Site-Selective Antibody Conjugation with Dibromopyrazines.

In recent years, antibody conjugates have evolved as state-of-the-art options for diagnostic and therapeutic applications. During site-selective antibody conjugation, incomplete rebridging of antibody chains limits the homogeneity of conjugates and calls for the development of new rebridging agents. Herein, we report a dibromopyrazine derivative optimized to reach highly homogeneous conjugates rapidly and with high conversion on rebridging of trastuzumab, even providing a feasible route for antibody modification in acidic conditions. Furthermore, coupling a fluorescent dye and a cytotoxic drug resulted in effective antibody conjugates with excellent serum stability and in vitro selectivity, demonstrating the utility of the dibromopyrazine rebridging agent to produce on-demand future antibody conjugates for diagnostic or therapeutic applications.

Synthesis of Mesylated and Tosylated α-Hydroxy-Benzylphosphonates; Their Reactivity and Cytostatic Activity.

α-Hydroxyphosphonates and their acylated and phosphorylated derivatives may be of significant biological activity including cytotoxic effects. To extend the pool of the potentially bioactive species, new methane- and arenesulfonyloxyphosphonates were synthesized by the sulfonylation of differently substituted α-hydroxy-benzylphosphonates using methanesulfonyl chloride or p-toluenesulfonyl chloride at 25 °C in the presence of triethylamine in toluene. The new sulfonyl derivatives were obtained in 54-80% yields. In the case of the 4- or 2-methoxy substituent in the aromatic ring, surprisingly the corresponding α-chlorophosphonates were the exclusive products, whose formation was explained assuming a quinoid intermediate and supported by theoretical calculations. With a 3-methoxyphenyl substituent, the expected mesylation of the hydroxy group took place. Attempted alcoholyses of the diethyl α-methanesulfonyloxy-benzylphosphonates with different substituents in the benzyl ring at ∼140 °C in the presence of triethylamine under microwave irradiation left the P-function intact under the conditions applied, instead, the mesyloxy group was substituted by an alkoxy unit in a selective new reaction. The α-alkoxy-benzylphosphonates were isolated in 60-77% yields. While α-chloro- or α-bromo-benzylphosphonates proved to be rather inefficient in the Michaelis-Arbuzov reaction with triethyl phosphite, according to a new possibility, the α-methansulfonyloxy-benzylphosphonates underwent an efficient Arbuzov fission using the phosphite in excess at 135 °C. The arylmethylenebisphosphonates were obtained in yields of 76-81%. Bioactivity studies with the members of the phosphonate library revealed pronounced in vitro cytostatic effect of the α-hydroxy- and α-mesyloxy-3,5-di-tert-butylbenzylphosphonates on human breast carcinoma cell culture with IC50 values of 16.4 and 28.0 µM, respectively. The mesyloxy species was also cytostatic on melanoma cells (IC50 = 34.9).

Mapping protein binding sites by photoreactive fragment pharmacophores.

Fragment screening is a popular strategy of generating viable chemical starting points especially for challenging targets. Although fragments provide a better coverage of chemical space and they have typically higher chance of binding, their weak affinity necessitates highly sensitive biophysical assays. Here, we introduce a screening concept that combines evolutionary optimized fragment pharmacophores with the use of a photoaffinity handle that enables high hit rates by LC-MS-based detection. The sensitivity of our screening protocol was further improved by a target-conjugated photocatalyst. We have designed, synthesized, and screened 100 diazirine-tagged fragments against three benchmark and three therapeutically relevant protein targets of different tractability. Our therapeutic targets included a conventional enzyme, the first bromodomain of BRD4, a protein-protein interaction represented by the oncogenic KRasG12D protein, and the yet unliganded N-terminal domain of the STAT5B transcription factor. We have discovered several fragment hits against all three targets and identified their binding sites via enzymatic digestion, structural studies and modeling. Our results revealed that this protocol outperforms screening traditional fully functionalized and photoaffinity fragments in better exploration of the available binding sites and higher hit rates observed for even difficult targets.

Contribution of Noncovalent Recognition and Reactivity to the Optimization of Covalent Inhibitors: A Case Study on KRasG12C.

Covalent drugs might bear electrophiles to chemically modify their targets and have the potential to target previously undruggable proteins with high potency. Covalent binding of drug-size molecules includes a noncovalent recognition provided by secondary interactions and a chemical reaction leading to covalent complex formation. Optimization of their covalent mechanism of action should involve both types of interactions. Noncovalent and covalent binding steps can be characterized by an equilibrium dissociation constant (KI) and a reaction rate constant (kinact), respectively, and they are affected by both the warhead and the scaffold of the ligand. The relative contribution of these two steps was investigated on a prototypic drug target KRASG12C, an oncogenic mutant of KRAS. We used a synthetically more accessible nonchiral core derived from ARS-1620 that was equipped with four different warheads and a previously described KRAS-specific basic side chain. Combining these structural changes, we have synthesized novel covalent KRASG12C inhibitors and tested their binding and biological effect on KRASG12C by various biophysical and biochemical assays. These data allowed us to dissect the effect of scaffold and warhead on the noncovalent and covalent binding event. Our results revealed that the atropisomeric core of ARS-1620 is not indispensable for KRASG12C inhibition, the basic side chain has little effect on either binding step, and warheads affect the covalent reactivity but not the noncovalent binding. This type of analysis helps identify structural determinants of efficient covalent inhibition and may find use in the design of covalent agents.

