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International audience; AbstractThe simultaneous wing movement by multiple worker bees in a colony produces a hissing sound, which is a novel acoustic and vibrational signal of the honey bees. Hissing of honey bees is thought to be a response to direct, threatening stimuli. However, we discovered Japanese honey bees (Apis cerana japonica) can hiss even without obvious disturbances in previous study. In this study, to understand the temporal characteristics of honey bee hissing, we conducted 24-h sound recordings over 7 months in 2015 and investigated when A. cerana japonica hissed every day. Additionally, we also investigated the relationship of hissing onset and offset times with sunrise and sunset times, and environmental factors. We found that honey bees hiss daily during daytime and most frequently at dawn, with hissing onset/offset occurring mostly within 30 min of sunrise/sunset time. Hissing onset and offset were significantly related to sunrise and sunset times, respectively, and also to solar radiation intensity. The findings reveal that A. cerana japonica hissing has unique temporal patterns, and also shed a new light on vibrational collective behavior in honey bees.

Hissing behavior of honey bees is simultaneous wing movement of multiple honey bees, and loud sounds or vibrations are produced from their movements. In previous the study, hissing sound of honey bees has historically been considered as an aposematic signal. This is because hissing is generally observed when disturbance occur, such as mechanical disturbance or visually disturbing stimuli like hornets approaching the colony. Few studies, however, have investigated situations in which honey bees hiss. In this study, 24-h sound recording and video monitoring were conducted to investigate when the Japanese honey bees (Apis cerana japonica) hissed and checked the external environment of the colony when they hissed. As a result, we found that A. cerana japonica hissed not only when hornets approached but also when there was no obvious threatening stimulus outside the colony. In addition, clear diurnal patterns of hissing were observed. It is still not clear whether hissing in A. cerana japonica is more than aposematic signal or not, but our findings of unique occurrence patterns of their hissing raise question about the potential function of hissing behavior in A. cerana japonica.

The intestinal microbiome is known to affect host health through various effects on nutrition and immunity. The oriental honey buzzard (OHB) is a raptor that feeds on bees and wasps. Due to its restricted diet, we reasoned that the OHB may have a unique microbiome. The aim of this study was to characterize the structure of the intestinal flora of oriental honey buzzards and to investigate the difference of intestinal bacterial flora between individuals in the wild and those reared in captivity. We investigated the intestinal microbiome of seven wild buzzards (Wild), one zoo-reared (Zoo), and one individual reared in captivity for one month (Rearing). Average operational taxonomic units in Wild and Rearing were 69.4 and 113, respectively. Diversity indices such as ACE, Chao 1, Shannon, and Alpha were significantly lower in the Wild than in the Rearing samples. These results suggest that the variety of Wild microbiome is remarkably low. At the phylum level, the composition of the microbiome was similar in all three groups, with firmicutes and bacteroidetes predominating. The third most abundant bacterium in Wild was Proteobacteria, whereas it was Actinobacteria in Rearing and unclassified bacteria in Zoo. Thus, microbiome composition is affected even with just one month of human rearing.

The flower of the oriental orchid Cymbidium floribundum is known to attract the Japanese honeybee Apis cerana japonica. This effect is observed not only in workers but also drones and queens; that is, it attracts even swarming and absconding bees. A mixture of 3-hydroxyoctanoic acid (3-HOAA) and 10-hydroxy-(E)-2-decenoic acid (10-HDA) was identified as the active principles from the orchid flower, whereas these compounds individually have no such activity. Both compounds are also mandibular gland components of worker honeybees with related compounds. This strongly supports the idea that orchid flowers mimic bee secretions, although the ecological consequences of this relationship remain unknown. Because the flower is used to capture swarms, the present identification may contribute to the development of new techniques in traditional beekeeping for Japanese bees as well as A. cerana in Southeast Asia.

