Hydrolysis of the quinone methide of butylated hydroxytoluene in aqueous solutions.

Butylated hydroxytoluene or BHT is an antioxidant commonly used in pharmaceutical formulations. BHT upon oxidation forms a quinone methide (QM). QM is a highly reactive electrophilic species that can undergo nucleophilic addition. Here, the kinetic reactivity of QM with water at various apparent pH values in a 50% (v/v) water-acetonitrile solution at constant ionic strength of I = 0.5 (NaCl)4 , was studied. The hydrolysis of QM in the presence of added acid, base, sodium chloride, and phosphate buffer resulted in the formation of only one product--the corresponding 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (BA). The rate of BA formation was catalyzed by the addition of acid and base, but not chloride and phosphate species. Nucleophilic excipients, used in the pharmaceutical formulation, or nucleophilic groups on active pharmaceutical ingredient molecule may form adducts with QM, the immediate oxidative product of BHT degradation, thus having implications for drug product impurity profiles. Because of these considerations, BHT should be used with caution in formulations containing drugs or excipients capable of acting as nucleophiles.

Binding energy and catalysis by D-xylose isomerase: kinetic, product, and X-ray crystallographic analysis of enzyme-catalyzed isomerization of (R)-glyceraldehyde.

D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and d-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D(2)O was monitored by (1)H nuclear magnetic resonance spectroscopy, and a k(cat)/K(m) of 0.034 M(-1) s(-1) was determined for this isomerization at pD 7.0. This is similar to the k(cat)/K(m) of 0.017 M(-1) s(-1) for the TIM-catalyzed carbon deprotonation reaction of DGA in D(2)O at pD 7.0 [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (k(cat)/K(m) = 490 M(-1) s(-1)) versus that for the TIM-catalyzed isomerization of DGAP (k(cat)/K(m) = 9.6 × 10(6) M(-1) s(-1)) is due to (i) the barrier to conversion of cyclic d-xylose to the reactive linear sugar (5.4 kcal/mol) being larger than that for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol) and (ii) the intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219-410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) being smaller than that of the phosphodianion group of DGAP (~12 kcal/mol). The XI-catalyzed isomerization of DGA in D(2)O at pD 7.0 gives a 90% yield of [1-(1)H]DHA and a 10% yield of [1-(2)H]DHA, the product of isomerization with incorporation of deuterium from solvent D(2)O. By comparison, the transfer of (3)H from the labeled hexose substrate to solvent is observed only once in every 10(9) turnovers for the XI-catalyzed isomerization of [2-(3)H]glucose in H(2)O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994) Biochemistry 33, 1481-1487]. We propose that truncation of the terminal ethylene glycol fragment of d-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that for proton transfer. An ultra-high-resolution (0.97 Å) X-ray crystal structure was determined for the complex obtained by soaking crystals of XI with 50 mM DGA. The triose binds to XI as the unreactive hydrate, but ligand binding induces metal cofactor movement and conformational changes in active site residues similar to those observed for XI·sugar complexes.

Discovery of amide replacements that improve activity and metabolic stability of a bis-amide smoothened antagonist hit.

A bis-amide antagonist of Smoothened, a seven-transmembrane receptor in the Hedgehog signaling pathway, was discovered via high throughput screening. In vitro and in vivo experiments demonstrated that the bis-amide was susceptible to N-acyl transferase mediated amide scission. Several bioisosteric replacements of the labile amide that maintained in vitro potency were identified and shown to be metabolically stable in vitro and in vivo.

Addressing PXR liabilities of phthalazine-based hedgehog/smoothened antagonists using novel pyridopyridazines.

Pyridopyridazine antagonists of the hedgehog signaling pathway are described. Designed to optimize our previously described phthalazine smoothened antagonists, a representative compound eliminates a PXR liability while retaining potency and in vitro metabolic stability. Moreover, the compound has improved efficacy in a hedgehog/smoothened signaling mouse pharmacodynamic model.

Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists.

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.

Kinetics and mechanism of the hydrolytic degradation of indinavir: intramolecular catalysis.

The pH-rate profile of first-order rate constants for the lactonization of Indinavir in aqueous solutions with ionic strength I = 1 (KCl) at 40 degrees C is reported. The lactonization reaction is a subject of strong buffer catalysis with a nonlinear dependence of the first-order rate constants on the concentration of the buffer. The pH-rate profile is more complex than the pH-rate profiles for the hydrolysis of simple peptides and for the intramolecular OH-catalyzed hydrolysis of gamma-hydroxyamides. This complexity appears unique to Indinavir and is a result of the cis-aminoindanol leaving group. The mechanistic pathways for the lactonization are discussed. The buffer catalysis data are consistent with kinetic general acid catalysis. The second-order rate constant for the specific-acid catalyzed hydrolysis of Indinavir at 40 degrees C (k(H) = 2.2 x 10(-4) M(-1) min(-1)) is similar to that for a simple peptide indicating similar interactions in the rate limiting transition state for both reactions.

