TWI759316B - Combination of a bcl-2 inhibitor and a mcl1 inhibitor, uses and pharmaceutical compositions thereof - Google Patents

Combination of a bcl-2 inhibitor and a mcl1 inhibitor, uses and pharmaceutical compositions thereof Download PDF

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TWI759316B
TWI759316B TW106124599A TW106124599A TWI759316B TW I759316 B TWI759316 B TW I759316B TW 106124599 A TW106124599 A TW 106124599A TW 106124599 A TW106124599 A TW 106124599A TW I759316 B TWI759316 B TW I759316B
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安德魯 魏
多妮亞 穆舟歐德
喬凡娜 普米力歐
安娜 萊蒂西亞 莫雷葛諾
奧莉薇 居內斯特
奧黛莉 克拉伯龍
海科 馬艾克
安薩 哈利洛維奇
戴爾 波特
艾立克 莫利斯
幼真 王
斯納 桑海維
普拉卡什 米斯崔
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法商施維雅藥廠
瑞士商諾華公司
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Abstract

A combination comprising a BCL-2 inhibitor and a MCL1 inhibitor, and compositions and uses thereof.

Description

BCL-2抑制劑及MCL1抑制劑之組合、其用途及醫藥組合物Combination of BCL-2 inhibitor and MCL1 inhibitor, use thereof and pharmaceutical composition

本發明係關於一種BCL-2抑制劑及MCL1抑制劑之組合。本發明亦關於一種該組合在治療癌症中的用途，特定言之，白血病、淋巴瘤、多發性骨髓瘤、神經母細胞瘤及肺癌，且更尤其，急性骨髓白血病、T細胞急性淋巴母細胞白血病、B細胞急性淋巴母細胞白血病、套細胞淋巴瘤、彌漫性大B細胞淋巴瘤及小細胞肺癌。亦提供適用於投與該等組合之醫藥調配物。The present invention relates to a combination of a BCL-2 inhibitor and an MCL1 inhibitor. The invention also relates to the use of a combination in the treatment of cancer, in particular leukemia, lymphoma, multiple myeloma, neuroblastoma and lung cancer, and more particularly acute myeloid leukemia, T cell acute lymphoblastic leukemia , B-cell acute lymphoblastic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma and small cell lung cancer. Pharmaceutical formulations suitable for administration of these combinations are also provided.

細胞凋亡係藉由各種細胞毒性刺激起始之高度調節的細胞死亡路徑，包括致癌壓力及化學治療劑。已表明，逃避細胞凋亡係癌症之特點且多種化學治療劑之療效視固有粒線體路徑的活化而定。BCL-2家族蛋白質之3個相異子群控制固有細胞凋亡路徑： (i) 僅促凋亡BH3 (BCL-2同源3)蛋白質；(ii) 促存活成員，諸如BCL-2自身、BCL-XL、Bcl-w、MCL1及BCL-2a1；及(iii) 促凋亡效應子蛋白質BAX及BAK (Czabotar等人,Nature Reviews Molecular cell biology 2014第15卷:49-63)。在多種癌症中觀測到BCL-2家族之抗凋亡成員的過度表現，特定言之在血液惡性病中，諸如套細胞淋巴瘤(MCL)、濾泡性淋巴瘤/彌漫性大B細胞淋巴瘤(FL/D)及多發性骨髓瘤中(Adams及CoryOncogene 2007第26卷:1324-1337)。近來研發之BH3模擬藥物(諸如ABT-199及ABT-263)對抗凋亡蛋白質BCL-2、BCL-XL及Bcl-w的藥理學抑制成為治療性策略，以誘使細胞凋亡及導致癌症中的腫瘤消退(Zhang等人,Drug Resist Updat 2007第10卷(6):207-17)。儘管如此，已觀測且研究此等藥物的耐受性機制(Choudhary GS等人,Cell Death and Disease 2015第6卷，e1593；doi:10.1038/cddis.2014.525)。 急性骨髓白血病(AML)係由造血幹細胞之純系轉型引起的快速致命血癌，其導致正常骨髓功能的麻痹及因嚴重全部血球減少症之併發症所致的死亡。AML占所有成人白血病之25%，其中最高發病率發生在美國、澳大利亞及歐洲(WHO. GLOBOCAN 2012，2012年世界範圍內估算癌症發病率、死亡率及患病率，國際癌症研究機構)。在全球範圍內，每年大致有88,000個新診斷案例。AML維持所有白血病之最低存活率，僅24%預期有5年的存活期。儘管40多年前早已構思用於AML之標準療法(阿糖胞苷(cytarabine)結合蒽環黴素(anthracycline))，但此疾病之成功靶向療法的引入仍為難以實現的目標。此外，對於患有AML的患者而言，仍需要一種無化學療法的治療選擇。自此疾病實現發展成為多純系層次，AML之靶向療法的構思已受妨礙，其中白血病次純系之快速過度生長成為抗藥性及疾病復發的主要原因(Ding L等人,Nature 2012 481:506-10)。最新臨床研究已表明BCL-2抑制劑在治療AML中的療效(Konopleva M等人,American Society of Hematology 2014:118)。儘管此等抑制劑臨床上起作用，但很可能將需要靶向其他BCL-2家族成員以增強對AML的整體療效。除了BCL-2之外，MCL1亦被鑑定為AML中細胞存活之重要的調節因子(Glaser SP等人,Genes & development 2012 26:120-5)。 多發性骨髓瘤(MM)係罕見且不可治癒的疾病，其特徵為骨髓(BM)中之純系漿細胞的積聚，且占所有血液惡性病之10%。在歐洲，每年大致有27,800個新案例。近年來，由於可利用包括硼替佐米(bortezomib)及來那度胺(lenalidomide)之新藥劑，及自體幹細胞移植(ASCT)，存活率得到提高。然而，對此等新藥劑的反應常常係不持久的，且其成為需要新治療的一種跡象，尤其對於復發/難治癒患者以及具有不利預後(不利細胞遺傳學特徵)之患者而言。近來研究表明BCL-2抑制劑在多發性骨髓瘤患者之子群中具有可行的活性(Touzeau C, Dousset C, Le Gouill S等人,Leukemia . 2014; 28(1):210-212)。MCL1亦已鑑定為多發性骨髓瘤中細胞存活之重要的調節因子(Derenne S, Monia B, Dean NM等人,Blood . 2002;100(1):194-199；Zhang B, Gojo I, Fenton RG.Blood . 2002;99(6):1885-1893)。 彌漫性大B細胞淋巴瘤(DLBCL)係非霍奇金淋巴瘤(Non-Hodgkin Lymphoma)之最常見類型(25-35%)，每年有24 000個新患者。DLBCL係具有超過12種次型之異質疾病，包括雙重打擊/MYC移位、活化B細胞(ABC)以及生發中心B細胞(GCB)。現代免疫化學療法(R-CHOP)治癒大致60%之患有DLBCL的患者，但對於其餘的40%而言，存在極少的治療性選擇且預後係不良的。因此，存在較高之增加諸如ABC次型(DLBCL之35%)之高危DLBCL的治癒比率及臨床結果的醫藥需要，其展示促存活性NF-κB路徑的組成性活化。 神經母細胞瘤(NB)係嬰兒及兒童中最常見的顱骨外實心腫瘤，代表當前分級為較低風險，中度風險或較高風險之所有兒童腫瘤的8%-10%。其大致占兒科群體中所有癌症相關死亡的15%。15歲年齡以下之兒童的NB發病率為每百萬10.2個案例，且每年報導接近500個新案例。診斷之中值年齡係22個月。在過去的30年間，患有NB之患者的結果穩定地改善，其中5年的存活率自52%上升至74%。然而，經預測高風險組中50%至60%之患者經歷復發，且由此發現其死亡率僅適度降低。復發的中值時間係13.2個月，且73%之復發的彼等時間係18個月或大於18個月。總而言之，NB的整體存活率仍為極其深不可測的(約20%為5年)，即使有更多積極療法(Colon及Chung，Adv Pediatr 2013 58:297-311)。主體治療由化學療法、手術割除及/或放射線療法組成。然而，多種攻擊性NB已發展為對化學治療劑具有耐受性，使復發可能性變得相當高(Pinto等人,J Clin Oncol 201533:3008-11)。視風險層級而定之NB的標準護理常常係卡鉑(carboplatin)、順鉑環磷醯胺(cisplatin cyclophosphamide)、小紅莓(doxorubicin)、依託泊苷(etoposide)、細胞介素(GM-CSF及IL2)以及長春新鹼(vincristine)。在對化學療法之初始反應之後的復發係治療失效的主要原因，尤其在高風險之NB中。 化學抗性可衍生自促存活性BCL-2蛋白質(例如BCL-2及MCL1蛋白質)之活化。NB表現較高水準之BCL-2及MCL1以及較低水準之BCL-XL。BCL-2之抑制使細胞敏感至死亡且誘使NB腫瘤活體內消退(Ham等人,Cancer Cell 29:159-172)。BCL-2及MCL1之拮抗作用恢復對較高風險NB的化學療法(Lestini等人,Cancer Biol Ther 2009 8:1587-1595；Tanos 等人,BMC Cancer 2016 16:97)。因此，將BCL-2及MCL1抑制劑組合於未經治療或抗性患者中係極其合理的。 本發明提供一種BCL-2抑制劑及MCL1抑制劑之新穎組合。結果展示，隨著靶向BCL-2及MCL1之有效小分子的發展，高度協同促凋亡活性揭露於原發性人類AML樣品(圖2A及圖17)以及AML(圖9、圖13及圖14)、多發性骨髓瘤(實例4)、淋巴瘤(圖4及圖12)、神經母細胞瘤(圖10)、T-ALL、B-ALL細胞株(圖11)、及小細胞肺癌細胞株(圖15(a)至圖15(e))中。吾人亦展示靶向活體內之組合的BCL-2及MCL1在以耐受劑量時對小鼠及大鼠內的AML及淋巴瘤異種移植模型係有效的(圖2、圖5、圖6、圖7、圖8及圖16)，且顯著增加了AML的復發時間(圖2B及圖2C)。此外，在細胞群落檢定中，吾人表明BCL-2+MCL1靶向對致白血病細胞係尤其有毒的，但對正常造血幹細胞非如此(圖3)，此與之前小鼠的MCL1基因靶向實驗形成對比。在此等有效及選擇性抑制劑研發之前，靶向BCL-2及MCL1兩者的可行性仍為不確定的。先前特定譜系缺失模型指示由MCL1之長期切除造成之以下各者的潛在性風險：心肌(Wang X等人,Genes & development . 2013;27(12):1351-1364；Thomas RL等人,Genes & development . 2013;27(12):1365-1377)，顆粒球/造血(Opferman J等人,Science ' s STKE . 2005;307(5712):1101；Dzhagalov I等人,Blood . 2007;109(4):1620-1626；Steimer DA等人,Blood . 2009;113(12):2805-2815)，胸腺細胞(Dunkle A等人,Cell Death & Differentiation . 2010;17(6):994-1002)，神經元(Arbour N等人,Journal of Neuroscience . 2008;28(24):6068-6078)以及肝功能(Hikita H等人,Hepatology . 2009;50(4):1217-1226；Vick B等人,Hepatology . 2009;49(2):627-636)。即使有此等擔憂，近來，每週一次、每週兩次及甚至每天一次(在5個連續日期間)靜脈內遞送MCL1之新型有效的短時間作用的藥理學抑制劑仍展示良好的耐受性且對抵抗活體內一系列癌症起作用，包括AML (Kotschy A等人,Nature . 2016;538(7626):477-482；WO 2015/097123)。MCL1蛋白質之短暫的半衰期可留出足夠的時間以用於其在關鍵器官中再生，由此使得對MCL1抑制劑短期暴露具有生理學耐受性(Yang T等人,Journal of cellular physiology . 1996;166(3):523-536)。迄今為止，仿效類似藥物效應之BCL-2及MCL1的脈衝式抑制尚無可能使用基因工程改造途徑達成。根據本發明之使用BCL-2及MCL1抑制劑的研究提供以下概念驗證論證：對此等藥物的間歇性暴露可足以觸發細胞凋亡及高度敏感疾病當中(諸如AML)的臨床反應，同時對主要器官系統無併發毒性。 在靶向兩者抗凋亡蛋白質時，活體外及活體內靶向BCL-2及MCL1兩者的協同效應及對正常骨髓產生的無毒性僅經由有效小分子抑制劑之組合來表明。此等態樣並非由基因靶向實驗的結果預料，其預測造血幹細胞會不太耐受MCL1缺失。Apoptosis is a highly regulated cell death pathway initiated by various cytotoxic stimuli, including oncogenic stressors and chemotherapeutic agents. It has been shown that evasion of apoptosis is a hallmark of cancer and the efficacy of various chemotherapeutic agents is contingent on activation of the intrinsic mitochondrial pathway. Three distinct subgroups of BCL-2 family proteins control the intrinsic apoptotic pathway: (i) only pro-apoptotic BH3 (BCL-2 homolog 3) proteins; (ii) pro-survival members such as BCL-2 itself, BCL-XL, Bcl-w, MCL1 and BCL-2a1; and (iii) the pro-apoptotic effector proteins BAX and BAK (Czabotar et al., Nature Reviews Molecular cell biology 2014 Vol. 15:49-63). Overexpression of anti-apoptotic members of the BCL-2 family has been observed in a variety of cancers, specifically in hematological malignancies such as mantle cell lymphoma (MCL), follicular lymphoma/diffuse large B-cell lymphoma (FL/D) and in multiple myeloma (Adams and Cory Oncogene 2007 Vol 26: 1324-1337). Pharmacological inhibition of the anti-apoptotic proteins BCL-2, BCL-XL and Bcl-w by recently developed BH3 mimetic drugs such as ABT-199 and ABT-263 has emerged as a therapeutic strategy to induce apoptosis and lead to cancer of tumor regression (Zhang et al., Drug Resist Updat 2007 Vol. 10(6):207-17). Nonetheless, mechanisms of tolerance to these drugs have been observed and studied (Choudhary GS et al., Cell Death and Disease 2015 Vol. 6, e1593; doi: 10.1038/cddis.2014.525). Acute myeloid leukemia (AML) is a rapidly lethal blood cancer caused by pure line transformation of hematopoietic stem cells, which results in paralysis of normal bone marrow function and death from complications of severe pancytopenia. AML accounts for 25% of all adult leukemias, with the highest incidence occurring in the United States, Australia and Europe (WHO. GLOBOCAN 2012, 2012 Worldwide Estimates of Cancer Incidence, Mortality and Prevalence, International Agency for Research on Cancer). Globally, there are roughly 88,000 new diagnoses each year. AML maintains the lowest survival rate of all leukemias, with only 24% expected to survive 5 years. Although the standard therapy for AML (cytarabine combined with anthracycline) was conceived more than 40 years ago, the introduction of successful targeted therapy for this disease remains an elusive goal. In addition, there remains a need for a chemotherapy-free treatment option for patients with AML. Since then the disease has developed into a polyclonal hierarchy, the concept of targeted therapy for AML has been hampered, in which rapid overgrowth of hypoclonal leukemia is a major cause of drug resistance and disease relapse (Ding L et al., Nature 2012 481:506- 10). Recent clinical studies have demonstrated the efficacy of BCL-2 inhibitors in the treatment of AML (Konopleva M et al., American Society of Hematology 2014:118). Although these inhibitors work clinically, it is likely that targeting other BCL-2 family members will be required to enhance overall efficacy in AML. In addition to BCL-2, MCL1 has also been identified as an important regulator of cell survival in AML (Glaser SP et al., Genes & development 2012 26:120-5). Multiple myeloma (MM) is a rare and incurable disease characterized by the accumulation of pure-line plasma cells in the bone marrow (BM) and accounts for 10% of all hematological malignancies. In Europe, there are roughly 27,800 new cases each year. In recent years, survival rates have improved due to the availability of new agents including bortezomib and lenalidomide, and autologous stem cell transplantation (ASCT). However, the response to these new agents is often not durable, and it is a sign that new treatments are needed, especially for relapsed/refractory patients and those with unfavorable prognosis (unfavorable cytogenetic profile). Recent studies have shown that BCL-2 inhibitors have viable activity in a subgroup of patients with multiple myeloma (Touzeau C, Dousset C, Le Gouill S et al, Leukemia . 2014; 28(1):210-212). MCL1 has also been identified as an important regulator of cell survival in multiple myeloma (Derenne S, Monia B, Dean NM et al, Blood . 2002;100(1):194-199; Zhang B, Gojo I, Fenton RG . Blood . 2002;99(6):1885-1893). Diffuse large B-cell lymphoma (DLBCL) is the most common type (25-35%) of Non-Hodgkin Lymphoma, with 24,000 new patients per year. The DLBCL lineage has a heterogeneous disease with more than 12 subtypes, including double hit/MYC translocation, activated B cells (ABC), and germinal center B cells (GCB). Modern immunochemotherapy (R-CHOP) cures approximately 60% of patients with DLBCL, but for the remaining 40%, few therapeutic options exist and the prognosis is poor. Therefore, there is a high medical need to increase the cure rate and clinical outcome of high-risk DLBCL such as the ABC subtype (35% of DLBCL), which exhibit constitutive activation of the pro-survival NF-κB pathway. Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, representing 8%-10% of all childhood tumors currently classified as low, intermediate, or high risk. It accounts for roughly 15% of all cancer-related deaths in the pediatric population. The incidence of NB in children under the age of 15 is 10.2 cases per million, and nearly 500 new cases are reported each year. The median age at diagnosis was 22 months. Outcomes for patients with NB have steadily improved over the past 30 years, with the 5-year survival rate increasing from 52% to 74%. However, 50% to 60% of patients in the high risk group are predicted to experience relapse and thus find only a modest reduction in mortality. The median time to relapse was 13.2 months, and 73% of the time to relapse was 18 months or greater. In conclusion, the overall survival rate for NB remains extremely unfathomable (about 20% at 5 years), even with more aggressive therapies (Colon & Chung, Adv Pediatr 2013 58:297-311). The main treatment consisted of chemotherapy, surgical excision and/or radiation therapy. However, many aggressive NBs have developed resistance to chemotherapeutic agents, making the likelihood of relapse quite high (Pinto et al, J Clin Oncol 2015 33:3008-11). The standard of care for NB, depending on the level of risk, is often carboplatin, cisplatin cyclophosphamide, doxorubicin, etoposide, GM-CSF and IL2) and vincristine. Relapse after the initial response to chemotherapy is a major cause of treatment failure, especially in high-risk NB. Chemoresistance can be derived from the activation of pro-survival BCL-2 proteins, such as BCL-2 and MCL1 proteins. NB performed higher levels of BCL-2 and MCL1 and lower levels of BCL-XL. Inhibition of BCL-2 sensitized cells to death and induced regression of NB tumors in vivo (Ham et al., Cancer Cell 29:159-172). Antagonism of BCL-2 and MCL1 restores chemotherapy to higher risk NB (Lestini et al, Cancer Biol Ther 2009 8:1587-1595; Tanos et al, BMC Cancer 2016 16:97). Therefore, it is extremely reasonable to combine BCL-2 and MCL1 inhibitors in treatment-naive or resistant patients. The present invention provides a novel combination of BCL-2 inhibitor and MCL1 inhibitor. The results show that with the development of potent small molecules targeting BCL-2 and MCL1, highly synergistic pro-apoptotic activity was revealed in primary human AML samples (FIG. 2A and FIG. 17) and AML (FIG. 9, FIG. 13 and FIG. 14), multiple myeloma (Example 4), lymphoma (Figure 4 and Figure 12), neuroblastoma (Figure 10), T-ALL, B-ALL cell lines (Figure 11), and small cell lung cancer cells strains (Fig. 15(a) to Fig. 15(e)). We have also shown that BCL-2 and MCL1 targeting the combination in vivo are effective at tolerated doses in AML and lymphoma xenograft model lines in mice and rats (Figure 2, Figure 5, Figure 6, Figure 2). 7, Figure 8 and Figure 16), and significantly increased the time to relapse of AML (Figure 2B and Figure 2C). Furthermore, in a cell colony assay, we showed that BCL-2+MCL1 targeting was particularly toxic to leukemic cell lines, but not normal hematopoietic stem cells (Fig. 3), in line with previous experiments with MCL1 gene targeting in mice Compared. Until such potent and selective inhibitors are developed, the feasibility of targeting both BCL-2 and MCL1 remains uncertain. A previous model of specific lineage deletion indicated a potential risk of the following from long-term resection of MCL1: Myocardium (Wang X et al, Genes & development . 2013;27(12):1351-1364; Thomas RL et al, Genes & development . development . 2013;27(12):1365-1377), Granules/Hematopoiesis ( Opferman J et al, Science 's STKE . 2005;307(5712):1101; Dzhagalov I et al, Blood . 2007;109(4 ): 1620-1626; Steimer DA et al, Blood . 2009;113(12):2805-2815), thymocytes (Dunkle A et al, Cell Death & Differentiation . 2010;17(6):994-1002), Neurons (Arbour N et al., Journal of Neuroscience . 2008;28(24):6068-6078) and liver function (Hikita H et al., Hepatology . 2009;50(4):1217-1226; Vick B et al., Hepatology . 2009;49(2):627-636). Despite these concerns, novel potent short-acting pharmacological inhibitors of MCL1 have recently been shown to be well tolerated by intravenous delivery of weekly, twice-weekly, and even daily (during 5 consecutive days) Sexual and active against a range of cancers in vivo, including AML (Kotschy A et al., Nature . 2016;538(7626):477-482; WO 2015/097123). The short half-life of the MCL1 protein allows sufficient time for its regeneration in key organs, thereby rendering physiological tolerance to short-term exposure to MCL1 inhibitors (Yang T et al., Journal of cellular physiology . 1996; 166(3):523-536). To date, pulsatile inhibition of BCL-2 and MCL1 mimicking drug-like effects has not been possible using a genetically engineered approach. Studies using BCL-2 and MCL1 inhibitors in accordance with the present invention provide proof-of-concept demonstrations that intermittent exposure to these drugs may be sufficient to trigger apoptosis and clinical responses in highly sensitive diseases, such as AML, while inhibiting major Organ system without concurrent toxicity. In targeting both anti-apoptotic proteins, the synergistic effect of targeting both BCL-2 and MCL1 in vitro and in vivo and the lack of toxicity to normal bone marrow production were only demonstrated by a combination of potent small molecule inhibitors. These aspects were not expected from the results of gene targeting experiments, which predicted that hematopoietic stem cells would be less resistant to MCL1 deletion.

