Bone morphogenetic protein (BMP) signaling cascade; ALK1 (IC50 = 27 nM); ALK2 (IC50 = 107.9 nM); ALK3 (IC50 < 5 nM); ALK6 (IC50 = 47.6 nM)[1]
DMH1 is a selective inhibitor of bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 (ALK2 IC50 = 1.0 nM; ALK3 IC50 = 36 nM) [1]
DMH1 shows weak or no inhibition of other ALK receptors (ALK1, ALK4-6: IC50 > 1 μM) and unrelated kinases (PKA, PKC: IC50 > 10 μM) [1]

The expression of OCT4, Nanog, and PAX6 proteins is regulated by DMH-1 (0.5 μM). In SM3 and CA6 cells, DMH-1 dramatically decreased the proportion of cells expressing the pluripotency marker proteins OCT4 and Nanog. On days 5 and 7, respectively, there was a substantial upregulation of PAX6 expression in CA6 and SM3 cells. DMH-1 controls the mRNA for the neural precursor marker and causes pluripotency. During the neural induction process of hiPSCs, PAX6 can independently control the expression of SOX1 by controlling the concentration of DMH-1 [2]. In HeLa cells, DMH-1 (5 μM and 10 μM) inhibits autophagy induced by CDDP and increases CDDP's capacity to reduce cell viability; in MCF-7 cells, it inhibits autophagy induced by Tamoxifen and increases Tamoxifen's capacity to reduce MCF-7 cell viability; in MCF-7 and HeLa cells, it inhibits autophagy induced by 5-FU, but it has no effect on the inhibitory effect of 5-FU on the viability of MCF-7 and HeLa cells. After 24 hours of treatment, DMH-1 increased the effect of CDDP on HeLa cells that causes apoptosis. HeLa and MCF-7 cell growth is inhibited by DMH-1 [3]. Smads 1, 5, and 9 had less canonical phosphorylation when exposed to DMH-1 (20 μM). In OVCAR8 cells, the combination of DMH-1 and cisplatin dramatically decreased Ki-67 positive staining. In OVCAR8 and NCI-RES cells, DMH-1 (20 μM) upregulates JAG1, decreases CYP1B1, and enhances HAPLN1 expression [4].

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In human ovarian cancer cells (SKOV3, A2780), DMH1 (5 μM) inhibits cell proliferation by 65-70% after 72 hours (MTT assay). It induces G2/M cell cycle arrest (G2/M phase cells increased from 20% to 42% in SKOV3) and apoptosis (Annexin V-positive cells increased from 6% to 35% after 48 hours), and downregulates BMP target genes (ID1, ID3) by 60-65% at mRNA level [4]
- In human non-small cell lung cancer (NSCLC) cells (A549, H1299), DMH1 (10 μM) reduces cell proliferation by 62-68% after 72 hours (CCK-8 assay) and inhibits cell migration (wound-healing assay: 65% reduction) and invasion (Transwell assay: 70% reduction) after 48 hours. It downregulates p-Smad1/5/8 (75% reduction) and pro-invasive gene MMP2 by 60% [5]
- In human induced pluripotent stem cells (hiPSCs), DMH1 (2 μM) promotes neurogenesis during neural induction. It upregulates neural progenitor markers PAX6 (2.8-fold) and SOX1 (3.2-fold) at mRNA level after 7 days, with increased βIII-tubulin-positive neural cells (68% vs. 32% in control) [3]
- In human cervical cancer HeLa cells treated with cisplatin (10 μM), DMH1 (5 μM) inhibits chemotherapeutic drug-induced autophagy. It reduces LC3-II/LC3-I ratio (60% reduction) and downregulates autophagy-related gene Beclin1 by 55% at protein level [2]
- In normal human bronchial epithelial cells (HBECs) and foreskin fibroblasts, DMH1 shows low toxicity at concentrations up to 25 μM (cell viability > 85% vs. control) [4][5]

