Pharmacological Effects of Panduratin A on Renal Cyst Development in In Vitro and In Vivo Models of Polycystic Kidney Disease.

Renal cyst expansion in polycystic kidney disease (PKD) involves abnormalities in both cyst-lining-cell proliferation and fluid accumulation. Suppression of these processes may retard the progression of PKD. Evidence suggests that the activation of 5' AMP-activated protein kinase (AMPK) inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride secretion, leading to reduced progression of PKD. Here we investigated the pharmacological effects of panduratin A, a bioactive compound known as an AMPK activator, on CFTR-mediated chloride secretion and renal cyst development using in vitro and animal models of PKD. We demonstrated that AMPK was activated in immortalized normal renal cells and autosomal dominant polycystic kidney disease (ADPKD) cells following treatment with panduratin A. Treatment with panduratin A reduced the number of renal cyst colonies corresponding with a decrease in cell proliferation and phosphorylated p70/S6K, a downstream target of mTOR signaling. Additionally, panduratin A slowed cyst expansion via inhibition of the protein expression and transport function of CFTR. In heterozygous Han:Sprague-Dawley (Cy/+) rats, an animal model of PKD, intraperitoneal administration of panduratin A (25 mg/kg BW) for 5 weeks significantly decreased the kidney weight per body weight ratios and the cystic index. Panduratin A also reduced collagen deposition in renal tissue. Intraperitoneal administration of panduratin A caused abdominal bleeding and reduced body weight. However, 25 mg/kg BW of panduratin A via oral administration in the PCK rats, another non-orthologous PKD model, showed a significant decrease in the cystic index without severe adverse effects, indicating that the route of administration is critical in preventing adverse effects while still slowing disease progression. These findings reveal that panduratin A might hold therapeutic properties for the treatment of PKD.

Protective Effect of Panduratin A on Cisplatin-Induced Apoptosis of Human Renal Proximal Tubular Cells and Acute Kidney Injury in Mice.

BACKGROUND: Cisplatin is an effective chemotherapy but its main side effect, acute kidney injury, limits its use. Panduratin A, a bioactive compound extracted from Boesenbergia rotunda, shows several biological activities such as anti-oxidative effects. The present study investigated the nephroprotective effect of panduratin A on cisplatin-induced renal injury. METHODS: We investigated the effect of panduratin A on the toxicity of cisplatin in both mice and human renal cell cultures using RPTEC/TERT1 cells. RESULTS: The results demonstrated that panduratin A ameliorates cisplatin-induced renal toxicity in both mice and RPTEC/TERT1 cells by reducing apoptosis. Mice treated with a single intraperitoneal (i.p.) injection of cisplatin (20 mg/kg body weight (BW)) exhibited renal tubule injury and impaired kidney function as shown by histological examination and increased serum creatinine. Co-administration of panduratin A (50 mg/kg BW) orally improved kidney function and ameliorated renal tubule injury of cisplatin by inhibiting activation of extracellular signal-regulated kinase (ERK)1/2 and caspase 3. In human renal proximal tubular cells, cisplatin induced cell apoptosis by activating pro-apoptotic proteins (ERK1/2 and caspase 3), and reducing the anti-apoptotic protein (Bcl-2). These effects were significantly ameliorated by co-treatment with panduratin A. Interestingly, panduratin A did not alter intracellular accumulation of cisplatin. It did not alter the anti-cancer efficacy of cisplatin in either human colon or non-small cell lung cancer cell lines. CONCLUSIONS: The present study highlights panduratin A has a potential protective effect on cisplatin's nephrotoxicity.

Panduratin A Derivative Protects against Cisplatin-Induced Apoptosis of Renal Proximal Tubular Cells and Kidney Injury in Mice.

