ETA (Ki = 0.679 nM); ETB (Ki = 569 nM)

In a concentration-dependent manner, nebentan potassium blocks the specific binding of [125I] endothelin-1 to endothelin ETA and ETB receptors; the Ki values for human and rat endothelin ETA receptors are 0.697 nM and 1.53 nM, respectively. By comparison, YM598 has low affinities, with Ki values of 569 nM and 155 nM for rat and human endothelin ETB receptors, respectively[1]. Nebentan potassium concentration-dependently suppresses the increase in [Ca2+]i induced by 10 nM endothelin-1 in both CHO and A10 cells when measuring intracellular Ca2+ concentration; the IC50 values for CHO cells are 26.2 nM and 26.7 nM, respectively[1].

---

Binding assays [1]
The potency of Nebentan (YM598) in inhibiting [125I]endothelin-1 binding to endothelin ETA and ETB receptors was examined using membranes from CHO cells expressing human endothelin ETA and ETB receptors, and rat cells and tissues. YM598 inhibited the specific binding of [125I]endothelin-1 to endothelin ETA and ETB receptors in a concentration-dependent manner (Fig. 2A and B). Ki values of YM598 were 0.697±0.132 nM (n=6) and 1.53±0.16 nM (n=4) for human and rat endothelin ETA receptors, respectively. In contrast, YM598 exhibited low affinities for human and rat endothelin ETB receptors, with Ki values of 569±90 nM (n=6) and 155±11 nM (n=4), respectively. Binding experiments were also performed for bosentan (Fig. 2A and B). Ki values of bosentan were 2.28±0.26 nM (n=6) and 7.99±1.68 nM (n=4) for human and rat endothelin ETA receptors, and those for human and rat endothelin ETB receptors were 174±19 nM (n=6) and 34.9±3.0 nM (n=4), respectively.
To further evaluate the properties of the interaction between Nebentan (YM598) and the endothelin ETA receptor, [125I]endothelin-1 saturation binding studies were performed using membranes from CHO cells expressing human endothelin ETA receptor. It was evident that increasing the concentration of YM598 caused successive decreases in the slope of the lines (Fig. 2C), indicating a kind of change in the Kd value of [125I]endothelin-1 binding. YM598 did not have a significant effect on the Bmax (maximal [125I]endothelin-1 binding).

---

Receptor specificity [1]
The specificity of Nebentan (YM598) was determined in 59 radioligand binding assays. Table 1 shows that Nebentan (YM598) had no significant inhibiting activity at 10 μM, except at the diltiazem binding site. However, in a functional assay (KCl-induced contraction in rabbit aorta), YM598 (up to 10 μM) did not inhibit L-type Ca2+-channel-mediated (KCl-induced) contraction, whereas diltiazem did (data not shown).

---

In vitro functional inhibitory potency: measurement of intracellular Ca2+ concentration [1]
Addition of endothelin-1 to Fura 2-loaded CHO cells expressing human endothelin ETA receptor and rat A10 cells resulted in a concentration-dependent increase in [Ca2+]i, whereas sarafotoxin S6c did not exert any effect at concentrations up to 100 nM. EC50 values of endothelin-1 were 10.5±4.3 nM (n=6) and 6.1±3.8 nM (n=6) in CHO cells expressing human endothelin ETA receptor and rat A10 cells, respectively. The maximal effects were attained at 100 nM endothelin-1 (CHO cells: 386±103 nM, rat A10 cells: 207±32 nM, n=6 in each cell) and their quantities were considered to be almost the same as those induced by other agonists (Roullet et al., 1997). Nebentan (YM598) concentration-dependently inhibited the increase in [Ca2+]i induced by 10 nM endothelin-1 in both CHO cells and A10 cells, with IC50 values of 26.2±3.6 nM for CHO cells (n=6) and 26.7±8.2 nM for A10 cells (n=6), respectively (Fig. 3). Bosentan also inhibited the [Ca2+]i increases in both cells with IC50 values of 53.5±9.2 nM for CHO cells (n=6) and 39.4±5.5 nM for A10 cells (n=6), respectively (Fig. 3). YM598 and bosentan did not show any agonistic or antagonistic effects on the basal [Ca2+]i.

