Structure–activity relationships of cyclic and linear peptide T analogues

Using the potent cyclic peptide T analog-Thr-Thr-Asn-Tyr-Thr-Asp- as parent compound, a series of analogues were synthesized and their potencies in a monocyte chemotaxis assay were compared with those of correspondingly modified linear peptides. Structure-activity relationships observed with cyclic compounds did not always parallel those determined with linear analogues. -Thr-Hse-Asn-Tyr-Thr-Asp- showed the highest affinity to CD4 receptor of monocytes of any peptide thus far studied. It also proved to be highly resistant to degradation by plasma or brain enzymes.     

Design of 16-residue peptides possessing antimicrobial and hemolytic activities or only antimicrobial activity from an inactive peptide

We have explored the possibility of generating peptides having antimicrobial and hemolytic activities or only antimicrobial activity, from a 16-residue peptide, GFFALIPKIISSPLFK, corresponding to the N-terminal region of the toxin pardaxin. This peptide does not exibit these activities, although it can permeabilize model membranes. Peptides were synthesized wherein either A4 or P7 were substituted by K and S11 replaced by K , Peptides in which P7 and S11 were replaced with K, ( AK ) and A4 and S11 replaced with K and A instead of P at position 7, ( KA ) showed potent antimicrobial and hemolytic activities. However, the peptide where S11 and A4 were replaced with K, ( KP ) showed pronounced antimicrobial activity with very weak hemolytic activity. Circular dichroism studies indicated that peptides AK and KA had a strong propensity to occur in a helical conformation, whereas KP did not. Peptides AK and KA were very effective in permeabilizing model membranes, whereas KP was relatively ineffective. Our studies thus suggest the requirements for a peptide to have only antimicrobial activity and also that selectivity in activity can be rationalized on the basis of biophysical principles. Thus, by judicious positioning of amino acids, especially positively charged ones, it should be possible to generate biologically active peptides without taking recourse to a combinatorial approach. © Munksgaard 1995.     

Solid-phase peptide synthesis and biological activity of bovine thymopoietin II (bTP–11)

Bovine thymopoietin (bTP), a 49 amino acid polypeptide, was synthesized using Merrifield's solid-phase peptide synthesis methodology. The polypeptide was purified using anion-exchange chromatography and reversed-phase HPLC and characterized by mass spectrometry and amino acid analysis of the full-length peptide and of products derived from digestion with Staphylococcus aureus V8 protease. The biological activity of the synthesized product was tested in several assay systems. Synthetic bTP was found to induce the expression of Thy 1.2 antigen on T-lymphocytes from athymic mice, in agreement with previous studies on the biological activity of endogenous bTP. Biological activity at skeletal muscle and neuronal nicotinic acetylcholine receptor sites, as reported by others for bTP, could not be confirmed in our studies. The absence of biological activity at nicotinic receptor sites may be related to the results of a recent report demonstrating the presence of a cobratoxin-like molecule in preparations of natural bTP. These data indicate that synthetic peptides have an important role for the evaluation of the specificity of the biological activity of polypeptides.     

Enhanced antitumor activity and selectivity of lactoferrin‐derived peptides

Abstract: A number of peptide analogs derived from the N‐terminal α‐helical region of bovine lactoferrin (LFB 14–31), were designed in order to investigate how deviating numbers and positions of positively charged residues and numbers of aromatic residues affected their activity against prokaryotic, normal and transformed eukaryotic cells. Most of the LFB derivatives were highly active against both Escherichia coli and Staphylococcus aureus. The peptides were more active against the tumor cell lines MethA, HT‐29 and MT‐1 than normal eukaryotic cells. The peptides that were most active against the tumor cell lines had all cationic residues concentrated in one sector of the helical structure. These peptides were less selective against the tumor cell lines than against normal fibroblasts. Quantitative structure?activity relationship studies showed that certain structural parameters affected toxicity against the tumor cell lines more than against fibroblasts. Peptides encompassing these parameters were slightly less active against tumor cells, but gained significant selectivity.     

