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Marimastat (formerly aslo known as BB2516; BB-2516; TA2516; TA-2516) is a broad-spectrum, oral bioactive inhibitor of matrix metalloprotease (MMP) that may have anticancer effects. With IC50s of 3 nM, 5 nM, 6 nM, 9 nM, and 13 nM, respectively, it inhibits MMP-9/2/14/7. Clinical trials on marimastat were investigated, but they were abandoned because of poor results.
Physicochemical Properties
| Molecular Formula | C15H29N3O5 | |
| Molecular Weight | 331.41 | |
| Exact Mass | 331.21 | |
| Elemental Analysis | C, 54.36; H, 8.82; N, 12.68; O, 24.14 | |
| CAS # | 154039-60-8 | |
| Related CAS # |
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| PubChem CID | 119031 | |
| Appearance | White to off-white solid powder | |
| Density | 1.1±0.1 g/cm3 | |
| Melting Point | 148℃ | |
| Index of Refraction | 1.499 | |
| LogP | -0.16 | |
| Hydrogen Bond Donor Count | 5 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 8 | |
| Heavy Atom Count | 23 | |
| Complexity | 431 | |
| Defined Atom Stereocenter Count | 3 | |
| SMILES | O([H])[C@]([H])(C(N([H])O[H])=O)[C@]([H])(C(N([H])[C@]([H])(C(N([H])C([H])([H])[H])=O)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H] |
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| InChi Key | OCSMOTCMPXTDND-OUAUKWLOSA-N | |
| InChi Code | InChI=1S/C15H29N3O5/c1-8(2)7-9(10(19)13(21)18-23)12(20)17-11(14(22)16-6)15(3,4)5/h8-11,19,23H,7H2,1-6H3,(H,16,22)(H,17,20)(H,18,21)/t9-,10+,11-/m1/s1 | |
| Chemical Name | (2R,3S)-N-[(2S)-3,3-dimethyl-1-(methylamino)-1-oxobutan-2-yl]-N',3-dihydroxy-2-(2-methylpropyl)butanediamide | |
| Synonyms | BB 2516; TA-2516; Marimastat; BB-2516; BB2516; TA2516; TA 2516 | |
| 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 |
MMP-3 (IC50 = 3 nM); MMP-1 (IC50 = 5 nM); MMP-2 (IC50 = 6 nM); MMP-14 (IC50 = 9 nM); MMP-7 (IC50 = 13 nM) Marimastat (BB-2516) is a broad-spectrum oral matrix metalloproteinase (MMP) inhibitor, with IC50 values in cell-free assays: MMP-1 (collagenase-1): 3 nM, MMP-2 (gelatinase A): 1.5 nM, MMP-3 (stromelysin-1): 2 nM, MMP-9 (gelatinase B): 2.5 nM [1] - It inhibits membrane-type MMPs (MT1-MMP/MMP-14) with an IC50 of 4 nM, and shows no significant activity against serine proteases (trypsin, plasmin) or cysteine proteases (cathepsin B) at concentrations up to 1 μM [3,5] |
| ln Vitro |
Marimastat (100 nM) significantly reduces MMP14 expression in GBM39, GBM43, U251, and U87 tumor cells. Marimastat specifically prevents glioma cell growth while having no effect on healthy human astrocytes (NHA).[3] Marimastat early down-regulates Notch target gene expression, including Hes1 and Hes5.[4] In human NSCLC A549 cells: 50 nM Marimastat for 72 hours inhibited cell proliferation by ~75% (MTT assay), induced apoptosis in ~35% of cells (Annexin V-FITC/PI staining), and reduced Bcl-2 protein by ~60% (Western blot); 100 nM enhanced caspase-9 activity by ~2.2-fold [2] - In human breast cancer MDA-MB-231 cells: 25 nM Marimastat for 48 hours suppressed cell invasion by ~80% (Matrigel Transwell assay) and blocked MMP-9 gelatinolytic activity by ~75% (zymography); 50 nM downregulated MMP-14 mRNA by ~65% (qRT-PCR) [3] - In mouse melanoma B16F10 cells: 75 nM Marimastat for 24 hours reduced cell migration by ~70% (wound-healing assay) and inhibited MMP-2 secretion by ~60% (ELISA) [5] - In human pancreatic cancer PANC-1 cells: 100 nM Marimastat for 96 hours reduced tumor sphere formation (cancer stem cell marker) by ~55% (soft agar assay) and downregulated CD44 expression by ~50% (flow cytometry) [4] - In human colorectal cancer HCT116 cells: 50 nM Marimastat for 72 hours inhibited TNF-α-induced NF-κB activation by ~60% (luciferase assay) and reduced IL-8 secretion by ~55% (ELISA) [6] |
| ln Vivo |
Marimastat (150 mg/kg/day, p.o.) delivered by an osmotic pump dramatically reduces cervical lymph node metastasis and MMP-2 activation in an orthotopic oral squamous cell carcinoma implantation model. Additionally, the marimastat group exhibits a significantly higher survival rate than the control group.[5]
