Novel n-benzyl phthalimides for the treatment of pain. methods of preparation and therapeutic use

Abstract

The present invention relates to an N-benzyl phthalimide derivative compound of formula I, a pharmaceutical composition comprising the same, a medicament, its use, combination, and methods for treating acute and chronic pain, primarily focusing on neuropathic disorders and syndromes associated with neuropathic pain.

Description

DescriptionTitle of Invention: NOVEL N-BENZYL PHTHALIMIDES FOR THE TREATMENT OF PAIN. METHODS OF PREPARATION AND THERAPEUTIC USEField of Invention

[0001] The present invention is related to medicine, pharmacy and chemistry. The present disclosure is directed to methods for preventing, treating, and managing different types of acute and chronic pain, mainly neuropathic pain. The present invention comprises novel substituted N-benzyl phthalimide compounds endowed with analgesic properties and used to treat or manage acute and chronic pain, as well as their medicinal applications and compositions containing these derivatives.Background of the invention

[0002] Pain is a distressing feeling often caused by intense or damaging stimuli. The International Association for the Study of Pain defines pain as "an unpleasant sensory and emotional experience associated with or resembling that associated with actual or potential tissue damage." Pain motivates the individual to withdraw from damaging situations, to protect a damaged body part while it heals, and to avoid similar experiences in the future. Most pain resolves once the noxious stimulus is removed and the body has healed, but sometimes it may persist and become chronic despite removing the stimulus and apparent healing and becoming a chronic disease.

[0003] Pain is usually transitory, lasting until the noxious stimulus is removed or the underlying damage or pathology has healed. Still, some painful conditions, such as rheumatoid arthritis, peripheral neuropathy, cancer, and idiopathic pain, among others, may persist for years. Pain that lasts a long time is called "chronic" or "persistent," and pain that resolves quickly is called "acute." Traditionally, the distinction between acute and chronic pain has relied upon an arbitrary interval between onset and resolution; the most used markers are three months for acute pain and six months or more for chronic or persistent pain since the beginning of symptoms.

[0004] The International Classification of Diseases (ICD) of the World Health Organization (WHO) category for “Chronic Pain” comprises the most common clinically relevant disorders. These disorders were divided into seven groups: (1 ) chronic primary pain, (2) chronic cancer pain, (3) chronic posttraumatic and postsurgical pain, (4) chronic neuropathic pain, (5) chronic headache and orofacial pain, (6) chronic visceral pain, and (7) chronic musculoskeletal pain.

[0005] Neuropathic pain resulting from injuries to the nervous system due to disease, trauma, or neurotoxins poses a significant challenge in terms of treatment. Clinicians and patients frequently find themselves having to manage neuropathic pain using opioids. However, this approach is limited by the eventual tolerance and loss in efficacy, the occurrence of severe side effects with prolonged opioid use, besides the strong potential for abuse and addiction.

[0006] Pain is the main reason people seek medical care, with three of the top ten reasons being osteoarthritis, back pain, and headaches. Among the four leading causes of years lost to disability, three (back pain, musculoskeletal disorders, and neck pain) are chronic pain conditions.

[0007] Prevalence rates of chronic pain occur in all ages but are more prevalent in people aged 50 years or older. Chronic pain varies between 11 % and 40% of the population, with a study by the US Centers for Disease Control and Prevention (CDC) estimating the point prevalence at 31 %. In the UK, the chronic pain prevalence rate is 43.5%, with moderate-to-severe disabling pain ranging from 10.4% to 14.3%. In the United States alone, it is estimated that about 80 million adults experience some chronic pain. The annual cost spent in the United States to treat pain and the loss of productivity is estimated to be over 700 billion dollars. It is more significant than the annual cost to treat heart disease, cancer, or diabetes. The available therapies to treat pain, including nonsteroidal anti-inflammatory drugs, opioids, anti-depressants, local anesthetics, anti-convulsants, and antiarrhythmics, do not offer satisfactory relief for all types of acute and chronic pain. Furthermore, all the availableanalgesic drugs used in clinics have a narrow therapeutic window and produce serious side effects that limit their use.

[0008] Due to the social and economic impact of chronic pain, developing novel non-narcotic analgesics to treat chronic pain is still a high-demand necessity.Summary of the Invention

[0009] According to a first aspect of the present invention, it is provided a substituted A / -benzyl phthalimide compound of formula (I) or a pharmaceutically acceptable salt, crystal, hydrate, prodrug, metabolite or solvate thereof:

[0010] [Chem.]WhereinW, X, Y and Z are independently selected from N and C;R1 is selected from the group comprising COOH, CN or bioisosteres of carboxylate and its precursors, such as esters and amides, which can be considered prodrugs;A, B, C, and D, are independently selected from CH, CD, CR and N;R2, R6, R7, and R8, are independently selected from H, halogen, CF3, NO2, CN, D, OH, SO3H, SO2NH2, C1 -C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, OR where R is C1 -C30 alkyl, alkenyl or aryl, NR2 where each R is the same or different and each is independently selected from H, C1 - C30 alkyl, C2-C30 alkenyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, heterocycloaliphatic amines;R3 is selected from H, halogen, CF3, NO2, CN, D, OH, C1 -C30 alkyl, C2- C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, OR where R is C1 -C30 alkyl, alkenyl or aryl, NR2 where each R is the same or different and each is C1 -C30 alkyl, C2- C30 alkenyl, C3-C8 cycloalkyl or aryl, mono, di, tri or tetra substituted aryl or heteroaryl;R5 and R4 are the same or different and can be H, D, CF3, halogen, C1 - C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl or aryl; or a pharmaceutically acceptable salt thereof.

[0011] In a second aspect, it is provided a combination comprising the compound of general formula (I) and at least a second pharmaceutically active compound.

[0012] In a third aspect, it is provided a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof, or a combination as defined in the second aspect of the invention, and a pharmaceutically acceptable excipient.

[0013] In a fourth aspect, it is provided a medicament comprising a compound of formula (I) for use in treating pain in a disease or condition.

[0014] In a fifth aspect, it is provided the use of a compound of formula (I) for the manufacture of a pharmaceutical composition for treating pain in a disease or condition.

[0015] It is a sixth aspect of the present invention to provide a method for treating pain in a disease or condition, the treatment comprising administering to the patient in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), as defined above or its pharmaceutically acceptable salt, crystal, hydrate, prodrug, metabolite or solvate thereof.Brief description of the figures

[0016] [Figure 1 ] describes the stability analysis of MB-611 , MB-657, and MB-658 involved exposure to varying pH levels (2.2, 4.4, 6.8, or 7.4) and incubation in rat plasma for several time intervals (0, 0.083, 0.25, 0.5, 1 , 2, and 4 hours). A) Stability of MB-611 (at 1 pg / mL) in pH conditions of 2.2, 4.4, and 6.8. B)Stability of MB-657 (at 1 pg / mL) in pH conditions of 2.2, 4.4, 6.8, and 7.4. C) Stability of MB-658 (at 1 pg / mL) in pH conditions of 2.2, 4.4, 6.8, and 7.4. D) Plasma stability of MB-611 . E) Investigating the plasma stability of MB-657. F) Plasma stability of MB-658. Data is presented as a percentage of the compound's initial concentration (baseline) at time zero.

[0017] [Figure 2] shows the pharmacokinetic profiles for MB-611 assessed through both oral and intravenous administration in mice and rats. Blood samples were obtained from each animal at specific time points, including 0.083, 0.25, 0.5, 1 , 2, and 4 hours following intravenous administration and 0.25, 0.5, 1 , 2, 4, 8, and 24 hours after oral administration. A) Single intravenous (i.v.) dose of MB-611 (1 mg / kg, i.v.) in mice plasma; B) Single oral dose (p.o.) of MB-611 (3 mg / kg) in mice plasma; C) Single dose of MB-611 (3 mg / kg, p.o.) in mice brain; D) Single intravenous (i.v.) dose of MB-611 (1 mg / kg, i.v.) in rat plasma; B) Single dose of MB-611 (3, 10 and 30 mg / kg, p.o.) in rat plasma; C) Single dose of MB-611 (3, 10 and 30 mg / kg, p.o.) in rat brain. Each point represents the means ± standard error mean (SEM) (n = 2 to 6 mice per group).

[0018] [Figure 3] shows the pharmacokinetic assessments for MB-657 and MB- 662 conducted in mice, encompassing both oral and intravenous administration. Blood samples were drawn from the same animals at consistent intervals: 0.083, 0.5, 1 , 2, and 4 hours following intravenous administration and 0.25, 1 , 2, 4, 6, 8, and 24 hours after oral administration. A) Single intravenous (i.v.) dose of MB-657 (1 mg / kg, i.v.) in mice plasma and evaluation of MB-657 and MB-658; B) Single oral dose (p.o.) of MB-657 (3 mg / kg) in mice plasma and evaluation of MB-657 and MB-658; C) Single dose of MB-657 (3 mg / kg, p.o.) in mice and evaluation of MB-657 and MB-658 in brain; D) Single dose of MB-657 (3 mg / kg, p.o.) in mice and evaluation of MB- 657 and MB-658 in spinal cord; E) Single dose of MB-662 (3 mg / kg, p.o.) in mice and evaluation of MB-657 and MB-658 in plasma; Each point represents the means ± standard error mean (SEM) (n = 2 to 6 mice per group).

[0019] [Figure 4] indicates the effects of MB-611 (A and B), MB-657 (C and D) or MB-662 (E and F) on a neuropathy animal model induced by partial sciatic nerve ligation (PSNL). Mice were kept anesthetized and the PSNL was performed. The mechanical sensitivity represented as paw withdrawal threshold was assessed with von Frey hairs before and 10 days after the PSNL. Gabapentin was used as a reference drug. The animals that became neuropathic were treated with MB-611 (10, 30 or 60 mg / kg, p.o.), MB-657 (0.3,1 or 3 mg / kg, p.o.), MB-662 (0.3, 1 and 3 mg / kg, p.o.), gabapentin (70 mg / kg, p.o.), or with vehicle (10 ml / kg, p.o.) and mechanical sensitivity was measured 0.5, 1 , 2, 4, and 6 hours after treatment with MB-611 and 0.5, 1 , 2, 4, 6 and 8 hours after treatment with MB-657 and MB-662. Each point represents the mean ± SEM of the paw withdrawal threshold (in Log) to mechanical stimulation and the median effective dose (EDso) was determined (A, C and E). Two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test. The area under the curve (AUC) was calculated (B, D and F) and the data in the graph were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test * indicates significant statistical difference (p<0.05) when compared to vehicle group.

[0020] [Figure 5] indicates the effects of MB-611 (A and B) and MB-657 (C and D) on a neuropathy animal model induced by paclitaxel (PTX). Mice received2 mg / kg of PTX intraperitoneally in four alternate days. The mechanical sensitivity represented as paw withdrawal threshold was assessed with electronic von Frey (A) and von Frey hairs (C) before and 14 days after PTX. Gabapentin was used as a reference drug. The animals that became neuropathic were treated with MB-611 (30 mg / kg, p.o.), MB-657 (10 mg / kg, p.o.), gabapentin (70 mg / kg, p.o.), or with vehicle (10 ml / kg, p.o.) and mechanical sensitivity was measured 0.5, 1 , 2, 4, and 6 hours after treatment with MB-611 and 0.5, 1 , 2, 4, 6 and 8 hours after treatment with MB-657. In A and C, each point represents the mean ± SEM of the paw withdrawal threshold (in g for the electronic von Frey and in Log for the von Frey hairs Log) to mechanical stimulation. Two-way analysis of variance (ANOVA) wasperformed, followed by the Bonferroni post hoc test. The area under the curve (AUC) was calculated (B and D) and the data in the graph were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test.#indicates significant statistical difference (p<0.05) when compared to vehicle group. * Indicates significant statistical difference (p < 0.05) when compared to PTX + vehicle group.

[0021] [Figure 6] shows the effects of MB-611 when administered via iintrathecal (A and B) and intracerebroventricular (C and D) routes on a neuropathy animal model induced by paclitaxel (PTX). Mice received 2 mg / kg of PTX intraperitoneally in four alternate days. The mechanical sensitivity represented as paw withdrawal threshold was assessed with von Frey hairs before and 14 days after PTX. Gabapentin was used as a reference drug. To assess the analgesic effect of MB-611 administered to the spinal cord, neuropathic mice were treated with MB-611 (75 and 150 ng / site, 5 pL, i.t.), gabapentin (30,000 ng / site, 5 pL, i.t.) or with vehicle (5 pL / site, i.t.) and mechanical hypersensitivity was measured 0.25, 0.5, 1 , 2, 4, 6 and 8 hours after treatment (A and B). To assess the analgesic effect of MB-611 administered to the brain, animals that became neuropathic following PTX treatment were treated with MB-611 (150 and 300 ng / site, 5 pL, i.c.v.), gabapentin (30,000 ng / site, 5 pL, i.c.v.) or vehicle (5 pL / site, i.c.v.) and mechanical hypersensitivity was measured 0.25, 0.5, 1 , 2, 4 and 6 hours after treatment (C and D). In A and C, each point represents the mean ± SEM of the paw withdrawal threshold (in Log) to mechanical stimulation. Two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test. The area under the curve (AUC) was calculated (B and D), and the data in the graph were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test. * Indicates significant statistical difference (p<0.05) when compared to PTX + vehicle group.

[0022] [Figure 7] indicates the effects of MB-658 when administered via intrathecal (A and B) and intracerebroventricular (C and D) routes on a neuropathy animal model induced by paclitaxel (PTX). Mice received 2 mg / kg of PTX intraperitoneally in four alternate days. The mechanical sensitivityrepresented as paw withdrawal threshold was assessed with von Frey hairs before and 14 days after PTX. Gabapentin was used as a reference drug. To assess the analgesic effect of MB-658 administered to the spinal cord, neuropathic mice were treated with MB-658 (12.5, 25 and 50 ng / site, 5 pL, i.t.), gabapentin (30,000 ng / site, 5 pL, i.t.) or with vehicle (5 pL / site, i.t.) and mechanical hypersensitivity was measured 0.25, 0.5, 1 , 2, 4, 6 and 8 hours after treatment (A and B). To assess the analgesic effect of MB-658 administered to the brain, animals that became neuropathic following PTX treatment were treated with MB-658 (25, 50 and 100 ng / site, 5 pL, i.c.v.), gabapentin (30,000 ng / site, 5 pL, i.c.v.), or vehicle (5 pL / site, i.c.v.) and mechanical hypersensitivity was measured 0.083, 1 , 2, 4, 6 and 8 hours after treatment (C and D). In A and C, each point represents the mean ± SEM of the paw withdrawal threshold (in Log) to mechanical stimulation and the median effective dose (EDso) was determined. Two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test. The area under the curve (AUC) was calculated (B and D) and the data in the graph were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test. * Indicates significant statistical difference (p<0.05) when compared to PTX + vehicle group.

[0023] [Figure 8] shows the effects of MB-662 on a post-operative pain model induced by plantar incision in mice. Mice were kept anesthetized, and the incision was performed. The mechanical sensitivity represented as paw withdrawal threshold was assessed with von Frey hairs before and one day after the incision (A and B). Heat hyperalgesia was assessed on a hot-plate test and time to withdrawal (latency) was recorded after 48h post-incision, before and after treatment (C). Morphine was used as a reference drug. Animals that underwent surgery (or sham procedure) were treated with MB- 662 (10 mg / kg, p.o.), morphine (10 mg / kg, s.c.), or with vehicle (10 ml / Kg, p.o.) and mechanical sensitivity was measured at 0, 1 , 2, 4, and 6 hours after treatment with MB-662. Heat hyperalgesia was measured at 3 hours after treatment with MB-662 and 1 hour after treatment with morphine. Each point represents the mean ± SEM of the paw withdrawal threshold (in Log) aftermechanical stimulation (A) or latency to withdrawal (in seconds) after thermal stimulation (C). For mechanical sensitivity, a two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test. The area under the curve (AUC) was calculated (B), and the data in the graph were expressed as mean ± SEM. For thermal sensitivity one-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test.#indicates a significant statistical difference (p<0.05) when compared to the sham group. * Indicates significant statistical difference (p<0.05) when compared to Paw incision + vehicle group.

[0024] [Figure 9] shows the effects of MB-611 on gene expression in cortex and spinal cord samples from a neuropathy animal model induced by partial sciatic nerve ligation (PSNL). Mice were kept anesthetized and the PSNL was performed. The animals that became neuropathic were treated for 14 days with MB-611 (30 mg / kg, p.o.) or with vehicle (10 ml / kg, p.o.). Sham animals received vehicle only. After this period, cortex and sciatic nerve samples were collected and submitted for analysis by PCR array. Data shown in the graphs refer to normalized RNAm expression of (A) Ptegr4 in cortex and (B) Ccr2, (C) Ngf, (D) P2x4 and (E) P2x7 in sciatic nerve.

[0025] [Figure 10] shows the effects of MB-611 (A and B) and MB-657 (C-F) on prostaglandin E2 (PGE2)-induced mechanical hyperalgesia in rats. For testing of MB-611 , male and female rats were pretreated with MB-611 (10, 30 and 100 mg / kg, p.o.), gabapentin (70 mg / kg, p.o.) or vehicle (10 ml / kg, p.o.). Then, 1 hour after treatment, the animals received an intraplantar injection of PGE2 (100 ng / paw, 50 pL), and mechanical sensitivity was measured after 0.5, 1 , 2, 4, and 6 hours through electronic von Frey. Each point represents the mean ± SEM of the paw withdrawal threshold (in g) for mechanical stimulation (A). For testing of MB-657, male and female rats were pretreated with MB-657 (10 mg / kg, p.o.), Dipyrone (100 mg / kg, p.o.) or Vehicle (10 ml / kg, p.o.) followed 1 hour after by intraplantar injection of PGE2 as described previously. Mechanical and thermal (heat) sensitivity was assessed through the Hargreaves apparatus or electronic von Frey, respectively, before and after the injection of PGE2. Thermal sensitivity was assessed at 1 , 2, 4, 6, and 8hours after PGE2, and mechanical sensitivity was assessed at 0.5, 1 , 2, 4, 6, and 8 hours. Each point represents the mean ± SEM of latency to withdrawal (in seconds) or the paw withdrawal threshold (in g) after thermal (B) or mechanical (C) stimulation, respectively. Statistical significance was assessed through a two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test (A, C, and E). In addition, the area under the curve (AUC) was calculated, and data in the graph were expressed as mean ± SEM (B, D, and F). Statistical significance was assessed through one-way ANOVA followed by Bonferroni post hoc test. * Indicates a significant statistical difference (p < 0.05) when compared to the vehicle group.

[0026] [Figure 11 ] shows the effects of MB-657 on carrageenan-induced thermal hyperalgesia and paw edema in rats. All animals were evaluated for baseline levels of thermal sensitivity (Hargreaves apparatus) and paw volume (mL) (Plethysmometer). Next, pretreatment with MB-657 (10 mg / kg, p.o.), indomethacin (5 mg / kg, s.c.), or vehicle (10 ml / kg, p.o.) was performed. Following 1 hour, the animals received an intraplantar injection of 100 pL of carrageenan (300 pg / paw), and thermal sensitivity and paw volume were measured 1 , 3, 5, and 7 hours after treatment. Each point represents the mean ± SEM of latency to withdrawal (in seconds) or paw volume (in mL) (A and B). Two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test. The area under the curve (AUC) was calculated (B and D), and the data in the graph were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test. * Indicates a significant statistical difference (p<0.05) when compared to the vehicle group.

[0027] [Figure 12] shows the effects of MB-611 on hERG channel functional assay. HEK293 cells transfected with human hERG were plated at a density of 4 x 104cells per well in a black 96-well flat plate with a transparent bottom and incubated with 50 pL of the fluorescent probe containing probenecid in the final concentration of 2.5 mM. After 1 hour, 25 pL of solution with MB-611 (30, 100, and 300 pM) was added to the wells, and the plates were incubated for 30 minutes. Then, the previously optimized stimulus buffer (50 pL of 1 mMthallium + 10 mM potassium) was added to each column through automated pipetting, and the signal was acquired at intervals of 1.52 seconds for approximately 140 seconds per column at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The results were expressed as the percentage of inhibition of the hERG channel and the mean inhibitory concentration (IC50), and the respective 95% confidence intervals were calculated using linear regression.

[0028] [Figure 13] shows the effects of MB-657 and MB-658 on hERG channel functional assay. HEK293 cells transfected with human hERG were plated at a density of 4 x 104cells per well in a black 96-well flat plate with a transparent bottom and incubated with 50 pL of the fluorescent probe containing probenecid in the final concentration of 2.5 mM. After 1 hour, 25 pL of solution with MB-657 (0.3, 3, and 30 pM) and MB-658 (3, 30, and 300 pM) were added to the wells, and the plates were incubated for 30 minutes. Then, the previously optimized stimulus buffer (50 pL of 1 mM thallium + 10 mM potassium) was added to each column through automated pipetting, and the signal was acquired at intervals of 1.52 seconds for approximately 140 seconds per column at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The results were expressed as a percentage of inhibition of the hERG channel, and the mean inhibitory concentration (IC50) and the respective 95% confidence intervals were calculated using linear regression.Definitions

[0029] The term "prodrug," as used herein, is a pharmacologically inactive compound that can be administered and metabolized within the body to yield an active therapeutic agent. This approach allows for the circumvention of specific physiological barriers or limitations that may impede the effective delivery of the active compound. By strategically modifying the chemical structure of the prodrug, factors such as solubility, stability, and bioavailability can be optimized, leading to enhanced therapeutic outcomes.

[0030] The term "bioisostere," as used herein, refers to a structurally similar chemical or molecular entity that exhibits comparable biological activity to atarget compound. These bioisosteres are strategically designed to mimic the physicochemical properties of the original compound while often possessing improved pharmacokinetic or pharmacodynamic characteristics.

[0031] The term "alkyl," as used herein, refers to an unbranched or branched chain, saturated, monovalent hydrocarbon residue containing 1 to 30 carbon atoms. Examples of alkyl groups include but are not limited to, lower alkyl groups, including methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t-butyl, or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

[0032] The term "alkenyl," as used herein, refers to an unsubstituted hydrocarbon chain radical having from 2 to 10 carbon atoms having one or two olefinic double bonds, preferably one olefinic double bond. Examples include but are not limited to, vinyl, 1-propenyl, 2-propenyl (allyl), or 2-butenyl (crotyl).

[0033] The term "heterocycle," as used herein, refers to an unsubstituted or substituted heterocycle containing carbon, hydrogen, and at least one of N, O, and S, where the C and N can be trivalent or tetravalent, i.e., sp2or sp3hybridized. Examples of heterocycles include, but are not limited to, aziridine, azetidine, pyrrolidine, piperidine, imidazole, oxazole, piperazine, etc.

[0034] The term "alkynyl," as used herein, refers to an unbranched or branched hydrocarbon chain radical with 2 to 10 carbon atoms, preferably 2 to 5 carbon atoms, and one triple bond. Examples include, but are limited to, ethynyl, -1 propynyl, 2-propynyl, 1 -butynyl, 2-butynyl, or 3-butynyl.

