Selective inhibition of MDM2-amplified cancers using targeted protein degraders

Abstract

The present invention relates to compounds useful for selective inhibition of MDM2-amplified cancers and pharmaceutical compositions thereof.

Description

SELECTIVE INHIBITION OF MDM2-AMPLIFIED CANCERS USING TARGETEDPROTEIN DEGRADERSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 587,927, filed on 4 October 2023. This prior provisional application is expressly incorporated herein by reference.BACKGROUND OF THE INVENTION

[0002] Tumor suppressor, p53, plays a central role in preventing tumor formation. Careful control of the activity of p53 is necessary for mammalian survival. Known as the ‘cellular gatekeeper” or “guardian of the genome,” p53 is widely recognized as the most frequently mutated gene in human cancer; its inactivation occurs in nearly half of all human tumors. Fluctuation in p53 levels can be problematic, such that high p53 levels can be lethal and low p53 levels can permit tumorigenesis.

[0003] Murine double minute 2 (MDM2), is an E3 ubiquitin ligase that is over-expressed in many cancers and regulates target proteins through ubiquitination. An oncogene that is critical for the control of p53 activity, MDM2 has both p53-dependent andp53- independent oncogenic activities. MDM2 is a key negative regulator of the p53 protein and forms an auto-regulatory feedback loop with p53. The p53 / MDM2 feedback loop is one of the best-studied examples of a negative feedback loop comprised of a transcription arm and a protein-interaction arm. P53 transcriptionally activates MDM2 which in turn targets p53 for degradation. Under normal conditions, the p53 / MDM2 feedback loop keeps p53 levels relatively low, until stress, such as DNA damage, is applied to the cell. These stress signals block MDM2 and / or promote degradation. The resulting decrease in the activity of MDM2 in the feedback loop leads to stabilization of p53 levels which in turn leads to increased transcription of MDM2 by p53. Overexpression of MDM2 via copy number amplification can lead to increased cancer risk and / or accelerated tumor formation and progress. The overexpression of MDM2 has been observed in human tumors, such as sarcomas (liposarcoma, leiomyosarcoma, osteosarcoma, rhabdomyosarcoma), leukemia, melanoma, and cancers affecting the gastrointestinal tract, pancreas, breast, bladder, and lungs.

[0004] The inhibition of MDM2 represents a treatment approach for cancers with wild-type or functional p53. MDM2 inhibitors, such as navtemadlin, have shown promising clinical efficacy in patients with liposarcoma, relapsed / refractory solid tumors, and acute myeloid leukemia. See WO 2011 / 153509. However, conventional MDM2 inhibitors simply disrupt the p53 / MDM2 feedback loop causing p53 levels to get stabilized quickly which in turn cause MDM2 levels to increase rapidly. Thus, in order to continue p53 suppression, the MDM2 inhibitor is forced to outcompete the resulting upregulation of MDM2, which requires increased dosing of the inhibitor. The required dosing results in on-target toxicity to normal cells that severely limits the clinical efficacy of MDM2 inhibitors. Further, primary and acquired resistance have limited the potential benefits. There are two routes to resistance. The more dominant mechanism is acquisition of a p53 inactivating mutation. The alternate mechanism is excessive MDM2 copy number, which results in resistance and is likely enhanced by the feedback loop. Likewise, as resistance becomes an issue, higher doses of the MDM2 inhibitors are required to outcompete the accumulation of MDM2.

[0005] Further, as targeted protein degradation becomes an emerging modality with the potential to target disease-causing proteins that are challenging to conventional small molecules, proteolysis-targeting chimera (PROTAC) technology generally harnesses the ubiquitin-proteasome system by recruiting E3 ligases, such as cereblon (CRBN) or von Hippel-Landau (VHL), as another approach that causes cell death and / or tumor growth inhibition. For example, dBET6 is a commercially available BRD4 degrader with the E3 ligase, CRBN. See US Patent No. 9694084. However, relying on E3 ligase production from CRBN or VHL will cause degradation equally across tumor and normal cells resulting in unintended on-target toxicity in the normal cells.

[0006] Therefore, there is a need to discover new oncolytic compounds that activate p53 levels without disrupting the p53 / MDM2 feedback loop. Additionally, the need also exists to selectively target cancer cells while causing minimal toxicity to normal cells.

[0007] The present invention provides new protein degraders that activate the production of p53 by targeting MDM2 -amplified cancers. Further, the compounds of the present invention benefit from the p53 / MDM2 feedback loop rather than disrupting it. MDM2- based PROTACs display enhanced therapeutic index compared to MDM2 inhibitors alone. Additionally, by leveraging the oncogenic activity of MDM2, the compounds of the present invention selectively direct degradation to protein targets critical to MDM2 -amplifiedtumors only; there is minimal effect on healthy cells. Moreover, pretreatment with MDM2 inhibitors enhances sensitivity of the MDM2 -amplified cell lines to the MDM2 -based degraders. The compounds of the present invention also target bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extraterminal (BET) protein family. The BRD4 degradation occurs at lower doses.BRIEF DESCRIPTION OF DRAWINGS

[0008] FIG. 1 shows that neither the MDM2 binders MI- 1061, idasanutlin, and brigimadlin nor BRD4 binders JQ-1 and JQ35 display a significant therapeutic index in LPS853 and T449 cell lines for MDM2 amplified liposarcoma and U2OS and A375 for MDM2 wild type tumor cell lines.

[0009] FIG. 2 shows that the therapeutic index of the MDM2 -based degraders, QL- MDM2-17, QL-MDM2-18 and QL-MDM2-19, can be enhanced using a less potent MDM2 inhibitor component, MI 1061, as compared to the idasanutlin based MDM2 -degraders, A1874, QL-MDM2-01, QL-MDM2-05 and QL-MDM2-06 regardless of which BRD4 binder is used. Also, incorporation of the more potent MDM2 binder brigimadlin increases the overall potency while maintaining a strong therapeutic index of the MDM2 -degrader, QL-MDM2-48.

[0010] FIG 3. shows that commercially available BRD4 degraders dBET6 and ARV771 as well as control MDM2 -based degraders included in the instant, (R)-QL-MDM2-01, (R)- QL-MDM2-18, QL-MDM2-45, QL-MDM2-47 and QL-MDM2-49 do not show selective activity toward LPS853 and T449 cell lines for MDM2 amplified liposarcoma and U2OS and A375 for MDM2 wild type tumor cell lines.

[0011] FIG 4. shows the therapeutic index of the MDM2 -based degrader, QL-MDM2-18, in direct comparison to control MDM2 -based degraders, (R)-QL-MDM2-18 and QL- MDM2-45, and confirms the degradation-based mechanism of action in the LPS853 cell line.

[0012] FIG 5. shows that QL-MDM2-18 induced BRD4 degradation requires ternary complex formation as seen with the progressive overload of JQ35 and MI- 1061 and proteosome activity as seen with addition of proteosome inhibitor bortezomib.

[0013] FIG. 6 shows that the MDM2-based degrader, QL-MDM2-01, has greater growth inhibitory activity in MDM2 amplified liposarcoma cells, LPS853 and T449, than in nontumor cell lines, melanocytes and WI-38.

[0014] FIG. 7 shows that BRD4 degradation with MDM2-based degraders, QL-MDM2-01 , QL-MDM2-17, QL-MDM2-18, QL-MDM2-19, and QL-MDM2-48 occurs at lower doses in MDM2 amplified LPS cells, LPS853, as compared to MDM2 wild-type models, A375 and normal fibroblast, preceding p53 activation.

[0015] FIG. 8 represent the quantification of the western blot data of FIG. 7. FIG. 10 shows that p53 knockout cells are resistant to idasanutlin but remain sensitive to the MDM2 -based degrader, QL-MDM2-01, which has the MDM2 inhibitor component, idasanutlin. Similarly, p53 knockout cells are resistant to MI- 1061 but remain sensitive to the MDM2-based degrader, QL-MDM2-18, which has the MDM2 inhibitor component, Mi- 1061. These data are consistent with clinical trials that show p53 inactivating mutations are a dominant mechanism of MDM2 inhibitor resistance and show MDM2- degraders maintain activity in the presence of p53 mutations.

