Secondary Amine Functions Research Articles

Diphenylamine: An unusual antioxidant

Diphenylamine (DPA) has been utilized as an antioxidant in studies of lipid peroxidation. Using peroxidizing red blood cell (RBC) membranes, we find that DPA actually promotes lipid hydroperoxide (LOOH) formation and oxygen consumption while markedly inhibiting generation of thiobarbituric acid reactive substances (TBARS). As a consequence, DPA increases the prelytic RBC K leak that results from peroxidative stress and potentiates a known nonprelytic but LOOH-dependent K leak pathway in RBC. In contrast, DPA abolishes formation of cyclooxygenase-dependent conversion products of arachidonase. DPA is almost as efficient as BHT in inhibiting peroxyl radical mediated destruction of phycoerythrin fluorescence. Study of DPA analogues shows that the antioxidant effect of DPA lies in its secondary amine function. Presumably, this results in intermediate formation of a nitrogen-based radical so that redox cycling of this aromatic amine stimulates further peroxidation. This dramatically illustrates the hazard of relying solely on TBARS measurements for assessment of peroxidation.

Stereospecific High-Performance Liquid Chromatographic Assay for the Enantiomers of Phenylpropanolamine in Human Plasma

A stereospecific high-performance liquid chromatographic assay for the enantiomers of phenylpropanolamine (PPA) in human plasma has been developed. The method is based on reaction of extracted PPA with the chiral derivatizing agent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate to form diastereomeric thiourea derivatives. These thiourea derivatives are stable for at least 24 h, readily separable by reversed-phase chromatography, and amenable to ultraviolet detection at 254 nm. Standard curves for each of the PPA enantiomers are linear over the concentration range 10-250 micrograms/L. Over the approximate range of the standard curve, precision (intra- and interday) ranged from 2.2-7.0% while accuracy ranged from 99.2-107.3%. The method has been shown to be free from interference from endogenous plasma constituents and from a range of drugs containing a primary or secondary amine function. Individual PPA enantiomer plasma concentration-time profiles measured for a subject administered racemic PPA are included to demonstrate the utility of the assay.

Precolumn derivatization technique for high-performance liquid chromatographic determination of penicillins with fluorescence detection

A precolumn derivatization method was developed for the high-performance liquid chromatographic (HPLC) determination of penicillins using fluorescence detection. Penicillins were derivatized by a two-step reaction, the β-lactam ring being opened by hydrolysis in aqueous sodium carbonate solution in the first step to give a secondary amine functionality, and the secondary amino group being reacted with 7-fluoro-4-nitrobenzo-2-oxa- 1,3-diazole in the second step to give a fluorescent derivative. The resulting reaction mixture was injected directly onto a reversed-phase column and analysed by HPLC. At a penicillin concentration of 10 μ/ml, the precision (relative standard deviation) ranged from 1.49 to 2.20%. In the concentration range 0.2–100 μ/ml, a linear response was observed. The detection limits of this method were 30–85 ng/ml for five different penicillins at a signal-to-noise ratio of 3:1. The proposed method was applied to the determination of penicillins added to serum following pretreatment by deproteinization and removal of compounds containing amino functionalities with a cation-exchange resin.

Synthesis and evaluation of 3-substituted analogues of 1,2,3,4-tetrahydroisoquinoline as inhibitors of phenylethanolamine N-methyltransferase.

