Leishmania amazonensis(EC50 = 13.63 μM)

The size of parasitophorous vacuoles (LPVs) decreases significantly with increasing concentrations of DHQZ 36; at 50 μM, vacuole sizes are reduced by 30%. There is a decrease in the quantity of parasites for every macrophage due to these parasites. After using DHQZ 36, significant parasite loss is seen at as low as 5 μM. When DHQZ 36 is applied at concentrations of 12.5 μM or higher, parasites cannot grow again.
Over 40% less parasite protein is secreted when DHQZ 36 is present. After LPS activation, DHQZ 36 reverses the suppression of IL-6 release by infected cells caused by Leishmania[1].

Promastigote drug susceptibility assay [1]
To determine the EC50 of Retro-2cycl and its analogs on axenic parasites, an MTT Cell Viability Assay Kit was used. Early stationary phase promastigotes were seeded at 1x105 parasites/well in a 96-well tissue culture plate and allowed to grow for 48 hrs at room temperature in the presence of Retro-2cycl, DHQZ-36 or DHQZ 36.1 with concentrations ranging from 0–100μM for L. amazonensis treatments and 0–200μM for L. donovani treatments. Miltefosine treatments of axenic promastigotes were in concentrations ranging from 0–100μM. Parasite susceptibility to the DMSO vehicle alone was assessed by treating parasites at an equal concentration of DMSO to the highest concentration of drug used in each experiment. Promastigotes were incubated with MTT for 2 hrs and formazan product was read at 570 nm wavelength and a 630 nm background wavelength as described by the Biotium protocol. Viable parasites were estimated from an MTT standard curve that was made from serial dilutions of parasites and correlation of those values to the relative amount of formazan product. Plots of % Cell Viability as compared to controls vs. Log Molar Concentration were generated in GraphPad Prism 7. EC50s were calculated by nonlinear regression analysis of the sigmoidal curves that were generated. Significance of the differences in parasite growth was determined using the multiple t-tests function. Statistical significance between time points of each concentration was measured using a two-way ANOVA in GraphPad Prism 7 with the Holm-Sidak posthoc test for multiple comparisons

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Promastigote recovery [1]
To determine if the inhibitory effect of Retro-2cycl and its SAR analogs on promastigote parasites was transient, a recovery experiment was performed on promastigotes. Promastigotes were seeded at 1x105 parasites per well and treated with concentrations of Retro-2cycl, DHQZ-36, DHQZ 36.1 and miltefosine ranging from 0–100μM for 72 hrs. After treatment, parasites were collected and spun down. Drugs were washed from parasites with fresh parasite medium and resuspended in 100μL fresh parasite medium. 50μL of the parasites were used for MTT assay for the 72 hrs time point and the other 50μL was plated in a new 96-well plate with fresh parasite medium. These cells were allowed to grow for 48 hrs before performing the MTT assay. Cell numbers were estimated by comparison of formazan product to a standard generated at time 0 with known numbers of parasites. Formazan products at 48 hrs after recovery was then compared to amounts produced at 72 hrs of treatment to determine if growth occurred. The experiment was repeated at least three times with three replicates per concentration. Graphs were generated in GraphPad Prism 7 and significance of differences in growth was determined using a using a two-way ANOVA with the Holm-Sidak posthoc test for multiple comparisons.

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Drug treatment of infected RAW264.7murine macrophages [1]
These experiments were performed as described previously. Briefly, macrophages were plated in 100 mm Petri dishes containing sterile glass coverslips and allowed to adhere overnight in complete DMEM media at 37°C with 5% CO2. Macrophages were infected with mid-stationary phase promastigotes at 1:10 or 1:20 ratio for 24 hrs. Coverslips were then washed and treated with the varying concentrations of Retro-2cycl, DHQZ-36, DHQZ 36.1 and miltefosine as described in the experiments. Some coverslips were incubated in DMSO as vehicle control. After incubation with drugs for an additional 24 hrs at 34°C, coverslips were fixed in methanol for 5 minutes prior to Giemsa staining. After methanol fixation, infected cells were stained with Wright-Giemsa stain at a 1:20 dilution for 15 minutes made fresh with NanoPure diH2O. Cells were then washed twice with NanoPure diH2O and allowed to dry for 1–2 hrs. Coverslips were dehydrated with xylenes for 1 minute and mounted in Permount on glass slides for viewing under a bright field light microscope. The percentage of infected macrophages and the average number of parasites was determined by counting at least 200 macrophages per coverslip. Counts were done in duplicate over at least three experiments and EC50 values were determined in GraphPad Prism using a three parameter dose-response best-fit curve line. Statistical significance between treated infected cells and the control was measured using a one-way ANOVA in GraphPad Prism 7.

