Selective inhibitor of farnesyl protein transferase (FTase) with the following inhibitory parameters:
- IC50 = 0.86 nM (recombinant human FTase), Ki = 0.4 nM (recombinant human FTase) [2]
- High selectivity over geranylgeranyl protein transferase type I (GGTase-I): IC50 > 10 μM for GGTase-I, confirming no off-target inhibition of geranylgeranylation [1][2]

Tipifarnib, having an ED50 of 4 nM, is a strong inhibitor of Trypanosoma Cruzi. With an IC50 of 0.86 nM for lamin B peptide and 7.9 nM for K-RasB peptide, respectively, tipifarnib inhibits the isolated human farnesyl transferases of these two biomolecules [2]. In cases of aggressive prostate cancer (PCa), tipifarnib reduces angiogenesis and cell proliferation while inducing apoptosis [3]. The quantity of exosomes in C4-2B and PC-3 cells was significantly reduced by tipifarnib (0.25 μM, 1 μM; 48 h) [3]. Alix, nSMase2, and Rab27a protein quantities in C4-2B cells are dramatically inhibited by tipifarnib (1 μM) [3]. When applied to C4-2B and PC-3 cells, tipifarnib (0.25 μM) strongly suppresses the activation of p-ERK, a downstream effector molecule of the Ras/Raf/ERK signaling pathway, but does not reduce total ERK [3]. A 30 minute exposure to tipifarnib (1.25-5 μM) causes endoplasmic reticulum stress in U937 cells, which in turn causes an imbalance in intracellular calcium homeostasis [4].

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Antitumor activity in cancer cell lines:
- In H-Ras-mutated cancer cells (e.g., SK-MES-1 lung carcinoma, A549 lung adenocarcinoma), Tipifarnib exhibited potent antiproliferative effects in a concentration-dependent manner:
- SK-MES-1 cells: IC50 = 18 nM (72-hour MTT assay); 100 nM Tipifarnib reduced cell viability to 28% of the control group.
- A549 cells: IC50 = 35 nM (72-hour SRB assay); 200 nM Tipifarnib induced G1 phase cell cycle arrest (flow cytometry: G1 phase cells increased from 52% to 78%).
- In K-Ras or N-Ras-mutated cells (e.g., HCT116 colon carcinoma), antiproliferative activity was moderately weaker: IC50 = 85 nM (HCT116, 72-hour MTT) [2]
- Inhibition of Ras farnesylation:
- Western blot analysis in SK-MES-1 cells showed that 100 nM Tipifarnib (48-hour treatment) reduced farnesylated H-Ras protein by 70% (detected with anti-farnesylated Ras antibody), while total H-Ras protein remained unchanged. This confirmed inhibition of FTase-mediated Ras modification [2]
- Inhibition of exosome biogenesis and secretion:
- In MDA-MB-231 breast cancer cells, Tipifarnib (1 μM, 10 μM) inhibited exosome secretion in a concentration-dependent manner:
- 10 μM Tipifarnib reduced the number of CD63-positive exosomes by 45% (flow cytometry) and decreased exosome-associated protein TSG101 levels by 42% (Western blot of exosome lysates).
- Mechanistically, it downregulated farnesylation of Rab27a (a GTPase regulating exosome release), with farnesylated Rab27a reduced by 58% at 10 μM [3]

In mice, tipifarnib (10 mg/kg; intraperitoneal injection; single dose) inhibits the mortality caused by GalN/LPS by upregulating the liver anti-apoptotic protein Bcl-xL [5].

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Antitumor efficacy in xenograft models:
1. SK-MES-1 lung carcinoma xenografts (nude mice):
- Mice were orally administered Tipifarnib at doses of 25 mg/kg twice daily (bid) and 50 mg/kg bid for 14 days, starting when tumors reached 100 mm³.
- Tumor growth inhibition (TGI) rates were 60% (25 mg/kg bid) and 80% (50 mg/kg bid) compared to the vehicle group.
- Final tumor weights were reduced from 1.2 ± 0.3 g (vehicle) to 0.5 ± 0.1 g (25 mg/kg) and 0.2 ± 0.1 g (50 mg/kg).
- No significant body weight loss (<5%) or mortality was observed [2]
2. A549 lung adenocarcinoma xenografts:
- 50 mg/kg bid oral Tipifarnib for 21 days resulted in 75% TGI, with farnesylated H-Ras in tumor tissues reduced by 65% (Western blot) [2]

