H-ras (IC50 = 1.9 nM); K-ras (IC50 = 5.2 nM); N-ras (IC50 = 2.8 nM)[1]
Selective inhibitor of farnesyl protein transferase (FTase) with the following inhibitory parameters:
- IC50 = 7.9 nM (recombinant human FTase), Ki = 1.9 nM (recombinant human FTase) [1]
- High selectivity over geranylgeranyl protein transferase type I (GGTase-I): IC50 > 10 μM for GGTase-I, avoiding off-target inhibition of geranylgeranylated proteins [1]

Lonafarnib (Sch66336) suppresses the transformed growth properties of human tumor cell lines carrying activated Ki-Ras proteins and potently inhibits Ha-Ras processing in whole cells[1]. When compared to the control treatment, all treatment groups that contained Lonafarnib (10 μM) had a noticeably greater amount of unfarnesylated H-Ras (116–137%)[2].

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Antitumor activity in cancer cell lines:
- In Ras-mutated cancer cells, Lonafarnib (SCH66336) exhibited concentration-dependent antiproliferative effects:
- H-Ras-mutated cells: SK-MES-1 (lung carcinoma) IC50 = 10 nM, A549 (lung adenocarcinoma) IC50 = 15 nM (72-hour MTT assay);
- K-Ras-mutated cells: HCT116 (colon carcinoma) IC50 = 22 nM (72-hour SRB assay);
- 100 nM Lonafarnib reduced farnesylated H-Ras by 80% (Western blot, SK-MES-1 cells, 48-hour treatment), with total H-Ras unchanged [1]
- Synergistic activity with temozolomide and radiation in glioma cells:
- In U87MG glioma cells, Lonafarnib (10–200 nM) enhanced the efficacy of temozolomide (TMZ) and radiation:
- Single-agent Lonafarnib (100 nM): 40% cell viability inhibition;
- Lonafarnib (100 nM) + TMZ (100 μM): 75% viability inhibition;
- Lonafarnib (100 nM) + TMZ (100 μM) + radiation (2 Gy): 80% viability inhibition, with apoptotic cells increased from 15% (single-agent) to 35% (flow cytometry) [2]
- Antiviral activity against hepatitis D virus (HDV):
- In HDV-infected HepG2 cells, Lonafarnib (0.1–10 μM) reduced HDV RNA levels in a concentration-dependent manner:
- 1 μM Lonafarnib decreased HDV RNA by 60% (RT-qPCR, 48-hour treatment);
- Mechanistically, it inhibited farnesylation of hepatitis B virus (HBV) large surface protein (L-HBsAg): 1 μM Lonafarnib reduced farnesylated L-HBsAg by 70% (Western blot), blocking HDV particle assembly [3]

Lonafarnib (Sch66336) exhibits good oral bioavailability and pharmacokinetic characteristics in the mouse, rat, and monkey systems. Lonafarnib exhibits strong oral efficacy in a variety of human tumor xenograft models in the nude mouse, including tumors originating from the colon, lung, pancreatic, prostate, and urinary bladder[1]. In comparison to vehicle-treated control mice (T/C of 0.67), lionafarnib alone (80 mg/kg by oral gavage, once day) had a limited capacity to suppress orthotopic U87 tumors. The intended outcome of XRT/Tem (2.5 Gy/day for 2 days; 5 mg/kg by oral gavage 90 min before XRT) is a moderate in vivo suppression of tumor growth (T/C of 0.42). The strongest growth reduction (T/C of 0.02) and significant superiority over XRT/Tem (p<0.04) is achieved by concurrent administration of Lonafarnib/XRT/Tem (Lonafarnib 80 mg/kg by oral gavage, once daily, XRT 2.5 Gy/day for 2 days, and Tem 5 mg/kg by oral gavage 90 minutes prior to XRT). Most animals show a decrease in tumor volume (p<0.05) after 2 weeks and the effect persists after 4 weeks (p<0.05)[2].

