Chem Biol Drug Des 2013; 82: 630–634 Research Letter
Synthesis, Analytical Analysis, and Medicinal Aspect of Novel Benzimidazoles and their Metal Complexes Sangeeta Agrawal1, Rishi Raj Bhatnagar2, Anjani Tiwari3, Rakesh Srivastava4 and Upasana sharma1,* 1
Department of chemistry, S.S.V (P.G) College, Hapur 245101, India 2 Department of Chemistry, Christ Church College, Kanpur 208001, India 3 Pharmaceutical Department, INMAS, Delhi 110057, India 4 Department of Chemistry, NGF College of Engg. & Technology, Palwal, Haryana 125001, India *Corresponding author: Upasana sharma, [email protected]; [email protected] Benzimidazole and their metal analogs that can act as multimodal agent and have non-peptidic CCK-B receptor antagonist were synthesized and characterized on the basis of spectroscopic techniques such as FT-IR, NMR, FAB-MS and also evaluated for biologic efficacy. The ligands showed binding to most of the organs, known to express CCK receptors in biodistribution studies. Cholecystokinin (CCK1 and CCK2) receptor binding affinities of these analogs (IC50) are 0.802 0.007 for compound C and 0.326 0.012 for compound D in rat pancreatic acini. These studies have provided a new template for further development of novel agents for various related diseases. Key words: benzimidazole, cholecystokinin, non-peptidic and metal sorption Received 8 May 2013, revised 27 June 2013 and accepted for publication 18 July 2013
Benzimidazole analogs are known to be pharmacologically active and find their application in the treatment of various medical applications such as epilepsy, diabetics, and antifertility (1,2). Recently, many researches elucidated that benzimidazole analogs can be suitably modified by the introduction of different heterocyclic moieties to exhibit a broad spectrum of biologic activities such as potent anticancerous and microbial activities (3–6). Keeping this factor in our mind, we have modified benzimidazole analogs in such a way that would bring them into preferred orientation. This may provide a more potent cholecystokinin (CCK) antagonist. 630
Cholecystokinin is very important for regulatory functions, as neurotransmitters in the brain and as regulators of various functions of the gastrointestinal tract, preliminary at the level of the stomach, pancreas, and gallbladder (7). In addition to that, they can act as an important growth factor in most parts of the gastrointestinal tract (8–10). Gastrin and CCK possess the same five amino acids at their COOH terminus, which is a biologically active site. Their actions are mediated by two different receptor types, CCK1 and CCK2 (11,12). CCK2 receptors are present in the gut mucosa and in the brain (7,13,14), whereas CCK1 receptors are present in the gallbladder, pancreas, and brain (7,15,16). The importance of selective antagonist is that they are potential tools to visualize various tumors (17,18). In vivo autoradiography studies have shown that cholecystokinin CCK-B/gastrin receptors are expressed not only in more than 90% of medullary thyroid carcinomas (MTC) (19) but also in high percentage of small-cell lung cancer, some ovarian cancer, astrocytomas, and potentially in a variety of adenocarcinomas, gastrointestinal tumors, and colorectal cell lines. In this work, we have designed multimodal analogs of benzimidazoles and studied it with various metal complexes to see their effect in applied as well as in medicinal chemistry, as its intermediate is synthesized according to previous literaturea and labeled with specific metal to explore binding aspect with CCK receptor. The initial results have shown good results for further evaluation.
Experimental All chemicals were purchased from well-known commercial sources and used as received. 1H NMR spectra were recorded on a NMR spectrometer with known internal standard tetramethylsilane (TMS). Mass peak was calculated by fast atom bombardment mass spectra (FAB-MS) on a JEOL NMS-SX102 spectrometer. All the details of material and method are given in reference and short notes part.b Synthesis of benzimidazole analog [Bzm] is presented in scheme 1 for the synthesis. The first two steps are carried out and as mentioned in short notes.a,b Compounds C and D were evaluated for their ability to displace [125I] (BH)-CCK8 (sulfated) from isolated rat pancreatic acini (CCK1) and guinea-pig cerebral cortex membranes (CCK2) according to established protocols. ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12201
Benzimidazoles and Metal Complexes
Scheme 1: Chemical scheme for synthesis of Bzm (where R = NH2 and CH3 in compound C & D respectively).
Table 1: CCK receptor binding data of the target compounds
Complexes
(R)
Rat pancreatic acini (CCK1), IC50 (lM)
Ref. (VL-0494) Compound (C) Compound (D)
Ph NH2 CH3
0.197 0.107 0.802 0.007 0.326 0.012
Guinea-pig brain Cortex (CCK2), IC50 (lM) 16.40 3.12 1.94
Binding affinities expressed as IC50 are reported in Table 1 along with reference compound 3-ureido-1,4-benzodiazepine (Merck L-365260). Values without standard errors were obtained from no more than two experiments.
