Synthesis of New 2,3‐Dihydroquinazolin‐4(1H)‐one Derivatives for Analgesic and Anti‐inflammatory Evaluation

Starting from isatoic anhydrides, several new 2,3‐dihydroquinazolin‐4(1H)‐one derivatives bearing chalcone or pyrazole or thiazole moieties at the third position were synthesized. The analgesic and anti‐inflammatory activities for most compounds were studied at a dose level of 50 mg/kg via the acetic‐acid‐induced writhing‐response method and carrageenan‐induced edema method, respectively. The study showed that the chalcones bearing a 4‐chlorophenyl group 4c or 4‐nitrophenyl group 4b were the most active ones as analgesics. Both chalcone 4c and N‐phenyl pyrazole bearing 4‐methoxy phenyl group 5b showed a higher anti‐inflammatory activity than celecoxib but still lower than that of diclofenac sodium. Moreover, the chalcone 4c has nearly the same ulcerogenic index as the selective cyclooxygenase‐2 inhibitor celecoxib.     

Synthesis and Antibacterial Activity of a New Series of 3‐[3‐(Substituted Phenyl)‐1‐Isonicotinoyl‐1H‐Pyrazol‐5‐yl]‐2H‐Chromen‐2‐one Derivatives

A novel series of 3‐[3‐(substituted phenyl)‐1‐isonicotinoyl‐1H‐pyrazol‐5‐yl]‐2H‐chromen‐2‐one derivatives 4a – k have been synthesized by the reaction of 3‐[2,3‐dibromo‐3‐(substituted phenyl) propanoyl]‐2H‐chromen‐2‐one 3a – k and isonicotinic acid hydrazide in the presence of triethylamine in absolute ethanol, characterized by spectral data and screened for their in‐vitro antibacterial activity against Gram‐positive and Gram‐negative bacteria. Among the series, compounds 4e , 4i , and 4k displayed an encouraging antibacterial activity profile as compared to the reference drug ampicillin against tested bacterial strains.     

Synthesis of N‐(piperidin‐1‐yl)‐5‐(4‐methoxyphenyl)‐1‐(2‐chlorophenyl)‐4‐[18F]fluoro‐1H‐pyrazole‐3‐carboxamide by nucleophilic [18F] fluorination: a PET radiotracer for studying CB1 cannabinoid receptors

The feasibility of nucleophilic displacement of bromide in the 4‐bromopyrazole ring with [¹⁸F]fluoride has been demonstrated by the synthesis of two radiolabeled compounds: N‐(piperidin‐1‐yl)‐5‐(4‐methoxyphenyl)‐1‐(2‐chlorophenyl)‐4‐[¹⁸F]fluoro‐1H‐pyrazole‐3‐carboxamide, ([¹⁸F] NIDA‐42033) 1b and 1‐(2‐chlorophenyl)‐4‐[¹⁸F]fluoro‐5‐(4‐methoxyphenyl)‐1H‐pyrazole‐3‐carboxylic acid, ethyl ester 4 . The radiochemical yields were in the range of 1–6%. [¹⁸F]NIDA‐42033, a potential radiotracer for the study of CB₁ cannabinoid receptors in the animal brain by positron emission tomography, has been synthesized in sufficient quantities with specific radioactivity greater than 2500 mCi/μmol and radiochemical purity >95%. Copyright © 2002 John Wiley & Sons, Ltd.     

Effectiveness of Novel 5‐(5‐amino‐1‐aryl‐1H‐pyrazol‐4‐yl)‐1H‐tetrazole Derivatives Against Promastigotes and Amastigotes of Leishmania amazonensis

In this research, a series of substituted 5‐(5‐amino‐1‐aryl‐1H‐pyrazol‐4‐yl)‐1H‐tetrazoles were synthesized and evaluated for in vitro antileishmanial activity. Among the derivatives, examined compounds 3b and 3l exhibited promising activity against promastigotes and amastigotes forms of Leishmania amazonensis. The cytotoxicity of these compounds was evaluated on murine cells, giving access to the corresponding selectivity index (SI).     

