Synthesis and Preclinical Evaluation of an 18F-Labeled Synaptic Vesicle Glycoprotein 2A PET Imaging Probe: [18F]SynVesT-2.

Synaptic vesicle glycoprotein 2A (SV2A) is a 12-pass transmembrane glycoprotein ubiquitously expressed in presynaptic vesicles. In vivo imaging of SV2A using PET has potential applications in the diagnosis and prognosis of a variety of neuropsychiatric diseases, e.g., Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, autism, epilepsy, stroke, traumatic brain injury, post-traumatic stress disorder, depression, etc. Herein, we report the synthesis and evaluation of a new 18F-labeled SV2A PET imaging probe, [18F]SynVesT-2, which possesses fast in vivo binding kinetics and high specific binding signals in non-human primate brain.

A modification to improve the reliability of [11 C]CN- production in the GE radiochemistry system.

We report herein a modification to a component of the GE radiochemistry system that increases the yield and reliability of [11 C]CN- production from [11 C]carbon dioxide.

In vivo variation in same-day estimates of metabotropic glutamate receptor subtype 5 binding using [11C]ABP688 and [18F]FPEB.

Positron emission tomography tracers [11C]ABP688 and [18F]FPEB target the metabotropic glutamate receptor subtype 5 providing quantification of the brain glutamatergic system in vivo. Previous [11C]ABP688 positron emission tomography human test-retest studies indicate that, when performed on the same day, significant binding increases are observed; however, little deviation is reported when scans are >7 days apart. Due to the small cohorts examined previously (eight and five males, respectively), we aimed to replicate the same-day test-retest studies in a larger cohort including both males and females. Results confirmed large within-subject binding differences (ranging from -23% to 108%), suggesting that measurements are greatly affected by study design. We further investigated whether this phenomenon was specific to [11C]ABP688. Using [18F]FPEB and methodology that accounts for residual radioactivity from the test scan, four subjects were scanned twice on the same day. In these subjects, binding estimates increased between 5% and 39% between scans. Consistent with [11C]ABP688, mean absolute test-retest variability was previously reported as <12% when scans were >21 days apart. This replication study and pilot extension to [18F]FPEB suggest that observed within-day binding variation may be due to characteristics of mGluR5; for example, diurnal variation in mGluR5 may affect measurement of this receptor.

3-ketosteroid reductase activity and expression by fetal rat osteoblasts.

In addition to reproductive tissue, sex hormones induce transcriptional events in many connective tissue cells, including osteoblasts. Some sex hormone receptor modulators with bone sparing effects selectively target estrogen or androgen receptors, whereas others appear more promiscuous, in part through enzymatic metabolism. Rat osteoblasts express significant oxidative 3alpha-hydroxysteroid dehydrogenase activity, which can convert precursor substrates to potent androgen receptor agonists. Here we show that they also express 3-ketosteroid reductase activity, exemplified by 7-methyl-17-ethynyl-19-norandrostan-5 (10)en-3-one (tibolone) conversion to potent estrogen receptor alpha agonists. Conversion was rapid and quantitative, with 3alpha-hydroxytibolone as the primary metabolite. Consistently, tibolone induced estrogen receptor alpha-dependent gene promoter activity through cis-acting estrogen response elements, increased the stimulatory effect of TGF-beta on Smad-dependent gene promoter activity, and enhanced prostaglandin E2-induced activity of transcription factor Runx2. Rat osteoblasts express the 3-ketosteroid reductase AKR1C9, an aldo-keto reductase gene family member. Exposure to prostaglandin E2 increased AKR1C9 gene promoter activity and mRNA expression. AKR1C9 promoter activity was also enhanced by overexpression of protein kinase A catalytic subunit or transcription factor C/EBPdelta, and the effect of PGE2 was reduced by dominant negative C/EBPdelta competition or C/EBPdelta antisense expression. Moreover, prostaglandin E2 increased the amount of functional endogenous nuclear C/EBPdelta that could bind specifically to a distinct domain approximately 1.8-kb upstream from the start site of AKR1C9 transcription. In summary, in addition to 3alpha-hydroxysteroid dehydrogenase, rat osteoblasts express significant and regulatable 3-ketosteroid reductase activity. Through these enzymes, they may selectively metabolize precursor compounds into potent steroid receptor agonists locally within bone.

Microwave-enhanced nucleophilic fluorination in the synthesis of fluoropyridyl derivatives of [3,2-c]pyrazolo-corticosteroids, potential glucocorticoid receptor-mediated imaging agents.