BACKGROUND: Panduratin A is a bioactive cyclohexanyl chalcone exhibiting several pharmacological activities, such as anti-inflammatory, anti-oxidative, and anti-cancer activities. Recently, the nephroprotective effect of panduratin A in cisplatin (CDDP) treatment was revealed. The present study examined the potential of certain compounds derived from panduratin A to protect against CDDP-induced nephrotoxicity. METHODS: Three derivatives of panduratin A (DD-217, DD-218, and DD-219) were semi-synthesized from panduratin A. We investigated the effects and corresponding mechanisms of the derivatives of panduratin A for preventing nephrotoxicity of CDDP in both immortalized human renal proximal tubular cells (RPTEC/TERT1 cells) and mice. RESULTS: Treating the cell with 10 µM panduratin A significantly reduced the viability of RPTEC/TERT1 cells compared to control (panduratin A: 72% ± 4.85%). Interestingly, DD-217, DD-218, and DD-219 at the same concentration did not significantly affect cell viability (92% ± 8.44%, 90% ± 7.50%, and 87 ± 5.2%, respectively). Among those derivatives, DD-218 exhibited the most protective effect against CDDP-induced renal proximal tubular cell apoptosis (control: 57% ± 1.23%; DD-218: 19% ± 10.14%; DD-219: 33% ± 14.06%). The cytoprotective effect of DD-218 was mediated via decreases in CDDP-induced mitochondria dysfunction, intracellular reactive oxygen species (ROS) generation, activation of ERK1/2, and cleaved-caspase 3 and 7. In addition, DD-218 attenuated CDDP-induced nephrotoxicity by a decrease in renal injury and improved in renal dysfunction in C57BL/6 mice. Importantly, DD-218 did not attenuate the anti-cancer efficacy of CDDP in non-small-cell lung cancer cells or colon cancer cells. CONCLUSIONS: This finding suggests that DD-218, a derivative of panduratin A, holds promise as an adjuvant therapy in patients receiving CDDP.

Preparation and Characterizations of RSPP050-Loaded Polymeric Micelles Using Poly(ethylene glycol)-b-Poly(ε-caprolactone) and Poly(ethylene glycol)-b-Poly(D,L-lactide).

RSPP050 (AG50) is one of the semi-synthetic andrographolide that is isolated from Andrographis paniculata NEES (Acanthaceae). The anti-proliferation effects of AG50 against cholangiocarcinoma (HuCCT1) were displayed high cytotoxicity. Unfortunately, poor water solubility of AG50 limited its clinical applications. This study aimed to increase the concentration of AG50 in water and drug loading and release study in phosphate-buffered saline (PBS) in the absence/presence of pig liver esterase enzyme. Cytotoxicity of AG50-loaded polymeric micelles was evaluated against HuCCT1. AG50 loaded micelles were prepared by film sonication and encapsulated by polymers including poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) or poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-b-PLA). Micelle properties were characterized such as solubility, drug loading, drug release and in vitro cytotoxicity against HuCTT1. AG50 was successfully loaded into both types of polymeric micelles. The best drug-polymer (D/P) ratio was 1 : 9. AG50/PCL and AG50/PLA-micelles had small particle size (36.4±5.1, 49.0±2.7 nm, respectively) and high yield (58.2±1.8, 58.8±2.9, respectively). AG50/PLA-micelles (IC50=2.42 µg/mL) showed higher cytotoxicity against HuCCT1 than AG50/PCL-micelles (IC50=4.40 µg/mL) due to the higher amount of AG50 released. Nanoencapsulation of AG50 could provide a promising development in clinical use for cholangiocarcinoma treatment.

Protective Effects of a Diarylheptanoid from Curcuma comosa Against Hydrogen Peroxide-Induced Astroglial Cell Death.

Oxidative stress is one of the major mechanisms causing neuronal and astroglial cell death in various neurological disorders such as Alzheimer's disease, Parkinson's disease, and brain ischemia. Two diarylheptanoids, (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (ASPP 049) and (3S)-7-(3,4-dihydroxyphenyl)-1-phenyl-(1E)-1-hepten-3-ol (ASPP 092), isolated from Curcuma comosa were investigated for cytoprotective effects on C6 astroglial cells using hydrogen peroxide (H2O2) exposure as a model of oxidative stress. ASPP 092 demonstrated free radical scavenging activity comparable to that of vitamin C, while ASPP 049 showed no antioxidant activity. Treatment with H2O2 at 400 µM for 12 h caused 79 % C6 astroglial cell death which was significantly reduced to 37 % by pretreatment with ASPP 092 (5 µM). In addition, ASPP 092 attenuated the increase in reactive oxygen species production and the decrease in total glutathione level induced by H2O2. The mechanism of ASPP 092 protection against H2O2-induced apoptotic signaling appeared to involve prevention of increase in the level of phosphorylated p53 and the Bax/Bcl-2 ratio as well as cleaved caspase-3. These findings provide new evidence that the diarylheptanoid ASPP 092 from C. comosa possesses antiapoptotic properties and could be further developed as a potential treatment for oxidative stress-related neuronal diseases.

