Pharmaceutical development and specification of stereoisomers

The pharmaceutical development of chiral drugs requires the activities of many different research and development groups. Guidelines which help to coordinate the activities of these groups and assist in the successful development of compounds with either single or multiple chiral centers are outlined and discussed.     

Metabolism of 2,3-butanediol stereoisomers in the perfused rat liver

The identification of 2,3-butanediol in sera of alcoholics led to the hypothesis that it may be a specific marker of alcohol abuse. We have investigated the metabolism of the individual isomers of 2,3-butanediol (2R,3R-, 2S,3S-, meso-2,3-butanediol and racemic 2,3-butanediol) in perfused livers from fed rats. Rates of uptake of the isomers decrease in the order (i) 2R,3R-, (ii) meso-, (iii) 2S,3S-2,3-butanediol. We observed interconversion of isomers and oxidation to acetoin with 2R,3R- and meso- but not with 2S,3S-2,3-butanediol. In perfusions conducted in deuterium oxide, interconversion of isomers was accompanied by incorporation of deuterium. Thus, interconversion of isomers occurs via a reversible oxidation to acetoin with incorporation of hydrogen from water. In perfusions with either 2R,3R- or meso-[2-14C]2,3-butanediol, the substrates were converted to labeled acetate, R-3-hydroxybutyrate and CO2, suggesting that 2,3-butanediol is oxidized to acetyl-CoA via acetoin.     

Amphetamine-like discrimination stimulus effects of ephedrine and its stereoisomers in pigeons.

This study assessed the discriminative stimulus effects of (±)-ephedrine and its stereoisomers in pigeons discriminating 1.0 mg/kg of amphetamine from saline. Amphetamine, (±)-, (-)-, and (+)-ephedrine, and cocaine occasioned greater than 80% drug-key responding with the following rank order of potency: amphetamine > cocaine > (-)-ephedrine ≥ (±)-ephedrine ≥ (+)-ephedrine. Neither the α-adrenergic antagonist, phentolamine, nor the β-adrenergic antagonist, propranolol, antagonized the effects of amphetamine or (±)-ephedrine. In contrast, the dopamine receptor antagonist, haloperidol, antagonized the discriminative stimulus effects of amphetamine and (±)-ephedrine as well as those of (-)- and (+)-ephedrine. These results indicate that, like cocaine, (±)-ephedrine and its stereoisomers share discriminative stimulus effects with amphetamine. Moreover, these effects appear to be the result of increased activity in dopaminergic systems. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Differential influence of stereoisomers of estradiol on sexual behavior of female hamsters.

Morphological and behavioral responses to estradiol-17β (E?-17β) and estradiol-17α (E?-17α) were examined in a series of 3 experiments with golden hamsters. The E?-17β augmented uterine growth to a greater extent than E?-17α. Lordosis in ovariectomized adults was elicited by treatment with E?-17β but not with E?-17α (each tested in combination with progesterone). When administered neonatally, only E?-17β disrupted estrous cyclicity in the intact female and induced the ability to mount in ovariectomized, androgen-treated adults. Results suggest the existence of a stereospecific response to estrogenic stimulation in neural tissue comparable with that occurring in the uterus. (15 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Simultaneous determination of the stereoisomers of guggulsterone in serum by high-performance liquid chromatography

Simultaneous separation of E- and Z-guggulsterone, which is the main ingredient of 'Guggulip', an ayurvedic drug, was accomplished by HPLC on a C18 column using methanol, acetonitrile, buffer and tetrahydrofuran as a mobile phase. The compounds were monitored at 248 nm on a photodiode array detector. The assay method was used for the simultaneous determination of stereoisomers (E and Z) of guggulsterone in spiked serum and dosed (50 mg/kg, p.o.) rats. The recoveries of E- and Z-isomers from serum samples were always greater than 90%. The calibration graph was linear over the range of 25-2500 ng/ml for Z- and E-isomers. Lowest quantitation limit of Z- and E-guggulsterones was 25 ng/ml.     

