Ryan E. Looper

Transformation by the ( R )-enantiomer of 2-hydroxyglutarate linked to EGLN activation

The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG, but not (S)-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy.

(R)-2-Hydroxyglutarate Is Sufficient to Promote Leukemogenesis and Its Effects Are Reversible

Focusing on the Right Metabolite A variety of human cancers, including acute leukemias and brain tumors, have mutations in the genes encoding isocitrate dehydrogenase 1 or 2 (IDH1, IDH2), which cause overproduction of a metabolite called 2-hydroxyglutarate (2HG). Losman et al. (p. 1621, published online 7 February) show that the R- but not the S-enantiomer of 2HG can transform cells and that R-2HG mediates transformation at least in part through effects on protein modifying EglN prolyl hydroxylases. Importantly, the transforming activity of R-2HG was reversible, suggesting that therapeutic strategies focusing on inhibition of R-2HG production or inhibition of EglN prolyl hydroxylases merit further investigation. A metabolite specific to certain cancers, and of therapeutic interest, exists in two forms, only one of which is oncogenic. Mutations in IDH1 and IDH2, the genes coding for isocitrate dehydrogenases 1 and 2, are common in several human cancers, including leukemias, and result in overproduction of the (R)-enantiomer of 2-hydroxyglutarate [(R)-2HG]. Elucidation of the role of IDH mutations and (R)-2HG in leukemogenesis has been hampered by a lack of appropriate cell-based models. Here, we show that a canonical IDH1 mutant, IDH1 R132H, promotes cytokine independence and blocks differentiation in hematopoietic cells. These effects can be recapitulated by (R)-2HG, but not (S)-2HG, despite the fact that (S)-2HG more potently inhibits enzymes, such as the 5′-methylcytosine hydroxylase TET2, that have previously been linked to the pathogenesis of IDH mutant tumors. We provide evidence that this paradox relates to the ability of (S)-2HG, but not (R)-2HG, to inhibit the EglN prolyl hydroxylases. Additionally, we show that transformation by (R)-2HG is reversible.

Glutamine-dependent anapleurosis dictates glucose uptake and cell growth by regulating MondoA transcriptional activity

Glucose and glutamine are abundant nutrients required for cell growth, yet how cells sense and adapt to changes in their levels is not well understood. The MondoA transcription factor forms a heterocomplex with its obligate partner Mlx to regulate ≈75% of glucose-dependent transcription. By mediating glucose-induced activation of thioredoxin-interacting protein (TXNIP), MondoA:Mlx complexes directly repress glucose uptake. We show here that glutamine inhibits transcriptional activation of TXNIP by triggering the recruitment of a histone deacetylase-dependent corepressor to the amino terminus of MondoA. Therefore, in the presence of both glucose and glutamine, TXNIP expression is low, which favors glucose uptake and aerobic glycolysis; the Warburg effect. Consistent with MondoA functioning upstream of TXNIP, MondoA knockdown reduces TXNIP expression, elevates glucose uptake and stimulates cell proliferation. Although glutamine has many intracellular fates, a cell permeable analog of a tricarboxylic acid cycle (TCA) intermediate, α-ketoglutarate, also blocks the transcriptional activity of MondoA at the TXNIP promoter and stimulates glucose uptake. Together our data suggest that glutamine-dependent mitochondrial anapleurosis dictates glucose uptake and aerobic glycolysis by blocking MondoA:Mlx-dependent transcriptional activation of TXNIP. We propose that this previously unappreciated coordination between glutamine and glucose utilization defines a metabolic checkpoint that restricts cell growth when subthreshold levels of these essential nutrients are available.

Addition-Hydroamination Reactions of Propargyl Cyanamides : Rapid Access to Highly Substituted 2-Aminoimidazoles

A valuable pharmacophore, the 2-aminoimidazole moiety, can be accessed with a variety of substitution patterns through an addition-hydroamination-isomerization sequence (see scheme; R(1),R(4),R(5)=alkyl; R(3)=alkyl, aryl; R(2)=H, alkyl, aryl). The synthesis of the propargyl cyanamide precursors through a three-component coupling enables the preparation of this important heterocyclic core structure in just three steps.

