Chemical Catalysis Guides Structural Identification for the Major In Vivo Metabolite of the BET Inhibitor JQ1.

The bromodomain inhibitor (+)-JQ1 is a highly validated chemical probe; however, it exhibits poor in vivo pharmacokinetics. To guide efforts toward improving its pharmacological properties, we identified the (+)-JQ1 primary metabolite using chemical catalysis methods. Treatment of (+)-JQ1 with tetrabutylammonium decatungstate under photochemical conditions resulted in selective formation of an aldehyde at the 2-position of the thiophene ring [(+)-JQ1-CHO], which was further reduced to the 2-hydroxymethyl analog [(+)-JQ1-OH]. Comparative LC/MS analysis of (+)-JQ1-OH to the product obtained from liver microsomes suggested (+)-JQ1-OH as the major metabolite of (+)-JQ1. The 2-thienyl position was then substituted to generate a trideuterated (-CD3, (+)-JQ1-D) analog having half-lives that were 1.8- and 2.8-fold longer in mouse and human liver microsomes, respectively. This result unambiguously confirmed (+)-JQ1-OH as the major metabolite of (+)-JQ1. These studies demonstrate an efficient process for studying drug metabolism and identifying the metabolic soft spots of bioactive compounds.

Design and construction of a stereochemically diverse piperazine-based DNA-encoded chemical library.

Here we report the successful construction of a novel, stereochemically diverse DNA-Encoded Chemical Library (DECL) by utilizing 24 enantiomerically pure trifunctional 2, 6- di-substituted piperazines as central cores. We introduce the concept of positional diversity by placing the DNA attachment at either of two possible sites on the piperazine scaffold. Using a wide range of building blocks, a diverse library of 77 million compounds was produced. Cheminformatic analysis demonstrates that this library occupies a wide swath of chemical space, and that the piperazine scaffolds confers different shape diversity compared to the commonly used triazine core.

Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery.

Despite the established roles of the epigenetic factor UHRF1 in oncogenesis, no UHRF1-targeting therapeutics have been reported to date. In this study, we use fragment-based ligand discovery to identify novel scaffolds for targeting the isolated UHRF1 tandem Tudor domain (TTD), which recognizes the heterochromatin-associated histone mark H3K9me3 and supports intramolecular contacts with other regions of UHRF1. Using both binding-based and function-based screens of a ~ 2300-fragment library in parallel, we identified 2,4-lutidine as a hit for follow-up NMR and X-ray crystallography studies. Unlike previous reported ligands, 2,4-lutidine binds to two binding pockets that are in close proximity on TTD and so has the potential to be evolved into more potent inhibitors using a fragment-linking strategy. Our study provides a useful starting point for developing potent chemical probes against UHRF1.

Practical and scalable synthesis of orthogonally protected-2-substituted chiral piperazines.

A synthetic route to orthogonally protected, enantiomerically pure 2-substituted piperazines is described. Starting from α-amino acids, within four steps chiral 2-substituted piperazines are obtained. The key transformation involves an aza-Michael addition between an orthogonally bis-protected chiral 1,2-diamine and the in situ generated vinyl diphenyl sulfonium salt derived from 2-bromoethyl-diphenylsulfonium triflate. Further validation using different protecting groups as well as synthesis on multigram scale was performed. The method was also applied to the construction of chiral 1,4-diazepanes and 1,4-diazocanes. Additionally, the method was utilized in a formal synthesis of chiral mirtazapine.

Metabolism of JQ1, an inhibitor of bromodomain and extra terminal bromodomain proteins, in human and mouse liver microsomes†.

JQ1 is a small-molecule inhibitor of the bromodomain and extra terminal (BET) protein family that potently inhibits the bromodomain testis-specific protein (BRDT), which is essential for spermatogenesis. JQ1 treatment produces a reversible contraceptive effect by targeting the activity of BRDT in mouse male germ cells, validating BRDT as a male contraceptive target. Although JQ1 possesses favourable physical properties, it exhibits a short half-life. Because the details of xenobiotic metabolism play important roles in the optimization of drug candidates and in determining the role of metabolism in drug efficacy, we investigated the metabolism of JQ1 in human and mouse liver microsomes. We present the first comprehensive view of JQ1 metabolism in liver microsomes, distinguishing nine JQ1 metabolites, including three monohydroxylated, one de-tert-butylated, two dihydroxylated, one monohydroxylated/dehydrogenated, one monohydroxylated-de-tert-butylated and one dihydroxylated/dehydrogenated variant of JQ1. The dominant metabolite (M1) in both human and mouse liver microsomes is monohydroxylated on the fused three-ring core. Using recombinant cytochrome P450 (CYP) enzymes, chemical inhibitors and the liver S9 fraction of Cyp3a-null mice, we identify enzymes that contribute to the formation of these metabolites. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is the main contributor to the production of JQ1 metabolites in vitro, and the CYP3A4/5 inhibitor ketoconazole strongly inhibits JQ1 metabolism in both human and mouse liver microsomes. Our findings suggest that JQ1 half-life and efficacy might be improved in vivo by co-administration of a selective CYP inhibitor, thereby impacting the use of JQ1 as a probe for BRDT activity in spermatogenesis and as a probe or therapeutic in other systems.