[0035] The term "cycloalkyl," as used herein, refers to a saturated carbocyclic ring comprising 3 to 8 carbon atoms, i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl.

[0036] The term "aryl," as used herein and unless otherwise specified, refers to substituted or unsubstituted phenyl (Ph), biphenyl, or naphthyl, preferably the term aryl refers to substituted or unsubstituted phenyl. The aryl group can be substituted with one or more moieties selected from among hydroxyl, F, Cl, Br, I, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate, either unprotected or protected as necessary, as known to those skilled in the art,

[0037] The term "amino acid," as used herein, includes naturally occurring and synthetic a, 0, y, or 6 amino acids and includes, but is not limited to, amino acids found in proteins, i.e., glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine, and histidine. In a preferred embodiment, the amino acid is in the D or L- configurations.

[0038] The term “allodynia,” as used herein, refers to a condition in which a normally non-painful stimulus or sensation, such as light touch or a gentle brush against the skin, elicits a painful response. In other words, allodynia is a perception of pain or discomfort in response to a stimulus that should not typically be painful. This increased sensitivity to non-painful stimuli is often associated with various pain-related conditions, including neuropathic pain, fibromyalgia, and certain types of chronic pain syndromes. Allodynia can manifest as a wide range of sensations, such as burning, tingling, or aching.

[0039] The term “single enantiomer”, as used herein, is intended to include a compound that comprises more than 50% of a single enantiomer (i.e., enantiomeric excess more than 60%, more than 70%, more than 80%, more than 90%, or up to 100% pure enantiomer).

[0040] The term “pharmaceutically acceptable,” as used herein, means that the referenced substance, such as a compound of the present invention or a formulation containing a compound of the present invention or a particular excipient, is suitable for administration to a patient. Moreover, the term “pharmaceutically acceptable salt”, as used herein, is intended to include nontoxic salts prepared from the parent compound, which contains a basic or acidic moiety by conventional chemical methods.

[0041] The term “central pain,” as used herein, refers to a subset of chronic pain that originates in the central nervous system (CNS), which includes the brain and spinal cord. This type of pain is often the result of damage or dysfunction within the CNS itself.

[0042] The term “hyperalgesia,” as used herein, refers to an increased sensitivity to painful stimuli. It refers to a condition where a person experiences more intense pain in response to a stimulus that would typically be mildly painful. In other words, hyperalgesia is an exaggerated or increased response to painful sensations resulting from damaged peripheral pain fibers, and it can be categorized as primary or secondary.

[0043] The term “neuropathic pain,” as used herein, refers to pain caused by a lesion or disease of the somatosensory nervous system. Neuropathic pain is defined as pain resulting from injury to, or dysfunction of, the somatosensory system.

[0044] The term “nociception,” as used herein, refers to the neural responses of encoding and processing noxious stimuli.

[0045] The term “therapeutically effective amount”, as used herein, means an amount of a compound that ameliorates, attenuates or eliminates one or more symptoms of a particular disease or condition or prevents or delays the onset of one or more symptoms of a specific disease or condition.

[0046] The term "patient,” as used herein, may refer to animals such as dogs, cats, cows, horses, sheep, or humans. Preferred patients are mammals. The term patient includes males and females.

[0047] The terms “treating,” “treat1or “treatment,” and the like include preventative (e.g., prophylactic) and palliative treatment.

[0048] The term “patient in need thereof,” as used herein, refers to a patient who has or is at risk of having a disease and / or condition that results in pain, such as chronic pain.

[0049] The terms “active compound” or “active ingredient”, as used interchangeably herein, refer to a compound of the present invention or its pharmaceutically acceptable salt used as the active pharmaceutical ingredient in a given pharmaceutical composition.

[0050] The term "excipient,” as used herein, means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or an ingredient other than theactive pharmaceutical ingredient, which is typically included for formulation and / or administration to a patient.

[0051] The term "parenteral administration", as used herein, refers to modes of administration other than enteral and topical, usually by injection or infusion, and includes, without limitation, subcutaneous, intraperitoneal, intrathecal, intraarticular, intramuscular, and intravenous administration.

[0052] The term “combined therapy,” as used herein, refers to two or more pharmaceutically active ingredients administered simultaneously or in any order to a patient, intending to treat the same or different symptoms of a disease or condition, intending to treat different diseases or conditions that may occur concurrently, or intending to modify, increase or reduce effects associated with one or more ingredients administered.Detailed Description of the Invention

[0053] The present invention provides compounds of general formula (I) or a pharmaceutically acceptable salt, crystal, hydrate, prodrug, metabolite or solvate thereof:

[0054] [Chem.]wherein:• X, Y, Z, and W are independently selected from N or C;• A, B, C, and D are independently selected from C or N;• R1 is COOH, CN or bioisostere of carboxylate and its precursors, such as esters and amides;• R2, R6, R7, and R8 are independently selected from H, halogen, CF3, NO2, CN, D, OH, SO3H, SO2NH2, C1 -C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, OR where R is C1-C30 alkyl, alkenyl or aryl, NR2 where each R is the same or different and each is independently selected from H, C1 -C30 alkyl, C2-C30 alkenyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl and heterocycloaliphatic amines;• R3 is selected from H, halogen, CF3, NO2, CN, D, OH, C1 -C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, OR where R is C1 -C30 alkyl, alkenyl or aryl, NR2 where each R is the same or different and each is independently selected from H, C1 -C30 alkyl, C2-C30 alkenyl, C3-C8 cycloalkyl or aryl, mono, di, tri or tetra substituted aryl or heteroaryl;• R5, R4 are the same or different and can be H, D, CF3, halogen, C1 -C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl or aryl.

[0055] The present invention also provides pharmaceutical compositions comprising compounds of formula (I) or pharmaceutically acceptable salts thereof, a medicament, the use of the compound of formula (I) and methods of treating diseases and / or conditions, such as pain, using compounds of formula (I) or pharmaceutically acceptable salts thereof.

[0056] In one embodiment, the bioisosteres of carboxylate and its precursors, such as esters and amides of R1is optionally selected from COOR, where R is C1-C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, Cs-Cs cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, which can be considered as prodrugs of their respective carboxylic acid.

[0057] In another embodiment, the bioisosteres of carboxylate and its precursors, such as esters and amides of R1is optionally selected from CONR9R10where each R9and R10is the same or different, and each is H, C1-C30 alkyl, C2-C30 alkenyl, Cs-Cs cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, amino acid or amino acid derivative, which can be considered as prodrugs of respective carboxylic acid.

[0058] In another embodiment, the compound of formula (I) is selected from the group comprising of:

[0059] [Chem. Group]

[0060] In one embodiment, the compound or suitable salt, crystal, hydrate, prodrug, metabolite or solvate thereof of formula (I) can have at least one stereocenter; the compound or suitable salt can exist in the racemic form, in the form of its pure optical isomers, or in the form of a mixture wherein one isomer is enriched relative to the other in any proportion.

[0061] Suitable bases include inorganic bases such as alkali and earth metal bases, such as those containing metallic cations, such as sodium, potassium, magnesium, or calcium. Non-limiting examples of suitable bases include sodium and potassium hydroxides, carbonates, and the like. Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids such as p-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, maleic acid, tartaric acid, fatty acids, long chain fatty acids, and the like. The compounds of the present invention containing an acidic or basic moiety are efficacious in the form of the free base or acid or the form of a pharmaceutically acceptable salt thereof.

[0062] The counterion forming a part of any salt of this invention is usually not critical since the salt is pharmacologically acceptable if the counterion does not contribute undesired qualities to the salt. Pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, or allergic response and are commensurate with a reasonable benefit / risk ratio. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-1 9 (1977).

[0063] It is further understood that the above compounds and salts may form solvates or exist in a substantially uncomplexed form, such as the anhydrousform. As used herein, "solvate" refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice. When the solvent contained in the solvate is water, the molecular complex is called a hydrate. Pharmaceutically acceptable solvates include hydrates, alcoholates, aceton itrilates, and the like. These compounds can also exist in polymorphic forms.

[0064] The compounds of the present invention may be used to manufacture a medicament to treat a disease or condition, such as pain. The treatment involves administering a pharmaceutical composition comprising a compound of the present invention, or its pharmaceutically acceptable salt, to a patient in need. The preferable type of disease or condition to be treated includes acute and chronic pain and can be selected without limitation from the following group: neuropathic pain, inflammatory pain, visceral pain, cancer pain, chemotherapy pain, trauma pain, surgical pain, post-surgical pain, childbirth pain, labor pain, chronic pain, persistent pain, peripherally mediated pain, centrally mediated pain, chronic headache, migraine headache, sinus headache, tension headache, phantom limb pain, dental pain or peripheral nerve injury.

[0065] In another aspect, the compounds of the present invention may also be used to treat pain in any disease or condition in which pain exists as a symptom. Such diseases or conditions include, but are not limited to, the following group: HIV, HIV treatment-induced neuropathy, trigeminal neuralgia, post-herpetic neuralgia, eudynia, primary or secondary otalgia, ulcerative colitis, heat sensitivity, sarcoidosis, irritable bowel syndrome, Crohn’s disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), diabetic neuropathy, peripheral neuropathy, arthritis, rheumatoid arthritis, osteoarthritis, osteoporosis, atherosclerosis, paroxysmal dystonia, myasthenia syndromes, myotonia, malignant hyperthermia, lupus, kidney stones, cystic fibrosis, pseudoaldosteronism, rhabdomyolysis, hypothyroidism, depression, anxiety, schizophrenia, sodium channel toxin-related illnesses, familial erythromelalgia, primary erythromelalgia, paroxysmal extreme pain disorder, cancer, epilepsy, partial and general tonic seizures, restless leg syndrome,arrhythmias, fibromyalgia, ischemic conditions caused by stroke or neural trauma, tachycardia, atrial fibrillation, ventricular fibrillation or a combination thereof.

[0066] In another aspect, some methods of use of compounds of the present invention require formulating said compounds in a pharmaceutically acceptable formulation suitable for the desired route of administration. The pharmaceutically acceptable formulation is typically prepared by mixing a compound of the present invention and a diluent, carrier or excipient, and is limited only by physicochemical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration. It is appreciated by one of skill in the art that the compounds of the present invention can be administered to a patient through different routes of administration, which includes oral, dental, topical, rectal, vaginal, urethral, otic (middle and inner ear), transdermal, subcutaneous, intraperitoneal, inhalatory, intranasal, intrapulmonary, intrathoracic, intratracheal, transtracheal, intradermal, intrathecal, epidural, intraspinal, intratendinous, intraosseous, intraarticular, intrabursal, intralesional, intramuscular, intravenous, intraarterial, intracardiac, intragingival, intracerebral, subarachnoid, intraocular, intracapsular or subcapsular administration, in single or multiple dosages. All methods that are used by those skilled in the art to administer a pharmaceutically active ingredient are contemplated.

[0067] Other than one or more active ingredients, pharmaceutically acceptable formulation components described herein, such as vehicles, adjuvants, excipients, or diluents, are well-known and readily available to those skilled in the art. It is preferred that a selected formulation component be chemically inert to the active compounds and does not cause detrimental side effects or toxicity under the conditions of use. The choice of components will be determined partly by the pharmaceutically active ingredient and the method used to administer the pharmaceutical composition. Accordingly, there is a wide variety of suitable formulations related to the present invention for a pharmaceutical composition. The following formulations for oral, topical, inhalatory, subcutaneous, intravenous, intraarterial, intramuscular,intraperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting.

[0068] Dosage forms suitable for oral administration of a compound of the present invention may include liquid, solid and semisolid forms. Liquid dosage forms include solutions, emulsions, colloids, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1 ,3-butylene glycol, dim ethylformamide, oils (in particular, cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil, and sesame seed oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances, and the like. Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Suspensions, in addition to the active compound, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, or mixtures of these substances, and the like.

[0069] Solid dosage forms for oral administration include tablets, capsules, pills, powders, granules, draggers, lozenges, and troches. Semisolid dosage forms may include pastes and gels. In such solid and semisolid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate, and / or any of the following: (a) fillers or extenders, as for example, starches, lactose, sucrose, mannitol, and silicic acid; (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, as for example, glycerol; (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds;(g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, such as kaolin and bentonite; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. The dosage forms may also comprise buffering agents in the case of capsules and tablets. Solid compositions may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols, and the like.

[0070] Solid dosage forms such as tablets, draggers, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may also be formulated to provide slow or controlled release of the active ingredient(s) therein, using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes, and / or microspheres. These compositions may also contain opacifying agents and can also be of such composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract and optionally in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. If appropriate, the active compounds can also be in microencapsulated form with one or more of the above-mentioned excipients.

[0071] To those of skill in the art, it is well-known that the compounds of the present invention may also be prepared into topical formulations. Dosage forms for topical administration include creams, ointments, gels, lotions, pastes, foams, patches, plasters, sprays, suppositories, and liquid solutions such as eye drops or ear drops. The active compound is admixed with a physiologically acceptable excipient and any preservatives or buffers that may be required.

[0072] The topical formulations may also contain other substances, including but not limited to, those known by one skilled in the art as useful to increase or extend absorption or penetration of the active ingredient through the skin or other affected areas. Topical / transdermal drug delivery systems or devicesmay also be used. One example is the use of patches, whose components include, for example, a backing, an active compound reservoir, a control membrane, a liner, and a contact adhesive. Such transdermal patches may be used to provide continuous pulsatile or on-demand delivery of the compounds of the present invention as desired. Ophthalmic formulations such as ointments, powders, sprays, solutions, and suspensions are also contemplated as being within the scope of this invention.

[0073] The compounds of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations include the use of acceptable pressurized propellants, such as hydrofluoroalkane or similarly suitable substances, and administration through a medical device such as an inhaler, which may include, for example, meter-dosed inhalers, dry powder inhalers or soft mist inhalers. Compounds of the present invention may also be formulated for non-pressured applications, such as in a nebulizer or an atomizer.

[0074] Formulations suitable for parenteral administration (comprising, for example, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal or intrathecal administration), include aqueous and nonaqueous sterile injection solutions, suspensions, colloids or emulsions, and sterile powder for reconstitution into a sterile injection solution These may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, in addition to suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compounds can be administered in a physiologically acceptable diluent in a pharmaceutical vehicle, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose or suitable related sugar-based solutions, alcohols, ethers, oils, fatty acid, fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, or emulsifying agents and other pharmaceutical adjuvants. The requirements for effective pharmaceutical vehicles for injectable compositions are well known to those of ordinary skill inthe art. See, for example, Pharmaceutics and Pharmacy Practice, J B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622- 630 (1986).

[0075] Additionally, the compounds of the present invention may be prepared into suppositories for rectal or vaginal administration by mixing the compounds with suitable non-irritating excipients or carries such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound(s). Other formulations suitable for vaginal or rectal administration may exist as suspensions, solutions, creams, gels, ointments, pastes, foams, sprays, or other aforementioned forms containing, in addition to one or more pharmaceutically active ingredients, such carriers as are known in the art to be appropriate. It is also contemplated within the scope of the invention the use of techniques to promote sustained release of the compounds into the vaginal or rectal cavity, which can be achieved, for example, through suppositories with higher melting point excipients, by increasing the viscosity of the melted base, or by incorporating polymers that slow drug diffusion.

[0076] The dose of a compound or its pharmaceutically acceptable salt administered to a patient, in accordance with the present invention, should comprise an amount considered enough to achieve the desired response. Such response includes the reversal and / or prevention of the occurrence / recurrence of different manifestations of pain, such as neuropathic pain. One skilled in the art can recognize that dosage depends upon a variety of factors, including the patient's age, condition, and body weight, as well as the source of the disease and extent of the disease in said patient. The dose size is also determined by the route, timing, and frequency of administration, as well as the existence, nature, and extent of any side effects and the desired physiological effect. Those skilled in the art are familiar with a range of general considerations considered when determining the 'effective amount', all of which are thoroughly described. See, for example, Gilman et al., eds.,Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference. It is also appreciated by those skilled in the art that various conditions or diseases may require different routes of administration or prolonged treatment involving multiple administrations.

[0077] Typically, doses will be in the range of 0.0001 mg / kg to 500 mg / kg of the subject's body weight, and more typically between about 0.0005 mg / kg and 150 mg / kg, such as from about 0.002 to about 100 mg / kg, from about 0.5 to about 50 mg / kg or from about 1 to about 40 mg / kg of the subject's body weight. Thus, unit dosage forms can be formulated based on the suitable ranges and the subject's body weight. The term "unit dosage form" as used herein, refers to a physically discrete unit of therapeutic agent appropriate for the issue to be treated.

[0078] In one aspect of topical applications, it is desired to administer an effective amount of a pharmaceutical composition, according to the invention, to a target area, e.g., skin surfaces, mucous membranes, and the like, which are adjacent to peripheral neurons that are to be treated. This amount will generally range from about 0.0001 mg to about 1 g of a compound of the invention per application, depending upon the area to be treated, whether the use is diagnostic, prophylactic, or therapeutic, the severity of the symptoms, and the nature of the topical vehicle employed. A preferred topical preparation is an ointment, wherein about 0.001 to about 50 mg of active ingredient is used per cc of ointment base.

[0079] In another aspect of the invention, the compounds of the present invention may be administered alone, in combination with other compounds of the present invention, or with one or more other pharmaceutically active ingredients in a combined therapy regimen. The other pharmaceutically active ingredients can be intended to treat the same disease or condition as the compounds of the present invention or a different disease or condition. The other pharmaceutically active ingredients may also be intended to modify theeffects of a compound of the present invention to increase the presence or duration of desired effects or decrease the presence or duration of undesired effects. If the patient is to receive or is receiving multiple pharmaceutically active compounds, the compounds can be administered simultaneously or sequentially. For example, in the case of tablets, the active compounds may be found in one tablet or in separate tablets, which can be administered at once or sequentially in any order. In addition, it should be recognized that the compositions might be in different forms. For example, one or more compounds may be delivered by a tablet, while another is administered by injection or orally as a syrup. All combinations, delivery methods, and administration sequences are contemplated. In addition, it is noted that the terms “pharmaceutically active compounds” and “pharmaceutically active ingredient” may also include biologies, such as proteins, antibodies, and peptides.

[0080] In another aspect of the invention, the compounds of the present invention may be used in combination with other pharmaceutically active compounds that are used to treat pain and / or inflammation, such as non-steroidal antiinflammatory drugs (NSAIDS), steroidal anti-inflammatory drugs, cyclooxygenase inhibitors, and opioid analgesics. Examples of such other compounds include, but are not limited to, aspirin, buphenorphine, celecoxib, diclofenac, hydrocodone, indomethacin, oxycodone, methadone, codeine, fentanyl, ibuprofen, ketoprofen, flurbiprophen, naproxen, acetaminophen, gabapentin, pregabalin, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, mometasone, budesonide, mefenamic acid, etoricoxib, acetylsalicylic acid, ketorolac, meloxicam, tenoxicam, piroxicam, etodolac, sulindac, diflunisal, tolmetin, fenoprofen, nabumetone, acemetacin, amfenac, ampiroxicam, emorfazone, epirizole, ethezamide, flufenamate, lonorxicam, loxoprofen, mofezolac, nabumetone, oxaprozin, pranoprofen, salicylamide, sodium salicylate, tiaprofenic acid, tiaramide, zaltoprofen, proglumetacin, fentiazac, cinnoxicam, nimesulide, aceclonenac, hydrocortisone, triamcinolone and deflazacort.

[0081] In another aspect of the invention, the compounds of the present invention can be administered in combination with other pharmaceutically active compounds intended to treat diseases or conditions where pain exists as a symptom. Examples of such diseases or conditions may include, without limitation, HIV, HIV treatment-induced neuropathy, sarcoidosis, irritable bowel syndrome, Crohn’s disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), ulcerative colitis, diabetes, arthritis, rheumatoid arthritis, osteoarthritis, osteoporosis, atherosclerosis, paroxysmal dystonia, myasthenia syndromes, myotonia, malignant hyperthermia, lupus, kidney stones, cystic fibrosis, pseudoaldosteronism, rhabdomyolysis, hypothyroidism, depression, anxiety, schizophrenia, familial erythromelalgia, primary erythromelalgia, paroxysmal extreme pain disorder, cancer, epilepsy, partial and general tonic seizures, restless leg syndrome, arrhythmias, fibromyalgia, ischemic conditions caused by stroke or neural trauma, tachycardia, atrial fibrillation, ventricular fibrillation or a combination thereof.

[0082] In another aspect of the invention, compounds of the present invention can be administered in combination with different classes of pharmaceutically active ingredients. These include, without limitation, analgesics, antibiotics, anticoagulants, antiemetics, antipsychotics, antipyretics, antidepressants, antidiabetic agents, anticancer agents, antiepileptic agents, antifungals, antihistamines, antihypertensive agents, anti-inflammatory agents, antivirals, bronchodilators, diuretics, hormone replacement therapy, immunosuppressants, laxatives, mood stabilizers, statins, sedatives or hypnotics, anxiolytics, and stimulants.

[0083] Since aspects of the present invention contemplate the treatment of disease / conditions with a combination of pharmaceutically active compounds that may be administered separately, the invention further relates to combining separate pharmaceutical compositions in kit form. The kit comprises two separate pharmaceutical compositions: a compound of the present invention and a second pharmaceutical compound. The kit comprises a container containing separate compositions, such as a divided bottle or a divided foil packet. Additional examples of containers include syringes, boxes, and bags.Typically, the kit comprises directions for the use of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral, and parenteral), are administered at different dosage intervals, or when the prescribing physician or veterinarian desires titration of the combination's individual components.

[0084] An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for packaging pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses, and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil, which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably, the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses, whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed by said opening.

[0085] It may be desirable to provide a memory aid on the kit, for example, in the form of numbers next to the tablets or capsules, whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested. Another example of such a memory aid is a calendar printed on the card, and other variations of memory aids will be readily apparent. A "daily dose” can be a single tablet or capsule or several pills or capsules to be taken on a given day. Also, a daily dose of a compound of the present invention can consist of one tablet or capsule, while a daily dose of the second compound can consist of several tablets or capsules and vice versa. The memory aid should reflect this and aid in the correct administration of the active agents.

[0086] In another aspect of the invention, a dispenser designed to dispense the daily doses one at a time in the order of their intended use is provided. Preferably, the dispenser is equipped with a memory-aid to further facilitate regimen compliance. An example of such a memory aid is a mechanical counter, which indicates the number of daily doses that have been dispensed. Another example of such a memory aid is a battery-powered microchip memory coupled with a liquid crystal readout, or audible reminder signal, which, for example, reads out the date that the last daily dose has been taken and / or reminds one when the next dose is to be taken.

[0087] The formulations containing compounds of the present invention, whether alone or in combination with other pharmaceutically active ingredients, can be packaged in unit-dose or multi-dose containers.

[0088] Those skilled in the art will recognize that the compound names and structures contained herein may be based on a particular tautomer of a compound. While the name or structure for only a particular tautomer may be used, all tautomers are intended to be encompassed by the present invention unless stated otherwise. It is also intended that the present invention encompass compounds that are synthesized using laboratory techniques, such as those well-known to synthetic chemists. The present invention also includes isotopically-labeled compounds, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, Such as 2H, 3H, 13C, 14C, 15N, 160, 170, 31 P, 32P, 35S, 18F and 36C In

[0089] Some of the compounds disclosed herein also can be known by the codes described in Table I.

