Effect of the rigidification of propranolol, a mixed ß-adrenoceptor and 5-HT1AR antagonist.

Propranolol is a popular ß adrenergic antagonists that, together with pindolol, binds also to serotoninergic receptors, namely 5-HT1A/B. In this work the rigidification of the propranolol structure by locking its hydroxyl group within a 1,3-dioxolane ring was investigated. Constrained derivatives of propranolol were synthesized, fully characterized and tested for their affinity at ß-adrenoreceptors and 5-HT1A/B/C receptors using radioligand binding assay. The constrained derivatives were inactive, as expected, at ß1/2/3 adrenergic receptors. Although less expected, these derivatives failed to bind also to 5-HT1A/B/C receptors. The rigidification of propranolol is detrimental for 5-HT1AR activity.

Preparation and In vitro Evaluation of FDM 3D-Printed Ellipsoid-Shaped Gastric Floating Tablets with Low Infill Percentages.

The aim of the study is to investigate the feasibility of fabricating FDM 3D-printed gastric floating tablets with low infill percentages and the effect of infill percentage on the properties of gastric floating tablets in vitro. Propranolol hydrochloride was selected as a model drug, and drug-loaded polyvinyl alcohol (PVA) filaments were produced by hot melt extrusion (HME). Ellipsoid-shaped gastric floating tablets with low infill percentage of 15% and 25% (namely E-15 and E-25) were then prepared respectively by feeding the extruded filaments to FDM 3D printer. Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), and scanning electron microscopy (SEM) were employed to characterize the filaments and 3D-printed tablets, and a series of evaluations were performed to the 3D-printed tablets, including the weight variation, drug content, hardness, in vitro floating behavior, and drug release of the tablets. The SEM results showed that the drug-loaded filaments and 3D-printed tablets appeared intact without defects, and the printed tablets were composed of filaments deposited uniformly layer by layer. The model drug and the excipients were thermally stable under the process temperature of extruding and printing, with a small amount of drug crystals dispersing in the drug-loaded filaments and 3D-printed tablets. Both E-15 and E-25 could float on artificial gastric fluids without any lag time and released in a sustained manner. Compared with E-15, the E-25 presented less weight variation, higher tablet hardness, shorter floating time, and longer drug release time.

Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4 Is Highly Active toward 4-Methylumbelliferone and 1-Naphthol Glucuronides, while MRP3 Exhibits Stereoselective Propranolol Glucuronide Transport.

Xenobiotic and endobiotic glucuronides, which are generated in hepatic and intestinal epithelial cells, are excreted via efflux transporters. Multidrug resistance proteins 2-4 (MRP2-MRP4) and the breast cancer resistance protein (BCRP) are efflux transporters that are expressed in these polarized cells, on either the basolateral or apical membranes. Their localization, along with expression levels, affects the glucuronide excretion pathways. We have studied the transport of three planar cyclic glucuronides and glucuronides of the two propranolol enantiomers, by the vesicular transport assay, using vesicles from baculovirus-infected insect cells expressing human MRP2, MRP3, MRP4, or BCRP. The transport of estradiol-17ß-glucuronide by recombinant MRP2-4 and BCRP, as demonstrated by kinetic values, were within the ranges previously reported. Our results revealed high transport rates and apparent affinity of MRP4 toward the glucuronides of 4-methylumbelliferone, 1-naphthol, and 1-hydroxypyrene (Km values of 168, 13, and 3 µM, respectively) in comparison to MRP3 (Km values of 278, 98, and 8 µM, respectively). MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with higher affinity toward the R-enantiomer than the S-enantiomer (Km values 154 vs 434 µM). The glucuronide of propranolol R-enantiomer was not significantly transported by either MRP2, MRP4, or BCRP. Of the tested small glucuronides in this study, BCRP transported only 1-hydroxypyrene glucuronide, at very high rates and high apparent affinity (Vmax and Km values of 4400 pmol/mg/min and 11 µM). The transport activity of MRP2 with all of the studied small glucuronides was relatively very low, even though it transported the reference compound, estradiol-17ß-glucuronide, at a high rate (Vmax = 3500 pmol/mg/min). Our results provide new information, at the molecular level, of efflux transport of the tested glucuronides, which could explain their disposition in vivo, as well as provide new tools for in vitro studies of MRP3, MRP4, and BCRP.

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates.

Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other ß-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates.

Multifunctional Cinnamic Acid Derivatives.

