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
NOD/SCID mice between 6–12 weeks of age Formulation: 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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Tumor Cell Line Xenografts[2]
Tumor 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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In Vivo Imaging[2]
Mice 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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Glioma Neurosphere Assay[2]
Collection 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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Nude mouse xenograft model :
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]
2. 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]
3. Drug preparation: Lonafarnib was dissolved in 0.5% CMC-Na (sonicated for 5 minutes) to form a suspension [1]
4. 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]
- Orthotopic glioma model :
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]
2. 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]
3. 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]
4. Endpoints: Body weight (every 3 days), median survival, tumor volume (via histopathology), and Ki-67 expression (immunohistochemistry) [2]
- Phase 2A clinical trial (chronic HDV patients, Literature [3]):
1. Subjects: 45 patients (18–65 years old) with chronic HDV infection (HDV RNA >1000 IU/mL, normal/elevated ALT) [3]
2. Randomization: 1:1:1 into placebo, Lonafarnib 100 mg bid, 200 mg bid (oral, 28 days), followed by 28-day follow-up [3]
3. 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 lonafarnib is unknown; in healthy subjects administration of either 75 or 100 mg of lonafarnib twice daily resulted in mean peak plasma concentrations (%CV) of 834 (32%) and 964 (32%) ng/mL, respectively. Twice daily administration of 115 mg/m2 lonafarnib in HGPS patients resulted in a median tmax of 2 hours (range 0-6), mean Cmax of 1777 ± 1083 ng/mL, mean AUC0-8hr of 9869 ± 6327 ng\hr/mL, and a mean AUCtau of 12365 ± 9135 ng\hr/mL. The corresponding values for a dose of 150 mg/m2 are: 4 hours (range 0-12), 2695 ± 1090 ng/mL, 16020 ± 4978 ng\hr/mL, and 19539 ± 6434 ng\hr/mL, respectively. Following a single oral dose of 75 mg in healthy subjects, the Cmax of lonafarnib decreased by 55% and 25%, and the AUC decreased by 29% and 21% for a high/low-fat meal compared to fasted conditions.
Up to 240 hours following oral administration of 104 mg [14C]-lonafarnib in fasted healthy subjects, approximately 62% and <1% of the initial radiolabeled dose was recovered in feces and urine, respectively. The two most prevalent metabolites were the active HM21 and HM17, which account for 14% and 15% of plasma radioactivity.
In healthy patients administered either 75 or 100 mg lonafarnib twice daily, the steady-state apparent volumes of distribution were 97.4 L and 87.8 L, respectively.

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Metabolism / Metabolites
Lonafarnib is metabolized _in vitro_ primarily by CYP3A4/5 and partially by CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1. Formation of the primary metabolites involves oxidation and subsequent dehydration in the pendant piperidine ring.

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Biological Half-Life
Lonafarnib has a mean half-life of approximately 4-6 hours following oral administration of 100 mg twice daily in healthy subjects.

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Oral absorption:
- Rats: Oral administration of Lonafarnib (10 mg/kg) showed oral bioavailability (F) = 42%, Tmax = 1.2 hours, Cmax = 95 ng/mL [1]
- Distribution:
- Nude mice: 2 hours post-oral Lonafarnib (30 mg/kg), tumor concentration (180 ng/g) was 2.25-fold higher than plasma concentration (80 ng/mL) [1]
- Elimination:
- Rats: Intravenous (iv) Lonafarnib (5 mg/kg) had an elimination half-life (t1/2) = 5.1 hours; 72-hour excretion: 68% via feces, 15% via urine [1]
- Plasma protein binding:
- Human plasma: >98% protein binding (equilibrium dialysis, 37°C, pH 7.4) [1]

Hepatotoxicity
In the small prelicensure clinical trials conducted in children with progeria, serum aminotransferase elevations occurred in 35% of lonafarnib treated subjects but were usually mild and self-limited, rising to above 3 times the upper limit of normal (ULN) in only 5%. There were no liver related serious adverse events and no patient had a concurrent elevation in serum aminotransferase and bilirubin levels. Since approval of lonafarnib, there have been no published reports of drug induced liver injury associated with its use, although clinical experience with the drug, particularly with long term therapy, has been limited.
Likelihood score: E (unproven but suspected rare cause of clinically apparent liver injury).

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Protein Binding
Lonafarnib exhibits _in vitro_ plasma protein binding of ≥99% over a concentration range of 0.5-40.0 μg/mL.

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Animal toxicity:
- Nude mice (30 mg/kg bid, 14 days): No significant changes in serum ALT, AST, BUN, creatinine; body weight loss <4% [1]
- wap-ras transgenic mice (25 mg/kg bid, 21 days): No lethargy, diarrhea, or organ histopathological damage [1]
- Orthotopic glioma mice (combination treatment): Body weight loss <5%; no liver/kidney toxicity [2]
- Human toxicity (phase 2A trial):
- Mild-to-moderate adverse events (AEs):
- 100 mg bid group: Nausea (20%), diarrhea (15%);
- 200 mg bid group: Nausea (35%), vomiting (25%), fatigue (20%);
- No severe AEs (e.g., liver failure, myelosuppression); no significant changes in CBC, 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
Lonafarnib is a direct farnesyl transferase inhibitor that reduces the farnesylation of numerous cellular proteins, including progerin, the aberrantly truncated form of lamin A that accumulates in progeroid laminopathies such as Hutchinson-Gilford progeria syndrome. Treatment with lonafarnib has been associated with electrolyte abnormalities, myelosuppression, and increased liver enzyme levels (AST/ALT), although causation remains unclear. Also, lonafarnib is known to cause nephrotoxicity in rats and rod-dependent low-light vision decline in monkeys at plasma levels similar to those achieved under recommended dosing guidelines in humans; patients taking lonafarnib should undergo regular monitoring for both renal and ophthalmological function. In addition, based on observations from animal studies with rats, monkeys, and rabbits with plasma drug concentrations approximately equal to those attained in humans, lonafarnib may cause both male and female fertility impairment and embryo-fetal toxicity.

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Lonafarnib (SCH66336) is the first orally bioavailable tricyclic farnesyl protein transferase (FTase) inhibitor, initially developed for Ras-mutated cancers and later repurposed for chronic hepatitis D virus (HDV) infection [1][3]
- Antitumor mechanism: Inhibits FTase-mediated farnesylation of Ras proteins (H-Ras/K-Ras), blocking their membrane localization and activation of oncogenic pathways (e.g., MAPK/ERK), thereby suppressing tumor proliferation and inducing G1 cell cycle arrest [1][2]
- Antiviral mechanism: Inhibits farnesylation of HBV large surface protein (L-HBsAg), a critical step for HDV particle assembly and secretion, reducing HDV viremia [3]
- Clinical value: The phase 2A trial confirmed its antiviral efficacy in chronic HDV (a disease with limited treatments), while preclinical studies demonstrated synergistic antitumor activity with TMZ/radiation, supporting its potential 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.)