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| Targets |
N-Boc-dolaproine dicyclohexylamine 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 exerts its antitumor activity by binding to tubulin, inhibiting tubulin polymerization and mitosis. The structure of Dolastatin 10, composed of amino acids including dolaproine, has been demonstrated to possess potent tubulin inhibition and cytotoxicity, forming the structural basis for the widely used auristatin-class payloads in ADC drugs.
N-Boc-dolaproine dicyclohexylamine exerts its effects as a precursor to the amino acid dolaproine, a key component of the pentapeptide Dolastatin 10. Dolastatin 10 is a potent antimitotic agent that binds to tubulin at the vinca alkaloid-binding site. It potently inhibits microtubule polymerization, leading to the disruption of the mitotic spindle and arrest of the cell cycle at the G2/M phase. Consequently, this triggers apoptotic cell death in rapidly dividing cells, forming the basis of its potent anticancer activity. The N-Boc protecting group safeguards the amino functionality during synthesis, making this compound an essential building block for constructing tubulin-targeting drugs and ADCs. |
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| ln Vitro |
N-Boc-dolaproine dicyclohexylamine, 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, and its derived auristatin-class compounds (e.g., MMAE, MMAF) are among the most widely used cytotoxic payloads in ADC drugs today, exhibiting picomolar to nanomolar cytotoxicity against various tumor cell lines.
Dolastatin 10, of which this compound is the amino acid residue, has been shown to have potent anticancer activity in preclinical models, with IC50 values typically in the picomolar to low nanomolar range against various human cancer cell lines. It potently inhibits the proliferation of leukemia, lymphoma, and solid tumor cell lines by binding to tubulin and causing G2/M cell cycle arrest. The pentapeptide is a potent inhibitor of tubulin polymerization and mitosis. N-Boc-dolaproine dicyclohexylamine itself is a synthetic building block and is not typically tested for direct in vitro activity; its activity is assessed after incorporation into the full Dolastatin 10 peptide sequence. |
| ln Vivo |
As an amino acid component of the complete pentapeptide Dolastatin 10, 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. ADC drugs containing dolaproine structural units have demonstrated in vivo antitumor efficacy in both preclinical and clinical studies.
The in vivo activity is attributed to the parent pentapeptide Dolastatin 10, which has demonstrated significant antitumor activity in various murine xenograft models of human cancers, including melanoma, ovarian carcinoma, and lung carcinoma. In vivo, Dolastatin 10 potently suppresses tumor growth at low doses, primarily through its antimitotic mechanism and anti-angiogenic effects. N-Boc-dolaproine dicyclohexylamine is used in the synthesis of tubulin inhibitors and ADCs that demonstrate in vivo antitumor efficacy and are designed to reduce systemic toxicity by targeting the payload to tumor cells. |
| Enzyme Assay |
N-Boc-dolaproine dicyclohexylamine 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, then coupled to the growing peptide chain via condensation reactions. Its purity can be analyzed by HPLC, and its structure can be characterized by NMR and mass spectrometry.
As it is a synthetic building block, typical binding assays are not performed on this intermediate. However, the parent compound Dolastatin 10's binding can be measured using a tubulin polymerization assay. In this assay, purified tubulin is incubated with varying concentrations of the compound in a buffer containing GTP. Polymerization is induced by warming the mixture to 37degC, and the turbidity increase is measured spectrophotometrically at 340-350 nm. Inhibition of microtubule assembly is determined by comparing the absorbance to a control group. Additionally, colchicine or vinblastine binding assays can be performed to determine the binding site on tubulin. For the N-Boc-dolaproine dicyclohexylamine building block, purity is confirmed by HPLC and LC-MS. |
| Cell Assay |
N-Boc-dolaproine dicyclohexylamine, 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.
The cellular activity is typically evaluated using the pentapeptide Dolastatin 10 in cell proliferation assays. Human cancer cell lines (e.g., HeLa, MCF-7, U251) are seeded in 96-well plates and treated with serial dilutions of the compound for 48-96 hours. Cell viability is then assessed using a colorimetric MTT, XTT, or resazurin-based assay. The IC50 value is calculated from the dose-response curve. Cell cycle analysis is performed by flow cytometry after propidium iodide staining to detect the accumulation of cells in the G2/M phase. Apoptosis is confirmed by Annexin V-FITC and PI double staining followed by flow cytometry analysis. |
| Animal Protocol |
N-Boc-dolaproine dicyclohexylamine, as a synthetic intermediate, is not used in in vivo animal experiments. 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.
