the family of ethylene cross - bridged tetraamine macrocyclic ligands ( figure 1a ) has become valuable as metal chelators in coordination , catalytic , as well as medicinal chemistry . their pendant - armed derivatives have been especially useful in endowing corresponding metal complexes with remarkable kinetic inertness . one lead bifunctional chelator for copper radiometals has been the bis - carboxymethyl pendant - armed cross - bridged cyclam , cb - te2a ( figure 1b ) . its cu - labeled bioconjugates have significantly improved in vivo behavior and superior inertness toward radiometal loss and are of interest for positron emission tomography ( pet ) imaging applications . we have been developing a second generation of cross - bridged ligands aimed at improving the kinetics of complexation and decreasing nontarget organ accumulation.(4 ) springborg and co - workers previously reported an interesting series of trimethylene cross - bridged tetraazamacrocycles and their coordination chemistry ( figure 1c , 1d ) . ( a ) cross - bridged tetraazamacrocycles ; ( b ) cb - te2a ; ( cd ) trimethylene cross - bridged tetraazamacrocycles . a limited number of pendant - armed derivatives of these ligands have been investigated , though none with ionizable functional groups . we were intrigued whether such a trimethylene cross - bridged tetraamine derivative would have distinct in vitro or in vivo behavior compared to its most closely related ethylene cross - bridged analogues . we report here the synthesis and characterization of a dicarboxymethyl pendant - armed cyclen with such a cross - bridge , c3b - do2a ( 1 ) , as well as its isomeric ethylene cross - bridged homocyclen ligand , cb - tr2a ( 2 ) ( figure 2 ) . finally , cu radiolabeling and animal biodistribution studies have been carried out for comparison with each other , and with the widely used chelator cb - te2a , with the overall goal of developing the optimal cu - cross - bridged complex for conjugation to biomolecules as potential pet imaging agents . the ligand c3b - do2a ( 1 ) was synthesized by n - alkylation of the parent trimethylene cross - bridged springborg ligand ( 3 , c3b - cyclen)(6 ) with t - butyl bromoacetate ( scheme 1 ) to give diester 4 as a hydrobromide salt . deprotection of this product with excess trifluoroacetic acid ( tfa ) provided h2-c3b - do2a , also as a hydrobromide ( h2 - 1hbr ) . the synthetic route used for preparation of cb - tr2a ( 2 ) the approach from homocyclen to cross - bridged homocyclen has been communicated previously,(8 ) but we include synthetic details and characterization data herein . condensation of homocyclen with aqueous glyoxal in mecn leads to a mixture of two constitutionally isomeric cis - fused tetracyclic bisaminals , 5 ( major ) and 6 ( minor ) , which were separated chromatographically.(9 ) the major isomer ( 5 ) , the desired compound , was shown to be cis - fused by virtue of dynamic nmr spectra indicative of enantiomerization ( selected variable temperature c{h } nmr spectral data are included in the experimental details for compound 5 ) . highly regio- and stereoselective dibenzylation of 5 gives rac salt 7 through benzylation on the most sterically available nitrogen lone pairs . double reductive ring expansion with nabh4 gives dibenzyl cross - bridged homocyclen 8 , which is debenzylated to cross - bridged homocyclen 9 by hydrogenolysis . in analogy to our synthesis of cb - te2a and c3b - do2a ( 1 ) ( vide supra ) , 9 was di - n - alkylated with t - butyl bromoacetate to give bis - ester - armed derivative 10 , which was deprotected with tfa to give the desired ligand as h2-cb - tr2a ( h2 - 2 ) as a tfa salt . the respective copper(ii ) complexes of 1 and 2 were prepared by refluxing cu(clo4)2 overnight with the ligand in a 95% ethanol solution that was adjusted to ph 8 . the h and c{h } nmr spectral data for c3b - do2a ( 1 ) and cb - tr2a ( 2 ) are detailed in the experimental section . the nmr spectra of h2 - 1 exhibit the expected time - averaged 2-fold symmetry in solution . the c{h } nmr spectrum exhibits 6 resonances and the h nmr spectrum shows the expected singlet for the enantiotopic hydrogens of the acetate methylenes ( n - ch2-co2h ) . on the other hand , h2 - 2 ( tfa salt ) is completely asymmetric as shown by nmr spectroscopy . the solid - state ir spectra of cu - c3b - do2a ( 11 ) and cu - cb - tr2a ( 12 ) showed carboxylate stretching bands at 1616 and 1609 cm , respectively , both consistent with coordinated