WO2010043010A1 - N-acyl hydrazine derivatives, method for producing n-acyl hydrazine compounds, pharmaceutical compositions containing same, uses thereof and methods for treatment - Google Patents

N-acyl hydrazine derivatives, method for producing n-acyl hydrazine compounds, pharmaceutical compositions containing same, uses thereof and methods for treatment Download PDF

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WO2010043010A1
WO2010043010A1 PCT/BR2009/000320 BR2009000320W WO2010043010A1 WO 2010043010 A1 WO2010043010 A1 WO 2010043010A1 BR 2009000320 W BR2009000320 W BR 2009000320W WO 2010043010 A1 WO2010043010 A1 WO 2010043010A1
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compound
human
treatment
formula
alkyl
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French (fr)
Portuguese (pt)
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Carlos Alberto Manssour Fraga
Eliezer Jesus De Lacerda Barreiro
Roberto Takashi Sudo
Hallen Jannisy Vieira Beirai
Arthr Eugen KÜMMERIE
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Universidade Federal Do Rio De Janeiro
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to compounds of the N-acylhydrazone family capable of activating nitric oxide (NO) biosynthesis in mammalian blood vessels and acting as muscle relaxants.
  • the present invention also relates to the process of preparing said N-acylhydrazonic compounds, pharmaceutical compositions containing such compounds and their uses, and methods of treatment involving the administration of these compounds to human or non-human mammals.
  • AH Hypertension
  • Endothelial cells play a relevant role in the control of vascular tone, regulating vasomotricity, vascular permeability, metabolism of endogenous and exogenous substances and platelet and leukocyte activity (Zanesnco and Antunes, 2005). Furchgott and Zawasdski (1980) were the first researchers to demonstrate the importance of endothelium in vascular tone control, reporting ACh (muscarinic agonist) -induced vasodilation dependent on the presence of a functional endothelium, and that endothelial cells released a relaxation factor. .
  • Endothelium-derived relaxing factors may be: nitric oxide (NO), prostacyclin (PGfe), and hyperpolarizing factor endothelium derivative (EDHF) (Furchgott and Zawasdski, 1980; Vanhoutte, 2003).
  • NO is a potent vasodilator and thus its role in blood pressure control is extremely relevant (Moncada et al 1991; Vanhoutte 2003).
  • LASSBio-294 has been described as a cardiac positive inotropic agent (Sudo et al., 2001) with vasodilatory property (Silva et al., 2002), and also capable of promoting increased contractility of both frog skeletal muscles (Gonzalez-Serratos et al., 2001) and rat (Zapata-Sudo et al., 2003).
  • the increase in cardiac contractility caused by LASSBio-294 was not related to the activation of ⁇ -adrenergic receptors, since the increase in muscle shake amplitude was not modified with pretreatment of propranolol, a non-selective blocker of these receptors.
  • LASSBio-294 could be increasing the sensitivity of Ca 2+ contractile proteins in the heart muscle was ruled out due to no change in the [Ca 2+ ] curve versus stress in denuded heart fibers (Sudo et al 2001). .
  • the probable mechanism of action of LASSBio-294 was attributed to higher Ca 2+ accumulation in the sarcoplasmic reticulum (RS) due to the higher Ca 2+ uptake by this organelle.
  • cAMP cyclic adenosine monophosphate
  • LASSBio-294 also reversed both norepinephrine and KCI-induced aortic ring contracture (Silva et al., 2002), an effect observed in preparations with intact endothelium.
  • the involvement of prostanoid release by LASSBio-294 to explain the effect of vascular relaxation was discarded as relaxation remained unchanged even in the presence of a cyclooxygenase inhibitor.
  • Reversal of relaxation by guanylate cyclase inhibition suggested the involvement of cGMP in the mechanism of LASSBio-294 in promoting vasodilation.
  • Figure 1 shows the effect of LASSBio-1289 on the inhibition of 10 ⁇ 10 phenylephrine-induced rat aorta segment contracture with and without preserved endothelium at cumulative concentrations (0.05-1 ⁇ ).
  • Figure 2 shows the relaxation-dependent concentration of intact endothelial aorta rings caused by the LASSBio-897 derivative following 10 ⁇ phenylephrine-induced contracture. Data represent the mean ⁇ SEM of 6 experiments. * P ⁇ 0.05 vs control.
  • Figure 3 shows the effect of LASSBio-897 and 1029 on cGMP production in endothelial aortic rings of normotensive rats, - * * P ⁇ 0.05.
  • LASSBio-897 and 029 vs DMSO.
  • # LASSBio-897 and 029 vs sildenafil citrate.
  • Figure 4 shows the effect of LASSBio-897 on indirect quantitation of nitric oxide in endothelial aorta rings of hypertensive rats in the absence and presence of L-NAME and Atropine.
  • # P ⁇ 0.05 * LASSBio-897 vs Aorta and DMSO. # L-NAME + LASSBio-897 and Atropine + LASSBio-897 vs LASSBio-897.
  • Figure 5 shows the effect of LASSBio-897 on indirect quantitation of nitric oxide in endothelial aorta rings of normotensive rats in the absence and presence of L-NAME and Atropine. * # P ⁇ 0.05. * LASSBio-897 vs Aorta and DMSO. # L-NAME + LASSBio-897 and Atropine + LASSBio-897 vs LASSBio-897.
  • Figure 6 shows the comparative effect between LASSBio-897 in the presence and absence of atropine on 10 ⁇ phenylephrine-induced aortic ring contracture.
  • Figure 7 A and B show the effect of LASSBio-897 on 10 ⁇ phenylephrine-induced endothelial aortic ring contracture in the presence of L-NAME (A) and ODQ (B). * P ⁇ 0.05. Data represent the mean ⁇ SEM of 6 experiments. * Endothelial aorta vs pre-incubated aorta with L-NAME (100 ⁇ ) and ODQ (10 ⁇ ).
  • Figure 8 shows the representative record of 10 ⁇ fen phenylephrine-induced rat aortic segment contracture of endothelium in the presence of L-NAME. After the plateau is reached, cumulative concentrations (0.05-1 ⁇ ) of LASSBio-897 were added.
  • Figure 9 shows the representative record of endothelial rat aortic segment contracture induced by 10 ⁇ phenylephrine in the presence of L-NAME (A) and ODQ (B). After the plateau is reached, cumulative concentrations (0.05-1 ⁇ ) of LASSBio-897 were added.
  • Figure 10A and B show the effect of LASSBio-897 on PS (A) and PD (B) at doses of 1 and 5 mg / kg during 14-day treatment of intraperitoneally normotensive rats. Data represent the mean measurements of PS and PD in mmHg ⁇ SEM of 6 animals.
  • Figure 11 shows the effect of LASSBio-897 on PS (A) and PD (B) at doses of 1 and 5 mg / kg during 14-day treatment of intraperitoneally hypertensive rats.
  • Data represent the mean measurements of PS and PD in mmHg ⁇ SEM of 6 animals. * P ⁇ 0.05 when compared to DMSO.
  • vascular endothelium capable of inducing nitric oxide biosynthesis through activation of muscarinic receptors present in the vascular endothelium thus causing, among other effects, muscle relaxation in human and non-human mammals, in a muscle group comprising smooth muscle and striated muscle.
  • skeletal preferably, relaxation occurs in the smooth muscle and; more preferably still in the vascular smooth muscle.
  • a second object of the present invention is a process for producing said V-acylhydrazonic derivative compounds of molecular formula.
  • Another object of the present invention is a pharmaceutical composition containing said derivative / V-acylhydrazonic compounds of
  • non-pharmaceutically acceptable compounds active in the treatment of disorders related to nitric oxide synthesis such as erectile dysfunction, hypertension, angina, myocardial infarction, insufficiency heart disease, pulmonary hypertension among others in human or non-human mammal.
  • compositions containing such V-acylhydrazonic derivative compounds of molecular formula (I) are also an object of the present invention.
  • Another object of the present invention is the use of the V-acylhydrazonic derivative compounds of molecular formula (I).
  • Another object of the present invention is the use of the V-acylhydrazonic derivative compounds of molecular formula (I).
  • the seventh object of the present invention is a method of treating diseases and dysfunctions linked to nitric oxide biosynthesis based on the administration of a compound of formula (I).
  • V-acylhydrazonic derivative compounds which are the subject of this invention have the general formula (I) below.
  • R1 and R2 may be H, alkyl group of 1 to 6 carbon atoms, or phenyl; wherein such radicals may further be optionally substituted, unsaturated and / or branched;
  • R3, R4 and R5 may be H, alkyl group of 1 to 6 carbon atoms, phenyl, amino (secondary, tertiary or quaternary), nitro, ester [RCOO-], acid [COOH], hydroxyl [-OH], alkoxy [-OR], halide [F, Cl, Br], cyano [-CN], sulfonamide [-SO 2 NH 2 ] or methylsulfone [-SO 2 CH 3]; wherein such radicals may further be optionally substituted, unsaturated and / or branched.
  • R1 and R2 may be H or an alkyl group containing 1 to 6 carbon atoms and;
  • R3, R4 and R5 may be H, alkyl group containing from 1 to 6 carbon atoms phenyl and substituted phenyl, amino (secondary, tertiary or quaternary), nitro, RCOO-, COOH, hydroxyl, alkoxy, halide, cyano, sulfonamide or methyl sulfone wherein such radicals may be optionally substituted, unsaturated and / or branched.
  • the compounds described herein have activity dependent on endothelial cell functionality and no cardiac inotropic activity; besides having tissue selectivity and differentiated pattern of substitution in its molecule when compared to the other N-acylhydrazonic derivatives contained in the state of the art.
  • V-acylhydrazonic derivatives of this invention have great potency, being up to 200 times more effective than the other V-acylhydrazonic derivatives belonging to the state of the art.
  • the said W-acylhydrazonic derivatives objects of this invention are not capable of altering the isometric tension of the cardiac muscle and tracheal smooth muscle, being effective, however, in the alteration of the isometric tension of the vascular smooth muscle, a fact that confers to the N-acylhydrazonic derivatives derived objects. of this invention a high ability to promote blood pressure reduction in human or non-human mammals.
  • Step a) occurs by reacting the piperonal to a solution containing an alcohol, a strong base and a halogen, occurring at a temperature between -10 ° C to + 25 ° C for a period of time between 20 to 240 minutes.
  • an ester of general formula (IV) is formed:
  • step a occurs at a temperature between -5 ° C to + 10 ° C within a maximum of 120 minutes, followed by step b.
  • step b The hydrazinolysis of ester (IV) occurs in step b by the addition of ester (IV) in an alcoholic solution containing hydrazine in a condensing apparatus with a temperature between 50 ° C and 130 ° C.
  • step b takes place in a reflux condenser between 70 ° C to 100 ° C.
  • the pH is adjusted and the solution is cooled to a temperature between -5 ° C to + 15 ° C to precipitate the compound.
  • step c the compound of formula (II) is condensed by the reaction of compound (II) with compounds of the 3-acylthiophenes family, functionalized at R 2 and / or R 3 and / or R and / or R 5.
  • the groups R2, R3, R and R5 are functionalized independently or together, resulting in the formation of the compound of formula (III).
  • the condensation reaction occurs under stirring in the presence of a primary or secondary alcohol, preferably a primary alcohol such as ethanol, at room temperature at acidic pH.
  • Compound III is then alkylated by the addition of the desired alkyl halide. This reaction promotes the addition of a regioselective alkyl radical and a saturated or unsaturated and / or branched alkyl may be employed.
  • said diseases and / or dysfunctions related to nitric oxide synthesis are all those related to insufficient NO production, such as erectile dysfunction, arterial hypertension, angina, myocardial infarction, heart failure and pulmonary hypertension among others.
  • compositions for the treatment of muscle disorders in human and / or non-human mammals.
  • the muscle dysfunctions contemplated by this invention are those related to muscles belonging to the tissue group, such as cardiac muscle, smooth muscle and skeletal muscle.
  • the pharmaceutical compositions of this invention may be employed in promoting smooth muscle relaxation.
  • non-active compounds which may be found in said pharmaceutical compositions are all those known to one of ordinary skill in the pharmaceutical art, such as adjuvants, stabilizers, preservatives, diluents, pH adjusters, colorants, flavorings, thickeners and the like.
  • V-acylhydrazonic compounds of formula (I) are also provided by this invention.
  • Another intended use of the present invention is the use of the V-acylhydrazonic compounds of formula (I)
  • Also provided by the present invention is a method of treating diseases and disorders associated with nitric oxide synthesis based on the administration of a compound of formula (I)
  • the last object described by the present invention is a method of treatment, muscle disorders and dysfunctions characterized by the administration of the compound of formula (I)
  • thoracic aortic rings dissected in a modified Tyrode nutrient solution were used. After removal of the adipose and connective tissues surrounding the vessel, rings measuring between 2 and 3 mm were cleaved and placed in the experimental vessel and the tension generated by the muscle was transformed into a digitized electrical signal (Digidata 1322A) and stored in a computer for further analysis using the axoscope 8.0 program (Axon Instruments. Inc). The rings were only mounted on rods as shown in the diagram below, allowing the measurement of transverse tension.
  • the first phase of the experiment began, which was to verify the integrity of the endothelium function.
  • vascular For this, the aortas were contracted with 10 ⁇ phenylephrine (Fe) and then exposed to 10 ⁇ acetylcholine (ACh) (Kaya et al., 2003). Endothelial integrity was confirmed in those aortic rings where relaxation obtained after Fe-induced contracture was> 80%. However, the absence of endothelium was considered when relaxation ⁇ 10%.
  • the preparation was washed for 30 min with the nutrient solution and a new contracture was induced by exposure to 10 ⁇ Fe. After the formation of the maximum Fe-induced contracture plateau, increasing concentrations of the derivatives as well as DMSO were added to the preparations.
  • EXAMPLE 2 Derivative-induced aorta relaxation
  • the vasodilatory activity of the LASSBio-1289 derivative was investigated in preserved endothelial aortic rings.
  • aortic rings were pre-contracted with 10 ⁇ phenylephrine and then After the contracture plateau, these rings were exposed to 10 ⁇ ACh.
  • the relaxation observed in the presence of ACh indicated the functional integrity of the endothelium.
