Pyridyl hydrazones for the treatment of tuberculosis and related diseases
09593103 · 2017-03-14
Assignee
Inventors
- Ana Lucia Almeida Ferreira (São Gregório CLD, PT)
- Ana Sofia Lopes (Loures, PT)
- Augusto Eugenio Pardal Filipe (Lisboa, PT)
- Carlos Caixado (Mafra, PT)
- Pedro Filipe Eufrasio Pedroso (Lisboa, PT)
- Susana Marques De Almeida Pecorelli (Alcabideche, PT)
Cpc classification
C07D409/12
CHEMISTRY; METALLURGY
A61K31/4436
HUMAN NECESSITIES
A61K31/4402
HUMAN NECESSITIES
International classification
C07D409/12
CHEMISTRY; METALLURGY
A61K31/4436
HUMAN NECESSITIES
Abstract
The invention provides pyridyl-hydfazones of Formula I, or pharmaceutically acceptable salts, esters, solvates, isprriers and prodrugs thereof as well as pharmaceutical compositions containing these compounds for use in the prophylactic and/or therapeutic treatment of tuberculosis and related diseases, such as, diseases caused by nontuberculous mycobacteria and/or caused by Micobacterium leprae.
Claims
1. A compound represented by the following Formula (Ib): ##STR00013## wherein R.sup.4 and R.sup.5 are each (C.sub.1-C.sub.4)alkyl, or a pharmaceutically acceptable salt or solvate, thereof.
2. The compound according to claim 1, wherein it is a pharmaceutically acceptable salt or solvate thereof.
3. A pharmaceutical composition for use in the treatment and/or prevention of tuberculosis and related diseases, as an antimicrobial drug, comprising: a compound according to claim 1; and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is at least one selected from the group consisting of binders, disintegrants, surfactants, glidants, lubricants, antioxidants, sequestrants, opacifiers or plasticizers.
4. The pharmaceutical composition according to claim 3, wherein tuberculosis and related diseases includes disease caused by Mycobacterium tuberculosis Complex (MTbC), Mycobacterium leprae and nontuberculous mycobacteria (NTM).
5. The pharmaceutical composition according to claim 3, wherein the tuberculosis related disease is selected from the group consisting of primary tuberculosis disease, post-primary pleuropulmonary tuberculosis disease, post-primary extra-pulmonary tuberculosis disease involving at least one organ or system of a mammal.
6. The pharmaceutical composition according to claim 4, wherein diseases caused by Mycobacterium tuberculosis complex (MTbC) includes infection caused by organisms of the group selected from Mycobacterium tuberculosis (Mtb), Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetii and Mycobacterium microti.
7. The pharmaceutical composition according to claim 4, wherein diseases caused by non-tuberculous mycobacteria (NTM) includes infection caused by organisms of the group selected from Mycobacterium avium complex (MAC), Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium terrae, Mycobacterium scrofulaceum, Mycobacterium vaccae, Mycobacterium marinum, Mycobacterium lentiflavum, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium intracellulare and Mycobacterium avium.
8. The pharmaceutical composition according to claim 4, wherein the treatment groups are children or elderly patients.
9. The pharmaceutical composition according to claim 4, wherein the treatment group are patients with any immunosuppressive condition.
10. The pharmaceutical composition according to claim 4, wherein the composition is in the form of a tablet, coated tablet, microcapsule, soft capsule, hard capsule, pellet, suppository, powder, solution, suspension, aerosol, syrup, drops, cream, paste, gel, ointment, tincture, lipstick or spray.
11. The pharmaceutical composition according to claim 4, wherein the treatment comprises administering the pharmaceutical composition to a mammal at a daily dosage of between 0.01 mg/kg body weight and 5000 mg/kg body weight.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
DETAILED DESCRIPTION OF THE INVENTION
(3) In one aspect of the present invention, there is provided a compound represented by the following Formula I:
(4) ##STR00006##
where Q is
(5) ##STR00007##
and A is a 5-6 member substituted or unsubstituted aryl or heteroaryl ring selected from:
(6) ##STR00008##
wherein
R.sup.1, R.sup.2 and R.sup.3 are each independently of one another, hydrogen, (C.sub.1-C.sub.4)alkyl, hydroxyl, halogen, methoxy or methoxy-acetic acid;
and
R.sup.4 and R.sup.5 are each independently of one another, hydrogen or (C.sub.1-C.sub.4)alkyl,
and pharmaceutical acceptable salts, esters, solvates, isomers and prodrugs thereof.
(7) In the context of the present invention the term (C.sub.1-C.sub.4)alkyl means methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl or tert-butyl, and the term halogen means fluorine, chlorine, bromine or iodine.
