Separation and structural analysis of vinyl- and isovinyl-azobilirubin derivatives

The coupling reaction of bilirubin with the diazonium salts of ethyl anthranilate or of aniline yields two isomeric azopigments. These can be separated by t.l.c. as their methyl esters. The mass spectra of each pair of azopigments are very similar, showing that they are isomers. Proton-magnetic-resonance spectrometric studies show that they differ in the positions of the substituents on the pyrrolenone end ring; in one compound the methyl and vinyl groups are interposed compared with the other compound. These azo compounds were used as reference standards for determination of the site of conjugation in bilirubin monoglucuronide prepared enzymically. Analysis showed that conjugation occurs at the carboxyethyl side chain of both sides of the bilirubin molecule. During the preparation of the ethyl anthranilate reference compounds a series of minor azopigments were isolated by t.l.c. Analysis of the mass spectra of many of these showed that three side reactions can occur: (1) methylation of the imide carbonyl group; (2) addition of methanol or water to the vinyl substituent; (3) transmethylation of the ethoxycarbonyl group.     

Heterogeneity of bile pigments formed by the breakdown of haemoglobin in the isolated male-dog kidney.

Normothermic perfused isolated male-dog kidneys formed radioactive bile pigments by the breakdown of radioactive haemoglobin prepared from [2-14C]glycin. After column chromatographic separation and preparation of dipyrrolic azopigments, 86.3 +/- 2.2% of the bile pigments seemed to be conjugated bilirubin. Thin-layer chromatographic separation of the azopigments of ethyl anthranilate revealed a good correlation between photometric scanning, radiochromatographic scanning and the radioactivity of the azopigments scraped off the thin-layer glass plates and counted in a liquid scintillation counter. Although the same heterogenity of the azopigments was observed as in dog bile, the isolated male-dog kidney formed significantly less alpha2- and significantly more gamma-fractions.     

Glucuronic acid conjugates of bilirubin-IXα in normal bile compared with post-obstructive bile. Transformation of the 1-O-acylglucuronide into 2-, 3-, and 4-O-acylglucuronides

Structures have been determined for bilirubin-IXalpha conjugates in freshly collected bile of normal rats, dogs and man and in post-obstructive bile of man and rats. The originally secreted conjugate has been characterized as azopigment (I), i.e. a 1-O-acyl-beta-d-glucopyranuronic acid glycoside. Conversion of the acetylated methyl ester of azopigment (I) into methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-beta-d-glucopyranuronate (V) indicates the pyranose ring structure for the carbohydrate and a C-1 attachment for the bilirubin-IXalpha acyl group. Alternative procedures for deconjugation of azopigment (I) and its derivatives are also described. In post-obstructive bile, the 1-O-acylglucuronide is converted into 2-, 3- and 4-O-acylglucuronides via sequential intramolecular migrations of the bilirubin acyl group. The following approach was utilized. (1) The tetrapyrrole conjugates were cleaved to dipyrrolic aniline and ethyl anthranilate azopigments, and the azopigments were separated as the acids or methyl esters. (2) The isomeric methyl esters were characterized by mass spectral analysis of the acetates and silyl ethers. (3) The free glycosidic function was demonstrated by 1-oxime and 1-methoxime derivative formation. (4) The position of the dipyrrolic O-acyl group was determined for the methyl esters by protecting the free hydroxyl groups of the glucuronic acid moieties as the acetals formed with ethyl vinyl ether and by further conversion of the carbohydrates into partially methylated alditol acetates. These were analysed by using g.l.c.-mass spectrometry. The relevance of the present results with regard to previous reports on disaccharidic conjugates is discussed. Details of procedures for the formation of chemical derivatives for g.l.c. and mass spectrometry have been deposited as Supplementary Publication SUP 50081 (15 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5.     

Heterogeneity of bile pigment conjugates as revealed by chromatography of their anthranilate azopigments

1. Azopigments derived from conjugated bile pigments by coupling with the diazonium salt of ethyl anthranilate are analysed conveniently by quantitative t.l.c. or by column chromatography on CM-cellulose. 2. By chromatographic studies combined with a series of chemical tests six groups of azopigments were demonstrable in preparations from bile and from icteric urine of man. Azobilirubin and its β-d-monoglucuronide have hitherto been considered to be the only major derivatives that can be obtained from human bile pigments. In the present work, other azopigments accounted for 30–40% of the total azopigment material, and the amounts of these showed considerable variation among biological fluids. 3. The divergence of the present results from earlier work is probably related to the use of milder diazotization conditions and of chromatographic techniques with a high resolving power. 4. The thin-layer chromatographic systems developed allow rapid and quantitative analysis of azopigments derived from bile pigments.     