  • increasing concentrations of the derivative were added to the experimental wells soon after the formation of the phenylephrine-induced contracture plateau.
  • LASSBio-1289 in phenylephrine pre-contracted functional endothelial aortic rings (10 ⁇ ) was able to produce concentration-dependent relaxation.
  • the mean inhibitory concentration (IC50) ie the concentration required to reduce by 50% the maximum phenylephrine-induced contracture was 0.69 ⁇ 0.18 ⁇ for LASSBio-1289 ( Figure 2).
  • cGMP concentration For the determination of cGMP concentration, the aortic rings were homogenized in trichloracetic acid (6%) and the homogenate was centrifuged (2000xg for 15 min at 4 ° C). The pellet was used to determine protein content according to the method adapted for insoluble proteins (Lowry et al 1951). The supernatant was washed four times with five volumes of water-saturated ethyl ether. The upper layer was discarded after each wash. The aqueous phase was then evaporated by drying (Delpy et al., 1996).
  • the dried extract was dissolved in an appropriate volume of the assay buffer and the cGMP content was determined colorimetrically using an Enzymeimmunoassay Biotrak (EIA) System cGMP kit. Positive control was sildenafil citrate (0, 1 ⁇ ) and negative control the vehicle at the volume in which the derivatives were dissolved (DMSO). Data were expressed in fmol / pg- 1 cGMP protein ( Figure 3).
  • NO production by the aorta was analyzed by measuring nitrite accumulation in the culture medium using the Gr ⁇ ess reaction (Green et al., 1982; Ding et al., 1988).
  • Thoracic aorta of normotensive and SHR Wistar rats weighing between (250 to 300 g) were dissected in phosphate buffered saline (PBS), pH 7.2, 37 ° C, where the connective tissues were removed so as not to damage the endothelium. .
  • PBS phosphate buffered saline
  • the aorta was then segmented into approximately 2-3 mm rings and each ring was placed into a well of a 24 well plate already containing 500 ⁇ PBS. After this procedure, the aorta rings were washed three times with PBS and after the last wash DMEM medium was incubated in each well of the plate.
  • the medium was exchanged for one containing ACh (10 ⁇ ), collected 50 ⁇ from each well and placed in a 96-well plate; 2.
  • the 24 well plate was again washed three times with PBS and each well was incubated with medium containing the derivatives LASSBio-897 (0.5 and 1 ⁇ ), LASSBio-1029 (8 and 50 ⁇ ), their respective vehicles ( DMSO) and pharmacological tools of interest as well as atropine (10 ⁇ ) and L-NAME (100 ⁇ ) 30 minutes before the derivatives.
  • 50 ⁇ was collected from each well, placed in a 96-well plate.
  • EXAMPLE 5 Blood pressure (BP) and electrocardiogram (ECG) recording in rats: acute treatment
  • SBP systolic
  • DBP diastolic
  • MBP mean blood pressure
  • a plethysmograph (model LE 5001 Pressure Meter) was used.
  • the animals were previously acclimatized in the containment vessel for a period of 3 days, 30 min / day and the mean SBP, DBP and MBP of the three days was considered day zero (day when the animal had not yet been treated with the derivatives).
  • This vessel allowed the animal's tail to be externalized so that the cuff and blood flow sensor were positioned in the proximal portion of the tail.
  • Prior to BP measurement the animals were kept under a radiant heat source (about 39 ° C) for vasodilation. Measurements were made 3 and 24 hours after administration of the derivatives. The measurement after 3 hours was performed only for an experimental group treated with LASSBio-1029 (5 mg / kg).
  • the aorta, spleen, brain, heart, esophagus, stomach, liver, lung, kidney and the solear (SOL) and long finger extensor (EDL) skeletal muscles were quickly dissected for analysis of the following macroscopic parameters: size , shape, coloring and mass (given obtained by the percentage of the organ mass in relation to the body mass of the animal). Then, for histotological analysis the dissected tissues were cleaved into blocks, which did not exceed the size of 1 X 1 cm and their thickness did not exceed 5 mm. The skeletal muscles were attached at the ends to a support so that they did not contract during fixation.
  • Tissue fixation was performed in 10% formaldehyde in phosphate buffer solution, whose fixative volume was 20 times greater than the sample volume, for a period of 5 days. After this period, the tissues were dehydrated in a sequence of increasing concentrations of ethyl alcohol (50, 70, 80, 90, 95 and 100%) where each step had a determined time of 30 min. Once the samples were dehydrated, they were clarified, that is, the replacement of the dehydrating agent with another one, called the intermediate liquid, in this case xylene, which made possible the inclusion (because the paraffin is miscible), next protocol step.
  • the intermediate liquid in this case xylene
  • Inclusion was the step in which it occurred gradually (xylene-paraffin in the ratio of one to one, xylene-paraffin in the ratio of 1 to 2 and pure paraffin) the paraffin infiltration in the samples. This step was performed at a temperature of 60 ° C kept constantly in a greenhouse. Finally, the solid block was fabricated using metal molds in which the histological sample was placed and embedded with liquid paraffin for rapid cooling and consequent solidification. The paraffin block containing the tissue for study was sectioned into a microtome (model MRP-03, Lupe Ind ⁇ stria e Comércio LTda.) With seven micron sections.
  • the papillary muscles were mounted in 10 ml internal volume experimental vats filled with a nutrient solution maintained at 37 ° C and constantly oxygenated with a carbogenic mixture (95% O 2 and 5% CO 2 ).
  • the muscles were electrically stimulated with supramaximal voltage (50 V) through a stimulator (Grass mod. S88) at a frequency of 1.0 Hz.
  • Muscle shake recording was obtained by digitizing the electrical signal captured by the transducer (Digidata 1322A ) and stored on a computer for later analysis using the AXOSCOPE 8.0 program (Axon Instruments, Inc).
  • LASSBio-897 The hypothesis to be confirmed that the formation of nitric oxide in endothelial cells could be mediated by muscarinic receptor activation by LASSBio-897 was evaluated using the non-selective muscarinic antagonist atropine.
  • LASSBio-897-induced vasodilation was sensitive to atropine (0.1 ⁇ ) because when aortic preparation with functional endothelium was preincubated with atropine and kept in the experimental well during the addition of increasing concentrations of the derivative after the plateau of Phenylephrine-induced contracture, no effect of vascular relaxation was observed.
  • EXAMPLE 9 Inhibition of the enzymes nitric oxide synthase and guanylate cyclase
  • Nitric oxide is capable of activating the soluble guanylate cyclase enzyme, responsible for the synthesis of cGMP, a nucleotide that promotes smooth muscle relaxation.
  • L-NAME 100 ⁇
  • nitric oxide synthase inhibitor and ODQ 10 ⁇
  • guanylate cyclase inhibitor were used 30 minutes before. phenylephrine-induced contraction and the addition of increasing concentrations of the derivatives.
  • Figures 8 and 9 show a typical outline of the incubation of aortic rings with the LASSBio-897 derivative.
  • EXAMPLE 10 Effect of LASSBio Compound Effect on Blood Pressure and Heart Rate - Acute Treatment
  • LASSBio-897 5 mg / kg venous injection into SHR reduced PS from 195.0 ⁇ 18.0 to 175.0 ⁇ 26.4 mmHg and PD from 145.0 ⁇ 5.0 to 120.0 ⁇ 8.6 mmHg without changing HR (Table 6). Effect of LSSBio-897 on systolic and diastolic pressure in hypertensive rats

Abstract

The present invention relates to N-acyl hydrazine compounds capable of promoting vascular smooth muscle relaxation by activation of the biosynthesis of nitric oxide (NO) and having the general formula (I), and to the method for producing same, to pharmaceutical compositions containing same, to uses thereof and methods for treatment of dysfunctions related to NO synthesis in human and non-human mammals, and to the treatment of muscle disorders.

Description

DERIVADOS /V-ACILIDRAZÒNICOS, PROCESSO DE PRODUÇÃO DE COMPOSTOS /V-ACILIDRAZÔNICOS, COMPOSIÇÕES FARMACÊUTICAS CONTENDO OS MESMOS, USOS E MÉTODOS DE TRATAMENTO. Campo da Invenção  V-ACYLIDRAZONIC DERIVATIVES, COMPOSITION / V-ACYLIDRAZONIC PROCESS, PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME, USES AND TREATMENT METHODS. Field of the Invention
A presente invenção se refere aos compostos da família das N- acilidrazonas capazes de ativar a biossíntese do óxido nítrico (NO) em vasos sanguíneos de mamíferos e atuarem como relaxantes musculares. A presente invenção se refere também ao processo de preparação dos ditos compostos N- acilidrazônicos, às composições farmacêuticas contendo tais compostos e seus usos, bem como aos métodos de tratamento envolvendo a administração destes compostos a mamífero humano ou não humano.  The present invention relates to compounds of the N-acylhydrazone family capable of activating nitric oxide (NO) biosynthesis in mammalian blood vessels and acting as muscle relaxants. The present invention also relates to the process of preparing said N-acylhydrazonic compounds, pharmaceutical compositions containing such compounds and their uses, and methods of treatment involving the administration of these compounds to human or non-human mammals.
Antecedentes da Invenção Background of the Invention
A hipertensão arterial (HA) é uma doença que possui alta incidência na população mundial e sua etiologia é multifatorial, envolvendo fatores genéticos, ambientais e psicológicos (Dórea e Lotufo, 2004; MacKay e Mensah, 2004). Sua prevalência tem aumentado de maneira crescente, sobretudo entre mulheres, negros e idosos. Mais de 50% dos indivíduos entre 60 e 69 anos e aproximadamente 3/4 da população acima dos 70 anos são afetados pela HA (Dórea e Lotufo, 2004). Além disso, existe uma correlação positiva e direta entre os níveis de pressão arterial elevada e o risco de doenças cardiovasculares, independentemente do sexo, faixa etária e etnia (Dórea e Lotufo, 2004; MacKay e Mensah, 2004).  Hypertension (AH) is a disease that has a high incidence in the world population and its etiology is multifactorial, involving genetic, environmental and psychological factors (Dórea and Lotufo, 2004; MacKay and Mensah, 2004). Its prevalence is increasing, especially among women, blacks and the elderly. More than 50% of individuals aged 60 to 69 and approximately 3/4 of the population over 70 are affected by hypertension (Dórea and Lotufo, 2004). In addition, there is a positive and direct correlation between high blood pressure levels and the risk of cardiovascular disease, regardless of gender, age and ethnicity (Dórea and Lotufo, 2004; MacKay and Mensah, 2004).
As células endoteliais desempenham relevante papel no controle do tônus vascular, regulando a vasomotricidade, a permeabilidade vascular, o metabolismo de substâncias endógenas e exógenas e a atividade plaquetária e leucocitária (Zanesnco e Antunes, 2005). Furchgott e Zawasdski (1980) foram os primeiros pesquisadores a demonstrar a importância do endotélio no controle do tônus vascular, relatando a vasodilatação induzida pela ACh (agonista muscarínico) dependente da presença de um endotélio funcionai, e que as células endoteliais liberavam um fator de relaxamento. Os fatores relaxantes derivados do endotélio podem ser: o óxido nítrico (NO), a prostaciclina (PGfe) e o fator hiperpolarizante derivado do endotélio (EDHF) (Furchgott e Zawasdski, 1980; Vanhoutte, 2003). O NO é um potente vasodilatador e assim seu papel no controle da pressão arterial é extremamente relevante (Moncada e cols., 1991 ; Vanhoutte, 2003). Endothelial cells play a relevant role in the control of vascular tone, regulating vasomotricity, vascular permeability, metabolism of endogenous and exogenous substances and platelet and leukocyte activity (Zanesnco and Antunes, 2005). Furchgott and Zawasdski (1980) were the first researchers to demonstrate the importance of endothelium in vascular tone control, reporting ACh (muscarinic agonist) -induced vasodilation dependent on the presence of a functional endothelium, and that endothelial cells released a relaxation factor. . Endothelium-derived relaxing factors may be: nitric oxide (NO), prostacyclin (PGfe), and hyperpolarizing factor endothelium derivative (EDHF) (Furchgott and Zawasdski, 1980; Vanhoutte, 2003). NO is a potent vasodilator and thus its role in blood pressure control is extremely relevant (Moncada et al 1991; Vanhoutte 2003).
Assim, o desenvolvimento de novos fármacos e a compreensão dos seus mecanismos celulares e moleculares envolvidos na génese da HA, é fundamental para a realização de medidas terapêuticas e/ou medidas preventivas com o objetivo de controlar os níveis pressoricos e, consequentemente, reduzir os riscos cardiovasculares associados à HA.  Thus, the development of new drugs and the understanding of their cellular and molecular mechanisms involved in the genesis of hypertension is fundamental for therapeutic and / or preventive measures to control blood pressure levels and, consequently, to reduce the risks. cardiovascular diseases associated with hypertension.
Recentemente, novos compostos bioativos pertencentes à classe das N- acilidrazonas foram sintetizados usando-se como material de partida o safrol, um produto natural brasileiro obtido do óleo de sassafrás (Ocotea pretiosa) (Figueiredo e cols., 2000). Uma propriedade farmacológica desta ciasse seria o efeito analgésico, já que a ligação hidrazona presente nas moléculas, possui importante papel na inibição das cicloxigenases (Todeschini e cols., 1998). A substituição do anel furânico da molécula W-heteroarilidrazona pelo seu bioisóstero tiofênico originou o 3,4-metilenodioxibenzoil-2-tienilidrazona, mostrado na estrutura abaixo, denominado LASSBio-294 e identificado como um bioisóstero de inibidores da fosfodiesterase (PDE) do tipo 4 (Piaz e cols., 1997), uma isoforma da enzima responsável pela degradação de nucleotídeos cíclicos.  Recently, new bioactive compounds belonging to the N-acylhydrazone class have been synthesized using safrol as a starting material, a Brazilian natural product obtained from sassafras oil (Ocotea pretiosa) (Figueiredo et al 2000). A pharmacological property of this ciasis would be the analgesic effect, since the hydrazone binding present in the molecules plays an important role in the inhibition of cyclooxygenases (Todeschini et al 1998). Replacing the furanic ring of the W-heteroarylhydrazone molecule with its thiophene bioisoster gave 3,4-methylenedioxybenzoyl-2-thienylhydrazone, shown in the structure below, named LASSBio-294 and identified as a type 4 phosphodiesterase inhibitor (PDE) bioisoster (Piaz et al., 1997), an isoform of the enzyme responsible for the degradation of cyclic nucleotides.
