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Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.

Two diastereomers of the protonated diketopiperazine (DKP) dipeptide cyclo(Tyr-Tyr), namely, cyclo(LTyr-LTyr)H

Nocardioazines A and B are prenylated, bioactive pyrroloindoline natural products, isolated from

Pyrroloindolines bearing a C3-N linkage comprise the core of many biologically active natural products, but many methods toward their synthesis are limited by the sterics or electronics of the product. We report a single electron-based approach for the synthesis of this scaffold and demonstrate high-yielding aminations, regardless of electronic or steric demands. The transformation uses copper wire and isopropanol to promote the reaction. The broad synthetic utility of this heterogeneous copper-catalyzed approach to access pyrroloindolines, diketopiperazine, furoindoline, and (+)-asperazine is included, along with experiments to provide insight into the mechanism of this new process.

Diketopiperazine (DKP) formation is an important degradation pathway for peptides and proteins. It can occur during synthesis and storage in either solution or the solid state. The kinetics of peptide cleavage through DKP formation have been analyzed for the model peptides Xaa

We have previously reported that neoechinulin B (1a), a prenylated indole diketopiperazine alkaloid, shows antiviral activities against hepatitis C virus (HCV) via the inactivation of the liver X receptors (LXRs) and the resultant disruption of double-membrane vesicles. In this study, a two-step synthesis of the diketopiperazine scaffold of 1a was achieved by the base-induced coupling of 1,4-diacetyl-3-{[(tert-butyldimethylsilyl)oxy]methyl}piperazine-2,5-dione with aldehydes, followed by the treatment of the resultant coupling products with tetra-n-butylammonium fluoride. Compound 1a and its 16 derivatives 1b–q were prepared using this method. Furthermore, variecolorin H, a related alkaloid, was obtained by the acid treatment of 1a in MeOH. The antiviral evaluation of 1a and its derivatives revealed that 1a, 1c, 1d, 1h, 1j, 1l, and 1o exhibited both anti-HCV and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activities. The results of this study indicate that the exomethylene moiety on the diketopiperazine ring is important for the antiviral activities. The antiviral compounds can inhibit the production of HCV and SARS-CoV-2 by inactivating LXRs.

Asymmetric construction of dithiodiketopiperazines on otherwise achiral scaffolds remains a pivotal synthetic challenge encountered in many biologically significant natural products. Herein, we report the first total syntheses of (−)-glionitrin A/B and revise the absolute configurations. Emerging from the study is a novel oxidative sulfenylation of triketopiperazines that enables asymmetric formation of dithiodiketopiperazines on sensitive substrates. The concise route paves the way for further studies on the potent antimicrobial and antitumor activities of glionitrin A and the intriguing ability of glionitrin B to inhibit invasive ability of cancer cells.

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. P450-mediated biocatalysis offers a practical avenue to access their structural diversity; however, many of these enzymes are insoluble in Escherichia coli and difficult to operate in Streptomyces. Through validation of the functions of two pairs Mycobacterium smegmatis sourced redox partners in vitro, and comparing the efficiency of different biocatalytic systems with tricky P450s in vivo, we herein demonstrated that M. smegmatis is much more efficient, robust, and cleaner in metabolites background than the regularly used E. coli or Streptomyces systems. The M. smegmatis-based system can completely convert 1 g L-1 of cyclodipeptide into HTDKPs within 18 h with minimal background metabolites. On the basis of this efficient system, 12 novel HTDPKs were readily obtained by using two HTDKP-forming P450s (NasbB and NASS1868). Among them, five compounds have neuroprotective properties. Our study significantly expands the bioactive chemical scope of HTDKPs and provides an excellent biocatalysis platform for dealing with problematic enzymes from Actinomycetes.

We conducted global genome mining of 162,672 bacterial genomes and identified 829 cyclodipeptide (CDP) biosynthesis gene clusters (BGC) containing a cytochrome P450 gene. Heterologous expression of BGC from Saccharopolyspora hirsuta DSM 44795 led to the identification of two novel crownlike CDPs, cyctetryptomycin A (4) and B (5), which possess unprecedented complex macrocycle and show neuroprotective activity. The two cytochrome P450s found in the BGC catalyze sequential reactions leading to the cyclization of diketopiperazine dimers.