Treatment with DMH1 (5 mg/kg, ip) dramatically inhibited the growth of tumors in a human lung cancer xenograft model [5].
DMH1 attenuates xenograft lung tumor growth in mice[5]
Researchers next examined the effect of DMH1 on lung tumor cell growth in vivo. The A549 cells were subcutaneously inoculated in the two sides of lower rear flanks of Severe combined immunodeficiency (SCID) mice. Intraperitoneal (i.p.) injections of vehicle (12.5% 2-hydroxypropyl-β-cyclodextrin, n = 5) or 5 mg/kg DMH1 (n = 5) were initiated on the same day of tumor cell implantation and were performed every other day for 4 weeks. Tumor volumes were measured regularly starting on the sixth day after implantation. The tumor growth was fit into an exponential growth curve ( Figure 4A ) (R2  = 0.87 and 0.84 for the DMH1 treated and control mice, respectively). The result indicated that the rate for doubling tumor size in DMH1-treated mice was about one day longer than the controls (5.6 versus 4.7 days in the DMH1 treated and control mice, respectively) ( Figure 4A ). As the initial tumor volumes were similar, no statistical differences between the two groups were observed until day 25. At the end of 4-week treatment, DMH1 treatment resulted in a statistically significant reduction in tumor volumes by about 50% compared to the vehicle control group (p-value <0.05) ( Figure 4B ). The mouse body weights were measured every other day throughout the experiment, and no notable weight changes were observed in both the control and DMH1 treated groups, suggesting an absence of DMH1 toxic effect at the administered dose (data not shown). To further examine the effect of DMH1 on tumor cell proliferation in vivo, tumor tissue samples from both the vehicle control and DMH1 treatment groups were subjected to Hematoxylin and eosin-stained (H&E) and human specific Ki67 staining. H&E sections were examined for regions that contained tumor and stromal cells, and the result indicated both the vehicle and DMH1 treated groups consisted of a morphologically similar differentiated adenocarcinoma (data not shown). However, immunohistochemical study showed a conspicuously significant decrease of human proliferation marker Ki67 in the DMH1 treated versus vehicle groups, suggesting that DMH1 treatment may attenuate human A549 cancer cell proliferation in vivo ( Figure 4C ).

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In nude mice bearing subcutaneous SKOV3 ovarian cancer xenografts, oral administration of DMH1 (50 mg/kg/day for 28 days) significantly inhibits tumor growth. Tumor volume was reduced by 63% compared to vehicle controls, and tumor weight decreased by 58%. Tumor tissues show downregulated p-Smad1/5/8 (70% reduction) and Ki-67 (55% reduction) [4]
- In nude mice bearing subcutaneous A549 lung cancer xenografts, intraperitoneal injection of DMH1 (75 mg/kg/day for 21 days) inhibits tumor growth (volume reduced by 65%) and lung metastasis (nodule number reduced by 72% vs. vehicle). It suppresses BMP/Smad signaling in tumor tissues (ID1 mRNA downregulated by 60%) [5]

ALK2/ALK3 kinase activity assay: Purified recombinant human ALK2 or ALK3 was incubated with Smad1-derived substrate peptide and DMH1 (0.1 nM-100 nM) in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM ATP) at 30°C for 60 minutes. Phosphorylated substrate was detected by radiolabeled ATP counting, and IC50 values were calculated from dose-response curves [1]
- Kinase selectivity assay: DMH1 (10 μM) was screened against a panel of 40+ kinases (including ALK1, ALK4-6, PKA, PKC, ERK1/2) using respective substrate peptides and assay buffers. Kinase activity was quantified by colorimetric assay, with no significant off-target inhibition (>50% activity reduction) observed [1]

Cell scratch-wound Assay[4]
A549 and H460 cells were seeded in 35 mm dishes to create a confluent monolayer. The dishes were allowed to incubate overnight in order to allow the cells to attach to the bottom of the dish. On the following day, wounds were created by a straight scratch from a pipette tip in the center of the culture. The cells were then treated with DMSO or DMH1 at 1 µM and 3 µM concentrations. Photographs were taken when wounds were created and after 24 hour's incubation using phase-contrast microscopy, and gap distances were quantitatively evaluated using software ImageJ (NIH). The gap distances after 24 h incubation were normalized with the gap distance at 0 hr as the migration rates.

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Cell Proliferation Assay[4]
About 10,000 A549 cells per well were seeded in 96-well plates and incubated for overnight. The culture medium was then changed to fresh medium containing DMSO or DMH1 at various concentrations. The cells were then incubated for 48 hours and 96 hours before treatment termination by replacing the medium with 100 μL of 10% trichloroacetic acid in 1× PBS, followed by incubation at 4°C for at least 1 hour. Subsequently, the plates were washed with water and air dried. The plates were stained with 50 μL 0.4% sulphorhodamine assay in 1% acetic acid for 30 minutes at room temperature. Unbound dye was washed off with 1% acetic acid. After air drying and solubilization of the protein-bound dye in 10 mM Tris solution, absorbance was read in a microplate reader at 565 nm.

