Selective inhibition of MDM2 amplified cancers using targeted protein degradation agents

Abstract

The present invention relates to compounds useful for selective inhibition of MDM2 amplification cancer and pharmaceutical compositions thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 587,927, filed October 4, 2023. That earlier provisional application is expressly incorporated herein by reference. Background Technology

[0003] The tumor suppressor p53 plays a central role in preventing tumor formation. Careful control of p53 activity is essential for mammalian survival. Known as the "gatekeeper of the cell" or the "guardian of the genome," p53 is widely considered the most frequently mutated gene in human cancers; its inactivation occurs in almost half of all human tumors. Fluctuations in p53 levels can be problematic, making high p53 levels potentially fatal, while low p53 levels may allow tumors to develop.

[0004] Mouse double microsome 2 (MDM2) is an E3 ubiquitin ligase that is overexpressed in many cancers and regulates target proteins through ubiquitination. The oncogene MDM2, crucial for the control of p53 activity, possesses both p53-dependent and p53-independent oncogenic activities. MDM2 is a key negative regulator of the p53 protein and forms an autoregulatory feedback loop with p53. The p53 / MDM2 feedback loop is a prime example of a negative feedback loop encompassing both transcriptional and protein-protein interaction arms. p53 transcriptionally activates MDM2, which then targets p53 for degradation. Under normal conditions, the p53 / MDM2 feedback loop maintains relatively low p53 levels until cellular stress (such as DNA damage) is applied. These stress signals block MDM2 and / or promote degradation. The resulting decrease in MDM2 activity in the feedback loop leads to stabilization of p53 levels, which in turn results in increased p53 transcription of MDM2. Overexpression of MDM2 via copy number amplification can lead to an increased risk of cancer and / or accelerated tumor formation and progression. 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 lung.

[0005] Inhibition of MDM2 represents a therapeutic approach targeting 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, leading to a rapid stabilization of p53 levels, which in turn causes a rapid increase in MDM2 levels. Therefore, to continue p53 inhibition, the MDM2 inhibitor is forced to overcome the resulting MDM2 upregulation, requiring increased inhibitor dosage. The required dosage results in on-target toxicity to normal cells, which severely limits the clinical efficacy of MDM2 inhibitors. Furthermore, primary and acquired resistance limit potential benefits. Two pathways lead to resistance. The more prevalent mechanism is the acquisition of p53 inactivating mutations. Another mechanism is an excess of MDM2 copy number, which leads to resistance and can be enhanced through the feedback loop. Similarly, because resistance becomes a problem, higher doses of MDM2 inhibitors are needed to overcome the accumulation of MDM2.

[0006] Furthermore, as targeted protein degradation has emerged as an emerging modality with the potential to target pathogenic proteins that are challenging for conventional small molecules, proteolytic-targeting chimeric (PROTAC) technology, as an alternative approach to induce cell death and / or tumor growth inhibition, typically utilizes the ubiquitin-proteasome system by recruiting E3 ligases such as cereblon (CRBN) or von Hippel-Landau (VHL) as an alternative method to induce cell death and / or tumor growth inhibition. For example, dBET6 is a commercially available BRD4 degrader containing the E3 ligase CRBN. See U.S. Patent No. 9,694,084. However, E3 ligase production dependent on CRBN or VHL will induce equivalent degradation across tumor and normal cells, resulting in unintended on-target toxicity in normal cells.

[0007] Therefore, there is a need to discover novel oncolytic compounds that can activate p53 levels without disrupting the p53 / MDM2 feedback loop. Furthermore, these compounds need to selectively target cancer cells while exhibiting minimal toxicity to normal cells.

[0008] This invention provides novel protein degraders that activate p53 production by targeting MDM2-amplified cancers. Furthermore, the compounds of this invention benefit from the p53 / MDM2 feedback loop rather than disrupting it. Compared to MDM2 inhibitors alone, MDM2-based PROTACs exhibit an enhanced therapeutic index. Additionally, by utilizing the oncogenic activity of MDM2, the compounds of this invention selectively guide the degradation of protein targets essential only for MDM2-amplified tumors; with minimal impact on healthy cells. Moreover, pretreatment with an MDM2 inhibitor enhances the sensitivity of MDM2-amplified cell lines to MDM2-based degraders. The compounds of this invention also target protein 4 (BRD4), which contains a bromine domain and is a member of the bromine domain and terminal extraterminal domain (BET) protein family. BRD4 degradation occurs at lower doses. Attached Figure Description

[0009] Figure 1 The results showed that the MDM2 conjugates MI-1061, idasanutlin, and brigidallin, as well as the BRD4 conjugates JQ-1 and JQ35, did not show a significant therapeutic index against MDM2-amplified liposarcoma in the LPS853 and T449 cell lines, nor against MDM2 wild-type tumor cell lines in the U2OS and A375 cell lines.

[0010] Figure 2 The results show that, compared to the edanulin-based MDM2 degraders A1874, QL-MDM2-01, QL-MDM2-05, and QL-MDM2-06, the use of the lower-potency MDM2 inhibitor component MI1061, regardless of the BRD4 conjugate, enhances the therapeutic index of the MDM2-based degraders QL-MDM2-17, QL-MDM2-18, and QL-MDM2-19. Furthermore, the incorporation of the higher-potency MDM2 conjugate brimdalene increases overall potency while maintaining the strong therapeutic index of the MDM2 degrader QL-MDM2-48.

[0011] Figure 3 It is shown that commercially available BRD4 degraders dBET6 and ARV771, as well as the control MDM2-based degraders (R)-QL-MDM2-01, (R)-QL-MDM2-18, QL-MDM2-45, QL-MDM2-47, and QL-MDM2-49 included herein, did not show selective activity against MDM2-amplified liposarcoma in LPS853 and T449 cell lines, nor selective activity against MDM2 wild-type tumor cell lines in U2OS and A375.

[0012] Figure 4The therapeutic index of the MDM2-based degrader QL-MDM2-18 was shown in direct comparison with the control MDM2-based degraders (R)-QL-MDM2-18 and QL-MDM2-45, and the degradation-based mechanism of action in the LPS853 cell line was confirmed.

[0013] Figure 5 It is shown that QL-MDM2-18-induced BRD4 degradation requires ternary complex formation (as seen in the case of progressive overload of JQ35 and MI-1061) and proteosome activity (as seen in the case of addition of the proteosome inhibitor bortezomib).

[0014] Figure 6 The results show that the MDM2-based degrader QL-MDM2-01 exhibits greater growth-inhibiting activity in MDM2-amplified liposarcoma cells LPS853 and T449 than in non-tumor cell lines melanocytes and WI-38.

[0015] Figure 7 The results show that, compared with the MDM2 wild-type model A375 and normal fibroblasts, BRD4 degradation occurred at a lower dose in MDM2-expanded LPS cells LPS853 before p53 activation, using the MDM2-based degraders QL-MDM2-01, QL-MDM2-17, QL-MDM2-18, QL-MDM2-19, and QL-MDM2-48.

