molecules Article New Bioconjugated Technetium and Rhenium Folates Synthesized by Transmetallation Reaction with Zinc Derivatives Jordi Borràs 1 , Julie Foster 2 , Roxana Kashani 2 , Laura Meléndez-Alafort 3 , Jane Sosabowski 2 , Joan Suades 1, * and Ramon Barnadas-Rodríguez 4, * 1 Departament de Química, Edifici C, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Catalonia, Spain; jborrasamoraga@gmail.com 2 Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 5P, UK; j.m.foster@qmul.ac.uk (J.F.); roxana.kashani@qmul.ac.uk (R.K.); j.k.sosabowski@qmul.ac.uk (J.S.) 3 Istituto Oncologico Veneto IRCCS, 35128 Padova, Italy; laura.melendezalafort@iov.veneto.it 4 Unitat de Biofísica/Centre d’Estudis en Biofísica, Facultat de Medicina, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Catalonia, Spain * Correspondence: joan.suades@uab.cat (J.S.); ramon.barnadas@uab.cat (R.B.-R.) Abstract: The zinc dithiocarbamates functionalized with folic acid 2Zn and 3Zn were synthesized with a simple straightforward method, using an appropriated folic acid derivative and a functionalized  zinc dithiocarbamate (1Zn ). Zinc complexes 2Zn and 3Zn show very low solubilities in water, making  them useful for preparing Tc-99m radiopharmaceuticals with a potentially high molar activity. Thus, Citation: Borràs, J.; Foster, J.; the transmetallation reaction in water medium between the zinc complexes 2Zn or 3Zn and the cation Kashani, R.; Meléndez-Alafort, L.; fac-[99m Tc(H2 O)3 (CO)3 ]+ , in the presence of the monodentate ligand TPPTS, leads to the formation Sosabowski, J.; Suades, J.; of the 2 + 1 complexes fac-[99m Tc(CO)3 (SS)(P)] bioconjugated to folic acid (2Tc and 3Tc ). In spite of Barnadas-Rodríguez, R. New the low solubility of 2Zn and 3Zn in water, the reaction yield is higher than 95%, and the excess zinc Bioconjugated Technetium and Rhenium Folates Synthesized by reagent is easily removed by centrifugation. The Tc-99m complexes were characterized by comparing Transmetallation Reaction with Zinc their HPLC with those of the homologous rhenium complexes (2Re and 3Re ) previously synthesized Derivatives. Molecules 2021, 26, 2373. and characterized by standard methods. Preliminary in vivo studies with 2Tc and 3Tc indicate low https://doi.org/10.3390/ specific binding to folate receptors. In summary, Tc-99m folates 2Tc and 3Tc were prepared in high molecules26082373 yields, using a one-pot transmetallation reaction with low soluble zinc dithiocarbamates (>1 ppm), at moderate temperature, without needing a subsequent purification step. Academic Editor: Jason Holland Keywords: technetium; carbonyl; folic; transmetallation; radiopharmaceutical; rhenium Received: 22 March 2021 Accepted: 16 April 2021 Published: 19 April 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in The bioconjugation of biomolecules to metallic fragments is an important topic with a published maps and institutional affil- wide range of potential applications, in particular, the development of new metal drugs for iations. diagnosis and therapy [1–3]. In this context, we are developing a new approach based on a transmetallation reaction between zinc complexes and transition metal atoms [4–6]. The transmetallation reaction has been applied for many years as a useful reaction for preparing new compounds by the simple transfer of one ligand between different metals [7–9]. The Zn(II) complexes are particularly useful for this goal because it is well-known that the Copyright: © 2021 by the authors. transmetallation reaction between Zn(II) and transition metals is usually favored from Licensee MDPI, Basel, Switzerland. This article is an open access article the thermodynamic point of view, due to the higher stability of bonds with a metal in distributed under the terms and dn configuration (n = 1–9) compared with those to a metal in a d10 configuration as conditions of the Creative Commons Zn(II) [4–6,10]. Attribution (CC BY) license (https:// Our strategy is based on using a functionalized Zn(II) dithiocarbamate complex creativecommons.org/licenses/by/ (complex 1Zn , Figure 1) which contains succinimidyl ester groups that make possible 4.0/). bioconjugation to a wide range of biomolecules [4–6]. This complex can be considered Molecules 2021, 26, 2373. https://doi.org/10.3390/molecules26082373 https://www.mdpi.com/journal/molecules Our strategy is based on using a functionalized Zn(II) dithiocarbamate c Molecules 2021, 26, 2373 (complex 1Zn, Figure 1) which contains succinimidyl ester groups that2make of 15 poss conjugation to a wide range of biomolecules [4–6]. This complex can be considere ful tool for the bioconjugation of transition metals because it can be conjugated to aecules useful with a very tool for simple experimental the bioconjugation of transitionprocedure. Theitobtained metals because zinc complex can be conjugated to al biomolecules the formation with of athe very simple experimental bioconjugated complexprocedure. with theThe obtained desired zinc complex transition metal by a allows for the formation of the bioconjugated complex with the desired transition metal by the simple transmetallation reaction. It should be highlighted that this compound applying the simple transmetallation reaction. It should be highlighted that this compound ready has been already employed been employedby by another research another research group group for preparing for preparing bioconjugated bioconjugated gold go plexes [11], complexes [11],which which is is aa very verydifferent different goalgoal toresearch to our our research objectives. objectives. Figure1.1.Molecular Figure Molecularstructure of 1Zn .of structure 1Zn. Our approach is focused on preparing of Tc-99m complexes for radiopharmaceutical Our approach applications. The Tc-99m is focused radionuclideon preparing is widely used of for Tc-99m in vivocomplexes for radiopharma SPECT (single-photon 99 99m Tc applications. emission computed Thetomography) Tc-99m radionuclide imaging because is widely it is easilyused for in accessible fromvivoMo/ SPECT (single generators, it shows an appropriate half-life ( ≈ 6 h) and emission computed tomography) imaging because it is easily accessible from 99Mit has very suitable emission properties and detectable gamma rays [12–14]. A key point that should be kept in mind generators, it shows an appropriate half-life (≈ 6 h) and it has very suitable emissio in the radiopharmaceutical chemistry of bioconjugated complexes with Tc-99m is that the erties and detectable concentration gamma of this radioactive rays metal [12–14]. in the labeling Areactions key point thatlow is very should (10−6 –10be−kept 8 M), in min radiopharmaceutical and consequently, although chemistry of bioconjugated the concentration complexes of the derivatized with Tc-99m biomolecule used in is that the centration of this radioactive metal in the labeling reactions is very lowit(10 labeling reaction to form the corresponding metal complex could be very low, is −6–10−8 usually several orders of magnitude higher than the concentration of the radioactive consequently, although the concentration of the derivatized biomolecule