A trypsin inhibitor from amaranth (Amaranthus cruentus) leaves

A protein that inhibited the proteolytic activity of trypsin was isolated from amaranth leaves (Amaranthus cruentus) by affinity chromatography on trypsin-Sepharose. The inhibition was noncompetitive (withp-nitroanilide-N-α-benzoyl-DL-arginine as substrate) and had aK _i, of 1.87 × 10⁻⁷ M. The protein caused a weaker inhibitory effect on chymotrypsin, had no effect on subtilisin, displayed a molecular weight of 8 kDa, and contained no cysteine residues.     

Affinity chromatography: purification of bovine trypsin and thrombin

Bovine trypsin has been purified by affinity chromatography on agarose beads containing covalently bound p-aminophenylguanidine, p-aminobenzamidine, or m-aminobenzamidine. Bovine thrombin was purified on a m-aminobenzamidine-agarose column containing a high concentration of the inhibitor. The values of the inhibition constant, K_i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer for thrombin than for trypsin. Only those benzamidines with low K_i values and coupled in high concentration to the agarose matrix were satisfactory for thrombin purification. Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration.     

Characterization of [3H]LS‐3‐134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand

LS‐3‐134 is a substituted N‐phenylpiperazine derivative that has been reported to exhibit: (i) high‐affinity binding (K_i value 0.2 nM) at human D3 dopamine receptors, (ii) > 100‐fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low‐affinity binding (K_i > 5000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin‐dependent activation of the adenylyl cyclase inhibition assay, LS‐3‐134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [³H]‐labeled LS‐3‐134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10–15 min at 37°C) and binds with high affinity to both human (K_d = 0.06 ± 0.01 nM) and rat (K_d = 0.2 ± 0.02 nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [³H]LS‐3‐134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [³H]LS‐3‐134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.     

Novel N‐propylphthalimide‐ and 4‐vinylbenzyl‐substituted benzimidazole salts: Synthesis,characterization, and determination of their metal chelating effects and inhibition profiles against acetylcholinesterase and carbonic anhydrase enzymes

The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted N‐heterocyclic carbene (NHC) precursors were synthesized by N‐substituted benzimidazolium with aryl halides. The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted NHC precursors have been characterized by using ¹H NMR, ¹³C NMR, FTIR spectroscopy, and elemental analysis techniques. They were tested for the inhibition of AChE and hCA enzymes and demonstrated efficient inhibition profiles with K_i values in the range of 351.0–1269.9 nM against hCA I, 346.6–1193.1 nM against hCA II, and 19.0–76.3 nM against AChE. On the other hand, acetazolamide, a clinically used molecule, utilized as CA inhibitor, obtained a K_i value of 1246.7 nM against hCA I and 1407.6 nM against hCA II. Additionally, tacrine inhibited AChE and obtained a K_i value of 174.6 nM.     

Design,Synthesis, and Anti-Tumor Activity of 4′-Thionucleosides as Potent and Selective Agonists at the Human A3 Adenosine Receptor

On the basis of potent and selective binding affinity of Cl-IB-MECA to the human A₃ adenosine receptor, its 4′-thioadenosine derivatives were efficiently synthesized starting from D-gulonic γ -lactone. Among compounds tested, 2-chloro-N ⁶-(3-iodobenzyl)- and 2-chloro-N ⁶-methyl-4′ -thioadenosine-5′ -methyluronamides (7a and 7b) exhibited nanomolar range of binding affinity (K _i = 0.38 nM and 0.28 nM, respectively) at the human A₃AR. These compounds showed anti-growth effects on HL-60 leukemia cell, which resulted from the inhibition of Wnt signaling pathway.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Protease Inhibitors. Part 2. Weakly Basic Thrombin Inhibitors Incorporating Sulfonyl-Aminoguanidine Moieties as S1 Anchoring Groups: Synthesis and Structure-Activity Correlations

