Synthesis of apicidin-derived quinolone derivatives: parasite-selective histone deacetylase inhibitors and antiproliferative agents.

Apicidin's indole was efficiently converted into a series of N-substituted quinolone derivatives by indole N-alkylation followed by a two-step, one-pot, ozonolysis/aldol condensation protocol. The new quinolones exhibited good parasite selectivity and potency both at the level of their molecular target, histone deacetylase, and in their whole cell antiproliferative activity in vitro.

High temporal resolution ECG-gated scintigraphic angiocardiography.

The cardiac blood pool is visualized with high temporal resolution during a complete, average, cardiac cycle. The technique yields both qualitative and quantitative measures of cardiac performance.

Technetium-99m pyrophosphate myocardial uptake in patients with stable angina pectoris.

99m-technetium (Tc) pyrophosphate myocardial scintigrams of 55 patients with stable angina pectoris were compared with those of 13 normal subjects. The mean scintigraphic score, obtained by averaging the blinded interpretations of four readers scoring on an integral scale from 0 to 4, was significantly higher for the patients with angina than for the control subjects (1.36 compared with 0.48, P less than 0.001). Among the patients with angina, those who had a prior myocardial infarction had a higher mean scintigraphic grade than those without a previous infarction (1.73 versus 1.15, P less than 0.005), and the mean grade in both groups was higher than that of control subjects (P less than 0.001). Radionuclide uptake was predominantly diffuse in the patients with angina pectoris (70%), although in those with greater uptake accumulation tended to be localized. Three of the 68 subjects had high levels of radionuclide uptake but no clinical evidence of acute myocardial injury. This study demonstrates that excess myocardial accumulation of 99m-Tc pyrophosphate can occur in patients with stable angina pectoris.

Whole-body PET imaging with [18F]fluorodeoxyglucose in management of recurrent colorectal cancer.

HYPOTHESIS: Metabolic imaging by positron emission tomography (PET) using [18F]fluorodeoxyglucose will be more accurate than anatomic imaging by computed tomography (CT) for detection of recurrent colorectal cancer. More accurate staging of recurrent tumor by PET will lead to more appropriate management decisions. DESIGN: Prospective blinded study comparing PET with CT, using histologic diagnosis, serial CT imaging, and clinical follow-up as criterion standards, with a fully blinded, retrospective reinterpretation of PET studies. Changes in diagnosis resulting from PET findings were correlated with subsequent treatment and surgical findings. Potential cost savings resulting from use of PET for preoperative staging were calculated. SETTING: Private practice in an outpatient tertiary referral center. PATIENTS: A group of 155 consecutive patients with imaging for diagnosis or staging of recurrent colorectal cancer. Twenty-one patient (14%) were excluded due to lack of a criterion standard. Computed tomographic scans were available for comparison for 115 patients. RESULTS: Positron emission tomographic scan sensitivity and specificity were 93% and 98%, respectively, compared with 69% and 96% for CT. Ninety-five percent confidence intervals for the differences between the modalities were 16% to 32% for sensitivity and 1% to 5% for specificity. The sensitivity of both modalities varied with anatomic site of recurrence. Positron emission tomographic scans were true positive in 12 (67%) of 18 patients with elevated serum carcinoembryonic antigen levels and negative CT findings. In 23 (29%) of 78 preoperative studies in which CT showed a single site of recurrence, PET showed tumor at additional sites. At surgery, nonresectable, PET-negative tumor was found in 7 (17%) of 42 patients who had PET evidence of localized recurrence only. Potential savings resulting from demonstration of nonresectable tumor by PET were calculated at $3003 per preoperative study. CONCLUSIONS: Positron emission tomography was more sensitive and specific than CT for detection of recurrent colorectal cancer. Preoperative detection of nonresectable tumor by PET may avoid unnecessary surgery, and thereby reduce the cost of patient treatment.

Staging non-small cell lung cancer by whole-body positron emission tomographic imaging.

BACKGROUND: A need exists for an accurate, noninvasive means of staging non-small cell lung cancer. METHODS: A prospective evaluation of regional and whole-body positron emission tomography (PET) imaging for staging lung cancer was carried out in 99 patients. Mediastinal PET and computed tomography findings were compared with results of surgical staging in 76 patients. Those PET and computed tomography findings that indicated possible distant metastasis were compared with biopsy results and the results of clinical and imaging follow-up. RESULTS: Sensitivity and specificity for the diagnosis of N2 disease were 83% and 94% for PET and 63% and 73% for computed tomography, respectively. Positron emission tomography showed previously unsuspected distant metastasis in 11 patients (11%), with no demonstrated false-positive results. Normal PET findings were obtained at distant sites of computed tomography abnormality in 19 patients (19%). Clinical and imaging follow-up in 14 of these patients showed no evidence of metastasis. In 1 case, the PET result proved to be falsely negative. CONCLUSIONS: Imaging with PET was more accurate than computed tomography for diagnosis of mediastinal and distant metastasis. Detection of unsuspected metastatic disease by PET may permit reduction in the number of thoracotomies performed for nonresectable disease.