本發明係關於一種組合，其包含(a)式(I)之BCL-2抑制劑：

Figure 02_image001
其中： ¨ X及Y表示碳原子或氮原子，應理解其可不同時表示兩個碳原子或兩個氮原子， ¨ A1 及A2 與其攜帶之原子一起形成視情況經取代之由5、6或7個環成員構成的芳族或非芳族雜環Het，除了表示為X或Y的氮之外，其亦可含有1至3個獨立地選自氧、硫及氮之雜原子，應理解所討論之氮可經表示氫原子、直鏈或分支鏈(C1 -C6 )烷基、或基團-C(O)-O-Alk的基團取代，其中Alk為直鏈或分支鏈(C1 -C6 )烷基，或A1 及A2 彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )多鹵烷基、直鏈或分支鏈(C1 -C6 )烷基或環烷基， ¨ T表示氫原子、視情況經1至3個鹵素原子取代之直鏈或分支鏈(C1 -C6 )烷基、基團(C1 -C4 )烷基-NR1 R2 或基團(C1 -C4 )烷基-OR6 ， ¨ R1 及R2 彼此獨立地表示氫原子或直鏈或分支鏈(C1 -C6 )烷基， 或R1 及R2 與攜帶其之氮原子形成雜環烷基， ¨ R3 表示直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、環烷基、(C3 -C10 )環烷基-(C1 -C6 )烷基，其中烷基部分係直鏈或分支鏈雜環烷基、芳基或雜芳基， 應理解，前述基團之碳原子或其可能的取代基之碳原子中之一或多者可氘化， ¨ R4 表示芳基、雜芳基、環烷基或直鏈或分支鏈(C1 -C6 )烷基，應理解前述基團之碳原子或其可能的取代基之碳原子中之一或多者可氘化， ¨ R5 表示氫或鹵素原子、直鏈或分支鏈(C1 -C6 )烷基或直鏈或分支鏈(C1 -C6 )烷氧基， ¨ R6 表示氫原子或直鏈或分支鏈(C1 -C6 )烷基， ¨ Ra 、Rb 、Rc 及Rd 彼此各獨立地表示R7 、鹵素原子、直鏈或分支鏈(C1 -C6 )烷氧基、羥基、直鏈或分支鏈(C1 -C6 )多鹵烷基、三氟甲氧基、-NR7 R7 '、硝基、R7 -CO-(C0 -C6 )烷基-、R7 -CO-NH-(C0 -C6 )烷基-、NR7 R7 '-CO-(C0 -C6 )烷基-、NR7 R7 '-CO-(C0 -C6 )烷基-O-、R7 -SO2 -NH-(C0 -C6 )烷基-、R7 -NH-CO-NH-(C0 -C6 )烷基-、R7 -O-CO-NH-(C0 -C6 )烷基-、雜環烷基或對(Ra 、Rb )、(Rb 、Rc )或(Rc 、Rd )中之一者的取代基與攜帶其的碳原子一起形成由5至7個環成員構成的環，其可含有1至2個選自氧及硫之雜原子，亦應理解上文定義之環的一或多個碳原子可氘化或經1至3個選自鹵素及直鏈或分支鏈(C1 -C6 )烷基的基團取代， ¨ R7 及R7 '各彼此獨立地表示氫、直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、芳基或雜芳基，或R7 及R7 '與攜帶其之氮原子一起形成由5至7個環成員構成之雜環， 應理解，當式(I)之化合物含有羥基時，後者可視情況轉化為以下基團中之一者：-OPO(OM)(OM')、-OPO(OM)(O- M1 + )、-OPO(O- M1 + )(O- M2 + )、-OPO(O- )(O- )M3 2 + 、-OPO(OM)(O[CH2 CH2 O]n CH3 )或-OPO(O- M1 + )(O[CH2 CH2 O]n CH3 )，其中M及M'彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、環烷基或雜環烷基，兩者皆由5至6環成員構成，同時M1 + 及M2 + 彼此獨立地表示醫藥學上可接受之單價陽離子，M3 2 + 表示醫藥學上可接受之二價陽離子，且n為1至5之整數， 應理解： - 「芳基」意指苯基、萘基、聯苯基或茚基， - 「雜芳基」意指任何由5至10個環成員構成之單環基或雙環基，其具有至少一個芳族部分且含有1至4個選自氧、硫及氮(包括四級氮)之雜原子， - 「環烷基」意指任何含有3至10個環成員之單環或雙環非芳族碳環基， - 「雜環烷基」意指任何由3至10個環成員構成，且含有1至3個選自氧、硫、SO、SO2 及氮之雜原子的單環或雙環非芳族稠合基團或螺基， 有可能如此定義之芳基、雜芳基、環烷基及雜環烷基，及基團烷基、烯基、炔基及烷氧基由1至3個選自以下各者之基團取代：視情況經羥基、嗎啉、3-3-二氟哌啶或3-3-二氟吡咯啶取代之直鏈或分支鏈(C1 -C6 )烷基；(C3 -C6 )螺環；視情況經嗎啉取代之直鏈或分支鏈(C1 -C6 )烷氧基；(C1 -C6 )烷基-S-；羥基；側氧基；N -氧化物；硝基；氰基；-COOR'；-OCOR'；NR'R"；直鏈或分支鏈(C1 -C6 )多鹵烷基；三氟甲氧基；(C1 -C6 )烷基磺醯基；鹵素；視情況經一或多個鹵素取代之芳基；雜芳基；芳氧基；芳基硫基；環烷基；視情況經一或多個鹵素原子或烷基取代之雜環烷基，其中R'及R"彼此獨立地表示氫原子或視情況經甲氧基取代之直鏈或分支鏈(C1 -C6 )烷基， 式(I)中定義之Het基團可能經1至3個選自直鏈或分支鏈(C1 -C6 )烷基、羥基、直鏈或分支鏈(C1 -C6 )烷氧基、NR1 'R1 "及鹵素之基團取代，應理解R1 '及R1 "如關於上文提及之基團R'及R"所定義， 或其對映異構體、非對映異構體，或其與醫藥學上可接受之酸或鹼的加成鹽， 及(b) MCL1抑制劑。 式(I)之該等化合物、其合成、其在治療癌症中的用途及其醫藥調配物描述於WO 2013/110890、WO 2015/011397、WO 2015/011399及WO 2015/011400中，其內容以引用的方式併入。 在某些實施例中，MCL1抑制劑係選自A-1210477 (Cell Death and Disease 2015 6, e1590; doi:10.1038/cddis.2014.561)及描述於WO 2015/097123、WO 2016/207216、WO 2016/207217、WO 2016/207225、WO 2016/207226或WO 2016/033486中的化合物，其內容以引用的方式併入。 本發明亦關於一種包含(a) BCL-2抑制劑及(b)式(II)之MCL1抑制劑的組合：
Figure 02_image004
其中： ¨ A表示直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、直鏈或分支鏈(C1 -C6 )烷氧基、-S-(C1 -C6 )烷基、直鏈或分支鏈(C1 -C6 )多鹵烷基、羥基、氰基、-NW10 W10 '、-Cy6 或鹵素原子， ¨ W1 、W2 、W3 、W4 及W5 彼此獨立地表示氫原子、鹵素原子、直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、直鏈或分支鏈(C1 -C6 )多鹵烷基、羥基、直鏈或分支鏈(C1 -C6 )烷氧基、-S-(C1 -C6 )烷基、氰基、硝基、-烷基(C0 -C6 )-NW8 W8 '、-O-Cy1 、-烷基(C0 -C6 )-Cy1 、-烯基(C2 -C6 )-Cy1 、-炔基(C2 -C6 )-Cy1 、-O-烷基(C1 -C6 )-W9 、-C(O)-OW8 、-O-C(O)-W8 、-C(O)-NW8 W8 '、-NW8 -C(O)-W8 '、-NW8 -C(O)-OW8 '、-烷基(C1 -C6 )-NW8 -C(O)-W8 '、-SO2 -NW8 W8 '、-SO2 -烷基(C1 -C6 )，或當接枝至兩個鄰近碳原子上時，對(W1 、W2 )、(W2 、W3 )、(W1 、W3 )、(W4 、W5 )中之一者的取代基與攜帶其的碳原子一起形成由5至7個環成員構成的芳族或非芳族環，其可含有1至3個選自氧、硫及氮之雜原子，應理解所得環可經選自直鏈或分支鏈(C1 -C6 )烷基、-NW10 W10 '、-烷基(C0 -C6 )-Cy1 或側氧基之基團取代， ¨ X'表示碳原子或氮原子， ¨ W6 表示氫、直鏈或分支鏈(C1 -C8 )烷基、芳基、雜芳基、芳基烷基(C1 -C6 )基團、雜芳基烷基(C1 -C6 )基團， ¨ W7 表示直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、-Cy3 、-烷基(C1 -C6 )-Cy3 、-烯基(C2 -C6 )-Cy3 、-炔基(C2 -C6 )-Cy3 、-Cy3 -Cy4 、-炔基(C2 -C6 )-O-Cy3 、-Cy3 -烷基(C0 -C6 )-O-烷基(C0 -C6 )-Cy4 、鹵素原子、氰基、-C(O)-W11 或-C(O)-NW11 W11 '， ¨ W8 及W8 '彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )烷基或-烷基(C0 -C6 )-Cy1 ，或(W8 、W8 ')與攜帶其之氮原子一起形成由5至7個環成員構成的芳族或非芳族環，除氮原子之外，其亦可含有1至3個選自氧、硫及氮之雜原子，應理解所討論之氮可經表示氫原子、或直鏈或分支鏈(C1 -C6 )烷基的基團取代，且應理解可能之取代基的一或多個碳原子可氘化， ¨ W9 表示-Cy1 、-Cy1 -烷基(C0 -C6 )-Cy2 、-Cy1 -烷基(C0 -C6 )-O-烷基(C0 -C6 )-Cy2 、-Cy1 -烷基(C0 -C6 )-NW8 -烷基(C0 -C6 )-Cy2 、-Cy1 -Cy2 -O-烷基(C0 -C6 )-Cy5 、-C(O)-NW8 W8 '、-NW8 W8 '、-OW8 、-NW8 -C(O)-W8 '、-O-烷基(C1 -C6 )-OW8 、-SO2 -W8 、-C(O)-OW8 、-NH-C(O)-NH-W8 、
Figure 02_image006
、
Figure 02_image008
或
Figure 02_image010
，如此定義之銨有可能以兩性離子的形式存在或具有單價陰離子相對離子， ¨ W10 、W10 '、W11 及W11 '彼此獨立地表示氫原子或直鏈或分支鏈(C1 -C6 )烷基， ¨ W12 表示氫或羥基， ¨ W13 表示氫原子或直鏈或分支鏈(C1 -C6 )烷基， ¨ W14 表示-O-P(O)(O- )(O- )基團、-O-P(O)(O- )(OW16 )基團、-O-P(O)(OW16 )(OW16 ')基團、-O-SO2 -O- 基團、-O-SO2 -OW16 基團、-Cy7 、-O-C(O)-W15 基團、-O-C(O)-OW15 基團或-O-C(O)-NW15 W15 '基團， ¨ W15 及W15 '彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )烷基或直鏈或分支鏈胺基(C1 -C6 )烷基， ¨ W16 及W16 '彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )烷基或芳基烷基(C1 -C6 )基團， ¨ Cy1 、Cy2 、Cy3 、Cy4 、Cy5 、Cy6 及Cy7 彼此獨立地表示環烷基、雜環烷基、芳基或雜芳基， ¨ n為等於0或1之整數， 應理解： - 「芳基」意指苯基、萘基、聯苯基、二氫茚基或茚基， - 「雜芳基」意謂任何由5至10個環成員構成之單環基或雙環基，其具有至少一個芳族部分且含有1至3個選自氧、硫及氮之雜原子， - 「環烷基」意指任何含有3至10個環成員之單環或雙環非芳族碳環基， - 「雜環烷基」意指任何含有3至10個環成員且含有1至3個選自氧、硫及氮之雜原子的單環或雙環非芳族碳環基，其可包括稠合、橋聯或螺環系統， 有可能如此定義之芳基、雜芳基、環烷基及雜環烷基，及烷基、烯基、炔基、烷氧基經1至4個選自以下各者之基團取代：直鏈或分支鏈(C1 -C6 )烷基，其可由表示直鏈或分支鏈(C1 -C6 )烷氧基之基團取代，該直鏈或分支鏈(C1 -C6 )烷氧基可由直鏈或分支鏈(C1 -C6 )烷氧基、直鏈或分支鏈(C1 -C6 )多鹵烷基、羥基、鹵素、側氧基、-NW'W"、-O-C(O)-W'或-CO-NW'W"取代；直鏈或分支鏈(C2 -C6 )烯基；可由表示直鏈或分支鏈(C1 -C6 )烷氧基之基團取代的直鏈或分支鏈(C2 -C6 )炔基；可由表示直鏈或分支鏈(C1 -C6 )烷氧基、直鏈或分支鏈(C1 -C6 )多鹵烷基、直鏈或分支鏈(C2 -C6 )炔基、-NW'W"或羥基之基團取代的直鏈或分支鏈(C1 -C6 )烷氧基；可由表示直鏈或分支鏈(C1 -C6 )烷氧基之基團取代的(C1 -C6 )烷基-S-；羥基；側氧基；N -氧化物；硝基；氰基；-C(O)-OW'；-O-C(O)-W'；-CO-NW'W"；-NW'W"；-(C=NW')-OW"；直鏈或分支鏈(C1 -C6 )多鹵烷基；三氟甲氧基；或鹵素； 應理解W'及W"彼此獨立地表示氫原子或可經表示直鏈或分支鏈(C1 -C6 )烷氧基之基團取代的直鏈或分支鏈(C1 -C6 )烷基；且應理解前述可能的取代基之一或多個碳原子可氘化， 其對映異構體、非對映異構體或構型異構體，或其與醫藥學上可接受之酸或鹼的加成鹽。 式(II)之該等化合物、其合成、其在治療癌症中的用途及其醫藥調配物描述於WO 2015/097123中，其內容以引用的方式併入。 在某些實施例中，BCL-2抑制劑係選自以下化合物： 4-(4-{[2-(4-氯苯基)-4,4-二甲基環己-1-烯-1-基]甲基}哌嗪-1-基)-N -[(3-硝基-4-{[(噁烷-4-基)甲基]胺基}苯基)碸基]-2-[(1H -吡咯并[2,3-b ]吡啶-5-基)氧基]苯甲醯胺(維納妥拉(venetoclax)或ABT-199)；4-(4-{[2-(4-氯苯基)-5,5-二甲基環己-1-烯-1-基]甲基}哌嗪-1-基)-N -(4-{[(2R )-4-(嗎啉-4-基)-1-(苯基硫基)丁-2-基]胺基}-3-(三氟甲磺醯基)苯磺醯基]苯甲醯胺(利妥昔(navitoclax)或ABT-263)；奧利默森(oblimersen) (G3139)；奧巴克拉(obatoclax) (GX15-070)；HA14-1；(±)-棉籽酚(BL-193)；(-)-棉籽酚(AT-101)；阿樸棉籽酚(apogossypol)；TW-37；抗微素(antimycin) A、ABT-737 (Oltersdorf T等人,Nature 2005年6月2日;435(7042):677-81)，及描述於WO 2013/110890、WO 2015/011397、WO 2015/011399及WO 2015/011400中的化合物，其內容以引用的方式併入。 根據本發明之第一態樣，提供一種組合，其包含： (a) 如本文所描述之式(I)的BCL-2抑制劑，及 (b) 如本文所描述之式(II)的MCL1抑制劑。 在另一實施例中，本發明提供一種組合，其包含： (a) 化合物1：N -(4-羥苯基)-3-{6-[((3S )-3-(4-嗎啉基甲基)-3,4-二氫-2(1H )-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N -苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺，或其醫藥學上可接受之鹽，及 (b) MCL1抑制劑， 同時、依序或分開使用。 在另一實施例中，本發明提供一種組合，其包含： (a) 化合物4：5-(5-氯-2-{[(3S )-3-(嗎啉-4-基甲基)-3,4-二氫異喹啉-2(1H )-基]羰基}苯基)-N -(5-氰基-1,2-二甲基-1H -吡咯-3-基)-N -(4-羥苯基)-1,2-二甲基-1H -吡咯-3-甲醯胺，或其醫藥學上可接受之鹽，及 (b) MCL1抑制劑， 同時、依序或分開使用。 可替代地，本發明提供一種組合，其包含： (a) BCL-2抑制劑，及 (b) 化合物2：(2R )-2-{[(5Sa )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(5-氟呋喃-2-基)噻吩并[2,3-d ]嘧啶-4-基]氧基}-3-(2-{[1-(2,2,2-三氟乙基)-1H-吡唑-5-基]甲氧基}苯基)丙酸， 同時、依序或分開使用。 在另一實施例中，本發明提供一種組合，其包含： (a) BCL-2抑制劑，及 (b) 化合物3：(2R )-2-{[(5Sa )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(4-氟苯基)噻吩并[2,3- d ]嘧啶-4-基]氧基}-3-(2-{[2-(2-甲氧基苯基)嘧啶-4-基]甲氧基}苯基)丙酸， 同時、依序或分開使用。 在另一實施例中，本發明提供一種如本文所描述之組合以用於治療癌症。 在另一實施例中，本發明提供如本文所描述之組合在製造治療癌症之藥物中的用途。 在另一實施例中，本發明提供一種分開地或一起地含有以下各者之藥劑， (a) 式(I)之BCL-2抑制劑及 (b) MCL1抑制劑， 或 (a) BCL-2抑制劑及 (b) 式(II)之MCL1抑制劑， 用於同時、依序或分開投與，且其中BCL-2抑制劑及MCL1抑制劑以有效量提供以用於治療癌症。 在另一實施例中，本發明提供一種治療癌症的方法，其包含向需要其之個體投與共同治療有效量之以下各者： (a) 式(I)之BCL-2抑制劑及 (b) MCL1抑制劑， 或 (a) BCL-2抑制劑及 (b) 式(II)之MCL1抑制劑。 在另一實施例中，本發明提供一種用於使(i)難以用至少一種化學療法治療或(ii)用化學療法治療之後復發，或(i)及(ii)兩者的患者敏感的方法，其中該方法包括向該患者投與共同治療有效量之以下各者： (a) 式(I)之BCL-2抑制劑及 (b) MCL1抑制劑， 或 (a) BCL-2抑制劑及 (b) 式(II)之MCL1抑制劑。 在一特定實施例中，BCL-2抑制劑係N -(4-羥苯基)-3-{6-[((3S )-3-(4-嗎啉基甲基)-3,4-二氫-2(1H )-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N -苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺鹽酸鹽(化合物1，HCl)。 在一特定實施例中，BCL-2抑制劑係5-(5-氯-2-{[(3S )-3-(嗎啉-4-基甲基)-3,4-二氫異喹啉-2(1H )-基]羰基}苯基)-N -(5-氰基-1,2-二甲基-1H -吡咯-3-基)-N -(4-羥苯基)-1,2-二甲基-1H -吡咯-3-甲醯胺鹽酸鹽(化合物4，HCl)。 在另一實施例中，BCL-2抑制劑係ABT-199。 在另一實施例中，MCL1抑制劑係(2R )-2-{[(5Sa )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(5-氟呋喃-2-基)噻吩并[2,3-d ]嘧啶-4-基]氧基}-3-(2-{[1-(2,2,2-三氟乙基)-1H -吡唑-5-基]甲氧基}苯基)丙酸(化合物2)。 在另一實施例中，MCL1抑制劑係(2R )-2-{[(5Sa )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(4-氟苯基)噻吩并[2,3-d ]嘧啶-4-基]氧基}-3-(2-{[2-(2-甲氧基苯基)嘧啶-4-基]甲氧基}苯基)丙酸(化合物3)。The present invention relates to a combination comprising (a) a BCL-2 inhibitor of formula (I):
Figure 02_image001
Wherein: ¨ X and Y represent carbon atoms or nitrogen atoms, it should be understood that they may not represent two carbon atoms or two nitrogen atoms at the same time, ¨ A 1 and A 2 together with the atoms they carry form a substituted by 5, 6 depending on the situation Or an aromatic or non-aromatic heterocycle Het composed of 7 ring members, in addition to the nitrogen represented as X or Y, it may also contain 1 to 3 heteroatoms independently selected from oxygen, sulfur and nitrogen, which should be It is understood that the nitrogen in question may be substituted with a group representing a hydrogen atom, a straight or branched chain (C 1 -C 6 )alkyl, or the group -C(O)-O-Alk, where Alk is straight or branched Chain (C 1 -C 6 ) alkyl, or A 1 and A 2 independently of each other represent a hydrogen atom, a linear or branched (C 1 -C 6 ) polyhaloalkyl, linear or branched (C 1 -C 6 ) polyhaloalkyl C 6 ) alkyl or cycloalkyl, ¨ T represents a hydrogen atom, a straight or branched chain (C 1 -C 6 ) alkyl group optionally substituted with 1 to 3 halogen atoms, a group (C 1 -C 4 ) ) alkyl-NR 1 R 2 or group (C 1 -C 4 ) alkyl-OR 6 , ¨ R 1 and R 2 independently of each other represent a hydrogen atom or a straight or branched chain (C 1 -C 6 )alkane group, or R 1 and R 2 form a heterocycloalkyl with the nitrogen atom carrying it, ¨ R 3 represents straight or branched (C 1 -C 6 ) alkyl, straight or branched (C 2 -C 6 ) alkyl groups ) alkenyl, straight or branched chain (C 2 -C 6 )alkynyl, cycloalkyl, (C 3 -C 10 )cycloalkyl-(C 1 -C 6 )alkyl, wherein the alkyl moiety is straight Chain or branched chain heterocycloalkyl, aryl or heteroaryl, it should be understood that one or more of the carbon atoms of the aforementioned groups or the carbon atoms of their possible substituents can be deuterated, ¨ R 4 represents an aryl group , heteroaryl, cycloalkyl or straight-chain or branched (C 1 -C 6 ) alkyl, it being understood that one or more of the carbon atoms of the aforementioned groups or the carbon atoms of their possible substituents may be deuterated , ¨ R 5 represents hydrogen or halogen atom, straight chain or branched chain (C 1 -C 6 ) alkyl group or straight chain or branched chain (C 1 -C 6 ) alkoxy group, ¨ R 6 represents hydrogen atom or straight chain or branched chain (C 1 -C 6 ) alkyl, ¨R a , R b , R c and R d each independently represent R 7 , a halogen atom, a straight or branched chain (C 1 -C 6 )alkoxy radical, hydroxyl, linear or branched (C 1 -C 6 ) polyhaloalkyl, trifluoromethoxy, -NR 7 R 7 ', nitro, R 7 -CO-(C 0 -C 6 )alkane base-, R 7 -CO-NH-(C 0 -C 6 )alkyl-, NR 7 R 7 '-CO-(C 0 -C 6 )alkyl-, NR 7 R 7 '-CO-(C 0 - C6 )alkyl-O-, R7 -SO2 - NH-( C0 - C6 )alkyl-, R 7 -NH-CO-NH-(C 0 -C 6 )alkyl-, R 7 -O-CO-NH-(C 0 -C 6 )alkyl-, heterocycloalkyl or p-(R a , Substituents of one of R b ), (R b , R c ) or (R c , R d ) form, together with the carbon atom carrying it, a ring of 5 to 7 ring members, which may contain 1 to 2 heteroatoms selected from oxygen and sulphur, it is also understood that one or more carbon atoms of the ring as defined above may be deuterated or 1 to 3 selected from halogen and straight or branched chain (C 1 -C 6 ) alkyl group substitution, ¨ R 7 and R 7 ' each independently represent hydrogen, straight-chain or branched-chain (C 1 -C 6 ) alkyl, straight-chain or branched-chain (C 2 -C 6 ) alkene radical, straight-chain or branched-chain (C 2 -C 6 )alkynyl, aryl or heteroaryl, or R 7 and R 7 ' together with the nitrogen atom carrying them form a heterocycle consisting of 5 to 7 ring members , it should be understood that when the compound of formula (I) contains a hydroxyl group, the latter can be converted into one of the following groups as appropriate: -OPO(OM)(OM'), -OPO(OM)(O - M 1 + ) , -OPO(O - M 1 + )(O - M 2 + ), -OPO(O - )(O - )M 3 2 + , -OPO(OM)(O[CH 2 CH 2 O] n CH 3 ) or -OPO(O - M 1 + )(O[CH 2 CH 2 O] n CH 3 ), wherein M and M' independently of each other represent a hydrogen atom, a straight or branched chain (C 1 -C 6 )alkane radical, straight-chain or branched-chain (C2 - C6 )alkenyl, straight-chain or branched-chain (C2 - C6 )alkynyl, cycloalkyl or heterocycloalkyl, both of which consist of 5 to 6 ring members composition, while M 1 + and M 2 + independently of each other represent a pharmaceutically acceptable monovalent cation, M 3 2 + represents a pharmaceutically acceptable divalent cation, and n is an integer from 1 to 5, it is understood that: - "Aryl" means phenyl, naphthyl, biphenyl or indenyl, - "Heteroaryl" means any monocyclic or bicyclic group of 5 to 10 ring members having at least one aryl group family moiety and contains 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen (including quaternary nitrogen), - "cycloalkyl" means any monocyclic or bicyclic non-aromatic carbon containing 3 to 10 ring members Cyclyl, - "heterocycloalkyl" means any monocyclic or bicyclic non-aromatic monocyclic or bicyclic non-aromatic group consisting of 3 to 10 ring members and containing 1 to 3 heteroatoms selected from oxygen, sulfur, SO, SO and nitrogen fused groups or spiro groups, aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups as may be so defined, and groups alkyl, alkenyl, alkynyl and alkoxy from 1 to 3 Substituted with a group selected from: linear or branched (C 1 -C 6 ) optionally substituted with hydroxy, morpholine, 3-3-difluoropiperidine or 3-3-difluoropyrrolidine ) alkyl; (C 3 -C 6 ) spiro; optionally morpholine-substituted linear or branched (C 1 -C 6 )alkoxy; (C 1 -C 6 )alkyl-S-; Hydroxyl; pendant oxy; N - oxide ; nitro; cyano; -COOR';-OCOR';NR'R";Oxy; (C 1 -C 6 )alkylsulfonyl; halogen; aryl optionally substituted with one or more halogens; heteroaryl; aryloxy; arylthio; cycloalkyl; as appropriate Heterocycloalkyl substituted with one or more halogen atoms or alkyl, wherein R' and R" independently of each other represent a hydrogen atom or a straight or branched chain (C 1 -C 6 ) optionally substituted with methoxy Alkyl, the Het group as defined in formula (I) may be through 1 to 3 selected from linear or branched (C 1 -C 6 ) alkyl, hydroxy, linear or branched (C 1 -C 6 ) Group substitution of alkoxy, NR 1 'R 1 " and halogen, it is understood that R 1 ' and R 1 " are as defined for the groups R' and R" mentioned above, or their enantiomers , a diastereomer, or an addition salt thereof with a pharmaceutically acceptable acid or base, and (b) an MCL1 inhibitor. These compounds of formula (I), their synthesis, their use in the treatment of cancer and their pharmaceutical formulations are described in WO 2013/110890, WO 2015/011397, WO 2015/011399 and WO 2015/011400, the contents of which are Incorporated by reference. In certain embodiments, the MCL1 inhibitor is selected from A-1210477 ( Cell Death and Disease 2015 6, e1590; doi: 10.1038/cddis.2014.561) and described in WO 2015/097123, WO 2016/207216, WO 2016/ 207217, WO 2016/207225, WO 2016/207226 or WO 2016/033486, the contents of which are incorporated by reference. The present invention also relates to a combination comprising (a) a BCL-2 inhibitor and (b) an MCL1 inhibitor of formula (II):
Figure 02_image004
Wherein: ¨ A represents straight chain or branched chain (C 1 -C 6 ) alkyl, straight chain or branched chain (C 2 -C 6 ) alkenyl, straight chain or branched chain (C 2 -C 6 ) alkynyl, Linear or branched (C 1 -C 6 )alkoxy, -S-(C 1 -C 6 )alkyl, linear or branched (C 1 -C 6 )polyhaloalkyl, hydroxyl, cyano , -NW 10 W 10 ', -Cy 6 or a halogen atom, ¨W 1 , W 2 , W 3 , W 4 and W 5 independently represent a hydrogen atom, a halogen atom, a straight chain or a branched chain (C 1 -C 6 ) Alkyl, straight-chain or branched-chain (C2 - C6 ) alkenyl, straight-chain or branched-chain (C2 - C6 ) alkynyl, straight-chain or branched-chain ( C1 - C6 ) polyhaloalkane group, hydroxyl, straight or branched chain (C 1 -C 6 )alkoxy, -S-(C 1 -C 6 )alkyl, cyano, nitro, -alkyl(C 0 -C 6 )- NW 8 W 8 ', -O-Cy 1 , -alkyl(C 0 -C 6 )-Cy 1 , -alkenyl(C 2 -C 6 )-Cy 1 , -alkynyl(C 2 -C 6 ) -Cy 1 , -O-alkyl(C 1 -C 6 )-W 9 , -C(O)-OW 8 , -OC(O)-W 8 , -C(O)-NW 8 W 8 ', -NW 8 -C(O)-W 8 ', -NW 8 -C(O)-OW 8 ', -Alkyl(C 1 -C 6 )-NW 8 -C(O)-W 8 ', - SO 2 -NW 8 W 8 ', -SO 2 -alkyl (C 1 -C 6 ), or when grafted to two adjacent carbon atoms, for (W 1 , W 2 ), (W 2 , W 3 ), (W 1 , W 3 ), (W 4 , W 5 ), the substituents of one of them form together with the carbon atom carrying it an aromatic or non-aromatic ring composed of 5 to 7 ring members, It may contain 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it being understood that the resulting ring may be selected from linear or branched (C 1 -C 6 ) alkyl, -NW 10 W 10 ', -alkane Substituted with radical (C 0 -C 6 )-Cy 1 or a pendant oxy group, ¨ X' represents carbon atom or nitrogen atom, ¨ W 6 represents hydrogen, straight chain or branched chain (C 1 -C 8 ) alkyl group , aryl, heteroaryl, arylalkyl (C 1 -C 6 ) group, heteroarylalkyl (C 1 -C 6 ) group, ¨ W 7 represents straight or branched chain (C 1 -C 6 ) C 6 ) alkyl, straight or branched chain (C 2 -C 6 ) alkenyl, straight or branched (C 2 -C 6 )alkynyl, -Cy 3 , -alkyl (C 1 -C 6 ) -Cy 3 , -Alkenyl(C 2 -C 6 )-Cy 3 , -alkynyl(C 2 -C 6 )-Cy 3 , -Cy 3 -Cy 4 , -alkynyl(C 2 -C 6 )-O-Cy 3 , -Cy 3 -alkyl(C 0 -C 6 ) -O-Alkyl (C 0 -C 6 )-Cy 4 , halogen atom, cyano group, -C(O)-W 11 or -C(O)-NW 11 W 11 ', ¨W 8 and W 8 ' independently of each other represent a hydrogen atom, a straight-chain or branched chain (C 1 -C 6 )alkyl or -alkyl(C 0 -C 6 )-Cy 1 , or (W 8 , W 8 ') and the nitrogen carrying it The atoms together form an aromatic or non-aromatic ring consisting of 5 to 7 ring members, which may contain, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it being understood that the discussion The nitrogen may be substituted with a group representing a hydrogen atom, or a straight or branched (C 1 -C 6 ) alkyl group, with the understanding that one or more carbon atoms of a possible substituent may be deuterated, ¨ W 9 representing - Cy 1 , -Cy 1 -alkyl(C 0 -C 6 )-Cy 2 , -Cy 1 -alkyl(C 0 -C 6 )-O-alkyl(C 0 -C 6 )-Cy 2 , - Cy 1 -Alkyl(C 0 -C 6 )-NW 8 -Alkyl(C 0 -C 6 )-Cy 2 , -Cy 1 -Cy 2 -O-Alkyl(C 0 -C 6 )-Cy 5 , -C(O)-NW 8 W 8 ', -NW 8 W 8 ', -OW 8 , -NW 8 -C(O)-W 8 ', -O-alkyl(C 1 -C 6 )- OW 8 , -SO 2 -W 8 , -C(O)-OW 8 , -NH-C(O)-NH-W 8 ,
Figure 02_image006
,
Figure 02_image008
or
Figure 02_image010
, the ammonium so defined may exist in the form of a zwitterion or have a monovalent anion opposite ion, ¨ W 10 , W 10 ', W 11 and W 11 ' independently of each other represent a hydrogen atom or a straight or branched chain (C 1 - C 6 ) alkyl group, ¨W 12 represents hydrogen or hydroxyl group, ¨W 13 represents hydrogen atom or straight or branched chain (C 1 -C 6 ) alkyl group, ¨W 14 represents -OP(O)(O - )( O - ) group, -OP(O)(O - )(OW 16 ) group, -OP(O)(OW 16 )(OW 16 ') group, -O-SO 2 -O - group, -O-SO 2 -OW 16 group, -Cy 7 , -OC(O)-W 15 group, -OC(O)-OW 15 group or -OC(O)-NW 15 W 15 ' group , ¨W 15 and W 15 ' independently of each other represent a hydrogen atom, a straight-chain or branched chain (C 1 -C 6 ) alkyl group or a straight-chain or branched chain amine group (C 1 -C 6 ) alkyl group, ¨W 16 and W 16 ' independently of each other represent a hydrogen atom, a straight or branched chain (C 1 -C 6 ) alkyl group or an arylalkyl (C 1 -C 6 ) group, ¨Cy 1 , Cy 2 , Cy 3 , Cy 4 , Cy 5 , Cy 6 and Cy 7 independently represent cycloalkyl, heterocycloalkyl, aryl or heteroaryl, ¨ n is an integer equal to 0 or 1, it should be understood that: - "aryl" means refers to phenyl, naphthyl, biphenyl, indenyl or indenyl, - "heteroaryl" means any monocyclic or bicyclic group of 5 to 10 ring members having at least one aromatic part and containing 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, - "cycloalkyl" means any monocyclic or bicyclic non-aromatic carbocyclic group containing 3 to 10 ring members, - "heterocyclic "Alkyl" means any monocyclic or bicyclic non-aromatic carbocyclic group containing 3 to 10 ring members and 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, which may include fused, bridged or Spiro ring systems, aryl, heteroaryl, cycloalkyl and heterocycloalkyl as may be so defined, and alkyl, alkenyl, alkynyl, alkoxy via 1 to 4 groups selected from the group consisting of Group substitution: linear or branched (C 1 -C 6 ) alkyl, which may be substituted by a group representing a linear or branched (C 1 -C 6 ) alkoxy group, the linear or branched (C 1 ) -C 6 )alkoxy can be linear or branched (C 1 -C 6 )alkoxy, linear or branched (C 1 -C 6 ) polyhaloalkyl, hydroxy, halogen, pendant oxy, - NW'W", -OC(O)-W' or -CO-NW'W"substituted; straight or branched (C 2 -C 6 ) alkenyl; may be represented by straight or branched (C 1 -C ) alkenyl 6 ) Linear or branched chain (C 2 -C 6 ) alkynyl substituted by alkoxy groups; Chain or branched (C 1 -C 6 )alkoxy, straight or branched (C 1 -C 6 ) polyhaloalkyl, straight or branched (C 2 -C 6 )alkynyl, -NW' Linear or branched chain (C 1 -C 6 ) alkoxy substituted with a group of W" or hydroxyl ; -C6 ) alkyl-S-; hydroxyl; pendant oxy; N -oxide; nitro; cyano; -C(O)-OW';-OC(O)-W';-CO-NW'W";-NW'W";-(C=NW')-OW"; linear or branched (C 1 -C 6 ) polyhaloalkyl; trifluoromethoxy; or halogen; W' is to be understood and W" independently of each other represent a hydrogen atom or a linear or branched (C 1 -C 6 )alkyl group which may be substituted by a group representing a linear or branched (C 1 -C 6 )alkoxy group; and should be It is understood that one or more of the carbon atoms of the foregoing possible substituents may be deuterated, enantiomers, diastereomers or configurational isomers thereof, or combinations thereof with pharmaceutically acceptable acids or bases. Add salt. These compounds of formula (II), their synthesis, their use in the treatment of cancer and their pharmaceutical formulations are described in WO 2015/097123, the contents of which are incorporated by reference. In certain embodiments, the BCL-2 inhibitor is selected from the following compounds: 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-ene-1 -yl]methyl}piperazin-1-yl) -N -[(3-nitro-4-{[(oxan-4-yl)methyl]amino}phenyl)sulfanyl]-2- [( 1H -pyrrolo[2,3- b ]pyridin-5-yl)oxy]benzamide (venetoclax or ABT-199); 4-(4-{[2- (4-Chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1 - yl)-N-(4-{[(2 R )-4 -(Morpholin-4-yl)-1-(phenylthio)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzenesulfonyl]benzamide (rital (navitoclax or ABT-263); oblimersen (G3139); obatoclax (GX15-070); HA14-1; (±)-gossypol (BL-193); ( -)-gossypol (AT-101); apogossypol; TW-37; antimycin A, ABT-737 (Oltersdorf T et al., Nature 2005 Jun 2; 435 ( 7042):677-81), and the compounds described in WO 2013/110890, WO 2015/011397, WO 2015/011399 and WO 2015/011400, the contents of which are incorporated by reference. According to the first state of the present invention Thus, there is provided a combination comprising: (a) a BCL-2 inhibitor of formula (I) as described herein, and (b) an MCL1 inhibitor of formula (II) as described herein. For example, the present invention provides a combination comprising: (a) Compound 1: N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl) )-3,4-dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6, 7,8-Tetrahydro-1-indolazinecarboxamide, or a pharmaceutically acceptable salt thereof, and (b) an MCL1 inhibitor, used simultaneously, sequentially or separately. In another embodiment, the present The invention provides a combination comprising: (a) Compound 4: 5-(5-Chloro-2-{[( 3S )-3-(morpholin-4-ylmethyl)-3,4-dihydroiso Quinolin-2( 1H )-yl]carbonyl}phenyl)-N-(5-cyano-1,2- dimethyl - 1H -pyrrol - 3-yl)-N-(4-hydroxybenzene base)-1,2-dimethyl- 1H -pyrrole-3-carboxamide, or a pharmaceutically acceptable salt thereof, and (b) MCL1 inhibitors, used simultaneously, sequentially or separately. Alternatively, the present invention provides a combination comprising: (a) a BCL-2 inhibitor, and (b) Compound 2: ( 2R )-2-{[( 5S a )-5-{3-chloro-2 -Methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3- d ] Pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propionic acid , used simultaneously, sequentially or separately. In another embodiment, the present invention provides a combination comprising: (a) a BCL-2 inhibitor, and (b) Compound 3: ( 2R )-2-{[( 5S a )-5-{3- Chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3 - d ] Pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid, simultaneously, sequentially or separately use. In another embodiment, the present invention provides a combination as described herein for use in the treatment of cancer. In another embodiment, the present invention provides the use of a combination as described herein in the manufacture of a medicament for the treatment of cancer. In another embodiment, the present invention provides a medicament comprising, separately or together, (a) a BCL-2 inhibitor of formula (I) and (b) an MCL1 inhibitor, or (a) a BCL- 2 Inhibitors and (b) MCL1 inhibitors of formula (II) for simultaneous, sequential or separate administration, and wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in effective amounts for the treatment of cancer. In another embodiment, the present invention provides a method of treating cancer comprising administering to an individual in need thereof a co-therapeutically effective amount of each of: (a) a BCL-2 inhibitor of formula (I) and (b) ) an MCL1 inhibitor, or (a) a BCL-2 inhibitor and (b) an MCL1 inhibitor of formula (II). In another embodiment, the present invention provides a method for sensitizing a patient who is (i) refractory to treatment with at least one chemotherapy or (ii) relapsed after treatment with chemotherapy, or both (i) and (ii) , wherein the method comprises administering to the patient a co-therapeutically effective amount of each of: (a) a BCL-2 inhibitor of formula (I) and (b) an MCL1 inhibitor, or (a) a BCL-2 inhibitor and (b) MCL1 inhibitors of formula (II). In a specific embodiment, the BCL-2 inhibitor is N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)-3,4 -Dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7,8-tetra Hydro-1-indolazinecarboxamide hydrochloride (compound 1, HCl). In a specific embodiment, the BCL-2 inhibitor is 5-(5-chloro-2-{[( 3S )-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinoline Lin-2( 1H )-yl]carbonyl}phenyl)-N-(5-cyano-1,2- dimethyl - 1H -pyrrol - 3-yl)-N-(4-hydroxyphenyl) )-1,2-dimethyl- 1H -pyrrole-3-carboxamide hydrochloride (compound 4, HCl). In another embodiment, the BCL-2 inhibitor is ABT-199. In another embodiment, the MCL1 inhibitor is ( 2R )-2-{[( 5Sa )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazine-1 -yl)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3-(2-{[1 -(2,2,2-Trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propionic acid (compound 2). In another embodiment, the MCL1 inhibitor is ( 2R )-2-{[( 5Sa )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazine-1 -yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3-(2-{[2-(2 -Methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid (compound 3).

