Engineered Imine Reductase for Asymmetric Synthesis of Dextromethorphan Key Intermediate.

(S)-1-(4-Methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-1-(4-methoxybenzyl)-OHIQ) is the key intermediate of the nonopioid antitussive dextromethorphan. In this study, (S)-IR61-V69Y/P123A/W179G/F182I/L212V (M4) was identified with a 766-fold improvement in catalytic efficiency compared with wide-type IR61 through enzyme engineering. M4 could completely convert 200 mM of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline into (S)-1-(4-methoxybenzyl)-OHIQ in 77% isolated yield, with >99% enantiomeric excess and a high space-time yield of 542 g L-1 day-1, demonstrating a great potential for the synthesis of dextromethorphan intermediate in industrial applications.

Preparation of Naringenin Nanosuspension and Its Antitussive and Expectorant Effects.

Naringenin (NRG) is a natural flavonoid compound abundantly present in citrus fruits and has the potential to treat respiratory disorders. However, the clinical therapeutic effect of NRG is limited by its low bioavailability due to poor solubility. To enhance the solubility, naringenin nanosuspensions (NRG-NSps) were prepared by applying tocopherol polyethylene glycol succinate (TPGS) as the nanocarrier via the media-milling method. The particle size, morphology, and drug-loading content of NRG-NSps were examined, and the stability was evaluated by detecting particle size changes in different physiological media. NRG-NSps exhibited a flaky appearance with a mean diameter of 216.9 nm, and the drug-loading content was 66.7%. NRG-NSps exhibited good storage stability and media stability. NRG-NSps presented a sustainable release profile, and the cumulative drug-release rate approached approximately 95% within 7 d. NRG-NSps improved the antitussive effect significantly compared with the original NRG, the cough frequency was decreased from 22 to 15 times, and the cough incubation period was prolonged from 85.3 to 121.6 s. Besides, NRG-NSps also enhanced expectorant effects significantly, and phenol red secretion was increased from 1.02 to 1.45 µg/mL. These results indicate that NRG-NSps could enhance the bioavailability of NRG significantly and possess a potential clinical application.

Pharmaceutical evaluation of Graphirine syrup formulated from some common indigenous herbs of Pakistan.

Since ancient times humans have relied on traditional medicine and herbal formulation for the cure of various diseases and disorders. Globally, interest in use of herbal medicine has increased; as drawbacks of modern medicine and have started getting more prominent. Most of the traditional system of medicine; all in their formulations depends on plants. Traditional herbal cough products have considered to be mild, nontoxic, non-sedative and even harmless. As Syrup is commonly taken for the relief of cough; we aimed to formulate a cough syrup (Graphirine) from some common indigenous herbs; Adhatoda vasica, Piper longum and Rosa Damasena and evaluate its physicochemical parameters along with the changes in accelerated stability testing. Preformulation studies, i.e. moisture content, ash, water and ethanol extractive values of all three herbs were defined. Physicochemical factors, quantitative spectral measurement, FTIR spectrum for identification of compounds; along with accelerated stability and possible microbial fungal growth; were also checked for the formulated syrup. Each and every parameter complied with the specifications.

Antitussive and Anti-inflammatory Dual-active Agents Developed from Natural Product Lead Compound 1-Methylhydantoin.

Natural products play an important role in drug discovery. This work employed a natural product 1-methylhydantoin as the lead compound to develop novel dual-active drugs. 1-Methylhydantoin was isolated from Oviductus Ranae, which is a traditional Chinese medicine that has been used for tussive and inflammation treatment for a long time. An in silico study screened the more active 1-methylhydantoin derivatives. Antitussive assessment indicated that the newly synthesized agent had similar bioactivity with the natural product. An anti-inflammatory model used xylene induced ear edema model. At the same dosage (100 mg/Kg), the newly prepared agent had an inhibition rate 53.18% which was much higher than that of the lead compound (22.69%). The results might be ascribed to the cyclooxygenases-1 (COX-1) and cyclooxygenases-2 (COX-2) selectivity, and the fitness of the compound, and the binding pocket. The anti-particulate matter (PM 2.5) acute pneumonia was evaluated through an in vivo model constructed by nasal instillation with PM 2.5 suspension. The results of the above models suggested that this novel agent had remarkable antitussive, anti-inflammatory, and anti-PM 2.5 acute pneumonia activities.

