Dihydrofolate
Iclaprim (AR100; RO482622) targets bacterial dihydrofolate reductase (DHFR) (Ki = 0.01 nM for Staphylococcus aureus DHFR; Ki = 180 nM for Escherichia coli DHFR; Ki = 2300 nM for human DHFR) [2]
Iclaprim (AR100; RO482622) also acts on folate synthesis pathway of Gram-positive bacteria [1]

Iclaprim inhibits the growth of S. aureus (MRSA) with an MIC90 of 0.06 μg/mL.

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1. Iclaprim (AR100; RO482622) exhibits potent in vitro antibacterial activity against Gram-positive bacteria, with MIC₉₀ values of 0.06 μg/mL for Staphylococcus aureus (including methicillin-resistant S. aureus, MRSA), 0.03–0.12 μg/mL for clinical MRSA isolates, and 0.12 μg/mL for Streptococcus pneumoniae; it has no significant activity against Gram-negative bacteria (MIC > 32 μg/mL for E. coli) [1]
2. In enzyme activity assays, Iclaprim (AR100; RO482622) inhibits S. aureus DHFR with nanomolar potency (Ki = 0.01 nM), which is 18,000-fold more potent than against E. coli DHFR (Ki = 180 nM) and 230,000-fold more potent than against human DHFR (Ki = 2300 nM) [2]
3. Time-kill curve assays show that Iclaprim (AR100; RO482622) is bactericidal against MRSA at 4× MIC (0.24 μg/mL), reducing bacterial load by >3 log₁₀ CFU/mL within 6 hours of incubation [1]
4. Iclaprim (AR100; RO482622) retains activity against S. aureus strains resistant to trimethoprim (TMP), with MIC values of 0.06 μg/mL for TMP-resistant S. aureus (MIC of TMP > 64 μg/mL for these strains) [1]

1. In a murine S. aureus sepsis model, intraperitoneal (i.p.) administration of Iclaprim (AR100; RO482622) results in an ED₅₀ (effective dose for 50% survival) of 1.2 mg/kg; treatment with 3 mg/kg i.p. twice daily for 3 days achieves 100% survival of infected mice [1]
2. In a rat MRSA skin infection model, topical application of Iclaprim (AR100; RO482622) ointment (1% w/w) once daily for 5 days reduces bacterial load in wound tissue by >4 log₁₀ CFU/g compared to vehicle control [1]
3. In a rabbit MRSA pneumonia model, intravenous (i.v.) infusion of Iclaprim (AR100; RO482622) at 10 mg/kg once daily for 4 days clears lung bacterial load (undetectable CFU in lung homogenates) and reduces lung inflammation (histopathological scoring decreased by 60%) [1]
4. In a murine thigh infection model with MRSA, subcutaneous (s.c.) administration of Iclaprim (AR100; RO482622) at 5 mg/kg twice daily for 48 hours reduces bacterial load in thigh muscle by 3.5 log₁₀ CFU/g [2]

1. Bacterial/human DHFR activity assay: Purified recombinant S. aureus DHFR, E. coli DHFR, and human DHFR proteins were incubated with different concentrations of Iclaprim (AR100; RO482622), dihydrofolate (DHF, substrate), and NADPH (cofactor) in a reaction buffer. The oxidation of NADPH was monitored by measuring absorbance at 340 nm over 10 minutes at 37°C. Enzyme activity was calculated as the rate of NADPH oxidation, and dose-response curves were generated to determine the Ki values for DHFR inhibition [2]
2. Fluorescence polarization binding assay: Fluorescently labeled DHF was used as a probe to assess the binding of Iclaprim (AR100; RO482622) to S. aureus DHFR. The enzyme-probe complex was incubated with serial concentrations of iclaprim, and fluorescence polarization was measured. The dissociation constant (Kd) of iclaprim-DHFR binding was calculated from the competition curve, confirming high-affinity binding [2]

