WO2012089876A1 - Pyrimidine urea‑derived compound for treating inflammatory diseases - Google Patents

Pyrimidine urea‑derived compound for treating inflammatory diseases Download PDF

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WO2012089876A1
WO2012089876A1 PCT/ES2011/070822 ES2011070822W WO2012089876A1 WO 2012089876 A1 WO2012089876 A1 WO 2012089876A1 ES 2011070822 W ES2011070822 W ES 2011070822W WO 2012089876 A1 WO2012089876 A1 WO 2012089876A1
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substituted
unsubstituted
pharmaceutical composition
compound
composition according
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PCT/ES2011/070822
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Spanish (es)
French (fr)
Inventor
Jesús VILLAR HERNANDEZ
José Manuel PADRON CARRILLO
Milena CASULA
Nuria Esther CABRERA BENITEZ
Angela María RAMOS NUEZ
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Ciber De Enfermedades Respiratorias
Servicio Canario De Salud
Universidad De La Laguna
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to compounds derived from pyrimidine urea that have anti-inflammatory activity.
  • Such compounds are useful for preparing pharmaceutical compositions for the treatment of inflammatory diseases, preferably sepsis, arthritis, kidney infections, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis, acute hepatitis, acute myocardial dysfunction induced by septic shock and necrotizing enterecolitis.
  • inflammatory diseases preferably sepsis, arthritis, kidney infections, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis, acute hepatitis, acute myocardial dysfunction induced by septic shock and necrotizing enterecolitis.
  • LPS Lipopolysaccharides
  • nosina which reduces the toxicity of cytokines in lung cells in in vitro experiments and suppresses LPS-induced lung inflammation in in vivo experiments.
  • WO00 / 43384 describes aromatic heterocyclic ureas that inhibit the production of cytokines involved in inflammatory processes.
  • US 2005/023981 1 A1 explains the use of trisubstituted ureas N-1, 1, 3 that inhibit the release of cytokines by lipopolysaccharides.
  • the present invention is a pyrimidine urea derivative compound of formula (I):
  • R a is selected from the group consisting of H, carboxyl, CF 3 , F, Cl, Br, I, substituted or unsubstituted CC 10 alkyl ether, substituted or unsubstituted C 2 -C 10 alkenyl ether, ether of substituted or unsubstituted C 2 -Ci 0 alkynyl, substituted or unsubstituted CC 0 alkyl, substituted or unsubstituted C 2 -C 0 alkenyl, substituted or unsubstituted C 2 -C 0 alkynyl, substituted C 3 -C 0 cycloatkyl or unsubstituted, substituted or unsubstituted C5-C10 cycloalkenyl, substituted or unsubstituted C 8 -C 10 cycloalkynyl, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted C
  • inflammatory diseases is understood as diseases that occur with inflammation.
  • infections caused by bacteria that occur with inflammation, such as sepsis.
  • arthritis kidney infections, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis, acute hepatitis, acute myocardial dysfunction induced by septic shock and necrotizing enterecolitis .
  • the compounds derived from pyrimidine urea described in the present invention possess anti-inflammatory activity since they are capable of decreasing the anti-proliferative effects induced by LPS of Gram-negative bacteria on the A549 cell line, inhibiting the increase of pro-cytokines inflammatory IL-8 and IL-1 /? induced by LPS and to inhibit the increase in expression of the TLR4 receptor protein and to inhibit the decrease of the NF- ⁇ transcription factor inhibitor protein induced by LPS.
  • a preferable embodiment is a compound of the invention, where R is benzene.
  • Another embodiment of the invention is a compound of the invention, where R is
  • Another embodiment of the invention is a compound of the invention, where R is
  • Another embodiment of the invention is a compound of the invention, where R is
  • Another embodiment of the invention is a compound of the invention, where R is
  • Another embodiment of the invention is a compound of the invention, where R is
  • a preferable embodiment is a pharmaceutical composition comprising at least one compound of the invention, together with pharmaceutically acceptable excipients, for the treatment of inflammatory diseases.
  • Another more preferable embodiment is a pharmaceutical composition of the invention in a dosage unit. And another embodiment is that said dosage unit is a tablet.
  • a preferable embodiment is the pharmaceutical composition of the invention, which is administered orally. And another embodiment is that said composition is administered intravenously, muscularly and / or intramuscularly.
  • a preferable embodiment is a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of the invention, together with pharmaceutically acceptable excipients and at least one other therapeutic agent for the treatment of inflammatory diseases.
  • Another embodiment is the pharmaceutical composition of the invention, wherein said inflammatory disease is caused by bacteria. And another embodiment is that said bacteria are Gram-negative bacteria.
  • a further embodiment is the pharmaceutical composition of the invention, wherein said inflammatory disease is a sepsis.
  • said inflammatory disease is selected from the group consisting of arthritis, renal infections, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, peritonitis septic, acute hepatitis, acute myocardial dysfunction induced by septic shock and necrotizing enterecolitis.
  • the most preferable embodiment of the invention is an intravenously administered composition, comprising at least one compound derived from pyrimidine urea of formula (I):
  • 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.
  • Figure 1 shows the effect of compounds 1-7 on the production of the pro-inflammatory cytokine IL-8 when the A549 cell line was stimulated with 300 micrograms / milliliter of E. coli LPS and treated with 100 microMolar of the compounds 1-7.
  • the results of the control cells (C), A549 cells treated with E. coli LPS (LPS) and A549 cells treated with E. coli LPS plus 100 microMolar of compounds 1-7 (Compounds 1-7) are shown.
  • This figure represents the statistical symbology: *** p ⁇ 0.001 vs. C; ⁇ p ⁇ 0.001 vs. LPS
  • Figure 2 shows the effect of compounds 1-7 on the production of the pro-inflammatory cytokine IL-1 /?
  • Figure 3 shows the bands corresponding to the Western Blot assays after the extraction of total proteins from control A549 cells (C), A549 cells under treatment with 300 micrograms / milliliter of LPS from E. Coli (LPS) and under the treatment of 300 micrograms / milliliter of E.Coli LPS plus 100 microMolar of compounds 1-7 (Compounds 1-7). Homogeneity in the protein concentration of each well by Beta-actin is also shown.
  • Figure 4 shows the expression changes of TLR4 and ⁇ proteins (normalized based on Beta-actin load control) when A549 cells were used as control (C), A549 cells under treatment with 300 micrograms / milliliter of LPS of E. Coli (LPS) and under the treatment of 300 micrograms / milliliter of LPS of E. Coli plus 100 microMolar of compounds 1-7 (Compounds 1-7).
  • Figure 5 shows the PS values (%) and the chemical structure of different urea compounds.
  • R- ⁇ , R 2 , and 3 being the groups specified in the following table:
  • Compound 7 has the formula:
  • Anti-microbial activity was determined in Gram-positive bacteria Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212) and Gram-negative bacteria Escher ⁇ chia coli (ATCC 35218), Klebsiella pneumoniae (ATCC 700603) and Pseudomonas aer53inosa (ATCC 700603) , 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) and the cytotoxic activity in HeLa cell lines (human cervical cancer cell line), Ishikawa (human uterine endometrial cancer cell line), SW1573 (human epithelial cancer cell line pulmonary), T-47D (human breast cancer cell line), WiDr (human colon cancer cell line) and A549 (human pulmonary epi
  • Bacterial and fungal species were grown on agar plates at 35-37 ° C. After 24 hours of incubation, the species were resuspended in saline solution at a concentration of approximately 5 x 10 5 cfu / ml (colony forming units per milliliter) for bacterial species and 1-5 x 10 3 for Candida species.
