N-hydroxyl Group Research Articles

2-(3-Indolyl)acetamides and their oxazoline analogues: Anticancer SAR study

We previously studied 2-aryl-2-(3-indolyl)acetohydroxamates as potential agents against melanoma. These compounds were ineffective in a mouse melanoma xenograft model, most likely due to unfavorable metabolic properties, specifically due to glucuronidation of the N-hydroxyl of the hydoxamic moiety. In the present work, we prepared a series of analogues, 2-aryl-2-(3-indolyl)acetamides and their oxazoline derivatives, which do not contain the N-hydroxyl group. We investigated the structure–activity relationship in both series of compounds and found that the 2-naphthyl is a preferred group at C-2 of the indole in the amide series, whereas the tetralin moiety is favorable in the same location in the oxazoline series. Overall, three compounds in the amide series have GI50 values as low as 0.2–0.3 µM and the results clearly indicate that the N-hydroxyl group is not necessary for high potency in vitro.

Cell active and functionally-relevant small-molecule agonists of calcitonin receptor.

The natural calcitonin (CT) receptor and its peptide agonists are considered validated targets for drug discovery. A small molecule agonist, SUN-B8155, has previously been shown to efficiently activate cellular CTR. Herein, we report the synthesis of a series of compounds (S8155 1-9) derived from SUN-B8155, and investigate the structural-functional relationship, bias properties and their cellular activity profile. We discover that the N-hydroxyl group from the pyridone ring is required for G protein activity and its affinity to the CT receptor. Among the compounds studied, S8155-7 exhibits improved G protein activity while S8155-4 displays a significant β-arrestin-2 signaling bias. Finally, we show that both S8155-4 and S8155-7 inhibit tumour cell invasion through CTR activation. These two compounds are anticipated to find extensive applications in chemical biology research as well drug development efforts targeting CT receptor.

The Disappearing Director: The Case of Directed N-Arylation via a Removable Hydroxyl Group.

A facile and broadly applicable method for the regiospecific N-arylation of benzotriazoles is reported. Copper-mediated reactions of diverse 1-hydroxy-1H-benzotriazoles with aryl boronic acids lead to 1-aryl-1H-benzotriazole 3-oxides. A N1-OH → N3 prototropy in the 1-hydroxy-1H-benzotriazoles is plausibly the underlying basis, where the tautomer is captured by the boronic acid, leading to C-N (not C-O) bond formation. Because the N-O bond in amine N-oxides and 1-hydroxy-1H-benzotriazoles can be easily reduced by diboron reagents such as (pinB)2 and B2(OH)4, exposure of the 1-aryl-1H-benzotriazole 3-oxides to B2(OH)4 then leads to facile reduction of the N-O bond resulting in diverse, regiospecifically-arylated benzotriazoles. Thus, the N-hydroxyl group in 1-hydroxy-1H-benzotriazoles acts as a disposable arylation director.

Reductive Approach to Nitrones from N-Siloxyamides and N-Hydroxyamides

Abstract This article describes the full details of our reductive approach to nitrones from amides. Reduction of N-siloxyamides with the Schwartz reagent [Cp2ZrHCl], followed by addition of an acid provided functionalized nitrones. The developed conditions were then extended to a catalytic version with the Vaska complex [IrCl(CO)(PPh3)2] and (Me2HSi)2O starting from N-hydroxyamides. 1H NMR studies of the Ir-catalyzed reaction revealed that the developed conditions promoted two different types of catalytic reactions including dehydrosilylation of an N-hydroxyl group and subsequent hydrosilylation of an amide carbonyl. A salient feature of our methods is their high chemoselectivity in the presence of a variety of functional groups. In addition, our reductive methods enabled concise synthesis of cyclic and macrocyclic nitrones, which are known to be challenging compounds to access by conventional methods.

Development of Allosteric Hydrazide-Containing Class I Histone Deacetylase Inhibitors for Use in Acute Myeloid Leukemia.

