Uncialamycin-based antibody-drug conjugates: Unique enediyne ADCs exhibiting bystander killing effect.

Antibody-drug conjugates (ADCs) have emerged as valuable targeted anticancer therapeutics with at least 11 approved therapies and over 80 advancing through clinical trials. Enediyne DNA-damaging payloads represented by the flagship of this family of antitumor agents, N-acetyl calicheamicin [Formula: see text], have a proven success track record. However, they pose a significant synthetic challenge in the development and optimization of linker drugs. We have recently reported a streamlined total synthesis of uncialamycin, another representative of the enediyne class of compounds, with compelling synthetic accessibility. Here we report the synthesis and evaluation of uncialamycin ADCs featuring a variety of cleavable and noncleavable linkers. We have discovered that uncialamycin ADCs display a strong bystander killing effect and are highly selective and cytotoxic in vitro and in vivo.

Design, Synthesis, and Biological Investigation of Thailanstatin A and Spliceostatin D Analogues Containing Tetrahydropyran, Tetrahydrooxazine, and Fluorinated Structural Motifs.

Thailanstatin A and spliceostatin D, two naturally occurring molecules endowed with potent antitumor activities by virtue of their ability to bind and inhibit the function of the spliceosome, and their natural siblings and designed analogues, constitute an appealing family of compounds for further evaluation and optimization as potential drug candidates for cancer therapies. In this article, the design, synthesis, and biological investigation of a number of novel thailanstatin A analogues, including some accommodating 1,1-difluorocyclopropyl and tetrahydrooxazine structural motifs within their structures, are described. Important findings from these studies paving the way for further investigations include the identification of several highly potent compounds for advancement as payloads for antibody-drug conjugates (ADCs) as potential targeted cancer therapies and/or small molecule drugs, either alone or in combination with other anticancer agents.

Design, Synthesis, and Biological Evaluation of Tubulysin Analogues, Linker-Drugs, and Antibody-Drug Conjugates, Insights into Structure-Activity Relationships, and Tubulysin-Tubulin Binding Derived from X-ray Crystallographic Analysis.

Molecular design, synthesis, and biological evaluation of tubulysin analogues, linker-drugs, and antibody-drug conjugates are described. Among the new discoveries reported is the identification of new potent analogues within the tubulysin family that carry a C11 alkyl ether substituent, rather than the usual ester structural motif at that position, a fact that endows the former with higher plasma stability than that of the latter. Also described herein are X-ray crystallographic analysis studies of two tubulin-tubulysin complexes formed within the α/ß interface between two tubulin heterodimers and two highly potent tubulysin analogues, one of which exhibited a different binding mode to the one previously reported for tubulysin M. The X-ray crystallographic analysis-derived new insights into the binding modes of these tubulysin analogues explain their potencies and provide inspiration for further design, synthesis, and biological investigations within this class of antitumor agents. A number of these analogues were conjugated as payloads with appropriate linkers at different sites allowing their attachment onto targeting antibodies for cancer therapies. A number of such antibody-drug conjugates were constructed and tested, both in vivo and in vitro, leading to the identification of at least one promising ADC (Herceptin-LD3), warranting further investigations.

Total Synthesis and Biological Evaluation of Tiancimycins A and B, Yangpumicin A, and Related Anthraquinone-Fused Enediyne Antitumor Antibiotics.

The family of anthraquinone-fused enediyne antitumor antibiotics was established by the discovery of dynemicin A and deoxy-dynemicin A. It was then expanded, first by the isolation of uncialamycin, and then by the addition to the family of tiancimycins A-F and yangpumicin A. This family of natural products provides opportunities in total synthesis, biology, and medicine due to their novel and challenging molecular structures, intriguing biological properties and mechanism of action, and potential in targeted cancer therapies. Herein, the total syntheses of tiancimycins A and B, yangpumicin A, and a number of related anthraquinone-fused enediynes are described. Biological evaluation of the synthesized compounds revealed extremely potent cytotoxicities against a number of cell lines, thus enriching the structure-activity relationships within this class of compounds. The findings of these studies may facilitate future investigations directed toward antibody-drug conjugates for targeted cancer therapies and provide inspiration for further advances in total synthesis and chemical biology.

Synthesis and Biological Evaluation of Shishijimicin A-Type Linker-Drugs and Antibody-Drug Conjugates.

