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Pepducins are a class of lipidated peptide G-protein coupled receptor (GPCR) allosteric modulators that exhibit unique pharmacological properties. Because of their rational design, pepducins can potentially be used to target all members of this gene family. In addition to enormous therapeutic potential, pepducins are proving to be valuable tools to elucidate the molecular mechanisms involved in GPCR signaling. In this review, we focus on the characterization of pepducin biology and discuss their utility as novel therapeutics.

The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4- and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341–mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.

The amphiphilic gadolinium complex MS-325 ((trisodium-{(2-(R)-[(4,4-diphenylcyclohexyl) phosphonooxymethyl] diethylenetriaminepentaacetato) (aquo)gadolinium(III)}) is a contrast agent for magnetic resonance angiography (MRA). MS-325 consists of two slowly interconverting diastereoisomers, A and B (65:35 ratio), which can be isolated at pH8.5 (TyeklAr, Z.; Dunham, S. U.; Midelfort, K.; Scott, D. M.; Sajiki, H.; Ong, K.; Lauffer, R. B.; Caravan, P.; McMurry, T. J. Inorg. Chem. 2007, 46, 6621-6631). MS-325 binds to human serum albumin (HSA) in plasma resulting in an extended plasma half-life, retention of the agent within the blood compartment, and an increased relaxation rate of water protons in plasma. Under physiological conditions (37 degrees C, pH 7.4, phosphate buffered saline (PBS), 4.5% HSA, 0.05 mM complex), there is no statistical difference in HSA affinity or relaxivity between the two isomers (A 88.6 +/- 0.6% bound, r1 = 42.0 +/- 1.0 mM(-1) s(-1) at 20 MHz; B 90.2 +/- 0.6% bound, r1 = 38.3 +/- 1.0 mM(-1) s(-1) at 20 MHz; errors represent 1 standard deviation). At lower temperatures, isomer A has a higher relaxivity than isomer B. The water exchange rates in the absence of HSA at 298 K, kA298 = 5.9 +/- 2.8 x 10(6) s(-1), kB298 = 3.2 +/- 1.8 x 10(6) s(-1), and heats of activation, DeltaHA = 56 +/- 8 kJ/mol, DeltaHB = 59 +/- 11 kJ/mol, were determined by variable-temperature 17O NMR at 7.05 T. Proton nuclear magnetic relaxation dispersion (NMRD) profiles were recorded over the frequency range of 0.01-50 MHz at 5, 15, 25, and 35 degrees C in a 4.5% HSA in PBS solution for each isomer (0.1 mM). Differences in the relaxivity in HSA between the two isomers could be attributed to the differing water exchange rates.

The synthesis of a novel ligand, based on N-methyl-diethylenetriaminetetraacetate and containing a diphenylcyclohexyl serum albumin binding group (L1) is described and the coordination chemistry and biophysical properties of its Gd(III) complex Gd-L1 are reported. The Gd(III) complex of the diethylenetriaminepentaacetate analogue of the ligand described here (L2) is the MRI contrast agent MS-325. The effect of converting an acetate to a methyl group on metal-ligand stability, hydration number, water-exchange rate, relaxivity, and binding to the protein human serum albumin (HSA) is explored. The complex Gd-L1 has two coordinated water molecules in solution, that is, [Gd(L1)(H2O)2]2- as shown by D-band proton ENDOR spectroscopy and implied by 1H and 17O NMR relaxation rate measurements. The Gd-H(water) distance of the coordinated waters was found to be identical to that found for Gd-L2, 3.08 A. Loss of the acetate group destabilizes the Gd(III) complex by 1.7 log units (log K(ML) = 20.34) relative to the complex with L2. The affinity of Gd-L1 for HSA is essentially the same as that of Gd-L2. The water-exchange rate of the two coordinated waters on Gd-L1 (k(ex) = 4.4x10(5) s(-1)) is slowed by an order of magnitude relative to Gd-L2. As a result of this slow water-exchange rate, the observed proton relaxivity of Gd-L1 is much lower in a solution of HSA under physiological conditions (r1(obs) = 22.0 mM(-1) s(-1) for 0.1 mM Gd-L1 in 0.67 mM HSA, HEPES buffer, pH 7.4, 35 degrees C at 20 MHz) than that of Gd-L2 (r1(obs) = 41.5 mM(-1) s(-1)) measured under the same conditions. Despite having two exchangeable water molecules, slow water exchange limits the potential efficacy of Gd-L1 as an MRI contrast agent.

