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FRA expression on regrown MORAb-202-treated LXFA-737 non-small cell lung carcinoma PDX tumors

Microtubule-targeting agents (MTA) have been investigated for many years as payloads for antibody–drug conjugates (ADC). In many cases, these ADCs have shown limited benefits due to lack of efficacy or significant toxicity, which has spurred continued investigation into novel MTA payloads for next-generation ADCs. In this study, we have developed ADCs using the MTA eribulin, a derivative of the macrocyclic polyether natural product halichondrin B, as a payload. Eribulin ADCs demonstrated in vitro potency and specificity using various linkers and two different conjugation approaches. MORAb-202 is an investigational agent that consists of the humanized anti-human folate receptor alpha (FRA) antibody farletuzumab conjugated via reduced interchain disulfide bonds to maleimido-PEG2-valine-citrulline-p-aminobenzylcarbamyl-eribulin at a drug-to-antibody ratio of 4.0. MORAb-202 displayed preferable biophysical properties and broad potency across a number of FRA-positive tumor cell lines as well as demonstrated improved specificity in vitro compared with farletuzumab conjugated with a number of other MTA payloads, including MMAE, MMAF, and the reducible maytansine linker-payload sulfo-SPDB-DM4. A single-dose administration of MORAb-202 in FRA-positive human tumor cell line xenograft and patient-derived tumor xenograft models elicited a robust and durable antitumor response. These data support further investigation of MORAb-202 as a potential new treatment modality for FRA-positive cancers, using the novel MTA eribulin as a payload.

A strategy for creating potent and pan-genotypic stimulator of interferon genes (STING) agonists is described. Locking a bioactive U-shaped conformation of cyclic dinucleotides by introducing a transannular macrocyclic bridge between the nucleic acid bases leads to a topologically novel macrocycle-bridged STING agonist (MBSA). In addition to substantially enhanced potency, the newly designed MBSAs, exemplified by clinical candidate E7766, exhibit broad pan-genotypic activity in all major human STING variants. E7766 is shown to have potent antitumor activity with long lasting immune memory response in a mouse liver metastatic tumor model. Two complementary stereoselective synthetic routes to E7766 are also described.

Microtubule-targeting agents (MTA) have been investigated for many years as payloads for antibody–drug conjugates (ADC). In many cases, these ADCs have shown limited benefits due to lack of efficacy or significant toxicity, which has spurred continued investigation into novel MTA payloads for next-generation ADCs. In this study, we have developed ADCs using the MTA eribulin, a derivative of the macrocyclic polyether natural product halichondrin B, as a payload. Eribulin ADCs demonstrated in vitro potency and specificity using various linkers and two different conjugation approaches. MORAb-202 is an investigational agent that consists of the humanized anti-human folate receptor alpha (FRA) antibody farletuzumab conjugated via reduced interchain disulfide bonds to maleimido-PEG2-valine-citrulline-p-aminobenzylcarbamyl-eribulin at a drug-to-antibody ratio of 4.0. MORAb-202 displayed preferable biophysical properties and broad potency across a number of FRA-positive tumor cell lines as well as demonstrated improved specificity in vitro compared with farletuzumab conjugated with a number of other MTA payloads, including MMAE, MMAF, and the reducible maytansine linker-payload sulfo-SPDB-DM4. A single-dose administration of MORAb-202 in FRA-positive human tumor cell line xenograft and patient-derived tumor xenograft models elicited a robust and durable antitumor response. These data support further investigation of MORAb-202 as a potential new treatment modality for FRA-positive cancers, using the novel MTA eribulin as a payload.

