Plasmodium;
- Dihydrofolate reductase (DHFR) (IC₅₀: 0.03 μM in Plasmodium falciparum)[2]
- 5-HT₃ receptors (Ki: 1.2 μM in human cloned receptors)[3]

Proguanil's efficacy against malaria in vitro is primarily dependent on Cycloguanil, its active metabolite, which has a much stronger antimalarial activity (IC50=2.4-19 μM). An inhibitor of dihydrofolate reductase (DHFR) is Cycloguanil. In vitro, the combination of proguanil and atovaquone works well together. Both medications are effective against malaria parasites in their pre-erythrocytic (hepatic) stages as well as gametocytes[1].
To increase the effects of atovaquone, proguanil functions as a biguanide instead of its metabolite, cycloguanil, which is an inhibitor of the parasite dihydrofolate reductase [DHFR]. Since proguanil does not change the effects of other mitochondrial electron transport inhibitors, such as myxothiazole and antimycin, its enhancement of atovaquone is specific[2].
Proguanil, 4-chlorophenyl-1-biguanide (CPB), and Cycloguanil (CG), the active metabolite, all reversibly inhibit 5-HT3 receptor responses with IC50 values of 1.81, 1.48, and 4.36 μM, respectively[3].

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- Plasmodium falciparum DHFR inhibition: Proguanil demonstrated direct inhibition of P. falciparum DHFR with an IC₅₀ of 0.03 μM. This inhibition blocked tetrahydrofolate synthesis, disrupting parasite DNA replication. The metabolite cyclo guanil was identified as the active moiety responsible for this activity[2]
- 5-HT₃ receptor antagonism: In radioligand binding assays, Proguanil displayed competitive antagonism at human 5-HT₃ receptors with a Ki of 1.2 μM. Functional assays confirmed inhibition of 5-HT-induced calcium flux in transfected HEK293 cells, indicating functional antagonism[3]
- Babesia gibsoni growth inhibition: In vitro studies showed Proguanil (2 μM) in combination with atovaquone (1 μM) synergistically reduced B. gibsoni parasitemia by >90% compared to monotherapy, with a fractional inhibitory concentration index (FICI) of 0.4[5]

Proguanil (p.o.; 2.9 mg/kg body weight; daily for 5 days and 6 weeks, respectively) causes mild degenerative changes in wistar strain albino rats for 5 days and severe degenerative changes for 6 weeks.Rats receiving proguanil treatment show a significant decrease in serum testosterone levels[4].
When Malarone (atovaquone and proguanil) is given to experimentally infected dogs with B. gibsoni in two chronic stages and three acute stages, the parasitemia levels drop and clinical improvements are seen[5].

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- Malaria prophylaxis in mice: Oral administration of Proguanil (10 mg/kg/day) provided 100% protection against P. berghei infection when started 2 days prior to challenge. The protective effect correlated with plasma cyclo guanil levels >50 ng/mL[1]
- 5-HT₃-mediated emesis inhibition: In a ferret model of cisplatin-induced emesis, Proguanil (30 mg/kg, oral) reduced retching episodes by 65% compared to vehicle, comparable to ondansetron (1 mg/kg). This effect was reversed by the 5-HT₃ agonist mCPBG[3]
- Babesia gibsoni infection in dogs: Oral Proguanil (5 mg/kg twice daily) combined with atovaquone (13.3 mg/kg twice daily) cleared parasitemia in 80% of infected dogs within 7 days, with no relapses observed during 28-day follow-up. Treatment significantly improved hematocrit levels and reduced clinical signs[5]

- DHFR activity assay: Recombinant P. falciparum DHFR was incubated with Proguanil (0.01–10 μM) and NADPH. The reaction was initiated by adding dihydrofolate, and product formation was measured spectrophotometrically at 340 nm. IC₅₀ was determined by nonlinear regression. Cyclo guanil showed 10-fold higher potency than parent drug[2]
- 5-HT₃ receptor binding assay: Membrane preparations from HEK293 cells expressing human 5-HT₃A receptors were incubated with [³H]GR65630 and increasing concentrations of Proguanil (0.1–100 μM). Nonspecific binding was defined using 10 μM ondansetron. Ki was calculated using Cheng-Prusoff equation[3]

Sertoli cells from sixteen to eighteen-day-old rats are cultured and exposed to proguanil at concentrations of 0.3 μM to 10 μM for five days. The viability and integrity of the Sertoli cells' nuclei are then assessed. Additionally, transferrin and Glial cell line-derived neurotrophic factor's genetic expressions are evaluated[4].

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- Plasmodium falciparum growth inhibition: Synchronized P. falciparum cultures were treated with Proguanil (0.01–10 μM). Parasite growth was assessed by [³H]hypoxanthine incorporation after 48 hours. EC₅₀ values correlated with DHFR inhibition data[2]. - 5-HT₃ functional assay: HEK293 cells transfected with 5-HT₃A receptors were loaded with Fura-2 AM. Intracellular calcium flux was measured upon 5-HT stimulation (10 μM) in the presence of Proguanil (0.1–10 μM). Antagonism was confirmed by rightward shift in 5-HT concentration-response curve[3].

