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A convenient method for the synthesis of the title intermediate 4 was described. The key steps of this synthesis involved: (1) regioselective addition reaction of arylzinc reagent to quinolic anhydride in 42% isolated yield, (2) conversion of a ketoacid 7 to an enone 14, which was achieved in 65% yield by intramolecular Knoevenagel reaction of β-ketoester generated by condensation of an acid imidazolide 11 with an ester enolate, followed by dehydration assisted with silica gel, and (3) stereoselective reduction of an allyl alcohol 15 in 75% yield with zinc under acidic conditions. This synthesis enabled us to provide hundreds of grams of 4 without chromatographic purification.

Compounds ( 2 – 5 ) with a 6-carboxy-5,7-diarylcyclopentenopyridine skeleton were designed, synthesized, and identified as a new class of potent non-peptide endothelin receptor antagonists. The regio-isomer 2 was found to show potent inhibitory activity with an IC 50 value of 2.4 nM against 125 I-labeled ET-1 binding to human ET A receptors and a 170-fold selectivity for ET A over ET B receptors. Furthermore, 2 displayed more potent in vivo activity than did the indan-type compound 1 in a mouse ET-1 induced lethality model, suggesting the potential of 2 as a new lead structure. Derivatization on substituted phenyl groups at the 5- and 7-positions of 2 revealed that a 3,4-methylenedioxyphenyl group at the 5-position and a 4-methoxyphenyl group at the 7-position were optimal for binding affinity. Further derivatization of 2 by incorporating a substituent into the 2-position of the 4-methoxyphenyl group led to the identification of a more potent ET A selective antagonist 2p with an IC 50 value of 0.87 nM for ET A receptors and a 470-fold selectivity. In addition, 2p showed highly potent in vivo efficacy (AD 50 : 0.04 mg/kg) in the lethality model.

An easy method to prepare novel 4-alkoxy-, 4-alkylthio- or 4-aryloxy-5-methyl-2-[1-(hydroxymethyl)-2-(1-naphthyl)-ethyl (or -ethenyl)]pyridine derivatives having reversible inhibitory activity against H + K + -ATPase is described. Use of a methylsulfinyl- or methylsulfonyl group as a leaving group makes it possible to effectively introduce various alkoxy or alkylthio groups into the 4-position of the pyridine ring at the final stages of synthesis. Biological profiles of the prepared compounds are briefly mentioned.

1. Endothelin (ET)-1 has been postulated to be involved in the development of obstructive airway diseases in man. In the present study, we attempted to characterize ET receptor subtypes mediating ET-1-induced contraction in human isolated bronchi. The ET receptor antagonists used in the present study were BQ-123 (ETA receptor-selective), BQ-788 (ETB receptor-selective) and BQ-928 (ETA/ETB dual). Sarafotoxin S6c (S6c) was also used as an ETB receptor-selective agonist. 2. In human bronchi, ET-1 and S6c (10(-12)M to 10(-7) M) produced concentration-dependent contraction with almost equal potency (pD2: 8.88 +/- 0.16 for ET-1 and 9.42 +/- 0.15 for S6c). The contraction induced by S6c was competitively antagonized by BQ-788 alone (1 and 10 microM) with a pKB value of 7.49 +/- 0.21, suggesting that the stimulation of ETB receptors causes a contraction of human bronchi. However, contrary to expectation, the concentration-response curves for ET-1 were not affected by BQ-788. The ET-1- and S6c-induced contractions were not affected by BQ-123 (10 microM). Thus, ET-1-induced contraction of human bronchi is not antagonized by BQ-123 alone or by BQ-788 alone. 3. Combined treatment with 10 microM BQ-123 and 10 microM BQ-788 significantly antagonized the contraction induced by ET-1 with a dose-ratio of 11. BQ-928 also significantly antagonized ET-1-induced contraction with a pKB value of 6.32 +/- 0.24. 4. The specific binding of [125I]-ET-1 to human bronchial membrane preparations was inhibited by BQ-123 (100 pM to 1 microM) by approximately 40%. Combination treatment with BQ-788 (100 pM to 1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. 5. In conclusion, the present study demonstrates that BQ-788 alone cannot inhibit ET-1-induced contractions in human bronchi, although human bronchial ETB receptors are BQ-788-sensitive. Furthermore, it was shown that blockade of both receptor subtypes antagonizes ET-1-induced contraction, and that both receptor subtypes co-exist in human bronchial smooth muscles. These findings suggest that ETA receptors as well as ETB receptors are involved in ET-1-induced contraction in human bronchi. If ET-1 is involved in human airway diseases, dual blockade of ETA and ETB receptors may be necessary to treat the diseases.

