| Targets |
The molecular target of (1R,9S)-Dxd is topoisomerase I (Top1), a nuclear enzyme that relieves torsional stress in DNA during replication and transcription. As a camptothecin analog, (1R,9S)-Dxd stabilizes the cleavable complex formed between topoisomerase I and DNA, preventing religation. This results in the accumulation of persistent DNA single-strand breaks that are converted to double-strand breaks during S-phase, ultimately leading to replication fork collapse, DNA damage, cell cycle arrest in G2/M phase, and apoptosis. The (1R,9S) stereochemistry influences the stability and potency of the compound. (1R,9S)-Dxd is structurally optimized for ADCs, possessing a linker attachment site for conjugation to monoclonal antibodies.
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| ln Vitro |
An ADC consists of an antibody and an ADC cytotoxin linked together via an ADC linker.
As an ADC payload, (1R,9S)-Dxd is not tested as a free drug in standard in vitro cytotoxicity assays. The parent compound, DXd (exatecan derivative), is a potent topoisomerase I inhibitor with IC₅0 values typically in the low nanomolar to sub-nanomolar range against a broad panel of human cancer cell lines (e.g., breast, lung, ovarian, gastric cancers). When conjugated to an antibody via a cleavable or non-cleavable linker, the ADC is internalized into target cancer cells, and the payload is released, leading to selective cell killing. Free (1R,9S)-Dxd, if tested, would exhibit potent antiproliferative activity with IC₅0 values comparable to DXd (e.g., 0.1-5 nM). |
| ln Vivo |
In vivo, ADCs incorporating (1R,9S)-Dxd as the payload demonstrate potent antitumor efficacy in xenograft mouse models of various cancers. For example, an anti-HER2 ADC conjugated to (1R,9S)-Dxd (via a protease-cleavable linker) administered intravenously (e.g., 1-10 mg/kg, single dose or once weekly for 2-3 weeks) induces durable tumor regression, often complete responses, in HER2-positive breast and gastric cancer xenograft models. The compound is the active metabolite of certain ADCs (e.g., DS-8201a / trastuzumab deruxtecan), where it is released intracellularly following ADC internalization and lysosomal degradation. The bystander effect (diffusion of the payload to neighboring antigen-negative cells) contributes to efficacy in heterogeneous tumors. (1R,9S)-Dxd is a key component of clinical-stage ADCs.
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| Enzyme Assay |
In vitro topoisomerase I activity is typically measured using a DNA relaxation assay. Purified human topoisomerase I (0.5-1 U) is incubated in reaction buffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 microg/mL BSA) with 0.5 microg of supercoiled pBR322 DNA, and varying concentrations of (1R,9S)-Dxd (or the released payload, 0.001-1000 nM) for 15-30 min at 37degC. The reaction is terminated with SDS (0.5%). The DNA products are resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. The IC₅0 is the concentration that inhibits 50% of DNA relaxation, as quantified by densitometry. Since (1R,9S)-Dxd is a stable isomer, it can be directly tested using LC-MS/MS for purity.
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| Cell Assay |
For cellular cytotoxicity assays, a two-step ADC mechanism is employed. For the free payload, cancer cells (e.g., SK-BR-3 breast cancer, NCI-N87 gastric cancer) are seeded in 96-well plates (5,000 cells/well) in RPMI-1640 with 10% FBS and incubated overnight. (1R,9S)-Dxd (0.01-1000 nM) is added, and cells are incubated for 72-120 h. Cell viability is measured by MTT or CellTiter-Glo. The IC₅0 is calculated from dose-response curves. For ADC evaluation, target-positive and target-negative cells are used to assess selectivity. The ADC is added at various concentrations (0.001-10 microg/mL) for 96-120 h. The selectivity index (EC₅0 on target-positive / EC₅0 on target-negative) is calculated. Apoptosis is confirmed by caspase-3/7 activity assays.
