z Medicinal Chemistry & Drug Discovery ‘4 + 1’ Mixed Ligand Strategy for the Preparation of 99m Tc-Radiopharmaceuticals for Hypoxia Detecting Applications Kusum Vats, [a, e] Madhava B. Mallia,* [a] Anupam Mathur, [c] Haladhar D. Sarma, [b] and Sharmila Banerjee* [d, e] The ‘4 + 1’ mixed ligand strategy enables radiolabeling of biomolecules with 99m Tc at low ligand concentration without affecting it’s in vivo biological activity. The ‘4 + 1’ mixed-ligand complexes consist of central Tc(III) metal atom coordinated to a tetradentate tripodal chelator 2,2’,2’’-nitrilotriethanethiol (NS 3 ) and a monodentate isocyanide group tethered to a biomole- cule. The present work describes the use of ‘4 + 1’ mixed ligand strategy to design radiopharmaceuticals for the detection of tissue hypoxia in vivo. Isocyanide derivatives of two nitro- imidazoles, viz. 2-nitroimidazole (2NimNC) and metronidazole (MetNC), were synthesized and radiolabeled with 99m Tc using ‘4 + 1’ mixed ligand approach. The complexes [ 99m Tc(NS 3 )(2NimNC)] and [ 99m Tc(NS 3 )(MetNC)] could be prepared in excellent yield (> 90%). The structure of 99m Tc-complexes prepared at the no-carrier-added (nca) level was corroborated by spectroscopic analysis of corresponding rhenium analogues at the macroscopic level. Preliminary biological evaluation of the two nitroimidazole-‘4 + 1’ mixed ligand complexes in Swiss mice bearing fibrosarcoma tumor showed uptake and retention of the complexes in tumor. Introduction Hypoxia is a pathophysiological condition in a locally advanced solid tumor arising from an imbalance between the oxygen supply it receives and consumption. [1] Presence of hypoxia in tumors is one of the major reasons for its resistance to conventional radiotherapy and chemotherapy. [2,3] Therefore, detection of hypoxia in cancerous lesions assumes great significance as it may help in patient selection and therapy planning, resulting in better clinical outcome. Radiolabeled nitroimidazoles are potential markers of hypoxia and offers a non-invasive technique for the detection of tissue hypoxia in vivo. Selective, oxygen dependent, reduction of nitroimidazole in hypoxic cells is taken advantage of to differentiate them from normoxic cells. The mechanism underlying hypoxia selectivity demonstrated by nitroimidazole has been reviewed elsewhere. [4,5] The single electron reduction potential (SERP) is an important molecular parameter, which determines the effi- ciency of reduction of nitroimidazoles in hypoxic cells. The SERP values of bare 2-, 4- and 5-nitroimidazoles are in the order -418 mV, -527 mVand -450 mV, respectively, with respect to the standard hydrogen electrode. [6] Considering the SERP values alone, the 2-nitroimidazole seems to be the most suitable vector for targeting hypoxic tissues. [6,7] However, there are instances where 4- or 5-nitroimidazole complexes, having lower SERP than 2-nitroimidazole, had shown hypoxic cell targeting in vivo. [8,9] Several nitroimidazole radiopharmaceuticals labelled with PET or SPECT radioisotope have been evaluated for in vivo detection of hypoxia. [10-21] At present, [ 18 F]Fluoromisonidazole ([ 18 F]FMISO), a 2-nitroimidazole radiotracer, is the radiopharma- ceutical of choice for the clinical imaging of tumor hypoxia. [12,22] However, being a cyclotron produced radiopharmaceutical, its clinical use is generally limited to nuclear medicine centers situated at the close proximity to the cyclotron centers. This apparent limitation for all PET radiopharmaceuticals in general, and [ 18 F]FMISO in particular, could be overcome to a significant extent by developing a SPECT radiopharmaceutical as a viable option for hypoxia imaging application. While selecting a SPECT radioisotope, Technetium-99m, with its favorable nuclear char- acteristics such as half-life, 140 keV gamma emissions with 88% [a] K. Vats, Ph. D. M. B. Mallia Radiopharmaceuticals Division Bhabha Atomic Research Centre Mumbai-400085 Telephone: 91-22-25590746 E-mail: mallia@barc.gov.in [b] H. D. Sarma Radiation Biology and Health Science Division Bhabha Atomic Research Centre Mumbai-400085 [c] A. Mathur Radiopharmaceuticals Program Board of Radiation and Isotope Technology Mumbai-400705, India [d] Ph. D. S. Banerjee Radiation Medicine Centre Parel, Mumbai-400012, India Telephone: 91-22-2415-7098 Fax: 91-22-415-7098 E-mail: sharmila@barc.gov.in [e] K. Vats, Ph. D. S. Banerjee Homi Bhabha National Institute Anushakti Nagar, Mumbai-400094, India Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/slct.201700150 Full Papers DOI: 10.1002/slct.201700150 2910 ChemistrySelect 2017, 2, 2910 – 2916  2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim