Comparative biological activities of potent active-site analogues of alpha-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several alpha-melanotropin (alpha-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr4]-alpha-MSH4-10-NH2 and Ac-[Tyr4]-alpha-MSH4-11-NH2 were compared with alpha-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr4]-alpha-MSH4-10-NH2 was found to be less active than Ac-[Tyr4]-alpha-MSH4-11-NH2 on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr4]-alpha-MSH4-10-NH2 exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr4]-alpha-MSH4-11-NH2 was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr4]-alpha-MSH4-10-NH2 over Ac-[Tyr4]-alpha-MSH4-11-NH2 on frog melanocytes may be related to the fact that the shorter 4-10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of alpha-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle4]-alpha-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiosynthesis of [11C]BBAC and [11C]BBPC as potential PET tracers for orexin2 receptors

Radiosynthesis of [N-methyl-(11)C](S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide ([(11)C]BBAC or [(11)C]3) and [N-methyl-(11)C] (S)-N-([1,1'-biphenyl]-2-yl)-1-(3-(1-methyl-1H-benzo[d]imidazol-2-yl)propanoyl)pyrrolidine-2-carboxamide ([(11)C]BBPC or [(11)C]-4), two potential PET tracers for orexin2 receptors are described. Syntheses of non-radioactive standards 3, 4 and corresponding desmethyl precursors 1, 2 were achieved from common intermediate (S)-2-([1,1'-biphenyl]-2-yl)-1-(pyrrolidin-2-yl)ethanone. Methylation using [(11)C]CH(3)OTf in the presence of base in acetone afforded [(11)C]3 and [(11)C]4 in 30±5% yield (EOS) with >99 % radiochemical purities with a specific activity ranged from 2.5±0.5 Ci/μmol (EOB). The logP of [(11)C]3 and [(11)C]4 were determined as 3.4 and 2.8, respectively. The total synthesis time was 30 min from EOB. However, PET scans performed in a rhesus monkey did not show tracer retention or appropriate brain uptake. Hence [(11)C]3 and [(11)C]4 cannot be used as PET tracers for imaging orexin2 receptors.     

Synthesis and in vitro bioactivity of human growth hormone-releasing factor analogs substituted at position-1

Eight position-1 analogs of the 40-amino acid fragment and two position-1 analogs of human growth hormone-releasing factor were synthesized by solid phase methodology and their capacity to release growth hormone was determined using rat anterior pituitary cells in monolayer culture. Relative to hGRF(1-40)OH, which was arbitrarily assigned a potency value of 1, [D-Tyr1]hGRF(1-40)OH, [Phe1]hGRF(1-40)OH, [Trp1]hGRF(1-40)OH, [His1]hGRF(1-40)OH, [Ala1]hGRF(1-40)OH, [(-Ac)Tyr1]hGRF(1-40)OH, Arg0-hGRF(1-40)OH and Ala0-hGRF(1-40)OH have potencies of 0.022, 0.038, 0.003, 0.351, 0.010, 0.032, 0.002 and 0.007 respectively. Relative to hGRF(1-44)NH2 = 1, [(3-Me)His1]hGRF(1-44)NH2 and [(O-Me)Tyr1]hGRF(1-44)NH2 have potencies of 0.132 and 0.001 respectively. These results demonstrate the prerequisite for an aromatic residue at position-1 for potent biological activity and also suggest that the capacity for hydrogen bond formation with the first residue is required for full receptor-ligand interaction.     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

Evaluation of [11C]hemicholinium-15 and [18F]hemicholinium-15 as new potential PET tracers for the high-affinity choline uptake system in the heart