Improved preparations of some per-O-acetylated aldohexopyranosyl cyanides.

3,4,6-Tri-O-acetyl-1,2-O-[1-(exo-, endo-cyano)ethylidene]-alpha-D- galacto- (1a/b), -alpha-D-gluco- (2a/b), and -beta-D-manno-pyranose (3a/b) were stereoselectively isomerized to the corresponding per-O-acetylated 1,2-trans-aldohexopyranosyl cyanides in 75, 16, and 62% yield, respectively, by treatment with boron trifluoride etherate in dry nitromethane. The corresponding per-O-acetylated 1,2-cis-aldohexopyranosyl cyanides were obtained concurrently in respective yields of 1.9, 0.9, and 4.8%. The per-O-acetylaldohexopyranosyl cyanide products were found stable to the reaction conditions and were readily isolated following completion of the rearrangement. It had previously been proved that reaction of 2,3,4,6-tetra-O-acetyl-alpha-D-manno- and -gluco-pyranosyl bromide with mercuric cyanide in nitromethane generates, in the ratio of approximately 1:1, the desired 1,2-trans-glycosyl cyanides and the corresponding 1,2-O-(1-cyanoethylidene) isomers (3a/b and 2a/b, respectively). Treatment of these reaction-mixtures with boron trifluoride etherate in nitromethane effected the rearrangement of 3a/b and 2a/b, thereby facilitating the isolation, and increasing the overall yields, of the per-O-acetylated 1,2-trans-D-manno- and -gluco-pyranosyl cyanides (58 and 30% total yield, respectively) relative to the earlier procedures. The boron trifluoride etherate-mediated reaction of per-O-acetyl-alpha- and -beta-D-galacto-, -alpha- and -beta-D-gluco-, -alpha-D-manno-, and -2-deoxy-2-phthalimido-beta-D-gluco-pyranoses with trimethylsilyl cyanide in nitromethane was also investigated. This reaction provides a "one-flask" synthesis of the corresponding per-O-acetylated 1,2-trans-aldohexopyranosyl cyanides in which 1,2-O-(1-cyanoethylidene) derivatives are isomerized in situ. Finally, improved preparations of the (not readily accessible) per-O-acetylated 1,2-cis-D-manno- and -gluco-pyranosyl cyanides are described. Thus, 2,3,4,6-tetra-O-acetyl-alpha- and -beta-D-mannopyranosyl cyanide (48 and 16% total yield, respectively) and -alpha- and -beta-D-glucopyranosyl cyanide (12 and 39% total yield, respectively) were synthesized by fusion of the corresponding alpha-D-glycosyl bromides with mercuric cyanide.

Synthesis and characterization of some anomeric pairs of per-O-acetylated aldohexopyranosyl cyanides (per-O-acetylated 2,6-anhydroheptononitriles). On the reaction of per-O-acetylaldohexopyranosyl bromides with mercuric cyanide in nitromethane.

The synthesis and characterization of the anomeric pairs of the per-O-acetylaldohexopyranosyl cyanides of D-galactose, L-fucose, D-glucose, and D-mannose, as well as of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl cyanide, are described. Cyanation of the readily available, per-O-acetylaldohexopyranosyl bromides with mercuric cyanide in nitromethane, and subsequent purification, gave the corresponding, crystalline glycosyl cyanides with a high degree of 1,2-trans stereoselectivity. Thus, per-O-acetylated aldohexopyranosyl cyanides of the 1,2-trans configuration were obtained in yields ranging from 20 to 79%, whereas the corresponding 1,2-cis anomers were obtained in yields of less than or equal to 8.4%, the ratios of the 1,2-trans:1,2-cis anomers so prepared being greater than or equal to 8.5:1. The principal by-products of these irreversible, cyanation reactions were the per-O-acetylated 1,2-O-[1-(exo- and endo-cyano)ethylidene]aldohexopyranoses, obtained in yields of up to 40%. The structural assignments of the per-O-acetylaldohexopyranosyl cyanides were unequivocally established by elemental analysis, chemical transformation, vibrational spectroscopy, and 13C- and 1H-nuclear magnetic resonance spectroscopy. Correlations between the physical properties and the anomeric configurations of these C-aldohexopyranosyl compounds are described.

