Solid-Phase Synthesis of a Pyrrole Library and Identification of Bioactive Compounds

Abstract

Pyrrole derivatives represent a privileged scaffold in medicinal chemistry due to their frequent presence in biologically active compounds. Herein, we report the solid-phase synthesis of a combinatorial library consisting of 211 pyrrole derivatives using a split-and-pool strategy based on the Hantzsch pyrrole synthesis. The pooled compounds were evaluated in a cell proliferation assay using the human lymphoblastoid cell line P493–6, a model with Myc-regulated growth. Four pools exhibited notable inhibitory activity, and subsequent deconvolution and synthesis of 16 individual pyrrole derivatives led to the identification of several compounds with potent antiproliferative profiles.

Graphical Abstract

Pyrrole scaffolds represent an important class of structures in medicinal chemistry, owing to their prevalence in a wide range of bioactive natural products and synthetic pharmaceuticals. These compounds have been reported to exhibit diverse biological activities, including anticancer, antimalarial, and antiviral effects. (¹, ²) These findings have led to continuous interest in the synthesis and screening of novel pyrrole derivatives for therapeutic applications. To facilitate the discovery of novel pyrrole derivatives with biological activity, various synthetic strategies have been explored. In this context, solid-phase synthesis offers a particularly effective approach for preparing structurally diverse pyrrole compounds. (³, ⁴, ⁵, ⁶, ⁷) The use of solid-phase methods, especially in combination with split-and-pool strategies, enables the parallel synthesis of multiple compounds in a time and cost-efficient manner. Among the synthetic methods toward pyrrole, the Hantzsch pyrrole synthesis is well suited for the solid-phase synthesis. (⁸) This reaction involves the condensation of 1,3-dicarbonyl compounds with primary amine followed by cyclization with α-bromoketone. The reaction proceeds under relatively mild conditions with good functional group tolerance and broad substrate scope. Furthermore, the use of commercially available and structurally diverse primary amine and α-bromoketone allows for easy access to chemically large and diverse libraries.

In the past, our research group successfully prepared a Kröhnke pyridine library using combinatorial chemistry and identified novel Myc inhibitor KJ-Pyr-9 and other bioactive molecules. (⁹, ¹⁰, ¹¹) This example clearly demonstrates that solid-phase synthesis is a powerful platform for generating chemically diverse compound libraries, which can serve as valuable starting points for drug discovery campaigns. Importantly, libraries derived from solid-phase reactions often consist of unbiased small molecules that are not restricted by preconceived notions of druglikeness, thereby allowing access to previously unexplored chemical space. As a result, such approaches can enable the identification of bioactive compounds for so-called “undrugabble” targets, highlighting the unique advantage of solid-phase synthesis in medicinal chemistry. (^{12, 13, 14})

Based on the precedent example of identification of novel bioactive compounds from the synthesized library, herein we sought to expand our heterocycle scope to include pyrrole derivatives. (¹⁵, ¹⁶, ¹⁷) We hypothesized that a similarly structured pyrrole library could yield compounds with distinct biological profiles, including ones with the potential to act against traditionally undruggable targets such as Myc.

We designed a pyrrole library using a split-and-pool solid-phase synthesis strategy starting from resin-bound acetoacetamide (Scheme 1a). We postulated that this split-and-pool strategy would give compound pools having unique pyrrole compounds, and it would streamline the biological screening process in solution phase (Scheme 1b). The synthesis involved two key steps: imine formation between the resin-bound acetoacetamide and a primary amine, followed by intermolecular cyclization with an α-bromoketone to form the pyrrole ring. Using 60 primary amines and four α-bromoketones (Scheme 1c and 1d), we generated 60 pools of compounds with each pool containing between 2 to 4 unique pyrrole derivatives, depending on compatibility and coupling efficiency of the reaction. Various amines including aliphatic amine, allylic amine, and chiral amine were well tolerated in our reaction to provide desired pyrrole compounds. However, certain amines such as aniline and 1-adamantylamine did not perform efficiently under the reaction condition, likely due to reduced nucleophilicity and steric hindrance. In addition, some amines bearing highly reactive functional groups, including Michael acceptors, failed to yield desired compounds, possibly due to competing side reactions. On the α-bromoketone side, substituted phenyl bromoketone, bromopinacolone, and desyl bromide were well tolerated. These building blocks afforded final pyrrole compounds with varied substituent patterns. The two-step synthesis was followed by trifluoroacetic acid (TFA)-mediated cleavage from the solid support. Crude products were obtained and analyzed using LC-MS and the overall synthesis successfully delivered compound pools in an average yield of 85% with 75% purity. (For details, see the supplementary information).

Scheme 1.

Solid-phase synthesis of a pyrrole library via a split-and-pool strategy. a) Overall synthetic scheme. b) Split-and-pool synthesis process. c) Primary amine building blocks: amines in red were not tolerated under the reaction conditions; the amine in purple did not react with bromoketone 1. d) α-bromoketone building blocks.

