Combined Organic Layers Research Articles

An Eco-friendly Strategy for the Synthesis of Spiro-benzimidazoquinazolinone and Spiro-benzothiazoloquinazolinone Derivatives using β-cyclodextrin as a Supramolecular Catalyst.

Cyclodextrins selectively bind with reactants and facilitate chemical reactions through supramolecular catalysis, similar to the mechanisms employed by enzymes. In this paper, β-cyclodextrin was used as a supramolecular catalyst in water as a green, reusable, and ecofriendly solvent system to synthesize spiro-benzimidazoquinazolinones and spiro-benzothiazoloquinazolinones. A supramolecular catalyst β-cyclodextrin (β-CD) is used to synthesize spiro- benzimidazoquinazolinones and spiro-benzothiazoloquinazolinones via multicomponent reaction involving the condensation of dimedone, isatin, and 2-aminobenzimidazole/2-aminobenzothiazole. In a 50 mL round bottom flask were added the respective mixture of substituted isatin (1 mmol), dimedone (1mmol), and 2-aminobenzimidazole/2-aminobenzothiazole (1 mmol) in water (5 ml) containing β-CD (113 mg, 10 mol. %) was stirred at 60oC for 30 min. The desired product was obtained with excellent yield. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with water and extracted with ethyl acetate (4X5 ml). The combined organic layers were washed with brine solution, dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude product was purified by silica gel chromatography. β-cyclodextrin catalyst showed very good efficiency in the synthesis of the desired compounds and can be easily recovered and reused at least five times with minimal deactivation in catalytic activity. The catalyst demonstrated remarkable effectiveness in producing the target compounds and conducting the reaction with different initial substances, resulting in excellent yields of the products, thereby confirming the broad applicability and versatility of this method.

Martin Silicates as Partners in Photoredox/Ni Dual Catalysis for the Installation of CH3, CH2D, CD2H, CD3and13CH3Groups onto (Hetero)Arenes

Martin Silicates as Partners in Photoredox/Ni Dual Catalysis for the Installation of CH₃, CH₂D, CD₂H, CD₃and¹³CH₃Groups onto (Hetero)Arenes

Study and optimization of Diels-Alder reaction of piperine in aqueous ionic solutions using Gn.HCl as a catalyst

Study and optimization of Diels-Alder reaction of piperine in aqueous ionic solutions using Gn.HCl as a catalyst

Copper (I)–Organic Frameworks for Catalysis: Networking Metal Clusters with Dynamic Covalent Chemistry

Metal clusters exhibit diverse structures, emerging functions, and applications; thus, incorporating clusters into metal–organic frameworks (MOFs) brings tremendous merits. Although the constructio...

DBU Catalysis: An Efficient Synthetic Strategy for 5,7-disubstituted-1,2,4- triazolo[1,5-a]pyrimidines.

An efficient and facile DBU catalysed synthesis of highly significant motif 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines under solvent-free condition has been reported. To a round bottom flask, 1.0 mmol of chalcone (1), 1.5 mmol of 3-amino-1,2,4- triazole (2) and 30 mol% of DBU were added at 70 °C and stirred in solvent-free condition. After the completion of the reaction (monitored by TLC), water (10 ml) was added. The aqueous layer was extracted with ethyl acetate (3 ×10 ml). The combined organic layers were dried over anhydrous Na2SO4. The combined organic layers were evaporated under reduced pressure and the resulting crude product was purified by column chromatography by using ethyl acetate and hexane as eluent. Reaction using chalcone and 3-amino-1,2,4-triazole as model substrates were carried out under different reaction conditions and it was observed that 30 mol% of DBU under the solvent-free condition at 70 °C was the optimum temperature for the proposed synthesis. Use of DBU (an organocatalyst) as a base, operational simplicity, high yield of products and short reaction time are some of the significant advantages associated with the proposed strategy.

