A preliminary disease survey in the wild Nile crocodile (Crocodylus niloticus) population in the Okavango Delta, Botswana.

The objective of this study was to conduct a preliminary survey of diseases that might be present in the wild Nile crocodile population in the Okavango Delta, Botswana. Blood samples were collected from crocodiles ranging in size from 34.0 cm to 463.0 cm total length. Samples were examined for blood parasites and underwent a haematological analysis. Before release the crocodiles were examined for various clinical abnormalities. Of the 144 crocodiles examined, none were visibly sick or displayed any signs of disease. No antibodies to Mycoplasma crocodyli were detected. Hepatozoon pettiti was present in 55.3% of blood smears examined, but there was no significant difference in any of the haematological values between the infected and uninfected crocodiles, and a high prevalence of Hepatozoon infection is not uncommon in other species. Only 7.6% of the examined crocodiles were infested with leeches. Further research is required for several of the crocodilian diseases, in particular to elucidate the role of wild crocodilians as reservoirs of infection.

Normal intestinal flora of wild Nile crocodiles (Crocodylus niloticus) in the Okavango Delta, Botswana.

Bacterial and fungal cultures were performed from cloacal swabs collected from 29 wild Nile crocodiles, captured in the Okavango Delta, Botswana. Sixteen species of bacteria and 6 fungal species were cultured. Individual crocodiles yielded 1-4 bacterial species, and 0-2 fungal species. The most commonly isolated bacteria were Microbacterium, Enterococcus faecalis, Aeromonas hydrophila, and Escherichia coli. No salmonellae were cultured. The most commonly occurring fungus was Cladosporium. Several of the bacterial and fungal species isolated have been implicated in cases of septicaemia in crocodilians. Knowledge of the normal intestinal flora will contribute towards the development of a crocodile-specific probiotic for use in farmed crocodiles.

Normal haematology and blood biochemistry of wild Nile crocodiles (Crocodylus niloticus) in the Okavango Delta, Botswana.

Wild Nile crocodiles (Crocodylus niloticus) of various size classes were captured in the Okavango Delta, Botswana. Blood was collected from the post occipital sinus and used for the determination of a wide range of haematological and biochemical parameters. These values were compared between the sexes and between 3 size classes. The values were also compared with the limited data available from farmed Nile crocodiles, as well as from other wild Nile crocodiles. The Okavango crocodiles were comparatively anaemic, and had comparatively low total protein and blood glucose levels. There was a high prevalence of Hepatozoon pettiti infection, however, there was no significant difference in haematological values between the infected and uninfected crocodiles. The values reported here will be useful in diagnostic investigations in both zoo and farmed Nile crocodiles.

2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics.

An estradiol metabolite, 2-methoxyestradiol (2-MeOE(2)), has shown antiproliferative effects in both hormone-dependent and hormone-independent breast cancer cells. Previously, a series of 2-hydroxyalkyl estradiol analogs had been synthesized in our laboratories as potential probes for comparison of estrogen receptor (ER)-mediated versus non-ER-mediated effects in breast cancer cells. A methoxy derivative of 2-hydroxymethyl estradiol was prepared for biological evaluation and comparison with 2-MeOE(2). Estrogenic activity of the synthetic analogs was evaluated in two ways, one by examining affinity of the analogs for the estrogen receptor in MCF-7 cells and the other by examining the ability of the analogs to induce estrogen-responsive gene expression. The analog, 2-methoxymethyl estradiol (2-MeOMeE(2)), demonstrated weak affinity for the estrogen receptor (0.9% of estradiol) and weak ability to stimulate estrogen-induced expression of the pS2 gene (0.02% of estradiol). Antitumor activity was evaluated both in vitro and in vivo. The steroidal nucleus seems to be an attractive target for developing novel tubulin polymerization inhibitors. Additionally, such steroidal compounds may have low toxicity compared to the natural products known to interact with tubulin. Interestingly, 2-MeOMeE(2) inhibited tubulin polymerization in vitro at concentrations of 1 and 3 microM and was more effective than 2-MeOE(2). In cells, 2-MeOMeE(2) was effective in suppressing growth and inducing cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. The cytotoxic effects of 2-MeOMeE(2) are associated with alterations in tubulin dynamics, with the frequent appearance of misaligned chromosomes, a significant mitotic delay, and the formation of multinucleated cells. In comparison, 2-MeOE(2) was more effective than 2-MeOMeE(2) in producing cytotoxicity and altering tubulin dynamics in intact cells. Assessment of in vivo antitumor activity was performed in athymic mice containing human breast tumor xenografts. Nude mice bearing MDA-MB-435 tumor xenografts were treated i.p. with 50 mg/kg per day of 2-MeOMeE(2) or vehicle control for 45 days. Treatment with 2-MeOMeE(2) resulted in an approximate 50% reduction in mean tumor volume at treatment day 45 when compared to control animals and had no effect on animal weight. Thus, 2-MeOMeE(2) is an estrogen analog with minimal estrogenic properties that demonstrates antiproliferative effects both in vitro and in the human xenograft animal model of human breast cancer.

