Pyrrolo[2,3-d]pyrimidines and pyrido[2,3-d]pyrimidines as conformationally restricted analogues of the antibacterial agent trimethoprim.

Conformationally restricted analogues of the antibacterial agent trimethoprim (TMP) were designed to mimic the conformation of drug observed in its complex with bacterial dihydrofolate reductase (DHFR). This conformation of TMP was achieved by linking the 4-amino function to the methylene group by one- and two-carbon bridges. A pyrrolo[2,3-d]pyrimidine, a dihydro analogue, and a tetrahydropyrido[2,3-d]pyrimidine were synthesized and tested as inhibitors of DHFR. One analogue showed activity equivalent to that of TMP against DHFR from three species of bacteria. An X-ray crystal structure of this inhibitor bound to Escherichia coli DHFR was determined to evaluate the structural consequences of the conformational restriction.

Quantitative structure-metabolism relationships for substituted benzoic acids in the rabbit: prediction of urinary excretion of glycine and glucuronide conjugates.

1. Quantitative relationships between molecular physicochemical properties of 24 substituted benzoic acids and their metabolic fate in the rabbit have been investigated using computational chemistry and multivariate statistical methods. 2. A total of 34 molecular properties were calculated for each compound using computational chemistry and were related statistically to the % molar recovery of glycine and glucuronide conjugates excreted in the urine of rabbits for the 24 compounds. 3. Compounds were successfully classified according to their dominant metabolic fate based on urinary excretion data, where stepwise linear regression analysis of the theoretical property data achieved good predictive fits for observed versus predicted % molar urinary recovery as glucuronide conjugates (r2 = 0.79) and % molar urinary recovery as glycine conjugates (r2 = 0.66). 4. Quantitative prediction of the urinary excretion of glucuronide and glycine conjugates of the parent compounds was achieved based on a statistical model using calculated molecular physicochemical parameters. Interpretation of the molecular properties, which are important for predicting the metabolic class, should give new insights into basic mechanisms of drug metabolism and underlying molecular recognition events that determine disposition and metabolism.

Binding measurements of indolocarbazole derivatives to immobilised human serum albumin by high-performance liquid chromatography.

The binding properties of six indolocarbazole derivative have been measured using immobilised human serum albumin (HSA) in an HPLC column. The compounds showed very strong binding to HSA which necessitated the application of a 30 to 40% concentration of 2-propanol in the mobile phase. This represents a much higher concentration than is recommended by the column manufacturers. This HSA column had not changed its binding property when it was used again with 4% 2-propanol and 96% phosphate buffer. The binding parameters were estimated by extrapolation to 0% 2-propanol and were above 99% for each indolocarbazole derivative. The correlation analysis, including the calculated octanol/water partition coefficient (log P), pKa values as well as measured reversed-phase retention data of the compounds revealed that the extremely strong binding can be explained by the hydrophobic and acidic properties of the compounds.

Prediction of urinary sulphate and glucuronide conjugate excretion for substituted phenols in the rat using quantitative structure-metabolism relationships.

1. The quantitative urinary excretion of the sulphate and glucuronide metabolites of 15 substituted phenols dosed to rat has been determined using high resolution 19F-nmr spectroscopy. 2. The urinary metabolic fate of each of the compounds was related to a series of calculated physicochemical properties for each compound to produce quantitative structure-metabolism relationships (QSMRs). Using these calculated molecular properties it was possible to predict the urinary recovery of xenobiotic material as a percentage of the administered dose, to classify the compounds according to their 'dominant' metabolite pattern and to predict quantitatively the proportions of glucuronide and sulphate conjugates in the urine by the use of multiple linear regression. 3. The quantitative predictions were tested by cross-validation and good prediction of total xenobiotic urinary recovery as a percentage of the administered dose was achieved based on an equation involving the electrophilic superdelocalizability at C4 (para to the hydroxyl function), the smallest principal ellipsoid axis dimension and the heat of formation. The largest moment of inertia and the electrophilic superdelocalizability at C3 were found to be the most significant factors for the prediction of the percentage glucuronide in the urine, and the urinary excretion of sulphate conjugates as a percentage of total urinary recovery was negatively correlated with the glucuronide excretion as little parent compound was excreted.

