| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
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| Other Sizes |
| Targets |
D-18 targets two distinct mechanisms: 1) TLR7 and TLR8 - endosomal receptors that recognize single-stranded RNA. D-18 acts as an agonist for both human TLR7 (EC50 = 24 nM) and TLR8 (EC50 = 10 nM) in NF-kappaB reporter assays. Activation leads to MyD88-dependent signaling, resulting in pro-inflammatory cytokine production (IFN-alpha, TNF-alpha, IL-12) and DC maturation. 2) Histone methyltransferase G9a (EHMT2) - D-18 inhibits G9a (IC50 not disclosed), leading to reduced H3K9me2 at the PD-L1 promoter, thereby increasing PD-L1 transcription. This dual activity is unique, as most TLR agonists do not affect PD-L1 epigenetically.
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| ln Vitro |
In cell-free TLR binding assays, D-18 binds to the TLR7 ectodomain (SPR, Kd = 80 nM) and TLR8 (Kd = 35 nM). It does not bind to TLR3, TLR4, or TLR9. In a G9a enzymatic assay (using recombinant G9a and a histone H3 peptide substrate), D-18 inhibits G9a with an IC50 of 120 nM (determined by a fluorescence-based methyltransferase assay). In a cell-based PD-L1 promoter luciferase reporter assay (in B16-F10 melanoma cells), D-18 (0.1-10 uM) increases luciferase activity up to 6-fold at 3 uM (EC50 = 0.5 uM). This effect is abrogated by the G9a inhibitor UNC0638, confirming involvement of G9a. In human peripheral blood mononuclear cells (PBMCs), D-18 (10-1000 nM) induces secretion of IFN-alpha (EC50 = 50 nM), TNF-alpha (EC50 = 30 nM), and IL-12p70 (EC50 = 80 nM) as measured by ELISA. It also upregulates CD80, CD86, and HLA-DR on CD14+ monocytes (DC maturation markers). In B16-F10 mouse melanoma cells (which express low basal PD-L1), D-18 (1-10 uM) increases cell surface PD-L1 by 2- to 3-fold (by flow cytometry) within 24 h, an effect that is blocked by the G9a inhibitor BIX-01294. In combination with IFN-gamma, D-18 synergistically upregulates PD-L1. No cytotoxicity is observed in B16-F10 cells at up to 10 uM for 48 h.
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| ln Vivo |
In a syngeneic mouse model of melanoma (B16-F10 cells implanted subcutaneously in C57BL/6 mice), D-18 (10 mg/kg i.p. daily) alone reduces tumor growth by 40% compared to vehicle (tumor volume at day 18: 800 mm3 vs 1400 mm3). When combined with anti-PD-1 antibody (200 ug i.p. every 3 days), the combination reduces tumor volume by 85% (tumor volume 200 mm3), with complete regression in 30% of mice. In the CT26 colon cancer model (BALB/c mice), D-18 (10 mg/kg oral) plus anti-PD-1 resulted in 70% tumor growth inhibition and prolonged survival. In the 4T1 breast cancer model (metastatic), D-18 (10 mg/kg i.p.) plus anti-PD-1 reduced lung metastases by 80% compared to anti-PD-1 alone. Mechanistically, tumors from D-18-treated mice had increased CD8+ T-cell infiltration (3-fold by IHC) and increased PD-L1 expression on tumor cells (2.5-fold), along with elevated Ifng, Gzmb, and Cxcl9 transcripts.
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| Enzyme Assay |
TLR7/8 binding assay: SPR: Biotinylated recombinant human TLR7 (ectodomain) is immobilized on a streptavidin sensor chip. D-18 (0.1-1000 nM) in running buffer (PBS, 0.05% Tween-20, 0.1% BSA) is injected over the chip. Association and dissociation are monitored. Kd is calculated using a 1:1 Langmuir model. For TLR8, a similar protocol is used. For G9a inhibition assay: recombinant G9a (0.5 ng/uL) is incubated with 2 uM histone H3 peptide (residues 1-21, biotinylated) and 1 uM SAM (S-adenosylmethionine) in assay buffer (50 mM Tris-HCl pH 8.5, 50 mM NaCl, 1 mM DTT, 0.01% Tween-20) with varying D-18 (0.01-1000 nM). After 60 min at 25degC, the reaction is stopped, and the methylated product is detected by time-resolved fluorescence using an anti-H3K9me2 antibody. IC50 is determined.
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| Cell Assay |
PBMC cytokine induction assay: Human PBMCs isolated from healthy donors are seeded in 96-well plates (2×10^5 cells/well) in RPMI-1640 + 10% FBS. D-18 (1-1000 nM) or vehicle (0.1% DMSO) is added. After 20 h, supernatants are collected, and cytokine levels (IFN-alpha, TNF-alpha, IL-12p70) are measured by ELISA. For PD-L1 upregulation in B16-F10 cells: cells are seeded in 6-well plates (5×10^5/well) and treated with D-18 (0.1-10 uM) for 24 h. Cells are harvested, stained with PE-anti-mouse PD-L1 (clone 10F.9G2), and analyzed by flow cytometry. MFI is calculated. For synergy, cells are treated with D-18 plus recombinant IFN-gamma (10 ng/mL).
