Method of preparation of Enantiomers of spiro-oxindole compounds

The present invention is directed to a process for the preparation of the (S) 1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole-7 enantiomer, 3'-indol] -2 '(1'H) -one.

BACKGROUND OF THE INVENTION [0002] Published PCT Patent Application No. WO 2006/110917 discloses certain spiro-indole compounds, in particular, 1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole7.3'-indolo] -2 '(1'H) -one, i.e., a compound of the following formula (I):

<img file="PL2789617T3_D0001.tif" />

These compounds are disclosed herein as being useful in the treatment of diseases or conditions, such as pain, in mammals, preferably humans, which are alleviated or reduced by the modulation, preferably inhibition, of voltage-gated sodium channels.

SUMMARY OF THE INVENTION [0003] The present invention is directed to a process for preparing (S) 1 '- {[5 (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole-7 enantiomer. , 3'-indolo] -2 '(1'H) -one having

<img file="PL2789617T3_D0002.tif" />

wherein the method comprises isolating the (S) 1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3f] [1,3] benzodioxole-7,3'-indolo] -2 enantiomer '(1'H) -one from a compound of formula (I):

<img file="PL2789617T3_D0003.tif" />

In chiral high pressure liquid chromatography or under conditions of chromatography with simulated bed displacement.

BRIEF DESCRIPTION OF THE DRAWINGS [0004] The following drawings form part of the present description and are incorporated to further illustrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with a detailed description of the specific embodiments set forth herein.

Figure 1 shows the concentration-response relationships for (S) and (R) enantiomers in the guanidine influx study from Biological Example 1 herein.

Figure 2 shows a comparison of the efficacy of (S) and (R) enantiomers with oral dosing in a model of inflammatory pain of Biological Example 3 herein.

Figure 3 shows a comparison of the efficacy of (S) and (R) enantiomers for topical administration in the neuropathic pain model of Biological Example 3 herein.

Figure 4 shows the time course of histamine-induced pruritus in untreated mice by the in vivo test described in Biological Example 7. Data are expressed as the mean ± SD of itch seizures. Figure 5 shows anti-histamine-induced pruritus with locally applied ointment containing 8% (w / v) of the (S) enantiomer. Data are expressed as the mean ± SD of itching attacks. Figure 6 shows the efficacy of the (S) enantiomer against histamine-induced pruritus when administered orally instead of topically. Data are expressed as the mean ± SD of itching attacks.

DETAILED DESCRIPTION OF THE INVENTION

Definitions [0005] As used in the specification and appended claims, the following terms have, unless otherwise indicated, the following meanings:

"Analgesia" means no pain in response to a stimulus that would normally be painful.

"Allodynia" means a condition in which normally harmless sensations, such as pressure or light touch, are perceived as extremely painful.

The term "enantiomers" refers to asymmetric molecules that can exist in two isomeric forms that have different configurations in space. Other terms used to designate or refer to enantiomers include "stereoisomers" (due to the different distribution or stereochemistry around the chiral center, although all enantiomers are stereoisomers, not all stereoisomers are enantiomers) or "optical isomers" (due to the optical activity of the pure enantiomers) , i.e. the ability of various pure enantiomers to twist the plane of polarization of polarized light in different directions). Because they do not have a plane of symmetry, the enantiomers are not identical to their mirror reflections; molecules that occur in two enantiomeric forms are chiral, which means that they can be treated as occurring in "left" and "righteous" forms. The most common cause of chirality in organic molecules is the presence of a tetrahedral coal associated with four different substituents or groups. Such coal 2

This is called a chiral center or a stereogenic center. The method of indicating the three-dimensional arrangement of atoms (or configurations) in the stereogenic center is with respect to the group priority system when the group with the lowest priority is oriented away from the hypothetical observer: If the arrangement of the other three groups from higher to lower priority is clockwise, the center stereogenic has the "R" (or "D") configuration; if the system is left, the stereogenic center has the "S" (or "L") configuration.

[0006] Enantiomers have the same empirical chemical formula and are generally chemically identical in terms of their reactions, their physical properties and their spectroscopic properties. However, the enantiomers show a different chemical reactivity to other asymmetric compounds and react differently to asymmetric physical disturbances. The most common asymmetric disorder is polarized light.

The enantiomer may twist the plane of polarization of polarized light; in this way, the enantiomer is optically active. Two different enantiomers of the same compound will twist the polarization polarized light plane in the opposite direction; in this way the light can be twisted to the left or in the opposite direction to the hypothetical observer (it's left-handed or "I" or minus, or it can be turned right or in the opposite direction (it's clockwise or "d" or plus "+"). The sign of optical (+) or (-) rotation is not related to the R, S designation. A mixture of equal amounts of two chiral enantiomers is called a racemic mixture or a racemate and is indicated by the symbol (+/-) or the prefix "d, I" indicating the mixture the dextrorotatory and levorotatory forms The compound of formula (I) as described herein, is a racemate. Racemates or racemic mixtures exhibit no optical rotation because the amounts of these (+) and (-) forms themselves are present. In general, the presence of a single enantiomer twists the polarized light in only one direction; in this way, the single enantiomer is further optically pure.

[0007] The designations "R" and "S" are used to denote the absolute configuration of the molecule around its center of chirality (S). Designations may appear as an additive or as a suffix; they may or may not be separated from the name of the enantiomer by a hyphen; they may or may not be separated by a thinkin; and may or may not be in parentheses.

[0008] The designations or prefixes "(+) and (-)" are used herein to designate the sign of rotation of the polarization plane of the polarized light of the compound, with (-) indicating that the compound is left-handed (turning to the left). The relationship to the (+) prefix is ​​clockwise (rotates to the right).

[0009] "Decomposition" or "separation" when used in reference to a racemic form of a compound or mixture, refers to the resolution of the racemate into its two enantiomeric forms (i.e., the forms (+) and (-), (R) and ( S)).

[0010] "Enantiomeric excess" or "ee" refers to a product in which one enantiomer is in excess relative to the other and is defined as the absolute difference in molar fraction of each enantiomer. The excess of the enantiomer is usually expressed as a percentage of the enantiomer present in the mixture relative to the other enantiomer. For purposes of the present invention, the (S) enantiomer of the invention is considered "substantially free" from the (R) enantiomer when the (S) -enantiomer is present in an enantiomeric excess of greater than 80%, preferably greater than 90%, more preferably greater than 95%. , most preferably greater than 99%.

[0011] The chemical naming of protocols and the structural schemes used in this document are a modified form of the I.UPAC nomenclature system using the software version ACD / name version 9.07. For example, the compound of formula (I) as set forth above in the summary of the invention is

Referred to herein as 1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3f] [1,3] benzodioxole-7,3'-indole] -2 '(1'H) -one. The corresponding (S) enantiomer, i.e. the (S) -enantiomer of formula (IS) as set forth above in the Summary of the Invention, is referred to herein as (S) 1 '- {[5- (trifluoromethyl) furan-2-yl] methyl } spiro [furo [2,3-f] [1,3] benzodioxole-7,3'-indol] -2 '(1'H) -one. Corresponding (R) enantiomer, (R) enantiomer of the following formula (IR):

<img file="PL2789617T3_D0004.tif" />

or a pharmaceutically acceptable solvate thereof is referred to herein as (R) -1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole-7 , 3'-indol] -2 '(1'H) -one.

[0012] By "prodrugs" is meant compounds that can be transformed under physiological conditions or by solvolysis into a biologically active compound of the invention. Thus, the term "prodrug" refers to a metabolic precursor of a compound prepared in accordance with the method of the invention that is pharmaceutically acceptable. The prodrug may be inactive when administered to a patient in need thereof but is converted in vivo into the active compound of the invention. Prodrugs are usually rapidly converted in vivo, e.g. by hydrolysis in the blood, to give the parent compound of the invention. The prodrug often presents benefits in terms of solubility, tissue compatibility or delayed release in the mammalian body (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 2124 (Elsevier, Amsterdam)). A discussion of prodrugs is contained in Higuchi, T., et al., "Pro-drugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0013] By "prodrug" is also meant any covalently bonded carriers that release the active compound of the invention in vivo when such a prodrug is administered to mammals. Prodrugs of compounds prepared according to the method of the invention can be prepared by modifying functional groups present in a compound of the invention in a way that modifications of the compound are cleaved, both routinely and in vivo, to the parent compound of the invention.

