Diiodohydroxyquinoline demonstrated potent anti-SARS-CoV-2 activity in vitro. The 50% maximal effective concentration (EC50) determined by plaque reduction assay was 1.38 μM. The 50% cytotoxic concentration (CC50) determined by CellTiterGlo luminescent assay in VeroE6 cells at 48 hours post-infection was >100 μM, resulting in a selectivity index (CC50/EC50) of >72.46. [3]
In a viral load reduction assay using VeroE6 cells infected with SARS-CoV-2 (MOI = 0.01) and treated with diiodohydroxyquinoline for 48 hours, dose-dependent reduction of viral RNA was observed, with approximately 3 logs reduction at 10 μM. Similar dose-dependent viral load reduction was also observed in SARS-CoV-2-infected Caco2 cell culture supernatants and cell lysates. [3]
In a cytopathic effect (CPE) inhibition assay, treatment of SARS-CoV-2-infected VeroE6 cells with 10 μM diiodohydroxyquinoline achieved up to ~70% inhibition of CPE development at 72 hours post-infection. [3]
Immunofluorescence staining of SARS-CoV-2-infected VeroE6 cells (MOI = 0.100) treated with 10 μM diiodohydroxyquinoline for 24 hours showed a marked reduction in viral nucleocapsid (N) protein expression compared to untreated controls. [3]
A time-of-drug-addition assay indicated that diiodohydroxyquinoline disrupts post-entry events of the SARS-CoV-2 replication cycle, as its addition at 0 and 3 hours post-infection (hpi), but not during pre-treatment of host cells or co-infection (virus adsorption), significantly reduced viral replication. [3]

Cell Viability and Cytotoxicity Assay: VeroE6 cells (4×10⁴ cells/well) were incubated with various concentrations of diiodohydroxyquinoline for 48 hours. Cell viability was determined using a luminescent CellTiterGlo assay, which measures ATP content as an indicator of metabolically active cells. The 50% cytotoxic concentration (CC50) was calculated from the dose-response data. [3]
CPE Inhibition Assay: VeroE6 cells seeded in 96-well plates were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for 1 hour. After washing, fresh medium containing serially diluted diiodohydroxyquinoline was added. Cell viability was assessed at 3 days post-infection using the CellTiterGlo assay to evaluate the compound's protective effect against virus-induced cytopathic effects. [3]
Viral Load Reduction Assay (qRT-PCR): VeroE6 or Caco2 cells were infected with SARS-CoV-2 and treated with different concentrations of diiodohydroxyquinoline. Supernatant samples were collected at 48 hours post-infection. Viral RNA was extracted and quantified by one-step quantitative reverse transcription PCR (qRT-PCR) targeting the SARS-CoV-2 RNA-dependent RNA polymerase/helicase (RdRp/Hel) gene. Intracellular viral loads in cell lysates were normalized to a human housekeeping gene (β-actin). [3]
Plaque Reduction Assay: VeroE6 cells were seeded in 12-well plates. The next day, 50 plaque-forming units (PFU) of SARS-CoV-2 were added to cell monolayers with or without diiodohydroxyquinoline and incubated for 1 hour. Unbound virus was removed, and cells were overlaid with medium containing low melting agarose and the compound. After 72 hours, cells were fixed, stained with crystal violet, and plaques were counted. The EC50 was calculated from the percentage of plaque inhibition relative to the control. [3]
Time-of-Drug-Addition Assay: VeroE6 cells in 24-well plates were infected with SARS-CoV-2 (MOI = 0.500). Diiodohydroxyquinoline was added at different time points: pre-treatment (-2 to -1 hpi), during virus adsorption/co-infection (-1 to 0 hpi), or post-infection (0 to +1 hpi). Viral titers in the culture supernatant were measured at 9 hpi. [3]
Immunofluorescence Staining: SARS-CoV-2-infected VeroE6 cells (MOI = 0.100) were treated with diiodohydroxyquinoline (10 μM) for 24 hours, then fixed. Viral N protein was detected using a cross-reactive rabbit antiserum against SARS-CoV-N, followed by an appropriate fluorescent secondary antibody. Cell nuclei were counterstained with DAPI. [3]

Absorption, Distribution and Excretion
After oral administration, a certain proportion (but varying considerably) of the ingested dose is absorbed. ...Diiodohydroxyquine has the lowest absorption rate, only one-third that of iodochlorohydroxyquine. Most of these drugs are excreted in the feces.

Iodoquinoline can cause adverse reactions such as headache, nausea, vomiting, and optic nerve damage. [1]
The half-maximal cytotoxic concentration (CC50) of diiodohydroxyquinoline in VeroE6 cells after 48 hours of treatment is >100 μM. [3]

[1]. Flavonoids as a Natural Treatment Against Entamoeba histolytica. Front Cell Infect Microbiol. 2018 Jun 22;8:209.

[2]. In vivo antimutagenic effect of ascorbic acid against mutagenicity of the common antiamebic drug diiodohydroxyquinoline. Mutat Res. 1989 Mar;222(3):219-22.

