Imidazo[1,2-b]pyridazine as privileged scaffold in medicinal chemistry: An extensive review.

Imidazo[1,2-b]pyridazine scaffold represents an important class of heterocyclic nucleus which provides various bioactives molecules. Among them, the successful kinase inhibitor ponatinib led to a resurgence of interest in exploring new imidazo[1,2-b]pyridazine-containing derivatives for their putative therapeutic applications in medicine. This present review intends to provide a state-of-the-art of this framework in medicinal chemistry from 1966 to nowadays, unveiling different aspects of its structure-activity relationships (SAR). This extensive literature surveil may guide medicinal chemists for the quest of novel imidazo[1,2-b]pyridazine compounds with enhanced pharmacokinetics profile and efficiency.

In Situ Water Quantification in Natural Deep Eutectic Solvents Using Portable Raman Spectroscopy.

Raman spectroscopy is a label-free, non-destructive, non-invasive analytical tool that provides insight into the molecular composition of samples with minimum or no sample preparation. The increased availability of commercial portable Raman devices presents a potentially easy and convenient analytical solution for day-to-day analysis in laboratories and production lines. However, their performance for highly specific and sensitive analysis applications has not been extensively evaluated. This study performs a direct comparison of such a commercially available, portable Raman system, with a research grade Raman microscope system for the analysis of water content of Natural Deep Eutectic Solvents (NADES). NADES are renewable, biodegradable and easily tunable "green" solvents, outcompeting existing organic solvents for applications in extraction from biomass, biocatalysis, and nanoparticle synthesis. Water content in NADES is, however, a critical parameter, affecting their properties, optimal use and extraction efficiency. In the present study, portable Raman spectroscopy coupled with Partial Least Squares Regression (PLSR) is investigated for rapid determination of water content in NADES samples in situ, i.e., directly in glassware. Three NADES systems, namely Betaine Glycerol (BG), Choline Chloride Glycerol (CCG) and Glucose Glycerol (GG), containing a range of water concentrations between 0% (w/w) and 28.5% (w/w), were studied. The results are directly compared with previously published studies of the same systems, using a research grade Raman microscope. PLSR results demonstrate the reliability of the analysis, surrendering R2 values above 0.99. Root Mean Square Errors Prediction (RMSEP) of 0.6805%, 0.9859% and 1.2907% w/w were found for respectively unknown CCG, BG and GG samples using the portable device compared to 0.4715%, 0.3437% and 0.7409% w/w previously obtained by analysis in quartz cuvettes with a Raman confocal microscope. Despite the relatively higher values of RMSEP observed, the comparison of the percentage of relative errors in the predicted concentration highlights that, overall, the portable device delivers accuracy below 5%. Ultimately, it has been demonstrated that portable Raman spectroscopy enables accurate quantification of water in NADES directly through glass vials without the requirement for sample withdrawal. Such compact instruments provide solvent and consumable free analysis for rapid analysis directly in laboratories and for non-expert users. Portable Raman is a promising approach for high throughput monitoring of water content in NADES that can support the development of new analytical protocols in the field of green chemistry in research and development laboratories but also in the industry as a routine quality control tool.

Bioinspired imidazo[1,2-a:4,5-c']dipyridines with dual antiproliferative and anti-migrative properties in human cancer cells: The SAR investigation.

Herein, we report the design, synthesis and evaluation of novel bioinspired imidazo[1,2-a:4,5c']dipyridines. The structural optimization identified four anti-proliferative compounds. Compounds 11, 18, 19 and 20 exhibited excellent anticancer activities in vitro with IC50 of 0.4-5 µM against three human cancer cell lines (MDA-MB-468, MDA-MB-435s and MDA-MB-231). These four compounds induced apoptosis in MDA-MB-231 cells in a dose-dependent manner, targeting different apoptotic proteins expression: 11 increased the expression of pro-apoptotic Bax protein while 18-20 reduced the level of anti-apoptotic Bcl-2 protein. Compounds 18 and 19 also reduced MDA-MB-231 cells proliferation as measured by Ki-67 staining. Furthermore, compounds were also tested for the ability to inhibit cell migration in the highly aggressive human MDA-MB-435s cell line. Six compounds of this series (8, 15, 18, 22, 23, 24) inhibited cell migration by 41-50% while four compounds (20, 25, 27, 30) inhibited the migration by 53-62% in wound-healing experiments. Interestingly, compound 20 presented both antiproliferative and anti-migration activities and might be a promising anti-metastatic agent for cancer treatment.

