Nucleoside Di- and Triphosphates As a New Generation of Anti-HIV Pronucleotides

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Review Nucleoside Di- and Triphosphates as a New Generation of Anti-HIV Pronucleotides. Chemical and Biological Aspects

Marta Rachwalak 1,2,* , Joanna Romanowska 1, Michal Sobkowski 1 and Jacek Stawinski 1,3,*

1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Pozna´n,Poland; [email protected] (J.R.); [email protected] (M.S.) 2 Mellon College of Science, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA 3 Arrhenius Laboratory, Department of Organic Chemistry, Stockholm University, S-10691 Stockholm, Sweden * Correspondence: [email protected] (M.R.); [email protected] (J.S.); Tel.: +14-12-268-5453 (M.R.); +48-61-852-8502 (ext. 1549) (J.S.)

Abstract: This review provides a short account of the chemical synthesis of nucleoside di- and triphosphates on a historical background, together with the use of this class of compounds as potential pronucleotides in anti-HIV therapy.

Keywords: nucleoside di- and triphosphates; anti-HIV pronucleotides; synthetic strategies; biological activity

1. Introduction

Citation: Rachwalak, M.; Phosphorylated nucleosides, collectively called nucleotides, play pivotal roles in Romanowska, J.; Sobkowski, M.; various biological processes. Naturally occurring deoxy- and ribonucleoside triphosphates Stawinski, J. Nucleoside Di- and are basic building blocks for the enzymatic synthesis of DNA and RNA and participate Triphosphates as a New Generation also in energy transfer processes, intracellular signaling, and the regulation of proteins’ of Anti-HIV Pronucleotides. biological activity [1]. Scientists have long been interested in synthetic nucleotide analogs Chemical and Biological Aspects. because they allow the study of complex biological systems and themselves have potential Appl. Sci. 2021, 11, 2248. https:// therapeutic and diagnostic value [2,3]. doi.org/10.3390/app11052248 Di- and triphosphates of modified nucleosides are often used as compounds of potential antiviral (e.g., anti-HIV) activity. The core of this class of compounds consists Academic Editor: Qi-Huang Zheng of 20,30-dideoxynucleoside 50-triphosphates (ddN, e.g., AZT, d4T, ddI, ddC, 3TC, FTC), which are inhibitors of reverse transcription (RT) during HIV replication [4,5]. However, Received: 2 February 2021 direct administration of such compounds to patients is pointless, because as negatively Accepted: 26 February 2021 charged molecules they are not effectively transported through the cell membrane [6], Published: 4 March 2021 which practically precludes their bioavailability [7–9]. The simplest solution would be to administer their prodrugs, dideoxynucleosides, which in vivo would be phosphorylated Publisher’s Note: MDPI stays neutral to the corresponding triphosphates. Although this approach works in many cases (e.g., with regard to jurisdictional claims in AZT), it turned out that some dideoxynucleosides (e.g., ddU), which are precursors of the published maps and institutional affil- very potent anti-HIV compounds (e.g., ddU 50-triphosphate), are practically not phospho- iations. rylated to nucleoside 50-monophospate (NMP) in the cell. Such dideoxynucleosides, when administered in the form of free nucleosides, usually showed no anti-HIV activity [4,5,7,8]. Moreover, AZT also suffers several drawbacks associated with the unfavorable kinetics of its successive in vivo phosphorylations (leading in consequence to serious adverse effects Copyright: © 2021 by the authors. of the AZT therapy) [10] and the existence of the thymidine kinase-deficient cells, in which Licensee MDPI, Basel, Switzerland. AZT is not phosphorylated and remains as an inactive prodrug prior to clearance (such This article is an open access article cells become reservoirs of viruses inaccessible for the therapy) [11]. Therefore, knowing distributed under the terms and the limitations of the use of 20,30-dideoxynucleosides as a potential anti-HIV therapeutics, conditions of the Creative Commons the pronucleotide idea was born. Attribution (CC BY) license (https:// Pronucleotides in their basic concept are electrically neutral phosphate derivatives creativecommons.org/licenses/by/ of biologically active nucleosides, in which protective groups on the phosphate residues 4.0/).

Appl. Sci. 2021, 11, 2248. https://doi.org/10.3390/app11052248 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 30

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Pronucleotides in their basic concept are electrically neutral phosphate derivatives of biologically active nucleosides, in which protective groups on the phosphate residues neu- neutralize electrical charges. In such form, phosphorylated nucleosides can easily pen- tralize electricaletrate charges. cell membranes.In such form, Inside phosphorylated the cell, the nucleosides masking groups can easily are removedpenetrate via enzymatic cell membranes.and/or Inside chemical the cell, hydrolysis,the masking which groups lead are to removed the release via of enzymatic the corresponding and/or nucleoside chemical hydrolysis,50-monophosphate which lead to (ddNMP). the release Then, of the cellular corresponding kinases in nucleoside a stepwise 5 manner′-mono- phosphorylate phosphate (ddNMP).ddNMP Then, ultimately cellular to kinases its 20,30 in-dideoxynucleoside a stepwise manner 5 phosphorylate0-triphosphate (ddNTP),ddNMP an anti-HIV ultimately to itsactive 2′,3′-dideoxynucleoside species (Scheme1)[ 95,′12-triphosphate,13]. (ddNTP), an anti-HIV active species (Scheme 1) [9,12,13].

Scheme 1. A general principle of the monophosphate pronucleotides mode of action. Scheme 1. A general principle of the monophosphate pronucleotides mode of action.

Initially, whenInitially, developing when the developing concept of thepronucleotides, concept of pronucleotides, it was assumed it as was an assumedabso- as an abso- lutely necessarylutely condition necessary that condition the electric that charges the electric in the charges prodrug in the molecule prodrug moleculewere com- were completely pletely masked.masked. Following Following this paradigm, this paradigm, various various pronucleotide pronucleotide strategies strategies were were pro- proposed, which posed, which werewere based based mainly mainly on on esters esters or or amides amides as aspotential potential protective protective groups groups for for5′- 50-phosphate phosphate moietymoiety of ddNMP of ddNMP (e.g., (e.g., studies studies of McGuigan of McGuigan [11,14,15], [11,14 Imbach,15], Imbach [16–19], [16 Meier–19], Meier [20,21], [20,21], KraszewskiKraszewski [22,23], [ 22and,23 others], and othersgroups). groups). However, However, over the over years, the it years, has been it has ob- been observed served that certainthat certaintypes of types pronucleotides of pronucleotides endowed endowed with negative with negative charges charges could also could ef- also effectively fectively penetratepenetrate cell membranes, cell membranes, and showed and showed high biological high biological activity activity (e.g., the (e.g., research the research of the of the WagnerWagner [24–28] and [24– 28Kraszewski] and Kraszewski groups [2 groups3,29,30]). [23 This,29,30 challenged]). This challenged the validity the of validity of the the generally acceptedgenerally postulate accepted of postulate electrical of neutrality electrical as neutrality the basic as criterion the basic for criterion the effec- for the effective tive transport transportof pronucleotides of pronucleotides through cell through membranes, cell membranes, but at the same but at time the sameit opened time it opened up up new possibilitiesnew possibilities for extending for the extending spectrum the of spectrum the designed of the pronucleotides designed pronucleotides to include to include new new ionic structuralionic structural features. features. The pronucleotideThe pronucleotidestrategy for delivering strategy monophosphorylated for delivering monophosphorylated antiviral nucleosides antiviral nucleosides to the cell did not,to the however, cell did completely not, however, solve completely the problem solve of the the effective problem in of vivo the genera- effective in vivo gen- tion of an activeeration metabolite, of an activeddNTP. metabolite, Sometimes ddNTP. the enzymatic Sometimes phosphorylation the enzymatic of phosphorylation 2′,3′- of dideoxynucleoside20,30-dideoxynucleoside monophosphates to monophosphates di- and ultimately to to di- triphosphates and ultimately is very to triphosphates diffi- is very cult or has undesirabledifficult or kinetics. has undesirable Typically, kinetics. such problems Typically, arise such during problems the arise conversion during theof conversion of a monophosphorylateda monophosphorylated nucleoside into nucleoside its corresponding into its corresponding diphosphate. For diphosphate. example, AZT For example, AZT is a good substrateis a good for thymidine substrate forkinase, thymidine similarly kinase, to natural similarly thymidine, to natural but thymidine, thymidylate but thymidylate kinase convertskinase AZTMP converts into AZTDP AZTMP much into AZTDPless efficiently much lessthan efficiently TMP into than TDP TMP [10]. intoThus, TDP [10]. Thus, in such situationsin such it would situations be advantageous it would be advantageous to deliver the to pronucleotides deliver the pronucleotides to the cells toin the cells in the the form of suitablyform of protected suitably protecteddiphosphates diphosphates of antiviral of antiviralnucleosides nucleosides in order into orderbypass to bypass enzy- enzymatic phosphorylationmatic phosphorylation to ddNDP. to While ddNDP. the While final phosphorylation the final phosphorylation of dideoxynucle- of dideoxynucleoside oside diphosphatesdiphosphates to the corresponding to the corresponding triphosphates triphosphates is usually is fast usually and effective, fast and effective,it was it was also also tempting temptingto develop to triphosphate develop triphosphate pronucleotides pronucleotides so that in sovivo that generationin vivo generation of the of the final final active metaboliteactive metabolite would bewould completely be completely independent independent of cellular of phosphorylation cellular phosphorylation pro- processes. cesses. Despite the apparent advantages of such approaches, the first studies on the use of di- Despite theand apparent triphosphate advantages pronucleotides of such a againstpproaches, HIV the appeared first studies two decadeson the use after of the concept of di- and triphosphateanti-HIV pronucleotides monophosphate against pronucleotides HIV appeared emerged two decades [31]. This after delay, the concept most likely, was due of anti-HIV monophosphateto synthetic difficulties pronucleotides and the em extremeerged [31]. lability This of delay, the anhydride most likely, bond was in uncharged di- due to syntheticand difficulties triphosphates, and the containing extreme lability masking of groupsthe anhydride in aqueous bond media. in uncharged However, the reports from the Wagner group [24–28], showing that monophosphate pronucleotides with electric charges are both stable and can cross the cell membranes, revived the interest of both Appl. Sci. 2021, 11, 2248 3 of 28

chemists and biologists in 20,30-dideoxynucleoside di- and triphosphates as potential pronucleotides. In this review, we present chemical methods for the formation of the anhydride polyphos- phate linkages together with their applications to the synthesis of 20,30-dideoxynucleoside di- and triphosphates, as a new generation of anti-HIV pronucleotides. For recent reviews on the related topics, see also Hollenstein (2012) [32] Hou et al. (2014) [33], Sherstyuk and Abramova (2015) [34], H. Tanaka (2015) [35], Ahmadipour and Miller (2017) [36], Kaczynski and Chmielewski (2017) [37], and Camarasa (2018) [38].

2. Chemical Synthesis of Nucleoside Di- and Triphosphates Due to the plethora of biological functions of nucleoside di- and triphosphates, it is not surprising that there has been a long-lasting interest in synthetic methods for the efficient preparation of this class of compounds. However, due to the presence of multiple reactive centers in nucleotide derivatives, developing efficient synthetic protocols is still a challenging task. There are two general synthetic approaches to nucleoside di- and triphosphates. His- torically, the first one exploits the chemistry of P(V) compounds and is based on increasing the electrophilicity of the phosphorous center through its activation with condensing agents to make it more susceptible to nucleophilic attack. The second approach makes use of more reactive P(III) compounds, which can be either coupled with a nucleophile and subsequently oxidized, or coupled and oxidized simultaneously in a reaction called an “oxidative coupling”. This opens up additional synthetic possibilities in the preparation of nucleoside di- and triphosphates and their analogs.

2.1. Early Methods for the Preparation of Nucleoside Di- and Triphosphates The first chemical synthesis of di- and triphosphates served mainly to determine and confirm the structure of nucleotides isolated from biological materials. The pioneering research in this field was carried out by Lord Alexander Todd, who, together with his collaborators, in the late 1940s and early 1950s, published a series of fundamental papers on the synthesis of an anhydride bond in nucleoside di- and triphosphates. Years of basic research have led to the development of new methods for the phosphorylation of alcohols and phenols with dibenzyl chlorophosphate (DBCP). It was found that this reagent could be used for both the phosphorylation of nucleosides and nucleotides (Scheme2). Thus, when DBCP was allowed to react with a free 5 0-OH group of the appro- priately masked adenosine, dibenzyl adenosine 50-monophosphate 1 was obtained. This compound could be partly deprotected by a careful treatment with diluted sulfuric acid in ethanol to form monobenzylated AMP 2. The phosphorylation of this intermediate, again with DBCP, followed by hydrogenation, led to the preparation of the first synthetic nucleoside diphosphate, ADP (Scheme2). It is noteworthy that the intermediate product 3 appeared to be a mixture of di- and tribenzyl diphosphates 3a and 3b, since one benzyl group was partly lost under the reaction conditions. Fortunately, there was no need for the separation of these products or a conversion of 3a into 3b, since the catalytic hydrogenation of this mixture furnished the desired adenosine 5’-diphosphate (ADP), which was isolated as an acridinium salt. After various modifications of this synthetic protocol, the product could be obtained in a 55% yield [39]. Appl. Sci. 2021, 11, 2248 4 of 28 Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 30

SchemeScheme 2.2. SynthesisSynthesis of of adenosine adenosine 5’-diphosphate 5’-diphosphate (ADP (ADP)) [39] [and39] adenosine and adenosine 5′-triphosphat 50-triphosphatee (ATP) (ATP)[40] according [40] according to Todd to Toddet al. et al.

ToddToddet et al.al. [[4040,,4141]] also developed a a method method for for the the preparation preparation of of nucleoside nucleoside 5′- 5tri-0- triphosphates.phosphates. The The first first synthesized synthesized compound compound was wasadenosine adenosine 5′-triphosphate 50-triphosphate (ATP), (ATP), which whichhelped helped confirm confirm the assumed the assumed structure structure of a compound of a compound (tentatively (tentatively identified identified as ATP) iso- as ATP)lated isolated from muscle from muscleextracts extracts15 years 15 earlier. years earlier.For this Forpurpose, this purpose, the aforementioned the aforementioned mixture mixtureof di- and of di-tribenzyl and tribenzyl diphosphates diphosphates 3a and 3b3a andwas 3busedwas asused the starting as the starting material. material. In the first In thestep first, fully step, protected fully protected diphosph diphosphateate 3a was converted3a was converted into dibenzyl into dibenzylderivative derivative 3b. For selec-3b. Fortive selective monodebenzylation monodebenzylation of the terminal of the terminalphosphate phosphate residue (P residueβ), a new (P reagentβ), a new was reagent devel- wasoped, developed, N-methylmorpholineN-methylmorpholine [42]. After [the42]. short After treatment the short (15 treatment min) of the (15 3a min)/3b mixture of the 3awith/3b thismixture amine, with Bn2 thisADP amine, 3b could Bn2 ADPbe isolated3b could from be the isolated reaction from mixture. the reaction This was mixture. phos- Thisphorylated was phosphorylated with DBCP to with an electrically DBCP to an neutral electrically tetrabenzyl neutral ATP tetrabenzyl that was ATPhydrogenated that was hydrogenatedin order to remove in order the tomasking remove groups, the masking and togroups, furnish andthe final to furnish ATP as the an final acridinium ATP as salt an acridiniumwith 37% yield. salt with The 37%relatively yield. low The yield relatively of this low method yield ofwas this accounted method wasfor by accounted a rapid deg- for 0 byradation a rapid of degradation the anhydride ofthe bond anhydride in the completely bond in the masked completely 5′-triphosphate masked 5 molecule-triphosphate, since 0 molecule,adenosine since 5′-diphosphate adenosine 5was-diphosphate observed as was a by observed-product as [40 a] by-product. [40]. AnotherAnother nucleotide,nucleotide, whichwhich structurestructure waswasconfirmed confirmed byby chemicalchemical synthesis,synthesis, waswas uri-uri- 0 dinedine 5 5′-diphosphate-diphosphate (UDP).(UDP). Initially,Initially, an an attempt attempt was was made made to to use use the the same same method method as as for for ATP, but it proved to be ineffective as it resulted in large amounts of by-products, prob- Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 30 Appl. Sci. 2021, 11, 2248 5 of 28

ATP, but it proved to be ineffective as it resulted in large amounts of by-products, proba- ablybly due due to to the the more more acidic acidic nature nature of of the the uracil uracil residue residue [ [4343]].. In In this this situation, situation, Todd Todd et al. developeddeveloped aa new new method method for for the the formation formation of theof the P–O–P P–O bond–P bond system system in this in particularthis particular compound,compound, starting starting from from benzyl benzyl uridine uridine H-phosphonate H-phosphonate diester diester4 as a substrate4 as a substrate (Scheme (Scheme3)[ 44] 0 0 (preparation3) [44] (preparation of H-phosphonate of H-phosphonate4 from 4 2 from,3 -O -isopropylideneuridine2′,3′-O-isopropylideneuridine and dibenzyl and dibenzyl pyro- H-phosphonatepyro-H-phosphonate was described was described in an accompanyingin an accompanying paper, paper, also by alsoTodd by etTodd al. [ 45et ]).al.Thus, [45]). 0 nucleosideThus, nucleoside 5 -H-phosphonate 5′-H-phosphonate4 was oxidized4 was oxidized with N-chlorosuccinimide with N-chlorosuccinimide (NCS) toward (NCS) theto- correspondingward the corresponding chlorophosphate chlorophosphate5, which was allowed5, which to react was with allowed triethylammonium to react with salt tri- 0 ofethylammonium dibenzyl phosphate salt of6 .dibenzyl This led tophosphate the formation 6. This of led a uridine to the 5formation-diphosphate of a derivativeuridine 5′- withdiphosphate a fully masked derivative charge with at a thefully phosphate masked charge moieties. at the The phosphate benzyl groups moieties. were The removed benzyl bygroups heating were with removed LiCl in ethoxyethanol,by heating with while LiCl thein ethoxyethanol, isopropylidene while group, the by isopropylidene treatment with angroup, acid. by Using treatment the precipitation with an acid. technique, Using the the precipitation product UDP technique, was isolated the product as a barium UDP was salt inisolated a total as yield a barium of ca. 25%salt in [44 a]. total yield of ca. 25% [44].

SchemeScheme 3. 3. SynthesisSynthesis of uridine of uridine 5′-diphosphate 50-diphosphate (UDP) (UDP) using uridine using uridine5′-H-phosphonate 50-H-phosphonate as a sub- as astrate substrate [44]. [44].

ToTo sum up this this part, part, the the early early syntheses syntheses of ofnucleotides nucleotides developed developed by byTodd Todd et al. et fo- al. focusedcused primarily primarily on on the the preparation preparation of ofthe the appropriate appropriate di- di-or triphosphates or triphosphates of natural of natural nu- nucleosidescleosides in order in order to verify to verify the structural the structural assignment assignment of compounds of compounds isolated isolated from biolog- from biologicalical materials. materials. However, However, due to due many to manydrawbacks, drawbacks, e.g., the e.g., significant the signiﬁcant formatio formationn of side of- side-products,products, low low yields, yields, and and synthetic synthetic inconveniences, inconveniences, these these approaches approaches have have not found found widerwider applicationapplication inin thethe synthesissynthesis ofof nucleosidenucleoside di-di- andand triphosphates.triphosphates.