Our research to discover potential new multitarget agents led to the synthesis of 10 novel derivatives of cinnamic acids and propranolol, atenolol, 1-adamantanol, naphth-1-ol, and (benzylamino) ethan-1-ol. The synthesized molecules were evaluated as trypsin, lipoxygenase and lipid peroxidation inhibitors and for their cytotoxicity. Compound 2b derived from phenoxyphenyl cinnamic acid and propranolol showed the highest lipoxygenase (LOX) inhibition (IC50 = 6 µΜ) and antiproteolytic activity (IC50 = 0.425 µΜ). The conjugate 1a of simple cinnamic acid with propranolol showed the higher antiproteolytic activity (IC50 = 0.315 µΜ) and good LOX inhibitory activity (IC50 = 66 µΜ). Compounds 3a and 3b, derived from methoxylated caffeic acid present a promising combination of in vitro inhibitory and antioxidative activities. The S isomer of 2b also presented an interesting multitarget biological profile in vitro. Molecular docking studies point to the fact that the theoretical results for LOX-inhibitor binding are identical to those from preliminary in vitro study.

Minor enantiomer recycling: application to enantioselective syntheses of beta blockers.

Continuous recycling of the minor product enantiomer obtained from the acetylcyanation of prochiral aldehydes provided access to highly enantiomerically enriched products. Cyanohydrin derivatives, which under normal conditions are obtained with modest or poor enantiomeric ratios, were formed with high enantiomeric purity by using a reinforcing combination of a chiral Lewis acid catalyst and a biocatalyst. The primarily obtained products were transformed into ß-adrenergic antagonists (S)-propanolol, (R)-dichloroisoproterenol, and (R)-pronethalol by means of a two-step procedure.

Asymmetric aminolytic kinetic resolution of racemic epoxides using recyclable chiral polymeric Co(III)-salen complexes: a protocol for total utilization of racemic epoxide in the synthesis of (R)-Naftopidil and (S)-Propranolol.

Chiral polymeric Co(III) salen complexes with chiral ((R)/(S)-BINOL, diethyl tartrate) and achiral (piperazine and trigol) linkers with varying stereogenic centers were synthesized for the first time and used as catalysts for aminolytic kinetic resolution (AKR) of a variety of terminal epoxides and glycidyl ethers to get enantio-pure epoxides (ee, 99%) and N-protected ß-amino alcohols (ee, 99%) with quantitative yield in 16 h at RT under optimized reaction conditions. This protocol was also used for the synthesis of two enantiomerically pure drug molecules (R)-Naftopidil (α1-blocker) and (S)-Propranolol (ß-blocker) as a key step via AKR of single racemic naphthylglycidyl ether with Boc-protected isoproylamine with 100% epoxide utilization at 1 g level. The catalyst 1 was successfully recycled for a number of times.

Synthesis of novel isoflavene-propranolol hybrids as anti-tumor agents.

Isoflavene-propranolol hybrid molecules were developed as potentially novel anti-tumour agents. Isoflavene itself has potent anti-cancer activity while propranolol can enhance anti-proliferative and anti-angiogenic properties of 5-fluorouracil and paclitaxel. The hybrids were produced via nucleophilic addition of substituted amine groups to a dioxiran intermediate, which was in turn generated from the Williamson-type reaction of isoflavene with (±)-epichlorohydrin. These analogues were tested in anti-cancer cell viability assays against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines, and were found to exhibit potent anti-proliferative activities. These compounds also displayed anti-angiogenic and anti-proliferative effects in HMEC-1 human microvascular endothelial cell lines. Notably, the most potent hybrid molecules synthesized in this work showed enhanced potency against cancer cell lines compared to either isoflavene or propranolol alone, while retaining significant selectivity for cancer cells over MRC-5 normal lung fibroblast cells.

Asymmetric hydrolytic kinetic resolution with recyclable macrocyclic Co(III)-salen complexes: a practical strategy in the preparation of (R)-mexiletine and (S)-propranolol.

A chiral cobalt(III) complex (1e) was synthesized by the interaction of cobalt(II) acetate and ferrocenium hexafluorophosphate with a chiral dinuclear macrocyclic salen ligand that was derived from 1R,2R-(-)-1,2-diaminocyclohexane with trigol bis-aldehyde. A variety of epoxides and glycidyl ethers were suitable substrates for the reaction with water in the presence of chiral macrocyclic salen complex 1e at room temperature to afford chiral epoxides and diols by hydrolytic kinetic resolution (HKR). Excellent yields (47% with respect to the epoxides, 53% with respect to the diols) and high enantioselectivity (ee>99% for the epoxides, up to 96% for the diols) were achieved in 2.5-16 h. The Co(III) macrocyclic salen complex (1e) maintained its performance on a multigram scale and was expediently recycled a number of times. We further extended our study of chiral epoxides that were synthesized by using HKR to the synthesis of chiral drug molecules (R)-mexiletine and (S)-propranolol.

