Synthesis, Analytical Analysis, and Medicinal Aspect ofNovel Benzimidazoles and their Metal Complexes

Sangeeta Agrawal1, Rishi Raj Bhatnagar2,Anjani Tiwari3, Rakesh Srivastava4 andUpasana sharma1,*

1Department of chemistry, S.S.V (P.G) College, Hapur245101, India2Department of Chemistry, Christ Church College, Kanpur208001, India3Pharmaceutical Department, INMAS, Delhi 110057, India4Department of Chemistry, NGF College of Engg. &Technology, Palwal, Haryana 125001, India*Corresponding author: Upasana sharma,upasanasharma001@rediffmail.com;upasanasharma7@gmail.com

Benzimidazole and their metal analogs that can act asmultimodal agent and have non-peptidic CCK-B recep-tor antagonist were synthesized and characterized onthe 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 stud-ies. Cholecystokinin (CCK1 and CCK2) receptor bindingaffinities of these analogs (IC50) are 0.802 � 0.007 forcompound C and 0.326 � 0.012 for compound D in ratpancreatic acini. These studies have provided a newtemplate for further development of novel agents forvarious related diseases.

Key words: benzimidazole, cholecystokinin, non-peptidic andmetal sorption

Received 8 May 2013, revised 27 June 2013 and acceptedfor publication 18 July 2013

Benzimidazole analogs are known to be pharmacologicallyactive and find their application in the treatment of variousmedical applications such as epilepsy, diabetics, andantifertility (1,2). Recently, many researches elucidated thatbenzimidazole analogs can be suitably modified by theintroduction of different heterocyclic moieties to exhibit abroad spectrum of biologic activities such as potentanticancerous and microbial activities (3–6). Keeping thisfactor in our mind, we have modified benzimidazoleanalogs in such a way that would bring them intopreferred orientation. This may provide a more potentcholecystokinin (CCK) antagonist.

Cholecystokinin is very important for regulatory functions,as neurotransmitters in the brain and as regulators ofvarious functions of the gastrointestinal tract, preliminary atthe level of the stomach, pancreas, and gallbladder (7). Inaddition to that, they can act as an important growth factorin most parts of the gastrointestinal tract (8–10). Gastrinand CCK possess the same five amino acids at their COOHterminus, which is a biologically active site. Their actions aremediated by two different receptor types, CCK1 and CCK2

(11,12). CCK2 receptors are present in the gut mucosa andin the brain (7,13,14), whereas CCK1 receptors are presentin the gallbladder, pancreas, and brain (7,15,16).

The importance of selective antagonist is that they arepotential tools to visualize various tumors (17,18). In vivo

autoradiography studies have shown that cholecystokininCCK-B/gastrin receptors are expressed not only in morethan 90% of medullary thyroid carcinomas (MTC) (19) butalso in high percentage of small-cell lung cancer, someovarian cancer, astrocytomas, and potentially in a variety ofadenocarcinomas, gastrointestinal tumors, and colorectalcell lines. In this work, we have designed multimodal ana-logs of benzimidazoles and studied it with various metalcomplexes to see their effect in applied as well as inmedicinal chemistry, as its intermediate is synthesizedaccording to previous literaturea and labeled with specificmetal to explore binding aspect with CCK receptor. Theinitial results have shown good results for further evaluation.

Experimental

All chemicals were purchased from well-known commer-cial sources and used as received. 1H NMR spectra wererecorded on a NMR spectrometer with known internalstandard tetramethylsilane (TMS). Mass peak was calcu-lated by fast atom bombardment mass spectra (FAB-MS)on a JEOL NMS-SX102 spectrometer. All the details ofmaterial and method are given in reference and shortnotes part.b Synthesis of benzimidazole analog [Bzm] ispresented in scheme 1 for the synthesis. The first twosteps are carried out and as mentioned in short notes.a,b

Compounds C and D were evaluated for their ability todisplace [125I] (BH)-CCK8 (sulfated) from isolated ratpancreatic acini (CCK1) and guinea-pig cerebral cortexmembranes (CCK2) according to established protocols.

