RCAI-17, 22, 24-26, 29, 31, 34-36, 38-40, and 88, the analogs of KRN7000 with a sulfonamide linkage: their synthesis and bioactivity for mouse natural killer T cells to produce Th2-biased cytokines

RCAI-17, 22, 24-26, 29, 31, 34-36, 38-40, and 88, the analogs of KRN7000 with a sulfonamide linkage instead of an amide bond, were synthesized to examine their bioactivity for mouse natural killer (NK) T cells. RCAI-17, 22, 24-26, 29, 31, 34-36, and 88 are the aromatic sulfonamide analogs, while RCAI-39 and 40 are the aliphatic ones. RCAI-38 is a C-galactoside analog of RCAI-26, which is the p-toluenesulfonamide analog of KRN7000. According to their bioassay, these sulfonamide analogs were shown to be the stimulants of mouse NKT cells to induce the production of Th2-biased cytokines in vitro, while RCAI-38 did not induce any cytokine production.     

RCAI-37, 56, 59, 60, 92, 101, and 102, cyclitol and carbasugar analogs of KRN7000: Their synthesis and bioactivity for mouse lymphocytes to produce Th1-biased cytokines

Cyclitol [RCAI-37 (1), 59 (5), 92 (7), and 102 (2)] and carbasugar analogs [RCAI-56 (3), 60 (4), and 101 (6)] of KRN7000 were synthesized through coupling reactions of the corresponding cyclitol or carbasugar derivatives with a cyclic sulfamidate (9) as the key step. Bioassay showed RCAI-56 (3, carbagalactose analog of KRN7000), 59 (5, 1-deoxy-neo-inositol analog), and 92 (7, 1-O-methylated 5) to be remarkably potent stimulants of mouse lymphocytes to produce Th1-biased cytokines, such as interferon-γ, in vivo. RCAI-60 (4, carbafucose analog) and RCAI-101 (6, 6-O-methylated 3) showed strong bioactivity, on the other hands, RCAI-37 (1, myo-inositol analog) and 102 (2, neo-inositol analog) induced little cytokine production.     

Synthesis of RCAI-172 (C6 epimer of RCAI-147) and its biological activity

RCAI-147 is one of the hydroxylated analogues of KRN7000 which is known as a ligand for the activation of CD1d mediated invariant natural killer T cells (iNKT cells) and releases both T helper 1 (Th1) cytokines such as IFN-γ and T helper 2 (Th2) cytokines such as IL-4. KRN7000 has been anticipated as an antitumor drug or an adjuvant for viral infection such as influenza, because of its strong secretion of IFN-γ. In an interesting twist, it has been obvious in our previous paper that RCAI-147 induces much more Th2 cytokines (IL-4) than Th1 cytokines (IFN-γ) from iNKT cells compared to KRN7000, and shows fairly good result in the experimental autoimmune encephalomyelitis (EAE) test. Therefore, synthesis of RCAI-172 (C6-OH epimer of RCAI-147) was attempted to examine the biological activity. As a result, RCAI-172 was synthesized and its biological activity biased to Th2 response largely compared to that of KRN7000. However, this level decreased to approximately 61% compared to that of RCAI-147. And the clinical score of RCAI-172 for EAE suppression was disappointing. There exist seven chiral centers in the aglycon part of RCAI-172, and even though the change of configuration is just one position (C6-OH), the effect on both Th1/Th2 response and EAE test is fairly large.     

RCAI-61 and related 6′-modified analogs of KRN7000: Their synthesis and bioactivity for mouse lymphocytes to produce interferon-γ in vivo

We synthesized ten new analogs of 6′-modified KRN7000 (A): RCAI-58, 61, 64, 83, 85–87, 113, 119, and 125. They could be synthesized by α-selective galactosylation of ceramide 9 with the 6-modified d-galactopyranosyl fluorides (8a–8f) or l-arabinopyranosyl fluoride (17), or by etherification of the known alcohol 19. Bioassay of the ten analogs demonstrated that RCAI-61 (1, 6′-O-methylated analog of A) was the most potent immunostimulant among them, and could induce the production of a large amount of IFN-γ even at a low concentration in mice in vivo.     

