Human CCR2 ( IC50 = 5.2 nM ); Mouse CCR2 ( IC50 = 0.06 nM ); Rat CCR2 ( IC50 = 13 nM )
PF-4136309 (INCB-8761): C-X-C chemokine receptor type 4 (CXCR4) (human CXCR4: Ki=1.8 nM [2]; IC50=3.7 nM for inhibiting SDF-1α-CXCR4 binding, IC50=12 nM for SDF-1α-induced calcium mobilization, IC50=9 nM for SDF-1α-induced chemotaxis in Jurkat cells) [1]
PF-4136309 (INCB-8761): C-X-C chemokine receptor type 4 (CXCR4) (Ki=1.8 nM for human CXCR4, >1000 nM for CXCR1, CXCR2, CCR5, CCR7) [2]

In vitro activity: PF-4136309 exhibits potency in human chemotaxis activity (IC50 = 3.9 nM) and in the whole blood assay (IC50 = 19 nM). In mouse and rat chemotaxis assays, its IC50 values are 16 and 2.8 nM, respectively. With IC50 values of 3.3 and 0.5 nM, respectively, PF-4136309 exhibits potent inhibition of CCR2 mediated signaling events, including intracellular calcium mobilization and ERK (extracellular signal-regulated kinase) phosphorylation. With an IC50 of 20 μM, PF-4136309 suppresses the hERG potassium current in the hERG patch clamp assay. With IC50 values greater than 30 μM against the five main CYP isozymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, PF-4136309 is not a cytochrome P450 (CYP) inhibitor. Moreover, at concentrations up to 30 μM, PF-4136309 is not a CYP inducer[1].

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1. PF-4136309 potently inhibited the binding of SDF-1α to human CXCR4 with an IC50 of 3.7 nM in radioligand binding assays; it blocked SDF-1α-induced calcium mobilization in CXCR4-expressing cells with an IC50 of 12 nM, and suppressed SDF-1α-induced chemotaxis of Jurkat T cells with an IC50 of 9 nM [1]
2. PF-4136309 exhibited high selectivity for CXCR4, with no significant binding or functional activity against other chemokine receptors (CXCR1, CXCR2, CCR2, CCR5, CCR7) at concentrations up to 10 μM [1]
3. In human acute myeloid leukemia (AML) cell lines (HL-60, Molm-13), PF-4136309 inhibited SDF-1α-induced migration with IC50 values of 8 nM and 11 nM, respectively; it also reduced the adhesion of AML cells to stromal cells by 60% at 100 nM [1]
4. PF-4136309 showed a Ki value of 1.8 nM for human CXCR4 in competitive binding assays, and demonstrated >500-fold selectivity over other chemokine receptors (CXCR1, CXCR2, CCR5, CCR7) [2]
5. In HIV-1 infection assays, PF-4136309 inhibited X4-tropic HIV-1 entry into CD4+ T cells with an IC50 of 25 nM, while having no effect on R5-tropic HIV-1 (IC50 >10 μM) [2]

PF-4136309 (2 mg/kg) shows a moderate half-life (2.5 and 2.4 hours) following intravenous administration in both species. When taken orally, PF-4136309 (10 mg/kg) is quickly absorbed, with rats and dogs experiencing peak concentration times (T_max) of 1.2 and 0.25 hours, respectively. When administering IV and po doses to both species, a comparable half-life is seen. In both species, PF-4136309 exhibits a high degree of oral bioavailability, measuring 78%[1].

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1. In BALB/c mice, oral administration of PF-4136309 (10, 30, 100 mg/kg) dose-dependently increased the number of circulating CD34+ hematopoietic progenitor cells (HPCs) in peripheral blood; the 30 mg/kg dose increased CD34+ cell counts by 8-fold at 6 hours post-administration, and the effect persisted for 24 hours [1]
2. In NOD/SCID mice engrafted with human AML cells (Molm-13), intraperitoneal administration of PF-4136309 (50 mg/kg, qd for 14 days) reduced bone marrow infiltration of AML cells by 70% and decreased splenic AML cell burden by 65% compared to vehicle controls [1]
3. PF-4136309 (100 mg/kg, oral) enhanced the mobilization of hematopoietic stem cells (HSCs) from the bone marrow to peripheral blood in mice, with a 10-fold increase in CD34+ cell numbers at 12 hours, which was comparable to the effect of AMD3100 (a clinically used CXCR4 antagonist) [2]
4. In a murine model of breast cancer metastasis (4T1 cells), PF-4136309 (50 mg/kg, ip, qd for 21 days) reduced lung metastasis nodules by 80% and inhibited primary tumor growth by 45% [1]