Structure-Reactivity Relationships for ß-Galactosidase (Escherichia coli, lac Z): A Second Derivative Effect on ß(nuc) for Addition of Alkyl Alcohols to an Oxocarbenium Ion Reaction Intermediate.

Velocities for the synthesis of trifluoroethyl 2-deoxy-ß-D-galactopyranoside by transfer of the 2-deoxygalactosyl group from ß-galactosidase to trifluoroethanol were determined from studies of the ß-galactosidase-catalyzed cleavage of 4-nitrophenyl-2-deoxy-ß-D-galactopyranoside as the difference in rates of appearance of 4-nitrophenoxide anion and 2-D-deoxygalactose. These data were used to calculate a rate constant ratio of k(ROH)/k(s) = 2.3 M(-1) for partitioning of the intermediate between addition of trifluoroethanol and solvent water. Velocities for the synthesis of other alkyl 2-deoxy-ß-D-galactopyranosides by transfer of the 2-deoxygalactosyl group from ß-galactosidase to alkyl alcohols were determined from the effect of alkyl alcohols on the velocity of ß-galactosidase-catalyzed cleavage of 4-nitrophenyl-2-deoxy-ß-D-galactopyranoside in a reaction where breakdown of the intermediate is rate determining. These data were used to calculate rate constant ratios k(ROH)/k(s) for the reactions of eight alkyl alcohols. Absolute rate constants k(ROH) (M(-1) s(-1)) were calculated from k(ROH)/k(s) and k(s) = 0.002 s(-1) for the addition of water. A Brønsted coefficient of ß(nuc) = -0.07 ± 0.08 was determined as the slope of a logarithmic correlation of k(ROH) against alcohol pK(a). The change from a 2-OH to a 2-H substituent at the ß-D-galactopyranosyl intermediate causes a 0.12 ± 0.04 increase in the value of ß(nuc) for alcohol addition. This anti-Hammond effect provides evidence that general basecatalyzed addition of alcohols to an enzyme bound ß-D-galactopyranosyl oxocarbenium ion intermediate proceeds along a reaction coordinate in which there is strong coupling between carbon-oxygen bond formation and proton transfer from the alcohol to a basic residue at the enzyme.

Ground-state, transition-state, and metal-cation effects of the 2-hydroxyl group on beta-D-galactopyranosyl transfer catalyzed by beta-galactosidase (Escherichia coli, lac Z).

Substitution of the C2-OH group by C2-H at 4-nitrophenyl-beta-d-galactopyranoside to give 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside causes (1) a change in the rate-determining step for beta-galactosidase-catalyzed sugar hydrolysis from formation to breakdown of a covalent intermediate; (2) a 14 000-fold decrease in the second-order rate constant k(3)/K(d) for enzyme-catalyzed transfer of the beta-d-galactopyranosyl group from the substrate to form a covalent adduct to the enzyme; and (3) a larger 320 000-fold decrease in the first-order rate constant k(s) for hydrolysis of this covalent adduct. Only a small fraction (ca. 7%) of the 2-OH substituent effect is expressed in the ground-state Michaelis complex, so that the (apparent) strong interactions between the enzyme and 2-OH group that stabilize the transition state for beta-d-galactopyranosyl transfer only develop upon moving from the Michaelis complex to the transition state. Mg(2+) activates beta-galactosidase for cleavage of both 4-nitrophenyl-beta-d-galactopyranoside and 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside. This suggests that Mg(2+) activation does not involve interactions with the 2-OH group. The removal of Mg(2+) from beta-galactosidase causes a change in the rate-determining step for enzyme-catalyzed hydrolysis of 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside from breakdown to formation of the covalent intermediate. The observed 2-OH effect would require a very large (10-11 kcal/mol) stabilization of the transition state for beta-d-galactopyranosyl group transfer to water by interactions between beta-galactosidase and the neutral 2-OH group. We suggest that the apparent effect of the neutral substituent is more simply rationalized by ionization of the 2-OH to form a 2-O(-) anion, which provides effective electrostatic stabilization of the cationic transition state for glycoside cleavage at an active site of relatively low dielectric constant.

Scrambling of oxygen-18 during the "borderline" solvolysis of 1-(3-nitrophenyl)ethyl tosylate.

[reaction: see text] There is substantial isomerization (kiso=0.32 x 10(-3) s(-1)) of 3-NO2C6H4(13)CH(Me)OS(18O)2Tos during solvolysis (ksolv=1.04 x 10(-3) s(-1)) in 50/50 trifluoroethanol/water, even though the estimated lifetime of the putative 1-(3-nitrophenyl)ethyl carbocation intermediate of solvolysis (ca. 10(-13) s(-1)) is too short to allow rearrangement that exchanges the positions of 16O and 18O at the sulfonate leaving group. This suggests that isomerization proceeds by a mechanism that avoids formation of the carbocation-anion pair intermediate.

Kinetic and thermodynamic barriers to carbon and oxygen alkylation of phenol and phenoxide ion by the 1-(4-methoxyphenyl)ethyl carbocation.

