CD147
CD147 (basigin, EMMPRIN) [2]

Treatment with AC-73 (5-10 μM; 24 hours) dramatically decreased the capacity of SMMC-7721 and Huh-7 cells to migrate in a dose-dependent manner, as well as the invasion of both HCC cells. The effects were seen within 24 hours. Treatment with AC-73 decreases HCC metastases. The treatment of two HCC cells with AC-73 at a maximum dosage of 20 μM did not significantly affect the viability of the cells. Glu64 and Glu73 in the N-terminal IgC2 domain, which are situated in the dimer interface of CD147, are potential binding sites for AC-73 on CD147 [1]. At a dose of 10 μM, AC-73 (5-10 μM; 24 hours; SMMC-7721 cells) treatment can considerably reduce MMP-2 and MMP-9 mRNA expression, with MMP-2 being the most affected, but not MMP-2. Impact. 1. MMP-11, MMP-13, MMP-7, or MMP-8. Gelatin zymography tests and RT-qPCR analysis demonstrated that AC-73 can dose-dependently lower MMP-2 mRNA expression and protein secretion [1]. The phosphorylation of ERK1/2 and STAT3 is dose-dependently inhibited by AC-73 (5–20 μM; 6 hours; SMMC-7721 cells) treatment [1].

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AC-73 inhibits leukemic cell proliferation in a time- and dose-dependent manner. In AML cell lines (U937, NB4, HL-60, NB4-R4, MV4-11, Kasumi-1), treatment with AC-73 at 2.5, 5, and 10 μM for 1–4 days reduces cell growth. NB4 and NB4-R4 cells are more sensitive (significant growth inhibition at 2.5 μM), while MV4-11 and Kasumi-1 require higher concentrations (5–10 μM) or longer treatment (3–4 days) for significant inhibition. [2]
- AC-73 induces apoptosis in leukemic cells in a dose-dependent manner, as measured by Annexin V/PI flow cytometry. NB4 and NB4-R4 show higher apoptosis at low-dose (2.5 μM) compared to U937 and HL-60. [2]
- AC-73 reduces cell viability in AML cell lines. For NB4 and NB4-R4, 2.5 μM AC-73 decreases viability over time (day 1–3). For U937 and HL-60, higher doses (5–10 μM) are required. [2]
- AC-73 does not affect cell cycle distribution in leukemic cell lines. [2]
- AC-73 does not induce differentiation in leukemic cell lines (no significant change in CD11b, CD14, CD15 expression). [2]
- AC-73 (5 μM) decreases the in vitro colony formation of U937 and NB-4 cells, indicating inhibition of clonogenic capacity. [2]
- AC-73 induces autophagy in leukemic cells. Western blot shows dose-dependent increase of LC3-II/LC3-I ratio in U937 and NB4 cells. Flow cytometry with Cyto-ID assay confirms dose-dependent autophagy induction (0, 2.5, 5, 10 μM AC-73 for 72 h) in CD147-expressing leukemic cells but not in CHO cells (CD147-negative). [2]
- AC-73 (5 μM for 3 days) decreases phospho-ERK1/2 and phospho-STAT3 (S727) levels without affecting total ERK and STAT3 in leukemic cell lines (U937, NB4, MV4-11). [2]
- AC-73 (2.5 μM for 24 h followed by 48 h co-treatment with Ara-C or ATO) enhances sensitivity of leukemic cells to chemotherapy. Combination with Ara-C (0.01–1 μM) or ATO (0.01–1 μM) significantly decreases cell viability compared to single agents in all cell lines (U937, HL-60, NB4, NB4-R4, MV4-11, Kasumi-1). AC-73 potentiates Ara-C sensitivity even in low-sensitivity lines (MV4-11, Kasumi-1). [2]
- AC-73 combined with ATO increases autophagy flux in both M3 (NB4) and non-M3 (U937) leukemic cells compared to single agent or control. [2]
- In primary AML blasts (M2, M3, M5 subtypes), AC-73 alone at 10 μM induces apoptosis, but lower doses (2.5, 5 μM) do not. However, AC-73 (2.5–5 μM) induces autophagy (increased LC3-II/LC3-I ratio). Combination of low-dose AC-73 (2.5 μM) with ATO or Ara-C (0.1–1 μM) more efficiently decreases blast viability than single agents. [2]
- AC-73 (5 μM) has moderate growth inhibition on normal CD34+ HPCs without affecting cell cycling or differentiation. Morphology analysis shows normal progression along granulocytic and monocytic differentiation. [2]
- AC-73 has no effect on cell growth and apoptosis of CHO cells (CD147-negative). [2]

The incidence of metastases in nude mice was dramatically decreased by AC-73 therapy (25–50 mg/kg; 4 weeks; male BALB/c nu/nu mice). In a dose-dependent manner, AC-73 suppresses the phosphorylation of STAT3 and ERK1/2. Moreover, AC-73 lowers MMP-2. In vivo tumor cell growth cannot be inhibited by AC-73 [1].

