New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models.

During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolyloxazoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral dosing. This compound, and others of its structural class, may prove to be useful in the development of new chemotherapeutic agents to treat human cancers.

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 5. Highly selective, potent, and orally active ET(A) antagonists.

The synthesis and structure-activity relationships (SAR) of a series of pyrrolidine-3-carboxylic acids as endothelin antagonists are described. The data shows an increase in selectivity when the methoxy of Atrasentan (ABT-627) is replaced with methyl, and the benzodioxole is replaced with dihydrobenzofuran. Adding a fluorine further increases the binding activity and provides a metabolically stable and orally bioavailable ET(A)-selective antagonist.

Identification and characterization of A-105972, an antineoplastic agent.

A high-throughput screening assay was designed to select compounds that inhibit the growth of cultured mammalian cells. After screening more than 60,000 compounds, A-105972 was identified and selected for further testing. A-105972 is a small molecule that inhibits the growth of breast, central nervous system, colon, liver, lung, and prostate cancer cell lines, including multidrug-resistant cells. The cytotoxic IC50 values of A-105972 were between 20 and 200 nM, depending on the specific cell type. The potency of A-105972 is similar in cells expressing wild-type or mutant p53. A majority of cells treated with A-105972 were trapped in the G2-M phases, suggesting that A-105972 inhibits the progression of the cell cycle. Using [3H]A-105972, we found that A-105972 bound to purified tubulin. Unlabeled A-105972 competed with [3H]A-105972 binding with an IC50 value of 3.6 microL. Colchicine partially inhibited [3H]A-105972 binding with an IC50 value of approximately 90 microM, whereas paclitaxel and vinblastine had no significant effect. Tumor cells treated with A-105972 were observed to contain abnormal microtubule arrangement and apoptotic bodies. DNA ladder studies also indicated that A-105972 induced apoptosis. A-105972 caused a mobility shift of bcl-2 on SDS-PAGE, suggesting that A-105972 induced bcl-2 phosphorylation. A-105972 treatment increased the life span of mice inoculated with B16 melanoma, P388 leukemia, and Adriamycin-resistant P388. These results suggest that A-105972 is a small molecule that interacts with microtubules, arrests cells in G2-M phases, and induces apoptosis in both multidrug resistance-negative and multidrug resistance-positive cancer cells. A-105972 and its analogues may be useful for treating cell proliferative disorders such as cancer.

'Irreversible' endothelin-1 binding does not prohibit ABT-627 from reversing endothelin-1-induced effects.

Endothelin-1 (ET-1) is thought to play a role in a wide range of pathological conditions. One of the distinct characteristics of ET-1 is its long-lasting vasoconstrictor action, which is presumably caused by the irreversible binding of ET-1 to ET receptors and by the functional effects of internalized ET receptors. ABT-627 is a potent endothelin-A (ET(A))-selective antagonist with a Ki value at 0.034 nM for the human ET(A) receptor, and is currently being used in clinical studies for prostate cancer. Unlike ET-1, the binding of 125I-labeled ABT-627 to human ET(A) receptors expressed in Chinese hamster ovary (CHO) cells is reversible, and the dissociation half-life for the ligand/receptor complex is 2 h. Interestingly, the binding of both ET-1 and ABT-627 to the ET(A)-receptor results in partial receptor internalization but only ET-1 is capable of triggering intracellular functional responses. Although ABT-627 binding to membranes is more reversible than ET-1 binding, ABT-627 is able to reverse an ET-1-induced contraction in rat aortic rings in a dose-dependent manner, and at 1 microM produces nearly complete reversal of the constrictor effects of 10 nM ET-1 within 60 min. Similarly, in vivo studies show that ABT-627 (0.01 and 0.1 mg/kg/min i.v.) reverses the ET-1-induced increase in arterial pressure in anesthetized, ganglionic-blocked rats, and after 60 min, ABT-627 essentially normalizes pressure. Our data show that ABT-627 is capable of reversing an established response induced by ET-1 and is useful in reversing pathological conditions involving ET-1.

Internalization of type-A endothelin receptor.

