Synthesis and characterisation of polyamine-poly(ethylene glycol) constructs for DNA binding and gene delivery

Improved non-viral vector systems are needed for efficient delivery of DNA to target cell nuclei in gene therapy. A series of linear polyamine poly(ethylene glycol) (PEG) constructs has been synthesised by reaction of appropriately Boc-protected thermine derivatives with omega-methoxyPEG oxiranylmethyl ethers. Constructs carrying 1-3 MeOPEG units and 0, 2 or 4 N-methyl groups have been prepared by this method. H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NHBoc was prepared efficiently by mono-trifluoroacetylation of thermine, attachment of Boc and removal of the trifluoroacetyl group in one pot. A similar process gave H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NH2. BocMeN(CH2)3NHMe was alkylated by 1,3-dibromopropane to give BocMeN(CH2)3NMe(CH2)3NMe(CH2)3NMeBoc. A cyanoethylation/reduction sequence extended H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NH2 to give H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NBoc(CH2)3NBoc(CH2) 3NH2, which was converted to its mono- and di-MeOPEG550 derivatives. Deprotection gave the linear polyamine MeOPEG constructs. A branched triamine-poly(ethylene glycol) construct was prepared by acylation of (BocHN(CH2)3)2N(CH2)3NH2 with omega-methoxyPEG 550 chloroformate, followed by deprotection. A cyanoethylation/reduction/protection sequence from (H2N(CH2)3)2 N(CH2)3NHBoc gave a protected pentamine. Alkylation with Br(CH2)5CONH(CH2)2NHBoc, deprotection, acylation with MeOPEG chloroformate and deprotection gave a pentamine MeOPEG construct in which the MeOPEG is attached through a linker to the central amine. The linear hexamine construct carrying MeOPEG550 at only one terminus was the most effective DNA-interactive member of the two series in an ethidium displacement assay and was effective in delivering a reporter gene to RIF-1 tumours.     

Effects of polyamines and analogs on staphylococcal nuclease.

The promoting activity of polyamine analogs (IV approximately XV) on staphylococcal nuclease with DNA as the substrate was compared with that of natural polyamines (I APPROXIMATELY III): I. NH2(CH2)3NH(CH2)4NH(CH2)3NH2(spermine); II. NH2(CH2)3NH(CH2)3NH(CH2)3NH2(thermine); III. NH2(CH2)4NH2 (putrescine); IV. CN(CH2)2NH(CH2)4NH(CH2)2CN; V. HOOC(CH2)2NH(CH2)4NH(CH2)2COOH; VI. C2H5OOC(CH2)2NH(CH2)4NH(CH2)2COOC2H5; VII. HO(CH2)3NH(CH2)4HH(CH2)3OH; VIII. CH3COHH(CH2)3NH(CH2)4NH(CH2)3NHCOCH3; IX. C2H5NH(CH2)3NH(CH2)4NH(CH2)3NHC2H5; X. NH2(CH2)3S(CH2)4S(CH2)3NH2; XI. NH2(CH2)3NH(CH2)2O(CH2)2NH(CH2)3NH2; XII. NH2(CH2)3NCH3(CH2)4HCH3(CH2)3NH2; XIII. CN(CH2)2NCH3(CH2)4NCH3(CH2)2CN; XIV. (CH3)2N(CH2)3NCH3(CH2)4NCH3(CH2)3N(CH3)2; XV. NH2(CH2)2O(CH2)2NH2 Replacement of the terminal groups by CN, COOH, COOEt, NHAc, NHEt, or N(CH3)2 remarkably decreased the activity. The compound VII with terminal hydroxyl groups had a lower promoting activity at low concentrations, but revealed higher activity at higher concentrations and, in contrast to spermine, no inhibition at all even at very high concentrations. Replacement of both internal amino groups by sulfur or NCH3 decreased the activity. The introduction of an ether bond into the internal methylene groups (compound XI) highly decreased the activity. Based upon these findings the possible relationship between structure and activity is discussed.     

