Diversity-Oriented Library Synthesis from Steviol and Isosteviol-Derived Scaffolds

Abstract

The readily available natural product stevioside provides a unique diterpene core structure that can be explored for small molecule library development by diversity-oriented synthesis and functional group transformations. Validation arrays were prepared from steviol, isosteviol and related analogs, derived from stevioside, to produce over 90 compounds. These compounds were submitted to the NIH Molecular Libraries Small Molecule Repository for screening in the Molecular Libraries Screening Center Network. Micromolar hits were identified in multiple high throughput assays for several library members. A cheminformatics analysis of the compounds was performed that verified the expected diversity and complexity of this set of compounds. The screening results indicate that scaffolds derived natural products can provide screening hits against multiple target proteins.

INTRODUCTION

Diversity-oriented synthesis (DOS) is an efficient approach to design and develop multiple small molecules that populate structurally diverse areas of chemical space. First introduced by Schreiber in early 2000,¹ this method provides rapid access to structurally different and complex compound libraries for biological screening. In contrast to target-oriented synthesis (TOS), which focuses on the synthesis of closely related structural analogs (focused libraries), DOS explores a broad area of chemical space that features compounds with different skeletal and stereochemical moieties.² Through DOS it is possible to create libraries of compounds from a single building block (scaffold) or collections of compounds from structurally similar scaffolds.

Natural products can serve as excellent scaffolds for chemical library development because of their inherent structural diversity and complexity, as compared to synthetic products.^3–5 In addition, natural products have been shown to occupy areas of chemical space that are generally not well covered by synthetic compounds.^6–7 Furthermore, natural products, their analogs, and natural product-inspired bioactive compounds have been sources of new drugs over many decades.⁸ Given the continuous research and effectiveness of exploring natural products for drug discovery and development, our group has investigated the library development of stevioside-derived scaffolds for biological evaluation. Our libraries were prepared under the Pilot Scales Libraries Initiative to synthesize and submit compounds to became part of the NIH Molecular Libraries Small Molecule Repository (MLSMR) and to be evaluated in the NIH-funded Molecular Libraries Screening Center Network centers.⁹

Stevioside is the primary natural product isolated from the Stevia rebaudiana Bertoni plant, indigenous to South America (Scheme 1).^10–11 Unlike many natural products, stevioside, which is used as a sweetener, is readily available in kilogram quantities and therefore ideally suited for the development of natural product-derived combinatorial libraries. Library synthesis is facilitated because stevioside chemistry has been explored extensively and chemical transformations leading to different types of scaffolds are well known.^12–14 Since stevioside, its analogs,^15–16 and related natural products such as gibberellic acid derivatives possess a variety of biological activities, we hypothesized that stevioside-derived libraries would provide promising opportunities to generate hits in screening campaigns.^17–20 The structure of stevioside contains an ent-kaurene core attached to three glucose molecules (Scheme 1). Upon enzymatic hydrolysis of the glycosidic bonds, steviol (1) is formed. Its structural analog isosteviol (2), which is stereochemically quite different, can be obtained through an acid-catalyzed Wagner-Meerwein rearrangement either from stevioside or steviol (Scheme 1).²¹ Both compounds provide stereochemically complex templates for library synthesis and can be converted to other diverse scaffolds in a few synthetic steps (Figure 1).^{12, 14, 22}

Scheme 1.

Synthesis of steviol and isosteviol from stevioside

Figure 1.

DOS approach to the ent-kaurene scaffold of steviol by D-ring modifications.

RESULTS AND DISCUSSION

Figure 1 illustrates several approaches that can be taken. For path II, the Wagner-Meerwein rearrangement, as discussed above, is achieved with hydrochloric acid to yield isosteviol. From isosteviol (2), several different scaffolds can be generated after one or two synthetic steps. Through route III, a Baeyer-Villiger oxidation of isosteviol (2) produces lactone 4.²³ Formation of an oxime and subsequent Beckmann rearrangement (path IV) yields lactam 5,¹² and photo-Beckman rearrangement leads to the corresponding amide regioisomer 6 (path V).¹² As shown for path VI, the Beckman fragmentation product 7 can also be obtained depending on reaction conditions.¹² Optimized protocols for isosteviol oxime formation, Beckman rearrangement and Beckmann fragmentation have been reported by us.¹² In addition to forming novel scaffolds, the functional groups in the molecules can be easily modified for library synthesis.¹⁴ Path I involves an allylic oxidation of the C17 methylene of steviol to furnish diol 3a, which can be further oxidized to the C15 ketone analog 3b.²⁴ Reductive amination of isosteviol (2) via route VII yields amine 8.^24–25 Other obvious modifications involve the carboxylic acid moiety, which can be reduced to the corresponding alcohol or a methyl group.^26–27 Curtius rearrangement of the C4 acyl azide leads to the formation of a 4-amine analog.¹⁶ The double bond of steviol can be transformed to an epoxide,²⁸ which then can be transformed to a diol,²⁸ and ozonolysis of the double bond provides the corresponding ketone.¹⁴