Figure imgf000004_0001
Figure imgf000004_0001
LASSBio-294  LASSBio-294
O LASSBio-294 foi descrito como um agente inotrópico positivo cardíaco (Sudo e cols., 2001) com propriedade vasodilatadora (Silva e cols., 2002), e também capaz de promover aumento da contratilidade da musculatura esquelética tanto de rã (Gonzalez-Serratos e cols., 2001 ) quanto de rato (Zapata- Sudo e cols., 2003). O aumento da contratilidade cardíaca provocada pelo LASSBio-294 não foi relacionado com a ativação de receptores β-adrenérgicos, uma vez que, o incremento da amplitude dos abalos musculares não foi modificado com o tratamento prévio de propranolol, um bloquedor não seletivo destes receptores. A possibilidade de que LASSBio-294 poderia estar aumentando a sensibilidade das proteínas contráteis ao Ca2+ no músculo cardíaco foi descartada devido a não alteração da curva de [Ca2+] versus tensão em fibras cardíacas desnudas (Sudo e cols., 2001 ). O provável mecanismo de ação do LASSBio-294 foi atribuído a maior acúmulo de Ca2+ no retículo sarcoplasmático (RS) em função da maior captação de Ca2+ por esta organela. Não se pode eliminar também a possibilidade de a inibição da PDE promover a elevação de adenosina monofosfato cíclica (AMPc) o que levaria a maior captação de Ca + pelo RS através da fosforilação da fosfolamban presente na bomba Ca2+-ATPase (SERCA, "sarcoplasmic reticulum Ca-ATPase"). LASSBio-294 has been described as a cardiac positive inotropic agent (Sudo et al., 2001) with vasodilatory property (Silva et al., 2002), and also capable of promoting increased contractility of both frog skeletal muscles (Gonzalez-Serratos et al., 2001) and rat (Zapata-Sudo et al., 2003). The increase in cardiac contractility caused by LASSBio-294 was not related to the activation of β-adrenergic receptors, since the increase in muscle shake amplitude was not modified with pretreatment of propranolol, a non-selective blocker of these receptors. The possibility that LASSBio-294 could be increasing the sensitivity of Ca 2+ contractile proteins in the heart muscle was ruled out due to no change in the [Ca 2+ ] curve versus stress in denuded heart fibers (Sudo et al 2001). . The probable mechanism of action of LASSBio-294 was attributed to higher Ca 2+ accumulation in the sarcoplasmic reticulum (RS) due to the higher Ca 2+ uptake by this organelle. Nor can the possibility of PDE inhibition promote the elevation of cyclic adenosine monophosphate (cAMP) which would lead to increased Ca + uptake by RS through phospholamban present in the Ca 2+ -ATPase pump (SERCA, " sarcoplasmic reticulum Ca-ATPase ").
O LASSBio-294 também reverteu a contratura de anéis de aorta induzida tanto por noradrenalina quanto por KCI (Silva e cols., 2002), efeito este observado em preparações com endotélio íntegro. O envolvimento da liberação de prostanóides pelo LASSBio-294 para explicar o efeito de relaxamento vascular foi descartado já que, o relaxamento permaneceu inalterado mesmo na presença de um inibidor da cicloxigenase. A reversão do relaxamento pela inibição da guanilato ciclase sugeriu o envolvimento de GMPc no mecanismo do LASSBio- 294 em promover vasodilatação.  LASSBio-294 also reversed both norepinephrine and KCI-induced aortic ring contracture (Silva et al., 2002), an effect observed in preparations with intact endothelium. The involvement of prostanoid release by LASSBio-294 to explain the effect of vascular relaxation was discarded as relaxation remained unchanged even in the presence of a cyclooxygenase inhibitor. Reversal of relaxation by guanylate cyclase inhibition suggested the involvement of cGMP in the mechanism of LASSBio-294 in promoting vasodilation.
Descrição das Figuras Description of the Figures
A Figura 1 mostra o efeito de LASSBio-1289 na inibição da contratura do segmento de aorta de rato com e sem endotélio preservado, induzida por fenilefrina 10 μΜ em concentrações cumulativas (0,05-1 μΜ).  Figure 1 shows the effect of LASSBio-1289 on the inhibition of 10 μ 10 phenylephrine-induced rat aorta segment contracture with and without preserved endothelium at cumulative concentrations (0.05-1 μΜ).
A Figura 2 mostra o relaxamento concentração dependente de anéis de aorta com endotélio intacto causado pelo derivado LASSBio-897, após contratura induzida por fenilefrina 10 μΜ. Dados representam a média ± EPM de 6 experimentos. *P<0,05 vs controle. Figure 2 shows the relaxation-dependent concentration of intact endothelial aorta rings caused by the LASSBio-897 derivative following 10 μΜ phenylephrine-induced contracture. Data represent the mean ± SEM of 6 experiments. * P <0.05 vs control.
A Figura 3 mostra o efeito de LASSBio-897 e 1029, na produção de GMPc em anéis de aorta com endotélio, de ratos normotensos,-** P<0,05. *LASSBio- 897 e 029 vs DMSO. # LASSBio-897 e 029 vs citrato de sildenafila. A Figura 4 mostra o efeito de LASSBio-897 na quantificação indireta de óxido nítrico em anéis de aorta com endotélio, de ratos hipertensos, na ausência e na presença de L-NAME e Atropina. *# P<0,05. *LASSBio-897 vs aorta e DMSO. # L-NAME + LASSBio-897 e Atropina + LASSBio-897 vs LASSBio-897. Figure 3 shows the effect of LASSBio-897 and 1029 on cGMP production in endothelial aortic rings of normotensive rats, - * * P <0.05. * LASSBio-897 and 029 vs DMSO. # LASSBio-897 and 029 vs sildenafil citrate. Figure 4 shows the effect of LASSBio-897 on indirect quantitation of nitric oxide in endothelial aorta rings of hypertensive rats in the absence and presence of L-NAME and Atropine. * # P <0.05. * LASSBio-897 vs Aorta and DMSO. # L-NAME + LASSBio-897 and Atropine + LASSBio-897 vs LASSBio-897.
A Figura 5 mostra o efeito de LASSBio-897 na quantificação indireta de óxido nítrico em anéis de aorta com endotélio, de ratos normotensos, na ausência e na presença de L-NAME e Atropina. *# P<0,05. *LASSBio-897 vs aorta e DMSO. # L-NAME + LASSBio-897 e Atropina + LASSBio-897 vs LASSBio-897.  Figure 5 shows the effect of LASSBio-897 on indirect quantitation of nitric oxide in endothelial aorta rings of normotensive rats in the absence and presence of L-NAME and Atropine. * # P <0.05. * LASSBio-897 vs Aorta and DMSO. # L-NAME + LASSBio-897 and Atropine + LASSBio-897 vs LASSBio-897.
A Figura 6 mostra o efeito comparativo entre LASSBio-897 na presença e na ausência de atropina na contratura de anéis de aorta com endotélio induzida pela fenilefrina 10 μΜ.  Figure 6 shows the comparative effect between LASSBio-897 in the presence and absence of atropine on 10 μΜ phenylephrine-induced aortic ring contracture.
A Figura 7 A e B mostra o efeito de LASSBio-897 na contratura de anéis de aorta com endotélio induzida pela fenilefrina 10 μΜ na presença de L-NAME (A) e ODQ (B). *P<0,05. Dados representam a média ± EPM de 6 experimentos. *Aorta com endotélio vs aorta pré-incubada com L-NAME (100 μΜ) e ODQ (10 μΜ).  Figure 7 A and B show the effect of LASSBio-897 on 10 μΜ phenylephrine-induced endothelial aortic ring contracture in the presence of L-NAME (A) and ODQ (B). * P <0.05. Data represent the mean ± SEM of 6 experiments. * Endothelial aorta vs pre-incubated aorta with L-NAME (100 μΜ) and ODQ (10 μΜ).
A Figura 8 mostra o registro representativo da contratura do segmento de aorta de rato com endotélio, induzida por fenilefrina 10 μΜ na presença de L- NAME. Após o platô ser atingido, concentrações cumulativas (0,05-1 μΜ) de LASSBio-897 foram adicionadas.  Figure 8 shows the representative record of 10 μ fen phenylephrine-induced rat aortic segment contracture of endothelium in the presence of L-NAME. After the plateau is reached, cumulative concentrations (0.05-1 μΜ) of LASSBio-897 were added.
A Figura 9 mostra o registro representativo da contratura do segmento de aorta de rato com endotélio, induzida por fenilefrina 10 μΜ na presença de L- NAME (A) e ODQ (B). Após o platô ser atingido, concentrações cumulativas (0,05- 1 μΜ) de LASSBio-897 foram adicionadas.  Figure 9 shows the representative record of endothelial rat aortic segment contracture induced by 10 μΜ phenylephrine in the presence of L-NAME (A) and ODQ (B). After the plateau is reached, cumulative concentrations (0.05-1 μΜ) of LASSBio-897 were added.
A Figura 10 A e B mostra o efeito de LASSBio-897 sobre as PS (A) e PD (B), nas doses de 1 e 5 mg/kg, durante o tratamento por 14 dias de ratos normotensos pela via intraperitoneal. Dados representam a média das medidas das PS e PD em mmHg ± EPM de 6 animais.  Figure 10A and B show the effect of LASSBio-897 on PS (A) and PD (B) at doses of 1 and 5 mg / kg during 14-day treatment of intraperitoneally normotensive rats. Data represent the mean measurements of PS and PD in mmHg ± SEM of 6 animals.
A Figura 1 1 mostra o efeito de LASSBio-897 sobre as PS (A) e PD (B), nas doses de 1 e 5 mg/kg, durante o tratamento por 14 dias de ratos hipertensos pela via intraperitoneal. Dados representam a média das medidas das PS e PD em mmHg ± EPM de 6 animais. *P<0,05 quando comparado ao DMSO. Sumário da invenção Figure 11 shows the effect of LASSBio-897 on PS (A) and PD (B) at doses of 1 and 5 mg / kg during 14-day treatment of intraperitoneally hypertensive rats. Data represent the mean measurements of PS and PD in mmHg ± SEM of 6 animals. * P <0.05 when compared to DMSO. Summary of the Invention
É um objeto da presente invenção, compostos derivados /V-acilidrazônicos de fórmula molecular (I)  It is an object of the present invention V-acylhydrazonic derivative compounds of molecular formula (I)
Figure imgf000007_0001
, capazes de induzir a biossíntese de óxido nítrico através da ativação de receptores muscarínicos presentes no endotélio vascular causando desta forma, dentre outros efeitos, o relaxamento muscular em mamíferos humanos e não humanos, em um grupo de músculos compreendendo o músculo liso e o músculo estriado esquelético; preferencialmente, o relaxamento ocorre na musculatura lisa e; mais preferencialmente ainda, na musculatura lisa vascular.
Figure imgf000007_0001
, capable of inducing nitric oxide biosynthesis through activation of muscarinic receptors present in the vascular endothelium thus causing, among other effects, muscle relaxation in human and non-human mammals, in a muscle group comprising smooth muscle and striated muscle. skeletal; preferably, relaxation occurs in the smooth muscle and; more preferably still in the vascular smooth muscle.
Um segundo objeto da presente invenção trata-se de um processo para produção dos ditos compostos derivados V-acilidrazônicos, de fórmula molecular  A second object of the present invention is a process for producing said V-acylhydrazonic derivative compounds of molecular formula.
Figure imgf000007_0002
Figure imgf000007_0002
Um outro objeto da presente invenção trata-se de uma composição farmacêutica contendo os ditos compostos derivados /V-acilidrazônicos, de Another object of the present invention is a pharmaceutical composition containing said derivative / V-acylhydrazonic compounds of
fórmula molecular (I)
Figure imgf000008_0001
molecular formula (I)
Figure imgf000008_0001
e/ou seus sais, solvatos e racematos farmaceuticamente aceitáveis, além de compostos não ativos farmaceuticamente aceitáveis voltada para o tratamento de disfunções relacionadas à síntese do óxido nítrico, tais como: a disfunção erétil, a hipertensão arterial, angina, infarto do miocárdio, insuficiência cardíaca, hipertensão pulmonar dentre outras em mamífero humano ou não humano. and / or pharmaceutically acceptable salts, solvates and racemates thereof and non-pharmaceutically acceptable compounds active in the treatment of disorders related to nitric oxide synthesis, such as erectile dysfunction, hypertension, angina, myocardial infarction, insufficiency heart disease, pulmonary hypertension among others in human or non-human mammal.
É também um objeto da presente invenção composições farmacêuticas contendo tais compostos derivados /V-acilidrazônicos, de fórmula molecular (I)  It is also an object of the present invention pharmaceutical compositions containing such V-acylhydrazonic derivative compounds of molecular formula (I)
Figure imgf000008_0002
e/ou seus sais, solvatos e racematos farmaceuticamente aceitáveis; além de compostos não ativos farmaceuticamente aceitáveis voltada para o tratamento de distúrbios musculares, em animais mamíferos humanos e/ou não humanos.
Figure imgf000008_0002
and / or pharmaceutically acceptable salts, solvates and racemates thereof; in addition to pharmaceutically acceptable non-active compounds for the treatment of muscle disorders in human and / or non-human mammalian animals.
Outro objeto da presente invenção se trata do uso dos compostos derivados /V-acilidrazônicos, de fórmula molecular (I)  Another object of the present invention is the use of the V-acylhydrazonic derivative compounds of molecular formula (I).
Figure imgf000008_0003
seus sais, solvatos e racematos, para a preparação de um medicamento destinado tratamento ou prevenção de disfunções relativas à síntese do óxido nítrico em mamíferos humanos e/ou não humanos.
Figure imgf000008_0003
their salts, solvates and racemates for the preparation of a medicament for treatment or prevention of dysfunctions relating to nitric oxide synthesis in human and / or non-human mammals.
