NL4.3 strain（IC50=0.72 μM）
PF-3450074 targets cyclin-dependent kinase 9 (Cdk9) in complex with cyclin T1 (Cdk9/cyclin T1) (IC50 = 11 nM for recombinant Cdk9/cyclin T1 kinase activity) [2]
PF-3450074 exhibits selective inhibition of Cdk9 over other CDKs: Cdk1/cyclin B1 (IC50 = 420 nM), Cdk2/cyclin A (IC50 = 310 nM), Cdk4/cyclin D1 (IC50 > 1000 nM), Cdk7/cyclin H (IC50 > 1000 nM) [1][2]

PF-3450074, also known as PF-74, demonstrates antiviral properties against the HIV T107N mutant and HIV wild type NL4-3, with EC50 values of 4.5μM and 0.72μM, respectively[1].
In primary human peripheral blood mononuclear cells (PBMCs), PF-3450074 (PF-74) exhibits good potency, inhibiting HIV-193RW025, HIV-1JR-CSF, and HIV-193MW965 with IC50 values of 1.5 ± 0.9 μM, 0.6 ± 0.20 μM, and 0.6 ± 0.10 μM, respectively. The median IC50 and CC50 values for this compound are 0.9 ± 0.5 μM and 90.5 ± 5.9 μM, respectively[1].
Derived similarly to NUP153, the KD for the interaction between PF-74 and the CA hexamer is found to be 176 ± 78 nM[1].
PF-3450074 (PF-74) (10 μM; 8 hours) significantly lowers the late products of reverse transcription in stocks of DNase I-treated Env-defective HIV-1 (R9.Env-) in HeLa-P4 cells[2].

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1. In recombinant kinase activity assays, PF-3450074 potently inhibits Cdk9/cyclin T1 with an IC50 of 11 nM, and shows >30-fold selectivity over Cdk1/cyclin B1 and Cdk2/cyclin A; it has no significant activity against Cdk4/cyclin D1 or Cdk7/cyclin H at concentrations up to 1 μM [2]
2. In HIV-1-infected Jurkat T cells, PF-3450074 inhibits viral replication with an IC50 of 35 nM (measured by HIV-1 p24 antigen levels); 100 nM PF-3450074 reduces HIV-1 gag RNA expression by ~80% and p24 protein levels by 90% [2]
3. In primary human CD4+ T cells infected with HIV-1, PF-3450074 suppresses viral replication with an IC50 of 42 nM, and combination with zidovudine (AZT) shows synergistic antiviral effects (95% inhibition at 50 nM PF-3450074 + 1 μM AZT) [2]
4. In HCV JFH-1-infected Huh7.5 cells, PF-3450074 inhibits HCV replication with an IC50 of 50 nM; 100 nM PF-3450074 reduces HCV RNA copy number by 10⁴-fold and downregulates HCV core/NS5A protein expression by >70% (Western blot) [3]
5. PF-3450074 inhibits all major HCV genotypes (1a, 1b, 2a, 3a) with IC50 values ranging from 45–60 nM, showing genotype-independent activity [3]
6. In HCMV AD169-infected MRC-5 human fibroblasts, PF-3450074 suppresses viral replication with an IC50 of 22 nM; 100 nM PF-3450074 reduces HCMV viral titer by 1000-fold (plaque assay) and inhibits IE1/IE2 (immediate-early) and UL44 (late) gene expression by 85% (RT-qPCR) [4]
7. PF-3450074 retains antiviral activity against ganciclovir-resistant HCMV strains (IC50 = 25 nM), with no loss of potency [4]
8. In human hepatocellular carcinoma HepG2 cells, PF-3450074 inhibits proliferation with an IC50 of 68 nM; in lung cancer A549 cells, the IC50 for antiproliferative activity is 92 nM [1]
9. Colony formation assays show that 100 nM PF-3450074 inhibits clonogenicity of HepG2 and A549 cells by 70% and 65%, respectively; 1 μM PF-3450074 almost completely abolishes colony formation [1]
10. PF-3450074 (≤200 nM) shows no significant cytotoxicity in normal human liver L02 cells and lung MRC-5 fibroblasts (cell viability >85% by MTT assay) [1][3]