We have found that giant hornets (Vespa mandarinia japonica) are killed in less than 10 min when they are trapped in a bee ball created by the Japanese honeybees Apis cerana japonica, but their death cannot be solely accounted for by the elevated temperature in the bee ball. In controlled experiments, hornets can survive for 10 min at the temperature up to 47 degrees C, whereas the temperature inside the bee balls does not rise higher than 45.9 degrees C. We have found here that the CO2 concentration inside the bee ball also reaches a maximum (3.6 +/- 0.2%) in the initial 0-5 min phase after bee ball formation. The lethal temperature of the hornet (45-46 degrees C) under conditions of CO2 concentration (3.7 +/- 0.44%) produced using human expiratory air is almost the same as that in the bee ball. The lethal temperature of the honeybee is 50-51 degrees C under the same air conditions. We concluded that CO2 produced inside the bee ball by honeybees is a major factor together with the temperature involved in defense against giant hornets.

Aim: To compare the efficacy of primary Helicobacter pylori eradication therapy between PPI/AC therapy (proton-pump inhibitor; lansoprazole, amoxicillin, and clarithromycin) and PPI/AM therapy (lansoprazole, amoxicillin, and metronidazole). Methods: The subjects were patients with Helicobacter pylori infection who received initial eradication treatment at a family doctor in Katori City, Japan. Infection of Helicobacter pylori was initially evaluated pathologically using gastric mucosal specimens obtained by biopsy through upper endoscopy. Ninety-two patients underwent PPI/AC therapy and 28 patients underwent PPI/AM for primary eradication. Success or failure of eradication was determined by a urea breath test, and the primary eradication rates were compared between the two therapies. Patients who underwent PPI/AC but failed to achieve eradication were recommended to undergo secondary eradication treatment with PPI/AM, and the secondary eradication rate was assessed. Results: In primary eradication therapy, successful eradication rates were 79.3% (73/92) in PPI/AC and 96.4% in PPI/AM, indicating that PPI/AM had a significantly higher eradication success rate (P=0.034). The 19 patients who failed to achieve eradication by PPI/AC underwent secondary eradication by PPI/AM. All 19 cases showed effective eradication (100%). Ultimately, eradication was successful in 99.2% (119/120) patients. Successful eradication rate in the total of primary and secondary PPI/AM treatments was also significantly higher than primary PPI/AC (46/47 vs. 73/92: P=0.007). Neither therapy was associated with any notable side effects. Conclusion: In this regional study, PPI/AM therapy showed a favorable eradication rate, significantly higher than PPI/AC therapy. Thus, PPI/AM therapy is recommended as the primary eradication therapy in the region.

A simple and reproducible method for the analysis of ampicillin in human serum was developed. Serum samples were extracted using solid-phase extraction disk cartridges containing a sorbent of styrene divinyl/benzene. Extracts were separated by reversed-phase C18 high-performance liquid chromatography with UV detection at 220 nm. The mobile phase consisted of acetonitrile-10 mM NaH2PO4 (6.5:93.5, v/v). Using this extraction procedure, recovery from serum was 98.4+/-5.6%. The quantitation limit was 0.19 microg/ml using 0.5 ml of serum. The calibration curves from 0.19 to 9.41 microg/ml were linear with correlation coefficients of 0.999. This method is suitable for therapeutic drug monitoring of ampicillin (ABPC) after oral administration of lenampicillin hydrochloride.

The metabolism of a new antiplatelet agent, ethyl 2-[4, 5-bis(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetate (KBT 3022), was studied in rats, mice, dogs and humans. We found 12 metabolites (M1-M12) after oral administration of KBT-3022 in rat's feces and bile, and identified M3, M5, M6 and M9. After KBT-3022 was hydrolyzed to M9, the demethylation of the methoxyphenyl groups in 4-or/and 5-positions of thiazol ring of M9 resulted in the formation of M3, M5, M6. Furtheremore, these metabolites may be conjugated to glucuronides or sulfates. M8 is probably a glucuronide of M9 as revealed by the results of treatments of with β-glucuronidase. There is the species difference in the rate of the demethylation, the rate of the demethylation in mouse and dog was slower than that in rat. In human, the unconjugated M9 was only detected in plasma, similarly as in mouse, while the unconjugated M9 and glucuronides of M3 and M5 were found in urine. Urinary excretion of the metabolites accounted for 0.32% of dose in human. We considered that the metabolism of KBT-3022 in human was close to that in mouse.