630 ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12201

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Research Letter

Binding affinities expressed as IC50 are reported in Table 1along with reference compound 3-ureido-1,4-benzodiaze-pine (Merck L-365260). Values without standard errorswere obtained from no more than two experiments.

Distribution studies with various metalsThe relative affinities of the compound for three metal ionswere studied by batch equilibrium method. The chelationproperty of the compound in its complex form with Ni, Zn,and Cu was determined by batch equilibrium methodinvolving metal ions in various electrolyte viz. KCl, in differ-ent concentrations such as 0.01, 0.1, and 0.5 M.

To know the appropriate time required for the maximumadsorption of the metals, the sorption capacity was deter-mined by shaking the complex with the metal ion solutionfor different time intervals. The physicochemical propertiesshow that the chelating compounds in H+ also behave asa weak cation exchanger. The sorption capacities weredetermined for seven metal ions in three concentrations.

In vivo studies[125I]BH-CCK-8 receptor binding assay in isolated ratpancreatic acinar cells was carried out by isolated

pancreatic acini prepared by enzymatic digestion ofpancreas. Drug-displacing experiments were carried outby incubating acinar cells [125I]BH-CCK-8 (25 pM final con-centration) and competitors in 0.5 mL total volume at37 °C for 30 min, in a shaking bath. At the end of incuba-tion, 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. Thesupernatant was aspirated, and the radioactivity associ-ated with the pellet measured. The non-specific bindingwas estimated in the presence of 1 jjM CCK-8, accountingfor 15% of total binding.

[125I]BH-CCK-8 receptor binding assay in membranesfrom guinea-pig cerebral cortices was carried out bydetermining the protein concentration using bovineserum albumin (BSA) as standard. The binding experi-ments 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 suspensionwith labeled ligand and inhibitors was carried out in a96-well microtiter filter plate (Multiscreen; Millipore Inc,Bedford, MA, USA) with integral Whatman GF/B mem-brane filters. Aliquot of membranes (0.5 mg of proteinper mL) was added to each well, containing [125I]BH-CCK8 (25 pM), in a final volume of 250 jjl. The non-spe-cific binding of iodinated peptide was defined in thepresence of 1 pM CCK-8, accounting for 20% of totalbinding. Non-specific binding of [125I]BH-CCK-8 to mem-brane filters (blank) measured in wells containing anequal amount of labeled ligand, but no membranes, was0.2% of total radioligand added. After 120 min at 25 °C,the 96-well plate was placed on the vacuum filtrationapparatus (Millipore Inc.).

Table 1: CCK receptor binding data of the target compounds

Complexes (R)

Ratpancreatic acini(CCK1), IC50 (lM)

Guinea-pigbrain Cortex(CCK2), IC50 (lM)

Ref. (VL-0494) Ph 0.197 � 0.107 16.40Compound (C) NH2 0.802 � 0.007 3.12Compound (D) CH3 0.326 � 0.012 1.94

Scheme 1: Chemical scheme forsynthesis of Bzm (where R = NH2and CH3 in compound C & Drespectively).

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Benzimidazoles and Metal Complexes

Labeling of the compounds [Bzm] has been carried out bytaking 100 lL of 0.03 nM solution of the compound [Bzm]dissolved in DMSO in a shielded vial, and 60 lL of1 9 10�2

M Sncl2.2H2O (dissolved in N2-purged 1 mL10% acetic acid) was added followed by addition of (<1 h)freshly eluted saline solution of sodium pertechnetate(NaTcO4) (74 MBq, 100 mL). The pH of the reaction mix-ture was adjusted to 6.5 with 0.1 M NaHCO3 solution andshook to mix the contents. The vial was allowed to standfor 45 min at room temp. In vitro serum stability assaywas carried out in fresh human serum, which was pre-pared by allowing blood collected from healthy volunteersto clot for 1 h at 37 °C in a humidified incubator main-tained at 5% carbon dioxide and 95% air. Then, the sam-ple was centrifuged at 400, and the serum was filteredthrough 0.22 micron syringe filter into sterile plastic culturetubes. The above freshly prepared technetium radiocomplex was incubated in fresh human serum at physio-logic conditions, that is, at 30 °C at a concentration of100 nM/mL, and then analyzed by ITLC-SG at differenttime intervals to detect any dissociation of complex. Per-centage of free pertechnetate at a particular time-pointwas estimated using saline and acetone as mobile phase,which represents percentage dissociation of the complexat that particular time-point in serum.