Molecular cloning of mouse type 2 and type 3 inositol 1,4,5-trisphosphate receptors and identification of a novel type 2 receptor splice variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SI(m2), at 176-208 of IP(3)R2. The long form (IP(3)R2 SI(m2)(+)) was dominant, but the short form (IP(3)R2 SI(m2)(-)) was detected in all tissues examined. IP(3)R2 SI(m2)(-) has neither IP(3) binding activity nor Ca(2+) releasing activity. In addition to its reticular distribution, IP(3)R2 SI(m2)(+) is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca(2+) ionophore stimulation, most of the IP(3)R2 SI(m2)(+) is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca(2+) ionophore stimulation. IP(3)R2 SI(m2)(-) does not form clusters in either resting or stimulated cells. IP(3) binding-deficient site-directed mutants of IP(3)R2 SI(m2)(+) and IP(3)R3 fail to form clusters, indicating that IP(3) binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SI(m2)(-) prevents stimulus-induced IP(3)R clustering, suggesting that IP(3)R2 SI(m2)(-) functions as a negative coordinator of stimulus-induced IP(3)R clustering. Expression of IP(3)R2 SI(m2)(-) in CHO-K1 cells significantly reduced ATP-induced Ca(2+) entry, but not Ca(2+) release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca(2+) signals.     

Isolation and comparative analysis of multiple isoenzymes of glutathione-S-transferase from the liver of intact rats and rats administered phenobarbital, 3-methylcholanthrene and butylhydroxytoluene

Seven glutathione-S-transferase (GST) isozymes were purified from liver cytosol of intact male Wistar rats: 1-1(A), 1-1(B), 1-2, 2-2, 3-3, 3-4, 4-4. Treatment of rats with butylated hydroxytoluene (BHT) led to the induction of isozymes GST 1-1(A), 1-1(B) (2-fold), 3-3 (3.5-fold) as well as to the appearance of two new isozymes--1-3 and 4-4(A). Phenobarbital (PB) induced isozymes GST 1-1(A), 1-1(B) (2-fold) and 3-3 (1.5-fold). BHT and PB caused an increase in the specific activity of isozymes 1-1(A), 1-1(B), 3-3, 3-4 towards 1-chloro-2.4-dinitrobenzene and 1.2-dichloro-4-nitrobenzene. 3-Methylcholanthrene (MC) induced isozymes 1-2 (1.5-fold), 2-2 (2-fold) and 4-4 (3-fold). A conclusion was drawn that BHT and PB induced the GST subunits 1 and 3, whereas MC--subunits 2 and 4.     

Expeditious and convenient synthesis of pregnanes and its glycosides as potential anti-dyslipidemic and anti-oxidant agents

A series of new pregnane derivatives and its glycosides were synthesized in order to find new 'leads' against some important targets. The 3beta-hydroxy-16alpha-(2-hydroxy ethoxy) pregn-5-en-20-one (5) was synthesized from 3beta-hydroxy-5,16-pregnadiene-20-one (2) by adopting general modified procedure using BF(3):Et(2)O as a catalyst. Reduction of 5, with sodium borohydride yielded 3beta,20beta-dihydroxy-16alpha-(2-hydroxy ethoxy) pregn-5-en (7) as the major isolable product. O-alkylation of the C-20-oxime-pregnadiene (9) with 1,5-dibromopentane yielded 20-(O-5-bromopentyl)-oximino-3beta-hydroxy-pregn-5,16-diene (11). Synthesis of C-16 substituted pregnane glycosides (20) and (21) were accomplished with the imidate method using BF(3):Et(2)O. The synthesis of 4-chlorobenzoate (3) and 2-chlorobenzoate (4), derivatives of 2 were also accomplished. These compounds were evaluated for their anti-dyslipidemic and anti-oxidant activity and amongst them compounds 3 and 7 showed more lipid lowering and anti-oxidant activity.     

Synthesis and stereostructure-activity relationship of three asymmetric center pyrethroids: 2-methyl-3-phenylcyclopropylmethyl 3-phenoxybenzyl ether and cyanohydrin ester

2-Methyl-3-phenylcyclopropylmethyl 3-phenoxybenzyl ether 2 and cyanohydrin ester 3, a couple of pyrethroids with three asymmetric centers, were synthesized. Of each of the four diastereomers of 2 and 3, only the (1R*,2R*,3R*)-2a and 3a showed significant insecticidal activities. Dual sets of enantiomers [(1R,2R,3R)-(-)-2a and (1S,2S,3S)-(+)-2a] and [(1R,2R,3R)-(-)-3a and (1S,2S,3S)-(+)-3a] were synthesized through the asymmetric cyclopropanation using the Aratani catalyst. Significant separations of insecticidal activities were observed between both the enantiomers against the tobacco cutworm (Spodoptera litura) and the common mosquito (Culex pipiens pallens); (1S,2S,3S)-(+)-2a and (+)-3a showed higher activities than their antipodes (1R,2R,3R)(-)-2a and (-)-3a. This result is the second example of such synthetic pyrethroids with three asymmetric centers.     

