Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate.

The present study was carried out to evaluate the effects of different activation protocols, enucleation methods, and culture media on the development of parthenogenetic and nuclear transfer (NT) rabbit embryos. Electroporation of 25 mM inositol 1,4, 5-trisphosphate (IP3) in calcium- and magnesium-free PBS immediately induced a single intracellular calcium transient in 6 out of 14 metaphase II-stage rabbit oocytes evaluated during a 10-min recording period. The percentage of oocytes treated with IP3 followed by 6-dimethylaminopurine (IP3 + DMAP) that cleaved (83.9%) and reached the blastocyst stage (50%) was significantly higher (p < 0.05) than those activated with multiple pulses (61.6% and 30.1%, respectively) or treated with ionomycin + DMAP (52.9% and 5.7%, respectively). Development of IP3 + DMAP-activated rabbit oocytes and in vivo-fertilized zygotes in different culture media was studied. Development of activated oocytes to the blastocyst stage in Earle's balanced salt solution (EBSS) supplemented with MEM nonessential amino acids, basal medium Eagle amino acids, 1 mM L-glutamine, 0.4 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (EBSS-complete) (40.6%) was significantly higher (p < 0.05) than those that developed in either Dulbecco's Modified Eagle's medium (DMEM)/RPMI + 10% FBS (15.5%) or CR1aa + 10% FBS (4%) medium. In addition, 100% of in vivo-fertilized rabbit zygotes developed to the blastocyst stage in EBSS-complete. A third set of experiments was carried out to study the efficiency of blind versus stained (Hoechst 33342) enucleation of oocytes. Twenty-nine of 48 blind enucleated and IP3 + DMAP-activated oocytes cleaved (60.4%), and 15 (31.2%) subsequently reached the blastocyst stage, whereas 9 of 52 oocytes enucleated using epifluorescence (17.3%) cleaved, and none of these reached the blastocyst stage. When the above parameters that yielded the highest blastocysts were combined in an NT experiment using adult rabbit fibroblast nuclei, 72.2% (39 of 54) of the fused nuclear transplant embryos cleaved and 29.6% (16 of 54) reached the blastocyst stage.

Spiranes 6. Ring A homologues of N-benzyloxy-2-azaspiro[4.4]nonane-1,3-dione. Synthesis, X-ray analysis and anticonvulsant evaluation.

A series of spirosuccinimides was synthesized and evaluated for anticonvulsant activity. The study was designed to determine the effect of varying the carbocyclic (ring A) nucleus, while maintaining the heterocyclic ring constant, on anticonvulsant activity. Results indicate that maximum activity was obtained with the ring A comprised of a six-membered spiro ring system, 2a, one methylene group greater than that previously reported for N-(benzyloxy)-2-azaspiro[4.4]nonane-1,3-dione, 1, the prototype analogue. Compound 2a was active in the MES test providing protection at 100 mg/kg, as was the spirododecane analog 2g. X-ray analysis revealed significant differences between active 2a, and the inactive spirooctane analogue, 2f. However these differences could not explain the unexpected activity demonstrated by the spirododecane analog 2g.

Synthesis and anticonvulsant activity of enaminones. 3. Investigations on 4'-, 3'-, and 2'-substituted and polysubstituted anilino compounds, sodium channel binding studies, and toxicity evaluations.

In a continuing evaluation of the aniline-substituted enaminones, the synthesis of additional para-substituted analogs has been made in an attempt to further quantify the electronic (sigma) and lipophilic (pi) requirements for anticonvulsant activity in this series. In addition, meta- and ortho-substituted and polysubstituted compounds have been synthesized and evaluated for anticonvulsant activity. In the para-substituted series, 4-cyano analogs (32 and 33) (+ sigma, - pi), which were highly active via intraperitoneal (ip) injection in mice, were inactive on oral (po) administration in rats. The para-substituted trifluoromethoxy (+ sigma, + pi) analog (8) had significant potency by both routes. Meta substitution limited the activity due to steric factors. Bromo and iodo substituents produced active para-substituted analogs (5 and 17) but were inactive when substituted in the meta position (37 and 41, respectively). Ortho substitution provided no clear relationship due to nonparametric deviations. Neither 1, the prototype enaminone, nor 2, the putative metabolite, produced significant nephrotoxicity or hepatotoxicity. Sodium channel binding of 1 and 8 indicated that 8 displayed relatively potent sodium channel binding but 1 showed weaker effects with IC50 values of 489 and 170 microM respectively against [3H]batrachotoxinin A 20 alpha-benzoate ([3H]BTX-B).

Cyclization reactions leading to beta-hydroxyketo esters.

The purpose of the research was to synthesize beta-diketo esters and to evaluate them for anticonvulsant activity. The reaction of methyl vinyl ketone with dimethyl malonate in the presence of potassium carbonate gave an uncyclized product that underwent a Claisen condensation to yield methyl 2-hydroxy-4-oxocyclohex-2-en-1-oate (5a). Similarly, other cyclized beta-hydroxyketo esters were prepared, and their spectrometric data confirmed that the enol tautomers were preferred to the beta-diketo tautomers. The synthetic work clarified the reaction pathway for the Michael addition of malonate esters to enones. Of the intermediates and products tested for anticonvulsant activity, dimethyl 2,2-bis-(3-oxobutyl)malonate (9a) was found to possess anticonvulsant property. However, it is emphasized that the beta-hydroxyketo esters could be useful intermediates in the synthesis of enaminone anticonvulsants.