Structure elucidation of the designer drug N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide and the relevance of predicted 13C NMR shifts – a case study

The detailed structure elucidation process of the new cannabimimetic designer drug, N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, with a highly substituted pyrazole skeleton, using nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) techniques is described. After a first analysis of the NMR spectra and comparison with 48 possible pyrazole and imidazole structures, a subset of six positional isomeric pyrazoles and six imidazoles remained conceivable. Four substituents of the heterocyclic skeleton were identified: a proton bound to a pyrazole ring carbon atom; a 5‐fluoropentyl group; a 4‐fluorophenyl substituent; and a carbamoyl group, which is N‐substituted with a methyl residue carrying a tert.‐butyl and a carbamoyl substituent. The 5‐fluoropentyl residue is situated at the nitrogen ring atom. Additional NMR experiments like the ¹H,¹³C HMBC were performed, but due to the small number of signals based on long‐range couplings, the comparison of predicted and observed ¹³C chemical shifts became necessary. The open access Internet shift prediction programs NMRDB, NMRSHIFTDB2, and CSEARCH were employed for the prediction of ¹³C shift values which allowed an efficient and unambiguous structure determination. For the identified N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, the best agreement between predicted ¹³C shifts and the observed chemical shifts and long‐range couplings for the pyrazole ring carbon atoms, with a standard error of about 2 ppm, was found with each of the predictions. For the comparison of measured and predicted chemical shifts model compounds with simple substituents proved helpful. The identified compound is a homologue of AZ‐037 which is offered by Internet suppliers. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis of 3‐[[4‐(4‐[18F] fluorophenyl)piperazin‐1‐yl]methyl]‐1H‐pyrrolo[2,3‐b]pyridine

3‐[[4hyphen;(4‐[¹⁸F]fluorophenyl)piperazin‐1‐yl] methyl] ‐1H‐pyrrolo[2,3‐b]pyridine, acandidate to image dopamine D₄ receptors, was synthesised via electrophilic fluorination of a trimethylstannyl precursor with high specific radioactivity [¹⁸F]F₂. The precursor was obtained by a facile four‐step synthetic approach; the trimethylstannyl leaving group was introduced by displacement of iodine utilising palladium catalysis and hexamethyldistannane in an inert solvent. The total radiosynthesis time was 50 min, including purification and formulation for injection. Decay corrected radiochemical yield was <1% as calculated from the amount of [¹⁸F]F^? produced. Specific radioactivity at the end of synthesis was 12.8–16.4 GBq/μmol. Radiochemical purity was 88–92%. Ex vivo studies in rats showed homogeneous distribution of radioactivity within rat brain. Copyright © 2002 John Wiley & Sons, Ltd.     

Facile synthesis of stable isotope‐labeled antibacterial agent RWJ‐416457 and its metabolite

Synthesis of multiple stable isotope‐labeled antibacterial agent RWJ‐416457, (N‐{3‐[3‐fluoro‐4‐(2‐methyl‐2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide), and its major metabolite, N‐{3‐[4‐(2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐3‐fluoro‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide, is described. The stable isotope‐labeled [¹³CD₃]RWJ‐416457 was prepared readily by acetylation of the precursor amine, 5‐aminomethyl‐3‐[3‐fluoro‐4‐(2‐methyl‐2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐phenyl]‐oxazolidin‐2‐one with CD₃¹³COCl in pyridine. Synthesis of the stable isotope‐labeled metabolite involved a construction of multiple isotope‐labeled pyrazole ring. N,N‐dimethyl(formyl‐¹³C,D)amide dimethyl acetal was first prepared by treating N,N‐dimethyl(formyl‐¹³C,D)amide with dimethyl sulfate, followed by sodium methoxide. Then, N‐{3‐[3‐fluoro‐4‐(3‐oxo‐pyrrolidin‐1‐yl)‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide was condensed with N,N‐dimethyl(formyl‐¹³C,D)amide dimethyl acetal, and the resultant β‐ketoenamine intermediate underwent pyrazole ring formation with hydrazine‐¹⁵N₂, to give the [¹³C¹⁵N₂D]‐labeled metabolite. Copyright © 2012 John Wiley & Sons, Ltd.     