(8) In a further aspect of the present invention, there is provided compounds falling under the definition of the general Formula I and represented by the following Formula Ia and Formula Ib:
(9) Formula (Ia) compounds are represented by:
(10) ##STR00009## wherein R.sup.1, R.sup.2 and R.sup.3 have the same meaning as defined above.
(11) And, Formula (Ib) compounds are represented by:
(12) ##STR00010##
wherein R.sup.4 and R.sup.5 have the same meaning as defined above.
(13) In yet another aspect of the present invention, there are provided compounds of Formula I selected from the group consisting of: N-pyridin-2-yl-N-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine, N-pyridin-2-yl-N-5-methyl-2-hydroxyphenylmethylene-hydrazine, N-pyridin-2-yl-N-5-bromo-2-hydroxyphenylmethylene-hydrazine and N-pyridin-2-yl-N-thiophen-2-ylmethylene-hydrazine.
(14) In a further aspect of the present invention, there is provided a compound of Formula I, Ia and/or Ib, or a pharmaceutical acceptable salt, ester, solvate, isomer or prodrug thereof, useful in the treatment of a disease in a mammal, such as a human.
(15) In yet another aspect of the present invention there is provided a pharmaceutical composition comprising: (a) a therapeutically effective amount of a compound of Formula I, Ia, and/or Ib; or a pharmaceutical acceptable salt, ester, solvate, isomer or prodrug thereof, and (b) a pharmaceutically acceptable excipient useful for the treatment of a disease in a mammal, such as human.
(16) In a further aspect of the present invention, there is provided a compound of Formula I, Ia and/or Ib, or a pharmaceutical acceptable salt, ester, solvate, isomer or prodrug thereof, or a pharmaceutical composition comprising said compound for inhibiting pantothenate synthetase enzyme activity.
(17) In a yet a further aspect of the present invention, there is provided a compound of Formula I, Ia and/or Ib, or a pharmaceutical acceptable salt, ester, solvate, isomer or prodrug thereof or a pharmaceutical composition comprising said compound for preventing and/or treating tuberculosis and related diseases, such as diseases caused by nontuberculous mycobacteria and/or caused by Micobacterium leprae.
(18) The present invention is intended to encompass all pharmaceutically acceptable ionized forms (e.g., salts) and solvates (e.g., hydrates) of the compounds of Formula I, Ia and Ib, regardless of whether such forms and solvates are specified, as it is well known in the art that pharmaceutical agents in an ionized or solvated form may be used.
(19) Compounds of Formula I, Ia and Ib may contain one or more chiral centers and exist in optically active forms. When a compound of Formula I, Ia and/or Ib or a salt thereof contains a single chiral center, it may exist in two enantiomeric forms. The present invention includes individual enantiomers and mixtures of these enantiomers, which may be obtained by methods known to those skilled in the art.
(20) When a compound of Formula I, Ia and Ib or a salt thereof contains more than one chiral center it may exist in diastereomeric forms. The present invention includes each diastereomer and mixtures of these diastereomers, which may be obtained by methods known to those skilled in the art.
(21) The compounds under Formula I, Ia and Ib may form organic and inorganic salts, for example, with inorganic or organic acids, e.g., hydrochloric acid, hydrobromic acid, fumaric acid, tartaric acid, citric acid, sulfuric acid, maleic acid, acetic acid, succinic acid, benzoic acid, palmitic acid, dodecanoic acid and acidic amino acids, such as glutamic acid, alkali metal hydroxides, e.g., sodium hydroxide, with amino acids, e.g., lysine or arginine. The salts formed with compounds under Formula I, Ia and Ib, provided that they are pharmaceutically acceptable may be used in the present invention. Such salts and corresponding solvates also fall within the scope of the present invention.
(22) Prodrugs of the compounds under Formula I, Ia and Ib are also the subject of the present invention. As is known in the art, prodrugs are altered in vivo and become a compound of the present invention. All standard methods of using the compounds of the present invention are intended, whether prodrug delivery is specified, to encompass the administration of a prodrug that is converted in vivo to a compound according to the present invention.
(23) A variety of routes of administration of the compounds and compositions of the present invention are possible including, but not necessarily limited to parenteral (e.g., intravenous, intra-arterial, intramuscular, subcutaneous injection), oral (e.g., dietary or by inhalation), topical, nasal, rectal, or via slow release micro-carriers, depending on the disease or condition to be treated. Oral, parenteral and intravenous administrations are preferred modes of administration. The formulation of the compounds of the present invention to be administered will vary according to the route of administration selected (e.g., solution, emulsion, gel, aerosol, capsule). Further dosage forms according to the present invention are, for example, solutions, suspensions, ointments, creams, pastes, gels, tinctures, lip-sticks, drops, syrups, aerosols and sprays.