Synthesis of diazomethyl beta-D-galactopyranosyl and beta-D-glucopyranosyl ketones. Potential affinity-labeling reagents for carbohydrate-binding proteins.

3,7-Anhydro-1-deoxy-1-diazo-D-glycero-L-manno-2-octulose (6a; diazomethyl beta-D-galactopyranosyl ketone) and 3,7-anhydro-1-deoxy-1-diazo-D-glycero-D-gulo-2-octulose (6b; diazomethyl beta-D-glucopyranosyl ketone) have been prepared. Readily available C-glycosyl compounds possessing the appropriate stereo-chemistry and hydroxyl-group protection, viz., per-O-acetyl-2,6-anhydroheptononitriles and per-O-acetyl-2,6-anhydroheptonamides, were employed as precursors to per-O-acetyl-2,6-anhydroheptonic acids. These key intermediates were then converted into mixed carboxylic-carbonic acid anhydrides, and these caused to react with diazomethane, to give the corresponding per-O-acetyl-3,7-anhydro-1-deoxy-1-diazo-2-octuloses. Zemplén deacetylation gave, stereospecifically, the crystalline target-molecules in good overall yield. It is proposed that such C-glycosyl compounds as 6a and 6b, which possess the diazoacetyl functional groups as their "aglycon", will be useful as enzyme-activated irreversible inhibitors (suicide substrates) of glycosidases, and as photoaffinity-labeling reagents and classical affinity-labeling reagents for carbohydrate-binding proteins.

The crystal structure of diazomethyl beta-D-galactopyranosyl ketone.

The title compound (C8H12N2O6) crystallizes in the orthorhombic space group P2(1)2(1)2(1) (Z = 4), with a = 4.871(1), b = 11.136(2), c = 18.301(2) A. The structure was solved by the multi-solution technique and refined by full-matrix least-squares to a final R-index of 0.042. The compound adopts the 4C1(D) conformation. Bond lengths in the diazoacetyl group are consistent with the presence of a zwitterion.

Suicide-substrate inactivation of beta-galactosidase by diazomethyl beta-D-galactopyranosyl ketone.

Diazomethyl beta-D-galactopyranosyl ketone (1) has been proven to be a mechanism-based, irreversible (suicide-substrate) inactivator of Aspergillus oryzae beta-D-galactosidase, but not an inactivator of E. coli lacZ beta-D-galactosidase. Compound 1 is stable in buffers of normal physiological pH. It is decomposed by H+, but not by nucleophiles. Inactivation of A. oryzae beta-D-galactopyranosyl ketone (2) nor diazomethyl alpha-D-galactopyranosyl ketone inactivated the enzyme and therefore inactivation is stereospecific, excess inhibitor could be separated from inactive enzyme without regain of activity and therefore it is bound irreversibly, and a second pulse of enzyme is inactivated at the same rate as enzyme inactivated to 95% activity by the first pulse. Diazomethyl beta-D-glucopyranosyl ketone (2) inhibited sweet almond beta-D-glucosidase.

Reliability and reproducibility of interpretation of 99mtechnetium pyrophosphate myocardial scintigrams.

The interpretations of 156 99mtechnetium pyrophosphate myocardial scintigrams by four observers were analyzed in order to determine the reliability and reproducibility of the subjective process of reading scintigrams. The scintigrams were scored on an integral scale from 0 to 4, depending upon the degree of myocardial radionuclide accumulation, and the site and nature of uptake were specified. Exact agreement upon score was generally poor but approximate concurrence of interpretation was good (90.4 and 92.5% inter- and intra-observer agreement, respectively). There was somewhat less agreement on scintigrams with the higher scores of 3 and 4 (83.3 and 78.0%, respectively). A high level of concurrence upon the differentiation between diffuse and localized uptake, and upon the site of uptake, was found. We conclude that only approximate rather than exact agreement of individual readers' interpretations can be expected in this subjective technique, that scintigrams with higher degrees of radionuclide accumulation produce slightly greater observer disagreement, and that variability of interpretation could account for some of the diagnostic inaccuracy of 99mtechnetium pyrophosphate myocardial scintigraphy.

Nitrophenide (Megasul) blocks Eimeria tenella development by inhibiting the mannitol cycle enzyme mannitol-1-phosphate dehydrogenase.

