Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ( ⅐ NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO ؊ ). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO 2 ؊ ), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ( ⅐ NO 2 ), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO ؊ , have been reported. Using multiple distinct models of acute inflammation with EPO-and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of heliumswept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate ⅐ NO 2 formation using H 2 O 2 and NO 2 ؊ as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO 2 ؊ -dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO ؊ but not ⅐ NO 2 . Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO 2 ؊ is the one-electron oxidation product, ⅐ NO 2 ; 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO 2 ؊
Two routes for the synthesis of cis-N-protected-3-methylamino-4-methylpiperidine (3) were examined: a route hinging on the
electrochemical oxidation of carbamate 1 to install a ketone at
the 3 position of the piperidine followed by reductive amination
(disconnection A), and a route involving the hydrogenation of
an appropriately functionalized pyridine (disconnection B).
While both routes to the desired compound were ultimately
successful, the pyridine hydrogenation approach proved to be
more amenable to kilogram-scale preparations due to the
crystallinity and purity of intermediates in that route.
The synthesis of the anti-cancer compound 2-methoxy-N-(3-{4-[3-methyl-4-(6-methyl-pyridin-3-yloxy)phenylamino]quinazolin-6-yl}-E-allyl)acetamide (CP-724,714) (1) on multikilogram
scale using several different synthetic routes is described.
Application of the Sonogashira, Suzuki, and Heck couplings to
this synthesis was investigated to identify a safe, environmentally
friendly, and robust process for the production of this drug
candidate. A convergent and selective synthesis of the candidate
was identified which utilizes a Heck coupling of a protected
allylamine to install the critical olefin.
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