Endothelins (ETs) are 21 amino acid peptides which bind to ET(A)- and ET(B)-receptors to evoke diverse physiological responses. This report studies the internalization of ET(A)-receptor in Chinese hamster ovary (CHO) cells which were stably transfected with ET(A)-receptor cDNA. ET-1 binding induced ET(A) internalization in a time-dependent manner with 40% of ET(A)-receptors internalized at 37 degrees C after 30 min. To localize internalized ET(A)-receptor, cells were immunostained using a polyclonal antibody against the extracellular loop between IV and V transmembrane segments of the ET(A)-receptor. To examine the fate of internalized ET-1, cells were treated with 10 nM biotinylated ET-1 coupled with Texas Red-labeled streptavidin. In the absence of ET-1, a majority of ET(A) was localized on the surface of cells. After ET-1 treatment for 60 min, internalized ET(A)-receptors were localized in a perinuclear structure. ET-1 remained bound to ET(A)-receptor after internalization for up to 60 min and then dissociated from the receptor. After dissociation, ET-1 possibly became degraded and ET(A) recycled back to the cell surface. Protein kinase inhibitors such as KT5926 and staurosporine partially inhibited ET(A)-receptor internalization. The results of this study may facilitate the understanding of pathways involved in ET-1-induced receptor internalization.

Extracellular signal-regulated kinases are involved in the antiapoptotic effect of endothelin-1.

An imbalance between proliferation and apoptosis is an important causal factor for disorders involving abnormal cell accumulation. The role and mechanism of how G protein-coupled receptors are linked to apoptosis are poorly understood. Endothelin-1 (ET-1), a 21-amino acid peptide that binds to G protein-coupled receptors with mitogenic and vasoconstricting activities, suppressed apoptosis of human prostatic smooth muscle cells induced by paclitaxel treatment or serum withdrawal. Serum withdrawal or paclitaxel (1-10 microM) treatment for 48 h resulted in DNA fragmentation, a characteristic of apoptosis. The addition of ET-1 attenuated DNA fragmentation. The attenuating effect of ET-1 on DNA fragmentation was not affected by wortmannin, bisindolylmaleimide I, tyrphostin AG490, or AG1478. However, PD98059, an inhibitor for the extracellular signal-regulated kinase (ERK) kinase, induced apoptosis, potentiated the effect of serum withdrawal on inducing apoptosis, and blocked the antiapoptotic effect of ET-1. The ERK1/2 activity in these cells decreased rapidly after paclitaxel treatment or serum withdrawal, but was maintained at a 2-fold higher level in the presence of ET-1 for at least 4 h. These results suggest that the ERK1/2 pathway is activated by ET-1, and blocking this pathway abolishes the antiapoptotic effect of ET-1.

Direct determination of endothelin receptor antagonist levels in plasma using a scintillation proximity assay.

An assay using scintillation proximity bead technology has been developed suitable for the quantitation of endothelin (ET) receptor antagonists in preclinical and clinical samples of plasma. The assay measures the competitive inhibition of radiolabelled ET-1 binding to ET(A) receptor membranes bound to wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads in the presence of plasma containing A-127722, a potent orally active, ET(A) selective ET antagonist. The assay requires as little as 50 microl plasma and no extraction procedure is needed. The SPA methodology eliminates the need for the separation of bound from free ligand. Using this method, A-127722 could be directly quantified in rat plasma with a detection limit of 1 ng/ml.

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 4. Side chain conformational restriction leads to ET(B) selectivity.

When the dialkylacetamide side chain of the ET(A)-selective antagonist ABT-627 is replaced with a 2,6-dialkylacetanilide, the resultant analogues show a complete reversal of receptor selectivity, preferring ET(B) over ET(A). By optimizing the aniline substitution pattern, as well as the alkoxy group on the 2-aryl substituent, it is possible to prepare antagonists with subnanomolar affinity for ET(B) and with selectivities in excess of 4000-fold. A number of these compounds also show promising pharmacokinetic profiles; a useful balance of properties is found in A-192621 (38). Pharmacology studies with A-192621 serve to reveal the role of the ET(B) receptor in modulating blood pressure; the observed hypertensive response to persistent ET(B) blockade is consistent with previous postulates and indicates that ET(B)-selective antagonists may not be suitable as agents for long-term systemic therapy.

Design, synthesis, and activity of a series of pyrrolidine-3-carboxylic acid-based, highly specific, orally active ET(B) antagonists containing a diphenylmethylamine acetamide side chain.

The endothelin (ET)-B receptor subtype is expressed on vascular endothelial and smooth muscle cells and mediates both vasodilation and vasoconstriction. On the basis of the pharmacophore of the previously reported ET(A)-specific antagonist 1, (ABT-627), we are reporting the discovery of a novel series of highly specific, orally active ET(B) receptor antagonists. Replacing the dibutylaminoacetamide group of 1 with a diphenylmethylaminoacetamide group resulted in antagonist 2 with a complete reversal of receptor specificity. Structure-activity relationship studies revealed that ortho-alkylation of the phenyl rings could further increase ET(B) affinity and also boost the ET(A)/ET(B) activity ratio of the resulting antagonists. A similar antagonism selectivity profile could also be achieved when one of the phenyl rings of the acetamide side chain was replaced with an alkyl group, preferably a tert-butyl group (10h). Combining these features with modification of the 2-aryl group of the pyrrolidine core, we have identified a potent antagonist (9k, A-308165) with over 27 000-fold selectivity favoring the ET(B) receptor and an acceptable pharmacokinetic profile (F = 24%) in rats.

