Preclinical Characterization of Catabolic Pathways and Metabolism of ABBV-011, a Novel Calicheamicin-Based SEZ6-Targeting Antibody-Drug Conjugate.

Antibody-drug conjugates (ADC) have gained momentum for treatment of cancers, with 14 ADCs currently approved for commercial use worldwide. Calicheamicin is one of the payloads contributing to this trend, being used for both gemtuzumab ozogamicin (GO; trade name: Mylotarg) and inotuzumab ozogamicin (IO; trade name: Besponsa). Here we discuss the catabolic pathway and metabolism of ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC being investigated for the treatment of small cell lung cancer (SCLC). Specifically, our investigation has found that disulfide bond cleavage in N-acetyl-γ-calicheamicin payload is a key liability that potentially impacts overall stability of the ADC. To our knowledge, there have been no reported observations of disulfide bond cleavage of calicheamicin ADCs. ABBV-011 utilizes a novel linker structure, leading to a distinct metabolic profile when compared with GO and IO. Despite this difference in linker structures, we propose that this liability may also be relevant for other calicheamicin ADCs. Multiple data sets supporting our investigation were acquired as part of the preclinical development of ABBV-011 and demonstrate the utility of in vitro experiments to characterize potential ADC candidates prior to clinical trials. SIGNIFICANCE STATEMENT: Several in vitro and in vivo stability studies of ABBV-011, a calicheamicin-based antibody-drug conjugate (ADC), identified circulating metabolites and catabolites and suggested that disulfide cleavage may be a key liability for the conjugated linker-payload. These observations may be relevant to other disulfide-linked ADCs such as gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin (Besponsa), both of which have reported similar half-lives that possibly indicate instability.

Selective Reduction of Cysteine Mutant Antibodies for Site-Specific Antibody-Drug Conjugates.

Developing site-specific conjugation technologies for antibody-drug conjugates (ADCs) aims to produce more homogeneous and controlled drug-loaded ADCs to reduce variability and thereby improve the therapeutic index. This article presents a technology that uses cysteine mutant antibodies and mild phosphine-based reductants to prepare site-specific ADCs. The two types of cysteine mutant antibodies, designated C6v1 and C6v2, have one of the interchain disulfide-forming cysteines in the Fab region in the light chain (LC214) or in the heavy chain (HC220) substituted by alanine (or other amino acids), respectively. Certain phosphine-based reductants were found to selectively reduce the "unpaired" cysteines, at the heavy chain (HC220) for C6v1 or at the light chain (LC214) for C6v2 while keeping the interchain disulfide bonds in the hinge region intact, resulting in 90% of DAR2 species and more than 95% of the desired specific conjugation at HC or LC following conjugation to maleimide moieties. The reduction method shows consistent selectivity toward various C6v1 or C6v2 antibody backbones. Sensitivity toward buffer pH for some reductants can be used to optimize reductant reactivity and selectivity. The technology can be further expanded to generate site-specific DAR4 or dual-payload ADCs based on C6v1 or C6v2 antibodies. This technology offers a method to control drug-loading and conjugation sites using a mild one-pot process, as compared to the reduction-oxidation methods used in technologies such as THIOMAB, and shows superior DAR profiles and process simplification as compared to other selective reduction methods.

Deuterated active pharmaceutical ingredients: A science-based proposal for synthesis, analysis, and control. Part 1: Framing the problem.

The International Consortium for Innovation & Quality (IQ) in Pharmaceutical Development recently established a working group focused on the development of a guidance to address Deuterated Active Pharmaceutical Ingredients. Deuteration of an Active Pharmaceutical Ingredient (API) in some cases can retard and/or alter API metabolism by exploiting the primary kinetic isotope effect. Several deuterated APIs have entered into the clinic, and one has recently been approved. In most cases, it is very difficult to nearly impossible to synthesize a 100% isotopically pure compound. This raises synthetic, analytical, and regulatory questions that warrant a science-based assessment and recommendations for synthetic methods, analytical methods, and specifications. A cross functional team of scientists with expertise in isotope chemistry, process chemistry, analytical chemistry, and drug metabolism and pharmacokinetics have been meeting under the auspices of IQ to define and address these questions. This paper strives to frame chemistry, manufacturing, and controls challenges.

Synthesis of ( R)-Boc-2-methylproline via a Memory of Chirality Cyclization. Application to the Synthesis of Veliparib, a Poly(ADP-ribose) Polymerase Inhibitor.