Acute effects of ethionine stereoisomers on hepatic RNA and protein synthesis in swiss mice

The acute biochemical effects of ethionine have been well studied in rats but not in mice. These results show that both hepatic RNA and protein synthesis in Swiss mice were inhibited by DL-ethionine. Protein synthesis was inhibited 30 to 40 percent 3 hr after 2500 mg/kg DL-ethionine while RNA synthesis was inhibited 80 to 90 percent 3 hr after 625 mg/kg DL-ethionine. Thus ethionine was a far more potent inhibitor of RNA synthesis than of protein synthesis. There was no sex difference in either of these responses. While both stereoisomers were active, the L isomer was a more potent inhibitor of RNA synthesis than was the D isomer. In male mice, 3 hr after 20 mg/kg L-ethionine, RNA synthesis was inhibited 80%, while after D-ethionine it was inhibited only 51%.     

Pharmacokinetics and pharmacodynamics of mivacurium stereoisomers in beagle dogs using twitch height and train-of-four response

Mivacurium, a non-depolarizing neuromuscular blocking agent, consists of three isomers; trans-trans (57%), cis-trans (36%) and cis-cis (7%). The purpose of this study was to characterize the pharmacokinetics and pharmacodynamics of mivacurium after various inputs. Four beagle dogs weighing between 7.95 and 9.89 kg were anesthetized with isofluorane (5%) and received a bolus dose (0.010-0.020 mg kg(-1)) and two constant rate infusions (1.0-1.5 microg kg(-1) min(-1)) of mivacurium via the saphenous vein. Single twitch height (TH) and train-of-four (TOF) were evaluated every 15 and 30 s, respectively. Arterial blood samples were collected, processed and analysed for mivacurium using a stereospecific HPLC-fluorescence method. The disposition of mivacurium isomers was best described by a two compartment model. Mean Cl for the cis-trans, trans-trans and cis-cis isomers were 19.98, 13.53 and 3.47 mL min(-1) kg(-1) respectively and the corresponding mean Vdss were 0.29, 0.24 and 1.00 L kg(-1). The measurement of onset showed dose dependence as evidenced by a rapid onset at the higher doses. TOF measurements were more sensitive to the onset of action and required a longer period of time to recover to baseline values as compared with TH measurements.     

Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 microM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 microM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7ADR cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH stereoisomers is associated with inhibition of PKC-mediated phosphorylation of P-gp.     

Conformations of cis- and trans-2,3-methanomethionine stereoisomers in the tripeptide mimic system Ac[cyclo-M]NHiPr

Coronary artery bypass grafting (CABG) is the most common procedure performed in adult cardiovascular surgery today. In our Department of Surgery at Baylor College of Medicine, we have experienced, as have most large programs, a trend to older patients, more comorbidity, worse ventricular function, and more redo CABG procedures. Along with this shift has come an evolution in surgical techniques, cardioplegia and choice of coronary bypass graft conduits. The benefit of using an internal mammary artery (IMA) to the left anterior descending coronary artery (LAD) appears irrefutable at this time. Data for multiple arterial grafts is still evolving and conduit choice for other than the IMA to LAD graft is often debated. The purpose of this article is to review the current literature on conduit choice and to allow a rational, data driven approach to graft choice.     

Reversed-phase high-performance liquid chromatographic identification of lutein and zeaxanthin stereoisomers in bovine retina using a C30 bonded phase

There is a considerable degree of variation in the amount of potentiation induced in different animals following the induction of long-term potentiation (LTP). This variation provided us with the opportunity to determine what types of synaptic changes were dependent upon the degree of induced potentiation. To examine possible 'degree of potentiation' effects on synapses, we conducted a multiple regression analysis examining the relationship between the degree of potentiation in LTP animals and a series of synaptic structural measures. We examined synapses in the middle third of the molecular layer (MML) of the rat dentate gyrus following repeated high frequency tetanization of the perforant path. LTP was induced over a 4 h period, and the animals were sacrificed 24 h after the final stimulation. Synapses from the ipsilateral inner third of the dentate molecular layer (IML) and from implanted only animals were also examined for comparison. Ultrastructural quantification included the total number of synapses per neuron, synaptic curvature, the presence of synaptic perforations, and the maximum length of the synaptic apposition. The only structural change that was significantly associated with the degree of potentiation was a positive correlation between the degree of LTP and the number of synapses per neuron. Therefore, synaptic number, while not appearing to be significantly associated with the induction of LTP, appears to be important for the degree of LTP expressed.     