One of the biggest hurdles yet to be overcome for the continued improvement of histone deacetylase (HDAC) inhibitors is finding alternative motifs equipotent to the classic and ubiquitously used hydroxamic acid. The N-hydroxyl group of this motif is highly subject to sulfation/glucoronidation-based inactivation in humans; compounds containing this motif require much higher dosing in clinic to achieve therapeutic concentrations. With the goal of developing a second generation of HDAC inhibitors lacking this hydroxamate, we designed a series of potent and selective class I HDAC inhibitors using a hydrazide motif. These inhibitors are impervious to glucuronidation and demonstrate allosteric inhibition. In vitro and ex vivo characterization of our lead analogues' efficacy, selectivity, and toxicity profiles demonstrate that they possess low nanomolar activity against models of acute myeloid leukemia (AML) and are at least 100-fold more selective for AML than solid immortalized cells such as HEK293 or human peripheral blood mononuclear cells.

DFT Study on the Radical Anions Formed by Primaquine and Its Derivatives

The electron affinities (EA) of the 8-aminoquinoline antimalarial drug primaquine and several of its metabolites were studied using the density functional theory method. We first considered six substituents at the 5-position, -CH(3), -OH, -OCH(3), -Ph, -OPh, and -CHO. We found that in the gas phase the adiabatic EAs are similar to that of the parent primaquine for the -CH(3), -OH, and -OCH(3) substituents. In contrast, the -Ph, -OPh, and -CHO substituents all markedly increase the adiabatic EA. However, only the -CHO substituted compound is predicted to form a stable covalently bound radical anion in the gas phase due to its significant positive vertical EA relative to that of the parent primaquine. In addition, when the 8-position is substituted by the N-hydroxyl group or a quinone-imine structure is formed, the electron capture ability is significantly increased. In aqueous solution, all these molecules have significantly larger adiabatic EAs than in the gas phase. In addition, all of the vertical EAs are positive in aqueous solution. The implications of these findings for contributing to our mechanistic understanding of the red cell toxicity of 8-aminoquinoline compounds are further discussed.

Structure-based design and synthesis of N(omega)-nitro-L-arginine-containing peptidomimetics as selective inhibitors of neuronal nitric oxide synthase. Displacement of the heme structural water.

The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N'-nitroguanidine (1) and 4-N-(Nomega-nitro-l-argininyl)-trans-4-amino-l-proline amide (2), led to the discovery of a conserved structural water molecule that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). On the basis of this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of 2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues 3-5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized. X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety (Figures 5 and 6). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1). Rationalizations for the small increase in potency are consistent with other changes in the crystal structures.

Elusive Forms and Structures of N-Hydroxyphthalimide: The Colorless and Yellow Crystal Forms of N-Hydroxyphthalimide

A comprehensive structural characterization of the colorless and yellow forms of N-hydroxyphthalimide (NHP), the deuterated form (NDP), and the ethoxylated form (ethoxy-NHP) has been carried out using single-crystal X-ray diffraction, FTIR and Raman spectroscopies, and scanning electron microscopy. Both NHP and NDP forms crystallize in the monoclinic space group (P21/c, No. 14). The various forms of NHP differ in the way in which the molecules adjoin one another through their N-hydroxyl groups and how the carbonyls of the isoindole-1,3-dione ring differ through intermolecular hydrogen bonding. Although the hydrogen bonding about the b axis is virtually the same, the isoindole-1,3-dione ring experiences different twists for the two NHP forms. Both the colorless and yellow forms of NHP exhibit strong intermolecular hydrogen bonding between O(3) and H(1). In the yellow form, the N-hydroxyl group is significantly out of the plane (approximately 1.19 degrees ), but the N-hydroxyl group in the colorless form is only approximately 0.06 degrees out of the plane. Both forms of NHP reveal an infinite chain of intermolecular hydrogen-bonded molecules in the direction of the b axis; however, the molecules are ordered differently within the unit cells. The hydrogen-bond geometry for the yellow form of NHP is O(2)-H(1)...O(3), with an angle of 185 degrees , intermolecular distances of O(2)...O(3) = 2.68 A and H(1)...O(3) = 1.70 A, and an intramolecular hydrogen bond of O(1)...H(1) = 1.17 A. The colorless form of NHP shows an intermolecular hydrogen-bond geometry between O(3) and H(1) with a distance of 1.78 A; the O(2)-O(3) distance is 2.71 A. The O(2)-H(1)...O(3) angle is 159 degrees, and the intramolecular distance is O(1)...H(1) = 0.97 A. The N-ethoxy derivative of NHP crystallizes in an orthorhombic space group (Pnma, No. 62) and exhibits no hydrogen bonding, displaying a strong head-to-tail stacking of the planar rings along the needle axis direction.