---

In vitro functional inhibitory potency: aortic ring contraction [1]
Endothelin-1 induced contraction of rings prepared from rat thoracic aorta in a concentration-dependent manner (Fig. 4). The contractile response induced by endothelin-1 in this tissue is mediated by the endothelin ETA receptor (Panel et al., 1992). Nebentan (YM598) (10–1000 nM) antagonized this endothelin-1-induced vasoconstriction without reducing the maximum response (Fig. 4), but had no direct effect on basal tone even at 1000 nM. The pA2 value of YM598 analyzed by the Schild plot was 7.6 (95% confidence interval: 6.5–7.8), with a slope of 0.84 (n=6–8).

When taken orally for four weeks at a dose of 0.1–1 mg/kg, nebentan potassium dramatically slows the advancement of pulmonary hypertension and right ventricular hypertrophy[2]. Nebentan potassium (oral; 1 mg/kg; 30 weeks) considerably lowers both pulmonary congestion and both ventricle hypertrophy, which in turn improves the poor survival rate of CHF rats[2].

---

In vivo functional inhibitory potency: effects on big endothelin-1- or sarafotoxin S6c-induced changes in blood pressure in conscious normotensive rats [1]
Effects of Nebentan (YM598) and bosentan on big endothelin-1-induced pressor response were investigated in conscious normotensive rats. Intravenous administration of big endothelin-1 (0.5 nmol/kg) elicited an increase in mean blood pressure (113.1±3.3 to 169.5±3.7 mm Hg) that reached a peak 10 to 15 min after injection and returned to the baseline within 1 h in conscious rats. Mean blood pressure was not altered 30 min after the oral administration of YM598 (0.1, 0.3, 1 mg/kg). Oral administration of YM598 (0.1, 0.3, 1 mg/kg) dose-dependently inhibited this big endothelin-1-induced pressor response, with maximum inhibitory effect observed 30 min after oral administration at every dose (Fig. 6A). At a dose of 1 mg/kg, p.o., YM598 produced approximately 80% inhibition of the big endothelin-1-induced pressor response, and approximately 60% inhibition was sustained for at least 6.5 h. Moreover, there was approximately 25% inhibition even at 24 h after oral administration at a dose of 1 mg/kg (vehicle: 89.7±6.8%, YM598: 65.0±9.7%, Fig. 6A). Oral administration of bosentan (3, 10, 30 mg/kg) also dose-dependently inhibited the pressor response induced by big endothelin-1 (0.5 nmol/kg), with maximum inhibitory effect observed 30 min after oral dosing (Fig. 6B). However, at least a 30-fold higher oral dose (30 mg/kg) of bosentan, compared to YM598, was required to produce a similar extent and duration of inhibition of the pressor response to big endothelin-1 (Fig. 6B).

---

In vivo functional inhibitory potency: inhibition of pressor response to big endothelin-1 in pithed rats [1]
The i.v. administration of big endothelin-1 (0.1–3.2 nmol/kg), precursor peptide of endothelin-1, induced dose-dependent pressor responses and elicited a maximum increase in diastolic blood pressure of about 100 mm Hg in pithed male Wistar rats (Fig. 5). Nebentan (YM598) (0.1, 0.3, 1 mg/kg) dose-dependently inhibited this big endothelin-1-induced pressor response and produced a parallel rightward shift of it following both i.v. and p.o. administration, with DR2 values of 0.53 mg/kg, i.v. and 0.77 mg/kg, p.o., respectively (Fig. 5). The calculated i.v./p.o. ratio from these DR2 values was 0.69. On the other hand, DR2 values of bosentan were 5.1 mg/kg, i.v. and 25.2 mg/kg, p.o., and the calculated i.v./p.o. ratio of bosentan was 0.20. [1]
Effects of Nebentan (YM598) and K-8794, a selective ETB receptor antagonist (Sonoki et al., 1997), on depressor and pressor responses induced by the selective ETB receptor agonist sarafotoxin S6c were also investigated in conscious normotensive rats. Intravenous administration of sarafotoxin S6c (0.3 nmol/kg) produced a transient depressor response (−23.9±1.7 mm Hg) followed by a sustained pressor response (30.6±2.1 mm Hg). Oral administration of YM598 (1, 3, 10 mg/kg, p.o.) did not significantly inhibit the depressor and pressor responses induced by sarafotoxin S6c at all doses (two-way ANOVA repeated measures) (Fig. 7A,B). On the other hand, oral administration of K-8794 (0.3 to 30 mg/kg) dose-dependently inhibited the initial depressor and pressor responses induced by sarafotoxin S6c (Fig. 7C,D). Bosentan at 30 mg/kg p.o. also inhibited the depressor response to sarafotoxin S6c but not the pressor response to sarafotoxin S6c (data not shown).