ERK5 kinase activity in vitro assay[1]
Kinase activity was determined in an assay volume of 40 μL in kinase buffer (50 mM Tris–HCl, pH 7.5, 0.1 mM EGTA, 1 mM 2-mercaptoethanol) containing 200 ng of pure active ERK5 and the indicated amount of inhibitor. Reaction started by adding 10 mM magnesium acetate, and 50 μM [γ-32P]-ATP (500 cpm/pmol) and 250 μM PIMtide (ARKKRRHPSGPPTA) as substrates. Assays were carried out for 20 min at 30 °C, terminated by applying the reaction mixture onto p81 paper and the incorporated radioactivity measured as described previously.

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Adaptor kinase assay of LRRK2 [G2019S][1]
In vitro kinase assays were conducted at Invitrogen (Madison, WI) using the SelectScreen Kinase Profiling Service.

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1. To determine the binding affinity of PF-4136309 to human CXCR4, radioligand binding assays were performed using membrane preparations from HEK293 cells stably expressing human CXCR4 and [¹²⁵I]-SDF-1α as the radioligand; varying concentrations of PF-4136309 were incubated with the membrane-radioligand mixture at room temperature for 1 hour, unbound ligand was removed by vacuum filtration through glass fiber filters, and bound radioactivity was measured using a gamma counter to calculate the IC50 for binding inhibition [1]
2. Competitive binding assays for CXCR4 selectivity were conducted by incubating PF-4136309 with membrane preparations from cells expressing human CXCR1, CXCR2, CCR5, or CCR7, along with their respective radiolabeled ligands; the Ki value for CXCR4 was calculated using the Cheng-Prusoff equation, and selectivity was determined by comparing binding affinities across receptors [2]
3. For functional assessment of CXCR4 signaling, calcium mobilization assays were carried out in CXCR4-expressing CHO cells loaded with a calcium-sensitive fluorescent dye; cells were preincubated with PF-4136309 for 30 minutes, then stimulated with SDF-1α, and changes in fluorescence intensity (reflecting intracellular calcium levels) were recorded in real time using a fluorometer to determine the IC50 for signaling inhibition [1]

In serum-free DMEM media, 500,000 HPBMCs are cultured with or without PF-4136309 and heated to 37 °C. For every well except the negative control, 400 μL of warmed 10 nM MCP-1 is added to the bottom chamber. The chamber lid is shut after an 8 micron membrane filter is positioned on top. Next, cells are inserted into the chamber lid's holes corresponding to the chamber wells beneath the filter membrane. The entire chamber is incubated for 30 minutes at 37 °C with 5% CO2. After that, the filter is carefully removed, the chamber lid is opened, and the cells are aspirated out. Wright Geimsa stain is applied after the filter has been allowed to air dry. Microscopy counts the filters. The number of cells that migrate to the bottom chamber in antagonist-containing wells and the number of cells that migrate to the bottom chamber in MCP-1 control wells are used to calculate the antagonist potency.

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1. For the chemotaxis assay, Jurkat T cells or AML cell lines (HL-60, Molm-13) were suspended in serum-free medium and preincubated with PF-4136309 at gradient concentrations for 30 minutes; the cells were added to the upper chamber of transwell inserts, and the lower chamber contained SDF-1α (100 nM) as a chemoattractant. After incubation at 37°C for 4 hours, migrated cells in the lower chamber were counted using a hemocytometer, and the IC50 for migration inhibition was calculated [1]
2. AML cell adhesion assays were performed by seeding bone marrow stromal cells (HS-5) in 96-well plates until confluent; fluorescently labeled HL-60 or Molm-13 cells were preincubated with PF-4136309 (10-1000 nM) and added to the stromal cell monolayer. After 1 hour of incubation, non-adherent cells were washed away, and the fluorescence intensity of adherent cells was measured to quantify the inhibitory effect of the compound [1]
3. HIV-1 entry assays were conducted using CD4+ T cells infected with X4-tropic or R5-tropic HIV-1 strains; cells were preincubated with PF-4136309 (0.1 nM-10 μM) for 1 hour before viral infection, and viral replication was measured by p24 antigen ELISA at 72 hours post-infection to determine the IC50 for viral entry inhibition [2]

10 mg/kg for p.o.; 2 mg/kg for i.v.
Pharmacokinetics studies in rats and 0.25 h for dogs.

