WO2014138833A1 - Compounds, use, pharmaceutical composition for inhibiting reductase in microorganisms, inha binder and method for obtaining inha binders - Google Patents

Compounds, use, pharmaceutical composition for inhibiting reductase in microorganisms, inha binder and method for obtaining inha binders Download PDF

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WO2014138833A1
WO2014138833A1 PCT/BR2014/000076 BR2014000076W WO2014138833A1 WO 2014138833 A1 WO2014138833 A1 WO 2014138833A1 BR 2014000076 W BR2014000076 W BR 2014000076W WO 2014138833 A1 WO2014138833 A1 WO 2014138833A1
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inha
formula
carbons
alkyl chain
chain containing
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PCT/BR2014/000076
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French (fr)
Portuguese (pt)
Inventor
Ivani PAULI
Osmar Norberto de SOUZA
Rafael Victório Carvalho GUIDO
Adriano Defini ANDRICOPULO
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União Brasileira De Educação E Assistência , Mantenedora Da Pucrs
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
    • C07D285/12—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/18—Halogen atoms or nitro radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/08—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D277/10—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D411/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms
    • C07D411/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D411/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention is in the field of biology, chemistry and pharmacy. More specifically, the present invention features novel enzyme inhibiting compounds in microorganisms. In one embodiment, the compounds of the present invention are useful for inhibiting the enzyme 2-transenoyl-ACP (CoA) reductase in microorganisms.
  • CoA 2-transenoyl-ACP
  • Tuberculosis is the fourth cause of death in the country from infectious diseases and the first cause of death in patients with AIDS (National Program for Tuberculosis Control, Secretary of Health Surveillance, Ministry of Health).
  • the present invention solves the problem of finding new drugs for the treatment of tuberculosis (TB) using the enzyme InhA, targeting the main drug employed in the treatment of this disease (isoniazid) (INH), as a therapeutic target.
  • InhA the enzyme employed in the treatment of this disease
  • IH isoniazid
  • the compounds of the present invention also have the potential to inhibit the InhA enzyme of these organisms.
  • Mycobacterium causes tuberculosis, leprosy, ulcers, and other infections
  • Plasmodium causes malaria, in particular cerebral malaria
  • Toxoplasma causes toxoplasmosis
  • Trypanosoma Causes Chagas disease
  • Klebsiella causes nosocomial infections, pneumonia
  • Chlamydia causes trachoma or chlamydia and chronic infections
  • Helycobacter. causes gastritis, ulcer and stomach cancer
  • Pseudomonas Causes Infections cholera and other
  • Yersinia Causes Black Death; Staphylococcus: causes nosocomial infection; Salmonella: Causes salmonellosis; Neisseria: causes venereal disease (gonorrhoeae) and meningitis (meningitidis).
  • US 2011/0092536 describes novel anti-infectious derivatives, method of production thereof, pharmaceutical compositions containing the compounds and their use in antimicrobial treatment
  • the derivatives comprise a general formula containing the combination of i) pyridine, pyridinium or dihydropyridine structures and structures related derivatives of isoniazid active metabolites; and ii) hydrophobic substituent targeting the active substrate site.
  • fatty acid inhibitors as potential antimicrobial agents in which an essential step of action of possible antimicrobial agents comprises being administered into a microbial cell to allow inhibition of an enzyme (synthase) related to the production of fatty acids.
  • the present invention provides novel microorganism reductase inhibiting compounds useful for inhibiting the enzyme InhA (2-fransenoyl-ACP (CoA) reductase) such as Mycobacterium tuberculosis InhA (EC: 1.3.1.9).
  • InhA 2-fransenoyl-ACP (CoA) reductase
  • Mycobacterium tuberculosis InhA EC: 1.3.1.9
  • any alkyl chain is a straight or branched chain, saturated or unsaturated, cyclic or not.
  • microorganism reductase inhibitor compound selected from the group comprising:
  • R1 and R4 being the same or distinct, comprise an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ;
  • R2 comprises an alkyl chain containing from 1 to 10 carbons or an oxygen atom
  • R3 and R5 comprise an alkyl chain containing from 1 to 10 carbons or a hydrogen atom
  • R1-R2 being the same or distinct, comprising an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ;
  • R3 comprising an alkyl chain containing from 1 to 10 carbons or an oxygen atom
  • R1 comprises an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ; and R2 comprises an alkyl chain containing from 1 to 10 carbons or an oxygen atom;
  • R1 and R3 may, being the same or distinct, comprise an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ;
  • R2 comprises an alkyl chain containing from 1 to 10 carbons or a hydrogen atom
  • a further object of the present invention is also a selected inhA binder a compound selected from the group comprising formula I, II, III and / or IV above, or combinations thereof.
  • a further object of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • Figure 1 Analysis of intermolecular interactions between the InhA enzyme of Mycobacterium tuberculosis and N- (2-phenoxyphenyl) -2- [2- (2-pyridyl) thiazol-4-yl] acetamide.
  • Figure 3 Analysis of intermolecular interactions between Mycobacterium tuberculosis enzyme InhA and 4- (7-chloro-4-quinolyl) -2- (diethylaminomethyl) phenol.
  • the present invention has as a common inventive concept the process or method for obtaining inhA binders. From said common inventive concept, different molecular entities are obtained and solve the technical problem of providing new inhA ligands.
  • the process or method for obtaining inhA binders of the present invention comprises at least three steps:
  • the process or method for obtaining inhA binders of the present invention comprises:
  • an inhA binder is a compound selected from the above-selected group comprising formula I, II, III and / or IV, or combinations thereof, as well as their salts, solvates and / or hydrates.
  • Said binder is particularly useful for in vitro and / or in vivo detection of inhA, notably when said compounds are endowed with some radioactive marker and / or colorimetric reaction.
  • Those skilled in the art will readily be able to identify said markers together with the binders of the invention, thereby providing detection and / or quantification of inhA in biological samples.
  • pharmaceutically acceptable carrier any carrier that provides insertion into a formulation containing acceptable excipients and carriers known to those skilled in the art, as well as at doses and treatments suitable for use in particular compositions.
  • Said carrier may be described in a number of treatment regimens including oral, parenteral, intravenous, intranasal, intravitreal and intramuscular, intracerebral, intracerebroventricular and intraocular or other means of administration and / or formulation.
  • the present invention provides the use of a compound selected from the group comprising formula I, II, III and / or IV described above, or combinations thereof, for the manufacture of antimicrobially acting medicament.
  • R 1 and R 5 are hydrogen atoms
  • R 2 is CH 2; R3 are ethyl, R4 is a chlorine atom.
  • R 1 is a hydrogen atom; R 3 is -CH 2 and R 2 is a methyl in the ortho position.
  • R 1 is a hydrogen atom and R 2 is a -CH 2 .
  • R 1 is a hydrogen atom
  • R 2 is a propyl
  • R 3 is a methyl in the ortho position.
  • InhA or Mycobacterium tuberculosis 2-fransenoyl-ACP (CoA) reductase (EC: 1.3.1.9) is an attractive enzyme for drug discovery efforts due to its validation as an effective biological target for tuberculosis therapy.
  • two distinct in silico ligand screening approaches were used to identify novel InhA inhibitors from a selected ligand library from the ZINC database.
  • a pre-selection of compounds to be selected was performed using the Database - INGr Molecular Filters - 2D Identify molecules with favorable characteristics to interact with the InhA binding cavity: 4 ⁇ LogP ⁇ 7; rotary connections ⁇ 6; hydrogen bridge acceptors ⁇ 8; polar surface area ⁇ 40 ⁇ 2 ; and molecular weight between 250 Da and 400 Da.
  • the final set of compounds comprising the cited characteristics comprised 999,853 compounds and were used in both approaches described below.
  • Hydrogen bridges and hydrophobic contacts were also analyzed by LIGPLOT to obtain a more robust interaction pattern profile between MtlnhA and its crystallographic ligands.
  • Structural analysis of MtlnhA-complexed-ligands-bound to the substrate binding cavity indicates several key amino acid residues that may be involved in the molecular recognition process. Based on this, a four-point 3-D pharmacophoric model was developed.
  • the 3-D structure molecule library was transferred to a UNITY database in SYBYL 8.0.
  • the 3-D search with UNITY was based on the generated pharmacophoric model.
  • Donor and receptor atoms were defined by connecting the donor and receptor atoms of the ligand via partial correspondence.
  • the hydrophobic characteristic was partially characterized based on the two rings present in the ligand structure. The bead diameters were adjusted so that a test sample could be obtained, composed of MtlnhA crystallographic ligands.
  • a flexible UNITY 3-D search was performed.
  • the UNITY algorithm scores high for molecules that match the pharmacophoric points in their flexible searches, and because of this, the optimized geometry of the compounds under analysis may be affected. Thus, the molecular structures of the selected set of compounds were minimized to avoid false positive results. THE Energy minimization of the molecules was performed using the same protocol described in the Computational Approach section.
  • the GOLD software suite was used to perform molecular docking simulations.
  • the MtInhA crystal structure used as a receptor (PDB ID, 1 P44) was chosen from among the 36 available structures because of its better relationship between the binding cavity volume (3.007.8 A 3 ) and protein resolution (2.7 ⁇ ).