Distribution studies with various metals The relative affinities of the compound for three metal ions were studied by batch equilibrium method. The chelation property of the compound in its complex form with Ni, Zn, and Cu was determined by batch equilibrium method involving metal ions in various electrolyte viz. KCl, in different concentrations such as 0.01, 0.1, and 0.5 M. To know the appropriate time required for the maximum adsorption of the metals, the sorption capacity was determined by shaking the complex with the metal ion solution for different time intervals. The physicochemical properties show that the chelating compounds in H+ also behave as a weak cation exchanger. The sorption capacities were determined for seven metal ions in three concentrations.
In vivo studies [125I]BH-CCK-8 receptor binding assay in isolated rat pancreatic acinar cells was carried out by isolated Chem Biol Drug Des 2013; 82: 630–634
pancreatic acini prepared by enzymatic digestion of pancreas. Drug-displacing experiments were carried out by incubating acinar cells [125I]BH-CCK-8 (25 pM final concentration) and competitors in 0.5 mL total volume at 37 °C for 30 min, in a shaking bath. At the end of incubation, 1 mL of ice-cold Hepes–Ringer buffer (10 mM Hepes, 118 mM NaCl, 1.13 mM MgCl2, 1.28 mM CaCl2, 1% BSA, 0.2 mg/mL Soybean trypsin inhibitor, pH 7.4) was added, and the tubes were centrifuged for 5 min at 12 500 g. The supernatant was aspirated, and the radioactivity associated with the pellet measured. The non-specific binding was estimated in the presence of 1 jjM CCK-8, accounting for 15% of total binding. [125I]BH-CCK-8 receptor binding assay in membranes from guinea-pig cerebral cortices was carried out by determining the protein concentration using bovine serum albumin (BSA) as standard. The binding experiments were performed in assay buffer containing 10 mM Hepes, 118 mM NaCl, 4.7 mM KCl, 5.0 mM MgCl2, 1.0 mM EGTA, pH 6.5 and supplemented with 0.2 mg/ mL bacitracin. The incubation of membrane suspension with labeled ligand and inhibitors was carried out in a 96-well microtiter filter plate (Multiscreen; Millipore Inc, Bedford, MA, USA) with integral Whatman GF/B membrane filters. Aliquot of membranes (0.5 mg of protein per mL) was added to each well, containing [125I]BHCCK8 (25 pM), in a final volume of 250 jjl. The non-specific binding of iodinated peptide was defined in the presence of 1 pM CCK-8, accounting for 20% of total binding. Non-specific binding of [125I]BH-CCK-8 to membrane filters (blank) measured in wells containing an equal amount of labeled ligand, but no membranes, was 0.2% of total radioligand added. After 120 min at 25 °C, the 96-well plate was placed on the vacuum filtration apparatus (Millipore Inc.). 631
Agrawal et al.
Labeling of the compounds [Bzm] has been carried out by taking 100 lL of 0.03 nM solution of the compound [Bzm] dissolved in DMSO in a shielded vial, and 60 lL of 1 9 10 2 M Sncl2.2H2O (dissolved in N2-purged 1 mL 10% acetic acid) was added followed by addition of ( Co2+ > Cu2+ > Ni2+ > Zn2+ > Ba2+ > Pb2+. The sorption was found to increase with an increase in pH; however, the capacity order for the investigated metal ions remained unchanged. The variation in uptake capacities for different metal ions may be explained on the basis of difference in stability of metal Bzm complexes under experimental conditions.
Figure 3: Metal ions uptake study with Bzm (C).
different time intervals, about 0.5 mL of blood samples was withdrawn from the dorsal vein of other ear and radioactivity was measured in the gamma-counter. The data from the experiment were expressed as percentage of administered dose at each time interval in Figures 1 and 2.
Thus, the benzimidazole ring structure fused to pyridine ring might provide lead for new type of compounds having CCK-B receptor affinity. Some non-specific uptake in other tissues can be overcome by changing the basic skeleton by different R groups. The therapeutic potential of these complexes can be extend further by applying these in different animal models and cell lines.
Conflict of Interest The authors state that there is no conflict of interest.
