Chemistry

A study of the kinetics and mechanism of the reaction between the dinuclear Pt complex [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1) and the 14-mer duplex 5'-d(ATACATG(7)G(8)TACATA)-3'.5'-d(TATG(25)TACCATG(18)TAT)-3' is reported. [(1)H,(15)N]-HSQC NMR was used to follow the reaction at 298 K, pH 5.4. The product is primarily the 5'-5' 1,4-interstrand cross-link between G(8) and G(18) bases and exists in two conformational forms. No evidence for the possible 1,2-intrastrand G(7)G(8) adduct was seen, confirming the preferential formation of interstrand cross-links by these dinuclear complexes. An initial electrostatic association of (15)N-1 with the duplex is indicated by changes in its (1)H/(15)N chemical shifts, followed by aquation of 1 to form the monoaqua monochloro species 2, with a rate constant of 4.00+/-0.03x10(-5) s(-1). Monofunctional binding to the duplex occurs primarily at G(8), the…

2,6,8-Trichloro-7-methylpurine (3) was converted to 2-chloro-8,9-dihydro-7-methyl-8-thioxopurin-6(1H)-one (5) by utilizing the difference in reactivity of the 2-, 6-, and 8-positions in the trichloropurine ring system to nucleophilic displacement. Compound 5 was subsequently glycosylated with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose according to the Vorbrüggen procedure to yield 2-chloro-8,9-dihydro-7-methyl-9-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosy l)-8- thioxopurin-6(1H)-one (6). Removal of the benzoyl protecting groups, followed by amination of 7 with liquid ammonia at 150 degrees C, gave 7,8-dihydro-7-methyl-8-thioxoguanosine (2). The structure of compound 2 was confirmed by X-ray crystallographic analysis. Compounds 1 (7,8-dihydro-7-methyl-8-oxoguanosine) and 2 were evaluated for activity in various animal virus infection models. Against banzi, Semliki Forest, and San Angelo viruses in mice, 2 was highly active when administered before virus inoculation.

2,6,8-Trichloro-7-methylpurine (3) was converted to 2-chloro-8,9-dihydro-7-methyl-8-thioxopurin-6(1H)-one (5) by utilizing the difference in reactivity of the 2-, 6-, and 8-positions in the trichloropurine ring system to nucleophilic displacement. Compound 5 was subsequently glycosylated with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose according to the Vorbrüggen procedure to yield 2-chloro-8,9-dihydro-7-methyl-9-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosy l)-8- thioxopurin-6(1H)-one (6). Removal of the benzoyl protecting groups, followed by amination of 7 with liquid ammonia at 150 degrees C, gave 7,8-dihydro-7-methyl-8-thioxoguanosine (2). The structure of compound 2 was confirmed by X-ray crystallographic analysis. Compounds 1 (7,8-dihydro-7-methyl-8-oxoguanosine) and 2 were evaluated for activity in various animal virus infection models. Against banzi, Semliki Forest, and San Angelo viruses in mice, 2 was highly active when administered before virus inoculation.

Novel analogues of the naturally occurring purine nucleosides were synthesized in the thiazolo[4,5-d]pyrimidine ring system to determine the immunomodulatory effects of insertion of a sulfur atom in place of nitrogen at position 7 of the purine ring. In particular, 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (7, guanosine analogue), 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,5,7(3H,4H,6H) trione (8, xanthosine analogue), 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione (10, inosine analogue), and 7-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidin-2(3H)-one (32, adenosine analogue) were prepared, as well as the 8-mercaptoguanosine (14) and 6-mercaptoguanosine (17) analogues. Single-crystal X-ray studies confirmed the structural assignment of 17 and 32 as having the beta-configuration with the site of glycosylation at N3. The nucleosides were evaluated for their ability to potentiate various murine immune functions in direct comparison to the known active agents 8-bromoguanosine (1), 8-mercaptoguanosine (2), and 7-methyl-8-oxoguanosine (3). Two of the guanosine analogues, 7 and 14, were found to exhibit significant immunoactivity relative to the positive control compounds (1-3), while the adenosine, inosine, xanthosine, and 6-mercaptoguanosine analogues were devoid of activity. Compound 7 exhibited greater immunoactivity than any of the other guanosine analogues and derivatives in all test systems. Specifically, 7 was shown to be about twice as potent as 3 in the murine spleen cell mitogenicity assay. In addition, treatment with 7 produced about a 4-fold increase in natural killer cell cytotoxicity, while treatment with 3 afforded a 3-fold increase over controls. Finally, 7 provided excellent protection (92% survivors compared to 0% for placebo controls) against Semliki Forest virus in mice. Induction of interferon may account for the major mode of action of these guanosine analogues.