[0016] FIG. 9 shows that pretreatment with MDM2 inhibitor, HDM201, enhances the therapeutic index of the MDM2 -based degrader, QL-MDM2-1, in the amplified LPS cell line but not in the MDM2 wild type cell line, A375.

[0017] FIG. 10 shows that p53 knockout cells are resistant to idasanutlin but remain sensitive to the MDM2 -based degrader, QL-MDM2-01, which has the MDM2 inhibitor component, idasanutlin. Similarly, p53 knockout cells are resistant to MI- 1061 but remain sensitive to the MDM2-based degrader, QL-MDM2-18, which has the MDM2 inhibitor component, Mi-1061. These data are consistent with clinical trials that show p53 inactivating mutations are a dominant mechanism of MDM2 inhibitor resistance and show MDM2-degraders maintain activity in the presence of p53 mutations.

[0018] FIG. 11 shows that MI- 1061 is approximately 1 log less potent than idasanutlin.

[0019] FIG. 12 shows mean plasma concentration-time profiles of QL-MDM2-18 and QL-MDM2-48 in male CD1 mice over 24 hours.

[0020] FIG. 13 shows a reduction in BRD4-positive tumor cells in LPS3 liposarcoma patient-derived xenografts following treatment with 15 mg / kg QL-MDM2-18.DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides a compound of the formula:or a pharmaceutical acceptable salt thereof.

[0022] The Binder (B) of the present invention is a BRD4 binder. Further, the BRD4 binder may be derived from JQ-1 (B-a) or JQ-35 (B-b),JQ-1 (CAS No. 1268524-70-4) and JQ-35 (CAS No. 1349719-98-7) are BET inhibitors. They are commercially available or can be made according to the procedures set forth in WO 2011 / 43669, which is incorporated herein by reference. Preferably, the BRD4 Binder is derived from JQ-35.

[0023] The linker (L) may be hydrocarbon of varying length (L-a) or a polyethylene glycol (PEG) chain of varying length (L-b or L-c). The linker is C2-C10 alkyl wherein x is 2 to 10 or a PEG chain having 1 to 5 PEG units, wherein y or z is 1 to 5:Preferably, x is 3 to 7, y is 1 to 4, and z is 3. Most preferably, x is 3 to 6 and z is 3.

[0024] The Inhibitor (I) is derived from an MDM2 inhibitor. Further, the inhibitor may be derived from idasanutlin (l-a), MI- 1061 (l-b), or brigimadlin (l-c),Idasanutlin (also known as RG-7388 or RO-5503781), (CAS No. 1229705-06-9) is a known potent and selective MDM2 inhibitor. It is commercially available or can be made according to the procedures set forth in WO 2010 / 031713, which is incorporated herein by reference. MI-1061 (CAS No. 1410737-34-6) is a known potent and selective MDM2 inhibitor. It is commercially available or can be made according to the procedures set forth in WO 2012 / 155066, which is incorporated herein by reference. Preferably, the Inhibitor is derived from MI- 106. Brigimadlin, (also known as BI-907828), (CAS No. 2095116-40-6) is a known potent and selective MDM2 inhibitor. It is commercially available or can be made according to procedures set forth in WO 2017060431, which is incorporated herein by reference.

[0025] A compound of the present invention is not the following compound,

[0026] A preferred embodiment of the invention relates to compounds wherein B isx is 6; y is 1 to 4; z is 3; andpharmaceutically acceptable salt thereof.

[0027] Another preferred embodiment of the invention relates to compounds wherein B isy is 3; z is 3; and

[0028] Pretreatment with an MDM2 inhibitor may enhance the therapeutic index of an MDM2-based degrader of the present invention. MDM2 inhibitors are well known in the art and include siremadlin (also known as HDM201), idasanutlin (also known as RG-7388 and RO-5503781), milademetan (also known as DS-3032 and DS-3032b), brigimadlin (also known as BI 907828), navtemadlin (aslo known as KRT-232 and AMG232), ALRN- 6924, STEMVAC (also known as EP-101), UBX-0101, ASTX-295, CGM-097, KT-253, RG-7775 (also known as RO 6839921), alrizomadlin (also known as APG-115), BI-MDM2 (also known as GBM-108 and GBM-14), APG-265, BI-0252, BI-0282, JW-1-283, MA242, MD-222, MD-224, ADO-21, MI-1061, MX69-114b, OM-301, SP-141, UNP-6457, WB- 214, NU-8165, MK-8242 (also known as SCH-900242), Nutlin-3a (also known as RO- 4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI-773), DS-5272, ATSP7041, Japonicone A, SAH-p53-8, SAR-299155, SAR-305801, serdemetan (also known as JNJ-1 and JNJ-26854165), SJ-2011, or VG-0604a.

[0029] The present invention provides a method of activating the production of p53 by targeting MDM2-amplified cancers comprising administering an effective amount of a compound of the invention or a pharmaceutical acceptable salt thereof. The present invention also provides a method of promoting degradation of MDM2-amplified cancers comprising administering an effective amount of a compound of the invention or a pharmaceutical acceptable salt thereof.

[0030] The present invention provides a method of treating cancer comprising administering an effective amount of a compound of the invention or a pharmaceutical acceptable salt thereof. In a further embodiment, the patient is also administered an effective amount of an MDM2 inhibitor, or a pharmaceutically acceptable salt thereof. In another embodiment, the effective amount of the MDM2 inhibitor may be administered in combination or separately, and if separately, simultaneously or sequentially. Preferably, the MDM2 inhibitor is administered separately and before the administration of the MDM2-degrader of the present invention. In another preferred embodiment, the MDM2 inhibitor is siremadlin (also known as HDM201), idasanutlin (also known as RG-7388 and RO-5503781), milademetan (also known as DS-3032 and DS-3032b), brigimadlin (also known as BI 907828), navtemadlin (aslo known as KRT-232 and AMG232), ALRN-6924, STEMVAC (also known as EP-101), UBX-0101, ASTX-295, CGM-097, KT-253, RG- 7775 (also known as RO 6839921), alrizomadlin (also known as APG-115), BI-MDM2(also known as GBM-108 and GBM-14), APG-265, BI-0252, BI-0282, JW-1-283, MA242, MD-222, MD-224, ADO-21, MI-1061, MX69-114b, OM-301, SP-141, UNP-6457, WB- 214, NU-8165, MK-8242 (also known as SCH-900242), Nutlin-3a (also known as RO- 4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI-773), DS-5272, ATSP7041, Japonicone A, SAH-p53-8, SAR-299155, SAR-305801, serdemetan (also known as JNJ-1 and JNJ-26854165), SJ-2011, or VG-0604a.

[0031] The present invention additionally provides a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In a particular embodiment, the composition further comprises one or more other therapeutic agents. In a further embodiment, the therapeutic agent is an MDM2 inhibitor, or a pharmaceutically acceptable salt thereof. In another embodiment, the MDM2 inhibitor may be administered in combination or separately, and if separately, simultaneously or sequentially. Preferably, the MDM2 inhibitor is administered separately and before the administration of the MDM2-degrader of the present invention. In another preferred embodiment the MDM2- inhibitor is siremadlin (also known as HDM201), idasanutlin (also known as RG-7388 and RO-5503781), milademetan (also known as DS-3032 and DS-3032b), brigimadlin (also known as BI 907828), navtemadlin (aslo known as KRT-232 and AMG232), ALRN-6924, STEMVAC (also known as EP-101), UBX-0101, ASTX-295, CGM-097, KT-253, RG- 7775 (also known as RO 6839921), alrizomadlin (also known as APG-115), BI-MDM2 (also known as GBM-108 and GBM-14), APG-265, BI-0252, BI-0282, JW-1-283, MA242, MD-222, MD-224, ADO-21, MI-1061, MX69-114b, OM-301, SP-141, UNP-6457, WB- 214, NU-8165, MK-8242 (also known as SCH-900242), Nutlin-3a (also known as RO- 4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI-773), DS-5272, ATSP7041, Japonicone A, SAH-p53-8, SAR-299155, SAR-305801, serdemetan (also known as JNJ-1 and JNJ-26854165), SJ-2011, or VG-0604a.