A novel impinging aerosols method for production of propranolol hydrochloride-loaded alginate gel microspheres for oral delivery.

Propranolol hydrochloride was directly encapsulated in alginate gel microspheres (40-50 µm in diameter) using a novel method involving impinging aerosols of CaCl(2) cross-linking solution and sodium alginate solution containing the drug. Microspheres formulated using 0.1 M CaCl(2) exhibited the highest drug loading (14%, w/w of dry microspheres) with 66.5% encapsulation efficiency. Less than 4% and 35% propranolol release occurred from hydrated and dried microspheres, respectively, in 2 h in simulated gastric fluid (SGF). The majority of the drug load (90%) was released in 5 and 7 h from hydrated and dried microspheres, respectively, in simulated intestinal fluid (SIF). Prior incubation of hydrated microspheres (cross-linked using 0.5 M CaCl(2)) in SGF prolonged the time of release in SIF to 10 h, which has implications for the design of protocols and correlation with in vivo release behaviour. Restricted propranolol release in SGF and complete extraction in SIF demonstrate the potential of alginate gel microspheres for oral delivery of pharmaceuticals.

Synthesis and biochemical evaluation of Aminopropanolyl-Thymine tri-Phosphate (ap-TTP).

Deoxyribonucleoside triphosphates (dNTPs) are building blocks for the biosynthesis of DNA. Various modified dNTPs' analogs have synthesized by structural changes of nucleoside's susgar and nucleobases and employed for synthesis of modified DNA. A very few modified dNTPs have prepared from non-sugar nucleoside analogs. This report describes the synthesis of acyclic nucleoside triphosphate (NTP) analog from amino acid L-Serine as aminopropanolyl-thymine triphosphate (ap-TTP) and demonstrate its biochemical evaluation as enzymatic incorporation of ap-TTP into DNA with DNA polymerases with primer extension methods. Alanyl peptide nucleicacids (Ala-PNA) are the analogs of DNA which contains alanyl backbone. Aminopropanolyl - analogs are derivatives of alanyl back bone. Ap-TTP analog is nucleoside triphosphate analog derived from Ala-PNA. Importantly, this report also sheds light on the crystal packing arrangement of alaninyl thymine ester derivative in solid-state and reveals the formation of self-duplex assembly in anti-parallel fashion via reverse Watson-Crick hydrogen bonding and π-π interactions. Hence, ap-TTP is a useful analog which also generates the free amine functional group at the terminal of DNA oligonucleotide after incorporation.

Edible solid foams as porous substrates for inkjet-printable pharmaceuticals.

The aim of this study was to investigate new porous flexible substrates, i.e., solid foams that would serve as a carrier with a high ink absorption potential for inkjet printable pharmaceuticals. Propranolol hydrochloride was used as a model active pharmaceutical ingredient (API). Pharmaceutically approved and edible cellulose derivatives and gums together with different additives were evaluated as a base for the substrate. Different methods for preparation of a solid foam such as freeze-drying, vacuum oven drying and drying at room temperature were explored. Only freeze-drying of the polymeric solutions resulted in the desired porous and flexible, but mechanically stable, soft sponge-like substrates with hydroxypropyl methylcellulose (HPMC)-based solid foams being the most suitable for the use in continuous inkjet printing. The plasticized HPMC foams had a superior absorption capacity and fast penetration speed for the different solvents due to the open cell pore structure and higher porosity as compared to nonplasticized additive-free foams, although, the latter were less hygroscopic. The produced solid foams were well suited for inkjet printing of high volumes of API-containing ink. The inkjet-printed API was immediately released from the dosage forms upon contact with the dissolution medium. This work demonstrates that the fabricated solid foams, based on plasticized HPMC, show a great potential as porous carriers in the fabrication of high dose dosage forms by inkjet printing.

Irreversible inactivation of beta-adrenergic receptors of C6 glioma cells. Synthesis and study of a thiol derivative of propranolol.

