An Efficient Synthesis of β-L-5-[(E)-2-bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3- (dioxolane-4-yl) uracil)] (L-BHDU) via Chiral Pure L-Dioxolane [001] Field of the Invention [002] The present invention relates to novel methods for synthesizing L-BDHU and related starting materials and intermediate compounds from (R)-2,2-dimethyl-1,3-dioxolane-4- carboxylate (7) in as few as 7 steps. The present invention eliminates expensive chiral purification of L-dioxolane (11), resulting in an enantiomerically enriched (ee > 99%) compound. The optically pure compound 11 was utilized to construct L-BHDU (15) and its monophosphate prodrug (POM-L-BHDU, 22). Novel methods of synthesis and novel intermediate compounds represent additional embodiments of the present invention. [003] Related Applications [004] This application claims the benefit of priority of United States provisional application serial numbers us63/621,722, filed 17 January 2024 and us63/716,343, filed 5 November 2024, the entire contents of each application in their entirety being incorporated by reference in this application. [005] Background and Overview of the Invention [006] Varicella zoster virus (VZV) is a highly contagious alpha herpesvirus that causes chickenpox (varicella) on primary infection and shingles (herpes zoster) on reactivation from latency.1 The Centers for Disease Control and Prevention (CDC) has estimated that in the U.S., there are annually approximately one million cases of zoster.2 People over the age of 50, immunocompromised persons, HIV-infected people, and organ transplant patients are at a higher risk of VZV infections.3 The VZV infection induces significant complications, it causes painful skin vesicular rash that contains abundant infectious virus. A significant problem of shingles is postherpetic neuralgia, which is long-lasting pain that persists for months or years even after the rashes healed.4 Accordingly, there is a need for effective and safe new antivirals for the treatment and management of VZV infection. However, vaccination is available for both stages of VZV infections, and adjuvant subunit vaccine is approved to prevent shingles, but these only apply for use in healthy persons.5
[007] Acyclovir (ACV), valacyclovir (VACV), and famciclovir (FCV, FIGURE 1) are approved drugs and are being used to treat VZV infections.6 However, these approved drugs are not highly effective against VZV, and large doses and long-term use are required, which usually promotes drug resistance.7 Cidofovir (CDV), a broad-spectrum antiviral, is active against VZV, but it is associated with nephrotoxicity and lacks oral administration.6 Another antiviral drug, brivudine (BVDU), is approved to treat VZV infection in Europe.8, 9 A major drawback associated with BVDU is that it metabolizes in the liver into bromovinyl-uracil (BVU).8 BVU impedes the activity of dihydropyridine dehydrogenase (DPD), which catabolizes thymidine and uracil. Thus, cancer treating patients with 5-fluorouracil (5-FU) creates a life threatening concern with the co-treatment of brivudine.10 Due to the significant adverse effects of currently approved drugs, there is an urgent need for new antivirals to treat VZV and its resistant strains. [008] For the last two decades, in search of better antiviral compounds against VZV, herpes simplex viruses 1 & 2, and Epstein Barr virus (EBV), the research group of the present inventors have been involved in the discovery of modified D and L-dioxolane-nucleos(t)ides. In these efforts, the inventors have invented, 2-(hydroxymethyl)-1,3-dioxolan-4-yl]5-vinyl uracil ( -HDVD, Figure 2)11 L 2-bromovinyl-2-(hydroxymethyl)-1,3 dioxolan uracil (L- BHDU)12, 13 with potent antiviral activity against EBV and VZV & HSV1. L-HDVD is highly active against EBV (EC50 value of 0.01 μM) and Kaposi’s sarcoma-associated herpesvirus (KSHV) (EC50 = 0.09 μM).11 L-BHDU has demonstrated an EC50 value of 0.25 μM without any cytotoxic in human foreskin fibroblasts (HFFs) up to 200 μM, with a selectivity index (SI) of >909.12 In in vivo studies, L-BHDU significantly reduces the viral load in comparison to the ACV and VACV. It is noteworthy to mention that in the metabolic studies, it was found that L-BHDU does not inhibit DPD and would have a better safety and antiviral profile than brivudine.12 [009] Recently, to increase cellular bioavailability and uptake of L-BHDU with enhanced pharmacokinetic properties, inventors has developed L-BHDU monophosphate pivaloyloxymethyl ester prodrug (POM- -BHDU-MP, FIGURE 2).14, 15 L POM-L-BHDU-MP has expressed similar antiviral activity to L-BHDU in infected cells. In contrast, POM-L- BHDU-MP was superior to L-BHDU in a VZV mouse model.16 The pharmacokinetic (PK) studies revealed that POM- -BHDU-MP had better oral absorption compared to -BHDU.14 L L
Recent conducted PK studies also revealed that POM-L-BHDU-MP releases almost equal concentration of L-BHDU in blood either via IV route or oral administration. This proves that the POM-L-BHDU-MP is orally demonstrate equally effect in compared to IV administration. Repeated assay in humanized mice showed that POM-L-BHDU-MP was effective orally given once per day at 11.3 mg/kg and higher, which indicates its potency and bioavailability in vivo.16 POM-L-BHDU-MP was evaluated as a topical treatment for VZV and HSV infections in a human skin explant model, and it was highly effective against both viruses at 0.2% formulated in cocoa butter.16 Based on these findings, POM-L-BHDU-MP was selected for the preclinical development against the VZV & HSV-1 infections. [010] To develop POM-L-BHDU-MP as a preclinical candidate, further extended biological, pharmacokinetic, and toxicological studies are required, which demands a significant amount of POM-L-BHDU-MP. Consequently, the development of a robust, practical, and cost-effective synthetic procedure of POM-L-BHDU-MP was needed. Earlier synthesis of L-BHDU was carried out by the L-dioxolane key intermediate (5, FIGURE 3, Scheme 1). The procedure reported by Sznaidman et al.17, 18 was used for the synthesis of L- dioxane 5, which is a critical intermediate for the synthesis of L-modified nucleoside analogs. [011] Scheme 1. Synthesis of chiral pure D & L dioxolane via chiral separation of racemic
Reagents and Conditions: (a) DMAP, Et3N, tert-butylmethyl ether, rt; (b) BF3.Et2O, acetonitrile 0°C-rt. [012] However, this reported process has involved the chiral chromatographic separation of racemic dioxolane (4), which limits the scalable and commercial utility of Scheme 1.
Furthermore, during the large-scale synthesis, in the presence of excess boron trifluoride diethyl etherate (BF3.Et2O), the decomposition of intermediate 4 was observed. Chiral pure D & L-dioxolane (5 & 6) also revealed stability issues in courses of chiral separation, and undesired impurities were formed during the concentration or lyophilization of collected chiral pure fractions. These downsides of Scheme 1 limit the process for a large-scale synthesis of both L & D dioxolane (5 & 6). To overcome the challenges of Scheme 1, herein inventors report a scalable, practical synthesis of L-BHDU in 7 steps from the methyl (R)-2,2- dimethyl-1,3-dioxolane-4-carboxylate, 7. However, the reported process in this communication is a revised procedure, especially for the synthesis of L-Dioxolane, which was previously reported by Bera S. et al 19 Earlier reported procedure and patent encountered many unclear information and challenges medicinal chemists for the specific synthesis of chiral pure L-dioxolane (enantiomeric excess ≥ 99%).20 Nevertheless, the present application is directed to a straightforward synthesis and purification of chiral pure L-dioxolane via diastereomeric chiral amine salt formation. This novel procedure also eliminates chiral separation and certain step column chromatography, which makes the process feasible for the scalable synthesis of POM-L-BHDU-MP for clinical and commercial applications. [013] Brief Description of the FIGURES [014] FIGURE 1 shows the chemical structures of a number of antiviral drugs for the treatment of VZV infection. [015] FIGURE 2 shows the chemical structures of L-HDVD, L-BHDU and POM-L-BHDU- MP [016] FIGURE 3, SCHEME 1, shows the chemical synthesis of chiral pure D & L dioxolane via chiral separation of racemic dioxolane
Reagents and Conditions: (a) DMAP, Et3N, tert-butylmethylether, rt; (b) BF3∙ET2O, acetonitrile 0º C-rt. [017] FIGURE 4, SCHEME 2, shows the synthesis of L-BHDU via chiral pure critical intermediate L-dioxolane (11). Reagents and Conditions: (a) p-TSA, or Dowex 50W X8 toluene 80 °C; (b) 1 M aqueous LiOH, THF, 0 °C- rt; (c) i) (S)-phenylethylamine, acetonitrile 75 °C- rt; BF3.Et2O, acetonitrile 0 °C-rt; (ii) EtOAc:IPA (3:1), rt; (d) 1 N aqueous
HCl, rt; (e) manganese diacetate (Mn (II)(OAc)2), diacetoxy iodobenzene , acetic acid, 2-Me- THF 65 °C, or alternatively Pb(OAc)4, pyridine, acetonitrile, 0 °C-rt; (f) Bromo vinyl uracil, TBDMSOTf, TMSI, DCM, rt; (g) BCl3, DCM, -78 °C. [018] FIGURE 5 shows (a) ORTEP diagram of compound 10 confirms the relative stereochemistry of L-dioxolane. (b) Shows the stereo structure of D and L-dioxolane; in the case of D-dioxolane C-2 and C-4 hydrogen are cis, whereas in L-dioxolane C-2 and C-4 hydrogen atoms are trans to each other. [019] FIGURE 6, SCHEME 3 shows a revised synthesis of L-BHDU via intermediate 18 from L-dioxolane 11. Reagents and Conditions: (a) 5% Pd/C, H2 gas, MeOH, r.t.; (b) Isobutyric anhydride, pyridine, DMAP, DCM, 0 ℃ - rt; (c) Mn(OAc)2, diacetoxy iodobenzene , acetic acid, 2-Me-THF, 65°C, or alternatively Pb(OAc)4, pyridine, acetonitrile, 0°C-rt ; (d) Bromovinyl-uracil, TBDMSOTf, TMSI, DCM, rt.; (e) Solution of 7 N NH3 in methanol rt. [020] FIGURE 7, SCHEME 4 shows the synthesis of POM-L-BHDU-MP prodrug via L- BHDU (15). Reagents and conditions: (a) NMI, THF, 0 °C to rt, 3 h. [021] Brief Description of the Invention [022] In embodiments, the present invention is directed to methods of synthesizing L- BDHU (15) through chemical precursors and chemical intermediates resulting in efficient syntheses of L-BHDU (15), POM-L-BHDU-MP (22) and a number of precursors and/or chemical intermediates which are used in the syntheses of these compounds. [023] In an embodiment, the present invention is directed to a method for synthesizing L- BDHU (15) from compound (7) pursuant to the synthetic steps which are described in enclosed FIGURE 4, Scheme 2. Compound 7 is first converted to pure key intermediate L- dioxolane acid (11) in four steps and L-dioxolane acid 11 is thereafter converted to L-BHDU (15) by one of two synthetic routes as disclosed herein below. Pursuant to a first embodiment
