Design, synthesis, biological evaluation and molecular docking of 1,7-dibenzyl substituted theophylline derivatives as novel BRD4-BD1 selective inhibitors

Bromodomain-containing protein 4 (BRD4), an epigenetic reader, has been recognized as a target with therapeutic potential in several types of cancer and cardiovascular diseases. In this study, a series of 1,7-dibenzyl substituted theophylline derivatives were synthesized and their BRD4 inhibitor activities were evaluated. Most of the compounds showed detectable activities with IC 50 values in the range of 2.51–10.50 µM. Therein, compound 6e showed significant selectivity for two bromodomains of BRD4, the inhibition of BD1 (IC 50 = 2.51 µM) was 20 times greater than that of BD2 (IC 50 > 50 µM). Similarly, compounds 6b – 6d, 6f, 6j and 8a also displayed favorable BRD4-BD1 selectivity. In addition, molecular docking of compound 6e was performed to predict con-ceivable binding patterns of it with BRD4, prompting residue Ile146 might be crucial to the observed selectivity of BRD4-BD1. These findings will be of great value and significance for the development of novel BRD4-BD1 inhibitors.


Structure-activity relationship and molecular docking
The BRD4-BD1 inhibitory activity of synthesized compounds was evaluated by HTRF assay and compound A which was identified as an excellent selective BRD4-BD1 inhibitor with no observed inhibition against BD2 was chosen as a positive control [33]. Firstly, as shown in Table 1, when there existed no substituent at R2 part (5a), no activity could be detected, illustrating the substituent at corresponding position was indispensable for activity of benzyl substituted theophylline derivatives. To preliminarily explore the preference of substituents at position-8, hydrophilic and hydrophobic side chains were introduced, the obtained result was that a slight increase in lipophilicity would be helpful to the improvement of activity (compare 1a, 3a and 4a). Subsequently, introduction of 3,5-dimethoxy and nitro to benzyl group at position-7 led to compounds 3b and 3c, but their activities declined somewhat compared with 3a, showing that electron-withdrawing group (EWG) or electron-donating group (EDG) on benzyl group had no effect on activity. Unexpectedly, compound 2a which was a by-product of N-alkylation of 4 with 3-bromobenzyl iodide revealed a promising consequence (IC50 = 18.73 µM). Therefore, we turned our attention to 1,7-dibenzyl substituted theophylline derivatives.
In accord with monosubstituted derivatives, compound 5b displayed no observed activity in the absence of any substituent at position-8. To further improve the activity towards BRD4-BD1, introduction of moderately hydrophobic groups (aromatic heterocycle, thioether and ether) to position-8 gave compounds 7a-7d and 8a-8e, respectively. However, only compound 8a exhibited detectable inhibitory activity with an IC50 value of 9.66 µM ( Table 2). A reasonable explanation was that alkyl chains with inappropriate lengths or aromatic heterocycles would be incompatible to the BRD4-BD1 pocket.
For interpreting the difference of activities between monosubstituted and disubstituted theophylline derivatives, we performed molecular docking studies for compounds 3a and 8a. As depicted in the Fig. 2A and 2B, both of their conformations could be stabilized in the pocket by theophylline moiety forming hydrogen bonds with Asn140, furthermore, proper substituents at position-8 provided steric hindrance to clash with substituted benzyl groups, making them fold downward the WPF region. However, due to that introduction of an extra 2-bromobenzyl group at position-1 of compound 8a led to a 180-degree flip of the ring plane, 8a could fit more closely with WPF region and form a π-H interaction with Ile146 by the benzyl group, as well as exhibit better inhibitor activity. showed moderate BRD4 inhibitory activity with an IC50 value of 8.34 µM. Subsequently, compounds 6c-6j were synthesized to explore the influence of type and position for benzylic substituents towards activity. The results indicated that inhibitory activity was inversely proportional with electronegativity of halogen substituents (6c-6g) as well as their activities along para-position, ortho-and meta-decreased in turn. Such as compounds 6c-6e with IC50 values of 9.21 µM, 6.67 µM and 2.51 µM, respectively ( Table 2). For the other substituents, the hydrophobic group was superior to hydrophilic group for enhancement of activity (compare 6h-6j) and para-substitution (6j) also exerted better activity.
To probe into the difference of potency, compounds 6b-6e were selected for molecular docking studies (Fig. 3). The results indicated that they contacted with BRD4-BD1 by π-H interactions which were established by different benzyl groups at position-1 with residue Ile146 (Fig. 3A, 3B, 3C and 3D). Therein, the hydrogen bond formed by theophylline scaffold with Asn140 was found in the docking conformations of 6b, 6d and 6e except for 6c, which provided a reasonable explanation for the fact that meta-substitution exhibited lower activity than ortho-and para-, but it was also possible that there existed a weaker water-mediated hydrogen bond in the actual combination. Nevertheless, π-H and T-shaped stacking interactions formed by 3-bromobenzyl group of 6c with Leu92 and Trp81 made up for the decrease of activity to some extent. More than that, similar T-shaped stacking interaction was discovered in the docking conformation of compound 6e with BRD4-BD1, not in 6b and 6d, which explained better activities of para-substituted benzyl derivatives than that of others.
To explore the mechanism about BRD4-BD1 selectivity of 1,7-dibenzyl substituted theophylline derivatives, we also researched the binding pattern of compound 6e with BRD4-BD2 (Fig. 3E), showing that 6e matched poorly with the binding pocket and interacted only with BRD4-BD2 by a π-H interaction assembled by Leu385 and substituted benzyl group of position-7, leading to an unstable binding mode and no obvious activity. By comparing the amino acid residues that could form interactions with compound 6e in binding pockets of BD1 and BD2, it was apparent to find that only this pair of amino acids (Ile 146 of BD1 and Val439 of BD2) showed significant differences. Additionally, a recent study showed that Ile146 plays a vital role for BRD4-BD1 selectivity of MS402 [35]. Hence, the observed selectivity might be attributed to that larger bulk of Ile146 made BRD4-BD1 binding pocket more compact and afforded π-H interactions for binding of compounds compared with Val439, leading to more matched and stable binding patterns of them with BD1.

Conclusion
In summary, 1,7-dibenzyl substituted theophylline derivatives as BRD4-BD1 inhibitors were discovered for the first time. Among them, compound 6e possessed favorable BRD4-BD1 inhibitory activity (IC50 = 2.51 µM) and significant selectivity for BRD4-BD1 which was twentyfold higher than that of BD2 (IC50 > 50 µM). Likewise, 6b-6d, 6f, 6j and 8a also exerted obvious selectivity of BRD4-BD1. At the same time, we explored the potential binding patterns of 1,7-dibenzyl substituted theophylline derivatives with BRD4 through molecular docking studies, showing that residue Asn140, Trp81, Leu92 and Ile146 in the binding pocket of BRD4-BD1 made an important contribution on inhibitory activity as well as Ile146 might be the key to fulfill selectivity of BRD4-BD1. Therefore, the 1,7-dibenzyl substituted theophylline scaffold is anticipated to be developed into a class of superb BRD4-BD1 inhibitors for drug discovery.