Structural and charge requirements for antimicrobial and hemolytic activity in the peptide PKLLETFLSKWIG,corresponding to the hydrophobic region of the antimicrobial protein bovine seminalplasmin

Several analogs of the 13-residue antimicrobial and hemolytic peptide PKLLETFLSKWIG (SPF), which is the most hydrophobic region of the 47-residue antimicrobial protein seminalplasmin [Sitaram, N. & Nagaraj, R. (1990) J. Biol. Chem. 265, 10438-104423 have been synthesized. The antimicrobial and hemolytic properties of the peptides were investigated with a view to gain an insight into the structural and charge requirements for these activities of SPF. Peptides in which E was replaced by K exhibited considerably improved antimicrobial activity with no concomitant increase in hemolytic activity. A peptide in which the aromatic amino acids were replaced by leucine exhibited antimicrobial activity like those of the peptides which had aromatic amino acids. Interchange in the positions of E and K and total replacement of K by E resulted in complete loss of activity. The peptides having antimicrobial activities showed appreciable helical content in a hydrophobic environment, whereas inactive peptides did not. Thus, by suitable‘engineering’ the biological activity of a short 13-residue peptide can be altered to yield peptides specifically having only antimicrobial activity with increased potency. © Munksgaard 1995.     

Structure–activity relationship of neuropeptide γ derived from mammalian and fish

Abstract: This study of relationship between structure and biologic activity was performed using five neuropeptide γs [NPγ; mammalian‐NPγ (M‐NPγ), trout‐NPγ (T‐NPγ), goldfish‐NPγ (G‐NPγ), bowfin‐NPγ (B‐NPγ), and shark‐NPγ (S‐NPγ)]. Circular dichroism (CD) spectra showed that all peptides took random structure in buffer solution. In neutral and acidic liposomes, M‐NPγ, T‐NPγ, B‐NPγ, and S‐NPγ still adopted random structure, while G‐NPγ had an α‐helical structure. The biologic activity of NPγs has been estimated by their effects on the intestinal motility and arterial relaxation. The intestinal motility was investigated with rat duodenum (RD), carp intestine (CI), and guinea‐pig ileum (GPI). The arterial relaxing effect was tested with guinea‐pig aorta (GPA) and rat mesenteric artery (RMA). In RD, the order of potency compared with the EC₅₀ value was M‐NPγ > S‐NPγ > B‐NPγ > G‐NPγ > T‐NPγ. G‐NPγ was the most contractile agent in CI. S‐NPγ was the most contractile agent in GPI. Using an arterial relaxing test, the order of potency was G‐NPγ > T‐NPγ > B‐NPγ > S‐NPγ > M‐NPγ in GPA, and all NPγs remarkably reduced relaxing activity in RMA. Despite their structural similarities to NPγs, G‐NPγ has high affinity to tachykinin receptor‐binding sites in GPA and CI, indicating an α‐helical structure may have a critical role for receptor binding. However, an α‐helical structure does not play a critical role in recognizing receptor‐binding sites in RD and GPI.     

Structure–activity studies of normal and retro pig cecropin–melittin hybrids

Chimeric analogs of cecropin P1 and melittin with normal and retro sequences were synthesized to explore the effect of sequence, amide bond direction (helical dipole), charge, amphipathicity and hydrophobicity on their antibacterial activity and channel-forming ability. When viewed from the opposite end by rotation in the plane 180° retro analogs have the same sequence as the parent with reversed amide bond and helical dipole directions. The expected activities were related to the important structural features and a series of assumptions were made. Retro analogs are expected to be inactive if both sequence and amide bond direction make critical contributions to the activity. CP1(1–10)M(2–9) amide, (SWLSKTAKKLIGAVLKVL), showed a broad antibacterial spectrum with high activity against the two Gram-negative and three Gram-positive bacteria tested. Retro-CP1(1–10)M(2–9) was less active compared to its normal peptide. CP1(1–9)M(1–8) and CP1(1–9)M(2–8) amides were found to be active against Gram-negative Escherichia coli and also Gram-positive Streptococcus pyogenes, but inactive against the other test organisms. The corresponding retro analogs were inactive against all the five bacteria tested. These results suggest that both sequence and amide bond direction (helix dipole) are important structural requirements for the activity of CP1-M hybrids. Acetylation of the N-terminal amine in both normal and retro analogs lowered their activity, indicating the contribution of free amine to the activity. These analogs form ion-conducting channels in lipid bilayers. The action of the peptides may be explained by self-aggregation and formation of ion-conducting pores across bacterial membranes. Conformational analysis obtained from CD measurements showed that all analogs form amphipathic α-helices in presence of 12–20% hexafluoro isopropanol. The retro CP1(1–10)M(2–9) amide showed higher helicity and is more potent compared to other retro analogs synthesized. These studies show the effect of small sequence modifications on the biological activity of the peptides and on their α-helical conformation in HFIP, the structure-inducing organic solvent.     

