Potent Nonsteroidal Aromatase Inhibitor Research Articles

Synthesis and Evaluation of New Brominated Azaflavones and Azaflavanone Derivatives as Cytotoxic agents against Breast Cancer Cell Line (MCF-7)

Abstract: Background: Flavonoids encompasses flavones, isoflavones, flavanones and flavanols each possessing the benzopyranone ring system as the common structural feature, were identified as potent nonsteroidal aromatase inhibitors (NSAIs). Purpose: Azaflavones which were isosteric structural scaffolds of flavonoids were also proven to be potent NSAIs. In order to develop new NSAIs as cytotoxic agents for breast cancer, we designed some 6-bromo-2-substituted azaflavanones and azaflavone derivatives. Method: Azaflavones and Azaflavonones were synthesized by a reaction of 2-amino-6-bromoacetophenone and various aromatic aldehydes to result in different chalcones (4) using Claisen-Schmidt condensation. Further cyclization of chalcones (4), led to tetrahydroquinoline-4-ones (5) using orthophosphoric acid. In the final oxidative step, the desired dihydroquinoline-4- ones (6) were obtained. Results: All the synthesized compounds were characterized by using IR, 1H NMR and ESI-MS data and were evaluated for cytotoxic activity by using MTT assay on MCF-7 cell lines. Conclusion: Compounds with furoyl and pyridyl groups as substituents were found to be potent. Key words: Azaflavones, Azaflavanones, Claisen-Schmidt condensation, Cytotoxicity, MTT assay, FT-IR, NMR.

Pharmacophore modeling, virtual screening, and 3D-QSAR studies on a series of non-steroidal aromatase inhibitors

Aromatase inhibitors are the most important targets in treatment of estrogen-dependent cancers. In order to search for potent non-steroidal aromatase inhibitors (NSAIs) with lower side effects and overcome cellular resistance, Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Database was used to derive 3D pharmacophore models. The obtained best pharmacophore model contains one acceptor atom, one donor atom, and two hydrophobes, which was used in effective alignment of dataset. In succession, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on 84 structurally diverse NSAIs to build 3D-QSAR models based on both pharmacophore and docking alignments. The CoMFA and CoMSIA models based on the pharmacophore alignment show better statistical results (CoMFA: q2 = 0.634, rncv2 = 0.986, rpred2 = 0.737; CoMSIA: q2 = 0.668, rncv2 = 0.926, rpred2 = 0.708). This 3D-QSAR approach provides significant insights that can be used to develop novel and potent NSAIs. In addition, the best pharmacophore model was used as a 3D query for virtual screening against NCI2000 database. The hit compounds were further filtered by docking, and their biological activities were predicted by the CoMFA and CoMSIA models, and six structurally diverse compounds with good predicted pIC50 values were obtained, which are expected to design novel NSAIs with new skeletons.

Effects of Non-Steroidal Aromatase Inhibitor Letrozole on Sex Inversion and Spermatogenesis in Yellow CatfishPelteobagrus fulvidraco

The effects of letrozole (LZ), a potent nonsteroidal aromatase inhibitor (AI), on growth performance, sex inversion, and sex changes were investigated in yellow catfish (Pelteobagrus fulvidraco), which display sexual dimorphic growth. Growth performance was promoted significantly in the low-dose LZ treatment, compared with the control. Four LZ treatments produced dose-dependent male proportions that were significantly higher than that of the control. Histological examination of testes treated by LZ displayed a large amount of spermatozoa and enlarged lobule lumens, indicating that LZ treatments can potentially stimulate spermatogenesis. Changes of sex proportions 45 days after the end of the LZ treatments prove that the female germ cells possess a certain degree of bipotentiality. These results suggest that aromatase activity plays a vital role in sex differentiation, as in other teleosts, with inhibition of aromatase activity by AI bringing about sex inversion.