Mass spectrometry of the humanized monoclonal antibody CAMPATH 1H.

Two mass spectrometric techniques, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) have been used to study the intact humanized monoclonal antibody CAMPATH 1H, its fully and partially deglycosylated species, and 13 fragments prepared from it. The transformed ESI mass spectra of the glycosylated species gave complex patterns of molecular masses (M(r's). These have been substantially assigned to the presence of a mixture of glycoforms, each resulting from the combination of a single protein species with specific glycans of four distinct masses. The MALDI mass spectra of the glycosylated species, with the exception of that of the smallest fragment Fc/2, which indicated the presence of three of the glycans, gave single M(r) values comparable to the mean M(r) calculated from the ESI results. The M(r) values for the 10 prepared nonglycosylated species support the validity of the published amino acid sequence for the antibody and define the cleavage sites for the enzymic fragmentations. It is concluded that mass measurement of the Fc/2 fragment using ESI techniques provides a convenient means of preliminary assessment of the major glycosylated entities.

Automatic data reduction and pattern recognition methods for analysis of 1H nuclear magnetic resonance spectra of human urine from normal and pathological states.

Multivariate data analysis techniques have been used to compare 600-MHz 1H nuclear magnetic resonance (NMR) spectra of urine obtained from patients with inborn errors of metabolism (IEM) and urine obtained from healthy subjects. These spectra are very complex; each contains many thousands of resonances with a high dynamic range. A consistent method of reducing this wealth of data to manageable proportions is presented as a two-stage process. Computer-based spectral descriptors are automatically generated and then reduced to two-dimensional maps for visualization of clustering. Data-scaling methodology has been developed to achieve complete separation between spectra from control adults and those from adult patients with independently diagnosed IEM. The methods were refined by relating IEM samples to the mean of the control samples and applying supervised learning techniques to identify descriptors contributing to class separation. This approach allowed separation of the various classes of IEM and achieved optimal separation of patients with cystinuria from those with oxalic aciduria; the principal metabolites responsible for this separation were determined as lysine and glyoxalate. The methods developed were then extended by application to the more subtle problem of classifying urine collected from healthy subjects under different physiological conditions (i.e., pre- and post-exercise and in different stages of hydration) where, unlike the IEM case, any underlying biochemical differences were not known at the outset. Fluid-loaded and fluid-deprived samples could be partially separated as well as fluid-deprived and fluid-restored samples. Partial classification of samples on the basis of subject was also observed. Therefore, intersubject differences were liable to obscure the separation by physiological state. However, by relating each sample to a mean of the normal daily urine samples for the same person and applying a form of "range scaling" to exclude data which contributed least to class separation, improved classification of the hydration states resulted, from which it was possible to deduce those biochemical substances which were altered. These novel techniques for the data reduction and classification of NMR spectra make comprehensive use of all of the NMR spectral information and have clear potential to assist in clinical diagnosis.

Pattern recognition classification of the site of nephrotoxicity based on metabolic data derived from proton nuclear magnetic resonance spectra of urine.