Cardioprotective activity of dexrazoxane (ICRF-187), the only clinically approved drug against anthracycline-induced cardiotoxicity, has traditionally been attributed to its iron-chelating metabolite. However, recent experimental evidence suggested that the inhibition and/or depletion of topoisomerase IIβ (TOP2B) by dexrazoxane could be cardioprotective. Hence, we evaluated a series of dexrazoxane analogues and found that their cardioprotective activity strongly correlated with their interaction with TOP2B in cardiomyocytes, but was independent of their iron chelation ability. Very tight structure-activity relationships were demonstrated on stereoisomeric forms of 4,4'-(butane-2,3-diyl)bis(piperazine-2,6-dione). In contrast to its rac-form 12, meso-derivative 11 (ICRF-193) showed a favorable binding mode to topoisomerase II in silico, inhibited and depleted TOP2B in cardiomyocytes more efficiently than dexrazoxane, and showed the highest cardioprotective efficiency. Importantly, the observed ICRF-193 cardioprotection did not interfere with the antiproliferative activity of anthracycline. Hence, this study identifies ICRF-193 as the new lead compound in the development of efficient cardioprotective agents.

The dimeric diketopiperazine (DKPs) alkaloids are a diverse family of natural products (NPs) whose unique structural architectures and biological activities have inspired the development of new synthetic methodology to access these molecules. However, catalyst-controlled methods that enable the selective formation of constitutional and stereoisomeric dimers from a single monomer are lacking. To resolve this long-standing synthetic challenge, we sought to characterize the biosynthetic enzymes that assemble these NPs for application in biocatalytic syntheses. Genome mining enabled identification of the cytochrome P450, NzeB (derived from Streptomyces sp. NRRL F-5053), which catalyzes both intermolecular carbon-carbon (C–C) and carbon-nitrogen (C–N) bond formation, generating all currently known DKP dimer scaffolds isolated from bacterial sources. To identify the molecular basis for the flexible site-, stereo-, and chemoselectivity of NzeB, we obtained high-resolution crystal structures (1.5Å) of the protein in complex with native and non-native substrates. This, to our knowledge, represents the first crystal structure of an oxidase catalyzing direct, intermolecular C–H amination. Site-directed mutagenesis was employed to assess the role individual active site residues play in guiding selective DKP dimerization. Finally, computational approaches were employed to evaluate plausible mechanisms regarding NzeB function and its ability to catalyze both C–C and C–N bond formation. These results provide a structural and computational rationale for the catalytic versatility of NzeB, as well as new insights into variables that control selectivity of CYP450 diketopiperazine dimerases.

This study reveals an alternative sequence for the synthesis of compounds that contain the pyrrolodiketopiperazine structural motif. Starting with a diketopiperazine precursor, a mild aldol condensation precedes pyrrole annulation and bicyclic ring fusion. The derived intermediate aldol condensation products, which bear either a protected carbonyl or a functionalized alkyne, can be cyclized to the pyrrolodiketopiperazine by protic or gold Lewis acid catalysis.

An efficient synthesis of diketopiperazinoindolines through an indium-catalyzed intramolecular 5-exo-dig cyclization of ortho-alkynyl diketopiperazines has been reported. The formation of diketopip...

We report the solid-phase synthesis of N,N′-di(acylamino)-2,5-diketopiperazine, an acylhydrazide-based conformationally rigid 2,5-DKP scaffold having exocyclic N–N bonds. We also show that different combinations of acylhydrazides, carbazates, semicarbazides, amino acids, and primary amines can be used to synthesize a highly diverse collection of hybrid DKP molecules via the solid-phase submonomer synthesis route. Finally, we show incorporation of a methyl substituent in one of the carbon atoms of the DKP ring to generate chiral daa- and hybrid-DKPs without compromising the synthetic efficiency.

Two new indolyl diketopiperazines, gartryprostatins A and B (1 and 2), with an unusual 2,3-furan-fused pyrano[2,3-g]pyrrolo[1″,2″:4',5']pyrazino[1',2':1,5]pyrrolo[2,3-b]indole nucleus, along with a new naturally occurring compound (gartryprostatin C, 3) were isolated from the solid culture of Aspergillus sp. GZWMJZ-258, an endophyte from Garcinia multiflora (Guttiferae). The structures of compounds 1-3 were determined by nuclear magnetic resonance, mass spectrometry, Marfey's analysis of amino acids, and chemical calculation. Compounds 1-3 displayed selective inhibition on human FLT3-ITD mutant AML cell line, MV4-11, with IC50 values of 7.2, 10.0, and 0.22 μM, respectively.

Photopiperazines A-D (1-4), unsaturated diketopiperazine derivatives, were isolated from the culture broth of a rare, marine-derived actinomycete bacterium, strain AJS-327. This strain shows very poor 16S rRNA sequence similarity to other members of the actinomycete family Streptomycetaceae, indicating it is likely a new lineage within this group. The structures of the photopiperazines were defined by analysis of HR-ESI-TOF-MS spectra in conjunction with the interpretation of 1D and 2D NMR data. The photopiperazines are sensitive to light, causing interconversion among the four olefin geometrical isomers, which made purification of each isomer challenging. The photopiperazines are highly cytotoxic metabolites that show selective toxicity toward U87 glioblastoma and SKOV3 ovarian cancer cell lines.

