AZD-5991 (AZD5991) is a novel, potent, rationally designed macrocyclic molecule and selective MCL-1 inhibitor with anticancer activity. With an IC50 of 0.7 nM in the FRET assay and a Kd of 0.17 nM in the surface plasmon resonance (SPR) assay, it inhibits MCL-1. The drug AZD5991, which is currently in clinical development, has a high affinity and selectivity for Mcl-1. By activating the Bak-dependent mitochondrial apoptotic pathway, AZD5991 binds directly to Mcl-1 and causes rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia. After a single well-tolerated dose, AZD5991 exhibits strong antitumor activity in vivo with complete tumor regression in a number of models of multiple myeloma and acute myeloid leukemia when used alone or in combination with bortezomib or venetoclax. A Phase I clinical trial (NCT03218683) has been started to evaluate AZD5991 in patients with hematological malignancies based on these encouraging data.

Physicochemical Properties

Molecular Formula	C35H34CLN5O3S2
Molecular Weight	672.2592
Exact Mass	671.18
Elemental Analysis	C, 62.53; H, 5.10; Cl, 5.27; N, 10.42; O, 7.14; S, 9.54
CAS #	2143061-81-6
Related CAS #	AZD-5991 Racemate;2143010-83-5;AZD-5991 (S-enantiomer);2143061-82-7
PubChem CID	131634760
Appearance	White to off-white solid powder
LogP	6.8
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	1
Heavy Atom Count	46
Complexity	1060
Defined Atom Stereocenter Count	0
InChi Key	KBQCEQAXHPIRTF-UHFFFAOYSA-N
InChi Code	InChI=1S/C35H34ClN5O3S2/c1-20-31-29(38-40(20)3)19-45-17-22-15-23(41(4)37-22)18-46-24-14-21-8-5-6-9-25(21)30(16-24)44-13-7-10-26-27-11-12-28(36)32(31)33(27)39(2)34(26)35(42)43/h5-6,8-9,11-12,14-16H,7,10,13,17-19H2,1-4H3,(H,42,43)
Chemical Name	17-chloro-5,13,14,22-tetramethyl-28-oxa-2,9-dithia-5,6,12,13,22-pentazaheptacyclo[27.7.1.14,7.011,15.016,21.020,24.030,35]octatriaconta-1(36),4(38),6,11,14,16,18,20,23,29(37),30,32,34-tridecaene-23-carboxylic acid
Synonyms	AZD 5991; AZD-5991; AZD5991
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	Mcl-1 (IC50 = 0.7 nM); Mcl-1 (Kd = 0.17 nM)
ln Vitro	AZD5991 is a potent and direct inhibitor of Mcl-1 with high selectivity versus other Bcl-2 family proteins. Apoptosis is triggered quickly in cancer cells by AZD5991 by binding directly to Mcl-1 and activating the Bak-dependent mitochondrial apoptotic pathway, most notably in myeloma and acute myeloid leukemia (GI50 100nM) cells. Hematological cells are preferentially killed by AZD5991 in a panel of cancer-derived cell lines with hematological or solid tumor origins[1][3].
ln Vivo	After a single well-tolerated dose, AZD5991 exhibits strong antitumor activity in vivo with complete tumor regression in a number of models of multiple myeloma and acute myeloid leukemia when used alone or in combination with bortezomib or venetoclax. As shown by the cleavage of caspase-3 and PARP in these in vivo studies, AZD5991's cytotoxic activity closely correlates with the activation of the mitochondrial apoptotic pathway[1].
Cell Assay	MOLP-8 cells are treated with AZD5991 or DMSO ontrol for 30 min. The samples are then centrifuged, and the pellet is resuspended in ice-cold lysis buffer and incubated for 20 min.on ice with vortexing every 5 min. After centrifuging the samples, the protein concentration was measured. Before incubating with anti-Mcl-1 antibody overnight at 4 °C with rotation, samples are pre-cleared for 30 min at 4 °C using rotation and a 50% slurry of Protein A/G magnetic beads. After that, Protein A/G magnetic beads are added and rotated for 1 hour at 4 °C. Each IP pellet is given a 10% sample reducing agent addition before being washed four times with lysis buffer/PBS (1:1) and then being subjected to a western blotting analysis.
Animal Protocol	Mice and Rats[1] In mice, drugs (e.g., AZD5991; 10-100 mg/kg) are dosed intravenously in a volume of 5 mL/kg except for Venetoclax that is dosed orally in a volume of 10 mL/kg. One million MV4-11, five million MOLP-8, ten million NCI-H929 or five million OCI-AML3 cells are injected subcutaneously in the right flank of mice in a volume of 0.1 mL. In rats, AZD5991 (10-100 mg/kg) is dosed intravenously in a volume of 10 mL/kg. Ten million MV4-11 cells are injected subcutaneously in the right flank of rats in a volume of 0.1 mL. Tumor volumes (measured by caliper), animal body weight, and tumor condition are recorded twice weekly for the duration of the study. The tumor volume is calculated[1].
References	[1]. Nat Commun . 2018 Dec 17;9(1):5341. [2]. Curr Opin Chem Biol . 2017 Aug:39:133-142. [3] AACR Cancer Res. 2018, 78(13 Suppl):Abstract nr 302.
Additional Infomation	AZD-5991 is under investigation in clinical trial NCT03218683 (Study of AZD5991 in Relapsed or Refractory Haematologic Malignancies.). Mcl-1 Inhibitor AZD5991 is an inhibitor of induced myeloid leukemia cell differentiation protein (myeloid cell leukemia-1; Mcl-1; Bcl2-L-3), with potential pro-apoptotic and antineoplastic activities. Upon administration, AZD5991 binds to Mcl-1, thereby preventing the binding of Mcl-1 to and inactivation of certain pro-apoptotic proteins, and promoting apoptosis of cells overexpressing Mcl-1. Mcl-1, an anti-apoptotic protein belonging to the Bcl-2 family of proteins, is upregulated in cancer cells and promotes tumor cell survival.

Solubility Data

Solubility (In Vitro)

DMSO: 100~250 mg/mL (148.8~371.9 mM）

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.09 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (3.09 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. Solubility in Formulation 3: 2.08 mg/mL (3.09 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 ultrasonication. 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 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 4: 2.08 mg/mL (3.09 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 ultrasonication. 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 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 5: ≥ 2.08 mg/mL (3.09 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. Solubility in Formulation 6: ≥ 2.08 mg/mL (3.09 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.4875 mL	7.4376 mL	14.8752 mL
	5 mM	0.2975 mL	1.4875 mL	2.9750 mL
	10 mM	0.1488 mL	0.7438 mL	1.4875 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.