---

The effects of the novel, selective endothelin-A (ETA) receptor antagonist Nebentan (YM598) on both-side heart failure were investigated. Right-side heart failure secondary to pulmonary hypertension was produced by a single subcutaneous injection of 60 mg/kg monocrotaline, and post-ischemic congestive left-side heart failure (CHF) produced by surgical left coronary artery ligation. In right-side heart failure rats, oral Nebentan (YM598) (0.1 and 1 mg/kg for 4 weeks), but not bosentan (30 mg/kg), significantly inhibited the progression of pulmonary hypertension and the development of right ventricular hypertrophy. YM598 also improved hypoxemia and morphological pulmonary lesions in these rats. In CHF rats, moreover, long-term oral administration of Nebentan (YM598) (1 mg/kg/day for approximately 30 weeks) significantly ameliorated their poor survival rate (P < 0.05). In the measurement of cardio-hemodynamic parameters, YM598 improved the contractile/diastolic capacity of the left ventricle and the preload in the right ventricle to the levels seen in sham-operated rats. YM598 also markedly inhibited both ventricular hypertrophy and pulmonary congestion, as well as lowering high plasma brain natriuretic peptide levels in CHF rats. These findings suggest that YM598 may have a clinical benefit with regards to ameliorating the cardiopulmonary changes of right-side heart failure, and the cardiac dysfunction and mortality/morbidity of CHF [2].

Binding assay [1]
Endothelin receptor binding assays were performed according to the method of Webb et al. (1993) as described previously with modifications. Briefly, competition studies were performed in a total volume of 250 μl containing 25 μl [125I]endothelin-1 (200 pM for recombinant human endothelin ETA and ETB receptors, 500 pM for rat A10 and cerebellum), 25 μl competing compounds or 100 nM endothelin-1 to define nonspecific binding, and incubation buffer (50 mM Tris–HCl, pH 7.4, 10 mM MgCl2 and 0.01% bovine serum albumin). Reaction was initiated by the addition of 200 μl of plasma membrane suspension reconstructed by incubation buffer, which contained 0.2 μg (recombinant human endothelin ETA and ETB receptor), 24 μg (rat A10), or 2.4 μg (rat cerebellum) of membrane protein. After the incubation period (3 h for recombinant human endothelin ETA and ETB receptor, 2 h for rat A10 and cerebellum, room temperature), the reaction was terminated by the addition of 3 ml of ice-cold incubation buffer followed by rapid filtration through Whatman GF/C filters. The filters were rinsed twice and the radioactivity retained on the filters was counted using a gamma counter at 60% efficiency. Each assay was performed 4–6 times in duplicate. For saturation binding studies, each plasma membrane preparation was incubated with various concentrations of [125I]endothelin-1 (1.5–800 pM) in the absence or presence of different concentrations of Nebentan (YM598). Assay conditions were the same as those described for competition binding. Maximal specific binding was calculated as total binding minus nonspecific binding. The concentration of test compound that caused 50% inhibition (IC50) of the specific binding of [125I]endothelin-1 was determined by regression analysis of displacement curves. Inhibitory dissociation constant (Ki) was calculated from the following formula: Ki=IC50/(1+[C]/Kd), where [C] is the concentration of radioligand present in the tubes and Kd is the dissociation constant of radioligand obtained from the Scatchard plot (Cheng and Prusoff, 1973).

