Forging Odd-Membered Rings: Palladium-Catalyzed Asymmetric Cycloadditions of Trimethylenemethane.

The catalytic asymmetric synthesis of complex molecules has been the focus of our research program for several decades because such strategies have significant utility for the construction of chiral building blocks for drug development as well as the total synthesis of natural products. Cycloaddition reactions are very powerful transformations in organic synthesis providing access to highly functionalized motifs from simple starting materials. In concert with this central interest, four decades ago, we reported the palladium-catalyzed trimethylenemethane (TMM) cycloaddition for forging odd-membered ring systems. In recent years, we focused our attention on the development of powerful ligand scaffolds which enable the preparation of valuable products with complete control of chemo-, regio-, diastereo-, and enantioselectivity, thereby addressing several limitations in the field of palladium-catalyzed asymmetric cycloadditions. The first section of this Account will outline the discovery of a new class of highly modular pyrrolidine-based phosphoramidite and diamidophosphite chiral ligands which facilitate [3 + 2] cycloadditions of TMM donors, opening a new area in asymmetric construction of five-membered rings.The formation of the Pd-TMM zwitterionic intermediates is driven by the unique charge distribution of the cationic π-allyl motif, in which the most electropositive central carbon stabilizes the neighboring carbanion generated by either desilylation or deprotonation. The second section of this Account summarizes the scope of cycloadditions between Pd-TMM zwitterionic intermediates generated via desilylation and a variety of electron-deficient acceptors to access cyclopentanes, pyrrolidines and tetrahydrofurans. This section also includes the use of nitrile-, vinyl-, alkynyl- and allene-substituted TMM donors to rapidly generate cycloadducts with high molecular complexity. The extension of this strategy to include [6 + 3] cycloadditions and dearomative processes will also be presented. The third section will discuss a new generation of TMM donors substituted with electron-withdrawing groups such as nitrile, benzophenone imine, trifluoromethyl, and phosphonate, where the Pd-TMM zwitterionic intermediates are generated via deprotonation of the acidic C-H bond adjacent to the π-allyl motif. This new strategy has enabled the synthesis of heterocycles with increased numbers of functional groups in highly asymmetric and atom-economic fashion.Throughout this Account, we will describe the implementation of these transformations toward the rapid assembly of drug candidates and the total synthesis of natural products such as (-)-marcfortine C. We will also give details of mechanistic studies regarding relevant intermediates within the catalytic cycles of the different strategies, which allowed us to better understand the origin of selectivity with various donors.

A Highly Fluorescent Nucleobase Molecular Rotor.

Fluorescent base analogs (FBAs) are powerful probes of nucleic acids' structures and dynamics. However, previously reported FBAs exhibit relatively low brightness and therefore limited sensitivity of detection. Here we report the hitherto brightest FBA that has ideal molecular rotor properties for detecting local dynamic motions associated with base pair mismatches. The new trans-stilbene annulated uracil derivative "tsT" exhibits bright fluorescence emissions in various solvents (ε × Φ = 3400-29 700 cm-1 M-1) and is highly sensitive to mechanical motions in duplex DNA (ε × Φ = 150-4250 cm-1 M-1). tsT is thereby a "smart" thymidine analog, exhibiting a 28-fold brighter fluorescence intensity when base paired with A as compared to T or C. Time-correlated single photon counting revealed that the fluorescence lifetime of tsT (τ = 4-11 ns) was shorter than its anisotropy decay in well-matched duplex DNA (θ = 20 ns), yet longer than the dynamic motions of base pair mismatches (0.1-10 ns). These properties enable unprecedented sensitivity in detecting local dynamics of nucleic acids.

HgII binds to C-T mismatches with high affinity.

Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C-T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C-T mismatches (kon ≈ 105 M-1 s-1, koff ≈ 10-3 s-1) as compared to T-T mismatches (kon ≈ 104 M-1 s-1, koff ≈ 10-4 s-1), resulting in very similar equilibrium binding affinities for both types of 'all natural' metallo base pairs (Kd ≈ 10-150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T-T mismatches exhibited much larger increases in thermal stability upon addition of HgII (ΔTm = 6-19°C), as compared to those containing C-T mismatches (ΔTm = 1-4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C-HgII-T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs - even in situations where metal binding has little or no impact on the thermal stability of the duplex.