Molecular Modeling Evaluation of Non‐Steroidal Aromatase Inhibitors†

A recent discovery of aromatase crystal structure triggered the efforts to design novel aromatase inhibitors for breast cancer therapy. While correlating docking scores with inhibitory potencies of known ligands, feeble robustness of scoring functions toward prediction was observed. This prompted us to develop new prediction models using stepwise regression analysis based on consensus of different docking and their scoring methods (GOLD, LIGANDFIT, and GLIDE). Quantitative structure-activity relationships were developed between the aromatase inhibitory activity (pIC(50) ) of flavonoid derivatives (n=39) and docking scores and docking descriptors. QSAR models have been validated internally [using leave-one-out cross-validated r(2)(cv) (LOO-Q2))] and externally to ensure the predictive capacity of the models. Model 2 [M2] developed using consensus of docking scores of scoring functions viz. ASP, potential of mean force and DOCK Score (r(2)(cv)=0.850, r(2) = 0.870, r(2)(pred) = 0.633, RMSE = 0.363 μm, r(2)(m(test)) =0.831, r(2)(m(overall)) =0.832) was found to be better in predicting aromatase inhibitory potency (pIC(50) ) compared to the Model 1 [M1] based on docking descriptors (r(2)(cv)= 0.848, r(2) = 0.825, r(2)(pred) =0.788, RMSE=0.421μm, r(2)(m(test)) =0.808, r(2)(m(overall)) =0.821). It has been observed that the natural flavonoids and their derivatives were less potent compared to these scaffolds with imidazolylmethyl substitution owing to the interaction of nitrogen atom of the imidazole ring toward the heme (Fe(3+) ) of the aromatase. Results confirm the potential of our methodology for the design of new potent non-steroidal aromatase inhibitors.

Effect of Nonsteroidal Aromatase Inhibitor on Sex Reversal of Oreochromis mossambicus (Peters, 1852)

In the present study, the efficacy of letrozole, a potent nonsteroidal aromatase inhibitor, and 17a-methyltestosterone on gondal sex differentiation and sex reversal was examined in Mozambique tilapia (Oreochromis mossambicus). Among the doses tried in the experiment, 100 mg letrozole, 200 mg letrozole, and 50 mg 17a-methyltestosterone/kg produced male dominated populations (97-100% males). The combination of 100 mg letrozole+25 mg 17a-methyltestoster/kg yielded 100% males while the combination of 50 mg letrozole+25 mg 17a-methyltestosterone/kg yielded 92% males. The control group consisted of 48.05% males and 51.95% females, close to the normal sex ratio of 1:1. Survival ranged 85.61-94.31% in the treated groups, significantly higher than 73.83% in the control (p<0.05). In general, the letrozole and combination treatments resulted in a slight alteration in the gonadosomatic index of the tilapia. Administration of letrozole alone or in combination with 17a-MT did not adversely affect the proximate composition of the muscle. Results suggest that letrozole and 17a-methyltestosterone have the potential to produce 100% male populations in Mozambique tilapia and that inhibition of aromatase activity influences sex differentiation in Mozambique tilapia.

PET of Aromatase in Gastric Parietal Cells Using 11C-Vorozole

Aromatase is a rate-limiting enzyme for estrogen biosynthesis and has been implicated in pathophysiological states of various diseases via estrogen production. This enzyme is known to be widely distributed in extragonadal and gonadal tissues including the stomach. In contrast to circulating estrogen, the functional role of gastric aromatase/estrogen has not been elucidated in detail, because there is no efficient methodology to investigate spatiotemporal changes of gastric aromatase/estrogen in vivo. Recently, (S)-(11)C-6-[(4-chlorophenyl)(1H-1,2,4-triazole-1-yl)methyl]-1-methyl-1H-benzotriazole ((11)C-labeled vorozole), based on a potent nonsteroidal aromatase inhibitor, has been developed as a tracer to investigate aromatase distribution in living animals and humans using a noninvasive PET technique. In the present study, we investigated gastric aromatase expression by means of PET with (11)C-vorozole. After bolus injection of (11)C-vorozole into the tail vein, emission scans were obtained for 90 min on male and female rats under isoflurane anesthesia. Displacement studies with unlabeled vorozole and autoradiographic analysis were conducted for demonstration of specific binding. Immunohistochemistry was performed to confirm aromatase expression. PET scans revealed that (11)C-vorozole highly accumulated in the stomach and adrenal glands. Displacement studies and autoradiography demonstrated that aromatase was expressed in the stomach but that the accumulation of (11)C-vorozole in the adrenal glands might be through nonspecific binding. Immunohistochemical analysis revealed that aromatase is expressed in gastric parietal cells but not in adrenal glands. Moreover, the accumulation of (11)C-vorozole in the stomach was significantly increased in fatigued rats. These results suggest that the (11)C-vorozole PET technique is a useful tool for evaluation of gastric aromatase dynamics in vivo, which may provide important information for understanding the molecular mechanisms of gastric aromatase/estrogen-related pathophysiological processes and for the development of new drugs.

