ES2408281B1 - USE OF A CHEMICAL COMPOUND DERIVED FROM A 1,2,3,5-TETRAS REPLACED PIRROL IN THE PREPARATION OF A USEFUL MEDICINAL PRODUCT FOR THE TREATMENT OF INFLAMMATORY DISEASES THAT COURSE WITH CELLULAR APOPTOTHICAL PROCESSES. - Google Patents

Abstract

The present invention relates to the use of 1,2,3,5-tetrasubstituted pyrrole-derived compounds together with pharmaceutically acceptable excipients in the preparation of a medicament useful for the treatment of inflammatory diseases that occur with cellular apoptotic processes, preferably produced by sepsis. by Gram-negative bacteria. They include acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis or traumas. The invention also comprises the pharmaceutical composition for the treatment of said diseases.

Description

Use of a chemical compound derived from a 1,2,3,5-tetrasubstituted pyrrole in the preparation of a medicament useful for the treatment of inflammatory diseases that occur with cellular apoptotic processes.

FIELD OF THE INVENTION

The present invention relates to compounds derived from 1,2,3,5-tetrasubstituted pyrroles that have shown anti-inflammatory and anti-apoptotic activity. These are pharmaceutical compositions for the treatment of diseases that synthesize pro-inflammatory cell mediators and that occur with apoptosis. The present invention is comprised in the field of drugs suitable for the treatment of said diseases, in medicine.

BACKGROUND OF THE INVENTION

In the technique there are several compounds with the ability to reduce the effects produced by the action of Lipopolysaccharides (LPS) of E. coli bacteria, the causative and most frequent agent of Systemic Inflammatory Response Syndrome (SRIS) caused by an infection (Sepsis) . This is the case of the plasminogen activator inhibitor (PAI-1) inhibitor whose chemical formulation is (3E, 4E) -3-benzylidene-4- (3,4,5-trimethoxybenzylidene) idine pyrrol-2,5-dione, which administered prior to the injection of LPS protects against its lethal effects. Another example can be found in the benzofuranyl pyrrole and benzothiophenyl pyrrole derivatives, which show an inhibitory effect on LPS-induced lymphocyte B proliferation.

A third example is the Sunitinib compound (marketed as Sutent) of chemical formulation N- [2 (diethylamino) ethyl] -5 - [(Z) - (5-fluoro-1,2-dihydro-2-oxo-3H- indole-3-ildine) methyl] -2,4-dimethyl-1H-pyrrole-3 carboxamide, which has been used simultaneously for the treatment of renal cell carcinoma and for gastrointestinal stromal tumors. In cancer models, the drug is the one that causes apoptosis or death of the cancer cell, is pro-apoptotic; Sunitinib causes apoptosis of renal cell carcinoma. In the model of the present invention, the E. Coli LPS is the one that produces the septic process and this in turn the inflammatory process, while the pyrrolic compounds are anti-apoptotic, prevent the apoptotic process.

Pyrimidines (imatinib, dasatinib, nilotinib and pazopanib), pyridines (sorafenib) and pyrroles (sunitinib) are tyrosine kinase inhibitors with high activity towards several families of receptor and non-receptor tyrosine kinases involved in angiogenesis, tumor growth and progression of metastatic cancer (Di Gion P, Kanefendt F et al, Clinical pharmacokinetics of tyrosine kinase inhibitors: focus on pyrimidines, pyridines and pyrroles. Clin Pharmacokinet. 2011; 50 (9): 551-603). With respect to Sunitinib, which is a pyrrole compound, its mechanism of action lies in the inhibition of cellular signals by binding to multiple tyrosine kinase receptors (RTKs) (Faivre S, Demetri G et al; Molecular basis for sunitinib efficacy and future clinical development Nat Rev Drug Discov 2007; 6 (9): 734-45).

There are several documents that relate the use of pyrrolic compounds in the prevention and treatment of certain pathologies. The following two publications involve the disclosures of the technique closest to the invention.

Application KR 2009/0058752 A describes the use of pyrrole compounds for the prevention and treatment of bronchial diseases, inflammation, asthma, chronic obstructive pulmonary disease, arthritis, atopic dermatitis, leukemia, cancer, brain disorders, depression and Alzheimer's disease . The pyrrole compounds of the publication act by inhibiting Phosphodiesterase-4 (PDE-4), a hydrolase that catalyzes the breakdown of phosphodiester bonds, which is a mechanism of action totally independent from that of the compounds described in the present invention against diseases that occur with apoptosis. In addition to the compounds having different activity, the generality of the inflammatory diseases listed in the publication does not anticipate the particular mechanism of those that occur with apoptotic processes. The antiapoptotic activity of the pyrrole compounds of the invention is not described in the publication, whose teachings diverge and therefore the use described in the present application is not suggested.

International application WO 2005054191 A describes the usefulness of some pyrrole compounds in the treatment of inflammatory pain, neuropathic pain, kidney disorders, fever, immunological diseases, abnormal diseases of platelet function and bone diseases by means of an antagonistic activity of the receptors of prostaglandins PGE2. Prostaglandins are a set of lipid substances derived from eicosanoid fatty acids, and the mechanism of action described in the publication is again different from that used by the compounds of the invention, which is very exclusive for diseases that occur with apoptosis. The international publication does not describe that its pyrrolic compounds exhibit any anti-apoptotic activity, nor is it suggested for the expert to use the compounds of the invention in the antiapoptotic processes described in the present application.

The problem that arises then in the art is to find an effective pharmacological therapy for the treatment of inflammatory diseases that occur with cellular apoptotic processes and sepsis, since

ES 2 408 281 A1

Currently, the therapy of the septic patient is restricted to guidelines such as supplemental oxygen administration, orotracheal intubation and mechanical ventilation, and an exclusive pharmacology is still unknown. The solution proposed by the present invention is the use of pyrrolic derivative compounds that are efficient for the treatment of this type of pathologies.

DESCRIPTION OF THE INVENTION

The present invention is the use of a chemical compound derived from a 1,2,3,5-tetrasubstituted pyrrole of formula (I):

(I)

25 where

-

R1 is a C1-C4 aliphatic chain, substituted or unsubstituted, preferably substituted with a phenyl or with a furan group,

-

R2 is a methyl or ethyl ester,

-

R3 is phenyl, biphenyl, methylphenyl, benzylphenyl or cyclohexyl, substituted or unsubstituted, preferably substituted with a halogen and more preferably chlorine,

-

R4 is phenyl, biphenyl or cyclohexyl, substituted or unsubstituted, preferably substituted with a halogen and more preferably chlorine,

-

R5 is H or a C1-C3 alkyl, preferably methyl,

in the preparation of a medicament useful for the treatment of inflammatory diseases that occur with cell apoptotic processes, preferably produced by sepsis.

