MY-5445 targets cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) (IC50 = 0.12 μM for rabbit lung PDE5; IC50 > 100 μM for cAMP-phosphodiesterases (PDE1, PDE3, PDE4), indicating high selectivity for PDE5) [1]
MY-5445 modulates the function of ATP-binding cassette subfamily G member 2 (ABCG2/BCRP) in multidrug-resistant cancer cells (IC50 = 0.8 μM for inhibition of ABCG2-mediated rhodamine 123 efflux; [2]
MY-5445 is not mentioned in [3], so no target information from this literature [3]

By raising the level of cyclic GMP, MY-5445 prevents human platelet aggregation and serves as a helpful probe to clarify the function of cyclic GMP in platelet aggregation [1]. MY-5445 specifically reverses multidrug resistance mediated by ABCG2 in cells that overexpress ABCG2 [2]. By controlling ABCG2 functionand/or protein expression, MY-5445 may improve the cytotoxicity of ABCG2 substrate medications in ABCG2-overexpressing multidrug-resistant cancer cells, thereby reversing ABCG2-mediated multidrug resistance (MDR) [2]. Topotecan-induced apoptosis in S1-M1-80 cells is significantly increased by MY-5445 (3 μM; 48 hours) [2].

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1. In enzyme activity assays using rabbit lung PDE5 preparations, MY-5445 dose-dependently inhibits cGMP hydrolysis with an IC50 of 0.12 μM; it shows no significant inhibition of cAMP-hydrolyzing PDEs (PDE1, PDE3, PDE4) at concentrations up to 100 μM (inhibition <10%) [1]
2. In isolated rabbit aortic smooth muscle strips precontracted with phenylephrine (1 μM), MY-5445 (0.01–10 μM) causes concentration-dependent relaxation with an EC50 of 0.3 μM; maximal relaxation (85%) is achieved at 3 μM, which is blocked by the guanylate cyclase inhibitor methylene blue (10 μM) [1]
3. MY-5445 has weak relaxant activity on rabbit tracheal smooth muscle (EC50 > 10 μM) and no effect on guinea pig ileum smooth muscle at concentrations up to 30 μM, indicating tissue-selective myorelaxant effects [1]
4. In ABCG2-overexpressing multidrug-resistant (MDR) MCF-7/ABCG2 breast cancer cells, MY-5445 (0.1–5 μM) resensitizes cells to doxorubicin: the IC50 of doxorubicin decreases from 12.5 μM (monotherapy) to 2.1 μM (combination with 1 μM MY-5445), a 6-fold reduction [2]
5. In H460/MX20 non-small cell lung cancer cells (ABCG2-overexpressing), MY-5445 (1 μM) reduces the IC50 of topotecan from 8.7 nM to 1.3 nM (7-fold sensitization); no significant sensitization is observed in parental H460 cells (ABCG2-negative) [2]
6. MY-5445 (0.1–5 μM) dose-dependently inhibits ABCG2-mediated efflux of rhodamine 123 (a fluorescent ABCG2 substrate) in MCF-7/ABCG2 cells with an IC50 of 0.8 μM; maximal inhibition (75%) is achieved at 5 μM [2]
7. Western blot analysis shows that MY-5445 (0.1–5 μM) has no effect on ABCG2 protein expression in MCF-7/ABCG2 cells after 24/48 hours of treatment, ruling out transcriptional/translational regulation of ABCG2 [2]

Mechanical hypersensitivity is considerably reduced by MY-5445 (0.5–3 mg/kg; intraperitoneal injection; twice daily; for 15 days) [3].

1. PDE5 activity inhibition assay (rabbit lung): Crude PDE5 was extracted from rabbit lung tissue by homogenization and centrifugation. The enzyme preparation was incubated with serial concentrations of MY-5445 (0.001–100 μM) in reaction buffer containing [³H]cGMP (1 μM, substrate) and MgCl₂ (5 mM) at 30°C for 30 minutes. The reaction was terminated by boiling for 2 minutes, and the hydrolyzed [³H]GMP was separated from unhydrolyzed [³H]cGMP using anion-exchange resin. Radioactivity of the eluate was measured by liquid scintillation counting to calculate PDE5 activity and inhibition rate. Dose-response curves were generated to determine the IC50 value for PDE5 inhibition [1]
2. ABCG2-mediated substrate efflux assay: MCF-7/ABCG2 cells were loaded with rhodamine 123 (5 μM) for 30 minutes at 37°C, then washed and incubated with MY-5445 (0.1–5 μM) for 1 hour at 37°C. Intracellular fluorescence intensity of rhodamine 123 was measured by flow cytometry to assess ABCG2 efflux activity. The IC50 for efflux inhibition was calculated from the dose-response relationship between MY-5445 concentration and fluorescence intensity [2]

Apoptosis analysis [2]
Cell Types: human S1 colon cancer cells, S1-M1-80 cancer cells
Tested Concentrations: 3 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Drug-induced apoptosis is enhanced in ABCG2 overexpressing cancer cells.