The Clinical Pharmacology of Anastrozole

Anastrozole is a potent non-steroidal aromatase inhibitor that selectively inhibits aromatase. It is the first aromatase inhibitor approved by the US Food and Drug Administration as an adjuvant hormonal therapy in early-stage breast cancer. With most patients needing to take anastrozole for two to five years, it is important for physicians to understand the clinical pharmacology of anastrozole. Anastrozole has excellent oral bioavailability and is widely distributed throughout the body. It is extensively metabolised by the liver, with a half-life of 40–50 hours. Anastrozole is primarily excreted through the faecal route. Steady-state concentrations are achieved after seven days of once-daily administration. The standard dosage of anastrozole is 1mg/day orally. No dose adjustments are necessary based on hepatic or renal dysfunction. No significant drug–drug interaction has been reported with anastrozole use. This article is to summarise the existing knowledge of the pharmacokinetics of anastrozole.

Effects of a nonsteroidal aromatase inhibitor on gonadal differentiation of bluegill sunfish Lepomis macrochirus

In the present study, the efficacy of Letrozole, a potent nonsteroidal aromatase inhibitor (AI), on gonadal sex differentiation and sex reversal was examined in bluegill sunfish (Lepomis macrochirus). In Experiment 1, using AI diet treatments (50, 150, 250 and 500 mg kg−1) from 30 to 90 days posthatch (dph), AI interrupted ovarian cavity formation at a dose of 500 mg kg1 diet and one intersex fish was identified in this group. The proportions of males in all the treated groups were significantly higher than those in the control group. In Experiment 2, using AI immersion treatments (250, 500 and 1000 μg L−1) during 30–50 dph, the treated groups of 500 and 1000 μg L−1 produced significantly more males than the control and 250 μg L−1 groups. Histological examination revealed no differences in ovary or testis tissue between control and AI-treated fish. There were no significant differences detected in body weight and length among the AI treated and control groups (P>0.05) for both experiments. The results from these two experiments suggest that inhibition of aromatase activity by AI could influence sex differentiation in bluegill sunfish.

Letrozole Co-Treatment in Infertile Women 40 Years Old and Older Receiving Controlled Ovarian Stimulation and Intrauterine Insemination

Controlled ovarian hyperstimulation (COH) using the gonadotropin, follicle stimulating hormone (FSH), combined with IUI (COH-IUI) achieve higher pregnancy rates (PR) than IUI alone in treatment of infertile older women. Letrozole, a highly potent nonsteroidal aromatase inhibitor, has been increasingly used in COH protocols. This retrospective case-control study investigated the effect of adding letrozole to FSH in COH-IUI cycles in a population of infertile women at or more than 40 years old. Two comparison groups were used: group I (n = 90) was comprised of infertile women who received letrozole combined with FSH and group II (n = 69) included patients who received FSH alone (n = 69). The primary study outcome was primary rate (PR). Secondary outcomes examined included mature follicles and serum levels of estradiol (E 2 ), progesterone (P), luteinizing hormone (LH), endometrial thickness, and rates of cycle cancellation. No significant difference in PR was found between the letrozole-FSH co-treatment group and the FSH alone group. Compared with the FSH alone group, the letrozole co-treatment group had significantly fewer cancelled cycles and more completed cycles. The serum E 2 levels and the number of mature follicles were significantly higher in the FSH only group than in the combined letrozole-FSH group (P < 0.001 for both). On cycle day 7, the serum level of LH was significantly higher in the letrozole co-treatment group (P < 0.001); LH serum levels were similar in both groups on all other cycle monitoring days. Serum P levels were significantly higher in the FSH alone group compared with the letrozole co-treatment group on the day of human chorionic gonadotropin administration (P < 0.001). These findings show that letrozole co-treatment has several beneficial modulator effects on gonadotropin-stimulated cycles without a negative impact on PR. The investigators believe that the addition of letrozole to gonadotropins in combination with the COH-IUI protocol could be a promising initial option for older infertile women.

The aromatase inhibitor letrozole and inhibitors of insulin-like growth factor I receptor synergistically induce apoptosis in in vitro models of estrogen-dependent breast cancer