Synthesis of Enantiomerically Pure 5-Substituted Piperazine-2-Acetic Acid Esters as Intermediates for Library Production.

The piperazine heterocycle is housed within a large number of FDA-approved drugs and biological probe compounds. Structurally, however, these compounds are mostly confined to substitutions on the two ring nitrogen atoms, rationalizing the expansion of piperazine chemical diversity through carbon substitutions. On the basis of the concept of systematic chemical diversity, a divergent six-step synthesis was developed in which chiral amino acids were transformed, with high diastereoselectivity, into either cis or trans 5-substituted piperazine-2-acetic acid esters that could be chromatographically rendered diastereomerically homogeneous. Starting from six commercially available amino acids or their respective amino alcohols (both antipodes), we obtained a complete set of 24 protected chiral 2,5-disubstituted piperazines, as single stereoisomers in multigram quantities. These diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as starting materials for parallel library synthesis and as intermediates for the targeted production of more complex C-substituted piperazine compounds.

Synthesis of Enantiomerically Pure 3-Substituted Piperazine-2-acetic Acid Esters as Intermediates for Library Production.

The piperazine heterocycle is broadly exploited in FDA-approved drugs and biologically active compounds, but its chemical diversity is usually limited to ring nitrogen substitutions, leaving the four carbon atoms underutilized. Using an efficient six-step synthesis, chiral amino acids were transformed into 3-substituted piperazine-2-acetic acid esters as diastereomeric mixtures whose cis and trans products (dr 0.56 → 2.2:1, respectively) could be chromatographically separated. From five amino acids (both antipodes) was obtained a complete matrix of 20 monoprotected chiral 2,3-disubstituted piperazines, each as a single absolute stereoisomer, all but one in multigram quantities. In keeping with our overall purpose of constructing more Csp3-enriched compound libraries for drug discovery, these diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as scaffolds for parallel library synthesis and as intermediates for the production of novel piperazine compounds.

Synthesis of Enantiomerically Pure 6-Substituted-Piperazine-2-Acetic Acid Esters as Intermediates for Library Production.

The piperazine heterocycle is broadly exploited in FDA-approved drugs and biologically active compounds, but its chemical diversity is usually limited to ring nitrogen substitutions, leaving the four carbon atoms underutilized. Using an efficient four-step synthesis, chiral amino acids were transformed into 6-substituted piperazine-2-acetic acid esters as diastereomeric mixtures whose cis and trans products could be chromatographically separated. From six amino acids (both antipodes), a complete matrix of 24 monoprotected chiral 2,6-disubstituted piperazines was obtained, each as a single absolute stereoisomer in multigram quantities. These diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as scaffolds for parallel library synthesis or intermediates for the production of novel piperazine compounds.

Mechanisms by which synthetic 6,7-annulated-4-substituted indole compounds with anti-proliferative activity disrupt mitosis and block cytokinesis in human HL-60 tumor cells in vitro.

BACKGROUND: Synthetic 6,7-annulated-4-substituted indole compounds, which elicit interesting antitumor effects in murine L1210 leukemia cells, were tested for their ability to inhibit human HL-60 tumor cell proliferation, disrupt mitosis and cytokinesis, and interfere with tubulin and actin polymerization in vitro. MATERIALS AND METHODS: Various markers of metabolic activity, mitotic disruption and cytokinesis were used to assess the effectiveness of the drugs in the HL-60 tumor cell system. The ability of annulated indoles to alter the polymerizations of purified tubulin and actin were monitored in cell-free assays and were compared to the effects of drugs known to disrupt the dynamic structures of the mitotic spindle and cleavage furrow. RESULTS: With one exception, annulated indoles inhibited the metabolic activity of HL-60 tumor cells in the low-micromolar range after two and four days in culture but these anti-proliferative effects were weaker than those of jasplakinolide, a known actin binder that blocks cytokinesis. After 24-48 h, antiproliferative concentrations of annulated indoles increased the mitotic index of HL-60 cells similarly to vincristine and stimulated the formation of many bi-nucleated cells, multi-nucleated cells and micronuclei, similarly to taxol and jasplakinolide, suggesting that these antitumor compounds might increase mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis. Since annulated indoles mimicked the effect of vincristine on tubulin polymerization, but not that of taxol, these compounds might represent a new class of microtubule de-stabilizing agents that inhibit tubulin polymerization. Moreover, annulated indoles remarkably increased the rate and level of actin polymerization similarly to jasplakinolide, suggesting that they might also stabilize the cleavage furrow to block cytokinesis. CONCLUSION: Although novel derivatives with different substitutions must be synthesized to elucidate structure-activity relationships, identify more potent antitumor compounds and investigate different molecular targets, annulated indoles appear to interact with both tubulin to reduce microtubule assembly and actin to block cytokinesis, thereby inducing bi- and multinucleation, resulting in genomic instability and apoptosis.

