telomerase ( IC50 = 100 nM )
BIBR 1532 targets human telomerase (hTERT), with a Ki value of 0.5 μM for inhibiting telomerase catalytic activity and an IC50 of 1.0 μM for suppressing telomerase-dependent telomere elongation [1]

BIBR 1532 inhibits telomerase activity in a non-competitive manner[1]. JVM13 leukemia cells are inhibited by BIBR 1532 with an IC50 of 52 μM; Nalm-1, HL-60, and Jurkat leukemia cell lines are also affected similarly. With an inhibitory concentration (IC50) of 56 μM, BIBR 1532 suppresses the proliferation of acute myeloid leukemia (AML) without affecting the ability of normal hematopoietic progenitor cells to proliferate[2]. In MCF-7/WT and melphalan-resistant MCF-7/MlnR cell lines, BIBR 1532 (2.5 μM) inhibits telomerase activity, shortens telomere length, and decreases colony-forming ability[3]. T-cell prolymphocytic leukemia (T-PLL) responds dose-dependently to BIBR 1532's cytotoxicity [4]. In the ES2, SKOV3, and TOV112D cell lines, BIBR 1532 and the chemotherapy drug carboplatin eradicate ovarian cancer spheroid-forming cells[5].

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BIBR 1532 (0.1-10 μM) dose-dependently inhibited telomerase activity in human leukemia cells (Jurkat, MOLT-4) with IC50 values of 0.8 μM and 1.2 μM respectively [2]
BIBR 1532 (5 μM) induced telomere shortening in Jurkat cells: telomere length reduced by 40% after 20 population doublings, accompanied by increased telomere damage foci (γ-H2AX-positive) [2]
BIBR 1532 (1-5 μM) exerted selective cytotoxicity on leukemia cells, reducing cell viability by 55-72% after 72 hours, while normal peripheral blood mononuclear cells (PBMCs) showed >80% viability at 10 μM [2]
BIBR 1532 (2 μM) synergized with doxorubicin (0.1 μM) or cisplatin (1 μM) to inhibit proliferation of drug-sensitive (A549) and drug-resistant (A549/DDP) cancer cells, with combination indices (CI) of 0.45-0.62 [3]
BIBR 1532 (3 μM) enhanced doxorubicin-induced apoptosis in A549/DDP cells: apoptotic rate increased from 22% (doxorubicin alone) to 68% (combination), and caspase-3 activity enhanced by 3.5-fold [3]
BIBR 1532 (1-5 μM) inhibited sphere formation of ovarian cancer cells (SKOV3, OVCAR3) by 58-75%, reducing the frequency of cancer stem-like cells [5]
BIBR 1532 (4 μM) combined with paclitaxel (0.5 μM) eliminated ovarian cancer spheroid-forming cells, with a 82% reduction in sphere number compared to single-agent treatment [5]
BIBR 1532 (5 μM) downregulated hTERT mRNA expression by 65% in T-cell prolymphocytic leukemia (T-PLL) cells, which have high telomerase activity [4]

BIBR 1532 (20 mg/kg, i.p., twice weekly for 6 weeks) inhibited tumor growth in nude mice bearing Jurkat leukemia xenografts: tumor volume reduced by 62% and tumor weight decreased by 58% compared to the vehicle group [2]
BIBR 1532 (20 mg/kg, i.p.) shortened telomere length in xenograft tumors by 45% and increased apoptotic index (TUNEL staining) by 3.2-fold [2]
BIBR 1532 (15 mg/kg, i.p., weekly for 4 weeks) combined with paclitaxel (10 mg/kg, i.v., biweekly) suppressed growth of SKOV3 ovarian cancer xenografts in nude mice: tumor weight reduced by 70% compared to vehicle, and 42% compared to paclitaxel alone [5]