[0090] General methods for providing the compounds of the present invention are well known in the art or described in the chemical literature, using the methods described herein or by a combination thereof.

[0091] General synthetic schemes for preparing representative compounds of the present invention are described below. These schemes are illustrative and are not meant to limit the possible techniques one skilled in the art may use to prepare the compounds disclosed herein.

[0092] Different methods to prepare the compounds of the present invention will be evident to those skilled in the art. Additionally, the various steps in the synthesis may be performed in an alternate sequence to give the desired compound or compounds.

[0093] The following abbreviations are used in the synthetic schemes detailed herein:DCM: Dichloromethane;Py: Pyridine;THF: tetrahydrofuran;EtOH: Ethanol;MeOH: Methanol;BnOH: Benzyl alcohol; iPrOH: Isopropyl alcohol;MeCN: AcetonitrileTEA: Triethylamine;DIAD: Disopropyl azodicarboxylate;PPh3: Triphenylphosphine;LiAIH4: Lithium aluminium hydride;PTSA: p-Toluenesulfonic acid;CSA: Camphorsulfonic acid;NBS: N-Bromosuccinimide;AIBN: Azobisisobutyronitrile;DMAP: 4-Dimethylaminopyridine;DIC: N,N'-Diisopropylcarbodiimide;AcOEt: Ethyl Acetate;DMF: Dimethylformamide;MW: Microwave assisted reaction;IC50 = Half-maximal inhibitory concentration;ED50 = Median effective dose;Schemes 1 -7 depict exemplary syntheses of embodiments of the invention.

[0094] Scheme 1 : Reagents and Conditions: (a) NBS, AIBN(cat), AcOEt, 65°C, 6 h, 75% (2A) or 68% (2B) or 73% (2C) or 70% (2D) or 77% (2E); (b) BnOH, DMAP, DIC, DCM / THF, 25°C, 6 h, 76% (3A) or 70% (3B) or 68% (3C) or 65%(3D) or 75% (3E); (c) 4A-D, K2CO3, Kl(cat), DMF, 25°C, 4 h, 85% (5A) or 80%(5B) or 80% (5C) or 82% (5D) or 76% (5E) or 78% (5F) or 72% (5G) or 82%(5H); (d) Pd / C, H2, THF / MeOH, 25°C,18 h, 98% (7A) or 90% (7D) or 99% (7H) or 88% (71); (e) SnCk, DCM, 40°C, 4 h, 86% (6B) or 75% (6C) or 82% (6E) or92% (6F) or 78% (6G).

[0095] Scheme 2: Reagents and Conditions: (a) 9, H2SO4, 0°C - 25°C, 4 - 12 h, 82% (10A) or 65% (10B) or 78% (10C) or 62% (10D) or 58% (10E) or 73% (10F) or 42% (10G) or 47% (10H); BCI3, DCM, -78°C - 0°C, 4h, 65% (101).

[0096] Scheme 3: Reagents and Conditions: (a) 11A-E, Na2COs, 10 mol%Pd(PPh3)4, dioxane, 100 °C, 24 h, 53%(12A) or 40% (12B) or 44% (12C) or 53% (12D) or 53% (12E) or 62% (12F); (b) Pd / C, H2, THF / MeOH, 25°C,18 h,87%(13A) or 74% (13C) or 68% (13D) or 32% (13E) or 62% (13F); (c) SnCI4,DCM, 40°C, 4 h, 88% (14B).

[0097] Scheme 4: Reagents and Conditions: (a) 15A-C, 10 mol% XPhos, 1052% (15C); (b) Pd / C, H2, THF / MeOH, 25°C,18 h, 68% (17A) or 57% (17B) or65% (17C).

[0098] Scheme 5: Reagents and Conditions: (a) SOCI2, 76°C, 18 h, MeOH, TEA 0°C, 1 h, 95%; (b) NaBH4, MeOH, 0°C, 4 h, 60%; (c) 4A-B, PPh3, DIAD, THF, 0 - 25°C, 65% (21 A) or 67% (21 B); (d) HCI, THF: H2O, 85°C, 95%; (e) H2SO4, EtOH for 23A or / PrOH for 23B, reflux, 18 h, 80% (23A) or 75% (23B).[for 24B, reflux, 20 h, 87% (24A) or 82% (24B); (b) SOCI2, THF, 50°C, 2h, then NH4OH for (25A) or morpholine and TEA for (25C) or piperidine and TEA for (25D), 0°C - 25°C, 12 h, 25% (25A) or 50% (25C) or 20% (25D); (c)CICOCOCI, MeCN, O=PPh3, TEA, 25°C, 33%; (d) DMF, SOCI2, 150°C, 23 h, 15%.

[0100] Scheme 7: Reagents and Conditions: (a) 1. SOCI2, EtOH,70% (28); (b)NaBH4MeOH 0°C 3h. 68 % (29); (c) 1. SOCI2, 2. Phthalimide, K2CO3, DMF, 65% (30); (d) boronic acid, Pd(PPh3)4, K2CO3, DMF, MW, 1.5h, 65% (32A) or 68% (32B) or 56% (32C) or 71 % (32D) or 62% (32E) or 46% (32F) or 82% (32G) or 54% (32H) or 53% (32I) or 65% (32J); (e) 4 eq. Fe°, 0.6 eq NH4CI, EtOH, 85°C, 2h, 95% (32K)Examples

[0101] The examples presented below illustrate specific embodiments of the present invention. These examples are meant to be representative and are not intended to limit the scope of the claims in any manner.

[0102] [Table 1 ] The following codes can also name some substances in this present invention in a preferred embodiment.Method

[0103] Reagents and instrumentation. All reagents and solvents were purchased from Sigma-Aldrich, Comb-Blocks, TCI Chemicals, Alfa Aesar, and Chempex. The1H NMR spectra were recorded at 80, 400, 500, and 600 MHz for 1 H in Broker Fourier 80, 400 MHz Broker, 500 MHz Broker, and Varian 600 MHz. Chemical shifts for1H are reported in ppm (5) relative to the residual signals for the solvents (5 = 2.50 for DMSO-cfe, 5 = 3.31 for CDsOD and 5 = 7.26 for CDCI3). Chemical shifts for13C are reported in ppm (5) relative to the signal of the deuterated solvent (5 = 39.52 for DMSO-cfe, 5 = 49.00 for CD3OD and 5 = 79.16 for CDCI3). The following abbreviations explained the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet, sept= septet. TLC analysis was performed on aluminum sheets precoated with silica gel F254 (0.2 mm) from Sigma-Aldrich. The purity of compounds was corroborated by High-Performance Liquid Chromatography (LC- 20AD / Shimadzu®) and prepared through dilution of mixture reaction or the final compound in ACN / 10mL 95% H2O / ACN. The injection volume was 10 pL, flow of 1.2 mL / min and the mobile phase was gradient 5-100% of two phases: phase B [90% ACN / 10% H2O (0,1 % TFA)] and phase A [90% H2O (0,1 % TFA) / 10% ACN], with an oven temperature of 30 °C on a column XTerra RP18 (250 x 4.6 mm 5pm). The melting was obtained in Fisatom® apparatus or FP62-Mettler (Mettler Toledo®). Peaks were detected by UV absorption with a diode array detector at 230, 254, and 280 nm. Al derivatives tested for biological activity showed >98.5% purity by HPLC analysis (detection at 254nm). The mass spectra were obtained on Liquid Chromatography tandemMass Spectrometry (LCMS-8045 / Shimadzu®) using ESI as ionization method. Infrared (IR) spectra were recorded on a FT-IR Spirit-T Spectrometer (Shimadzu®).Purity analysis

[0104] The purity of compounds was corroborated by High-Performance Liquid Chromatography (LC-20AD / Shimadzu®) and prepared through dilution of mixture reaction or the final compound in ACN / 10mL 95% H2O / ACN. The injection volume was 10 pL, flow of 1.2 mL / min and the mobile phase was gradient 5-100% of two phases: phase B [90% ACN / 10% H2O (0,1 % TFA)] and phase A [90% H2O (0,1 % TFA) / 10% ACN], with an oven temperature of 30 °C on a column XTerra RP18 (250 x 4.6 mm 5pm).

[0105] The melting points f were obtained in Fisatom® apparatus and the starting materials were purchased from commercial suppliers and used without further purification.Example 1 - General procedure for the synthesis of 3-((1,3- dioxoisoindolin-2-yl)methyl)benzoic acid derivatives 6 and 7.

[0106] Step a: To a 250 ml round bottom flask equipped with a magnetic string bar charged with benzoic acid 1 A-E (367 mmol), AIBN (1 ,62g, 18,3 mmol) and NBS (98g, 550 mmol) was added the 500 mL of concentrated ethyl acetate,the reaction refluxed in an oil bath for 6 hours. The reaction temperature was brought to it, followed by adding 200 ml of brine and stirring for one hour. The organic phase was separated, and the aqueous phase was twice extracted with ethyl acetate (2x 50 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. 75% 2A) or 68% (2B) or 73% (2C) or 70% (2D) or 77% (2E)

[0107] Step b: To a 500ml round bottom flask equipped with a magnetic stirring bar was added the 3-(bromomethyl) benzoic acid 2A-E (47 mmol), benzyl alcohol (11 ,8 mL, 118 mmol). The reaction was stirred for 6h at 30 °C. After cooling to r.t. the solvent was removed from the reaction mixture in vacuo. Ethyl acetate (100 mL) was added to the residue, and the organics were washed with H2O (50 mL) and saturated aqueous NaHCOs (2 x 50 mL). The organic layer was washed with brine (50 mL) and dried over Na2SO4, filtered and concentrated in vacuo. 76% (3A) or 70% (3B) or 68% (3C) or 65% (3D) or 75% (3E)

[0108] Step c: To a 250ml round bottom flask equipped with a magnetic stirring bar charged with 3-(bromomethyl)benzoate 3a-e (32 mmol), K2CO3 (11 ,32g , 81 mmol), KI (543 mg, 3,8 mmol) and phthalimide 4A-D (48 mmol) followed by the addition of 160ml of DMF. The reaction was stirred for 4h at rt, followed by the addition of 300 ml of ethyl acetate the organic phase was washed with brine (100 ml) and thrice whit H2O (3 x 50 mL) and dried over Na2SO4, filtered and concentrated in vacuo. The pure product was obtained by recrystallization in ethanol. 5A) or 80% (5B) or 80% (5C) or 82% (5D) or 76% (5E) or 78% (5F) or 72% (5G) or 82% (5H)

[0109] Step d: Compound 5A-H (0,4mmol) was charged in a 10 ml flask equipped with a stirring bar, followed by 2ml of methanol and Pd / C 10% (46 mg 0,04mmol). The reaction flask was capped and the atmosphere replaced by H2. The reaction was stirred overnight with two hydrogen balloons at room temperature. The reaction was dissolved in 30ml of methanol / THF (1 :1 ) and filtered through a one-inch silica pad and concentrated under reduced pressure. The resulting solid was recrystallized in hot ethanol.

[0110] Step e: Compound 5A- H (0,4mmol) was charged in a 10 ml flask equipped with a stirring bar, followed by 2ml of DCM and SnCk (0,56 ml, 4,85mmol). The reaction flask was. The stirred at 40 °C for 4h. The reaction was dissolved in 20ml DCM, the organic phase was washed with brine (100 ml) and twice with H2O (3 x 50 mL) and dried over Na2SO4, filtered and concentrated in vacuo. The pure product was obtained by recrystallization in ethanol.3-bromo-5-((1,3-dioxoisoindolin-2-yl)methyl)benzoic acid 6B

[0111] White solid (128 mg, 90%); mp. 255 °C;1H NMR (80 MHz, DMSO) 5 8.03 (d, J = 2.2 Hz, 1 H), 7.90 (m, 4H), 7.68 (dd, J = 8.4, 2.2 Hz, 1 H), 7.18 (d, J = 8.4 Hz, 1 H), 5.11 (s, 2H); [M + H]+= 360.0 m / z3-((1,3-dioxoisoindolin-2-yl)methyl)-5-fluorobenzoic acidQC

[0112] White solid (98mg, 82%); mp. 235-240 °C (dec);1H NMR (80 MHz, DMSO- d6) 5 7.89 (d, J = 1.2 Hz, 4H), 7.71 (d, J = 1.5 Hz, 1 H), 7.52 (td, J = 7.9, 2.1 Hz, 2H), 4.85 (s, 2H).; [M + H]+= 300.1 m / z.2-bromo-3-((1,3-dioxoisoindolin-2-yl)methyl)benzoic acid 6E

[0113] White solid (131 mg, 92%); mp. 272 oC; 1 H NMR (80 MHz, DMSO) 5 13.49 (s, 1 H), 7.92 (q, J = 1 .0 Hz, 4H), 7.66 - 7.44 (m, 1 H), 7.42 - 7.21 (m, 2H); [M + H]+= 360.0 m / z3-((4-fluoro-1,3-dioxoisoindolin-2-yl)methyl)benzoic acid 6F

[0114] White solid (93 mg, 78%); mp. 260 oC. 1 H NMR (300 MHz, DMSO) 5 7.94 - 7.79 (m, 3H), 7.77 - 7.62 (m, 2H), 7.60 - 7.53 (m, 1 H), 7.44 (t, J = 7.7 Hz, 1 H), 4.79 (s, 2H); [M - H]- = 298 m / z3-((4-nitro-1,3-dioxoisoindolin-2-yl)methyl)benzoic acid &G

[0115] White solid (101 mg, 78%); mp. 230-240 oC (dec); 1 H NMR (80 MHz, DMSO-d6) 5 8.16 - 7.35 (m, 7H), 7.35 - 6.59 (m, 2H), 4.81 (s, 2H); [M - H]- = 325 m / z3-((4-amino-1,3-dioxoisoindolin-2-yl)methyl)benzoic acid 7\

[0116] Yellow solid (132 mg, 88%); mp.261 °C;1H NMR (300 MHz, DMSO-d6) 57.84 (d, J = 6.0 Hz, 2H), 7.59 - 7.39 (m, 3H), 6.99 (d, J = 7.8 Hz, 2H), 6.51 (s, 2H), 4.76 (s, 2H).; [M + H]+= 297 m / z3-((1,3-dioxoisoindoiin-2-yi)methyi)benzoic acid 7A

[0117] White solid (1 g, 61 %); mp. 236 °C.1H NMR (80 MHz, DMSO-d6) 5 7.87(s, 6H), 7.48 (d, J = 7.5 Hz, 2H), 4.83 (s, 2H); [M + H]+= 282.1 m / z3-((1,3-dioxoisoindolin-2-yl)methyl)-5-(trifluoromethyl)benzoic acid 7D

[0118] White solid (138 mg, 99%); mp. 233 °C;1H NMR (80 MHz, DMSO-d6) 58.10 (d, J = 4.0 Hz, 2H), 8.00 (s, 1 H), 7.89 (s, 4H), 4.94 (s, 2H).; [M + H]+= 350.1 m / z3-((1 ,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl)methyl)benzoic acid 7H

[0119] White solid (1 g, 88%); mp. 212 °C.1H NMR (80 MHz, DMSO-d6) 5 8.40 - 7.93 (m, 3H), 7.95 - 7.72 (m, 2H), 7.71 - 7.29 (m, 2H), 4.86 (s, 2H).; [M + H]+= 350 m / zExample 2 - General procedure for the synthesis of 3-((1,3- dioxoisoindolin-2-yl)methyl)benzoic acid 10

[0120] To a 25,0 ml round bottom flask equipped with a magnetic string bar was added the benzoic acid 8A-G (24,8 mmol), followed by 13,3 mL (24,3g 24,9 mmol), of concentrated sulfuric acid, the reaction was stirred in an ice bath until the complete dissolution of the solid, then was added 2- (hydroxymethyl)isoindoline-l , 3-dione 9 in one portion (5,3g, 29,8mmol or 10,6g, 59,6 mmol for the acid 8H). After 1 hour the ice bath was removed and the reaction was stirred at room temperature for another 18h. The reaction was poured in a 500 mL backer flask containing 50,0 ml of H2O previously heated to 65 °C equipped with a magnetic stirring bar. The suspension was stirrer for one hour and then filtered in a Buchner funnel and dried under vacuum. The solid was recrystallized from hot ethanol.3-((1,3-dioxoisoindolin-2-yl)methyl)-5-methoxybenzoic acid 10A

[0122] White solid (6,1 g, 65%); mp. 230 °C;1H NMR (600 MHz, CDCI3) 5 7.92 (d, J = 2.2 Hz, 1 H), 7.82 (dd, J = 5.4, 3.1 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 7.44 (dd, J = 8.3, 2.3 Hz, 1 H), 7.36 (d, J = 8.3 Hz, 1 H), 7.25 (s, 1 H), 4.80 (s, 2H); [M + H]+= 316.0 m / z3-chloro-5-((1,3-dioxoisoindolin-2-yl)methyl)benzoic acid 10C

[0123] White solid (9,8g, 78%); mp. 259 °C.1H NMR (400 MHz, CDCIs) 5 7.76 (dd, J = 29.4, 2.1 Hz, 1 H), 7.74 (dd, J = 5.4, 3.1 Hz, 2H), 7.70 (d, J = 2.0 Hz, 1 H), 7.63 (dd, J = 5.5, 3.1 Hz, 2H), 7.31 (d, J = 8.3 Hz, 1 H), 4.85 (s, 2H).; [M + H]+= 316.0 m / z

[0125] White solid (7,0g, 58%); mp. 230-240 °C (dec).1H NMR (600 MHz, CDCI3) 5 7.82 (d, J = 8.1 Hz, 1 H), 7.80 (d, J = 2.2 Hz, 1 H), 7.75 (dd, J = 5.5, 3.0 Hz, 2H), 7.64 (dd, J = 5.5, 3.0 Hz, 2H), 7.09 (dd, J = 8.2, 2.3 Hz, 1 H), 4.71 (s, 2H);[M + H]+= 408 m / z3-((1,3-dioxoisoindolin-2-yl)methyl)-5-methylbenzoic acid 10F

[0126] White solid (8,8g, 73%); mp. 235 °C.1H NMR (400 MHz, CDCI3) 5 7.83 -7.74 (m, 4H), 7.71 - 7.62 (m, 2H), 7.16 (d, J = 7.9 Hz, 1 H), 4.80 (s, 2H); [M + H]+= 296 m / z

[0128] White solid (0.753 g, 45%); mp. 273 °C.1H NMR (600 MHz, CDCI3) 5 11.58 (s, 1 H), 7.83 - 7.77 (m, 3H), 7.77 - 7.72 (m, 2H), 7.67 (ddd, J = 5.2, 3.1 , 1 .7 Hz, 2H), 7.64 (ddd, J = 5.2, 3.1 , 1.8 Hz, 2H), 7.46 (d, J = 2.4 Hz, 1 H), 4.84 (s, 2H), 4.67 (s, 2H); [M + H]+= 457 m / zExample 3 Procedure for the synthesis of 3-((1,3- dioxoisoindolin-2-yl)methyl)-4-hydroxybenzoic acid 101

[0129] A 10 ml flask equipped with a magnetic string bar was added 10a (2g, 6,4 mmol), the flask was sealed, and dichloromethane (20 mL) was added under an argon atmosphere. The reaction was cooled to -78°C in a dry-ice / acetone bath and BCh 1 M in DCM was slowly added (9,64 mL, 9,64 mmol), then thecooling bath was removed, and the reaction was stirred for 2 hours. The solution was diluted in 10ml DCM; the organic phase was washed with brine (10 ml) and twice with H2O (2 x 10 mL) and dried over Na2SO4, filtered, and concentrated in vacuo. The pure product was obtained by recrystallization in ethanol.

