Pirarubicin (THP) 72496-41-4_Topoisomerase_DNA Damage_Signaling Pathways_Products

Pirarubicin (THP; THPADM; THPDOX; 1609RB; THP-Adriamycin; THP-Doxorubicin; Pinorubicin; Theprubicine; Therarubicin) is an analog of anthracycline with potential antitumor activity. It has been used as a topoisomerase II inhibitor to treat a number of cancers.

Physicochemical Properties

Molecular Formula	C32H37NO12
Molecular Weight	627.64
Exact Mass	627.231
Elemental Analysis	C, 61.24; H, 5.94; N, 2.23; O, 30.59
CAS #	72496-41-4
Related CAS #	95343-20-7
PubChem CID	11296583
Appearance	Pink to red solid powder
Density	1.5±0.1 g/cm3
Boiling Point	834.7±65.0 °C at 760 mmHg
Melting Point	188-192ºC (dec.)
Flash Point	458.6±34.3 °C
Vapour Pressure	0.0±3.2 mmHg at 25°C
Index of Refraction	1.671
LogP	3.85
Hydrogen Bond Donor Count	5
Hydrogen Bond Acceptor Count	13
Rotatable Bond Count	7
Heavy Atom Count	45
Complexity	1120
Defined Atom Stereocenter Count	7
SMILES	O=C1C2=C(C=CC=C2OC)C(C3=C(O)C4=C([C@@H](O[C@@]5([H])C[C@H](N)[C@H](O[C@@]6([H])OCCCC6)[C@H](C)O5)C[C@@](C(CO)=O)(O)C4)C(O)=C31)=O
InChi Key	KMSKQZKKOZQFFG-YXRRJAAWSA-N
InChi Code	InChI=1S/C32H37NO12/c1-14-31(45-21-8-3-4-9-42-21)17(33)10-22(43-14)44-19-12-32(40,20(35)13-34)11-16-24(19)30(39)26-25(28(16)37)27(36)15-6-5-7-18(41-2)23(15)29(26)38/h5-7,14,17,19,21-22,31,34,37,39-40H,3-4,8-13,33H2,1-2H3/t14-,17-,19-,21+,22-,31+,32-/m0/s1
Chemical Name	(7S,9S)-7-[(2R,4S,5S,6S)-4-amino-6-methyl-5-[(2R)-oxan-2-yl]oxyoxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione
Synonyms	THP; THPADM; THPDOX; 1609RB; Tepirubicin; Tetrahydropyranyl-Doxorubicin; THP-Adriamycin; THP-Doxorubicin; brand names: Pinorubicin; Theprubicine; THP-ADM; Adriamycin, tetrahydropyranyl; 4'-O-Tetrahydropyranyladriamycin; DTXSID2046755; D58G680W0G; Therarubicin
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). This product is not stable in solution, please use freshly prepared working solution for optimal results.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets

Topoisomerase II

ln Vitro

In vitro activity: Pirarubicin is a topoisomerase II inhibitor[1]. Pirarubicin demonstrates inhibitory effects on Ehrlich and M5076 cells, with IC50 values of 0.078 and 0.366 μM, respectively. Because topoisomerase II is expressed much less in M5076 cells than in Ehrlich cells, pirarubicin is less cytotoxic to M5076 cells than to Ehrlich cells[2]. The effects of pirarubicin (2.5, 5, 10 μg/mL) on bladder cancer (T24, EJ, 5637, J82, and UM-UC-3) cells show a significant dose-dependent induction of autophagy. Furthermore, in bladder cancer cells, pirarubicin (5 μg/mL) induces apoptosis by inhibiting mTOR/p70S6K/4E-BP1, and this effect is amplified by inhibiting autophagy[3].

ln Vivo

Pirarubicin (18 mg/kg, i.v.) dramatically increases serum levels of BNP, CK-MB, CTnT, LDH, and MDA when compared to the control group in rats with acute cardiac toxicity. In the acute cardiac toxicity model, pirarubicin also diminishes heart rate, depresses R-wave voltage, and prolongs QT intervals [4].

Enzyme Assay

Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients[3].

Cell Assay

Cell survival analysis is done using MTS. In short, 96-well plates are used to plate cells in triplicate, with 2 × 10³ cells per well, and the cells are then cultured in growth medium. Following that, cells are exposed to pirarubicin for 24 hours at three different concentrations: 2.5 μg/mL, 5 μg/mL, and 10 μg/mL. Once added, the MTS reagent (5 mg/mL) is incubated for 4 hours at 37°C. A microplate reader is used to measure the absorbance at 490 nm[3].

Animal Protocol

A single injection of 18 mg/kg pirarubicin into the caudal vein creates an acute cardiac toxicity model. Six groups are randomly assigned, each consisting of thirty-six rats: the normal control group, the cardiac injury (THP) model, the low-dose rutin group (25 mg/kg), the middle-dose rutin group (50 mg/kg), and the high-dose rutin group (100 mg/kg). For seven days, rats in the rutin-treated group receive varying doses of rutin and CMC-Na by gavage, along with a single injection of 18 mg/kg pirarubicin into the caudal vein. For six days, sodium carboxymethylcellulose (CMC-Na) is gavaged into the rats in the dexrazoxane-treated group. On day seven, rats receive an intraperitoneal injection of 40 mg/kg dexrazoxane and an injection of 18 mg/kg pirarubicin into the caudal vein. After seven days of CMC-Na gavage, rats in the THP model group receive an injection of 18 mg/kg of pirarubicin into their caudal vein on day seven. Rats in the usual control group are given CMC-Na by gavage for seven days, and on day seven, they are given saline injections into their caudal veins[4].

References

[1]. Cytotoxic effect of topoisomerase II inhibitors against adriamycin- and etoposide-resistant small cell lung cancer sublines. Gan To Kagaku Ryoho. 1993 May;20(7):929-35.

[2]. Relationships between the in vitro cytotoxicity and transport characteristics of pirarubicin and doxorubicin in M5076 ovarian sarcoma cells, and comparison with those in Ehrlich ascites carcinoma cells. Cancer Chemother Pharmacol. 2002 Mar;49(3):244-50. Epub 2002 Jan 8.

[3]. Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells. Biochem Biophys Res Commun. 2015 May 1;460(2):380-5.

[4]. Cardioprotective effects of rutin in rats exposed to pirarubicin toxicity. J Asian Nat Prod Res. 2017 Oct 27:1-13.

Additional Infomation

(7S,9S)-7-[[(2R,4S,5S,6S)-4-amino-6-methyl-5-[[(2R)-2-oxanyl]oxy]-2-oxanyl]oxy]-6,9,11-trihydroxy-9-(2-hydroxy-1-oxoethyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione is an anthracycline.
Pirarubicin is an analogue of the anthracycline antineoplastic antibiotic doxorubicin. Pirarubicin intercalates into DNA and interacts with topoisomerase II, thereby inhibiting DNA replication and repair and RNA and protein synthesis. This agent is less cardiotoxic than doxorubicin and exhibits activity against some doxorubicin-resistant cell lines. (NCI04)

Solubility Data

Solubility (In Vitro)

DMSO: 7~10 mg/mL (11.2~15.9 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In Vivo)

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 Formulations
(e.g. IP/IV/IM/SC) 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).
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 Formulations 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).
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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.5933 mL	7.9664 mL	15.9327 mL
	5 mM	0.3187 mL	1.5933 mL	3.1865 mL
	10 mM	0.1593 mL	0.7966 mL	1.5933 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

PeptideDB

Pirarubicin (THP) 72496-41-4

CAS No.: 72496-41-4

Physicochemical Properties

Biological Activity

Solubility Data