Patent Application
20100196476
The present invention refers to the field of 4-quinolinones and derived quinolines, to be used in pharmaceutical formulations as synthetic protease inhibitors, such as haemorrhagic metalloprotease resulting from bites of the snakes of the Bothrops genus and other applications.
Snake venoms comprise complex protein mixtures including phospholipases A2, myotoxins, haemorrhagic metalloprotease, coagulant serine protease, cytotoxins, cardiotoxins and others. The pathophysiology of poisoning by snakes involves a complex series of events that depend on the combined action of these components. (Gutiérrez, J. M. “Comprendiendo los venenos de serpientes: 50 años de investigaciones en América Latina” [Understanding snake venoms: 50 years of research in Latin América]. Rev. Biol. Trop., 50:377, 2002). Both phospholipases A2 and proteases are abundantly present in snake venom, as well as having a digestive role in the hydrolysis of phospholipids and proteins. These enzymes may present a broad variety of pharmacological activities, such as neurotoxicity, myotoxicity as well as oedematogenic, haemorrhagic and coagulant activities, amongst others (Gutiérrez, J. M. & Lomonte, B. “Phospholipase A2, myotoxins from Bothrops snake venoms”. Toxicon, 33:1405, 1995.; Ownby C. L. J. “Structure, function and biophysical aspects of the myotoxins from snake venoms”. Toxicol.-Toxins Review, 17:213, 1998.; Ownby, C. L.; Araujo, H. S. S.; White, S. P. & Fletcher, J. E. “Lysine 49 phospholipase A2 proteins”. Toxicon, 37: 411, 1999.; Soares, A. M.; Fontes, M. R. M. & Giglio, J. R. Phospholipase A2 myotoxins from Bothrops snake venoms: Structure-Function relationship. Review. Curr. Org. Chem., 8: 1677, 2004.).
Pharmacological studies have demonstrated that the extracts and fractions of certain plants used in traditional medicine possess anti-inflammatory, antiviral and antivenin properties (Martz, W. “Plants with a reputation against snakebite”. Toxicon, 30:1131, 1992.; Mors, W. B.; Nascimento, M. C.; Pereira, B. M. R. & Pereira, N. A. “Plant natural products active against snake bite—the molecular approach”. Phytochemistry, 55: 627, 2000.; Soares, A. M.; Ticli, F. K.; Marcussi, S.; Lourenço, M. V.; Januário, A. H.; Sampaio, S. V.; Giglio, J. R.; Lomonte, B. & Pereira, P. S. “Medicinal plants with inhibitory properties against snake venoms”. Curr. Med. Chem., 12:2625, 2005.; Soares, A. M.; Januário, A. H.; Lourenço, M. V.; Pereira, A. M. S.; Pereira, P. S. Drugs Future 29:1105, 2004.)
Patent application US2004/0242639 presents the activity of a phospholipase C inhibitor as therapy for inflammatory diseases. On the other hand, quinolinones with N-heteroamino in position 5 of the quinolinone ring described in publication WO94/10163 (and the corresponding U.S. Pat. No. 5,646,163 and Brazilian patent applications BR 9307347 and BR 9507553) present antimicrobial activity, while the quinolinones substituted in position 3 described in this invention present inhibitor activity for phospholipase C.
Patent document EP0304158 claims quinolones possessing anti-bacterial activity with variations of the substitutes in positions and 3, while patent application JP2000273086 describes quinolinones only substituted in position 2.
Despite the structures described in publication CN1817880A being of the type 4-quinolinones, the substitute in position 2 differs from those included in the claims herein.
Patent document WO01/53266 describes type 4-quinolinone structures substituted in position 3 that are useful for treating diseases associated to white blood cell disorders such as autoimmune and inflammatory diseases but, however, are distinct from the structures proposed in the present invention.
The US patent application published as US2003/0124120 describes quinolinones with antagonistic activity in vitronectin receptors and presents variants that may be either in positions 2 or 3.
Certain 2-quinolinone compounds having serine protease activity were described in U.S. Pat. No. 6,855,726. Yet other quinolinone structures were claimed in patent document WO01/70698.
The international publication WO2004/007461 describes a method for the treatment or prophylaxis of a neurological condition—more specifically neurodegenerative disorders—that consists administering an effective quantity of a composition of the formula (I) to a patient requiring treatment. The standard skeleton of the molecule that is the object of that publication is similar to the compounds of the present invention but, however, despite the many variations of the substitutes described, no skeleton suggests or describes compounds similar to the compounds described herein.