Outro objeto da presente invenção se trata do uso dos compostos derivados /V-acilidrazônicos, de fórmula molecular (I)  Another object of the present invention is the use of the V-acylhydrazonic derivative compounds of molecular formula (I).
Figure imgf000009_0001
seus sais, solvatos e racematos, para a preparação de um medicamento para o tratamento de distúrbios musculares em mamíferos humanos e/ou não humanos.
Figure imgf000009_0001
their salts, solvates and racemates for the preparation of a medicament for the treatment of muscle disorders in human and / or non-human mammals.
O sétimo objeto da presente invenção se trata de um método de tratamento de doenças e disfunções ligadas à biossíntese do óxido nítrico, baseado na administração de um composto de fórmula geral (I)  The seventh object of the present invention is a method of treating diseases and dysfunctions linked to nitric oxide biosynthesis based on the administration of a compound of formula (I).
Figure imgf000009_0002
e/ou seus sais, solvatos e racematos a um mamífero humano e/ou não humano que apresente biossíntese reduzida ou insuficiente de óxido nítrico.
Figure imgf000009_0002
and / or their salts, solvates and racemates to a human and / or non-human mammal having reduced or insufficient nitric oxide biosynthesis.
O último objeto da presente invenção se trata de um método de tratamento de doenças e disfunções musculares caracterizado por se administrar o composto  The last object of the present invention is a method of treating muscle diseases and dysfunctions characterized by administering the compound
de fórmula geral (I)
Figure imgf000009_0003
,e/ou seus sais, solvatos e racematos a um animal mamífero humano ou não humano. of general formula (I)
Figure imgf000009_0003
and / or their salts, solvates and racemates to a human or non-human mammalian animal.
Descrição Detalhada da Invenção  Detailed Description of the Invention
Os compostos derivados /V-acilidrazônicos, que são alvo desta invenção, apresentam a fórmula geral (I) abaixo.  The V-acylhydrazonic derivative compounds which are the subject of this invention have the general formula (I) below.
Figure imgf000010_0001
Figure imgf000010_0001
onde R1 e R2 podem ser H, grupo alquila de 1 a 6 átomos de carbono, ou fenila; sendo que tais radicais podem ainda ser opcionalmente substituídos, insaturados e/ou ramificados;  where R1 and R2 may be H, alkyl group of 1 to 6 carbon atoms, or phenyl; wherein such radicals may further be optionally substituted, unsaturated and / or branched;
R3, R4 e R5 podem ser H, grupo alquila de 1 a 6 átomos de carbono, fenila, amino (secundário, terciário ou quaternário), nitro, éster [RCOO-], ácido [COOH], hidroxila [-OH], alcoxila [-OR], haleto [F, Cl, Br], ciano [-CN], sulfonamida [-SO2NH2] ou metilsulfona [-SO2CH3]; sendo que tais radicais podem ainda ser opcionalmente substituídos, insaturados e/ou ramificados. R3, R4 and R5 may be H, alkyl group of 1 to 6 carbon atoms, phenyl, amino (secondary, tertiary or quaternary), nitro, ester [RCOO-], acid [COOH], hydroxyl [-OH], alkoxy [-OR], halide [F, Cl, Br], cyano [-CN], sulfonamide [-SO 2 NH 2 ] or methylsulfone [-SO 2 CH 3]; wherein such radicals may further be optionally substituted, unsaturated and / or branched.
Preferivelmente, R1 e R2 podem ser H ou um grupo alquila contendo 1 a 6 átomos de carbono e;  Preferably R1 and R2 may be H or an alkyl group containing 1 to 6 carbon atoms and;
R3, R4 e R5 podem ser H, grupo alquila contendo entre 1 a 6 átomos de carbono fenila e fenila substituída, amino (secundário, terciário ou quaternário), nitro, RCOO-, COOH, hidroxila, alcoxila, haleto, ciano, sulfonamida ou metilsulfona sendo que tais radicais podem ser opcionalmente substituídos, insaturados e/ou ramificados.  R3, R4 and R5 may be H, alkyl group containing from 1 to 6 carbon atoms phenyl and substituted phenyl, amino (secondary, tertiary or quaternary), nitro, RCOO-, COOH, hydroxyl, alkoxy, halide, cyano, sulfonamide or methyl sulfone wherein such radicals may be optionally substituted, unsaturated and / or branched.
Os compostos aqui descritos possuem atividade dependente da funcionalidade das células endoteliais e» não apresentam atividade inotropica cardíaca; além de possuírem seletividade tissular e padrão diferenciado de substituição em sua molécula, quando comparado aos demais derivados N- acilidrazônicos contidos no estado da técnica. The compounds described herein have activity dependent on endothelial cell functionality and no cardiac inotropic activity; besides having tissue selectivity and differentiated pattern of substitution in its molecule when compared to the other N-acylhydrazonic derivatives contained in the state of the art.
Devido a estas qualidades, estes derivados /V-acilidrazônicos, de fórmula  Because of these qualities, these V-acylhydrazonic derivatives of formula
molecular (I)
Figure imgf000011_0001
são capazes de induzir a biossíntese de óxido nítrico pelos vasos sanguíneos de um mamífero humano ou não humano, sendo também capazes de atuarem como relaxantes musculares, atuando em pelo menos um dos tecidos do grupo consistido de músculo cardíaco, músculo liso e músculo esquelético. Os ditos derivados /V-acilidrazônicos objetos desta invenção, possuem grande potência, sendo até 200 vezes mais efetivos que os demais compostos derivados /V- ãcilidrazônicos pertencentes ao estado da técnica. molecular (I)
Figure imgf000011_0001
They are capable of inducing nitric oxide biosynthesis by the blood vessels of a human or non-human mammal, and are also capable of acting as muscle relaxants, acting in at least one of the tissues of the heart muscle, smooth muscle and skeletal muscle tissues. Said V-acylhydrazonic derivatives of this invention have great potency, being up to 200 times more effective than the other V-acylhydrazonic derivatives belonging to the state of the art.
Os derivados V-acilidrazônicos, de fórmula molecular (I)  V-acylhydrazonic derivatives of molecular formula (I)
Figure imgf000011_0002
e objetos desta invenção, são capazes de induzir a biossíntese do NO em células endoteliais funcionais, provavelmente pela ativação de receptores muscarínicos com consequente formação de GMPc no músculo liso vascular.
Figure imgf000011_0002
and objects of this invention, are capable of inducing NO biosynthesis in functional endothelial cells, probably by activating muscarinic receptors with consequent formation of cGMP in vascular smooth muscle.
Para aferir capacidade de indução da síntese do óxido nítrico e da capacidade de efetuar o relaxamento muscular, foram efetuados testes com diferentes derivados /V-acilidrazônicos objetos desta invenção. Tais testes foram realizados de acordo com o estado da técnica e comprovam uma elevada capacidade em induzir o relaxamento do tecido muscular liso comprovando também sua elevada capacidade de induzir a síntese de NO em vasos sanguíneos, fatos que, sozinhos ou somados, conferem efeitos altamente hipotensivos, sendo aproximadamente 200 mais efetivos que os demais compostos derivados A/-acilidrazônicos anteriormente descritos. To assess the ability to induce nitric oxide synthesis and the ability to effect muscle relaxation, tests were performed with different derivatives / V-acylhydrazonic objects of this invention. Such tests were performed according to the state of the art and prove a high ability to induce relaxation of smooth muscle tissue proving also its high capacity to induce NO synthesis in blood vessels, facts that, alone or together, confer highly hypotensive effects, being approximately 200 more effective than the other A / -acylidrazonic derivatives previously described.
Os ditos derivados W-acilidrazônicos objetos desta invenção não são capazes de alterar a tensão isométrica do músculo cardíaco e músculo liso traqueal, sendo entretanto, efetivo na alteração da tensão isométrica do músculo liso vascular, fato que confere aos compostos derivados N-acilidrazônicos, objetos desta invenção, uma elevada capacidade em promover a redução da pressão arterial em mamíferos humanos ou não humanos.  The said W-acylhydrazonic derivatives objects of this invention are not capable of altering the isometric tension of the cardiac muscle and tracheal smooth muscle, being effective, however, in the alteration of the isometric tension of the vascular smooth muscle, a fact that confers to the N-acylhydrazonic derivatives derived objects. of this invention a high ability to promote blood pressure reduction in human or non-human mammals.
Os testes acima referidos serão pormenorizados nos exemplos que serão apresentados adiante. Estes testes também comprovam a capacidade que os derivados /V-acilidrazônicos aqui descritos e reivindicados possuem de alterar a produção de GMPc pelos vasos sanguíneos de mamíferos; reduzindo assim a pressão arterial em mamíferos no período agudo da hipertensão; bem como, durante um período prolongado de tratamento, além de verificar seus efeitos toxicológicos.  The above tests will be detailed in the examples below. These tests also prove the ability of the V-acylhydrazonic derivatives described and claimed herein to alter the production of cGMP by mammalian blood vessels; thus reducing blood pressure in mammals in the acute period of hypertension; as well as over an extended period of treatment, as well as checking its toxicological effects.
Os resultados deste testes demonstram que os derivados /V-acilidrazônicos objetos desta invenção, podem ser úteis no tratamento de doenças e disfunções relacionadas à síntese do óxido nítrico, bem como, no tratamento de doenças associadas ao tecido vascular.  The results of these tests demonstrate that the objects / V-acylhydrazonic derivatives of this invention may be useful in the treatment of diseases and dysfunctions related to nitric oxide synthesis, as well as in the treatment of vascular tissue associated diseases.
O processo de obtenção dos derivados N-acilidrazônicos de fórmula (I)  The process of obtaining the N-acylhydrazonic derivatives of formula (I)
Figure imgf000012_0001
, envolveu o emprego das seguintes etapas:
Figure imgf000012_0001
, involved the use of the following steps:
a) Esterificação do piperonal para obtenção do compostos de fórmula (IV); b) Hidrazinólise do compostos de fórmula (IV) produzido em a); c) Condensação do composto de fórmula (II); a) Piperonal esterification to obtain the compounds of formula (IV); b) Hydrazinolysis of the compounds of formula (IV) produced in a); c) Condensation of the compound of formula (II);
d) Alquilação do composto de fórmula (Hl).  d) Alkylation of the compound of formula (H1).
A etapa a) ocorre pela reação do piperonal a uma solução contendo um álcool, uma base forte e um halogênio, ocorrendo em uma temperatura entre - 10°C a + 25°C, durante um período de tempo entre 20 a 240 minutos. Ao final desta etapa, é formado um éster de formula geral (IV):  Step a) occurs by reacting the piperonal to a solution containing an alcohol, a strong base and a halogen, occurring at a temperature between -10 ° C to + 25 ° C for a period of time between 20 to 240 minutes. At the end of this step, an ester of general formula (IV) is formed:
Figure imgf000013_0001
Figure imgf000013_0001
Preferivelmente, a etapa a ocorre a uma temperatura entre -5°C a +10°C em no máximo 120 minutos, sendo seguida da etapa b.  Preferably, step a occurs at a temperature between -5 ° C to + 10 ° C within a maximum of 120 minutes, followed by step b.
A hidrazinólise do éster (IV), ocorre na etapa b, pela adição do éster (IV) em uma solução alcoólica contendo hidrazina, em um aparelho condensador com temperatura entre 50°C e 130°C. Preferivelmente a etapa b ocorre em um condensador de refluxo, entre 70°C a 100°C.  The hydrazinolysis of ester (IV) occurs in step b by the addition of ester (IV) in an alcoholic solution containing hydrazine in a condensing apparatus with a temperature between 50 ° C and 130 ° C. Preferably step b takes place in a reflux condenser between 70 ° C to 100 ° C.
Ao término da hidrazinólise, o pH é ajustado, e, a solução é resfriada a uma temperatura entre -5°C a +15°C, para que ocorra a precipitação do composto  At the end of hydrazinolysis, the pH is adjusted and the solution is cooled to a temperature between -5 ° C to + 15 ° C to precipitate the compound.
Figure imgf000013_0002
Figure imgf000013_0002
de fórmula (II).  of formula (II).
Na etapa c, o composto de fórmula (II), é condensado, pela reação do composto (II) com compostos da família 3-acil-tiofenos, funcionalizados em R2 e/ou R3 e/ou R e/ou R5, podendo os grupamentos R2, R3, R e R5 estarem funcionalizados de forma independente ou conjunta, resultando na formação do composto de fórmula (III). A reação de condensação ocorre sob agitação, na presença de um álcool primário ou secundário, preferencialmente, um álcool primário como o etanol, à temperatura ambiente, em pH ácido. In step c, the compound of formula (II) is condensed by the reaction of compound (II) with compounds of the 3-acylthiophenes family, functionalized at R 2 and / or R 3 and / or R and / or R 5. the groups R2, R3, R and R5 are functionalized independently or together, resulting in the formation of the compound of formula (III). The condensation reaction occurs under stirring in the presence of a primary or secondary alcohol, preferably a primary alcohol such as ethanol, at room temperature at acidic pH.
Esta reação ocorre entre 20 a 120 minutos, ocorrendo precipitação do  This reaction occurs between 20 to 120 minutes, with precipitation of the
composto
Figure imgf000014_0001
fórmula (III) funcionalizado, no final da reação. Preferencialmente, essa reação ocorre entre 35 a 100 minutos. compound
Figure imgf000014_0001
functionalized formula (III) at the end of the reaction. Preferably, this reaction occurs within 35 to 100 minutes.
O composto III é então alquilado, pela a adição do haleto de alquila desejado. Esta reação promove a adição de um radical alquila, de forma regiosseletiva, podendo ser empregada uma alquila saturada ou insaturada e/ou ramificada.  Compound III is then alkylated by the addition of the desired alkyl halide. This reaction promotes the addition of a regioselective alkyl radical and a saturated or unsaturated and / or branched alkyl may be employed.
As composições farmacêuticas contendo os compostos /V-acilidrazônicos de fórmula molecular (I)  Pharmaceutical compositions containing the / V-acylhydrazonic compounds of formula (I)
Figure imgf000014_0002
e/ou seus sais, solvatos e racematos farmaceuticamente aceitáveis e, compostos não ativos farmaceuticamente aceitáveis voltada para o tratamento de doenças e/ou disfunções relacionadas à síntese do óxido nítrico em mamífero humano ou não humano, são também um objeto desta invenção.