used in metal [15]. Hence, to avoid receptor site saturation, the concentration of the unlabeled beling reaction derivatized to form biomolecule thebe should corresponding as low as possible metal at thecomplex end of thecould process.beUsing veryZn- low, it is several orders of magnitude higher than the concentration dithiocarbamates is useful for this goal because they show very low solubilities in water, so of the radioactive me Hence, the to avoid concentration receptor biomolecule of unlabeled site saturation, stays verythelow concentration in the reactionof the unlabeled medium (water) der and the insoluble Zn-dithiocarbamate can be easily removed biomolecule should be as low as possible at the end of the process. Using Zn-dith by filtration. In addition, Zn-dithiocarbamates are useful compounds for transmetallation reactions, a property that mates is useful for this goal because they show very low solubilities in water, so has been reported for different applications [4–6,10,11,16]. centration In previousof papers, unlabeled biomolecule we have stays very shown the viability of this low in the approach withreaction medium (wa the homologous the insoluble rhenium complexesZn-dithiocarbamate and with Tc-99m in the canusual be easily removed experimental by filtration. conditions employedInfor addition the preparation of radiopharmaceuticals [4–6]. In this paper thiocarbamates are useful compounds for transmetallation reactions, a property we have studied the viability of this approach for preparing the folate derivatives of rhenium and technetium-99m for been reported for different applications [4–6,10,11,16]. radiopharmaceutical applications. Folic acid (Figure 2) is a vitamin (B9) necessary for cell growth Inbecause previous papers, in it is involved we thehave shown synthesis the viability of purine and thymine of this approach nucleotides with the h and the gous rhenium synthesis complexes of the amino and with acid methionine Tc-99m [17,18]. in the The fast usual growth andexperimental proliferation ofconditions cancer em for the cells preparation requires of radiopharmaceuticals a high consumption of folic acid, and, for [4–6]. this In this the reason, paper folate we have studied receptors are bility of this approach for preparing the folate derivatives of rheniumand over-expressed in many cancer cells (ovarian, breast, cervical, nasopharyngeal and tech colon) [19,20]. Consequently, different methods to label folic acid with Tc-99m have been 99m for radiopharmaceutical applications. Folic acid (Figure 2) is a vitamin (B9) ne studied by different research groups [21–26]. In the present paper, we report the preparation fortwo of cellnewgrowth becausebioconjugated Zn(II) complexes it is involved in the to folic acid,synthesis of purine using two different andbetween linkers thymine nuc andfolic the theacid synthesis of the amino acid and the dithiocarbamate group.methionine We studied the [17,18]. The fast growth transmetallation reaction and of proli these complexes with rhenium and technetium-99m. Preliminary of cancer cells requires a high consumption of folic acid, and, for this reason, th biological studies of the new Tc-99m complexes were also made to analyze whether these compounds could lead to receptors are over-expressed in many cancer cells (ovarian, breast, cervical, naso new radiopharmaceuticals with high molar activity. geal and colon) [19,20]. Consequently, different methods to label folic acid with have been studied by different research groups [21–26]. In the present paper, w the preparation of two new Zn(II) complexes bioconjugated to folic acid, using tw ent linkers between the folic acid and the dithiocarbamate group. We stud transmetallation reaction of these complexes with rhenium and technetium-99m. inary biological studies of the new Tc-99m complexes were also made to analyze these compounds could lead to new radiopharmaceuticals with high molar activi Molecules 2021, Molecules 2021, 26, 26, 2373 x FOR PEER REVIEW 33 of of 15 15 Molecules 2021, 26, x FOR PEER REVIEW 3 of 15 Figure 2. Folic acid. Figure 2. Figure 2. Folic acid. 2. Results 2. 2. Results Results 2.1. Zinc Complexes 2.1. Zinc Complexes The first step to obtain obtain the the bioconjugated bioconjugated zinc complexes complexes was toto derivatize derivatize the the folic folic The first step to obtain the bioconjugated zinc complexes was to derivatize the folic acid in order to obtain a molecule that contains a primary aminoamino group, group, which which can can lead lead to to acid in order to obtain a molecule that contains a primary amino group, which can lead to peptide bond the formation of the peptide bond with with the the activated activated ester ester groups groups of ofcomplex complex11ZnZn. Thus, the formation of the peptide bond with the activated ester groups of complex 1Zn. Thus, folicacid the folic acidderivatives derivativesshown shown in in Figure Figure 3 were 3 were synthesized synthesized by using by using previously previously reportedre- the folic acid derivatives shown in Figure 3 were synthesized by using previously re- ported methods methods [27,28]. [27,28]. ported methods [27,28]. Figure 3. Folic acid derivatives. Figure 3. Folic acid derivatives. These organic compounds were characterized by the usual spectroscopic and spec- Thesemethods, trometric organic compounds and all datawere characterized are consistent with bythose the usual spectroscopic previously reported and spec- [27,28]. trometric methods, These compounds compounds were and all werechosen data chosenbecause are consistent becausethey theycontainwith contain those both previously reported [27,28]. both anan amine amine group group thatthat allows allows the These the compounds biomolecule were chosen because they contain both an amine group that allows the biomolecule to betolinked be linked withwith complex complex 1Zn ,aand 1Zn, and a spacer spacer that isthat is useful useful to distance to distance the folatethe biomolecule folate to be linked with complex 1 Zn, and a spacer that is useful to distance the folate group group from the from thecenter, metal metal center, to limitto thelimit the possible possible steric hindrance steric hindrance of the center of the metallic metallic in group in center from thethe metal center, interaction with tothe limit thereceptor. folate possible Furthermore, steric hindrance of the metallic center in the interaction with the folate receptor. Furthermore, we chosewe twochose two derivatives derivatives with dif- the interactionpolarities with with the folate receptor. Furthermore, we chose twoan derivatives withand dif- ferentdifferent polarities (see Figure (see3).Figure The top 3).molecule The top contains molecule contains an alkylic chain, alkylic and chain, the bottom ferent the polarities bottom (see molecule Figure 3). The contains chain. top a polyether molecule chain. contains The an use of twoalkylic chain, different and the linkers is bottom molecule contains a polyether The use of two different linkers is interesting to inter- com- molecule esting contains athe polyether chain. The use of two