Interaction of acylated and substituted antimicrobial peptide analogs with phospholipid-polydiacetylene vesicles. Correlation with their biological properties

A series of peptide analogs based on region 6-22 of Plantaricin 149 sequence were synthesized. The interaction between these analogs and phospholipid-polydiacetylene vesicles was investigated to evaluate the ability of the bioassay to detect differences in the interaction of the peptides with dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles, associated with amino acid substitution and N-terminal conjugation of the sequences with short fatty acids (8 and 12 carbon atoms). Fatty acid conjugation of peptides with low antimicrobial activity resulted in lipopeptides with improved activity against strains of Staphylococcus aureus and Listeria monocytogenes. The length of the fatty acid determined the bacterial specificity, and the conjugation with n-octanoic acid yielded the most active analog (C8-CT) against Staphylococcus aureus strain (MIC: 1.0 μm) while the conjugation with n-dodecanoic acid (C12-CT) was optimal for Listeria monocytogenes strain (MIC: 2.0 μm). In contrast, the substitution of Phe by Trp had an unfavorable effect on the antimicrobial activity. Hemolysis tests and membrane interaction studies with dipalmitoylphosphatidylcholine-polydiacetylene vesicles showed that lipopeptides interact to a greater extent with both biological and biomimetic membranes. Also, a good correlation was found between antimicrobial activity against Staphylococcus aureus strain and % colorimetric response values with dipalmitoylphosphatidylglycerol-polydiacetylene vesicles.     

Structure−function studies on the amphibian peptide brevinin 1E: translocating the cationic segment from the C‐terminal end to a central position favors selective antibacterial activity

Abstract: Brevinin 1E, which has the sequence FLPLLAGLAANFLPKIFCKITRKC , is an antimicrobial peptide isolated from the skin secretions of the European frog Rana esculenta. Both the linear and the disulfide‐bridged forms have relatively broad‐spectrum antibacterial as well as hemolytic activities. The antibacterial and hemolytic activities and biophysical properties of synthetic peptides corresponding to brevinin 1E and its analog in which the segment CKITRKC has been transposed to a central location resulting in the sequence FLPLLAGLCKITRKC AANFLPKIF have been investigated. Our studies indicate that the analog peptide has antibacterial activity comparable with brevinin 1E, but with considerably reduced hemolytic activity. The linear variant of the analog has no hemolytic activity, unlike the linear form of brevinin 1E. The biological activities can be explained on the basis of relative affinities for anionic and zwitterionic lipids. A cluster of cationic amino acids flanked on one side by a hydrophobic stretch of amino acids and another side composed of apolar amino acids appears to favor preferential antibacterial activity.     

牛和羊胎盘肽的制备及其生物活性的研究

目的探索一种新型的免疫调节剂,充分利用动物胎盘。方法利用超滤法从牛和羊胎盘中提取胎盘肽,采用改良的加脲Tricine-SDS-PAGE法测定其相对分子质量;利用紫外光扫描其最大吸收峰;并通过建立体外抑制兔淋巴细胞模型,利用四甲基偶氮唑盐(MTT)比色法测定所提取胎盘肽的免疫调节作用。结果从牛和羊胎盘中成功提取出了胎盘肽,其相对分子质量分别为4 785和4 386;其最大吸收峰分别为210.5和225.4 nm;牛和羊胎盘肽均可使顺铂抑制的兔外周血T淋巴细胞的转化率显著提高,其中牛胎盘肽以1∶1×103、1∶1×1042组吸收度明显高于抑制组(P<0.05);羊胎盘肽以1∶1×102、1∶1×103和1∶1×1043组效果最为明显(P<0.01)。结论牛和羊胎盘中含有的胎盘肽可成功提取出来,其可明显提高淋巴细胞的转化率,是一种较为理想的免疫调节剂。     

Structure–activity relationships for nicotine analogs comparing competition for [3H]nicotine binding and psychotropic potency

