A new species in the Simulium vernum group from the Northern Limestone Alps of Austria: Simulium hasekei (Diptera: Simuliidae).

Simulium hasekei new species is described from the Alps of Austria. It is characterized by a broad trapezoidal ventral plate in the male, long straight arms of the genital fork in the female, a well-developed anterodorsal projection on the weakly woven cocoon, and a deep rounded postgenal cleft and strong paralateral hypostomal teeth in the larva. The species is chromosomally most similar to the Holarctic S. bicorne Dorogostaisky, Rubtsov Vlasenko and S. fontinale Radzivilovskaya and the Nearctic S. craigi Adler Currie. It is known from one small ponor brook, in association with S. aureum (Fries) s. s., S. beltukovae (Rubtsov), and Simulium vernum Macquart s. s.

A new species in the Simulium vernum group from the Alps of southern Germany: Simulium arminii (Diptera: Simuliidae).

Simulium arminii new species is described from the Alps of southern Germany. It is characterized by a broad trapezoidal ventral plate in the male, long slender arms of the genital fork in the female, a weakly developed or absent anterodorsal projection on the thinly woven cocoon, and a long median hypostomal tooth in the larva. The species is chromosomally similar to Simulium beltukovae (Rubtsov) but differs most conspicuously by the absence of a chromocenter. It is known from two small, springfed streams above the timber line, in association with Prosimulium latimucro (Enderlein) and Simulium bavaricum Seitz and Adler.

Genetic Diversity and Identification of Palearctic Black Flies in the Subgenus Wilhelmia (Diptera: Simuliidae).

Accurate species identifications are the essential first step in understanding the medical, economic, and ecological importance of black flies. The utility of DNA barcoding based on cytochrome c oxidase subunit 1 (COI) sequences was evaluated for identifying six common species of Palearctic black flies in the subgenus Wilhelmia, including several that are virulent pests. Chromosomally identified larvae from Turkey and Germany and COI sequences in GenBank were analyzed. Intraspecific genetic divergence was 0.7-3.5% (mean 1.6%), whereas interspecific genetic divergence was 2.7-16.9%. On the basis of COI barcodes, the six nominal species of Simulium (Wilhelmia) were clustered in three distinct clades with high levels of genetic divergence, using maximum likelihood and Bayesian analyses. All specimens of Simulium equinum (L.), Simulium pseudequinum Séguy, and Simulium paraequinum Puri were correctly identified. However, >75% of identifications were ambiguous for Simulium lineatum (Meigen) and Simulium turgaicum Rubtsov (Meigen) because of overlapping intra- and interspecific divergence of the two species and Simulium balcanicum (Enderlein), all three of which are chromosomally similar and nearly isomorphic. Phylogenetic evaluation showed that S. balcanicum, S. equinum, S. pseudequinum, and S. paraequinum were monophyletic, with high bootstrap and posterior probability values, but it also showed that S. lineatum and S. turgaicum were paraphyletic, each clustering in two distinct groups, suggesting the presence of cryptic taxa. Although DNA barcoding provided a partial means of identification and indications of additional biodiversity, other molecular markers are needed to clarify the limits of all pest species of the subgenus Wilhelmia.

Cryptic Biodiversity and the Origins of Pest Status Revealed in the Macrogenome of Simulium colombaschense (Diptera: Simuliidae), History's Most Destructive Black Fly.

