One pot synthesis of pyrimidine and bispyrimidine derivatives and their evaluation for anti-inflammatory and analgesic activities

A number of pyrimidine derivatives (1-10) have been synthesized by condensation of 4-isothiocyanato-4-methylpentan-2-one with furfurylamine, histamine, 1-(3-aminopropyl)imidazole, 1-(3-aminopropyl)-2-pyrrolidinone, 2-aminobenzonitrile and 3-isothiocyanatobutanal with 1-(3-aminopropyl)-2-pyrrolidinone and 2-hydrazinopyridine under different reaction conditions. Various bispyrimidine derivatives (11-15) were obtained by condensation of 4-isothiocyanato-4-methylpentan-2-one with 2,4,8,10-tetraoxaspiro[5,5]undecane3,9-dipropamine (11'), 1,4-bis(3-aminopropyl)piperazine (13'), 3,5-diamino 1,2,4-triazole (15') and 3-isothiocyanatobutanal with 2,4,8,10-tetraoxaspiro[5,5]undecane 3,9-dipropamine, 1,4-bis(3-aminopropyl)piperazine. All these compounds were characterized by correct FT-IR, (1)H NMR, MS and elemental analysis. These compounds were screened for anti-inflammatory and analgesic activities. Anti-inflammatory activity of 3 is comparable while analgesic activity was found to be better than that of standard drug.     

Chemoenzymatic synthesis of the sialyl-alpha-(2-->3')-lactosamine trisaccharide with a 3-aminopropyl group as a spacer at the reducing end

The trisaccharide, 3-aminopropyl 5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid-(2-->3)-beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside has been synthesized chemoenzymatically for the first time. First, the acceptor, 3-aminopropyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside was synthesized in a conventional chemical manner, and then it was coupled with CMP-sialic acid using alpha-(2-->3)-(N)-sialyltransferase to afford the desired trisaccharide by an enzymatically stereocontrolled manner.     

Preparation of azacrown-functionalized 2'-O-methyl oligoribonucleotides, potential artificial RNases

An improved synthesis for 3-(3-aminopropyl)- and 3-(3-mercaptopropyl)-1,5,9-triazacyclododecane has been developed and alternative methods for their conjugation to oligonucleotides have been described. Accordingly, the 3-aminopropyl azacrown and its N-(3-aminopropanoyl)-3-aminopropyl analogue have been tethered to the 3'-terminus of a 2'-O-methyloligoribonucleotide by aminolytic cleavage of the thioester linker utilized for the chain assembly. Studies on a monomeric model compound verify that the reaction proceeds solely by the attack of the primary amino group. 5'-Conjugation has been achieved by introducing a 2-benzylthio-2-oxoethyl group to the 5'-terminus as a phosphoramidite reagent and cleaving the thioester bond with the 3-aminopropyl azacrown. For intrachain conjugation, a phosphoramidite reagent derived from 1-deoxy-1-(2-benzylthio-2-oxoethyl)-beta-d-erythro-pentofuranose has been inserted in a desired position within the chain and subjected to on-support aminolysis with the 3-aminopropyl azacrown or its N-(3-aminopropanoyl)-3-aminopropyl and N-(6-aminohexanoyl)-3-aminopropyl analogues. The 3-mercaptopropyl-derivatized azacrown has been tetherd by a disulfide bond to a 3'-(3-mercaptoalkyl)phosphate-tailed oligonucleotide. The 3'- and intrachain-tethered conjugates have been shown to cleave as their Zn(II) chelate complementary oligoribonucleotide sequences.     

Reactions of plant copper/topaquinone amine oxidases with N6-aminoalkyl derivatives of adenine

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N6-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N6-(3-aminopropyl)adenine, N6-(4-aminobutyl)adenine, N6-(4-amino-trans-but-2-enyl) adenine, N6-(4-amino-cis-but-2-enyl) adenine and N6-(4-aminobut-2-ynyl) adenine. From these, N6-(4-aminobutyl) adenine and N6-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (Km approximately 10(-4)M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N6-(3-aminopropyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N6-(3-aminopropyl)adenine and N6-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (Ki approximately 10(-5) - 10(-4) M) whereas, N6-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.     