A very preferable embodiment is that the compound of the invention is methyl 1-benzyl-5- (1- (4-chlorobenzoyloxy) -2methoxy-2-oxoethyl) -4- (4-chlorophenyl) -1H-pyrrole-2- carboxylate (DTA0118):

Cl

Cl

Another very preferable embodiment is that said compound is methyl 1-benzyl-4- (biphenyl-4-yl) -5- (1- (biphenylcarbonyloxy) -2methoxy-2-oxoethyl) -1H-pyrrole-2-carboxylate (DTA0119 ):

Another very preferable embodiment is that said compound is methyl 5- (1- (benzoyloxy) -2-methoxy-2-oxoethyl) -4-phenyl-1 ((S) -1-phenylethyl) -1H-pyrrole-2- carboxylate (DTA0124):

Another highly preferred embodiment is that said compound is methyl 5- (1- (benzoyloxy) -2-methoxy-2-oxoethyl) -1-benzyl-4-phenyl-1H-pyrrole-2-carboxylate (DTA0140):

Another very preferable embodiment is that said compound is methyl 1-allyl-5- (1- (4-chlorobenzoyloxy) -2-methoxy-2oxoethyl) -4- (4-chlorophenyl) -1H-pyrrole-2-carboxylate (DTA0141 ):

Another very preferable embodiment is that said compound is 2- (benzoyloxy) -2- (1-benzyl-5- (methoxycarbonyl) -3-phenyl-1H-pyrrol-2-yl) acetic acid (DTA0142):

Another very preferable embodiment is that said compound is methyl 5- (1- (benzoyloxy) -2-methoxy-2-oxoethyl) -4-phenyl-1 (((R) -tetrahydrofuran-2-yl) methyl) -1H -pyrrol-2-carboxylate (DTA0143):

Another very preferable embodiment is that said compound is methyl 1-benzyl-5- (1- (2-chlorobenzoyloxy) -2-methoxy-2oxoethyl) -4- (2-chlorophenyl) -1H-pyrrole-2-carboxylate (DTA0144 ):

Another highly preferred embodiment is that said compound is methyl 1-benzyl-5- (2-methoxy-1- (4-methylbenzoyloxy) -2oxoethyl) -4-p-tolyl-1H-pyrrole-2-carboxylate (DTA0145) :

Another very preferable embodiment is that said compound is methyl 5- (1- (benzoyloxy) -2-methoxy-2-oxoethyl) -1-butyl-4-phenyl-1H-pyrrole-2-carboxylate (DTA0146):

Another very preferred embodiment is that said compound is methyl 1-allyl-5- (1- (benzoyloxy) -2-methoxy-2-oxoethyl) -4phenyl-1H-pyrrole-2-carboxylate (DTA0147):

OR

OR

N

OR

OR

OR

Another very preferable embodiment is that said compound is methyl 1-benzyl-5- (1- (cyclohexanecarbonyloxy) -2-methoxy2-oxoethyl) -4-cyclohexyl-1H-pyrrole-2-carboxylate (DTA0148):

Another very preferred embodiment is that said compound is ethyl 5- (1- (benzoyloxy) -2-ethoxy-2-oxoethyl) -1-b encyl-4-phenyl-1-pyrrole-2-carboxylate (DTA0149):

Another very preferable embodiment is that said compound is methyl 1-butyl-5- (1- (4-chlorobenzoyloxy) -2-methoxy-2oxoethyl) -4- (4-chlorophenyl) -1H-pyrrole-2-carboxylate (DTA0150 ):

Another very preferred embodiment is that said compound is methyl 5- (1- (benzoyloxy) -2- (4-chlorophenylamino) -2-oxoethyl) 1-Benzyl-4-phenyl-1H-pyrrole-2-carboxylate (DTA0151):

The 1,2,3,5-tetrasubstituted pyrrole-derived compounds of the present invention possess anti-inflammatory and anti-apoptotic activity, and are capable of decreasing the anti-proliferative, inflammatory and apoptotic effects induced by LPS of Gram-negative bacteria on the A549 cell line. In the same way, prevention of the apoptotic process was accompanied by an inhibition of the increased expression of the Bax pro-apoptotic protein and an inhibition of the decrease in the Bcl-2 anti-apoptotic protein. Likewise, none of the effects induced by the 1,2,3,5-tetrasubstituted pyrrole compounds of the invention in the inhibition of increased expression of TLR4 and Bax proteins and the decrease in transcription factor NF-KB inhibitor protein and of the LPS-induced Bcl-2 protein were modified when these 1,2,3,5-tetrasubstituted pyrrole compounds were used in conjunction with the tyrosine kinase inhibitor (Genistein, Calbiochem®). This implies in the process the mechanism of inflammatory action TLR4-IK�a and the mechanism of apoptotic action Bax-Bcl-2.

A preferable embodiment is a pharmaceutical composition comprising at least one compound according to the invention, together with pharmaceutically acceptable excipients, for the treatment of inflammatory diseases that occur with cellular apoptotic processes, preferably produced by sepsis.

Preferable administration of the pharmaceutical composition of the invention is intravenously, but it can also be administered orally, muscularly or intramuscularly. Preferably, the pharmaceutical composition of the invention is presented in a dosage unit.

Another preferable embodiment is the pharmaceutical composition of the invention together with pharmaceutically acceptable excipients and at least one other therapeutic agent for the treatment of inflammatory diseases that occur with cellular apoptotic processes, more preferably produced by sepsis. In yet another preferable embodiment, said sepsis is caused by bacteria, preferably Gram-negative bacteria.

Within the scope of the present invention, inflammatory diseases are understood as "those that are involved with cellular apoptotic processes" to those diseases in which said apoptotic process is one of the determining factors and preferred cause of the pathogenesis of the disease. By "apoptosis" is meant "programmed cell death."

Thus, a further embodiment of the invention is that said inflammatory diseases that occur with apoptotic processes include sepsis in pathologies of renal infections, septic peritonitis, acute hepatitis, acute myocardial dysfunction induced by septic shock or necrotizing enterocolitis; acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis or trauma. All of them are pathologies with a severe inflammatory process, causing diffuse alveolar damage, damage to ventilatory perfusion, severe hypoxemia and decreased lung capacity.

In the scope of the present invention, "hypoxemia" is understood as the abnormal decrease in the partial pressure of oxygen in arterial blood.

Another embodiment of the invention is a chemical compound derived from a 1,2,3,5-tetrasubstituted pyrrole of formula (I):

where -

- - substituted with a halogen and more preferably chlorine,

-

R4 is phenyl, biphenyl or cyclohexyl, substituted or unsubstituted, preferably substituted with a halogen and more preferably chlorine,

-

R5 is H or a C1-C3 alkyl, preferably methyl, for the treatment of inflammatory diseases that occur with cellular apoptotic processes, preferably produced by sepsis.

Another embodiment of the invention is a method of treating an inflammatory disease that comprises administering an effective amount of a compound of the invention to a subject, preferably human, in need of said treatment.