method, methyl-(R-)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (compound 7) is used to produce L-dioxolane compound 11 by first being reacted with 2-(benzyloxy)acetaldehyde in solvent (preferably toluene) with a catalytic amount of acid (preferably, toluene/p-toluene sulfonic acid or Dowex 50W X8)) at elevated temperature (above room temperature, e.g.60- 100 ºC for several hours) to provide a diastereomeric mixture (often 1:1) of benzyl-protected dioxolane ester compound 8 in high yield (often >70%) as depicted herein below. Compound 8 is thereafter subjected to ester hydrolysis in aqueous solvent/base (e.g.1 M LiOH/THF) to remove the ester group and provide the corresponding benzyl protected carboxylate compound 9, often in greater than 90% yield. Compound 9 is complexed with (S)- phenylethylamine via formation of acid base salt to provide substantially pure (i.e., de >90%, often de>99%) compound 10 by first complexing the benzyl protected racemic carboxylate compound 9 with (S)-phenylethylamine in solvent (e.g. acetonitrile, preferably conducted for about 2 hours at an elevated temperature of 70 ºC followed by cooling to room temperature for another 2 hours) to provide a salt of compound 10 which contains a mixture of both D and L isomers (15% D, 85% L, de > 70%, referenced as compound mixture 10M) which was precipitated and/or crystallized to provide a salt mixture of D and L isomers of acid amine 10 (referred to as compound 10M), which mixture was taken up (preferably, several times) in a 3:1 volume ratio of ethyl acetate:isopropyl alcohol, stirred at room temperature for several hours (e.g., 3, 4 or 5, often 4 hours) and precipitated. The mixture was taken up from 2-5 times, often 3-5 times, often 3 times in preferred embodiments) to provide substantially pure (de >90%, often >99%) (S)-phenylethylaminium salt (acid amine salt) 10. The (S)- phenylethylaminium acid amine salt 10 is then taken up in methylene chloride (DCM) and exposed/sequentially washed with cold aqueous acid (e.g., 1 N aq. Solution of HCl), brine and dried (e.g. sodium sulfate) to provide compound 11 (L-dioxolane) as a diasteromerically pure or substantially diastereomerically pure compound (de>90%, de>95%, often >99%, most often 99+%-100%). Compound 11 (as the trans/L form) is thus obtained from compound 9 by precipitation in the absence of chromatographic separation in high purity. It is noted that one or more of the aforementioned steps is conducted in separate steps or in a single pot reaction vessel.
[024] In an embodiment, compound 11 is converted to compound 15 through compounds 12 and 13 as described herein below and in enclosed FIGURE 4, SCHEME 2. In this embodiment, compound 11 is first reacted with Mn(II)OAc, diacetoxy iodobenzene , acetic acid in 2-Me-THF at 65 °C (or alternatively, lead tetraacetate Pb(OAc)4 in acetonitrile/base, often pyridine or trimethylamine, more often pyridine with the components combined at 0 °C, followed by rt for 16 hours, step not shown in Scheme, below) and chromatographic separation (preferably using column chromatography, elution with ethyl acetate/hexanes) to provide a mixture of α and β anomers (α/β ratio often of about 2:1) of benzyl protected dioxolane acetate compound 12. Compound 12 (as a mixture of α and β anomers) is coupled to bromovinyl uracil in dry solvent (e.g. dichloromethane (DCM)) using tert- butyldimethylsilyl trifluoromethanesulfonate (TBDMSOTf), iodotrimethylsilane (TMSI) and 2,4,6-collidine and thereafter subjected to chromatography (e.g. silica gel column, 1:4 EtOAc/hexane as eluent) to provide a minor component of α-isomer compound 14 (about 10- 20% yield, undesired) and desired β-isomer compound 13 in >50% yield (often 55% or more). Compound 13 is then subjected to deprotection of the benzyl group (e.g. in DCM using BCl3 at low temperature, e.g. -78 ºC) to provide compound 15 in >50% yield. Each of the aforementioned synthetic steps may be conducted in separate steps or together in a single reaction vessel.