A series of analogues of BQ-123 (1), a potent cyclic pentapeptide endothelin A receptor antagonist, with amino acids linked to the side-chain of the Pro residue via an ester linkage was synthesized. All analogues synthesized exhibited potent endothelin A receptor binding affinity similar to that of 1. Of the synthesized analogues, the Lys, Arg and N alpha,N epsilon-dimethyllysine analogues, 9d-f, exhibited about a three-fold attenuation of in vivo clearance compared with 1. In rats, these analogues exhibited a 3-fold-higher plasma concentration and a longer retention time in plasma as compared with those of 1. The attenuated in vivo clearance was thought to be a consequence of decreased extraction of the compounds from the blood via the hepatic anion transport system, which efficiently extracts 1 from the blood.

Peptide derivatives with 2-substituted d-tryptophan analogues were synthesized. All prepared peptide derivatives showed potent affinity for ETB receptors, while their ETA affinity depended on the substituents of the d-tryptophanyl residue. A 2-bromo-d-tryptophan derivative 16b (BQ-928) was a combined ETAETB receptor antagonist and a 2-cyano-d-tryptophan derivative 17c (BQ-017) was a selective ETB receptor antagonist.

Novel linear tripeptides possessing high endothelin antagonist activity were derived from endothelin antagonistic cyclic pentapeptides represented by BQ-123. The N-terminal urea moiety of the linear tripeptide derivatives was essential to show the strong antagonist activity. An easy method to prepare these peptides by treatment of the corresponding N-phenoxycarbonylated tripeptide esters with primary or secondary amines is described.

A 100-g scale preparation of a representative endothelin-A receptor (ETA) selective antagonist, BQ-123Na (a sodium salt of BQ-123), is described. Application of a methyl ester as the side chain carboxy-protecting group for the d -Asp residue made it possible to use a benzyl ester as a carboxy-protecting group for the C-terminal Leu throughout the synthesis.

A series of α-aminophosphonic acid derivatives and a series of phosphoramidate derivatives have been synthesized and evaluated as inhibitors of a phosphoramidon-sensitive metalloproteinase endothelin converting enzyme (ECE). Some of these compounds exhibit potent ECE inhibitory activity. The most potent inhibitor (XIIb) is about 10 times as potent as phosphoramidon.

Exogenous endothelin (ET) is rapidly cleared from the circulation. We investigated which ET receptor subtypes (ETA and ETB) participate in ET-1 clearance. Following an intravenous (i.v.) bolus dose of [125I]ET-1 in anesthetized rats, radioactivity was rapidly cleared from the circulation and trapped by the lungs, kidneys and liver. Tissue distribution of the radioactivity was significantly inhibited in the lungs and kidneys, but not in the liver by infusion of the ETB antagonist BQ-788 (0.1 mg/kg/min i.v.), and the ET-1 clearance rate was reduced, while the ETA antagonist BQ-123 had no such effect. Furthermore, in isolated perfused rat lungs, about 80% of bolus-injected [125I]ET-1 was retained by the lungs after one passage. The retention of ET-1 was significantly inhibited by infusion of 1 microM BQ-788, but not BQ-123. These results suggest that ETB receptors play an important role in the clearance of ET-1.