(24) An appropriate composition of the present invention comprising the compound or compounds of Formula I, Ia and/or Ib can be prepared in a physiologically acceptable vehicle or carrier and optional adjuvant and preservatives. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, sterile water, creams, ointments, lotions, oils, pastes and solid carriers. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers. (See Remington's Pharmaceutical Science, 16.sup.th Edition, Mack, Ed. (1980))
(25) The preferred compositions for parenteral administration are under the form of solutions, suspensions, emulsions, dispersions and lyophilized compositions of the compounds of the invention, preferably in the form of isotonic aqueous solutions, dispersions, emulsions or suspensions. These compositions are preferably sterile, either being processed in a sterile environment during their whole preparation process or by being sterilized in the end of said process. Furthermore, their manufacture is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers. These compositions may be ready to apply or be presented under solid form (for example as a lyophilizate) requiring reconstitution prior application.
(26) Parenteral compositions according to the present invention may comprise excipients, for example vehicles, stabilizers (reducing agents, anti-oxidants and/or sequestering agents), buffering agents, preservatives, isotonizing agents, emulsifiers, solubilizers, viscosity increasing agents, and/or bulking agents and are prepared by conventional processes well known to those knowledgeable of the art.
(27) Non-limiting examples of vehicles, in the context of the present invention, include water for injections, oily vehicles, polyethylene glycol, benzyl alcohol, ethanol and glycerol.
(28) Non-limiting examples of oily vehicles in the context of the present invention include fatty acid esters and mixtures of fatty acid esters, vegetable oils, synthetic oils and semisynthetic oils, almond oil, castor oil, cottonseed oil, groundnut oil, olive oil, sesame oil, and soybean oil.
(29) Non-limiting examples of reducing agents in the context of the present invention include sodium sulfite, sodium bisulfite and sodium metabisulfite.
(30) Non-limiting examples of anti-oxidants in the context of the present invention include butylated hydroxyanisole, gallic acid esters and tocopherols.
(31) Non-limiting examples of sequestering agents in the context of the present invention include ethylenediaminetetraacetic acid in the form of sodium salt (EDTA), tartaric acid, thiourea and monothioglycerol.
(32) Non-limiting examples of buffering agents in the context of the present invention include the combination of monosodium phosphate with disodium itself, trisodium phosphate, urea, sodium borate and sodium citrate.
(33) Non-limiting examples of preservatives in the context of the present invention include methylparaben, the cresols, benzyl alcohol and phenyllic alcohol.
(34) Non-limiting examples of isotonizing agents in the context of the present invention include boric acid, calcium gluconate, chlorobutanol, potassium chloride, sodium citrate, sodium borate, sodium phosphate, sodium chloride and sodium lactate.
(35) Non-limiting examples of emulsifiers in the context of the present invention include lecithins, monoglycerides, polyethylene polymers and polypropylene polymers.
(36) Non-limiting examples of solubilizers in the context of the present invention include ethanol, polypropylene glycol, N,N-dimethylacetamide or polyoxyethylene sorbitan esters.
(37) Non-limiting examples of viscosity-increasing agents in the context of the present invention include sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatins.
(38) Non-limiting examples of bulking agents in the context of the present invention include mannitol, lactose, sucrose, trehalose, sorbitol, glucose, raffinose, arginine, glycine, histidine, dextran or polyethylene glycol.
(39) Pharmaceutical oral compositions in the solid oral form (tablets, soft capsules, hard capsules or any other) according to the present invention comprise excipients, provided they are compatible with the active ingredient of the composition, including, but not limited to, diluents, binders, disintegrants, surfactants, glidants, lubricants, antioxidants or free radicals sequestrants, coating components, opacifiers or plasticizers.
(40) Soft capsules in the context of the present invention consist of gelatin or any other suitable substance containing the compounds of the invention dissolved, emulsified or suspended in a suitable soft capsule vehicle and optionally excipients such as stabilizers (reducing agents, anti-oxidants and/or sequestering agents, as defined above), solubilizers (as defined above), plasticizers or others. Hard capsules in the context of the present invention may in addition to the compounds of the invention also optionally contain excipients such as fillers, glidantes or others.
(41) Non-limiting examples of diluents in the context of the present invention include cellulose preparations, calcium phosphates, anhydrous lactose, monohydrate lactose, dihydrate lactose, sorbitol, starch, pregelatinized starch, sucrose and mannitol.