Fluoropyridyl derivatives of [3,2-c]pyrazolo-corticosteroids have high affinity for the glucocorticoid receptor (GR) and are highly active glucocorticoids. They are thus considered to be excellent candidates for PET imaging of GR containing tissues when labeled with fluorine-18 (t(1/2)=110 min). Previously reported syntheses of these fluorinated glucocorticoids were accomplished by conventional thermal nucleophilic halogen exchange reactions with chloropyridyl precursors. These reactions were found to proceed at rates too slow for feasible application to radiosynthesis using [(18)F]fluoride. We have applied microwave-heating methods to these reactions and found that significant rate enhancements can be realized. Kinetic experiments showed an average relative rate ratio of 3/1 for microwave versus conventional heating and preparative experiments showed an average relative conversion ratio of 4.5/1 during the initial 120 min, a period approximating one half-life of the isotope. The microwave method described was used to prepare previously unreported 2'-(2-fluoro-4-pyridyl)-11beta,17,21-trihydroxy-16alpha-methyl-20-oxo-pregn-4-eno-[3,2-c]-pyrazole, which was evaluated for biological activity.

Nonpolar and short side chain groups at C-11beta of estradiol result in antiestrogens.

We have previously found that esters of 11beta-estradiol carboxylates are transformed from an estrogen into an antiestrogen when the 11beta-side chain is increased in length from four to five non-hydrogen atoms (n > or = 5). To understand the structural requirements for this transformation and obtain metabolically stable analogues that are not susceptible to esterase cleavage, we have synthesized other compounds having an 11beta-side chain composed of other functional groups: ketones, amides, ethers, and thiono esters. With the exception of amides, which bind poorly to the estrogen receptor (ER), all of these compounds exhibit antiestrogenic action when the side chain length is n > or = 5. Ethers (n > or = 5), studied in more detail, inhibit the action of estradiol with either ERalpha or ERbeta. In rat uteri they are estrogen antagonists/weak agonists and decrease the concentration of cholesterol in blood (an hepatic estrogenic action). Thus, these short chain and nonpolar 11beta-analogues of estradiol have tissue specific antiestrogenic/estrogenic actions, characteristics of selective estrogen receptor modulators.

Estrogen to antiestrogen with a single methylene group resulting in an unusual steroidal selective estrogen receptor modulator.

Selective estrogen receptor (ER) modulators (SERMs) are important therapeutic agents for breast cancer prevention and treatment. We have synthesized two analogs, E11-2,1 [methyl-(3,17beta-dihydroxyestra-1,3,5(10)-triene-11beta-yl)acetate] and E11-2,2 [ethyl-(3,17beta-dihydroxyestra-1,3,5(10)-triene-11beta-yl)acetate], the methyl and ethyl esters of an estradiol analog, substituted in the B ring at C-11beta with a carboxymethyl group. The shorter methyl ester, E11-2,1, has high ER affinity and high estrogenic potency in the Ishikawa estrogen cell bioassay, whereas the longer ethyl ester, E11-2,2, has even higher ER affinity, but little or no estrogenic activity. We found that this minor change of one methylene group transforms a potent estrogenic agonist into an antagonist in vitro with either ER alpha or beta. In the rat, E11-2,2 acts as a SERM in the uterus, where it inhibits estradiol-induced proliferation, and as an estrogen agonist in the liver and skeleton, where it decreases plasma cholesterol and increases bone growth. The characteristic feature of antiestrogens, including SERMs, is a long and polar side-chain that prevents agonist-induced conformation of helix 12 of ER. E11-2,2 with its short, nonpolar side-chain, lacks this critical structure, presenting the possibility that it might act through a unique mechanism.

Estren (4-estren-3alpha,17beta-diol) is a prohormone that regulates both androgenic and estrogenic transcriptional effects through the androgen receptor.

Alternative mechanisms of steroid action, through both traditional nuclear receptors and indirect pathways of gene activation, are emerging. Recent studies suggest that the synthetic steroid, 4-estrene-3alpha,17beta-diol (estren), has nongenotropic as well as sex-nonspecific osteogenic effects in ovariectomized and orchidectomized mice. We found limited estrogen receptor-dependent effects by estren on gene expression in primary osteoblast cultures and showed that it binds poorly to estrogen and androgen receptors in vitro. However, estren potently regulated direct and indirect androgen receptor-dependent effects on gene expression by osteoblasts. Consistent with this, osteoblasts produced the potent androgen 19-nortestosterone from estren by way of a 3alpha-hydroxysteroid dehydrogenase-like activity. Moreover, recombinant 3alpha-hydroxysteroid dehydrogenase (AKR1C9) and osteoblast-derived cell lysate each effectively converted estren to 19-nortestosterone in vitro, and mRNA encoding this enzyme occurs in osteoblasts. In addition to its androgenic activity, estren potently stimulated androgen receptor-dependent effects on gene expression through conventional estrogen-sensitive transcriptional elements in osteoblasts. Therefore, through local metabolism, estren indirectly activates the androgen receptor to regulate both androgen- and estrogen-like transcriptional responses by bone-forming cells.

