RESUMEN
Introduction of fluorine into bioactive molecules has attracted much attention in drug development. For example, tetrafluorination of the phthalimide moiety of immunomodulatory drugs (IMiDs) has a strong beneficial effect on the ability to inhibit angiogenesis. The neomorphic activity of E3 ligase complexes is induced by the binding of IMiDs to cereblon. We investigated that a set of eight thalidomide analogs, comprising non- and tetrafluorinated counterparts, did not induce the degradation of neomorphic substrates (IKZF3, GSPT1, CK1α, SALL4). Hence, the antiangiogenic activity of fluorinated IMiDs was not triggered by neosubstrate degradation features. A fluorine scanning of non-traditional IMiDs of the benzamido glutarimide chemotype was performed. By measuring the endothelial cell tube formation, no angiogenesis inhibitors were identified, confirming the narrow structure-activity window of IMiD-induced antiangiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis , Talidomida , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/síntesis química , Talidomida/farmacología , Talidomida/química , Talidomida/análogos & derivados , Talidomida/síntesis química , Humanos , Relación Estructura-Actividad , Estructura Molecular , Halogenación , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Immunomodulatory imide drugs (IMiDs) play a crucial role in the treatment landscape across various stages of multiple myeloma. Despite their evident efficacy, some patients may exhibit primary resistance to IMiD therapy, and acquired resistance commonly arises over time leading to inevitable relapse. It is critical to develop novel therapeutic options to add to the treatment arsenal to overcome IMiD resistance. We designed, synthesized, and screened a new class of polyfluorinated thalidomide analogs and investigated their anti-cancer, anti-angiogenic, and anti-inflammatory activity using in vitro and ex vivo biological assays. We identified four lead compounds that exhibit potent anti-myeloma, anti-angiogenic, anti-inflammatory properties using three-dimensional tumor spheroid models, in vitro tube formation, and ex vivo human saphenous vein angiogenesis assays, as well as the THP-1 inflammatory assay. Western blot analyses investigating the expression of proteins downstream of cereblon (CRBN) reveal that Gu1215, our primary lead candidate, exerts its activity through a CRBN-independent mechanism. Our findings demonstrate that the lead compound Gu1215 is a promising candidate for further preclinical development to overcome intrinsic and acquired IMiD resistance in multiple myeloma.
Asunto(s)
Resistencia a Antineoplásicos , Mieloma Múltiple , Talidomida , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Humanos , Talidomida/análogos & derivados , Talidomida/farmacología , Talidomida/química , Resistencia a Antineoplásicos/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/química , Línea Celular Tumoral , Antiinflamatorios/farmacología , Antiinflamatorios/química , Evaluación Preclínica de MedicamentosRESUMEN
BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other haematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.
Asunto(s)
Diseño de Fármacos , Proteolisis , Proteínas Proto-Oncogénicas c-bcl-2 , Ubiquitina-Proteína Ligasas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Humanos , Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacología , Sulfonamidas/síntesis química , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , Quimera Dirigida a la ProteólisisRESUMEN
A titrant for the SARS-CoV-2 main protease (Mpro) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active Mpro by active-site titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme-titrant complex. Four fluorogenic substrates of Mpro, including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions. By exploiting the new titrant, key kinetic parameters for the Mpro-catalyzed cleavage of these substrates were determined.
RESUMEN
Immunomodulatory imide drugs (IMiDs) such as thalidomide, pomalidomide, and lenalidomide are the most common cereblon (CRBN) recruiters in proteolysis-targeting chimera (PROTAC) design. However, these CRBN ligands induce the degradation of IMiD neosubstrates and are inherently unstable, degrading hydrolytically under moderate conditions. In this work, we simultaneously optimized physiochemical properties, stability, on-target affinity, and off-target neosubstrate modulation features to develop novel nonphthalimide CRBN binders. These efforts led to the discovery of conformationally locked benzamide-type derivatives that replicate the interactions of the natural CRBN degron, exhibit enhanced chemical stability, and display a favorable selectivity profile in terms of neosubstrate recruitment. The utility of the most potent ligands was demonstrated by their transformation into potent degraders of BRD4 and HDAC6 that outperform previously described reference PROTACs. Together with their significantly decreased neomorphic ligase activity on IKZF1/3 and SALL4, these ligands provide opportunities for the design of highly selective and potent chemically inert proximity-inducing compounds.