[0032] Further, the present invention also provides compounds of the invention or pharmaceutically acceptable salts thereof for use in therapy, in particular for the treatment of cancer. In a further embodiment, the patient is also administered an MDM2 inhibitor, or a pharmaceutically acceptable salt thereof. In another embodiment, the MDM2 inhibitor may be administered in combination or separately, and if separately, simultaneously or sequentially. Preferably, the MDM2 inhibitor is administered separately and before theadministration of the MDM2-degrader of the present invention, n another preferred embodiment the MDM2-inhibitor is siremadlin (also known as HDM201), idasanutlin (also known as RG-7388 and RO-5503781), milademetan (also known as DS-3032 and DS-3032b), brigimadlin (also known as BI 907828), navtemadlin (aslo known as KRT-232 and AMG232), ALRN-6924, STEMVAC (also known as EP-101), UBX-0101, ASTX-295, CGM-097, KT-253, RG-7775 (also known as RO 6839921), alrizomadlin (also known as APG-115), BI-MDM2 (also known as GBM-108 and GBM-14), APG-265, BI-0252, BI- 0282, JW-1-283, MA242, MD-222, MD-224, ADO-21, MI-1061, MX69-114b, OM-301, SP-141, UNP-6457, WB-214, NU-8165, MK-8242 (also known as SCH-900242), Nutlin- 3a (also known as RO-4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI- 773), DS-5272, ATSP7041, Japonicone A, SAH-p53-8, SAR-299155, SAR-305801, serdemetan (also known as JNJ-1 and JNJ-26854165), SJ-2011, or VG-0604a. In another embodiment, the present invention provides the use of activating the production of p53 by targeting MDM2-amplified cancers comprising administering an effective amount of a compound of the invention or a pharmaceutical acceptable salt thereof.

[0033] Furthermore, the present invention provides compounds of the invention or pharmaceutically acceptable salts thereof for use in the treatment of cancer. In a further embodiment, the patient is also administered an MDM2 inhibitor, or a pharmaceutically acceptable salt thereof. In another embodiment, the MDM2 inhibitor may be administered in combination or separately, and if separately, simultaneously or sequentially. Preferably, the MDM2 inhibitor is administered separately and before the administration of the MDM2-degrader of the present invention. In another preferred embodiment the MDM2 inhibitor is siremadlin (also known as HDM201), idasanutlin (also known as RG-7388 and RO-5503781), milademetan (also known as DS-3032 and DS-3032b), brigimadlin (also known as BI 907828), navtemadlin (aslo known as KRT-232 and AMG232), ALRN-6924, STEMVAC (also known as EP-101), UBX-0101, ASTX-295, CGM-097, KT-253, RG- 7775 (also known as RO 6839921), alrizomadlin (also known as APG-115), BI-MDM2 (also known as GBM-108 and GBM-14), APG-265, BI-0252, BI-0282, JW-1-283, MA242, MD-222, MD-224, ADO-21, MI-1061, MX69-114b, OM-301, SP-141, UNP-6457, WB- 214, NU-8165, MK-8242 (also known as SCH-900242), Nutlin-3a (also known as RO- 4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI-773), DS-5272,ATSP7041, Japonicone A, SAH-p53-8, SAR-299155, SAR-305801, serdemetan (also known as JNJ-1 and JNJ-26854165), SJ-2011, or VG-0604a.

[0034] Even further, the present invention provides the use of a compound of the invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of cancer. In another embodiment, the present invention provides the use of activating the production of p53 by targeting MDM2-amplified cancers comprising administering an effective amount of a compound of the invention or a pharmaceutical acceptable salt thereof.

[0035] The present invention provides preferred embodiments of the methods and uses as described herein, in which cancer is any highly MDM2 -amplified cancer. Preferred cancers are neoplasms, lymphoma, sarcoma, solid tumors, and myeloid tumors. More preferred cancers are neoplasms, including hematologic neoplasms and salivary gland neoplasms, sarcoma, including liposarcoma and advanced liposarcoma, leukemia, including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, mixed phenotype acute leukemia, and t-cell prolymphocytic leukemia, melanoma, such as uveal melanoma, multiple myeloma, polycythemia vera, primary myelofibosis, endometrial carcinoma, merkel cell carcinoma, adenoid cystic carcinoma, transitional cell carcimoma, primary myelofibrosis, brain cancers, such as neurofibrosarcoma, neuroblastoma, and glioblastoma, eye cancer, such as retinobalastoma, gastrointestinal cancer, such as stomach cancer and colorectal cancer, pancreatic cancer, breast cancer, bladder cancer, prostate cancer, and lung cancer, such as extensive stage small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer, non-small cell lung cancer, non-sqamous non-small cell lung cancer . Especially preferred cancers are neoplasms, including hematologic neoplasms and salivary gland neoplasms, sarcoma, including liposarcoma and advanced liposarcoma, leukemia, including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, mixed phenotype acute leukemia, and t-cell prolymphocytic leukemia, melanoma, such as uveal melanoma, multiple myeloma, polycythemia vera, primary myelofibosis, endometrial carcinoma, merkel cell carcinoma, adenoid cystic carcinoma, transitional cell carcimoma, primary myelofibrosis, brain cancers, such as neurofibrosarcoma, neuroblastoma, and glioblastoma, eye cancer, such as retinobalastoma, gastrointestinal cancer, such as stomach cancer and colorectal cancer,breast cancer, prostate cancer, and lung cancer, such as extensive stage small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer, non-small cell lung cancer, non-sqamous non-small cell lung cancer .

[0036] The present invention provides a method of treating radiation injuries comprising administering an effective amount of a compound of the invention or a pharmaceutical acceptable salt thereof. In a further embodiment, the effective amount of a compound of the present invention may be administered in combination with radiation treatment or separately, and if separately, simultaneously or sequentially.

[0037] The present invention also provides compounds of the invention or pharmaceutically acceptable salts thereof for use in the treatment of radiation injuries. In a further embodiment, the effective amount of a compound of the present invention may be administered in combination with radiation treatment or separately, and if separately, simultaneously or sequentially.

[0038] The present invention provides the use of a compound of the invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of radiation injuries. In a further embodiment, the effective amount of a compound of the present invention may be administered in combination with radiation treatment or separately, and if separately, simultaneously or sequentially.

[0039] The present invention provides a method of treating degenerative diseases, including osteoarthritis, macular dystrophy, and macular degeneration comprising administering an effective amount of a compound of the invention or a pharmaceutical acceptable salt thereof. The present invention also provides compounds of the invention or pharmaceutically acceptable salts thereof for use in the treatment of degenerative diseases, including osteoarthritis, macular dystrophy, and macular degeneration. The present invention provides the use of a compound of the invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of degenerative diseases, including osteoarthritis, macular dystrophy, and macular degeneration.

[0040] Although all of the exemplified compounds of the invention are useful

[0041] Compounds of the present invention may be administered as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd 30 Revised Edition (Wiley-VCR, 2011); S. M.Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1,Jan. 1977.

[0042] Compounds of the present invention are preferably formulated as pharmaceutical compositions administered by a variety of routes. Such pharmaceutical compositions and processes for preparing the same are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy(I) (A. Gennaro, et al., eds., 21st ed., Mack Publishing Co., 2005).

[0043] The compounds of the present invention are generally effective over a wide dosage range. It will be understood that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound or compounds administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.

[0044] As used herein, the term “therapeutic index” refers to the range of doses that induce at least 50% growth inhibition across MDM2 amplified lines but not MDM2 wild type lines.

[0045] As used herein, the term “treating” (or “treat” or “treatment”) refers to restraining, slowing, stopping, or reversing the progression or severity of an existing symptom, condition, or disorder.

[0046] As used herein, the phrase "effective amount" means an amount of a compound of the invention that is sufficient to treat in one or more doses a condition or detrimental effect thereof herein described or an amount of a compound of the invention that is sufficient to inhibit MDM2 or BRD4 to achieve the objectives of the invention.

[0047] As used herein, the phrase “administering” (or “administer” or “administration”) means the act of giving a compound of the invention or the direct application of a compound of the invention by ingestion, inhalation, injection, or any other means, to the body of a patient. Two or more compounds of the invention may be administered in combination or separately, and if separately, simultaneously or sequentially over a period of time as determined by a qualified care giver.

[0048] As used herein, "patient" refers to a mammal, preferably human.