Bidirectional metal‐free ROMP from difunctional organic initiators

Bidirectional metal‐free ROMP from difunctional organic initiators

Asymmetric Total Synthesis and Absolute Configuration Reassignment of Indole Alkaloid (+)-Alsmaphorazine D

The first asymmetric total synthesis of (+)-alsmaphorazine D has been achieved through a traceless chirality transfer strategy, which also enabled absolute configuration reassignment of the natural product. Key steps of this efficient approach entail a catalytic oxidative cyclization (To a solution of indoline ester 8 (5.82 g, 10 mmol) in AcOH (100 mL) were added CAN (550 mg, 1 mmol) and NaOAc (1.64 g, 20 mmol). The reaction vessel was exposed to air through a CaCl2 tube. The resulting mixture was stirred at 110 ℃ for 12 h before it was concentrated in vacuo. The residue was diluted with H2O, neutralized with NaHCO3 (sat. aq.) and extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO4) and concentrated in vacuo. Flash column chromatography (silica gel, hexanes/EtOAc, V∶V=4∶1) afforded δ-lactamindole 7 (4.34 g, 75%) as a white amorphous solid), a diastereoselective oxidative cyclic aminal formation (To a stirred solution of mono-ester 13 (5.20 g, 10 mmol, dr=1∶1) in acetone (100 mL) at 0 ℃ was added NaHCO3 (100 mL, sat. aq.). The resulting mixture was stirred for 0.5 h before it was added oxone (12.28 g, 20 mmol) slowly. The reaction mix- ture was stirred at 0 ℃ for an additional 2 h before it was diluted with H2O. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried (Na2SO4) and concentrated in vacuo. Flash column chromatog- raphy (silica gel, hexanes/EtOAc, V∶V=3∶1) afforded pyrroloindole 6' (1.61 g, 71%) as a white amorphous solid) and an intramolecular radical cyclization (To a stirred solution of ent-5 (250 mg, 0.47 mmol) in benzene (5 mL) at 80 ℃ were add- ed n-Bu3SnH (152 μL, 0.56 mmol) and AIBN (5 μL, 0.047 mmol). The resulting mixture was stirred for 0.5 h before it was concentrated in vacuo. Flash column chromatography (silica gel, hexanes/EtOAc, V∶V=1∶1) afforded C(16)-epi-ent-4 (213 mg, 72%) as a colorless oil.). Keywords alkaloid; total synthesis; tetracyclic system; oxidative cyclization; radical reaction

Estimation and Validation of Propanil Residues in Rice and Soil Samples by Gas Liquid Chromatography with Electron Capture Detector.

The purpose of this analysis was to develop an efficient analysis for the estimation of residues of propanil in rice grain, husk, straw and soil samples. A liquid-liquid partitioning method was used for the extraction of propanil residues from the rice grains and its contents. For this, representative 10 g samples of blended rice grain, husk, straw and soil were soaked in acetone for 24 h, and the contents then partitioned two times into 100 and 50 mL dichloromethane and one time with 100 mL hexane. The combined organic layers were collected and were concentrated to 10 mL acetone using a rotary vacuum evaporator at 40°C. The extract was then subjected to cleanup by dispersive solid phase extraction. The final extract was injected onto a GLC column, where the propanil residues were determined by electron capture detector. The percentage recoveries were ranged from 84.9 % to 98.3 % when samples were spiked at the levels of 0.05, 0.25 and 0.50 mg/kg. The limits of quantification and detection were 0.05 and 0.017 mg/kg, respectively.