The previous studies have shown that there was the species difference between rat and mouse inthe metabolic fate of KBT-3022, and that the O-demethylation of two methoxyphenyl groups of desethyl KBT-3022 (M9) was main metabolic pathway. The study of metabolism of M9 by rat liver, lung, kidney and small intestine 9, 000×g supernatant suggested that liver was the most active metabolizing organ. In this study, we have investigated the in vitro metabolism of M9 using 9, 000×g supern atant of liver homogenates of male rats and mice in order to clarify the species difference in the metabolic fate of M9. The metabolic activity of M9 and the O-demethylase activity of 9, 000×g supernatant of liver homogenates of rats were much higher than those of mice. The activity of demethylation of 5 methoxyphenyl group of M9 was higher than that of 4-methoxyphenyl group, both in rats and mice. However, in the demethylation of monodesmethylated M9 in rats, the activity of demethylation of 4-methoxyphenyl group was higher than that of 5-methoxyphenyl group. These results suggest that the species difference in the demethylation of M9 by 9, 000×g supernatant of liver homogenates relates to the species difference in the metabolic fate of KBT-3022.

A novel tetracyclic pyridone carboxylic acid with a thiazolidine ring, 1,2-dihydro-9,1-(epoxymethano)-7-fluoro-8-(4-methyl-1-piperazinyl)-5-oxo -5H- thiazolo[3,2-a]quinoline-4-carboxylic acid (4a), and variants with a nitrogen atom (4b) or carbonyl group (4c) in the place of the 10-position oxygen atom of 4a were prepared and tested for antibacterial activity and inhibitory activity on DNA gyrase from Escherichia coli KL-16. The in vitro antibacterial potency with regard to the 10-position atom was found to be of the following order; O > NCH3 = C = O. The IC50 values for DNA gyrase inhibition activity for the 4a, 4b, and 4c compounds were 0.33, 0.53, and 0.67 g/mL, respectively. The activity of 4a, in which the C-3 methyl group and C-5 of ofloxacin (2a) were connected with a sulfur atom to restrict the conformation of 2a, was more potent than that of 2a against both Gram-positive and -negative bacteria, except for Pseudomonas aeruginosa. Compared to the tetracyclic pyridone carboxylic acid 1a, which has a flat thiazole ring, compound 4a showed comparable or slightly more potent activity against both Gram-positive and -negative bacteria, except for P. aeruginosa.

Various 4-arylthiomethyl-2-oxo-1, 3-dioxole derivatives IIIa-o were synthesized. Their hydrolysis rates by arylesterase (EC 3.1.1.2) and cholinesterase (EC 3.1.1.8) in human serum were evaluated. Some of them were not hydrolyzed by cholinesterase, but were hydrolyzed easily by arylesterase.Among the substrates, sodium 4-((5-methyl-2-oxo-1, 3-dioxol-4-yl)methylthio)benzenesulfonate (IIIg) was selected for its substrate reactivity toward arylesterase and its good water solubility. In addition, neither aliesterase (EC 3.1.1.1), acetylesterase (EC 3.1.1.6) nor cholesterol esterase (EC 3.1.1.13)hydrolyzed the compound. IIIg is thus concluded to be a specific substrate for arylesterase.Our assay system for serum arylesterase using IIIg can be readily applied to an automatic analyzer in the diagnosis of liver cirrhosis.