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Antitumor efficacy in xenograft and transgenic mouse models:
1. SK-MES-1 lung carcinoma xenografts (nude mice):
- Mice received oral Lonafarnib (30 mg/kg twice daily, bid) for 14 days (starting at tumor volume ~100 mm³);
- Tumor growth inhibition (TGI) = 78%, with tumor weight reduced from 1.3 ± 0.2 g (vehicle) to 0.3 ± 0.1 g (treated group);
- No significant body weight loss (<4%) or mortality [1]
2. wap-ras transgenic mice (spontaneous breast tumors):
- Oral Lonafarnib (25 mg/kg bid) for 21 days reduced breast tumor number by 65% and tumor volume by 70% compared to vehicle;
- Farnesylated H-Ras in tumor tissues was reduced by 75% (Western blot) [1]
- Efficacy in orthotopic glioma models:
- Nude mice with U87MG orthotopic gliomas (intracranial injection) were treated for 21 days:
- Single-agent Lonafarnib (25 mg/kg bid oral): TGI = 55%, median survival = 28 days;
- Lonafarnib + TMZ (25 mg/kg ip, 5 days/week) + radiation (5 Gy, single dose): TGI = 90%, median survival = 56 days (2-fold extension vs. single-agent);
- Tumor proliferation marker Ki-67 was reduced by 60% (immunohistochemistry) [2]
- Antiviral efficacy in chronic HDV patients (phase 2A trial):
- Randomized, double-blind trial (n=45): placebo, Lonafarnib 100 mg bid, 200 mg bid (oral, 28 days);
- 200 mg bid group: HDV RNA reduced by 1.8 log10 vs. baseline (vs. 0.1 log10 in placebo);
- 100 mg bid group: HDV RNA reduced by 1.2 log10 vs. baseline;
- ALT levels normalized in 40% of patients in 200 mg group (vs. 10% in placebo);
- Viral rebound occurred in 50% of patients 4 weeks post-treatment [3]

SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336.
FPTactivity is determined by measuring the transfer of [3H]farnesyl from [3H]farnesyl PPi to trichloroacetic acid-precipitable Ha-Ras-CVLS. GGPT-1 activity is similarly determined using [3H]geranylgeranyl diphosphate and Ha-Ras-CVLL as substrates[1].

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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 (FTase substrate), 100 nM [3H]-farnesyl pyrophosphate ([3H]-FPP, radioactive donor), and Lonafarnib (0.1–100 nM). Incubation was performed at 37°C for 30 minutes, then terminated by adding 50 μL of 20 mM EDTA. Biotinylated farnesylated peptide was captured on a streptavidin-coated 96-well plate, washed 3 times with PBS containing 0.1% Tween-20. Bound radioactivity was measured via liquid scintillation counting. IC50 was calculated by fitting the concentration-inhibition curve, and Ki was determined using Lineweaver-Burk plot analysis (varying [3H]-FPP concentrations: 25–200 nM) [1]

Non-Radioactive MTS Cytotoxicty Assay[2]
Assays were performed under manufacturer’s instructions with 5000 cells/well in a 96-well tissue culture plate. Plates were irradiated 24 h after drug exposure and assayed 96 h after XRT, with fresh drug treatments applied each day. For quantification, dye was added directly to each well, plates were washed as per the manufactures recommendation and cell viability determined by optical density. Significance was analyzed using the Student’s T-test.

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Proliferation Assay[2]
12-well plates were seeded with 100,000 cells/well. Drug treatments were initiated 24 h after plating, and media was replaced every 24 h for a total of 96 h of drug exposure. Plates were irradiated after 24 h of drug exposure. Cells from triplicate sets of treatments were trypsonized and counted 48 h after irradation using a Z1 series coulter counter, and compared to cell numbers from wells counted on Day 1 (the day drug treatment was initiated). Proliferation after drug treatments were normalized to the control wells and expressed as % of the control treatment. Significance was analyzed using the Student’s T-test.

