Detection and structure elucidation of the new degradation impurities in the pharmaceutical formulations of ruxolitinib hydrobromide.

New degradation impurities at m/z 327.15 and m/z 311.16 using gradient UHPLC method with UV detection and highly selective QDa mass detection were observed during the ruxolitinib hydrobromide (RUX.HBr) : excipient binary mixture degradation study. High mass resolution LC-MS and nuclear magnetic resonance (NMR) techniques were employed to identify and fully characterize the degradation compounds. The degradation impurities were unambiguously identified as (R)-4-amino-6-(1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)pyrimidine-5-carboxylic acid and (R)-3-(4-(6-amino-5-formylpyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile and mechanism of their formation was proposed. It has been confirmed that the degradation products are formed in mixtures of RUX.HBr with some excipients in the presence of oxygen. Based on the forced degradation study, the chemically stable of pharmaceutical formulations were prepared to eliminate the formation of these impurities.

Radical and Nitrenoid Reactivity of 3-Halo-3-phenyldiazirines.

3-Halo-3-phenyl-3H-diazirines (halogen = Br or Cl) undergo a dissociative single-electron transfer from alkyllithiums (RLi) in THF-based solvent mixtures. The resulting 3-phenyldiazirinyl radical, observed by EPR spectroscopy, is eventually transformed to benzonitrile. In Et2O, 2 equiv of RLi add to both nitrogens of halodiazirine N═N bond, affording N,N'-dialkylbenzamidines. The nitrenoid reactivity of some N-alkyl-1H-diazirine intermediates is manifested by their insertion into the α-C-H bond of THF or Et2O.

Evidence for the cyclic CN2 carbene in the gas phase.

3-Halodiazirine-3-carboxylic acids (c-CN2XCOOH, X = Cl or Br) were prepared from their esters and converted to the corresponding sodium salts. Collision-induced dissociation (CID) of the carboxylate ions led exclusively to the loss of CO2 and the resulting c-CN2X(-) ions dissociated to c-CN2 carbene at low energies. The bond dissociation energy (BDE) for c-CN2Br(-) was found to be less than 8 kcal/mol using CID of the anion generated by electrospray ionization of the carboxylate. The analogous difluoro system (CF2XCOOH/CF2X(-)/CF2) exhibits similar dissociative behavior. All experimental BDEs are in very good agreement with MP4/aug-cc-pVTZ calculations.

Evidence for the cyclic CN2 carbene in solution.

Diazirinylidene (c-CN2) is formally the simplest of the N-heterocyclic carbenes. The intermediacy of this elusive species in the fragmentation of butyl 3-bromodiazirine-3-carboxylate (1a) with pent-4-en-1-ols and their sodium alkoxides in DMF is supported by the formation of 2-oxabicyclo[4.1.0]heptanes and dipentenoxymethanes. These products result from an intramolecular [2 + 1] cycloaddition and O-H insertion, respectively, of pentenoxymethylenes suggested to originate from the reaction of the electrophilic c-CN2 with an alkoxide ion. The reaction of 1a with primary or secondary amines in methanol affords the corresponding 3-bromodiazirine-3-carboxamides.

Reactivity of 1-chloro-3-phenyldiazirines.

p-Substituted 1-chloro-3-phenyldiazirines (5), the putative intermediates of the reaction of N,N,N'-trichlorobenzamidines (10) with excess of bromide ions, react further to afford mixtures of 3-bromo- (4) and 3-chloro-3-phenyldiazirines (6). The 6:4 ratios inversely correlate with the Hammett sigma(p) and sigma(p)+ constants of the p-substituents. The formation of 4, proposed to proceed by anti-S(N)2' mechanism, is predominant with electron-withdrawing p-substituents. Compounds 6, the major products with electron-donating p-substituents, may arise from 5 by a [1,3]-sigmatropic shift of chlorine proceeding via polar transition structures 12. The results of a gas-phase DFT (B3LYP/6-31+G*) study on the two mechanisms are consistent with experiment.

Synthesis of carboalkoxychloro- and bromodiazirines.

The first known 3H-diazirines bearing a carbonyl group and a halogen atom on C-3 have been prepared by a novel synthetic method. Carboalkoxychloro- and bromodiazirines 1a-d are formed in up to 45% yields by reductive dechlorination of carboalkoxy-N,N,N'-trichloroformamidines 9a,b using chloride or bromide ion. This method constitutes the first example of the use of N,N,N'-trichloroamidines as starting materials in organic synthesis.