Halogenated benzoate derivatives of altholactone with improved anti-fungal activity.

Altholactone exhibited the anti-fungal activity with a high MIC value of 128 µg ml(-1) against Cryptococcus neoformans and Saccharomyces cerevisiae. Fifteen ester derivatives of altholactone 1-15 were modified by esterification and their structures were confirmed by spectroscopic methods. Most of the ester derivatives exhibited stronger anti-fungal activities than that of the precursor altholactone. 3-Bromo- and 2,4-dichlorobenzoates (7 and 15) exhibited the lowest minimal inhibitory concentration (MIC) values against C. neoformans at 16 µg ml(-1), while the 4-bromo-, 4-iodo-, and 1-bromo-3-chlorobenzoates (11-13) displayed potent activity against S. cerevisiae with MIC values of 1 µg ml(-1). In conclusion, this analysis indicates that the anti-fungal activity of altholactone is enhanced by addition of halogenated benzoyl group to the 3-OH group.

Chitosan-functionalised poly(2-hydroxyethyl methacrylate) core-shell microgels as drug delivery carriers: salicylic acid loading and release.

This work presents the evaluation of chitosan-functionalised poly(2-hydroxyethyl methacrylate) (CS/PHEMA) core-shell microgels as drug delivery carriers. CS/PHEMA microgels were prepared by emulsifier-free emulsion polymerisation with N,N '-methylenebisacrylamide (MBA) as a crosslinker. The study on drug loading, using salicylic acid (SA) as a model drug, was performed. The results showed that the encapsulation efficiency (EE) increased as drug-to-microgel ratio was increased. Higher EE can be achieved with the increase in degree of crosslinking, by increasing the amount of MBA from 0.01 g to 0.03 g. In addition, the highest EE (61.1%) was observed at pH 3. The highest release of SA (60%) was noticed at pH 2.4, while the lowest one (49.4%) was obtained at pH 7.4. Moreover, the highest release of SA was enhanced by the presence of 0.2 M NaCl. The pH- and ionic-sensitivity of CS/PHEMA could be useful as a sustained release delivery device, especially for oral delivery.

Protective effect of diarylheptanoids from Curcuma comosa on primary rat hepatocytes against t-butyl hydroperoxide-induced toxicity.

CONTEXT: Curcuma comosa Roxb. (Zingiberaceae) has traditionally been used as an anti-inflammatory agent in liver, and recent study has shown its hepatoprotective effect against CCl4-induced liver injury in vivo. OBJECTIVE: This study further assesses the protective effect of C. comosa extracts and its isolated compounds against tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in isolated primary rat hepatocytes. MATERIALS AND METHODS: Isolated primary hepatocytes were pretreated with either ethanol (5-50 µg/ml) or hexane extract (1-50 µg/ml), or two diarylheptanoids (4-35 µM): compound D-91 [1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol] and compound D-92 [(3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol], from C. comosa for 2 h prior to exposure to 1.5 mM t-BHP for 15 and 30 min. Their hepatoprotective activities were then determined. RESULTS: t-BHP markedly caused the formation of MDA and ALT leakage from the hepatocytes. Pretreatment with the C. comosa ethanol extract showed greater protective effect than the hexane extract, and the effect was concentration related. Treating the hepatocytes with compound D-92 provided greater protective effect than compound D-91. IC50 values of compounds D-91, D-92, and silymarin for the protection of ALT leakage at 30 min were 32.7 ± 1.1, 9.8 ± 0.7, and 160 ± 8 µM, respectively. Further investigation showed that compound D-92 was more effective in maintaining the intracellular glutathione content in the t-BHP treated group, whereas the reduction in antioxidant enzymes, glutathione peroxidase and glutathione-S-transferase activities, were not improved. DISCUSSION AND CONCLUSION: Results suggest that diarylheptanoids are the active principles that provide protection against t-BHP-induced injury. Their ability to maintain intracellular glutathione content is the main mechanisms underlying the protective action.