International audience; The conformational landscape of the diketopiperazine (DKP) dipeptide built on tyrosine and proline, namely, cyclo Tyr-Pro, is studied by combining resonance-enhanced multiphoton ionisation, double resonance IR-UV spectroscopy, and quantum chemical calculations. Despite the geometrical constraints due the two aliphatic rings, DKP and proline, cyclo Tyr-Pro is a flexible molecule. For both diastereoisomers, cyclo LTyr-LPro and cylo LTyr-DTyr, two structural families coexist under supersonic jet conditions. In the most stable conformation, the aromatic tyrosine substituent is folded over the DKP ring (g + geometry of the aromatic ring) as it is in the solid state. The other structure is completely extended (g-geometry of the aromatic ring) and resembles that proposed for the vapor phase. IR-UV results are not sufficient for unambiguous assignment of the observed spectra to either folded or extended conformations and the simulation of the vibronic pattern of the S0-S1 transition is necessary. Still, the comparison between IR-UV results and anharmonic calculations allow explaining the minor structural differences between cyclo LTyr-LPro and cylo LTyr-DPro in terms of different NH… and CH… interactions. 2

The NAD+-dependent deacetylase SIRT2 represents an attractive target for drug development. Here, we designed and synthesized drug-like SIRT2-selective inhibitors based on an analysis of the putative binding modes of recently reported SIRT2-selective inhibitors and evaluated their SIRT2-inhibitory activity. This led us to develop a more drug-like diketopiperazine structure as a "hydrogen bond (H-bond) hunter" to target the substrate-binding site of SIRT2. Thioamide 53, a conjugate of diketopiperazine and 2-anilinobenzamide which is expected to occupy the "selectivity pocket" of SIRT2, exhibited potent SIRT2-selective inhibition. Inhibition of SIRT2 by 53 was mediated by the formation of a 53-ADP-ribose conjugate, suggesting that 53 is a mechanism-based inhibitor targeting the "selectivity pocket", substrate-binding site, and NAD+-binding site. Furthermore, 53 showed potent antiproliferative activity toward breast cancer cells and promoted neurite outgrowth of Neuro-2a cells. These findings should pave the way for the discovery of novel therapeutic agents for cancer and neurological disorders.

Eight new diketopiperazine-type alkaloids including four oxepin-containing diketopiperazine-type alkaloids, oxepinamides H-K (1-4), and four 4-quinazolinone alkaloids, puniceloids A-D (5-8), together with two known analogues (9 and 10), were isolated from the culture broth extracts of the deep-sea-derived fungus Aspergillus puniceus SCSIO z021. Their structures were elucidated by spectroscopic analyses, and their absolute configurations were determined by Marfey's method along with comparison of their specific rotations and ECD spectra. The absolute configurations of 4 and 5 were further confirmed by a single-crystal X-ray diffraction analysis. Compounds 1-8 showed significant transcriptional activation of liver X receptor α with EC50 values of 1.7-50 μM, and 7 and 8 were the most potent agonists.

Dipeptides are known to spontaneously cyclize to diketopiperazines, and in some cases these cyclic dipeptides have been shown to self-assemble to form supramolecular nanostructures. Herein, we demonstrate the in situ cyclization of dipeptide methyl esters in aqueous buffer by intramolecular aminolysis, leading to the formation of diverse supramolecular nanostructures. The chemical nature of the amino acid side chains dictates the supramolecular arrangement and resulting nanoscale architectures. For c[LF], supramolecular gels are formed, and the concentration of starting materials influences the mechanical properties of hydrogels. Moreover, by adding metalloporphyrin to the starting dipeptide ester solution, these become incorporated through cooperative assembly, resulting in the formation of nanofibers able to catalyse the oxidation of organic phenol in water. The approach taken here, which combines the chemically activated assembly with the versatility of short peptides might pave the way for achieving the spontaneous formation of supramolecular order and function using simple building blocks.

Herein, we report a convenient synthesis of unprecedented aza-diketopiperazines (aza-DKPs). The strategy is based on selective diversification of bicyclic aza-DKP scaffolds by click reaction, N-acylation, and/or N-alkylation. These scaffolds containing either azido or amino groups were obtained by a key Rh(I)-catalyzed hydroformylative cyclohydrocarbonylation reaction of allyl-substituted aza-DKP. The methodology is readily amenable to the parallel synthesis of original aza-DKPs to enlarge the chemical diversity of aza-heterocycles.