Asunto(s)
Quimera Dirigida a la Proteólisis , Ubiquitina-Proteína Ligasas , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ligandos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Hypoxia-inducible factor-1α (HIF-1α) constitutes the principal mediator of cellular adaptation to hypoxia in humans. The HIF-1α protein level and activity are tightly regulated by the ubiquitin E3 ligase von Hippel-Lindau (VHL). Here, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors. Our iterative and combinatorial strategy focused on chemical variability at the phenylene unit and encompassed further points of diversity. The exploitation of tailored phenylene fragments and the stereoselective installation of the benzylic methyl group provided potent VHL ligands. Three high-resolution structures of VHL-ligand complexes were determined, and bioactive conformations of these ligands were explored. The most potent inhibitor (30) exhibited dissociation constants lower than 40 nM, independently determined by fluorescence polarization and surface plasmon resonance and an enhanced cellular potency, as evidenced by its superior ability to induce HIF-1α transcriptional activity. Our work is anticipated to inspire future efforts toward HIF-1α stabilizers and new ligands for proteolysis-targeting chimera (PROTAC) degraders.
Asunto(s)
Ubiquitina-Proteína Ligasas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Ligandos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ubiquitina/metabolismo , HipoxiaRESUMEN
The Petasis borono-Mannich reaction was employed for an alternative entry towards three-branched cereblon ligands. Such compounds are capabable of making multiple interactions with the protein surface and possess a suitable linker exit vector. The high-affinity ligands were used to assemble prototypic new molecular glues and proteolysis targeting chimeras (PROTACs) targeting BRD4 for degradation. Our results highlight the importance of multicomponent reactions (MCRs) in drug discovery and add new insights into the rapidly growing field of protein degraders.
RESUMEN
Proteolysis targeting chimeras (PROTACs) represent a new pharmacological modality to inactivate disease-causing proteins. PROTACs operate via recruiting E3 ubiquitin ligases, which enable the transfer of ubiquitin tags onto their target proteins, leading to proteasomal degradation. However, several E3 ligases are validated pharmacological targets themselves, of which inhibitor of apoptosis (IAP) proteins are considered druggable in cancer. Here, we report three series of heterobifunctional PROTACs, which consist of an IAP antagonist linked to either von Hippel-Lindau- or cereblon-recruiting ligands. Hijacking E3 ligases against each other led to potent, rapid, and preferential depletion of cellular IAPs. In addition, these compounds caused complete X-chromosome-linked IAP knockdown, which was rarely observed for monovalent and homobivalent IAP antagonists. In cellular assays, hit degrader 9 outperformed antagonists and showed potent inhibition of cancer cell viability. The hetero-PROTACs disclosed herein are valuable tools to facilitate studies of the biological roles of IAPs and will stimulate further efforts toward E3-targeting therapies.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis , Neoplasias , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Neoplasias/metabolismo , LigandosRESUMEN
PROteolysis TArgeting Chimaeras (PROTACs) offer new opportunities in modern medicine by targeting proteins that are undruggable to classic inhibitors. However, due to their hydrophobic structure, PROTACs typically suffer from low solubility, and oral bioavailability remains challenging. At the same time, due to their investigative state, the drug supply is meager, leading to limited possibilities in terms of formulation development. Therefore, we investigated the solubility enhancement employing mini-scale formulations of amorphous solid dispersions (ASDs) and liquisolid formulations of the prototypic PROTAC ARCC-4. Based on preliminary supersaturation testing, HPMCAS (L Grade) and Eudragit® L 100-55 (EL 100-55) were demonstrated to be suitable polymers for supersaturation stabilization of ARCC-4. These two polymers were selected for preparing ASDs via vacuum compression molding (VCM), using drug loads of 10 and 20%, respectively. The ASDs were subsequently characterized with respect to their solid state via differential scanning calorimetry (DSC). Non-sink dissolution testing revealed that the physical mixtures (PMs) did not improve dissolution. At the same time, all ASDs enabled pronounced supersaturation of ARCC-4 without precipitation for the entire dissolution period. In contrast, liquisolid formulations failed in increasing ARCC-4 solubility. Hence, we demonstrated that ASD formation is a promising principle to overcome the low solubility of PROTACs.
RESUMEN
The targeted degradation of histone deacetylase 6 (HDAC6) by heterobifunctional degraders constitutes a promising approach to treat HDAC6-driven diseases. Previous HDAC6 selective degraders utilised a hydroxamic acid as a zinc-binding group (ZBG) which features mutagenic and genotoxic potential. Here we report the development of a new class of selective HDAC6 degraders based on a difluoromethyl-1,3,4-oxadiazole warhead as ZBG.