Albino mice strain was used for the tissue distributionstudies. Animal handling and experimentation were car-ried out as per the guidelines of the Institutional AnimalEthics Committee. For biodistribution studies, albino micestrain (A) was used as animal model and complex oflabeled (Bzm) was used. An equal dose of test com-pound was injected in mice through tail vein of each ani-mal. Similarly, in another group of animals, CCK-B(1 mg/kg by weight of animal model) was injected 10 minbefore injection of the radiolabeled test compound, andbiodistribution was studied under receptor saturation con-ditions. At different time intervals, mice were killed, bloodwas collected, and different tissue and organs were dis-sected and analyzed (Tables 2 and 3). The actual amountof drug administered to each animal was calculated bysubtracting the activity left in the tail from the activity

injected. Total volume of the blood was calculated as 7%of the body weight.

Blood kinetics studiesThe blood clearance study was performed in albino NewZealand rabbits weighing approximately 2.5–3.0 kg afteradministration of drug [Bzm] in 0.3 mL via the ear vein. At

Table 2: CCK-B receptor expressing organs (at the time intervalsof 1, 2, 4, 6, 8 h) for compound [C]

Organs

Uptake (% ID/g)

After 1 h After 2 h After 4 h After 6 h After 8 h

Stomach 1 2.5 3 6 7.5Intestine 0.5 0.4 0.2 2 2Brain 0 0.2 0.45 1 1Liver 17 21 15 14 13Spleen 11 15 14 12 11Kidney 5 7 2 5 5Blood 3 2 2 0.4 0.4Heart 1 0.4 0.3 0.3 0.1

Table 3: CCK-B receptor expressing organs (at the time intervalsof 1, 2, 4, 6, 8 h) for compound [D]

Organs

Uptake (% ID/g)

After 1 h After 2 h After 4 h After 6 h After 8 h

Stomach 1 2.0 3 7 7.3Intestine 0.5 0.5 0.2 1 1Brain 0 0.1 0.45 1 1Liver 16 23 13 14 13Spleen 11 13 17 16 15Kidney 5.5 6.0 3 4.5 4.5Blood 2.5 2 2 2.5 2.1Heart 0.8 0.5 0.2 0.1 0.2

Figure 2: Percentage uptake studies in various organs bytreatment of Bzm (D).

Figure 1: Percentage uptake studies in various organs bytreatment of Bzm (C).

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different time intervals, about 0.5 mL of blood samples waswithdrawn from the dorsal vein of other ear and radioactivitywas measured in the gamma-counter. The data from theexperiment were expressed as percentage of administereddose at each time interval in Figures 1 and 2.

Result and Discussion

The synthesized compound was characterized by IR (COnear 1600/cm, OH 3200–3600, and aromatic below900/cm), NMR (aromatic proton in range from 6 to8.5 ppm and heterofunctional group via D2O exchange),MASS spectroscopic studies, and Rf values of therelevant complexes (Rf values). Labeling efficiencieswere more than 97%, and complex was stable about12 h at 30 °C. Results of serum stability studies showedthat the metal ion was intact under physiologicconditions, and unbound radioactivity was less than0.5% in 8 h.

Biodistribution studies were determined by injectedradiolabeled compound, intravenously as a function oftime, differentiating those tissues which are known orexpected to express CCK receptors, organs involved inblood pool, and excretion of the ligand. Higher uptake wasfound in liver, spleen, and stomach, but retention waslonger in stomach (Figures 1 and 2). Very low activity wasfound in brain probably due to inability of crossing theblood–brain barrier. The rapid blood clearance was alsoevident from the blood kinetics study.