In the same manner, U.S. Pat. No. 5,444,071 and patent application US2006/0217322 describe compounds having pharmaceutical activity with basic skeletons similar to those of the compounds proposed herein but none of these structures anticipates those of the present invention.
U.S. Pat. No. 5,102,892 describes compounds with a quinoline structure similar to those described in the present invention but, however, varying the position 4—from Oxygen to Nitrogen.
International publication WO2006/068617 presents a methodology for the preparation of enamine using dimethyl acetylenedicarboxylate (DMAD) but, however, the input materials and products obtained are different from those that constitute the object of the present invention. No intermediate similar to the compounds of that invention is suggested or described.
The publications BR1100774, WO02/200625 and WO97/21680 describe other classes of quinolines different from those comprising the object of the present invention.
In the compounds described in U.S. Pat. No. 5,789,419, U.S. Pat. No. 4,412,075 and U.S. Pat. No. 4,593,101, the skeletons present a difference compared to the double conjugate that is not found in the compounds of the present invention and neither use the conjugate system proposed herein. Furthermore, patent document U.S. Pat. No. 5,789,419 describes compounds intended to treat bronchial e circulatory system disorders.
The structures described in patent EP1458718 are of the type 2-quinolinones and the activity is present in various receptors.
U.S. Pat. No. 4,859,669, U.S. Pat. No. 6,271,416, CN1594295, U.S. Pat. No. 6,855,726, patent application US2005/0054672, publications EP1574501, EP1097139B1, EP1270006 and patent application US 2005/0209247 describe 2-quinolinones structurally distinct from those object of the present invention.
Patent documents EP1245566 and U.S. Pat. No. 6,645,983 describe 4-quinolinones but, however, the structure described does not include a substitute in position 2 and the biological activities are respectively described as being anti-microbial and intended for the treatment of cancers.
Publication EP0251308 describes a fluoroquinolinone as substitute in positions 2 and 3 of the quinolinone system. R2 is H or S linked to the N of the ring by an ethylene bridge.
Thus, the literature shows that despite considerable technological development, there is still great need for new 4-quinolinones obtained through processes using derivates of borane as ester reducing agents in the presence of enone, with these compounds being useful as protease, lipase and phospholipase inhibitors as well as having other medicinal applications such as for disorders affecting the haemostatic system or white blood cells. The process for the preparation, the compounds and the pharmaceutical formulations of the 4-quinolinones and their quinoline derivates are described and claimed herein.
Overall, the present invention refers to derivates of 4-quinolinone in accordance with the formula below:
Whereby:
R1 and R2 are selected independently of each other, with H, OH, an alkyl group of C1-C4, an alkoxy group of C1-C5, a —OCO—R7 group, and a group derived from a saccharide, optionally R1 and R2 together forming a methylenedioxy group, a phenyl group or a phenyl group substituted in 1 and 3 with groups selected from H, an alkoxy group in C1-C4, a —OCOR7 group, a —O—SO2—R7 group, halogen, an alkyl or CF3 group, and —NR7R8 group, in which R7 and R5 are selected independently of each other, from hydrogen, alkyl group in C1-C5, alkenyl group, alkyl phenyl group (C1-C4), dimethylamine, rings with 4-6 member heterocycles, optionally with one or more heteroatoms selected from oxygen, nitrogen and sulphur or a methylpiperazinyl group;
R3 is selected from H, alkyl group in C1-C4, alkenyl group, a —CO—R8 group and a -A-R9 group, —CO2R9′ group in which R9′ is a benzyl group, branched or linear alkyl group, p-methoxy benzyl group, —NH2, —CHCH2CH2, R8 is an alkyl group in C1-C4; A is an alkylene group in C1-C4; R9 is selected from heterocycle groups with 5 or 6 members containing 1 to 4 heteroatoms of oxygen, sulphur and nitrogen, CN, hydroxyl, —COOR10 and CONR11R12 groups, a —NR13R14 group, a —COR15 group and a OSO2R16 group;
R10, R11, R12, R14 and R15 are independently selected from hydrogen, alkyl groups in C1-C4, halogen and alkyl phenyl group (C1-C4), R16 is selected from the phenyl group and the alkyl phenyl group (C1-C4);
R4═OH, halogens, alkoxy group in C1-C6, alkoxy benzyl group, —CO—R17 in which R17 is alkyl C1-C6 or p-methoxy benzyl, —O—SO2—R7′ in which R7′ is an alkyl group or CF3 group, group derived from saccharide; R5 is H, halogen, phenyl group or phenyl substituted 1 or 3 times with groups selected from H, alkoxy groups C1-C4, a —OCOR7 group, a —O—SO2—R7′ group in which R7′ is an alkyl group or CF3 group, benzylamine group, and group derived from saccharide, alkyl group, —COOH, or salts, hydrates and pharmacologically acceptable pro-pharmacons.