[0130] White solid (1 ,23g, 65%); mp. 271 °C.1H N MR (300 MHz, DMSO) 5 12.48 (s, 1 H), 10.65 (s, 1 H), 7.91 (dt, J = 12.3, 4.3 Hz, 4H), 7.72 (d, J = 8.3 Hz, 1 H), 7.54 (s, 1 H), 6.89 (d, J = 8.4 Hz, 1 H), 4.74 (s, 2H); [M + H]+= 298 m / zExample 4 - Procedure for the synthesis of 3-((1,3-7a 25b

[0131] A solution containing 7A (0.50 g, 1.65 mmol) in anhydrous CH2CI2 (5 ml) was cooled to 0 °C, followed by dropwise addition of SOCI2 (0.3 g, 0.18 ml, 2.5 mmol) and heated until reflux for 1.5 h. After the mixture was cooled to 0 °C, followed by dropwise addition of DMF (20 ml). The reaction mixture was stirred at room temperature for 2 h, quenched with water (5 mL) and extracted with CH2CI2 (2 x 30 mL). The combined organic layers were washed with10% HCI (10 mL), sat. NaHCOs (10 mL), and brine (10 mL), and dried with Na2SO4, and concentrated in vacuo to give a brown oil. The residue was purified by flash chromatography (SiC>2, cyclohexane: acetone) giving a white solid and 15 % yield.1H NMR (80 MHz, CDCI3) 5 7.85 - 7.52 (m, 4H), 7.48 - 7.13 (m, 4H), 4.72 (s, 2H), 2.93 (d, J = 9.1 Hz, 6H), [M + H]+= 309 m / zExample 5 - Procedure for the synthesis of 3-((1,3- dioxoisoindolin-2-yl)methyl)benzamide 25Aa) SOCI2

[0132] A solution containing 7A (0.50 g, 1.65 mmol) in anhydrous CH2CI2 (5 ml) was cooled to 0 °C, followed by dropwise addition of SOCI2 (0.3 g, 0.18 ml, 2.5 mmol) and heated until reflux for 1.5 h. After the mixture was cooled to 0 °C, followed by dropwise addition of NH4OH (30 %, 6.57 mL, 49.50 mmol). The reaction mixture was stirred at room temperature for 2 h, quenched with water (5 mL) and extracted with CH2CI2 (2 x 30 mL). The combined organic layers were washed with10% HCI (10 mL), sat. NaHCOs (10 mL), and brine (10 mL), and dried with Na2SO4, and concentrated in vacuo to give a brown oil. The residue was purified by flash chromatography (SiC>2, cyclohexane: acetone) giving a white solid with 23 % yield.1H NMR (80 MHz, DMSO-d6) 5 7.98 - 7.70 (m, 6H), 7.48 (h, J = 2.4 Hz, 2H), 4.85 (s, 2H), 3.51 (s, 2H); [M + H]+= 281 m / zExample 6 - Procedure for the synthesis of 3-((1,3- dioxoisoindolin-2-yl)methyl)benzonitrile 26A

[0133] To a solution of Ph3PO (0.05 g, 0.016 mmol 1.0 %) and 25A (0.46 g, 1.65 mmol) in anhydrous MeCN (8 ml) was added EtsN (0.50 g, 0.27 m, 3.30 mmol). The resulting solution was treated dropwise with neat oxalyl chloride (0.42, 0.27 ml, 3.30 mmol) using an adjustable volume pipette (0.1 -1.0 mL) and the mixture was stirred for 10 min at rt. The reaction progress was monitored byHPLC. After the reaction was complete, the solution was filtered and concentrated in vacuum. The crude mixture thus obtained was purified by flash chromatography (SiO2, cyclohexane: acetone) yielding a transparent solid in 33 % yield.1H NMR (80 MHz, DMSO-d6) 5 8.35 - 7.75 (m, 8H), 5.17 (s, 2H); [M + H]+= 263 m / zExample 7 - Procedure for the synthesis of 2-(3-(morpholine-4- carbonyl)benzyl)isoindoline-1 , 3-dione 25C a) SOCI7a 25c

[0134] A solution containing 7A (0.50 g, 1.65 mmol) in anhydrous CH2CI2 (5 ml) was cooled to 0 °C, followed by dropwise addition of SOCI2 (0.3 g, 0.18 ml, 2.5 mmol) and heated until reflux for 1.5 h. After the mixture was cooled to 0 °C, followed by dropwise addition of EtsN (0.51 g, 0.70 mL, 5.0 mmol) and morpholine (0.18 g, 0.18 mL, 2.1 mmol) in CH2CI2 (1 ml). The reaction mixture was stirred at room temperature for 2 h, quenched with water (5 mL) and extracted with CH2CI2 (2 x 15 mL). The combined organic layers were washed with 10% HCI (10 mL), sat. NaHCOs (10 mL), and brine (10 mL), and dried with Na2SO4, and concentrated in vacuo to give a brown oil. The residue was purified by flash chromatography (SiO2, cyclohexane: acetone) and recrystallized in MeOH giving a white pale solid in 45 % yield.1H NMR (80 MHz, CDCI3) 5 7.75 (t, J = 4.7, 4.1 Hz, 4H), 7.52 - 7.07 (m, 4H), 3.50 (d, J = 21.0 Hz, 4H), 1.61 (s, 6H); [M + H]+= 351 m / z.Example 8 - Procedure for the synthesis of 2-(3-(piperidine-1- carbonyl)benzyl)isoindoline-1 , 3-dione 25D

[0135] A solution containing 7A (0.50 g, 1.65 mmol) in anhydrous CH2CI2 (5 ml) was cooled to 0 °C, followed by dropwise addition of SOCI2 (0.3 g, 0.18 ml, 2.5 mmol) and heated until reflux for 1.5 h. After the mixture was cooled to 0 °C, followed by dropwise addition of EtsN (0.51 g, 0.70 mL, 5.0 mmol) and piperidine (0.20 g, 0.23 mL, 2.3 mmol) in CH2CI2 (1 ml). The reaction mixture was stirred at room temperature for 2 h, quenched with water (5 mL) and extracted with CH2CI2 (2 x 30 mL). The combined organic layers were washed with10% HCI (10 mL), sat. NaHCOs (10 mL), and brine (10 mL), and dried with Na2SO4, and concentrated in vacuo to give a yellow oil. The residue was purified by flash chromatography (SiC>2, cyclohexane: acetone) giving a white solid in 20 % yield.1H NMR (80 MHz, CDCI3) 5 7.75 (dd, J = 4.7, 4.1 Hz, 4H), 7.52 - 7.07 (m, 4H), 3.50 (d, J = 21 .0 Hz, 4H), 1 .61 (s, 6H); [M + H]+= 349 m / zExample 9 - isopropyl 3-((1,3-dioxoisoindolin-2- yl)methyl)benzoate 24A

[0136] To a 10 ml flask equipped with a magnetic string bar was added 7A (2g, 7, 14 mmol), isopropanol (20 mL) followed by sulfuric acid (0,2ml), the reactions was refluxed overnight then cooled, diluted in 50ml DCM, the organic phase was washed with saturated sodium bicarbonate solution (10 ml) and twice with H2O (2 x 10 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The pure product was obtained by recrystallization in hot methanol. White solid (1 ,73 g, 75%); mp 97 °C,1H NMR (600 MHz, CDCI3) 5 8.06 (s, 1 H), 7.92 (d, J = 7.8 Hz, 1 H), 7.85 - 7.82 (m, 3H), 7.71 - 7.68 (m, 3H), 7.58 (d, J = 7.7 Hz,1 H), 7.37 (t, J = 7.7 Hz, 2H), 5.21 (sept, J = 6.3 Hz, 1 H), 4.88 (s, 2H), 1.34 (d, J = 6.3 Hz, 6H), [M + H]+= 324 m / z.Example 10 - methyl 3-(( 1,3-dioxoisoindolin-2- yl) methyl) benzoate 24B

[0137] To a 10 ml flask equipped with a magnetic string bar was added 7A (2g, 7,14 mmol), Methanol (20 mL) followed by sulfuric acid (0,2ml), the reactions was refluxed overnight then cooled, diluted in 50ml DCM, the organic phase was washed with saturated sodium bicarbonate solution (10 ml) and twice with H2O (2 x 10 mL), dried over Na2SO4, filtered, and concentrated in vacuum. The pure product was obtained by recrystallization in hot methanol. White solid (1 ,75 g, 82%); mp. 120°C,1H NMR ((80 MHz, CDCI3) 5 8.31 (d, J = 1.8 Hz, 8H), 5.13 (s, 2H), 4.14 (s, 3H); [M + H]+= 296 m / zExample 11 - General procedure for the synthesis of (3-((1,3- dioxoisoindolin-2-yl)methyl-benzoic acid 17A-C

[0138] Step a: To a 50 ml flask equipped with a magnetic string bar was added 5B (2g, 4,5 mmol), Pd(dba)2 (255 mg, 0.4mmol), XPhos (211 ,7 mg, 0.4mmol), cesium carbonates (2.89 mg, 8,9 mmol). The flask was sealed, and toluene (20 mL) was added under argon atmosphere. The reactions were stirred for 18 hours at 100 °C in an oil bath. The solution was cooled at room temperature, diluted in 50ml acetone, and filtered through a one-inch silica pad andconcentrated under reduced pressure. The resulting solid was recrystallized in hot methanol. 60% (15A) or 62% (15B) or 52% (15C).

[0139] Step b: Compound 16A-C (0,4mmol) was charged in a 10 ml flask equipped with a stirring bar, followed by 2ml of methanol and Pd / C 10% (46 mg 0,04mmol). The reaction flask was capped, and the atmosphere replaced by H2. The reaction was stirred overnight at room temperature. The reaction was dissolved in 30ml of methanol / THF (1 :1 ) and filtered through a one-inch silica pad and concentrated under reduced pressure. The resulting solid was recrystallized in hot methanol.

[0141] White solid (83 mg, 57%); 1 H NMR (80 MHz, D2O) 5 12.90 (s, 1 H), 7.88 (d, J = 1.2 Hz, 4H), 7.51 - 7.03 (m, 3H), 3.71 (d, J = 5.4 Hz, 4H), 3.15 (d, J = 5.2 Hz, 4H); [M + H]+= 367 m / z.3-((1,3-dioxoisoindolin-2-yl)methyl)-5-(4-methylpiperazin-1-yl)benzoic acid 17C

[0142] White solid (98 mg, 65%); mp. 210 °C, 1 H NMR (400 MHz, DMSO-d6) 5 7.96 - 7.83 (m, 4H), 7.42 (d, J = 2.7 Hz, 1 H), 7.03 (dd, J = 8.7, 2.7 Hz, 1 H), 6.95 (d, J = 8.6 Hz, 1 H), 5.06 (s, 2H), 3.13 (d, J = 5.2 Hz, 2H), 2.51 - 2.44 (m, 4H), 2.24 (s, 3H); [M + H]+= 380 m / zExample 12 - General procedure for the synthesis of 5-((1,3- dioxoisoindolin-2-yl)methyl)-[ 1, 1 '-biphenyl]-3-carboxylic acid13A,C-E; 14B-C

[0143] Step a: To a 50 ml flask equipped with a magnetic string bar was added 5B (2g, 4,5 mmol), Pd(PPh3)4 (462 mg, 0.4mmol), K2CO3 (920 mg, 6,6 mmol). The flask was sealed and dry dioxane (20 mL) was added under argon atmosphere. The reaction was stirred for 18 hours at 100 °C in an oil bath. The solution was cooled at room temperature, diluted in 50ml acetone and filtered through a one-inch silica pad and concentrated under reduced pressure. The resulting solid was recrystallized in hot methanol.

[0144] Step b: Compound 12A-E (0,4 mmol) was charged in a 10 ml flask equipped with a stirring bar, followed by 2ml of methanol and Pd / C 10% (46 mg 0,04mmol). The reaction flask was capped, and the atmosphere replaced by H2. The reaction was stirred overnight at room temperature. The reaction was dissolved in 30ml of methanol / THF (1 :1 ) and filtered through a one-inch silica pad and concentrated under reduced pressure. The resulting solid was recrystallized in hot methanol.5-((1 ,3-dioxoisoindolin-2-yl)methyl)-[ 1, 1 '-biphenyl]-3-carboxylic acid 13A

[0145] White solid (124 mg, 87%); mp. 220 °C.1H NMR (80 MHz, DMSO-d6) 5 8.53 - 6.88 (m, 13H), 5.17 (s, 2H) ppm; [M + H]+= 358.1 m / z5-((1,3-dioxoisoindolin-2-yl)methyl)-4'-fluoro-[ 1, 1 '-biphenyl]-3-carboxylic acid 14B

[0146] White solid (132 mg, 88%); mp. 275 °C;1H NMR (80 MHz, DMSO-d6) 5 13.31 (si, 1 H), 8.23 - 7.53 (m, 8H), 7.30 (t, J = 8.4 Hz, 3H), 5.19 (s, 2H) ppm; [M + H]+= 376.1 m / z5-((1,3-dioxoisoindolin-2-yl)methyl)-4'-methoxy-[ 1, 1 '-biphenyl]-3-carboxylic acid 13E

[0147] White solid (50 mg, 32%); mp. 230 °C (decomposition); 5 1 H NMR (80 MHz, DMSO-d6) 5 8.03 (d, J = 16.9 Hz, 5H), 7.78 - 7.23 (m, 5H), 7.05 (d, J = 8.6 Hz, 1 H), 5.17 (s, 2H), 3.80 (s, 3H).; [M + H]+= 388.4 m / z5-((1,3-dioxoisoindolin-2-yl)methyl)-4 '-(trifluoromethyl)-[ 1, 1 '-biphenyl]-3- carboxylic acid. 13D

[0148] White solid (115,6 mg, 68%); mp. 275 °C.1H NMR (80 MHz, DMSO-d6) 5 8.23 (d, J = 1.9 Hz, 1 H), 7.91 -7.78 (m, 9H), 7.32 (d, J = 8.2 Hz, 1 H), 5.20 (s, 2H) ppm; [M + H]+= 426 m / z4 '-amino-5-((1,3-dioxoisoindolin-2-yl)methyl)-[ 1, 1 '-biphenyl]-3-carboxylic acid 13C

[0149] White solid (110 mg, 74%); mp. 220 °C (decomposition);1H NMR (80 MHz, DMSO-d6) 5 8.05 (d, J = 2.0 Hz, 1 H), 7.91 (s, 4H), 7.80 - 7.46 (m, 1 H), 7.24 (dd, J = 17.5, 8.2 Hz, 3H), 6.64 (d, J = 8.3 Hz, 2H), 5.15 (s, 2H).; [M + H]+= 373 m / z.Example 13 - General procedure for the synthesis of 2-carboxy-

[0150] Step a: To a 500 ml round bottom flask equipped with a magnetic stirring bar charged with picolinic acid 18 (50 g, 0,3 mol), was added thionyl chloride (100ml, 1 ,5 mol), the reaction was refluxed for 12 hours, until all the solid was completely dissolved. The excess of thionyl chloride was distilled from the rection mixture until the formation of a red slurry. The flask was transferred to an ice bath and 300 ml of a mixture of methanol / TEA (2:1 ) was slowly added. The suspension was stirrer for one hour and then filtered in a Buchner funnel and dried under vacuum. The solid was dissolved in 300 ml of dichloromethane, filtered in a Buchner funnel and the resulting solution was concentrated under vacuum, and the resulting solid dried under vacuum. The product dimethyl pyridine-2,6-dicarboxylate (51 g, 261 mmol, 87% yield) was pure enough for the next step.

[0151] Step b: To a 1 L flask equipped with a magnetic string bar charged with dimethyl pyridine-2,6-dicarboxylate 19 (51 g, 261 mmol) was added 500 ml of methanol and 200 ml of dichloromethane. The flask was cooled in an ice bath and sodium borohydride (36g, 0,9 mol) was divided in 4 portions, each added at hourly intervals. One hour after the addition of the last portion 40 ml of acetone was poured in the reaction and it was stirred for an extra hour. Acetic acid (40ml) was added the reaction and it was stirred for 12 hours. The solvent was removed under reduced precure, and an extra 300 ml of methanol wasadded to the slurry solid, and the solvent was again removed under reduced pressure. The remaining solid was dissolved in dichloromethane (300 mL) and the solution was alkalized with sodium bicarbonate to pH 7. The organic phase was separated, and the aqueous phase was extracted two times with dichloromethane (50 mL), the combined organic phases were concentrated under vacuum and the resulting solid was recrystallized in methanol. White solid (31 mg, 63%, two steps); Rr = 0.55 (MeOH / DCM 2:8).

[0152] Step c: To a 500 mL flask equipped with a magnetic string bar was added the methyl 6-(hydroxymethyl) picolinate (10 g, 60 mmol), phthalimide 4 (89 mmol) and triphenyl phosphine (23,5 g, 89 mmol) followed by the addition of THF 100 ml. The reaction was cooled in an ice-bath and DIAD (89 ml, 90 mmol) was added dropwise for 30 minutes. The ice bath was removed, and the reaction was stirred for 18 hours at room temperature. The solvent was removed under vacuum. The slurry solid was recrystallized twice from hot methanol.2-carboxy-6-((1,3-dioxoisoindolin-2-yl)methyl)pyridin-1-ium 21 A

[0154] White solid (11 g, 60%); Rf= 0.52 (MeOH / DCM 1 :9); mp. 135 °C.1H NMR 1 H (80 MHz, CDCh) 5 8.40 - 7.31 (m, 6H), 5.32 (s, 2H), 4.15 (s, 3H); [M + H]+= 315 m / zExample 14 - Procedure for the synthesis of 6-((1,3- dioxoisoindolin-2-yl)methyl)picolinic acid 22

[0155] To a 20 mL flask equipped with a magnetic string bar charged with 2- carboxy-6-((1 ,3-dioxoisoindolin-2-yl)methyl)pyridin-1 -ium 21 A (1g, 3,4 mmol) was added 10 ml of HCL 4M and 10 ml of THF the reactions was capped and heated in an oil-bath at 80 °C for 18h. the THF was removed under vacuum and the resulting solid was filtered. The pure product was obtained after hot ethanol recrystallization. White solid (850 mg, 83%); mp. 140 °C;1H NMR (80 MHz, DMSO-d6): 5 8.10 - 7.50 (m, 7H), 4.96 (s, 2H); [M + H]+= 283 m / zExample 15 - Procedure for the synthesis ethyl 6-((1,3- dioxoisoindolin-2-yl)methyl)picolinate 23A

[0156] To 250 ml round bottom flask equipped with a magnetic string bar charged with 21 A (1 g, 3,4 mmol) was added 100 ml of EtOH, followed by the dropwise addition of sulfuric acid 1 mL the reactions was refluxed in an oil-bath after 18h. After cooling the solution was reduced to 10 ml under vacuum and the resulting solid was filtered and the pure product was obtained after hot ethanol recrystallization. White solid (850 mg, 81 %); mp 132 °C;1H NMR (80 MHz, CDCh) 5 8.22 - 7.64 (m, 6H), 7.43 (dd, J = 7.9, 1 .5 Hz, 1 H), 5.23 (s, 2H), 4.48 (q, J = 7.1 Hz, 2H), 1 .41 (t, J = 7.1 Hz, 3H); [M + H]+= 311 m / zExample 16 - Procedure for the synthesis isopropyl 6-((1,3- dioxoisoindolin-2-yl)methyl)picolinate 23B

[0157] To 250 ml round bottom flask equipped with a magnetic string bar charged with 21 A (1g, 3,4 mmol) was added 100 ml of / -PrOH, followed by the dropwise addition of sulfuric acid 1 mL the reactions was refluxed in an oil-bath after 18h.After cooling the solution was reduced to 10 ml under vacuum and the resulting solid was filtered and the pure product was obtained after hot methanol recrystallization. White solid (730 mg, 76%); mp. 105 °C ;1H NMR (80 MHz, CDCh) 5 8.41 - 7.66 (m, 6H), 7.63 - 7.27 (m, 1 H), 5.34 (d, J = 6.5 Hz, 3H), 1 .45 (d, J = 6.1 Hz, 6H); [M + H]+= 325 m / z.Example 17 - Procedure for the synthesis of derivatives 32A-K.

[0158] Step a: In a 10 mL tube equipped with a magnetic stirrer with 2.5 g of chelidamic acid (27) (1 eq), 14.85 mL of SOCI2 (15 eq) at 0 °C was added, followed by 1 mL of anhydrous DMF and the resulting reaction was refluxed for 4 hours at 80°C under an argon atmosphere. Subsequently, the thionyl chloride was removed by evaporation. To the resulting orange solid, 5 mL of anhydrous EtOH was added, and the system was stirred at 55 °C for 2 h andthen at room temperature overnight. The crude mixture was evaporated, and purification by flash chromatography was performed in an isocratic system Hex:AcOEt (30%) affording 2600 mg of (28) as a white solid (74%).

[0159] Step b: To diethyl 4-chloropyridine-2,6-dicarboxylate (28) (3000 mg, 1 eq), stirred in EtOH (15 mL) and 5 mL of DCM at 0 °C, NaBH4 (309 mg, 0.7 eq) was added. The reaction was stirred at 0 °C for one hour and left at room temperature for another hour. After monitoring by TLC, one portion of 30 mg of NaBH4 was added, and the complete reduction was monitored by TLC. For the work-up, acetone (20 ml) was added and stirred for 30 minutes; then, the reaction was concentrated in vacuo. The organic extracts were dissolved in EtOAc (100 mL), washed with water (3 x 10 mL), and concentrated to give a colorless oil that later solidified into an orange solid, affording a CAKE with 87 % purity. After column chromatography purification (Hex:AcOEt 1 :1 ), the reduction product (29) was obtained as a yellow solid (1.76 grams) in 70 % yield.

[0160] Step c: 1. Thoroughly dried ethyl 4-chloro-6- (hydroxymethyl)picolinate (29) (3230 mg, 1 eq) was dissolved in DCM (15 mL), then SOCI2 (5 eq, 5 mL) was added dropwise at 0 °C. After 90 min, the solution reached room temperature, and the excess of SOCI2 was removed under reduced pressure without heating. DCM (20 mL) was added to the oily residue, and the solution was washed with saturated aqueous of NaHCOs and dried over Na2SO4. Evaporation of the solvent afforded the product as a beige solid (3400 mg, 96%), which was used in the next step without purification. 2. In a 10 mL tube equipped with a magnetic stirrer with chlorinated substrate (1150 mg, 1 .0 eq) and dry potassium carbonate (750 mg, 1.1 eq) were added to 3 mL of DMF. Phthalimide (689 mg, 0.95 eq) was added gradually over 30 minutes. The reaction was stirred for 18 hours at room temperature and monitored by TLC (Hex: EtOAc 40%). After 18 hours of reaction, the reaction was complete. The workup was carried out with precipitation on ice (100 mL H2O:AcOH 0.05% m / m), followed by vacuum filtration. The crude CAKE was crystalized in ethanol (30 mL), affording the product 30 in 70% yield (1300 g) as a white solid.

[0161] Step d: In a 5 mL tube equipped with a magnetic stirrer with 2,6- dicarboxylatodiethyl 4-chloropyridine (1.0 eq), Pd(PPh3)4 (0.06 eq), K2CO3 (1.5 eq), and the Corresponding boronic acid (1.3 eq) was added. The tube was sealed and dry DMF (3 mL) was added under N2 atmosphere. The reaction was adjusted to the indicated temperature in a biotage microwave for 1 .3 hours. After this time, the mixture was poured onto ice, and the precipitate was subsequently vacuum-filtered. The precipitate was dissolved in DCM and dried with Na2SO4. The solvent was removed, and the crude product was purified by column chromatography (DCM:EtOH 5%. ) and recrystallized in EtOH.

[0162] Step e: The nitro derivative (32C) derivative 70 mg (1.0 eq), iron (4.0 eq), and ammonium chloride (0.6 eq) were suspended in 1 mL of EtOH: 1 mL of H2O. The reaction was heated to 85 °C and kept under stirring for 1 .5 h. Subsequently, the reaction was extracted with DCM, dried with Na2SO4, and evaporated under reduced pressure; a yellow solid was obtained. The product was purified by reverse-phase column chromatography (ACN:H2O), and 62 mg (95 % yield) of the aniline (32K) was obtained as a yellow amorphous solid.Ethyl 6-((1,3-dioxoisoindolin-2-yl)methyl)-4-phenylpicolinate 32A

[0163] White solid (75 mg, 68 %); mp. 127-129 °C;1H NMR (500 MHz, CDCI3): 5 ppm 5 8.24 (d, J = 1.6 Hz, 1 H, H-4), 7.93 (dd, J = 5.4, 3.1 Hz, 2H, H-25, H-28), 7.78 (dd, J = 5.5, 3.1 Hz, 2H, H-26, H-27), 7.65 - 7.61 (m, 2H, H- 8, H-12), 7.56 (d, J = 1.6 Hz, 1 H, H-2), 7.51 - 7.45 (m, 3H, H-9, H-10, H-11 ), 5.21 (s, 2H, H-18), 4.45 (q, J = 7.1 Hz, 2H, H-16), 1.37 (t, J = 7.1 Hz, 3H, H- 17); [M + H]+= 387 m / zEthyl 6-((1,3-dioxoisoindolin-2-yl)methyl)-4-(4-fluorophenyl)picolinate 32B.

[0164] White amorphous solid (72 g, 68 %); mp. 125-126 °C;1H NMR (500 MHz, CDCk): 5 ppm 5 8.20 (d, J = 1.7 Hz, 1 H, H-4), 7.93 (dt, J = 7.5, 3.7 Hz, 2H, H-25, H-28), 7.79 (dd, J = 5.5, 3.1 Hz, 2H, H-26, H-27), 7.65 - 7.58 (m, 2H, H-8, H-12), 7.52 (d, J = 1.7 Hz, 1 H, H-2), 7.18 (t, J = 8.6 Hz, 2H, H-9, H- 11 ), 5.20 (s, 2H, H-18), 4.44 (q, J = 7.1 Hz, 2H, H-16), 1.37 (t, J = 7.1 Hz, 3H, H-17); [M + H]+= 405 m / z.Ethyl 6-((1,3-dioxoisoindolin-2-yl)methyl)-4-(4-nitrophenyl)picolinate 32C.