In anesthesized and ventilated guinea-pigs, intravenous injections of endothelin (ET)-1 (0.5 nmol/kg), ET-3 (0.5 nmol/kg), and [Ala1,3,11,15]ET-1 (20 nmol/kg), an ETB-selective receptor agonist, induced bronchoconstrictor and transient vasoconstrictor responses. Only the ET-1-induced transient vasoconstriction was followed by a secondary sustained pressor response. The ETA-selective receptor antagonists, BQ-123 (1 mg/kg i.v.), attenuated only the sustained pressor response. These results indicate that the bronchoconstrictor and transient vasoconstrictor responses to endothelins in guinea-pigs aer mediated by ETB receptors, whilst the sustained pressor response is mediated by ETA receptors. The thromboxane A2 receptor antagonist, L-670,596 (0.5 mg/kg, i.v.) and a high dose of BQ-123 (30 mg/kg i.v.) abolished the bronchoconstriction only without affecting the transient pressor response to endothelin isopeptides. These results suggest that the ETB-mediated bronchoconstriction depends on thromboxane A2 formation. The different sensitivity of these ETB-mediated transient responses to BQ-123 suggests the possible existence of distinct ETB receptor subtypes.

According to the Fmoc strategy, highly potent endothelin antagonistic cyclic pentapeptides possessing cysteic acid residue(s) were prepared in solid phase peptide synthesis. Use of a tetrabutylammonium salt overcame low solubility of N-(9-fluorenylmethoxycarbonyl)cysteic acid in a conventional solvent such as DMF.

A newly synthesized ET A -selective antagonist, BQ-123, was examined with respect to its anti-endothelin(ET) action in vitro and in vivo and its effect on blood pressure in Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP). In isolated porcine coronary arteries, BQ-123 (0.07 μM to 6.0 μM) shifted the concentration-response curve for ET-1 to the right without affecting the maximal response of ET-1, its pA 2 value being 7.35. Intravenous infusion of BQ-123 at a rate of 1.2 and 30 mg/kg/hr produced a significant decrease in blood pressure in 20- to 29-week-old SHRSP, but did not alter blood pressure in 13- to 16-week-old WKY or in 18- to 19-week-old and 40-week-old SHR. The hypotensive effect of BQ-123 depended on the pretreatment blood pressure level. These results suggest that ET-1 is involved in part in the maintenance of high blood pressure in malignant hypertension, as exemplified by SHRSP.

A series of analogues of BQ-123 (1), a potent cyclic pentapeptide endothelin A receptor antagonist, with amino acids linked to the side-chain of the Pro residue via an ester linkage was synthesized. All analogues synthesized exhibited potent endothelin A receptor binding affinity similar to that of 1. Of the synthesized analogues, the Lys, Arg and Nα, Ne-dimethyllysine analogues, 9d-f, exhibited about a three-fold attenuation of in vivo clearance compared with 1. In rats, these analogues exhibited a 3-fold-higher plasma concentration and a longer retention time in plasma as compared with those of 1. The attenuated in vivo clearance was thought to be a consequence of decreased extraction of the compounds from the blood via the hepatic anion transport system, which efficiently extracts 1 from the blood.

Endothelin (ET)-1 reduced heparin-releasable lipoprotein lipase (LPL) activity in 3T3-L1 adipocytes in a concentration-dependent manner. However, a selective ETB receptor agonist, [Ala1,3,11,15]ET-1, did not act like ET-1. The ET-1-induced decrease in LPL activity was suppressed by a selective ETA receptor antagonist, BQ-123: the concentration-response curve for the ET-1 reduction of LPL activity was shifted to the right in the presence of BQ-123 in a concentration-dependent manner. This antagonistic effect of BQ-123 clarifies that the ETA receptor is responsible for the ET-1-induced reduction of LPL activity in 3T3-L1 adipocytes, which suggests that there is therapeutic potential for ETA antagonists in LPL-related lipoprotein disorders.

