 × g for 22 minutes at 4 ℃. The final pellets (pellet) were stored at-80 ℃ until use. For binding experiments, membranes were thawed and resuspended in assay buffer (10mM MES-KOH pH6, containing 10mM MgCl)₂And 1mM CaCl₂) To obtain a concentration of 1mg protein/ml. The protein content of the membrane suspension was determined using bovine serum albumin as a standard.

Binding test:

in the experiment, 10. mu.l aliquots of membrane were incubated with radioligand [3H ] at 1nM concentration in the absence or presence of different concentrations of the compound to be tested]Prostaglandin E2 (PGE)₂) And incubating together. Non-specific binding to 1 μ M PGE₂Is determined in the presence of (a). Incubation in a final volume of 0.1ml in a 96 deep well microplate was performed at 25 ℃ for 90 minutes. Separation of free radioligand from radioligand bound to the receptor was performed by rapid filtration under vacuum using a Unifilter GFB glass fiber filter microplate pre-wetted with 0.3% polyethylenimine dissolved in assay buffer, followed by 3 washes with cold buffer (50mM HEPES, NaCl 500mM, BSA 0.1%, pH 7.4). The filter discs were then dried at 30 ℃, 0.05ml of Microscint-20 scintillation fluid (Perkin Elmer) was added to each filter disc, and finally the radioactivity present was measured after stabilization for at least one hour.

The compound/radioligand competition curves were analyzed using a "nonlinear curve fitting" program (Graph Pad, 7 th edition for Windows) that allowed the IC of each compound evaluated to be calculated₅₀Values (concentration that inhibits 50% of radioligand binding to the receptor in the experiment (under administration)).

Results: the results thus obtained are denoted IC₅₀Values, and are shown in table 1 below.

Table 1: binds to the recombinant human EP4 receptor.

From the data shown in the table, it can be seen how both compound 1 and CR6086 exhibit strong activity in inhibiting binding to the EP4 receptor; compound 1 shows p PGE₂Inhibitory activity of ligand binding, which is practically identical to that obtained with the comparative compound CR 6086. The inventors have noted and considered interesting that the EP4 antagonist activity of compound 1 is stereospecific, as compound 2, which is the reference "S form" enantiomer of compound 1, i.e. (S) -4- (1- (1- (4- (trifluoromethyl) benzyl) pyrrolidine-2-carboxamide) cyclopropyl) benzoic acid, is inhibiting PGE₂The activity of the ligand in binding to the human EP4 receptor was about 50-fold lower.

Example 8:

determination of cAMP in CHO cells transfected with the human EP4 receptor (functional test).

The method comprises the following steps: evaluation of agonist and antagonist Activity of Compound 1 in human EP4 receptor expressed in CHO cells transfected with human EP4 receptor by measuring the absence and presence of endogenous agonist PGE from Compound 1₂The effect on cAMP production in the case of (2).

CHO cells were suspended in HBSS buffer (Invitrogen) supplemented with HEPES 20mM (pH 7.4) and IBMX 500mM, then distributed in microplates at a density of 20000 cells per well and at room temperature without incubationIncubation in the presence (control) or in the presence of the compound to be examined was continued for 10 minutes. As a reference control for agonist activity, 1 μ M of PGE was added₂Added to individual dose wells. In the case of evaluating antagonism, the agonist PGE will be referred to₂Instead to a final concentration of 30 nM. After 10 min incubation at room temperature, cells were lysed and a fluorescent acceptor (cAMP labeled D2) and a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) were added. After 60 minutes at room temperature, the fluorescence transfer was measured using a microplate reader (Envison, Perkin Elmer) at an excitation wavelength of 337nm and at emission wavelengths of 620nm and 665 nm. The cAMP concentration was determined by dividing the signal measured at 665nm by the signal measured at 620nm (ratio). In the case of evaluating the agonist effect, the results are expressed as PGE at 1. mu.M₂While in the case of evaluating the effect of the antagonist, the results are expressed as PGE at 30nM₂Percent inhibition of response to the control agonist. To determine antagonist activity, the apparent dissociation constant (Kb) was determined by applying a modified Cheng Prusoff equation (Kb ═ IC)₅₀/(1+A/EC₅₀A) To calculate; wherein A is a reference agonist PGE₂And EC of₅₀EC of a ═ reference agonist₅₀The value is obtained.