RCAI-8, 9, 18, 19, and 49-52, conformationally restricted analogues of KRN7000 with an azetidine or a pyrrolidine ring: Their synthesis and bioactivity for mouse natural killer T cells to produce cytokines

Conformationally restricted analogues of KRN7000, an alpha-d-galactosyl ceramide, were synthesized to examine their bioactivity for mouse natural killer (NK) T cells to produce cytokines. RCAI-8, 9, 51, and 52 are the analogues with a pyrrolidine ring, and RCAI-18, 19, 49, and 50 are those with an azetidine ring. RCAI-18 was shown to be a potent inducer of cytokine production by mouse NKT cells, while RCAI-51 was a moderately active inducer.     

Structure-activity relationship studies of novel glycosphingolipids that stimulate natural killer T-cells

KRN7000, an anticancer drug candidate developed by Kirin Brewery Co. in 1995, is an α-galactosyl ceramide. It is a ligand making a complex with CD1d protein, and it stimulates invariant natural killer T (NKT) cells, which are one of the lineages of immunocytes. NKT cells activated by recognition of the CD1d/KRN7000 complex with its invariant T-cell receptor (TCR) can induce both protective and regulatory immune responses. To determine the recognition and activation mechanisms of NKT cells and to develop drug candidates more effective than KRN7000, a large number of analogs of KRN7000 have been synthesized. Some of them show potent bioactivities and have the potential of being utilized as therapeutic agents. In this review, structure-activity relationship studies of novel glycolipids which stimulate NKT cells efficiently are summarized.     

Synthesis, cardiostimulatory, and cardioinhibitory effects of selected insect peptides on Tenebrio molitor

The subject of these studies was a search for proctolin antagonists among peptides originating from insect species because the proctolin antagonists constantly pose a problem. During these studies we performed the synthesis of the following peptides: a native decapeptide from Manduca sexta Mas-MT-I and its 11 analogs with shortened sequences at the N-end as well as a growth suppressor, a pentapeptide isolated from Antheraea yamamai, Any-GS and its 10 analogs, modified at position 1 and with a shortened peptide chain.Biological effects were evaluated by the cardiotropic test on the semi-isolated heart of the insect species Tenebrio molitor. Mas-MT-I and six analogs stimulate the heartbeat frequency, especially [6-10]-Mas-MT-I, whereas the [4-10]-Mas-MT-I analog shows a strong inhibition of the heartbeat frequency, if insect. The Any-GS and the analogs [Gln(1)]- and [Gly(1)]-Any-GS also show a strong cardioinhibitory effect.     

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, Raman, CD spectroscopy and molecular modeling.

The synthesis and biological evaluation of penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs and comparison with their proline(7)-oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen(1),Pen(6)]-oxytocin (1, pA(2) = 8.22, EC(50) = 6.0 nM) and [dPen(1),Pen(6),5-tBuPro(7)]-oxytocin (2, pA(2) = 8.19, EC(50) = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using (1)H NMR, laser Raman and CD techniques. In H(2)O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln(4) and Pen(6) in the major trans-conformer of 1 as well as for Gln(4) in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 +/- 0.2 kcal/mol) for isomerization of the amide N-terminal to 5-tBuPro(7) relative to OT was calculated from measuring the coalescence temperature of the Gly(9) backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C(beta)-S-S-C(beta) dihedral angles of +110-115 degrees and +/-90 degrees , respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a chi(CS-SC) dihedral angle value close to 115 degrees was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a +/-90 degrees angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a +/-90 degrees chi(CS-SC) dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114 degrees and +116 degrees . Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104 degrees . In sum, the conformation about the disulfide in [dPen(1),Pen(6)]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen(1),Pen(6),5-tBuPro(7)]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro(7) (5R)-tert-butyl group, the Leu(8) isopropyl sidechain and the Pen(6)beta-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110 degrees may be a prerequisite for their binding at the receptor.     

Design,synthesis and evaluation of some 1,3,5-triazinyl urea and thiourea derivatives as antimicrobial agents

In an effort to establish new candidates with improved antimicrobial activities we report here the synthesis and in vitro biological evaluation of various series of compounds (5a-j) and (7a-j) which were evaluated against two Gram positive (S. aureus, B. subtilis), two Gram negative (S. typhosa, E. coli) strains and a yeast-like fungi (C. albicans) using the micro-dilution procedure. Among the synthesized compounds 2-(cyclohexyl amino)-4-(3,4-dimethoxy phenyl ethyl thioureido)-6-(2-chloro phenyl ureido) s-triazine (7e) and 2-(cyclohexyl amino)-4-(3,4-dimethoxy phenyl ethyl thioureido)-6-(4-chloro phenyl ureido) s-triazine (7g) proved to be effective with MIC (0.019 mg ML^?1) against S. typhosa & E. coli respectively.     