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In an orthotopic transplant nude mouse model (SMMC-7721 cells injected into left liver lobe), treatment started 1 week after implantation. AC-73 (25 or 50 mg/kg/day) significantly decreased the incidence of intrahepatic metastatic foci compared to vehicle (Cremophor EL/ethanol), evaluated by gross pathology [1].
- AC-73 inhibited the phosphorylation of ERK1/2 and STAT3 in original orthotopic tumors in a dose-dependent manner, as shown by both Western blot and immunohistochemistry (IHC) analyses [1].
- Expression of MMP-2 was also reduced by AC-73 in tissue (IHC), while CD147 expression showed no significant difference among groups [1].
- AC-73 did not inhibit tumor cell proliferation in vivo, as determined by calculating the maximum tumor diameter of in situ focal [1].

Surface plasmon resonance (SPR) assay[1]
SPR measurements were performed using the ProteOn XPR36 system with ProteOn GLH sensor chips. ProteOn PBS/Tween running buffer (phosphate-buffered saline, pH 7.4, with 0.005% Tween 20) containing 0.1% DMSO was used as a running buffer throughout, and all experiments were performed at 25°C. Purified CD147wt or CD147mt was immobilized on a GLH chip. Each compound was used at the same concentration (100 μM) and simultaneously injected in the horizontal direction, with running buffer injected as a control. Dissociation was monitored for 15 min. The data were analyzed using ProteOn Manager Software, version 2.0.

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Non-denaturing SDS-PAGE assay[1]
To evaluate whether AC-73 could inhibit the dimerization of CD147 in a Prokaryotic expression system, non-denaturing SDS-PAGE followed by a Western blot assay was used as previously described. Both CD147wt and CD147mts were purified, and 5 μg of each was added to various concentrations of AC-73 (0, 0.1, 0.25, or 0.5 μM) and mixed with 5× Laemmli sample buffer lacking SDS, followed by resolution by 10% SDS-PAGE without boiling and immunoblotting with anti-His6 antibody.

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Non-denaturing SDS-PAGE assay: To evaluate whether AC-73 could inhibit CD147 dimerization in a prokaryotic expression system, wild-type CD147 (CD147wt) and CD147 mutants (CD147mts) were purified. 5 μg of each protein was added to various concentrations of AC-73 (0, 0.1, 0.25, or 0.5 μM) and mixed with non-denaturing loading buffer, followed by resolution by 10% SDS-PAGE without boiling and immunoblotting with anti-His antibody. Two major bands for CD147wt (monomer at 21 kDa and dimer at 42 kDa) were observed [1].
- Surface Plasmon Resonance (SPR) assay: SPR measurements were performed using a ProteOn XPR36 system with GLH sensor chips. Purified CD147wt or CD147mt was immobilized on a GLH chip. Each compound was used at the same concentration (100 μM) and simultaneously injected. Dissociation was monitored for 15 min. The binding of AC-73 to CD147wt presented high Response Units (RU), while binding to CD147mts (E64A, E73A) was negligible [1].
- Molecular docking and energy calculations: The binding simulation revealed multiple hydrogen bonds between AC-73 and two amino acid residues (Glu64 and Glu73) in the active site. Per-residue interaction scores for residues within 10 Å of AC-73 were calculated using Glide software, showing Glu64 and Glu73 presented the greatest predicted energy contributions (-49.4590 and -35.8100, respectively) [1].

RT-PCR[1]
Cell Types: SMMC-7721 Cell
Tested Concentrations: 5 μM or 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: MMP-2 and MMP-9 mRNA expression was Dramatically inhibited at 10 μM concentration. MMP-2 expression at the mRNA level and secretion at the protein level were dose-dependently diminished using RT-qPCR analysis and gelatin zymography experiments.

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Western Blot Analysis [1]
Cell Types: SMMC-7721 Cell
Tested Concentrations: 5 μM, 10 μM or 20 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: The phosphorylation of ERK1/2 and STAT3 was dose-dependently inhibited.

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Cell culture and AC-73 treatment: Human AML cell lines (U937, NB4, HL-60, NB4-R4, MV4-11, Kasumi-1) were grown in RPMI medium with 10% FCS. AC-73 was dissolved in 20% DMSO and diluted in DMEM (final DMSO ≤0.2%). For dose-response and time-course, cells were treated with AC-73 at 1.0, 2.5, 5.0, and 10 μM for 1 to 4 days, with AC-73 added every 2 days to maintain activity. Control cells received 0.2% DMSO. [2]
- Cell growth, viability and apoptosis: Cell growth was assessed by cell counting. Viability was measured using trypan blue exclusion or similar assays, presented as percentage of viable cells relative to control. Apoptosis was detected by Annexin V/PI staining and flow cytometry. [2]
- Cell cycle analysis: Cell cycle profile was analyzed by flow cytometry after propidium iodide staining. [2]
- Differentiation analysis: Expression of CD11b, CD14, CD15 was measured by flow cytometry in AC-73-treated leukemic cells. [2]
- Clonogenic assay: U937 and NB-4 cells were treated with 5 μM AC-73, and colony formation was assessed in semi-solid media. [2]
- Autophagy detection: Autophagy flux was monitored using Cyto-ID autophagy detection kit by flow cytometry. Cells were treated with AC-73 for 72 hours. Additionally, western blot for LC3-I to LC3-II conversion was performed. [2]
- Western blot: Protein expression of CD147, pERK, ERK, pSTAT3(S727), STAT3, LC3, and actin was analyzed. Cells were treated with AC-73 (5 μM for 3 days) alone or in combination with Ara-C (1 μM) or ATO (1 μM). [2]
- Combination treatment: Cells were pretreated with AC-73 (2.5 μM) for 24 hours, then co-treated with Ara-C (0.01, 0.1, 1 μM) or ATO (0.01, 0.1, 1 μM) for additional 1 day (for NB4, NB4-R4) or 2 days (for U937, HL-60, MV4-11, Kasumi-1). Cell viability was then assessed. [2]
- Primary AML blast culture: Human primary AML blasts were maintained in Iscoe medium supplemented with 10% FCS, GM-CSF (10 ng/mL), SCF (50 ng/mL), IL-3 (10 ng/mL). AC-73 treatment was performed similarly. [2]
- Normal HPC culture and AC-73 treatment: Cord blood CD34+ HPCs were purified and cultured under granulocytic or monocytic differentiation conditions. AC-73 (5 μM) was added every 2 days. Cell growth, cycle, phenotype (CD11b, CD15, CD14), and morphology (May-Grünwald-Giemsa staining) were analyzed. [2]

Animal/Disease Models: Male BALB/c nu/nu (nude) mice (4-6 weeks) with SMMC-7721 cells [1]
Doses: 25 mg/kg, 50 mg/kg
Route of Administration: injection; daily; 3 consecutive Weekly
Experimental Results: Dramatically diminished the incidence of metastasis in nude mice. Inhibits the phosphorylation of ERK1/2 and STAT3 in a dose-dependent manner. MMP-2 is also diminished.

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For in vivo experiments, AC-73 was dissolved in Cremophor EL/ethanol (50:50; Cremophor EL, 95% ethyl alcohol) at 4-fold of the highest dose and stored at room temperature.[1]
Establishment of the orthotopic transplant nude mouse model of HCC metastasis[1]
Male BALB/c nu/nu mice, 4 to 6 weeks of age, were provided by the Laboratory Animal Research Center of FMMU, and the animal study was reviewed and approved by the FMMU Animal Care and Use Committee. The mice were housed in a standard animal laboratory under constant environmental conditions, including a 12-h light and dark cycle, with free access to water and food. A mixture of SMMC-7721 cells (1 × 106) in 0.1 ml culture medium and the same volume of diluted Matrigel was injected into the left liver lobe of the nude mice. The treatment was started 1 week after implantation. The mice were divided into three groups: vehicle control (Cremophor EL/ethanol), AC-73 (25 mg/kg/day) and AC-73 (50 mg/kg/day). The mice were sacrificed 4 weeks after implantation. The number of intrahepatic metastases was calculated and statistically analyzed. Tumor tissues were then fixed, embedded in paraffin, and serially sectioned at a thickness of 4 mm. IHC staining was performed, and the sections were examined by a pathologist to verify the presence of tumors.

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Toxicity test[1]
6-week-old male nude mice were divided into 4 groups randomly (n = 5). Mice were injected with normal saline (control), Cremophor EL/ethanol (vehicle), 25 mg/kg/day and 50 mg/kg/day of AC-73. The body weight of each mouse was recorded daily. After 20 more days, mice were killed and selected tissues were fixed in4% paraformaldehyde. Serial histologic sections of their removed hearts, lungs, testis, spleens, kidneys and livers were stained by Hematoxylin and eosin (H&E). The concentration of the serum GPT/ALT and GOT/AST were measured by extracting the eyeball blood using a commercial AST or ALT assay kit. Liver tissue apoptosis were detected by tunel stain (Tunel stain kit).

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Orthotopic transplant nude mouse model of HCC metastasis: Male BALB/c nu/nu mice (4-6 weeks old) were used. A mixture of SMMC-7721 cells (1 × 10^6) in 0.1 ml culture medium and Matrigel was injected into the left liver lobe. Treatment started 1 week after implantation. Mice were divided into three groups: vehicle control (Cremophor EL/ethanol), AC-73 (25 mg/kg/day), and AC-73 (50 mg/kg/day). Mice were sacrificed 4 weeks after implantation. The number of intrahepatic metastases was calculated. Tumor tissues were fixed, embedded in paraffin, sectioned, and examined by IHC [1].
- Toxicity test: 6-week-old male nude mice were divided into 4 groups (n=5) and injected with normal saline (control), Cremophor EL/ethanol (vehicle), 25 mg/kg/day or 50 mg/kg/day of AC-73. Body weight was recorded daily. After 20 days, mice were killed. Tissues (heart, lungs, testis, spleens, kidneys, livers) were fixed, sectioned, and stained with H&E. Serum ALT and AST levels were measured. Liver tissue apoptosis was detected by TUNEL staining [1].

In normal CD34+ HPCs, AC-73 at 5 μM moderately decreases cell growth but does not affect cell cycling, differentiation, or cause morphological abnormalities. [2]
- AC-73 has no detectable effect on cell growth and apoptosis of CHO cells (CD147-negative cell line). [2]

[1]. A novel small-molecule compound targeting CD147 inhibits the motility and invasion of hepatocellular carcinoma cells. Oncotarget. 2016 Feb 23;7(8):9429-47.

[2]. The small-molecule compound AC-73 targeting CD147 inhibits leukemic cell proliferation, induces autophagy and increases the chemotherapeutic sensitivity of acute myeloid leukemia cells. Haematologica. 2019 May;104(5):973-985.

AC-73 is a small-molecule compound (3-[2-[(1,1'-biphenyl]-4-ylmethyl)amino]-1-hydroxyethyl]phenol) proposed as a specific inhibitor of CD147. [2]
- The compound inhibits leukemic cell proliferation by suppressing the ERK/STAT3 activation pathway and by activating non-apoptotic autophagic cell death. [2]
- AC-73 enhances chemotherapeutic sensitivity of AML cells to Ara-C and ATO, allowing lower concentrations of these drugs. [2]
- AC-73 increases ATO-induced autophagy in both M3 and non-M3 leukemic cells. [2]
- CD147 is expressed in CD34+CD371+ AML cells (potential leukemic stem cells), suggesting AC-73 may target LSCs. [2]
- The study proposes AC-73 as a potential anti-leukemic drug for AML treatment, especially in combination with conventional chemotherapy. Future toxicological and pharmacodynamic studies in animal models are required for preclinical evaluation. [2]

C21H21NO2

319.396945714951

319.157

C, 78.97; H, 6.63; N, 4.39; O, 10.02

775294-71-8

775294-71-8;

2989791

White to off-white solid powder

2.8

3

3

6

24

348

0

C1=CC=C(C=C1)C2=CC=C(C=C2)CNCC(C3=CC(=CC=C3)O)O

UECKKYNEEBRMIL-UHFFFAOYSA-N

InChI=1S/C21H21NO2/c23-20-8-4-7-19(13-20)21(24)15-22-14-16-9-11-18(12-10-16)17-5-2-1-3-6-17/h1-13,21-24H,14-15H2

3-{2-[(Biphenyl-4-ylmethyl)-amino]-1-hydroxy-ethyl}-phenol

AC-73; AC 73; AC73

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 : ≥ 250 mg/mL (~782.72 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.51 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 (6.51 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (6.51 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.)