Antifungal activities of two new azasordarins, GW471552 and GW471558, in experimental models of oral and vulvovaginal candidiasis in immunosuppressed rats.

Sordarins constitute a new class of antifungal agents with a novel mechanism of action involving the selective inhibition of fungal protein synthesis. A further evolution of this class of antifungals has led to a new family of sordarin derivatives called azasordarins. The therapeutic efficacies of two new azasordarins, GW471552 and GW471558, were studied in experimental models of oral and vulvovaginal candidiasis in immunosuppressed rats. In all cases rats were immunosuppressed with dexamethasone in the drinking water. Oral candidiasis was established by inoculating 0.1 ml of a yeast suspension containing 5 x 10(8) cells of Candida albicans 4711E with a cotton swab on three alternate days. Vulvovaginal candidiasis was established in ovariectomized and estrus-induced rats by intravaginal inoculation of 10(7) CFU of C. albicans 4711E in 0.1 ml of saline. GW471552 and GW471558 were administered at 1, 5, and 10 mg/kg of body weight via the subcutaneous route. In oral candidiasis, azasordarins were administered each 8 h for 7 consecutive days, while in vaginal candidiasis the compounds were given each 4 h for 3 consecutive days. Antifungal activity of azasordarins was assessed by colony counts and by histological examination 1 day after treatment. In the oral infection model, GW471552 and GW471558 administered at 5 mg/kg significantly reduced (P < 0.05) the number of CFU of C. albicans compared with untreated controls. In addition, GW471552 and GW471558 given at 10 mg/kg eradicated C. albicans from the oral cavities of 100% of infected animals. Against vulvovaginal infection, both compounds showed significant therapeutic efficacy. GW471552 was able to eradicate the vaginal fungal burden at a dose of 10 mg/kg, and it significantly reduced the number of CFU of C. albicans in vaginas of rats treated with a dose of 5 mg/kg (P < 0.05). GW471558 showed greater efficacy, eradicating the fungal burden of 100% of infected rats at a dose of 5 mg/kg and significantly reducing (P < 0.05) the C. albicans vaginal counts even at a dose of 1 mg/kg. In both therapeutic efficacy studies, the histological findings confirmed the microbiological results. The experimental results presented show that the tested azasordarins are effective against oral and vulvovaginal candidiasis in immunosuppressed rats and could be promising antifungal agents for use in humans.

Nitric oxide-producing CD11b(+)Ly-6G(Gr-1)(+)CD31(ER-MP12)(+) cells in the spleen of cyclophosphamide-treated mice: implications for T-cell responses in immunosuppressed mice.

During recovery from intensive chemotherapy with cyclophosphamide (CTX), mice suffer a severe but transitory impairment in spleen cell proliferation to T-cell mitogens (Con A or anti-CD3 plus IL-2). Although CTX treatment reduced spleen T-cell cellularity, this cannot fully account for T-cell unresponsiveness. The results showed that CTX induces the colonization of spleen by an immature myeloid CD11b(+)Ly-6G(+)CD31(+) population. Its presence closely correlated with the maximum inhibition of T-cell proliferation. Moreover, this suppressive activity was dependent on nitric oxide (NO) production in cultures since (1) higher amounts of nitric oxide and inducible nitric oxide synthase (iNOS) mRNA were produced in CTX spleen cells (CTX-SC) than in control splenocyte cultures and (2) NOS inhibitors greatly improved the proliferation of T lymphocytes. Nitric oxide production and suppressive activity were also dependent on endogenous interferon-gamma (IFN-gamma) production since anti-IFN-gamma abrogated both effects. Finally, iNOS protein expression was restricted to a heterogeneous population of CD31(+) cells in which CD11b(+)Ly-6G(+) cells were required to suppress T-cell proliferation. These results indicated that CTX might also cause immunosuppression by a mechanism involving the presence of immature myeloid cells with suppressor activity. This may have implications in clinical praxis since inappropriate immunotherapies in patients treated with intensive chemotherapy could lead to deleterious T-cell responses. (Blood. 2000;95:212-220)

Activities of sordarins in murine histoplasmosis.