New method for the preparation of 3'- and 2'-O-phosphoramidites of 2'- and 3'-difluoromethyluridine derivatives

Hydrogenation of 2'-deoxy-2'-difluoromethylene-5'-O-dimethoxytrityluridine (1) and 3'-deoxy-3'-difluoromethylene-5'-O-dimethoxytrityluridine (7), gave the corresponding 2'- and 3'-difluoromethyluridine derivatives 2a and 8a. Detritylation of compounds 2a, 2b and 8a, 8b resulted in the formation of 1-(2-deoxy-2-C-difluoromethyl-beta-D-arabino-pentofuranosyl)uracil (3a) and 1-(3-deoxy-3-C-difluoromethyl-beta-D-xylo-pento furanosyl)- uracil (9a) as well as corresponding minor isomers 3b and 9b. Compounds 3a and 3b were also obtained from 2'-deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (4). Finally, phosphitylation of 2a and 8a provided the title 2'- and 3'-O-phosphoramidites 6 and 10.     

Regulation of Akt and Wnt signaling by the group II metabotropic glutamate receptor antagonist LY341495 and agonist LY379268

Metabotropic glutamate receptors 2/3 (mGlu(2/3)) have been implicated in schizophrenia and as a novel treatment target for schizophrenia. The current study examined whether mGlu(2/3) regulates Akt (protein kinase B) and Wnt (Wingless/Int-1) signaling, two cascades associated with schizophrenia and modified by antipsychotics. Western blotting revealed increases in phosphorylated Akt (pAkt) and phosphorylated glycogen synthase kinase-3 (pGSK-3) following acute and repeated treatment of LY379268 (mGlu(2/3) agonist), whereas increases in dishevelled-2 (Dvl-2), dishevelled-3 (Dvl-3), GSK-3 and β-catenin were only observed following repeated treatment. LY341495 (mGlu(2/3) antagonist) induced the opposite response compared with LY379268. Co-immunoprecipitation experiments showed an association between the mGlu(2/3) complex and Dvl-2 providing a possible mechanism to explain how the mGlu(2/3) can mediate changes in Wnt signaling. However, there was no association between the mGlu(2/3) complex and Akt suggesting that changes in Akt signaling following LY341495 and LY379268 treatments may not be directly mediated by the mGlu(2/3) . Finally, an increase in locomotor activity induced by LY341495 treatment correlated with increased pAkt and pGSK-3 levels and was attenuated by the administration of the GSK-3 inhibitor, SB216763. Overall, the results suggest that mGlu(2/3) regulates Akt and Wnt signaling and LY379268 treatment has overlapping effects with D(2) dopamine receptor antagonists (antipsychotic drugs).     

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.     

Preparation and configuration of racemic and optically active analgesic cycloaminoalkylnaphthalenes

Cycloaminoalkylnaphthalene 3 shows interesting opioid‐like analgesic properties. It possesses two chiral centers and can exist as two racemic pairs and four diastereomers. Since the binding of opioids with receptors is stereoselective, it was important to have the two racemic pairs as well as the four diastereomers. In this paper the synthesis of the (2R,3S/2S,3R) racemate and the (2R,3S) and (2S,3R) enantiomers of the 1,2‐dimethyl‐3‐[2‐(6‐hydroxynaphthyl)]‐3‐hydroxypyrrolidine 3 is considered and the determination of absolute configuration is described. The (2R,3S/2S,3R)‐ 3 racemate and the (2R,3S)‐ 3 and (2S,3R)‐ 3 enantiomers were prepared by reaction of the racemic and optically active 1,2‐dimethyl‐3‐pyrrolidone 2, respectively, with the lithiation product obtained from 2‐bromo‐6‐tetrahydropyranyloxy‐naphthalene 1 and acidic hydrolysis. The above‐mentioned enantiomers of 3 were also obtained by optical resolution via fractional crystallization of the salts with d ‐ and l ‐tartaric acids. The configuration of the optically active compounds was determined by X‐ray analysis of a crystal of (−)‐(2S,3R)‐ 3 · HCl · H₂O. The pharmacological test HPT showed that (−)‐(2S,3R)‐ 3 · HCl · H₂O enantiomer is able to induce opioid‐like analgesia with a relative potency 1.5 times that of (2R,3S/2S,3R)‐ 3 and ∼1.5 times that of morphine. Chirality 11:21–28, 1999. © 1999 Wiley‐Liss, Inc.     