[0049] The compounds of the present invention, or salts thereof, may be prepared by a variety of procedures known in the art, some of which are illustrated in the Preparationsand Examples below. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different preparations or examples, to prepare compounds or salts of the present invention. The products of each step in the Preparations and Examples below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the Preparations and Examples below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art.

[0050] Additionally, certain intermediates described in the following Preparations and Examples may contain one or more nitrogen protecting groups. The variable protecting group may be the same or different in each occurrence depending on the particular reaction conditions and the particular transformations to be performed. The protection and deprotection conditions are well known to the skilled artisan and are described in the literature (See for example “Green ’s Protective Groups in Organic Synthesis” , Fourth Edition, by Peter G. M. Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).

[0051] The skilled artisan will appreciate that compounds of the present invention are comprised of a core that contains at least one chiral center. Certain stereochemical centers may have been left unspecified and certain substituents may have been eliminated in the following Preparations and Examples for the sake of clarity and are not intended to limit the teaching of the schemes in any way. Single enantiomers or diastereomers may be prepared beginning with chiral reagents or by stereoselective or stereospecific synthetic techniques. Alternatively, the single enantiomers or racemates may be isolated from mixtures by standard chiral chromatographic or crystallization techniques at any convenient point in the synthesis of compounds of the invention by methods such as selective crystallization techniques or chiral chromatography (See for example, J. Jacques, et al., “Enantiomers, Racemates, and Resolutions”, John Wiley and Sons, Inc., 1981, and E. L. Eliel and S. H. Wilen, “Stereochemistry of Organic Compounds”, Wileylnterscience, 1994).

[0052] Some intermediates or compounds of the present invention may have one or more chiral centers. The present invention contemplates all individual enantiomers or diastereomers, as well as mixtures of the enantiomers and diastereomers of said compounds including racemates. It is preferred that compounds of the present invention containing atleast one chiral center exist as a single enantiomer or diastereomer. The single enantiomer or diastereomer may be prepared beginning with chiral reagents or by stereoselective or stereospecific synthetic techniques. Alternatively, the single enantiomer or diastereomer may be isolated from mixtures by standard chiral chromatographic or crystallization techniques. The skilled artisan will appreciate that in some circumstances the elution order of enantiomers or diastereomers may be different due to different chromatographic columns and mobile phases. The abbreviations used herein are defined according to Aldrichimica Acta, vol. 17, No. 1, 1984. Other abbreviations are defined as follows: “AcOH” refers to acetic acid; “BCA” refers to bicinchoninic acid; “Boc” refers to tertbutyloxycarbonyl; “CRISPR” refers to Clustered Regularly Interspaced Short Palindromic Repeats; “DCM” refers to dichloromethane; “DIPEA” refers to diisopropylethylamine;“DMEM” refers to Dulbecco’s Modified Eagle Medium; “DMF” refers to dimethylformamide; “DMSO” refers to dimethylsulfoxide; “EMEM” refers to Eagle’s Minimum Essential Medium; “ESIMS” refers to Electrospray Ionization Mass Spectrometry; “FBS” refers to fetal bovine serum; “HATU” refers to 1 - [bis(dimethylamino)methylene]-l / f-l,2,3-triazolo[4,5-6]pyridinium 3-oxid hexafluorophosphate; “HPLC” refers to High Performance Liquid Chromatography; “MeOH” refers to methanol; “NMR” refers to nuclear magnetic resonance spectroscopy; “PBS” refers to phosphate-buffered saline; “Pic-BHs” refers to a-picoline borane; “RIP A” refers to radioimmunoprecipitation assay; “TEA” refers to triethylamine; “Tris” refers to 2- amino-2-(hydroxymethyl)propane- 1 ,3 -diol.Schemes

[0053] In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are either commercially available or may be prepared by methods well known to one of ordinary skill in the art, some of which are presented in the preparations below. Without limiting the scope of the invention, the following schemes, preparations, and examples are provided to further illustrate the invention.

[0054] Scheme 1.

[0055] Scheme 1 depicts the preparation of compounds of the present disclosure beginning with (S)-JQ1 acid which can undergo amide coupling in step 1 and step 2 with [i] and [iii] respectively using HATU and Hiinig’s base or TEA in an appropriate solvent such as DMF to give [ii] and [v]. The variable X may represent an alkyl or PEG chain as represented by the linkers defined herein. One skilled in the art would recognize a variety of reagents and conditions could be utilized for the activation of carboxylic acids and coupling of amines. Forexample, DCC / DMAP, EDC / HOBt, CDI / TEA can be used for amidation. Intermediate [ii] can further undergo deprotection under acidic conditions and subsequent reductive amination with [iv] using 2-picoline-borane in an appropriate polar solvent to give [vi]. The variable X may represent an alkyl or PEG chain as represented by the linkers defined herein. Once skilled in the art would recognize a variety of reducing agents could be utilized for amination. For example, STAB, sodium cyanoborohydride, and hydride reducing agents. Intermediates [v] and [vi] can both undergo deprotection under acidic conditions and subsequent amide coupling with the carboxylic acid moiety of the MDM2 inhibitors defined herein to give products [vii] and [viii].PREPARATIONS AND EXAMPLES

[0056] The following Preparations and Examples further illustrate the invention and represent typical synthesis of the compound of the invention. The reagents and starting materials are readily available or may be readily synthesized by one of ordinary skill in the art. It should be understood that the Preparations and Examples are set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

[0057] The (7?) or (S) configuration of the compound of the invention may be determined by standard techniques such as X-ray analysis and correlation with chiral -HPLC retention time. The naming of the following Preparations and Examples is generally performed using the IUPAC naming feature in CHEMDRAW® version 22.2.0.

[0058] Unless otherwise specified, percentages in the below Preparations and Examples refer to percent yield.Preparation 1 tert-butyl (5)-4-(3 -(2-(4-(4-chlorophenyl)-2,3 ,9-trimethyl-677-thieno[3 ,2-f [ 1 ,2,4]triazolo[4,3-a] [ 1 ,4] diazepin-6-yl)acetamido)propyl)piperazine-l -carboxylate

[0059] In a 25 mL vial fitted with a stir bar, (5)-JQl acid (434 mg), tert-butyl 4-(3- aminopropyl)piperazine-l -carboxylate (265 mg), and HATU (454 mg) are dissolved in DMF (3 mL), followed by addition of DIPEA (0.78 mL). Then the resulting mixture is stirred at 25 °C for 2 h. After the reaction is completed, the mixture is directly purified by silica gel chromatography (eluent: DCM / MeOH) to obtain the title compound (609 mg) as an oil. ESI-MS: calculated C3iH4i35ClN7O3S [M+l]+: 626.27, found: 626.63.Preparation 2 tert-butyl (S)-(6-(4-(3-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6 / / -thieno[3,2- f\ [ 1 ,2,4]triazolo[4,3-a] [1 ,4]diazepin-6-yl)acetamido)propyl)piperazin-l -yl)hexyl)carbamate

[0060] (1) In a 25 mL vial fitted with a stir bar, [1] (94 mg) is dissolved in DCM (1 mL) followed by dropwise addition of HC1 (4 M in 1,4-dioxane, 1 mL). The reaction is stirred at 25 °C overnight, and the solvent is removed. The residue is subjected to the next step reaction without further purification. (2) In a 4 mL vial fitted with a stir bar, the residuefrom the above step and tert-butyl (6-oxohexyl)carbamate [2] (66 mg) are dissolved in a mixed solvent (MeOH:AcOH = 10:1, 0.45 mL), followed by Pic-BH3(23 mg). The resulting mixture is stirred at 25 °C overnight and the solvent is removed. The residue is directly purified by silica gel chromatography (eluent: DCM / MeOH, with 1 vol% NH3, 7 M in MeOH) to obtain the title compound (76 mg) as an oil. ESI-MS: calculated C37H5435C1N8O3S [M+l]+: 725.37, found: 725.57.Example 1QL-MDM2-01(21?,3S,47?,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-7V-(4-((6-(4-(3-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-677-thieno[3,2- / ][l,2,4]triazolo[4,3- a] [ 1 ,4]diazepin-6-yl)acetamido)propyl)piperazin- 1 -yl)hexyl)carbamoyl)-2- methoxyphenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamide

[0061] (1) In a 4 mL vial fited with a stir bar, [3] (16 mg) is dissolved in DCM (0.1 ml) followed by dropwise addition of HC1 (4 M in 1,4-dioxane, 0.1 mL). The reaction is stirred at 25 °C overnight and the solvent is removed. The residue is subjected to the next step reaction without further purification. (2) In a 4 mL vial fited with a stir bar, the residue from the above step, idasanutlin [4] (14 mg), and HATU (14 mg) are dissolved in DMF (0.1 mL), followed by addition of DIPEA (20 pL). The resulting mixture is stirred at 25 °C for 2 h. After the reaction is completed, the solvent is removed. The residue is directlypurified by silica gel chromatography (eluent: DCM / MeOH, with 1 vol% NH3 7 M in MeOH) to obtain the title compound (20 mg) as a white powder. ESI-MS: calculated C63H7335C13F2N11O4S [M+l]+: 1222.46, found: 1222.66.1H NMR (500 MHz, DMSO-<Z6) 8 10.40 (s, 1 H), 8.44-8.36 (m, 1 H), 8.31 (d, J= 8.5 Hz, 1 H), 8.19 (t, J= 5.3 Hz, 1 H), 7.73 (t, J= 7.0 Hz, 1 H), 7.61-7.29 (m, 10 H), 6.78 (br, 2 H), 4.63-4.54 (m, 2 H), 4.50 (t, J= 7.0 Hz, 1 H), 4.42-4.31 (m, 1 H), 4.00-3.86 (m, 4 H), 3.27-3.02 (m, 9 H), 2.59 (s, 3 H), 2.40 (s, 3 H), 2.37-2.15 (m, 6 H), 1.62 (s, 3 H), 1.71-1.16 (m, 14 H), 0.97 (s, 9 H).

[0062] Prepare Examples 2-6 below by essentially following the procedures described in Preparation 1, Preparation 2, and Example 1 using the appropriate enantiomer of JQ-1 acid of Preparation 1 and the appropriate aldehyde [2] of Preparation 2.Example 2(7?)-QL-MDM2-01(27?,35,47?,55)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-7V-(4-((6-(4-(3-(2-((l?)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- / ][l,2,4]triazolo[4,3- a] [1 ,4]diazepin-6-yl)acetamido)propyl)piperazin-l -yl)hexyl)carbamoyl)-2- methoxyphenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamideQL-MDM2-05 (27?,3*S'>47?,55)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-7V-(4-((2-(2-(4-(3- (2-((5)-4-(4-chlorophenyl)-2,3,9-trimethyl-6 / 7-thieno[3,2- / ][l,2,4]triazolo[4,3- a] [ 1 ,4]diazepin-6-yl)acetamido)propyl)piperazin- 1 -yl)ethoxy)ethyl)carbamoyl)-2- methoxyphenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamideExample 4QL-MDM2-06(27?,3S,47?,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-7V-(4-((2-(2-(2-(4-(3-(2-((5)-4-(4-chlorophenyl)-2,3,9-trimethyl-6 / 7-thieno[3,2- / |[l,2,4]triazolo[4,3- a] [ 1 ,4]diazepin-6-yl)acetamido)propyl)piperazin- 1 -yl)ethoxy)ethoxy)ethyl)carbamoyl)-2- methoxyphenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamideExample 5 QL-MDM2-11 (27?,35',47?,51S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-Ar-(4-((2-(2-(2-(2- (4-(3-(2-((5)-4-(4-chlorophenyl)-2,3,9-trimethyl-677-thieno[3,2- / |[l,2,4]triazolo[4,3- a] [ 1 ,4]diazepin-6-yl)acetamido)propyl)piperazin- 1 - yl)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-2-methoxyphenyl)-4-cyano-5- neopentylpyrrolidine-2-carboxamideExample 6 QL-MDM2-12 (27?, 3S,^R, 5S)-3 -(3 -chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-jV-(4-(( 14-(4-(3-(2- ((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-677-thieno[3,2- / ][l,2,4]triazolo[4,3- a] [ 1 ,4]diazepin-6-yl)acetamido)propyl)piperazin- 1 -y 1) -3 ,6,9, 12- tetraoxatetradecyl)carbamoyl)-2-methoxyphenyl)-4-cyano-5-neopentylpyrrolidine-2- carboxamidePreparation 3 tert-butyl (5)-(l-(4-(4-chlorophenyl)-2,3,9-trimethyl-67 / -thieno[3,2- / ][l,2,4]triazolo[4,3- a] [ 1 ,4]diazepin-6-yl)-2-oxo-6,9, 12-trioxa-3 -azatetradecan- 14-yl)carbamate

[0063] In a 4 mL vial fitted with a stir bar, (S)-JQ1 acid (10 mg), [5] (7.3 mg), and HATU (10.5 mg) are dissolved in DMF (0.1 mL), followed by addition of DIPEA (22 pL). The resulting mixture is stirred at 25 °C for 2 h. After the reaction is completed, the mixture is directly purified by silica gel chromatography (eluent: DCM / MeOH) to obtain the title compound (17 mg) as a light-yellow powder. ESI-MS: calculated CsiHw^ClNeOeS [M+l]+: 675.27, found: 675.46.Example 7 QL-MDM2-17 (3'7?,4'S',5'7?)-6"-chloro-4'-(3-chloro-2-fluorophenyl)-A-(4-((l-((5)-4-(4-chlorophenyl)- 2,3,9-trimethyl-6 / f-thieno[3,2- / | [1 ,2,4]triazolo[4,3-a] [1 ,4]diazepin-6-yl)-2-oxo-6,9, 12- trioxa-3-azatetradecan-14-yl)carbamoyl)phenyl)-2"-oxodispiro[cyclohexane-l,2'- pyrrolidine-3 ',3 "-indoline] -5'-carboxamide

[0064] (1) In a 4 mL vial fited with a stir bar, [6] (9 mg) is dissolved in DCM (0.13 mL) followed by dropwise addition of HC1 (4 M in 1,4-dioxane, 0.13 mL). The reaction is stirred at 25 °C overnight, and the solvent is removed. The residue is subjected to the next step reaction without further purification. (2) In a 4 mL vial fitted with a stir bar, the residue from the above step, MI- 1061 [7] (8.5 mg), and HATU (6 mg) are dissolved in DMF (0.1 mL), followed by addition of DIPEA (13 pL). The resulting mixture is stirred at 25 °C for 2 h. After the reaction is completed, the solvent is removed. The residue is directly purified by silica gel chromatography (eluent: DCM / MeOH, with 1 vol% NH3 7 M in MeOH) to obtain the title compound (11 mg) as a white powder. ESI-MS: calculated C57H6O35C13FN907S [M+l]+: 1138.34, found: 1138.52.Example 8 QL-MDM2-18 (3 ?,4'S,5' / ?)-6"-chloro-4'-(3-chloro-2-fluorophenyl)-2V-(4-((6-(4-(3-(2-((5)-4-(4- chlorophenyl)-2,3,9-trimethyl-677-thieno[3,2- / | [1 ,2,4]triazolo[4,3-a] [1 ,4]diazepin-6- yl)acetamido)propyl)piperazin-l-yl)hexyl)carbamoyl)phenyl)-2"-oxodispiro[cyclohexane- l,2'-pyrrolidine-3',3"-indoline]-5'-carboxamide

[0065] (1) In a 4 mL vial fitted with a stir bar, [3] (52 mg) is dissolved in DCM (0.1 ml) followed by dropwise addition of HC1 (4 M in 1,4-dioxane, 0.36 mL). The reaction is stirred at 25 °C overnight, and the solvent is removed. The residue is subjected to the next step reaction without further purification. (2) In a 4 mL vial fitted with a stir bar, the residue from the above step, MI-1061 [7] (42 mg), and HATU (30 mg) are dissolved in DMF (0.22 mL), followed by addition of DIPEA (63 pL). Then the resulting mixture is stirred at 25 °C for 2 h. After the reaction is completed, the mixture is directly purified by silica gel chromatography (eluent: DCM / MeOH, with 1 vol% NH3 7 M in MeOH) to obtain the title compound (62 mg) as a white powder. ESI-MS: calculated C62H7035C13FNIIO4S [M+l]+: 1188.44, found: 1188.56.NMR (500 MHz, DMSO-rZ6) 8 10.59 (s, 1 H), 10.22 (s, 1 H), 8.33 (t, J= 5.5 Hz, 1 H), 8.19 (t, J= 5.5 Hz, 1 H), 7.81 (d, J= 8.5 Hz, 2 H), 7.68 (d, J= 8.5 Hz, 2 H), 7.63 (t, J= 6.9 Hz, 1 H), 7.51-7.39 (m, 5 H), 7.35 (t, J= 7.5 Hz, 1 H), 7.14 (t, J= 8.1 Hz, 1 H), 7.04 (dd, Ji = 7.0 Hz, J2= 2.0 Hz, 1 H), 6.69 (d, J= 1.8 Hz, 1 H), 4.76 (d, J= 9.5 Hz, 1 H), 4.72-4.64 (m, 1 H), 4.50 (t, J= 7.0 Hz, 1 H), 3.69 (br, 1 H), 3.26- 3.03 (m, 6 H), 2.59 (s, 3 H), 2.40 (s, 3 H), 2.45-2.15 (m, 11 H), 2.07-2.00 (m, 1 H), 1.62 (s, 3 H), 1.70-0.78 (m, 20 H).

