Patent Grant
8969384
The present invention relates to flavonoid compounds and a process for preparation thereof. The present invention relates to the demonstration of anti Helicobacter pylori activity and gastric antisecretory activity of a semisynthetically designed flavonoid compounds, to be used for the prevention and treatment of gastroduodenal disorders in general and peptic ulcer diseases in particular.
The aetiopathology of peptic ulcer disease is best understood in terms of an imbalance between mucosal defense factors (bicarbonate, mucin, prostaglandin, nitric oxide, some peptides and growth factors) and aggressive factors (acid, pepsin and H. pylori). Peptic ulcers do not occur when there is a balance between the aggressive factors and the defensive factors, but when the attacking factors become stronger than normal or when the defensive factors weaken, peptic ulcers can occur (Hoogerwerf and Pasricha, 2001).
Helicobacter pylori (H. pylori) is a ubiquitous, gram negative, highly motile, S-shaped, microaerophilic bacterium which colonizes the human gastric mucosa for extended time period. H. pylori infection is widespread with seroprevalence in the developed world between 30-60% (Everhart, 2000). Infection with the bacterium is usually contracted during childhood and patients remain infected for life unless treated. H. pylori infection has been shown to result in the development of chronic gastritis, gastro esophageal reflux disorder (GERD), and peptic ulcer diseases including gastric and duodenal ulcers. It is also linked to mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma (Go, 2000).
The other major etiology of peptic ulcer diseases is hyperacid secretion. For ulcers that are not caused by H. pylori, acid suppressive therapy alone with antisecretory agents is recommended in the form of H2-receptor antagonists or proton pump inhibitors, besides simple use of acid-neutralizing agents like antacids (Hoogerwerf and Pasricha, 2001). reflux
The eradication of the bacterium by triple therapies consisting of two antibiotics and a proton pump inhibitor in infected patients has resulted in good healing rates for both active gastritis and peptic ulcer diseases (Graham et. al., 1992). However, rising prevalence of acquired resistance of H. pylori to some antibiotics (Glupczynski et al., 2001), ulcer recurrence and the relatively high incidence of side effects (Wermeille et al., 2002) are the major causes for concern in recent times. Strains resistant to clarithromycin (CLR) and metronidazole (MNZ) have been well documented (Mégraud, 2004) while resistance to amoxicillin (AMX) and tetracycline was mainly reported in Asian countries (Wu et al., 2000, Kwon et al., 2000). On the other hand, as far as the use of antisecretory agents H2 receptor blockers and proton pump inhibitors are concerned, a number of side effects are reported. The adverse drug interaction of the cytochrome P450 system with H2-receptor blocker, hypersensitivity and damage of the liver by proton pump inhibitors, requirements of multiple doses of antacids to alleviate symptomatic-only relief, coupled with ulcer recurrence problems (Bullard, 1997) necessitate searching for better therapeutic management of hypersecretory disorders.
Regarding the treatment of H. pylori infection, reference may be made to an U.S. patent (Borody, 1993) which described a method consisting of the administration of a bismuth compound, an antibiotic belonging to the groups of penicillin and tetracycline, and a second antibiotic, such as metronidazole. The relevant therapy thus consists of the administration of three medications several times a day. There are other patents describing multiple therapies for the eradication of H. pylori, such as Neeman et al., 1995, 1996; Shell, 1996). None of these however eliminate the need to administer complex medications.
As antisecretory agents, several patents disclose the use of diverse molecular structures like fumagillol (Yanai et al. 1998), diphenyl ether phosphate esters (Catrenich et al., 1995), heterocyclyl-phenyl-(sulfonyl- or phosphonyl)-amidines (Cereda et al., 1987), N-alkylated benzo- and hetero-fused compounds (Schiehser et al., 1986), N-aryl-N′-(1,4,5,6-tetrahydropyrimidin-2-yl)ureas (Ramussen, 1984), 4,5,6,7-tetrahydroimidazo-[4,5-c]-pyridine (Arcari et al., 1980), 1-(4-chlorophenyl)-3-(1-ureido)-2-imidazolidinone (Schwan et al., 1978), 1,3-dimethyl-1H-pyrazolo(4,3-d)pyrimidine-7(6H)-ones (Ratajczyk et al., 1976), 4-acetoxy-1,2,3,4-tetrahydro-2,2-dimethyl-6,7-methylenedioxy isoquinolinium iodide (Schwan et al., 1978), furan or thiophene derivatives of iminomethyl piperidine (Scott, 1986), and 0-(carboxymethyl)-4-chromanone oxime (Wright et al., 1978).
Flavonoids, a class of polyphenols compounds, are present in many fruits and vegetables and offer a large number of biological activities. The antimicrobial activity of different types of flavonoids, either isolated from different plants or their chemically modified analogues, have been reported (Cushnie and Lamb, 2005). Anti peptic ulcer activity including anti H. pylori activity of naturally occurring flavonoids have also been documented (Bell et al., 1995; Ohsaki et al., 1999; Fukai et al., 2002; Park et al., 2004).
Three U.S. patents have disclosed the use of flavone or flavanone compounds for preventing or treating damages to the mucosal lining of the gastrointestinal tract (Ares et al., 1995; Yoo et al., 0.2000; Xu, 2004) while a Chinese patent (CN1615947) has implicated the use of flavones for treating oral ulcer, gastric ulcer, burn, scald and traumatic infection. A recent patent application from our group has disclosed the use of flavonoids for the treatment of gastrointestinal toxicity, associated symptoms and ulcers (Rao et al., 2007).
A Japanese patent (JP11228407) and an U.S. patent (Higuchi et al., 2001) disclosed the use of flavones for increasing the activity of beta lactam antibiotics against methicillin-resistant Staphylococcus aureus. An U.S. patent disclosed the use of biflavonoids in treating viral infection (Lin et al., 2002), while another U.S. patent disclosed the use of 3-methylene flavanones and 3-methylene chromanones having activity against microorganisms (Buckler et al., 1980). The use of flavones as antibacterial agents exhibiting suppressing effect against indigenous dermatic bacteria has been disclosed in the patent JP62145017. The antibacterial activity of an antibiotic flavone has also been disclosed in an US patent (Richards et al., 1972). Use of chrysin as antibacterial, antiviral and immunostimulatory agents has been patented (Markonius, 1995, 1999). Use of oroxylin A as inducible nitric oxide synthase inhibitor, cyclooxygenase-2 inhibitor and potassium channel activator has also been patented (Lee et al., 2004).
In view of the efforts towards searching for flavonoids which could be active against antibiotic-resistant bacterial strains or which would not impart resistance to otherwise susceptible strains (Xu and Lee, 2001; Iinuma et al., 1994; Liu et al., 2001), the inventors were interested in designing, synthesizing and bioevaluating a series of chrysin and oroxylin A derivatives with a view to imparting both anti H. pylori as well as antisecretory property in the flavone core structure.
The main objective of the present invention is to provide novel flavonoid compounds and process for preparation thereof.
Another objective of the present invention is to develop compounds, which could be therapeutically safe and useful in treating peptic ulcer diseases.