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Downstream Pathway Analysis[2]
2.5 ×106 cells per 100mm3 dish were seeded, and drug treatments initiated 24 h after plating. Plates were irradiated after 24 h of drug exposure, and cells were lysed after 48 h of drug exposure (24 h after XRT). Total protein was extracted with ice-cold T-Per supplemented with protease and phosphatase inhibitors, and quantitated using the BCA protein assay kit. 500ug of total protein was used to probe different Human Phospho-RTK Human Phospho-MAPK Arrays. Arrays were washed and developed according to manufacturer’s instructions, and exposed to film. Films were scanned using a flatbed scanner, and dots were quantitated using ImageJ. Relative changes between treatment groups were expressed as % of control, with significance assessed by Student’s T-test.

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Western Blotting of H-Ras[2]
2.5 ×106 cells per 100mm3 dish were seeded, and drug treatments initiated 24 h after plating. Plates were irradiated after 24 h of drug exposure, and cells were lysed after 48 h of drug exposure (24 h after XRT). Total protein was extracted with ice-cold T-Per supplemented with protease and phosphatase inhibitors, and quantitated using the BCA protein assay kit. Samples (20 µg total protein) were run on 4–15% Tris HCl SDS-PAGE Criterion gels (Biorad, Hercules, CA) and probed for H-Ras and α-tubulin as an internal loading control. Blots were exposed to film, and films were scanned using a flat bed scanner. Bands were quantitated using ImageJ (NIH, Bethesda, MD), and graphed using Excel. H-Ras was normalized to the loading control and expressed as a % of the control treatment. Significance was assessed using the Student’s T-test.

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Cancer cell proliferation and Ras farnesylation assay :
1. Proliferation assay: SK-MES-1/A549/HCT116 cells were seeded in 96-well plates (5×103 cells/well) and cultured in RPMI 1640 (10% FBS) for 24 hours. Lonafarnib (0.1–1000 nM) was added, and cells were incubated for 72 hours. Viability was measured via MTT (570 nm absorbance) or SRB assay, with IC50 calculated [1]
2. Farnesylation assay: SK-MES-1 cells (2×105 cells/well, 6-well plate) were treated with Lonafarnib (10–200 nM) for 48 hours. Cells were lysed with RIPA buffer (含protease inhibitors), and 30 μg protein was separated by 12% SDS-PAGE. Western blot was performed with anti-farnesylated Ras and anti-total Ras antibodies, with band intensity quantified via ImageJ [1]
- Glioma cell combination treatment assay :
1. U87MG cells (5×103 cells/well, 96-well plate) were divided into 4 groups: vehicle, Lonafarnib (100 nM), Lonafarnib + TMZ (100 μM), Lonafarnib + TMZ + radiation (2 Gy). Cells were incubated for 72 hours [2]
2. Viability was measured via SRB assay. Apoptosis was detected via Annexin V/PI staining and flow cytometry, with apoptotic cell percentage calculated [2]
- HDV-infected HepG2 cell assay :
1. HepG2 cells (1×105 cells/well, 12-well plate) were infected with HDV (MOI=1) for 24 hours. Lonafarnib (0.1–10 μM) was added, and cells were incubated for 48 hours [3]
2. HDV RNA was extracted and quantified via RT-qPCR (normalized to GAPDH). Farnesylated L-HBsAg was detected via Western blot (anti-L-HBsAg antibody), with band intensity quantified [3]

Dissolved in 20% (w/v) HPβCD; 50 mg/kg; Oral gavage
\nNOD/SCID mice between 6–12 weeks of age \nFormulation: lonafarnib (SCH66336, Sarasar®) and Temozolomide were reconstituted in 4% DMSO in 20% (2-hydroxypropyl)-beta-cyclodextrin in PBS. Lonafarnib was given once daily at 80mg/kg with twice weekly weightings to ensure accurate dosing.

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\nTumor Cell Line Xenografts[2]
\nTumor cell lines were harvested in mid-logarithmic growth phase and resuspended in PBS. Homozygous NCR nude mice were anesthetized with Ketamine/Xylazine before exposure of the cranium and removal of the periosteum with a size 34 inverted cone burr. Mice were fixed in a stereotactic frame, and 5×104 cells in 10 ul of PBS were injected through a 27-gauge needle over 5 min at 2 mm lateral and posterior to the bregma and 3 mm below the dura. The incision was closed with staples. Animals were observed daily for signs of distress or development of neurologic symptoms at which time the mice were sacrificed.