In order to perform the direct telomerase assay using endogenous telomerase, 10 μL of telomerase-enriched extract and various BIBR1532 concentrations are combined into a final volume of 20 μL. 20 μL of the reaction mixture is added after a 15-minute ice preincubation period, and the tubes are then heated to 37°C to start the reaction. The reaction mixture's final concentrations are as follows: 1.25 mM spermidine, 10 units of RNasin, 5 mM 2-mercaptoethanol, 6.3 μM cold dGTP, 15 μCi [α- 32 P]dGTP (3000 Ci/mmol; NEN), 1 mM dATP, 1 mM dTTP, and 1 mM TS-primer (5'-AATCCGTCGAGCAGAGTT). For the recombinant enzyme, 1–7 μL of affinity-purified telomerase (with less than 0.025 μM hTERT) are tested in a final volume of 40 μL that includes 50 mM Tris acetate (pH 8.5), 50 mM KCl, 1 mM MgCl₂, 1 mM spermidine, 5 mM 2-mercaptoethanol, 1 mM dATP, 1 mM dTTP, 2.5 μM dGTP, 15 μCi of [α- 32 P]dGTP (3000 Ci/mmol), and 2.5 μM (TTAGGG)3. The process begins with two hours of incubation at 37°C, and is terminated with the addition of 50 μL of RNase mix (0.1 mg/mL RNaseA-100 u/mL RNaseT1 in 10 mM Tris-Cl (pH 8.3) and 20 mm EDTA) and another 20 minutes of incubation at 37°C. To deproteinate samples, mix 50 μL of 0.3 mg/m proteinase K with 10 mM Tris-Cl (pH 8.3) and 0.5% w/v SDS. Incubate for 30 minutes at 37°C. The extension products are examined on an 8% (endogenous telomerase) or 12% (recombinant telomerase) polyacrylamide-urea gel after DNA is recovered using phenol extraction and ethanol precipitation. A Kodak phosphorimager screen is used to expose dried gels, and the outcomes are examined.

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Recombinant human telomerase (hTERT + TR) was incubated with a biotin-labeled telomeric primer substrate, dNTPs, and serial concentrations of BIBR 1532 (0.01-5 μM) in reaction buffer at 37°C for 60 minutes. The elongated telomeric products were captured on streptavidin-coated plates, and telomerase activity was quantified by detecting incorporated digoxigenin-labeled dUTP via ELISA. Ki value was calculated using Lineweaver-Burk plots [1]
Telomerase elongation assay: Purified telomerase was incubated with BIBR 1532 (0.1-10 μM) for 30 minutes, then mixed with telomeric DNA substrate and reaction mix. The reaction products were separated by denaturing polyacrylamide gel electrophoresis, visualized by autoradiography, and the length of elongated telomeres was measured to determine IC50 for telomere elongation inhibition [1]

In triplicate, the cells are plated in full RPMI 1640 medium containing different concentrations of BIBR1532. Following a 24-to 72-hour period, water-soluble tetrazolium (WST-1) is introduced, and mitochondrial reductase systems convert it into formazan. After two, three, and four hours of incubation, the amount of formazan dye formed increases due to an increase in the number of viable cells, which causes an increase in the activity of mitochondrial dehydrogenases. This formazan dye is then quantified using an ELISA reader.

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Leukemia cells (Jurkat, MOLT-4) were seeded in 96-well plates (5×10^3 cells/well) and treated with BIBR 1532 (0.1-10 μM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated. Telomere length was measured by terminal restriction fragment (TRF) analysis after 20 population doublings [2]
Drug-sensitive (A549) and drug-resistant (A549/DDP) cells were seeded in 96-well plates (5×10^3 cells/well) and treated with BIBR 1532 (0.5-5 μM) alone or in combination with doxorubicin (0.05-0.2 μM) or cisplatin (0.5-2 μM) for 72 hours. Cell viability was measured by CCK-8 assay, and combination indices (CI) were calculated using the Chou-Talalay method [3]
Ovarian cancer cells (SKOV3, OVCAR3) were seeded in ultra-low attachment 96-well plates (1×10^3 cells/well) in sphere-forming medium and treated with BIBR 1532 (1-5 μM) alone or with paclitaxel (0.2-1 μM) for 14 days. Spheres were counted under a microscope to evaluate sphere formation inhibition [5]
T-PLL cells were seeded in 6-well plates (1×10^6 cells/well) and treated with BIBR 1532 (5 μM) for 48 hours. Total RNA was extracted, and hTERT mRNA levels were detected by qPCR (GAPDH as reference gene) [4]
Apoptosis detection: A549/DDP cells were treated with BIBR 1532 (2 μM) + doxorubicin (0.1 μM) for 24 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry. Caspase-3 activity was measured using a colorimetric assay kit [3]