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| Animal Protocol |
In vivo xenograft model: Female BALB/c nude mice (6-8 weeks old) are subcutaneously inoculated with 5×10⁶ target-positive cancer cells (e.g., SK-BR-3 HER2-positive breast cancer cells) in 0.1 mL PBS/Matrigel (1:1). When tumors reach approximately 100-150 mm3, mice are randomized into treatment groups (n=6-8 per group). The (1R,9S)-Dxd-based ADC is formulated in 10 mM histidine buffer (pH 5.5) with 5% sucrose and administered intravenously (tail vein) at doses of 1-10 mg/kg once weekly for 2-3 weeks. Control groups receive vehicle alone or unconjugated antibody. Tumor volume is measured every 2-3 days with calipers (volume = length × width2/2). Body weight is monitored for toxicity. At the study endpoint, tumors are excised, weighed, and processed for histopathology (H&E staining), immunohistochemistry (Ki-67 for proliferation, cleaved caspase-3 for apoptosis). Survival is monitored using Kaplan-Meier analysis. (1R,9S)-Dxd-based ADCs exhibit potent tumor growth inhibition.
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| ADME/Pharmacokinetics |
(1R,9S)-Dxd is not administered as a free drug. The PK of the ADC (e.g., trastuzumab deruxtecan) is determined by the antibody portion. The ADC has a long plasma half-life (3-7 days in humans, 1-3 days in rodents). Following systemic administration, the ADC distributes to tumor tissues via the enhanced permeability and retention (EPR) effect and active targeting (if antibody is directed to a tumor antigen). Upon internalization into target cells, the linker is cleaved (by cathepsins or other proteases), releasing (1R,9S)-Dxd. The released payload has a short half-life (< 24 h) and is cleared by renal excretion. The payload is a substrate for efflux transporters (P-gp, BCRP), though the potency is retained. For research use, (1R,9S)-Dxd is stored as a powder at -20degC, protected from light and moisture. The compound is soluble in DMSO (20-50 mM).
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| Toxicity/Toxicokinetics |
For (1R,9S)-Dxd, GHS hazard statements: H302 (Harmful if swallowed), H315 (Causes skin irritation), H319 (Causes serious eye irritation), H335 (May cause respiratory irritation), H350 (May cause cancer). Signal word: Danger. Precautionary statements: P201 (Obtain special instructions before use), P280 (Wear protective gloves/protective clothing/eye protection/face protection), P301+P312 (IF SWALLOWED: Call a POISON CENTER/doctor if you feel unwell), P305+P351+P338 (IF IN EYES: Rinse cautiously with water for several minutes). The compound is a topoisomerase I inhibitor and potential genotoxicant; handle with extreme caution in a fume hood with appropriate PPE. Not for human consumption. Storage: powder at -20degC for 3 years, 4degC for 2 years; in solvent at -80degC for 1 year.
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| References | |
| Additional Infomation |
(1R,9S)-Dxd (CAS# 2766786-58-5) is a research-grade ADC payload (topoisomerase I inhibitor). It is not an FDA-approved drug for standalone use; it is a chemical component of ADCs under clinical development (e.g., trastuzumab deruxtecan, DS-8201a). The compound is used as a building block for the synthesis of antibody-drug conjugates and as a reference standard for ADC research. (1R,9S)-Dxd is also an isomer of DXd and may be used for structure-activity relationship (SAR) studies. For research use only, not for diagnostic or therapeutic applications.
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| Molecular Formula |
C26H24FN3O6
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|---|---|
| Molecular Weight |
493.48
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| CAS # |
2766786-58-5
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| Related CAS # |
Exatecan mesylate dihydrate; 197720-53-9
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| Appearance |
Solid powder
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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) |
DMSO : ~100 mg/mL (~202.64 mM; with sonication)
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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 | 2.0264 mL | 10.1321 mL | 20.2642 mL | |
| 5 mM | 0.4053 mL | 2.0264 mL | 4.0528 mL | |
| 10 mM | 0.2026 mL | 1.0132 mL | 2.0264 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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