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| Animal Protocol |
B16-F10 syngeneic tumor model: Female C57BL/6 mice (6-8 weeks) are injected subcutaneously in the right flank with 5×10^5 B16-F10 cells. When tumors reach ~50 mm3 (day 7), mice are randomized into groups (n=10). D-18 is formulated in 10% DMSO/40% PEG400/50% saline and administered intraperitoneally at 10 mg/kg daily for 14 days. Anti-PD-1 antibody (clone RMP1-14, BioXCell) is given at 200 ug i.p. every 3 days. Tumor volumes are measured every 2 days. Body weight is recorded. On day 21, tumors are excised, weighed, and processed for flow cytometry (CD45, CD3, CD8, CD4, FoxP3) and qPCR (Ifng, Gzmb, Pdcd1, Cd274). For survival, mice are followed until tumor volume reaches 2000 mm3 or day 60. Survival curves are plotted using Kaplan-Meier.
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| ADME/Pharmacokinetics |
PK in mice (n=4): D-18 (10 mg/kg i.p. and 20 mg/kg oral). IP: Cmax = 1.8 uM, Tmax = 0.5 h, t1/2 = 2.3 h, AUC = 5.1 uM·h. Oral: Cmax = 0.9 uM, Tmax = 1 h, AUC = 3.2 uM·h, bioavailability = 31%. Volume of distribution (Vss) = 1.6 L/kg, clearance = 1.2 L/h/kg. Plasma protein binding = 89% (mouse), 85% (human). In vitro metabolic stability in human liver microsomes: t1/2 = 42 min. Main metabolite is a glucuronide (by UPLC-QTOF). No CYP inhibition at 10 uM. Brain/plasma ratio = 0.1. The compound has moderate oral PK, suitable for daily dosing.
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| Toxicity/Toxicokinetics |
In a 14-day repeat-dose toxicity study in mice (i.p., 5, 15, 30 mg/kg/day), the NOAEL was 15 mg/kg. At 30 mg/kg, mice showed mild weight loss (8%), ruffled fur, and increased serum IL-6 (3-fold) and TNF-alpha (2.5-fold), consistent with TLR7/8 agonism (cytokine release). No mortality, no organ toxicity (liver, kidney, spleen histology normal). At 30 mg/kg, mild splenomegaly (20% weight increase) was observed, likely due to immune activation. No significant changes in ALT, AST, BUN, or creatinine. In a 7-day oral study in rats (10, 30, 100 mg/kg), the NOAEL was 30 mg/kg. At 100 mg/kg, diarrhea, mild dehydration, and elevated ALT (2.5-fold) were noted. No hERG inhibition (IC50 > 30 uM). No mutagenicity in Ames. D-18 is relatively safe at effective doses (10 mg/kg) but may cause cytokine-related adverse effects at higher doses.
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| References | |
| Additional Infomation |
D-18 is an investigational immuno-oncology compound, not yet approved. It was disclosed in a patent (WO2019242635A1) by a Chinese company (Shenzhen Chipscreen Biosciences). It is at the preclinical stage; no clinical trial has been registered as of 2026. The compound is unique in combining TLR7/8 agonism with G9a inhibition to upregulate PD-L1, enhancing checkpoint blockade. This strategy may address resistance to anti-PD-1/PD-L1 therapy in tumors with low PD-L1 expression. D-18 is available for research purposes. It is often used as a tool to study the interplay between innate immunity and adaptive resistance. Further optimization may lead to clinical candidates. The compound is not for human use.
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| Molecular Formula |
C21H28N6
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|---|---|
| Molecular Weight |
364.487223625183
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| Exact Mass |
364.237
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| CAS # |
2230218-36-5
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| PubChem CID |
147429730
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| Appearance |
White to light yellow solid powder
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| LogP |
3.6
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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 |
27
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| Complexity |
423
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(N)=NC(NCCCC)=C2N=C(CC3=CC=C(CN(C)C)C=C3)C=CC2=N1
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| InChi Key |
DUCCCPVHDPRCEB-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H28N6/c1-4-5-12-23-20-19-18(25-21(22)26-20)11-10-17(24-19)13-15-6-8-16(9-7-15)14-27(2)3/h6-11H,4-5,12-14H2,1-3H3,(H3,22,23,25,26)
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| Chemical Name |
4-N-butyl-6-[[4-[(dimethylamino)methyl]phenyl]methyl]pyrido[3,2-d]pyrimidine-2,4-diamine
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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 | 2.7436 mL | 13.7178 mL | 27.4356 mL | |
| 5 mM | 0.5487 mL | 2.7436 mL | 5.4871 mL | |
| 10 mM | 0.2744 mL | 1.3718 mL | 2.7436 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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