Chloroquine Phosphate 50-63-5_ATM(ATR)_DNA Damage_Signaling Pathways_Products

Chloroquine phosphate is reported to be highly effective in combating SARS-CoV-2 (COVID-19, CoronaVirus, or the COVID-19 pandemic) infection in vitro. It functions as a strong inhibitor of autophagy and toll-like receptors (TLRs) and a 4-aminoquinoline antimalarial drug that is used to treat and prevent malaria in regions where the disease is known to be susceptible to its effects. In addition to being an ATM activator, it also has anti-rheumatoid properties. Chloroquine diphosphate has been documented as an adjuvant for chemotherapy and radiation therapy to induce autophagy in cells and prevent the proliferation or metastasis of anti-cancer cells. The process by which chloroquine diphosphate induces autophagy in cells involves arresting them in the G1 phase, which up-regulates the expression of p53 and p27 and down-regulates the expression of CDK2 and cyclin D1.

Physicochemical Properties

Molecular Formula

C18H32CLN3O8P2

Molecular Weight

515.8625

Exact Mass

515.14

Elemental Analysis

C, 41.91; H, 6.25; Cl, 6.87; N, 8.15; O, 24.81; P, 12.01

CAS #

50-63-5

Related CAS #

Chloroquine phosphate;50-63-5;Chloroquine-d5;1854126-41-2;Chloroquine dihydrochloride;3545-67-3;Chloroquine-d5 diphosphate; 132-73-0 (sulfate); 1854126-42-3; 54-05-7 ;151-69-9 (acetate) ; 1446-17-9 (phosphate); 3545-67-3 (HCl) ; 50-63-5 (diphosphate) ;

PubChem CID

64927

Appearance

White to light yellow crystalline powder

Boiling Point

460.6ºC at 760 mmHg

Melting Point

200 °C

Flash Point

232.3ºC

LogP

3.03

Hydrogen Bond Donor Count

Hydrogen Bond Acceptor Count

Rotatable Bond Count

Heavy Atom Count

Complexity

359

Defined Atom Stereocenter Count

InChi Key

QKICWELGRMTQCR-UHFFFAOYSA-N

InChi Code

InChI=1S/C18H26ClN3.2H3O4P/c1-4-22(5-2)12-6-7-14(3)21-17-10-11-20-18-13-15(19)8-9-16(17)18;2*1-5(2,3)4/h8-11,13-14H,4-7,12H2,1-3H3,(H,20,21);2*(H3,1,2,3,4)

Chemical Name

4-N-(7-chloroquinolin-4-yl)-1-N,1-N-diethylpentane-1,4-diamine;phosphoric acid

Synonyms

Quingamine; NSC 14050; Khingamin; Ipsen 225; Gontochin phosphate; Chloroquine dihydrogen phosphate (1:2); Chlorochin diphosphate; Chingamin phosphate; Bemaphate; Aralen diphosphate; Chloroquine; Chloroquine diphosphate; Chingamin; Miniquine, Resochin; Chloroquine; Aralen; Arechin; Tanakan; Chloroquine phosphate; Chloroquine diphosphate; Aralen phosphate; Avloclor; Chingaminum; Chloroquin diphosphate; Delagil; Nivaquine B; Resoquine

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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

Plasmodium; Malaria; TLRs; SARS-COV-2; HIV-1

ln Vitro

In vitro activity: Chloroquine is a chemotherapeutic drug used in the clinical management of malaria. Because chloroquine can bind to DNA, it can prevent RNA synthesis and DNA replication, which ultimately leads to cell death. The creation of a toxic heme-chloroquine complex could potentially be connected to the effects of chloroquine. By raising the pH of vacuoles and blocking the actions of vacuolar phospholipase, vacuolar proteases, and heme polymerase, chloroquine prevents the breakdown of trophozoite hemoglobin. There are specific antirheumatic properties of chloroquine. Due to its immuno-modulatory properties, chloroquine inhibits the release and synthesis of interleukin 6 and tumour necrosis factor. Chloroquine also has direct antiviral effects, preventing the replication of various viruses, including retroviruses, coronaviruses, and flaviviruses, at pH-dependent stages. The effects it has on HIV replication have received the most research. Through the process of ion trapping, chloroquine can accumulate within the macrophage phagolysosome, where it possesses strong antifungal properties against both Cryptococcus neoformans and Histoplasma capsulatum through different mechanisms. While chloroquine directly poisons C. neoformans, it inhibits the growth of H. capsulatum through pH-dependent iron deprivation.

ln Vivo

Chloroquine (80 mg/kg, i.p.) has no effect on the growth of triple-negative MDA-MB-231 cells with high or low TLR9 expression levels in the orthotopic mouse model. The mouse xenograft model shows significant tumor growth inhibition upon TLR7 and TLR9 inhibition with IRS-954 or chloroquine. Chloroquine also significantly inhibits the development of HCC in the DEN/NMOR rat model. In a mouse model using a subcutaneous xenograft of 4T1 cells, treatment with chloroquine diphosphate dramatically reduced tumor growth and tumor cell metastasis to the lung, improving the mice's survival. Chloroquine diphosphate worked in concert with 5-FU to significantly increase the tumor growth inhibition caused by 5-FU in BALB/c mice that received subcutaneous injections of colon26 cells.This was achieved by increasing the proportion of apoptotic cells.