The compounds of Formula (I) produce the quinolines of Formula (II) in the presence of a K2CO3 base and alkylating agents:
Whereby:
The invention also refers to a process for preparing the compounds of formula (I) and formula (II), with the above mentioned processes comprising the stages of:
The invention provides compounds 1, 2, 10, 11 and the general formula (I) and (II) as being protease, lipase, phospholipase and enzyme inhibitors.
The invention provides compounds 1, 2, 10, 11 and the general formula (I) and (II) to be used in the broad aspect of inflammatory, antirheumatic, analgesic, autoimmune, antivenin, antithrombotic, anti-allergic and expectorant activities, as well as white blood cell disorders and haemostatic system disorders amongst other possible pharmaceutical applications.
The invention provides compositions, formulations or medicines containing effective amounts of compounds 1, 2, 10, 11 and the general formula (I) and (II) or their pharmaceutically acceptable salts.
The invention also provides compositions including the compounds 1, 2, 10, 11 and the general formula (I) and (II) to be used in the broad aspect of inflammatory, antirheumatic, analgesic, autoimmune, antivenin, antithrombotic, anti-allergic and expectorant activities, as well as white blood cell disorders and haemostatic system disorders amongst other possible pharmaceutical applications.
The invention also provides pharmaceutical formulations prepared in the form of pills, coated pills, capsules, inhalable powder, effervescent tablets, sublingual pills, syrups and oral solutions, injectable solutions, ointments, creams, gels and other pharmaceutical preparations known in pharmaceutical techniques.
The invention also provides the administration of the above mentioned formulations by oral, rectal, topical or parenteral route, with the active principle in a quantity not less than 0.001% of the composition's final weight together with at least one pharmaceutically appropriate excipient.
The invention also provides pharmaceutical formulations that comprise: a) a compound of general formula (I) as an active principle in a quantity not less than 0.001% of the composition's final weight, and b) at least one pharmaceutically appropriate excipient.
The invention also provides pharmaceutical formulations that comprise: a) a compound of general formula (II) as an active principle in a quantity not less than 0.001% of the composition's final weight, and b) at least one pharmaceutically appropriate excipient.
Furthermore, the invention provides pharmaceutical formulations that comprise the 4-quinolinones and quinoline derivates of the present invention to be administered to animals and/or humans.
A first aspect of the invention are the 4-quinolinone compounds and quinoline derivates in accordance to formula (I) and formula (II).
A second aspect of the present invention relates to the preparation process of the 4-quinolinones with pharmacological activity and, more specifically, activity against the venom of the Bothrops genus snakes.
A third aspect of the invention are the medicinal formulations containing an efficient quantity of a 4-quinolinone compound (or a quinoline derivate) of the invention.
A fourth aspect of the invention is the use of the formulated compounds as inhibitors of metalloprotease, serine protease, autoimmune and inflammatory diseases including rheumatism as well as for anti-coagulant, antivenin, analgesic and antithrombotic purposes amongst other pharmaceutical applications.
According to the process of the invention, the 4-quinolinones of the invention, 2-hydroxymethyl-1,4-dihydro-4-quinolinone (compound 1) and 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (compound 2), are synthetised using a methodology that uses BH3.5Me2 as reducer agent and uses 4-oxo-1,4-dihydro-2-quinoline methyl carboxylate (compound 3) and 6-methoxy-4-oxo-1,4-dihydro-2-quinoline methyl carboxylate (compound 4) as initial material. Furthermore, 1 and 2 respectively provide the quinoline compounds 10 and 11 by reaction with K2CO3, DMF, EtBr 80% followed by NaH, EtBr 80%.
The reduction reaction using borane-dimethyl sulphide occurs selectively in the ester group rather than in the enone group of the 4-quinolinone system. Apart from these novel compounds, it is further possible to prepare intermediates and derivates.
