An Activatable and Switchable Nanoaggregate Probe for Detecting H2 S and Its Application in Mice Brains.

Employing a sequentially activated probe design method, an activatable, switchable and dual-mode probe was designed. This nanoprobe, HSDPP, could be effectively activated by H2 S to form H-type aggregates with green emission; subsequently, the aggregates could bind to mtDNA to form monomers and the emIssion color switched from green to deep-red. We exploited HSDPP to image exogenous and endogenous H2 S in living cells. Of note, for the first time, this novel nanoprobe with an optimal partition coefficient value (LogP=1.269) was successfully applied for tracking the endogenous H2 S upregulation stimulated by cystathionase activator S-adenosyl-L-methionine (SAM) in mice brains. Finally, our work provides an invaluable chemical tool for probing endogenous H2 S upregulation in vitro/vivo and, importantly, affords a prospective design strategy for developing switchable chemosensors to unveil the relationship between biomolecules and DNA in mitochondria in many promising areas.

Synthesis, structure and in vitro cytotoxicity testing of some 1,3,4-oxadiazoline derivatives from 2-hydroxy-5-iodobenzoic acid.

The syntheses of nine new 5-iodosalicylic acid-based 1,3,4-oxadiazoline derivatives starting from methyl salicylate are described. These compounds are 2-[4-acetyl-5-methyl-5-(3-nitrophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6a), 2-[4-acetyl-5-methyl-5-(4-nitrophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6b), 2-(4-acetyl-5-methyl-5-phenyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)-4-iodophenyl acetate, C19H17IN2O4 (6c), 2-[4-acetyl-5-(4-fluorophenyl)-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate, C19H16FIN2O4 (6d), 2-[4-acetyl-5-(4-chlorophenyl)-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate, C19H16ClIN2O4 (6e), 2-[4-acetyl-5-(3-bromophenyl)-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6f), 2-[4-acetyl-5-(4-bromophenyl)-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6g), 2-[4-acetyl-5-methyl-5-(4-methylphenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6h) and 2-[5-(4-acetamidophenyl)-4-acetyl-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6i). The compounds were characterized by mass, 1H NMR and 13C NMR spectroscopies. Single-crystal X-ray diffraction studies were also carried out for 6c, 6d and 6e. Compounds 6c and 6d are isomorphous, with the 1,3,4-oxadiazoline ring having an envelope conformation, where the disubstituted C atom is the flap. The packing is determined by C-H...O, C-H...π and I...π interactions. For 6e, the 1,3,4-oxadiazoline ring is almost planar. In the packing, Cl...π interactions are observed, while the I atom is not involved in short interactions. Compounds 6d, 6e, 6f and 6h show good inhibiting abilities on the human cancer cell lines KB and Hep-G2, with IC50 values of 0.9-4.5 µM.

Synthesis and Structure of Hypervalent Iodine(III) Reagents Containing Phthalimidate and Application to Oxidative Amination Reactions.

A new class of hypervalent iodine reagents containing phthalimidate was synthesized, and structurally characterized by X-ray analysis. The benziodoxole-based reagent displays satisfactory solubility in common organic solvents and is reasonably stable in solution as well as in the solid state. The reagent was used for the oxidative amination of the C(sp(3))-H bond of N,N-dimethylanilines. In addition, the reagent was also applicable to oxidative amination with rearrangement of trialkylamines as well as enamines that were prepared in situ from secondary amines and aldehydes.

Preparation and X-ray structural study of 1-arylbenziodoxolones.

Various 1-arylbenziodoxolones can be conveniently prepared from 2-iodobenzoic acid and arenes by a one-pot procedure using Oxone (2KHSO5·KHSO4·K2SO4) as an inexpensive and environmentally safe oxidant. This procedure is also applicable for the synthesis of the 7-methylbenziodoxolone ring system using 2-iodo-3-methylbenzoic acid as starting compound. Structures of four 1-arylbenziodoxolone derivatives were established by single-crystal X-ray diffraction analysis. An enhanced reactivity of 1-aryl-7-methylbenziodoxolones toward nucleophiles compared to unsubstituted 1-arylbenziodoxolones has been found.

SIB-DOTA: a trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies.