different linkers is interesting to com- pare the behavior of the possible radiopharmaceuticals in vivo, since it is well-known it to compare behavior of the possible radiopharmaceuticals in vivo, since is that pare the behavior well-known that of the more possible polar radiopharmaceuticals compounds show a lower in vivo, since interaction with it blood is well-known proteins that and more polar compounds show a lower interaction with blood proteins and favor elimina- more elimination favor polar compounds by renal show a lower excretion interaction with blood proteins and favor elimina- [24,29,30]. tion by renal excretion [24,29,30]. tion by Therenal excretion [24,29,30]. The next next step stepwaswasthethereaction reactionofofthe thetwotwocompounds compounds of of Figure Figure3 with 3 withthethe zinczinc complex com- Zn toThe 1plex next step new was zincthe reaction bioconjugated complexes of the two compounds with folicof 1Zn to yield the new zinc complexes bioconjugated with folic acid 2Zn and 3Zn, in yield the Figure acid 2 Zn 3 with and 3 Zn the , as zinc shown com- as plex 1Zn1.to yield the new zinc complexes bioconjugated with folic acid 2Zn and 3Zn, as Scheme shown in Scheme 1. shown in Scheme 1. were performed in similar experimental conditions to those previ- These These reactions reactions were performed in similar experimental conditions to those previ- ouslyThese reactions reported were [4,5]. The performed only modificationsin similar wereexperimental that the solvent conditions was changed to those previ- to DMSO ously reported [4,5]. The only modifications were that the solvent was changed to DMSO ously reported (dimethyl [4,5]. because sulfoxide) The onlyfolicmodifications acid showswere that the solvent low solubility in DMFwas andchanged the reactionto DMSOtime (dimethyl sulfoxide) because folic acid shows low solubility in DMF and the reaction time (dimethyl sulfoxide) because folic acid shows low solubility in was longer. The final products 2Zn and 3Zn were easily separated from unreacted folic acidDMF and the reaction time was longer. The final products 2Zn and 3Zn were easily separated from unreacted folic acid was longer. because the The newfinalzincproducts complexes 2Znareandless 3Zn soluble were easily separated in water than from unreacted folic acid. The two folicnew acid because the new zinc complexes are less soluble in water than folic acid. The two new because thewere complexes newcharacterized zinc complexes by theareusual less soluble in water spectroscopic and than folic acid. methods spectrometric The two new (11H, complexes were characterized by the usual spectroscopic and spectrometric methods 13 1 H NMR ( 1H, C NMR, IR complexes wereandcharacterized HRMS can bebyfound as Supplementary the usual spectroscopic and 13C NMR, IR and HRMS can be found as Supplementary Materials). Thus, the 1H NMR Materials). Thus, the spectrometric methods ( H, spectra 13C NMR, are IR consistent and HRMS withcan the beformation found as of Supplementary the bioconjugated zinc complexes, Materials). Thus, the showing 1H NMR the spectra are consistent with the formation of the bioconjugated zinc complexes, showing characteristic signals ofwith spectra are consistent the isonipecotic the formation ringofafter the bioconjugation bioconjugated zinc (4.83–4.86 ppm, assigned complexes, showing Molecules 2021, 26, x FOR PEER REVIEW Molecules 2021, 26, 2373 the characteristic signals of the isonipecotic ring after bioconjugation 4(4.83–4.86 of 15 p signed to the two equatorial protons) and the absence of the signals of the reag Furthermore, the presence of the characteristic aromatic signals of folic acid fragm the to absences the of theprotons) two equatorial signal andof the the primary absence ofamine group the signals arereagent of the in accordance with the 1Zn . Further- more, the presence of the characteristic aromatic signals of folic acid fragment esis that the folic derivative has reacted and it is not present as a contaminant in and the absences of the signal of the primary amine group are in accordance with the hypothesis products. The 13C NMR spectra are also consistent with the formation of the t that the folic derivative has reacted and it is not present as a contaminant in the final complexes, products. Theshowing 13 C NMR the characteristic spectra signalwith are also consistent of the dithiocarbamate the formation of the twogroup new at 20 and all expected signals of the different organic groups. Finally, complexes, showing the characteristic signal of the dithiocarbamate group at 203.2 ppm the elemental an and all expected signals in accordance of thecalculated with the different organic groups. Finally, stoichiometry, andthethe elemental analysis analysis of theis in two com accordance with the calculated stoichiometry, and the analysis of the two compounds by− by ESI-HRMS shows the expected signals for 2Zn (m/e; 787.1798; − − 2H+[M] − L − 2H+) ESI-HRMS shows the expected signals for 2 (m/e; 787.1798; [M] − L +) in agreement with the theoretical calculated Zn Zn ) and 3 (m/e;1579.4329; (m/e; 1579.4329;[M][M] − H− +) H in agreement with the theoretical calculated values (787.1792 values (7 and1579.4293, and 1579.4293, respectively). respectively). Scheme1. 1. Scheme Synthesis Synthesis of complexes of zinc zinc complexes bioconjugated bioconjugated with with folic acid folic (2Zn andacid 3Zn ). (2Zn and 3Zn). 2.2. Rhenium Complexes 2.2. Before Rhenium Complexes studying the transmetallation reaction with Tc-99m, it is necessary to synthesize Before studying and characterize the transmetallation the homologous rhenium complexes. reaction with Tc-99m Tc-99m,areit prepared complexes is necessary to at a high size anddilution of Tc-99m: characterize theThe concentration homologous (10 –10−8 M) − 6 is so lowcomplexes. rhenium that it iscomplexes Tc-99m not possible to use standard methods to analyze the complexes. However, it is well-known that pared at a high dilution of Tc-99m: The concentration is so low (10−6–10−8 M) that they can be characterized by comparing their retention times in HPLC with those of their possible torhenium homologues use standard complexes methods to analyze because they show similartheretention complexes. However,The times [12–14,31]. it is well that they 3can fac-[Re(CO) 2 − Br3 ] bereagent characterized was chosenby forcomparing their retention this purpose because it is usefultimes in HPLC with for synthesizing rhenium carbonyl complexes homologues of the Tc-99m complexes their homologues rhenium complexes because they show similar retention obtained with the cation tim fac-[99m Tc(H2 O)3 (CO)3 ]+ [32,33]. This Tc-99m complex is particularly useful for preparing 14,31]. The fac-[Re(CO)3Br3] reagent was chosen for this purpose because it is us 2− new radiopharmaceuticals because the easy substitution of the labile water molecules synthesizing leads to very inertrhenium carbonyl and stable complexes Tc-99m complexes homologues that are useful forof the Tc-99m complexes o radiopharmaceutical with the cation purposes [32,33]. fac-[ 99mTc(H2O)3(CO)3]+ [32,33]. This Tc-99m complex is