(42) Non-limiting examples of binders in the context of the present invention include sodium carboxymethyl cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, methylcellulose, hydroxypropylcellulose, povidone, a starch paste, pregelatinized starch and sucrose.
(43) Non-limiting examples of disintegrants in the context of the present invention include sodium carboxymethyl cellulose, microcrystalline cellulose, croscarmellose sodium, crospovidone, hydroxypropylcellulose, povidone, poloxamer, sodium lauryl sulfate, starch, pregelatinized starch sodium glycolate, alginic acid or a salt thereof, such as sodium alginate.
(44) Non-limiting examples of surfactants in the context of the present invention include poloxamer and sodium lauryl sulfate.
(45) Non-limiting examples of glidants in the context of the present invention include calcium silicate, starch, talc, colloidal silicon dioxide, magnesium stearate and sodium aluminum silicate.
(46) Non-limiting examples of lubricants in the context of the present invention include sodium stearyl fumarate, sodium lauryl sulfate, talc, silicic acid, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
(47) Non-limiting examples of antioxidants and free radical scavengers in the context of the present invention include butilhidroxiltoluen, butilhidroxilanisol, citric acid and citrate salts, ascorbate salts and ascorbate, alpha-tocopherol, sodium acetate, sodium sulphite and compound with organic thiol function.
(48) Non-limiting examples of coating components in the context of the present invention include concentrated sugar solutions, gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, titanium dioxide, coating solutions in suitable organic or mixed solvents, cellulose phtalates (acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate).
(49) Non-limiting examples of plasticizers in the context of the present invention include glycerol and sorbitol.
(50) Non-limiting examples of soft capsule vehicles in the context of the present invention include fatty oils, paraffin oil, liquid polyethylene glycols or ethylene/propylene glycol fatty acid esters.
(51) Suppositories according to the present invention comprise a compound of the present invention admixtured with a suppository base and optionally further excipients.
(52) Non-limiting examples of suppository bases in the context of the present invention include natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols (alkanols with at least eight carbon atoms).
(53) The term pharmaceutically acceptable carrier as used in the present invention includes any solvent, dispersion media, coatings, antibacterial, and antifungal agents, isotonic and absorption delaying agents, and the like which are compatible with the activity of the compounds and are physiologically acceptable to the subject.
(54) Effective amount a used in the present invention includes the amount of the compound or a pharmaceutically acceptable salt thereof, ester, isomer, solvate, or prodrug thereof which allows it to perform its intended function, i.e., prevention of onset or treatment of tuberculosis or related diseases such as caused by nontuberculous mycobacteria and/or caused by Mycobacterium leprae.
(55) A therapeutically effective amount of the active substance of the present invention can be administered by an appropriate route in a single dose or multiple doses.
(56) The therapeutically effective amount will depend upon a number of factors, including biological activity, mode of administration, frequency of treatment, type of concurrent treatment, if any, age, body weight, sex, general health, severity of the condition to be treated, as well as appropriate pharmacokinetic properties. One skilled in the art can determine the appropriate dosage based on the above factors.
(57) The compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, satisfactory results may be obtained when the compounds of the invention are administered to a human at a daily dosage of between 0.01 mg and 5000 mg (measured as the solid form). A preferred dose ranges between 0.01 and 750 mg/Kg, more preferably between 0.05 and 150 mg/Kg.
(58) The compound can be administered in the form of pharmaceutical compositions comprising the compound once a day or at different times within the day, prophylactically or therapeutically, preferably in an amount effective against tuberculosis or the related disease, to a mammal, for example a human, requiring such treatment. In the case of an individual having a bodyweight of about 70 kg, the daily dose of the mixture administered is from approximately 0.01 g to approximately 50 g, preferably from approximately 0.05 g to approximately 10 g, of a compound of Formula I, Ia and/or Ib.
(59) The pharmaceutical compositions of the present invention comprise from approximately 5% to approximately 95% of a mixture of a compound of formula I.
(60) The pharmaceutical compositions of the present invention may, if desired, be formulated so as to provide an immediate or modified release of the active ingredient after administration to the patient.
(61) Unit dose administration forms according to the present invention comprise from approximately 20% to approximately 90% of the compound of formula I, and forms that are non-unit dose type from approximately 5% to approximately 20% of the mentioned compound. Unit dose forms according to the present invention refer to, for example, coated and uncoated tablets, microcapsules, soft and hard capsules, pellets, powdered doses, ampoules, vials and suppositories.