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Cytokine secretion of RAW264.7 macrophages during infection [1]
Macrophages were plated in 6-well plates at a density of 5x105 cells/mL overnight for adherence. The cells were then infected at a 1:20 ratio with stationary L. amazonensis promastigotes as described above. After 24 hrs infected wells were treated with Retro-2cycl, DHQZ-36, DHQZ 36.1 at 12.5, 50 or 100μM or miltefosine at 1, 5 or 10μM for 24 hrs in complete DMEM supplemented with 100ng/ml or 500ng/mL LPS and 100ng/mL IFN-γ. Controls were treated with complete DMEM alone or LPS/IFN-γ without drugs or with drugs without LPS/IFN-γ stimulation. Media was collected from plates after 24 hrs of treatment and spun down to remove any cell debris. Supernatants fluids were then assessed in ELISA as described previously.

[1]. Structurally optimized analogs of the retrograde trafficking inhibitor Retro-2cycl limit Leishmania infections. PLoS Negl Trop Dis. 2017 May 15;11(5):e0005556.

In infected mammalian cells, Leishmania parasites reside within specialized compartments called parasitophorous vacuoles (LPVs). We have previously shown that Retro-2, a member of a novel class of small retrograde pathway inhibitors caused reduced LPV sizes and lower parasite numbers during experimental L. mexicana sp. infections. The purpose of this study was to determine if structural analogs of Retro-2cycl reported to have superior potency in the inhibition of retrograde pathway-dependent phenomena (i.e., polyomavirus cellular infection by polyomavrius and Shiga toxin trafficking in cells) are also more effective than the parent compound at controlling Leishmania infections. In addition to their effects on LPV development, we show that two optimized analogs of Retro-2cycl, DHQZ 36 and DHQZ 36.1 limit Leishmania amazonensis infection in macrophages at EC50 of 13.63+/-2.58μM and10.57+/-2.66μM, respectively, which is significantly lower than 40.15μM the EC50 of Retro-2cycl. In addition, these analogs caused a reversal in Leishmania induced suppression of IL-6 release by infected cells after LPS activation. Moreover, we show that in contrast to Retro-2cycl that is Leishmania static, the analogs can kill Leishmania parasites in axenic cultures, which is a desirable attribute for any drug to treat Leishmania infections. Together, these studies validate and extend the published structure-activity relationship analyses of Retro-2cycl.[1]

C21H18F2N2OS

384.442230701447

384.11

C, 65.61; H, 4.72; F, 9.88; N, 7.29; O, 4.16; S, 8.34

1542098-94-1

1542098-94-1;

72720908

Solid powder

5

1

5

4

27

528

0

SVXVTSKHYHQIPJ-UHFFFAOYSA-N

InChI=1S/C21H18F2N2OS/c1-2-16-8-10-19(27-16)20-24-18-9-7-15(23)11-17(18)21(26)25(20)12-13-3-5-14(22)6-4-13/h3-11,20,24H,2,12H2,1H3

2-(5-Ethyl-thiophen-2-yl)-6-fluoro-3-(4-fluoro-benzyl)-2,3-dihydro-1H-quinazolin-4-one

DHQZ-36; DHQZ 36; DHQZ 36; 1542098-94-1; 2-(5-ethylthiophen-2-yl)-6-fluoro-3-[(4-fluorophenyl)methyl]-1,2-dihydroquinazolin-4-one; 2-(5-Ethylthiophene-2-yl)-6-fluoro-3-(4-fluorobenzyl)-2,3-dihydroquinazoline-4(1H)-one; CHEMBL3322091; SCHEMBL15616349; SLC09894; DHQZ36;

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)

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

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.

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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).

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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)

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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).

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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

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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.

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