Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene.

Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC50 value of 34.2 nM. Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.

Preparation, characterization and in vivo evaluation of a formulation of dantrolene sodium with hydroxypropyl-ß-cyclodextrin.

Dantrolene sodium (Da) is an effective skeletal muscle relaxant. However, its pharmacological effects are severely limited owing to its poor solubility and low oral bioavailability. In order to solve these problems, an inclusion complex using hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to improve the oral bioavailability of Da was prepared successfully by freeze-drying. The prepared complex was characterized by Powder X-ray diffractometry (PXRD), Fourier transform infrared spectroscopy (FTIR) and evaluated by a dissolution test and a pharmacokinetic study. The results of PXRD and FTIR proved the formation of a complex between Da and HP-ß-CD. The dissolution rate of Da was markedly improved from inclusion complex with more than 90% being released within 5min. The in vivo pharmacokinetics of Da and dantrolene sodium-hydroxypropyl-ß-cyclodextrin (Da-HP-ß-CD) inclusion complex were investigated in rats using a UPLC/MS/MS method. The Cmax and AUC0-t of the Da-HP-ß-CD inclusion complex were 5- and 3-fold higher than that of the Da. These results suggested that the Da-HP-ß-CD inclusion complex markedly improved the dissolution rate and bioavailability of Da.

Radiosynthesis of [13N]dantrolene, a positron emission tomography probe for breast cancer resistant protein, using no-carrier-added [13N]ammonia.

Dantrolene (1) is a substrate for breast cancer resistant protein, which is widely distributed in the blood-brain-barrier, intestine, gall bladder, and liver. PET study with 1 labeled with a positron emitter can be used to visualize BCRP and to elucidate the effect of BCRP on the pharmacokinetics of drugs. The objective of this study was to label 1 using nitrogen-13 ((13)N, a positron emitter; half-life: 9.9min). Using no-carrier-added [(13)N]NH(3) as the labeling agent, we synthesized [(13)N]dantrolene ([(13)N]1) for the first time. The reaction of carbomyl chloride 2b with [(13)N]NH(3) gave an unsymmetrical urea [(13)N]3, followed by cyclization of [(13)N]3 to afford [(13)N]1. Due to its instability, 2b was prepared in situ by treating amine 5 with triphosgene in a ratio of 4 to 1 and used for subsequent [(13)N]ammonolysis without purification.

Design of dantrolene-derived probes for radioisotope-free photoaffinity labeling of proteins involved in the physiological Ca2+ release from sarcoplasmic reticulum of skeletal muscle.

Bifunctional dantrolene derivatives have been synthesized as probes for radioisotope-free photoaffinity labeling with the aim of elucidating the molecular mechanism of skeletal muscle contraction. GIF-0430 and GIF-0665 are aromatic azido-functionalized derivatives that were designed to selectively inhibit physiological Ca2+ release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal muscle without a strong effect on Ca2+-induced Ca2+ release (CICR). These photoaffinity probes consist of either an azidomethyl or an ethynyl group, respectively, which could function as a tag for introduction of an optional detectable marker unit by an appropriate chemoselective ligation method after the photo-cross-linking operation. Actually, the former probe worked to photolabel its target proteins specifically as confirmed by subsequent fluorescent visualization.

Dantrolene analogues revisited: general synthesis and specific functions capable of discriminating two kinds of Ca2+ release from sarcoplasmic reticulum of mouse skeletal muscle.

The general synthesis of dantrolene analogues with various substituents on its phenyl ring has been developed via palladium-catalyzed cross-coupling reactions, the Stille or Suzuki reaction, as the key step. The effects of synthesized analogues have been evaluated by two kinds of Ca(2+) release modes from sarcoplasmic reticulum (SR) of mouse skeletal muscle fibers based on: (1) the measurement of twitch contraction caused by the physiological Ca(2+) release (PCR) of intact skeletal muscle and (2) the rate of Ca(2+)-induced Ca(2+) release (CICR) in saponin-treated skinned muscle fibers. Although dantrolene, a lead compound, inhibits both twitch contraction and CICR, some structurally modified analogues exhibit one or the other of these effects. The methoxy congener, GIF-0185, potently inhibits the twitch contraction without affecting the CICR, while GIF-0166 and GIF-0248, the ortho-nitro regioisomer and ortho, ortho-dinitro substituted analogues, respectively, doubly potentiate the CICR exclusively.

[(125)I]-N-[(3-azido-5-iodo)benzyl]dantrolene and [(125)I]-N-[[3-iodo-5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl]dantrolene: photoaffinity probes specific for the physiological Ca(2+) release from sarcoplasmic reticulum of skeletal muscle.

