Natural quinazolinone alkaloid
The antimalarial activity of ISOFEBRIFUGINE is evaluated against Plasmodium falciparum (FCR-3 strain) in vitro

In this study, the antimalarial activities of the synthesized febrifugine and isofebrifugine, and their antipodes, were examined. It was revealed that the activities and selectivities of natural febrifugine and isofebrifugine were much higher than those of the antipodes[1].

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The EC50 value of synthetic natural ISOFEBRIFUGINE (2', (2'S,3'S)) against Plasmodium falciparum (FCR-3 strain) in vitro is (2.9 ± 0.3) × 10^{-10} M. [1]
The EC50 value of its antipode (2, (2'R,3'R)) against Plasmodium falciparum in vitro is (1.6 ± 0.2) × 10^{-7} M. [1]
The cytotoxicity of ISOFEBRIFUGINE (2') against mouse mammary tumor FM3A cells (wild-type, subclone F28-7) shows an EC50 value of (7.3 ± 1.4) × 10^{-7} M, with a selectivity index (EC50 FM3A / EC50 P. falciparum) of 2517. [1]
The antipode (2) shows an EC50 value of (1.9 ± 0.1) × 10^{-5} M against FM3A cells, with a selectivity index of 119. [1]
Natural isofebrifugine obtained from Dichroa febrifuga has been reported to have EC50 values of approximately 10^{-9} M for P. falciparum in vitro (citing unpublished data). [1]

Antimalarial Assay:  [1]
In vitro Antimalarial Activity of Febrifugine, Isofebrifugine, and Their Enantiomers: The following procedures were used for assay of antimalarial activity. Various concentrations of compounds in ethanol were prepared. A 5 μL amount of each solution was added to individual wells of a multidish 24 wells arrangement. Erythrocytes with 0.3% parasitemia were added to each well containing 995 μL of culture medium to give a final hematocrit level of 3%. The plates were incubated at 37 °C for 72 h in a CO2−O2−N2 incubator (5% CO2, 5% O2, and 90% N2 atmosphere). To evaluate the antimalarial activity of a compound, we prepared thin blood films from each culture and stained them with Giemsa. A total of 10 000 erythrocytes per one thin blood film were examined by microscopy. All of the test compounds were assayed in duplicate at each concentration. Drug-free control cultures were run simultaneously. All data points represent the mean of three experiments. The EC50 value refers to the concentration of the compound necessary to inhibit the increase in parasite density at 72 h by 50% of control.[1]

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Toxicity against Mammalian Cell Line.[1]
FM3A cells grew with a doubling time of about 12 h. Prior to exposure to drugs, cell density was adjusted to 5 × 104 cells/mL. A cell suspension of 995 μL was dispensed to the test plate, and compounds at various concentrations suspended in ethanol (5.0 μL) were added to individual wells of a multidish 24 well arrangement. The plates were incubated at 37 °C in 5% CO2 atmosphere for 48 h. All the test compounds were assayed in duplicate at each concentration. Cell numbers were measured using a microcell counter CC-130. All data points represent the mean of three experiments. The EC50 value refers to the concentration of the compound necessary to inhibit the increase in cell density at 48 h by 50% control.

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Antimalarial activity assay: Various concentrations of ISOFEBRIGUGINE (and its antipode) in ethanol were prepared. Erythrocytes with 0.3% parasitemia (Plasmodium falciparum, FCR-3 strain) were added to each well containing 995 μL of culture medium to give a final hematocrit level of 3%, after adding 5 μL of compound solution to multidish 24-well plates. The plates were incubated at 37 °C for 72 h in a CO2-O2-N2 incubator (5% CO2, 5% O2, 90% N2 atmosphere). Thin blood films were prepared from each culture, stained with Giemsa, and 10,000 erythrocytes per film were examined by microscopy. The EC50 value is the concentration necessary to inhibit the increase in parasite density at 72 h by 50% of control. All test compounds were assayed in duplicate at each concentration, and data points represent the mean of three experiments. [1]
Cytotoxicity assay against mammalian cell line: Mouse mammary tumor FM3A cells (wild-type, subclone F28-7) were grown with a doubling time of about 12 h. Prior to exposure to drugs, cell density was adjusted to 5 × 10^4 cells/mL. A cell suspension of 995 μL was dispensed to test plates, and 5.0 μL of ISOFEBRIGUGINE (or antipode) solutions in ethanol were added to individual wells of a multidish 24-well plate. The plates were incubated at 37 °C in 5% CO2 atmosphere for 48 h. All test compounds were assayed in duplicate at each concentration. Cell numbers were measured using a microcell counter. The EC50 value is the concentration necessary to inhibit the increase in cell density at 48 h by 50% of control. Data points represent the mean of three experiments. [1]

[1]. Catalytic Asymmetric Synthesis of Antimalarial Alkaloids Febrifugine and Isofebrifugine and Their Biological Activity.J Org Chem. 1999 Sep 3;64(18):6833-6841.

Isofebrin belongs to the quinazoline class of compounds. It has been reported that isoflavones are found in hydrangeas (Hydrangea macrophylla) and (Hydrangea febrifuga), and relevant data are available for reference.

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The absolute configuration of ISOFEBRIGUGINE was revised based on asymmetric total synthesis: the previously reported (2'R,3'R) configuration was corrected to (2'S,3'S) for the natural enantiomer. The synthetic natural enantiomer (2') exhibits much higher antimalarial activity and selectivity than its antipode (2). The EC50 for the natural isofebrifugine (2') is 1/552 of that for the antipode (2). [1]

C16H19N3O3

301.3404

301.143

32434-44-9

9851693

White to off-white solid

1.48±0.1 g/cm3 (20 ºC 760 Torr)

519.8±58.0 °C(Predicted)

138-139 ºC

Chinese medicinal plant, Chang Shan (Dichroa febrifuga)

0.954

2

5

2

22

483

2

C1C[C@H]2[C@H](CC(O2)(CN3C=NC4=CC=CC=C4C3=O)O)NC1

YLYLCQRQSRDSQR-UHFFFAOYSA-N

InChI=1S/C16H19N3O3/c20-15-11-4-1-2-5-12(11)18-10-19(15)9-16(21)8-13-14(22-16)6-3-7-17-13/h1-2,4-5,10,13-14,17,21H,3,6-9H2

3-[(2-hydroxy-3a,4,5,6,7,7a-hexahydro-3H-furo[3,2-b]pyridin-2-yl)methyl]quinazolin-4-one

Isofebrifugine; 32434-44-9; 3-[[(3aS,7aS)-2-hydroxy-3a,4,5,6,7,7a-hexahydro-3H-furo[3,2-b]pyridin-2-yl]methyl]quinazolin-4-one; 3-[(2-hydroxy-3a,4,5,6,7,7a-hexahydro-3H-furo[3,2-b]pyridin-2-yl)methyl]quinazolin-4-one; ISOFEBRIFUGINE (B604866K055); CHEMBL105789; C10698; SureCN12033795;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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.)