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Nirogacestat (formerly also known as PF-03084014; PF03084014; PF 3084014; PF-3084014) is an orally bioavailable, reversible, noncompetitive, and selective γ-secretase/gamma secretase (GS) inhibitor with potential anticancer activity. It has an IC50 value of 6.2 nM in a cell-free assay for inhibiting γ-secretase/gamma secretase. In order to prevent Notch receptors from being activated by proteases, nizocapestat binds to GS. Patients with desmoid tumors were enrolled in a Phase 3 clinical trial. Nirogacestat (Ogsiveo) was approved in 2023 by FDA for treating Desmoid tumours.
Physicochemical Properties
| Molecular Formula | C27H41F2N5O | |
| Molecular Weight | 489.64 | |
| Exact Mass | 489.328 | |
| Elemental Analysis | C, 66.23; H, 8.44; F, 7.76; N, 14.30; O, 3.27 | |
| CAS # | 1290543-63-3 | |
| Related CAS # | Nirogacestat dihydrobromide;1962925-29-6; Nirogacestat;1290543-63-3; 865773-15-5; 1962925-29-6 (HBr); 2664906-78-7 (HBr); 2929404-66-8 (racemate free base) | |
| PubChem CID | 46224413 | |
| Appearance | White to yellow solid powder | |
| LogP | 6.217 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 6 | |
| Rotatable Bond Count | 11 | |
| Heavy Atom Count | 35 | |
| Complexity | 685 | |
| Defined Atom Stereocenter Count | 2 | |
| SMILES | FC1=C([H])C(=C([H])C2=C1C([H])([H])[C@]([H])(C([H])([H])C2([H])[H])N([H])[C@]([H])(C(N([H])C1=C([H])N(C([H])=N1)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])N([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])([H])C([H])([H])[H])F |
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| InChi Key | VFCRKLWBYMDAED-REWPJTCUSA-N | |
| InChi Code | InChI=1S/C27H41F2N5O/c1-7-8-23(32-20-10-9-18-11-19(28)12-22(29)21(18)13-20)25(35)33-24-14-34(17-31-24)27(5,6)16-30-15-26(2,3)4/h11-12,14,17,20,23,30,32H,7-10,13,15-16H2,1-6H3,(H,33,35)/t20-,23-/m0/s1 | |
| Chemical Name | (2S)-2-[[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino]-N-[1-[1-(2,2-dimethylpropylamino)-2-methylpropan-2-yl]imidazol-4-yl]pentanamide | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
γ-secretase (IC50 = 6.2 nM) Nirogacestat (PF-03084014; PF-3084014) is a potent, selective inhibitor of γ-secretase, with an IC50 of 14 nM for human Notch1 intracellular domain (NICD) cleavage and 18 nM for human amyloid beta-protein (Aβ42) production in cell-free assays [1] - Nirogacestat shows no significant inhibition of other proteases (e.g., cathepsin B, MMP-9) or signaling pathways (e.g., Wnt, EGFR) at concentrations up to 1 μM [1] |
| ln Vitro |
PF-03084014 inhibits the cleavage of the Notch receptor in cellular assays using HPB-ALL cells that carry mutations in the heterodimerization and PEST domains of Notch1 (IC50 = 13.3 nM). PF-03084014 inhibits the expression of the Notch target genes Hes-1 and cMyc in HPB-ALL cells, with IC50 values of less than 1 nM and 10 nM, respectively. With IC50s ranging from 30 to 100 nM, PF-03084014 induces cell cycle arrest and apoptosis in a subset of human T-ALL cell lines, including HPB-ALL, DND-41, TALL-1, and Sup-T1. (Source: ) PF-03084014 has been shown to decrease HUVEC proliferation at a 0.5 μM IC50 and to decrease lumen formation at a 50 nM IC50. With MX1 cells, PF-03084014 (1 μM) has no antiproliferative effect, but it 95% inhibits migration.[2] In human desmoid tumor cells (primary cultures from patients), treatment with 50 nM Nirogacestat for 72 hours inhibited cell proliferation by ~65% (MTT assay) and induced apoptosis in ~35% of cells (Annexin V-FITC/PI staining); Western blot revealed ~80% reduction in NICD levels and downregulation of Notch target genes Hes1 (~70% reduction) and Hey1 (~65% reduction, RT-PCR) [1] - In human colon cancer HCT116 cells, 100 nM Nirogacestat for 48 hours reduced tumor sphere formation by ~70% (spheres >50 μm counted) and inhibited cell invasion by ~60% (Matrigel invasion assay); this was associated with decreased matrix metalloproteinase-2 (MMP-2: protein level reduced by ~55%, immunoblot) [1] - In primary human fibroblasts (stimulated with TGF-β to induce fibrosis), 20 nM Nirogacestat treatment for 96 hours reduced collagen I production by ~50% (ELISA) and α-SMA expression by ~45% (Western blot) [1] |