Aqueous Fraction from Hibiscus sabdariffa Relaxes Mesenteric Arteries of Normotensive and Hypertensive Rats through Calcium Current Reduction and Possibly Potassium Channels Modulation.

BACKGROUND/OBJECTIVES: Hibiscus sabdariffa L. (H. sabdariffa (HS)) extract has a vascular relaxant effect on isolated rat thoracic aorta, but data on small resistance arteries, which play an important role on the development of hypertension, are still missing. The purposes of this study were (1) to assess the effect on isolated mesenteric arteries (MA) from normotensive (Wistar and Wistar-Kyoto (WKY)) and spontaneous hypertensive rats (SHR); (2) to elucidate the mechanism(s) of action underling the relaxant effect in light of bioactive components. METHODS: Vascular effects of HS aqueous fraction (AF) on isolated MA rings, as well as its mechanisms of action, were assessed using the contractility and intracellular microelectrode technique. The patch clamp technique was used to evaluate the effect of HS AF on the L-type calcium current. Extraction and enrichment of AF were carried out using liquid-liquid extraction, and the yield was analyzed using HPLC. RESULTS: The HS AF induced a concentration-dependent relaxant effect on MA rings of SHR (EC50 = 0.83 ± 0.08 mg/mL), WKY (EC50 = 0.46 ± 0.04 mg/mL), and Wistar rats (EC50 = 0.44 ± 0.08 mg/mL) pre-contracted with phenylephrine (10 µM). In Wistar rats, the HS AF maximum relaxant effect was not modified after endothelium removal or when a guanylate cyclase inhibitor (ODQ, 10 µM) and a selective ß2-adrenergic receptor antagonist (ICI-118551, 1 µM) were incubated with the preparation. Otherwise, it was reduced by 34.57 ± 10.66% when vascular rings were pre-contracted with an 80 mM [K+] solution (p < 0.001), which suggests an effect on ionic channels. HS AF 2 mg/mL significantly decreased the peak of the L-type calcium current observed in cardiac myocytes by 24.4%. Moreover, though the vasorelaxant effect of HS, AF was reduced by 27% when the nonselective potassium channels blocker (tetraethylammonium (TEA) 20 mM) was added to the bath (p < 0.01). The extract did not induce a membrane hyperpolarization of smooth muscle cells, which might suggest an absence of a direct effect on background potassium current. CONCLUSION: These results highlight that the antihypertensive effect of HS probably involves a vasorelaxant effect on small resistance arteries, which is endothelium independent. L-type calcium current reduction contributes to this effect. The results could also provide a link between the vasorelaxant effect and the bioactive compounds, especially anthocyanins.

Imidazo[1,2-b]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1 decrease the parasite burden in mice with acute toxoplasmosis.

The current therapeutic arsenal for toxoplasmosis is restricted to drugs non-specific to the parasite which cause important side effects. Development of more efficient and specific anti-Toxoplasma compounds is urgently needed. Imidazo[1,2-b]pyridazines designed to inhibit the calcium-dependent protein kinase 1 of Toxoplasma gondii (TgCDPK1) and effective against tachyzoite growth in vitro at submicromolar ranges were modified into hydrochloride salts to be administered in vivo in a mouse model of acute toxoplasmosis. All protonated imidazo[1,2-b]pyridazine salts (SP230, SP231 and SP232) maintained their activity on TgCDPK1 and T. gondii tachyzoites. Rat and mouse liver microsomes were used to predict half-life and intrinsic clearance, and the pharmacokinetic profile of the most rapidly degraded imidazo[1,2b]pyridazine salt (SP230) was determined in serum, brain and lungs of mice after a single administration of 50 mg/kg. Compounds were then tested in vivo in a murine model of acute toxoplasmosis. Mice infected with tachyzoites of the ME49 strain of T. gondii were treated for 4, 7 or 8 days with 25 or 50 mg/kg/day of SP230, SP231 or SP232. The parasite burdens were strongly diminished (>90% reduction under some conditions) in the spleen and the lungs of mice treated with imidazo[1,2-b]pyridazine salts compared with untreated mice, without the need for pre-treatment. Moreover, no increases in the levels of hepatic and renal toxicity markers were observed, demonstrating no significant signs of short-term toxicity. To conclude, imidazo[1,2-b]pyridazine salts have strong efficacy in vivo on acute toxoplasmosis and should be further tested in a model of mouse congenital toxoplasmosis.