[0016] Figure 8 express Figure 7 Quantification of protein blot data. Figure 10 The results show that p53 knockout cells are resistant to edanulin but remain sensitive to the MDM2-based degrader QL-MDM2-01 (which contains the MDM2 inhibitor component edanulin). Similarly, p53 knockout cells are resistant to MI-1061 but remain sensitive to the MDM2-based degrader QL-MDM2-18 (which contains the MDM2 inhibitor component Mi-1061). These data are consistent with clinical trials that demonstrated that p53 inactivation mutations are the primary mechanism of MDM2 inhibitor resistance and that MDM2 degraders remain active in the presence of p53 mutations.

[0017] Figure 9 It is shown that pretreatment with the MDM2 inhibitor HDM201 enhanced the therapeutic index of the MDM2-based degrader QL-MDM2-1 in expanded LPS cell lines, but did not enhance the therapeutic index of the degrader in the MDM2 wild-type cell line A375.

[0018] Figure 10The results show that p53 knockout cells are resistant to edanulin but remain sensitive to the MDM2-based degrader QL-MDM2-01 (which contains the MDM2 inhibitor component edanulin). Similarly, p53 knockout cells are resistant to MI-1061 but remain sensitive to the MDM2-based degrader QL-MDM2-18 (which contains the MDM2 inhibitor component Mi-1061). These data are consistent with clinical trials that demonstrated that p53 inactivation mutations are the primary mechanism of MDM2 inhibitor resistance and that MDM2 degraders remain active in the presence of p53 mutations.

[0019] Figure 11 It is shown that MI-1061 has lower potency than edanulin, by a factor of approximately 1 log.

[0020] Figure 12 The mean plasma concentration-time curves of QL-MDM2-18 and QL-MDM2-48 over 24 hours are shown in male CD1 mice.

[0021] Figure 13 This study demonstrated a reduction in BRD4-positive tumor cells in xenografts derived from LPS3 liposarcoma patients after treatment with 15 mg / kg QL-MDM2-18. Detailed Implementation

[0022] This invention provides a compound or a pharmaceutically acceptable salt thereof, the compound having the following formula:

[0023] .

[0024] The conjugate (B) of this invention is a BRD4 conjugate. Alternatively, the BRD4 conjugate may be derived from JQ-1 (Ba) or JQ-35 (Bb).

[0025] ,

[0026] B-aB-b

[0027] 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 prepared according to the procedures set forth in WO 2011 / 43669 (which is incorporated herein by reference). Preferably, the BRD4 conjugate is derived from JQ-35.

[0028] The connector (L) can be a hydrocarbon (La) of varying lengths or a polyethylene glycol (PEG) chain of varying lengths (Lb or Lc). The connector is C2-C. 10Alkyl groups (where x is 2 to 10) or PEG chains having 1 to 5 PEG units (where y or z is 1 to 5):

[0029] , or

[0030] L-aL-bL-c

[0031] 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.

[0032] Inhibitor (I) is derived from MDM2 inhibitors. Alternatively, inhibitors may be derived from edanulin (Ia), MI-1061 (Ib), or brimdalene (Ic).

[0033] , ,

[0034] I-aI-b

[0035]

[0036] Ic

[0037] Idanulin (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 prepared 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 prepared according to the procedures set forth in WO 2012 / 155066 (which is incorporated herein by reference). Preferably, the inhibitor is derived from MI-106. Brimadleline (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 prepared according to the procedures described in WO2017060431 (which is incorporated herein by reference).

[0038] The compounds of this invention are not the following compounds:

[0039]

[0040] A preferred embodiment of the present invention relates to a compound or a pharmaceutically acceptable salt thereof, wherein B is... ;

[0041] L is selected from , and ;

[0042] x is 6;

[0043] y ranges from 1 to 4;

[0044] z is 3; and

[0045] I selected , and .

[0046] Another preferred embodiment of the invention relates to a compound or a pharmaceutically acceptable salt thereof, wherein B is...

[0047] ;

[0048] L is selected from and ;

[0049] y is 3;

[0050] z is 3; and

[0051] I am .

[0052] Pretreatment with an MDM2 inhibitor can enhance the therapeutic index of the MDM2-based degrader of the present invention. MDM2 inhibitors are well known in the art and include siremadlin (also known as HDM201), edanulin (also known as RG-7388 and RO-5503781), milademetan (also known as DS-3032 and DS-3032b), brimmadelin (also known as BI 907828), navidlin (also 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-1 41. 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.

[0053] This invention provides a method for activating p53 production by targeting MDM2-amplified cancers, the method comprising administering an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof. This invention also provides a method for promoting the degradation of MDM2-amplified cancers, the method comprising administering an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0054] This invention provides a method for treating cancer, comprising administering an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof. In another embodiment, an effective amount of an MDM2 inhibitor or a pharmaceutically acceptable salt thereof is also administered to the patient. In yet another embodiment, the effective amount of the MDM2 inhibitor may be administered in combination or alone, and if administered alone, may be administered simultaneously or sequentially. Preferably, the MDM2 inhibitor is administered alone and prior to the administration of the MDM2 degrading agent of the invention. In another preferred embodiment, the MDM2 inhibitor is ciremadelin (also known as HDM201), edanulin (also known as RG-7388 and RO-5503781), miradtan (also known as DS-3032 and DS-3032b), brimmadeline (also known as BI 907828), navidelin (also 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), Alizomaderin (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...) It is known as SCH-900242), Natrin-3a (also known as RO-4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI-773), DS-5272, ATSP7041, Bisinura lactone A, SAH-p53-8, SAR-299155, SAR-305801, Sheldermetan (also known as JNJ-1 and JNJ-26854165), SJ-2011 or VG-0604a.

[0055] The present invention further provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In one specific embodiment, the composition further comprises one or more other therapeutic agents. In another embodiment, the therapeutic agent is an MDM2 inhibitor or a pharmaceutically acceptable salt thereof. In yet another embodiment, the MDM2 inhibitor may be administered in combination or alone, and if administered alone, may be administered simultaneously or sequentially. Preferably, the MDM2 inhibitor is administered alone and prior to the administration of the MDM2 degrading agent of the present invention. In another preferred embodiment, the MDM2 inhibitor is ciremadelin (also known as HDM201), edanulin (also known as RG-7388 and RO-5503781), miradtan (also known as DS-3032 and DS-3032b), brimmadeline (also known as BI 907828), navidelin (also 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), Alizomaderin (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...) It is known as SCH-900242), Natrin-3a (also known as RO-4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI-773), DS-5272, ATSP7041, Bisinura lactone A, SAH-p53-8, SAR-299155, SAR-305801, Sheldermetan (also known as JNJ-1 and JNJ-26854165), SJ-2011 or VG-0604a.

