We aim the power of C–H bond functionalization at a problem of major practical consequence—namely, scalable access to drug metabolites and their analogs. In this context, we pursue the development of simple C–H oxidation methods that tolerate a wide range of functional groups and structural features found in pharmaceuticals (‘chemotransformations’). In this Letter, we focus on N,N-dimethylamines as they are frequently found in pharmaceuticals and undergo CYP-mediated N-demethylation reactions in vivo. The direct access to these in vivo metabolites, from the parent drugs, has remained a significant synthetic challenge for which a broad solution had not previously been found. We here show that a simple copper–oxygen catalytic system provides a scalable route to complex N-demethylated drug metabolites.

The iboga alkaloids have attracted considerable attention in both the scientific community and popular media due to their reported ability to reverse or markedly diminish cravings for, and self-administration of, the major drugs of abuse. We have developed three new intramolecular C–H functionalization procedures leading to the core seven-membered ring of the iboga skeleton, a cyclization that proved to be highly challenging. The electrophilic palladium salt Pd(CH3CN)4(BF4)2 was effective for the cyclization of diverse N-(2-arylethyl)isoquinuclidines with yields of 10–35%. A two-step, bromination-reductive Heck reaction protocol was also effective for the synthesis of ibogamine in 42% yield. Finally, a direct Ni(0)-catalyzed C–H functionalization provided the benzofuran analogues of ibogamine (74%) and epi-ibogamine (38%). Although each approach suffers from significant shortcomings, in combination, the methods described provide practical routes to diverse ibogamine analogues.

Vesicular monoamine transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent Km of FFN206 at VMAT2 was found to be 1.16 ± 0.10 μM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z′-factor of 0.7–0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture.

We recently introduced fluorescent false neurotransmitters (FFNs) as optical tracers that enable the visualization of neurotransmitter release at individual presynaptic terminals. Here, we describe a pH-responsive FFN probe, FFN102, which as a polar dopamine transporter substrate selectively labels dopamine cell bodies and dendrites in ventral midbrain and dopaminergic synaptic terminals in dorsal striatum. FFN102 exhibits greater fluorescence emission in neutral than acidic environments, and thus affords a means to optically measure evoked release of synaptic vesicle content into the extracellular space. Simultaneously, FFN102 allows the measurement of individual synaptic terminal activity by following fluorescence loss upon stimulation. Thus, FFN102 enables not only the identification of dopamine cells and their processes in brain tissue, but also the optical measurement of functional parameters including dopamine transporter activity and dopamine release at the level of individual synapses. As such, the development of FFN102 demonstrates that, by bringing together organic chemistry and neuroscience, molecular entities can be generated that match the endogenous transmitters in selectivity and distribution, allowing for the study of both the microanatomy and functional plasticity of the normal and diseased nervous system.

We describe a general approach for the synthesis of complex aryl 1,2,4-triazoles. The electronic character of the C–H bonds and the triazole ring allows for the regioselective C–H arylation of 1-alkyl- and 4-alkyltriazoles under catalytic conditions. We have also developed the SEM and THP switch as well as trans-N-alkylation, which enable sequential arylation of the triazole ring to prepare 3,5-diaryltriazoles. This new strategy provides rapid access to a variety of arylated 1,2,4-triazoles and well complements existing cyclization methods.

We here report a study of the intramolecular amination of sp3 C–H bonds via the hydride transfer cyclization of N-tosylimines (HT-amination). In this transformation, 5-aryl aldehydes are subjected to N-toluenesulfonamide in the presence of BF3·OEt2 to effect imine formation and HT-cyclization, leading to 2-arylpiperidines and 3-aryl-1,2,3,4-tetrahydroisoquinolines in a one-pot procedure. We examined the reactivity of a range of aldehyde substrates as a function of their conformational flexibility. Substrates of higher conformational rigidity were more reactive, giving higher yields of the desired products. However, a single substituent on the alkyl chain linking the N-tosylimine and the benzylic sp3 C–H bonds was sufficient for HT-cyclization to occur. In addition, an examination of various arenes revealed that the electronic character of the hydridic C–H bonds dramatically affects the efficiency of the reaction. We also found that this transformation is highly stereoselective; 2-substituted aldehydes yield cis-2,5-disubstituted piperidines, while 3-substituted aldehydes afford trans-2,4-disubstituted piperidines. The stereoselectivity is a consequence of thermodynamic control. The pseudoallylic strain between the arene and tosyl group on the piperidine ring is proposed to rationalize the greater stability of the isomer with the aryl ring in the axial position. This preferential placement of the arene is proposed to affect the observed stereoselectivity.