Introduction STING (stimulator of interferon genes) is an emerging target for cancer immunotherapy. 2’,3'-cGAMP, a natural cyclic dinucleotide (CDN) STING agonist, and its phosphorothioate analogs, have drawn broad attention as lead molecules for STING targeted drug discovery. These CDNs, however, lack efficacy in some common STING genotypes disproportionally represented in non-Caucasians. Moreover, such CDNs have not fully addressed liability in chemical/metabolic stability. Here we report our chemistry approach to control STING agonist conformation to enhance binding affinity across all common STING genotypes and broaden the therapeutic potential of such compounds. Methods Our SBDD approach started with analysis of the binding pocket and key protein-ligand interactions to prioritize a focused set of analogs for chemical synthesis. Systematic SAR was built upon in vitro assays for STING binding affinity and activation of STING genotypes. X-ray single crystal structures were established for STING and diverse analogs, in free and bound states, to provide structural insight for rational analog design. Results Structural modeling was refined to evaluate different binding modes and dynamic conformational changes in the STING-ligand interface. We observed that STING-bound CDNs had the two ancillary nucleobases specifically oriented in close proximity with parallel pi-pi stacking and discovered that covalently linking the nucleobases advantageously pre-organize the bioactive constrained conformation for enhanced STING affinity. Our discovery established a novel class of macrocycle-bridged STING agonists (MBSAs). E7766, a representative of Eisai MBSA platform, shows superior in vitro activity against all the major human STING genotypes over reference CDNs, most distinctly in STINGREF. E7766 co-crystal structures with STINGWT and STINGREF provide structural basis for the added benefit of the topological novelty. The macrocyclic linker bridging the top of nucleobases perturbs the STING lid loop conformation and create new and specific interactions with both genotypes. In twelve subcutaneous tumor models in immune competent mice, single intra-tumoral injections achieved either complete regression or significant tumor growth delay with no serious adverse effect. E7766 also shows excellent chemical and metabolic stability, presumably conferred by conformational rigidity of the unique macrocycle bridge. More biological characterization of E7766 can be found in abstract #. Conclusion Eisai successfully discovered E7766, a representative of a novel class of macrocycle-bridged STING agonist topologically distinct from conventional STING agonists. E7766 demonstrated pan-genotypic STING activation, potent anti-cancer activities and excellent chemical and metabolic stability for further development. Citation Format: Atsushi ENDO, Dae-Shik Kim, Kuan-Chun Huang, Ming-Hong Hao, Steven Mathieu, Hyeong-wook Choi, Utpal Majumder, Xiaojie Zhu, Yongchun Shen, Kristen Sanders, Thomas Noland, Dinesh Chandra, Yu Chen, Karen TenDyke, Kara Loiacono, Donna Kolber-Simonds, Rongrong Jiang, Vaishali Dixit, Janna Hutz, John Wang, Xingfeng Bao, Francis Fang, Nadeem Sarwar. Discovery of E7766: A representative of a novel class of macrocycle-bridged STING agonists (MBSAs) with superior potency and pan-genotypic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4456.

Chlorotoxin is an established tumor targeting peptide that naturally occurs in scorpion venom. 131I-labelled chlorotoxin assessed in early phase human glioma clinical trials achieved promising results. A peptide drug conjugate (PDC) composed of chlorotoxin peptide linked to a cytotoxic payload (cryptophycin analog) was used as a tool to probe the tumor targeting mechanism of chlorotoxin. The PDC proved efficacious, yet differential sensitivity was observed in multiple human tumor models. Previously described chlorotoxin targets did not align with observed PDC activity; therefore, studies to further elucidate its mechanism were undertaken. PDC treatment of distinct xenograft models led to a wide range of antitumor activity even though similar levels of active metabolite were present in tumor lysates; thus it was hypothesized that a role for chlorotoxin in uptake may be relevant. We identified that the endocytic receptor Neuropilin1 (NRP1) binds to chlorotoxin peptide fragments following proteolytic digestion in vitro. NRP1 binding was selective for peptides with a free C-terminal arginine, while native chlorotoxin which has an amidated C-terminal arginine did not bind. Recovery of chlorotoxin from ex vivo tumor lysate revealed its metabolism to a carboxylated C-terminal arginine version of the peptide, capable of binding to NRP1. These data suggest that chlorotoxin acts as a cryptic peptide incapable of binding to NRP1 systemically and only when metabolized in the tumor microenvironment is NRP1 binding revealed. The expression level of human NRP1 in tumors correlated to the PDC antitumor activity in multiple xenograft models; a wider therapeutic window was observed when NRP1 was highly expressed. Reduction of NRP1 levels in vivo through administration of NRP1 blocking antibodies or by NRP1 knockout in tumor cells blunted PDC antitumor activity while not affecting activity of the cytotoxic payload alone. Reduced PDC antitumor activity correlated with significantly lower levels of active metabolite detected in NRP1-deficient tumors. Together, our findings suggest that chlorotoxin metabolized in the tumor microenvironment binds NRP1 on tumor cells to increase uptake of active metabolite into cells, resulting in enhanced antitumor activity. The identification of NRP1 as an uptake mechanism for chlorotoxin will enable selection of tumors for treatment with chlorotoxin-based therapeutics. Citation Format: Donna Kolber-Simonds, Jiayi Wu, Utpal Majumder, Daniel Custar, Danyang Li, Hong Du, Maarten H. Postema, Thomas Noland, Andrew Hart, George Lai, Sean Eckley, Vaishali Dixit, Karen Tendyke, Kenichi Nomoto, Mary Woodall-Jappe, Sharon McGonigle. Role for neuropilin1 in mode of action of chlorotoxin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3961.