1,2,3,4-Tetrahydroisoquinoline (THIQ) and aryl-substituted derivatives of THIQ are potent inhibitors of the enzyme that catalyzes the formation of epinephrine--phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28). In previous studies, we found that substitution of the 3-position of THIQ with a methyl group resulted in enhanced activity as an inhibitor for 3-methyl-THIQ with respect to THIQ itself. To more fully delineate this region of the PNMT active site, we have synthesized and evaluated other 3-substituted THIQ analogues that vary in both steric and electronic character. Extension of the methyl side chain in 8 by a single methylene unit results in diminished potency for 3-ethyl-THIQ, suggesting that this zone of the active site is spatially compact; furthermore, the region of steric intolerance may be located principally on only "one side" of the 3-position of bound THIQs, since the carbonyl containing (bent) analogues 3-(methoxycarbonyl)-THIQ and 3-(aminocarbonyl)-THIQ are much less capable of forming a strong enzyme-inhibitor dissociable complex compared to straight-chain derivatives possessing a similar steric component. The good activity of 3-(hydroxymethyl)-THIQ as a PNMT inhibitor cannot be explained solely by steric tolerance for this side chain. We believe that an active-site amino acid residue capable of specific (i.e., hydrogen bond) interactions is located in close proximity to the 3-position of bound THIQs and that association of the OH functionality with this active-site residue results in the enhanced in vitro potency of this analogue (Ki = 2.4 microM) compared to that of THIQ (Ki = 10.3 microM). Incorporation of a hydroxymethyl substituent onto the 3-position of the potent PNMT inhibitor 7,8-dichloro-THIQ (SKF 64139, Ki = 0.24 microM) did not result in the same enhancement in inhibitor potency for 17 (Ki = 0.38 microM). This result suggests that simultaneous binding in an optimal orientation of the aromatic halogens, secondary amine, and side-chain hydroxyl functionalities to the PNMT active site is not allowed in this analogue.

Enzymatic oxidative activation and transformation of the antitumor agent mitoxantrone

Ambient temperature incubation of the anticancer agent mitoxantrone with horseradish peroxidase and hydrogen peroxide converts it into a hexahydronaphtho[2,3-f]quinoxaline-7,112-dione in which one side chain has cyclized to the chromophore. The structure of this cyclic metabolite was secured by independent synthesis. This peroxidative conversion of mitoxantrone, the progress of which can be followed spectrophotometrically, is accompanied by formation of a free radical species. The EPR characteristics, and dependence on pH of the latter, suggest it exists as a radical cation. The enzymatic oxidation of mitoxantrone is totally irreversible. The purified cyclic metabolite is a substrate for the peroxidase affording the unstable fully oxidized diimino compound and this reaction is fully reversible upon addition of ascorbate or other biological reductants. Admixture of the fully oxidized diimino product with the reduced cyclic metabolite generates the corresponding radical cation species by disproportionation-comproportionation processes. Independent kinetic studies confirm that reaction of the peroxidase with the cyclic metabolite proceeds more rapidly than with mitoxantrone itself. A derivative of mitoxantrone, in which the side-chain secondary amine functions are acylated, generates a radical cation upon treatment with the peroxidase-H 2O 2 system but does not cyclize subsequently. Derivatives without phenolic hydroxyls or those in which the phenolic hydroxyls are blocked also undergo peroxidative reaction. These observations suggest that initial peroxidative attack occurs at the aromatic nitrogens of mitoxantrone. The possible relevance of these results to the anticancer action of mitoxantrone and the implications for suppression of lipid peroxidation in vivo are discussed.

An infrared spectroscopic investigation of the curing reactions of the EPON 828/meta-phenylenediamine system

Mid- and near-infrared (IR) spectroscopy has been used to study the curing of a bisphenol-A based epoxy resin (EPON-828) with a tetrafunctional curing agent, viz., meta-phenylenediamine (MPDA). Three different cure cycles were used in the study. Primary amine functionality was observed to react relatively rapidly; none remained after curing for 2 h at 75°C. Secondary amine functionality was exhausted in epoxy rich samples subjected to the standard cure cycle (2 h at 75°C followed by 2 h at 125°C). In samples with stoichiometric amount or higher MPDA, complete reaction of secondary amine or epoxy groups was not observed. In amine-rich samples subjected to post curing (6 h at 175°C), evidence was seen for the reaction of hydroxyl and epoxy groups, resulting in a considerable increase in the crosslink density of these samples.