Figure imgf000014_0002
and / or pharmaceutically acceptable salts, solvates and racemates thereof, and non-pharmaceutically acceptable compounds active in the treatment of diseases and / or dysfunctions related to nitric oxide synthesis in human or non-human mammals are also an object of this invention.
Para esta invenção, as ditas doenças e/ou disfunções relacionadas à síntese do óxido nítrico são todas aquelas relacionadas à produção insuficiente do NO, tais como a disfunção erétil, a hipertensão arterial, angina, infarto do miocárdio, insuficiência cardíaca e hipertensão pulmonar dentre outras. compostos /V-acilidrazônicos, de fórmula molecular For this invention, said diseases and / or dysfunctions related to nitric oxide synthesis are all those related to insufficient NO production, such as erectile dysfunction, arterial hypertension, angina, myocardial infarction, heart failure and pulmonary hypertension among others. . V-acylhydrazonic compounds of molecular formula
Figure imgf000015_0001
e/ou seus sais, solvatos e racematos farmaceuticamente aceitáveis; além de compostos não ativos farmaceuticamente aceitáveis, podem também serem empregados em composições farmacêuticas voltadas para o tratamento de distúrbios musculares, em mamíferos humanos e/ou não humanos.
Figure imgf000015_0001
and / or pharmaceutically acceptable salts, solvates and racemates thereof; In addition to pharmaceutically acceptable non-active compounds, they may also be employed in pharmaceutical compositions for the treatment of muscle disorders in human and / or non-human mammals.
As disfunções musculares previstas por esta invenção são aquelas relacionadas a músculos pertencentes ao grupo de tecidos, tais como o músculo cardíaco, músculo liso e músculo esquelético. Preferencialmente, as composições farmacêuticas desta invenção podem ser empregadas na promoção do relaxamento no músculo liso.  The muscle dysfunctions contemplated by this invention are those related to muscles belonging to the tissue group, such as cardiac muscle, smooth muscle and skeletal muscle. Preferably, the pharmaceutical compositions of this invention may be employed in promoting smooth muscle relaxation.
Para esta invenção compostos não ativos que podem ser encontrados nas ditas composições farmacêuticas são todos aqueles conhecidos por um especialista da área farmacêutica, como por exemplo: adjuvantes, estabilizantes, conservantes, diluentes, ajustadores de pH, corantes, flavorizantes, espessantes e outros.  For this invention non-active compounds which may be found in said pharmaceutical compositions are all those known to one of ordinary skill in the pharmaceutical art, such as adjuvants, stabilizers, preservatives, diluents, pH adjusters, colorants, flavorings, thickeners and the like.
O uso dos compostos /V-acilidrazônicos, de fórmula molecular (I)  The use of / V-acylhydrazonic compounds of molecular formula (I)
Figure imgf000015_0002
,e/ou seus sais, solvatos e racematos, para a preparação de um medicamento destinado ao tratamento ou prevenção de disfunções relativas à síntese do óxido nítrico em mamíferos humanos e/ou não humanos também é previsto por esta invenção. Um outro uso previsto pela presente invenção, se trata do uso dos compostos V-acilidrazônicos, de fórmula molecular (I)
Figure imgf000015_0002
, and / or salts, solvates and racemates thereof, for the preparation of a medicament for the treatment or prevention of nitric oxide synthesis dysfunction in human and / or non-human mammals is also provided by this invention. Another intended use of the present invention is the use of the V-acylhydrazonic compounds of formula (I)
Figure imgf000016_0001
seus sais, solvatos e racematos, para a preparação de um medicamento para o tratamento de distúrbios musculares em mamíferos humanos e/ou não humanos.
Figure imgf000016_0001
their salts, solvates and racemates for the preparation of a medicament for the treatment of muscle disorders in human and / or non-human mammals.
Também é previsto pela presente invenção, um método de tratamento de doenças e disfunções ligadas à síntese do óxido nítrico, baseado na administração de um composto de fórmula geral (I)  Also provided by the present invention is a method of treating diseases and disorders associated with nitric oxide synthesis based on the administration of a compound of formula (I)
Figure imgf000016_0002
e/ou seus sais, solvatos e racematos a um mamífero humano e/ou não humano que apresente síntese reduzida ou insuficiente de óxido nítrico.
Figure imgf000016_0002
and / or their salts, solvates and racemates to a human and / or non-human mammal having reduced or insufficient nitric oxide synthesis.
Dentre as doenças relativas à biossíntese reduzida ou insuficiente de óxido nítrico previstas pela presente invenção, podemos citar a disfunção erétil, a angina, hipertensão arterial, infarto do miocárdio, insuficiência cardíaca, hipertensão pulmonar.  Among the diseases related to reduced or insufficient nitric oxide biosynthesis predicted by the present invention, we can mention erectile dysfunction, angina, arterial hypertension, myocardial infarction, heart failure, pulmonary hypertension.
O último objeto descrito pela presente invenção se trata de um método de tratamento, de dpenças e disfunções musculares caracterizado por se administrar o composto de fórmula geral (I) The last object described by the present invention is a method of treatment, muscle disorders and dysfunctions characterized by the administration of the compound of formula (I)
Figure imgf000017_0001
,e/ou seus sais, solvatos e racematos a um animal mamífero humano ou não humano.
Figure imgf000017_0001
and / or their salts, solvates and racemates to a human or non-human mammalian animal.
Os exemplos que serão dados a seguir são meramente ilustrativos, e, tem o objetivo apenas demonstrar as concretizações realizadas, não devendo portanto, serem utilizados na delimitação dos direitos dos inventores.  The following examples are for illustration purposes only and are intended only to demonstrate the embodiments made and should not be used to delimit the rights of inventors.
Compostos testados: Tested Compounds:
Os exemplos aqui descritos ilustram os testes realizados com 2 compostos obtidos, que apresentam entre si, diferenças em relação ao substituinte Ri. Estes compostos doravante serão chamados de LASSBio-897 e LASSBio-1289. Os demais derivados LASSBio citados são pertencentes ao estado da técnica.  The examples described here illustrate the tests carried out with 2 compounds obtained which differ from the substituent R 1. These compounds will henceforth be called LASSBio-897 and LASSBio-1289. The other LASSBio derivatives mentioned are from the state of the art.
EXEMPLO 1 : Registro de tensão isométrica em músculo liso vascular EXAMPLE 1: Record of Isometric Tension in Vascular Smooth Muscle
Para a avaliação dos derivados no músculo liso vascular foram utilizados anéis de aorta torácica dissecados em solução nutridora Tyrode modificada. Após a remoção dos tecidos, adiposo e conjuntivo que circundam o vaso, anéis medindo entre 2 e 3 mm foram clivados e posicionados na cuba experimental e a tensão gerada pelo músculo foi transformada em um sinal elétrico digitalizado (Digidata 1322A) e armazenado em computador para posterior análise usando o programa axoscope 8.0 (Axon Instruments. Inc). Os anéis foram apenas montados em hastes como mostra o esquema abaixo, permitindo a medida da tensão transversa.  For the evaluation of vascular smooth muscle derivatives, thoracic aortic rings dissected in a modified Tyrode nutrient solution were used. After removal of the adipose and connective tissues surrounding the vessel, rings measuring between 2 and 3 mm were cleaved and placed in the experimental vessel and the tension generated by the muscle was transformed into a digitized electrical signal (Digidata 1322A) and stored in a computer for further analysis using the axoscope 8.0 program (Axon Instruments. Inc). The rings were only mounted on rods as shown in the diagram below, allowing the measurement of transverse tension.
Logo após a colocação dos músculos em cubas experimentais, foram mantidos durante 120 minutos para equilíbrio da preparação após um estiramento correspondente a 1 ,0 g de tensão. A solução nutridora durante esta fase foi trocada constantemente (a cada 30 minutos).  Immediately after placing the muscles in experimental vats, they were kept for 120 minutes to balance the preparation after stretching corresponding to 1.0 g tension. The nourishing solution during this phase was constantly changed (every 30 minutes).
Terminado o período de equilíbrio, iniciava-se a primeira fase do experimento, que se tratava da verificação da integridade da função do endotélio vascular. Para tal, as aortas foram contraídas com 10 μΜ de fenilefrina (Fe) e em seguida expostas a 10 μΜ de acetilcolina (ACh) (Kaya e cols., 2003). A integridade do endotélio foi confirmada naqueles anéis de aorta em que o relaxamento obtido após a contratura induzida pela Fe fosse > a 80%. No entanto, a ausência do endotélio foi considerada quando o relaxamento < a 10%. After the equilibrium period, the first phase of the experiment began, which was to verify the integrity of the endothelium function. vascular. For this, the aortas were contracted with 10 μΜ phenylephrine (Fe) and then exposed to 10 μΜ acetylcholine (ACh) (Kaya et al., 2003). Endothelial integrity was confirmed in those aortic rings where relaxation obtained after Fe-induced contracture was> 80%. However, the absence of endothelium was considered when relaxation <10%.
Após esta etapa, a preparação foi lavada durante 30 min com a solução nutridora e uma nova contratura foi induzida através da exposição a 10 μΜ de Fe. Após a formação do platô da contratura máxima induzida pela Fe, concentrações crescentes dos derivados, assim como de DMSO foram adicionadas às preparações.  After this step, the preparation was washed for 30 min with the nutrient solution and a new contracture was induced by exposure to 10 μΜ Fe. After the formation of the maximum Fe-induced contracture plateau, increasing concentrations of the derivatives as well as DMSO were added to the preparations.
Ao final do experimento, a reversibilidade do efeito farmacológico foi observada com a lavagem da preparação por 30 minutos seguida de nova exposição à Fe.  At the end of the experiment, the reversibility of the pharmacological effect was observed by washing the preparation for 30 minutes followed by further exposure to Fe.
Observando o efeito vasodilatador dos derivados (LASSBio- 029 e LASSBio-897) em relação a substância protótipo (LASSBio-294) nas preparações de músculo liso vascular, alguns procedimentos experimentais foram realizados tais como: 1. em alguns anéis de aorta, o endotélio foi mecanicamente removido utilizando-se uma haste de polietileno para a avaliação do seu envolvimento no efeito dos derivados; 2. ferramentas farmacológicas foram aplicadas para possível identificação do alvo e da via pela qual os derivados poderiam estar agindo, onde as aortas foram pré-incubadas durante 30 minutos com o antagonista de receptor muscarínico (atropina 10 μ ), com um inibidor da óxido nítrico sintase (L-NAME 100 μΜ) e com um inibidor da guanilato ciclase (ODQ 10 μΜ) seguida de nova contratura produzida por 10 μΜ de Fe e a curva concentração-resposta dos derivados obtida; 3. alguns anéis de aorta foram incubados por 10 min com 8 e 50 μΜ de LASSBio-1029 e 0,5 e 1 μΜ de LASSBio-897 e imediatamente congelados em nitrogénio líquido para quantificação da produção de G Pc.  Observing the vasodilatory effect of the derivatives (LASSBio-029 and LASSBio-897) on the prototype substance (LASSBio-294) in vascular smooth muscle preparations, some experimental procedures were performed such as: 1. In some aortic rings, the endothelium was mechanically removed using a polyethylene rod to evaluate its involvement in the effect of the derivatives; 2. pharmacological tools were applied for possible identification of the target and the route by which the derivatives could be acting, where the aortas were preincubated for 30 minutes with the muscarinic receptor antagonist (atropine 10 μ), with a nitric oxide inhibitor. synthase (L-NAME 100 μΜ) and a guanylate cyclase inhibitor (ODQ 10 μΜ) followed by a new contracture produced by 10 μΜ Fe and the concentration-response curve of the derivatives obtained; 3. Some aorta rings were incubated for 10 min with 8 and 50 μΜ LASSBio-1029 and 0.5 and 1 μΜ LASSBio-897 and immediately frozen in liquid nitrogen to quantify G Pc production.
EXEMPLO 2: Relaxamento da aorta induzido pelos derivados EXAMPLE 2: Derivative-induced aorta relaxation
A atividade vasodilatadora do derivado LASSBio-1289 foi investigada em anéis de aorta com endotélio preservado. Para verificar a funcionalidade do endotélio, os anéis de aorta foram pré-contraídos com 10 μΜ de fenilefrina e logo após o platô da contratura, estes anéis foram expostos a 10 μΜ de ACh. O relaxamento observado na presença da ACh indicou a integridade funcional do endotélio. Trinta minutos depois da lavagem das preparações, foram adicionadas às cubas experimentais concentrações crescentes do derivado logo após a formação do platô da contratura induzida pela fenilefrina. LASSBio-1289 em anéis de aorta com endotélio funcional pré-contraídos com fenilefrina (10 μΜ), foi capaz de produzir um relaxamento concentração-dependente. A concentração inibitória média (CI50), ou seja, a concentração necessária para reduzir em 50% a contratura máxima induzida pela fenilefrina foi de 0,69 ± 0,18 μΜ para LASSBio- 1289 (figura 2). The vasodilatory activity of the LASSBio-1289 derivative was investigated in preserved endothelial aortic rings. To verify endothelial functionality, aortic rings were pre-contracted with 10 μΜ phenylephrine and then After the contracture plateau, these rings were exposed to 10 μΜ ACh. The relaxation observed in the presence of ACh indicated the functional integrity of the endothelium. Thirty minutes after washing the preparations, increasing concentrations of the derivative were added to the experimental wells soon after the formation of the phenylephrine-induced contracture plateau. LASSBio-1289 in phenylephrine pre-contracted functional endothelial aortic rings (10 μΜ) was able to produce concentration-dependent relaxation. The mean inhibitory concentration (IC50), ie the concentration required to reduce by 50% the maximum phenylephrine-induced contracture was 0.69 ± 0.18 μΜ for LASSBio-1289 (Figure 2).