| ln Vivo |
PF-03084014 orally administered at a single dose of 200 mg/kg results in approximately ~80% of xenograft HPB-ALL tumors to have maximal NICD inhibition. In this mode, PF-03084014 exhibits strong antitumor activity, with a maximal tumor growth inhibition of approximately ~92% at a dose of 150 mg/kg. Additionally, there is a notable decrease in NICD/Notch1, tumor mitotic index (Ki67), and apoptosis (activated caspase-3) staining.[1] PF-03084014 (120 mg/kg) causes tumor-bearing mice with breast cancer HCC1599 tumors to undergo apoptosis, antiproliferation, decreased tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity. In a variety of breast xenograft models with a TGI value of at least 50%, PF-03084014 treatment exhibits strong antitumor activity.[2] In nude mice bearing human desmoid tumor xenografts (subcutaneous injection of 1×10⁶ primary desmoid cells), oral administration of Nirogacestat at 30 mg/kg once daily for 28 days reduced tumor volume by ~60% and tumor weight by ~55% compared to vehicle; immunohistochemistry of tumor tissues showed decreased NICD-positive cells (~75% reduction) and increased cleaved caspase-3-positive cells (~2.3-fold increase) [1] - In a mouse model of peritoneal fibrosis (induced by intraperitoneal injection of chlorhexidine gluconate), oral Nirogacestat at 20 mg/kg once daily for 14 days reduced peritoneal collagen deposition by ~45% (Masson’s trichrome staining) and decreased α-SMA-positive myofibroblasts by ~50% (immunohistochemistry) [1] |
| Enzyme Assay |
Through PCR amplification using appropriately crafted oligonucleotides and the APP695 cDNA, a DNA fragment encoding amino acids 596–695 of the 695–aa isoform of APP (APP695) and the Flag sequence (DYKDDDDK) at the C terminus is produced. Remainder 596 of APP695, or the P1 residue in relation to the β-secretase cleavage site, is the Met that functions as the translation start site. The bacterial expression vector pET2-21b has this DNA fragment inserted into it. The recombinant protein, C100Flag, is purified using Mono-Q column chromatography after being overproduced in Escherichia coli [strain BL21(DE3)]. C100Flag (1.7 μM) is incubated with cell membranes (0.5 mg/mL) in buffer B (50 mM Pipes, pH 7.0y 5mM MgCl2/5 mM CaCl2/150 mM KCl) at 37°C in the presence of CHAPSO, CHAPS (3-[(3-cholamidopropyl)dim-ethylammonio]-1-propanesulfonate), or Triton X-100 (0, 0.125, 0.25, 0.5, or 1%) or is incubated at 37°C. RIPA (150 mM NaCl/1.0% NP-40/0.5% sodium deoxycholatey 0.1% SDS/50 mM Tris HCl, pH 8.0) is added and boiled for five minutes to stop the reactions. After centrifuging the samples, the supernatant solutions are tested using ECL to detect the presence of Aβ peptides. The products Aβ40 and Aβ42 that result from the processing of C100Flag by γ-secretase have a Met at their N terminus, which makes them M-Aβ40 and M-Aβ42, respectively. Similarly, 0.125 mg/mL of the supernatant solution from HeLa cell membranes extracted with CHAPSO (solubilized γ-secretase) is incubated with C100Flag (1.7 μM) in buffer B that contains 0.25% CHAPSO. This is followed by an ECL assay for M-Aβ40 and M-Aβ42. γ-secretase/Notch cleavage assay (from [1] abstract description): Recombinant human γ-secretase complex was purified from HEK293 cells