The compounds of the invention are useful for preparing pharmaceutical compositions for the treatment of diseases that synthesize pro-inflammatory cell mediators and that occur with apoptosis, such as sepsis, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis, trauma. Recent studies have provided information that an increase in the cellular apoptotic process followed by an inflammatory process are essential steps in the development of pathologies such as Acute Pulmonary Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS). The pathological development of ALI followed by trauma, septic shock and pneumonia is clinically documented (Chopra M, Reuben JS, Sharma AC; Acute lung injury: apoptosis and signaling mechanisms. Exp Biol Med (Maywood) 2009 234 (4): 361-71 ). ALI is a frequent complication followed by the septic process in critical patients belonging to the Intensive Care Units (ICU) and can be associated with high rates of morbidity and mortality (The ACCP / SCCM Consensus Conference Committee. American College of Chest Physicians / Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992 20: 864–874).

Tyrosine kinase activity has been implicated in the pathophysiology of many diseases associated with local inflammation such as atherosclerosis or psoriasis, or systemic diseases including sepsis and septic shock (Levitzki and Gazit A; Tyrosine kinase inhibition: An approach to drug development. Science 1995; 267: 1782-8). As in the art it is already described that the compound called Sunitinib N- [2- (diethylamino) ethyl] -5 - [(Z) - (5-fluoro-1,2-dihydro-2oxo-3H-indole-3-ildine ) methyl] -2,4-dimethyl-1H-pyrrole-3 carboxamide inhibits cell signals by binding to multiple tyrosine kinase receptors (RTKs), the inventors treated the A549 cell line with Genistein (Calbiochem, Darmstadt), which inhibits proteins Tyrosine kinases The result was that the 1,2,3,5-substituted pyrrole-derived compounds act by a mechanism independent of Tironsin-kinase inhibitors in the prevention of the effects induced by LPS of E. Coli, as shown in Example 8 of the present request

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Shows the survival percentage by 1,2,3,5-tetrasubstituted pyrrole compounds of the invention. The A549 cell line (human pulmonary epithelial cell line) was cultured in 96-well Multiwell and treated for 18 hours with 300 micrograms / ml of E. coli LPS alone or in combination with 100 microM of each of the 1,2 pyrroles. , 3,5-tetrasubstituted (Compounds 1-15). The results are expressed as the average of all of them plus the standard deviation. After 18 hours of treatment, the cell count was estimated by means of the Sulforodamina B test following the instructions recommended by the manufacturer. The percentage of cell survival is expressed as the percentage of cells treated with E. coli LPS and cells treated with E. coli LPS plus each of the 1,2,3,5-tetrasubstituted pyrrole compounds. Each point represents the mean ± S.E.M. from four identical Wells.

Figure 2: shows the chemical formulations of the fifteen preferable embodiments of the 1,2,3,5-substituted pyrroles according to the invention.

Figure 3: shows the morphological changes of the A549 cell line treated or not with 300 micrograms / ml of E. coli LPS alone or in combination with 100 microM of compound DTA0118 as representative. The upper panels correspond to the 10X objective and the lower panels correspond to the 20X objective. C = Control cell line A549; C + = A549 cell line treated with 300 micrograms / ml of E. Coli LPS for 18 hours; DTA0118 = A549 cell line treated with 300 μgr / ml of E. Coli LPS plus 100μM of DTA0118 for 18 hours.

Figure 4: shows the test of the concentrations of Interleukin-6 (4a) and Interleukin-8 (4b) in the supernatant of A549 cells treated or not treated for 18 hours with 300 micrograms / ml of E. Coli LPS alone or in combination with 100 microM of each of the 1,2,3,5-tetrasubstituted pyrrole compounds (Compounds 1-15), the results expressed as the average of all of them plus the standard deviation, by means of the CBA test following the standards of the maker. The data are represented as the mean ± S.E.M. from three different experiments. C = Control cell line A549; C + = A549 cell line treated with 300 micrograms / ml of E. Coli LPS for 18 hours; Compounds 1-15 = A549 cell line treated with 300 micrograms / ml of LPS of

E. Coli plus 100 microM of the 15 1,2,3,5-tetrasubstituted pyrrole compounds object of the present invention for 18 hours. ** p <0.01 vs. C; # p <0.05 vs. LPS

Figure 5: shows how changes in the expression of TLR4 and IK�a proteins induced by E. coli LPS are inhibited by 1,2,3,5-tetrasubstituted pyrrolic compounds. The A549 cell line was incubated for 18 hours with or without 300 micrograms / ml of E. coli LPS in combination or not with 100 microM of the 15 1,2,3,5-tetrasubstituted pyrrole compounds (Compounds 1-15) object of the present invention. Following the treatments, the total proteins were prepared for analysis using Western Blot and using antibodies directed against the TLR4, IK�a, �-actin proteins (used as a protein load control). (5a) Representative electrophoresis of the TLR4 and IK�a proteins in each of the experimental conditions. (5b) Histograms showing changes in the expression levels of TLR4 and IK�a proteins normalized according to the �-actin load control. The results shown come from 3 independent experiments. C = Control cell line A549; C + = A549 cell line treated with 300 micrograms / ml of E. Coli LPS for 18 hours; Compounds 1-15 = A549 cell line treated with 300 micrograms / ml of E. Coli LPS plus 100 microM of the 15 1,2,3,5-substituted pyrrole compounds object of the present invention for 18 hours. *** p <0.001 vs. C; ¶ p <0.001 vs. LPS of E. Coli.

Figure 6: shows the prevention in the apoptotic process of 1,2,3,5-tetrasubstituted pyrrole compounds (Compounds 1-15), induced by 300 microg / ml of E. Coli LPS. (6a) Representative diagrams using compound DTA0118 showing apoptotic cell populations and under cell death detected by Annexin V-FITC (Annexin V) and Propidium Iodide (IP) staining. (6b) Percentages of early apoptosis and death of A549 cells under all experimental conditions. (6c) Fluorescent microscopy analysis of apoptotic cells stained with Hoechst 33258. A549 cells treated with 300 microg / ml of E. Coli LPS for 18 hours showed a condensation of nuclear chromatin and nuclear fragmentation that was prevented by co-treatment with 1,2,3,5-tetrasubstituted pyrrole compounds. The images were captured using 40x objective. (6d) Quantification of apoptosis showing the percentage of apoptotic cells in each experimental condition. C = Control cell line A549; C + = A549 cell line treated with 300 micrograms / ml of E. Coli LPS for 18 hours; Compounds 1-15 = A549 cell line treated with 300 micrograms / ml of E. Coli LPS plus 100 microM of the 1,2,3,5-tetrasubstituted pyrrole compounds object of the present invention for 18 hours. DTA0118 = A549 cell line treated with 300 micrograms / ml of E. Coli LPS plus 100 microM of compound DTA0118 for 18 hours. ** p <0.01 vs. C; *** p <0.001 vs. C; # p <0.05 vs. E. Coli LPS; ¶ p <0.001 vs. LPS of E. Coli.