Dinuclear Metal-ProPhenol Catalysts: Development and Synthetic Applications.

The ProPhenol ligand is a member of the chiral aza-crown family that spontaneously forms a bimetallic complex upon treatment with alkyl metal reagents, such as Et2 Zn and Bu2 Mg. The resulting complex features Lewis acidic and Brønsted basic sites, enabling simultaneous activation of both nucleophile and electrophile in the same chiral environment. Since the initial report in 2000, metal-ProPhenol catalysts have been used to facilitate a broad range of asymmetric transformations, including aldol, Mannich, and Henry reactions, as well as alkynylations and conjugation additions. By promoting such a diverse array of reactions, these complexes provide rapid and atom-economical access to valuable complex building blocks. In this Review, we describe in detail the development and synthetic applications of these versatile catalysts with a special focus on recent efforts to improve reactivity and selectivity through ligand design and structural modification.

Total Synthesis of Callyspongiolide, Part 2: The Ynoate Metathesis/cis-Reduction Strategy.

The macrocyclic core of the cytotoxic marine natural product callyspongiolide (1) was forged by ring-closing alkyne metathesis (RCAM) of an ynoate precursor using a molybdenum alkylidyne complex endowed with triarylsilanolate ligands as the catalyst. This result is remarkable in view of the failed attempts documented in the literature at converting electron deficient alkynes with the aid of more classical catalysts. The subsequent Z-selective semi-reduction of the resulting cycloalkyne by hydrogenation over nickel boride required careful optimization in order to minimize overreduction and competing dehalogenation of the compound's alkenyl iodide terminus as needed for final attachment of the side chain of 1 by Sonogashira coupling. The required cyclization precursor itself was prepared via Kocienski olefination.

Synthesis and Molecular Editing of Callyspongiolide, Part 1: The Alkyne Metathesis/trans-Reduction Strategy.

A path-scouting investigation into the highly cytotoxic marine macrolide callyspongiolide is reported that capitalizes on the selective formation of the C10-C11 alkene site. While the closure of the macrocycle by ring closing alkyne metathesis (RCAM) with the aid of a molybdenum alkylidyne complex was high yielding, the envisaged semi-reduction of the cycloalkyne to the corresponding E-alkene proved challenging. The reasons are likely steric in origin, in that the methyl branches on either side of the alkyne seem to prevent effective coordination of the substrate to the ruthenium catalyst, which must carry a bulky Cp* ligand to ensure high trans-selectivity. This notion is supported by the preparation of a callyspongiolide analogue, in which the two methyl groups in question are excised; its formation by RCAM followed by trans-hydrostannation/proto-destannation was straightforward. In parallel work the formation of the fully functional building block 54 showed that the presence of an unprotected -OH group allows even hindered substrates to be processed: the protic group adjacent to the triple bond engages with a chloride ligand on the ruthenium catalyst in hydrogen bonding and hence assists in substrate binding. Moreover, the preparation of an alkynylogous callyspongiolide analogue is described.

Enantioselective Palladium-Catalyzed [3+2]†Cycloaddition of Trimethylenemethane and Fluorinated Ketones.

A nitrile-substituted trimethylenemethane (TMM) donor undergoes palladium-catalyzed [3+2] cycloadditions with fluorinated ketones to generate tetrasubstituted trifluoromethylated centers in high enantioselectivity under mild conditions. The generation of the palladium-TMM complex was achieved by a self-deprotonation strategy, which shows remarkable improvements in regiocontrol, efficiency, and atom economy of asymmetric [3+2] cycloadditions. Moreover, the versatility of the nitrile group provides direct access to a variety of synthetically useful intermediates, including amides, aldehydes, and esters. The developed reaction conditions allow for the synthesis of a wide variety of aromatic, heteroaromatic, and aliphatic fluorinated dihydrofurans in excellent regio- and enantioselectivities.

Enantio- and Diastereoselective Synthesis of Chiral Allenes by Palladium-Catalyzed Asymmetric [3+2] Cycloaddition Reactions.