  • the antimicrobial activity of the compounds was determined in 96-well plates using ueller Hinton broth medium for bacterial species and RP I-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 microMolar.
  • the 96-well plates were incubated at 35-37 ° C in a dark and humid chamber. After incubation, the species were precipitated in 25 microliters of 50% (weight / volume) TCA (trichloroacetic acid) and fixed for 60 minutes at 4 ° C. Then, the colorimetric assay described in Skehan, P. e ⁇ al., New colorimetric cytotoxicity assay for anticancer-drug screening was carried out. J Nati Cancer Inst 1990 (82) 1107-1112. After the test, agitation of the 96-well plates occurred and by spectrophotometry (BioTek ' s PowerWave XS Absorbance Microplate Reader) optical density values were obtained.
  • TCA trichloroacetic acid
  • the 96-well plates corresponding to the bacterial species were read at 630 nanometers after 24 hours of incubation.
  • the 96-well plates corresponding to the fungal species were read at 490 nanometers after 48 hours of incubation.
  • the MCI value minimum inhibitory concentration
  • Compounds 1 to 7 did not exhibit anti-microbial activity on the bacteria and fungal species tested, since all the compounds were inactive (MCI values greater than 100 microMolar).
  • cell lines were grown in 25 cm 2 cell culture bottles in RPMI-1640 culture medium supplemented with 5% fetal bovine serum and 2 milliMolar glutamine in an incubator with the following conditions Experimental: 37 ° C, 5% C0 2 and 95% humid air. Exponentially growing cells were trypsinized and resuspended in culture medium containing antibiotics (100 units of penicillin and 0.1 milligrams of streptomycin per milliliter). The cell suspension with a percentage greater than 97% viability by the Trypan Blue staining and exclusion technique was counted. After counting, cell dilutions were made for 96-well plate inoculation.
  • the cells were seeded at a concentration of 1 x 10 4 (for the SW1573 cell line), 1.5 x 10 4 (for the HeLa, Ishikawa and T-47D cell lines) and 2 x 10 4 (for the WiDr cell line ) cells per well. Each compound was tested in triplicate at different dilutions in the range of 1 -100 micro olar. 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% (weight / volume) TCA (trichloroacetic acid) and fixed for 60 minutes at 4 ° C. Then, the colorimetric assay described in Skehan, P. et al.
  • the optical density of each well was read at 492 nanometers using the BioTek ' s PowerWave XS Absorbance Microplate Reader spectrophotometer.
  • the percentage growth (PG) was calculated with respect to the untreated control cells (C) in each of the concentrations of the compounds based on the difference in optical density at the beginning (T 0 ) and at the end of the exposure of the compounds (T), 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. Nati. Cancer Inst. 1991 (83) 757-766.
  • the concentration at which PG is +50, 0 and -50 represents, respectively, 50% growth inhibition (Gl 50 ), total growth inhibition (TGI) and 50% cell death (LC 50 ). 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.
  • the compounds used in the present invention did not possess cytotoxic activity on the cell lines tested since all the compounds were inactive (Gl 50 growth inhibition values greater than 100 microMolar).
  • Example 3 Determination of the expression levels of IL-8, IL- ⁇ , TLR4 and ⁇ .
  • the A549 cell line human pulmonary epithelial cell line
  • 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 commercially distributable by the Sigma-Aldrich ® company) together with 100 microMolar of the synthesis products described in the present invention for 18 hours . After the treatment time, the culture medium was collected in sterile Falcon 15 milliliter test tubes and centrifuged at 1,200 RPM (revolutions per minute) for 10 minutes at 4 ° C.
  • TLR4 membrane receptor protein
  • NF- ⁇ transcription factor inhibitor protein
  • electrophoresis of each of the samples was performed in SDS-PAGE gels (10%) for 2 hours.
  • the SDS-PAGE gels were transferred into polyvinylidene difluoride (PVDF) membranes and blocked for 1 hour at room temperature in the TBST buffer (Tr ⁇ s-buffered saline Tween-20) plus 10% of Milk lacking in fat content.
  • the anti-TLR4 (Santa Cruz Biotechnology Inc.) and anti-NF- ⁇ inhibitor (Santa Cruz Biotechnology Inc.) antibodies were incubated for 2 hours in TBST plus 5% milk lacking in fat.
  • the same PVDF membranes were re-incubated with the primary Beta-actin antibody (from the Cell Signallng Technology company) for 2 hours and with the same secondary antibody and the same experimental conditions of anti-TLR4 and anti-transcription factor inhibitor protein called kappa B nuclear factor.
  • Figure 1 shows that compounds 1-7 were able to reduce the levels of IL-8 pro-inflammatory cytokine induced under LPS treatment of E Coli (p ⁇ 0.001) in a significant way (pO.001).
  • Figure 2 shows that the compounds 1-7 object 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-1/9 (p ⁇ 0.01 ).
  • Figures 3 and 4 show that TLR4 protein expression was significantly increased under treatment with E. coli LPS (p ⁇ 0.001).
  • the A549 cell line was stimulated with 300 microgramds / milliliter of E. coli LPS in combination with each of the compounds at a single dose (00 microMolar).
  • the incubation time of the compounds was 18 hours, after which the cells were precipitated in 25 microliters of 50% (weight / volume) TCA (trichloroacetic acid) and fixed for 60 minutes at 4 ° C.
  • TCA trichloroacetic acid

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Abstract

The present invention relates to a pyrimidine urea‑derived compound for treating inflammatory diseases. The compound of the invention can be used for preparing a pharmaceutical composition for treating said inflammatory diseases, preferably sepsis caused by Gram‑negative bacteria, arthritis, renal infections, acute sepsis‑induced pulmonary diseases, acute sepsis‑induced respiratory distress syndrome, endotoxic or endotoxemic shock, septic peritonitis, acute hepatitis, acute septic‑shock‑induced myocardial dysfunction and necrotizing enterocolitis.