[(11)C]Hemicholinium-15 ([(11)C]HC-15) and [(18)F]hemicholinium-15 ([(18)F]HC-15) have been synthesized as new potential PET tracers for the heart high-affinity choline uptake (HACU) system. [(11)C]HC-15 was prepared by N-[(11)C]methylation of the appropriate precursor, 4-methyl-2-phenyl-morpholin-2-ol, using [(11)C]CH(3)OTf in 55-70% radiochemical yield decay corrected to end of bombardment (EOB) and 2-3Ci/mumol specific activity at end of synthesis (EOS). [(18)F]HC-15 was prepared by N-[(18)F]fluoromethylation of the precursor using [(18)F]FCH(2)OTf in 20-30% radiochemical yield decay corrected to EOB and >1.0Ci/mumol specific activity at EOS. The biodistribution of both compounds was determined in rats at 20min post-intravenous injection, and the results show the heart region uptakes 1.32+/-0.75%ID/g in R-ventricle for [(11)C]HC-15 and 1.28+/-0.81%ID/g in L-ventricle for [(18)F]HC-15, respectively. The dynamic PET imaging studies of [(11)C]HC-15 in rats were acquired 60min post-intravenous injection of the tracer using the IndyPET-II scanner. For the blocking experiments, the rats were intravenously pretreated with 3.0mg/kg of unlabeled HC-15 prior to [(11)C]HC-15 injection. [(11)C]HC-15 rat heart PET studies show rapid heart uptake to give clear heart images. The rat heart PET blocking studies found no significant blocking effect. The dynamic PET studies in normal and ablated dogs were performed using Siemens PET scanner with [(13)N]NH(3), [(11)C]HC-15, and [(18)F]HC-15. PET studies in dogs of both [(11)C]HC-15 and [(18)F]HC-15 also show significant heart uptake and give images of the heart. However, there is no significant change in [(11)C]HC-15 L-ventricle uptake following radiofrequency ablation in the dog. These results suggest that the localization of HC-15 tracers in the heart is mediated by non-specific processes, and the visualization of HC-15 tracers on the heart is related to non-specific binding of HACU.     

Penile erection and yawning induced by oxytocin and related peptides: structure-activity relationship

The potency of several oxytocin-related peptides in inducing penile erection and yawning after injection into a lateral ventricle of male rats was compared. Substitution of two amino acids in the oxytocin molecule or deletion of the C-terminal glycinamide as in des-GlyNH2-oxytocin [oxytocin(1-8)] reduced oxytocin potency in inducing both effects, the rank order being: oxytocin greater than [Thr4,Gly7]-oxytocin congruent to isotocin [( Ser4,Ile8]-oxytocin) greater than vasopressin [( Phe3,Arg8]-oxytocin) greater than des-GlyNH2-oxytocin. Oxytocin's ability to induce penile erection and yawning was abolished by permanent opening of the disulfide bridge by reduction and carboxymethylation. Oxytocin(1-6) and oxytocin(7-9) were also inactive. Penile erection and yawning induced by oxytocin-related peptides were antagonized in a dose-dependent manner by nonapeptide antagonists with a rank order of potency that follows their antioxytocic activity (d[(CH2)5Tyr(Me)Orn8]-vasotocin congruent to [Pen1,Phe(Me)2,Thr4,Orn8]-oxytocin greater than d[(CH2)5Tyr(Me)Arg8]-vasopression). Carboxymethylated oxytocin, oxytocin(1-6), and oxytocin(7-9) were devoid of antagonistic activity. The present results suggest that central oxytocin receptors mediating the expression of penile erection and yawning are structurally related to those present in the uterus and in the mammary gland.     

The influence of substance P and its fragments on endogenous phosphorylation of synaptosomal membrane protein (synapsin) from cerebral cortex of rat brain.

1. The effects of substance P and its fragments and analogue of a C-terminal fragment on cyclic AMP-dependent phosphorylation of synapsin I in synaptosomal membranes (SM) from cerebral cortex were investigated. 2. SP(I-II) and SP(1-4) at 10(-3) M caused a marked stimulation of synapsin I phosphorylation. 3. A C-terminal fragment of SP (SP6-11) had no effect on phosphorylation of synapsin 1. 4. Analogue of C-terminal fragment [(Tyr8)SP6-11] at 10(-3) M distinctly inhibits phosphorylation of synapsin I. 5. These data suggest that SPI-II and its C- and N-terminal fragments have a modulator function against the phosphorylation of some rat brain proteins.     

PET imaging and optical imaging with D-luciferin [11C]methyl ester and D-luciferin [11C]methyl ether of luciferase gene expression in tumor xenografts of living mice

New carbon-11 labeled D-luciferin analogs D-luciferin [(11)C]methyl ester ([(11)C]LMEster, [(11)C]1) and D-luciferin [(11)C]methyl ether ([(11)C]LMEther, [(11)C]2) were synthesized in 25-55% radiochemical yield. PET studies with [(11)C]LMEster and [(11)C]LMEther demonstrate a lower retention of the C-11 label at 45 min post-injection in luciferase expression tumor. Optical imaging with unlabeled substrate D-luciferin and radiotracers [(11)C]LMEster and [(11)C]LMEther gave tumor luciferase images within a few minutes of photon counting.     

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.     