Asymmetric synthesis of gem-difluoromethylenated linear triquinanes via cascade gem-difluoroalkyl radical cyclization.

An asymmetric synthesis of gem-difluoromethylenated linear triquinanes is described exploiting the synthetic utilities of PhSCF2TMS (5) as a "(•)CF2(-)'' building block. The strategy involves fluoride-catalyzed nucleophilic addition of PhSCF2TMS (5) to chiral ketocyclopentenes 6 to provide silylated adducts 9 or alcohol derivatives 10 and 11. Subsequent cascade radical cyclization of the gem-difluoroalkyl radical generated from silylated adducts 9 or alcohols 10 and 11 afforded gem-difluoromethylenated linear triquinanes 16 as an approximate 1:1 mixture of two diastereomers (16A and 16B). Alternatively, a convenient asymmetric synthesis of gem-difluoromethylenated linear triquinanes 16A can be accomplished by oxidation of 16a (R = H) to provide ketotriquinane 17 followed by a highly stereoselective nucleophilic addition to 17 employing DIBAL, NaBH4, and various Grignard reagents.

Synthesis of gem-difluoromethylenated polycyclic cage compounds.

The synthesis of gem-difluoromethylenated polycyclic cage compounds, utilizing PhSCF2SiMe3 as a gem-difluoromethylene building block, is described. The fluoride-catalyzed nucleophilic addition of PhSCF2SiMe3 to both maleic anhydride-cyclopentadiene and maleic anhydride-cyclohexadiene adducts was accomplished with high stereoselectivity to provide the corresponding adducts that were treated with Grignard reagents, followed by acid-catalyzed lactonization to afford the corresponding γ-butyrolactones, each as a single isomer. These γ-butyrolactones underwent intramolecular radical cyclization to give the corresponding tetracyclic cage γ-butyrolactones, which were employed as precursors for the synthesis of gem-difluoromethylenated tetracyclic cage lactols or tetracyclic cage furans, upon treatment with Grignard reagents.

Spirotetronate antibiotics with anti-Clostridium activity from Actinomadura sp. 2EPS.

The rare actinomycetes strain 2EPS was isolated from soil and analysis of cultural, morphological characteristics, diaminopimelic acid content of its cell wall, and 16S rRNA gene sequence indicates that 2EPS belongs to genus Actinomadura. In addition, neighbor-joining phylogenetic tree also confirmed the relationships of this strain to other members of Actinomadura. A butanol extract with antibacterial activity was purified by reversed-phase chromatography to obtain three bioactive compounds, designated as compounds 1, 2 and 3. The structures of these compounds were determined using spectroscopic analysis ((1)H-NMR and (13)C-NMR) and mass spectrometric analysis (HR-TOF-MS). Compounds 1-3 were identified and found to be the same as those included in the Japanese patent number JP 09227587 for spirotetronate antibiotics and are BE-45722A (1), BE-45722B (2) and BE-45722C (3), respectively. All compounds were active against Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579, and B. subtilis ATCC 6633) with low MIC values between 0.08 and 5.0 µg/ml. Moreover, both 1 and 3 also exhibited strong activity, with similar MIC values, against Clostridium perfringens S107 at 0.63 µg/ml and C. difficile 630 at 0.08 µg/ml. These results suggest the identified spirotetronate compounds may have potential in the treatment of Clostridium infections. Overall, this analysis demonstrates that rare actinomycetes are a promising source for discovery of antimicrobial compounds.

Formal synthesis of (+)-3-epi-eupomatilone-6 and the 3,5-bis-epimer.

The formal synthesis of (+)-3-epi-eupomatilone-6 () and the 3,5-bis-epimer () has been accomplished. The key synthetic strategy involved the stereoselective construction of (3R,4S,5R)- and (3R,4S,5S)-trisubstituted γ-butyrolactones and from (2R,3R)-2,3-dimethyl-4-pentenoic acid derivative , which was readily obtained via stereoselective conjugate addition of vinylmagnesium chloride to a chiral α,ß-unsaturated N-acyl oxazolidinone (Evans' auxiliary) followed by α-methylation.