Result and Discussion The synthesized compound was characterized by IR (CO near 1600/cm, OH 3200–3600, and aromatic below 900/cm), NMR (aromatic proton in range from 6 to 8.5 ppm and heterofunctional group via D2O exchange), MASS spectroscopic studies, and Rf values of the relevant complexes (Rf values). Labeling efficiencies were more than 97%, and complex was stable about 12 h at 30 °C. Results of serum stability studies showed that the metal ion was intact under physiologic conditions, and unbound radioactivity was less than 0.5% in 8 h. Biodistribution studies were determined by injected radiolabeled compound, intravenously as a function of time, differentiating those tissues which are known or expected to express CCK receptors, organs involved in blood pool, and excretion of the ligand. Higher uptake was found in liver, spleen, and stomach, but retention was longer in stomach (Figures 1 and 2). Very low activity was found in brain probably due to inability of crossing the blood–brain barrier. The rapid blood clearance was also evident from the blood kinetics study. The result of pretreatment studies of labeled [Bzm] by blocking with 1 mg/kg of CCK2, 15 min before the injection of radio complex, reduced the accumulation in stomach where the activity in the intestine was reduced. There was increased accumulation of activity in liver, but reduction in case of rest of the organs studied. The metal ion uptake study are given in Figure 3. This shows that Bzm in H+ also behaves as a weak exchanger. The results of rate of sorption experiments showed that time required for maximum sorption capacity is 45 min. Chem Biol Drug Des 2013; 82: 630–634
References 1. Singh S., Ojha H., Tiwari A.K., Kumar N., Singh B., Mishra A.K. (2010) Design, synthesis, and in vitro antiproliferative activity of benzimidazole analogues for radiopharmaceutical efficacy. Cancer Biother Radiopharm;25:245–250. 2. Shukla G., Tiwari A.K., Singh V.K., Bajpai A., Chandra H., Mishra A.K. (2008) Effect of a novel series of benzothiazolo-quinazolones on epidermal growth factor receptor (EGFR) and biological evaluations. Chem Biol Drug Des;72:533–539. 3. Tiwari A.K., Mishra A.K., Bajpai A., Mishra P., Sharma R.K., Pandey V.K., Singh V.K. (2006a) Synthesis and pharmacological study of novel pyrido-quinazolone analogues as anti-fungal, antibacterial, and anticancer agents. Bioorg Med Chem Lett;16:4581–4585. 4. Dandia A., Singh R. (2005) Synthesis of novel quinazolinone and fused quinazolinones. J Flour Chem;126: 307–312. 5. Srivastava V., Srivastava A.M., Tiwari A.K., Srivastava R., Sharma R., Sharma H., Singh V.K. (2009) Disubstituted 4(3H) quinazolones: A novel class of antitumor agents. Chem Biol Drug Des;74:297–301. 6. Malecki N., Caroto P., Rigo B., Goossens J.H., Henchart J.P. (2004) Synthesis of condensed quinolines and quinazolines as DNA ligands. Bioorg Med Chem;12:641. 7. Tanwar J., Datta A., Tiwari A.K., Thirumal M., Chuttani K., Mishra A.K. (2011) Preclinical evaluation of DO3PAME-DO3P: A polyazamacrocyclic methylene phosphonate for diagnosis and therapy of skeletal metastases. Bioconjug Chem;22:244–255. 8. Kakkar D., Tiwari A.K., Chuttani K., Kaul A., Singh H., Mishra A.K. (2010) Comparative evaluation of gluta633
Agrawal et al.
mate-sensitive radiopharmaceuticals: Technetium-99mglutamic acid and technetium-99m-diethylenetriaminepentaacetic acid-bis(glutamate) conjugate for tumor imaging. Cancer Biother Radiopharm;25:645–655. 9. Tiwari A.K., Singh V.K., Bajpai A., Shukla G., Singh S., Mishra A.K. (2007) Synthesis and biological properties of 4-(3H)-quinazolone derivatives. Eur J Med Chem;42:1234–1238. 10. Ram V.J., Farhanullah M., Tripathi B.K., Srivastava A.K. (2003) Synthesis and antihyperglycemic activity of suitably functionalized 3H-quinazolin-4-ones. Bioorg Med Chem;11:2439–2444. 11. Srivastava P., Tiwari A.K., Chadha N., Chuttani K., Mishra A.K. (2013) Synthesis and biological evaluation of newly designed phosphonate based bone-seeking agent. Eur J Med Chem;65:12–20. 