OAC1 (BAS 00287861) targets Oct4 (POU5F1) by activating its promoter activity [1]
OAC1 (BAS 00287861) indirectly regulates HOXB4 expression through Oct4 activation, [2]

OAC1 (10 μM; 7 d) improves the effectiveness of reprogramming. OAC1 promotes the production of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs) and speeds up the development of colonies that resemble iPSCs[1]. Human IMR90 fibroblast cells) stimulate the production of Tet1, Oct4, Nanog, and Sox2 endogenously[1]. The number of functional hematopoietic progenitor cells (HPC) and phenotypic hematopoietic stem cells (HSC) is increased by OAC1 (500 nM; 4 d; CD34+ cells)[2]. Hematopoietic stem cells (HSC) can be expanded by OAC1 (500 nM; 4 d; CD34+ cells) activating OCT4 through elevation of HOXB4 expression[2].

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In mouse embryonic fibroblasts (MEFs) undergoing reprogramming to induced pluripotent stem cells (iPSCs) with OSKM (Oct4, Sox2, Klf4, c-Myc) factors, treatment with OAC1 (BAS 00287861) (0.1–10 μM) dose-dependently enhanced reprogramming efficiency. At 5 μM, the number of alkaline phosphatase (AP)-positive colonies increased by ~2.8-fold compared to the vehicle control. It also upregulated Oct4 mRNA (by ~3.2-fold) and protein levels (by ~2.5-fold) at 5 μM, as detected by RT-PCR and Western blot [1]
- OAC1 (BAS 00287861) (1–5 μM) activated the Oct4 promoter in HEK293T cells transfected with an Oct4-luciferase reporter plasmid: luciferase activity was increased by ~2.1-fold (1 μM) and ~3.5-fold (5 μM) compared to control. This activation was specific to Oct4, as it did not affect the promoters of Sox2, Nanog, or c-Myc [1]
- In human cord blood (hCB) hematopoietic stem and progenitor cells (HSPCs), OAC1 (BAS 00287861) (0.5–5 μM) promoted ex vivo expansion. After 7 days of culture, 2 μM OAC1 (BAS 00287861) increased total nucleated cells (TNCs) by ~3.6-fold, CD34+ cells by ~2.9-fold, and CD34+CD38– primitive progenitor cells by ~4.2-fold compared to the control group [2]
- RT-PCR analysis showed that OAC1 (BAS 00287861) (2 μM) upregulated HOXB4 mRNA expression by ~3.1-fold in hCB HSPCs, while Oct4 mRNA levels were increased by ~2.7-fold. Western blot confirmed that HOXB4 and Oct4 protein levels were elevated by ~2.3-fold and ~2.1-fold, respectively [2]
- OAC1 (BAS 00287861) (0.5–5 μM) did not affect the viability of hCB HSPCs (viability >90% at all tested concentrations) or induce apoptosis, as shown by Annexin V-FITC/PI staining [2]

In irradiated NSG mice, OAC1 (50000 CB CD34+ cells with OAC1(500 nM); i.e., 16 weeks) improves both the short- and long-term engrafting of hematopoietic stem cells (HSC)[2].

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In NOD/SCID mice transplanted with OAC1 (BAS 00287861)-pretreated hCB HSPCs (2 μM for 7 days in vitro), the engraftment efficiency of human cells in the bone marrow (BM) was significantly increased. At 8 weeks post-transplantation, the percentage of human CD45+ cells in BM was ~18.6% (OAC1 group) vs. ~7.2% (control group). The number of human CD34+ cells in BM was ~3.1-fold higher in the OAC1 group [2]
- OAC1 (BAS 00287861) pretreatment enhanced the multilineage reconstitution capacity of hCB HSPCs: the percentages of human CD33+ (myeloid), CD19+ (B lymphoid), and CD3+ (T lymphoid) cells in mouse BM were significantly higher in the OAC1 group compared to control, with no bias towards a specific lineage [2]

Oct4 promoter activation assay: HEK293T cells were seeded in 24-well plates and transfected with an Oct4-luciferase reporter plasmid (containing the human Oct4 promoter region) and a Renilla luciferase plasmid (as internal control). After 24 hours of transfection, OAC1 (BAS 00287861) (0.1–10 μM) was added, and cells were cultured for another 24 hours. Luciferase activity was measured using a dual-luciferase assay system, with relative luciferase activity (RLA) calculated as the ratio of firefly luciferase to Renilla luciferase activity [1]

Cell Proliferation Assay[2]
Cell Types: CD34+ cells from human cord blood
Tested Concentrations: 500 nM
Incubation Duration: 4 days
Experimental Results: Increased the numbers of CD34+CD38- cells. Increased the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ cells. Increased in the number of phenotypic hematopoietic stem cells (HSC) compared to control vectors. Enhanced expansion of HPC with both full and suboptimal cytokine doses in the expansion and colony forming phases.

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MEF reprogramming assay: MEFs were isolated from E13.5 mouse embryos and seeded in 6-well plates. The next day, cells were transduced with retroviruses expressing OSKM factors. After 24 hours, OAC1 (BAS 00287861) (0.1–10 μM) was added to the culture medium, which was refreshed every 2 days. On day 14, cells were stained for alkaline phosphatase (AP), and AP-positive colonies were counted to assess reprogramming efficiency. RT-PCR and Western blot were performed to detect Oct4, Sox2, and Nanog expression [1]
- hCB HSPC expansion assay: hCB samples were processed to isolate mononuclear cells (MNCs) by density gradient centrifugation. CD34+ HSPCs were enriched and seeded in 24-well plates at 1×104 cells/well in serum-free expansion medium. OAC1 (BAS 00287861) (0.5–5 μM) was added, and cells were cultured at 37°C with 5% CO2 for 7 days. Total nucleated cells (TNCs) were counted using a hemocytometer, and CD34+、CD34+CD38– cell populations were analyzed by flow cytometry [2]
- Gene expression analysis: After in vitro treatment with OAC1 (BAS 00287861), total RNA was extracted from MEFs or hCB HSPCs, reverse-transcribed to cDNA, and RT-PCR was performed to quantify Oct4, HOXB4, Sox2, and Nanog mRNA levels. For Western blot, cell lysates were prepared, proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies against Oct4, HOXB4, and β-actin (internal control) [1,2]
- Apoptosis assay: hCB HSPCs were treated with OAC1 (BAS 00287861) (0.5–5 μM) for 7 days, then harvested and stained with Annexin V-FITC and PI. Apoptosis rate was analyzed by flow cytometry to evaluate cell survival [2]

Animal/Disease Models: Irradiated NSG mice with OAC1 or vehicle control treated CB CD34+ cells within 24h after irradiation[2]
Doses: 16 weeks
Route of Administration: subcutaneous (sc) injection OAC1(500 nM) treated CB CD34+ cells
Experimental Results: Increased cord blood (CB) cells in primary recipients, compared to the vehicle control group, with the board increase for human B cells, T cells, and myeloid cells in the bone marrow (BM) of primary recipients, resulting in a significant expansion of SRC numbers.

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NOD/SCID mouse transplantation model: 6–8 weeks old NOD/SCID mice were irradiated with a sublethal dose (3.5 Gy) 24 hours before transplantation. hCB HSPCs were pre-treated with OAC1 (BAS 00287861) (2 μM) in expansion medium for 7 days in vitro. The pre-treated HSPCs (1×105 CD34+ cells/mouse) were intravenously injected via the tail vein. Control mice received HSPCs without OAC1 pretreatment. At 8 weeks post-transplantation, mice were sacrificed, and bone marrow cells were isolated. Flow cytometry was used to detect the percentage of human CD45+、CD34+、CD33+、CD19+、and CD3+ cells to assess engraftment and multilineage reconstitution [2]

In vitro toxicity: OAC1 (BAS 00287861) (0.1–10 μM) had no effect on the viability of MEF, HEK293T cells or hCB HSPCs, and cell viability remained above 90% at all tested concentrations [1,2]
- In vivo toxicity: During the 8-week observation period, no significant side effects (e.g., weight loss, lethargy, organ abnormalities) were observed in NOD/SCID mice transplanted with HSPCs pretreated with OAC1 (BAS 00287861) [2]

[1]. Identification of Oct4-activating compounds that enhance reprogramming efficiency. Proc Natl Acad Sci U S A. 2012 Dec 18;109(51):20853-8.

[2]. Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression. Leukemia. 2016 Jan;30(1):144-53.

OAC1 (BAS 00287861) is a small molecule activator of Oct4, discovered through high-throughput screening of compounds that enhance the activity of the Oct4 promoter [1]. The mechanism of action of OAC1 (BAS 00287861) involves direct activation of the Oct4 promoter, thereby upregulating Oct4 expression and subsequently promoting the expression of pluripotency-related genes (e.g., Nanog) during iPSC reprogramming [1]. In human cord blood hematopoietic stem cells (hCB HSPCs), OAC1 (BAS 00287861) enhances in vitro expansion and transplantation capacity by activating Oct4-mediated upregulation of HOXB4 without altering the pluripotency of HSPCs [2]. OAC1 (BAS 00287861) shows potential application value in regenerative medicine, including inducing pluripotent stem cell generation and hematopoietic stem cell transplantation for the treatment of hematological diseases [1,2].

C14H11N3O

237.26

237.09

300586-90-7

789882

Off-white to gray solid powder

1.4±0.1 g/cm3

386.6±22.0 °C at 760 mmHg

187.6±22.3 °C

0.0±0.9 mmHg at 25°C

1.755

1.19

2

2

2

18

301

0

HWJRIFZDXJKJJN-UHFFFAOYSA-N

InChI=1S/C14H11N3O/c18-14(10-4-2-1-3-5-10)17-13-8-11-6-7-15-12(11)9-16-13/h1-9,15H,(H,16,17,18)

N-(1H-pyrrolo[2,3-c]pyridin-5-yl)benzamide

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 (10.54 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 (10.54 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 25.0 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: ≥ 2.5 mg/mL (10.54 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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 (Please use freshly prepared in vivo formulations for optimal results.)