The biological evaluation of these compounds was for antivenin activity. Snake venoms comprise complex mixtures of proteins including phospholipases A2 (PPA2), myotoxins, haemorrhagic metalloproteases, coagulant serine proteases, cytotoxins, cardiotoxins and others. The pathophysiology of snake poisoning involves a complex series of events that depends on the combined action of these components (Gutiérrez, J. M. “Comprendiendo los venenos de serpientes: 50 años de investigaciones en América Latina” [Understanding snake venoms: 50 years of research in Latin América]. Rev. Biol. Trop., 50:377, 2002). Both phospholipases A2 and proteases are abundantly present in snake venom, as well as having a digestive role in the hydrolysis of phospholipids and proteins. These enzymes may present a broad variety of pharmacological activities, such as neurotoxicity, myotoxicity as well as oedematogenic, haemorrhagic and coagulant activities, amongst others (Gutiérrez, J. M. & Lomonte, B. “Phospholipase A2, myotoxins from Bothrops snake venoms”. Toxicon, 33:1405, 1995.; Ownby C. L. J. “Structure, function and biophysical aspects of the myotoxins from snake venoms”. Toxicol.-Toxins Review, 17:213, 1998.; Ownby, C. L.; Araujo, H. S. S.; White, S. P. & Fletcher, J. E. “Lysine 49 phospholipase A2 proteins”. Toxicon, 37: 411, 1999.; Soares, A. M.; Januário, A. H.; Lourenço, M. V.; Pereira, A. M. S.; Pereira, P. S. Drugs Future 29:1105, 2004).
The preparation processes of the compounds of the present invention are summarily described in Diagram 1 below:
The details of each stage are explained below.
The enamines 5 and 6 are obtained based on the methodology described by Edmont, D.; Rocher, R.; Plisson, C. & Chenault, J. “Synthesis and evaluation of quinoline carboxyguanidines as antidiabetic agents” Bioorg. Med. Chem. Lett., 10: 1831, 2000, with a 50% yield.
The preparation process of the compounds 5 and 6 consists of the following experimental procedure:
A balloon flask containing anilin 3 (5.0 g, 0.054 mol) in dry MeOH (54 mL), has dimethyl acetylenodicarboxylate (DMAD) (7.7 g, 0.054 mol) added under N2 at 55° C. The reaction is monitored by Fine Layer Chromatography (FLC) using n-hexane as an eluent. At the end of the reaction the MeOH is evaporated, after which CH2Cl2 (30 mL) is added for extraction and the organic phase is washed in a saturated solution of NH4Cl (3×10 mL) followed by water (3×10 mL). The organic phase is dried and evaporated. Compound 5 is obtained with a yield of 50% (6.3 g).
RMN 1H (200 MHz, CDCl3) δ: 9.67 (sl, 1H); 7.32-7.25 (m, 2H); 7.13-7.05 (m, 1H); 6.92-6.88 (m, 2H); 5.39 (s, 1H); 3.74 (s; 3H); 3.70 (s, 3H).
RMN 13C (50 MHz, CDCl3) δ: 169.75; 164.72; 147.91; 140.18; 129.03; 124.13; 120.60; 116.56; 93.46; 52.62; 51.07.
EM (relative intensity %) m/z: 235.25 (6.9); 144.15 (93); 77.10 (100).
IV (νmax, KBr) cm−1: 3457; 3380; 2953; 1739; 1668; 1282; 1031.
A balloon flask containing anisiline 4 (5.0 g, 0.041 mol) in dry MeOH (41 mL), has (DMAD) (5.8 g, 0.041 mol) added under N2 at 55° C. The reaction is monitored by (FLC) using n-hexane as an eluent. At the end of the reaction the MeOH is evaporated, after which CH2Cl2 (30 mL) is added for extraction and the organic phase is washed in a saturated solution of NH4Cl (3×10 mL) followed by water (3×10 mL). The organic phase is dried and evaporated. Compound 6 is obtained with a yield of 50% (5.5 g).
RMN 1H (200 MHz, CDCl3) δ: 9.57 (sl, 1H); 6.91-679 (m, 4H); 5.30 (s, 1H); 3.78 (s, 3H); 3.73 (s, 3H); 3.67 (s, 3H). RMN 13C (50 MHz, CDCl3) δ: 182.44; 170.0; 164.76; 156.87; 148.99; 133.39; 122.96; 114.34; 91.66; 55.40; 52.61; 21.01.
IV (νmax, KBr) cm−1: 3284; 3210; 2952; 2836; 1742; 1637; 1033.
EM (relative intensity %) m/z: 265.50 (13); 146.15 (75); 77.15 (95).
Intramolecular cyclisation is achieved through reaction at high temperature. The nucleophilic attack on the ester carbonyl is directed by the nitrogen which is an ortholpara director and thus closes the ring.
In laboratory, a balloon flask containing a reflux condenser has ether diphenyl (8 mL) and reflux added, after which enamine 5 (1 g, 4.25 mmol) is added and after a specific time this system is removed from the sand bath and immersed in ice with the precipitation of the substrates being observed. A pre-purification is performed using dry-flash separation with a gradient elution of n-hexane to methanol. Compound 7 is obtained with a yield of 70% (600 mg) following recrystallisation.