12. Hynes J.B., Buch J.M., Freisheim J.H. (1975) Quinazolines as inhibitors of dihydrofolate reductase. 3. Analogs of pteroic and isopteroic acids. J Med Chem;18:1191. 13. Alagarsamy V., Murugananthan G., Venkateshperumal R. (2003) Synthesis of some novel 2-mercapto-3-(substituted amino)-5,6,7,8-tetrahydro-3H-benzo[4,5]thieno [2,3-d]pyrimidin-4-ones as analgesic and anti-inflammatory agents. Biol Pharm Bull;26:1711. 14. Tiwari A.K., Singh Rathore V., Sinha D., Datta A., Sehgal N., Chuttani K., Mishra A.K. (2012) Design and docking studies of [diethylenetriaminepentaacetic acid(amino acid)2] with acetylcholine receptor as a molecular imaging agent for single-photon emission computed tomographic application. Mol Imaging;11:240–250. 15. Sinha D., Tiwari A.K., Singh S., Shukla G., Mishra P., Chandra H., Mishra A.K. (2008) Synthesis, characterization and biological activity of schiff base analogues of indole-3-carboxaldehyde. Eur J Med Chem;43:160– 165. 16. Fukunaga J.Y., Hansch C., Steller E.E. (1976) Inhibition of dihydrofolate reductase. Structure-activity correlations of quinazolines. J Med Chem;19:605–611. 17. Elslager E.F., Johnson J.L., Werbel L.M. (1983) Folate antagonists.20. Synthesis and antitumor and antimalarial properties of trimetrexate and related 6-[(phenylamino) methyl]-2, 4-quinazolinediamines. J Med Chem; 26:1753–1760. 18. Kung P.P., Casper M.D., Cook K.L., Wilson-Lingardo L. (1999) Structure-activity relationships of novel 2-substituted quinazoline antibacterial agents. J Med Chem;42:4705–4713. 19. Saeki T., Adachi Y. (1995) A selective type V phosphodiesterase inhibitor, E4021, dilates porcine large coronary artery. J Pharmacol Exp Ther;272:825–831.
634
Notes a
Synthesis of 4-methyl,7-hydroxyl 8 [N-(methyl phthalimide)] benzo-[1,2,b] pyrane 2 one] [A] as 4-methyl-7-hydroxycoumarin (0.05 mole) and N-methylol-phthalimide (0.08 mole) were dissolved in 8 N sulphuric Acid (100 mL) by stirring vigorously and carefully; while dissolving, the contents were occasionally cooled (as exothermic reaction occurred) in order to avoid the decomposition of the reactants. After refluxing for 1 h, the dark-coloured solution keft under refrigeration overnight and after that it was poured into cold water (250 mL). A solid separated out was filtered off and washed with water and a small portion treated with sodium bicarbonate solution (10%) to ensure the completion of reaction by ceasing of effervescence. The crude material was recrystallized by glacial acetic acid (yield 72%) m.p. 153 °C. IR (KBr pellets, per cm) 3385, 3290, 1640, 1715, 1196, 782. 1HNMR (200 MHz, CDCl3) d ppm; 7.69– 8.13 (m 4H, phthalimide aryl ring), 4.89(S, 2H, N-substituted methylene), 5.00 (S, 1H, OH exchanged with D2O), 6.57–7.27 (m, 3H, coumarin ring), 1.71(S, 3H, coumarin ring). Anal. Calculated for C15H13NO5, 67.8% C (theoretical C 66.52%), 3.89% H (theoretical H 4.03%), 5.02% N (theoretical N 4.88%). Found: M/Z 334[M]+, 160, 105, 77. b Synthesis of 4-methyl,7-hydroxy-8(N-methyl phthalimide) quinoline (1,5c) benzimidazole [B] was carried out by mixing 4 methyl, 7 hydroxy [N-(methyl phthalimide)] benzo[1,2,b] pyrane 2 one [A] (0.01 mole) and o-phenylenediamine (0.015 mole) in dry pyridine (50 mL). The refluxing reaction mixture was stirred for 6–7 h. This reaction was monitored by TLC; on completion of reaction, the solvent was removed in vacuo and the reaction mixture was cooled and then poured into ice-cold diluted HCl (50 mL); on neutralization, it was purified by column chromatography [column of Sio2 (80 g) pre-adsorption of the residue at SiO2 with ethyl acetate, elution with petroleum ether/ethyl acetate = 60: 40 (v/v) to obtain the product]. (Yield 81%) m.p.101 °C.IR (KBr pellets per cm) 3461, 3355, 2988, 1657,1278,742. 1H NMR (200 MHz, CDCl3) d ppm; 7.69–8.13 (m, 3H, quinoline ring). 5.02 (S, 1H, OH exchanged with D2O), 6.4–7.39 (m, 8H, aromatic rings). Anal. Calculated for C25H17N3O3, 71.42% C (theoretical C 73.71%), 4.1% H (theoretical H 4.18%), 9.87% N (theoretical N 10.12). Found: M/Z 404 [M]+, 405, 254, 77.
