666-15 is a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 µM). It is a weak inhibitor of the CREB-CBP interaction (IC50 = 18.27 ± 2.81 µM) and does not directly bind to CREB or CBP. [1]

The consequences of MSN overexpression, such as cell ischemia, weakening, soft agar colony capability, and production of CREB downstream genes, are significantly blocked by 666-15 (73 nM) when given for a 12-hour period. 666-15 Prevents MSN-induced CREB phosphorylation 666-15 (1 μM; wait two hours) efficiently prevents PE-induced CREB phosphorylation. 666-15 suppresses middle ER activation, including PE + ylation, and dramatically lowers the protein production of ANP and β-MHC [2]. The phosphorylation of IRE1 and the expression of GRP78, CHOP, and ATF6 in the siRNA + 666-15 group and PE + si-CTRP3 + 666-15 group [3]. Template growth is substantially inhibited by 666-15. In MDA-MB-231 and MDA-MB-468 cells, the GI50 of 666-15 was 73 and 46 nM, respectively. The majority of actions were observed in A549 and MCF-7 cells, with GI50 values of 0.47 and 0.31 μM, respectively. With an IC50 of 18.27 μM, 666-15 was also discovered to have a relatively poor CREB-CBP binding reaction. In live cells, 666-15 transcribes CREB transcriptional activity without the need for direct binding reactions to CBP or CREB. Nuclear trap-related protein 1 (Nurr1/NR4A2) transcript levels are one of the endogenous CREB target genes that are inhibited by 666-15, which also transcribes CREB [1].

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666-15 potently inhibits CREB-mediated gene transcription in a CRE Renilla luciferase reporter assay in HEK 293T cells with an IC50 of 81 nM, representing a 28-fold improvement over the parent compound 3a. [1]
It selectively inhibits forskolin-stimulated expression of the endogenous CREB target gene Nurr1/NR4A2 in HEK 293T cells in a dose-dependent manner, with significant inhibition observed at 50 nM. [1]
In transcription factor selectivity assays, 666-15 showed much weaker inhibition of VP16-CREB, p53-mediated, and NF-κB-mediated gene transcription, indicating selectivity for CREB-mediated transcription. [1]
666-15 exhibits potent antiproliferative activity against a panel of cancer cell lines, with GI50 values of 0.47 µM (A549 lung cancer), 0.31 µM (MCF-7 breast cancer), 0.073 µM (MDA-MB-231 breast cancer), and 0.046 µM (MDA-MB-468 breast cancer). [1]
It shows selective toxicity towards cancer cells. No significant growth inhibition was observed in normal human mammary epithelial cells (HMEC) and human foreskin fibroblasts (HFF) at concentrations up to 1 µM, which is more than 10-fold higher than its GI50 in MDA-MB-231 and MDA-MB-468 cells. [1]

Results: It has a good effect on the growth of breast cancer. inhibitory effect. 666-15 (10 mg/kg; intraperitoneal injection; once a week; 11 consecutive weeks) can have a good effect on cell proliferation when used alone, and the effect is better when combined with RP-56976 (DOC) [2]. Animal model: 1-month-old female nude mice, MDA-MB-231 or T47D cells [2]. Dosage: 10 mg/kg Administration method: IP; once a week; for 11 consecutive weeks.

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In an MDA-MB-468 xenograft model in nude mice, intraperitoneal administration of 666-15 at 10 mg/kg once daily, 5 days per week for 5 weeks, completely suppressed tumor growth (causing tumor stasis) compared to the vehicle-treated group, where tumor volume more than tripled. [1]

The inhibition of CREB-mediated gene transcription was assessed using a CREB Renilla luciferase reporter assay. HEK 293T cells were transfected with a Renilla luciferase reporter under the control of a synthetic promoter containing three copies of cAMP-response elements (CRE). Transfected cells were treated with different concentrations of compounds for 30 minutes. Subsequently, forskolin (10 µM), an activator of adenylate cyclase, was added to stimulate CREB's transcription activity and induce luciferase synthesis. Luciferase activity was measured and normalized to total protein content. The IC50 values were derived from nonlinear regression analysis of the concentration-response curves. [1]

Cell Proliferation Assay[2]
Cell Types: CTRL or MSN overexpression MDA-MB-231 Cell
Tested Concentrations: 73 nM
Incubation Duration: 12 hrs (hours)
Experimental Results: Dramatically blocked cell proliferation caused by MSN overexpression.

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Western Blot Analysis [3]
Cell Types: NRCMs
Tested Concentrations: 1 μM
Incubation Duration: 2 hrs (hours) (pretreatment)
Experimental Results: Effectively inhibited PE-induced CREB phosphorylation.