P.F.=215-225° C.
RMN 1H (400 MHz, CDCl3) δ: 12.03 (s, 1H); 8.13 (d, J=8.0 Hz, 1H); 7.95 (d, J=10 Hz, 1H); 7.66-7.63 (m, 1H); 7.35-7.32 (m, 1H); 6.73 (s; 1H); 3.99 (s; 3H).
RMN 13C (100 MHz, CDCl3) δ: 176.39; 161.09; 138.44; 135.75; 130.55; 124.37; 123.04; 122.09; 117.89; 108.77; 51.53.
IV (νmax, KBr) cm−1: 3436; 2925; 2886; 1733; 1639; 1538; 1033.
EM (relative intensity %) m/z: 203 (30); 143 (100); 115.15 (73); 89.15 (66).
In a balloon flask containing a reflux condenser ether diphenyl (8 mL) was added, refluxed, after which enamine 6 (1.0 g, 3.8 mmol) was added and after a determined time the reflux was ended and the balloon was immersed in ice, observing the precipitation of the substrates. A pre-purification is performed using dry-flash separation with a gradient elution of hexane to methanol. Compound 8 is obtained with a yield of 60% (530 mg) following recrystallisation.
P.F.=250-260° C.
RMN 1H (400 MHz, CDCl3) δ: 12.14 (s, 1H); 7.91 (d, J=9.1 Hz, 1H); 7.46 (d, J=2.8 Hz, 1H); 7.66 (dd, J=9.0 e 2.8 Hz, 1H); 6.67 (s, 1H); 3.96 (s, 3H); 3.85 (s, 3H).
RMN 13C (100 MHz, CDCl3) δ: 176.30; 162.28; 155.78; 136.18; 134.16; 126.73; 122.84; 120.91; 108.34; 103.19; 54.94; 52.90.
IV (νmax, KBr) cm−1: 3440; 2935; 2865; 1729; 1639; 1552; 1024.
EM (relative intensity %) m/z: 207.50 (54); 173.2 (91); 73.2 (100).
The compounds obtained were characterised using RMN 1H and 13C and it was observed that one of the methoxyls of enamines 5 and 6 disappeared. The shift rate of the ester methoxyls to the closed system was at 3.99 and 3.96 ppm respectively for compounds 7 and 8.
The hydrogen in position 3 shifted at 6.73 and 6.67 ppm respectively for 5 and 6. It is possible to observe the hydrogens linked to the nitrogens (position 1) at 12.03 and 12.14 ppm respectively for 7 and 8.
The presence of methoxyl modifies the coupling systems in the aromatic system and in the case of 7 ortho couplings with J at 8.0 and 10 Hz are observed with the signal at 8.14 ppm (J=8.0 Hz) referring to H 9 while H 6 shows a shift at 7.97 ppm (J=10.0 Hz), H 8 shows a shift at 7.35 ppm while the H 7 signal is observed at 7.66 ppm.
The spectrum of RMN 13C for 7 shows a carbonyl signal (in carbon C4) α,β-insaturated at 176.39 ppm while C3 shows a shift at 108.76 ppm.
However, in the case of compound 8, a duplicate was observed at 7.9 ppm with J=9 Hz referring to H 8 and the dd at 7.39 ppm, with J of 9.0 and 2.8 Hz referring to H 7, since it has an ortho coupling with H 8 and a meta coupling with H 6. The spectrum of RMN 13C for 7 shows a carbonyl (C4) α,β-insaturated at 176.29 ppm while C3 shows a shift at 103.76 ppm.
Selective reductions of functional carbonyl groups are important reactions in organic synthesis and several reducer agents have been developed. The borane-dimethyl sulphide (BH3.5Me2) complex is used in the reduction of esters with a strong preference for the group located in the a position of the hydroxyl groups (Saito, S., Ishikawa, T., Kuroda, A., Koga, K. & Moriwake, T. “A revised mechanism for chemoselective reduction of esters with borane-dimethyl sulfide complex and catalytic sodium tetrahydroborate directed by adjacent hydroxyl group”. Tetrahedron, 48: 4067, 1992).
It is acknowledged that the reduction of ketones in the presence of enones is possible using BH3.5Me2, while the ester group may be reduced with diisobutyl aluminium hydride (DIBAL-H) in the presence of enones (Larock, R. C. “Comprehensive organic transformations” A guide to functional group preparations p 537, 1989). There does not seem to be any prior mention in the literature for the type of system present in compounds 7 and 8.