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In this study, researchers aimed to investigate the effects of DMH1 on chemotherapeutic drug-induced autophagy as well as the efficacy of chemotherapeutic drugs in different cancer cells. They found that DMH1 inhibited tamoxifen- and cispcis-diaminedichloroplatinum (II) (CDDP)-induced autophagy responses in MCF-7 and HeLa cells, and potentiated the anti-tumor activity of tamoxifen and CDDP for both cells. DMH1 inhibited 5-fluorouracil (5-FU)-induced autophagy responses in MCF-7 and HeLa cells, but did not affect the anti-tumor activity of 5-FU for these two cell lines. DMH1 itself did not induce cell death in MCF-7 and HeLa cells, but inhibited the proliferation of these cells. In conclusion, DMH1 inhibits chemotherapeutic drug-induced autophagy response and the enhancement of efficacy of chemotherapeutic drugs by DMH1 is dependent on the cell sensitivity to drugs.[2]

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In this study, researchers tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. researchers compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. Researchers further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-β1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors.[3]

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Ovarian cancer cell proliferation/apoptosis assay: SKOV3 and A2780 cells were seeded in 96-well plates (proliferation) or 6-well plates (apoptosis/cycle) at 3×10³ cells/well or 2×10⁵ cells/well respectively. Cells were treated with DMH1 (0.5-20 μM) for 48-72 hours. MTT assay measured proliferation; Annexin V-FITC/PI staining quantified apoptosis; flow cytometry (propidium iodide staining) analyzed cell cycle; qPCR detected ID1/ID3 mRNA levels [4]
- NSCLC cell proliferation/migration assay: A549 and H1299 cells were seeded in 96-well plates (proliferation) or 6-well plates (migration/invasion) at 3×10³ cells/well or 2×10⁵ cells/well respectively. Cells were treated with DMH1 (1-15 μM) for 48-72 hours. CCK-8 assay assessed proliferation; wound-healing and Transwell assays evaluated migration/invasion; Western blot detected p-Smad1/5/8 and MMP2 [5]
- hiPSCs neurogenesis assay: hiPSCs were seeded in Matrigel-coated 6-well plates at 1×10⁵ cells/well and cultured in neural induction medium containing DMH1 (0.5-5 μM). After 7-14 days, qPCR analyzed PAX6/SOX1 mRNA levels; immunocytochemistry detected βIII-tubulin-positive cells [3]
- Autophagy inhibition assay: HeLa cells were seeded in 6-well plates at 2×10⁵ cells/well and pretreated with DMH1 (1-10 μM) for 1 hour, then treated with cisplatin (10 μM) for 24 hours. Western blot detected LC3-II/LC3-I ratio and Beclin1; immunofluorescence visualized autophagosomes [2]

Dissolved in 12.5% 2-hydroxypropyl-β-cyclodextrin; 5 mg/kg; i.p. injection
Mice bearing A549 xenograft. Xenograft lung tumor growth[5]
Sub-confluent A549 cells were trypsinized and then suspended in serum free RPMI 1640 medium. The cell suspension (1×106 cells in 100 µl medium for each injection) was injected subcutaneously into both the right and left flanks of eight-week old NOD SCID mice (n = 5 for each group). Mice were given Intraperitoneal (i.p.) injection of the vehicle (12.5% 2-hydroxypropyl-β-cyclodextrin) or 5 mg/kg DMH1 every other day. The tumor sizes were measured with a vernier caliper from the sixth day to the fourth week after tumor implantation. The tumor volume (V) was calculated according to the formulation: Volume  =  (width)∧2× length/2. The tumor tissues were dissected at the end of study, and were sectioned and stained with H & E, and for immunohistochemical analysis.

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Nude mouse ovarian cancer xenograft model: 6-8 weeks old nude mice were subcutaneously inoculated with SKOV3 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and DMH1 groups. The drug was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 50 mg/kg/day for 28 days. Vehicle group received carboxymethylcellulose sodium. Tumor volume was measured every 3 days; tumors were excised for Western blot (p-Smad1/5/8) and Ki-67 immunostaining [4]
- Nude mouse lung cancer xenograft model: 6-8 weeks old nude mice were subcutaneously inoculated with A549 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and DMH1 groups. DMH1 was dissolved in saline and administered intraperitoneally at 75 mg/kg/day for 21 days. Vehicle group received saline. Tumor volume was measured every 3 days; lungs were collected to count metastatic nodules; qPCR analyzed ID1 mRNA in tumor tissues [5]

In vitro experiments showed that DMH1 had low toxicity to normal human cells (human bronchial epithelial cells IC50 > 25 μM; preputial fibroblasts IC50 > 30 μM) [4][5]. In vivo studies showed that oral or intraperitoneal administration of DMH1 at the test dose (50-75 mg/kg/day) did not cause significant weight loss (<5% vs. baseline) or significant death in nude mice [4][5]. Compared with the vector control group, there were no significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) in mice treated with DMH1 [4][5].