In this report, we focus on steviol (1) and D ring modified compounds 2, 5, 7, and 8 as the scaffolds for library development. We describe the synthesis of several pilot scale libraries, their evaluation in multiple high throughput screens and an in silico analysis of the chemical and drug-like properties of the compounds prepared in this study.

RESULTS AND DISCUSSION

Hydrolysis of stevioside under well-known reaction conditions provided steviol (1) and isosteviol (2) in good yields (Scheme 1).^29–30 These two molecules served as scaffolds for library development with reactions aimed at transforming the carboxylic acid moiety on ring A in both compounds to amides. We set up validation arrays for the steviol and isosteviol scaffolds for amide bond formation utilizing both solution and solid phase chemistry (only the isosteviol validation array is shown in Scheme 2).

Scheme 2.

Validation array for amide formation from isosteviol 2 by solution phase (A) and solid phase synthesis (B)

These reactions were carried out to compare product yields, ease of work-up, and purity of products. For the solution phase chemistry, isosteviol was converted to the corresponding acid chloride by reaction with oxalyl chloride and then reacted with amines 9{1-4} to furnish amides 10{1-4}. For the second array, we used polymer-bound 1-hydroxybenzotriazole (HOBt), a reagent that reacts with the acid moiety to form a reactive ester intermediate. Isosteviol was loaded onto the polymer using the coupling reagent EDCI in the presence of DMAP. The polymer-bound activated ester was purified by several washes and then reacted with amines 9{1-4} to form the corresponding amides 10{1-4}. Use of the amine as the limiting reagent in this reaction assures high purity of the resulting amide reaction products. After careful analysis and comparison of both the solution and solid phase approaches, we decided to carry out polymer-supported parallel synthesis to construct the library. The resin-bound synthesis approach allowed for a simple filtration to obtain the final products in high purity. Using parallel solid phase synthesis, we then prepared a small library of approximately 60 amides from steviol, isosteviol, lactam acid 12, nitrile acid 14, and amine acid 16 with amines 9 (Scheme 3). A selection of diverse primary amines was used in the library production.

Scheme 3.

Amide library formation by combination of 2, 1, 12, 14, and 16 with amines 9

After modifying the carboxylic acid moiety on the A ring, modifications of the C and D rings were explored through alkylation of the hydroxyl group of steviol, reduction of the ketone of isosteviol, and alkylation of the D-ring lactam of scaffold 5 (Scheme 4). We converted the acid functionalities of scaffolds 1, 2, 5, and 8 to the corresponding methyl esters. Reacting the hydroxyl group of the steviol methyl ester with alkylating agents 18{1-9} provided ethers 19{1-9}. The C16 ketone group of the isosteviol methyl ester was reduced with sodium borohydride to yield the corresponding secondary alcohol, which then was alkylated with 13{1-6} to provide ethers 20{1-6}.²⁴ N-Alkylated lactams 21{1-9} were obtained by reaction of lactam 5 with 13{1-9}, while N-alkylated amines 22{1-5} were generated from reacting amine 8 with 13{1-5}. The alkylations were carried out with sodium hydride, because a strong base was necessary to facilitate the removal of the hydroxyl proton for ether formation and the amide proton for lactam alkylation. These routes created a 33-membered library of O- and N-alkylated reaction products.

Scheme 4.

Synthesis of an O- and N-alkyl library

Bifunctional libraries can be prepared using alkylated products 19-22 to synthesize amides. Scheme 5 shows a representative example of one such library. Reaction of N-methyl amide analog 21{1} with amines 9{1-4} by first hydrolyzing the ester and then standard amide formation from the corresponding acid chlorides yielded amides 23{1-4}.

Scheme 5.