[0056] In addition, the present invention provides compounds of the present invention or pharmaceutically acceptable salts thereof for use in therapy, particularly for the treatment of cancer. In another embodiment, an MDM2 inhibitor or a pharmaceutically acceptable salt thereof is also administered to the patient. In yet another embodiment, the MDM2 inhibitor may be administered in combination or alone, and if administered alone, may be administered simultaneously or sequentially. Preferably, the MDM2 inhibitor is administered alone and prior to the administration of the MDM2 degrading agent of the present invention. In another preferred embodiment, the MDM2 inhibitor is ciremadelin (also known as HDM201), edanulin (also known as RG-7388 and RO-5503781), miradtan (also known as DS-3032 and DS-3032b), brimmadeline (also known as BI 907828), navidelin (also 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), Alizomaderin (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...) These compounds are known as SCH-900242, Natrin-3a (also known as RO-4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI-773), DS-5272, ATSP7041, Bistriol A, SAH-p53-8, SAR-299155, SAR-305801, Sheldermetan (also known as JNJ-1 and JNJ-26854165), SJ-2011, or VG-0604a. In another embodiment, the present invention provides the use of activating p53 production by targeting MDM2-amplified cancers, the use comprising administering an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0057] Furthermore, the present invention provides compounds of the present invention or pharmaceutically acceptable salts thereof for use in the treatment of cancer. In another embodiment, an MDM2 inhibitor or a pharmaceutically acceptable salt thereof is also administered to the patient. In yet another embodiment, the MDM2 inhibitor may be administered in combination or alone, and if administered alone, may be administered simultaneously or sequentially. Preferably, the MDM2 inhibitor is administered alone and prior to the administration of the MDM2 degrading agent of the present invention. In another preferred embodiment, the MDM2 inhibitor is ciremadelin (also known as HDM201), edanulin (also known as RG-7388 and RO-5503781), miradtan (also known as DS-3032 and DS-3032b), brimmadeline (also known as BI 907828), navidelin (also 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), Natrin-3a (also known as RO-4435385), NW-8-153, RG-7112, SAR-405838 (also known as MI-773), DS-5272, ATSP7041, Bisinura lactone A, SAH-p53-8, SAR-299155, SAR-305801, Sheldermetan (also known as JNJ-1 and JNJ-26854165), SJ-2011 or VG-0604a.

[0058] Furthermore, the present invention provides the use of the compounds of the present invention or pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of cancer. In another embodiment, the present invention provides the use of activating p53 production by targeting MDM2 amplification in cancer, the use comprising administering an effective amount of the compounds of the present invention or pharmaceutically acceptable salts thereof.

[0059] This invention provides preferred embodiments of the methods and uses described herein, wherein the cancer is any cancer with high MDM2 amplification. Preferred cancers include growths, lymphomas, sarcomas, solid tumors, and myeloid tumors. More preferred cancers include: growths (including hematologic growths and salivary gland growths), sarcomas (including liposarcoma and advanced liposarcoma), leukemias (including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic myeloid monocytic leukemia, mixed phenotype acute leukemia, and T-cell prolymphocytic leukemia), melanomas (such as uveal melanoma), multiple myeloma, polycythemia vera, primary myelofibrosis, endometrial cancer, Merkel cell carcinoma, adenoid cystic carcinoma, transitional cell carcinoma, primary myelofibrosis, brain cancers (such as neurofibrosarcoma, neuroblastoma, and glioblastoma), eye cancers (such as retinoblastoma), gastrointestinal cancers (such as gastric cancer and colorectal cancer), pancreatic cancer, breast cancer, bladder cancer, prostate cancer, and lung cancers (such as extensive-stage small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer, non-small cell lung cancer, and non-squamous non-small cell lung cancer). Especially preferred cancers include: growths (including hematologic growths and salivary gland growths), sarcomas (including liposarcoma and advanced liposarcoma), leukemias (including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic myeloid monocytic leukemia, mixed phenotype acute leukemia, and T-cell prolymphocytic leukemia), melanomas (such as uveal melanoma), multiple myeloma, polycythemia vera, primary myelofibrosis, endometrial cancer, Merkel cell carcinoma, adenoid cystic carcinoma, transitional cell carcinoma, primary myelofibrosis, brain cancers (such as neurofibrosarcoma, neuroblastoma, and glioblastoma), eye cancers (such as retinoblastoma), gastrointestinal cancers (such as gastric cancer and colorectal cancer), breast cancer, prostate cancer, and lung cancers (such as extensive-stage small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer, non-small cell lung cancer, and non-squamous non-small cell lung cancer).

[0060] This invention provides a method for treating radiation injury, comprising administering an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof. In another embodiment, the effective amount of the compound of the invention may be administered in combination with or alone with radiotherapy, and if administered alone, may be administered simultaneously or sequentially.

[0061] The present invention also provides compounds of the present invention or pharmaceutically acceptable salts thereof for use in the treatment of radiation injury. In another embodiment, an effective amount of the compound of the present invention may be administered in combination with or alone with radiation therapy, and if administered alone, simultaneously or sequentially.

[0062] This invention provides the use of the compounds of the invention or pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of radiation injury. In another embodiment, an effective amount of the compound of the invention may be administered in combination with or alone with radiation therapy, and if administered alone, simultaneously or sequentially.

[0063] This invention provides a method for treating degenerative diseases, including osteoarthritis, macular dystrophy, and macular degeneration, comprising administering an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof. The invention also provides a compound of the invention or a pharmaceutically acceptable salt thereof for use in the treatment of degenerative diseases, including osteoarthritis, macular dystrophy, and macular degeneration. Furthermore, the invention provides the use of a compound of the invention or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of degenerative diseases, including osteoarthritis, macular dystrophy, and macular degeneration.

[0064] Although all exemplary compounds of the present invention are available,

[0065] However, the compounds of the present invention can be administered as pharmaceutically acceptable salts. Pharmaceutically acceptable salts and common methods for their preparation are well known in the art. See, for example, P. Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd edition, 30th revision (Wiley-VCR, 2011); SM Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.

[0066] The compounds of the present invention are preferably formulated as pharmaceutical compositions for administration via multiple routes. Such pharmaceutical compositions and methods for their preparation are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy (I) (edited by A. Gennaro et al., 21st ed., Mack Publishing, 2005).

[0067] The compounds of this invention are generally effective over a wide dosage range. It should be understood that the actual amount of compound administered will be determined by the physician based on relevant circumstances, including the condition to be treated, the route of administration chosen, one or more actual compounds administered, the individual patient's age, weight and response, and the severity of the patient's symptoms.

[0068] As used herein, the term “therapeutic index” refers to the dose range that induces at least 50% growth inhibition in trans-MDM2 amplified lines rather than MDM2 wild-type lines.

[0069] As used in this article, the term "treatment" refers to the containment, mitigation, cessation, or reversal of the progression or severity of existing symptoms, conditions, or disorders.

[0070] As used herein, the phrase “effective amount” means an amount of the compound of the invention sufficient to treat the condition described herein or its adverse effects at one or more doses, or an amount of the compound of the invention sufficient to inhibit MDM2 or BRD4 to achieve the purpose of the invention.