Selective method for plasma quantitation of the stereoisomers of a new aminotetralin by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method is described for the quantitation in plasma of the four stereoisomers of a new aminotetralin, (SRR, RSS)(SRS, RSR)-5,6-dimethoxy-2-[3'-(p-hydroxyphenyl)-3'-hydroxy-2'- propyl]aminotetralin (CHF 1255, internal code). After liquid-liquid extraction of the drug, separation was obtained after chiral derivatization with R-(+)-alpha-methylbenzyl isocyanate. The selective derivatization of the amino group was obtained by controlling the pH of the reaction medium at 7.5. The reaction was quantitative after a period of 16 h. The structures of the urea derivatives were confirmed by proton nuclear magnetic resonance spectroscopy and high-performance liquid chromatography with mass spectrometric detection. The use of an electrochemical detector, operating in the oxidative mode, allows the quantitation in plasma of all four urea derivatives at the nanogram level. The method was demonstrated to be precise, reproducible and applicable to pharmacokinetics studies after administration of the two epimeric racemates.     

Stereospecific induction of apoptosis in U937 cells by N-octanoyl-sphingosine stereoisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis

We investigated the ability of N-octanoyl-sphingosine (C8-Cer) stereoisomers, N-octanoyl-DL-erythro-dihydrosphingosine (DL-e-DHC8-Cer), and a new ceramide derivative, N-octyl-D-erythro-sphingosine (D-e-C8-Ceramine), to induce apoptosis in U937 cells. We found the C8-Cer stereoisomers to be stereospecific with the D- and L-threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D-t-C8-Cer = L-t-C8-Cer > L-e-C8-Cer > D-e-C8-Cer > DL-e-DHC8-Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D-e-C8-Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D-e-C8-Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide-activated protein kinase (CAPK). While the potent DNA fragmentation-inducing compounds D-e-C8-Ceramine and L-t-C8-Cer failed to increase the cellular ceramide levels, D-e-C8-Cer, D-t-C8-Cer and D-e-C8-Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation-inducing ability of the threo stereoisomers and D-e-C8-Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D-e-C8-Ceramine and L-t-C8-Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D-e-C8-Cer.     

Synthesis and structural determination of stereoisomers of muscarinic ligands of the (3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycloalkane type

Methods for the synthesis of each of the four stereoisomers of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]oc tane (10, 11, 12, and 13) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]he ptane (18, 19, 20, and 21), and the two stereoisomers of 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]oc tane (27 and 28) were developed. The relative configuration of the compounds was determined on the basis of previously described 1H NOE experiments, and the absolute configuration of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]oc tanes (10, 11, 12, and 13) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]oc tane (27 and 28) was determined by single crystal X-ray crystallography. Optical purity was determined by capillary electrophoresis (CE) using chiral selectors as trimethyl-beta-cyclodextrin and heparin dissolved in the running buffer. All the 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycles had low nanomolar affinity for muscarinic receptors as determined by displacement of radiolabelled oxotremorine-M (3H-Oxo-M) and pirenzepine (3H-Pz) from cortical rat brain homogenates. The binding assay discriminated between diastereomers, but only a minor degree of enantioselectivity was observed in the binding assays.     