  • hydrogen atoms were added to the protein with standard geometry using the Biopolymer module implemented in SYBYL 8.0.
  • the active site was defined as incorporating all amino acid residues with a 8.0 A sphere radius centered on the bound crystallographic ligand (Genz-10850).
  • Coenzyme NADH was treated as part of the protein in all docking simulations.
  • GOLD Genetic Optimization for Ligand Docking
  • the program gives some options related to the scoring function and among them we have chosen GoldScore to be used in our simulations.
  • FlexX was implemented as Incremental Construction Algorithm, where the base fragment (the center of the binder) is automatically selected and placed within the active site using an algorithmic approach based on a pattern recognition technique called pose clustering. After a good set of locations has been obtained as a base, the remaining portions of the binder are divided into small fragments and incrementally "grow" within alternative bases.
  • the Surflex-Dock algorithm applies the Hammerhead empirical scoring function and uses an idealized active site ligand, or "protomol", to generate ligands by incremental construction, and also uses an information crossing procedure that combines pieces with distinct configurations. Protomols were computed using the cognate ligand position to define the binding site.
  • the protein receptor chosen from the MtlnhA simulations was that with the PDB code 1 P44 and coenzyme NADH was treated as part of the receptor. In all docking simulations the receiver structure was kept rigid while the ligands were treated with complete flexibility.
  • the set of molecules was the same as that used for the pharmacophore-based approach.
  • OSIRIS drug discovery computer system
  • OS ⁇ RIS Property Explorer available at http://www.organic-chemistry.org/prog/peo/
  • OS ⁇ RIS Property Explorer is an integral part of Actelion's internal substance registration system. It calculates drug-relevant properties simultaneously while the user draws the molecule.
  • the molecules selected by both The approaches used in the present invention were subjected to OSIRIS and all those with unfavorable characteristics were removed from the final set of molecules for in vitro testing.
  • Equation 1 is adequate for the behavior of non-competitive inhibitors, with K values of 24 (+ 3) ⁇ and 20 ( ⁇ 2) ⁇ for NADH and 2-fraA7S-dodecenoil-CoA, respectively.
  • This inhibition profile indicates that the inhibitor binds exclusively to the enzyme-substrate (ES) complex, leading to inactivation of the enzyme-substrate-inhibitor (ESI) complex, and the affinity of the inhibitor is maximum at substrate saturating concentrations.

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Abstract

The present invention describes new compounds for binding and/or inhibiting enzymes in microorganisms. In one embodiment, the compounds of the present invention are used to inhibit the 2-trans-enoyl-ACP (CoA) reductase enzyme in microorganisms, and are therefore useful as antimicrobial agents.

Description

Relatório Descritivo de Patente de Invenção  Patent Invention Descriptive Report
COMPOSTOS, USO, COMPOSIÇÃO FARMACÊUTICA INIBIDORA DE REDUTASE EM MICRORGANISMOS, LIGANTE DE INHA, E, MÉTODO PARA  COMPOSITIONS, USE, PHARMACEUTICAL COMPOSITION INHIBITOR OF MICROORGANISMS REDUCER, INHA BINDER, AND METHOD FOR
OBTENÇÃO DE LIGANTES DE INHA  OBTAINING YELLOW LINKS
Campo da Invenção Field of the Invention
A presente invenção se situa no campo da biologia, da química e da farmácia. Mais especificamente, a presente invenção apresenta novos compostos inibidores enzimáticos em microrganismos. Em uma concretização, os compostos da presente invenção são úteis para inibir a enzima 2-trans- enoil-ACP (CoA) redutase em microrganismos.  The present invention is in the field of biology, chemistry and pharmacy. More specifically, the present invention features novel enzyme inhibiting compounds in microorganisms. In one embodiment, the compounds of the present invention are useful for inhibiting the enzyme 2-transenoyl-ACP (CoA) reductase in microorganisms.
Antecedentes da Invenção Background of the Invention
Mais de dois bilhões de pessoas estão infectadas com o agente causador da tuberculose, o Mycobacterium tuberculosis. Em 2010 houve 8,8 milhões (faixa: 8,5-9,2 milhões) de casos de TB no mundo. Desses, 1 ,1 milhão (faixa: 0,9-1 ,2 milhão) morreram de TB apenas e 0,35 milhão (faixa: 0,32-0,39 milhão) morreram de TB associada a HIV/AIDS. Surgem a cada ano 500 mil novos casos de TB multirresistente a fármacos (MDR). São 10 milhões de crianças órfãs devido à morte dos pais por TB (WHO, Global Report, 2011). No Brasil, 71 mil casos de TB foram notificados em 2010 com 4,6 mil mortes por ano. Tuberculose é a 4a causa de mortes no país por doenças infecciosas e a 1a causa de morte de pacientes com AIDS (Programa Nacional de Controle da Tuberculose, Secretária de Vigilância da Saúde, Ministério da Saúde). More than two billion people are infected with the causative agent of tuberculosis, Mycobacterium tuberculosis. In 2010 there were 8.8 million (range: 8.5-9.2 million) TB cases worldwide. Of these, 1.1 million (range: 0.9-1, 2 million) died from TB alone and 0.35 million (range: 0.32-0.39 million) died from HIV / AIDS-associated TB. 500,000 new cases of multidrug-resistant TB (MDR) appear each year. There are 10 million orphaned children due to their parents' death from TB (WHO, Global Report, 2011). In Brazil, 71,000 cases of TB were reported in 2010 with 4,600 deaths per year. Tuberculosis is the fourth cause of death in the country from infectious diseases and the first cause of death in patients with AIDS (National Program for Tuberculosis Control, Secretary of Health Surveillance, Ministry of Health).
Assim, novos compostos para o controle da tuberculose são necessários para o combate da doença. A invenção presente resolve o problema de se encontrar novos fármacos para o tratamento da tuberculose (TB), utilizando a enzima InhA, alvo do principal fármaco empregado no tratamento deste doença (isoniazida) (INH), como alvo terapêutico. Adicionalmente, é importante salientar que, por causa da clara relação evolutiva entre a InhA de Mycobacterium tuberculosis e suas homólogas em outros organismos, os compostos da presente invenção também possuem o potencial de inibir a enzima InhA destes organismos. Dentre esses, destacamos os géneros: Mycobacterium: causa tuberculose, lepra, úlceras, e outras infecções; 5 Plasmodium: causa malária, em particular malária cerebral; Toxoplasma: causa toxoplasmose; Trypanosoma: causa doença de chagas; Klebsiella: causa infecções hospitalares, pneumonia; Chlamydia: causa tracoma ou clamidíase e infecções crónicas; Helycobacter. causa gastrite, úlcera e câncer estomacal; Pseudomonas: causa infecções
Figure imgf000004_0001
-cólera e outrasThus, new compounds for tuberculosis control are needed to fight the disease. The present invention solves the problem of finding new drugs for the treatment of tuberculosis (TB) using the enzyme InhA, targeting the main drug employed in the treatment of this disease (isoniazid) (INH), as a therapeutic target. In addition, it is important to note that because of the clear evolutionary relationship between InhA Mycobacterium tuberculosis and homologues thereof in other organisms, the compounds of the present invention also have the potential to inhibit the InhA enzyme of these organisms. Among these, we highlight the genera: Mycobacterium: causes tuberculosis, leprosy, ulcers, and other infections; 5 Plasmodium: causes malaria, in particular cerebral malaria; Toxoplasma: causes toxoplasmosis; Trypanosoma: Causes Chagas disease; Klebsiella: causes nosocomial infections, pneumonia; Chlamydia: causes trachoma or chlamydia and chronic infections; Helycobacter. causes gastritis, ulcer and stomach cancer; Pseudomonas: Causes Infections
Figure imgf000004_0001
cholera and other
-ío infecçõês7T3ãc7/7us: intoxicação alimentar (cereus) e carbúnculo (anthracis); - infections7T3c7 / 7us: food poisoning (cereus) and carbuncle (anthracis);
Yersinia: causa a peste negra; Staphylococcus: causa infecção hospitalar; Salmonella: causa salmonelose; Neisseria: causa doença venérea (gonorrhoeae) e meningite (meningitidis).  Yersinia: Causes Black Death; Staphylococcus: causes nosocomial infection; Salmonella: Causes salmonellosis; Neisseria: causes venereal disease (gonorrhoeae) and meningitis (meningitidis).
A busca na literatura científica e patentária apontou alguns documentos The search in the scientific and patent literature pointed to some documents
15 parcialmente relevantes para a presente invenção, os quais serão descritos a seguir. 15 partially relevant to the present invention, which will be described below.
O artigo científico intitulado "High affinity InhA inhibitors with activity against drug-resistant strains of Mycobacterium tuberculosis" de Sullivan et al (2006) descreve a necessidade de novos quimioterápicos serem utilizados The scientific article entitled "High affinity inhibitors with activity against drug-resistant strains of Mycobacterium tuberculosis" by Sullivan et al (2006) describes the need for new chemotherapy to be used.
20 contra cepas de Mycobacterium tuberculosis resistentes à fármacos utilizando design racional baseado em estrutura para desenvolvimento de uma série de éteres alquil difenil que são inibidores não-competitivos de InhA. 20 against drug resistant Mycobacterium tuberculosis strains using rational structure-based design for development of a series of alkyl diphenyl ethers that are non-competitive InhA inhibitors.
