ARD-61 is a highly efficient and specific PROTAC androgen receptor (AR) degrader. ARD-61 potently and efficiently induces AR and progesterone receptor (PR) degradation in AR+ cancer cell lines. ARD-61 causes apoptosis and also effectively inhibits tumor growth in the mouse MDA-MB-453 xenograft model. ARD-61 is a reagent for click chemistry. It contains Alkyne groups and could undergo CuAAc (copper-catalyzed azide-alkyne cycloaddition reaction) with compounds bearing an Azide group.

Physicochemical Properties

Molecular Formula	C61H71CLN8O7S
Molecular Weight	1095.78385281563
Exact Mass	1094.485
CAS #	2316837-08-6
PubChem CID	154704831
Appearance	White to light yellow solid powder
LogP	9.3
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	15
Heavy Atom Count	78
Complexity	2200
Defined Atom Stereocenter Count	4
SMILES	ClC1=C(C#N)C=CC(=C1)OC1C(C)(C)C(C1(C)C)NC(C1C=CC(C#CC2CCN(CC2)C2CCN(C(C[C@@H](C3C=CC(C4=C(C)N=CS4)=CC=3)NC([C@@H]3C[C@H](CN3C([C@@H](C3=CC(C)=NO3)C(C)C)=O)O)=O)=O)CC2)=CC=1)=O
InChi Key	RGPUSLXMPBTXNU-CUPIBCLHSA-N
InChi Code	InChI=1S/C61H71ClN8O7S/c1-36(2)53(51-29-37(3)67-77-51)57(75)70-34-46(71)30-50(70)56(74)65-49(41-15-17-42(18-16-41)54-38(4)64-35-78-54)32-52(72)69-27-23-45(24-28-69)68-25-21-40(22-26-68)10-9-39-11-13-43(14-12-39)55(73)66-58-60(5,6)59(61(58,7)8)76-47-20-19-44(33-63)48(62)31-47/h11-20,29,31,35-36,40,45-46,49-50,53,58-59,71H,21-28,30,32,34H2,1-8H3,(H,65,74)(H,66,73)/t46-,49+,50+,53-,58?,59?/m1/s1
Chemical Name	(2S,4R)-N-[(1S)-3-[4-[4-[2-[4-[[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]carbamoyl]phenyl]ethynyl]piperidin-1-yl]piperidin-1-yl]-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]-3-oxopropyl]-4-hydroxy-1-[(2R)-3-methyl-2-(3-methyl-1,2-oxazol-5-yl)butanoyl]pyrrolidine-2-carboxamide
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	VHL
ln Vitro	In order to recruit AR protein to cullin 2 for ubiquitination, followed by proteasome-dependent AR degradation, ARD-61 binds to AR protein through its AR antagonist portion and von Hippel-Lindau (VHL)/cullin 2 E3 ligase through its VHL ligand portion[1]. ARD-61 (0.001-100 μM; for 7 days) has IC50 values of 235 nM and 121 nM in the MDA-MB-453 and HCC1428 cell lines, which have the highest AR expression, respectively. With IC50 values of 39, 147, and 380 nM, respectively, ARD-61 exhibits partial inhibition of cell growth in the MCF-7, BT-549, and MDA-MB-415 cell lines, all of which have a moderate level of AR protein[1]. In all three of these AR+ breast cancer cell lines, ARD-61 (25–1000 nM; 6-72 h) induces G2/M cell cycle arrest in a dose- and time-dependent manner[1]. The MDA-MB-453 and HCC1428 cell lines undergo apoptosis when exposed to ARD-61 (25–1000000 nM; 72 h)[1]. The potency and effectiveness of ARD-61 (0.01-1000 nM; 6 h) in lowering AR protein levels is very high. In the T47D cells, ARD-61 (0.01-1000 nM; 6 h) nM; 24 h) lowers the level of PR protein with a DC50 value of 0.15 nM. The ER and GR proteins are not visibly affected by ARD-61[1]. ARD-61 (1 µM) blocks the MYC and Wnt/β-catenin signaling pathways efficiently over a 24-hour period. In addition to decreasing phosphorylated and unphosphorylated HER2 and HER3 proteins, ARD-61 (1-1000 nM) is also effective against both for a 24-hour period[1]. Both MDA-MB-453 and MCF-7 cell lines exhibit complete resistance to AR degradation induced by ARD-61 (100 nM; 24 h) when VHL is effectively knocked down[1].
ln Vivo	In male SCID mice, ARD-61 (25, 50 mg/kg/day; ip; for 75 days) efficiently suppresses tumor growth in the MDA-MB-453 xenograft tumor model[1]. The AR protein in the MDA-MB-453 xenograft tissue is quickly and efficiently reduced by ARD-61 (25 mg/kg; ip; for 75 days; single dose), with the effect lasting for at least 24 hours. In a time-dependent way, ARD-61 is particularly effective in lowering the mRNA level of WNT7B[1].
Cell Assay	Cell Viability Assay[1] Cell Types: MDA-MB-453 and HCC1428 cell lines Tested Tested Concentrations: 0.001, 0.01, 0.1, 1, 10, 100 μM Incubation Duration: 7 days Experimental Results: Achieves near complete inhibition of cell growth. Cell Cycle Analysis[1] Cell Types: MDA-MB-453, HCC1428 and MCF-7 cell lines Tested Tested Concentrations: 25, 250, 500, 1000, 10000, 100000 nM Incubation Duration: 6-72 hrs (hours) Experimental Results: Induced G2/M cell cycle arrest in a dose- and time-dependent manner in each of these three AR+ breast cancer cell lines. Apoptosis Analysis[1] Cell Types: MDA-MB-453 and HCC1428 cell lines Tested Tested Concentrations: 25, 250, 500, 1000, 10000, 100000 nM Incubation Duration: 6-72 hrs (hours) Experimental Results: Induced apoptosis in the MDA -MB-453 and HCC1428 cell lines in a dose-dependent manner. Western Blot Analysis[1] Cell Types: MDA-MB-453, MCF-7, BT549, MDA-MB-415 and HCC1428 cell lines Tested Tested Concentrations: 0.01, 0.03 , 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 nM Incubation Duration: 6 hrs (hours) Experimental Results: diminished AR protein levels in the MDA-MB-453 (DC50=0.44 nM), MCF-7 (DC50= 1.8
Animal Protocol	Animal/Disease Models: MDA-MB-453 xenograft tumor model in male SCID mice[1] Doses: 25, 50 mg/kg Route of Administration: IP; daily; for 75 days Experimental Results: Effectively inhibited tumor growth.
References	[1]. A highly potent PROTAC androgen receptor (AR) degrader ARD-61 effectively inhibits AR-positive breast cancer cell growth in vitro and tumor growth in vivo. Neoplasia. 2020 Oct;22(10):522-532.

Solubility Data

Solubility (In Vitro)

DMSO: 100 mg/mL (91.26 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.28 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (2.28 mM) (saturation unknown) in 10% DMSO + 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 25.0 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 3: ≥ 2.5 mg/mL (2.28 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	0.9126 mL	4.5630 mL	9.1259 mL
	5 mM	0.1825 mL	0.9126 mL	1.8252 mL
	10 mM	0.0913 mL	0.4563 mL	0.9126 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.