[025] In an embodiment, the present invention is directed to a method for synthesizing compound 15 from L-dioxolane acid 11 through compounds 16-20 as depicted herein below and in enclosed FIGURE 6, SCHEME 3. In this embodiment of the present invention, compound 11 is subjected to benzyl deprotection (preferably utilizing reductive cleavage, often with Pd/C, hydrogen gas, in MeOH at r.t) to provide unprotected dioxolane acid compound 16 in high yield which is subsequently protected on the free hydroxyl group with an isobutyrate group. This step is performed, for example, by reacting compound 16 with isobutyric anhydride in base (e.g. pyridine and dimethylamino pyridine (DMAP)) in dichloromethane solvent, initially at 0 ºC followed by reaction at RT (e.g. overnight, from 12- 20 hours, often 16 hours) to provide isobutyrate protected compound 17, often in greater than 80-85% yield. Compound 17 was acylated to form compound 18 as a mixture of diastereomers using Mn(II)OAc, diacetoxy iodobenzene , acetic acid in 2-Me-THF(e.g. initially at 0 ºC, followed by RT overnight or from 12-20 hours, often 16 hours, then at 65 °C for 4h). Alternatively, lead tetraacetate Pb(OAc)4 in acetonitrile/base, often pyridine or trimethylamine, more often pyridine with the components combined at 0°C, followed by rt for 16 hours can be used (alternative step not shown in Scheme, below). Compound 18 was
thereafter coupled to bromovinyluracil in dichloromethane (DCM) using tert- butyldimethylsilyl trifluoromethanesulfonate (TBDMSOTf), iodotrimethylsilane (TMSI) and 2,4,6-collidine which provided a diasteromeric mixture of compound 19 and 20, which mixture was thereafter subjected to chromatography (e.g. silica gel using 15-18% EtOAc/hexanes) to provide compound 20, often in greater than about 45-50% yield, which could be readily converted to L-BHDU (15) by exposure to base in solvent (e.g. NH3/MeOH). Each of the aforementioned synthetic steps may be conducted in separate steps or together in a single reaction vessel.
[026] In an embodiment, the present invention is directed to the synthesis of benzyl protected dioxolane acid 11 from benzyl protected dioxolane acid 9 by reacting (S)- phenylethamine with compound 9 in acetonitrile at room temperature for several hours (e.g.
often about 4 hours) to form a mixture of D and L isomeric benzyl protected phenylethylaminium salts 10 (the mixture of isomeric compounds is also referred to as 10M) which solid mixture is resuspended in a 3:1 ratio (volume) of ethylacetate/isopropanyl for 4 hours and filtered. This resuspension process is optionally and preferably repeated several times until the undesired D isomer is removed from the salt mixture) to provide the substantially pure L-isomer compound 10 (de of at least 90%, often at least 95%, at least 99%, 99+%, 99.3+%). The L-isomer (compound 10) is exposed to aqueous acid (e.g. 1N aqueous HCl) at room temperature for a time sufficient to provide the benzyl protected L- dioxolane acid 11. Compound 11 is readily converted to the deprotected L-dioxolane acid 16 by removing the benzyl protecting group under reduction conditions (e.g. Pd/C, H2).
[027] In an embodiment, the present invention is directed to a method for synthesizing isobutyrate protected nucleoside compound 20 from isobutyrate protected L-dioxolane acid 17 by acylating compound 17 using Mn (II) OAc, diacetoxy iodobenzene , acetic acid in 2- Me-THF (or alternatively, lead tetraacetate Pb(OAc)4 in acetonitrile/base, often pyridine or trimethylamine, more often pyridine with the components combined at 0°C, followed by rt for 16 hours, not shown in the Scheme below) to form a diastereomeric mixture of the acylated protected L-dioxolane compound 18. Compound 18 is then coupled with bromovinyl-uracil in dichloromethane (DCM) using tert-butyldimethylsilyl trifluoromethanesulfonate (TBDMSOTf), followed by iodotrimethylsilane (TMSI) to provide a diasteromeric mixture of compounds 19 and 20. The diastereomeric mixture of compounds 19 and 20 is subjected to column chromatography to afford compound 20. Compound 20 is
then exposed to ammonia/methanol (or other appropriate base) to remove the isobutyrate protecting group to afford final compound L-BHDU 15. Each of the aforementioned synthetic steps may be conducted in separate steps or together in a single reaction vessel.
[028] In an embodiment, as presented in FIGURE 7, SCHEME 4, L-BHDU (15) is converted to POM-L-BHDU-MP (22) by reacting compound 15 with bis(POM)phosphorochloridate (21) in THF solvent in the presence of weak base (e.g. N- methylimidazole, NMI) and purified (e.g. chromatography) and crystallized to afford compound 22 in greater than 40% yield. [029] In embodiments, the present invention is also directed to any one or more of the compounds, including intermediate compounds identified above, or a salt, including a pharmaceutically acceptable salt, thereof.