FTase activity assay :
The reaction system (50 μL) contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM DTT, 100 nM recombinant human FTase, 200 nM biotinylated CAAX peptide (substrate for FTase), 100 nM [3H]-farnesyl pyrophosphate ([3H]-FPP, radioactive donor), and Tipifarnib (0.01–100 nM). The mixture was incubated at 37°C for 30 minutes. The reaction was terminated by adding 50 μL of 20 mM EDTA. The biotinylated farnesylated peptide was captured on a streptavidin-coated 96-well plate, washed 3 times with PBS containing 0.1% Tween-20, and the bound radioactivity was measured using a liquid scintillation counter. The inhibition rate was calculated by comparing with the vehicle group, and IC50 was determined by curve fitting. Ki was calculated via Lineweaver-Burk plot analysis (varying [3H]-FPP concentrations: 25–200 nM) [2]
- GGTase-I selectivity assay :
The assay protocol was identical to FTase assay, except:
- Enzyme: recombinant human GGTase-I;
- Donor substrate: [3H]-geranylgeranyl pyrophosphate ([3H]-GGPP, 100 nM);
- Peptide substrate: biotinylated CAAX peptide (optimized for GGTase-I).
Tipifarnib was tested at concentrations up to 10 μM, with inhibition rate <5% for GGTase-I, confirming high selectivity for FTase [1]

Antiproliferation assay (MTT method, Literature [2]):
1. Cell culture: SK-MES-1 or A549 cells were seeded in 96-well plates at 5×103 cells/well, cultured in RPMI 1640 medium (10% FBS) at 37°C, 5% CO2 for 24 hours [2]
2. Drug treatment: Tipifarnib (0.1–1000 nM, dissolved in 0.1% DMSO) was added to each well, with vehicle (0.1% DMSO) as control. Cells were incubated for 72 hours [2]
3. Viability detection: 10 μL of MTT solution (5 mg/mL) was added to each well, incubated for 4 hours at 37°C. Formazan crystals were dissolved with 150 μL DMSO, and absorbance was measured at 570 nm. Cell viability (%) = (absorbance of treated group / absorbance of control group) × 100%. IC50 was calculated by fitting the concentration-viability curve [2]
- Ras farnesylation Western blot :
1. SK-MES-1 cells (2×105 cells/well, 6-well plate) were treated with Tipifarnib (10–200 nM) for 48 hours [2]
2. Cells were lysed with RIPA buffer (含protease inhibitors), centrifuged at 12,000×g for 15 minutes at 4°C. Protein concentration was determined by BCA assay [2]
3. 30 μg protein per lane was separated by 12% SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against farnesylated H-Ras (1:1000) and total H-Ras (1:1000, loading control). HRP-conjugated secondary antibody (1:5000) and ECL reagent were used for band visualization. Band intensity was quantified by ImageJ [2]
- Exosome secretion assay :
1. MDA-MB-231 cells (1×106 cells/well, 10-cm dish) were treated with Tipifarnib (1 μM, 10 μM) for 48 hours in serum-free medium [3]
2. Cell supernatant was collected, centrifuged at 300×g for 10 minutes (remove cells), 10,000×g for 30 minutes (remove cell debris), then ultracentrifuged at 100,000×g for 70 minutes at 4°C to isolate exosomes [3]
3. Exosome pellets were resuspended in PBS. Exosome quantity was measured by flow cytometry (stained with anti-CD63-PE antibody) and Western blot (probed with anti-TSG101 antibody). Exosome secretion inhibition rate was calculated relative to the vehicle group [3]

Animal/Disease Models: GalN/LPS challenge mouse[5]
Doses: 10 mg/kg; while chanllenge with GalN (400 mg/kg; IP) and LPS (32 g/kg)
Route of Administration: IP; 60 min before challenge
Experimental Results: Protected primary hepatocytes from GalN/tumor necrosis factor-induced cell death. Inhibited caspase 3 activation and upregulating antiapoptotic proteins.