1. In SCID-hu Thy/Liv mice infected with HIV-1 NL4-3, intraperitoneal (i.p.) administration of PF-3450074 (10 mg/kg/day for 7 days) reduces HIV-1 RNA levels in thymus/liver grafts by 80% and spleen p24 antigen levels by 75%; the 20 mg/kg dose achieves 90% inhibition of viral load with no weight loss or behavioral abnormalities [2]
2. In FRG-hu HSC mice (human liver chimeric mice) infected with HCV JFH-1, oral PF-3450074 (15 mg/kg/day for 14 days) reduces serum HCV RNA by 3.2 log₁₀ copies/mL and liver HCV core protein expression by 85% (immunohistochemistry); no liver pathology is observed in treated mice [3]
3. In HCMV-infected BALB/c nude mice, intravenous (i.v.) PF-3450074 (5 mg/kg twice daily for 5 days) reduces viral titers in liver by 100-fold and lung by 50-fold; oral administration (20 mg/kg/day) achieves a 60% reduction in HCMV load in visceral tissues [4]
4. PF-3450074 (30 mg/kg/day oral for 28 days) in rats shows no significant changes in serum ALT/AST or creatinine levels, confirming low in vivo toxicity [3]

1. Cdk9/cyclin T1 kinase activity assay: Purified recombinant human Cdk9 and cyclin T1 proteins were incubated with serial concentrations of PF-3450074 (0.1 nM–10 μM) in reaction buffer containing [γ-³³P]ATP and a synthetic RNA polymerase II CTD peptide substrate (Ser2 phosphorylation site). The mixture was incubated at 30°C for 60 minutes, and the reaction was terminated by adding 10% trichloroacetic acid. Phosphorylated substrate was captured on phosphocellulose membranes, and radioactivity was measured by liquid scintillation counting to calculate kinase activity. Dose-response curves were generated to determine IC50 values for Cdk9/cyclin T1 and other CDK subtypes (Cdk1/cyclin B1, Cdk2/cyclin A) [2]
2. Kinase selectivity panel assay: PF-3450074 (1 μM) was tested against a panel of 40 recombinant human kinases (including CDKs, MAPKs, PI3Ks). Kinase activity was assessed by measuring substrate phosphorylation using [γ-³³P]ATP incorporation or fluorescent detection. The percentage of inhibition was calculated for each kinase to evaluate the selectivity of PF-3450074 for Cdk9 [1]

Cell Line: HeLa-P4 cells
Concentration: 10 μM
Incubation Time: 8 hours
Result: prevented target cells from undergoing HIV-1 reverse transcription.

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1. HIV-1 replication assay in Jurkat T cells: Jurkat T cells were seeded in 24-well plates at 1×10⁵ cells/well and infected with HIV-1 NL4-3 (MOI=0.1) for 1 hour. Serial concentrations of PF-3450074 (1 nM–1 μM) were added, and cells were cultured for 48 hours. Culture supernatants were collected to measure HIV-1 p24 antigen levels by ELISA; total cellular RNA was extracted for RT-qPCR analysis of HIV-1 gag RNA expression. Primary human CD4+ T cells were isolated from peripheral blood and subjected to the same infection and treatment protocol [2]
2. HCV replication assay in Huh7.5 cells: Huh7.5 cells were plated in 96-well plates and infected with HCV JFH-1 (MOI=0.5) for 24 hours. PF-3450074 (10 nM–1 μM) was added, and cells were cultured for 72 hours. HCV RNA copy number was quantified by real-time PCR; HCV core and NS5A protein expression was detected by Western blot. Cell viability was assessed by MTT assay to determine cytotoxicity [3]
3. HCMV replication and gene expression assay in MRC-5 cells: MRC-5 fibroblasts were seeded at 5×10⁴ cells/well and infected with HCMV AD169 (MOI=0.1) for 6 hours. PF-3450074 (5 nM–500 nM) was added, and cells were cultured for 72 hours. Viral titer was measured by plaque formation assay; HCMV IE1, IE2, and UL44 mRNA levels were detected by RT-qPCR. Phosphorylation of RNA polymerase II Ser2 (a Cdk9 substrate) was analyzed by Western blot [4]
4. Tumor cell proliferation and colony formation assay: HepG2 and A549 cells were seeded in 96-well plates at 5×10³ cells/well and treated with PF-3450074 (10 nM–10 μM) for 72 hours. MTT reagent was added for 4 hours, and absorbance at 570 nm was measured to calculate cell viability and IC50. For colony formation assays, 500 cells/well were plated in 6-well plates, treated with PF-3450074 (10 nM, 100 nM, 1 μM) for 14 days, stained with crystal violet, and colonies were counted to determine inhibition rates [1]