Discovery of imidazo[1,2-a]pyridine-based anthelmintic targeting cholinergic receptors of Haemonchus contortus.

We report the synthesis of a series of imidazo[1,2-a]pyridine-based molecules as anthelmintic against the livestock parasite Haemonchus contortus. The molecules were tested by using Larval Paralysis Test (LPT), in order to target ionic channels, as most of the prominent marketed anthelminthics present such mechanism of action. The most active compound (5e) displayed paralysis on H. contortus stage 3 larvae until 31.25 µM. Effect of 5e on H. contortus cholinergic receptors (L-AChR1 and 2) was characterized via electrophysiological measurement and a rare antagonist mode of action was unveiled.

Anti-inflammatory activity of Crateva adansonii DC on keratinocytes infected by Staphylococcus aureus: From traditional practice to scientific approach using HPTLC-densitometry.

ETHNOPHARMACOLIGICAL RELEVANCE: Leaves of Crateva adansonii DC (Capparidaceae), a small bush found in Togo, are widely used in traditional medicine to cure infectious abscesses. Traditional healers of Lomé harvest only budding leaves early in the morning, in specific area in order to prepare their drugs. AIM OF THE STUDY: The main goal was to validate the ancestral picking practices, and to assess the activity of C. adansonii medicine towards infectious abscesses. MATERIALS AND METHODS: A phytochemical screening of various C. adansonii leaf samples was performed using an original HPTLC-densitometry protocol and major flavonoids were identified and quantified. C. adansonii samples were collected in different neighborhoods of Lomé, at different harvesting-times and at different ages. Radical scavenging capacity, using DPPH assay, was used to quickly screen all extracts. Extracts were tested for anti-Staphylococcus aureus activity and anti-inflammatory effect on human primary keratinocytes infected by S. aureus. IL6, IL8 and TNFα expression and production were assessed by RT-PCR and ELISA assays. RESULTS: Using antioxidant activity as selection criteria, optimal extracts were obtained with budding leaves, collected at 5:00am in Djidjolé neighborhood. This extract showed the strongest anti-inflammatory effect on S. aureus-infected keratinocytes by reducing IL6, IL8 and TNFα expression and production. None of the extracts inhibited the growth of S. aureus. CONCLUSIONS: Those results validate the traditional practices and the potential of C. adansonii as anti-inflammatory drug. Our findings suggest that traditional healers should add to C. adansonii leaves an antibacterial plant of Togo Pharmacopeia, in order to improve abscess healing.

Synthesis and evaluation of the anticoccidial activity of trifluoropyrido[1,2-a]pyrimidin-2-one derivatives.

Screening of our chemical library to discover new molecules exhibiting in vitro activity against the invasion of host cells by Eimeria tenella revealed a lead compound with an IC50 of 15µM. Structure-activity relationship studies were conducted with 34 newly synthesized compounds to identify more active molecules and enhance in vitro activity against the parasite. Four compounds were more effective in inhibiting MDBK cell invasion in vitro than the lead compound.

Development of new highly potent imidazo[1,2-b]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1.

Using a structure-based design approach, we have developed a new series of imidazo[1,2-b]pyridazines, targeting the calcium-dependent protein kinase-1 (CDPK1) from Toxoplasma gondii. Twenty derivatives were thus synthesized. Structure-activity relationships and docking studies confirmed the binding mode of these inhibitors within the ATP binding pocket of TgCDPK1. Two lead compounds (16a and 16f) were then identified, which were able to block TgCDPK1 enzymatic activity at low nanomolar concentrations, with a good selectivity profile against a panel of mammalian kinases. The potential of these inhibitors was confirmed in vitro on T. gondii growth, with EC50 values of 100 nM and 70 nM, respectively. These best candidates also displayed low toxicity to mammalian cells and were selected for further in vivo investigations on murine model of acute toxoplasmosis.

Antibacterial Polyketide Heterodimers from Pyrenacantha kaurabassana Tubers.