(62) The present invention relates especially to the use of a compound of formula I, Ia and/or Ib or a pharmaceutical acceptable salt, ester, isomer, solvate and/or prodrug, as such or in the form of a pharmaceutical formulation with at least one pharmaceutically acceptable carrier for the therapeutic and also prophylactic treatment of tuberculosis.
(63) The pharmaceutical compositions of the invention are not only useful for the prevention and treatment of tuberculosis, i.e. a disease caused by Mycobacterium tuberculosis complex (MTbC), but also for the treatment of diseases caused by related bacteria, in particular Mycobacterium leprae, and caused by nontuberculous mycobacteria (NTM).
(64) Mycobacterial diseases are caused by organisms of the Mycobacterium tuberculosis complex (MtbC) like Mycobacterium tuberculosis (Mtb), Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetii and Mycobacterium microti. Mycobacteria other than MtbC and Mycobacterium leprae are known as non-tuberculous mycobacteria (NTM) and can cause also human and animal diseases as is the case for Mycobacterium avium complex (MAC), Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium terrae, Mycobacterium scrofulaceum, Mycobacterium vaccae, Mycobacterium marinum, Mycobacterium lentiflavum, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium intracellulare and Mycobacterium avium.
(65) As above mentioned, multidrug-resistant TB (MDR-TB) is defined by resistance to the two most commonly used drugs in the current four-drug (or first-line) regimen, INH and RMP. Drug-resistant TB is the man-made result of interrupted, erratic, or inadequate TB therapy, and its spread is undermining efforts to control the global TB epidemic. Multidrug-resistant (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) develop when the long, complex, decades-old TB drug regimen is improperly administered, or when people with TB stop taking their medicines before the disease has been fully eradicated from their body. Extensively drug resistant tuberculosis (XDR-TB) is defined as TB that is resistant to any fluoroquinolone, and at least one of three injectable second-line drugs (capreomycin, kanamycin, and amikacin), in addition to INH and RMP.
(66) The pharmaceutical compositions of the invention are effective in the treatment and prevention of all forms of tuberculosis such as primary tuberculosis disease, post-primary pleuropulmonary tuberculosis disease, post-primary extra-pulmonary tuberculosis disease involving at least one organ or system such as, but not restricted, to lymph nodes, kidney, central nervous system, osteoarticular systems, gastrointestinal system tract, eye, skin and soft tissues or urogenital system, disseminated tuberculosis and reactivated tuberculosis.
(67) The pharmaceutical compositions of the invention are effective in the treatment and prevention of all forms of tuberculosis in adults, children and elderly patients, with or without immunodepression conditions such as, but not restricted to, diabetes mellitus, chronic renal insufficiency, malnutrition, alcoholism, human immunodeficiency virus infection (HIV)/acquired immunodeficiency syndrome (AIDS), silicosis, paracoccidioidomycosis, leukemias, solid tumors, immunosuppressive drug treatments and hereditary diseases or syndromes.
(68) The pharmaceutical compositions of the invention are effective in the treatment and prevention of diseases caused by non-tuberculous mycobacteria that included at least one organ or system such as, but not restricted to, lungs and endobronchial tree, lymph nodes, kidney, central nervous system, osteoarticular system, gastrointestinal system, eye, skin and soft tissues, urogenital system, disseminated and reactivated forms of disease.
(69) The pharmaceutical compositions of the invention are effective in the treatment and prevention of diseases caused by nontuberculous mycobacteria in adults, children and elderly patients, with or without immunosuppressive conditions such as, but not restricted to, diabetes mellitus, chronic renal insufficiency, malnutrition, alcoholism, human immunodeficiency virus infection (HIV)/acquired immunodeficiency syndrome (AIDS), leukemias, solid tumors, immunosuppressive drug treatments and hereditary diseases or syndromes.
(70) The pharmaceutical compositions of the invention are effective in the treatment and prevention of diseases caused by Mycobacterium leprae that included at least one organ or system such as, but not restricted to, skin and soft tissues, urogenital system and central nervous system, disseminated and reactivated forms of disease.
(71) The pharmaceutical compositions of the invention are effective in the treatment and prevention of diseases caused by Mycobacterium leprae in adults, children and elderly patients, with or without immunosuppressive conditions such as, but not restricted to, diabetes mellitus, chronic renal insufficiency, malnutrition, alcoholism, human immunodeficiency virus infection (HIV)/acquired immunodeficiency syndrome (AIDS), leukemias, solid tumors, immunosuppressive drug treatments and hereditary diseases or syndromes.