A series of 8-substituted-9,1-(epoxymethano)-7-fluoro-5-oxo-5H- thiazolo[3,2-a]quinoline-4-carboxylic acids having a novel tetracyclic structure was synthesized and tested for antibacterial activity. The nature of the heteroatom (N, O, or S) substituted at the 8-position had little influence on the antibacterial activity. Among the six pyrrolidinyl derivatives and the five piperazinyl derivatives, the 8-(3-hydroxy-1-pyrrolidinyl) derivative 6h and the hydrochloride of the 8-(4-methyl-1-piperazinyl) derivative 6l showed the most potent activity against both Gram-positive and Gram-negative bacteria. Against nalidixic acid resistant strains, isolated from Escherichia coli KC-14, compound 6h was less potent than 6l. Replacement of the piperazinyl nitrogen atom by a carbon atom, an oxygen atom, or a sulfur atom (corresponding to the piperidino, morpholino, or thiomorpholino group, respectively) enhanced the activity against Gram-positive bacteria, but reduced the activity against Gram-negative bacteria. Compound 6l also showed potent in vivo antibacterial activity against Gram-positive and Gram-negative bacteria, and did not cause convulsions in mice with the concomitant administration of fenbufen. Replacement of the carboxy group by a sulfonic acid group in 6l resulted in a complete loss of antibacterial activity.

A novel series of 8-(2-substituted morpholino)-9,1-[(N-methylimino) methano]-7-fluoro-5-oxo-5H-thiazolo[3,2-a]quinoline-4-carboxylic acids, designated 8a-j, with a unique tetracyclic structure were synthesized, and the in vitro and in vivo antibacterial activities against Gram-positive strains, including methicillin-resistant Staphylococcus aureus isolates (MRSA), and Gram-negative strains were evaluated. These morpholino derivatives, 8a-j, showed excellent in vitro antibacterial activities against Gram-positive bacteria. The substitutions at the C-2 position of the 8-morpholino moiety of compound 8 play an important role in the enhancement of in vivo antibacterial activity. The unsubstituted morpholino derivative 8a, the 2,6-dimethyl derivative 8c, and the 2-ethylmorpholino derivative 8d showed poor in vivo antibacterial activity, while 8b, 8f-h, and 8j exhibited good activities. The 2-(methoxymethyl)morpholino derivative, 8h, showed the most potent activity in vivo. The therapeutic effects of 8h on systemic infection against S. aureus IID 803 were over 10-fold more potent than that of ofloxacin. Compound 8h, which showed superior oral bioavailability, has a chiral center. The enantiomers of 8h were synthesized, and the in vitro and in vivo antibacterial activities were evaluated. Both enantiomers, (S)-8h and (R)-8h, and the racemic compound 8 exhibited similar activities in vitro and in vivo. Compounds 8b and 8f-h also showed good levels of antibacterial activity against MRSA strains. The morpholino derivatives with unique tetracyclic structures are characterized by strong antibacterial activities against MRSA strains.

It is known that thiazolo[3,2-a][1,8]naphthyridine derivatives (3a) exhibit good antibacterial activity. Accordingly, several analogues of 3a, viz. oxazolo- and imidazolo[3,2-a][1,8]naphthyridine derivatives 3b and 3c, were synthesized and evaluated for antibacterial activity in vitro and for inhibitory activity against DNA gyrase of Escherichia coli K-12 C600. Compound 3a exhibited antibacterial activity comparable to that of ofloxacin and enoxacin against Gram-positive and Gram-negative bacteria and displayed antibacterial activity superior to that of 3b and 3c. The antibacterial activities of 3b and 3c decreased in that order. DNA gyrase inhibitory activities of 3a-c in E. coli K-12 C600 paralleled their in vitro antibacterial activity. It was found that enhancement of the DNA gyrase inhibitory activity of 3a was dependent on a certain feature of the sulfur atom of the thiazole ring.

Baicalin (BG) is one of the major components of Sho-Saiko-To. We found a new substance as the metabolite of BG in human plasma after oral administration of Sho-Saiko-To. The metabolite was identified as baicalein 6-O-sulfate (BS) by comparing its retention time in HPLC and electrospray ionization mass spectra (ESI-MS)/MS methods with that of an authentic sample. Time profiles for the plasma concentrations of BS and BG after oral administration of Sho-Saiko-To (EK-9) at a daily dose (6 g), were investigated in 14 healthy male volunteers. The determination for the concentrations of BS and BG in human plasma was developed by the HPLC method using electrochemical detector (ECD). Each 1 ml of the plasma specimen was used for the solid phase extraction. The calibration curves of BS and BG showed a good linearity between 5 and 300 ng/ml. The quantitative limits of BS and BG in human plasma were 5 ng/ml. Using this method, BS was detected after 1 h, reached a maximum level at 5 h and then decreased to the level less than the quantitative limit after 36 h, and the plasma level of BS showed a slight peak at 24 h. BG was detected after 1 h, reached a maximum level at 5 h and then decreased to the level less than the quantitative limit after 36 h, and the plasma level of BG showed two peaks at 12 h and 24 h.