[0066] Prepare Example 9 below by essentially following the procedures described in Example 8 and using the appropriate enantiomer of [3]. The appropriate enantiomer of [3]may be prepared by using the appropriate enantiomer of JQ-1 acid and following the procedure described in Preparation 1 and Preparation 2.Example 9 (7?)-QL-MDM2-18 (3'J?,4'S,5'J?)-6"-chloro-4'-(3-chloro-2-fluorophenyl)-7V’-(4-((6-(4-(3-(2-((J?)-4-(4- chlorophenyl)-2,3,9-trimethyl-677-thieno[3,2- / | [1 ,2,4]triazolo[4,3-a] [1 ,4]diazepin-6- yl)acetamido)propyl)piperazin-l-yl)hexyl)carbamoyl)phenyl)-2"-oxodispiro[cyclohexane- l,2'-pyrrolidine-3',3"-indoline]-5'-carboxamideESI-MS: calculated C62H7035C13FNHO4S [M+l]+: 1188.79, found: 1188.56.Preparation 4(Relates to Example 10 below) tert-butyl (S)-(l-(4-(4-chlorophenyl)-2,3,9-trimethyl-6 / f-thieno[3,2- / ][l,2,4]triazolo[4,3- a] [ 1 ,4]diazepin-6-yl)-2-oxo-7, 10, 13 -trioxa-3 -azahexadecan- 16-yl)carbamate(5)-JQl acid [9]

[0067] In a 4 mL vial fited with a stir bar, (S)-JQ1 acid (15 mg), [8] (12 mg), and HATU (14 mg) are dissolved in DMF (0.2 mL), followed by addition of TEA (26 pL). Then the resulting mixture is stirred at 25 °C for 2 h. After the reaction is completed, the mixture is directly purified by silica gel chromatography (eluent: DCM / MeOH) to obtain the title compound (14 mg) as an oil. ESI-MS: calculated C34H4835C1N6C>6S [M+l]+: 703.30, found: 703.42.Example 10QL-MDM2-19(3'7?,4'S',5'7?)-6"-chloro-4'-(3-chloro-2-fluorophenyl)-A-(4-((l-((5)-4-(4-chlorophenyl)- 2,3,9-trimethyl-677-thieno[3,2- / ] [ 1 ,2,4]triazolo[4,3-a] [ 1 ,4]diazepin-6-yl)-2-oxo-7, 10,13- trioxa-3-azahexadecan-16-yl)carbamoyl)phenyl)-2"-oxodispiro[cyclohexane-l,2'- pyrrolidine-3 ',3 "-indoline] -5'-carboxamide

[0068] (1) In a 4 mL vial fited with a stir bar, [9] (14 mg) is dissolved in DCM (0.1 ml) followed by dropwise addition of HC1 (4 M in 1,4-dioxane, 0.1 mL). The reaction is stirred at 25 °C overnight, and the solvent is removed. The residue is subjected to the next step reaction without further purification. (2) In a 4 mL vial fited with a stir bar, the residue from the above step, MI-1061 [7] (12 mg), and HATU (8 mg) are dissolved in DMF (0.1mL), followed by addition of DIPEA (18 uL). Then the resulting mixture is stirred at 25 °C for 2 h. After the reaction is completed, the solvent is removed. The residue is directly purified by silica gel chromatography (eluent: DCM / MeOH, with 1 vol% NH3 7 M in MeOH) to obtain the title compound (17 mg) as a white powder. ESI-MS: calculated C59H6435C13FN9O7S [M+l]+: 1166.37, found: 1166.60.Example 11QL-MDM2-45(3'S,4'R,5'S)-6"-chloro-4'-(3-chloro-2-fluorophenyl)-N-(4-((6-(4-(3-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f] [1 ,2,4]triazolo[4,3-a] [1 ,4]diazepin-6- yl)acetamido)propyl)piperazin- 1 -yl)hexyl)carbamoyl)phenyl)-2"-oxodispiro[cyclohexane-l ,2'- pyrrolidine-3 ',3 "-indoline] -5'-carboxamide

[0069] (1) In a 4 mL vial fited with a stir bar, [3] (14 mg) is dissolved in DCM (0.1 mL) followed by dropwise addition of HC1 (4 M in 1,4-dioxane, 0.1 mL). The reaction is stirred at 25 °C overnight, and the solvent is removed. The residue is subjected to the next step reaction without further purification. (2) To a solution of ENT-MI-1061

[0010] (12 mg, 0.02 mmol) in DMF (5 mL) was added DIPEA (13 mg, 0.104 mmol) and HATU (9 mg, 0.022 mmol) at 0 °C. The mixture was stirred for 0.5 h. Then, the residue from the above step (13 mg, 0.02 mmol) was added at 0 °C. The mixture was stirred at room temperature for 1 h. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (30 mL x 2), dried over NaiSCL andconcentrated. The residue was purified by Prep-HPLC (acetonitrile with 0.1 % of NH3-H2O in water 5% to 95%) to obtain the title compound (6.35 mg, 26.5% yield) as a white solid. LC-MS [M+H]+: 1188.6.1H NMR (400 MHz, DMSO-6) 5 10.59 (s, 1H), 10.22 (s, 1H), 8.36 - 8.31 (m, 1H), 8.29 (s, 1H), 8.21 - 8.16 (m, 1H), 7.81 (d, J= 8.7 Hz, 2H), 7.68 (d, J= 8.8 Hz, 2H), 7.65 - 7.59 (m, 1H), 7.50 - 7.40 (m, 5H), 7.35 (dd, 1H), 7.17 - 7.11 (m, 1H), 7.03 (dd, J = 8.1, 2.0 Hz, 1H), 6.71 - 6.67 (m, 1H), 4.76 (d, J= 9.3 Hz, 1H), 4.70 (d, 1H), 4.50 (dd, J= 7.9, 6.3 Hz, 1H), 3.24 - 3.19 (m, 4H), 3.14 - 3.05 (m, 2H), 2.59 (s, 3H), 2.40 (s, 3H), 2.38 - 2.19 (m, 11H), 2.05 (d, 1H), 1.88 - 1.76 (m, 1H), 1.70 - 1.53 (m, 10H), 1.53 - 1.45 (m, 3H), 1.44 - 1.36 (m, 3H), 1.34 - 1.21 (m, 5H), 1.04 - 0.91 (m, 1H), 0.84 (dd, J= 12.9, 8.7 Hz, 1H).