O artigo científico intitulado "A slow, tight inhibitor of InhA, the enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis" de Luckner et al The scientific article entitled "A slow, tight inhibitor of InhA, the enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis" by Luckner et al
25 (2010) descreve um novo inibidor, 2-(o-tolioxi-5-hexilfenol) (PT70) como um inibidor ideal para enoil-ACP redutase de Mycobacterium tuberculosis. 25 (2010) describes a new inhibitor, 2- (o-tolioxy-5-hexylphenol) (PT70) as an ideal inhibitor for Mycobacterium tuberculosis enoyl-ACP reductase.
O documento US 2011/0092536 descreve novos derivados anti- infecciosos, método de produção dos mesmos, composições farmacêuticas contendo os compostos e o uso dos mesmos no tratamento antimicrobiano, US 2011/0092536 describes novel anti-infectious derivatives, method of production thereof, pharmaceutical compositions containing the compounds and their use in antimicrobial treatment,
30 sendo que os derivados compreendem uma fórmula geral contendo a combinação de i) estruturas piridina, piridínio ou dihidropiridina e estruturas relacionadas derivada dos metabólitos ativos de isoniazida; e ii) substituinte hidrofóbico cujo alvo é o sítio ativo do substrato. Wherein the derivatives comprise a general formula containing the combination of i) pyridine, pyridinium or dihydropyridine structures and structures related derivatives of isoniazid active metabolites; and ii) hydrophobic substituent targeting the active substrate site.
O documento US 5,614,551 descreve inibidores de ácido graxo como potenciais agentes antimicrobianos no qual uma etapa essencial de ação dos possíveis agentes antimicrobianos compreende ser administrado em uma célula microbiana a fim de permitir a inibição de uma enzima (sintase) relacionada à produção de ácidos graxos.  US 5,614,551 describes fatty acid inhibitors as potential antimicrobial agents in which an essential step of action of possible antimicrobial agents comprises being administered into a microbial cell to allow inhibition of an enzyme (synthase) related to the production of fatty acids.
Os documentos acima referidos apresentam compostos potencialmente relevantes para a inibição de enzimas em microrganismos-Entretanto, nenhum dos documentos apontados descreve ou sugere os compostos da presente invenção e tampouco seu uso como inibidores enzimáticos, preferencialmente de enoil-ACP redutase em microrganismos, preferencialmente em Mycobacterium tuberculosis.  The above documents contain compounds potentially relevant for inhibition of enzymes in microorganisms. However, none of the above documents describe or suggest the compounds of the present invention and their use as enzyme inhibitors, preferably of enoyl-ACP reductase in microorganisms, preferably in Mycobacterium. tuberculosis.
Do que se depreende da literatura pesquisada, não foram encontrados documentos antecipando ou sugerindo os ensinamentos da presente invenção, de forma que a solução aqui proposta, aos olhos dos inventores, possui novidade e atividade inventiva frente ao estado da técnica.  From what can be inferred from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here, in the eyes of the inventors, has novelty and inventive activity in the state of the art.
Sumário da Invenção Summary of the Invention
É um dos objetos da invenção proporcionar um processo ou método para obtenção de ligantes de inhA, referido método compreendendo as etapas de:  It is an object of the invention to provide a process or method for obtaining inhA binders, said method comprising the steps of:
- avaliação in silico de potenciais ligantes de inhA;  - in silico evaluation of potential inhA ligands;
- predição in silico de toxicidade e/ou dos referidos ligantes; e  - in silico prediction of toxicity and / or said binders; and
- testes in vitro dos referidos ligantes, notadamente estudos cinéticos e/ou de inibição de inhA.  - in vitro testing of said binders, notably kinetic and / or inhibition studies of inhA.
Em um aspecto, a presente invenção proporciona novos compostos inibidores de redutase de microrganismos, úteis para inibir a enzima InhA (2- frans-enoil-ACP (CoA) redutase), como a InhA de Mycobacterium tuberculosis (EC: 1.3.1.9). Para fins da presente invenção, entende-se como qualquer cadeia alquílica, uma cadeia linear ou ramificada, saturada ou insaturada, cíclica ou não. In one aspect, the present invention provides novel microorganism reductase inhibiting compounds useful for inhibiting the enzyme InhA (2-fransenoyl-ACP (CoA) reductase) such as Mycobacterium tuberculosis InhA (EC: 1.3.1.9). For purposes of the present invention, any alkyl chain is a straight or branched chain, saturated or unsaturated, cyclic or not.
É um objeto da presente invenção um composto inibidor de redutase em microrganismo ser selecionado do grupo que compreende:  It is an object of the present invention for a microorganism reductase inhibitor compound to be selected from the group comprising:
a) fórmula I:  a) formula I:
Figure imgf000006_0001
onde R1 e R4, sendo os mesmos iguais ou distintos, compreendem uma cadeia alquílica contendo de 1 a 10 carbonos, um átomo de hidrogénio, um átomo do grupo dos halogênios, OH ou O";
Figure imgf000006_0001
wherein R1 and R4, being the same or distinct, comprise an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ;
R2 compreende uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de oxigénio; e  R2 comprises an alkyl chain containing from 1 to 10 carbons or an oxygen atom; and
R3 e R5 compreendem uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de hidrogénio;  R3 and R5 comprise an alkyl chain containing from 1 to 10 carbons or a hydrogen atom;
b) fórmula II: b) formula II:
R2R2
Figure imgf000007_0001
onde R1-R2, sendo os mesmos ou distintos, compreendendo uma cadeia alquílica contendo de 1 a 10 carbonos, um átomo de hidrogénio, um átomo do grupo dos halogênios, OH ou O"; e
Figure imgf000007_0001
where R1-R2, being the same or distinct, comprising an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ; and
R3 compreendendo uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de oxigénio;  R3 comprising an alkyl chain containing from 1 to 10 carbons or an oxygen atom;
c) fórmula III:  c) formula III:
Figure imgf000007_0002
onde R1 compreende uma cadeia alquílica contendo de 1 a 10 carbonos, um átomo de hidrogénio, um átomo do grupo dos halogênios, OH ou O"; e R2 compreende uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de oxigénio;
Figure imgf000007_0002
wherein R1 comprises an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ; and R2 comprises an alkyl chain containing from 1 to 10 carbons or an oxygen atom;
d) fórmula IV:  d) formula IV:
Figure imgf000008_0001
Figure imgf000008_0001
onde R1 e R3 podem, sendo os mesmos iguais ou distintos, compreender uma cadeia alquílica contendo de 1 a 10 carbonos, um átomo de hidrogénio, um átomo do grupo dos halogênios, OH ou O"; e wherein R1 and R3 may, being the same or distinct, comprise an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ; and
R2 compreende uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de hidrogénio;  R2 comprises an alkyl chain containing from 1 to 10 carbons or a hydrogen atom;
e) ou sais, solvatos e/ou hidratos farmaceuticamente aceitáveis dos mesmos.  e) or pharmaceutically acceptable salts, solvates and / or hydrates thereof.
É também um objeto adicional da presente invenção um ligante de inhA selecionado um composto selecionado do grupo compreendendo fórmula I, II, III e/ou IV acima, ou combinações dos mesmos.  A further object of the present invention is also a selected inhA binder a compound selected from the group comprising formula I, II, III and / or IV above, or combinations thereof.
É também um objeto adicional da presente invenção uma composição farmacêutica compreendendo:  A further object of the present invention is a pharmaceutical composition comprising:
- um composto selecionado do grupo compreendendo fórmula I, II, III e/ou IV acima, ou combinações dos mesmos; e  a compound selected from the group comprising formula I, II, III and / or IV above, or combinations thereof; and
- um veículo farmaceuticamente aceitável.  a pharmaceutically acceptable carrier.
É um objeto adicional da presente invenção o uso de um composto selecionado do grupo compreendendo fórmula I, II, III e/ou IV acima, ou combinações dos mesmos, para fabricação de medicamento com ação antimicrobiana.  It is a further object of the present invention to use a compound selected from the group comprising formula I, II, III and / or IV above, or combinations thereof, for the manufacture of antimicrobially acting medicaments.
Estes e outros objetos da invenção serão imediatamente valorizados pelos versados na arte e pelas empresas com interesses no segmento, e serão descritos em detalhes suficientes para sua reprodução na descrição a seguir. These and other objects of the invention will be immediately appreciated by those skilled in the art and companies with interests in the segment, and will be described in sufficient detail for reproduction in the following description.
Breve descrição das figuras Brief Description of the Figures
Figura 1. Análise das interações intermoleculares entre a enzima InhA de Mycobacterium tuberculosis e N-(2-fenoxifenil)-2-[2-(2-piridil)tiazol-4-il] acetamida.  Figure 1. Analysis of intermolecular interactions between the InhA enzyme of Mycobacterium tuberculosis and N- (2-phenoxyphenyl) -2- [2- (2-pyridyl) thiazol-4-yl] acetamide.