---

Receptor specificity [1]
The specificity of Nebentan (YM598) for endothelin receptors was examined by measuring the ability of YM598 to compete with receptor-specific ligands in 59 different ligand binding assays. YM598 was tested at 10 μM.

In vitro functional inhibitory potency: measurement of intracellular Ca2+ concentration [1]
Measurement of intracellular Ca2+ concentration ([Ca2+]i) was performed according to the method described previously Grynkiewicz et al., 1985, Tahara et al., 1998 with minor modification. CHO cells expressing human endothelin ETA receptor and A10 cells were plated on cover glasses (13.5 mm in diameter) and serum-starved for 12 h. Cell monolayers were loaded in Hank's balanced salt solution (HBSS: 140 mM NaCl, 4 mM KCl, 1 mM K2HPO4, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 20 mM HEPES, pH 7.4) with Fura 2-AM (4 μM/cover glass) for 1 h at 37 °C. They were then washed, transferred to Fura 2-free HBSS and incubated for an additional 30 min at 37 °C. The cover glass was placed into a quartz cuvette containing 2 ml HBSS buffer and maintained at 37 °C with continuous stirring. When thermal equilibrium was reached, the fluorescence signal was recorded with a CAF-110 spectrofluorometer with 340/380 nm excitation and 500 nm emission wavelengths. After recording the baseline signal for a short while, vehicle or test compound was added to the cuvette. Two minutes after the test compounds were added, endothelin-1 was added to the cuvette to stimulate the mobilization of [Ca2+]i in the presence or absence of the test compounds. Fluorescence measurements were converted to [Ca2+]i by determining maximal fluorescence (Rmax) with the nonfluorescent Ca2+ ionophore, ionomycin (25 μM), after which minimal fluorescence (Rmin) was obtained by adding 3 mM EGTA. From the ratio of fluorescence at 340 and 380 nm, [Ca2+]i was calculated using the following equation: [Ca2+]i (nM)=Kd×[(R−Rmin)/(Rmax−R)]×b. The term b is the ratio of fluorescence of Fura 2 at 380 nm in zero and saturation Ca2+. Rmax, Rmin, and the value of b were yielded twice, at the beginning and end of the experiment, and the mean values were used in calculation. Kd is the dissociation constant of Fura 2 for Ca2+, assumed to be 224 nM. The activity of the test compound was evaluated by expressing the increase in [Ca2+]i as the percentage of that to treatment with endothelin-1 (10 nM), determined in each experiment using the same preparation of cells. IC50 values of test compound were determined by regression analysis.

---

In vitro functional inhibitory potency: rat aortic ring contraction [1]
Antagonism of endothelin-1-induced vasoconstriction was evaluated with isolated rat aortic rings because this response is mediated by endothelin ETA receptors in this tissue (Panek et al., 1992). Male Wistar rats (320–370 g) were anesthetized with sodium pentobarbital (60 mg/kg i.p.) and the thoracic aorta was quickly removed and placed in a Krebs–Henseleit solution (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2, 11.1 mM glucose) with 95:5 O2/CO2 to maintain pH 7.4. The endothelium was removed by gentle rubbing of the intimal surface using a small cotton ball and each ring was suspended in a 10-ml isolated siliconized organ chamber containing gassed (95:5 O2/CO2) and warmed (37 °C) Krebs–Henseleit solution. Vessel segments were attached to an isometric force transducer linked to a physiographic recorder for monitoring tension change. Baseline tension was set at 1.0 g and the tissues were allowed to equilibrate for 1 h. The tissues were maximally contracted with phenylephrine (1 μM) followed by challenge with acetylcholine (1 μM). A negative relaxant response to acetylcholine confirmed the absence of endothelium. After washing out these agents, the rings were stimulated to contract with 60 mM KCl repeatedly until the contractile response to KCl became stable before starting experiments. Cumulative concentration–response curves to endothelin-1 were performed in the presence or absence of Nebentan (YM598) after a 30-min pretreatment period. Contractile responses were expressed as a percentage of the response elicited by 60 mM KCl. The effective concentration of endothelin-1 causing 50% maximum response (EC50) in the presence or absence of Nebentan (YM598) was determined by regression analysis. The negative logarithm of the molar concentration of antagonist required to produce a 2-fold rightward shift of concentration–response curves to agonist (pA2) value was determined as an index of potency by the equation: pA2=log (concentration ratio−1)−log [B], where concentration ratio is the ratio of EC50 values with and without antagonist and [B] is the concentration of antagonist. Regression analysis of the plot log (concentration ratio−1) against the log [B] (Schild plot) allowed us to confirm the competitive nature of the antagonist by assessing its slope.