Therefore, the research required for the present invention was initially directed at testing reduction using BH3.5Me2 due to the simplicity of the work-up for this reagent when compared to DIBAL-H since borane may be removed from the reagent medium through distillation with anhydrous MeOH without the addition of water and it is also possible to remove any other impurities through recrystallisation.
The reduction reaction of the ester group to the hydroxyl group using borane proves to be chemoselective and it is possible to obtain both compounds 1 and 2 with a yield of 70%.
Experimentally, a balloon flask containing compound 7 (500 mg, 2.5 mmol) in anhydrous THF (10 mL) under N2 atmosphere at 0° C. has pure BH3.5Me2 complex (233 μL, 2.5 mmol) in a solution of THF (3 mL) added drop-by-drop. When the entire solution has been added, the reactor flask is left at room temperature. The reaction is monitored by FLC using AcOEt as eluent. Anhydrous MeOH (10 mL) is then added after 24 hours of reaction and the solution is distilled. Anhydrous MeOH (3×15 mL) is then added again. Compound 1 is obtained with a characterized yield of 70% (300 mg).
P.F.=230-240° C.
RMN 1H (200 MHz, CDCl3) δ: 8.04 (d, J=8 Hz, 1H); 7.69-7.56 (m, 2H); 7.31-7.23 (m, 1H); 6.02 (s, 1H); 4.48 (s, 2H).
RMN 13C (50 MHz, CDCl3) δ: 153.49; 140.42; 131.56; 125.25; 124.95; 122.88; 118.59; 105.59; 60.47.
IV (νmax, KBr) cm−1: 3384; 2946; 2917; 1619; 1359; 1083.
A balloon flask containing compound 8 (500 mg, 2.16 mmol) in anhydrous THF (10 mL) under N2 atmosphere at 0° C. has pure BH3.5Me2 complex (204 μL, 2.16 mmol) in a solution of THF (2 mL) added drop-by-drop. When the entire solution has been added, the reactor flask is left at room temperature. The reaction is monitored by FLC using AcOEt as eluent. Anhydrous MeOH (10 mL) is then added after 24 hours of reaction and the solution is distilled. Anhydrous MeOH (3×15 mL) is then added again, since the distillation process removes the remaining residues and impurities of BH3SMe2. Compound 2 is obtained with a characterized yield of 70% (300 mg).
P.F.=250-260° C.
RMN 1H (400 MHz, CDCl3) δ: 7.95-7.92 (m, 1H); 7.52-7.41 (m, 2H); 6.63 (s, 1H); 4.72 (s, 2H); 3.89 (s, 3H).
RMN 13C (100 MHz, CDCl3) δ: 171.50; 156.79; 155.51; 134.45; 124.16; 123.15; 120.97; 103.11; 102.71; 60.00; 55.61.
IV (νmax, KBr) cm−1: 3448; 2921; 2852; 1637; 1504; 1035.
The compounds are characterised by RMN 1H, whereby the forming of compound 1 is confirmed by the disappearance of the methoxyl group signal (3.99 ppm) and the appearance of the carbonylic methylene signal at 4.48 ppm, while the carbonylic methylene signal appears on the spectrum of RMN 13C at 60.47 ppm.
In the case of compound 2, the signal appearing at 4.72 ppm relates to carbinolic methylene and the disappearance pf methoxyl at 3.96 ppm.
The preparation of the quinolinic derivates occurs by a fast and clean reaction using DMF as solvent and K2CO3 as base and, depending on the intended product, using the alkylating agents EtBr or MeI (Edmont, D.; Rocher, R.; Plisson, C. & Chenault, J. “Synthesis and evaluation of quinoline carboxyguanidines as antidiabetic agents” Bioorg. Med. Chem. Lett., 10: 1831, 2000). The balance is shifted to form the O-alkylated product, with the selectivity depending on factors such as the alkyl halid structure, ring substitutes and the solvent (Comins, D. L. & Jianhua, G. “N- vs O-alkylation in the Mitsunobu reaction of 2-pyridone”. Tetrahedron Lett., 35: 2819, 1994.). All compounds are obtained using the same reaction conditions and the yields were moderate to good (60-80%).
A flask containing compound 8 (20 mg, 0.1 mmol) and K2CO3 (20.7 mg, 0.15 mmol) has added anhydrous DMF (50 μL) and MeI (9.5 μL, 0.15 mmol). The flask is agitated for a period of 12 hours at ambient temperature. The solution is then filtered using silica so as to remove the precipitate and the concentrated solvent following which the resulting material is purified using column chromatography using a gradient of n-hexane—methanol as eluent resulting in 17 mg of product 9 (80% yield).
P.F.: 105-107° C.