Synthesis and evaluation of B-, C-, and D-ring-substituted estradiol carboxylic acid esters as locally active estrogens.

We have synthesized derivatives of estradiol that are structurally modified to serve as "soft" estrogens and act within a geographically limited area of the body; estrogens without systemic action. We have previously shown with 16alpha-substituted analogues of estradiol that carboxylates proximal to the steroid ring neither bind to the estrogen receptor nor activate estrogen-responsive genes. However, when the carboxylic acid is masked as an ester, they bind to the receptor and stimulate estrogenic responses. Enzymatic hydrolysis through nonspecific esterases can inactivate these estrogens and thereby limit their area of action. Here, we describe our continued studies to design "soft" estrogens by synthesizing carboxylic acid esters of estradiol at the 7alpha-, 11beta-, and 15alpha-positions in the steroid nucleus at which bulky substituents are accommodated by the estrogen receptor. These compounds were tested for estrogen receptor binding (estrogen receptors alpha and beta), stimulation of an estrogen sensitive gene in Ishikawa cells in culture, and as substrates for enzymatic hydrolysis. Likely candidates were tested in in vivo assays for systemic and local estrogenic action. The biological studies showed that regardless of the point of attachment, all of the short-chain carboxylic acids, C-1 to C-3, were devoid of hormonal action, while many of the esters were estrogenic. The site on the steroid nucleus had great influence on hormonal activity and esterase hydrolysis. Formate esters at 7alpha and 15alpha were good estrogens, but lengthening the chain to acetate dramatically decreased hormonal activity. However, the 7alpha-formate esters were not enzymatically hydrolyzed. At 11beta, the acetate (methyl ester) was an effective estrogen, but increasing the chain length to propionate dramatically reduced hormonal activity. In general, the length of the alcohol from methyl to butyl had only a small effect on receptor binding, and as the size of the alcohol increased, so did esterase hydrolysis. One exception was the 11beta-acetate esters where increasing the alcohol moiety from methyl to ethyl eliminated estrogenic activity (Ishikawa cells) without affecting estrogen receptor binding. Several of the esters were tested in vivo, and two, the methyl and ethyl esters of estradiol-15alpha-formate, appeared to have the requisite properties (high local and low systemic activity) of superior "soft" estrogens.

Synthesis of halogen-substituted pyridyl and pyrimidyl derivatives of [3,2-c]pyrazolo corticosteroids: strategies for the development of glucocorticoid receptor mediated imaging agents.

Ligands for the glucocorticoid receptor labeled with high-energy isotopes are highly desired for their potential applications in nuclear medical studies of the brain where the dysregulation of this receptor system is thought to be involved in various neurodegenerative disorders. Analogues of the glucocorticoid cortivazol have previously been prepared as target compounds for labeling with high-energy isotopes. However, the phenyl rings of arylpyrazoles of this type are not sufficiently activated for nucleophilic substitution reactions that are generally required for the synthesis of radiohalogenated analogues. Since suitably substituted aromatic nitrogen heterocyclic groups are amenable to nucleophilic substitution, the goal of this study was the synthesis of pyridylpyrazolo and pyrimidylpyrazolo analogues similar to cortivazol that could be labeled with radiohalogens in the pyridine or pyrimidine rings. We describe the synthesis of several [3,2-c]pyrazolo steroids containing pyridyl, halopyridyl, and pyrimidyl substituents at the 2' position of the pyrazole ring. These compounds were tested for binding to the glucocorticoid receptor and for biological activity in glucocorticoid responsive HeLa cells grown in tissue culture. Of the pyridyl and pyrimidyl derivatives, 2'-(3-pyridyl)-11 beta,17,21-trihydroxy-16 alpha-methyl-20-oxopregn-4-eno[3,2-c]pyrazole showed superior activity in both assays and it was used as the basis for the synthesis of several analogues that were halogenated in the pyridine ring. These halogenated compounds were all tested for their binding to the glucocorticoid receptor and for their biological activity. One, a fluorinated compound 2'-(2-fluoro-5-pyridyl)-11 beta,17,21-trihydroxy-16 alpha-methyl-20-oxopregn-4-eno[3,2-c]pyrazole had excellent activity, considerably better than the potent glucocorticoid dexamethasone. Most importantly, fluorination was achieved using a nucleophilic exchange reaction, a method that is adaptable to radiolabeling with the positron-emitting isotope fluorine-18. Thus, considering its superior biological activity and adaptability for facile radiosynthesis, this target compound has the potential for imaging of glucocorticoid receptor containing tissues using positron emission tomography.