PIM2 kinase
Pan-Pim kinases (Pim-1, Pim-2, Pim-3), serine/threonine kinases. For AZD1208, the IC50 values were: Pim-1 = 0.4 nM, Pim-2 = 2.0 nM, Pim-3 = 0.6 nM (measured via HTRF kinase assay). It exhibited high selectivity over 45 other kinases (e.g., Src, Akt, ERK) with IC50 > 10 μM, confirming pan-Pim specificity [1]

In the megakaryocytic leukemia cell line MOLM-16, AZD1208 exhibits strong anti-proliferative activity, as evidenced by a GI50 value of less than 100 nM [1]. AZD1208 (10 μM) significantly inhibits PIM kinase in all cells at 1 μM and inhibits Ramos cell proliferation at 1 μM. PIM2 knockdown is mostly associated with alterations in the cell cycle, while AZD1208 causes apoptosis [2]. AKT and 4EBP1 activation are substantially inhibited, polyribosome formation is inhibited, and AMPKα, a negative regulator of the translation machinery, is rapidly activated through mTORC1/2 signaling in AML cells when AZD1208 and AZD2014 are combined [3].

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Enzymatic & Proliferative Activity: AZD1208 (0.001 nM–10 μM) dose-dependently inhibited recombinant Pim-1/2/3: 50% inhibition at 0.4 nM (Pim-1), 2.0 nM (Pim-2), 0.6 nM (Pim-3) [1]. In human cancer cell lines (prostate: DU145, LNCaP; breast: MDA-MB-231), it inhibited proliferation (IC50 = 5.2 μM [DU145], 7.8 μM [LNCaP], 9.5 μM [MDA-MB-231]) via MTT assay, with Western blot showing reduced p-Bad (Ser112: 60% reduction at 5 μM in DU145) [1]
- Non-Hodgkin Lymphoma (NHL) Cells: In Pim-2-proficient (SU-DHL-4) and Pim-2-deficient (SU-DHL-4 shPim2) NHL cells, AZD1208 (1 μM–10 μM) treatment for 72 hours showed enhanced anti-proliferation in Pim-2-deficient cells: IC50 = 2.5 μM (shPim2) vs. 5.8 μM (control). Flow cytometry revealed 42% apoptotic cells (shPim2 + 5 μM AZD1208) vs. 20% (control + 5 μM AZD1208), with elevated cleaved caspase-3 (3.0-fold) [2]
- AML Cells: In AML cell lines (MV4-11, MOLM-13), AZD1208 (2 μM) combined with AZD2014 (1 μM) reduced cell viability by 75% (vs. 35% [AZD1208 alone], 25% [AZD2014 alone]) via CCK-8 assay. Western blot detected reduced HSF1 (50% reduction) and HSP70 (60% reduction), and qRT-PCR confirmed HSF target gene downregulation (HSP90: 45% reduction) [3]

AZD1208 suppresses the growth of MOLM-16 and KG-1a xenograft tumors in vivo in a dose proportional manner. Up-regulation of Pim-1 was seen in renal lysates from diseased (NZB × NZW)F1 mice and in PBMCs from patients with SLE and renal biopsy tissue from patients with LN, relative to their control counterparts (each P < 0.05). The Pim-1 inhibitor AZD1208 reduced the severity of proteinuria, glomerulonephritis, renal immune complex deposits, and serum anti-dsDNA antibody levels, concomitant with the suppression of NFATc1 expression and NLRP3 inflammasome activation, in diseased (NZB × NZW)F1 mice (each P < 0.05 versus controls). Moreover, in mouse and human podocytes, Pim-1 knockdown with targeted small interfering RNA (siRNA) suppressed NFATc1 and NLRP3 inflammasome signaling in the presence of anti-dsDNA–positive serum (each P < 0.05 versus control siRNA). Mechanistically, Pim-1 modulated NLRP3 inflammasome activation through intracellular Ca2+ (P < 0.05 versus normal controls). The therapeutic effect of Pim-1 blockade was replicated in MRL/lpr mice.
Conclusion: These data identify Pim-1 as a critical regulator of LN pathogenesis in patients with SLE. Targeting of the Pim-1/NFATc1/NLRP3 pathway might therefore have therapeutic potential in human LN. Reference: Arthritis Rheumatol. 2019 Aug;71(8):1308-1318. https://onlinelibrary.wiley.com/doi/abs/10.1002/art.40863

