Mcl-1 (IC50 = 0.7 nM); Mcl-1 (Kd = 0.17 nM)
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].

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].

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AZD5991 induces rapid apoptosis in Mcl-1-dependent cancer cell lines. In the sensitive multiple myeloma cell line MOLP-8, treatment with 10 nM AZD5991 dissociated Mcl-1 from the pro-apoptotic protein Bak within 15 minutes, as shown by co-immunoprecipitation. This was followed by mitochondrial outer membrane permeabilization (MOMP), caspase-3/7 activation, phosphatidylserine externalization, loss of cellular ATP, and ultimately cell membrane permeability within 20 hours. [1]
Cellular Thermal Shift Assay (CETSA) in MV4-11 cells demonstrated that AZD5991 engages and stabilizes Mcl-1 protein in cells with an EC₅₀ of 13 nM (95% CI 0.004328–0.03125 nM). [1]
The cytotoxic activity of AZD5991 is Bak-dependent. Depletion of Bak in NCI-H23 cells conferred resistance to AZD5991-induced caspase-3/7 activation and cell death. Overexpression of other anti-apoptotic proteins (Bcl-2, Bcl-xL, Bfl-1/A1, Bcl-w) blocked its activity in Eu-Myc lymphoma cells, confirming on-target, Mcl-1-specific mechanism. [1]
In a broad panel of cancer cell lines, hematological malignancies (e.g., multiple myeloma, acute myeloid leukemia) were particularly sensitive to AZD5991. Its ability to inhibit cell growth closely correlated with its capacity to activate caspase-3/7. [1]
Ex vivo treatment of primary mononuclear cells isolated from bone marrow of 48 multiple myeloma patients with AZD5991 for 24 hours showed that 71% of samples had an EC₅₀ for apoptosis induction below 100 nM. [1]
In vitro combination studies showed that AZD5991 synergizes with the proteasome inhibitor bortezomib (which upregulates the pro-apoptotic protein Bim) and the Bcl-2 inhibitor venetoclax in inducing caspase activation, particularly in cell lines resistant to single agents. [1]

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].

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In a subcutaneous MOLP-8 (multiple myeloma) xenograft model in mice, a single intravenous dose of AZD5991 induced dose-dependent anti-tumor activity. Doses of 10 and 30 mg/kg resulted in 52% and 93% tumor growth inhibition (TGI), respectively, at day 10. A single dose of 60 mg/kg led to 99% tumor regression (TR) with no detectable tumors in 6 out of 7 mice, and a 100 mg/kg dose caused complete TR in all mice. Tumor regression correlated with induction of cleaved caspase-3 in tumors. [1]
In an NCI-H929 multiple myeloma xenograft model, a single intravenous dose of AZD5991 at 100 mg/kg also resulted in complete tumor regression. [1]
In an MV4-11 (acute myeloid leukemia, AML) xenograft model, a single intravenous dose of AZD5991 (10, 30, 100 mg/kg) caused dose-dependent anti-tumor activity, with 100 mg/kg inducing complete tumor regression in all mice within 7 days. Efficacy correlated with increased cleaved caspase-3 and PARP in tumors. [1]
In a disseminated MOLM-13 AML model in mice, weekly intravenous AZD5991 (60 mg/kg) significantly reduced the percentage of leukemic cells in both peripheral blood and bone marrow after 10 days of treatment. [1]
Combination therapy in vivo demonstrated enhanced efficacy. In the NCI-H929 model, a sub-efficacious dose of AZD5991 (30 mg/kg i.v.) combined with bortezomib (1 mg/kg i.v.) induced 88% tumor regression, whereas single agents showed minimal activity. [1]
In the OCI-AML3 xenograft model (inherently resistant to single agents), the combination of AZD5991 (60 mg/kg i.v., weekly) and venetoclax (100 mg/kg p.o., daily) led to tumor regression in all mice, while monotherapies showed minimal growth inhibition. [1]

The binding affinity and selectivity of AZD5991 for Mcl-1 and other Bcl-2 family proteins were determined using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) competitive binding assays. Recombinant GST- or His-tagged proteins (Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bfl-1) were incubated with a Europium-labeled antibody and a fluorescently-labeled peptide (Bim or Bak peptide). Test compounds were serially diluted and added to the reaction. After incubation, the ratio of emission at 665 nm (acceptor) to 612 nm (donor) was measured. Percent inhibition was calculated relative to DMSO and peptide-only controls, and IC₅₀ values were derived. Inhibition constants (K_i) were calculated using the Cheng-Prusoff equation with known peptide K_d values. [1]

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.

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For caspase activation and cell viability assays, cells were plated in 384-well plates. For caspase-3/7 activation, cells were treated with AZD5991 for 2 or 6 hours, followed by addition of Caspase-Glo 3/7 reagent, and luminescence was measured. For cell viability, cells were treated for 24 hours, followed by addition of CellTiter-Glo reagent, and luminescence was measured. Dose-response curves were plotted to determine EC₅₀ (caspase) or GL₅₀ (viability) values. [1]
For apoptosis kinetic analysis in MOLP-8 cells, markers were assessed over time after AZD5991 treatment: Mitochondrial outer membrane permeabilization (MOMP) was measured by TMRE staining and flow cytometry; phosphatidylserine exposure was measured by Annexin V staining; cell membrane permeability was measured by 7-AAD staining. [1]
For cellular target engagement, a Cellular Thermal Shift Assay (CETSA) was performed. MV4-11 cells were treated with AZD5991 or DMSO, subjected to a heat challenge, lysed, and Mcl-1 protein levels in the soluble fraction were analyzed by Western blot. Isothermal dose-response curves at 48°C were used to determine the EC₅₀ for Mcl-1 stabilization. [1]
For co-immunoprecipitation (co-IP) to assess Mcl-1:Bak complex disruption, MOLP-8 cells were treated with AZD5991, lysed, and Mcl-1 was immunoprecipitated. Co-precipitated Bak and levels of Mcl-1, Bim, and cleaved PARP in the lysate were detected by Western blot. [1]

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].