A protocol for the asymmetric synthesis of highly substituted chiral allenes with control of point and axial chirality has been developed. A palladium-catalyzed [3+2] cycloaddition using readily available racemic allenes gives access to densely functionalized chiral allenes with excellent yields and functional group tolerance. The catalytic asymmetric protocol utilizes a broad range of allenyl TMM (trimethylenemethane) donors to form cyclopentanes, pyrrolidines, and spirocycles with very good control of regio-, enantio-, and diastereoselectivity. The chiral allene moiety is shown to be a valuable functional group for rapid elaboration towards complex targets.

Fluorescent Base Analogue Reveals T-HgII-T Base Pairs Have High Kinetic Stabilities That Perturb DNA Metabolism.

The thymidine analogue DMAT was used for the first fluorescence-based study of direct, site-specific metal binding reactions involving unmodified nucleobases in duplex DNA. The fluorescence properties of DMAT-A base pairs were highly sensitive to mercury binding reactions at T-T mismatches located at an adjacent site or one base pair away. This allowed for precise determination of the local kinetic and thermodynamic parameters of T-HgII-T binding reactions. The on- and off-rates of HgII were surprisingly slow, with association rate constants (kon) ≈ 104-105 M-1 s-1, and dissociation rate constants (koff) ≈ 10-4-10-3 s-1; giving equilibrium dissociation constants (Kd) = 8-50 nM. In contrast, duplexes lacking a T-T mismatch exhibited local, nonspecific HgII binding affinities in the range of Kd = 0.2-2.0 µM, depending on the buffer conditions. The exceptionally high kinetic stabilities of T-HgII-T metallo-base pairs (half-lives = 0.3-1.3 h) perturbed dynamic processes including DNA strand displacement and primer extension by DNA polymerases that resulted in premature chain termination of DNA synthesis. In addition to providing the first detailed kinetic and thermodynamic parameters of site-specific T-HgII-T binding reactions in duplex DNA, these results demonstrate that T-HgII-T base pairs have a high potential to disrupt DNA metabolism in vivo.

Fluorescent probe for proton-coupled DNA folding revealing slow exchange of i-motif and duplex structures.

Ionized nucleobases participate in pairing interactions outside of Watson and Crick's rules. Base pairing and ionization can be coupled via global conformational changes to raise the apparent pKa of protonated nucleobases to values above physiological pH. To provide the first specific reporter of proton-coupled DNA folding, we developed a "push-pull" fluorescent nucleoside analog composed of dimethylaniline (DMA) fused to deoxycytidine. "(DMA)C" exhibits the same pKa and base pairing characteristics as native cytosine residues in the human telomeric repeat sequence, where it causes little or no perturbation of DNA structure or stability. Upon protonation of (DMA)C, enhanced charge transfer results in large red shifting (+40 nm) of its excitation/emission maxima. (DMA)C's fluorescence intensity, anisotropy, and energy transfer properties can be used to track conformational changes in real time. Strand displacement assays were conducted by mixing (DMA)C-labeled duplexes containing a 5' single-stranded overhang with an excess of unlabeled DNA to initiate thermodynamically favorable unfolding-refolding reactions that release the (DMA)C-labeled strand from its complement. Rate constants for strand displacement upon addition of i-motif DNA (k = 1.0 M(-1) s(-1), t1/2 ≈ 12 h) were 320-fold lower than those measured upon addition of unfolded DNA (k = 3.2 × 10(2) M(-1) s(-1), t1/2 ≈ 2 min). These results reveal that i-motif structures having only marginal thermodynamic stabilities (Tm < 40 °C) can still pose large kinetic barriers to duplex formation under near-physiological conditions of pH (5.75), temperature (25 °C), and salt (100 mM NaCl).

Artificial nucleobase-amino acid conjugates: a new class of TAR RNA binding agents.

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is involved in the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop structure, which is encoded by the 5' leader sequence found in all HIV-1 mRNAs. Herein, we report the rational design, synthesis, and in vitro evaluation of new RNA binding agents that were conceived in order to bind strongly and selectively to the stem-loop structure of TAR RNA and, thus, inhibit the Tat/TAR interaction. We have demonstrated that the conjugation of modified nucleobases, able to interact specifically with an RNA base pair, and various amino acids allows these motifs to bind the target RNA selectively and in a cooperative manner that leads to the inhibition of viral replication in HIV-infected cells.

