 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C28H41N3O4S |
| Molecular Weight | 515.7078 |
| Exact Mass | 515.282 |
| Elemental Analysis | C, 65.21; H, 8.01; N, 8.15; O, 12.41; S, 6.22 |
| CAS # | 1019640-33-5 |
| Related CAS # | 1019640-33-5;57695-04-2 (free); |
| PubChem CID | 24769161 |
| Appearance | Typically exists as solid at room temperature |
| LogP | 7.261 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 12 |
| Heavy Atom Count | 36 |
| Complexity | 554 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | S(C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1[H])(=O)(=O)O[H].O(C([H])([H])[H])C1=C([H])C2=C(C([H])([H])[H])C([H])=C([H])N=C2C(=C1[H])N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])[H] |
| InChi Key | LOBPWLFCZDGYKW-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C21H33N3O.C7H8O3S/c1-5-24(6-2)14-10-8-7-9-12-22-20-16-18(25-4)15-19-17(3)11-13-23-21(19)20;1-6-2-4-7(5-3-6)11(8,9)10/h11,13,15-16,22H,5-10,12,14H2,1-4H3;2-5H,1H3,(H,8,9,10) |
| Chemical Name | N',N'-diethyl-N-(6-methoxy-4-methylquinolin-8-yl)hexane-1,6-diamine;4-methylbenzenesulfonic acid |
| Synonyms | Sitamaquine tosylate; 1019640-33-5; Sitamaquine (tosylate); N1,N1-Diethyl-N6-(6-methoxy-4-methyl-8-quinolinyl)-1,6-hexanediamine 4-methylbenzenesulfonate; N',N'-Diethyl-N-(6-methoxy-4-methylquinolin-8-yl)hexane-1,6-diamine;4-methylbenzenesulfonic acid; WR 6026 tosylate; SCHEMBL2933465; CHEMBL4544369; |
| 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 |
| 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 | Leishmania |
| ln Vitro | The mechanism of plasmodicidal action of sitamaquine is not completely understood. Like other quinoline derivatives, it is thought to inhibit heme polymerase activity. This results in accumulation of free heme, which is toxic to the parasites. |
| ln Vivo | Sitamaquine (WR6026), an 8-aminoquinoline derivative, is a new antileishmanial oral drug. As a lipophilic weak base, it rapidly accumulates in acidic compartments, represented mainly by acidocalcisomes. In this work, we show that the antileishmanial action of sitamaquine is unrelated to its level of accumulation in these acidic vesicles. We have observed significant differences in sitamaquine sensitivity and accumulation between Leishmania species and strains, and interestingly, there is no correlation between them. However, there is a relationship between the levels of accumulation of sitamaquine and acidotropic probes, acidocalcisomes size, and polyphosphate levels. The Leishmania major AP3delta-null mutant line, in which acidocalcisomes are devoid of their usual polyphosphate and proton content, is unable to accumulate sitamaquine; however, both the parental strain and the AP3delta-null mutants showed similar sensitivities to sitamaquine. Our findings provide clear evidence that the antileishmanial action of sitamaquine is unrelated to its accumulation in acidocalcisomes[1]. |
| Enzyme Assay |
Amastigote sensitivity in vitro.[1] Late-stage promastigotes of wild-type and AP3δ-null mutant L. major lines were used to infect peritoneal macrophages from BALB/c mice. at a ratio of 1:5 macrophages/parasites, as previously described. After 4 h of infection, excess parasites were removed by washing with serum-free medium. The infected macrophage cultures were maintained at 37°C with 5% CO2 with different sitamaquine concentrations in RPMI 1640 medium plus 10% heat-inactivated fetal bovine serum. After 72 h, samples were fixed for 20 min at 4°C with 2% (wt/vol) paraformaldehyde in PBS, followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min. Intracellular parasites were detected by nuclear staining (Prolong-Gold antifade reagent with DAPI). The percentage of infection and the mean number of amastigotes by infected macrophages were calculated in 200 macrophages/well. Three independent experiments were performed with duplicates. Determination of polyP levels in Leishmania lines.[1] Fluorescence staining using DAPI is commonly used for nucleic acid detection (using an excitation wavelength at 360 nm, with a peak of emission wavelength at 475 nm), but it is known that DAPI also binds and stains other polyanions such as polyP, using an excitation wavelength at 415 nm with a peak of emission wavelength at 525 nm. We used DAPI staining to quantify the acidocalcisomal polyP content in different Leishmania species and strains. Leishmania parasites (2 × 107) were resuspended in 1 ml of PBS and incubated for 10 min at room temperature with 10 μg/ml DAPI. After two washes with PBS, parasites in 2 ml of PBS were transferred into magnetically stirred four-window cuvettes at 28°C. Cell density determined at 600 nm was equilibrated in all the samples before fluorescence measurement. Sample fluorescence was calculated by an emission spectrum (from 450 to 650 nm) using excitation at 415 nm in an Aminco-Bowman series 2 spectrometer. |
| Cell Assay |
Flow cytometry analysis.[1] Parasites (4 × 106 cells/ml) were labeled with 100 nM of the acidotropic dye Lysotracker Green DND-26 in HBS buffer at 28°C. After a 10-min incubation, parasites were treated with 20 mM NH4Cl for 1, 5, and 8 min or with 1, 10, and 30 μM sitamaquine for 15 min at 28°C. Washed parasites were resuspended in PBS, and the cellular fluorescence intensity of the probe was measured by flow cytometry in a FACScan flow cytometer equipped with an argon laser operating at 488 nm. The cells were gated to eliminate dead cells and debris, and the cell fluorescence was quantified by scanning the emissions between 515 and 545 nm (FL-1) by using Cell Quest software. |