The computer-based pattern recognition procedures of nonlinear mapping and principal-component analysis have been applied to analyze 1H NMR-generated metabolic data on the biochemical effects of 15 acute nephrotoxin treatments affecting the renal cortex and/or renal medulla in rats. The 1H NMR signal intensities for 16 urinary metabolites representative of several major intermediary biochemical pathways were estimated using either a simple semiquantitative scoring system or complete peak intensity quantitation. NMR-derived data were treated as input coordinates in a multidimensional metabolic space and were analyzed by pattern recognition methods through which the dimensionality was reduced for display and categorization purposes. Different nephrotoxin treatments were initially classified using semiquantitative metabolite scores on the basis of their 1H NMR-detectable biochemical effects, and a good separation of renal cortical toxin treatments from renal medullary toxin treatments was achieved. The refinement of using exact peak heights rather than metabolic data scores utilized the available metabolic information more fully and provided a unique classification of each type of toxin according to its pattern of biochemical effects and site of toxic action. Principal-component analysis provided consistently better results than did nonlinear mapping in terms of discrimination between different sites of toxicity, and maps generated from correlation matrices gave improved discrimination, compared with those based directly on the original metabolic data. A comparison between the use of an added internal quantitation standard (3-trimethylsilyl-[2,2,3,3-2H4]-1-propionate) and independently determined glucose excretion rates for scaling to the NMR-detected urinary glucose levels demonstrated that the consistent classification of site-specific nephrotoxicity was independent of the quantitation standard used. This study has provided a rigorous assessment of data processing, relative quantitation, and pattern recognition methods, and the utility of applying these methods to the classification of NMR-derived toxicological data. The considerable potential of the NMR-pattern recognition approach in the assessment of nephrotoxicity has also been confirmed with the discovery of new combinations of molecular markers of renal cellular damage.

On the analysis of bovine trypsin by electrospray-mass spectrometry.

In this paper the results obtained from an electrospray mass spectrometric (ES-MS) analytical study of commercial grade bovine trypsin are presented and discussed. It is proven, somewhat contrary to an earlier report, that ES-MS analysis may be performed routinely on a triple quadrupole mass spectrometer using the normal ES-MS raw data transformation procedures to identify and quantify the three forms of trypsin, namely, beta, alpha and psi, present in the samples. Further, it was found that all of the samples analysed contained small amounts of two peptides of M(r) = 5447 and 17,882 Da, which are postulated to originate from catalytic cleavage of alpha-trypsin by beta-trypsin.

Rapid validation of molecular structures of biological samples by electrospray-mass spectrometry.

A short account is presented of the method of measuring molecular masses (M(r)) of pure biological samples by electrospray ionisation mass spectrometry. It is demonstrated that the technique yields M(r) values with an effective accuracy equal to or better than 0.008% of the calculated M(r), provided that the correct molecular structure is employed in the calculation. It is therefore recommended that this method of measuring M(r)'s should be considered to form an essential part of all studies aimed at elucidating the molecular structure of purified biological macromolecules or for confirming the identity of labelled samples of such molecules.

Studies on the comparative toxicity of S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2-dichlorovinyl)-L-homocysteine and 1,1,2-trichloro-3,3,3-trifluoro-1-propene in the Fischer 344 rat.

The renal tubular toxicity of various halogenated xenobiotics has been attributed to their enzymatic bioactivation to reactive intermediates by S-conjugation. A combination of high resolution proton nuclear magnetic resonance (1H NMR) spectroscopy of urine, renal histopathology and more routinely used clinical chemistry methods has been used to explore the acute toxic and biochemical effects of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) and 1,1,2-trichloro-3,3,3-trifluoro-1-propene (TCTFP) up to 48 h following their administration to male Fischer 344 (F344) rats. In the absence of gross renal pathology, 1H NMR urinalysis revealed increased excretion of the tricarboxylic acid cycle intermediates citrate and succinate following DCVC administration. In contrast, both DCVHC and TCTFP produced functional defects in the S2 and S3 segments of the proximal tubule that were confirmed histologically. In these cases, 1H NMR urinalysis revealed increased excretion of glucose, L-lactate, acetate and 3-D-hydroxybutyrate (HB) as well as selective amino aciduria (alanine, valine, glutamate and glutamine). The significance of the proximal nephropathies induced by DCVHC and TCTFP is discussed in relation to biochemical observations on other xenobiotics that are toxic by similar mechanisms.

Studies of the biochemical toxicology of uranyl nitrate in the rat.