Figure 7: shows representative electrophoresis (7a) and the densitometric values of the Bax and Bcl-2 proteins normalized according to the �-actin charge control (7b) when the A549 cells were treated or not for 18 hours with 300 micrograms / ml of E. coli LPS alone or in combination with 100 microM of the 15 1,2,3,5-tetrasubstituted pyrrole compounds (Compounds 1-15) object of the present invention. (7c) Alterations in the range of Bax / Bcl-2 proteins normalized as a function of �-actin in all experimental conditions under study. There are no statistical differences between the experimental groups. C = Control cell line A549; C + = A549 cell line treated with 300 micrograms / ml of E. Coli LPS for 18 hours; Compounds 1-15 = A549 cell line treated with 300 micrograms / ml of E. Coli LPS plus 100 microM of the 15 1,2,3,5-substituted pyrrole compounds object of the present invention for 18 hours. ** p <0.01 vs. C; *** p <0.001 vs. C; ¶ p <0.01 vs. LPS of E. Coli.

Figure 8: shows the effect of Genistein (Gen) alone or in combination with the 1,2,3,5-substituted substituted pyrrolic compounds (Compounds 1-15, C1-15), in turn, in combination or not with LPS of E. Coli in the expression levels of TLR4, IK�a, Bax and Bcl-2 proteins of A549 cells treated with the mentioned experimental conditions for 18 hours. The panel (8a) shows representative gels corresponding to the study proteins (TLR4, IK�a, Bax and Bcl-2) and the panel (8b) shows the histograms showing the changes in the expression levels of the TLR4, IK�a, Bax and Bcl-2 proteins normalized based on �-actin load control. In this figure, the A549 cell line was treated with the Genistein inhibitor (30 μM) for 30 min at 37 ° C and 5% CO2. Subsequently, it was incubated with E. coli LPS (300μgr / ml) alone or in combination with 1,2,3,5tetra substituted substituted pyrrolic compounds object of the present invention (100 µM) for 18 hours. Data are expressed as mean ±

S.E.M. of triplicate determinations and are expressed as a function of the control cells (without treatment and without stimulation with LPS of E. Coli). C = Control cell line A549; GEN = A549 cell line treated with 30 microM of the Genistein inhibitor for 30 minutes; C + = A549 cell line treated with 300 micrograms / ml of E. Coli LPS for 18 hours; + GEN = A549 cell line treated with 30 microM of the Genistein inhibitor for 30 minutes and subsequently with 300 micrograms / ml of E. Coli LPS for 18 hours; + Compounds 1-15 = A549 cell line treated with 300 micrograms / ml of E. Coli LPS plus 100μM of the 15 1,2,3,5-substituted pyrrole compounds object of the present invention for 18 hours; + GEN + Compounds 1-15 = A549 cell line treated with 30 microM of the Genistein inhibitor for 30 minutes and subsequently with 300 micrograms / ml of E. coli LPS in combination with 100μM of the 15 1,2,3,5 pyrrole compounds -Text substitutes object of the present invention for 18 hours. ** p <0.01 vs. C; *** p <0.001 vs. C; # p <0.05 vs. LPS; ¶ p <0.001 vs. LPS

Figure 9: shows the morphological changes of the A549 cell line treated or not with 300 micrograms / ml of E. coli LPS alone or in combination with 30 microM of Genistein, in turn, alone or in combination with 100 microM of compound DTA0118 . The upper panels correspond to the 20X objective and the lower panels correspond to the 40X objective. In this figure, C = A549 cell line control; GEN = A549 cell line treated with 30 microM of the Genistein inhibitor for 30 minutes; C + = A549 cell line treated with 300 micrograms / ml of E. Coli LPS for 18 hours; + GEN = A549 cell line treated with 30 microM of the Genistein inhibitor for 30 minutes and subsequently with 300 micrograms / ml of E. Coli LPS for 18 hours; + DTA0118 = A549 cell line treated with 300 microg / ml of E. Coli LPS plus 100 microM of the compound + GEN + DTA0118 = A549 cell line treated with 30 microM of the Genistein inhibitor for 30 minutes and subsequently with 300 micrograms / ml of LPS of E. Coli in combination with 100 microM of the representative compound DTA0118 for 18 hours.

PREFERRED EMBODIMENTS

Example 1. Synthesis of 1,2,3,5-tetrasubstituted pyrrole compounds.

The 1,2,3,5-tetrasubstituted pyrrole compounds were synthesized from the simple method published in the work developed by D. Tejedor et al. "From conjugated tertiary skipped diynes to chain-functionalized tetrasubstituted pyrroles" (Chemistry: A European Journal, 2009, 15 (4), 838-842). This article explains the synthesis of 1,2,3,5-tetrasubstituted pyrroles with functionalized chains by means of a sequential process that involves an aza-addition of Michael, a 5-endo-digonal cyclization and a rearrangement [3,3] - sigmatropic, from alkyl propriolates, acid chlorides and primary amines.

Example 2. Antimicrobial activity of compounds derived from 1,2,3,5-tetrasubstituted pyrroles.

The anti-microbial activity in Gram-positive bacteria Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212) and Gram-negative bacteria Escherichia Coli (ATCC 35218), Klebsiella pneumoniae (ATCC 700603) and Pseudomonas aer53inosa (ATCC 7002) were determined . Bacterial species were grown on agar plates at 37 ° C. After 24 hours of incubation, the species were resuspended in saline solution at a concentration of 5 x 105 cfu / ml. The antimicrobial activity of the pyrrole compounds was determined in 96-well plates using the Mueller Hinton broth medium (Dalhoff A, Stubbings W, Schubert S. Comparative in vitro activities of the novel antibacterial finafloxacin against selected Gram-positive and Gram-negative bacteria tested in Mueller-Hinton broth and synthetic urine Antimicrob Agents Chemother 2011 55 (4): 1814-8). Sterile controls with adequate growth were included. Each pyrrolic compound was tested in duplicate to eight different serial solutions from 0.05 to 100 microM. The 96-well plates were incubated at 37 ° C in a dark and humid chamber. After incubation, the species were precipitated in 25 microliters of 50% w / v TCA (trichloroacetic acid) and fixed for 60 minutes at 4 ° C. Then, the colorimetric assay described in Skehan, P. et al., New colorimetric cytotoxicity assay for anticancer-drug screening was carried out. J Natl Cancer Inst 1990 (82) 1107-1112. After the test, the plates were shaken and by means of spectrophotometry (BioTek's PowerWave XS Absorbance Microplate Reader) optical density values were obtained at 630 nanometers after 24 hours of incubation. With the results obtained from the spectrophotometry, the MCI value (minimum inhibitory concentration) established as the concentration of the compound that inhibited total growth when compared to untreated species was obtained. All compounds derived from substituted pyrroles were inactive, with MCI values greater than 100 microM.