MS-325 is a novel blood pool contrast agent for magnetic resonance imaging currently undergoing clinical trials to assess blockage in arteries. MS-325 functions by binding to human serum albumin (HSA) in plasma. Binding to HSA serves to prolong plasma half-life, retain the agent in the blood pool, and increase the relaxation rate of water protons in plasma. Ultrafiltration studies with a 5 kDa molecular weight cutoff filter show that MS-325 binds to HSA with stepwise stoichiometric affinity constants (mM(-1)) of K(a1) = 11.0 +/- 2.7, K(a2) = 0.84 +/- 0.16, K(a3) = 0.26 +/- 0.14, and K(a4) = 0.43 +/- 0.24. Under the conditions 0.1 mM MS-325, 4.5% HSA, pH 7.4 (phosphate-buffered saline), and 37 degrees C, 88 +/- 2% of MS-325 is bound to albumin. Fluorescent probe displacement studies show that MS-325 can displace dansyl sarcosine and dansyl-L-asparagine from HSA with inhibition constants (K(i)) of 85 +/- 3 microM and 1500 +/- 850 microM, respectively; however, MS-325 is unable to displace warfarin. These results suggest that MS-325 binds primarily to site II on HSA. The relaxivity of MS-325 when bound to HSA is shown to be site dependent. The Eu(III) analogue of MS-325 is shown to contain one inner-sphere water molecule in the presence and in the absence of HSA. The synthesis of an MS-325 analogue, 5, containing no inner-sphere water molecules is described. Compound 5 is used to estimate the contribution to relaxivity from the outer-sphere water molecules surrounding MS-325. The high relaxivity of MS-325 bound to HSA is primarily because of a 60-100-fold increase in the rotational correlation time of the molecule upon binding (tau(R) = 10.1 +/- 2.6 ns bound vs 115 ps free). Analysis of the nuclear magnetic relaxation dispersion (T(1) and T(2)) profiles also suggests a decrease in the electronic relaxation rate (1/T(1e) at 20 MHz = 2.0 x 10(8) s(-1) bound vs 1.1 x 10(9) s(-1) free) and an increase in the inner-sphere water residency time (tau(m) = 170 +/- 40 ns bound vs 69 +/- 20 ns free).

Clinically relevant relaxivity enhancement of a magnetic resonance imaging (MRI) contrast agent has been achieved by using prodrug Gd3+ complexes (see picture, DTPA=diethylenetriaminepentaaceto). Enzymatic cleavage of lysine residues from the prodrug exposes a group that has a high affinity to human serum albumin and promotes enhanced relaxivity, thus enabling the detection of targets at submicromolar concentrations.

A convenient synthesis of enantiomerically enriched 1-hydroxymethyl-DTPA (diethylenetriamine pentaacetic acid) penta-t-butyl esters 3 has been achieved for the preparation of chiral synthones of new paramagnetic gadolinium complexes.

The acqueous solution chemistry of lead(II) complexes of DOTA DOTA4− = 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetate) has been investigated in the course of developing 203Pb radiolabeled monoclonal antibodies as imaging agents. The stability and protonation constant of the 1:1 complex of Pb(DOTA)2− were estimated to be 1024.3±0.2 and 103.3±0.1 at 25 °C, I = 0.10 M (NaClO4), respectively. Formation reaction kinetics between Pb(II) and DOTA were investigated by stopped-flow methods. Possible mechanisms developed for the reaction involve (i) the rapid formation of a protonated intermediate which subsequently loses a proton during the rate-limiting step and (ii) formation of a rapid ‘dead-end’ equilibrium, with reactants reacting by a second-order process to give products. Dissociation of Pb(DOTA)2− to form the aquo Pb(II) cation and protonated DOTA was studied in perchloric acid. These results, when applied to the radiolabelling of monoclonal antibody B72.3 conjugates of DOTA with 203Pb, led to the biological evaluation of the 203Pb(DOTA)-B72.3 radioimmunoconjugate in mice. Biodistribution studies reported here suggest that 203Pb(DOTA) labeled monoclonal antibodies may be useful for tumor localization of Pb(II) radionuclides.