C–H bond functionalization offers strategically novel approaches to complex organic compounds. However, many C–H functionalization reactions suffer from poor compatibility with Lewis basic functional groups, especially amines, which are often essential for biological activity. This study describes a systematic examination of the substrate scope of catalytic hydroarylation in the context of complex amino coumarin synthesis. The choice of substrates was guided by the design and development of the next generation of fluorescent false neurotransmitters (FFNs), neuroimaging probes we recently introduced for optical imaging of neurotransmission in the brain. Comparison of two mild protocols using catalytic PtCl4 or Au(PPh3)Cl/AgSbF6revealed that each method has a broad and mutually complementary substrate scope. The relatively less active platinum system out-performed the gold catalyst with indole substrates lacking substitution at the C-3 position and provided higher regioselectivity in the case of carbazole-based substrates. On the other hand, the more active gold catalyst demonstrated excellent functional group tolerance, and the ability to catalyze the formation of strained, helical products. The development of these two protocols offers enhanced substrate scope and provides versatile synthetic tools required for the structure–activity examination of FFN neuroimaging probes as well as for the synthesis of complex coumarins in general.

We report a new catalytic protocol for highly selective C–H arylation of pyridines containing common and synthetically versatile electron-withdrawing substituents (NO2, CN, F and Cl). The new protocol expands the scope of catalytic azine functionalization as the excellent regioselectivity at the 3- and 4-positions well complements the existing methods for C–H arylation and Ir-catalyzed borylation, as well as classical functionalization of pyridines. Another important feature of the new method is its flexibility to adapt to challenging substrates by a simple modification of the carboxylic acid ligand or the use of silver salts. The regioselectivity can be rationalized on the basis of the key electronic effects (repulsion between the nitrogen lone pair and polarized C–Pd bond at C2-/C6-positions and acidity of the C–H bond) in combination with steric effects (sensitivity to bulky substituents).

We introduce pH-responsive fluorescent false neurotransmitters (pH-responsive FFNs) as novel probes that act as vesicular monoamine transporter (VMAT) substrates and ratiometric fluorescent pH sensors. The development of these agents was achieved by systematic molecular design that integrated several structural elements, including the aminoethyl group (VMAT recognition), halogenated hydroxy-coumarin core (ratiometric optical pH sensing in the desired pH range), and N- or C-alkylation (modulation of lipophilicity). Of 14 compounds that were synthesized, the probe Mini202 was selected based on the highest uptake in VMAT2-transfected HEK cells and desirable optical properties. Using Mini202, we measured the pH of catecholamine secretory vesicles in PC-12 cells (pH ≈ 5.9) via two-photon fluorescence microscopy. Incubation with methamphetamine led to an increase in vesicular pH (pH ≈ 6.4), consistent with a proposed mechanism of action of this psychostimulant, and eventually to redistribution of vesicular content (including Mini202) from vesicles to cytoplasm. Mini202 is sufficiently bright, photostable, and suitable for two-photon microscopy. This probe will enable fundamental neuroscience and neuroendocrine research as well as drug screening efforts.

The study of dynamic properties of metabolic and signaling networks is hindered by the lack of methods for imaging metabolic fluxes in individual intact cells. We describe a novel optical approach for measuring the changes of metabolic fluxes in cells, based on a two-substrate competition between a physiological substrate and a fluorogenic reporter substrate. We have constructed a model cell system for a two-step metabolic pathway involved in the metabolism of testosterone. Potent androgen testosterone is converted by steroid 5α-reductase to DHT (5α-dihydrotestosterone), which is subsequently metabolized to 3α-diol (3α,17β-androstanediol) by the reductase AKR1C2 (aldo-ketoreductase 1C2), for which we have previously developed the fluorogenic reporter substrate Coumberone. Despite the medicinal importance of 5α-reductase, there are presently no probes or methods for the continuous activity readout of this enzyme in cells. We show that the activity of 5α-R1 (5α-reductase type 1) can be measured in COS-1 cells via the changes of DHT flux. Our system enables a measurement of 5α-reductase activity in cells, via either fluorimetry or fluorescence microscopy, with a wide dynamic range of activities, and provides a continuous optical assay for evaluation of small molecule inhibitors for this important enzyme. Furthermore, this paper demonstrates a novel optical approach to measuring metabolic flux changes in living cells and expands the utility of fluorogenic enzyme reporter substrates: optical reporters can measure not only the activity of the target enzyme but also the activity of other enzymes upstream in the pathway, for which there are no probes available.

We report a catalytic intramolecular coupling between terminal unactivated alkynes and sp3 C−H bonds via through-space hydride transfer (HT-cyclization of alkynes). This method enables one-step preparation of complex heterocyclic compounds by α-alkenylation of readily available cyclic ethers and amines. We show that PtI4 is an effective Lewis acid catalyst for the activation of terminal alkynes for hydride attack and subsequent C−C bond formation. In addition, we have shown that the activity of neutral platinum salts (PtXn) can be modulated by the halide ligands. This modulation in turn allows for fine-tuning of the platinum center reactivity to match the reactivity and stability of selected substrates and products.