Unusual base‐catalyzed exchange in the synthesis of deuterated PF‐2413873a

The preparation of deuterated PF‐2413873 (4‐[3‐cyclopropyl‐1‐(methanesulfonylmethyl)‐5‐methyl‐1H‐pyrazol‐4‐yl]oxy‐2,6‐dimethylbenzonitrile, 1) is described for use as a bioanalytical standard in clinical trials. Two strategies were investigated. The sulfone‐containing substituent was labelled by base‐catalyzed exchange, but unacceptable deuterium loss was noted under assay conditions. Alternatively, labelling 4‐cyano‐3,5‐dimethylphenol was achieved by heating with deuterium oxide over platinum oxide. After building up the pyrazole ring we discovered that, during the subsequent alkylation to attach the methylthiomethyl group, the base, potassium t‐butoxide, caused unwanted scrambling of deuteriums on the aromatic portion and the methylthiomethyl group. Thus, it was necessary to remove all base‐labile hydrogens to prevent their exchange. This was accomplished by alkylating the pyrazole with per‐deuterated chloromethyl methylsulfide, oxidation to the sulfone, and selective removal of its deuteriums by treatment with sodium hydroxide. The unusual sensitivity and selectivity of these base‐promoted exchange reactions are discussed. Thus, 4‐[3‐cyclopropyl‐1‐(methanesulfonylmethyl)‐5‐methyl‐1H‐pyrazol‐4‐yl]oxy‐[²H₆]2,6‐dimethyl‐[3,5‐²H]benzonitrile (17) was obtained, labelled with eight deuterium atoms and an acceptable D₀/D₈ ratio. Copyright © 2009 John Wiley & Sons, Ltd.     

The synthesis of [1‐14C]2‐(1H‐tetrazol‐5‐yl)acetic acid

Tetrazoles are a common heterocyclic functionality in many biologically active molecules. [1‐¹⁴C]2‐(1H‐Tetrazol‐5‐yl)acetic acid was required as an intermediate in the synthesis of a development candidate as part of a discovery phase program to complete metabolic profiling studies. [1‐¹⁴C]2‐(1H‐Tetrazol‐5‐yl)acetic acid was prepared in 4 steps overall and in 3 radiochemical steps from K¹⁴CN in an overall 32% radiochemical yield.     

The synthesis and structures of deuterium‐labeled 5‐substituted 1H‐tetrazoles

The synthesis and crystal structures of deuterium‐labeled 5‐substituted 1H‐tetrazoles, 5‐[²H₅]phenyl‐1H‐tetrazole (I), 5‐[²H₇]tolyl‐1H‐tetrazole (II), and 5‐[²H₇]benzyl‐1H‐tetrazole (III) are reported. All syntheses were carried out using simple, facile steps and the products were obtained in high yields. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis,radiosynthesis and preliminary in vivo evaluation of [123I]‐(4‐fluorophenyl) {1‐[2‐(2‐iodophenyl)ethyl]piperidin‐4‐yl}methanone,a potential 5‐HT2A‐antagonist for SPECT brain imaging

Many people suffer from psychiatric illnesses like depression and anorexia. Relevant to these diseases is amongst others a malfunctioning of brain 5‐HT_2A‐receptors. To allow in vivo quantification of these receptors with Single Photon Emission Computerized Tomography (SPECT), a radiolabelled ligand with high 5‐HT_2A affinity is needed. This work reports the radiosynthesis of [¹²³I]‐(4‐fluorophenyl) {1‐[2‐(2‐iodophenyl)ethyl]piperidin‐4‐yl}methanone, the synthesis of its precursor, (4‐fluorophenyl) {1‐[2‐(2‐bromophenyl)ethyl]piperidin‐4‐yl}methanone, and the preliminary in vivo evaluation of the tracer. The precursor was synthesized with a total yield of 40%. Radiolabelling was performed using a halogen exchange reaction and the yield was 70%. Radiochemical purity was >95%, and specific activity was at least 2.4 Ci/µmol. Log P was measured to be 2.52. The tracer showed uptake in mice brain (3.5% I.D./g tissue at 3 min post injection) and therefore will be evaluated further by regional brain biodistribution and displacement studies in rabbits. Copyright © 2004 John Wiley & Sons, Ltd.     