Description

COMPUESTO DERIVADO DE UREA PIRÍMIDÍNICA PARA EL TRATAMIENTO DE COMPOUND DERIVED FROM PYRIMIDINE UREA FOR THE TREATMENT OF
ENFERMEDADES INFLAMATORIASINFLAMMATORY DISEASES
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se refiere a compuestos derivados de urea pirimidínica que tienen actividad anti-inflamatoria. Dichos compuestos son útiles para preparar composiciones farmacéuticas para el tratamiento de enfermedades inflamatorias, preferiblemente sepsis, artritis, infecciones renales, enfermedades pulmonares agudas inducidas por sepsis, síndrome de distrés respiratorio agudo inducido por sepsis, shock endotóxico o endotoxemia, peritonitis séptica, hepatitis aguda, disfunción miocárdica aguda inducida por shock séptico y enterecolitis necrotizante. The present invention relates to compounds derived from pyrimidine urea that have anti-inflammatory activity. Such compounds are useful for preparing pharmaceutical compositions for the treatment of inflammatory diseases, preferably sepsis, arthritis, kidney infections, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis, acute hepatitis, acute myocardial dysfunction induced by septic shock and necrotizing enterecolitis.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
En la técnica existen diversos compuestos con capacidad para disminuir los efectos inflamatorios producidos por acción de los Lipopolisacáridos (LPS). Es el caso de la ¡nosina, que reduce la toxicidad de las citoquinas en células pulmonares en experimentos in vitro y suprime la inflamación pulmonar inducida por LPS en experimentos in vivo. Existen varios documentos que relacionan el uso de ureas con una posible acción anti-inflamatoria. El documento WO00/43384 describe .ureas heterocíclicas aromáticas que inhiben la producción de citoquinas involucradas en los procesos inflamatorios. El documento US 2005/023981 1 A1 explica el uso de ureas N-1 , 1, 3 trisustituídas que inhiben la liberación de citoquinas por parte de los lipopolisacáridos. El documento US 2003/0216396 A1 describe el uso de compuestos ureas conteniendo una piridina, quinolina o isoquinolina y que funcionalmente serían de utilidad en el tratamiento de enfermedades inflamatorias como la osteoporosls o en los desórdenes patológicos relacionados con la angiogénesis.  In the art there are several compounds with the capacity to reduce the inflammatory effects produced by the action of Lipopolysaccharides (LPS). This is the case of nosina, which reduces the toxicity of cytokines in lung cells in in vitro experiments and suppresses LPS-induced lung inflammation in in vivo experiments. There are several documents that relate the use of ureas with a possible anti-inflammatory action. WO00 / 43384 describes aromatic heterocyclic ureas that inhibit the production of cytokines involved in inflammatory processes. US 2005/023981 1 A1 explains the use of trisubstituted ureas N-1, 1, 3 that inhibit the release of cytokines by lipopolysaccharides. US 2003/0216396 A1 describes the use of urea compounds containing a pyridine, quinoline or isoquinoline and which would functionally be useful in the treatment of inflammatory diseases such as osteoporosls or in pathological disorders related to angiogenesis.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención es un compuesto derivado de urea pirimidínica de fórmula (I):
Figure imgf000004_0001
The present invention is a pyrimidine urea derivative compound of formula (I):
Figure imgf000004_0001
donde: where:
- R es  - R is
Figure imgf000004_0002
Figure imgf000004_0002
- donde Ra está seleccionado entre el grupo formado por H, carboxilo, CF3, F, Cl, Br, I, éter de alquilo C C10 sustituido o no sustituido, éter de alquenilo C2-C10 sustituido o no sustituido, éter de alquinilo C2-Ci0 sustituido o no sustituido, alquilo C C 0 sustituido o no sustituido, alquenilo C2-Ci0 sustituido o no sustituido, alquinilo C2-C 0 sustituido o no sustituido, cicloatquilo C3-C 0 sustituido o no sustituido, cicloalquenilo C5-C10 sustituido o no sustituido, cicloalquinilo C8-C10 sustituido o no sustituido, arilo C6-C10 sustituido o no sustituido, aralquilo C7-C10 sustituido o no sustituido, heterociclilo de 3-10 miembros de anillo sustituido o no sustituido y heteroarilalquilo sustituido o no sustituido de 2 a 10 átomos de carbono, y ambos heterociclos de 1 a 3 átomos diferentes al carbono, - where R a is selected from the group consisting of H, carboxyl, CF 3 , F, Cl, Br, I, substituted or unsubstituted CC 10 alkyl ether, substituted or unsubstituted C 2 -C 10 alkenyl ether, ether of substituted or unsubstituted C 2 -Ci 0 alkynyl, substituted or unsubstituted CC 0 alkyl, substituted or unsubstituted C 2 -C 0 alkenyl, substituted or unsubstituted C 2 -C 0 alkynyl, substituted C 3 -C 0 cycloatkyl or unsubstituted, substituted or unsubstituted C5-C10 cycloalkenyl, substituted or unsubstituted C 8 -C 10 cycloalkynyl, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted C 7 -C 10 aralkyl, 3-10 heterocyclyl substituted or unsubstituted ring members and substituted or unsubstituted heteroarylalkyl of 2 to 10 carbon atoms, and both heterocycles of 1 to 3 atoms other than carbon,
para el tratamiento de enfermedades inflamatorias. for the treatment of inflammatory diseases.
En la presente invención, se entiende por "enfermedades inflamatorias" a enfermedades que cursan con inflamación. En este grupo se encuentran las infecciones causadas por bacterias que cursan con inflamación, como por ejemplo, la sepsis. También se encuentran dentro de este grupo la artritis, infecciones renales, enfermedades pulmonares agudas inducidas por sepsis, síndrome de distrés respiratorio agudo inducido por sepsis, shock endotóxico o endotoxemia, peritonitis séptica, hepatitis aguda, disfunción miocárdica aguda inducida por shock séptico y enterecolitis necrotizante. Los compuestos derivados de urea pirimidínica descritos en la presente invención poseen actividad anti-inflamatoria ya que son capaces de disminuir los efectos anti-- proliferativos inducidos por LPS de bacterias Gram-negativas sobre la línea celular A549, inhibir el aumento de las citoquinas pro-inflamatorias IL-8 e IL-1/? inducidas por LPS y de inhibir el aumento de expresión de la proteína receptora TLR4 y de inhibir la disminución de la proteína inhibidora del factor de transcripción NF-κΒ inducidos por LPS. In the present invention, "inflammatory diseases" is understood as diseases that occur with inflammation. In this group are infections caused by bacteria that occur with inflammation, such as sepsis. Also included in this group are arthritis, kidney infections, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, septic peritonitis, acute hepatitis, acute myocardial dysfunction induced by septic shock and necrotizing enterecolitis . The compounds derived from pyrimidine urea described in the present invention possess anti-inflammatory activity since they are capable of decreasing the anti-proliferative effects induced by LPS of Gram-negative bacteria on the A549 cell line, inhibiting the increase of pro-cytokines inflammatory IL-8 and IL-1 /? induced by LPS and to inhibit the increase in expression of the TLR4 receptor protein and to inhibit the decrease of the NF-κΒ transcription factor inhibitor protein induced by LPS.
Una realización preferible es un compuesto de la invención, donde R es benceno Otra realización de la invención es un com uesto de la invención, donde R es  A preferable embodiment is a compound of the invention, where R is benzene. Another embodiment of the invention is a compound of the invention, where R is
Otra realización de la invención e la invención, donde R es Another embodiment of the invention and the invention, where R is
Figure imgf000005_0001
Figure imgf000005_0001
Otra realización de la invención es un compuesto de la invención, donde R es
Figure imgf000005_0002
Another embodiment of the invention is a compound of the invention, where R is
Figure imgf000005_0002
Otra realización de la invención es un compuesto de la invención, donde R es Another embodiment of the invention is a compound of the invention, where R is
Figure imgf000005_0003
Figure imgf000005_0003
Otra realización de la invención es un compuesto de la invención, donde R es
Figure imgf000006_0001
Another embodiment of the invention is a compound of the invention, where R is
Figure imgf000006_0001
Otra realización de la invención es un compuesto de la invención, donde R es Another embodiment of the invention is a compound of the invention, where R is
Figure imgf000006_0002
Figure imgf000006_0002
Una realización preferible es una composición farmacéutica que comprende al menos un compuesto de la invención, junto con excipientes farmacéuticamente aceptables, para el tratamiento de enfermedades inflamatorias. A preferable embodiment is a pharmaceutical composition comprising at least one compound of the invention, together with pharmaceutically acceptable excipients, for the treatment of inflammatory diseases.
Otra realización más preferible es una composición farmacéutica de la invención en una unidad de dosificación. Y otra realización es que dicha unidad de dosificación sea un comprimido. Another more preferable embodiment is a pharmaceutical composition of the invention in a dosage unit. And another embodiment is that said dosage unit is a tablet.
Una realización preferible es la composición farmacéutica de la invención, que se administra por vía oral. Y otra realización es que dicha composición se administra por vía intravenosa, muscular y/o intramuscular. A preferable embodiment is the pharmaceutical composition of the invention, which is administered orally. And another embodiment is that said composition is administered intravenously, muscularly and / or intramuscularly.