Another objective of the present invention is to design and develop such single compound(s) that should possess both anti H. pylori activity as well as gastric antisecretory activity so as to mitigate the two major aetiopathologies of peptic ulcer diseases, namely, the bug and the acid, thereby reducing the requirement of triple/quadruple therapy currently in practice.
Yet another objective of the present invention is to impart H. pylori inhibiting property and also gastric antisecretory property to a class of semi-synthetic flavonoids by structural modification of the side chain of the natural compounds chrysin and oroxylin A, along with simultaneously increasing the lipophilicity of the molecule, and by linking with another natural product molecule.
Still another objective of the present invention is to provide detailed experimental evidence for anti H. pylori activity and gastric antisecretory activity in one designed molecule 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-4H-benzpyran-4-one or, 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-chromen-4-one or in short, 7-O-(6-piperidin-1-ylhexyl)-chrysin (abbreviated in this document as CPP-1).
Accordingly, the present invention provides a flavonoid compound of general formula-X1, wherein R1 is selected from a group consisting of morpholinyl, N-methyl piperizinyl, piperidinyl and N,N′-dimethylamino groups, and value of n is 3-6, or a pharmaceutically acceptable salt thereof.
In an embodiment of the present invention, the structure of representative compounds of
In an embodiment of the present invention the representative compounds comprising:
In an embodiment of the present invention, the pharmaceutically acceptable salt is any addition salt of an acid selected from chloride, sulphate, maleate, tartrate, phosphate and acetate.
In an embodiment of the present invention, the compounds are useful as potential anti Helicobacter pylori as well as gastric antisecretory agent which could be a monotherapy drug candidate for prevention and treatment of peptic ulcer diseases.
In still another embodiment of the present invention, said compounds show bacteriostatic as well as bactericidal activity against clinical as well as ATCC® standard strains of H. pylori.
In an embodiment of the present invention, said compounds are equally effective at acidic pH, the gastric lumen environmental pH where these are supposed to act.
In an embodiment of the present invention, said compounds do not develop resistance to several subcultures of H. pylori, in clinical strain or with standard strain upon long term use.
In an embodiment of the present invention, 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-4H-benzpyran-4-one (CPP-1), has been demonstrated to induct irreversible morphological deformation of H. pylori at its MIC/MBC dose range of 6.25-12.5 μg/mL, effectively kill the bacteria at pH ranging from 4-6 unlike clarithromycin, and remain potentially active towards the bacteria upon repeated exposure unlike metronidazole.
In an embodiment of the present invention, said compound, 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-4H-benzpyran-4-one (CPP-1), inhibits gastric H+ pump activity with an IC50 value of 10 (±2) μg/mL.
In an embodiment of the present invention, 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyioxy)-4H-benzpyran-4-one (CPP-1), inhibits acid hypersecretion in gastric parietal cells.
In an embodiment of the present invention, 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-4H-benzpyran-4-one (CPP-1), inhibits basal acid secretion in gastric parietal cells in a dose-dependent manner with IC50 value of 5 (±1)μg/mL.
In an embodiment of the present invention, 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-4H-benzpyran-4-one (CPP-1), inhibits histamine-stimulated acid secretion in gastric parietal cells in a dose-dependent manner with IC50 value of 15 (±3) μg/mL.
Accordingly the present invention provides a process for preparation of compounds of general formula X1, wherein the process steps comprising:
Present invention also provides a pharmaceutical composition for treating or preventing H. pylori infection and gastric hypersecretion in a subject comprising a pharmaceutically effective amount of the compound of formula X1 optionally along with one or more pharmaceutically acceptable carriers, additives, lubricants and diluents, wherein the ratio of the compound of general formula X1 to the additives ranging from 1-10: 10-1.
In an embodiment of the present invention, the carriers used are selected from the group consisting of proteins, carbohydrates, sugar, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and pharmaceutically acceptable excipients, diluents or solvents.
In an embodiment of the present invention, the composition to be administered in human at a dose ranging between 20-60 mg twice per day.
In an embodiment of the present invention, the composition is useful for treating or preventing H. pylori infection and gastric hypersecretion leading to management of peptic ulcer diseases, which include one or more of the following disorders: gastric peptic ulcer, duodenal peptic ulcer, chronic and acute gastritis, chronic and acute duodenitis, non-ulcer dyspepsia, gastro esophageal reflux disorders, mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma.
The present invention deals with the development of a designed molecule, 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-4H-benzpyran-4-one, that effectively kills the bacterium Helicobacter pylori as well as inhibits gastric hypersecretion, giving rise to an unique single compound therapy for the management of peptic ulcer diseases.
Table 1a exhibits representative compounds of 7-O-alkylamino derivative of chrysin (Formula V).
Table 1b shows representative compounds of 7-O-acyl derivative of oroxylin A (Formula X2).
The IUPAC name was obtained using AutoNom 1.0 add-in for ISIS draw from MDL Information Systems, Inc.
Table 2 shows anti H. pylori activity of the compound analogues.
The discs were impregnated with 100 and 200 mg of each of the compounds, dissolved either in ethanol or DMSO, and the zone of inhibition was measured after 72 h of growth under microaerophilic conditions at 37° C. Inhibition zone diameter ranging 10-20 mm was given a score of +, 20-30 mm as ++ and that of >30 mm as +++, as obtained employing dose range of 100 and 200 mg/disc. The detailed structural information of the molecules is provided under ‘Description of the compounds’.
Table 3 shows anti H. pylori spectrum of the compounds, and Table 3a summarizes MIC range and MIC50 values of such compounds.
MIC was determined using agar dilution assay following the CLSI-t guidelines. Six clinical isolates and four standard strains were used for this study. Five microliter of freshly grown 3-day old culture (˜1×108 CFU/mL) was streaked in plates containing 2-fold serial dilution of the compounds ranging 1.56-100 mg/mL, and incubated under microaerophilic condition at 37° C. After 72 h, the plates were inspected visually for absence of growth to assess the MIC values. The experiment was performed in duplicates. MIC50 is defined as the dose (μg/m) required for killing 50% of the strains.
Table 4 exhibits MIC and MBC values of four most active compounds, determined by microbroth dilution method.
The compounds, serially diluted (3.125-100 mg/mL) in Muller Hinton broth containing 5% FCS, were incubated in 96-well microtitre plate containing fresh cultures of HP001 (μ5.5×106 CFU/mL) or ATCC® 43504 (2.65×106 CFU/ml) in a final volume of 200 ml. After 72-h of incubation under microaerophilic condition at 37° C., 100 ml of cultures wherein no growth had been detected were streaked on fresh brain heart infusion agar plates for MBC determination.
Table 5 shows anti H. pylori activity evaluation of the spacer molecules by disc diffusion sensitivity assay and microbroth dilution assay.
Methods and protocols for the determination of inhibition zone diameter are as stated under Experiment 1, and that of MIC values by microbroth dilution assay as in Experiment 3. ‘Nil’ means zone of inhibition below 10 mm.
Table 6 exhibits acid stability study of CPP-1, NMC-1, NMC-2 and CPP-3.
Details of experimental protocol are described under Experiment 7. MIC/MBC values were determined by microbroth dilution assay.
Table 7 shows the effect of CPP-1 on pig gastric H+K+-ATPase activity.