Copper-Catalyzed Cyclization of Oxime Acetates with 2-Benzylidenemalononitriles for Synthesis of 2?Aminonicotinonitriles

The 2-aminopyridine is the core structure of many potential drug candidates for the treatment of a variety of dis- orders. It is widely used as building block for the synthesis of series nitrogen-containing heterocycles. However, the traditional synthesis of 2-aminopyridines relies on amination of 2-unsubstituted pyridines with sodium amide which is limited to low yield and narrow scope for the using of strongly basic conditions. Oximes and their derivatives are a class of powerful building blocks in organic synthesis. Recently, a series of reports are focus on copper-catalyzed cyclization of oxime esters, and various methods are developed for the construction of five- or six-membered heterocycles. Based on our sustained efforts to copper-catalyzed cyclization for access to heterocyles, we herein report a novel process of copper-catalyzed cyclization of oxime esters with 2-benzylidenemalononitrile for the construction of 2-aminopyridines. Various 2-aminopyridines were ob- tained in middle to good yields under the optimized reaction conditions. In this case, the N—O bond cleavage would occur for oxime esters reduced by copper(I) catalyst, followed by the formation of nucleophilic copper(II) enamide which serve as a precursor in heterocycle synthesis. A representative procedure for the copper-catalyzed construction of 2-aminopyridines by cyclization of oxime esters with 2-benzylidenemalononitrile is as following: The substrate acetophenone O-acetyl oxime (1a, 0.5 mmol, 0.0885 g), 2-benzylidenemalononitrile (2a, 0.5 mmol, 0.0770 g), CuCl (0.1 mmol, 0.0989 g, 20 mol%), and Na- HSO3 (1.0 mmol, 0.1041 g) were added to a 10 mL Schlenk tube, followed by addition of DMF (dried by calcium hydride and freshly distilled, 3 mL). The mixture was stirred at 60 ℃ as monitored by TLC. The solution was then quenched by H2O and extracted with EtOAc (20 mL×3), the combined organic layers were dried over Na2SO4, filtered, and evaporated under vaccum. The residue was purified by column chromatography on silica gel (eluent: light petroleum ether∶ethyl acetate, V∶ V=5∶1) to afford the desired product 4,6-diphenylpyridin-2-amine 3a. Keywords copper; oxime esters; 2-benzylidenemalononitrile; cyclization; 2-aminopyridine

A novel and efficient method for N-acylation of sulfonamides and carbamates: Their biological evaluation towards anti Malassezia activity

BackgroundThe N-acylation of sulfonamides and carbamates with acid anhydrides in the presence of Lewis catalysis has been described. The N-acylation of various sulfonamides and carbamates with carboxylic acid anhydrides were effectively promoted by catalytic amount of CeCl3 under solvent-free conditions to produce the corresponding N-acyl products in good to excellent yields. The synthesized compounds were tested for their antifungal activity. MethodsTo a mixture of sulfonamide and anhydride, and anhydrous CeCl3 was added and the reaction was stirred for the given time. After completion of the reaction, the reaction mixture was diluted with dichloromethane and washed with water and brine solution. The combined organic layers were dried over Na2SO4 and evaporated in vacuum. The crude compound was purified by column chromatography to afford the corresponding N-acylated product. ResultsSynthesized compounds were screened for their antifungal activity against two virulent organisms Malassezia furfur and Malassezia pachydermatis. The compounds were tested in different concentrations ranging from 200 uM to 1 uM and taking their growth observations at OD 600 nm at different time intervals. The growth in the treated wells was compared with the growth in the untreated wells. Ketoconazole was used as control, among the compounds screened 2a, 2i and 4a showed activity than the standard antifungal drug i.e. Ketoconazole. ConclusionSynthesized compounds were evaluated for their antifungal activity and three compounds 2a, 2i, and 4a showed very good activity against both the organisms, for the first time N-acyl sulfonamides and carbamates classes were evaluated as potential anti-Malassezia agents.

Mitsunobu Reaction of 1,2,3‐NH‐Triazoles: A Regio‐ and Stereoselective Approach to Functionalized Triazole Derivatives

Mitsunobu Reaction of 1,2,3‐NH‐Triazoles: A Regio‐ and Stereoselective Approach to Functionalized Triazole Derivatives

Small Molecule Activator of the Human Epithelial Sodium Channel

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.

