In vivo Assessment of the Impact of Molecular Weight on Constructs of 68Ga-DOTA-Manocept in a Syngeneic Mouse Tumor Model.

PURPOSE: Manocept™ constructs are mannosylated amine dextrans (MADs) that bind with high affinity to the mannose receptor, CD206. Tumor-associated macrophages (TAMs) are the most numerous immune cells in the tumor microenvironment and a recognized target for tumor imaging and cancer immunotherapies. Most TAMs express CD206, suggesting utility of MADs to deliver imaging moieties or therapeutics to TAMs. The liver Kupffer cells also express CD206, making them an off-target localization site when targeting CD206 on TAMs. We evaluated TAM targeting strategies using two novel MADs differing in molecular weight in a syngeneic mouse tumor model to determine how varying MAD molecular weights would impact tumor localization. Increased mass dose of the non-labeled construct or a higher molecular weight (HMW) construct were also used to block liver localization and enhance tumor to liver ratios. PROCEDURES: Two MADs, 8.7 kDa and 22.6 kDa modified with DOTA chelators, were synthesized and radiolabeled with 68Ga. A HMW MAD (300 kDa) was also synthesized as a competitive blocking agent for Kupffer cell localization. Balb/c mice, with and without CT26 tumors, underwent dynamic PET imaging for 90 min followed by biodistribution analyses in selected tissues. RESULTS: The new constructs were readily synthesized and labeled with 68Ga with ≥ 95% radiochemical purity in 15 min at 65 °C. When injected at doses of 0.57 nmol, the 8.7 kDa MAD provided 7-fold higher 68Ga tumor uptake compared to the 22.6 kDa MAD (2.87 ± 0.73%ID/g vs. 0.41 ± 0.02%ID/g). Studies with increased mass of unlabeled competitors showed reduced liver localization of the [68Ga]MAD-8.7 to varying degrees without significant reductions in tumor localization, resulting in enhanced tumor to liver signal ratios. CONCLUSION: Novel [68Ga]Manocept constructs were synthesized and studied in in vivo applications, showing that the smaller MAD localized to CT26 tumors more effectively than the larger MAD and that the unlabeled HMW construct could selectively block liver binding of [68Ga]MAD-8.7 without diminishing the localization to tumors. Promising results using the [68Ga]MAD-8.7 show a potential path to clinical applications.

Rhodium-catalyzed sequential allylic amination and olefin hydroacylation reactions: enantioselective synthesis of seven-membered nitrogen heterocycles.

Dynamic kinetic asymmetric amination of branched allylic acetimidates has been applied to the synthesis of 2-alkyl-dihydrobenzoazepin-5-ones. These seven-membered-ring aza ketones are prepared in good yield with high enantiomeric excess by rhodium-catalyzed allylic substitution with 2-amino aryl aldehydes followed by intramolecular olefin hydroacylation of the resulting alkenals. This two-step procedure is amenable to varied functionality and proves useful for the enantioselective preparation of these ring systems.

Rhodium-catalyzed regio- and enantioselective amination of racemic secondary allylic trichloroacetimidates with N-methyl anilines.

We report the chiral diene ligated rhodium-catalyzed dynamic kinetic asymmetric transformation (DYKAT) of racemic secondary allylic trichloroacetimidates with a variety of N-methyl anilines, providing allylic N-methyl arylamines in high yields, regioselectivity, and enantiomeric excess. The rhodium-catalyzed DYKAT method addresses limitations previously associated with this particular class of aromatic nitrogen nucleophiles.

Rhodium-catalyzed dynamic kinetic asymmetric transformations of racemic tertiary allylic trichloroacetimidates with anilines.

The rhodium-catalyzed regio- and enantioselective amination of racemic tertiary allylic trichloroacetimidates with a variety of aniline nucleophiles is a direct and efficient route to chiral α,α-disubstituted allylic N-arylamines. We describe the first dynamic kinetic asymmetric transformations of racemic tertiary allylic electrophiles with anilines utilizing a chiral diene-ligated rhodium catalyst. The method allows for the formation of α,α-disubstituted allylic N-arylamines in moderate to good yields with good to excellent levels of regio- and enantioselectivity.

Synthesis of α,α-disubstituted aryl amines by rhodium-catalyzed amination of tertiary allylic trichloroacetimidates.

The rhodium-catalyzed regioselective amination of tertiary allylic trichloroacetimidates with unactivated aromatic amines is a direct and efficient approach to the preparation of α,α-disubstituted allylic aryl amines in good yield and with excellent regioselectivity. This method is applicable to a variety of unactivated primary and secondary amines and allows for the preparation of reverse prenylated indoles in two steps.

The acyl coenzymeA:monoacylglycerol acyltransferase 3 (MGAT3) gene is a pseudogene in mice but encodes a functional enzyme in rats.

Triglyceride (TAG) absorption involves its initial hydrolysis to fatty acids and monoacylglycerol (MAG), which are resynthesized back to diacylglycerol (DAG) and TAG within enterocytes. The resynthesis of DAG is facilitated by fatty acyl-CoA dependent monoacylglycerol acyltransferases (MGATs). Three MGAT enzymes have been isolated in humans and the expression of MGAT2 and MGAT3 in the intestines suggests their functional role in the TAG absorption. In this paper, we report that the Mogat3 gene appears to be a pseudogene in mice while it is a functional gene in rats. Examination of the mouse genomic Mogat3 sequence revealed multiple changes that would result in a translational stop codon or frameshifts. The rat Mogat3 gene, however, is predicted to encode a functional enzyme of 362 amino acids. Expression of rat MGAT3 in human embryonic kidney 293 (HEK293) cells led to the formation of a 36-kDa protein that displayed significant MGAT but not DGAT activity. Tissue expression analysis of rat MGAT3 by real-time PCR analysis indicated that rat MGAT3 has a high level of expression in intestines and pancreas. Our results thus provide the molecular basis to understand the relative functional role of MGAT2 and MGAT3 and also for future exploration of MGAT3 function in animal models.

Rhodium-catalyzed regioselective amination of secondary allylic trichloroacetimidates with unactivated aromatic amines.

The use of unactivated aromatic amines in the rhodium-catalyzed regioselective amination of secondary allylic trichloroacetimidates is explored. The desired N-arylamines are obtained in high yields and regioselectivity, favoring the branched amination products. The presence of the trichloroacetimidate leaving group was found to be critical for successful regioselective amination reactions with unactivated aromatic amines. Control studies show that rhodium is not simply acting as a Lewis acid to activate the trichloroacetimidate leaving group.

The 'Ethereal' nature of TLR4 agonism and antagonism in the AGP class of lipid A mimetics.

To overcome the chemical and metabolic instability of the secondary fatty acyl residues in the AGP class of lipid A mimetics, the secondary ether lipid analogs of the potent TLR4 agonist CRX-527 (2) and TLR4 antagonist CRX-526 (3) were synthesized and evaluated along with their ester counterparts for agonist/antagonist activity in both in vitro and in vivo models. Like CRX-527, the secondary ether lipid 4 showed potent agonist activity in both murine and human models. Ether lipid 5, on the other hand, showed potent TLR4 antagonist activity similar to CRX-526 in human cell assays, but did not display any antagonist activity in murine models and, in fact, was weakly agonistic. Glycolipids 2, 4, and 5 were synthesized via a new highly convergent method utilizing a common advanced intermediate strategy. A new method for preparing (R)-3-alkyloxytetradecanoic acids, a key component of ether lipids 4 and 5, is also described.