α1A-adrenoceptor (pKi = 9.6); α1D-adrenoceptor (pKi = 7.9); (α1B-adrenoceptor (pKi = 7.5)

Human lower urinary tract (LUT) tissue, specifically RS100329 hydrochloride, inhibits norepinephrine contraction in a concentration-dependent and surmountable manner [2].

---

In competition studies Ro 70-0004, RS100329 hydrochloride, prazosin and tamsulosin all competed to the same extent for [3H]-prazosin binding: calculated affinity estimates (pKi) are shown in Table 1. Prazosin and tamsulosin demonstrated limited α1-AR subtype selectivity (<10 fold), whereas significant selectivities for the α1A- over the α1B- and α1D-AR subtypes were evident for Ro 70-0004 (60 and 50 fold respectively) and RS100329 hydrochloride (126 and 50 fold respectively). [2]
Prazosin showed some selectivity for the α1B- and α1D-AR subtypes over the α1A-AR subtype, whereas tamsulosin showed approximately 10 fold selectivity for the α1A-AR subtype over the α1B-, but not the α1D-AR subtype. Ro 70-0004 (Figure 2a) was 30 and 80 fold selective for the α1A-subtype over the α1B- and α1D-subtypes respectively, and RS100329 hydrochloride (Figure 2b) also showed significant α1A-adrenoceptor selectivity (60 and 50 fold respectively). [2]
In human LUT tissues, contractions to NA were antagonized in a surmountable and concentration-dependent manner by Ro 70-0004, RS100329 hydrochloride and prazosin with similar affinity (pA2 ∼9). [2]
In contrast, both Ro 70-0004 and RS100329 hydrochloride were approximately 100 fold weaker in antagonizing the contractile response to NA in human renal artery. The affinities of prazosin and tamsulosin did not differ from those observed in studies using human LUT tissues (Table 2). In studies using rat aortic rings, Ro 70-0004, RS100329 hydrochloride and tamsulosin antagonized contractile responses to NA with affinities close to those observed for human renal artery. Prazosin however, was approximately 10 fold more potent in rat aorta when compared to human renal artery. [2]

Sprague Dawley rats administered with RS100329 hydrochloride (0.01-0.1 mg/kg) intravenously have lower baseline urethral pressure and less reflex urethral contractions [1].

---

The effects of the α1-adrenoceptor antagonists doxazosin (0.1 – 2 mg kg−1), RS100329 hydrochloride (α1A; 0.01 – 1 mg kg−1), RS-513815 (Ro 151-3815, α1B; 0.3 – 3 mg kg−1) and BMY 7378 (α1D; 0.1 – 1 mg kg−1), the 5-HT1A receptor agonist, 8-OH-DPAT (0.03 – 0.3 mg kg−1) and antagonist WAY-100635 (0.03 – 0.3 mg kg−1) were investigated (i.v.) on the ‘micturition reflex' in the urethane anaesthetized male rat. [1]
Reflex-evoked urethra contractions were most sensitive to the inhibitory action of RS100329 hydrochloride, followed by doxazosin, BMY 7378 and WAY-100635 and then RS-513815. The maximum inhibition was 66, 63, 54, 46 and 22% at doses of 0.3, 0.5, 0.3, 0.3 and 3 mg kg−1 respectively. [1]
BMY 7378 and 8-OH-DPAT decreased, while WAY-100635 increased, the pressure threshold to induce bladder contraction. WAY-100635 (0.01 mg kg−1) blocked the effects of BMY 7378 (1 mg kg−1) on bladder pressure and volume threshold. [1]
Doxazosin, RS100329 hydrochloride and BMY 7378 had a similar potency in inducing a fall in arterial blood pressure while WAY-100635 only caused a fall at the highest dose. [1]
Therefore, reflex-evoked urethral contraction involves the activation of α1A/1D-adrenoceptors, as BMY 7378 and RS100329 hydrochloride are similarly potent in attenuating this effect. The ability of WAY-100635 to attenuate this contraction may suggest that 5-HT1A receptors are also involved. However, as this inhibition occurred at the highest dose of WAY-100635, which also caused a fall in arterial blood pressure; this effect is considered to be due to blockade of α1-adrenoceptors not 5-HT1A receptors. Nevertheless the initiation of the ‘micturition reflex' involves the activation of 5-HT1A receptors.