因此，本發明在實施例E1中提供一種組合，其包含(a)式(I)之BCL-2抑制劑：

Figure 02_image012
其中： ¨ X及Y表示碳原子或氮原子，應理解其可不同時表示兩個碳原子或兩個氮原子， ¨ A1 及A2 與攜帶其之原子一起形成視情況經取代之由5、6或7個環成員構成的芳族或非芳族雜環Het，除了表示為X或Y的氮之外，其亦可含有1至3個獨立地選自氧、硫及氮之雜原子，應理解所討論之氮可經表示氫原子、直鏈或分支鏈(C1 -C6 )烷基、或基團-C(O)-O-Alk的基團取代，其中Alk為直鏈或分支鏈(C1 -C6 )烷基， 或A1 及A2 彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )多鹵烷基、直鏈或分支鏈(C1 -C6 )烷基或環烷基， ¨ T表示氫原子、視情況經1至3個鹵素原子取代之直鏈或分支鏈(C1 -C6 )烷基、基團(C1 -C4 )烷基-NR1 R2 或基團(C1 -C4 )烷基-OR6 ， ¨ R1 及R2 彼此獨立地表示氫原子或直鏈或分支鏈(C1 -C6 )烷基，或R1 及R2 與攜帶其之氮原子一起形成雜環烷基， ¨ R3 表示直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、環烷基、(C3 -C10 )環烷基-(C1 -C6 )烷基，其中烷基部分為直鏈或分支鏈的、雜環烷基、芳基或雜芳基，應理解前述基團之碳原子或其可能的取代基之碳原子中之一或多者可氘化， ¨ R4 表示芳基、雜芳基、環烷基或直鏈或分支鏈(C1 -C6 )烷基，應理解前述基團之碳原子或其可能的取代基之碳原子中之一或多者可氘化， ¨ R5 表示氫或鹵素原子、直鏈或分支鏈(C1 -C6 )烷基或直鏈或分支鏈(C1 -C6 )烷氧基， ¨ R6 表示氫原子或直鏈或分支鏈(C1 -C6 )烷基， ¨ Ra 、Rb 、Rc 及Rd 各彼此獨立地表示R7 、鹵素原子、直鏈或分支鏈(C1 -C6 )烷氧基、羥基、直鏈或分支鏈(C1 -C6 )多鹵烷基、三氟甲氧基、-NR7 R7 '、硝基、R7 -CO-(C0 -C6 )烷基-、R7 -CO-NH-(C0 -C6 )烷基-、NR7 R7 '-CO-(C0 -C6 )烷基-、R7 -SO2 -NH-(C0 -C6 )烷基-、R7 -NH-CO-NH-(C0 -C6 )烷基-、R7 -O-CO-NH-(C0 -C6 )烷基-、雜環烷基，或對(Ra 、Rb )、(Rb 、Rc )或(Rc 、Rd )中之一者的取代基與攜帶其的碳原子一起形成由5至7個環成員構成的環，其可含有1至2個選自氧及硫的雜原子，亦應理解上文定義之環的一或多個碳原子可氘化或經1至3個選自鹵素及直鏈或分支鏈(C1 -C6 )烷基的基團取代， ¨ R7 及R7 '彼此獨立地表示氫、直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、芳基或雜芳基，或R7 及R7 '與攜帶其之氮原子一起形成由5至7個環成員構成之雜環， 應理解，當式(I)之化合物含有羥基時，後者可視情況轉化為以下基團中之一者：-OPO(OM)(OM')、-OPO(OM)(O- M1 + )、-OPO(O- M1 + )(O- M2 + )、-OPO(O- )(O- )M3 2 + 、-OPO(OM)(O[CH2 CH2 O]n CH3 )或-OPO(O- M1 + )(O[CH2 CH2 O]n CH3 )，其中M及M'彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、環烷基或雜環烷基，兩者皆由5至6環成員構成，同時M1 + 及M2 + 彼此獨立地表示醫藥學上可接受之單價陽離子，M3 2 + 表示醫藥學上可接受之二價陽離子，且n為1至5之整數， 應理解： - 「芳基」意指苯基、萘基、聯苯基或茚基， - 「雜芳基」意指任何由5至10個環成員構成之單環基或雙環基，其具有至少一個芳族部分且含有1至4個選自氧、硫及氮(包括四級氮)之雜原子， - 「環烷基」意指任何含有3至10個環成員之單環或雙環非芳族碳環基， - 「雜環烷基」意指任何由3至10個環成員構成且含有1至3個選自氧、硫、SO、SO2 及氮之雜原子的單環或雙環非芳族稠合基團或螺基， 有可能如此定義之芳基、雜芳基、環烷基及雜環烷基，及基團烷基、烯基、炔基及烷氧基由1至3個選自以下各者之基團取代：視情況經羥基、嗎啉、3-3-二氟哌啶或3-3-二氟吡咯啶取代之直鏈或分支鏈(C1 -C6 )烷基；(C3 -C6 )螺環；視情況經嗎啉取代之直鏈或分支鏈(C1 -C6 )烷氧基；(C1 -C6 )烷基-S-；羥基；側氧基；N -氧化物；硝基；氰基；-COOR'；-OCOR'；NR'R"；直鏈或分支鏈(C1 -C6 )多鹵烷基；三氟甲氧基；(C1 -C6 )烷基磺醯基；鹵素；視情況經一或多個鹵素取代之芳基；雜芳基；芳氧基；芳基硫基；環烷基；視情況經一或多個鹵素原子或烷基取代之雜環烷基，其中R'及R"彼此獨立地表示氫原子或視情況經甲氧基取代之直鏈或分支鏈(C1 -C6 )烷基， 式(I)中定義的Het基團可能經1至3個選自直鏈或分支鏈(C1 -C6 )烷基、羥基、直鏈或分支鏈(C1 -C6 )烷氧基、NR1 'R1 "及鹵素之基團取代，應理解R1 '及R1 "如關於上文提及之基團R'及R"所定義， 或其對映異構體、非對映異構體，或其與醫藥學上可接受之酸或鹼的加成鹽， 及(b) MCL1抑制劑， 同時、依序或分開使用。 本發明亦在實施例E2中提供一種包含(a) BCL-2抑制劑及(b)式(II)之MCL1抑制劑的組合：
Figure 02_image014
其中： ¨ A表示直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、直鏈或分支鏈(C1 -C6 )烷氧基、-S-(C1 -C6 )烷基、直鏈或分支鏈(C1 -C6 )多鹵烷基、羥基、氰基、-NW10 W10 '、-Cy6 或鹵素原子， ¨ W1 、W2 、W3 、W4 及W5 彼此獨立地表示氫原子、鹵素原子、直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、直鏈或分支鏈(C1 -C6 )多鹵烷基、羥基、直鏈或分支鏈(C1 -C6 )烷氧基、-S-(C1 -C6 )烷基、氰基、硝基、-烷基(C0 -C6 )-NW8 W8 '、-O-Cy1 、-烷基(C0 -C6 )-Cy1 、-烯基(C2 -C6 )-Cy1 、-炔基(C2 -C6 )-Cy1 、-O-烷基(C1 -C6 )-W9 、-C(O)-OW8 、-O-C(O)-W8 、-C(O)-NW8 W8 '、-NW8 -C(O)-W8 '、-NW8 -C(O)-OW8 '、-烷基(C1 -C6 )-NW8 -C(O)-W8 '、-SO2 -NW8 W8 '、-SO2 -烷基(C1 -C6 )，或當接枝至兩個鄰近碳原子上，對(W1 、W2 )、(W2 、W3 )、(W1 、W3 )、(W4 、W5 )中之一者的取代基，與攜帶其的碳原子一起形成由5至7個環成員構成的芳族或非芳族環，其可含有1至3個選自氧、硫及氮之雜原子，應理解所得環可經選自直鏈或分支鏈(C1 -C6 )烷基、-NW10 W10 '、-烷基(C0 -C6 )-Cy1 或側氧基之基團取代， ¨ X'表示碳原子或氮原子， ¨ W6 表示氫、直鏈或分支鏈(C1 -C8 )烷基、芳基、雜芳基、芳基烷基(C1 -C6 )基團、雜芳基烷基(C1 -C6 )基團， ¨ W7 表示直鏈或分支鏈(C1 -C6 )烷基、直鏈或分支鏈(C2 -C6 )烯基、直鏈或分支鏈(C2 -C6 )炔基、-Cy3 、-烷基(C1 -C6 )-Cy3 、-烯基(C2 -C6 )-Cy3 、-炔基(C2 -C6 )-Cy3 、-Cy3 -Cy4 、-炔基(C2 -C6 )-O-Cy3 、-Cy3 -烷基(C0 -C6 )-O-烷基(C0 -C6 )-Cy4 、鹵素原子、氰基、-C(O)-W11 或-C(O)-NW11 W11 '， ¨ W8 及W8 '彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )烷基或-烷基(C0 -C6 )-Cy1 ，或(W8 、W8 ')與攜帶其之氮原子一起形成由5至7個環成員構成的芳族或非芳族環，其可含有1至3個除氮原子之外的選自氧、硫及氮之雜原子，應理解所討論之氮可由表示氫原子、或直鏈或分支鏈(C1 -C6 )烷基的基團取代，且應理解可能之取代基的一或多個碳原子可氘化， ¨ W9 表示-Cy1 、-Cy1 -烷基(C0 -C6 )-Cy2 、-Cy1 -烷基(C0 -C6 )-O-烷基(C0 -C6 )-Cy2 、-Cy1 -烷基(C0 -C6 )-NW8 -烷基(C0 -C6 )-Cy2 、-Cy1 -Cy2 -O-烷基(C0 -C6 )-Cy5 、-C(O)-NW8 W8 '、-NW8 W8 '、-OW8 、-NW8 -C(O)-W8 '、-O-烷基(C1 -C6 )-OW8 、-SO2 -W8 、-C(O)-OW8 、-NH-C(O)-NH-W8 、
Figure 02_image016
、
Figure 02_image018
或
Figure 02_image020
，如此定義之銨有可能以兩性離子的形式存在或具有單價陰離子相對離子， ¨ W10 、W10 '、W11 及W11 '彼此獨立地表示氫原子或直鏈或分支鏈(C1 -C6 )烷基， ¨ W12 表示氫或羥基， ¨ W13 表示氫原子或直鏈或分支鏈(C1 -C6 )烷基， ¨ W14 表示-O-P(O)(O- )(O- )基團、-O-P(O)(O- )(OW16 )基團、-O-P(O)(OW16 )(OW16 ')基團、-O-SO2 -O- 基團、-O-SO2 -OW16 基團、-Cy7 、-O-C(O)-W15 基團、-O-C(O)-OW15 基團或-O-C(O)-NW15 W15 '基團， ¨ W15 及W15 '彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )烷基或直鏈或分支鏈胺基(C1 -C6 )烷基， ¨ W16 及W16 '彼此獨立地表示氫原子、直鏈或分支鏈(C1 -C6 )烷基或芳基烷基(C1 -C6 )基團， ¨ Cy1 、Cy2 、Cy3 、Cy4 、Cy5 、Cy6 及Cy7 彼此獨立地表示環烷基、雜環烷基、芳基或雜芳基， ¨ n為等於0或1之整數， 應理解： - 「芳基」意指苯基、萘基、聯苯基、二氫茚基或茚基， - 「雜芳基」意謂任何由5至10個環成員構成之單環基或雙環基，其具有至少一個芳族部分且含有1至3個選自氧、硫及氮之雜原子， - 「環烷基」意指任何含有3至10個環成員之單環或雙環非芳族碳環基， - 「雜環烷基」意指任何含有3至10個環成員且含有1至3個選自氧、硫及氮之雜原子的單環或雙環非芳族碳環基，其可包括稠合、橋聯或螺環系統， 有可能如此定義之芳基、雜芳基、環烷基及雜環烷基，及烷基、烯基、炔基、烷氧基由1至4個選自以下各者之基團取代：直鏈或分支鏈(C1 -C6 )烷基，其可經表示直鏈或分支鏈(C1 -C6 )烷氧基之基團取代，該直鏈或分支鏈(C1 -C6 )烷氧基可經直鏈或分支鏈(C1 -C6 )烷氧基、直鏈或分支鏈(C1 -C6 )多鹵烷基、羥基、鹵素、側氧基、-NW'W"、-O-C(O)-W'或-CO-NW'W''取代；直鏈或分支鏈(C2 -C6 )烯基；可經表示直鏈或分支鏈(C1 -C6 )烷氧基之基團取代的直鏈或分支鏈(C2 -C6 )炔基；可經表示直鏈或分支鏈(C1 -C6 )烷氧基、直鏈或分支鏈(C1 -C6 )多鹵烷基、直鏈或分支鏈(C2 -C6 )炔基、-NW'W''或羥基之基團取代的直鏈或分支鏈(C1 -C6 )烷氧基；可經表示直鏈或分支鏈(C1 -C6 )烷氧基之基團取代的(C1 -C6 )烷基-S-；羥基；側氧基；N -氧化物；硝基；氰基；-C(O)-OW'；-O-C(O)-W'；-CO-NW'W''；-NW'W''；-(C=NW')-OW''；直鏈或分支鏈(C1 -C6 )多鹵烷基；三氟甲氧基；或鹵素；應理解W'及W''彼此獨立地表示氫原子或可由表示直鏈或分支鏈(C1 -C6 )烷氧基之基團取代的直鏈或分支鏈(C1 -C6 )烷基；且應理解前述可能的取代基之一或多個碳原子可氘化， 其對映異構體、非對映異構體或構型異構體，或其與醫藥學上可接受之酸或鹼的加成鹽， 同時、依序或分開使用。 本文描述本發明之進一步所列舉的實施例(E)。應認識到在各實施例中指定之特徵可與其他指定特徵組合以提供本發明之其他實施例。 E3.一種根據E1之組合，其中MCL1抑制劑係如E2中所定義之式(II)化合物。 E4.一種根據E1至E3中之任一者的組合，其中BCL-2抑制劑係N -(4-羥苯基)-3-{6-[((3S )-3-(4-嗎啉基甲基)-3,4-二氫-2(1H )-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N -苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺。 E5.一種根據E1至E3中之任一者的組合，其中BCL-2抑制劑係5-(5-氯-2-{[(3S )-3-(嗎啉-4-基甲基)-3,4-二氫異喹啉-2(1H )-基]羰基}苯基)-N -(5-氰基-1,2-二甲基-1H -吡咯-3-基)-N -(4-羥苯基)-1,2-二甲基-1H -吡咯-3-甲醯胺。 E6.一種根據E4之組合，其中N -(4-羥苯基)-3-{6-[((3S )-3-(4-嗎啉基甲基)-3,4-二氫-2(1H )-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N -苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺呈鹽酸鹽的形式。 E7.一種根據E5之組合，其中5-(5-氯-2-{[(3S )-3-(嗎啉-4-基甲基)-3,4-二氫異喹啉-2(1H )-基]羰基}苯基)-N -(5-氰基-1,2-二甲基-1H-吡咯-3-基)-N -(4-羥苯基)-1,2-二甲基-1H -吡咯-3-甲醯胺呈鹽酸鹽的形式。 E8.一種根據E4或E6的組合，其中在組合治療期間，N -(4-羥苯基)-3-{6-[((3S )-3-(4-嗎啉基甲基)-3,4-二氫-2(1H )-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N -苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺的劑量為50 mg至1500 mg。 E9.一種根據E1至E8中之任一者的組合，其中BCL-2抑制劑一週投與一次。 E10.一種根據E6或E8的組合，其中在組合治療期間，N -(4-羥苯基)-3-{6-[((3S )-3-(4-嗎啉基甲基)-3,4-二氫-2(1H )-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N -苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺一日投與一次。 E11.一種根據E1至E3中之任一者的組合，其中BCL-2抑制劑係ABT-199。 E12.一種根據E1至E11中之任一者的組合，其中MCL1抑制劑係(2R )-2-{[(5Sa )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(5-氟呋喃-2-基)噻吩并[2,3-d ]嘧啶-4-基]氧基}-3-(2-{[1-(2,2,2-三氟乙基)-1H-吡唑-5-基]甲氧基}苯基)丙酸。 E13.一種根據E1至E11中之任一者的組合，其中MCL1抑制劑係(2R )-2-{[(5Sa )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(4-氟苯基)噻吩并[2,3-d ]嘧啶-4-基]氧基}-3-(2-{[2-(2-甲氧基苯基)嘧啶-4-基]甲氧基}苯基)丙酸。 E14.一種根據E1至E13中之任一者的組合，其中BCL-2抑制劑及MCL1抑制劑經口投與。 E15.一種根據E1至E13中之任一者的組合，其中BCL-2抑制劑經口投與且MCL1抑制劑經靜脈內投與。 E16.一種根據E1至E13中之任一者的組合，其中BCL-2抑制劑及MCL1抑制劑經靜脈內投與。 E17.一種根據E1至E16中之任一者的組合，其用於治療癌症。 E18.根據E17之供使用的組合，其中BCL-2抑制劑及MCL1抑制劑以共同治療有效的量提供以用於治療癌症。 E19.根據E17之供使用的組合，其中BCL-2抑制劑及MCL1抑制劑以協同有效量提供以用於治療癌症。 E20.根據E17之供使用的組合，其中BCL-2抑制劑及MCL1抑制劑以實現癌症治療中各化合物的所需劑量減少的協同有效量提供，同時提供有效的癌症治療，以及最終降低副作用。 E21.根據E17至E20中之任一者之以使用的組合，其中癌症係白血病。 E22.根據E21之供使用的組合，其中癌症係急性骨髓白血病、T-ALL或B-ALL。 E23.根據E17至E20中之任一者之供使用的組合，其中癌症係骨髓發育不良症候群或骨髓增生疾病。 E24.根據E17至E20中之任一者之供使用的組合，其中癌症係淋巴瘤。 E25.根據E24中之任一者之供使用的組合，其中淋巴瘤係非霍奇金淋巴瘤。 E26.根據E25中之任一者之供使用的組合，其中非霍奇金淋巴瘤係彌漫性大B細胞淋巴瘤或套細胞淋巴瘤(mantle-cell lymphoma)。 E27.根據E17至E20中之任一者之供使用的組合，其中癌症係多發性骨髓瘤。 E28.根據E17至E20中之任一者之供使用的組合，其中癌症係神經母細胞瘤。 E29.根據E17至E20中之任一者之供使用的組合，其中癌症係小細胞肺癌。 E30.一種根據E1至E16中之任一者的組合，進一步包含一或多種賦形劑。 E31.一種根據E1至E16中之任一者之組合在製造用於治療癌症之藥物中的用途。 E32.根據E31之用途，其中癌症係白血病。 E33.根據E32之用途，其中癌症係急性骨髓白血病、T-ALL或B-ALL。 E34.根據E31之用途，其中癌症係骨髓發育不良症候群或骨髓增生疾病。 E35.根據E31之用途，其中癌症係淋巴瘤。 E36.根據E35之用途，其中淋巴瘤係非霍奇金淋巴瘤。 E37.根據E36之用途，其中非霍奇金淋巴瘤係彌漫性大B細胞淋巴瘤或套細胞淋巴瘤。 E38.根據E31之用途，其中癌症係多發性骨髓瘤。 E39.根據E31之用途，其中癌症係神經母細胞瘤。 E40.根據E31之用途，其中癌症係小細胞肺癌。 E41.一種藥物，其分開地或共同含有， (a) 如E1中所定義之式(I)的BCL-2抑制劑，及 (b) MCL1抑制劑， 用於同時、依序或分開投與，且其中BCL-2抑制劑及MCL1抑制劑以有效量提供以用於治療癌症。 E42.一種藥物，其分開地或共同含有， (a) BCL-2抑制劑，及 (b) 如E2中所定義之式(II)的MCL1抑制劑， 用於同時、依序或分開投與，且其中BCL-2抑制劑及MCL1抑制劑以有效量提供以用於治療癌症。 E43.一種治療癌症之方法，其包含向需要其之個體投與共同治療有效量之(a)如E1中所定義之式(I)的BCL-2抑制劑，及(b)MCL1抑制劑。 E44.一種治療癌症之方法，其包含向需要其之個體投與共同治療有效量之(a) BCL-2抑制劑，及(b)如E2中所定義之式(II)的MCL1抑制劑。 E45.一種用於使(i)難以用至少一種化學療法治療或(ii)用化學療法治療之後復發，或(i)及(ii)兩者的患者敏感的方法，其中該方法包含向該患者投與共同治療有效量之(a)如E1中所定義之式(I)的BCL-2抑制劑，及(b) MCL1抑制劑。 E46.一種用於使(i)難以用至少一種化學療法治療或(ii)用化學療法治療之後復發，或(i)及(ii)兩者的患者敏感的方法，其中該方法包含向患者投與共同治療有效量之(a) BCL-2抑制劑，及(b)如E2中所定義之式(II)的MCL1抑制劑。 「組合」係指以一個單位劑型(例如膠囊、錠劑或藥囊)之固定劑量組合、不固定劑量組合或分裝部分之套組以用於組合投與，其中本發明化合物及一或多種組合搭配物(例如下文所解釋之另一藥物，亦稱作「治療劑」或「輔劑」)可同時獨立地投與或在時間間隔內分開投與，尤其在此等時間間隔使得組合搭配物顯現合作，例如協同效應時。 如本文所用之術語「共投與」或「組合投與」或其類似術語意欲涵蓋向有需要之單一個體(例如患者)投與所選擇之組合搭配物，且意欲包括藥劑不一定藉由相同投與途徑投與或同時投與之治療方案。 術語「固定劑量組合」意指活性成分，例如式(I)化合物及一或多種組合搭配物均以單一實體或劑量的形式同時向患者投與。 術語「不固定劑量組合」意指活性成分，例如本發明化合物及一或多種組合搭配物均以單獨實體的形式同時或依序地向患者投與，無特定時間限制，其中該投與提供治療有效量之兩種化合物至患者體內。後者亦適用於混合物療法，例如投與3種或大於3種活性成分。 「癌症」意指細胞群呈現不可控生長的一類疾病。癌症類型包括血液癌症(淋巴瘤及白血病)及包括癌瘤、肉瘤或母細胞瘤的實體腫瘤。特定言之，「癌症」係指白血病、淋巴瘤或多發性骨髓瘤，且更尤其係指急性骨髓白血病。 術語「共同治療有效」意指治療劑可在其偏好在溫血動物(尤其待治療之人類)內仍顯現(較佳協同)相互作用(共同治療效應)的此類時間間隔內分開給與(以時間順序錯開方式，尤其特定順序方式)。不管此是否係可尤其藉由根據血液含量測定的情況，顯現兩種化合物至少在某些時間間隔期間內皆存在於待治療之人類血液中。 「協同有效」或「協同」意指在投與兩種或大於兩種藥劑後所觀測到的治療性效果大於投與各單一藥劑之後所觀測到的總治療性效果。 如本文所用，術語「治療(treat/treating/treatment)」任何疾病或病症在一個實施例中係指改善疾病或病症(亦即，減緩或停滯或減少疾病或其至少一個臨床症狀之發展)。在另一實施例中，「治療(treat/treating/treatment)」係指緩解或改善至少一個生理參數，包括患者可能無法辯別之生理參數。在又一實施例中，「治療(treat/treating/treatment)」係指在身體上(例如，可辯別症狀之穩定化)、生理上(例如生理參數之穩定化)或在這兩方面調節疾病或病症。 如本文中所用，若個體將在生物學、醫學或生活品質上受益於治療，則該個體「需要」該治療。 在另一態樣中，提供一種用於使(i)難以用至少一種化學療法治療或(ii)用化學療法治療之後復發，或(i)及(ii)兩者的人類敏感的方法，其中該方法包含向患者投與如本文所描述之式(I)的BCL-2抑制劑，以及MCL1抑制劑。敏感的患者為回應於涉及投與如本文所描述之式(I)的BCL-2抑制劑，以及MCL1抑制劑的治療的患者，或對此治療尚未發展有耐受性的患者。 「藥物」意指醫藥組合物，或若干個醫藥組合物之組合，其在一或多種賦形劑存在下含有一或多種活性成分。 『AML』意指急性骨髓白血病。 『T-ALL』及『B-ALL』意指T細胞急性淋巴母細胞白血病及B細胞急性淋巴母細胞白血病。 『游離鹼』係指尚未呈鹽形式的化合物。 在根據本發明之醫藥組合物中，按重量計(組合物之總重量中的活性成分重量)活性成分的比例為5至50%。 在根據本發明之醫藥組合物中，更尤其將使用適於經口、非經腸且尤其靜脈內、全皮膚或反皮膚、鼻、直腸、經舌、眼或呼吸道投與的彼等物，更確切而言，錠劑、糖衣丸劑、舌下錠劑、硬明膠膠囊、直腸給藥劑型、膠囊、口含錠、可注射製劑、噴霧劑、眼或鼻滴劑、栓劑、乳膏、軟膏、經皮凝膠等。 根據本發明之醫藥組成物包含一或多種選自以下之賦形劑或載劑：稀釋劑、潤滑劑、黏合劑、崩解劑、穩定劑、防腐劑、吸附劑、著色劑、甜味劑、調味劑等。藉助於非限制性實例，可提及： w作為稀釋劑 ：乳糖、右旋糖、蔗糖、甘露糖醇、山梨糖醇、纖維素、甘油， w作為潤滑劑 ：二氧化矽、滑石、硬脂酸及其鎂及鈣鹽、聚乙二醇， w作為黏合劑 ：矽酸鎂鋁、澱粉、明膠、黃蓍、甲基纖維素、羧甲基纖維素鈉及聚乙烯吡咯啶酮， w作為崩解劑 ：瓊脂、海藻酸及其鈉鹽、起泡性混合物。 組合之化合物可同時或依序投與。投與途徑較佳係經口途徑，且相應醫藥組合物可允許活性成分瞬時或延緩釋放。此外，組合之化合物可以各含有活性成分中之一者的兩個獨立醫藥組合物形式投與，或以活性成分呈混合物形式之單一醫藥組合物形式投與。 優先考慮呈錠劑之醫藥組合物。含有 50 mg 及 100 mg 之原料藥的化合物 1 鹽酸 鹽包覆膜衣錠劑的醫藥組合物
Figure 106124599-A0304-0001
 藥理學資料 實例 1 - 3 之材料及方法 ： 原發性 AML 患者樣品 ： 在知情同意之後，根據由阿爾弗雷德醫院人類研究倫理委員會(Alfred Hospital Human research ethics committees)核准之指南自患有AML之患者採集骨髓或末梢血液樣品。單核細胞藉由菲科爾-帕克(Ficoll-Paque) (GE Healthcare, VIC, Aus)密度梯度離心來分離，隨後紅血球在37℃下在氯化銨(NH4 Cl)裂解緩衝液中消耗10分鐘。隨後，細胞再懸浮於含有2%胎牛血清之磷酸鹽緩衝生理食鹽水中(Sigma, NSW, Aus)。隨後，單核細胞懸浮於含有青黴素及鏈黴素(GIBCO)以及非熱活化之胎牛血清15% (Sigma)的RPMI-1640(GIBCO VIC, Aus)培養物中。 細胞株、細胞培養及產生螢光素酶報導細胞株 ： 細胞株MV4;11、OCI-AML3、HL-60、HEL、K562、KG-1及EOL-1在37℃，5% CO2 下保持在補充有10% (v/v)胎牛血清(Sigma)及青黴素以及鏈黴素(GIBCO)的RPMI-1640 (GIBCO)中。MV4;11螢光素酶細胞株由慢病毒轉導(lentiviral transduction)產生。 抗體 ： 用於西方墨點分析(western blot analysis)之原發性抗體為MCL1、BCL-2、Bax、Bak、Bim、BCL-XL(內部WEHI產生)及微管蛋白質(T-9026, Sigma)。 細胞存活率 ： 將來自AML患者樣品之新純化單核細胞調節至2.5×105 /ml之濃度及每孔100μL細胞等分入96孔盤(Sigma)。隨後，細胞用1 nM至10 μM之跨越6對數濃度範圍的化合物1，HCl、化合物2、ABT-199 (Active Biochem, NJ, USA)或艾達黴素(idarubicin) (Sigma)處理48小時。為了組合分析，以1:1比率自1 nM至10 μM添加藥物，且在37℃、5% CO2 下培養。隨後，細胞用sytox藍色核酸染料(sytox blue nucleic acid stain) (Invitrogen, VIC, Aus)及螢光染色，該螢光使用LSR-II Fortessa(Becton Dickinson, NSW, Aus)藉由流動式細胞量測分析量測。FACSDiva軟體用以資料收集，且FlowJo軟體用於分析。母細胞使用向前及側面分散特性門控。對於各藥物，在6個濃度下測定排除sytox藍的活細胞，且測定50%的致死性濃度(LC50 ，以μM為單位)。 LC50 測定及協同 ： 格拉夫帕德稜鏡(Graphpad Prism)用於使用非線性回歸來計算LC50 。協同藉由基於所描述之Chou Talalay方法計算複合指數(CI)來測定(Chou Cancer Res; 70(2), 2010年1月15日)。 群落分析 ： 群落形成分析在來自AML患者之新純化及凍結的單核部分上實施。初級細胞在35 mm培養皿(Griener-bio, Germany)中以1×104 至1×105 個雙重複培養。細胞以2:1:1比率之0.6%瓊脂(Difco NSW, Aus):AIMDM 2×(IMDM 粉末-Invitrogen)，補充有NaHCO3 、聚葡萄糖、青黴素/鏈黴素、B巰基乙醇及天冬醯胺):胎牛血清(Sigma)塗鋪。為了最佳生長條件，所有盤皆含有GM-CSF(每盤100 ng)、IL-3 (100 ng/盤R&D系統，USA)SCF(100 ng/盤R&D系統)以及EPO (4U/盤)(在存在及不存在藥物下，在37℃，5% CO2 下，在高濕度培養箱中生長2至3週。在培育之後，盤在生理食鹽水中利用2.5%戊二醛固定且使用來自Oxford Optronix (Abingdon, United Kingdom)之GelCount計數。 西方墨點法 ： 在補充有蛋白質酶抑制劑混合物(Roche, Dee Why, NSW, Australia)之NP40裂解緩衝液(10 mM Tris-HCl pH為7.4，137 mM NaCl，10%甘油，1% NP40)中製備裂解物。蛋白質樣品在降低負載之染料中沸騰，隨後在4%至12% Bis-Tris聚丙烯醯胺凝膠(Invitrogen, Mulgrave, VIC, Australia)上分離，且傳送至Hybond C硝化纖維素膜(GE, Rydalmere, NSW, Australia)以用於與特定抗體一起培育。所有膜阻斷步驟及抗體稀釋使用5% (v/v)脫脂牛奶在含有0.1% (v/v)吐溫(Tween)-20磷酸鹽緩衝生理食鹽水(PBST)或Tris緩衝生理食鹽水之PBS中實施，且利用PBST或TBST進行沖洗步驟。西方墨點藉由經強化之化學發光(GE)觀測。 活體內實驗 AML 移植 ： 動物研究在由阿爾弗雷德醫藥研究及教育領域動物倫理學委員會核准的機構指南下實施，將利用螢光素酶報導基因(pLUC2)轉導的MV4;11細胞以1×105 個細胞靜脈內注射入如先前所描述之經輻射(100Rad)非肥胖糖尿病/重度聯合免疫缺乏(NOD/SCID/IL2rγnull)的小鼠中(Jin等人,Cell Stem Cell 2 July 2009, 第5卷, 1期, 第31-42頁)。在第7天時藉由流式細胞量測術及藉由生物發光MV4;11細胞之IVIS成像來量化PB中hCD45+細胞的百分比來量測移植。在第10天時，小鼠每日口服管飼40:10:60之溶解於PEG400 (Sigma)中的化合物1，HCl (表現為游離鹼之200 µL 100 mg/kg劑量)、無水乙醇(Sigma)以及蒸餾水，或每週兩次接收溶解於50% 2-羥丙基)-β-環糊精(Sigma)之化合物2 (200 µL 25 mg/kg)以及50% 50 mM HCl或藥物組合或媒劑，歷時4週。使用血液學分析器(BioRad, Gladesville, NSW)測定血球計數。 IVIS 成像 ： 生物發光成像使用測徑規IVIS Lumina III XR成像系統來實施。小鼠利用異氟醚麻醉且腹膜內注入100 µL之125 mg/kg螢光素(Perkin Elmer, Springvale, VIC)。實例 4 之材料及方法 ： 細胞株 ： 人類骨髓瘤細胞株(HMCL)衍生自培養於RPMI 1640培養物中之原發性骨髓瘤細胞，該培養物補充有5%胎牛血清及3 ng/ml用於IL-6相關細胞株之重組IL-6。HMCL為患者回應於療法之表現型及基因組異質性以及變異的代表。 MTT 分析 ： 細胞存活率使用MTT (溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鹽)比色存活分析來量測。細胞與化合物一起每次在含有100 µl/孔之最終體積的96孔盤中培育。根據單一藥劑敏感度，以9個不同濃度使用(2R )-2-{[(5Sa )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(5-氟呋喃-2-基)噻吩并[2,3-d ]嘧啶-4-基]氧基}-3-(2-{[1-(2,2,2-三氟乙基)-1H -吡唑-5-基]甲氧基}苯基)丙酸(化合物2)。以1 µM之固定劑量使用N -(4-羥苯基)-3-{6-[((3S )-3-(4-嗎啉基甲基)-3,4-二氫-2(1H )-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N -苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺鹽酸鹽(化合物1，HCl)。在各處理結束時，細胞與1 mg/mL MTT (50 µl MTT溶液每孔2.5 mg/ml)在37℃下一起培育3個小時，使得MTT代謝。將裂解緩衝液(100 µl裂解緩衝液:DMF (2:3)/SDS (1:3))添加入各孔以溶解甲䐶結晶(formazan cristal)，且在培育18小時之後，使用分光光度計量測570 nm處的活細胞中之吸光度。 作為對照，細胞僅用培養物培育及用含有0.1% DMSO之培養物培育。作為骨髓瘤細胞生長對照，每日記錄骨髓瘤細胞吸光度(D0、D1、D2、D3及D4)。 所有實驗重複3次，且在各實驗中各實驗條件至少重複三次孔。 用下式計算抑制效應： 抑制效應(%)=(1-所處理細胞之吸光度值/對照細胞之吸光度值)*100實例 1 ： BCL - 2 及 MCL1 為表現於 AML 中之顯性促存活蛋白質 對具有＞70%母細胞之7個AML細胞株及13個原發性AML樣品進行免疫墨點法以用於圖1中之指定蛋白質。 如圖1中所說明，AML中BCL-2家族成員之表現的蛋白質組測量展示，除了BCL-2之外，大多數原發性AML樣品及AML細胞株共表現促存活蛋白質MCL1。BCL-XL不常在AML中表現。實例 2 ： 組合 BCL - 2 及 MCL1 靶向在 AML 中顯示協同殺滅 54個AML患者樣品用6對數濃度範圍之化合物1(鹽酸鹽)、化合物2或1:1濃度在RPMI/15% FCS中培育48個小時且測定LC50 (圖2A)。 大致20%之原發性AML樣品對化合物1或化合物2高度敏感，其中需要在48小時之後殺死50%之原發性AML母細胞之藥物的致死性濃度(LC50 )在低奈莫耳範圍內(LC50 ＜10 nM)(圖2A)。對比而言，當化合物1及化合物2組合時，敏感AML樣品的比例顯著增加至70%，指示當BCL-2及MCL1同時靶向時的協同活性(圖2A)。一些結果展示於圖17中。 為驗證此途徑之活體內活性，將表現螢光素酶之MV4;11 AML細胞移植入NSG小鼠中且僅用化合物1(鹽酸鹽)或化合物2或組合治療，且在療法14天及21天之後評估腫瘤負荷(圖2B)。在完成28天的療法時，小鼠繼續存活(圖2C)。此等實驗示出化合物1及化合物2之組合為活體內高度有效的，驗證了使用原發性AML細胞在活體外所觀測到之給人深刻印象的活性。 此處呈現在圖2A至圖2C中之資料表明AML中化合物1，HCl與化合物2之間的協同組合活性。實例 3 ： 組合 BCL - 2 及 MCL1 抑制標靶白血病 ， 但無正常祖細胞功能 為分析BCL-2抑制組合MCL1抑制對正常人類CD34+細胞或來自患有AML之患者的菲科爾法分離(ficolled)母細胞的毒性，細胞群落潛在性在暴露至組合療法的2週之後分析。群落生長在補充有10% FCS、IL3、SCF、GM-CSF及EPO的瓊脂中，歷時14天，且群落用自動Gelcount®分析器計數。原發性AML樣品的分析以兩份且平均化的方式實施。CD34+之誤差表示2個獨立正常供體樣品的平均值+/-SD。結果相對於DMSO對照中所計數的群落數目進行校正。所指定藥物濃度塗鋪在D1上。特別地，化合物1+化合物2在不影響正常CD34+群落生長的功能的情況下遏制AML群落形成活性。 總而言之，實例2及實例3展示BCL-2及MCL1之雙重藥理學抑制為治療AML的一種新穎方法，其不需要額外化學療法且利用可接受治療性安全窗口。實例 4 ： 回應於作為單一藥劑之 MCL1 抑制劑或與 BCL - 2 抑制劑組合之多發性骨髓瘤細胞存活的活體外評估 27個人類多發性骨髓瘤細胞株對化合物1、化合物2或在1 µM之化合物1存在下的化合物2的靈敏度藉由使用MTT細胞存活率檢定來分析。測定50%抑制濃度(IC50 ，以nM為單位)。 結果展示於下表中：
Figure 106124599-A0304-0002
 當使化合物1與化合物2組合時，在大部分細胞株中表明了相比於單獨化合物之較強的協同活性。實例 5 ： 組合 MCL1 抑制劑與 BCL - 2 抑制劑在 17 個彌漫性大 B 細胞淋巴瘤 ( DLBCL ) 細胞株組中對增殖的活體外影響 材料及方法 細胞株來源於且保持在如表1所指示之補充有FCS(胎牛血清)的鹼性培養物中。另外，所有培養物皆含有青黴素(100 IU/ml)、鏈黴素(100 µg/ml)及L-麩醯胺酸(2 mM)。除非另外提及，否則培養物及補充劑來自Amimed/Bioconcept (Allschwil, Switzerland)。 細胞株在37℃下在含有5% CO2 的潮濕氛圍中培養且在T-75燒瓶中擴增。在所有情況下，細胞自凍結儲備液解凍，使用合適稀釋液擴增≥1代，使用CASY細胞計數器(Omni Life Science, Bremen, Germany)計數且檢定存活率，隨後以表1中所指定的密度塗鋪25 μl/孔至384孔盤(Corning)中。所有細胞株藉由在Idexx Radil (Columbia, MO, USA)處實施的PCR檢定確定為無黴漿菌污染物，且藉由在Asuragen (Austin, TX, USA)或內部檢定48個小核苷酸多態性(SNP)組排除辨識錯誤。 化合物之儲備溶液以10 mM之濃度在DMSO (Sigma)中製備且儲存於-20℃下。當需要得到全部劑量-反應曲線時，儲備溶液在DMSO中預稀釋至所需起始濃度的1'000倍(參見表2)。在細胞接種之後的那天，使用非接觸300D數位施配器(TECAN, Männedorf, Switzerland)以單獨地或以棋盤格方式之所有可能的排列直接將各化合物之8個2.5倍的連續稀釋液施配至細胞分析盤中，如圖4中所概述。所有孔中之最終DMSO濃度為0.2%。 在培育2天之後，分析單一藥劑以及其棋盤格組合對細胞存活率的影響，該分析在37℃/5% CO2 下藉由使用CellTiterGlo (Promega, Madison, WI, USA)根據條件以25 μL反應劑/孔及n=2個複寫盤量化細胞ATP含量來進行。發光在M1000多用途讀盤儀(TECAN, Männedorf, Switzerland)上量化。在化合物添加時同樣地分析細胞的數目/存活率且用於分析特定細胞株之群體倍增時間的程度。 使用標準四參數曲線擬合計算單一藥劑IC50 。化合物組合之間的潛在協同相互作用根據洛伊相加模型(Loewe additivity model)使用過量抑制2D矩陣來分析，且報導為協同作用分值(Synergy Score) (Lehar等人,Nat Biotechnol .2009 年 7月 ; 27(7): 659-666)。所有計算使用組合分析模組(Combination Analysis Module)內部軟體來實施。IC50 定義為CTG信號減少至媒劑(DMSO)對照所量測之50%時的化合物濃度。 協同作用分值之解釋如下： SS ~ 0 → 相加性 SS ＞1 → 弱協同 SS ＞2 → 協同表 1 . 用於組合實驗中之17個彌漫性大B細胞淋巴瘤細胞株的識別及分析條件。
Figure 106124599-A0304-0003
 *此培養物進一步補充有50 µM 2-巰基乙醇。基於結束時相比於化合物培育開始時之ATP含量的差值來計算倍增時間。表 2. 指定化合物3及化合物1，HCl之單一藥劑IC50 值，以及其組合之協同作用分值。當觀測之分值≥ 2.0時，認為相互作用為協同的。
Figure 106124599-A0304-0004
 「起始濃度(Start conc)」意指起始濃度。 「無水IC50 (Abs IC50 )」意指無水IC50 。 「最大抑制(Max Inh)」意指最大抑制。 結果 在17個彌漫性大B細胞淋巴瘤(DLBCL)細胞株組中分析組合MCL1抑制劑化合物3與BCL-2抑制劑化合物1，HCl對增殖的影響。 化合物3作為單一藥劑強有力地抑制了所測試之17個DLBCL細胞株中大部分的生長(表1)。因此，14個細胞株顯示小於100 nM的IC50 ，且額外1個細胞株顯示100 nM與1μ M之間的IC50 。僅2個細胞株顯示大於1μ M的IC50 。 化合物1，HCl作為單一藥劑亦抑制所測試之17個DLBCL細胞株中大部分的生長，儘管不太有效(表2)。因此，2個細胞株顯示小於100 nM之IC50 ，且6個細胞株顯示100 nM與1μ M之間的IC50 。9個細胞株顯示大於1μ M之IC50 (其中4個大於10μ M)。 化合物3及化合物1，HCl的組合處理造成所測試之17個中之16個DLBCL細胞株的協同生長抑制(亦即高於2之協同作用分值-Lehar等人,Nat Biotechnol .2009 July ; 27(7): 659-666)(表2)。在5個細胞株中，用5與10之間的協同作用分值標記協同效應。在4個細胞株中，協同效應為優越的，獲得10與17.3之間的協同作用分值。重要的是，協同不依賴於單一藥劑抗增殖效應，且事實上在化合物3及化合物1自身不展示抗增殖效應之濃度下協同為尤其強的。舉例而言，在DB細胞中，所測試之第二最低濃度的化合物3及化合物1分別引發僅1%及2%的生長抑制，而兩個化合物之對應組合獲得96%的生長抑制(圖4A，左圖)，因此比基於單一藥劑活性所計算之相加性高91%(圖4A，右圖)。作為另一實例，在Toledo細胞中，其中化合物3不太有效且在所測試之最高濃度下僅獲得部分生長抑制(46%)，與化合物1之第二最低濃度之組合造成98%之協同生長抑制(圖4B，左圖)，因此比基於單一藥劑活動所計算之相加性高52%(圖4B，右圖)。 此外，值得注意的是，協同效應在遍及大範圍之單一藥劑濃度內出現，此應證明關於給藥含量及安排之靈活性方面在活體內為有益的。 總而言之，化合物3及化合物1之組合在大部分所測試之DLBCL細胞株中獲得較強至優越的協同生長抑制。實例 6 ： MCL1 抑制劑 ( 化合物 3 ) 及 BCL - 2 抑制劑 ( 化合物 1 ) 之組合對 Karpas422 異種移植物的活體內療效 材料及方法 腫瘤細胞培養及細胞接種 Karpas 422人類B細胞非霍奇金氏淋巴瘤(NHL)細胞株自患有抗化學治療NHL之患者的肋膜積液建立。細胞由DSMZ細胞庫獲得，且在空氣中含5% CO2 之氛圍中，37℃下，在補充有10% FCS (BioConcept Ltd. Amimed)、2 mM L-麩醯胺酸(BioConcept Ltd. Amimed)、1 mM丙酮酸鈉(BioConcept Ltd. Amimed)及10 mM HEPES (Gibco)的RPMI-1640培養物(BioConcept Ltd. Amimed,)中培養。細胞保持在0.5×106與1.5×106個細胞/mL之間。為建立Karpas 422，收集異種移植物細胞且再懸浮於HBSS (Gibco)中且與基質膠(BD Bioscience) (1:1 v/v)混合，隨後在用異氟醚麻醉的動物的右側腹皮下注射含有1×107個細胞的200 µL。在細胞接種之前的二十四小時，所有動物皆使用ɤ-照射器用5Gy照射超過2分鐘。腫瘤生長 細胞接種後定期監測腫瘤生長且在腫瘤體積達到合適體積時將動物隨機分佈入治療組(n=5)。在治療時段期間，約一週兩次使用測徑規量測腫瘤體積。以mm3 為單位之腫瘤尺寸由(L×W2×π/6)計算。其中W=腫瘤之寬度且L=腫瘤之長度。治療 攜帶腫瘤之動物(大鼠)在其腫瘤達到合適尺寸時登記入治療組(n=5)以形成具有約450 mm3 之平均腫瘤體積的組。治療組如表3中所概述。在用於化合物3之媒劑或化合物3藉由15分鐘iv 輸液投與之前的1 h，用於化合物1，HCl之媒劑或化合物1，HCl藉由口服(po )管飼投與。對於iv 輸液，動物用異氟醚/O2 麻醉，且媒劑或化合物3經由在尾部靜脈中之插管投與。動物在給藥日稱重且根據體重調節劑量，對於兩種化合物而言，給藥量為10 ml/kg。體重 動物每週至少稱重2次，且常常檢測任何不良效應的明顯病徵。資料分析及統計評估 使用GraphPad Prism 7.00 (GraphPad Software)以統計方式分析腫瘤資料。若資料中之變動為正常分佈，則資料使用具有事後鄧尼特測試(post hoc Dunnett's test)的單因子變異數分析(one-way ANOVA)來分析，用於治療組與對照組之對比。事後杜凱氏測試(post hoc Tukey's test)用於對比。或者，使用Kruskal-Wallis評級的事後鄧恩測試(post hoc Dunn's test)。適當時，結果呈現為平均值±SEM。 作為療效之量測，T/C%值在實驗結束時根據下式計算： (Δ腫瘤體積治療 /腫瘤體積對照 )*100 腫瘤消退根據下式計算： -(Δ腫瘤體積治療 /腫瘤體積開始治療時 )*100 其中Δ腫瘤體積表示評估日的平均腫瘤體積減去實驗開始時的平均腫瘤體積。表 3 . 用於攜帶Karpass422異種移植物大鼠之組合療效的治療組
Figure 106124599-A0304-0005
 當平均腫瘤體積為約450 mm3 時起始治療。化合物1，HCl在PEG400/EtOH/Phosal 50 PG (30/10/60)中調配且化合物3置放於溶液中。 QW意指每週一次。 結果 在以20 mg/kgiv 輸液化合物3之前1 h以150 mg/kgpo 化合物1游離鹼的組合治療引起自治療開始的第30天所有Karpas422腫瘤的完全消退(圖5)。在治療於第35天至第90天停止之後，治療組中之所有動物保持無腫瘤。在組合組中觀測到相比於單一藥劑活性的積極組合效應。在第34天，單一藥劑化合物3及組合組中之腫瘤反應明顯不同於媒劑組(p＜0.05)。組合治療基於體重變化具有良好的耐受性(圖6)。實例 7 ： MCL1 抑制劑 ( 化合物 3 ) 及 BCL - 2 抑制劑 ( 化合物 1 ， HCl) 之組合對 DLBCL Toledo 異種移植物的活體內療效 材料及方法 細胞植入 異種移植模型藉由將具有50%基質膠的3百萬托萊多細胞懸浮液直接皮下(sc)植入SCID/米色小鼠之皮下區域建立。所有程序使用無菌技術進行。在整個程序時段期間麻醉小鼠。 一般而言，總計每組6個動物參與療效研究。對於單一藥劑及組合研究而言，動物經由口服管飼(po)給藥化合物1，且經由尾部靜脈靜脈內(iv)給藥化合物3。化合物1，HCl調配為PEG300/EtOH/水(40/10/50)中之溶液，且化合物3置放於溶液中。當腫瘤在細胞植入後第26天達到大致220 mm3 時，攜帶腫瘤之小鼠隨機分佈入治療組。 包括所有治療組之劑量安排之研究設計概述於下表中。在給藥日稱重動物且根據體重調節劑量，給藥量為10 ml/kg。在隨機化時收集腫瘤尺寸及體重且其後在研究期間每週收集兩次。各天收集資料之後提供以下資料：死亡發生率、個體及組平均體重以及個體及組平均腫瘤體積。
Figure 106124599-A0304-0006
 對於在托萊多模型中研究而言，在細胞植入之後的第26天，當平均腫瘤體積為約218至228 mm3 時，開始治療。 QW意指每週一次。體重 ( BW ) 如下計算體重變化百分比：(BW當前 - BW初始 )/(BW初始 ) × 100。資料呈現為自治療開始之日起的體重變化百分比。腫瘤體積及研究中剩餘小鼠百分比 治療/對照(T/C)百分比值使用下式計算： T/C% = 100 ´ DT/DC若DT ＞0 消退% = 100 ´ DT/T0 若DT ＜0 其中： T =最終研究日之藥物治療組的平均腫瘤體積； DT = 最終研究日之藥物治療組之平均腫瘤體積-初始給藥日之藥物治療組之平均腫瘤體積； T0 =定組日之藥物治療組之平均腫瘤體積； C =最終研究日之對照組的平均腫瘤體積；及 DC = 最終研究日之對照組之平均腫瘤體積-初始給藥日之對照組之平均腫瘤體積。 研究中剩餘小鼠百分比=6-達至終點之小鼠數目/6*100統計分析 所有資料表現為平均值±平均值標準誤差(SEM)。△腫瘤體積及體重變化百分比用於統計分析。使用單因數ANOVA，隨後使用事後圖克測試(post hoc Tukey test)進行組間之比較。對於所有統計評估，顯著性水準設定為p＜0.05。除非另外說明，否則報導相比於媒劑對照組之顯著性。 結果
Figure 106124599-A0304-0007
 在托萊多模型中，100 mg/kg之化合物1游離鹼產生37% T/C之統計學上顯著之抗腫瘤效應。25 mg/kg之化合物3產生102% T/C之無抗腫瘤效應(圖7)。化合物1+化合物3之組合產生3% T/C之腫瘤停滯，此相比於媒劑、化合物1及化合物3治療之腫瘤為統計學上顯著的(p＜0.05，藉由單因子變異數分析測試(one-way ANOVA test))。 因此，BCL-2及MCL1在DLBCL中之組合抑制可產生臨床治療效益。另外，圖8展示針對托萊多之平均體重變化。化合物1，HCl及化合物3之小鼠治療呈現體重增加(分別1.081%及2.3%)。組合組展示少量體重損失(-3.2%)。在此研究中未觀測到其他不良事件病徵。整個研究中的所有6個動物存活。 總而言之，實例2、實例6及實例7展示MCL1抑制劑及BCL-2抑制劑之組合在攜帶源自急性骨髓白血病及人類淋巴瘤之細胞株的異種移植物的小鼠及大鼠中在耐受劑量下為有效的，此表明可利用此組合在此等疾病中實現適合之治療窗。實例 8 ： 組合 MCL1 抑制劑及 BCL - 2 抑制劑於 13 個急性骨髓白血病 ( AML ) 細胞株組中對增殖的活體外影響。 材料及方法 細胞株來源於且保持在如表1所指示之補充有FBS(胎牛血清)的鹼性培養物中。另外，所有培養物皆含有青黴素(100 IU/ml)、鏈黴素(100 µg/ml)及L-麩醯胺酸(2 mM)。 細胞株在37℃下在含有5% CO2 的潮濕氛圍中培養且在T-150燒瓶中擴增。在所有情況下自冷凍儲備液解凍細胞，使用合適的稀釋液擴增≥1代，使用CASY細胞計數器計數且檢定存活率，隨後以表1中所指示之密度塗鋪150 μl/孔至96孔盤中。所有細胞株皆測定為內部無黴漿菌污染物。 化合物之儲備溶液以在DMSO中5 mM之濃度製備且儲存於-20℃下。 為分析化合物作為單一藥劑的活性，細胞接種且用各單獨直接施配至細胞分析盤中之化合物的9個2倍連續稀釋液處理。在培育3天之後，分析化合物對細胞存活率的影響，該分析在37℃/5% CO2 下藉由使用CellTiterGlo以75 μL反應劑/孔定量細胞ATP含量來進行。所有實驗皆重複實施三次。在多用途盤讀取器上量化發光。使用標準四參數曲線擬合計算單一藥劑IC50 。IC50 定義為CTG信號減少至媒劑(DMSO)對照所量測之50%時的化合物濃度。 為了分析以組合形式的化合物活性，細胞接種且用各化合物之7或8個3.16倍的連續稀釋液處理，各化合物單獨地或以棋盤格方式之所有可能的排列直接施配至細胞分析盤中，如圖9所指示。在培育3天之後，分析單一藥劑以及其棋盤格組合對細胞存活率的影響，該分析在37℃/5% CO2 下藉由使用CellTiterGlo以75 μL反應劑/孔量化細胞ATP含量來進行。實施兩個獨立實驗，各者重複兩次實施。在多用途盤讀取器上量化發光。 化合物組合之間的潛在協同相互作用根據洛伊相加模型(Loewe additivity model)使用過量抑制2D矩陣來分析，且報導為協同作用分值(Lehar等人,Nat Biotechnol .2009 年 7 月 ; 27(7): 659-666)。所有計算皆使用ClaliceTM 生物資訊軟體來實施。 表3中所指示之倍增時間為自細胞解凍至其在96孔盤中接種之實施的不同代(在T-150燒瓶中)中所獲得之倍增時間的平均值。 協同作用分值之解釋如下： SS ~ 0 → 相加性 SS ＞1 → 弱協同 SS ＞2 → 協同表 3. 用於組合實驗中之13個急性骨髓白血病(AML)細胞株的識別及分析條件。
Figure 106124599-A0304-0008
 表 4a. 指示13個AML細胞株中化合物3、化合物1，HCl及ABT-199的單一藥劑IC50 值。化合物在3天期間與細胞一起培育。
Figure 106124599-A0304-0009
 表 4b. 指示5個AML細胞株中化合物4，HCl的單一藥劑IC50 值。化合物在3天期間與細胞一起培育。
Figure 106124599-A0304-0010
 表 5a. 指示13個AML細胞株中化合物3及化合物1組合的協同作用分值。當觀測之分值≥ 2.0時，認為相互作用為協同的。指示化合物之起始濃度、最大抑制之平均值及協同作用分值之標準差(sd)。化合物在3天期間與細胞一起培育。
Figure 106124599-A0304-0011
 表 5b. 指示8個AML細胞株中化合物3及ABT-199組合的協同作用分值。當觀測之分值≥ 2.0時，認為相互作用為協同的。指示化合物之起始濃度、最大抑制之平均值及協同作用分值之標準差(sd)。化合物在3天期間與細胞一起培育。
Figure 106124599-A0304-0012
 表 5c. 指示5個AML細胞株中化合物3及化合物4，HCl組合的協同作用分值。當觀測之分值≥ 2.0時，認為相互作用為協同的。指示化合物之起始濃度、最大抑制之平均值及協同作用分值之標準差(sd)。化合物在3天期間與細胞一起培育。
Figure 106124599-A0304-0013
 結果 組合 ( a ). 組合MCL1抑制劑化合物3及BCL-2抑制劑化合物1對增殖的影響在13個急性骨髓白血病(AML)細胞株組中分析。 化合物3作為單一藥劑強有力地抑制了所測試之13個AML細胞株中大部分的生長(表4a)。因此，10個細胞株展示小於100 nM的IC50 ，且額外2個細胞株展示100 nM與1 μM之間的IC50 。僅1個細胞株展示大於1 μM的IC50 。 化合物1，HCl作為單一藥劑亦抑制若干個所測試之AML細胞株的生長，儘管不太有效(表4a)。因此，5個細胞株顯示小於100 nM之IC50 ，且2個細胞株顯示100 nM與1 μM之間的IC50 。6個細胞株展示大於1 μM的IC50 。 化合物3及化合物1，HCl組合治療造成所測試之整體13個細胞株的協同生長抑制(亦即高於2之協同作用分值)(表5a)。在2個細胞株中，用5與10之間的協同作用分值標記協同效應。在10個細胞株中，協同效應為優越的，獲得10與19.8之間的協同作用分值。重要的是，協同不依賴於單一藥劑抗增殖效應，且事實上在化合物3及化合物1自身不具有抗增殖效應之濃度下協同為尤其強的。舉例而言，在OCI-AML3細胞中，所測試之第三最低濃度的化合物3及化合物1分別引發5%及1%的生長抑制，而兩種化合物之對應組合獲得84%的生長抑制(圖9A，左上圖)，因此比基於單一藥劑活性所計算之相加性高79%(圖9A，右上圖)。 此外，值得注意的是，協同效應在遍及大範圍之單一藥劑濃度內出現，此應證明關於給藥含量及安排之靈活性方面在活體內為有益的。 總而言之，化合物3及化合物1之組合在所有測試之13個AML細胞株中提供協同生長抑制。重要的是，在所測試之大部分AML細胞株中觀測到優越的協同生長抑制(10/13)。 組合 (b). 組合MCL1抑制劑化合物3及BCL-2抑制劑ABT-199對增殖的影響在8個急性骨髓白血病(AML)細胞株組中分析。 化合物3作為單一藥劑強有力地抑制了所測試之8個AML細胞株中大部分的生長(表4a)。因此，5個細胞株展示小於100 nM的IC50 ，且額外2個細胞株展示100 nM與1 μM之間的IC50 。僅1個細胞株展示大於1 μM的IC50 。 ABT-199作為單一藥劑亦抑制AML細胞株之生長，儘管不太有效(表4a)。因此，僅1個細胞株顯示小於100 nM之IC50 ，且2個細胞株顯示100 nM與1 μM之間的IC50 。5個細胞株展示大於1 μM的IC50 。 MCL1抑制劑及ABT-199組合治療造成所測試之8個細胞株整組的協同生長抑制(亦即高於2之協同作用分值)(表5b)。在大部分細胞株中，協同效應為優越的，獲得10與17.6之間的協同作用分值。重要的是，協同不依賴於單一藥劑抗增殖效應，且事實上在MCL1抑制劑及ABT-199自身不具有抗增殖效應之濃度下協同為尤其強的。舉例而言，在OCI-AML3細胞中，所測試之第三最低濃度的MCL1及ABT-199分別引發26%及18%的生長抑制，而兩個化合物之對應組合獲得91%的生長抑制(圖13，左上圖)。 此外，值得注意的是，協同效應在遍及大範圍之單一藥劑濃度內出現，此應證明關於給藥含量及安排之靈活性方面在活體內為有益的。 總而言之，化合物3及ABT-199之組合獲得所有測試之8個AML細胞株的協同生長抑制。重要的是，在所測試之大部分AML細胞株中觀測到優越的協同生長抑制(7/8)。 組合 (c). 組合MCL1抑制劑化合物3及BCL-2抑制劑化合物4對增殖的影響在5個急性骨髓白血病(AML)細胞株組中分析。 化合物3作為單一藥劑強有力地抑制所測試之5個AML細胞株的生長(表4b)。因此，所有細胞株皆展示小於200 nM的IC50 。化合物4，HCl作為單一藥劑亦抑制所測試5個中之4個細胞株的生長，具有小於或等於40 nM之IC50 ，一個細胞株對具有10 µM之IC50 的化合物4具有耐受性。化合物3及化合物4，HCl的組合治療造成所測試之整體5個細胞株的協同生長抑制(亦即高於2之協同作用分值)(表5c)。在2個細胞株中，用5與10之間的協同作用分值標記協同效應。在1個細胞株中，協同效應為優越的，獲得16.5之協同作用分值。重要的是，協同不依賴於單一藥劑抗增殖效應，且事實上在化合物4，HCl及化合物3自身不具有或具有較低之抗增殖效應的濃度下協同為尤其強的。舉例而言，在OCI-AML3細胞中，所測試之第三最低濃度的化合物4，HCl及化合物3分別引發1%及40%的生長抑制，而兩個化合物之對應組合獲得98%之生長抑制(圖1A，左圖；代表兩個獨立實驗)；因此比基於單一藥劑活性所計算之相加性高53%(圖式14A，右圖)。在ML-2中，所測試之第五最低濃度的化合物4，HCl及化合物3分別引發18%及26%的生長抑制，而兩個化合物之對應組合獲得100%的生長抑制(圖14B，左圖；代表兩個獨立實驗)，因此比基於單一藥劑活性所計算之相加性高51%(圖15，右圖)。 總而言之，化合物4及化合物3之組合獲得所有測試之5個AML細胞株的協同生長抑制。實例 9 ： 組合 MCL1 抑制劑及 BCL - 2 抑制劑於 12 個神經母細胞瘤 ( NB ) 細胞株組中對增殖的活體外影響 材料及方法 細胞株來源於且保持在表1中所指示之補充有FBS的鹼性培養物中。另外，所有培養物皆含有青黴素(100 IU/ml)、鏈黴素(100 µg/ml)及L-麩醯胺酸(2 mM)。細胞株在37℃下在含有5% CO2 的潮濕氛圍中培養且在T-150燒瓶中擴增。在所有情況下，自冷凍儲備液解凍細胞，使用合適的稀釋液擴增≥1代，使用CASY細胞計數器計數且分析存活率，隨後以表6中所指示之密度塗鋪150 μl/孔至96孔盤中。所有細胞株皆測定為內部無黴漿菌污染物。 化合物之儲備溶液以在DMSO中5 mM之濃度製備且儲存於-20℃下。為分析化合物作為單一藥劑的活性，細胞接種且用各單獨直接施配至細胞分析盤中之化合物的9個3.16倍連續稀釋液處理。在培育2或3天(如表6所指示)之後，分析化合物對細胞存活率的影響，該分析在37℃/5% CO2 下藉由使用CellTiterGlo以150 μL反應劑/孔量化細胞ATP含量來進行。實施兩個獨立實驗，各者重複實施兩次。所有實驗皆重複實施三次。在多用途盤讀取器上量化發光。使用標準四參數曲線擬合計算單一藥劑IC50 。IC50 定義為CTG信號減少至媒劑(DMSO)對照所量測之50%時的化合物濃度。 實施相同實驗以分析化合物組合之間的潛在協同相互作用。使用過量抑制2D矩陣根據洛伊相加性模型分析協同作用分值(Lehar等人,Nat Biotechnol .2009 年 7月; 27(7): 659-666)。所有計算皆使用Chalice TM生物資訊軟體來進行。 表6中所指示之倍增時間為自細胞解凍至其在96孔盤中接種之實施的不同代(在T-150燒瓶中)中所獲得之倍增時間的平均值。 協同作用分值之解釋如下： SS ~ 0 → 相加性 SS ＞1 → 弱協同 SS ＞2 → 協同表 6. 用於組合實驗中之12個神經母細胞瘤(NB)細胞株的識別及分析條件。
Figure 106124599-A0304-0014
 表 7. 指示化合物3及化合物1，HCl的單一藥劑IC50 值。化合物在2或3天期間與細胞一起培育。
Figure 106124599-A0304-0015
 表 8. 指示與化合物3及化合物1，HCl組合的協同作用分值。當觀測之分值≥ 2.0時，認為相互作用為協同的。化合物在2或3天期間與細胞一起培育。
Figure 106124599-A0304-0016
 結果 MCL1抑制劑化合物3與BCL-2抑制劑化合物1之組合對增殖的影響在12個神經母細胞瘤細胞株組中分析。所測試之12個中之3個細胞株對作為單一藥劑之化合物3敏感(表7)。1個細胞株展示小於100 nM的IC50 ，且額外2個細胞株展示100 nM與1 μM之間的IC50 。 所有細胞株皆對化合物1，HCl作為單一藥劑具有耐受性，其中所測試之所有細胞株展示大於1 µM的IC50 。化合物3及化合物1的組合治療造成所測試之12中之11個NB細胞株的協同生長抑制(亦即高於2之協同作用分值-Lehar等人,Nat Biotechnol .2009 年 7月 ; 27(7): 659-666)(表8)。在5個細胞株中，協同效應為優越的，獲得10與17.81之間的協同作用分值。重要的是，協同不依賴於單一藥劑抗增殖效應，且事實上在化合物3及化合物1，HCl自身不展示抗增殖效應之濃度下協同為尤其強的。舉例而言，在LAN-6細胞中，630 nM之化合物3及化合物1，HCl分別引發僅12%及0%的生長抑制，而兩個化合物之對應組合獲得95%的生長抑制(圖10，左上圖)，因此比基於單一藥劑活性所計算之相加性大76%(圖10，右上圖)。總而言之，化合物3及化合物1之組合獲得大部分所測試之神經母細胞瘤細胞株的較強至優越的協同生長抑制。實例 10 ： 組合 MCL1 抑制劑及 BCL - 2 抑制劑在 8 個 B 細胞急性淋巴母細胞性白血病組 ( B - ALL ) 及 10 個 T 細胞急性淋巴母細胞性白血病組 ( T - ALL ) 細胞株中對增殖的活體外影響。 材料及方法 細胞株來源於且保持在表1中所指示之補充有FBS的鹼性培養物中。另外，所有培養物皆含有青黴素(100 IU/ml)、鏈黴素(100 μg/ml)及L麩醯胺酸(2 mM)。細胞株在37℃下在含有5% CO2 的潮濕氛圍中培養且在T-150燒瓶中擴增。在所有情況下，自冷凍儲備液解凍細胞，使用合適的稀釋液擴增≥1代，使用CASY細胞計數器計數且分析存活率，隨後以表9中所指示之密度塗鋪150 μl/孔至96孔盤中。所有細胞株皆測定為內部無黴漿菌污染物。 化合物之儲備溶液以5 mM之濃度在DMSO中製備且儲存於-20℃下。為分析化合物作為單一藥劑的活性，細胞接種且用各單獨直接施配至細胞分析盤中之化合物的9個2倍連續稀釋液處理。在培育3天之後，分析化合物對細胞存活率的影響，該分析在37℃/5% CO2 下藉由使用CellTiterGlo以75 μL反應劑/孔量化細胞ATP含量來進行。所有條件進行重複三次測試。在多用途盤讀取器上量化發光。使用標準四參數曲線擬合計算單一藥劑IC50 。IC50 定義為CTG信號減少至媒劑(DMSO)對照所量測之50%時的化合物濃度。 為了分析以組合形式的化合物活性，細胞接種且用各化合物之7或8個3.16倍的連續稀釋液處理，各化合物單獨地或以棋盤格方式之所有可能的排列直接施配至細胞檢定盤中，如圖1所指示。在培育3天之後，分析單一藥劑及其棋盤格組合對細胞存活率的影響，該分析在37℃/5% CO2 下藉由使用CellTiterGlo以75 μL反應劑/孔量化細胞ATP含量來進行。對於B-ALL細胞株而言，實施兩個獨立實驗，所實施的各個實驗重複兩次。對於T-ALL細胞株而言，實施一個實驗，其重複三次實施。在多用途盤讀取器上量化發光。 化合物組合之間的潛在協同相互作用根據洛伊相加模型(Loewe additivity model)使用過量抑制2D矩陣來分析，且報導為協同作用分值(Lehar等人,Nat Biotechnol . 2009 年 7月 ; 27(7): 659-666)。所有計算使用在地平線網站(Horizon website)中可獲得之Chalice TM生物資訊軟體來實施。 表9中所指示之倍增時間為自細胞解凍至其在96孔盤中接種之實施的不同代(在T-150燒瓶中)中所獲得之倍增時間的平均值。 協同作用分值之解釋如下： SS ~ 0 → 相加性 SS ＞1 →弱協同 SS ＞2 → 協同表 9. 用於組合實驗中之8個B-ALL及10個T-ALL細胞株的識別及分析條件。
Figure 106124599-A0304-0017
 表 10. 指示8個B-ALL及10個T-ALL細胞株中化合物3及化合物1，HCl的單一藥劑IC50 值。化合物在3天期間與細胞一起培育。
Figure 106124599-A0304-0018
 表 11. 指示8個B-ALL及10個T-ALL細胞株中化合物3及化合物1，HCl組合的協同作用分值。當觀測之分值≥ 2.0時，認為相互作用為協同的。指示化合物之起始濃度、最大抑制之平均值及協同作用分值之標準差(sd)。化合物在3天期間與細胞一起培育。
Figure 106124599-A0304-0019
 結果 分析組合MCL1抑制劑及BCL-2抑制劑於8個B-ALL及10個T-ALL細胞株組中對增殖的影響。 MCL1抑制劑作為單一藥劑強有力地抑制了所測試之大部分ALL細胞株的生長(表10)。因此，13個ALL細胞株展示小於100 nM的IC50 ，且額外2個ALL細胞株展示100 nM與1 μM之間的IC50 。僅3個ALL細胞株展示大於1 μM的IC50 。 BCL-2抑制劑作為單一藥劑亦抑制了所測試之若干個ALL細胞株的生長，儘管其不太有效(表10)。因此，5個細胞株顯示小於100 nM之IC50 ，且2個細胞株顯示100 nM與1 μM之間的IC50 。11個ALL細胞株展示大於1 μM的IC50 。 MCL1抑制劑及BCL-2抑制劑組合治療造成所測試之總體17/18之ALL細胞株的協同生長抑制(亦即高於2之協同作用分值-Lehar等人,Nat Biotechnol .2009 年 7月 ; 27(7): 659-666)(表11)。在6個細胞株中，用5與10之間的協同作用分值標記協同效應。在5個細胞株中，協同效應為優越的，獲得10與15.9之間的協同作用分值。重要的是，協同不依賴於單一藥劑抗增殖效應，且事實上在MCL1抑制劑及BCL-2抑制劑自身不具有抗增殖效應之濃度下協同為尤其強的。舉例而言，在NALM-6細胞中，所測試之第四最低濃度的MCL1抑制劑及BCL-2抑制劑分別引發6%及8%的生長抑制，而兩種化合物之對應組合獲得61%的生長抑制(圖11，左上圖)。 此外，值得注意的是，協同效應在遍及大範圍之單一藥劑濃度內出現，此應證明關於給藥含量及安排之靈活性方面在活體內為有益的。 總而言之，MCL1抑制劑及BCL-2抑制劑之組合獲得大部分(17/18)所測試之ALL細胞株的協同生長抑制。重要的是，在所測試之5/18的ALL細胞株中觀測到優越的協同生長抑制。實例 11 ： 組合 MCL1 抑制劑及 BCL - 2 抑制劑於 5 個套細胞淋巴瘤 ( MCL ) 細胞株組中對增殖的活體外影響。 材料及方法 細胞株來源於且保持在表12中所指示之補充有FBS的鹼性培養物中。另外，所有培養物皆含有青黴素(100 IU/ml)、鏈黴素(100 µg/ml)及L-麩醯胺酸(2 mM)。 細胞株在37℃下在含有5% CO2 的潮濕氛圍中培養且在T-150燒瓶中擴增。在所有情況下，自冷凍儲備液解凍細胞，使用合適的稀釋液擴增≥1代，使用CASY細胞計數器計數且分析存活率，隨後以表12中所指示之密度塗鋪150 μl/孔至96孔盤中。所有細胞株皆測定為內部無黴漿菌污染物。 化合物之儲備溶液以在DMSO中5 mM之濃度製備且儲存於-20℃下。為了分析以單一藥劑或組合形式的化合物活性，細胞接種且用各化合物之7或8個3.16倍的連續稀釋液處理，各化合物單獨地或以棋盤格方式之所有可能的排列直接施配至細胞分析盤中。在培育2天之後，分析單一藥劑及其棋盤格組合對細胞存活率的影響，該分析在37℃/5% CO2 下藉由使用CellTiterGlo以150 μL反應劑/孔量化細胞ATP含量來進行。所有條件進行重複三次測試。在多用途盤讀取器上量化發光。 化合物組合之間的潛在協同相互作用根據洛伊相加模型(Loewe additivity model)使用過量抑制2D矩陣來分析，且報導為協同作用分值(Lehar等人, Nat Biotechnol.2009 年 7月 ; 27(7): 659-666)。所有計算使用可在地平線網站中獲得之ChaliceTM 生物資訊軟體來實施。 使用標準四參數曲線擬合計算單一藥劑IC50 。IC50 定義為CTG信號減少至媒劑(DMSO)對照所量測之50%時的化合物濃度。 表12中所指示之倍增時間為自細胞解凍至其在96孔盤中接種之實施的不同代(在T-150燒瓶中)中所獲得之倍增時間的平均值。協同作用分值 SS ~ 0 → 相加性 SS ＞1 → 弱協同 SS ＞2 →協同表 12. 用於組合實驗中之5個套細胞淋巴瘤細胞株的識別及分析條件。表 13. 指示5個套細胞淋巴瘤細胞株中化合物3及化合物1，HCl的單一藥劑IC50 值。化合物在2天期間與細胞一起培育。
Figure 106124599-A0304-0021
 表 14. 指示5個套細胞淋巴瘤細胞株中化合物3及化合物1，HCl組合的協同作用分值。當觀測之分值≥ 2.0時，認為相互作用為協同的。指示化合物之起始濃度、最大抑制及協同作用分值。化合物在2天期間與細胞一起培育。
Figure 106124599-A0304-0022
 結果 分析組合MCL1抑制劑及BCL-2抑制劑於5個套細胞淋巴瘤細胞株組中對增殖的活體外影響。 作為單一藥劑，MCL1抑制劑展示相比於BCL-2抑制劑更優越的活性。因此，針對MCL1抑制劑3個細胞株展示小於100 nM的IC50 ，然而針對BCL-2抑制劑僅一個細胞株展示小於100 nM的IC50 (表13)。 MCL1抑制劑及BCL-2抑制劑組合治療造成所有測試之細胞株的協同生長抑制(表14)(亦即高於2之協同作用分值-Lehar等人,Nat Biotechnol .2009 年 7月 ; 27(7): 659-666)，如圖12中所例示。重要的是，在4/5個細胞株中，用高於5之協同作用分值標記協同效應。實例 12 ： 組合 MCL1 抑制劑及 BCL - 2 抑制劑於 5 個小細胞肺癌 ( SCLC ) 細胞株組中對增殖的活體外影響。 所有細胞株由ATCC獲得。補充有10% FBS(海克隆；HyClone)之含有RPMI1640(英維羅根；Invitrogen)的培養物用於COR-L95、NCI-H146、NCI-H211、SHP-77、SW1271、NCI-H1339、NCI-H1963及NCI-H889。具有10% FBS之含有Waymouth's MB 752/1(英維羅根)的培養物用於DMS-273。含5% FBS之含有DMEM/F12(英維羅根)且補充有0.005 mg/ml胰島素、0.01 mg/ml運鐵蛋白質及30 nM亞硒酸鈉溶液(英維羅根)、10 nM氫皮質酮(Sigma)、10 nM β-雌二醇(Sigma)及2 mM L-麩醯胺酸(海克隆)的培養物用於NCI-H1105。 在37℃及5% CO2 培養箱中培養且在T-75燒瓶中擴增細胞株。在所有情況下，自冷凍儲備液解凍細胞，使用1:3稀釋液擴增≥1代，使用ViCell計數器(Beckman-Coulter)計數且分析存活率，隨後塗鋪於384孔盤中。為了分離及擴增細胞株，細胞使用0.25%胰蛋白質酶-EDTA (GIBCO)自燒瓶移出。如藉由在Idexx Radil (Columbia, MO, USA)中進行之PCR偵測方法所測定且藉由SNP組偵測恰當地確認，所有細胞株測定為無黴漿菌污染物。 細胞增殖在72小時之CellTiter-Glo™ (CTG)分析(Promega G7571)中量測，且所顯示之所有結果為至少重複三次量測的結果。為了CellTiter-Glo™分析，細胞施配至經組織培養物處理的384孔盤(Corning 3707)，其具有35 μL之最終體積的培養物且呈每孔5000個細胞的密度。塗鋪24小時之後，將5 μL各化合物稀釋組轉移至含有細胞之盤中，產生0至10 μM範圍內的化合物濃度及0.16%之最終DMSO (Sigma D8418)濃度。將盤培育72小時且使用CellTiter-GloTM 發光細胞存活率分析(Promega G7571)及Envision盤讀取器(Perkin Elmer)測定化合物對細胞增殖之影響。 CellTiter-Glo®發光細胞存活率分析為基於所存在之ATP數量(其指示代謝活性細胞之存在)測定培養物中活細胞之數目的均質方法。該方法詳細描述於Technical Bulletin, TB288 Promega中。簡言之，在如上所述之培養物中在不透明壁多孔盤中塗鋪細胞。亦製備含有培養物而不含細胞之對照孔以獲得背景發光值。隨後添加15 μL之CellTiter-Glo®反應劑且在定軌振盪器(orbital shaker)上混合內含物10分鐘以誘使細胞分解。隨後，使用盤讀取器記錄發光。 使用Chalice軟體(CombinatoRx, Cambridge MA)分析生長抑制及過量抑制百分比。在經板標記之抑制中顯示相對於DMSO之生長抑制百分比，且抑制量超過經板標記之ADD過量抑制中之預期量(圖15(a)至圖15(e))。沿自左至右之底部列展示化合物1，HCl之濃度及沿自底部至頂部之最左邊行展示化合物3之增加濃度。網格顯示器中之所有剩餘點由對應於兩個軸上指示之單一藥劑濃度的兩種抑制劑的組合產生。細胞增殖之資料分析使用描述於Lehar等人,Nat Biotechnol .2009 年 7月 ; 27(7): 659-666中之Chalice Analyser 實施。使用Loewe協同模型計算過量抑制，其量測對生長之影響，該生長相對於兩種藥物以一種劑量添加方式表現時預期之生長。正數表示增加協同之區域。協同作用分值 SS ~ 0 → 劑量相加性 SS ＞2 →協同 SS ＞1 → 弱協同 結果 化合物1及化合物3組合治療造成8/10小細胞肺癌細胞株的協同生長抑制(亦即高於2之協同作用分值)。重要的是，在6個細胞株中，用高於6之協同作用分值標記協同效應。實例 13 ： MCL1 抑制劑 ( 化合物 3 ) 及 BCL - 2 抑制劑 ( 化合物 1 ， HCl 或 ABT - 199 ) 之組合對源自患者之原發性 AML 模型 HAMLX5343 的活體內療效 材料及方法 材料 動物 在操作之前，使稱重為17至27公克(傑克遜實驗室(Jackson Laboratories))的免疫不全γ (NOD scid gamma；NSG)雌性小鼠隨意取用食物及水3天以適應新環境。原發腫瘤模型 攜帶KRAS 突變之源自患者之原發性AML模型HAMLX5343及野生型FLT3 自Dana Farber癌症研究所獲得。測試化合物 ， 調配物 化合物1，HCl在5%乙醇，20% Dexolve-7中調配為溶液以用於靜脈內投與，或在PEG300/EtOH/水(40/10/50)中調配以用於口服。ABT-199在PEG300/EtOH/水(40/10/50)中調配以用於口服。其所有在4℃下穩定至少一週。化合物3在脂質調配物中調配為溶液以用於靜脈內調配物，該調配物在4℃下穩定3週。按需要製備媒劑及化合物給藥溶液。所有動物以10 mL/kg給藥化合物1(表現為游離鹼)或ABT-199，或以5 mL/kg給藥化合物3。方法 研究設計 8個治療組用於研究7844HAMLX5343-XEF中，如表15中所概述。當平均腫瘤負荷(CD-45陽性細胞%)在8%與15%之間時，起始所有治療。 在此研究中，作為單一藥劑，化合物1藉由口服管飼(po)或靜脈內投與以50 mg/kg一週投與一次，ABT-199藉由口服管飼(po)以25 mg/kg一週投與一次，或組合化合物3以12.5 mg/kg一週投與一次，持續18天。 化合物1(表現為游離鹼)及ABT-199兩者以10 mL/kg投與。化合物3以5 mL/kg投與。劑量根據體重調節。體重每週記錄兩次且腫瘤負荷每週記錄一次。 表15. 7844HAMLX5343-XEF之劑量*及劑量安排