A major drawback of internalizing monoclonal antibodies (mAbs) radioiodinated with direct electrophilic approaches is that tumor retention of radioactivity is compromised by the rapid washout of iodo-tyrosine, the primary labeled catabolite for mAbs labeled via this strategy. In our continuing efforts to develop more versatile residualizing labels that could overcome this problem, we have designed SIB-DOTA, a prosthetic labeling template that combines the features of the prototypical, dehalogenation-resistant N-succinimidyl 3-iodobenzoate (SIB) with DOTA, a useful macrocyclic chelator for labeling with radiometals. Herein we describe the synthesis of the unlabeled standard of this prosthetic moiety, its protected tin precursor, and radioiodinated SIB-DOTA. An anti-EGFRvIII-reactive mAb, L8A4 was radiolabeled with [(131)I]SIB-DOTA in 27.1±6.2% (n=2) conjugation yields and its targeting properties to the same mAb labeled with [(125)I]SGMIB both in vitro and in vivo using U87MG·ΔEGFR cells and xenografts were compared. In vitro paired-label internalization assays showed that the intracellular radioactivity from [(131)I]SIB-DOTA-L8A4 was 21.4±0.5% and 26.2±1.1% of initially bound radioactivity at 16 and 24h, respectively. In comparison, these values for [(125)I]SGMIB-L8A4 were 16.7±0.5% and 14.9±1.1%. Similarly, the SIB-DOTA prosthetic group provided better tumor targeting in vivo than SGMIB over 8 d period. These results suggest that SIB-DOTA warrants further evaluation as a residualizing agent for labeling internalizing mAbs including those targeted to EGFRvIII.

Synthesis and preliminary evaluation of piperidinyl and pyrrolidinyl iodobenzoates as imaging agents for butyrylcholinesterase.

PURPOSE: The purpose of this study is to synthesize and evaluate specific agents for molecular imaging of butyrylcholinesterase (BuChE), known to be associated with neuritic plaques and neurofibrillary tangles in Alzheimer's disease (AD). In this study, these agents were tested in a normal rat model. The distribution of radiolabel was compared with known BuChE histochemical distribution in the rat brain. PROCEDURES: Iodobenzoate esters were synthesized and tested, through spectrophotometric analysis, as specific substrates for BuChE. These compounds were converted to the corresponding (123)I esters from tributyltin intermediates and purified for studies in the rat model. Whole body dynamic scintigraphic images were obtained for biodistribution studies. Autoradiograms of brain sections were obtained and compared to histochemical distribution of the enzyme in this model system. RESULTS: The three iodobenzoate esters studied were specific substrates for BuChE. Whole body biodistribution studies with (123)I-labeled compounds showed rapid disappearance from the body while radioactivity was retained in the head region. Brain section autoradiography of animals injected with these labeled compounds indicated that most areas known to contain BuChE corresponded to areas of radioactivity accumulation. CONCLUSION: BuChE-specific radiolabeled iodobenzoates enter the brain and, in general, label areas known to exhibit BuChE activity in histochemical studies. Such molecules may represent a new direction for the development of agents for the molecular imaging of BuChE in the living brain, especially in regions where BuChE-containing neuropathological structures appear in AD.

Pincer thioamide and pincer thioimide palladium complexes catalyze highly efficient Negishi coupling of primary and secondary alkyl zinc reagents at room temperature.

Pincer thioamide Pd(II) complex 2 was prepared, and its reaction with cyclohexylzinc chloride yielded novel pincer thioimide Pd(II) complex 3 besides Pd(0) species. The structures of complexes 2 and 3 were confirmed by X-ray analysis. Both complexes are efficient catalysts for Negishi couplings involving primary and secondary alkyl zinc reagents bearing beta-hydrogen atoms. At a concentration of 0.1-0.5 mol % both catalysts readily promoted reactions at room temperature or even at 0 degrees C. The operational simplicity of these processes, in conjunction with the easy accessibility of both catalysts and substrates, promises synthetic utility of this new methodology. An experiment on a scale of 19.35 g carried out at very low catalyst loading of 2 (turnover number: 6,100,000) highlighted the potential application of the catalytic system. Monoalkyl and dialkyl zinc reagents displayed different reactivities and selectivities in reactions with aryl iodides catalyzed by complexes 2 or 3, and isomerization in reactions involving acyclic secondary alkyl zinc derivatives was suppressed by using appropriate amounts of dialkyl zinc reagents. Based on preliminary kinetic profiles and reaction evidence, three possible pathways are proposed for the reactions involving acyclic secondary alkyl zinc reagents to rationalize the difference between mono-alkyl and dialkyl zinc derivatives.