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Antiproliferative activity was evaluated using the colorimetric MTT assay. Cells were plated in 96-well plates, allowed to attach overnight, and then treated with various concentrations of compounds for 72 hours. MTT reagent was added and incubated for 3 hours. The formed formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. The percentage of growth was calculated relative to control and initial cell density, and GI50 values were determined from concentration-response curves. [1]
The effect on endogenous CREB target gene expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). HEK 293T cells were treated with compounds for 1 hour, followed by stimulation with forskolin (10 µM) or vehicle for 45 minutes. Total RNA was isolated, treated with DNase I, and reverse transcribed into cDNA. Quantitative real-time PCR was performed using specific primers for the target gene Nurr1/NR4A2, with hypoxanthine phosphoribosyltransferase 1 (HPRT) as the reference gene. Relative mRNA levels were calculated using the 2^(-ΔΔCT) method. [1]

Animal/Disease Models: 1-month-old female nude mice, MDA-MB-231 or T47D cells [2]
Doses: 10 mg/kg
Route of Administration: IP; once a week; for 11 consecutive weeks.
Experimental Results: It has a good effect on the growth of breast cancer. inhibitory effect.

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For the in vivo antitumor efficacy study, female BALB/c nude mice (6-8 weeks old) were inoculated subcutaneously at the right flank with MDA-MB-468 cells (5 × 10^6 cells) suspended in a 1:1 mixture of HBSS and Matrigel. When the average tumor volume reached approximately 100 mm³, mice were randomized into treatment groups. 666-15 was dissolved in a vehicle containing 1% N-methylpyrrolidone (NMP) and 5% Tween-80 in water. Mice received either vehicle or666-15 at a dose of 10 mg/kg via intraperitoneal injection once daily, 5 consecutive days per week, for a total of 5 weeks. Tumor dimensions were measured 2-3 times per week using a digital caliper, and tumor volume was calculated. Body weights were also monitored. [1]

In vitro experiments showed that compound 666-15 at concentrations up to 1 µM did not significantly inhibit the growth of normal human cell lines (HMEC and HFF). [1] In an in vivo study of MDA-MB-468 xenografts, mice injected intraperitoneally with 10 mg/kg of compound 666-15 for 5 weeks did not show significant weight loss or toxicity. [1]

[1]. Identification of a Potent Inhibitor of CREB-Mediated Gene Transcription with Efficacious in Vivo Anticancer Activity. J Med Chem. 2015 Jun 25;58(12):5075-87.

[2]. Interfering MSN-NONO complex-activated CREB signaling serves as a therapeutic strategy for triple-negative breast cancer. Sci Adv. 2020 Feb 19;6(8):eaaw9960.

[3]. C1q-TNF-related protein-3 attenuates pressure overload-induced cardiac hypertrophy by suppressing the p38/CREB pathway and p38-induced ER stress. Cell Death Dis. 2019 Jul 8;10(7):520.

Compound 666-15 (also known as compound 3i) is a naphthamide derivative optimized from lead compound 3a. Structure-activity relationship studies showed that the phenolic hydroxyl group on the chlorobenzene ring, the primary amino group on the side chain, and the two naphthyl rings are crucial to the activity. In addition, the specific length of the carbon linker between the naphthyl ring and the side chain is also crucial to the optimal efficacy. Molecular modeling suggests that highly efficient analogs like compound 666-15 have a compact conformation and intramolecular hydrogen bonding and π-π stacking, which may contribute to their activity. This study supports CREB as a promising target for cancer therapy. [1]

C33H31CL2N3O5

620.5223

619.164

1433286-70-4

71566396

White to off-white solid powder

5

6

11

43

879

0

LELLCPHHYHLWBH-UHFFFAOYSA-N

InChI=1S/C33H30ClN3O5.ClH/c34-25-10-11-28(29(38)20-25)37-33(40)27-17-22-7-2-4-9-24(22)19-31(27)42-15-13-36-32(39)26-16-21-6-1-3-8-23(21)18-30(26)41-14-5-12-35;/h1-4,6-11,16-20,38H,5,12-15,35H2,(H,36,39)(H,37,40);1H

3-(3-Aminopropoxy)-N-[2-[[3-[[(4-chloro-2-hydroxyphenyl)amino]carbonyl]-2-naphthalenyl]oxy]ethyl]-2-naphthalenecarboxamide hydrochloride

66615; 666 15; 666-15; Compound 3i

2934.99.9001

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, avoid exposure to moisture.

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

DMSO : ~125 mg/mL (~201.44 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (3.35 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.35 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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