The European black fly Simulium (Simulium) colombaschense (Scopoli), once responsible for as many as 22,000 livestock deaths per year, is chromosomally mapped, permitting its evolutionary relationships and pest drivers to be inferred. The species is 12 fixed inversions removed from the standard sequence of the subgenus Simulium. Three of these fixed inversions, 38 autosomal polymorphisms, and a complex set of 12 X and 6 Y chromosomes in 29 zygotic combinations uniquely characterize S. colombaschense and reveal 5 cytoforms: 'A' in the Danube watershed, 'B' in Italy's Adige River, 'C' in the Aliakmonas River of Greece, 'D' in the Aoös drainage in Greece, and 'E' in the Belá River of Slovakia. 'C' and 'D' are reproductively isolated from one another, and 'B' is considered a cytotype of 'A,' the probable name bearer of colombaschense. The species status of 'E' cannot be determined without additional collections. Three derived polytene sequences, based on outgroup comparisons, place S. colombaschense in a clade of species composed of the S. jenningsi, S. malyschevi, and S. reptans species groups. Only cytoforms 'A' and 'B' are pests. Within the Simuliidae, pest status is reached through one of two principal pathways, both of which promote the production of large populations of blood-seeking flies: (1) colonization of the world's largest rivers (habitat specialization) or (2) colonization of multiple habitat types (habitat generalization). Evolutionary acquisition of the ability to colonize large rivers by an ancestor of the S. jenningsi-malyschevi-reptans clade set the scene for the pest status of S. colombaschense and other big-river members of the clade. In an ironic twist, the macrogenome of S. colombaschense reveals that the name associated with history's worst simuliid pest represents a complex of species, two or more of which are nonpests potentially vulnerable to loss of their limited habitat.

Chromosomal relationships of Simulium armoricanum and its undescribed sister species in the Simulium vernum species group (Diptera: Simuliidae).

The banding sequence of the polytene chromosomes of Simulium armoricanum Doby & David from England, Portugal, and Spain was resolved relative to the standard map for the S. vernum group. The species is characterized by 11 fixed inversions, one nearly fixed inversion, and three common polymorphisms. The sister species of S. armoricanum is proposed as a formally undescribed species discovered in samples from Portugal. It shares one unique inversion with S. armoricanum, but otherwise differs by eight fixed or nearly fixed rearrangements. Simulium armoricanum and its newly discovered sister species, informally referred to as Simulium 'IL-8', are members of a larger clade of Palearctic species defined by a small pericentric inversion in chromosome III. Among the simuliid species occupying the same streams with S. armoricanum was the first record, chromosomally confirmed, of S. aureum Fries sensu stricto in Portugal. Successful chromosomal analysis of samples of S. armoricanum 17 years after initial fixation demonstrates the importance of storing cytologically fixed larvae at subzero temperatures.

A new species, Simulium (Nevermannia) berchtesgadense (Diptera: Simuliidae), and its chromosomes, from the Alps of southeastern Germany.

Simulium (Nevermannia) berchtesgadense nov. spec. is described from the Alps of southeastern Germany. The morphology and diagnostic characters for all life stages except the egg are given, and the polytene chromosomes are compared with those of other members of the Simulium (Nevermannia) vernum group. The species is chromosomally similar to Simulium (Nevermannia) cryophilum cytoform 'A' but differs morphologically in each life stage. Bionomic information and the associated simuliid fauna are presented.

3D QSAR analyses-guided rational design of novel ligands for the (alpha4)2(beta2)3 nicotinic acetylcholine receptor.

Three-dimensional quantitative structure-activity relationship methods, the comparative molecular field analysis (CoMFA) and the comparative molecular similarity indices analysis (CoMSIA), were applied using a training set of 45 ligands of the (alpha4)2(beta2)3 nicotinic acetylcholine receptor (nAChR). All compounds are related to (-)-epibatidine, (-)-cytisine, (+)-anatoxin-a, and (-)-ferruginine, and additionally, novel diazabicyclo[4.2.1]nonane- and quinuclidin-2-ene-based structures were included. Their biological data have been determined by utilizing the same experimental protocol. Statistically reliable models of good predictive power (CoMFA r2 = 0.928, q2 = 0.692, no. of components = 3; CoMSIA r2 = 0.899, q2 = 0.701, no. of components = 3) were achieved. The results obtained were graphically interpreted in terms of field contribution maps. Hence, physicochemical determinants of binding, such as steric and electrostatic and, for the first time, hydrophobic, hydrogen bond donor, and hydrogen bond acceptor properties, were mapped back onto the molecular structures of a set of nAChR modulators. In particular, changes in the binding affinity of the modulators as a result of modifications in the aromatic ring systems could be rationalized by the steric, electrostatic, hydrophobic, and hydrogen bond acceptor properties. These results were used to guide the rational design of new nAChR ligands such as 48-52 and 54, which were subsequently synthesized for the first time and tested. Key steps of our synthetic approaches were successfully applied Stille and Suzuki cross-coupling reactions. Predictive r2 values of 0.614 and 0.660 for CoMFA and CoMSIA, respectively, obtained for 22 in part previously unknown ligands for the (alpha4)2(beta2)3 subtype, demonstrate the high quality of the 3D QSAR models.