Several guanosine analogues were synthesized in the pyrazolo[3,4-d]pyrimidine ring system with various substituents at the 3-position. The new analogues prepared here include the CH3 (2-amino-3-methyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13a), the phenyl (2-amino-3-phenyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13b), and the NH2 (3,6-diamino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4(5H)- one, 17) substituted derivatives. These new agents, as well as several other 3-substituted derivatives including H, Br, OCH3, COOH, and oxo, were evaluated for their ability to potentiate certain murine immune functions relative to the known active agent 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (4, 7-thia-8-oxoguanosine). The biological evaluation included the (1) ex vivo determination of increased natural killer cell function and (2) in vivo antiviral protection against a lethal challenge of Semliki Forest virus. The 3...

The in vitro antiviral activity of 28 nucleosides against the parainfluenza virus type 3 has been analyzed by using a novel computer aided receptor modeling procedure. The method involves an extensive modification of our earlier work (Ghose, A. K.; Crippen, G. M. J. Med. Chem. 1985, 28, 333). It presents a more straightforward algorithm for the steps that suffered from subjectivity in the earlier method. The method first determines the possible low-energy conformations of the nucleosides, and assigns a priority value for each conformation of each molecule. It then performs the following steps repeatedly, until it finds an acceptable solution. Starting from the conformation of highest priority, the various energetically allowed conformations of the other molecules are superimposed on it. On the basis of the physicochemical property matching (or overlapping), the best superposition is determined. The superimposed molecules are dissected into a minimum number of parts and the local physicochemical properties at different regions are correlated with their binding data (antiviral activity). A modified version of distance geometry has been used for geometric comparison of the structure of the molecules. On the basis of the virus rating (VR) of 28 ribonucleosides, this procedure hypothesized the minimum-energy conformation of 6-(methylthio)-9-beta-D-ribofuranosylpurine as a reference conformation and used three physicochemical properties, namely hydrophobicity, molar refractivity, and formal charge density for property matching. The binding-site cavity was divided into seven regions or pockets to differentiate the nature of interaction quantitatively. The model suggests that the 2- and 3-positions of the purine ring and the corresponding atoms of the other rings get some steric repulsion, and nucleosides having a single five-membered heterocyclic ring will better fit this virus. The methylthio group gets a strong attraction from dispersive interaction. Both hydrophilic and dispersive groups are attractive here. Although our calculation supports the previously suggested active conformation of ribavirin, it shows that it is not the global minimum-energy conformation. The difference lies in the orientation of the amide group. The calculated viral rating from this model showed a correlation coefficient of 0.971 with the observed values, and the explained variance and the standard deviation of the fit were 0.880 and 0.125, respectively.

A novel and direct synthesis of the antiviral and antitumor agent 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (ARPP, 8) and its alpha-anomer (11) has been developed. Treatment of 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine (1) with 2,3-O-isopropylidene-D-ribofuranosylamine gave an anomeric mixture of 2,4,6-trichloro-8-(2,3-O-isopropylidene-beta- and -alpha-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidines (3 and 4) in a ratio of 1.0:0.7. A nucleophilic displacement of the 4-chloro group of 3 and 4 with NH3 furnished 4-amino-2,6-dichloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino ] pyrimido[5,4-d]pyrimidine (6) and its alpha-anomer (9), respectively. Catalytic hydrogenation of 6 and 9, followed by deisopropylidenation gave ARPP (8) and the alpha-anomer 11, respectively. Similarly, 3 and 4 have been transformed to 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine (MRPP, 14) and its alpha-anomer (17). Application of this procedure to 3 with NH2Me or NHMe2 resulted in the synthesis of 4-(methylamino)- and 4-(dimethylamino)-8-(beta-D-ribofuranosylamino)pyrimido [5,4-d]pyrimidine (24 and 27, respectively). A synthesis of 8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidin-4(3H)-one (21) has also been accomplished from 3 in three steps. Selective hydrogenation of 6 furnished 4-amino-6-chloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino] pyrimido[5,4-d]pyrimidine (36), the structure of which was established by single-crystal X-ray diffraction analysis. Deisopropylidenation of 36 gave 6-chloro-ARPP (37). Extended treatment of 36 with NH3 furnished 4,6-diamino-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino]pyrimido [5,4-d]pyrimidine (34), which on deisopropylidenation gave 6-amino-ARPP (35). An unambiguous synthesis of 34 and 36 has also been accomplished by the reaction of 4,6,8-trichloropyrimido[5,4-d]pyrimidine (28) with 2, followed by the treatment with NH3. Nucleophilic displacement studies with 1, 6, and 28 indicated the reactivity of the halogens in these compounds is in the order of 8 greater than 4 greater than 6 greater than 2. The structures of 3 and 9 have been assigned on the basis of 1H NMR data and further confirmed by single-crystal X-ray diffraction analysis. The exocyclic aminonucleosides synthesized during this study were tested for their activity against several RNA and DNA viruses in vitro and against L1210, WI-L2, and LoVo/L in cell culture. The effect of these compounds on the de novo nucleic acid biosynthesis has been studied. Compound 14 (MRPP) exhibited enhanced activity against L1210 in vivo, when compared to ARPP (8).