A novel five‐lipoxygenase activity protein inhibitor labeled with carbon‐14 and deuterium

2‐[4‐(3‐{(1R)‐1‐[4‐(2‐Aminopyrimidin‐5‐yl)phenyl]‐1‐cyclopropylethyl}‐1,2,4‐oxadiazol‐5‐yl)‐1H‐pyrazol‐1‐yl]‐N,N‐dimethylacetamide (1), is a novel and selective five‐lipoxygenase activity protein (FLAP) inhibitor with excellent pharmacokinetics properties. The availability of a key chiral intermediate allowed the synthesis of [¹⁴C]‐(1) in six radiochemical steps and in 47% overall radiochemical yield with a specific activity of 51 mCi/mmol using carbon‐14 zinc cyanide. 2‐Chloro‐N,N‐dimethyl‐²H₆‐acetamide was prepared and condensed with a penultimate intermediate to give [²H₆]‐(1) in very high yield and in more than 99% isotopic enrichment.     

Synthesis and Antimicrobial Activity of Some Novel 2‐[4‐(Substituted Piperazin‐/Piperidin‐1‐ylcarbonyl)phenyl]‐1H‐benzimidazole Derivatives

In this study, we report the synthesis and antimicrobial evaluation of several new 4‐(1H‐benzimidazol‐2‐yl)benzamides ( 11 – 30 ) and 5‐chloro‐1‐(p‐fluorobenzyl)‐2‐{4‐[(4‐methylpiperazin‐1‐yl)carbonyl]phenyl}‐1H‐benzimidazole ( 33 ). Compound 20 exhibited the best antibacterial activity with MIC value of 6.25 μg/mL against Staphylococcus aureus and methicillin‐resistant Staphylococcus aureus (MRSA). Significant antifungal activities were obtained with the compounds 13 , 14, 18 , 19, and 33 with MIC values of 3.12 μg/mL which are close to fluconazole.     

Synthesis and Structure‐Activity Relationship Studies of Pyrazole‐based Heterocycles as Antitumor Agents

Several 4‐cyano‐1,5‐diphenylpyrazoles attached to different heterocyclic ring systems at position 3 were synthesized starting from ethyl 4‐cyano‐1,5‐diphenyl‐1H‐pyrazole‐3‐carboxylate 1 . The newly synthesized compounds were tested in vivo for their anti‐estrogenic effects and evaluated in vitro for their cytotoxic properties against estrogen‐dependent tumors. 3‐(5‐Mercapto‐1,3,4‐oxadiazole‐2‐yl)‐1,5‐diphenyl‐1H‐pyrazole‐4‐carbonitrile 13 revealed the highest cytotoxic activity with a GI₅₀ value equal to 40 nM against the IGROVI ovarian tumor cell line. It also showed an anti‐estrogen activity 1.6 more effective than the reference drug, in addition to a high tolerable dose. 3‐(5‐(Methylthio)‐4‐phenyl‐4H‐1,2,4‐triazol‐3‐yl)‐1,5‐diphenyl‐1H‐pyrazole‐4‐carbonitrile 7 was found to have the highest anti‐estrogenic activity, while 1,5‐diphenyl‐3‐[5‐(phenylamino)‐1,3,4‐thiadiazol‐2‐yl]‐1H‐pyrazole‐4‐carbonitrile 11 showed the lowest activity. The oral LD₅₀ values revealed that most of the tested compounds are relatively nontoxic.     