Synthesis of a bifunctional library from amide 21{1}

All compounds were submitted to the NIH’s Molecular Libraries Small Molecule Repository (MLSMR).⁹ All submitted compounds were analyzed for identity and purity (≥90%) by the MLSMR. Supplemental Information Table S1 provides a listing of the 22 compounds that were selected for screening by the NIH MLSMR. These compounds were evaluated in 56 assays by the Molecular Libraries Screening Center Network. Several library members, including previously prepared compounds 21{1} and 24-27 (Table 1),¹² displayed single or double digit micromolar inhibition of target proteins and are shown in Table 1, which shows a representative sample of current PubChem data on our submitted compounds that have been tested by the MLSMR.^31–32 A table of all currently-tested compounds is available in the supporting information section that only shows results from active primary screens or confirmatory assays; inconclusive or inactive results, which make up a majority of the data, are not listed (Table S1). Some assays in which steviol and isosteviol and their corresponding libraries showed activity include target proteins neuropilin-1 (NRP-1); tyrosyl-DNA phosphodiesterase 1 (TDP1); NACHT, leucine-rich repeat, Pyrin domain-containing-3 (NALP3); hepatitis C virus (HCV); glucagon-like peptide-1 (GLP1); and lipid storage modulators. NRP-1 is a protein receptor for vascular endothelial growth factor, thus playing a role in angiogenesis and is being studied for its role in tumor growth progression for anticancer research.^33–34 The TDP1 enzyme is also being investigated for anticancer development due to its mechanism of DNA repair and treatment for spinocerebellar ataxia, which is caused by a TDP1 mutation.³⁵ Furthermore, assays also identified NALP3, a protein involved in the inflammatory process of the human innate immune system.³⁶ Primary screens for inhibitors of the NALP3 protein can provide potential starting points for anti-inflammatory therapeutic development.³⁷ Compound 21{3} exhibited low micromolar activity in a confirmatory inhibitor assay for HCV (Table 1). Recently, Lin et al. created 33 ureido- and amide-substituted steviol analogs, similar to amide product 11 of steviol, to study their inhibitory effects against the Hepatitis B Virus (HBV), which similar to HCV infections can lead to liver cirrhosis and cancer.¹⁶ Their work demonstrates the importance of using natural products and their analogs as sources for therapeutic research as well as implies the potential for isosteviol analogs to be further investigated in HBV drug development. Finally, both GLP1 and lipid storage modulators have been sought in diabetes and anti-obesity research.^38–39

Table 1.

Activity of Steviol and Isosteviol Analogs

^aIC₅₀ = concentration of an inhibitor required for 50% inhibition of maximum control response;

^bAbsAC = Absolute active concentration with compounds below 10 μM to be considered active hits;

^cAC₅₀ = concentration required to elicit a 50% response in an in vitro assay;

^dEC₅₀ = concentration of an agonist required to produce 50% maximum (effective) response

A PMI (principal moment of inertia) plot shows that this library of compounds is both drug-like and diverse (Figure 2). This type of plot places rod shaped molecules (e.g., acetylene) at the upper left corner (A), pancake shaped molecules (e.g. benzene) at the lower apex (B), and spherical molecules (e.g. adamantane) in the upper right corner (C). As described previously calculated PMI for most drugs tend to congregate along the A-B axis from rod to pancake shape.⁴⁰ This plot demonstrates that our library lies in relevant three-dimensional space near this axis and that the library features shape diversity relative to commercially available lead-finding libraries (e.g. Maybridge HitFinder Collection; 14,400 compounds selected to represent the overall diversity of the screening collection, maybridge.com).

Figure 2.

PMI plot of stevioside library overlayed with a commercial “diversity” library demonstrating that the stevioside library is both drug-like and diverse.

More detailed comparisons of these stevioside-derived compounds with commercial libraries (cLogP, Fsp³, number of stereocenters, and pairwise similarity) confirm the uniqueness of this library (see Supporting Information).

CONCLUSION

We have developed a small library of compounds using a diversity-oriented synthesis approach based on the steviol and isosteviol scaffolds. Testing of the libraries provided screening hits in several assays against diverse protein targets. We analyzed the compounds using an in silico technique to determine their complexity and structural diversity relative to two commercial libraries. This analysis supported our hypothesis that the structures of the compounds we prepared are complex and diverse. Further analysis showed that our libraries are drug-like.

Supplementary Material

The Supporting Information is available free of charge on the ACS Publications website at DOI:

Calculations of drug like properties of compounds, experimental procedures and compound characterization data of 20 library members that were tested, including full ¹H and ¹³C NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.