SY-5609 (SY5609; CDK7-IN-3) is a novel, selective, non-covalent and orally bioavailable CDK7 inhibitor (KD = 0.059 nM) with anticancer activity. SY-5609 shows high selectivity for CDK7 over CDK2 (K_i=2600 nM), CDK9 (K_i=960 nM), CDK12 (K_i=870 nM). CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.

Physicochemical Properties

Molecular Formula	C23H26F3N6OP
Molecular Weight	490.4611
Exact Mass	490.185
CAS #	2417302-07-7
PubChem CID	146662729
Appearance	White to light yellow solid powder
LogP	2.7
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	4
Heavy Atom Count	34
Complexity	838
Defined Atom Stereocenter Count	1
SMILES	P(C([H])([H])[H])(C([H])([H])[H])(C1=C(C#N)C([H])=C([H])C2=C1N([H])C([H])=C2C1C(C(F)(F)F)=C([H])N=C(N=1)N([H])[C@]1([H])C([H])([H])N([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])C1([H])[H])=O
InChi Key	JDJOUBVVSQDIRC-AWEZNQCLSA-N
InChi Code	InChI=1S/C23H26F3N6OP/c1-22(2)8-7-14(10-30-22)31-21-29-12-17(23(24,25)26)18(32-21)16-11-28-19-15(16)6-5-13(9-27)20(19)34(3,4)33/h5-6,11-12,14,28,30H,7-8,10H2,1-4H3,(H,29,31,32)/t14-/m0/s1
Chemical Name	7-dimethylphosphoryl-3-[2-[[(3S)-6,6-dimethylpiperidin-3-yl]amino]-5-(trifluoromethyl)pyrimidin-4-yl]-1H-indole-6-carbonitrile
Synonyms	SY 5609SY-5609 SY5609
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	SY-5609 (0.01-10000 nM; 72 hours) has strong antiproliferative effects on ovarian (OVA) and triple-negative breast cancer (TNBC) cells [1]. Apoptosis is induced by SY-5609 (100–500 nM; 48–72 hours) [1]. In HCC70 cells, SY-5609 (100–500 nM; 48 hours) causes G2/M cell cycle arrest [1]. Through CAK loss of function, SY-5609 (25–500 nM; 6-48 hours) suppresses CDK2 phosphorylation at Thr160 [1]. In the HCC70 cell line, SY-5609 (Compound 101; 126.4 pM-4 µM; 72 hours) has an EC50 of 5.6 nM [2].
ln Vivo	SY-5609 (2 mg/kg/day; oral; for 21 days) causes tumor regression throughout a 21-day dosage period [1]. Mice were orally treated 2 mg/kg SY-5609 daily, the plasma exposure was 261.28 ng h/mL, the Cmax was 50.67 ng/mL (103 nM), and the elimination half-life was 3.33 h[1].
Cell Assay	Cell Proliferation Assay[1] Cell Types: HCC70, MDA-MB453, COV504, A2780, OVCAR3, CAOV3 Cell Tested Concentrations: 0.01-10000 nM Incubation Duration: 72 hrs (hours) Experimental Results: Displayed strong anti-proliferative effect with IC50 of 1-6 nM . Apoptosis analysis[1] Cell Types: HCC70, MDA-MB-468, CAOV3 and OVCAR3 Cell Tested Concentrations: 100, 250, 500 nM Incubation Duration: 48 and 72 hrs (hours) Experimental Results: Induction of apoptosis. Cell cycle analysis[1] Cell Types: HCC70 Cell Tested Concentrations: 100, 250, 500 nM Incubation Duration: 48 hrs (hours) Experimental Results: Induced G2/M cell cycle arrest. Western Blot Analysis[1] Cell Types: HCC70 Cell Tested Concentrations: 25, 50, 100, 250, 500 nM Incubation Duration: 6, 24, 48 hrs (hours) Experimental Results: Inhibition of CDK2 phosphorylation at Thr160 via loss of CAK function 24 and 48 Hour.
Animal Protocol	Animal/Disease Models: 6 to 8 weeks old HCC70 cell line Balb/c nude female mice [1] Doses: 2 mg/kg Route of Administration: oral; daily; 21 days Experimental Results: Tumor induction over 21 days of dosing period subsided and was well tolerated. No tumor regrowth was observed until day 28.
References	[1]. Discovery of SY-5609: A Selective, Noncovalent Inhibitor of CDK7. J Med Chem. 2021 Nov 2. [2]. Inhibitors of cyclin-dependent kinase 7 (cdk7). WO2020093011A1.
Additional Infomation	CDK7 Inhibitor SY-5609 is an orally bioavailable, selective inhibitor of cyclin-dependent kinase 7 (CDK7), with potential antineoplastic activity. Upon oral administration, SY-5609 selectively targets, binds to and inhibits the activity of CDK7, thereby inhibiting CDK7-mediated signaling. Specifically, inhibition of CDK7 prevents phosphorylation of the carboxy-terminal domain (CTD) of RNA Polymerase II, thereby preventing transcription of important cancer-promoting genes. In addition, it prevents phosphorylation of the cell cycle kinases CDK1, 2, 4, and 6, thereby disrupting uncontrolled cell cycle progression. Altogether, this may induce apoptosis, cause cell cycle arrest, inhibit DNA damage repair and inhibit tumor cell proliferation in certain cancers that are dependent on CDK7-mediated transcriptional regulation and signaling. CDK7, a serine/threonine kinase, plays a role in controlling cell cycle progression, transcriptional regulation, and promotes the expression of key oncogenes such as c-Myc and beta-catenin, through the phosphorylation of RNA polymerase II.

Solubility Data

Solubility (In Vitro)

DMSO : ~40 mg/mL (~81.56 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 4 mg/mL (8.16 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 sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 40.0 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. Solubility in Formulation 2: ≥ 4 mg/mL (8.16 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 40.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: ≥ 2.5 mg/mL (5.10 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0389 mL	10.1945 mL	20.3890 mL
	5 mM	0.4078 mL	2.0389 mL	4.0778 mL
	10 mM	0.2039 mL	1.0195 mL	2.0389 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.