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.     

Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats

Bilinoid pigments in bile of homozygous Gunn rats (jj) were analysed either after formation of dipyrrolic ethyl anthranilate azo derivatives or as the unmodified parent tetrapyrroles. 1. T.l.c. of the azo derivatives revealed seven major unconjugated components which were structurally characterized by chemical tests, spectrophotometry and mass spectrometry. In addition, two minor components were identified as azodipyrrole (A+B)-glucoside and azodipyrrole (A+B)-β-d-glucuronide. 2. Extraction and t.l.c. of the tetrapyrrolic pigments showed 13 major yellow diazo-positive bands. Four of them, accounting for 59% of total diazo-positive material, were identified as unconjugated bilirubin-IXα, -IXβ, -IXγ and -IXδ. A fifth band (16%) was characterized as a mixture of two isomeric monohydroxyl derivatives and another band (8%) as a dihydroxyl derivative of bilirubin-IXα. 3. Although unconjugated bilirubin-IXα constitutes one-third of total diazo-positive material in bile of our strain of Gunn rats, the daily amount excreted represented only about 3–4% of daily bilirubin production. 4. Phototherapy caused a 2.2-fold increase in the biliary output of diazo-positive bilinoids, but did not affect markedly their composition. However, an additional diazo-negative pigment, accounting for one-third of total yellow colour, was observed but was not identified. Mass-spectral data on two dipyrrolic azopigments have been deposited as Supplementary Publication SUP 50076 (3 pages) with the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1977) 161, 1.     

Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile.

1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.     

Mass-spectrometric structure elucidation of dog bile azopigments as the acyl glycosides of glucopyranose and xylopyranose

1. The structures of the alpha(2)- and alpha(3)-azopigments, prepared by diazotization of dog bile with ethyl anthranilate, were shown by mass spectrometry and g.l.c. to correspond to azobilirubin beta-d-xylopyranoside and azobilirubin beta-d-glucopyranoside respectively. 2. Both azopigments consist of a mixture of two methyl vinyl isomers having structures (IIIa) and (IIIb) for the alpha(2)-azopigment and structures (IVa) and (IVb) for the alpha(3)-azopigment. Separation of methyl vinyl isomers was obtained by t.l.c. or column chromatography performed on the acetylated azopigments. Hydrolysis of the less polar acetates derived from components (IIIa) and (IVa) gave rise to the azopigment (Ia), whereas hydrolysis of the more polar acetates derived from components (IIIb) and (IVb) gave rise to the azopigment acid (Ib). The positions of methyl and vinyl substituents in compounds (Ia) and (Ib) were assigned on the basis of their n.m.r. spectra. 3. Molecular ions in the mass spectra of the trimethylsilyl and acetyl derivatives of the azopigments indicated the presence of a pentose and a hexose conjugating sugar. 4. The ester functions linking the sugars to the propionic acid side chain of azobilirubin were demonstrated by ammonolysis and identification of the amide of azobilirubin as the aglycone derivative. 5. The sugar moieties were shown to occur as xylopyranose (alpha(2)) and glucopyranose (alpha(3)), bound at C-1, by application of a sequence of reactions performed on a micro-scale. The sugar hydroxyl groups were acetylated and the 1-acyl aglycone removed selectively by treatment with hydrogen bromide in acetic acid. Hydrolysis of the 1-bromo sugar acetates followed by acetylation afforded the alpha- and beta-xylopyranose tetra-acetates and alpha- and beta-glucopyranose penta-acetates, identified by a combination of g.l.c. and mass spectrometry. 6. The validity of this degradation scheme was confirmed (a) by g.l.c.-mass spectrometry identification of the alpha- and beta-1-propionyl derivatives of glucopyranose tetra-acetate, obtained from the alpha(3)-azopigment after final reaction with propionic anhydride; (b) by subjecting the acetates of alphabeta-glucopyranose, alphabeta-xylofuranose and alphabeta-glucofuranose to the same sequence of reactions.     

Inaccuracies in measurement of conjugated and unconjugated bilirubin in bile with ethyl anthranilate diazo and solvent-partition methods.