A shortened synthesis of 4-(3-aminopropyl) pyrazole, an affinity ligand for alcohol dehydrogenase purification

The most efficient, specific and rapid procedures for alcohol dehydrogenase (ADH) purification utilize immobilized 4-(3-aminopropyl) pyrazole to which pyrazole sensitive ADHs, i.e. class I isozymes, bind. Because of the length of the reported synthesis of this affinity resin, we synthesized the 4-(3-aminopropyl) pyrazole ligand by a new method in two steps from commercially available nicotinaldehyde. The ligand synthesized by this simplified procedure was directly coupled to the chain-extended support, Activated CH-Sepharose 4B, to yield the same ligand-spacer combination as reported by L.G. Lange and B.L. Vallee (Biochem. 15: 4681-4686, 1976). Human and hamster class I ADHs purified using this resin were homogeneous by SDS-PAGE followed by silver staining. Specific activity and recovery of human class I ADH were comparable to those previously reported.     

Newly discovered polyamine, 2-hydroxyspermidine, in Pseudomonas acidovorans.

A previously unknown hydroxylated polyamine has been recovered from Pseudomonas acidovorans 29. It has been identified as 2-hydroxyspermidine, N4-(3-aminopropyl)-1,4-diaminobutane-2-ol, by its chromatographic behavior, electrophoretic mobility, and reaction with metaperiodate. It can be synthesized enzymatically from 2-hydroxyputrescine by cell-free preparations from Escherichia coli or P. acidovorans 29 which contain propylamine transferase. It is interesting to note that the naturally occurring compound is the 2-hydroxyspermidine and not the 3-hydroxyspermidine, N1-(3-aminopropyl)-1,4-diaminobutane-2-ol, indicating that the propylamine transferase reacts preferentially with the amine distal to the hydroxyl group. A mixture of 2- and 3-hydroxyspermidines and hydroxyspermine was synthesized by reacting acrylonitrile with 2-hydroxyspermidine and catalytic reduction of the products with hydrogen. N-(gamma-aminopropyl)-beta-alanine, used to help identify the hydroxyspermidines, was synthesized from N-(3-aminopropyl)-3-aminopropanenitrile by hydrolysis with 10% NaOH.     

Urinary metabolites of polyamines in rats

Urinary metabolites of polyamines in rats were studied systematically by the intraperitoneal injection of radioactive polyamines. Urinary metabolites were fractionated into 4 fractions containing non-polar and acidic compounds, acidic and neutral ampholytes, basic ampholytes and polyamines. A large amount of radioactivity was detected in the fractions containing non-polar and acidic compounds and polyamines of urine of rats injected with radioactive putrescine, while in the case of the injection of radioactive spermidine or spermine a relatively large amount of radioactivity was found in the basic ampholyte fraction as well as in the polyamine fraction. Analysis of these fractions indicated that gamma-aminobutyric acid, N-monoacetylputrescine, 2(3)-hydroxyputrescine, putreanine, N-(3-aminopropyl)-4-aminobutyric acid (isoputreanine), spermic acid, N-(3-aminopropyl), N'-(2-carboxyethyl)-1,4-diaminobutane, and N-monoacetylspermidine A and B were excreted as urinary metabolites of the polyamines in addition to putrescine, spermidine and spermine.     

Synthesis and selective tumor targeting properties of water soluble porphyrin-Pt(II) conjugates

We have designed and synthesized a series of novel water soluble porphyrins and their platinum(II) conjugates, cis-[(Por)Pt(dmso)X], where Por=5-(4-pyridyl)-10,15,20-tris(4-sulfonatophenyl)porphyrin) (PyTPPS) or 5-[4-(3-aminopropyl)pyridiniumyl]-10,15,20-tris(4-sulphonatophenyl)porphyrin (PyTPPS-NPn), X=2Cl, 1,1-cyclobutanedicarboxylic acid, oxalate, or malonate. Their biodistribution in tumor bearing mouse was examined along with their antitumor activity against murine leukemia L1210 cell line. The representative complex 1 exhibited a significant accumulation in tumor tissue with a tumor/muscle ratio of 7 after 24 h post injection. The antitumor activity of the title compounds was marginal (T/C: 95-117%), but it was found that platinum(II) coordination to the porphyrin periphery did not affect the tumor accumulating properties of the porphyrin permitting further derivatization for efficient delivery of the Pt(II) antitumor agent.     