Modulation of pharmacokinetics of radioiodinated sugar-conjugated somatostatin analogues by variation of peptide net charge and carbohydration chemistry

Sugar conjugation of biooactive peptides has been shown to be a powerful tool to modulate peptide pharmacokinetics. In the case of radiolabeled somatostatin analogues developed for in vivo scintigraphy of somatostatin receptor (sst) expressing tumors, it generally led to tracers with predominant renal excretion and low uptake in nontarget organs, and in some cases also with enhanced tumor accumulation. Especially with respect to endoradiotherapeutic applicability of these tracers, however, understanding the structural requirements for minimal kidney accumulation and maximal tumor uptake is important. The aim of this study was therefore the evaluation of the potential of specific glycoside structures in combination with reduced peptide net charge to reduce kidney accumulation without affecting tumor accumulation. Three glyco analogues of radioiodinated Tyr(3)-octreotate (TOCA) with z = 0 were evaluated in a comparative study using [(125)I]Mtr-TOCA (z = +1), the maltotriose-Amadori analogue of [(125)I]TOCA, as a reference, [(125)I]Glucuron-TOCA, the Amadori conjugate with glucuronic acid, and [(125)I]Gluc-S- and [(125)I]Gal-S-TOCA, the coupling products with glucosyl- and mannosyl-mercaptopropionate. In cells transfected with sst(1)-sst(5), all three new analogues show sst-subtype binding profiles similar to I-Mtr-TOCA with high, but somewhat reduced, affinity for sst(2). In contrast, internalization into sst(2)-expressing cells (in % of [(125)I]Tyr(3)-octreotide ([(125)I]TOC)) as well as the EC(50,R) of unlabeled TOC for internalization determined in dual-tracer experiments are substantially enhanced for [(123)I]Gal-S-TOCA and [(123)I]Gluc-S-TOCA (internalization, 190% +/- 12% and 265% +/- 20%, respectively, vs 168% +/- 6% of [(125)I]TOC for [(123)I]Mtr-TOCA; EC(50,R), 2.62 +/- 0.07 and 2.96 +/- 0.14, respectively, vs 1.81 +/- 0.07 for [(123)I]Mtr-TOCA). The tumor accumulation of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA in AR42J tumor-bearing nude mice 1 h p.i. is consequently very high (22.6 +/- 2.2 and 26.2 +/- 5.6%ID/g) and comparable to that of [(125)I]Mtr-TOCA (25.1 +/- 4.4%ID/g). [(125)I]Glucuron-TOCA showed lower uptake in sst-expressing tissues than did [(125)I]Mtr-TOCA, but considerably enhanced accumulation in nontarget organs such as liver, intestine, and kidney. Due to increased lipophilicity, hepatic and intestinal uptake 1 and 4 h p.i. of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA was also slightly higher than that of [(125)I]Mtr-TOCA. Kidney accumulation, however, was reduced by approximately 50% for both compounds (2.6 +/- 0.3 and 2.2 +/- 0.4, respectively, vs 4.0 +/- 0.7%ID/g at 1 h p.i.). Because no sugar-specific effect was detected in the latter case, it is concluded that general ligand pharmacokinetics and especially kidney accumulation of the tracers investigated are mainly determined by physicochemical characteristics such as lipophilicity, net charge, and also charge distribution ([(125)I]Glucuron-TOCA vs [(125)I]Gal-S- and [(125)I]Gluc-S-TOCA). With respect to receptor targeting, however, the structure of the carbohydrate moiety plays an important role, leading to dramatically enhanced ligand internalization, especially in the case of [(123)I]Gluc-S-TOCA. Taking into account the combined effects of the Gluc-S-moiety both on kidney and on tumor accumulation, this group seems to be a promising synthon for the synthesis of other radiolabeled peptide analogues with improved pharmacokinetics.     

Synthesis and biological evaluation of novel carbon-11-labelled analogues of citalopram as potential radioligands for the serotonin transporter

Three serotonin reuptake inhibitors where the 5-cyano group in citalopram [1-(3-dimethylamino-propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile (1)] was replaced with a methyl, acetyl and piperidinyl carbonyl group, respectively, were synthesized. In a Stille reaction applying [(11)C]methyl iodide the labelled compound [5-methyl-(11)C][3-[1-(4-fluorophenyl)-5-methyl-1,3-dihydroisobenzofuran-1-yl]-propyl]-dimethylamine ([(11)C]-2) was synthesized in 60-90% radiochemical yield. [5-carbonyl-(11)C][1-[1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl]-1-piperidin-1-yl-methanone] ([(11)C]-3) was synthesized in 62% radiochemical yield in a palladium mediated cross-coupling reaction utilizing [(11)C]carbon monoxide. The specific activity of [(11)C]-2 was highly dependent on whether the corresponding trimethyltin or tributyltin precursor was applied. In ex vivo rodent studies compound [(11)C]-2 exhibited a good blood-brain barrier (BBB) penetration whereas [(11)C]-3 did not. The brain distribution of [(11)C]-2 was investigated in a non-human primate using PET. There was a rapid uptake of radioactivity into the brain. Accumulation of the radiotracer was in agreement with the known distribution of serotonin transporters. The maximal thalamus to cerebellum ratio of 1.3 was reached after 85 min and the specific binding was partly blocked after pre-treatment with citalopram. Thus, [(11)C]-2 does not exhibit appropriate properties as radioligand for visualization of the serotonin transporter in vivo.     