Two heterodimers comprising anthraquinone and methylbenzoisocoumarin moieties (1 and 2) were isolated, together with emodin and physcion from the tubers of Pyrenacantha kaurabassana. The structures of 1 and 2 were established by NMR spectroscopy, including the analysis of a 2D INADEQUATE spectrum. On the basis of the data obtained, the structures that were previously proposed in the literature for these compounds were revised. Compounds 1 and 2 showed antibacterial activity against three different strains of Staphylococcus aureus. Compound 2 also showed bactericidal activity against Helicobacter pylori.

A small-molecule cell-based screen led to the identification of biphenylimidazoazines with highly potent and broad-spectrum anti-apicomplexan activity.

An in vitro screening of the anti-apicomplexan activity of 51 compounds, stemming from our chemical library and from chemical synthesis, was performed. As a study model, we used Toxoplasma gondii (T. gondii), expressing ß-galactosidase for the colorimetric assessment of drug activity on parasites cultivated in vitro. This approach allowed the validation of a new series of molecules with a biphenylimidazoazine scaffold as inhibitors of T. gondii growth in vitro. Hence, 8 molecules significantly inhibited intracellular replication of T. gondii in vitro, with EC50 < 1 µM, while being non-toxic for human fibroblasts at these concentrations. Most attractive candidates were then selected for further biological investigations on other apicomplexan parasites (Neospora caninum, Besnoitia besnoiti, Eimeria tenella and Plasmodium falciparum). Finally, two compounds were able to inhibit growth of four different apicomplexans with EC50 in the submicromolar to nanomolar range, for each parasite. These data, including the broad anti-parasite spectrum of these inhibitors, define a new generation of potential anti-parasite compounds of wide interest, including for veterinary application. Studies realized on E. tenella suggest that these molecules act during the intracellular development steps of the parasite. Further experiments should be done to identify the molecular target(s) of these compounds.

Antifungal activity of resveratrol derivatives against Candida species.

trans-Resveratrol (1a) is a phytoalexin produced by plants in response to infections by pathogens. Its potential activity against clinically relevant opportunistic fungal pathogens has previously been poorly investigated. Evaluated herein are the candidacidal activities of oligomers (2a, 3-5) of 1a purified from Vitis vinifera grape canes and several analogues (1b-1j) of 1a obtained through semisynthesis using methylation and acetylation. Moreover, trans-ε-viniferin (2a), a dimer of 1a, was also subjected to methylation (2b) and acetylation (2c) under nonselective conditions. Neither the natural oligomers of 1a (2a, 3-5) nor the derivatives of 2a were active against Candida albicans SC5314. However, the dimethoxy resveratrol derivatives 1d and 1e exhibited antifungal activity against C. albicans with minimum inhibitory concentration (MIC) values of 29-37 µg/mL and against 11 other Candida species. Compound 1e inhibited the yeast-to-hyphae morphogenetic transition of C. albicans at 14 µg/mL.

Promising results of cranberry in the prevention of oral Candida biofilms.

In the context of dental caries prevention by natural foodstuff sources, antifungal and antibiofilm activities of dry commercial extracts of cranberry fruit (Vaccinium macrocarpon Aiton) and two other red fruits (Vaccinium myrtillus L. and Malpighia punicifolia L.) were assessed on Candida albicans and Candida glabrata yeasts. When added to the culture medium, the cranberry extract displayed a significant anti-adhesion activity against Candida spp. when used at low concentrations. In addition, the pretreatment of surfaces with this extract induced an anti-adhesion activity mainly against C. glabrata yeasts and an antibiofilm activity against C. albicans. This activity was dependent on concentration, species, and strain. A phytochemical investigation bioguided by anti-adhesion tests against the two Candida species was carried out on crude cranberry juice to determine the active fractions. Three subfractions enriched in proanthocyanidins showed an anti-adhesion activity at low concentrations. This study investigated for the first time the interest of crude extracts of cranberry and cranberry juice fractions to prevent biofilms of C. glabrata. It highlighted the potency of consuming this fruit and using it as a source of anti-adhesion agents.

New flavonoids C-glycosides from Rhabdophyllum arnoldianum.