Peptides that bind to fibrin but not to fibrinogen or serum albumin were selected from phage display libraries as targeting moieties for thrombus molecular imaging probes. Three classes of cyclic peptides (cyclized via disulfide bond between two Cys) were identified with consensus sequences XArXCPY(G/D)LCArIX (Ar = aromatic, Tn6), X(2)CXYYGTCLX (Tn7), and NHGCYNSYGVPYCDYS (Tn10). These peptides bound to fibrin at ∼2 sites with K(d) = 4.1 μM, 4.0 μM, and 8.7 μM, respectively, whereas binding to fibrinogen was at least 100-fold weaker. The peptides also bind to the fibrin degradation product DD(E) with similar affinity to that measured for fibrin. The Tn7 and Tn10 peptides bind to the same site on fibrin, while the Tn6 peptides bind to a unique site. Alanine scanning identified the N- and C-terminal ends of the Tn6 and Tn7 peptides as most tolerant to modification. Peptide conjugates with either fluorescein or diethylenetriaminepentaaceto gadolinium(III) (GdDTPA) at the N-terminus were prepared for potential imaging applications, and these retained fibrin binding affinity and specificity in plasma. Relaxivity and binding studies on the GdDTPA derivatives revealed that an N-terminal glycyl linker had a modest effect on fibrin affinity but resulted in lower fibrin-bound relaxivity.

The biodistribution of indium-111/yttrium-88-labeled B3 monoclonal antibody, a murine IgG1k, was evaluated in non-tumor-bearing mice. B3 was conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1B4M) or 2-(p-SCN-Bz)-1,4,7,10 tetraazacyclododecane tetra-acetic acid (2B-DOTA) and labeled with 111In at 1.4-2.4 mCi/mg and 88Y at 0.1-0.3 mCi/mg. Non-tumor-bearing nude mice were co-injected i.v. with 5-10 microCi/4-10 micrograms of 111In/88Y-labeled B3 conjugates and sacrificed at 6 h and daily up to 168 h post-injection. Mice injected with 111In/88Y-(1B4M)-B3 showed a similar biodistribution of the two radiolabels in all tissues except the bones, where significantly higher accretion of 88Y than 111In was observed, with 2.8% +/- 0.2% vs 1.3% +/- 0.16% ID/g in the femur at 168 h, respectively (P0.0001). In contrast, mice receiving the 111In/88Y-(DOTA)-B3 conjugate showed significantly higher accumulation of 111In than 88Y in most tissues, including the bones, with 2.0% +/- 0.1% vs 1.2% +/- 0.09% ID/g in the femur at 168 h, respectively (P0.0001). Whereas the ratios of the areas underneath the curve (%ID x h/g) in the blood, liver, kidney and bone were 0.96, 1.12, 1.13, and 0.74 for 111In/88Y-(1B4M)-B3 and 0.84, 1.23, 1.56, and 1.31 for 111In/88Y-(DOTA)-B3, respectively, ratios approximately 1 were observed between 111In-(1B4M)-B3 and 88Y-(DOTA)-B3. In summary, while neither 1B4M nor DOTA was equally stable for 111In and 88Y, the fate of 88Y-(DOTA)-B3 could be closely traced by that of 111In-(1B4M)-B3.

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.

A convenient synthesis of a bifunctional derivative of the ligand NOTA is described. The synthesis offers advantages over current literature methods for making similar molecules by starting with the easily prepared ortho-amide of 1,4,7-triazacyclanonane which can be sequentially alkylated to provide a bifunctional NOTA derivative in 9% yield overall. The replacement of preparative HPLC with simple ion-exchange chromatography provides analytically pure ligand suitable for clinical applications.