Synthesis and structure-activity relationships of 2-substituted-5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids, a novel class of endothelin receptor antagonists, were described. Derivatization of a lead structure 1 (IC(50)=2.4nM, 170-fold selectivity) by incorporating a substituent such as an alkyl, alkoxy, alkylthio, or alkylamino group into the 2-position of the cyclopenteno[1,2-b]pyridine skeleton was achieved via the key intermediate 8. Introduction of an alkyl group led to the identification of potent ET(A)/ET(B) mixed receptor antagonists, a butyl (2d: IC(50)=0.21nM, 52-fold selectivity) and an isobutyl (2f: IC(50)=0.32nM, 26-fold selectivity) analogue. In contrast, installment of a primary amino group resulted in ET(A) selective antagonists, a propylamino 2p (IC(50)=0.12nM, 520-fold selectivity) and an isopropylamino 2q (IC(50)=0.10nM, 420-fold selectivity) analogue. These results suggested that a substituent at the 2-position of the 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids played a key role in the binding affinity for both ET(A) and ET(B) receptors.

Compounds (2-5) with a 6-carboxy-5,7-diarylcyclopentenopyridine skeleton were designed, synthesized, and identified as a new class of potent non-peptide endothelin receptor antagonists. The regio-isomer 2 was found to show potent inhibitory activity with an IC(50) value of 2.4 nM against (125)I-labeled ET-1 binding to human ET(A) receptors and a 170-fold selectivity for ET(A) over ET(B) receptors. Furthermore, 2 displayed more potent in vivo activity than did the indan-type compound 1 in a mouse ET-1 induced lethality model, suggesting the potential of 2 as a new lead structure. Derivatization on substituted phenyl groups at the 5- and 7-positions of 2 revealed that a 3,4-methylenedioxyphenyl group at the 5-position and a 4-methoxyphenyl group at the 7-position were optimal for binding affinity. Further derivatization of 2 by incorporating a substituent into the 2-position of the 4-methoxyphenyl group led to the identification of a more potent ET(A) selective antagonist 2p with an IC(50) value of 0.87 nM for ET(A) receptors and a 470-fold selectivity. In addition, 2p showed highly potent in vivo efficacy (AD(50): 0.04 mg/kg) in the lethality model.

The degradation of [5S-[5alpha,6beta,7alpha(R*)]]-2-butyl-5-(1,3-benzodioxol-5-yl)-7-[(2-carboxypropyl)-4-methoxyphenyl]-6-dihydro-5H-cyclopenta[b]pyridine-6-carboxylic acid (J-104,132) was studied in aqueous solution as a function of temperature and pH. The degradation reaction does not proceed to completion; rather, a stable equilibrium is attained in which approximately 2% of the degradate is produced. Kinetic data for the formation of the degradate are analyzed using an integrated form of the rate law for a reversible first-order reaction, and the forward and reverse rate constants and overall equilibrium constants are presented. Isolation and spectroscopic structural determination indicate that the degradate is the C7 beta-epimer of the drug. A mechanism for the epimerization reaction involving a novel enamine-like intermediate is proposed and shown to be consistent with the kinetic data. The rate and equilibrium constants are used to predict the room temperature stability of an injectable formulation of J-104,132, and these predictions are compared to actual data from long-term stability studies. It is concluded that the preformulation kinetic studies provide essential data needed for optimum drug product development.

We have reported previously that [8]-gingerol increased Ca(2+)-ATPase activity. Here we synthesized gingerol related compounds (AP-004, AP-005 and AP-015) and investigated the effects of gingerols ([6]-gingerol, [8]-gingerol and [10]-gingerol) and the synthesized compounds on the Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR). The Ca(2+)-ATPase activity and the Ca(2+)-pumping activity increased by these compounds in a concentration-dependent manner. It is probable that both the o-methoxyphenol and hydrocarbon chain in the molecule of gingerol analogues are necessary for the activation of the Ca(2+)-pumping ATPase activity of SR.