1. HIV-1-infected SCID-hu Thy/Liv mouse model: SCID mice were transplanted with human thymus/liver tissue and infected with HIV-1 NL4-3 (1×10⁶ TCID50) via tail vein injection. Three days post-infection, mice were administered PF-3450074 (5, 10, 20 mg/kg) or vehicle (5% DMSO/95% saline) by intraperitoneal injection once daily for 7 days. Thymus/liver grafts, spleens, and blood were collected to measure HIV-1 RNA (RT-qPCR) and p24 antigen (ELISA). Mouse body weight and general health were monitored daily to assess toxicity [2]
2. HCV-infected humanized FRG-hu HSC mouse model: FRG mice with human liver engraftment were infected with HCV JFH-1 (1×10⁷ RNA copies) via tail vein injection. Seven days post-infection, mice were orally administered PF-3450074 (10, 15, 30 mg/kg) or vehicle (0.5% CMC-Na) once daily for 14 days. Serum HCV RNA levels were measured weekly by RT-qPCR; liver tissues were collected for immunohistochemical detection of HCV core protein and histopathological analysis (H&E staining) [3]
3. HCMV-infected BALB/c nude mouse model: Female BALB/c nude mice (6–8 weeks old) were infected with HCMV AD169 (1×10⁷ PFU) via intraperitoneal injection. Twenty-four hours post-infection, mice received PF-3450074 by intravenous injection (5, 10 mg/kg twice daily) or oral gavage (10, 20 mg/kg once daily) for 5 days. Liver, lung, and spleen tissues were homogenized for viral titer measurement (plaque assay). Serum ALT/AST levels were measured to evaluate hepatotoxicity, and mouse weight/survival was recorded [4]

1. Pharmacokinetics in mice: After oral administration of PF-3450074 (20 mg/kg) to CD-1 mice, the peak plasma concentration (Cmax) was 1.5 μg/mL (Tmax = 1 hour), the elimination half-life (t₁/₂) was 4.2 hours, and the absolute oral bioavailability was 35% [1]
2. Pharmacokinetics of mice after intravenous injection: After intravenous injection of PF-3450074 (5 mg/kg) to mice, the volume of distribution (Vd) was 1.8 L/kg, and the plasma clearance (CL) was 10 mL/min/kg [1]
3. Pharmacokinetics in rats: After oral administration of PF-3450074 (30 mg/kg) to Sprague-Dawley rats, the peak plasma concentration (Cmax) was 2.1 μg/mL, the half-life was 5.1 hours, and the bioavailability was 28%; the AUC₀-∞ of intravenous injection (10 mg/kg) was 28% 25 μg·h/mL [4]
4. Tissue distribution: PF-3450074 is mainly distributed in the liver, spleen and lungs of mice (tissue/plasma ratio of 2.5–3.0), with low brain permeability (brain/plasma ratio of 0.12) [4]
5. Plasma protein binding rate: PF-3450074 has a plasma protein binding rate of 92% in human plasma and 90% in mouse plasma (determined by ultrafiltration) [2]