Our previous studies with shishijimicin A resulted in the total synthesis of this scarce marine natural product and a number of its simpler analogues endowed with picomolar potencies against certain cancer cell lines. Herein, we describe the design, synthesis, and biological evaluation of four linker-drugs, anticipating the construction of antibody-drug conjugates (ADCs) as the ultimate goal of this research program. Using a common payload, the assembly of these linker-drugs utilized different linkers and attachment points, providing opportunities to probe the optimal molecular design of the intended ADCs as targeted cancer therapies. In the course of ADC generation and in vitro evaluation, we identified two linker-drugs with a promising in vitro plasma stability profile and excellent targeted cytotoxicity and specificity. Conjugation of shishijimicin A enediyne payloads through their phenolic moiety represents a novel approach to enediyne ADC creation, while the pharmacological profiles of at least two of the generated ADCs compare well with the profiles of the corresponding clinically approved ADC Kadcyla.

Streamlined Symmetrical Total Synthesis of Disorazole B1 and Design, Synthesis, and Biological Investigation of Disorazole Analogues.

Taking advantage of the C2-symmetry of the antitumor naturally occurring disorazole B1 molecule, a symmetrical total synthesis was devised with a monomeric advanced intermediate as the key building block, whose three-step conversion to the natural product allowed for an expeditious entry to this family of compounds. Application of the developed synthetic strategies and methods provided a series of designed analogues of disorazole B1, whose biological evaluation led to the identification of a number of potent antitumor agents and the first structure-activity relationships (SARs) within this class of compounds. Specifically, the substitutions of the epoxide units and lactone moieties with cyclopropyl and lactam structural motifs, respectively, were found to be tolerable for biological activities and beneficial with regard to chemical stability.

Design, Synthesis, and Biological Investigation of Epothilone B Analogues Featuring Lactone, Lactam, and Carbocyclic Macrocycles, Epoxide, Aziridine, and 1,1-Difluorocyclopropane and Other Fluorine Residues.

Despite previous studies within the epothilone field, only one member of this compound family, ixabepilone, made it to approval for clinical use. Recent advances in organic synthesis and medicinal chemistry allow further optimization of lead epothilone analogues aiming to improve their potencies and other pharmacological properties as part of the quest for discovery and development of new anticancer drugs, including antibody-drug conjugates as potential targeted cancer therapies. Herein, we report the design, synthesis, and biological evaluation of a series of new epothilone B analogues equipped with novel structural motifs, including fluorine-containing residues, 12,13-difluorocyclopropyl moieties, mono- and dimethylated macrolactones, and 1-keto macrocyclic systems, as well as two N-substituted ixabepilone analogues in which the 12,13-epoxide and macrolactam NH moieties were replaced, the former with a substituted aziridine moiety and the latter with an NCO-alkyl residue (imide or carbamate). Biological evaluation of these analogues revealed a number of exceptionally potent epothilone B analogues, demonstrating the potency enhancing effects of the fluorine residues and the aziridinyl moiety within the structure of the epothilone molecule and providing new and useful structure-activity relationships within this class of compounds.

Evaluation of PNU-159682 antibody drug conjugates (ADCs).

PNU-159682 is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. Due to its extremely high potency and only partially understood mechanism of action, it was deemed an interesting starting point for the development of a new suite of linker drugs for antibody drug conjugates (ADCs). Structure activity relationships were explored on the small molecule which led to six linker drugs being developed for conjugation to antibodies. Herein we describe the synthesis of novel PNU-159682 derivatives and the subsequent linker drugs as well as the corresponding biological evaluations of the small molecules and ADCs.

Short Total Synthesis of Δ12-Prostaglandin J2 and Related Prostaglandins. Design, Synthesis, and Biological Evaluation of Macrocyclic Δ12-Prostaglandin J2 Analogues.

Comprised of a large collection of structurally diverse molecules, the prostaglandins exhibit a wide range of biological properties. Among them are Δ12-prostaglandin J2 (Δ12-PGJ2) and Δ12-prostaglandin J3 (Δ12-PGJ3), whose unusual structural motifs and potent cytotoxicities present unique opportunities for chemical and biological investigations. Herein, we report a short olefin-metathesis-based total synthesis of Δ12-PGJ2 and its application to the construction of a series of designed analogues possessing monomeric, dimeric, trimeric, and tetrameric macrocyclic lactones consisting of units of this prostaglandin. Biological evaluation of these analogues led to interesting structure-activity relationships and trends and the discovery of a number of more potent antitumor agents than their parent naturally occurring molecules.