Noscapine comes of age.

Noscapine is a phthalideisoquinoline alkaloid, which represents a class of plant specialized metabolites within the large and structurally diverse group of benzylisoquinoline alkaloids. Along with the narcotic analgesic morphine, noscapine is a major alkaloid in the latex of opium poppy (Papaver somniferum) that has long been used as a cough suppressant and has undergone extensive investigation as a potential anticancer drug. Cultivated opium poppy plants remain the only commercial source of noscapine. Despite its isolation from opium more than two centuries ago, the almost complete biosynthesis of noscapine has only recently been established based on an impressive combination of molecular genetics, functional genomics, and metabolic biochemistry. In this review, we provide a historical account of noscapine from its discovery through to initial investigations of its formation in opium poppy. We also describe recent breakthroughs that have led to an elucidation of the noscapine biosynthetic pathway, and we discuss the pharmacological properties that have prompted intensive evaluation of the potential pharmaceutical applications of noscapine and several semi-synthetic derivatives. Finally, we speculate on the future potential for the production of noscapine using metabolic engineering and synthetic biology in plants and microbes.

Synthesis and biological evaluation of N-substituted noscapine analogues.

Noscapine is a phthalideisoquinoline alkaloid isolated from the opium poppy Papaver somniferum. It has long been used as an antitussive agent, but has more recently been found to possess microtubule-modulating properties and anticancer activity. Herein we report the synthesis and pharmacological evaluation of a series of 6'-substituted noscapine derivatives. To underpin this structure-activity study, an efficient synthesis of N-nornoscapine and its subsequent reduction to the cyclic ether derivative of N-nornoscapine was developed. Reaction of the latter with a range of alkyl halides, acid chlorides, isocyanates, thioisocyanates, and chloroformate reagents resulted in the formation of the corresponding N-alkyl, N-acyl, N-carbamoyl, N-thiocarbamoyl, and N-carbamate derivatives, respectively. The ability of these compounds to inhibit cell proliferation was assessed in cell-cycle cytotoxicity assays using prostate cancer (PC3), breast cancer (MCF-7), and colon cancer (Caco-2) cell lines. Compounds that showed activity in the cell-cycle assay were further evaluated in cell viability assays using PC3 and MCF-7 cells.

Synthesis and receptor profiling of Stemona alkaloid analogues reveal a potent class of sigma ligands.

Reported biological activities of Stemona natural products, such as antitussive activity, inspired the development of synthetic methods to access several alkaloids within this family and in so doing develop a general route to the core skeleta shared by the class of natural products. The chemistry was subsequently adapted to afford a series of analogue sets bearing simplified, diverse Stemona-inspired skeleta. Over 100 of these analogues were subjected to general G protein-coupled receptor profiling along with the known antitussive compound, neostenine; this led to the identification of hit compounds targeting several receptor types. The particularly rich hit subset for sigma receptors was expanded with two focused library sets, which resulted in the discovery of a fully synthetic, potent chemotype of sigma ligands. This collaborative effort combined the development of synthetic methods with extensive, flexible screening resources and exemplifies the role of natural products in bioactivity mining.

Addictive evaluation of cholic acid-verticinone ester, a potential cough therapeutic agent with agonist action of opioid receptor.

AIM: The purpose of this work was to search for potential drugs with potent antitussive and expectorant activities as well as a low toxicity, but without addictive properties. Cholic acid-verticinone ester (CA-Ver) was synthesized based on the clearly elucidated antitussive and expectorant activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. In our previous study, CA-Ver showed a much more potent activity than codeine phosphate. This study was carried out to investigate the central antitussive mechanism and the addictive evaluation of CA-Ver. METHODS: Testing on a capsaicin-induced cough model of mice pretreated with naloxone, a non-selective opioid receptor antagonist, was performed for the observation of CA-Ver's central antitussive mechanism. We then took naloxone-induced withdrawal tests of mice for the judgment of CA-Ver's addiction. Lastly, we determined the opioid dependence of CA-Ver in the guinea pig ileum. RESULTS: The test on the capsaicin-induced cough model showed that naloxone could block the antitussive effect of CA-Ver, suggesting the antitussive mechanism of CA-Ver was related to the central opioid receptors. The naloxone-urged withdrawal tests of the mice showed that CA-Ver was not addictive, and the test of the opioid dependence in the guinea pig ileum showed that CA-Ver had no withdrawal response. CONCLUSION: These findings suggested that CA-Ver deserved attention for its potent antitussive effects related to the central opioid receptors, but without addiction, and had a good development perspective.