Single- and double-headed analogs of pyridoxamine 5′-phosphate as probes for pyridoxamine 5′-phosphate utilizing enzymes

Single- and double-headed analogs of pyridoxamine 5′-phosphate were synthesized as cleft-depth probes for the catalytic sites of two types of enzymes, viz. those which use pyridoxamine 5′-phosphate as a substrate, i.e., pyridoxamine (pyridoxine) 5′-phosphate oxidase and those which use pyridoxamine 5′-phosphate as coenzyme, i.e., aspartate amino-transferase. These probes were prepared by borohydride reduction of the Schiff's bases formed by the interaction of pyridoxal 5′-phosphate with one or both ends of 1, n-diaminoalkanes (where n = 2, 4, 6, 8, or 12 carbons). Kinetic studies using these analogs as substrates for the oxidase revealed that the single-headed analogs exhibit values ranging from 3 K m to K m for pyridoxamine 5′-phosphate in a curvilinear relationship with chain lengths for n = 2 to 12. The short-chain substrates ( n = 2, 4) exhibit V m values greater than for pyridoxamine 5′-phosphate, whereas the long-chain ones exhibit lesser V m values. These observations may indicate the increasing hydrophobicity of the substrate with increasing chain length and thus poorer turnover. Lineweaver-Burk plots of the double-headed substrates were bilinear for the shorter chains ( n = 2, 4, 6) and linear for the longer chains ( n = 8, 12) over the concentration range of study (1 to 500 μ m). This indicates that the catalytic cleft may impede reorientation for further substrate action of the single-headed amine released from the double-headed substrate. In both cases (>50 μ m), the high K m values may reflect the ionic repulsion forces established by the different secondary amine functionalities, (CH 2) n NH 3 + for single-headed and 5′-phosphopyridoxyl for the double-headed, with active site residues thus effecting binding and ultimate turnover. These same analogs do not act as coenzymes for apo-aspartate aminotransferase or compete with pyridoxamine 5′-phosphate at the micromolar level. They do appear to bind weakly to the enzyme as observed by fluorescence quenching.

Magnetic semi-permeable polyethyleneimine microcapsules for monitoring of N-nitrosation in the gastrointestinal tract.

Semi-permeable polyethyleneimine (PEI) microcapsules were developed recently for trapping unstable electrophilic products in the gastrointestinal tract. Their N-nitrosation has been investigated in vitro and in vivo in the present study. At acid pH N-nitrosation was found to be linearly dependent on nitrite concentration, without a pH maximum. Up to 70% of the nitrosating agent was converted to N-nitroso products that were retained in the microcapsules and detected by a total N-nitroso assay procedure. Microcapsules prepared with different formulations (in order to vary membrane characteristics) provided a limit of detection in the range 1-10 nmol N-nitrosating agent and were also found to have a different capacity for N-nitrosation. Based on n.m.r. data it seems that such N-nitrosation is favoured by the incomplete protonation of this polyamine at acid pH and that nitrosation occurs at the least hindered secondary amine functions. Microcapsules were administered intragastrically in doses of 2 or 6 million to rats also receiving nitrite in the drinking water and were recovered magnetically from faeces. Relative to the maximum possible yield of N-nitrosated microcapsules that could have been excreted within the first 24 h, the excretion (maximum 4.9%) was found to depend on number of capsules administered and the time of administration relative to access to nitrite. Although performed with only a few animals, these preliminary data indicate a performance possibly superior to existing endogenous nitrosation indicators, and a dual purpose system to trap both nitrosating agents and their direct-acting DNA-damaging N-nitroso products.

Poly(2‐imidazolin‐5‐ones)—A new class of heterocyclic polymers

AbstractThe reaction of bisazlactones (2‐oxazolin‐5‐ones) with primary diamines containing additional secondary or tertiary amine functionality (e.g., diethylenetriamine, triethylenetetramine, or N‐methyliminobispropylamine) readily produces polyamides which serve as precursors to a new class of heterocyclic polymers. Thermal cyclodehydration takes place under relatively mild conditions (180–200°C) to produce water‐soluble polymers containing the 2‐imidazolin‐5‐one heterocycle. Model reactions have been studied to verify this mode of cyclization and confirm the proposed polymer structure.