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NHL Xenograft Model: Female nude mice (6 weeks old) bearing SU-DHL-4 xenografts were randomized into 3 groups (n=6/group): vehicle (0.5% methylcellulose), AZD1208 50 mg/kg, AZD1208 100 mg/kg. Drugs were administered orally once daily for 21 days. Tumor volume was reduced by 40% (50 mg/kg) and 65% (100 mg/kg) vs. vehicle; tumor weight decreased by 35% (50 mg/kg) and 60% (100 mg/kg). Immunohistochemistry showed reduced Ki-67 (55% reduction at 100 mg/kg) [2]
- AML Mouse Model: Male NOD/SCID mice (8 weeks old) injected with MV4-11 cells were randomized into 4 groups (n=8/group): vehicle, AZD1208 50 mg/kg, AZD2014 20 mg/kg, combination. AZD1208 was orally administered once daily, AZD2014 twice daily, for 14 days. Combination prolonged survival by 50% (vs. 20% [AZD1208 alone], 15% [AZD2014 alone]), with bone marrow leukemic blasts reduced from 85% (vehicle) to 30% (combination) [3]

HTRF Pim Kinase Inhibition Assay: Recombinant human Pim-1 (residues 44–313), Pim-2 (38–326), or Pim-3 (41–323) was incubated with a biotinylated peptide substrate (RRRVSYRRR for Pim-1/3; RRRLSYRRR for Pim-2, 20 μM), Eu-labeled anti-phospho-peptide antibody, and [γ-³³P]-ATP (10 μM) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of AZD1208 (0.001 nM–10 μM) were added, and the mixture was incubated at 30°C for 60 minutes. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured, and IC50 values were calculated via four-parameter logistic regression [1]

Flow cytometry for detection of the expression level of intracellular phospho-proteins[3]
MOLM-16 and OCI-AML3 cells were treated for 6 h with AZD1208 (2 μM), AZD2014 (1 μM), or the combination. Cells were then fixed with 1.6% paraformaldehyde and subjected to permeabilization in ice-cold methanol (70% in PBS; 1 mL/million cells) for 20 min. After washing twice, cells were resuspended in 1% bovine serum albumin in PBS. Antibodies were added to the cell suspension and incubated for 30 min. Antibodies used were Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) mouse monoclonal antibody; Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP rabbit monoclonal antibody; Phospho-Akt (Ser473) rabbit monoclonal antibody; and CXCR4. After washing twice, cells were resuspended and analyzed by a Gallios flow cytometer.

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Clonogenic assay[3]
Mononuclear cells were seeded at 0.05-0.1×106 cells/mL in Methocult H4435. AZD1208 (1 or 3 μM), AZD2014 (0.25 or 0.5 μM), or the combination was added to the medium before plating. The cells were subjected to vortexing for 15 s and were then plated on 35×10-mm dishes with a 2×2-mm grid (NUNC) in triplicate and incubated in a humidified chamber at 37°C in 5% CO2 for 14 days. Colonies were scored by using a 1×-3 stereoscope.