The data are averages of 3-4 determinations each carried out in triplicate. The assay system contained 10 mM PIPES-Tris (pH 6.8), 2 mM each of ATP and MgCl2 with or without 10 mM KCl and about 10 mg membrane protein in a final volume of 1 mL. Both blank and experimental tubes with or without CPP-1 at different concentrations were preincubated with otherwise complete assay mixture at 37° C. for 10 min before initiating the reaction with the substrate ATP. K+-stimulated activity referred to as H+,K+-ATPase activity, was calculated as the difference between the activity in presence of Mg2+ plus K+ and the basal activity (Mg2+-ATPase) in presence of Mg2+ alone. The specific activity of H+,K+-ATPase in control sets was in the range of 40-50 μmole Pi/mg protein/h.
Left-side panels (a-e) HP001; right-side panels (f-j) ATCC® 43504. The time and dose-kill curves for each of the compounds represent, whenever applicable, 0.5×MIC (filled circles), 1×MIC (filled squares), 2×MIC (filled triangles) and 4×MIC (filled hexagons). Control (open circles), bacterial suspension without compound or antibiotic, contained either ethanol or DMSO at the concentration used. MICs of CPP-1 (panels a and f) and NMC-1 (panels b and g) and that of NMC-2 (panels c and h) and CPP-3 (panels d and i), against both the strains, were 6.25 and 12.5 μg/mL respectively. The MICs of clarithromycin against HP001 (panel e) and ATCC® 43504 (panel j) were 0.01 μg/mL and 0.04 μg/mL respectively.
a shows time kill analysis of CPP-1 at different pH values of 4.0, 5.0, 6.0 and 7.2.
The bacterial suspension (ATCC® 43504) was incubated in biphasic culture seeded with CPP-1 at different concentrations under microaerophilic atmosphere at 37° C. The samples were taken for viable count at 0, 3, 6, 9, 12 and 24 h. Symbols: ◯ control; ● 6.25 mg/mL; ▪ 12.5 mg/mL; ▴ 25 mg/mL.
b shows short-time kill, kinetics study of CPP-1 at different acidic pH values.
Employing ATCC® 43504 strain in shake culture, short time killing kinetics with the most potent molecule CPP-1 was investigated at pH 4.0, 5.0 and 6.0. Control (open circle); CPP-1 50 μg/mL (filled circle); CPP-1 100 μg/mL (filled square); CPP-1 150 μg/mL (filled triangle).
A: Control; B: 3.125 mg/mL CPP-1; C: 6.25 mg/mL CPP-1 and D: 12.5 mg/mL CPP-1. Acridine orange fluorescence microscopic evidence is provided. The details of experimental procedures are provided under Experiment 9.
H. pylori ATCC® 43504 43504 cells were treated with CPP-1 for 24 h under microaerophilic condition at 37° C. in the absence (A-1), and the presence of 6.25 μg/mL (B-1) and 12.5 (μg/mL (C-1) of CPP-1. The left panel shows 8,200× magnifications of the electron micrographs and the right panel depicts higher magnifications (16,500×) of the correspondingly same micrographs (A-2 control; B-2 6.25 μg/mL; and C-2 12.5 μg/mL).
a demonstrates the effect of CPP-1 on basal acid secretion in gastric parietal cell. Dose-dependent inhibition of aminopyrine (AP) uptake ratio in parietal cell suspension after CPP-1 treatment was measured.
b shows the effect of CPP-1 on histamine-stimulated (0.1 mM) acid secretion in gastric parietal cell. Dose-dependent inhibition of histamine-stimulated aminopyrine (AP) uptake ratio in parietal cell suspension after CPP-1 treatment was measured.
CMD: cimetidine; OPZL: omeprazole. In all experiments, the data represent averages of 2-3 determinations each carried out in triplicate.
The present invention relates to the development of a semi-synthetic hetero-dimer of chrysin and piperidine, clicked via (CH2)n spacer linkage, namely CPP-1 and analogues thereof, as anti H. pylori as well as gastric antisecretory agent, which is being envisaged as a potential mono-therapy drug for the prevention and treatment of peptic ulcer diseases. Thus, the conferred bi-functional activity within a single molecule has been disclosed here for the development of novel antiulcer therapeutics.
It is to be borne in mind that the current world-wide therapeutic regimen is typically triple/quadruple therapy comprising of one or two antibiotics to kill the bacteria (like clarithromycin, amoxicillin and/or metronidazole, 1-2 antisecretory agent (either in the form of H2 receptor blocker like ranitidine, famotidine etc or gastric proton pump inhibitor like omeprazole, rabeprazole, pantoprazole etc), occasionally with a mucus coating agent also like bismuth tricitrate. The present invention disclosed both the anti H. pylori and the antisecretory activity in one molecule. Thus, this invention has immense commercial implications besides being extremely novel in its approach.
In an effort to design molecules that could possess both anti Helicobacter pylori activity and also gastric antisecretory potential, to be used for the treatment of gastroduodenal ulcers, the inventors synthesized a series of semisynthetic flavonoids, examined for their anti H. pylori properties, picked up the best molecule, and finally demonstrated gastric antisecretory activity in such potent anti H. pylori compound. Two series of flavone analogues, 7-O-alkylamino derivatives of chrysin and 7-O-acyl derivatives of oroxylin A, and the parent chrysin and oroxylin A obtained from the medicinal plant Oroxylum indicum, were evaluated in vitro for anti H. pylori activity as a function of several necessary parameters, employing a panel of clinical isolates as well as standard strains. The most potent molecule 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-4H-benzpyran-4-one (CPP-1) showed MIC range of 3.125-25 μg/mL, MIC50 of 3.125 μg/mL and MBC 6.25-12.5 μg/mL with demonstrated efficacy against drug-resistant as well as sensitive strains, could kill the bacteria within 12 h at 12.5 μg/mL doses, was functionally active at acidic pH unlike clarithromycin, and did not develop drug resistance unlike metronidazole. Neither the chrysin/oroxylin A core structure nor the spacer-linked n-alkylated amine ring system alone could contribute to the activity that such designed hetero-dimers manifest. The compound CPP-1 exhibited in vitro anti gastric H+,K+-ATPase activity with an IC50 value of 10 μg/mL, demonstrated dose-dependent inhibition of basal acid secretion (IC50 5 μg/mL) as well as histamine-stimulated acid secretion (IC50 15 μg/mL) in rabbit gastric parietal cell, indicating thereby its potential as gastric antisecretory principle. The conferred bi-functional activity within a single molecule has been disclosed here for the development of newer antiulcer therapeutics.
For the development of newer therapeutic agents, the inventors investigated a wide range of Indian biodiversity including plant and microbial resources (Jayaraman, 2003). As a result, some of the plants and their parts were primarily considered as ‘leads’ for the development of anti peptic ulcer agent (Rao et al., 2007; Das et al, 2007a, b). Oroxylum indicum is one such medicinally important plant of Indian biodiversity that showed hints of anti ulcer activity in several preclinical experimental models (Khandhar et al., 2006). Most parts of the tree are used medicinally. The fresh root bark is a well known drug in Ayurvedic medicine as an ingredient of the compound formulation Dashamularishta “concoction of ten roots” (Jabbar et al., 2004). It is also reported that the powdered stem bark is given in dropsy and eruptive fevers, and the stem bark mixed with neem powder is used to treat fevers among tribal inhabitants of southern Bihar (Singh, 2002). Chrysin and oroxylin A are two naturally occurring flavones that are abundantly present in O. indicum (Ali et al., 1998; Babu et al., 2005, 2006).