ChemInform Abstract: 1‐AZACYCLOALKYL‐1,4‐BENZODIAZEPIN‐2‐ONES WITH ANTIANXIETY‐ANTIDEPRESSANT ACTIONS

A series of 1-azacycloalkyl-1,4-benzodiazepin-2-ones were synthesized from 1-azacycloalkyl-2-benzoylanilines and corresponding imines and then evaluated for their central nervous system activities. Pharmacological data showed that some of these compounds have potent antidepressant properties, as assessed by their antagonism of tetrabenzine (TBZ) induced ptosis and their inhibition of [3H]norepinephrine uptake into rat brain synaptosomes, as well as their moderate antianxiety properties of preventing of pentylenetetrazol (PTZ) convulsion, suppressing conflict behavior, and displacing potential for [3H]diazepam binding. Introduction of a halogen substituent at position 7 of the 1,4-benzodiazepine ring lengthened the anti-PTZ effects, although the peak effect was slightly reduced and clearly enhanced the anti-PTZ and anticonflict properties. Introduction of Cl to the ortho position of the phenyl ring at position 5 greatly reduced the antidepressant properties. The secondary amine function of the azacyclic ring at position 1 was essential for the production of the antidepressant properties. Of these new series, 7-fluoro-5-(2-fluorophenyl)-1,3-dihydro-1-(4-piperidinyl)-2H-1,4-benzodi azepin-2 -one has the potential to become a useful antidepressant drug with a moderate antianxiety property.

ChemInform Abstract: SYNTHESIS OF POLYAZA MACROCYCLES WITH ONE DIFFERENTIATED SECONDARY AMINE FUNCTION

AbstractDurch die im Formelschema aufgeführte Synthesemethode gelingt es, ausgehend von der Reaktion zwischen den Tritosylaten (1) und den Trimesy1aten(II) die Makrocyclen (IV) zu synthetisieren.

1-Azacycloalkyl-1,4-benzodiazepin-2-ones with antianxiety-antidepressant actions.

A series of 1-azacycloalkyl-1,4-benzodiazepin-2-ones were synthesized from 1-azacycloalkyl-2-benzoylanilines and corresponding imines and then evaluated for their central nervous system activities. Pharmacological data showed that some of these compounds have potent antidepressant properties, as assessed by their antagonism of tetrabenzine (TBZ) induced ptosis and their inhibition of [3H]norepinephrine uptake into rat brain synaptosomes, as well as their moderate antianxiety properties of preventing of pentylenetetrazol (PTZ) convulsion, suppressing conflict behavior, and displacing potential for [3H]diazepam binding. Introduction of a halogen substituent at position 7 of the 1,4-benzodiazepine ring lengthened the anti-PTZ effects, although the peak effect was slightly reduced and clearly enhanced the anti-PTZ and anticonflict properties. Introduction of Cl to the ortho position of the phenyl ring at position 5 greatly reduced the antidepressant properties. The secondary amine function of the azacyclic ring at position 1 was essential for the production of the antidepressant properties. Of these new series, 7-fluoro-5-(2-fluorophenyl)-1,3-dihydro-1-(4-piperidinyl)-2H-1,4-benzodi azepin-2 -one has the potential to become a useful antidepressant drug with a moderate antianxiety property.

GLC Analysis of Loxapine, Amoxapine, and Their Metabolites in Serum and Urine

A GLC analysis is presented for loxapine, amoxapine, and their major metabolites in serum and urine. Electron-capture detection is employed for serum analysis, and flame ionization is used for urine analysis. The procedure includes trifluoroacetylation of secondary amine functions, followed by trimethylsilylation of phenolic groups after ethyl acetate extraction of the sample. Urine requires prior enzymatic hydrolysis of conjugates. Data indicating the utility of the procedure in hospitalized patients and normal volunteers are presented.

On the mechanism of the Hg2+ and base induced hydrolysis reactions of the β2‐(RR, SS)‐Co (trien) (glyOR)Cl2+ions (R = H, C2H5), evidence for the site of deprotonation in the reactive conjugate base