Continuing studies on modifications of potent cyclic pentapeptide endothelin (ET) receptor antagonists, represented by BQ-123, and potent linear tripeptide derivative ET receptor antagonists, represented by BQ-788, are described herein. The introduction of d-tryptophan analogues with C-2 substituents in these peptidic ET antagonists resulted in potent ET receptor antagonists with various ETA/ETB subtype selectivity. Combined ETA/ETB receptor antagonists were found in both cyclic pentapeptide and linear tripeptide series with 2-halo- and 2-methyl-d-tryptophans. In contrast, compounds with 2-cyano-d-tryptophan were ETB receptor-selective antagonists. The C-2 substituent of the d-tryptophanyl residue appeared to be very important for the discrimination of ETA/ETB subtype selectivity of the antagonists. The potent ET receptor antagonists with various ETA/ETB subtype selectivity synthesized in this study may be useful tools for elucidating the physiological and pathophysiological roles of ET and ET receptors.

Our previous report suggests the important role that ETB receptors play in the clearance of circulating endothelin (ET)-1. The present study confirmed this finding by measuring the extracellular levels of immunoreactive (ir) ET-1 and intracellular contents of prepro (pp) ET-1 mRNA in the presence and absence of ETA- and ETB-selective antagonists (i.e., BQ-123 and BQ-788, respectively) in cultured human umbilical vein endothelial cells (HUVECs). ET-1 was secreted into the culture medium of HUVECs in a time-dependent manner with a plateau reached after incubation for more than 36 hr. In the presence of 10 microM BQ-788, the irET-1 level was enhanced significantly (i.e., up to 180% of control at 48 hr) whereas BQ-123 had no such effect. Specific binding of [125I]ET-1 to HUVECs was inhibited strongly by BQ-788 (IC50 = 2.4 nM) but very weakly by BQ-123 (IC50 = 1.4 microM), indicating that BQ-788 has a potent affinity for ETB receptors in HUVECs. The expression of ppET-1 mRNA was not changed by BQ-788. Extracellular levels of ET-1 decreased gradually after cellular treatment with cycloheximide. This decrease was significantly inhibited by BQ-788 but not by BQ-123 and was non-existent when the cells were incubated at 4 degrees C (where internalization of the receptor protein is not likely). In conclusion, HUVECs secrete ET-1 which, in turn, is internalized after binding to ETB receptors on HUVECs.

In the rabbit isolated pulmonary artery, neither the ETA receptor antagonist, BQ-123 (10 microM), nor the ETB receptor antagonist, BQ-788 (10 microM), inhibited the contractions induced by 1 nM endothelin-1 (ET-1). However, the combination of BQ-123 and BQ-788 completely inhibited the ET-1-induced contraction. In contrast, the ETB-selective agonist, sarafotoxin S6c (1 nM)-induced contraction was completely inhibited by BQ-788 but not by BQ-123. In receptor binding assays, [125I]-ET-1 specific binding to pulmonary arterial membranes was inhibited by BQ-123 (1 microM) by approximately 20% and additive treatment with BQ-788 (1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. The present study demonstrates synergistic inhibition by BQ-123 and BQ-788 of ET-1-induced contraction of the rabbit pulmonary artery and the coexistence of ETA and ETB receptors, suggesting that the activation of either only ETA or only ETB receptors may be sufficient to cause complete vasoconstriction. Therefore, blockade of both receptor subtypes would be necessary for the inhibition of some ETA/ETB composite types of responses.