1. In vitro cytotoxicity: PF-3450074 (≤1 μM) showed no significant cytotoxicity to normal human L02 hepatocytes and MRC-5 fibroblasts (cell viability >85%); in tumor cells, cytotoxicity was observed only at concentrations >5 μM (IC50 of L02/MRC-5 > 5 μM) [1][3] 2. Acute in vivo toxicity: A single intraperitoneal injection of PF-3450074 (200 mg/kg) in mice did not cause death or behavioral abnormalities (sleepiness/ataxia) within 7 days; a single oral administration (500 mg/kg) resulted in transient decreased activity, which was completely recovered within 24 hours (LD50 > 500 mg/kg orally) [2] 3. Chronic in vivo toxicity: Treatment with PF-3450074 (30 mg/kg/day orally) for 28 days In rats, serum ALT/AST, creatinine, or blood urea nitrogen levels remained unchanged for 1 day; histopathological analysis of liver/kidney tissue showed no abnormalities. High-dose treatment (100 mg/kg/day) can cause mild leukopenia (reversible after discontinuation) [3]
4. Drug interactions: PF-3450074 (≤1 μM) does not inhibit human CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) in vitro, and no pharmacokinetic interactions with antiretroviral drugs (AZT) or antiHCV drugs (sofosbuvir) were observed [2][4]

[1]. J Drug Des Res. 2018;5(2). pii: 1070. Epub 2018 Aug 13.

[2]. J Virol. 2011 Jan;85(1):542-9.

[3]. J Virol.2016 May 27;90(12):5808-5823.

[4]. J Virol.2018 Sep 26;92(20). pii: e00845-18.

1. PF-3450074 is a selective Cdk9 small molecule inhibitor developed by Pfizer. It was initially considered an anticancer drug candidate and was later reused in antiviral research [1][2]. 2. PF-3450074 exerts its antiviral activity by inhibiting Cdk9/cyclin T1-mediated phosphorylation of RNA polymerase II Ser2, thereby blocking the transcriptional elongation of host Cdk9-dependent viral genes (HIV-1, HCV, HCMV) [2][3][4]. 3. PF-3450074 has broad-spectrum antiviral activity against retroviruses (HIV-1), flaviviruses (HCV), and herpesviruses (HCMV), including drug-resistant strains [2][3][4]. 4. In preclinical studies, PF-3450074 has shown synergistic effects with existing antiviral drugs, such as AZT for HIV-1 and sofosbuvir for HCV, suggesting the existence of potential combination therapy strategies [2][3]

C27H27N3O2

425.53

425.21

C, 76.21; H, 6.40; N, 9.87; O, 7.52

1352879-65-2

1352879-65-2;

49800090

Light yellow to yellow solid powder

4.8

2

2

7

32

626

1

N1C2=C(C=CC=C2)C(CC(N[C@@H](CC2=CC=CC=C2)C(N(C)C2=CC=CC=C2)=O)=O)=C1C

ACDFWSNAQWFRRF-VWLOTQADSA-N

InChI=1S/C27H27N3O2/c1-19-23(22-15-9-10-16-24(22)28-19)18-26(31)29-25(17-20-11-5-3-6-12-20)27(32)30(2)21-13-7-4-8-14-21/h3-16,25,28H,17-18H2,1-2H3,(H,29,31)/t25-/m0/s1

2-Methyl-N-[(1S)-2-(methylphenylamino)-2-oxo-1-(phenylmethyl)ethyl]- 1H-indole-3-acetamide

PF74; PF-74; PF 74; PF-3450074; PF 3450074; PF3450074;

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 (~587.52 mM）

Solubility in Formulation 1: ≥ 6.25 mg/mL (14.69 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 2: 10% DMSO+90% Corn Oil: ≥ 6.25 mg/mL (14.69 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)