| Size | Price | Stock | Qty |
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| 100mg |
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| Other Sizes |
| Targets |
N-Boc-dolaproine-methyl itself, as an amino acid residue of Dolastatin 10, does not directly act on a specific target. Its activity is derived from the antitumor mechanism of the complete pentapeptide Dolastatin 10: Dolastatin 10 binds to tubulin, inhibiting tubulin polymerization and mitosis, thereby exerting its antitumor activity. Auristatin-class compounds containing the dolaproine structural unit form the structural basis for the cytotoxic payloads widely used in antibody-drug conjugates (ADCs).
N-Boc-dolaproine-methyl serves as the protected dolaproine residue, which is an essential component of the pentapeptide Dolastatin 10. Dolastatin 10 potently inhibits tubulin polymerization by binding to the vinca alkaloid binding site on tubulin, leading to disruption of microtubule dynamics and blockade of the cell cycle at the G2/M phase. This ultimately results in apoptotic death of rapidly dividing cancer cells. The methyl ester protects the carboxyl group during peptide coupling reactions, enabling the incorporation of dolaproine into larger peptide sequences. The Boc group is acid-labile and removed during the final steps of synthesis to generate the active drug. |
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| ln Vitro |
N-Boc-dolaproine-methyl, as a synthetic building block of Dolastatin 10, has not been reported to possess direct in vitro cellular activity. However, Dolastatin 10 and auristatin analogs containing the dolaproine residue demonstrate extremely potent antiproliferative activity in vitro. Dolastatin 10 exerts its effects by inhibiting tubulin polymerization, exhibiting picomolar to nanomolar cytotoxicity against various tumor cell lines.
The in vitro antiproliferative activity is attributed to the full Dolastatin 10 peptide. Dolastatin 10 has shown extremely potent cytotoxicity against a broad panel of human cancer cell lines, with IC50 values typically in the picomolar (e.g., 0.1-0.5 nM) range against leukemia (e.g., CCRF-CEM, HL-60), lymphoma, and various solid tumor cell lines (e.g., NCI-H460, LOX IMVI). This activity is mediated through the disruption of the microtubule cytoskeleton, leading to G2/M cell cycle arrest and subsequent apoptosis. N-Boc-dolaproine-methyl is a synthetic intermediate; thus, its direct activity is generally not assessed. |
| ln Vivo |
This compound is merely a synthetic intermediate, and its pharmacological activity derives from the final constructed antitumor molecule. Dolastatin 10 and its auristatin derivatives demonstrate significant tumor growth inhibition in animal xenograft models, and ADC drugs containing dolaproine structural units have demonstrated in vivo antitumor efficacy in both preclinical and clinical studies.
The in vivo efficacy is demonstrated by the parent Dolastatin 10 in animal models. In murine xenograft studies using human tumor cell lines, including LOX IMVI melanoma and MV-4-11 leukemia, Dolastatin 10 has shown significant tumor growth inhibition (TGI) and tumor regression at well-tolerated doses. The compound is typically administered intravenously or intraperitoneally. The potent antimitotic effect translates into in vivo antitumor activity. N-Boc-dolaproine-methyl is not directly tested in vivo; rather, it is used to synthesize active pharmaceutical ingredients (APIs) for in vivo studies. |
| Enzyme Assay |
N-Boc-dolaproine-methyl itself, as a synthetic intermediate, is not directly used in enzyme/receptor binding assays. This compound is primarily used as a raw material for introducing the dolaproine amino acid residue in solid-phase or liquid-phase peptide synthesis. In relevant applications, the compound is first subjected to Boc deprotection and methyl ester hydrolysis, then coupled to the growing peptide chain via condensation reactions. Its purity can be analyzed by HPLC (supplier purity ≥95%), and its structure can be characterized by NMR and mass spectrometry.
