1,2,4,5-Tetraoxacycloalkanes: synthesis and antimalarial activity.

In this short review the methods of preparation of novel 1,2,4,5-tetraoxacycloalkanes and the related peroxides are summarized, with the emphasis on the usefulness of 1,1-bishydroperoxides as the precursor. Also, their antimalarial activities in vitro and in vivo are discussed.

A sero-epidemiological survey of gelatin sensitization in young Japanese children during the 1979-1996 period.

Recently, an increasing number of adverse reactions in children inoculated with live attenuated virus vaccines containing gelatin have been reported. However, the distribution, magnitude and rate of gelatin sensitization in the Japanese population have not been established. Here, the purpose was to investigate the distribution of children with positive gelatin immunoglobulin G (IgG) and/or IgE in Japan and to ascertain whether the incidence of positive antigelatin antibody cases among the general population, as reflected in the sample employed here, has been increasing during the period in question. The presence of IgE and IgG antibodies were measured against gelatin in 1600 panel sera collected from different age groups of Japanese children in Hokkaido/Sapporo from 1979 through 1996. Among the subjects, 39 had gelatin IgG- and/or IgE-positive sera, and these were correlated with the time of obtaining the sera as well as with the age of the subjects. The older the subject and the later the period, the higher the sero-incidence. Japanese children have become increasingly sensitized to gelatin, especially since the mid-1990s.

Simple isoquinoline and benzylisoquinoline alkaloids as potential antimicrobial, antimalarial, cytotoxic, and anti-HIV agents.

Twenty-six simple isoquinolines and 21 benzylisoquinolines were tested for antimicrobial, antimalarial, cytotoxic, and anti-HIV activities. Some simple isoquinoline alkaloids were significantly active in each assay, and may be useful as lead compounds for developing potential chemotherapeutic agents. These compounds include 13 (antimicrobial), 25, 26, and 42 (antimalarial), 13 and 25 (cytotoxic), and 28 and 29 (anti-HIV). A quaternary nitrogen atom of isoquinolium or dihydroisoquinolinium type may contribute to enhanced potency in the first three types of activities. In contrast, anti-HIV activity was found with tetrahydroisoquinoline and 6,7-dihydroxyisoquinolium salts.

Cloning and expression of uridine/cytidine kinase cDNA from human fibrosarcoma cells.

Uridine/cytidine kinase which converts uridine and cytidine to their corresponding monophosphates is a rate-limiting enzyme involved in the salvage pathway of pyrimidine synthesis. We isolated cDNA encoding the enzyme from human fibrosarcoma cells, then determined its nucleotide sequence by the 5'-RACE method followed by confirmation employing the human genome DNA library. The isolated uridine/cytidine kinase cDNA (UCK cDNA) consisted of 786 nucleotides encoding 261 amino acids and was found to have approximately 70% homology with mouse UCK cDNA. Northern blot analysis of human leukemia RNAs with labeled UCK gene showed a single band at 1.6 kb to be UCK mRNA, and southern blot analysis of the UCK cDNA after digestion with BamHI, SacI and XbaI enzymes showed four band signals, suggesting the UCK gene to have at least 4 exons. A truncated form of UCK cDNA was expressed as the His-tag conjugated protein in Escherichia coli. The expressed and purified protein specifically converted uridine and cytidine to their corresponding monophosphates and also phosphorylated antitumor nucleosides such as 5-fluorouridine, cyclopentenyl-cytosine and 3'-C-ethynylcytidine. The present results suggest that our cloned human UCK cDNA encodes the correct amino acid sequence for UCK protein, showing high intracellular phosphorylation activity forward natural and synthetic pyrimidine nucleosides.

Synthesis and antimalarial activity of febrifugine derivatives.

The regioisomers (2a,b) of the piperidine ring of febrifugine (1a) and isofebrifugine (1b) were synthesized from 4-allyl-3-piperidone (5). Reduction of 5 afforded a mixture of the trans and cis alcohols (6a,b) without diastereoselectivity; this result differentiated it from the reduction of 2-allyl-3-piperidone (14). The antimalarial activity of 2a,b and related compounds was tested.

Antimalarial and cytotoxic activities of bicycl.