Constrained TRPV1 agonists synthesized via silver-mediated intramolecular azo-methine ylide cycloaddition of α-iminoamides.

As part of an effort to identify agonists of TRPV1, a peripheral sensory nerve ion channel, high throughput screening of the NIH Small Molecule Repository (SMR) collection identified MLS002174161, a pentacyclic benzodiazepine. A synthesis effort was initiated that ultimately afforded racemic seco analogs 12 of the SMR compound via a silver mediated intramolecular [3+2] cycloaddition of an azo-methine ylide generated from α-iminoamides 11. The cycloaddition set four contiguous stereocenters and, in some cases, also spontaneously afforded imides 13 from 12. The synthesis of compounds 12, the features that facilitated the conversion of 12-13, and their partial agonist activity against TRPV1 are discussed.

Diversity by divergence: Solution-phase parallel synthesis of a library of N-diversified 1-oxa-7-azaspiro[4.5]decan-2-yl-propanes and -butanes.

The synthesis of a 162-member compound library derived from a single precursor via a multistage divergence strategy is described. Divergence is sequentially introduced in three ways: (1) by early preparation of two separable spirocyclic diastereomers, (2) by elaboration of each spirocyclic diastereomer to a different scaffold using four Horner-Emmons-Wadsworth reagents, and (3) by employing three different modes of nitrogen diversification with each scaffold to afford the final compounds. This 2 diastereomers × 4 reagents × 3 modes of diversification strategy leads to 24 unique synthetic pathways that ultimately afforded, in parallel format, the 162-compound set.

Synthesis of a family of spirocyclic scaffolds: building blocks for the exploration of chemical space.

This report describes the preparation of a series of 17 novel racemic spirocyclic scaffolds that are intended for the creation of compound libraries by parallel synthesis for biological screening. Each scaffold features two points of orthogonal diversification. The scaffolds are related to each other in four ways: (1) through stepwise changes in the size of the nitrogen-bearing ring; (2) through the oxidation state of the carbon-centered point of diversification; (3) through the relative stereochemical orientation of the two diversification sites in those members that are stereogenic; and (4) through the provision of both saturated and unsaturated versions of the furan ring in the scaffold series derived from 3-piperidone. The scaffolds provide incremental changes in the relative orientation of the diversity components that would be introduced onto them. The scaffolds feature high sp(3) carbon content which is essential for the three-dimensional exploration of chemical space. This characteristic is particularly evident in those members of this family that bear two stereocenters, i.e., the two series derived from 3-piperidone and 3-pyrrolidinone. In the series derived from 3-piperidone we were able to "split the difference" between the two diastereomers by preparation of their corresponding unsaturated version.

Solution-phase synthesis of a diverse library of benzisoxazoles utilizing the [3 + 2] cycloaddition of in situ-generated nitrile oxides and arynes.

A library of benzisoxazoles has been synthesized by the [3 + 2] cycloaddition of nitrile oxides with arynes and further diversified by acylation/sulfonylation and palladium-catalyzed coupling processes. The eight key intermediate benzisoxazoles have been prepared by the reaction of o-(trimethylsilyl)aryl triflates and chlorooximes in the presence of CsF in good to excellent yields under mild reaction conditions. These building blocks have been used as the key components of a diverse set of 3,5,6-trisubstituted benzisoxazoles.

Skeletal diversification via heteroatom linkage control: preparation of bicyclic and spirocyclic scaffolds from N-substituted homopropargyl alcohols.

The discovery and application of a new branching pathway synthesis strategy that rapidly produces skeletally diverse scaffolds is described. Two different scaffold types, one a bicyclic iodo-vinylidene tertiary amine/tertiary alcohol and the other, a spirocyclic 3-furanone, are each obtained using a two-step sequence featuring a common first step. Both scaffold types lead to intermediates that can be orthogonally diversified using the same final components. One of the scaffold types was obtained in sufficiently high yield that it was immediately used to produce a 97-compound library.

Antitumor effects of synthetic 6,7-annulated-4-substituted indole compounds in L1210 leukemic cells in vitro.