Synthesis of bridged medium-sized rings through the intramolecular Pauson-Khand reaction.

[reaction: see text] A series of aromatic enynes containing steric buttressing elements were prepared and evaluated in the NMO-mediated Pauson-Khand cyclization. O-Allyl systems led to the expected angularly fused products, whereas the O-butenyl and O-pentenyl derivatives afforded the unprecedented bridge systems.

Synthesis and Diels-Alder reactions of 4-vinylimidazoles.

[structure in text] The synthesis of several 4-vinylimidazoles via Stille cross-coupling reactions of the corresponding protected 4-iodoimidazoles with tributylvinylstannane is described. These heterocyclic dienes are shown to be effective partners in the Diels-Alder reaction, providing adducts in good yield and exhibiting useful levels of isomer selectivity and stereoselectivity (endo/exo).

Synthesis and biological evaluation of 4-(hydroxyalkyl)estradiols and related compounds.

A series of synthetic estrogens containing hydroxyalkyl side chains at the C-4 position of the A ring were designed as metabolically stable analogs of 4-hydroxyestradiol, a catechol estrogen. These synthetic steroids would facilitate investigations on the potential biological role of catechol estrogens and also enable further examination of the structural and electronic constraints on the A ring in the interaction of estrogens with the estrogen receptor. Catechol estrogens are implicated as possible causative agents in estrogen-induced tumorigenesis. 4-Hydroxyestradiol has weaker affinity for the estrogen receptor and exhibits lower estrogenic activity in vivo; on the other hand, the catechol estrogens are prone to further oxidative metabolism and can form reactive intermediates. This report describes the synthesis and initial biochemical evaluation of 4-(hydroxyalkyl)estrogens and 4-(aminoalkyl)estradiols. The 4-(hydroxyalkyl)estrogens were prepared by oxidative hydroboration of 4-alkenylestradiols. The alkenylestradiols were obtained via a Stille cross-coupling between a MOM-protected 4-bromoestradiol and an alkenylstannane. The (4-aminoalkyl)estrogens were prepared from the hydroxyalkyl derivatives with phthalimide under Mitsunobu conditions. The substituted estradiols were evaluated for estrogen receptor binding activity in MCF-7 human mammary carcinoma cells, and 4-(hydroxymethyl)estradiol 1 exhibited the highest affinity with an apparent EC50 value of 364 nM. The relative activities for mRNA induction of the pS2 gene in MCF-7 cell cultures by the 4-(hydroxyalkyl)estrogens closely parallel the relative binding affinities. 4-(Hydroxymethyl)estradiol 1 did not stimulate the growth of MCF-7 cells at concentrations up to 1 microM. Thus, 4-(hydroxymethyl)estradiol 1 exhibited similar estrogen receptor affinity as the catechol estrogen, 4-hydroxyestradiol, and may prove useful in the examination of the biological effects of 4-hydroxyestrogens.

Aromatase inhibition in JAr choriocarcinoma cells by 7alpha-arylaliphatic androgens.

