C-6α- vs C-7α-Substituted Steroidal Aromatase Inhibitors: Which Is Better? Synthesis, Biochemical Evaluation, Docking Studies, and Structure-Activity Relationships.

C-6α and C-7α androstanes were studied to disclose which position among them is more convenient to functionalize to reach superior aromatase inhibition. In the first series, the study of C-6 versus C-7 methyl derivatives led to the very active compound 9 with IC50 of 0.06 µM and Ki = 0.025 µM (competitive inhibition). In the second series, the study of C-6 versus C-7 allyl derivatives led to the best aromatase inhibitor 13 of this work with IC50 of 0.055 µM and Ki = 0.0225 µM (irreversible inhibition). Beyond these findings, it was concluded that position C-6α is better to functionalize than C-7α, except when there is a C-4 substituent simultaneously. In addition, the methyl group was the best substituent, followed by the allyl group and next by the hydroxyl group. To rationalize the structure-activity relationship of the best inhibitor 13, molecular modeling studies were carried out.

Exploring new chemical functionalities to improve aromatase inhibition of steroids.

In this work, new potent steroidal aromatase inhibitors both in microsomes and in breast cancer cells have been found. The synthesis of the 3,4-(ethylenedioxy)androsta-3,5-dien-17-one (12), a new steroid containing a heterocycle dioxene fused in the A-ring, led to the discovery of a new reaction for which a mechanism is proposed. New structure-activity relationships were established. Some 5ß-steroids, such as compound 4ß,5ß-epoxyandrostan-17-one (9), showed aromatase inhibitory activity, because they adopt a similar A-ring conformation as those of androstenedione, the natural substrate of aromatase. Moreover, new chemical features to increase planarity were disclosed, specifically the 3α,4α-cyclopropane ring, as in 3α,4α-methylen-5α-androstan-17-one (5) (IC50=0.11µM), and the Δ(9-11) double bond in the C-ring, as in androsta-4,9(11)-diene-3,17-dione (13) (IC50=0.25µM). In addition, induced-fit docking (IFD) simulations and site of metabolism (SoM) predictions helped to explain the recognition of new potent steroidal aromatase inhibitors within the enzyme. These insights can be valuable tools for the understanding of the molecular recognition process by the aromatase and for the future design of new steroidal inhibitors.

New structure-activity relationships of A- and D-ring modified steroidal aromatase inhibitors: design, synthesis, and biochemical evaluation.

A- and D-ring androstenedione derivatives were synthesized and tested for their abilities to inhibit aromatase. In one series, C-3 hydroxyl derivatives were studied leading to a very active compound, when the C-3 hydroxyl group assumes 3ß stereochemistry (1, IC(50) = 0.18 µM). In a second series, the influence of double bonds or epoxide functions in different positions along the A-ring was studied. Among epoxides, the 3,4-epoxide 15 showed the best activity (IC(50) = 0.145 µM) revealing the possibility of the 3,4-oxiran oxygen resembling the C-3 carbonyl group of androstenedione. Among olefins, the 4,5-olefin 12 (IC(50) = 0.135 µM) revealed the best activity, pointing out the importance of planarity in the A,B-ring junction near C-5. C-4 acetoxy and acetylsalicyloxy derivatives were also studied showing that bulky substituents in C-4 diminish the activity. In addition, IFD simulations helped to explain the recognition of the C-3 hydroxyl derivatives (1 and 2) as well as 15 within the enzyme.

Synthesis and biochemical studies of 17-substituted androst-3-enes and 3,4-epoxyandrostanes as aromatase inhibitors.

A series of 5alpha-androst-3-enes and 3alpha,4alpha-epoxy-5alpha-androstanes were synthesized and tested for their abilities to inhibit aromatase in human placental microsomes. In these series the original C-17 carbonyl group was replaced by hydroxyl, acetyl and hydroxyimine groups. Inhibition kinetic analysis on the most potent steroid of these series revealed that it inhibits the enzyme in a competitive manner (IC(50)=6.5 microM). The achieved data pointed out the importance of the C-17 carbonyl group in the D-ring of the studied steroids as a structural feature required to reach maximum aromatase inhibitory activity. Further, at least one carbonyl group (C-3 or C-17) seems to be essential to effective aromatase inhibition.

Structure-activity relationships of new A,D-ring modified steroids as aromatase inhibitors: design, synthesis, and biological activity evaluation.

Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New A,D-ring modified steroid analogues of formestane and testolactone were designed and synthesized and their biochemical activity was investigated in vitro in an attempt to find new aromatase inhibitors and to gain insight into their structure-activity relationships (SAR). All compounds tested were less active than formestane. However, the 3-deoxy steroidal olefin 3a and its epoxide derivative 4a proved to be strong competitive aromatase inhibitors (K(i) = 50 and 38 nM and IC50 = 225 and 145 nM, respectively). According to our findings, the C-3 carbonyl group is not essential for anti-aromatase activity, but 5alpha-stereochemistry and some planarity in the steroidal framework is required. Furthermore, modification of the steroidal cyclopentanone D-ring, by construction of a delta-lactone six-membered ring, decreases the inhibitory potency. From the results obtained, it may be concluded that the binding pocket of the active site of aromatase requires planarity in the region of the steroid A,B-rings and the D-ring structure is critical for the binding.