In vivo functional inhibitory potency: inhibition of pressor response to big endothelin-1 in pithed rats [1]
In vivo antagonistic activity in pithed rats was evaluated according to the method of Clozel et al. (1994) as described previously. Briefly, after tracheal intubation, male Wistar rats (280–320 g) were pithed with a steel rod under sodium pentobarbital anesthesia (60 mg/kg, i.p.) and artificially ventilated with room air. The right common carotid artery and the left femoral vein were cannulated for blood pressure measurements and i.v. administration of drugs, respectively. After stabilization of blood pressure, various doses of (1 ml/kg) Nebentan (YM598) or vehicle (distilled water) were injected. Five minutes later, the first dose of big endothelin-1 was injected intravenously in a volume of 0.5 ml/kg. Increasing doses were injected in a cumulative manner (0.1–3.2 nmol/kg, i.v.), with each dose being given after stabilization of the effect of the previous dose on blood pressure. In another series of experiments, the oral activity of Nebentan (YM598) was tested. Various doses of (5 ml/kg) Nebentan (YM598) or vehicle (0.5% methyl cellulose) were orally administered by gastric gavage with a cannula. About 20 min later, the rats were anesthetized with sodium pentobarbital, and 30 min after dosing were pithed and ventilated. About 1 h after oral administration of YM598, waiting for stabilization of blood pressure, the first dose of big endothelin-1 was injected intravenously. In this study, DR2 value was defined as the dose of YM598 required to produce a 2-fold rightward shift of dose–response curves to big endothelin-1 in diastolic blood pressure.

---

In vivo functional inhibitory potency: effect on big endothelin-1- or sarafotoxin S6c-induced changes in blood pressure in conscious normotensive rats [1]
Male Wistar rats (270–360 g) were anesthetized with sodium pentobarbital (60 mg/kg i.p.). The right common carotid artery and the left jugular vein were cannulated with a polyethylene tube for determination of blood pressure and heart rate, and for i.v. administration of big endothelin-1. The animals were allowed to recover for 2 days after the operation, during which time they were housed in individual cages with free access to rat chow and water. Rats were then placed in individual cages, and big endothelin-1 (0.5 nmol/kg) was intravenously administered three times at intervals of 1 h. Thirty minutes after the third administration of big endothelin-1, various doses of Nebentan (YM598), bosentan or vehicle (0.5% methyl cellulose) (5 ml/kg) were orally administered by gastric gavage with a cannula. Repeated doses of big endothelin-1 were administered 30 min later, followed every 60 min over a 6-h period and finally again 24 h after administration with Nebentan (YM598), bosentan, or vehicle. The activity of the test compound was evaluated by expressing the pressor response in mean blood pressure as the percentage of that to the third administration of big endothelin-1. In another set of experiments, the effects on sarafotoxin S6c-induced depressor and pressor responses were also tested. Instead of big endothelin-1, sarafotoxin S6c (0.3 nmol/kg) was intravenously administered and various doses of YM598, K-8794, a selective endothelin ETB receptor antagonist (Sonoki et al., 1997), and vehicle were orally administered. The activity of the test compound was evaluated by expressing the depressor and pressor response as the percentage of those to the third administration of sarafotoxin S6c.

[1]. Pharmacological Characterization of YM598, an Orally Active and Highly Potent Selective Endothelin ET(A) Receptor Antagonist. Eur J Pharmacol. 2003 Sep 30;478(1):61-71.

[2]. YM598, an Orally Active ET(A) Receptor Antagonist, Ameliorates the Progression of Cardiopulmonary Changes and Both-Side Heart Failure in Rats With Cor Pulmonale and Myocardial Infarction. J Cardiovasc Pharmacol. 2004 Nov:44 Suppl 1:S354-7.