Figura 2. Análise das interações intermoleculares entre a enzima InhA de Mycobacterium tuberculosis e 3-(o-tolil)-1-[5-(4-propoxifenil)-1 ,3,4-tiadiazol- Figure 2. Analysis of intermolecular interactions between Mycobacterium tuberculosis enzyme InhA and 3- (o-tolyl) -1- [5- (4-propoxyphenyl) -1,3,4-thiadiazole
2- il]urea...... 2- il] urea ......
Figura 3. Análise das interações intermoleculares entre a enzima InhA de Mycobacterium tuberculosis e 4-(7-cloro-4-quinolil)-2-(dietilaminometil)fenol.  Figure 3. Analysis of intermolecular interactions between Mycobacterium tuberculosis enzyme InhA and 4- (7-chloro-4-quinolyl) -2- (diethylaminomethyl) phenol.
Figura 4. Análise das interações intermoleculares entre a enzima InhA de Mycobacterium tuberculosis e N-(4-{5-[(2-metilfenoxi)metil]-1 ,2,4-oxadiazol- Figure 4. Analysis of intermolecular interactions between Mycobacterium tuberculosis InhA enzyme and N- (4- {5 - [(2-methylphenoxy) methyl] -1,4,4-oxadiazole
3- yl}phenyl)thiophene-2-carboxamide. 3-yl} phenyl) thiophene-2-carboxamide.
Descrição Detalhada da Invenção Detailed Description of the Invention
A presente invenção tem como conceito inventivo comum o processo ou método para obtenção de ligantes de inhA. A partir do referido conceito inventivo comum, diferentes entidades moleculares são obtidas e resolvem o problema técnico de proporcionar novos ligantes de inhA. O processo ou método para obtenção de ligantes de inhA dã presente invenção compreende ao menos três etapas:  The present invention has as a common inventive concept the process or method for obtaining inhA binders. From said common inventive concept, different molecular entities are obtained and solve the technical problem of providing new inhA ligands. The process or method for obtaining inhA binders of the present invention comprises at least three steps:
- avaliação in silico dê potenciais ligantes de inhA;  - in silico evaluation of potential inhA ligands;
- predição in silico de toxicidade e/ou dos referidos ligantes; e  - in silico prediction of toxicity and / or said binders; and
- testes in vitro dos referidos ligantes, notadamente estudos cinéticos e/ou de inibição de inhA.  - in vitro testing of said binders, notably kinetic and / or inhibition studies of inhA.
Em uma concretização, o processo ou método para obtenção de ligantes de inhA da presente invenção compreende:  In one embodiment, the process or method for obtaining inhA binders of the present invention comprises:
- seleção da biblioteca de moléculas;  - selection of the library of molecules;
- geração do modelo farmacofórico;  - generation of the pharmacophoric model;
- geração do farmacóforo 3-D e busca em bancos de dados; - modelagem molecular; - 3-D pharmacophore generation and database searching; - molecular modeling;
- testes in silico de docagem molecular;  - in silico tests of molecular docking;
- predição in silico de toxicidade;  - in silico prediction of toxicity;
- medidas de inibição enzimática de MtlnhA in vitro;  - enzymatic inhibition measures of MtlnhA in vitro;
- seleção de moléculas para testes in vitro; e  - selection of molecules for in vitro tests; and
- estudo de inibição por cinética do estado estacionário.  - steady state kinetic inhibition study.
Ligante Binder
Na presente invenção, um ligante de inhA é um composto selecionado um .compostO-selecionado-do-grupo-Gompreendendo-fórmula I, II, III e/ou IV acima, ou combinações dos mesmos, bem como seus sais, solvatos e/ou hidratos. Referido ligante é particularmente útil para a detecção in vitro e/ou in vivo de inhA, notadamente quando os referidos compostos são dotados de algum marcador radioativo e/ou de reação colorimétrica. Os versados na arte imediatamente saberão identificar os referidos marcadores em conjunto com os ligantes da invenção, proporcionando com isso a detecção e/ou quantificação de inhA em amostras biológicas.  In the present invention, an inhA binder is a compound selected from the above-selected group comprising formula I, II, III and / or IV, or combinations thereof, as well as their salts, solvates and / or hydrates. Said binder is particularly useful for in vitro and / or in vivo detection of inhA, notably when said compounds are endowed with some radioactive marker and / or colorimetric reaction. Those skilled in the art will readily be able to identify said markers together with the binders of the invention, thereby providing detection and / or quantification of inhA in biological samples.
Veículo farmaceuticamente aceitável Pharmaceutically acceptable vehicle
Na presente invenção entende-se por "veículo farmaceuticamente aceitável" qualquer veículo que proporcione a inserção em uma formulação, contendo excipientes e carreadores aceitáveis conhecidos por técnicos no assunto, assim como em doses e tratamentos convenientes para uso em composições particulares. O dito veículo pode ser descrito em uma série de regimentos de tratamento, incluindo oral, parenteral, intravenoso, intranasal, intravítreo e intramuscular, intracerebral, intracerebroventricular e intraocular ou demais meios de administração e/ou formulação.  By the present invention "pharmaceutically acceptable carrier" is meant any carrier that provides insertion into a formulation containing acceptable excipients and carriers known to those skilled in the art, as well as at doses and treatments suitable for use in particular compositions. Said carrier may be described in a number of treatment regimens including oral, parenteral, intravenous, intranasal, intravitreal and intramuscular, intracerebral, intracerebroventricular and intraocular or other means of administration and / or formulation.
Uso Use
A presente invenção proporciona o uso de um composto selecionado do grupo compreendendo fórmula I, II, III e/ou IV descritos acima, ou combinações dos mesmos, para a fabricação dè medicamento com ação antimicrobiana.  The present invention provides the use of a compound selected from the group comprising formula I, II, III and / or IV described above, or combinations thereof, for the manufacture of antimicrobially acting medicament.
Em uma concretização, na fórmula I, R1 e R5 são átomos de hidrogénio; In one embodiment, in formula I, R 1 and R 5 are hydrogen atoms;
R2 é -CH2; R3 serem etilas, R4 é um átomo de cloro. Em uma concretização, na fórmula II, R1 é um átomo de hidrogénio; R3 é um -CH2 e R2 é uma metila na posição orto. R 2 is CH 2; R3 are ethyl, R4 is a chlorine atom. In one embodiment, in formula II, R 1 is a hydrogen atom; R 3 is -CH 2 and R 2 is a methyl in the ortho position.
Em uma concretização, na fórmula III, R1 é um átomo de hidrogénio e R2 é um -CH2. In one embodiment, in formula III, R 1 is a hydrogen atom and R 2 is a -CH 2 .
Em uma concretização, na fórmula IV, R1 é um átomo de hidrogénio, R2 é uma propila e R3 é uma metila na posição orto.  In one embodiment, in formula IV, R 1 is a hydrogen atom, R 2 is a propyl and R 3 is a methyl in the ortho position.
Na preparação de composições farmacêuticas antimicrobianas, sais, solvatos e/ou hidratos farmaceuticamente aceitáveis dos compostos de fórmula ' > JLL" e/ou IV descritos_acima,_QU_combinações-dos-mesmos, também podem ser usados.  In the preparation of pharmaceutically acceptable antimicrobial pharmaceutical compositions, salts, solvates and / or hydrates of the above-described compounds of formula III and / or IV may also be used.
Os exemplos aqui mostrados têm o intuito somente de exemplificar uma das inúmeras maneiras de se realizar a invenção, contudo, sem limitar o. escopo da mesma.  The examples shown herein are intended solely to exemplify one of the numerous ways of carrying out the invention, however, without limiting the invention. scope of it.
Exemplo 1. Identificação de inibidores de InhA  Example 1. Identification of InhA Inhibitors
InhA ou 2-frans-enoil-ACP (CoA) redutase de Mycobacterium tuberculosis (EC: 1.3.1.9) é uma enzima atrativa para realizar esforços de descoberta de fármacos devido a sua validação como alvo biológico efetivo para terapia de tuberculose. Na presente invenção, duas abordagens distintas de triagem in silico de ligantes foram utilizadas a fim de identificar novos inibidores InhA a partir de uma biblioteca de ligantes selecionada do banco de dados ZINC.  InhA or Mycobacterium tuberculosis 2-fransenoyl-ACP (CoA) reductase (EC: 1.3.1.9) is an attractive enzyme for drug discovery efforts due to its validation as an effective biological target for tuberculosis therapy. In the present invention, two distinct in silico ligand screening approaches were used to identify novel InhA inhibitors from a selected ligand library from the ZINC database.