RMN 1H (400 MHz, CDCl3) δ: 8.11 (d, J=9.2 Hz, 1H); 7.55 (s, 1H); 7.46 (d, J=2.8 Hz, 1H); 7.48 (dd, J=9.2, 2.8 Hz, 2H); 4.36 (q, J=7 Hz, 2H); 4.06 (s, 3H); 3.96 (s, 3H); 1.60 (t, J=7 Hz, 3H).
RMN 13C (100 MHz, CDCl3) δ: 166.46; 161.29; 158.91; 146.60; 144.42; 131.82; 123.42; 123.02; 101.01; 99.61; 64.53; 55.64; 53.08; 14.48.
IV (νmax, KBr) cm−1: 2933; 2856; 1730; 1639; 1483; 1236; 1024
Compound 10 is produced following a procedure analogous to that described above for compound 9.
The compound 4-ethoxy-2-ethoxymethyl-6-methoxyquinoline (11) is prepared using NaH in DMF and DME to ascertain the influence of the hydroxyl group on the biological activity (Osornio, Y. M.; Miranda, L. D.; Cruz-Almanza, R. & Muchowski, J. M. “Radical cyclizations to quinolone and isoquinolone systems under oxidative and reductive reductions” Tetrahedron. Lett., 45:2855, 2004.).
Compound 11 is characterised by RMN 1H and its formation is confirmed by the appearance of two carbonillic methylene signs at 4.33 and 3.67 ppm apart from the methyls at 1.59 and 1.31 ppm, while the spectrum of RMN 13C shows the appearance of carbonillic methylenes at 74.39; 66.35; 64.09 ppm.
The compounds selected for the initial screening are shown in
This first assay established that 10, 1, 2, 11 and 13 presented partial inhibition properties over coagulant serine proteases with 2 being outstanding due to the added potential of inhibiting the haemorrhagic metalloprotease of the venoms and thus proving the most promising of the tested compounds illustrated in
Therefore, further assays were performed to determine the efficiency of compound 2 for various other activities.
The inhibition of haemorrhagic activity suggests interaction of the inhibitor with a metal and/or metalloprotease, thus neutralising effects. Likewise, compound 2 significantly inhibits proteolytic activity on casein and coagulants in human plasma induced by snake venoms and isolated enzymes, metalloprotease (
The proteolytic activity induced by Class I (neuwiedase isolated from B. neuwiedi) and III (BjussuMP-I isolated from B. jararacussu) metalloproteases was inhibited by compound 2 by approximately 67 and 70%, respectively, at a ratio of 1:10 protease:inhibitor (m/m).
The results for compound 2 show that it displays powerful coagulation action for the venoms of B. jararacussu and C. d. terrificus with this activity probably being due to the interaction of the active principle with the thrombin type enzymes BjussuSP-I and gyroxin respectively isolated from these same venoms (
The inhibition of fibrinogenolytic activity induced by serine protease and metalloprotease enzymes is shown in
In the assays for myotoxicity, oedema, and activity for phospholipase in vivo, compound 2 partially reduced these effects when induced by the venoms of B. jararacussu and C. durissus terrificus and isolated PLA2s (A
The muscle damage inflicted by the venom of Bothrops is partially caused by a group of proteins having PLA2 structures (Gutiérrez, J. M. & Lomonte, B. “Phospholipase A2, myotoxins from Bothrops snake venoms”. Toxicon, 33:1405, 1995; Soares, A. M. & Giglio, J. R. Chemical modifications of phospholipases A2 from snake venoms: Effects on catalytic and pharmacological properties. Review. Toxicon, 42: 855, 2003).
Compound 2 inhibited the myotoxic activity of both enzymes of Asp49 BthTX-II and Lys49 BthTX-I phospholipases A2 of B. jararacussu. Compound 2 proved more efficient in neutralising PLA2 activity induced by basic Asp49 PLA2s (BthTX-II and CB) in A
These data suggest a more specific link with basic PLA2s, intermediated by interactions of probable electrostatic cause and supports various authors who have pointed out the distinct or partial power of the overlap of catalytic sites and another pharmacologic one. (Soares, A. M. & Giglio, J. R. Chemical modifications of phospholipases A2 from snake venoms: Effects on catalytic and pharmacological properties. Review. Toxicon, 42: 855, 2003.).
In conclusion, compound 2 inhibits haemorrhages, enhances coagulation, proteolytic activity, oedema and myotoxicity induced by the venom of the Bothrops and Crotalus snakes and isolated metalloprotease, serine protease and phospholipases A2 enzymes demonstrating that the inhibitor is a good tool having potential antivenin activity.