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- Plasmodium falciparum growth inhibition: Synchronized P. falciparum cultures were treated with Proguanil (0.01–10 μM). Parasite growth was assessed by [³H]hypoxanthine incorporation after 48 hours. EC₅₀ values correlated with DHFR inhibition data[2]
- 5-HT₃ functional assay: HEK293 cells transfected with 5-HT₃A receptors were loaded with Fura-2 AM. Intracellular calcium flux was measured upon 5-HT stimulation (10 μM) in the presence of Proguanil (0.1–10 μM). Antagonism was confirmed by rightward shift in 5-HT concentration-response curve[3]

Rats: Proguanil (2.9 mg/kg body weight) is given daily to groups of ten to twelve-week-old rats for five days and six weeks, respectively. Following that, weights of the body and reproductive organs are recorded, sperm parameters are examined, and testicular and epididymal histology is performed. Moreover, serum concentrations of follicle stimulating hormone, luteinizing hormone, and testosterone are measured[4].

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- Malaria prophylaxis study: C57BL/6 mice were infected with P. berghei via intraperitoneal injection. Proguanil was administered orally (10 mg/kg/day) starting 2 days prior to infection and continuing for 7 days. Parasitemia was monitored by blood smears, and survival was recorded[1]
- Reproductive toxicity study: Male Sprague-Dawley rats received Proguanil (0, 25, 50, 100 mg/kg/day) via oral gavage for 90 days. Testicular weight, sperm count/motility, and histopathology were evaluated. Significant dose-dependent decreases in sperm parameters were observed at ≥50 mg/kg[4]
- Babesia gibsoni treatment: Infected dogs received Proguanil (5 mg/kg) and atovaquone (13.3 mg/kg) orally twice daily for 7 days. Blood samples were collected daily for parasitemia quantification and hematology[5]

Absorption, Distribution and Excretion
Following oral doses of 50 to 500 mg, guanidine is rapidly and well absorbed in the human body. Metabolism/Metabolites It is metabolized in the liver by cytochrome P450 isoenzymes to the active triazine metabolite cyclic guanidine, but the degree of metabolism varies. This metabolic variation in guanidine may have important clinical significance for populations with slower metabolism (e.g., Asian and African populations susceptible to malaria). For these populations, prophylaxis with guanidine may be ineffective because even with multiple doses, they may not achieve sufficient therapeutic concentrations of the active compound cyclic guanidine. Known metabolites of guanidine include cyclic guanidine and 4-chlorophenylbiguanidine. Biological Half-Life Approximately 20 hours. Absorption: After oral administration of guanidine (100 mg), the time to peak concentration (Tmax) in the human body is 2–4 hours, with a bioavailability of 70–80%. Food can increase Cmax by 30%, but does not affect AUC[1]
- Metabolism: In the liver, it is mainly metabolized by CYP2C19 and CYP3A4 to cyclic guanidine (the main active metabolite) and other inactive conjugates. The plasma half-life of guanidine is 14–16 hours, while that of cyclic guanidine is 16–20 hours[1]
- Excretion: Approximately 60% of the dose is excreted in the urine as metabolites, and 30% is excreted in the feces. Less than 5% of the original drug is detected in the urine[1]

Hepatotoxicity
Atorvaquinone and guanidine combination therapy has been associated with transient and mild elevations in serum transaminases in a small number of patients. More importantly, there have been a few reports of specific acute liver injury in patients taking atorvaquinone/guanidine, but the number of cases is too small to determine a typical clinical course. In one reported case, the injury occurred 3 weeks after administration, presenting as fatigue, jaundice, and cholestatic elevations in serum enzymes. The injury resolved within 2 months of discontinuation (Case 1). In another case report of chloroquine and guanidine, liver injury occurred within days of initiation of combination therapy, with a mixed pattern of elevated serum enzymes. In both cases, allergic symptoms were mild, and no autoantibodies were detected. Both cases involved combination therapy, and either drug could have been a cause of the injury. Atorvaquinone and guanidine have also been associated with rare cases of Stevens-Johnson syndrome, which is typically accompanied by mild liver injury or elevated liver enzymes. Probability Score: E (Unproven, but sometimes suspected as a cause of clinically apparent liver injury).

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Protein binding
Approximately 75% - Acute toxicity: LD₅₀ >2000 mg/kg (oral) in rats. Clinical symptoms included sedation and gastrointestinal disturbances [1] - Reproductive toxicity: In male rats, guanidine (50 mg/kg/day) caused testicular atrophy, decreased spermatogenesis, and increased abnormal sperm morphology after 90 days. These effects were reversible after 4 weeks of washout [4] - Hematologic effects: In vitro human lymphocyte studies showed that guanidine (520 ng/mL) induced dose-dependent DNA damage (40% increase in comet tail moment) without affecting cell viability. Metabolic activation of the S9 mixture enhanced its genotoxicity [8]

[1]. Atovaquone and proguanil hydrochloride: a review of nonclinical studies. J Travel Med. 1999 May;6 Suppl 1:S8-12.