[0070] Prepare Example 12 below by essentially following the procedures described in Example 11 and using the appropriate enantiomer of [3]. The appropriate enantiomer of [3] may be prepared by using the appropriate enantiomer of JQ-1 acid and following the procedure described in Preparation 1 and Preparation 2.Example 12 QL-MDM2-47 (3'5',4'7?,5,S)-6"-chloro-4'-(3-chloro-2-fluorophenyl)-JV-(4-((6-(4-(3-(2-((R)-4-(4- chlorophenyl)-2,3,9-trimethyl-677-thieno[3,2- / ] [1 ,2,4]triazolo[4,3-a] [1 ,4]diazepin-6- yl)acetamido)propyl)piperazin-l-yl)hexyl)carbamoyl)phenyl)-2"-oxodispiro[cyclohexane-l,2'-pyrrolidine-3',3"-indoline]-5'-carboxamideLCMS: [M+H]+=1188.6.Preparation 5 Ent-Brigimadlin(3 'R, 3a' R, 10a'7?)-6-chloro-3 '-(3 -chloro-2-fluorophenyl)- 1 '-(cyclopropylmethyl)-6'-methyl-2- oxo-3',3a',10',10a'-tetrahydro-r / 7-spiro[indoline-3,2'-pyrrolo[2',3':4,5]pyrrolo[l,2- b]indazole]-7'-carboxylic acid

[0071] Preparation 5 and 6 were produced as outlined in WO 2017 / 060431 Al as a racemic mixture (2 g) and separated by SFC (column: DAICELCHIRALCEL®OJ (250*30 mm, 10 pm); mobile phase A: Supercritical CO2; mobile phase B: MeOH (+0.1% 7.0 mol / L Ammonia in MeOH)) to afford the title compound (750 mg, 75% yield) as a white solid. LC-MS [M+H]+: 591.1. ’H NMR (400 MHz, CD3OD) 5 7.77 - 7.63 (m, 1H), 7.58 - 7.46 (m, 2H), 7.43 - 7.30 (m, 2H), 7.19 (t, J= 8.0 Hz, 1H), 7.03 (dd, J= 8.1, 1.9 Hz, 1H), 6.66(d, J= 1.8 Hz, 1H), 6.01 (t, J= 8.3 Hz, 1H), 5.20 (td, J= 8.0, 2.5 Hz, 1H), 4.20 (d, J= 8.4 Hz, 1H), 3.75 - 3.64 (m, 1H), 3.58 - 3.49 (m, 1H), 2.67 (s, 3H), 2.51 - 2.41 (m, 1H), 2.37 - 2.27 (m, 1H), 0.93 - 0.80 (m, 1H), 0.67 - 0.55 (m, 1H), 0.49 - 0.38 (m, 1H), 0.26 - 0.14 (m, 1H), 0.05 - 0.01 (m, 1H).Example 13 QL-MDM2-48 (3a'5,10a'5)-6-chloro-3'-(3-chloro-2-fluorophenyl)-JV-(6-(4-(3-(2-((5)-4-(4-chlorophenyl)- 2,3,9-trimethyl-6H-thieno[3,2- / | [1 ,2,4]triazolo[4,3-a] [1 ,4]diazepin-6- yl)acetamido)propyl)piperazin- 1 -yl)hexyl)- 1 '-(cyclopropylmethyl)-6'-methyl-2-oxo- 3',3a', 10', lOa'-tetrahydro- 177-spiro[indoline-3,2'-pyrrolo[2',3':4,5]pyrrolo[l ,2 -b] indazole] - 7'-carboxamide

[0072] (1) In a 4 mL vial fited with a stir bar, [3] (41 mg) is dissolved in DCM (0.3 mL) followed by dropwise addition of HC1 (4 M in 1,4-dioxane, 0.3 mL). The reaction is stirred at 25 °C overnight, and the solvent is removed. The residue is subjected to the next step reaction without further purification. (2) To a solution of brigimadlin

[0011] (33 mg, 0.06 mmol) in DMF (5 mL) was added DIPEA (36 mg, 0.28 mmol) and HATU (27 mg, 0.022 mmol) at 0 °C. The mixture was stirred for 0.5 h. Then, the residue from the above step (35 mg, 0.056 mmol) was added at 0 °C. The mixture was stirred at room temperature for 1 h. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (30 mL x 2), dried over NaiSCL andconcentrated. The residue was purified by Prep-HPLC (acetonitrile with 0.1 % of NH3-H2O in water 5% to 95%) to obtain the title compound (7.72 mg, 11.5% yield) as a white solid. LC-MS [M+H]+: 1197.6.1H NMR (400 MHz, DMSO-6) 8 10.49 (s, 1H), 8.33 (s, 1H), 8.21 - 8.16 (m, 1H), 8.13 - 8.08 (m, 1H), 7.73 - 7.67 (m, 1H), 7.53 - 7.46 (m, 4H), 7.42 (d, J= 8.6 Hz, 2H), 7.35 - 7.26 (m, 2H), 7.01 (dd, J= 8.0, 1.9 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 6.63 (d, J= 1.9 Hz, 1H), 6.00 - 5.94 (m, 1H), 5.08 (dd, J= 10.9, 5.2 Hz, 1H), 4.50 (dd, J= 7.9, 6.3 Hz, 1H), 4.03 (d, J= 8.4 Hz, 1H), 3.66 (dd, J= 16.9, 7.7 Hz, 1H), 3.44 - 3.40 (m, 2H), 3.20 (dd, J= 10.4, 7.0 Hz, 4H), 3.15 - 3.08 (m, 2H), 2.59 (s, 3H), 2.40 (s, 3H), 2.37 - 2.16 (m, 16H), 1.62 (s, 3H), 1.60 - 1.54 (m, 2H), 1.53 - 1.46 (m, 2H), 1.45 - 1.37 (m, 2H), 1.36 - 1.25 (m, 4H), 0.83 - 0.75 (m, 1H), 0.57 - 0.49 (m, 1H), 0.40 - 0.32 (m, 1H), 0.18 (dd, J= 9.3, 4.7 Hz, 1H), -0.04 (dd, J= 9.8, 4.6 Hz, 1H).

[0073] Prepare Example 14 below by essentially following the procedures described in Example 13 and using the appropriate enantiomer of

[0011] .Example 14 QL-MDM2-49 (3a'S,10a'S)-6-chloro-3'-(3-chloro-2-fluorophenyl)-JV-(6-(4-(3-(2-((S)-4-(4-chlorophenyl)- 2,3,9-trimethyl-677-thieno[3,2- / | [1 ,2,4]triazolo[4,3-a] [1 ,4]diazepin-6- yl)acetamido)propyl)piperazin- 1 -yl)hexyl)- 1 '-(cyclopropylmethyl)-6'-methyl-2-oxo- 3',3a',10',10a'-tetrahydro-r77-spiro[indoline-3,2'-pyrrolo[2',3':4,5]pyrrolo[l,2-b]indazole]- 7'-carboxamide6.3 mg, 9.38% yield. LC-MS [M+H]+: 1197.6. H NMR (400 MHz, DMSO- <fc) 5 10.49 (s,1H), 8.21 (s, 1H), 8.21 - 8.17 (m, 1H), 8.13 - 8.08 (m, 1H), 7.73 - 7.66 (m, 1H), 7.53 - 7.46 (m, 4H), 7.42 (d, J= 8.6 Hz, 2H), 7.35 - 7.27 (m, 2H), 7.01 (dd, J= 8.0, 1.9 Hz, 1H), 6.94 (d, J= 8.6 Hz, 1H), 6.63 (d, J= 1.9 Hz, 1H), 6.01 - 5.94 (m, 1H), 5.08 (dd, J= 11.1, 5.3 Hz, 1H), 4.50 (dd, J= 7.9, 6.3 Hz, 1H), 4.04 (d, J= 8.4 Hz, 1H), 3.69 - 3.63 (m, 1H), 3.44 (s, 2H), 3.21 (dd, J= 9.9, 7.0 Hz, 4H), 3.14 3.06 (m, 2H), 2.59 (s, 3H), 2.40 (s, 3H), 2.38 - 2.16 (m, 16H), 1.62 (s, 3H), 1.60 - 1.55 (m, 2H), 1.53 - 1.47 (m, 2H), 1.45 - 1.37 (m, 2H), 1.36 - 1.26 (m, 4H), 0.85 - 0.75 (m, 1H), 0.58 - 0.49 (m, 1H), 0.41 - 0.32 (m, 1H), 0.18 (dd, J= 9.5, 4.7 Hz, 1H), -0.04 (dd, J= 9.7, 5.0 Hz, 1H).ASSAYSCell lines and cell culture