After preparation of our pyrrole compound library, we tested whether the pyrrole compound pools could be utilized for biological screening (Figure 1). Toward this goal, we evaluated the inhibitory activity of compound pools using the human B-cell line P493–6, engineered to express Myc under the control of a tetracycline-regulated promoter (Tet-off system). (¹⁰, ¹⁸, ¹⁹, ²⁰) In the absence of doxycycline, Myc is robustly expressed, leading to rapid cell proliferation. Conversely, addition of doxycycline suppresses Myc expression in a stringently controlled and dose-dependent manner. Therefore, this system has been widely used to assess the Myc-dependence of compound-induced inhibition of proliferation. Each compound pool was tested at a final concentration of 40 μM (10 μM per each compound) in a 96-well plate format. In this early primary screen, cell proliferation profiles and corresponding cell growth inhibition for each pool were compared to non-treated control wells, as well as known inhibitors, KJ-Pyr-9 and compound 5 (²¹). Clearly, treatment with doxycycline effectively inhibited cell proliferation in our P493–6 cell assay as anticipated. The well-characterized Myc inhibitor KJ-Pyr-9 and previously identified compound 5 also demonstrated expected levels of growth suppression. Among the 60 pyrrole compound pools tested, several showed measurable inhibition of cell proliferation. Notably, four pools, designated as F3, F4, M1, and N1, displayed strongest antiproliferative effects, giving >50% growth inhibition. Therefore, these pools were prioritized for further study and subjected to deconvolution to reveal the individual compounds responsible for the observed biological activity.

Figure 1.

Antiproliferation activity of 60 pyrrole compound pools using P493–6 cells and a 96-well resazurin-based cell assay. Cells were incubated with compound pools (40 μM, 10 μM per each compound) for 48 h followed by addition of resazurin, and fluorescence was measured after 2 h. Dox (doxycycline), KJ-Pyr-9, and 5 were used as controls.

To identify the active compounds within the pools, we synthesized all 16 pyrrole derivatives individually from the F3, F4, M1, and N1 hit pools using solution-phase conditions that closely mirrored the solid-phase protocol (Scheme 2). From acetoacetamide, one-pot synthesis comprising imine condensation and cyclization reaction provided individual pyrrole compounds in a concise manner. The single compounds were then reevaluated in the P493–6 cell proliferation assay at an initial concentration of 10 μM. Cells were treated with compound in 12-well plates and the number of live cells were counted every 24 h for 4 d, resulting in pronounced growth inhibition (Figure 2 and Supplementary Figure 2). Doxycycline and the known Myc inhibitors, KJ-Pyr-9 and compound 5, remained effective showing an expected cell growth inhibitory profile. Among the 16 synthesized pyrrole derivatives, 4 compounds, F33, F43, M13, and N12, exhibited strongest inhibitory activity, significantly reducing cell growth ≥80% under Myc-driven conditions. Structural comparison of these active compounds revealed common features. All active compounds contained a two-carbon linker between the nitrogen atom of the pyrrole ring and a substituted phenyl group. Additionally, compounds bearing aryl or diaryl substituents on the pyrrole scaffold were more active than those with bulky aliphatic groups, such as tert-butyl group. These observations suggest a preliminary structure-activity relationship (SAR) and may inform future optimization efforts.

Scheme 2.

Synthesis of 16 individual pyrrole compounds from the hit pools.

Figure 2.

Antiproliferation activity of individual pyrrole compounds in P493–6 cells after treatment for 72 h. Cells were incubated with compound (10 μM) in 12-well plates, and the number of cells per well were quantified and compared to untreated cells. Dox (doxycycline), KJ-Pyr-9, and 5 were used as controls.

To further characterize the activity of the four lead compounds, dose-response assays were performed to determine IC₅₀ values (Figure 3). P493–6 cells were treated with serial dilutions of F33, F43, M13, and N12, and inhibition of cell growth was monitored. Among the four pyrrole compounds, M13 showed the highest inhibitory potency with an IC₅₀ value of 0.06 μM. Compounds F43, F33, and N12 exhibited single digit IC₅₀ values of 0.7 μM, 1.4 μM, and 1.4 μM, respectively. These results indicated that our pyrrole compounds show moderate to strong inhibitory activity in the sub-micromolar range in P493–6 cells. Finally, resazurin was added to the cells to rule out potential off-target general cellular toxicity (Supplementary Figure 4 and 5). No significant decrease in cell viability was observed at compound concentration ranges around their IC₅₀ values, suggesting that the identified pyrrole compounds exhibit specific drug-induced growth rate inhibition in P493–6 cells. (²²)

Figure 3.

Dose-dependent inhibition of P493–6 cell growth by four lead compounds after treatment for 72 h. Cells were incubated with varying concentrations of compound in 12-well plates, and the number of cells per well were quantified and compared to untreated cells. The corresponding IC₅₀ values for each compound are in parentheses.

In summary, we synthesized a structurally diverse library of pyrrole derivatives using a split-and-pool solid-phase synthesis strategy based on Hantzsch pyrrole synthesis. A total of 211 compounds were prepared from 60 primary amines and four α-bromoketones, resulting in 60 compound pools. The compound pools were first screened in a Myc-driven cell proliferation assay using P493–6 cells. Four pools were identified as biologically active and shown to inhibit cell growth. Deconvolution and resynthesis of 16 individual compounds led to the identification of 4 pyrrole derivatives with significant antiproliferative activity with M13 demonstrating nanomolar potency. Preliminary structure-activity analysis indicated the importance of specific substituent patterns for biological activity. These results demonstrate the utility of solid-phase synthesis for generating libraries of bioactive compounds and provide a starting point for the development of pyrrole-based Myc inhibitors. Future work will focus on structural optimization and mechanistic studies to further evaluate their therapeutic potential.

Supplementary Material

Appendix A. Supplementary data

Supplementary data to this article can be found online at xxx.

Highlights.

• Solid-phase synthesis using a split-and-pool strategy efficiently generated diverse pyrrole compound libraries.

• The resulting pyrrole compound pools were employed in biological screening to identify potent lead compounds.

• Individual hit compounds F33, F43, M13, and N12 exhibited strong inhibitory effects in a cell proliferation assay.

Data availability

Data will be made available on request.