Since these compounds are abundantly present in various plants, they can be obtained by extraction from plants. In particular, these compounds are present in the plant Oroxylum indicum belonging to the family Bignoniaceae, in a large amount and, therefore, they can be obtained easily by extraction from such plants. Alternatively, these compounds can also be prepared by synthesis.
The present invention relates generally to development of molecules for inhibiting the growth of pathogenic bacteria and gastroduodenal pathogenesis due to Helicobacter species, such as H. pylori. In a preferred embodiment, the methods and compositions are designed so as to substantially eradicate ulcer-causing bacteria. The term substantially eradicates preferably means at least 50%, more preferably 75%, and most preferably 95% of the H. pylori are killed.
The present invention additionally relates to the demonstration of anti gastric acid secretion property of one or more of such compounds as well as establishing the method of affecting gastric acid secretion and formulating pharmaceutical preparations containing one or more of the said compounds.
Description of the Compounds
The present invention provides a composition comprising of a compound having a chrysin moiety (5, 7-dihydroxy-2 phenyl chromen-4-one) that is bridged to different varieties of heterocyclic ring system at 7 position, preferably N-methyl piperazine, piperidine, morpholine, or to dimethylamine, through an ether linkage, via 2-6 methylene spacers in between, or a pharmaceutically acceptable, non-toxic, acid-addition salt thereof in a therapeutically effective amount. The present invention also provides a composition comprising of a compound having 7-O-acyl derivatives of oroxylin A, or a pharmaceutically acceptable, non-toxic, acid-addition salt thereof in a therapeutically effective amount.
One embodiment of the present invention relates to anti H. pylori activity evaluation of a series of semisynthetic 7-O-alkylamino derivatives of chrysin (I), represented by the formula X1 wherein R1 is selected from morpholinyl, N-methyl piperizinyl, piperidinyl and N,N′-dimethylamino, and n is 3-6, or a pharmaceutically acceptable salt thereof.
In another embodiment, the present invention relates to anti H. pylori activity evaluation of 7-O-acyl derivatives of oroxylin A (II) represented by the formula X2, wherein R2 is selected from C17 alkyl or 4-tolyl, or a pharmaceutically acceptable salts thereof.
Representative compounds of Formula X1 and Formula X2 are described in Table 1a and 1b respectively below.
The methods of isolation and/or synthesis, and structural characterization of chrysin and oroxylin A and the synthetic analogues as in entry no. 5-14 (Tables 1a, 1b) are disclosed in patent application WO/2007/080484.
Method of Making a Few Designed Compounds of the Present Invention
In general, the compounds of this invention may be prepared by standard techniques known in the art, by known processes analogues thereto, and/or by the processes discussed below, using starting materials which are either naturally obtained, commercially available, producible according to routine, conventional chemical methods, and/or the synthesis of which are described herein (Babu et al., 2005, 2006; Rao et al., 2007).
However, based on the objectives of this invention and the observations disclosed in the earlier patent (Rao et al., 2007), we wanted to design synthesize such molecules that would impart both anti H. pylori activity and gastric antisecretory activity, so as to mechanistically mitigate the two major causes of peptic ulcer diseases (the bug H. pylori and the acid HCl), thereby effectively achieving the central objective of this invention that a single molecule can be designed which would replace the currently prevailing triple therapy.
Thus, the chromen-4-one derivatives of Formula X1 where R1 is morpholinyl, N-methyl piperizinyl, piperidinyl and N,N′-dimethylamino, can be prepared by the method outlined below in Scheme I and as described in Babu et al. (2006).
In Scheme I, the 7-O-hexyl chrysin derivatives of formula X1 where R1 is morpholinyl, N-methyl piperizinyl, piperidinyl and N,N′-dimethylamino may be synthesized by alkylation of chrysin (I) using 1,6 dibromo hexane in presence of a base such as potassium carbonate in a solvent such as acetone under reflux condition to afford 7-O-(6-bromohexyl)-chrysin (III). The appropriate amine (morpholine/N-methyl piperazine/piperidine/N,N′-dimethyl amine) was alkylated with compound (III) in presence of a base such as potassium carbonate in a solvent such as anhydrous acetonitrile under reflux condition to afford the desired compounds of 7-O-hexyl chrysin derivatives (CHM-1, NMC-1, CPP-1, CHN-1).
A compound of formula X2 where R2 is C17 alkyl or 4-tolyl can generally be synthesized from oroxylin A (II) as described in Babu et al. (2005).
Following examples are given by way of illustrations and should not construed the scope of the present invention.
Step 1:
To a mixture of chrysin I (1 g, 3.93 mole) and anhydrous potassium carbonate (0.81 g, 5.8 mmol) in 20 mL acetone, 1,6-dibromohexane was added. The mixture was refluxed under nitrogen atmosphere for 4 h. After completion of the reaction, potassium carbonate was filtered and washed with excess of acetone (2×50 mL). The combined acetone layers were concentrated under vacuum. The residue was purified by column chromatography on silica gel (60-120 mesh) to yield 7-(6-bromohexyl) chrysin.
Step 2:
To a mixture of 7-(6-bromohexyl) chrysin and anhydrous potassium carbonate (2.41 g, 17.2 mmole) in 20 mL acetonitrile, piperidine was added. The mixture was refluxed under nitrogen atmosphere for 3 h. After completion of the reaction, the reaction mixture was brought to room temperature (25° C.) and was poured into ice water and washed with methylene chloride (2×10 mL). The combined organic layers were dried over anhydrous sodium sulphate and concentrated under vacuum. The residue was purified by column chromatography on silica gel (60-120 mesh) to give 5-hydroxy-2-phenyl-7-(6-piperidin-1-yl-hexyloxy)-chromen-4-one (CPP-1).
Using the method of Example 1, and appropriate reagents, compounds CHM-1, NMC-1 and CHN-1 were similarly prepared.
In another embodiment of the present invention of synthetic analogues, 7-O-hexyl (morpholino) chrysin (Entry No. 1, CHM-1)) have the following spectrochemical and physical properties: Pale yellow solid, mp 103° C., 1H NMR (400 MHz, CDCl3): δ 12.74 (1H, s, OH-5), 7.92-7.82 (2H, m, H-2′, 6′), 7.58-7.44 (3H, m, H-3′,4′,5′), 6.70 (1H, s, H-3), 6.58 (1H, d, J=1.8 Hz, H-6), 6.40 (1H, d, J=1.8 Hz, H-6), 4.20 (2H, t, J=6 Hz, H-1″), 3.78 (4H, t, H-3′″,5′″), 2.40 (4H, t, J=4 Hz, H-2′″, 6′″), 2.38-2.30 (2H, m, H-6″), 2.0-1.80 (4H, m, H-2″), 1.60-1.55 (2H, m, H-3″), 1.42-1.38 (4H, m, H-4″, 5″). FABMS: 434 (M++1).