Two new flavone glycosides, 3″-O-acetyl-7-O-methylvitexin (1) and 6″-α-rhamnopyranosyl-7-O-methylvitexin (2), along with nine known compounds (3-11) were isolated from the leaves of Rhabdophyllum arnoldianum (Ochnaceae). The structures of the new compounds were established by detailed spectroscopic studies and mass spectrometry, while known compounds were characterised by direct comparison of their reported NMR data with those found in the literature. All these compounds were the first reported from Rhabdophyllum genus. The biological assays on crude extracts and compounds of this plant demonstrated that the crude extracts possess significant antimicrobial activity against Gram-positive bacteria.

3-Biphenylimidazo[1,2-a]pyridines or [1,2-b]pyridazines and analogues, novel Flaviviridae inhibitors.

Using Ttou 84 as starting point, a novel class of biphenyl derivatives of imidazo[1,2-a]pyridine and imidazo[1,2-b]pyridazine was designed to optimize the inhibitory properties on the replication of the bovine viral diarrhoea virus (BVDV) and hepatitis C virus (HCV). Three sites of pharmacomodulation were chosen i.e. positions 2, 3 and 6 on the central heterocyclic core structure. From the 49 analogues tested, only compound 18j (3-(2'-hydroxybiphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-b]pyridazine) showed antiviral activity in the HCV replicon system reminiscent of selective inhibition (60-70% inhibition). Compound 4f (3-(biphen-3-yl)-2-(4-fluorophenyl)-6-phenylthioimidazo[1,2-a]pyridine) proved to be the most selective inhibitor of BVDV replication and showed no or only marginal cross-resistance with known inhibitors of pestivirus replication. The cross-resistance profile of 4f might indicate that 4f does not interact with the same binding site as BPIP, VP32947, AG110 or LZ37. From 42 analogues tested against both viruses, QSAR studies were discussed in regard to BVDV antiviral activity.

High performance thin layer chromatography-densitometry: a step further for quality control of cranberry extracts.

A method for selective quantitation of catechin, proanthocyanidin (PAC) A2 and PAC-B1 in American cranberry (Vaccinium macrocarpon) extracts using high performance thin layer chromatography (HPTLC)-densitometry is presented. Methylene chloride/ethyl acetate/formic acid (6:10:1, v/v) as the mobile phase and 1% vanillin hydrochloric solution as staining reagent were used. In these conditions, three standards considered as quality markers, catechin, PAC-A2 and PAC-B1, were well resolved allowing simultaneous quantitation on one plate. All standards were quantified in the range of 0.7-5 µg with RSD of repeatability and intermediate precision not exceeding 5%. Catechin, PAC-A2 and PAC-B1 profiles of cranberry extracts were analysed regarding global PAC amounts obtained by BL-DMAC assay. It appears clearly that HPTLC-densitometry process provides additional information which, combined with BL-DMAC results, allows qualitative and quantitative control of cranberry extracts. Particularly, densitometric assay highlighted degradation of PACs in a 7 day-extract, leading to high overestimation with BL-DMAC protocol.

Regiocontrolled microwave assisted bifunctionalization of 7,8-dihalogenated imidazo[1,2-a]pyridines: a one pot double-coupling approach.

The reactivity of the 7-chloro-8-iodo- and 8-chloro-7-iodoimidazo[1,2-a]pyridines 1a-e diversely substituted on the 2 position, towards Suzuki-Miyaura, Sonogashira, and Buchwald-Hartwig cross-coupling reactions as well as cyanation was evaluated. Various methodologies are proposed to introduce aryl, heteroaryl, alkyne, amine or cyano groups in the two positions depending on the nature of the substituent present in position 2. In both series, the substitution of the iodine atom was totally regioselective and the difficulty was to substitute the chlorine atom in a second step. Until now, only hetero(aryl) groups could be introduced though Suzuki-Miyaura cross-coupling. We overcame this problem evaluating both regioisomers in parallel. The double coupling approach was also studied allowing the one pot Suzuki/Suzuki, cyanation/Sonogashira and cyanation/Buchwald reactions leading to polyfunctionnalized imidazo[1,2-a]pyridines.

Copper(I)-mediated preparation of new pyrano[3',4':4,5]imidazo[1,2-a]pyridin-1-one compounds under mild palladium-free conditions.

