Antimicrobial and antitumor activity of N-heteroimmine-1,2,3-dithiazoles and their transformation in triazolo-, imidazo-, and pyrazolopirimidines.

The reaction of Appel's salt with o-amino nitrile heterocycles 10-19 gave the corresponding 4-chloro-5-heteroimmine-1,2,3-dithiazoles 20-29 which were evaluated for their antibacterial, antifungal and antitumor activity. Although all these N-heteroimines were devoid of significant antibacterial activity, they showed significant antifungal activity. Moreover, the same derivatives represent highly versatile intermediates in heterocyclic synthesis, in fact the pyrazoleimino dithiazoles 20-26 can be converted in one step into 2-cyano derivatives of the corresponding 4-methoxy-pyrazolo[3,4-d]pyrimidines 30-35 by sodium methoxide in refluxing methanol. This provides a general and attractive route to 4-methoxy-6-cyano pyrazolo[3,4-d]pyrimidines from 1-substituted 5-amino pyrazoles 10-19 in two simple steps. Finally, the isosteric replacement of the pyrazole ring atoms to give the imidazole[3,4-d]pyrimidine and triazole [4,5-d] pyrimidine ring systems was examined.     

A divergent synthesis of [1-14C]-mono-E isomers of fatty acids

A convenient synthesis of [1-14C]-mono-trans fatty acid using olefin inversion as a key-step is described. This methodology allows for a facile synthesis of [1-14C]-labelled mono-trans analogues of oleic, linoleic and linolenic acids. As an example, only eleven steps were necessary to obtain the [1-14C]-mono-E isomers of linolenic acid from its commercial all-Z form. In the first step, Barton's decarboxylation procedure yielded a bromo intermediate. Epoxidation of this compound resulted in the formation of three monoepoxides, which could be separated by HPLC. After identification by 1H NMR and MS, the pure monoepoxides were then subjected to inversion consisting of a stereospecific deoxygenation followed by a beta-elimination step. Finally, the labelling was introduced by substitution of the bromine by a [14C]-cyano group followed by hydrolysis.     

7-deazainosine derivatives: synthesis and characterization of 7- and 7,8-substituted pyrrolo [2,3-d]pyrimidine ribonucleosides

The synthesis of model 7 deazapurine derivatives related to tubercidin and toyocamycin has been performed. Tubercidin derivatives were obtained by simple conversion of the amino group of the heterocyclic moiety of the starting 7-deazadenosine compounds, into a hydroxyl group. Preparation of toyocamycin derivatives was accomplished by treatment of the silylated 6-bromo-5-cyanopyrrolo[2,3-d]pyrimidin-4-one with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-d-ribofuranose. The glycosylation reaction afforded a mixture of 8-bromo 7-cyano 2',3',5' tri-O-benzoyl 7-deazainosine and 6-bromo-5-cyano-3-(2',3',5'-tri-O-benzoyl-beta-d-ribofuranosyl)pyrrolo[2,3-d]-pyrimidin-4-one isomers: The structures were assigned on the basis of NMR spectroscopy studies. Next deprotection treatment gave the novel 7-deazainosine ribonucleosides.     

Synthesis,Antiproliferative, and Antiviral Evaluation of Certain Acyclic 6-Substituted Pyrrolo[2,3-D]-pyrimidine Nucleoside Analogs Related to Sangivamycin and Toyocamycin