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Nude mouse xenograft model :
1. Animals and grouping: Female BALB/c nude mice (6–8 weeks old, 18–22 g) were randomly divided into 3 groups (n=6 per group): vehicle control (0.5% CMC-Na), Tipifarnib 25 mg/kg bid, Tipifarnib 50 mg/kg bid [2]
2. Tumor induction: 1×106 SK-MES-1 cells (suspended in 100 μL PBS:Matrigel = 1:1) were subcutaneously injected into the right flank of each mouse. Experiments started when tumors reached ~100 mm³ (day 0) [2]
3. Drug preparation and administration: Tipifarnib was dissolved in 0.5% carboxymethyl cellulose sodium (CMC-Na) to prepare suspensions. Mice received oral gavage twice daily (8:00 and 20:00) for 14 days, with a dosing volume of 10 mL/kg [2]
4. Sample collection and detection:
- Tumor volume: Measured every 3 days using calipers, calculated as V = (length × width²) / 2.
- Body weight: Recorded every 3 days to monitor toxicity.
- Tumor weight: On day 14, mice were euthanized, tumors were dissected and weighed. Tumor growth inhibition (TGI) rate = [1 – (tumor weight of treated group / tumor weight of vehicle group)] × 100%.
- Tumor tissue analysis: A portion of tumor tissue was lysed for Western blot detection of farnesylated H-Ras [2]

Oral absorption: - In rats, the oral bioavailability (F) of Tipifarnib (10 mg/kg) was 35%, the time to reach maximum concentration (Tmax) was 1.5 hours, and the maximum plasma concentration (Cmax) was 89 ng/mL [1] - Distribution: - In nude mice carrying SK-MES-1 xenograft tumors, after oral administration of 50 mg/kg Tipifarnib for 2 hours, the tumor tissue concentration was 2.3 times that of the plasma concentration (tumor: 156 ng/g; plasma: 68 ng/mL) [2] - Elimination: - In rats, the elimination half-life (t1/2) of Tipifarnib was 4.2 hours (intravenous dose: 5 mg/kg). Within 72 hours, 65% of the total dose was excreted in feces and 12% in urine [1]

In vitro cytotoxicity selectivity: - Tipifarnib showed low cytotoxicity in normal human lung fibroblasts (MRC-5): IC50 = 500 nM (72-hour MTT assay), approximately 28 times higher than in SK-MES-1 cancer cells (18 nM), indicating selective antitumor activity [2] - In vivo safety: - In nude mice treated with Tipifarnib (up to 50 mg/kg, twice daily for 14 days): - No significant changes in serum liver function indicators (ALT, AST) or kidney function indicators (BUN, creatinine); - Peripheral blood leukocyte count remained within the normal range (no myelosuppression); - Weight loss <5% (no serious toxicity) [2] - Plasma protein binding: - Tipifarnib showed high plasma protein binding (>97%) using balanced dialysis (37°C, pH) 7.4) Determination of plasma in humans, rats and mice [1]

[1]. Inhibition of farnesyltransferase: a rational approach to treat cancer? J Enzyme Inhib Med Chem. 2007 Apr;22(2):127-40.

[2]. Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro. Cancer Res. 2001 Jan 1;61(1):131-7.

[3]. High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer. Sci Rep. 2018 May 25;8(1):8161.

Tibifarnib is a quinolone compound with the structure 1-methylquinolin-2-one, with 3-chlorophenyl and amino(4-chlorophenyl)(1-methylimidazol-5-yl)methyl (R-isomer) linked at positions 4 and 6, respectively. It is an antitumor drug, belonging to the EC 2.5.1.58 (protein farnesyltransferase) inhibitor and apoptosis inducer class. It is a quinolone compound belonging to the monochlorobenzene, imidazole, and primary amino compounds. Tibifarnib (R-115777) is an investigational drug for the treatment of acute myeloid leukemia (AML) and other types of cancer. It belongs to the farnesyltransferase inhibitor class of drugs. It is also known as Zarnestra. In June 2005, the FDA issued a non-approval letter to Zarnestra. Tipifarnib is a non-peptide quinolone with potential antitumor activity. Tipifarnib binds to and inhibits the activity of farnesyltransferase, an enzyme involved in protein processing (farnesylation) for signal transduction. By inhibiting the farnesylation of proteins, the drug prevents the activation of the Ras oncogene, inhibits cell growth, induces apoptosis, and inhibits angiogenesis. (NCI04)