Discovery of a series of pyrrolidine-based endothelin receptor antagonists with enhanced ET(A) receptor selectivity.

Endothelins, ET-1, ET-2, and ET-3 are potent vasoconstricting and mitogenic 21-amino acid bicyclic peptides, which exert their effects upon binding to the ET(A) and ET(B) receptors. The ET(A) receptor mediates vasoconstriction and smooth muscle cell proliferation, and the ET(B) receptor mediates different effects in different tissues, including nitric oxide release from endothelial cells, and vasoconstriction in certain vascular cell types. Selective antagonists of endothelin receptor subtypes may prove useful in determining the role of endothelin in various tissue types and disease states, and hence as therapeutic agents for such diseases. The pyrrolidine carboxylic acid A-127722 has been disclosed as a potent and ET(A)-selective antagonist, and is currently undergoing clinical trials. In our efforts to find antagonists with altered selectivity (ET(A)-selective, ET(B)-selective, or nonselective), we investigated the SAR of the 2-substituent on the pyrrolidine. Compounds with alkyl groups at the 2-position possessed ET(A) selectivity improved over A-127722 (1400-fold selective), with the best of these compounds showing nearly 19,000-fold selectivity.

SV40 virus transformation down-regulates endothelin receptor.

Simian virus 40 (SV40) is an oncogenic DNA virus that induces malignant transformation. Endothelin (ET), a 21 amino acid peptide with mitogenic and anti-apoptotic effects, binds to G-protein coupled ETA and ETB receptors. This report examines the effect of SV40 transformation on the expression of ET receptors. Results from receptor binding and reverse transcription (RT)-polymerase chain reaction (PCR) studies show that human lung fibroblasts IMR90 and WI38 express both ETA and ETB receptors, and that the expression of both receptors is significantly down-regulated in IMR90-SV40 and WI38-SV40, cell lines derived from IMR90 and WI38 with SV40 virus transformation. Receptor binding and RT-PCR analysis of 3A(tPA-30-1), a cell line derived from human placenta that expresses a higher level of SV40 large T-antigen at the permissive temperature (33 degrees C) than at the restrictive temperature (40 degrees C), further demonstrates that there is an inverse correlation between the expression of SV40 T-antigen and the expression of ET receptor. ET-1 and fetal bovine serum stimulate DNA synthesis in non-transformed cells; however, proliferation of transformed cells is independent of either fetal bovine serum or ET-1. We conclude that SV40 transformation down-regulates the expression of ET receptors, and that expression of ET receptors is inversely correlated with expression of SV40 large T-antigen.

Endothelin stimulates glucose uptake and GLUT4 translocation via activation of endothelin ETA receptor in 3T3-L1 adipocytes.

Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed approximately 560,000 sites/cell of ETA receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ETA receptor. ET-1 treatment did not induce phosphorylation of insulin receptor beta-subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 microM), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ETA receptor.

Pharmacology of A-216546: a highly selective antagonist for endothelin ET(A) receptor.

Endothelins, 21-amino acid peptides involved in the pathogenesis of various diseases, bind to endothelin ET(A) and ET(B) receptors to initiate their effects. Here, we characterize the pharmacology of A-216546 ([2S-(2,2-dimethylpentyl)-4S-(7-methoxy-1,3-benzodioxol-5-yl )-1-(N,N-di(n-butyl) aminocarbonylmethyl)-pyrrolidine-3R-carboxylic acid), a potent antagonist with > 25,000-fold selectivity for the endothelin ET(A) receptor. A-216546 inhibited [125I]endothelin-1 binding to cloned human endothelin ET(A) and ET(B) receptors competitively with Ki of 0.46 and 13,000 nM, and blocked endothelin-1-induced arachidonic acid release and phosphatidylinositol hydrolysis with IC50 of 0.59 and 3 nM, respectively. In isolated vessels, A-216546 inhibited endothelin ET(A) receptor-mediated endothelin-1-induced vasoconstriction, and endothelin ET(B) receptor-mediated sarafotoxin 6c-induced vasoconstriction with pA2 of 8.29 and 4.57, respectively. A-216546 was orally available in rat, dog and monkey. In vivo, A-216546 dose-dependently blocked endothelin-1-induced pressor response in conscious rats. Maximal inhibition remained constant for at least 8 h after dosing. In conclusion, A-216546 is a potent, highly endothelin ET(A) receptor-selective and orally available antagonist, and will be useful for treating endothelin-1-mediated diseases.