pendant arms . their d - d electronic spectral maxima in aqueous solutions are at 614 nm ( 85 m cm ) and 608 nm ( 32 m cm ) respectively , also typical of six - coordinate cu(ii ) with n4o2 coordination spheres . n sodium acetate revealed an irreversible reduction wave with a peak potential at 1.05 v ( ag / agcl , scan rate 200 mv / s ) with a large copper stripping peak in the return scan . under the same conditions , cu - cb - tr2a ( 12 ) exhibited a quasi - reversible reduction wave at 0.95 v with a peak - to - peak separation of 138 mv . our previous results indicated that cu(ii)-complexes of ethylene cross - bridged cyclam and its derivatives typically have quasi - reversible reductions while cyclen analogues display irreversible reductions . thus , electrochemically cu - c3b - do2a ( 11 ) behaves as expected for a cross - bridged cyclen complex while the isomeric homocyclen - based complex cu - cb - tr2a ( 12 ) is more similar to cross - bridged cyclam complexes . the possible relevance of cu(ii ) reduction potentials to in vivo radio - copper loss from tetraazamacrocyclic chelators and their bioconjugates has been postulated.(12 ) it was hypothesized that cu(ii ) reduction potentials lower than about 0.6 v ( ag / agcl ) may be necessary to avoid reduction by common bioreductants . if indeed so , neither of these complexes should be susceptible to in vivo cu(ii ) reduction . acid inertness data of cu(ii)-tetraamine complexes provide a convenient measure of their resistance toward demetalation in aqueous solution . we have found acid inertness half - lives obtained under pseudo first - order conditions to be useful first predictors for in vivo viability of cu - labeled chelator complexes.(2 ) in particular , cu - cb - te2a was shown to have a half - life of more than 6 d even in 5 m hcl at 90 c.(11 ) studies in 5 m hcl solutions at 30 c revealed that cu - c3b - do2a ( 11 ) is indefinitely inert while cu - cb - tr2a ( 12 ) dissociated with a halflife of 10.8(4 ) h. remarkably , the cu - c3b - do2a ( 11 ) complex only demetalated in 12 m hcl at 90 c with a half - life of 1.1(1 ) d. to our knowledge , this represents the most acid - inert copper amine complex studied . since springborg reported the half - life of the parent trimethylene cross - bridged copper complex in 5 m hcl at 25 c to be almost 6 days while we have found the isomeric cu - cb - homocyclen complex to be significantly less inert ( half - life only 4.8 h in 1 m hcl , 30 c ) , the trimethylene bridge does appear to impart greater acid inertness . here , attachment of the two enveloping carboxymethyl pendant arms further enhanced the acid inertness of cu - c3b - do2a ( 11 ) even beyond that of cu - cb - te2a . a summary of crystal data for the two x - ray diffraction studies can be found in table 1 . while the quality of the cu - cb - tr2a ( 12 ) crystal used was relatively poor , nonetheless three distinct six - coordinated distorted octahedral complexes with n4o2 coordination spheres were found . each of these provides a pendant arm oxygen to bridge two of three sodium cations which together form a central core for this assembly . two of these , complexes 12a and 12b are well - defined ( figure 3 ) . complex 12a has a / ligand conformation while complex 12b adopts instead a / conformation , the distinction mainly being the sign of the nccn torsion angle of the short bridge . complex 12c is conformationally disordered but possesses the same basic cu coordination sphere as 12a and 12b . for full details , see the supporting information . x - ray structure of 12 : views of complexes 12a and 12b ( 50% thermal ellipsoids ; hydrogens omitted for clarity ) . ncun angles of 191.3(2) and 183.2(2) respectively , with each cation sunken into the ligand cavity but with the / ligand conformation engulfing the cation more . corresponding  equatorial jahnteller elongations in both structures are along select ncuo axes ; n(8)cu(2)o(6 ) in 12a and n(3)cu(1)o(2 ) in 12b . coppernitrogen bond distances in both structures range from 1.96 to 2.24  while copperoxygen distances are between 1.96 and 2.51  ( table 2 ) . the x - ray structure of cu - c3b - do2a ( 11 ) is shown in figure 4 . similar numbers were found in a cu(ii ) complex of the parent trimethylene cross - bridged cyclen ( 170.5 and 99.0).