particularl for preparing Complexes 2new Re and radiopharmaceuticals 3Re were synthesized by usingbecause themethod a similar easy substitution of the labi to that previously reported [4–6] for the transmetallation reaction between the bioconjugated molecules leads to very inert and stable Tc-99m complexes that are useful for rad zinc complexes, the rhenium metal complex [NEt4 ]2 [Re(CO)3 Br3 ] and the mono-dentated ligand TPPTS maceutical purposes [32,33]. (triphenylphosphine trisulphonate), by means of the 2 + 1 reaction shown in Scheme 2. Complexes The co-ligand TPPTS 2Rewas and 3Re were selected synthesizedligand as monodentate by using becausea similar it formsmethod to that pre very stable reported with complexes [4–6]Tc-99m for theandtransmetallation reaction between it increases the hydrophilicity the bioconjugated of the complex, favoring its zi plexes, the rhenium metal complex [NEt4]2[Re(CO)3Br3] and the mono-dentated TPPTS (triphenylphosphine trisulphonate), by means of the 2 + 1 reaction sh Scheme 2. The co-ligand TPPTS was selected as monodentate ligand because it for stable complexes with Tc-99m and it increases the hydrophilicity of the complex, f Molecules 2021, 26, 2373 5 of 15 Molecules 2021, 26, x FOR PEER REVIEW excretion by the renal-urinary system [34,35]. This monodentate ligand has also been used in other studies of Tc-99m with folate derivatives [22,23,25]. Scheme Scheme 2. Synthesis of rhenium 2. Synthesis complexescomplexes of rhenium 2Re and 3Re . 2Re and 3Re. Complexes 2Re and 3Re were characterized by common spectroscopic and spectro- metric Complexes methods (1 H, 13 2Re C, and 3Re were 31 P NMR, IR andcharacterized HRMS can be found by common spectroscopic as Supplementary Ma- an terials). One of the ric methods (1H,most 13C, characteristic 31P NMR, signals IR and of HRMS the formation can beof the 2 + 1 as found complexes Supplement fac-[Re(CO)3 (SS)(P)] is a signal in the 31 P NMR spectra at around 17 ppm, assigned to the One ligand TPPTS of thecoordinated most characteristic to the metal in this signals of the coordinative formation set, which is observedof for the both 2 + 1 c [Re(CO) metal 3(SS)(P)] complexes. is a symptomatic Another signal in the signal,Pwhich 31 NMR spectra shows attransmetallation that the around 17 ppm, a reaction TPPTS ligand coordinated to the metal in this coordinativeprotons has occurred, is the absence of the signal assigned to the equatorial of the is obs set, which isonipecotic group that are nearest to the dithiocarbamate fragment in the zinc complexes 2metal complexes. Another symptomatic signal, which shows that the trans Zn and 3Zn (4.83–4.86 ppm), and the presence of a new signal with a shift of around action 0.7 has occurred, ppm (4.05–4.12 ppm). This is the shift absence of the signal has been previously observedassigned totransmetal- after similar the equatorial lation reactions [4–6]. The IR spectroscopy is also very isonipecotic group that are nearest to the dithiocarbamate fragment revealing because the signals of thein the z rhenium carbonyl fac-[Re(CO)3 Br3 ]2− cannot be observed in the ν(CO) region, and they are 2Zn and 3by substituted Zn (4.83–4.86 ppm), and the presence of a new signal with a shift the characteristic pattern of the fac-[Re(CO)3 (SS)(P)] coordinative set [4–6,36]. ppm (4.05–4.12 Finally, ESI-HRMS shows ppm). This shift the expected has been rhenium fragmentpreviously observed in the negative after region (m/e; 2Re :similar 1541.1235 calculated for [M] − Na + , found 1541.1226; 3 : 775.0620 calculated for [M] − tion reactions [4–6]. The IR spectroscopy isRealso very revealing because the 2Na+ , found 775.0614). rhenium carbonyl fac-[Re(CO)3Br3]2− cannot be observed in the ν(CO) region In order to obtain more information about the possibilities of the transmetallation substituted reaction by the we with rhenium, characteristic made a study with pattern of the fac-[Re(CO) experimental conditions close 3(SS)(P)] to those coord used for preparing radiopharmaceuticals. Thus, 6,36]. Finally, ESI-HRMS shows the expected rhenium fragment the transmetallation reaction was car- in the n ried out in a water medium at a low concentration (20 ppm) of the cationic complex (m/e; 2Re: 1541.1235 calculated for [M] − Na , found 1541.1226; 3Re: 775.0620 + fac-[Re(H2 O)3 (CO)3 ]+ (Scheme 3). The analysis of the reaction mixture by ESI-HRMS, after [M] − 2Na separation , found of +the unreacted775.0614). 2Zn complex by centrifugation, confirmed the formation of the expected bioconjugated In order to obtain more rhenium complex 2Re . These results information about indicate that this reactionof the possibilities is the tra viable for preparing the homologous Tc-99m. They are also informative about possible stud- reaction with rhenium, we made a study with experimental conditions close ies for preparing the metal complexes with the β-emitting radionuclides Re-186/Re-188, for preparing which are useful forradiopharmaceuticals. therapeutic purposes [29,37,38]. Thus, the transmetallation reaction w in a water medium at a low concentration (20 ppm) of the cationic [Re(H2O)3(CO)3]+ (Scheme 3). The analysis of the reaction mixture by ESI-HR aration of the unreacted 2Zn complex by centrifugation, confirmed the for expected bioconjugated rhenium complex 2Re. These results indicate that t viable for preparing the homologous Tc-99m. They are also informative a studies for preparing the metal complexes with the β-emitting radionuclid 188, which are useful for therapeutic purposes [29,37,38]. [Re(H2O)3(CO)3]+ (Scheme 3). The analysis of the reaction mixture by ESI-HRMS, after sep aration of the unreacted 2Zn complex by centrifugation, confirmed the formation of the expected bioconjugated rhenium complex 2Re. These results indicate that this reaction i viable for preparing the homologous Tc-99m. They are also informative about possible Molecules 2021, 26, 2373 6 of 15 studies for preparing the metal complexes with the β-emitting radionuclides Re-186/Re 188, which are useful for therapeutic purposes [29,37,38]. Molecules 2021, 26, x FOR PEER REVIEW 6 of 15 Scheme 3. Synthesis of 2Re in water medium at low concentration (20 ppm). Scheme 3. Synthesis of 2Re in water medium at low concentration (20 ppm). 