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.     

Structure and cytotoxic activity of enzymatic hydrolysis products of secoiridoid glucosides, isoligustroside and isooleuropein

Hydrolysis of isoligustroside (1) and isooleuropein (2), secoiridoid glucosides, in the presence of β-glucosidase provided 2-(4-hydroxyphenyl)methyl (2R,3S,4S)-3-formyl-3,4-dihydro-4-(2-methoxy-2-oxoethyl)-2-methyl-2H-pyran-5-carboxylate (3) and 2-(3,4-dihydroxyphenyl)methyl (2R,3S,4S)-3-formyl-3,4-dihydro-4-(2-methoxy-2-oxoethyl)-2-methyl-2H-pyran-5-carboxylate (4), respectively. The structures of 3 and 4 were elucidated on the basis of extensive spectral analyses, including 2D-NMR experiments. Compounds 3 and 4 were found to be new rearrangement products of the aglycones of 1 and 2. The cytotoxic activities of 3 and 4 were evaluated using a disease-oriented panel of 39 human cancer cell lines and showed moderate cytotoxic activity for 4, while 3 exhibited weaker activity compared to that of 4.     

Synthesis and structure determination of 6-methylbenzo[a]pyrene-deoxyribonucleoside adducts and their identification and quantitation in vitro and in mouse skin

Activation of the moderate carcinogen 6-methylbenzo[a]pyrene (6-CH(3)BP) by one-electron oxidation to form DNA adducts was studied. Iodine oxidation of 6-CH(3)BP in the presence of dGuo produces BP-6-CH(2)-N(2)dGuo, BP-6-CH(2)-N7Gua and a mixture of 6-CH(3)BP-(1&3)-N7Gua, whereas in the presence of Ade the adducts BP-6-CH(2)-N1Ade, BP-6-CH(2)-N3Ade, BP-6-CH(2)-N7Ade and 6-CH(3)BP-(1&3)-N1Ade are obtained. Furthermore, for the first time an aromatic hydrocarbon radical cation afforded an adduct with dThd, the stable adduct BP-6-CH(2)-N3dThd. Formation of these adducts indicates that the 6-CH(3)BP radical cation has charge localized at the 6, 1 and 3 position. When 6-CH(3)BP was activated by horseradish peroxidase in the presence of DNA, two depurinating adducts were identified, BP-6-CH(2)-N7Gua (48%) and 6-CH(3)BP-(1&3)-N7Gua (23%), with 29% unidentified stable adducts. In the binding of 6-CH(3)BP catalyzed by rat liver microsomes, the same two depurinating adducts, BP-6-CH(2)-N7Gua (22%) and 6-CH(3)BP-(1&3)-N7Gua (10%), were identified, with 68% unidentified stable adducts. In 6-CH(3)BP-treated mouse skin, the two depurinating adducts, BP-6-CH(2)-N7Gua and 6-CH(3)BP-(1&3)-N7Gua, were identified. Although quantitation of these two adducts was not possible due to coelution of metabolites on HPLC, they appeared to be the major adducts found in mouse skin. These results show that 6-CH(3)BP forms depurinating adducts only with the guanine base of DNA, both in vitro and in mouse skin. The weaker reactivity of 6-CH(3)BP radical cation vs. BP radical cation could account for the weaker tumor-initiating activity of 6-CH(3)BP in comparison to that of BP.     

Spectroscopic and electrochemical characterization of gold(I) and gold(III) complexes with glyoxaldehyde bis(thiosemicarbazones): cytotoxicity against human tumor cell lines and inhibition of thioredoxin reductase activity

Complexes [Au(2)(H(2)Gy3DH)(2)]Cl(2) (1), [Au(H(2)Gy3Me)]Cl(3) (2) and [Au(H(2)Gy3Et)]Cl(3) (3) were obtained with glyoxaldehyde bis(thiosemicarbazone) (H(2)Gy3DH) and its N(3)-methyl (H(2)Gy3Me) and N(3)-ethyl (H(2)Gy3Et) derivatives. The bis(thiosemicarbazones) and their gold(I) and gold(III) complexes exhibited anti-proliferative activity against HL-60, Jurkat (leukemia) and MCF-7 (breast cancer) cells at 10 μmol L(-1). Complex (2) was able to in vitro inhibit thioredoxin reductase (TrxR) activity, which suggests that inhibition of TrxR could be part of its mechanism of action.     