EXEMPLO 3: Dosagem de GMPc  EXAMPLE 3: Dosing of cGMP
Para a determinação da concentração de GMPc, os anéis de aorta foram homogeneizados em ácido tricloracético (6%) e o homogenado foi centrifugado (2000xg por 15 min em 4°C). O pellet foi usado para determinar o conteúdo de proteína de acordo com o método adaptado para proteínas insolúveis (Lowry e cols., 1951 ). O sobrenadante foi lavado quatro vezes com cinco volumes de água- éter etílico saturado. A camada superior foi descartada depois de cada lavagem. A fase aquosa foi então evaporada por secagem (Delpy e cols., 1996). O extrato seco foi dissolvido em um volume adequado do tampão de análise e o conteúdo de GMPc foi determinado colorimetricamente usando um kit para imunoanálise enzimática (kit cGMP Enzymeimmunoassay Biotrak (EIA) System). Como controle positivo foi utilizado o citrato de sildenafila (0, 1 μΜ) e controle negativo o veículo no volume em que os derivados foram dissolvidos (DMSO). Os dados foram expressos em fmol/pg"1 da proteína GMPc (figura 3). For the determination of cGMP concentration, the aortic rings were homogenized in trichloracetic acid (6%) and the homogenate was centrifuged (2000xg for 15 min at 4 ° C). The pellet was used to determine protein content according to the method adapted for insoluble proteins (Lowry et al 1951). The supernatant was washed four times with five volumes of water-saturated ethyl ether. The upper layer was discarded after each wash. The aqueous phase was then evaporated by drying (Delpy et al., 1996). The dried extract was dissolved in an appropriate volume of the assay buffer and the cGMP content was determined colorimetrically using an Enzymeimmunoassay Biotrak (EIA) System cGMP kit. Positive control was sildenafil citrate (0, 1 μΜ) and negative control the vehicle at the volume in which the derivatives were dissolved (DMSO). Data were expressed in fmol / pg- 1 cGMP protein (Figure 3).
EXEMPLO 4: Dosagem de óxido nítrico (NO) EXAMPLE 4: Nitric Oxide (NO) Dosage
A produção de NO pela aorta foi analisada pela medida do acúmulo de nitrito no meio de cultura usando a reação de Gríess (Green e cols., 1982; Ding e cols., 1988). Aorta torácica de ratos Wistar normotensos e de SHR pesando entre (250 a 300 g), foram dissecados em tampão fosfato salina (PBS), pH 7,2, 37°C, onde os tecidos conectivos foram retirados de forma a não danificar o endotélio. A aorta foi então segmentada em anéis de aproximadamente 2-3 mm e cada anel foi colocado dentro de um poço de uma placa de 24 poços já contendo 500 μΙ de PBS. Terminado este procedimento, os anéis de aorta foram lavados três vezes com PBS e após a última lavada foi incubado meio DMEM em cada poço da placa. NO production by the aorta was analyzed by measuring nitrite accumulation in the culture medium using the Gríess reaction (Green et al., 1982; Ding et al., 1988). Thoracic aorta of normotensive and SHR Wistar rats weighing between (250 to 300 g) were dissected in phosphate buffered saline (PBS), pH 7.2, 37 ° C, where the connective tissues were removed so as not to damage the endothelium. . The aorta was then segmented into approximately 2-3 mm rings and each ring was placed into a well of a 24 well plate already containing 500 μΙ PBS. After this procedure, the aorta rings were washed three times with PBS and after the last wash DMEM medium was incubated in each well of the plate.
Foi esperado um período de 60 min para estabilizar a preparação e dois procedimentos diferentes foram realizados: 1. o meio foi trocado por outro contendo ACh (10 μΜ), coletado 50 μΙ de cada poço e colocado em uma placa de 96 poços; 2. a placa de 24 poços foi novamente lavada três vezes com PBS e cada poço foi incubado com meio contendo os derivados LASSBio-897 (0,5 e 1 μΜ), LASSBio-1029 (8 e 50 μΜ), seus respectivos veículos (DMSO) e as ferramentas farmacológicas de interesse bem como atropina (10 μΜ) e L-NAME (100 μΜ) 30 minutos antes dos derivados. Foi coletado 50μΙ de cada poço, colocado em uma placa de 96 poços. Concluído estes dois procedimentos foi adicionado o mesmo volume do reagente de Griess (1% sulfanilamida, 0,1% dicloreto de nafitiletilenodiamina, 5% H3P04) em cada poço da placa de 96 poços e depois de dez minutos em temperatura ambiente, a densidade óptica (DO) da amostra foi lida em 550 nm utilizando um espectrofotômetro (modelo Titertek Multiskan MCC/340 MK) e a quantidade de NO produzida foi expressa em pmol.L" 1. Nitrito de sódio a 1 mM foi utilizado para fazer as soluções da curva padrão, como mostra a tabela 2. Este experimento foi realizado em triplicata e a média aritmética de cada dado foi utilizada para plotar o gráfico. A period of 60 min was expected to stabilize the preparation and two different procedures were performed: 1. The medium was exchanged for one containing ACh (10 μΜ), collected 50 μΙ from each well and placed in a 96-well plate; 2. The 24 well plate was again washed three times with PBS and each well was incubated with medium containing the derivatives LASSBio-897 (0.5 and 1 μΜ), LASSBio-1029 (8 and 50 μΜ), their respective vehicles ( DMSO) and pharmacological tools of interest as well as atropine (10 μΜ) and L-NAME (100 μΜ) 30 minutes before the derivatives. 50μΙ was collected from each well, placed in a 96-well plate. After completing these two procedures the same volume of Griess reagent (1% sulfanylamide, 0.1% naphthylethylenediamine dichloride, 5% H 3 P0 4 ) was added to each well of the 96-well plate and after ten minutes at room temperature, the optical density (OD) of the sample was read at 550 nm using a spectrophotometer (Titertek Multiskan MCC / 340 MK model) and the amount of NO produced was expressed in pmol.L " 1. 1 mM sodium nitrite was used to make the solutions of the standard curve, as shown in table 2. This experiment was performed in triplicate and the arithmetic mean of each data was used to plot the graph.
EXEMPLO 5: Registro da pressão arterial (PA) e eletrocardioqrama (ECG) em ratos: tratamento agudo  EXAMPLE 5: Blood pressure (BP) and electrocardiogram (ECG) recording in rats: acute treatment
5.1 - Preparo do animal:  5.1 - Preparation of the animal:
Ratos Wistar machos (250 a 300 g) e espontaneamente hipertensos (SHR, Male Wistar rats (250 to 300 g) and spontaneously hypertensive (SHR,
250 a 300 g) foram anestesiados por injeção i.p. de pentobarbital sódio (50 mg.kg" 1) para que a artéria carótida direita fosse dissecada e canulizada para registro da pressão arterial (PA). Os cateteres usados no procedimento foram previamente heparinizados (40 U/mL) para evitar a formação de coágulos durante o experimento. Eletrodos foram posicionados e fixados na região precordial do animal para registro eletrocardiográfico (ECG) em derivação Dl durante todo o experimento, permitindo assim avaliar a influência dos derivados na frequência cardíaca (FC). A veia jugular externa foi isolada e cateterizada para a injeção in bolus dos derivados LASSBio-897 e LASSBio-1029 nas doses iniciais de 1 ; 5; 7,5 e 10 mg/Kg. Após o preparo do animal foi alocado em caixa de contenção e o experimento iniciava-se quando foi observada a recuperação completa da anestesia, ou seja, quando o animal recuperava a sua postura normal. Registros da PA e ECG foram obtidos antes e depois da injeção in bolus dos derivados. 250 to 300 g) were anesthetized by ip injection of pentobarbital sodium (50 mg.kg " 1 ) so that the right carotid artery was dissected and cannulated to record blood pressure. The catheters used in the procedure were previously heparinized (40 U / mL) to avoid clot formation during the experiment Electrodes were positioned and fixed in the animal's precordial region for electrocardiographic recording (ECG) in Dl lead throughout the experiment, thus allowing to evaluate the influence of the derivatives on the frequency. heart rate (HR). The external jugular vein was isolated and catheterized for in-bolus injection of LASSBio-897 and LASSBio-1029 derivatives at initial doses of 1; 5; 7.5 and 10 mg / kg. After preparation of the animal, it was allocated in a containment box and the experiment started when the complete recovery of anesthesia was observed, that is, when the animal recovered its normal posture. BP and ECG records were obtained before and after in-bolus injection of the derivatives.
5.2 - Medida não invasiva da pressão arterial (PA) em ratos:  5.2 - Noninvasive blood pressure (BP) measurement in rats:
Para o tratamento crónico, foram utilizados ratos Wistar machos e SHR com 11 semanas de idade (seis ratos para cada grupo experimental). O tratamento foi realizado diariamente, com uma única dose, por via intraperitoneal ou oral, durante sete ou quatorze dias com os derivados LASSBio-897 (1 e 5 mg/kg) e 1029 (5 e 10 mg/kg). Em um grupo experimental (LASSBio-897, 1 mg/kg) o tratamento foi realizado por sete dias e suspenso por mais sete dias, para avaliar a reversibilidade do efeito antihipertensivo. A pressão arterial sistólica (PAS), diastólica (PAD) e média (PAM), além da frequência cardíaca (FC), foram medidas através do método indireto de forma não invasiva, na cauda do animal. Para este protocolo experimental foi utilizado um pletismógrafo (modelo LE 5001 Pressure Meter). Os animais foram aclimatizados previamente no recipiente de contenção por um período de 3 dias, 30 min/dia e a média da PAS, PAD e PAM dos três dias foi considerada o dia zero (dia em que o animal ainda não tinha sido tratado com os derivados). Este recipiente permitiu a exteriorização da cauda do animal para que o manguito e o sensor de fluxo sanguíneo fossem posicionados na porção proximal da cauda. Antes da aferição da PA, os animais foram mantidos sob uma fonte de calor irradiante (cerca de 39°C) para produção de vasodilatação. As medidas foram feitas após 3 e 24 horas da administração dos derivados. A medida após 3 horas foi realizada somente para um grupo experimental com tratamento de LASSBio-1029 (5 mg/kg).  For chronic treatment, 11-week-old male Wistar and SHR rats (six rats for each experimental group) were used. Treatment was performed daily, with a single dose, intraperitoneally or orally, for seven or fourteen days with derivatives LASSBio-897 (1 and 5 mg / kg) and 1029 (5 and 10 mg / kg). In an experimental group (LASSBio-897, 1 mg / kg) the treatment was performed for seven days and suspended for another seven days to assess the reversibility of the antihypertensive effect. Systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure, as well as heart rate (HR), were measured by the non-invasive indirect method at the tail of the animal. For this experimental protocol a plethysmograph (model LE 5001 Pressure Meter) was used. The animals were previously acclimatized in the containment vessel for a period of 3 days, 30 min / day and the mean SBP, DBP and MBP of the three days was considered day zero (day when the animal had not yet been treated with the derivatives). This vessel allowed the animal's tail to be externalized so that the cuff and blood flow sensor were positioned in the proximal portion of the tail. Prior to BP measurement, the animals were kept under a radiant heat source (about 39 ° C) for vasodilation. Measurements were made 3 and 24 hours after administration of the derivatives. The measurement after 3 hours was performed only for an experimental group treated with LASSBio-1029 (5 mg / kg).
EXEMPLO 6: Técnica histológica de rotina para microscopia óptica EXAMPLE 6: Routine Histological Technique for Optical Microscopy
Após o sacrifício dos animais, aorta, baço, cérebro, coração, esôfago estômago, fígado, pulmão, rim e os músculos esqueléticos solear (SOL) e extensor longo dos dedos (EDL), foram rapidamente dissecados para análise dos seguintes parâmetros macroscópicos: tamanho, forma, coloração e massa (dado obtido através da porcentagem da massa do órgão em relação a massa corpórea do animal). Em seguida, para a análise histotológica os tecidos dissecados foram clivados em blocos, que não excederam o tamanho de 1 X 1 cm e sua espessura não ultrapassou 5 mm. Os músculos esqueléticos foram presos pelas extremidades em um suporte para que não contraíssem durante a fixação. A fixação dos tecidos foi realizada em formaldeído a 10% em solução de tampão fosfato, cujo volume do fixador foi 20 vezes maior que o volume da amostra, por um período de 5 dias. Após este período, os tecidos foram desidratados em uma sequência de concentrações crescentes de álcool etílico (50, 70, 80, 90, 95 e 100%) onde cada passo teve o tempo determinado de 30 min. Uma vez que as amostras foram desidratadas, procedeu-se o seu clareamento, isto é, a substituição do agente desidratante por outro, chamado de líquido intermediário, neste caso o xileno, o qual possibilitou a inclusão (por ser miscível a parafina), próxima etapa do protocolo. A inclusão foi a etapa em que ocorreu de forma gradativa (xileno-parafina na proporção de um para um, xileno-parafina na proporção de 1 para 2 e parafina pura) a infiltração da parafina nas amostras. Esta etapa foi realizada à temperatura de 60°C mantida constantemente em estufa. Finalmente, foi realizada a fabricação do bloco sólido utilizando moldes metálicos nos quais foi colocada a amostra histológica e embebidas com parafina líquida para resfriamento rápido e consequente solidificação. O bloco de parafina contendo em seu interior o tecido para estudo foi seccionado em micrótomo (modelo MRP-03, Lupe Indústria e Comércio LTda.) com cortes de sete micra. Essas frações foram fixadas em lâminas histológicas (desparafinização), sendo todos os tecidos corados com hematoxilina e eosina. Os cortes foram lavados em água destilada, desidratados em soluções crescentes de etanol, clareados em xileno, adicionadas algumas gotas de entellan (meio para montagem) e cobertos com uma lamínula. As imagens dos tecidos foram obtidas por uma câmera digital (modelo Canon Power Shot A 620/610) acoplada ao microscópio óptico de luz (modelo Zeiss Axiostar plus). After the animals were sacrificed, the aorta, spleen, brain, heart, esophagus, stomach, liver, lung, kidney and the solear (SOL) and long finger extensor (EDL) skeletal muscles were quickly dissected for analysis of the following macroscopic parameters: size , shape, coloring and mass (given obtained by the percentage of the organ mass in relation to the body mass of the animal). Then, for histotological analysis the dissected tissues were cleaved into blocks, which did not exceed the size of 1 X 1 cm and their thickness did not exceed 5 mm. The skeletal muscles were attached at the ends to a support so that they did not contract during fixation. Tissue fixation was performed in 10% formaldehyde in phosphate buffer solution, whose fixative volume was 20 times greater than the sample volume, for a period of 5 days. After this period, the tissues were dehydrated in a sequence of increasing concentrations of ethyl alcohol (50, 70, 80, 90, 95 and 100%) where each step had a determined time of 30 min. Once the samples were dehydrated, they were clarified, that is, the replacement of the dehydrating agent with another one, called the intermediate liquid, in this case xylene, which made possible the inclusion (because the paraffin is miscible), next protocol step. Inclusion was the step in which it occurred gradually (xylene-paraffin in the ratio of one to one, xylene-paraffin in the ratio of 1 to 2 and pure paraffin) the paraffin infiltration in the samples. This step was performed at a temperature of 60 ° C kept constantly in a greenhouse. Finally, the solid block was fabricated using metal molds in which the histological sample was placed and embedded with liquid paraffin for rapid cooling and consequent solidification. The paraffin block containing the tissue for study was sectioned into a microtome (model MRP-03, Lupe Indústria e Comércio LTda.) With seven micron sections. These fractions were fixed in histological slides (deparaffinization), and all tissues were stained with hematoxylin and eosin. The sections were washed in distilled water, dehydrated in rising ethanol solutions, bleached in xylene, added a few drops of entellan (mounting medium) and covered with a coverslip. Tissue images were taken by a digital camera (Canon Power Shot A 620/610) coupled to the light optical microscope (Zeiss Axiostar plus).