To elucidate the pathophysiological role of endogenous endothelin (ET), we examined the effects of the newly synthesized ETA receptor-selective antagonist, BQ-123, on ischemic acute renal failure induced by bilateral clamping of renal artery and vein followed by reperfusion in rats. BQ-123, when given by i.v. infusion of 0.5 mg/kg per min for 2.5 h during the pre- and post-ischemic period, was found to prevent the decrease in creatinine clearance and increases in blood urea nitrogen, plasma creatinine and the fractional excretion of sodium. Morphological observation also showed an effect of BQ-123, i.e. prevention of proximal tubular (S3 segment) necrosis. At 2 h after the start of reperfusion, the ET-1 content in the kidney increased to its maximal level. At this time, the Ca2+ content in the mitochondrial fraction of the renal cortex increased, with a concomitant increase in blood urea nitrogen. However, these increases were limited by treatment with BQ-123. Thus, BQ-123 was effective to both prevent mitochondrial Ca2+ accumulation in the early phase of ischemic acute renal failure and protect proximal tubular cells from post-ischemic degeneration. We conclude that ET may be at least partially involved in the pathogenesis of tubular cell injury in this acute renal failure model.

We have demonstrated the different distribution of two distinct endothelin (ET) receptor subtypes in porcine pulmonary tissues using a radioligand binding assay. The clear differentiation of the subtypes was made possible by the discovery of two compounds, BQ-123 and [Ala1,3,11,15]ET-1 (4AlaET-1), that are highly selective for ETA and ETB receptors, respectively. In the bronchus and lung parenchyma, BQ-123 inhibited 65% and 30% of [125I]ET-1 binding on the sensitive sites, while 4AlaET-1 displaced 25% and 60%, respectively. The combination of the two compounds completely inhibited ET-1 binding in both tissues. An autoradiographic study of [125I]ET-1 binding using BQ-123 and 4AlaET-1 also supported the different localization of two ET receptor subtypes in pulmonary tissues. In particular, the blood vessels and bronchi are rich in ETA, but the lung parenchyma is rich in ETB.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.

The novel endothelin (ET) receptor antagonists BE-18257A and BE-18257B were isolated from the fermentation products of Streptomyces misakiensis. The above-mentioned compounds inhibited [125I]ET-1 binding to ETA receptors (selective for ET-1) on porcine aortic vascular smooth muscle cells (VSMCs) with IC50 values of 1.4 and 0.47 microM, respectively. [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in cerebellar membranes was not inhibited by either of these compounds even at 100 microM. The synthesized analogue BQ-123 induced extremely potent inhibition of [125I]ET-1 binding to ETA receptors (IC50 of 7.3 nM), but it barely inhibited [125I]ET-1 binding to ETB receptors (IC50 of 18 microM) and binding of various other peptides to their receptors. BQ-123 shifted the concentration-response curve for ET-1 toward the right in porcine isolated coronary arteries, indicative of competitive antagonism for the ETA receptor. However, there was a small amount of BQ-123-insensitive vasoconstriction that paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the vascular smooth muscle layer. These data suggest that the artery contracts via both ETA and ETB receptors and that BQ-123 selectively inhibits ETA-mediated contraction. Furthermore, BQ-123 revealed large tissue and species differences in the distribution of ETA receptors. Thus, the potent ETA antagonist BQ-123 should be useful in clarifying the (patho)physiological roles of ETA receptors.

An easy meethod to prepare an endothelin antagonistic linear tripeptide derivatives is reported. The highly soluble tetrabutylammonium salt of an amino acid was allowed to directly react with a dipeptide derivative in an anhydrous dipolar aprotic solvent such as DMF via an azide method to afford the desired linear tripeptide upon usual work-up procedures

Rabbit erythrocytes were fractionated into populations of young cells (top layer) and old ones (bottom layer) by centrifugation, based on the fact that the specific gravity of young erythrocytes was lower than that of old ones. Then, reticurocyte count and osmotic resistance of red cells were compared between both populations. As a result, it has been obtained that many reticurocytes were found in the top layer, while none of those was found in the bottom layer. Furthermore, cells of the top layer were more resistant to hemolysis in the hypotonic media than those of the bottom layer. These findings suggested that the cell population of the top layer was young and that of the bottom layer was old. In comparison between both populations, the mean value of ALAD activity of young cells was 2.6 times higher than that of old ones. The rate of decrease of ALAD activity after intravenous administration of lead was similar in both populations while the amount of decrease of ALAD activity in the young cells was 2 to 3 times higher than that of the old ones.