Stereochemical influence on the stability of radio-metal complexes in vivo. Synthesis and evaluation of the four stereoisomers of 2-(p-nitrobenzyl)-trans-CyDTPA

Distinct differences in in vivo stability of the two diastereomeric C-Functionalized CyDTPA chelating agents, (CHX-A DTPA and CHX-B DTPA, both racemates), as recently reported prompted further investigation as to why differences in configuration produced striking effects on the in vivo stability of their yttrium complexes. To this end, the four individual component stereoisomers of CHX-A and CHX-B were synthesized and ability to bind yttrium was investigated both in vitro and in vivo.     

Biodegradation of [S,S], [R,R] and mixed stereoisomers of ethylene diamine disuccinic acid (EDDS), a transition metal chelator

An in-depth biodegradation test program was executed on the hexadentate ligand Ethylene Diamine Di Succinate (EDDS). The EDDS structure contains two chiral carbon atoms, and has three stereoisomers ([R,R], [R,S]/[S,R], [S,S]). Our research has focused on the isomer mixture (i.e. 25%[S,S]; 25%[R,R]; 50%[S,R]/[R,S], as produced from the reaction of ethylene diamine with maleic anhydride) and on the single [S,S]- and [R,R]-isomers. Biodegradation screening of the 14C-labelled EDDS isomer mixture in a Batch Activated Sludge (BAS) test with various inocula revealed incomplete mineralization, up to ca. 65% after 28 days. N-(2-aminoethyl) aspartic acid (AEAA), probably the d-isomer, was identified as the major portion of the 14C-material remaining in solution. Further testing revealed that the [S,S]-isomer is rapidly and completely mineralized in all test systems. By contrast, [R,R]-EDDS remained undegraded in a Sturm (OECD 301B) test, but was very slowly biotransformed into the recalcitrant metabolite AEAA in a BAS test. The [S,R]/[R,S] form undergoes biotransformation to AEAA in both high and low biomass systems. In a sewage treatment simulation test (OECD 303) the steady state DOC removal of mixture-EDDS in a CAS test was limited to 25-35%, even after extensive pre-acclimation, while the [S,S]-isomer achieved nearly complete removal (96%). This study illustrates the importance stereospecificity may have on the biodegradation and metabolite formation of a chemical. A biodegradation scheme for the different EDDS stereoisomers is proposed.     

Induced circular dichroism of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide stereoisomers covalently bound to deoxyribooligonucleotides used to probe equilibrium distribution between groove binding and intercalative adduct conformations

Binding conformations of single anti-BPDE-N2-dG adducts in oligonucleotides of varying base composition have been studied by induced circular dichroism (ICD). The sign of the ICD around 350 nm of single-stranded oligonucleotide adducts and the sign of an exciton type of CD component at 260 nm in both single strand and duplex forms of adducts correlate with the absolute configuration of the cyclohexyl moiety of the adduct. Changes in magnitude and sign of the ICD around 350 nm were observed upon duplex formation. The results show that adducts displaying external (minor groove) binding characteristics are associated with a significant positive ICD. Conversely, adducts displaying intercalation binding characteristics were found to have a positive or negative ICD. The magnitude of the ICD is dependent on the sequence context and the particular adduct isomer studied. Duplexes with (+)-trans-anti-BPDE-N2-dG in 5'-d(CCTATCGCTATCC) or 5'-d(CCTATAGATATCC) exhibit a relatively strong positive ICD. In contrast, the duplexes with (+)-trans-anti-BPDE-N2-dG in 5'-d(CCTATTGCTATCC) and 5'-d(CCTATTGTTATCC) display a small positive and negative ICD, respectively, in both cases suggesting conformational heterogeneity. Partially complementary duplexes (dA, dT, or dG) localized opposite the (+)-trans-anti-BPDE-N2-dG adduct in 5'-d(CCTATCGCTATCC) or 5'-d(CCTATAGATATCC) also demonstrated negative ICD. These results together with light absorption characteristics suggest a preferred conformation of intercalation for the mismatched duplexes. Evidence of an equilibrium between the external and intercalative adduct conformation is provided by the results from the temperature dependence of the near-UV absorption and ICD characteristics of (+)-trans-anti-BPDE-N2-dG complex in a 5'-d(CCTATAGATATCC) duplex.