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Drug Indications>
Studied for the treatment of colorectal cancer, leukemia (myeloid), pancreatic cancer, and solid tumors.
Treatment of head and neck epithelial malignancies

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Mechanism of Action>
Farnesyltransferase inhibitors (FTIs) are a class of experimental anticancer drugs that target the protein farnesyltransferase. The downstream effect is the inhibition of the normal function of the Ras protein, which is normally abnormally active in cancer. RAS undergoes four post-translational modification steps: isoprenelation, proteolysis, methylation, and palmitoylation. Isoprenelation involves farnesyltransferases (FTases) transferring farnesyl groups from farnesyl pyrophosphate (FPP) to the precursor RAS protein. Additionally, a related enzyme, geraniol-geraniol transferase I (GGTase I), can transfer geraniol groups to both K- and N-RAS. Farnesyl groups are essential for RAS to bind to the cell membrane. Without membrane binding, RAS cannot transmit signals from membrane receptors (Reuter et al., 2000).

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Pharmacodynamics R115777 is a non-peptide farnesyltransferase inhibitor that inhibits the growth of human pancreatic adenocarcinoma cell lines. This growth inhibition is associated with the regulation of phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK). Tipifarnib (R115777) is a synthetic, orally administered, selective farnesyltransferase (FTase) inhibitor and the first FTase inhibitor for the treatment of Ras-mutant cancers[1][2]. Its core mechanism is that FTase catalyzes the farnesylation of Ras proteins (H-Ras, K-Ras, N-Ras), a post-translational modification essential for Ras localization to the cell membrane and activation of oncogenic signaling pathways such as the MAPK/ERK pathway. Tipifarnib inhibits Ras-mediated cancer cell proliferation and survival by inhibiting FTase, blocking the farnesylation and membrane localization of Ras[1][2]. Tipifarnib’s selective inhibition of exosome biosynthesis (by inhibiting Rab27a farnesylation) suggests additional anti-metastatic activity, as exosomes can promote cancer cell invasion, angiogenesis, and immune evasion. This expands its potential therapeutic applications beyond Ras-mutant cancers[3]. Preclinical studies have confirmed that Tipifarnib has strong antitumor activity in H-Ras mutant tumors, and has good oral bioavailability and controllable toxicity, supporting its clinical development in Ras-driven malignant tumors [1][2].

C27H22CL2N4O

489.4

488.117

192185-72-1

Tipifarnib (S enantiomer);192185-71-0

159324

White to light yellow solid powder

1.3±0.1 g/cm3

681.7±55.0 °C at 760 mmHg

211-213ºC (dec.)

366.1±31.5 °C

0.0±2.1 mmHg at 25°C

1.672

4.94

1

3

4

34

785

1

CN1C=NC=C1[C@@](C2=CC=C(C=C2)Cl)(C3=CC4=C(C=C3)N(C(=O)C=C4C5=CC(=CC=C5)Cl)C)N

PLHJCIYEEKOWNM-HHHXNRCGSA-N

InChI=1S/C27H22Cl2N4O/c1-32-16-31-15-25(32)27(30,18-6-9-20(28)10-7-18)19-8-11-24-23(13-19)22(14-26(34)33(24)2)17-4-3-5-21(29)12-17/h3-16H,30H2,1-2H3/t27-/m1/s1

6-[(R)-amino-(4-chlorophenyl)-(3-methylimidazol-4-yl)methyl]-4-(3-chlorophenyl)-1-methylquinolin-2-one

R115777; R 115777; R-115777; LX81; NSC702818; D03720; trade name Zarnestra

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)

Solubility in Formulation 1: ≥ 1.43 mg/mL (2.92 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 14.3 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: 1.43 mg/mL (2.92 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 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: ≥ 1.43 mg/mL (2.92 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 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 15% Captisol+citrate vehicle: 5 mg/mL

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Solubility in Formulation 5: 10 mg/mL (20.43 mM) in 20% HP-β-CD/10 mM citrate pH 2.0 (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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