The effects of diminishing albumin binding to some Endothelin receptor antagonists.

As a pharmacological class, Endothelin-A receptor (ET(A)) antagonists are highly bound (>98%) to serum albumin. In the presence of physiological concentrations of albumin, their affinities for ET(A) decrease 10 to 100 fold. We have prepared ET(A) antagonists which exhibit lower degrees of binding to albumin, while maintaining potency and selectivity for the ET(A) receptor. The protein induced IC50 shift is reduced or eliminated in this new series of compounds. The compounds also display altered in vivo and pharmacokinetic profiles which may be consistent with their lower degree of protein binding.

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 3. Discovery of a potent, 2-nonaryl, highly selective ETA antagonist (A-216546).

Previously we have reported the discovery of ABT-627 (1, A-147627, active enantiomer of A-127722), a 2,4-diaryl substituted pyrrolidine-3-carboxylic acid based endothelin receptor-A antagonist. This compound binds to the ETA receptor with an affinity (Ki) of 0. 034 nM and with a 2000-fold selectivity for the ETA receptor versus the ETB receptor. We have expanded our structure-activity studies in this series, in an attempt to further increase the ETA selectivity. When the p-anisyl group of 1 was replaced by an n-pentyl group, the resultant antagonist 3 exhibited substantially increased ETB/ETA activity ratio, but a decreased ETA affinity. Structure-activity studies revealed that substitution and geometry of this alkyl group, and substitution on the benzodioxolyl ring, are important in optimizing this series of highly ETA selective antagonists. In particular, the combination of a (E)-2,2-dimethyl-3-pentenyl group and a 7-methoxy-1,3-benzodioxol-5-yl group provided hydrophobic compound 10b with subnanomolar affinity for human ETA receptor subtype and with an ETB/ETA activity ratio of over 130000. Meanwhile, synthetic efforts en route to olefinic compounds led to the discovery that 2-pyridylethyl (9o) and 2-(2-oxopyrrolidinyl)ethyl (9u) replacement of the p-anisyl group of 1yielded very hydrophilic ETA antagonists with potency and selectivity equal to those of 10b. On the basis of overall superior affinity, high selectivity for the ETA receptor (Ki, 0.46 nM for ETA and 13000 nM for ETB), and good oral bioavailability (48% in rats), A-216546 (10a) was selected as a potential clinical backup for 1.

Endothelin receptor antagonists: effect of serum albumin on potency and comparison of pharmacological characteristics.

Endothelins (ETs) are 21-amino acid peptides that bind to membrane receptors to initiate pathophysiological effects. Two types of ET receptors, ETA and ETB, have been identified. Various ET receptor antagonists are being developed as therapeutic agents. This report examines the effects of bovine serum albumin (BSA) on the potency of ET receptor antagonists and compares five ET receptor antagonists. Competition studies show that in the absence of BSA, A-127722 and L-749329 inhibited ET-1 binding to ETA receptor with the same IC50 value of 0.09 nM. Addition of increasing concentrations of BSA incrementally decreased the potency of the antagonists: in the presence of 5% BSA, the IC50 values increased to 4.3 and 820 nM, respectively. Similarly, addition of BSA decreased the potency of antagonists in inhibiting ET-1-stimulated phosphatidylinositol hydrolysis. These results suggest that serum albumin has profound effects on the potencies of ET receptor antagonists. FR139317, PD-156707, L-749329, Ro-47-0203 and A-127722 were then selected for direct comparison under identical experimental conditions with 0.2% BSA. The potency of antagonists was assessed by binding studies for the determination of IC50 and Ki values and by ET-1-stimulated phosphatidylinositol hydrolysis and arachidonic acid release for the determination of IC50 and pA2 values. All five antagonists inhibited ET binding and the biological effects exerted by ET in a competitive mode. The Ki values for A-127722, PD-156707, FR139317, Ro-47-0203 and L-749329 for the ETA receptor were 0.07, 0.38, 0.80, 3.67 and 33.6 nM, respectively. A similar hierarchy was revealed by the functional assays. Our results suggest that the rank order of potency of the antagonists is A-127722 > or = PD-156707 > or = FR139317 > Ro-47-0203 > L-749329.

Potent and selective non-benzodioxole-containing endothelin-A receptor antagonists.