[030] These and/or other embodiments of the present invention may be readily gleaned from a review of the presentation of the specification and the identified discussion and examples which follow. [031] DETAILED DESCIPTION OF THE INVENTION [032] The following terms are used to describe the present invention. In instances where a term is left undefined, the term is given its art recognized meaning. In accordance with the present invention there may be employed conventional chemical synthetic methods and other biological and pharmaceutical techniques within the skill of the art. Such techniques are well- known and are otherwise explained fully in the literature. [033] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon atoms in which case each carbon atom number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. [034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. [035] It is to be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The term “about” when used, signifies an amount within +5% of the amount or number specifically set forth.
[036] The term “compound”, as used herein, unless otherwise indicated, refers to any specific chemical compound or intermediate disclosed herein, and often refers to β-L nucleoside analogs or D & L dioxolane intermediates used to produce these nucleoside compounds using the synthetic steps described herein, but may include, within context, tautomers, regioisomers, geometric isomers, anomers, and where applicable, optical isomers (enantiomers) or often diastereomers (two chiral centers or more) thereof of these compounds, as well as pharmaceutically acceptable salts thereof, solvates and/or polymorphs thereof. Within its use in context, the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures and/or diastereomers as described herein) as well as specific enantiomers, enantiomerically enriched or individual diastereomers or mixtures of these disclosed compounds. It is noted that if a carbon range is provided for a compound, that range signifies that each and every carbon individually is considered part of the range. For example, a C1-C20 group describes a group with a single carbon, two carbon atoms, three carbon atoms, four carbon atoms, etc. up to twenty carbons. [037] The term “salt” or “pharmaceutically acceptable salt” is used throughout the specification to describe, where applicable, a salt form of one or more of the compounds described herein which are presented to increase the outcome of a synthetic reaction, or alternatively, the solubility of a compound, in certain embodiments where administration has been effected, in the gastric juices of the patient's gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, where applicable. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids well known in the pharmaceutical art. Sodium and potassium salts are particularly preferred as neutralization salts of the phosphates according to the present invention, where relevant.
[038] The term "pharmaceutically acceptable derivative" is used throughout the specification to describe any pharmaceutically acceptable prodrug form (such as an ester, ether or amide, phosphoramidate, phosphate ester, phosphonate or other prodrug group) which, upon administration to a patient, provides directly or indirectly the present compound or an active metabolite of the present compound. [039] The term “D-configuration” as used in the context of the present invention refers to the configuration of the nucleoside compounds as well as other intermediates according to the present invention which mimics the natural configuration of sugar moieties as opposed to the unnatural occurring nucleosides or “L” configuration, which describes most of the compounds used in the chemical syntheses or as final products as set forth herein. The term “β” or “β anomer” is used to describe intermediates or nucleoside analogs according to the present invention in which the nucleoside base is configured (disposed) above the plane of the dioxolane moiety in the compound. [040] The term “diastereomeric excess”, “de” or “enantiomeric excess” , “ee” is used throughout the specification to describe an intermediate compound or a nucleoside compound which has a diastereomeric or enantiomeric excess of at least about 90%, often at least about 95%, preferably at least about 96%, more preferably at least about 97%, even more preferably, at least about 98%, and even more preferably at least about 100% or more of a single diastereomer or a single enantiomer of that compound. Dioxolane nucleoside compounds such as L-BHDU or its derivatives according to the present invention are generally β-L-nucleoside compound analogs. When the present compounds according to the present invention are referred to in this specification, it is presumed that the intermediates or final nucleoside compounds have the L-nucleoside configuration and are diasteromerically and/or enantiomerically enriched (preferably, about 100% of the L-nucleoside), unless otherwise stated. The term “diasteromerically pure”, “diasteromerically enriched”, or “diastereomeric excess” or de” is used to describe a single diastereomer of a compound according to the present invention which contains an excess of at least 90%, 95%, 96%, 97%, 98%, 99%, 99+%, 99.5% or 100% by weight of that diastereomer to the exclusion of other possible diastereomers.