Unsporulated oocysts of the protozoan parasite Eimeria tenella contain high levels of mannitol, which is thought to be the principal energy source for the process of sporulation. Biosynthesis and utilization of this sugar alcohol occurs via a metabolic pathway known as the mannitol cycle. Here, results are presented that suggest that 3-nitrophenyl disulfide (nitrophenide, Megasul), an anticoccidial drug commercially used in the 1950s, inhibits mannitol-1-phosphate dehydrogenase (M1PDH), which catalyzes the committed enzymatic step in the mannitol cycle. Treatment of E. tenella-infected chickens with nitrophenide resulted in a 90% reduction in oocyst shedding. The remaining oocysts displayed significant morphological abnormalities and were largely incapable of further development. Nitrophenide treatment did not affect parasite asexual reproduction, suggesting specificity for the sexual stage of the life cycle. Isolated oocysts from chickens treated with nitrophenide exhibited a dose-dependent reduction in mannitol, suggesting in vivo inhibition of parasite mannitol biosynthesis. Nitrophenide-mediated inhibition of MIPDH was observed in vitro using purified native enzyme. Moreover, MIPDH activity immunoprecipitated from E. tenella-infected cecal tissues was significantly lower in nitrophenide-treated compared with untreated chickens. Western blot analysis and immunohistochemistry showed that parasites from nitrophenide-treated and untreated chickens contained similar enzyme levels. These data suggest that nitrophenide blocks parasite development at the sexual stages by targeting M1PDH. Thus, targeting of the mannitol cycle with drugs could provide an avenue for controlling the spread of E. tenella in commercial production facilities by preventing oocyst shedding.

Biosynthesis and catabolism of mannitol is developmentally regulated in the protozoan parasite Eimeria tenella.

The mannitol cycle is a metabolic branch of the glycolytic pathway found in Eimeria tenella. In this paper, we describe the biosynthesis and consumption of mannitol during parasite development. Low micromolar levels of mannitol were detected in all of the asexual stages and mannitol production increased sharply during the sexual phase of the life cycle. Unsporulated oocysts had high mannitol content (300 mM or 25% of the oocyst mass). Mannitol-1-phosphate dehydrogenase (M1PDH), the first committed step of the mannitol cycle, was also elevated in sexual stages and this coincides with mannitol levels. Approximately 90% of the mannitol present in unsporulated oocysts was consumed in the first 15 hr of sporulation, and levels continued to drop until the sporulation process was complete at approximately 35 hr. Thus, mannitol appears to be the "fuel" for sporulation during the vegetative stage of the parasite life cycle. Evaluation of oocyst extracts from 6 additional Eimeria species for mannitol content and the presence of M1PDH indicated that the mannitol cycle was broadly present in this genus. This finding combined with the lack of mannitol metabolism in higher eukaryotes makes this pathway an attractive chemotherapeutic target.

EKG gated first-transit radionuclide angiocardiography.

Radioisotopic assessment of left ventricular ejection fraction and wall motion has previously required either EKG gated blood pool imaging or nongated single-transit angiography. A simplified technique involving EKG gated image acquisition during the first cardiac transit of an isotope bolus was evaluated in 36 patients and compared to results obtained from EKG gated blood pool imaging and contrast ventriculography. The method required rapid intravenous injection of 20 mCi of any Tc-99m agent. EKG gated scintillation camera data collection was started after activity entered the left ventricle and resulted in simultaneous acquisition of end-diastolic and end-systolic images on a dedicated computer system. Thus, images representing summed intervals from eight to ten beats were immediately available for calculation of ejection fraction and subjective evaluation of wall motion. Ejection fraction from the gated first-pass images using the area length method was well correlated (r = .95, P less than .001) but tended to be slightly underestimated when compared to contrast values. Abnormal wall motion was identified with a sensitivity of 92%, a specificity of 100%, and an accuracy of 95%. When compared to EKG gated blood pool imaging, inferior wall motion abnormalities, in particular, were more evident on the first-pass study. This method provided simple and accurate assessment of ejection fraction and wall motion and appears to be a useful technique for the noninvasive assessment of left ventricular function.

Angiocardiography with the seven-pinhole collimator: evaluation of methodology and accuracy in assessing global and regional left ventricular function.