Thaxtomins are a group of phytotoxic diketopiperazines produced by tens of plant pathogenic Streptomyces strains and have received considerable attention as bioherbicide. To synthesize thaxtomin analogue libraries for herbicide development, we here develop an in vitro biocombinatorial approach. We first prepared two recombinant single-module multifunctional nonribosomal peptide synthetases (NRPSs), TxtA and TxtB. Biochemical studies revealed that TxtA and TxtB tolerated small substituents on the aromatic moiety of l-Phe and l-Trp, respectively. Intriguingly, TxtB showed a control of the substrate scopes of TxtA and a previously characterized, pathway-specific nitration promoting P450 TxtE. We further demonstrated that the methyltransferase (MT) domain of TxtA and TxtB posed a minimal influence on the assembly of the diketopiperazine core by other domains of these two enzymes, providing a way for structural diversification. The pathway-specific bifunctional P450 TxtC was then recombinantly produced in Esch...

The incorporation of the recently discovered reciprocal n → π* interactions in 2,5-diketopiperazines (DKPs) is reported to design a novel N, N'-di(acylamino)-2,5-diketopiperazine (daa-DKP) scaffold. The design, synthesis, and structural features of daa-DKPs and the effect of reciprocal n → π* interactions in their structural rigidity is discussed.

The bis(pyridine)silver (I) permanganate promoted hydroxylation of diketopiperazines has served as a pivotal transformation in the synthesis of complex epipolythiodiketopiperazine alkaloids. This late-stage C–H oxidation chemistry is strategically critical to access N-acyl iminium ion intermediates necessary for nucleophilic thiolation of advanced diketopiperazines en route to potent epipolythiodiketopiperazine anticancer compounds. In this study, we develop an informative mathematical model using hydantoin derivatives as a training set of substrates by relating the relative rates of oxidation to various calculated molecular descriptors. The model prioritizes Hammett values and percent buried volume as key contributing factors in the hydantoin series while correctly predicting the experimentally observed oxidation sites in various complex diketopiperazine case studies. Thus, a method is presented by which to use simplified training molecules and resulting correlations to explain and predict reaction behavior for more complex substrates.

The first enantioselective total synthesis of the epipolythiodiketopiperazine (ETP) natural product (−)-acetylapoaranotin (3) is reported. The concise synthesis was enabled by an eight-step synthesis of a key cyclohexadienol-containing amino ester building block. The absolute stereochemistry of both amino ester building blocks used in the synthesis is set through catalytic asymmetric (1,3)-dipolar cycloaddition reactions. The formal syntheses of (−)-emethallicin E and (−)-haemotocin are also achieved through the preparation of a symmetric cyclohexadienol-containing diketopiperazine.

Chemical profiling of extracts from a mud dauber wasp-associated fungus, Aspergillus sp. (CMB-W031), revealed a remarkably diverse array of secondary metabolites, with many biosynthetic gene clusters being transcriptionally responsive to specific culture conditions. Chemical fractionation of a jasmine rice cultivation yielded many known fungal metabolites, including the highly cytotoxic (-)-stephacidin B and an unprecedented nonribosomal peptide synthase derived nitro depsi-tetrapeptide diketopiperazine, waspergillamide A (1). All structures were assigned by detailed spectroscopic analysis and, where appropriate, chemical degradation and Marfey's analysis.

The structure of a C2-symmetric epidithiodiketopiperazine alkaloid, SCH 64874, was determined by semisynthesis. The relative stereochemistry of the β-hydroxy carboxylic acid chain having three chiral centers was determined by comparison of the NMR data of the four possible diastereomeric β-hydroxy carboxylic acid fragments with those of SCH 64874. Condensation of the (−)-deacetylaranotin core with two enantiomeric β-hydroxy carboxylic acids revealed the relative stereochemistry of SCH 64874. The relative stereochemistry of the β-keto carboxylic acid chain of the analogous alkaloid hirsutellomycin was determined in a stepwise manner. The C4′–C6′ syn relationships were predicted by comparing the NMR data of the corresponding ester fragments with that of hirsutellomycin. The relative stereochemistry of the whole molecule, including the epimerizable C2′ stereocenter, was determined by introduction of four possible side chains into the bisdethiodi(methylthio)deacetylaranotin core. We found that the stereochemi...

Parkinson's disease (PD) is a neurodegenerative disorder, which can be modeled using the neurotoxin 6-hydroxydopamine (6-OHDA) to generate oxidative stress. Here, we studied the effects of the antioxidants deferricoprogen (DFC) and dimerumic acid (DMA), produced by rice fermented with Monascus purpureus NTU 568, on 6-OHDA-induced apoptosis in SH-SY5Y cells and their potential protective mechanisms. DMA and DFC inhibited 6-OHDA-induced apoptosis and cellular reactive oxygen species (ROS) in SH-SY5Y human neuroblastoma cells. Molecular analysis demonstrated associated upregulation of the Ak mouse strain thymoma (Akt), heme oxygenase-1 (HO-1), and signal-regulated kinase (ERK) pathways along with inhibited phosphorylation of c-Jun N-terminal kinase (JNK) and p38 pathways and altered homodimeric glycoprotein, N-methyl-d-aspartate (NMDA) receptor, and immunoglobulin Fc receptor gene expression. These results suggested that the neuroprotection elicited by DMA and DFC against 6-OHDA-induced neurotoxicity was associated with the Akt, MAPK, and HO-1 pathways via regulating the gene expression of NMDA receptor, homodimeric glycoprotein, and immunoglobulin Fc receptor.