Figure 106124599-A0304-0023
 * 劑量表現為游離鹼 原發性 AML 模型 對此實驗，32個小鼠植入有原發性AML株HAMLX5343。小鼠靜脈內注射2.0百萬個白血病細胞。當腫瘤負荷在8%至15%之間時，動物隨機分佈成8個組，各組四個小鼠，各用於媒劑、化合物1 (po)、化合物1 (iv)、ABT-199、化合物3或組合治療。在治療18天之後，當腫瘤負荷達到99%時終止研究。腫瘤負荷藉由FACS分析量測。動物監測 每日兩次監測動物健康及行為，包括清整及移動。監測小鼠的總體健康且每日記錄死亡率。殺死任何垂死動物。腫瘤量測 小鼠每週一次經由剪尾抽血。血液分成96孔盤之IgG對照孔及CD33/CD45孔。血液在室溫下用200 µl RBC裂解緩衝液裂解兩次，隨後用FACS緩衝液洗滌一次(PBS中具有5% FBS)。隨後，樣品在100 µl阻斷緩衝液(5%小鼠Fc嵌段+5%人類Fc嵌段+90% FACS緩衝液)中在4℃下培養10至30分鐘。將20 µl IgG對照混合物(2.5 µl小鼠igG1 K同型對照-PE+2.5 µl小鼠igG1 K同型對照-APC+15 µl FACS緩衝液)添加至IgG對照孔及20 µl CD33/CD45混合物(2.5 µl小鼠抗人類CD33-PE+2.5 µl小鼠抗人類CD45-APC+15 µl FACS緩衝液)。在分析之前，樣品在4℃下培養30至60分鐘，隨後洗滌兩次。樣品在具有FACSDiva軟體之Canto上操作。用FloJo軟體進行分析。CD45-陽性活體單細胞之百分比報導為腫瘤負荷。資料分析 治療/對照(T/C)百分比值使用下式計算： %T/C = 100 ´ DT/DC若DT ＞0 %消退= 100 ´ DT/T初始 ，若DT ＜0 其中： T=最終研究日之藥物治療組的平均腫瘤負荷； DT=最終研究日之藥物治療組之平均腫瘤負荷-初始給藥日之藥物治療組之平均腫瘤負荷； T初始 =初始給藥日之藥物治療組的平均腫瘤負荷； C=最終研究日之對照組的平均腫瘤負荷；及 DC=最終研究日之對照組的平均腫瘤負荷-初始給藥日之對照組的平均腫瘤負荷。 所有資料皆表現為平均值±SEM。△腫瘤負荷及體重用以統計分析。最終量測值之組之間的對比使用杜凱氏測試之ANOVA實施。使用GraphPad Prism進行統計分析。統計分析 所有資料表現為平均值±平均值標準誤差(SEM)。△腫瘤體積及體重用以統計分析。使用Kruskal-Wallis ANOVA進行組之間的對比，隨後進行事後鄧恩測試(post hoc Dunn's test)或杜凱氏測試(Tukey's test)。對於所有統計評估，顯著性水準設定為p＜0.05。除非另外說明，否則報導相比於媒劑對照組之顯著性。用於藥理學研究之標準協定未預先確定功效以展現組合相較於對應單一藥劑治療之統計上的顯著優越性。統計功效常常受有效單一藥劑反應及/或模型變異限制。然而，提供用於組合對比單一藥劑治療的p值。 結果 組合之 MCL1 及 BCL - 2 抑制的協同抗腫瘤效應 在7844HAMLX5343-XEF研究中，在一週一次分別以50 mg/kg(口服或iv )、25 mg/kg(口服)或12.5 mg/kg (iv )投與時，僅化合物1、ABT-199或化合物3在攜帶KRAS 突變之HAMLX5343模型中不顯示抗腫瘤活性(分別98%、92%、98%或99%的T/C%，p＞0.05)。 在此模型中，當一週一次以50 mg/kg之化合物1或以25 mg/kg之ABT-199結合化合物3 (12.5 mg/kgiv )經口投與時，導致腫瘤停滯(分別3%或6%之T/C%，p＜0.05)。 另一方面，靜脈內投與化合物1與化合物3之組合引起腫瘤幾乎完全消退(100%之消退%)，此明顯不同於單一藥劑(p＜0.05)或化合物1/化合物3 po/iv 組合。對於18天治療時段針對時間標繪各治療組的平均腫瘤負荷，如圖1中所示。腫瘤負荷、T/C%或消退%的變化呈現於表16及圖16(a)至16(b)中。表 16. 7844HAMLX5343-XEF研究中抗腫瘤效應的概述
Figure 106124599-A0304-0024
 * p ＜ 0.05對比媒劑及單一藥劑 (ANOVA，杜凱氏測試(Tukey's test)) ** p ＜ 0.05對比po / iv 組合 (ANOVA，杜凱氏測試) 結論 AML為攻擊性及異質的惡性血液病，其由因獲得基因改變之造血祖細胞的轉型造成(Patel等人,New England Journal of Medicine 2012 366:1079-1089)。AML之5年存活率由於缺乏有效療法而較低。細胞凋亡之逃避為癌症之特點(Hanahan等人Cell 2000 100:57-70) 癌細胞藉由其逃避細胞凋亡的主要手段中之一者為藉由上調促存活BCL-2家族蛋白質，諸如BCL-2、BCL-xL及MCL1。 MCL1基因為癌症患者中最常見的擴增基因。(Beroukhim等人, Nature 2010 463:899-905)。此外，BCL-2及MCL1兩者在AML中高度表現。因此，化合物1 (BCL-2i)及化合物3 (MCL1)之組合可藉由增強促凋亡信號提供協同以作為通用機制抵抗AML。 此處吾人表明BCL-2抑制劑化合物1或ABT-199結合化合物3(MCL1抑制劑)在治療具有KRAS突變(wt FLT3)之AML異種移植模型中的AML中具有顯著的協同效應。iv / iv 化合物1/化合物3組合優於相同劑量之po / iv 組合治療。結果表明BCL-2及MCL1抑制劑之組合將為AML的有效療法。Accordingly, the present invention provides in embodiment E1 a combination comprising (a) a BCL-2 inhibitor of formula (I):
Figure 02_image012
Wherein: ¨ X and Y represent carbon atoms or nitrogen atoms, it should be understood that they may not represent two carbon atoms or two nitrogen atoms at the same time, ¨ A 1 and A 2 together with the atoms carrying them form a substituted by 5, Aromatic or non-aromatic heterocycle Het consisting of 6 or 7 ring members, in addition to the nitrogen represented by X or Y, it may also contain 1 to 3 heteroatoms independently selected from oxygen, sulfur and nitrogen, It should be understood that the nitrogen in question may be substituted with a group representing a hydrogen atom, a straight or branched chain (C 1 -C 6 )alkyl, or the group -C(O)-O-Alk, where Alk is straight chain or Branched-chain (C 1 -C 6 ) alkyl, or A 1 and A 2 independently of each other represent a hydrogen atom, linear or branched (C 1 -C 6 ) polyhaloalkyl, linear or branched (C 1 ) -C 6 ) alkyl or cycloalkyl, ¨ T represents a hydrogen atom, a straight-chain or branched (C 1 -C 6 ) alkyl group optionally substituted with 1 to 3 halogen atoms, a group (C 1 -C 6 ) 4 ) alkyl-NR 1 R 2 or group (C 1 -C 4 ) alkyl-OR 6 , ¨ R 1 and R 2 independently represent a hydrogen atom or a straight or branched chain (C 1 -C 6 ) Alkyl, or R 1 and R 2 together with the nitrogen atom carrying it form a heterocycloalkyl group, ¨ R 3 represents straight or branched (C 1 -C 6 ) alkyl, straight or branched (C 2 - C 6 ) alkenyl, straight or branched chain (C 2 -C 6 )alkynyl, cycloalkyl, (C 3 -C 10 )cycloalkyl-(C 1 -C 6 )alkyl, wherein the alkyl moiety is straight-chain or branched, heterocycloalkyl, aryl or heteroaryl, it being understood that one or more of the carbon atoms of the aforementioned groups or the carbon atoms of their possible substituents may be deuterated, ¨ R 4 represents aryl, heteroaryl, cycloalkyl or straight or branched chain (C 1 -C 6 )alkyl, it being understood that one or more of the carbon atoms of the aforementioned groups or the carbon atoms of their possible substituents Deuterated, ¨ R 5 represents hydrogen or halogen atom, straight or branched (C 1 -C 6 ) alkyl or straight or branched (C 1 -C 6 ) alkoxy, ¨ R 6 represents hydrogen atom or straight chain or branched chain (C 1 -C 6 ) alkyl, ¨R a , R b , R c and R d each independently represent R 7 , a halogen atom, straight chain or branched chain (C 1 -C 6 ) ) alkoxy, hydroxyl, straight or branched chain (C 1 -C 6 ) polyhaloalkyl, trifluoromethoxy, -NR 7 R 7 ', nitro, R 7 -CO-(C 0 -C 6 ) alkyl-, R 7 -CO-NH-(C 0 -C 6 ) alkyl-, NR 7 R 7 '-CO-(C 0 -C 6 ) alkyl-, R 7 -SO 2 -NH -(C 0 -C 6 )alkyl-, R 7 -NH-CO-NH-(C 0 -C 6 )alkyl-, R 7 -O-CO-NH-(C 0 -C 6 )alkyl-, heterocycloalkyl, or para (R a , R b ), (R b , R c ) or (R c , R d ) The substituents of one of them, together with the carbon atoms that carry it, form a ring of 5 to 7 ring members, which may contain 1 to 2 heteroatoms selected from oxygen and sulfur, and the ring as defined above is also to be understood One or more of the carbon atoms may be deuterated or substituted with 1 to 3 groups selected from halogen and straight or branched (C 1 -C 6 ) alkyl groups, ¨ R 7 and R 7 ' represent independently of each other hydrogen, straight or branched (C 1 -C 6 ) alkyl, straight or branched (C 2 -C 6 ) alkenyl, straight or branched (C 2 -C 6 ) alkynyl, aryl or Heteroaryl, or R 7 and R 7 ', together with the nitrogen atom carrying it, form a heterocycle composed of 5 to 7 ring members, it should be understood that when the compound of formula (I) contains a hydroxyl group, the latter can be converted into One of the following groups: -OPO(OM)(OM'), -OPO(OM)(O - M 1 + ), -OPO(O - M 1 + )(O - M 2 + ), -OPO (O - )(O - )M 3 2 + , -OPO(OM)(O[CH 2 CH 2 O] n CH 3 ) or -OPO(O - M 1 + )(O[CH 2 CH 2 O] n CH 3 ), wherein M and M' independently of one another represent a hydrogen atom, a straight-chain or branched-chain (C 1 -C 6 )alkyl, straight-chain or branched (C 2 -C 6 )alkenyl, straight-chain or Branched (C 2 -C 6 )alkynyl, cycloalkyl or heterocycloalkyl, both of which consist of 5 to 6 ring members, while M 1 + and M 2 + independently of each other represent a pharmaceutically acceptable a monovalent cation, M 3 2 + represents a pharmaceutically acceptable divalent cation, and n is an integer from 1 to 5, it being understood that: - "aryl" means phenyl, naphthyl, biphenyl or indenyl, - "Heteroaryl" means any monocyclic or bicyclic group of 5 to 10 ring members having at least one aromatic moiety and containing 1 to 4 atoms selected from the group consisting of oxygen, sulfur and nitrogen (including quaternary nitrogen) ), - "cycloalkyl" means any monocyclic or bicyclic non-aromatic carbocyclic group containing from 3 to 10 ring members, - "heterocycloalkyl" means any ring from 3 to 10 ring members Monocyclic or bicyclic non-aromatic fused or spiro groups consisting of and containing 1 to 3 heteroatoms selected from the group consisting of oxygen, sulfur, SO, SO and nitrogen, aryl, heteroaryl, Cycloalkyl and heterocycloalkyl, and the groups alkyl, alkenyl, alkynyl and alkoxy are substituted with 1 to 3 groups selected from hydroxy, morpholine, 3-3 as appropriate - Linear or branched chain (C 1 -C 6 ) alkyl substituted with difluoropiperidine or 3-3-difluoropyrrolidine; (C 3 -C 6 ) spiro; optionally substituted with morpholine or branched chain (C 1 -C 6 )alkoxy; (C 1 -C 6 )alkyl-S-; hydroxyl; pendant oxy; N -oxide; nitro; cyano; -COOR'; -OCOR ';NR'R"; straight or branched chain (C 1 -C 6 ) polyhaloalkyl; trifluoromethoxy; (C 1 -C 6 ) alkylsulfonyl; halogen; Aryl substituted with multiple halogens; heteroaryl; aryloxy; arylthio; cycloalkyl; optionally substituted with one or more halogen atoms or alkyl, where R' and R" independently of each other represent a hydrogen atom or a straight-chain or branched-chain (C 1 -C 6 )alkyl group optionally substituted by methoxy, the Het group as defined in formula (I) may be selected from straight-chain via 1 to 3 Or branched (C 1 -C 6 ) alkyl, hydroxyl, straight or branched (C 1 -C 6 ) alkoxy, NR 1 'R 1 " and halogen group substitution, it should be understood that R 1 ' and R 1 "is as defined for the groups R' and R" mentioned above, or an enantiomer, diastereomer, or addition thereof to a pharmaceutically acceptable acid or base The salt, and (b) the MCL1 inhibitor, are used simultaneously, sequentially or separately. The present invention also provides in embodiment E2 a combination comprising (a) a BCL-2 inhibitor and (b) an MCL1 inhibitor of formula (II):
Figure 02_image014
Wherein: ¨ A represents straight chain or branched chain (C 1 -C 6 ) alkyl, straight chain or branched chain (C 2 -C 6 ) alkenyl, straight chain or branched chain (C 2 -C 6 ) alkynyl, Linear or branched (C 1 -C 6 )alkoxy, -S-(C 1 -C 6 )alkyl, linear or branched (C 1 -C 6 )polyhaloalkyl, hydroxyl, cyano , -NW 10 W 10 ', -Cy 6 or a halogen atom, ¨W 1 , W 2 , W 3 , W 4 and W 5 independently represent a hydrogen atom, a halogen atom, a straight chain or a branched chain (C 1 -C 6 ) Alkyl, straight-chain or branched-chain (C2 - C6 ) alkenyl, straight-chain or branched-chain (C2 - C6 ) alkynyl, straight-chain or branched-chain ( C1 - C6 ) polyhaloalkane group, hydroxyl, straight or branched chain (C 1 -C 6 )alkoxy, -S-(C 1 -C 6 )alkyl, cyano, nitro, -alkyl(C 0 -C 6 )- NW 8 W 8 ', -O-Cy 1 , -alkyl(C 0 -C 6 )-Cy 1 , -alkenyl(C 2 -C 6 )-Cy 1 , -alkynyl(C 2 -C 6 ) -Cy 1 , -O-alkyl(C 1 -C 6 )-W 9 , -C(O)-OW 8 , -OC(O)-W 8 , -C(O)-NW 8 W 8 ', -NW 8 -C(O)-W 8 ', -NW 8 -C(O)-OW 8 ', -Alkyl(C 1 -C 6 )-NW 8 -C(O)-W 8 ', - SO 2 -NW 8 W 8 ', -SO 2 -alkyl (C 1 -C 6 ), or when grafted to two adjacent carbon atoms, for (W 1 , W 2 ), (W 2 , W 3 ), (W 1 , W 3 ), (W 4 , W 5 ) a substituent which, together with the carbon atom carrying it, forms an aromatic or non-aromatic ring consisting of 5 to 7 ring members, It may contain 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it being understood that the resulting ring may be selected from linear or branched (C 1 -C 6 ) alkyl, -NW 10 W 10 ', -alkane Substituted with radical (C 0 -C 6 )-Cy 1 or a pendant oxy group, ¨ X' represents carbon atom or nitrogen atom, ¨ W 6 represents hydrogen, straight chain or branched chain (C 1 -C 8 ) alkyl group , aryl, heteroaryl, arylalkyl (C 1 -C 6 ) group, heteroarylalkyl (C 1 -C 6 ) group, ¨ W 7 represents straight or branched chain (C 1 -C 6 ) C 6 ) alkyl, straight or branched chain (C 2 -C 6 ) alkenyl, straight or branched (C 2 -C 6 )alkynyl, -Cy 3 , -alkyl (C 1 -C 6 ) -Cy 3 , -Alkenyl(C 2 -C 6 )-Cy 3 , -alkynyl(C 2 -C 6 )-Cy 3 , -Cy 3 -Cy 4 , -alkynyl(C 2 -C 6 )-O-Cy 3 , -Cy 3 -alkyl(C 0 -C 6 ) -O-Alkyl (C 0 -C 6 )-Cy 4 , halogen atom, cyano group, -C(O)-W 11 or -C(O)-NW 11 W 11 ', ¨W 8 and W 8 ' independently of each other represent a hydrogen atom, a straight-chain or branched chain (C 1 -C 6 )alkyl or -alkyl(C 0 -C 6 )-Cy 1 , or (W 8 , W 8 ') and the nitrogen carrying it The atoms together form an aromatic or non-aromatic ring of 5 to 7 ring members, which may contain 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen in addition to the nitrogen atom, it being understood that the nitrogen in question Can be substituted by a group representing a hydrogen atom, or a straight or branched chain (C 1 -C 6 ) alkyl group, with the understanding that one or more carbon atoms of a possible substituent may be deuterated, ¨ W 9 represents -Cy 1 , -Cy 1 -alkyl(C 0 -C 6 )-Cy 2 , -Cy 1 -alkyl(C 0 -C 6 )-O-alkyl(C 0 -C 6 )-Cy 2 , -Cy 1 -Alkyl(C 0 -C 6 )-NW 8 -Alkyl(C 0 -C 6 )-Cy 2 , -Cy 1 -Cy 2 -O-Alkyl(C 0 -C 6 )-Cy 5 , - C(O)-NW 8 W 8 ', -NW 8 W 8 ', -OW 8 , -NW 8 -C(O)-W 8 ', -O-alkyl(C 1 -C 6 )-OW 8 , -SO 2 -W 8 , -C(O)-OW 8 , -NH-C(O)-NH-W 8 ,
Figure 02_image016
,
Figure 02_image018
or
Figure 02_image020
, the ammonium so defined may exist in the form of a zwitterion or have a monovalent anion opposite ion, ¨ W 10 , W 10 ', W 11 and W 11 ' independently of each other represent a hydrogen atom or a straight or branched chain (C 1 - C 6 ) alkyl group, ¨W 12 represents hydrogen or hydroxyl group, ¨W 13 represents hydrogen atom or straight or branched chain (C 1 -C 6 ) alkyl group, ¨W 14 represents -OP(O)(O - )( O - ) group, -OP(O)(O - )(OW 16 ) group, -OP(O)(OW 16 )(OW 16 ') group, -O-SO 2 -O - group, -O-SO 2 -OW 16 group, -Cy 7 , -OC(O)-W 15 group, -OC(O)-OW 15 group or -OC(O)-NW 15 W 15 ' group , ¨W 15 and W 15 ' independently of each other represent a hydrogen atom, a straight-chain or branched chain (C 1 -C 6 ) alkyl group or a straight-chain or branched chain amine group (C 1 -C 6 ) alkyl group, ¨W 16 and W 16 ' independently of each other represent a hydrogen atom, a straight or branched chain (C 1 -C 6 ) alkyl group or an arylalkyl (C 1 -C 6 ) group, ¨Cy 1 , Cy 2 , Cy 3 , Cy 4 , Cy 5 , Cy 6 and Cy 7 independently represent cycloalkyl, heterocycloalkyl, aryl or heteroaryl, ¨ n is an integer equal to 0 or 1, it should be understood that: - "aryl" means refers to phenyl, naphthyl, biphenyl, indenyl or indenyl, - "heteroaryl" means any monocyclic or bicyclic group of 5 to 10 ring members having at least one aromatic part and containing 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, - "cycloalkyl" means any monocyclic or bicyclic non-aromatic carbocyclic group containing 3 to 10 ring members, - "heterocyclic "Alkyl" means any monocyclic or bicyclic non-aromatic carbocyclic group containing 3 to 10 ring members and 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, which may include fused, bridged or Spiro ring systems, aryl, heteroaryl, cycloalkyl and heterocycloalkyl as may be so defined, and alkyl, alkenyl, alkynyl, alkoxy groups from 1 to 4 groups selected from Group substitution: linear or branched (C 1 -C 6 ) alkyl, which may be substituted with a group representing a linear or branched (C 1 -C 6 ) alkoxy group, the linear or branched (C 1 -C 6 ) alkoxy group 1 - C6 )alkoxy can be via straight-chain or branched-chain ( C1 - C6 )alkoxy, straight-chain or branched-chain ( C1 - C6 )polyhaloalkyl, hydroxy, halogen, pendant oxy , -NW'W", -OC(O)-W' or -CO-NW'W''substituted; linear or branched (C 2 -C 6 ) alkenyl; may be represented by a linear or branched chain ( C 1 -C 6 )alkoxy group-substituted straight or branched chain (C 2 -C 6 )alkynyl; may be represented by Linear or branched (C 1 -C 6 )alkoxy, linear or branched (C 1 -C 6 ) polyhaloalkyl, linear or branched (C 2 -C 6 )alkynyl, -NW Linear or branched (C 1 -C 6 )alkoxy groups substituted with 'W'' or hydroxyl groups; may be substituted with groups representing linear or branched (C 1 -C 6 )alkoxy groups (C 1 -C 6 )alkyl-S-; hydroxy; pendant oxy; N -oxide; nitro; cyano; -C(O)-OW';-OC(O)-W'; -CO -NW'W'';-NW'W'';-(C=NW')-OW''; linear or branched (C 1 -C 6 ) polyhaloalkyl; trifluoromethoxy; or halogen; it is to be understood that W' and W'' independently of each other represent a hydrogen atom or a linear or branched chain (C 1 -C 6 ) which may be substituted by a group representing a linear or branched (C 1 -C 6 )alkoxy group ) alkyl; and it should be understood that one or more of the carbon atoms of the foregoing possible substituents may be deuterated, its enantiomer, diastereomer or configurational isomer, or its pharmaceutically acceptable The addition salts of the accepted acids or bases are used simultaneously, sequentially or separately. A further enumerated embodiment (E) of the invention is described herein. It will be appreciated that features specified in each embodiment may be combined with other specified features to provide further embodiments of the invention. E3. A combination according to E1, wherein the MCL1 inhibitor is a compound of formula (II) as defined in E2. E4. A combination according to any one of E1 to E3, wherein the BCL-2 inhibitor is N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4- olinylmethyl)-3,4-dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl- 5,6,7,8-Tetrahydro-1-indolizinecarboxamide. E5. A combination according to any one of E1 to E3, wherein the BCL-2 inhibitor is 5-(5-chloro-2-{[( 3S )-3-(morpholin-4-ylmethyl) -3,4-Dihydroisoquinolin-2( 1H )-yl]carbonyl}phenyl)-N-(5-cyano-1,2- dimethyl - 1H -pyrrol-3-yl) - N- (4-hydroxyphenyl)-1,2-dimethyl- 1H -pyrrole-3-carboxamide. E6. A combination according to E4, wherein N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)-3,4-dihydro- 2( 1H )-Isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7,8-tetrahydro-1- Indolizine carboxamide is in the form of the hydrochloride salt. E7. A combination according to E5, wherein 5-(5-chloro-2-{[( 3S )-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinoline-2( 1 H )-yl]carbonyl}phenyl)-N-(5-cyano-1,2- dimethyl -1H-pyrrol - 3-yl)-N-(4-hydroxyphenyl)-1,2 -Dimethyl- 1H -pyrrole-3-carboxamide in the form of the hydrochloride salt. E8. A combination according to E4 or E6, wherein during combination therapy, N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)- 3,4-Dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7, 8-Tetrahydro-1-indolazinecarboxamide is available in doses ranging from 50 mg to 1500 mg. E9. A combination according to any of El to E8, wherein the BCL-2 inhibitor is administered once a week. E10. A combination according to E6 or E8, wherein during combination therapy, N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)- 3,4-Dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7, 8-Tetrahydro-1-indolizine carboxamide is administered once a day. E11. A combination according to any one of E1 to E3, wherein the BCL-2 inhibitor is ABT-199. E12. A combination according to any one of E1 to E11, wherein the MCL1 inhibitor is ( 2R )-2-{[( 5S a )-5-{3-chloro-2-methyl-4-[2- (4-Methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3- d ]pyrimidin-4-yl]oxy} -3-(2-{[1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propionic acid. E13. A combination according to any one of E1 to E11, wherein the MCL1 inhibitor is ( 2R )-2-{[( 5S a )-5-{3-chloro-2-methyl-4-[2- (4-Methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3- (2-{[2-(2-Methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid. E14. A combination according to any one of E1 to E13, wherein the BCL-2 inhibitor and the MCL1 inhibitor are administered orally. E15. A combination according to any one of E1 to E13, wherein the BCL-2 inhibitor is administered orally and the MCL1 inhibitor is administered intravenously. E16. A combination according to any one of E1 to E13, wherein the BCL-2 inhibitor and the MCL1 inhibitor are administered intravenously. E17. A combination according to any one of E1 to E16 for use in the treatment of cancer. E18. The combination for use according to E17, wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in a co-therapeutically effective amount for the treatment of cancer. E19. The combination for use according to E17, wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in synergistically effective amounts for the treatment of cancer. E20. The combination for use according to E17, wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in synergistically effective amounts to achieve a reduction in the required dose of each compound in cancer therapy, while providing effective cancer therapy, and ultimately reduced side effects. E21. The combination for use according to any one of E17 to E20, wherein the cancer is leukemia. E22. The combination for use according to E21, wherein the cancer is acute myeloid leukemia, T-ALL or B-ALL. E23. The combination for use according to any one of E17 to E20, wherein the cancer is myelodysplastic syndrome or a myeloproliferative disease. E24. The combination for use according to any one of E17 to E20, wherein the cancer is lymphoma. E25. The combination for use according to any of E24, wherein the lymphoma is non-Hodgkin's lymphoma. E26. The combination for use according to any one of E25, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma or mantle-cell lymphoma. E27. The combination for use according to any one of E17 to E20, wherein the cancer is multiple myeloma. E28. The combination for use according to any one of E17 to E20, wherein the cancer is neuroblastoma. E29. The combination for use according to any one of E17 to E20, wherein the cancer is small cell lung cancer. E30. A combination according to any one of E1 to E16, further comprising one or more excipients. E31. Use of a combination according to any one of E1 to E16 in the manufacture of a medicament for the treatment of cancer. E32. The use according to E31, wherein the cancer is leukemia. E33. The use according to E32, wherein the cancer is acute myeloid leukemia, T-ALL or B-ALL. E34. The use according to E31, wherein the cancer is myelodysplastic syndrome or myeloproliferative disease. E35. The use according to E31, wherein the cancer is lymphoma. E36. The use according to E35, wherein the lymphoma is non-Hodgkin's lymphoma. E37. The use according to E36, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma or mantle cell lymphoma. E38. The use according to E31, wherein the cancer is multiple myeloma. E39. The use according to E31, wherein the cancer is neuroblastoma. E40. The use according to E31, wherein the cancer is small cell lung cancer. E41. A medicament comprising, separately or jointly, (a) a BCL-2 inhibitor of formula (I) as defined in E1, and (b) an MCL1 inhibitor, for simultaneous, sequential or separate administration , and wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in effective amounts for the treatment of cancer. E42. A medicament comprising, separately or jointly, (a) a BCL-2 inhibitor, and (b) an MCL1 inhibitor of formula (II) as defined in E2, for simultaneous, sequential or separate administration , and wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in effective amounts for the treatment of cancer. E43. A method of treating cancer comprising administering to an individual in need thereof a co-therapeutically effective amount of (a) a BCL-2 inhibitor of formula (I) as defined in El, and (b) an MCL1 inhibitor. E44. A method of treating cancer comprising administering to an individual in need thereof a co-therapeutically effective amount of (a) a BCL-2 inhibitor, and (b) a MCL1 inhibitor of formula (II) as defined in E2. E45. A method for sensitizing a patient who is (i) refractory to treatment with at least one chemotherapy or (ii) relapses after treatment with chemotherapy, or both (i) and (ii), wherein the method comprises administering to the patient A co-therapeutically effective amount of (a) a BCL-2 inhibitor of formula (I) as defined in E1, and (b) an MCL1 inhibitor is administered. E46. A method for sensitizing a patient who is (i) refractory to treatment with at least one chemotherapy or (ii) relapses after treatment with chemotherapy, or both (i) and (ii), wherein the method comprises administering to the patient With a co-therapeutically effective amount of (a) a BCL-2 inhibitor, and (b) an MCL1 inhibitor of formula (II) as defined in E2. "Combination" means a fixed-dose combination, a variable-dose combination, or a set of sub-parts in one unit dosage form (eg, a capsule, lozenge, or sachet) for combined administration, wherein a compound of the present invention and one or more Combination partners (such as another drug, also referred to as a "therapeutic agent" or "adjuvant" as explained below) can be administered independently at the same time or administered separately at time intervals, especially when such time intervals allow the combination When things show cooperation, such as when a synergistic effect occurs. The terms "co-administered" or "administered in combination" or similar terms as used herein are intended to encompass the administration of a selected combination partner to a single individual (eg, a patient) in need thereof, and is intended to include that the agents are not necessarily delivered by the same Route of Administration The treatment regimen is administered or concurrently administered. The term "fixed dose combination" means that the active ingredients, eg, a compound of formula (I) and one or more combination partners are both administered to a patient simultaneously in a single entity or dose. The term "variable-dose combination" means that the active ingredients, such as a compound of the invention and one or more combination partners, are administered to a patient simultaneously or sequentially, without a specific time limit, as separate entities, wherein the administration provides therapy An effective amount of both compounds is administered to the patient. The latter also applies to mixture therapy, eg the administration of 3 or more active ingredients. "Cancer" means a class of diseases in which a population of cells exhibits uncontrolled growth. Cancer types include blood cancers (lymphomas and leukemias) and solid tumors including carcinomas, sarcomas or blastomas. In particular, "cancer" refers to leukemia, lymphoma or multiple myeloma, and more particularly to acute myeloid leukemia. The term "co-therapeutically effective" means that the therapeutic agents can be administered separately ( staggered in chronological order, especially in specific order). Whether or not this is the case, in particular, by blood content determination, it can be shown that both compounds are present in the blood of the human being treated, at least during certain time intervals. "Synergistically effective" or "synergistic" means that the therapeutic effect observed following administration of two or more than two agents is greater than the total therapeutic effect observed following administration of each single agent. As used herein, the term "treat/treating/treatment" of any disease or disorder refers in one embodiment to ameliorating the disease or disorder (ie, slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof). In another embodiment, "treat/treating/treatment" refers to alleviating or ameliorating at least one physiological parameter, including a physiological parameter that may not be discernible by a patient. In yet another embodiment, "treat/treating/treatment" refers to modulating physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physiological parameters), or both disease or condition. As used herein, an individual "requires" a treatment if the individual would benefit from the treatment biologically, medically, or in quality of life. In another aspect, there is provided a method for sensitizing a human (i) refractory to treatment with at least one chemotherapy or (ii) relapse following treatment with chemotherapy, or both (i) and (ii), wherein The method comprises administering to a patient a BCL-2 inhibitor of formula (I), as described herein, and an MCL1 inhibitor. Sensitive patients are those who have responded to therapy involving the administration of a BCL-2 inhibitor of formula (I), as described herein, and an MCL1 inhibitor, or who have not developed resistance to such therapy. "Drug" means a pharmaceutical composition, or a combination of several pharmaceutical compositions, containing one or more active ingredients in the presence of one or more excipients. "AML" means acute myeloid leukemia. "T-ALL" and "B-ALL" mean T-cell acute lymphoblastic leukemia and B-cell acute lymphoblastic leukemia. "Free base" refers to a compound that is not yet in the form of a salt. In the pharmaceutical composition according to the present invention, the proportion of the active ingredient by weight (the weight of the active ingredient in the total weight of the composition) is 5 to 50%. In the pharmaceutical compositions according to the present invention, more particularly those suitable for oral, parenteral and especially intravenous, whole- or anti-dermal, nasal, rectal, lingual, ocular or respiratory administration will be used, More precisely, lozenges, dragees, sublingual lozenges, hard gelatin capsules, rectal dosage forms, capsules, lozenges, injectables, sprays, eye or nasal drops, suppositories, creams, ointments , transdermal gel, etc. The pharmaceutical composition according to the present invention comprises one or more excipients or carriers selected from the group consisting of diluents, lubricants, binders, disintegrants, stabilizers, preservatives, adsorbents, colorants, sweeteners , flavoring agents, etc. By way of non-limiting example, mention may be made of: w as diluents : lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycerol, w as lubricants : silica, talc, stearin Acid and its magnesium and calcium salts, polyethylene glycol, w as binder : magnesium aluminum silicate, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone, w as Disintegrating agent : agar, alginic acid and its sodium salt, foaming mixture. The combined compounds can be administered simultaneously or sequentially. The route of administration is preferably the oral route, and the corresponding pharmaceutical composition may allow for immediate or delayed release of the active ingredient. Furthermore, the combined compounds can be administered in two separate pharmaceutical compositions each containing one of the active ingredients, or in a single pharmaceutical composition in which the active ingredients are in a mixture. Preference is given to pharmaceutical compositions in the form of lozenges. Pharmaceutical composition of compound 1 hydrochloride in film-coated tablet containing 50 mg and 100 mg of drug substance
Figure 106124599-A0304-0001