[041] The term “stereoselective” is used to describe a synthetic step or series of steps in which a single reactant produces a particular isomer, enantiomer or diastereomer (of at least two possible isomers or diastereomers) in greater quantities than one or more possible isomer(s) or diastereomer(s) from that reactant. In some instances, the stereoselectivity of a reaction may be close to 100%. [042] The term “protecting group” or “blocking group” is used to describe a chemical group or moiety which is introduced into a molecule by chemical modification of a functional group to obtain chemo selectivity and/or stereoselectivity in a subsequent chemical reaction. The protecting group/blocking group plays an important role in providing precursors to chemical components which provide compounds according to the present invention. Blocking groups are often used to protect hydroxyl groups on the intermediate based in order to form compounds according to the present invention. Typical blocking groups are typically used on alcohol groups or phosphate groups in the present invention and include principally benzyl groups (removed by hydrogenolysis), isobutyrate groups (removed by base), methyl ester groups (removed by base), acetyl groups (removed in coupling reactions) and pivaloylmethyl ester (POM) groups on phosphate group intermediates. Pivaloyloxymethyl ester (POM) groups are also used on phosphate groups in final compound POM-L-BHDU-MP to instill prodrug characteristics to nucleoside compounds to enhance bioavailability. These and other/or groups are described herein. [043] CHEMICAL SYNTHESIS OVERVIEW AND RESULTS [044] Numerous efforts by various groups to construct chiral pure L-dioxolane often lead to a racemic mixture of D & L dioxolane and demand expensive chiral SFC purification to obtain chiral pure and dioxolane. To remove the chiral SFC separation, Bera S. et a 19 D L l. reported the synthesis of chiral dioxolane via methyl-(R)-2,2-dimethyl-1,3-dioxalane-4- carboxylate. However, the reported procedure was ambiguous and required a close analytical technique to confirm the D & L stereoisomers, which may sometimes occur to the end of the final undesired conformational product. In the present instance, the inventors obtained an L- BHDU authentic sample that was matched with synthesized L-BHDU via the reported protocol of Bera S. al. After that, the inventors found that this procedure without an authentic sample and chiral HPLC proved to be very challenging to furnish the desired isomers of the
targeted compound. To accomplish the scalable synthesis of L-BHDU, a new protocol for the synthesis of exclusively chiral pure L-dioxolane was urgently needed, thus precipitating studies which led to the present invention. The present invention is principally focused on the synthesis of chiral L-dioxolane with an excellent enantiomeric excess (e.e. ≥ 99%). Notably, this procedure may be suitable for the large-scale synthesis of L-derived dioxolane nucleosides such as troxacitabine.21 It may also expedite the extended structure-activity relationship (SAR) studies of both D & L-dioxane-derived molecules of therapeutic interest. [045] The synthesis of chiral pure L-dioxolane (11) key intermediate was initiated by condensation of commercially available methyl (R)-2,2-dimethyl-1,3-dioxolane-4- carboxylate (7) with 2-benzyloxyacetaldehyde via trans ketalization in the presence of p-TSA to give the racemic 1,3-dioxolane derivative in 1:1 diastereomeric mixture dioxolane esters, 8 (FIGURE 4, SCHEME 2) in 72% yield. Next, hydrolysis of methyl ester (8) was carried out by the 1 M aqueous solution of LiOH in THF to afford a diastereomeric mixture of acids (9) in 92% yield. [046] Thereafter the goal was to obtain pure L-dioxolane acid (11) in high enantiomeric purity (e.e. > 99%) by avoiding the chromatographic separation of racemic acid (9). However, the selective separation of L-dioxolane was also challenging. One approach was to insert chiral auxiliary via esterification of carboxy acid 9, followed by a fractional crystallization to render enantiomeric pure L-dioxolane. However, this strategy unnecessarily increases the additional two-step process of esterification and hydrolysis, which is not efficient for the large-scale synthesis of L-dioxolane. To reduce the additional steps, it was thought to perform resolution of racemic 9 via a diastereomeric salt formation with a chiral amine. Noteworthy to mention is that compound 9 is oily in nature, and other fractional crystallization techniques may not be appropriate for the resolution of chiral pure L-dioxolane (11). Singh et al. reported chiral resolution of racemic amines via chiral pure L-(+)-tartaric acid.22 Taking the lead from the reported procedure by Singh et al. it was thought to adopt a reverse strategy, chiral resolution of racemic acid (9) via salt formation with a chiral pure amine.
[047] To achieve diastereomerically pure salt formation of racemic 9, several (R) or (S) chiral amines were utilized (see Table 1, below) in various solvent systems. In the investigational resolution of racemic acid 9, diastereomeric salt formation of 9 with (S)- phenylethylamine produced pure chiral dioxolane salt 10 with a diastereomeric excess (de) of 99.37% (entry 4 Table 1). Racemic acid 9 was taken in acetonitrile and treated with 0.8 eq. of (S)-phenylethylamine which gives a precipitate of white salt of acid amine. The next goal was to determine the chiral purity of the precipitated salt as well as the stereo conformation of diastereomeric salt. The precipitated diastereomeric salt was treated with 1 N aq. HCl to afford free diastereomeric dioxolane. The chiral purity of obtained free dioxolane was determined by chiral HPLC, which indicates the optical purity of precipitated isomers is 85% and 15% presence of another isomer. The obtained results of diastereomeric salt formation via resolution of racemic 9 were not encouraging because, for the construction of optically pure L-BHDU, there was a need for L-dioxolane with the optical purity of more than 99% (ee ≥ 99%). [048] Hence, the process of the diastereomeric salt formation of racemic 9 was revisited. To enrich the diastereomeric excess (de) of precipitated salt (via acetonitrile), the obtained salt was re-suspended in EtOAc-IPA (3:1) and stirred for 4 h at room temperature, then filtered and neutralized by 1 N aqueous HCL aqueous solution. The afforded, dioxolane was reexamined for the chiral purity via chiral HPLC, which demonstrated ee of more than 99% (ee ≥ 99.3%) in 38% yield. Therefore, it was concluded that treatment of a double solvent system, first in acetonitrile followed by EtOAc: isopropanol (3:1), enhances the diastereomeric excess (de) of precipitate salt (de ≥ 99.3%), an unexpected result. The subsequent objective was the stereo conformation determination of precipitated salt, either D or L-form of dioxolane. The X-ray crystal of precipitated salt was developed, and it confirmed the formed diastereomeric salt matches with the stereo conformation of L-dioxolane (10). The X-ray structure revealed that hydrogen atoms present at carbon-2 (C-2) and C-3 are trans (see FIGURE 5b) to each other, which confirms L-dioxolane salt precipitation. [049] However, a single solvent salt formation approach was tried with various chiral amines mentioned in Table 1, below, but in each case, the results of de were unsatisfactory.