The result of pretreatment studies of labeled [Bzm] byblocking with 1 mg/kg of CCK2, 15 min before the injec-tion of radio complex, reduced the accumulation instomach where the activity in the intestine was reduced.There was increased accumulation of activity in liver, butreduction in case of rest of the organs studied.

The metal ion uptake study are given in Figure 3. Thisshows that Bzm in H+ also behaves as a weak exchanger.The results of rate of sorption experiments showed thattime required for maximum sorption capacity is 45 min.

The uptake capacities determined for seven metal ions inthree concentration systems resulted in the followingorder: Mn2+ > Co2+ > Cu2+ > Ni2+ > Zn2+ > Ba2+ > Pb2+.The sorption was found to increase with an increase inpH; however, the capacity order for the investigated metalions remained unchanged. The variation in uptakecapacities for different metal ions may be explained on thebasis of difference in stability of metal Bzm complexesunder experimental conditions.

Thus, the benzimidazole ring structure fused to pyridinering might provide lead for new type of compounds havingCCK-B receptor affinity. Some non-specific uptake in othertissues can be overcome by changing the basic skeletonby different R groups. The therapeutic potential of thesecomplexes can be extend further by applying these in dif-ferent animal models and cell lines.

Conflict of Interest

The authors state that there is no conflict of interest.

References

1. Singh S., Ojha H., Tiwari A.K., Kumar N., Singh B.,Mishra A.K. (2010) Design, synthesis, and in vitro anti-proliferative activity of benzimidazole analogues forradiopharmaceutical efficacy. Cancer Biother Radiop-harm;25:245–250.

2. Shukla G., Tiwari A.K., Singh V.K., Bajpai A., ChandraH., Mishra A.K. (2008) Effect of a novel series of ben-zothiazolo-quinazolones on epidermal growth factorreceptor (EGFR) and biological evaluations. Chem BiolDrug Des;72:533–539.

3. Tiwari A.K., Mishra A.K., Bajpai A., Mishra P., SharmaR.K., Pandey V.K., Singh V.K. (2006a) Synthesis andpharmacological study of novel pyrido-quinazoloneanalogues as anti-fungal, antibacterial, and anticanceragents. Bioorg Med Chem Lett;16:4581–4585.

4. Dandia A., Singh R. (2005) Synthesis of novel quinazo-linone and fused quinazolinones. J Flour Chem;126:307–312.

5. Srivastava V., Srivastava A.M., Tiwari A.K., SrivastavaR., Sharma R., Sharma H., Singh V.K. (2009) Disubsti-tuted 4(3H) quinazolones: A novel class of antitumoragents. Chem Biol Drug Des;74:297–301.

6. Malecki N., Caroto P., Rigo B., Goossens J.H., HenchartJ.P. (2004) Synthesis of condensed quinolines and qui-nazolines as DNA ligands. Bioorg Med Chem;12:641.

7. Tanwar J., Datta A., Tiwari A.K., Thirumal M., ChuttaniK., Mishra A.K. (2011) Preclinical evaluation of DO3P-AME-DO3P: A polyazamacrocyclic methylene phos-phonate for diagnosis and therapy of skeletal metas-tases. Bioconjug Chem;22:244–255.

8. Kakkar D., Tiwari A.K., Chuttani K., Kaul A., Singh H.,Mishra A.K. (2010) Comparative evaluation of gluta-

Figure 3: Metal ions uptake study with Bzm (C).

Chem Biol Drug Des 2013; 82: 630–634 633

Benzimidazoles and Metal Complexes

mate-sensitive radiopharmaceuticals: Technetium-99m-glutamic acid and technetium-99m-diethylenetriamine-pentaacetic acid-bis(glutamate) conjugate for tumorimaging. 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 propertiesof 4-(3H)-quinazolone derivatives. Eur J MedChem;42:1234–1238.

10. Ram V.J., Farhanullah M., Tripathi B.K., SrivastavaA.K. (2003) Synthesis and antihyperglycemic activity ofsuitably functionalized 3H-quinazolin-4-ones. BioorgMed Chem;11:2439–2444.