EXEMPLO 7: Registro de tensão isométrica de musculo cardíaco EXAMPLE 7: Cardiac Muscle Isometric Stress Record
Para avaliação da contratilidade cardíaca, músculos papilares do ventrículo esquerdo de rato foram dissecados após rápida retirada do coração. Durante este procedimento, o órgão foi mantido submerso numa solução nutridora (Tyrode) previamente oxigenada com mistura carbogênica (mistura 95% 021 CO2), pH 7.4, cujos constituintes encontram-se descritos na Tabela 3. A parte inferior do músculo papilar foi fixada a uma haste metálica presa a um eletrodo de estimulação, e a parte superior a uma haste longa e móvel, conectada a um transdutor de força (Grass mod. FT 03), como mostra o esquema representativo abaixo. To assess cardiac contractility, papillary muscles of the left ventricular rat were dissected after rapid removal of the heart. During this In this procedure, the organ was kept submerged in a previously oxygenated nutrient solution (Tyrode) with carbogenic mixture (95% 0 2 1 CO 2 mixture), pH 7.4, whose constituents are described in Table 3. The lower part of the papillary muscle was fixed. a metal rod attached to a stimulation electrode, and the top to a long, mobile rod connected to a force transducer (Grass mod. FT 03), as shown in the representative diagram below.
Os músculos papilares foram montados em cubas experimentais de volume interno de 10 ml preenchidas com solução nutridora mantida à 37°C e oxigenadas constantemente com mistura carbogênica (95% O2 e 5% CO2). Os músculos foram estimulados eletricamente com voltagem supramaximal (50 V) através de um estimulador (Grass mod. S88) à uma frequência de 1,0 Hz. O registro dos abalos musculares foi obtido através da digitalização do sinal elétrico captado pelo transdutor (Digidata 1322A) e armazenado em computador para posterior análise, usando o programa AXOSCOPE 8.0 (Axon Instruments, Inc). The papillary muscles were mounted in 10 ml internal volume experimental vats filled with a nutrient solution maintained at 37 ° C and constantly oxygenated with a carbogenic mixture (95% O 2 and 5% CO 2 ). The muscles were electrically stimulated with supramaximal voltage (50 V) through a stimulator (Grass mod. S88) at a frequency of 1.0 Hz. Muscle shake recording was obtained by digitizing the electrical signal captured by the transducer (Digidata 1322A ) and stored on a computer for later analysis using the AXOSCOPE 8.0 program (Axon Instruments, Inc).
Após 30 minutos de estabilização dos abalos musculares, os registros foram realizados na presença de concentrações crescentes dos derivados do LASSBio- 294 (0,05-300 μΜ). Para solubilização das substâncias foi utilizado o dimetilsulfóxido (DMSO). Curvas concentração-resposta foram construídas com cada um dos derivados e com DMSO separadamente, no volume correspondente a cada concentração dos compostos em teste, para verificar sua influência na atividade contrátil do músculo cardíaco.  After 30 minutes of muscle shake stabilization, recordings were performed in the presence of increasing concentrations of LASSBio-294 derivatives (0.05-300 μΜ). For solubilization of the substances, dimethyl sulfoxide (DMSO) was used. Concentration-response curves were constructed with each derivative and with DMSO separately, at the volume corresponding to each concentration of the test compounds, to verify their influence on the contractile activity of the heart muscle.
EXEMPLO 8: Via de formação de óxido nítrico EXAMPLE 8: Nitric Oxide Formation Pathway
A hipótese a ser confirmada de que a formação de óxido nítrico nas células endoteliais poderia ser mediada pela ativação de receptores muscarínicos pelo LASSBio-897 foi avaliada utilizando-se o antagonista muscarínico não seletivo, atropina. A vasodilatação induzida pelo LASSBio-897 foi sensível a atropina (0,1 μΜ) pois quando a preparação de aorta com endotélio funcional foi pré-incubada com a atropina e mantida na cuba experimental durante a adição das concentrações crescentes do derivado após o platô da contratura induzida pela fenilefrina, nenhum efeito de relaxamento vascular foi observado. Porém o efeito inibitório da atropina foi revertido quando concentrações maiores (>1 μΜ) de LASSBio-897 foram adicionadas à preparação de aorta, indicando que este derivado poderia estar deslocando o antagonista (atropina) da sua ligação ao receptor muscarínico nas células endoteliais (Figura 11). Observou-se que a concentração inibitória média CI50 aumentou de 0,35 ± 0,02 μΜ na ausência da atropina para 10,15 ± 3,19 μ na presença deste antagonista muscarínico. Estes resultados indicaram que atropina promoveu um antagonismo do tipo competitivo, pois diminuiu a potência do LASSBio-897 sem alterar sua eficácia farmacológica. The hypothesis to be confirmed that the formation of nitric oxide in endothelial cells could be mediated by muscarinic receptor activation by LASSBio-897 was evaluated using the non-selective muscarinic antagonist atropine. LASSBio-897-induced vasodilation was sensitive to atropine (0.1 μΜ) because when aortic preparation with functional endothelium was preincubated with atropine and kept in the experimental well during the addition of increasing concentrations of the derivative after the plateau of Phenylephrine-induced contracture, no effect of vascular relaxation was observed. However, the atropine inhibitory effect was reversed when higher concentrations (> 1 μΜ) of LASSBio-897 were added to the aorta preparation, indicating that this derivative could be displacing the antagonist (atropine) from its binding to the muscarinic receptor in endothelial cells (Figure 11). The mean inhibitory concentration IC50 increased from 0.35 ± 0.02 μΜ in the absence of atropine to 10.15 ± 3.19 μ in the presence of this muscarinic antagonist. These results indicated that atropine promoted a competitive antagonism, as it decreased the potency of LASSBio-897 without altering its pharmacological efficacy.
EXEMPLO 9: Inibição das enzimas óxido nítrico sintase e guanilato ciclase EXAMPLE 9: Inhibition of the enzymes nitric oxide synthase and guanylate cyclase
O óxido nítrico é capaz de ativar a enzima guanilato ciclase solúvel, responsável pela síntese de GMPc, nucleotídeo que promove relaxamento da musculatura lisa. Com o objetivo de inibir a síntese de óxido nítrico e GMPc em anéis de aorta com endotélio funcional, foi usado L-NAME (100 μΜ), inibidor da óxido nítrico sintase e ODQ (10 μ ), inibidor da guanilato ciclase, trinta minutos antes da contração induzida pela fenilefrina e da adição das concentrações crescentes dos derivados. As Figuras 8 e 9, representam um traçado típico da incubação dos anéis de aorta com o derivado LASSBio-897.  Nitric oxide is capable of activating the soluble guanylate cyclase enzyme, responsible for the synthesis of cGMP, a nucleotide that promotes smooth muscle relaxation. In order to inhibit the synthesis of nitric oxide and cGMP in functional endothelial aorta rings, L-NAME (100 μΜ), nitric oxide synthase inhibitor and ODQ (10 μ), guanylate cyclase inhibitor, were used 30 minutes before. phenylephrine-induced contraction and the addition of increasing concentrations of the derivatives. Figures 8 and 9 show a typical outline of the incubation of aortic rings with the LASSBio-897 derivative.
Com este protocolo experimental foi percebido que LASSBio-897 não induziu o relaxamento vascular em nenhuma das concentrações testadas, tanto nos experimentos em que o inibidor da enzima óxido nítrico sintase quanto o inibidor da enzima guanilato ciclase foram utilizados (Figuras 8, 9 e 10 A e B). Estes dados estão de acordo com aqueles obtidos quando foi quantificado indiretamente o óxido nítrico através da reação de Griess e quando a quantificação de GMPc foi realizada de forma direta através do kit Imunoensaioenzimático para GMPc Sistema Biotrak (EIA).  With this experimental protocol it was found that LASSBio-897 did not induce vascular relaxation at any of the concentrations tested, either in experiments where nitric oxide synthase enzyme inhibitor or guanylate cyclase enzyme inhibitor were used (Figures 8, 9 and 10 A and B). These data are in agreement with those obtained when the nitric oxide was indirectly quantified by the Griess reaction and when the cGMP quantification was performed directly through the Biotrak System (EIA) cGMP Immunoassay Kit.
EXEMPLO 10: Efeito do efeito dos composto LASSBio na pressão arterial e frequência cardíaca - Tratamento agudo EXAMPLE 10: Effect of LASSBio Compound Effect on Blood Pressure and Heart Rate - Acute Treatment
Para verificar o efeito do LASSBio na pressão arterial e frequência cardíaca em animais normotensos e hipertensos, utilizou-se o protocolo experimental em que a artéria carótida e a veia jugular externa foram dissecadas e canulizadas para medida da pressão arterial sistólica (PS) e diastólica (PD) bem como para a administração intravenosa in ÒO/L S de LASSBionas doses de 1 e 5 mg/kg. LASSBio-897, administrado na dose de 5 mg/kg em ratos normotensos promoveu alteração significativa das PS e PD sem interferir com o padrão do ECG (Figura 8). To verify the effect of LASSBio on blood pressure and heart rate in normotensive and hypertensive animals, we used the experimental protocol in which the carotid artery and external jugular vein were dissected and cannulated to measure systolic (PS) and diastolic blood pressure ( PD) as well as for intravenous in ÒO / LS administration of LASSBionas doses of 1 and 5 mg / kg. LASSBio-897 administered at a dose of 5 mg / kg in normotensive rats promoted significant change in PS and PD without interfering with the ECG pattern (Figure 8).
Conforme pode ser observado na Tabela 5, a redução da pressão arterial média (PAM) induzida pelo LASSBio-897 em ratos normotensos não foi acompanhada por alteração da FC. A PS reduziu de 124,6 ± 2,8 para 51,7 ± 2,8 mmHg e a PD de 91 ,4 ± 5,7 para 23,3 ± 5,7 mmHg quando os animais normotensos foram tratados com 5 mg/kg desta substância.  As can be seen in Table 5, the reduction in LASSBio-897-induced mean arterial pressure (MAP) in normotensive rats was not accompanied by change in HR. PS decreased from 124.6 ± 2.8 to 51.7 ± 2.8 mmHg and PD from 91.4 ± 5.7 to 23.3 ± 5.7 mmHg when normotensive animals were treated with 5 mg / kg of this substance.
Efeito do LASSBio-897 na pressão sistólica e diastóiica de ratos normotensos Effect of LASSBio-897 on systolic and diastolic pressure of normotensive rats
Figure imgf000025_0001
Figure imgf000025_0001
Tabela 1. Injeção in bolus, em ratos normotensos, de LASSBio-897 nas doses de 1 e 5 mg/kg. Dados representam a média ± EP de 6 experimentos. *P<0,05 vs controle.  Table 1. In-bolus injection in normotensive rats of LASSBio-897 at doses of 1 and 5 mg / kg. Data represent the mean ± SE of 6 experiments. * P <0.05 vs control.
A injeção venosa de 5 mg/kg de LASSBio-897 nos SHR reduziu a PS de 195,0 ± 18,0 para 175,0 ± 26,4 mmHg e a PD de 145,0 ± 5,0 para 120,0 ± 8,6 mmHg sem alterar a FC (Tabela 6). Efeito do LÂSSBio-897 na pressão sistólica e diastólica de ratos hipertensos LASSBio-897 5 mg / kg venous injection into SHR reduced PS from 195.0 ± 18.0 to 175.0 ± 26.4 mmHg and PD from 145.0 ± 5.0 to 120.0 ± 8.6 mmHg without changing HR (Table 6). Effect of LSSBio-897 on systolic and diastolic pressure in hypertensive rats
Figure imgf000026_0001
Figure imgf000026_0001
Tabela 2. Injeção in bolus, em ratos hipertensos, de LASSBio-897 nas doses de 1 e 5 mg/kg. Dados representam a média ± EPM de 6 experimentos. *P<0,05 vs controle.  Table 2. In-bolus injection in hypertensive rats of LASSBio-897 at doses of 1 and 5 mg / kg. Data represent the mean ± SEM of 6 experiments. * P <0.05 vs control.

Claims

REIVINDICAÇÕES
Compostos derivados /V-acilidrazônico compreendendo a fórmula geral V-acylhydrazonic derivative compounds comprising the general formula
Figure imgf000027_0001
Figure imgf000027_0001
R1 e R2 podem ser H, alquilaC1-C6, alquenil C1-C6 ou arilalquil, sendo que tais radicais podem ser opcionalmente substituídos, insaturados e/ou ramificados e;  R 1 and R 2 may be H, C 1 -C 6 alkyl, C 1 -C 6 alkenyl or arylalkyl, such radicals may be optionally substituted, unsaturated and / or branched and;
R3, R4 e R5 podem ser H, grupo alquila contendo entre 1 a 6 átomos de carbono, fenila e fenila substituída, amino (secundário, terciário ou quaternário), nitro, RCOO-, -COOH, hidroxila, alcoxila, haleto, ciano, sulfonamtda, metilsulfonamida.  R3, R4 and R5 may be H, alkyl group containing from 1 to 6 carbon atoms, phenyl and substituted phenyl, amino (secondary, tertiary or quaternary), nitro, RCOO-, -COOH, hydroxyl, alkoxy, halide, cyano, sulfonamide, methyl sulfonamide.