Curcumin I mediates neuroprotective effect through attenuation of quinoprotein formation, p-p38 MAPK expression, and caspase-3 activation in 6-hydroxydopamine treated SH-SY5Y cells.

6-Hydroxydopamine (6-OHDA) selectively enters dopaminergic neurons and undergoes auto-oxidation resulting in the generation of reactive oxygen species and dopamine quinones, subsequently leading to apoptosis. This mechanism mimics the pathogenesis of Parkinson's disease and has been used to induce experimental Parkinsonism in both in vitro and in vivo systems. In this study, we investigated the effects of curcumin I (diferuloylmethane) purified from Curcuma longa on quinoprotein production, phosphorylation of p38 MAPK (p-p38), and caspase-3 activation in 6-OHDA-treated SH-SY5Y dopaminergic cells. Pretreatment of SH-SY5Y with curcumin I at concentrations of 1, 5, 10, and 20 µM, significantly decreased the formation of quinoprotein and reduced the levels of p-p38 and cleaved caspase-3 in a dose-dependent manner. Moreover, the levels of the dopaminergic neuron marker, phospho-tyrosine hydroxylase (p-TH), were also dose-dependently increased upon treatment with curcumin I. Our results clearly demonstrated that curcumin I protects neurons against oxidative damage, as shown by attenuation of p-p38 expression, caspase-3-activation, and toxic quinoprotein formation, together with the restoration of p-TH levels. This study provides evidence for the therapeutic potential of curcumin I in the chemoprevention of oxidative stress-related neurodegeneration.

Radical cyclization/ipso-1,4-aryl migration cascade: asymmetric synthesis of 3,3-difluoro-2-propanoylbicyclo[3.3.0]octanes.

A novel method for the asymmetric synthesis of 3,3-difluoro-2-propanoylbicyclo-[3.3.0]octanes involves an unprecedented intramolecular radical cyclization/ipso-1,4-aryl migration cascade.

Fluoride-catalyzed nucleophilic addition of PhSCF2SiMe3 to anhydrides: synthesis of γ-difluoromethylated γ-lactams.

PhSCF2SiMe3 () underwent fluoride-catalyzed nucleophilic addition to the carbonyl group of anhydrides to provide the corresponding γ-difluoro(phenylsulfanyl)methyl γ-lactols, which were employed for the synthesis of γ-difluoromethylated γ-lactams.

A synthesis of γ-trifluoromethyl α,ß-unsaturated γ-butyrolactones using CF(3)SiMe(3) as a trifluoromethylating agent.

A general synthesis of γ-trifluoromethyl α,ß-unsaturated γ-butyrolactones is described. The fluoride-catalyzed nucleophilic addition of a trifluoromethyl (CF3) group generated from (trifluoromethyl)trimethylsilane (CF3SiMe3, Ruppert-Prakash reagent) to a masked maleic anhydride 1 (cyclopentadiene-maleic anhydride adduct) provides the corresponding adducts 2 with high stereoselectivity. The γ-trifluoromethyl α,ß-unsaturated γ-butyrolactones 4 were obtained after treatment of the adducts 2 with Grignard reagents, followed by flash-vacuum pyrolysis.

Cytotoxic, antitopoisomerase IIα, and anti-HIV-1 activities of triterpenoids isolated from leaves and twigs of Gardenia carinata.

Eight new cycloartane triterpenoids (1-8), named carinatins A-H, and the known compounds secaubryolide (9) and dikamaliartane D (10) were isolated from the leaves and twigs of Gardenia carinata. Their structures were determined on the basis of spectroscopic methods. Cytotoxic, antitopoisomerase IIα, and anti-HIV-1 activities of compounds 1-7, 9, and 10 were investigated.

Black ginger extract and its active compound, 5,7-dimethoxyflavone, increase intestinal drug absorption via efflux drug transporter inhibitions.