Sordarins are new antifungals which inhibit fungal protein synthesis by blocking elongation factor 2. Three compounds were evaluated in a murine model of histoplasmosis. Immune-competent mice were infected intravenously with 10(6) to 10(8) CFU of Histoplasma capsulatum yeast cells. Mice were treated either orally with sordarins or fluconazole from day 2 through 8 after infection or intraperitoneally with amphotericin B during the same period. Protection was measured by increased rates of survival for 30 days after infection or reduction of lung or kidney tissue counts 9 days after infection. All three of the antifungal drugs tested were protective compared with controls. Sordarins were effective at doses as low as 2 mg/kg of body weight/day. This novel class of drugs compared favorably with amphotericin B and fluconazole for the treatment of histoplasmosis.

Sordarins: in vitro activities of new antifungal derivatives against pathogenic yeasts, Pneumocystis carinii, and filamentous fungi.

GM 193663, GM 211676, GM 222712, and GM 237354 are new semisynthetic derivatives of the sordarin class. The in vitro antifungal activities of GM 193663, GM 211676, GM 222712, and GM 237354 against 111 clinical yeast isolates of Candida albicans, Candida kefyr, Candida glabrata, Candida parapsilosis, Candida krusei, and Cryptococcus neoformans were compared. The in vitro activities of some of these compounds against Pneumocystis carinii, 20 isolates each of Aspergillus fumigatus and Aspergillus flavus, and 30 isolates of emerging less-common mold pathogens and dermatophytes were also compared. The MICs of GM 193663, GM 211676, GM 222712, and GM 237354 at which 90% of the isolates were inhibited (MIC90s) were 0.03, 0.03, 0.004, and 0.015 microg/ml, respectively, for C. albicans, including strains with decreased susceptibility to fluconazole; 0.5, 0.5, 0.06, and 0.12 microg/ml, respectively, for C. tropicalis; and 0.004, 0.015, 0.008, and 0.03 microg/ml, respectively, for C. kefyr. GM 222712 and GM 237354 were the most active compounds against C. glabrata, C. parapsilosis, and Cryptococcus neoformans. Against C. glabrata and C. parapsilosis, the MIC90s of GM 222712 and GM 237354 were 0.5 and 4 microg/ml and 1 and 16 microg/ml, respectively. The MIC90s of GM 222712 and GM 237354 against Cryptococcus neoformans were 0.5 and 0.25 microg/ml, respectively. GM 193663, GM 211676, GM 222712, and GM 237354 were extremely active against P. carinii. The efficacies of sordarin derivatives against this organism were determined by measuring the inhibition of the uptake and incorporation of radiolabelled methionine into newly synthesized proteins. All compounds tested showed 50% inhibitory concentrations of <0.008 microg/ml. Against A. flavus and A. fumigatus, the MIC90s of GM 222712 and GM 237354 were 1 and 32 microg/ml and 32 and >64 microg/ml, respectively. In addition, GM 237354 was tested against the most important emerging fungal pathogens which affect immunocompromised patients. Cladosporium carrioni, Pseudallescheria boydii, and the yeast-like fungi Blastoschizomyces capitatus and Geotrichum clavatum were the most susceptible of the fungi to GM 237354, with MICs ranging from /=2 microg/ml. In summary, we concluded that some sordarin derivatives, such as GM 222712 and GM 237354, showed excellent in vitro activities against a wide range of pathogenic fungi, including Candida spp., Cryptococcus neoformans, P. carinii, and some filamentous fungi and emerging invasive fungal pathogens.

Synthesis and antiviral activity of 3'-C-cyano-3'-deoxynucleosides.