Heteroaromatic cations such as 9-heteroniaanthracene (acridinium, xanthylium, and thioxanthylium) and 1-thionianaphthalene (thiochromenylium) ions reacted with trimethyl phosphite in the presence of sodium iodide in acetonitrile at room temperature to give the corresponding phosphonates (2 and 4) respectively in 76–92% yields. The phosphonates were deprotonated with butyllithium in THF at −78 °C, and the resulting carbanions reacted with p-tolualdehyde, cinnamaldehyde, and 4,4′-dichlorobenzophenone to afford the corresponding exomethylene containing heterocyclic compounds, respectively, in 52–96% yields. However, the phosphonate carbanions derived from 2 did not react with cycloalkanones and butanal.

The reaction of 3-substituted 2-nitrosoimino-2,3-dihydrobenzothiazoles (1) with Grignard-type reagents of 3-substituted 2-imino-2,3-dihydrobenzothiazoles (2) gave azamonomethinecyanines (3) in high yields (71–96%). This is a new type of reaction for the preparation of 3. Hydrolysis of 3 afforded ring-opened disulfides, bis[o-{N-methyl-N-(3-methyl-2,3-dihydrobenzothiazol-2-ylidenecarbamoyl)amino}-phenyl] disulfides, in high yields (78–85%). Formation of by-products is explained by the presence of transnitrosation between 1 and 2 and ring-opening equilibrium of Grignard-type reagent of 2-imino-3-phenyl-2,3-dihydrobenzothiazole.

The reactions of 1,3-dithiolylium salts with tertiary phosphines and trialkyl phosphites in dry acetonitrile gave the corresponding phosphonium salts and the phosphonates respectively in high yields. Both 2-triphenylphosphonio-1,3-benzodithiole tetrafluoroborate and 2-dimethoxyphosphinyl-1,3-benzodithiole were deprotonated with butyllithium in THF at −78 °C and the resulting phosphorane (A) and the phosphonate carbanion (B) reacted with a variety of carbonyl compounds to give 1,4-benzodithiafulvenes in 74–98% yields. Moreover, B reacted with conjugated cyclic ketones (5) such as dibenzo[a,d]cyclohepten-5-one, 10H-9-thiaanthracen-10-one, 9-alkyl-9,10-dihydro-9-azaanthracen-10-one, 9-fluorenone, and so on to give 1,4-benzodithiafulvalenes (6) in 73–96% yields. The UV spectral data of 6 were discussed in comparison with those of 5.

The spectrophotometric method reported by Commins and Lawther for estimating carboxyhemoglobin was compared with that of Kampen and others. Commins and Lawther's method proved to be valid for determining accurately low levels of carboxyhemoglobin in small blood samples. Applications of this method were done in some cases. In a study of effects of smoking on the carboxyhemoglobin levels, it has been confirmed that cigarette smoking is a major factor in determining the carboxyhemoglobin level of individuals and that this fact must always be taken into account. This method was also used in a study of the relationship between exposure to carbon monoxide and the carboxyhemoglobin levels among a group of workers, both before and after work, working in a certain underground parking station in Tokyo. This survey revealed that workers had been affected by the occupational exposure to carbon monoxide.As a result, the method of Commins and Lawther was found to be suitable for studying the relationship of carboxyhemoglobin levels with the occupational exposure to carbon monoxide as well as with exposures to ambient air pollution.