High resolution 1H NMR spectroscopy of urine and plasma, conventional clinical chemical methods and histopathology have been applied to investigate the effects of uranyl nitrate (UN) on renal function and biochemistry in the Fischer 344 (F344) rat. Administration of UN (5-20 mg/kg) to male F344 rats resulted in a dose-related proximal nephropathy assessed conventionally by histopathology and urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), and related to changes in the patterns of low MW metabolites observed in 400 MHz 1H NMR spectra of urine. The changes in urinary metabolite profiles included elevations in glucose accompanied by minor elevations in certain amino acids (alanine, valine and glutamate). 1H NMR urinalysis also revealed altered excretion of low MW metabolites which are not routinely measured, such as L-lactate, acetate, citrate, succinate and 2-oxoglutarate (2-OG). In addition, the striking appearance of high concentrations of 3-D-hydroxybutyrate (HB) in the urine was noted, in the absence of acetoacetate or acetone, and it is suggested that this may provide a new marker of proximal tubular damage for certain types of nephrotoxic mechanism. Broadening of the 1H NMR signals of citrate following 10 mg/kg UN was shown to be due to a dynamic exchange process involving chelation with urinary Ca2+ and Mg2+ ions. Conventional biochemical analysis of plasma from UN-treated rats revealed dose-related increases in creatinine, urea and HB concentrations. 1H NMR-detected evidence of raised alanine amino-transferase (ALT) levels in rats administered the highest dose of UN was indicated by the partial deuteration of alanine in lyophilised plasma reconstituted in 2H2O. The degree of 1H NMR-detected abnormalities agreed well with histopathological observations and conventional biochemical indices of nephrotoxicity and more fully characterised the renal changes produced by UN. The significance of HB-uria in UN-induced proximal nephropathy is discussed in relation to biochemical observations on other proximal nephrotoxins.

Raised transaminase activity of blood plasma from rats with experimentally-induced kidney damage detected by spin-echo 1H-NMR spectroscopy.

We report the application of spin-echo 1H-NMR spectroscopy to the detection of raised plasma transaminase activity in rats treated with the nephrotoxic cephalosporin antibiotic cephaloridine (CPH). Spin-echo 1H-NMR analysis of lyophilized plasma, reconstituted in H2O reveals a doublet at delta 1.48 for alanine. However when samples were reconstituted with 2H2O we noted that in samples from CPH-treated rats (but not in control samples) there was a variable degree of appearance of a singlet at delta 1.47 together with a reduction in the doublet at delta 1.48. We suggest that this is due to the release of transaminases from damaged tissue which, via a reversible conversion of alanine to pyruvate, causes selective deuteration of alanine at the alpha-hydrogen (alpha-CH) position. This observation suggests that these 1H-NMR spectral patterns are dependent on the level of plasma transaminases and this may provide a novel indicator of tissue damage.

On the purity of 3X-recrystallized bovine alpha-chymotrypsin.

The results of an electrospray-mass spectrometric analytical study of aqueous solutions of fifteen commercial samples of 3X-recrystallized bovine alpha-chymotrypsin are presented and discussed. It was found that only six samples were predominantly alpha-chymotrypsin and that two samples contained no alpha-chymotrypsin at all. The remaining seven samples were found to be mixtures of alpha-chymotrypsin with other chymotrypsins and, in some cases, neochymotrypsinogens. The majority of the results are rationalised in terms of previously postulated and/or observed products of proteolytic activation of bovine chymotrypsinogen A. However, evidence is also presented for the presence in many of the samples of three new serine proteases, of significantly lower molecular masses than alpha-chymotrypsin, which cannot at present be explained. The paper is concluded with a brief discussion of the implications of the analytical findings for enzymological studies.

NMR spectroscopy of human post mortem cerebrospinal fluid: distinction of Alzheimer's disease from control using pattern recognition and statistics.

1H NMR spectra have been measured at 500 and 600 MHz on 23 human cerebrospinal fluid samples obtained at autopsy from Alzheimer's disease patients and controls. The spectra at 500 MHz were quantified using 42 descriptors based on NMR peak heights and it was shown that differences between the two classes were apparent in the delta 2.4-2.9 region. Remeasured at 600 MHz a detailed examination of this chemical shift range identified citrate, aspartate, N-acetyl aspartate, methionine and glutamate in this region of the spectra. Principal components analysis showed that a separation of the two classes was possible and detailed statistics indicated that citrate level was the principal marker. Patient age and the interval between death and autopsy (parameters not closely matched between the two groups) were examined statistically to establish whether these might account for the citrate differences. Although they could possibly account for them to some extent, the relationship between citrate levels and disease state remained significant at p < 0.05. The data invite a test of the importance of citrate levels in Alzheimer's disease using samples taken ex vivo.