Chem Biol Drug Des 2013; 82: 630–634
Synthesis, Analytical Analysis, and Medicinal Aspect of Novel Benzimidazoles and their Metal Complexes Sangeeta Agrawal1, Rishi Raj Bhatnagar2, Anjani Tiwari3, Rakesh Srivastava4 and Upasana sharma1,* 1
Department of chemistry, S.S.V (P.G) College, Hapur 245101, India 2 Department of Chemistry, Christ Church College, Kanpur 208001, India 3 Pharmaceutical Department, INMAS, Delhi 110057, India 4 Department of Chemistry, NGF College of Engg. & Technology, Palwal, Haryana 125001, India *Corresponding author: Upasana sharma, [email protected]; [email protected] Benzimidazole and their metal analogs that can act as multimodal agent and have non-peptidic CCK-B receptor antagonist were synthesized and characterized on the basis of spectroscopic techniques such as FT-IR, NMR, FAB-MS and also evaluated for biologic efficacy. The ligands showed binding to most of the organs, known to express CCK receptors in biodistribution studies. Cholecystokinin (CCK1 and CCK2) receptor binding affinities of these analogs (IC50) are 0.802 0.007 for compound C and 0.326 0.012 for compound D in rat pancreatic acini. These studies have provided a new template for further development of novel agents for various related diseases. Key words: benzimidazole, cholecystokinin, non-peptidic and metal sorption Received 8 May 2013, revised 27 June 2013 and accepted for publication 18 July 2013
Benzimidazole analogs are known to be pharmacologically active and find their application in the treatment of various medical applications such as epilepsy, diabetics, and antifertility (1,2). Recently, many researches elucidated that benzimidazole analogs can be suitably modified by the introduction of different heterocyclic moieties to exhibit a broad spectrum of biologic activities such as potent anticancerous and microbial activities (3–6). Keeping this factor in our mind, we have modified benzimidazole analogs in such a way that would bring them into preferred orientation. This may provide a more potent cholecystokinin (CCK) antagonist. 630
Cholecystokinin is very important for regulatory functions, as neurotransmitters in the brain and as regulators of various functions of the gastrointestinal tract, preliminary at the level of the stomach, pancreas, and gallbladder (7). In addition to that, they can act as an important growth factor in most parts of the gastrointestinal tract (8–10). Gastrin and CCK possess the same five amino acids at their COOH terminus, which is a biologically active site. Their actions are mediated by two different receptor types, CCK1 and CCK2 (11,12). CCK2 receptors are present in the gut mucosa and in the brain (7,13,14), whereas CCK1 receptors are present in the gallbladder, pancreas, and brain (7,15,16). The importance of selective antagonist is that they are potential tools to visualize various tumors (17,18). In vivo autoradiography studies have shown that cholecystokinin CCK-B/gastrin receptors are expressed not only in more than 90% of medullary thyroid carcinomas (MTC) (19) but also in high percentage of small-cell lung cancer, some ovarian cancer, astrocytomas, and potentially in a variety of adenocarcinomas, gastrointestinal tumors, and colorectal cell lines. In this work, we have designed multimodal analogs of benzimidazoles and studied it with various metal complexes to see their effect in applied as well as in medicinal chemistry, as its intermediate is synthesized according to previous literaturea and labeled with specific metal to explore binding aspect with CCK receptor. The initial results have shown good results for further evaluation.
Experimental All chemicals were purchased from well-known commercial sources and used as received. 1H NMR spectra were recorded on a NMR spectrometer with known internal standard tetramethylsilane (TMS). Mass peak was calculated by fast atom bombardment mass spectra (FAB-MS) on a JEOL NMS-SX102 spectrometer. All the details of material and method are given in reference and short notes part.b Synthesis of benzimidazole analog [Bzm] is presented in scheme 1 for the synthesis. The first two steps are carried out and as mentioned in short notes.a,b Compounds C and D were evaluated for their ability to displace [125I] (BH)-CCK8 (sulfated) from isolated rat pancreatic acini (CCK1) and guinea-pig cerebral cortex membranes (CCK2) according to established protocols. ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12201
Benzimidazoles and Metal Complexes
Scheme 1: Chemical scheme for synthesis of Bzm (where R = NH2 and CH3 in compound C & D respectively).
Table 1: CCK receptor binding data of the target compounds
Complexes
(R)
Rat pancreatic acini (CCK1), IC50 (lM)
Ref. (VL-0494) Compound (C) Compound (D)
Ph NH2 CH3
0.197 0.107 0.802 0.007 0.326 0.012
Guinea-pig brain Cortex (CCK2), IC50 (lM) 16.40 3.12 1.94
Binding affinities expressed as IC50 are reported in Table 1 along with reference compound 3-ureido-1,4-benzodiazepine (Merck L-365260). Values without standard errors were obtained from no more than two experiments.
