Current ADC Linker Chemistry.

The list of ADCs in the clinic continues to grow, bolstered by the success of first two marketed ADCs: ADCETRIS® and Kadcyla®. Currently, there are 40 ADCs in various phases of clinical development. However, only 34 of these have published their structures. Of the 34 disclosed structures, 24 of them use a linkage to the thiol of cysteines on the monoclonal antibody. The remaining 10 candidates utilize chemistry to surface lysines of the antibody. Due to the inherent heterogeneity of conjugation to the multiple lysines or cysteines found in mAbs, significant research efforts are now being directed toward the production of discrete, homogeneous ADC products, via site-specific conjugation. These site-specific conjugations may involve genetic engineering of the mAb to introduce discrete, available cysteines or non-natural amino acids with an orthogonally-reactive functional group handle such as an aldehyde, ketone, azido, or alkynyl tag. These site-specific approaches not only increase the homogeneity of ADCs but also enable novel bio-orthogonal chemistries that utilize reactive moieties other than thiol or amine. This broadens the diversity of linkers that can be utilized which will lead to better linker design in future generations of ADCs.

Antibody-Oligonucleotide Conjugates: A Twist to Antibody-Drug Conjugates.

A summary of the key technological advancements in the preparation of antibody-oligonucleotide conjugates (AOCs) and the distinct advantages and disadvantages of AOCs as novel therapeutics are presented. The merits and demerits of the different approaches to conjugating oligonucleotides to antibodies, antibody fragments or other proteins, mainly from the perspective of AOC purification and analytical characterizations, are assessed. The lessons learned from in vitro and in vivo studies, especially the findings related to silencing, trafficking, and cytotoxicity of the conjugates, are also summarized.

Synthesis and SAR study of novel pseudo-steroids as potent and selective progesterone receptor antagonists.

Synthesis of novel 7-pseudo-steroids 1c has been achieved from trenbolone 3 via an efficient 14 step sequence with overall yields of 10-15%. Various substitutions were incorporated at both the aromatic side chain as well as the D ring. The orientation of aromatic side chain at C10 plays a crucial role for progesterone receptor (PR) activity. Compound 2a (T47D=1nM) with -NMe(2) para to the aromatic group along with spirofurane groups in the D ring was the optimal substitution. All compounds were also evaluated for glucocorticoid receptor (GR) antagonist activities in vivo in a rat and found efficacious in uterine complement C3 assay via the oral route of administrations.

Insight from molecular modeling into different conformation and SAR of natural steroids and unnatural 7-oxa-steroids.

Replacement of the 7-CH2 group of natural steroid with an oxygen atom led to identification of unnatural 7-oxa-steroids as potent and selective progesterone receptor antagonists. The unnatural 7-oxa-steroids exhibited a different structure-activity relationship (SAR) from natural steroids. Molecular modeling demonstrated that the switch of carbon to oxygen in the B-ring results in a subtle conformational change of the tetracyclic skeleton and induces a remarkable spatial shift at the terminal end of the side chain of the D-ring. This shift causes the phenyl ring on the D-ring to form a perfect parallel-displaced form of pi-pi interaction with the phenyl ring of Phe794. The unnatural 7-oxa-steroids were orally active in a rat complement C3 assay and showed comparable pharmacokinetic and metabolic profiles to those of the natural steroid, mifepristone.

Design and Synthesis of Isoquinolidinobenzodiazepine Dimers, a Novel Class of Antibody-Drug Conjugate Payload.

Antibody-drug conjugates (ADCs) represent an important class of emerging cancer therapeutics. Recent ADC development efforts highlighted the use of pyrrolobenzodiazepine (PBD) dimer payload for the treatment of several cancers. We identified the isoquinolidinobenzodiazepine (IQB) payload (D211), a new class of PBD dimer family of DNA damaging payloads. We have successfully synthesized all three IQB stereoisomers, experimentally showed that the purified (S,S)-D211 isomer is functionally more active than (R,R)-D221 and (S,R)-D231 isomers by >50,000-fold and ∼200-fold, respectively. We also synthesized a linker-payload (D212) that uses (S,S)-D211 payload with a cathepsin cleavable linker, a hydrophilic PEG8 spacer, and a thiol reactive maleimide. In addition, homogeneous ADCs generated using D212 linker-payload exhibited ideal physicochemical properties, and anti-CD33 ADC displayed a robust target-specific potency on AML cell lines. These results demonstrate that D212 linker-payload described here can be utilized for developing novel ADC therapeutics for targeted cancer therapy.

Synthesis, structure-activity relationship and in vivo antiinflammatory efficacy of substituted dipiperidines as CCR2 antagonists.

A series of substituted dipiperidine compounds have been synthesized and identified as selective CCR2 antagonists. Combining the most favorable substituents led to the discovery of remarkably potent CCR2 antagonists displaying IC50 values in the nanomolar range. Compound 7a had outstanding selectivity over CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR8 and showed excellent efficacy in adjuvant-induced arthritis model, collagen-induced arthritis model, and allergic asthma model.

Eradication of Tumors through Simultaneous Ablation of CD276/B7-H3-Positive Tumor Cells and Tumor Vasculature.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.

Novel chromene-derived selective estrogen receptor modulators useful for alleviating hot flushes and vaginal dryness.

