Qinheng Zheng

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA

Click chemistry and drug discovery are a perfect match

One of the most profound advancements of modern chemistry is click chemistry. Ranked top of the most cited papers in the history of chemistry is the 2001 published Manifesto by Kolb, Finn and Sharpless¹. It has set the logical and strategical foundation to search for molecular chemical functions through a few good chemical reactions that are efficient and selective at a zone apparently transcending the normal reactivity. Representative click reactions^2-4 including the prototypic copper-catalyzed azide-alkyne cycloaddition (CuAAC) are as follows.

The core idea about click chemistry is to make covalent, intermolecular connections through a few good reactions. It has been well argued from an thermodynamic perspective in the Manifesto¹ that the formation of a carbon-heteroatom (C–X) or heteroatom-heteroatom (X–X) bond is favored over that of a carbon-carbon (C–C) bond. Another obvious advantage of making C–X or X–X lies in the predominant abundance of useful heteroatom synthons or building blocks (amines, acids, alcohols, etc) compared to reactive carbon-based synthons.

Implanted within the concept of connection are two logically interdependent key ideas: modularity and connectivity. The former concerns about the strategy of chemical entities being linked, while the latter describes the reactivity and chemical content within the linker. With a reliable connectivity, e.g. 1,4-triazole, a combinatorial library of reactive modules should result a library with its quantity of entries squared (Figure 1).⁵ The expectation is not only to double the possibility to find a hit, but also to achieve functions better than the mathematical sum (or product) of the efficacy of single modules. Such approach has proven viable in a few cases by the Sharpless Lab^6-9 and others¹⁰.

The following features of the “click” reaction make it especially useful in the construction of screening libraries:

1. the reaction is not significantly affected by the steric and/or electronic properties of both coupling partners;
2. the reaction works well or even better in water;
3. the reaction crude product is minimally contaminated in terms of by-product or catalyst or organic co-solvent;
4. the reaction is efficient at or slightly above room temperature;
5. The linker generated from the reaction is stable and retains or improves the pharmacological properties of substrates.

Click chemistry has had a significant impact on drug discovery¹¹, but also has become far more than originally intended, rapidly evolving as a “go-to” technology in almost every corner of all practical branches of chemistry and related sciences^11-20. The current review focuses on the “2nd generation” click chemistry, namely sulfur fluoride exchange (SuFEx).

SuFEx’s edge

W. Steinkopf’s authoritative series papers^21-22 published in the Journal für Praktische Chemie marked the dawn of the organic chemistry of hexavalent sulfur fluoride compounds. One important, yet often neglected experimental fact in the 1927 paper titled Über Aromatische Sulfofluoride²¹ is that “benzenesulfonyl fluoride…refluxing with aniline for several hour causes no change”²³. Now known as the refluxing aniline test to us²⁴, the scope of electrophilic groups able to resist refluxing aniline are extremely rare; in fact, sulfur(VI) fluoride groups are the only functionalities among a panel of electrophiles, including epoxide, acrylamide, vinyl sulfone, α-chloroketone(amide), β-lactam, maleimide and fluorophosphate that stand intact in the test. The realization of the inertness led to the conclusion that the reactivity of S^VI–F bonds stays at the far fringe of the acid-base chemistry⁴.

It is the realization that almost all sulfur(VI) compounds could be traced to one or a couple of basic, connective S^VI-cores, and the so-called periphery nucleophilic fragments prompted us to imagine a bottom-up strategy. Sulfur(VI) provides an ideal hub for the modular, multivalent ligand display. This approach of combinatorial library construction was named the hub-and-plugin model. Sulfur(VI) centers can display four distinct covalently attached ligands with tetrahedral arrangements. The modularity about the S^VI-hubs instantly opens a largely unexplored chemical space that parallels and, in the sense of multivalency, transcends the prevailing carbonyl world. SuFEx embodies the efficient methodologies of developing hubs with a few selectively stageable bis- or multi-electrophiles (e.g., O=SF₄, SO₂F₂, CH₂=CHSO₂F) which are at different levels of substitution. Sequential anchoring nucleophilic fragments to the S^VI-hubs have yielded unprecedented libraries of various hexavalent sulfur fluorides, organic sulfates (4-V), sulfamides (3-VII), and sulfamate esters (4-VI), to name a few.

Scheme 3. Landscape of Multidimensional SuFEx Click Reaction Sequences.

All known transformations have been summarized in the landscape of SuFEx reaction sequences (Scheme 3). These libraries are expected to improve the hit discovery and lead optimization processes of traditional medicinal chemistry by providing high quality and large quantity of molecules. This article aims to review the successful medicinal chemistry campaigns based on aryl fluorosulfate (3-III), iminosulfur oxydifluoride (2-II) and their downstream derivatives.