Inicialmente, um modelo farmacofórico tridimensional foi construído baseado em 36 estruturais de cristais MtlnhA disponíveis. Pela combinação de informação baseada em estrutura e baseada em ligantes, quatro pontos farmacofóricos foram desenhados para selecionar moléculas com habilidade para satisfazer as características de ligação da cavidade de ligação do substrato da MtlnhA. A segunda abordagem consiste em usar quatro programas bem estabelecidos de docagem molecular, com diferentes algoritmos de busca, para comparar o modo de ligação e escore das moléculas selecionadas na biblioteca acima mencionada. Depois de uma análise detalhada dos resultados, quatro ligantes foram selecionados para análise in vitro. Desses, três apresentaram resultados satisfatórios de atividade inibitória com IC50 de 20 μΜ to 83 μΜ. O melhor composto apresentou inibição não competitiva aos substratos NADH e 2-fraA7s-dodecenoil-CoA, com Kt de 24 μΜ e 20 μΜ, respectivamente. Esses resultados validaram o método desenvolvido. Material e métodos Initially, a three-dimensional pharmacophoric model was constructed based on 36 available structural MtlnhA crystals. By combining structure-based and ligand-based information, four pharmacophoric points were designed to select molecules capable of satisfying the binding characteristics of the MtlnhA substrate binding cavity. The second approach is to use four well-established molecular docking programs with different search algorithms to compare the binding mode and score of the selected molecules in the aforementioned library. After a detailed analysis of the results, four ligands were selected for in vitro analysis. vitro. Of these, three presented satisfactory results of inhibitory activity with IC 50 from 20 μΜ to 83 μΜ. The best compound showed noncompetitive inhibition to NADH and 2-fraA7s-dodecenoil-CoA substrates, with K t of 24 μΜ and 20 μΜ, respectively. These results validated the developed method. Material and methods
Seleção da biblioteca de moléculas  Molecule Library Selection
Baseado na analise estrutural e físico química da MtlnhA, uma pré- seleção de compostos a serem selecionados foi realizada utilizando o Banco de- Dados— INGr Filtros moleculares- 2D idêTitificafám moléculas com características favoráveis para interagir com a cavidade de ligação da InhA: 4 < LogP < 7; ligações rotatórias < 6; aceptores de pontes de hidrogénio < 8; área de superfície polar < 40 Ã2 ; e peso molecular entre 250 Da e 400 Da. O conjunto final de compostos compreendendo as características citadas compreendeu 999.853 compostos e foram utilizados em ambas abordagens descritas a seguir. Based on the structural and physical chemical analysis of MtlnhA, a pre-selection of compounds to be selected was performed using the Database - INGr Molecular Filters - 2D Identify molecules with favorable characteristics to interact with the InhA binding cavity: 4 < LogP <7; rotary connections <6; hydrogen bridge acceptors <8; polar surface area <40 Ã 2 ; and molecular weight between 250 Da and 400 Da. The final set of compounds comprising the cited characteristics comprised 999,853 compounds and were used in both approaches described below.
ABORDAGEM 1 : Geração de farmacóforo baseado em estrutura  APPROACH 1: Structure-Based Pharmacophore Generation
Abordagem computacional Computational approach
A busca pelo farmacóforo foi realizada utilizando-se o pacote de programas SYBL 8.0 (Tripos Inc., St Louis, MO) utilizado em estacões Red Hat Enterprise Linux. As estruturas 3-D dos inibidores foram geradas usando parâmetros geométricos padrões do pacote de modelagem molecular do pacote de programas SYBL 8.0. Cada conformação otimizada de uma molécula do conjunto de dados foi energeticamente minimizada utilizando o campo de força Tripos e o algoritmo de gradiente conjugado Powell com um critério de convergência de 0,005 kcal/mol. Á. Cargas atómicas parciais foram calculadas pelo método Gasteiger-Huckel. As análises, cálculos e visualizações foram realizadas utilizando os programas SYBYL 8.0 e PyMol.  The search for the pharmacophorus was performed using the SYBL 8.0 software package (Tripos Inc., St Louis, MO) used at Red Hat Enterprise Linux stations. Inhibitor 3-D structures were generated using standard geometric parameters from the SYBL 8.0 software package molecular modeling package. Each optimized conformation of a molecule in the dataset was energetically minimized using the Tripos force field and the Powell conjugate gradient algorithm with a convergence criterion of 0.005 kcal / mol. THE. Partial atomic charges were calculated by the Gasteiger-Huckel method. Analyzes, calculations and visualizations were performed using the SYBYL 8.0 and PyMol programs.
Geração do modelo farmacofórico Generation of the pharmacophoric model
Atualmente, 36 estruturas cristalinas de MtlnhA estão disponíveis no banco de dados Protein Data Bank (PDB). Entre elas, encontramos o tipo selvagem de InhA e seus mutantes relacionados a resistência a drogas, na forma apo (sem ligante) e complexados com NADH (coenzima), análogos de substrato e diversos ligantes. As 36 estruturas cristais foram sobrepostas através de seus átomos Ca pelo método dos mínimos quadrados utilizando o programa TopMatch disponível em (http://topmatch.services.came.sbg.ac.at/). Os volumes das cavidades de ligação foram calculados com CASTp (http://sts.bioengr.uic.edu/castp/). Pontes de hidrogénio e contatos hidrofóbicos também foram analisados pelo LIGPLOT para obtenção de um perfil mais robusto de padrão de interação entre MtlnhA e seus ligantes cristalográficos. A análise-estrutural-de-MtlnhA-complexado-com-ligantes-ligados a cavidade de ligação do substrato indicam diversos resíduos de aminoácido chaves, que podem estar envolvidos no processo de reconhecimento molecular. Com base nisso, um modelo farmacofórico 3-D de quatro pontos foi desenvolvido. Currently 36 MtlnhA crystal structures are available in the Protein Data Bank (PDB) database. These include the wild type of InhA and its drug resistance mutants in apo form (no binder) and complexed with NADH (coenzyme), substrate analogs and various binders. The 36 crystal structures were superimposed through their Ca atoms by the least squares method using the TopMatch program available at (http://topmatch.services.came.sbg.ac.at/). Binding well volumes were calculated with CASTp (http://sts.bioengr.uic.edu/castp/). Hydrogen bridges and hydrophobic contacts were also analyzed by LIGPLOT to obtain a more robust interaction pattern profile between MtlnhA and its crystallographic ligands. Structural analysis of MtlnhA-complexed-ligands-bound to the substrate binding cavity indicates several key amino acid residues that may be involved in the molecular recognition process. Based on this, a four-point 3-D pharmacophoric model was developed.
Geração do farmacóforo 3-D e busca em bancos de dados 3-D pharmacophore generation and database searching
A biblioteca de moléculas de estruturas 3-D foi transferida para um banco de dados UNITY no SYBYL 8.0. A busca 3-D com UNITY foi baseada no modelo farmacofórico gerado. A fim de aplicar o modelo farmacofórico com restrições 3-D, utilizamos coordenadas espaciais dos cristais do ligante 5-{[4- (9H-fluoreno-9-yl)piperazina-1-il]carbonil}-1 H-indol (Genz-10850), ligado a MtlnhA (PDB ID:1 P44). Os átomos doadores e receptores foram definidos pela conexão dos átomos doadores e receptores do ligante via correspondência parcial. A característica hidrofóbica foi parcialmente caracterizada baseada nos dois anéis presentes na estrutura do ligante. Os diâmetros das esferas foram ajustados de forma que uma amostra teste pode ser obtida, composta de ligantes cristalográficos MtlnhA. Pára realizar a seleção na biblioteca de moléculas, foi realizada uma busca flexível UNITY 3-D.  The 3-D structure molecule library was transferred to a UNITY database in SYBYL 8.0. The 3-D search with UNITY was based on the generated pharmacophoric model. In order to apply the 3-D constrained pharmacophoric model, we used spatial coordinates of 5 - {[4- (9H-fluorene-9-yl) piperazine-1-yl] carbonyl} -1H-indole ligand crystals (Genz -10850), linked to MtlnhA (PDB ID: 1 P44). Donor and receptor atoms were defined by connecting the donor and receptor atoms of the ligand via partial correspondence. The hydrophobic characteristic was partially characterized based on the two rings present in the ligand structure. The bead diameters were adjusted so that a test sample could be obtained, composed of MtlnhA crystallographic ligands. To perform the selection in the molecule library, a flexible UNITY 3-D search was performed.
Modelagem molecular Molecular modeling
O algoritmo UNITY assinala alta pontuação para moléculas que combinam com os pontos farmacofóricos em suas buscas flexíveis e, por conta disso, a geometria otimizada dos compostos sob analise podem ser afetados. Dessa forma, as estruturas moleculares do conjunto de compostos selecionados foram minimizadas a fim de evitar resultados falso positivos. A minimização energética das moléculas foi realizada utilizando o mesmo protocolo descrito na seção de Abordagem Computacional. The UNITY algorithm scores high for molecules that match the pharmacophoric points in their flexible searches, and because of this, the optimized geometry of the compounds under analysis may be affected. Thus, the molecular structures of the selected set of compounds were minimized to avoid false positive results. THE Energy minimization of the molecules was performed using the same protocol described in the Computational Approach section.