The JAr choriocarcinoma cell cultures have demonstrated high levels of aromatase activity and have been useful for assaying a wide variety of aromatase inhibitors for aromatase inhibition in intact cells. Recently, several 7alpha-arylaliphatic androgens have shown effective inhibition of human placental microsomal aromatase in vitro, with apparent Ki values ranging from 10 to 20 nM. A series of 7alpha-arylaliphatic androst-4-ene-3,17-dione compounds demonstrated potent competitive inhibition, and 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones were enzyme-activated irreversible inhibitors. Both series of these potent inhibitors were investigated for the ability to inhibit aromatase activity in JAr cells by measuring the conversion of [1beta-3H]-androstenedione to 3H2O and unlabelled estrone. JAr cell cultures were incubated for 2 h at 37 degrees C with the aromatase inhibitors at concentrations of 10 pM to 10 microM, the percentage of enzyme inhibition was determined, and IC50 values for inhibitors were calculated. Both series of synthetic compounds demonstrated good to excellent aromatase inhibition, and the most effective inhibitors in both series were those compounds with a phenylpropyl substituent at the 7alpha-position of the steroid nucleus. The 7alpha-arylaliphatic androst-4-ene-3,17-diones exhibited inhibition of JAr aromatase activity with IC50 values from 300 to 434 nM. More potent aromatase inhibition was observed with the 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones, which exhibited IC50 values from 64 to 232 nM. Enhanced efficacy of steroidal enzyme-activated irreversible inhibitors compared to competitive inhibitors was observed in these studies and is consistent with previous reports. These results suggest that JAr choriocarcinoma cells with high levels of aromatase activity may be useful in differentiating steroidal aromatase inhibitors exhibiting different mechanisms of enzyme inhibition. In summary, the 7alpha-phenylpropyl androsta-1,4-diene-3,17-dione analogs, which are enzyme-activated irreversible inhibitors, demonstrated the most effective inhibition of aromatase activity present in the JAr cell cultures among the various 7alpha-arylaliphatic androgens.

Biochemistry and pharmacology of 7alpha-substituted androstenediones as aromatase inhibitors.

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7alpha-substituted androstenediones. Numerous 7alpha-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7alpha-(4'-aminophenylthio)-androsta-1,4-diene-3,17-di one (7alpha-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7alpha-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7alpha-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7alpha position with a carbon-carbon linkage. Several 7alpha-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent Kis ranging between 13 and 19 nM. Extension of the research on these 7alpha-arylaliphatic androgens includes the introduction of a C1-C2 double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7alpha-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k(inact)s ranging from 4.4 x 10(-4) to 1.90 x 10(-3) s(-1). The best inactivator of the series was 7alpha-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T(1/2) of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC50 values of 64-328 nM. The 7alpha-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7alpha-position of the steroid in the active site of aromatase. The size of the 7alpha-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.

2-(Hydroxyalkyl)estradiols: synthesis and biological evaluation.

Synthetic estrogens possessing hydroxyalkyl side chains at the C-2 position of the A-ring were designed in order to further elucidate the structural and electronic requirements of the estrogen receptor to A-ring modifications. Furthermore, these compounds were envisaged as being stable analogs of the estradiol metabolite 2-hydroxyestradiol. The homologous series of 2-(hydroxyalkyl)estradiols 1-3 has been prepared by chain extension of 2-formylestradiol 6, which, in turn, was prepared via ortholithiation of estradiol. The substituted estradiols 1-3 were assayed for their abilities to bind to the estrogen receptor in MCF-7 cells and induce estrogen-responsive gene expression. The estradiol homologs exhibited significantly weaker affinity than estradiol for the MCF-7 cell estrogen receptor, with relative binding affinities (estradiol = 100) ranging from 1.11 for 2-(hydroxymethyl)estradiol (1) to 0.073 for 2-(hydroxypropyl)estradiol (3). The relative activities for mRNA induction of the pS2 gene by the estradiol homologs closely parallel the relative binding affinities for the estrogen receptor in MCF-7 cells. 2-(Hydroxymethyl)-estradiol exhibited similar estrogen receptor affinity and pS2 gene induction to the catechol estrogen 2-hydroxyestradiol and may prove useful in examination of the further biological effects of 2-hydroxyestrogen homologs.