As a synthetic building block, specific in vitro binding assays are not performed on this compound. The binding of the final active payload is evaluated using a standard tubulin polymerization assay. In this assay, purified bovine or porcine brain tubulin is incubated with the test compound in a buffer containing GTP. Polymerization is initiated by warming to 37degC, and the increase in optical density is measured at 340 nm to monitor microtubule assembly. The compound's inhibitory effect is quantified by calculating the half-maximal inhibitory concentration (IC50). To determine the binding site, a competitive binding assay with [3H]vinblastine or [3H]colchicine can be used. Purity is verified by HPLC and NMR. |
| Cell Assay |
N-Boc-dolaproine-methyl, as a synthetic intermediate, is not used in direct in vitro cell assays. Cytotoxicity evaluation of complete Dolastatin 10 or auristatin-class compounds containing the dolaproine structure typically follows this protocol: Exponentially growing tumor cells (e.g., human leukemia cells or solid tumor cell lines) are seeded into 96-well culture plates at densities of 5,000-10,000 cells/well, cultured overnight, then treated with various concentrations of the test compound (0.001-100 nM) for 72-96 hours. Cell viability is assessed using MTT or CellTiter-Glo luminescent assays, and GI₅₀ values are calculated.
Cellular activity is tested using the final compound in various cancer cell lines. Cells are seeded in 96-well plates and treated with serial dilutions of the compound for 48-96 hours. Cell viability is measured using an MTT, CellTiter-Glo, or other colorimetric/fluorometric assay. Cell cycle distribution is analyzed by flow cytometry after propidium iodide (PI) staining to evaluate G2/M phase arrest. Apoptosis is quantified using Annexin V-FITC/PI double staining. Additionally, immunocytochemistry can be performed to visualize microtubule organization and tubulin polymerization status in fixed cells. |
| Animal Protocol |
This compound must undergo multi-step synthesis to assemble into the complete active molecule before being used for in vivo pharmacodynamic studies. In vivo experiments for complete Dolastatin 10 or auristatin-class ADC drugs typically use 6-8-week-old female nude mice subcutaneously inoculated with human tumor cell lines (5×10⁶ cells/100 μL PBS). When tumor volumes reach approximately 100-150 mm³, animals are randomly assigned to treatment groups, with tumor volume and body weight measured 2-3 times weekly to calculate tumor inhibition rates.
In vivo experiments are performed with the complete active peptide. For xenograft models, immunocompromised mice (e.g., nude or SCID) are implanted subcutaneously with human tumor cells (e.g., MV-4-11, MDA-MB-231). When tumors reach a predefined size (e.g., 100-200 mm3), mice are randomized and treated with the compound via intravenous (IV), intraperitoneal (IP), or subcutaneous (SC) injection. Dosing schedules, such as qd x5, q4d x3, or qw x3, are selected based on the compound's pharmacokinetics. Tumor volumes are measured twice weekly with calipers, and body weight is monitored to assess tolerability. At study termination, tumors are excised and analyzed for pharmacodynamic markers like phospho-histone H3 (pHH3) to confirm mitotic block. |
| ADME/Pharmacokinetics |
N-Boc-dolaproine-methyl is a synthetic intermediate and is not directly used for pharmacokinetic studies. The pharmacokinetics of auristatin-class ADC drugs containing dolaproine units have been reported in preclinical and clinical studies. Taking MMAE as an example, following intravenous administration in tumor-bearing mice, the half-life is approximately 2.5 hours, plasma clearance is approximately 60 mL/h, tissue distribution shows the highest concentration in the liver, metabolism is primarily mediated by CYP3A4, and excretion occurs via feces and urine. This compound itself is not used directly as a drug, and its pharmacokinetic parameters do not possess independent pharmacological significance.