(72) Obtaining the Compounds of the Present Invention
(73) All compounds are available at Chembridge Corporation (www.chembridge.com), however the skilled technician can easily obtain them by applying various synthetic methods described in the literature:
(74) ##STR00011##
Method 1) for Obtaining Compounds of Formula I, in Particular, Compounds of Formula (Ia)
(75) Reaction of 2-chloropyridine with hydrazine hydrate at a reflux temperature results in the formation of 2-hydrazinopyridine. Further condensation with different aldehydes (for instance, in ethanol at a reflux temperature) gives the corresponding hydrazone. The synthesis method is described in Chaur, Manuel N. et al.; ChemistryA European Journal; vol. 17; nb. 1; (2011); p. 248-258; Padalkar, Vikas S. Et al.; Synthetic Communications; vol. 41; nb.6; (2011); p. 925-938; Fargher; Furness; Journal of the Chemical Society; vol. 107; (1915); p.695
(76) Method 2) for Obtaining Compounds of Formula I, in Particular Compounds of Formula (Ib)
(77) These compounds can be obtained with good overall yield from 2-aminopyridine which is transformed in the corresponding 2-bromo derivative by treatment with bromine and bromidric acid. The 2-bromo compound obtained is then treated in SNAr conditions with hydrazine hydrate to provide the desired 2-hydrazinepyridine derivative. Finally, the compounds are prepared by condensation of the 2-hydrazinepyridine derivative (in acidic ethanolic solution) at room temperature with the appropriated heteroarylaldehyde, producing the desired compounds in good yield. The synthesis method is described in Todeschini, Adriane R. et al.; European Journal of Medicinal Chemistry; vol. 33; nb.3; (1998); p. 189-200.
(78) Obtaining the Pharmaceutical Compositions of the Present Invention
(79) The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving, emulsifying or lyophilizing processes. Optionally, the manufacture of the compositions according to the present invention includes more steps such as liposomal encapsulation.
(80) In particular, a tablet may be made by compression and molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound of the present invention in a free-flowing form, e.g., a powder or granules, optionally mixed with ingredients, such as, binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.
(81) In particular, a syrup or suspension may be made by adding the active compound of the present invention to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which also any accessory ingredient may be added. Such accessory ingredients may include, flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.
(82) Formulations for rectal administration may be made with a conventional carrier, e.g., cocoa butter or Witepsol S55 (commercial registered trademark). Specific details related to particular aspects of conventional processes of galenic development can be found in Swarbrick and Boylan's Encyclopedia of pharmaceutical technology (1988-2001 NY, Published by M. Dekker).
(83) Alternatively, the compounds of the present invention may be made in liposomes or microspheres (or microparticles), such methods essentially comprising dissolving the compounds of the present invention in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary. Liposomal encapsulation techniques detailed in Claudio Nastruzzi's book Lipospheres in drug targets and delivery: approaches, methods, and applications (Boca Raton 2005, published by CRC Press) and in Lasic and Papahadjopoulos' Lipospheres in drug targets and delivery: approaches, methods, and applications (1998 Amsterdam, N.Y., Published by Elsevier).
(84) Development and Validation of a High-Throughput Screening (HTS) Platform
(85) Regarding the experimental phase, the present invention included the development and validation of a high-throughput screening (HTS) platform that involves the targeting of pantothenate synthetase, an enzyme essential to the metabolic pathways of Mycobacterium tuberculosis, by using small molecules as potential inhibitors of this enzyme.
(86) The HTS platform was developed, validated and used to screen 50.000 compounds that were obtained from a diverse chemical library supplied by a vendor called Chembridge.
(87) The HTS platform consists in a yeast strain genetically modified to express the Mycobacterium tuberculosis's pantothenate synthetase gene (PanC) instead of the regular Pan6 of the wild-type yeast. Additionally, this PanC yeast strain is completely dependent on the activity of the expressed pantothenate synthetase to survive.
(88) The validation of the platform demonstrated that (in normal conditions): Wild-type yeast (Control)shows normal growth; Yeast without Pan6shows reduced growth; Yeast without Pan6 and complemented with PanCshows normal growth (similar to control); Yeast without Pan6 and complemented with Pan6shows normal growth (similar to control which proves that the genetic intervention does not compromise the metabolic processes of the yeast).
(89) Therefore, a potential inhibitor of the pantothenate synthetase enzyme would result in a reduced growth of the PanC yeast strain.
(90) At the end of this phase the obtained results allowed the classification of a ranking of compounds that included the most potent in inhibiting the growth of the yeast that carried the target, pantothenate synthetase of Mycobacterium tuberculosis.