BACKGROUND: Because annulated indoles have almost no representation in the PubChem or MLSMR databases, an unprecedented class of an indole-based library was constructed, using the indole aryne methodology, and screened for antitumor activity. Sixty-six novel 6,7-annulated-4-substituted indole compounds were synthesized, using a strategic combination of 6,7-indolyne cycloaddition and cross-coupling reactions under both Suzuki-Miyaura and Buchwald-Hartwig conditions, and tested for their effectiveness against murine L1210 tumor cell proliferation in vitro. MATERIALS AND METHODS: Various markers of tumor cell metabolism, DNA degradation, mitotic disruption, cytokinesis and apoptosis were assayed in vitro to evaluate drug cytotoxicity. RESULTS: Most compounds inhibited the metabolic activity of leukemic cells in a time- and concentration-dependent manner but only 9 of them were sufficiently potent to inhibit L1210 tumor cell proliferation by 50% in the low-µM range after 2 (IC(50): 4.5-20.4 µM) and 4 days (0.5-4.0 µM) in culture. However, the antiproliferative compounds that were the most effective at day 4 were not necessarily the most potent at day 2, suggesting different speeds of action. A 3-h treatment with antiproliferative annulated indole was sufficient to inhibit, in a concentration-dependent manner, the rate of DNA synthesis measured in L1210 cells over a 0.5-h period of pulse-labeling with (3)H-thymidine. Four of the antiproliferative compounds had weak DNA-binding activities but one compound reduced the fluorescence of the ethidium bromide-DNA complex by up to 53%, suggesting that some annulated indoles might directly interact with double-stranded DNA to disrupt its integrity and prevent the dye from intercalating into DNA base pairs. However, all 9 antiproliferative compounds induced DNA cleavage at 24 h in L1210 cells, containing (3)H-thymidine-prelabeled DNA, suggesting that these antitumor annulated indoles might trigger an apoptotic pathway of DNA fragmentation. Indeed the antiproliferative annulated indoles caused a time-dependent increase of caspase-3 activity with a peak at 6 h. Interestingly, the compounds with the most potent antiproliferative IC(50) values at day 2 were consistently the most effective at inhibiting DNA synthesis at 3 h and inducing DNA fragmentation at 24 h. After 24-48 h, antiproliferative concentrations of annulated indoles increased the mitotic index of L1210 cells and stimulated the formation of many bi-nucleated cells, multi-nucleated cells, apoptotic cells and micronuclei, suggesting that these antitumor compounds might enhance mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis to induce apoptosis. CONCLUSION: Although annulated indoles may have interesting bioactivity, novel derivatives with different substitutions must be synthesized to elucidate structure-activity relationships, identify more potent antitumor lead compounds, and investigate their molecular targets and mechanisms of action.

Automated synthesis of a library of triazolated 1,2,5-thiadiazepane 1,1-dioxides via a double aza-Michael strategy.

The construction of a 96-member library of triazolated 1,2,5-thiadiazepane 1,1-dioxides was performed on a Chemspeed Accelerator (SLT-100) automated parallel synthesis platform, culminating in the successful preparation of 94 out of 96 possible products. The key step, a one-pot, sequential elimination, double-aza-Michael reaction, and [3 + 2] Huisgen cycloaddition pathway has been automated and utilized in the production of two sets of triazolated sultam products.

Synthesis of amino-benzothiaoxazepine-1,1-dioxides utilizing a microwave-assisted, S(N)Ar protocol.

The development of a microwave-assisted, intermolecular S(N)Ar protocol for the synthesis of a 126-member benzothiaoxazepine-1,1-dioxide library is reported. Diversification of 12 benzothiaoxazepine-1,1-dioxides was achieved in rapid fashion utilizing a variety of 2° amines and amino alcohols to generate an 80-member library. A second 48-member library was subsequently generated via a two-step alkylation, intermolecular S(N)Ar diversification protocol.

Triazole-containing isothiazolidine 1,1-dioxide library synthesis: one-pot, multi-component protocols for small molecular probe discovery.

The construction of two libraries of triazole-containing isothiazolidine 1,1-dioxides is reported utilizing either a one-pot click/aza-Michael or click/OACC esterification protocol. One core dihydroisothiazole 1,1-dioxide scaffold was prepared rapidly on multigram scale via ring-closing metathesis (RCM) and was subjected to a one-pot multicomponent click/aza-Michael protocol with an array of amines and azides for the generation of a 180-member triazole-containing isothiazolidine 1,1-dioxide library. Alternatively, three daughter scaffolds were generated via the aza-Michael of three amino alcohols, followed by a one-pot, multicomponent click/esterification protocol utilizing a ring-opening metathesis polymerization (ROMP)-derived coupling reagent, oligomeric alkyl carbodiimide (OACC) to generate a 41-member library of triazole-containing isothiazole 1,1-dioxides.