2.3. Technetium-99m 2.3. Technetium-99mComplexes Complexes The Tc-99m The Tc-99m complexes complexes 22Tc Tc and and 33Tc Tc (Scheme (Scheme4)4)were wereprepared preparedunderunderthetheexperimental experimen- conditions commonly used for synthesizing Tc-99m radiopharmaceuticals. tal conditions commonly used for synthesizing Tc-99m radiopharmaceuticals. Reactions Reactions were carried were carried out out by by adding adding aa suspension suspension of of aa small small amount amount of of the the appropriate appropriate zinc zinc com- com- plex (2 plex (2Zn Zn and 3 3Zn Zn,, ~1 ~1 mg) and monodentate ligand (TPPTS) to an an aqueous aqueous solution solution of of [[99m Tc(H2 O)3 (CO)3 ]+ and 99mTc(H2O)3(CO)3]+ and posterior posteriorheating heatingat moderate at moderate temperatures temperatures (45–65 ◦ C)a for °C) for (45–65 shorta time (~25 short timemin). After the (~25 min). suspension After had cooled the suspension down, down, had cooled it was centrifuged, and theand it was centrifuged, ob- tained the water water obtained solution was ready solution was to be used ready to befor the for used biological studies.studies. the biological The lowThe reaction low temperature reaction and the short temperature reaction and the short time make reaction thismake time procedure appropriate this procedure for preparing appropriate for Tc-99m compounds preparing for radiopharmaceutical Tc-99m compounds purposes, for radiopharmaceutical since long purposes, reaction since times are long reaction timesin- are incompatible compatible with with the decay the decay of theofradionuclides. the radionuclides. Scheme 4. Scheme 4. Synthesis Synthesis of of Tc-99m Tc-99mcomplexes complexes22TcTc and and 33Tc Tc. Figure Figure44shows showsthe thechromatograms chromatogramsofof22TcTcand Tc compared and33Tc compared with with those thoseofof the the rhenium rhenium homologues 2 and 3 . As can be observed, the retention times of the homologues 2Re and 3Re. As can be observed, the retention times of the homologous com- Re Re homologous complexes plexes are are veryvery similar, similar, and and thethe small small differencesare differences aredue duetotothe the different different times times that that the the sample needs for arriving to the two different detectors (UV-detector for rhenium sample needs for arriving to the two different detectors (UV-detector for rhenium com- complexes and gamma plexes ray–detector and gamma for Tc-99m ray–detector complexes). for Tc-99m Hence, complexes). this result Hence, confirms this result that that confirms the the homologous Tc-99m complexes 2Tc and 3Tc can be obtained by the simple transmetal- lation reaction with 2Zn and 3Zn. Furthermore, the yields of the radiosynthesis were higher than 95% in both cases, without any purification procedure. It was only necessary to cen- trifuge the reaction mixture to remove the excess of the insoluble zinc compound to obtain Molecules 2021, 26, 2373 7 of 15 Molecules 2021, 26, x FOR PEER REVIEW 7 of 15 homologous Tc-99m complexes 2Tc and 3Tc can be obtained by the simple transmetallation reaction with 2Zn and 3Zn . Furthermore, the yields of the radiosynthesis were higher than 95% in both cases, without any purification procedure. It was only necessary to centrifuge <0.2 ppm because the reaction mixtureistobelow remove thethe detection excess oflimit of the equipment; the insoluble 3Zn, 0.61 zinc compound ppm).a These to obtain water results confirm solution usefulthat it is possible for biological to prepare studies. This 2isTc aand 3Tc with simple high yields procedure that in a medium could be usedwith for apreparing very low new concentration of the unlabeled radiopharmaceuticals [4–6]. biomolecule. Figure 4. Figure Chromatogramsof 4. Chromatograms ofTc-99m Tc-99m complexes complexes(black(blackcolor, color, radiometric radiometricdetector; detector;(left)(left)22TcTc, ,(right) (right) 33TcTc))and andtheir theirhomologous homologousrhenium rheniumcomplexes complexes(red (redcolor, color,UV UVdetector; detector;(left) (left) , (right) 2Re2,Re(right) 3Re3).ReUV ). UV signal signal of of 33Re Re chromatograms chromatograms was was obtained obtained from from the the complex complex prepared prepared in in DMSO DMSO atat milligram milligram level; level; therefore, therefore, only only in thisincase thisiscase the is the at peak peak 10.0atmin 10.0evident min evident whichwhich corresponds corresponds to the presence to the presence of 3Zn . of 3Zn. In the Introduction, it was highlighted that a relevant point of the transmetallation 2.4. Serumwith reaction Stability Studies zinc dithiocarbamate complexes is the low solubility of the zinc dithiocarba- mates. This fact makes the bioconjugation The stability of the coordinative set [99m ofTc(CO) the Tc-99m possible 3(SS)(P)] in a medium in amino withand acid media a very se- low concentration of the unlabeled biomolecule, increasing the molar rum has been previously demonstrated [6]. However, the stability of 2Tc and 3Tc was as- activity (defined as the ratio sessed in between serum to the radioactivity confirm of a labeled their stability biomolecule in physiological and the before conditions total amount carryingof out the biomolecule the in moles [39]) of the potential radiopharmaceuticals. Thus, the water solu- in vitro studies. bilities of complexes Compounds 2Zn 3and 2Tc and 3Zn were Tc were addedmeasured by inductively to fresh mouse serum and coupled incubatedplasma–optical at 37 °C for emission spectrometry (ICP–OES), obtaining very low values for both 6 h. Aliquots were removed at time points 1, 2, 3, 4 and 6 h, and the proteins were precip- complexes (2Zn , <0.2 ppm because is below the detection limit of the equipment; 3 Zn itated with acetonitrile. The RCP (radiochemical purity) of each aliquot was measured by , 0.61 ppm). These results confirm RP-HPLC. that it is The results possible (Figure to prepare 5) showed that and 2Tc2Tc and3Tc3Tcwith high yields complexes haveinenough a medium with stability a very low concentration of the unlabeled in serum to perform the in vitro and in vivo studies. biomolecule. 2.4. Serum Stability Studies The stability of the coordinative set [99m Tc(CO)3 (SS)(P)] in amino acid media and serum has been previously demonstrated [6]. However, the stability of 2Tc and 3Tc was assessed in serum to confirm their stability in physiological conditions before carrying out the in vitro studies. Compounds 2Tc and 3Tc were added to fresh mouse serum and incubated at 37 ◦ C for 6 h. Aliquots were removed at time points 1, 2, 3, 4 and 6 h, and the proteins were precipi- tated with acetonitrile. The RCP (radiochemical purity) of each aliquot was measured by RP-HPLC. The results (Figure 5) showed that 2Tc and 3Tc complexes have enough stability in serum to perform the in vitro and in vivo studies. Figure 5. Stability study of complexes 2Tc and 3Tc in mouse serum (mean ± SD; n = 3). sessed in serum to confirm their stability in physiological conditions before carrying out the in vitro studies. Compounds 2Tc and 3Tc were added to fresh mouse serum and incubated at 37 °C for 6 h. Aliquots were removed at time points 1, 2, 3, 4 and 6 h, and the proteins were precip- itated with acetonitrile. The RCP (radiochemical purity) of each aliquot was measured by Molecules 2021, 26, 2373 8 of 15 RP-HPLC. The results (Figure 5) showed that 2Tc and 3Tc complexes have enough stability in serum to perform the in vitro and in vivo studies. Molecules 2021, 26, x FOR PEER REVIEW 8 of 15 Figure Figure 5. 5. Stability Stability study study of of complexes complexes 22Tc Tc and and33TcTcin inmouse mouseserum serum(mean (mean±±SD; SD;n n= =3).3). 