Although several approaches for making antibody-drug conjugates (ADC) have been developed, it has yet to be reported that an antibody binding peptide such as Z33 from protein A is utilized as the pivotal unit to generate the noncovalent-type ADC (NC-ADC). Herein we aim to establish a novel probe for NC-ADC by synthesizing the Z33-conjugated antitumor agent, plinabulin. Due to the different solubility of two components, including hydrophobic plinabulin and hydrophilic Z33, an innovative method with a solid-supported disulfide coupling reagent is required for the synthesis of the target compounds with prominent efficiency (29% isolated yield). We demonstrate that the synthesized hybrid exhibits a binding affinity against the anti-HER2 antibody (Herceptin) and the anti-CD71 antibody (6E1) (Kd = 46.6 ± 0.5 nM and 4.5 ± 0.56 μM, respectively) in the surface plasmon resonance (SPR) assay. In the cell-based assays, the hybrid provides a significant cytotoxicity in the presence of Herceptin against HER2 overexpressing SKBR-3 cells, but not against HER2 low-expressing MCF-7 cells. Further, it is noteworthy that the hybrid in combination with Herceptin induces cytotoxicity against Herceptin-resistant SKBR-3 (SKBR-3HR) cells. Similar results are obtained with the 6E1 antibody, suggesting that the synthesized hybrid can be widely applicable for NC-ADC using the antibody of interest. In summary, a series of evidence presented here strongly indicate that NC-ADCs have high potential for the next generation of antitumor agents.

The concise, enantioselective total syntheses of (+)-asperazine (1), (+)-iso-pestalazine A (2), and (+)-pestalazine A (3) have been achieved by the development of a late-stage C3-C8' Friedel-Crafts union of polycyclic diketopiperazines. Our modular strategy enables the union of complex polycyclic diketopiperazines in virtually their final forms, thus providing rapid and highly convergent assembly at the challenging quaternary stereocenter of these dimeric alkaloids. The significance of this carbon-carbon bond formation can be gauged by the manifold constraints that were efficiently overcome, namely the substantial steric crowding at both reactive sites, the nucleophilic addition of C8' over N1' to the C3 carbocation, and the multitude of reactivity posed by the use of complex diketopiperazine fragments in the coupling event. The success of the indoline π-nucleophile that evolved through our studies is notable given the paucity of competing reaction pathways observed in the presence of the highly reactive C3 carbocation generated. This first total synthesis of (+)-pestalazine A also allowed us to revise the molecular structure for this natural alkaloid.

An approach to the dihydrooxepino[4,3-b]pyrrole core of diketopiperazine natural products which utilizes a vinyl pyrrole epoxide Cope rearrangement was investigated. It was found that an ester substituent on the epoxide was essential for the [3,3]-rearrangement to occur. Density functional calculations with M06-2X provided explanations for the effects of the pyrrole and ester groups on these rearrangements.

The cis-trans isomerization and conformer specificity of δ-azaproline and its carbamate-protected form in linear and cyclic peptides were investigated using NMR and α-chymotrypsin assay. Comparisons of the chemical shift value of the α-hydrogen in each case of δ-azaproline-containing peptides with conformer-specific locked diketopiperazines reveal the fact that an upfield chemical shift value corresponds to cis conformer and a downfield value corresponds to a trans conformer. δ-Azaproline adopts cis-conformation in simple amides, dipeptides, and tripeptides whereas its carbamate-protected form adopts trans-conformation. In the case of longer, linear or cyclic peptides, vice versa results are obtained. Interestingly, in all these peptides exclusively one conformer, either cis or trans, is stabilized. This cis-trans isomerization is independent of both temperature and solvents; only the δ-nitrogen protecting group plays key role in the isomerization. δ-Azaproline is conformer-specific in either of its protected or deprotected forms, which is a unique property of this proline. Unlike other covalently modified proline surrogates, this isomerization of δ-azaproline can be tuned easily by a protecting group. The mechanism of cis-trans isomerization of δ-azaproline during deprotection and reprotection is supported by theoretical calculations.

A one-pot sequence for the synthesis of aza-diketopiperazines is reported, involving carbazate acylation with chloroacetyl chloride, SN2 with a primary amine, N-isocyanate formation, and cyclization. Nitrogen-substituted isocyanates (N-isocyanates) are a rare class of amphoteric isocyanate with high, but severely underdeveloped synthetic potential. This approach highlights that βN-acyl carbazates can act as blocked (masked) N-isocyanates, thus allowing a challenging intermolecular SN2 reaction of a primary amine to proceed while the N-isocyanate is "protected", and then cyclization once it is unmasked. Control experiments show that the alternate pathway--N-isocyanate substitution and then cyclization by an intramolecular SN2 reaction--is not operating.