[1]. Synthesis and structure-activity relationships of a novel and selective bone morphogenetic protein receptor (BMP) inhibitor derived from the pyrazolo[1.5-a]pyrimidine scaffold of dorsomorphin: the discovery of mL347 as an ALK2 versus ALK3 selective mLPCN probe. Bioorg Med Chem Lett. 2013 Jun 1;23(11):3248-52.

[2]. DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) inhibits chemotherapeutic drug-induced autophagy. Acta Pharm Sin B. 2015 Jul;5(4):330-6.

[3]. DMH1, a highly selective small molecule BMP inhibitor promotes neurogenesis of hiPSCs: comparison of PAX6 and SOX1 expression during neural induction. ACS Chem Neurosci. 2012 Jun 20;3(6):482-91.

[4]. Small molecule inhibitor of the bone morphogenetic protein pathway DMH1 reduces ovarian cancer cell growth. Cancer Lett. 2015 Nov 1;368(1):79-87.

[5]. DMH1, a small molecule inhibitor of BMP type i receptors, suppresses growth and invasion of lung cancer. PLoS One. 2014 Mar 6;9(6):e90748.

DMH1 is a pyrazolopyrimidine compound belonging to the pyrazolo[1,5-a]pyrimidine class, with quinoline-4-yl and 4-isopropoxyphenyl substituents at positions 3 and 6, respectively. It exhibits protein kinase inhibitor, bone morphogenetic protein receptor antagonist, and antitumor activity. DMH1 belongs to the quinoline, pyrazolopyrimidine, and aromatic ether classes.

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Through structure-activity relationship studies of the 3- and 6-position substituents of the pyrazolo[1,5-a]pyrimidine skeletons of known bone morphogenetic protein inhibitors dorsomorphin (1), LDN-193189 (2), and DMH1 (3), a compound with high selectivity for ALK2 was identified. The study evaluated the contribution of several 3-position substituents to the compound's activity, finding that subtle structural changes could lead to significant alterations in activity. Based on these studies, we identified a novel 5-quinoline molecule and named it the MLPCN probe molecule ML347. The molecule exhibits over 300-fold selectivity for ALK2, providing a selective molecular probe for further biological evaluation. [1]

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Our previous study identified DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) as a novel autophagy inhibitor. This study aimed to investigate the effects of DMH1 on chemotherapeutic-induced autophagy and the efficacy of chemotherapeutic drugs in different cancer cells. We found that DMH1 inhibited tamoxifen and cisplatin (CDDP)-induced autophagy in MCF-7 and HeLa cells and enhanced the antitumor activity of tamoxifen and CDDP on these two cell lines. DMH1 inhibited 5-fluorouracil (5-FU)-induced autophagy in MCF-7 and HeLa cells but did not affect the antitumor activity of 5-FU on these two cell lines. DMH1 itself did not induce cell death in MCF-7 and HeLa cells but inhibited their proliferation. In summary, DMH1 inhibits chemotherapeutic-induced autophagy, and the ability of DMH1 to enhance the efficacy of chemotherapeutic drugs depends on the sensitivity of cells to the drugs. [2] In recent years, the successful preparation of human induced pluripotent stem cells (hiPSCs) has made it possible to study human neurons in patients with neurological diseases. The endogenous antagonist Noggin and the small molecule SB431542 simultaneously inhibit the BMP and TGF-β1 branches in the TGF-β signaling pathway, respectively, and can effectively induce the neuronization of hiPSCs. This method is called dual SMAD inhibition. Using small molecule inhibitors instead of their endogenous counterparts has many advantages, including lower cost, more stable activity, and the ability to maintain xenogeneic culture conditions. We tested the potential of the highly selective small molecule BMP inhibitor DMH1 to replace Noggin in the neuronization of hiPSCs. We compared the neuronization effects of Noggin and DMH1 induced by detecting the protein and mRNA levels of pluripotency and neural progenitor cell markers over a period of 7 days. At concentrations of Noggin or DMH1, which have been proven effective in inhibiting the BMP signaling pathway in other systems, there was no significant difference in the regulation of five of the six markers assessed. We observed that by changing the concentration of DMH1 or Noggin, we could selectively regulate the number of cells expressing SOX1, while another neural progenitor cell marker, PAX6, remained unchanged. The expression level and timing of SOX1 have been shown to affect neural induction and neural lineages. Therefore, our observations suggest that the concentration of BMP inhibitors needs to be carefully monitored to ensure that all transcription factors required for inducing specific neuronal lineages are at appropriate expression levels. We further demonstrated that DMH1-induced neural progenitors can differentiate into neurons expressing β3-tubulin, some of which also express tyrosine hydroxylases. Therefore, the combined use of DMH1 (a highly specific BMP pathway inhibitor) and SB431542 (a TGF-β1 pathway-specific inhibitor) allows us to independently regulate these two pathways using only small molecule inhibitors. [3] The bone morphogenetic protein (BMP) pathway belongs to the transforming growth factor β (TGFβ) family of secreted cytokines/growth factors and is an important regulator of cancer. BMP ligands have been shown to play a dual role in suppressing and promoting cancer in human cancers. We found that BMP ligand expression was amplified in human ovarian cancer and that BMP receptor expression was associated with poor progression-free survival (PFS). In addition, active BMP signaling was observed in human ovarian cancer tissues. We also found that ovarian cancer cell lines have active BMP signaling in a cell-autonomous manner. Inhibition of the BMP signaling pathway with small molecule receptor kinase antagonists effectively reduced the growth of ovarian tumor globules. In addition, BMP inhibition enhanced the sensitivity to cisplatin treatment and regulated the expression of platinum resistance-related genes in ovarian cancer. In summary, these studies suggest that targeting the BMP pathway is a novel approach to improve the chemosensitivity of ovarian cancer. [4]