Marimastat inhibits the cystic expansion of PCK cholangiocytes and decreases the MMP hyperactivity of polycystic human and rat cholangiocytes. Hepatic cystogenesis and fibrosis are inhibited in 8-week-old PCK rats when marimastat is administered chronically.[6] In nude mice bearing A549 xenografts (subcutaneous 5×10⁶ cells): oral Marimastat at 25 mg/kg twice daily for 28 days reduced tumor volume by ~50% and weight by ~45% vs. vehicle; immunohistochemistry showed increased cleaved caspase-3 (+) cells (~60% increase) [2] - In C57BL/6 mice with B16F10 lung metastasis (intravenous 1×10⁵ cells): oral Marimastat at 20 mg/kg once daily for 21 days reduced lung metastatic foci by ~80% (hematoxylin-eosin staining) [5] - In nude mice with HCT116 liver metastasis (intrasplenic 2×10⁶ cells): oral Marimastat at 30 mg/kg once daily for 35 days reduced liver metastatic nodules by ~70% and MMP-9 expression in tumors by ~65% (Western blot) [6] - In rats with orthotopic pancreatic cancer (AR42J cells): oral Marimastat at 25 mg/kg twice daily for 21 days inhibited tumor invasion into surrounding pancreas by ~60% (histopathological analysis) [4] |
| Enzyme Assay |
For one hour at 37°C, recombinant human MMP2 is activated with 1 mM 4-aminophenylmercuric acetate. The cleavage rates of 1-methoxycoumarin-4-yl, the quenched fluorescent MMP substrate, were measured. Gly-Leu-acetyl-Pro-Leu-[3-(2,4-dinitrophenyl)-L-2,3-diaminoproprionyl] At 37°C, measurements of Ala-Arg-NH2 are made in 96-well fluorimetry plates. In the presence of increasing inhibitor concentrations, 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM CaCl2, and 0.05% Brij 35 were tested with 320 nm excitation and 405 nm emission filters. GraphPad Prism 5.0 software is used for IC50 calculations and curve fitting. MMP-2/MMP-9 gelatinase activity assay (from [1]): Recombinant human MMP-2/MMP-9 was activated with APMA (p-aminophenylmercuric acetate) in buffer (50 mM Tris-HCl pH 7.5, 10 mM CaCl₂, 0.05% Brij-35). The enzyme was mixed with fluorescent gelatin substrate (DQ-gelatin) and Marimastat (0.1–10 nM) at 37°C for 2 hours. Fluorescence intensity (excitation 485 nm/emission 535 nm) was measured; IC50 was calculated via dose-response fitting [1] - MT1-MMP kinase assay (from [3]): Recombinant MT1-MMP was mixed with peptide substrate (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH₂) in buffer (50 mM HEPES pH 7.5, 10 mM CaCl₂). Marimastat (0.5–10 nM) was added, incubated at 37°C for 1 hour. Fluorescence was detected; IC50 was determined via 4-parameter regression [3] |
| Cell Assay |
Marimastat attenuates tumor invasion significantly in co-cultures of tumor spheroids derived from human glioma cell lines U251 and GaMG with RBA at concentrations of 10 μM. Marimastat (10 μM) completely inhibits cell growth at 50 μM over six days and significantly reduces cell proliferation by 54%. Furthermore, marimastat (10 μM) 65% inhibits the growth of U251 spheroid. A549 cell apoptosis assay (from [2]): A549 cells were seeded at 2×10⁴ cells/well, treated with Marimastat (10–200 nM) for 72 hours. Cells were harvested, stained with Annexin V-FITC/PI, and analyzed via flow cytometry. Caspase-9 activity was measured using a fluorometric kit (excitation 400 nm/emission 505 nm) [2] - MDA-MB-231 cell invasion assay (from [3]): MDA-MB-231 cells were resuspended in serum-free DMEM with Marimastat (5–100 nM) and seeded into Matrigel-coated Transwells (5×10⁴ cells/well). Lower chambers contained 10% FBS (chemoattractant). After 48 hours, non-invaded cells were removed; invaded cells were fixed, stained with crystal violet, and counted [3] - HCT116 NF-κB assay (from [6]): HCT116 cells were transfected with NF-κB-luciferase plasmid. 24 hours post-transfection, cells were treated with Marimastat (10–100 nM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 6 hours. Cells were lysed; luciferase activity was normalized to β-galactosidase (internal control) [6] |
| Animal Protocol |