Radioligand binding studies [2]
Affinity estimates (pKi) were made from competition curves (using ten concentrations of displacing agent) using intact CHO-K1 cells stably expressing human cloned α1A-, α1B- and α1D-adrenoceptors. Cells were grown as described above and harvested by incubating with Dulbecco's phosphate buffered saline (PBS) containing EDTA (30 μM) for 10 min at 37°C. Harvested cells were washed twice by centrifugation and resuspension in Ham's medium, and finally resuspended in Ham's medium at approximately 0.2×106 cells ml−1. [2]
[3H]-Prazosin (0.3–0.4 nM; specific activity 82 Ci mmole−1) was used as the radioligand and specific binding was defined using 10 μM phentolamine. Assay tubes contained 100 μl competing compound, 100 μl [3H]-prazosin and 300 μl cell suspension. All equilibrations were carried out for 30 min at 37°C in Ham's culture medium (pH 7.4), and were terminated by vacuum filtration through glass fibre filters. Bound radioactivity was determined using liquid scintillation spectroscopy. Concentrations of competing agent producing 50% reduction of specific [3H]-prazosin binding (IC50) were calculated using non-linear iterative curve-fitting methodologies, and affinity estimates (pKi) were estimated according to Cheng & Prusoff (1973). Estimates of pKD were made by saturation analysis using 10–12 concentrations of [3H]-prazosin (1 pM–3 nM).

In vitro tissue bath studies [2]
Tissue bath studies were conducted at 37°C in 10 ml organ baths and used Krebs' buffer (mM: Na+ 143.5, K+ 6.0, Ca2+ 2.5, Mg2+ 1.2, Cl− 126, HCO3− 25, H2PO4− 1.2, SO42− 1.2), pH 7.4, gassed with 95% O2, 5% CO2, and supplemented with cocaine (30 μM), corticosterone (30 μM), propranolol (1 μM), idazoxan (0.3 μM), ascorbate (100 μM) and nitrendipine (1 μM: rat aorta only, see Blue et al., 1995) to ensure equilibrium conditions and α1-adrenoceptor isolation.[2]
Human lower urinary tract tissues (LUT) were obtained from patients undergoing transurethral resection of the prostate (TURP). Samples were kept in cold Krebs' solution until use in functional studies, which were performed later the same day or early the following day. Human renal artery sections (3 mm×6 mm) were obtained from a transplant donor bank, and were usually received within 24–36 h of removal. Rabbit bladder neck strips (2 mm×6 mm) were from male New Zealand White rabbits (2.5–3.5 kg) and rat aortic rings (2 mm width) were from male Sprague-Dawley rats (350–500 g), both euthanized with carbon dioxide. Vascular tissues were endothelium-denuded prior to study.[2]
Tissues were mounted under 0.5 g (human LUT, renal artery) or 1.0 g (rabbit bladder neck, rat aorta) resting tension and allowed to equilibrate for at least 1 h. Cumulative concentration-response curves to NA were constructed in the absence and presence (following a 1 h equilibration) of various concentrations of antagonist. Responses were measured as changes in isometric tension. Iterative curve fitting was used to determine EC50 values for the agonist in the absence and presence of various concentrations of each antagonist. Schild plots (Arunlakshana & Schild, 1959) were constructed in order to estimate antagonist affinity estimates.

Animal/Disease Models: Sprague Dawley rat (300-390 g) [1]
Doses: 0.01-0.1 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: Caused a decrease in baseline urethral pressure up to 23%.

[1]. The role of alpha(1)-adrenoceptors and 5-HT(1A) receptors in the control of the micturition reflex in male anaesthetized rats. Br J Pharmacol. 2001 May;133(1):61-72.

[2]. In vitro alpha1-adrenoceptor pharmacology of Ro 70-0004 and RS-100329, novel alpha1A-adrenoceptor selective antagonists. Br J Pharmacol. 1999;127(1):252-258.