Studies on the effects of L(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) on 4-aminophenol-induced nephrotoxicity in the Fischer 344 rat.

4-Aminophenol (para-aminophenol; PAP) causes selective necrosis to the S3 segment of the proximal tubule in experimental animals. The mechanism of PAP nephrotoxicity has not been fully elucidated, although it has been suggested to involve glutathione (GSH)-dependent S-conjugation followed by processing by the enzyme gamma-glutamyl transpeptidase (gamma GT) to the corresponding cysteine S-conjugate. This proposed toxicity mechanism was probed further by administering L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), a potent gamma GT inhibitor, to Fischer 344 (F344) rats before treatment with PAP (100 mg/kg). AT-125 pretreatment did not appear to protect against PAP-induced nephrotoxicity as assessed by renal histopathology, clinical chemistry and proton nuclear magnetic resonance (1H NMR) spectroscopy of urine. These data suggest that renal gamma GT activity is not a prerequisite for PAP nephrotoxicity and that the generation of a cysteine S-conjugate is not a unique requirement for the induction of PAP nephrotoxicity.

Nuclear magnetic resonance spectroscopy and pattern recognition analysis of the biochemical processes associated with the progression of and recovery from nephrotoxic lesions in the rat induced by mercury(II) chloride and 2-bromoethanamine.

Nephrotoxic lesions were induced in Fischer 344 rats using HgCl2, a proximal tubular toxin, and 2-bromoethanamine (BEA), a medullary toxin. Biochemical effects of these toxins on urinary composition were observed by high resolution 1H NMR spectroscopy over 9 days after dosing. The onset of, progression of, and recovery from the induced toxic lesions were also followed histopathologically and related to the perturbed urinary biochemistry. Urinary concentrations of 20 endogenous substances were measured simultaneously by NMR at eight time points, to provide a time-related 20-dimensional description of the urinary biochemistry for each rat. Principal components analysis and nonlinear mapping were used to reduce the biochemical parameter spaces for each rat to two or three dimensions for display and classification purposes. An investigation of alternative data-presentation methods was made, and taking interanimal means of the map coordinates at each time point yielded a novel type of metabolic trajectory diagram with which the biochemical abnormalities associated with the HgCl2 and BEA lesions could be related to the progression and recovery phases of the toxic lesions. The time-course trajectories showed characteristically different paths for each toxin. These trajectories allowed the time points at which there were maximum metabolic differences to be determined and provided the visualization of net movements of the treatment group populations in time in relation to interanimal variation. Control animal urine samples subjected to this analysis showed simple clustering, with no evidence of metabolic trajectory. The trajectory for BEA showed different routes for onset of and recovery from toxicity, whereas for HgCl2 the outward trajectory (onset) mapped a space similar to the inward trajectory (recovery phase). This suggests that the NMR-detectable biochemical abnormalities after mercury toxicity mainly reflect the proportions of functional cells lining the nephron, whereas the biochemical abnormalities associated with renal medullary insult probably relate to functional integrity. An examination has been made for those metabolites that are most responsible for defining the trajectories, i.e., the discrimination of renal cortical and medullary toxicity from each other and from controls. These discriminatory metabolites (using paired t test, p < 0.001) included valine, taurine, trimethylamine N-oxide, and glucose for HgCl2 and acetate, methylamine, dimethylamine, lactate, and creatine for BEA, whereas citrate, succinate, N-acetyl resonances from as yet unidentified metabolites, hippurate, alanine, and 2-oxoglutarate played an important role in defining the biochemically perturbed trajectory of both toxins.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative structure-metabolism relationships for substituted benzoic acids in the rat. Computational chemistry, NMR spectroscopy and pattern recognition studies.