Subsequentemente, os candidatos a ligantes selecionados foram encaixados dentro da cavidade de ligação do substrato MtInhA. O conjunto de programas GOLD foi utilizado para realizar as simulações de docagem molecular. A estrutura cristal MtInhA usada como receptor (PDB ID, 1 P44) foi escolhida dentre as 36 estruturas disponíveis devido a sua melhor relação entre o volume da cavidade de ligação (3,007.8 A3) e a resolução da proteína (2,7 Á).._P_ara_.os_cálculos,-átomos-de-hidrogênio foram adicionados a proteína com geometria padrão utilizando o modulo Biopolymer implementado no SYBYL 8.0. O sitio ativo foi definido como incorporando todos os resíduos de aminoácidos com um raio de esfera de 8.0 A centralizado no ligante cristalográfico ligado (Genz-10850). A coenzima NADH foi tratada como parte da proteína em todas as simulações de docagem. Estudos de modelagem molecular foram realizados utilizando os parâmetros padrões do GOLD. A função de alinhamento do GoldScore foi aplicada para selecionar uma configuração representativa para cada composto. As soluções foram visualmente checadas para checar se a configuração obtida por docagem ainda estava de acordo com a hipótese do farmacóforo original. A inspeção visual é uma prática comum e importante para evitar o teste de falsos positivos devido a suposições e deficiências dos- métodos de docagem funções de pontuação. Na inspeção visual, nós criticamente acessamos a conformação de ligação sugerida, a superfície mutual de complementariedade do ligante e da proteína, e a possível presença de espaços não preenchidos ao longo da interface proteína-ligante. Somente essas moléculas que preencheram todos os quatro pontos farmacofóricos após as simulações de docagem foram selecionadas para o próximo teste (predição de toxicidade in silico). Subsequently, selected binder candidates were seated within the MtInhA substrate binding cavity. The GOLD software suite was used to perform molecular docking simulations. The MtInhA crystal structure used as a receptor (PDB ID, 1 P44) was chosen from among the 36 available structures because of its better relationship between the binding cavity volume (3.007.8 A 3 ) and protein resolution (2.7 Á). For the calculations, hydrogen atoms were added to the protein with standard geometry using the Biopolymer module implemented in SYBYL 8.0. The active site was defined as incorporating all amino acid residues with a 8.0 A sphere radius centered on the bound crystallographic ligand (Genz-10850). Coenzyme NADH was treated as part of the protein in all docking simulations. Molecular modeling studies were performed using standard GOLD parameters. GoldScore's alignment function has been applied to select a representative setting for each compound. The solutions were visually checked to verify that the configuration obtained by docking was still in accordance with the original pharmacophore hypothesis. Visual inspection is a common and important practice to avoid testing false positives due to assumptions and shortcomings in docking methods. On visual inspection, we critically accessed the suggested binding conformation, the mutual surface of ligand and protein complementarity, and the possible presence of unfilled spaces along the protein-ligand interface. Only those molecules that filled all four pharmacophoric points after docking simulations were selected for the next test (prediction of in silico toxicity).
ABORDAGEM 2: Simulações exaustivas de docagem utilizando diferentes algoritmos  APPROACH 2: Comprehensive docking simulations using different algorithms
Algoritmos de docagem molecular Em paralelo a abordagem baseada em estrutura do farmacóforo, simulações de docagem molecular foram realizadas utilizando quatro diferentes e bem estabelecidos programas: GOLD, AutoDock, FlexX e Surflex-Dock. Molecular Docking Algorithms In parallel to the structure-based approach of the pharmacophore, molecular docking simulations were performed using four different and well-established programs: GOLD, AutoDock, FlexX and Surflex-Dock.
GOLD (Generic Optimization for Ligand Docagem) é um programa que aplica um algoritmo genético para docagem de ligantes flexíveis em sítios de ligação de proteínas utilizando Algoritmo Genético. O programa dá algumas opções relacionadas à função de pontuação e, entre elas, elegemos o GoldScore para ser utilizado em nossas simulações.  GOLD (Generic Optimization for Ligand Docking) is a program that applies a genetic algorithm for docking flexible ligands at protein binding sites using Genetic Algorithm. The program gives some options related to the scoring function and among them we have chosen GoldScore to be used in our simulations.
AutoDock-utiliza um-campo-de força-semi-empírico de-energia livre para avaliar conformações durante as simulações de docagem e proporciona muitos métodos para realização da busca conformacional. O Algoritmo Genético Lamarckiano, uma modificação do Algoritmo Genético tradicional, foi nossa escolha para este estudo.  AutoDock-utilizes a free-energy semi-empirical force-field to evaluate conformations during docking simulations and provides many methods for performing conformational searching. The Lamarckian Genetic Algorithm, a modification of the traditional Genetic Algorithm, was our choice for this study.
FlexX foi implementado como Algoritmo de Construção Incremental, onde o fragmento base (o centro do ligante) é automaticamente selecionado e colocado dentro do sitio ativo usando uma abordagem algorítmica baseada em uma técnica de reconhecimento de padrão chamada de agrupamento de configurações (pose clustering). Após um bom conjunto de localizações ter sido obtido como base, as porções remanescentes do ligante são divididas em pequenos fragmentos e incrementalmente "crescem" dentro de bases alternativas.  FlexX was implemented as Incremental Construction Algorithm, where the base fragment (the center of the binder) is automatically selected and placed within the active site using an algorithmic approach based on a pattern recognition technique called pose clustering. After a good set of locations has been obtained as a base, the remaining portions of the binder are divided into small fragments and incrementally "grow" within alternative bases.
O algoritmo Surflex-Dock aplica a função de pontuação empírica Hammerhead e usa um ligante de sitio ativo idealizada, ou "protomol", para gerar ligantes pela construção incremental, e também usa um procedimento de cruzamento de informações que combina pedaços com configurações distintas. Protomols foram computadas usando a posição do ligante cognato para definir o sitio de ligação.  The Surflex-Dock algorithm applies the Hammerhead empirical scoring function and uses an idealized active site ligand, or "protomol", to generate ligands by incremental construction, and also uses an information crossing procedure that combines pieces with distinct configurations. Protomols were computed using the cognate ligand position to define the binding site.
Receptor Proteico Protein Receiver
O receptor proteico escolhido das simulações com MtlnhA foi aquele com o código PDB 1 P44 e a coenzima NADH foi tratada como parte do receptor. Em todas as simulações de docagem a estrutura do receptor foi mantida rígida, enquanto os ligantes foram tratados com total flexibilidade. The protein receptor chosen from the MtlnhA simulations was that with the PDB code 1 P44 and coenzyme NADH was treated as part of the receptor. In all docking simulations the receiver structure was kept rigid while the ligands were treated with complete flexibility.
Conjunto de moléculas Molecules set
O conjunto de moléculas foi o mesmo utilizado para a abordagem baseada no farmacóforo.  The set of molecules was the same as that used for the pharmacophore-based approach.
Simulações de docagem Docking Simulations
Inicialmente, foi realizado docagem com todas as moléculas do conjunto de dados inicial dentro de MtlnhA usando GOLD. O sítio ativo foi definido como incorporando- todos os-resíduos-de-aminoácidos com uma esfera de raio 8.0 À centralizada no ligante cristalográfico ligado no 1 P44 (Genz-10850). As 100 moléculas mais bem ranqueadas pelo GOLD foram selecionadas para serem submetidas a outros três programas, AutoDock, FlexX e Surflex-Dock, usando os mesmos parâmetros do GOLD, sendo somente diferente os algoritmos para busca e pontuação das melhores configurações do ligante, os quais são característicos de cada programa. Os ligantes que obtiveram uma conformação muito similar, em termos de posicionamento dos grupos funcionais, em pelo menos três dos quatro algoritmos, foram selecionados para o próximo passo (predição de toxicidade in silico).  Initially, docking was performed with all molecules from the initial dataset within MtlnhA using GOLD. The active site was defined as incorporating all amino acid residues with an 8.0 A radius sphere centered on the 1 P44-bound crystallographic ligand (Genz-10850). The top 100 molecules ranked by GOLD were selected to be submitted to three other programs, AutoDock, FlexX and Surflex-Dock, using the same parameters as GOLD, with only the search and scoring algorithms of the best ligand configurations being different. are characteristic of each program. Binders that obtained a very similar conformation in terms of functional group positioning in at least three of the four algorithms were selected for the next step (prediction of in silico toxicity).
Predição de toxicidade in silico Prediction of in silico toxicity
Toxicidade não aceitável ainda é o maior gargalo no processo de descoberta de fármacos. A fim de superar esse problema, técnicas de predição de toxicidade in silico são rápidas são alternativas de baixo custo (ou suplementos) de ensaios biológicos para a identificação de efeitos tóxicos em um estágio inicial de desenvolvimento de produto. Portanto, como uma analise adicional in silico para todas as moléculas selecionadas pela primeira e pela segunda abordagem, nós utilizamos o sistema de informática de descoberta de fármacos OSÍRIS para predizer as características de toxicidade. O OSÍRIS Property Explorer (disponível em http://www.organic-chemistry.org/prog/peo/) é parte integrante do sistema de registro de substâncias interno do Actelion. Ele calcula as propriedades relevantes para fármacos simultaneamente enquanto o usuário desenha a molécula. As moléculas selecionadas por ambas abordagens usadas na presente invenção foram submetidas ao OSÍRIS e todas aquelas que apresentaram características desfavoráveis foram removidas do conjunto final de moléculas para serem testadas in vitro. Unacceptable toxicity is still the biggest bottleneck in the drug discovery process. In order to overcome this problem, rapid silicon toxicity prediction techniques are low cost alternatives (or supplements) to biological testing for the identification of toxic effects at an early stage of product development. Therefore, as an additional in silico analysis for all molecules selected by the first and second approaches, we use the OSIRIS drug discovery computer system to predict toxicity characteristics. OSÍRIS Property Explorer (available at http://www.organic-chemistry.org/prog/peo/) is an integral part of Actelion's internal substance registration system. It calculates drug-relevant properties simultaneously while the user draws the molecule. The molecules selected by both The approaches used in the present invention were subjected to OSIRIS and all those with unfavorable characteristics were removed from the final set of molecules for in vitro testing.