The pharmacological efficiency of compound 2 is superior in inhibiting the proteases induced by PLA2s and thus provided information for development of therapeutic agents for the treatment of haemostatic diseases. Furthermore, the inhibitor has potential use as a complementary antivenin and is an alternative for treating poisoning caused by snake bite.
Results have demonstrated that the compounds may be used as antirheumatics, analgesics, immunosuppressors, antivenins, antithrombotics, anti-allergics and expectorants as well as for the treatment of white blood cell disorders and haemostatic system disorders amongst other therapeutic applications; in illnesses related to white blood cell disorders, such as autoimmune and inflammatory diseases including rheumatism amongst others, as well as anti-coagulants, antivenin, analgesics, antithrombotics and other therapeutical applications.
In the assays for myotoxicity, oedema, and activity for phospholipase in vivo, compound 2 partially reduced these effects when induced by the venoms of B. jararacussu and C. durissus terrificus and isolated PLA2s (A
The muscle damage inflicted by the venom of Bothrops is partially caused by a group of proteins having PLA2 structures.
Compound 2 inhibits the myotoxic activity of both enzymes Asp49 BthTX-II and Lys49 BthTX-I phospholipases A2 of B. jararacussu. Compound 2 proved more efficient in neutralising PLA2 activity induced by basic Asp49 PLA2s (BthTX-II and CB) in A
This data suggests a more specific link with basic PLA2s, intermediated by interactions of probable electrostatic cause and supports various authors who have pointed out the distinct or partial power of the overlap of the catalytic sites and another pharmacologic one, as related in the article by Soares, A. M. & Giglio, J. R. Chemical modifications of phospholipases A2 from snake venoms: Effects on catalytic and pharmacological properties. Review. Toxicon, 42: 855, 2003.
Therefore, compound 2 inhibits haemorrhages, enhances coagulation, proteolytic activity, oedema and myotoxicity induced by the venom of the Bothrops and Crotalus snakes and isolated metalloprotease, serine protease and phospholipases A2 enzymes demonstrating that the inhibitor is a good tool having potential antivenin activity.
The compounds were tested against snake venom in accordance with the procedures described below:
Snake venom or isolated metalloprotease (40 μg) is incubated with casein 1% (w/v) (1.0 mL) in a buffer solution of 0.1 M Tris-HCl (pH 8.0) for 30 minutes at 37° C. The reaction is ended through the addition of a trichloroacetic acid solution at 5% (v/v) (1.0 mL) and the mixture is left standing for 30 minutes at ambient temperature and then centrifuged (2000 rpm) for 5 minutes at 25° C. The proteolytic activity is estimated by the measurement of the absorbance of the supernatant at 280 nm.
Aliquots of plasma (0.2 mL) were incubated with venom or venom/inhibitor (50 μL) in varying proportions (m/m) during a period of 30 minutes at 37° C. and the coagulation time was verified. The control tubes included plasma incubated with phosphate buffered saline solution (PBS)+calcium or dimethyl sulphoxide or only the compound.
The oedemas are induced by the direct injection of venom (20 μg) and purified proteins (20 μg) into the right leg of male Swiss mice (18-22 g). Inhibition activity is verified incubating the venom or isolated protein with the inhibitor in varying concentrations (m/m). The control groups are injected with 50 μL of phosphate buffered saline solution (PBS, pH=7.2) alone, dimethyl sulphoxide (DMSO) or the compounds. Oedema progression is assessed through measurement of the decrease in blood pressure using a pachymeter (Mitutoyo, Japan) at intervals of 30 and 60 minute after injection.
Male Swiss mice (18-22 g) are injected with solutions containing 25 μg/50 μL doses of venom or toxin intra-muscularly at the right leg. The mixtures of venom or toxin/inhibitor (m/m) are then verified. The controls receive phosphate buffered saline solution or just inhibitor. The mice are bled at the tail 3 hours after injection and the blood is collected in capillary tubes containing heparin. The creatin kinase (CK) activity of the plasma is determined using a Bioclin Kit (Bioclin, Brasil). This activity is expressed in units/L, a corresponding unit for the production of one micromol of nicotinamide adenine dinucleotide (NADH)/min at 30° C.
Indirect haemolytic activity is tested using agarose-erythrocyte-egg yolk gel as a substrate. The compounds are tested following incubation with the pure venoms or PLA2 in varying ratios (m/m).
The data was analysed according P the value of <0.05, which was considered as indicative of significance.
| Number | Date | Country | Kind |
|---|---|---|---|
| P10701664-6 | May 2007 | BR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/BR08/00151 | 5/23/2008 | WO | 00 | 4/16/2010 |