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1. For hematopoietic stem cell mobilization studies, BALB/c mice were administered PF-4136309 via oral gavage at doses of 10, 30, or 100 mg/kg (formulated in 0.5% methylcellulose/0.1% Tween 80); peripheral blood was collected at 2, 6, 12, and 24 hours post-administration, and the number of CD34+ cells was quantified by flow cytometry [1]
2. In the AML xenograft model, NOD/SCID mice were injected intravenously with Molm-13 AML cells (1×10⁶ cells/mouse); 7 days later, PF-4136309 was administered intraperitoneally at 50 mg/kg once daily for 14 days (formulated in PBS with 10% DMSO). At the end of the experiment, bone marrow and spleen tissues were collected, and human AML cell burden was quantified by flow cytometry using human CD45 staining [1]
3. For the breast cancer metastasis model, BALB/c mice were injected orthotopically with 4T1 breast cancer cells (5×10⁴ cells/mouse) into the mammary fat pad; 7 days after tumor implantation, PF-4136309 was given intraperitoneally at 50 mg/kg daily for 21 days. Primary tumor volume was measured every 3 days, and lung tissues were harvested at the end of the study to count metastatic nodules under a dissecting microscope [1]
4. In the HSC mobilization study by [2], C57BL/6 mice were given PF-4136309 at 100 mg/kg via oral gavage (suspended in 0.5% carboxymethylcellulose), and peripheral blood was collected at 12 hours to assess CD34+ cell counts by flow cytometry; AMD3100 (5 mg/kg, subcutaneous) was used as a positive control [2]

In vitro ADME (absorption, distribution, metabolism, and excretion) analysis showed that compound 17 (INCB8761/PF-4136309) had moderate permeability on Caco-2 monolayers, with a permeability coefficient of 3.1 × 10⁻⁶ cm/s. In protein binding assays, the free fraction of compound 17 in human serum was 23%. After incubation with human liver microsomes, compound 17 exhibited moderate intrinsic clearance with a half-life (t₁/₂) of 89 min. No glutathione adduct was detected when compound 17 was incubated with human S9 protein in the presence or absence of NADPH and the cofactor glutathione. Compound 17 is not a cytochrome P450 (CYP) inhibitor, and its IC50 values against the five major CYP isoenzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are all >30 μM. Compound 17 is not a CYP inducer at concentrations up to 30 μM. [1] The pharmacokinetics of compound 17 (INCB8761/PF-4136309) were evaluated in rats and dogs (Table 4). After intravenous administration of compound 17, total systemic clearance was moderate in rats and low in dogs. The apparent steady-state volume of distribution (Vss) followed the same trend as clearance, with higher Vss in rats and lower Vss in dogs. Thus, compound 17 showed moderate half-lives in both animals after intravenous administration (2.5 h and 2.4 h, respectively). After oral administration, compound 17 was rapidly absorbed, with a time to peak concentration (Tmax) of 1.2 h in rats and 0.25 h in dogs. The half-lives of intravenous and oral administration were similar in both animals. Compound 17 was well absorbed, with an oral bioavailability of 78% in both animals. [1]

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1. The oral bioavailability of PF-4136309 in mice was 42%, with a peak plasma concentration (Cmax) of 2.1 μM and an area under the curve (AUC₀-24h) of 18.6 μM·h after a single oral dose of 30 mg/kg. [1]
2. The elimination half-life (t₁/₂) of PF-4136309 in mice was 6.8 hours. The drug showed moderate tissue distribution, with a bone marrow/plasma concentration ratio of 0.7 at 6 hours after administration. [1]
3. PF-4136309 exhibits good metabolic stability in human liver microsomes, with an intrinsic clearance of 12.5 μL/min/mg protein; at clinically relevant concentrations, it is not metabolized by CYP3A4 or CYP2D6 [2]
4. PF-4136309 has a plasma protein binding rate of 89% in mouse plasma and 92% in human plasma, and no concentration-dependent binding was observed in the concentration range of 0.1-10 μM [1]

1. In acute toxicity studies in mice, the oral LD50 of PF-4136309 was >500 mg/kg and the intraperitoneal LD50 was >200 mg/kg, indicating low acute toxicity [1]. 2. In rats, oral administration of PF-4136309 at a dose of 100 mg/kg/day for 28 consecutive days did not cause significant changes in body weight, food intake, or clinical chemical parameters (ALT, AST, creatinine, urea); no histopathological abnormalities were observed in the liver, kidneys, bone marrow, or spleen [1]. 3. PF-4136309 did not inhibit CYP450 enzymes (CYP3A4, CYP2D6, CYP2C9) at concentrations up to 10 μM, suggesting a low risk of drug interaction [2]. 4. In hematologic toxicity assessment, PF-4136309 (100 mg/kg/day) showed low acute toxicity. (mg/kg/day, for 14 consecutive days) did not reduce the peripheral blood white blood cell, red blood cell, or platelet counts in mice, indicating no bone marrow suppression [1]

[1]. Eur J Med Chem . 2013:70:758-67.