[0074] All liposarcoma cell lines used in this study are commercially available from Cellosaurus: LPS141 (RRID:CVCL_M823), LPS853 (RRID:CVCL_V415), T449 (RRID:CVCL_M807), T778 (RRID:CVCL_M808). The Cellosaurus is developed by Amos Bairoch of the CALIPHO group at the SIB - Swiss Institute of Bioinformatics as part of the neXtProt project. All liposarcoma cell lines are maintained in RPMI-1640 medium (Gibco, ThermoFisher Scientific) supplemented with 15% FBS, 1% GLUTAMAX™ (Gibco), and 1,000 Units / mL Penicillin and 1 mg / mL Streptomycin (Gibco). A-375, WI- 38, andU-2 OS cell lines are purchased from ATCC. A-375 (Cat# CRL-1619) cells aremaintained in DMEM medium (Gibco, ThermoFisher Scientific) with 10% FBS, 1,000 Units / mL Penicillin and 1 mg / mL Streptomycin (Gibco). WI-38 (Cat# CCL-75) cells are maintained in EMEM medium (Gibco, ThermoFisher Scientific) with 10% FBS, 1,000 Units / mL Penicillin and 1 mg / mL Streptomycin (Gibco). U-2 OS (Cat# HTB-96) cells are maintained in McCoy’s 5A medium (Gibco, ThermoFisher Scientific) with 10% FBS, 1,000 Units / mL Penicillin and 1 mg / mL Streptomycin (Gibco). Normal melanocytes are purchased from ThermoFisher (Cat# C0025C, lot / donor# 2291033), maintained in supplemented Medium 254 (Cat# M-254-500) and cultured in hypoxic conditions (5% CO2, and 5% 02). All cell lines are regularly screened for mycoplasma.Dose response curves

[0075] Cells are seeded in 96 well plates at the following densities: LPS853 = 750 cells / well, LPS141 = 1250 cells / well, T449 = 1000 cells / well, T778 = 250 cells / well, U2OS = 500 cells / well, A375 = 250 cells / well, WI-38 = 500 cells / well, melanocytes = 5000 cells / well. The following day, regular growth media is replaced with drug-containing media. The optimal dose range for each drug is determined for each cell line and is tested across 9 half-log doses. Each plate includes 0.1% DMSO as a negative control for growth inhibition. Drug media is changed 48 h after initial treatment. Plates are incubated in drug at 37 °C for a total of 96 h or 120 h and lysed by adding 20 pL CELL TITER-GLO® reagent (Promega, Cat. No. G7570) to 100 ml cell media. Luminescence is measured using a GLOMAX® explorer (Promega) plate reader and growth inhibition is calculated relative to DMSO treated wells. All experiments are performed in biological duplicates and, within a given experiment, at least 3 technical replicates are tested per condition. The drm R package is used to fit data to a four-parameter logistic curve using the LL.4() model. From this curve IC50 and EC50 values were obtained for the following cell lines as illustrated in Table 1, Table 2, Table 3, and Table 4 below.Table 1Table 2Table 3Table 4Western Blotting

[0076] Cells are plated and, the following day, normal growth media is removed and replaced with DMSO or drug-containing media. Cells are incubated in drug media for 24 h and washed two times with lx PBS and scraped on ice in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS) supplemented with fresh protease (HALT™, Thermofisher) and phosphatase inhibitors (Thermofisher). Protein is quantified and normalized using the BCA Protein Assay kit (Thermo #23227) according to the manufacturer’s protocol. Total signal for each protein is quantified using FIJI® and normalized to an actin loading control. Quantifications are fitted to dose response curves using the drm R package (fct = LL.4Q) and plotted in R.Generation of LPS853 P53 knockout cell line

[0077] TP53 knockout experiments utilize pXPR_044, all-in-one CRISPR-Cas9 vector carrying mCherry and sgRNA. The following CRISPR sgRNAs are cloned into the vector: safe harbor 5’-GGCTAAATTCCTCTTATTCA-3’; TP53 start site 5’- TCGACGCTAGGATCTGACTG-3’. LPS853 cells are transfected with 3 pg / mL of lentivirus plasmid and 5 pL / mL of TransIT-LTl reagent (MirusBio, MIR2300). The following day, cells are selected using fluorescent activated cell sorting (Sony, SH800). The gating strategy first involves selecting the live population with forward and side scatter. Untreated cells are used to set the mCherry threshold gate. Cells expressing the highest mCherry (top 50%) are selected. Genomic DNA is isolated and the TP53 start site is amplified using the following primer set: F 5’-CCCAACCCTTGTCCTTACCA-3’; R 5’- CAACATGCAAAGCCCTGTCT-3’. CRISPR-mediated disruption of this amplified region is confirmed via sanger sequencing using the TIDE tool.Pharmacokinetic measurements

[0078] For pharmacokinetic characterization of QL-MDM2-18 and QL-MDM2-48, 50 mg / kg of drug was administered to 3 CD1 mice. QL-MDM2-18 was prepared in 10% DMSO, 90% (10% HP-P-CD in sterile water) at 5 mg / mL for IP dosing. QL-MDM2-48 was prepared in 10% DMSO, 10% Solutol HS 15, 80% (10% HP-P-CD in sterile water) at 5 mg / mL for IP dosing. Approx. 25 pL of blood were taken into K2EDTA tubes from the animals via facial vein. 20 pL of blood was diluted with 60 pL diH2O immediately after sampling and vortexed until mixed well and stored at -70C. Plasma drug concentrations were measured via LC-MS / MS. PK parameters were estimated by non-compartmental model using WinNonlin 8.2.Pharmacodynamic measurements in patient-derived xenografts

[0079] In vivo drug treatments using QL-MDM2-18 were performed in male NSG mice harboring LPS3 PDX tumors, which were injected subcutaneously. The compound QL- MDM2-18 was prepared in 10% Solutol HS 15, 80% (10% HP-P-CD in sterile water) at 5 mg / mL for IP dosing at 15 mg / kg QD, based on good toleration in toxicity studies. Twomice were treated for 16 or 20 doses and three mice were given lOul / g of vehicle for 14 doses. Tumor tissue was harvested by flash freezing 24 hours after the last dose of drug or vehicle was administered. Flash frozen samples were post-fixed in 4% formaldehyde at 4°C and then cryoprotected in sucrose solution prior to re-freezing. 10 uM sections were prepared on a cryostat and then permeabilized, blocked and stained with a human-specific CDK4 antibody (cat# MA5-41178) and a BRD4 (cat# CST63759) antibody at 1 :250 dilutions. Samples were imaged on a Leica DMi8 imaging system using a 40x objective with oil immersion and images were processed using the THUNDER computational clearing method. QuPath software was used to first select tumor cells marked by CDK4 staining, then quantify the number of tumor cells that were positive for BRD4 staining.

Claims

We claim:

1. A compound of the formula:or a pharmaceutical acceptable salt thereof, wherein:B-a, B-bL is independently a hydrocarbon or polyethylene glycol linker selected fromwherein x is 2-10, y is 1-5, z is 1-5; and I is independently selected from:

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein B is3. The compound of any of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wher4. The compound of any of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein L is5. The compound of any of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein x is 2 to 10.

6. The compound of claim 5, or a pharmaceutically acceptable salt thereof, wherein x is 3 to7.

7. The compound of any of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein L is8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein y or z is 1 to 5.

9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein y or z is 3.

10. The compound of any of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein B isC1L is selected from x is 4; y is 1 to 4; z is 3; and11. The compound of any of claim 1, or a pharmaceutically acceptable salt thereof, wherein B12. The compound of any of claims 1 to 10, wherein the compound is selected fromor a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition comprising a compound any of claims 1 to 12, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

14. A method for treating cancer comprising administering an effective amount of a compound of any of Claims 1 to 12, or a pharmaceutically acceptable salt thereof, to a patient in need thereof.

15. The method of claim 14, wherein the cancer is an MDM2 -amplified cancer.

16. The method of claim 14 or 15, wherein the cancer is selected from neoplasms, lymphoma, sarcoma, bladder cancer, adenoid cystic carcinoma, glioblastoma, solid tumors, and myeloid tumors.

17. A method of activating the production of p53 by targeting MDM2 -amplified cancers comprising administering an effective amount of a compound of any of Claims 1 to 12, or a pharmaceutical acceptable salt thereof.

18. A compound of any of Claims 1 to 12, or a pharmaceutically acceptable salt thereof, for use in therapy.

19. A compound of any of Claims 1 to 12, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

20. A compound of any of Claims 1 to 12, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of cancer.

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