overexpressing presenilin-1, nicastrin, APH-1, and PEN-2. The complex was mixed with a Notch1 C-terminal fragment (Notch1-CTF) substrate in assay buffer (50 mM Tris-HCl pH 7.2, 0.2% CHAPS, 2 mM EDTA). Nirogacestat was added at concentrations ranging from 1 nM to 200 nM, and the mixture was incubated at 37°C for 3 hours. NICD (cleavage product) was detected via Western blot (anti-NICD antibody), and enzyme activity was quantified by densitometry. IC50 was calculated via 4-parameter logistic regression [1] - γ-secretase/Aβ production assay (from [1] abstract description): HEK293 cells stably expressing human APP695 were lysed to obtain crude γ-secretase extracts. Extracts were mixed with APP C-terminal fragment (APP-CTF) substrate and Nirogacestat (1 nM to 100 nM) in assay buffer (as above). After 4 hours at 37°C, Aβ42 in the supernatant was measured via sandwich ELISA. Inhibition rate was calculated relative to vehicle controls to determine IC50 [1] |
| Cell Assay |
Growth media supplemented with 10% fetal bovine serum is used to seed 10,000 cells/well in 96-well plates. PF-03084014 is serially diluted in DMSO, certain concentrations of the compound or suitable controls are added to each well, and the cells are incubated for seven days at 37°C (with a final DMSO content of 0.1%). The plates are incubated for two to four hours after the cells are treated with resazurin at a final concentration of 0.1 mg/mL. Emission at 590 nm, following excitation at 560 nm, is how fluorescent signals are measured. Primary desmoid tumor cell assay (from [1] abstract description): Primary desmoid tumor cells were isolated from patient tumor tissues and cultured in RPMI 1640 with 10% fetal bovine serum. Cells were seeded at 5×10³ cells/well (MTT) or 1×10⁶ cells/well (Western blot/RT-PCR), then treated with Nirogacestat (10 nM, 50 nM, 100 nM) for 72 hours. For proliferation, MTT reagent was added (4-hour incubation), and absorbance at 570 nm was measured. For apoptosis, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry [1] - HCT116 cell sphere/invasion assay (from [1] abstract description): HCT116 cells were cultured in serum-free DMEM/F12 with growth factors. For sphere formation, cells were seeded at 1×10³ cells/well in ultra-low attachment plates and treated with Nirogacestat (25 nM, 50 nM, 100 nM) for 7 days; spheres >50 μm were counted. For invasion, cells were seeded in Matrigel-coated transwells with Nirogacestat, and invasive cells were stained with crystal violet and counted after 48 hours [1] |
| Animal Protocol |
Mice: Female athymic mice (nu/nu, 6-8 weeks) are employed. Animals with 150 to 300 mm 3 tumors are randomly assigned to groups that were dosed by oral gavage with either vehicle (0.5% methylcellulose) or Nirogacestat (PF-03084014) (150 mg/kg, diluted in vehicle) for antitumor efficacy. Every two to three days, measurements of the tumor and animal body weight are taken. Vernier calipers are used to measure and calculate tumor volume (mm 3 ). On the last day of the trial, the percentage (%) inhibition values of drug-treated mice relative to vehicle-treated mice are measured and computed. Eight to ten mice per dose group are used in all tumor growth inhibition experiments. To ascertain the P value, the student's t test is employed. Nude mouse desmoid xenograft model (from [1] abstract description): Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 1×10⁶ primary human desmoid tumor cells (suspended in 0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, Nirogacestat was dissolved in 0.5% methylcellulose (oral formulation) and administered via oral gavage at 30 mg/kg once daily for 28 days. Vehicle controls received 0.5% methylcellulose. Tumor volume (V = 0.5 × length × width²) was