A criticial evaluation was made of the ethyl anthranilate diazo and two solvent-partition methods for the determination of conjugated and unconjugated bilirubin in human and rat bile. The ethyl anthranilate diazo reagent, which reacts only with conjugated bilirubin in serum, also diazotized a variable proportion of unconjugated bilirubin in bile and thus overestimated the concentration of monoconjugates. With the Weber-Schalm and modified Folsch solvent-partition methods applied to human or rat bile, 4--9% of added 14C-labelled unconjugated bilirubin partitioned with the conjugated bilirubin in the upper phase, and 4--9% of added 14C-labelled conjugated bilirubin partitioned into the lower phase. With dog bile, the spill-over of 14C-labelled bilirubin into the lower phase was 9--11%. Analysis of azopigments from the Weber-Schalm partition confirmed that over two-thirds of the bilirubin in the lower phase represents monoconjugates, principally the less-polar monoxylosides and monoglucosides. These solvent-partition methods thus overestimate the concentration of unconjugated bilirubin in bile.     

Synthesis and separation by thin-layer chromatography of bilirubin-IX isomers. Their identification as tetrapyrroles and dipyrrolic ethyl anthranilate azo derivatives.

Procedures for the synthesis, separation and determination of structure of the bilirubin-IX isomers are described. 1. The four biliverdin-IX isomers were prepared by oxidative cleavage of haemin and were separated as their dimethyl esters. The individual esters were reduced with NaBH4, and the bilirubin esters obtained were subjected to alkaline hydrolysis yielding the corresponding bilirubin-IX isomers. 2. The bilirubin-IX isomers were structurally characterized (a) at the tetrapyrrolic stage by mass spectrometry of their trimethylsilyl derivatives and (b) by formation and structural analysis of their dipyrrolic ethyl anthranilate azo derivatives. 3. The absorption spectrum of bilirubin-IX alpha differed strikingly from the spectra of the other isomers. The presence of a pronounced shoulder around 453 nm in the spectrum of bilirubin-IXbeta allows easy differentiation from bilirubin-IXdelta. Methylation of the carboxyl groups largely eliminates the spectral differences between the IXalpha- and non-alpha isomers. 4. The bilirubin-IX isomers are conveniently separated by t.l.c. Detection and unequivocal identification is possible on a micro-scale by (a) t.l.c. with respect to reference compounds and (b) subsequent formation and t.l.c. of the more stable ethyl anthranilate azopigments. 5. Pronounced differences in polarity, i.e. solvent distribution, between the bilirubin-IX isomers indicate that a re-evaluation of conclusions reached previously with regard to the presence in, or absence from, biological fluids of some isomers and their relative amounts is needed.     

The formation and distribution of bilirubin monoglucuronide and diglucuronide in rat liver slices.

1. Bilirubin conjugation in rat liver slices was reassessed by using analysis of ethyl anthranilate azopigments to estimate separately the formation of bilirubin mono- and di-glucuronides. 2. Conjugation in slices resembles the situation in vivo more closely than does microsomal conjugation, in that diglucuronide is formed in appreciable quantity. 3. Both bilirubin mono- and di-glucuronides were present in slices in approximately equal amounts, but the monoglucuronide was the major product found in the incubation medium. 4. These results are discussed in relation to recent theories on the relationship between bilirubin mono- and di-glucuronide formation in vivo.     

The isolation of an azobilirubin β-d-monoglucoside from dog gall-bladder bile

An ethyl anthranilate azopigment of bilirubin conjugated to beta-d-monoglucoside was isolated from dog gall-bladder bile. Glucose was cleaved from the azopigment by treatment with beta-glucosidase and beta-glucuronidase. Mild alkaline hydrolysis of the compound by sodium methoxide yielded two kinds of compounds, water-soluble and organic-soluble. The former were shown, by enzymic analysis, t.l.c., nuclear magnetic resonance, and combined g.l.c. and mass spectrometry, to contain glucose. No evidence was obtained from these data that a disaccharide was present in this fraction. The organic-soluble compounds formed during this methanolysis were shown, by t.l.c. and mass spectrometry, to be the isomeric dipyrrole azopigments of bilirubin. These findings contribute further evidence to the controversy surrounding the nature of conjugated bilirubin.     