[2]. A mechanism for the synergistic antimalarial action of atovaquone and proguanil. Antimicrob Agents Chemother. 1999 Jun;43(6):1334-9.

[3]. The antimalarial drug proguanil is an antagonist at 5-HT3 receptors. J Pharmacol Exp Ther. 2014 Dec;351(3):674-84.

[4]. Prolonged administration of proguanil induces reproductive toxicity in male rats. J Toxicol Sci. 2011 Oct;36(5):587-99.

[5]. The in vitro interactions and in vivo efficacy of atovaquone and proguanil against Babesia gibsoni infection in dogs. Vet Parasitol. 2013 Nov 8;197(3-4):527-33.

Proguanil is a biguanide compound with an isopropyl group and a p-chlorophenyl substituent at its terminal nitrogen atom. As a prophylactic antimalarial drug, it works by inhibiting dihydrofolate reductase, an enzyme involved in the reproduction of Plasmodium parasites (Plasmodium falciparum and Plasmodium vivax) within red blood cells. Proguanil has a dual action: antimalarial, antiprotozoal, and an EC 1.5.1.3 (dihydrofolate reductase) inhibitor. It belongs to the biguanide and monochlorobenzene classes.
Proguanil is a prophylactic antimalarial drug whose mechanism of action is to prevent the reproduction of Plasmodium parasites (Plasmodium falciparum and Plasmodium vivax) within red blood cells. It achieves this effect by inhibiting dihydrofolate reductase, an enzyme involved in the reproduction of Plasmodium.
Proguanil is an antimalarial drug. The mechanism of action of Proguanil is as a dihydrofolate reductase inhibitor.
Proguanil is a biguanide derivative, effective against various protozoa, and is often used in combination with atovaquinone and chloroquine for the prevention and treatment of malaria. Proguanil as a single agent has not been extensively evaluated, but its combination with atovaquinone or chloroquine has been used to treat malaria and has been associated with elevated serum enzymes during treatment and rare cases of clinically significant acute liver injury. Proguanil is a biguanide compound that metabolizes in the body to cyclic guanidine, an antimalarial drug. Indications: For the prevention and suppression of malaria caused by susceptible strains of Plasmodium falciparum and other Plasmodium species found in certain parts of the world. Mechanism of Action: Proguanil inhibits dihydrofolate reductase in Plasmodium, thereby blocking the biosynthesis of purines and pyrimidines, which are essential for DNA synthesis and cell proliferation. This leads to the failure of nuclear division during schizont formation in erythrocytes and the liver. Pharmacodynamics: Proguanil is a biguanide derivative that is converted into its active metabolite, cyclic guanidine. It exerts its antimalarial effect by inhibiting parasite dihydrofolate reductase. It has pathogenicity prevention and inhibition effects against Plasmodium falciparum and can cure acute infections. It can also effectively suppress clinical attacks of vivax malaria. However, its action is slower compared to 4-aminoquinoline drugs.

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- Mechanism of action: Proguanil is a prodrug that can be converted to cyclic guanidine, which inhibits dihydrofolate reductase (DHFR), thereby blocking folate metabolism. Synergistic effect with atovaquinone involves dual inhibition of the mitochondrial electron transport chain (atorvaquinone) and dihydrofolate reductase (DHFR, cyclic guanidine) [2]
- Resistance mechanism: Point mutations in the DHFR of Plasmodium falciparum (e.g., C59R, S108N) confer resistance to Proguanil. Cross-resistance with pyrimethamine is common [2]
- FDA label: Approved for use in combination with atovaquinone for malaria prevention. Due to the risk of hemolysis, it is contraindicated in patients with G6PD deficiency[1]
- Off-label use: It has been studied for the treatment of toxoplasmosis and babesiosis, usually in combination with atovaquine[5]

C11H16CLN5

253.73

253.109

C, 52.07; H, 6.36; Cl, 13.97; N, 27.60

500-92-5

Proguanil-d6;Proguanil hydrochloride;637-32-1;Proguanil-d4;1189805-15-9

6178111

White to off-white solid powder

1.29g/cm3

402.7ºC at 760mmHg

129°

197.4ºC

1.6110 (estimate)

3.263

3

1

4

17

292

0

ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])/C(/N([H])[H])=N/C(/N([H])[H])=N/C([H])(C([H])([H])[H])C([H])([H])[H]

SSOLNOMRVKKSON-UHFFFAOYSA-N

InChI=1S/C11H16ClN5/c1-7(2)15-10(13)17-11(14)16-9-5-3-8(12)4-6-9/h3-7H,1-2H3,(H5,13,14,15,16,17)

Biguanide, 1-(p-chlorophenyl)-5-isopropyl-

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 : 51~130 mg/mL ( 201.0~512.36 mM )
Ethanol : ~51 mg/mL

Solubility in Formulation 1: ≥ 2.17 mg/mL (8.55 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 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.

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Solubility in Formulation 2: ≥ 2.17 mg/mL (8.55 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.17 mg/mL (8.55 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.17 mg/mL (8.55 mM)

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