Hisham G. Lutfi

Transcriptional regulation of estrogen-responsive genes by non-steroidal estrogens: Doisynolic and allenolic acids

Estrogen receptor (ER), a member of the nuclear receptor superfamily, exerts prominent physiological roles in both humans and other species by acting directly as a transcription factor, altering nuclear gene expression. One peculiarity of estrogenic regulation is that it is affected by a wide variety of non-steroidal compounds in addition to the natural hormone, estradiol. Doisynolic and allenolic acid compounds are non-steroidal compounds that act as potent estrogens in animal studies, yet bind to ER extremely poorly in competitive binding assays, raising the possibility of alternative molecular mechanisms for the observed estrogenic effects. In this work we demonstrate that (+/-)-Z-bisdehydrodoisynolic acid, (+/-)-Z-bisdehydrodoisynolic acid 3-methyl ether, and (-) allenolic acid can interact directly with ER. These compounds all serve as ligands for ER in mechanism-specific tissue culture-based reporter gene assays for both positive and negative gene regulation. We have also used a novel assay based on electromobility shift by ER for directly determining relative binding affinities for ER. In addition, we show cell-type-specific activity differences for (+/-)-Z-bisdehydrodoisynolic acid 3-methyl ether, supporting clinical observations indicating a higher potency of this compound in female animals than in humans.

Racemic 1,2,4-Trimethylurazole

The structural and stereochemical parameters of 1,2,4-trimethyl-1,2,4-triazolidine-3,5-dione, C5H9N3O2, (1), were examined in view of the contrast between its physical properties and those of urazoles having one or more unsubstituted NH groups. The X-ray structure shows that (1) is devoid of intermolecular hydrogen bonding but retains the structural features of strongly hydrogen-bonded urazoles. Its trans H3C—N—N—CH3 torsion angle, 72.3 (5)°, is slightly larger than the corresponding angle in urazoles having an unsubstituted or monomethylated N—N function, but smaller than that in urazoles monosubstituted with bulkier groups. Like other crystalline urazoles, (1) is racemic by virtue of N1 and N2 chirality. In its packing pattern of parallel molecular sheets, two pass through the unit cell; within a sheet, each molecule is surrounded by two of its own chirality and four of opposite chirality. While physical properties of crystalline urazoles can be associated with their degree of intermolecular hydrogen bonding, this study shows that their chirality is an intrinsic molecular phenomena.

(1R-2R)-4,4-Dimethylpyrazolidine-3,5-dione-α-D-pyranosyl-2-deoxyriboside

The first example of a crystalline pyrazolidinedione nucleoside has been synthesized from the reaction of 2-deoxy-D-ribose with 4,4-dimethylpyrazolidine-3,5- dione and characterized by X-ray crystallography as a single α-pyranoside diastereomer [IUPAC name: 1- (2-deoxy-α-D-erythro-pentopyranosyl) 1(R),2(R)-4,4-dimethylpyrazolidine-3,5-dione], C 10 H 16 N 2 O 5 . Although the pyrazolidinedione ring is essentially planar, the two hydrazidic N atoms are pyramidal and chiral, their respective pyranosyl and H-atom substituents being trans-R,R configured. The intermolecular hydrogen bonding involves pyranose-pyranose and pyranosepyrazolidinedione interactions. Each molecule is linked via six hydrogen bonds to four surrounding molecules in which the pyrazolidinedione hydrazidic N(H) atom is a donor and its adjacent carbonyl O atom is an acceptor, and the pyranose hydroxylic O atoms are donors as well as acceptors. The second carbonyl O atom has no hydrogen-bonding interactions with OH or NH, but exhibits a weak C-H...O intermolecular interaction with the pyranose ring. The pyranose ring O atom does not participate in hydrogen bonding. Substituting the OH groups with OD and the NH with ND resulted in no measurable changes in the structure (within error), including the hydrogen-bonding parameters.