Mass-spectrometric study of the azopigments obtained from bile pigments with diazotized ethyl anthranilate

The structures of some azopigments obtained by diazotization of conjugated and unconjugated bile pigments with diazotized ethyl anthranilate were studied by mass spectrometry. The alpha(0)-azopigments derived from rat bile and human bile were shown to be identical (t.l.c. and mass spectra) with azobilirubin derived from unconjugated bilirubin. The presence of two methyl vinyl isomers (Ia) and (Ib) in equal amounts was shown by t.l.c. and mass spectrometry. The structure of the delta-azopigment derived from rat bile was studied by two methods: (a) ammonolysis gave rise to an amide having a CH(2).CH(2).CO.NH(2) side chain as shown by its mass spectrum; (b) the mass spectrum of a trimethylsilyl derivative of the delta-azopigment methyl ester confirmed the ester to be a beta-d-monoglucuronide ester of azobilirubin I.     

Excretion in dog bile of glucose and xylose conjugates of bilirubin

1. T.l.c. with neutral solvent systems of ethyl anthranilate azopigments derived from bile of man, dog and rat revealed pronounced species variation. The less polar components (α-group) could be separated conveniently by development with chloroform–methanol (17:3, v/v). 2. The azopigment material derived from gallbladder bile of dog contained about 10% of azobilirubin β-d-monoxyloside (azopigment α₂) and 30% of azobilirubin β-d-monoglucoside (azopigment α₃). The sugar moieties were identified by t.l.c. with acidic, neutral and basic solvent systems and by anion-exchange column chromatography of their boric acid complexes. Treatment of the purified azopigments with ammonia vapour led to the formation of the amide of azobilirubin, indicating that both pigments are ester glycosides. The β-d configuration was demonstrated by enzymic studies with emulsin (an adequate source of β-glucosidase activity) and with Mylase-P (an adequate source of β-glucosidase and β-xylosidase activities). 3. Hydrolysis studies with model substrates and with the α₂- and α₃-azopigments suggested that in Mylase-P the β-glucosidase and β-xylosidase activities reside in separate enzymes. 4. Compared with the accepted conjugation with glucuronic acid as a major route of detoxication in mammals, the detection of large amounts of xylose and glucose conjugates of bilirubin in dog bile suggests that the underlying biosynthetic pathways may be important alternative routes of detoxication.     

Extra hepatic formation of bilirubin glucuronides in the rat

After total hepatectomy in the rat, the presence of conjugated bilirubin in the plasma has been demonstrated using an extraction technique. This is particularly striking after loading the animals with unconjugated bilirubin. The identification of bilirubin glucuronide has been achieved using thin-layer chromatography of the azopigments formed (1) with ethyl anthranilate and (2) with p-iodoaniline.     

High-performance liquid chromatographic separation of bilirubin conjugates

A fast sensitive method for the isolation and quantitation of biliary bile pigments by reverse-phase high-performance liquid chromatography has been developed. Nine conjugates of bilirubin as well as unconjugated bilirubin and an internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution. The following sequence of eluted compounds was chemically identified by separating their ethyl anthranilate derivatives by thin-layer chromatography and by their enzymatic formation with UDP-bilirubin transferase and cosubstrate: bilirubin diglucuronide, bilirubin monoglucuronide monoglucoside, bilirubin monoglucuronide monoxyloside, bilirubin monoglucuronide (C-8, C-12), bilirubin diglucoside, bilirubin monoglucoside monoxyloside, bilirubin dixyloside, bilirubin monoglucoside (C-8, C-12), and bilirubin monoxyloside. The use of the commercially available mesobilirubin IX alpha as an internal standard was found to facilitate quantitation of the bilirubin conjugates.     

Design, synthesis, and biological evaluation of (E)-3-(4-methanesulfonylphenyl)-2-(aryl)acrylic acids as dual inhibitors of cyclooxygenases and lipoxygenases