The synthesis of the thio analogue (thio-DHPG, 2) of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) is described. The synthesis of 2 proceeded via the condensation of acetoxymethyl sulfide 9 with diacetylguanine 10 to give the protected nucleoside analogue 11. Although catalytic hydrogenolysis failed, the benzyl ether functionalities of 11 were successfully cleaved by an acetolysis reaction to furnish 14. Ammonolysis of 14 gave 2, which was also transformed to sulfoxide 15 and sulfone 16. Preliminary in vitro screening indicated that 2 exhibited comparable activity to DHPG against herpes simplex virus type 1 (HSV-1) but was less active against the type 2 virus (HSV-2) and human cytomegalovirus (HCMV). In a mouse encephalitis model (HSV-2), subcutaneous treatment with 2 led to a 53% reduction in mortality at a dose of 100 mg/kg per day.

2,6,8-Trichloro-7-methylpurine (3) was converted to 2-chloro-8,9-dihydro-7-methyl-8-thioxopurin-6(1H)-one (5) by utilizing the difference in reactivity of the 2-, 6-, and 8-positions in the trichloropurine ring system to nucleophilic displacement. Compound 5 was subsequently glycosylated with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose according to the Vorbrüggen procedure to yield 2-chloro-8,9-dihydro-7-methyl-9-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosy l)-8- thioxopurin-6(1H)-one (6). Removal of the benzoyl protecting groups, followed by amination of 7 with liquid ammonia at 150 degrees C, gave 7,8-dihydro-7-methyl-8-thioxoguanosine (2). The structure of compound 2 was confirmed by X-ray crystallographic analysis. Compounds 1 (7,8-dihydro-7-methyl-8-oxoguanosine) and 2 were evaluated for activity in various animal virus infection models. Against banzi, Semliki Forest, and San Angelo viruses in mice, 2 was highly active when administered before virus inoculation.

We have previously reported the antioxidant and neuroprotective activities of the aqueous extract of four varieties of Ficus deltoidea (Fd) (Moraceae) namely var kunstleri (Fdvk), var angustifolia (Fdva), var deltoidea (Fdvd) and var intermedia (Fdvi). In this study, flavonoid constituents in aqueous leaf and fig extracts of the four varieties were analyzed and characterized using liquid chromatography mass spectrometer quadrupole-time of flight (LCMS-Q-TOF) via hierarchical cluster analysis (HCA) technique. The HCA dendrogram revealed that the abundant flavonoids among the eight samples are epicatechin, quercetin-3-rutinoside, quercetin 5,4'-di-O-beta-D-glucopyranoside, myricetin and naringenin. The study found that the distribution of the flavonoids differed between the four varieties and varied within the plant parts. To date, the flavonoid distribution of the different plant parts of the four varieties has not been documented. A positive correlation was observed between flav...

In clinical practice, deep brain stimulation (DBS) is effective for treatment of motor symptoms in Parkinson's disease (PD). However, the mechanisms have not been understood completely. There are some reports that electrical stimulation exerts neuroprotective effects on the central nervous system diseases including cerebral ischemia, head trauma, epilepsy and PD, although there are a few reports on neuroprotective effects of spinal cord stimulation (SCS). We investigated the neuroprotective effects of high cervical SCS on PD model of rats. Adult female Sprague-Dawley rats received hour-long SCS (2, 50 or 200 Hz) with an epidural electrode at C1-2 level for 16 consecutive days. At 2 days after initial SCS, 6-hydroxydopamine (6-OHDA) was injected into the right striatum of rats. Behavioral evaluations of PD symptoms were employed, including cylinder test and amphetamine-induced rotation test performed at 1 and 2 weeks after 6-OHDA injection. Animals were subsequently euthanized fo...