In another embodiment of the present invention of synthetic analogues, 7-O-hexyl (N-methyl piperazinyl) chrysin (Entry No. 2, NMC-1) have the following spectrochemical and physical properties: yellow solid, mp 107° C., 2H NMR (400 MHz, CDC3): δ 12.60 (1H, s, OH-5), 7.94-7.82 (2H, m, H-2′,6′), 7.58-7.44 (3H, m, H-3′,4′,5′), 6.62 (1H, s, H-3), 6.42 (1H, d, J=1.8 Hz, H-6), 6.26 (1H, d, J=1.8 Hz, H-8), 4.18 (2H, t, J=6 Hz, H-1″), 2.58-2.30 (8H, m, H-3′″, 5′″ and H-2′″, 6′″), 2.40 (2H, t, J=4 Hz, H-6″), 2.22 (3H, Me), 1.82-1.78 (2H, m, H-2″), 1.56-1.42 (4H, m, H-4″, H-5″), 1.42-1.38 (2H, m, H-3″). FABMS: 447 (M++1).
In another embodiment of the present invention of synthetic analogues, 7-O-hexyl (piperidinyl) chrysin (Entry No. 3, CPP-1) have the following spectrochemical and physical properties: Pale yellow solid, mp 108.8° C., 1H NMR (300 MHz, CDC3): 6 12.71 (1H, s, OH-5), 7.90-7.86 (2H, m, H-2′, 6′), 7.58-7.51 (3H, m, H-3′, 4′, 5′), 6.67 (1H, s, H-3), 6.50 (1H, d, J=1.5 Hz, H-6), 6.36 (1H, d, J=1.5 Hz, H-8), 4.0 (2H, t, J=6 Hz, H-1″), 2.80-2.60 (6H, m, H-2′″, 6′″ and H-6″), 1.80-1.60 (4H, m, H-2″, H-5″), 1.60-1.20 (10H, m, H-3′″, 5′″, 4′″). FABMS: 422 (M++1).
In another embodiment of the present invention of synthetic analogues, 7-O-hexyl (N,N′-dimethylamino) chrysin (Entry No. 4, CHN-1) have the following spectrochemical and physical properties: pale yellow solid, mp 85° C., 1H NMR (400 MHz, CDC3): δ 12.70 (1H, s, OH-5), 7.84-7.88 (2H, m, H-2′,6′), 7.52-7.58 (3H, m, H-3′,4′,5′), 6.70 (1H, s, H-8), 6.48 (1H, s, H-3), 6.38 (1H, s, H-6), 4.12 (2H, t, H-1″), 2.22-2.38 (4H, m, H-2″, 6″), 2.0 (6H, s, 2×Me), 1.78-1.82 (2H, m, H-5″), 1.44-1.58 (2H, m, H-3″), 1.38-1.42 (2H, m, H-4″). FABMS: 382 (M++1).
Pharmaceutically acceptable salts of the compounds are also within the scope of this invention. The term ‘pharmaceutically acceptable salts’ refer to an inorganic or organic salt of a compound of the present invention that has properties acceptable for therapeutic use.
Biological Activity
The present invention relates to conferring anti H. pylori activity in some designed semisynthetic flavonoids like 7-O-alkylamino chrysin derivatives (formula X1) and 7-O-acyl oroxylin A derivatives (formula X2). Accordingly, in an embodiment of the present invention, the anti H. pylori activity of 16 such molecules, 2 naturally isolated and 14 semisynthetically designed, were screened by disc diffusion sensitivity assay employing ATCC® standard strains as well as clinical isolates. Based on the observed inhibition zone diameter (see Table 2) as obtained with two doses of the molecules and with majority of the strains, the compounds were grouped into four classes, viz., compounds CPP-1 and CPP-3 as strongly active (3+, diameter >30 mm), compounds CHN-2, CHM-3 and CHN-3 as intermediate active (2+, ˜20-30 mm diameter), compounds CHM-1, NMC-1, NMC-2, CPP-2 and NMC-3 as moderately active (1+, ˜10-20 mm diameter), and the two natural compounds chrysin and oroxylin A as well as CHN-1, CHM-2, ORC-16 and ORPM-1 as not active (<10 mm diameter). Thus, lack of activity in natural molecules was surmounted by inventing appropriate substitutions like piperidinyl group in R2 position of chrysin core structure as in Formula X1.
In another embodiment of the present invention, the MIC values of all the 10 compounds that have shown some activity in disc diffusion susceptibility test (zone of inhibition ≧10 mm) were determined by agar dilution method following CLSI® antimicrobial susceptibility testing protocol and employing a panel of clinical isolates and ATCC® standard strains (Table 3a). Compounds like NMC-1, CPP-1, NMC-2, CPP-2 and CPP-3 showed high bacteriostatic activity, CPP-1 being the most potent. An assessment of the MIC range vis-à-vis MIC50 values of some potent compounds gave a rank order as: CPP-1 (MIC range 3.125-25 μg/mL, MIC50 μ3.125 μg/mL)>NMC-1 (MIC range 6.25-12.5, μg/mL MIC50˜6.25 μg/mL)>NMC-2=CPP-2=CPP-3 (MIC range 6.25-100, μg/mL MIC50˜12.5 μg/mL). Among the other molecules, such values of CHM-1, CHN-2, NMC-3 and CHN-3 were about 6.25-25 μg/mL and 25 μg/mL respectively, and that of CHM-3 was 50-100 μg/mL and 50 μg/mL respectively (Table 3b). The natural molecules chrysin and oroxylin A however showed much higher MIC range and MIC50 values. CPP-1, NMC-1, NMC-2 and CPP-3 exerted potential anti H. pylori activity even in metronidazole-resistant clinical strains, at similar doses, as with other sensitive strains. Such compounds are therefore expected to be effective against drug-resistant as well as sensitive strains of H. pylori thereby achieving one of the objectives of this invention.
In another embodiment of the present invention, the MIC and the MBC values of the four most active compounds, namely, NMC-1, CPP-1, NMC-2 and CPP-3, were further examined by microbroth dilution assay employing one each of clinical strain and ATCC® standard strain (Table 4). Based on both bacteriostatic as well as bactericidal activity, as judged by the two methods and using several H. pylori strains, such four compounds were rank ordered in terms of efficacy as CPP-1>NMC-1>CPP-3>NMC-2.
In yet another embodiment of the present invention, the role of flavone moiety or the n-alkylated amine ring system in manifesting the activity was ascertained. The inhibition zone diameter and the MIC values were determined for five selected molecules containing either the flavone component with ether linked spacer or the n-alkylated amine components attached with spacer (Table 5). All five compounds showed negligible activity confirming the necessity of both the chrysin core structure as well the spacer containing ring systems in manifesting the anti H. pylori activity.