Synthesis and nicotinic binding studies on enantiopure diazine analogues of the novel (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene UB-165.

As part of our program aimed at optimizing therapeutic effects over toxic effects (as observed in the naturally occurring nicotinic acetylcholine receptor modulators (-)-nicotine, (-)-epibatidine, (-)-ferruginine, and (+)-anatoxin-a), we investigated the bioisosteric potential of diazines in the field of (+)-anatoxin-a-type structures. In the series of diazine analogues of deschloro-UB-165 (DUB-165, 6), bioisosteric replacement of the 3-pyridyl pharmacophoric element by a 4-pyridazinyl, 5-pyrimidinyl, or 2-pyrazinyl moiety resulted in novel nAChR ligands 7, 8, and 9. A palladium-catalyzed Suzuki cross-coupling of the 3-diethylboranylpyridine (14) and a Stille cross-coupling of the corresponding tributylstannyl diazines 15-17 with the vinyl triflate 13 of the N-protected 9-azabicyclo[4.2.1]nonan-2-one 12 constitute the key steps in the syntheses of these enantiopure anatoxinoids 6-9. Studies of the in vitro affinity for (alpha4)(2)(beta2)(3), alpha3(beta)4, and alpha7 nAChR subtypes by radioligand binding assays demonstrated that the diazine analogues 7-9 can be considered as pharmacologically attractive bioisosteres of DUB-165 (6) but with different effects on the binding affinity with regard to the diazine moiety. The pyrimidine-containing bioisostere 8 turned out to be the most active diazine analogue, which interacts potently (K(i) = 0.14 nM) with the (alpha4)(2)(beta2)(3) subtype and differentiates significantly among the nAChR subtypes investigated. The nitrogens in this anatoxinoid 8 show by far the most negative atomic charges (calculated using the AM1 Hamiltonian). This qualitatively correlates with the highest binding affinity observed for 8 for all subtypes under consideration.

Ferro- and Antiferromagnetic Coupling through 1,n-Dithiosquarate Bridges (n = 2, 3) in Copper(II) Dimers. Syntheses, Crystal Structures, and Magnetic Properties of [Cu(2)(bpca)(2)(1,2-dtsq)(H(2)O)].2H(2)O and [Cu(2)(bpca)(2)(1,3-dtsq)].2H(2)O.