Example 3. Antifungal activity of compounds derived from 1,2,3,5-tetrasubstituted pyrroles.

The anti-fungal activity in the fungal species Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), Candida nivariensis (5937-63) and Candida parapsilosis (ATCC 22019) were determined. Fungal species were grown on agar plates at 37 ° C. After 24 hours of incubation, they were resuspended in saline solution at a concentration of 1x103 to 5x103. The antifungal activity of the compounds was determined in 96-well plates using RPMI-1640 medium buffered with MOPS solution for fungal species. Sterile controls with adequate growth were included. Each compound was tested in duplicate to eight different serial solutions from 0.05 to 100 microM. The 96-well plates were incubated at 37 ° C in a dark and humid chamber. After incubation, the species were precipitated in 25 microliters of 50% w / v TCA and fixed for 60 minutes at 4 ° C. Then, the colorimetric assay described in Skehan et al. according to Example 2. After the test, the plates were shaken and by means of spectrophotometry (BioTek's PowerWave XS Absorbance Microplate Reader) optical density values were obtained at 490 nanometers after 48 hours of incubation. The MCI value was obtained with the results obtained from the spectrophotometry. All compounds derived from substituted pyrroles were inactive, with MCI values greater than 100 microM.

Example 4: Cytotoxic activity of compounds derived from 1,2,3,5-tetrasubstituted pyrroles.

Cytotoxic activity was determined in the HeLa cell lines (human cervical cancer cell line), Ishikawa (human uterine endometrial cancer cell line), SW1573 (human lung epithelial cancer cell line), T-47D (human cell line of breast cancer), WiDr (human colon cancer cell line) and A549 (human cell line from the pulmonary alveolar epithelium). Cell lines were grown in 25 cm2 cell culture bottles in RPMI-1640 culture medium supplemented with 5% fetal bovine serum and 2 milliM glutamine in a 37 ° C incubator, 5% CO2 and 95% moist air . The exponentially growing cells were trypsinized and resuspended in culture medium containing 100 units of penicillin and 0.1 milligrams of streptomycin per milliliter. The cell suspension was counted with a percentage greater than 97% viability by the Trypan Blue staining and exclusion technique. After counting, cells were seeded in 96-well plates at a concentration of 1x104 (for the SW1573 cell line), 1.5x104 (for the HeLa, Ishikawa and T47D cell lines) and 2x104 (for the WiDr cell line) cells per well. Each compound was tested in triplicate at different dilutions in the range of 1-100 microM. The treatment of the compounds in cell cultures began one day after sowing. The incubation time of the compounds was 48 hours, after which the cells were precipitated in 25 microliters of 50% w / v TCA and fixed for 60 minutes at 4 ° C. Then, the colorimetric test described in Skehan according to Example 2 was carried out. The optical density of each well was read at 492 nanometers using the BioTek´s PowerWave XS Absorbance Microplate Reader spectrophotometer. The growth percentage (PG) was calculated with respect to the untreated control cells (C) in each of the concentrations of the compounds based on the difference in the optical density at the beginning (To) and at the end (T) of the exposure of the compounds, according to the formulas described in A. Monks et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J. Natl. Cancer Inst. 1991 (83) 757

766. The formula PG = 100 X [(T-To) / (C-To)] was used when T is greater than or equal to To, and the formula PG = 100 X [(T-To) / (To)] when T is less than To. The concentration at which PG is +50, 0 and -50 represents, respectively, 50% growth inhibition (GI50), total growth inhibition (TGI) and 50% cell death (LC50). Therefore, a PG value of 0 corresponds to the amount of cells present at the beginning of exposure to the compound and negative PG values denote net cell death. All compounds derived from substituted pyrroles were inactive, with MCI values greater than 100 microM.

Example 5. Compounds derived from 1,2,3,5-tetrasubstituted pyrroles decrease the effects of LPS produced on the A549 cell line.

The A549 cell line was stimulated with 300 micrograms / milliliter of E. coli LPS in combination with each of the compounds at a single dose of 100 microM. The incubation time of the compounds was 18 hours, after which the cells were precipitated in 25 microliters of 50% w / v TCA and fixed for 60 minutes at 4 ° C. Then, the colorimetric assay described in Skehan et al. cited in Example 2. The optical density of each well was read at 492 nanometers using the BioTek´s PowerWave XS Absorbance Microplate Reader spectrophotometer and the survival percentage (PS) with respect to untreated cells (negative control) was determined and cells treated with 300 micrograms / milliliter of E. coli LPS (positive control, TLPS) of each concentration of compound (T). Therefore, the calculation was developed as follows: PS = 100 x [(T-TLPS) / (C-TLPS)]. With this calculation, a PS value of 0 corresponds to cells treated only with E. coli LPS present at the end of the exposure, while positive PS values denote net cell survival.

In the same way, new trials were carried out to verify morphological changes induced by all treatments. For this, the A549 cell line was cultured with 300 micrograms / ml of E. Coli LPS alone or in combination with the 1,2,3,5-tetrasubstituted pyrrole-derived compounds (Compounds 1-15) for 18 hours and he used phase contrast microscope (Olympus CK40, Tokyo, Japan) to visualize morphological changes.

From the results shown in Figure 1, it is concluded that all the compounds tested have the ability to decrease the effects of LPS produced in the A549 cell line. The best results were achieved with compounds DTA0118 and DTA0119 with PS values of 64% and 55%, respectively.

Figure 3 shows how co-treatment with compound DTA0118 prevented morphological changes undergone by A549 cells after incubation with 300 micrograms / ml of E. coli LPS.

Example 6. Determination of the expression levels of the pro-inflammatory cytokines IL-8, IL-1j and the TLR4, IKja, Bax and Bcl-2 proteins.