Preparation and configuration of racemic and optically active analgesic dialkylaminoalkylnaphthalenes

The alkylaminoalkylnaphthalene 3 shows interesting opioid-like analgesic properties, μ-selective ligand competition, and enkephalin hydrolyzing enzyme inhibition. 3 possesses two chiral centers and can exist as two racemic pairs and four diastereomers. Since the binding of opioids with the receptor is stereoselective, it was important to have the two racemic pairs as well as the four diastereomers. In this paper the synthesis of the (1R,2R/1S,2S)- and (1R,2S/1S,2R)-racemates and the (1R,2R)- and (1S,2S)-enantiomers of the 1-ethyl-1-hydroxy-1-[2-(6-hydroxynaphthyl)]-2-methyl-3-dimethylaminopropane 3 is considered and the determination of absolute configuration is described. The (1R,2R/1S,2S)- 3 and (1R,2S/1S,2R)- 3 racemates and the (1R,2R)- 3 and (1S,2S)- 3 enantiomers were prepared by reaction of the racemic and optically active 1-dimethylamino-2-methylpentan-3-one 2 , respectively, with the lithiation product obtained from 2-bromo-6-tetrahydropyranyloxynaphthalene and acidic hydrolysis. The optical resolution of aminoketone 2 was carried out via fractional crystallization of salts (+)- and (?)-dibenzoyltartrates. The configuration of the optically active compounds was determined by X-ray analysis of a crystal of (+)-(1R,2R)- 3 · HCl · H₂O. Preliminary pharmachological tests showed that (+)-(1R,2R)- 3 enantiomer is able to induce opioid-like analgesia with a relative potency 2.5 times that of (1R,2R/1S,2S)- 3 and about 4 times that of morphine. © 1994 Wiley-Liss, Inc.     

Ca2(+)-mobilising properties of synthetic fluoro-analogues of myo-inositol 1,4,5-trisphosphate and their interaction with myo-inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase

The ability of two fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. DL-2-deoxy-2-fluoro-scyllo-Ins(1,4,5)P3 (2F-Ins(1,4,5)P3) and DL-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 (2,2-F2-Ins(1,4,5)P3) were full agonists (EC50s 0.77 and 0.41 microM respectively) and slightly less potent than D-Ins(1,4,5)P3 (EC50 0.13 microM), indicating that the axial 2-hydroxyl group of Ins(1,4,5)P3 is relatively unimportant in receptor binding and stimulation of Ca2+ release. Both analogues mobilized Ca2+ with broadly similar kinetics and were substrates for Ins(1,4,5)P3 3-kinase but, qualitatively, were slightly poorer than Ins(1,4,5)P3. 2F-Ins(1,4,5)P3 was a weak substrate for Ins(1,4,5)P3 5-phosphatase but 2,2-F2-Ins(1,4,5)P3 was apparently not hydrolysed by this enzyme, although it inhibited its activity potently (Ki = 26 microM).     

Activation of telomerase and cyclooxygenase-2 in PDGF and FGF inhibition of C2-ceramide-induced apoptosis

In the present study, the roles of telomerase and prostaglandin E(2) (PGE(2)) in platelet-derived growth factor (PDGF's) and fibroblast growth factor-2 (FGF-2's) effects against C(2)-ceramide-induced cell death were investigated. C(2)-ceramide reduced the viability of NIH3T3 cells in a condition without calf serum (CS) in accordance with decreasing telomerase activity according to the TRAP assay. The addition of CS significantly protected cells from C(2)-ceramide-induced apoptosis through increased telomerase activity, and the phosphorylations of PDGF and the FGF-2-like receptor in NIH3T3 cells were detected. Adding PDGF and FGF-2 decreased the cytotoxic effect elicited by C(2)-ceramide through stimulating telomerase activity, which was blocked by adding a telomerase inhibitor (TI). Activations of ERKs and JNKs were detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities induced by PDGF and FGF were respectively inhibited by the addition of the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125. Accordingly, induction of cyclooxygenase-2 (COX-2) protein expression and PGE(2) production was detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities stimulated by PDGF and FGF were reduced by adding a specific COX-2 inhibitor, NS398, through a decrease in PGE(2) production. Incubation of cells with PGE(2) or the EP1 agonist, 17-PT, but not the EP2 agonist, sulprostone, the EP3 agonist, butaprost, or the EP4 agonist, PGE(1) alcohol, significantly enhanced the telomerase activity of NIH3T3 cells. PGE(2) protection of NIH3T3 cells against C(2)-ceramide-induced cell death was identified by the MTT and LDH-release assays, and it was inhibited by adding the EP1 antagonist, SC-19220. Ceramide metabolites including ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P), and a standard control of exogenous ceramide C(2)-dihydroceramide show no effect on the telomerase activity and viability of NIH3T3 cells. The involvement of COX-2/PGE(2)-mediated telomerase activation by PDGF and FGF-2 against C(2)-ceramide-induced cell death is first demonstrated herein.     