A synthesis of the bifunctional chelator 2-(p-thiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [2-(p-NCS-Bz)-NOTA] is described which illustrates the especial utility of the (4-methoxy-2,3,6-trimethylphenyl)sulfonyl (Mtr) protecting group as an alternative to the p-tolylsulfonyl (Ts) moiety commonly used for Richman-Atkins type cyclizations. Reaction of N,N'-bis(p-tolylsulfonyl)-1-(p-benzamidobenzyl)ethylenediami ne with N,N-bis[2-[(p-tolylsulfonyl)oxy]ethyl]-p-toluenesulfonamide gave 2-(p-benzamidobenzyl)-1,4,7-tris(p-tolylsulfonyl)-1,4,7-tria zacyclononane in 55% yield, whereas the analogous reaction using the Mtr-protected starting materials gave the corresponding Mtr-protected macrocycle in 34% yield. However, deprotection of the Ts- and Mtr-protected macrocycles (H2SO4, 90 degrees C) afforded 2-(p-benzamidobenzyl)-1,4,7-triazacyclononane in 23% and 60% yield, respectively, illustrating the relatively facile cleavage of the Mtr moiety. A modest improvement in overall percent conversion of (p-nitrobenzyl)ethylenediamine into substituted macrocyclic polyamine was observed when comparing the Mtr vs Ts protection (12.6 vs 10.6%). The macrocyclic triamine was converted to 2-(p-NCS-Bz)-NOTA by alkylation with bromoacetic acid (pH 9, 73%) followed by hydrolysis of the benzamide protecting group (6 M HCl, 70 degrees C, 87%) and reaction with thiophosgene (90%). The serum stability of the 67Cu complexes of 1,4,7-triazacyclononane (I), 2-(p-nitrobenzyl)-1,4,7,10-tetraazacy-clododecane (II), 2-(p-nitrobenzyl)-1,4,8,11-tetraazacyclotetradecane (III), 2-(p-PhCONH-Bz)-NOTA (IV), 2-(p-nitrobenzyl)-1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid (V), 2-(p-nitrobenzyl)-1,4,8,11-tetraazatetradecane-1,4,8,11-tetraaceti c acid (VI), and the acyclic ligand 1-(p-nitrobenzyl)-4-methyldiethylenetriamine-N,N,N',N", N"-pentaacetic acid (VII) was measured at 37 degrees C (5% CO2) and showed the following order of relative stability: I < VII << VI << IV < V approximately equal to II approximately equal to III.

A convenient synthesis of 4-nitrobenzyl-substituted macrocyclic tetraamines and their conversion to bifunctional poly(amino carboxylate) chelating agents is described. Cyclization of (4-nitrobenzyl)-ethylenediamine with appropriate BOC-protected amino disuccinimido esters in dioxane at 90 degrees C resulted in the formation of 12- and 14-membered ring diamides in 40% and 44% yield, respectively. A 12-membered macrocyclic triamide was also prepared in 44% yield by cyclization of N-(2-aminoethyl)-4-nitrophenylalaninamide with disuccinimidyl N-(tert-butoxycarbonyl)iminodiacetate. Deprotection (HCl/dioxane) and reduction with borane gave the substituted macrocyclic amines which were then alkylated with either bromoacetic acid or tert-butyl bromoacetate. Preparation of the isothiocyanate derivatives and 14C labeled chelating agents are described. Attempts to prepare a 9-membered macrocyclic diamide using this cyclization technique resulted instead in a 20% yield of a 10:1 mixture of isomeric fused 5,6 ring acylamidines. Deprotection (HCl/dioxane) and reduction with borane gave a substituted piperazine derivative in 55% yield.

A convenient synthesis of enantiomerically enriched 1-hydroxymethyl-DTPA (diethylenetriamine pentaacetic acid) penta-t-butyl esters 3 has been achieved for the preparation of chiral synthones of new paramagnetic gadolinium complexes.

A. Water Exchange 2326 B. Proton Exchange 2327 C. Electronic Relaxation 2327 D. Relaxivity 2331 E. Outerand Second-Sphere Relaxivity 2334 F. Methods of Improving Relaxivity 2336 V. Macromolecular Conjugates 2336 A. Introduction 2336 B. General Conjugation Methods 2336 C. Synthetic Linear Polymers 2336 D. Synthetic Dendrimer-Based Agents 2338 E. Naturally Occurring Polymers (Proteins, Polysaccharides, and Nucleic Acids) 2339

A strategy for preparing high relaxivity, metabolically-stable, peptide-based MR contrast agents is described.

The amphiphilic gadolinium complex MS-325 ((trisodium-{(2-(R)-[(4,4-diphenylcyclohexyl) phosphonooxymethyl] diethylenetriaminepentaacetato) (aquo)gadolinium(III)}) is a contrast agent for magnetic resonance angiography (MRA). MS-325 comprises a GdDTPA core with an appended phosphodiester moiety linked to a diphenylcyclohexyl group to facilitate noncovalent binding to serum albumin and extension of the plasma half-life in vivo. The chiral DTPA ligand (R) was derived from L-serine, and upon complexation with gadolinium, forms two interconvertible diastereomers, denoted herein as isomers A and B. X-ray crystallography of the tris(ethylenediamine)cobalt(III) salt derivative of isomer A revealed a structure in the polar acentric space group P32. The structure consisted of three independent molecules of the gadolinium complex in the asymmetric unit along with three Delta-[Co(en)3]3+ cations, and it represents an unusual example of spontaneous Pasteur resolution of the cobalt cation. The geometry of the coordination core was best described as a distorted trigonal prism, and the final R factor was 5.6%. The configuration of the chiral central nitrogen of the DTPA core was S. The Gd-water (2.47-2.48 A), the Gd-acetate oxygens (2.34-2.42 A), and the Gd-N bond distances (central N, 2.59-2.63 A; terminal N, 2.74-2.80 A) were similar to other reported GdDTPA structures. The structurally characterized single crystal was one of two interconvertable diastereomers (isomers A and B) that equilibrated to a ratio of 1.81 to 1 at pH 7.4 and were separable at elevated pH by ion-exchange chromatography. The rate of isomerization was highly pH dependent: k1 = (1.45 +/- 0.08) x 102[H+] + (4.16 +/- 0.30) x 105[H+]2; k-1 = (2.57 +/- 0.17) x 102[H+] + (7.54 +/- 0.60) x 105[H+]2.