11. Srivastava P., Tiwari A.K., Chadha N., Chuttani K.,Mishra A.K. (2013) Synthesis and biological evaluationof newly designed phosphonate based bone-seekingagent. Eur J Med Chem;65:12–20.

12. Hynes J.B., Buch J.M., Freisheim J.H. (1975) Quinazo-lines as inhibitors of dihydrofolate reductase. 3.Analogs of pteroic and isopteroic acids. J MedChem;18:1191.

13. Alagarsamy V., Murugananthan G., VenkateshperumalR. (2003) Synthesis of some novel 2-mercapto-3-(sub-stituted amino)-5,6,7,8-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyrimidin-4-ones as analgesic and anti-inflam-matory 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 anddocking studies of [diethylenetriaminepentaacetic acid-(amino acid)2] with acetylcholine receptor as a molecu-lar imaging agent for single-photon emission computedtomographic 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, characteri-zation and biological activity of schiff base analoguesof indole-3-carboxaldehyde. Eur J Med Chem;43:160–165.

16. Fukunaga J.Y., Hansch C., Steller E.E. (1976) Inhibitionof dihydrofolate reductase. Structure-activity correla-tions of quinazolines. J Med Chem;19:605–611.

17. Elslager E.F., Johnson J.L., Werbel L.M. (1983) Folateantagonists.20. Synthesis and antitumor and antimalar-ial properties of trimetrexate and related 6-[(phenylami-no) methyl]-2, 4-quinazolinediamines. J Med Chem;26:1753–1760.

18. Kung P.P., Casper M.D., Cook K.L., Wilson-LingardoL. (1999) Structure-activity relationships of novel2-substituted quinazoline antibacterial agents. J MedChem;42:4705–4713.

19. Saeki T., Adachi Y. (1995) A selective type V phospho-diesterase inhibitor, E4021, dilates porcine largecoronary artery. J Pharmacol Exp Ther;272:825–831.

Notes

aSynthesis of 4-methyl,7-hydroxyl 8 [N-(methyl phthali-mide)] benzo-[1,2,b] pyrane 2 one] [A] as 4-methyl-7-hy-droxycoumarin (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, thecontents were occasionally cooled (as exothermic reactionoccurred) in order to avoid the decomposition of the reac-tants. After refluxing for 1 h, the dark-coloured solution keftunder refrigeration overnight and after that it was pouredinto cold water (250 mL). A solid separated out was filteredoff and washed with water and a small portion treated withsodium bicarbonate solution (10%) to ensure the comple-tion of reaction by ceasing of effervescence. The crudematerial 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-substi-tuted methylene), 5.00 (S, 1H, OH exchanged with D2O),6.57–7.27 (m, 3H, coumarin ring), 1.71(S, 3H, coumarinring). Anal. Calculated for C15H13NO5, 67.8% C (theoreticalC 66.52%), 3.89% H (theoretical H 4.03%), 5.02% N (theo-retical N 4.88%). Found: M/Z 334[M]+, 160, 105, 77.bSynthesis of 4-methyl,7-hydroxy-8(N-methyl phthalimide)quinoline (1,5c) benzimidazole [B] was carried out by mix-ing 4 methyl, 7 hydroxy [N-(methyl phthalimide)] benzo-[1,2,b] pyrane 2 one [A] (0.01 mole) and o-phenylenedi-amine (0.015 mole) in dry pyridine (50 mL). The refluxingreaction mixture was stirred for 6–7 h. This reaction wasmonitored by TLC; on completion of reaction, the solventwas removed in vacuo and the reaction mixture wascooled and then poured into ice-cold diluted HCl(50 mL); on neutralization, it was purified by columnchromatography [column of Sio2 (80 g) pre-adsorption ofthe residue at SiO2 with ethyl acetate, elution withpetroleum ether/ethyl acetate = 60: 40 (v/v) to obtain theproduct]. (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, aromaticrings). 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.

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