The structure–activity relationships were established for nicotine analogs and related agents comparing their competition for [³H]nicotine binding to rat brain membranes, Torpedo membranes, and transfected insect cells with their ability to produce prostration in rats following administration into the rat lateral ventricles. A total of 59 compounds were investigated, consisting of pyridine- and pyrrolidine-substituted analogs of nicotine, other tobacco alkaloids and related molecules, various aminoalkylpyridines, and ring-shifted analogs of nicotine. Some of the compounds were also evaluated for [³H]nicotine binding to Torpedo electroplax membranes and to Sf9 cells expressing an α4β2 nicotinic cholinergic receptor subtype. Linear regression plots of Ki vs ED₅₀ values for prostration of the various classes of the compounds yielded correlation of determination (R²) of 0.923 for the pyridine-substituted analogs, 0.725 for the pyrrolidine-substituted analogs, 0.947 for other tobacco alkaloids and related compounds, and 0.537 for all 59 compounds. An excellent correlation was observed comparing Torpedo Ki values with both prostration ED₅₀ values and rat brain Ki values. Within the pyridine-substituted series, methyl substiuents in the 6-position resulted in over three-fold greater potency compared to nicotine, whereas potency decreased markedly with bulkier alkyl and cycloalkyl substituents. Within the pyrrolidine-substituted series, methyl groups in the 2′-position only slightly reduced potency compared to nicotine, whereas 3′- and 5′-addition markedly reduces potency. A linear regression plot of the Ki values of brain vs. those of Sf9 cells expressing the α4β2 nicotinic cholinergic receptor subtype yielded a coefficient of correlation of 0.981; a finding which is consistent with the notion that the α4β2 subtype comprises over 90% of total rat brain receptor. Drug Dev. Res. 45:10–16, 1998. © 1998 Wiley-Liss, Inc.     

Structure–activity relationships of astemizole derivatives for inhibition of store operated Ca channels and exocytosis

The effects of a series of analogues of the antiallergic drug astemizole on the exocytosis of the enzyme β-hexosaminidase were studied in a mast cell model, the rat basophilic leukemia (RBL-2H3) cell. Besides differences in the effects on FcεRI receptor-stimulated exocytosis, changes were also observed in Ca²⁺ influx and in the perturbation of the cell membrane. A strong correlation was found between the effects on antigen- and thapsigargin-stimulated

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influx was correlated with the inhibition of β-hexosaminidase release and membrane stabilization. It is concluded that the astemizole analogues are capable of inhibiting mast cell β-hexosaminidase release through inhibition of Ca²⁺-store-operated Ca²⁺ channels (SOC). Compounds with high lipophilicity also released Ca²⁺ from intracellular stores. Lowering of the hydrophobicity by introduction of nitrogens or truncation at different sites in the astemizole structure decreased inhibitory activity on SOC channels. The inhibition of SOC channels cannot completely be ascribed to non-specific membrane effects. The piperidinyl–benzimidazole moiety was found to be important for inhibition of SOC channels. The observed differences in activity possibly depend on the way the compounds penetrate the membrane bilayer. Astemizole is an interesting new tool to study SOC channels and can be a lead for the design of mast cell-stabilizing antiallergic drugs.     

Structure–activity relationships of arodyn,a novel acetylated kappa opioid receptor antagonist*,†

Abstract: We previously reported that the novel dynorphin A (Dyn A, Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln) analog arodyn (Ac[Phe^1,2,3,Arg⁴,d ‐Ala⁸]Dyn A‐(1–11)NH₂, Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617–5619) is a κ opioid receptor‐selective peptide [K_i(κ) = 10 nm , K_i ratio (κ/μ/δ) = 1/174/583] which exhibits antagonist activity at κ opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure–activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d ‐amino acid substitution in the N‐terminal ‘message’ sequence, NMePhe substitution individually in positions 1–3, and modifications in position 1. The results for the Ala‐substituted derivatives indicated that Arg⁶ and Arg⁷ are the most important residues for arodyn's nanomolar binding affinity for κ opioid receptors. Ala substitution of the other basic residues (Arg⁴, Arg⁹ and Lys¹¹) resulted in lower decreases in affinity for κ opioid receptors (three‐ to fivefold compared with arodyn). Of particular interest, while [Ala¹⁰]arodyn exhibits similar κ opioid receptor binding as arodyn, it displays higher κ vs. μ opioid receptor selectivity [K_i ratio (κ/μ) = 1/350] than arodyn because of a twofold loss in affinity at μ opioid receptors. Surprisingly, the Tyr¹ analog exhibits a sevenfold decrease in κ opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr¹]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing κ opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe¹]arodyn which exhibits high affinity [K_i(κ) = 4.56 nm ] and exceptional selectivity for κ opioid receptors [K_i ratio (κ/μ/δ) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A‐(1–13)NH₂. Thus [NMePhe¹]arodyn is a highly κ opioid receptor‐selective antagonist that could be a useful pharmacological tool to study κ opioid receptor‐mediated activities.     