Magnetic resonance imaging (MRI) contrast agents are utilized adjunctively to enhance the contrast between normal and abnormal structures on MRI scans. Along with the rapid evolution of the field has come a new appreciation for the medicinal chemistry of this unique class of metallopharmaceuticals. The efficacy of MRI agents is a complex function of chemical, biophysical, and pharmacological properties, which must be married in a package of exquisite safety. This report illustrates the wide range of medicinal chemistry relevant to existing agents that are either approved or in clinical development, as well as concepts, which may result in exciting new pharmaceuticals in the future.

The manganese(II) ion has many favorable properties that lead to its potential use as an MRI contrast agent: high spin number, long electronic relaxation time, labile water exchange. The present work describes the design, synthesis, and evaluation of a novel Mn(II) complex (MnL1) based on EDTA and also contains a moiety that noncovalently binds the complex to serum albumin, the same moiety used in the gadolinium based contrast agent MS-325. Ultrafiltration albumin binding measurements (0.1 mM, pH 7.4, 37 degrees C) indicated that the complex binds well to plasma proteins (rabbit: 96 +/- 2% bound, human: 93 +/- 2% bound), and most likely to serum albumin (rabbit: 89 +/- 2% bound, human 98 +/- 2% bound). Observed relaxivities (+/- 5%) of the complex were measured (20 MHz, 37 degrees C, 0.1 mM, pH 7.4) in HEPES buffer (r(1) = 5.8 mM(-)(1) s(-)(1)), rabbit plasma (r(1) = 51 mM(-)(1) s(-)(1)), human plasma (r(1) = 46 mM(-)(1) s(-)(1)), 4.5% rabbit serum albumin (r(1) = 47 mM(-)(1) s(-)(1)), and 4.5% human serum albumin (r(1) = 48 mM(-)(1) s(-)(1)). The water exchange rate was near optimal for an MRI contrast agent (k(298) = 2.3 +/- 0.9 x 10(8) s(-)(1)). Variable temperature NMRD profiles indicated that the high relaxivity was due to slow tumbling of the albumin-bound complex and fast exchange of the inner sphere water. The concept of a high relaxivity Mn(II)-based contrast agent was validated by imaging at 1.5 T. In a rabbit model of carotid artery injury, MnL1 clearly delineated both arteries and veins while also distinguishing between healthy tissue and regions of vessel damage.