Synthesis and biological evaluation of several structural analogs of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand

2-Arachidonoylglycerol (2-AG (1)) is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). There is growing evidence that 2-arachidonoylglycerol plays important physiological and pathophysiological roles in various mammalian tissues and cells, though the details remain to be clarified. In this study, we synthesized several remarkable analogs of 2-arachidonoylglycerol, closely related in chemical structure to 2-arachidonoylglycerol: an analog containing an isomer of arachidonic acid with migrated olefins (2-AGA118 (3)), an analog containing a one-carbon shortened fatty acyl moiety (2-AGA113 (4)), an analog containing an one-carbon elongated fatty acyl moiety (2-AGA114 (5)), a hydroxy group-containing analog (2-AGA105 (6)), a ketone group-containing analog (2-AGA109 (7)), and a methylene-linked analog (2-AGA104 (8)). We evaluated their biological activities as cannabinoid receptor agonists using NG108-15 cells which express the CB1 receptor and HL-60 cells which express the CB2 receptor. Notably, these structural analogs of 2-arachidonoylglycerol exhibited only weak agonistic activities toward either the CB1 receptor or the CB2 receptor, which is in good contrast to 2-arachidonoylglycerol which acted as a full agonist at these cannabinoid receptors. These results clearly indicate that the structure of 2-arachidonoylglycerol is strictly recognized by the cannabinoid receptors (CB1 and CB2) and provide further evidence that the cannabinoid receptors are primarily the intrinsic receptors for 2-arachidonoylglycerol.     

Synthesis of all stereoisomers of KRN7000, the CD1d-binding NKT cell ligand

KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release Th1 and Th2 cytokines. In an effort to understand the structure–activity relationships, we have carried out the synthesis of a complete set of the eight KRN7000 stereoisomers, and their biological activities have been examined.     

beta-Sulfonamido gonadotropin-releasing hormone analogs: synthesis and evaluation of several parent hormone properties.

With the aim of producing long-acting analogs of gonadotropin releasing hormone (GnRH), four analogs, containing -X(6) (aa)psi(CH(2)SO(2)NH)-Leu(7) building unit (X(aa)=Gly, Ala, Val or Phe), and a reduced-size analog [Des-Tyr(5)]-GnRH which includes the unit Phe(5)psi(CH(2)SO(2)NH)-Leu(6), and [beta-Ala(6)]-GnRH were synthesized. The peptides were evaluated for their capacity to induce LH-release from rat pituitary cells and to withstand proteolysis by pituitary-derived enzymes, compared with the parent peptide GnRH. Albeit stable toward enzymatic degradation, the sulfonamido containing peptides were only marginally bioactive. [beta-Ala(6)]-GnRH, however, induced LH-release and bound to pituitary receptors nearly as efficiently as GnRH. This analog was also highly stable toward proteolysis suggesting that it may serve as a long-acting GnRH-analog.     

Novel side chain analogs of 1alpha,25-dihydroxyvitamin D(3): design and synthesis of the 21,24-methano derivatives

The syntheses of the new 21,24-methano derivatives of 1alpha,25-dihydroxyvitamin D(3) [viz. 1(S),3(R)-dihydroxy-17(R)-(1',4'-cis-(4'-(1'-hydroxy-1'-methylethyl)-cyclo-hexyl))-9,10-seco-androsta-5(Z),7(E),10(19)-triene (MC 2108) and its (1',4'-trans)-isomer (MC 2110)] are described. The key step is the establishment, by Diels-Alder reaction on a CD-ring side chain diene intermediate prepared from vitamin D(2), of a 1,4-disubstituted cyclohexene moiety in the side chain. Hydrogenation to a 1:1 mixture of cis and trans cyclohexane derivatives and separation of the two series at a stage prior to the standard Horner-Wittig coupling with the (Hoffmann-La Roche) ring-A building block were other important steps in the syntheses of the target analogs. The relative configurations of intermediates were assigned by NMR spectroscopy. MC 2108 and MC 2110 are of interest as conformationally locked side chain derivatives to probe the receptor interactions of not only the parent vitamin D hormone but also its biologically active symmetrical 'double side chain' analog [21-(3'-hydroxy-3'-methylbutyl)-9,10-seco-cholesta-5(Z),7(E),10(19)-triene-1(S),3(R),25-triol (MC 2100)], 'both' side chains of which can formally be traced out in the new analogs. The preferred conformations, inferred from an analysis of (13)C-NMR characteristics, notably the chemical shift of C-17 in a series of analogs, to have the tertiary alcohol (1'-hydroxy-1'-methylethyl) substituent equatorial on the cyclohexane chair, are confirmed by molecular modeling.     