The discovery of tropane derivatives as nociceptin receptor ligands for the management of cough and anxiety.

The discovery of 1 as a high-affinity ligand for the nociceptin receptor has led to the synthesis of a series of tropane (8-methyl-8-azabicyclo[3.2.1]octane) derivatives as optimized ligands. These compounds exhibit high affinity for the nociceptin receptor, moderate to excellent selectivity over the opioid mu receptor, and behave as full agonists. In this Letter, we present the synthesis and highlight the structure-activity relationship of tropane derivatives culminating in the identification of 24 and 32 as potent and orally active antitussive and anxiolytic agents. The in vitro and in vivo activities, pharmacokinetic profile, and the hPXR activity, which predicts the potential 3A4 induction in human, are disclosed.

Structural analysis and antitussive evaluation of five novel esters of verticinone and bile acids.

Shedan-Chuanbei powder, a complex of traditional Chinese medicine preparation, which consists of Snake Bile (Chinese name "Shedan") and Fritillariae Cirrhosae (Chinese name "Chuanbei"), is the most popular antitussive and expectorant formulation in Chinese communities. However, the clinical application of Shedan-Chuanbei powder is now stringently limited because of the shortage of the two crude medicinal materials, especially for the sake of animal protection. In addition, the inherent defects of the most of the complex of traditional Chinese medicine such as the indistinct basal pharmacodynamic materials and the difficulties in quality control had blocked them heading into the international medicinal market. So we attempted to seek new substitute for Shedan-Chuanbei powder for antitussive drugs. In order to gain some new compounds with better bioactivity and attenuated toxicity, we tried to combine two kinds of drugs through ester bond. Enlightened with "combination principle" in drug discovery, we synthesized five novel esters of verticinone and bile acids, both of which are the major bioactive components in Shedan-Chuanbei powder. We then evaluated the antitussive activity and the acute toxicity of the five ester-linked compounds. The five ester-linked compounds had much more potent antitussive activity and expectorant activity than single bile acids at the same doses, and had equivalent antitussive activity and expectorant activity in comparison with about double moles dose of the monomer verticinone. Especially, cholic acid-verticinone ester had much more potent antitussive effects than the monomer verticinone or cholic acid at the same dose. A further acute toxicity study showed that the LD(50) values of the five ester-linked compounds exceeded 3.5g/kg by intraperitoneal injection in mice. Based on the studies of pharmacology and acute toxicity, the five ester-linked compounds have synergic pharmacodynamic action and attenuated toxicity compared with single verticinone and single bile acids.

Design and synthesis of a metabolically stable and potent antitussive agent, a novel delta opioid receptor antagonist, TRK-851.

We have previously reported on antitussive effect of (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-5',6'-dihydro-3-methoxy-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-14-ol(1b) methanesulfonate (TRK-850), a selective delta opioid receptor antagonist which markedly reduced the number of coughs in a rat cough model. We designed TRK-850 based on naltrindole (NTI), a typical delta opioid receptor antagonist, to improve its permeability through the blood-brain barrier by introducing hydrophobic moieties to NTI. The ED(50) values of NTI and compound 1b by intraperitoneal injections were 104 microg/kg and 2.07 microg/kg, respectively. This increased antitussive potency probably resulted from the improved brain exposure of compound 1b. However, 1b was extremely unstable toward metabolism by cytochrome P450. In this study, we designed and synthesized compound 1b derivatives to improve the metabolic instability, which resulted in affording highly potent and metabolically stable oral antitussive agent (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-8'-fluoro-5',6'-dihydro-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-3,14-diol (1c) methanesulfonate (TRK-851).