Synthesis,In Vitro Protoporphyrinogen Oxidase Inhibition,and Herbicidal Activity of N‐(Benzothiazol‐5‐yl)hexahydro‐1H‐isoindole‐1,3‐diones and N‐(Benzothiazol‐5‐yl)hexahydro‐1H‐isoindol‐1‐ones

Protoporphyrinogen oxidase ( EC 1.3.3.4 ) is one of the most significant targets for a large family of herbicides. As part of our continuous efforts to search for novel protoporphyrinogen oxidase‐inhibiting herbicides, N‐(benzothiazol‐5‐yl)tetrahydroisoindole‐1,3‐dione was selected as a lead compound for structural optimization, leading to the syntheses of a series of novel N‐(benzothiazol‐5‐yl)hexahydro‐1H‐isoindole‐1,3‐diones ( 1a – o ) and N‐(benzothiazol‐5‐yl)hexahydro‐1H‐isoindol‐1‐ones ( 2a – i ). These newly prepared compounds were characterized by elemental analyses, ¹H NMR, and ESI‐MS, and the structures of 1h and 2h were further confirmed by X‐ray diffraction analyses. The bioassays indicated that some compounds displayed comparable or higher protoporphyrinogen oxidase inhibition activities in comparison with the commercial control. Very promising, compound 2a , ethyl 2‐((6‐fluoro‐5‐(4,5,6,7‐tetrahydro‐1‐oxo‐1H‐isoindol‐2(3H)‐yl)benzo[d]thiazol‐2‐yl)‐sulfanyl)acetate, was recognized as the most potent candidate with K_i value of 0.0091 μm . Further greenhouse screening results demonstrated that some compounds exhibited good herbicidal activity against Chenopodium album at the dosage of 150 g/ha.     

Anxiolytic‐like effect of 2‐(4‐((1‐phenyl‐1H‐pyrazol‐4‐yl)methyl)piperazin‐1‐yl)ethan‐1‐ol is mediated through the benzodiazepine and nicotinic pathways

In this study, we proposed the design, synthesis of a new compound 2‐(4‐((1‐phenyl‐1H‐pyrazol‐4‐yl)methyl)piperazin‐1‐yl)ethan‐1‐ol (LQFM032), and pharmacological evaluation of its anxiolytic‐like effect. This new compound was subjected to pharmacological screening referred to as Irwin test, prior to sodium pentobarbital‐induced sleep, open‐field and wire tests. The anxiolytic‐like effect of this compound was evaluated using elevated plus maze and light–dark box tests. In addition, the mnemonic activity was evaluated through step‐down test. In sodium pentobarbital‐induced sleep test, LQFM032 decreased latency and increased duration of sleep. In the open‐field test, LQFM032 altered behavioral parameter, that suggested anxiolytic‐like activity, as increased in crossings and time spent at the center of open field. In the plus maze test and light–dark box test, the LQFM032 showed anxiolytic‐like activity, increased entries and time spent on open arms, and increased in number of transitions and time spent on light area, respectively. Those effects was antagonized by flumazenil but not with 1‐(2‐Methoxyphenyl)‐4‐(4‐phthalimidobutyl)piperazine (NAN‐190). The LQFM032 did not alter mnemonic activity. Moreover, the anxiolytic‐like activity of LQFM032 was antagonized by mecamylamine. In summary, LQFM032 showed benzodiazepine and nicotinic pathways mediated anxiolytic‐like activity without altering the mnemonic activity.     

The synthesis of three isotopomers of 2‐methyl‐2‐(4‐[3‐[1‐(4‐methylbenzyl)‐5‐oxo‐4,5‐dihydro‐1H‐[1,2,4]triazol‐3‐yl]propyl]phenoxy)propionic acid,a potent and selective peroxisome proliferator‐activated receptor alpha agonist