Synthesis and evaluation of 18F- and 11C-labeled phenyl-galactopyranosides as potential probes for in vivo visualization of LacZ gene expression using positron emission tomography

3-Hydroxy-2-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, a derivative of the chromogenic beta-galactosidase (beta-gal) substrate o-nitrophenyl beta-D-galactopyranoside (ONPG) was synthesized using a Koenigs-Knorr glycosylation reaction. It was alkylated with 2-[(18)F]fluoroethyl triflate or [(11)C]methyl triflate, followed by deacetylation of the sugar hydroxyl groups to obtain radiolabeled 3-(2'-[(18)F]fluoroethoxy)-2-nitrophenyl beta-D-galactopyranoside ([(18)F]-2c) and 3-[(11)C]methoxy-2-nitrophenyl beta- d-galactopyranoside ([(11)C]-3c), which were evaluated as potential reporter probes for in vivo visualization of LacZ gene expression with positron emission tomography (PET). In vitro, [(18)F]- 2c and [(11)C]-3c were good substrates of beta-gal and showed, respectively, a 7.5- and 2.5-fold higher uptake into beta-gal expressing cells (LacZ cells) compared to control cells. However, reversed-phase HPLC analysis of the LacZ cell lysate and supernatant showed that labeled 3-(2'-[(18)F]fluoroethoxy)-2-nitrophenol, the hydrolysis product formed by beta-gal-mediated cleavage of [(18)F]-2c, substantially leaked out of the cells, which would lead to loss of PET signal. In a microPET study of [(18)F]-2c in a mouse with a beta-gal expressing tumor, high retention was observed in liver and kidneys, but only negligible accumulation was seen in the tumor. As a general conclusion, it can be stated that the synthesized PET tracers [ (18)F]-2c and [(11)C]-3c are not suitable for use as LacZ reporter probes. Further structural modifications to improve the diffusion over the tumor cell membrane and to increase retention in beta-gal expressing cells may lead to more favorable in vivo imaging probes.     

Facile synthesis and PET imaging of a novel potential heart acetylcholinesterase tracer N-[11C]methyl-3-[[(dimethylamino)carbonyl]oxy]-2-(2',2'-diphenylpropionoxymethyl)pyridinium

A new AChE tracer N-[(11)C]methyl-3-[[(dimethylamino)carbonyl]oxy]-2-(2',2'-diphenylpropionoxymethyl)pyridinium ([(11)C]MDDP, [(11)C]1) has been synthesized in 40-65% radiochemical yield. Initial PET dynamic studies of [(11)C]MDDP in rat heart showed rapid heart uptake and blood pool clearance to give high-quality heart images. Blocking studies of [(11)C]MDDP with pretreatment drug neostigmine in rats found only minor reductions in rat heart [(11)C]MDDP retention. The results suggest that [(11)C]MDDP delineates the heart very clearly, and the uptakes of [(11)C]MDDP in rat heart might be related to non-specific binding.     

5-(5-(6-[(11)C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole as a potential PET radioligand for imaging cerebral α7-nAChR in mice

The radiosynthesis and in vivo evaluation of 5-(5-(6-[(11)C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole [(11)C]rac-(1), a potential PET tracer for α7 nicotinic acetylcholine receptors (α7-nAChR), are described. Syntheses of the nonradioactive standard rac-1 and corresponding desmethyl precursor 7 were achieved in several reaction steps. Radiomethylation of 7 with [(11)C]CH(3)I afforded [(11)C]rac-1 in an average radiochemical yield of 30 ± 5% (n=5) with high radiochemical purity and an average specific radioactivity of 444 ± 74 GBq/μmol (n=5). The total synthesis time was 30 min from end-of-bombardment. Biodistribution studies in mice showed that [(11)C]rac-1 penetrates the blood-brain barrier and specifically labels neuronal α7-nAChRs.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