An extensive set of computed molecular properties, both steric and electronic, have been calculated using molecular orbital and empirical methods for benzoic acid (1) and a congeneric series of substituted benzoic acids, i.e. 2-, 3- and 4-fluorobenzoic acids (2-4), 2-, 3- and 4-trifluoromethyl benzoic acids (5-7), 2-, 3- and 4-methylbenzoic acids (8-10), 4-amino benzoic acid (11), 2-fluoro-4-trifluoromethyl benzoic acid (12), 4-fluoro-2-trifluoromethyl benzoic acid (13), 3-trifluoromethyl-4-fluorobenzoic acid (14). We have monitored the urinary excretion profiles and determined the metabolic fate of compounds 2-7, 12-14 in the rat using high resolution 1H and 19F NMR spectroscopy. Corresponding data for compounds 1,8-11 are taken from the literature. In all cases phase II glucuronidation or glycine conjugation reactions dominated the metabolism of these compounds. Compounds 5, 7, 12, 13 have ester glucuronides as their major metabolites; the rest primarily form glycine conjugates. Compounds (1-12) have been classified according to their calculated physicochemical properties using pattern recognition methods and principal components maps have been used as a novel type of structure-metabolism diagram. The maps of compounds in the physicochemical property space served to separate the compounds into the two major classes which related to their principal metabolic fate in vivo, namely glucuronidation versus glycine conjugation. Compounds 13 and 14 were used as further probes of the property space, and dominant metabolic fates of glucuronidation and glycine conjugation, respectively, were predicted from the previous "training set map". The metabolic fate of compounds 1-14 can thus be classified according to a simple set of physicochemical rules. Investigation of the physicochemical properties which are important in distinguishing the metabolic fate of the compounds may give insight into key features of the drug-metabolizing enzyme active sites and hence provide information on basic mechanisms of benzoate metabolism.

Cephaloridine-induced nephrotoxicity in the Fischer 344 rat: proton NMR spectroscopic studies of urine and plasma in relation to conventional clinical chemical and histopathological assessments of nephronal damage.

The acute toxicological effects of the nephrotoxic antibiotic cephaloridine (CPH, 0-1500 mg/kg) in male Fischer 344 (F344) rats, have been investigated over 48 h using clinical chemistry, histopathology and proton nuclear magnetic resonance (1H NMR) spectroscopy of urine and plasma. High field (400 and 600 MHz)1H NMR urinalysis revealed increased excretion of lactic acid, acetoacetate, alanine, valine, lysine, glutamine and glutamate and a severe, time-dependent glycosuria. A major change observed in urine of CPH-treated animals was the dose-dependent increase in HB which may relate to altered energy metabolism. CPH also caused dose-dependent decreases in the urinary excretion of hippurate, allantoin and protein (conventional assay). This abnormal metabolic profile is consistent with a functional defect in the S1/S2 regions of the proximal tubule, and was confirmed by histology post mortem. Functional changes observed included elevations in blood urea nitrogen (BUN) and urine flow rate (UFR) and dose-related decreases in urine osmolality. Spin-echo 1H NMR spectroscopic analysis of lyophilised plasma, reconstituted with 2H2O revealed an abnormal phase modulation of the methyl signal from free alanine and it is postulated that this is due to the release of transaminases from damaged tissue which via a reversible conversion to pyruvate, cause variable deuteration of alanine at the alpha-CH position. This observation suggests that 1H NMR spectral patterns are also dependent on the level of plasma transaminases and this may provide a novel indicator of tissue damage.

Some electrospray mass spectrometric evidence for the existence of covalent O-acyl enzyme intermediates.

Electrospray mass spectrometry has been used to measure the masses of the species present in solutions of three serine proteases (alpha-chymotrypsin, subtilisin Carlsberg and subtilisin BPN') before, during and after completion of the hydrolytic reaction with cinnamoyl imidazole and indole acryloyl imidazole. The masses measured during the reaction demonstrated that covalent O-acyl enzyme intermediates had been formed.