Asymmetric Electrophilic Fluorination of β-Keto Phosphonates in Ionic Liquid Media Catalyzed by Chiral Palladium Complexes

The chemistry of bioactive organofluorine compounds is a rapidly developing area of research because of their importance in biochemical and medicinal application. Chiral organofluorine compounds containing a fluorine atom bonded directly to a stereogenic center have been utilized in studies of enzyme mechanisms and as intermediates in asymmetric syntheses. The development of effective methodologies for the preparation of new selectively fluorinated, stereochemically defined compounds is critical to further advances of fluorine chemistry. The catalytic enantioselective construction of fluorinated stereogenic centers is still important challenge in modern organic chemistry. α-Fluoroalkylphosphonates are better mimics of natural phosphates with matched 2nd pKa vaules (~6.5). The enantioselective construction of α-fluoroalkylphosphonates is extremely important because the stereochemistry of αcarbon does affect enzyme binding. Although there have been several reports for the asymmetric synthesis of αfluoroalkylphosphonates, synthetic methods of chiral αfluoro β-keto phosphonates are limited. The employment of ionic liquids as solvents for chemical reactions has been received increased attention because they have essentially no vapor pressure and provide high solubility for a wide range for organic and organometallic compounds. These solvents are reusable, enhance the reactivity of chemical transformations, simplify product isolation and allow for catalysts recycling. As part of research program related to the development of synthetic methods for the enantioselective construction of stereogenic carbon centers, we report the catalytic enantioselective fluorination of β-keto phosphonate excellent enantioselectivity (87-97% ee) promoted by a chiral palladium complexes. In this letter, we wish to report the catalytic enantioseletive electrophilic fluorination of β-keto phosphonates using chiral palladium complexes 1 in ionic liquid media. We first examined electrophilic fluorination of β-keto phosphonate 2a with NFSI in the presence of 5 mol% of 1 using [bmim]BF4 at room temperature (Table 1, entry 2). After stirring for 10 h at room temperature, the reaction was quenched by extraction with ether (5 × 5 mL). The combined organic layers concentrated and the crude product purified by silica-gel chromatography to afford α-fluoro βketo phosphonate 3a in 95% yield and 93% ee. Concerning the ionic liquids, the use of [bmim]BF4 and [bmim]OTf gave the best results, whereas the fluorination in [bmim]PF6, [bmim]SbF6 and [emim]BF4 led to lower yields and enantioselectivities or no reaction. The chemical yield and enantioselectivity were almost similar as those obtained in MeOH (8 h, 93% yield, 97% ee), a longer reaction time was necessary for the completion of the reaction. The reaction of phosphonates 2b-2d afforded the corresponding fluorinated phosphonates 3b-3d in 88-91% yield and 90-93% ee. We also tested recycling of the catalyst (Table 2). The reaction was performed seven times without affecting the yield and selectivity. The pale pink color [bmim]BF4 layer and colorless ether layer indicated that chiral palladium catalyst was retained in ionic liquid layer. In the 2nd Table 1. Catalytic enantioselective fluorination of β-keto phosphonates in ionic liquid

Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting Steps

Testosterone 6beta-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6beta-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6beta-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-(2)H(5); (ii) 6,6-(2)H(2); (iii) 6alpha-(2)H; (iv) 6beta-(2)H; and (v) 6beta-(3)H testosterone. Only the 6beta-hydrogen was removed by P450 3A4 and not the 6alpha, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the beta face. Analysis of the rates of hydroxylation of 6beta-(1)H-, 6beta-(2)H-, and 6beta-(3)H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ((D)k) of 15. The deuterium isotope effects on k(cat) and k(cat)/K(m) in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6beta-(2)H substitution. The high (D)k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high (D)k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high (D)k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.