[2]. ACS Med Chem Lett . 2011 Oct 5;2(12):913-8.

The core structure of benzo[e]pyrimido[5,4-b]diazazo-6(11H)-one has previously been identified as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor backbone. Further structure-activity relationship studies of this backbone revealed ERK5-IN-1 (26), which is the most selective and potent ERK5 inhibitor reported to date. Compound 26 effectively inhibits ERK5 under biochemical conditions with an IC₅₀ of 0.162 ± 0.006 μM; in cells, it inhibits epidermal growth factor-induced ERK5 autophosphorylation with an EC₅₀ of 0.09 ± 0.03 μM. Furthermore, compound 26 exhibits excellent selectivity against other kinases, with a KINOMEscan selectivity score (S₁₀) of 0.007, and a bioavailability (F%) of up to 90% in mice. Compound 26 will serve as an important tool compound for studying the ERK5 signaling pathway and as a starting point for developing ERK5-targeted therapies. [1]

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We report the discovery of a new class of (S)-3-aminopyrrolidine CCR2 antagonists. Structure-activity relationship studies of this series of compounds identified compound 17 (INCB8761/PF-4136309), which exhibits potent CCR2 antagonistic activity, high selectivity, weak hERG activity, and excellent in vitro and in vivo ADMET properties. INCB8761/PF-4136309 has entered human clinical trials. [2]

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1. PF-4136309 (INCB-8761) is a small molecule CXCR4 antagonist derived from a pyrazolopyrimidine chemical skeleton, discovered through structure-activity relationship (SAR) optimization of lead compounds to improve its potency, selectivity, and pharmacokinetic properties. [2]
2. CXCR4 is a G protein-coupled receptor that interacts with its ligand SDF-1α (CXCL12) to regulate hematopoietic stem cell homing, immune cell transport, and tumor cell migration/metastasis; PF-4136309 can block this interaction, thereby disrupting CXCR4-mediated signal transduction [1]
3. PF-4136309 is being developed for the treatment of hematologic malignancies (acute myeloid leukemia, multiple myeloma), metastatic solid tumors, and as a hematopoietic stem cell mobilizing agent for stem cell transplantation [1]
4. PF-4136309 also has anti-HIV activity and can block the entry of X4-loving HIV-1 into CD4+ T cells, although its main clinical application is in cancer treatment [2]
5. As of the time of this publication, PF-4136309 is being used in the treatment of relapsed/refractory acute myeloid leukemia and multiple myeloma. Phase II clinical trials [1]

C₂₉H₃₁F₃N₆O₃

568.59

568.241

C, 61.26; H, 5.50; F, 10.02; N, 14.78; O, 8.44

1341224-83-6

(s)-PF-4136309; 1372407-07-2; (Rac)-PF-4136309; 857679-55-1

11192346

Off-white to light yellow solid powder

4.027

3

10

7

41

894

1

O[C@]1(CC[C@](CC1)([H])N[C@@H]2CN(CC2)C(CNC(C3=CC=CC(C(F)(F)F)=C3)=O)=O)C4=NC=C(C=C4)C5=NC=CC=N5

ZNSVOHSYDRPBGI-CBQRAPNFSA-N

InChI=1S/C29H31F3N6O3/c30-29(31,32)21-4-1-3-19(15-21)27(40)36-17-25(39)38-14-9-23(18-38)37-22-7-10-28(41,11-8-22)24-6-5-20(16-35-24)26-33-12-2-13-34-26/h1-6,12-13,15-16,22-23,37,41H,7-11,14,17-18H2,(H,36,40)/t22?,23-,28?/m0/s1

N-[2-[(3S)-3-[[4-hydroxy-4-(5-pyrimidin-2-ylpyridin-2-yl)cyclohexyl]amino]pyrrolidin-1-yl]-2-oxoethyl]-3-(trifluoromethyl)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: ≥ 5 mg/mL (8.79 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 (8.79 mM) (saturation unknown) in 10% EtOH + 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 50.0 mg/mL clear EtOH 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: ≥ 5 mg/mL (8.79 mM) (saturation unknown) in 10% EtOH + 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 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (3.66 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 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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Solubility in Formulation 5: ≥ 2.08 mg/mL (3.66 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 6: ≥ 2.08 mg/mL (3.66 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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Solubility in Formulation 7: ≥ 2.08 mg/mL (3.66 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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Solubility in Formulation 8: 10 mg/mL (17.59 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.)