Endothelin Receptor Type A Antagonist YM598 is an orally active synthetic substituted phenylethenesulfonamide. As a selective endothelin A receptor antagonist, YM598 inhibits endothelin-mediated mechanisms involved in tumor cell growth and progression, angiogenesis, and metastasis.

---

We describe here the pharmacology of (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), a novel selective endothelin ET(A) receptor antagonist synthesized through the modification of the ET(A)/ET(B) non-selective antagonist, bosentan. YM598 inhibited [125I]endothelin-1 binding to cloned human endothelin ET(A) and ET(B) receptor, with K(i) of 0.697 and 569 nM, and inhibited endothelin-1-induced increases in intracellular Ca(2+) concentration in human and rat endothelin ET(A) receptor. YM598 also inhibited endothelin-1-induced vasoconstriction in isolated rat aorta with a pA(2) value of 7.6. In vivo, YM598 inhibited the pressor response to big endothelin-1, a precursor peptide of endothelin-1. DR(2) values of YM598 in pithed rats were 0.53 mg/kg, i.v. and 0.77 mg/kg, p.o., and its antagonism in conscious rats was maintained for more than 6.5 h at 1 mg/kg, p.o. In contrast, YM598 had no effect on the sarafotoxin S6c-induced depressor or pressor responses. YM598 showed not only superior antagonistic activity and higher-selectivity for endothelin ET(A) receptor in vitro, but at least a 30-fold higher potency in vivo than bosentan. In conclusion, YM598 is a potent and orally active selective endothelin ET(A) receptor antagonist. [1]

---

To assess the specificity of YM598 for the endothelin receptor, YM598 was tested at 10 μM in a variety of radioligand competition assays using 59 receptors. YM598 did not affect radioligand binding except at the diltiazem binding site. In a functional assay (KCl-induced contraction in rabbit aorta), however, YM598 did not inhibit KCl-induced contraction. These data indicate that YM598 might not provide functional inhibition of the effects of L-type Ca2+-channel blockers, including diltiazem. As a point of reference, a 10-μM concentration is approximately 10,000-fold higher than the Ki of YM598 for binding at the endothelin ETA receptor. These data show the specificity of YM598 for the endothelin receptor.

The ability of YM598 to antagonize endothelin-1-induced functional responses in vitro was investigated by measuring the inhibitory effects of endothelin-1 on the increases in [Ca2+]i in both CHO cells expressing human endothelin ETA receptor and rat A10 cells. YM598 concentration-dependently antagonized the increase in [Ca2+]i stimulated by 10 nM endothelin-1. Almost identical IC50 values in both cells indicate that the antagonistic activity of YM598 in humans is equal to that in rats. We also evaluated the effect of YM598 on the contractile response of isolated rat aorta for endothelin ETA receptor-mediated contraction, because endothelin-1 is a potent constrictor of smooth muscle and the endothelin ETA receptor is the predominant mediator of endothelin-1 activity in rat aorta (Panek et al., 1992). YM598 produced a parallel and rightward shift of the endothelin-1 concentration–response curve without affecting the maximal force to yield a pA2 value of 7.6. These results indicate that YM598 is a functional endothelin ETA receptor antagonist.