AbstractAcid hydrolysis of the ester function in Δ‐(−)589‐β2‐(RR)‐[Co (trien) (glyOEt) Cl]2+ ((−)‐1) produces optically pure Δ‐(−)589‐(RR)‐[Co (trien) (glyOH)Cl]2+ ((−)‐4). Hg2+ induced removal of chloride in (−)‐4 follows the rate law kobs = kHg [Hg2+] with kHg = (1.36 ± 0.03) × 10−2 M−1s−1, 25°, μ=1.0, and produces optically pure Δ‐(−)589‐β2‐(RR)‐[Co(trien) (glyO)]2+ ((−)‐2). Competition by NO occurs in this reaction ([NO], 1M, 3%) indicating a path whereby external nucleophiles (YNO, H2O) compete with the intramolecular carboxylate function for an intermediate of reduced coordination number. Rapid ring closure to 2 must ensue for Y  H2O. Base hydrolysis of chloride in (±)‐1 produces (±)‐2 together with its diastereoisomer β2‐(RS, SR)‐[Co(trien) (glyO)]2+, ((±)‐3), in which one secondary amine function has an inverted configuration. 2 and 3 incorporate 18O‐labelled solvent into the Co‐O position of the coordinated carboxylate moiety (2: 9.0%; 3: 12.3%) indicating that at least part of the product arises via intramolecular hydrolysis in β2‐hydroxo ethylglycinate intermediates (Fig. 4). Base hydrolysis of (−)‐4 follows the rate law Kobs = kOH[OH−] with kOH = (6.3 ± 0.6) × 10−4M−1 S−1, 25°, μ = 1.0 producing (−)‐2 (37‐45%) and (−)‐3 (63‐55%), the ratio being somewhat medium dependent. Competition by added N (1M) occurs using (±) ‐4 forming β2‐(RR, SS)‐[Co (trien) (glyO)N3]+ (∼2%) and β2‐(RS, SR)‐[Co (trien) (glyO)N3]+ (∼ 13%). Mutarotation at the secondary nitrogen centre is shown to occur after the rate determining loss of Cl− in 1 and 4 and before the formation of 2 and 3. It is concluded that this secondary nitrogen is the site of deprotonation in the reactive conjugate bases of 1 and 4, and possible mechanisms for the mutarotation process are considered.

Structure activity relations for inhibition of neurotransmission in rat vas deferens by adenosine

Adenosine inhibits the isometric contractions of the rat vas deferens in response to field stimulation in vitro by presynaptic inhibition of transmitter release. In the present study the structure activity relations for the inhibition of neurotramsmission in the rat vas deferens by adenosine were examined. Adenosine and adenosine-N 1-oxide were the most potent inhibitors studied. 6-Methylaminopurine riboside, 6-hydroxylaminopurine riboside, 5′-deoxyadenosine and 5′-nitroadenosine were slightly less potent inhibitors. The common structural requirements for activity include a primary or secondary amine function at C 6 of the purine ring with little tolerance for major steric changes or substitutions on the sugar moiety. None of the analogues studied prevented the presynaptic inhibitory action of adenosine.

Degradation of Phenylephrine Hydrochloride in Tablet Formulations Containing Aspirin

The breakdown of aspirin in tablet formulations containing phenylephrine was found to result in a concurrent loss of phenylephrine activity. Specific functional group analysis of the secondary amine function on phenylephrine was necessary to follow degradation in tablet formulations. Analyses with methods based on the phenolic function of phenylephrine did not show similar activity loss. With the use of thin-layer chromatography and comparative chromatograms with synthetic acetylated phenylephrine derivatives, three acetylated phenylephrine degradation products could be identified in tablet formulations. At room temperature the primary degradation pathway was the acetylation of the secondary amine function, but at elevated temperatures, acetylation was found to have progressed to phenylephrine's phenolic and alcoholic groups.

Prediction of Stability in Pharmaceutical Preparations IX: Solution degradation and chemical assay of the antibiotic actinospectacin

The kinetics of degradation of the antibiotic actinospectacin11The trade name of The Upjohn Co. for the antibiotic actinospectacin is Trobicin. in solution have demonstrated that the material is unstable in the alkaline region and that its degradation is catalyzed by buffer anions such as borate, phosphate, and carbonate. Equations have been obtained to characterize degradation as a function of hydroxyl ion concentration and temperature. A chemical assay has been devised based on the formation of the copper complex of a derived dithiocarbamic acid which is extractable and spectrophotometrically measurable in organic solvent. Optimum conditions are described to measure the unique secondary amine functions of the active antibiotic and the change in solubility characteristics of the copper complex of the derived dithiocarbamic acids. Bioassay has been correlated with this chemical assay and optimum conditions defined for its use.