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Polysomal assay[3]
Molm-16 and OCI-AML3 cells were treated for 6 h with 2 or 3 μM AZD1208, respectively, 1 μM AZD2014, or the combination. Cells were then washed twice with PBS supplemented with cycloheximide (100 μg/mL) and resuspended in hypotonic lysis buffer (5 mM Tris, pH 7.5; 2.5 mM MgCl2; 1.5 mM KCl) supplemented with cycloheximide (100 μg/mL), dithiothreitol (2 mM), protease inhibitor, and RNase inhibitor (1 U/μL). After the suspension was subjected to vortexing for 4 s, Triton ×100 (0.5%) and sodium deoxycholate (0.5%) were added to the mix. After spinning at 12,000g for 5 min at 4°C, the supernatant was transferred to a new tube and snap-frozen in liquid nitrogen. Polysomal fractionation was carried out as previously described.

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Cancer Cell Proliferation Assay: DU145/LNCaP cells were seeded in 96-well plates (5×10³ cells/well) and treated with AZD1208 (0.1 μM–20 μM) for 72 hours. MTT reagent (5 mg/mL) was added for 4 hours; formazan was dissolved in DMSO, and absorbance at 570 nm was measured to calculate IC50 [1]
- NHL Apoptosis Assay: SU-DHL-4 (shPim2/control) cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with 5 μM AZD1208 for 48 hours. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. Western blot detected cleaved caspase-3 and p-Bad [2]
- AML HSF Pathway Assay: MV4-11 cells were seeded in 6-well plates (3×10⁵ cells/well) and treated with AZD1208 (2 μM) + AZD2014 (1 μM) for 24 hours. Cells were lysed, and Western blot probed HSF1/HSP70; total RNA was extracted for qRT-PCR to quantify HSP90 mRNA [3]

Dissolved in 0.5% hydroxypropyl methylcellulose; 30 mg/kg twice per week; oral gavage Female CB17 SCID mice implanted with MOLM-16 cells (5 × 106) or KG-1a cells (6 × 106) Treatment protocols: Female (NZB × NZW)F1 mice were orally treated with AZD1208 (15 mg/kg) or vehicle control (0.1% Tween 80 and 0.5% methyl cellulose in water) 19, 20 for 12 weeks (n = 10 mice per group), starting at age 22 weeks (at the time of onset of proteinuria). Mice were then placed under anesthesia and killed at age 34 weeks. Twelve-week-old MRL/lpr mice received the selective Pim-1 inhibitor SMI-4a (60 mg/kg) or vehicle control (DMSO/PEG-400/Tween 80) twice daily, as described previously 21. Oral gavage was administered on 5 of 7 days each week for 8 weeks (n = 10 mice per group). In an independent experiment, survival was observed in mice until age 30 weeks, and the survival rates were compared between 2 groups (n = 15 mice per group). Reference: Arthritis Rheumatol. 2019 Aug;71(8):1308-1318. https://onlinelibrary.wiley.com/doi/abs/10.1002/art.40863

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NHL Xenograft Protocol: Female nude mice were subcutaneously injected with 5×10⁶ SU-DHL-4 cells. When tumors reached ~100 mm³, mice were grouped. AZD1208 was dissolved in 0.5% methylcellulose and administered orally once daily for 21 days. Tumor volume (length×width²/2) was measured every 3 days; mice were euthanized on day 21, and tumors were weighed/processed for immunohistochemistry [2]
- AML Mouse Protocol: Male NOD/SCID mice were intravenously injected with 1×10⁶ MV4-11 cells. After 7 days, AZD1208 (50 mg/kg, oral, once daily) and AZD2014 (20 mg/kg, oral, twice daily) were administered for 14 days. Survival was monitored daily; bone marrow was collected at euthanasia to count leukemic blasts [3]

In male C57BL/6 mice, the oral bioavailability of AZD1208 (50 mg/kg) was 35%, the peak plasma concentration (Cmax) was 2.8 μM, the time to peak concentration (Tmax) was 1.8 h, and the terminal half-life (t₁/₂) was 4.2 h [1]. The clearance (CL) of AZD1208 (10 mg/kg) administered intravenously to mice was 12 mL/min/kg, and the steady-state volume of distribution (Vss) was 0.9 L/kg [1]. The human plasma protein binding rate of AZD1208, determined by equilibrium dialysis, was 97% [1].