Asunto(s)
Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Oxadiazoles , Zinc/metabolismoRESUMEN
Targeting deubiquitinating enzymes (DUBs) has emerged as a promising therapeutic approach in several human cancers and other diseases. DUB inhibitors are exciting pharmacological tools but often exhibit limited cellular potency. Here we report PROTACs based on a ubiquitin-specific protease 7 (USP7) inhibitor scaffold to degrade USP7. By investigating several linker and E3 ligand types, including novel cereblon recruiters, we discovered a highly selective USP7 degrader tool compound that induced apoptosis of USP7-dependent cancer cells. This work represents one of the first DUB degraders and unlocks a new drug target class for protein degradation.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Neoplasias , Apoptosis , Humanos , Neoplasias/tratamiento farmacológico , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
Various BRAF kinase inhibitors were developed to treat cancers carrying the BRAFV600E mutation. First-generation BRAF inhibitors could lead to paradoxical activation of the MAPK pathway, limiting their clinical usefulness. Here, we show the development of two series of BRAFV600E-targeting PROTACs and demonstrate that the exchange of the inhibitor scaffold from vemurafenib to paradox-breaker ligands resulted in BRAFV600E degraders that did not cause paradoxical ERK activation.
RESUMEN
In recent years, proteolysis-targeting chimeras (PROTACs), capable of achieving targeted protein degradation, have proven their great therapeutic potential and usefulness as molecular biology tools. These heterobifunctional compounds are comprised of a protein-targeting ligand, an appropriate linker, and a ligand binding to the E3 ligase of choice. A successful PROTAC induces the formation of a ternary complex, leading to the E3 ligase-mediated ubiquitination of the targeted protein and its proteasomal degradation. In over 20 years since the concept was first demonstrated, the field has grown substantially, mainly due to the advancements in the discovery of non-peptidic E3 ligase ligands. Development of small-molecule E3 binders with favourable physicochemical profiles aided the design of PROTACs, which are known for breaking the rules of established guidelines for discovering small molecules. Synthetic accessibility of the ligands and numerous successful applications led to the prevalent use of cereblon and von Hippel-Lindau as the hijacked E3 ligase. However, the pool of over 600 human E3 ligases is full of untapped potential, which is why expanding the artillery of E3 ligands could contribute to broadening the scope of targeted protein degradation. In this comprehensive review, we focus on the chemistry aspect of the PROTAC design process by providing an overview of liganded E3 ligases, their chemistries, appropriate derivatisation, and synthetic approaches towards their incorporation into heterobifunctional degraders. By covering syntheses of both established and underexploited E3 ligases, this review can serve as a chemistry blueprint for PROTAC researchers during their future ventures into the complex field of targeted protein degradation.
Asunto(s)
Ubiquitina-Proteína Ligasas , Humanos , Ligandos , Proteolisis , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide are highly effective treatments for multiple myeloma. However, virtually all patients eventually relapse due to acquired drug resistance with resistance-causing genetic alterations being found only in a small subset of cases. To identify non-genetic mechanisms of drug resistance, we here perform integrated global quantitative tandem mass tag (TMT)-based proteomic and phosphoproteomic analyses and RNA sequencing in five paired pre-treatment and relapse samples from multiple myeloma patients. These analyses reveal a CDK6-governed protein resistance signature that includes myeloma high-risk factors such as TRIP13 and RRM1. Overexpression of CDK6 in multiple myeloma cell lines reduces sensitivity to IMiDs while CDK6 inhibition by palbociclib or CDK6 degradation by proteolysis targeting chimeras (PROTACs) is highly synergistic with IMiDs in vitro and in vivo. This work identifies CDK6 upregulation as a druggable target in IMiD-resistant multiple myeloma and highlights the use of proteomic studies to uncover non-genetic resistance mechanisms in cancer.
Asunto(s)
Quinasa 6 Dependiente de la Ciclina , Lenalidomida , Mieloma Múltiple , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos , Humanos , Factores Inmunológicos/farmacología , Lenalidomida/farmacología , Lenalidomida/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Recurrencia Local de Neoplasia/tratamiento farmacológico , Proteómica , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia ArribaRESUMEN
Although the androgen receptor (AR) is a validated target for the treatment of prostate cancer, resistance to antiandrogens necessitates the development of new therapeutic modalities. Exploiting the ubiquitin-proteasome system with proteolysis-targeting chimeras (PROTACs) has become a practical approach to degrade specific proteins and thus to extend the portfolio of small molecules used for the treatment of a broader spectrum of diseases. Herein, we present three subgroups of enzalutamide-based PROTACs in which only the exit vector was modified. By recruiting cereblon, we were able to demonstrate the potent degradation of AR in lung cancer cells. Furthermore, the initial evaluation enabled the design of an optimized PROTAC with a rigid linker that degraded AR with a DC50 value in the nanomolar range. These results provide novel AR-directed PROTACs and a clear rationale for further investigating AR involvement in lung cancer models.