The synthesis and biological evaluation of orally active inhibitors of matrix metalloproteinase are reported. Modifications of the P2' position and the alpha-substituent of hydroxamic acid derivatives were carried out, and revealed that the P2' substituent influenced the MMP inhibitory activities in vitro and in plasma after oral administration. The hydroxamates with phenylglycine at the P2' position were absorbed well orally. Compound 15e, which exhibited the longest duration of inhibitory activity in plasma after oral administration among the phenylglycine derivatives (5a-5d, 15a, 15c, 15e), was evaluated in a rat adjuvant arthritis model. A reduction in hind foot pad swelling and improvements of some inflammatory parameters were demonstrated when the compound was administered orally. These results indicate the potential of MMP inhibitors for rheumatoid arthritis.

Various structural transformation processes observed in a number of past developmental examples of pharmaceuticals and agrochemicals are regarded as being invaluable precedents for the prospective analog design. In certain cases, (sub)structural transformation patterns are interchangeable among various compound series in spite of differences in their pharmacological category. Thus, the patterns extracted with a computer-readable format could be accumulated and integrated as a database for potential “rules” for bioanalogous molecular transformations. EMIL is a system that incorporates the database and a data-processing engine constructed to release “higher-ordered” candidate structures from a “lower-ordered” input structure “automatically”. Conceptual background for the database construction and the procedure for the database collection are presented on the basis of some lead evolution examples among pharmaceutical and agrochemical series of compounds.

A novel series of 8-(2-substituted morpholino)-9,1-[(N-methylimino) methano]-7-fluoro-5-oxo-5H-thiazolo[3,2-a]quinoline-4-carboxylic acids, designated 8a-j, with a unique tetracyclic structure were synthesized, and the in vitro and in vivo antibacterial activities against Gram-positive strains, including methicillin-resistant Staphylococcus aureus isolates (MRSA), and Gram-negative strains were evaluated. These morpholino derivatives, 8a-j, showed excellent in vitro antibacterial activities against Gram-positive bacteria. The substitutions at the C-2 position of the 8-morpholino moiety of compound 8 play an important role in the enhancement of in vivo antibacterial activity. The unsubstituted morpholino derivative 8a, the 2,6-dimethyl derivative 8c, and the 2-ethylmorpholino derivative 8d showed poor in vivo antibacterial activity, while 8b, 8f-h, and 8j exhibited good activities. The 2-(methoxymethyl)morpholino derivative, 8h, showed the most potent activity in vivo. The therapeutic effects of 8h on systemic infection against S. aureus IID 803 were over 10-fold more potent than that of ofloxacin. Compound 8h, which showed superior oral bioavailability, has a chiral center. The enantiomers of 8h were synthesized, and the in vitro and in vivo antibacterial activities were evaluated. Both enantiomers, (S)-8h and (R)-8h, and the racemic compound 8 exhibited similar activities in vitro and in vivo. Compounds 8b and 8f-h also showed good levels of antibacterial activity against MRSA strains. The morpholino derivatives with unique tetracyclic structures are characterized by strong antibacterial activities against MRSA strains.

A series of novel pyridone carboxylic acids having a 4-hydroxypiperazin-1-yl, a 4-hydroxy-3-methylpiperazin-1-yl, and a 4-hydroxy-3,5-dimethylpiperazin-1-yl group was prepared, and their metabolism to corresponding piperazinyl derivatives after oral administration to mice and rats was studied. This reductive metabolism appeared to be more extensive in mice than in rats. Moreover, the introduction of a methyl group into the alpha-position of the 4-hydroxy group depressed the metabolism in both species.