2. Composto de acordo com a reivindicação 1 , onde preferencialmente R1 e R2 poderem ser H ou alquila C1-C6 e;  A compound according to claim 1, wherein preferably R 1 and R 2 may be H or C 1 -C 6 alkyl and;
R3, R4 e R5 podem ser H, grupo alquila contendo entre 1 a 6 átomos de carbono fenila e fenila substituída, amino (secundário, terciário ou quaternário), nitro, RCOO-, -COOH, hidroxila, alcoxila, haleto, ciano, sulfonamida, metilsulfonamida.  R3, R4 and R5 may be H, alkyl group containing from 1 to 6 carbon atoms phenyl and substituted phenyl, amino (secondary, tertiary or quaternary), nitro, RCOO-, -COOH, hydroxyl, alkoxy, halide, cyano, sulfonamide methylsulfonamide.
3. Composto de acordo com a reivindicação 1 úteis no tratamento de doenças e disfunções relacionadas à síntese do óxido nítrico, bem como, no tratamento de doenças associadas ao tecido muscular.  A compound according to claim 1 useful in the treatment of diseases and disorders related to nitric oxide synthesis as well as in the treatment of diseases associated with muscle tissue.
4. Composto de acordo com a reivindicação 3 compreendendo a capacidade de induzir relaxamento muscular seietivo em animais mamíferos humanos e não humanos. A compound according to claim 3 comprising the ability to induce selective muscle relaxation in human and non-human mammalian animals.
5. Processo de produção do composto de fórmula geral (I): 5. Production process of the compound of formula (I):
Figure imgf000028_0001
compreendendo as seguintes etapas:
Figure imgf000028_0001
comprising the following steps:
a) Esteriftcação do piperonal;  (a) piperonal esterification;
b) Hidrazinólise do éster (IV) produzido em a);  b) Hydrazinolysis of the ester (IV) produced in a);
c) Condensação do composto de fórmula (II);  c) Condensation of the compound of formula (II);
d) Alquilação do composto de fórmula (III).  d) Alkylation of the compound of formula (III).
6. Processo de acordo com a reivindicação 5 em que a etapa a) ocorre pela adição do piperonal a uma solução contendo um álcool, uma base forte e um halogênio, ocorrendo em uma temperatura entre -10°C a + 25°C, durante um período de tempo entre 20 a 240 minutos, sendo formado um éster.  The process according to claim 5 wherein step a) occurs by adding the piperonal to a solution containing an alcohol, a strong base and a halogen occurring at a temperature between -10 ° C to + 25 ° C for 20 to 240 minutes, an ester being formed.
7. Processo de acordo com a reivindicação 5 em que a etapa b) ocorre pela adição do éster formado na etapa a) em uma solução alcoólica contendo hidrazina, em um aparelho condensador com temperatura entre 50°C e 130°C.  A process according to claim 5 wherein step b) occurs by the addition of the ester formed in step a) in an alcoholic solution containing hydrazine in a condensing apparatus with a temperature between 50 ° C and 130 ° C.
8. Processo de acordo com a reivindicação 7 em que ao término da reação a solução é resfriada a uma temperatura entre -5°C a +15°C, para que ocorra a precipitação do composto obtido.  A process according to claim 7 wherein at the end of the reaction the solution is cooled to a temperature between -5 ° C to + 15 ° C to precipitate the obtained compound.
9. Processo de acordo com a reivindicação 5 em que a etapa c) ocorre a condensação da 1 ,3-benzodioxolil-5-carboidrazida com um composto da família 3- acil-tiofenos, funcionalizados em R2 e/ou R3 e/ou R4 e/ou R5, podendo os grupamentos R2, R3, R4 e R5 estarem funcionalizados de forma independente ou conjunta.  The process according to claim 5 wherein step c) condensing 1,3-benzodioxolyl-5-carbohydrazide with a compound of the family 3-acylthiophenes functionalized at R2 and / or R3 and / or R4 and / or R5, and the groups R2, R3, R4 and R5 may be independently or jointly functionalized.
10. Processo de acordo com a reivindicação 9 em que a reação ocorre sob agitação, na presença de um álcool primário ou secundário, à temperatura ambiente, em pH ácido em um período entre 20 a 120 minutos, ocorrendo precipitação do composto (III) no final da reação. A process according to claim 9 wherein the reaction takes place under agitation in the presence of a primary or secondary alcohol at room temperature. at pH 20 to 120 minutes, with precipitation of compound (III) occurring at the end of the reaction.
11. Processo de acordo com a reivindicação 5 em que na etapa d) ocorre a adição de um haleto de alquila, podendo ser empregada uma alquila saturada ou insaturada, ramificada ou não ramificada.  A process according to claim 5 wherein in step d) the addition of an alkyl halide occurs, a saturated or unsaturated, branched or unbranched alkyl may be employed.
12. Composição farmacêutica compreendendo um composto de fórmula geral (I):  A pharmaceutical composition comprising a compound of formula (I):
Figure imgf000029_0001
Figure imgf000029_0001
R1 e R2 podem ser H, alquilaC1-C6, alquenil C1-C6 ou arilalquil, sendo que tais radicais podem ser opcionalmente substituídos, insaturados e/ou ramificados e;  R 1 and R 2 may be H, C 1 -C 6 alkyl, C 1 -C 6 alkenyl or arylalkyl, such radicals may be optionally substituted, unsaturated and / or branched and;
R3, R4 e R5 podem ser H, grupo alquila contendo entre 1 a 6 átomos de carbono, fenila e fenila substituída, amino (secundário, teciário opu quaternário), nitro, RCOO-, -COOH, hidroxila, alcoxila, haleto, ciano, sulfonamida, metilsulfonamida;  R3, R4 and R5 may be H, alkyl group containing from 1 to 6 carbon atoms, phenyl and substituted phenyl, amino (secondary, opu quaternary weaver), nitro, RCOO-, -COOH, hydroxyl, alkoxy, halide, cyano, sulfonamide, methyl sulfonamide;
e seus sais, solvatos e racematos farmaceuticamente aceitáveis e; compostos não ativos farmaceuticamente aceitáveis voltada para o tratamento de doenças e/ou disfunções relacionadas à síntese do óxido nítrico em um animal mamífero humano ou não humano, são também um objeto desta invenção..  and pharmaceutically acceptable salts, solvates and racemates thereof and; Pharmaceutically acceptable non-active compounds directed to the treatment of diseases and / or dysfunctions related to nitric oxide synthesis in a human or non-human mammalian animal are also an object of this invention.
12. Composição de acordo com a reivindicação 11 , onde as disfunções relativas à síntese do óxido nítrico podem ser a disfunção erétil, a hipertensão arterial, angina, infarto dentre outras. A composition according to claim 11, wherein the dysfunctions related to nitric oxide synthesis may be erectile dysfunction, hypertension, angina, infarction among others.
13. Composição farmacêutica compreendendo compostos derivados N- acilidrazônicos, de fórmula molecular (I) A pharmaceutical composition comprising N-acylhydrazonic derivative compounds of molecular formula (I)
Figure imgf000030_0001
onde:
Figure imgf000030_0001
Where:
R2 podem ser H, alquilaC1-C6, alquenil C1-C6 ou arilalquil, sendo que tais radicais podem ser opcionalmente substituídos, insaturados e/ou ramificados e;  R 2 may be H, C 1 -C 6 alkyl, C 1 -C 6 alkenyl or arylalkyl, such radicals may be optionally substituted, unsaturated and / or branched and;
R3, R4 e R5 podem ser H, grupo alquila contendo entre 1 a 6 átomos de carbono, fenila e fenila substituída, amino (secundário, teciário opu quaternário), nitro, RCOO-, -COOH, hidroxila, alcoxila, haleto, ciano, sulfonamida, metilsulfonamida;  R3, R4 and R5 may be H, alkyl group containing from 1 to 6 carbon atoms, phenyl and substituted phenyl, amino (secondary, opu quaternary weaver), nitro, RCOO-, -COOH, hydroxyl, alkoxy, halide, cyano, sulfonamide, methyl sulfonamide;
e/ou seus sais, solvatos e racematos farmaceuticamente aceitáveis; além de compostos não ativos farmaceuticamente aceitáveis voltadas para o tratamento de distúrbios musculares, em animais mamíferos humanos e/ou não humanos.  and / or pharmaceutically acceptable salts, solvates and racemates thereof; in addition to pharmaceutically acceptable non-active compounds for the treatment of muscle disorders in human and / or non-human mammalian animals.
1 . Uso de um composto compreendendo a fórmula geral (I):  1 . Use of a compound comprising the general formula (I):
Figure imgf000030_0002
onde:
Figure imgf000030_0002
Where:
R1 e R2 podem ser H, alquilaC1-C6, alquenil C1-C6 ou arilalquil, sendo que tais radicais podem ser opcionalmente substituídos, insaturados e/ou ramificados e; R3, R4 e R5 podem ser H, grupo alquila contendo entre 1 a 6 átomos de carbono, fenila e fenila substituída, amino (secundário, teciário opu quaternário), nitro, RCOO-, -COOH, hidroxila, alcoxila, haleto, ciano, sulfonamida, metilsulfonamida; R 1 and R 2 may be H, C 1 -C 6 alkyl, C 1 -C 6 alkenyl or arylalkyl, such radicals may be optionally substituted, unsaturated and / or branched and; R3, R4 and R5 may be H, alkyl group containing from 1 to 6 carbon atoms, phenyl and substituted phenyl, amino (secondary, opu quaternary weaver), nitro, RCOO-, -COOH, hydroxyl, alkoxy, halide, cyano, sulfonamide, methyl sulfonamide;
e seus sais, derivados e/ou solvatos farmacêuticamente aceitáveis; na preparação de um medicamento destinado ao tratamento ou prevenção de disfunções relativas à síntese do óxido nítrico em mamíferos.  and pharmaceutically acceptable salts, derivatives and / or solvates thereof; in the preparation of a medicament for the treatment or prevention of nitric oxide synthesis disorders in mammals.
15. Uso de um composto compreendendo a fórmula geral (I):  Use of a compound comprising the general formula (I):
Figure imgf000031_0001
Figure imgf000031_0001
R1 e R2 podem ser H, alquilaC1-C6, alquenil C1-C6 ou arilalquil, sendo que tais radicais podem ser opcionalmente substituídos, insaturados e/ou ramificados e;  R 1 and R 2 may be H, C 1 -C 6 alkyl, C 1 -C 6 alkenyl or arylalkyl, such radicals may be optionally substituted, unsaturated and / or branched and;
R3, R4 e R5 podem ser H, grupo alquila contendo entre 1 a 6 átomos de carbono, fenila e fenila substituída, amino (secundário, teciário opu quaternário), nitro, RCOO-, -COOH, hidroxila, alcoxila, haleto, ciano, sulfonamida, metilsulfonamida; e seus sais, derivados e/ou solvatos farmacêuticamente aceitáveis; na preparação de um medicamento destinado ao tratamento e/ou prevenção de doenças associadas ao tecido muscular.  R3, R4 and R5 may be H, alkyl group containing from 1 to 6 carbon atoms, phenyl and substituted phenyl, amino (secondary, opu quaternary weaver), nitro, RCOO-, -COOH, hydroxyl, alkoxy, halide, cyano, sulfonamide, methyl sulfonamide; and pharmaceutically acceptable salts, derivatives and / or solvates thereof; in the preparation of a medicament for the treatment and / or prevention of diseases associated with muscle tissue.
16. Uso, de acordo com a reivindicação 15, onde as doenças são escolhidas do grupo que compreende a insuficiência coronariana, espasmos do músculo liso vascular sistémico, dificuldades de ereção, espasmos de músculos esqueléticos e espasmos da musculatura lisa respiratória.  Use according to claim 15, wherein the diseases are chosen from the group comprising coronary insufficiency, systemic vascular smooth muscle spasms, erection difficulties, skeletal muscle spasms, and respiratory smooth muscle spasms.
17. Método de tratamento de doenças e disfunções ligadas à síntese do óxido nítrico compreendendo a administração de um composto de fórmula geral A method of treating nitric oxide synthesis related disorders and disorders comprising administering a compound of formula
(I)
Figure imgf000032_0001
, e/ou seus sais, solvatos e racematos a um mamífero humano e/ou não humano que apresente síntese reduzida ou insuficiente de óxido nítrico. (I)
Figure imgf000032_0001
and / or their salts, solvates and racemates to a human and / or non-human mammal having reduced or insufficient nitric oxide synthesis.
18. Método de tratamento de doenças de doenças associadas ao tecido muscular compreendendo administração de um composto de fórmula geral (I):  A method of treating diseases of muscle tissue associated diseases comprising administering a compound of formula (I):
Figure imgf000032_0002
Figure imgf000032_0002
sais, solvatos e racematos a um animal mamífero humano ou não humano. salts, solvates and racemates to a human or non-human mammalian animal.
PCT/BR2009/000320 2008-10-16 2009-10-15 N-acyl hydrazine derivatives, method for producing n-acyl hydrazine compounds, pharmaceutical compositions containing same, uses thereof and methods for treatment WO2010043010A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0403363A (en) * 2004-08-20 2006-05-02 Univ Rio De Janeiro selective muscle relaxants and pharmaceutical compositions
US7091238B1 (en) * 1999-06-21 2006-08-15 University Of Maryland Thienylhydrazon with digitalis-like properties (positive inotropic effects)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7091238B1 (en) * 1999-06-21 2006-08-15 University Of Maryland Thienylhydrazon with digitalis-like properties (positive inotropic effects)
BRPI0403363A (en) * 2004-08-20 2006-05-02 Univ Rio De Janeiro selective muscle relaxants and pharmaceutical compositions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SILVA, A.G. ET AL.: "Synthesis and vasodilatory activity of new N-acylhydrazone derivatives, designed as LASSBio-294 analogues.", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 13, 2005, pages 3431 - 3437 *

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