Structure activity relationship studies on C2 side chain substituted 1,1-bis(4-methoxyphenyl)-2-phenylalkenes and 1,1,2-tris(4-methoxyphenyl)alkenes

1,1-bis(4-Methoxyphenyl)-2-phenylalkenes (1a-9a) and 1,1,2-tris(4-methoxyphenyl)alkenes (1b-9b) with various C2-substituents (H (1a, 1b), methyl (2a, 2b), ethyl (3a, 3b), propyl (4a, 4b), butyl (5a, 5b), 2-cyanoethyl (6a, 6b), 3-cyanopropyl (7a, 7b), 3-aminopropyl (8a, 8b), 3-carboxypropyl (9a, 9b)) were tested for cytotoxic effects on hormone dependent MCF-7 cells. The effects were correlated with agonistic and antagonist properties determined on the MCF-7-2a cell line stably transfected with the plasmid ERE(wtc)luc. We demonstrated that the antiproliferative effects did not result from an interaction with the estrogen receptor (ER). The most cytotoxic compounds 5,5-bis(4-methoxyphenyl)-4-phenylpent-4-enylamine (8a) and 4,5,5-tris(4-methoxyphenyl)pent-4-enyl (8b) showed cytocidal effects without having significant agonistic and antagonistic properties.     

A new method of chemical modification of N6-amino group in adenine nucleotides with formaldehyde and a thiol and its application to preparing immobilized ADP and ATP.

Reaction of AMP with formaldehyde and 3-mercaptopropionic acid at pH 11.7 gave a new AMP derivative, N6-[(2-carboxyethyl)thiomethyl]-AMP (I) in 91% yield and reaction at pH 3.1 gave another new derivative, N6,N6-bis[(2-carboxyethyl)thiomethyl]-AMP (II) in 57% yield. The structures were determined by their 13C and 1H nuclear magnetic resonance spectra coupled with those of the simple analogues, N6-[(2-carboxyethyl)thiomethyl]-9-methyladenine (III) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-9-methyladenine (IV) which were synthesized from 9-methyladenine in the same way as for derivatives I and II. ADP and ATP were treated in the same way as AMP to afford the corresponding carboxyl derivatives, N6-[(2-carboxyethyl)thiomethyl]-ADP (V), N6-[(2-carboxyethyl)thiomethyl]-ATP (VI), N6,N6-bis[(2-carboxyethyl)thiomethyl]-ADP (X) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-ATP (XI) in 71%, 75%, 53% and 40% yield, respectively. These compounds were coupled to 1,3-diaminopropane with a water-soluble carbodiimide to give the corresponding amino derivatives, N6-([N-3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (VIII), N6-(N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (IX), N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (XIII), and N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (XIV), which were further bound to CNBr-activated dextran to give new polymer-bound derivatives of ADP and ATP. These free and bo-nd derivatives were tested for their coenzymic activities against several kinases. The activities of the ADP derivatives, V, VIII, X, XIII, dextran-bound VIII, and dextran-bound XIII against acetate kinase were 82%, 81%, 68%, 55%, 35%, and 15%, respectively, relative to ADP and those of the ATP derivatives, VI, IX, XI, XIV, dextran-bound IX, and dextran-bound XIV against hexokinase were 88%, 94%, 60%, 81%, 58%, and 49%, respectively, relative to ATP.     

Synthesis of (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) derivatives modified at the carbamoyl moiety as a new class of NMDA receptor antagonists

(1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC, ), which is a conformationally restricted analogue of the antidepressant milnacipran [(+/-)-1], represents a new class of potent NMDA receptor antagonists. A series of PPDC analogues modified at the carbamoyl moiety were synthesized. Among these, (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-dipropylcyclopropanecarboxamide (4d) was identified as the most potent NMDA receptor antagonist in this series and clearly reduced the MMDA receptor mediated potentiation of rat hippocampal slices, a model of long-term potentiation (LTP). The three-dimensional structure of 4d was also analyzed in detail to clarify the receptor-binding conformation.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

Construction of polyamine-modified uridine and adenosine derivatives--evaluation of DNA binding capacity and cytotoxicity in vitro

We here report the synthesis of the two polyamine-based nucleoside derivatives 5-{[bis-(3-aminopropyl)amino]acetamido-1-propynyl}uridine and 2-{[bis-(3-aminopropyl)amino]-acetamido-1-propynyl}adenosine. The various polyamine derivatives have been used in thermal melting analysis using DNA from herring testes, and in cellular studies using four different cell lines. The compounds were all found to be non-toxic, thus holding good promise for future use as siRNA building blocks.     