Complementary to the earlier successful diversity-oriented synthesis (DOS) by S. L. Schreiber^25-29, this SuFEx plug-and-play model utilize only the most reliable processes to join fragments at a minimal, indivisible S^VI-center; which is to date impossible for a quaternary carbon hub. On the same direction escaping the flatland^30-32, a common misunderstanding in the chemistry community is reflected by the zealousness for high sp³-carbon (Fsp³) fraction skeletons. In fact, a fragment screen by D. E. Shaw³³ revealed that achiral, planar aromatics yielded more productive binding against a diverse set of protein pockets in terms of shapes and chemical characteristics. Moreover, non-planarity could be easily introduced by three-dimensional connections of planar fragments, e.g. heteroatomic linker (S^VI), a small portion of hydrocarbon saturation, and even a simple carbon-carbon bond between rings. Otherwise, the additional affinity, if any, introduced can hardly compensate the large entropy lost. The unique SuFEx chemistry approach leveraging the rigidity and multidimensionality could be an edge. Planar plugin nucleophiles such as phenols and anilines will be prioritized and assembled to a tetrahedral S^VI-hub. Diversity of molecules could be achieved with the overall rigidity retained.

SuFEx-enabled high-throughput hit-to-lead optimization

High-throughput screening (HTS) of existing chemical libraries has become an effective method for the early-stage discovery of bioactive hit molecules. In a drug development pipeline, the follow-up hit-to-lead optimization, where new analogs are synthesized and tested (structure-activity relationship, SAR) with the expectation of improved target specificity, potency, and stability, often represents a more demanding process. Its extensive time and labor costs remain significant hurdles for the rapid discovery of affordable medicines.

The near perfect property of iminosulfur oxydifluoride as a multi-electrophilic hub was first realized by Dr. Suhua Li of the Sharpless Lab. In the 2017 Angewandte Chemie paper³⁶, it was demonstrated that this rarely studied functional group reacted efficiently with phenols and amines under defined conditions (Scheme 4). More impressively, the two fluorine atoms could be subsequently replaced in a stageable manner. Toward the end, a fully substituted S(VI)-center would be obtained. It was also noted in the paper that an aqueous buffer could promote the SuFEx reaction.

 

The realization that SuFEx reactions, especially the two water compatible sulfamide syntheses(Scheme 5) led to the idea of bridging SuFEx and medicinal chemistry, especially that empowered with high-throughput potential. While the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has been used in proof-of-concept studies on lead optimization, including the direct evaluation of biological potency^38-43, there are only a couple of drugs that contain the 1,2,3-triazole linkage. However, the potential of SuFEx to unite diverse modules using an O=SF₄ hub has not been explored. Not to mention, the resulting sulfamide linkers appear attractive^44-51.

It could be posited that the biocompatible reaction conditions would enable us to measure the potency of products directly using in vitro enzyme assays to prioritize the molecules (Scheme 6). Additionally, the rapid and diverse analog synthesis from the most available starting materials (i.e., primary and secondary amines) and the non-planer 3-dimensional structures, multiple hydrogen-bond donors/acceptors, drug-like lipophilicity, and stability in biological conditions of the products are ideal for medicinal chemistry⁵².

Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease of the papain-fold protease family (clan CA)⁵³ secreted by the majority of Streptococcus pyogenes strains, a Gram-positive bacterium that only infects humans⁵⁴. S. pyogenes infections lead to many ailments, such as tonsillitis, scarlet fever, and meningitis. Deep penetration into tissues by the bacterium can lead to more serious diseases, including necrotizing fasciitis and sepsis⁵⁵. To date, there are no S. pyogenes-specific treatments for infection, and high mortality rates are associated with invasive infections. An earlier HTS-identified inhibitor (5, IC₅₀ = 14 µM) of SpeB⁵⁶ was picked as the starting point.

Although peptidic SpeB inhibitors have been reported, such as E64^57-58, potent small molecule inhibitors have not been developed against SpeB. In preliminary SAR studies, introduction of an (S)-benzyl moiety (6) improved the potency to 2.1 µM. The SpeB:5 co-complex x-ray structure⁵⁶ and initial SAR campaign have suggested that additional surface pockets on SpeB were accessible for compound optimization via extension of 6 from the meta-positions of both benzene rings (See arrows in Figure 2). To verify the structural information, chemically synthesized ortho-, and para-substituted analogs were tested. Much diminished potency justified the idea to focus on both meta-position modification of the left or/and right benzene rings.

An iminosulfur oxydifluoride diversification handle was therefore introduced at the meta-position of either benzyl moieties of 6 to give 7 and 8. These molecules with an difluoride hub were separately coupled with a panel of 240 amines (170 primary amines, 70 secondary amines) to generate 480 analogs overnight using an equivolume mixture of DMSO and phosphate-buffered saline (PBS),incubated at 37°C. The well plate based parallel reactions mostly gave good to excellent yields of corresponding sulfamide type molecules.

The addition of PBS was essential for the full conversion (Figure 3); sole organic solvent with 5 equivalents of Hünig’s base at the same temperature could only yield poor conversion (41% in DMSO; 32% in MeCN). This condition originally developed for DNA-encoded library was found incredibly useful in the preparation of a customized library for medicinal chemistry. 