Black ginger is used as an herbal medicine for self-care and health promotion. Black ginger extract has been shown to alter the function of transporters in several cell types. This study demonstrates the interaction between the extract and 5,7-dimethoxyflavone (DMF) on drug efflux mediated by breast cancer resistance proteins (BCRP) and P-glycoprotein (P-gp) in Caco-2 cells and heterologous cell systems [Madin-Darby canine kidney type II (MDCKII) stably transfected with human BCRP (MDCKII/BCRP) or human P-gp (MDCKII/P-gp)]. The transepithelial flux of 3H-Digoxin and 3H-Estrone sulfate, prototypic substrates of P-gp, and BCRP, respectively, across Caco-2 cell monolayers, MDCKII/BCRP, and MDCKII/P-gp cells were determined. The results demonstrate that black ginger extract (10 µg/ml) significantly increases 3H-Digoxin and 3H-Estrone sulfate transport from the apical to basolateral side while decreasing transport from the basolateral to apical side of Caco-2 cells and MDCKII cell overexpression of BCRP or P-gp. The effect of the extract on 3H-Digoxin and 3H-Estrone sulfate transport was related to a decrease in efflux ratio. Likewise, DMF (5 µM) significantly increased 3H-Digoxin and 3H-Estrone sulfate absorption with a decreased efflux ratio compared to the control. Interestingly, the extract also significantly increased absorption of paclitaxel, an anti-cancer drug, which has poor oral absorption. Taken together, co-administration of drugs as substrates of BCRP and P-gp, with the black ginger extract containing DMF, might alter the pharmacokinetic profiles of the medicine.

ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation.

BACKGROUND: Cleistanthin A (CA), extracted from Phyllanthus taxodiifolius Beille, was previously reported as a potential V-ATPase inhibitor relevant to cancer cell survival. In the present study, ECDD-S16, a derivative of cleistanthin A, was investigated and found to interfere with pyroptosis induction via V-ATPase inhibition. OBJECTIVE: This study examined the ability of ECDD-S16 to inhibit endolysosome acidification leading to the attenuation of pyroptosis in Raw264.7 macrophages activated by both surface and endosomal TLR ligands. METHODS: To elucidate the activity of ECDD-S16 on pyroptosis-induced inflammation, Raw264.7 cells were pretreated with the compound before stimulation with surface and endosomal TLR ligands. The release of lactate dehydrogenase (LDH) was determined by LDH assay. Additionally, the production of cytokines and the expression of pyroptosis markers were examined by ELISA and immunoblotting. Moreover, molecular docking was performed to demonstrate the binding of ECDD-S16 to the vacuolar (V-)ATPase. RESULTS: This study showed that ECDD-S16 could inhibit pyroptosis in Raw264.7 cells activated with surface and endosomal TLR ligands. The attenuation of pyroptosis by ECDD-S16 was due to the impairment of endosome acidification, which also led to decreased Reactive Oxygen Species (ROS) production. Furthermore, molecular docking also showed the possibility of inhibiting endosome acidification by the binding of ECDD-S16 to the vacuolar (V-)ATPase in the region of V0. CONCLUSION: Our findings indicate the potential of ECDD-S16 for inhibiting pyroptosis and prove that vacuolar H+ ATPase is essential for pyroptosis induced by TLR ligands.

Asymmetric synthesis of gem-difluoromethylenated dihydroxypyrrolizidines and indolizidines.

An asymmetric synthesis of gem-difluoromethylenated dihydroxypyrrolizidines and indolizidines is described. The fluoride-catalyzed nucleophilic addition of PhSCF(2)SiMe(3) (1) to chiral imides was achieved in satisfactory yields to provide mixtures of syn- and anti-isomers 6-9 with moderate to good diastereoselectivities. Reductive cleavage of the phenylsulfanyl group followed by intramolecular radical cyclization of the syn-isomers 6-9 occurred under refluxing conditions to afford the corresponding gem-difluoromethylenated 1-azabicyclic compounds 10-13 in moderate yields as a separable mixture of cis- and trans-isomers. The cis-isomers of compounds 10 and 12 and trans-13 were readily transformed to gem-difluoromethylenated dihydroxypyrrolizidines 20 and 27 and indolizidine 28, respectively, by reductive cleavage of the hydroxyl group and organometallic addition followed by hydrogenolysis.