The design of potent, selective, non-covalent, peptide thrombin inhibitors utilizing imidazole as a S1 binding element.

Modeling of neutral or mildly basic functional groups in the S1 site of thrombin led to the targeting of imidazole as a S1 binding element and correctly predicted the optimal chain length for connecting this group with the S2 and S3 binding elements. Derivatives of 4-(3-aminopropyl)-imidazole can be selective inhibitors of thrombin demonstrating potent anticoagulant activity.     

Whole-cell uptake and nuclear localization of 1,25-dihydroxycholecalciferol by breast cancer cells (T47 D) in culture

1. The specificity of rat prostatic spermidine synthase and spermine synthase with respect to the amine acceptor of the propylamine group was studied. 2. Spermidine synthase could use cadaverine (1,5-diaminopentane) instead of putrescine, but the Km for cadaverine was much greater and the rate with 1mM-cadaverine was only 10% of that with putrescine. 1,3-Diaminopropane was even less active (2% of the rate with putrescine) and no other compound tested (including longer alpha,omega-diamines, spermidine and its homologues and monoacetyl derivatives) was active. 3. Spermine synthase was equally specific. The only compounds tested that showed any activity were 1,8-diamino-octane, sym-homospermidine, sym-norspermidine and N-(3-aminopropyl)-cadaverine, which at 1mM gave rates 2, 17, 3 and 4% of the rate with spermidine respectively. 4. The formation of polyamine derivatives of cadaverine and to a very small extent of 1,3-diaminopropane was confirmed by exposing transformed mouse fibroblasts to these diamines when synthesis of putrescine was prevented by alpha-difluoromethylornithine. Under these conditions the cells accumulated significant amounts of N-(3-aminopropyl)cadaverine and NN'-bis(3-aminopropyl)cadaverine when exposed to cadaverine and small amounts of sym-norspermidine and sym-norspermine when exposed to 1,3-diaminopropane.     

Sequestration of biogenic amines by alginic and fulvic acids

The interaction of natural (alginic and fulvic acids) and synthetic (polyacrylic acid 2.0 kDa) polyelectrolytes with some protonated polyamines [diamines: ethylendiamine, 1,4-diaminobutane (or putrescine), 1,5-diaminopentane (or cadaverine); triamines: N-(3-aminopropyl)-1,4-diaminobutane (or spermidine), diethylenetriamine; tetramine: N,N'-bis(3-aminopropyl)-1,4-diaminobutane (or spermine); pentamine: tetraethylene-pentamine; hexamine: pentaethylenehexamine] was studied at T=25 degrees C by potentiometry and calorimetry. Measurements were performed without supporting electrolyte, in order to avoid interference, and results were reported at I=0 mol L(-)(1). For all the systems, the formation of (am)L(2)H(i) species was found (am=amine; L=polyelectrolyte; i=1...4, depending on the amine considered). The stability of polyanion-polyammonium cation complexes is always significant, and for high-charged polycations, we observe a stability comparable to that of strong metal complexes. For example, by considering the formation reaction (am)H(i)+2L=(am)L(2)H(i) we found log K(i)=6.0, 6.5 and 10.8 for i=1, 2 and 3, respectively, in the system alginate-spermidine. Low and positive formation DeltaH(degrees) values indicate that the main contribution to the stability is entropic in nature. The sequestering ability of polyelectrolytes toward amines was modelled by a sigmoid Boltzman type equation. Some empirical relationships between stability, charges and DeltaG(degrees) and TDeltaS(degrees) are reported. Mean values per salt bridge of formation thermodynamic parameters (DeltaX(degrees) (n)) are DeltaG(degrees) (n)=-5.8+/-0.4, DeltaH degrees (n)=0.7+/-0.5 and TDeltaS(degrees) (n)=6.5+/-0.5 kJmol(-)(1) for all the systems studied in this work.     