The preparation, crystal structures, and variable-temperature magnetic susceptibility data for two dithiosquarate-bridged copper(II) complexes of formulas [Cu(2)(bpca)(2)(1,2-dtsq)(H(2)O)].2H(2)O (1) and [Cu(2)(bpca)(2)(1,3-dtsq)].2H(2)O (2) (where bpca = the bis(2-pyridylcarbonyl)amide anion, 1,2-dtsq = dianion of 3,4-dimercapto-3-cyclobutene-1,2-dione, and 1,3-dtsq = dianion of 3-hydroxy-4-mercapto-2-thioxo-3-cyclobuten-1-one) are reported herein. 1 and 2 crystallize in the triclinic system, space group P&onemacr;, with a = 9.3243(4) Å, b = 10.9036(5) Å, c = 15.5432(4) Å, alpha = 90.276(2) degrees, beta = 100.009(2) degrees, gamma = 94.139(2) degrees, and Z = 2 for 1 and a = 7.656(2) Å, b = 9.351(3) Å, c = 11.587(2) Å, alpha = 109.02(2) degrees, beta = 97.40(2) degrees, gamma = 110.27(2) degrees, and Z = 1 for 2. Their structures consist of the neutral dithiosquarate-bridged copper(II) units [Cu(2)(bpca)(2)(1,2-dtsq)(H(2)O)] (1) and [Cu(2)(bpca)(2)(1,3-dtsq)] (2) and water molecules of crystallization. The copper environment is distorted square pyramidal CuN(3)SO in both compounds. Bpca acts as a tridentate end-cap ligand with its three nitrogen atoms occupying three of the four basal positions. 1,3-Dithiosquarate adopts a centrosymmetric bis-bidentate bridging mode with the sulfur atom filling the fourth equatorial position around each copper atom and the oxygen at the apical site. 1,2-Dithiosquarate acts also as a bridging ligand but in an asymmetrical manner, chelating through the two sulfur atoms at one copper atom and unidentate through one sulfur at the other copper atom. The bridging sulfur occupies one equatorial position at a copper atom and the apical site at the other one. The metal-metal separations through the extended 1,2- and 1,3-dtsq bridges are 5.1654(8) and 7.212(3) Å, respectively. The exchange pathways accounting for the intermediate ferro- (1, J = +32.4 cm(-)(1)) and antiferromagnetic (2, J = -33.5 cm(-)(1)) couplings (H = -JS(A).S(B)) observed in these dithiosquarate-bridged compounds are analyzed and discussed in the light of density functional theory (DFT) calculations.

Two novel series of allocolchicinoids with modified seven membered B-rings: design, synthesis, inhibition of tubulin assembly and cytotoxicity.

Two new attractive series of allocolchicinoids were designed as inhibitors of tubulin assembly using the potent ketone 4 and the tetracyclic, pyrazole annulated NCME variant 7 (NCME = N-acetyl colchinol-O-methylether (2)) as lead structures. The first group of inhibitors of type 6 with novel oxepine and azepine B-ring structures belongs to the NCME-series and was synthesized via a multistep total synthesis starting from simple and cheap 3-methoxybenzaldehyde (12) and 3,4,5-trimethoxybenzaldehyde (13). Biaryl-coupling of the starting materials 12 and 13 was accomplished via Ziegler-Ullmann-reaction to furnish the biphenyl 11 equipped with two carbaldehyde functions. The subsequent Cannizzaro reaction of this dicarbaldehyde 11 proceeded with high regioselectivity to yield almost exclusively the key compound, the hydroxymethyl carboxylic acid 9. Ring closure to the o,o'-bridged biphenyls was accomplished by two routes: on the one hand, treatment of 9 with aqueous hydrochloric acid yielded the lactone 15. On the other hand, a four step sequence starting from the isomeric mixture 9/10 furnished the constitutionally isomeric lactams 23 and 24; these could be converted to the corresponding thiolactams 25 and 26 and to the tetrazole annulated NCME-type derivatives 27 and 28. The second series of bioactive compounds are congeners of allocolchicine (3). The well known desacetyl allocolchicine (29) was easily oxidized to the oxime 30, which was further transformed to the corresponding ketone 31. This served as key precursor for the syntheses of various tetracyclic allocolchicine modifications 33-36 annulated with a pyrazole, isoxazole, pyrimidine or 2-aminopyrimidine heterocycle, respectively. Unexpectedly, all the NCME-variants with a substituent in position 7 like in NCME (2) inhibited the tubulin assembly only moderately. In contrast, the new series of allocolchicine modifications proved to be highly potent antimicrotubule agents. Inhibition of tubulin assembly occurred at lower concentrations compared to those measured for the reference colchicine (1). Surprisingly, these promising results could not be confirmed in the cytotoxicity tests against the human MCF-7 breast cancer cell line, where an unexpected loss of effectiveness compared to the corresponding NCME-derivatives was observed.

Halogenated and isosteric cytisine derivatives with increased affinity and functional activity at nicotinic acetylcholine receptors.