Amphiphilic gadolinium complexes were investigated as potential magnetic resonance imaging (MRI) contrast agents. A series of complexes was synthesized in order to study the effect of hydrophilic phosphodiester groups on albumin binding, relaxivity, and blood half-life in rats. Thus, compound 11a, a diethylenetriaminepentaacetato aquo gadolinium(III) (Gd-DTPA) derivative with an octyl substituent, was synthesized and compared to 5b, the analogous octyl derivative containing a phosphodiester linkage between the gadolinium chelate and the alkyl chain. Likewise, 11b, a naphthyl Gd-DTPA derivative, was compared to the naphthyl phosphodiester derivative 5c. A direct comparison is not available for 5a, a 4,4-diphenylcyclohexyl phosphodiester Gd-DTPA derivative; however, its pharmacokinetic properties mirror those of the other phosphodiester derivatives. Although the introduction of the phosphodiester moiety decreased log P by approximately 1.7 units, albumin binding data obtained in 4.5% human serum albumin (HSA) indicated that derivatives containing the phosphodiester group exhibited somewhat higher albumin affinity than their alkyl analogues (54 +/- 5 and 44 +/- 4% for 5b and 11a, respectively; 40 +/- 4 and 30 +/- 3% for 5c and 11b, respectively). Both classes of agents were characterized by enhanced relaxivity in the presence of 4.5% HSA (r1 = 16-42 mM(-1) s(-1) at 20 MHz and 37 degrees C) as compared with the relaxivity values measured in phosphate-buffered saline (PBS) alone (r1 = 4.6-6.6 mM(-1) s(-1) at 20 MHz and 37 degrees C). Pharmacokinetic data indicated that compound 5b had a half-life of 14.3 +/- 1.8 min in the rat as compared with a half-life of 6.20 +/- 0.04 min for the non-phosphodiester analogue 11a. Similarly, the half-life obtained for the phosphodiester 5c was 14.3 +/- 1.7 min as compared with a half-life of 6.80 +/- 0.03 min for 11b. The percent biliary excretion was significantly lower for the phosphodiester compounds than for non-phosphodiester analogues (17.7 +/- 4.0 and 66.9 +/- 3.4% for 5b and 11a, respectively; 17.0 +/- 1.6 and 64.3 +/- 9.0% for 5c and 11b, respectively). The percent biliary excretion (15.8 +/- 4.4%) and plasma half-life in the rat (23.1 +/- 2.9 min) for 5a are consistent with the extended plasma half-life of the other phosphodiester derivatives. Taken together, the enhanced relaxivity and extended blood half-life of the phosphodiester derivatives support the concept of using endogenous albumin binding to achieve blood pool-like properties for small-molecule magnetic resonance imaging (MRI) contrast agents.

A. Water Exchange 2326 B. Proton Exchange 2327 C. Electronic Relaxation 2327 D. Relaxivity 2331 E. Outerand Second-Sphere Relaxivity 2334 F. Methods of Improving Relaxivity 2336 V. Macromolecular Conjugates 2336 A. Introduction 2336 B. General Conjugation Methods 2336 C. Synthetic Linear Polymers 2336 D. Synthetic Dendrimer-Based Agents 2338 E. Naturally Occurring Polymers (Proteins, Polysaccharides, and Nucleic Acids) 2339

Clinically relevant relaxivity enhancement of a magnetic resonance imaging (MRI) contrast agent has been achieved by using prodrug Gd

The solution equilibria, acid dissociation, and serum stability of a series of Y(III) complexes of DTPA ligands functionalized with p-nitrobenzyl, methyl, and trans-cyclohexyl substituents were studied. The thermodynamic stability of the complexes studied ranged from log K = 21.53 to 24.7. Acid dissociation rates were found to decrease as the substitution on the carbon backbone increased, and significant differences in dissociation rates were observed for the Y(III) complexes of a pair of diasteriomeric cyclohexyl-DTPA ligands. While one diastereomer was found to have the slowest acid dissociation rate of the entire DTPA series, it was remarkably labile in both serum stability and in vivo studies.

A simple spectrophotometric method was developed to quantitate micromolar concentrations of a bifunctional DTPA ligand in DTPA monoclonal antibody (mAb) conjugates. Titration of a brightly colored 1:2 yttrium (III) complex of arsenazo III with the ligand 1B4M-DTPA obeyed Beer's law over the concentration range 0-2.0 microM 1B4M-DTPA at 652 nm. From a calibration plot of absorbance versus 1B4M molarity, concentrations of 1B4M-DTPA conjugated to mAb were determined. Mole ratios of 1B4M-DTPA to mAb agreed satisfactorily with the ratios obtained by a radioanalytical technique using carbon-14-labeled 1B4M-DTPA and a binding assay using 111In. The spectrophotometric method was applied successfully to the preparation of 1B4M-DTPA mAb anti-TAC, a mAb conjugate used in clinical trials of 90Y radioimmunotherapy.

Tiron (1,2-dihydroxybenzene-3,5-disulfonate), a non-toxic chelator of a variety of metals, is used to alleviate acute metal overload in animals. It is also oxidized to the EPR-detectable semiquinone radical by various biologically relevant oxidants, such as .OH, Ofx98-12 alkyl, and alkoxyl radicals. Since Tiron reacts with potentially toxic intracellular species and is also a metal chelator, we evaluated its protective effects in V79 cells subjected to various types of oxidative damage and attempted to distinguish the protection due to direct detoxification of intracellular radicals from that resulting from chelation of redox-active transition metals. We found that Tiron protects Chinese hamster V79 cells against both Ofx98-22-induced (and H2O2 via dismutation of Ofx98-32 and H2O2-induced cytotoxicity as measured by clonogenic assays. In experiments where Tiron was incubated with V79 cells and rinsed prior to exposure to HX XO or H2O2, cytoprotection was observed, indicating that it protects against intracellular oxidative damage. On the other hand, Tiron did not protect V79 cells against the damage caused by ionizing radiation under aerobic conditions, which is predominantly mediated by H., .OH, and hydrated electrons in a metal-independent fashion. We demonstrate also that in in vitro studies, Tiron protects supercoiled DNA from metal-mediated superoxide-dependent strand breaks. We conclude that Tiron is a potentially useful protecting agent against the lethal effects of oxidative stress and suggest that it offers protection by chelating redox-active transition metal ions, in contrast to earlier reports where the protection by this compound in cellular systems subjected to oxidative damage has been interpreted as due to radical scavenging alone.