[0071] As used herein, the phrase “administration” means the act of giving a compound of the invention to a patient’s body by ingestion, inhalation, injection, or any other means, or the direct application of a compound of the invention to a patient’s body. Two or more compounds of the invention may be administered in combination or alone, and if administered alone, they may be administered simultaneously or sequentially over a period of time as determined by a qualified caregiver.

[0072] As used in this article, "patient" refers to a mammal, preferably a human.

[0073] The compounds of the present invention or their salts can be prepared by a variety of processes known in the art, some of which are illustrated in the following "Preparation and Examples". The specific synthetic steps of each of the described pathways can be combined in different ways, or combined with steps from different preparations or examples, to prepare the compounds or salts of the present invention. The products of each step in the following "Preparation and Examples" can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding, and crystallization. In the following "Preparation and Examples", unless otherwise specified, all substituents are as previously defined. Reagents and starting materials are readily available to those skilled in the art.

[0074] Additionally, some intermediates described in the “Preparation and Examples” section below may contain one or more nitrogen protecting groups. Depending on the specific reaction conditions and the specific transformation to be performed, the protecting groups may be the same or different each time they appear. Protecting and deprotecting conditions are well known to those skilled in the art and are described in the literature (see, for example, Peter GM Wuts and Theodora W. Greene, “Green’s Protective Groups in Organic Synthesis,” 4th edition, John Wiley and Sons, 2007).

[0075] Those skilled in the art will understand that the compounds of the present invention comprise a core containing at least one chiral center. For clarity, certain stereochemical centers may not be specified and certain substituents may be omitted in the following “Preparation and Examples”, and this is not intended to limit the teachings in any way. Monoenantiomers or diastereomers may be prepared from chiral reagents or by stereoselective or stereodirectional synthetic techniques. Alternatively, monoenantiomers or racemates may be separated from the mixture at any convenient point in the synthesis of the compounds of the present invention by methods such as selective crystallization or chiral chromatography (see, for example, J. Jacques et al., “Enantiomers, Racemates, and Resolutions”, John Wiley and Sons, 1981; and E.L. Eliel and S.H. Wilen, “Stereochemistry of Organic Compounds”, Wiley Interscience, 1994).

[0076] Some intermediates or compounds of the present invention may have one or more chiral centers. The present invention envisions all individual enantiomers or diastereomers, as well as mixtures of enantiomers and diastereomers of said compounds (including racemates). Preferably, compounds of the present invention containing at least one chiral center exist as a single enantiomer or diastereomer. A single enantiomer or diastereomer can be prepared from a chiral reagent or by stereoselective or stereodirectional synthetic techniques. Alternatively, a single enantiomer or diastereomer can be separated from the mixture by standard chiral chromatography or crystallization techniques. Those skilled in the art will understand that in some cases, the elution order of enantiomers or diastereomers may differ 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 dioctoctanic acid; "Boc" refers to tert-butoxycarbonyl; "CRISPR" refers to clustered, regularly spaced short palindromic repeats; "DCM" refers to dichloromethane; "DIPEA" refers to diisopropylethylamine; "DMEM" refers to Durbeco modified Eagle medium; "DMF" refers to dimethylformamide; "DMSO" refers to dimethyl sulfoxide; "EMEM" refers to Eagle minimum essential medium; "ESIMS" refers to electrospray ionization mass spectrometry; "FBS" refers to... Fetal bovine serum; "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide 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-BH3" refers to α-methylpyridineborane; "RIPA" refers to radioimmunoprecipitation assay; "TEA" refers to triethylamine; "Tris" refers to 2-amino-2-(hydroxymethyl)propane-1,3-diol.

[0077] plan

[0078] In the following schemes, all substituents are as previously defined unless otherwise specified. Reagents and starting materials are commercially available or can be prepared by methods well known to those skilled in the art, some of which are presented in the following preparations. The following schemes, preparations, and examples are provided to further illustrate the invention without limiting its scope.

[0079] Option 1.

[0080]

[0081] Scheme 1 describes the preparation of the compounds of this disclosure, which begins with an (S)-JQ1 acid, which is amide-coupled with [i] and [iii] in steps 1 and 2, respectively, in a suitable solvent (such as DMF) using HATU and Hünig bases or TEA, to yield [ii] and [v]. Variable X may represent an alkyl or PEG chain as defined herein by a linker. Those skilled in the art will recognize that a variety of reagents and conditions can be used for the activation of carboxylic acids and the coupling of amines. For example, DCC / DMAP, EDC / HOBt, and CDI / TEA can be used for amidation. Intermediate [ii] may be further deprotected under acidic conditions and subsequently reductively amination with [iv] in a suitable polar solvent using 2-methylpyridine-borane to yield [vi]. Variable X may represent an alkyl or PEG chain as defined herein by a linker. Those skilled in the art will recognize that a variety of reducing agents can be used for amination. For example, STAB, sodium cyanoborohydride, and hydride reducing agents. Both intermediates [v] and [vi] can be deprotected under acidic conditions and then amide-coupled with the carboxylic acid moiety of the MDM2 inhibitor as defined herein to give products [vii] and [viii].

[0082] Preparation and Examples

[0083] The following "Preparation and Examples" further exemplifies the invention and illustrates typical synthesis of the compounds of the invention. Reagents and starting materials are readily available to those skilled in the art, or can be readily synthesized by those skilled in the art. It should be understood that the "Preparation and Examples" are described by way of illustration and not limitation, and various modifications can be made by those skilled in the art.

[0084] The (R) or (S) configuration of the compounds of this invention can be determined by standard techniques such as X-ray analysis and correlation with chiral-HPLC retention times. The nomenclature in the following "Preparation and Examples" generally uses CHEMDRAW. ® The naming features in version 22.2.0 are used.

[0085] Unless otherwise specified, the percentages in the following “Preparation and Examples” refer to yield percentages.

[0086] Preparation 1

[0087] (S)-4-(3-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f] [1,2,4]triazolo[] [4,3-a][1,4]diazaphen-6-yl)acetamyl)propyl)piperazine-1-carboxylic acid tert-butyl ester

[0088]

[0089] In a 25 mL vial equipped with a stir bar, (S)-JQ1 acid (434 mg), tert-butyl 4-(3-aminopropyl)piperazine-1-carboxylate (265 mg), and HATU (454 mg) were dissolved in DMF (3 mL), followed by the addition of DIPEA (0.78 mL). The resulting mixture was then stirred at 25 °C for 2 h. After the reaction was complete, the mixture was purified directly by silica gel chromatography (elution: DCM / MeOH) to obtain the title compound (609 mg) in oil form. ESI-MS: Calculated C 31 H 41 35 ClN7O3S [M+1] + : 626.27, measured value: 626.63.