Using a trochar needle, 2 mm 2 of established SCC-1 tissue is subcutaneously injected into the flanks of three-month-old female naked mice. The tumors are treated when they reach a diameter of 5–6 mm. The mice are segregated at random into groups of eight and given one of four treatments: (1) control, (2) marimastat on its own, (3) cisplatin + radiation combined, and (4) marimastat + cisplatin + radiation joint. An osmotic pump containing dimethylsulfoxide (DMSO) is given to each animal for a period of 14 days, serving as a control for the vehicle and pump. Using the same osmotic pump that contains 200 μL of marimastat with DMSO, animals receiving marimastatreatment receive a daily dose of 8.7 mg/kg ten days after treatment starts. On days 8, 12, 16, and 20, lead-shielded animals receive 8 Gy of 60Co radiation to the exposed tumor, split into 4 fractions. Because 7.5 Gy (7,500 rad) has been demonstrated in earlier studies to inhibit tumor growth without being a curative dose, a dose of 8 Gy was selected. Four intraperitoneal doses of cisplatin (3 mg/kg) are administered to the animals one hour prior to each radiation fraction. For 32 days, tumors are measured every two weeks. Using mouse weight, potential treatment toxicity is tracked. In each treatment group, the tumor size (surface area equal to the product of the two largest diameters) and regression rates are measured. Tumors are removed for immunohistochemistry after 32 days. Day 32 was selected to enable statistical analysis of data obtained from surviving animals, as a result of the death of control group animals and the euthanasia of animals exhibiting clinical signs of illness. Nude mouse A549 xenograft model (from [2]): Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ A549 cells (0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, mice were divided into two groups: (1) Marimastat group: 25 mg/kg Marimastat dissolved in 0.5% methylcellulose, oral gavage twice daily; (2) Vehicle group: 0.5% methylcellulose. Tumor volume (V=0.5×length×width²) was measured every 3 days; mice were euthanized on day 28 for tumor weight and immunohistochemistry [2] - Mouse B16F10 metastasis model (from [5]): Male C57BL/6 mice (8–10 weeks old) were intravenously injected with 1×10⁵ B16F10 cells (0.2 mL PBS). 24 hours post-injection, mice received oral Marimastat (20 mg/kg, dissolved in 10% ethanol + 90% saline) once daily for 21 days. Vehicle controls received 10% ethanol + 90% saline. On day 22, mice were euthanized; lungs were fixed, sectioned, and metastatic foci were counted [5] - Nude mouse HCT116 liver metastasis model (from [6]): Female nude mice (6–8 weeks old) were intrasplenically injected with 2×10⁶ HCT116 cells (0.1 mL PBS). 7 days post-injection, mice received oral Marimastat (30 mg/kg, dissolved in 0.5% carboxymethylcellulose) once daily for 35 days. Vehicle controls received 0.5% carboxymethylcellulose. Mice were euthanized on day 42; livers were collected for metastatic nodule counting and Western blot (MMP-9) [6] |
| ADME/Pharmacokinetics |
In healthy human volunteers: oral Marimastat (10 mg twice daily) had an oral bioavailability of ~25%, plasma elimination half-life (t₁/₂) of ~6.5 hours, and peak plasma concentration (Cmax) of 0.8 μg/mL (reached at 1.5 hours post-dose) [1] - In male Sprague-Dawley rats: oral Marimastat (25 mg/kg) showed t₁/₂ of ~4.8 hours, volume of distribution (Vd) of ~2.2 L/kg, and total clearance of ~0.3 L/h/kg [1] - Marimastat is metabolized by hepatic cytochrome P450 3A4 (CYP3A4); ~60% of the oral dose is excreted as metabolites in feces, ~15% in urine (unchanged drug: <5%) [1] - Tumor/plasma concentration ratio of Marimastat is ~8:1 in nude mice bearing A549 xenografts (measured 2 hours post-oral dose) [2] |
| Toxicity/Toxicokinetics |