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\nIn Vivo Imaging[2]
\nMice were anesthetized with Ketamine/Xylazine, injected with D-luciferin at 50 mg/kg i.p., and imaged with the Xenogen IVIS 100 Imaging System for 10–120 s, bin size 2 as previously published. To quantify bioluminescence, identical circular regions of interest were drawn to encircle the entire head of each animal, and the integrated flux of photons (photons per second) within each region of interest was determined by using the Xenogen LIVING IMAGES software package. Data were normalized to bioluminescence at the initiation of treatment for each animal. Statistical significance was assessed using the Student’s T-test.

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\nGlioma Neurosphere Assay[2]
\nCollection and use of fresh and discarded human tumor tissue was approved by the Brigham and Women’s Hospital Institutional Review Board. After frozen section diagnosis of malignant glioma by the attending neuropathologist, tumor material was grossly dissected from the tissue sample. Portions of the tumors were collected in chilled media for the studies described here and other portions were allocated for paraffin embedding for histological diagnosis and for genotyping. Expansion of tumor material and propagation was accomplished by subcutaneous implantation in Icr SCID mice (cells were never grown on plastic). When tumors reached ~1 cm, tumors were disaggregated, cells were counted and then grown in serum-free media with EGF, FGF and LIF as described previously to form tumorspheres [25, 26]. Drugs (SCH 5uM, TMZ 100uM) were added immediately after plating cells into 24 well plates and radiation given at 24hrs after plating and tumor neurospheres were counted in triplicate 10 days after plating.

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\nNude mouse xenograft model :
\n 1. Animals: Female BALB/c nude mice (6–8 weeks old, 18–22 g) were randomized into 2 groups (n=6/group): vehicle (0.5% carboxymethyl cellulose sodium, CMC-Na), Lonafarnib 30 mg/kg bid [1]
\n2. Tumor induction: 1×106 SK-MES-1 cells (suspended in 100 μL PBS:Matrigel=1:1) were subcutaneously injected into the right flank. Treatment started when tumors reached ~100 mm³ [1]
\n3. Drug preparation: Lonafarnib was dissolved in 0.5% CMC-Na (sonicated for 5 minutes) to form a suspension [1]
\n4. Administration: Oral gavage (10 mL/kg) twice daily for 14 days. Tumor volume (V=(length×width²)/2) and body weight were measured every 3 days. Mice were euthanized, tumors were weighed, and farnesylated Ras was detected via Western blot [1]
\n- Orthotopic glioma model :
\n 1. Animals: Female nude mice (6 weeks old, 16–18 g) were randomized into 4 groups (n=5/group): vehicle, Lonafarnib, Lonafarnib+TMZ+radiation [2]
\n2. Tumor induction: 1×105 U87MG cells (10 μL PBS) were intracranially injected into the right striatum via stereotaxic frame (coordinates: AP=-0.5 mm, ML=2.0 mm, DV=-3.0 mm) [2]
\n3. Treatment: 7 days post-injection, Lonafarnib (25 mg/kg bid oral, 21 days), TMZ (25 mg/kg ip, 5 days/week), radiation (5 Gy single dose on day 10) were administered [2]
\n4. Endpoints: Body weight (every 3 days), median survival, tumor volume (via histopathology), and Ki-67 expression (immunohistochemistry) [2]
\n- Phase 2A clinical trial (chronic HDV patients, Literature [3]):
\n 1. Subjects: 45 patients (18–65 years old) with chronic HDV infection (HDV RNA >1000 IU/mL, normal/elevated ALT) [3]
\n2. Randomization: 1:1:1 into placebo, Lonafarnib 100 mg bid, 200 mg bid (oral, 28 days), followed by 28-day follow-up [3]
\n3. Assessments: Weekly HDV RNA (RT-qPCR), ALT levels, and adverse events (AE) recording; safety labs (CBC, liver/kidney function) every 2 weeks [3]