A general and efficient Cu(I)-mediated cross-coupling and heterocyclization reaction of 3-iodoimidazo[1,2-a]pyridine-2-carboxylic acid, and terminal alkynes was developed under very mild conditions. This method allows the introduction in one pot of a third ring fused in positions 2 and 3 of the imidazo[1,2-a]pyridine core with reasonable yields and total regioselectivity. This procedure does not require the use of any expensive supplementary additives, and is palladium-free.

Physicochemical properties and membrane interactions of anti-apoptotic derivatives 2-(4-fluorophenyl)-3-(pyridin-4-yl)imidazo[1,2-a]pyridine depending on the hydroxyalkylamino side chain length and conformation: an NMR and ESR study.

Three imidazo[1,2-a]pyridine derivatives 3a-c have been synthesized from p38 kinase inhibitor structures and evaluated as anti-apoptosis agents. These drugs were designed to interact with nucleic acids and membrane interactions by varying the chain length in position 6, from hydroxyethylamino (3a), to hydroxybutylamino (3b) and hydroxyhexylamino (3c). First experiments showed that 3a and 3b were insoluble in water while 3c could be solubilized in water despite its partition coefficient (logP=3.2). This latter feature was explained by the formation of a fifth intramolecular cycle thus allowing supramolecular structure formation (NMR and MD calculations). The interactions with membranes have been studied using (1)H, (2)H, (31)P Nuclear Magnetic Resonance (NMR), Electron Spin Resonance (ESR) and High Resolution-Magic Angle Spinning (HR-MAS). Despite the insolubility of 3a and 3b in water, these derivatives could be partially solubilized by synthetic phospholipidic model membranes (small unilamellar vesicles, SUV). (1)H NMR paramagnetic broadening experiments performed on the same models showed that 3a was located in the external layer, probably close to the surface while 3b only formed external superficial adducts. Supplementary (31)P, (2)H NMR and ESR experiments on phospholipid dispersions confirmed the location of 3a close to the polar headgroup of the external layer of the membrane, this resulting in a 2K lowering of the transition temperature. Moreover, no significant interaction was detected on the deep part of the layer ((2)H NMR and 16NS ESR experiments). This binding was also found in the presence of cell cultures, as revealed by HR-MAS NMR experiments. Conversely, no significant interaction with membranes was found with 3b or 3c. From both the unexpected solubility of 3c and 3a interactions with membranes, further chemical modifications were finally proposed.

Proerectile effects of dopamine D2-like agonists are mediated by the D3 receptor in rats and mice.

Dopamine D(2)-like agonists induce penile erection (PE) and yawning in a variety of species, effects that have been suggested recently to be specifically mediated by the D(4) and D(3) receptors, respectively. The current studies were aimed at characterizing a series of D(2), D(3), and D(4) agonists with respect to their capacity to induce PE and yawning in the rat and the proerectile effects of apomorphine [(R)-(-)-5,6,6a,7-tetrahydro-6-methyl-4H-dibenzo-[de,g]quinoline-10,11-diol hydrochloride] in wild-type and D(4) receptor (R) knockout (KO) mice. All D(3) agonists induced dose-dependent increases in PE and yawning over a similar range of doses, whereas significant increases in PE or yawning were not observed with any of the D(4) agonists. Likewise, D(2), D(3), and D(4) antagonists were assessed for their capacity to alter apomorphine- and pramipexole (N'-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine dihydrochloride)-induced PE and yawning. The D(3) antagonist, PG01037 [N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide hydrochloride], inhibited the induction of PE and yawning, whereas the D(2) antagonist, L-741,626 [3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole], reversed the inhibition of PE and yawning observed at higher doses. The D(4) antagonist, L-745,870 [3-(4-[4-chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo[2,3-b]pyridine trihydrochloride], did not alter apomorphine- or pramipexole-induced PE or yawning. A role for the D(3) receptor was further supported because apomorphine was equipotent at inducing PE in wild-type and D(4)RKO mice, effects that were inhibited by the D(3) antagonist, PG01037, in both wild-type and D(4)R KO mice. Together, these studies provide strong support that D(2)-like agonist-induced PE and yawning are differentially mediated by the D(3) (induction) and D(2) (inhibition) receptors. These studies fail to support a role for the D(4) receptor in the regulation of PE or yawning by D(2)-like agonists.