Regioselective Rhodium(II)‐Catalyzed Hydroaminations of Propargylguanidines

Cyclic and polycyclic guanidinium ion natural products have been shown to modulate a variety of important biological processes and their activities are often reliant on the unique hydrogen bond donor-acceptor topologies that these substructures display.1 While most synthetic methods to prepare guanidines rely on the addition of an amine (guanylation) of an activated thiourea or urea,2 alternative methodologies that generates peripheral C-N bonds from an intact guanidine nucleus have proven powerful for the preparation of polycyclic guanidine natural products.3a-e Our interest in the biological activity of guanidine natural products has prompted us to develop a synthetic platform that is capable of delivering cyclic guanidines having multiple ring sizes, substitution patterns, and oxidation states in short order. To this end we have been interested in the addition of guanidine N-H bonds across C-C π systems and recently reported a La(III) catalyzed tandem addition-hydroamination reaction of propargylcyanamides which required forcing conditions, and resulted in exclusive 5-exo-dig cyclization. 4 This led us to study the hydroamination of preformed di-Boc protected propargylguanidines of the type 1, in hopes of finding a 6-endo-dig selective process. Traditionally, metal catalyzed cyclizations on alkynes favor a 5-exo-dig pathway. This can be seen in Au(I) and Au(III) catalyzed cyclization of propargylcarbamates, propargylureas, and propargylamides, 5,6 as well as the Ti(IV)-amide hydroamination reactions of homopropargylamines. 7,8 Examples of cyclization of heteroatom nucleophiles onto alkynes leading to 6-endo-dig cyclization, although as synthetically significant as the 5-exo-dig products, are nevertheless much less common and usually observed with substrates in which the tether is largely sp2 hybridized. 9,10 During the preparation of this manuscript, Gin and co-workers described the AuCl3 catalyzed hydroamination of alkynes with 2-aminopyrimidines en route to the synthesis of crambedine.11 Their gold-catalyzed 6-exo-dig cyclization highlights the power of this approach in total synthesis. Achieving selectivity in these hydroaminations, particularly 5-exo-dig vs. 6-endo-dig, would present a valuable tool for the synthesis of complex guanidine containing natural products. This manuscript highlights the discovery of an unusual reactivity of dirhodium(II)-carboxylates as highly 6-endo-dig selective hydroamination catalysts in the cyclization of propargylguanidines, while Ag(I) is typically 5-exo-dig selective. We first examined the ability of traditional π-Lewis acids to catalyze the hydroamination of 1a (Table 1). Initial experiments highlighted that there were multiple reaction pathways available (entry 1). Cyclization of 1a in the presence of Cu(I) salts reliably gave a mixture of 6-endo-dig and 5-exo-dig products, 2a and 3a respectively, that are easily identified by the magnitude of either 3J or 4J coupling. From nOe experiments it was also confirmed that the 5-exo product 3a carried the Z-alkene configuration. The unanticipated product was the yne-guanidine 4, which may arise from a [1,3]-prototropic shift followed by isomerization as detailed by Gevorgyan for propargyl acetates.12 Table 1 Catalyst screen for propargylguanidine hydroamination. Ag(I) catalysis proved to be optimal for the generation of 3a with AgOAc giving a 1 : >20 ratio of 2a : 3a in 55% yield. It was further found that addition of AcOH to the AgOAc catalyzed reaction gave significant rate enhancements suggesting that protonolysis of the vinyl metal species was rate limiting, consistent with the known electrophilic hydroamination mechanism 13 Our first success in reversing the selectivity was found with gold (III) catalysts that favored the 6-endo product (entries 6-7). The sluggishness of the Au catalyzed reactions directed our attention to Rh(II). Dirhodium(II)-carboxylates have rarely been used to activate alkynes, however Murai has shown that Rh2(tfa)4 is a competent ene-ene-yne cycloisomerization catalyst and gives different product distributions than Pt(II) or Ru(II).14 Gratifyingly, Rh2(tfa)4 turned the selectivity over favoring the 6-endo product (entry 8). Quite to our surprise, given its decreased Lewis-acidity, Rh2(OAc)4 was a very selective catalyst favoring the 6-endo product 13 : 1 (entry 9). The reaction times were long and we suspected that this was due to catalyst solubility. The reaction was enhanced with Rh2(Oct)4 giving the 6-endo product in 81% isolated yield after 16 hours in > 20 : 1 (2a : 3a)selectivity as judged by 1H NMR (entry 10). Having identified a selective catalyst set, a substrate-selectivity profile was developed for the reaction (Table 2) and it was immediately apparent that the reactivity of the dirhodium(II)-carboxylates was quite unique. For example, cyclization of the p-methoxyphenyl