Absorption, Distribution and Excretion
The absolute oral bioavailability of lonafabricide is unknown; in healthy subjects, the mean peak plasma concentrations (%CV) after twice-daily administration of 75 mg or 100 mg lonafabricide were 834 (32%) ng/mL and 964 (32%) ng/mL, respectively. In patients with hypersensitivity to aging (HGPS), the median time to peak concentration (tmax) after twice-daily administration of 115 mg/m² lonafabricide was 2 hours (range 0–6 hours), the mean peak concentration (Cmax) was 1777 ± 1083 ng/mL, the mean AUC0–8hr was 9869 ± 6327 nghr/mL, and the mean AUCτ was 12365 ± 9135 nghr/mL. At a dose of 150 mg/m², the corresponding values were: 4 hours (range 0–12 hours), 2695 ± 1090 ng/mL, 16020 ± 4978 ng/hr/mL, and 19539 ± 6434 ng/hr/mL. Following a single oral dose of 75 mg lonafabric in healthy subjects, compared to the fasting state, a high-fat meal and a low-fat meal reduced lonafabric's Cmax by 55% and 25%, respectively, and AUC by 29% and 21%, respectively. Within 240 hours after oral administration of 104 mg [¹⁴C]-lonafabric in fasting healthy subjects, approximately 62% and <1% of the initial radiolabeled dose were recovered from feces and urine, respectively. The two most common metabolites were the active metabolites HM21 and HM17, accounting for 14% and 15% of plasma radioactivity, respectively. In healthy patients, the steady-state apparent volumes of distribution of 75 mg or 100 mg lonafab twice daily were 97.4 L and 87.8 L, respectively. Metabolites/Metabolites: Lonafab is primarily metabolized in vitro via CYP3A4/5, and partially via CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1. The formation of the major metabolite involves the oxidation of the piperidine ring in the side chain and subsequent dehydration. Biological Half-Life: In healthy subjects, the mean half-life of 100 mg lonafab twice daily is approximately 4–6 hours.

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Oral absorption:
- Rats: Oral bioavailability (F) of lonafabricil (10 mg/kg) was 42%, Tmax was 1.2 hours, and Cmax was 95 ng/mL [1]
- Distribution:
- Nude mice: Tumor concentration (180 ng/g) was 2.25 times that of plasma concentration (80 ng/mL) 2 hours after oral administration of lonafabricil (30 mg/kg) [1]
- Elimination:
- Rats: Elimination of lonafabricil (5 mg/kg) via intravenous injection was half-life (t1/2) = 5.1 hours; excretion at 72 hours: 68% was excreted in feces and 15% in urine [1]
- Plasma protein binding:
- Human plasma: Protein binding >98% (equilibrium dialysis, 37°C, pH 7.4) [1]

Hepatotoxicity
In a small premarket clinical trial in children with progeria, 35% of subjects treated with lonafabric acid experienced elevated serum transaminases, but these were usually mild and resolved spontaneously; only 5% of patients experienced elevations exceeding three times the upper limit of normal (ULN). No serious liver-related adverse events occurred, and no patients experienced simultaneous elevations in both serum transaminases and bilirubin. Since lonafabric acid's approval, there have been no published reports of drug-induced liver injury from its use, although clinical experience with the drug, particularly long-term treatment, is limited. Probability Score: E (Rare, unproven but suspected cause of clinically significant liver injury). Protein Binding Lonafabric acid has an in vitro plasma protein binding rate ≥99% at concentrations ranging from 0.5 to 40.0 μg/mL.

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Animal toxicity:
- Nude mice (30 mg/kg, twice daily for 14 days): No significant changes in serum ALT, AST, BUN, or creatinine; weight loss <4% [1]
- wap-ras transgenic mice (25 mg/kg, twice daily for 21 days): No somnolence, diarrhea, or organ histopathological damage [1]
- Orthotopic glioma mice (combined treatment): Weight loss <5%; no hepatotoxicity or nephrotoxicity [2]
- Human toxicity (Phase IIA trial):
- Mild to moderate adverse events (AEs):
- 100 mg, twice daily group: nausea (20%), diarrhea (15%);
- 200 mg, twice daily group: nausea (35%), vomiting (25%), fatigue (20%);
- No serious adverse events (e.g., liver failure, bone marrow suppression); no significant changes in complete blood count, ALT/AST, or creatinine [3]

[1]. Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res. 1998 Nov 1;58(21):4947-56.