Formation and identification of rotameric 9-pivaloylfluorene oximes. Structure of ap-(Z)-9-pivaloylfluorene oxime

Reaction of hydroxylamine with sterically hindered ap-9-pivaloylfluorene (I) produced the rotameric antiperiplanar (ap) and synperiplanar (sp) oximes (40% yield) as determined by 1 H NMR (CDCl 3 or DMSOd 6 ). Their E/Z configurations (substituent geometry in C = N) could not be ascertained this way. Evaporation of solutions containing both rotamers consistently provided only one solid oxime which was shown to be the ap-(Z) oxime (IIa) [ap-(Z)-9-pivaloylfluorine oxime, C 18 H 19 NO] by single-crystal X-ray diffraction. When crystalline (IIa) was redissolved (CDCl 3 or DMSO-d 6 ) both rotamers were present in ratios dependent on the concentration. The proximity of the oxime O atom to the fluorene-ring plane in (IIa), 2.598 A, results in a repulsion which raises the ground-state energy, thereby lowereing the rotational barrier leading to the isomeric sp-(Z) oxime (IIIa)

Racemic 1-methyl-1,2,4-triazolidine-3,5-dione (1-methylurazole). Definitive characterization of pyramidal hydrazide N atoms and racemate intermolecular hydrogen bonding

In contrast to the rapid formation of ribosides of urazole and 4-methylurazole through a hydrazide N atom, 1-methylurazole, C 3 H 5 N 3 O 2 (I), fails to exhibit any reactivity with ribose; this has been evaluated crystallography. The X-ray structure of (I) shows the imide N atom to be trigonal but both hydrazide N atoms to be pyramidal, with N1-C6 and N2-H trans, affording a torsion angle C6-N1-N2-H2 of −49(2) o . These data suggest that attack on the N2 electron lobe of (I) must be cis to the N1 methyl, invoking a sterically hindered, relatively high-energy transition state and intermediate

The surprising sp rotameric structure of 9-methyl-9-pivaloylfluorene

Methylation of 9-lithiated ap-9-pivaloylfluorene, (I), as well as pivaloylation of 9-lithiated 9-methylfluorene provided rotationally stable sp-9-methyl-9-pivaloylfluorene, (III), C(19)H(20)O, which lies about a crystallographic mirror plane. Fluorene (I) exists exclusively in the ap configuration in solution (NMR) as well as in the crystalline state, reflecting the unfavorable interaction between the tert-butyl and fluorene-ring pi electrons in the sp configuration. The existence of (III) exclusively in the sp configuration indicates that, in this case, the interaction between the tert-butyl group and the fluorene-ring pi electrons provides relatively more thermodynamic stability than the steric interaction between the tert-butyl and 9-methyl groups (ap configuration).

Exclusivity of the sp Rotamers of 9-(o-tert-Butylphenyl)fluorene and 9-(o-tert-Butylphenyl)-9-fluorenol in Solution and the Crystalline State

Both 9-(o-tert-butylphenyl)fluorene (C 23 H 22 ) and 9-(o-tert-butylphenyl)-9-fluorenol (C 23 H 22 O) maintained sp rotameric structures exclusively in crystalline form as well as in solution. This result is in contrast to that obtained for the corresponding 9-(o-isopropylphenyl)-fluorene and 9-(o-isopropylphenyl)-9-fluorenol. In-plane sterically imposed distortion of the tert-butyl group is exhibited in sp-9-(o-tert-butylphenyl)fluorene and to a much larger extent in sp-9-(o-tert-butylphenyl)-9-fluorenol. The asymmetric unit of the latter contains two crystallographically distinct, but nearly identical, molecules which are hydrogen bonded to each other via their respective OH groups; hydrogen bonding between the crystallographically equivalent molecules is not observed.

Acetone 4,4-Dimethyl-5-oxo-2-pyrazolin-3-ylhydrazone

The title compound, C 8 H 14 N 4 O, exhibits an overall molecular coplanarity and trigonal planar geometry of all four N atoms, suggesting resonance interaction extending over eight contiguous atoms. Strong hydrogen bonding between hydrazidic-N donor and carbonyl-O acceptor atoms results in an almost-planar octagonal bridge between two molecules. These dimers are interconnected through a second octagonal bridge of weaker hydrogen bonds between side-chain hydrazine-N donor and ring-N acceptor atoms, leading to an almost-flat linear polymeric structure. The acetone-hydrazone N atom does not participate in hydrogen bonding. From the structure of the title compound, the unequivocal identification of 4,4-dimethyl-5-oxo-2-pyrazolin-3-ylhydrazine, an unexpected byproduct of the reaction of 4,4-dimethyl-pyrazolidine-3,5-dione with hydrazine, was made.