A library of noviosylated coumarin analogues.

The DNA gyrase inhibitor, novobiocin, was recently shown to inhibit Hsp90 via a previously unrecognized C-terminal ATP-binding site. Previous structure-activity relationship studies identified key moieties that appear important for Hsp90 inhibitory activity. In an effort to provide a more efficacious lead compound, a parallel library of noviosylated coumarin analogues was prepared.

Optimization of a convenient route to produce N-succinimidyl 4-radiodobenzoate for radioiodination of proteins.

The preparation of N-succinimidyl-4-[131I]iodobenzoate (SIB) has been optimized using an alternative technique employing Cu(I)-assisted radioiododebromination that produces p-[131I]iodobenzoic acid. The reaction conditions were optimized and radiochemical purity of more than 90% was obtained when using 160 degrees C, 60 min reaction time and a [CuCl]/[p-bromobenzoic acid] relation of about 10(-2). After purification, the p-[131I]iodobenzoic acid reacted with TSTU to produce the SIB in a radiochemical yield greater than 98%. Protein conjugation using SIB resulted in a relatively low radiochemical yield. Biological distribution studies evidenced the in vivo stability of the labeled protein.

A novel route to radioiodinated [123I]-N-succinimidyl-3-iodobenzoate, a reagent for radioiodination of bioactive peptides.

Radiolabeled peptides continue to emerge as potential radiopharmaceuticals for targeting several diseases such as cancer, infection and inflammation and even tissue and organ rejection. The classical method for labeling these molecules has been the electrophilic route. Evidence suggests that most molecules labeled via this route perturb their biological activity. Moreover, this method is not applicable to peptides lacking a tyrosine moiety in their structure. Hence, there is the need to develop alternate methods such as the prosthetic approach. We have optimized a solid-state radioiodination by exchange to produce [123I]-metaiodobenzylguanidine ([123I]-mIBG). The mIBG served as a precursor to obtain an activated N-succinimidyl ester for efficient coupling to amine functions in peptides, preferably the lysine group(s). The method was used to label a model chemotactic peptide and evaluated in vivo.

A convenient synthesis of N-succinimidyl-3-iodo-[125I]benzoate, a reagent for protein iodination.

A convenient procedure has been developed for the synthesis of N-succinimidyl-3-iodo-[125I]benzoate. The procedure involved the synthesis of chloromercuribenzoic acid, its esterification with N-hydroxysuccinimide (NHS) and exchanging the mercury moiety with radioactive iodine in the presence of an oxidant. The obtained product was attached to human serum albumin and its stability was compared with Chloramine-T (Ch-T) radioiodinated protein. The results indicated that the reagent-radiolabeled protein was stable for longer periods and the deiodination rate was significantly lower.

Radioiodination of antibodies via N-succinimidyl 2,4-dimethoxy-3-(trialkylstannyl)benzoates.

We have previously shown that use of N-succinimidyl 3-iodobenzoate (SIB) for radioiodination of monoclonal antibodies (MAbs) decreases the loss of radioiodine in vivo compared to MAbs labeled by using conventional methods. Herein, the synthesis of N-succinimidyl 2,4-dimethoxy-3-(trialkylstannyl)benzoates (alkyl = Me, Bu) are described as is their use as precursors for the radiosynthesis of N-succinimidyl 2,4-dimethoxy-3-iodobenzoate (SDMIB). A MAb F(ab')2 fragment labeled with SDMIB retained its ability to bind specifically to tumor homogenates. Paired-label tissue distribution studies indicate that the thyroid uptake (an indicator of deiodination) of hydrolyzed SDMIB was about 20 times that of hydrolyzed SIB. In contrast, thyroid uptake for SDMIB, when conjugated to a MAb, was only 1.4-2.8 times that for SIB and was considerably lower than levels reported in the literature for MAbs labeled by using direct, electrophilic iodination methods. Although MAbs labeled with SDMIB are significantly more inert to dehalogenation than those labeled by conventional methods, compared to the original SIB reagent, addition of two methoxy groups decreased retention of label in vivo.