A novel SERM (selective estrogen receptor modulators), 1-(R), a chromene-derived bisbenzopyran, was discovered to alleviate hot flushes and effectively increase vaginal fluidity in rats. Moreover, 1-(R) was found to have beneficial effects on plasma cholesterol and bone metabolism while maintaining antiestrogenic activity in the uterus. The biological profile of its enantiomer 1-(S) was also evaluated.

Total Synthesis of Vancomycin Aglycon-Part 1: Synthesis of Amino Acids 4-7 and Construction of the AB-COD Ring Skeleton.

A triazene-based synthetic strategy for the construction of the complex biaryl ethers and a Suzuki coupling reaction were the key steps in the synthesis of precursor 1 of the aglycon of vancomycin, which already contains the complete skeleton of the target compound. The cleavage of the triazene unit from the D ring and the removal of the other protecting groups led to the aglycon of vancomycin. These strategies should be particularly valuable for the synthesis of other naturally occurring glycopeptide antibiotics and offer opportunities for the synthesis of combinatorial libraries of compounds of the vancomycin family for chemical biology studies.

Total Synthesis of Vancomycin Aglycon-Part 2: Synthesis of Amino Acids 1-3 and Construction of the AB-COD-DOE Ring Skeleton.

A triazene-based synthetic strategy for the construction of the complex biaryl ethers and a Suzuki coupling reaction were the key steps in the synthesis of precursor 1 of the aglycon of vancomycin, which already contains the complete skeleton of the target compound. The cleavage of the triazene unit from the D ring and the removal of the other protecting groups led to the aglycon of vancomycin. These strategies should be particularly valuable for the synthesis of other naturally occurring glycopeptide antibiotics and offer opportunities for the synthesis of combinatorial libraries of compounds of the vancomycin family for chemical biology studies.

Total Synthesis of Vancomycin Aglycon-Part 3: Final Stages.

A triazene-based synthetic strategy for the construction of the complex biaryl ethers and a Suzuki coupling reaction were the key steps in the synthesis of precursor 1 of the aglycon of vancomycin, which already contains the complete skeleton of the target compound. The cleavage of the triazene unit from the D ring and the removal of the other protecting groups led to the aglycon of vancomycin. These strategies should be particularly valuable for the synthesis of other naturally occurring glycopeptide antibiotics and offer opportunities for the synthesis of combinatorial libraries of compounds of the vancomycin family for chemical biology studies.

Identification and structure-activity relationships of chromene-derived selective estrogen receptor modulators for treatment of postmenopausal symptoms.

As part of a program aimed at the development of selective estrogen receptor modulators (SERMs), novel chromene scaffolds, benzopyranobenzoxapanes, were discovered. Many compounds showed binding affinity as low as 1.6-200 nM, displayed antagonist behaviors in the MCF-7 human breast adenocarcinoma cell line as well in Ishikawa cell line with IC(50) values in the range 0.2-360 nM. On the basis of the side chain substitution, various compounds demonstrated strong inhibitory activity in anti-uterotropic assay. Compound 7-(R) and its major metabolites 5-(R) and 6-(R) were evaluated in several in vivo models of estrogen action. Relative to a full estrogen agonist (ethynyl estradiol) and the SERM raloxifene, 7-(R) was found to be a potent SERM that behaved as antagonist in the uterus and exhibited estrogen agonistic activity on bone, plasma lipids, hot flush, and vagina. The overall pharmacokinetic profile and stability were significantly improved compared to those of the phase 2 development compound 9-(R).

Parallel synthesis and SAR study of novel oxa-steroids as potent and selective progesterone receptor antagonists.

Efficient parallel synthesis of novel 7-oxa-steroids 4 has been achieved from the key intermediate 3 via a one-pot four-step sequence. oxa-Steroids 4 with various ortho-, meta-, and para-monosubstituents on the phenyl ring, as well as disubstituted phenyl and heterocycles, were evaluated for progesterone receptor (PR) and glucocorticoid receptor (GR) antagonist activities. SAR study demonstrated that the para-fluorinated substituents on the phenyl ring not only increased the potency for PR in a T47D cell functional assay, but also improved the selectivity over GR in an A549 cell functional assay. The para-fluorophenyl oxa-steroid 4l and the para-trifluoromethylphenyl oxa-steroid 4p were found to be remarkably more potent and more selective PR antagonists than mifepristone, with subnanomolar potency and about 140-fold selectivity over GR. Molecular modeling of the oxa-steroid bound to PR provided meaningful insight for the SAR study. oxa-Steroids 4a and 4b were found to be more efficacious than mifepristone in vivo in a rat uterine complement C3 assay via the oral route, although they were less than or equally potent to mifepristone in the T47D assay.

Synthesis and identification of novel oxa-steroids as progesterone receptor antagonists.

A novel series of oxa-steroids 6 derived from (8S, 13S, 14R)-7-oxa-estra-4,9-diene-3,17-dione 1 have been synthesized and identified as potent and selective progesterone receptor antagonists. These novel oxa-steroids showed similar potency to mifepristone. Preliminary SAR study resulted in the most potent 17-phenylethynyl oxa-steroid 6i wih an IC(50) of 1.4nM. In contrast to the equipotent mifepristone toward the progesterone receptor (PR) and glucocorticoid receptor (GR), compound 6i had over 200-fold selectivity for PR over GR.