The in vivo efficacy is typically evaluated using the complete pentapeptide Dolastatin 10 in murine xenograft models. Immunodeficient mice (e.g., nude mice) are subcutaneously inoculated with human tumor cells (e.g., MV-4-11 leukemia, MDA-MB-231 breast, or A549 lung). Once tumors reach a certain size (~100 mm3), mice are randomized and treated intravenously or intraperitoneally with the compound or vehicle control, typically on a qd, qod, or q4d schedule. Tumor volumes are measured with calipers, and body weight is recorded. Efficacy is determined by comparing tumor growth inhibition in treated vs. control groups. Tolerability is assessed by monitoring body weight loss and other signs of toxicity. Ex vivo analysis may include immunohistochemistry for tubulin or Ki-67 to assess proliferation. |
| ADME/Pharmacokinetics |
N-Boc-dolaproine dicyclohexylamine 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 building block, PK studies focus on the final drug product. The N-Boc protecting group is generally labile and is cleaved in acidic environments, which can occur in vivo. The compound is likely highly lipophilic due to the dicyclohexylamine salt and Boc group. Following incorporation into an ADC, the ADC circulates in the bloodstream, and upon internalization into target cancer cells, the payload is released. The payload (e.g., MMAE or Dolastatin 10 derivative) then distributes into tissues and is metabolized, primarily by CYP3A4 enzymes in the liver. The half-life of an ADC can range from days to weeks, while the payload half-life is generally shorter, on the order of hours. Excretion is primarily through the hepatobiliary and renal routes. |
| Toxicity/Toxicokinetics |
N-Boc-dolaproine dicyclohexylamine, 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) along with some gastrointestinal reactions. The presence of the dicyclohexylamine salt indicates that this compound may have some irritant properties, and standard laboratory safety practices should be followed during handling.
No specific toxicology data is reported for the building block, but the parent Dolastatin 10 exhibits classical microtubule inhibitor-related toxicities, including dose-limiting neutropenia, peripheral neuropathy, and gastrointestinal effects. Due to its mechanism of action as a potent antimitotic agent, toxicity is largely driven by effects on rapidly dividing cells in the bone marrow and gastrointestinal tract. The dicyclohexylamine component has known toxicity, including potential irritation and sensitization. When used as a building block, rigorous purity and safety standards are applied, and the final product is managed under appropriate chemical safety protocols. |
| 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 dicyclohexylamine (CAS#: 1369427-40-6) is a key intermediate in the synthesis of Dolastatin 10 and its analogs, which are potent antimitotic agents. Dolastatin 10 is a naturally occurring pentapeptide isolated from the marine sea hare Dolabella auricularia and serves as the lead compound for the development of a class of anticancer agents called auristatins. Auristatins, such as monomethyl auristatin E (MMAE), are widely used as the cytotoxic payloads in clinically approved ADCs like brentuximab vedotin (Adcetris®) and enfortumab vedotin (Padcev®). This building block enables the efficient synthesis of these highly potent payloads, which are among the most successful classes of ADC warheads. This compound is for research use only and is not for human consumption.
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| Molecular Formula |
C26H48N2O5
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| Molecular Weight |
468.67
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| Exact Mass |
468.356
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| CAS # |
1369427-40-6
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| Related CAS # |
N-Boc-dolaproine;120205-50-7
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| PubChem CID |
89824205
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| Appearance |
White to off-white solid at room temperature
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
33
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| Complexity |
479
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| Defined Atom Stereocenter Count |
3
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| SMILES |
O(C([H])([H])[H])[C@]([H])([C@]([H])(C(=O)O[H])C([H])([H])[H])[C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C(=O)OC(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H].N([H])(C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]
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| InChi Key |
PKZALTWOMMNNOL-QJQMQQLTSA-N
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| InChi Code |
InChI=1S/C14H25NO5.C12H23N/c1-9(12(16)17)11(19-5)10-7-6-8-15(10)13(18)20-14(2,3)4;1-3-7-11(8-4-1)13-12-9-5-2-6-10-12/h9-11H,6-8H2,1-5H3,(H,16,17);11-13H,1-10H2/t9-,10+,11-;/m1./s1
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| Chemical Name |
N-cyclohexylcyclohexanamine;(2R,3R)-3-methoxy-2-methyl-3-[(2S)-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidin-2-yl]propanoic acid
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| Synonyms |
1369427-40-6; dicyclohexylamine (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate; MFCD31560448; N-Boc-dolaproine (dicyclohexylamine); N-cyclohexylcyclohexanamine;(2R,3R)-3-methoxy-2-methyl-3-[(2S)-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidin-2-yl]propanoic acid;
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
Ethanol: 100 mg/mL (213.4 mM)
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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 | 2.1337 mL | 10.6685 mL | 21.3370 mL | |
| 5 mM | 0.4267 mL | 2.1337 mL | 4.2674 mL | |
| 10 mM | 0.2134 mL | 1.0668 mL | 2.1337 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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