Una realización preferible es una composición farmacéutica que comprende al menos un compuesto de la invención, junto con excipientes farmacéuticamente aceptables y al menos otro agente terapéutico para el tratamiento de enfermedades inflamatorias. A preferable embodiment is a pharmaceutical composition comprising at least one compound of the invention, together with pharmaceutically acceptable excipients and at least one other therapeutic agent for the treatment of inflammatory diseases.
Otra realización más es la composición farmacéutica de la invención, donde dicha enfermedad inflamatoria está causada por bacterias. Y otra realización es que dichas bacterias sean bacterias Gram-negativas. Another embodiment is the pharmaceutical composition of the invention, wherein said inflammatory disease is caused by bacteria. And another embodiment is that said bacteria are Gram-negative bacteria.
Una realización más es la composición farmacéutica de la invención, donde dicha enfermedad inflamatoria es una sepsis. Una realización preferible es la composición farmacéutica de la invención, donde dicha enfermedad inflamatoria está seleccionada entre el grupo compuesto por la artritis, infecciones renales, enfermedades pulmonares agudas inducidas por sepsis, síndrome de distrés respiratorio agudo inducido por sepsis, shock endotóxico o endotoxemia, peritonitis séptica, hepatitis aguda, disfunción miocárdica aguda inducida por shock séptico y enterecolitis necrotizante. A further embodiment is the pharmaceutical composition of the invention, wherein said inflammatory disease is a sepsis. A preferred embodiment is the pharmaceutical composition of the invention, wherein said inflammatory disease is selected from the group consisting of arthritis, renal infections, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or endotoxemia, peritonitis septic, acute hepatitis, acute myocardial dysfunction induced by septic shock and necrotizing enterecolitis.
La realización más preferible de la invención es una composición administrada por vía intravenosa, que comprende al menos un compuesto derivado de urea pirimidínica de fórmula (I): The most preferable embodiment of the invention is an intravenously administered composition, comprising at least one compound derived from pyrimidine urea of formula (I):
Figure imgf000007_0001
Figure imgf000007_0001
para el tratamiento de enfermedades inflamatorias. for the treatment of inflammatory diseases.
Otra realización de la invención es un método de tratamiento de una enfermedad inflamatoria que comprende administrar una cantidad efectiva de un compuesto de la invención a un sujeto, preferiblemente humano, en necesidad de dicho tratamiento. 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.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
La figura 1 muestra el efecto de los compuestos 1-7 en la producción de la citoquina pro-inflamatoria IL-8 cuando la línea celular A549 fue estimulada con 300 microgramos/mililitro de LPS de E. Coli y tratada con 100 microMolar de los compuestos 1-7. Se muestran los resultados de las células control (C), células A549 tratadas con LPS de E. Coli (LPS) y células A549 tratadas con LPS de E. Coli más 100 microMolar de los compuestos 1-7 (Compuestos 1-7). En esta figura se representa la simbología estadística: ***p<0.001 vs. C;† p<0.001 vs. LPS. La figura 2 muestra el efecto de los compuestos 1-7 en la producción de la citoquina pro-inflamatoria IL-1/? cuando la línea celular A549 fue considerada como control (C), tratada con 300 microgramos/mililitro de LPS de E. Coli (LPS) y tratada con 300 microgramos/militro de LPS de E. Coli más 100 microMolar de los compuestos 1-7 (Compuestos 1-7). En esta figura, **p<0.01 vs. C; 1| p<0.01 vs. LPS. Figure 1 shows the effect of compounds 1-7 on the production of the pro-inflammatory cytokine IL-8 when the A549 cell line was stimulated with 300 micrograms / milliliter of E. coli LPS and treated with 100 microMolar of the compounds 1-7. The results of the control cells (C), A549 cells treated with E. coli LPS (LPS) and A549 cells treated with E. coli LPS plus 100 microMolar of compounds 1-7 (Compounds 1-7) are shown. This figure represents the statistical symbology: *** p <0.001 vs. C; † p <0.001 vs. LPS Figure 2 shows the effect of compounds 1-7 on the production of the pro-inflammatory cytokine IL-1 /? when the A549 cell line was considered as control (C), treated with 300 micrograms / milliliter of LPS from E. Coli (LPS) and treated with 300 micrograms / milliliter of LPS from E. Coli plus 100 microMolar of compounds 1-7 (Compounds 1-7). In this figure, ** p <0.01 vs. C; 1 | p <0.01 vs. LPS
La figura 3 muestra la bandas correspondientes a los ensayos de Western Blot tras la realización de la extracción de las proteínas totales de células A549 control (C), células A549 bajo el tratamiento con 300 microgramos/mililitro de LPS de E. Coli (LPS) y bajo el tratamiento de 300 microgramos/mililitro de LPS de E.Coli más 100 microMolar de los compuestos 1-7 (Compuestos 1 -7). También se muestra la homogeneidad en la concentración proteica de cada pocilio mediante la Beta-actina. Figure 3 shows the bands corresponding to the Western Blot assays after the extraction of total proteins from control A549 cells (C), A549 cells under treatment with 300 micrograms / milliliter of LPS from E. Coli (LPS) and under the treatment of 300 micrograms / milliliter of E.Coli LPS plus 100 microMolar of compounds 1-7 (Compounds 1-7). Homogeneity in the protein concentration of each well by Beta-actin is also shown.
La figura 4 muestra los cambios de expresión de las proteínas TLR4 e \Κβα (normalizados en función del control de carga Beta-actina) cuando células A549 se emplearon como control (C), células A549 bajo el tratamiento con 300 microgramos/mililitro de LPS de E. Coli (LPS) y bajo el tratamiento de 300 microgramos/mililitro de LPS de E. Coli más 100 microMolar de los compuestos 1-7 (Compuestos 1-7). En esta figura, ** p<0.01 vs. C; ***p<0.001 vs. C;† p<0.001 vs. LPS; 1j p<0.05 vs. LPS: Figure 4 shows the expression changes of TLR4 and \βα proteins (normalized based on Beta-actin load control) when A549 cells were used as control (C), A549 cells under treatment with 300 micrograms / milliliter of LPS of E. Coli (LPS) and under the treatment of 300 micrograms / milliliter of LPS of E. Coli plus 100 microMolar of compounds 1-7 (Compounds 1-7). In this figure, ** p <0.01 vs. C; ** * p <0.001 vs. C; † p <0.001 vs. LPS; 1j p <0.05 vs. LPS:
La figura 5 muestra los valores PS (%) y la estructura química de diferentes compuestos urea. MODOS DE REALIZACIÓN PREFERENTE Figure 5 shows the PS values (%) and the chemical structure of different urea compounds. PREFERRED EMBODIMENTS
Ejemplo 1. Síntesis de los compuestos 1-7 Example 1. Synthesis of compounds 1-7
Se sintetizaron los compuestos -1 a 7, derivados de urea pirimidínica, desde los compuestos (pirimidin-4-iloxi) - y (pirimidin-3-il) acetohidrazidas, tal y como se describe en Jakubkiene, V. et al., Synthesis of (pyrimidin4-yloxy)-and (pyrimidin-3- yl)acetyl azides and their rearrangement to carbamates and ureas. Arkivoc 2010 (11 ) 39-48. Los compuestos puros fueron disueltos en dimetil sulfóxido.  Compounds -1 to 7, derived from pyrimidine urea, were synthesized from the compounds (pyrimidin-4-yloxy) - and (pyrimidin-3-yl) acetohydrazides, as described in Jakubkiene, V. et al., Synthesis of (pyrimidin4-yloxy) -and (pyrimidin-3- yl) acetyl azides and their rearrangement to carbamates and ureas. Arkivoc 2010 (11) 39-48. The pure compounds were dissolved in dimethyl sulfoxide.