 Pharmacological Information Materials and Methods for Examples 1 - 3 : Primary AML Patient Samples : After informed consent, self-developed with AML according to guidelines approved by the Alfred Hospital Human research ethics committees Bone marrow or peripheral blood samples were collected from the patients. Monocytes were isolated by Ficoll-Paque (GE Healthcare, VIC, Aus) density gradient centrifugation, followed by depletion of erythrocytes in ammonium chloride ( NH4Cl ) lysis buffer at 37°C for 10 minute. Subsequently, cells were resuspended in phosphate buffered saline (Sigma, NSW, Aus) containing 2% fetal bovine serum. Subsequently, monocytes were suspended in RPMI-1640 (GIBCO VIC, Aus) cultures containing penicillin and streptomycin (GIBCO) and non-heat-activated fetal bovine serum 15% (Sigma). Cell lines, cell culture and production of luciferase reporter cell lines : cell lines MV4;11, OCI-AML3, HL-60, HEL, K562, KG-1 and EOL- 1 maintained at 37°C, 5% CO In RPMI-1640 (GIBCO) supplemented with 10% (v/v) fetal bovine serum (Sigma) and penicillin and streptomycin (GIBCO). The MV4;11 luciferase cell line was generated by lentiviral transduction. Antibodies : Primary antibodies used for western blot analysis were MCL1, BCL-2, Bax, Bak, Bim, BCL-XL (in-house WEHI generated) and tubulin (T-9026, Sigma) . Cell viability : Freshly purified monocytes from AML patient samples were adjusted to a concentration of 2.5 x 105/ml and 100 [mu]L cells per well were aliquoted into 96-well plates (Sigma). Cells were then treated with Compound 1, HCl, Compound 2, ABT-199 (Active Biochem, NJ, USA) or idarubicin (Sigma) spanning a 6-log concentration range from 1 nM to 10 μM for 48 hours. For combinatorial analysis, drugs were added in a 1:1 ratio from 1 nM to 10 μM and incubated at 37°C, 5% CO 2 . Cells were subsequently stained with sytox blue nucleic acid stain (Invitrogen, VIC, Aus) and fluorescein by flow cytometry using LSR-II Fortessa (Becton Dickinson, NSW, Aus). Measurement analysis measurement. FACSDiva software was used for data collection and FlowJo software was used for analysis. Blast cells were gated using forward and lateral dispersion properties. For each drug, viable cells depleted of sytox blue were determined at 6 concentrations, and the 50 % lethal concentration (LC50 in μM) was determined. LC50 Determination and Synergy : Graphpad Prism was used to calculate LC50 using nonlinear regression. Synergy was determined by calculating the composite index (CI) based on the method of Chou Talalay as described (Chou Cancer Res; 70(2), Jan 15, 2010). Colony Analysis : Colony formation assays were performed on freshly purified and frozen mononuclear fractions from AML patients. Primary cells were cultured in 1×10 4 to 1×10 5 double replicates in 35 mm dishes (Griener-bio, Germany). Cells were plated in 2:1:1 ratio of 0.6% agar (Difco NSW, Aus):AIMDM 2x (IMDM powder - Invitrogen), supplemented with NaHCO3 , polydextrose, penicillin/streptomycin, B mercaptoethanol and aspartame amine): fetal bovine serum (Sigma) coating. For optimal growth conditions, all plates contained GM-CSF (100 ng per plate), IL-3 (100 ng/dish R&D system, USA) SCF (100 ng/dish R&D system) and EPO (4U/dish) ( Grow in a high humidity incubator for 2 to 3 weeks in the presence and absence of drug at 37°C, 5% CO. After incubation, plates were fixed with 2.5% glutaraldehyde in normal saline and used from Oxford GelCount count by Optronix (Abingdon, United Kingdom). Western blotting method : in NP40 lysis buffer (10 mM Tris-HCl pH 7.4, 137) supplemented with protease inhibitor cocktail (Roche, Dee Why, NSW, Australia) Lysates were prepared in mM NaCl, 10% glycerol, 1% NP40. Protein samples were boiled in reduced loading dye followed by 4% to 12% Bis-Tris polyacrylamide gels (Invitrogen, Mulgrave, VIC, Australia). ) and transferred to Hybond C nitrocellulose membranes (GE, Rydalmere, NSW, Australia) for incubation with specific antibodies. All membrane blocking steps and antibody dilutions were performed using 5% (v/v) skim milk in Performed in PBS containing 0.1% (v/v) Tween-20 Phosphate Buffered Saline (PBST) or Tris-buffered Saline, and washed steps with PBST or TBST. Western blotting was performed by Enhanced Chemiluminescence (GE) Observation. In Vivo AML Transplantation : Animal studies were conducted under institutional guidelines approved by the Alfred Committee on Animal Ethics for Pharmaceutical Research and Education and will utilize a luciferase reporter gene (pLUC2) Transduced MV4;11 cells were injected intravenously at 1 x 10 cells into irradiated (100Rad) non-obese diabetic/severe combined immunodeficiency (NOD/SCID/IL2rγnull) mice as previously described (Jin et al. Human, Cell Stem Cell 2 July 2009, Vol. 5, Issue 1, pp. 31-42). Quantified at day 7 by flow cytometry and by IVIS imaging of bioluminescent MV4;11 cells Engraftment was measured by the percentage of hCD45+ cells in PB. On day 10, mice were orally gavaged daily with Compound 1 dissolved in PEG400 (Sigma) at 40:10:60, HCl (200 µL as free base) 100 mg/kg dose), absolute ethanol (Sigma), and distilled water, or twice weekly received in 50% 2-hydroxypropyl)-β-cyclodextrin (Sigma) Compound 2 (200 µL of 25 mg/kg) and 50% 50 mM HCl or drug combination or vehicle for 4 weeks. Blood counts were determined using a hematology analyzer (BioRad, Gladesville, NSW). IVIS imaging : Bioluminescence imaging was performed using the caliper IVIS Lumina III XR imaging system. Mice were anesthetized with isoflurane and 100 µL of 125 mg/kg luciferin (Perkin Elmer, Springvale, VIC) was injected intraperitoneally. Materials and Methods for Example 4 : Cell Line : Human Myeloma Cell Line (HMCL) was derived from primary myeloma cells grown in RPMI 1640 culture supplemented with 5% fetal bovine serum and 3 ng/ml Recombinant IL-6 for IL-6 related cell lines. HMCL is representative of phenotypic and genomic heterogeneity and variation in patient response to therapy. MTT assay : Cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric viability assay. Cells were incubated with compound each in 96-well plates containing a final volume of 100 µl/well. ( 2R )-2-{[( 5Sa )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazine-1 was used at 9 different concentrations based on single agent sensitivity -yl)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3-(2-{[1 -(2,2,2-Trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propionic acid (compound 2). N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)-3,4-dihydro-2( 1 H )-Isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7,8-tetrahydro-1-indole Oxacarboxamide hydrochloride (Compound 1, HCl). At the end of each treatment, cells were incubated with 1 mg/mL MTT (50 μl MTT solution 2.5 mg/ml per well) for 3 hours at 37°C to allow MTT to metabolize. Lysis buffer (100 µl lysis buffer:DMF (2:3)/SDS (1:3)) was added to each well to dissolve formazan cristal, and after 18 hours of incubation, a spectrophotometer was used. Absorbance was measured in living cells at 570 nm. As controls, cells were grown with culture alone and with culture containing 0.1% DMSO. As a myeloma cell growth control, the absorbance of myeloma cells (D0, D1, D2, D3 and D4) was recorded daily. All experiments were repeated 3 times, and each experimental condition was replicated at least 3 times in each experiment well. The inhibitory effect was calculated using the following formula: Inhibitory effect (%)=(1-absorbance value of treated cells/absorbance value of control cells)*100 Example 1 : BCL - 2 and MCL1 are dominant pro-survival proteins expressed in AML Seven AML cell lines with >70% blasts and 13 primary AML samples were immunoblotted for the indicated proteins in Figure 1 . As illustrated in Figure 1, proteomic measurements of the expression of BCL-2 family members in AML show that, in addition to BCL-2, most primary AML samples and AML cell lines co-express the pro-survival protein MCL1. BCL-XL is infrequently manifested in AML. Example 2 : Combination of BCL - 2 and MCL1 targeting in AML shows synergistic killing of 54 AML patient samples with a 6-log concentration range of Compound 1 (HCl), Compound 2, or 1:1 concentrations in RPMI/15% FCS were incubated for 48 hours and the LC50 was determined (FIG. 2A). Approximately 20% of primary AML samples were highly sensitive to Compound 1 or Compound 2, with lethal concentrations (LC50) of the drug required to kill 50 % of primary AML blasts after 48 hours at low nanomolar levels range (LC50 &lt ; 10 nM) (Fig. 2A). In contrast, when Compound 1 and Compound 2 were combined, the proportion of sensitive AML samples increased significantly to 70%, indicating synergistic activity when BCL-2 and MCL1 were simultaneously targeted (Figure 2A). Some results are shown in Figure 17. To verify the in vivo activity of this pathway, luciferase-expressing MV4;11 AML cells were transplanted into NSG mice and treated with Compound 1 (HCl) or Compound 2 alone, or a combination, and were treated at 14 days and Tumor burden was assessed after 21 days (Figure 2B). At the completion of 28 days of therapy, the mice continued to survive (Figure 2C). These experiments show that the combination of Compound 1 and Compound 2 is highly effective in vivo, validating the impressive activity observed in vitro using primary AML cells. The data presented here in Figures 2A-2C demonstrate synergistic combined activity between Compound 1, HCl and Compound 2 in AML. Example 3 : Combination of BCL - 2 and MCL1 inhibits target leukemia but no normal progenitor cell function for analysis of BCL-2 inhibition in combination with MCL1 inhibition on normal human CD34+ cells or ficolled from patients with AML Toxicity of blast cells, cell colony potential was analyzed after 2 weeks of exposure to combination therapy. Colonies were grown in agar supplemented with 10% FCS, IL3, SCF, GM-CSF and EPO for 14 days and colonies were counted with an automated Gelcount® analyzer. Analysis of primary AML samples was performed in duplicate and averaged. Error in CD34+ represents mean +/- SD of 2 independent normal donor samples. Results were corrected for the number of colonies counted in the DMSO control. The indicated drug concentrations were spread on D1. In particular, Compound 1 + Compound 2 suppressed AML colony-forming activity without affecting the function of normal CD34+ colony growth. In conclusion, Examples 2 and 3 show that dual pharmacological inhibition of BCL-2 and MCL1 is a novel approach to the treatment of AML that does not require additional chemotherapy and utilizes an acceptable therapeutic safety window. Example 4 : In Vitro Evaluation of Multiple Myeloma Cell Survival in Response to MCL1 Inhibitor as Single Agent or in Combination with a BCL - 2 Inhibitor in 27 Human Multiple Myeloma Cell Lines The sensitivity of Compound 2 in the presence of Compound 1 was analyzed by using the MTT cell viability assay. The 50% inhibitory concentration ( IC50 in nM) was determined. The results are shown in the table below:
Figure 106124599-A0304-0002
 When compound 1 was combined with compound 2, stronger synergistic activity was demonstrated in most cell lines compared to compounds alone. Example 5 : In Vitro Effects of Combining MCL1 Inhibitors with BCL - 2 Inhibitors on Proliferation in a Panel of 17 Diffuse Large B -Cell Lymphoma ( DLBCL ) Cell Lines Materials and Methods Cell lines were derived and maintained as shown in Table 1 In alkaline cultures supplemented with FCS (fetal calf serum) as indicated. In addition, all cultures contained penicillin (100 IU/ml), streptomycin (100 µg/ml) and L-glutamic acid (2 mM). Cultures and supplements were from Amimed/Bioconcept (Allschwil, Switzerland) unless otherwise mentioned. Cell lines were grown at 37°C in a humidified atmosphere containing 5% CO 2 and expanded in T-75 flasks. In all cases, cells were thawed from frozen stocks, expanded for ≥ 1 passage using appropriate dilutions, counted and assayed for viability using a CASY cytometer (Omni Life Science, Bremen, Germany), and subsequently at the densities specified in Table 1 Plate 25 μl/well into a 384-well plate (Corning). All cell lines were determined to be free of Mycoplasma contaminants by PCR assays performed at Idexx Radil (Columbia, MO, USA) and 48 small nucleotides by PCR assays performed at Asuragen (Austin, TX, USA) or in-house Polymorphism (SNP) groups exclude identification errors. Stock solutions of compounds were prepared at a concentration of 10 mM in DMSO (Sigma) and stored at -20°C. When required to obtain full dose-response curves, stock solutions were pre-diluted in DMSO to 1'000-fold the desired starting concentration (see Table 2). On the day following cell seeding, eight 2.5-fold serial dilutions of each compound were dispensed directly into the Cells were analyzed in the dish as outlined in Figure 4. The final DMSO concentration in all wells was 0.2%. After 2 days of incubation, the effects of single agents and their checkerboard combinations on cell viability were analyzed by using CellTiterGlo (Promega, Madison, WI, USA) at 37°C/5% CO 2 according to the conditions in 25 μL Reagents/well and n=2 replicate dishes were performed to quantify cellular ATP content. Luminescence was quantified on an M1000 multipurpose plate reader (TECAN, Männedorf, Switzerland). The number/viability of cells was also analyzed at the time of compound addition and was used to analyze the extent of population doubling time for a particular cell line. Single agent IC50s were calculated using standard four parameter curve fitting. Potential synergistic interactions between compound combinations were analyzed according to the Loewe additivity model using excess inhibition 2D matrix and reported as Synergy Score (Lehar et al, Nat Biotechnol . 2009 7 Jan; 27(7): 659-666). All calculations were performed using the Combination Analysis Module internal software. IC50 is defined as the concentration of compound at which CTG signal is reduced to 50% of that measured by vehicle (DMSO) control. Interpretation of synergy scores is as follows: SS ~ 0 → Additive SS > 1 → Weak synergy SS > 2 → Synergy Table 1. Identification and analysis of 17 diffuse large B-cell lymphoma cell lines used in combination experiments condition.
Figure 106124599-A0304-0003
 *This culture was further supplemented with 50 µM 2-mercaptoethanol. Doubling time was calculated based on the difference in ATP content at the end compared to the start of compound incubation. Table 2. Single agent IC50 values for specified Compound 3 and Compound 1, HCl, and synergy scores for their combinations. An interaction was considered synergistic when the observed score was ≥ 2.0.
Figure 106124599-A0304-0004
 "Start conc" means the starting concentration. "Anhydrous IC50 (Abs IC50 )" means anhydrous IC50 . "Max Inh" means maximum inhibition. Results The effect of combining MCL1 inhibitor compound 3 with BCL-2 inhibitor compound 1, HCl on proliferation was analyzed in 17 diffuse large B-cell lymphoma (DLBCL) cell line groups. Compound 3 as a single agent potently inhibited the growth of most of the 17 DLBCL cell lines tested (Table 1). Thus, 14 cell lines showed IC50s of less than 100 nM and an additional cell line showed IC50s between 100 nM and 1 μM . Only 2 cell lines showed IC50s greater than 1 μM . Compound 1, HCl as a single agent also inhibited growth in most of the 17 DLBCL cell lines tested, although less effectively (Table 2). Thus, 2 cell lines showed IC50s of less than 100 nM and 6 cell lines showed IC50s between 100 nM and 1 μM . Nine cell lines showed IC50s greater than 1 μM (4 of which were greater than 10 μM ). Combination treatment of Compound 3 and Compound 1, HCl caused synergistic growth inhibition (ie, synergy scores above 2) of 16 of the 17 DLBCL cell lines tested - Lehar et al., Nat Biotechnol . 2009 July; 27 (7): 659-666) (Table 2). Synergy was marked with a synergy score between 5 and 10 in 5 cell lines. In 4 cell lines, synergy was superior, with a synergy score between 10 and 17.3. Importantly, synergy was not dependent on a single agent antiproliferative effect, and in fact synergy was particularly strong at concentrations where Compound 3 and Compound 1 themselves did not exhibit an antiproliferative effect. For example, in DB cells, the second lowest concentration of Compound 3 and Compound 1 tested elicited only 1% and 2% growth inhibition, respectively, while the corresponding combination of the two compounds achieved 96% growth inhibition (Fig. 4A). , left panel), thus being 91% more additive than calculated based on single agent activity (Fig. 4A, right panel). As another example, in Toledo cells, where Compound 3 was less effective and achieved only partial growth inhibition (46%) at the highest concentration tested, the combination with the second lowest concentration of Compound 1 resulted in 98% synergistic growth Inhibition (Fig. 4B, left panel), and thus 52% higher than that calculated based on single agent activity (Fig. 4B, right panel). Furthermore, it is worth noting that synergistic effects occur over a wide range of single agent concentrations, which should prove beneficial in vivo with regard to flexibility in dosing levels and schedules. In conclusion, the combination of Compound 3 and Compound 1 resulted in strong to superior synergistic growth inhibition in most of the DLBCL cell lines tested. Example 6 : In vivo efficacy of a combination of MCL1 inhibitor ( Compound 3 ) and BCL - 2 inhibitor ( Compound 1 ) on Karpas 422 xenografts Materials and Methods Tumor Cell Culture and Cell Seeding Karpas 422 Human B Cells Non-Hodgkin's Lymphoma (NHL) cell lines were established from pleural effusions in patients with chemotherapy-resistant NHL. Cells were obtained from the DSMZ cell bank and were incubated with 10% FCS (BioConcept Ltd. Amimed), 2 mM L-glutamic acid (BioConcept Ltd. Amimed) at 37°C in an atmosphere of 5% CO in air. ), 1 mM sodium pyruvate (BioConcept Ltd. Amimed) and 10 mM HEPES (Gibco) in RPMI-1640 cultures (BioConcept Ltd. Amimed,). Cells were maintained between 0.5 x 106 and 1.5 x 106 cells/mL. To establish Karpas 422, xenograft cells were harvested and resuspended in HBSS (Gibco) and mixed with Matrigel (BD Bioscience) (1:1 v/v), then subcutaneously in the right flank of animals anesthetized with isoflurane. Inject 200 µL containing 1 x 107 cells. Twenty-four hours prior to cell seeding, all animals were irradiated with 5 Gy for more than 2 minutes using an ɤ-irradiator. Tumor Growth Tumor growth was monitored periodically after cell inoculation and animals were randomized into treatment groups (n=5) when tumor volume reached an appropriate volume. During the treatment period, tumor volume was measured using a caliper approximately twice a week. Tumor size in mm 3 was calculated from (L×W2×π/6). where W=width of tumor and L=length of tumor. Treatment Tumor bearing animals (rats) were enrolled into treatment groups (n=5) when their tumors reached the appropriate size to form groups with mean tumor volumes of approximately 450 mm3 . Treatment groups are summarized in Table 3. Vehicle for Compound 1, HCl or Compound 1, HCl was administered by oral ( po ) gavage 1 h prior to vehicle for Compound 3 or Compound 3 by 15 min iv infusion. For iv infusion, animals were anesthetized with isoflurane/O 2 and vehicle or compound 3 was administered via a cannula in the tail vein. Animals were weighed on the day of dosing and doses were adjusted according to body weight, 10 ml/kg for both compounds. Body Weight Animals are weighed at least twice a week and are often checked for obvious signs of any adverse effects. Data Analysis and Statistical Evaluation Tumor data were statistically analyzed using GraphPad Prism 7.00 (GraphPad Software). If the variance in the data was normally distributed, the data were analyzed using one-way ANOVA with a post hoc Dunnett's test for comparison of treatment and control groups. A post hoc Tukey's test was used for comparison. Alternatively, use the post hoc Dunn's test with Kruskal-Wallis ratings. Where appropriate, results are presented as mean ± SEM. As a measure of efficacy, the T/C% value was calculated at the end of the experiment according to the following formula: (Δtumor volume treated /tumor volume control )*100 Tumor regression was calculated according to the following formula: -(Δtumor volume treated /tumor volume started treatment h )*100 where Δtumor volume represents the mean tumor volume on the evaluation day minus the mean tumor volume at the start of the experiment. Table 3. Treatment Groups for Combination Efficacy in Karpass422 Xenograft Bearing Rats
Figure 106124599-A0304-0005
 Treatment was initiated when the mean tumor volume was approximately 450 mm3 . Compound 1, HCl was formulated in PEG400/EtOH/Phosal 50 PG (30/10/60) and compound 3 was placed in solution. QW means once a week. Results Combination treatment of Compound 1 free base at 150 mg/kg po 1 h prior to the iv infusion of Compound 3 at 20 mg/kg resulted in complete regression of all Karpas422 tumors from day 30 of treatment initiation (Figure 5). All animals in the treatment groups remained tumor free after treatment was discontinued on days 35 to 90. A positive combination effect was observed in the combination group compared to single agent activity. On day 34, tumor responses in the single-agent Compound 3 and combination groups were significantly different from those in the vehicle group (p<0.05). Combination therapy was well tolerated based on body weight change (Figure 6). Example 7 : In vivo efficacy of a combination of MCL1 inhibitor ( Compound 3 ) and BCL - 2 inhibitor ( Compound 1 , HCl) on DLBCL Toledo xenografts Materials and Methods A 3 million Toledo cell suspension of the gel was established directly subcutaneously (sc) into the subcutaneous area of SCID/beige mice. All procedures are performed using aseptic technique. Mice were anesthetized during the entire procedural period. In general, a total of 6 animals per group participated in efficacy studies. For single agent and combination studies, animals were dosed with Compound 1 via oral gavage (po) and Compound 3 administered intravenously (iv) via the tail vein. Compound 1, HCl was formulated as a solution in PEG300/EtOH/water (40/10/50) and compound 3 was placed in solution. When tumors reached approximately 220 mm3 on day 26 after cell implantation, tumor-bearing mice were randomized into treatment groups. The study design including dosing schedule for all treatment groups is summarized in the table below. Animals were weighed on the day of dosing and doses were adjusted according to body weight at 10 ml/kg. Tumor size and body weight were collected at randomization and thereafter twice weekly for the duration of the study. The following data were provided after data collection on each day: incidence of death, individual and group mean body weight, and individual and group mean tumor volume.
Figure 106124599-A0304-0006
 For studies in the Toledo model, treatment was started on day 26 after cell implantation when the mean tumor volume was approximately 218 to 228 mm3 . QW means once a week. Body weight ( BW ) was calculated as the percent change in body weight as: (BW current - BW initial )/(BW initial ) x 100. Data are presented as percent body weight change since the start of treatment. Tumor volume and percent of mice remaining in the study The treated/control (T/C) percent value was calculated using the following formula: T/C % = 100 ´ DT/DC if DT > 0 % regression = 100 ´ DT/T 0 if DT < 0 Where: T = mean tumor volume of drug treatment group on final study day; DT = mean tumor volume of drug treatment group on final study day - mean tumor volume of drug treatment group on initial dosing day; T 0 = group day The mean tumor volume of the drug-treated group; C = the mean tumor volume of the control group on the final study day; and DC = the mean tumor volume of the control group on the final study day - the mean tumor volume of the control group on the initial dosing day. Percentage of mice remaining in study = 6-number of mice reaching endpoint/6*100 Statistical analysis All data are presented as mean ± standard error of the mean (SEM). △Tumor volume and percentage change in body weight were used for statistical analysis. Comparisons between groups were performed using a one-way ANOVA followed by a post hoc Tukey test. For all statistical evaluations, the level of significance was set at p<0.05. Significance compared to the vehicle control group is reported unless otherwise stated. result
Figure 106124599-A0304-0007
 In the Toledo model, 100 mg/kg of Compound 1 free base produced a statistically significant antitumor effect of 37% T/C. Compound 3 at 25 mg/kg produced no antitumor effect of 102% T/C (Figure 7). The combination of Compound 1 + Compound 3 produced tumor stasis of 3% T/C, which was statistically significant compared to vehicle, Compound 1 and Compound 3 treated tumors (p<0.05 by one-way analysis of variance) test (one-way ANOVA test). Therefore, combined inhibition of BCL-2 and MCL1 in DLBCL may yield clinical therapeutic benefit. Additionally, Figure 8 shows the mean body weight change for Toledo. Treatment of mice with Compound 1, HCl and Compound 3 exhibited weight gain (1.081% and 2.3%, respectively). The combination group exhibited small weight loss (-3.2%). No other symptoms of adverse events were observed in this study. All 6 animals survived throughout the study. In summary, Example 2, Example 6, and Example 7 show that the combination of MCL1 inhibitor and BCL-2 inhibitor is well tolerated in mice and rats bearing xenografts derived from cell lines derived from acute myeloid leukemia and human lymphoma. The doses are effective, indicating that a suitable therapeutic window can be achieved in these diseases with this combination. Example 8 : In vitro effect of combined MCL1 inhibitor and BCL - 2 inhibitor on proliferation in a panel of 13 acute myeloid leukemia ( AML ) cell lines. Materials and Methods Cell lines were derived and maintained in alkaline culture supplemented with FBS (fetal bovine serum) as indicated in Table 1. In addition, all cultures contained penicillin (100 IU/ml), streptomycin (100 µg/ml) and L-glutamic acid (2 mM). Cell lines were grown at 37°C in a humidified atmosphere containing 5% CO 2 and expanded in T-150 flasks. In all cases cells were thawed from frozen stocks, expanded for ≥ 1 passage using appropriate dilutions, counted and assayed for viability using a CASY cytometer, and then plated with 150 μl/well to 96 wells at the densities indicated in Table 1 on the plate. All cell lines were determined to be free of mycoplasma contamination internally. Stock solutions of compounds were prepared at a concentration of 5 mM in DMSO and stored at -20°C. To assay the activity of compounds as single agents, cells were seeded and treated with nine 2-fold serial dilutions of each compound dispensed individually directly into cell assay dishes. After 3 days of incubation, compounds were assayed for their effect on cell viability by quantifying cellular ATP content using CellTiterGlo at 75 μL reagent/well at 37°C/5% CO 2 . All experiments were performed in triplicate. Luminescence was quantified on a multipurpose disc reader. Single agent IC50s were calculated using standard four parameter curve fitting. IC50 is defined as the concentration of compound at which CTG signal is reduced to 50% of that measured by vehicle (DMSO) control. To analyze compound activity in combination, cells were seeded and treated with 7 or 8 3.16-fold serial dilutions of each compound dispensed either individually or in all possible arrangements of a checkerboard directly into cell assay dishes , as indicated in Figure 9. After 3 days of incubation, the effects of single agents and their checkerboard combinations on cell viability were analyzed by quantifying cellular ATP content using CellTiterGlo at 75 μL reagents/well at 37°C/5% CO 2 . Two independent experiments were performed, each performed in duplicate. Luminescence was quantified on a multipurpose disc reader. Potential synergistic interactions between compound combinations were analyzed according to the Loewe additivity model using the excess inhibition 2D matrix and reported as synergy scores (Lehar et al. , Nat Biotechnol . 2009 Jul ; 27 ( 7): 659-666). All calculations were performed using Clalice ™ bioinformatics software. The doubling times indicated in Table 3 are the average of the doubling times obtained in the different passages (in T-150 flasks) performed from thawing of cells to their seeding in 96-well plates. Interpretation of synergy scores is as follows: SS ~ 0 → Additive SS > 1 → Weak synergy SS > 2 → Synergy Table 3. Identification and analysis conditions of 13 acute myeloid leukemia (AML) cell lines used in combination experiments .
Figure 106124599-A0304-0008
 Table 4a. Indicates the single agent IC50 values of Compound 3, Compound 1, HCl and ABT-199 in 13 AML cell lines. Compounds were incubated with cells over a 3 day period.
Figure 106124599-A0304-0009
 Table 4b. Indicates the single agent IC50 values of Compound 4, HCl in 5 AML cell lines. Compounds were incubated with cells over a 3 day period.
Figure 106124599-A0304-0010
 Table 5a. Indicates synergy scores for the combination of Compound 3 and Compound 1 in 13 AML cell lines. An interaction was considered synergistic when the observed score was ≥ 2.0. The initial concentration of the indicated compounds, the mean of maximal inhibition, and the standard deviation (sd) of the synergy score. Compounds were incubated with cells over a 3 day period.
Figure 106124599-A0304-0011
 Table 5b. Indicates synergy scores for the combination of Compound 3 and ABT-199 in 8 AML cell lines. An interaction was considered synergistic when the observed score was ≥ 2.0. The initial concentration of the indicated compounds, the mean of maximal inhibition, and the standard deviation (sd) of the synergy score. Compounds were incubated with cells over a 3 day period.
Figure 106124599-A0304-0012
 Table 5c. Indicates the synergy scores for the combination of Compound 3 and Compound 4, HCl in 5 AML cell lines. An interaction was considered synergistic when the observed score was ≥ 2.0. The initial concentration of the indicated compounds, the mean of maximal inhibition, and the standard deviation (sd) of the synergy score. Compounds were incubated with cells over a 3 day period.
Figure 106124599-A0304-0013
