Preparation and properties of ryodipine, an effective antihypertensive agent

A novel effective antihypertensive agent, 2,6-dimethyl-3,5-dimethoxycarbonyl-4-(o-difluoromethoxyphenyl++ +)-1,4-dihydropyridine (ryodipine, PP-1466) has been obtained. A method for ryosidine synthesis has been developed, side products of PP-1466 synthesis have been isolated and identified. Special characteristics (IRS, UVS, NMR, MS) and chromatography data (TLC, HPLC) are cited.     

Nitrosation of glycine ethyl ester and ethyl diazoacetate to give the alkylating agent and mutagen ethyl chloro(hydroximino)acetate

Whereas nitrosation of secondary amines produces nitrosamines, amino acids with primary amino groups and glycine ethyl ester were reported to react with nitrite to give unidentified agents that alkylated 4-(p-nitrobenzyl)pyridine to produce purple dyes and be direct mutagens in the Ames test. We report here that treatment of glycine ethyl ester at 37 degrees C with excess nitrite acidified with HCl, followed by ether extraction, gave 30-40% yields of a product identified as ethyl chloro(hydroximino)acetate [ClC(=NOH)COOEt, ECHA] and a 9% yield of ethyl chloroacetate. The ECHA was identical to that synthesized by a known method from ethyl acetoacetate, strongly alkylated nitrobenzylpyridine, and may have arisen by N-nitrosation of glycine ethyl ester to give ethyl diazoacetate, which was C-nitrosated and reacted with chloride to give ECHA. Nitrosation of ethyl diazoacetate also yielded ECHA. Ethyl nitroacetate was not an intermediate as its nitrosation did not produce ECHA. ECHA reacted with aniline to give ethyl (hydroxamino)(phenylimino)acetate [PhN=C(NHOH)CO2Et]. This product was different from ethyl [(phenylamino)carbonyl]carbamate [PhNHC(=O)NHCO2Et], which was synthesized by reacting ethyl isocyanatoformate (OCN.CO2Et) with aniline. ECHA reacted with guanosine to give a derivative, which may have been a guanine-C(=NOH)CO2Et derivative. ECHA showed moderate toxicity and weak but significant mutagenicity without activation in Salmonella typhimurium TA-100 (mean, 1.31 x control value for 12-18 microg/plats) and for V79 mammalian cells (1.5-1.7 x control value for 60-100 microM). In conclusion, gastric nitrosation of glycine derivatives such as peptides with a N-terminal glycine might produce ECHA analogues that alkylate bases of gastric mucosal DNA and thereby initiate gastric cancer.     

Biological properties of 2,4-dioxo-3H-quinoline-3-carboxylic acid and its ethyl ester

Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 26, No. 2, pp. 33–35, February, 1992.     

Pharmacokinetics of cysteine ethyl ester in rat

1. The pharmacokinetics of the mucolytic compound 35S-cysteine ethyl ester in rat show that it is completely absorbed after oral administration, with a bioavailability of 0.59. 2. By autoradiography, tissue distribution of 35S-cysteine ethyl ester showed a high affinity for the lung, different to that observed with 35S-cysteine. 3. Unchanged cysteine ethyl ester, cysteine and inorganic sulphates were present in the lung. 4. After oral or i.v. administration of 35S-cysteine ethyl ester the total radioactivity excreted in the urine amounted to 16% dose and 40% was excreted in the faeces, following biliary excretion. It was metabolized to inorganic sulphate, cysteine and taurine, which were excreted in the urine.     

Effect of creatine, creatinine, and creatine ethyl ester on TLR expression in macrophages

Despite the widespread availability and use of dietary supplements, minimal work has been performed to assess the potential dangers many of these supplements may have on the host's well-being, in particular the host's ability to respond to infection. One supplement extensively used by both adolescents and adults is creatine. Using Real-time PCR, we examined the impact of short-term exposure of a mouse macrophage cell line (RAW 264.7 cells) to two readily available forms of creatine used in supplements--creatine monohydrate (CR) and creatine ethyl ester (CEE) as well as the end product of creatine metabolism, creatinine (CRN), on expression of toll-like receptor-2 (TLR-2), TLR-3, TLR-4, and TLR-7. CR down-regulated TLR-2, TLR-3, TLR-4 and TLR-7 mRNA levels in RAW cells. Similar results were observed following exposure of RAW cells to CRN. Conversely CEE appears to possess immunostimulatory properties and increases expression of TLR-2, TLR-3, TLR-4, and TLR-7 in RAW cells. These data are supported by immunostaining using antibodies specific for the individual TLRs before and after exposure of RAW cells to CR, CRN, or CEE. To extend these findings, we isolated murine splenocytes and exposed the cells to CR, CEE, or CRN for 24 hours and performed immunofluorescent staining for TLR-2, TLR-3, TLR-4 and TLR-7. The results obtained from this study with primary splenocytes were consistent with the studies using RAW cells. Together, these data suggest that creatine and creatine derivatives may impact the ability of immune cells to sense a wide array of viral and bacterial pathogens. Of great interest, CRN--largely considered to be a waste product of the argenine biosynthesis pathway may also have immunosuppressive properties similar to those of CR.     