To evaluate the antagonistic effect of YM598 in vivo, we examined the effects of both intravenous and oral administration of YM598 in rats. The pressor response induced by exogenous big endothelin-1 is considered to be mediated by the endothelin ETA receptor in rats (Haleen et al., 1993). In pithed rats, intravenous and oral administration of YM598 produced a parallel rightward shift of big endothelin-1-induced dose–response curves, with DR2 values of 0.53 mg/kg, i.v. and 0.77 mg/kg, p.o. The calculated i.v./p.o. ratio from these DR2 values was 0.69. In contrast, DR2 values of i.v. and p.o. administration and calculated i.v./p.o. ratio of bosentan were 5.1 mg/kg, 25.2 mg/kg, and 0.20, which are almost equal to previously reported data (Clozel et al., 1994). The relative potencies of YM598 to bosentan in inhibiting exogenous big endothelin-1 were approximately 10-fold on i.v. and 30-fold on p.o. administration. These results indicate that YM598 has potent antagonistic activity in vivo as well as in vitro and may be easily absorbed by oral administration with a higher oral bioavailability than bosentan. We also evaluated the inhibitory effect of YM598 on the pressor response to exogenous big endothelin-1 in conscious normotensive rats at the same doses used in pithed rats. The magnitude of maximum inhibition was about 55%, 70%, and 80% at the doses of 0.1, 0.3, and 1 mg/kg of YM598, 30 min after oral administration. At the same doses, the duration of action was 2.5 h, over 6.5 h, and over 24 h, while a 30-fold higher oral dose of bosentan was needed to bring about a response equal to that of YM598. These results suggest that YM598 has a long-lasting effect and that its oral antagonistic activity to endothelin response via the endothelin ETA receptor is 30-fold more potent than that of bosentan in vivo.

Finally, to ascertain whether YM598 retains endothelin ETA receptor selectivity in vivo, we investigated the effects of YM598 on sarafotoxin S6c-induced responses in conscious normotensive rats, and compared the results with those of K-8794, a selective endothelin ETB receptor antagonist. Oral administration of YM598 at up to 10 mg/kg affected neither the depressor nor pressor responses to sarafotoxin S6c. However, K-8794 potently suppressed depressor and pressor responses to sarafotoxin S6c at 1 to 10 mg/kg oral administration. This result is consistent with those previously reported Sonoki et al., 1997, Sawaki et al., 2000, which showed K-8794 to be ineffective on the pressor response to big endothelin-1 at 30 mg/kg in rats. These results indicate that YM598 is a selective endothelin ETA receptor antagonist in vivo, at least up to 10 mg/kg.

In conclusion, we have developed a new selective endothelin ETA receptor antagonist, YM598, through the modification of bosentan. YM598 is a potent, orally active endothelin ETA receptor antagonist with a long duration of action. This compound blocks endothelin ETA receptors but not endothelin ETB receptors both in vitro and in vivo, and its endothelin ETA receptor antagonistic activities were more potent than those of bosentan. Although it has not been clarified whether blockade of endothelin ETA receptors only or of both endothelin ETA and ETB receptors is more beneficial in the long-term treatment of diseases associated with endothelins, the potency and selectivity of YM598 provide a new tool in analyzing the pathophysiological role of the endothelin ETA receptor in various disorders in which endothelins are implicated. [1]

C24H20KN5O5S

529.61

529.082

C, 54.43; H, 3.81; K, 7.38; N, 13.22; O, 15.10; S, 6.05

342005-82-7

Nebentan;403604-85-3

12093171

Off-white to light yellow solid powder

5.829

0

10

9

36

773

0

COC1=CC=CC=C1OC2=C(N=C(N=C2OC)C3=NC=CC=N3)[N-]S(=O)(=O)/C=C/C4=CC=CC=C4.[K+]

WOPWEXSDEXIRNG-ZUQRMPMESA-N

InChI=1S/C24H20N5O5S.K/c1-32-18-11-6-7-12-19(18)34-20-21(29-35(30,31)16-13-17-9-4-3-5-10-17)27-23(28-24(20)33-2)22-25-14-8-15-26-22;/h3-16H,1-2H3;/q-1;+1/b16-13+;

potassium;[6-methoxy-5-(2-methoxyphenoxy)-2-pyrimidin-2-ylpyrimidin-4-yl]-[(E)-2-phenylethenyl]sulfonylazanide

Nebentan potassium; 342005-82-7; YM598 potassium; OK6K3MDZ98; YM-598 potassium; 403604-85-3; Nebentan [INN]; UNII-IJ670B0H4A; IJ670B0H4A; HE-11; DTXSID50193350; (E)-N-(6-Methoxy-5-(2-methoxyphenoxy)-2,2'-bipyrimidin-4-yl)-2-phenylethenesulfonamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 125 mg/mL (236.02 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 1.67 mg/mL (3.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 1.67 mg/mL (3.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)