Diversity of Alkylproline Moieties in Pyrrolobenzodiazepines Arises from Postcondensation Modifications of a Unified Building Block.

Anticancer pyrrolobenzodiazepines (PBDs) are one of several groups of natural products that contain unusual 4-alkyl-l-proline derivatives (APDs) in their structure. APD moieties of PBDs are characterized by high structural diversity achieved through unknown biosynthetic machinery. Based on LC-MS analysis of culture broths, feeding experiments, and protein assays, we show that APDs are not incorporated into PBDs in their final form as was previously hypothesized. Instead, a uniform building block, 4-propylidene-l-proline or 4-ethylidene-l-proline, enters the condensation reaction. The subsequent postcondensation steps are initiated by the introduction of an additional double bond catalyzed by a FAD-dependent oxidoreductase, which we demonstrated with Orf7 from anthramycin biosynthesis. The resulting double bond arrangement presumably represents a prerequisite for further modifications of the APD moieties. Our study gives general insight into the diversification of APD moieties of natural PBDs and provides proof-of-principle for precursor directed and combinatorial biosynthesis of new PBD-based antitumor compounds.

The Effects of Class-Specific Histone Deacetylase Inhibitors on the Development of Limbs During Organogenesis.

Histone deacetylases (HDACs) play a major role in chromatin remodeling, gene regulation, and cellular signaling. While the role of each class of HDAC during normal development is unclear, several HDAC inhibitors are embryotoxic; the mechanisms leading to the teratogenicity of HDAC inhibitors are not known. Here, we investigated the effects of class-specific HDAC inhibitors on the development of organogenesis-stage murine limbs. Timed-pregnant COL2A1-ECFP, COL10A1-mCherry, and COL1A1-YFP CD1 reporter mice were euthanized on gestation day 12; embryonic forelimbs were excised and cultured in vitro for 1, 3, and 6 days in the presence or absence of MS275 (a class I HDAC inhibitor), MC1568 (a class III HDAC inhibitor), Sirtinol (a class II HDAC inhibitor), or valproic acid, our positive control. Fluorescently tagged COL2A1, COL10A1, and COL1A1 served as markers of the differentiation of proliferative chondrocytes, hypertrophic chondrocytes, and osteoblasts, respectively. MS275 and valproic acid caused a reduction in expression of all three markers, suggesting effects on both chondrogenesis and osteogenesis. MC1568 had no effect on chondrocyte markers and mildly inhibited COL1A1 expression at 6 days. Sirtinol had no effect on COL2A1 expression or chondrocyte differentiation 1 day following exposure; however, it caused a drastic regression in limb cartilage and reduced the expression of all three differentiation markers to nearly undetectable levels at 6 days. MS275 and Sirtinol caused a 2.2- and 2.7-fold increase, respectively, in cleaved-caspase 3, a marker of apoptosis, suggesting embryotoxicity. These data demonstrate that inhibition of class I or III HDACs causes severe developmental toxicity and is highly teratogenic.

Tetrahydropyrrolo[3,2-c]azepin-4-ones as a new class of cytotoxic compounds.

Pyrroloazepinones 8a-j and 9a-j were designed by structural modification of lead compound 3. These compounds were tested on five tumor cell lines to determine the role of the azeto ring and the 2-methyl substituent in the cytotoxicity of compound 3. Our results show that compounds 8a-j (R1=CH3) have dramatically reduced cytotoxicity, resulting from the loss of the azeto moiety of lead compound 3. By contrast, azepinones 9a-j (R1=4-nitrophenyl) inhibited the proliferation of almost all cancer cell lines tested even though they lack the azeto ring. Preliminary SAR studies with these compounds revealed the importance of halogens at the para- or meta-position of the 1-phenyl moiety. Additionally, derivatives 9a (R2=H), 9e (R2=4-F), and 9g (R2=4-OMe) were selectively cytotoxic to U-251 cells. However, none of the pyrroloazepinones inhibited the enzymatic activity of CDK1/cyclin B, CDK5/p25, and GSK-3.

Permeability characteristics of novel aldose reductase inhibitors using rat jejunum in vitro.

The aim of this study was to estimate in vivo permeability and bioavailability of epalrestat and newly synthesized compounds with possible therapeutic activity as aldose enzyme inhibitors (ARIs). For this purpose permeability in vitro using rat jejunum mounted in side-by-side diffusion cells was determined. Tested substances were found to be low and moderately permeable and some of them were also substrates for efflux transporters. It was shown, that the higher efflux for some derivatives was due to MRP-2, but not Pgp involvement. Tested ARIs do not share the same efflux transporter with epalrestat, the only ARI currently on the market in Japan. The most permeable compound, a 2,6-difluoro-4-pyrrol-1ylphenol derivative, is not a substrate for efflux transporters and would therefore be the most promising lead compound for further investigation of potent ARIs.

Antibacterial activity of (-)-deoxypseudophrynaminol versus its racemate and derivatives.