In order to capture and identify key molecules that regulate the release of Ca(2+) from the sarcoplasmic reticulum (SR) of skeletal muscle, we designed specific photoaffinity probes based on the structural modification of dantrolene. Thus, GIF-0082 and GIF-0276 possessing azido- and trifluoromethyldiazirinyl-benzyl groups, respectively, at the hydantoin moiety were found to have a highly selective inhibitory effect on physiological Ca(2+) release (PCR) without affecting Ca(2+)-induced Ca(2+) release (CICR). Successful realization of the sharp discrimination between PCR and CICR has led to the creation of [(125)I]GIF-0082 and [(125)I]GIF-0276, which were synthesized by substituting a stannyl group with (125)I in the corresponding phenylstannane precursors.

[3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum.

Dantrolene sodium is a medically important hydantoin derivative that interferes with release of Ca2+ from intracellular stores of skeletal muscle by an unknown mechanism. Identification of the molecular target of dantrolene would greatly aid in understanding both the mechanism of action of the drug and the dynamics of intracellular Ca2+ release in muscle. [3H]Azidodantrolene was designed and synthesized as a photoaffinity analogue in order to identify a putative dantrolene receptor in skeletal muscle. Introduction of 1 mole-atom of tritium into aldehyde 5b was required during radioligand synthesis in order to ensure high enough specific activity for detection of photo-cross-linked proteins by fluorographic methods. This was accomplished by reduction of ester 3 with custom synthesized, 100% tritium-labeled lithium triethylborotritide, followed by oxidation to 5b by manganese(IV) oxide. Compound 6b was demonstrated to be >/=95% tritium-labeled at the imine position by NMR spectroscopy, and the specific radioactivity of [3H]azidodantrolene sodium was empirically determined by HPLC and liquid scintillation counting to be 24.4 Ci/mmol, approximately 85% of theoretical maximum. [3H]Azidodantrolene was found to be pharmacologically active in ligand-receptor binding studies with skeletal muscle sarcoplasmic reticulum membranes. Photo-cross-linking experiments analyzed by SDS-PAGE and tritium fluorography have identified a approximately 160-kDa specifically labeled protein as the putative, intracellular, skeletal muscle dantrolene receptor. This photolabeled protein comigrates with a protein in Western blots immunologically cross-reactive to a polyclonal anti-rabbit skeletal muscle ryanodine receptor antibody. Thus, the putative dantrolene receptor may be related to the skeletal muscle ryanodine receptor.

Synthesis and skeletal muscle relaxant activity of 3-(aminoacyl)-1-[[[5-(substituted phenyl)-2-furanyl]methylene]amino] -2,4-imidazolidinediones.

A series of 3-(aminoacyl)-1-[[[5-(substituted phenyl)-2-furanyl] methylene]amino]-2,4-imidazolidinediones was synthesized and evaluated for skeletal muscle relaxant activity. All compounds were active by the intravenous route, and nine of 11 were active orally. One compound was very active when evaluated by the mouse Straub tail and rotarod tests; its efficacy index (ED50 rotarod/ED50 Straub tail) was 2.0, while its therapeutic index (LD50/ED50 Straub tail) was > 225.

Synthesis and skeletal muscle relaxant activity of quaternary ammonium salts of dantrolene and clodanolene.

A series of quaternary ammonium salts of dantrolene and clodanolene was prepared and evaluated for skeletal muscle relaxant activity. The quaternary ammonium salts exhibit greater aqueous solubility and, therefore, facilitate intravenous administration. One member of this series, although less effective orally, exhibited greater aqueous solubility than the sodium salt. When administered intravenously, it was a more potent antagonist of skeletal muscle contraction and yielded comparable therapeutic and muscle relaxant efficacy indexes.

Synthesis of 5-hydroxy-1-((5-(p-nitrophenyl)furfurylidene)-amino) hydantoin, a metabolite of dantrolene.

The synthesis and structural elucidation of 5-hydroxyl-1-[[5-(p-nitrophenyl)furfurylidene]amino]hydantoin, a compound proposed as a metabolite of dantrolene sodium, are reported. In addition, a chromatographic comparison of the biological and synthesized materials is made.

Positional isomer of the muscle relaxant dantrolene.

3-[[5-(P-Nitrophenyl) furfurylidene]amino]hydantoin, a position isomer of dantrolene, was synthesized and evaluated for skeletal muscle relaxant activity.

5-chloro-2-pyrimidinyl analog of dantrolene.

1-[5-(5-Chloro-2-pyrimidinyl)furfurylidene]amino-hydantoin, a structural analog of the skeletal muscle contraction antagonist dantrolene, was synthesized and found to have no skeletal muscle relaxant activity.