As a synthetic building block, the pharmacokinetic (PK) properties refer to the final drug. The Boc group is labile under acidic conditions, but the methyl ester is typically stable in circulation. When used in ADC synthesis, the linker-payload circulates in the bloodstream and, upon tumor cell internalization, the payload is released. The released auristatin derivative then distributes into tissues, is metabolized by liver CYP3A4 enzymes, and is excreted. ADCs have a long circulation half-life of days, while the released small molecule has a shorter half-life of hours. The methyl ester may be hydrolyzed by esterases in vivo to the active acid form. The high lipophilicity of the auristatin payload often contributes to high plasma protein binding and a large volume of distribution. |
| Toxicity/Toxicokinetics |
N-Boc-dolaproine-methyl, as a synthetic intermediate, has not been systematically studied for its toxicological profile. This product is explicitly intended for research use only and is not for human therapeutic applications. The toxicity of auristatin-class payloads derived from it has been evaluated in relevant ADC drug studies, primarily manifesting as dose-limiting hematological toxicities (such as neutropenia and thrombocytopenia). Standard laboratory safety practices should be followed during handling. The powder can be stored at -20°C for 3 years, and in solution at -80°C for 6 months.
The toxicity profile is attributed to the final tubulin-targeting payload. Common toxicities of auristatin-based antimitotic agents include dose-limiting neutropenia, peripheral neuropathy, fatigue, and gastrointestinal disturbances (e.g., nausea, diarrhea). These are on-target effects resulting from the disruption of microtubule function in rapidly proliferating cells. The dicyclohexylamine component has its own toxicity profile, including potential irritation and sensitization. As an intermediate, it is handled with standard chemical safety precautions. The final ADC is designed to deliver the potent payload specifically to tumor cells to mitigate these systemic toxicities. |
| References |
[1]. An easy and stereoselective synthesis of N-Boc-dolaproine via the Baylis–Hillman reaction[J]. Tetrahedron letters, 2003, 44(5): 937-940.
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| Additional Infomation |
N-Boc-dolaproine-methyl is a specialized synthetic intermediate for constructing the dolaproine (Dap) residue of Dolastatin 10. This marine natural product is a highly potent antimitotic agent, 100-1000 times more active than traditional chemotherapeutics, and serves as the foundation for the auristatin class of cytotoxic drugs. The most prominent derivatives, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), are the "warheads" in several commercially approved and investigational ADCs, including brentuximab vedotin (Adcetris®), enfortumab vedotin (Padcev®), and polatuzumab vedotin (Polivy®). The orthogonal protection strategy of this intermediate allows for efficient solid-phase and solution-phase synthesis of these complex molecules, which are too unstable to be effectively used in their natural form. This product is strictly for research and development purposes only.
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| Molecular Formula |
C14H25NO5
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|---|---|
| Molecular Weight |
287.35
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| Exact Mass |
287.173
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| CAS # |
164456-57-9
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| PubChem CID |
11346731
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| Appearance |
Colorless to light yellow liquid
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| LogP |
1.493
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
20
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| Complexity |
363
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| Defined Atom Stereocenter Count |
3
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| SMILES |
COC([C@H](C)[C@@H](O)[C@H]1N(C(OC(C)(C)C)=O)CCC1)=O
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| InChi Key |
YESVEVMVKPHDFA-OUAUKWLOSA-N
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| InChi Code |
InChI=1S/C14H25NO5/c1-9(12(17)19-5)11(16)10-7-6-8-15(10)13(18)20-14(2,3)4/h9-11,16H,6-8H2,1-5H3/t9-,10+,11-/m1/s1
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| Chemical Name |
tert-butyl (2S)-2-[(1R,2R)-1-hydroxy-3-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylate
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| Synonyms |
N-Boc-dolaproine-methyl; 164456-57-9; (S)-tert-butyl2-((1R,2R)-1-hydroxy-3-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate; tert-butyl (2S)-2-[(1R,2R)-1-hydroxy-3-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylate; tert-Butyl (S)-2-((1R,2R)-1-hydroxy-3-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4801 mL | 17.4004 mL | 34.8008 mL | |
| 5 mM | 0.6960 mL | 3.4801 mL | 6.9602 mL | |
| 10 mM | 0.3480 mL | 1.7400 mL | 3.4801 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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