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Supplementary Figures 1-4, Methods from Halogenated Benzimidazole Carboxamides Target Integrin α4β1 on T-Cell and B-Cell Lymphomas

Integrin α4β1 is an attractive but poorly understood target for selective diagnosis and treatment of T-cell and B-cell lymphomas. This report focuses on the rapid microwave preparation, structure-activity relationships, and biological evaluation of medicinally pertinent benzimidazole heterocycles as integrin α4β1 antagonists. We documented tumor uptake of derivatives labeled with 125I in xenograft murine models of B-cell lymphoma. Molecular homology models of integrin α4β1 predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. The high-affinity halogenated ligands identified offer attractive tools for medicinal and biological use, including fluoro and iodo derivatives with potential radiodiagnostic (18F) or radiotherapeutic (131I) applications, or chloro and bromo analogues that could provide structural insights into integrin-ligand interactions through photoaffinity, cross-linking/mass spectroscopy, and X-ray crystallographic studies. Cancer Res; 70(13); 5448–56. ©2010 AACR.

Establishing improved methods for the radiolabeling of peptides with fluorine-18 via solid-phase peptide synthesis (SPPS) is desirable for the efficient synthesis of peptide-based molecular imaging agents. This work focuses on the development of a standardized platform to facilitate the reliable and efficient synthesis of high-purity fluorine-18 radiolabeled peptides for in vivo imaging with positron emission tomography (PET). Seven commercially available resins were selected for solid-phase radiolabeling of the model peptide VQAAIDYING with 4-[(18)F]fluorobenzoic acid ([(18)F]FBA). A wide range of radiochemical yields (18.8 ± 1.5% to 41.2 ± 5.3%) was obtained using standard conditions (coupling: 3 eq amino acid, 3 eq HATU, 6 eq DIPEA, 1.5 h, r.t.; cleavage: 94% TFA, 3 h, r.t.). After modification of coupling reagents and employing heated reactions to 37°C, radiochemical yields were improved by as much as 35.3% over standard conditions. When the optimized conditions were applied to the synthesis of [(18)F]FBA-PEG(28)-A20FMDV2, which targets the α(v)β(6) integrin in vivo, radiochemical yields improved by as much as 73.4% over those obtained using standard coupling and cleavage conditions. This platform can be utilized to improve the synthesis of peptide-based molecular probes for molecular imaging with PET.

Triazacryptand (TAC)-based fluorescent K + sensors have broad biomedical utility, yet their advancement has been hindered because of their challenging synthesis. Herein, an efficient synthesis is reported that delivers a didesmethyl triazacryptand (ddTAC) K + sensor in twofold fewer steps and ninefold higher overall yield than the original TAC synthesis. Our synthesis utilizes a C-O dianionic oxidative macrocyclization and reports new examples of aminoarylations and a microwave route to xanthythilium chromophores. The K + sensitivity and selectivity of the ddTAC-based sensor are comparable to the TAC-based sensor.

Current cancer chemotherapeutic agents clinically deployed today are designed to be indiscriminately cytotoxic, however, achieving selective targeting of cancer malignancies would allow for improved diagnostic and chemotherapeutic tools. Integrin alpha(4)beta(1), a heterodimeric cell surface receptor, is believed to have a low-affinity conformation in resting normal lymphocytes and an activated high-affinity conformation in cancerous cells, specifically T- and B-cell lymphomas. This highly attractive yet poorly understood receptor has been selectively targeted with the bisaryl urea peptidomimetic antagonist 1. However, concerns regarding its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzothiazole moiety, resulting in an analogue with improved physicochemical properties, solubility, and kidney:tumor ratio while maintaining potency (6; IC(50) = 53 pM). The results presented herein utilized heterocyclic and solid-phase chemistry, cell adhesion assay, and in vivo optical imaging using the cyanine dye Cy5.5 conjugate.

A library of novel, propeller-shaped dispirotriheterocyclic isoxazolinopiperidinochromanones is reported. Each rigid dispirotriheterocycle was prepared in five linear steps from commercially available tert-butyl 4-oxopiperidine-1-carboxylate and various derivatives of 1-(2-hydroxyphenyl)ethanone, benzaldehyde oxime, and carboxylic acids. Computational chemistry was employed to analyze the three-dimensional geometries of these dispirotriheterocycles, as well as to generate chemoinformatic bioavailability data. X-ray crystallographic structure determination verified the regioselectivity of the nitrile oxide 1,3-dipolar cycloaddition reaction. The resulting library of compounds has been added to the National Institutes of Health repository (approximately 10 mg of each withor =90% purity) for pilot-scale biomedical studies with bioassay data available at the National Center for Biotechnology Information PubChem database.

Using carbodiimide reagents [1,3-diisopropylcarbodiimide or N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)], we have developed a mild, generalized, one-pot method that delivers N-2-arylaminobenzimidazole esters from commercially available aryl isothiocyanates and o-phenylenediamines. Following saponification and acidifying, the benzimidazole acids were isolated in overall yields ranging from 75 to 88% from the starting aryl isothiocyanates. Nine benzimidazole acids were converted into a library consisting of 180 benzimidazole amides following EDC coupling with commercially available amines. The National Institute of General Medical Science will dispense these benzimidazole amides to academia groups for pilot scale biomedical studies. Using these mild conditions and environmentally safe reagents, we demonstrated that these pharmaceutically ornate heterocycles can also be constructed on solid support.