Results：

Functional cAMP assay showed that compound 1 is a pure antagonist of the human EP4 receptor (Kb value: 15nM compound 1 vs 7.8nM CR6086) with no agonist activity.

Compound 1

Agonist action: is inactive until 10^-6M

Antagonist action: IC (integrated circuit)₅₀＝1.6×10^-7M；Kb＝1.5×10^-8M

CR6086

Agonist action: is inactive until 10^-6M

Antagonist action: IC (integrated circuit)₅₀＝8.2×10^-8M；Kb＝7.8×10^-9M

Example 9:

ex vivo functional assay evaluation of the occupancy of EP4 receptor by Compound 1(30mg/kg) compared to CR6086(30mg/kg) in rats

The method comprises the following steps: induction by endotoxin lipopolysaccharide (from e.coli) and by increasing PGE was assessed by cells present in rat whole blood and expressing the EP4 receptor₂(ii) the release of the cytokine TNF α. As is known from the literature, PGE₂The compounds tested, which modulate this release by inhibiting it and are selective for the EP4 receptor, if present in the blood after their uptake, bind to the receptor and prevent PGE₂Inhibiting the release of cytokines. This reversal of the modulation of TNF α release can be considered as a biomarker of antagonism of the EP4 receptor (antagonism).

Male Wistar rats weighing 250-275g (Charles River, Italy) were used and were treated orally with vehicle or Compound 1(30mg/kg) or CR6086(30 mg/kg). Each treatment group consisted of 6 animals.

Measurement of Ex vivo Effect in rat Whole blood：

The effect of receptor occupancy of the compound examined was evaluated one hour after its administration and compared to the effect measured in the presence of the vehicle alone. After gas anesthesia with isoflurane, blood was taken from the abdominal aorta and transferred to a tube containing heparin (0.1-0.2U/ml). A 0.4ml aliquot of blood was then taken and distributed into a series of tubes, which were pre-heated at 37 ℃ for 10 minutes. To the samples were added LPS (0.1. mu.g/ml final concentration), which represents the test control, or LPS mixture (0.1. mu.g/ml final concentration) + PGE at various concentrations₂. In samples representing the basal release of cytokines, LPS was replaced by sterile Phosphate Buffered Saline (PBS). The samples were incubated at 37 ℃ for 4 hours, then 40. mu.l of 10mM EDTA was added and the samples were transferred to ice. The samples were then centrifuged at 1500 × g for 10 minutes at 4 ℃ and the plasma thus obtained was removed and stored at-80 ℃ until the time of administration. The content of TNF alpha in blood plasma is aided byMeasured with a commercial kit.

The final concentration of TNF α was calculated for each treated animal and for each experimental condition, from which the increase due to PGE was obtained compared to the sample containing only LPS₂The percent inhibition caused by (c). The cytokine release baseline results were below the ELISA assay sensitivity limit indicated by the manufacturer. Thus, IC₅₀Values were calculated from the inhibition curves. For each treatment group, a PGE was constructed₂Inhibition curves (mean of inhibition. + -. SD vs PGE)₂Concentration) as shown in fig. 1. The mean TNF α values of LPS-only samples in the different treatment groups were analyzed to verify the possible intrinsic effect of the compounds examined. The results thus obtained are illustrated in fig. 1.

As can be seen in FIG. 1, the PGE was increased as expected₂In the range of 10-1000nM, inhibits TNF α release in a concentration-dependent manner. PGE was observed after treatment with CR6086 or Compound 1 due to the presence of a selective receptor antagonist₂Statistically significant reversal of the effects of (a) (. p)<0.01 relative to vehicle group; two-way ANOVA). PGE calculated for each treatment group with Compound₂IC of₅₀The values were significantly higher than those calculated after treatment with vehicle, indicating the same PGE due to the presence of antagonist₂Efficacy change (power shift). No difference was observed between the effects caused by these two compounds, and neither showed a significant effect on TNF α release by itself (in the absence of PGE)₂In the case of (1).

Example 10:

collagen arthritis (CIA) in mice compared to CR6086 in a model of rheumatoid arthritis in mice Effect of Compound 1

The method comprises the following steps: arthritis was induced in mice by intradermal injection of heterologous collagen emulsified in freund's complete adjuvant (CFA). The developed arthritis is characterized by a significant destruction of the articular cartilage, with immune complex deposition, synovitis and periarticular inflammation. CollagenActs as an antigen and induces an immune response involving both T and B lymphocytes, wherein anti-collagen antibodies are produced. Studies in mice using this experimental model allowed the identification of many cytokines and chemokines involved in the pathogenesis of human rheumatoid arthritis. In the presence of isoflurane/O₂After gas anaesthesia, adult male DBA/1 mice were immunized by intradermal injection of 100 μ l of an emulsion containing 200 μ g of bovine collagen type II in CFA at the base of the tail. The CFA in turn contained 3mg/ml M.tuberculosis suspended in a mixture of paraffin oil and mannide monooleate (mono-oleate mannide). One group of non-immunized animals was used as a healthy control group. Mice were included in the study at the onset of the first clinical symptoms of the condition, such as edema and redness of the hind and forepaws. At different times since induction of the pathology, the extent of the pathology was assessed by assigning a clinical score according to the following scale: 0 for normal, 1 for mild redness and swelling of the ankle, 2 for marked edema, and 3 for severe edema and rigidity; the clinical score value assigned to each paw was then added. The volume of 4 paws was also measured using a thickness meter (micro-guard) and summed. At the end of the study, animals were sacrificed and 4 paws were taken, fixed in 10% formalin and processed for histological analysis. The articles were analyzed using light microscopy to assess cartilage and bone degradation, inflammatory cell infiltration, and synovial inflammation. To this end, a score is assigned to quantify the severity of the individual parameter being evaluated. A drug to be tested is considered effective if it is able to combat the progression of the disease by reducing edema, lowering clinical scores and reducing tissue damage to the joint. Compound 1(45-90mg/kg) and CR6086(30mg/kg) dissolved in distilled water were administered orally for 16 days during the progression of the disease state.

And (5) carrying out statistical analysis.Values are expressed as group means ± standard error and analyzed using GraphPad Prism software version 6.0. Statistical analysis was performed using different measurements taken at 24 hours on day 16 after final treatment. ANOVA one-way analysis of variance was performed followed by Dunnett's test for multiple comparisons of edema values. To analyze the clinical score valueAnd histopathological score values, the sum of scores of 4 paws was analyzed by means of ANOVA-Kruskall-Wallis non-parametric test, followed by Dunn test for multiple comparisons. For the statistical analysis performed, values below P0.05 were considered significant.

As a result:

compound 1 administered orally at doses of 45mg/kg and 90mg/kg was evaluated for therapeutic effect on the progression of collagen-induced arthritis in mice. Compound 1 significantly and in a dose-dependent manner reduced the evolution (evolution) of the pathology over 16 days of drug treatment. The results thus obtained are graphically summarized in fig. 2 and 3, fig. 2 showing CIA edema assessment and fig. 3 reporting CIA clinical assessment.

This effect was present in all parameters analyzed, i.e. edema (. P <0.001 vs. vehicle; Dunnett test), clinical and histopathological scores (. P <0.05,. P <0.001 vs. vehicle; Dunnett test).

Comparing the results associated with edema (figure 2) and clinical score (figure 3) with those obtained in a similar study for the selective EP4 antagonist CR6086 at the 30mg/kg dose, there was a greater effect of compound 1 at the 45mg/kg dose on reversal of clinical symptoms (swelling of the paw). This result allows to draw the conclusion that: both compounds at the same dose may exhibit the same strong therapeutic activity on the progression of arthritis in mice.

Example 11

Pharmacokinetics

Compound 1 administered to Wistar rats exhibited excellent oral bioavailability. In fact, when administered at a dose of 5mg/kg, Compound 1 exhibited a C of 4270ng/ml_MAXAnd an AUC of 5460ngxh/ml (0-24 h). 4270ng/ml of C_MAXThe concentration corresponds to a plasma concentration of about 9000nM, i.e. the ratio of the IC obtainable from Table 1₅₀Values of about 200 times the concentration. This result is a good demonstration of the activity of compound 1 in inhibiting inflammation (paw swelling) in the CIA model shown in figure 2.