[0165] Yellow amorphous solid (140 mg, 56%); mp. 216-217 °C;1H NMR (500 MHz, CDCk): 5 ppm 8.38 - 8.33 (m, 2H, H-9, H-11 ), 8.26 (s, 1 H, H-4), 7.94 (dt, J = 5.7, 2.7 Hz, 2H, H-25, H-28), 7.80 (d, J = 7.5 Hz, 4H, H-8, H-12, H-26, H-27), 7.59 (s, 1 H, H-2), 5.24 (s, 2H, H-18), 4.50 - 4.42 (m, 2H, H-16), 1.41 - 1 .34 (m, 3H, H-17); [M + H]+= 432 m / zEthyl 6-((1,3-dioxoisoindolin-2-yl) methyl) -4- (4-(trifluoromethyl) phenyl) picolinate 32D.32D

[0166] White amorphous solid (97 mg, 71 %); mp. 151 °C;1H NMR (500 MHz, CDCk): 5 ppm 8.24 (s, 1 H, H-4), 7.94 (dd, J = 6.0, 2.7 Hz, 2H, H-25, H- 28), 7.83 - 7.78 (m, 2H, H-26, H-27), 7.75 (s, 4H, H-8, H-9, H-11 , H-12), 7.56 (s, 1 H, H-2), 5.23 (s, 2H, H-18), 4.45 (q, J = 7.2 Hz, 2H, H-16), 1.37 (t, J = 7.1 Hz, 3H, H-17).; [M + H]+= 455 m / z.Ethyl6-((1,3-dioxoisoindolin-2-yl)methyl)-4-(4-methoxyphenyl) picolinate 32E

[0167] White solid (32 mg, 66%); mp. 170-171 °C.1H NMR (500 MHz, CDCk): 5 ppm 8.21 (d, J = 1.7 Hz, 1 H, H-4), 7.93 (dd, J = 5.4, 3.1 Hz, 2H, H- 25, H-28), 7.78 (dd, J = 5.5, 3.0 Hz, 2H, H-26, H-27), 7.63 - 7.56 (m, 2H, H-8, H-12), 7.52 (d, J = 1.7 Hz, 1 H, H-2), 7.03 - 6.96 (m, 2H, H-9, H-11 ), 5.20 (s, 2H, H-18), 4.45 (q, J = 7.1 Hz, 2H, H-16), 1.37 (t, J = 7.1 Hz, 3H, H-31 ).; [M + H]+= 417 m / z.Ethyl 4-(4-acetylphenyl)-6-((1,3-dioxoisoindolin-2-yl)methyl)picolinate 32F.

[0168] White amorphous solid (110 mg, 46%); mp. 163-164 °C.1H NMR (500 MHz, CDCk): 5 ppm 8.26 (d, J = 1.8 Hz, 1 H, H-4), 8.07 (d, J = 8.0 Hz, 2H, H-25, H-28), 7.94 (dd, J = 5.5, 2.9 Hz, 2H, H-26, H-27), 7.79 (dd, J = 5.5, 3.0 Hz, 2H, H-8, H-12), 7.73 (d, J = 8.0 Hz, 2H, H-9, H-11 ), 7.59 (d, J = 1.8 Hz, 1 H, H-2), 5.23 (s, 2H, H-18), 4.45 (q, J = 7.2 Hz, 2H, H, H-16), 2.66 (s, 2H, H- 33), 1.37 (t, J = 7.2 Hz, 3H, H-17); [M + H]+= 412 m / zEthyl 4-(4-chlorophenyl)-6-((1,3-dioxoisoindolin-2-yl)methyl)picolinate 32G.

[0169] White solid (350 mg, 82%); mp. 141-142 °C.1H NMR (500 MHz, CDCIs): 5 ppm 8.21 (d, J = 1 .6 Hz, 1 H, H-4), 7.98 - 7.90 (m, 2H, H-25, H-28), 7.83 - 7.75 (m, 2H, H-26, H-27), 7.60 - 7.54 (m, 2H, H-8, H-12), 7.53 (d, J = 1 .6 Hz, 1 H, H-2), 7.50 - 7.43 (m, 2H, H-9, H-11 ), 5.21 (s, 2H, H-18), 4.45 (q, J = 7.1 Hz, 2H, H-16), 1.37 (t, J = 7.1 Hz, 3H, H-17); [M + H]+= 421 m / zEthyl 6-((1,3-dioxoisoindolin-2-yl)methyl)-4-(p-tolyl)picolinate 32H.

[0170] White amorphous solid (120 g, 54%); mp. 149-150 °C.1H NMR (500 MHz, CDCI3): 5 ppm 8.23 (d, J = 1.7 Hz, 1 H, H-4), 7.93 (dd, J = 5.3, 3.1 Hz, 2H, H-25, H-28), 7.78 (dd, J = 5.4, 3.1 Hz, 2H, H-26, H-27), 7.56 - 7.51 (m, 3H, H-2, H-8, H-12), 7.29 (d, J = 4.8 Hz, 2H, H-9, H-11 ), 5.20 (s, 2H, H-18), 4.44 (q, J = 7.1 Hz, 2H, H-16), 2.41 (s, 3H, H-20), 1.37 (t, J = 7.1 Hz, 3H, H- 17); [M + H]+= 401 m / zEthyl 4-(4-acetylphenyl)-6-((1,3-dioxoisoindolin-2-yl)methyl)picolinate 32I.

[0171] White amorphous solid (109 g, 53%); mp. 163-164 °C.1H NMR (500 MHz, CDCIs): 5 ppm 8.26 (d, J = 1.8 Hz, 1 H, H-4), 8.07 (d, J = 8.0 Hz, 2H, H- 25, H-28), 7.94 (dd, J = 5.5, 2.9 Hz, 2H, H-26, H-27), 7.79 (dd, J = 5.5, 3.0 Hz, 2H, H-8, H-12), 7.73 (d, J = 8.0 Hz, 2H, H-9, H-11 ), 7.59 (d, J = 1.8 Hz, 1 H, H- 2), 5.23 (s, 2H, H-18), 4.45 (q, J = 7.2 Hz, 2H, H, H-16), 2.66 (s, 2H, H-33), 1.37 (t, J = 7.2 Hz, 3H, H-17); [M + H]+= 429 m / zEthyl 6-((1,3-dioxoisoindolin-2-yl)methyl)-[4,4'-bipyridine]-2-carboxylate 32 J.

[0172] White solid (200 mg, 65%); mp. 153-155 °C.1H NMR (500 MHz, CDCI3): 5 ppm 5 8.75 (d, J = 5.0 Hz, 2H, H-9, H-11 ), 8.26 (d, J = 1.6 Hz, 1 H, H-4), 7.94 (dd, J = 5.4, 3.0 Hz, 2H, H-25, H-28), 7.80 (dd, J = 5.5, 3.1 Hz, 2H, H-26, H-27), 7.60 (d, J = 1 .6 Hz, 1 H, H-2), 7.56 - 7.52 (m, 2H, H-8, H-12), 5.23 (s, 2H, H-18), 4.45 (q, J = 7.1 Hz, 2H, H-16), 1.37 (t, J = 7.1 Hz, 3H, H-17); [M + H]+= 389 m / zEthyl 4-(4-aminophenyl)-6-((1,3-dioxoisoindolin-2-yl)methyl)picolinate 32k

[0173] Yellow solid (62 mg, 95%); mp. 224-225 °C.1H NMR (500 MHz, CDCI3): 5 ppm 5 8.18 (s, 1 H, H-4), 7.93 (dd, J = 5.4, 3.1 Hz, 2H, H-25, H-28), 7.78 (dd, J = 5.5, 3.1 Hz, 2H, H-26, H-27), 7.50 (s, 1 H, H-2), 7.49 (d, J = 2.6 Hz, 2H, H-8, H-12), 6.84 (d, J = 8.1 Hz, 2H, H-9, H-11 ), 5.17 (s, 2H, H-18), 4.44 (q, J = 7.1 Hz, 2H, H-16), 1.37 (t, J = 7.1 Hz, 3H, H-17).; [M + H]+= 402 m / zPharmacological assays

[0174] A series of non-clinical pharmacology and toxicology studies have been performed to support the clinical evaluation of the compounds according to the present disclosure in human subjects. These studies were performed in accordance with internationally recognized guidelines for study design and in compliance with the requirements of Good Laboratory Practice (GLP) unless otherwise noted.Example 18 - Stability of MB-611, MB-657 and MB-658 in several pHs and plasma.Methods:

[0175] According to the present invention, the stability of MB-611 was evaluated at three different pH levels (2.2, 4.4, and 6.8), and MB-657 and MB- 658 at four different pH levels (2.2, 4.4, 6.8, and 7.4). Additionally, MB-611 , MB-657, and MB-658 were analyzed for plasma stability. The compound solutions were incubated at 37°C in a water bath, and aliquots were collected at 5, 15, 30, 60, 120 and 240 minutes, as well as after 24 hours (MB-611 ) of incubation. At each collection time, two samples were taken from each test solution at different pH levels and also in plasma. The chromatographic peak area ratio of the compounds to the previously quantified Internal Standard peak area, set at 100% during the pre-test phase, served as the baseline for calculating the stability percentage across various pH levels and in plasma.Results:

[0176] In the current innovation, the MB-611 compound exhibited good stability over 24 hours of incubation at pH levels of 2.2 and 4.4, with no significant degradation. However, when exposed to pH 6.8, approximately 40% of the initial compound degraded after the same incubation period (Figure 1 A). Besides, MB-657 and MB-658 displayed robust stability for a 4-hour period when exposed to pH levels of 2.2 and 4.4. Nevertheless, their stability sharply declined when subjected to varying pH conditions, namely pH 6.8 and pH 7.4 (See Figure 1 , Panels B and C). When introduced to plasma, MB-611 initially maintained stability for 1 -hour post-incubation, with its levels graduallydiminishing until reaching a steady state between 4 and 24 hours after the initial incubation (See Figure 1 , Panel D). In contrast, within just 5 minutes of incubation in plasma, MB-657 underwent rapid degradation, while the concentration of MB-658 underwent a continuous, gradual reduction over the course of 4 hours (See Figure 1 , Panel E). Furthermore, MB-658 concentration exhibited a consistent, gradual decrease over the 4-hour observation period (See Figure 1 , Panel F).Animals

[0177] All experiments were approved by the Animal Ethics Committee of Center of Innovation and Pre-Clinical Studies (CIEnP) under the numbers: 212, 213, 240, 246, 248, 268, 274. Adult male and female Sprague Dawley rats (weight 250 - 300 g) or CD1 , Swiss and C57BL / 6 mice strains (20-40 g) were provided by CIEnP and placed in a humidity control room at a temperature of 23 ± 2 °C with light / dark cycle for 12 hours. All animals were maintained under SPF (Specific Pathogen Free) conditions and were obtained from CIEnP facility, whose breeding colonies were purchased from Charles River Laboratories (USA). Animals had free access to food and water and were randomly divided into the several groups. The experiments were carried blindly, i.e. the experimenter was unaware of the treatments of the animals.Example 19 - Oral and intravenous pharmacokinetics for MB-611 in mice and rats.Methods:

[0178] According to the present invention, the pharmacokinetic profile was assessed following the intravenous or oral administration of MB-611 in both mice and rats. Animals were subjected to various treatments, including intravenous (i.v.) and oral (p.o.) gavage administration of MB-611 as per the current invention. The doses used were 1 mg / kg (i.v.) for mice and rats, 3 mg / kg (p.o.) for mice, and 3, 10, and 30 mg / kg (p.o.) for rats. Plasma was collected by caudal vein puncture at the following time points: 0.083; 0.25; 0.5; 1 ; 2 and 4 hours after intravenous administration and 0.25; 0.5; 1 ; 2; 4; 8 and 24 hours after oral administration. The MB-611 was prepared and quantifiedusing an analytical method previously developed and validated by means of UPLC-MSMS. The concentration of MB-611 was evaluated in both plasma and brain tissues.Results:

[0179] The MB-611 (30 mg / kg, p.o.) exhibited excellent oral absorption in mice and rats, achieving a plasma concentration in mice of 15.37 pg / mL and it is able to cross the blood-brain barrier to reach a maximum concentration of 206.08 ng / mL in the brains (Figure 2 and Table 2). When rats were administered a single dose of MB-611 at 30 mg / kg orally, it resulted in the highest plasma concentration of 14.73 pg / mL. Furthermore, the compound efficiently crosses the blood-brain barrier, reaching a peak concentration of 227.53 ng / mL in the brain. The pharmacokinetic parameters are presented in Table 2.Table 2: Pharmacokinetic parameters for MB-611 in mice and rats.Cmax: Peak concentration; Tmax: Time to reach Cmax; T1 / 2: half-life; AUCall area under de curve (all); F: bioavailability; NA: Not applicable; NC: Not calculated.Example 20 - Oral and intravenous pharmacokinetics for MB-657 and MB-662 in mice.Methods:

[0181] According to the present invention, the pharmacokinetic profile was evaluated after intravenous or oral administration with MB-657 and after oral administration of MB-662 in mice. Animals were treated with MB-657 (1 mg / kg, i.v.) or (3 mg / kg, p.o.) or MB-657 and MB-658 and their quantifications were assessed in plasma, brain, and spinal cord. Also, animals treated with MB-662 (3 mg / kg, p.o.), and the concentrations of MB-662 and MB-658 were assessed in plasma. Plasma was collected by caudal vein puncture at the following time points: 0.083; 0.5; 1 ; 2 and 4 hours after intravenous administration and 0.25; 1 ; 2; 4; 6; 8 and 24 hours after oral administration. The methodology used to assess the plasma and brain concentration of these compounds is the same as shown in example 19.Results:

[0182] The MB-657 and MB-662 were prepared and quantified using an analytical method previously developed and validated by means of UPLC- MSMS. MB-657 and MB-622 were not detectable in plasma, brain and spinal cord while MB-658 was detectable and quantified. The MB-658 reach micromolar plasma concentration (Figure 3). In addition, MB easily cross the blood-brain barrier and reach high concentration in the brain, and notably in the spinal cord, of 46.29 and 132,88 pg per gram of tissue, respectively. All pharmacokinetic parameters are presented in table 3.Table 3: Pharmacokinetic parameters for MB-657 and MB-662 in mice.Cmax: Peak concentration; Tmax: Time to reach Cmax; T1 / 2: half-life; AUCall area under de curve (all); F: bioavailability; ND: Not detectable; NC: Not calculated.Example 21 - Screening of N-benzyl phthalimides compounds in neuropathic pain model induced by partial sciatic nerve ligation (PSNL), in miceMethods:

[0184] The efficacy of the different N-benzyl phthalimides compounds, according to the present invention, on mechanical hypersensitivity in a neuropathy animal model induced by partial sciatic nerve ligation (PSNL), was performed according to Malmberg AB, Basbaum Al. Pain 76(1 -2):215-222, 1998 and its efficacy was compared with Gabapentin, a reference drug commonly used to treat neuropathic pain due to nerve damage. For this, male and female mice were kept anesthetized by the inhalation of 3% isoflurane plus 3% oxygen. PSNL was performed by tying a tight ligature with 8.0 silk suture (Shalon®) around approximately 1 / 3 to 1 / 2 the diameter of the sciatic nerve. In sham-operated mice the nerve was exposed, but not ligated. The mechanical sensitivity represented as paw withdrawal threshold was assessed with von Frey hairs (North Coast Medicals, Gilroy, CA, EUA) using the up- down paradigm (Bonin RP et al. Molecular Pain 10:26, 2014) before and after the PSNL. The animals that became neuropathic were treated with the different compounds (Table 1 ) 10 or 30 mg / Kg, p.o. or with Gabapentin (70 mg / kg, p.o.) a reference item, or with Vehicle (10 ml / Kg, p.o.) and mechanical sensitivity was measured 0.5, 1 , 2, 4, 6 and 8 hours after treatment. The compounds were solubilized in DMSO and Saline (1 :1 ).Results:

[0185] The percentage of reversion of the mechanical hyperalgesia induced by the nerve ligation are shown on Table 1 .Example 22 - Evaluation of the efficacy of MB-611 according to the present invention in neuropathic pain model induced by partial sciatic nerve ligation (PSNL), in miceMethods:

[0186] The efficacy of the MB-611 according to the present invention, was assessed on mechanical hypersensitivity in the same animal model of neuropathy (PSNL) described in the example 21. The mechanicalhypersensitivity represented as paw withdrawal threshold was assessed with von Frey hairs using the up-down paradigm (Bonin RP et al. Molecular Pain 10:26, 2014) before and after the PSNL. The animals that became neuropathic were treated with MB-611 (10, 30 or 60 mg / Kg, p.o.) or with Gabapentin (70 mg / kg, p.o.), a reference item, or with Vehicle (10 ml / Kg, p.o.) and mechanical sensitivity was measured 0.5, 1 , 2, 4, and 6 hours after treatment.Results:

[0187] The MB-611 (30 and 60 mg / Kg, p.o.) decreased significantly the mechanical sensitivity (hyperalgesia) by 51 and 83 %, respectively, when compared to the Vehicle group (p < 0.05). Gabapentin (70 mg / Kg, p.o.) also reduced the same extent the mechanical hyperalgesia, when compared with the Vehicle group. The mean EDso value for MB-611 in suppressing mechanical hyperalgesia in the 4thhour was 29.67 (23.73 - 36.03) mg / kg (Figure 4, Panels A and B).Example 23 - Evaluation of the analgesic efficacy of MB-657 according to the present invention in neuropathic pain model induced by partial sciatic nerve ligation (PSNL) in miceMethods:

[0188] The efficacy of the MB-657 according to the present invention, was assessed on mechanical hypersensitivity in the same animal model of neuropathy (PSNL) described in the example 21. The mechanical hypersensitivity represented as paw withdrawal threshold was assessed with von Frey hairs using the up-down paradigm (Bonin RP et al. Molecular Pain 10:26, 2014) before and after the PSNL. The animals that became neuropathic were treated with MB-657 according to the present invention (0.3, 1 or 3 mg / Kg, p.o.) or with Gabapentin (70 mg / Kg, p.o.), a reference item, or with Vehicle (10 ml / Kg, p.o.) and mechanical sensitivity was measured 0.5, 1 , 2, 4, 6 and 8 hours after treatment.Results:

[0189] The MB-657 (1 and 3 mg / Kg, p.o.) decreased the mechanical sensitivity (hyperalgesia) by 57 and 76 %, respectively, when compared to the Vehicle group (p < 0.05). Gabapentin (70 mg / Kg, p.o.) also reduced the sameextent (83 %) the mechanical hyperalgesia when compared with the Vehicle group. The mean ED50 value for MB 657 for suppressing mechanical hyperalgesia at the 4thhour was 1 .06 (0.70 - 1 .67) mg / kg (Figure 4, Panels C and D).Example 24 - Evaluation of the analgesic efficacy of MB-662 according to the present invention in neuropathic pain model induced by partial sciatic nerve ligation (PSNL) in miceMethods:

[0190] The efficacy of the MB-662 according to the present invention, was performed on mechanical hypersensitivity in the same animal model of neuropathy (PSNL) described in the example 21. The mechanical hypersensitivity represented as paw withdrawal threshold was assessed with von Frey hairs using the up-down paradigm (Bonin RP et al. Molecular Pain 10:26, 2014) before and after the PSNL. The animals that became neuropathic were treated with MB-662 according to the present invention (0.3, 1 and 3 mg / Kg, p.o.) or with Gabapentin (70 mg / Kg, p.o.), a reference item, or with Vehicle (10 ml / Kg, p.o.) and mechanical sensitivity was measured 0.5, 1 , 2, 4, 6 and 8 hours after treatment.Results:

[0191] The MB-662 (1 and 3 mg / Kg, p.o.) decreased the mechanical sensitivity (hyperalgesia) by 51 and 62 %, respectively, when compared to the Vehicle group (p < 0.05). Gabapentin (70 mg / Kg, p.o.) also reduced the same extent (70 %) the mechanical hyperalgesia when compared with the Vehicle group. The mean EDso value for MB 662 in suppressing the mechanical hyperalgesia at 4thhour was 2.15 (0.01 - 1.81 ) mg / kg (Figure 4, Panels E and F).Example 25 - Evaluation of the efficacy of MB-611 according to the present invention in neuropathic pain model induced by chemotherapy in miceMethods:

[0192] According to the present invention, the effect of MB-611 on mechanical hypersensitivity in a neuropathic pain animal model induced bychemotherapy (Paclitaxel) was studied, and the activity compared with Gabapentin, a reference drug used to treat neuropathic pain. Mice received 2 mg / kg of Paclitaxel intraperitoneally in four alternate days (cumulative dose of 8 mg / kg), according to Polomano RC et al. Pain, 94 (3): 293-304, 2001 and adapted for use in mice by Costa R et al. British Journal of Pharmacology, 164: 681-693, 2011. The presence of mechanical hyperalgesia was confirmed in the hindpaws of the mice 14 days after the first Paclitaxel administration. Sham-treated mice received saline solution intraperitoneally instead Paclitaxel. The mechanical hypersensitivity thresholds were measured by means of electronic von Frey (Analgesimetro digital EFF-302, Insight Pesquisa e Ensino, Sao Paulo, Brazil) before and after the Paclitaxel treatment. The animals that became neuropathic following paclitaxel treatment were treated with a single dose of MB-611 (30 mg / Kg, p.o.), Gabapentin (70 mg / Kg, p.o.), a reference item, or with Vehicle (10 ml / Kg, p.o.) and mechanical sensitivity was measured 0.5, 1 , 2, 4, and 6 hours after treatment.Results:

[0193] The MB-611 (30 mg / Kg, p.o.) significantly decreased the mechanical sensitivity (hyperalgesia) in mice treated with paclitaxel by 68% when compared to the Vehicle group (p < 0.05). Gabapentin (70 mg / Kg, p.o.) also reduced the mechanical hyperalgesia by 77% when compared with the Vehicle group (p<0.05) (Figure 5, Panels A and B).Example 26 - Evaluation of the analgesic action of MB-657 according to the present invention in neuropathic pain model induced by chemotherapy in miceMethods:

[0194] The efficacy of the MB-657 according to the present invention, was performed on mechanical hypersensitivity in the same animal model of neuropathy induced by chemotherapy (paclitaxel) described in the example 25. The activity of MB-657 was compared with Gabapentin, a reference drug used to treat neuropathic pain. The mechanical hypersensitivity represented as paw withdrawal threshold was assessed with von Frey hairs using the up- down paradigm (Bonin RP et al. Molecular Pain 10:26, 2014) before and afterthe Paclitaxel treatment. The animals that became neuropathic following paclitaxel treatment were treated with MB-657 (10 mg / Kg, p.o.), Gabapentin (70 mg / Kg, p.o.), a reference item, or with Vehicle (10 ml / Kg, p.o.) and mechanical hypersensitivity was measured 0.5, 1 , 2, 4, 6 and 8 hours after treatment.Results:

[0195] The MB-657 (10 mg / Kg, p.o.) decreased the mechanical sensitivity (hyperalgesia) by 99 %, when compared to the Vehicle group (p < 0.05). Gabapentin (70 mg / Kg, p.o.) also reduced the mechanical hyperalgesia by 95 % when compared with the Vehicle group (p<0.05) (Figure 5, Panels C and D).Example 27 - Evaluation of the analgesic effect of MB-611 according to the present invention, when administered in brain or spinal cord, in neuropathic pain model induced by chemotherapy in miceMethods:

[0196] According to the PK data, the MB-611 was found in the brain. Taking this into account, we decided to inject MB-611 directly into the brain and into the spinal cord and assess neuropathic pain in mice. For this purpose, the same animal model of neuropathy induced by chemotherapy (paclitaxel) described in the example 25 was used. The analgesic effect of MB-611 was compared with Gabapentin, a reference drug used to treat neuropathic pain. The mechanical hypersensitivity represented as paw withdrawal threshold was assessed with von Frey hairs using the up-down paradigm before and after the Paclitaxel treatment.