The beta-adrenergic receptor of C6 glioma cells contains a disulfide bridge which can be reduced by dithiothreitol (DTT). On intact cells, N-ethylmaleimide (NEM) (5 mM) does not change the affinity of [3H] H2-alprenolol ([3H] DHA) but reduces the total number of beta-adrenergic cell receptors by 21 +/- 3 per cent ; (N = 3). After receptor reduction by DTT, NEM irreversibly blocks the accessibility of the beta-adrenergic receptors to [3H]DHA. On isolated membranes, incubation in the presence of either NEM (5 mM) or isoproterenol (5.10(-7) M) does not significantly modify the total number of beta-adrenergic receptors accessible to [3H]DHA. Incubation of membranes with both NEM and isoproterenol reduces the number of binding sites by 33 +/- 2 per cent ; (N = 3). A thiol derivative of propranolol was synthetized. Its affinity is 10 times lower than that of propranolol. This sulfur derivative reduces the total number of beta-adrenergic receptors by 22 +/- 3 per cent (N = 3) when incubated with the native receptor and by 55 +/- 4 per cent (N = 4) when incubated with the reduced receptor. DTT does not significantly reverse the blockade induced by propranolol-SH. A model is proposed for explaining these results.

Synthesis and chromatographic separation of the glucuronides of (R)- and (S)-propranolol.

One of the major metabolites of propranolol (Inderal) is the O-glucuronide. In order to further study its disposition, possible metabolism, and contribution to the antihypertensive effect of propranolol, we have synthesized and separated the two diastereomeric propranolol O-beta-D-glucuronides (9a,b). These compounds were prepared by reaction of naphthol with epichlorohydrin and treatment of the resulting (2RS)-1'-(2,3-epoxypropoxy)naphthalene (2) with sodium azide to give (2RS)-1-(1'-naphthoxy)-3-azido-2-propanol (3). Alkylation of 3 with methyl (2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranosid)uronate (4) gave methyl (2RS)-[1-(1'-naphthoxy)-3-azido-2-propyl-2",3",4"-tri-O-acetyl-beta-D- glucopyranosid]uronate (5a,b). Reductive alkylation, followed by HPLC separation of the diastereomers, gave methyl (2R)- and (2S)-[1-(1'-naphthoxy)-3-(isopropylamino)-2-propyl-2",3",4"-tri-O-acetyl- beta-D-glucopyranosid]uronate (6a,b). Hydrolytic removal of the acetyl and methyl protecting groups gave the free glucuronides, which were then converted to the sodium salts, 9a,b. The stereochemistry of the glycoside linkage was deduced from the 400-MHz 1H NMR spectra. The absolute configuration of the aglycon portion was determined after Glusulase hydrolysis by derivatization with (R)-(+)- or -(-)-alpha-methylbenzyl isocyanate and comparison of the HPLC retention volumes with those of derivatized reference (R)- and (S)-propranolols.

Synthesis of 4'-hydroxypropranolol sulfate, a major non-beta-blocking propranolol metabolite in man.

4'-Hydroxypropranolol sulfate was recently identified as a major metabolite of propranolol (Inderal). In order to confirm the structure and to further study disposition and biological activity, we have synthesized 8 with use of 1,4-naphthoquinone as the starting material. Reduction and alkylation with benzyl iodide gave 4-(benzyloxy)naphthol. Sulfation and chlorosulfuric acid in N,N-dimethylaniline gave potassium 1-(benzyloxy)-4-naphthol sulfate. Catalytic hydrogenation, alkylation with [[[(trifluoromethyl)sulfonyl]oxy]methyl]oxirane, and amination in isopropylamine gave 8. Racemic 8 was found to be 100-1000 times less potent than racemic propranolol as a beta-adrenergic receptor blocking agent in the dog.

Relationship of nonspecific antiarrhythmic and negative inotropic activity with physicochemical parameters of propranolol analogues.

In an attempt to separate the nonspecific antiarrhythmic activity of propranolol from its negative inotropic effects, analogues containing hydrophilic and lipophilic substituents on the nitrogen and on the naphthyl ring were prepared and tested in an isolated tissue preparation. Though it had been predicted that analogues containing a very hydrophilic group on the nitrogen would have the highest antiarrhythmic/negative inotropic effect ratio, it was found that both effects increased identically when the lipophilicity of either the nitrogen or ring substituent was increased.

The 3,4-catechol derivative of propranolol, a minor dihydroxylated metabolite.

The O,O-dibenzyl ether of the 3,4-catechol derivative of propranolol (11) was prepared to determine whether the catechol is a product of metabolic hydroxylation. 4-(Allyloxy)-1,2-naphthoquinone (5) was reduced with sodium dithionite and alkylated with benzyl chloride to produce ether 7. Osmium tetroxide oxidation of 7 afforded glycol 8. Subsequent monotosylation, oxirane formation with KOH, and opening with isopropylamine afforded benzyl ether 11. Although hydrogenolysis was successful, catechol 3 was rapidly oxidized to the corresponding o-quinone (12). Reduction of 12 with sodium bisulfite afforded 3, which was derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) to serve as a standard for the metabolic experiments. Gas chromatography-mass spectrometry of the Me3Si ethers of the products of metabolism of pseudoracemic propranolol (made up of equal molar (2R)-propranolol-d0/(2S)-propranolol-3',3'-d2) in the presence of the rat liver 9000g supernatant fraction showed four dihydroxylated metabolites, of which catechol 3 was in smallest amount, approximately 9% of the sum of dihydroxylated metabolites. Each of the four dihydroxylated propranolols arises stereoselectively from the 2R enantiomer of propranolol (by 1.15- to 2-fold), as determined by parent ion intensities at m/z 507 vs. 509. Quinone 12 was a nonselective competitive beta-adrenoceptor antagonist, being about 16-fold less potent than propranolol in both beta 1 and beta 2 assays.