In another embodiment of the present invention, the bactericidal activity of the four potent molecules, CPP-1, NMC-1, NMC-2 and CPP-3, were investigated using time kill assay employing one each of clinical and standard strains of H. pylori (
In yet another embodiment of the present invention, it is demonstrated that a clinical strain HP001 and a standard strain ATCC® 43504, both resistant to metronidazole (MIC for metronidazole are 12.5 and 50 μg/mL respectively), remained susceptible to CPP-1 treatment in vitro. Following repeated, exposure for 10 growth cycles in the presence of sub-MIC dose of CPP-1, there was no significant increase in MIC values, meaning chances of resistance development with CPP-1 treatment is minimal (
In another embodiment of the present invention, the anti H. pylori potential of CPP-1, NMC-1, NMC-2 and CPP-3 was examined at acidic pH with a view to ascertain their efficacy in stomach acidic environment. Accordingly, the acid stability of the four compounds were examined by exposing them at acidic pH of 2.0 for 2 hours, and then diluting in 96-well microtitre plate to see the changes in MIC and MBC values, if any (Table 6). The activity of CPP-1 remained almost unchanged upon prolonged exposure to acid as evident from just about 2-fold increase in MIC and MBC values, while the other compounds showed relatively higher increase of their MIC/MBC values. This provided clue as to the putative efficacy of CPP-1 in the gastric acidic environment. It was therefore of further interest to examine the killing potential of the most active compound CPP-1 at variable stomach pH ranging 4.0 to 7.2.
In still another embodiment of the present invention, H. pylori standard strain ATCC® 43504 was grown in pH range of 4.0-7.2 by two methods, biphasic culture to investigate long-term killing kinetics and short-time killing kinetics in shake culture media where the liquid media was shaken at 150 rpm to facilitate quick growth of the bacteria. The evidence suggests that the compound CPP-1 is quite effective in killing H. pylori at lower pH values. In biphasic culture media where MBC concentration ranges can be used to demonstrate killing phenomenon, the initial rate of killing at all the concentrations was found to be progressively increasing as the pH goes down indicating strongly that the compound CPP-1 is equally active, if not more, at acidic pH of the stomach (
In yet another embodiment of the present invention, the morphological transformation of H. pylori cells from live, rod and spiral shape to non-alive coccoid shape upon exposure to CPP-1 during growth was examined by acridine orange fluorescence staining method (
In still another embodiment of the present invention, such morphological alterations of H. pylori cells exposed to CPP-1 were examined in transmission electron microscope. Most of the untreated control cells (ATCC® 43504) appeared as slightly curved or straight bacilli. Cultures exposed to two concentrations of CPP-1 for 24 h under microaerophilic conditions at 37° C. revealed only few bacteria with intact shape and structure at 6.25 μg/mL, and at higher dose (12.5 μg/mL), most cells were found to be progressively swollen and destroyed (
Further, in an embodiment of the present invention, the effect of the compound CPP-1 was examined for its capacity to inhibit gastric proton pump. Gastric H+ pump (H+,K+-ATPase) is one of the important targets in designing antisecretory drugs. Using H+,K+-ATPase rich tubulovesicular and apical membranes, isolated from freshly slaughtered pig gastric mucosa, the effects of different concentrations of CPP-1 on such membrane H+,K+-ATPase activity was examined in vitro. The observation suggests that the compound CPP-1 at the dose range of 1-10 μg/assay can inhibit H+,K+-ATPase to an extent of 10-50% (Table 7). Under identical experimental condition omeprazole, the standard medicine used for inhibiting gastric H+,K+-ATPase activity, inhibits 20-80% of enzyme activity in the same dose range of 1-10 μg/assay. This evidence was taken to mean that the compound has the potential to effectively block gastric H+,K+-ATPase, and thereby can act as anti-secretory agent.
In yet another embodiment of the present invention, the compound CPP-1 was examined for its capacity to reduce gastric acid secretion. Such experiments can be performed using gastric acid secreting cells, called parietal cell. Live, pure and stimulation-competent gastric parietal cells have been prepared from rabbit gastric mucosa to examine the effect of different concentrations of CPP-1 on acid secreting processes, both basal acid secretion and stimulated acid secretion. Histamine (0.1 mM) was used as physiological secretagogue to stimulate freshly prepared parietal cells in experimental set up. Concentration-dependent strong inhibition of both basal (
Further, in an embodiment of the present invention, the compound CPP-1 has been demonstrated to be non-toxic. In acute toxicity study using Swiss albino mice, it is demonstrated that CPP-1 up to a dose of 0.5 g/kg of body weight, did not show any mortality, and the animals remained completely healthy after 15 days.
The anti H. pylori activity of all 16 flavonoid molecules, 2 naturally isolated and 14 synthetic derivatives, was screened by disc diffusion sensitivity assay employing 2 clinical strains HP001 and HP002, and three standard strains ATCC® 700392, 43504 and 49503. A 0.5 mL inoculum (˜108 CFU/mL) for each bacterial strain tested was flooded on freshly prepared Brain Heart Infusion (BHI) agar plates supplemented with 7% FCS, 0.5% IsoVitalex and 0.0025% DENT® and the discs (5 mm diameter) containing different compounds or antibiotics (dissolved either in ethanol or DMSO) were placed on the agar surface. After incubation for 3 days in a microaerophilic atmosphere at 37° C., the diameter of the zone of inhibition was measured (Glupczynsky, 1996; McNulty et al., 2002). Clarithromycin showed inhibition zone of 18 mm for the strains HP001 and HP002 at 0.01 mg/disc and that of 28, 20 and 15 mm respectively with strains 700392 (0.4 mg/disc), 43504 (0.04 mg/disc) and 49503 (0.005 mg/disc). Amoxycillin gave inhibition zone diameter of 10-16 mm with clinical strains at 0.16 mg/disc and about 13-15 mm with standard strains at 0.5-1.0 mg/disc.
Based on the observed inhibition zone diameter as obtained with two doses of the molecules (100 and 200 μg/disc) and with majority of the strains (Table 2), the compounds were grouped into four classes: (i) compounds CPP-1 and CPP-3 as strongly active (diameter>30 mm) and were given a score of 3+, (ii) compounds CHN-2, CHM-3 and CHN-3 as intermediate active 20-30 mm diameter) and were given a score of 2+, (iii) compounds CHM-1, NMC-1, NMC-2, CPP-2 and NMC-3 as moderately active (˜10-20 mm diameter) and were given a score of 1+, and (iv) the two natural compounds chrysin, and oroxylin A as well as CHN-1, CHM-2, ORC-16 and ORPM-1 as not active (>10 mm diameter). There was not much strain specific variation with all the molecules, excepting with compounds CPP-1, CHN-2, CPP-3 and CHN-3 wherein we found a maximum of about 10 mm difference in zone diameters (Table 2).
MICs were determined against a panel of 6 clinical isolates (HP001, HP002, HP003, HP004, HP005 and HP006), and 4 standard strains in BHI agar plates following essentially CLSI® antimicrobial susceptibility testing method (Glupczyriski et al., 2002; Best et al., 2003). Essentially, the strains employed for the evaluation were clarithromycin and amoxicillin sensitive. The standard strain ATCC® 43504 and all six clinical strains were metronidazole resistant, but standard strains ATCC® 700392, 49503, and 43629 were metronidazole sensitive. Plates contained two-fold serial dilutions of the compounds ranging from 1.56-100 μg/mL. Five microliters of 3-day old freshly grown H. pylori culture (˜108 CFU/mL) was inoculated onto drug-containing BHI agar plates supplemented with 7% FCS, 0.5% IsoVitalex and 0.0025% DENT®, which were then incubated for 3 days under microaerophilic condition at 37° C. The MIC was defined as the lowest concentrations that completely inhibited the development of visible growth on the agar plates, and were determined in duplicate for each strain.