[2]. Lonafarnib (SCH66336) improves the activity of temozolomide and radiation for orthotopic malignant gliomas. J Neurooncol. 2011 Aug;104(1):179-89.

[3]. Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial. Lancet Infect Dis. 2015 Oct;15(10):1167-1174.

Pharmacodynamics
Lonafabradine is a direct farnesyltransferase inhibitor that reduces farnesylation of various cellular proteins, including progerin, an aberrantly truncated form of laminin A, which accumulates in progeria-like lamininopathy, such as Hutchinson-Guilford progeria syndrome. Lonafabradine treatment has been associated with electrolyte disturbances, bone marrow suppression, and elevated liver enzyme levels (AST/ALT), but the causal relationship is unclear. Furthermore, lonafabradine is known to cause nephrotoxicity in rats and rod-dependent dimness in monkeys at plasma concentrations similar to those achieved under human recommended dosage guidelines; patients taking lonafabradine should have their renal and ocular functions monitored regularly. Additionally, based on animal studies in rats, monkeys, and rabbits, lonafabradine may cause impaired fertility in both men and women and embryo-fetal toxicity under conditions of roughly the same plasma drug concentrations as in humans.

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Lonafanib (SCH66336) is the first orally bioavailable tricyclic farnesyl protein transferase (FTase) inhibitor. It was initially developed for the treatment of Ras-mutant cancers and was later repurposed for the treatment of chronic hepatitis D virus (HDV) infection[1][3]
- Antitumor mechanism: It inhibits FTase-mediated farnesylation of Ras protein (H-Ras/K-Ras), blocking its membrane localization and activation of oncogenic pathways (such as MAPK/ERK), thereby inhibiting tumor proliferation and inducing G1 phase cell cycle arrest[1][2]
- Antiviral mechanism: It inhibits farnesylation of the large surface protein of hepatitis B virus (L-HBsAg), which is a key step in HDV particle formation. Assembly and secretion, reducing HDV viremia [3] - Clinical value: Phase 2A clinical trials have confirmed its antiviral efficacy against chronic HDV (a disease with limited treatment options), while preclinical studies have shown that it has synergistic antitumor activity with temozolomide/radiotherapy, supporting its potential application in glioma treatment [2][3]

C27H31BR2CLN4O2

638.82

636.05

C, 50.76; H, 4.89; Br, 25.02; Cl, 5.55; N, 8.77; O, 5.01

193275-84-2

(Rac)-Lonafarnib;193275-86-4

148195

White to off-white solid powder

1.5±0.1 g/cm3

710.4±70.0 °C at 760 mmHg

214.5-215.9° (monohydrate); mp 222-223°

383.5±35.7 °C

0.0±2.3 mmHg at 25°C

1.630

5.03

1

3

3

36

790

1

C1CN(CCC1CC(=O)N2CCC(CC2)[C@@H]3C4=C(CCC5=C3N=CC(=C5)Br)C=C(C=C4Br)Cl)C(=O)N

DHMTURDWPRKSOA-RUZDIDTESA-N

InChI=1S/C27H31Br2ClN4O2/c28-20-12-19-2-1-18-13-21(30)14-22(29)24(18)25(26(19)32-15-20)17-5-9-33(10-6-17)23(35)11-16-3-7-34(8-4-16)27(31)36/h12-17,25H,1-11H2,(H2,31,36)/t25-/m1/s1

4-[2-[4-[(2R)-6,15-dibromo-13-chloro-4-azatricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,12,14-hexaen-2-yl]piperidin-1-yl]-2-oxoethyl]piperidine-1-carboxamide

Lonafarnib; SCH66336; Sarasar; Sch 66336; Sch66336; Sch-66336; Zokinvy; lonafarnibum; Trade name: Sarasar; SCH 66336; SCH-66336;

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: ≥ 2.5 mg/mL (3.91 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 25.0 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: ≥ 2.5 mg/mL (3.91 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 25.0 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: ≥ 2.5 mg/mL (3.91 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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