IntroductionEndocrine-dependent, estrogen receptor positive breast cancer cells proliferate in response to estrogens, synthesized by the cytochrome p450 aromatase enzyme. Letrozole is a potent nonsteroidal aromatase inhibitor that is registered for the treatment of postmenopausal women with advanced metastatic breast cancers and in the neoadjuvant, early, and extended adjuvant indications. Because crosstalk exists between estrogen receptor and insulin-like growth factor I receptor (IGF-IR), the effect of combining a selective IGF-IR inhibitor (NVP-AEW541) with letrozole was assessed in two independent in vitro models of estrogen-dependent breast cancer.MethodsMCF7 and T47D cells stably expressing aromatase (MCF7/Aro and T47D/Aro) were used as in vitro models of aromatase-driven breast cancer. The role of the IGF-IR pathway in breast cancer cells stimulated only by 17β-estradiol or androstenedione was assessed by proliferation assays. The combination of letrozole and NVP-AEW541 was assessed for synergy in inhibiting cell proliferation using Chou-Talalay derived equations. Finally, combination or single agent effects on proliferation and apoptosis were assessed using proliferation assays, flow cytometry, and immunoblotting.ResultsBoth MCF7 and T47D cells, as well as MCF7/Aro and T47D/Aro, exhibited sensitivity to inhibition of 17β-estradiol dependent proliferation by NVP-AEW541. Letrozole combined with NVP-AEW541 synergistically inhibited androstenedione-dependent proliferation in aromatase-expressing cells with combination index values of 0.6 or less. Synergistic combination effects correlated with higher levels of apoptosis as compared with cells treated with the single agent alone. Treatment with either agent also appeared to inhibit IGF-IR signalling via phosphoinositide 3-kinase. Notably, IGF-IR inhibition had limited effect on estrogen-dependent proliferation in the cell lines, but was clearly required for survival, suggesting that the combination of letrozole and IGF-IR inhibition sensitizes cells to apoptosis.ConclusionInhibition of the IGF-IR pathway and aromatase was synergistic in two independent estrogen-dependent in vitro models of breast cancer. Moreover, synergism of NVP-AEW541 and letrozole correlated with induction of apoptosis, but not cell cycle arrest, in the cell lines tested. Combination of IGF-IR inhibitors and letrozole may hold promise for the treatment of patients with estrogen-dependent breast cancers.

Letrozole induction of ovulation in women with clomiphene citrate–resistant polycystic ovary syndrome may not depend on the period of infertility, the body mass index, or the luteinizing hormone/follicle-stimulating hormone ratio

Letrozole induction of ovulation in clomiphene citrate-resistant women with polycystic ovary syndrome is associated with an ovulation rate of 54.6% and pregnancy rate of 25%. There was no significant difference between letrozole responders and nonresponders in age, period of infertility, body mass index, waist circumference, LH, FSH, or LH/FSH ratio.

Sex reversal in Nile tilapia (Oreochromis niloticus) using a nonsteroidal aromatase inhibitor.

Several studies have demonstrated that steroid hormones can influence sex differentiation in nonmammalian vertebrates and it has been hypothesized that male and female sex differentiation are driven by androgen and estrogen hormones, respectively. Estrogen biosynthesis is mediated by the steroidogenic enzyme cytochrome P450 aromatase, which converts androgens to estrogens. In the present study we examined the efficacy of a potent nonsteroidal aromatase inhibitor incorporated into the food, on sex reversal of Nile tilapia (Oreochromis niloticus) larvae. Nile tilapia larvae were divided in seven groups, which were fed with diets containing different amounts of the aromatase inhibitor Fadrozole (0, 50, 75 and 100 mg/kg) during 15 and 30 days, starting 9 days after hatching. Independent of the period, the proportion of males was significantly higher in the treated groups. Treatment with the highest doses (75 and 100 mg/kg) for 30 days produced 100% males. Histological examination revealed no differences in gonadal tissues between control males and treated fish. Furthermore, one intersex fish was identified in the group treated with 50 mg Fadrozole/kg for 30 days. This study reports that a 100% Nile tilapia male population can be obtained by suppressing aromatase activity and suggests that besides steroid hormones, nonsteroidal compounds, such as aromatase inhibitors, have potential for production of monosex population in tilapia. J. Exp. Zool. 290:177-181, 2001.

New aromatase inhibitors. Synthesis and biological activity of aryl-substituted pyrrolizine and indolizine derivatives

We report herein the design and the synthesis of some aryl-substituted pyrrolizine and indolizine derivatives, on the basis of a hypothetical pharmacophore structure designed to fit the catalytic site of the human cytochrome P450 aromatase. The in vitro biological evaluation of these compounds allowed us to point out two new potent non-steroidal aromatase inhibitors, MR 20494 and MR 20492, with IC50 values in the range of 0.1μM.

Effect of chronic administration of an aromatase inhibitor to adult male rats on pituitary and testicular function and fertility.