Enzyme Assay

Chloroquine suppressed matrix metalloproteinase (MMP)-2 and MMP-9 mRNA expression and protein activity, whereas MMP-13 mRNA expression and proteolytic activity were increased. Despite enhancing TLR9 mRNA expression, chloroquine suppressed TLR9 protein expression in vitro.[2]

Cell Assay

The cells are cultivated in 6-well plates using normal culture medium with either vehicle or 25 or 50 μM chloroquine until they are almost confluent. Afterward, they are rinsed with sterile phosphate-buffered saline (PBS) and continue to culture in serum-free culture medium for the specified durations. Centrifugation is used to quickly harvest the cells in lysis buffer and clarify them after the culture medium is discarded at the predetermined time points. Once the supernatants have been boiled in reducing sodium dodecyl sulphate (SDS) sample buffer, 100 μg of protein is loaded into each lane. The samples are then electrophoresed into 10 or 4–20% gradient polyacrylamide SDS gels before being placed onto a nitrocellulose membrane. TLR9 is detected by incubating the blots with anti-TLR9 antibodies diluted 1:500 in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for an overnight period at 4°C. Polyclonal rabbit anti-actin is used to confirm equal loading. Horseradish peroxidase-linked secondary antibodies are used in secondary detection. Using an ECL kit, the protein bands can be seen through chemiluminescence.

Animal Protocol

Mice with impaired immunity, four weeks old, have their mammary fat pads injected with a combination of control and TLR9 siRNA MDA-MB-231 cells (5×10⁵ cells in 100 μL) (athymic nude/nu Foxn1). Seven days after the tumor cell inoculation, treatments begin. Every day, the mice receive either a vehicle (PBS) or intraperitoneal (i.p.) chloroquine (80 mg/kg). Every day, the animals are observed for clinical indications. Tumor volumes are estimated using the formula V=(π/6) (d1×d2)^3/2, where d1 and d2 are the perpendicular tumor diameters. Tumor measurements are carried out twice a week. The mice are killed and the tumors are removed for one last measurement after the tumors are left to grow for 22 days. The animals are kept in controlled, pathogen-free environments with 20–21°C temperatures, 30–60% relative humidity, and a 12-hour lighting cycle throughout the trials. Small-animal food pellets are fed to the mice, and they are given access to sterile water whenever they need it.

Toxicity/Toxicokinetics

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Very small amounts of chloroquine are excreted in breast milk; when given once weekly, the amount of drug is not sufficient to harm the infant nor is the quantity sufficient to protect the child from malaria. United Kingdom malaria treatment guidelines recommend that weekly chloroquine 500 mg be given until breastfeeding is completed and primaquine can be given. Breastfeeding infants should receive the recommended dosages of chloroquine for malaria prophylaxis.In HIV-infected women, elevated viral HIV loads in milk were decreased after treatment with chloroquine to a greater extent than other women who were treated with the combination of sulfadoxine and pyrimethamine. Because no information is available on the daily use of chloroquine during breastfeeding, hydroxychloroquine or another agent may be preferred in this situation, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Several authors have pointed out that malaria prophylaxis in nursing mothers with chloroquine is common in endemic areas. As of the revision date, no reports of adverse reactions in breastfed infants have been published.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

References

[1]. Chloroquine promotes IL-17 production by CD4+ T cells via p38-dependent IL-23 release by monocyte-derived Langerhans-like cells. J Immunol. 2014 Dec 15;193(12):6135-43.

[2]. Chloroquine has tumor-inhibitory and tumor-promoting effects in triple-negative breast cancer. Oncol Lett. 2013 Dec;6(6):1665-1672.

[3]. Effect of toll-like receptor 7 and 9 targeted therapy to prevent the development of hepatocellular carcinoma. Liver Int. 2014 Jul 2. doi: 10.1111/liv.12626.

[4]. Chloroquine and hydroxychloroquine as available weapons to fight COVID-19. Int J Antimicrob Agents. 2020;55(4):105932.

[5]. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020 Mar;30(3):269-271.

[6]. The anti-HIV-1 activity of chloroquine. J Clin Virol. 2001;20(3):131-135.

Additional Infomation

Chloroquine Phosphate is the phosphate salt of chloroquine, a quinoline compound with antimalarial and anti-inflammatory properties. Chloroquine is the most widely used drug against malaria, except for those cases caused by chloroquine resistant Plasmodium falciparum. Although the mechanism of action is not fully understood, chloroquine is shown to inhibit the parasitic enzyme heme polymerase that converts the toxic heme into non-toxic hemazoin, thereby resulting in the accumulation of toxic heme within the parasite. Chloroquine may also interfere with the biosynthesis of nucleic acids.
See also: Chloroquine (has active moiety); Chloroquine phosphate; primaquine phosphate (component of); Chloroquine Phosphate; Embutramide; Lidocaine (component of).

Solubility Data

Solubility (In Vitro)

DMSO: <1 mg/mL

Water: ~100 mg/mL (~193.9 mM)

Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: 100 mg/mL (193.85 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.9385 mL	9.6926 mL	19.3851 mL
	5 mM	0.3877 mL	1.9385 mL	3.8770 mL
	10 mM	0.1939 mL	0.9693 mL	1.9385 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

Chloroquine Phosphate 50-63-5

CAS No.: 50-63-5

Physicochemical Properties

Biological Activity

Solubility Data