[0090] Preparation 2

[0091] (S)-(6-(4-(3-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]tri) Azo[4,3-a][1,4]diazaphen-6-yl)acetamyl)propyl)piperazin-1-yl)hexyl)tert-butyl carbamate

[0092]

[0093] (1) In a 25 mL vial equipped with a stir bar, [1] (94 mg) was dissolved in DCM (1 mL), followed by dropwise addition of HCl (4 M, in 1,4-dioxane, 1 mL). The reaction was stirred overnight at 25 °C and the solvent was removed. The residue was subjected to the next step of the reaction without further purification. (2) In a 4 mL vial equipped with a stir bar, the residue from the above step and tert-butyl carbamate [2] (66 mg) were dissolved in a mixed solvent (MeOH:AcOH = 10:1, 0.45 mL), followed by Pic-BH3 (23 mg). The resulting mixture was stirred overnight at 25 °C and the solvent was removed. The residue was purified directly by silica gel chromatography (elution: DCM / MeOH, containing 1 v / v NH3, 7 M, in MeOH) to obtain the title compound (76 mg) in oil form. ESI-MS: Calculated C 37 H 54 35 ClN8O3S [M+1] + : 725.37, measured value: 725.57.

[0094] Example 1

[0095] QL-MDM2-01

[0096]

[0097] (2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-N-(4-((6-(4-(3-(2- ((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazine (Hydro-6-yl)acetamyl)propyl)piperazin-1-yl)hexyl)carbamoyl)-2-methoxyphenyl)-4-cyano-5-neo Pentylpyrrolidine-2-carboxamide

[0098] (1) In a 4 mL vial equipped with a stir bar, [3] (16 mg) was dissolved in DCM (0.1 mL), followed by dropwise addition of HCl (4 M, in 1,4-dioxane, 0.1 mL). The reaction was stirred overnight at 25 °C and the solvent was removed. The residue was subjected to the next step of the reaction without further purification. (2) In a 4 mL vial equipped with a stir bar, the residue from the above steps, edanulin [4] (14 mg) and HATU (14 mg) were dissolved in DMF (0.1 mL), followed by addition of DIPEA (20 µL). The resulting mixture was stirred at 25 °C for 2 h. After the reaction was complete, the solvent was removed. The residue was purified directly by silica gel chromatography (elution: DCM / MeOH, containing 1 v / v NH3, 7 M, in MeOH) to obtain the title compound (20 mg) as a white powder. ESI-MS: Calculated C 63 H 73 35 Cl3F2N 11 O4S [M+1] + : 1222.46, measured value: 1222.66. 1 HNMR (500MHz, DMSO-d6) δ 10.40 (s, 1 H), 8.44-8.36 (m, 1 H), 8.31 (d, J =8.5Hz, 1 H), 8.19 (t, J = 5.3Hz, 1 H), 7.73 (t, J = 7.0Hz, 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 = 7.0Hz, 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).

[0099] Examples 2 to 6 were prepared by essentially following the procedures described in Preparation 1, Preparation 2 and Example 1, using a suitable enantiomeric form of the JQ-1 acid of Preparation 1 and a suitable aldehyde of Preparation 2 [2].

[0100] Example 2

[0101] (R)-QL-MDM2-01

[0102] (2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-N-(4-((6-(4-(3-(2- ((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazide (Hydro-6-yl)acetamyl)propyl)piperazin-1-yl)hexyl)carbamoyl)-2-methoxyphenyl)-4-cyano-5-neo Pentylpyrrolidine-2-carboxamide

[0103]

[0104] Example 3

[0105] QL-MDM2-05

[0106] (2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-N-(4-((2-(2-(4-(3-) (2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]di) (Zazo-6-yl)acetamyl)propyl)piperazin-1-yl)ethoxy)ethyl)carbamoyl)-2-methoxyphenyl)-4- Cyano-5-neopentylpyrrolidine-2-carboxamide

[0107]

[0108] Example 4

[0109] QL-MDM2-06

[0110] (2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-N-(4-((2-(2-(2-(4-) (3-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1, 4] diazaphen-6-yl)acetamyl)propyl)piperazin-1-yl)ethoxy)ethoxy)ethyl)carbamoyl)-2-methoxy (2-(4-cyano-5-neopentylpyrrolidine-2-carboxamide))

[0111]

[0112] Example 5

[0113] QL-MDM2-11

[0114] (2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-N-(4-((2-(2-(2-(2-) (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]diazazo-6-yl)acetamyl)propyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl (2-methoxyphenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamide

[0115]

[0116] Example 6

[0117] QL-MDM2-12

[0118] (2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-N-(4-((14-(4-(3-(2- ((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazine (Hydroxano-6-yl)acetamyl)propyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecyl)carbamoyl)-2-methoxy (2-(4-cyano-5-neopentylpyrrolidine-2-carboxamide))

[0119]

[0120] Preparation 3

[0121] (S)-(1-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- [1,4]diazaphen-6-yl)-2-oxo-6,9,12-trioxa-3-azatetradecane-14-yl)carbamate tert-butyl

[0122]

[0123] In a 4 mL vial equipped with a stir bar, (S)-JQ1 acid (10 mg), [5] (7.3 mg), and HATU (10.5 mg) were dissolved in DMF (0.1 mL), followed by the addition of DIPEA (22 µL). The resulting mixture was stirred at 25 °C for 2 h. After the reaction was complete, the mixture was purified directly by silica gel chromatography (elution: DCM / MeOH) to obtain the title compound (17 mg) as a pale yellow powder. ESI-MS: Calculated C 32 H 44 35 ClN6O6S [M+1] + : 675.27, measured value: 675.46.

[0124] Example 7

[0125] QL-MDM2-17

[0126] (3'R,4'S,5'R)-6''-chloro-4'-(3-chloro-2-fluorophenyl)-N-(4-((1-((S)-4-(4-chlorophenyl)-2, 3,9-Trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl)-2-oxo-6, 9,12-Trioxa-3-azatetradecane-14-yl)carbamoyl)phenyl)-2''-oxodispiro[cyclohexane-1,2'-pyrrole] [alkyl-3',3''-dihydroindole]-5'-formamide

[0127]

[0128] (1) In a 4 mL vial equipped with a stir bar, [6] (9 mg) was dissolved in DCM (0.13 mL), followed by dropwise addition of HCl (4 M, in 1,4-dioxane, 0.13 mL). The reaction was stirred overnight at 25 °C, and the solvent was removed. The residue was subjected to the next step of the reaction without further purification. (2) In a 4 mL vial equipped with a stir bar, the residue from the above steps, MI-1061 [7] (8.5 mg) and HATU (6 mg) were dissolved in DMF (0.1 mL), followed by addition of DIPEA (13 µL). The resulting mixture was stirred at 25 °C for 2 h. After the reaction was complete, the solvent was removed. The residue was purified directly by silica gel chromatography (elution: DCM / MeOH, containing 1 v / v NH3, 7 M, in MeOH) to obtain the title compound (11 mg) as a white powder. ESI-MS: Calculated C 57 H 60 35 Cl3FN9O7S [M+1] + : 1138.34, measured value: 1138.52.