Stereoselective N-glycosylation of 2-deoxythioribosides for fluorescent nucleoside synthesis.

An efficient method for the N-2-deoxyribosylation of modified nucleobases by 2-deoxythioriboside donors is reported. In the presence of an in situ silylated nucleobase, thioglycosides can be activated with NIS/HOTf to give nucleosides in high yields and with good ß-selectivity. By tuning the protecting groups on the C3 and C5 hydroxyls, α/ß ratios ranging from 1.0:4.0 to 4.5:1.0 can be obtained. This strategy is applicable to the synthesis of various nucleosides, including ring-expanded pyrimidine derivatives containing sulfur that have previously been reported in low yields. The utility of this approach is further demonstrated by the synthesis of fluorescent nucleosides analogues such as quinazoline and oxophenothiazine that should find broad utility in DNA-folding and recognition studies.

Design, Synthesis, and Structure-Activity Relationship Optimization of Pyrazolopyrimidine Amide Inhibitors of Phosphoinositide 3-Kinase γ (PI3Kγ).

Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

Direct Enantio- and Diastereoselective Zn-ProPhenol-Catalyzed Mannich Reactions of CF3- and SCF3-Substituted Ketones.

Enantioselective incorporation of trifluoromethyl (-CF3) and trifuoromethylthio (-SCF3) groups in small molecules is of high interest to modulate the potency and pharmacological properties of drug candidates. Herein, we report a Zn-ProPhenol catalyzed diastereo- and enantioselective Mannich addition of α-trifluoromethyl- and α-trifuoromethylthio-substituted ketones. This transformation uses cyclic and acyclic ketones and generates quaternary trifluoromethyl and tetrasubstituted trifuoromethylthio stereogenic centers in excellent yields and selectivities.

Discovery of Potent and Selective 7-Azaindole Isoindolinone-Based PI3Kγ Inhibitors.

The successful application of immunotherapy in the treatment of cancer relies on effective engagement of immune cells in the tumor microenvironment. Phosphoinositide 3-kinase γ (PI3Kγ) is highly expressed in tumor-associated macrophages, and its expression levels are associated with tumor immunosuppression and growth. Selective inhibition of PI3Kγ offers a promising strategy in immuno-oncology, which has led to the development of numerous potent PI3Kγ inhibitors with variable selectivity profiles. To facilitate further investigation of the therapeutic potential of PI3Kγ inhibition, we required a potent and PI3Kγ-selective tool compound with sufficient metabolic stability for use in future in vivo studies. Herein, we describe some of our efforts to realize this goal through the systematic study of SARs within a series of 7-azaindole-based PI3Kγ inhibitors. The large volume of data generated from this study helped guide our subsequent lead optimization efforts and will inform further development of PI3Kγ-selective inhibitors for use in immunomodulation.

Discovery of Potent and Selective PI3Kγ Inhibitors.

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 µM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

A fluorescent surrogate of thymidine in duplex DNA.

is a new fluorescent thymidine mimic composed of 2'-deoxyuridine fused to dimethylaniline. exhibits the same pKa and base pairing characteristics as native thymidine residues, and its fluorescence properties are highly sensitive to nucleobase ionization, base pairing and metal binding.

Synthesis and solvatochromic fluorescence of biaryl pyrimidine nucleosides.

Fluorescent pyrimidine analogs containing a fused biphenyl unit were prepared in high yields using stereoselective N-glycosylation and Suzuki-Miyaura cross-coupling reactions. The resulting "push-pull" fluorophores exhibit highly solvatochromic emissions from twisted intramolecular charge-transfer (TICT) states.

Synthesis and structure of a hydrogenated zinc hemiporphyrazine.

Palladium-catalyzed hydrogenation of an octahedral zinc trans-ditriflate hemiporphyrazine "HpH2Zn(OTf)2" furnishes a new macrocycle "HpH6Zn(OTf)2". This reaction is fully reversible upon heating in nitrobenzene, and the conversion is easily monitored by changes in color and fluorescence properties. The reversible cycling between these molecules may find future applications in hemiporphyrazine-based catalysts and/or hydrogen storage devices.