A series of 3'-C-cyano-3'-deoxynucleosides have been synthesized and evaluated as antiviral agents. Reaction of 2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-erythro-pentofuranos- 3'-ulosyl derivatives of uracil, 4-N-acetylcytosine, and adenine with sodium cyanide gave a mixture of epimeric cyanohydrins, which after 3'-deoxygenation yielded the corresponding 3'-C-cyano-3'-deoxy-beta-D-xylo-pentofuranosyl derivatives 10. These compounds were epimerized to the corresponding beta-D-ribo-pentofuranosyl derivatives 11. Desilylation of 10 and 11 gave the deprotected 3'-C-cyano-3'-deoxy-beta-D-xylo- and -ribo-pentofuranosyl nucleosides. These derivatives of uridine, cytidine, and adenine, as well as the 3'-C-cyano-3'-deoxy-beta-D-xylo- and -ribo-pentofuranosyl, 3'-C-cyano-2',3'-dideoxy-beta-D-threo- and -erythro-pentofuranosyl, and 3'-C-cyano-2',3'-dideoxy-beta-D-glycero-pent-2'-enofuranosyl derivatives of thymine, were evaluated for their antiviral activity. None of the compounds proved active against the replication of retroviruses (human immunodeficiency virus, murine sarcoma virus) at concentrations that were not toxic to the host cells. However, the 3'-C-cyano-3'-deoxy-beta-D-xylo- (12e) and -ribo-pentofuranosyl (13e) derivatives of adenine showed activity against some DNA (i.e., vaccinia) and RNA (i.e., Sindbis, Semliki forest) viruses at concentrations well below the cytotoxicity threshold.

Effect of a uridine 5'-diphosphate glucose analogue on herpes simplex keratitis in rabbits and vaginal infection in guinea pigs.

A uridine 5'-diphosphate glucose analogue, active in vitro against herpes simplex type 1 and 2 viruses, was assayed in rabbit infected corneas with the above viruses. The infected eyes were treated by drug instillation thrice daily and evaluation of ocular lesion was performed by slit-lamp biomicroscopy. The compound [[[5'-(2'',3'',4'',6''-tetra-O-benzoyl-alpha-D-glucopyranosyl)oxy] carbonyl]amino)sulfonyl]uridine shows a moderate antiviral activity, resulting in a reduction in the severity of clinical illness during acute infection. Vaginal infection of guinea pigs with herpes simplex type 2 virus was treated topically by instillation twice daily with the compound. The effect on clinical evolution was related to viral shedding from the genital tract, and a moderate reduction of both parameters in respect to the infected untreated controls was observed.

Synthesis and antiviral activity of 5'-O-(substituted) sulfamoyl pyrimidine nucleosides.

5'-O-[N-(Aminoacyl or isobutyryl)sulfamoyl]uridines 4a-e, 5a-e and 5'-O-[N-(isopropyl)sulfamoyl]cytidines 7-9 have been synthesized and tested against herpes simplex virus type 2. Condensation of 2',3'-O-isopropylidene-5'-O-sulfamoyluridine with the N-hydroxysuccinimide esters of Boc-Gly, Boc-L-Ala, Boc-D-Ala and Boc-L-Phe, gave 4a-d which, on deprotection under acidic conditions, provided 5a-d. A similar condensation of 2',3'-di-O-acetyl-5'-O-sulfamoyluridine with the N-hydroxysuccinimide ester of isobutyric acid afforded 4e which on deacylation led to 5e. 5'-O-[N-(Isopropyl)sulfamoyl]-2',3'-O-isopropylidenecytidine (7) was prepared by reaction of 2',3'-O-isopropylidene-4-N-[(dimethylamino)methylene] cytidine with N-isopropylsulfamoyl chloride. Acidic hydrolysis of 7 provided 8 which, upon acetylation, gave the corresponding 2',3'-di-O-acetyl derivative 9. Compounds 7-9 show antiviral effect. Structure-activity relationships are discussed.

Mode of action of a new type of UDP-glucose analog against herpesvirus replication.