[0129] Example 8

[0130] QL-MDM2-18

[0131] (3'R,4'S,5'R)-6''-chloro-4'-(3-chloro-2-fluorophenyl)-N-(4-((6-(4-(3-(2-((S)-4-(4-) (chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl) Acetamidopropylpiperazin-1-ylhexylcarbamoylphenyl-2''-oxodispiro[cyclohexane-1,2'-pyrrole] [alkyl-3',3''-dihydroindole]-5'-formamide

[0132]

[0133] (1) In a 4 mL vial equipped with a stir bar, [3] (52 mg) was dissolved in DCM (0.1 mL), followed by dropwise addition of HCl (4 M, in 1,4-dioxane, 0.36 mL). The reaction was stirred overnight at 25 °C and the solvent was removed. The residue was subjected to the next step of the reaction without further purification. (2) In a 4 mL vial equipped with a stir bar, the residue from the above steps, MI-1061 [7] (42 mg) and HATU (30 mg) were dissolved in DMF (0.22 mL), followed by addition of DIPEA (63 µL). The resulting mixture was then stirred at 25 °C for 2 h. After the reaction was complete, the mixture was purified directly by silica gel chromatography (elution: DCM / MeOH, containing 1 v / v NH3, 7 M, in MeOH) to obtain the title compound (62 mg) as a white powder. ESI-MS: Calculated C 62 H 70 35 Cl3FN 11 O4S [M+1] + : 1188.44, measured value: 1188.56. 1H NMR(500MHz, DMSO-d6) δ 10.59 (s, 1 H), 10.22 (s, 1 H), 8.33 (t, J = 5.5Hz, 1 H), 8.19 (t, J = 5.5Hz, 1 H), 7.81 (d, J = 8.5Hz, 2 H), 7.68 (d, J = 8.5Hz, 2 H), 7.63 (t, J = 6.9Hz, 1 H), 7.51-7.39 (m, 5 H), 7.35 (t, J = 7.5Hz, 1 H), 7.14 (t, J = 8.1Hz, 1 H), 7.04 (dd, J1 = 7.0Hz, J2 = 2.0Hz, 1 H), 6.69 (d, J =1.8Hz, 1 H), 4.76 (d, J = 9.5Hz, 1 H), 4.72-4.64 (m, 1 H), 4.50 (t, J =7.0Hz, 1 H), 3.69 (br, 1 H), 3.26-3.03 (m, 6 H), 2.59 (s, 3 H), 2.40 (s, 3H), 2.45-2.15 (m, 11H), 2.07-2.00 (m, 1H), 1.62 (s, 3H), 1.70-0.78 (m, 20H).

[0134] The following Example 9 was prepared by substantially following the procedures described in Example 8 and using a suitable enantiomer of [3]. A suitable enantiomer of [3] could be prepared by using a suitable enantiomer of JQ-1 acid and following the procedures described in Preparation 1 and Preparation 2.

[0135] Example 9

[0136] (R)-QL-MDM2-18

[0137] (3'R,4'S,5'R)-6''-chloro-4'-(3-chloro-2-fluorophenyl)-N-(4-((6-(4-(3-(2-((R)-4-(4-) (chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl) Acetamidopropylpiperazin-1-ylhexylcarbamoylphenyl-2''-oxodispiro[cyclohexane-1,2'-pyrrole] [alkyl-3',3''-dihydroindole]-5'-formamide

[0138]

[0139] ESI-MS: Calculated value C62H7O35Cl3FN11O4S [M+1]+: 1188.79, Measured value: 1188.56.

[0140] Preparation 4

[0141] (This relates to the following Example 10)

[0142] (S)-(1-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- [1,4]diazaphen-6-yl)-2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl) tert-butyl carbamate

[0143]

[0144] In a 4 mL vial equipped with a stir bar, (S)-JQ1 acid (15 mg), [8] (12 mg), and HATU (14 mg) were dissolved in DMF (0.2 mL), followed by the addition of TEA (26 µL). The resulting mixture was then stirred at 25 °C for 2 h. After the reaction was complete, the mixture was purified directly by silica gel chromatography (elution: DCM / MeOH) to obtain the title compound (14 mg) in oil form. ESI-MS: Calculated C 34 H 48 35 ClN6O6S [M+1] + : 703.30, measured value: 703.42.

[0145] Example 10

[0146] QL-MDM2-19

[0147] (3'R,4'S,5'R)-6''-chloro-4'-(3-chloro-2-fluorophenyl)-N-(4-((1-((S)-4-(4-chlorophenyl)-2, 3,9-Trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl)-2-oxo-7, 10,13-Trioxa-3-azahexadecane-16-yl)carbamoyl)phenyl)-2''-oxodispiro[cyclohexane-1,2'-pyridine] [[3',3''-dihydroindole]-5'-formamide]

[0148]

[0149] (1) In a 4 mL vial equipped with a stir bar, [9] (14 mg) was dissolved in DCM (0.1 mL), followed by dropwise addition of HCl (4 M, in 1,4-dioxane, 0.1 mL). The reaction was stirred overnight at 25 °C and the solvent was removed. The residue was subjected to the next step of the reaction without further purification. (2) In a 4 mL vial equipped with a stir bar, the residue from the above steps, MI-1061 [7] (12 mg) and HATU (8 mg) were dissolved in DMF (0.1 mL), followed by addition of DIPEA (18 μL). The resulting mixture was then stirred at 25 °C for 2 h. After the reaction was complete, the solvent was removed. The residue was purified directly by silica gel chromatography (elution: DCM / MeOH, containing 1 v / v NH3, 7 M, in MeOH) to obtain the title compound (17 mg) as a white powder. ESI-MS: Calculated C 59 H 64 35 Cl3FN9O7S [M+1] + : 1166.37, measured value: 1166.60.

[0150] Example 11

[0151] QL-MDM2-45

[0152] (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-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl) Acetamidopropylpiperazin-1-ylhexylcarbamoylphenyl-2''-oxodispiro[cyclohexane-1,2'-pyrrole] [alkyl-3',3''-dihydroindole]-5'-formamide

[0153]

[0154] (1) In a 4 mL vial equipped with a stir bar, [3] (14 mg) was dissolved in DCM (0.1 mL), followed by dropwise addition of HCl (4 M, in 1,4-dioxane, 0.1 mL). The reaction was stirred overnight at 25 °C and the solvent was removed. The residue was subjected to the next step of the reaction without further purification. (2) DIPEA (13 mg, 0.104 mmol) and HATU (9 mg, 0.022 mmol) were added to a solution of ENT-MI-1061

[10] (12 mg, 0.02 mmol) in DMF (5 mL) 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 Na2SO4 and concentrated. The residue was purified by preparative HPLC (acetonitrile, containing 0.1% NH3·H2O in water, 5% to 95%) to obtain the title compound as a white solid (6.35 mg, 26.5% yield). LC-MS [M+H] + :1188.6. 1H NMR (400MHz, DMSO- d6) δ 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 =8.7Hz, 2H), 7.68 (d, J = 8.8Hz, 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, 2.0Hz, 1H), 6.71– 6.67 (m, 1H), 4.76 (d, J = 9.3Hz, 1H), 4.70 (d, 1H), 4.50 (dd, J = 7.9,6.3Hz, 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, 8.7Hz, 1H).