Abstract

Two series of derivatives have been prepared and assayed as inhibitors of two physiologically relevant serine proteases, human thrombin and human trypsin. The first series includes alkyl-/ aralkyl-/aryl- and hetarylsulfonyl-aminoguanidines. It was thus observed that sulfanilyl-aminoguanidine possesses moderate but intrinsically selective thrombin inhibitory properties, with K_i values around 90 and 1400 nM against thrombin and trypsin respectively. Further elaboration of this molecule afforded compounds that inhibited thrombin with K_i values in the range 10–50 nM, whereas affinity for trypsin remained relatively low. Such compounds were obtained either by attaching benzyloxycarbonyl- or 4-toluenesulfonylureido-protected amino acids (such as D-Phe, L-Pro) or dipeptides (such as Phe-Pro, Gly-His, β-Ala-His or Pro-Gly) to the N-4 atom of the lead molecule, sulfanilyl-aminoguanidine, or by attaching substituted-pyridinium-propylcarboxamido moieties to this lead. Thus, this study brings novel insights regarding a novel non-basic S1 anchoring moiety (i, e., SO₂NHNHC(=NH)NH₂), and new types of peptidomimetic scaffolds obtained by incorporating tosylureido-amino acids/pyridinium-substituted-GABA moieties in the hydrophobic binding site(s). Structure-activity correlations of the new serine protease inhibitors are also discussed based on a QSAR model described previously for a large series of structurally-related derivatives (Supuran et al. (1999) J. Med. Chem., in press).     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Imprinted polymers as tools for the recovery of secondary metabolites produced by fermentation

Imprinted polymers were synthesized using a mixture of pigments, N‐glutamyl‐rubropuctamine, and N‐glutamyl‐monascorubramine (I) as the template, and 2‐methacrylamido‐6‐picoline or 4‐aminostyrene as functional monomers, to obtain recognition materials capable of forming hydrogen bonds and charge interactions, respectively, with carboxyl groups of target I in the binding sites. The polymers were prepared thermally at a template loading of 5 mol% using ethylene‐glycol dimethacrylate or trimethylolpropane trimethacrylate as crosslinkers and acetonitrile or tetrahydrofuran as porogens. The selective binding of I to both types of polymer was demonstrated, although aminostyrene‐based materials showed higher overall adsorption and were studied in more detail. It was shown that the kinetics of binding of I from ethyl‐acetate extracts of fermented Monascus sp. was very rapid and virtually all the pigment adsorbed can be released by washing the polymer with ethanol–water mixtures. The feasibility of reusing imprinted polymer in consecutive adsorption/desorption cycles was also demonstrated. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 232–239, 1999.     

Computational neural network analysis of the affinity of N-n-alkylnicotinium salts for the α4β2* nicotinic acetylcholine receptor

Based on an 85 molecule database, linear regression with different size datasets and an artificial neural network approach have been used to build mathematical relationships to fit experimentally obtained affinity values (K_i) of a series of mono- and bis-quaternary ammonium salts from [³H]nicotine binding assays using rat striatal membrane preparations. The fitted results were then used to analyze the pattern among the experimental K_i values of a set of N-n-alkylnicotinium analogs with increasing n-alkyl chain length from 1 to 20 carbons. The affinity of these N-n-alkylnicotinium compounds was shown to parabolically vary with increasing numbers of carbon atoms in the n-alkyl chain, with a local minimum for the C₄ (n-butyl) analogue. A decrease in K_i value between C₁₂ and C₁₃ was also observed. The statistical results for the best neural network fit of the 85 experimental K_i values are r² = 0.84, rmsd = 0.39; r_cv² = 0.68, and loormsd = 0.56. The generated neural network model with the 85 molecule training set may also be of value for future predictions of K_i values for new virtual compounds, which can then be identified, subsequently synthesized, and tested experimentally.     

Recognition of specific and nonspecific DNA by human lactoferrin

The general principles of recognition of nucleic acids by proteins are among the most exciting problems of molecular biology. Human lactoferrin (LF) is a remarkable protein possessing many independent biological functions, including interaction with DNA. In human milk, LF is a major DNase featuring two DNA‐binding sites with different affinities for DNA. The mechanism of DNA recognition by LF was studied here for the first time. Electrophoretic mobility shift assay and fluorescence measurements were used to probe for interactions of the high‐affinity DNA‐binding site of LF with a series of model‐specific and nonspecific DNA ligands, and the structural determinants of DNA recognition by LF were characterized quantitatively. The minimal ligands for this binding site were orthophosphate (K_i = 5 mM), deoxyribose 5'‐phosphate (K_i = 3 mM), and different dNMPs (K_i = 0.56–1.6 mM). LF interacted additionally with 9–12 nucleotides or nucleotide pairs of single‐ and double‐stranded ribo‐ and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleoside phosphate groups. Such nonspecific interactions of LF with noncognate single‐ and double‐stranded d(pN)₁₀ provided ~6 to ~7.5 orders of magnitude of the enzyme affinity for any DNA. This corresponds to the Gibbs free energy of binding (ΔG⁰) of ?8.5 to ?10.0 kcal/mol. Formation of specific contacts between the LF and its cognate DNA results in an increase of the DNA affinity for the enzyme by approximately 1 order of magnitude (K_d = 10 nM; ΔG⁰ ≈ ?11.1 kcal/mol). A general function for the LF affinity for nonspecific d(pN)_n of different sequences and lengths was obtained, giving the K_d values comparable with the experimentally measured ones. A thermodynamic model was constructed to describe the interactions of LF with DNA. Copyright © 2013 John Wiley & Sons, Ltd.     