Structure–activity relationship of butyrate analogues on apoptosis,proliferation and histone deacetylase activity in HCT‐116 human colorectal cancer cells

1. Butyrate, a bacteria fermentative product in the colonic lumen, has been shown to produce a wide variety of biological effects in human cancer cells in vitro. However, there are pharmacological drawbacks associated with the use of butyrate therapy and there are limited published data on the structure–activity relationship of butyrate analogues in colorectal cancer cells. Previously, we determined structure–activity relationship using HT‐29 human colorectal cancer cells. However, it was viewed as important to explore similar relationships in another colorectal cancer cell line. 2. Therefore, in the present study, the in vitro structure–activity relationship of butyrate analogues was examined by investigating their effects on apoptosis, cell proliferation, histone deacetylase (HDAC) activity and lactate dehydrogenase (LDH) leakage as a measure of cell toxicity in HCT‐116 human colorectal cancer cells. 3. Of the 32 analogues tested, only 4‐benzoylbutyrate, 3‐benzo‐ylpropionate, 4‐(4‐nitrophenyl)butyrate and 3‐(4‐fluorobenzoyl)propionate exhibited comparable biological effects to butyrate. The common structural properties of the compounds of interest were to lack amino or hydroxyl substitutions at the 2‐, 3‐ and/or 4‐position of the aliphatic moiety of butyrate. 4. The present study reveals a dissociation between the induction of apoptosis, inhibition of cell proliferation, HDAC activity and LDH leakage. The results indicate differential responses of butyrate analogues in HT‐29 and HCT‐116 colorectal cancer cells.     

Syntheses and structure–membrane active antimicrobial activity relationship of alkylamino‐modified glucose,maltooligosaccharide, and amylose

Antimicrobial alkylamine‐modified sugars were prepared. Microwave‐assisted click reaction efficiently achieved poly‐functionalization of oligo‐ and polysaccharides. The sugars exhibited a unique relationship of their bacterial membrane permeabilization and antimicrobial activity with the number of functional groups and the structure of the molecular scaffold. It shows that the assembly of the functional groups is necessary for being antimicrobial. The amylose derivatives also exhibited synergy to minimize the necessary dose of conventional antibiotics and increase their antimicrobial potency.     

Studies on the peptide corresponding to residues 34–47 of bovine factor X

Calcium binding studies of a 14-residue peptide corresponding to the 37–46 sequence of bovine factor X were performed using calcium ion selective electrode titrations and equilibrium dialysis. The presence of γ-carboxyglutamic acid residues at positions 36 and 40 coupled with the assumption that the peptide would bind calcium ions also prompted an investigation of possible secondary conformational changes in the peptide by use of circular dichroism spectroscopy. Equilibrium dialysis revealed a single relatively weak calcium binding site (log K_a= 2.39); an ion selective electrode experiment confirmed this result (log K_a= 2.17). The peptide maintained a random coil conformation throughout the calcium ion titrations as measured by circular dichroism.     