The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent

An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 microM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure. A micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry (microLC-microESI-MS/MS) method detected glutathione ethyl ester conjugates using selected reaction monitoring (SRM) to simultaneously monitor for multiple MH+ to [MH - 129]+ transitions, where the 129 mass unit (Da) represents the neutral loss of the pyroglutamate moiety from GSH-EE. The multiple MH+ to [MH - 129]+ transitions (SRM mass table) were generated for potential reactive intermediates of each compound. Glutathione (GSH) and GSH-EE conjugate standards were used to evaluate MS detection sensitivity. Based on direct comparison of standard curve data, an approximate 10-fold increase in sensitivity was observed for conjugates containing GSH-EE moiety versus GSH. In vitro experiments were conducted using literature substrates acetaminophen, rosiglitazone, clozapine, diclofenac and either GSH-EE or GSH as a reactive intermediate conjugating agent. An increase in detection sensitivity was observed for each GSH-EE conjugate and in the case of acetaminophen-GSH-EE the peak area increase was approximately 80-fold. Twelve drug compounds, each having known biotransformation mechanisms, were used to further test the detection capabilities of the assay and establish a concordance to literature data. When GSH was used in the assay, conjugates were detected for 4 out of the 12 test compounds (33%). When GSH-EE was used in the assay, conjugates were detected for 10 out of the 12 test compounds (83%).     

Effects of an agent inducing dominant lethals on rat sperm--examination with ethyl methanesulfonate

Ethyl methanesulfonate (EMS), an alkylating agent which induces dominant lethals, was administered in oral doses of 100 mg/kg to Crj:CD(SD)IGS male rats for 5 consecutive days. At the termination of treatment and after a 28-day withdrawal, mating with untreated females and sperm analysis (motion, number, and morphology) were performed. The copulated females were sacrificed at 20 days of gestation. At the termination of treatment, no clinical signs related to EMS were observed except for a decrease in body weight. Gross pathology and sperm analysis revealed no abnormalities in treated males. However, females mated at the termination of treatment had a clearly higher fetal mortality. Females mated after the 28-day withdrawal exhibited lower fetal mortality than females mated at the termination of treatment. On the other hand, females mated after the 28-day withdrawal exhibited a lower implantation rate that was not observed in females mated at the termination of treatment. For males after a 28-day withdrawal, sperm analysis revealed both a decrease in sperm motion and number and an increase in morphological change. These findings indicate that two types of male reproductive toxicity induced by EMS can be distinguished. One induces a low implantation rate that can be detected by sperm analysis, while the other induces fetal lethals that could not be detected by sperm analysis in this study.     

3-m-bromoacetylamino benzoic acid ethyl ester: a new cancericidal agent that activates the apoptotic pathway through caspase-9

The mechanism underlying the cancericidal activity of 3-m-bromoacetylamino benzoic acid ethyl ester (3-BAABE) was investigated. 3-BAABE exerted a strong cancericidal effect on human leukemia and lymphoma cells (IC(50) < 0.2 microgram/mL) and on cell lines of prostate, colon, ductal, and kidney cancer (IC(50) 0.8 to 0.88 microgram/mL). Multiple drug resistance (MDR) had no effect on the susceptibility of human lymphoma cells to 3-BAABE, since Daudi/MDR(20) and wild-type Daudi cells had a similar susceptibility to the cytotoxic effect of 3-BAABE. The cancericidal effect of 3-BAABE, which was not associated with changes in the cell cycle, was mediated by apoptosis. Thus, cells exposed to 3-BAABE displayed the DNA fragmentation ladder characteristic for apoptosis, associated with a marked increase of the activity of apoptosis effector caspases-3 and -6, which was followed by proteolytic cleavage of DNA fragmentation factor (DFF) and poly(ADP-ribose) polymerase (PARP). Exposure of tumor cells to 3-BAABE increased the activity of apical caspase-9, but had no effect on caspase-8. Complete inhibition of 3-BAABE-induced apoptosis was exerted by LEHD-FMK, a caspase-9 inhibitor. DEVD-FMK, a caspase-3 inhibitor, and VEID-FMK, a caspase-6 inhibitor, partially inhibited 3-BAABE-induced apoptosis, whereas exposure to IETD-FMK, a caspase-8 inhibitor, had no effect. The fragmentation and elevated activity of caspase-9 in 3-BAABE-treated cells and the fact that only an inhibitor of caspase-9 abrogated 3-BAABE-induced apoptosis indicate that 3-BAABE is a distinctive compound that elicits apoptosis through a pathway that is limited specifically to activation of apical caspase-9.     