A series of novel tetracyclic pyridone carboxylic acids replacing the 10-position oxygen atom of 9,1-(epoxymethano)-7-fluoro-8-(4-methyl-1-piperazinyl)-5-oxo-5H-thiazolo [3,2-alpha]quinoline-4-carboxylic acid by imino groups (NR; R = Me, Et, c-Pr, allyl, Ph, benzyl), a sulfur atom, or a carbonyl group was prepared and evaluated for antibacterial activity and inhibitory activity on DNA gyrase isolated from E. coli KL-16. The in vitro antibacterial potency and DNA gyrase inhibitory activity were found to be in the following order: NMeor = OSC = O. Moreover, a methyl group was the optimal alkyl substituent at the 10-position nitrogen atom for antibacterial activity and for DNA gyrase inhibitory activity. 7-Fluoro-9,1-[(N-methylimino)methano]-8- (4-methyl-1-piperazinyl)-5-oxo-5H-thiazolo[3,2-alpha]quinoline-4-carboxy lic acid (10-NCH3) showed potent in vivo antibacterial activity.

A series of 8-substituted-9,1-(epoxymethano)-7-fluoro-5-oxo-5H- thiazolo[3,2-a]quinoline-4-carboxylic acids having a novel tetracyclic structure was synthesized and tested for antibacterial activity. The nature of the heteroatom (N, O, or S) substituted at the 8-position had little influence on the antibacterial activity. Among the six pyrrolidinyl derivatives and the five piperazinyl derivatives, the 8-(3-hydroxy-1-pyrrolidinyl) derivative 6h and the hydrochloride of the 8-(4-methyl-1-piperazinyl) derivative 6l showed the most potent activity against both Gram-positive and Gram-negative bacteria. Against nalidixic acid resistant strains, isolated from Escherichia coli KC-14, compound 6h was less potent than 6l. Replacement of the piperazinyl nitrogen atom by a carbon atom, an oxygen atom, or a sulfur atom (corresponding to the piperidino, morpholino, or thiomorpholino group, respectively) enhanced the activity against Gram-positive bacteria, but reduced the activity against Gram-negative bacteria. Compound 6l also showed potent in vivo antibacterial activity against Gram-positive and Gram-negative bacteria, and did not cause convulsions in mice with the concomitant administration of fenbufen. Replacement of the carboxy group by a sulfonic acid group in 6l resulted in a complete loss of antibacterial activity.

(5-Substituted 2-oxo-1, 3-dioxolen-4-yl) methyl esters were designed as a new type of ampicillin prodrug. These esters were prepared and confirmed to produce higher blood levels of ampicillin than ampicillin trihydrate itself after oral administration to mice. The compounds which produced particularly high blood levels of ampicillin were found to be hydrolyzed readily in blood in vitro. Ampicillin (5-methyl-2-oxo-1, 3-dioxolen-4-yl) methyl ester hydrochloride (KBT-1585) showed the best oral absorbability in mice.

Several 3-formylquinolone derivatives (8a-c) were synthesized to assay the antibacterial activity both in vitro and in vivo. In vitro, all of the compounds 8a-c showed lower activity than that of the corresponding 3-carboxyl compounds 1a-c, and in vivo, they showed higher activity than that of compounds 1a-c. After oral administration of 3-formyl compounds 8a-c to mice, the compounds were rapidly metabolized into 3-carboxyl compounds 1a-c. In particular, the 3-formyl derivative (8a) of norfloxacin (NFLX, 1a) gave a 2-fold higher serum level than that of NFLX and functioned as a prodrug of NFLX.