2.5. In Vitro Binding Assays 2.5. In Vitro Binding Assays In vitro studies of the binding affinity of radiocompounds 2Tc and 3Tc with cells were In vitro studies of the binding affinity of radiocompounds 2Tc and 3Tc with cells were carried out on SKOV3 ovarian cancer cells characterized by their overexpression of the carried out on SKOV3 ovarian cancer cells characterized by their overexpression of the folate folate receptor receptor [40,41]. [40,41]. Cells Cellswere weregrown grownin in media mediafree free of of folic folic acid acid in in order order to to avoid avoid the the blocking blocking of the folate receptors. The comparison of blocked and unblocked binding assays of the folate receptors. The comparison of blocked and unblocked binding assays carried carriedoutoutininparallel paralleldemonstrated demonstrated only a minimal only a minimalstatistically significant statistically difference significant (see difference Figure 6), which indicates low specific binding to the folate receptors. However, (see Figure 6), which indicates low specific binding to the folate receptors. However, the the non- specific binding non-specific determined binding on blocked determined cells was on blocked cells higher than than was higher that reported by other that reported bio- by other conjugated bioconjugated folate Tc-99m folate complexes Tc-99m [21,25]. complexes [21,25]. Figure6. Figure Resultsof 6. Results ofbinding bindingassay assayfor for22TcTcand and33TcTccarried carriedout outon onSKOV3 SKOV3ovarian ovariancancer cancercells. cells. 2.6. Biodistribution Studies 2.6. Biodistribution Studies Six-week-old female athymic mice bearing a SKOV3 tumor on the right flank (n = 3) Six-week-old female athymic mice bearing a SKOV3 tumor on the right flank (n = 3) were used for bio-distribution studies. Animals were injected with a high molar activity so- were used for bio-distribution studies. Animals were injected with a high molar activity lution of 2Tc or 3Tc and sacrificed 4 h post-injection. Considering the activity injected and the solution of 2of solubilities Tc or 3Tc and sacrificed 4 h post-injection. Considering the activity2injected and 2Zn and 3Zn the values of molar activity calculated were 6.7 × 10 MBq/nmol the solubilities 3 of 2Zn and and 2.1 × 10 MBq/nmol for3complexes Zn the values of molar activity calculated were 6.7×102 2Tc and 3Tc , respectively. These values are higher MBq/nmol than 3.7 × and102 2.1 × 10 MBq/nmol MBq/nmol 3 for complexes (10 Ci/µmol), 2Tc and the highest 3Tc, activity molar respectively. valueThese values reported for are 99mhigher than 3.7 × 10 2 MBq/nmol (10 Ci/μmol), the highest molar activity value reported Tc-radiopharmaceuticals as far as we know. [42]. for 99mTc-radiopharmaceuticals as far as we know. [42]. The results in Table 1 show that both radiocomplexes were highly accumulated in the intestines due to the lipophilic nature of the 99mTc(CO)3 core, which promotes the hepatobiliary excretion of the radiocompound [30,43,44]. Nevertheless, the incorporation of the polar ligand TPPTS to the 2Tc and 3Tc radiocompounds increases their hydrophilic character, which enhances their renal clearance. Consequently, the significant kidney up- Molecules 2021, 26, 2373 9 of 15 The results in Table 1 show that both radiocomplexes were highly accumulated in the intestines due to the lipophilic nature of the 99m Tc(CO)3 core, which promotes the hepatobiliary excretion of the radiocompound [30,43,44]. Nevertheless, the incorporation of the polar ligand TPPTS to the 2Tc and 3Tc radiocompounds increases their hydrophilic character, which enhances their renal clearance. Consequently, the significant kidney uptake is due to both the presence of folate receptors in this organ [45] and the renal clearance of the complexes. Table 1. Biodistribution results of complexes 2Tc and 3Tc in athymic mice bearing a SKOV3 tumor at 4 h post-injection. Organs and Tissues 2Tc 3Tc Tumor 0.58 ± 0.06 0.48 ± 0.11 Intestine 52.16 ± 4.21 38.85 ± 1.41 Pancreas 0.13 ± 0.03 0.12 ± 0.02 Spleen 0.17 ± 0.01 0.15 ± 0.03 Kidney 10.51 ± 1.76 11.77 ± 1.87 Stomach 1.63 ± 0.10 1.49 ± 0.20 Liver 2.88 ± 0.37 2.41 ± 0.25 Heart 0.31 ± 0.05 0.24 ± 0.03 Lung 0.62 ± 0.13 0.76 ± 0.16 Blood 0.39 ± 0.07 0.67 ± 0.15 Muscle 0.14 ± 0.04 0.24 ± 0.04 Data depict the amount of tracer uptake (%ID/g) and are presented as an average from 3 animals ± SEM. Tumor uptake is only from two to four times higher than muscle, which are quite low values to be considered good diagnosis agents. These results are consistent with the low specific binding to the folate receptors found in vitro and could be associated with the molecular structure of the complexes. In order to improve the specific binding and the tumor uptake, further studies involving changing the complex structure are necessary. 3. Materials and Methods 3.1. General All reactions were performed under nitrogen, using standard Schlenk tube techniques. Complex 1Zn [4], and folic acid derivatives [CAS 62682-21-7] [27] and [CAS 1099829-15-8] [28] were synthesized by using previously reported methods. The NMR spectra were recorded in the Servei de Ressonància Magnètica Nuclear at the Universitat Autònoma de Barcelona (UAB, Cerdanyola del Vallès, Spain), on a Bruker DPX-250, DPX-360 and AV400 instru- ments. Infrared spectroscopy, microanalyses and mass spectrometry measurements were performed by the Servei d’Anàlisi Química del Departament de Química (UAB, Cerdanyola del Vallès, Spain). Attenuated total reflection infrared spectra (ATR-IR) were registered in Tenson 72 equipment from Bruker. Microanalysis was performed with Thermo Fisher Scientific Flash EA 2000 CHNS equipment, and zinc concentrations were determined by inductively coupled plasma–optical emission spectroscopy (ICP–OES) with the equipment optima 4300DV System (Perkin-Elmer). High-resolution electrospray ionization mass spectrometry (ESI-HRMS) analyses were recorded by means of liquid chromatography equipment 1200 RR from Agilent Technologies, coupled with a microTOF-Q time-of-flight detector from the company Bruker Daltoniks. 3.2. Synthesis of 2Zn Folic acid derivative [CAS 62682-21-7] (2.38 g, 4.4 mmol) was dissolved in DMSO (55 mL) by slightly heating. Complex 1Zn (1.22 g, 1.8 mmol) and DIPEA (N, N-diisopropylethylamine; 1.7 mL, 9.9 mmol) were added to the previous solution, and the resulting suspension was stirred at room temperature, overnight. After the addition of 150 mL of THF, the product was precipitated as a yellow solid. It was collected by filtration and washed with a basic water solution (NaOH; pH 9–10) (4 × 15 mL) and Molecules 2021, 26, 2373 10 of 15 acetone (2 × 20 mL). Yield: 2.37 g (85%). IR (ATR, cm−1 ): 3294 (N-H); 1604 (C=O); 3271 (N-H); 1490 (C-N, S2 CN). 1 H-NMR (DMSO-d6 , δ in ppm): 8.64; 7.65; 6.66 (10H, ArH); 8.01; 7.81 (6H, RNHCOR); 6.92 (s, 2H, ArNH-R); 4.86 (d, 4H, S2 CNCH,eq); 4.49 (d, 2H, ArCH2 NHAr); 4.31 (s, 2H, NHCHCOOH); 3.02 (s, 4H, OCNHCH2 CH2 ); 2.24–2.06 (m, 8H, COOHCHCH2 CH2 CO); 1.76 (s, 4H, S2 CNCH2 CH,eq); 1.59 (s, 4H, S2 CNCH2 CH,ax); 1.36–1.09 (16H, alkyl). 13 C-NMR (DMSO-d6, δ in ppm): 203.2 (S2 CN); 175.5 (COOH); 173.8 (RCONH, isonipecotic); 172.3 (CH2 CH2 CONH); 166.6–166.1 (HNCOCN, pteridine ring) and CONH (aminobenzoic acid); 156.6 (NH2 CNHN, pteridine ring); 151.1 (HNC, aminoben- zoic acid); 148.8 (NHCCHN, pteridine ring); 129.1 (HNCCHCHCCONH (aminoben- zoic acid) and NHCOCN (pteridine ring)); 121.9 (ArCCONH, aminobenzoic acid); 111.7 (HNCCHCHCCONH, aminobenzoic acid); 53.8 (NHCCOOH); 51.0 (S2 CNCH2 ); 46.3 (ArCH2 NHAr); 32.2–15.2 (alkyl). ESI-HRMS (negative mode, m/z): calcd. for C32 H39 N10 O6 S2 Zn ([M] − L− − 2H+ ), 787.1792, found 787.1798. Anal. Elem for C64 H82 N20 O12 S4 Zn·3H2 O: C, 48.9; H, 5.65; N, 17.5. Experimental: C, 48.8; H, 5.82; N, 17.5. 