Análise de similaridades com fármacos e alvos existentes Similarity analysis with existing drugs and targets
Para os ligantes que passaram nos testes de análise de toxicidade, foi realizada uma busca utilizando a ferramenta online SEA (Similarity Ensemble Approach - http://sea.bkslab.org/search/) para acessar as relações entre nossos escolhidos e moléculas que já possuem propriedades e alvos descritos.  For the ligands that passed the toxicity analysis tests, a search was performed using the SEA (Similarity Ensemble Approach - http://sea.bkslab.org/search/) online tool to access the relationships between our chosen and molecules that have already have properties and targets described.
Exemplo 2. Medidas de inibição enzimática de MtlnhA in vitro Example 2. In vitro inhibition measures of MtlnhA in vitro
Para avaliar a potência relativa de cada ligante selecionado, medidas de velocidades em estado estacionário na presença de ligantes foram realizadas utilizando espectrofotômetro visível no UV UV-2550 (Shimadzu®), monitorando a oxidação de NADH em 340 nm (ερ-NADH = 6.22 x1Cr3 M~ 1) devido à redução do substrato 2-írans-dodecenoil-CoA. Experimentos foram realizados a 25°C, em 100 mM NaH2PO4 pH 7.5 e medidas das reações químicas de catálise enzimática começaram com a adição de MtlnhA à mistura. Resumidamente, diferentes concentrações (1 - 20 μΜ) de compostos selecionados foram adicionados à mistura reacional e a velocidade enzimática foi analisada como a % de inibição. A taxa máxima de reação enzimática (100% de atividade MtlnhA) foi determinada na ausência do inibidor, e na presença de concentrações fixas não saturantes de NADH (60 μΜ = Km) e 2- fra/?s-dodecenoil-CoA (45 μΜ = m). To assess the relative power of each selected ligand, steady-state velocity measurements in the presence of ligands were performed using UV-2550 visible spectrophotometer (Shimadzu ® ), monitoring NADH oxidation at 340 nm (ερ-NADH = 6.22 x1Cr 3 M ~ 1 ) due to the reduction of the 2-trans-dodecenoyl-CoA substrate. Experiments were performed at 25 ° C in 100 mM NaH 2 PO 4 pH 7.5 and measurements of enzymatic catalysis chemical reactions began with the addition of MtlnhA to the mixture. Briefly, different concentrations (1 - 20 μΜ) of selected compounds were added to the reaction mixture and the enzymatic rate was analyzed as the% inhibition. The maximum enzyme reaction rate (100% MtlnhA activity) was determined in the absence of the inhibitor, and in the presence of fixed non-saturating concentrations of NADH (60 μΜ = K m ) and 2-fra /? S-dodecenoil-CoA (45 μΜ = m ).
Para determinar o modo de inibição e constante de inibição (Kj), taxas iniciais foram medidas como uma função da concentração de NADH em uma concentração fixa não saturante de 2-írans-dodecenoil-CoA (45 μΜ) e concentrações fixas variantes de inibidor (0, 1 , 10 e 20 μΜ). Estudos de inibição foram também realizados na presença de concentração fixa não saturante de NADH (60 μΜ) e concentrações fixas variantes de inibidor (0, 6, 10 e 20 μΜ), e 2-trans-dodecenoil-CoA como substrato variável. Os valores de K para ambos substratos foram calculados ajustando os dados da equação descrevendo a inibição não competitiva (Eq. 1), na qual [I] é a concentração do inibidor, [S] é a concentração do substrato, Km é a constante de Michaelis- Menten, \Zmax é a velocidade máxima e Ki é a constante de inibição total.
Figure imgf000018_0001
To determine inhibition mode and inhibition constant (Kj), baseline rates were measured as a function of NADH concentration at a fixed non-saturating concentration of 2-transdodecenoyl-CoA (45 μΜ) and fixed inhibitor variants ( 0, 1, 10 and 20 μΜ). Inhibition studies were also performed in the presence of fixed non-saturating NADH concentration (60 μΜ) and fixed inhibitor variants (0, 6, 10 and 20 μΜ), and 2-trans-dodecenoyl-CoA as the variable substrate. K values for both substrates were calculated by fitting the equation data describing non-competitive inhibition (Eq. 1), where [I] is the concentration of the inhibitor, [S] is substrate concentration, K m is the Michaelis-Menten constant, \ Z max is the maximum velocity and Ki is the total inhibition constant.
Figure imgf000018_0001
Seleção de moléculas para testes in vitro Selection of molecules for in vitro tests
A seleção final para moléculas testadas in vitro para verificação de sua atividade inibitória contra MtInhA foi baseada em todas análises descritas acima. Das 19 moléculas- restantes— quatro foram- escolhidas para serem testadas in vitro, a fim de abordar diferentes modos de interação com a cavidade de ligação com o substrato da MtinhA. Para a primeira abordagem baseada em farmacóforo, foi escolhido ZINC22559057, o qual interage com a cavidade de ligação principalmente por meio de contatos hidrofóbicos. ZINC09137707 não está ligado pelo hidrogénio a NADH; entretanto, ele realiza duas pontes de hidrogénio com Tyr158 e diversas interações hidrofóbicas.  The final selection for in vitro tested molecules for verification of their inhibitory activity against MtInhA was based on all analyzes described above. Of the remaining 19 molecules - four were - chosen to be tested in vitro to address different modes of interaction with the MtinhA substrate-binding cavity. For the first pharmacophore-based approach, ZINC22559057 was chosen, which interacts with the binding cavity primarily through hydrophobic contacts. ZINC09137707 is not hydrogen bound to NADH; however, it performs two hydrogen bridges with Tyr158 and various hydrophobic interactions.
Tabela 1 - Informação sobre as moléculas selecionadas pela Abordagem 1 Table 1 - Information on the molecules selected by Approach 1
Figure imgf000018_0002
zs
Figure imgf000018_0002
zs
3-(o-tolil)-1-[5- (4-propoxifenil) 3- (o-tolyl) -1- [5- (4-propoxyphenyl)
ZINC 4.74 1 6 -1 367.454 7  ZINC 4.74 1 6 -1 367.454 7
-1 ,3,4-tiadiazol- 09137707  -1,3,4-thiadiazole-09137707
2-il]urea  2-il] urea
Tabela 2 - Informação sobre as moléculas selecionadas pela Abordagem 2 Table 2 - Information on the molecules selected by Approach 2
Figure imgf000019_0001
Exemplo 3. Estudo de inibição por cinética do estado estacionário
Figure imgf000019_0001
Example 3. Steady state kinetic inhibition study
Uma escolha inicial mostrou que três dos seis compostos selecionados apresentaram uma porcentagem satisfatória de inibição (%), de forma que foram realizadas outras analises complementares com abordagens diferentes. O composto ZINC02931014 com uma concentração final de 2 μΜ mostrou 5,5% de inibição da atividade enzimática de MtlnhA, enquanto ZINC12509636 em uma concentração de 4 μΜ mostrou 2,5% de inibição. O decaimento linear da atividade enzimática de MtlnhA em função do aumento das concentrações de ZINC02931014 e ZINC12509636 permitiram uma estimativa de valores de IC50 de cerca de 24 μΜ e 83 μΜ, respectivamente. O composto ZINC09137707 com uma concentração final de 1 μΜ mostrou 15% de inibição enzimática de MtlnhA. Dessa forma, o comp_QSto.ZIMCQ9-137707-foi selecionado para estudos posteriores. O modo de inibição e os valores da constante de inibição (Kj) foram derivados a partir de taxas iniciais medidas conforme descrito na seção Material e Métodos. Gráficos de dupla reciprocidade com concentrações diferentes de ZINC09137707 demonstram um padrão de linhas paralelas, sugerindo que esse composto age como inibidor não-competitivo de NADH e 2- fra/7s-dodecenoil-CoA, no qual os valores de Vmax e Km foram simultaneamente reduzidos. An initial choice showed that three of the six selected compounds had a satisfactory percentage of inhibition (%), so that Other complementary analyzes were performed with different approaches. Compound ZINC02931014 with a final concentration of 2 μΜ showed 5.5% inhibition of MtlnhA enzymatic activity, while ZINC12509636 at a concentration of 4 μΜ showed 2.5% inhibition. The linear decay of MtlnhA enzymatic activity as a function of increasing concentrations of ZINC02931014 and ZINC12509636 allowed an estimated IC 50 values of about 24 μΜ and 83 μΜ, respectively. Compound ZINC09137707 with a final concentration of 1 μΜ showed 15% enzymatic inhibition of MtlnhA. Thus, comp_QSto.ZIMCQ9-137707-was selected for further studies. Inhibition mode and inhibition constant (Kj) values were derived from initial measured rates as described in the Material and Methods section. Double reciprocity plots with different concentrations of ZINC09137707 demonstrate a parallel line pattern, suggesting that this compound acts as a non-competitive inhibitor of NADH and 2 fra / 7s-dodecenoyl-CoA, in which Vmax and K m values were simultaneously reduced.