To improve the yield of intermediate 11, an altered ratio of EtOAc & IPA was tried, but in all efforts, either less yield or less chiral purity of intermediate 11 was achieved. Table 1: Diastereomeric salt formation results of racemic compounds 10 with various chiral amines Entry Chiral mine Solvent Temp. Time Result Yield Salt contains both D & L isomers in 1:1 1 (R)-phenylethylamine EtOAc:IPA rt 4h >10% (2:0.5) (de ~50%) Salt contains both D >10% & L isomers in 1:1 2 (R)-phenylethylamine EtOAc:IPA 45 ℃ to rt 4h (2:1)
Salt contains both D 15% & L isomers in 1:1 3 (R)-phenylethylamine Acetonitrile 70 ℃ to rt 4h (de ~50%) Salt contains both D (15%) & L isomer Acetonitrile 70 ℃ to rt 4h (85%) (de ≥70% ) (Step-1) Obtained salt 38% from step-1 4 (S)-phenylethylamine was after both resuspended in step 1& 2 Yielded salt contain with L-isomers (de ≥ rt 4h EtOAc:IPA 99.3%) (3:1) (Step-2) Salt formation was but moister sensitive 5 (S)-phenylethylamine EtOAc:IPA 60 °C -rt 4h during filtration
(1:1) 6 (S)-phenylethylamine EtOAc:IPA 60°C - rt 4h Salt formation was NA but moister sensitive (1:2) during filtration 7 (S)-phenylethylamine EtOA:IPA rt 6h Yielded salt contain 23% L-isomers (de ≥ 3:1 90.0%)
8 (S)-(+)-1-(1-Napthyl) Acetonitrile 70 ℃ to rt 78h No salt formation NA ethylamine 9 (R)-(+)-1-(1-Napthyl) Acetonitrile 70 ℃ to rt 16h No salt formation NA ethylamine 10 (S)-(-)-N-benzyl-1- Acetonitrile 70 ℃ to rt 32h No salt formation NA phenylethylamine [050] L-dioxolane compound 11 served as the key intermediate for the synthesis of L- BHDU. Treatment of 11 with Mn(II)OAc, acetic acid, diacetoxy iodobenzene in 2-methyl THF, or alternatively lead (IV) acetate Pb(OAc)4, in the presence of pyridine in acetonitrile, converts the 2-carboxyl group of 11 to the 2-acetoxy intermediate 12 in an α/β ratio of 2:1 (racemic) in 74% yield. Furthermore, glycosylation coupling of 12 with bromovinyl-uracil was carried out under Vorbrüggen coupling condition.23 Bromouracil base treated with trimethylsilyl trifluoromethanesulfonate (TMSOTf) followed by (iodotrimethylsilane) TMSI, which in situ generates silylated bromo vinyl uracil and condenses with acetate intermediate 12 (α/β ratio 2:1) to render coupled β and α isomer of 13 (in 55% yield) and 14 (in 18% yield) in ratio of 3:1. Separation of isomers by column chromatography afforded desired β-isomer in 55% yield and α-isomer in 18% yield. Finally, deprotection of the 5ʹ-benzyl group of coupled product 13 was performed by 1 M solution of boron trichloride (BCl3) in DCM to furnish targeted compound L-BHDU in 60% yield. This series of reactions is presented in accompanying FIGURE 4, SCHEME 2. [051] It was determined that the exothermic final deprotection of benzyl group of compound 13 using BCl3 was not suitable for large-scale synthesis. To avoid the use of BCl3, a protection group replacement strategy was adopted, and the 5-O-benzyl of intermediate 11 was replaced with isobutyric ester as a hydroxyl protecting group. The debenzylation of 11 was carried out with 5% Pd/C under hydrogenation conditions to afford intermediate 16 in 91% yield. Intermediate 16 was treated with isobutyric anhydride in the presence of pyridine to produce compound 17 in 88% yield. Acylation of compound 17 followed by the coupling with bromovinyl-uracil afforded coupled desired (β) nucleoside 20 (54% yield) and undesired (α) nucleoside 19 (yield 22%) in approximately 2:1 ratio (see FIGURE 6, SCHEME 3).
