(+)-JQ1 PA (also known as (+)-JQ1 propargyl amide) is a novel propargyl amide derivative of (+)-JQ1 with IC50 of 10.4 nM for Bromodomain and extra-terminal (BET). It was created as a functionally conserved compound that is amenable to click chemistry and can be used as molecular probes in vitro and in vivo. (+)-JQ1 is a potent and highly specific BET (Bromodomain and extra terminal domain) bromodomain inhibitor, with IC50 of 77 nM and 33 nM for BRD4(1/2) in enzymatic assays. (−)-JQ1 shows no significant interaction with any bromodomain. Besides, (−)-JQ1 enantiomer is comparatively inactive in nuclear protein in testis (NUT) midline carcinoma (NMC). (+)-JQ1 has high specificity for BET in that it only binds to bromodomains of the BET family, but not to any bromodomains of non-BET family. (+)-JQ1 has potential antineoplastic activity against various cancers such as MM (Multiple myeloma), pancreatic ductal adenocarcinoma and ovarian cancer etc. Its mechanism of action is to inhibit c-MYC and upregulate p21. (+)-JQ1 has been used as a chemical probe to investigate the role of BET bromodomains in the transcriptional regulation of oncogenesis.

Physicochemical Properties

Molecular Formula	C22H20CLN5OS
Molecular Weight	437.945101737976
Exact Mass	437.11
Elemental Analysis	C, 60.34; H, 4.60; Cl, 8.09; N, 15.99; O, 3.65; S, 7.32
CAS #	2115701-93-2
Related CAS #	2115701-93-2 [(+)-JQ1 PA];
PubChem CID	134821687
Appearance	Typically exists as Light yellow to yellow solids
LogP	3.4
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	30
Complexity	730
Defined Atom Stereocenter Count	1
SMILES	CC1=C(SC2=C1C(=N[C@H](C3=NN=C(N32)C)CC(=O)NCC#C)C4=CC=C(C=C4)Cl)C
InChi Key	ZLSCJWMPQYKVKU-KRWDZBQOSA-N
InChi Code	InChI=1S/C22H20ClN5OS/c1-5-10-24-18(29)11-17-21-27-26-14(4)28(21)22-19(12(2)13(3)30-22)20(25-17)15-6-8-16(23)9-7-15/h1,6-9,17H,10-11H2,2-4H3,(H,24,29)/t17-/m0/s1
Chemical Name	2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]-N-prop-2-ynylacetamide
Synonyms	(+)-JQ-1 PA;(+)-JQ 1 PA;(+)-JQ1 PA;(+)-JQ1 PA; 2115701-93-2; (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(prop-2-yn-1-yl)acetamide; 2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]-N-prop-2-ynylacetamide; (6S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-N-2-propyn-1-yl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide; SCHEMBL24861365; (+)-JQ1 PA?; (+)-JQ1 propargyl amide
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
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

Targets	BET ( IC50 = 10.4 nM)[1].
ln Vitro	(+)-JQ1 PA is a derivative of JQ1 and an inhibitor of BET. The IC50 of (+)-JQ1 PA against BET is 10.4 nM, whereas the IC50 of JQ1 in MV4;11 cells is 14.3 nM [1].
ln Vivo	(+)-JQ1 (50 mg/kg) inhibits tumors growth in mice with NMC 797 xenografts. (+)-JQ1 (50 mg/kg) results in effacement of NUT nuclear speckles in mice with NMC 797 xenografts, consistent with competitive binding to nuclear chromatin. (+)-JQ1 (50 mg/kg) induces strong (grade 31) keratin expression in NMC 797 xenografts. (+)-JQ1 (50 mg/kg) promotes differentiation, tumor regression and prolonged survival in mice models of NMC xenografts. (+)-JQ1 (50 mg/kg) results in a significant prolongation in overall survival of SCID-beige mice orthotopically xenografted after intravenous injection with MM.1S-luc+ cells compared to vehicle-treated animals. (+)-JQ1 (50 mg/kg i.p.) leads to a highly significant increase in survival of mice bearing Raji xenografts.
Enzyme Assay	Enhancers with differential JQ1–PA occupancy[1] Coverage of ChIP-seq and click-seq reads were calculated with BEDtools(32) and normalised by size of region and library size. Plots were drawn in R(33) with 15 ggplot2. The relative click-seq coverage to BRD4 ChIP-seq coverage at each enhancer was calculated by the LFC of JQ1–PA normalised reads to BRD4 normalised reads. The resulting LFC were used to divide enhancers into 5 equal sized groups of differing JQ1–PA occupancy.
Cell Assay	RNA-seq [1] MV4;11 cells were cultured with JQ1 or JQ1-PA (0.5 μM) for 6 hours. RNA extracted as previously described. Reads were aligned to the human genome (G1k V37) with Tophat2 and Bowtie2, and reads were assigned to genes with htseq-count. Differential expression was calculated with edgeR in the R statistical programming language. Genes with a false discovery rate (corrected for multiple testing with the method of Benjamini and Hochberg) below 0.05 and a log2 fold change (LFC) greater than one were considered to be significantly differentially expressed. Correlation plot and heatmap of RNA-seq data were drawn in R with ggplot2. In vitro Click-chem Fluorescence. [1] (Cu(I) dependant) MV4;11 cells treated with JQ1-PA , 50nM-5μM (3μM for microscopy) or vehicle; 5μM JQ1 in culture for 3 hrs, fixed with 4% PFA (EMS) for 10 minutes, permeabilized (0.1% Triton-X) and added to Cu+ dependant Click Master Mix; (488 Alexa-Fluor azide 5μM, E301 5mM, and 4mM CuSO4). Cells were then washed 3× in 16 PBST buffer and either mounted onto Poly-L-lysine coated class slide and/or assessed by flow cytometry. Cells imaged by microscopy were also probed for BRD4 as per protocol above and imaged on Leica TCS SP5 Confocal Microscope with 63× oil objectives.
Animal Protocol	In vivo formulations used (reported): 1. Dissolved in 5% dextrose; 50 mg/kg; i.p. injection; Nature. 2010 Dec 23;468(7327):1067-73 2. Dissolved in 10% DMSO and 90% of a 10% 2-hydroxypropyl-β-cyclodextrin solution; Leukemia. 2017 Oct;31(10):2037-2047 3. Dissolved in 1% DMSO+5% Glucose+ddH2O; Cell. 2018 Sep 20;175(1):186-199.e19 4. Dissolved in 20% hydroxypropyl-β-cyclodextrin, 5% DMSO, 0.2% Tween-80 in saline; Mol Cancer Ther. 2016 Jun;15(6):1217-26 5. Dissolved in 1:1 propylene glycol:water; J Biol Chem. 2016 Nov 4;291(45):23756-23768 6. Dissolved in 5% DMSO in 10% 2-hydroxypropyl-β-cyclodextrin solution; Cancer Lett. 2017 Aug 28;402:100-109
References	[1]. Click chemistry enables preclinical evaluation of targeted epigenetic therapies. Science. 2017 Jun 30;356(6345):1397-1401.
Additional Infomation	The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.[1]

Solubility Data

Solubility (In Vitro)

DMSO: 10 mM Water: Ethanol:

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2834 mL	11.4168 mL	22.8337 mL
	5 mM	0.4567 mL	2.2834 mL	4.5667 mL
	10 mM	0.2283 mL	1.1417 mL	2.2834 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.