The mode of action of a new type of UDP-glucose analog against herpes simplex virus type 1 (HSV-1) replication was examined. The analog showed good selectivity and potent activity. At 10 micrograms/ml, P-536 inhibited the formation of infectious HSV-1 by more than 90%, whereas at 100 micrograms/ml it had no cytotoxic effects, as evidenced by phase-contrast microscopy. P-536 showed a wide spectrum of action and was active against HSV-1, adenovirus type 5, vaccinia virus, poliovirus type 1, encephalomyocarditis virus, vesicular stomatitis virus, influenza virus, and measles virus, irrespective of whether these viruses have lipidic envelopes or not. P-536 clearly inhibited protein glycosylation if added at the time when late viral proteins were being synthesized. Moreover, it also interfered with the synthesis of nucleic acids and the phosphorylation of nucleosides. If P-536 was present from the beginning of infection, HSV-1 replication was blocked at an early step and the infected cells continued to synthesize cellular proteins for long periods.

Synthesis of some derivatives of 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl)-3-quino linecarbo xylic acid (norfloxacin) as potential antibacterial agents.

Twelve new fluoroquinolones, structurally related to norfloxacin, have been synthesized in order to investigate the effect of substituents at the secondary nitrogen of the piperazine ring on antimicrobial activity. The new substances (carbamates, isoureas, guanidines, ureas and cyanamides) tested on a variety of gram-positive and gram-negative organisms showed lower activity than the model compound.

Antiviral activity of uridine 5'-diphosphate glucose analogues against some enveloped viruses in cell culture.

Twenty five analogues of uridine 5'-diphosphate glucose were screened against herpes simplex type 2, vaccinia virus, Sindbis virus and African swine fever virus. After screening, the compound 5'-[[[[(2",3",4",6"-tetra-O-benzoyl-alpha-D- glucopyranosyl)oxi]carbonyl]amino]sulfonyl]uridine (2), the synthesis of which has been reported (Camarasa et al., J. Med. Chem. 28, 40-46, 1985), was selected for further study. This compound showed in vitro activity against all viruses tested. The replication of herpes virus type 2 and African swine fever virus was completely inhibited at 100 micrograms/ml and 150 micrograms/ml respectively; vaccinia virus and Sindbis virus were inhibited to a lesser extent. The compound may inhibit several steps in the viral replication process.

Synthesis and antiviral evaluation of nucleosides of 5-methylimidazole-4-carboxamide.

Due to the antiviral activity of certain 5-substituted imidazole nucleosides related to ribavirin, 5-methylimidazole-4-carboxamide nucleosides having beta-D-ribofuranosyl, 2-deoxy-beta- and -alpha-D-ribofuranosyl, and (2-hydroxyethoxy)methyl moieties have been prepared and tested as antiviral agents. 1-beta-D-Ribofuranosyl-5-methylimidazole-4-carboxamide was obtained by deacetylation of the corresponding tri-O-acetyl nucleoside 11 or by deacetylation and ammonolysis of the blocked ethyl 5-methylimidazole-4-carboxylate nucleoside 10, which was prepared from the stannic chloride catalyzed condensation of the trimethylsilyl derivative of ethyl 4(5)-methylimidazole-5(4)-carboxylate. Glycosylation of 4(5)-methylimidazole-5(4)-carboxamide with 3,5-di-O-p-toluoyl-2-deoxy-D-erythro-pentofuranosyl chloride via mercuric cyanide method provided an anomeric mixture of the blocked 5-methylimidazole-4-carboxamide deoxynucleoside 14 along with an anomeric mixture of the 4-methyl 5-carboxamide isomer 15. Separation of compound 14 into the corresponding beta and alpha anomers was achieved by conversion to the 3',5'-di-O-acetyl derivatives 17 and 18, which after chromatographic separation were deacetylated to give 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-methylimidazole-4-carboxa mid e and its alpha anomer 20. 1-[(2-Hydroxyethoxy)methyl]-5-methylimidazole-4-carboxamide was prepared by alkylation of the imidazole 13 with (2-acetoxyethoxy)methyl bromide followed by treatment with methanolic ammonia. All these imidazole nucleosides were tested in HeLa cell cultures against type 1 herpes simplex and vesicular stomatitis viruses. The ribofuranosyl derivative 12 showed a significant activity against type 1 herpes simplex virus.

Uridine 5'-diphosphate glucose analogues. Inhibitors of protein glycosylation that show antiviral activity.