(-)-Deoxypseudophrynaminol 1 possesses 43-fold greater antibacterial potency than the racemate toward Staphylococcus aureus, indicating that the (-)-enantiomer is the biologically active isomer in this assay. Comparison of the percent growth inhibition by derivatives of 1 indicates that prenylation of N8 and replacement of N1-methyl by methyl carbamate are detrimental to antibacterial potency. (-)-1 is a promising lead structure for the development of the novel hexahydropyrrolo[2,3-b]indole class of antibacterial agents.

Different types of hydrogen bonds in 2-substituted pyrroles and 1-vinyl pyrroles as monitored by (1)H, (13)C and (15)N NMR spectroscopy and ab initio calculations.

According to the (1)H, (13)C and (15)N NMR spectroscopic data and ab initio calculations, the strong N--H...O intramolecular hydrogen bond in the Z-isomers of 2-(2-acylethenyl)pyrroles causes the decrease in the absolute size of the (1)J(N,H) coupling constant by 2 Hz in CDCl(3) and by 4.5 Hz in DMSO-d(6), the deshielding of the proton and nitrogen by 5-6 and 15 ppm, respectively, and the lengthening of the N--H link by 0.025 A. The N--H...N intramolecular hydrogen bond in the 2(2'-pyridyl)pyrrole leads to the increase of the (1)J(N,H) coupling constant by 3 Hz, the deshielding of the proton by 1.5 ppm and the lengthening of the N--H link by 0.004 A. The C--H...N intramolecular hydrogen bond in the 1-vinyl-2-(2'-pyridyl)-pyrrole results in the increase of the (1)J(C,H) coupling constant by 5 Hz, the deshielding of the proton by 1 ppm and the shortening of the C--H link by 0.003 A. Different behavior of the coupling constants and length of the covalent links under the hydrogen bond influence originate from the different nature of the hydrogen bonding (predominantly covalent or electrostatic), which depends in turn on the geometry of the hydrogen bridge. The Fermi-contact mechanism only is responsible for the increase of the coupling constant in the case of the predominantly electrostatic hydrogen bonding, whereas both Fermi-contact and paramagnetic spin-orbital mechanisms bring about the decrease of coupling constant in the case of the predominantly covalent hydrogen bonding.

Phenylpyrroles, a new chemolibrary virtual screening class of 5-HT7 receptor ligands.

Virtual screening studies have identified a series of phenylpyrroles as novel 5-HT7 receptor ligands. The synthesis and the affinity for the 5-HT7 receptor of these phenylpyrroles are described. Some of these compounds exhibited high affinity for the 5-HT7 receptors.

Pyrrolo[1,2-a][1,4]benzodiazepine: a novel class of non-azole anti-dermatophyte anti-fungal agents.

Broad screening revealed compound 1a to be a novel anti-fungal agent with high specificity towards dermatophytes. The anti-fungal structure-activity relationship of this novel class of 5,6-dihydro-4H-pyrrolo[1,2-a][1,4]benzodiazepines is described together with its mode of action that appeared to be the inhibition of squalene epoxidase. Preliminary in vivo results of the most active compounds are also reported.

Mass spectral study of meso-alkyl and meso-cycloalkyl calix(4)pyrroles under electron impact conditions.

A series of meso-cycloalkyl calix(4)pyrroles (I) and meso-dialkyl calix(4)pyrroles (II) has been studied under electron ionization (EI) mass spectral conditions. All the calix(4)pyrroles showed prominent molecular ions. The cleavage of the C--C bond linked at position 2 of the pyrrole ring (beta-cleavage) is the foremost and dominant fragmentation process. The beta-cleavage process, either through ring opening or directly, results in the loss of an alkyl radical from the molecular ion. The collision-induced dissociation (CID) spectra of I showed specific sequential expulsion of pyrrole and/or cycloalkyl rings from the molecular ion with or without hydrogen migrations, revealing more information on the structure of individual compounds than was available from the EI spectra. The isomeric cycloalkyl calix(4)pyrroles showed distinguishable CID spectra, indicating structure specificity in initial ring opening whereas, in the case of II, the EI mass spectrum contains all the structure-indicative fragment ions. The CID spectra of II resulted in a dominant [M-R]+ ion, with other characteristic ions being less abundant.

Class-specific pro-apoptotic effect of statins on human vascular endothelial cells.

Neonangiogenesis represents an important step in tumor development and propagation. Statins may have anticancerogenic potential by blocking vascular endothelial cell growth. The antiproliferative effect of four statins on human endothelial cells was compared, concomitantly delineating a possible pro-apoptotic process. All four statins tested, i. e. atorvastatin, fluvastatin, lovastatin, and simvastatin inhibited cell proliferation. Nearly complete blocking of cell proliferation was achieved at a concentration of 10 microM. We were able to demonstrate that the antiproliferative effect of the statins is not due to cytotoxicity but rather to an apoptotic effect as demonstrated by comparison of cytotoxicity assay and apoptosis assay. The apoptotic mechanism seems to involve caspases, since the statins significantly enhanced caspase activity at dosages of 10 and 20 microM. Further experiments revealed a downregulation of the pro-apoptotic protein Bcl-2. Our data indicate that statins may class-specific inhibit angiogenesis at high dosages which can contribute to prevention of tumor development and progression.