Synthesis, structural characterization and biological study of new organotin(IV), silver(I) and antimony(III) complexes with thioamides

An overview of our work on the synthesis and biological activity of a series of tin(IV), silver(I) and antimony(III) complexes with thioamides is reported. Organotin(IV) complexes of formulae (n-Bu)2Sn(MBZT)2 (1), Me2Sn(CMBZT)(2) (2), {(Ph3Sn)2(MNA) (Me2CO)} (3), Ph3Sn(MBZT) (4), Ph3Sn(MBZO) (5), Ph3Sn(CMBZT) (6), Ph2Sn(CMBZT)2 (7) and (n-Bu)2Sn(CMBZT)2 (8), Me2Sn(PMT)2 (9), (n-Bu)2Sn(PMT)2 (10), Ph2Sn(PMT)2 (11), Ph3Sn(PMT) (12) {where MBZT=2-mercapto-benzothiazole, CMBZT=5-chloro-2-mercapto-benzothiazole, H2MNA=2-mercapto-nicotinic acid, MBZO=2-mercapto-benzoxazole and PMTH=2-mercapto-pyrimidine} were characterized by spectroscopic (NMR, IR, Mossbauer, etc.) and X-ray diffraction techniques and their influence on the peroxidation of oleic acid was studied. They were found to inhibit strongly the peroxidation of linoleic acid by the enzyme lipoxygenase. In addition, organotin(IV) complexes were found to exhibit stronger cytotoxic activity in vitro, against leiomyosarcoma cells, than cisplatin. The antiproliferative activity of the organotin complexes studied, against leiomyosarcoma cells follow the same order of LOX activity inhibition. This is, 3>12>7>6 approximately 8 approximately 10>5 approximately 4>2>9. Thus, among organotin(IV)-CMBZT complexes, 7 exhibits higher activity than the others and this is explained by a free radical mechanism, as it is revealed by an EPR study. The results are compared with the corresponding ones found for the silver(I) complexes of formulae complexes {[Ag6(mu3-HMNA)4(mu3-MNA)2](2-).[(Et(3)NH)+]2.(DMSO)2.(H2O)} (13), {[Ag4Cl4(mu3-STHPMH2)4]n} (14), {[Ag6(mu2-Br)6(mu2-STHPMH2)4(mu3-STHPMH2)2]n} (15), {[Ag4(mu2STHPMH2)6](NO3)4}(n) (16), {[AgCl(TPTP)]4} (17), [AgX(TPTP)3] with X=Cl (18), Br (19), I (20) (where STHPMH2=2-mercapto-3,4,5,6-tetrahydro-pyrimidine, TPTP=tri(p-toly)phosphine) and those of antimony(III) complexes {[SbCl2(MBZIM)4](+).Cl(-).2H2O.(CH3OH)} (21), {[SbCl2(MBZIM)4]+.Cl(-).3H2O.(CH3CN)} (22), [SbCl3(MBZIM)2] (23), [SbCl3(EMBZIM)2] (24), [SbCl3(MTZD)2] (25), {[SbCl3(THPMT)2]} (26) and {[Sb(PMT)3].0.5(CH3OH)} (27) (where MBZIM is 2-mercapto-benzimidazole, EMBZIM=5-ethoxy-2-mercapto-benzimidazole and MTZD is 2-mercapto-thiazolidine), which they have characterized with similar techniques as in case of organotin(IV) complexes. Silver(I) and antimony(III) complexes were found to be cytotoxic against various cancer cell lines.     