5-Methyl-3-[3-[4-[2-(2,2,2-trifluoroethoxy)phenyl]-1-piperazinyl]propyl]-1H-pyrimidin-2,4-dione is a piperazine compound. The fact that RS-100329 and BMY 7378 have similar potency in lowering arterial blood pressure suggests that the maintenance of sympathetic vasoconstriction mediated by α1-adrenergic receptors is co-mediated by α1A- and α1D-adrenergic receptors, and a comparison of the potency differences of these receptor antagonists (see above) indicates that α1D-adrenergic receptors dominate this effect. In this regard, α1D-adrenergic receptors have been shown to be involved in the contraction of the rat aorta, mesenteric artery, and pulmonary artery (Hussain & Marshall, 1997; Williams et al., 1999), while α1A-adrenergic receptors have been shown to mediate the contraction of the rat caudal artery (Lachnit et al., 1997). However, RS-513815 failed to affect arterial blood pressure, indicating that α1B-adrenergic receptors, at least in this study's urethane-anesthetized rat model, do not play a role in maintaining baseline arterial blood pressure. Interestingly, high-dose RS-100329 caused a significant decrease in arterial blood pressure, and the decrease was greater than that of high-dose doxazosin, suggesting that the mechanism by which RS-100329 interferes is not α1-adrenergic receptors, but other mechanisms equally important for maintaining arterial blood pressure. Furthermore, somewhat surprisingly, 8-OH-DPAT failed to induce a decrease in arterial blood pressure in this model. However, in rats, activation of central 5-HT1A receptors can induce both sympathetic inhibition (Gradin et al., 1985; Fozard et al., 1987) and sympathetic excitation (Anderson et al., 1995; 1996). [1]

---

Some studies have hypothesized that selective antagonism of α1A adrenergic receptor-mediated lower urinary tract tissue contraction in patients with benign prostatic hyperplasia can improve symptoms by selectively relieving urinary tract obstruction. This study describes the α1-AR subtype selectivity of two novel α1-adrenergic receptor (α1-AR) antagonists Ro 70-0004 (also known as RS-100975) and its structure-related compound RS-100329, and compares them with the selectivity of prazosin and tamsulosin. Radioligand binding and second messenger studies in intact CHO-K1 cells expressing human clonal α1A, α1B, and α1D adrenergic receptors (ARs) showed that Ro 70-0004 (pKi 8.9: 60-fold and 50-fold selectivity for α1A AR, respectively) and RS-100329 (pKi 9.6: 126-fold and 50-fold selectivity for α1A AR, respectively) both possessed nanomolar affinities and significant selectivity for the α1A AR isotype, while their selectivity for the α1B AR and α1D AR isotypes was 126-fold and 50-fold higher, respectively. In contrast, prazosin and tamsulosin showed very low isotype selectivity. Norepinephrine-induced contraction of the human lower urinary tract (LUT) or rabbit bladder neck was competitively antagonized by Ro 70-0004 (pA2 values of 8.8 and 8.9, respectively), RS-100329 (pA2 values of 9.2 and 9.2, respectively), tamsulosin (pA2 values of 10.4 and 9.8, respectively), and prazosin (pA2 values of 8.7 and 8.3, respectively). The estimated affinity of tamsulosin and prazosin for antagonizing α1-adrenergic receptor-mediated contraction of the human renal artery (HRA) and rat aorta (RA) was similar to that observed in LUT tissue, while the potency of Ro 70-0004 and RS-100329 was approximately 100-fold lower (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA, respectively). Ro 70-0004 and RS-100329 both exhibit alpha1A-AR subtype selectivity in both clonal and natural systems, which should allow for the evaluation of the clinical value of "urinary tract selective" drugs in the treatment of symptoms associated with benign prostatic hyperplasia. [2]

C20H25F3N4O3

426.43

462.165

232953-52-5

1215654-26-4 (HCl)

3567002

Typically exists as solid at room temperature

545.8ºC at 760 mmHg

283.9ºC

3.01E-12mmHg at 25°C

2.803

1

8

7

30

648

0

CC1=CNC(=O)N(C1=O)CCCN2CCN(CC2)C3=CC=CC=C3OCC(F)(F)F

RCOBWVAGWYRNHZ-UHFFFAOYSA-N

InChI=1S/C20H25F3N4O3/c1-15-13-24-19(29)27(18(15)28)8-4-7-25-9-11-26(12-10-25)16-5-2-3-6-17(16)30-14-20(21,22)23/h2-3,5-6,13H,4,7-12,14H2,1H3,(H,24,29)

5-methyl-3-[3-[4-[2-(2,2,2-trifluoroethoxy)phenyl]piperazin-1-yl]propyl]-1H-pyrimidine-2,4-dione

RS-100329; RS 100329; 5-methyl-3-[3-[4-[2-(2,2,2-trifluoroethoxy)phenyl]piperazin-1-yl]propyl]-1H-pyrimidine-2,4-dione; 232953-52-5; CHEMBL56331;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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