Human telomeric G-quadruplex (ΔT_m = 26.6°C at 1 μM); HSP90 promoter G-quadruplex (HSP90A: ΔT_m = 33.1°C, HSP90B: ΔT_m = 28.6°C at 1 μM) [1]

3d exhibits potent antiproliferative activity against multiple cancer cell lines, with IC₅₀ values of 10 nM in MIA PaCa-2 (pancreatic), 70 nM in MCF-7 (breast), and 230 nM in WI-38 (normal fibroblasts). In MIA PaCa-2 cells, it induces cellular senescence (50–60% senescent cells at 3–4 nM after 7 days) but does not inhibit telomerase activity even at 10 μM, as confirmed by TRAP-LIG assay.

qPCR analysis reveals dose-dependent transcriptional modulation: significant upregulation of DNA damage response genes (CDKN1A, DDT3, GADD45A/G, PPM1D) and downregulation of telomere maintenance genes (hPOT1, PARP1) at 20–80 nM concentrations after 24 h exposure.

FRET melting assays demonstrate high stabilization of human telomeric quadruplex (ΔT_m = 26.6°C) and HSP90 promoter quadruplexes (ΔT_m = 33.1°C/28.6°C) at 1 μM, with superior quadruplex-vs-duplex selectivity compared to earlier analogues.

FRET-based melting assays were performed using FAM/TAMRA-labeled oligonucleotides (human telomeric sequence: 5′-FAM-d[AGGG(TTAGGG)₃]-TAMRA-3′; HSP90 promoter sequences HSP90A/B). Compounds (1 μM final) in 60 mM K⁺ cacodylate buffer (pH 7.4) were heated from 30°C to 100°C at 0.5°C intervals. Fluorescence was monitored (ex: 450–495 nm; em: 515–545 nm), and ΔT_m values calculated from triplicate data.

TRAP-LIG telomerase activity assay: MIA PaCa-2 cell extracts (1 μg protein) were incubated with TS primer, elongated products purified, PCR-amplified with ACX primer, and analyzed via PAGE/SYBR green staining. No telomerase inhibition was observed at ≤10 μM 3d.

Cell proliferation was assessed by SRB assay. Cells (MIA PaCa-2, MCF-7, A549, WI-38) were treated with 2-fold serial dilutions of 3d for 96 h. Absorbance at 540 nm measured cell viability, and IC₅₀ values were calculated relative to untreated controls.

Senescence detection: MIA PaCa-2 cells treated with 3d (3.3–6.6 nM) for 7 days were fixed, stained with β-galactosidase kit, and senescent (blue) cells counted microscopically.

qPCR arrays: Total RNA from 3d-treated MIA PaCa-2 cells (20–160 nM, 24 h) was extracted, reverse-transcribed, and analyzed using pathway-focused arrays (telomere maintenance/DNA damage response). Data normalized to housekeeping genes; fold-changes calculated via ΔΔC_T method.

[1]. Structure-based design and evaluation of naphthalene diimide G-quadruplex ligands as telomere targeting agents in pancreatic cancer cells. J Med Chem. 2013 Apr 11;56(7):2959-74.

3d is a dimorpholine-substituted naphthalene diimide formulated through structure-based optimization. Its crystal structure (PDB: 3UYH) confirms its binding to the 3′ G-tetramer parallel to the human telomere quadruplex, with the morpholine group maintaining groove interactions via water-mediated hydrogen bonding. In pancreatic cancer cells, it exhibits 10-fold greater potency than tetra-N-methylpiperazine analogs (4), likely due to its reduced cationic properties, thereby enhancing cellular uptake. Mechanistically, it disrupts telomere maintenance by substituting POT1 and induces cellular senescence without inhibiting telomerase, triggering various DNA damage responses. No in vivo or ADME data have been reported. Terminally positively charged tetrasubstituted naphthalene diimide (ND) derivatives are potent stabilizers of human telomeres and gene promoter DNA quadruplexes and inhibit the growth of human cancer cells in vitro and in vivo. This study reports the enhancement of the pharmacological properties of early ND compounds through structure-based drug design. Crystal structures of three intramolecular tetras of human telomere showed that two of the four strongly basic N-methylpiperazine groups could be replaced by weaker basic morpholine groups without losing the intermolecular interactions in the tetrasand grooves. The new compound maintained a high affinity for human telomere tetrasand DNA, but its inhibitory activity against the MIA PaCa-2 pancreatic cancer cell line was increased by 10-fold, with an IC50 of approximately 10 nM. The lead compound induced cellular senescence, but did not inhibit telomerase activity at the nanomolar dose level required to inhibit cell proliferation. Gene chip qPCR analysis of MIA PaCa-2 cells treated with the lead compound showed that the expression of a specific set of genes was significantly dose-dependently regulated, including the expression of genes that strongly induce DNA damage response, such as CDKN1A, DDIT3, GADD45A/G, and PPM1D, and the expression of genes involved in telomere maintenance, such as hPOT1 and PARP1, were inhibited. [1]

C44H66N10O6

831.06

830.5167

C, 63.59; H, 8.01; N, 16.85; O, 11.55

1429028-96-5

135566608

Blue to purple solid powder

-0.4

2

14

17

60

1810

0

C12=CC(NCCCN3CCN(C)CC3)=C3C4C1=C(C(NCCCN1CCN(C)CC1)=CC=4C(=O)N(CCCN1CCOCC1)C3=O)C(=O)N(CCCN1CCOCC1)C2=O

KOQWCTULEQQTBE-UHFFFAOYSA-N

InChI=1S/C44H66N10O6/c1-47-15-19-49(20-16-47)9-3-7-45-35-31-33-38-37-34(42(56)53(43(57)39(35)37)13-5-11-51-23-27-59-28-24-51)32-36(46-8-4-10-50-21-17-48(2)18-22-50)40(38)44(58)54(41(33)55)14-6-12-52-25-29-60-30-26-52/h31-32,45,55H,3-30H2,1-2H3

14-hydroxy-3-[3-(4-methylpiperazin-1-yl)propylamino]-10-[3-(4-methylpiperazin-1-yl)propylimino]-6,13-bis(3-morpholin-4-ylpropyl)-6,13-diazatetracyclo[6.6.2.04,16.011,15]hexadeca-1(14),2,4(16),8,11(15)-pentaene-5,7,12-trione

MM41; 1429028-96-5; MM-41; compound 3d; 4,9-bis((3-(4-methylpiperazin-1-yl)propyl)amino)-2,7-bis(3-morpholinopropyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; 4,9-Bis{[3-(4-Methylpiperazin-1-Yl)propyl]amino}-2,7-Bis[3-(Morpholin-4-Yl)propyl]benzo[lmn][3,8]phenanthroline-1,3,6,8(2h,7h)-Tetrone; 0DX; CHEMBL2335138; SCHEMBL16048175;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~33.33 mg/mL (~40.11 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)