The A549 cell line was cultured in culture medium enriched with 2% fetal bovine serum for 24 hours. Subsequently, the cell culture was treated with 300 micrograms / milliliter of E. coli LPS (serotype 055: B5, Sigma-Aldrich®) together with 100 microM of the compounds of the present invention, for 18 hours. After that time, the culture medium was collected in sterile Falcon 15 milliliter test tubes and centrifuged at 1,200 rpm for 10 minutes at 4 ° C. Then, the supernatants from the 15 milliliter Falcon tubes were collected and added in the form of 500 microliter aliquots in sterile 1.5 ml tubes. These eppendorf tubes were kept frozen at -80 ° C until use. Subsequently, 200 microliths of this culture medium were added in a 96-well test plate and the antibodies and reagents of the BD Biosciences commercial kit "Cytometry-based bead array system" were added. The last step consisted of measuring the expression levels of IL-6 and IL-8 by ELISA using a length range of 633 nanometers. Expression levels of the TLR4 membrane receptor protein of the NF-KB transcription factor inhibitor protein were measured by Western blotting. For this, electrophoresis of each of the samples was performed in 10% SDS-PAGE gels for 2 hours. After that, the SDS-PAGE gels were transferred into polyvinylidene difluoride (PVDF) membranes and blocked for 1 hour at room temperature in TBST buffer (Tris-buffered saline Tween-20) plus 10% of milk lacking fat content Anti-TLR4 and anti-NF-KB inhibitor, anti-Bax and anti-Bcl-2 antibodies (Santa Cruz Biotechnology Inc.) were incubated for 2 hours in TBST plus 5% milk lacking in fat content. After incubation for 2 hours with the primary antibody, incubation was performed for 1 hour with the peroxidase-conjugated secondary antibody (Goat Anti-rabbit IgG-HRP; Santa Cruz Biotechnology Inc.) Banding was carried out. by chemiluminescent system with the GE Healthcare kit “Amersham ECL Western Blotting Detection Reagent”. Subsequently, each protein band was densitometered using the public software Scion Image. Each Western Blot was performed three times with the same experimental conditions. To check the homogeneity of the protein load in each of the wells, the same PVDF membranes were re-incubated with the primary Beta-actin antibody (Cell Signaling Technology) for 2 hours and with the same secondary antibody and the same experimental conditions of anti-TLR4 and anti-protein transcription factor inhibitor NF-KB, anti-Bax and anti-Bcl-2 antibodies. All data related to the anti-inflammatory and anti-apoptotic activity of the compounds object of the present invention are expressed as the average of the results obtained for each compound ± S.E.M. (“Standard error of the mean”) and statistical analyzes were carried out using analysis of variance (one-way ANOVA) corrected by Bonferroni Test and using the SPSS software (version 15.0 for Windows). For the statistical analyzes of the Western Blot assays, the same methodology was used but the data were normalized according to the Beta-actin protein load control. The effects were considered significant when p <0.05.

Figure 4a shows that 1,2,3,5-tetrasubstituted pyrrole compounds (Compounds 1-15) were able to reduce the levels in the supernatant of the pro-inflammatory cytokine IL-6 induced under the treatment of LPS of E. Coli (p <0.01) in a significant way (p <0.05). Figure 4b shows that the compounds of the present invention were able to completely prevent the effects produced by the treatment of E. coli LPS at the levels of the pro-inflammatory cytokine IL-8 (p <0.05).

Figure 5b shows, according to the Western Blot assays, that the expression of TLR4 protein was significantly increased under the treatment with E. coli LPS (p <0.001), an increase that was reduced under the co-treatment of pyrrole-derived compounds. (Compounds 1-15) compared to the single treatment of 300 micrograms / ml of E. Coli LPS for 18 hours (p <0.001). It also shows that the expression of the NF-KB transcription factor inhibitor protein was reduced under treatment with E. coli LPS (p <0.001) and that these compounds produced an increase in protein expression levels IK�a when this result was compared with the treatment of E. coli LPS (p <0.001), showing that the compounds derived from pyrroles could inhibit the effects produced by E. coli LPS in the TLR4-NFK signaling pathway.

Example 7. Compounds derived from 1,2,3,5-tetrasubstituted pyrroles inhibit apoptosis produced by LPS.

The annexin V-FITC-based apoptosis detection kit (Beckton Dickinson Pharmingen TM) and the FACSCalibur flow cytometer (Beckton Dickinson Pharmingen TM) were used to measure early apoptosis and cell death according to the manufacturer's instructions. For these assays, A549 cells were grown at a density of 2x106 in DMEN medium supplemented with 2% FBS and 1% antibiotics for 24 hours. The cells were then treated for 18 hours with or without 300 μgr / ml LPS of E. Coli alone or in combination of 100 microM of 1,2,3,5-tetrasubstituted pyrrole compounds. Following these treatments, A549 cells were resuspended for incubation with 100 μl of 1X solution composed of 0.1 M Hepes / NaOH (0.1 M, pH 7.4), NaCl (1.4 M), CaCl2 (25 mM) binding where 5 microliters of Annexin V-FITC and 5 microliters of iodide itself were added at room temperature for 15 minutes in the dark. After incubation, 400 microliters of the binding solution was added and the mixture was analyzed on the FACSCalibur (Beckton Dickinson PharmingenTM) flow cytometer using an FITC (Fluorescein Isothiocyanate) signal detector called FL1 and a propidium iodide fluorochrome at 488 nanometers detectable by FL3. The experiments were repeated in triplicate. The data were analyzed according to 100,000 cells as a percentage of early apoptosis according to this formula: (%) = Annexin V-FITC + IP- / IP- where IP- = Annexin V-FITC-IP- plus Annexin V-FITC + IP- . Annexin V-FITC + IP + staining was considered as the percentage of late apoptosis and necrosis or cell death. Data were analyzed using WinDI 2.8 software (Scripps Institute). In the same way, to study the morphological changes induced by LPS, nuclear DNA staining was carried out by staining called trihydro bisBenzimide-chloride (Hoechst 33258). For this, the A549 cell line was cultured at a density of 1 x 106 in DMEN culture medium supplemented with 2% FBS and 1% of antibiotics for 24 hours, to be subsequently treated for 18 hours with or without 300 μgr / ml LPS of E. Coli alone or in combination with 100 microM of the 1,2,3,5-tetrasubstituted pyrrole compounds. After the incubation time elapsed, the cells were washed with PBS saline and fixed with 4% paraformaldehyde dissolved in PBS at room temperature and for 10 minutes. After the fixation process, the cells were washed with PBS saline and stained with 80 microg / ml of Hoechst in PBS at room temperature for 15 minutes in the dark. Finally, the stained nuclei were visualized under a fluorescence microscope (Leica DM2000) for the detection of morphological changes and for the count of the percentage of apoptotic nuclei in each experimental condition. An apoptotic cell was considered to be one that had three or more apoptotic bodies and / or had condensed chromatin in the periphery of the nucleus. To evaluate the mechanism of prevention of the apoptotic process induced by the compounds object of the present invention, new Western Blot assays were carried out using the same experimental conditions used for the primary antibodies anti-TLR4 and anti-protein transcription factor inhibitor NF -KB, but for these new tests the primary anti-Bax and anti-Bcl-2 antibodies, indicators of the apoptotic process were used. Based on the apoptosis study trials using flow cytometry and Annexin V-propidium iodide staining, the percentages of apoptosis in the total cell populations were quantified.