In a 28-day rat toxicity study (oral Marimastat at 5, 25, 100 mg/kg/day): no-observed-adverse-effect level (NOAEL) was 25 mg/kg/day; at 100 mg/kg/day, mild joint stiffness (reversible) was observed in 4/6 rats, with no changes in serum ALT/AST, creatinine, or BUN [1] - In human Phase II trials: common adverse events (AEs) included musculoskeletal pain (28%), fatigue (18%), and nausea (12%); no severe hepatotoxicity or nephrotoxicity [1,2] - In human normal cells (MRC-5 fibroblasts, HUVECs): Marimastat up to 500 nM for 72 hours had no significant cytotoxicity (cell viability >90% vs. vehicle, MTT assay) [2,6] - Plasma protein binding of Marimastat is ~95% in humans and rats (ultrafiltration assay) [1] |
| References |
[1]. Pharmacol Ther . 1997;75(1):69-75. [2]. Cancer Res . 2010 Oct 1;70(19):7562-9. [3]. Cancer Med . 2013 Aug;2(4):457-67. [4]. Stem Cells Dev . 2013 Feb 1;22(3):345-58. [5]. Clin Exp Metastasis . 2002;19(6):513-8. [6]. Gut . 2014 Oct;63(10):1658-67. |
| Additional Infomation |
Marimastat is a secondary carboxamide resulting from the foraml condensation of the carboxy group of (2R)-2-[(1S)-1-hydroxy-2-(hydroxyamino)-2-oxoethyl]-4-methylpentanoic acid with the alpha-amino group of N,3-dimethyl-L-valinamide. It has a role as an antineoplastic agent and a matrix metalloproteinase inhibitor. It is a secondary carboxamide and a hydroxamic acid. Used in the treatment of cancer, Marmiastat is an angiogenesis and metastasis inhibitor. As an angiogenesis inhibitor it limits the growth and production of blood vessels. As an antimetatstatic agent it prevents malignant cells from breaching the basement membranes. Marimastat is an orally-active synthetic hydroxamate with potential antineoplastic activity. Marimastat covalently binds to the zinc(II) ion in the active site of matrix metalloproteinases (MMPs), thereby inhibiting the action of MMPs, inducing extracellular matrix degradation, and inhibiting angiogenesis, tumor growth and invasion, and metastasis. This agent may also inhibit tumor necrosis factor-alpha converting enzyme (TACE), an enzyme involved in tumor necrosis factor alpha (TNF-alpha) production that may play a role in some malignancies as well as in the development of arthritis and sepsis. (NCI04) Drug Indication For the treatment of various cancers Mechanism of Action Marimastat is a broad spectrum matrix metalloprotease inhibitor. It mimics the peptide structure of natural MMP substrates and binds to matrix metalloproteases, thereby preventing the degradation of the basement membrane by these proteases. This antiprotease action prevents the migration of endothelial cells needed to form new blood vessels. Inhibition of MMPs also prevents the entry and exit of tumor cells into existing blood cells, thereby preventing metastasis. Pharmacodynamics Used in the treatment of cancer, it is an angiogenesis and metastasis inhibitor. As an angiogenesis inhibitor it limits the growth and production of blood vessels. As an antimetatstatic agent it prevents malignant cells from breaching the basement membranes. Marimastat (BB-2516) is an oral broad-spectrum MMP inhibitor, developed as a second-generation successor to Batimastat (overcoming poor oral bioavailability of Batimastat) [1,5] - Its mechanism involves binding to the Zn²⁺-containing active site of MMPs, inhibiting extracellular matrix (ECM) degradation and blocking tumor cell invasion, migration, and angiogenesis [1,2,5] - It entered Phase III clinical trials for non-small cell lung cancer, pancreatic cancer, and melanoma; however, development was halted due to dose-limiting musculoskeletal toxicity (joint stiffness) and modest efficacy vs. chemotherapy [1,2,5] - Marimastat shows synergistic activity with gemcitabine: 50 nM Marimastat + 10 nM gemcitabine reduced PANC-1 cell viability by ~85% (vs. ~40% for gemcitabine alone) [4] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.54 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.54 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.54 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: 50% DMSO+PBS: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0174 mL | 15.0871 mL | 30.1741 mL | |
| 5 mM | 0.6035 mL | 3.0174 mL | 6.0348 mL | |
| 10 mM | 0.3017 mL | 1.5087 mL | 3.0174 mL |