Abstract

A number of 6-substituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine and 7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3-d]pyrimidine derivatives related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested for their biological activity. Treatment of 2-amino-5-bromo-3,4-dicyanopyrrole (2) with triethylorthoformate, followed by alkylation via the sodium salt method with either 2-(acetoxyethoxy)methyl bromide or (1,3-diacetoxy-2-propoxy)methyl bromide, furnished the corresponding N-substituted pyrroles 3a and 3b. These compounds were then smoothly converted to the requisite deprotected 4-amino-6-bromopyrrolo[2,3-d]-pyrimidine-5-carbonitriles 5a and 5b (toyocamycin analogs) by methanolic ammonia. The 6-amino-derivatives were obtained by a displacement of the bromo group with liquid ammonia. Conventional functional group transformations involving the 5-cyano group furnished the 5-carboxamide (sangivamycin) and 5-thioamide analogs. Compounds substituted at the 7-position with a ribosyl moiety were active against human cytomegalovirus (HCMV) at micromolar concentrations, but the apparent activity was not selective. The 7-ribosyl compounds also had no activity against human immunodeficiency virus (HIV), though they were all cytotoxic. The new compounds were also evaluated against HCMV, herpes simplex virus type I (HSV-1), HIV, and also for their ability to inhibit the growth of L1210 murine leukemic cells in vitro. None of these compounds with (2-hydroxyethoxy)methyl substituents or 7-(1,3-dihydroxy-2-propoxy)methyl substituent at N-7 showed significant cytotoxicity toward L1210, or toward uninfected human foreskin fibroblasts (HFF cells), and KB cells. Nor were they cytotoxic in human lines CEM or MT2. Only compound 4a was found to be active against HCMV, having an IC₅₀ of 32 μM.     

Syntheses and anti-cancer activities of 2-[1-(indol-3-yl-/pyrimidin-5-yl-/pyridine-2-yl-/quinolin-2-yl)-but-3-enylamino]-2-phenyl-ethanols

Schiff bases prepared by the reactions of substituted amines with indole-/, pyrimidine-/, pyridine-/, and quinoline-aldehydes are made to undergo indium mediated allylation whereby a (substituted amine, allyl)methyl group has been introduced at C-3 of indole, C-5 of pyrimidine, and C-2 of pyridine and quinoline. Amongst the 16 compounds investigated for anti-cancer activities at 59 human tumor cell lines 3, 9-12, and 14 show appreciable activities. The structure-activity relationship studies point that the contribution of phenylglycinol moiety as a part of side chain at C-3 of indole and C-5 of pyrimidine seems to be crucial for exhibiting anti-cancer activities.     

Novel and orally active 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives as selective FLT3 inhibitors

5-(1,3,4-Oxadiazol-2-yl)pyrimidine derivative 1 was identified as a new class of FLT3 inhibitor from our compound library. With the aim of enhancement of antitumor activity of 2 prepared by minor modification of 1, structure optimization of side chains at the 2-, 4-, and 5-positions of the pyrimidine ring of 2 was performed to improve the metabolic stability. Introduction of polar substituents on the 1,3,4-oxadiazolyl group contributed to a significant increase in the metabolic stability. As a result, a series of compounds showed increased efficacy against MOLM-13 xenograft model in mice by oral administration.     

Synthesis of 5-chloroformycin A, 5-chloro-2''-deoxyformycin A and certain related 5,7-disubstituted 3-beta-D-ribofuranosylpyrazolo[4,3-d] pyrimidines from formycin A.

A facile synthesis of 7-amino-5-chloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5-chloroformycin A, 6), 7-amino-5-chloro-3-(2-deoxy-beta-D-erythro-pentofuranosyl) pyrazolo [4,3-d]-pyrimidine (5-chloro-2'-deoxyformycin A, 13) and certain related 5,7-disubstituted pyrazolo[4,3-d]pyrimidine ribonucleosides is described starting with formycin A. Thiation of tri-O-acetyloxoformycin B (4b) with phosphorus pentasulfide, followed 3-beta-D-ribofuranosyl-7-thioxopyrazolo[4,3-d] pyrimidin-5(1H,4H,6H)-one (3b) in excellent yield. Chlorination of 4b with either phosphorus oxychloride or phenyl phosphonicdichloride furnished the key intermediate 5,7-dichloro-3-(2,3, 5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (5a), which on deacetylation afforded 5,7-dichloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5b). Ammonolysis of 5a with liquid ammonia gave 6, whereas with MeOH/NH3, a mixture of 6 and 7-methoxy-5-chloro-3-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine (7) was obtained. Reaction of 6 with lithium azide and subsequent hydrogenation afforded 5-aminoformycin A (10). Treatment of 5a with thiourea gave 5-chloro-3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) pyrazolo[4,3-d]pyrimidine-7(1H,6H)-thione (8a), which on further reaction with sodium hydrosulfide furnished 3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine-5,7(1H,4H,6H)-dithione (11). The four-step deoxygenation procedure using phenoxythiocarbonylation of the 2'-hydroxy group of the 3', 5'-protected 6 gave 5-chloro-2'-deoxyformycin A (13).     