Los compuestos 1 a 6 tienen la fórmula: Compounds 1 to 6 have the formula:
Figure imgf000009_0001
siendo R-ι, R2, y 3 los grupos que se especifican en la siguiente tabla:
Figure imgf000009_0001
R-ι, R 2 , and 3 being the groups specified in the following table:
Figure imgf000009_0003
Figure imgf000009_0003
El compuesto 7 tiene la fórmula: Compound 7 has the formula:
Figure imgf000009_0002
Ejemplo 2. Actividad anti-microbiana, anti-fúngica y citotóxica de los compuestos
Figure imgf000009_0002
Example 2. Anti-microbial, anti-fungal and cytotoxic activity of the compounds
Se determinó la actividad anti-microbiana en bacterias Gram-positivas Staphylococcus aureus (ATCC 29213) y Enterococcus faecalis (ATCC 29212) y bacterias Gram-negativas Escheríchia coli (ATCC 35218), Klebsiella pneumoniae (ATCC 700603) y Pseudomonas aeruginosa (ATCC 27853), la actividad anti-fúngica en las especies fúngicas Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), Candida nivariensis (5937-63) y Candida parapsilosis (ATCC 22019) y la actividad citotóxica en las líneas celulares HeLa (línea celular humana de cáncer de cérvix), Ishikawa (línea celular humana de cáncer de endometrio uterino), SW1573 (línea celular humana de cáncer epitelial pulmonar), T-47D (línea celular humana de cáncer de pecho), WiDr (Línea celular humana de cáncer de colon) y A549 (línea celular humana epiteliar pulmonar). Anti-microbial activity was determined in Gram-positive bacteria Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212) and Gram-negative bacteria Escheríchia coli (ATCC 35218), Klebsiella pneumoniae (ATCC 700603) and Pseudomonas aer53inosa (ATCC 700603) , 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) and the cytotoxic activity in HeLa cell lines (human cervical cancer cell line), Ishikawa (human uterine endometrial cancer cell line), SW1573 (human epithelial cancer cell line pulmonary), T-47D (human breast cancer cell line), WiDr (human colon cancer cell line) and A549 (human pulmonary epithelial cell line).
Las especies bacterianas y fúngicas fueron crecidas en placas de agar a 35-37°C. Después de 24 horas de incubación, las especies fueron resuspendidas en solución salina a una concentración aproximadamente de 5 x 105 cfu/ml (unidades formadoras de colonias por mililitro) para las especies bacterianas y 1-5 x 103 para las especies Candida. La actividad antimicrobiana de los compuestos fue determinada en placas de 96 pocilios usando el medio ueller Hinton broth para las especies bacterianas y el medio RP I-1640 tamponado con . solución MOPS para las especies fúngicas. Se incluyeron controles estériles y con crecimiento adecuado. Cada compuesto fue - testado por duplicado a ocho diferentes soluciones seriadas desde 0,05 a 100 microMolar. Las placas de 96 pocilios fueron incubadas a 35-37°C en una cámara oscura y húmeda. Después de la incubación, las especies fueron precipitadas en 25 microlitros de 50% (peso/volumen) TCA (Ácido tricloroacético) y fijadas durante 60 minutos a 4°C. Después, se llevó a cabo el ensayo colorimétrico descrito en Skehan, P. eí al., New colorimetric cytotoxicity assay for anticancer-drug screening. J Nati Cáncer Inst 1990 (82) 1107-1112. Después del ensayo, se produjo la agitación de las placas de 96 pocilios y mediante espectrofotometría (BioTek's PowerWave XS Absorbance Microplate Reader) se obtuvieron valores de densidad óptica. Las placas de 96 pocilios correspondientes a las especies bacterianas fueron leídas a 630 nanómetros después de 24 horas de incubación. Las placas de 96 pocilios correspondientes a las especies fúngicas fueron leídas a 490 nanómetros después de 48 horas de incubación. Con los resultados obtenidos de la espectrofotometría se obtuvo el valor MCI (mínima concentración inhibitoria) y fue establecido como la concentración del compuesto que inhibió el crecimiento total cuando se comparó con las especies no tratadas. Bacterial and fungal species were grown on agar plates at 35-37 ° C. After 24 hours of incubation, the species were resuspended in saline solution at a concentration of approximately 5 x 10 5 cfu / ml (colony forming units per milliliter) for bacterial species and 1-5 x 10 3 for Candida species. The antimicrobial activity of the compounds was determined in 96-well plates using ueller Hinton broth medium for bacterial species and RP I-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 microMolar. The 96-well plates were incubated at 35-37 ° C in a dark and humid chamber. After incubation, the species were precipitated in 25 microliters of 50% (weight / volume) TCA (trichloroacetic acid) and fixed for 60 minutes at 4 ° C. Then, the colorimetric assay described in Skehan, P. eí al., New colorimetric cytotoxicity assay for anticancer-drug screening was carried out. J Nati Cancer Inst 1990 (82) 1107-1112. After the test, agitation of the 96-well plates occurred and by spectrophotometry (BioTek ' s PowerWave XS Absorbance Microplate Reader) optical density values were obtained. The 96-well plates corresponding to the bacterial species were read at 630 nanometers after 24 hours of incubation. The 96-well plates corresponding to the fungal species were read at 490 nanometers after 48 hours of incubation. With the results obtained from the spectrophotometry, the MCI value (minimum inhibitory concentration) was obtained and was established as the concentration of the compound that inhibited total growth when compared to untreated species.
Los compuestos 1 a 7 no presentaban actividad anti-microbiana sobre las bacterias y especies fúngicas ensayadas, ya que todos los compuestos fueron inactivos (valores MCI superiores a 100 microMolar). Compounds 1 to 7 did not exhibit anti-microbial activity on the bacteria and fungal species tested, since all the compounds were inactive (MCI values greater than 100 microMolar).