Metal‐Free, Organocatalytic Asymmetric Transfer Hydrogenation of α,β‐Unsaturated Aldehydes

Selective reduction without metals: An imidazolidinone salt effectively catalyzes the highly enantioselective biomimetic transfer hydrogenation of α,β-unsaturated aldehydes to give the saturated analogues using a synthetic dihydropyridine cofactor (see scheme). Remarkably only one enantiomer forms regardless of the configuration of the enal starting material. Asymmetric catalytic hydrogenations are used in the large-scale industrial production of pharmaceuticals and fine chemicals and also by all living organisms. While chemical hydrogenations require metal catalysts or the use of stoichiometric amounts of metal hydrides,1 living organisms typically rely on organic cofactors such as nicotinamide adenine dinucleotide (NADH) in combination with metalloenzymes.2 Until now, metal-free catalytic asymmetric hydrogenations have been unknown in chemical synthesis and seem to be rare in nature.3 Here we show that a small organic molecule effectively catalyzes a highly enantioselective biomimetic transfer hydrogenation of α,β-unsaturated aldehydes using a synthetic dihydropyridine cofactor. This reaction is the first example of a completely metal-free transfer hydrogenation of olefins.7 We could also show that enantioselective iminium catalysis of the reaction is in principle possible. Iminium catalysis has recently been introduced as a powerful organocatalytic method for carbonyl transformations such as conjugate additions and cycloadditions.8 We have now completed an extensive screening of several synthetic and commercially available Hantzsch dihydropyridines and chiral ammonium salt catalysts and report here on an efficient enantioselective variant of our transfer hydrogenation. Entry Starting Product Yield [%] e.r. material 1 1 1 77[a] 95:5 2 1 1 89 98:2 3 1 1 83 (from (E)-5 c) 97:3 80 (from (Z)-5 c) 97:3 81 (from (E)/(Z)- 5 c (1:1)) 97:3 4 1 1 90 97:3 5 1 1 85 97:3 6 1 1 86 96:4 Like our nonasymmetric variant, the enantioselective reactions are generally clean and highly chemoselective, and carbonyl reduction or aldolization products were not detected. We also investigated the influence of the stereochemistry at the double bond. Remarkably, when we subjected both the isolated pure E or Z isomers of 4-nitro-substituted derivative 5 c to our reaction conditions, the same R enantiomer of product 8 c was obtained and with the same enantiomeric ratio of 97:3. Similarly, (E)/(Z)-5 c mixtures always gave the same result and, independent of their exact ratio, all furnished (R)-8 c in 97:3 e.r. Thus, our process is enantioconvergent, a highly desirable yet rare feature of a catalytic asymmetric reaction, where a mixture of stereoisomers furnishes only one product enantiomer. As a practical consequence of this feature, the unsaturated aldehyde starting material of our reaction may be used as a mixture of E and Z isomers as obtained from common synthetic procedures such as the Wittig reaction. Mechanistically, we assume the reaction to proceed by formation of iminium ion 9, which presumably isomerizes quickly via dienamine 10 (Scheme 1). The following rate-determining hydride transfer from dihydropyridine 6 to enal (E)-9 via transition state A proceeds faster than to (Z)-9 [k(E)>k(Z)] and, as a result, saturated aldehyde (R)-8 is formed predominantly. Proposed mechanism of the organocatalytic asymmetric transfer hydrogenation. In summary we have described the first completely metal-free catalytic asymmetric transfer hydrogenation. In our iminium catalytic reaction α,β-unsaturated aldehydes are highly efficiently reduced by means of transfer hydrogenation from a dihydropyridine. Attractive features of the process are 1) its high yields, chemo-, and enantioselectivities, 2) its enantioconvergence, and 3) its simplicity and practicability. Applications in the synthesis of natural products, pharmaceuticals, and fine chemicals may be envisioned. General procedure for the asymmetric transfer hydrogenation reaction: To a stirred solution of α,β-unsaturated aldehyde 5 (0.5 mmol) in dioxane (7 mL) at 13 °C was added catalyst 7 (20.4 mg, 0.05 mmol, 10 mol %) and, after five minutes, crystalline dihydropyridine 6 (129.2 mg, 0.51 mmol). After a reaction time of 48 h the mixture was poured into distilled water (20 mL) and extracted with dichloromethane (2×15 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated. The product was isolated by flash chromatography (SiO2, ethyl acetate/hexane) to give the saturated aldehyde product 8. Aldehydes 5 a–f were synthesized according to previously reported methods and their analytical data as well of those of aldehydes 8 match literature values.9 The absolute configuration of (R)-8 f was determined by measurement of its optical rotation and comparision to the literature value.10 Enantiomeric ratios were determined by chiral stationary phase GC-analysis.