Potential tumor- or organ-imaging agents. 29. Radioiodinated esters and amides of 20-hydroxy- and 20-aminopregn-5-en-3 beta-ols.

Radioiodinated benzoyl esters and amides of epimeric 20-hydroxy- and 20-aminopregn-5-en-3 beta-ols were synthesized in an effort to find an agent that would be rapidly and selectively taken up by adrenal cortical tissue. Achievement of such a goal would provide a basis for the development of adrenal imaging agents superior to those currently available for clinical use. The iodobenzoyl derivatives were obtained by treating the appropriate epimer with 2-iodobenzoic acid in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine. The resulting esters and amides were readily labeled with radioiodine by isotope exchange with sodium iodide-125 in pivalic acid. Tissue distribution studies in female rats revealed that only the esters displayed appreciable adrenal specificity, and the ester having the same configuration at C-20 as cholesterol was significantly better than the corresponding C-20 epimer.

Labeling proteins using aryl iodide acylation agents: influence of meta vs para substitution on in vivo stability.

The effect of para vs meta substitution on the biological behavior of an intact antibody and an F(ab')2 fragment was investigated. Paired-label studies were performed using 81C6 IgG and OC 125 F(ab')2 labeled using the N-succinimidyl esters of both p-[125I]- and m-[131I]iodobenzoate as well as with the potential catabolites, p-[125I]- and m-[131I]iodobenzoic acid. In all 3 studies, up to 55% lower uptake of 131I in thyroid and stomach was observed, suggesting that the m-substituted species were more inert to dehalogenation in vivo.

Synthesis of 125I labeled N-succinimidyl p-iodobenzoate for use in radiolabeling antibodies.

A new method is reported for the synthesis of N-succinimidyl p-([125/127I]iodobenzoate (NS-p-IB) from N-succinimidyl p-(tri-n-butylstannyl)benzoate via an iodination-destannylation reaction. The tin precursor was obtained in 70% overall yield from p-bromobenzoyl chloride after a four-step synthesis. The radiochemical yield of NS-p-[125I]IB was 75%. Conjugation of NS-p-[125I]IB to rabbit IgG or bovine serum albumin gave yields of 52 and 60% respectively. In vitro the radiolabeled proteins in serum at 37 degrees C showed less than 1% deiodination by 24 h. In vivo the stability of mouse IgG (MIgG) labeled with NS-p-[125I]IB was superior to MIgG radioiodinated in the presence of chloramine-T.

Potential tumor or organ-imaging agents, 24. Radioiodinated pregnenolone esters.

A series of radioiodinated pregnenolone esters was prepared in an effort to find an agent that would be rapidly and selectively taken up by adrenal cortical tissue. Achievement of such a goal would provide the basis for the development of an adrenal imaging agent having several advantages over those agents currently available for clinical use. The radioiodinated esters for this study were readily prepared by treating pregnenolone with the appropriate iodobenzoic acid in the presence of dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-pyridine (DMAP). The resulting esters were readily labeled with radioiodine by isotope exchange with sodium iodide-125 in pivalic acid. Subsequent tissue distribution studies in rats revealed that those esters displaying the most stability towards hydrolysis achieved the highest concentration in adrenal cortical tissue. For example, the 2,3,5-triiodobenzoate (6) showed an adrenal uptake of 23% of administered dose per gram of tissue at 0.5 hours. The achievement of high levels of radioactivity in the adrenal with this agent at early time periods warrants further evaluation of this agent in other animals.

O-(4-Diazo-3,5-di[125I]iodobenzoyl)sucrose, a novel radioactive label for determining organ sites of catabolism of plasma proteins.

A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose (DD125IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M4, and the long-lived protein bovine serum albumin. Derivatization with DD125IBS did not alter the clearance of either protein. Uptake of DD125IBS-labelled lactate dehydrogenase, isoenzyme M4, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20 h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD125IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins.