2‐amino‐4‐oxo‐6‐substituted‐pyrrolo[2,3‐d]pynmidines as potential inhibitors of thymidylate synthase

Classical, antifolate inhibitors of thymidylate synthase often suffer from a number of potential disadvantages when used as antitumor agents. These include impaired uptake due to an alteration of the active transport system required for cellular uptake, as well as the formation of long acting, non‐effluxing polygluta‐mates via folypolyglutamate synthetase, which are responsible for toxicity to normal cells. To overcome some of the disadvantages of classical thymidylate synthase inhibitors, there has been considerable interest in the synthesis and evaluation of nonclassical inhibitors, which could enter cells via passive diffusion and are not substrates for folypolyglutamate synthetase. A series of eight nonclassical 6‐substituted 2‐amino‐4‐oxo‐pyrrolo[2,3‐d]pyrimidines 2a‐2h were designed as potential inhibitors of thymidylate synthase. The synthesis of the target compounds 2a‐2h was achieved via regioselective iodination at the 6‐position of 5 , palladium‐catalyzed coupling with the appropriate phenylacetylenes, reduction of the C8‐C9 triple bond followed by saponification. Preliminary biological results indicated that none of the target compounds showed inhibitory activities against thymidylate synthase from Escherichia coli, Lactobacillus casei, rat or human thymidylate synthase at the concentrations tested. None of the target compounds showed inhibitory activity against dihydrofolate reductase from Escherichia coli, Lactobacillus casei, rat or human at 3.0 × 10^?5 M. However, 50% inhibition of dihydrofolate reductase from Pneumocystis carinii and from Toxoplasma gondii was achieved with compound 2d and with compound 2g at 3.0 × 10^?5 M.     

A Dehydratase Domain in Ambruticin Biosynthesis Displays Additional Activity as a Pyran‐Forming Cyclase

Hydropyran rings are a common structural motif in reduced polyketides. Information on their biosynthetic formation and particularly the biochemical characterization of the responsible enzymes has only been reported in few cases. The dehydratase domain AmbDH3 from the ambruticin polyketide synthase was investigated. Through in vitro assay of the recombinant domain with synthetically‐derived substrate surrogates, it was shown that it has a second catalytic activity as a cyclase that performs oxa‐conjugate addition. Probing AmbDH3 with synthetic substrate analogues revealed stereoselectivity and substrate tolerance in both substeps. This is the first characterization of a pyran‐forming cyclase from a cis‐AT PKS system and the first report of a polyketide synthase domain with this kind of dual activity. Finally, it was revealed that this domain shows potential for application in chemoenzymatic synthesis.     

A novel preventive mechanism of gentamicin‐induced nephrotoxicity by atorvastatin

Atorvastatin (ATO) inhibits the synthesis of nonsteroidal isoprenoid compounds and possesses a pleiotropic effect. However, the detailed mechanism of ATO in preventing gentamicin (GM)‐induced renal injury remains obscure. Although underlying multifaceted mechanisms involving GM‐induced nephrotoxicity were well known, further work on elucidating the essential mechanism was needed. Using a fluorogenic derivatization–liquid chromatography tandem mass spectrometry proteomic method (FD‐LC–MS/MS method), we investigated the effects and mechanisms of ATO treatment on GM‐induced nephrotoxicity in rats. Consequently, 49 differentially expressed proteins were identified. The most significant mechanisms of nephrotoxicity caused by GM were mitochondrial dysfunction, fatty acid metabolism and oxidative stress. Their upstream regulator was found to be PPARα. The proteins involved in GM nephrotoxicity were sodium–hydrogen exchanger regulatory factor (SLC9A3R1), cathepsin V (CTSV), macrophage migration inhibitory factor (MIF) and RhoGDP dissociation inhibitor alpha (ARHGDIA). After ATO intervention, we observed a reversed enrichment pattern of their expression, especially in CTSV and SLC9A3R1 (P‐value<0.05). We predicted that ATO may improve abnormal phospholipid metabolism and phospholipidosis caused by GM and also alleviate cell volume homeostasis and reverse the interference of GM with the transporter. Furthermore, proteomic results also provided clues as to GM‐induced nephrotoxicity biomarkers such as CTSV and transthyretin.     

Insights into the Dual Activity of a Bifunctional Dehydratase‐Cyclase Domain

Oxygen‐containing heterocycles are a common structural motif in polyketide natural products and contribute significantly to their biological activity. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratase (DH) and pyran‐forming cyclase in ambruticin biosynthesis. AmbDH3 is similar to monofunctional DH domains, using H51 and D215 for dehydration. V173 was confirmed as a diagnostic residue for cyclization activity by a mutational study and enzymatic in vitro experiments. Similar motifs were observed in the seemingly monofunctional AmbDH2, which also shows an unexpected cyclase activity. Our results pave the way for mining of hidden cyclases in biosynthetic pathways. They also open interesting prospects for the generation of novel biocatalysts for chemoenzymatic synthesis and pyran‐polyketides by combinatorial biosynthesis.     

Acceptor Reactivity in the Total Synthesis of Alginate Fragments Containing α‐L‐Guluronic Acid and β‐D‐Mannuronic Acid

The total synthesis of mixed‐sequence alginate oligosaccharides, featuring both β‐D ‐mannuronic acid (M) and α‐L ‐guluronic acid (G), is reported for the first time. A set of GM, GMG, GMGM, GMGMG, GMGMGM, GMGMGMG, and GMGGMG alginates was assembled using GM building blocks, having a guluronic acid acceptor part and a mannuronic acid donor side to allow the fully stereoselective construction of the cis‐glycosidic linkages. It was found that the nature of the reducing‐end anomeric center, which is ten atoms away from the reacting alcohol group in the key disaccharide acceptor, had a tremendous effect on the efficiency with which the building blocks were united. This chiral center determines the overall shape of the acceptor and it is revealed that the conformational flexibility of the acceptor is an all‐important factor in determining the outcome of a glycosylation reaction.     

Synthesis of novel,nonclassical 2‐amino‐4‐oxo‐6‐(arylthio)ethylpyrrolo[2,3‐d] pyrimidines as potential inhibitors of thymidylate synthase

A novel series of 14 nonclassical 6‐substituted pyrrolo[2,3‐d]pyrimidines 2a ‐ 2n were designed as potential inhibitors of thymidylate synthase, based on previously reported 2‐amino‐4‐oxopyrrolo[2,3‐d]‐pyrimidines 1a and 1b . The synthesis of the target compounds 2a‐2n was accomplished by nucleophilic displacement of the mesylate 11 with appropriately substituted aromatic thiols. Most of the target compounds did not show inhibition of either Escherichia coli thymidylate synthase or recombinant human thymidylate synthase at the concentrations tested. However, compounds 2h (2,4‐dichloro), 2j (3,4‐dichloro) and 2m (4‐nitro) did show 25%, 40% and 35% inhibition of human thymidylate synthase at 23 μM, 23 μM and 24 μM, respectively. These observations are in accordance with previous reports, which suggest that strong electron withdrawing substituents on the side chain aromatic ring are conducive to inhibition of thymidylate synthase.     

An Improved Preparation of D‐Glyceraldehyde 3‐Phosphate and Its Use in the Synthesis of 1‐Deoxy‐D‐xylulose 5‐Phosphate

D ‐Glyceraldehyde 3‐phosphate (=D ‐GAP; 2 ) was prepared by an improved chemical method (Scheme 2), and it was then employed to synthesize 1‐deoxy‐D ‐xylulose 5‐phosphate (=DXP; 3 ) which is enzymatically one of the key intermediates in the MEP ( 4 ) terpenoid biosynthetic pathway (Scheme 1). The recombinant DXP synthase of Rhodobacter capsulatus was used to catalyze the condensation of D ‐glyceraldehyde 3‐phosphate ( 2 ) and pyruvate (=2‐oxopropanoate; 1 ) to produce the sugar phosphate 3 (Scheme 2). The simple two‐step chemoenzymatic route described affords DXP ( 3 ) with more than 70% overall yield and higher than 95% purity. The procedure may also be used for the synthesis of isotope‐labeled DXP ( 3 ) by using isotope‐labeled pyruvate.     

Synthesis,one‐ and two‐photon properties of poly[9,10‐bis(3,4‐bis(2‐ethylhexyl‐oxy)phenyl)‐2,6‐anthracenevinylene‐alt‐N‐octyl‐3,6‐/2,7‐carbazolevinylene]

The synthesis, one‐ and two‐photon absorption (TPA) and emission properties of two novel 2,6‐anthracenevinylene‐based copolymers, poly[9,10‐bis(3,4‐bis(2‐ethylhexyloxy)phenyl)‐2,6‐anthracenevinylene‐alt‐N‐octyl‐3,6‐carbazolevinyl‐ene] ( P1 ) and poly[9,10‐bis(3,4‐bis(2‐ethylhexyloxy)phenyl)‐2,6‐anthracenevinyl‐ene‐alt‐N‐octyl‐2,7‐carbazolevinylene] ( P2 ) were reported. The as‐synthesized polymers have the number‐average molecular weights of 1.56 × 10⁴ for P1 and 1.85 × 10⁴ g mol^?1 for P2 and are readily soluble in common organic solvents. They emit strong bluish‐green one‐ and two‐photon excitation fluorescence in dilute toluene solution (? _P1 = 0.85, ? _P2 = 0.78, λ_em( P1 ) = 491 nm, λ_em( P2 ) = 483 nm). The maximal TPA cross‐sections of P1 and P2 measured by the two‐photon‐induced fluorescence method using femtosecond laser pulses in toluene are 840 and 490 GM per repeating unit, respectively, which are obviously larger than that (210 GM) of poly[9,10‐bis‐(3,4‐bis(2‐ethylhexyloxy) phenyl)‐2,6‐anthracenevinylene], indicating that the poly(2,6‐anthracenevinylene) derivatives with large TPA cross‐sections can be obtained by inserting electron‐donating moieties into the polymer backbone. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 463–470, 2010     

A FRET Probe for Cell‐Based Imaging of Ganglioside‐Processing Enzyme Activity and High‐Throughput Screening

Gangliosides are important signaling molecules in the cell membrane and are processed by several enzymes. Deficiencies in these enzymes can cause human lysosomal storage diseases. Building an understanding of the pathways of glycosphingolipid catabolism requires methods for the analysis of these enzymatic activities A GM3‐derived FRET probe was synthesized chemoenzymatically for the detection and quantitation of a range of ganglioside‐degrading enzymes, both in cell lysates and in living cells. This is the first substrate that enables the ratiometric fluorogenic assay of sphingolipid ceramide N‐deacylase and endoglycoceramidase and can detect and localize neuraminidase activity in living cells. It is therefore a valuable tool for building a better understanding of membrane‐confined enzymology. It also enables the robust and reliable assay of ganglioside‐degrading enzymes in a microtiter plate, thus opening the door to screening for novel or engineered biocatalysts or for new inhibitors.     

The Synthesis and Biological Evaluation of N‐Substituted 1H‐Benzimidazol‐2‐yl‐1H‐pyrazole‐3,5‐diamines

The synthesis of 1H‐benzimidazol‐2‐yl‐1H‐pyrazole‐3,5‐diamines has been developed. Synthesized bisheteroaryls contain two privileged medicinal scaffolds, aminopyrazole and benzimidazole, with two diversity positions at N1 of benzimidazole and C3 of pyrazole, respectively. The three‐step synthesis includes the Mitsunobu N‐alkylation of benzimidazole and subsequent one‐pot formation of aminopyrazole involving substitution of methylthio groups with amine and hydrazine followed with final ring closure. Inhibitory activity toward cyclin‐dependent kinase 2/cyclin E and cytotoxicity against two cancer cell lines were evaluated for all novel pyrazoles. Two compounds showed modest cyclin‐dependent kinase inhibition activity and cytotoxicity against cancer cell lines K562 and MCF7.     

Discovery of a Novel Mycobacterial F‐ATP Synthase Inhibitor and its Potency in Combination with Diarylquinolines

The F₁F_O‐ATP synthase is required for growth and viability of Mycobacterium tuberculosis and is a validated clinical target. A mycobacterium‐specific loop of the enzyme's rotary γ subunit plays a role in the coupling of ATP synthesis within the enzyme complex. We report the discovery of a novel antimycobacterial, termed GaMF1, that targets this γ subunit loop. Biochemical and NMR studies show that GaMF1 inhibits ATP synthase activity by binding to the loop. GaMF1 is bactericidal and is active against multidrug‐ as well as bedaquiline‐resistant strains. Chemistry efforts on the scaffold revealed a dynamic structure activity relationship and delivered analogues with nanomolar potencies. Combining GaMF1 with bedaquiline or novel diarylquinoline analogues showed potentiation without inducing genotoxicity or phenotypic changes in a human embryonic stem cell reporter assay. These results suggest that GaMF1 presents an attractive lead for the discovery of a novel class of anti‐tuberculosis F‐ATP synthase inhibitors.     

Alternating copolymers of 2,6‐bis(p‐dihexylaminostyryl)anthracene‐9,10‐diyl and N‐octylcarbazole with large two‐photon cross sections

We report the synthesis, thermal, one‐ and two‐photon properties of poly(2,6‐bis(p‐dihexylaminostyryl)anthracene‐9,10‐diyl‐alt‐N‐octylcarbazole‐3,6‐/2,7‐diyl) ( P1/P2 ). The as‐synthesized polymers exhibit number‐average molecular weights of 1.7 × 10⁴ for P1 and 2.1 × 10⁴ g/mol for P2 . They emit strong one‐ and two‐photon excitation fluorescence with the peak around 502 nm, and the fluorescence quantum yields around 0.76 in chloroform. In film state, P1 and P2 show different red‐shift emission with the peaks at 512 nm and 523 nm, respectively. The DSC measurement reveals that as‐synthesized polymers are all amorphous aggregates with the glass transition temperatures of 131 °C for P1 and 152 °C for P2 . The solution two‐photon absorption (TPA) properties of P1 and P2 in chloroform are measured by the two‐photon‐induced fluorescence method using femtosecond laser pulses (120 fs). The TPA cross sections (δ) are measured over the range of 700–900 nm. The maximal δ of P1 and P2 all appear at ～800 nm and are 1010 GM and 940 GM per repeating unit, respectively. This suggests that no notable interactions among structure units that impair their fluorescence and TPA properties, and the polymers with large δ can be obtained by using the high TPA‐active units as building blocks. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Two‐Photon STED Spectral Determination for a New V‐Shaped Organic Fluorescent Probe with Efficient Two‐Photon Absorption

Two‐photon stimulated emission depletion (STED) cross sections were determined over a broad spectral range for a novel two‐photon absorbing organic molecule, representing the first such report. The synthesis, comprehensive linear photophysical, two‐photon absorption (2PA), and stimulated emission properties of a new fluorene‐based compound, (E)‐2‐{3‐[2‐(7‐(diphenylamino)‐9,9‐diethyl‐9H‐fluoren‐2‐yl)vinyl]‐5‐methyl‐4‐oxocyclohexa‐2,5‐dienylidene} malononitrile ( 1 ), are presented. Linear spectral parameters, including excitation anisotropy and fluorescence lifetimes, were obtained over a broad range of organic solvents at room temperature. The degenerate two‐photon absorption (2PA) spectrum of 1 was determined with a combination of the direct open‐aperture Z‐scan and relative two‐photon‐induced fluorescence methods using 1 kHz femtosecond excitation. The maximum value of the 2PA cross section ～1700 GM was observed in the main, long wavelength, one‐photon absorption band. One‐ and two‐photon stimulated emission spectra of 1 were obtained over a broad spectral range using a femtosecond pump–probe technique, resulting in relatively high two‐photon stimulated emission depletion cross sections (～1200 GM). A potential application of 1 in bioimaging was demonstrated through one‐ and two‐photon fluorescence microscopy images of HCT 116 cells incubated with micelle‐encapsulated dye.     