The first total synthesis of (+)-luteoalbusins A and B is described. Highly regio- and diastereoselective chemical transformations in our syntheses include a Friedel-Crafts C3-indole addition to a cyclotryptophan-derived diketopiperazine, a late-stage diketopiperazine dihydroxylation, and a C11-sulfidation sequence, in addition to congener-specific polysulfane synthesis and cyclization to the corresponding epipolythiodiketopiperazine. We also report the cytoxicity of both alkaloids, and closely related derivatives, against A549, HeLa, HCT116, and MCF7 human cancer cell lines.

Phenotype-guided natural products discovery is emerging as a useful new discovery tool that addresses challenges in early, unbiased natural product biological annotation. These high-content approaches yield screening results that report directly on the impact of test compounds on cellular processes in target organisms and can be used to predict the modes of action (MOAs) of bioactive constituents from primary screening data. In this study we explored the use of our recently implemented cytological profiling (CP) platform for the isolation of compounds with a specific, predefined mode of action, namely induction of mitotic arrest. Screening of a microbially-derived extract library revealed six extracts whose cytological profiles clustered closely with those of known antimitotic agents from the pure compound training set. Subsequent examination of one of these extracts revealed the presence of two separate bioactive constituents, each of which possessed a unique cytological profile. The first, diketopiperazine XR334 (3), recapitulated the observed antimitotic phenotype of the original extract, demonstrating that cytological profiling can be used for the targeted isolation of compounds with specific modes of action. The second, nocapyrone L (6), possessed a cytological profile that clustered with known calcium channel modulators, in line with previous published activities for this compound class, indicating that cytological profiling is a flexible and powerful platform for the de novo characterization of compound modes of action.

A method for the preparation of 3,5-bridged piperazin-2-ones from a tryptophan-proline-based diketopiperazine is described using diphosgene to induce the ring closure. Density functional theory calculations were conducted to study the mechanism of this C-C bond formation. Several derivatives of the thus obtained α-chloroamine were synthesized by substitution of the chlorine atom using a range of O-, N-, S-, and C-nucleophiles. This novel class of brevianamide F analogues possess interesting breast cancer resistance protein inhibitory activity.

Chemical investigation of the marine-sponge-derived fungus Penicillium adametzioides AS-53 resulted in the identification of two new bisthiodiketopiperazine derivatives, adametizines A (1) and B (2), from cultivation in a liquid potato-dextrose broth (PDB) culture medium, whereas two new acorane sesquiterpenes, adametacorenols A (3) and B (4), were isolated from a rice solid culture medium. The structures of these compounds were elucidated on the basis of spectroscopic analysis. The absolute configuration of compound 1 was determined by X-ray crystallographic analysis, and that of 3 was determined by modified Mosher's method. Compound 1 exhibited lethality against brine shrimp (Artemia salina) with an LD50 value of 4.8 mu M and inhibitory activities against Staphyloccocus aureus, Aeromonas hydrophilia, Vibrio spp. V. harveyi and V. parahaemolyticus, and Gaeumannomyces graminis with minimum inhibitory concentration values of 8, 8, 32, 8, and 16 mu g/mL, respectively. Chlorination at C-7 significantly increased the brine shrimp lethality and antimicrobial activity of the bisthiodiketopiperazines.

The factors determining diastereoselectivity observed in the multicomponent conversion of amino acids, aziridine aldehyde dimers, and isocyanides into chiral piperazinones have been investigated. Amino acid-dependent selectivity for either trans- or cis-substituted piperazinone products has been achieved. An experimentally determined diastereoselectivity model for the three-component reaction driven by aziridine aldehyde dimers has predictive value for different substrate classes. Moreover, this model is useful in reconciling the previously reported observations in multicomponent reactions between isocyanides, α-amino acids, and monofunctional aldehydes.

Aromatic prenyltransferases are an actively mined enzymatic class whose biosynthetic repertoire is growing. Indole prenyltransferases catalyze the formation of a diverse set of prenylated tryptophan and diketopiperazines, leading to the formation of fungal toxins with prolific biological activities. At a fundamental level, the mechanism of C4-prenylation of l-tryptophan recently has surfaced to engage a debate between a "direct" electrophilic alkylation mechanism (for wt DMATS and FgaPT2) versus an indole C3-C4 "Cope" rearrangement followed by rearomatization (for mutant FgaPT2). Herein we provide the first series of regioselectively tunable conditions for a Cope rearrangement between C3 and C4 positions. Biomimetic conditions are reported that effect a [3,3]-sigmatropic shift whose two-step process is interrogated for intramolecularity and rate-limiting general base-promoted mechanism. Solvent polarity serves a crucial role in changing the regioselectivity, resulting in sole [1,3]-shifts under decalin. An intermolecular variant is also reported that effectively prenylates the C3 position of l-tryptophan, resulting in products that mimic the structures accessed by bacterial indole prenyltransferases. We report an elaborate investigation that includes screening various substituents and measuring steric and electronic effects and stereoselectivity with synthetically useful transformations.

Chemical analysis of an Australian marine sediment-derived Aspergillus sp. (CMB-M081F) yielded the new diketomorpholine (DKM) shornephine A (1) together with two known and one new diketopiperazine (DKP), 15b-β-hydroxy-5-N-acetyladreemin (2), 5-N-acetyladreemin (3), and 15b-β-methoxy-5-N-acetyladreemin (4), respectively. Structure elucidation of 1–4 was achieved by detailed spectroscopic analysis, supported by chemical degradation and derivatization, and biosynthetic considerations. The DKM (1) underwent a facile (auto) acid-mediated methanolysis to yield seco-shornephine A methyl ester (1a). Our mechanistic explanation of this transformation prompted us to demonstrate that the acid-labile and solvolytically unstable DKM scaffold can be stabilized by N-alkylation. Furthermore, we demonstrate that at 20 μM shornephine A (1) is a noncytotoxic inhibitor of P-glycoprotein-mediated drug efflux in multidrug-resistant human colon cancer cells.

International audience; Oxidative nucleophilic addition of ethylenediamine and guanidine derivatives to pyrrole-amino acid diketopiperazines was shown to provide substituted 5,6-dihydro-2(1H)-piperazinones, quinoxalinones, and 2-aminoimidazolones. On the basis of this methodology, a concise approach to natural products didebromohamacanthin A and demethylaplysinopsine has been demonstrated.

A general strategy for the conversion of [2.2.2]-diazabicyclic alkene structures to 2-pyridone aromatic heterocyclic products is reported. The reaction sequence starts from 2,5-diketopiperazine (DKP) derivatives, is compatible with both aromatic and aliphatic aldehyde components, and can intercept either intra- or intermolecular cycloaddition manifolds. Priming of one aza-bridging function in the intermediate [2.2.2]-DKP scaffold permits cycloreversion (microwave heating) and selective extrusion of cyanate derivatives leading to the formation of 2-pyridone structures. Progress toward the synthesis of louisianin A and B, antiproliferative 2-pyridone natural products, is also disclosed.

Three new compounds named leporizines A-C have been isolated from an Aspergillus sp. strain. Their structures were elucidated by analysis of 1D and 2D NMR spectra. Leporizines A and B were isolated during dereplication of hits from a high-throughput screening campaign for correctors of the cystic fibrosis transmembrane conductance regulator (CFTR), and leporizine C was isolated while preparing additional material for characterization of leporizines A and B. CFTR activity observed for leporizines A and B was highly correlated with cell toxicity and was determined to be a nonspecific effect. Leporizine C was not cytotoxic to cells and did not elicit a response in the CFTR assays. To the best of our knowledge, leporizines A-C represent the first examples of this unusual epithiodiketopiperazine skeleton.

We present a general approach to describe the structure-activity relationships (SAR) of combinatorial data sets with activity for two biological endpoints with emphasis on the rapid identification of substitutions that have a large impact on activity and selectivity. The approach uses Dual-Activity Difference (DAD) maps that represent a visual and quantitative analysis of all pairwise comparisons of one, two, or more substitutions around a molecular template. Scanning the SAR of data sets using DAD maps allows the visual and quantitative identification of activity switches defined as specific substitutions that have an opposite effect on the activity of the compounds against two targets. The approach also rapidly identifies single- and double-target R-cliffs, i.e., compounds where a single or double substitution around the central scaffold dramatically modifies the activity for one or two targets, respectively. The approach introduced in this report can be applied to any analogue series with two biological activity endpoints. To illustrate the approach, we discuss the SAR of 106 pyrrolidine bis-diketopiperazines tested against two formylpeptide receptors obtained from positional scanning deconvolution methods of mixture-based libraries.

An efficient approach for the synthesis of indole- and pyrrole-fused diketopiperazines has been developed. This protocol involves the Ugi four-component reaction (U-4CR) followed by an intramolecular cyclization of the Ugi products at room temperature to afford the desired products in good to excellent yields. In addition, it is interesting to report the subsequent regioselective ring-opening of diketopiperazine unit occurring via an intermolecular transamidation reaction under mild condition, resulting in the formation of highly functionalized indole-2-carboxamides and pyrrole-2-carboxamides.