alkyne (entry 1) with Ag(I) favored the 5-exo product, however with poor selectivity, consistent with the ability of this substituent to competitively stabilize a vinyl cation for 6-endo cyclization. In contrast, Rh(II) gave exclusively the 6-endo product 2b. Cyclization of electron poor aryl alkynes showed the opposite trend, with Ag(I) giving high 5-exo selectivity while Rh(II) was more modestly selective for the 6-endo product 2c (entry 2). Alkyl substituted alkynes gave the 6-endo products exclusively with Rh(II) (entries 3-7). This is in direct contrast to the Ag(I) catalyzed reactions, where modest selectivities for the 5-exo products are seen when the alkyne termini are electronically indistinguishable. Importantly for subsequent annulations and access to polycyclic skeletons, Rh2(Oct)4 tolerates useful functional groups (e.g. alcohols and alkyl chlorides, entries 4-7). These primary alkyl chlorides were also cleanly cyclized to the 5-exo-dig isomers 2g,h with Ag(I). Substitution is also permitted at the propargylic position wherein good selectivities are seen with Ag(I) for the 5-exo product and with Rh(II) for the 6-endo product (entry 8). Substituents on the guanidine nitrogen had modest effects on selectivity, with the 6-endo product always favored under Rh(II) catalysis (entries 9,10). The t-butyl alkyne 1l (entry 11) reacted well in the Ag(I) catalyzed process but was unreactive toward Rh(II), presumably due to the size of the Rh(II) catalyst. Table 2 Substrate scope for the Rh(II)-catalyzed hydroamination of propargylguanidines. The resultant cyclic eneguanidines contain a rich functional group handle for further manipulations (Scheme 1). For example bromination proceeds to give the 5-bromodihydropyrimidine 5 the structure of which was further confirmed by X-ray crystallography (Eq. 1). Oxidative cyclization of pendant nucleophiles gives the spirocyclic hemiaminal 6, reminiscent of the crambescidins (Eq. 2). Deprotection of the 5-membered ene-guanidine 2a under acidic conditions was accompanied by isomerization to give the 2-aminoimidazole 7 (Eq. 3). Reduction was also cleanly performed on 2b to give tetrahydropyrimidine 8 (Eq. 4). Scheme 1 Transformations of the resultant ene-guanidines. Examples of alkyne activation by dirhodium(II)-carboxylates are quite rare.8,15 Given the fact that reactions of internal alkynes have only been observed with Rh2(tfa)4 16 and that Rh2(Oct)4 is not able to activate alkynes independently,8 we were surprised that the dirhodium(II)-alkylcarboxylates were competent catalysts for this transformation especially at room temperature.17 This was especially true for the cyclization of electron deficient alkynes such as the pCF3Ph substituted alkyne 1c. We were even more surprised by the observation that 1c reacted ten times faster than the p-methoxy substituted alkyne 1b. Regardless of the poor π-Lewis aciditiy of these catalysts, this suggested that coordination of the alkyne was not rate limiting. Addition of 3 equivalents of acetic acid changed the selectivity from 5:1 to 1:1.7 (2c:3c, table 3 compare entries 1 and 2) suggesting that protonation of a vinyl-rhodium intermediate might be involved in the product determining step. The use of Rh2(tfa)4 as a catalyst also favored the 5-exo-dig product with 1c (entry 3). Taken together this may suggest a Curtin-Hammett scenario wherein the initial cyclization to form a 5- or 6-membered vinyl-rhodium anion is highly reversible and that perturbation of this pre-equilibrium by stabilizing the kinetically favored 5-exo-dig intermediate leads to poor selectivity.18 Table 3 Condition effects on selectivity. Mechanistically it is difficult to rationalize oxidation state changes in the dirhodium(II)-carboxylates upon arriving at a vinylrhodium intermediate (in this case the formation of Rh(I) in the dimer). This prompted us to examine better defined catalysts known to proceed through vinyl-rhodium intermediate.19 Although sluggish, Wilkinson’s catalyst promoted the cyclization and favored the 6-endo product in 10:1 selectivity (entry 4). Cationic Rh(I) however reversed the selectivity favoring the 5-exo product in 10:1 selectivity (entry 5). Again this may support the necessity for the initial cyclization event to be reversible in order to access the 6-endo product. More importantly this suggests access to more defined vinyl-rhodium intermediates that might be exploited for cascade reactivity. In conclusion we have demonstrated the unique ability of dirhodium(II)-carboxylates to catalyze the 6-endo-dig selective hydroamination of propargylguanidines. The resultant cyclic eneguanidines contain a rich latent functional group for the preparation of skeletally diverse cyclic guanidine natural product substructures. Studies to further understand the reactivity of dirhodium(II)-carboxylates and the application of these products to more complex targets are ongoing and will be reported in due course.

2-Aminoimidazoles from Leucetta Sponges: Synthesis and Biology of an Important Pharmacophore

This review will focus on the ability of the 2-aminoimidazole to occupy a unique subset of chemical space which makes it an ideal pharmacophore for the development of small molecule collections for discovery based research. These observations rely both on the use of 2-aminoimidazoles as building blocks in medicinal chemistry as well as the recent discovery of alkaloids from sponges of the genus Leucetta which exhibit a diverse range of biological activities around a relatively limited structural core. The preparation of these compounds will also be highlighted. In particular, marine natural products derived from sponges have provided valuable leads for therapeutic small molecules (3, 4). Surprisingly the large majority of these compounds have been isolated from organisms of the class Dermospongiae. In the mid 1980s chemists noted that the other major sponge class, Calcarea, had rarely been subject to chemical investigations. A flurry of efforts through the mid-1990s helped to establish biogenetic relationships among these sponges. Isolated to explore these inter- connections and not necessarily for specific biological responses the activities of these natural products have remained largely uncovered. Since these initial inves- tigations, an emerging structural class has recurrently been identified through bioassay guided isolation which contains the 2-aminoimidazole core. From the viewpoint of small molecule discovery this review will highlight alkaloids isolated from Leucetta sp. This small skeletal family has been shown to interrogate an incredibly diverse range of biological processes and thus represents an important discovery scaffold for both medicinal and discovery based research.

Total synthesis of (±)-heliannuol D, an allelochemical from Helianthus annuus

Abstract The total synthesis of (±)-heliannuol D and its epimer has been completed in 9 steps and 12% overall yield from 2-methylanisole. The benzoxepane moiety of the title compound, a common structural feature in the heliannuol family of natural products, is prepared by a biomimetic opening of an epoxide by a phenol.

A stereocontrolled synthesis of (+)-saxitoxin

A concise stereoselective total synthesis of (+)-saxitoxin is described. A silver(I)-initiated hydroamination cascade constructs the bicyclic guanidinium ion core from a alkynyl bisguanidine. This sequence creates two C-N bonds, one C-O bond, and three rings and forms a single stereoisomer in a single synthetic transformation. This process enabled us to complete the synthesis of (+)-saxitoxin in 14 steps from N-Boc-l-serine methyl ester.

Human phosphoglycerate dehydrogenase produces the oncometabolite D-2-hydroxyglutarate.

Human d-3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in the serine biosynthetic pathway, is genomically amplified in tumors including breast cancer and melanoma. In PHGDH-amplified cancer cells, knockdown of PHGDH is not fully rescued by exogenous serine, suggesting possible additional growth-promoting roles for the enzyme. Here we show that, in addition to catalyzing oxidation of 3-phosphoglycerate, PHGDH catalyzes NADH-dependent reduction of α-ketoglutarate (AKG) to the oncometabolite d-2-hydroxyglutarate (d-2HG). Knockdown of PHGDH decreased cellular 2HG by approximately 50% in the PHGDH-amplified breast cancer cell lines MDA-MB-468 (normal concentration 93 μM) and BT-20 (normal concentration 35 μM) and overexpression of PHGDH increased cellular 2HG by over 2-fold in non-PHGDH-amplified MDA-MB-231 breast cancer cells, which normally display very low PHGDH expression. The reduced 2HG level in PHGDH knockdown cell lines can be rescued by PHGDH re-expression, but not by a catalytically inactive PHGDH mutant. The initial connection between cancer and d-2HG involved production of high levels of d-2HG by mutant isocitrate dehydrogenase. More recently, however, elevated d-2HG has been observed in breast cancer tumors without isocitrate dehydrogenase mutation. Our results suggest that PHGDH is one source of this d-2HG.

Construction of the A-ring of cylindrospermopsin via an intramolecular oxazinone-N-oxide dipolar cycloaddition

Abstract The efficient synthesis of an A-ring synthon for the marine hepatatoxin cylindrospermopsin has been achieved. The key step features an intramolecular oxazinone N-oxide/alkene dipolar cycloaddition resulting in the establishment of the three contiguous stereogenic centers in the A-ring from one pre-existing stereogenic center in a single step.