Biological evaluations of bicyclo[6.4.0]dodecenone derivatives on antimalarial activity in vitro against Plasmodium falciparum and cytotoxicity against human KB cells were made. (+/-)-(1R*,4S*,7R*,8S*)-4-tert-Butyl-dimethylsiloxy-5,5-dimethyl-1-methyl-9-methylene-7-phenylsulfonylbicyclo[6.4.0]dodec-2,11-dien-10-one (15) exhibited potent antimalarial activity, whereas (+/-)-(1R*,7R*,8S*)-1-methyl-9-methylene-7-phenylsulfonylbicyclo[6.4.0]dodec-2,11-dien-10-one (14) showed significant cytotoxic activity in human KB cells. Both 14 and 15 possess, as a structural character, the exo-methylene moiety in their 6-membered ring of the 8-6 fused ring system.

Synthesis and antimalarial activity of novel medium-sized 1,2,4,5-tetraoxacycloalkanes.

CsOH- or Ag(2)O-mediated cycloalkylation of (alkylidene)bisperoxides 3 and 1,n-dihaloalkanes (n = 3-8) provided the corresponding medium-sized 1,2,4,5-tetraoxacycloalkanes 4-8 in moderate yields. Subsequent evaluation of the antimalarial activity of the cyclic peroxides 4-8 in vitro and in vivo revealed that 1,2,6,7-tetraoxaspiro[7.11]nonadecane 4a has considerable potential as a new, inexpensive, and potent antimalarial drug.

A strong association between HLA-DR9 and gelatin allergy in the Japanese population.

The frequency of HLA class I and II phenotypes was determined among 23 patients with positive gelatin IgE, eight of whom developed anaphylaxis, 18 patients who did not have gelatin IgE but who experienced non-immediate reactions after exposure to gelatin. HLA-DR9, which is unique to Orientals, was present in 56.5% of the gelatin IgE positive patients, as compared to control population frequency of 24% (P < 0.002). In the non-immediate reaction group, who did not generate IgE, phenotype distribution resembled controls. HLA-DR9 positive individuals have a relative risk of 4.1 for developing gelatin allergy with positive IgE.

A homologue of N-ethylmaleimide-sensitive factor in the malaria parasite Plasmodium falciparum is exported and localized in vesicular structures in the cytoplasm of infected erythrocytes in the brefeldin A-sensitive pathway.

N-Ethylmaleimide-sensitive factor (NSF) and its homologues play a central role in vesicular trafficking in eukaryotic cells. We have identified a NSF homologue in Plasmodium falciparum (PfNSF). The reported PfNSF gene sequence (GenBank accession number CAB10575) indicated that PfNSF comprises 783 amino acids with a calculated molecular weight of 89,133. The overall identities of its gene and amino acid sequences with those of rat NSF are 50.9 and 48.8%, respectively. Reverse transcription-polymerase chain reaction analysis and Northern blotting with total P. falciparum RNA indicated expression of the PfNSF gene. Polyclonal antibodies against a conserved region of NSF specifically recognized an 89-kDa polypeptide in the parasite cells. After homogenization of the parasite cells, approximately 90% of an 89-kDa polypeptide is associated with particulate fraction, suggesting membrane-bound nature of PfNSF. PfNSF was present within both the parasite cells and the vesicular structure outside of the parasite cells. The export of PfNSF outside of the parasite cells appears to occur at the early trophozoite stage and to terminate at the merozoite stage. The export of PfNSF is inhibited by brefeldin A, with 9 microM causing 50% inhibition. Immunoelectromicroscopy indicated that intracellular PfNSF was associated with organelles such as food vacuoles and that extracellular PfNSF was associated with vesicular structures in the erythrocyte cytoplasm. These results indicate that PfNSF expressed in the malaria parasite is exported to the extracellular space and then localized in intraerythrocytic vesicles in a brefeldin A-sensitive manner. It is suggested that a vesicular transport mechanism is involved in protein export targeted to erythrocyte membranes during intraerythrocytic development of the malaria parasite.

Analysis of Pfmdr 1 gene in mefloquine-resistant Plasmodium falciparum.

Drug resistance in Plasmodium falciparum is a serious problem in most endemic areas. Recent studies have suggested the potential involvement of genes in the MDR gene family in resistance to quinoline-containing compounds in P. falciparum. In our present studies, a molecular analysis of pfmdr 1 in isolate strain of P. falciparum, 523a R, from Japanese mefloquine-resistant patient was done to determine the reported association of pfmdr 1 intragenic alleles and mefloquine resistance, and to examine the antimalarial activities of several antimalarial agents against the P. falciparum strain. The antimalarial activities against the strain was decreased susceptibility to mefloquine, artemisinin and halofantrine, in contrast increased susceptibility to chloroquine. The DNA sequence analysis of pfmdr 1 gene in a strain reveled no association of intragenic alleles with mefloquine resistance. Furthermore, the overexpression of pfmdr1 mRNA have been observed and it is about 7.2 times higher than sensitive strain. Our data shows that overexpression of pfmdr1 gene may be associated in mefloquine-resistance mechanism.