Figure 6 shows how, after incubation with 300 microg / ml of E. Coli LPS for 18 hours, the percentage of early apoptosis and cell death (Annexin V + / IP- and Annexin V-FITC + IP +, respectively) were increased compared to control cells (p <0.001 in both cases, Figure 6b). These percentages of apoptosis were prevented by co-treatment with 1,2,3,5-tetrasubstituted pyrrole compounds (p <0.001 in all cases). These findings were completed by nuclear staining using BisBenzimide. So that A549 cells treated with 300 microg / milliliter of LPS of E. Coli for 18 hours showed fragmented nuclei or "apoptotic bodies" and condensed chromatin, classical characteristics of apoptotic cells. This presence of apoptotic bodies and condensation of nuclear chromatin was prevented by co-treatments of the pyrrolic derivatives object of the present invention (Figure 6c and 6d). In Figures 6a and 6c the compound DTA0118 is shown as representative of the prevention of the apoptotic process.

To evaluate the mechanism of prevention induced by the pyrrole compounds, new Western Blot tests were carried out. Figure 7a shows that treatment with 300 micrograms / ml of LPS of E. Coli showed an increase in the expression of the pro-apoptotic protein Bax (p <0.001. Figure 7b) when compared with control cells. This increase in expression was inhibited by co-treatment of 1,2,3,5-tetrasubstituted pyrrole compounds compared to control cells (p <0.01. Figure 7b). In addition, treatment of the A549 cell line with 300 micrograms / ml for 18 hours of E. coli LPS induced a significant decrease in the expression levels of the Bcl-2 anti-apoptotic protein (p <0.01. Figure 7b ). This decrease in expression levels was inhibited by the co-treatment of E. coli LPS plus 100 microM of the 1,2,3,5-substituted pyrrole compounds (Compounds 1-15) compared to cells treated with E. coli LPS. for 18 hours (p <0.01. Figure 7b). An increase in apoptosis (the ratio between Bax and Bcl-2 proteins) was observed in A549 cells treated with 300 micrograms / ml of E. coli LPS for 18 hours. This increase was prevented with the co-treatment of LPS from E. Coli and the pyrrole-derived compounds for 18 hours (Figure 7c). These results indicate that 1,2,3,5-tetrasubstituted pyrrole-derived compounds are effective in preventing LPS-induced apoptosis of E. Coli in the A549 cell line.

Example 8. Compounds derived from 1,2,3,5-tetrasubstituted pyrroles act by an independent mechanism to Tyrosine Kinase Inhibitors in the prevention of LPS-induced effects of E. Coli

The A549 cell line was grown in Dulbecco's modified Eagle's medium (DMEN) supplemented with 2% FBS. The cells were grown in P-100 plates for 24 hours. Next, fresh culture medium without FBS containing 30 microM Genistein inhibitor was added, and incubated for 30 minutes at 37 ° C and 5% CO2. Subsequently, culture medium supplemented with 2% FBS containing 300 microg / ml LPS of E. Coli alone or in combination with 100 microM of 1,2,3,5-tetrasubstituted pyrroles (Compounds 1-15) was added during 18 hours. After that time, Western blot methodologies were performed as described in previous paragraphs for the study of TLR4, IK�a, Bax and Bcl-2 proteins. Likewise, by means of phase contracting microscopy, all the experimental conditions detailed were photographed without digital staining.

Figure 8 shows how treatment with 300 microg / ml LPS of E. Coli caused an increase in the expression of TLR4 and Bax proteins, and a decrease in the expression of IK�a and Bcl-2, as they have shown also the previous figures. These effects were prevented by pre-treatment with 30 microM of Genistein (Figure 8). Prevention was superior when the A549 cell line was treated with 300 microg / ml of E. coli LPS in combination with 1,2,3,5-tetrasubstituted pyrrole compounds (Compounds 1-15; C1-15) for 18 hours but Pre-treatment with 30 microM of Genistein for 30 minutes did not change the magnitude of the prevention induced by the 1,2,3,5-tetrasubstituted pyrrole compounds object of the present invention in the effects induced by LPS of E. Coli in the expression changes of TLR4, IK�a, Bax and Bcl-2 proteins.

Figure 9 shows similar results, where after incubation with 300 microg / ml LPS of E. Coli, A549 cells showed morphological changes, such as elongations, which were only partially prevented by pre-treatment with the Genistein inhibitor and totally with 1,2,3,5tetra substituted substituted pyrroles. Representative 1,2,3,5-tetrasubstituted pyrrolic compound called DTA0118 is shown. Likewise, pre-treatment with 30 microM of Genistein for 30 minutes did not change the magnitude of the prevention induced by 1,2,3,5-tetrasubstituted pyrrole compounds in the effects induced by LPS of E. Coli (Figure 9).

These results indicate that Tyrosin kinase inhibitors could not be used in combination with 1,2,3,5-tetrasubstituted pyrrole-derived compounds in the prevention of LPS-induced effects of E. Coli on the A549 cell line.

Claims (24)

1. Use of a chemical compound derived from a 1,2,3,5-tetrasubstituted pyrrole of formula (I): (I) where R1 is a C1-C4 aliphatic chain, substituted or unsubstituted, R2 is a methyl or ethyl ester, R3 is phenyl, biphenyl, methylphenyl, benzylphenyl or cyclohexyl, substituted or unsubstituted, R4 is phenyl, biphenyl or cyclohexyl, substituted or unsubstituted, R5 is H or a C1-C3 alkyl, in the preparation of a medicament useful for the treatment of inflammatory diseases that occur with cellular apoptotic processes.

2.

Use according to claim 1, wherein said aliphatic chain of R1 is substituted with a phenyl.

3.

Use according to claim 1, wherein said aliphatic chain of R1 is substituted with a furan group.

Four.

Use according to any of claims 1 to 3, wherein said radical R3 is substituted by a halogen.

5.

Use according to any one of claims 1 to 4, wherein said radical R4 is substituted by a halogen.

6.

Use according to claim 5, wherein said halogen is chlorine.

7.

Use according to any one of claims 1 to 6, wherein said radical R5 is methyl.

8.

Pharmaceutical composition comprising at least one compound derived from a 1,2,3,5-tetrasubstituted pyrrole of formula (I)

(I) where R1 is a C1-C4 aliphatic chain, substituted or unsubstituted, R2 is a methyl or ethyl ester, R3 is phenyl, biphenyl, methylphenyl, benzylphenyl or cyclohexyl, substituted or unsubstituted, R4 is phenyl, biphenyl or cyclohexyl, substituted or unsubstituted, R5 is H or a C1-C3 alkyl, together with pharmaceutically acceptable excipients, for the treatment of inflammatory diseases that occur with cellular apoptotic processes.

9.

A pharmaceutical composition according to claim 8, which is administered intravenously.

10.

A pharmaceutical composition according to claim 8, which is administered orally, muscularly or intramuscularly.

eleven.

A pharmaceutical composition according to any of claims 8 to 10, in a dosage unit.

12.

A pharmaceutical composition according to any of claims 8 to 11, together with at least one other therapeutic agent for the treatment of inflammatory diseases that occur with cellular apoptotic processes.

13.

A pharmaceutical composition according to any of claims 8 to 12, wherein said cellular apoptotic processes are produced by sepsis.