The benzodioxole ((methylenedioxy)benzene) group is present in a number of endothelin (ET) receptor antagonists thus far reported. As part of our own endothelin antagonist program we have developed (2R*,3R*,4S*)-1-(N,N-dibutylacetamido)-4-(1,3-benzodioxol-5- yl)-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (A-127722). This is a potent antagonist, binding to the ETA and ETB receptor subtypes with affinities (IC50) of 0.4 and 520 nM, respectively, and also contains the aforementioned benzodioxole. While this compound was seemingly optimized at its N-terminus, no effort had been directed toward understanding the contributions to binding affinity or receptor subtype selectivity conferred by the benzodioxole. Substitution by 1- or 2-naphthyl yielded weak antagonists. Oxygenated benzenes, such as p-anisyl, were potent compounds with IC50s in the low-nanomolar range. Simple deletion of either of the two oxygen atoms (dihydrobenzofurans) yielded extremely potent agents, possessing subnanomolar affinity for the ETA receptor. Additionally, the compounds showed enhanced selectivity, binding to the ETB receptor subtype in the micromolar range. This paper describes the development of this novel class of compounds.

Dissociation characteristics of endothelin receptor agonists and antagonists in cloned human type-B endothelin receptor.

The human type-B endothelin receptor (h-ETB) was cloned from human lung poly A+RNA and stably expressed in CHO cells. Endothelin (ET) receptor binding and stimulation of PI hydrolysis demonstrated that the cloned h-ETB receptor is functional and linked to intracellular signal transduction pathways in CHO cells. The molecular mass of the h-ETB receptor was determined to be 65 KDa, and Bmax and Kd were 0.36 pmol/mg and 80 pM, respectively. Competition studies employing receptor ligands revealed that the potencies of the test ligands (IRL1620, PD142893, and Ro46-2005) were dependent on the length of the incubation time, whereas the natural agonists (ET-1 and ET-3) were not. When competing with ET-1 in the h-ETB receptor binding, the IC50 increased from 1.2 nM to 8.2 nM for IRL1620, 0.068 microM to 1.9 microM for PD142893, and 0.76 microM to 12.7 microM for Ro46-2005, as the incubation time increased from 1 hr to 24 hr. These time-induced changes are likely due to differences in the dissociation characteristics between the artificial ligands and the natural ligands.

Endothelin receptor in benign prostatic hyperplastic cells. Binding and functional studies.

Endothelins (ETs) are 21-amino acid peptides that bind to membrane receptors to initiate pathophysiological effects. This report characterizes ET receptors in benign prostatic hyperplasia-1 (BPH-1) cells, a prostate cell line isolated from a specimen of a 60-yr-old man with benign prostatic hyperplasia. [(125)I]ET-1 or -3 binding was of high affinity, with B(max) and K(d) values of 48 fmol/1 x 10(6) cells and 0.16 nM for ET-1, and 2.9 fmol/1 x 10(6) cells and 0.033 nM for ET-3, respectively. ET-1, ET-3, FR139317, Ro 46-2005, and IRL1620 inhibited [(125)I]ET-1 binding to these cells with IC50 values of 0.22, 186, 0.20, 52.8, and 772.3 nM, respectively. Reverse transcription-polymerase chain reaction confirmed that BPH-1 cells expressed more ET(A) than ET(B) receptors. ET-1 did not have any effect on arachidonic acid release, but caused a modest stimulation of phosphatidylinositol hydrolysis, and induced a prominent, sustained elevation in intracellular Ca2+ concentrations. The functional effects of ET-1 were completely inhibited by the ET(A)-selective antagonists FR139317 and A-127722, suggesting that the effects were mediated by the ET(A) receptor. These results suggest that ET may play functional roles in benign prostatic hyperplasia.

Endothelin attenuates apoptosis in human smooth muscle cells.

An imbalance between proliferation and apoptosis is an important causal factor for disorders involving abnormal cell accumulation. Endothelin (ET)-1, a 21-amino-acid peptide with mitogenic and vasoconstricting activities, not only acts as a mitogen, but also attenuates paclitaxel-induced apoptosis in smooth muscle cells. In both human pericardial and prostatic smooth muscle cells, addition of ET-1 reduced paclitaxel-induced DNA fragmentation and phosphatidylserine on the cell surface, two characteristics of apoptosis. By comparison, angiotensin II, another vasoactive peptide, did not have a significant effect on apoptosis. The effect of ET-1 was dose-dependent with an EC50 of 1 nM. These results suggest that ET is a potential survival factor for smooth muscle cells, and that altered activity of the ET system in disease states has potential to contribute to aberrant cell growth.