(5 ) the first angle indicates that the cation is protruding slightly from the ligand cavity while the latter value is substantially larger than those found in reported ethylene cross - bridged ligand cu(ii ) complexes ( typically around 80 ) as a result of its trimethylene cross - bridging . its jahnteller elongation is along the n(1)cu(1)o(3 ) axis which deviates substantially from linearity at 159. the coppernitrogen bond distances range from 2.0 to 2.2  , similar to those found in cu - cb - tr2a ( 12 ) , while the cuo distances are 2.00 and 2.45  . in light of these observations , no significant structural insight can be gleaned to account for the very disparate acid inertness between these two complexes . x - ray structure of complex 11 ( 50% thermal ellipsoids ; hydrogens omitted for clarity ) . consistent with the extremely high acid inertness of its cu - complex , radiolabeling of c3b - do2a was found to be very challenging and ultimately required rather harsh conditions for successful labeling . radiolabeling was attempted under a variety of aqueous conditions , varying concentration ( 0.51 g/l ) , buffer ( 0.11.25 m nh4oac , 0.10.5 m nh4-citrate , and 0.10.5 na2hpo4 ) , ph ( 4.58 ) , and temperature ( 2595 c ) . all reactions had very disappointing yields that rarely exceeded 50% within 2 h , even at high temperature . at longer time points ( 318 h ) furthermore , instability of the cu - c3b - do2a complex was observed in the form of secondary peak formation . although in our hands , radiolysis has not been commonly observed for small molecules , reactions were carried out in the presence of two different radiolytic scavengers with no significant benefit . after many failed attempts at radiolabeling 1 with high yields under aqueous conditions , the standard conditions for radiolabeling cb - te2a were attempted,(16 ) by dissolving the compound ( 0.53 mg ) in ethanol ( 50100 ml ) , preincubating it in the presence of an excess of cs2co3 and then heating the reaction at 95 c with 100130 ci of cu . unfortunately , the yields were still relatively low ( 3070% after 1 h ) and did not exceed 85% even after several hours . replacing cs2co3 with li2co3 resulted in extremely low yields ( 36% ) at all time points ( 118 h ) . in an attempt to speed up the reaction kinetics , a microwave reactor was employed . in all reactions 1.0 g c3b - do2a was incubated with 100 ci cu in a total volume of 300 l . many different reactions were performed where solvent , ph , temperature , and reaction time were varied , and it was found that the shortest reaction that consistently provided a greater than 97% radiochemical purity consisted of microwave heating a solution of 1 in milli - q water at ph = 3 to 100 c for 2 h. in contrast , cb - tr2a was successfully labeled with cu by the method employed for cb - te2a(16 ) ( radiochemical purity 98% ) at room temperature in basic ethanolic solution . a single peak corresponding to cu - cb - tr2a was confirmed by radio - tlc . the biodistribution of cu - c3b - do2a and cu - cb - tr2a were determined in normal , juvenile lewis rats to examine their in vivo properties . the blood , liver , and kidney clearance of these agents are plotted in figure 5 along with previously published data of cu - cb - do2a and cu - cb - te2a for comparison.(18 ) although cu - cb - tr2a demonstrated rapid clearance from the blood suggesting that it is stable for the limited time it remains in the bloodstream , significantly more activity was observed in the blood at 24 h for cu - cb - tr2a compared to cu - cb - te2a ( cu - cb - te2a vs cu - cb - tr2a ( % id / g ; p value ) : 0.003  0.0006 vs 0.01  0.002 ; p = 0.003 ) . levels of cu - cb - tr2a remaining in the blood at 24 h were comparable to cu - cb - do2a ( 0.01  0.002 vs 0.011  0.004 ) . biodistribution of cu-11 and cu-12 compared to published data for cu - cb - do2a , cu - cb - te2a . additionally , more tissue associated radioactivity was observed in the kidneys of rats receiving cu - cb - tr2a at 24 h p.i . compared to the other two cb chelators ( cu - cb - tr2a vs cu - cb - te2a vs cu - cb - do2a : 0.18  0.05 , 0.012  0.003 and 0.064  0.007%id / g , respectively ; p < 0.0001 ) . similarly , there was significantly greater accumulation in the liver of mice receiving cu - cb - tr2a compared to cu - cb - do2a or cu - cb - te2a ( 0.075  0.014 , 0.024  0.002 , and 0.056 0.012%id / g , respectively ; p = 0.0003 ) . while the increased cu in the liver from cu - cb - tr2a may be attributed to hepatobilary clearance , transchelation of copper to liver proteins is also a likely possibility.(17 ) boswell et al . reported the transchelation of radio - copper to liver proteins such as superoxide dismutase and metallotheinein after injection of cu labeled cb - chelators.