[3]. Discovery of the FDA-approved drugs bexarotene, cetilistat, diiodohydroxyquinoline, and abiraterone as potential COVID-19 treatments with a robust two-tier screening system. Pharmacol Res. 2020 Sep;159:104960.

[4]. Efficacy and safety of metronidazole versus a combination of metronidazole and diiodohydroxyquinoline for the treatment of patients with intestinal amebiasis: a primary care physician research group study. 1995 July, 56(7): 678-683.

[5]. Cohlan SQ. Chronic nonspecific diarrhea in infants and children treated with diiodohydroxyquinoline. Pediatrics. 1956 Sep;18(3):424-32.

Iodoquinoline is a monohydroxyquinoline, namely quinoline-8-ol, in which the hydrogen atoms at the 5 and 7 positions are replaced by iodine atoms. It is considered the drug of choice for treating asymptomatic or moderate amebiasis. It has anti-amoebic, antibacterial, disinfectant, and antigenic effects on animals. It is a monohydroxyquinoline and an organic iodine compound. Diiodohydroxyquinoline, also known as iodoquinoline or iodoquinoline, is a quinoline derivative used to treat amebiasis. Its exact mechanism of action is unknown. Currently, iodoquinoline is not listed in any FDA-approved products. A halogenated 8-quinoline alcohol widely used as an intestinal disinfectant (especially for anti-amoebic purposes). It is also used to treat other infections and may cause central nervous system and eye damage. It has many similar trade names worldwide. Drug Indications: For the treatment of amebiasis. Mechanism of Action: Unknown. It is unclear whether their efficacy against intestinal amebiasis is solely due to their presence in the intestinal lumen or also partly due to their presence in the bloodstream. /8-Hydroxyquinoline/
……8-Hydroxyquinolines have a direct killing effect on amoebae. They are effective against both motile and cystic amoebae, but their efficacy in clearing cysts may be based on their ability to destroy trophozoites. They are only effective against intestinal amoebae and are ineffective against amoeboid abscesses and hepatitis. /8-Hydroxyquinoline/

---

Therapeutic Uses>
Amoeboid agent; Topical anti-infective agent
……Can be used for the topical and systemic treatment of trichomoniasis and infections caused by Trichomonas vaginalis. It is used topically to treat certain fungal skin infections and eczema with fungal infection.
……It has been used to treat various skin diseases, and high-dose oral administration has also been used to treat a rare and potentially fatal childhood disease—enteropathic acrodermatitis.
……Effective against intestinal amoebae and…can be used to treat asymptomatic cyst discharge.…Suitable for outpatient and group treatment. Inexpensive. It has therapeutic value for quinacrine-resistant laparocystis infections, baranche dysentery, and intestinal infections caused by Entamoeba fragilis. /8-Hydroxyquinolines/
For more complete data on the therapeutic uses of diiodohydroxyquinolines (8 in total), please visit the HSDB record page.

---

Drug Warnings>
While…effective in patients with expelled cysts, its efficacy in treating acute amoebic dysentery is much lower.
These drugs are not recommended for the treatment of “traveler’s diarrhea” and chronic nonspecific diarrhea in children/Chinese/or adults, as these conditions are self-limiting and any possible therapeutic benefit is insufficient to offset the risk of serious neurotoxicity. /8-Hydroxyquinolines/
These drugs are contraindicated in patients with hepatic impairment or iodine intolerance. …Because these drugs contain iodine, they may interfere with certain thyroid function tests (e.g., hypothyroidism). /8-Hydroxyquinolines/
...It now appears that the gastrointestinal neurological syndrome of unknown etiology commonly seen in areas where diiodohydroxyquinoline is used is caused by this drug...Therefore, Japan has withdrawn this type of drug from the market.
Veterinarian: Commercially available pregabalin may cause yellow-brown staining of white fur. Use of this product may alter serum protein-bound iodine test results.

---

Iodoquinoline is used to treat amoebic colitis at a dose of 650 mg orally three times daily. It is described as highly selective. [1]

C9H5NOI2

396.9501

396.846

C, 27.23; H, 1.27; I, 63.94; N, 3.53; O, 4.03

83-73-8

53341-25-6 (sodium);83-73-8;

3728

Light yellow to brown crystalline solid.

2.5±0.1 g/cm3

401.8±45.0 °C at 760 mmHg

>200 °C (dec.)(lit.)

196.8±28.7 °C

0.0±1.0 mmHg at 25°C

1.828

4.11

1

2

0

13

191

0

IC1C(O)=C2C(C=CC=N2)=C(I)C=1

UXZFQZANDVDGMM-UHFFFAOYSA-N

InChI=1S/C9H5I2NO/c10-6-4-7(11)9(13)8-5(6)2-1-3-12-8/h1-4,13H

5,7-diiodoquinolin-8-ol

5,7-Diiodo-8-hydroxyquinoline; 5,7-Diiodo-8-quinolinol;Diiodohydroxyquin, Diiodoquin, Diquinol, Sebaquin

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)

DMSO : 13~20.83 mg/mL ( 32.74~52.48 mM)
H2O : ~0.67 mg/mL (~1.69 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.30 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 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: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2.5 mg/mL (6.30 mM)

---

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