Modification of the 4-phenylbutyl side chain of potent 3-benzazepine-based GluN2B receptor antagonists

Excitotoxicity driven by overactivation of NMDA receptors represents a major mechanism of acute and chronic neurological and neurodegenerative disorders. Negative allosteric modulators interacting with the ifenprodil binding site of the NMDA receptor are able to interrupt this ongoing neurodamaging process. Starting from the potent 3-benzazepine-1,7-diol 4a novel NMDA receptor antagonists were designed by modification of the N-(4-phenylbutyl) side chain. With respect to developing novel fluorinated PET tracers, regioisomeric fluoroethoxy derivatives 11, 12, 14, and 15 were synthesized. Analogs 19 and 20 with various heteroaryl moieties at the end of the N-side chain were prepared by Sonogashira reaction and nucleophilic substitution. The fluoroethyl triazole 37 was obtained by 1,3-dipolar cycloaddition. In several new ligands, the flexibility of the (hetero)arylbutyl side chain was restricted by incorporation of a triple bond. The affinity towards the ifenprodil binding site was tested in an established competition assay using [³H]ifenprodil as radioligand. Introduction of a fluoroethoxy moiety at the terminal phenyl ring, replacement of the terminal phenyl ring by a heteroaryl ring and incorporation of a triple bond into the butyl spacer led to considerable reduction of GluN2B affinity. The phenol 15 (K_i = 193 nM) bearing a p-fluoroethoxy moiety at the terminal phenyl ring represents the most promising GluN2B ligand of this series of compounds. With exception of 15 showing moderate σ₂ affinity (K_i = 79 nM), the interaction of synthesized 3-benzazepines towards the PCP binding site of the NMDA receptor, σ₁ and σ₂ receptors was rather low (K_i > 100 nM).     

N,N –Disubstituted piperazines and homopiperazines: Synthesis and affinities at α4β2* and α7* neuronal nicotinic acetylcholine receptors

A series of N, N– disubstituted piperazines and homopiperazines were prepared and evaluated for binding to natural α4β2* and α7* neuronal nicotinic acetylcholine receptors (nAChRs) using whole brain membrane. Some compounds exhibited good selectivity for α4β2* nAChRs and did not interact with the α7* nAChRs subtype. The most potent analogs were compounds 8-19 (K_i = 10.4 μM), 8–13 (K_i = 12.0 μM), and 8–24 (K_i = 12.8 μM). Thus, linking together a pyridine π-system and a cyclic amine moiety via a homopiperazine ring affords compounds with low affinity but with good selectivity for α4β2* nAChRs.     

Hydrophobic binding sites of human liver alanine aminopeptidase

Fatty acids, alkyl amines, and amides of α-amino fatty acids inhibit human liver alanine aminopeptidase apparently by binding to residue binding site 1 of the active center, i.e., the N-terminal binding site. The pK_i values of the acids, amines, and amides increase until the overall chain length reaches eight carbons. The pK_i values are the same for members of the series with chain lengths longer than eight carbon atoms. Assuming an extended structure of the inhibitors, this site will accommodate amino acid side chains of not longer than 11.7 Å from the α-carbon to the end of the chain. Long chain amino acids inhibit by binding apparently at residue site 3. The pK_i values of dl-α-amino acids from α-aminobutyric acid to α-aminodecanoic acid increase with the addition of each methylene unit. Thus, site 3 will accommodate amino acid side chains which are at least 13.0 Å from the α-carbon to the end of the chain. Methanol and other organic solvents reversibly inhibit the binding of substrates at pH 6.9 without affecting the maximum rate of catalysis. At lower pH values, the maximum rate of catalysis is lowered. Sodium chloride also inhibits substrate hydrolysis at pH 6.9 but does not affect the maximum rate of catalysis. The pK_i values of fatty acids, alkyl amines, and amino acids are strongly decreased by methanol and slightly increased by sodium chloride. These data indicate that a major portion of the interactions of the enzyme with fatty acids, amines, and amino acids is of a hydrophobic nature.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Design,Synthesis, Lipophilic Properties,and Binding Affinities of Potential Ligands in Positron Emission Tomography (PET) for Visualization of Brain Dopamine D4 Receptors