 Results Combination ( a ). The effect of combining MCL1 inhibitor compound 3 and BCL-2 inhibitor compound 1 on proliferation was analyzed in 13 acute myeloid leukemia (AML) cell line groups. Compound 3 as a single agent potently inhibited the growth of most of the 13 AML cell lines tested (Table 4a). Thus, 10 cell lines exhibited IC50s of less than 100 nM and an additional 2 cell lines exhibited IC50s between 100 nM and 1 μM . Only 1 cell line exhibited an IC50 greater than 1 μM. Compound 1, HCl as a single agent also inhibited the growth of several of the tested AML cell lines, although less effectively (Table 4a). Thus, 5 cell lines showed IC50s of less than 100 nM, and 2 cell lines showed IC50s between 100 nM and 1 μM. Six cell lines exhibited IC50s greater than 1 μM. Compound 3 and Compound 1, HCl combination treatment resulted in synergistic growth inhibition (ie, a synergy score higher than 2) of the overall 13 cell lines tested (Table 5a). In both cell lines, synergy was marked with a synergy score between 5 and 10. In 10 cell lines, synergy was superior, with a synergy score between 10 and 19.8. Importantly, the synergy was independent of a single agent antiproliferative effect, and in fact synergy was particularly strong at concentrations where Compound 3 and Compound 1 had no antiproliferative effect on their own. For example, in OCI-AML3 cells, the third lowest concentration of Compound 3 and Compound 1 tested elicited 5% and 1% growth inhibition, respectively, while the corresponding combination of the two compounds achieved 84% growth inhibition (Fig. 9A, top left panel), and thus 79% higher than the additivity calculated based on single agent activity (FIG. 9A, top right panel). Furthermore, it is worth noting that synergistic effects occur over a wide range of single agent concentrations, which should prove beneficial in vivo with regard to flexibility in dosing levels and schedules. In conclusion, the combination of Compound 3 and Compound 1 provided synergistic growth inhibition in all 13 AML cell lines tested. Importantly, superior synergistic growth inhibition was observed in most of the AML cell lines tested (10/13). Combination (b). The effect of combining the MCL1 inhibitor Compound 3 and the BCL-2 inhibitor ABT-199 on proliferation was analyzed in groups of 8 acute myeloid leukemia (AML) cell lines. Compound 3 as a single agent potently inhibited the growth of most of the eight AML cell lines tested (Table 4a). Thus, 5 cell lines exhibited IC50s of less than 100 nM and an additional 2 cell lines exhibited IC50s between 100 nM and 1 μM. Only 1 cell line exhibited an IC50 greater than 1 μM. ABT-199 as a single agent also inhibited the growth of AML cell lines, although less effectively (Table 4a). Therefore, only 1 cell line showed an IC50 of less than 100 nM, and 2 cell lines showed an IC50 of between 100 nM and 1 μM. Five cell lines exhibited IC50s greater than 1 μM. Combination treatment with MCL1 inhibitor and ABT-199 resulted in synergistic growth inhibition (ie, a synergy score above 2) of the entire group of 8 cell lines tested (Table 5b). In most cell lines, synergy was superior, obtaining a synergy score between 10 and 17.6. Importantly, synergy was independent of a single agent antiproliferative effect, and in fact synergy was particularly strong at concentrations where the MCL1 inhibitor and ABT-199 itself did not have an antiproliferative effect. For example, in OCI-AML3 cells, the third lowest concentration of MCL1 and ABT-199 tested elicited 26% and 18% growth inhibition, respectively, while the corresponding combination of the two compounds achieved 91% growth inhibition (Fig. 13, top left). Furthermore, it is worth noting that synergistic effects occur over a wide range of single agent concentrations, which should prove beneficial in vivo with regard to flexibility in dosing levels and schedules. In conclusion, the combination of Compound 3 and ABT-199 achieved synergistic growth inhibition of all 8 AML cell lines tested. Importantly, superior synergistic growth inhibition was observed in most of the AML cell lines tested (7/8). Combination (c). The effect of the combination of MCL1 inhibitor compound 3 and BCL-2 inhibitor compound 4 on proliferation was analyzed in 5 acute myeloid leukemia (AML) cell line groups. Compound 3 as a single agent potently inhibited the growth of the five AML cell lines tested (Table 4b). Therefore, all cell lines exhibited IC50s of less than 200 nM. Compound 4, HCl as a single agent also inhibited the growth of 4 of the 5 cell lines tested with an IC50 of less than or equal to 40 nM, and one cell line was resistant to Compound 4 with an IC50 of 10 µM. Combination treatment of Compound 3 and Compound 4, HCl resulted in synergistic growth inhibition (ie, a synergy score higher than 2) of the overall 5 cell lines tested (Table 5c). In both cell lines, synergy was marked with a synergy score between 5 and 10. In 1 cell line, the synergistic effect was superior, obtaining a synergy score of 16.5. Importantly, synergy was independent of single agent antiproliferative effects, and in fact synergy was particularly strong at concentrations where Compound 4, HCl, and Compound 3 had no or lower antiproliferative effects on their own. For example, in OCI-AML3 cells, the third lowest concentration tested, Compound 4, HCl, and Compound 3 elicited 1% and 40% growth inhibition, respectively, while the corresponding combination of the two compounds achieved 98% growth inhibition (FIG. 1A, left panel; representative of two independent experiments); thus 53% more additive than calculated based on single agent activity (Scheme 14A, right panel). In ML-2, the fifth lowest concentration tested, Compound 4, HCl, and Compound 3 elicited 18% and 26% growth inhibition, respectively, while the corresponding combination of the two compounds achieved 100% growth inhibition (Figure 14B, left Figure; representative of two independent experiments), thus 51% more additive than calculated based on single agent activity (Figure 15, right panel). In conclusion, the combination of Compound 4 and Compound 3 achieved synergistic growth inhibition of all 5 AML cell lines tested. Example 9 : In Vitro Effects of Combining MCL1 and BCL - 2 Inhibitors on Proliferation in a Panel of 12 Neuroblastoma ( NB ) Cell Lines Materials and Methods Cell lines were derived and maintained as indicated in Table 1 for the supplement in alkaline cultures with FBS. In addition, all cultures contained penicillin (100 IU/ml), streptomycin (100 µg/ml) and L-glutamic acid (2 mM). Cell lines were grown at 37°C in a humidified atmosphere containing 5% CO 2 and expanded in T-150 flasks. In all cases, cells were thawed from frozen stocks, expanded for ≥ 1 passage using appropriate dilutions, counted using a CASY cytometer and analyzed for viability, followed by plating 150 μl/well to 96 at the densities indicated in Table 6 in the hole plate. All cell lines were determined to be free of mycoplasma contamination internally. Stock solutions of compounds were prepared at a concentration of 5 mM in DMSO and stored at -20°C. To assay the activity of compounds as single agents, cells were seeded and treated with nine 3.16-fold serial dilutions of each compound dispensed individually directly into the cell assay dish. After 2 or 3 days of incubation (as indicated in Table 6), the effect of compounds on cell viability was analyzed by quantifying cellular ATP content at 37°C/5% CO by using CellTiterGlo at 150 μL reagent/well to proceed. Two independent experiments were performed, each performed in duplicate. All experiments were performed in triplicate. Luminescence was quantified on a multipurpose disc reader. Single agent IC50s were calculated using standard four parameter curve fitting. IC50 is defined as the concentration of compound at which CTG signal is reduced to 50% of that measured by vehicle (DMSO) control. The same experiments were performed to analyze potential synergistic interactions between compound combinations. Synergy scores were analyzed according to the Lowy additive model using an excess inhibition 2D matrix (Lehar et al., Nat Biotechnol . 2009 Jul; 27 (7): 659-666). All calculations were performed using Chalice™ bioinformatics software. The doubling times indicated in Table 6 are the average of the doubling times obtained in the different passages (in T-150 flasks) performed from thawing of cells to their seeding in 96-well plates. Interpretation of synergy scores is as follows: SS ~ 0 → Additive SS > 1 → Weak synergy SS > 2 → Synergy Table 6. Identification and Analysis of 12 Neuroblastoma (NB) Cell Lines Used in Combination Experiments condition.
Figure 106124599-A0304-0014
 Table 7. Indicated single agent IC50 values for Compound 3 and Compound 1, HCl. Compounds were incubated with cells over a 2 or 3 day period.
Figure 106124599-A0304-0015
 Table 8. Indicates synergy scores in combination with Compound 3 and Compound 1, HCl. An interaction was considered synergistic when the observed score was ≥ 2.0. Compounds were incubated with cells over a 2 or 3 day period.
Figure 106124599-A0304-0016
 Results The effect of the combination of the MCL1 inhibitor compound 3 and the BCL-2 inhibitor compound 1 on proliferation was analyzed in 12 neuroblastoma cell line groups. Three of the 12 cell lines tested were sensitive to compound 3 as a single agent (Table 7). 1 cell line displayed an IC50 of less than 100 nM and an additional 2 cell lines displayed an IC50 of between 100 nM and 1 μM. All cell lines were resistant to Compound 1, HCl as a single agent, with all cell lines tested exhibiting an IC50 greater than 1 μM. Combination treatment of Compound 3 and Compound 1 resulted in synergistic growth inhibition (ie, synergy scores above 2) of 11 of the 12 NB cell lines tested - Lehar et al. , Nat Biotechnol . 2009 Jul;27( 7): 659-666) (Table 8). Among the 5 cell lines, synergy was superior, obtaining a synergy score between 10 and 17.81. Importantly, the synergy was not dependent on a single agent antiproliferative effect, and in fact synergy was particularly strong at concentrations where Compound 3 and Compound 1, HCl itself did not exhibit an antiproliferative effect. For example, in LAN-6 cells, 630 nM of Compound 3 and Compound 1, HCl elicited only 12% and 0% growth inhibition, respectively, while the corresponding combination of the two compounds achieved 95% growth inhibition (Figure 10, upper left panel), and thus 76% greater than the additivity calculated based on single agent activity (Figure 10, upper right panel). In conclusion, the combination of Compound 3 and Compound 1 resulted in strong to superior synergistic growth inhibition for most neuroblastoma cell lines tested. Example 10 : Combination of MCL1 inhibitor and BCL - 2 inhibitor in 8 B - cell acute lymphoblastic leukemia group ( B - ALL ) and 10 T - cell acute lymphoblastic leukemia group ( T - ALL ) cell lines In vitro effects on proliferation. Materials and Methods Cell lines were derived and maintained in alkaline cultures supplemented with FBS as indicated in Table 1. In addition, all cultures contained penicillin (100 IU/ml), streptomycin (100 μg/ml) and L-glutamic acid (2 mM). Cell lines were grown at 37°C in a humidified atmosphere containing 5% CO 2 and expanded in T-150 flasks. In all cases, cells were thawed from frozen stocks, expanded for > 1 passage using appropriate dilutions, counted using a CASY cytometer and analyzed for viability, followed by plating 150 μl/well to 96 at the densities indicated in Table 9 in the hole plate. All cell lines were determined to be free of mycoplasma contamination internally. Stock solutions of compounds were prepared at a concentration of 5 mM in DMSO and stored at -20°C. To assay the activity of compounds as single agents, cells were seeded and treated with nine 2-fold serial dilutions of each compound dispensed individually directly into cell assay dishes. After 3 days of incubation, compounds were assayed for effect on cell viability by quantifying cellular ATP content using CellTiterGlo at 75 μL reagent/well at 37°C/5% CO 2 . All conditions were tested in triplicate. Luminescence was quantified on a multipurpose disc reader. Single agent IC50s were calculated using standard four parameter curve fitting. IC50 is defined as the concentration of compound at which CTG signal is reduced to 50% of that measured by vehicle (DMSO) control. To analyze compound activity in combination, cells were seeded and treated with 7 or 8 3.16-fold serial dilutions of each compound dispensed either individually or in all possible permutations of a checkerboard directly into cell assay plates , as indicated in Figure 1. After 3 days of incubation, the effects of single agents and their checkerboard combinations on cell viability were analyzed by quantifying cellular ATP content using CellTiterGlo at 75 μL reagent/well at 37°C/5% CO 2 . For the B-ALL cell line, two independent experiments were performed, each performed in duplicate. For the T-ALL cell line, one experiment was performed in triplicate. Luminescence was quantified on a multipurpose disc reader. Potential synergistic interactions between compound combinations were analyzed according to the Loewe additivity model using the excess inhibition 2D matrix and reported as synergy scores (Lehar et al. , Nat Biotechnol . 2009 Jul;27( 7): 659-666). All calculations were performed using the Chalice™ bioinformatics software available on the Horizon website. The doubling times indicated in Table 9 are the average of the doubling times obtained in the different passages (in T-150 flasks) performed from thawing of cells to their seeding in 96-well plates. Interpretation of synergy scores is as follows: SS ~ 0 → Additive SS > 1 → Weak synergy SS > 2 → Synergy Table 9. Identification of 8 B-ALL and 10 T-ALL cell lines used in combination experiments and analysis conditions.
Figure 106124599-A0304-0017
 Table 10. Indicated single agent IC50 values for Compound 3 and Compound 1, HCl in 8 B-ALL and 10 T-ALL cell lines. Compounds were incubated with cells over a 3 day period.
Figure 106124599-A0304-0018
 Table 11. Indicates synergy scores for the combination of Compound 3 and Compound 1, HCl in 8 B-ALL and 10 T-ALL cell lines. An interaction was considered synergistic when the observed score was ≥ 2.0. The initial concentration of the indicated compounds, the mean of maximal inhibition, and the standard deviation (sd) of the synergy score. Compounds were incubated with cells over a 3 day period.
Figure 106124599-A0304-0019
 Results The effects of combined MCL1 inhibitor and BCL-2 inhibitor on proliferation in 8 B-ALL and 10 T-ALL cell line groups were analyzed. The MCL1 inhibitor as a single agent potently inhibited the growth of most ALL cell lines tested (Table 10). Thus, 13 ALL cell lines displayed IC50s of less than 100 nM and an additional 2 ALL cell lines displayed IC50s between 100 nM and 1 μM . Only 3 ALL cell lines exhibited IC50s greater than 1 μM. The BCL-2 inhibitor as a single agent also inhibited the growth of several ALL cell lines tested, although it was less effective (Table 10). Thus, 5 cell lines showed IC50s of less than 100 nM, and 2 cell lines showed IC50s between 100 nM and 1 μM. Eleven ALL cell lines displayed IC50s greater than 1 μM. Combination treatment with MCL1 inhibitor and BCL-2 inhibitor resulted in synergistic growth inhibition (ie, a synergy score above 2) of the overall 17/18 ALL cell lines tested - Lehar et al. , Nat Biotechnol . Jul 2009 ; 27(7):659-666) (Table 11). Synergy was marked with a synergy score between 5 and 10 in 6 cell lines. Among the 5 cell lines, synergy was superior, obtaining a synergy score between 10 and 15.9. Importantly, synergy was independent of single agent antiproliferative effects, and in fact synergy was particularly strong at concentrations where MCL1 inhibitors and BCL-2 inhibitors themselves did not have antiproliferative effects. For example, in NALM-6 cells, the fourth lowest concentration of MCL1 inhibitor and BCL-2 inhibitor tested elicited 6% and 8% growth inhibition, respectively, while the corresponding combination of the two compounds achieved 61% Growth inhibition (Figure 11, upper left panel). Furthermore, it is worth noting that synergistic effects occur over a wide range of single agent concentrations, which should prove beneficial in vivo with regard to flexibility in dosing levels and schedules. In conclusion, the combination of MCL1 inhibitor and BCL-2 inhibitor resulted in synergistic growth inhibition of most (17/18) ALL cell lines tested. Importantly, superior synergistic growth inhibition was observed in 5/18 of the ALL cell lines tested. Example 11 : In vitro effect of combined MCL1 inhibitor and BCL - 2 inhibitor on proliferation in a panel of 5 mantle cell lymphoma ( MCL ) cell lines. Materials and Methods Cell lines were derived and maintained in alkaline cultures supplemented with FBS as indicated in Table 12. In addition, all cultures contained penicillin (100 IU/ml), streptomycin (100 µg/ml) and L-glutamic acid (2 mM). Cell lines were grown at 37°C in a humidified atmosphere containing 5% CO 2 and expanded in T-150 flasks. In all cases, cells were thawed from frozen stocks, expanded for ≥ 1 passage using appropriate dilutions, counted and analyzed for viability using a CASY cytometer, and then plated at 150 μl/well to 96 at the densities indicated in Table 12 in the hole plate. All cell lines were determined to be free of mycoplasma contamination internally. Stock solutions of compounds were prepared at a concentration of 5 mM in DMSO and stored at -20°C. To analyze compound activity as a single agent or in combination, cells were seeded and treated with 7 or 8 3.16-fold serial dilutions of each compound, administered either individually or directly to cells in all possible arrangements in a checkerboard fashion Analysis disk. After 2 days of incubation, the effects of single agents and their checkerboard combinations on cell viability were analyzed by quantifying cellular ATP content using CellTiterGlo at 150 μL reagent/well at 37°C/5% CO 2 . All conditions were tested in triplicate. Luminescence was quantified on a multipurpose disc reader. Potential synergistic interactions between compound combinations were analyzed according to the Loewe additivity model using an excess inhibition 2D matrix and reported as synergy scores (Lehar et al. , Nat Biotechnol. 2009 Jul; 27( 7): 659-666). All calculations were performed using Chalice (TM) bioinformatics software available on the Horizon website. Single agent IC50s were calculated using standard four parameter curve fitting. IC50 is defined as the concentration of compound at which CTG signal is reduced to 50% of that measured by vehicle (DMSO) control. The doubling times indicated in Table 12 are the average of the doubling times obtained in the different passages (in T-150 flasks) performed from thawing of cells to their seeding in 96-well plates. Synergy score SS ~ 0 → Additive SS > 1 → Weak synergy SS > 2 → Synergy Table 12. Conditions for identification and analysis of 5 mantle cell lymphoma cell lines used in combination experiments. Table 13. Indicated single agent IC50 values for Compound 3 and Compound 1, HCl in 5 mantle cell lymphoma cell lines. Compounds were incubated with cells over a 2-day period.
Figure 106124599-A0304-0021
 Table 14. Indicated synergy scores for the combination of Compound 3 and Compound 1, HCl in 5 mantle cell lymphoma cell lines. An interaction was considered synergistic when the observed score was ≥ 2.0. The initial concentration, maximal inhibition and synergy scores of the indicated compounds are indicated. Compounds were incubated with cells over a 2-day period.
Figure 106124599-A0304-0022
 Results The in vitro effects of combined MCL1 inhibitor and BCL-2 inhibitor on proliferation in five mantle cell lymphoma cell line groups were analyzed. As a single agent, MCL1 inhibitors exhibit superior activity compared to BCL-2 inhibitors. Thus, three cell lines exhibited IC50s of less than 100 nM against MCL1 inhibitors, whereas only one cell line exhibited IC50s of less than 100 nM against BCL-2 inhibitors (Table 13). Combination treatment with MCL1 inhibitor and BCL-2 inhibitor resulted in synergistic growth inhibition of all tested cell lines (Table 14) (ie, a synergy score above 2 - Lehar et al. , Nat Biotechnol . 2009 Jul; 27 (7): 659-666), as illustrated in Figure 12. Importantly, synergy was marked with a synergy score above 5 in 4/5 cell lines. Example 12 : In vitro effect of combined MCL1 inhibitor and BCL - 2 inhibitor on proliferation in a panel of 5 small cell lung cancer ( SCLC ) cell lines. All cell lines were obtained from ATCC. Cultures containing RPMI1640 (Invitrogen) supplemented with 10% FBS (HyClone) for COR-L95, NCI-H146, NCI-H211, SHP-77, SW1271, NCI-H1339, NCI -H1963 and NCI-H889. Cultures containing Waymouth's MB 752/1 (Inverrogen) with 10% FBS were used for DMS-273. DMEM/F12 (Inverlogan) with 5% FBS supplemented with 0.005 mg/ml insulin, 0.01 mg/ml transferrin and 30 nM sodium selenite solution (Inverlogan), 10 nM Hydrocortisone Cultures of ketone (Sigma), 10 nM β-estradiol (Sigma) and 2 mM L-glutamic acid (Haclon) were used for NCI-H1105. Cell lines were grown in a 37°C and 5% CO2 incubator and expanded in T-75 flasks. In all cases, cells were thawed from frozen stocks, expanded >1 passage using 1:3 dilutions, counted and analyzed for viability using a ViCell counter (Beckman-Coulter), and then plated in 384-well plates. To isolate and expand cell lines, cells were removed from flasks using 0.25% trypsin-EDTA (GIBCO). All cell lines were determined to be free of Mycoplasma contaminants as determined by PCR detection methods performed in Idexx Radil (Columbia, MO, USA) and properly confirmed by SNP panel detection. Cell proliferation was measured in a 72-hour CellTiter-Glo™ (CTG) assay (Promega G7571), and all results shown are the results of at least three replicate measurements. For CellTiter-Glo™ analysis, cells were dispensed into tissue culture treated 384-well plates (Corning 3707) with a final volume of 35 μL of culture and at a density of 5000 cells per well. Twenty-four hours after plating, 5 μL of each compound dilution was transferred to dishes containing cells, resulting in compound concentrations ranging from 0 to 10 μM and a final DMSO (Sigma D8418) concentration of 0.16%. The discs were incubated for 72 hours and the effect of compounds on cell proliferation was determined using a CellTiter-Glo ™ Luminescent Cell Viability Assay (Promega G7571) and an Envision Disc Reader (Perkin Elmer). The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method for determining the number of viable cells in culture based on the amount of ATP present, which is indicative of the presence of metabolically active cells. The method is described in detail in Technical Bulletin, TB288 Promega. Briefly, cells were plated in opaque walled multi-well dishes in culture as described above. Control wells containing cultures but no cells were also prepared to obtain background luminescence values. 15 μL of CellTiter-Glo® reagent was then added and the contents mixed on an orbital shaker for 10 minutes to induce cell disintegration. Subsequently, the luminescence was recorded using a disc reader. Growth inhibition and percent excess inhibition were analyzed using Chalice software (CombinatoRx, Cambridge MA). The percent growth inhibition relative to DMSO is shown in plated inhibition, and the amount of inhibition exceeds that expected in plated ADD excess inhibition (Figures 15(a)-15(e)). The concentrations of Compound 1, HCl, are shown along the bottom column from left to right and increasing concentrations of Compound 3 are shown along the leftmost row from bottom to top. All remaining points in the grid display result from the combination of the two inhibitors corresponding to the single agent concentrations indicated on the two axes. Data analysis of cell proliferation was performed using the Chalice Analyser described in Lehar et al., Nat Biotechnol . 2009 Jul;27(7):659-666. Excess inhibition was calculated using the Loewe synergy model, which measures the effect on growth relative to what would be expected if both drugs behaved in a dosing fashion. Positive numbers indicate areas of increased synergy. Synergy score SS ~ 0 → dose additive SS > 2 → synergistic SS > 1 → weak synergistic result The combination of compound 1 and compound 3 caused synergistic growth inhibition of 8/10 small cell lung cancer cell lines (ie, higher than 2 synergy score). Importantly, synergy was marked with a synergy score above 6 in 6 cell lines. Example 13 : In vivo efficacy of a combination of an MCL1 inhibitor ( Compound 3 ) and a BCL - 2 inhibitor ( Compound 1 , HCl or ABT - 199 ) on a patient-derived primary AML model HAMLX5343 Materials and Methods Materials Animals are operated on Immunocompromised gamma (NOD scid gamma; NSG) female mice weighing 17 to 27 grams (Jackson Laboratories) were previously acclimated to the new environment with ad libitum access to food and water for 3 days. Primary Tumor Models A patient-derived primary AML model HAMLX5343 and wild-type FLT3 carrying KRAS mutations were obtained from the Dana Farber Cancer Institute. Test Compound , Formulation Compound 1, HCl was formulated as a solution in 5% ethanol, 20% Dexolve-7 for intravenous administration, or in PEG300/EtOH/water (40/10/50) for oral. ABT-199 was formulated in PEG300/EtOH/water (40/10/50) for oral administration. All are stable at 4°C for at least one week. Compound 3 was formulated as a solution in lipid formulations for intravenous formulations that were stable at 4°C for 3 weeks. Vehicles and compound administration solutions are prepared as desired. All animals were dosed with Compound 1 (expressed as free base) or ABT-199 at 10 mL/kg, or Compound 3 at 5 mL/kg. Methods Eight treatment arms were designed for study 7844HAMLX5343-XEF as outlined in Table 15. All treatments were initiated when the mean tumor burden (% CD-45 positive cells) was between 8% and 15%. In this study, as a single agent, Compound 1 was administered by oral gavage (po) or intravenous administration at 50 mg/kg once a week and ABT-199 was administered by oral gavage (po) at 25 mg/kg Administered once a week, or Combination Compound 3 was administered at 12.5 mg/kg once a week for 18 days. Both Compound 1 (presented as the free base) and ABT-199 were administered at 10 mL/kg. Compound 3 was administered at 5 mL/kg. Dosage is adjusted according to body weight. Body weight was recorded twice weekly and tumor burden was recorded weekly. Table 15. Dosage* and Dosing Schedule of 7844HAMLX5343-XEF
Figure 106124599-A0304-0023
 * Dose is shown for free base primary AML model For this experiment, 32 mice were implanted with the primary AML strain HAMLX5343. Mice were injected intravenously with 2.0 million leukemia cells. When tumor burden was between 8% and 15%, animals were randomly distributed into 8 groups of four mice each for vehicle, compound 1 (po), compound 1 (iv), ABT-199, Compound 3 or combination therapy. After 18 days of treatment, the study was terminated when tumor burden reached 99%. Tumor burden was measured by FACS analysis. Animal Monitoring Animal health and behavior, including cleaning and movement, were monitored twice daily. The general health of the mice was monitored and mortality was recorded daily. Kill any dying animals. Tumor Measurements Mice were bled via tail clipping once a week. Blood was divided into IgG control wells and CD33/CD45 wells of 96-well plates. Blood was lysed twice with 200 μl RBC lysis buffer at room temperature, followed by one wash with FACS buffer (5% FBS in PBS). Subsequently, the samples were incubated in 100 µl of blocking buffer (5% mouse Fc block + 5% human Fc block + 90% FACS buffer) at 4°C for 10 to 30 minutes. Add 20 µl IgG control mix (2.5 µl mouse igG1 K isotype control - PE + 2.5 µl mouse igG1 K isotype control - APC + 15 µl FACS buffer) to IgG control wells and 20 µl CD33/CD45 mix (2.5 µl Mouse anti-human CD33-PE + 2.5 µl mouse anti-human CD45-APC + 15 µl FACS buffer). Before analysis, samples were incubated at 4°C for 30 to 60 minutes, followed by two washes. Samples were run on Canto with FACSDiva software. Analysis was performed with FloJo software. The percentage of CD45-positive viable single cells is reported as tumor burden. Data Analysis Treatment/control (T/C) percentage values were calculated using the following formula: %T/C = 100 ´ DT/DC if DT > 0 % resolved = 100 ´ DT/T initial , if DT < 0 where: T = final The mean tumor burden of the drug treatment group on the study day; DT = the mean tumor burden of the drug treatment group on the final study day - the average tumor burden of the drug treatment group on the initial dosing day; T initial = the drug treatment group on the initial dosing day mean tumor burden; C = mean tumor burden of control group on final study day; and DC = mean tumor burden of control group on final study day - mean tumor burden of control group on initial dosing day. All data are presented as mean ± SEM. △Tumor burden and body weight were used for statistical analysis. Comparisons between groups of final measurements were performed using ANOVA of the Ducati test. Statistical analysis was performed using GraphPad Prism. Statistical Analysis All data are presented as mean ± standard error of the mean (SEM). △Tumor volume and body weight were used for statistical analysis. Comparisons between groups were performed using Kruskal-Wallis ANOVA followed by post hoc Dunn's test or Tukey's test. For all statistical evaluations, the level of significance was set at p<0.05. Significance compared to the vehicle control group is reported unless otherwise stated. Standard protocols used in pharmacological studies do not predetermine efficacy to demonstrate statistically significant superiority of the combination over the corresponding single agent treatment. Statistical power is often limited by effective single agent response and/or model variability. However, p-values are provided for combination versus single agent treatments. Results Synergistic antitumor effect of combined MCL1 and BCL - 2 inhibition In the 7844HAMLX5343-XEF study, once weekly doses of 50 mg/kg (oral or iv ), 25 mg/kg (oral) or 12.5 mg/kg ( iv ) ) when administered, only Compound 1, ABT-199, or Compound 3 showed no antitumor activity in the KRAS -mutated HAMLX5343 model (98%, 92%, 98%, or 99% T/C%, respectively, p>0.05 ). In this model, once weekly oral administration of Compound 1 at 50 mg/kg or ABT-199 at 25 mg/kg in combination with Compound 3 (12.5 mg/kg iv ) resulted in tumor stasis (3% or 3%, respectively). 6% T/C%, p<0.05). On the other hand, intravenous administration of the combination of Compound 1 and Compound 3 resulted in almost complete tumor regression (% regression of 100%), which was significantly different from the single agent (p<0.05) or the Compound 1/Compound 3 po/ iv combination. The mean tumor burden for each treatment group was plotted against time for the 18-day treatment period, as shown in FIG. 1 . Changes in tumor burden, T/C% or % regression are presented in Table 16 and Figures 16(a)-16(b). Table 16. Summary of antitumor effects in the 7844HAMLX5343-XEF study
Figure 106124599-A0304-0024
 *p < 0.05 vs. vehicle and single agent (ANOVA, Tukey's test) **p < 0.05 vs. po / iv combination (ANOVA, Tukey's test) Conclusion AML is an aggressive and heterogeneous hematological malignancy disease caused by transformation of hematopoietic progenitor cells that acquire genetic alterations (Patel et al., New England Journal of Medicine 2012 366:1079-1089). The 5-year survival rate for AML is low due to the lack of effective therapy. Evasion of apoptosis is a hallmark of cancer (Hanahan et al. Cell 2000 100:57-70) One of the major means by which cancer cells escape apoptosis is by upregulating pro-survival BCL-2 family proteins such as BCL-2, BCL-xL and MCL1. The MCL1 gene is the most commonly amplified gene in cancer patients. (Beroukhim et al., Nature 2010 463:899-905). Furthermore, both BCL-2 and MCL1 are highly expressed in AML. Thus, the combination of Compound 1 (BCL-2i) and Compound 3 (MCL1) may provide synergy as a general mechanism against AML by enhancing pro-apoptotic signaling. Here we show that the BCL-2 inhibitor Compound 1 or ABT-199 combined with Compound 3 (MCL1 inhibitor) has a significant synergistic effect in the treatment of AML in an AML xenograft model with a KRAS mutation (wt FLT3). The iv / iv Compound 1/Compound 3 combination was superior to the po / iv combination therapy at the same dose. The results suggest that the combination of BCL-2 and MCL1 inhibitors would be an effective therapy for AML.