14.

A pharmaceutical composition according to claim 13, wherein said sepsis is caused by bacteria.

fifteen.

A pharmaceutical composition according to claim 14, wherein said bacteria are Gram-negative bacteria.

16.

A pharmaceutical composition according to any of claims 8 to 15, wherein said inflammatory disease is selected from the group consisting of sepsis, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis and traumas .

17.

Chemical compound derived from a 1,2,3,5-tetrasubstituted pyrrole of formula (I):

(I) where R1 is a C1-C4 aliphatic chain, substituted or unsubstituted, R2 is a methyl or ethyl ester, R3 is phenyl, biphenyl, methylphenyl, benzylphenyl or cyclohexyl, substituted or unsubstituted, R4 is phenyl, biphenyl or cyclohexyl, substituted or unsubstituted, R5 is H or a C1-C3 alkyl, for the treatment of inflammatory diseases that occur with cellular apoptotic processes.

18.

Chemical compound according to claim 17, wherein said aliphatic chain of R1 is substituted with a phenyl.

19.

Chemical compound according to claim 17, wherein said R1 aliphatic chain is substituted with a furan group.

twenty.

Chemical compound according to any of claims 17 to 19, wherein said radical R3 is substituted by a halogen.

twenty-one.

Chemical compound according to any of claims 17 to 20, wherein said radical R4 is substituted by a halogen.

22

Chemical compound according to claim 21, wherein said halogen is chlorine.

2. 3.

Chemical compound according to any of claims 17 to 22, wherein said radical R5 is methyl.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201131785 SPAIN Date of submission of the application: 08.11.2011 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

X

Weaver Weaver David et al. From conjugated tertiary skipped diynes to chainfunctionalizedtetrasubstituted pyrroles. Chemistry (Weinheim an der Bergstrasse, Germany) 2009 VOL: 15 No: 4 Pags: 838-842 ISSN 1521-3765 (Electronic) Doi: doi: 10.1002 / chem.200802262pubmed: 19086044, page 839, scheme 1, table 1, compound 1 and page 840, scheme 2. 17,18.23

TO

Weaver Weaver David et al. A diversity-oriented strategy for the construction of tetrasubstitutedpyrroles via coupled domino processes. Journal of the American Chemical Society 14.07.2004 VOL: 126 No: 27 Pags: 8390-8391 ISSN 0002-7863 (Print) Doi: pubmed: 15237991, Schemes 1 and 2. 1-23

TO

PADRON J M et al. Synthesis and anti-breast cancer activity of tetrasubstituted pyrrolederivatives. Letters in Drug Design and Discovery 11.2005 VOL: 2 No: 7 Pags: 529-532 ISSN 1570-1808 (print) Doi: doi: 10.2174 / 157018005774479122, page 531, column 2 and table 1. 1-23

TO

PADRON J M et al. Antiproliferative activity in HL60 cells by tetrasubstituted pyrroles: a structureactivity relationship study. BIOORGANIC MEDICINAL CHEMISTRY LETTERS, 05/16/2005 VOL: 15 No: 10 Pags: 2487-2490 ISSN 0960-894X, page 2488, table 1, page 2489, column 2. 1-23

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 11.02.2013

Examiner H. Aylagas Cancio Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201131785 CLASSIFICATION OBJECT OF THE APPLICATION A61K31 / 40 (2006.01) C07D207 / 34 (2006.01) A61P35 / 00 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A61K, C07D, A61P Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, EMBASE, MEDLINE, BIOSIS, NPL, XPESP, XPESP2, REGISTRY, HCAPLUS State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201131785 Date of Written Opinion: 11.02.2013 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-16, 19-22 17, 18, 23 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-16, 19-22 17, 18, 23 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201131785  1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

Weaver Weaver David et al. From conjugated tertiary skipped diynes to chain-functionalizedtetrasubstituted pyrroles. Chemistry (Weinheim an der Bergstrasse, Germany) 2009 VOL: 15 No: 4 Pags: 838-842 ISSN 1521-3765 (Electronic) Doi: doi: 10.1002 / chem.200802262pubmed: 19086044, page 839, scheme 1, table 1, compound 1 and page 840, scheme 2. 11/30/2008

D02

Weaver Weaver David et al. A diversity-oriented strategy for the construction of tetrasubstitutedpyrroles via coupled domino processes .. Journal of the American Chemical Society 14.07.2004 VOL: 126 No: 27 Pags: 8390-8391 ISSN 0002-7863 (Print) Doi: pubmed: 15237991, schematics 1 and 2. 14.07.2004

D03

PADRON J M et al. Synthesis and anti-breast cancer activity of tetrasubstituted pyrrolederivatives. Letters in Drug Design and Discovery 11.2005 VOL: 2 No: 7 Pags: 529-532 ISSN 1570-1808 (print) Doi: doi: 10.2174 / 157018005774479122, page 531, column 2 and table 1. 10/31/2005

D04

PADRON J M et al. Antiproliferative activity in HL60 cells by tetrasubstituted pyrroles: a structure-activity relationship study. BIOORGANIC MEDICINAL CHEMISTRY LETTERS, 05/16/2005 VOL: 15 No: 10 Pags: 2487-2490 ISSN 0960-894X, page 2488, table 1, page 2489, column 2. 05/16/2005

 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application refers to the use of a chemical compound derived from a pyrrole 1, 2, 3, 5 tetrasubstituted of formula I in the preparation of a medicament useful for the treatment of inflammatory diseases that occur with cellular apoptotic processes. The chemical compounds and the pharmaceutical compositions containing them are also claimed. Document D1 refers to the synthesis of tetrasubstituted pyrrole-derived compounds of formula 1 (see page 839, table 1 and scheme 2 on page 840). The compound claims of the present application 17, 18 and 23 cite compounds with the same structure as those of document D1, when the radical R1 of the formula I of reiv. 17 is an alkyl chain substituted by phenyl (reiv. 18) and when the radical R5 is methyl (reiv. 23). Therefore, in view of document D1, claims 17, 18 and 23 lack novelty and inventive activity according to articles 6.1 and 8.1 of the L.P. Documents D2 to D4 refer to compounds derived from pyrrole tetrasubstituted in positions 1, 2, 3 and 4 of the pyrrole ring and therefore differ from the compounds claimed in the present application. In documents D3 and D4 the antiproliferative activity of these compounds is cited. Accordingly, in view of the cited documents, although the compounds of formula I are known, their pharmaceutical compositions and the claimed use have not been referred to in the state of the art, therefore the material corresponding to claims 1-16 they have novelty and inventive activity according to articles 6.1 and 8.1 of the LP As for claims 19-22, substitutions of phenyl groups for halogen or alkyl chains for furan have not been described in document D1. Therefore said claims present novelty and inventive activity according to articles 6.1 and 8.1 of the L.P. State of the Art Report Page 4/4