Anticancer molecular mechanism of 3'-ethynylcytidine (ECyd).

The antitumor ribonucleoside analogue 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd), synthesized in 1995, has strong antitumor activity. In mouse mammary tumor FM3A cells, ECyd was rapidly phosphorylated to ECyd-triphosphate (ECTP) as the final product, strongly inhibiting RNA synthesis. The ultimate metabolite of ECyd, ECTP, is stable in cultured FM3A cells with a half-life of 21 hr; ECyd is on a "closed" metabolic pathway to ECTP. Deaminated ECyd derivatives were minor metabolites in the cells treated with Ecyd; therefore cytidine forms probably were not converted to uridine forms at the nucleoside or nucleotide stage. The characteristics of ECyd may be important for the antitumor activity. RNA polymerase in the nucleus was inhibited competitively by ECTP; the ki value was 21 nM. ECyd induced DNA and 28S ribosomal RNA fragnetations. The cleavage pattern of rRNA resembled in that mediated by RNase L. The results suggested that RNase L related mechanisms might be involved in the antitumor activity of ECyd.

Synthesis of novel ferrocenyl sugars and their antimalarial activities.

The synthesis of twelve types of novel ferrocenyl sugars and their biological properties towards the malaria parasite (P. falciparum) and mouse cancer cell (FM3A) are described.

A trial for a DNA diagnosis of Plasmodium vivax malaria recently reemerging in the Republic of Korea using microtiter plate hybridization assay.

The polymerase chain reaction-based microtiter plate hybridization (PCR-MPH) assay was utilized for a DNA diagnosis of Plasmodium vivax malaria, which has recently reemerged in the Republic of Korea. The subjects were 18 parasite-proven patients and 5 healthy controls. Follow-up blood samples were collected from 4 patients after a standard course of treatment. Polymerase chain reaction and electrophoresis of all the patients' blood showed a prominent band at the 138 base pair area, but not in the controls or after treating the patients. Hybridization of the PCR products with known species-specific probes of the 18S rRNA of various malaria species revealed strong positive reactions against the Plasmodium vivax-specific probe (absorbance 1.30-1.90 at 405 nm) in all of the patients. The absorbance was positively correlated with the degree of blood parasitemia, but with a borderline significance. Sequencing of the probe region of the Korean P. vivax revealed no significant variations from the typical P. vivax. The results show that the PCR-MPH is a highly useful technique for the DNA diagnosis of Korean vivax malaria.

Potent in vivo antimalarial activity of 3,15-di-O-acetylbruceolide against Plasmodium berghei infection in mice.

The antimalarial activity of the O-acylated bruceolide derivative, 3,15-di-O-acetylbruceolide, was evaluated against Plasmodium berghei in vivo. The concentration of 3,15-di-O-acetylbruceolide required for 50% suppression (ED50) of P. berghei in mice was 0.46 +/- 0.06 mg/kg/day, whereas bruceolide was only half as effective as 3,15-di-O-acetylbruceolide. Two antimalarial drugs used clinically, chloroquine and artemisinin, demonstrated only low activity corresponding to 1/4 and 1/12 of the ED50 value of 3,15-di-O-acetylbruceolide, respectively. These results may be helpful in the design of better chemotherapeutic bruceolides against falciparum malaria.

Cytotoxic mechanisms of inhibitor of RNA synthesis, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106).

We investigated the molecular mechanisms of cell death induced by 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106: Figure 1), a potent inhibitor of RNA synthesis, using mouse mammary tumor FM3A cells and human fibrosarcoma HT1080 cells. ECyd induced the characteristics of apoptosis on these cells, such as morphological changes, DNA fragmentations and caspase-3-like protease activation. General caspases inhibitor (Z-Asp-CH2-DCB) inhibited cell death. Interestingly, we also found that ECyd induced rRNA fragmentation with the size of 3.2, 2.8 and 1.5 kb, and which might be caused by inhibition of RNA synthesis. rRNA fragmentation was mainly occurred in D8 domain of 28 S rRNA, and the end of 5'-terminal sequence of 1.5 kb fragment was C3220pC3221p or C3221pG3222p, that was identical to the recognition sequence of RNase L. Furthermore, the fragmentation patterns of rRNA digested with RNase L resembled that of ECyd treated cells in shape. These results indicate that antitumor mechanisms of ECyd are involved in activation of RNase L. rRNA fragmentation may be one of the death events as a result of inhibition of RNA synthesis and play an important role in the antitumor activity of ECyd.