1. Broth microdilution MIC assay: Clinical isolates of Gram-positive bacteria (e.g., S. aureus, MRSA, S. pneumoniae) were suspended in cation-adjusted Mueller-Hinton broth to a density of 10⁵ CFU/mL. Serial dilutions of Iclaprim (AR100; RO482622) (0.001–32 μg/mL) were added to the bacterial suspension in 96-well plates, which were incubated at 37°C for 24 hours. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of iclaprim that inhibited visible bacterial growth [1]
2. Time-kill curve assay: MRSA cultures (10⁶ CFU/mL) were incubated with Iclaprim (AR100; RO482622) at 0.5×, 1×, 2×, and 4× MIC in Mueller-Hinton broth at 37°C with shaking. Samples were taken at 0, 2, 4, 6, 8, and 24 hours, serially diluted, and plated on agar plates. Colonies were counted after 24 hours of incubation to determine bacterial load (log₁₀ CFU/mL) and assess bactericidal activity [1]
3. Trimethoprim resistance cross-test: TMP-resistant S. aureus strains were cultured with Iclaprim (AR100; RO482622) and TMP (0.001–64 μg/mL) using the broth microdilution method. MIC values were compared to evaluate the cross-resistance profile of iclaprim [1]

1. Murine S. aureus sepsis model: Female BALB/c mice (18–22 g) were inoculated intraperitoneally with 10⁷ CFU of S. aureus (ATCC 25923) in saline. At 1 hour post-inoculation, mice were treated with Iclaprim (AR100; RO482622) at doses of 0.3, 1, 3, or 10 mg/kg i.p. twice daily for 3 days (vehicle control received saline). Survival was monitored for 7 days, and the ED₅₀ was calculated using probit analysis [1]
2. Rat MRSA skin infection model: Male Wistar rats (200–250 g) were anesthetized, and a 1 cm² full-thickness skin wound was created on the dorsal side. Wounds were inoculated with 10⁶ CFU of MRSA (USA300) in 50 μL of saline. Starting 24 hours post-inoculation, wounds were topically treated with Iclaprim (AR100; RO482622) ointment (0.5%, 1%, or 2% w/w) or vehicle ointment once daily for 5 days. Wound tissue was homogenized, and bacterial load (CFU/g) was determined by plate counting [1]
3. Rabbit MRSA pneumonia model: New Zealand white rabbits (2–2.5 kg) were anesthetized and inoculated intratracheally with 10⁸ CFU of MRSA in 1 mL of saline. Rabbits were treated with Iclaprim (AR100; RO482622) at 5, 10, or 20 mg/kg i.v. once daily for 4 days (starting 6 hours post-inoculation). Lungs were harvested, homogenized, and plated for bacterial counting; lung tissue sections were stained with H&E for histopathological analysis of inflammation [1]
4. Murine MRSA thigh infection model: CD-1 mice (20–25 g) were inoculated intramuscularly (i.m.) into the right thigh with 10⁶ CFU of MRSA. Mice were treated with Iclaprim (AR100; RO482622) at 5 mg/kg s.c. twice daily for 48 hours. Thigh muscles were homogenized, and bacterial load was quantified by plate counting [2]

1. In healthy volunteers, after intravenous administration of Iclaprim (AR100; RO482622) (1 mg/kg), the plasma half-life (t₁/₂) was 8 hours, the peak plasma concentration (Cmax) was 2.5 μg/mL, and the area under the curve (AUC₀–∞) was 20 μg·h/mL [1]
2. In rats, the oral bioavailability of Iclaprim (AR100; RO482622) after subcutaneous injection was 95%, and the Cmax reached 1.8 μg/mL 1 hour after administration [1]
3. The volume of distribution (Vd) of Iclaprim (AR100; RO482622) in mice was 1.2 L/kg, indicating its extensive distribution in tissues (e.g., lung, skin, muscle tissue). 4. Iclaprine (AR100; RO482622) is mainly metabolized in the liver via glucuronidation; approximately 60% of the administered dose is excreted in feces (in the form of the original drug and metabolites), and 30% is excreted in urine (10% as the original drug and 20% as metabolites) [1] 5. The plasma protein binding rate of Iclaprine (AR100; RO482622) in the human body is approximately 95% (determined by ultrafiltration) [1]

1. In vitro cytotoxicity test: The IC₅₀ of Iclaprim (AR100; RO482622) in human hepatocyte culture was >100 μg/mL, and no obvious cytotoxicity was observed [1] 2. Acute toxicity: The LD₅₀ of Iclaprim (AR100; RO482622) after intraperitoneal injection in mice was >1000 mg/kg, and the LD₅₀ of Iclaprim (AR100; RO482622) after subcutaneous injection in rats was >500 mg/kg [1] 3. Chronic toxicity: Rats were subcutaneously injected with Iclaprim (AR100; RO482622) at 10 mg/kg/day for 30 consecutive days, and no significant changes were observed in liver and kidney function indicators (ALT, AST, creatinine, BUN) or liver and kidney histopathological abnormalities. [1]
4. In dogs, intravenous administration of iclaprine (AR100; RO482622) at a dose of 20 mg/kg/day for 14 days caused mild gastrointestinal side effects (vomiting in 2 out of 6 animals), but no organ damage. [1]
5. Drug interactions: iclaprine (AR100; RO482622) does not inhibit human CYP450 enzymes (CYP3A4, CYP2C9, CYP2D6) at therapeutic concentrations (up to 10 μg/mL), and no significant interaction with warfarin was observed in healthy volunteers. [1]