New synthetic methods for the construction of novel heterocycles and tryptamines are described. Thus, N-Boc anilines (I) are sequentially converted to heterocycles II ((3-(2-aminophenyl)pyrrolidin-3-ol) derivatives), III (substituted 2-oxo-1,2-dihydrospirobenzo[d][1,3]oxazine-4,3'-pyrrolidines), and VI (2-(4,5-dihydro-1H-pyrrol-3-yl)aniline) derivatives through a route involving t-BuLi induced ortho-metalation/LaCl(3).2LiCl metal exchange, reaction with N-Boc pyrrolidin-3-one (5), and subsequent decarboxylative fragmentation. Labile intermediates VI are effectively converted to tryptamines Xa and Xb under controlled protic acid conditions. In addition to providing expedient access to the 2-oxo-1,2-dihydrospirobenzo[d][1,3]oxazine-4,3'-pyrrolidines (III), the method is applicable to the synthesis of the corresponding 2-oxo-1,2-dihydrospirobenzo[d][1,3]oxazine-4,3'-piperidine series of spirocycles (e.g., 42) and their precursors (3-(2-aminophenyl)piperidin-3-ol derivatives, e.g., 43) by using N-Boc-protected piperidin-3-one (40). Applications of the developed synthetic technologies to the synthesis of regioisomeric spirocycles 87 and 90, tryptamines 88 and 91, Corey's aspidophytine tryptamine (97), and efavirenz (1) are also described.

The preceding manuscript detailed our synthesis of the gambierol A-C and F-H ring precursors. Reported herein is a description of the coupling of the two precursors and the conversion of the coupled material into gambierol. Coupling of the subunits involved ester formation, enol ether RCM, and mixed thioketal formation and reduction. By employing this strategy we were able to bring highly advanced subunits into the coupling and, as a result, we were able to minimize the number of post-coupling transformations required to complete gambierol. At the completion of the synthesis, we generated 7.5 mg (1.5 % overall yield) of (-)-gambierol in 44 steps (longest linear sequence).

[structure: see text] This manuscript describes our synthesis of the F-H subunit of gambierol. In addition to the synthesis of the tricycle, of note is an interesting protecting group influence on the generation of a C(23) C-glycoside as well as the use of ring-closing metathesis to generate a tetrasubstituted enol ether.

Reactions of various diketo compounds with Deoxofluor [(CH3OCH2CH2)2NSF3] have been investigated. When reacted with Deoxofluor, α-diketones, R1COCOR2 (R1 = R2 = Ph; R1 = R2 = 4-Me-C6H4; R1 = Ph, R2 = Me; R1 = Me, R2 = Et) (1a−d) formed difluoro derivatives (2a−d) in the presence of a catalytic amount of HF and/or tetrafluoro (3a−d) products depending on the reaction conditions and stoichiometry used. Reactions of β-diketones, R3COCH2COR4 (R3 = R4 = Ph; R3 = R4 = Me; R3 = Me, R4 = Ph) (4e−g), with Deoxofluor in the presence of a catalytic amount of HF led to the formation of difluoroalkenones as a mixture of E (5e−g) and Z (6e−g) isomers in good yield. Reaction of other diones, R5CO-X-COR6 (R5 = R6 = Ph, X = −CHCH−; R5 = R6 = Me, X = −C6H4C6H4−; R5 = R6 = Ph, X = −CH2CH2CH2−; R5 = R6 = Me, X = −CH2CH2−) (7h−k) with Deoxofluor produced mainly difluoro products (8h−k) with low yields of tertrafluoro derivatives (9h−k). Acyclic α-keto amides react poorly to give the corresponding difluoro derivatives, whereas...

New synthetic methods for the construction of novel heterocycles and tryptamines are described. Thus, N-Boc anilines (I) are sequentially converted to heterocycles II ((3-(2-aminophenyl)pyrrolidin-3-ol) derivatives), III (substituted 2-oxo-1,2-dihydrospirobenzo[d][1,3]oxazine-4,3′-pyrrolidines), and VI (2-(4,5-dihydro-1H-pyrrol-3-yl)aniline) derivatives through a route involving t-BuLi induced ortho-metalation/LaCl3·2LiCl metal exchange, reaction with N-Boc pyrrolidin-3-one (5), and subsequent decarboxylative fragmentation. Labile intermediates VI are effectively converted to tryptamines Xa and Xb under controlled protic acid conditions. In addition to providing expedient access to the 2-oxo-1,2-dihydrospirobenzo[d][1,3]oxazine-4,3′-pyrrolidines (III), the method is applicable to the synthesis of the corresponding 2-oxo-1,2-dihydrospirobenzo[d][1,3]oxazine-4,3′-piperidine series of spirocycles (e.g., 42) and their precursors (3-(2-aminophenyl)piperidin-3-ol derivatives, e.g., 43) by using N-Boc-protected ...

An expedient asymmetric total synthesis of aspidophytine is reported. A highly convergent strategy involving the sequential annulation of vinyl iodide 5 with indole 6 exploits varying modes of indole reactivity to provide aspidophytine in 23% over six steps from 5.

Gambierol, a representative of the marine ladder toxin family, consists of eight ether rings, 18 stereocenters, and two challenging pyranyl rings having methyl groups that are in a 1,3-diaxial orientation to one another. Herein we describe the generation of gambierol's A-C and F-H ring systems and demonstrate the versatility of the glycosyl anhydride, enol ether-olefin RCM strategy to fused polycyclic ethers. This work has both enabled us to generate sufficient quantities of the gambierol precursors and has enabled us to better understand the chemical transformations that were key to these efforts. Fundamental work included efforts to C-glycosides and C-ketosides, Claisen rearrangements, and enol ether-olefin RCM reactions.

The synthesis and characterization of the first stable trialkyl(difluoroamino)silane, R3SiNF2, as well as of R3SiNHF and R3SiN(CH3)F in moderate yields are reported. The (difluoroamino)silane has promise as a new synthon for the introduction of the −NF2 group into a variety of electrophilic inorganic and organic substrates. Activation barriers and relative energies were calculated for the unimolecular decompositions of Me3SiCF3 and t-Bu3SiNF2 using density functional theory (B3LYP/6-31G*). The calculated activation energies confirm the long-assumed kinetic stability of Me3SiCF3.

Reactions of various diketo compounds with Deoxofluor [(CH(3)OCH(2)CH(2))(2)NSF(3)] have been investigated. When reacted with Deoxofluor, alpha-diketones, R(1)COCOR(2) (R(1) = R(2) = Ph; R(1) = R(2) = 4-Me-C(6)H(4); R(1) = Ph, R(2) = Me; R(1) = Me, R(2) = Et) (1a-d) formed difluoro derivatives (2a-d) in the presence of a catalytic amount of HF and/or tetrafluoro (3a-d) products depending on the reaction conditions and stoichiometry used. Reactions of beta-diketones, R(3)COCH(2)COR(4) (R(3) = R(4) = Ph; R(3) = R(4) = Me; R(3) = Me, R(4) = Ph) (4e-g), with Deoxofluor in the presence of a catalytic amount of HF led to the formation of difluoroalkenones as a mixture of E (5e-g) and Z (6e-g) isomers in good yield. Reaction of other diones, R(5)CO-X-COR(6) (R(5) = R(6) = Ph, X = -CH=CH-; R(5) = R(6) = Me, X = -C(6)H(4)C(6)H(4)-; R(5) = R(6) = Ph, X = -CH(2)CH(2)CH(2)-; R(5) = R(6) = Me, X = -CH(2)CH(2)-) (7h-k) with Deoxofluor produced mainly difluoro products (8h-k) with low yields of tertrafluoro derivatives (9h-k). Acyclic alpha-keto amides react poorly to give the corresponding difluoro derivatives, whereas cyclic alpha-keto amides (10l-p) react smoothly under very mild conditions to produce the corresponding difluoro products (11l-p) in88% isolated yield.

This communication describes the total synthesis of the marine polyether toxin, gambierol. This work couples our iterative C-glycoside/enol ether-olefin metathesis strategy to the subunits with a unique olefin metathesis/carbonyl olefination reaction to bring the subunits together.