An improved sulfenylation method for the preparation of epidithio-, epitetrathio- and bis-(methylthio)diketopiperazines from diketopiperazines has been developed. Employing NaHMDS and related bases and elemental sulfur or bis[bis(trimethylsilyl)amino]trisulfide (23) in THF, the developed method was applied to the synthesis of a series of natural and designed molecules, including epicoccin G (1), 8,8′-epi-ent-rostratin B (2), gliotoxin (3), gliotoxin G (4), emethallicin E (5) and haematocin (6). Biological screening of selected synthesized compounds led to the discovery of a number of nanomolar anti poliovirus agents (i.e. 46, 2,2′-epi-46 and 61, Table 5) and several low micromolar anti Plasmodium falciparum lead compounds (i.e. 46, 2,2′-epi-46, 58, 61 and 1, Table 5).

The histone acetyltransferase (HAT) activity of p300 is essential for androgen receptor (AR) function. Androgen-independent prostate cancer cells require AR-mediated transcriptional activation for their growth. These observations indicate that p300 HAT is a promising target to overcome such hormone-resistant cancer cells. We sought p300 HAT inhibitors among microbial metabolites. By culturing a production strain belonging to Penicillium, we identified two new compounds, NK13650A and NK13650B, which were obtained as specific p300 HAT inhibitors. Structural analyses of these compounds elucidated that NK13650s have novel chemical structures comprising several amino acids and citrate. We applied a newly developed biosynthesis-based method to reveal the absolute configuration at the citrate quaternary carbon. This was accomplished by feeding a (13)C-labeled biosynthetic precursor of citrate. NK13650s selectively inhibited the activity of p300 HAT but not that of Tip60 HAT. NK13650s showed inhibitory activity against agonist-induced AR transcriptional activation, and NK13650A treatment inhibited hormone-dependent and -independent growth of prostate cancer cells.

International audience; Studies of peptide fragment ion structures are important to aid in the accurate kinetic modeling and prediction of peptide fragmentation pathways for a given sequence. Peptide b(2)(+) ion structures have been of recent interest. While previously studied b(2)(+) ions that contain only aliphatic or simple aromatic residues are oxazolone structures, the HA b(2)(+) ion consists of both oxazolone and diketopiperazine structures. The structures of a series of histidine-analogue-containing Xxx-Ala b(2)(+) ions were studied by using action infrared multiphoton dissociation (IRMPD) spectroscopy, fragment ion hydrogen-deuterium exchange (HDX), and density functional theory (DFT) calculations to systematically probe the influence of different side chain structural elements on the resulting b(2)(+) ion structures formed. The b(2)(+) ions studied include His-Ala (HA), methylated histidine analogues, including π-methyl-HA and τ-methyl-HA, pyridylalanine (pa) analogues, including 2-(pa)A, 3-(pa)A, and 4-(pa)A, and linear analogues, including diaminobutanoic acid-Ala (DabA) and Lys-Ala (KA). The location and accessibility of the histidine π-nitrogen, or an amino nitrogen on an aliphatic side chain, were seen to be essential for diketopiperazine formation in addition to the more typical oxazolone structure formation, while blocking or removal of the τ-nitrogen did not change the b(2)(+) ion structures formed. Linear histidine analogues, DabA and KA, formed only diketopiperazine structures, suggesting that a steric interaction in the HisAla case may interfere with the complete trans-cis isomerization of the first amide bond that is necessary for diketopiperazine formation.

An efficient solution-phase synthesis of aza-diketopiperazines (aza-DKP, triazinediones) is reported. A structurally diverse collection of c-[aza-alkylGly-Pro] derivatives and yet unreported 2,4,5-trisubstituted-1,2,4-triazine-3,6-diones has been synthesized starting from Fmoc-l-Pro-OH and various Fmoc-l-amino acids. To extend the practical value of this class of dipeptidomimetics, a general solid-phase synthesis approach amenable to library production was developed on both Wang-PS and HMBA-PS resins. The final acidic treatment of the resins in TFA/water mixture at room temperature enabled the rapid and quantitative cyclization/release highly pure triazinediones. The conformational preferences and the spatial organization of the three substituents of a representative 2,4,5-trisubstituted-1,2,4-triazine-3,6-dione were investigated by X-ray diffraction and (1)H NMR spectroscopy.

Plinabulin (11, NPI-2358) is a potent microtubule-targeting agent derived from the natural diketopiperazine "phenylahistin" (1) with a colchicine-like tubulin depolymerization activity. Compound 11 was recently developed as VDA and is now under phase II clinical trials as an anticancer drug. To develop more potent antimicrotubule and cytotoxic derivatives based on the didehydro-DKP skeleton, we performed further modification on the tert-butyl or phenyl groups of 11, and evaluated their cytotoxic and tubulin-binding activities. In the SAR study, we developed more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively. The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable second-generation derivative with both vascular disrupting and cytotoxic activities.