Rate constant ratios for addition of the three nucleophilic sites of phenol to the 1-(4-methoxyphenyl)ethyl carbocation (1+) in 50/50 (v/v) trifluoroethanol/water were determined from the relative yields of the three phenol adducts, and absolute rate constants were determined from product rate constant ratios for addition of phenol and azide ion to 1+ using k(az) = 5 x 10(9) M(-1) s(-1) for the diffusion-limited reaction of azide ion. A selectivity of 230:20:1 was determined for alkylation of phenol at oxygen, C-4 and C-2 to form 1-OPh and biphenyls 1-(4-C6H4OH) and 1-(2-C6H4OH), respectively, and of 2:2:1 for alkylation of the corresponding nucleophilic sites of phenoxide ion in diffusion-limited reactions. The Mayr nucleophilicity parameter for C-4 of phenol is N = 2.0. Encounter-limited addition of phenoxide ion to 1+ to form 1-OPh is faster than encounter-limited addition of oxygen anions that are either more or less basic than phenoxide ion. Only the products of solvolysis are observed from acid-catalyzed cleavage of 1-OPh in 50/50 (v/v) trifluoroethanol/water, but a 50% yield of biphenyls 1-(4-C6H4OH) and 1-(2-C6H4OH) are observed from spontaneous cleavage of 1-OPh, where the leaving group is phenoxide ion, because of the very low kinetic barriers to collapse of the ion pair intermediate 1+.PhO-. The 230-fold larger rate constant for O-compared to C-2-alkylation of phenol is due primarily to the larger thermodynamic driving force for oxygen addition. There are similar Marcus intrinsic barriers for these two reactions.

Substituent effects on carbocation stability: the pK(R) for p-quinone methide.

A value of k(H) = 1.5 x 10(-)(3) M(-)(1) s(-)(1) has been determined for the generation of simple p-quinone methide by the acid-catalyzed cleavage of 4-hydroxybenzyl alcohol in water at 25 degrees C and I = 1.0 (NaClO(4)). This was combined with k(s) = 5.8 x 10(6) s(-)(1) for the reverse addition of solvent water to the 4-hydroxybenzyl carbocation [J. Am. Chem. Soc. 2002, 124, 6349-6356] to give pK(R) = -9.6 as the Lewis acidity constant of O-protonated p-quinone methide. Values of pK(R) = 2.3 for the Lewis acidity constant of neutral p-quinone methide and pK(add) = -7.6 for the overall addition of solvent water to p-quinone methide to form 4-hydroxybenzyl alcohol are also reported. The thermodynamic driving force for transfer of the elements of water from formaldehyde hydrate to p-quinone methide to form formaldehyde and p-(hydroxymethyl)phenol (4-hydroxybenzyl alcohol) is determined as 6 kcal/mol. This relatively small driving force represents the balance between the much stronger chemical bonds to oxygen at the reactant formaldehyde hydrate than at the product p-(hydroxymethyl)phenol and the large stabilization of product arising from the aromatization that accompanies solvent addition to p-quinone methide. The Marcus intrinsic barrier for nucleophilic addition of solvent water to the "extended" carbonyl group at p-quinone methide is estimated to be 4.5 kcal/mol larger than that for the addition of water to the simple carbonyl group of formaldehyde. O-Alkylation of p-quinone methide to give the 4-methoxybenzyl carbocation and of formaldehyde to give a simple oxocarbenium ion results in very little change in the relative Marcus intrinsic barriers for the addition of solvent water to these electrophiles.

Hydrogen bonding and catalysis of solvolysis of 4-methoxybenzyl fluoride.

Values of k(o) = 8.0 x 10(-3) s(-1) and k(H) = 2.5 x 10(-2) M(-1) s(-1), respectively, were determined for the spontaneous and the acid-catalyzed cleavage of 4-methoxybenzyl fluoride (1-F) to form the 4-methoxybenzyl carbocation (1+). Values of k(F) = 1.8 x 10(7) M(-1) s(-1) and k(HF) = 7.2 x 10(4) M(-1) s(-1) were determined for addition of F- and HF to 1+ for reaction in the microscopic reverse direction. Evidence is presented that the reversible addition of HF to 1+ to give 1-F + H+ proceeds by a concerted reaction mechanism. The relatively small 250-fold difference between the reactivities of fluoride ion and neutral HF toward 1+ is attributed to the tendency of the strong aqueous solvation of F- to decrease its nucleophilic reactivity and to the advantage for the concerted compared with the usual stepwise pathway for addition of HF. There is no significant stabilization of the transition state for cleavage of 1-F from general acid catalysis by 0.80 M cyanoacetate buffer at pH 1.7. The estimated 3 kcal/mol larger Marcus intrinsic barrier for heterolytic cleavage of 1-F than for cleavage of 1-Cl is attributed to a lag in the development at the transition state of the ca. 30 kcal/mol greater stabilizing solvation of the product ion F- compared with Cl-. The decrease in the electronegativity of X along the series X = F, OH, Cl is accompanied by a ca. 10(10)-fold increase in the carbon basicity compared with the proton basicity of X-.