The MIC values of all the 10 molecules that have shown some activity were evaluated by agar dilution method (Table 3a). The results indicated potential bacteriostatic activity in compounds like NMC-1, CPP-1, NMC-2, CPP-2 and CPP-3. Somewhat good activity of the molecules NMC-3 and CHN-3 was also noted. Such MIC data correlated well with the values obtained by disk diffusion sensitivity assay. An assessment of the MIC50 values (Table 3b) of some potent compounds gave a rank order: CPP-1 (˜3.125 μg/mL)>NMC-1 (˜6.25 μg/mL)>NMC-2=CPP-3=CPP-2 (˜12.5 μg/mL). Among the other molecules, such MIC50 value of the compounds CHM-1, CHN-2, CHN-3 and NMC-3 was about 25 μg/mL, and that of compound CHM-3 were approximately 50-100 μg/mL. The molecules chrysin, oroxylin A and its two semisynthetic derivatives, showed much higher MIC50 values. We have also determined MIC values of the standard antibiotics, clarithromycin, amoxycillin and metronidazole against the clinical isolates and the standard strains (Table 3a).
Based on disc diffusion sensitivity and MIC values both, the four most active compounds, namely, NMC-1, CPP-1, NMC-2 and CPP-3 were considered for further evaluation. In microbroth dilution assay employing one clinical and one standard strain, both the bacteriostatic and the bactericidal values were determined for all four compounds as well as for clarithromycin (Table 4). Two-fold serial dilutions were prepared in 96-well microtitre plate containing a total volume of 0.1 mL Mueller Hinton broth supplemented with 5% FCS. A 3-day old liquid culture was diluted 10 times in Mueller Hinton broth and 0.1 mL of these cultures was inoculated into each well to give a final concentration of ˜106 CFU/mL. The plates were incubated for 3 days in a microaerophilic atmosphere at 37° C. Following incubation, the plates were examined visually, and the lowest concentration showing complete inhibition of growth was recorded as the MIC of the respective compound (Hachem et al., 1996). Aliquots (0.1 mL) of 72-h culture in which no growth had been detected were taken from the wells of the above microtitre plates and used to streak on fresh BHI agar plates. MBCs were determined by visual inspection of such plates after further incubation for 72 h at 37° C. and the titre-point where no growth (less than 10 colonies) appeared was considered as the MBC.
The compound CPP-1 showed lowest MIC and MBC values of ˜6.25 μg/mL with standard strain 43504 (Table 4). None of the other three molecules showed such low MIC and MBC values considering the data obtained with both the strains. However, with clinical strain HP001, both the compounds CPP-1 and NMC-1 appear to have shown similar potency (MIC 6.25 μg/mL and MBC 12.5 μg/mL) and the compounds NMC-2 and CPP-3 exhibited a 2-fold lower potency. The compound CPP-1 was found to kill the standard strain ATCC® 43504 below 6.25 μg/mL. But for killing the clinical strain a 2-fold dose i.e., 12.5 μg/mL was required. Employing the same dose, bacteriostatic but not the bactericidal activity could be achieved with other compounds like NMC-1, NMC-2 and CPP-3 (Table 4).
It was of necessity to determine the role of the substituted cyclohexane ring system attached to chrysin core structure through 3 C, 4 C and 6 C spacers in manifesting the anti H. pylori activity. Thus the activity of five such molecules was investigated against H. pylori employing disc diffusion sensitivity assay and MIC determination by microbroth dilution assay. As evident from Table 5, none of the spacers bearing morpholinyl, N-methyl piperazinyl or piperidinyl showed any anti H. pylori activity. Also, the withdrawal of the OH group from the 5-position of the parent chrysin ring system resulted loss in anti H. pylori activity.
The bactericidal activity of the four potent molecules was further investigated using time kill assay employing one clinical and a standard strain (
With clinical strain HP001, clarithromycin could not produce any inhibition of cell growth within 6 h (
Using standard strain ATCC® 43504, the time kill assay with same 4 compounds CPP-1, NMC-1, NMC-2 and CPP-3 along with clarithromycin was carried out. Clarithromycin at 0.08 μg/mL did exert complete killing (4-log decrease in cell count) within 24 h. Compound CPP-1, at 0.5×MIC (3.125 μg/mL), could produce only 1-log decrease in cell count within 36 h while at 1×MIC (6.25 μg/mL) and at MBC value (i.e., 2×MIC), it exhibited complete killing within 36 h and 24 h respectively (panel f). Compound NMC-1 produced only 1-log decrease in cell count within 36 h at MIC value (6.25 μg/mL) while at 2×MIC (i.e., MBC; 12.5 μg/mL), complete killing of bacteria was observed within 24 hour (panel g). The MIC dose of compound NMC-2 (12.5 μg/mL) could not produce complete killing of the standard strain, but at MBC (25 μg/mL) a complete killing effect was observed well within 12 h (panel h). Compound CPP-3 has no killing potency at MIC (12.5 μg/mL). The next two higher doses (25 μg/mL and 50 μg/mL) could however kill the bacteria totally in about 12 h, the rate of killing being slightly different between the low and high dose (panel i).
To investigate the development of drug resistance, CPP-1, NMC-1, NMC-2 and CPP-3 were prepared as 2-fold serial dilutions with medium in 96-well microtitre plates. H. pylori strains, one clinical HP001 and one standard ATCC® 43504, were inoculated into each dilution series at an inoculum size of ˜106 CFU/well. After 3 days of incubation, the culture from each series with the highest concentrations of the compound and also showing turbidity was subcultured in a fresh series of the same drug. This procedure was repeated for up to 10 cycles, and alterations in MIC values during the course of continued exposure were determined (Iwao et al., 2004).
The repeated transfer (10 times) of the standard as well as the clinical strain (both metronidazole resistant) in the drug containing media produced no significant increase in MIC values for CPP-1 (only 2-fold increase in MIC value), while somewhat higher increase of the MIC values was observed with other 3 compounds (
A 100 μL stock of CPP-1, NMC-1, NMC-2 and CPP-3 (1-2 mg/mL) was treated with 20-30 μL of 0.12 NHCl to bring the pH of the solution to ˜2.0, and kept at 25° C. for 2 h (Funatogawa et al., 2004). Such acid-treated samples were then serially diluted (2 fold) with medium in 96-well microtitre plates to determine the MIC and the MBC values by microbroth dilution assay as detailed under Experiment 3.
The activity of CPP-1 remained almost unchanged upon prolonged exposure to acid as evident from just about 2-fold increase in MIC and MBC values, while the other compounds showed relatively higher increase of their MIC/MBC values (Table 6).
The decrease in viable cell count during exposure to CPP-1 was evaluated as a function of time at different low pH values (citrate buffer for pH 4.0 and 5.0 and citrate-phosphate buffer for pH 6.0) using biphasic culture. A 24-h culture broth of H. pylori ATCC® 43504 (obtained from biphasic culture) was inoculated into Brucella broth containing CPP-1 at different concentrations in biphasic culture. The bacterial suspension was incubated in microaerophilic environment at 37° C., and samples were taken to determine viable counts at 0, 3, 6, 9, 12 and 24 h after drug exposure.