Synthesis of Purpurin Imide Derivatives by Modification of Peripheral Functional Groups of Methyl Pheophorbide-a

A series of novel purpurin-18 imide derivatives exhibiting long wavelength absorption and amphiphilicity were obtained from methyl pheophorbide-a (MPa) by modification of the peripheral functional groups. The vinyl group at 3-position was oxidized with OsO4 and NaIO4 to form the formyl group and the Grignard reaction of this aldehyde with the alkyl magnesium bromide was carried out to give the corresponding 3-(1-hydroxylalkyl) pheophorbide-a. The E-ring of these chlorines was converted into anhydride ring to give purpurin derivatives by air oxidation. The trans-32-alkyl purpurin derivatives were obtained by dehydration of hydroxyl group at 31-position. The N-hydroxyl purpurin imide was generated by treatment of the anhydride ring with hydroxylamine hydrochloride. The 31-alkylacyl-N-hydroxyl purpurin imides were obtained by oxidation of hydroxyl group at 31-position. The acylation of N-hydroxyl group was completed to afford N-acyloxy purpurin imides. The photocytoxicity of several compounds in vitro were tested.

The Relative and Absolute Configuration of PF1140.

A potentially general approach for elucidating the absolute configuration of N-hydroxypyridone antibiotics has been developed. One member of this family of antibiotics, PF1140, was efficiently purified from a crude fungal extract following allylation of its N-hydroxyl group. Removal of the resultant allyl group permitted regeneration of the N-hydroxyl group as well as conversion into the corresponding pyridone derivative. The stereochemistry of PF1140 including the absolute configuration was established by X-ray crystallographic analysis of the S-2-methoxy-2-(1-naphthyl)propionic ester derivative.

Design and synthesis of phthalimide-type histone deacetylase inhibitors

Several hydroxamic acid derivatives with a substituted phthalimide group as a linker and/or cap structure, prepared during structural development studies based on thalidomide, were found to have histone deacetylase (HDAC)-inhibitory activity. Structure–activity relationship studies indicated that nature of the substituent introduced at the phthalimide nitrogen atom, introduction of a hydroxamic acid structure, and distance between the N-hydroxyl group and the cap structure are important for HDAC-inhibitory activity.

The Relative and Absolute Configuration of PF1140

A potentially general approach for elucidating the absolute configuration of N-hydroxypyridone antibiotics has been developed. One member of this family of antibiotics, PF1140, was efficiently purified from a crude fungal extract following allylation of its N-hydroxyl group. Removal of the resultant allyl group permitted regeneration of the N-hydroxyl group as well as conversion into the corresponding pyridone derivative. The stereochemistry of PF1140 including the absolute configuration was established by X-ray crystallographic analysis of the S-2-methoxy-2-(1-naphthyl)propionic ester derivative.

Heterologous expression of human N-acetyltransferases 1 and 2 and sulfotransferase 1A1 in Salmonella typhimurium for mutagenicity testing of heterocyclic amines