The aim of the present study was to evaluate the effects of the administration of a potent non-steroidal aromatase inhibitor, anastrozole, on male reproductive function in adult rats. As anastrozole was to be administered via the drinking water, a preliminary study was undertaken in female rats and showed that this route of administration was effective in causing a major decrease in uterine weight (P<0.02). In an initial study in male adult rats, anastrozole (100 mg/l or 400 mg/l) was administered via the drinking water for a period of 9 weeks. Treatment with either dose resulted in a significant increase ( approximately 10%) in testis weight and increase in plasma FSH concentrations (P<0.01) throughout the 9 weeks. Mating was altered in both groups of anastrozole-treated rats, as they failed to produce copulatory plugs. Histological evaluation of the testes from anastrozole-treated rats revealed that spermatogenesis was grossly normal. In a more detailed study, adult rats were treated with 200 mg/l anastrozole via the drinking water for periods ranging from 2 weeks to 1 year. Plasma FSH and testosterone concentrations were increased significantly (P<0.001) during the first 19 weeks of treatment. However, LH concentrations were increased only at 19 weeks (P<0.001) in anastrozole-treated rats, and this coincided with a further increase in circulating and intratesticular testosterone concentrations (P<0.05). No consistent change in inhibin-B concentrations was observed during the study. Suppression of plasma oestradiol concentrations could not be demonstrated in anastrozole-treated animals, but oestradiol concentrations in testicular interstitial fluid were reduced by 18% (P<0.01). Mating was again inhibited by anastrozole treatment, but could be restored by s.c. injection of oestrogen, enabling demonstration that rats treated for 10 weeks or 9 months were still fertile. Testis weight was increased by 19% and 6% after treatment for 19 weeks and 1 year, respectively. Body weight was significantly decreased (P<0.01) by 19 weeks of anastrozole treatment; after 1 year the animals appeared to have less fat as indicated by a 27% decrease in the weight of the gonadal fat pad. The majority of anastrozole-treated animals had testes with normal spermatogenesis but, occasionally, seminiferous tubules showed abnormal loss of germ cells or contained only Sertoli cells. Ten percent of anastrozole-treated animals had testes that appeared to contain only Sertoli cells, and one rat had 'giant' testes in which the tubule lumens were severely dilated. Morphometric analysis of the normal testes at 19 weeks showed no difference in the number of Sertoli cells or germ cells, or the percentage volumes of the seminiferous epithelium, tubule lumens and interstitium between control and anastrozole-treated rats. On the basis of the present findings, oestrogen appears to be involved in the regulation of FSH secretion and testosterone production, and is also essential for normal mating behaviour in male rats. Furthermore, these data suggest that the brain and the hypothalamo-pituitary axis are considerably more susceptible than is the testis to the effects of an aromatase inhibitor. Anastrozole treatment has resulted in a model of brain oestrogen insufficiency.

Evaluation of the racemate and the enantiomers of a new highly active and selective aromatase inhibitor of the aminoglutethimide type

Compound 1 [3-(4-aminophenyl)-3-cyclohexylpiperidine-2,6-dione] is a highly potent nonsteroidal aromatase inhibitor of the aminoglutethimide (AG)-type containing an asymmetric carbon atom. 1 and its enantiomers (+)- 1 and (−)- 1 inhibited human placental aromatase by 50% at 0.3, 0.15, and 4.6 μM, respectively (IC 50 AG = 37 μM). A competitive type of inhibition was observed for 1 and (+)- 1 ( K i 1 = 3.9 nM, K i (+)- 1 = 2.0 nM, K i AG = 408 nM ). Using solubilized high spin aromatase, 1 showed a type II difference spectrum indicating the interaction of the amino nitrogen with the central Fe(III)-ion of the cytochrome P 450 heme component. 1 and (+)- 1 inhibited cholesterol side chain cleavage enzyme (desmolase) by 50% at 67 and 82 μM, respectively (IC 50 AG = 29 μM). In ACTH-stimulated rat adrenal tissue in vitro, 1 was less active in inhibiting aldosterone and corticosterone production compared to AG (IC 50s, 1, 130 and 140 μM, AG, 80 and 50 μM, respectively). In vivo, 1 was superior to AG, too: it showed a stronger inhibition of the plasma estradiol concentration of pregnant mares' serum gonadotropin-primed SD rats, the activity residing mainly in the (+)-enantiomer [ovarian vein: (+)- 1, 0.31 mg/kg: 81% inhibition, (−)- 1, 0.31 mg/kg: 6%, AG, 1.25 mg/kg: 35%]. Furthermore 1 was much more active in inhibiting the testosterone-stimulated tumor growth of the ovariectomized 9,10-dimethyl-1,2-benzanthracene tumor-bearing SD rat (postmenopausal model). Up to a dose of 600 mg/kg of 1 no central nervous symptom depressive effects were observed in the motility test and the rotarod experiment, whereas AG exhibited ED 50s of 62 and 164 mg/kg, respectively.