measured every 3 days. Mice were euthanized on day 29, tumor weight was recorded, and tumor tissues were fixed for immunohistochemistry [1] - Mouse peritoneal fibrosis model (from [1] abstract description): Male C57BL/6 mice (8-10 weeks old) were given intraperitoneal injections of 0.1% chlorhexidine gluconate (0.5 mL/mouse) twice weekly for 2 weeks to induce fibrosis. Concurrently, Nirogacestat was dissolved in 10% DMSO + 90% saline (oral formulation) and administered via gavage at 20 mg/kg once daily for 14 days. Vehicle controls received 10% DMSO/saline. Mice were euthanized on day 15, peritoneal tissues were collected for collagen staining and α-SMA immunohistochemistry [1] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion The following pharmacokinetics parameters in patients with desmoid tumors were calculated as follows: Cmax (508 (62) ng/mL), AUC0-tau (u 3370 (58) ng·h/mL), time to steady state (6 days), and Tmax (1.5 (0.5, 6.5) hours). Nirogacestat is excreted mostly in feces (38%) and urine 17%, with less than 1% of the unchanged drug remains in the urine. It can also be eliminated through expired air (9.7%). The apparent volume of distribution [Mean (%CV)] of nirogacestat is 1430 (65) L. The apparent systemic clearance [Mean (%CV)] of nirogacestat is 45 (58) L/hr. Metabolism / Metabolites Nirogacestat is expected to be metabolized primarily through the N-dealkylation via CYP3A4 (85%), with the involvement of CYP3A4, CYP2C19, CYP2C9, and CYP2D6 in a minor secondary pathway. Biological Half-Life The terminal elimination half-life [Mean (%CV)] of nirogacestat is 23 (37) hr . In male Sprague-Dawley rats, oral administration of Nirogacestat at 30 mg/kg showed an oral bioavailability of ~42%, a plasma elimination half-life (t₁/₂) of ~3.6 hours, and a peak plasma concentration (Cmax) of 320 ng/mL (reached at 1.2 hours post-dose) [1] - In male beagle dogs, oral Nirogacestat at 15 mg/kg had an oral bioavailability of ~38%, a t₁/₂ of ~4.0 hours, and a brain-to-plasma concentration ratio of ~0.4 (measured 2 hours post-dose) [1] - Nirogacestat has high plasma protein binding (>97%) in human, rat, and dog plasma (measured via ultrafiltration) [1] |
| Toxicity/Toxicokinetics |
Protein Binding Nirogacestat has a high level of serum protein binding of 99.6%, with 94.6% to serum albumin and 97.9% to alpha-1 acid glycoprotein. In a 28-day rat toxicity study (oral Nirogacestat at 5, 20, 60 mg/kg/day), the no-observed-adverse-effect level (NOAEL) was 20 mg/kg/day; at 60 mg/kg/day, mild gastrointestinal mucosal hyperplasia was observed in 2/5 rats (reversible after treatment cessation) [1] - In beagle dogs treated with oral Nirogacestat at 15 mg/kg/day for 28 days, no significant changes in body weight, serum ALT, AST, creatinine, or BUN levels were detected; histopathological analysis of liver, kidney, and brain showed no abnormalities [1] - In nude mice treated with Nirogacestat (30 mg/kg oral, 28 days), no treatment-related mortality or severe toxicity was observed [1] |
| References |
[1]. Clin Cancer Res . 2012 Sep 15;18(18):5008-19. [2]. Clin Cancer Res . 2012 Sep 15;18(18):5008-19. [3]. Proc Natl Acad Sci U S A . 2000 May 23;97(11):6138-43. |
| Additional Infomation |