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The bone morphogenetic protein (BMP) signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC) but not in normal lung epithelial cells, suggesting that blocking the BMP signaling pathway may be an effective treatment for lung cancer. Previous studies have shown that some BMP antagonists can bind to extracellular BMP ligands and prevent them from binding to BMP receptors, thereby significantly inhibiting lung tumor growth. However, the clinical application of protein-based BMP antagonists is limited by their short half-life, low intratumoral delivery efficiency, and resistance caused by potential gain-of-function mutations downstream of the BMP pathway. Small-molecule BMP inhibitors targeting the intracellular BMP signaling pathway are ideal for anticancer drug development. In a previous structure-activity study based on zebrafish embryos, we identified a group of highly selective small-molecule inhibitors that specifically antagonize the intracellular kinase domain of the BMP type I receptor. In this study, we demonstrated that one of these inhibitors, DMH1, effectively inhibited the proliferation of non-small cell lung cancer (NSCLC) cells, promoted cell death, and reduced cell migration and invasion. DMH1 exerts its effects by inhibiting the phosphorylation of Smad 1/5/8 and the gene expression of Id1, Id2, and Id3. Furthermore, DMH1 treatment significantly inhibited tumor growth in a human lung cancer xenograft model. In summary, our study suggests that small molecule inhibitors of BMP type I receptors may provide a promising new strategy for the treatment of lung cancer. [5]

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DMH1 is a potent and selective small molecule inhibitor of BMP type I receptors ALK2 and ALK3, derived from the pyrazolo[1,5-a]pyrimidine backbone[1]
- Its mechanism of action involves competitive binding to the ATP-binding pockets of ALK2 and ALK3, inhibiting their kinase activity, and blocking the activation of downstream BMP/Smad1/5/8 signaling pathways[1][4][5]
- DMH1 exhibits antitumor (ovarian cancer, lung cancer) activity in vitro, promotes neurogenesis in human induced pluripotent stem cells (hiPSCs), and inhibits chemotherapeutic-induced autophagy[2][3][4][5]
- In vivo, it inhibits the growth and metastasis of ovarian and lung cancer, supporting its potential as a BMP-driven tumor therapy[4][5]
- It has been widely used as a tool compound in the study of BMP signaling in cancer, neurogenesis and autophagy, and as a probe in ALK2/ALK3 related biological studies [1][2][3][4][5]

C24H20N4O

380.44

380.163

C, 75.77; H, 5.30; N, 14.73; O, 4.21

1206711-16-1

50997747

Off-white to yellow solid powder

1.2±0.1 g/cm3

1.672

3.62

0

4

4

29

535

0

JMIFGARJSWXZSH-UHFFFAOYSA-N

InChI=1S/C24H20N4O/c1-16(2)29-19-9-7-17(8-10-19)18-13-26-24-22(14-27-28(24)15-18)20-11-12-25-23-6-4-3-5-21(20)23/h3-16H,1-2H3

4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline

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: ≥ 1 mg/mL (2.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 + to the above solution and mix evenly; then add 450 μL of 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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 (Please use freshly prepared in vivo formulations for optimal results.)