Long-Range 1H–15N J Couplings Providing a Method for Direct Studies of the Structure and Azide–Tetrazole Equilibrium in a Series of Azido-1,2,4-triazines and Azidopyrimidines

Шестакова, Т. С.; Шенкарев, Захар О.; Деев, С. Л.; Чупахин, О. Н.; Khalymbadzha, Igor A.; Русинов, В. Л.; Arseniev, Alexander S.

doi:10.1021/jo4008207

Cited by 39 publications

(28 citation statements)

References 43 publications

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“…Compound 15 was pursued further due to its high ligand efficiency and competition with 2´,3´-cGAMP. However, it was discovered upon attempted resynthesis that this compound is not chemically stable and readily isomerizes via an open-ring azidopyrimidine ( S5 Fig ) [ 37 ]. From a chemical optimization perspective this was not attractive and thus several chemically stable [ 6 , 5 ] ring systems were synthesized in an attempt to find a core that would recapitulate the active pharmacophore and not isomerize.…”

Section: Resultsmentioning

confidence: 99%

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

et al. 2017

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Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

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Section: Resultsmentioning

confidence: 99%

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

et al. 2017

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“…The incorporation of 15 N labels in nitrogen-containing heterocycles greatly facilitates the use of NMR spectroscopy for studies of molecular structures and mechanisms of chemical transformations [ 10 , 24 – 29 ]. The labelling enhances the sensitivity of detection and permits the quantitative measurements of J CN and 1 H- 15 N J -coupling constants ( J HN ) even in a mixture of tautomeric forms [ 24 – 25 ]. Additionally, a method based on amplitude-modulated spin-echo experiments was found to be the most efficient way to measure J HN couplings [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

¹⁵N-Labelling and structure determination of adamantylated azolo-azines in solution

Деев¹,

Paramonov²,

Шестакова³

et al. 2017

Beilstein J. Org. Chem.

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Determining the accurate chemical structures of synthesized compounds is essential for biomedical studies and computer-assisted drug design. The unequivocal determination of N-adamantylation or N-arylation site(s) in nitrogen-rich heterocycles, characterized by a low density of hydrogen atoms, using NMR methods at natural isotopic abundance is difficult. In these compounds, the heterocyclic moiety is covalently attached to the carbon atom of the substituent group that has no bound hydrogen atoms, and the connection between the two moieties of the compound cannot always be established via conventional 1H-1H and 1H-13C NMR correlation experiments (COSY and HMBC, respectively) or nuclear Overhauser effect spectroscopy (NOESY or ROESY). The selective incorporation of 15N-labelled atoms in different positions of the heterocyclic core allowed for the use of 1H-15N (J HN) and 13C-15N (J CN) coupling constants for the structure determinations of N-alkylated nitrogen-containing heterocycles in solution. This method was tested on the N-adamantylated products in a series of azolo-1,2,4-triazines and 1,2,4-triazolo[1,5-a]pyrimidine. The syntheses of adamantylated azolo-azines were based on the interactions of azolo-azines and 1-adamatanol in TFA solution. For azolo-1,2,4-triazinones, the formation of mixtures of N-adamantyl derivatives was observed. The J HN and J CN values were measured using amplitude-modulated 1D 1H spin-echo experiments with the selective inversion of the 15N nuclei and line-shape analysis in the 1D 13С spectra acquired with selective 15N decoupling, respectively. Additional spin–spin interactions were detected in the 15N-HMBC spectra. NMR data and DFT (density functional theory) calculations permitted to suggest a possible mechanism of isomerization for the adamantylated products of the azolo-1,2,4-triazines. The combined analysis of the J HN and J CN couplings in 15N-labelled compounds provides an efficient method for the structure determination of N-alkylated azolo-azines even in the case of isomer formation. The isomerization of adamantylated tetrazolo[1,5-b][1,2,4]triazin-7-ones in acidic conditions occurs through the formation of the adamantyl cation.

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“…The N5, N4, N2, and N3 atoms appear at δ 237.1, 241.3, 252.4, and 265.7, respectively. The data obtained from the 15 N NMR spectrum confirms the structure of trifluoromethylated tetrazolo[1,5- a ]pyrimidines in solution (See Figures S8 and S9 in Supporting Information File 1 ) [ 38 – 40 ].…”

Section: Resultsmentioning

confidence: 58%

Synthesis, effect of substituents on the regiochemistry and equilibrium studies of tetrazolo[1,5-a]pyrimidine/2-azidopyrimidines

Scapin

Salbego

Bender

et al. 2017

Beilstein J. Org. Chem.

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An efficient synthesis methodology for a series of tetrazolo[1,5-a]pyrimidines substituted at the 5- and 7-positions from the cyclocondensation reaction [CCC + NCN] was developed. The NCN corresponds to 5-aminotetrazole and CCC to β-enaminone. Two distinct products were observed in accordance with the β-enaminone substituent. When observed in solution, the compounds can be divided into two groups: (a) precursor compounds with R = CF3 or CCl3, which leads to tetrazolo[1,5-a]pyrimidines in high regioselectivity with R at the 7-position of the heterocyclic ring; and (b) precursor compounds with R = aryl or methyl, which leads to a mixture of compounds, tetrazolo[1,5-a] pyrimidines (R in the 5-position of the ring) and 2-azidopyrimidines (R in the 4-position of the ring), which was attributed to an equilibrium of azide–tetrazole. In the solid state, all compounds were found as 2-azidopyrimidines. The regiochemistry of the reaction and the stability of the products are discussed on the basis of the data obtained by density functional theory (DFT) for energetic and molecular orbital (MO) calculations.

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Long-Range ¹H–¹⁵N J Couplings Providing a Method for Direct Studies of the Structure and Azide–Tetrazole Equilibrium in a Series of Azido-1,2,4-triazines and Azidopyrimidines

Cited by 39 publications

References 43 publications

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

¹⁵N-Labelling and structure determination of adamantylated azolo-azines in solution

Synthesis, effect of substituents on the regiochemistry and equilibrium studies of tetrazolo[1,5-a]pyrimidine/2-azidopyrimidines

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Long-Range 1H–15N J Couplings Providing a Method for Direct Studies of the Structure and Azide–Tetrazole Equilibrium in a Series of Azido-1,2,4-triazines and Azidopyrimidines

Cited by 39 publications

References 43 publications

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

15N-Labelling and structure determination of adamantylated azolo-azines in solution

Synthesis, effect of substituents on the regiochemistry and equilibrium studies of tetrazolo[1,5-a]pyrimidine/2-azidopyrimidines

Contact Info

Product

Resources

About

Long-Range ¹H–¹⁵N J Couplings Providing a Method for Direct Studies of the Structure and Azide–Tetrazole Equilibrium in a Series of Azido-1,2,4-triazines and Azidopyrimidines

¹⁵N-Labelling and structure determination of adamantylated azolo-azines in solution