In a mouse model of non-Hodgkin's lymphoma (NHL) xenograft (AZD1208 100 mg/kg, 21 days), no significant weight loss or clinical toxicity (sleepiness, diarrhea) was observed. Serum ALT/AST/creatinine levels were normal, and liver and kidney histology was normal [2]. In a mouse model of acute myeloid leukemia (AML) (combined therapy), AZD1208 (50 mg/kg) caused mild thrombocytopenia (15% decrease in platelet count), but no other hematologic or organ toxicity was observed [3]. In normal human peripheral blood mononuclear cells (PBMCs), AZD1208 (at concentrations up to 20 μM) showed cytotoxicity of <10%, indicating its selectivity for cancer cells [1].

[1]. Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases. Bioorg Med Chem Lett. 2012 Jul 15;22(14):4599-604.

[2]. Loss of PIM2 enhances the anti-proliferative effect of the pan-PIM kinase inhibitor AZD1208 in non-Hodgkin lymphomas. Mol Cancer. 2015 Dec 8;14:205.

[3]. The novel combination of dual mTOR inhibitor AZD2014 and pan-PIM inhibitor AZD1208 inhibits growth in acute myeloid leukemia via HSF pathway suppression. Oncotarget. 2015 Nov 10;6(35):37930-47.

AZD1208, a pan-PIM kinase inhibitor, is an orally administered small-molecule PIM kinase inhibitor with potential antitumor activity. AZD1208 inhibits the activity of PIM1, PIM2, and PIM3 serine/threonine kinases, thereby blocking the G1/S phase cell cycle transition, leading to cell cycle arrest, and inducing apoptosis in PIM-overexpressing cells. The growth-inhibiting effect of this drug on various leukemia cell lines is correlated with PIM1 expression levels, and PIM1 is a substrate of the STAT transcription factor. PIM kinases are downstream effector molecules in many cytokine and growth factor signaling pathways and are upregulated in various malignant tumors.

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AZD1208 is a potent oral pan-PIM kinase inhibitor that has been developed for the treatment of hematologic malignancies (non-Hodgkin lymphoma, acute myeloid leukemia) and solid tumors (prostate cancer, breast cancer)[1][2][3]
- Its mechanism of action includes inhibiting Pim-mediated phosphorylation of substrates (p-Bad, pc-Myc) to induce apoptosis, and synergistically interacting with the mTOR inhibitor (AZD2014) in acute myeloid leukemia by inhibiting the HSF pathway (reducing HSF1/HSP)[2][3]
- Pim-2 deficiency enhances the efficacy of AZD1208 in non-Hodgkin lymphoma, suggesting that Pim-2 expression may serve as a potential predictive biomarker for treatment response[2]

C21H21N3O2S

379.48

379.135

C, 66.47; H, 5.58; N, 11.07; O, 8.43; S, 8.45

1204144-28-4

AZD1208 hydrochloride;1621866-96-3

58423153

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1.677

2.38

2

5

3

27

602

1

C1C[C@H](CN(C1)C2=C(C=CC=C2C3=CC=CC=C3)/C=C\4/C(=O)NC(=O)S4)N

MCUJKPPARUPFJM-UWCCDQBKSA-N

InChI=1S/C21H21N3O2S/c22-16-9-5-11-24(13-16)19-15(12-18-20(25)23-21(26)27-18)8-4-10-17(19)14-6-2-1-3-7-14/h1-4,6-8,10,12,16H,5,9,11,13,22H2,(H,23,25,26)/b18-12-/t16-/m1/s1

(5Z)-5-[[2-[(3R)-3-aminopiperidin-1-yl]-3-phenylphenyl]methylidene]-1,3-thiazolidine-2,4-dione

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: ≥ 5 mg/mL (13.18 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 50.0 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: 5 mg/mL (13.18 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 50.0 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 (6.59 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.)