An Improved Synthesis of 5-(2,6-Dichlorophenyl)-2-(phenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one (a VX-745 analog)

An improved procedure for the synthesis an analog of the p38 inhibitor compound VX- 745 is reported.

Synthesis of Cored Dendrimers with Internal Cross-Links This work was supported by the National Institute of Health (GM 39782). L.G.S. acknowledges the Department of Chemistry, University of Illinois, and Pharmacia & Upjohn for fellowship support.

The activity of dihydroceramide desaturase was determined in phosphate buffer (0.1m, pH 7.4), with d-erythro-N-octanoylsphingosine as substrate. The inhibitor (at the indicated concentrations) and the substrate (15 nmol) were solubilized (15 nmol of BSA in phosphate buffer/ethanol 9:1 v/v, 100 mL), combined with the microsome suspension (1 mg of protein) and NADH (30 mL, 1 mm in phosphate buffer), and made up to a final volume of 300 mL with phosphate buffer. The suspension was incubated for 30 min at 378C, and the reactions were terminated by the addition of CHCl3 (0.5 mL) containing d-erythro-N-hexanoylsphingosine (1 nmol) as an internal stan- dard for quantification. The lipids were extracted with CHCl3 (2 250 mL), the combined organic layers were evaporated under a stream of nitrogen, and the residue was derivatized with bistrimethylsilyltrifluoroacetamide (50 mL, 258C, 60 min). After derivatization, CHCl3 (50 mL) was added and the samples were stored atˇ 808C. Instrumental analyses were carried out by gas chromatography coupled to electron impact (70 eV) mass spec- trometry using a Fisons gas chromatograph (8000 series) coupled to a Fisons MD-800 mass-selective detector. The system was equipped with a nonpolar Hewlett - Packard HP-1 capillary column (30 m 0.20 mm i.d.), which was programmed from 1008C to 3408 Ca t 78C min ˇ1 . Analyses were carried out in the selected ion monitoring mode. Selected ions were m/za 311, 313, 230, and 258. Dwell (time for which a given mass is monitored) was set at 0.02 s and the mass span at 0.5.

β-Glucuronidase activation of latent aggression-promoting cues in mouse bladder urine

The present study was designed to investigate the chemical properties of the aggression-promoting cues present in bladder urine of male mice. The results of the first experiment confirmed earlier work by demonstrating the presence of an aggression-promoting chemosignal in bladder urine. In Experiment 2, behavioral assays were separately performed on the organic and aqueous layers of bladder urine obtained by repeated dichloromethane extractions. Only the combined organic layers of the initial three extractions demonstrated behavioral activity. A fourth extraction showed no behavioral activity for both organic and aqueous layers. However, the findings of Experiment 3 showed that incubation of the aqueous layer from the third CH 2Cl 2 extraction in β-glucuronidase can free additional aggression-promoting cues into a subsequent CH 2Cl 2 extraction. It is concluded that two forms of the aggression-promoting chemosignal are present in bladder urine. One is lipophilic and behaviorally active, whereas the other is conjugated, possessing latent chemosignal properties.