A series of analogues of uridine 5'-diphosphate glucose and uridine 5'-diphosphate glucosamine have been synthesized by reaction of 2,3,4,6-tetra-O-benzyl-, 2,3,4,6-tetra-O-benzoyl-, 2,3,4,6-tetra-O-acetyl-, and 2,3,4,6-tetra-O-palmitoyl-alpha-D-glucopyranose and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranose with chlorosulfonyl isocyanate and 2',3'-O-isopropylideneuridine. Isopropylidene and acetyl groups of the resulting 5'-O-[[[[(alpha-D-glucopyranosyl)oxy]carbonyl]amino]sulfonyl] -2',3'-O-isopropylideneuridine derivatives were removed by reaction with a TFA/water (5:1) mixture and methanolic ammonia, respectively. The 5'-O-[[[[(2",3",4",6"-tetra-O-benzyl-and 2",3",4",6"-tetra-O-benzoyl-alpha-D-glucopyranosyl)oxy]carbonyl] amino]sulfonyl]-2',3'-O-isopropylideneuridine (13 and 19) and the corresponding deisopropylidenated derivatives showed antiviral activity as determined by the inhibition of the cytopathic effect induced by HSV-1 replication and by the plaque assay method. Compound 13 inhibited glycosylation of proteins in HSV-1 infected HeLa cells.

[Design and synthesis of anticancer drugs]. / Diseño y síntesis de fármacos anticancerosos.

The authors write about the importance of rational design and organic synthesis for the development of anti-cancer drugs. They give some examples of new alkylating agents and nucleoside antimetabolites.

[Synthesis, cytostatic activity and mechanism of action of N-glycosylhalomethylazoles. A new class of alkylating agents]. / Sintesis, actividad citostática y mecanismo de acción de N-glicosil-halometil-azoles. Un nuevo tipo de agentes alquilantes.

A model to produce a new type of alkylating agents having anti-cancer activity is described. They consist of a halomethyl-azol group of the benzyl type as alkylating center, and a sugar moiety as a carrier fragment. Alkylating nucleosides were produced as halomethyl-pyrazole, -imidazole, 1.2.3-triazole, and 1.2.4-triazole derivatives following the model, and tested upon Ehrlich ascites cancer and P-388 lymphocytic leukemia.

Alkylating nucleosides 1. Synthesis and cytostatic activity of N-glycosyl(halomethyl)-1,2,3-triazoles. A new type of alkylating agent.

1,3-Dipolar cycloaddition of benzyl azide or peracetylated glucopyranosyl azides to propargyl halides or 1,4-dihalobutynes yielded 1-benzyl- or 1-glycosyl(halomethyl)-1,2,3-triazoles. Alkylating chloromethyl- bromomethyl- and iodomethyl-1,2,3-triazoles were also obtained from the corresponding hydroxymethyl derivatives by treatment with (C6H5)3P/CCl4, (C6H5O)3P/Br2, and (C6H5O)3P/I2, respectively. 1-Benzyl-4-(fluoromethyl)-1,2,3-triazole was obtained from 1-benzyl-4-(bromomethyl)-1,2,3-triazole by treatment with KF and 18-crown-6. Chloromethyl-, bromomethyl-, and iodomethyl-1,2,3-triazole derivatives inhibited the "in vitro" growth of HeLa cells. Some of these compounds increased the life span of mice bearing tumors.

Cytostatic quinones. 3. Synthesis of benzotriazolequinones by a new oxidation with m-chloroperbenzoic acid. Biochemical studies.

Oxidation of N-1 and N-2 alkylbenzotriazoles with m-chloroperbenzoic acid afforded 1-alkylbenzotriazole 3-oxides and 2-alkylbenzotriazole-4,4-diones, respectively. The quinonic compounds inhibited the "in vitro" growth of both HeLa and KB cells, the synthesis of macromolecules (DNA, RNA, and proteins), and the uptake of glucose by Ehrlich carcinoma ascites cells. A possible mode of action is suggested.