Synthesis, reactions, and preliminary evaluations of enaminone esters.

The objective of this work was to design enaminone esters that would possess potential medicinal properties. The reaction between beta-hydroxyketo esters and primary or secondary amines yielded secondary or tertiary enaminone esters, respectively. The UV spectra of the enaminone esters were determined in acidic, alkaline, and neutral media; the spectra have a hypsochromic shift in acidic media in comparison with neutral media. The enaminone esters provided nucleophilic and electrophilic sites for a variety of reactions. Thus, the enaminone esters were converted into enaminone amides and O-alkylation products exclusively. Although the enaminone esters were generally resistant to reduction by metal hydrides, one unhindered enaminone ester was reduced to an alcohol with sodium borohydride. Another enaminone ester reacted with guanidine to give the corresponding quinazolinone. Due to the variety of nucleophilic and electrophilic sites in the enaminone system, enaminone esters possess a great potential as reaction intermediates and medicinal compounds. Preliminary evaluations of the enaminone esters revealed a histaminergic effect, uterine relaxant properties, and anticonvulsant activity.

Synthesis and CLOGP correlation of imidooxy anticonvulsants.

Continuing structure-activity studies on the anticonvulsant activity of analogs of N-(benzyloxy)-2-azaspiro[4.4]nonane-1,3-dione (2a), which displayed anti-electroshock seizure (MES) activity and a protective index (TD50/ED50) of > 4.5 are reported. An in-depth analysis of this moiety was studied employing the Topliss structure activity and the Craig plot analytical approaches as well as a semiempirical method. CLOG P analysis was also applied to this series after experimentally determining the NOR fragment. All compounds were minimized and these physicochemical parameters correlated to anticonvulsant activity. Several interesting substituted benzyloxy compounds emerged from this study: the 2',4'-dichloro (2b), 4'-(trifluoromethyl) (2c), 2'-bromo (2d), 3'-chloro (2o), 2'-chloro (2r), 2'-fluoro (2p), and 3'-fluoro (2w) analogs, all of which had comparable, or better activity than the parent unsubstituted analog (2a). X-ray crystal analysis of the active 2a versus inactive N-benzyl-2-azaspiro[4.4]nonane-1,3-dione (10) is discussed.

Synthesis and anticonvulsant activity of enaminones. 2. Further structure-activity correlations.

This report continues the in-depth evaluation of methyl 4-[(p-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-oate , 1 (ADD 196022), and methyl 4-(benzylamino)-6-methyl-2-oxocyclohex-3-en-1-oate, 2, two potent anticonvulsant enaminones. These compounds were evaluated employing the amygdala kindling model. Neither 1 nor 2 was active against amygdala kindled seizures, further supporting the corneal kindled model as a definitive tool for antielectroshock seizure evaluation as previously reported. Additional intraperitoneal (ip) data on 1 revealed toxicity at 24 h at 100 mg/kg. Several active analogs have been prepared with the view to minimizing toxicity. In a special ip rat screen developed by the Antiepileptic Drug Development (ADD) Program, these newer analogs were evaluated for protection against maximal electroshock seizures (MES) at 10 mg/kg and neurotoxicity at 100 mg/kg. From this screen, several compounds were shown to be safer alternatives, the most notable was methyl 4-[(p-bromophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-oate, 13. Compound 13 had an ip ED50 of 4 mg/kg in the rat and a TD50 of 269 mg/kg, providing a protective index (TD50/ED50) of > 67. By variation in the ring size, additional aromatic substitutions and the synthesis of acyclic analogs, these newer compounds provide a more definitive insight into the structure-activity correlation. CLOGP evaluation and molecular modeling studies are also provided to further elaborate the molecular characteristics of potential anticonvulsant enaminones.

Synthesis and anticonvulsant activity of imidooxy derivatives.

Previous results of anticonvulsant activity in several imidooxy carboxylates related to (aminooxy)acetic acid in young chicks, prompted an in-depth reinvestigation of these analogues in mice. A series of 22 succinimidooxy, phthalimidooxy, and naphthalimidooxy carboxylates were synthesized and evaluated for anticonvulsant activity by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS). Methyl (succinimidooxy)acetate (2d), ethyl (succinimidooxy)acetate (2e), methyl (phthalimidooxy)acetate (3d), ethyl (phthalimidooxy)acetate (3e), and ethyl 2-(phthalimidooxy)propionate (3g), which were initially found to be active as anticonvulsants in young chicks were uniformly inactive in the Phase I seizure tests involving maximal electroshock (MES), pentylenetetrazol (scMet), or neurologic toxicity toxicity (Tox). Several newer analogues, ethyl (succinimidooxy)formate (2c) and methyl 3-(phthalimidooxy)-2-methylacrylate (4h) were found to be active in the scMet (3a) or both (4h) evaluations. Most interesting was the anticonvulsant results of N-(benzyloxy)-2-azaspiro[4,4] nonane-1,3-dione (5b), which displayed anti-MES activity and a protective index (TD50/ED50) of greater than 4.5.