3.3. Synthesis of 3Zn Folic acid derivative [CAS 1099829-15-8] (2.5 g, 4.4 mmol) was dissolved in DMSO (30 mL) by slightly heating. Complex 1Zn (1.22 g, 1.8 mmol) and DIPEA (1.5 mL, 8.8 mmol) were added to the previous solution, and the resulting suspension was stirred at room temperature, overnight. After the addition of 95 mL of THF, the product was precip- itated as a yellow solid. It was collected by filtration and washed with a basic water solution (NaOH; pH 9–10) (3 × 20 mL) and acetone (2 × 20 mL). Yield: 2.56 g (88%). IR (ATR, cm−1 ): 3282 (N-H); 1604 (C=O); 1510 (C-N, S2 CN). 1 H-NMR (DMSO-d6 , δ in ppm): 8.64; 7.63; 6.65 (10H, ArH); 8.02; 7.93; (s, 6H, RNHCOR)); 6.93; (s, 2H, ArNHR); 4.83 (d, 4H, S2 CNCH,eq); 4.49 (d, 4H, ArCH2 NHAr); 4.32 (s, 2H, NHCHCOOH); 3.19 (b, 24H, OCNHCH2 CH2 OCH2 CH2 OCH2 CH2 NHCO); 2.30–2.17 (m, 8H, COOHCHCH2 CH2 CO); 1.79 (s, 4H, S2 CNCH2 CH,eq); 1.56 (s, 4H, S2 CNCH2 CH,ax). 13 C-NMR (DMSO-d6 , δ in ppm): 203.2 (S2 CN); 174.0 (RCONH, isonipecotic); 172.4 (CH2 CH2 CONH); 166.6 (HNCOCN, pteridine ring; CONH, amino-benzoic acid); 156.7 (NH2 CNHN, pteridine ring); 151.4 (HNC, amino-benzoic acid); 149.0 (NHCCHN, pteridine ring); 129.5 (HNCCHCHCCONH, amino-benzoic acid; NHCOCN, pteridine ring); 121.8 (ArCCONH, amino–benzoic acid); 111.9 (HNCCHCHCCONH, amino-benzoic acid); 69.9–69.5 (RCH2 OCH2 CH2 OCH2 R); 65.6 (OCNHCH2 CH2 OCH2 CH2 OCH2 CH2 NHCO); 53.8 (NHCCOOH); 51.3 (S2 CNCH2 ); 46.4 (ArCH2 NHAr); 29.0 (COOHCHCH2 CH2 CONH); 19.5 (S2 CNCH2 CH2 ); 15.8 (COOHCHCH2 CH2 CONH). ESI-HRMS (negative mode, m/z): calcd. for C64 H81 N20 O16 S4 Zn ([M] − H+ ), 1579.4293, found 1579.4329. Anal. Elem for C64 H82 N20 O16 S4 Zn·2H2 O: C, 47.5; H, 5.36; N, 17.3. Experimental: C, 47.6; H, 5.54; N, 17.1. 3.4. Synthesis of 2Re The precursor [NEt4 ]2 [Re(CO)3 Br3 ] (89 mg, 0.11 mmol) was dissolved in degassed DMSO (2.5 mL) and added to a solution of 2Zn (100 mg, 66.1 µmol) in also degassed DMSO (2 mL). The red resulting mixture was heated at 110 ◦ C for 1 h. Next, TPPTS (69 mg, 0.12 mmol) was added, and the resulting suspension was heated for two additional hours. After cooling down the reaction solution at room temperature, 75 mL of absolute ethanol:methanol (3:1) mixture was added to yield a yellow precipitate, which was then collected by centrifugation and washed with cold ethanol (3 × 10 mL). Yield: 127 mg (70%). IR (ATR, cm−1 ): 3328 (NH); 2017, 1919; 2017 (C≡O); 1604 (C=O); 1501 (C-N, S2 CN). 1 H NMR (DMSO-d6 , δ in ppm): 8.65; 7.79–7.48; 6.65 (17H, ArH); 8.01; 7.81; 6.98; (s, 3H, RNHCOR); 4.48 (d, 2H, ArCH2 NHAr); 4.26 (s, 1H, NHCHCOOH); 4.05 (dd, 2H, S2 CNCH,eq); 3.02 (s, 4H, OCNHCH2 (CH2 )4 CH2 NHCO); 2.21 (s, 2H, S2 CNCH2 CH,eq); 1.82 (m, 4H, COOHCHCH2 CH2 CO); 1.59 (s, 2H, S2 CNCH2 CH,ax). 31 P NMR (DMSO-d6 , δ in ppm): 17.3, also observed 17.1. 13 C NMR (DMSO-d6 , δ in ppm): 193.1 (CO); 172.3 (CH2 CH2 CONH); 166.9 (HNCOCN, pteridine ring; CONH, amino-benzoic acid); 151.3 (HNC, amino-benzoic acid); 149.2–148.3 (NHCCHN, pteridine ring); 134.9–122.0 (TPPTS); Molecules 2021, 26, 2373 11 of 15 111.4 (HNCCHCHCCONH, amino-benzoic acid); 51.8 (S2 CNCH2 ); 46.3 (ArCH2 NHAr); 29.4–25.5 (alkyl). ESI-HRMS (negative mode, m/z): calcd. for C53 H53 N10 O18 S5 Na2 PRe ([M] − Na+ ), 1541.1235, found 1541.1226. Retention time RP-HPLC (min): 11.4. 3.5. Synthesis of 3Re The precursor [NEt4 ]2 [Re(CO)3 Br3 ] (127 mg, 0.16 mmol) was added to a solution of 3Zn (150 mg, 0.095 mmol) in degassed DMSO (1 mL), and the resulting solution was heated at 110 ◦ C for 1 h. At that point, TPPTS (98 mg, 0.17 mmol) was added to the previous red solution, which was then heated for an additional 2 h. At the end of the reaction, the solution was cooled at room temperature, and then absolute ethanol (10 mL) was added to the mixture to promote product precipitation, which was then collected by centrifugation and washed with cold ethanol (2 × 10 mL). Yield: 159 mg (70%). IR (ATR, cm−1 ): 3311 (NH); 2020, 1923; 1896 (C≡O); 1607 (C=O); 1510 (C-N, S2 CN). 1 H-NMR (DMSO-d6 ): 8.64; 7.82–7.45; 6.65 (13H, ArH); 7.98; 7.83; 6.98; (s, 3H, RNHCOR); 4.48 (d, 2H, ArCH2 NHAr); 4.37 (s, 1H, NHCHCOOH); 4.12 (dd, 2H, S2 CNCH,eq); 3.17 (s, 12H, OCNHCH2 CH2 OCH2 CH2 OCH2 CH2 NHCO); 2.23 (s, 2H, S2 CNCH2 CH,eq); 1.96–1.80 (m, 4H, COOHCHCH2 CH2 CO); 1.62 (s, 2H, S2 CNCH2 CH,ax). 31 P-NMR (DMSO-d6 , δ in ppm): 16.9, also observed 16.6. 13 C-NMR (DMSO d6 , δ in ppm): 149.1 (NHCCHN, pteri- dine ring); 129.5–121.9 (TPPTS); 112.4 (HNCCHCHCCONH, amino-benzoic acid); 69.3 (RCH2 OCH2 CH2 OCH2 R); 51.3 (S2 CNCH2 ); 46.4 (ArCH2NHAr); 27.9–25.6 (alkyl). ESI- HRMS (negative mode, m/z): calcd. for C53 H53 N10 O20 S5 NaPRe ([M] − 2Na+ ), 775.0620, found 775.0614. Retention time RP-HPLC (min): 11.9. 3.6. Preparation of Tc-99m Complexes Na [99m TcO4 ] was eluted and provided by CADISA and Barts Health NHS Trust from a 99 Mo/99m Tc generator, using 0.9% saline solution. The precursor fac-[99m Tc(CO)3 (H2 O)3 ]+ was synthetized either with the Isolink kit (Coviden) or with an alternative method [46]. Briefly, in a 20 mL vial was added sodium potassium tartrate (15 mg), NaBH4 (6.5 mg) and Na2 CO3 (6.0 mg), and then the vial was carefully sealed and flushed for 15 min with CO. Finally, 1 mL of NaTcO4 (37 MBq) was added and heated to 90 ◦ C for 25 min. For both methods, after the heating period, the vial was cooled down to room temperature, and the pH was adjusted to approximately 7.0 with HCl 0.1 M. The radiochemical yield was evaluated with an RP-HPLC. For the preparation of Tc-99m complexes 2Tc and 3Tc , 100 µL of fac-[99m Tc(CO)3 (H2 O)3 ]+ (10 –10−8 M) was added to a suspension of the zinc dithiocarbamate compound (2Zn or − 6 3Zn ) (150 µL, 10 mg/mL), previously sonicated for 15 min, with 10 µL of the solution of the TPPTS (10−3 M). The resulting suspension was heated to 40–65 ◦ C for 25 min. Afterwards, the reaction mixture was cooled down, and the solid was removed by centrifugation. Retention time RP-HPLC (min): 11.5 (2Tc ); 12.4 (3Tc ). Radiochemical yields: 96% (2Tc ); 98% (3Tc ). 3.7. Reverse-Phase High-Performance Liquid Chromatography The HPLC system featured at Barts Cancer Institute is a Beckman HPLC system Gold 128 with solvent module and a 166 UV detector module (monitoring at 280 nm). Radioac- tivity was detected by using a gamma-detector (Raytest). Compounds were separated on a Jupiter 5 µm C4 from company Phenomenex. Reverse phase analysis was carried out by using: (A) aqueous 0.1 M TEAP (triethylammonium phosphate solution) and (B) acetonitrile. Method: 0–5 min, 5% B; 3–15 min, 5–75% B; 15–20 min, 75% B; 20–25 min, 75–5% B; 25–28 min, 5% B. Flow rate: 1 mL min−1 . 