De la misma manera, las líneas celulares se cultivaron en frascos de cultivo celular de 25 cm2 en medio de cultivo RPMI-1640 suplementado con el 5% de suero fetal bovino y 2 miliMolar de glutamina en un incubador con las siguientes condiciones experimentales: 37°C, 5% de C02 y 95% de aire húmedo. Células en crecimiento exponencial fueron tripsinizadas y resuspendidas en medio de cultivo que contiene antibióticos (100 unidades de penicilina y 0,1 miligramos de estreptomicina por mililitro). La suspensión celular con un porcentaje superior al 97% de viabilidad por la técnica de exclusión y de tinción Azul Tripán fue contada. Después del contaje, se realizaron las diluciones celulares para su inoculación en placa de 96 pocilios. Las células fueron sembradas a una concentración de 1 x 104 (para la línea celular SW1573), 1 ,5 x 104 (para las líneas celulares HeLa, Ishikawa y T-47D) y 2 x 104 (para la línea celular WiDr) células por pocilio. Cada compuesto fue testado por triplicado a diferentes diluciones en el rango de 1 -100 micro olar. El tratamiento de los compuestos en los cultivos celulares comenzó un día después de la siembra. El tiempo de incubación .de los compuestos fue de 48 horas, después del cual las células fueron precipitadas en 25 microlitros de 50% (peso/volumen) TCA (Ácido tricloroacético) y fijadas durante 60 minutos a 4°C. Después, se llevó a cabo el ensayo colorimétrico descrito en Skehan, P. et al. La densidad óptica de cada pocilio se leyó a 492 nanómetros empleando el espectrofotómetro BioTek's PowerWave XS Absorbance Microplate Reader. El porcentaje de crecimiento (PG) fue calculado con respecto a las células control no tratadas (C) en cada una de las concentraciones de los compuestos basándose en la diferencia de la densidad óptica al comienzo (T0) y al final de la exposición de los compuestos (T), según las fórmulas descritas en A. Monks et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J. Nati. Cáncer Inst. 1991 (83) 757-766. Se empleó la fórmula PG= 100 X [(T-T0)/(C-T0)] cuando T es mayor o igual a T0> y la fórmula PG= 100 X [(T-T0)/(T0)j cuando T es menor que T0. La concentración a la cual PG es +50, 0 y -50 representa, respectivamente, un 50% de inhibición del crecimiento (Gl50), inhibición total del crecimiento (TGI) y 50% de muerte celular (LC50). Por tanto, un valor PG de 0 corresponde a la cantidad de células presente en el comienzo de la exposición al compuesto y valores negativos de PG denotan muerte celular neta. Los compuestos empleados en la presente invención no poseían actividad citotóxica sobre las líneas celulares ensayadas ya que todos los compuestos fueron inactivos (valores de inhibición del crecimiento Gl50 superiores a 100 microMolar). In the same way, cell lines were grown in 25 cm 2 cell culture bottles in RPMI-1640 culture medium supplemented with 5% fetal bovine serum and 2 milliMolar glutamine in an incubator with the following conditions Experimental: 37 ° C, 5% C0 2 and 95% humid air. Exponentially growing cells were trypsinized and resuspended in culture medium containing antibiotics (100 units of penicillin and 0.1 milligrams of streptomycin per milliliter). The cell suspension with a percentage greater than 97% viability by the Trypan Blue staining and exclusion technique was counted. After counting, cell dilutions were made for 96-well plate inoculation. The cells were seeded at a concentration of 1 x 10 4 (for the SW1573 cell line), 1.5 x 10 4 (for the HeLa, Ishikawa and T-47D cell lines) and 2 x 10 4 (for the WiDr cell line ) cells per well. Each compound was tested in triplicate at different dilutions in the range of 1 -100 micro olar. 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% (weight / volume) TCA (trichloroacetic acid) and fixed for 60 minutes at 4 ° C. Then, the colorimetric assay described in Skehan, P. et al. The optical density of each well was read at 492 nanometers using the BioTek ' s PowerWave XS Absorbance Microplate Reader spectrophotometer. The percentage growth (PG) was calculated with respect to the untreated control cells (C) in each of the concentrations of the compounds based on the difference in optical density at the beginning (T 0 ) and at the end of the exposure of the compounds (T), 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. Nati. Cancer Inst. 1991 (83) 757-766. The formula PG = 100 X [(TT 0 ) / (CT 0 )] was used when T is greater than or equal to T 0> and the formula PG = 100 X [(TT 0 ) / (T 0 ) j when T is less than T 0 . The concentration at which PG is +50, 0 and -50 represents, respectively, 50% growth inhibition (Gl 50 ), total growth inhibition (TGI) and 50% cell death (LC 50 ). 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. The compounds used in the present invention did not possess cytotoxic activity on the cell lines tested since all the compounds were inactive (Gl 50 growth inhibition values greater than 100 microMolar).
Ejemplo 3. Determinación de los niveles de expresión de IL-8, IL- β, TLR4 e ΙΚβα . Se cultivó la línea celular A549 (línea celular humana epiteliar pulmonar) en medio de cultivo enriquecido con el 2% de suero fetal bovino durante 24 horas. Posteriormente, se trató el cultivo celular con 300 microgramos/mililitro de LPS de E. Coli (serotipo 055: B5 distribuible comercialmente por la compañía Sigma-Aldrich ®) junto con 100 microMolar de los productos de síntesis descritos en la presente invención durante 18 horas. Pasado el tiempo de tratamiento, se recogió el medio de cultivo en tubos dé ensayo Falcon 15 mililitros estériles y se centrifugaron a 1.200 RPM (revoluciones por minuto) durante 10 minutos a 4°C. Después, se recogieron los sobrenadantes de los tubos Falcon 15 mililitros y se añadieron en forma de alícuotas de 500 microlitros en tubos eppendorf 1 ,5 mililitros estériles. Dichos tubos eppendorf se conservaron en congelación (-80°C) hasta su uso. Example 3. Determination of the expression levels of IL-8, IL-β, TLR4 and ΙΚβα. The A549 cell line (human pulmonary epithelial 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 commercially distributable by the Sigma-Aldrich ® company) together with 100 microMolar of the synthesis products described in the present invention for 18 hours . After the treatment time, the culture medium was collected in sterile Falcon 15 milliliter test tubes and centrifuged at 1,200 RPM (revolutions per minute) 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 1.5-5 milliliter eppendorf tubes. Said eppendorf tubes were kept frozen (-80 ° C) until use.
Posteriormente, se añadieron 200 microlitos de este medio de cultivo en una placa de ensayo de 96 pocilios y se añadieron los anticuerpos y reactivos del kit comercial de BD Biosciences "Cytometry-based bead array system". El último paso consistió en medir los niveles de expresión de IL-8 e IL-1 ? mediante ensayo ELISA a un rango de longitud de 633 nanómetros. 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 was to measure the expression levels of IL-8 and IL-1? by ELISA test at a length range of 633 nanometers.
Los niveles de expresión de la proteína de receptor de membrana denominada TLR4 y la proteína inhibidora del factor de transcripción denominado Factor nuclear kappa B (NF-κΒ) se midieron mediante Western Blot. Para ello, se realizó electroforesis de cada una de las muestras en geles de SDS-PAGE (10%) durante 2 horas. Después de ello, se realizó la transferencia de los geles SDS-PAGE en membranas de difluoruro de polivinilideno (PVDF) y bloqueadas durante 1 hora a temperatura ambiente en el tampón TBST (del inglés Trís-buffered saline Tween-20) más 10% de leche carente de contenido graso. Los anticuerpos anti-TLR4 (Santa Cruz Biotechnology Inc.) y anti-proteína inhibidora NF-κΒ (Santa Cruz Biotechnology Inc.) se incubaron durante 2 horas en TBST más 5% de leche carente de contenido graso. Después de la incubación durante 2 horas con el anticuerpo primario, se realizó la incubación durante 1 hora con el anticuerpo secundario conjugado con peroxidasa (Goaf Anti-rabbit IgG-HRP; Santa Cruz Biotechnology Inc.). La detección de las bandas se llevó a cabo por sistema quimioluminiscente con el kit de GE Healthcare "Amersham ECL Western Blotting Detection Reagenf. Posteriormente, se densitometró cada banda proteica mediante el software público Scion Image. Cada Western Blot se realizó tres veces con las mismas condiciones experimentales nombradas. Expression levels of the membrane receptor protein called TLR4 and the transcription factor inhibitor protein called Kappa B nuclear factor (NF-κΒ) were measured by Western Blot. For this, electrophoresis of each of the samples was performed in SDS-PAGE gels (10%) 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 the TBST buffer (Trís-buffered saline Tween-20) plus 10% of Milk lacking in fat content. The anti-TLR4 (Santa Cruz Biotechnology Inc.) and anti-NF-κΒ inhibitor (Santa Cruz Biotechnology Inc.) antibodies were incubated for 2 hours in TBST plus 5% milk lacking in fat. After incubation for 2 hours with the primary antibody, incubation was performed for 1 hour with the peroxidase-conjugated secondary antibody (Goaf Anti-rabbit IgG-HRP; Santa Cruz Biotechnology Inc.). The detection of the bands was carried out by chemiluminescent system with the GE Healthcare kit "Amersham ECL Western Blotting Detection Reagenf. Subsequently, each protein band was densitometered using the public software Scion Image. Western Blot was performed three times with the same experimental conditions named.
Para comprobar la homogeneidad de la carga proteica en cada uno de los pocilios, las mismas membranas PVDF se re-incubaron con el anticuerpo primario Beta-actina (de la compañía Cell Signallng Technology) durante 2 horas y con el mismo anticuerpo secundario y las mismas condiciones experimentales de los anticuerpos anti-TLR4 y anti-proteína inhibidora del factor de transcripción denominado Factor nuclear kappa B. 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 (from the Cell Signallng Technology company) for 2 hours and with the same secondary antibody and the same experimental conditions of anti-TLR4 and anti-transcription factor inhibitor protein called kappa B nuclear factor.
Todos los datos relacionados con la actividad anti-inflamatoria de los compuestos objeto de la presente invención son expresados como media de los resultados obtenidos para cada compuesto ± error estándar y los análisis estadísticos fueron llevados a cabo empleando los Test de Fisher y la T de Student para datos emparejados y datos no emparejados y empleando el software SPSS (versión 15.0 para Windows). Para los análisis estadísticos de los ensayos Western Blot se empleó la misma metodología pero los datos fueron normalizados en función del control de carga proteica Beta-actina. Los efectos se consideraron significantes cuando p<0.05. All data related to the anti-inflammatory activity of the compounds object of the present invention are expressed as the average of the results obtained for each compound ± standard error and the statistical analyzes were carried out using the Fisher Test and Student's T for paired data and unpaired data 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.
Basados en los ensayos de medición de los niveles de citoquinas pro-inflamatorias en el sobrenadante de la línea celular A549, la figura 1 muestra que los compuestos 1-7 fueron capaces de reducir los niveles de la citoquina pro-inflamatoria IL-8 inducidos bajo el tratamiento de LPS de E Coli (p<0.001 ) de una manera significativa (pO.001). La figura 2 muestra que los compuestos 1-7 objeto de la presente invención fueron capaces de prevenir completamente los efectos producidos por el tratamiento de LPS de E. Coli en los niveles de la citoquina pro- inflamatoria IL-1/9 (p<0.01). Basados en los ensayos de Western Blot, las figuras 3 y 4 muestran que la expresión de la proteína TLR4 se incrementó de manera significativa bajo el tratamiento con LPS de E. Coli (p<0.001 ). Incremento que fue reducido bajo el co-tratamiento de los compuestos 1-7 (p<0.001 ) y también muestran que la expresión de la proteína inhibidora dei factor de transcripción denominado Factor nuclear kappa B se redujo bajo el tratamiento con LPS de E. Coli de manera significativa (p<0.01 ) y que los compuestos 1-7 objeto de la presente invención produjeron un aumento en los niveles de expresión de la proteína \Κβο cuando se comparó este resultado con el tratamiento de LPS de E. Coli (p<0.05). Ejemplo 4. Los compuestos 1 a 7 disminuyen los efectos de LPS producidos en la línea celular A549 Based on the assays measuring the levels of pro-inflammatory cytokines in the supernatant of the A549 cell line, Figure 1 shows that compounds 1-7 were able to reduce the levels of IL-8 pro-inflammatory cytokine induced under LPS treatment of E Coli (p <0.001) in a significant way (pO.001). Figure 2 shows that the compounds 1-7 object 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-1/9 (p <0.01 ). Based on the Western Blot assays, Figures 3 and 4 show that TLR4 protein expression was significantly increased under treatment with E. coli LPS (p <0.001). Increase that was reduced under the co-treatment of compounds 1-7 (p <0.001) and also show that the expression of the transcription factor inhibitor protein called Kappa B nuclear factor was reduced under the treatment with E. coli LPS. significantly (p <0.01) and that the Compounds 1-7 object of the present invention produced an increase in the levels of \βο protein expression when this result was compared with the LPS treatment of E. Coli (p <0.05). Example 4. Compounds 1 to 7 decrease the effects of LPS produced on the A549 cell line
Se estimuló la línea celular A549 con 300 microgramds/mililitro de LPS de E. Coli en combinación con cada uno de los compuestos a una dosis simple ( 00 microMolar). El tiempo de incubación de los compuestos fue de 18 horas, después del cual las células fueron precipitadas en 25 microlitros de 50% (peso/volumen) TCA (Ácido tricloroacético) y fijadas durante 60 minutos a 4°C. Después, se llevó a cabo el ensayo colorimétrico descrito en Skehan, P. et al., New colorimetric cytotoxicity assay for anticancer-drug screening. J Nati Cáncer Inst 1990 (82) 1107-1112. La densidad óptica de cada pocilio se leyó a 492 nanómetros empleando el espectrofotómetro BioTek's PowerWave XS Absorbance Microplate Reader y se determinó el porcentaje de supervivencia (PS) con respecto a las células no tratadas (control negativo) y las células tratadas con 300 microgramos/mililitro de LPS de E. Cóli (control positivo, TLPS) de cada concentración de compuesto (T). Por tanto, el cálculo se desarrolló de la siguiente manera: PS= 100 x [(T- TLPS)/(C- ΤίΡ5)]. Con este cálculo, un valor de PS de 0 corresponde a células tratadas solamente con LPS de E. Coli presente al final de la exposición, mientras valores positivos de PS denotan muerte celular neta. En la figura 5 se detallan valores PS correspondientes a diversos derivados de ureas. De los resultados obtenidos, como se muestra en esta figura, se podría concluir que todos los compuestos ensayados poseen la habilidad para disminuir los efectos de LPS producidos en la línea celular A549. Los mejores resultados se alcanzaron con el compuesto 5 donde se alcanzó un valor de PS de 58%. The A549 cell line was stimulated with 300 microgramds / milliliter of E. coli LPS in combination with each of the compounds at a single dose (00 microMolar). The incubation time of the compounds was 18 hours, after which the cells were precipitated in 25 microliters of 50% (weight / volume) 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 Nati Cancer Inst 1990 (82) 1107-1112. 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) was determined with respect to untreated cells (negative control) and cells treated with 300 micrograms / milliliter of LPS of E. Cóli (positive control, T LPS ) of each concentration of compound (T). Therefore, the calculation was developed as follows: PS = 100 x [(T-T LPS ) / (C- Τ ίΡ5 )]. 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 death. Figure 5 shows PS values corresponding to various urea derivatives. From the results obtained, as shown in this figure, it could be concluded that all the compounds tested possess the ability to decrease the effects of LPS produced in the A549 cell line. The best results were achieved with compound 5 where a PS value of 58% was reached.