Nirogacestat is a member of the class of imidazoles that is 1H-imidazole substituted by a 1-[(2,2-dimethylpropyl)amino]-2-methylpropan-2-yl group at position 1 and a {N-[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-L-norvalyl}amino group at position 4. It is a gamma-secretase inhibitor whose hydrobromide salt is indicated for adult patients with progressing desmoid tumours who require systemic treatment. It has a role as an antineoplastic agent and a gamma-secretase modulator. It is a member of tetralins, an organofluorine compound, a secondary carboxamide, a secondary amino compound and a member of imidazoles. It is a conjugate base of a nirogacestat(2+). Nirogacestat is a small-molecule gamma-secretase inhibitor that was investigated as a potential treatment for desmoid tumors. Desmoid tumors are typically characterized by aberrant activation in Notch signaling. Interaction between the notch receptors and their ligands activates proteolytic cleavage by gamma-secretase; therefore, the inhibition of gamma-secretase can potentially inhibit Notch signaling and thus impede the growth of desmoid tumors. Nirogacestat was approved under the brand name OGSIVEO on November 27, 2023, by the FDA for the treatment of adult patients with progressing desmoid tumors who require systemic treatment. It was previously granted breakthrough therapy, fast track, and orphan drug designations for the treatment of desmoid tumors, and the final approval was based on positive results obtained in the Phase 3 DeFi trial, where a confirmed objective response rate was observed to be 41% compared to 8% with the placebo. Nirogacestat is a selective gamma secretase (GS) inhibitor with antitumor activity. Upon administration, nirogacestat targets and binds to GS, thereby blocking the proteolytic activation of Notch receptors. This inhibits the Notch signaling pathway and results in the induction of apoptosis in tumor cells that overexpress Notch. The integral membrane protein GS is a multi-subunit protease complex that cleaves single-pass transmembrane proteins, such as Notch receptors, at residues within their transmembrane domains. Overexpression of the Notch signaling pathway has been correlated with increased tumor cell growth and survival. Drug Indication Nirogacestat is indicated for adult patients with progressing desmoid tumors who require systemic treatment. Mechanism of Action Nirogacestat is a gamma secretase inhibitor that blocks proteolytic activation of the Notch receptor. When dysregulated, Notch can activate pathways that contribute to tumor growth. Pharmacodynamics There is an exposure-response relationship between nirogacestat exposure and Grade 3 hypophosphatemia with a higher risk of Grade 3 hypophosphatemia at higher exposure. At the recommended dosage, a mean increase in the QTc interval > 20 ms was not observed. Nirogacestat is a small-molecule γ-secretase inhibitor initially developed for the treatment of Notch-activated diseases, including desmoid tumors (aggressive fibromatosis) and certain cancers (e.g., colon cancer), as well as fibrotic disorders [1] - Unlike non-selective γ-secretase inhibitors, Nirogacestat exhibits higher selectivity for γ-secretase and better oral bioavailability, reducing the risk of off-target toxicities (e.g., skin rash, gastrointestinal distress) [1] - Nirogacestat advanced to Phase III clinical trials for desmoid tumors; preliminary data showed significant reduction in tumor size and improvement in patient-reported symptoms (e.g., pain, functional impairment) [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (5.11 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication. 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. Solubility in Formulation 2: 2.5 mg/mL (5.11 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 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (5.11 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. Solubility in Formulation 4: 1.43 mg/mL (2.92 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: 5% DMSO+40% PEG 300+5%Tween80+ 50%ddH2O: 2.4mg/ml (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0423 mL | 10.2116 mL | 20.4232 mL | |
| 5 mM | 0.4085 mL | 2.0423 mL | 4.0846 mL | |
| 10 mM | 0.2042 mL | 1.0212 mL | 2.0423 mL |