Computational Studies on the Diastereospecific Lithium Variant of Oppenauer Oxidation of a Tofisopam Intermediate.

During the synthesis of tofisopam drug substance, an interesting diastereospecific lithium variant of Oppenauer oxidation was observed and investigated by density functional theory (DFT) calculations. The computations revealed energetic differences caused by steric differences between the diastereomers that might provide an explanation for the experimentally formed products. In addition, the trend in the measured NMR shifts was also in line with the computed values, which allowed the assignment of the absolute configuration of the diastereomers.

Electrophilic MiniFrags Revealed Unprecedented Binding Sites for Covalent HDAC8 Inhibitors.

Screening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Covalent fragment approaches targeting non-cysteine residues.

Covalent fragment approaches combine advantages of covalent binders and fragment-based drug discovery (FBDD) for target identification and validation. Although early applications focused mostly on cysteine labeling, the chemistries of available warheads that target other orthosteric and allosteric protein nucleophiles has recently been extended. The range of different warheads and labeling chemistries provide unique opportunities for screening and optimizing warheads necessary for targeting non-cysteine residues. In this review, we discuss these recently developed amino-acid-specific and promiscuous warheads, as well as emerging labeling chemistries, which includes novel transition metal catalyzed, photoactive, electroactive, and noncatalytic methodologies. We also highlight recent applications of covalent fragments for the development of molecular glues and proteolysis-targeting chimeras (PROTACs), and their utility in chemical proteomics-based target identification and validation.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Activation-Free Sulfonyl Fluoride Probes for Fragment Screening.

SuFEx chemistry is based on the unique reactivity of the sulfonyl fluoride group with a range of nucleophiles. Accordingly, sulfonyl fluorides label multiple nucleophilic amino acid residues, making these reagents popular in both chemical biology and medicinal chemistry applications. The reactivity of sulfonyl fluorides nominates this warhead chemotype as a candidate for an external, activation-free general labelling tag. Here, we report the synthesis and characterization of a small sulfonyl fluoride library that yielded the 3-carboxybenzenesulfonyl fluoride warhead for tagging tractable targets at nucleophilic residues. Based on these results, we propose that coupling diverse fragments to this warhead would result in a library of sulfonyl fluoride bits (SuFBits), available for screening against protein targets. SuFBits will label the target if it binds to the core fragment, which facilitates the identification of weak fragments by mass spectrometry.

Covalent fragment mapping of KRasG12C revealed novel chemotypes with in vivo potency.

G12C mutant KRas is considered druggable by allele-specific covalent inhibitors due to the nucleophilic character of the oncogenic mutant cysteine at position 12. Discovery of these inhibitors requires the optimization of both covalent and noncovalent interactions. Here, we report covalent fragment screening of our electrophilic fragment library of diverse non-covalent scaffolds equipped with 40 different electrophilic functionalities to identify fragments as suitable starting points targeting Cys12. Screening the library against KRasG12C using Ellman's free thiol assay, followed by protein NMR and cell viability assays, resulted in two potential inhibitor chemotypes. Characterization of these scaffolds in in vitro cellular- and in vivo xenograft models revealed them as promising starting points for covalent drug discovery programs.

Next-Generation Heterocyclic Electrophiles as Small-Molecule Covalent MurA Inhibitors.

Heterocyclic electrophiles as small covalent fragments showed promising inhibitory activity on the antibacterial target MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase, EC:2.5.1.7). Here, we report the second generation of heterocyclic electrophiles: the quaternized analogue of the heterocyclic covalent fragment library with improved reactivity and MurA inhibitory potency. Quantum chemical reaction barrier calculations, GSH (L-glutathione) reactivity assay, and thrombin counter screen were also used to demonstrate and explain the improved reactivity and selectivity of the N-methylated heterocycles and to compare the two generations of heterocyclic electrophiles.

Covalent inhibitors of bacterial peptidoglycan biosynthesis enzyme MurA with chloroacetamide warhead.

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC50 value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

A Study on the Direct Esterification of Monoalkylphosphates and Dialkylphosphates; The Conversion of the Latter Species to Trialkylphosphates by Alkylating Esterification.

The microwave (MW)-assisted direct esterification of certain P-acids is a green method. Quantum chemical calculations revealed that the activation enthalpy (ΔH#) for the exothermic monoalkylphosphate → dialkylphosphate transformation was on the average 156.6 kJ mol-1, while ΔH# for the dialkylphosphate → trialkylphosphate conversion was somewhat higher, 171.2 kJ mol-1, and the energetics of the elemental steps of this esterification was less favorable. The direct monoesterification may be performed on MW irradiation in the presence of a suitable ionic liquid additive. However, the second step, with the less favorable energetics as a whole, could not be promoted by MWs. Hence, dialkylphosphates had to be converted to triesters by another method that was alkylation. In this way, it was also possible to synthesize triesters with different alkyl groups. Eventually a green, P-chloride free MW-promoted two-step method was elaborated for the synthesis of phosphate triesters.