Although fenofibrate ( 1a ) is commercially available and clinically effective in lowering serum triglycerides, its activity and sub‐type selectivity at the PPARα receptors are only moderate; therefore, there exists a need for more potent and sub‐type selective PPARα agonists. To that end, discovery efforts have identified 2‐methyl‐2‐(4‐[3‐[1‐(4‐methylbenzyl)‐5‐oxo‐4,5‐dihydro‐1H‐[1,2,4]triazol‐3‐yl]propyl]phenoxy)propionic acid ( 2 ), a potent and selective human PPARα receptor agonist. In support of pre‐clinical ADME studies and bioanalysis, three isotopomers of 2 have been synthesized. The results of these efforts are described below. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis and Positive Inotropic Evaluation of (E)‐2‐(4‐Cinnamylpiperazin‐1‐yl)‐N‐(1‐substituted‐4,5‐dihydro‐[1,2,4]triazolo[4,3‐a]quinolin‐7‐yl)acetamides

A series of (E)‐2‐(4‐cinnamylpiperazin‐1‐yl)‐N‐(1‐substituted‐4,5‐dihydro‐[1,2,4]triazolo[4,3‐a]quinolin‐7‐yl)acetamides were synthesized and evaluated for their positive inotropic activity by measuring the left atrium stroke volume on isolated rabbit heart preparations. This class of compounds presented favorable in vitro activity compared with the standard drug, milrinone, among which N‐(1‐(3‐chlorophenyl)‐4,5‐dihydro‐[1,2,4]triazolo[4,3‐a]quinolin‐7‐yl)‐2‐(4‐cinnamylpiperazin‐1‐yl)acetamide 5e was found to be the most potent with 16.58 ± 0.11% increased stroke volume (milrinone: 2.46 ± 0.07%) at a concentration of 3 × 10^?5 M. The chronotropic effects of the compounds having inotropic effects were also evaluated.     

Synthesis and in vivo evaluation in mice of [123I]‐(4‐fluorophenyl)[1‐(3‐iodophenethyl)piperidin‐4‐yl]methanone as a potential SPECT‐tracer for the serotonin 5‐HT2A receptor

This work reports the synthesis, radiolabelling and in vivo evaluation in NMRI mice of [¹²³I]‐(4‐fluorophenyl)[1‐(3‐iodophenethyl)piperidin‐4‐yl]methanone ([¹²³I]‐3‐I‐CO) as a potential SPECT tracer for the 5‐HT_2A receptor. The tributylstannylprecursor was synthesized with a 15% overall yield. Radiolabelling was performed using an electrophilic iododestannylation with yields of 85%. Radiochemical purity was always >95%. Log P was determined to be 3.10±0.10. The tracer showed good uptake in mouse brain (6.3±1.3% ID/g tissue at 10 min p.i., 2±0.36% ID/g tissue at 1 h p.i.). These results warrant further research in larger animals to determine suitability of [¹²³I]‐3‐I‐CO as a 5‐HT_2A tracer. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis and Positive Inotropic Evaluation of 2‐(4‐(4‐Substituted benzyloxy)‐3‐methoxybenzyl)‐1,4‐diazepan‐1‐yl)‐N‐(4,5‐dihydro‐1‐methyl[1,2,4]triazolo[4,3‐a]quinolin‐7‐yl)‐acetamides

In an attempt to search for more potent positive inotropic agents, a series of 2‐(4‐(4‐substituted benzyloxy)‐3‐methoxybenzyl)‐1,4‐diazepan‐1‐yl)‐N‐(4,5‐dihydro‐1‐methyl[1,2,4]triazolo[4,3‐a]quinolin‐7‐yl)acetamides was synthesized and their positive inotropic activities were evaluated by measuring left atrium stroke volume on isolated rabbit‐heart preparations. Several compounds showed favorable activity compared with the standard drug Milrinone among which 2‐(4‐(4‐(2‐chlorobenzyloxy)‐3‐methoxybenzyl)‐1,4‐diazepan‐1‐yl)‐N‐(4,5‐dihydro‐1‐methyl‐[1,2,4]triazolo[4,3‐a]quinolin‐7‐yl)acetamide 6e was found to have the most desirable potency with the 6.79 ± 0.18% increased stroke volume (Milrinone: 1.67 ± 0.64%) at a concentration of 1×10^–5 M in our in‐vitro study. The chronotropic effects of those compounds having inotropic effects were also evaluated in this work.