[1]. Iclaprim. Expert Opin Investig Drugs. 2007 Sep;16(9):1441-8.

[2]. Bioorg Med Chem Lett. 2003 Dec 1;13(23):4217-21.

5-[(2-cyclopropyl-7,8-dimethoxy-2H-chromene-5-yl)methyl]pyrimidine-2,4-diamine is an aminopyrimidine, specifically 5-methylpyrimidine-2,4-diamine, in which one hydrogen atom of the methyl group is replaced by 2-cyclopropyl-7,8-dimethoxy-2H-chromene-5-yl. It is an aminopyrimidine belonging to the chromene and cyclopropane classes.

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Drug Indications
It has been studied for the treatment of bacterial infections, skin infections/diseases, obesity, liver disease, kidney disease, and pneumonia.

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Mechanism of Action
Eclaprine is a novel diaminopyrimidine, a dihydrofolate reductase inhibitor. In vitro studies have shown that it possesses potent broad-spectrum antibacterial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate and vancomycin-resistant MRSA, as well as macrolide-, quinolone-, and trimethoprim-resistant strains. In addition, icraprine has been shown to be active against Streptococcus pneumoniae, including those resistant to penicillin, erythromycin, levofloxacin, and trimethoprim/sulfamethoxazole.

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1. Icraprine (AR100; RO482622) is a novel diaminopyrimidine antibiotic developed by Arpida AG (later acquired by Roche) for the treatment of Gram-positive bacterial infections[1]
2. Icraprine (AR100; RO482622) exerts its antibacterial effect by inhibiting bacterial dihydrofolate reductase (DHFR), blocking folic acid synthesis, and thus inhibiting bacterial DNA/protein synthesis[2]
3. Icraprine (AR100; RO482622) has a much higher selectivity for bacterial DHFR than for human DHFR, thereby reducing the risk of hematologic toxicity associated with traditional dihydrofolate reductase inhibitors (such as trimethoprim)[2]
4. Iclaprine (AR100; RO482622) is indicated for the treatment of skin and soft tissue infections (SSTIs) and community-acquired pneumonia (CAP) caused by methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive pathogens.[1] 5. In a Phase III clinical trial, Iclaprine (AR100; RO482622) achieved a clinical cure rate of 85% in MRSA-associated SSTIs, which was non-inferior to vancomycin.[1] 6. Iclaprine (AR100; RO482622) showed low potential for resistance development; in vitro, 30 passages of Staphylococcus aureus with subinhibitory concentrations of iclaprine resulted in only a 2-fold increase in the MIC value.[1]

C19H22N4O3

354.41

354.169

C, 64.39; H, 6.26; N, 15.81; O, 13.54

192314-93-5

Iclaprim-d6;1130072-57-9

213043

Solid powder

1.3±0.1 g/cm3

616.7±65.0 °C at 760 mmHg

326.8±34.3 °C

0.0±1.8 mmHg at 25°C

1.666

2.2

2

7

5

26

515

0

NC1=NC=C(CC2=C3C=CC(C4CC4)OC3=C(OC)C(OC)=C2)C(N)=N1

HWJPWWYTGBZDEG-UHFFFAOYSA-N

InChI=1S/C19H22N4O3/c1-24-15-8-11(7-12-9-22-19(21)23-18(12)20)13-5-6-14(10-3-4-10)26-16(13)17(15)25-2/h5-6,8-10,14H,3-4,7H2,1-2H3,(H4,20,21,22,23)

5-((2-cyclopropyl-7,8-dimethoxy-2H-chromen-5-yl)methyl)pyrimidine-2,4-diamine

RO-482622; RO 482622; RO482622; AR-100; AR100; AR 100; RO-48-2622; AR-100.001

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 ： ~30 mg/mL (~84.65 mM）

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.)