Role of Tyr-288 at the dioxygen reduction site of cytochrome bo studied by stable isotope labeling and resonance raman spectroscopy

To explore the role of a cross-link between side chains of Tyr-288 and His-284 at the heme-copper binuclear center, we prepared cytochrome bo where d(4)-Tyr, 1-[(13)C]Tyr, or 4-[(13)C]Tyr has been biosynthetically incorporated. Unexpectedly, the d(4)-Tyr-labeled enzyme showed a large decrease in the ubiquinol-1 oxidase and CO binding activities. Optical absorption and resonance Raman spectra identified the defect in the distal side of the heme-copper binuclear center. In the CO-bound d(4)-Tyr-labeled enzyme, a large fraction of the nu((Fe-C)) mode was shifted from the normal 520-cm(-1) band to a broad band centered around 491 cm(-1), as found for the Y288F mutant. Our results suggested that the substitution of ring hydrogens of Tyr-288 with deuteriums slows down the formation of the His-Tyr cross-link essential for dioxygen reduction at the binuclear center.     

Synthesis and evaluation in vitro and in vivo of a 11C-labeled cyclooxygenase-2 (COX-2) inhibitor

The radiosynthesis and radiopharmacological evaluation of 1-[(11)C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-1-enyl)-benzene [(11)C]5 as novel PET radiotracer for imaging of COX-2 expression is described. The radiotracer was prepared via O-methylation reaction with [(11)C]methyl iodide in 19% decay-corrected radiochemical yield at a specific activity of 20-25GBq/mumol at the end-of-synthesis within 35 min. The radiotracer [(11)C]5 was evaluated in vitro using various pro-inflammatory and tumor cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribution of compound [(11)C]5 was characterized in male Wistar rats. Compound [(11)C]5 was rapidly metabolized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioactivity uptake in the tumor was not COX-2 mediated.     

Utilization of [11C]phosgene for radiosynthesis of N-(2-{3-[3,5-bis(trifluoromethyl)]phenyl[11C]ureido}ethyl)glycyrrhetinamide, an inhibitory agent for proteasome and kinase in tumors

N-(2-{3-[3,5-Bis(trifluoromethyl)]phenylureido}ethyl)glycyrrhetinamide (2), an ureido-substituted derivative of glycyrrhetinic acid (1), has been reported to display potent inhibitory activity for proteasome and kinase, which are overexpressed in tumors. In this study, we labeled this unsymmetrical urea 2 using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]2 could become a positron emission tomography ligand for the imaging of proteasome and kinase in tumors. The strategy for the radiosynthesis of [(11)C]2 was to react hydrochloride of 3,5-bis(trifluoromethyl)aniline (4·HCl) with [(11)C]COCl(2) to possibly give isocyanate [(11)C]6, followed by the reaction of [(11)C]6 with N-(2-aminoethyl)glycyrrhetinamide (3).     

Synthesis and radiopharmacological characterization of [11C]AL-438 as a nonsteroidal ligand for imaging brain glucocorticoid receptors

The radiosynthesis and the radiopharmacological characterization of [(11)C]AL-438 as a nonsteroidal ligand for the glucocorticoid receptor (GR) is described. Radiolabeling of the corresponding desmethyl precursor 10 with [(11)C]MeI gave [(11)C]AL-438 in decay-corrected radiochemical yields of 30+/-4% (based upon [(11)C]CO(2)) within 35 min at a specific radioactivity of 10-15 GBq/micromol at the end-of-synthesis. The radiopharmacological evaluation of [(11)C]AL-438 involved biodistribution and small animal PET imaging in rats, and autoradiography studies using rat brain sections. Biodistribution studies were performed in male Wistar rats and demonstrated high radioactivity uptake in pituitary and brain. However, the inability of high dose corticosterone to block binding would suggest that the radioactivity accumulation in the brain was not receptor-mediated.     

A new high-yield synthetic route to PET CB1 radioligands [11C]OMAR and its analogs

OMAR analogs reference standards and their corresponding desmethylated precursors were synthesized from substituted anilines either in 4 and 5 steps with 27-32% and 24-31% yield, or in 3 and 4 steps with 21-30% and 19-28% yield, respectively. [(11)C]OMAR and its analog radioligands were prepared from their desmethylated precursors with [(11)C]CH(3)OTf through O-[(11)C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 50-65% radiochemical yields based on [(11)C]CO(2) and decay corrected to end of bombardment (EOB), with 370-740 GBq/μmol specific activity at EOB.