[0197] To assess the analgesic effect of MB-611 on the brain, the animals that became neuropathic following paclitaxel treatment were treated with MB- 611 (150 and 300 ng / site, 5pL, by intracerebroventricular route), Gabapentin (30,000 ng / site, 5 pL, by intracerebroventricular route), a reference item, or with Vehicle (5 pL, by intracerebroventricular route) and mechanical hypersensitivity was measured 0.25, 0.5, 1 , 2, 4 and 6 hours after treatment.

[0198] To assess the analgesic effect of MB-611 on the spinal cord, the neuropathic mice were treated with MB-611 (75 and 150 ng / site, 5pL, intrathecal route), Gabapentin (30,000 ng / site, 5pL, intrathecal route), a reference item, or with Vehicle (5 pL, intrathecal route) and mechanical hypersensitivity was measured 0.25, 0.5, 1 , 2, 4, 6 and 8 hours after treatment.Results:

[0199] The MB-611 (150 and 300 ng / site) injected by intracerebroventricular route, caused a significant reduction of the mechanical sensitivity (decrease of hyperalgesia) in 35 and 49%, respectively, when compared to the Vehicle group (p< 0.05). Gabapentin (30,000 ng / site, 5 pL, intracerebroventricular route) also reduced the mechanical hyperalgesia by 71 % when compared with the Vehicle group (p<0.05). (Figure 6, Panels A and B).

[0200] When injected by intrathecal route, the MB-658 (75 and 150 ng / site) significantly decreased the mechanical sensitivity (causing analgesia) in 72 and 60%, respectively, when compared to the Vehicle group (p< 0.05). Gabapentin (30,000 ng / site, 5 pL, intrathecal route) also reduced the mechanical hyperalgesia by 88% when compared with the Vehicle group (p<0.05). (Figure 6, Panels C and D).Example 28 - Evaluation of the analgesic effect of MB-658 according to the present invention, when administered in brain or spinal cord, in neuropathic pain model induced by chemotherapy in miceMethods:

[0201] According to the PK data, the MB-658 was found in the spinal cord and brain. With the knowledge that MB-657 is completely converted into MB- 658 after its administration, we decided to inject MB-658 directly into the spinal cord and into the brain and assess neuropathic pain in mice. For this purpose, the same animal model of neuropathy induced by chemotherapy (paclitaxel) described in the example 25 was used. The analgesic effect of MB-658 was compared with Gabapentin, a reference drug used to treat neuropathic pain. The mechanical hypersensitivity represented as paw withdrawal threshold wasassessed with von Frey hairs using the up-down paradigm before and after the Paclitaxel treatment.

[0202] To evaluate the analgesic effect of MB-658 on the spinal cord, the neuropathic mice were treated with MB-658 (12.5, 25 and 50 ng / site, 5 pL, intrathecal route), Gabapentin (30,000 ng / site, 5pL, intrathecal route), a reference item, or with Vehicle (5 pL, intrathecal route) and mechanical hypersensitivity was measured 0.083, 1 , 2, 4, 6 and 8 hours after treatment.

[0203] To assess the analgesic effect of MB-658 on the brain, the animals that became neuropathic following paclitaxel treatment, were treated with MB- 658 (25, 50 and 100 ng / site, 5 pL, by intracerebroventricular route), Gabapentin (30,000 ng / site, 5 pL, by intracerebroventricular route), a reference item, or with Vehicle (5 pL, by intracerebroventricular route) and mechanical hypersensitivity was measured 0.25, 0.5, 1 , 2, 4 and 6 hours after treatment.Results:

[0204] When injected by intrathecal route, the MB-658 (25 and 50 ng / site) significantly decreased the mechanical sensitivity (causing analgesia) in 56 and 77 %, respectively when compared to the Vehicle group (p < 0.05). Gabapentin (30,000 ng / site, 5 pL, intrathecal route) also reduced the mechanical hyperalgesia by 85 % when compared with the Vehicle group (p<0.05). The mean EDso value for MB 662 in suppressing the mechanical hyperalgesia at 4thhour was 23.94 (18.36 - 31 .06) ng / site (Figure 7, Panels A and B).

[0205] The MB-658 (25, 50 and 100 ng / site) injected by intracerebroventricular route, caused a significant and dose-dependent reduction of the mechanical sensitivity (decrease of hyperalgesia) in 32, 46 and 60 %, respectively when compared to the Vehicle group (p < 0.05). Gabapentin (30,000 ng / site, 5 pL, injected by intracerebroventricular route) also reduced the mechanical hyperalgesia by 78 % when compared with the Vehicle group (p<0.05). The mean EDso value for MB 662 in suppressing the mechanical hyperalgesia at 4thhour was 42.53 (2.60 - 95.35) ng / site (Figure 7, Panels C and D).Example 29 - Evaluation of the analgesic action of MB-662 according to the present invention in post-operative pain model induced by plantar incision in miceMethods:

[0206] The efficacy of the MB-662 according to the present invention, was carried out on the plantar incision model in mice (Brennan TJ et al. Pain 64: 493-502, 1996 and adapted to mice by Pogatzki EM, Raja SN. Anesthesiology 99: 1023-1027, 2003). Furthermore, its analgesic efficacy was compared with Morphine, a reference drug commonly used to treat post-operative pain. For this, male and female mice were kept anesthetized by the inhalation of 3% isoflurane plus 3% oxygen. Next, a 5 mm longitudinal incision, starting 2 mm from the proximal edge of the heel and extending toward the toes, was made with a number 11 blade (Advantive, Wuxi Xinda Medical Device Co. LTD, Jiangsu, CN) through the skin and fascia. With the aid of a curved forceps, the underlying muscle and tendons were carefully elevated, and then replaced to the normal anatomical position. After the skin was closed with a single suture 8.0 silk (Shalon®) in the middle of the incision. Sham mice underwent the anesthesia without an incision. The mechanical sensitivity was assessed with von Frey hairs using the up-down paradigm and was measured before and 24 h post-incision. Then, the animals were treated with MB-662 (10 mg / Kg, p.o.), or with Morphine (10 mg / Kg, s.c.), a reference item, or with Vehicle (10 ml / Kg, p.o.) and the mechanical sensitivity was newly measured 1 , 2, 4, and 6 hours after treatment. Before treatment, 24 hours after the incision, a reduction in the paw withdrawal threshold by 61 % was observed.

[0207] It was also assessed the thermal hyperalgesia to heat on hot-plate apparatus (Ugo Basile, Italy) at a constant temperature (52 ± 0.1 °C). The contact time of the incised hind paw with the hot plate was recorded until withdrawal response (licking, shaking, or guarding) and measured as latency time (s). A cut-off time of 30 s was used to avoid tissue damage. The thermal hyperalgesia was measured before testing (48 h post-incision), then the animals were treated with MB-662 (10 mg / Kg, p.o.), or with Morphine (10 mg / Kg, s.c.), a reference item, or with Vehicle (10 ml / Kg, p.o.) and the thermalhyperalgesia was newly measured 3 hours after treatment with MB-662 and 1 hour after treatment with Morphine.Results:

[0208] The MB-662 (10 mg / Kg, p.o.) significantly decreased the mechanical sensitivity (hyperalgesia) by 59 %, when compared to the Vehicle group (p < 0.05). Likewise, Morphine (10 mg / Kg, s.c.) also reduced the same extent (54 %) the mechanical hyperalgesia when compared with the Vehicle group (p < 0.05) (Figure 8, Panels A and B). The operated animals that were treated with the Vehicle showed a 28 % reduction in the latency time for withdrawal response (licking, shaking, or guarding) compared to the group of Sham animals. The MB-662 (10 mg / Kg, p.o.) was able to reverse the latency time (analgesia) by 30 % while Morphine reversed this effect by 183 % (p < 0.05) (Figure 8, Panel C).Example 30 - Evaluation of some mechanisms of action of the MB-611 according to the present invention in neuropathic pain induced by partial sciatic nerve ligation (PSNL) in the miceMethods:

[0209] According to the present invention, it was assessed the possible mechanism underlying the MB-611 analgesic effect in reducing mechanical hyperalgesia in a mouse model of neuropathic pain (PSNL). The same model described in the example 21 was used. However, before the treatment with the MB-611 (30 mg / Kg, p.o.), the animals were treated with voltage-dependent K+channel blocker (TEA, 4 mg / kg, intraperitoneally), or with the ATP -dependent K+channel blocker (Glibenclamide, 10 mg / kg, intraperitoneally), or with L type of Ca2+channel activator (BAYK-8644, 8 mg / kg, intraperitoneally) or with an opioid receptor antagonist (Naloxone, 5 mg / kg, intraperitonially). These treatments were performed with the aim of evaluating the participation of these targets / pathways in the analgesic effect promoted by MB-611 . The mechanical sensitivity thresholds were measured 0.5, 1 , 2, 4, 6, and 8 hours after treatments.

[0210] Other targets evaluated to assess the possible mechanism of action of MB-611 were P2X purinergic receptors and the AdenylateCyclase / cAMP / PKA signaling pathway. For that, mice were pretreated with MB-611 according to the present invention (30 mg / Kg, p.o.), or with TNP-ATP (10 pmol / kg, intraperitoneally), a P2X receptor antagonist, used as a positive control, or with Vehicle (10 ml / Kg, p.o.). After 1 hour, animals received an intraplantar injection of [3-5-Metileno ATP (200 nmol / paw), a P2X receptor agonist or Forskolin (50 nmol / paw) an Adenylate Cyclase (AC) enzyme activator. The mice were assessed by 5 minutes (after [3-5-Metileno ATP injection) and 10 minutes (after Forskolin injection), and the time that they spent licking the injected paw was recorded with a chronometer and considered to be indicative of nociception.

[0211] To evaluate the possible involvement of the voltage-gated Na+channels in the analgesic action of MB-611 , mice were pretreated with MB- 611 (30 mg / Kg, p.o.), or with Carbamazepine (30 mg / kg, p.o.), a voltage-gated Na+channels blocker, used as positive control or with Vehicle (10 ml / Kg, p.o.). After 1 hour, the animals received an intraplantar injection of Veratrine (0.3 pg / paw), a voltage-gated Na+channels activator and 1 hour after the mechanical sensitivity thresholds were measured by means of electronic von Frey.

[0212] To test if TRPV1 and TRPA1 receptors / signaling pathways constitute potential targets for the antinociceptive actions of MB-611 , mice were treated with MB-611 (30 mg / Kg, p.o.), or with Capsazepine (4 mg / kg, i.p.) a TRPVI receptor antagonist, used as positive control or with HC-030031 (100 mg / kg, i.p.) a TRPA1 receptor antagonist, used as positive control or with Vehicle (10 ml / Kg, p.o.). After 1 hour, the animals received an intraplantar injection of Capsaicin (1.6 pg / paw) or Cinnamaldehyde (100 nmol / paw). The mice were assessed by 5 minutes and the time that they spent licking the injected paw was recorded with a chronometer and considered to be indicative of nociception.

[0213] In another set of experiments, animals that became neuropathic after the PSNL, were treated during 14 days with MB-611 (30 mg / Kg, p.o.). Sham mice had the nerve localized but not ligated and received Vehicle (10 ml / Kg, p.o.) during 14 days. After this period, the animals were euthanizedand the cortex, spinal cord and sciatic nerve were collected and evaluated by PCR array (TaqMan Array 96 - Well Fast Plate, Neuropathic Pain Plate, FP002A1 L). For these analyses, total RNA from tissues was isolated and transcribed in cDNA using random primers and probes. The experimental procedures were carried out according to the manufacturer's instructions. Normalized gene expression was calculated as a ratio of Ct of target gene vs housekeeping gene (18S) transcripts (ACt) and changes of expression vs control (2-AACt).Results:

[0214] According to the present invention, MB-611 (30 mg / Kg, p.o.) promoted an increase in the paw withdrawal threshold (decreased the mechanical sensitivity and caused analgesia), especially in the time points of 1 to 4 hours after its oral administration. Pretreatment of animals with Glibenclamide, TEA, with BAYK-8644 or with Naloxone, significantly prevented the analgesic action of MB-611 characterized by increase in the paw withdrawal threshold by 31 , 63, 93 and 75 %, respectively. Taken together, these results further support the analgesic effect elicited by MB-611 in the PSNL-induced neuropathic pain model. Furthermore, they also suggest that this effect seems to be modulated, at least in part, by activation of endogenous opioid system, opening of voltage-dependent K+channels and / or blocking of Ca2+ channels (Table 4).

[0215] The MB-611 (30 mg / Kg, p.o.) significantly inhibited the nociceptive behavior response caused by [3-5-Metileno ATP by 82 % when compared to the Vehicle group (p < 0.05). TNP-ATP (10 pmol / kg, administered intraperitoneally), used as positive control, also reduced by 60 % the nociceptive behavior when compared with the Vehicle group (Table 4).

[0216] After the Veratrine injection in the mice paw it was observed that the MB-611 (30 mg / Kg, p.o.) elicited an increase in the paw withdrawal threshold (decreased the mechanical sensitivity) by 74%. The positive control Carbamazepine increased the paw withdrawal threshold (decreased the mechanical sensitivity) by 88 % when compared to the Vehicle group (p < 0.05) (Table 4).

[0217] Taking into account the experiments carried out in vivo, we cannot associate the TRPV1 and TRPA1 receptors and the Adenylate Cyclase / cAMP / PKA pathway with the analgesic effect of MB-611 (Table 4).

[0218] PCR-array allowed the evaluation of the expression of most genes related to neuropathic pain. The PCR-assay data in the cortex tissue showed an increase in the expression of Ptger4, in animals with neuropathy that received Vehicle. Ptger4 is the gene that encodes the EP4 receptor for PGE2, the blockade of the prostanoid EP4 receptor may represent a new therapeutic strategy in pain. The repeated treatment with MB-611 (30 mg / Kg, p.o.) reduced the gene expression at a lower level than that quantified in non-neuropathic animals (Sham) (Figure 9 A). In the spinal cord tissue, no relevant changes were found. On the other hand, in the sciatic nerve, neuropathic animals showed an increase in the expression of Ccr2, Ngf, P2rx4 and P2rx7 genes. After the repeated treatment with MB-611 (30 mg / Kg, p.o.), was observed a decrease of Ccr2 expression close to or smaller than seven folds when compared to the Sham group (Figure 9 B). Ccr2 is the gene that encodes the C-C Motif Chemokine Receptor 2, protein mediates agonist-dependent calcium mobilization and inhibition of adenylyl cyclase. In relation to Ngf, P2rx4 and P2rx7 genes, was observed a reduction of expression close to the basal values (Figure 9 C, D and F). The A / gfgene encodes the Nerve Growth Factor protein and has a nerve growth stimulating activity and the complex is involved in the regulation of growth and the differentiation of sympathetic and certain sensory neurons. Ionotropic ATP signaling via excitatory and calcium permeable P2X receptor channels is appropriately recognized as a crucial factor in the initiation and maintenance of pathological pain.Example 31 - Evaluation of some of the mechanisms of the analgesic action of the MB-657 and MB-662 according to the present invention in neuropathic pain induced by partial sciatic nerve ligation (PSNL) in the mice.Methods:

[0219] According to the present invention, it was assessed the possible mechanism underlying the MB-657 analgesic effect in reducing mechanical hyperalgesia in a mouse model of neuropathic pain (PSNL). The same modeldescribed in the example 21 was used. However, before the treatment with the MB-657 (10 mg / Kg, p.o.), the animals were treated with voltage-dependent K+channel blocker (TEA, 4 mg / kg, intraperitoneally), or with the ATP -dependent K+channel blocker (Glibenclamide, 10 mg / kg, intraperitoneally), or with or with an opioid receptor antagonist (Naloxone, 5 mg / kg, intraperitonially), or with a CB1 cannabinoid receptor antagonist (AM-251 , 3 mg / kg, intraperitoneally), or woth a CB2 cannabinoid receptor antagonist (AM-630, 3 mg / kg, intraperitoneally). Regarding the effects of MB-662, before the treatment with the MB-662, the animals were injected with 8-Cyclopentyl-1 ,3- dipropylxanthine, an A1 adenosine receptor antagonist (DPCPX, 3 mg / kg, intraperitoneally), or with a CB2 cannabinoid receptor antagonist (AM-630, 3 mg / kg, intraperitoneally), or with a L type of Ca2+channel activator (BAYK- 8644, 8 mg / kg, intraperitoneally). These treatments were performed with the aim of evaluating the participation of these targets / pathways in the analgesic effect promoted by MB-657 and MB-662. The mechanical sensitivity thresholds were measured 0.5, 1 , 2, 4, 6, and 8 hours after treatments.

[0220] Other targets evaluated to assess the analgesic mechanism of action of MB-657 were the voltage-gated Na+ channels. For that, the mice were pretreated with MB-657 according to the present invention (10 mg / Kg, p.o.), Carbamazepine (30 mg / kg, p.o.), a voltage-gated Na+ channels blocker, used as positive control or with Vehicle (10 ml / Kg, p.o.). After 1 hour the animals received an intraplantar injection of Veratrine (0.3 pg / paw), a voltagegated Na+ channels activator and 1 hour after the mechanical thresholds were assessed with von Frey hairs using the up-down paradigm.

[0221] To test if TRPV1 and TRPA1 receptors / signaling pathways constitute potential targets for the antinociceptive actions of MB-657, mice were treated with MB-657 (10 mg / Kg, p.o.), or with Capsazepine (4 mg / kg, i.p.) a TRPVI receptor antagonist, used as positive control or with HC-030031 (100 mg / kg, i.p.) a TRPA1 receptor antagonist, used as positive control or with Vehicle (10 ml / Kg, p.o.). After 1 hour, the animals received an intraplantar injection of Capsaicin (1.6 pg / paw) or Cinnamaldehyde (100 nmol / paw). The mice were assessed by 5 minutes and the time that they spent licking theinjected paw was recorded with a chronometer and considered to be indicative of nociception.

[0222] The Adenylate Cyclase / cAMP / PKA signaling pathway was also acessed. For this, mice were pretreated with MB-657 according to the present invention (10 mg / Kg, p.o.) or with Vehicle (10 ml / Kg, p.o.). After 1 hour, animals received an intraplantar injection of Forskolin (50 nmol / paw), an Adenylate Cyclase (AC) enzyme activator. The mice were assessed by 10 minutes and the time that they spent licking the injected paw was recorded with a chronometer and considered to be indicative of nociception.

[0223] The participation of P2X purinergic receptors in the analgesic activity of MB-662 was also evaluated. For that, mice were pretreated with MB- 662 according to the present invention (10 mg / Kg, p.o.), or with TNP-ATP (10 pmol / kg, intraperitoneally), a P2X receptor antagonist, used as a positive control, or with Vehicle (10 ml / Kg, p.o.). After 1 hour, animals received an intraplantar injection of [3-5-Metileno ATP (200 nmol / paw), a P2X receptor agonist. The mice were assessed by 5 minutes (after [3-5-Metileno ATP injection) and the time that they spent licking the injected paw was recorded with a chronometer and considered to be indicative of nociception.Results:

[0224] According to the present invention, MB-657 and MB-662 promoted an increase in the paw withdrawal threshold (decreased the mechanical hypersensitivity). Pretreatment of animals with TEA (voltage-dependent K+ channel blocker), Glibenclamide (an ATP-dependent K+ channel blocker), AM- 630 (a CB2 cannabinoid receptor antagonist) prevented the analgesic effect induced by the MB-657 by 80, 82 and 62 %, respectively. In the same way, the treatment of animals with AM-630 and BAYK-8644 (a L type of Ca2+ channel activator) prevented the analgesic effect induced by the MB-662 by 59 and 91 %, respectively. Furthermore, these data suggest that the analgesic effect of MB-657 and MB-662 seems to be modulated by pathways that involve CB2 cannabinoid mediated effects, the opening of voltage-dependent and ATP- dependent K+ channels or the block of L type of Ca2+ channel (Table 4).

[0225] After the pain caused by Veratrine injection in the mice paw it was observed that the MB-657 (10 mg / Kg, p.o.) elicited an increase in the paw withdrawal threshold (decreased the mechanical sensitivity) by 74 %, an effect that was quite similar to that caused by positive control Carbamazepine, when compared to the Vehicle group (p < 0.05) (Table 4). This data makes the voltage-gated Na+ channels another possible target for the analgesic effect of MB-657.

[0226] Moreover the treatment of animals with MB-657 inhibited by 74% the capsaicin induced nociception and the treatment of animals with MB-662 inhibited by 100 % the [3-5-Metileno ATP induced nociception (Table 4), which reinforces the possible participation of Ca2+ modulation in the analgesic effect of MB-657 and MB-662.