Structure-antitussive activity relationships of naltrindole derivatives. Identification of novel and potent antitussive agents.

We have previously reported antitussive effects of naltrindole (NTI), a typical delta opioid receptor antagonist, in a rat model. The ED50 values of NTI by intraperitoneal and peroral injections were 104 microg/kg and 1840 microg/kg, respectively, comparable to those of codeine. Codeine, one of the most reliable centrally acting antitussive drugs, has micro agonist activity and thus the same side effects as morphine, e.g., constipation, dependency, and respiratory depression. Because NTI is a delta opioid antagonist, its derivatives have potential as highly potent antitussives, free from the mu opioid agonist side effects. We attempted to optimize the NTI derivatives to develop novel antitussive agents. On the basis of the studies of structure-antitussive activity relationships of alkyl substituted NTI derivatives, we designed NTI derivatives with extra ring fused structures. As a clinical candidate, we identified a highly potent new compound, (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-5',6'-dihydro-3-methoxy-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-14-ol (5b) methanesulfonate (TRK-850) which was effective even by oral administration (ED50 6.40 microg/kg).

Synthesis and antitussive evaluation of verticinone-cholic acid salt, a novel and potential cough therapeutic agent.

AIM: To seek a novel and potent antitussive drug based on Shedan-Chuanbei powder, a complex of traditional Chinese medicine preparation for cough therapy. METHODS: Verticinone-cholic acid (Ver-CA) salt, a novel, salifying derivative of verticinone and cholic acid, both of which are the major bioactive components in Shedan-Chuanbei powder, was synthesized. We then evaluated the antitussive activity and the acute toxicity of the salt. RESULTS: The new compound, with good solubility in water, has much more potent antitussive activity in comparison with the same dose of single verticinone and single cholic acid. The administration 3 mg/kg of Ver-CA could result in over 50% reduction of a citric acid-induced cough. Pretreatment with naloxone (0.8 mg/kg, ip) can only partially antagonize its antitussive effect. On the other hand, glybenclamide (3 mg/kg, ip), an ATP-sensitive K+ channel blocker, can also significantly reduce the antitussive effect of Ver-CA. A further acute toxicity study showed that the LD(50) values of Ver-CA were 3 times that of verticinone. CONCLUSION: Based on the studies of pharmacology and acute toxicity, the salt has a synergic and attenuated toxicity compared with single verticinone and cholic acid. Moreover, the present study also suggests that Ver-CA, a potential novel antitussive agent, may exert its antitussive effect via both the peripheral (modulated by ATP-sensitive K+ channels) and central mechanisms (modulated by the opioid receptor).

Synthesis and structure-activity relationships of 4-hydroxy-4-phenylpiperidines as nociceptin receptor ligands: Part 2.

A series of 4-[2-(aminomethyl)phenyl]-1-[bis(2-chlorophenyl)methyl]-4-hydroxypiperidine analogs has been identified as nociceptin receptor ligands. These compounds display high affinity and functional activity at the nociceptin receptor. The synthesis and structure-activity relationships at the C-4 phenyl and N-1 positions are described and the antitussive activity of a selected compound is reported.

Synthesis and biological evaluation of a cyclic ether fluorinated noscapine analog.

We present here a novel semi-synthetic cyclic ether fluorinated noscapine analog (CEFNA) that shows potent antiproliferative and anticancer activity in both hormone-responsive (MCF-7) and hormone non-responsive (MDA-MB-231) breast cancer cells. Interestingly, it is also effective against MCF-7/Adr, an adriamycin-resistant variant of MCF-7 cells. Immunofluorescence experiments showed numerous micronuclei, indicative of apoptotic cell death triggered by this novel analog. Mechanistically, CEFNA exerts a strong antimitotic effect as revealed by cell-cycle studies that show a dose-dependent increase in G2/M population preceding a rising sub-G1 population, suggesting apoptosis.

Identification of some benproperine metabolites in humans and investigation of their antitussive effect.