The hydride transfer initiated cyclization (“HT-cyclization”) of aryl alkyl ethers, which leads to direct coupling of sp3 C−H bonds and activated alkenes, is reported. Readily available salicylaldehyde derived ethers are converted in one step to dihydrobenzopyrans, an important class of heteroarenes frequently found in biologically active compounds. This process has not been previously reported, in contrast to known HT-cyclizations of the corresponding tert-amines (“tert-amino effect” reactions).

We here present an optical method for monitoring the activity of the inducible aldo-keto reductases AKR1C2 and AKR1C3 in living human cells. The induction of these enzymes is regulated by the antioxidant response element (ARE), as demonstrated in recent literature, which in turn is dependent on the transcription factor Nrf2. The activation of ARE leads to the transcription of a coalition of cytoprotective enzymes and thus represents an important target for the development of new therapies in the area of neurodegenerative diseases and cancer. Through the use of Coumberone, a metabolic fluorogenic probe, and isoform-selective inhibitors, the upregulation of cellular stress markers AKR1C2 and AKR1C3 can be quantitatively measured in the presence of ARE activator compounds, via either a fluorimetric assay or fluorescence microscopy imaging of intact cells. The method has both high sensitivity and broad dynamic range, as demonstrated by induction studies in three cell lines with dramatically different metabolic capabilities (transfected monkey kidney COS-1 cells, human neuroblastoma IMR-32 cells, and human liver HepG2 cells). We applied the new method to examine a number of neurotrophic natural products (spirotenuipesine A, spirotenuipesine B, scabronine G-methylester, and panaxytriol), and discovered that panaxytriol, an active component of red ginseng extracts, is a potent ARE inducer. The upregulation of AKR1C enzymes, induced by chemically homogeneous panaxytriol, was partially dependent on PKC and PI3K kinases as demonstrated by the application of selective inhibitors. This cellular mechanism may account for panaxytriol’s neurotrophic, neuroprotective, and anticancer properties. The protective effects of ARE inducers against tumorgenesis and neurodegeneration fuel the growing interest in this area of research and the method described here will greatly enable these endeavors.

Abstract

Direct and selective replacement of carbon-hydrogen bonds with new bonds (such as C–C, C–O, and C–N) represents an important and long-standing goal in chemistry. These transformations have broad potential in synthesis because C–H bonds are ubiquitous in organic substances. At the same time, achieving selectivity among many different C–H bonds remains a challenge. Here, we focus on the functionalization of C–H bonds in complex organic substrates catalyzed by transition metal catalysts. We outline the key concepts and approaches aimed at achieving selectivity in complex settings and discuss the impact these reactions have on synthetic planning and strategy in organic synthesis.

A modular synthetic method for the differential incorporation of two lanthanide ions into a single molecular scaffold is reported; the mixed bimetallic Tb/Eu complex displays an interesting solvent polarity-dependent ratiometric luminescence.

The current arsenal of tools and methods for the continuous monitoring and imaging of redox metabolic pathways in the context of intact cells is limited. Fluorogenic substrates allow for direct measurement of enzyme activity in situ; however, in contrast to proteases and exo-glycosidases, there are no simple guidelines for the design of selective probes for redox metabolic enzymes. Here, we introduce redox probe 1 and demonstrate its high selectivity in living cells for human hydroxysteroid dehydrogenases (HSDs) of the aldo-keto reductase (AKR) superfamily. AKR1C isoforms perform multiple functions among which the metabolism of potent steroid hormones is well documented. Moreover, expression of these enzymes is responsive to cellular stress and pathogenesis, including cancer. Our probe design is based on redox-sensitive optical switches, which couple a ketone–alcohol redox event to a profound change in fluorescence. The high selectivity of phenyl ketone 1 for AKR1C2 over the many endogenous reductases present in mammalian cells was established by a quantitative comparison of the metabolic rates between null control cells (COS-1) and AKR1C2-transfected cells. Phenyl ketone 1 is a cell-permeable fluorogenic probe that permits a direct, real-time, and operationally simple readout of AKR1C2 enzyme activity in intact mammalian cells. Furthermore, it was demonstrated that probe 1 enables the quantitative examination of physiological substrate 5α-dihydrotestosterone (“dark substrate”) in situ by means of a two-substrate competitive assay. Similarly, inhibitor potency of physiological (ursodeoxycholate) and synthetic inhibitors (flufenamic acid, ibuprofen, and naproxen) was also readily evaluated.