At low pH, CPP-1 was found to provide potentiation of the H. pylori killing activity at its MIC/MBC concentration ranges; the efficacy was more in pH 4.0 and 5.0 as compared to pH 6.0 and 7.0. The MBC concentration of 12.5 μg/mL was enough to kill all the bacteria within 10-12 h at pH 4.0 while at higher pH values, the same concentration produced killing only after 24 h (
The decrease in viable cell count during exposure to high concentrations of CPP-1 in short time kill kinetics assay was also evaluated at different acidic pH values of 4.0, 5.0 and 6.0 (citrate buffer for pH 4.0 and 5.0 and citrate-phosphate buffer for pH 6.0). A 24-h culture broth of H. pylori ATCC® 43504 was inoculated into Brucella broth containing CPP-1 at different concentration. The bacterial suspension was incubated in microaerophilic environment at 37° C. with shaking at 150 rpm, and samples were taken at 0, 20, 40, 60, 80, 100 and 120 min after drug exposure to determine viable counts.
The effect of CPP-1 at higher concentrations 50, 100 and 150 mg/mL was studied at different pH upon short exposure to ATCC® 43504. At 50 mg/mL, a 3-log decrease of growth was observed in the pH 4.0, 5.0 and 6.0 whereas the bacteria survived at pH 7.2 at such concentration (
For the purpose of fluorescence staining, a bacterial smear was prepared, fixed with methanol for 2 min, stained with 0.1% acridine orange solution for 2-3 min, rinsed thoroughly with distilled water (Haqqani, 2001) and observed under fluorescence microscopy using 1-3 filter (emission wavelength 510 nm). The live organism appeared as orange color in the dark background and the non-viable cells as green (Shirai et al., 2000).
The morphological alterations of the clinical strain (HP001) exposed to 3.125, 6.25 and 12.5 mg/mL of CPP-1 for 24 h as observed under fluorescence microscopy are shown in
The morphological alteration of H. pylori cells (ATCC® 43504) exposed to various concentrations of CPP-1 was further examined using transmission electron microscopy (Dai et al., 2005). After exposure to CPP-1 at 0, 6.25 and 12.5 μg/mL for 24 h under microaerophilic conditions at 37° C., cells were harvested by centrifugation, fixed with glutaraldehyde and postfixed with osmium tetroxide. Samples were dehydrated, embedded in SPUR, sections cut and applied to copper grids. The grids were contrasted with uranyl acetate and lead citrate and the sections were examined on a transmission electron microscope.
The morphological alterations of H. pylori cells exposed to 6.25 μg/mL (B) and 12.5 μg/mL (C) of CPP-1 as compared with control (A) are evident (
The effect of CPP-1 on gastric H+K+-ATPase was investigated. Isolation of pig gastric mucosal membranes, rich in H+,K+-ATPase activity, was carried out by density gradient centrifugation technique (Ray, 1978). The enzyme assay system contained 10 mM PIPES-Tris (pH 6.8), 2 mM each of ATP and MgCl with or without 10 mM KCl and about 10 mg membrane protein in a final volume of 1 mL. Both blank and experimental tubes with or without CPP-1 at different concentrations were preincubated with otherwise complete assay mixture at 37° C. for 10 min before initiating the reaction with the substrate ATP. After appropriate time of incubation with ATP at 37° C., the reaction was terminated with the addition of 1 mL of ice-cold TCA (14%, w/v). The liberated inorganic phosphate (Pi) was estimated essentially by the method of Sanui (1974). K+-stimulated activity referred to as H+,K+-ATPase activity, was calculated as the difference between the activity in presence of Mg2+ plus K+ and the basal activity (Mg2+-ATPase) in presence of Mg2+ alone. The specific activity of H+,K+-ATPase was expressed as micromoles of Pi liberated per hour per milligram protein. The data are averages of 3-4 determinations each carried out in triplicate. The specific activity of H+,K+-ATPase in control sets was in the range of 40-50 μmole Pi/mg protein/h.
CPP-1 was found to inhibit gastric H+,K+-ATPase activity in vitro in a dose dependent manner. About 50% inhibition was observed in the concentration range of 10 mg/assay (Table 7). The standard medicine omeprazole, under similar experimental condition produced about 80% inhibition of such H+,K+-ATPase.
Parietal cells (PC) were isolated from New Zealand White rabbits (weighing ˜2.5 kg) essentially according to Berglindh (1984) and as modified by Mazzeo et al. (1988). The PC preparations were nearly 90% pure and viable. The effect of CPP-1 on basal and histamine stimulated acid secretion in parietal cells was examined as follows. The PC suspension was incubated for 10 min at 37° C. with gentle stirring and continuous slow top gassing with oxygen. The [14C]-aminopyrine (115 mCi/mmole) was added to the suspension at 0.1 mCi/mL, and incubation was continued for another 10 min. One-milliliter aliquot of PC (˜5×106 cells) was added to various concentrations of CPP-1 in the presence and the absence of histamine (0.1 mM) and incubated for 20 min at 37° C. with slow shaking and oxygen top gassing. At the end of the incubation, the PC suspensions were centrifuged, supernatant carefully aspired off and the pellets solubilized in scintillation fluid containing Triton X-100. The resulting solution was counted in a liquid scintillation counter. The aminopyrine accumulation was determined as the ratio of intra- to extracellular aminopyrine according to Mazzeo et al. (1988).
The effect of varied concentrations of CPP-1 on basal acid secretion in such freshly prepared parietal cells was examined. The basal acid secretion was decreased in a dose dependent manner upon exposure to CPP-1 (
The most potent compound CPP-1 was checked for mortality of the Swiss albino mice. Five animals of Swiss albino mice strain were used for this purpose. The dosage up to 0.5 g/kg body weight given orally per mice and the animals were kept for 15 days under observation. It was observed that the mice were not affected at the dose level of 0.5 g/kg body weight of CPP-1. No mortality and behavioral abnormality was noted.
Advantages
Advantages
| Number | Date | Country | Kind |
|---|---|---|---|
| 381/DEL/2009 | Feb 2009 | IN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IN2010/000117 | 2/26/2010 | WO | 00 | 8/26/2011 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2010/097816 | 9/2/2010 | WO | A |
| Number | Date | Country |
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| WO 2005011671 | Feb 2005 | WO |
| WO 2007080484 | Jul 2007 | WO |
| WO 2007080484 | Jul 2007 | WO |
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| “Treatment.” (2009). In Mosby's Dictionary of Medicine, Nursing, & Health Professions. Retrieved from <http://www.credoreference.com/entry/ehsmosbymed/treatment> on Nov. 18, 2010. |
| Suresh Babu et al., “Synthesis and Biological Evaluation of Novel C (7) Modified Chrysin Analogues as Antibacterial Agents,” Bioorg. Med. Chem. Lett. (2006), 16:221-224, Elsevier Ltd. |
| Number | Date | Country | |
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| 20120022062 A1 | Jan 2012 | US |