2.2.2.2. Synthesis ofof Nucleoside Di-Di- andand TriphosphatesTriphosphates UsingUsing CarbodiimidesCarbodiimides TheThe limitationslimitations of of the the benzyl benzyl chlorophosphate chlorophosphate strategy strategy in the insynthesis the synthesis of diphosphates of diphos- ledphate Khoranas led Khorana et al. to et the al. study to the of study carbodiimides of carbodiimides as coupling as coupling agents in agents the condensation in the conden- of mononucleotides with inorganic phosphate (Scheme4). sation of mononucleotides with inorganic phosphate (Scheme 4). Appl. Sci. 2021, 11, 2248 6 of 28 Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 30

Scheme 4. SchemeA mechanism 4. A mechanism for the formation for the formation of nucleoside of nucleoside 50-diphosphates 5′-diphosphates using dicyclohexylcarbodiimide using dicyclohexylcar- (DCC) as a condensingbodiimide agent. (DCC) as a condensing agent. 0 By using the mostBy usingcommon the carbodiimide, most common N,N carbodiimide,′-dicyclohexylcarbodiimideN,N -dicyclohexylcarbodiimide (DCC), it (DCC), was possible toit synthesize was possible a number to synthesize of di- and a number triphosphates of di- and of naturally triphosphates occurring of naturally nu- occurring cleosides (adenosine,nucleosides uridine, (adenosine, guanosine, uridine, thymidine, guanosine, and deoxycytidine thymidine,) in and isolated deoxycytidine) yields in isolated of 25–35% [46,47yields]. During of 25–35% the initial [46,47 studies]. During on the the reaction initial studiesmechanism on the using reaction pyridine mechanism using (Py) as a solvent,pyridine the formation (Py) as a ofsolvent, symmetrical the formation dinucleoside of symmetrical pyrophosphates dinucleoside was ob- pyrophosphates served. It is noteworthywas observed. that Itthe is noteworthy desired products, that the nucleoside desired products, diphosphates nucleoside, were diphosphates, not were formed when trinot-n- formedbutylamine when was tri- presentn-butylamine in the reaction was present mixture. in the This reaction suggested mixture. a cru- This suggested a cial role of the crucialprotonation role of of the DCC protonation in trigger ofing DCC this in reaction. triggering Consistent this reaction. with Consistent this, the with this, the presence of strongly basic amines in the reaction mixture prevented the condensation [48]. presence of strongly basic amines in the reaction mixture prevented the condensation [48]. An interesting observation was that using a 10-fold molar excess of phosphoric acid and An interesting observation was that using a 10-fold molar excess of phosphoric acid and a 50-molar excess of DCC over the nucleoside monophosphoate favored the formation a 50-molar excess of DCC over the nucleoside monophosphoate favored the formation of of nucleoside triphosphates over the corresponding diphosphates. For this, a credible nucleoside triphosphates over the corresponding diphosphates. For this, a credible mech- mechanistic explanation was provided [49]. Upon optimization of the method, various anistic explanation was provided [49]. Upon optimization of the method, various NTPs NTPs could be obtained in 40–80% yields, depending on the type of the mononucleotide could be obtained in 40–80% yields, depending on the type of the mononucleotide used used as a substrate [49]. as a substrate [49]. Unfortunately, the method was not free from some drawbacks. The main one was the Unfortunately, the method was not free from some drawbacks. The main one was formation of symmetrical diphosphates (inorganic and dinucleoside ones) as a result of the formation of symmetrical diphosphates (inorganic and dinucleoside ones) as a result the competing dimerization of mononucleotides and inorganic phosphate, and other di- of the competing dimerization of mononucleotides and inorganic phosphate, and other and triphosphates, which complicated the separation process. Currently, this method is di- and triphosphates,not used which very often.complicated the separation process. Currently, this method is not used very often. 2.3. Synthesis of Nucleoside Di- and Triphosphates via Phosphoramidate Intermediates 2.3. Synthesis of Nucleoside Di- and Triphosphates via Phosphoramidate Intermediates During studies on the activation of phosphate monoesters, nucleoside phosphorami- During studiesdates on emerged the activation as potential of phosphate synthetically monoesters, useful nucleoside intermediates. phosphorami- Preliminary studies by dates emergedKhorana as potential and Chambers synthetically showed useful that intermediates. phosphoramidic Preliminary acid rapidly studies hydrolyzed by to phospho- Khorana and Chambersric acid in showed an acidic that environment phosphoramidic [50]. Under acid these rapidly conditions, hydrolyzed the protonation to phos- of the amino phoric acid in anfunction acidic environment apparently transformed [50]. Under itthese into conditions, a good leaving the protonation group (ammonia), of the signiﬁcantly amino functionincreasing apparently the transformed electrophilicity it into of thea good phosphorus leaving group center. ( Thisammonia), could opensignifi- up new synthetic cantly increasingpossibilities. the electrophilicity The expected of the advantage phosphorus of the cente phosphoramidater. This could open strategy up new in the synthesis of synthetic possibilities.di- and triphosphatesThe expected comparedadvantage to of the the chlorophosphate phosphoramidate method strategy described in the above was the synthesis of di-possibility and triphosphates of using unprotectedcompared to phosphate the chlorophosphate groups, which method would described reduce the number of above was the possiblepossibility side of reactions,using unprotected mainly diphosphate and triphosphate groups, hydrolysis,which would during reduce the deprotection the number of possibleprocess. side An importantreactions, mainly point was di- alsoand tri thephosphate relatively hydrolysis, low reactivity during of phosphoramidates the deprotection process.towards An alcohols, important which point should was also alleviate the relatively the need tolow protect reactivity the 2 of0- andphos- 30-OH groups in phoramidates towardsthe ribose alcohols, ring [43 which,47]. should alleviate the need to protect the 2′- and 3′- OH groups in the riboseIn the ring initial [43, attempts47]. to synthesize di- and triphosphates by the phosphoramidate In the initialmethod, attempts an unsubstitutedto synthesize di amide- and group triphosphates P-NH2 was by used.the phosphoramidate However, the low reactivity of method, an unsubstitutedsuch compounds amide has group limited P-NH their2 was use used. to only However, a few cases, the e.g.,low ADP,reactivity ATP of and UDP [51,52]. such compoundsIn has order limited to increase their use the to reactivity only a few of phosphoramidatescases, e.g., ADP, ATP as welland asUDP their [51 solubility,52]. in organic Appl. 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In order to increaseIn order the to reactivity increase ofthe phosphoramidates reactivity of phosphoramidates as well as their as solubility well as their in or- solubility in organic solvents,ganicsolvents, derivatives solvents, derivatives of derivatives2° amines of 2◦ amines wereof 2° investigated,amines were investigated, were mainlyinvestigated, mainly phosphoromorphol- phosphoromorpholidatesmainly phosphoromorphol- and idates and phosphorimidazolides,idatesphosphorimidazolides, and phosphorimidazolides, generated generated from fromgenerated the thecorresponding corresponding from the corresponding NMPs. NMPs. NMPs. The first methodTheThe made first ﬁrst methoduse of phosphoromorpholidates mademade useuse of of phosphoromorpholidates phosphoromorpholidates of type 7 (Scheme of of type type 5)7 as (Scheme7 reac-(Scheme5) as5) reactiveas reactive intermediatestiveintermediates generatedintermediates generatedin situ generated, which in situ, werein situ which further, which were reacted were further further with reacted tetra reacted- withn-butylammo- with tetra- tetran-butylammonium-n-butylammonium salts of niummonosalts of-salts or mono- diphosphates of mono or diphosphates- or diphosphatesin pyridine in pyridine to inprovide pyridine to providenucleoside to provide nucleoside di -nucleoside or triphos- di- or di triphosphates,- or triphosphates, respectivelyphates,respectively [51 respectively], [although51], although [ 51 the], thealthough latter latter were were the obtained obtainedlatter were only only obtained in moderate moderate only yields yi inelds moderate (<60%). Further yields (<60%). Further(studies<60% studies). Further have have showed showed studies that thathave pyridinepyridine showed used usedthat pyridine asas a solvent used stimulate stimulates as a solvents equilib- equilibrium stimulates betweenequilib- rium between riumnucleosidenucleoside between triphosphatetriphosphate nucleoside (NTP) (triphosphateNTP) andand diphosphatediphosphate (NTP) and (NDP),( NDPdiphosphate), andand itsits (replacement NDPreplace-), and its by replace- DMSO ment by DMSOmentraised raised by the theDMSO yields yields raised of of NTPs NTPs the yields toto >80%>80% of (SchemeNTPs to 5>80%5))[ [53 (Scheme]].. This This method method 5) [53] of. ofThis synth synthesizing methode- of synth di- ande- sizing di- and sizingtriphosphates,triphosphates, di- and tri developeddevelopedphosphates, byby MoffattdevelopedMoffatt and and by Khorana Khorana Moffatt in inand the the earlyKhorana early 1960s, 1960s, in the was was early rather 1960s, universal was rather universalratherand and is universal stillis still in in use useand today; today; is still it works itin works use well today; well with withit aworks variety a variety well of nucleosidewithof nucleoside a variety substrates, sub-of nucleoside and is sub- also strates, and is alsostrates,used used to and make to ismake phosphonatealso phosphonateused to make mimics mimics phosphonate of the of triphosphatethe triphosphatemimics of group the group triphosphate [54– 56[54].–56] .group [54–56].

Scheme 5. SynthesisSchemeScheme of NTP 5. SynthesisSynthesis via phosphoromorpholidates, of of NTP NTP via via phosphoromorpholidates, according to Moffatt according and Khorana to Moffatt [53 and ]. Khorana [53].].

It is worth notingItIt is is thatworth in noting2013, Sun thatthat et inin al 2013,2013,. described Sun Sun et et al.a al novel. described described variant a a novel ofnovel this variant variantapproach, of of this this approach, approach, in in which the reactiveinwhich which thephosphoramidate the reactive reactive phosphoramidate phosphoramidate intermediate intermediate 8 intermediate was obtained8 was 8 fromwas obtained obtaineda suitably from from apro- suitably a suitably protected protected nucleosidetectednucleoside and nucleoside benzyl and N,N benzyl and-diisopropylchlorophosphoramidite benzylN,N-diisopropylchlorophosphoramidite N,N-diisopropylchlorophosphoramidite 9 (Scheme9 (Scheme 6). The9 (Scheme 6). The 6). phos- The phosphitylationphosphitylationphitylation was promoted was promotedwas by 1promotedH-tetraz by 1oleH by-tetrazole and 1H -thetetraz obtained andole the and obtained phosphoramidite the obtained phosphoramidite phosphoramidite inter- intermediate intermediate (structuremediate(structure not (structure shown) not shown) was not was hydrolyzed shown) hydrolyzed was to hydrolyzed to benzyl benzyl nucleoside nucleoside to benzyl H H-phosphonate - nucleosidephosphonate H - diester,phosphonate which was subjected to oxidative coupling using the CCl /Et N/piperidine system to afford phos- diester, which diesterwas subjected, which wasto oxidative subjected coupling to oxidative using coupling the CCl4 4 /Etusing33N/piperidine the CCl4/Et system3N/piperidine system to afford phosphoropiperidatetophoropiperidate afford phosphoropiperidate 8. Then, all benzylbe nzyl8. Then, groups all be (Bn(Bnnzyl andand groups Cbz)Cbz) were were(Bn and removedremoved Cbz) bywere by hydrogenation, removed by hydrogenation,hydrogenation,and and the the unprotected unprotected and phosphoramidate the phosphoramidate unprotected was phosphoramidate treated was treated with tris(tetrabutylammonium) with was tris(tetrabu- treated with tris(tetrabu- salts of tylammonium)tylammonium)phosphoric salts of phosphoric or pyrophosphoric salts ofor phosphoricpyrophosphoric acids or in pyrophosphoric the acids presence in the of presence 4,5-dicyanoimidazole acids in of the 4,5 presence-dicy- (DCI), of 4,5 which-dicyanoimidazole anoimidazole(DCI),acted aswhich a mild acted(DCI), proton as which a donormild acted proton that as catalyzed ado mildnor thatproton the catalyzed substitution donor thatthe reaction substitutioncatalyzed at the phosphorussubstitution center. This chemistry was used for the preparation of ribo- and deoxynucleoside di- and reaction at thereaction phosphorus at the center. phosphorus This chemistry center. This was chemistry used for thewas preparation used for the of preparation ribo- of ribo- triphosphates in good yields (68–81%) [57]. and deoxynucleosideand deoxynucleoside di- and triphosphates di- and triphosphatesin good yields in(6 8good–81%) yields [57]. (68–81%) [57].

SchemeScheme 6. Synthesis 6. SynthesisScheme of nucleoside of nucleoside 6. Synthesis di- anddi of- and triphosphatesnucleoside triphosphates di- via and phosphoropiperidatevia tri phosphophosphatesropiperidate via phospho as an as intermediate anropiperidate intermedi- [ 57as]. an intermediate [57]. ate [57]. Another route to nucleoside di- and triphosphates was proposed by Cramer et al., Another routewhoAnother showedto nucleoside thatroute phosphorimidazolides dito -nucleoside and triphosphates di- and10 triwas(Schemephosphates proposed7) could wasby Cramer alsoproposed be easilyet al.,by Cramer generated et byal., who showed thatwhothe phosphorimidazol reaction showed ofthat nucleoside phosphorimidazolides 10 phosphates (Schemeides 7) with 10could (Scheme carbonyldiimidazole also be 7) easilycould generatealso (CDI)be easilyd by (Scheme generate7),d and by the reaction ofthemay nucleoside reaction be useful ofphosphates intermediatesnucleoside with phosphates incarbonyldiimidazole the synthesis with carbonyldiimidazole of nucleoside (CDI) (Scheme pyrophosphates (CDI) 7), and (Scheme [58–60 7),]. and The may be useful mayinintermediates situ-generated be useful intermediatesin the phosphorimidazolides synthesis in ofthe nucleoside synthesis of type pyrophosphatesof nucleoside10 easily reacted pyrophosphates [58 with–60]. variousThe [58 phosphate–60]. The nucleophiles, leading to NDP or NTP. The reaction occurred under mild conditions and Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 30

in situ-generated phosphorimidazolides of type 10 easily reacted with various phosphate nucleophiles, leading to NDP or NTP. The reaction occurred under mild conditions and did not require the use of protective groups in the nucleoside moiety. This synthetic protocol was further developed by Hoard and Ott, who applied it to the preparation of 5′-O- triphosphates of deoxynucleosides and oligodeoxynucleotides. The yields of isolated Appl. Sci. 2021, 11, 2248products reached 70%, although in some cases were as low as 20% [61]. Phosphorimidaz- 8 of 28 olides 10 intermediates were used also for the synthesis of nucleotide derivatives, with various natural and unnatural sugar residues at the Pβ phosphate moiety (Scheme 7) [62]. An effective modification of this approach was proposed by Kore and colleagues [63]. For the formationdid not require of phosphorimidazolide the use of protective 10 groups they in used the nucleosidean imidazole/tri- moiety. This synthetic phenylphosphine/2,2′protocol-dithiopyridine was further developed reagent system by Hoard (B, Scheme and Ott, 7), whowhich applied was previously it to the preparation of 0 developed by Mukaiyama5 -O-triphosphates et al. [64 of]. deoxynucleosides After the generation and of oligodeoxynucleotides. nucleotide derivative The10, in- yields of isolated organic phosphateproducts and a reachedcatalyst 70%,(zinc althoughchloride, to in increase some cases the leaving were as group low as ability) 20% [61 were]. Phosphorimida- added. The condensationzolides 10 intermediatesto NDP was usually were used complete also for within the synthesisa few hours, of nucleotide and the final derivatives, with products were variousformed naturalin very andgood unnatural yields (95 sugar–97%) residues [63]. at the Pβ phosphate moiety (Scheme7)[ 62].

SchemeScheme 7. Synthesis 7. Synthesis of various of various nucleoside nucleoside di- and di- triphosphatesand triphosphates via phosphorimidazolide via phosphorimidazolide intermediates inter- 10. mediates 10. An effective modification of this approach was proposed by Kore and colleagues [63]. For the for- Currently,mation there ofare phosphorimidazolide several methods available10 they used for anthe imidazole/triphenylphosphine/2,2 synthesis of di- and triphos- 0-dithiopyridine phates from thereagent corresponding system (B, Schemephosphate7), which monoesters, was previously differing developed in the bychoiceMukaiyama of the rea- et al. [64]. After the gent used to generategeneration phosphorimidazolide of nucleotide derivative 1010 ,[3 inorganic,65]. One phosphate of the recent and a catalystproposals (zinc is chloride, 2- to increase imidazolyl-1,3-thedimethylimidazinium leaving group ability) werechloride added. [66 The] or condensation hexafluorophosphate to NDP was [ usually2] (ImIm, complete C, within a few Scheme 7), whichhours, were and both the final prepared products in were situ formedand were in very used good in yieldsthe synthesis (95–97%) of [63 NDPs,]. NTPs, symmetrical bisCurrently,-5′,5′-O-dinucleoside there are several diphosphates, methods availableand nucleoside for the di synthesis- and triphos- of di- and triphosphates with variousphates sugar from residues the corresponding attached to phosphate the terminal monoesters, phosphate. differing The advantage in the choice of of the reagent this strategy is usedthat u tonprotected generate phosphorimidazolide nucleotides (commercially10 [3,65 available]. One of sodium the recent or potassium proposals is 2-imidazolyl- salts) can be used1,3-dimethylimidazinium as starting materials, and chloride the reactions [66] or hexaﬂuorophosphate can take place in an [ 2aqueous] (ImIm, C, Scheme7), medium. The yieldswhich of were this bothmethod prepared are 20– in60%. situ and were used in the synthesis of NDPs, NTPs, sym- 0 0 To sum up,metrical in most bis-5 of the,5 methods-O-dinucleoside based on diphosphates, the activation and of nucleosidea phosphorus di- center and triphosphates in with the form of phosphoramidates,various sugar residues the transformation attached to the of terminalthe substrate phosphate. into the The desired advantage prod- of this strategy uct (NDP or NTP)is that takes unprotected quite a long nucleotides time (in some (commercially cases even available several days), sodium and or the potassium yield salts) can be of isolated compoundsused as startingis not always materials, satisfactory. and the reactions can take place in an aqueous medium. The yields of this method are 20–60%. 2.4. Synthesis of DiphosphatesTo sum up, Using in most 5′-O- ofTosylated the methods Nucleosides based on the activation of a phosphorus center in the form of phosphoramidates, the transformation of the substrate into the desired product A completely different synthetic approach was proposed in the mid-1980s by the (NDP or NTP) takes quite a long time (in some cases even several days), and the yield of Poulter group. They developed a method for the synthesis of nucleoside 5′-diphosphates isolated compounds is not always satisfactory. based on a one-step nucleophilic substitution (SN2) of the 5′-O-tosylyl group in nucleosides 11 in acetonitrile2.4. Synthesis by tetrabutylammonium of Diphosphates Using salt 50-O-Tosylated of pyrophosphate Nucleosides (Scheme 8). This method allowed the synthesis of different NDPs in a yield range of 43% to 83%, depending A completely different synthetic approach was proposed in the mid-1980s by the Poulter group. They developed a method for the synthesis of nucleoside 50-diphosphates 0 based on a one-step nucleophilic substitution (SN2) of the 5 -O-tosylyl group in nucleosides 11 in acetonitrile by tetrabutylammonium salt of pyrophosphate (Scheme8). This method allowed the synthesis of different NDPs in a yield range of 43% to 83%, depending on the type of a nucleoside used. It was also successful in the preparation of 50-diphosphate analogs containing a methylene group in the bridging position of the anhydride bond [67]. Appl. Sci. 2021,, 11,, xx FORFOR PEERPEER REVIEWREVIEW 9 of 30

Appl. Sci. 2021, 11, 2248 on the type of a nucleoside used. It was also successful in the preparation of 5′-diphos-9 of 28 phate analogs containing a methylene group in the bridging position of the anhydride bond [67].

SchemeScheme 8. 8.The The Poulter’sPoulter’s methodmethod forfor thethe synthesissynthesis ofof nucleosidenucleoside 55′0-diphosphates-diphosphates[ [6767].].

AA signiﬁcantsignificant improvement of of the the discussed discussed strategy strategy was was made made by byHodgson Hodgson and and col- collaboratorslaborators [68 [68,69,69]. ].In In their their approach, approach, instead instead of of typical typical tri tri-- or or tetraammonium tetraammonium salt ofof py-pyrophosphate,rophosphate, they they used used tris{(bis(triphenylphosphoranylidene)ammonium} tris{(bis(triphenylphosphoranylidene)ammonium} pyrophosphate pyrophos- (PPN,phate Figure (PPN,1 )[ Fig68ure]. This 1) [ modiﬁcation68]. This modification alleviated a alleviated persistent problema persistent of alkylammonium problem of al- pyrophosphates:kylammonium pyrophosphates extreme hygroscopicity: extreme hygroscopicity and the formation and ofthe heavily formation water-clathrated of heavily wa- structures.ter-clathrated In contrast, structures. PPN In pyrophosphate contrast, PPN pyrophosphate is not hygroscopic is not and hy cangroscopic be easily and obtained can be ter-clathrated structures. In contrast, PPN pyrophosphate is not hygroscopic0 and can be byeasily precipitation obtained by as precipitation a dry powder. as After a dry the powder. formation After of the the formation nucleoside of 5the-diphosphates, nucleoside 5′- excessdiphosphates, PPN pyrophosphate excess PPN pyrophosphate can be precipitated, can be precipitated, which greatly which facilitates greatly thefacilitates isolation the ofisolation products of [products69]. [69].

FigureFigure 1. 1.Tris{(bis(triphenylphosphoranylidene)ammonium} Tris{(bis(triphenylphosphoranylidene)ammonium} pyrophosphate pyrophosphate (PPN). (PPN).

2.5.2.5. SynthesisSynthesis ofof NucleosideNucleoside Di-Di-and and TriphosphatesTriphosphates via via Dichlorophosphate Dichlorophosphate Intermediate Intermediate LudwigLudwig andand co-workersco-workers describeddescribed anotheranother methodmethod forfor thethe synthesissynthesis ofof thetheP–O–P P–O–P bond.bond. TheyThey usedused thethe Yoshikawa’sYoshikawa’s protocolprotocol[ 70[70],], the the reaction reaction of of unprotected unprotected nucleosides nucleosides with POCl3 in trimethyl phosphate as a solvent, to generate reactive dichlorophosphate with POCl3 in trimethyl phosphate as a solvent, to generate reactive dichlorophosphate 12 intermediate. After adding tetraalkylammonium phosphate salts and hydrolysis in 12 intermediate. After adding tetraalkylammonium phosphate salts and hydrolysis in tri- triethylammonium bicarbonate (TEAB) buffer (pH 7.5), various nucleoside di- or triphos- ethylammonium bicarbonate (TEAB) buffer (pH 7.5), various nucleoside di- or triphosphates could be obtained (Scheme9)[ 71]. Ludwig’s approach was recently optimized phates could be obtained (Scheme 9) [71]. Ludwig’s approach was recently optimized us- using modern techniques to control the progress of the reaction and the application of ing modern techniques to control the progress of the reaction and the application of liquid liquid chromatography to isolate the products (NTPs, 51–74% yields) [72]. chromatography to isolate the products (NTPs, 51–74% yields) [72].

SchemeScheme 9. 9.The The synthesis synthesis of of nucleoside nucleoside 50-di- 5′-di and- and triphosphates triphosphates via dichlorophosphatevia dichlorophosphate intermediate intermedi- [71]. ate [71]. Interestingly, nucleoside 50-O-dichlorophosphates of type 12 were prepared pre- viouslyInterestingly, using tetrachloropyrophosphate nucleoside 5′-O-dichlorophosphates (P2O3Cl4) as aof phosphorylatingtype 12 were prepared agent; previ- how- 0 ever,ously they using were tetrachloropyrophosphate not converted into pyrophosphates, (P2O3Cl4) as a but phosphorylating hydrolyzed to agent nucleoside; however, 5 - monophosphatesthey were not converted [73,74]. into pyrophosphates, but hydrolyzed to nucleoside 5′-mono- phosphatesThe main [73 advantage,74]. of Ludwig’s approach lies in the use of unprotected nucleosides as substrates. Unfortunately, this method, due to a strong activation of the phosphorus center in intermediate 12, required difﬁcult-to-control conditions to give satisfactory results [72]. In order to overcome these drawbacks, Hodgson and co-workers proposed using PPN pyrophosphate (Figure1), which indeed remedied some of these problems [ 68]. Finally, Appl. Sci. 2021, 11, x FOR PEER REVIEW 10 of 30

The main advantage of Ludwig’s approach lies in the use of unprotected nucleosides

Appl. Sci. 2021, 11,as 2248 substrates. Unfortunately, this method, due to a strong activation of the phosphorus 10 of 28 center in intermediate 12, required difficult-to-control conditions to give satisfactory results [72]. In order to overcome these drawbacks, Hodgson and co-workers proposed using PPN pyrophosphate (Figure 1), which indeed remedied some of these problems [68]. Finally, replacingreplacing POCl3 with POCl PSCl3 with3 in PSClthe first3 in stage the ﬁrst of the stage synthesis of the allowed synthesis them allowed to ob- them to obtain tain various α-variousthiotriphosphateα-thiotriphosphate analogs of analogsNTPs (dAZTPαS, of NTPs (dAZTP dGTPαS,αS, UTPαS, dGTPα dTTPαS)S, UTPα S, dTTPαS) in in 26–60% yields26–60% [75,76 yields]. [75,76]. In order to obtainInorder analogs to obtainof P–CH analogs2–P diphosphates, of P–CH2–P Darzynkiewicz diphosphates, Darzynkiewicz et al. [77] used et al. [77] used the phosphorylationthe phosphorylation of nucleosides of under nucleosides Yoshikawa’s under conditions Yoshikawa’s with conditions methylenebis with methylenebis (phosphonic dichloride)(phosphonic 13 bearing dichloride) a methylene13 bearing bridge a methylene between bridge two phosphorus between two centers phosphorus centers (Scheme 10). Compound(Scheme 10 13). Compoundwas found to13 bewas more found reactive to be than more POCl reactive3, apparently than POCl due3, apparently to due to the presence of the -CH - group in the bridging position, which could not donate electrons the presence of the -CH2- group in the bridging2 position, which could not donate electrons by back donationby backand thus donation increased and thusthe electrophilicity increased the electrophilicity of the phosphorus of the center. phosphorus After center. After the generation theof trichlorodiphosphonate generation of trichlorodiphosphonate 14 and its hydrolysis14 and in its TEAB hydrolysis buffer in (pH TEAB 7.5), buffer (pH 7.5), analogs of nucleoside diphosphates (P–CH –P), which were difﬁcult to access in any other analogs of nucleoside diphosphates (P–CH2–P), which were2 difficult to access in any other way, were obtainedway, werein good obtained yields in(approx. good yields 70%) (approx.[77]. 70%) [77].

Scheme 10.SchemeSynthesis 10. of Synthesis nucleoside of pyrophosphonatenucleoside pyrophosphonate analogs with analogs the methylene with the group methylene in the bridging group in position, the developed by Darzynkiewiczbridging et position, al. [77]. developed by Darzynkiewicz et al. [77].

2.6. Synthesis of2.6. Nucleoside Synthesis Di of- a Nucleosidend Triphosphates Di- and via Triphosphates Cyclic Phosphite via Cyclic Triester Phosphite Intermediates Triester Intermediates The previouslyThe discussed previously methods discussed for di- methodsand triphosphate for di- and synthesis triphosphate were based synthesis on were based the use of reagentson the containing use of reagents the phosphorus containing atom the in phosphorus the V oxidation atom state in the (P(V) V oxidation com- state (P(V) pounds). At thecompounds). end of the 1980s, At the Ludwig end of the and 1980s, Eckstein Ludwig developed and Eckstein a different developed approach a different approach for the preparationfor the of preparation nucleoside of triphosphates nucleoside triphosphates and theirs andα-thio theirs-analogs,α-thio-analogs, which was which was based based on the reactivityon the reactivity of P(III)– ofP(V) P(III)–P(V) mixed anhydrides mixed anhydrides (Scheme 11). (Scheme In this 11 method). In this, nu- method, nucleo- cleosides must sidesbe properly must beprotected properly due protected to their non due-regioselective to their non-regioselective phosphitylation phosphitylation with with salicylchlorophosphitesalicylchlorophosphite 15. In the first stage15. In of the the ﬁrst synthesis, stage of nucleoside the synthesis, cyclic nucleoside phosphite cyclic phosphite 16 is formed, which16 is formed,undergoes which a reaction undergoes with a tributylammonium reaction with tributylammonium salt of pyrophosphate salt of pyrophosphate and is transformed into mixed P(III)–P(V) “trimeta” anhydride 17, a key intermediate for Appl. Sci. 2021, 11and, x FOR is transformedPEER REVIEW into mixed P(III)–P(V) “trimeta” anhydride 17, a key intermediate for 11 of 30 the whole process.the wholeIts oxidation, process. followed Its oxidation, by hydrolysis followed and by hydrolysisdeprotection and, gives deprotection, the final gives the ﬁnal 0 product, the correspondingproduct, the correspondingnucleoside 5′-triphosphate nucleoside 5 (Scheme-triphosphate 11) [78 (Scheme]. 11)[78].

SchemeScheme 11. 11.Synthesis Synthesis ofof NTPNTP andand NTPNTPαSαS using the Ludwig and and Eckstein Eckstein method method [78 [78].].

It is postulated that the first intermediate 16 undergoes a nucleophilic attack at the phosphorus center by pyrophosphate with the departure of a carboxylate, followed by the ring’s closure as a result of an intramolecular substitution of the aryloxy group [78]. The formed mixed anhydride 17 is then oxidized with iodine/water or sulfur, leading to nucleoside cyclic triphosphates 18 or their thio derivatives. Hydrolysis of these trimetaphosphate intermediates, followed by their deprotection (under basic or acidic conditions, depending on the protecting groups used), leads to the formation of nucleoside triphosphates in the ribo- and deoxy- series (oxidation with I2/H2O system) or their α-thio-analogs (oxidation with elemental sulfur). The ring opening proceeds with full regioselectivity towards NTPs/NTPαSs without the formation of branched isomers ΨNTPs/ΨNTPαSs (Scheme 11). After DEAE-Sephadex chromatography, the products were obtained in 60– 75% yields. Due to the high reactivity of salicylchlorophosphite 15 it is important to maintain strictly anhydrous conditions to minimize the formation of undesired side-products [65]. Exploring the further synthetic possibilities of this method, Ludwig and Eckstein proposed the use of thiopyrophosphate salts (Figure 2) as a nucleophile for the reaction with cyclic intermediate 16. This, combined with the oxidation of the P(III) center by sulfur, provided access to the P,Pβ-dithio analogs of nucleoside 5′-triphosphates. The ring opening was not fully chemoselective, and the formation of ca. 15% of Pα,Pγ-dithio isomer was also observed. This by-product could be removed by selective acidic hydrolysis toward nucleoside thiodiphosphate, followed by DEAE-Sephadex chromatography [79].

Figure 2. Thiopyrophosphate used as a nucleophile in the Ludwig and Eckstein method [79].

The above-described one-pot, three-steps protocol is currently one of the most popular methods for the preparation of nucleoside 5′-triphosphates NTPs and theirs various analogs [32].

2.7. Synthesis of Nucleoside Di- and Triphosphates Using Cyclic Phosphate Triesters In 2009, Meier et al. described a new, effective method for the synthesis of nucleoside di- and triphosphates based on his earlier concept of the cycloSal group [80]. CycloSal nucleotides, developed for the purpose of the pronucleotide approach, are derivatives of Appl. Sci. 2021, 11, x FOR PEER REVIEW 11 of 30

Appl. Sci. 2021, 11, 2248 11 of 28 Scheme 11. Synthesis of NTP and NTPαS using the Ludwig and Eckstein method [78].

It is postulated that the first intermediate 16 undergoes a nucleophilic attack at the phosphorusIt is postulated center by that pyrophosphate the ﬁrst intermediate with the departure16 undergoes of a carboxylate, a nucleophilic followed attack by at thethe phosphorusring’s closure center as a result by pyrophosphate of an intramolecular with the departuresubstitution of of a carboxylate,the aryloxy followedgroup [78 by]. The the ring’sformed closure mixed as anhydride a result of 17 an is intramolecular then oxidized substitutionwith iodine/water of the or aryloxy sulfur, group leading [78 ].to Thenu- formedcleoside mixed cyclic anhydridetriphosphates17 is 18 then or their oxidized thio withderivatives. iodine/water Hydrolysis or sulfur, of these leading trimetaphos- to nucleo- sidephate cyclic intermediates, triphosphates followed18 or theirby their thio deprotection derivatives. Hydrolysis(under basic of or these acidic trimetaphosphate conditions, de- intermediates,pending on the followed protecting by their groups deprotection used), leads (under to the basic formation or acidic ofconditions, nucleoside depending triphos- onphates the protectingin the ribo- groups and deoxy used),- series leads (oxidation to the formation with I2/H of2O nucleoside system) or triphosphates their α-thio-analogs in the ribo-(oxidation and deoxy- with elemental series (oxidation sulfur). The with ring I2 /Hopening2O system) proceeds or theirwith fullα-thio-analogs regioselectivity (oxida- to- tionwards with NTPs/NTP elementalαSs sulfur). without The the ring formation opening proceeds of branched with full isomers regioselectivity ΨNTPs/Ψ towardsNTPαSs NTPs/NTP(Scheme 11).αSs After without DEAE the-Sephadex formation chromatography of branched isomers, the ΨproductsNTPs/Ψ wereNTPα obtainedSs (Scheme in 1160).– After75% yields. DEAE-Sephadex Due to the high chromatography, reactivity of salicylchlorophosphite the products were obtained 15 it is important in 60–75% to yields. main- Duetain strictly to the high anhydrous reactivity conditions of salicylchlorophosphite to minimize the formation15 it is important of undesired to maintain side-products strictly anhydrous[65]. conditions to minimize the formation of undesired side-products [65]. Exploring the the further further synthetic synthetic possibilities possibilities of ofthis this method, method, Ludwig Ludwig and andEckstein Eckstein pro- proposedposed the theuse useof thiopyrophosphate of thiopyrophosphate salts salts (Figure (Figure 2) as2 )a asnucleophile a nucleophile for the for reaction the reaction with withcyclic cyclic intermediate intermediate 16. This,16. This, combined combined with with the the oxidation oxidation of ofthe the P(III) P(III) center center by by sulfur, sulfur, 0 provided accessaccess toto the the P,P P,Pβ-dithioβ-dithio analogs analogs of of nucleoside nucleoside 5 -triphosphates.5′-triphosphates. The The ring ring opening open- wasing was not not fully fully chemoselective, chemoselective, and and the the formation formation of ca.of ca. 15% 15% of of Pα P,Pα,Pγγ-dithio-dithio isomerisomer waswas also observed.observed. This by-productby-product could be removedremoved byby selectiveselective acidicacidic hydrolysishydrolysis towardtoward nucleoside thiodiphosphate, followedfollowed byby DEAE-SephadexDEAE-Sephadex chromatographychromatography [[7979].].

Figure 2. Thiopyrophosphate used as a nucleophile in thethe LudwigLudwig andand EcksteinEckstein methodmethod [[7979].].

The above-describedabove-described one-pot, one-pot, three-steps three-steps protocol protocol is currentlyis currently one one of theof the most most popular pop- methodsular method for thes for preparation the preparation of nucleoside of nucleoside 50-triphosphates 5′-triphosphates NTPs and theirsNTPsvarious and theirs analogs various [32]. analogs [32]. 2.7. Synthesis of Nucleoside Di- and Triphosphates Using Cyclic Phosphate Triesters Appl. Sci. 2021, 11, x FOR PEER REVIEW 12 of 30 2.7. SynthesisIn 2009, Meierof Nucleoside et al. described Di- and Triphosphates a new, effective Using method Cyclic for Phosphate the synthesis Triesters of nucleoside di- andIn 2009 triphosphates, Meier et al. based described on his a new, earlier effective concept method of the forcyclo theSal synthesis group [ 80of]. nucleosideCycloSal nucleotides,di- and triphosphates developed based for theon his purpose earlier of concept the pronucleotide of the cycloSal approach, group [80 are]. Cyclo derivativesSal nu- ofcleotides,NMPs NMPs in indevelopedwhich which the the phosphatefor phosphate the purpose group group of is is theesterified esteriﬁed pronucleotide with with salicyl approach, alcohol are (or(or derivatives “saligenin”),“saligenin of”), formingforming cyclic cyclic phosphate phosphate triesters triesters of of typetype 1919(Scheme (Scheme 12 12))[ 20[20,21,21].]. InIn the the reaction reaction with with monophosphatemonophosphate or diphosphate tetrabut tetrabutylammoniumylammonium salts salts,, these these species species form form the the corre- cor- respondingsponding NDPs NDPs or or NTPs NTPs in in40 40–80%–80% yie yields.lds. The The starting starting material, material, cyclocycloSal Saltriester triester 19, can19, canbe obtained be obtained by bythe the reaction reaction of ofphosphorus phosphorus trichloride trichloride (PCl (PCl3) 3with) with 2- 2-(hydroxymethyl)-(hydroxymethyl)-4- 4-nitro(chloro)phenol,nitro(chloro)phenol, followed followed by by ox oxidativeidative condensation condensation with with a protected a protected nucleoside. nucleoside. The Thereactivity reactivity of these of these compounds compounds can canbe modulated be modulated by the by introduction the introduction of -NO of -NO2 or 2-Clor sub- -Cl substituentsstituents in inthe the aromatic aromatic ring ring,, which which,, due due to to the the electron electron-withdrawing-withdrawing effect, makesmakes thethe phosphorus center more electrophilic and therefore more susceptible to nucleophilic attack. phosphorus center more electrophilic and therefore more susceptible to nucleophilic0 at- Thetack. Meier The Meier group group also used also compoundused compoun19 ford 19 the for preparation the preparation of nucleoside of nucleoside 5 -diphosphate 5′-diphos- analogsphate analogs with various with varioussugar residues sugar residues at the terminal at the phosphorus terminal phosphorus atom [81]. atomThe method [81]. The is rathermethod straightforward, is rather straightforward although the, although yields in the some yields cases in some are moderate cases are [ 80moderate]. [80].

Scheme 12. Synthesis of nucleoside di- and triphosphates via the cycloSal approach [80]. Scheme 12. Synthesis of nucleoside di- and triphosphates via the cycloSal approach [80].

2.8. Synthesis of Nucleoside Di- and Triphosphates via Mixed P(III)–P(V) Anhydrides One of the more recently proposed method for the synthesis of nucleoside di- and triphosphates was described by Jessen et al., which uses the high reactivity of P(III)–P(V) mixed anhydrides generated in situ (Scheme 13). In the first stage, an unprotected nucleoside phosphate (NMP) is allowed to react with a slight excess (1.1–1.4 equiv.) of bis(fluorenylmethyl) phosphoramidite 20 in the presence of a tetrazole-type of activator, leading to the formation of mixed anhydride 21, which was then oxidized in situ. After the removal of the phosphate-protecting groups (β-elimination under very mild conditions), nucleoside 5′-diphosphates could be obtained in 75–93% yields as piperidinium salts. If NDP is used as a substrate, the method permits the synthesis of the corresponding nucleoside 5′-triphosphates. Interestingly, despite the known high reactivity of phosphoramidites, no phosphitylation of the 3′-OH group of nucleosides with reagent 20 was observed, probably due to the steric hindrance imparted by two fluorenylmethyl groups [82]. This approach has many advantages, including, among others, the use of unprotected NMPs, short reaction times, the ease of purification of the products, and good yields. However, as in other methods based on P(III)’s chemistry, strictly anhydrous conditions are crucial for the successful reactions, as traces of water lead to the formation of various by-products. Appl. Sci. 2021, 11, 2248 12 of 28

2.8. Synthesis of Nucleoside Di- and Triphosphates via Mixed P(III)–P(V) Anhydrides One of the more recently proposed method for the synthesis of nucleoside di- and triphosphates was described by Jessen et al., which uses the high reactivity of P(III)–P(V) mixed anhydrides generated in situ (Scheme 13). In the first stage, an unprotected nucleoside phosphate (NMP) is allowed to react with a slight excess (1.1–1.4 equiv.) of bis(fluorenylmethyl) phosphoramidite 20 in the presence of a tetrazole-type of activator, leading to the formation of mixed anhydride 21, which was then oxidized in situ. After the removal of the phosphate-protecting groups (β-elimination under very mild conditions), nucleoside 50-diphosphates could be obtained in 75–93% yields as piperidinium salts. If NDP is used as a substrate, the method permits the synthesis of the corresponding nucleoside 50-triphosphates. Interestingly, despite the known high reactivity of phosphoramidites, Appl. Sci. 2021, 11, x FOR PEER REVIEW 0 13 of 30 no phosphitylation of the 3 -OH group of nucleosides with reagent 20 was observed, probably due to the steric hindrance imparted by two fluorenylmethyl groups [82].

SchemeScheme 13.13.Synthesis Synthesis ofofnucleoside nucleoside diphosphatesdiphosphates usingusing thethe mixedmixed P(III)–P(V)P(III)–P(V) anhydrides anhydrides [ 82[82].].

2.9. SynthesisThis approach of Nucleoside has many Di- advantages, and Triphosphates including, via Phosphobetaines among others, the use of unprotected NMPs,Further short developing reaction times, the phosphoramidate the ease of puriﬁcation strategy, of Borch the products, and colleagues and good proposed yields. a However, as in other methods based on P(III)’s chemistry, strictly anhydrous conditions new way to activate the phosphorus center in the form of a highly reactive pyrrolidinium are crucial for the successful reactions, as traces of water lead to the formation of various intermediate 22 (Scheme 14). The pyrrolidinium group in this betaine can be effectively by-products. substituted by phosphate nucleophiles, leading to the formation of nucleoside 5′-di- and 2.9.triphosphates Synthesis of, Nucleosideas well as Di-disubstituted and Triphosphates 5′-diphosphates via Phosphobetaines bearing sugar residues on the Pβ phosphates (Scheme 14) [83,84]. The reaction times were rather short; for example, 1 h for Further developing the phosphoramidate strategy, Borch and colleagues proposed a the reaction with carbohydrate phosphates or 10 min for the formation of nucleoside tri- new way to activate the phosphorus center in the form of a highly reactive pyrrolidinium phosphates. For carbohydrate–nucleoside diphosphates, the formation of small amounts intermediate 22 (Scheme 14). The pyrrolidinium group in this betaine can be effectively of nucleoside monophosphates was observed as a side product, presumably due to the substituted by phosphate nucleophiles, leading to the formation of nucleoside 50-di- and presence of traces of water in the reaction0 system [83]. Despite the promising good yields, triphosphates, as well as disubstituted 5 -diphosphates bearing sugar residues on the Pβ this method is not frequently used, probably due to the time-consuming and rather labo- phosphates (Scheme 14)[83,84]. The reaction times were rather short; for example, 1 h rious synthesis of the starting phosphoramidate diester 23 [32]. for the reaction with carbohydrate phosphates or 10 min for the formation of nucleoside triphosphates. For carbohydrate–nucleoside diphosphates, the formation of small amounts of nucleoside monophosphates was observed as a side product, presumably due to the presence of traces of water in the reaction system [83]. Despite the promising good yields, this method is not frequently used, probably due to the time-consuming and rather laborious synthesis of the starting phosphoramidate diester 23 [32].

Scheme 14. Synthesis of nucleoside di- and triphosphates using zwitterion intermediate, N- methylpyrrolidiniumphosphonate 22 [83,84]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 13 of 30

Scheme 13. Synthesis of nucleoside diphosphates using the mixed P(III)–P(V) anhydrides [82].

2.9. Synthesis of Nucleoside Di- and Triphosphates via Phosphobetaines Further developing the phosphoramidate strategy, Borch and colleagues proposed a new way to activate the phosphorus center in the form of a highly reactive pyrrolidinium intermediate 22 (Scheme 14). The pyrrolidinium group in this betaine can be effectively substituted by phosphate nucleophiles, leading to the formation of nucleoside 5′-di- and triphosphates, as well as disubstituted 5′-diphosphates bearing sugar residues on the Pβ phosphates (Scheme 14) [83,84]. The reaction times were rather short; for example, 1 h for the reaction with carbohydrate phosphates or 10 min for the formation of nucleoside triphosphates. For carbohydrate–nucleoside diphosphates, the formation of small amounts of nucleoside monophosphates was observed as a side product, presumably due to the Appl. Sci. 2021, 11, 2248presence of traces of water in the reaction system [83]. Despite the promising good yields, 13 of 28 this method is not frequently used, probably due to the time-consuming and rather laborious synthesis of the starting phosphoramidate diester 23 [32].

Appl. Sci. 2021, 11, x FOR PEER REVIEW 14 of 30

Scheme 14. SchemeSynthesis 14. of Synthesis nucleoside of dinucleoside and triphosphates di- and tri usingphosphates zwitterion using intermediate, zwitterionN intermediate,-methylpyrrolidiniumphosphonate N- 22 [83,84]. methylpyrrolidiniumphosphonate 22 [83,84]. AnotherAnother type of a phosphobetaine, phosphobetaine, which which potentially potentially can can be be used used in in the the synthesis synthesis of ofdi- di- and and triphosphate triphosphates,s, is nucleoside is nucleoside N-pyridiniumphosphonateN-pyridiniumphosphonate 25 (Scheme25 (Scheme 15). 15 The). Thefor- formationmation of ofsuch such compounds compounds (which (which may may be be considered considered as as a a pyridine pyridine adduct to nucleoside metaphosphate)metaphosphate) waswas postulatedpostulated byby MichelsonMichelson inin 1960s1960s duringduring studiesstudies onon thethe activationactivation ofof phosphatephosphate monoestersmonoesters [[8585],], butbut forfor manymany yearsyears theythey diddid notnot findfind aa practicalpractical applicationapplication inin thethe synthesissynthesis ofof phosphatephosphate anhydrides,anhydrides, mainlymainly duedue toto thethe lacklack ofof anan effectiveeffective methodmethod forfor thethe generationgeneration ofof this this type type of of zwitterions. zwitterions. At At the the end end of of the the 1990s, 1990s, Stawinski Stawinski and and co-workers co-work- developeders developed an efficientan efficient and and simple simple method method for for the the formation formation of of phosphobetaine phosphobetaine of of typetype 2525 fromfrom H-phosphonateH-phosphonate monoestersmonoesters 2626,, andand showedshowed itsits highhigh reactivityreactivity towardstowards differentdifferent nucleophiles,nucleophiles, e.g.,e.g., alcohols,alcohols, amines,amines, andand fluoridesfluorides [[8686––8888].]. DrawingDrawing onon thesethese findings,findings, inin 2008,2008, Sun et al. used phosphobetaine phosphobetaine 2525 forfor the the preparation preparation of of NTPs NTPs by by reacting reacting it it with with a atetrabutylammonium tetrabutylammonium salt salt of of pyrophosphate pyrophosphate (Scheme (Scheme 15).15). While While the the yields of of isolated isolated productsproducts werewere moderatemoderate (26–41%),(26–41%), thethe conversionconversion ofof H-phosphonatesH-phosphonates 2626 intointo NTPsNTPs waswas simplesimple andand rapid,rapid, asas waswas thethe purificationpurification ofof thethe productsproducts [[8989].].

SchemeScheme 15.15. SynthesisSynthesis ofof NTPsNTPs viavia NN-pyridiniumphosphonate-pyridiniumphosphonate intermediatesintermediates ofof typetype 2525 [[8989].].

TheThe same research group group took took advantage advantage of of the the ease ease of of the the in insitu situ gener generationation of ofN- Npyridiniumphosphonate-pyridiniumphosphonate 25 25andand its itshigh high susceptibility susceptibility to nucleophilic to nucleophilic substitution substitution in the in thesynthesis synthesis of symmetric of symmetric dinucleoside dinucleoside diphosphates. diphosphates. To this To end, this end,the generated the generated zwitterion zwit- terion25 was25 reactedwas reacted with 1 equiv. with 1 of equiv. water ofto waterfurnish to in furnish good yields in good (70– yields75%) ribo (70–75%)-homodinu- ribo- homodinucleotidescleotides (27a, Figure (27a 3), Figureas well3 )as as homodinucleotides well as homodinucleotides containing containing nucleosides nucleosides of known of knownantiviral antiviral activity activity (AZT and (AZT d4T and derivatives d4T derivatives 27b; Figure27b; Figure3). [90,391).] [ 90,91]

Figure 3. Homodinucleotides obtained by Sun et al. via pyridinium phosphobetaine intermediates.

Recently, Romanowska et al. described the use of orthophosphoric acid as a nucleophile in the reaction with N-pyridiniumphosphonate 25 as a simple and convenient method for the preparation of various ribo- and deoxyribonucleoside diphosphates and their analogs. The use of orthophosphoric acid instead of lipophilic phosphate salts (e.g., tetrabutylammonium salts) greatly simplified the purification process of the NDPs’ synthesis [92].

3. Anti-HIV Pronucleotides–Nucleotide Reverse-Transcriptase Inhibitors (NtRTIs) The antiviral activity of anti-HIV pronucleotides is based on the presence in its structure of 2′,3′-dideoxynucleosides (ddNs), which, due to the lack of the 3′-OH function in the dideoxynucleoside moiety, terminates the process of reverse transcription after the incorporation of a nucleotide analog into the growing viral DNA chain. This inhibits the synthesis of viral DNA and prevents virus replication (Scheme 16) [93]. Modified nucleosides are usually not biologically active as such, and must be phosphorylated in the cell to the appropriate 5’-triphosphate in order to exert a therapeutic effect [4]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 14 of 30

Another type of a phosphobetaine, which potentially can be used in the synthesis of di- and triphosphates, is nucleoside N-pyridiniumphosphonate 25 (Scheme 15). The formation of such compounds (which may be considered as a pyridine adduct to nucleoside metaphosphate) was postulated by Michelson in 1960s during studies on the activation of phosphate monoesters [85], but for many years they did not find a practical application in the synthesis of phosphate anhydrides, mainly due to the lack of an effective method for the generation of this type of zwitterions. At the end of the 1990s, Stawinski and co-workers developed an efficient and simple method for the formation of phosphobetaine of type 25 from H-phosphonate monoesters 26, and showed its high reactivity towards different nucleophiles, e.g., alcohols, amines, and fluorides [86–88]. Drawing on these findings, in 2008, Sun et al. used phosphobetaine 25 for the preparation of NTPs by reacting it with a tetrabutylammonium salt of pyrophosphate (Scheme 15). While the yields of isolated products were moderate (26–41%), the conversion of H-phosphonates 26 into NTPs was simple and rapid, as was the purification of the products [89].

Scheme 15. Synthesis of NTPs via N-pyridiniumphosphonate intermediates of type 25 [89].

The same research group took advantage of the ease of the in situ generation of N- pyridiniumphosphonate 25 and its high susceptibility to nucleophilic substitution in the synthesis of symmetric dinucleoside diphosphates. To this end, the generated zwitterion Appl. Sci. 2021, 11, 2248 25 was reacted with 1 equiv. of water to furnish in good yields (70–75%) ribo-homodinu-14 of 28 cleotides (27a, Figure 3) as well as homodinucleotides containing nucleosides of known antiviral activity (AZT and d4T derivatives 27b; Figure 3). [90,91]

FigureFigure 3.3. HomodinucleotidesHomodinucleotides obtainedobtained byby SunSun etet al.al. viavia pyridiniumpyridinium phosphobetainephosphobetaine intermediates.intermediates.

Recently,Recently, Romanowska Romanowska et et al. al. described described the the use use of orthophosphoricof orthophosphoric acid acid as a as nucleophile a nucleo- inphile the reaction in the reaction with N-pyridiniumphosphonate with N-pyridiniumphosphonate25 as a simple 25 andas convenienta simple and method convenient for the preparationmethod for of the various preparation ribo- and of deoxyribonucleosidevarious ribo- and deoxyribon diphosphatesucleoside and their diphospha analogs. Thetes useand oftheir orthophosphoric analogs. The aciduse of instead orthophosphoric of lipophilic phosphateacid instead salts of (e.g.,lipophilic tetrabutylammonium phosphate salts salts)(e.g., greatlytetrabutylammonium simplified the purification salts) greatly process simplified of the NDPs’ the purification synthesis [92 process]. of the NDPs’ synthesis [92]. 3. Anti-HIV Pronucleotides–Nucleotide Reverse-Transcriptase Inhibitors (NtRTIs) 3. AntiThe-HIV antiviral Pronucleotid activity ofes– anti-HIVNucleotide pronucleotides Reverse-Transcriptase is based on theInhibitors presence (Nt inRTI its struc-s) ture of 20,30-dideoxynucleosides (ddNs), which, due to the lack of the 30-OH function in The antiviral activity of anti-HIV pronucleotides is based on the presence in its struc- the dideoxynucleoside moiety, terminates the process of reverse transcription after the ture of 2′,3′-dideoxynucleosides (ddNs), which, due to the lack of the 3′-OH function in incorporation of a nucleotide analog into the growing viral DNA chain. This inhibits the dideoxynucleoside moiety, terminates the process of reverse transcription after the the synthesis of viral DNA and prevents virus replication (Scheme 16)[93]. Modiﬁed incorporation of a nucleotide analog into the growing viral DNA chain. This inhibits the Appl. Sci. 2021, 11, x FOR PEER REVIEWnucleosides are usually not biologically active as such, and must be phosphorylated15 in of the 30 synthesis of viral DNA and prevents virus replication (Scheme 16) [93]. Modified nucleo- cell to the appropriate 5’-triphosphate in order to exert a therapeutic effect [4]. sides are usually not biologically active as such, and must be phosphorylated in the cell to the appropriate 5’-triphosphate in order to exert a therapeutic effect [4].

SchemeScheme 16.16. AA generalgeneral mechanismmechanism for the inhibition of HIV replication via termination of of the the viral viral DNADNA chainchain synthesissynthesis catalyzedcatalyzed byby thethe viralviral reversereverse transcriptase.transcriptase.

TheThe efficacyefficacy ofof 22′,3′0,30-dideoxynucleosides-dideoxynucleosides inin antiretroviral therapytherapy is significantlysignificantly ham- peredpered byby theirtheir limitedlimited bioavailabilitybioavailability andand complexcomplex metabolicmetabolic pathwayspathways [[44].]. InIn orderorder toto increaseincrease thethe therapeutictherapeutic effecteffect ofof ddNs,ddNs, theirtheir administrationadministration inin aa phosphorylatedphosphorylated formform (i.e.,(i.e., asas nucleotides)nucleotides) seemedseemed toto bebe aa solutionsolution toto bypassbypass thethe first,first, oftenoften chimeric,chimeric, phasephase ofof thethe phosphorylationphosphorylation cascade.cascade. Unfortunately,Unfortunately, underunder physiologicalphysiological conditionsconditions nucleosidenucleoside phosphatesphosphates are are ionized, ionized, which which prevents prevents their their permeation permeation through through charged charged cell membranes. cell mem- Moreover,branes. Moreover, biological biological membranes membranes are rich are in phosphatases, rich in phosphatases, which rapidly which rapidly hydrolyze hydro- the P–O–Clyze the ester P–O linkages,–C ester linkages, leading toleading the reconstitution to the reconstitution of the parent of the nucleosides. parent nucleosides. This would This jeopardizewould jeopardize the whole the concept whole of concept improving of improving the pharmacokinetics the pharmacokinetics of a potential of drug a potential in this waydrug [ 6in,94 this–96 way]. [6,94–96]. DueDue toto thesethese limitations, limitations the, the idea idea of of pronucleotides pronucleotides was was born, born, according according to which to which antiviralantiviral nucleotides nucleotides should should be administered be administered in a formin a form able toable cross to cross the cell the membranes. cell membranes. Once Once inside the cell, the therapeutic nucleotide would be released through the use of chemical or enzymatic processes. Pronucleotides are thus prodrugs, which are not biologically active per se, but after chemical and/or enzymatic transformations, can generate a precursor of biologically active compound, the corresponding ddNMP (Scheme 1). Ini- tially, the pronucleotide approach assumed the delivery of suitably masked 2′,3′-dideoxynucleoside monophosphates, and over two decades, many monophosphate pronucleotide strategies were proposed and experimentally verified. In contrast, the concept of using protected nucleoside di- or triphosphates as possible prodrugs has long been of much less interest. This was mainly due to the fact that fully protected di- and triphosphates are unstable in the aqueous environment due to their high susceptibility to the hydrolysis of the uncharged P–O–P anhydride bond that basically excluded their use as therapeutic agents [97]. Only recently, it has emerged that partially protected pronucleotides are also apparently capable of entering the cell, as evidenced by their high antiviral activity. This opened a possibility of also exploring compounds with a di- or triphosphate skeleton as potential pronucleotides. The second part of this review will be focused on dedicated synthetic strategies that have been developed to prepare nucleoside di- and triphosphates useful in anti-HIV therapy as prodrugs (pronucleotide analogs of di- and triphosphates), along with the biological evaluation of the synthesized compounds.

3.1. Diphosphate Esters of 2′,3′-Dideoxynucleosides At the onset, the strategy of di- and triphosphate prodrugs was developed mainly for AZT derivatives, since for this particular 2′,3′-dideoxynucleoside, the second stage of Appl. Sci. 2021, 11, 2248 15 of 28

inside the cell, the therapeutic nucleotide would be released through the use of chemical or enzymatic processes. Pronucleotides are thus prodrugs, which are not biologically active per se, but after chemical and/or enzymatic transformations, can generate a precursor of biologically active compound, the corresponding ddNMP (Scheme1). Initially, the pronucleotide approach assumed the delivery of suitably masked 20,30-dideoxynucleoside monophosphates, and over two decades, many monophosphate pronucleotide strategies were proposed and experimentally veriﬁed. In contrast, the concept of using protected nucleoside di- or triphosphates as possible prodrugs has long been of much less interest. This was mainly due to the fact that fully protected di- and triphosphates are unstable in the aqueous environment due to their high susceptibility to the hydrolysis of the uncharged P–O–P anhydride bond that basically excluded their use as therapeutic agents [97]. Only recently, it has emerged that partially protected pronucleotides are also apparently capable of entering the cell, as evidenced by their high antiviral activity. This opened a possibility of also exploring compounds with a di- or triphosphate skeleton as potential pronucleotides. The second part of this review will be focused on dedicated synthetic strategies that have been developed to prepare nucleoside di- and triphosphates useful in anti-HIV therapy as prodrugs (pronucleotide analogs of di- and triphosphates), along with the biological evaluation of the synthesized compounds.

Appl. Sci. 2021, 11, x FOR PEER REVIEW3.1. Diphosphate Esters of 20,30-Dideoxynucleosides 16 of 30

At the onset, the strategy of di- and triphosphate prodrugs was developed mainly for AZT derivatives, since for this particular 20,30-dideoxynucleoside, the second stage of phosphorylationphosphorylation (conversion (conversion of of AZTMP AZTMP to AZTDP) to AZTDP) is the is most the most problematic problematic and is anda source is a ofsource the acute of the toxicity acute toxicity of the AZT of the therapy AZT therapy [7,8]. [7,8]. StudiesStudies onon diphosphatediphosphate pronucleotides date back to earlyearly 1990s1990s andand thethe worksworks ofof HostetlerHostetler andand collaborators,collaborators, who introducedintroduced lipidiclipidic moietiesmoieties atat thethe terminalterminal phosphatephosphate residueresidue ofof AZTDPAZTDP and and ddCDP, ddCDP, and and evaluated evaluated the the anti-HIV anti-HIV potential potential of such of su phospholipidch phospho- conjugateslipid conjugates (compounds (compounds of type of 28type, Scheme 28, Scheme 17)[98 17)–100 [98].–100 It should]. It should be noted, be noted, however, however, that thesethat these compounds compounds were designedwere designed as precursors as precursors of ddN of monophosphates, ddN monophosphates, not their not diphos- their phates.diphosphates. In order In to order obtain to theseobtain derivatives, these derivatives, suitable suitable phosphoromorpholidate phosphoromorpholidate derivatives de- (originallyrivatives (originally introduced introduced by Khorana by et Khorana al.) [101 ]et were al.) [ coupled101] were with coupled glycerophospholipids with glycerophos- to givepholipids diphosphates to give diphosphates of type 28. of type 28.

SchemeScheme 17.17. A postulated hydrolysis pathwayspathways forfor glycerideglyceride nucleosidenucleoside diphosphatesdiphosphates inin thethe cell.cell.

ItIt waswas arguedargued thatthat glycerideglyceride nucleosidenucleoside diphosphates,diphosphates, duedue toto thethe presencepresence ofof highlyhighly lipophiliclipophilic acylacyl groups, groups, should should easily easily penetrate penetrate the the cell cell membrane. membrane. Hostetler Hostetler et al. et predicted al. pre- andicted intracellular an intracellular metabolism metabolism of these of compounds these compounds to be, by to analogy be, by analogy to natural toglycerolipids, natural glyc- erolipids, initiated by phospholipase A or lysophospholipase. This should lead to the formation of a derivative of type 29, which then can be degraded by another enzyme (phosphodiesterase) to the corresponding ddNMP (path A, Scheme 17). Alternatively, compound 28 with the P–O–P anhydride bond could be directly hydrolyzed by cellular pyrophosphatases to generate nucleoside monophosphate and phosphatidic acid (path B, Scheme 17) [100]. Studies on the stability of the pyrophosphate anhydride bond in pyrophosphate 28 showed that path B of ddNMP formation was preferred [99]. The potential therapeutic value of the obtained diphosphates was rather disappointing, as the antiviral activity of the AZTDP derivative of type 28 (R = C15H31) in CEM cells was lower than that of AZT alone (EC50 = 7 μM vs. 0.2 μM, respectively) [100]. These studies were followed a few years later by Hong and collaborators. They reported on the synthesis of alkyl and thioalkyl glyceride ether derivatives of type 30 (Figure 4) [102]. These compounds were synthesized via nucleoside 5′-phosphoromorpholidates that were subjected to condensation with the corresponding glycerophospholipids. All the investigated compounds had a low cytotoxicity (CC50 > 200 μM), and the thioether derivative 30a showed greater anti-HIV activity (EC50 < 0.58 μM) than the ether analog 30b (EC50 = 57 μM) [102]. Appl. Sci. 2021, 11, 2248 16 of 28

initiated by phospholipase A or lysophospholipase. This should lead to the formation of a derivative of type 29, which then can be degraded by another enzyme (phosphodiesterase) to the corresponding ddNMP (path A, Scheme 17). Alternatively, compound 28 with the P–O–P anhydride bond could be directly hydrolyzed by cellular pyrophosphatases to generate nucleoside monophosphate and phosphatidic acid (path B, Scheme 17)[100]. Studies on the stability of the pyrophosphate anhydride bond in pyrophosphate 28 showed that path B of ddNMP formation was preferred [99]. The potential therapeutic value of the obtained diphosphates was rather disappointing, as the antiviral activity of the AZTDP derivative of type 28 (R = C15H31) in CEM cells was lower than that of AZT alone (EC50 = 7 µM vs. 0.2 µM, respectively) [100]. These studies were followed a few years later by Hong and collaborators. They reported on the synthesis of alkyl and thioalkyl glyceride ether derivatives of type 30 (Figure4 )[102]. These compounds were synthesized via nucleoside 50-phosphoromorpholidates that were Appl. Sci. 2021, 11, x FOR PEER REVIEW 17 of 30 subjected to condensation with the corresponding glycerophospholipids. All the investigated Appl. Sci. 2021, 11, x FOR PEER REVIEW µ 30a 17 of 30 compounds had a low cytotoxicity (CC50 > 200 M), and the thioether derivative showed greater anti-HIV activity (EC50 < 0.58 µM) than the ether analog 30b (EC50 = 57 µM) [102].

Figure 4. Lipophilic nucleoside diphosphate analogs designed by Hong et al. [102]. FigureFigure 4.4. Lipophilic nucleoside diphosphate analogsanalogs designeddesigned byby HongHong etet al.al. [[102102].]. At the same time, Huynh Dinh and collaborators designed mixed carboxylic–phosphoric anhydrides of type 31 as potential pronucleotides (Figure 5). These derivatives con- AtAt thethe samesame time, time Huynh, Huynh Dinh Dinh and and collaborators collaborators designed designed mixed mixed carboxylic–phosphoric carboxylic–phos- tain acyl groups with long alkyl chains at the terminal phosphate moiety, which should anhydridesphoric anhydrides of type of31 typeas potential 31 as potential pronucleotides pronucleotides (Figure5 (Fig). Theseure 5). derivatives These derivatives contain acylcon- provide appropriate lipophilicity, similarly to the above glycerolipid analogs [103,104]. groupstain acyl with groups long alkylwith chainslong alkyl at the chains terminal at the phosphate terminal moiety, phosphate which moiety, should providewhich should appro- Nucleotide derivatives of type 31 were synthetized from the corresponding carboxylic ac- priateprovide lipophilicity,similarly appropriate lipophilicity, to the above similarly glycerolipid to the analogs above [glycerolipid103,104]. Nucleotide analogs derivatives [103,104]. ids and nucleoside diphosphates using the carbodiimide approach for the formation of an ofNucleotide type 31 were derivatives synthetized of type from 31 the were corresponding synthetized carboxylic from the corresponding acids and nucleoside carboxylic diphos- ac- anhydride bond. phatesids and using nucleoside the carbodiimide diphosphates approach using forthethe carbodiimide formation of approach an anhydride for the bond. formation of an anhydride bond.

FigureFigure 5.5. ExamplesExamples ofof nucleosidenucleoside mono-, mono- di-,, di- and, and triphosphate triphosphate pronucleotides pronucleotides with with a structural a structural motif motif of the mixed carboxylic–phosphoric anhydrides [103,104]. ofFigure the mixed 5. Examples carboxylic–phosphoric of nucleoside mono anhydrides-, di-, and [103 triphosphate,104]. pronucleotides with a structural motif of the mixed carboxylic–phosphoric anhydrides [103,104]. DerivativesDerivatives of of type type31 31were were assumed assumed to penetrate to penetrate cell membranes cell membranes and release and ddNMP, release ddNDPddNMP, or ddNDP ddNTP or for ddNTPn = 1, for 2 or n = 3, 1, respectively, 2 or 3, respectively, as a result as a of result the expected of the expected preferential pref- Derivatives of type 31 were assumed to penetrate cell membranes and release hydrolysiserential hydrolysis of the mixed of the carboxylic–phosphoricmixed carboxylic–phosphoric acid anhydride acid anhydride bond bond (C-O-P) (C-O over-P) over the ddNMP, ddNDP or ddNTP for n = 1, 2 or 3, respectively, as a result of the expected pref- pyrophosphatethe pyrophosphate bond bond (P–O–P). (P–O– Indeed,P). Indeed, this cleavagethis cleavage order order was was conﬁrmed confirmed in both in both triethy- tri- erential hydrolysis of the mixed carboxylic–phosphoric acid anhydride bond (C-O-P) over lammoniumethylammonium acetate acetate buffer buffer at physiological at physiological pH and pH inand RPMI; in RPMI; however, however, in the in last the medium last me- the pyrophosphate bond (P–O–P). Indeed, this cleavage order was confirmed in both tri- thedium reaction the reaction was so was rapid so (trapid< (t 21/2 h) < that2 h) thethat lipophilic the lipophilic compounds compounds31 apparently 31 apparently did notdid ethylammonium acetate buffer1/2 at physiological pH and in RPMI; however, in the last me- havenot have enough enough time time to enter to enter the cellsthe cell [104s ].[104 The]. biologicalThe biological evaluation evaluation of the of activitythe activity of the of dium the reaction was so rapid (t1/2 < 2 h) that the lipophilic compounds 31 apparently did AZTthe AZT analogs analogs of 31 ofshowed 31 showed that their that ECtheir50 valuesEC50 values were ofwere the of same the ordersame asorder for the as parentfor the not have enough time to enter the cells [104]. The biological evaluation of the activity of AZTparent nucleoside, AZT nucleoside, and for and the for d4T the derivatives, d4T derivatives, even beingeven being 100 times 100 times lower lower than than that that for the AZT analogs of 31 showed that their EC50 values were of the same order as for the d4Tfor d4T nucleoside. nucleoside. parent AZT nucleoside, and for the d4T derivatives, even being 100 times lower than that TheThe strategy,strategy, basedbased onon thethe introductionintroduction ofof longlong aliphaticaliphatic chainschains intointo thethe nucleotides,nucleotides, for d4T nucleoside. waswas extendedextended toto alkoxyalkylalkoxyalkyl derivativesderivatives ofof acyclicacyclic nucleosidenucleoside phosphonatesphosphonates (ANPs)(ANPs) byby The strategy, based on the introduction of long aliphatic chains into the nucleotides, RuizRuiz etet al.,al., whowho developeddeveloped derivativesderivatives ofof typetype32 32as as antiviralantiviral agentsagents (Figure(Figure6 )[6) 105[105].].In In thethe was extended to alkoxyalkyl derivatives of acyclic nucleoside phosphonates (ANPs) by synthesis of this class of compounds, alkoxyalkyl phosphoromorpholidates were used as Ruiz et al., who developed derivatives of type 32 as antiviral agents (Figure 6) [105]. In the intermediates that were coupled with the appropriate C-phosphonates of antiviral acyclic synthesis of this class of compounds, alkoxyalkyl phosphoromorpholidates were used as nucleoside analogs, adefovir and cidofovir. intermediates that were coupled with the appropriate C-phosphonates of antiviral acyclic nucleoside analogs, adefovir and cidofovir.

Figure 6. Lipophilic acyclic nucleoside phosphonates (ANP) analogs 32 designed by Ruiz et al. [105]. Figure 6. Lipophilic acyclic nucleoside phosphonates (ANP) analogs 32 designed by Ruiz et al. [105]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 17 of 30

Figure 4. Lipophilic nucleoside diphosphate analogs designed by Hong et al. [102].

At the same time, Huynh Dinh and collaborators designed mixed carboxylic–phosphoric anhydrides of type 31 as potential pronucleotides (Figure 5). These derivatives contain acyl groups with long alkyl chains at the terminal phosphate moiety, which should provide appropriate lipophilicity, similarly to the above glycerolipid analogs [103,104]. Nucleotide derivatives of type 31 were synthetized from the corresponding carboxylic acids and nucleoside diphosphates using the carbodiimide approach for the formation of an anhydride bond.

Figure 5. Examples of nucleoside mono-, di-, and triphosphate pronucleotides with a structural motif of the mixed carboxylic–phosphoric anhydrides [103,104].

Derivatives of type 31 were assumed to penetrate cell membranes and release ddNMP, ddNDP or ddNTP for n = 1, 2 or 3, respectively, as a result of the expected preferential hydrolysis of the mixed carboxylic–phosphoric acid anhydride bond (C-O-P) over the pyrophosphate bond (P–O–P). Indeed, this cleavage order was confirmed in both triethylammonium acetate buffer at physiological pH and in RPMI; however, in the last medium the reaction was so rapid (t1/2 < 2 h) that the lipophilic compounds 31 apparently did not have enough time to enter the cells [104]. The biological evaluation of the activity of the AZT analogs of 31 showed that their EC50 values were of the same order as for the parent AZT nucleoside, and for the d4T derivatives, even being 100 times lower than that Appl. Sci. 2021, 11, 2248 for d4T nucleoside. 17 of 28 The strategy, based on the introduction of long aliphatic chains into the nucleotides, was extended to alkoxyalkyl derivatives of acyclic nucleoside phosphonates (ANPs) by Ruiz et al., who developed derivatives of type 32 as antiviral agents (Figure 6) [105]. In the synthesissynthesis ofof thisthis classclass ofof compounds,compounds, alkoxyalkylalkoxyalkyl phosphoromorpholidatesphosphoromorpholidates werewere usedused asas intermediatesintermediates thatthat werewere coupledcoupled withwith thethe appropriateappropriate C-phosphonatesC-phosphonates ofof antiviralantiviral acyclicacyclic nucleosidenucleoside analogs,analogs, adefoviradefovir andand cidofovir.cidofovir.

Appl. Sci. 2021, 11, x FOR PEER REVIEW 18 of 30

FigureFigure 6.6. LipophilicLipophilic acyclicacyclic nucleoside nucleoside phosphonates phosphonates (ANP) (ANP) analogs analogs32 32designed designed by by Ruiz Ruiz et al.et al. [105 ]. [105]. InIn vitro vitro biologicalbiological assays assays showed showed that that analoganalog 32a-HDP32a-HDP inhibitedinhibited HIV HIV replication replication much better (EC50 = 0.003 μM) than adefovir alone (EC50 = 1.3 μM); unfortunately, its cy- much better (EC50 = 0.003 µM) than adefovir alone (EC50 = 1.3 µM); unfortunately, its totoxicity in MT-2 cells was clearly higher (CC50 = 0.018 μM vs. CC50 = 157 μM), which cytotoxicity in MT-2 cells was clearly higher (CC50 = 0.018 µM vs. CC50 = 157 µM), resulted in the SI50 selectivity index for this compound being only 6 (SI50 = 121 for adefovir) which resulted in the SI50 selectivity index for this compound being only 6 (SI50 = 121 for[105 adefovir)]. Interestingly, [105]. Interestingly, adefovir esterified adefovir with esterified the HDP with group, the HDPhaving group, similar having cytotoxicity, similar 50 50 cytotoxicity,showed extremely showed high extremely activity high against activity HIV, against EC = HIV, 0.02 nM,EC50 which= 0.02 nMgave, which SI = gave3000. SICidofovir50 = 3000 . derivatives Cidofovir derivatives were tested were against tested HCMV, against HSV HCMV,-1, HSV-1,vaccinia, vaccinia, and cowpox and cowpox infec- infections.tions. Of the Of five the compounds five compounds tested tested in this in series, this series, the pyrophosphate the pyrophosphate derivative derivative was found was foundto be the to be superior the superior in one in case one. case. Derivative Derivative 32b-32b-ODEODE showedshowed the thestrongest strongest inhibition inhibition of ofHSV HSV (EC (EC50 =50 0.2= 0.2nM), nM while), while being being moderately moderately cytotoxic cytotoxic (EC (EC50 =50 9.5= 9.5μM),µM), which which resu resultedlted in inSI50 SI ≈50 500≈,000.500,000. No information No information on the on possible the possible mechanism mechanism of action of actionof this oftype this of typepronu- of pronucleotidescleotides was provided was provided by the by authors. the authors. TheThe most comprehensive comprehensive and and extensive extensive research research on on di-di- and and triphosphate triphosphate pronucleo- pronu- cleotidestides was was carried carried out out by byMeier Meier and and co co-workers.-workers. In In 2008, 2008, the the first first attempts attempts were were made toto adaptadapt the thecyclo cycloSalSal approach approach of pronucleotides of pronucleotides to diphosphate to diphosphate derivatives derivatives [106]. This[106] choice. This seemedchoice seemed justified justified considering considering the promising the promising biological biological results ofresults the cyclo of theSal-NMP cycloSal deriva--NMP tivesderivatives [21]. Unfortunately, [21]. Unfortu undernately, hydrolytic under hydrolytic conditions, conditions compounds, compounds of type 33 (Scheme of type 1833) hydrolyzed(Scheme 18) withhydrolyzed cleavage with of thecleavage P–O–P of bond the P (path–O–P Bbond), resulting (path B mainly), resulting in the mainly release in ofthe ddNMP release of rather ddNMP than rather the expected than the ddNDPexpected (path ddNDPA)[ (path106]. A Thus,) [106cyclo]. Thus,Sal-NDP cycloSal deriva--NDP tivesderivatives appeared appeared to be precursorsto be precursors of the of same the same nucleoside nucleoside monophosphates monophosphates as the as simplerthe sim- cyclopler Sal-NMPcycloSal-NMP constructs. constructs.

SchemeScheme 18.18. TheThe expectedexpected (A(A)) and and the the actual actual (B (B) hydrolysis) hydrolysis pathways pathways of ofcyclo cycloSal-NDPSal-NDP pronucleotides. pronucleotides. Due to the unfavorable course of the hydrolysis of cycloSal-NDP 33, Meier et al. developedDue to another the unfavorable pronucleotide course strategy, of the hydrolysis called DiPP ofro. cyclo InS thisal-NDP approach, 33, Meier the terminalet al. de- phosphateveloped another group of pronucleotide pronucleotide strategy,34 was protected called Di withPPro. stable In this benzyl-type approach groups,, the terminal which requiredphosphate the group action of cellular pronucleotide enzymes 34 to was release protected unmasked with phosphate. stable benzyl This- allowedtype groups, them towhich avoid required the unfavorable the action nucleophilic of cellular attackenzymes on to the release phosphorus unmasked center phosphate. (Scheme 19 This)[106 al-]. Thelowed protecting them togroups avoid containedthe unfavorable a carboxyester nucleophilic moiety attack that on was the prone phosphorus to cleavage center by cellular(Scheme carboxyesterases, 19) [106]. The protecting releasing groups the labile contained 4-hydroxybenzyl a carboxyester ester moiety in intermediate that was prone35, whichto cleavage spontaneously by cellular collapsedcarboxyesterases to diphosphate, releasing36 the. The labile second 4-hydroxybenzyl benzyl group ester could in bein- removedtermediate by 35 the, which same mechanism spontaneously or could collapsed be cleaved to diphosphate directly by phosphoesterases.36. The second benzyl This modegroup of could action be was removed analogous by the to thesame bis(4-acyloxybenzyl)-pronucleotide mechanism or could be cleaved strategydirectly proposedby phosphoesterases. This mode of action was analogous to the bis(4-acyloxybenzyl)-pronucleotide strategy proposed by Freeman et al. in 1993 to achieve the in-cell formation of ddMPs, which did not receive much attention at that time [107]. For DiPPro pronucleotides 34, it was indeed found that the desired ddNDPs were formed during incubation in cell extracts [106]. Although this route was dominant, some ddNMPs formation was also observed [108,109]. Appl. Sci. 2021, 11, 2248 18 of 28

by Freeman et al. in 1993 to achieve the in-cell formation of ddMPs, which did not receive much attention at that time [107]. For DiPPro pronucleotides 34, it was indeed found that Appl. Sci. 2021, 11, x FOR PEER REVIEW 19 of 30 the desired ddNDPs were formed during incubation in cell extracts [106]. Although this route was dominant, some ddNMPs formation was also observed [108,109].

Scheme 19.SchemeA postulated 19. A postulated mechanism mechanism for the releasing for the releasing of nucleoside of nucleoside diphosphates diphosphates from Di fromPPro Di pronucleotidesPPro [106]. pronucleotides [106]. Initially, the RC(O) acyl moieties of both benzyl-protecting groups at the Pβ center Initially,were the RC(O) identical, acyl and moieties the chain of both length benzyl of R,-protecting which correlated groups at with the thePβ center lipophilicity of R, was were identical,found and the to significantlychain length of affect R, which the hydrolyticcorrelated with properties the lipophilicity of DiPPro ofpronucleotides R, was 34. The found to significantlyanti-HIV activityaffect the of hydrolytic the d4T derivatives properties of of34 DiwasPPro similar pronucleotides or slightly 34 worse. The than that of d4T anti-HIV activityin CEM/0 of the cells;d4T derivatives however, in of TK 34- cells,was similar in which or d4Tslightly is completely worse than inactive, that of diphosphates 34 - d4T in CEM/0(R cells; = heptyl however, or phenyl) in TK retained cells, in their which activity. d4T is In completely contrast, Diinactive,PPro with dipho R =s- Me was probably phates 34 (R too= heptyl polar or to phenyl) penetrate retained the cell their membrane, activity. In and contrast, the one Di withPPro Rwith = tBu R = was Me speculated to be was probablyresistant too polar to to the penetrate second deprotection the cell membrane step (i.e.,, and36 the→ d4TDP) one with [106 R ].= t TheseBu was results, along with  speculated tothe be findingresistant that to the the second amount deprotection of undesirable step monophosphate (i.e., 36 d4TDP) formed [106]. fromThese pronucleotides 34 results, along with the finding that the amount of undesirable monophosphate formed closely correlated with the increasing chain length of the acyl residue, prompted the Meier from pronucleotides 34 closely correlated with the increasing chain length of the acyl res- group to design unsymmetrical DiPPro derivatives 34a (Figure7), which contained two idue, prompted the Meier group to design unsymmetrical DiPPro derivatives 34a (Figure different masking groups at the Pβ phosphate, differing in the length of the carbon chains 7), which contained two different masking groups at the Pβ phosphate, differing in the of the acyl residues [108,109]. The R1 group with a short aliphatic chain should undergo length of the carbon chains of the acyl residues [108,109]. The R1 group with a short ali- rapid hydrolysis in the presence of appropriate cellular enzymes, thus preventing cleavage phatic chain should undergo rapid hydrolysis in the presence of appropriate cellular en- of the P–O–P bond. In contrast, the acyl substituent in the second benzyl group, with a long zymes, thus preventing cleavage of the P–O–P bond. In contrast, the acyl substituent in the second benzylcarbon group, chain, with should a long ensure carbon the chain adequate, should lipophilicity ensure the adequate of the molecule. lipophilic- When tested under ity of the molecule.various When experimental tested under conditions, various the experimental unsymmetrical conditions, compounds the unsymmet-34a produced significantly Appl. Sci. 2021, 11rical, x FOR compounds PEER REVIEWless NMPs34a produced and more significantly NDPs in allless instances NMPs and compared more NDPs to the in previous all instances20 of symmetrical 30 DiPPro

compared to compoundsthe previous 34symmetrical. DiPPro compounds 34. The Meier group also designed bis(benzyloxybenzyl) derivatives of type 34b (Figure 7), containing in their structure electron-withdrawing and electron-donating substituents in the para position of the benzoyl residues. Stability studies in cell extract revealed that derivatives of type 34b with strong electron-withdrawing groups (e.g., CF3, CN, NO2) released only or mostly the expected ddNDP [110].

FigureFigure 7.7. StructuresStructures of the of developed the developed pronucleotides pronucleotides of DiPPro type of [ Di108PP]. ro type [108]. Studies on the therapeutical parameters of d4T derivatives of type 34 have shown that that these compounds had similar antiviral activities to the parent nucleosides [106]. Most of these analogs were effective in inhibiting HIV replication, and in some cases improved biological activity, as compared to free nucleosides, was even apparent. Moreover, a high antiviral activity was also observe in CEM/TK- cells, which supported the pronucleotide mode of action for these compounds [108]. Among the investigated compounds, the most active were analogs of type 34b [110]. For the synthesis of various types of DiPPro pronucleotides, the Meier group used a combination of P(III) and P(V) chemistries. The phosphitylating reagent 37 bearing two identical benzyl groups could be prepared relatively simply by reacting dichloro-N,N- diisopropylaminophosphoramidite with 2 equiv. of an appropriate benzyl alcohol [106,110,111]. In the case of an unsymmetrical version of reagent 37, this approach failed, and a more sophisticated protocol had to be developed. To this end, the first benzyl alcohol was reacted with PCl3 to give benzyl phosphorodichloridite intermediate, which was converted into the bis(N,N-diisopropylamidite) derivative, and subjected to the reaction with the second benzyl alcohol. The reaction of phosphoramidite 37 with tetraalkylammonium salt of a monophosphate or a diphosphate of antiviral nucleoside (e.g., d4T) resulted in the mixed P(III)–P(V) anhydride 38, the oxidation of which with tert-butyl hydroperoxide (tBuOOH) led to the desired NDP, with the masking groups at the terminal phosphate, in 31–41% yields (Scheme 20) [112,113].

Scheme 20. Synthesis of pronucleotides of the DiPPro type using the mixed anhydrides P(III)–P(V) method [106,108,110,112]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 20 of 30

Appl. Sci. 2021, 11, 2248 19 of 28

The Meier group also designed bis(benzyloxybenzyl) derivatives of type 34b (Figure7 ), containing in their structure electron-withdrawing and electron-donating substituents in the para position of the benzoyl residues. Stability studies in cell extract revealed that derivativesFigure 7. Structures of type of34b the developedwith strong pronucleotides electron-withdrawing of DiPPro type groups [108]. (e.g., CF3, CN, NO2) released only or mostly the expected ddNDP [110]. StudiesStudies onon the the therapeutical therapeutical parameters parameters of of d4T d4T derivatives derivatives of type of type34 have 34 have shown shown that thatthat thesethat these compounds compounds had similar had similar antiviral antiviral activities activities to the to parent the parent nucleosides nucleosides [106]. Most[106]. ofMost these of analogsthese analogs were effectivewere effective in inhibiting in inhibiting HIV replication, HIV replication, and in and some in cases some improved cases im- biologicalproved biological activity, activity, as compared as compared to free nucleosides, to free nucleosides, was even was apparent. even apparent. Moreover, Moreover, a high antivirala high antiviral activity activity was also was observe also observe in CEM/TK in CEM/TK- cells, which- cells, supportedwhich supported the pronucleotide the pronu- modecleotide of actionmode of for action these for compounds these compounds [108]. Among [108] the. Among investigated the investigated compounds, compounds the most, activethe most were active analogs were of analogs type 34b of [type110]. 34b [110]. ForFor thethe synthesissynthesis ofof variousvarious typestypes ofof DiDiPPPProro pronucleotides,pronucleotides, thethe MeierMeier groupgroup usedused aa combinationcombination ofof P(III)P(III) andand P(V)P(V) chemistries.chemistries. TheThe phosphitylatingphosphitylating reagentreagent 3737 bearingbearing twotwo identicalidentical benzylbenzyl groupsgroups couldcould bebe preparedprepared relativelyrelatively simply simply byby reactingreacting dichloro-dichloro-NN,,NN-- diisopropylaminophosphoramiditediisopropylaminophosphoramiditewith with 2 equiv. 2 equiv. of an appropriate of an appropriate benzyl alcohol benzyl [106 ,110 alcohol,111]. In[106 the,110 case,111 of]. anIn the unsymmetrical case of an unsymmetrical version of reagent version37 ,of this reagent approach 37, this failed, approach and a failed more, sophisticatedand a more sophisticated protocol had protocol to be developed. had to be To developed. this end, the To ﬁrst this benzyl end, the alcohol first wasbenzyl reacted alco- withhol was PCl reacted3 to give with benzyl PCl phosphorodichloridite3 to give benzyl phosphorodichloridite intermediate, which intermediate, was converted which intowas theconverted bis(N,N into-diisopropylamidite) the bis(N,N-diisopropylamidite) derivative, and subjectedderivative, to and the reactionsubjected with to the the reaction second benzylwith the alcohol. second Thebenzyl reaction alcohol. of The phosphoramidite reaction of phosphoramidite37 with tetraalkylammonium 37 with tetraalkylammo- salt of a monophosphatenium salt of a monophosphate or a diphosphate or a of diphosphate antiviral nucleoside of antiviral (e.g., nucleoside d4T) resulted (e.g., d4T) in the resulted mixed P(III)–P(V)in the mixed anhydride P(III)–P(V)38 ,anhydride the oxidation 38, the of which oxidation with oftert which-butyl with hydroperoxide tert-butyl hydroperox- (tBuOOH) ledide to(tB theuOOH) desired led to NDP, the desired with the NDP masking, with groupsthe masking at the groups terminal at the phosphate, terminal phosphate in 31–41%, yieldsin 31–41 (Scheme% yields 20 ()[Scheme112,113 20)]. [112,113].

SchemeScheme 20.20. SynthesisSynthesis ofof pronucleotidespronucleotides ofof thethe DiDiPPPProro typetype usingusing thethe mixedmixed anhydridesanhydrides P(III)–P(V)P(III)–P(V) methodmethod [[106106,,108108,,110110,,112112].].

3.2. Triphosphate Esters of 20,30-Dideoxynucleosides It has been long believed that the delivery of a triphosphate prodrug to the cell is impossible due to the high polarity of such compounds and the inherent lability of the anhydride bond in fully masked NTP. Additionally, due to susceptibility to phosphatases, it was assumed that these compounds are short-lived in vivo [109]. Despite these concerns, the Meier group, encouraged by the positive results of the DiPPro approach, synthesized triphosphate analogs, called TriPPPro pronucleotides, with a similar arrangement of masking groups on the terminal Pγ -phosphate residue as for the DiPPro derivatives, expecting their metabolism to lead directly to biologically active ddNTPs, without the involvement of cellular kinases (Scheme 21)[38,113–115]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 21 of 30

3.2. Triphosphate Esters of 2′,3′-Dideoxynucleosides It has been long believed that the delivery of a triphosphate prodrug to the cell is impossible due to the high polarity of such compounds and the inherent lability of the anhydride bond in fully masked NTP. Additionally, due to susceptibility to phosphatases, it was assumed that these compounds are short-lived in vivo [109]. Despite these concerns, the Meier group, encouraged by the positive results of the DiPPro approach, synthesized Appl. Sci. 2021, 11, 2248 triphosphate analogs, called TriPPPro pronucleotides, with a similar arrangement of 20 of 28 masking groups on the terminal Pγ -phosphate residue as for the DiPPro derivatives, expecting their metabolism to lead directly to biologically active ddNTPs, without the involvement of cellular kinases (Scheme 21) [38,113–115].

SchemeScheme 21. 21. TheThe TriPPP TriPPPro approachro approach to a kinase to- abypassing kinase-bypassing delivery of ddNTPs delivery to the of cell. ddNTPs to the cell. In the initial studies, symmetrical acyloxybenzyl-masking groups with C1–C17 alkyl, alkoxyl,In and the aminoalkyl initial studies, chains symmetricalwere used (compound acyloxybenzyl-masking of type 39a, R1 = R2, Scheme groups 21) with C1–C17 alkyl, 1 2 alkoxyl,[113–115] and. These aminoalkyl compounds chains were synthesized were used analogously (compound to ofthe type nucleoside39a,R diphospho-= R , Scheme 21) [113–115]. Thesenate analogs compounds discussed were above synthesized (Scheme 20),analogously using ddNDPs to (prepared the nucleoside via the cyclo diphosphonateSal ap- analogs dis- cussedproach, cf. above Scheme (Scheme 12) instead 20), of using ddNMPs, ddNDPs as the nucleotidic (prepared components via the cyclo [113Sal]. In approach, addi- cf. Scheme 12) insteadtion, triphosphates of ddNMPs, of type as the40 (Nucl nucleotidic = d4T) with components only one benzyl [113-].masking In addition, group, triphosphatesthe ex- of type 40 pected metabolites of compounds 39, were prepared to determine the stability under var- (Nuclious experimental = d4T) with conditions only one. Since benzyl-masking the route via a partial group, hydrolysis the expected of the corresponding metabolites of compounds 39, weredibenzyl prepared derivatives, to determine analogously the to stabilitymonobenzyl under compounds various in experimental the DiPPro series conditions. [111], Since the route viawas anot partial very effective, hydrolysis monobenzyl of the corresponding TriPPPro’s 40 were dibenzyl obtained derivatives, (albeit with analogously moderate to monobenzyl compoundsyields) by reacting in the benzyl DiPP cycloroS seriesal esters [111 with], wasddNDPs not [ very113]. effective, monobenzyl TriPPPro’s 40 were It was found that dibenzyl TriPPPros 39 (Nucl = d4T) bearing alkyl chains up to C11 obtainedwere efficiently (albeit cleaved with enzymatically moderate yields) to monobenzyl by reacting derivatives benzyl 40cyclo, andSal then esters to the with ex- ddNDPs [113]. pectedIt d4TNTPs. was found Although that theirdibenzyl anti-HIV Tri activityPPPros and39 cytotoxicity(Nucl = d4T) in CEM bearing cells were alkyl chains up to C11similar were to that efficiently of the parent cleaved nucleoside, enzymatically these compounds to retained monobenzyl their full derivativesanti-HIV po- 40, and then to thetency expected in TK- cells, d4TNTPs. where d4T was Although totally inactive their [ anti-HIV113]. These activityresults demonstrated and cytotoxicity that in CEM cells ionic nucleoside triphosphates with the appropriate lipophilic masking groups can enter were similar to that of the parent nucleoside, these compounds retained their full anti-HIV cells and deliver an active nucleotide, probably in the form of the corresponding nucleo- - potencyside triphosphate. in TK cells, where d4T was totally inactive [113]. These results demonstrated thatIn ionic follow nucleoside-up studies, Meier triphosphates et al. described with an alternative the appropriate method for lipophilic the preparation masking groups can enterof pronucleotides cells and of deliver type 39 (Scheme an active 22) nucleotide,[114,115]. A key probably reagent in inthis the method, form the of py- the corresponding nucleosiderophosphorylating triphosphate. agent of type 41, was obtained by the transesterification of diphenyl H-phosphonate [116] with the appropriate benzyl alcohol, followed by chlorination with N-chlorosuccinimideIn follow-up studies,and the reaction Meier with et al. inorganic described phosphate. an alternative Its activation method with tri- for the preparation offluoroacetic pronucleotides anhydride of and type the39 subsequent(Scheme reactions22)[114 with,115 ].N- Amethylimidazole key reagent and in this method, the pyrophosphorylatingddNMPs led to TriPPPro-nucleotides agent of typein 35–4185%, was yields. obtained by the transesterification of diphenyl H-phosphonate [116] with the appropriate benzyl alcohol, followed by chlorination with N-chlorosuccinimide and the reaction with inorganic phosphate. Its activation with trifluo- Appl. Sci. 2021, 11, x FOR PEER REVIEW 22 of 30 roacetic anhydride and the subsequent reactions with N-methylimidazole and ddNMPs led to TriPPPro-nucleotides in 35–85% yields.

SchemeScheme 22. HH-Phosphonate-Phosphonate approach approach to to the the preparation preparation of ofTri TriPPPPPPro-ro-nucleotidesnucleotides 39 [39115[]115. ].

The previous results on the highly selective conversion of unsymmetrical DiPPros into the corresponding ddNDPs encouraged Meier’s group to apply the same strategy also to TriPPPro-nucleotides. They synthetized a series of triphosphate derivatives of type 39b (Figure 8), in which one benzyl group contained an acyl with a short (C2–C6) alkyl chain, while the other benzyl group, an acyl with a long (C14–C17) alkyl chain, or triethyleneglycol linked via succinic or glutaric diester [117]. The shorter-chain acyl group was expected to be more susceptible to enzymatic hydrolysis than the longer-chain acyl group, thus providing the better pharmacokinetics of the pronucleotide. However, these expectations did not come true. Hydrolysis studies of such compounds revealed that apparently, there was no selective cleavage between the short-chain acyl ester and the long- chain acyl ester groups (in contrast to the DiPPro derivatives, vide supra), and thus unsymmetrical pronucleotides of type 39b did not have any advantage over the simpler, symmetrical TriPPPros 39a (R1 = R2) in terms of their anti-HIV activity. The same was observed also for TriPPPros 39 containing other known or potential antiviral nucleosides, e.g., AZT, AZU, ddC, ddG, ddI, carba-T, FddClU, FTC, 3TC, Abacavir, Carbovir, BVdU, which showed appreciable anti-HIV activity both in TK+ and TK- cells [114,115,118]. Appl. Sci. 2021, 11, 2248 21 of 28

The previous results on the highly selective conversion of unsymmetrical DiPPros into the corresponding ddNDPs encouraged Meier’s group to apply the same strategy also to TriPPPro-nucleotides. They synthetized a series of triphosphate derivatives of type 39b (Figure8), in which one benzyl group contained an acyl with a short (C2–C6) alkyl chain, while the other benzyl group, an acyl with a long (C14–C17) alkyl chain, or triethyleneglycol linked via succinic or glutaric diester [117]. The shorter-chain acyl group was expected to be more susceptible to enzymatic hydrolysis than the longer-chain acyl group, thus providing the better pharmacokinetics of the pronucleotide. However, these expectations did not come true. Hydrolysis studies of such compounds revealed that apparently, there was no selective cleavage between the short-chain acyl ester and the long-chain acyl ester groups (in contrast to the DiPPro derivatives, vide supra), and thus unsymmetrical pronucleotides of type 39b did not have any advantage over the simpler, symmetrical TriPPPros 39a (R1 = R2) in terms of their anti-HIV activity. The same was observed also for TriPPPros 39 containing other known or potential antiviral nucleosides, Appl. Sci. 2021, 11, x FOR PEER REVIEW e.g., AZT, AZU, ddC, ddG, ddI, carba-T, FddClU, FTC, 3TC, Abacavir,23 of Carbovir, 30 BVdU,

which showed appreciable anti-HIV activity both in TK+ and TK- cells [114,115,118].

Figure 8. ExamplesFigure 8. of Examples general structures of general of structures TriPPPro-nucleotides of TriPPPro- (nucleotides39a–e) synthesized (39a–e) bysynthesized the Meier by group the Meier [113,117 ,119]. group [113,117,119]. This approach was applied also for the preparation of the other unsymmetrical This approachTriPPPros was described applied later also in for the the text. preparation Products 39b–d of thewere other obtained unsymmetrical in 23–78% yields. Al- TriPPPros describedthough originallylater in the developed text. Products for the 39b triphosphorylation–d were obtained ofin d4T,23–78 it% was yields. used Alt- also for the syn- hough originallythesis developed of TriPPPro for prodrugs the triphosphorylation of a number of known of d4T, or potentialit was used antiviral also fornucleosides—AZT the , synthesis of TriAZU,PPP ddC,ro prodrugs ddG, ddI, of acarba number-T, FddClU, of known FTC, or potential 3TC, Abacavir, antiviral Carbovir, nucleosides BVdU,— and several AZT, AZU, ddC, ddG, ddI, carba-T, FddClU, FTC, 3TC, Abacavir, Carbovir, BVdU, and several their regioisomers and derivatives. Appreciably, most of these triphosphate pronucleotides were found to be active against HIV in both TK+ and TK- cells [114,115,118]. Although TriPPPro of type 39b apparently acted as true pronucleotides, the authors considered that their rate of conversion into the corresponding nucleoside triphosphates was too high and this compromised their anti-HIV potential. To modulate this process, specifically, the rate of the generation of monobenzyl intermediates of type 40 (Scheme 21) from 39b precursors, one or both of the acyloxybenzyl groups of symmetrical TriPP- Pro-nucleotides 39 were replaced with the alkoxycarbonyloxybenzyl ones (compounds 39c and 39d, Figure 8) [119]. It was argued that triphosphate derivatives of type 39c and 39d will still act as pronucleotides for nucleoside triphosphates (Scheme 23), but since the carbonate diesters should be less susceptible to degradation by esterases than the acyl esters, this would secure slower generation of, and provide higher cellular stability in, the monobenzyl intermediates (e.g., 40b, Scheme 23). Additionally, alkyl substituents R1–R3 of various chain lengths were used for the optimal cell membrane permeability and the kinetics of formation of d4TP in the cell. Appl. Sci. 2021, 11, 2248 22 of 28

their regioisomers and derivatives. Appreciably, most of these triphosphate pronucleotides were found to be active against HIV in both TK+ and TK- cells [114,115,118]. Although TriPPPro of type 39b apparently acted as true pronucleotides, the authors considered that their rate of conversion into the corresponding nucleoside triphosphates was too high and this compromised their anti-HIV potential. To modulate this process, speciﬁcally, the rate of the generation of monobenzyl intermediates of type 40 (Scheme 21) from 39b precursors, one or both of the acyloxybenzyl groups of symmetrical TriPPPro- nucleotides 39 were replaced with the alkoxycarbonyloxybenzyl ones (compounds 39c and 39d, Figure8)[ 119]. It was argued that triphosphate derivatives of type 39c and 39d will still act as pronucleotides for nucleoside triphosphates (Scheme 23), but since the carbonate diesters should be less susceptible to degradation by esterases than the acyl esters, this would secure slower generation of, and provide higher cellular stability in, the monobenzyl intermediates (e.g., 40b, Scheme 23). Additionally, alkyl substituents R1–R3 of various Appl. Sci. 2021, 11, x FOR PEER REVIEW 24 of 30 chain lengths were used for the optimal cell membrane permeability and the kinetics of formation of d4TP in the cell.

Scheme 23. Unsymmetrical dibenzyl prodrugs 39c/d39c/d andand theirtheir assumedassumed cellularcellular metabolismmetabolism toto d4TTPd4TTP [[119119].].

For thethe preparationpreparation of of such such unsymmetrical unsymmetrical TriPPP TrirosPPP39c/dros ,39c/d an analogous, an analogous H-phosphonate H-phos- strategyphonate to strategy that depicted to that in depicted Scheme 22 in was Scheme used [22119 was]. By used these [ means,119]. By variants these means,39e of Tri variantsPPPros were39e of also Tri prepared,PPPros were in which also prepared one of the, benzylin which groups one of was the replaced benzyl withgroups 2-cyanoethyl was replaced to enable with the2-cyanoethyl formation ofto monobenzylenable the formation intermediate of monobenzyl40b via a β-elimination intermediate process. 40b via a β-elimination process.TriPPP ro pronucleotides of type 39d had similar anti-HIV activity as d4T in nor- - mal cellsTriPPP (ECro50 pronucleotides≈ 0.2–0.3 µM) of and type also 39d in had TK similarcells (EC anti50-HIV≈ 1–3 activityµM), as conﬁrming d4T in normal their pronucleotidecells (EC50  0.2 mode–0.3 μ ofM) action. and also Interestingly, in TK- cells (EC pronucleotides50  1–3 μM),39c confirmingwere more their active pronucle- than 39dotideby mode roughly of action. an order Interestingly, of magnitude, pronucleotides both in TK +39cand were TK - morecells. active The highest than 39d activ- by + - 1 ityroughly (EC50 an= order 0.005 µofM magnitude,for TK and both 0.1 inµ TKM+for and TK TK)- cells. was foundThe highest for 39c activitywith R(EC=50 Et= 0.005 and 2 RμM= for C16 TKH33+ .and Bioassay 0.1 μM studies for TK-) were was alsofound carried for 39c out with for R the1 = Et monobenzyl and R2 = C16 triphosphatesH33. Bioassay ofstudies type 40bwere(bearing also carried C12 out and for C16 the alkyl monobenzyl chains), andtriphosphates these showed of type practically 40b (bearing the same C12 activityand C16 as alkyl that of chains), compounds and these39c. These showed clearly practically demonstrated the same that activity ionic compounds as that of com- with thepounds proper 39c lipophilic. These clearly handles demonstrated can efﬁciently that migrate ionic compounds through the with cell the membrane proper lipophilic [119]. 1 2 handlesPronucleotides can efficiently of migrate type 39c through(R = Et the or Bu;cell Rmembrane= C16H33 [119) but]. bearing other than d4T antiviralPronucleotides nucleoside of residues type 39c were (R1 also= Et prepared,or Bu; R2 and= C16 showedH33) but similarbearing or other even than slightly d4T + betterantiviral activities nucleoside against residues HIV-1 were and also HIV-2 prepared, than the and parent showed nucleosides similar or ineven both slightly TK andbet- - TKter activitiescells [118 against]. HIV-1 and HIV-2 than the parent nucleosides in both TK+ and TK- cells Despite[118]. the promising results obtained with pronucleotides TriPPPro of type 39 (bearing theDespite benzyl the groups promising derivatized results with obtained both with carboxy pronucleotides and/or carbonate TriPPP esterro of groups), type 39 (bearing the benzyl groups derivatized with both carboxy and/or carbonate ester groups), detailed analyses revealed some drawbacks of these constructs. The stability studies in enzymatic media showed that although the compounds 39 were admittedly liberated of the desired NTPs, their rapid intracellular dephosphorylation compromised their pronucleotide mode of action. Moreover, a careful scrutiny of the possible sources of the observed cytotoxicity strongly suggested that the released ddNTPs were not only substrates for the viral reverse transcriptase (RT), but also to some extent for cellular polymerases, particularly for the mitochondrial DNA polymerase γ [120]. To remedy these problems, Meier et al. developed another type of unsymmetrical triphosphate pronucleotides 43 (Figure 9) that contained only one biodegradable acyloxybenzyl group, as in the previous constructs (R = C1–C15), in combination with the simple alkyls phosphate esters of different chain length (C4, C15, C18). The aim of this was, on the one hand, to ensure the adequate lipophilicity of the pronucleotides, which would enable the efficient crossing of cell membranes, and, on the other hand, to protect the triphosphate residue against dephosphorylation by the presence of a more difficult- to-cleave γ-alkyl group. It was also expected that the presence of the γ-alkyl group would Appl. Sci. 2021, 11, 2248 23 of 28

Appl. Sci. 2021, 11, x FOR PEER REVIEW 25 of 30

detailed analyses revealed some drawbacks of these constructs. The stability studies in enzymatic media showed that although the compounds 39 were admittedly liberated of theprevent desired the NTPs,binding their of such rapid triphosphate intracellular analogs dephosphorylation by cellular polymerases, compromised but their not pronu- by the cleotidemore promiscuous mode of action. viral Moreover, RT enzyme a careful. scrutiny of the possible sources of the observed cytotoxicityFor the strongly synthesis suggested of prodrugs that the 43, released the same ddNTPs H-phosphonate were not only method substrates (Scheme for the22) vi-as ralfor reverseprodrugs transcriptase 39b–d (yields (RT), 33 but–63%) also was to some used, extent and the for expected cellular polymerases, products of their particularly cellular formetabolism, the mitochondrial γ-alkyl-NTPs DNA 44 polymerase, were obtainedγ [120 ].via β-elimination of the cyanoethyl group. The synthesizedTo remedy these pronucleotides problems, 43 Meier were et found al. developed to successfully another enter type the of cells, unsymmetrical followed by triphosphatea selective cleavage pronucleotides of the benzyl43 (Figure group.9) thatThe formed contained γ-alkyl only- oneNTPs biodegradable 44 appeared acyloxy-to be sta- benzylble in cell group, extracts, as in as the expected, previous and constructs acted as (R substrates = C1–C15), for in the combination viral RT only. with This the selectiv- simple alkylsity can phosphate form the basis esters for of differentfighting viral chain infection length (C4, in the C15, body C18). without The aim interfering of this was, with on thethe onevital hand, functions to ensure of the the cell. adequate Almost lipophilicityall pronucleotides of the 43 pronucleotides, obtained were which slightly would more enable active theagainst efﬁcient HIV crossing-1 and HIV of cell-2 than membranes, the parent and, nucleoside on the other d4T, hand, and some to protect of them the retained triphosphate their residuefull activity against in the dephosphorylation TK- cells. The γ-alkyl by the-NTPs presence 44 were of asimilarly more difﬁcult-to-cleave active in normalγ CEM/0-alkyl group.cells, while It was their also anti expected-HIV activity that the in presence TK- cells of varied the γ-alkyl dramatically group would depending prevent on thethe bind-alkyl ingchain of suchlength. triphosphate Thus, for analogsderivatives by cellular44 with polymerases, R = C4 or C11, but notno anti by the-HIV more activity promiscuous was ob- viralserved, RT while enzyme. for R = C18, the antiviral activity became very high (EC50 = 0.05 μM) [120].

FigureFigure 9.9. StructureStructure ofof triphosphatetriphosphate pronucleotides pronucleotides43 43containing containing alkyl alkyl chains chains at at the the P γPγmoiety moiety [ 120]. [120]. For the synthesis of prodrugs 43, the same H-phosphonate method (Scheme 22) as for4. Conclusions prodrugs 39b–d (yields 33–63%) was used, and the expected products of their cellular metabolism, γ-alkyl-NTPs 44, were obtained via β-elimination of the cyanoethyl group. The biological importance of phosphorylated nucleosides has been the driving force The synthesized pronucleotides 43 were found to successfully enter the cells, followed behind the chemical studies on phosphorus-containing natural products. Lord Todd’s by a selective cleavage of the benzyl group. The formed γ-alkyl-NTPs 44 appeared to seminal work in the middle of the last century, honored with the Nobel Prize in Chemistry be stable in cell extracts, as expected, and acted as substrates for the viral RT only. This in 1957, not only helped elucidate the chemical structure of first nucleoside di- and tri- selectivity can form the basis for fighting viral infection in the body without interfering with phosphates isolated from natural sources (e.g., ATP, UTP, etc.), but also was instrumental the vital functions of the cell. Almost all pronucleotides 43 obtained were slightly more in the development of the future chemical synthesis of oligonucleotide and the emergence active against HIV-1 and HIV-2 than the parent nucleoside d4T, and some of them retained of chemical biology. While most of the preparative chemical methods developed by this their full activity in the TK- cells. The γ-alkyl-NTPs 44 were similarly active in normal CEM/0group are cells, now while primarily their anti-HIVof historical activity value, in they TK- laidcells the varied foundation dramatically for nucleotide depending chem- on theistry alkyl as a chain separate length. field Thus, of phosphorus for derivatives chemistry.44 with RAfter = C4 several or C11, nodecad anti-HIVes of research, activity was the chemical synthesis of nucleoside di- and triphosphates is still not a trivial task, and the observed, while for R = C18, the antiviral activity became very high (EC50 = 0.05 µM) [120]. search for new synthesis strategies, more efficient and simpler methods, and simplified 4.purification Conclusions procedures, is still valid. In recent years, we have witnessed a growing interest inThe nucleoside biological di importance- and triphosphate of phosphorylated derivatives and nucleosides their analogs, has been for which the driving new meth- force behindods based, the inter chemical alia, studieson the activation on phosphorus-containing of P(V) compounds natural, or the products. use of the Lord reactivity Todd’s of seminalP(III) derivatives work in the, have middle been ofdeveloped the last century,and reviewed honored in this with paper. the Nobel This time, Prize the in Chem-stimuli istrycame in from 1957, extensive not only helped research elucidate on pronucleotides the chemical as structure potential of antiviral first nucleoside and anti di--tumor and triphosphatesagents. This called isolated for from novel natural structural sources variants (e.g., ATP,of the UTP, nucleoside etc.), but di also- and was tri instrumentalphosphates, inand the it developmentseems that chemistry of the future will chemicalmeet these synthesis new challenges of oligonucleotide again. and the emergence of chemical biology. While most of the preparative chemical methods developed by this groupAuthor are Contribution now primarilys: The ofcontribution historicals value, of the theyauthors laid M the.R., foundationJ.R., M.S. and for J.S. nucleotide in the preparation chem- istryof the as above a separate review are field nearly of phosphorus equal. All authors chemistry. have read After and several agreed decadesto the published of research, version the of the manuscript. chemical synthesis of nucleoside di- and triphosphates is still not a trivial task, and the Funding: Financial support from the National Centre for Research and Development, Poland, (grant LIDER/041/711/L-4/12/NCBR-2013 to J.R.) is gratefully acknowledged. Appl. Sci. 2021, 11, 2248 24 of 28

search for new synthesis strategies, more efficient and simpler methods, and simplified purification procedures, is still valid. In recent years, we have witnessed a growing interest in nucleoside di- and triphosphate derivatives and their analogs, for which new methods based, inter alia, on the activation of P(V) compounds, or the use of the reactivity of P(III) derivatives, have been developed and reviewed in this paper. This time, the stimuli came from extensive research on pronucleotides as potential antiviral and anti-tumor agents. This called for novel structural variants of the nucleoside di- and triphosphates, and it seems that chemistry will meet these new challenges again.

Author Contributions: The contributions of the authors M.R., J.R., M.S. and J.S. in the preparation of the above review are nearly equal. All authors have read and agreed to the published version of the manuscript. Funding: Financial support from the National Centre for Research and Development, Poland, (grant LIDER/041/711/L-4/12/NCBR-2013 to J.R.) is gratefully acknowledged. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We are indebted to Adam Kraszewski and Justyna Goł˛ebiewska(Institute of Bioorganic Chemistry PAS, Poznań,Poland) for their interest in this research. Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References 1. Berg, J.M.; Tymoczko, J.L.; Gatto, G.J.; Stryer, L. Biochemistry, 9th ed.; Palgrave Macmillan: London, UK, 2019. 2. Depaix, A.; Peyrottes, S.; Roy, B. One-pot synthesis of nucleotides in water medium. Phosphorus Sulfur Silicon Relat. Elem. 2018, 194, 335–336. [CrossRef] 3. Burgess, K.; Cook, D. Syntheses of Nucleoside Triphosphates. Chem. Rev. 2000, 100, 2047–2059. [CrossRef] 4. Balzarini, J. Metabolism and mechanism of antiretroviral action of purine and pyrimidine derivatives. Pharm. World Sci. 1994, 16, 113–126. [CrossRef][PubMed] 5. Arts, E.J.; Wainberg, M.A. Mechanisms of Nucleoside Analog Antiviral Activity and Resistance during Human Immunodeficiency Virus Reverse Transcription. Antimicrob. Agents Chemother. 1996, 20, 527–540. [CrossRef] 6. Leibman, K.C.; Heidelberger, C. The Metabolism of P32-labeled Ribonucletides in Tissue Slices and Cell Suspensions. J. Biol. Chem. 1955, 216, 823–830. [CrossRef] 7. Mansuri, M.M.; Hitchcock, M.J.; Buroker, R.A.; Bregman, C.L.; Ghazzouli, I.; Desiderio, J.V.; Starrett, J.E.; Sterzycki, R.Z.; Martin, J.C. Comparison of in vitro biological properties and mouse toxicities of three thymidine analogs active against human immunodeficiency virus. Antimicrob. Agents Chemother. 1990, 34, 637–641. [CrossRef][PubMed] 8. Tan, X.; Chu, C.K.; Boudinot, F.D. Development and Optimization of anti-HIV Nucleoside Analogs and Prodrugs: A Re- view of Their Cellular Pharmacology, Structure-Activity Relationships and Pharmacokinetics. Adv. Drug Deliv. Rev. 1999, 39, 117–151. [CrossRef] 9. Schultz, C. Prodrugs of biologically active phosphate esters. Bioorganic Med. Chem. 2003, 11, 885–898. [CrossRef] 10. Sommadossi, J.P.; Carlisle, R.; Zhou, Z. Cellular pharmacology of 30-azido-30-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells. Mol. Pharmacol. 1989, 36, 9–14. [PubMed] 11. McGuigan, C.; Pathirana, R.N.; Mahmood, N.; Devine, K.G.; Hay, A.J. Aryl phosphate derivatives of AZT retain activity against HIV1 in cell lines which are resistant to the action of AZT. Antivir. Res. 1992, 17, 311–321. [CrossRef] 12. Anastasi, C.; Quéléver, G.; Burlet, S.; Garino, C.; Souard, F.; Kraus, J.-L. New antiviral nucleoside prodrugs await application. Curr. Med. Chem. 2003, 10, 1825–1843. [CrossRef] 13. Wiemer, A.J.; Wiemer, D.F. Prodrugs of Phosphonates and Phosphates: Crossing the Membrane Barrier. Top. Curr. Chem. 2014, 360, 115–160. [CrossRef] 14. McGuigan, C.; Nicholls, S.R.; O’Connor, T.J.; Kinchington, D. Synthesis of Some Novel Dialkyl Phosphate Derivatives of 30-Modified Nucleosides as Potential Anti-AIDS Drugs. Antivir. Chem. Chemother. 1990, 1, 25–33. [CrossRef] 15. McGuigan, C.; O’Connor, T.J.; Nicholls, S.R.; Nickson, C.; Kinchington, D. Synthesis and anti-HIV Activity of Some Novel Substituted Dialkyl Phosphate Derivatives of AZT and ddCyd. Antivir. Chem. Chemother. 1990, 1, 355–360. [CrossRef] 16. Gosselin, G.; Girardet, J.L.; Perigaud, C.; Benzaria, S.; Lefebvre, I.; Schlienger, N.; Pompon, A.; Imbach, J.-L. New Insights Regard- ing the Potential of the Pronucleotide Approach in Antiviral Chemotherapy. Acta Biochim. Pol. 1996, 43, 195–208. [CrossRef] Appl. Sci. 2021, 11, 2248 25 of 28

17. Lefebvre, I.; Périgaud, C.; Pompon, A.; Aubertin, A.-M.; Girardet, J.-L.; Kirn, A.; Gosselin, G.; Imbach, J.-L. Mononucleoside Phosphotriester Derivatives with S-Acyl-2-thioethyl Bioreversible Phosphate-Protecting Groups: Intracellular Delivery of 30- Azido-20,30-dideoxythymidine 50-Monophosphate. J. Med. Chem. 1995, 38, 3941–3950. [CrossRef] 18. Perigaud, C.; Gosselin, G.; Lefebvre, I.; Girardet, J.-L.; Benzaria, S.; Barber, I.; Imbach, J.-L. Rational Design for Cytosolic Delivery of Nucleoside Monophosphates: “SATE” and “DTE” as Enzym-Labile Transient Phosphate Protecting Groups. Bioorg. Med. Chem. Lett. 1993, 3, 2521–2526. [CrossRef] 19. Peyrottes, S.; Egron, D.; Lefebvre, I.; Gosselin, G.; Imbach, J.-L.; Périgaud, C. SATE Pronucleotide Approaches: An Overview. Mini-Rev. Med. Chem. 2004, 4, 395–408. [CrossRef] 20. Meier, C. 2-Nucleos-50-O-yl-4H-1,3,2-benzodioxaphos-phinin-2-oxides—A New Concept for Lipophilic, Potential Prodrugs of Biologically Active Nucleoside Monophosphates. Angew. Chem. Int. Ed. 1996, 35, 70–72. [CrossRef] 21. Meier, C.; Lorey, M.; de Clercq, E.; Balzarini, J. cycloSal-20,30-dideoxy-20,30-didehydrothymidine Monophosphate (cycloSal- d4TMP): Synthesis and Antiviral Evaluation of a New d4TMP Delivery System. J. Med. Chem. 1998, 41, 1417–1427. [CrossRef] 22. Romanowska, J.; Szymańska-Michalak,A.; Boryski, J.; Stawinski, J.; Kraszewski, A.; Loddo, R.; Sanna, G.; Collu, G.; Secci, B.; la Colla, P. Aryl nucleoside H-phosphonates. Part 16: Synthesis and anti-HIV-1 activity of di-aryl nucleoside phosphotriesters. Bioorganic Med. Chem. 2009, 17, 3489–3498. [CrossRef][PubMed] 23. Kraszewski, A.; Sobkowski, M.; Stawinski, J. H-Phosphonate Chemistry in the Synthesis of Electrically Neutral and Charged Antiviral and Anticancer Pronucleotides. Front. Chem. 2020, 8, 8. [CrossRef] 24. Chang, S.-I.; Griesgraber, G.; Wagner, C.R. Comparision of the Antiviral Activity of Hydrophobic Amoni Acid Phosphoramidate Monoesters of 20,30-Dideoxyadenosine (ddA) and 30-Azido-30-Deoxythymidine (AZT). Nucleosides Nucleotides Nucleic Acids 2001, 20, 1571–1582. [CrossRef] 25. Drontle, D.P.; Wagner, C.R. Designing a Pronucleotide Stratagem: Lessons from Amino Acid Phosphoramidates of Anticancer and Antiviral Pyrimidines. Mini-Rev. Med. Chem. 2004, 4, 409–419. [CrossRef][PubMed] 26. Kim, J.; Chou, T.-F.; Griesgraber, G.W.; Wagner, C.R. Direct Measurement of Nucleoside Monophosphate Delivery from a Phos- phoramidate Pronucleotide by Stable Isotope Labeling and LC−ESI–MS/MS. Mol. Pharm. 2004, 1, 102–111. [CrossRef][PubMed] 27. McIntee, E.J.; Remmel, R.P.; Schinazi, R.F.; Abraham, T.W.; Wagner, C.R. Probing the Mechanism of Action and Decompo- sition of Amino Acid Phosphomonoester Amidates of Antiviral Nucleoside Prodrugs. J. Med. Chem. 1997, 40, 3323–3331. [CrossRef][PubMed] 28. Wagner, C.R.; Chang, S.-L.; Griesgraber, G.W.; Song, H.; Mcintee, E.J.; Zimmerman, C.L. Antiviral nucleoside drug delivery via amino acid phosphoramidates. Nucleosides Nucleotides 1999, 18, 913–919. [CrossRef] 29. Kolodziej, K.; Romanowska, J.; Stawinski, J.; Boryski, J.; D ˛abrowska, A.; Lipniacki, A.; Piasek, A.; Kraszewski, A.; Sobkowski, M. Aryl H-Phosphonates 18. Synthesis, properties, and biological activity of 20,30-dideoxynucleoside (N- heteroaryl)phosphoramidates of increased lipophilicity. Eur. J. Med. Chem. 2015, 100, 77–88. [CrossRef] 30. Romanowska, J.; Sobkowski, M.; Szymańska-Michalak,A.; Kołodziej, K.; D ˛abrowska, A.; Lipniacki, A.; Piasek, A.; Pietrusiewicz, Z.M.; Figlerowicz, M.; Guranowski, A.; et al. ArylH-Phosphonates 17: (N-Aryl)phosphoramidates of Pyrimidine Nucleoside Analogues and Their Synthesis, Selected Properties, and Anti-HIV Activity. J. Med. Chem. 2011, 54, 6482–6491. [CrossRef] 31. McGuigan, C.; Devine, K.G.; O’Connor, T.J.; Galpin, S.A.; Jeffries, D.J.; Kinchington, D. Synthesis and Evaluation of Some Novel Phosphoramidate Derivatives of 30-Azido-30-Deoxythymidine (AZT) as Anti-HIV Compounds. Antivir. Chem. Chemother. 1990, 1, 107–113. [CrossRef] 32. Hollenstein, M. Nucleoside Triphosphates—Building Blocks for the Modification of Nucleic Acids. Molecules 2012, 17, 13569–13591. [CrossRef] 33. Hou, S.; Qu, Z.; Tang, L.; Shuhua, Q.Z.H. Advances in the Synthesis of Organic Pyrophosphate. Chin. J. Org. Chem. 2014, 34, 54. [CrossRef] 34. Sherstyuk, Y.V.; Abramova, T.V. How to Form a Phosphate Anhydride Linkage in Nucleotide Derivatives. Chembiochem Eur. J. Chem. Biol. 2015, 16, 2562–2570. [CrossRef] 35. Tanaka, H. Recent Approaches to the Chemical Synthesis of Sugar Nucleoside Diphosphates. Trends Glycosci. Glycotechnol. 2015, 27, 99–110. [CrossRef] 36. Ahmadipour, S.; Miller, G.J. Recent advances in the chemical synthesis of sugar-nucleotides. Carbohydr. Res. 2017, 451, 95–109. [CrossRef] 37. Kaczynski, T.P.; Chmielewski, M.K. Mini-Review of the Phosphate Center Activation Strategy in Nucleoside Triphosphate Preparation. Mini Rev. Org. Chem. 2017, 14, 448–452. [CrossRef] 38. Camarasa, M.-J. Prodrugs of Nucleoside Triphosphates as a Sound and Challenging Approach: A Pioneering Work That Opens a New Era in the Direct Intracellular Delivery of Nucleoside Triphosphates. ChemMedChem 2018, 13, 1885–1889. [CrossRef] [PubMed] 39. Baddiley, J.; Todd, A.R. 122. Nucleotides. Part I. Muscle adenylic acid and adenosine diphosphate. J. Chem. Soc. 1947, 648–651. [CrossRef] 40. Baddiley, J.; Michelson, A.M.; Todd, A.R. Nucleotides. Part II. A Synthesis of Adenosine Triphosphate. J. Chem. Soc. 1949, 582–586. [CrossRef] 41. Baddiley, J.; Michelson, A.M.; Todd, A.R. Synthesis of Adenosine Triphosphate. Nat. Cell Biol. 1948, 161, 761–762. [CrossRef] Appl. Sci. 2021, 11, 2248 26 of 28

42. Baddiley, J.; Clark, V.M.; Michalski, J.J.; Todd, A.R. Studies on Phosphorylation. 5. the Reaction of Tertiary Bases with Esters of Phosphorous, Phosphoric, and Pyrophosphoric Acids—A New Method of Selective Debenzylation. J. Chem. Soc. 1949, 815–821. [CrossRef] 43. Anand, N.; Clark, V.M.; Hall, R.H.; Todd, A.R. Nucleotides. Part XV. A synthesis of Uridine-50 Pyrophsphte, a Breakdown Product of the Coenzyme “Uridine-diphosphateglucose”. J. Chem. Soc. 1952, 3665–3669. [CrossRef] 44. Kenner, G.W.; Todd, A.R.; Weymouth, F.J. 705. Nucleotides. Part XVII. N-Chloroamides as reagents for the chlorination of diesters of phosphorous acid. A new synthesis of uridine-5? Pyrophosphate. J. Chem. Soc. 1952, 3675–3681. [CrossRef] 45. Corby, N.S.; Kenner, G.W.; Todd, A.R. 704. Nucleotides. Part XVI. Ribonucleoside-5? phosphites. A new method for the preparation of mixed secondary phosphites. J. Chem. Soc. 1952, 16, 3669–3675. [CrossRef] 46. Khorana, G.; Todd, A.R. Studies on Phosphorylation. XI. The Reaction Between Carbodi-imides and Acid Esters of Phosphoric Acid—A New Method for the Preparation of Pyrophosphates. J. Chem. Soc. 1953, 2257–2260. [CrossRef] 47. Baddiley, J.; Hughes, N.A. The Synthesis of Nucleotide Coenzymes. In Advances in Enzymology and Related Areas of Molecular Biology; Wiley: Hoboken, NJ, USA, 2006; Volume 22, pp. 157–203. 48. Smith, M.; Moffat, J.G.; Khorana, H.G. Carbodiimides. VIII. Observations on the Reactions of Carbodiimides with Acids and Some New Applications in the Synthesis of Phosphoric Acid Esters. J. Am. Chem. Soc. 1958, 80, 6204–6212. [CrossRef] 49. Smith, M.; Khorana, H.G. Nucleoside Polyphosphates. VI. An Improved and General Method for the Synthesis of Ribo- and Deoxyribonucleoside 50-Triphosphates. J. Am. Chem. Soc. 1958, 80, 1141–1145. [CrossRef] 50. Chambers, R.W.; Khorana, H.G. Nucleoside Polyphosphates. VII. The Use of Phosphoramidic Acids in the Synthesis of Nucleoside-50 Pyrophosphates. J. Am. Chem. Soc. 1958, 80, 3749–3752. [CrossRef] 51. Moffatt, J.G.; Khorana, H.G. Nucleoside Polyphosphates. VIII. New and Improved Syntheses of Uridine Diphosphate Glucose and Flavin Adenine Dinucleotide Using Nucleoside-50-Phosphoramidates. J. Am. Chem. Soc. 1958, 80, 3756–3761. [CrossRef] 52. Clark, V.M.; Kirby, G.W.; Todd, A.R. Studies on Phosphorylation. Part XV. The Use of Phosphoramidic Esters in Acylation. A New Preparation of Adenosine-50-Pyrophsphate and Adenosine-50-Triphosphate. J. Chem. Soc. 1957, 79, 1497–1501. [CrossRef] 53. Moffatt, J.G. A General Synthesis of Nucleoside 50-Triphosphates. Can. J. Chem. 1964, 599–604. [CrossRef] 54. Hamilton, C.J.; Roberts, S.M.; Shipitsin, A. Synthesis of a Potent Inhibitor of HIV Reverse Transcriptase. Chem. Commun. 1998, 1087–1088. [CrossRef] 55. Hebei, D.; Kirk, K.L.; Kinjo, J.; Kovacs, T.; Lesiak, K.; Balzarini, J.; de Clercq, E.; Torrence, P.F. Synthesis of a Diﬂuoroethylenephos- phonate Analogue of AZT 50-Triphosphate and its Inhibition of HIV-1 Reverse Transcriptase. Bioorg. Med. Chem. Lett. 1991, 1, 357–360. [CrossRef] 56. Kosma, P. Occurrence, Synthesis and Biosynthesis of Bacterial Heptoses. Curr. Org. Chem. 2008, 12, 1021–1039. [CrossRef] 57. Sun, Q.; Gong, S.; Sun, J.; Liu, S.; Xiao, Q.; Pu, S. A P(V)–N Activation Strategy for the Synthesis of Nucleoside Polyphosphates. J. Org. Chem. 2013, 78, 8417–8426. [CrossRef][PubMed] 58. Cramer, F.; Schaller, H.; Staab, H.A. Darstellung von Imidazoliden der Phosphorsaure. Chem. Ber. 1961, 1612–1621. [CrossRef] 59. Schaller, H.; Staab, H.A.; Cramer, F. Zur Chemie der „energiereichen Phosphate”, XII. Phosphorylierungsreaktionen mit Salzen der Imidazolylphosphonate und Diimidazolylphosphinate. Eur. J. Inorg. Chem. 1961, 94, 1621–1633. [CrossRef] 60. Cramer, F.; Neunhoeffer, H. Rektionen von Adenosin-50-phosphorsaure-imidazolid—Eine neue Synthese von Adenosindiphos- phat und Flavin-adenin-dinucleotid. Chem. Ber. 1965, 95, 1664–1669. 61. Hoard, D.E.; Ott, D.G. Conversion of Mono- and Oligodeoxyribonucleotides to 50-Triphosphates1. J. Am. Chem. Soc. 1965, 87, 1785–1788. [CrossRef] 62. Cramer, F.; Neunhoeffer, H.; Scheit, K.H.; Schneider, G.; Tennigkeit, J. New Phosphorylation Reactions and Protecting Groups for Nucleotides. Angew. Chem. Int. Ed. 1962, 1, 331. [CrossRef] 63. Kore, A.R.; Parmar, G. Convenient Synthesis of Nucleoside-50-Diphosphates from the Corresponding Ribonucleoside-50- phosphoroimidazole. Synth. Commun. 2006, 36, 3393–3399. [CrossRef] 64. Mukaiyama, T.; Hashimoto, M. Phosphorylation by Oxidation-Reduction Condensation. Preparation of Active Phosphorylating Reagents. Bull. Chem. Soc. Jpn. 1971, 44, 2284. [CrossRef] 65. Roy, B.; Depaix, A.; Périgaud, C.; Peyrottes, S. Recent Trends in Nucleotide Synthesis. Chem. Rev. 2016, 116, 7854–7897. [CrossRef] 66. Tanaka, H.; Yoshimura, Y.; Jorgensen, M.R.; Cuesta-Seijo, J.A.; Hindsgaul, O. A Simple Synthesis of Sugar Nucleoside Diphos- phates by Chemical Coupling in Water. Angew. Chem. Int. Ed. 2012, 51, 11531–11534. [CrossRef] 67. Davisson, V.J.; Davis, D.R.; Dixit, V.M.; Poulter, C.D. Synthesis of nucleotide 50-diphosphates from 50-O-tosyl nucleosides. J. Org. Chem. 1987, 52, 1794–1801. [CrossRef] 68. Korhonen, H.J.; Bolt, H.L.; Vicente-Gines, L.; Perks, D.C.; Hodgson, D.R.W. PPN Pyrophosphate: A New Reagent for the Preparation of Nucleoside Triphosphates. Phosphorus Sulfur Silicon Relat. Elem. 2015, 190, 758–762. [CrossRef] 69. Korhonen, H.J.; Bolt, H.L.; Hodgson, D.R.W. A procedure for the preparation and isolation of nucleoside-50-diphosphates. Beilstein J. Org. Chem. 2015, 11, 469–472. [CrossRef][PubMed] 70. Yoshikawa, M.; Kato, T.; Takenishi, T. A novel method for phosphorylation of nucleosides to 50-nucleotides. Tetrahedron Lett. 1967, 8, 5065–5068. [CrossRef] 71. Ludwig, J. A new route to nucleoside 50-triphosphates. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131–133. [PubMed] 72. Gillerman, I.; Fischer, B. An Improved One-Pot Synthesis of Nucleoside 50-Triphosphate Analogues. Nucleosides Nucleotides Nucleic Acids 2010, 29, 245–256. [CrossRef] Appl. Sci. 2021, 11, 2248 27 of 28

73. Grunze, H.; Koransky, W. Phosphorylierung von Nucleosiden mit Dichlor-phosphorsäure-anhydrid. Angew. Chem. 1959, 71, 407. [CrossRef] 74. Koransky, W.; Grunze, H.; Münch, G. Phosphorylierung von Nucleosiden mit Pyrophosphorylchlorid. Z. Nat. B 1962, 17, 291–295. [CrossRef] 75. Arabshahi, A.; Frey, P.A. A Simplified Procedure for Synthesizing Nucleoside 1-Thiotriphosphates: dATPS, dGTPS, UTPS, and dTTPS. Biochem. Biophys. Res. Commun. 1994, 204, 150–155. [CrossRef] 76. Goody, R.S.; Isakov, H. Simple Synthesis and Separation of the Diastereomers of α-Thio Analogs of Ribo- and Deoxyribo- Di- and Triphosphates. Tetrahedron Lett. 1986, 27, 3599–3602. [CrossRef] 77. Kalek, M.; Jemielity, J.; Stepinski, J.; Stolarski, R.; Darzynkiewicz, E. A Direct Method for the Synthesis of Nucleoside 5- methylenebis(phopshonate)s from Nucleosides. Tetrahedron Lett. 2005, 46, 2417–2421. [CrossRef] 78. Ludwig, J.; Eckstein, F. Rapid and Efficient Synthesis of Nucleoside 50-O-(1-Thiotriphosphates), 50-Triphosphates and 20,30- Cyclophosphorothioates Using 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one. J. Org. Chem. 1989, 54, 631–635. [CrossRef] 79. Ludwig, J.; Eckstein, F. Synthesis of nucleoside 50-O-(1,3-dithiotriphosphates) and 50-O-(1,1-dithiotriphosphates). J. Org. Chem. 1991, 56, 1777–1783. [CrossRef] 80. Warnecke, S.; Meier, C. Synthesis of Nucleoside Di- and Triphosphates and Dinucleoside Polyphosphates with cycloSal- Nucleotides. J. Org. Chem. 2009, 74, 3024–3030. [CrossRef] 81. Wendicke, S.; Warnecke, S.; Meier, C. Efficient Synthesis of Nucleoside Diphosphate Glycopyranoses. Angew. Chem. Int. Ed. 2008, 47, 1500–1502. [CrossRef] 82. Cremosnik, G.S.; Hofer, A.; Jessen, H.J. Iterative Synthesis of Nucleoside Oligophosphates with Phosphoramidites. Angew. Chem. Int. Ed. 2013, 53, 286–289. [CrossRef] 83. Meyers, C.L.F.; Borch, R.F. A Novel Method for the Preparation of Nucleoside Diphosphates. Org. Lett. 2001, 3, 3765–3768. [CrossRef] 84. Wu, W.; Meyers, C.L.F.; Borch, R.F. A Novel Method for the Preparation of Nucleoside Triphosphates from Activated Nucleoside Phosphoramidates. Org. Lett. 2004, 6, 2257–2260. [CrossRef] 85. Michelson, A.M. Chemistry of Nucleotides. In The Chemistry of Nucleosides and Nucleotides; Academic Press, Inc.: Cambridge, MA, USA, 1963; pp. 98–152. 86. Bollmark, M.; Stawinski, J. A Facile Access to Nucleoside Phosphorofluoridate, Nucleoside Phosphorofluoridothioate, and Nucleoside Phosphorofluoridodithioate Monoesters. Tetrahedron Lett. 1996, 37, 5739–5742. [CrossRef] 87. Garegg, P.J.; Regberg, T.; Stawinski, J.; Strömberg, R. Nucleoside phosphonates: Part 7. Studies on the oxidation of nucleoside phosphonate esters. J. Chem. Soc. Perkin Trans. 1 1987, 1, 1269–1273. [CrossRef] 88. Kers, I.; Stawinski, J.; Kraszewski, A. A New Synthetic Method for the Preparation of Nucleoside Phosphoramidate Analogues with the Nitrogen Atom in Bridging Positions of the Phosphoramidate Linkage. Tetrahedron Lett. 1998, 39, 1219–1222. [CrossRef] 89. Sun, Q.; Edathil, J.P.; Wu, R.; Smidansky, E.D.; Cameron, C.E.; Peterson, B.R. One-Pot Synthesis of Nucleoside 50-Triphosphates from Nucleoside 50-H-Phosphonates. Org. Lett. 2008, 10, 1703–1706. [CrossRef] 90. Sun, Q.; Liu, S.; Sun, J.; Gong, S.-S. An H-phosphonate strategy for the synthesis of 20,30-dideoxynucleoside triphosphates and homodinucleotides. Chin. Chem. Lett. 2014, 25, 427–430. [CrossRef] 91. Sun, Q.; Liu, S.; Sun, J.; Gong, S.; Xiao, Q.; Shen, L. One-pot synthesis of symmetrical P1,P2-dinucleoside-50-diphosphates from nucleoside-50-H-phosphonates: Mechanistic insights into reaction path. Tetrahedron Lett. 2013, 54, 3842–3845. [CrossRef] 92. Rachwalak, M.; Rozniewska, M.; Golebiewska, J.; Jakubowski, T.; Sobkowski, M.; Romanowska, J. A practical synthesis of nucleoside 50-diphosphates from nucleoside 50-H-phosphonate monoesters. Synth. Commun. 2020, 50, 3836–3844. [CrossRef] 93. Tarrago-Litvak, L.; Andreola, M.L.; Fournier, M.; Nevinsky, G.A.; Parissi, V.; de Soultrait, V.R.; Litvak, S. Inhibitors of HIV-1 reverse transcriptase and integrase: Classical and emerging therapeutical approaches. Curr. Pharm. Des. 2002, 8, 125–133. [CrossRef] 94. Cohen, S.S. Commentary on the Therapeutic Use of Nucleosides and the Penetrability of Phosphorylated Compounds. Biochem. Pharmacol. 1975, 24, 1929–1932. [CrossRef] 95. Lichtenstein, J.; Barner, H.D.; Cohen, S.S. The Metabolism of Exogenously Supplied Nucleotides by Escherichia coli. J. Biol. Chem. 1960, 235, 457–465. [CrossRef] 96. Iglesias, L.E.; Lewkowicz, E.S.; Medici, R.; Bianchi, P.; Iribarren, A.M. Biocatalytic approaches applied to the synthesis of nucleoside prodrugs. Biotechnol. Adv. 2015, 33, 412–434. [CrossRef][PubMed] 97. Pradere, U.; Garnier-Amblard, E.C.; Coats, S.J.; Amblard, F.; Schinazi, R.F. Synthesis of Nucleoside Phosphate and Phosphonate Prodrugs. Chem. Rev. 2014, 114, 9154–9218. [CrossRef][PubMed] 98. Van Wijk, G.; Hostetler, K.; Schlame, M.; Bosch, H.V.D. Cytidine diphosphate diglyceride analogs of antiretroviral dideoxynucleosides: Evidence for release of dideoxynucleoside-monophosphates by phospholipid biosynthetic enzymes in rat liver subcellular fractions. Biochim. Biophys. Acta Lipids Lipid Metab. 1991, 1086, 99–105. [CrossRef] 99. Van Wijk, G.M.; Hostetler, K.Y.; Bosch, H.V.D. Lipid conjugates of antiretroviral agents: Release of antiretroviral nucleoside monophosphates by a nucleoside diphosphate diglyceride hydrolase activity from rat liver mitochondria. Biochim. Biophys. Acta Lipids Lipid Metab. 1991, 1084, 307–310. [CrossRef] 100. Hostetler, K.Y.; Stuhmiller, L.M.; Lenting, H.B.M.; van den Bosch, H.; Richman, D.D. Synthesis and Antiretroviral Activity of Phospholipid Analogs of Azidothymidine and Other Antiviral Nucleosides. J. Biol. Chem. 1990, 265, 6112–6117. [CrossRef] Appl. Sci. 2021, 11, 2248 28 of 28

101. Moffatt, J.G.; Khorana, H.G. Nucleoside Polyphosphates. X. The Synthesis and Some Reactions of Nucleoside-50- Phosphoromorpholidates and Related Compounds. Improved Methods for the Preparation of Nucleoside-50 Polyphosphates. J. Am. Chem. Soc. 1961, 649–658. [CrossRef] 102. Hong, C.I.; Nechaev, A.; Kirisits, A.J.; Vig, R.; West, C.R.; Manouilov, K.K.; Chu, C.K. Nucleoside Conjugates. 15. Synthesis and Biological Activity of Anti-HIV Nucleoside Conjugates of Ether and Thioether Phospholipids. J. Med. Chem. 1996, 39, 1771–1777. [CrossRef] 103. Bonnaffe, D.; Dupraz, B.; Ughetto-Monfrin, J.; Namane, A.; Dinh, T.H. Synthesis of acyl pyrophosphates. Application to the synthesis of nucleotide lipophilic prodrugs. Tetrahedron Lett. 1995, 36, 531–534. [CrossRef] 104. Bonnaffe, D.; Dupraz, B.; Ughetto-Monfrin, J.; Namane, A.; Henin, Y.; Dinh, T.H. Potential Lipophilic Nucleotide Prodrugs: Synthesis, Hydrolysis, and Antiretroviral Activity of AZT and d4T Acyl Nucleotides. J. Org. Chem. 1996, 61, 895–902. [CrossRef] 105. Ruiz, J.C.; Beadle, J.R.; Aldern, K.A.; Keith, K.A.; Hartline, C.B.; Kern, E.R.; Hostetler, K.Y. Synthesis and antiviral evaluation of alkoxyalkyl-phosphate conjugates of cidofovir and adefovir. Antivir. Res. 2007, 75, 87–90. [CrossRef] 106. Jessen, H.J.; Schulz, T.; Balzarini, J.; Meier, C. Bioreversible Protection of Nucleoside Diphosphates. Angew. Chem. Int. Ed. 2008, 47, 8719–8722. [CrossRef] 107. Thomson, W.; Nicholls, D.; Irwin, W.J.; Al-Mushadani, J.S.; Freeman, S.; Karpas, A.; Petrik, J.; Mahmood, N.; Hay, A.J. Synthesis, bioactivation and anti-HIV activity of the bis(4-acyloxybenzyl) and mono(4-acyloxybenzyl) esters of the 5?-monophosphate of AZT. J. Chem. Soc. Perkin Trans. 1 1993, 1, 1239–1245. [CrossRef] 108. Weinschenk, L.; Schols, D.; Balzarini, J.; Meier, C. Nucleoside Diphosphate Prodrugs: Nonsymmetric DiPPro-Nucleotides. J. Med. Chem. 2015, 58, 6114–6130. [CrossRef][PubMed] 109. Meier, C. Nucleoside diphosphate and triphosphate prodrugs—An unsolvable task? Antivir. Chem. Chemother. 2017, 25, 69–82. [CrossRef] 110. Weinschenk, L.; Gollnest, T.; Schols, D.; Balzarini, J.; Meier, C. Bis(benzoyloxybenzyl)-DiPPro Nucleoside Diphosphates of Anti-HIV Active Nucleoside Analogues. ChemMedChem 2015, 10, 891–900. [CrossRef] 111. Schulz, T.; Balzarini, J.; Meier, C. The DiPPro Approach: Synthesis, Hydrolysis, and Antiviral Activity of Lipophilic d4T Diphosphate Prodrugs. ChemMedChem 2014, 9, 762–775. [CrossRef][PubMed] 112. Pertenbreiter, F.; Balzarini, J.; Meier, C. Nucleoside Mono- and Diphosphate Prodrugs of 20,30-Dideoxyuridine and 20,30-Dideoxy- 20,30-didehydrouridine. ChemMedChem 2015, 10, 94–106. [CrossRef] 113. Gollnest, T.; de Oliveira, T.D.; Schols, D.; Balzarini, J.; Meier, C. Lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals. Nat. Commun. 2015, 6, 8716. [CrossRef][PubMed] 114. Weising, S.; Sterrenberg, V.; Schols, D.; Meier, C. Synthesis and Antiviral Evaluation of TriPPPro-AbacavirTP, TriPPProCarbovirTP and their 10,20-cis-disubstituted Analogues. ChemMedChem 2018, 13, 1771–1778. [CrossRef][PubMed] 115. Gollnest, T.; de Oliveira, T.D.; Rath, A.; Hauber, I.; Schols, D.; Balzarini, J.; Meier, C. Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues. Angew. Chem. Int. Ed. 2016, 55, 5255–5258. [CrossRef][PubMed] 116. Jankowska, J.; Sobkowski, M.; Stawinski, J.; Kraszewski, A. Studies on aryl H-phosphonates. 1. An efficient method for the preparation of deoxyribo- and ribonucleoside 30-H-phosphonate monoesters by transesterification of diphenyl H-phosphonate. Tetrahedron Lett. 1994, 35, 3355–3358. [CrossRef] 117. Zhao, C.; Jia, X.; Schols, D.; Balzarini, J.; Meier, C. γ-Non-Symmetrically Dimasked Tri PPPro Prodrugs as Potential Antiviral Agents against HIV. ChemMedChem 2021, 16, 499–512. [CrossRef][PubMed] 118. Jia, X.; Schols, D.; Meier, C. Lipophilic Triphosphate Prodrugs of Various Nucleoside Analogues. J. Med. Chem. 2020, 63, 6991–7007. [CrossRef][PubMed] 119. Jia, X.; Schols, D.; Meier, C. Anti-HIV-Active Nucleoside Triphosphate Prodrugs. J. Med. Chem. 2020, 63, 6003–6027. [CrossRef] 120. Zhao, C.L.; Weber, S.; Schols, D.; Balzarini, J.; Meier, C. Prodrugs of γ-Alkyl-modified Nucleoside Triphosphates Improved Inhibition of HIV Reverse Transcriptase. Angew. Chem. Int. Ed. 2020, 59, 22063–22071. [CrossRef]