A convenient synthesis of 4-nitrobenzyl-substituted macrocyclic tetraamines and their conversion to bifunctional poly(amino carboxylate) chelating agents is described. Cyclization of (4-nitrobenzyl)-ethylenediamine with appropriate BOC-protected amino disuccinimido esters in dioxane at 90 degrees C resulted in the formation of 12- and 14-membered ring diamides in 40% and 44% yield, respectively. A 12-membered macrocyclic triamide was also prepared in 44% yield by cyclization of N-(2-aminoethyl)-4-nitrophenylalaninamide with disuccinimidyl N-(tert-butoxycarbonyl)iminodiacetate. Deprotection (HCl/dioxane) and reduction with borane gave the substituted macrocyclic amines which were then alkylated with either bromoacetic acid or tert-butyl bromoacetate. Preparation of the isothiocyanate derivatives and 14C labeled chelating agents are described. Attempts to prepare a 9-membered macrocyclic diamide using this cyclization technique resulted instead in a 20% yield of a 10:1 mixture of isomeric fused 5,6 ring acylamidines. Deprotection (HCl/dioxane) and reduction with borane gave a substituted piperazine derivative in 55% yield.

EP-647 is a serum albumin-targeted magnetic resonance imaging contrast agent comprising a GdDTPA (DTPA, diethylenetriaminepentaacetate) chelate for magnetic resonance signal generation linked via a phosphodiester to a substituted biphenyl for albumin targeting. Albumin binding and relaxivity are higher than the benchmark magnetic resonance angiographic agent MS-325. EP-647 binds primarily to a unique site on serum albumin that is different from the MS-325 site and the binding sites of other drugs.

The ligand N-[2-amino-3-(p-isothiocyanatophenyl)propyl]-(±)-trans-1,2-diaminocyclohexane-N,N′,N″-pentaacetic acid has been synthesized and linked to IgG and to monoclonal antibody B72.3, and labelled with 206Bi and 212Bi to demonstrate the in vivo stability of the label and its utility for 212Bi-radioimmunotherapy.

Mossbauer spectra of a polycrystalline form of oxidized chloro‐5,10,15,20‐tetra(mesityl)porphyrin‐ atoiron(III) [Fe(TMP)Cl], compound A, were recorded over a range of temperatures (4.2–195 K) and magnetic fields (0–6 T). These spectra of compound A exhibit magnetic features which are markedly different from those of the analogous protein complexes, horse radish peroxidase compound I (HRP‐I) and compound ES of cytochrome c peroxidase, even though chemical evidence and optical spectroscopy indicate that compound A is similar to the others in comprising a Fe(IV) complex within a porphyrin cation radical. We interpret the data by employing a spin Hamiltonian model in which the central Fe(IV) complex, with S=1, is tightly coupled to a S=1/2 system of the oxidized porphyrin to yield a net S=3/2 system as suggested by the susceptibility measurements. The theoretical treatment yields information on the d‐electron energies which is similar to that more directly available in the peroxidase spectra. The strength of ...

X-ray absorption edge and extended x-ray absorption fine structure studies have been undertaken on resting (ferric) horseradish peroxidase, HRP compound I (HRP-I), HRP compound II (HRP-II), and several highly oxidized synthetic iron porphyrins that may have relevance as models for the iron site in horseradish peroxidase. The energies of the absorption edges are consistent with an Fe(IV) formulation for the highly oxidized species. The shapes of the absorption edges further support the assignment of HRP-I and one of the model compounds as Fe(IV)-porphyrin pi-cations. The edge shapes also demonstrate that the iron sites in the model porphyrins are not identical to the iron sites in the enzyme. Preliminary curve fitting analysis of the extended x-ray absorption fine structure clearly indicates the presence of two different nearest neighbor distances for the iron, both in HRP-I and HRP-II, as well as in two of the highly oxidized model porphyrins. These distances are interpreted as an iron-porphyrin nitrogen distance and as a short (approximately 1.6 A) iron-oxygen distance.