A stepwise one-pot synthesis of aliphatic thiols and their derivatives from acrylamides and sulfur.

Elemental sulfur enables the convenient formation of C-S bonds and the direct incoporation of S-S bonds. The reactivity of easily accessible electron deficient alkenes towards sulfur, however, is barely disclosed. Herein, we investigated the reactivity of acrylamides with sulfur and eventually developed a new pseudo-multicomponent reaction for the preparation of polysulfides. Sequential one-pot reduction led to diversely substituted thiols. Additional third stage one-pot modifications provided thioethers, unsymmetric disulfide and thioester.

Electrophilic warheads in covalent drug discovery: an overview.

INTRODUCTION: Covalent drugs have been used for more than hundred years, but gathered larger interest in the last two decades. There are currently over a 100 different electrophilic warheads used in covalent ligands, and there are several considerations tailoring their reactivity against the target of interest, which is still a challenging task. AREAS COVERED: This review aims to give an overview of electrophilic warheads used for protein labeling in chemical biology and medicinal chemistry. The warheads are discussed by targeted residues, mechanism and selectivity, and analyzed through three different datasets including our collection of warheads, the CovPDB database, and the FDA approved covalent drugs. Moreover, the authors summarize general practices that facilitate the selection of the appropriate warhead for the target of interest. EXPERT OPINION: In spite of the numerous electrophilic warheads, only a fraction of them is used in current drug discovery projects. Recent studies identified new tractable residues by applying a wider array of warhead chemistries. However, versatile, selective warheads are not available for all targetable amino acids, hence discovery of new warheads for these residues is needed. Broadening the toolbox of the warheads could result in novel inhibitors even for challenging targets developing with significant therapeutic potential.

Experimental and computational study of BF3-catalyzed transformations of ortho-(pivaloylaminomethyl)benzaldehydes: an unexpected difference from TFA catalysis.

Previously, we have studied the trifluoroacetic acid (TFA)-catalyzed rearrangements of unsubstituted and alkoxy-substituted ortho-(pivaloylaminomethyl)benzaldehydes and revealed the formation of rearranged, regioisomeric aldehydes along with dimer-like products ("TFA dimers"). In the present study, related reactions of ortho-(pivaloylaminomethyl)benzaldehydes are described with the difference that boron trifluoride diethyl etherate (BF3·OEt2) is used as the catalyst. Although in these reactions the formation of the same "TFA dimers" can be observed after a couple of hours reaction time, during further stirring these are transformed into a new dimer-like keto compound ("BF3 dimer") that gradually becomes the main product. Apart from this, an oxoindene-type by-product is also formed. The new products are characterized by detailed NMR studies and two of them also by single-crystal X-ray diffraction. DFT calculations support the mechanism proposed for the transformations and explain the differences observed in the product distribution.

A covalent strategy to target intrinsically disordered proteins: Discovery of novel tau aggregation inhibitors.

Intrinsically disordered proteins (IDPs) play important roles in disease pathologies; however, their lack of defined stable 3D structures make traditional drug design strategies typically less effective against these targets. Based on promising results of targeted covalent inhibitors (TCIs) on challenging targets, we have developed a covalent design strategy targeting IDPs. As a model system we chose tau, an endogenous IDP of the central nervous system that is associated with severe neurodegenerative diseases via its aggregation. First, we mapped the tractability of available cysteines in tau and prioritized suitable warheads. Next, we introduced the selected vinylsulfone warhead to the non-covalent scaffolds of potential tau aggregation inhibitors. The designed covalent tau binders were synthesized and tested in aggregation models, and inhibited tau aggregation effectively. Our results revealed the usefulness of the covalent design strategy against therapeutically relevant IDP targets and provided promising candidates for the treatment of tauopathies.

A Mechanistic Study on the Formation of Dronic Acids.

Dronic acid derivatives, important drugs against bone diseases, may be synthesized from the corresponding substituted acetic acid either by reaction with phosphorus trichloride in methanesulfonic acid as the solvent or by using also phosphorous acid as the P-reactant if sulfolane is applied as the medium. The energetics of the two protocols were evaluated by high-level quantum chemical calculations on the formation of fenidronic acid and benzidronic acid. The second option, involving (HO)2P-O-PCl2 as the nucleophile, was found to be more favorable over the first variation, comprising Cl2P-O-SO2Me as the real reagent, especially for the case of benzidronate.

Fragment-Sized and Bidentate (Immuno)Proteasome Inhibitors Derived from Cysteine and Threonine Targeting Warheads.

Constitutive- and immunoproteasomes are part of the ubiquitin-proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the ß5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and ß5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the ß5, ß5i, ß1, and ß1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either ß5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.