Difluorenyl carbo‐Benzenes: Synthesis,Electronic Structure,and Two‐Photon Absorption Properties of Hydrocarbon Quadrupolar Chromophores

The synthesis, crystal and electronic structures, and one‐ and two‐photon absorption properties of two quadrupolar fluorenyl‐substituted tetraphenyl carbo‐benzenes are described. These all‐hydrocarbon chromophores, differing in the nature of the linkers between the fluorenyl substituents and the carbo‐benzene core (C?C bonds for 3 a , C?C?C?C expanders for 3 b ), exhibit quasi–superimposable one‐photon absorption (1PA) spectra but different two‐photon absorption (2PA) cross‐sections σ_2PA. Z‐scan measurements (under NIR femtosecond excitation) indeed showed that the C?C expansion results in an approximately twofold increase in the σ_2PA value, from 336 to 656 GM (1 GM=10^?50 cm⁴ s molecule^?1 photon^?1) at λ=800 nm. The first excited states of A_u and A_g symmetry accounting for 1PA and 2PA, respectively, were calculated at the TDDFT level of theory and used for sum‐over‐state estimations of σ_2PA(λ_i), in which λ_i=2 hc/E_i, h is Planck’s constant, c is the speed of light, and E_i is the energy of the 2PA‐allowed transition. The calculated σ_2PA values of 227 GM at 687 nm for 3 a and 349 GM at 708 nm for 3 b are in agreement with the Z‐scan results.     

Polymer‐Assisted Solution‐Phase Synthesis and Neurite‐Outgrowth‐Promoting Activity of 15‐Deoxy‐Δ12,14‐PGJ2 Derivatives

An efficient solution‐phase synthesis of rac‐15‐deoxy‐Δ^12,14‐PGJ₂ (15dPGJ₂) derivatives that contain variable α and ω chains based on a polymer‐assisted strategy and their neurite‐outgrowth‐promoting activity are described. The strategy for the synthesis of PGJ₂ derivatives involves the use of a vinyl iodide bearing cyclopentenone as a key intermediate, which undergoes Suzuki–Miyaura coupling and subsequent Lewis acid catalyzed aldol condensation for incorporation of the ω and α chains, respectively. For easy access to the PGJ₂ derivatives, a polymer‐supported catalyst and scavengers were adapted for use in these four diverse steps, in which workup and purification can be performed by simple filtration of the solid‐supported reagents. By using this methodology, we succeeded in the synthesis of 16 PGJ₂ derivatives with four alkyl boranes and four aldehydes. The neurite‐outgrowth‐promoting activity of the 16 synthetic compounds in PC12 cells revealed that the side‐chains play a major role in modulating their biological activity. The carboxylic acid on the α chain improved the biological activity, although it was not absolutely required. Furthermore, a PGJ₂ derivative with a phenyl moiety on the ω chain was found to exhibit an activity comparable to that of natural 15dPGJ₂.     