Claims

REIVINDICACIONES
1. Compuesto derivado de urea pirimidínica de fórmula (I):  1. Compound derived from pyrimidine urea of formula (I):
Figure imgf000015_0001
donde Ra está seleccionado entre el grupo formado por H, carboxilo, CF3, F, Cl, Br, I, éter de alquilo C1-C10 sustituido o no sustituido, éter de alquenilo C2-C10 sustituido o no sustituido, éter de alquinilo C2-C 0 sustituido o no sustituido, alquilo C C10 sustituido o no sustituido, alquenilo C2-Ci0 sustituido o no sustituido, alquinilo C2-C10 sustituido o no sustituido, cicloalquilo C3-C10 sustituido o no sustituido, cicloalquenilo C5-C 0 sustituido o no sustituido, cicloalquinilo C8-C-|0 sustituido o no sustituido, arilo C6-C-|0 sustituido o no sustituido, aralquilo C7-C10 sustituido o no sustituido, heterociclilo de 3-10 miembros de anillo sustituido o no sustituido y heteroarilalquilo sustituido o no sustituido de 2 a 10 átomos de carbono, y ambos heterociclos de 1 a 3 átomos diferentes al carbono,
Figure imgf000015_0001
where R a is selected from the group consisting of H, carboxyl, CF 3 , F, Cl, Br, I, substituted or unsubstituted C1-C10 alkyl ether, substituted or unsubstituted C2-C10 alkenyl ether, alkynyl ether C 2 -C 0 substituted or unsubstituted, CC 10 substituted or unsubstituted alkyl, alkenyl C 2 Ci 0 substituted or unsubstituted C2-C10 substituted or unsubstituted, C3-C10 substituted or unsubstituted cycloalkenyl C 5 -C 0 substituted or unsubstituted, C 8 -C- cycloalkynyl | 0 substituted or unsubstituted, aryl C 6 -C- | 0 substituted or unsubstituted, C 7 -C 10 substituted or unsubstituted aralkyl, 3-10 substituted or unsubstituted heterocyclyl ring and substituted or unsubstituted heteroarylalkyl of 2 to 10 carbon atoms, and both heterocycles of 1 to 3 atoms other than carbon,
para el tratamiento de enfermedades inflamatorias.  for the treatment of inflammatory diseases.
2. Un compuesto según reivindicación 1 , donde R es benceno.  2. A compound according to claim 1, wherein R is benzene.
3. Un compuesto según reivindicación 1 , donde R es  3. A compound according to claim 1, wherein R is
Figure imgf000015_0002
Figure imgf000015_0002
4. Un compuesto según reivindicación 1 , donde R es
Figure imgf000016_0001
4. A compound according to claim 1, wherein R is
Figure imgf000016_0001
Un compuesto según reivindica R  A compound as claimed in R
6. Un compuesto según reivindi es 6. A compound according to claim is
Figure imgf000016_0002
Figure imgf000016_0002
7. Un compuesto según reivindicación 1 donde R es  7. A compound according to claim 1 wherein R is
Figure imgf000016_0003
Figure imgf000016_0003
8. Un compuesto según reivindicación 1 , donde R es  8. A compound according to claim 1, wherein R is
Figure imgf000016_0004
Figure imgf000016_0004
9. Composición farmacéutica que comprende al menos un compuesto según cualquiera de las reivindicaciones 1 a 8, junto con excipientes farmacéuticamente aceptables, para el tratamiento de enfermedades inflamatorias.  9. Pharmaceutical composition comprising at least one compound according to any of claims 1 to 8, together with pharmaceutically acceptable excipients, for the treatment of inflammatory diseases.
10. Una composición farmacéutica según la reivindicación 9, en una unidad de dosificación.  10. A pharmaceutical composition according to claim 9, in a dosage unit.
11. Una composición farmacéutica según la reivindicación 10, donde dicha unidad de dosificación es un comprimido.  11. A pharmaceutical composition according to claim 10, wherein said dosage unit is a tablet.
12. Una composición farmacéutica según una de las reivindicaciones 10 ó 1 1 , que se administra por vía oral.  12. A pharmaceutical composition according to one of claims 10 or 1, which is administered orally.
13. Una composición farmacéutica según la reivindicación 9, que se administra por vía intravenosa, muscular y/o intramuscular. 13. A pharmaceutical composition according to claim 9, which is administered intravenously, muscularly and / or intramuscularly.
14. Una composición farmacéutica que comprende al menos un compuesto según cualquiera de las reivindicaciones 1 a 8, junto con excipientes farmacéuticamente aceptables y al menos otro agente terapéutico para el tratamiento de enfermedades inflamatorias. 14. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 8, together with pharmaceutically acceptable excipients and at least one other therapeutic agent for the treatment of inflammatory diseases.
15. Una composición farmacéutica según cualquiera de las reivindicaciones 9 a 14, donde dicha enfermedad inflamatoria está causada por bacterias.  15. A pharmaceutical composition according to any of claims 9 to 14, wherein said inflammatory disease is caused by bacteria.
16. Una composición farmacéutica según reivindicación 15, donde dichas bacterias son bacterias Gram-negativas.  16. A pharmaceutical composition according to claim 15, wherein said bacteria are Gram-negative bacteria.
17. Una composición farmacéutica según una de las reivindicaciones 15 ó 16, donde dicha enfermedad inflamatoria es una sepsis.  17. A pharmaceutical composition according to one of claims 15 or 16, wherein said inflammatory disease is a sepsis.
18. Una composición farmacéutica según cualquiera de las reivindicaciones 9 a 14, donde dicha enfermedad inflamatoria está seleccionada entre el grupo compuesto por la artritis, infecciones renales, enfermedades pulmonares agudas inducidas por sepsis, síndrome de distrés respiratorio agudo inducido por sepsis, shock endotóxico o endotoxemia, peritonitis séptica, hepatitis aguda, disfunción miocárdica aguda inducida por shock séptico y enterecolitis necrotizante.  18. A pharmaceutical composition according to any of claims 9 to 14, wherein said inflammatory disease is selected from the group consisting of arthritis, renal infections, acute pulmonary diseases induced by sepsis, acute respiratory distress syndrome induced by sepsis, endotoxic shock or Endotoxemia, septic peritonitis, acute hepatitis, acute myocardial dysfunction induced by septic shock and necrotizing enterecolitis.
PCT/ES2011/070822 2010-12-28 2011-11-25 Pyrimidine urea‑derived compound for treating inflammatory diseases WO2012089876A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1466906A1 (en) * 1999-03-12 2004-10-13 Boehringer Ingelheim Pharmaceuticals Inc. Heterocyclic urea and related compounds useful as anti-inflammatory agents

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1466906A1 (en) * 1999-03-12 2004-10-13 Boehringer Ingelheim Pharmaceuticals Inc. Heterocyclic urea and related compounds useful as anti-inflammatory agents

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Title
BRUGEL, T.A. ET AL.: "Development of N-2,4-pyrimidine-N-alkyl -N'-phenylureas as inhibitors of tumor necrosis factor alpha (TNF-a) synthesis. Part I.", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 16, 2006, pages 3510 - 3513 *
JAKUBNIENE, V. ET AL.: "Synthesis of (pyrimidin-4-yloxy) and (pyrimidin-3-yl)acetyl azides and their rearrangement to carbamates and ureas", ARKIVOC, vol. 2010, pages 39 - 48 *
LATTHE, P.R. ET AL.: "Curtius rearrangement reactions of 3-(4-azidocarbonyl) phenylsydnone. Synthesis of 4-(sydnon-3-yl) phenyl carbamates, N-aryl-N'-[4-(sydnon-3-yl)] phenyl ureas and their antimicrobial and insecticidal activities", JOURNAL OF CHEMICAL SCIENCES, vol. 118, no. 3, 2006, pages 249 - 256 *
MAIER, J.A. ET AL.: "Development of N-4,6-pyrimidine-N-alkyl- N'-phenylureas as orally active inhibitors of lymphocyte specific tyrosine kinase.", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 16, 2006, pages 3646 - 3650 *

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