Determination of left ventricular function with tomographic radionuclide angiocardiography using a seven-pinhole collimator was evaluated by comparing results of invasive contrast studies and planar multigated blood pool imaging to the tomographic study of 25 patients. LAO, seven-pinhole multigated blood pool acquisition was reconstructed to produce eight slices from apex to base in each of eight segments of the cardiac cycle. After applying an edge detection routine, three-dimensional reconstruction of perimeters allowed cyclic viewing of the left ventricular angiogram in any projection. When planar and tomographic radionuclide techniques were compared to contrast studies, sensitivity and specificity for identification of segmental wall motion abnormalities were not different (93% and 90% for both). Ejection fraction was determined from tomography by integration of slices to produce a noncalibrated volume and from planar blood pool imaging and contrast ventriculograms by standard techniques. Ejection fraction as compared to contrast studies was accurately determined on the planar angiogram (R = .87, P less than .001) with tomographic analysis showing similar significant correlation (R = .79, P less than .001). Methodologic evaluation indicated that the tomographic study was readily positioned and acquired and provided the advantage of requiring little operator interaction. It suffers, however, from the disadvantage of long reconstruction times with current software and occasional difficulties in defining the superior extent of the left ventricle.

MMPI-2 items which correctly identified women with histories of childhood sexual abuse.

This study examined whether MMPI-2 items could be used to discriminate between 58 women who experienced childhood sexual abuse and 57 women who did not. A set of 48 items were identified which correctly classified 95% of the subjects.

Conversion of 5-S-ethyl-5-thio-D-ribose to ethionine in Klebsiella pneumoniae. Basis for the selective toxicity of 5-S-ethyl-5-thio-D-ribose.

5-S-Ethyl-5-thio-D-ribose (ethylthioribose) exhibits antiprotozoal activity against Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis, but is nontoxic to cultured human and murine bone marrow cells (Riscoe, M. K., Ferro, A. J., and Fitchen, J. H. (1988) Antimicrob. Agents Chemother. 32, 1904-1906). We propose the following mechanism to account for the observed selective toxicity of ethylthioribose. 1) The cytocidal action of ethylthioribose against protozoa is a result of its conversion to ethionine, a well-known cytotoxic agent. 2) This transformation occurs through the pathway which normally converts 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine. 3) Conversion of ethylthioribose to ethionine cannot occur in mammalian cells since these cells cannot phosphorylate methylthioribose (ethylthioribose), a first step in the pathway to methionine (ethionine). To test this hypothesis, [5-3H]ethylthioribose has been synthesized and its metabolism by cell-free extracts of Klebsiella pneumoniae and rat liver was examined. The pathway by which methylthioribose is converted to methionine in K. pneumoniae is well characterized. When supplemented with ATP and L-glutamine, the bacterial extract efficiently converted [5-3H]ethylthioribose to [3H]ethionine. By contrast, ethionine was not produced upon incubation of [5-3H]ethylthioribose, ATP, and L-glutamine with rat liver homogenate. The mammalian cell extract lacks a kinase activity capable of converting ethylthioribose to 1-phospho-5-S-ethyl-5-thio-alpha-D-ribofuranoside, an obligate intermediate in the biosynthesis of ethionine from ethylthioribose in K. pneumoniae. These results support our hypothesis and provide a basis for understanding the apparently selective toxicity of ethylthioribose.

Conversion of 5-S-methyl-5-thio-D-ribose to methionine in Klebsiella pneumoniae. Stable isotope incorporation studies of the terminal enzymatic reactions in the pathway.

Extracts of Klebsiella pneumoniae convert 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine and formate. To probe the terminal steps of this biotransformation, [1-13C]methylthioribose has been synthesized and its metabolism examined. When supplemented with Mg2+, ATP, L-glutamine, and dioxygen, cell-free extracts of K. pneumoniae converted 50% of the [1-13C]methylthioribose to [13C]formate. The formation of [13C]formate was established by 13C and 1H NMR spectroscopy studies of the purified formate, and by 13C and 1H NMR spectroscopy and mass spectrometry studies of its p-phenylphenacyl derivative. By contrast, no incorporation of label from [1-13C]methylthioribose into the biosynthesized methionine was detected by either mass spectrometry or 13C and 1H NMR spectroscopy. The most reasonable interpretation of these results is that C-1 of methylthioribose is converted directly to formate concomitant with the conversion of carbon atoms 2-5 to methionine. The penultimate step in the conversion of methylthioribose to methionine and formate is an oxidative carbon-carbon bond cleavage reaction in which an equivalent of dioxygen is consumed. To investigate the fate of the dioxygen utilized in this reaction, the metabolism of [1-13C]methylthioribose in the presence of 18O2 was also examined. Mass spectrometry revealed the biosynthesis of substantial amounts of both [18O1]methionine and [13C, 18O1]formate under these conditions. These results suggest that the oxidative transformation in the conversion of methylthioribose to methionine and formate may be catalyzed by a novel intramolecular dioxygenase. A mechanism for this dioxygenase is proposed.