Interaction of purine nucleotides with cobalt-hexammine, cobalt-pentammine and cobalt-tetrammine cations. Evidence for the rigidity of adenosine and flexibility of guanosine and deoxyguanosine sugar conformations.

The interaction of adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP) and 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) with the [Co(NH3)6]3+, [Co(NH3)5Cl]2+ and [Co(NH3)4Cl2]+ cations has been investigated in aqueous solution with metal/nucleotide ratios (r) of 1/2, 1 and 2 at neutral pH. The solid complexes have been isolated and characterized by FT-IR and 1H-NMR spectroscopy. The complexes are polymeric in nature both in the crystalline solid and aqueous solution. The binding of the cobalt-hexammine cation is indirectly (via NH3) through the N-7 and the PO3(2-) groups of the AMP and via O-6, N-7 and the PO3(2-) of the GMP and dGMP anions (outer-sphere). The cobalt-pentammine and cobalt-tetrammine bindings are through the phosphate groups (inner-sphere) and the N-7 site (outer-sphere) of these nucleotide anions. The ribose moiety shows C2'-endo/anti conformation, in the free AMP and GMP anions as well as in the cobalt-ammine-AMP complexes, whereas a mixture of teh C2'-endo/anti and C3'-endo/anti sugar puckers were observed for the Co(NH3)6-GMP, Co(NH3)5-GMP and a C3'-endo/anti conformer for the Co(NH3)4-GMP complexes. The deoxyribose showed an O4'-endo/anti conformation for the free dGMP anion and a C3'-endo/anti for the Co(NH3)6-dGMP, Co(NH3)5-dGMP and Co(NH3)4-dGMP complexes.     

Syntheses, crystal structures and antimicrobial activities of monomeric 8-coordinate, and dimeric and monomeric 7-coordinate bismuth(III) complexes with tridentate and pentadentate thiosemicarbazones and pentadentate semicarbazone ligands

Novel bismuth(III) complexes 1-4 with the tridentate thiosemicarbazone ligand of 2N1S donor atoms [Hmtsc (L1); 2-acetylpyridine (4N-morpholyl thiosemicarbazone)], the pentadentate double-armed thiosemicarbazone ligand of 3N2S donor atoms [H2dmtsc (L3); 2,6-diacetylpyridine bis(4N-morpholyl thiosemicarbazone)] and the pentadentate double-armed semicarbazone ligand of 3N2O donor atoms [H2dasc (L4b); 2,6-diacetylpyridine bis(semicarbazone)], were prepared by reactions of bismuth(III) nitrate or bismuth(III) chloride and characterized by elemental analysis, thermogravimetric and differential thermal analysis (TG/DTA), FTIR and NMR (1H and 13C) spectroscopy. The crystal and molecular structures of complexes 1, 2a, 2b and 4b, and the "free" ligand L1 were determined by single-crystal X-ray structure analysis. The dimeric 7-coordinate bismuth(III) complex [Bi(dmtsc)(NO3)]2, 1, and the monomeric 7-coordinate complexes [Bi(Hdasc)(H2O)](NO3)2.H2O (major product), 2a, and [Bi(dasc)(H2O)]NO3.H2O (minor product), 2b, all with pentagonal bipyramidal bismuth(III) centers, are depicted with one electron pair (6s2) of the bismuth(III) atom, deprotonated forms of multidentate thiosemicarbazone or semicarbazone ligands, and monodentate NO3 or H2O ligands, respectively. These complexes are related to the positional isomers of one electron pair of the bismuth(III) atom; 1 has an electron pair positioned in the pentagonal plane (basal position), while 2a and 2b have an electron pair in the apical position. The monomeric 8-coordinate complex [Bi(mtsc)2(NO3)], 4b, which was obtained by slow evaporation in MeOH of the 1.5 hydrates 4a, was depicted with one electron pair of the bismuth(III) atom, two deprotonated mtsc- ligand and one nitrate ion. On the other hand, crystals of the complex "[Bi(mtsc)Cl2]", 3, prepared by a reaction of BiCl3 with L1 showed several polymorphs (3a, 3b, 3c and 3d) due to coordination and/or solvation of dimethyl sulfoxide (DMSO) used in the crystallization. Bismuth(III) complexes 1 and 4a showed selective and effective antibacterial activities against Gram-positive bacteria. The structure-activity relationship was discussed.