Tri-catechol ligands form selective and strong complexes with the ferric ion, making them attractive candidates for use as iron decorporation pharmaceuticals. Recent work in our group has concentrated on the synthesis of macrocyclic and macrobicyclic ligands incorporating the 2.3-dihydroxyterephthalamide binding subunit, which has been shown to be the most efficient bidentate iron chelator known (pM's ranging from 21.1 to 22.7). Macrocyclic polycatechol ligands with e ndocyclic b inding subunits were synthesized using high dilution techniques in yields of up to 24%. A series of macrobicyclic tri-catechol ligands in which the ligand properties were varied by incorporating TREN, mesitylene triamine, and TPT backbones, were synthesized in 10-15% yield via a stepwise approach employing high dilution conditions for the tripod-tripod coupling cyclization. In addition, a template synthesis was developed which allowed the preparation of (Et,NH),(Fe(bicapped TRENCAM)) in 50% overall yield. Reversible e lectrochemical behavior was observed above pH 11.5 for Na,(Fe(bicapped TRENCAM)) (E1/2 --.95V vs. NHE) and above pH 8.3 for Na3(Fe(6icapped TRENMECAM)) (E1/2 --.92 V. vs. NHE). The crystal structure of Na,(Fe(bicapped TRENCAM)) was determined and showed the coordination geometry about the metal center to be trigonal prismatic, an unprecedented geometry for Fe(II1).

Preparation a partir d'un precurseur radicalaire cationique d'une porphyrine de Fer(III). Porphyrine: La tetramesitylporphyrine. Caracterisation par les spectres RPE, Mossbauer, RMN, visible et par susceptibilite magnetique

Extended X-ray absorption fine structure spectroscopy has been utilized to determine the structural environment of the heme iron sites in horseradish peroxidase compounds I and II. For comparison, analogous studies have been undertaken on putative ferryl (Fe/sup IV/=O) porphyrin model compounds and on crystallographically characterized Cr/sup IV/=O and Cr/sup V/ identical with N porphyrins. In a preliminary communication, they suggested that a short ca. 1.6 A Fe-O bond is present in the high valent forms of both the enzyme and the synthetic porphyrins. The present work demonstrates unambiguously that a short, ca. 1.64 A, Fe-O bond length is present both in HRP compounds I and II and in their synthetic analogues. This structure is consistent only with an oxo-ferryl (Fe=O) complex as the active oxygen species in horseradish peroxidase. The structural details, their implications for heme protein mediated oxygen activation, and the difference between their results and those recently published by other workers.

Extended X-ray absorption fine structure spectroscopy has been utilized to determine the structural environment of the heme iron sites in horseradish peroxidase compounds I and II. For comparison, analogous studies have been undertaken on putative ferryl (Fe/sup IV/=O) porphyrin model compounds and on crystallographically characterized Cr/sup IV/=O and Cr/sup V/ identical with N porphyrins. In a preliminary communication, they suggested that a short ca. 1.6 A Fe-O bond is present in the high valent forms of both the enzyme and the synthetic porphyrins. The present work demonstrates unambiguously that a short, ca. 1.64 A, Fe-O bond length is present both in HRP compounds I and II and in their synthetic analogues. This structure is consistent only with an oxo-ferryl (Fe=O) complex as the active oxygen species in horseradish peroxidase. The structural details, their implications for heme protein mediated oxygen activation, and the difference between their results and those recently published by other workers.

Methods for the design and synthesis of ligands intended to be specific for a metal ion have been a recent chemical development. This article describes how this process can be inverted so that the specifics of the coordination environment around the metal ion can be used as a template in large-scale ligand synthesis. The synthesis of macrobicyclic ligands for ferric ion has been accomplished by using active esters of catechol ligands in which catecholate coordination to iron is a prelude to the organic chemical reactions that link the coordination subunits together into one ligand system surrounding a central metal ion coordination site. The lanthanide(III) ions, which are among the most labile metal ions known, have coordination numbers of 8 or higher, and thus their encapsulation into a macrobicyclic structure is a challenging problem. Lanthanide amine complexes have been used as metal templates in the synthesis of such macrobicyclic lanthanide complexes. There is evidence that such a complex is inert to exchange in aqueous solution.