AIM: To identify 4 unknown metabolites of benproperine (BPP, 1) in human urine after a po dose, and to investigate the antitussive effect of monohydroxylate metabolites. METHODS: The putative metabolite references were prepared using chemical synthesis. Their structures were identified using 1H and 13C nuclear magnetic resonance, and mass spectrometry. The metabolites in human urine were separated and assayed using liquid chromatography-ion trap mass spectrometry (LC/MS/MS), and further confirmed by comparison of their mass spectra and chromatographic retention times with those of synthesized reference substances. The antitussive effects of metabolites were evaluated on coughs induced by 7.5% citric acid in conscious guinea pigs. RESULTS: 1-[1-Methyl-2-[2-(phenylmethyl)phenoxy]-ethyl]-4-piperidinol (2), 1-[1-methyl-2-[2-(phenylmethyl)phenoxy] ethyl]-3-piperidinol (3) and their glucuronides 4 and 5 were obtained from chemical synthesis. Four urinary metabolites in human urine showed peaks with the same chromatographic retention times and mass spectra in LC/MS/MS as synthetic substances 2, 3, 4 and 5. Phosphates of compounds 2 and 3 prolonged the latency of cough and reduced the number of coughs during the 3 min test using citric acid, but did not reduce the number of coughs during the 5 min immediately after the test in conscious guinea pigs. CONCLUSION: Compounds 2, 3, 4, and 5 were identified as the metabolites of BPP in human urine. Among them, compounds 2 and 3 are inactive in the antitussive effect.

Chemo-enzymatic synthesis of levodropropizine.

Levodropropizine, an antitussive drug, was prepared in high enantiomeric excess in three steps, starting from dichloroacetone (2). Monosubstitution of 2 with sodium benzoate and subsequent baker's yeast reduction stereoselectively afforded the corresponding chlorohydrin in 73% ee, which was converted to levodropropizine and enantiomerically enriched up to 95% ee by fractional crystallisation.

N-acyl-2-substituted-1,3-thiazolidines, a new class of non-narcotic antitussive agents: studies leading to the discovery of ethyl 2-[(2-methoxyphenoxy)methyl]-beta-oxothiazolidine-3-propanoate.

The synthesis of a novel class of antitussive agents is described. The compounds were examined for antitussive activity in guinea pig after cough induction by electrical or chemical stimulation. Ethyl 2-[(2-methoxyphenoxy)methyl]-beta-oxothiazolidine-3-propanoate (BBR 2173, moguisteine, 7) and other structurally related compounds showed a significant level of activity, comparable to that of codeine and dextromethorphan. The compounds presented in this paper are characterized by the N-acyl-2-substituted-1,3-thiazolidine moiety, which is a novel entry in the field of antitussive agents. The serendipitous discovery of the role played by the thiazolidine moiety in determining the antitussive effect promoted extensive investigations on these structures. This optimization process on N-acyl-2-substituted-1,3-thiazolidines led to the initial identification of 2-[(2-methoxypheoxy)methyl]-3-[2-(acetylthio)acetyl]- 1,3-thiazolidine (18a) as an interesting lead compound. The careful study of the rapid and very complicated metabolism of 18a provided further insights for the design of newer related derivatives. The observation that the metabolic oxidation on the lateral chain's sulfur of 18a to sulfoxide maintained the antitussive properties suggested the introduction of isosteric functional groups with respect to the sulfoxide moiety. Subsequent structural modifications showed that hydrolyzable malonic residues in the 3-position of the thiazolidine ring were able to assure high antitussive activity. This optimization ultimately led to the selection of moguisteine (7) as the most effective and safest representative of the series. Moguisteine is completely devoid of unwanted side effects (such as sedation and addiction), and its activity was demonstrated also in clinical studies.

Synthesis and some properties of two salsalate derivatives.

The synthesis of esters of 2-hydroxy benzoic acid-2-carboxyphenyl ester (salsalate) with guaiacol for the treatment of inflammatory bronchopneumopathies is reported. The antiinflammatory, analgesic and antipyretic activities of these derivatives were evaluated, together with their antioxidant, mucolytic and broncho-bacteriostatic properties in comparison to acetylsalicylic acid.