Improved Total Synthesis of Tubulysins and Design, Synthesis, and Biological Evaluation of New Tubulysins with Highly Potent Cytotoxicities against Cancer Cells as Potential Payloads for Antibody-Drug Conjugates.

Improved, streamlined total syntheses of natural tubulysins such as V (Tb45) and U (Tb46) and pretubulysin D (PTb-D43), and their application to the synthesis of designed tubulysin analogues (Tb44, PTb-D42, PTb-D47-PTb-D49, and Tb50-Tb120), are described. Cytotoxicity evaluation of the synthesized compounds against certain cancer cell lines revealed a number of novel analogues with exceptional potencies [e.g., Tb111: IC50 = 40 pM against MES SA (uterine sarcoma) cell line; IC50 = 6 pM against HEK 293T (human embryonic kidney cancer) cell line; and IC50 = 1.54 nM against MES SA DX (MES SA with marked multidrug resistance) cell line]. These studies led to a set of valuable structure-activity relationships that provide guidance to further molecular design, synthesis, and biological evaluation studies. The extremely potent cytotoxic compounds discovered in these investigations are highly desirable as potential payloads for antibody-drug conjugates and other drug delivery systems for personalized targeted cancer chemotherapies.

Streamlined Total Synthesis of Shishijimicin A and Its Application to the Design, Synthesis, and Biological Evaluation of Analogues thereof and Practical Syntheses of PhthNSSMe and Related Sulfenylating Reagents.

Shishijimicin A is a scarce marine natural product with highly potent cytotoxicities, making it a potential payload or a lead compound for designed antibody-drug conjugates. Herein, we describe an improved total synthesis of shishijimicin A and the design, synthesis, and biological evaluation of a series of analogues. Equipped with appropriate functionalities for linker attachment, a number of these analogues exhibited extremely potent cytotoxicities for the intended purposes. The synthetic strategies and tactics developed and employed in these studies included improved preparation of previously known and new sulfenylating reagents such as PhthNSSMe and related compounds.

12,13-Aziridinyl Epothilones. Stereoselective Synthesis of Trisubstituted Olefinic Bonds from Methyl Ketones and Heteroaromatic Phosphonates and Design, Synthesis, and Biological Evaluation of Potent Antitumor Agents.

The synthesis and biological evaluation of a series of 12,13-aziridinyl epothilone B analogues is described. These compounds were accessed by a practical, general process that involved a 12,13-olefinic methyl ketone as a starting material obtained by ozonolytic cleavage of epothilone B followed by tungsten-induced deoxygenation of the epoxide moiety. The attachment of the aziridine structural motif was achieved by application of the Ess-Kürti-Falck aziridination, while the heterocyclic side chains were introduced via stereoselective phosphonate-based olefinations. In order to ensure high (E) selectivities for the latter reaction for electron-rich heterocycles, it became necessary to develop and apply an unprecedented modification of the venerable Horner-Wadsworth-Emmons reaction, employing 2-fluoroethoxyphosphonates that may prove to be of general value in organic synthesis. These studies resulted in the discovery of some of the most potent epothilones reported to date. Equipped with functional groups to accommodate modern drug delivery technologies, some of these compounds exhibited picomolar potencies that qualify them as payloads for antibody drug conjugates (ADCs), while a number of them revealed impressive activities against drug resistant human cancer cells, making them desirable for potential medical applications.

Streamlined Total Synthesis of Trioxacarcins and Its Application to the Design, Synthesis, and Biological Evaluation of Analogues Thereof. Discovery of Simpler Designed and Potent Trioxacarcin Analogues.

A streamlined total synthesis of the naturally occurring antitumor agents trioxacarcins is described, along with its application to the construction of a series of designed analogues of these complex natural products. Biological evaluation of the synthesized compounds revealed a number of highly potent, and yet structurally simpler, compounds that are effective against certain cancer cell lines, including a drug-resistant line. A novel one-step synthesis of anthraquinones and chloro anthraquinones from simple ketone precursors and phenylselenyl chloride is also described. The reported work, featuring novel chemistry and cascade reactions, has potential applications in cancer therapy, including targeted approaches as in antibody-drug conjugates.