[0227] Taking into account the results of the experiments carried out in vivo with MB-657 and MB-662 we cannot associate the signaling pathways related with opioid receptors, CB1 cannabinoid receptors, TRPA1 receptors, A1 Adenosine receptors, as well as the Adenylate Cyclase / cAMP / PKA pathway with the analgesic effect of MB-657 (Table 4).Example 32 - Assessment of the mechanism of action of analgesic action of MB-611, MB-657 and MB-658 on hP2X3, hP2X4, hP2X7 and mTRPAI human receptors in HEK293 cells.Methods:

[0228] In order to establish a stable cell line expressing the proteins associated with chronic pain targets, the HEK293 cell line underwent transfection with vectors (hP2X3, hP2X4, hP2X7, and mTRPAI ) by using the FuGENE® transfection reagent (Promega, Madison, Wl, USA). Following cell thawing, they were seeded at a density of 1 x 104 cells per well in a 24-well plate with a flat, transparent bottom. The transfection protocol was initiated 24 hours post-seeding. The cells were maintained in a controlled environment at 37 ± 0.3 °C with a 5 ± 0.3% CO2 atmosphere using an appropriate culture medium. After 48 hours of transfection, a selection antibiotic was introduced to maintain the positive cells expressing the target proteins, including ion channels. Following one week of selection, the cells underwent a processinvolving limiting dilution, selection, and amplification of stable expression clones. For the evaluation of calcium influx, cells transfected with ion channels (hP2X3, hP2X4, hP2X7, and mTRPAI ) were subcultured in 96-well plates at a density of 5 x 104 cells per well with a flat-bottomed, black, transparent plate. Similar to the previous culture conditions, the cells were kept in an environment with a temperature of 37 ± 0.3 °C, 5 ± 0.3% CO2, and were subjected to the Fluo-4 Direct Calcium Assay kit protocol (Thermo Fisher Scientific, Waltham, MA, USA) 24 hours after plating. In this assay, the cells were initially incubated with the Fluo-4 probe (Thermo Fisher Scientific, Waltham, MA, USA) for approximately one hour at room temperature while protected from light. Subsequently, the plate was placed in the FlexStation 3® equipment (Molecular Devices, San Jose, CA, USA) for the automatic injection of MB-611 (at concentrations of 3 and 30 pM), MB-657 (at 10 pM), and MB-658 (at 10 pM) for the hP2X3, hP2X4, and hP2X7 targets, and MB-658 (at 10 pM) for the mTRPAI receptor. After a 15-m inute incubation, the channel-specific agonist was added via automatic pipetting using the FlexStation 3® equipment. The results were obtained and analyzed using the SoftMax® Pro Software.Results:

[0229] The date presented in Table 4, indicate that none of the tested compounds exhibited significant changes on the calcium influx caused by activation of hP2X3, hP2X4, hP2X7, or mTRPAI channels (Table 5).Example 33 - Evaluation of the analgesic effect MB-611 according to the present invention in Prostaglandin E2-induced mechanical hyperalgesia in rats.Methods:

[0230] According to the present invention, the effect of compound MB-611 on nociception caused by relevant inflammatory mediator PGE2 was assessed and its activity was compared with Gabapentin a reference drug used to treat chronic pain. For that, male and female rats were pretreated with MB-611 (10, 30 and 100 mg / Kg, p.o.), Gabapentin (70 mg / kg, p.o.), a reference item or with Vehicle (10 ml / Kg, p.o.). Following 1 hour after treatments, the animals received an intraplantar injection of 50 pL of prostaglandin E2 (PGE2: 100ng / paw) and mechanical sensitivity was measured 0.5, 1 , 2, 4, and 6 hours after treatment. The mechanical sensitivity thresholds were measured by means of electronic von Frey (Analgesimetro digital EFF-302, Insight Pesquisa e Ensino, Sao Paulo, Brazil) before and after the PGE2 injection.Results:

[0231] The intraplantar PGE2 injection elicited a time dependent and significant reduction in the paw withdrawal threshold to mechanical stimulus by 73 %. Treatment of animals with MB-611 (30 and 100 mg / Kg, p.o.), significantly reversed the paw withdrawal threshold (analgesia) to mechanical stimulus by 44 and 22 % (an increase of 56 and 78% on mechanical sensitivity, respectively) when compared to the Vehicle group (p < 0.05). Likewise, Gabapentin (70 mg / kg, p.o.) also reduced the same extent (81 %) the mechanical hyperalgesia caused by PGE2 when compared with the Vehicle group (p < 0.05) (Figure 10, Panels A and B).Example 34 - Evaluation of the analgesic action of MB-657 according to the present invention in Prostaglandin E2-induced mechanical and thermal (heat) hyperalgesia in rats.Methods:

[0232] According to the present invention, the effect of MB-657 on nociception caused by specific inflammatory mediator also was studied in rats, and the activity compared with Dipyrone, a reference drug used to treat acute pain. The efficacy of the MB-657 on thermal hyperalgesia was assessed by using a Hargreaves apparatus (Ugo Basile, Varese, Italy). Furthermore, mechanical hypersensitivity was also evaluated in the same animal model as described in the example 33. All animals, female and male rats, were evaluated for basal parameters of thermal and mechanical stimulus.Results:

[0233] The PGE2 injection into the rat paw promoted a reduction in the paw withdrawal threshold to thermal stimulus by 67 %. The MB-657 (10 mg / Kg, p.o.) caused an analgesic effect characterized by reversing the reaction time after thermal stimulus in PGE2 treated animals by 79 % when compared to the Vehicle group (p < 0.05). Dipyrone (100 mg / kg, p.o.) reversed the reaction timeby 87 % when compared with the Vehicle group (p < 0.05) (Figure 10, Panels C and D).

[0234] The PGE2 injection caused a time dependent and significant reduction in the paw withdrawal threshold to mechanical stimulus by 66%. Treatment of animals with MB-657 (10 mg / Kg, p.o.) 1 h before, caused an analgesic action characterized by reducing the paw withdrawal threshold to mechanical stimulus by 37 % (an increase of 63 % on mechanical sensitivity) when compared to the Vehicle group (p < 0.05). Likewise, Dipyrone (100 mg / kg, p.o.) also reduced by 75% the mechanical hyperalgesia when compared with the Vehicle group (p < 0.05) (Figure 10, Panels E and F).Example 35 - Evaluation of the efficacy of MB-657 according to the present invention in Carrageenan-induced thermal hyperalgesia and paw oedema in rats.Methods:

[0235] According to the present invention, the effect of MB-657 on nociception caused by inflammatory mediator Carrageenan also was studied, and the activity compared with Indomethacin, a reference drug used to treat inflammatory processes, including inflammatory pain. All animals were evaluated for basal parameters of thermal stimulus (Hargreaves apparatus - Ugo Basile, Varese, Italy) and paw volume (mL) (Plethysmometer - Ugo Basile, Varese, Italy). Next, the rats were pretreated with MB-657 (10 mg / Kg, p.o.), or with Indomethacin (5 mg / kg, s.c.), a reference item, or with Vehicle (10 ml / Kg, p.o.). Following 1 hour, the animals received an intraplantar injection of 100 pL of Carrageenan (300 pg / paw) and thermal sensitivity and paw volume were measured 1 , 3, 5, and 7 hours after treatment.Results:

[0236] The Carrageenan injection into the rat paw caused a time dependent reduction in the paw withdrawal threshold to thermal stimulus by 50 % (hyperalgesia). Treatment of animals with MB-657 (10 mg / Kg, p.o.) significantly reversed the reaction time after thermal stimulus by 92 % (analgesia) when compared to the Vehicle group (p < 0.05). In a similar way,Indomethacin (5 mg / kg, s.c.) reversed the reaction time by 119 % when compared with the Vehicle group (p < 0.05) (Figure 11 , Panels A and B).

[0237] Furthermore, the intraplantar injection of carrageenan produced a marked and time dependent paw oedema formation. The treatment of animals with MB-657 (10 mg / Kg, p.o.) significantly reduced the carrageenan-induced oedema, with inhibition of 76 %, when compared to the Vehicle group (p< 0,05). Indomethacin (5 mg / kg, s.c.) also reduced the same extent (78 %), the paw oedema formation, when compared with the Vehicle group (p < 0.05) (Figure 11 , Panels C and D).Example 36 - Assessment of MB-657 and MB-658 effects on IL- 1p release in murine macrophage.Methods:

[0238] To assess the impact of the compounds on IL-1 [3 release, Raw- 264.7 cells (murine macrophages) were seeded at a density of 1 x 105cells per well in a 96-well plate and incubated overnight under controlled conditions at 37 ± 0.2 °C with 5 ± 0.2% CO2. Subsequently, the cells were exposed to either compounds MB-657 (at 60 pM) and MB-658 (at 60 pM) or a vehicle control. After a 2-hour incubation, lipopolysaccharide (LPS) at a concentration of 100 ng / mL was introduced to the wells for 24 hours, followed by the addition of a 5 mM ATP solution to activate the NLRP3 inflammasome (for 60 minutes). Upon completion of the assay, the plates were centrifuged, and the supernatant was collected for the quantification of IL-1 [3 using an enzyme- linked immunosorbent assay (ELISA). The measurement of IL-1 [3 served as an indicator of NLRP3 inflammasome activity.Results:

[0239] As illustrated in Table 5, both compounds MB-657 and MB-658 failed to inhibit the release of IL-1 [3 caused by NLRP3 inflammasome activation with LPS + ATP in murine macrophages.Example 37 - Assessment of MB-611, MB-657, MB-658 and MB- 662 effects on COX-1 and COX-2 activities.Methods:

[0240] To evaluate the possible effect of the compounds on the enzymatic activities of cyclooxygenases 1 (COX-1 ) and 2 (COX-2), we followed the experimental procedures outlined in the respective commercial assay kits. Specifically, the COX-1 assay employed the fluorometric inhibition assay kit from Abeam, while the COX-2 assay utilized the fluorometric inhibition assay kit provided by Sigma-Aldrich. For these investigations, we incubated the compounds MB-611 (at concentrations ranging from 1 to 30 pM), MB-657 (at concentrations from 1 to 100 pM), MB-658 (at concentrations from 1 to 100 pM), and MB-662 (at concentrations from 1 to 100 pM) in 96-well plates, alongside other essential reagents and controls required to assess their impact on enzyme activity.Results:

[0241] As indicated in Table 5, none of the compounds demonstrated any relevant inhibitory effect on both the COX-1 and COX-2 activities. Moreover, both MB-611 and MB-657 failed to elicit any inhibition of COX-2 enzyme activity. In contrast, MB-662 exhibited a weak inhibitory effect, of about 20%, at a concentration of 100 pM in relation to COX-2 enzyme activity. Lastly, MB- 658 displayed a weak inhibitory effect on COX-2 enzyme activity, resulting in a 16% inhibition at a concentration of 10 pM, 23% at 30 pM, and 34% at 100 pM, with the inhibitory concentration at 50% (ICso) exceeding 100 pM.able 4: Possible in vivo mechanisms of action of MB-611 , MB-657 and MB-662.able 5: Investigation of the possible in vitro mechanisms of action of the MB-611 , MB-657, MB-658 and MB-662.Safety studies of MB-611 and MB-657Example 38 - Genotoxicity evaluation of MB-611 - AMES test.Methods:

[0242] Genotoxicity tests were developed to detect substances with the potential to induce damage to genetic material. These tests are recommended by regulatory agencies worldwide as part of the safety assessment of chemicals as they identify risks related to DNA damage. Substances found positive in these assays that detect genetic modifications are potentially carcinogenic and / or mutagenic to humans. Thus, the bacterial mutagenicity test is widely used as an initial screening to assess possible genotoxic activity, in particular, for point mutation-inducing activity. The reverse bacterial mutation assay was performed in accordance to OECD recommendations, guideline 471 - Guideline for Testing of Chemicals. Method 471 “Bacterial Reverse Mutation Test” (Adopted: 26 June 2020). The preliminary test with the strain TA 100, in the absence or presence of metabolic activation (S9) was conducted aiming at selecting adequate concentrations of the MB-611 for the definitive test.Results:

[0243] MB-611 showed no evidence of mutagenic activity in the reverse mutation test in Salmonella typhimirium bacteria, both in the absence and in the presence of metabolic activation (S9), in the TA 97a, TA 98, TA 100, TA 102 and TA 1535 (Table 6).Example 39 - Genotoxicity evaluation of MB-611 Micronucleous test.Methods:

[0244] The micronucleus test detects genetic changes resulting from chromosomal lesions and / or damage to the mitotic system. The formation of micronuclei is an indicative of irreversible losses to DNA and, its frequency can be used as an index of mutagenicity. It is already known that there is a positive correlation between the increase in the frequency of micronuclei and the appearance of tumors in rodents and humans. The micronucleus test in mouse bone marrow was carried out in a GLP compliant condition in accordance to the recommendations of OECD guideline 474 - Guideline for Testing of Chemicals.Results:

[0245] In the preliminary test the animals (male mice) were treated with MB-611 , for 3 days, with doses of 8, 50, 320 and 2,000 mg / kg, orally. No clinical signs related to severe toxicity were observed in animals that received doses of 8, 50 and 320 mg / kg. However, animals treated with the dose of 2,000 mg / kg showed drowsiness, then fell asleep and remained asleep until the 6thhour after administration of MB-611 . During sleep, the animals showed signs compatible with respiratory distress and hypothermia, compared to untreated animals. After the second administration of MB-611 , the dose of 2,000 mg / kg caused the death of the animals. Therefore, as lethality was identified with the highest dose (2,000 mg / kg), treatments with intermediate doses were carried out. Then, groups of male mice were treated, for 3 days, with a dose immediately lower than the dose that promoted mortality and intermediate doses (500, 800 and 1 ,250 mg / kg, orally). The same clinical signs (drowsiness, falling asleep, difficulty breathing and hypothermia) were observed with doses of 800 and 1 ,250 mg / kg. Additionally, mortality was identified at a dose of 1 ,250 mg / kg. Therefore, a group of female mice was treated with the maximum dose established for males (500 mg / kg, orally) and another group of females was treated with a dose above the maximum dose established for males (800 mg / kg, p.o.). No mortality was observed with any of the doses tested in females, however, the same clinical signs (drowsiness and falling asleep) wereobserved up to 3 hours after administration, after this period the animals showed normal behavior. For the definitive test, doses of 32, 125 and 500 mg / kg of MB-611 were selected, administered orally, for three consecutive days. None of the administered doses caused an increase in the number of micronuclei in immature erythrocytes (EPCs), when compared to the Vehicle (20% DMSO, 60% PEG 400, 5% Tween 80 and 15 % Propylene glycol, 0.1 mL / 10g, p.o.). The EPC / ENC ratio (ratio) has not changed. The reduction of EPCs in relation to the number of ENCs reflects toxicity and depression of bone marrow cells (cytotoxicity). Therefore, these data indicate that MB-611 did not present toxic action in relation to bone marrow cells. Treatment with Cyclophosphamide (25 mg / kg, intraperitoneally for 2 days), used as Positive Control of the test, increased the frequency of micronuclei in EPCs when compared to the Vehicle, validating the experiment. The results are presented in Table 7.

[0246] [Table 7] Incidence of micronucleated polychromatic erythrocytes (MNPCE) and the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes in mice treated with MB-611 .MNPCE / 4,000 Dose Ratio PCE / NCEGroup Route PCE(mg / Kg) (Mean t S.D.)(Mean ± S.D.)Vehicle 0 p.o. 6.60 ± 2.55 1.48 ± 0.2532 p.o. 8.10 ± 3.69 1.76 ± 0.50MB-611 125 p.o 9.90 ± 4.25 1.43 ± 0.39500 p.o 8.56 ± 1.88 1.64 ± 0.29Cyclophosphamide 25 i.p. 14.10 ± 5.30* 1.53 ± 0.24 p.o. = per os; i.p. = intraperitoneal; PCE = polychromatic erythrocytes; NCE = normochromatic erythrocytes; MNPCE = micronucleated polychromatic erythrocytes; S.D. = standard deviation. * Significant difference from vehicle (20% DMSO, 60% PEG 400, 5% Tween 80 and 15 % Propylene glycol by Kruskal-Wallis test: *p < 0.05.Example 40 - Maximum tolerated dose and dose selection of MB- 611, in miceMethods:

[0247] This assay was designed to investigate the safety and tolerability of MB-611. For this, two different phases were conducted: Phase I: to determine the maximum tolerated dose (MTD) of MB-611 , and Phase II) exploratory evaluation of the toxicity of repeated treatments with MB-611 for 7 days, as described below:

[0248] Phase I: Determination of MTD by a single oral administration of MB-611 in an escalating dose scheme. Animals were randomly distributed in five experimental groups (3 males and 3 females / group). Animals from the vehicle-treated group were treated with a formulation containing 20% DMSO, 60% PEG 400, 5% Tween 80 and 15 % Propylene glycol, 0.1 mL / 10g, oral route. Four additional groups were treated with different doses of MB-611 (175 mg / kg, 375 mg / kg, 550 mg / kg and 1 ,000 mg / kg). The animals in the first group (group 1 ) received the MB-611 at the dose of 175 mg / kg, in a single administration. The animals were observed for a period of 24 hours to detect possible signs of toxicity. As no signs of toxicity were observed in the group that received the dose of 175 mg / kg, a higher dose was administered to group 2 and so on. Each group was euthanized 14 days after the treatment, for subsequent analyses.

[0249] Phase II: 7-day repeated toxicity study. The protocol consisted of two experimental groups (5 males and 5 females / group). Group 1 was treated with the Vehicle and the group 2 was treated with MB-611 (300 mg / kg). All animals were treated by oral gavage once a day for 7 days. All animals were euthanized 14 days after the treatment for subsequent analyses. Clinical and behavior observations, as well as general macroscopic observations in the necropsy were carried out for animals from both phases even as the hematological and biochemical analyses for Phase II animals.Results:

[0250] The administration of a single dose of MB-611 at a dose of 550 mg / kg caused morbidity represented by lethargy, this effect was reversed 4hours after administration. The dose of 1 ,000 mg / kg caused prolonged lethargy and hypothermia, followed by death within 48 hours of evaluation. No morbidity and mortality were observed at doses of 175 mg / kg and 375 mg / kg. After the necropsy procedure, the weights (g) of the main organs (adrenal glands, spleen, brain, heart, kidney, thymus, liver, testicles, epididymis and ovary) were measured for each animal in all experimental groups. Doses of 375 mg / kg, 550 mg / kg and 1 ,000 mg / kg caused an apparent reduction in the absolute and relative weight of the epididymis (Tables 8 and 9). No statistical analysis was performed due to the experimental n (n = 3). Macroscopic evaluations carried out during the necropsy procedure of animals treated with MB-611 at doses of 175, 375, 550 and 1 ,000 mg / kg revealed no observable changes.

[0251] The repeated treatment with MB-611 (300 mg / kg) did not cause death nor clinical signs toxicity. In addition, no macroscopic changes were observed in organs and tissues collected from animals treated with MB-611 (300 mg / kg). Only a punctual reduction in platelets in male mice and an increase in the total number of cells in female mice, an increase in triglyceride levels and a reduction in glucose levels in male mice, compared to the Vehicle group (p<0.05). Thus, the dose of 300 mg / kg is the maximum dose to be tested to assess the subchronic and chronic toxicity of MB-611 (Tables 10, 11 and 12).

[0252] It is concluded, based on the results obtained, that MB-611 was safe within the experimental conditions evaluated, and the hematological and biochemical changes observed should be better explored and confirmed in longer-lasting toxicity studies.

[0253] [Table 8] Absolute weight (g) of the organs of male mice after a single dose of MB-611 , p.o. followed for 14 days without treatment - Phase I.&Standard Deviation, N=3.

[0254] [Table 9] Relative weight (%) of the organs of male mice after a single dose of MB-611 , p.o. followed for 14 days without treatment - Phase I.&Standard Deviation, N=3.

[0255] [Table 10] Analyses of hematological parameters of male mice after7 days of daily treatment with MB-611 , p.o. followed for 7 days without treatment - Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group; WBC - Total Cells; W-LCR - Large leukocyte (neutrophil) index; W-SCR - Small leukocyte (lymphocyte) index; W- MCR - Mean leukocyte (monocyte) index; RBC - Red blood cells; HGB - Hemoglobin; HCT - Hematocrit; MCV - Corpuscular Volume; MCH - Mean corpuscular hemoglobin; MCHC - Mean corpuscular hemoglobin concentration; PLT - Platelet count.

[0256] [Table 11] Analyses of hematological parameters of female mice after 7 days of daily treatment with MB-611 , p.o. followed for 7 days without treatment - Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group; WBC - Total Cells; W-LCR - Large leukocyte (neutrophil) index; W-SCR - Small leukocyte (lymphocyte) index; W- MCR - Mean leukocyte (monocyte) index; RBC - Red blood cells; HGB - Hemoglobin; HCT - Hematocrit; MCV - Corpuscular Volume; MCH - Mean corpuscular hemoglobin; MCHC - Mean corpuscular hemoglobin concentration; PLT - Platelet count.

[0257] [Table 12] Analyses of biochemical parameters of male mice after 7 days of daily treatment with MB-611 , p.o. followed for 7 days without treatment - Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group; ALT - Alanine aminotransferase; TRI - Triglycerides; TP - Total protein; CRE - Creatinine; ALB - Albumin; AST - Aspartate Aminotransferase; TC - Total cholesterol; AP - Alkaline phosphatase; P - Phosphorus; BT - Total Bilirubin.Example 41 - Maximum tolerated dose and dose selection of MB- 657, in miceMethods:

[0258] To assess the safety and tolerability of MB-657 the same assay described in the example 40 was used, with some modifications, as described below:

[0259] Phase I: Determination of MTD by a single oral administration of MB-657 in an escalating dose scheme. Animals were randomly distributed in three experimental groups (3 males and 3 females / group) and each group were treated with different doses of MB-657 (175, 550 e 1 ,500 mg / kg, p.o). The higher dose was limited by solubility. The animals in the first group (group 1 ) received the MB-657 at the dose of 175 mg / kg, in a single administration. The animals were observed for a period of 24 hours to detect possible signs of toxicity. As no signs of toxicity were observed in the group that received the dose of 175 mg / kg, a higher dose was administered to group 2 and so on. Each group was euthanized 24 hours after treatment, for subsequent analyses.

[0260] Phase II: 7-day repeated toxicity study. The protocol consisted of two experimental groups (5 males and 5 females / group). Group 1 was treated with the Vehicle (5% Ethanol, 45% PEG 400, 30% Propylene glycol, and 20% NaOH 0.9% - 0.1 mL / 10g, oral route) and the group 2 was treated with MB- 657 (200 mg / kg). All animals were treated by oral gavage once a day for 7 days. All animals were euthanized 24 hours after the last treatment, for subsequent analyses. Clinical and behavior observations, as well as general macroscopic observations in the necropsy were carried out for animals from both phases even as the hematological and biochemical analyses for Phase II animals.Results:

[0261] In phase I of the study, the Single administration of Test Item ST- 080.82 at doses of 175, 550 and 1500 mg / kg (step I) did not result in changes in general clinical signs in males. Females showed a slight reduction in grip strength in the first and second hour after administration of the 175 mg / kg dose. However, these signs were reversed and the remaining doses did not promote changes. Was also observed an increase in the absolute weight of the kidneys of male animals after a single administration of MB-657 (550 mg / kg), an increase of the heart of females after a single administration of MB- 657 at a dose of 1 ,500 mg / kg, and a reduction in the relative weight of the liver of female (single administration of 550 mg / kg) in comparison to the group of animals treated with a dose of 175 mg / kg (p<0.05) (Tables 13, 14 and 15).

[0262] [Table 13] Absolute weight (g) of the organs of male mice, 24 hours after single treatment with MB-657, p.o Phase I.&Standard Deviation. N=3.

[0263] [Table 14] Absolute weight (g) of the organs of female mice. 24 hours after single treatment with MB-657, p.o .- Phase I.&Standard Deviation. N=3.

[0264] [Table 15] Relative weight (%) of the organs of female mice. 24 hours after single treatment with MB-657, p.o Phase I.&Standard Deviation. N=3.

[0265] The repeated treatment with MB-657 (200 mg / kg) did not cause death nor clinical signs of toxicity. No changes in food consumption and body weight were observed. However, the repeated administration of MB-657 (200 mg / kg) promoted a reduction in the absolute and relative weight of the spleen in males and an increase in the absolute and relative weight of the spleen in females, when compared to the vehicle group (p<0.05 ) (Tables 16, 17, 18 and 19).

[0266] [Table 16] Absolute weight (g) of the organs of male mice after 7 days of daily treatment with MB-657, p.o .- Phase II.no&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group.

[0267] [Table 17] Relative weight (%) of the organs of male mice after 7 days of daily treatment with MB-657, p.o Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group.

[0268] [Table 18] Absolute weight (g) of the organs of female mice after 7 days of daily treatment with MB-657, p.o .- Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group.

[0269] [Table 19] Relative weight (g) of the organs of female mice after 7 days of daily treatment with MB-657, p.o Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group.

[0270] In relation to hematological analyses the repeated treatment with MB-657 caused a reduction in neutrophils, monocytes, platelets and an increase in lymphocyte, corpuscular volume and mean corpuscular hemoglobin in males. In females, an increase in red blood cells, hemoglobin, hematocrit and a reduction in the mean concentration of corpuscular hemoglobin were observed. The changes were compared with the vehicle group (p<0.05) (Tables 20 and 21 ). The results of biochemical analyses demonstrated a reduction in the levels of ALT, AST, albumin, urea, total bilirubin and an increase in phosphorus levels in male rats treated with MB- 657 (200 mg / kg) compared to the Vehicle group (p<0 .05). In females, a reduction in triglyceride levels was observed when compared to the vehicle group (p<0.05). Macroscopic evaluations performed during the necropsy procedure revealed no observable changes. The liver, spleen and kidneys were histopathologically examined and males and females presented mild non neoplastic lesions on the liver when compared to animals from the Vehicle control group (p<0.05) (Tables 22 and 23).

[0271] [Table 20] Analyses of hematological parameters of male rats after 7 days of daily treatment with MB-657, p.o .- Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group WBC - Total Cells; W-LCR - Large leukocyte (neutrophil) index; W-SCR - Small leukocyte (lymphocyte) index; W- MCR - Mean leukocyte (monocyte) index; RBC - Red blood cells; HGB - Hemoglobin; HCT - Hematocrit; MCV - Corpuscular Volume; MCH - Mean corpuscular hemoglobin; MCHC - Mean corpuscular hemoglobin concentration; PLT - Platelet count.

[0272] [Table 21] Analyses of hematological parameters of female rats after 7 days of daily treatment with MB-657, p.o Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group; WBC - Total Cells; W-LCR - Large leukocyte (neutrophil) index; W-SCR - Small leukocyte (lymphocyte) index; W- MCR - Mean leukocyte (monocyte) index; RBC - Red blood cells; HGB - Hemoglobin; HCT - Hematocrit; MCV - Corpuscular Volume; MCH - Mean corpuscular hemoglobin; MCHC - Mean corpuscular hemoglobin concentration; PLT - Platelet count.

[0273] [Table 22] Analyses of biochemical parameters of male mice after 7 days of daily treatment with MB-657. p.o Phase II.&Standard deviation; N=5; ‘Differs significantly in relation to the Vehicle group; ALT - Alanine aminotransferase; TRI - Triglycerides; PT - Total protein; CRE - Creatinine; ALB - Albumin; AST - Aspartate Aminotransferase; TC - Total cholesterol; FA - Alkaline phosphatase; P - Phosphorus; BT - Total Bilirubin.

[0274] [Table 23] Analyses of biochemical parameters of female mice after 7 days of daily treatment with MB-657. p.o .- Phase II.&Standard deviation; N=5; * Differs significantly in relation to the Vehicle group; ALT - Alanine aminotransferase; TRI - Triglycerides; PT - Total protein; CRE - Creatinine; ALB - Albumin; AST - Aspartate Aminotransferase; TC - Total cholesterol; FA - Alkaline phosphatase; P - Phosphorus; BT - Total Bilirubin.

[0275] Based on the results obtained The MB-657 did not promote relevant changes in parameters indicative of toxicity, such as mortality and / or morbidity and clinical signs. However, specific changes were detected in relation to the weight of some main organs, hematological, biochemical and histological parameters. These changes should be better explored and confirmed in longer-lasting toxicity studies.Example 42 - Exploratory repeated dose 14-day toxicity study of MB-611 in miceMethods:

[0276] The purpose of this study was to assess the potential toxicity after treatment with MB-611 administered by oral route for 14 days in mice. Male and female Swiss mice (8-11 weeks) were used.

[0277] Morbidity and mortality: Morbidity and mortality parameters were assessed twice daily during the 14 days of treatment with the Vehicle or with the different doses of the Test Item.

[0278] General and detailed clinical signs: General clinical signs were evaluated daily. Detailed clinical signs were performed once before the beginning of the treatments to verify the health status of the animals, and subsequently once a week until the end of the experimental protocol (three evaluations). General clinical signs included assessments of the animal’s general behavior, observations in the caudal region, abdomen and extremities. Weekly, the animals were weighed and feed consumption was evaluated. The Shimadzu semi-analytical balance model BL 3200 - H was used.

[0279] To evaluate detailed clinical signs, a scoring scale from 0 to 4 was used for the parameters of vocal fremitus, irritability, response to touch, general activity, grip strength, tearing and salivation, piloerection, general appearance, general activity, pinching tail, righting reflex, ataxia, ocular assessment. Defection / urination and breathing / heart rate.

[0280] Body weight changes and food consumption: Body weight and food consumption were measured once before the start of treatments (baseline) and then once a week (three evaluations).

[0281] Necropsy and postmortem analyses: On day 14 animals were subjected to necropsy. During necropsy, the outer surface of the body, orifices, cranial, thoracic and abdominal cavities, as well as their contents of each animal were examined. In the analysis of the body surface, a detailed evaluation was performed and the presence of lesions or deformities, size, color, texture, shape, severity, as well as weight and volume were noted and recorded.

[0282] Hematological analyses: Blood samples were collected by heart puncture after inhalation anesthesia and were evaluated with the assistance of an automatic hematology analyzer - model BC-5300vet, for: total cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCHC), mean corpuscular hemoglobin (MCH), platelet count (PLT), percentage of neutrophils, lymphocytes and monocytes.

[0283] Clinical chemistry: Blood samples were processed and evaluated with the assistance of a biochemical analyzer - Model BS 120, for: aspartateaminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, urea, creatinine, calcium, phosphorus, total proteins, albumin, glucose, total cholesterol and triglycerides.

[0284] Histopathological analyses: Histopathological analyses were carried out after the tissue fixation process and preparation of histological slides using a tissue processor - Model Excelsior AS, an embedding workstation - Model HistoStar. Semi-automatic microtome - Model HM 340E and an automatic slide stainer - Gemini AS, all from Thermo Scientific brand. The slides were observed under an optical microscope Model E200-LED, Nikon.Results:

[0285] Morbidity and mortality: No animal deaths were observed during the study. However, a male animal treated with a dose of 300 mg / kg of the Test Item was euthanized on the 11thday of the study as it showed signs of discomfort and pain. Furthermore, the penis was exposed and the abdominal region close to the member had a hardened appearance. These signs appear to be unrelated to the Test Item, as no signs of morbidity were observed in the other animals that received the same dose.

[0286] General and detailed clinical signs: The results obtained from these analyses did not show any clinical signs that would indicate toxicity of MB-611 (30 mg / kg and 300 mg / kg). However, a male animal treated with a dose of 300 mg / kg of the Test Item was euthanized on the 11thday of the study, as it showed signs of discomfort and pain, as mentioned in item above.

[0287] Body weight changes and food consumption: No significant changes in body weight and in food intake related to the treatment with MB- 611 (30 mg / kg or 300 mg / kg) were observed.

[0288] Absolute weight of organs: After the necropsy procedure, the absolute weight (g) of the kidneys, lungs, liver, pancreas and brain were measured for each animal, in all experimental groups. The results obtained demonstrated that there was no change in the absolute weight of the aforementioned organs, of males and females, when compared to the vehicle group.

[0289] Weight of organs relative (%) to body weight: The relative weight of the kidneys, lungs, liver, pancreas and brain was determined, calculating the percentage of the weight of each organ in relation to the terminal body weight (on the day of necropsy) for each animal. Repeated treatment with MB-611 (30 and 300 mg / kg) did not change the relative weight of the organs in both males and females.

[0290] Macroscopic analyses: In general, macroscopic evaluations performed during the necropsy procedure did not reveal changes related to repeated treatment for 14 days with MB-611 at doses of 30 and 300 mg / kg.

[0291] Hematological analyses: Repeated treatment with MB-611 (30 and 300 mg / kg) for 14 days, in general, did not cause significant changes in the hematological parameters analyzed in both sexes. However, a small increase in the number of platelets was observed in male animals treated with the dose of 30 mg / kg, in comparison with the vehicle group (p<0.05). Furthermore, a reduction in the number of total leukocytes was observed in male animals treated with the dose of 300 mg / kg (p<0.05) (Table 24). No hematological changes were observed in females.

[0292] Clinical chemistry: Repeated treatment with MB-611 caused a reduction in serum bilirubin levels in male animals treated with the dose of 300 mg / kg. In females, repeated treatment with MB-611 (300 mg / kg) caused an increase in glucose, cholesterol and urea levels. Furthermore, an increase in urea levels was observed in the group of females treated with a dose of 30 mg / kg. Comparisons were made with the vehicle control group (p<0.05) (Table 25 and 26).

[0293] Histopathological analyses: The results demonstrated that no effects related to repeated treatment with MB-611 (300 mg / kg) were observed in any of the organs examined, when compared to animals in the Vehicle group. All changes described in the raw data are commonly associated with spontaneous pathologies of the strain, gender and age of the animals used in the study and autolysis due to the necropsy procedure. Furthermore, these same changes were observed in animals that received the Vehicle. Due to the absence of effects related to treatment with the 300 mg / kg dose, organs fromthe group treated with the 30 mg / kg dose of MB-611 were not analyzed, as recommended by the OECD 408 guide.

[0294] Treatment with MB-611 orally at the doses analyzed did not show important changes in the various parameters indicative of toxicity, such as mortality and / or morbidity and relevant clinical signs. The changes related to treatments with MB-611 observed in some hematological and biochemical parameters were punctual, were not dose-dependent, nor were observed in male and female animals. Based on these results, it is concluded that MB-611 , in the doses and conditions evaluated, proved to be safe. The dose of MB-611 defined as NOEL (No-Observable-Effect-Level) was 300 mg / kg.

[0295] [Table 24] Analyses of hematological parameters of male rats after 14 days of daily treatment with MB-611 .&Standard deviation; N=4 to 5; ‘Differs significantly in relation to the Vehicle group; WBC - Total Cells; W-LCR - Large leukocyte (neutrophil) index; W-SCR - Small leukocyte (lymphocyte) index; W-MCR - Mean leukocyte (monocyte) index; RBC - Red blood cells; HGB - Hemoglobin; HCT - Hematocrit; MCV - Corpuscular Volume; MCH - Mean corpuscular hemoglobin; MCHC - Mean corpuscular hemoglobin concentration; PLT - Platelet count.

[0296] [Table 25] Analyses of biochemical parameters of male mice after 14 days of daily treatment with MB-611 .&Standard deviation; N=5; * Differs significantly in relation to the Vehicle group; ALT - Alanine aminotransferase; TRI - Triglycerides; TP - Total protein; CRE - Creatinine; ALB - Albumin; AST - Aspartate Aminotransferase; TC - Total cholesterol; FA - Alkaline phosphatase; P - Phosphorus; BT - Total Bilirubin.

[0297] [Table 26] Analyses of biochemical parameters of female mice after 14 days of daily treatment with MB-611 .&Standard deviation; N=5; * Differs significantly in relation to the Vehicle group; ALT - Alanine aminotransferase; TRI - Triglycerides; PT - Total protein; CRE - Creatinine; ALB - Albumin; AST - Aspartate Aminotransferase; TC - Total cholesterol; FA - Alkaline phosphatase; P - Phosphorus; BT - Total Bilirubin.Example 43 - Effect of MB-611, MB-657 or MB-658 on hERG channel functional assay.Methods:

[0298] The voltage-dependent potassium channels of the hERG type (human ether-a-go-go related) are essential for normal electrical activity in the heart. hERG channel dysfunction can cause long QT syndrome (LOTS). Characterized by delayed repolarization and prolongation of the QT interval of the cardiac cell’s action potential, which increases the risk of ventricular arrhythmias and sudden death. Thus, compounds that act in this channel and that has potential to cause long QT syndrome have been eliminated early in the process of non-clinical development in safety tests.

[0299] Studies that aim to evaluate the inhibition of the potassium channel hERG, are traditionally carried out through electrophysiology tests, using the patch clamp technique, which is considered the gold standard for ion channel studies; however, other methodologies have been developed in order to assess the influx of ions through ion channels transfected into immortalized cells. One of these methodologies consists in using the commercial kit called FLIPRR Potassium Assay, which is increasingly used for the evaluation and rapid and robust screening of compounds on ion channels, such as the hERGchannel. The method used in this test was based on the permeability of the hERG potassium channels to thallium, a component present in the commercial FLIPRR Potassium Assay kit. When the hERG potassium channels are opened by a stimulus, the influx of thallium from the external environment is detected by a highly sensitive indicator dye. The fluorogenic signal quantitatively reflects the activity of hERG ion channels that are permeate to thallium. The results of validating the methodology using the FLIPRR Potassium Assay, when compared to electrophysiology studies, demonstrated that both methods produce equivalent results on the hERG channel. Therefore, to assess the interaction with hERG, the commercial kit FLIPRR Potassium Assay (Molecular Devices) was used and the test was performed according to the manufacturer’s specifications.

[0300] The recombinant HEK293 cell line for the expression of the human hERG gene Kv11.1 was acquired from the company BPS Bioscience. For use in the present study, the cells were thawed and cultured according to the supplier’s specifications: hERG (Kv11.1 ) - HEK293 Recombinant Cell line Cat #: 60619 product sheets. The cells were kept in bottles containing supplemented culture medium, in a CO2 incubator, at 37 °C with 5 % and 0.2% CO2, until the time of the tests. For this, after thawing the HEK293 cells transfected with human hERG. They were plated at a density of 4 x 104cells per well in a black 96-well, flat, transparent bottom plate. After the confluence of the cells, the plate culture medium was aspirated and replaced with 50 pL of HBSS calcium and magnesium free. Then, the cells were incubated with 50 pL of the fluorescent probe present in the commercial kit, containing probenecid in the final concentration of 2.5 mM. After 1 hour of incubation at room temperature and in the dark, 25 pL of treatment with MB-611 (30, 100 and 300 pM), MB-657 (0.3, 3 and 30 pM) and MB-658 (3, 30 and 300 pM) were added to the wells, and the plates were incubated again for 30 minutes. The previously optimized stimulus buffer (50 pL of 1 mM thallium + 10 mM potassium) was added to each column through automated pipetting present in the FlexStation 3® equipment. The signal was acquired at intervals of 1.52 seconds for approximately 140 seconds per column. The data were obtained using the SoftMax®Pro Software, at an excitation wavelength of 485 nm andan emission wavelength of 538 nm. Data analysis was performed using SoftMax®Pro Software and GraphPad Prism®. The results were expressed as percentage of inhibition of the hERG channel and the mean inhibitory concentration (IC50) and the respective 95% confidence intervals were calculated using linear regression.Results:

[0301] As seen in Figure 12 the compound MB-611 (A) incubated in cells even at high concentrations (up to 300 pM) that largely exceed those observed in rodent plasma caused only very low inhibition (below 20%) in potassium permeation through the hERG channel. On the other hand, dofetilide (B), a reported selective inhibitor of hERG channel, produced a concentrationdependent inhibition of hERG channels. The estimated mean IC50 concentration of dofetilide of the hERG channel activity was 0.012 pM.

[0302] As depicted in Figure 13 (A), when the compounds MB-657 and MB-658 were incubated in cells, even at substantially higher concentrations (MB-657 with an IC50 up to 30 pM and MB-658 with an IC50 up to 300 pM), which significantly exceeded those observed in rodent plasma, they exhibited minimal inhibition of potassium permeation through the hERG channel. In contrast, the estimated mean IC50 concentration for dofetilide (B) for hERG channel inhibition was estimated as being 0.012 pM.

[0303] [Table 27] The following codes can also name some substances in this present invention. The screening of analgesic activity was done with the compounds solubilized in DMSO and Saline (1 :1 ) and tested in a neuropathy animal model induced by partial sciatic nerve ligation (PSNL). Efficacy is the percentage of reversion of the mechanical hyperalgesia induced by the nerve ligation.

Claims

Claims

1. A compound characterized in that it is of general formula (I) or a pharmaceutically acceptable salt, crystal, hydrate, prodrug, metabolite or solvate thereof,wherein:• X, Y, Z, and W are independently selected from N or C;• A, B, C, and D are independently selected from Cor N;• R1 is COOH, CN or bioisostere of carboxylate and its precursors, such as esters and amides;• R2, R6, R7, and R8 are independently selected from H, halogen, CF3, NO2, CN, D, OH, SO3H, SO2NH2, C1-C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, OR where R is C1 -C30 alkyl, alkenyl or aryl, NR2 where each R is the same or different and each is independently selected from H, C1 -C30 alkyl, C2-C30 alkenyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl and heterocycloaliphatic amines;• R3 is selected from H, halogen, CF3, NO2, CN, D, OH, C1 -C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, OR where R is C1 -C30 alkyl, alkenyl or aryl, NR2 where each R is the same or different and each is independently selected from H, C1 -C30 alkyl, C2-C30 alkenyl,C3-C8 cycloalkyl or aryl, mono, di, tri or tetra substituted aryl or heteroaryl;• R5, R4 are the same or different and can be H, D, CF3, halogen, C1 -C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl or aryl.

2. The compound, according to claim 1 , characterized in that the bioisostere of carboxylate and its precursors, such as esters and amides of R1 are optionally selected from COOR, where R is C1 -C30 alkyl, C2-C30 alkenyl, C2-C10 alkynyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, which can be considered as prodrugs of respective carboxylic acid; orR1 is CONR9R10 where each R9 and R10 is the same or different, and each is H, C1 -C30 alkyl, C2-C30 alkenyl, C3-C8 cycloalkyl, aryl, mono, di, tri or tetra substituted aryl or heteroaryl, amino acid or amino acid derivative, optionally they are prodrugs of the respective carboxylic acid.

3. The compound, according to claim 1 , or a pharmaceutically acceptable salt, crystal, hydrate, prodrug, metabolite or solvate thereof, characterized in that the compound is selected from the group comprising:

4. A combination characterized in that it comprises at least one compound as defined in claim 1 and at least a second pharmaceutically active compound.

5. The combination, according to claim 4, characterized in that said at least second pharmaceutically active compound is selected from analgesics, antibiotics, anticoagulants, antiemetics, antipsychotics, antipyretics, antidepressants, antidiabetic agents, anticancer agents, antiepileptic agents, antifungals, antihistamines, antihypertensive agents, anti-inflammatory agents, antivirals, bronchodilators, diuretics, hormone replacement therapy, immunosuppressants, laxatives, mood stabilizers, statins, sedatives or hypnotics, anxiolytics, and stimulants.

6. The combination, according to claims 4 or 5, characterized in that said second pharmaceutically active compound is analgesics and / or antiinflammatory agent selected from non-steroidal anti-inflammatory drugs (NSAIDS), steroidal anti-inflammatory drugs, cyclooxygenase inhibitors, or opioid analgesics.

7. A pharmaceutical composition characterized in that it comprises at least one compound as defined in claim 1 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

8. A pharmaceutical composition characterized in that it comprises the combination as defined in claim 4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claims 7 or 8, characterized in that their pharmaceutical dosage form comprises tablets, capsules, pills, powders, granules, dragees, lozenges, troches, solutions, emulsions, colloids, suspensions, syrups, elixirs, sprays, creams, ointments, gels, lotions, pastes, foams, plasters, patches, and suppositories.

10. A medicament characterized in that it comprises a compound of formula (I), as defined in claim 1 , for use in treating pain in a disease or condition.

11. A use of a compound of formula (I), as defined in claim 1 , characterized in that it is for the manufacture of a pharmaceutical composition for treating pain in a disease or condition.

12. A method for the treatment of pain in a disease or condition in a patient in need thereof, characterized in that it comprises administering to said patient a pharmaceutical composition comprising a therapeutically effective amount of a compound in accordance to claim 1 , or a pharmaceutically acceptable salt thereof.

13. The method, according to claim 12, characterized in that said disease or condition is selected from the group comprising neuropathic pain, inflammatory pain, visceral pain, cancer pain, chemotherapy pain, trauma pain, surgical pain, post-surgical pain, childbirth pain, labor pain, chronic pain, persistent pain, peripherally mediated pain, centrally mediated pain, chronic headache, migraine headache, sinus headache, tension headache, phantom limb pain, dental pain or peripheral nerve injury.

14. The method, according to claim 12, characterized in that said disease or condition is selected from the group comprising pain associated with HIV, HIV treatment-induced neuropathy, trigeminal neuralgia, postherpetic neuralgia, eudynia, primary or secondary otalgia, ulcerative colitis, heat sensitivity, sarcoidosis, irritable bowel syndrome, Crohn’s disease, pain associated with multiple sclerosis (MS) or amyotrophic lateral sclerosis (ALS), diabetic neuropathy, peripheral neuropathy, arthritis, rheumatoid arthritis, osteoarthritis, osteoporosis, atherosclerosis, paroxysmal dystonia, myasthenia syndromes, myotonia, malignant hyperthermia, lupus, kidney stones, cystic fibrosis, pseudoaldosteronism, rhabdomyolysis, hypothyroidism, depression, anxiety, schizophrenia, sodium channel toxin-related illnesses, familial erythromelalgia, primary erythromelalgia, paroxysmal extreme pain disorder, cancer, epilepsy, partial and general tonic seizures, restless leg syndrome, arrhythmias, fibromyalgia, ischemic conditions caused by stroke or neural trauma, tachycardia, atrial fibrillation, ventricular fibrillation or a combination thereof.

15. The method, according to claim 12 or 13, characterized in that said disease or condition is: chronic primary pain, chronic cancer pain, chronic posttraumatic and postsurgical pain, chronic neuropathic pain, chronic headache and orofacial pain, chronic visceral pain, and chronic musculoskeletal pain.

16. The method, according to claim 12, characterized in that said disease or condition may be treated by the inhibition of signaling of prostaglandins or its receptors, including but not limited to osteoarthritis, gout, rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, post-surgical pain, migraine, dysmenorrhea, thrombotic diseases, autoimmune diseases, metabolic syndrome, neurodegenerative diseases, chronic obstructive pulmonary diseases, and chronic inflammatory bowel disease.

17. The method, according to claim 12, characterized in that the compound is administered via the following routes: oral, dental, topical, rectal, vaginal, urethral, otic (inner and middle ear), transdermal, subcutaneous, intraperitoneal, inhalatory, intranasal, intrapulmonary, intrathoracic, intratracheal, transtracheal, intradermal, intrathecal, epidural, intraspinal, intratendinous, intraosseous, intraarticular, intrabursal, intralesional, intramuscular, intravenous, intraarterial, intracardiac, intragingival, intracerebral, subarachnoid, intraocular, intracapsular or subcapsular administration in single or multiple dosages.

18. The method, according to claim 12, characterized in that the administration of a compound of claim 1 , or its pharmaceutically acceptable salt, crystal, hydrate, prodrug, metabolite or solvate thereof, is preceded, simultaneous with, or followed by administration of at least one more pharmaceutically active ingredient in a combined therapy regimen.

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