3.8. Determination of Solubilities of Zinc Compounds A suspension in water (10 mL) of the appropriate zinc precursor (2Zn or 3Zn ) was heated at 40 ◦ C for 1 h. Afterwards, the suspension was filtered through a 0.2 µm and centrifuged at 13,000 rpm for 20 min. Finally, the solution was diluted in 1% HNO3 , and Molecules 2021, 26, 2373 12 of 15 the zinc content was analyzed by means of inductively coupled plasma–optical emission spectrometry (ICP–OES). 3.9. Serum Stability Studies Mice blood (4 mL) was centrifuged at 3000 rpm; the supernatant obtained (600 µL) was added to a 150 µL of the radiocompound solution (2Tc or 3Tc ) and incubated at 37 ◦ C. At each time point (0, 1, 2, 4 and 6 h), aliquots of 150 µL were taken and added to 150 µL of acetonitrile, to precipitate the proteins. Each sample was stirred for 5 min and centrifuged. The supernatant was analyzed by RP-HPLC to evaluate the stability of the radiocompound. 3.10. Cell Culture SKOV-3 cells (human ovarian adenocarcinoma) were cultured as monolayers at 37 ◦ C in a humidified atmosphere containing 5% CO2 . The study was performed with free-folate culture medium FFRPMI (modified RPMI, without folic acid, vitamin B12 and phenol red) supplemented with 10% (v/v) of FBS as the only source of folic acid. Cells were harvested in 75 cm2 flasks; when they achieve 80% of confluence, the medium was removed and cells were washed with the PBS buffer (10 mL). Then, 2 mL of trypsin-EDTA was added, and the flask was left in the incubator for 3 min, to remove cells from the flask wall. Finally, 4 mL of fresh media was added to deactivate the trypsin; cells suspensions were then diluted (1:3) and transferred into a new flask. This procedure was repeated approximately 2 or 3 times a week, depending on the dilution factor. 3.11. Binding Assay To a PBS solution with 2.7 × 106 cells in 0.5 mL, 250 µL (KBq) of the radiocomplex (2Tc or 3Tc ) was added, and then the mixture was incubated for 1.5 h at 37 ◦ C. Then samples were centrifuged, and the pellets were washed three times with PBS. Blocking assay were carried out in parallel, adding to 2.7 × 106 cells 250 µL radiocompound and 200 µL of folic acid (3 mM), at the same time, in order to block the folate receptors. Finally, the activity in all the pellets was measured with a gamma counter. The cell uptake of the radiocompound was given by the formula % uptake = (counts of cells/total counts) × 100. All the experiments were done in triplicate. 3.12. Biodistribution Studies Three nude mice (6 weeks old) fed with a folate-free rodent diet were inoculated subcutaneously with a suspension of SKOV3 Cells (5 × 106 cells in 100 µL PBS with 100 µL of Matrigel) into the right flank, 15–17 days before the study. Biodistribution studies were carried after injection of 200 µL of the radiocomplex (2Tc or 3Tc ) (7–12 MBq) into the tail vein. Animals were sacrificed 4 h post-injection; all the interested organs and tissues were explanted, weighted and measured with a gamma counter. The uptake was expressed as a percentage of the injected dose per gram of tissue. All experiments on animals were approved by the QMUL Animal Welfare and Ethical Review Body and were carried out under UK Home Office Project License PPL70/7493. 4. Conclusions This study confirmed that 1Zn is a very useful tool for the conjugation of biological molecules to transition metals. Using this compound, it was possible to obtain the zinc folate complexes 2Zn and 3Zn with a simple and convenient reaction, in which the products can be isolated as pure products of the reaction medium by precipitation. In addition, the study demonstrates the viability of the transmetallation reaction between bioconjugated zinc complexes and the technetium tricarbonyl cation to prepare Tc-99m compounds, which could be used for radiopharmaceutical purposes. Tc-99m folates 2Tc and 3Tc were prepared with high yields from 2Zn and 3Zn in a reaction medium that contains an extremely low concentration of the zinc reagents (lower than 1 ppm). The structures of 2Tc and 3Tc were corroborated by comparing them with the HPLC data of Molecules 2021, 26, 2373 13 of 15 their homologous rhenium complexes 2Re and 3Re . Therefore, it was concluded that this approach based on the transmetallation reaction with low soluble zinc complexes makes it possible to prepare Tc-radiopharmaceuticals with high molar activity in a simple one-pot reaction at a low temperature, without a posterior purification step. However, the in vitro and in vivo studies of Tc-99m folates 2Tc and 3Tc have shown a low specific uptake in tumor cells. This problem could be caused by the 3D molecular structure of the folate complexes that prevents its binding to the folate receptor. Further studies should be carried out to study how to modify the molecular structure, in order to increase the linker length, to improve the folate receptor recognition. Supplementary Materials: The following are available online. Figure S1: Spectroscopic data for 2(Zn) (1 H and 13 C NMR). Figure S1: Spectroscopic data for 2(Zn) (1 H and 13 C NMR). Figure S2: Spectroscopic data for 2(Zn) (IR). Figure S3: Spectrometric data for 2(Zn) (HRMS). Figure S4: Spec- troscopic data for 3(Zn) (1 H and 13 C NMR). Figure S5: Spectroscopic data for 3(Zn) (IR). Figure S6: Spectrometric data for 3(Zn) (HRMS). Figure S7: Spectroscopic data for 2(Re) (1 H and 13 C NMR). Figure S8: Spectroscopic data for 2(Re) (31 P). Figure S9: Spectroscopic data for 2(Re) (IR). Figure S10: Spectrometric data for 2(Re) (HRMS). Figure S11: Spectroscopic data for 3(Re) (1 H and 13 C NMR). Figure S12: Spectroscopic data for 3(Re) (31 P). Figure S13: Spectroscopic data for 3(Re) (IR). Figure S14: Spectrometric data for 3(Re) (HRMS). Author Contributions: Conceptualization, J.B., J.S. (Joan Suades) and R.B.-R.; methodology, J.B., J.S. (Jane Sosabowski) and J.S. (Joan Suades); investigation, J.B., J.F. and R.K.; analysis of results, J.B., L.M.-A., J.S. (Jane Sosabowski), J.S. (Joan Suades) and R.B.-R.; writing—original draft preparation, J.B., L.M.-A. and J.S. (Joan Suades); writing—review and editing, J.B., L.M.-A., J.S. (Joan Suades) and R.B.-R.; visualization, J.B., L.M.-A. and R.B.-R.; supervision, J.S. (Joan Suades), L.M.-A., J.S. (Jane Sosabowski) and R.B.-R.; project administration, J.S. (Joan Suades), L.M.-A. and J.S. (Jane Sosabowski); funding acquisition, J.B., J.S. (Joan Suades) and R.B.-R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Spanish MINECO-FEDER, grant numbers BIO2015-67358-C2- 2-P and CTQ2015-70371-REDT, and by Generalitat de Catalunya, grant number 2017SGR-795. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the QMUL Animal Welfare and Ethical Review Body, under UK Home Office Project License PPL70/7493. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article and supplementary material. Acknowledgments: We thank to Imma Ponte (Departament de Bioquímica, UAB) for allowing us to use the laboratory for handling radioactive substances. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds are not available from the authors. References 1. Arrowsmith, R.L.; Pascu, S.I.; Smugowski, H. New developments in the biomedical chemistry of metal complexes: From small molecules to nanotheranostic design. Organomet. Chem. 2012, 38, 1–35. 2. Monney, A.; Albrecht, M. 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