( R)-Boc-2-methylproline (3a) was synthesized in good yield with excellent stereochemical control from alanine benzyl ester hydrochloride 11. The process, which is based on a modification of one described by Kawabata, proceeds in four steps and requires no chromatography. The product ( R)-Boc-2-methylproline (3a) was then carried forward in three steps to produce veliparib 1, a poly(ADP-ribose) polymerase inhibitor.

Discovery and Development of Metal-Catalyzed Coupling Reactions in the Synthesis of Dasabuvir, an HCV-Polymerase Inhibitor.

Dasabuvir (1) is an HCV polymerase inhibitor which has been developed as a part of a three-component direct-acting antiviral combination therapy. During the course of the development of the synthetic route, two novel coupling reactions were developed. First, the copper-catalyzed coupling of uracil with aryl iodides, employing picolinamide 16 as the ligand, was discovered. Later, the palladium-catalyzed sulfonamidation of aryl nonaflate 33 was developed, promoted by electron-rich palladium complexes, including the novel phosphine ligand, VincePhos (50). This made possible a convergent, highly efficient synthesis of dasabuvir that significantly reduced the mutagenic impurity burden of the process.

Synthesis of a TRPV1 receptor antagonist.

A five-step synthesis of a TRPV1 receptor antagonist 1 is described. The key step involves a novel palladium-catalyzed amidation reaction of 4-chloro-1-methylindazole 8 with the benzyl urea 9 to form the unsymmetrically substituted urea 1.

A general method for the synthesis of unsymmetrically substituted ureas via palladium-catalyzed amidation.

A general and practical method for the preparation of unsymmetrically substituted ureas has been developed utilizing palladium-catalyzed amidation. Both aryl bromides and chlorides, as well as heteroaryl chlorides, have been coupled to aryl, benzyl, and aliphatic ureas by using a novel nonproprietary bipyrazole ligand (bippyphos).

Synthesis of erythromycin derivatives via the olefin cross-metathesis reaction.

Olefin cross metathesis (CM) was applied to the synthesis of 6-O-substituted erythromycin derivatives. The reactions were catalyzed by transition metal alkylidene complexes, particularly bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride (Grubbs' first-generation catalyst). This approach allowed for the elaboration of the 6-O-allyl group of highly functionalized macrolides at various stages of the synthetic sequence, affording 6-O-3-aryl-propenyl products with excellent E-selectivity. Little or no self-dimerization of the reacting components was found in the crude mixtures. Preliminary kinetic data accounts for the observed cross-selectivity based on substrate reactivity and steric factors.

Allylation of erythromycin derivatives: introduction of allyl substituents into highly hindered alcohols.

Functionalized erythromycin 9-oxime derivatives are 6-O-allylated under mild conditions using substituted allyl tert-butyl carbonates under palladium(0) catalysis. This allylation works well where traditional ether-forming protocols function poorly. Allyl tert-butyl carbonates provide higher yields in this reaction than lesser substituted carbonates such as ethyl or isopropyl. Aryl-substituted allyl carbonates or carbamates may be employed as well and, when used, produce trans-olefinic products.

General method for the palladium-catalyzed allylation of aliphatic alcohols.

A palladium catalysis-mediated approach to coupling aliphatic alcohols with allyl carbonates has been developed. The method allows for the allylation of primary, secondary, and tertiary alcohols efficiently under mild conditions. Limitations were explored as well as the asymmetric application of the chemistry. Regiochemical and olefin geometry was controlled in the coupling of unsymmetrical allylating agents. Transient allyl carbonates were observed in the coupling, which comprised the trans-carboxylation of the allyl-carbonate with the requisite alcohol.

Characterization and Synthesis of a 6-Substituted Benzo[b]thiophene.

An impurity observed during the synthesis of zileuton (Zyflo) has been isolated and characterized as a benzo[b]thiophene derivative that has undergone electrophilic substitution in the 6 position (4). A nine-step synthesis confirms the structural assignment. Key steps in the synthesis include a regioselective Friedel-Crafts coupling between 2-hydroxythioanisole, 8, and 1-(benzo[b]thien-2-yl)ethanol, 1, and formation of a benzo[b]thiophene from an o-methylthiobenzaldehyde, 14, and chloroacetone. The synthesis provides a potentially general route to substituted benzo[b]thiophenes.