Prodrugs include a compound prepared according to the method of the invention in which a hydroxyl, amino or mercapto group is attached to any group which, when the prodrug is administered to a mammal, cleaves to form a free hydroxyl group, a free amino group or a free mercapto group, respectively. . Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol functionality or amide derivatives of amine functional groups in the compounds of the invention, and the like.

[0014] The application also illustrates enantiomers (S) and radiolabelled (R) enantiomers in which one or more atoms are replaced by an atom having a different atomic mass or mass number.

Examples of isotopes that can be included in the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, such as, respectively, ²H ³H ¹¹C ¹³C ¹⁴C ¹³N ¹⁵N ¹⁵ABOUT, ¹⁷ABOUT,¹⁸O and ¹⁸F. These radiolabelled compounds may be useful in determining or measuring the efficacy of compounds by determining, for example, the locus or mode of action on voltage-gated sodium channels or

The binding affinity to the pharmacologically significant site of action in voltage-gated channels. Isotopically-labeled compounds of the invention, e.g. compounds containing a radioactive isotope, are useful in tissue and / or substrate tissue distribution studies. Radioactive isotopes, tritium, i.e.

³H and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose because of the ease of their introduction and detection. The radioligand (3H) -type tritium is particularly useful in membrane-ligand binding studies that contain voltage-gated sodium channels, because tritium has a long half-life of decomposition and the emission is of relatively low energy, and the radioisotope is therefore relatively safe.

Radioligand is usually prepared by exchanging tritium with hydrogen in an unlabeled relationship. The identification of active and inactive enantiomers of a particular racemate facilitates the development of a ligand binding assay, since an unlabeled active enantiomer may be added to the assay to reduce, eliminate or otherwise control the non-specific binding of the tritiated active enantiomer. Substitution with heavier isotopes such as deuterium, i.e.²H, may lead to certain therapeutic advantages resulting from greater metabolic stability, e.g. an increased in vivo half-life or reduced dosage, and may therefore be beneficial in some circumstances.

[0016] In positron emission tomography (PET) studies, a substitution with positron emitting isotopes may be useful to investigate the saturation of receptors with substrates, such as ¹¹C ¹⁸F ¹⁶O and ¹³N. The radiolabeled enantiomers of the invention can usually be prepared by standard methods known to those skilled in the art or analogously to those described herein using a suitable isotopically-labeled reagent in place of a non-labeled reagent previously used.

[0017] The application further illustrates in vivo metabolic products of the disclosed enantiomers. Such products may be formed by, for example, oxidation, reduction, hydrolysis, amidation, esterification, and the like, administered compounds, mainly due to enzymatic processes. Thus, the invention includes metabolic products made by a process comprising administering to the mammal an enantiomer of the present invention for a period sufficient to produce a metabolic product. Such metabolic products can be identified by administering an isotopically labeled enantiomer, at a detectable dose, to an animal such as a rat, mouse, guinea pig, monkey or human, waiting for a sufficient time for metabolic events, and isolating the metabolic product from urine, blood or other biological samples .

[0018] "Selectivity" and "selective" as used herein is a relative measure of the tendency of a compound of the invention to preferential association with one, as opposed to another (or other groups), as well as between or among voltage gated sodium channels. For example, selectivity can be determined by comparative measurements of the kinetics and equilibrium of binding affinity and / or functional measures of ion transport through voltage-gated sodium channels. The tendency of a voltage-gated association compound for a voltage-gated sodium channel can be measured in a number of different ways, and many types of compound are known to those skilled in the art, as described elsewhere herein. Selectivity means that in a particular type of association, measured in a certain way, the compound has a tendency or priority to associate with one voltage-gated sodium channel compared to one or more other voltage-gated sodium channels. This relationship may be different for different types of tests or different measurement methods.

[0019] A "stable enantiomer" and a "stable structure" indicate a compound that is sufficiently stable to

The process of surviving the isolation from the reaction mixture to a useful degree of purity and the process of formulation into an effective therapeutic agent survive.

[0020] "Mammal" includes humans and both domestic and farm animals, such as laboratory animals and domestic animals (e.g. cats, dogs, pigs, cattle, sheep, goats, horses and rabbits), as well as non-domestic animals, such as wild animals, and the like.

[0021] A "pharmaceutically acceptable carrier, diluent" includes any excipient, carrier, excipient, lubricant, sweetener, diluent, preservative, colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer , a tonicity agent, a solvent or an emulsifier that has been approved for use in humans or domestic and farm animals by, by way of non-limiting example, the US Food and Drug Administration (FDA), Health Canada or the European Medicines Agency.

[0022] A "pharmaceutical composition" refers to a formulation of a compound of the invention and an agent generally acceptable in the art for delivering a biologically active compound to mammals, e.g. humans. Such an agent includes all pharmaceutically acceptable carriers, diluents or excipients.

[0023] The pharmaceutical composition described in the application includes one or more pharmaceutically acceptable excipients which include, but is not limited to, any solvent, adjuvant enhancer, carrier, lubricant, sweetening agents, diluent, preservative, color / coloring agent, a flavoring agent, a surfactant, a wetting agent, a dispersing agent, a suspending agent, a stabilizer, an isotonic agent, a buffer and / or an emulsifier approved by, as a non-limitative, United States Food and Drug Administration, Health Canada or the European Medicines Agency, as acceptable for use in humans or pets. Examples of pharmaceutically acceptable excipients include, but are not limited to, the following:

benzyl alcohol benzyl benzoate caprylcaproil-macrogolgiglycerides (e.g. Labrasol®) dimethylamine ("DMA") ethanol

2- (2-ethoxyethoxy) ethanol (e.g., Transcutol®) glucose (solution) glycerol caprylate / caprate and PEG-8 (ethylene glycol), caprylate / caprate complex (e.g. Labrasol®) isopropyl alcohol

Lauroyl Macrogol-32 Glycerides (e.g. Gelucire® 44/14) macrogol-15 hydroxystearate (e.g., Solutol® HS15) medium chain triglycerides (e.g. Miglyol® 810, Miglyol® 840 or Miglyol® 812) peanut oil polysorbate 80 (e.g., Tween® 80) polyethylene glycol (PEG) polyethylene glycol 400 (PEG400, e.g. Lutrol® E 400) polyethylene glycol 6000

Polyoxy castor oil (e.g., Cremophor® EL) polyoxyl 40 hydrogenated castor oil (e.g., Cremophor® RH 40) propylene glycol (PG) propylene glycol monocaprylate (Capryol® 90) soybean sulfobutylether-e-cyclodextrin oil ( e.g. Capitsol®)

TPGS (α-tocopherol-polyethylene glycol, succinate) water

Additional pharmaceutically acceptable excipients are disclosed herein.

[0024] As a result of the crystallization process, a solvate of a compound of the invention is often obtained. As used herein, the term "solvate" refers to an aggregate that includes one or more molecules of a compound of the invention with one or more solvent molecules. The solvent may be water, in which case the hydrate will be the solvate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may be in the form of a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the like, as well as suitable solvated forms. The compounds of the invention may be "real" solvates, whereas in other cases,

[0025] The term "therapeutically effective amount" refers to that amount of a compound of the invention which, when administered to a mammal, preferably a human, is sufficient to effectively treat a disease or condition of interest in a mammal, preferably a human. The amount of a compound of the invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease condition and its severity, the mode of administration, the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on its knowledge and description.

[0026] The term "treat" or "treatment", as used herein, refers to the treatment of a disease or condition in a mammal, preferably a human, and includes:

(i) preventing a disease or condition in a mammal, in particular when such a mammal is predisposed to a given condition, but has not yet been diagnosed with it;

(ii) inhibiting the disease or condition, i.e. inhibiting its development;

(iii) relieving the disease or condition, i.e. causing regression of the disease or condition; or (iv) relieving symptoms resulting from the disease or condition, i.e., relieving pain without combating the underlying disease or condition.

[0027] As used herein, the terms "alleviate", "alleviate", "decrease" or "decrease" are given in their generally accepted definitions. For example, "alleviate" means generally improve or improve the condition relative to the condition prior to the mitigation event. "Reduce" generally means making the state more tolerable in relation to the state before the reduction event. The term "mitigation" as used herein

or "alleviate" may refer to a disease or condition that is improved or improved by administering a compound of the invention. The term "reducing" or "reducing" as used herein may refer to

A disease or condition that is rendered tolerable by administering a compound of the invention. For example, "reduction" of pain will include a reduction in the severity or amount of pain.