We report the synthesis of compounds structurally related to the high‐affinity dopamine D₄ receptor ligand N‐{2‐[4‐(3‐cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐methoxybenzamide ( 1e ). All compounds were specifically designed as potential PET radioligands for brain D₄ receptor visualization, having lipophilicity within a range for brain uptake and weak non‐specific binding (0.75<cLogP<3.15) and bearing a substituent for easy access to labeling with the positron emitter isotope ¹¹C or ¹⁸F. The best compound of the series, N‐{2‐[4‐(4‐chlorophenyl)piperazin‐1‐yl]ethyl}‐6‐fluoropyridine‐3‐carboxamide ( 7a ), displayed excellent selectivity over D₂ and D₃ receptors (>100‐fold), but its D₄ receptor affinity was suboptimal for imaging of brain D₄ receptors (K_i=30 nM ).     

In vitro effects of standard antioxidants on lactoperoxidase enzyme–A molecular docking approach

Lactoperoxidase (LPO), an antioxidant enzyme, is a natural antimicrobial system that eliminates the harmful effects of microorganisms in milk. It has a wide range of applications and is also preferred in cosmetic and clinical applications, as well as used in foods. The use of antioxidants is well recognized in the food and feed industries to improve the shelf life of products. This study aimed to determine the in vitro inhibition effects of Trolox, α‐tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate, which are commonly used as antioxidants in food and pharmaceutical products. For this purpose, LPO was first purified in a single step using sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity gel chromatography. Also, some inhibition parameters, including half‐maximal inhibitory concentration (IC₅₀), K_i values, and inhibition types, were calculated for each antioxidant molecule. The IC₅₀ values of these molecules, which exhibited competitive inhibition, varied between 377.7 and 3397.8 nM. Molecular docking studies were also performed for all compounds. According to the binding scores, α‐tocopherol was shown to exhibit the most effective inhibitor property (IC₅₀: 377.7 nM and K_i: 635.8 ± 16.8 nM) among the standard antioxidants used in this study. Inhibiting the LPO activity by standard antioxidants results in the weakening of the immune system during lactation, which is important for metabolism.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue α-amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 α-carbon in the d conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.     

Rational design and molecular engineering of peptide aptamers to target human pancreatic trypsin in acute pancreatitis

Human pancreatic trypsin (hPT) is an established target for acute pancreatitis (AP) therapeutics. Here, a bioinformatics protocol of protein docking, peptide refinement, dynamics simulation and affinity analysis was described to perform rational design and molecular engineering of hPT peptide aptamers. Protein docking was employed to model the intermolecular interactions between hPT and its cognate inhibitory protein, the human pancreatic trypsin inhibitor (hTI). A number of peptide fragments were cut out from the interaction sites of docked hPT–hTI complexes, from which a decapeptide fragment ¹³LNGCTLEYRP²² was found to exhibit potent inhibition against hPT (K _i = 5.3 ± 0.8 μM). We also carried out alanine scanning and virtual mutagenesis to systematically examine the independent contribution of peptide residues to binding affinity, and the harvested knowledge were then used to guide modification and optimization of the decapeptide fragment. Subsequently, inhibition studies of nine promising candidates against recombinant hPT were conducted, from which four samples were successfully identified to have high or moderate potency (K _i < 10 μM). In particular, the peptides LQVCTLEYCN and LQICTLEYCT were found to inhibit hPT activity significantly (K _i = 0.23 ± 0.04 and 0.85 ± 0.18 μM, respectively). Structural analysis of hPT–peptide complex systems unraveled diverse chemical interactions such as hydrogen bonds, salt bridges and hydrophobic forces across the complex interfaces.     

1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-Acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-Releasing factor-1 (CRF1) receptor antagonist activities

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF₁ receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF₁ receptor (K_i=9 nM).