A series of pyridone ring-modified derivatives of (7R,9S)-(-)-cytisine were evaluated for affinity and functional activity at neuromuscular alpha1beta1gammadelta, ganglionic alpha3beta4, and central neuronal alpha4beta2 subtypes of nicotinic receptors. Halogenation at the 3-position improved affinity and functional activity, while substitution at the 5-position led to modest decreases in both, and disubstitution led to near abolition of functional activities and could be correlated with the electron-withdrawing ability of the halogen. Subtype selectivities of the halogenated derivatives were altered relative to cytisine in a substitution-dependent manner. Caulophylline methiodide was less potent than cytisine, but retained significant activity. Thiocytisine was relatively weak in potency and efficacy, but was significantly selective for the alpha4beta2 subtype.

Syntheses and evaluation of pyridazine and pyrimidine containing bioisosteres of (+/-)-pyrido[3.4-b]homotropane and pyrido-[3.4-b]tropane as novel nAChR ligands.

Bioisosteric replacement of the pyridine pharmacophoric element in (+/-)-pyrido[3.4-b]homotropane (PHT) and pyrido[3.4-b]tropane with the pyridazine and pyrimidine nucleus resulted in hitherto unknown nAChR ligands such as 5-8. Inverse type Diels-Alder reactions constitute the key steps in the new routes to the pyridazine- or pyrimidine-annulated bioisosteres. The enantiopure (+)-2-tropinone (11) from the 'chiral pool' is transformed to the ring-expanded silyl enol ether 12 and to the enamine 15. Both proved to be highly dienophilic species in the inverse type [4+2] cycloaddition reactions with the 1,2,4,5-tetrazines 13 and 16a,b or with the 1,3,5-triazine 19 to provide the enantiopure target compounds 5-7. In the same way the racemic pyrimidine-annulated species 8 was obtained from 3-tropanone 21. The new ligands were tested for their in vitro affinity for (alpha4)2(beta2)3 and alpha7* nAChR subtype. In comparison to PHT, well known to exhibit affinity for agonist binding sites in rat brain approximately equivalent to that of (+)-anatoxin-a (1), replacement of the pyridine by the bioisosteric pyridazine resulted in 30-fold lower affinity at the (alpha4)2(beta2)3 subtype. The annulated diazinotropanes 6-8, ligands with ferruginine-like structures more or less retained the affinity of (-)-norferruginine (3) except of compound 7. Remarkably, all of the novel ligands are devoid of affinity at the alpha7* subtype.

Novel B-ring modified allocolchicinoids of the NCME series: design, synthesis, antimicrotubule activity and cytotoxicity.

Two new series of allocolchicinoids mimicking the structure of (-)-N-acetylcolchinol O-methyl ether (2, NCME) were synthesized and evaluated for their abilities to inhibit tubulin assembly. Possible antitumor properties resulting thereof were evaluated in vitro on the human MCF-7 breast cancer cell line. The first series of NCME-derivatives was brought about by extending the seven membered B-ring to novel semisynthetic variations with a nitrogen containing eight-membered B-ring similar, for example, to the artificial, potent steganacin aza-analogue 3. In the second series the seven-membered B-ring of NCME (2) was modified by annulation with a heterocyclic ring system. The racemic ketone 7a serving as key precursor involved in the syntheses of all the target NCME variants 9-13 and 15, 16 was easily transformed into the eight-membered B-ring lactams 9 and 10 via a Beckmann rearrangement of the corresponding E-oxime 8. The tetrazole annulated congener 11 was prepared via azidotrimethylsilane-mediated Schmidt rearrangement. Treatment of educt 7a with Bredereck's reagent led to the enamino ketone 14, which was easily converted into the pyrazole- or pyrimidine-annulated allocolchicinoids 15 and 16. Remarkably, all the allocolchicinoids 9-13 with an azocin-B-ring affected the tubulin/microtubule equilibrium only moderately. In contrast, the novel heterocycle annulated seven membered B-ring variants 15 and 16 proved to be highly potent tubulin-inhibitory, antimitotic agents. Interaction with tubulin occured at concentrations similar to those observed for colchicine (1) or the lead NCME (2). In all cases the antiproliferative effects correlated roughly with the inhibition of tubulin assembly.