Distribution studies with various metals The relative affinities of the compound for three metal ions were studied by batch equilibrium method. The chelation property of the compound in its complex form with Ni, Zn, and Cu was determined by batch equilibrium method involving metal ions in various electrolyte viz. KCl, in different concentrations such as 0.01, 0.1, and 0.5 M. To know the appropriate time required for the maximum adsorption of the metals, the sorption capacity was determined by shaking the complex with the metal ion solution for different time intervals. The physicochemical properties show that the chelating compounds in H+ also behave as a weak cation exchanger. The sorption capacities were determined for seven metal ions in three concentrations.
In vivo studies [125I]BH-CCK-8 receptor binding assay in isolated rat pancreatic acinar cells was carried out by isolated Chem Biol Drug Des 2013; 82: 630–634
pancreatic acini prepared by enzymatic digestion of pancreas. Drug-displacing experiments were carried out by incubating acinar cells [125I]BH-CCK-8 (25 pM final concentration) and competitors in 0.5 mL total volume at 37 °C for 30 min, in a shaking bath. At the end of incubation, 1 mL of ice-cold Hepes–Ringer buffer (10 mM Hepes, 118 mM NaCl, 1.13 mM MgCl2, 1.28 mM CaCl2, 1% BSA, 0.2 mg/mL Soybean trypsin inhibitor, pH 7.4) was added, and the tubes were centrifuged for 5 min at 12 500 g. The supernatant was aspirated, and the radioactivity associated with the pellet measured. The non-specific binding was estimated in the presence of 1 jjM CCK-8, accounting for 15% of total binding. [125I]BH-CCK-8 receptor binding assay in membranes from guinea-pig cerebral cortices was carried out by determining the protein concentration using bovine serum albumin (BSA) as standard. The binding experiments were performed in assay buffer containing 10 mM Hepes, 118 mM NaCl, 4.7 mM KCl, 5.0 mM MgCl2, 1.0 mM EGTA, pH 6.5 and supplemented with 0.2 mg/ mL bacitracin. The incubation of membrane suspension with labeled ligand and inhibitors was carried out in a 96-well microtiter filter plate (Multiscreen; Millipore Inc, Bedford, MA, USA) with integral Whatman GF/B membrane filters. Aliquot of membranes (0.5 mg of protein per mL) was added to each well, containing [125I]BHCCK8 (25 pM), in a final volume of 250 jjl. The non-specific binding of iodinated peptide was defined in the presence of 1 pM CCK-8, accounting for 20% of total binding. Non-specific binding of [125I]BH-CCK-8 to membrane filters (blank) measured in wells containing an equal amount of labeled ligand, but no membranes, was 0.2% of total radioligand added. After 120 min at 25 °C, the 96-well plate was placed on the vacuum filtration apparatus (Millipore Inc.). 631
Agrawal et al.
Labeling of the compounds [Bzm] has been carried out by taking 100 lL of 0.03 nM solution of the compound [Bzm] dissolved in DMSO in a shielded vial, and 60 lL of 1 9 10 2 M Sncl2.2H2O (dissolved in N2-purged 1 mL 10% acetic acid) was added followed by addition of ( Co2+ > Cu2+ > Ni2+ > Zn2+ > Ba2+ > Pb2+. The sorption was found to increase with an increase in pH; however, the capacity order for the investigated metal ions remained unchanged. The variation in uptake capacities for different metal ions may be explained on the basis of difference in stability of metal Bzm complexes under experimental conditions.
Figure 3: Metal ions uptake study with Bzm (C).
different time intervals, about 0.5 mL of blood samples was withdrawn from the dorsal vein of other ear and radioactivity was measured in the gamma-counter. The data from the experiment were expressed as percentage of administered dose at each time interval in Figures 1 and 2.
Thus, the benzimidazole ring structure fused to pyridine ring might provide lead for new type of compounds having CCK-B receptor affinity. Some non-specific uptake in other tissues can be overcome by changing the basic skeleton by different R groups. The therapeutic potential of these complexes can be extend further by applying these in different animal models and cell lines.
Conflict of Interest The authors state that there is no conflict of interest.