Figure 3. Representative UV-HPLC Traces of SuFEx Reactions in DMSO (A) and Aqueous Buffer (B).

The reaction products were directly screened for SpeB inhibition with an established kinetic fluorogenic substrate assay^56-57. Scatter plots of the screening results are shown in Figure 4. Additionally, no appreciable inhibition of SpeB was observed on the amines per se (absence of 7 or 8 in reaction), and the fluoride ion by-product. The SuFEx-based high throughput synthesis and screen has been proven applicable for functional assay, at least in this protease inhibitor development.

Molecules selected based on potency (Figure 4), lipophilicity, and molecular weight were synthesized and purified on milligram scale. There was an obvious correlation between potency estimated in the initial screen and those of re-synthesized compounds. Structures of representative molecules with improved IC₅₀ values are shown in Table 1.

#	Structure	IC50 (nM)
		SpeB	Papain	Caspase 3
5		14000	77
9		29 (± 4)	31	>100
10		71 (± 7)
11		93 (± 10)	10	> 100

Table 1. Validated SpeB Inhibitors.

The improved binding affinity was further validated by surface plasmon resonance and differential scanning fluorometry. The x-ray crystal structure of SpeB in complex with 9 was determined to elucidate the origin of improved inhibition (Figure 5). Interestingly, 9 binds SpeB in a U-shaped conformation with an intramolecular CH-π interaction^59-60 between the benzyl moiety and a hydrogen on the piperidyl group that likely contributes the binding confirmation. Compound 9 binds within the SpeB active site whereby the carbonyl oxygen of the carbamate moiety is oriented toward the SpeB oxyanion hole created by the main chain nitrogen atoms of residues Cys192 and Val193.

Figure 5. Co-Crystal Structure of SpeB:9.

The SuFEx high-throughput medicinal chemistry was further found compatible to miniaturization using an Echo Acoustic liquid handler. A correlation (R² = 0.88) in inhibitory potency was observed between the picomole-scale (1536-well, 2 µL, 200 µM of iminosulfur oxydifluoride, 400 pmol) and nanomole-scale (96-well, 50 µL, 10 nmol) syntheses, demonstrating the successful miniaturization of the library construction. Importantly, unlike previously reported nanoscale medicinal chemistry attempts⁶¹, the sub-nanomole-scale SuFEx-based library synthesis does not require specialized equipment, such as dry-boxed liquid handlers and highly sensitive mass spectrometry for the biological assay. Thus, the SuFEx-based libraries can be readily adapted in screening facilities with standard HTS robotics and liquid handler systems.

Figure 6. Correlation between Picomole-Scale (x-axis) and Nanomole-Scale (y-axis) Synthesis (R² = 0.88).

Based on biological stability and solubility in PBS, 11 was selected for further biological characterization. Compound 11 is stable against human liver microsomes in vitro (t_1/2 = 120 min), soluble in PBS, selective for SpeB (over other cysteine proteases, see Table 1), non-cytotoxic, and adheres to Lipinski’s rules⁶². The effect of inhibitor 11 was tested in an established neutrophil killing assay, wherein SpeB activity provides relative resistance to  S. pyogenes against human neutrophils^63-64. Wild-type (WT) S. pyogenes (M1 serotype strain 5448) and a corresponding isogenic mutant strain lacking SpeB (∆SpeB) were preincubated with 11 prior to introduction of freshly isolated neutrophils from human blood. The presence of 11 decreased the viability of WT S. pyogenes in a concentration-dependent manner, while no similar drug effect of 11 occurred in the ΔSpeB mutant strain (Figure 7).

Outlook

Acceleration of drug discovery is important to affordable drug discovery campaigns. The introduction of high-throughput screening robotics, liquid handler systems, and assay miniaturization have revolutionized screening of bioactive molecules. Via the proof-of-concept work aforementioned, inexpensive high-throughput synthesis and screening processes are now routinely used in cell-based and in vitro assays against biomedically relevant targets. This study highlights the utility of SuFEx chemistry for rapidly generating diversified molecules for hit-to-lead applications and shows the potential of the combination of click chemistry, miniaturized synthesis, and direct evaluation of biological potency.

The key idea to link chemical moieties and direct screening of reaction mixture or crude product without chromatography purification is deeply rooted in click chemistry. Sibling strategies have also proven successful in a few works not covered here. The premier examples include the fluorosulfation of phenolic drugs and direct screening against cancer cell lines⁶⁵, and the ultralarge azide library made by fluorosulfonyl azide-mediated diaozotransfer⁵.

Future directions include the merger of click chemistry and other large libraries, for example, DNA-encoded libraries⁶⁶ or cyclic peptide libraries⁶⁷ are promising for the purpose of plug-and-play in a high-throughput fashion.

Acknowledgement

The author acknowledges the guidance of Professor Barry Sharpless at The Scripps Research Institute, the collaboration of Professor Dennis Wolan and Dr. Seiya Kitamura, and the financial support of the Ellen Browning Scripps Foundation through a graduate fellowship.

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