As a prodrug approach to norfloxacin (NFLX, 2), we have prepared several N-masked NFLXs (1a-f) and studied the cleavage mechanism of the C-N bond of N-masked NFLXs utilizing the following experiments: (1) the oxidation of N-masked NFLXs (1a-f) with m-chloroperbenzoic acid (MCPBA) and their subsequent cleavage to 2 in chloroform at room temperature or at 50 degrees C; (2) the liberation of NFLX from N-masked NFLXs after oral administration in mice. It was found that the chemical oxidative dealkylation of N-masked NFLXs proceeded when anion-stabilizing groups (e.g., CN, COR, COOR) are present on the alpha carbon of the nitrogen atom. In in vivo experiments, N-masked NFLXs having acidic hydrogens on the alpha carbon to the nitrogen atom also liberated NFLX (2) after oral administration.

As a new type of norfloxacin (NFLX) prodrug, N-(5-substituted 2-oxo-1, 3-dioxol-4-yl) methyl NFLXs were designed. These N-masked NFLXs were prepared and confirmed to produce higher NFLX levels in blood than NFLX itself after oral administration to mice. N-(5-Methyl-2-oxo-1, 3-dioxol-4-yl) methyl NFLX was found to be smoothly hydrolyzed in mouse blood in vitro, and when administered orally, gave about 5-fold higher blood levels of NFLX than NFLX itself. Thus, the (5-methyl-2-oxo-1, 3-dioxol-4-yl) methyl group was confirmed to function as an amine-type promoiety.

In order to prepare ampicillin prodrugs conveniently for practical use, a new synthetic method for 6β-aminopenicillanic acid (6-APA) esters as key intermediates of various ampicillin prodrugs has been developed. Acylation of 6-APA esters to ampicillin esters was achieved in good yields by using phenylglycyl chloride hydrochloride in dichloromethane in the presence of sodium bicarbonate and amide, or ammonium bicarbonate alone. KBT-1585, bacampicillin, talampicillin and pivampicillin have been obtained in good yields by this procedure.

The chemical oxidation of N-[(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl] norfloxacin (2) was carried out to afford N-[(4-methyl-5-methylene-2-oxo-1,3-dioxolan-4-yl)oxy]norfloxacin (4). In vitro, 4 exhibited lower activity than that of norfloxacin (NFLX, 1) for both Gram-positive and Gram-negative bacteria. However, in vivo the activity of 4 was higher than that of NFLX. Bioavailability studies in mice showed that 4 liberated a higher concentration of NFLX in plasma than NFLX itself when administered orally. From these data, 4 obtained by the chemical oxidation of 2 functioned as a prodrug of NFLX as well as did 2. The mechanism of the formation of 4 is interpreted in terms of [2,3]-sigmatropic rearrangement.

Several N-(oxoalkyl)norfloxacin derivatives (3a-g) were synthesized and evaluated for antibacterial activity in vitro and in vivo. Most of the compounds exhibited in vitro activity comparable to that of norfloxacin for Gram-positive bacteria, whereas their activity was lower than for Gram-negative bacteria. N-(2-Oxopropyl)norfloxacin (3b) liberated norfloxacin in the blood after oral administration in mice, and the serum level of norfloxacin was about 3-fold higher than that of norfloxacin itself. Thus, 3b showed high antibacterial activity in vivo.

In order to prepare ampicillin prodrugs conveniently for practical use, a new synthetic method for 6β-aminopenicillanic acid (6-APA) esters as key intermediates of various ampicillin prodrugs has been developed. Acylation of 6-APA esters to ampicillin esters was achieved in good yields by using phenylglycyl chloride hydrochloride in dichloromethane in the presence of sodium bicarbonate and amide, or ammonium bicarbonate alone. KBT-1585, bacampicillin, talampicillin and pivampicillin have been obtained in good yields by this procedure.

In order to investigate a new type of promoiety for ampicillin prodrugs, acyloxyallyl esters were prepared and their oral absorbabilities, stabilities and hydrolyzabilities to parent ampicillin were studied. As starting materials for (3-phthalidylidene)ethyl esters, ethylidenephthalide and its derivatives were regiospecifically prepared by a new method using the Wittig reaction. (3-Phthalidylidene)ethyl esters (14a-c) given orally were well absorbed, but (thiophthalidylidene)ethyl ester (14d) and (isocoumarin-4-yl)methyl ester (14e) were poorly absorbed.