[0155] The following Example 12 was prepared by substantially following the procedures described in Example 11 and using a suitable enantiomer of [3]. The suitable enantiomer of [3] could be prepared by using a suitable enantiomer of JQ-1 acid and following the procedures described in Preparation 1 and Preparation 2.

[0156] Example 12

[0157] QL-MDM2-47

[0158] (3'S,4'R,5'S)-6''-chloro-4'-(3-chloro-2-fluorophenyl)-N-(4-((6-(4-(3-(2-((R)-4-(4-) (chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl) Acetamidopropylpiperazin-1-ylhexylcarbamoylphenyl-2''-oxodispiro[cyclohexane-1,2'-pyrrole] [alkyl-3',3''-dihydroindole]-5'-formamide

[0159]

[0160] LCMS: [M+H] + =1188.6.

[0161] Preparation 5

[0162] Ent-Brimadellin

[0163] (3'R,3a'R,10a'R)-6-chloro-3'-(3-chloro-2-fluorophenyl)-1'-(cyclopropylmethyl)-6'-methyl-2-oxo 3',3a',10',10a'-tetrahydro-1'H-spiro[dihydroindole-3,2'-pyrrolo[2',3':4,5]pyrrolo[1,2-b]indole [Azazole-7'-formic acid]

[0164]

[0165] Preparations 5 and 6 were made as a racemic mixture (2 g) as outlined in WO 2017 / 060431A1 and were obtained via SFC (column: DAICELCHIRALCEL). ® The title compound was separated by OJ (250*30mm, 10µm); mobile phase A: supercritical CO2; mobile phase B: MeOH (+0.1% 7.0 mol / L ammonia, in MeOH) to give a white solid (750 mg, 75% yield). LC-MS [M+H] + :591.1. 1 H NMR (400MHz, CD3OD) δ 7.77 – 7.63 (m, 1H), 7.58 –7.46 (m, 2H), 7.43 – 7.30 (m, 2H), 7.19 (t, J = 8.0Hz, 1H), 7.03 (dd, J =8.1, 1.9Hz, 1H), 6.66 (d, J = 1.8Hz, 1H), 6.01 (t, J = 8.3Hz, 1H), 5.20 (td,J = 8.0, 2.5Hz, 1H), 4.20 (d, J = 8.4Hz, 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).

[0166] Example 13

[0167] QL-MDM2-48

[0168] (3a'S,10a'S)-6-chloro-3'-(3-chloro-2-fluorophenyl)-N-(6-(4-(3-(2-((S)-4-(4-chlorophenyl)- 2,3,9-Trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl)acetamido) (propyl)piperazin-1-yl)hexyl)-1'-(cyclopropylmethyl)-6'-methyl-2-oxo-3',3a',10',10a'-tetrahydro-1'H- Spiro[dihydroindole-3,2'-pyrrolo[2',3':4,5]pyrrolo[1,2-b]indazole]-7'-formamide

[0169]

[0170] (1) In a 4 mL vial equipped with a stir bar, [3] (41 mg) was dissolved in DCM (0.3 mL), followed by dropwise addition of HCl (4 M, in 1,4-dioxane, 0.3 mL). The reaction was stirred overnight at 25 °C and the solvent was removed. The residue was subjected to the next step of the reaction without further purification. (2) DIPEA (36 mg, 0.28 mmol) and HATU (27 mg, 0.022 mmol) were added to a solution of brimmadeline

[11] (33 mg, 0.06 mmol) in DMF (5 mL) 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 × 2), dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC (acetonitrile, containing 0.1% NH3·H2O in water, 5% to 95%) to obtain the title compound as a white solid (7.72 mg, 11.5% yield). LC-MS [M+H] + :1197.6. 1H NMR (400MHz, DMSO- d6) δ 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 = 8.6Hz, 2H), 7.35 – 7.26 (m, 2H), 7.01 (dd, J =8.0, 1.9Hz, 1H), 6.94 (d, J = 8.6Hz, 1H), 6.63 (d, J = 1.9Hz, 1H), 6.00 –5.94 (m, 1H), 5.08 (dd, J = 10.9, 5.2Hz, 1H), 4.50 (dd, J = 7.9, 6.3Hz, 1H), 4.03 (d, J = 8.4Hz, 1H), 3.66 (dd, J = 16.9, 7.7Hz, 1H), 3.44 – 3.40 (m, 2H), 3.20 (dd, J = 10.4, 7.0Hz, 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, 4.7Hz, 1H), -0.04 (dd,J = 9.8, 4.6Hz, 1H).

[0171] The following Example 14 was prepared by substantially following the procedures described in Example 13 and using a suitable enantiomer of

[11] .

[0172] Example 14

[0173] QL-MDM2-49

[0174] (3a'S,10a'S)-6-chloro-3'-(3-chloro-2-fluorophenyl)-N-(6-(4-(3-(2-((S)-4-(4-chlorophenyl)- 2,3,9-Trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl)acetamido) (propyl)piperazin-1-yl)hexyl)-1'-(cyclopropylmethyl)-6'-methyl-2-oxo-3',3a',10',10a'-tetrahydro-1'H- Spiro[dihydroindole-3,2'-pyrrolo[2',3':4,5]pyrrolo[1,2-b]indazole]-7'-formamide

[0175]

[0176] 6.3 mg, 9.38% yield. LC-MS [M+H] + : 1197.6 1 H NMR (400 MHz, DMSO-d6) δ 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, ۲H), 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).

[0177] Measurement

[0178] Cell lines and cell culture

[0179] All liposarcoma cell lines used in this study were commercially available from Cellosaurus: LPS141 (RRID: CVCL_M823), LPS853 (RRID: CVCL_V415), T449 (RRID: CVCL_M807), and T778 (RRID: CVCL_M808). Cellosaurus was 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 were maintained supplemented with 15% FBS and 1% GLUTAMAX. ™ The A-375, WI-38, and U-2 OS cell lines were purchased from ATCC. A-375 (catalog number CRL-1619) cells were maintained in DMEM medium (Gibco, ThermoFisher Scientific) containing 10% FBS, 1,000 units / mL penicillin, and 1 mg / mL streptomycin (Gibco). WI-38 (catalog number CCL-75) cells were maintained in EMEM medium (Gibco, ThermoFisher Scientific) containing 10% FBS, 1,000 units / mL penicillin, and 1 mg / mL streptomycin (Gibco). U-2 OS (catalog number HTB-96) cells were maintained in McCoy 5A medium (Gibco, ThermoFisher Scientific) containing 10% FBS, 1,000 units / mL penicillin, and 1 mg / mL streptomycin (Gibco). Normal melanocytes were purchased from ThermoFisher (catalog number C0025C, lot / donor number 2291033), maintained in supplemented medium 254 (catalog number M-254-500), and cultured under hypoxic conditions (5% CO2 and 5% O2). All cell lines were periodically screened for mycoplasma.