A group of (E)-3-(4-methanesulfonylphenyl)acrylic acids possessing a substituted-phenyl ring (4-H, 4-Br, 3-Br, 4-F, 4-OH, 4-OMe, 4-OAc, and 4-NHAc) attached to the acrylic acid C-2 position were prepared using a stereospecific Perkin condensation reaction. A related group of compounds having 4- and 3-(4-isopropyloxyphenyl)phenyl, 4- and 3-(2,4-difluorophenyl)phenyl and 4- and 3-(4-methanesulfonylphenyl)phenyl substituents attached to the acrylic acid C-2 position were also synthesized, using a palladium-catalyzed Suzuki cross-coupling reaction, for evaluation as dual cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors. (E)-2-(3-Bromophenyl)-3-(4-methanesulfonylphenyl)acrylic acid (9h), and compounds having 4-(4-isopropyloxyphenyl-, 2,4-difluorophenyl-, or 4-methylsulfonylphenyl)phenyl moieties at the acrylic acid C-2 position (11a,b,d), were particularly potent COX-2 inhibitors with a high COX-2 selectivity index (COX-2 IC50 approximately 0.32 microM, SI > 316) similar to the reference drug rofecoxib (COX-2 IC50 = 0.5 microM, SI > 200). Acrylic acid analogs with a C-2 4-hydoxyphenyl (9d, IC50 = 0.56 microM), or 4-acetamidophenyl (9g, IC50 = 0.11 microM), substituent were particularly potent 5-LOX inhibitors that may participate in an additional specific hydrogen-bonding interaction. A number of compounds possessing a C-2 substituted-phenyl moiety (4-Br, 4-F, and 4-OH), or a 4- or 3-(2,4-difluorophenyl)phenyl moiety, showed potent 15-LOX inhibitory activity (IC50 values in the 0.31-0.49 microM range) relative to the reference drug luteolin (IC50 = 3.2 microM). Compounds having a C-2 4-acetylaminophenyl, or 4-(2,4-difluorophenyl)phenyl, moiety exhibited anti-inflammatory activities that were equipotent to aspirin, but less than that of celecoxib. The structure-activity data acquired indicate the acrylic acid moiety constitutes a suitable scaffold (template) to design novel acyclic dual inhibitors of the COX and LOX isozymes.     

Structure revision of disaccharidic conjugates of bilirubin-IX alpha in human bile and identification of phenylazo derivatives B4, B5, and B6 as 2-, 3- and 4-O-acylglucuronides.

Aniline azopigments B4, B5 and B6, derived from conjugates of bilirubin-IX alpha in human bile, and previously characterized as disaccharidic esters [Kuenzle (1970) Biochem. J. 119, 387-394 and 411-435], were analysed by using t.l.c. and mass spectrometry. The compounds were identified as partially separated mixtures of 2-, 3- and 4-O-acylglucuronide positional isomers. The 1-O-acylglucuronide was not detected in the mixtures and was the only compound hydrolysed with beta-glucuronidase. Further scrutiny of structural assignments made by Kuenzle [(1970) Biochem. J. 119, 411-435] led to identification of the lactone and hexuronic acid derivatives that were obtained from azopigment B5 along with glucuronolactone and glucuronic acid. A branched-chain structure, i.e. 3-C-hydroxy-methyl-D-riburonic acid, was assigned previously, but the derivatives have now been identified as various incompletely silylated forms of glucuronolactone and glucuronic acid. Several trimethylsilyl derivatives glucuronolactone were isolated and characterized by n.m.r. and mass spectrometry.     

Hantzsch 1,4-dihydropyridines containing a diazen-1-ium-1,2-diolate nitric oxide donor moiety to study calcium channel antagonist structure-activity relationships and nitric oxide release

A group of racemic 3-isopropyl 5-[(2-piperazin-1-yl)ethyl] 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (12a-c), 3-isopropyl 5-{2-[4-nitrosopiperazinyl]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (14a-c) and 3-isopropyl 5-{2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (22-30) were prepared using modified Hantzsch reactions. This group of compounds (12a-c, 14a-c, and 22-30) exhibited less potent calcium channel antagonist activity (IC(50)=0.11 to 3.35muM range) than the reference drug nifedipine (IC(50)=0.01 microM). The point of attachment of the isomeric C-4 substituent was a determinant of calcium channel antagonist activity providing the potency profile 2-pyridyl3-pyridyl4-pyridyl. The N-nitrosopiperazinyl compounds (14a-c) did not release nitric oxide. The prodrugs 22-30 that have a C-5 2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl ester substituent, upon incubation with guinea pig serum, undergo consecutive cleavage of the O(2)-acetoxymethyl moiety to give a nitric oxide donor diazenium-1-ium-1,2-diolate species that subsequently releases nitric oxide. The extent of nitric oxide released from the diazen-1-ium-1,2-diolate group is dependent upon the nature of the amino functionality attached directly to the diazen-1-ium N-1 position where the nitric oxide release profile is 1,4-piperazinyl>N-Et>N-(n-Bu)>N-Me upon exposure to guinea pig serum esterase(s). The results from this study suggest this class of hybrid calcium channel antagonist/nitric oxide donor prodrugs should release the vasodilator nitric oxide in vivo, preferentially in the vascular endothelium, to enhance the smooth muscle calcium channel antagonist effect to produce a combined synergistic antihypertensive effect.