A equação 1 se mostra adequada ao comportamento de inibidores não- competitivos, com valores de K, de 24 (+ 3) μΜ e 20 (± 2) μΜ para, respectivamente, NADH e 2-fraA7S-dodecenoil-CoA.  Equation 1 is adequate for the behavior of non-competitive inhibitors, with K values of 24 (+ 3) μΜ and 20 (± 2) μΜ for NADH and 2-fraA7S-dodecenoil-CoA, respectively.
Esse perfil de inibição indica que o inibidor se liga exclusivamente ao complexo enzima-substrato (ES), levando a inativação do complexo enzima- substrato-inibidor (ESI), e a afinidade do inibidor é máxima em concentrações saturantes de substrato.  This inhibition profile indicates that the inhibitor binds exclusively to the enzyme-substrate (ES) complex, leading to inactivation of the enzyme-substrate-inhibitor (ESI) complex, and the affinity of the inhibitor is maximum at substrate saturating concentrations.
De maneira interessante, para inibidores não competitivos, a inibição enzimática não pode ser superada por altas concentrações de substrato, o que é uma fraqueza dos inibidores competitivos. Esses estudos de inibição de bancada proporcionam suporte aos métodos de seleção in silico aqui descritos como ferramentas para mineração de compostos químicos com atividade inibitória da enzima MtlnhA e pode se mostrar útil para demais esforços de descoberta de fármacos. Os versados na arte valorizarão os conhecimentos aqui apresentados e poderão reproduzir a invenção nas modalidades apresentadas e em outros variantes, abrangidos no escopo das reivindicações anexas. Interestingly, for non-competitive inhibitors, enzyme inhibition cannot be overcome by high substrate concentrations, which is a weakness of competitive inhibitors. These bench-top inhibition studies support the in silico selection methods described herein as tools for mining chemical compounds with MtlnhA inhibitory activity and may prove useful for other drug discovery efforts. Those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the embodiments presented and in other embodiments within the scope of the appended claims.

Claims

Reivindicações COMPOSTOS, USO, COMPOSIÇÃO FARMACÊUTICA INIBIDORA DE REDUTASE EM MICRORGANISMOS, LIGANTE DE INHA, E, MÉTODO PARA OBTENÇÃO DE LIGANTES DE INHA COMPOSITIONS, USE, PHARMACEUTICAL COMPOSITION INHIBITOR OF MICROORGANISMS REDUCES, INHA BINDER, AND METHOD FOR OBTAINING INHA BINDS
1. Composto inibidor de redutase em microrganismo caracterizado por ser selecionado do grupo que compreende: 1. A microorganism reductase inhibitor compound which is selected from the group comprising:
a) fórmula I:  a) formula I:
Figure imgf000022_0001
onde R1 e R4, sendo os mesmos iguais ou distintos, compreendem uma cadeia alquílica contendo de 1 a 10 carbonos, um átomo de hidrogénio, um átomo do grupo dos halogênios, OH ou O";
Figure imgf000022_0001
wherein R1 and R4, being the same or distinct, comprise an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ;
R2 compreende uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de oxigénio; e  R2 comprises an alkyl chain containing from 1 to 10 carbons or an oxygen atom; and
R3 e R5 compreendem uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de hidrogénio;  R3 and R5 comprise an alkyl chain containing from 1 to 10 carbons or a hydrogen atom;
b) fórmula II: b) formula II:
Figure imgf000023_0001
Figure imgf000023_0001
onde R1-R2, sendo os mesmos ou distintos, compreendendo uma cadeia alquílica contendo de 1 a 10 carbonos, um átomo de hidrogénio, um átomo do grupo dos halogenios, OH ou O"; e where R1-R2, being the same or distinct, comprising an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ;
R3 compreendendo uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de oxigénio;  R3 comprising an alkyl chain containing from 1 to 10 carbons or an oxygen atom;
c) fórmula III:  c) formula III:
Figure imgf000023_0002
onde R1 compreende uma cadeia alquílica contendo de 1 a 10 carbonos, um átomo de hidrogénio, um átomo do grupo dos halogênios, OH ou O"; e R2 compreende uma cadeia alquílica contendo de 1 a 10 carbonos átomo de oxigénio;
Figure imgf000023_0002
wherein R1 comprises an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ; and R2 comprises an alkyl chain containing from 1 to 10 carbons oxygen atom;
d) fórmula IV:  d) formula IV:
Figure imgf000024_0001
Figure imgf000024_0001
onde R1 e R3 podem, sendo os mesmos iguais ou distintos, compreender uma cadeia alquílica contendo de 1 a 10 carbonos, um átomo de hidrogénio, um átomo do grupo dos halogênios, OH ou O"; e wherein R1 and R3 may, being the same or distinct, comprise an alkyl chain containing from 1 to 10 carbons, a hydrogen atom, a halogen group atom, OH or O " ; and
R2 compreende uma cadeia alquílica contendo de 1 a 10 carbonos ou um átomo de hidrogénio;  R2 comprises an alkyl chain containing from 1 to 10 carbons or a hydrogen atom;
e) ou sais, solvatos e/ou hidratos farmaceuticamente aceitáveis dos mesmos.  e) or pharmaceutically acceptable salts, solvates and / or hydrates thereof.
2. Composto de acordo com a reivindicação 1 caracterizado pelo fato de que na fórmula I, R1 e R5 são átomos de hidrogénio; R2 é -CH2; R3 serem etilas, R4 é um átomo de cloro. A compound according to claim 1 wherein in formula I, R 1 and R 5 are hydrogen atoms; R 2 is CH 2; R3 are ethyl, R4 is a chlorine atom.
3. Composto de acordo com a reivindicação 1 caracterizado pelo fato de que na fórmula II, R1 é um átomo de hidrogénio; R3 é um -CH2 e R2 é uma metila na posição orto. A compound according to claim 1 characterized in that in formula II, R 1 is a hydrogen atom; R 3 is -CH 2 and R 2 is a methyl in the ortho position.
4. Composto de acordo com reivindicação 1 caracterizado pelo fato de que na fórmula III, R1 é um átomo de hidrogénio e R2 é um -CH2. A compound according to claim 1 wherein in formula III, R 1 is a hydrogen atom and R 2 is a -CH 2 .
5. Composto de acordo com a reivindicação 1 caracterizado pelo fato de que na fórmula IV, R1 é um átomo de hidrogénio, R2 é uma propila e R3 é uma metila na posição orto.  Compound according to claim 1, characterized in that in formula IV, R 1 is a hydrogen atom, R 2 is a propyl and R 3 is an ortho methyl.
6. Composição farmacêutica antimicrobiana caracterizada por compreender:  6. Antimicrobial pharmaceutical composition comprising:
- um composto selecionado do grupo compreendendo fórmula I, II, III e/ou IV descritos na reivindicação 1 , ou combinações dos mesmos; e- a compound selected from the group comprising formula I, II, III and / or IV described in claim 1, or combinations thereof; and
- um veículo farmaceuticamente aceitável. a pharmaceutically acceptable carrier.
7. Uso de um composto selecionado do grupo compreendendo fórmula I, II, III e/ou IV descritos na reivindicação 1 , ou combinações dos mesmos caracterizado por ser para a fabricação de medicamento com ação antimicrobiana.  Use of a compound selected from the group comprising formula I, II, III and / or IV described in claim 1, or combinations thereof characterized for the manufacture of medicament with antimicrobial action.
8. Ligante de inhA caracterizado por ser um composto conforme definido em qualquer uma das reivindicações 1 a 5.  InhA binder characterized in that it is a compound as defined in any one of claims 1 to 5.
9. JVIétodo . par_a_obtenção_.de_ligantes -de inhA caracterizado por compreender:  9. The method. pair_of_attention_of_high binders, characterized in that it comprises:
- avaliação in silico de potenciais ligantes de inhA;  - in silico evaluation of potential inhA ligands;
- predição in silico de toxicidade e/ou dos referidos ligantes; e  - in silico prediction of toxicity and / or said binders; and
- testes iri vitro dos referidos ligântes, notadamente estudos cinéticos e/ou de inibição de inhA.  in vitro tests of said ligands, notably kinetic and / or inhibition studies of inhA.
10. Método de acordo com a reivindicação 9 caracterizado por adicionalmente compreender ao menos uma das etapas descritas abaixo:  A method according to claim 9 further comprising at least one of the steps described below:
- seleção da biblioteca de moléculas;  - selection of the library of molecules;
- geração do modelo farmacofórico;  - generation of the pharmacophoric model;
- geração do farmacóforo 3-D e busca em bancos de dados;  - 3-D pharmacophore generation and database searching;
- modelagem molecular;  - molecular modeling;
- testes in silico de docagem molecular;  - in silico tests of molecular docking;
- predição in silico de toxicidade;  - in silico prediction of toxicity;
- medidas de inibição enzimática de MtlnhA in vitro;  - enzymatic inhibition measures of MtlnhA in vitro;
- seleção de moléculas para testes in vitro; e  - selection of molecules for in vitro tests; and
- estudo de inibição por cinética do estado estacionário.  - steady state kinetic inhibition study.
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