(91) Based on the data obtained in the screening assay, a ranking of compounds was established and the top compounds were selected based on a curve fitting approach that allowed the determination of inhibitory concentrations (IC.sub.50 and IC.sub.90) for each screened compound.
(92) For each compound, a concentration-response curve was created using a four parameter logistic fit (available in the curve-fitting software IBDS XLfit curve fitting software) that allowed the calculation of 10.sub.50 and 10.sub.90 values for each compound.
(93) Additionally, an in vitro assay using Mycobacterium smegmatis (as a surrogate for anti-Mycobacterium tuberculosis activity) was developed, implemented and used to screen the compounds of interest in an experimental assay. It is important to mention that Mycobacterium smegmatis is considered to be a valuable tool for screening of compounds potentially active against Mycobacterium tuberculosis, not only because of its non-pathogenic profile but also because of the known gene homology between the two mycobacteria species (Andries K et al., A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science. 2005; 307:223-227; Chatuverdi V et al., Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis. J Gen Appl Microbiol. 2007; 53:333-337; Cho Y, Ioerger T R and Sacchettini. Discovery of Novel Nitrobenzothiazole Inhibitors for Mycobacterium tuberculosis ATP phosphoribosyl Transferase (HisG) through virtual screening. J Med Chem. 2008; 51:5984-5992).
(94) The experimental assay with Mycobacterium smegmatis involves the incubation of the test items and the control items with Mycobacterium smegmatis in 5 concentrations (1.24, 3.7, 11, 33 and 100 M) for 96 hours. In terms of control items, a blank control that will exhibit maximum growth and a positive control that will exhibit inhibited control are used in this assay. The microorganism growth is evaluated by the measurement of the suspension turbidity: the comparison between negative control and compound or positive control turbidity allowed for the evaluation of the growth inhibition due to the compound presence. The positive control compound used was rifampicin (RMP) that was determined to be the most effective against this strain of Mycobacterium smegmatis. Additionally, it is important to notice that RMP is a first-line treatment drug used against Mycobacterium tuberculosis in the clinical setting.
(95) After obtaining the concentration-response data for each of the compounds tested in this Mycobacterium smegmatis screening assay, a curve fitting approach based on a four parameter curve fit, allowed for the determination of inhibitory concentrations values such as the IC.sub.50 that represents the required concentration to inhibit 50% of the Mycobacterium smegmatis growth. This assay had the objective of confirming the superior inhibitory activity of the compounds of the present invention over those known from the prior art, such as RMP.
(96) The results obtained through these assays demonstrate that the following compound: 3,5-dibromo-2hydroxybenaldehyde (2-pyridinyl)hydrazine, denominated compound 42, show a superior inhibitory activity against Mycobacterium smegmatis than the prior art compound RMP.
(97) Further and in order to define analogues of the compound N-pyridin-2-yl-N-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine (compound 42) with the desired pharmacological activity, fingerprint and pharmacophore searching methods were carried out using browser-based search engines. In the case of the fingerprint searching method, 2D substructural fragments of N-pyridin-2-yl-N-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine (compound 42) were used to search online databases for other molecules that share the same 2D substructures and therefore calculate the similarity of both molecules as a function of the number of fragments that they have in common. For the pharmacophore search, the compound N-pyridin-2-yl-N-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine (compound 42) was used as the pharmacophore model, including the correspondent pharmacophoric pattern that involves its ensemble of steric and electrostatic features, and online databases with pre-computed conformations were searched for analogues. For both methodologies, only the analogues with a similarity higher than 90% were chosen and tested in the same Mycobacterium smegmatis assay described above.
(98) The results obtained from this assay locate additional compounds falling under the formula I, Ia and Ib bearing the desired pharmacological activity. These compounds are in particular: N-pyridin-2-yl-N-5-methyl-2-hydroxyphenylmethylene-hydrazine, N-pyridin-2-yl-N-5-bromo-2-hydroxyphenylmethylene-hydrazine and N-pyridin-2-yl-N-thiophen-2-ylmethylene-hydrazine.
EXAMPLES
(99) The invention will now be further described by the following working examples, which are preferred embodiments of the invention. These examples are illustrative rather than limiting and it is to be understood that there may be other embodiments that fall within the spirit and scope of the invention as defined by the claims appended hereto.
Example 1
Identification of Compounds with Pantothenate Synthetase Inhibiting Activity
(100) The screening step in the HTS platform consisted in the incubation of the library compounds (50.000 diverse and publically available chemical entities) with the yeast complemented with PanC and the comparison of its growth at the end of 72 hours with the growth observed in the control yeast. This was performed in a high-throughput manner using robotized procedures and 96-wells plaques for the compound and yeast incubation. The screening step involved the use of 5 concentrations of each compound (0.5, 1, 5, 10 and 20 M). The comparison of the growth observed for the control yeast with the growth observed for each screened compound allowed the determination of growth inhibition percentage for each tested concentration.