(18 ) at this time it is unclear why cb - tr2a was less stable in vivo than cb - do2a despite its comparatively low reduction potential and its relative kinetic acid inertness . cu - c3b - do2a exhibited very slow clearance from the liver compared to cb - tr2a , cb - do2a , and cb - te2a ( percent reduction at 22 or 24 h pi : 22.2 , 66.4 , 69.5 and 90.2% clearance , respectively ) . kidney clearance showed a similar pattern out to 22 or 24 h pi ( cu - c3b - do2a , 50.4% ; cu - cb - tr2a , 89.2% ; cu - cb - do2a , 95.6% ; and cu - cb - te2a , 99.1% ) . the poor bioclearance of cu - c3b - do2a compared to the other radiolabeled cross - bridged chelators is surprising . previously , in vitro assays of cu(ii)-complex acid inertness and resistance to cu(ii)/cu(i ) reduction have provided useful indicators of their in vivo stability and bioclearance behavior . the very slow hepatic clearance of cu - c3b - do2a observed here suggests significant radiometal loss in the liver from what was predicted to be a very inert complex . close examination of the x - ray structure of cu - c3b - do2a revealed that the trimethylene cross - bridge led to a larger equatorial ncun angle of 98 instead of the 8088 typical for its ethylene - bridged analogues but no other noteworthy differences in cu - chelator bonding parameters . the possibility that an incompletely formed , perhaps out - of - cavity , cu complex being the actual radiolabeled product can account for the observed radiocopper loss in vivo . however , the very harsh radiolabeling conditions ( 2 h , microwave 100 c , ph 3 ) mitigate against such a kinetic product . other contributing factors affecting in vivo clearance including lipophilicity , susceptibility to metabolic degradation , or formation of easily reduced ternary complexes as a consequence of this trimethylene cross - bridge may be implicated but will require additional sar studies for validation.(19 ) the trimethylene cross - bridged chelator c3b - do2a ( 1 ) and its ethylene cross - bridged isomer cb - tr2a ( 2 ) have been synthesized and characterized as were their copper(ii ) complexes . both complexes have distorted octahedral n4o2 coordination spheres and should be resistant to biological reduction . while cu - cb - tr2a ( 12 ) is reasonably resistant to acid decomplexation , cu - c3b - do2a ( 11 ) is remarkably inert . at the same time , successful radio - copper labeling of cb3b - do2a ( 11 ) required relatively harsh conditions . contrary to the highly inert in vitro behavior of cu - c3b - do2a ( 11 ) , biodistribution studies of cu - c3b - do2a revealed very poor in vivo clearance data , significantly inferior to those of cu - cb - te2a and even cu - cb - do2a .
ethylene cross - bridged tetraamine macrocycles are useful chelators in coordination , catalytic , medicinal , and radiopharmaceutical chemistry . springborg and co - workers developed trimethylene cross - bridged analogues , although their pendant - armed derivatives received little attention . 
 we report here the synthesis of a bis - carboxymethyl pendant - armed cyclen with a trimethylene cross - bridge ( c3b - do2a ) and its isomeric ethylene - cross - bridged homocyclen ligand ( cb - tr2a ) as well as their copper(ii ) complexes . 
 the in vitro and in vivo properties of these complexes are compared with respect to their potential application as 64cu - radiopharmaceuticals in positron emission tomography ( pet imaging ) . 
 the inertness of cu - c3b - do2a to decomplexation is remarkable , exceeding that of cu - cb - te2a . 
 electrochemical reduction of cu - cb - tr2a is quasi - reversible , whereas that of cu - c3b - do2a is irreversible . 
 the reaction conditions for preparing 64cu - c3b - do2a ( microwaving at high temperature ) are relatively harsh compared to 64cu - cb - tr2a ( basic ethanol ) . 
 the in vivo behavior of the 64cu complexes was evaluated in normal rats . 
 rapid and continual clearance of 64cu - cb - tr2a through the blood , liver , and kidneys suggests relatively good in vivo stability , albeit inferior to 64cu - cb - te2a . 
 although 64cu - c3b - do2a clears continually , the initial uptake is high and only about half is excreted within 22 h , suggesting poor stability and transchelation of 64cu to proteins in the blood and/or liver . 
 these data suggest that in vitro inertness of a chelator complex may not always be a good indicator of in vivo stability .