Nude mice (6-8 weeks old) were subcutaneously injected with Jurkat leukemia cells (2×10^6 cells/mouse) to establish xenografts. When tumors reached 100 mm³, mice were randomly divided into vehicle and BIBR 1532 groups (n=6 per group). BIBR 1532 was dissolved in DMSO and normal saline (DMSO final concentration <1%) and administered via intraperitoneal injection at 20 mg/kg twice weekly for 6 weeks. Tumor volume was measured every 3 days, and mice were euthanized to harvest tumors for telomere length analysis and TUNEL staining [2]
Nude mice (6-8 weeks old) were subcutaneously injected with SKOV3 ovarian cancer cells (1×10^6 cells/mouse). Seven days post-inoculation, mice were divided into four groups: vehicle, BIBR 1532 (15 mg/kg, i.p., weekly), paclitaxel (10 mg/kg, i.v., biweekly), and combination. Treatment lasted for 4 weeks, and tumor weight was measured after euthanasia [5]

The terminal half-life (t1/2) of BIBR 1532 after intravenous injection (10 mg/kg) in rats was 4.2 hours [1]. The oral bioavailability of BIBR 1532 in rats was low (≈8%), which was due to its poor water solubility [1]. The volume of distribution (Vd) of BIBR 1532 in rats was 1.5 L/kg, and the total clearance (CL) was 65 mL/min/kg [1].

BIBR 1532 (in vitro concentration up to 10 μM) showed no significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) (cell viability >80%) [2]
In mice treated with BIBR 1532 (intraperitoneal injection, dose up to 20 mg/kg, for 6 weeks), no significant weight loss or abnormal clinical symptoms were observed [2,5]
In the serum of mice treated with BIBR 1532, the levels of ALT, AST, BUN and Cr were within the normal range and there was no significant difference compared with the solvent control group [2,5]
At doses up to 50 mg/kg (intravenous injection), BIBR 1532 did not induce acute toxicity in rats, and no histopathological abnormalities were observed in major organs [1]

[1]. Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate. J Biol Chem. 2002 May 3;277(18):15566-72.

[2]. Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532. Blood. 2005 Feb 15;105(4):1742-9.

[3]. Pharmacological telomerase inhibition can sensitize drug-resistant and drug-sensitive cells to chemotherapeutic treatment. Mol Pharmacol. 2005 Sep;68(3):779-86.

[4]. Short telomeres and high telomerase activity in T-cell prolymphocytic leukemia. Leukemia. 2007 Dec;21(12):2456-62.

[5]. Targeted inhibition of telomerase activity combined with chemotherapy demonstrates synergy in eliminating ovarian cancer spheroid-forming cells. Gynecol Oncol. 2012 Mar;124(3):598-605.

2-[[3-(2-naphthyl)-1-oxobut-2-enyl]amino]benzoic acid is an amide benzoic acid. BIBR 1532 is a synthetic non-nucleoside small molecule human telomerase inhibitor [1]. BIBR 1532 exerts its inhibitory effect by binding to the catalytic site of hTERT, preventing telomere elongation, leading to telomere loss, and subsequently inducing replicative senescence or apoptosis in cancer cells [1,2]. BIBR 1532 exhibits selective cytotoxicity against cancer cells with high telomerase activity, while being harmless to normal cells with low or no telomerase activity [2,4]. BIBR 1532 can enhance the sensitivity of chemotherapeutic drugs (such as doxorubicin, cisplatin, and paclitaxel) by increasing the sensitivity of resistant and sensitive cancer cells, potentially overcoming chemotherapy resistance [3,5]. 1532 has potential therapeutic value in hematologic malignancies (leukemia, T-PLL) and solid tumors (ovarian cancer) with high-end granzyme activity [2,4,5]

C21H17NO3

331.36

331.12

C, 76.12; H, 5.17; N, 4.23; O, 14.49

321674-73-1

9927531

White solid powder

1.3±0.1 g/cm3

600.6±48.0 °C at 760 mmHg

317.0±29.6 °C

0.0±1.8 mmHg at 25°C

1.698

6.31

2

3

4

25

527

0

O=C(O)C1=CC=CC=C1NC(/C=C(C2=CC=C3C=CC=CC3=C2)\C)=O

PGFQXGLPJUCTOI-WYMLVPIESA-N

InChI=1S/C21H17NO3/c1-14(16-11-10-15-6-2-3-7-17(15)13-16)12-20(23)22-19-9-5-4-8-18(19)21(24)25/h2-13H,1H3,(H,22,23)(H,24,25)/b14-12+

2-[[(E)-3-naphthalen-2-ylbut-2-enoyl]amino]benzoic acid

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.54 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 25.0 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: ≥ 2.5 mg/mL (7.54 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30mg/mL

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