[0028] As used herein, the terms "disease" and "condition" can be used interchangeably or differently, because the causative factors of particular ailments or conditions may be unknown (and thus their aetiology is still unclear), thereby causing this disorder or the condition is not yet considered a disease, but only as an unwanted syndrome or condition in which doctors have identified a more or less specific set of symptoms.

USEFULNESS AND TEST OF THE COMPOUNDS OF THE INVENTION [0029] The present invention relates to a process for preparing (S) 1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole enantiomer. -7,3'-indol] -2 '(1'H) -one.

MANUFACTURE OF THE ENANCJOMER (S) FOR THE INVENTION [0030] The (S) enantiomer and the corresponding (R) enantiomer are prepared by separating the compound of formula (I) as set out above in the Summary of the invention using either chiral high-pressure liquid chromatography or chromatography chromatography. a simulated moving bed as described in the following reaction diagram, in which "chiral HPLC" refers to chiral high performance liquid chromatography, and "MSP" refers to a simulated moving bed chromatography:

REACTION SCHEME

<img file="PL2789617T3_D0005.tif" />

<img file="PL2789617T3_D0006.tif" />

[0031] The compound of formula (I) can be prepared by the methods disclosed in PCT Patent Application Publication No. WO 2006/110917, by methods described herein or by methods known to those skilled in the art.

[0032] One of ordinary skill in the art would recognize differences in the above reaction scheme that are suitable for the separation of the individual enantiomers.

Alternatively, the (S) -enantiomer of formula (IS) and the (R) -enantiomer of formula (IR) can be synthesized from starting materials that are known or can be easily prepared using a process analogous to those known.

[0034] Preferably, the (S) enantiomer obtained by the separation methods disclosed herein is substantially free of the (R) enantiomer or contains only traces of the (R) enantiomer.

[0035] The following synthetic examples serve to illustrate the separation methods of the reactions disclosed in the above schemes and are not intended to limit the scope of the invention.

SYNTHETIC EXAMPLE 1

Synthesis of 1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole-7,3'-indolo] 2' (1'H) -one (Compound of formula (I))

<img file="PL2789617T3_D0007.tif" />

[0037] To a suspension of spiro [furo [2,3-f] [1,3] benzodioxole-7,3'-indol] -2 '(1'H) -one (1.0 g, 3.6 mmol) which can be prepared according to the methods disclosed in the PCT patent application publication of patent application No. WO 2006/110917, and cesium carbonate (3.52 g, 11 mmol) in acetone (50 ml), 2-bromomethyl-5-trifluoromethylfuran (1.13 g) is added. , 3.9 mmol) in one portion and the reaction mixture was stirred at 55-60 ° C for 16 hours. After cooling to ambient temperature, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The residue was subjected to column chromatography, eluting with ethyl acetate / hexane (1/9 - 1/1), and 1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3f] [1.3 ] benzodioxole-7; 3'-indolo] -2 '(1'H) -one, i.e. a compound of formula (I) (1.17 g, 76%) as a white solid:¹H NMR (300 MHz, CDCl3) δ 7.32-6.97 (m, 5H), 6.72 (d, J = 3.3 Hz, 1H), 6.66 (s, 1H), 6.07 (s, 1H), 5.90-5.88 (m, 2H), 5.05, 4.86 (ABq, JAB = 16.1 Hz, 2H), 4.91 (d, J = 9.0 Hz; 1H), 4.66 (d, J = 9.0 Hz, 1H); ¹³C NMR (75 MHz, CDCl3) δ 176.9, 155.7, 153.5, 148.8, 142.2, 141.9, 140.8, 140.2, 139.7, 139.1, 132 , 1, 129.2, 124.7, 124.1, 123.7, 121.1, 120.1, 117.6, 114.5, 114.4, 110: 3, 109.7, 103.0 , 101.9, 93.8, 80.0, 57.8, 36.9; MS (ES +) m / z 430.2 (M + 1), 452.2 (M + 23); Cal'd for C22H14F3NO5: C, 61.54%; H, 3.29%; N, 3.26%; Found: C, 61.51%; H, 3.29%; N, 3.26%.

SYNTHETIC EXAMPLE 2

Resolution of a compound of formula (I) by chiral HPLC [0038] The compound of formula (I) is separated into the (S) enantiomer of the invention and the corresponding (R) enantiomer by means of chiral HPLC under the following conditions:

Column: Chiralcel ® OJ-RH; 20 mm ID χ 250 mm, 5 mic; Lot: OJRH CJ-EH001 (Daicel

Chemical Industries, Ltd)

Eluent: Acetonitrile / water (60/40, v / v, isocratic)

Flow rate: 10ml / min

Time: 60 minutes

Load: 100 mg of the compound of formula (I) in 1 ml of acetonitrile

EP 2 789 617 B1

Temperature: Ambient In the above conditions, the chiral HPLC (R) enantiomer of the compound of formula (I), i.e., (R) -1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo] 2,3-f] [1,3] -benzodioxole-7,3'-indolo] -2 '(1'H) -one, was isolated as the first fraction as a white solid; ee (enantiomeric excess)&gt; 99% (analytical OJ-RH, 55% acetonitrile in water); mp 103-105 ° C;¹H NMR (300 MHz, DMSO-d6) δ 7.32-6.99 (m, 5H), 6.71 (d, J = 3.4 Hz, 1 H), 6.67 (s, 1H); 6.05 (s, 1H), 5.89 (d, J = 6.2 Hz, 2H), 5.13, 5.02 (ABq, JAB = 16.4 Hz, 2H), 4.82, 4 72 (ABq, JAB = 9.4 Hz, 2H);¹³C NMR (75 MHz, CDCl 3) δ 177.2, 155.9 , 152.0, 149.0, 142.4, 142.0, 141.3, 132.0, 129.1, 123.9, 120 , 6. 119.2, 117.0, 112.6, 109.3, 108.9, 103.0, 101.6, 93.5, 80.3, 58.2, 36.9; MS (ES +) m / z 430.2 (M + 1), [α] D-17 ° (c 0.99, DMSO). The (S) -enantiomer of the compound of formula (I), i.e., (S) -1 '- {[5- (trifluoromethyl) furan-2-yl] methyl) spiro [furo [2,3-f] [1,3 ] benzodioxole-7,3'-indolo] -2 '(1'H) -one was isolated as a second fraction in the form of a white solid; ee&gt; 99% (analytical OJ-RH, 55% acetonitrile in water); mp 100-102 ° C;¹H NMR (300 MHz, DMSO-d6) δ 7.32-6.99 (m, 5H), 6.71 (d, J = 3.4 Hz, 1H), 6.67 (s, 1H), 6.05 (s, 1H), 5.89 (d, J = 6.3 Hz, 2H), 5.12, 5.02 (ABq, JAB = 16.4 Hz, 2H), 4.82, 4 72 (ABq, JAB = 9.4 Hz, 2H); ¹³C NMR (75 MHz, CDCl 3) δ 177.2, 155.9, 152.0, 149.0, 142.4, 142.0, 141.3, 132.0, 129.1, 123.9, 120 , 6. 119.2, 117.0, 112.6, 109.3, 108.9, 103.0, 101.6, 93.5, 80.3, 58.2, 36.9; MS (ES +) m / z 430.2 (M + 1), [α] D + 14.04 ° (c 0.99, DMSO).

SYNTHETIC EXAMPLE 3

Resolution of the compound of formula (I) by SMB chromatography [0040] The compound of formula (I) is separated into the (S) -enantiomer of the invention and the corresponding (R) -enantiomer by SMB chromatography under the following conditions:

Extract:

The raffinate:

eluent:

Power supply:

recovery:

Time:

Temperature:

Pressure:

147.05 ml / min 76.13 ml / min 183.18 ml / min 40 ml / min 407.88 ml / min 0.57 min. C bars [0041] A feed solution (25 g of a compound of formula (I), in 1.0 L mobile phase (25: 75: 0.1 (v / v: trifloroacetic acid mixture)) was fed continuously to the SMB (Novasep Licosep Lab Unit) system, which was fitted in eight identical columns in a 2-2-2-2 configuration containing 110 g (per column, 9.6 cm, 4.8 cm ID) ChiralPak-AD as the stationary phase. The first eluting enantiomer (the (R) enantiomer of the compound of formula (I)) was included in the raffinate stream and the second eluting enantiomer (S (enantiomer of the compound of formula (I)) was included in the extract stream. Data characterizing the enantiomer

(S) and the (R) enantiomer obtained SMBs were identical to those obtained above using chiral HPLC.