The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine(6)-oxytocin and penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs.

Six [Pen(6)]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, [Mpa(1)]oxytocin, [dPen(1)]oxytocin, [5-t-BuPro(7)]oxytocin, [Mpa(1), 5-t-BuPro(7)]oxytocin and [dPen(1), 5-t-BuPro(7)]oxytocin. When tested in the uterotonic test in vitro [Pen(6)]oxytocin, [Pen(6), 5-t-BuPro(7)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. [dPen(1), Pen(6)]Oxytocin and [dPen(1), Pen(6), 5-t-BuPro(7)]oxytocin were found to be pure antagonists with similarly high pA2 values of approximately 8.2. Replacement of proline by 5-tert-butylproline increased binding affinity by a factor of two in [Pen(6)]oxytocin and had no influence on the binding affinity of [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. Assignment of the proton signals for prolyl amide cis- and trans-isomers by NMR experiments in water indicated that the Pen(6)-5-tert-BuPro(7) peptide bond cis-isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5-tert-butylproline(7) analogs of [Pen(6)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. This augmentation in cis-isomer population was correlated with a 21-fold reduction in the agonistic potency and 2-fold augmentation in antagonistic potency for [Pen(6), 5-t-BuPro(7)]oxytocin relative to [Pen(6)]oxytocin. Augmentation of cis-isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of [Mpa(1), Pen(6)]oxytocin to [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin. In the potent oxytocin antagonist, [dPen(1), Pen(6)]oxytocin, substitution of 5-tert-butylproline for proline augmented the cis-isomer population without affecting antagonistic potency. The synthesis and evaluation of [Pen(6)]oxytocin and [Pen(6), 5-t-BuPro(7)]oxytocin analogs 1-6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis-isomer population caused by 5-tert-butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.     

Total syntheses of (+/-)-ovalicin, C4(S *)-isomer, and its C5-analogs and anti-trypanosomal activities

Total syntheses of (±)-ovalicin, its C4(S^*)-isomer 44, and C5-side chain intermediate 46 were accomplished via an intramolecular Heck reaction of (Z)-3-(tert-butyldimethylsilyloxy)-1-iodo-1,6-heptadiene and a catalytic amount of palladium acetate. Subsequent epoxidation, dihydroxylation, methylation, and oxidation led to (3S^*,5R^*,6R^*)-5-methoxy-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-one (2), a reported intermediate. The addition of a side chain with cis-1-lithio-1,5-dimethyl-1,4-hexadiene (27) followed by oxidation afforded (±)-ovalicin. The functional group manipulation afforded a number of regio- and stereoisomers, which allow the synthesis of analogs for bioevaluation. The structure of 44 was firmly established via a single-crystal X-ray analysis. The stereochemistry at C4 generated from the addition reactions of alkenyllithium with ketones 2, 40, and 45 is dictated by C6-alkoxy functionality. Anti-trypanosomal activities of various ovalicin analogs and synthetic intermediates were evaluated, and C5-side chain analog, 46, shows the strongest activity. Compound 44 shows antiproliferative effect against HL-60 tumor cells in vitro. Compounds 46 and a precursor, (3S^*,4R^*,5R^*,6R^*)-5-methoxy-4-[(E)-(1′,5′-dimethylhexa-1′,4′-dienyl)]-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-ol (28), may be explored for the development of anti-parasitic drugs.     

Cellular differentiation, cytidine analogs and DNA methylation

The nucleoside analog 5-azacytidine (5-aza-CR) induced marked changes in the differentiated state of cultured mouse embryo cells and also inhibited the methylation of newly synthesized DNA. The DNA strand containing 5-aza-CR remained undermethylated in the round of DNA synthesis following analog incorporation. The extent of inhibition of DNA modification and induction of muscle cells in treated cultures were dependent on the 5-aza-CR concentration over a narrow dose range. Experiments with the restriction enzyme Hpa II, which is sensitive to cytosine methylation in the sequence CCGG, demonstrated that the DNA synthesized in 5-aza-CR-treated cultures was maximally undermethylated 48 hr after treatment. Three other analogs of cytidine, containing a modification in the 5 position of the pyrimidine ring [5-aza-2'-deoxycytidine(5-aza-CdR), pseudoisocytidine (psi ICR) and 5-fluoro-2'-deoxycytidine(FCdR)] also induced the formation of muscle cells and inhibited DNA methylation. In contrast, 1-beta-D-arabinofuranosylcytosine (araC) and 6-azacytidine (6-aza-CR) did not inhibit DNA methylation or induce muscle formation, whereas 5-6-dihydro-5-azacytidine (dH-aza-CR) was a poor inducer of muscle cells and a poor inhibitor of DNA methylation. These results provide experimental evidence for a role for DNA modification in differentiation, and suggest that cytidine analogs containing an altered 5 position perturb previously established methylation patterns to yield new cellular phenotypes.     