A variety of carcinogenic heterocylic amines (HAs) are found in cooked food. They can be metabolised to reactive intermediates via N-hydroxylation catalysed by cytochrome P450 1A2, followed by conjugation of the resulting N-hydroxyl group by N-acetyltransferase (NAT) or sulfotransferase (SULT). In order to compare the role of O-acetylation and O-sulfonation by human enzymes in the activation of HAs, we have introduced the cDNAs for wild-type forms of human NAT1, NAT2 and SULT1A1 in the acetyltransferase-deficient Salmonella typhimurium strain TA1538/1,8-DNP. Functional expression of recombinant proteins was demonstrated using immunoblot analysis and determination of enzyme activity with characteristic substrates. The established strains were used to study the mutagenicity of the N-hydroxy derivatives of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). The results demonstrate that N-hydroxy-HAs are activated by different human enzymes. At the concentrations used in the mutagenicity assay, N-hydroxy-IQ was activated by human NAT2, but not by NAT1 or SULT1A1. In contrast, N-hydroxy-PhIP was activated specifically by human SULT1A1, but not by NAT1 or NAT2. Therefore, both O-acetylation and O-sulfonation by human enzymes have to be regarded as important determinants for HA genotoxicity in humans.

Studies of RNA Cleavage by Photolysis of N-Hydroxypyridine-2(1H)-thione. A New Photochemical Footprinting Method

N-Hydroxypyridine-2(1H)-thione (N-HPT) has been studied as a photochemical source of hydroxyl radicals for use in photoinitiated nucleic acid footprinting experiments. Steady-state photolysis of dilute aqueous solutions of N-HPT at 350 nm in the presence of a 385 nucleotide (32)P-labeled RNA, the Tetrahymena L-21 ribozyme, resulted in cleavage of the RNA at nucleotide resolution. No cleavage of the RNA occurred in the absence of light or in the absence of N-HPT. Photolysis of the analogous pyridine lacking the N-hydroxyl group did not result in detectable amounts of RNA cleavage. The addition of RNA to preirradiated solutions of N-HPT gave no apparent RNA cleavage products, suggesting that the photoproducts of N-HPT do not result in RNA modification. Cleavage of RNA, upon photolysis in the presence of N-HPT, occurred in a sequence-independent fashion with double-stranded RNA being cleaved as efficiently as single-stranded RNA. Based on these observations, we conclude that photochemically generated diffusable hydroxyl radicals are responsible for the RNA cleavage. Experiments involving the photolysis of N-HPT in the presence of the Tetrahymena ribozyme and magnesium showed a magnesium-dependent protection from RNA cleavage due to formation of a folded RNA tertiary structure. The locations and amount of protection were identical to those observed in footprinting experiments performed with other hydroxyl radical sources. The presence of N-HPT had no effect on either the rate of folding or the catalytic activity of the folded RNA, indicating that this reagent does not disrupt RNA tertiary structure or otherwise affect activity. Thus, N-HPT is established as a new reagent for use in photoinitiated RNA footprinting experiments.

The structure of the Aeromonas proteolytica aminopeptidase complexed with a hydroxamate inhibitor. Involvement in catalysis of Glu151 and two zinc ions of the co-catalytic unit.

The structure of the complex of Aeromonas proteolytica aminopeptidase, a two-zinc exopeptidase, with the inhibitor p-iodo-D-phenylalanine hydroxamate has been determined by X-ray crystallography. Refinement of the structure, which includes 220 water molecules, using data at 0.80-0.23-nm resolution resulted in a crystallographic residual R value of 16%. The hydroxamate group adopts a planar conformation whereby the two oxygen atoms interact with the zinc ions. The N-hydroxyl group of the inhibitor is located between the two zinc ions, a position which is close to that occupied by a water molecule in the native structure. The carbonyl oxygen of the inhibitor binds to Zn1, which becomes pentacoordinated while Zn2 remains tetracoordinated, in contrast to the native protein where both zinc ions were shown to be tetracoordinated and structurally equivalent. Interactions of the carboxylate oxygens of Glu151 with the hydroxamate group play an important role in the stabilization of the complex.