Determination of the biological activity and structure activity relationships of drugs based on the highly cytotoxic duocarmycins and CC-1065

The natural antibiotics CC‑1065 and the duocarmycins are highly cytotoxic compounds which however are not suitable for cancer therapy due to their general toxicity. We have developed glycosidic prodrugs of seco-analogues of these antibiotics for a selective cancer therapy using conjugates of glycohydrolases and tumour-selective monoclonal antibodies for the liberation of the drugs from the prodrugs predominantly at the tumour site. For the determination of structure activity relationships of the different seco-drugs, experiments addressing their interaction with synthetic DNA were performed. Using electro­spray mass spectrometry and high performance liquid chromatography, the experiments revealed a correlation of the stability of these drugs with their cytotoxicity in cell culture investigations. Furthermore, it was shown that the drugs bind to AT-rich regions of double-stranded DNA and the more cytotoxic drugs induce DNA fragmentation at room temperature in several of the selected DNA double-strands. Finally, an explanation for the very high cytotoxicity of CC-1065, the duocarmycins and analogous drugs is given.     

Exploring relationships between mimic configuration,peptide conformation and biological activity in indolizidin‐2‐one amino acid analogs of gramicidin S*

Abstract: Indolizidin‐2‐one amino acids (I²aas, 6S‐ and 6R‐ 1 ) possessing 6S‐ and 6R‐ring‐fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution‐phase and solid‐phase techniques were used to synthesize three analogs with I²aa residues in place of the d ‐Phe‐Pro residues at the turn regions of GS: [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ), [Lys^2,2′,(6S)‐I²aa^{4?5,4′?5′}]GS ( 3 ) and [(6R)‐I²aa^{4?5,4′?5′}]GS ( 4 ). Although conformational analysis of [I²aa^{4?5,4′?5′}]GS analogs 2?4 indicated that both ring‐fusion stereoisomers of I²aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)‐I²aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)‐I²aa analog 4 , suggesting a greater propensity for the (6S)‐diastereomer to adopt the β‐turn/antiparallel β‐pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)‐I²aa analog 2 exhibited significantly better potency than the (6R)‐I²aa diastereomer 4 . Relative to GS, [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)‐I²aa diastereomer. The synthesis and evaluation of GS analogs 2?4 has furnished new insight into the importance of ring‐fusion stereochemistry for turn mimicry by indolizidin‐2‐one amino acids as well as novel antimicrobial peptides.     

Structure–activity relationships of isoeugenol‐based chlorophenylpiperazine derivatives on serotonergic/adrenergic receptor,platelet aggregation,and lipid peroxidation

Three isoeugenol‐based eugenosedin chlorphenylpiperazine derivatives, Eu‐A, Eu‐B, and Eu‐C, were synthesized and tested for their serotonergic, adrenergic antagonist, antioxidant, and anti‐aggregation activities. In radioligand binding assays, all three agents displayed significant binding affinities on α₁, α₂, β₁, 5‐HT_1B, and 5‐HT_2A receptors. In human platelet, they inhibited epinephrine and 5‐HT‐induced aggregation, and in human platelet with α₂ and 5‐HT_2A receptors they had a competitive binding effect. Eu‐B and Eu‐C were more potent than Eu‐A. All compounds had antioxidant effects derived from aryloxypropanolamine. Eu‐ A, Eu‐B, or Eu‐C (1, 3, 5 mg/kg iv) given to normotensive Wistar rats produced a dose‐dependent decrease in mean arterial blood pressure and heart rate and when injected into the cisternum, Eu‐A, Eu‐B, or Eu‐C (0.3, 0.03 µmol) increased blood pressure within 15 min. Pretreatment with any of the three agents inhibited clonidine (38 pmol)‐induced hypotension. In vitro experiments, Eu‐A, Eu‐B, or Eu‐C (1, 10, and 100 µM) competitively antagonized norepinephrine‐, clonidine‐, and 5‐HT (10^?8–10^?4 M)‐induced vasocontraction in isolated rat aorta, and competitively antagonized isoproterenol (10^?8–10^?4 M)‐induced positive inotropic effects in a concentration‐dependent manner in the isolated rat left atrium. In isolated rabbit ear arteries sensitized with 16 mM K⁺, all three agents antagonized 5‐nonyloxytryptamine‐ and 5‐HT‐induced vasocontractions. These findings show that Eu‐A, Eu‐B, and Eu‐C possess functional α₁, α₂, β₁, 5‐HT_1B, and 5‐HT_2A receptor blocking activities. In conclusion, the changes in the position of chloride at phenylpiperazine influenced the serotonergic receptor, adrenoceptor antagonistic activities, but not anti‐aggregation and antioxidant activities. Drug Dev Res 71:1–9, 2010. © 2010 Wiley‐Liss, Inc.