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.     

Solid phase synthesis of structurally diverse pyrimido[4,5-d] pyrimidines for the potential use in combinatorial chemistry

An efficient solid phase synthesis of pyrimido[4,5-d]pyrimidine derivatives is described. Reaction of polymer-bound pyrimidine 1 with urea or thiourea followed by cleavage from the support provided 4-aminopyrimido[4,5-d]pyrimidines 4 and 5 while treatment of 6 with phenyl isocyanate or phenyl isothiocyanate followed by cleavage from resin afforded 3-phenylpyrimido[4,5-d]pyrimidines 9 and 10.     

4-Amino-3-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl)-5-pyrazole-carb oni trile. Synthesis and conversion into a sugar-blocked 5,7-disubstituted formycin analog

The title compound, a potential intermediate to protected C-nucleoside analogs related to formycin A, was synthesized via a new route wherein 2,3,5-tri-O-benzyl-1-O-p-nitrophenyl)-D-ribofuranose was converted to 2,5-anhydro-3,4,6-tri-O-benzyl-D-allonic acid, and further transformed into 4-(tert-butyloxycarbonyl)-5-ethoxycarbonyl-3-(2,3,5-tri-O-benzyl-beta-D- ribofuranosyl)pyrazole. After amidation and dehydration to form the 4-(tert-butyloxycarbonyl)-5-pyrazolecarbonitrile, acidolysis followed by a Curtius-type sequence afforded the 4-amino-5-pyrazole-carbonitrile nucleoside. Treatment of the latter with nitrous acid and copper chloride in a Sandmeyer-type reaction gave a diazonitrile rather than a chloronitrile. Attempts to convert either the aminonitrile or the diazonitrile to 5,7-diamino-3-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl)pyrazolo[4,3-d] pyrimidine (5-aminoformycin A) by condensation with guanidine or N,N-dimethylguanidine were unsuccessful. Condensation of the aminonitrile with carbon disulfide in pyridine provided access to the formycin system in the form of 3-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine-5,7 - dithione.     

Effects of molecular structures on reduction properties of formyl groups in chlorophylls and pheophytins prepared from oxygenic photosynthetic organisms

Reduction of the 7-formyl groups in chlorophyll (Chl) b and its demetalated compound pheophytin (Phe) b was kinetically analyzed by using tert-butylamine-borane complex (t-BuNH(2)·BH(3)), and was compared with that of the 3-formyl groups in Chl d and Phe d. Reduction kinetics of the 7-formyl group in Chl b was similar to that in Phe b in dichloromethane containing 5mM t-BuNH(2)·BH(3). Little difference of the reduction kinetics of the 7-formyl groups between Chl b and Phe b was in sharp contrast to the reduction kinetics of the 3-formyl groups in Chl d and Phe d: the 3-formyl group in Phe d was reduced 5.3-fold faster than that in Chl d. The 7-formyl groups in Chl b and Phe b were reduced more slowly than the 3-formyl groups in Chl d and Phe d, respectively. The difference of the reactivity between the 3- and 7-formyl groups was in line with (13)C NMR measurements of chlorophyllous pigments, in which the chemical shifts of carbon atoms in the 7-formyl groups of Chl b and Phe b were high-field shifted compared with those in the 3-formyl groups of Chl d and Phe d, respectively. These indicate that the 7-formyl groups in chlorophyllous pigments were less reactive for reduction to the corresponding hydroxymethyl groups than the 3-formyl groups due to the difference in electronic states of the formyl groups in the A- and B-rings of the chlorin macrocycle.     