Conformational perturbations induced by N-amination and N-hydroxylation of peptides

Amination and hydroxylation of the amide nitrogen in a peptide chain have little influence on the local geometry, but both affect the hydrogen-bonding network, and therefore the conformational properties of the modified peptide. An experimental study in solution (IR spectroscopy and 1 H-NMR) and in the solid state (X-ray diffraction) has been carried out on the N-amino and N-hydroxy analogues of the two RCO-Pro-NHMe and RCO-Pro-Gly-NHiPr peptides known to adopt preferentially the γ- and β-turn structures, respectively. The N-amino group is a weak proton donor which does not interact significantly with the peptide chain. On the contrary, the N-hydroxyl group is a strong proton donor giving close contacts with the peptide carbonyls

Maize Microsomal Benzoxazinone N-Monooxygenase

The benzoxazinones occur in hydroxamic acid and lactam forms in maize (Zea mays L.) tissue. The hydroxamic acid forms which possess a N-hydroxyl group are found in the highest concentration while the lactam members which lack the N-hydroxyl group occur in lower concentrations. The hydroxamic acid 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) has as its lactam counterpart 2-hydroxy-1,4-benzoxazin-3-one (HBOA). An enzyme has been identified in maize microsomal preparations which catalyzes the N-hydroxylation of HBOA to form DIBOA. The enzyme is initially observed in seedlings 2 days after imbibition which coincides with the onset of hydroxamic acid accumulation. The enzyme requires NADPH and is inhibited by sulfhydryl reagents, NADP, cytochrome c, cations, carbon monoxide, and nitrogen gas. The effect of nitrogen can be reversed by exposing the enzyme to air, while the effect of carbon monoxide can be reversed by exposing the enzyme to 450 nanometer light during the incubation period. The apparent K(m) values for HBOA and NADPH are 13 and 5 micromolar, respectively. The pH optimum is 7.5 and the temperature optimum for the enzyme is 35 degrees C. A 450 nanometer absorbance peak is observed when reduced microsomal preparations are exposed to carbon monoxide which in combination with other data presented supports the hypothesis that the enzyme is a cytochrome P-450 dependent N-monooxygenase.

Biogenesis of the Carbon Skeleton of Glycerinopyrin: A New Biosynthetic Pathway for Pyrroles

Ring closure of the amino acid leucine 1 and its esterification with glycerol 2 embodies a novel biogenesis of the naturally occurring glycerinopyrin 3. Such a methyl-substituted fivemembered nitrogen heterocycle occurs otherwise in nature only without the N-hydroxyl group in an ant phermone and might be formed in this way. The novel biosynthetic pathway to the pyrroles, the “Pigments of Life”, is of great biochemical interest.

Reaction of 1-hydroxy-5-phenyltetrazole with olefin oxides

1. 1-Hydroxy-5-phenyltetrazole adds to ethylene oxide and propylene oxide to give the products of O- and N-β-oxyalkylation in 2∶1 ratio. 2. Propylene oxide is opened by 1-hydroxy-5-phenyltetrazole predominantly at the sterically most available carbon atom; this direction is favored by a 3∶1 ratio. 3. The product of the O-β-oxyalkylation of 1-hydroxy-5-phenyltetrazole upon treatment with a strong base regenerates the initial N-hydroxytetrazole and, thus, the O-β-hydroxyalkyl-ation of N-hydroxytetrazoles may serve to protect the N-hydroxyl group in these compounds.

Transport of the iron ionophore ferrichrome inEscherichia coliK-12 andSalmonella typhimuriumLT2

Iron(III) is actively transported as a ferrichrome complex into the cells of Escherichia coli and Salmonella typhimurium [1,2]. We have shown that in E. coli the ligand is modified and excreted as the iron-free form into the medium [3]. The modified ligand could be converted back into ferrichrome by mild acid hydrolysis. 1 mol of acetate was released per mol of ferrichrome; therefore it was proposed that an N-hydroxyl group, one of the six binding sites of iron(Ill) in ferrichrome [4], was acetylated. This could explain the low affinity of the modified ligand for iron(III). It was likely that reduction before modification took place. We suggested that the inactivation of ferrichrome prevented the intracellular withdrawal of iron by the ligand from sites of synthesis and function of the many iron containing proteins. We now describe a cell-free system that modifies ferrichrome in the same way as transporting cells. Reduction was a prerequisite for modification to occur. Both reduction and modification were membrane-bound processes. It is therefore likely that ferrichrome does not have to enter the cytoplasm in order to function as an iron(III) ionophore. To test this conclusion, we converted an analogue of ferri-