Result and Discussion The synthesized compound was characterized by IR (CO near 1600/cm, OH 3200–3600, and aromatic below 900/cm), NMR (aromatic proton in range from 6 to 8.5 ppm and heterofunctional group via D2O exchange), MASS spectroscopic studies, and Rf values of the relevant complexes (Rf values). Labeling efficiencies were more than 97%, and complex was stable about 12 h at 30 °C. Results of serum stability studies showed that the metal ion was intact under physiologic conditions, and unbound radioactivity was less than 0.5% in 8 h. Biodistribution studies were determined by injected radiolabeled compound, intravenously as a function of time, differentiating those tissues which are known or expected to express CCK receptors, organs involved in blood pool, and excretion of the ligand. Higher uptake was found in liver, spleen, and stomach, but retention was longer in stomach (Figures 1 and 2). Very low activity was found in brain probably due to inability of crossing the blood–brain barrier. The rapid blood clearance was also evident from the blood kinetics study. The result of pretreatment studies of labeled [Bzm] by blocking with 1 mg/kg of CCK2, 15 min before the injection of radio complex, reduced the accumulation in stomach where the activity in the intestine was reduced. There was increased accumulation of activity in liver, but reduction in case of rest of the organs studied. The metal ion uptake study are given in Figure 3. This shows that Bzm in H+ also behaves as a weak exchanger. The results of rate of sorption experiments showed that time required for maximum sorption capacity is 45 min. Chem Biol Drug Des 2013; 82: 630–634
References 1. Singh S., Ojha H., Tiwari A.K., Kumar N., Singh B., Mishra A.K. (2010) Design, synthesis, and in vitro antiproliferative activity of benzimidazole analogues for radiopharmaceutical efficacy. Cancer Biother Radiopharm;25:245–250. 2. Shukla G., Tiwari A.K., Singh V.K., Bajpai A., Chandra H., Mishra A.K. (2008) Effect of a novel series of benzothiazolo-quinazolones on epidermal growth factor receptor (EGFR) and biological evaluations. Chem Biol Drug Des;72:533–539. 3. Tiwari A.K., Mishra A.K., Bajpai A., Mishra P., Sharma R.K., Pandey V.K., Singh V.K. (2006a) Synthesis and pharmacological study of novel pyrido-quinazolone analogues as anti-fungal, antibacterial, and anticancer agents. Bioorg Med Chem Lett;16:4581–4585. 4. Dandia A., Singh R. (2005) Synthesis of novel quinazolinone and fused quinazolinones. J Flour Chem;126: 307–312. 5. Srivastava V., Srivastava A.M., Tiwari A.K., Srivastava R., Sharma R., Sharma H., Singh V.K. (2009) Disubstituted 4(3H) quinazolones: A novel class of antitumor agents. Chem Biol Drug Des;74:297–301. 6. Malecki N., Caroto P., Rigo B., Goossens J.H., Henchart J.P. (2004) Synthesis of condensed quinolines and quinazolines as DNA ligands. Bioorg Med Chem;12:641. 7. Tanwar J., Datta A., Tiwari A.K., Thirumal M., Chuttani K., Mishra A.K. (2011) Preclinical evaluation of DO3PAME-DO3P: A polyazamacrocyclic methylene phosphonate for diagnosis and therapy of skeletal metastases. Bioconjug Chem;22:244–255. 8. Kakkar D., Tiwari A.K., Chuttani K., Kaul A., Singh H., Mishra A.K. (2010) Comparative evaluation of gluta633
Agrawal et al.
mate-sensitive radiopharmaceuticals: Technetium-99mglutamic acid and technetium-99m-diethylenetriaminepentaacetic acid-bis(glutamate) conjugate for tumor imaging. Cancer Biother Radiopharm;25:645–655. 9. Tiwari A.K., Singh V.K., Bajpai A., Shukla G., Singh S., Mishra A.K. (2007) Synthesis and biological properties of 4-(3H)-quinazolone derivatives. Eur J Med Chem;42:1234–1238. 10. Ram V.J., Farhanullah M., Tripathi B.K., Srivastava A.K. (2003) Synthesis and antihyperglycemic activity of suitably functionalized 3H-quinazolin-4-ones. Bioorg Med Chem;11:2439–2444. 11. Srivastava P., Tiwari A.K., Chadha N., Chuttani K., Mishra A.K. (2013) Synthesis and biological evaluation of newly designed phosphonate based bone-seeking agent. Eur J Med Chem;65:12–20. 