圖1.BCL - 2 及 MCL1 之表現在 AML 中普遍。 對具有＞70%母細胞之7個AML細胞株及13個原發性AML樣品的指定蛋白質進行免疫墨點法，其展示了主要表現BCL-2以及MCL1蛋白質，與在較低比例之樣品中表現的BCL-XL形成對比。 圖2.組合之 BCL - 2 / MCL1 靶向對 AML 具有活體外及活體內的協同活性。 (A) 54個原發性AML樣品與6對數濃度範圍之化合物1(鹽酸鹽)、化合物2或以1:1濃度在RPMI/15% FCS中培育48小時且測定LC50 (B) NSG小鼠之四個群組用表現螢光素酶之MV4;11細胞移植。腫瘤移植在第10天(基線)時驗證，且隨後化合物1，HCl以100 mg/d在工作日經口(表現為游離鹼)或化合物2以25 mg/kg IV每週兩次開始投與，持續4週。化合物2及與化合物1之組合的影響藉由起始治療後14天及28天螢光素酶體積的減少，及整體上存活期增加來證明(C)。 圖3.組合之 BCL - 2 / MCL1 靶向來自正常供體之正常 CD34 + 細胞或白血病母細胞的毒性檢定。 塗鋪分類之正常CD34+或白血病母細胞，且以指定濃度之1:1比率的化合物1，HCl及化合物2處理。組合之化合物1+化合物2對白血病有毒但對正常CD34+祖細胞無毒。 圖4.在 DB 細胞 ( A ) 及托萊多 (Toledo) 細胞 ( B ) 中由 化合物 3 以及 化合物 1 ， HCl 獲得之 細胞生長 ( 左 ) 抑制及洛伊 ( Loewe ) 過量抑制 ( 右 ) 的細胞生長抑制效應及協同組合矩陣。 效應矩陣之值範圍為0(不抑制)至100(完全抑制)。協同矩陣之值表示生長抑制超過基於所測試濃度之化合物3及化合物1，HCl的單一藥劑活性所計算的理論相加性的程度。協同效應在貫穿大範圍單一藥劑濃度內出現。 圖5.大鼠之淋巴瘤 Karpass422 異種移植模型中化合物 1 ， HCl 、化合物 3 及化合物 1 ， HCl+ 化合物 3 之組合的抗腫瘤效應。 圖6.大鼠之淋巴瘤 Karpass422 異種移植模型中經化合物 1 ， HCl 、化合物 3 及化合物 1 ， HCl + 化合物 3 之組合治療的動物的體重變化。 圖7.化合物 1 ， HCl 、化合物 3 及化合物 1 ， HCl+ 化合物 3 組合在小鼠之 DLBCL Toledo 異種移植模型中的抗腫瘤效應。 圖8.小鼠之 DLBCL Toledo 異種移植模型中經化合物 1 ， HCl 、化合物 3 及化合物 1 ， HCl+ 化合物 3 之組合治療的動物的體重變化。 圖9(A)及圖(B).在兩個獨立實驗中 ， 在 AML 細胞株 OCI - AML3 中由化合物 3 ( MCL1 抑制劑 ) 以及 化合物 1 ， HCl ( BCL - 2 抑制劑 ) 獲得之 細胞生長 ( 左 ) 抑制及洛伊過量抑制 ( 右 ) 的細胞生長抑制效應及協同組合矩陣。 效應矩陣之值範圍為0(不抑制)至100(完全抑制)。協同矩陣之值表示生長抑制超過基於所測試濃度之化合物3及化合物1，HCl的單一藥劑活性所計算的理論相加性的程度。協同效應在貫穿大範圍單一藥劑濃度內出現。 圖10(A)及圖(B).在兩個獨立實驗中 ， 在 NB 細胞株 LAN - 6 中由化合物 3 ( MCL1 抑制劑 ) 以及 化合物 1 ， HCl ( BCL - 2 抑制劑 ) 獲得之 細胞生長 ( 左 ) 抑制及洛伊過量抑制 ( 右 ) 的細胞生長抑制效應及協同組合矩陣 ( N1 ： 上圖 ； N2 ： 下圖 ) 。 效應矩陣之值範圍為0(不抑制)至100(完全抑制)。協同矩陣之值表示生長抑制超過基於所測試濃度之化合物3及化合物1，HCl的單一藥劑活性所計算的理論相加性的程度。 圖11.在兩個獨立實驗中 ， 在 B - ALL 細胞株 NALM - 6 中由化合物 3 ( MCL1 抑制劑 ) 以及 化合物 1 ， HCl ( BCL - 2 抑制劑 ) 獲得之 細胞生長 ( 左 ) 抑制及洛伊過量抑制 ( 右 ) 的細胞生長抑制效應及協同組合矩陣 ( N1 ： 上圖 ； N2 ： 下圖 ) 。 圖12.在 MCL 細胞株 Z - 138 中由化合物 3 ( MCL1 抑制劑 ) 以及 化合物 1 ， HCl ( BCL - 2 抑制劑 ) 獲得之 細胞生長 ( 左 ) 抑制及洛伊過量抑制 ( 右 ) 的細胞生長抑制效應及協同組合矩陣。 圖13.在兩個獨立實驗中 ， 在 AML 細胞株 OCI - AML3 中由化合物 3 ( MCL1 抑制劑 ) 以及 ABT - 199 ( BCL - 2 抑制劑 ) 獲得之 細胞生長 ( 左 ) 抑制及洛伊過量抑制 ( 右 ) 的細胞生長抑制效應及協同組合矩陣 ( N1 ： 上圖 ； N2 ： 下圖 ) 。 圖14.在 AML 細胞株中由化合物 3 ( MCL1 抑制劑 ) 以及 化合物 4 ， HCl ( BCL - 2 抑制劑 ) 獲得之 細胞生長 ( 左 ) 抑制及洛伊過量抑制 ( 右 ) 的例示性細胞生長抑制效應及協同組合矩陣 ( ML - 2 細胞在 A 中且 OCI - AML - 3 在 B 中 ) 。 圖15 (a)至圖(e).在 SCLC 細胞株組中由化合物 3 ( MCL1 抑制劑 ) 以及化合物 1 ， HCl ( BCL - 2 抑制劑 ) 獲得之抑制 ( 左 ) 、洛伊盈餘抑制 ( 中間 ) 及生長抑制的劑量矩陣。 圖16 (a)至圖(b).小鼠之源自患者的原發性 AML 模型 HAMLX5343 中化合物 1 ， HCl 、 ABT - 199 、化合物 3 及化合物 1 ， HCl 或 ABT - 199 + 化合物 3 之組合的抗腫瘤效應。 圖17.AML 對 BH3 - 模擬單藥療法、或藥物組合 ( 以 1 : 1 比率測試 ) 的 敏感性 ( LC50 ) 的熱圖對比 ， 相對於 48 小時暴露之後的化學治療 ( 艾達黴素 ； idarubicin ) 。展示各原發性 AML 樣品在 DMSO 中 48 小時之後的細胞存活率。 Figure 1. Expression of BCL - 2 and MCL1 is prevalent in AML . Immunoblotting was performed on the indicated proteins of 7 AML cell lines with >70% blasts and 13 primary AML samples, which showed predominantly BCL-2 and MCL1 proteins, compared with lower proportions of samples The performance of the BCL-XL is in contrast. Figure 2. Combined BCL - 2 / MCL1 targeting has synergistic activity against AML in vitro and in vivo. (A) 54 primary AML samples were incubated with 6 log concentration ranges of Compound 1 (HCl), Compound 2, or 1:1 in RPMI/15 % FCS for 48 hours and LC50 determined (B) NSG Four groups of mice were transplanted with luciferase expressing MV4;11 cells. Tumor engraftment was validated on day 10 (baseline) and subsequently Compound 1, HCl at 100 mg/d orally (expressed as free base) on weekdays or Compound 2 at 25 mg/kg IV twice weekly started , for 4 weeks. The effect of Compound 2 and in combination with Compound 1 was demonstrated by a reduction in luciferase volume 14 and 28 days after initiation of treatment, and an overall increase in survival (C). Figure 3. Toxicity assay of the combined BCL - 2 / MCL1 targeting normal CD34 + cells or leukemic blasts from normal donors . Sorted normal CD34+ or leukemic blasts were plated and treated with Compound 1, HCl and Compound 2 in a 1:1 ratio at the indicated concentrations. The combination of Compound 1 + Compound 2 is toxic to leukemia but not to normal CD34+ progenitor cells. Figure 4. Cell growth inhibition ( left ) and Loewe overinhibition ( right ) in DB cells ( A ) and Toledo cells ( B ) by compound 3 and compound 1 , HCl Growth inhibitory effect and synergistic combination matrix. The value of the effect matrix ranges from 0 (no suppression) to 100 (complete suppression). The values of the synergy matrix represent the extent to which growth inhibition exceeds the theoretical additivity calculated based on the single agent activity of Compound 3 and Compound 1, HCl, at the concentrations tested. Synergistic effects occur across a wide range of single agent concentrations. Figure 5. Antitumor effects of Compound 1 , HCl , Compound 3 and the combination of Compound 1 , HCl + Compound 3 in a rat lymphoma Karpass422 xenograft model . Figure 6. Body weight changes in animals treated with Compound 1 , HCl , Compound 3 and the combination of Compound 1 , HCl + Compound 3 in the rat lymphoma Karpass422 xenograft model . Figure 7. Antitumor effect of Compound 1 , HCl , Compound 3 and Compound 1 , HCl + Compound 3 combination in a DLBCL Toledo xenograft model in mice. Figure 8. Body weight change in animals treated with Compound 1 , HCl , Compound 3 and the combination of Compound 1 , HCl + Compound 3 in the DLBCL Toledo xenograft model in mice . Figure 9(A) and (B). Cell growth obtained with Compound 3 ( MCL1 inhibitor ) and Compound 1 , HCl ( BCL - 2 inhibitor ) in the AML cell line OCI - AML3 in two independent experiments ( Left ) Cytostatic effect and synergistic combination matrix of inhibition and Loy excess inhibition ( right ) . The value of the effect matrix ranges from 0 (no suppression) to 100 (complete suppression). The values of the synergy matrix represent the extent to which growth inhibition exceeds the theoretical additivity calculated based on the single agent activity of Compound 3 and Compound 1, HCl, at the concentrations tested. Synergistic effects occur across a wide range of single agent concentrations. Figure 10(A) and (B). Cell growth in NB cell line LAN - 6 obtained with Compound 3 ( MCL1 inhibitor ) and Compound 1 , HCl ( BCL - 2 inhibitor ) in two independent experiments ( Left ) Cytostatic effect of inhibition and Lloyd excess inhibition ( right ) and synergistic combination matrix ( N1 : upper panel ; N2 : lower panel ) . The value of the effect matrix ranges from 0 (no suppression) to 100 (complete suppression). The values of the synergy matrix represent the extent to which growth inhibition exceeds the theoretical additivity calculated based on the single agent activity of Compound 3 and Compound 1, HCl, at the concentrations tested. Figure 11. Inhibition of cell growth ( left ) and loss of cell growth by compound 3 ( MCL1 inhibitor ) and compound 1 , HCl ( BCL - 2 inhibitor ) in the B - ALL cell line NALM - 6 in two independent experiments Cell growth inhibitory effect and synergistic combination matrix ( N1 : upper panel ; N2 : lower panel ) of IL-oversuppression ( right ) . Figure 12. Inhibition of cell growth ( left ) and Lloyd excess inhibition ( right ) by compound 3 ( MCL1 inhibitor ) and compound 1 , HCl ( BCL - 2 inhibitor ) in MCL cell line Z - 138 Inhibitory effect and synergistic combination matrix. Figure 13. Inhibition of cell growth ( left ) and Loy excess inhibition by Compound 3 ( MCL1 inhibitor ) and ABT - 199 ( BCL - 2 inhibitor ) in the AML cell line OCI - AML3 in two independent experiments ( Right ) Cytostatic effect and synergistic combination matrix ( N1 : upper panel ; N2 : lower panel ) . Figure 14. Exemplary cell growth inhibition by Compound 3 ( MCL1 inhibitor ) and Compound 4 , HCl ( BCL - 2 inhibitor ) ( left ) and Loy excess inhibition ( right ) in AML cell lines Effector and synergistic combination matrix ( ML - 2 cells in A and OCI - AML - 3 in B ) . Figure 15(a) to (e). Inhibition by Compound 3 ( MCL1 inhibitor ) and Compound 1 , HCl ( BCL - 2 inhibitor ) ( left ) , Lowy surplus inhibition ( middle ) in SCLC cell line group ) and a dose matrix for growth inhibition. Figure 16(a)-(b). Compound 1 , HCl , ABT - 199 , Compound 3 and the combination of Compound 1 , HCl or ABT - 199 + Compound 3 in a patient-derived primary AML model HAMLX5343 in mice antitumor effect. Figure 17. Heatmap comparison of the sensitivity ( LC50 ) of AML to BH3 - mock monotherapy, or drug combination ( tested in a 1 : 1 ratio ) , relative to chemotherapy ( idamycin ; idarubicin ) . Cell viability after 48 hours in DMSO for each primary AML sample is shown.