5-Heteroatom substituted pyrazoles as canine COX-2 inhibitors. Part III: molecular modeling studies on binding contribution of 1-(5-methylsulfonyl)pyrid-2-yl and 4-nitrile

The structure-activity relationship toward canine COX-1 and COX-2 in vitro whole blood activity of 4-hydrogen versus 4-cyano substituted 5-aryl or 5-heteroatom substituted N-phenyl versus N-2-pyridyl sulfone pyrazoles is discussed. The differences between the pairs of compounds with the 4-nitrile pyrazole derivatives having substantially improved in vitro activity are highlighted for both COX-2 and COX-1. This difference in activity may be due to the contribution of the hydrogen bond of the 4-cyano group with Ser 530 as shown by our molecular modeling studies. In addition, our model suggests a potential contribution from hydrogen bonding of the pyridyl nitrogen to Tyr 355 for the increased activity over the phenyl sulfone analogs.     

Synthesis and cytotoxic activity of N-(2-diethylamino)ethylcarboxamide and other derivatives of 10H-quindoline

A series of mono- and dimeric N-methylquindoline carboxamides were prepared by Friedlander condensation between methyl 2-amino-3-formyl benzoate and 3-acetoxy-1-acetylindoles, followed by exhaustive methylation with methyl iodide to give N-methylquindoline esters. Direct amination of these, or hydrolysis to the acids and amine coupling via intermediate imidazolides gave the desired carboxamides. The compounds were evaluated in a panel of cell lines in culture. The monomeric compounds showed similar structure-activity relationships to the known indeno[1,2-b]quinolines, with a 4-methyl group increasing potency several-fold. Bis analogues linked through the carboxamide were more cytotoxic than the corresponding monomers in the human leukemia lines, but N-N linked dimers were generally less potent, except for a tetracationic derivative. The most potent monomeric analogue showed moderate growth delay (ca. 5 days) against sub-cutaneously implanted colon 38 tumors in mice.     

Artificially produced [7-formyl]-chlorophyll d functions as an antenna pigment in the photosystem II isolated from the chlorophyllide a oxygenase-expressing Acaryochloris marina

Acaryochloris marina, a chlorophyll (Chl) d-dominated cyanobacterium, is a model organism for studying photosynthesis driven by far-red light using Chl d. Furthermore, studies on A. marina may provide insights into understanding how the oxygenic photosynthetic organisms adapt after the acquisition of new Chl. To solve the reaction mechanism of its unique photosynthesis, photosystem (PS) II complexes were isolated from A. marina and analyzed. However, the lack of a molecular genetic method for A. marina prevented us from conducting further studies. We recently developed a transformation system for A. marina and we introduced a chlorophyllide a oxygenase gene into A. marina. The resultant transformant accumulated [7-formyl]-Chl d, which has never been found in nature. In the current study, we isolated PS II complexes that contained [7-formyl]-Chl d. The pigment composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96±0.04 Chl a, 53.21±1.00 Chl d, and 5.48±0.33 [7-formyl]-Chl d per two pheophytin a molecules. In contrast, the composition of the control PS II complexes was 2.01±0.06 Chl a and 62.96±2.49 Chl d. The steady-state fluorescence and excitation spectra of the PS II complexes revealed that energy transfer occurred from [7-formyl]-Chl d to the major Chl d species; however, the electron transfer was not affected by the presence of [7-formyl]-Chl d. These findings demonstrate that artificially produced [7-formyl]-Chl d molecules that are incorporated into PS II replace part of the Chl d molecules and function as the antenna. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Similar Effect of Rifampin and Other Rifamycin Derivatives on Vaccinia Virus Morphogenesis

Membrane-limited structures, resembling virus envelope precursors previously shown to form during the interruption of poxvirus assembly by rifampin, were now observed by electron microscopy in vaccinia-infected HeLa cells treated with a series of rifamycin derivatives. The active compounds N-demethyl rifampin, AF/DMI, and 3-formyl rifamycin SV lacked, respectively, a methyl group, the piperazine ring, and the hydrazone portion of rifampin. A vaccinia mutant selected only for resistance to rifampin was also resistant to the effect on morphogenesis produced by all of the rifamycin derivatives. We concluded that this antiviral effect was specific and was a property associated with the macrocyclic ring rather than the hydrazone-containing side chain of rifampin. In addition to their effects on vaccinia morphogenesis, 3-formyl rifamycin SV and AF/DMI had unusual cytotoxic effects.     