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For subcutaneous xenograft efficacy studies, female CB-17 SCID mice were implanted with tumor cells (e.g., 1x10^6 MV4-11, 5x10^6 MOLP-8). When tumors reached an average volume of approximately 230 mm³, mice were randomized into treatment groups. AZD5991 was formulated in 10% DMSO / 40% PEG400 / 50% citrate buffer (pH 4) and administered as a single intravenous bolus injection at doses ranging from 10 to 100 mg/kg in a volume of 5 mL/kg. Tumor volumes and body weights were monitored regularly. [1]
For combination studies with bortezomib in the NCI-H929 model, AZD5991 (30 mg/kg, i.v.) and bortezomib (1 mg/kg, i.v.) were administered as single doses. [1]
For combination studies with venetoclax in the OCI-AML3 model, AZD5991 was administered intravenously at 60 mg/kg once weekly, and venetoclax was administered orally at 100 mg/kg daily. Venetoclax was formulated in 10% ethanol, 30% PEG 400, 60% Phosal 50 PG. [1]
For the disseminated MOLM-13 AML model, CIEA-NOG mice were injected intravenously with leukemia cells. Treatment started 3 days later. AZD5991 (60 mg/kg) was administered intravenously once weekly. Bone marrow and peripheral blood were analyzed by flow cytometry for human CD45+ HLA-ABC+ leukemic cells on day 10. [1]
For pharmacokinetic analysis, plasma samples were collected from tumor-bearing mice at various time points after a single intravenous dose of AZD5991 (10-100 mg/kg). Samples were processed with acetonitrile containing internal standard and analyzed by LC-MS/MS against a calibration curve. [1]

AZD5991 exhibits high plasma protein binding, with only 0.1% of the free fraction detected in fetal bovine serum. This significantly affects its cellular viability, as evidenced by the increasing EC50 value of its induced caspase in MOLP-8 cells with increasing serum concentration (EC50 values were 0.001, 0.008, and 0.033 μM at 0%, 2%, and 10% serum concentrations, respectively). [1] In mice carrying MOLP-8 tumors, plasma exposure was dose-dependent following a single intravenous injection of AZD5991. We present plasma concentration-time curves over 24 hours at doses of 10, 30, 60, and 100 mg/kg. [1] The binding affinity of AZD5991 varies by species. Its dissociation constant K_d for mouse Mcl-1 is about 25 times lower than that for human Mcl-1, and about 4 times lower than that for rat Mcl-1. It has the same high affinity for human, dog and cynomolgus monkey Mcl-1. [1]

In mouse xenograft studies, single intravenous injections of AZD5991 at doses up to 100 mg/kg were well tolerated with no significant weight loss observed. However, the weak binding affinity to mouse Mcl-1 must be considered when interpreting tolerance in these models. [1] In the rat MV4-11 xenograft model, intravenous injection of AZD5991 at 30 mg/kg induced tumor regression without significant weight loss. [1] In the Eµ-Myc lymphoma homologous mouse model (where the host and tumor have similar sensitivity to Mcl-1), intravenous injection of AZD5991 at 100 mg/kg on days 3 and 10 post-transplantation cleared leukemia cells from peripheral blood and prolonged survival without serious toxicity, indicating that AZD5991 is active at tolerated doses in a pharmacologically relevant context. [1]

[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.

AZD-5991 is currently undergoing clinical trial NCT03218683 (Study on AZD5991 for the treatment of relapsed or refractory hematologic malignancies).
AZD5991, a Mcl-1 inhibitor, is an inhibitor of differentiation proteins inducible myeloid leukemia cells (Myeloid Leukemia-1; Mcl-1; Bcl2-L-3) with potential pro-apoptotic and anti-tumor activities. After administration, AZD5991 binds to Mcl-1, thereby preventing Mcl-1 from binding to and inactivating certain pro-apoptotic proteins, and promoting apoptosis in Mcl-1-overexpressing cells. Mcl-1 is an anti-apoptotic protein belonging to the Bcl-2 protein family, upregulated in cancer cells, and promotes tumor cell survival.
AZD5991 is a first-in-class, rationally designed cyclic molecule that directly and selectively inhibits the anti-apoptotic protein Mcl-1. Currently, AZD5991 is undergoing a Phase I clinical trial (NCT03218683) for patients with hematologic malignancies. [1]
The mechanism of action of this drug involves binding with a high affinity to Mcl-1, thereby replacing pro-apoptotic proteins such as Bak and Bim, and activating the endogenous (mitochondrial) apoptosis pathway in a Bak-dependent manner. [1]
AZD5991 has shown strong monotherapy activity in preclinical models of multiple myeloma and acute myeloid leukemia, and is expected to overcome resistance when used in combination with bortezomib or venetoclax. [1]

C35H34CLN5O3S2

672.2592

671.18

C, 62.53; H, 5.10; Cl, 5.27; N, 10.42; O, 7.14; S, 9.54

2143061-81-6

AZD-5991 Racemate;2143010-83-5;AZD-5991 (S-enantiomer);2143061-82-7

131634760

White to off-white solid powder

6.8

1

7

1

46

1060

0

KBQCEQAXHPIRTF-UHFFFAOYSA-N

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)

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

AZD 5991; AZD-5991; AZD5991

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: 100~250 mg/mL (148.8~371.9 mM）

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.

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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.

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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.

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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.

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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.

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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.

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