Figure 106124599-A0101-11-0002-1
Figure 106124599-A0101-11-0002-1

Claims (28)

一種組合，其包含：(a)BCL-2抑制劑，其係選自N-(4-羥苯基)-3-{6-[((3S)-3-(4-嗎啉基甲基)-3,4-二氫-2(1H)-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N-苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺或其與醫藥學上可接受之酸或鹼的加成鹽、5-(5-氯-2-{[(3S)-3-(嗎啉-4-基甲基)-3,4-二氫異喹啉-2(1H)-基]羰基}苯基)-N-(5-氰基-1,2-二甲基-1H-吡咯-3-基)-N-(4-羥苯基)-1,2-二甲基-1H-吡咯-3-甲醯胺或其與醫藥學上可接受之酸或鹼的加成鹽及4-(4-{[2-(4-氯苯基)-4,4-二甲基環己-1-烯-1-基]甲基}哌嗪-1-基)-N-[(3-硝基-4-{[(噁烷-4-基)甲基]胺基}苯基)碸基]-2-[(1H-吡咯并[2,3-b]吡啶-5-基)氧基]苯甲醯胺；及(b)MCL1抑制劑，其係選自：(2R)-2-{[(5S a )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(5-氟呋喃-2-基)噻吩并[2,3-d]嘧啶-4-基]氧基}-3-(2-{[1-(2,2,2-三氟乙基)-1H-吡唑-5-基]甲氧基}苯基)丙酸或其與醫藥學上可接受之酸或鹼的加成鹽及(2R)-2-{[(5S a )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(4-氟苯基)噻吩并[2,3-d]嘧啶-4-基]氧基}-3-(2-{[2-(2-甲氧基苯基)嘧啶-4-基]甲氧基}苯基)丙酸或其與醫藥學上可接受之酸或鹼的加成鹽，係用於同時、依序或分開使用。 A combination comprising: (a) a BCL-2 inhibitor selected from N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinomethyl) yl)-3,4-dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6 ,7,8-Tetrahydro-1-indolizinecarboxamide or its addition salt with a pharmaceutically acceptable acid or base, 5-(5-chloro-2-{[( 3S )-3 -(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin-2( 1H )-yl]carbonyl}phenyl) -N- (5-cyano-1,2-dimethyl yl- 1H -pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2- dimethyl - 1H -pyrrole-3-carboxamide or a pharmaceutically acceptable acid thereof or base addition salt and 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazine-1- yl) -N -[(3-nitro-4-{[(oxan-4-yl)methyl]amino}phenyl)teranyl]-2-[( 1H -pyrrolo[2,3 - b ]pyridin-5-yl)oxy]benzamide; and (b) an MCL1 inhibitor selected from: ( 2R )-2-{[ ( 5Sa )-5-{3-chloro- 2-Methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl) -1H -pyrazol-5-yl]methoxy}phenyl) Propionic acid or its addition salt with a pharmaceutically acceptable acid or base and ( 2R )-2-{[( 5S a )-5-{3-chloro-2-methyl-4-[2-( 4-Methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3-( 2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid or its addition salt with a pharmaceutically acceptable acid or base for use in Simultaneous, sequential or separate use. 如請求項1之組合，其中該BCL-2抑制劑係N-(4-羥苯基)-3-{6-[((3S)-3-(4-嗎啉基甲基)-3,4-二氫-2(1H)-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯 -5-基}-N-苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺。 The combination of claim 1, wherein the BCL-2 inhibitor is N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)-3 ,4-Dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7,8 - Tetrahydro-1-indolazinecarboxamide. 如請求項1之組合，其中該BCL-2抑制劑係5-(5-氯-2-{[(3S)-3-(嗎啉-4-基甲基)-3,4-二氫異喹啉-2(1H)-基]羰基}苯基)-N-(5-氰基-1,2-二甲基-1H-吡咯-3-基)-N-(4-羥苯基)-1,2-二甲基-1H-吡咯-3-甲醯胺。 The combination of claim 1, wherein the BCL-2 inhibitor is 5-(5-chloro-2-{[( 3S )-3-(morpholin-4-ylmethyl)-3,4-dihydro Isoquinolin-2( 1H )-yl]carbonyl}phenyl)-N-(5-cyano-1,2- dimethyl - 1H -pyrrol - 3-yl)-N-(4-hydroxy phenyl)-1,2-dimethyl- 1H -pyrrole-3-carboxamide. 如請求項2之組合，其中N-(4-羥苯基)-3-{6-[((3S)-3-(4-嗎啉基甲基)-3,4-二氫-2(1H)-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N-苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺呈鹽酸鹽的形式。 A combination as claimed in claim 2, wherein N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)-3,4-dihydro-2 ( 1H )-Isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7,8-tetrahydro-1-indone Tolazinecarboxamide is in the form of the hydrochloride salt. 如請求項3之組合，其中5-(5-氯-2-{[(3S)-3-(嗎啉-4-基甲基)-3,4-二氫異喹啉-2(1H)-基]羰基}苯基)-N-(5-氰基-1,2-二甲基-1H-吡咯-3-基)-N-(4-羥苯基)-1,2-二甲基-1H-吡咯-3-甲醯胺呈鹽酸鹽的形式。 The combination of claim 3, wherein 5-(5-chloro-2-{[( 3S )-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinoline-2(1 H )-yl]carbonyl}phenyl)-N-(5-cyano-1,2- dimethyl - 1H -pyrrol - 3-yl)-N-(4-hydroxyphenyl)-1,2 -Dimethyl- 1H -pyrrole-3-carboxamide in the form of the hydrochloride salt. 如請求項2之組合，其中在組合治療期間，N-(4-羥苯基)-3-{6-[((3S)-3-(4-嗎啉基甲基)-3,4-二氫-2(1H)-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N-苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺的劑量為50mg至1500mg。 The combination of claim 2, wherein during combination therapy, N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)-3,4 -Dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7,8-tetra The dose of hydro-1-indolizine carboxamide is 50 mg to 1500 mg. 如請求項1之組合，其中該BCL-2抑制劑係4-(4-{[2-(4-氯苯基)-4,4-二甲基環己-1-烯-1-基]甲基}哌嗪-1-基)-N-[(3-硝基-4-{[(噁烷-4-基)甲基]胺基}苯基)碸基]-2-[(1H-吡咯并[2,3-b]吡啶-5-基)氧基]苯甲醯胺(ABT-199)。 The combination of claim 1, wherein the BCL-2 inhibitor is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl] Methyl}piperazin-1-yl) -N -[(3-nitro-4-{[(oxan-4-yl)methyl]amino}phenyl)thiol]-2-[(1 H -pyrrolo[2,3- b ]pyridin-5-yl)oxy]benzamide (ABT-199). 如請求項1至7中任一項之組合，其中該MCL1抑制劑係(2R)-2-{[(5S a )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(5-氟呋喃-2-基)噻吩并[2,3-d]嘧啶-4-基]氧基}-3-(2-{[1-(2,2,2-三氟乙基)-1H-吡唑-5-基]甲氧基}苯基)丙酸。 The combination of any one of claims 1 to 7, wherein the MCL1 inhibitor is ( 2R )-2-{[ ( 5Sa )-5-{3-chloro-2-methyl-4-[2-( 4-Methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3- d ]pyrimidin-4-yl]oxy}- 3-(2-{[1-(2,2,2-Trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propionic acid . 如請求項1至7中任一項之組合，其中該MCL1抑制劑係(2R)-2-{[(5S a )-5-{3-氯-2-甲基-4-[2-(4-甲基哌嗪-1-基)乙氧基]苯基}-6-(4-氟苯基)噻吩并[2,3-d]嘧啶-4-基]氧基}-3-(2-{[2-(2-甲氧基苯基)嘧啶-4-基]甲氧基}苯基)丙酸。 The combination of any one of claims 1 to 7, wherein the MCL1 inhibitor is ( 2R )-2-{[( 5S a )-5-{3-chloro-2-methyl-4-[2-( 4-Methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3-( 2-{[2-(2-Methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid. 如請求項1至7中任一項之組合，其進一步包含一或多種賦形劑。 The combination of any one of claims 1 to 7, further comprising one or more excipients. 一種如請求項1至10中任一項之組合的用途，其係用於製造用於治療癌症之藥物。 A use of the combination of any one of claims 1 to 10 for the manufacture of a medicament for the treatment of cancer. 如請求項11之用途，其中該癌症係白血病。 The use of claim 11, wherein the cancer is leukemia. 如請求項12之用途，其中該白血病係急性骨髓白血病、T細胞急性淋巴母細胞白血病(T-ALL)或B細胞急性淋巴母細胞白血病(B-ALL)。 The use of claim 12, wherein the leukemia is acute myeloid leukemia, T-cell acute lymphoblastic leukemia (T-ALL) or B-cell acute lymphoblastic leukemia (B-ALL). 如請求項11之用途，其中該癌症係骨髓發育不良症候群或骨髓增生疾病。 The use of claim 11, wherein the cancer is myelodysplastic syndrome or myeloproliferative disease. 如請求項11之用途，其中該癌症係淋巴瘤。 The use of claim 11, wherein the cancer is lymphoma. 如請求項15之用途，其中該淋巴瘤係非霍奇金淋巴瘤。 The use of claim 15, wherein the lymphoma is non-Hodgkin's lymphoma. 如請求項16之用途，其中該非霍奇金淋巴瘤係彌漫性大B細胞淋巴瘤或套細胞淋巴瘤。 The use of claim 16, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma or mantle cell lymphoma. 如請求項11之用途，其中該癌症係多發性骨髓瘤。 The use of claim 11, wherein the cancer is multiple myeloma. 如請求項11之用途，其中該癌症係神經母細胞瘤。 The use of claim 11, wherein the cancer is neuroblastoma. 如請求項11之用途，其中該癌症係小細胞肺癌。 The use of claim 11, wherein the cancer is small cell lung cancer. 如請求項11之用途，其中該BCL-2抑制劑一週投與一次。 The use of claim 11, wherein the BCL-2 inhibitor is administered once a week. 如請求項11之用途，其中該BCL-2抑制劑為N-(4-羥苯基)-3-{6-[((3S)-3-(4-嗎啉基甲基)-3,4-二氫-2(1H)-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N-苯基-5,6,7,8-四氫-1-吲哚嗪甲醯胺且在該組合治療期間一日投與一次。 The use of claim 11, wherein the BCL-2 inhibitor is N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)-3 ,4-Dihydro-2( 1H )-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7,8 - Tetrahydro-1-indolizine carboxamide and administered once daily during the combination treatment. 如請求項22之用途，其中N-(4-羥苯基)-3-{6-[((3S)-3-(4-嗎啉基甲基)-3,4-二氫-2(1H)-異喹啉基)羰基]-1,3-苯并二氧雜環戊烯-5-基}-N-苯基 -5,6,7,8-四氫-1-吲哚嗪甲醯胺呈鹽酸鹽的形式。 Use as claimed in claim 22, wherein N- (4-hydroxyphenyl)-3-{6-[(( 3S )-3-(4-morpholinylmethyl)-3,4-dihydro-2 ( 1H )-Isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl} -N -phenyl-5,6,7,8-tetrahydro-1-indone Tolazinecarboxamide is in the form of the hydrochloride salt. 如請求項11之用途，其中該BCL-2抑制劑及該MCL1抑制劑經口投與。 The use of claim 11, wherein the BCL-2 inhibitor and the MCL1 inhibitor are administered orally. 如請求項11之用途，其中該BCL-2抑制劑經口投與且該MCL1抑制劑經靜脈內投與。 The use of claim 11, wherein the BCL-2 inhibitor is administered orally and the MCL1 inhibitor is administered intravenously. 如請求項11之用途，其中該BCL-2抑制劑及該MCL1抑制劑經靜脈內投與。 The use of claim 11, wherein the BCL-2 inhibitor and the MCL1 inhibitor are administered intravenously. 一種藥物，其分開地或共同含有：(a)如請求項1所定義之BCL-2抑制劑，及(b)如請求項1所定義之MCL1抑制劑，用於同時、依序或分開投與，且其中該BCL-2抑制劑及該MCL1抑制劑以有效量提供以用於治療癌症。 A medicament containing, separately or jointly: (a) a BCL-2 inhibitor as defined in claim 1, and (b) an MCL1 inhibitor as defined in claim 1, for simultaneous, sequential or separate administration and, and wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in effective amounts for the treatment of cancer. 一種如請求項1至10中任一項之組合的用途，其係用於製備用於使(i)難以用至少一種化學療法治療或(ii)用化學療法治療之後復發，或(i)及(ii)兩者的患者敏感的藥物，其中該敏感化包含向該患者投與共同治療有效量之(a)如請求項1中所定義之BCL-2抑制劑，及(b)如請求項1中所定義之MCL1抑制劑。 A use of the combination of any one of claims 1 to 10 for the preparation of (i) refractory to treatment with at least one chemotherapy or (ii) relapse after treatment with chemotherapy, or (i) and (ii) both a patient-sensitive drug, wherein the sensitization comprises administering to the patient a co-therapeutically effective amount of (a) a BCL-2 inhibitor as defined in claim 1, and (b) as claimed MCL1 inhibitor as defined in 1.
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