Design and synthesis of propranolol analogues as serotonergic agents.

Serotonin (5-HT) binds with nearly identical affinity at the various central 5-HT binding sites. Few agents bind with selectivity for 5-HT1A sites. The beta-adrenergic antagonist propranolol binds stereoselectively both at 5-HT1A and 5-HT1B sites (with a several-fold selectivity for the latter) and, whereas it is a 5-HT1A antagonist, it appears to be a 5-HT1B agonist. As such, it could serve as a lead compound for the development of new 5-HT1A and 5-HT1B agents. The purpose of the present study was to modify the structure of propranolol in such a manner so as to reduce its affinity for 5-HT1B and beta-adrenergic sites while, at the same time, retaining its affinity for 5-HT1A sites. Removal of the side-chain hydroxyl group of propranolol, and conversion of its secondary amine to a tertiary amine, reduced affinity for 5-HT1B and beta-adrenergic sites. In addition, shortening the side chain by one carbon atom resulted in compounds with affinity for hippocampal 5-HT1A sites comparable to that of racemic propranolol, but with a 30- to 500-fold lower affinity for 5-HT1B sites and a greater than 1000-fold lower affinity for beta-adrenergic sites. The results of these preliminary studies attest to the utility of this approach for the development of novel serotonergic agents.

Synthesis and beta-adrenergic antagonist activity of stereoisomeric practolol and propranolol derivatives.

A series of stereoisomeric practolol and propranolol derivatives has been synthesized in which the N-isopropyl group of the drug was replaced by an asymmetric heptanoic acid terminated by a substituted p-toluidide or p-(trifluoromethyl)anilide. The asymmetric epoxide, 3-(p-acetamidophenoxy)-1,2-epoxypropane, was allowed to react with a preformed enantiomeric 6-aminoheptanoic acid amide to yield the stereoisomeric practolol congener derivatives. An asymmetric drug precursor epoxide was prepared from p-acetamidophenol and enantiomeric 3-(tosyloxy)-1,2-epoxypropane 6-aminoheptanoic acid amides were allowed to react with one of the enantiomers of 3-(1-naphthyloxy)-1,2-epoxypropane. This drug precursor epoxide was prepared either by combining 1-naphthol with enantiomeric 3-(tosyloxy)-1,2-epoxypropane (the Sharpless epoxide) or by combining 1-naphthol with enantiomeric 3-(tosyloxy)-1,2-propanediol followed by epoxidation. Pharmacological studies carried out for the practolol derivatives demonstrated a significant dependence of enhanced potency and tissue/subreceptor specificity on both the configuration of the drug asymmetric carbon and the configuration of the spacer asymmetric carbon. The compounds containing the S configuration at the drug asymmetric center and the R configuration at the spacer asymmetric carbon exhibited an increase in potency over the other stereoisomeric congener derivatives and the progenitor drug. For the propranolol congener derivatives, a large decrease in potency was observed for all of the stereoisomers over the progenitor drug. The propranolol stereoisomers containing the S configuration at the drug asymmetric center were more active than those containing the R configuration at that center.

Synthesis and biologic distribution of radioiodinated beta-adrenergic antagonists.

Iodinated analogues 2, 7, and 8 were prepared from propranolol, practolol, and acebutolol in 30--50% yields. Radioisotopic exchange between carrier-free Na125I and the molten iodinated beta-adrenergic antagonist yielded the corresponding 125I-labeled product. The biodistribution in rats, determined at 15 and 60 min postinjection, indicated that the radioiodinated analogues of the cardioselective drugs practolol and acebutolol localized to a greater degree in the liver and heart than the analogue of propranolol. Conversely, [125I]iodopropranolol (2) was concentrated to a greater extent in the lungs than [125I]iodopractolol (7) or [125I]iodoacebutolol (8). Therefore, 123I- or 131I-labeled cardioselective beta-adrenergic antagonists, such as 7 or 8, may prove useful as radiodiagnostic agents for the external imaging of the myocardium.