| Animal Protocol |
Determination of sitamaquine accumulation.[1] Leishmania parasites washed twice with PBS were resuspended in HEPES-buffered saline (HBS; 21 mM HEPES, 0.7 mM Na2HPO4, 137 mM NaCl, 5 mM KCl, and 6 mM dextrose, adjusted to pH 7). A final concentration of 2 × 107 parasites per ml was incubated at 28°C or 4°C with 5 μM [14C]sitamaquine for 15 min in the presence or absence of different concentrations of nonradioactive sitamaquine. Afterwards, samples were removed and placed on ice. The parasites were spun down in a microcentrifuge and washed in PBS or in PBS containing 100 μΜ sitamaquine for 10 min on ice, followed by two washes with PBS to remove the radiolabeled drug adhered to the cell surface, as previously described for the chloroquine uptake assays in yeast. Finally, the cell pellet was resuspended in 0.1 ml of 1% Triton X-100. Eight microliters of the sample were used for protein determination with a Bradford kit (Bio-Rad), and the remaining volume was used to determine cell-associated radioactivity by liquid scintillation counting. Energy, protein, pH, and H+ gradient dependence in the sitamaquine uptake process.[1] Parasite suspensions were prepared as described above. For the energy depletion study, parasites were preincubated for 30 min at 28°C in HBS buffer without glucose, with 5 mM 2-deoxy-d-glucose and 20 mM sodium azide as previously described. For protein modification, parasites were treated with 1 mM N-ethylmaleimide (NEM) for 15 min on ice, centrifuged, and resuspended in fresh HBS as previously described. H+ gradient dependence was determined with parasites pretreated at 28°C in HBS with 20 mM NH4Cl for 1 min and 10 μM of the ionophores nigericin and monensin for 10 min. Finally, 2 × 107 parasites per ml were incubated at 28°C with 5 μM [14C]sitamaquine for 15 min in HBS for parasites pretreated with NEM or in HBS without glucose for energy depletion studies. Parasites preincubated with ionophores and NH4Cl were incubated with 5 μM [14C]sitamaquine for 5 min in HBS. The influence of extracellular pH in drug uptake was established with parasites incubated at 28°C with radiolabeled sitamaquine in HBS adjusted to different pHs. Samples were then removed and placed on ice. The parasites were spun down and washed in PBS containing 100 μΜ sitamaquine for 10 min on ice, followed by two washes with PBS. The amount of drug incorporated into the cells was determined as described above. Sitamaquine sensitivity assay.[1] The sensitivity of Leishmania parasites to sitamaquine was determined after a 72-h incubation at 28°C in the presence of increasing concentrations of sitamaquine. The concentration of sitamaquine necessary to inhibit the parasites growth by 50% (EC50) was calculated by the Alamar Blue method using a spectrofluorometer (Molecular Devices Ltd., Wokingham, United Kingdom) at an excitation and emission wavelength of 530 nm and 585 nm, respectively. This randomized, open label, multicenter study assessed the dose-response and safety profile for oral sitamaquine in 120 Indian subjects with visceral leishmaniasis (VL). Patients aged 5-64 years (mean age 21.2 years) received one of four sitamaquine doses (1.5, 1.75, 2.0, or 2.5 mg kg(-1) day(-1)) daily for 28 days. At Day 180 in the intent-to-treat population, final cure (primary efficacy outcome) was achieved in 92 of 106 (87%) patients overall and 25 of 31 (81%), 24 of 27 (89%), 23 of 23 (100%), and 20 of 25 (80%) patients at doses of 1.5, 1.75, 2.0, or 2.5 mg kg(-1) day(-1) sitamaquine, respectively. Sitamaquine was generally well tolerated. The most common adverse events during the active treatment phase were vomiting (8% [10 of 120]), dyspepsia (8% [9 of 120]) and cyanosis (3% [4 of 120]). Nephrotic syndrome (3% [3 of 120]) and glomerulonephritis (2% [2 of 120]) were also reported and require further investigation. Oral sitamaquine demonstrated efficacy in Indian VL and was well tolerated.[2] |
| References |
[1]. Sitamaquine sensitivity in Leishmania species is not mediated by drug accumulation in acidocalcisomes. Antimicrob Agents Chemother. 2008;52(11):4030-4036. [2]. A phase II dose-ranging study of sitamaquine for the treatment of visceral leishmaniasis in India. Am J Trop Med Hyg. 2005 Dec;73(6):1005-11. |
| Additional Infomation |
Sitamaquine (WR-6026) is an orally active 8-aminoquinoline analog in development by the Walter Reed Army Institute, in collaboration with GlaxoSmithKline (formerly SmithKline Beecham), for the potential treatment of visceral leishmaniasis. The aim of this study was to determine if there was a correlation between sitamaquine sensitivity and accumulation in different Leishmania species. Furthermore, we have found an explanation for the differences observed in sitamaquine accumulation between the Leishmania species L. donovani and L. tropica. We have identified the fact that acidocalcisomes play a key role in the accumulation of sitamaquine in nonpermeabilized parasites and that they can be considered the main factor which determines the differences shown by Leishmania strains in terms of sitamaquine accumulation but not the antileishmanial potency of the drug.[1] |
Solubility Data
| Solubility (In Vitro) | 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 |
| 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 | 1.9391 mL | 9.6954 mL | 19.3907 mL | |
| 5 mM | 0.3878 mL | 1.9391 mL | 3.8781 mL | |
| 10 mM | 0.1939 mL | 0.9695 mL | 1.9391 mL |