[0180] Dose response curve

[0181] Cells were 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, and melanocytes = 5000 cells / well. The following day, the standard growth medium was replaced with drug-containing medium. For each cell line, the optimal dose range for each drug was determined and tested across nine half-log doses. Each plate contained 0.1% DMSO as a negative control against growth inhibition. The drug-containing medium was replaced 48 h after initial treatment. The plates were incubated in the drug at 37°C for a total of 96 h or 120 h, and the culture was repeated by adding 20 µL LCELL TITER-GLO... ® The reagent (Promega, catalog number G7570) was added to 100 ml of cell culture medium for lysis. GLOMAX was used. ® An explorer (Promega) plate reader was used to measure luminescence and to calculate growth inhibition relative to DMSO-treated wells. All experiments were performed with biological replicates, and at least three technical replicates were conducted for each condition in a given experiment. The drm R package was used to fit the data to a four-parameter logistic curve using the LL.4() model. IC50 values ​​for the following cell lines were obtained from this curve. 50 and EC 50 The values ​​are illustrated in Tables 1, 2, 3 and 4 below.

[0182] Table 1

[0183]

[0184] Table 2

[0185]

[0186] Table 3

[0187]

[0188] Table 4

[0189]

[0190] Protein blot

[0191] Cell plates were seeded, and the normal growth medium was removed the following day and replaced with DMSO or drug-containing medium. Cells were incubated in the drug-containing medium for 24 h, washed twice with 1x PBS, and then supplemented with fresh HALT protease. ™ Scrape the protein onto ice in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS) containing Thermofisher protein and a phosphatase inhibitor (Thermofisher). Quantify and normalize the protein using the BCA Protein Assay Kit (Thermo No. 23227) according to the manufacturer's specifications. Use FIJI... ® The total signal for each protein was quantified and normalized to the actin loading control. The quantization was fitted to the dose-response curve using the drm R package (fct = LL.4()) and plotted in R.

[0192] Generation of LPS853 P53 knockout cell lines

[0193] The TP53 knockout assay utilized pXPR_044, an integrated CRISPR-Cas9 mediator carrying both mCherry and sgRNA. The following CRISPR sgRNAs were cloned into the mediator: safe harbor 5'-GGCTAAATTCCTCTTATTCA-3'; TP53 start site 5'-TCGACGCTAGGATCTGACTG-3'. LPS853 cells were transfected with 3 µg / mL lentiviral plasmid and 5 µL / mL TransIT-LT1 reagent (MirusBio, MIR2300). The following day, cells were selected using fluorescence-activated cell sorting (Sony, SH800). The gating strategy first involved selecting a live population with forward and side scattering. Untreated cells were used to set the mCherry threshold gate. Cells expressing the highest levels of mCherry (top 50%) were selected. Genomic DNA was isolated and the TP53 start site was amplified using the following primer set: F 5'-CCCAACCCTTGTCCTTACCA-3'; R 5'-CAACATGCAAAGCCCTGTCT-3'. CRISPR-mediated disruption of the amplified region was confirmed by Sanger sequencing using the TIDE tool.

[0194] Pharmacokinetic Measurement

[0195] For pharmacokinetic characterization of QL-MDM2-18 and QL-MDM2-48, 50 mg / kg of the drug was administered to three CD1 mice. QL-MDM2-18 was prepared at 5 mg / mL in 10% DMSO, 90% (10% HP-β-CD, in sterile water) for intraperitoneal (IP) administration. QL-MDM2-48 was prepared at 5 mg / mL in 10% DMSO, 10% Solutol HS 15, 80% (10% HP-β-CD, in sterile water) for IP administration. Approximately 25 μL of blood was collected from the animals via the facial vein and placed in a K2EDTA tube. Immediately after sampling, 20 µL of blood was diluted with 60 µL diH2O and vortexed until thoroughly mixed, then stored at -70°C. Plasma drug concentrations were measured by LC-MS / MS. PK parameters were estimated using a non-compartmental model with WinNonlin 8.2.

[0196] Pharmacokinetic measurements in xenografts derived from patients

[0197] In vivo drug treatment with QL-MDM2-18 was performed in male NSG mice carrying LPS3 PDX tumors injected subcutaneously. Based on good tolerability in toxicity studies, 5 mg / mL of compound QL-MDM2-18 was prepared in 10% Solutol HS 15, 80% (10% HP-β-CD, in sterile water) for intraperitoneal administration at 15 mg / kg QD. Two mice were treated for up to 16 or 20 doses, and three mice were given 10 μL / g of solvent for up to 14 doses. Tumor tissue was harvested by rapid freezing 24 hours after administration of the last dose of drug or solvent. The rapidly frozen samples were post-fixed in 4% formaldehyde at 4°C and subsequently cryoprotected in sucrose solution before refrozen. 10 μM sections were prepared on a cryostat, then permeabilized, blocked, and stained with human-specific CDK4 antibody (catalog number MA5-41178) and BRD4 antibody (catalog number CST63759) diluted 1:250. Samples were imaged on a Leica DMi8 imaging system using a 40x objective lens with oil immersion, and images were processed using the THUNDER computational clearance method. Tumor cells labeled with CDK4 were first selected using QuPath software, and then the number of BRD4-positive tumor cells was quantified.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, said compound having the following formula: ， in: B is selected independently and ; B-aB-b L is an independent connector for hydrocarbons or polyethylene glycol selected from the following: or or ; Where x ranges from 2 to 10, y ranges from 1 to 5, and z ranges from 1 to 5; and I independently selected from: , and I-aI-b Ic The prerequisite is that the compound is not a 。 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein B is... 。 3. The compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein I is... .

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein L is... 。 5. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein x is 2 to 10.

6. The compound of claim 5 or a pharmaceutically acceptable salt thereof, wherein x is 3 to 7.

7. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein L is... or .

8. 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 according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein B is... ; L is selected from , and ; x is 4; y ranges from 1 to 4; z is 3; and I selected , and .

11. The compound according to any one of claims 1, or a pharmaceutically acceptable salt thereof, wherein B is... ; L is selected from and ; y is 3; z is 3; and I am .

12. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from... 、 、 、 、 、 、 、 , and .

13. A pharmaceutical composition comprising a compound according to any one 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, the method comprising administering to a patient in need an effective amount of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12.

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

16. The method according to claim 14 or 15, wherein the cancer is selected from growths, lymphomas, sarcomas, bladder cancer, adenoid cystic carcinoma, glioblastoma, solid tumors, and myeloid tumors.

17. A method for activating p53 production by targeting MDM2 amplification in cancer, the method comprising administering an effective amount of the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof.

18. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein the compound or a pharmaceutically acceptable salt thereof is used in a therapeutic manner.

19. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein the compound or a pharmaceutically acceptable salt thereof is used in the treatment of cancer.

20. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein the compound or a pharmaceutically acceptable salt thereof is used in the manufacture of a medicament for treating cancer.

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