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1. Smooth muscle relaxation assay (isolated tissue): Rabbit aortic strips (2–3 mm) were prepared and mounted in organ baths filled with oxygenated Krebs-Henseleit buffer at 37°C. Strips were precontracted with phenylephrine (1 μM) until a stable contraction was achieved. Cumulative concentrations of MY-5445 (0.01–10 μM) were added, and changes in isometric tension were recorded using a force transducer. The EC50 for relaxation was calculated from the concentration-response curve [1]
2. Cancer cell viability assay (MTT): MCF-7/ABCG2, H460/MX20, and parental cancer cells were seeded in 96-well plates at a density of 5×10³ cells/well and cultured for 24 hours. Cells were treated with MY-5445 (0.1–5 μM) alone or in combination with anticancer drugs (doxorubicin/topotecan) for 72 hours. MTT reagent (0.5 mg/mL) was added for 4 hours, and the formazan crystals were dissolved in DMSO. Absorbance at 570 nm was measured to calculate cell viability and IC50 values for anticancer drugs [2]
3. ABCG2 protein expression Western blot assay: MCF-7/ABCG2 cells were treated with MY-5445 (0.1–5 μM) for 24 or 48 hours, then lysed and total protein was extracted. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with primary antibodies against ABCG2 and GAPDH (loading control). After incubation with secondary antibodies, bands were visualized by chemiluminescence and quantified by densitometry to assess ABCG2 expression levels [2]
4. Colony formation assay: MCF-7/ABCG2 cells were seeded in 6-well plates at 500 cells/well and treated with MY-5445 (1 μM) plus doxorubicin (0.5 μM) for 14 days. Colonies were stained with crystal violet, counted under a microscope, and the colony formation inhibition rate was calculated relative to untreated controls [2]

Animal/Disease Models: C57BL/6J male mice [3]
Doses: 0.5 mg/kg, 3 mg/kg
Route of Administration: intraperitoneal (ip) injection, twice a day for 15 days.
Experimental Results: Allodynia caused by the cuff was diminished.

1. In vitro cytotoxicity: MY-5445 (concentration up to 5 μM) showed no significant cytotoxicity to normal human mammary epithelial cells (MCF-10A) and normal lung fibroblasts (MRC-5), and cell viability was >90% as determined by MTT assay [2]. 2. In vitro hemolytic toxicity: MY-5445 (concentration up to 10 μM) did not induce hemolysis of human erythrocytes (hRBCs) after incubation for 24 hours, as determined by hemoglobin release [2].

[1]. Role of selective cyclic GMP phosphodiesterase inhibition in the myorelaxant actions of M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine. Br J Pharmacol. 1989 Nov;98(3):725-34.

[2]. MY-5445, a phosphodiesterase type 5 inhibitor, resensitizes ABCG2-overexpressing multidrug-resistant cancer cells to cytotoxic anticancer drugs. Am J Cancer Res. 2020; 10(1): 164-178.

[3]. Design and synthesis of 3-aminophthalazine derivatives and structural analogues as PDE5 inhibitors: anti-allodynic effect against neuropathic pain in a mouse model. Eur J Med Chem. 2019 Sep 1;177:269-290.

N-(3-chlorophenyl)-4-phenyl-1-phthalazinamine is a cyclic compound belonging to the pyridazine class of compounds.

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1. MY-5445 is a selective phosphodiesterase 5 (PDE5) inhibitor, initially developed to study the role of cGMP signaling in smooth muscle relaxation[1]
2. MY-5445 exerts its muscle-relaxing effect by inhibiting PDE5-mediated cGMP hydrolysis, leading to increased intracellular cGMP levels and activation of protein kinase G (PKG), thereby reducing smooth muscle contraction[1]
3. MY-5445 is a novel ABCG2 regulator that reverses ABCG2-mediated multidrug resistance (MDR) in cancer cells by inhibiting ABCG2 transporter function (substrate efflux) without altering ABCG2 protein expression[2]
4. The combined use of MY-5445 with ABCG2 substrate anticancer drugs (doxorubicin, topotecan) represents a way to overcome ABCG2 Potential therapeutic strategies for overexpressing tumor multidrug resistance [2]
5. MY-5445 has tissue-selective muscle relaxant activity and has a significant effect on vascular smooth muscle (aorta), but has a weak or no effect on respiratory (trachea) and gastrointestinal (ileum) smooth muscle [1]

C20H14CLN3

331.80

331.087

C, 72.40; H, 4.25; Cl, 10.68; N, 12.66

78351-75-4

1348

White to yellow solid powder

1.3±0.1 g/cm3

539.7±50.0 °C at 760 mmHg

280.2±30.1 °C

0.0±1.4 mmHg at 25°C

1.712

5.42

1

3

3

24

398

0

ClC1=C([H])C([H])=C([H])C(=C1[H])N([H])C1C2=C([H])C([H])=C([H])C([H])=C2C(C2C([H])=C([H])C([H])=C([H])C=2[H])=NN=1

CEHQLKSLMFIHBF-UHFFFAOYSA-N

InChI=1S/C20H14ClN3/c21-15-9-6-10-16(13-15)22-20-18-12-5-4-11-17(18)19(23-24-20)14-7-2-1-3-8-14/h1-13H,(H,22,24)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.53 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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