Asunto(s)
Neoplasias Pulmonares , Neoplasias de la Próstata , Receptores Androgénicos , Humanos , Masculino , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Proteolisis , Receptores Androgénicos/metabolismo , Relación Estructura-Actividad , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismoRESUMEN
Naturally occurring compounds represent a vast pool of pharmacologically active entities. One of such compounds is andrographolide, which is endowed with many beneficial properties, including the activity against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). To initiate a drug repurposing or hit optimization campaign, it is imperative to unravel the primary mechanism(s) of the antiviral action of andrographolide. Here, we showed by means of a reporter gene assay that andrographolide exerts its anti-SARS-CoV-2 effects by inhibiting the interaction between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2-related factor 2 (NRF2) causing NRF2 upregulation. Moreover, we demonstrated that subtle structural modifications of andrographolide could lead to derivatives with stronger on-target activities and improved physicochemical properties. Our results indicate that further optimization of this structural class is warranted to develop novel COVID-19 therapies.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Diterpenos/química , SARS-CoV-2/efectos de los fármacos , Animales , COVID-19/virología , Línea Celular , Chlorocebus aethiops , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Factor 2 Relacionado con NF-E2/metabolismo , SARS-CoV-2/fisiología , Células Vero , Replicación Viral , Tratamiento Farmacológico de COVID-19RESUMEN
Proteolysis targeting chimeras (PROTACs) hijacking the cereblon (CRBN) E3 ubiquitin ligase have emerged as a novel paradigm in drug development. Herein we found that linker attachment points of CRBN ligands highly affect their aqueous stability and neosubstrate degradation features. This work provides a blueprint for the assembly of future heterodimeric CRBN-based degraders with tailored properties.
RESUMEN
Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/-) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis.
Asunto(s)
Lesión Renal Aguda/patología , Ferroptosis/fisiología , Túbulos Renales/patología , Daño por Reperfusión/patología , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/etiología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Cisplatino/administración & dosificación , Cisplatino/toxicidad , Modelos Animales de Enfermedad , Células Epiteliales , Femenino , Ferroptosis/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HT29 , Trasplante de Corazón/efectos adversos , Humanos , Imidazoles/química , Imidazoles/farmacología , Imidazoles/uso terapéutico , Indoles/química , Indoles/farmacología , Indoles/uso terapéutico , Masculino , Ratones , Ratones Transgénicos , Microsomas Hepáticos , Proteínas Mitocondriales/metabolismo , Células 3T3 NIH , Necrosis/tratamiento farmacológico , Necrosis/etiología , Necrosis/patología , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/antagonistas & inhibidores , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Cultivo Primario de Células , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/etiologíaRESUMEN
Proteolysis-targeting chimeras (PROTACs) have received tremendous attention as a new and exciting class of therapeutic agents that promise to significantly impact drug discovery. These bifunctional molecules consist of a target binding unit, a linker, and an E3 ligase binding moiety. The chemically-induced formation of ternary complexes leads to ubiquitination and proteasomal degradation of target proteins. Among the plethora of E3 ligases, only a few have been utilized for the novel PROTAC technology. However, extensive knowledge on the preparation of E3 ligands and their utilization for PROTACs has already been acquired. This review provides an in-depth analysis of synthetic entries to functionalized ligands for the most relevant E3 ligase ligands, i.e. CRBN, VHL, IAP, and MDM2. Less commonly used E3 ligase and their ligands are also presented. We compare different preparative routes to E3 ligands with respect to feasibility and productivity. A particular focus was set on the chemistry of the linker attachment by discussing the synthetic opportunities to connect the E3 ligand at an appropriate exit vector with a linker to assemble the final PROTAC. This comprehensive review includes many facets involved in the synthesis of such complex molecules and is expected to serve as a compendium to support future synthetic attempts towards PROTACs.
RESUMEN
Aromatic scaffolds are an important part of biologically active compounds and molecular probes used to study biochemical pathways and the involved targeted proteins of interest. 1-Oxo-1H-phenalene-2,3-dicarbonitrile-based compounds have been described as inhibitors of the BCL-2 family of proteins, and this core structure represents numerous possibilities for modifications that could lead to improved inhibitory potencies. Many studies demonstrated intriguing characteristics of these compounds in terms of reactivity and, interestingly, some contradictory literature reports appeared about reaction outcomes to synthesize them. Here, we initially provide a condensed overview of transformations performed on the phenalene scaffold, followed by the resynthesis of a 6-phenoxy-substituted derivative. We show that the initial determination of this particular structure was wrong and provide two-dimensional nuclear magnetic resonance (NMR) evidence to assign the structure properly. When preparing new derivatives using the same synthetic route, we observed 6- and 7-substituted regioisomers. After confirming their structures by NMR experiments, the ability of these compounds to inhibit BCL-2 was evaluated. The most potent 1-oxo-1H-phenalene-2,3-dicarbonitrile derivatives inhibited BCL-2 in the nanomolar range and showed double-digit micromolar cytotoxicity against four different cancer cell lines.