12. Hynes J.B., Buch J.M., Freisheim J.H. (1975) Quinazolines as inhibitors of dihydrofolate reductase. 3. Analogs of pteroic and isopteroic acids. J Med Chem;18:1191. 13. Alagarsamy V., Murugananthan G., Venkateshperumal R. (2003) Synthesis of some novel 2-mercapto-3-(substituted amino)-5,6,7,8-tetrahydro-3H-benzo[4,5]thieno [2,3-d]pyrimidin-4-ones as analgesic and anti-inflammatory agents. Biol Pharm Bull;26:1711. 14. Tiwari A.K., Singh Rathore V., Sinha D., Datta A., Sehgal N., Chuttani K., Mishra A.K. (2012) Design and docking studies of [diethylenetriaminepentaacetic acid(amino acid)2] with acetylcholine receptor as a molecular imaging agent for single-photon emission computed tomographic application. Mol Imaging;11:240–250. 15. Sinha D., Tiwari A.K., Singh S., Shukla G., Mishra P., Chandra H., Mishra A.K. (2008) Synthesis, characterization and biological activity of schiff base analogues of indole-3-carboxaldehyde. Eur J Med Chem;43:160– 165. 16. Fukunaga J.Y., Hansch C., Steller E.E. (1976) Inhibition of dihydrofolate reductase. Structure-activity correlations of quinazolines. J Med Chem;19:605–611. 17. Elslager E.F., Johnson J.L., Werbel L.M. (1983) Folate antagonists.20. Synthesis and antitumor and antimalarial properties of trimetrexate and related 6-[(phenylamino) methyl]-2, 4-quinazolinediamines. J Med Chem; 26:1753–1760. 18. Kung P.P., Casper M.D., Cook K.L., Wilson-Lingardo L. (1999) Structure-activity relationships of novel 2-substituted quinazoline antibacterial agents. J Med Chem;42:4705–4713. 19. Saeki T., Adachi Y. (1995) A selective type V phosphodiesterase inhibitor, E4021, dilates porcine large coronary artery. J Pharmacol Exp Ther;272:825–831.
634
Notes a
Synthesis of 4-methyl,7-hydroxyl 8 [N-(methyl phthalimide)] benzo-[1,2,b] pyrane 2 one] [A] as 4-methyl-7-hydroxycoumarin (0.05 mole) and N-methylol-phthalimide (0.08 mole) were dissolved in 8 N sulphuric Acid (100 mL) by stirring vigorously and carefully; while dissolving, the contents were occasionally cooled (as exothermic reaction occurred) in order to avoid the decomposition of the reactants. After refluxing for 1 h, the dark-coloured solution keft under refrigeration overnight and after that it was poured into cold water (250 mL). A solid separated out was filtered off and washed with water and a small portion treated with sodium bicarbonate solution (10%) to ensure the completion of reaction by ceasing of effervescence. The crude material was recrystallized by glacial acetic acid (yield 72%) m.p. 153 °C. IR (KBr pellets, per cm) 3385, 3290, 1640, 1715, 1196, 782. 1HNMR (200 MHz, CDCl3) d ppm; 7.69– 8.13 (m 4H, phthalimide aryl ring), 4.89(S, 2H, N-substituted methylene), 5.00 (S, 1H, OH exchanged with D2O), 6.57–7.27 (m, 3H, coumarin ring), 1.71(S, 3H, coumarin ring). Anal. Calculated for C15H13NO5, 67.8% C (theoretical C 66.52%), 3.89% H (theoretical H 4.03%), 5.02% N (theoretical N 4.88%). Found: M/Z 334[M]+, 160, 105, 77. b Synthesis of 4-methyl,7-hydroxy-8(N-methyl phthalimide) quinoline (1,5c) benzimidazole [B] was carried out by mixing 4 methyl, 7 hydroxy [N-(methyl phthalimide)] benzo[1,2,b] pyrane 2 one [A] (0.01 mole) and o-phenylenediamine (0.015 mole) in dry pyridine (50 mL). The refluxing reaction mixture was stirred for 6–7 h. This reaction was monitored by TLC; on completion of reaction, the solvent was removed in vacuo and the reaction mixture was cooled and then poured into ice-cold diluted HCl (50 mL); on neutralization, it was purified by column chromatography [column of Sio2 (80 g) pre-adsorption of the residue at SiO2 with ethyl acetate, elution with petroleum ether/ethyl acetate = 60: 40 (v/v) to obtain the product]. (Yield 81%) m.p.101 °C.IR (KBr pellets per cm) 3461, 3355, 2988, 1657,1278,742. 1H NMR (200 MHz, CDCl3) d ppm; 7.69–8.13 (m, 3H, quinoline ring). 5.02 (S, 1H, OH exchanged with D2O), 6.4–7.39 (m, 8H, aromatic rings). Anal. Calculated for C25H17N3O3, 71.42% C (theoretical C 73.71%), 4.1% H (theoretical H 4.18%), 9.87% N (theoretical N 10.12). Found: M/Z 404 [M]+, 405, 254, 77.
Chem Biol Drug Des 2013; 82: 630–634