Synthesis of six novel N,N-dialkyl derivatives of spermidine and effects on growth of the fungal plant pathogen Pyrenophora avenae

Six novel N,N-dialkyl derivatives of spermidine were synthesised and examined for activity against the oat stripe pathogen Pyrenophora avenae. Two of these spermidine analogues, N,N-dimethyl-N1-(3-aminopropyl)-1,3-diaminopropane trihydrochloride (27) and N,N-dimethyl-N1-(3-aminopropyl)-1,4-diaminobutane trihydrochloride (28), reduced radial extension of P. avenae on plates when used at 2 mM, and caused more substantial reductions in fungal growth in liquid culture when used at 1 mM. Preliminary data suggest that neither compound affected polyamine biosynthesis, determined by following the incorporation of label from ornithine into polyamines and examining intracellular polyamine concentrations in fungal tissue.     

The amino aldehydes produced by spermine and spermidine oxidation with maize polyamine oxidase have anti-leishmanial effect.

Purified maize polyamine oxidase bound to hydroxylapatite was utilized as an enzymatic reactor to produce oxidized spermidine and spermine. The oxidation products showed a consistent inhibitory activity on Leishmania infantum in mice. 1,3-diaminopropane showed a much lower activity. Moreover chemically prepared 4-aminobutyraldehyde had a similar ED50 to that of oxidized spermidine. It is concluded that among the compounds arising from polyamine oxidation those with the greatest anti-leishmanial effect are 4-aminobutyraldehyde and 1-(3-aminopropyl)-4-aminobutyraldehyde.     

Comparison of the activities of variant forms of eIF-4D. The requirement for hypusine or deoxyhypusine.

Eukaryotic protein synthesis initiation factor 4D (eIF-4D) (current nomenclature, eIF-5A) contains the unique amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). The first step in hypusine biosynthesis, i.e. the formation of the intermediate, deoxyhypusine (N epsilon-(4-aminobutyl)lysine), was carried out in vitro using spermidine, deoxyhypusine synthase, and ec-eIF-4D(Lys), an eIF-4D precursor prepared by over-expression of human eIF-4D cDNA in Escherichia coli. In a parallel reaction, using N-(3-aminopropyl)cadaverine in place of spermidine, a variant form of eIF-4D containing homodeoxyhypusine (N epsilon-(5-aminopentyl)lysine) was prepared. Evidence that N-(3-aminopropyl)cadaverine can also act as the amine substrate for deoxyhypusine synthase in intact cells was obtained by incubating putrescine- and spermidine-depleted Chinese hamster ovary cells with [3H]cadaverine. In these cells, in which [3H]cadaverine is readily converted to N-(3-aminopropyl) [3H]cadaverine, small amounts of [3H]homodeoxyhypusine and another 3H-labeled compound, presumed to be N epsilon-(5-amino-2-hydroxy[3H]pentyl)lysine, were found. eIF-4D stimulates methionyl-puromycin synthesis, an in vitro model assay for translation initiation. Whereas the unmodified precursor ec-eIF-4D(Lys) appeared inactive, the deoxyhypusine-containing form provided a significant degree of stimulation. The variant form containing homodeoxyhypusine, on the other hand, showed little or no activity. These findings emphasize the importance of hypusine or deoxyhypusine for the biological activity of eIF-4D and demonstrate the influence of both the length and chemical nature of its amino alkyl side chain.     

Formation of cadaverine derivatives in Saccharomyces cerevisiae

Abstract The higher homologues of cadaverine, aminopropylcadaverine (APC) and N , N - bis (3-aminopropyl)cadaverine (3APC) were formed by a wild-type strain of Saccharomyces cerevisiae , and by two mutant strains, spe 3-1 and spe 4-1, exhibiting point mutations in the genes for spermidine synthase and spermine synthase, respectively. This, together with the incomplete inhibition of APC and 3 APC formation in the presence of inhibitors of 5-adenosylmethionine decarboxylase and spermidine synthase, suggests that the cadaverine derivatives are formed partly by the operation of a different route. However, the yeast strains were unable to utilise [¹⁴C]aspartate and lysine to form APC and 3APC. Since the ornithine decarboxylase inhibitor adifluoromethylomithine (DFMO) greatly reduced the formation of APC and 3APC, it is suggested that these compounds are formed preferentially in these yeast strains from cadaverine formed by ODC. APC and 3APC formation in the yeast strains was increased substantially following exposure to 37 °C for 2 h.