[0042] The compound of formula (I) was separated into its constituent enantiomers by a Waters LCMS apparatus of a preparative auto-purification system. The first enantiomer eluted from the chiral column was brominated (in place, well removed from the stereogenic center) to give the corresponding 5'-bromo derivative, which then crystallized to generate a single crystal suitable for X-ray crystallography. The crystal structure of this brominated derivative of the first eluted enantiomer was obtained and the absolute configuration was found to be the same as in the (R) -enantiomer of the invention. Hence the second enantiomer eluted from the chiral column (S) -enantiomer of the invention. In addition, the material obtained from the extract stream of SMB resolution had optical rotation with the same sign (positive, i.e. dextrorotary),

BIOLOGICAL STUDIES [0043] Various techniques are known in the art to test the activity of a compound of the invention or to determine their solubility in known pharmaceutically acceptable excipients. To enable a more complete understanding of the invention described herein, the following biological studies are provided below. It should be understood that these examples are illustrative only.

BIOLOGICAL EXAMPLE 1

Guanidine influx study (in vitro study) [0044] This example describes an in vitro test for testing and profiling test agents for human or rat voltage gated sodium channels stably expressed in cells of endogenous origin or from heterologous expression. The test is also useful for determining the IC 50 value of a voltage modulating gated, preferably blocking, sodium channel. The assay is based on the guanidine flow assay described by Reddy, NL, et al., J Med Chem (1998), 41 (17): 3298-302.

[0045] The guanidine influx test is a radiotracer flow study used to determine the ionic activity of voltage-gated sodium channels in a high-performance microplate based format. The study uses hydrochloride¹⁴C-guanidines in combination with various known voltage-gated sodium channel modulators that generate sustained flow to determine the potency of the agents being tested. The potency is determined by calculating the IC50 value. Selectivity is determined by comparing the potency of a voltage-blocking compound of a given sodium channel with its potency against other voltage-gated sodium channels (so-called "selectivity profiling").

[0046] Each of the test agents is tested on the cells in which the voltage gated channel is expressed. Voltage-gated sodium channels are characterized as sensitive or insensitive to TTX. This property is useful when assessing the activity of a given sodium channel

The voltage gated voltage is present when it is in a mixed population with other voltage-gated sodium channels. The following Table 1 lists cell lines useful in screening for the activity of a specific voltage-gated sodium channel in the presence or absence of TTX.

TABLE 1

<td>CELL LINE</td><td>Expression of mRNA</td><td>Functional characteristics</td>

<td rowspan="2">CHO-K1 (Hamster ovary cells Chinese; recommended host cell line) ATTC number: CCL-61 L6 (rat mioblast) number ATTC: CRL-1458</td><td>• Nav1.4 expression was demonstrated by the RTPCR method • No expression was found Nav</td><td rowspan="2">• 18- to 20-fold increase in inflow [¹⁴C] guanidine was completely blocked via TTX (Nav1.4 is sensitive to TTX) • 10- to -15-fold increase in inflow [¹⁴C] guanidine was only partially blocked via TTX at 100 nM (Nav1.5 is a channel resistant to TTX)</td>

<td>• Expression of Nav1,4 and 1.5</td>

<td>SH-SY5Y (human neuroma) number ATTC: CRL-2266</td><td>• Published expressing Nav1.9 and Nav1.7 (Blum et al.)</td><td>• 10- to 16-fold increase in inflow [¹⁴C] guanidine above the background has been partially blocked by TTX (Nav1.9 is a channel resistant to TTX)</td>

<td>SK-N-BE2C (human neuroma cell line, ATTC number: CRL2268)</td><td>• Expression NaV1.8</td><td>• Stimulation of BE2C cells with pyrethroid causes a 6-fold increase in inflow [¹⁴C] guanidines over background. • TTX partially blocked the inflow (Nav1.8 is a TTX-resistant channel)</td>

<td rowspan="2">PC12 (rat stump) number ATTC: CRL-1721 CHO-K1 (Hamster ovary cells Chinese; recommended host cell line) ATTC number: CCL-61</td><td>• Expression of Nav1.2</td><td>• 8 - up to -12-fold increase in inflow [¹⁴C] guanidine was completely blocked via TTX (Nav1.2 is a sensitive channel on TTX)</td>

<td>• Nav1.4 expression was demonstrated by the RTPCR method • No expression was found Nav</td><td>• 18- to 20-fold increase in inflow [¹⁴C] guanidine was completely blocked via TTX (Nav1.4 is sensitive to TTX)</td>

<td>L6 (rat mioblast) number ATTC: CRL-1458</td><td>• Expression of Nav1,4 and 1.5</td><td>• 10- to -15-fold increase in inflow [¹⁴C] guanidine was only partially blocked via TTX at 100 nM (Nav1.5 is a channel resistant to TTX)</td>

EP 2 789 617 B1

<td>HEK293 (human cells embryonic kidneys) ATTC Number CRL-1573</td><td>• Expressing hNaV1.7</td><td>• Nav1.7 is a TTX-resistant channel. TTX IC50 in a functional guanidine study is 8 nM.</td>

[0047] It is also possible to use immortalized cell lines expressing voltage-gated sodium channels. Methods for cloning, stable transfection and propagation of such cells are known to those skilled in the art (see, for example, Klugbauer, N, et al., EMBO J. (1995), 14 (6): 1084-90; and Lossin, C., et al., Neuron (2002), 34, pp. 877-884).

[0048] Cells expressing the voltage-gated sodium channel in question are grown according to the supplier's instructions or, in the case of a recombinant cell, in the presence of selective growth medium G418 (Gibco / Invitrogen). The cells are separated from the cell culture plates with the enzyme solution (1X) Trypsin / EDTA (Gibco / Invitrogen) and analyzed for density and viability using a hemocytometer (Neubauer). The cells are washed and resuspended in media and then plated on Poly-D-lysine-coated Scintiplates (Perkin Elmer) plates (approximately 100,000 cells / well) and incubated at 37 ° C / 5% CO 2. for 20-24 hours. After extensive washing with low-salt HEPES buffered saline (LNHBSS) (150 mM choline chloride, 20 nM HEPES (Sigma), 1 mM calcium chloride, 5 mM potassium chloride, 1 mM magnesium chloride, 10 mM glucose), assay dilutions diluted with LNHBSS (different concentrations of the test agent may be used) at appropriate concentrations are added to each well. The activation / radiolabelled mixture contains an alkaloid, such as veratridine, or aconitine (Sigma) or a pyrethroid, such as deltamethrin, such as Leiurus quinquestriatus hebraeus scorpion (Sigma) and hydrochloride¹⁴C-guanidines (ARC) to measure the flow through voltage-gated sodium channels.

[0049] After introduction of the test agent and the activation / radiolabelled mixture into the cells, the Scintiplates-coated Poly-D-lysine plates are incubated at ambient temperature. After incubation, the Scintiplates-coated Poly-D-lysine plaques are thoroughly washed with LNHBSS supplemented with guanidine (Sigma). The Scintiplates are dried and counted using a Wallac MicroBeta TriLux (PerkinElmer Life Sciences). The ability of the test agent to block voltage-gated sodium channel activity is determined by comparing the amount¹⁴C-guanidine present in cells expressing different voltage-gated sodium channels. Based on this data, various calculations may be used as given elsewhere in this specification to determine if the test agent is selective for a specific voltage-gated sodium channel.

[0050] Using the general method described above, the IC50 value of the test agent for a particular voltage-gated sodium channel can be determined. IC 50 can be determined using 3, 8, 10, 12 or 16 point (two or three repeats) curve at an initial concentration of 1.5 or 10 μΜ, serial dilution and final concentrations up to sub-nanomolar, nanomolar and low micromolar ranges. Typically, the middle concentration of the test agent is set to 1 μM, and subsequent concentrations are used with half the dilution of greater or less (e.g., 0.5 μM, 5 μM and 0.25 μM, 10 μM and 0.125 μM, 20 μM etc.). The IC50 value is calculated using the four-parameter logistic model or sigmoidal dose-response model (fit = (A + ((BA) / (1 + ((C / x)^ANDD)))).

[0051] The selectivity ratio (fold selectivity, factor of selectivity or multiple of selectivity) is calculated by dividing the IC50 values ​​of the voltage gated sodium channel tested by the voltage-gated reference voltage sodium channel, for example Nav1.5.

In view of the above, the compound of formula (I), the (S) enantiomer of the compound of formula (I), i.e., the (S) enantiomer of the invention and the (R) enantiomer of the compound of formula (I), tested in this test demonstrated the blocking activity of the voltage-gated sodium channel on hNaV1.7, as shown in Table 2 below:

TABLE 2

<td>Relationship</td><td>Chemical name</td><td>IC50 (ΜΜ)</td>

<td>(AND)</td><td>1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole-7,3'indolo] -2' (1'H) - he</td><td>0,007</td>

<td>(IR)</td><td>(R) -1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole-7,3'indolo] -2' (1 H) -one</td><td>4,200</td>

<td>(IS)</td><td>1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioxole-7,3'indolo] -2' (1'H) - he</td><td>0.003</td>

[0053] The concentration-response relationship for the (S) enantiomer of the invention and the (R) enantiomer is shown in Figure 1. The solid curves indicate the smallest best alignment of the squares with a 1: 1 binding isotherm; The IC50s that describe these curves are shown in Table 2. The (S) -enantiomer of the invention showed a significantly higher (e.g., 1000-fold) inhibitory potency for hNaV1.7 in this model compared to the inhibitory potency with the corresponding (R) -enantiomer.

[0054] These results favor the use of the (S) -enantiomer of the invention with respect to the (R) enantiomer or compound of formula (I) (racemate) for the uses described herein, because a higher pharmacological activity can be achieved at lower dosage levels with as few effects as possible. side. In addition, the (R) enantiomer is a very important tool for safety research because it allows to distinguish between mechanism-based effects (those mediated by a sodium channel block) and activities beyond the purpose of the invention that can be removed without impairing the efficacy of analogs. If the adverse effect is based on the mechanism, the (S) enantiomer will be much stronger than the (R) enantiomer,

BIOLOGICAL EXAMPLE 2

Electrophysiological study (in vitro study) [0055] HEK293 cells expressing Nav1.7 are cultured in DMEM culture medium (Gibco) with 0.5 mg / ml G418, +/- 1% PSG and 10% heat inactivated fetal bovine serum in 37C ° and 5% CO2. For electrophysiological readings, the cells are placed on 10 mm plates.

[0056] The registration is carried out in a whole-cell configuration with the whole cell voltage clamp technique (Bean et al., Op cit.), Using an Axopatch 200B amplifier and Clampex software (Axon Instruments, Union City, CA ). All experiments are carried out at room temperature. The electrodes are polished in fire to 2-4 ohms. Voltage and capacitance errors are minimized by, respectively, serial resistance compensation and capacitance compensation. Data is downloaded at 40 kHz and filtered at 5 kHz. Composition of external solution (bath): NaCl (140 mM), KCI (5 mM), CaCl2 (2 mM), MgCl2 (1 mM), HEPES (10 mM) at pH 7.4. Composition of the internal solution (pipette) (in mM): NaCl (5), CaCl2 (0,1) MgCl2 (2),

CsCl (10), CsF (120), HEPES (10), EGTA (10), pH 7.2.

[0057] In order to assess the affinity at steady state of the compounds for the dormant channel in the state of inactivation and inactivation (Kr and Ki respectively), 12.5 ms test impulses are used from the holding potential of -120 mV to depolarization voltages ranging from -60 mV to +90 mV, in order to construct current-voltage relationships (curves IV). Voltage close to the peak of curve IV (from -30 to 0 mV) is used as the test pulse voltage during the remainder of the experiment. Next, inactivity curves are constructed in the steady state (availability), measuring the current excited during the 8.75 ms test pulse and the next 1 s of the boost pulses to the potentials in the range from -120 to -10 mV.

[0058] The dependence on the binding voltage of the compound to the steady state sodium channel is determined by measuring the ionic current blocking for the two holding potentials. Binding to dormant channels is determined using a holding potential of -120 mV, in order to achieve maximum availability. Binding to channels in the inactivated state was evaluated at the holding potential such that only 10% of the channels were available for opening. The membrane potential is maintained at this voltage for at least 10 seconds, so that the binding of the drug can be established.

[0059] The apparent dissociation constant at each voltage is calculated using the equation:

<img file="PL2789617T3_D0008.tif" />

where K d is the dissociation constant (Kr or Ki), and [drug] is the concentration of the test compound.

Therefore, the compound of formula (I), the (S) enantiomer of the compound of formula (I), i.e. the (S) enantiomer of the invention and the (R) enantiomer of the compound of formula (I), tested in this model, showed affinity for resting / closed and inactivated state of hNaV1.7, as shown below in Table 3:

TABLE 3

<td>Relationship</td><td>Chemical name</td><td>Ki (ΜΜ)</td><td>Cr (ΜΜ)</td>

<td>(AND)</td><td>1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioksolo7,3'-indol] -2' (1'H) -one</td><td>0.142</td><td>&gt; 10um</td>

<td>(IR)</td><td>(R) -1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3f] [[1,3] benzodioxole-7,3'-indol] -2' (1 H) -one</td><td>0.869</td><td>&gt; 10um</td>

<td>(IS)</td><td>1 '- {[5- (trifluoromethyl) furan-2-yl] methyl} spiro [furo [2,3-f] [1,3] benzodioksolo7,3'-indol] -2' (1'H) -one</td><td>0.161</td><td>&gt; 10um</td>

[0061] As shown by these results, the (S) -enantiomer produced by the process of the invention is a hNaV1.7-dependent-voltage dependent modifier, with a low affinity to the resting / closed state and high affinity to the inactivated state. The results showed that the (S) -enantiomer is approximately 5-fold more strongly bound to hNaV1.7 in the inactivated state than the (R) enantiomer.

In addition, the results show that the (S) enantiomer is primarily responsible for the strength of the racemate i.e. the compound of the formula (I).

BIOLOGICAL EXAMPLE 3

In vivo studies

Acute Pain (Formalin Test) [0062] The fomalin test is used as an animal model of acute pain. In the formalin test, on the day before the experiment day, the animals are briefly acclimated to the plexiglass test chambers for 20 minutes. On the day of the test, the animals are randomly injected with the test substances. 30 minutes after drug administration, 50 μΐ of 10% formalin is injected subcutaneously in the plantar region of the left hind paw of rats. The collection of video data starts immediately after the administration of formalin and lasts 90 minutes.

[0063] Images are recorded using the Actimetrix Limelight program, which stores files with the extension * .Ilii, and then converts them to the MPEG-4 format. Then video recordings are analyzed using the "The Observer 5.1" behavioral analysis program, (version 5.0, Noldus Information Technology, Wageningen, the Netherlands). The video analysis is carried out by observing the behavior of the animals and awarding points for each behavior depending on its type, as well as determining the duration of the behavior (Dubuisson and Dennis, 1977). Targeted types of behavior include: (1) normal behavior, (2) paw removal, (3) lifting 46 paws, (4) licking / biting or scratching the paw. Lifting, relieving or excessive licking, biting and scratching the injected paw indicates a painful reaction.

[0064] The analysis of data from the formalin test is carried out with respect to two factors: (1) the percentage of the maximum possible inhibition effect (Percent Maximal Potential Effect Inhibitors,% MPIE) and (2) the score on the pain scale. The% MPIE values ​​are calculated in several stages, the first of which is to sum up the duration of abnormal behaviors (behavior 1, 2, 3) of each animal. A single value for the therapeutic group with vehicle is obtained by averaging all results within this therapeutic group. The following calculation gives the MPIE value for each animal:

MPIE (%) = 100 - [(sum for treatment groups / mean value for a vehicle group) X 100%] [0065] The score on the pain scale is counted using a weighted scale as described above. In order to determine the result on the pain scale for each animal, the duration of the behavior by the weight is multiplied (the severity of the reaction) and divided by the total duration of the observation. The calculations are represented by the following formula:

EP 2 789 617 B1

Pain rating = [D (To) + 1 (T1) + 2 (T2) + 3 (T3)] / (To + T1 + T2 + T3)

CFA-induced chronic inflammatory pain [0066] In this study, mechanical allodynia is assessed using calibrated von Frey filaments. After a full week of acclimation to the vivarium room, the plantar region of the left hind paw of the rats was injected subcutaneously, under light general anesthesia, isoflurane, 150 μΐ of Freund's complete adjuvant emulsion (CFA) (CFA suspended in oil-in-saline emulsion (1: 1 ), at a concentration of 0.5 mg / ml). Animals were allowed to recover from anesthesia and reference values ​​were established for mechanical and thermal nociceptive pain thresholds for all animals, one week after CFA administration.

On the day before the experiment started, all animals were habituated to the test devices for 20 minutes. Test and control products were administered to animals, and nociceptive thresholds were measured at predetermined time points after drug administration to determine analgesic responses to each of the six treatments available. Selected time points were determined earlier in order to show the highest analgesic effect for each of the tested compounds.

[0067] The (S) -enantiomer prepared by the method of the invention and the corresponding (R) enantiomer were compared using both oral and topical dosing. Figure 2 shows a comparison of the efficacy of (S) enantiomer and enantiomer (R) with oral dosing. Each enantiomer was dosed at 10, 30, 100 or 200 mg / kg. The plasma concentration at each dose was also determined, and the reversal of the pain response (as a% increase from the reference threshold) was plotted as a function of plasma concentration. [0068] The (S) enantiomer had a larger maximum effect when it was dosed at 200 mg / kg. The (R) enantiomer has significantly higher plasma concentrations at the equivalent dose level. It was an unexpected and unusual discovery. Consequently, the use of a racemate, i.e. a compound of formula (I), would result in a 10-fold excess of the non-active enantiomer, i.e. the (R) enantiomer.

[0069] The (S) -enantiomer prepared by the method of the invention was also administered topically to animals at various doses (1%, 2%, 4% and 8% (w / v)) and nociceptive thresholds were measured at set time points after drug administration. to determine the analgesic response to each of the six available therapies. Selected time points are defined earlier in order to show the highest analgesic effect for each tested compound.

[0070] After the Hargreaves test, the threshold values ​​of the animals' response to tactile stimuli were determined using the Electrovonfray estiometer model 2290 (IITC Life Science, Woodland Hills, CA). The animals were placed in suspended plexiglass chambers based on a wire mesh surface. After 15 minutes of acclimatization, previously calibrated von Frey filaments were applied perpendicular to the plantar space of the hind paws of the animals on the same side with sufficient force, measured in grams to induce a violent paw response. The answer indicated the withdrawal of the painful stimulus and set the end point of effectiveness. The test was continued until the filament was indicated, which, at the lowest applied force, caused a rapid withdrawal of the paw or until the limit of applied force was reached, amounting to about 20 g. This limit of force is applied because it is equal to about 10% of the body weight of the animals; this is to prevent the entire limb from being lifted from

Because of the use of stiffer filaments, which would change the nature of the stimulus. Data were expressed as a percentage increase compared to the initial threshold value measured in grams.

[0071] The (S) -enantiomer tested in this model showed analgesia as shown below in Table 4.

TABLE 4

<td></td><td colspan="4">% increase from baseline (CFB)</td>

<td>Relationship</td><td>1% locally</td><td>2% locally</td><td>4% locally</td><td>8% locally</td>

<td>(IS)</td><td>0.62</td><td>16.71</td><td>28.79</td><td>45,06</td>

[0072] The (S) enantiomer at 2%, 4% and 8% (w / v) showed an increase in the mechanical retraction thresholds of von Frey expressed as a percentage increase from baseline (IFB) to indicate analgesia. The analgesic effect of the (S) enantiomer increased with increasing doses up to the highest 8% dose tested (w / v), which showed that the maximum percentage of IFB at + 45.1%. A 1% (w / v) dosing group, however, did not show an appreciable increase in von Frey's mechanical withdrawal thresholds. The results show that the (S) enantiomer has an analgesic effect in the CFA-induced inflammatory pain model in the range of 2% to 8% (w / v).

Nociception model in the postoperative period [0073] In this model, cutaneous hyperalgesia in the plantar paw area is determined by subjecting the paw to a developing tactile stimulus until the paw withdraws from the stimulus applied. While the animals are under general anesthesia induced by 3,5% isoflurane administered intranasally, in the plantar area of ​​the left hind paw, using a blades number 10, a 1 cm long incision is made, crossing the skin and fascia, starting 0.5 cm from the proximal heel edge up to the fingers. After making the incision, the skin is sutured using a sterile silk suture 2, 30. The damaged area is covered with polysporin and betadine. The animals are allowed to regenerate in cages overnight.

[0074] The retraction threshold under the influence of the tactile touch on animals, the treated (ipsilateral) paw and the untreated (contra-lateral) paw are determined using the Electrovonfray estimate, model 2290 (IITC, Life Science, Woodland Hills, CA). The animals are placed in suspended plexiglass chambers based on a wire mesh surface. After at least 10 minutes of acclimatization, the previously calibrated von Frey filaments are applied perpendicular to the plantar space of both paws of animals in ascending order, starting with a 10 g filament, with a force sufficient to cause a slight deflection of the filament. The test was continued until the filament was indicated which, at the lowest applied force, causes the paw to be rapidly withdrawn or until in which a strength limit of about 20 g is reached. This force limit is applied because it is about 10% of the body weight of the animals; this is to prevent lifting the whole limb due to the use of stiffer filaments, which would change the nature of the stimulus.

Model of neuropathic pain: injury caused by constant pressure

[0075] In this model, at the height of half the left thigh of the animal, using a blade number 10, a 3 cm incision was made, cutting the skin and fascia. The left sciatic nerve was exposed by dividing the tissues bluntly through the biceps femoris, trying to minimize bleeding. Four ligatures were loosely placed around the sciatic nerve using a sterile non-absorbable 4-0 silk suture at a distance of 1 to 2 mm. The stress of loosely ligated ligatures is to be sufficient to cause slight sciatic nerve compression observed by a dissecting microscope in a 4-fold magnification. In the sham-operated animal, the left sciatic nerve was exposed without further manipulation. Antibacterial ointment was applied directly to the wound and the muscle was sutured using sterile sutures. Tumadine was applied to the muscle and surrounding tissues,

[0076] The animal response thresholds for tactile stimuli were determined using an Electrovonfray estimate, Model 2290 (IITC Life Science, Woodland Hills, CA). The animals are placed in suspended plexiglass chambers based on a wire mesh surface. After 10 minutes of acclimatization, previously calibrated von Frey filaments were applied perpendicular to the plantar space of both paws of the animals in ascending order, starting with a 0.1 g filament, with a force sufficient to cause a slight deflection of the filament. The test shall be continued until the filament is indicated which, at the lowest applied force, causes the paw to back violently or to a point where a force limit of approximately 20 g is reached. This limit of force is applied because it is about 10% of body weight animals;

[0077] The nociceptive thermal pain thresholds for the animals were determined using the Hargreaves test. After measuring the threshold of touch sensitivity, the animals were placed in suspended plexiglass chambers, the base of which is a glass platform with heating units. In all test samples, the temperature of the thermostated glass platform is set at approximately 24-26 ° C. After being placed in the chambers, the animals were allowed to adapt for 10 minutes in order to stop any exploratory behavior. A beam of infrared radiation was applied to the underside of the glass platform towards the plantar zone of the hind paws, using the Plantar / Tail Stimulator analgetic model 226 (IITC, Woodland Hills, CA). During all trials to prevent tissue damage,

[0078] The (S) -enantiomer was compared to the corresponding (R) -enantiomer and racemate (compound of formula (I)) in this CCI model using topical use as described for the CFA model (see Figure 3). Each test compound was administered as an ointment containing 2% (w / v). According to the various activities of these two enantiomers as voltage-gated sodium channel inhibitors, only the (S) enantiomer reversed the pain responses, while the (R) -enantiomer had a significant increase from the initial value. Both the (S) enantiomer and the racemate show a similar percentage increase from the beginning of the study, which seems to suggest that the (S) -enantiomer is responsible for analgesia.