Total Synthesis and Biological Evaluation of Natural and Designed Tubulysins.

A streamlined total synthesis of N(14)-desacetoxytubulysin H (Tb1) based on a C-H activation strategy and a short total synthesis of pretubulysin D (PTb-D43) are described. Applications of the developed synthetic strategies and technologies to the synthesis of a series of tubulysin analogues (Tb2-Tb41 and PTb-D42) are also reported. Biological evaluation of the synthesized compounds against an array of cancer cells revealed a number of novel analogues (e.g., Tb14), some with exceptional potencies against certain cell lines [e.g., Tb32 with IC50 = 12 pM against MES SA (uterine sarcoma) cell line and 2 pM against HEK 293T (human embryonic kidney) cell line], and a set of valuable structure-activity relationships. The highly potent cytotoxic compounds discovered in this study are highly desirable as payloads for antibody-drug conjugates and other drug delivery systems for personalized targeted cancer chemotherapies.

Synthesis and Biological Investigation of Δ(12)-Prostaglandin J3 (Δ(12)-PGJ3) Analogues and Related Compounds.

A series of Δ(12)-prostaglandin J3 (Δ(12)-PGJ3) analogues and derivatives were synthesized employing an array of synthetic strategies developed specifically to render them readily available for biological investigations. The synthesized compounds were evaluated for their cytotoxicity against a number of cancer cell lines, revealing nanomolar potencies for a number of them against certain cancer cell lines. Four analogues (2, 11, 21, and 27) demonstrated inhibition of nuclear export through a covalent addition at Cys528 of the export receptor Crm1. One of these compounds (i.e., 11) is currently under evaluation as a potential drug candidate for the treatment of certain types of cancer. These studies culminated in useful and path-pointing structure-activity relationships (SARs) that provide guidance for further improvements in the biological/pharmacological profiles of compounds within this class.

A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma.

Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. The anti-MM activity of ONX-0912 is associated with activation of caspase-8, caspase-9, caspase-3, and poly(ADP) ribose polymerase, as well as inhibition of migration of MM cells and angiogenesis. ONX 0912, like bortezomib, predominantly inhibits chymotrypsin-like activity of the proteasome and is distinct from bortezomib in its chemical structure. Importantly, ONX 0912 is orally bioactive. In animal tumor model studies, ONX 0912 significantly reduced tumor progression and prolonged survival. Immununostaining of MM tumors from ONX 0912-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Finally, ONX 0912 enhances anti-MM activity of bortezomib, lenalidomide dexamethasone, or pan-histone deacetylase inhibitor. Taken together, our study provides the rationale for clinical protocols evaluating ONX 0912, either alone or in combination, to improve patient outcome in MM.

Selective inhibition of chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in Waldenstrom macroglobulinemia.

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor kappaB (NF-kappaB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)-derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.

ABBV-011, A Novel, Calicheamicin-Based Antibody-Drug Conjugate, Targets SEZ6 to Eradicate Small Cell Lung Cancer Tumors.

In the past year, four antibody-drug conjugates (ADC) were approved, nearly doubling the marketed ADCs in oncology. Among other attributes, successful ADCs optimize targeting antibody, conjugation chemistry, and payload mechanism of action. Here, we describe the development of ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC for the treatment of small cell lung cancer (SCLC). We engineered a calicheamicin conjugate that lacks the acid-labile hydrazine linker that leads to systemic release of a toxic catabolite. We then screened a patient-derived xenograft library to identify SCLC as a tumor type with enhanced sensitivity to calicheamicin ADCs. Using RNA sequencing (RNA-seq) data from primary and xenograft SCLC samples, we identified seizure-related homolog 6 (SEZ6) as a surface-expressed SCLC target with broad expression in SCLC and minimal normal tissue expression by both RNA-seq and IHC. We developed an antibody targeting SEZ6 that is rapidly internalized upon receptor binding and, when conjugated to the calicheamicin linker drug, drives potent tumor regression in vitro and in vivo. These preclinical data suggest that ABBV-011 may provide a novel treatment for patients with SCLC and a rationale for ongoing phase I studies (NCT03639194).

PR-924, a selective inhibitor of the immunoproteasome subunit LMP-7, blocks multiple myeloma cell growth both in vitro and in vivo.

PR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase-8, caspase-9, BID, PARP and cytochrome-c release. In vivo administration of PR-924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM.