Palladium-Catalyzed Modification of Nucleosides, Nucleotides and Oligonucleotides

India

Chapter 1

The Future of Drug Discovery: The Importance of Modified Nucleosides, Nucleotides, and Oligonucleotides

Anant R. Kapdi*; Yogesh S. Sanghvi†    ⁎ Institute of Chemical Technology, Mumbai, India

† Rasayan Inc., Encinitas, CA, United States

Abstract

This chapter summarizes various Pd-catalyzed cross-coupling reactions enabling the synthesis of chemically modified nucleic acid analogs with potential applications in therapeutic and diagnostic field, including design and application of novel fluorescent nucleoside analogs. A brief discussion of all chapters in this book describing prominent cross-coupling reactions such as Stille, Suzuki-Miyaura, Sonogashira, Heck, Buchwald-Hartwig, and C-H activation is offered, where Pd-mediated chemistry has been employed during assembly of modified nucleosides, nucleotides, and oligonucleotides.

Keywords

Drug discovery; Modification; Nucleosides; Nucleotides; Oligonucleotides; Sonogashira; Suzuki; Heck

Contents

1.Introduction

2.Biologically Active Modified Nucleosides

3.Synthetic Fluorescent Nucleoside Analogs

4.Oligonucleotide Applications

5.An Overview of Other Cross-Coupling Reactions in This Book

6.Stille Cross-Coupling

7.Suzuki-Miyaura Cross-Coupling

8.Sonogashira Reactions

9.Heck Cross-Coupling

10.Buchwald-Hartwig Reactions

11.H Activation Reactions

12.Cross-Coupling of Nucleotides and Oligonucleotides

13.Summary and Future Directions

References

1 Introduction

O bond formation in highly efficient manner. Currently, Pd-catalyzed cross-coupling is perhaps the most efficient way to generate a library of diversified structures needed for drug discovery efforts.¹

During the past two decades, myriads of Pd-catalyzed cross-coupling reactions have been developed and utilized in the preparation of chemically modified nucleic acid analogs with potential application in both the therapeutic and diagnostic fields.² The purpose of this introductory chapter is to highlight some of the key examples in which Pd-mediated chemistry has been employed to perform cross-coupling reactions during the assembly of modified nucleosides, nucleotides, and oligonucleotides as useful pharmaceutical agents and diagnostic tools. The conventional cross-coupling catalysts are sterically bulky, electron-rich, phosphine Pd-complexes that readily undergo standard steps such as oxidative addition and reductive elimination. More recently, the trend has been to use air-stable Pd precatalysts containing one molar equivalent of an ancillary ligand to drive the cross-coupling reactions to higher efficiency.

Today, Pd is perhaps the most widely used metal in cross-coupling catalysis for several reasons. First, Pd has the propensity to undergo two- electron processes during cross-coupling reactions. Second, Pd complexes are inert to a variety of functional groups, leaving the molecule unharmed during reactions. Third, these reactions often proceed at room temperature and reach completion within a few hours. Finally, low precatalyst loading (< 1 mol%) is sufficient to drive the reactions in a facile manner. The objective for this book is to explore, elaborate, and demonstrate the power of Pd-catalyzed reactions in which reactivity and selectivity are maintained during complex nucleic acid transformations. For brevity, we only discuss important examples to illustrate the role of Pd-catalyzed transformations resulting in useful pharmaceutical and diagnostic products derived from nucleic acids.

The impressive list of various applications is summarized in Fig. 1, where the nucleic acid field has benefited due to Pd-catalyzed reactions. Clearly, the most important contribution has been toward the design and synthesis of chemically modified nucleosides as antiviral and antitumor agents. This topic is reviewed in greater detail in the forthcoming chapters of this book. Therefore, we plan to provide a snapshot of discoveries that enabled the assembly of these molecules.

Fig. 1 Progress and applications of palladium catalysis in nucleic acid chemistry.

2 Biologically Active Modified Nucleosides

It has been > 40 years since the discovery of (E)-5-(2-bromovinyl)-2′-deoxyuridine (1, BVDU) as a selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) infections and the modified nucleoside still in use as an effective antiviral drug. Therefore, we developed an improved protocol for the synthesis of BVDU utilizing a Pd-imidate complex under extremely mild conditions and high yields.³ We also demonstrated the possibility of cost reduction via recycling of the palladium catalyst. Additionally, a large number of C5-modified pyrimidine nucleosides have been synthesized via Pd-mediated cross-coupling reactions and products tested for their biological activity. Another important example is FV-100 (2, Fig. 2), a bicyclic nucleoside analog that has emerged as the most promising candidate for the inhibition of VZV, currently in Phase III clinical trials. Our ongoing effort in developing sustainable and green processes led to a one-pot sequential Sonogashira cyclization protocol using Pd(OAc)2 and water-soluble phosphatriazene ligands toward an efficient synthesis of FV-100.⁴

Fig. 2 Biologically active modified nucleosides.

Berdis and co-workers reported the synthesis of 3-ethynyl-5- nitroindolyl-2′-deoxynucleoside (3, Endosine) as a potential theranostic agent that may improve the dosing regimens for anticancer treatment.⁵ The synthesis of Endosine was accomplished via Pd-mediated Sonogashira reaction in high yield. During the last decade, Hocek and co-workers have prepared over 100 novel purine nucleoside analogs exploiting the power of Pd-catalyzed cross-coupling reactions. This effort has resulted in new methodologies for the synthesis of potent 6-heteroarylpurine nucleosides such as structure 4 with anti-HCV activity (EC90 = 0.1–1 μM).⁶

The importance of carbocyclic nucleoside analogs as antiviral and anticancer drugs has played a prominent role in drug discovery. Abacavir (5) remains a unique example of carbocyclic nucleoside for the treatment of HIV-1. The original synthesis of Abacavir was accomplished via a Pd-mediated coupling of purine base and the vinyl carbocyclic derivative.⁷ In 2017, Rhee and co-workers reported the first catalytic asymmetric synthesis of the anticancer drug (−)-Tegafur (6) employing a Pd-catalyzed addition reaction of an unprotected pyrimidine to alkoxyallene.⁸ This study further highlights the application of Pd-mediated reactions in an atom-efficient manner for the assembly of a nucleoside-based drug.

Recently, bio-orthogonal chemistry has emerged as a safer option for drug delivery. In this vein, Unciti-Broceta and co-workers were inspired to design an N-propargyloxycarbonyl (N-Poc) protected prodrug of gemcitabine 7.⁹ The authors were able to clearly demonstrate that compound 7 had reduced (> 23-fold) cytotoxicity and achieved effective release of the gemcitabine in-vivo. This feast was accomplished with the Pd-mediated dealkylation of the N-Poc group, releasing the active drug in an effective manner and offering an alternative for delivery of the cytotoxic compound. These protocols with palladium chemistry are expected to offer safer and effective tools for both therapeutics and chemical biology.

Cidofovir (HPMPC), adefovir (PMEA), and tenofovir (PMPA) are FDA-approved antiviral drug molecules in which sugar moiety has been exchanged with an acyclic phosphonate linker. The exciting opportunity with acyclic nucleosides has resulted in many attempts to generate new leads for drug discovery. Last year, Diaz and co-workers reported on the synthesis of acyclic nucleosides in high yield via palladium-catalyzed asymmetric allylic amination reaction starting with vinyl-epoxide and carbonate and nucleic acid bases as nucleophiles.¹⁰ A representative acyclic phosphonate molecule 8 is shown in Fig. 2.

There are > 40 synthetic nucleoside analogs that are approved by the FDA as therapeutic drugs as antiviral and anticancer agents. As indicated in this section, modification of the sugar moiety and/or base functionality has generated promising lead molecules over the years, and several are currently in clinical trials. One area of interest is installation of an alkynyl group on the bases to modulate the activity of the nucleoside. Komiotis et al. have pursued this avenue and described an efficient microwave-assisted synthesis of C8-alkynyl adenine pyranonucleosides 9 with antitumor activity (Fig. 2). The authors utilized a palladium-catalyzed Sonogashira reaction to install a variety of alkynyl groups starting with 8-halo-purine analogs under microwave irradiation (200 W).¹¹ The base modification of purine nucleosides continues to be an area of interest demonstrated by Burley et al. from GSK, a pharmaceutical company.¹² Interestingly, these authors employed readily accessible 6-chloropurine nucleoside as a starting material and successfully executed Sonogashira coupling employing KI as an additive for the iodine source. This study also performed a Pd-132-catalyzed Suzuki-Miyaura reaction in a two-step manner, resulting in facile synthesis of 6-heteroaryl- substituted nucleosides. Len and co-workers published an excellent review article summarizing palladium-catalyzed Suzuki-Miyaura cross-coupling in a continuous flow system.¹³ Due to the ever-increasing cost of therapeutic drugs, there is always a need for process improvements enabling the cost reduction of goods. This objective could be accomplished if some of the cross-coupling reactions described in this book could be carried out in a continuous flow system when the demand for these molecules rise.

3 Synthetic Fluorescent Nucleoside Analogs

The four natural 2′-deoxy-nucleic acid bases, thymine, cytosine, adenine, and guanine, lack appreciable fluorescence. Therefore, applications of unmodified nucleosides, nucleotides, and oligonucleotides are of limited value for imaging, biophysical and high-throughput biochemical protocols. This limitation inspired many researchers to design fluorescent probes that could be conjugated to a natural nucleic acid base without interrupting their function. Tor and his research group have been actively pursuing synthesis of fluorescent molecules in which the natural geometry of the nucleic acid bases is retained including the H-bonding groups. These fluorescent synthetic mimics of nucleic acids are called isomorphic molecules. Extensive literature is available on this topic from Tor laboratory and several other investigators. An excellent collection of review articles is assembled in a recently published book edited by Wilhelmsson and Tor.¹⁴ Therefore, the following discussion is intended to highlight some of the key accomplishments in the design, synthesis, and application of fluorescent functional molecules.

The ease of cross-coupling and high efficiency toward the C5-position of pyrimidine nucleosides has culminated into impressive number of publications. Several of these publications are discussed in various chapters included in this book. One example that stands out is the C5-modification of 6-aza-pyrimidine nucleosides 10a reported by Tor et al. They synthesized two uridine mimics 5-Fur6AzaU (10a; x = O) and 5-Thioph6AzaU (10a; x = S; Fig. 3) with significant increase in the fluorescent quantum yield compared to uridine with the same C5-substituent. The observed improvement was attributed to the insertion of a single nitrogen atom at the 6-position, enhancing the push-pull effects. These molecules are expected to serve as sensitive hybridization probes for DNA diagnostics. Additionally, Tor and co-workers synthesized 5-(pyr-2-yl)-uridine 10b in a high-yielding palladium-mediated Stille coupling. The single bond linking the two π- systems resembles a molecular rotor, offering sensitivity to viscosity—a desirable feature for an emissive surrogate of thymine base.

Fig. 3 Synthetic fluorescent nucleoside analogs.

The synthesis and application of artificial base pairs has become a powerful tool for site-specific incorporation in DNA as an expanded genetic alphabet beyond natural Watson-Crick base pairs as third base pairs. Hirao and co-workers have made excellent strides in this field and added several unnatural base pairs that offer fluorescence for the site-specific labeling of nucleic acids. Hirao’s efforts have led to several interesting purine-like bases that are modified to elicit fluorescence while retaining their hydrogen-bonding geometry. Among several examples, 7-(2,2′-bithien-5-yl)-imidazo[4,5-b]pyridine 11 (DSS fluorophore) was synthesized using palladium-catalyzed coupling. Incorporation of compound 11 in an oligonucleotide exhibited strong fluorescence emission centered at 456 nm with excitation at 385 nm. Compound 11 also exhibited pairing with 2-nitropyrrole (Pn) moiety generating a functional third base pair in replication.¹⁵

The Bag research group in India has been exploring the synthesis of environmentally sensitive fluorescent (ESF) nucleosides as probes for structural studies of nucleic acids. Therefore, a conceptual ESF structure 12 (Fig. 3) is shown in which the majority of the C8-modified purine nucleosides have been assembled via palladium(0)-mediated Sonogashira cross-coupling reaction. As an example, the Bag group synthesized 8-(4-N,N-dimethylaminophenylethynyl)-2′-deoxyadenosine (8-EDA) and demonstrated its utility as a probe for a single-stranded molecular beacon. A full account of the design, synthesis, and application of extrinsic synthetic fluorescent nucleobases with remarkable photophysical properties is presented in Chapter 4 of this book. The Hudson group in Canada is actively pursuing the design of fresh and innovative structural analogs of cytidine base that retains the ability to hydrogen bond and elicit fluorescent properties. For example, synthesis of a pyrrolocytosine analog 13 (PhpdC; Fig. 3) was easily achieved via Sonogashira cross-coupling employing palladium chemistry. Interestingly, placement of PhpdC in an oligonucleotide resulted in enhanced stability of the duplex, which is a desirable feature for a therapeutic sequence. Furthermore, incorporation of intrinsic fluorescence of 6-phenylpyrrolocytosine was exploited for improved understanding of siRNA uptake and intracellular distribution.¹⁶

In Switzerland, the Luedtke research group is also focused in the synthesis and design of fluorescent bases for probing bimolecular structures. Their efforts led to the synthesis of 8-(2-pyridyl)-2′-deoxyguanosine (2PyG, 14a; ). The synthesis of nucleoside 14a was accomplished via palladium- mediated coupling. Oligonucleotide containing 14a permitted evaluation of metal binding in G-quadruplex structures.¹⁷ Seela and his research group in Germany have been exploring the conjugation of fluorescent groups to 7-deazapurine bases through Sonogashira-type coupling. More recently, they described the synthesis of 8-phenyl-substituted 7-deazaguanine nucleoside 14b via S-M reaction. This nucleoside exhibited strong fluorescence in polar aprotic solvents accompanied by solvatochromism. The modified nucleoside was converted into an amidite and incorporated into oligonucleotides successfully.¹⁸

The Kool research group at Stanford has been both inventive and successful in the design of fluorescent C-glycosides synthesized via palladium- catalyzed cross-coupling of an activated alkene and an aryl halide of choice. The C-glycoside analog 15 was efficiently incorporated into oligonucleotides offering valuable tools (as dyes) and improved chemical stability compared to a natural nucleoside. These chemistry efforts have led to the design of several oligodeoxyfluorosides (ODFs) exhibiting unique properties such as red-shifted emission in biological and analytical applications.¹⁹ Inouye and collaborators have further extended the concept of C-glycoside by inserting an alkynyl moiety between the sugar and aromatic group to improve fluorescent properties via extended conjugation systems. Short oligonucleotides containing alkynyl-pyrene C-glycoside 16 exhibited red-shift in emission resulting from extended conjugation.

The repertoire of modified nucleosides exhibiting extraordinary fluorescent properties continues to expand with the help of palladium-catalyzed cross-coupling reactions. These molecules are expected to improve our understanding of the three-dimensional structure of biomolecules and continue to unravel the mystery of nucleic acid functions.

4 Oligonucleotide Applications

Six chemically modified oligonucleotides have been approved by the FDA as therapeutic drugs, and over hundred oligonucleotides are currently in active clinical trials for treatment of a variety of diseases. Often, an oligonucleotide is chemically modified to make it nuclease stable while maintaining desired affinity for the target. Therefore, the basic architecture of sugar, phosphate backbone, and natural bases must be altered in a way that brings about a drug-like character. These oligonucleotide-based drugs exert their activity based on several modalities such as antisense, siRNA, microRNA, lncRNA, aptamers, and others. Because the majority of the oligonucleotides require chemical modifications for their use in therapeutics or diagnostic applications, palladium-catalyzed cross-coupling has played an important role during synthesis of several interesting building-blocks. A short summary of selected molecules is presented herein highlighting these palladium- catalyzed reactions.