1,2,4,5-Tetrazines (prepared from aryl nitriles) condense with isoxazolylcyclobutanones (prepared from 3-benzenesulfonyl-3-vinylcyclobutanol) in methanolic KOH to give conformationally restricted 6-isoxazol-5-yl-6,7-dihydro-5H-[1,2]diazocin-4-ones. The solution 1H NMR spectra of dihydrodiazocinone 1a with phenyl moieties at C3 and C8 reveal two conformations of the eight-membered heterocycle that are non-interconverting on the NMR time scale at ambient temperature. The kinetics of the conversion process, followed by 1H NMR between 21 and 70 degrees C in DMSO solution, yield an activation energy of approximately 21 kcal/mol relative to the kinetic conformer and show an equilibrated ratio of approximately 5:1 of the thermodynamic to the kinetic conformers. The electronic structure calculations on a model dihydrodiazocinone predict geometries for the two conformations. One of these geometries agrees with the X-ray crystallographic analysis of the thermodynamic conformation of 1a.

Introduction Click chemistry, particularly the Huisgen 1,3-dipolar cycloaddition of an alkyne with an azide, has quickly become popular for site-specific radiolabeling. Recently, strain-promoted click chemistries have been developed, eliminating the need for potentially toxic copper catalysts. This study presents radiolabeling of an αvβ6 integrin targeting peptide (A20FMDV2) via strain-promoted click using a fluorine-18 prosthetic group, and in vitro and in vivo evaluation. Methods The radiotracer [18F]FBA-C6-ADIBON3-PEG7-A20FMDV2 (1) was prepared from [18F]FBA-C6-ADIBO (2) and N3-PEG7-A20FMDV2 (ethanol; 10 min; 35–45 °C). HPLC-purified and formulated radiotracer 1 was evaluated in vitro by cell binding (DX3puroβ6, αvβ6-positive; and DX3puro, αvβ6-negative control) and serum stability, and in vivo using PET/CT imaging and biodistribution studies in mice. Results The radiotracer 1 was readily prepared and purified (from 2 : 40 ± 4 min including HPLC, 11.9 ± 3.2% decay corrected isolated radiochemical yield, > 99% radiochemical purity, n = 4) and displayed good stability (1 h: > 99%, saline; 94.6%, serum). Strong αvβ6-targeted binding was observed in vitro (DX3puroβ6 cells, 15 min: 43.2% binding, > 6:1 for DX3puroβ6:DX3puro). In the mouse model DX3puroβ6-tumor binding was low (1 h: 0.47 ± 0.28% ID/g, 4 h: 0.14 ± 0.09% ID/g) and clearing from the bloodstream was via the renal and hepatobiliary routes (urine: 167 ± 84% ID/g at 1 h, 10.3 ± 4.8% ID/g at 4 h; gall bladder: 95 ± 33% ID/g at 1 h, 63 ± 11% ID/g at 4 h). Conclusion Copper-free, strain-promoted click chemistry is an attractive, straightforward approach to radiolabeling. Although the [18F]FBA-C6-ADBIO-based prosthetic group did not interfere with αvβ6-targeted binding in vitro, it did influence the pharmacokinetics, possibly due to its size and lipophilic nature.

The strain-promoted click 1,3-dipolar cycloaddition reactions involving azides and cyclooctynes for the synthesis of triazoles offer the advantage of being able to be performed in biological settings via copper-free chemistries. While strained reagents conjugated to optical dyes and radiometal conjugates have been reported, cyclooctyne reagents labeled with fluorine-18 ((18)F) and radiochemically evaluated in a copper-free click reaction have yet to be explored. This report describes the conversion of a bifunctional azadibenzocyclooctyne (ADIBO) amine to the (18)F-labeled cyclooctyne 4, the subsequent fast copper-free 1,3-dipolar cycloaddition reaction with alkyl azides at 37 °C (70% radiochemical conversion in 30 min), and biological evaluations (serum stability of95% at 2 h). These findings demonstrate the excellent reactivity of the (18)F-labeled cyclooctyne 4 with readily available azides that will allow future work focusing on rapid copper-free in vitro and in vivo click chemistries for PET imaging using (18)F-labeled cyclooctyne derivatives of ADIBO.

This protocol describes a rapid, high-yielding, microwave-mediated route that affords benzazole heterocycles in high crude purity and represents a significant advancement toward an environmentally friendly reaction. The reaction of aryl isothiocyanates with o-nucleophilic anilines produces thiourea intermediates that, in the presence of a carbodiimide-functionalized resin, cyclize to benzazoles with the safe removal of one equivalent of hydrogen sulfide. This procedure takes ∼ 8.5 h to complete: 1-3 h for setup, 4.5 h for benzazole formation and 2 h for workup and purification.

An efficient synthesis is reported that delivers in 5 steps and 52% overall yield a new structurally simplified fluorescent K(+) sensor with improved K(+) sensitivity and selectivity over existing K(+) sensors. The synthesis procedure utilizes a new template-directed oxidative C-N bond-forming macrocyclization reaction and reports new approaches to Pd(0), Sandmeyer-like and metal-free aminoarylations, as well as organotitanium additions to vinylogous sulfonates.

An effective route to benzimidazo[2,1-b]quinazolin-12(5H)-ones from commercially available o-aryl isothiocyanate esters and o-phenylenediamines is reported. This method accommodates a variety of substituents on either starting material and proceeds under microwave irradiation in the presence of barium hydroxide, conditions that do not hydrolyze methyl ester substituents. The pharmacologically pertinent benzimidazoquinazolinone heterocycle is delivered in excellent yield and purity via both solution- and solid-phase protocols, the latter involving traceless release from the resin.