BIOLOGICAL EXAMPLE 4

Examination of aconitine-induced arrhythmia

[0079] The anti-arrhythmic effect of the compounds of the invention is demonstrated in the following study. Arrhythmia is provoked by intravenous administration of aconitine (2.0 μg / kg) dissolved in physiological saline. Test compounds of the invention were administered intravenously 5 minutes after aconitine administration. The assessment of antiarrhythmic activity is performed by measuring the time from aconitine to extrasystole (ES) and the time from aconitine to ventricular tachycardia (VT).

[0080] In isoflurane anesthesis rats (1/4 to 1/3 2%), a tracheotomy is performed, first making an incision in the neck area, and then isolating the trachea and making a 2 mm incision to insert the 2 cm tracheal tube into the trachea, so that the opening of the cannula is placed just above the muzzle. The cannula is secured with sutures and connected to the respirator for the duration of the experiment.

[0081] In the thigh area, an incision is made (2.5 cm) and the thyroid vessels are extracted using a blunt probe. Both femoral veins are cannulated, one to maintain anesthesia with pentobarbital (0.02-0.05 ml), and the other for infusion and injection of drug and vehicle. The femoral artery is cannulated using a gel catheter to monitor blood pressure with the transmitter.

[0082] The ECG electrodes are connected to the pectoral muscle in the Lead II position (the upper right side above the heart - the white electrode and the lower one on the left below the heart - the red electrode). The electrodes are secured with seams.

[0083] All areas in which surgical procedures have been performed are coated with gauze soaked in 0.9% saline. Saline (1-1.5 ml 0.9% solution) is used to moisten areas after surgery. Animals were allowed to equilibrate the heart and breath for at least 30 minutes.

[0084] Arrhythmia is induced by infusion of 2 μg / kg / min aconitine for 5 minutes. During this time, the ECG is recorded and continuously monitored.

BIOLOGICAL EXAMPLE 5

Investigation of induced arrhythmia ischemia [0085] To study potential therapeutic agents for the treatment of atrial and ventricular arrhythmias in humans, rat models of ventricular arrhythmias in the paradigms of acute cardioversion and prophylaxis were used. Myocardial ischemia leading to myocardial infarction is a common cause of disease states and mortality. The ability of a compound to prevent ischemia-induced ventricular tachycardia and ventricular fibrillation is an accepted model for determining the efficacy of a compound in the clinical setting of atrial and ventricular tachycardia and atrial fibrillation and ventricles.

[0086] General anesthesia is first induced with pentobarbital (administered intraperitoneally) and is maintained by intravenous bolus. The trachea of ​​male SD rats are cannulated for mechanical ventilation with room air, with a stroke volume of 10 ml / kg, 60 strokes / minute. The right thigh artery and vein can be cannulated using a PE50 cannula to measure mean arterial blood pressure (MAP) and intravenous administration, respectively.

[0087] The chest opens at an elevation between the fourth and fifth ribs, forming a 1.5 cm opening revealing the heart. Each rat is placed on a corrugated platform and fastens the skeleton of the cage

Chest pectorus with metal handles, opening the thoracic cavity. A surgical needle is used to enter the ventricle just under the raised vestibule and diagonal (after pointing down) the exit from the ventricle, resulting in&gt; 30% and &lt;50% occlusion area (OZ). The starting place is ~ 0.5 cm below the point where the aorta connects to the left ventricle. The suture is clamped so as to form a loose loop (occluder) around the artery branch. Then, the chest is closed using the end of the occluder accessible from outside the chest.

[0088] The electrodes are placed in the Lead II position (from the right atrium to the apex) to measure the ECG as follows: one electrode is placed on the right right paw and the other electrode is placed on the back left paw.

[0089] During the experiment, the body temperature, mean MAP arterial pressure, ECG and heart rate are continuously measured. After these important parameters have stabilized, a 1-2 minute measurement is made to establish a reference value. After the reference value has been established, the compound prepared by the method of the invention or control is infused. After a 5-minute compound or control infusion, the suture is clamped to ligate the left coronary artery and cause ischemia in the left ventricle. For 20 minutes after ligation, important parameters are continuously recorded unless the MAP achieves a critical level of 20-30 mmHg (0.0266-0.0399 bar) for at least 3 minutes, in which case the record shall stop because it acknowledges that the death of the animal will occur, after which the animal is killed.

BIOLOGICAL EXAMPLE 6 [0090] Compared to the racemate, i.e. the compound of formula (I), the (S) enantiomer, substantially free of the (R) -enantiomer, has a better solubility profile in a wide variety of pharmaceutically acceptable excipients. Thus, the (S) enantiomer may be formulated in a smaller number of dosage units than the racemate. This property facilitates dosing to patients at a higher level if efficacy is to be achieved. Examples of the difference in solubility are shown in Table 5 below:

TABLE 5

<td>Additional substance</td><td>Compound of formula (I) (racemate)</td><td>Enantiomer (S)</td>

<td>Labrasol</td><td>72.5 mg / ml</td><td>231 mg / ml</td>

<td>Propylene glycol</td><td>2.7 mg / ml</td><td>9.8 mg / ml</td>

<td>PEG 400</td><td>&lt;50 mg / ml</td><td>&gt; 55 mg / ml</td>

<td>Capryol® 90</td><td>18.1 mg / ml</td><td>96 mg / ml</td>

<td>Tween® 80</td><td>64 mg / ml</td><td>&gt; 123 mg / ml</td>

<td>Ethanol</td><td>10.0 mg / ml</td><td>36.4 mg / ml</td>

<td>Labrasol® / PEG 400 60/40</td><td>70.4 mg / ml</td><td>182 mg / ml</td>

<td>Labrasol® / Capryol®90 60/40</td><td>44.4 mg / ml</td><td>191 mg / ml</td>

<td>Labrasol® / Transcutol® 60/40</td><td>74.2 mg / ml</td><td>186 mg / ml</td>

EP 2 789 617 B1

BIOLOGICAL EXAMPLE 7

In vivo study for the treatment of pruritis [0091] Histamine induces pruritus (itching) in humans. Accordingly, this test evaluates the efficacy of the orally administered and topical (S) -enantiomer of the invention for histamine-induced pruritus in male ICR mice.

[0092] The animals were randomly divided into test groups, including the untreated group, the group treated with the local pharmaceutical composition with 8% (w / v) of the (S) enantiomer and the group treated with the oral composition of 50 mg / kg of the (S) enantiomer. One day before the test, the scapular regions on animals were shaved with a hair clipper. On the day of testing, the animals were habituated for 60 minutes in a test chamber containing a transparent plastic ridge placed in a vertical position on a flat surface. After the habituation period, the animals were removed from the plastic tube, placed in a cage and injected with histamine in the shaved paddle region. The injections were made intradermally in small injection amounts (10 μθ using a Hamilton syringe. The solutions for injection consisted of histamine dissolved in physiological saline at a concentration of 100 μg / 10 ml (or 10 mg / ml). 10 μg of this solution was injected into each mouse. Immediately after the injection, the animals were returned to the test chambers and observed by cameras placed above the test chambers for a total of 50 minutes. The cameras were connected to a computer where digital video files were created, saved and analyzed.

[0093] The number of pruritus fits was assessed within 40 minutes. The "itching attack" was defined as lifting the hind leg, using it to scratch the scapular region and then placing it back on the ground. Alternatively, if instead of placing the back leg back on the ground licking of the mouse paw was observed, it was also counted as an itching attack.

[0094] In the untreated group, animals (n = 7) were habituated to the test chamber for 60 minutes prior to histamine injection. To assess the (S) -enantial enantiomer in histamine induced it, animals (n = 16 / group) were habituated to the test chamber for 30 minutes, followed by 50 mg, 8% (w / v), topical (S) enantiomer or carrier for the shaved region on the back. The animals return to the test chamber for a further 30 minutes of habituation before injecting histamine. To assess the oral (S) enantiomer, the animals (n = 8 / group) were orally administered by gavage at a dose of 50 mg / kg (S) enantiomer or vehicle and were then habituated in the test chamber for 60 minutes prior to the injection of histamine.

[0095] Data were analyzed using GraphPad Prism 5 software for statistical analysis and the unpaired t-test was used for one-dimensional analysis. The results are expressed as mean ± SEM. Values ​​that reached p &lt;0.05 significance level were considered statistically significant.

Results [0096] The injection of histamine into the skin resulted in the animals feeling sporadic itching in attacks that lasted 1-2 seconds. In the untreated group, itching attacks started immediately after the injection and lasted about 40 minutes after its completion (see Figure 4). A group treated with 8% (w / v) of the topical (S) enantiomer showed a significant decrease in pruritus (see Figure 5). Animals treated

Only the device had a total number of 134.3 ± 13.31 (n = 16) itching seizures, whereas in mice treated with the (S) -enantiomer enantiomer had 89.00 ± 10.51 (n = 16) itching attacks. . The difference between these groups was statistically significant at the p-value of 0.0122. In the 50 mg / kg group, the (S) -enantiomer orally showed a significant decrease in pruritus (see Figure 6). Animals treated with vehicle alone had a total of 42.88 ± 6.667 (n = 8) itching seizures, whereas mice treated with the (S) enantiomer had 17.25 ± 6.310 (n = 8) itching attacks. The difference between the orally treated group was statistically significant at a p-value of 0.0144. The results showed that the orally and topically enantiomer (S) reduced pruritus. In addition, it is evident that two common methods of drug delivery,

BIOLOGICAL EXAMPLE 8

Clinical trial in humans for primary / inherited erythromelegaly (IEM) treatment [0097] Primary / inherited erythromelia (IEM) is a rare pain inherited condition. The cause of IEM may be one or more of the potentiation of the mutation function in the NaV1.7 voltage-gated sodium channel, which has been shown to be inhibited by the (S) enantiomer of the invention.

[0098] Patients who are people with IEM have recurrent episodes of intense burning pain associated with redness and warmth in the hands and feet, but eventually the pain becomes permanent. The pain is replaced by cooling, but is largely immune to pharmacological intervention. However, there are reports of voltage-gated sodium channel blockers showing moderate to outstanding pain relief for this condition.

[0099] Clinical trials to determine the efficacy of the (S) -enantiomer of the invention in ameliorating or reducing IEM may be designed for a three-period, double-blind, multi-dose and cross-study study to minimize the rate of dropout of participants and will take into account that patients saved will be available only for a 10-day study. Each of the patients participating in the study will serve as their own control, receiving both placebo and 400 mg of the (S) -enantiomer of the invention twice a day in a cross-over fashion.

BIOLOGICAL EXAMPLE 9

Clinical study in humans in the treatment of toothache [0100] The aim of this clinical trial was to compare safety and efficacy (start time, relief duration and overall efficacy) with a single 500 mg dose of (S) -enantiomer produced in accordance with the invention relative to the placebo dose for the relief of pain after extraction of the third molar.

[0101] Sixty-one patients participated in the study. The average age of patients was 20.4 years and all patients were men. Most of the patients were Caucasian (95.1%).

[0102] Austerity and pain relief were measured using an 11-point numeric scale of pain intensity assessment (on a scale of 0 = no pain at all 10 = worst pain imaginable), (PINRS) and a 5-point scale

[0010] The pain relief (REL) is categorical. Patients completed the PINRS after surgery and prior to administration of the (S) enantiomer of the invention. The efficacy variables were from the REL and PINRS scores and included total pain relief (TOTPAR), pain intensity difference (PID) and peaked pain intensity difference (SPID) and evaluated in time points 4, 6, 8 and 12 hours after administration of the enantiomer (S ) made by the method of the invention.

[0103] However, all primary and secondary endpoints showed a consistent analgesic tendency with pronounced separation of (S) -enantiomer compared to placebo. These results suggest that the (S) enantiomer has analgesic properties, but statistical significance with placebo has not been achieved due to two main reasons: (1) a relatively high response rate to placebo and (2) slow onset of (S) enantiomer. The used dental model is designed and is best suited for the evaluation of fast-start medicines, such as the anti-inflammatory agents of the NSAID group. It is evident from this study that the (S) -enantiomer of the invention does not have such a NSAID-like rapid onset of action. However, the pain reduction shown by those patients who received the (S) enantiomer,

BIOLOGICAL EXAMPLE 10

Human S enantiomer (S) safety study in accordance with the invention [0104] This clinical study was Phase 1, a randomized, double-blind, placebo-controlled study in healthy volunteers to evaluate the safety and pharmacokinetics of a topical ointment containing (S) -enantiomer by invention.

[0105] The ointment with the (S) -enantiomer was applied daily for 21 days to determine the local skin toxicity / irritation of the (S) enantiomer. Systemic pharmacokinetics and local levels of the drug in the skin were also evaluated. Systemic exposure to the (S) enantiomer after topical administration and local skin irritation after multiple doses of the ointment with the (S) enantiomer were assessed. Each subject received 5 treatments over 21 consecutive days of (S) enantiomer in ointment from 4% to 8% (w / w) (1 x 100 μΙ, treatments A and B, respectively), with placebo as an ointment (treatment C ), saline (0.9%) (1 x 100 μL, negative control, Treatment D) and sodium lauryl sulfate (SLS) 0.1% solution (1 x 100 μ! positive control, treatment E). Treatments were applied in two different places on the upper back of each individual in a covered manner (five treatments) and partly covered (the first three treatments). Location for each treatment on each page (Treatments A, B, C, D and E on a covered site and treatment A,

B and C on a partially covered site) was randomized. Patients were limited to a clinical research center from about 18 hours before the first dosing on day 1 to about 8 hours after the second dose (Day 2). Patients returned each day for 19 consecutive days (from 3 to 21) as part of the dosing and testing procedures.

[0106] There were no serious adverse events (SAEs) or deaths. All adverse events (AEs) were mild or moderate in severity, with most adverse events associated with local skin reactions from the surgical patch to adhere to an occlusive dressing. All patients responded to a positive control. Positive control was stopped for

All patients on day 4 after complaints about excessive discomfort of the subjects. Skin irritation scores were low for all treatments administered (maximum score 3 measured on a scale of 0-7), indicating that the (S) enantiomer in the ointment is locally well tolerated. No differences were observed between cumulative irritation results for (S) enantiomer 4% (w / w), enantiomer (S) 8% (w / w) ointment, placebo and negative control (0.9% saline). Symptoms of irritation have completely disappeared by Day 28 (7 days after the last dose) for most patients.

[0107] Records of electrocardiography did not show clinically significant changes in the frequency of resting heart rate, quiescent state or QTc intervals in patients, and there were no clinically significant changes from the baseline in the examined vital signs, physical examinations or laboratory assessments. Systemic exposure to the (S) enantiomer was negligible because plasma concentrations of the (S) -enantiomer were below the lower limit of quantification (LLOQ) (0.1 ng / ml or 100 pg / ml) in most samples (489 at 546 = ~ 90% ). The highest level of (S) enantiomer observed in one patient in the dosing period (day 22) was 994 pg / ml. Based on the minimal local irritation and favorable safety profile, along with the low systemic exposure of the (S) enantiomer, it was found that

BIOLOGICAL EXAMPLE 11

Clinical trial in humans for the treatment of neuralgia [0108] Post-herpetic pain (PHN) is a well-established and recognized model for the study of neuropathic pain. In addition, PHN demonstrates strong evidence of the efficacy of the sodium channel blocker. The following study represents randomized, double-blind, placebo-controlled, two-way, cross-over studies to assess the safety, tolerability, pre-efficacy, and systemic exposure of the (S) enantiomer of the invention administered locally to patients with herpes zoster. The main goals are (a) to compare the efficacy and safety of an ointment containing (S) -enantiomer to those of placebo in relieving pain in patients with PHN, and (b) to assess the systemic exposure of (S) -enantiomer after topical application of the (S) -enantiomer in patients with PHN . Treatments consist of an ointment with an (S) enantiomer of 8% (w / w) and a corresponding placebo ointment. [0109] The test will include the following four periods:

1. Initial screening and leaching period (up to 3 weeks);

2. Single-blind study, placebo treatment period (1 week);

3. A period of cross-treatment, which will consist of 2 treatment periods, each lasting 3 weeks, separated by 2 weeks of leaching / single blank test, duration of placebo (a total of 8 weeks); and

4. The following safety period (2 weeks).