Antiallergic properties of an orally effective agent, [[3-(1H-tetrazol-5-yl)-phenyl] amino] oxoacetic acid n-butyl ester

A newly synthesized compound, [[3-(1H-tetrazol-5-yl)-phenyl] amino]oxoacetic acid n-butyl ester (MTB) has been demonstrated to be an orally active antiallergic agent. This compound inhibited the 48-hr passive cutaneous anaphylaxis (48-hr PCA) induced by IgE in rats. In guinea pigs, MTB also inhibited the 8-day passive cutaneous anaphylaxis (8-day PCA) and the 8-day passive systemic anaphylaxis induced by IgE. The compound partially inhibited the IgG-mediated 3-hr PCA in rats and guinea pigs, but failed to have any effect on the rabbit IgG-mediated 3-hr PCA in these animals. In the rat, MTB was not an antagonist of histamine or serotonin. The antiallergic effect of MTB was not mediated via any adrenergic mechanisms. MTB significantly inhibited histamine release from rat peritoneal cells induced by rat IgE in vitro.     

Preparation and crystal characterization of a polymorph, a monohydrate, and an ethyl acetate solvate of the antifungal fluconazole

The preparation and solid-state characterization of three crystalline modifications of the antifungal agent fluconazole [2-(2,4-difluorophenyl)-1,3-bis-(1H-124-triazol-1-yl)-propan-2-ol] are reported. Recrystallization of fluconazole from propan-2-ol yielded a polymorph (Form III), whereas the solvents water and ethyl acetate yielded the solvated products fluconazole monohydrate and fluconazole. (ethyl acetate)(0.25), respectively. These species were analyzed by thermogravimetry (TGA), differential scanning calorimetry (DSC), FTIR spectroscopy, powder X-ray diffractometry (PXRD), and single crystal X-ray diffraction. Availability of the hitherto unknown crystal structures facilitated interpretation of the thermal data and clarified previous findings relating to the polymorphism of this compound. Fluconazole was found to exist as a centrosymmetric hydrogen bonded dimer in Form III. For the solvated phases, the solvent locations within the drug host matrices were established as isolated sites for water molecules and constricted channels for ethyl acetate molecules. Desolvation of the monohydrate and ethyl acetate solvate yielded polymorphic Form I. Reference PXRD patterns computed from the refined single-crystal X-ray data for the title compounds are presented.     

Antitumor activity of the alkylating oligopeptides J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester) and P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester): comparison with melphalan

Peptichemio, a mixture of six short oligopeptides all comprising the alkylating amino acid m-L-sarcolysin, has shown clinical activity in several malignancies. Previous studies have suggested that activity mainly resides in one of the peptides, P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester). In the present study the in vitro activity of P2 was further investigated and compared to melphalan and the novel alkylating dipeptide J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester), which is structurally related to P2 and melphalan. Cytotoxic activity was studied using patient tumor cells in a non-clonogenic cytotoxicity assay, whereas cellular response, and kinetics thereof, were studied in the lymphoma cell line U-937 GTB. Cellular metabolism was studied using microphysiometry, kinetic effects on macromolecular synthesis by radiolabeled substrate incorporation and, finally, the microculture kinetic assay of apoptosis was used to monitor morphologic changes following drug exposure. The assays compared P2 favorably with melphalan. Interestingly J1 was even more cytotoxic, and produced more pronounced effects in the kinetic assays for macromolecular synthesis, metabolic activity and apoptosis. The results indicate that the delivery properties of J1 are improved compared to those of melphalan and P2.