A series of 5-(heteroaryl)-dU analogs were synthesized in good yield via palladium-catalyzed coupling of protected 5-iodo-2′-deoxyuridine with trialkyl-heteroarylstannanes and subsequently incorporated into antisense oligonucleotides exhibiting significant enhancement in thermal stability (Tm) for complimentary RNA.²⁰ Among several analogs tested, 5-(thiazol-2-yl)-dU modification 17 (x = S, Y = N; Fig. 4) proved to be the best, with an increase in the affinity of + 1.7ºC/substitution. The observed enhancement in affinity was attributed to increased base stacking interactions with the adjacent base pairs. This study was a cornerstone demonstrating that placement of a hydrophobic group at C5-position was beneficial in the design of high affinity oligonucleotides.

Fig. 4 Oligonucleotide applications.

Pedersen and co-workers reported an efficient post-synthetic on- column Sonogashira coupling while the DNA was attached to the solid support. This study allowed the synthesis and selective incorporation of para- and ortho-twisted intercalating nucleic acids 18 and 19, respectively (Fig. 4) with ethynylpyrene residues.²¹ Incorporation of 18 or 19 modifications resulted in modulation of thermal stability of parallel triplexes and antiparallel duplexes. In particular, ortho-TINA 19 modification has found excellent commercial utility in hybridization capture assays, target amplification systems, and qPCR.²² Additionally, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in the formation of an excimer band at 505 nm for ethynylpyrene analogs accompanied by higher thermal stability. A G4-decoy containing para-TINA showed promising activity for possible treatment of pancreatic cancer.²³

The researchers at Somalogic have pursued the design of modified nucleosides with ligands having protein-like side chains and demonstrated their utility in both the diagnostic and therapeutic arenas. They have carried out palladium-mediated carbonylation reactions for attachment of various functional groups at the C5-position of uracil base and incorporated these modified residues, such as 20 in aptamers. The placement of hydrophobic aromatic side chains created a unique intermolecular motif which offered interaction with functional proteins. More recently, this group combined sugar and base modification to develop aptamers exhibiting drug-like properties such as metabolic stability and favorable pharmacokinetic profiles.²⁴ The ease of synthesis and large-scale production of aptamers may hold promise as possible replacements for antibodies. Base-modified aptamers could act as a fluorescent probe, serving as biophysical analytical tools. In this vein, Fadock and Manderville recently reported the use of 8-thienyl-2′-deoxyguanosine 21 as a desirable modification of an internal emissive nucleobase probe for monitoring the interaction of small molecules and aptamer interactions. This study proposed the use of 21 in a sensor for detection of food toxins.²⁵

Kore and co-workers at ThermoFisher have published several protocols related to the synthesis of C5-modified pyrimidine 5′-triphosphates, such as 22, useful as a reagent of choice for numerous biological experiments. A chemoselective palladium-catalyzed Sonogashira coupling of 5-iodouridine-5′-triphosphate with propargylamine was developed furnishing 22 in high purity (> 99%) and good yield.²⁶ The incorporation of 22 into DNA offers an amino-terminus for further conjugation and labeling experiments. This protocol further demonstrates the power and potential of palladium-mediated reactions carried out under mild conditions in water without degradation of sensitive triphosphates analogs.

The fast-paced growth of palladium catalyzed reactions in combination with variety of novel precatalysts has opened the flood gates for the rapid assembly of modified nucleosides and oligonucleotides useful for diagnostic and biological applications that were not possible a decade ago. We expect further improvements to result in low catalyst loading and protocols for removal of palladium from the products where therapeutic potential is envisaged.

5 An Overview of Other Cross-Coupling Reactions in This Book

The advancement and future potential of palladium-catalyzed cross- coupling reactions encouraged us to assemble a collection of review articles written by leading investigators in this discipline. This book is a result of collective efforts that our colleagues and friends made possible. It is our hope that this book will serve as a source for inspiration and will catalyze conversation to further expand the application of this powerful reaction in the near future. The following discussion is an overview of nine outstanding chapters presented in this book, further expanding the horizons for students and young researchers indulging in the exciting world of nucleic acid