Syntheses and Antitumor Evaluation of C(6)‐Isobutyl‐ and C(6)‐Isobutenyl‐Substituted Pyrimidines,and Dihydropyrrolo[1,2‐c]pyrimidine‐1,3‐diones

A growing body of evidence supports that pyrimidine derivatives, in which the sugar residues have been replaced by acyclic side chains, might be developed as promising anticancer agents that interfere with tumor cell proliferation, survival, and metastatic formation. In this work, we prepared novel pyrimidines bearing i‐Bu (i.e., 3, 4 , and 7 – 9 ) and isobutenyl (i.e., 5 and 10 ) side chains at C(6) and examined their in vitro effects on tumor cell lines. The dihydropyrrolo[1,2‐c]pyrimidine‐1,3‐diones 6 and 11 were obtained as products of intramolecular cyclization, which occurred during the removal of Bn in 5 or MeO protecting groups in 10 . Fluorination of 3 with diethylaminosulfur trifluoride (DAST) and then dehydrohalogenation of the resulting fluorinated derivative 4 afforded 6‐isobut‐2′‐enyl pyrimidine derivative 5 with a C(2′)C(3′) bond. For the preparation of 6‐isobut‐1′‐en‐1‐yl pyrimidine 10 , a synthetic strategy involving acetylation of the 1,3‐diols was applied. Antitumor evaluation of compounds 3 – 11 showed that 2,4‐dimethoxypyrimidine containing 6‐[(1,3‐dibenzyloxy)‐2‐hydroxy]methyl side chain, 3 , exerted a strong antiproliferative effect on the studied tumor cell lines. Additionally, it was shown that the mechanism of antiproliferative effect of 3 in HeLa cells include early G2/M arrest and apoptosis, as well as a p53‐independent S‐phase arrest upon prolonged treatment.     

Synthesis and biological evaluation of 7-(aminoalkyl)pyrazolo[1,5-a]pyrimidine derivatives as cathepsin K inhibitors

A series of novel 7-aminoalkyl substituted pyrazolo[1,5-a]pyrimidine derivatives were synthesized and tested for inhibition of cathepsin K. The synthetic methodology comprises cyclization of 5-aminopyrazoles with N-Boc-α-amino acid-derived ynones followed by transformation of the ester and the Boc-amino functions. It allows for easy diversification of the pyrazolo[1,5-a]pyrimidine scaffold at various positions. Molecular docking studies with pyrazolo[1,5-a]pyrimidine derivatives were also performed to elucidate the binding mode in the active site of cathepsin K. The synthesized compounds exhibited moderate inhibition activity (K_i ≥ 77 μM).     

Cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity.

The enzyme 5-formyl tetrahydrofolate cyclodehydrase plays an important role in the conversion of 5-formyl tetrahydrofolate to 5,10-methenyl tetrahydrofolate. A second enzyme, cyclohydrolase, converts 5,10-methenyl tetrahydrofolate to 10-formyl tetrahydrofolate. These folate derivatives play a significant part in the biosynthesis of purines. A method has been devised for the cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity which uses 5-formyl tetrahydrofolate or 5,10-methenyl tetrahydrofolate as substrate respectively, blocking possible interferences by other enzymes, and allows the nonenzymatic reduction of nitro-blue tetrazolium by 5,10-methenyl tetrahydrofolate formed by the action of the cyclodehydrase on the substrate 5-formyl tetrahydrofolate, and by 10-formyl tetrahydrofolate formed by the action of cyclohydrolase on the substrate 5,10-methenyl tetrahydrofolate, thus revealing intracellular sites of enzyme activity. The methods appear to show only intracellular localization of the blue formazan deposits of reduced tetrazolium. The distribution of positivity in cells of human blood and bone marrow is described.     

Synthesis of 1′-C-Cyano Pyrimidine Nucleosides and Characterization as HCV Polymerase Inhibitors

Ribose modified 1′-C-cyano pyrimidine nucleosides were synthesized. A silver triflate mediated Vorbrüggen reaction was used to generate the nucleoside scaffold and follow-up chemistry provided specific ribose modified analogs. Nucleosides and phosphoramidate prodrugs were tested for their anti-HCV activity.