Synthesis and SAR of b-Annulated 1,4-Dihydropyridines Define Cardiomyogenic Compounds as Novel Inhibitors of TGFβ Signaling

Abstract

A medium-throughput murine embryonic stem cell (mESC)-based high-content screening of 17,000 small molecules for cardiogenesis led to the identification of a b-annulated 1,4-dihydropyridine (1,4-DHP) that inhibited Transforming Growth Factor β (TGFβ)/Smad signaling by clearing the type II TGFβ receptor from the cell surface. Since this is an unprecedented mechanism of action, we explored the series' structure activity relationship (SAR) based on TGFβ inhibition, and evaluated SAR aspects for cell-surface clearance of TGFβ receptor II (TGFBR2) and for biological activity in mESCs. We determined a pharmacophore and generated 1,4-DHPs with IC50's for TGFβ inhibition in the nanomolar range (e.g., compound 28, 170 nM). Stereochemical consequences of a chiral center at the 4-position was evaluated, revealing 10- to 15-fold more potent TGFβ inhibition for the (+)- than the (−) enantiomer. This stereopreference was not observed for the low level inhibition against Activin A signaling, and was reversed for effects on calcium handling in HL-1 cells.

Design, synthesis and 3D-QSAR studies of novel 1,4-dihydropyridines as TGFβ/Smad inhibitors

“…(7,8) Interestingly, these mechanistic studies also revealed that the stereoselective mode of action was reversed for calcium channel antagonism since only the (-)-enantiomers strongly inhibited calcium transients in rat cardiomyocytes whereas the TGFβ inhibiting (+)-enantiomers were weakly active. (8) One of the designated goals of our ongoing work is to define a detailed picture of the …”

“…Therefore, the newly synthesized compounds were designed to incorporate heteroatom-containing, polar functional groups but also to explore steric aspects of certain regions of the molecules. Compounds were evaluated in a TGFβ/Smad reporter gene assay in HEK293T as described before, (8) and the data is summarized in Table 1. (20a and 21a), a carboxylic acid group pointed out the limitation of introducing a negatively charged moiety in the 2-position since both compounds were inactive.…”

“…A complete list of all chemical structures and biological data used for the 3D-QSAR model is provided: training dataset: Experimental details regarding crystal structure, data collection and structure refinement is archived at CCDC (deposition number 1043330). (7) 11.2(6) -0.1(5) -0.5(5) 9.8(6) C (1) 27.8 (9) 30 (1) 25.0(8) -6.7(7) -4.0(7) 17.6(8) C (2) 19.8(8) 20.6(8) 15.3 (7) 0.0(6) -1.4(6) 9.0(7) C(3) 21.4(8) 20.6(8) 14.2(7) -2.1(6) -2.7(6) 10.5(7) C(4) 20.8 (8) 16.0 (7) 15.0(7) -1.0(6) 0.8 (6) 11.1(7) C (5) 18.6(8) 17.6(8) 13.8 (7) -0.6(6) 0.0(6) 9.7(6) C (6) 18.2(8) 17.8 (7) 12.3(7) 0.6(6) 0.7(6) 8.7(6) C (7) 16.7 (7) 19.4 (8) 13.8 (7) (9) 1.0(7) 2.7 (7) 12.1(7) C (14) 25.0(9) 18.7 (8) 22.0(8) -2.9(6) 3.4 (7) 9.3(7) C (15) 28.2(9) 28.6(9) 19.8(9) -1.0(7) (14) …”

“…An initial focused medicinal chemistry study was done which revealed a rather steep SAR profile for the observed biological activity. (8) Here, we report on the extension of these SARs with additional, more versatile substituted and functionalized derivatives addressing distinct shortcomings of our current SAR picture of these compounds ( Figure 1). Along these lines, it was key to determine the -up to now -unknown absolute configuration of the active (+)-enantiomers.…”

Regulatory aspects of small molecule drugs for heart regeneration

“…However, a recent study shows that degradation of the TGF-β type II receptor (TGFBR2) by ITD-1, a novel molecule that is a highly selective TGF-β pathway inhibitor, leads to differentiation of mouse ESC into cardiomyocytes suggesting a role of inhibition of TGF-β signaling in heart development and potential role in regeneration [39,40]. The effects on differentiation were dependent on timing of treatment: when the inhibitor was added at days 3-5 of differentiation, it resulted in formation of cardiac cells; in contrast treatment during days 1-3 of differentiation abolished this effect.…”