Defective DNA replication and repair associated with decreases in deoxyribonucleotide pools in a mouse cell mutant with thermolabile ubiquitin-activating enzyme E1.

Upon shift-up in temperature, mouse tsFS20 mutant cells with thermolabile ubiquitin-activating enzyme E1 immediately stopped DNA replication and showed cell cycle arrest in S-phase. In contrast, when the cells were permeabilized with lysolecithin after culture at the nonpermissive temperature, they exhibited a normal level of replicative DNA synthesis in vitro. In agreement with this, intracellular pools of deoxyribonucleoside triphosphates were significantly reduced in the cells cultured at the nonpermissive temperature. Even under the permissive conditions, tsFS20 cells were more sensitive to hydroxyurea and alkylating agents, and induced less mutation than the wild-type cells. These results suggest that the ubiquitin system affects DNA replication and repair.

New type of febrifugine analogues, bearing a quinolizidine moiety, show potent antimalarial activity against Plasmodium malaria parasite.

Febrifugine (1) and isofebrifugine (2), isolated from the roots of Dichroa febrifuga Lour. (Chinese name: Cháng Shan), are active principles against malaria. Adducts of 1 and 2 with acetone, Df-1 (3) and Df-2 (4), respectively, were obtained using silica gel and acetone. They showed high activity against P. falciparum malaria in vitro. Compound 3 was found to be equally effective against P. berghei in vivo as the clinically used drug chloroquine, whereas 4 showed only 1/24 of the activity of 3. Metabolism studies of these compounds revealed that compound 4 is readily metabolized in mouse liver. Accordingly, the dose of 4 must be higher than that of 3 to attain blood levels sufficient for a favorable therapeutic effect.

A new antitumor nucleoside, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd), is a potent inhibitor of RNA synthesis.

The antitumor activity, metabolism, and mechanism of action of a newly developed antitumor nucleoside, 1-(3-C-Ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd) are described.

Antitumor activity and novel DNA-self-strand-breaking mechanism of CNDAC (1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine) and its N4-palmitoyl derivative (CS-682).

We have studied the antitumor activity and the novel DNA-self-strand-breaking mechanism of CNDAC (1-(2-Ccyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine) and its N4-palmitoyl derivative (CS-682). In vitro, CS-682 showed strong cytotoxicity against human tumor cells comparable with that of CNDAC; both compounds displayed a similar broad spectrum. In vivo, however, orally administered CS-682 showed a more potent activity against human tumor xenografts than CNDAC, 5'-deoxy-5-fluorouridine, 5-fluorouracil and 2',2'-difluorodeoxycytidine. Moreover, CS-682 was effective against various human organ tumor xenografts at a wide dose range and with low toxicity, and was effective against P388 leukemic cells resistant to mitomycin-C, vincristine, 5-fluorouracil or cisplatin in syngeneic mice. CNDAC, an active metabolite of CS-682, had a prolonged plasma half-life after repeated oral administrations of CS-682 but not after oral administrations of CNDAC itself. This difference may partially explain the higher antitumor activity of CS-682 relative to CNDAC. In both CNDAC- and CS-682-treated carcinoma cells, CNDAC 5'-triphosphate (CNDACTP) was generated and incorporated into a DNA strand. High performance liquid chromatography (HPLC) and mass spectrometric analysis of the nucleosides prepared by digestion of the DNA from the CNDAC-treated cells detected ddCNC (2'-Ccyano-2',3 '-didehydro-2',3 '-dideoxycytidine), which was shown to be generated only when the self-strand-breakage of CNDACTP-incorporated DNA occurred. The cytotoxicity of CNDAC was completely abrogated by the addition of 2'-deoxycytidine and was low against cells with decreased deoxycytidine kinase. Our results suggest that CNDAC is converted to CNDACMP by deoxycytidine kinase and that the resulting CNDACTP incorporated into a DNA strand as CNDACMP may induce DNA-self-strand-breakage. This novel DNA-self-strand-breaking mechanism may contribute to the potent antitumor activity of CS-682.