(101) At the end of this phase the obtained results allowed the classification of a ranking of compounds that included the most potent in inhibiting the growth of the yeast that carried the target, pantothenate synthetase of Mycobacterium tuberculosis.
(102) From this ranking of compounds, the top compounds were selected based on a curve fitting approach that allowed the determination of inhibitory concentrations (IC.sub.50 and IC.sub.90) for each screened compound.
(103) For each compound, a concentration-response curve was created using a four parameter logistic fit (available in the curve-fitting software IBDS XLfit curve fitting software) that allowed the calculation of IC.sub.50 and IC.sub.90 values for each compound. The fit quality of each curve was determined by the calculation of curve fit quality values (r.sup.2).
Example 2
In Vitro Assay with Mycobacterium smegmatis for Screening Compounds with Anti Mycobacterium tuberculosis Activity
(104) Each of the compounds tested and the positive control were incubated with Mycobacterium smegmatis in 5 concentrations (100, 33, 11, 3.7 and 1.24 M) for 96 hours. Mycobacterium smegmatis incubation was performed in suspension mode, without agitation, in Middlebrook 7H9 media supplemented with ADC, at 37 C. The microorganism growth was evaluated by the measurement of the suspension turbidity: the comparison between negative control and compound or positive control turbidity allowed for the evaluation of the growth inhibition due to the compound presence. The positive control compound used was RMP that was determined to be the most effective against this strain of Mycobacterium smegmatis. Additionally, it is important to notice that RMP is a first-line treatment drug used against Mycobacterium tuberculosis in the clinical setting.
(105) Based on the results, which are shown in
Example 3
Obtaining Analogues of Most Effective Compound
(106) A series of analogues of the compound, 3,5-dibromo-2-hydroxybenzaldehyde(2-pyridinyl)hydrazine (compound 42) were obtained through fingerprint and pharmacophore searching methods and only the analogues with a similarity higher than 90% were chosen and tested in the same Mycobacterium smegmatis assay described above. The following table comprise some examples of compounds representative of the whole serie:
(107) TABLE-US-00001 TABLE 1 Example of Compounds of Formula Ia
(108) Table 2 and
(109) TABLE-US-00002 TABLE 2 Mycobacterium Smegmatis growth inhibition results Concen- % of Celular Growth at 96 hours tration Compound Compound Compound (M) Rifampicin 61 42 70 100.00 22.26% 24.45% 31.91% 38.9% 33.33 58.04% 20.08% 41.52% 44.2% 11.11 90.62% 83.84% 98.17% 80.0% 3.70 81.40% 69.76% 104.48% 96.6% 1.23 82.55% 75.35% 110.06% 95.9%
Example 4
Measurement of IC50 Value
(110) The inhibitory effect of a compound can be described by an IC.sub.50 value, that is the concentration of inhibitor at which half (50%) inhibition of the maximal (100%) inhibition occurs. IC.sub.50 values were determined by measuring the extent of inhibition over a range of concentrations of the compounds of interest, preferably a range where the degree of inhibition varied from no inhibition (0%) to complete inhibition (100%). The IC.sub.50 value can be estimated from a plot of % inhibition against a concentration of inhibitor, or can be calculated using data fitting programs, such as IDBS XLfit.
(111) Based on the data obtained in the Mycobacterium smegmatis screening assay, a curve fitting approach allowed for the determination of inhibitory concentrations values such as the IC.sub.50 that represents the required concentration to inhibit 50% of the Mycobacterium smegmatis growth. The lower the IC.sub.50, the higher the potency of the compounds to inhibit the Mycobacterium smegmatis. The IC.sub.50 values obtained for specific compounds under Formula Ia were depicted in Table 3:
(112) TABLE-US-00003 TABLE 3 IC50 Values Compound IC.sub.50 (M) Rifampicin 24.82 Compound 61 13.54 Compound 42 15.18 Compound 70 13.87
(113) These results showed that the compounds representative of the present invention (compound n 42 and its analogues, compounds n 61 and 70) exhibit better IC.sub.50 values than RMP which translate into more potency for Mycobacterium smegmatis inhibition.
(114) As the results demonstrate surprisingly, the compounds representative of the present invention exhibit better IC.sub.50 values and more potency than the active control, RMP, thereby confirming its superiority in terms of inhibition of Mycobacterium smegmatis.
(115) Lisbon, Nov. 19, 2013.