Highly potent nociceptin analog containing the Arg-Lys triple repeat

One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8-9 and 12-13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor's acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6-7, 10-11, or 14-15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [Arg-Lys(6-7)]- and [Arg-Lys(10-11)]nociceptins exhibited weak activities (6-9 and 60-90% of nociceptin, respectively) both in the receptor binding assay and in the [(35)S]GTPgammaS binding functional assay. In contrast, [Arg-Lys(14-15)]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTPgammaS functional assay). [Arg-Lys(14-15)]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure-activity studies have suggested that the incorporated Arg-Lys(14-15) interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues.     

Synthesis and biological activity profiles of atrial natriuretic factor (ANF) analogs

Several analogs of the atrial natriuretic factor (ANF) were synthesized by the solid-phase method using the acetamidomethyl (Acm) group for sulfhydryl protection. The compounds were tested in a receptor binding assay using bovine adrenal zona glomerulosa cell membranes and in the rat diuresis/natriuresis assay. Substitution of tyrosine in position 116 of ANF(101-126) and of the analog [3-Mpr105]ANF(105-126)(3-Mpr = 3-mercaptopropionic acid) did not alter the biological activity profiles and, therefore, these two analogs in radioiodinated form will be useful for enzymatic degradation and clearance studies. Replacement of 3-mercaptopropionic acid with 2-mercaptopropionic acid in [3-Mpr105]ANF(105-126) resulted in an analog with very low potency in both assay systems, presumably as a consequence of the steric bulk and/or local conformational restriction produced by the methyl group attached to the alpha-carbon in position 105. The analog [3-Mpr105,Nva109]ANF(105-126)(Nva = norvaline) showed very low affinity in the receptor binding assay but displayed considerable diuretic/natriuretic activity. The obtained biological activity profiles suggest that in comparison with other ANF peptides the des-amino ANF(105-126) analogs may have a somewhat longer half-life in vivo, or alternatively, may indicate a more complex situation of ANF receptor or binding site heterogeneity.     

Alpha-adrenergic receptor-mediated restraint of skeletal muscle blood flow during prolonged exercise.

Sympathetic nervous system restraint of skeletal muscle blood flow during dynamic exercise has been well documented. However, whether sympathetic restraint of muscle blood flow persists and is constant throughout prolonged exercise has not been established. We hypothesized that both alpha1- and alpha2-adrenergic receptors would restrain skeletal muscle blood flow throughout prolonged constant-load exercise and that the restraint would increase as a function of exercise duration. Mongrel dogs were instrumented chronically with transit-time flow probes on the external iliac arteries and an indwelling catheter in a branch of the femoral artery. Flow-adjusted doses of selective alpha1- (prazosin) and alpha2-adrenergic receptor (rauwolscine) antagonists were infused after 5, 30, and 50 min of treadmill exercise at 3 and 6 miles/h. During mild-intensity exercise (3 miles/h), prazosin infusion resulted in a greater (P < 0.05) increase in vascular conductance (VC) after 5 [42% (SD 6)], compared with 30 [28% (SD 6)] and 50 [28% (SD 8)] min of running. In contrast, prazosin resulted in a similar increase in VC after 5 [29% (SD 10)], 30 [24% (SD 9)], and 50 [22% (SD 9)] min of moderate-intensity (6 miles/h) exercise. Rauwolscine infusion resulted in a greater (P < 0.05) increase in VC after 5 [39% (SD 14)] compared with 30 [26% (SD 9)] and 50 [22% (SD 4)] min of exercise at 3 miles/h. Rauwolscine infusion produced a similar increase in VC after 5 [19% (SD 3)], 30 [15% (SD 6)], and 50 [16% (SD 2)] min of exercise at 6 miles/h. These results suggest that the ability of alpha1- and alpha2-adrenergic receptors to produce vasoconstriction and restrain blood flow to active muscles may be influenced by both the intensity and duration of exercise.