Concurrent Asymmetric Reactions Combining Photocatalysis and Enzyme Catalysis: Direct Enantioselective Synthesis of 2,2‐Disubstituted Indol‐3‐ones from 2‐Arylindoles

The combination of photoredox and enzymatic catalysis for the direct asymmetric one‐pot synthesis of 2,2‐disubstituted indol‐3‐ones from 2‐arylindoles through concurrent oxidization and alkylation reactions is described. 2‐Arylindoles can be photocatalytically oxidized to 2‐arylindol‐3‐one with subsequent enantioselective alkylation with ketones catalyzed by wheat germ lipase (WGL). The chiral quaternary carbon center at C2 of the indoles was directly constructed. This mode of concurrent photobiocatalysis provides a mild and powerful strategy for one‐pot enantioselective synthesis of complex compounds. The experiments proved that other lipases containing structurally analogous catalytic triad in the active site also can catalyze the reaction in the same way. This reaction is the first example of combining the non‐natural catalytic activity of hydrolases with visible‐light catalysis for enantioselective organic synthesis and it does not require any cofactors.     

Synthesis of a Monophosphoryl Derivative of Escherichia coli Lipid A and Its Efficient Coupling to a Tumor‐Associated Carbohydrate Antigen

Monophosphoryl lipid A is a safe and potent immunostimulant and vaccine adjuvant, which is potentially useful for the development of effective carbohydrate‐based conjugate vaccines. This paper presents a convergent and efficient synthesis of a monophosphoryl derivative of E. coli lipid A that has an alkyne functionality at the reducing end, which is suitable for coupling with various molecules. The coupling of this derivative to an N‐modified analogue of tumor‐associated antigen GM3 through click chemistry is also presented.     

Synthesis and Antimicrobial Activity of N‐Aryl‐4‐(cyano/alkoxycarbonyl)‐5‐(pyridin‐3‐yl)‐1H/3H‐1,2,3‐triazole Derivatives

A convenient synthesis of a new series of N‐aryl‐5‐(pyridin‐3‐yl)‐1H/3H‐1,2,3‐triazole‐4‐carbonitriles and alkyl N‐aryl‐5‐(pyridin‐3‐yl)‐1H/3H‐1,2,3‐triazole‐4‐carboxylic acid esters is reported. The newly synthesized 5‐(pyridin‐3‐yl)‐1,2,3‐triazole derivatives are evaluated for their antibacterial and antifungal activity. Some of these triazole derivatives have exhibited moderate antimicrobial activity.     

Cu‐free Sonogashira Type Cross‐Coupling of 6‐Halo‐2‐cyclopropyl‐3‐(pyridyl‐3‐ylmethyl) Quinazolin‐4(3H)‐ones as Potential Antimicrobial Agents

C(sp)–C(sp2) bond formation via Sonogashira cross‐coupling reactions on 6‐halo‐2‐cyclopropyl‐3‐(pyridyl‐3‐ylmethyl)quinazolin‐4(3H )‐ones with appropriate alkynes was explored. Optimization of reaction conditions with various catalysts, ligands, bases, and solvents was conducted. The combination of PdCl₂(MeCN)₂ with X‐Phos proved to be the best metal–ligand system for this conversion in the presence of triethylamine (Et₃N) in tetrahydrofuran at room temperature for iodosubstrates, at 80°C for the bromosubstrates in 8 h, and also for the chlorosubstrates in 16 h. We also demonstrated synthesis of a successful diversity‐oriented synthesis library of highly functionalized quinazolinones via Cu‐free Sonogashira coupling of diverse aryl halides and azido‐alkyne (“click”) ligation reactions with substituted azides. The library exhibited significant antimicrobial activity when screened against several microorganisms.     

Polyoxometalate immobilized on MOF‐5 as an environment‐friendly catalyst for the synthesis of poly‐functionalized 3‐pyrrolin‐2‐ones

A polyoxometalate immobilized on MOF‐5 (POM/MOF‐5) material has been synthesized and evaluated for the diversity‐oriented synthesis of poly‐functionalized 3‐pyrrolin‐2‐ones via pseudo‐four‐component reaction between dialkyl acetylenedicarboxylate, amines, and aldehyde. The catalyst can be separated from the reaction mixture and reused at least five times with superior activity.