[052] Deprotection of the isobutyl ester group of 20 was performed in base (7 N NH3 solution in MeOH) to give L-BHDU in 92% yield. Optical rotation and other analytical data of 15 were consistent with previously synthesized authentic -BHDU.24 L [053] To synthesize POM-L-BHDU-MP prodrug, first synthesis of bis(POM)phosphorochloridate (21) was carried out according to the reported protocol by Hawang Y. et al.25 Next, coupling of L-BHDU (15) was performed with POM chloride (21), in the presence of N-methyl imidazole (NMI) in THF to furnish POM-L-BHDU-MP (22) in 47% yield (FIGURE 7, SCHEME 4). [054] CONCLUSIONS DRAWN [055] To determine the full biological evaluation of POM-L-BHDU-MP, an efficient and scalable synthetic method of L-BHDU has been developed via commercially available methyl (R)-2,2-dimethyl-1,3-dioxolane-4-carboxylate, 7. The selective diastereomeric salt formation of racemic 9 via a chiral (S)-phenylethylamine followed by neutralization of 10 gave optically pure L-dioxolane, 11 (ee ≥ 99%). Compound 11 was converted to acetate intermediate 12, which was utilized for Vorbrüggen coupling with bromovinyl-uracil followed by the benzyl deprotection of coupled nucleoside afforded the target compound 15 (L-BHDU) in 7 steps with approximately 4.9 % overall yield. This process removes the expensive chiral separation of racemic D & L dioxolane and is more efficient than the previously reported method for the synthesis of L-BHDU. Further coupling of bis(POM)phosphorochloridate, 21 with L-BHDU produced L-BHDU-monophosphate ester prodrug 22 (POM-L-BHDU-MP). The removal of chiral separation and reduction of column chromatography in synthetic steps in comparison to the previously reported methods and the use of economical reagents make the current methodology amenable for large scale preparation of POM-L-BHDU-MP and selective synthesis of chiral pure L-dioxolane (11). [056] It was noted during chemical synthesis that the use of Mn(OAc)2 was found unexpectedly to be a particularly effective substitute for Pb(OAc)4 for forming compounds 12 and 18 (conversion of precursor carboxylic acid to acetate group at the 4-position of the dioxolanyl moiety) and has become the preferred reagent for conducting these steps in scale- up reactions of the present invention. The reactivity of Mn(OAc)2 turned out to be at least
equal to and in some instances somewhat greater than Pb(OAc)4 and has other attributes which make this reagent substantially more favorable than Pb(OAc)4 for use in the present invention despite the overwhelming prevalence of Pb(OAc)4 in conventional chemistry, particularly of this nature. Moreover, Mn(OAc)2 is about 25% of the cost of Pb(OAc)4 and offers substantial cost savings, especially in commercial applications. This reagent is also more readily available from commercial suppliers due to its far lower reagent toxicity compared to Pb(OAc)4. Reaction time for the conversion of the carboxylic acid group to the acetate in compounds 12 and 18, respectively turns out to be substantially faster for Mn(OAc)2 (4h) compared to Pb(OAc)4 (>14h), which will support the expedited synthesis of POM-L-BHDU-MP. Additionally, the handling of Mn(OAc)2 is far less challenging than is the handling of Pb(OAc)4 in commercial applications, inasmuch as Pb(OAc)4 is very hygroscopic (moisture sensitive) and in large settings (>25g) makes its use difficult often requiring special handling, whereas Mn(OAc)2 is very stable (not moisture sensitive) and easy to handle. [057] Pb(OAc)4 is highly exothermic in >50g scale reactions and in the present invention it was required to add reagent in portion wise to control the reaction temperature and to run the reaction under cooling conditions. This took considerably longer than with the use of Mn(OAc)2, because the reaction steps with Mn(OAc)2 is not exothermic and does not require external cooling. In the reactions utilizing Pb(OAc)4, pyridine is required in equimolar quantity and in the completion of the reaction and final work product removing pyridine is difficult and produces a particularly pungent smell in large scale reactions. The Mn(OAc)2 reactions do not require pyridine (or any amine), thus substantially reducing clean up and final processing of compound. Additional considerations include the fact that the product obtained from Pb(OAc)4 reaction must be purified by column chromatography to obtain pure enough to take to the next step, whereas Mn(OAc)2 results in an almost clean conversion and crude product may be readily used for the next step reaction, thus avoiding column chromatographic purifications. This is particularly important in commercial applications of the present invention. [058] In the present invention, in Pb(OAc)4 reaction conditions at large scale, colloidal solutions in the reaction mixture and during work up generate challenges to process the reaction mixture and break up the reaction mixture colloidal state. This colloidal solution formation can often compromise the yield of reaction. The Pb(OAc)4 reaction conditions are
not ecofriendly, whereas those utilizing Mn(OAc)2 are eco friendly and substantially safe for the environment. Accordingly, Mn(OAc)2 represents a substantially better reagent for the scalable synthesis for use in the present invention. [059] EXAMPLES [060] EXPERIMENTAL SECTION [061] General Analytical Methods [062] Reagents and anhydrous solvents were purchased from commercial sources and used without further purification. Moisture-sensitive reactions were performed using oven-dried glassware under a nitrogen or argon atmosphere. Reactions were monitored by thin-layer chromatography plates (TLC silica gel GF 250 microns) that were visualized using a Spectroline UV lamp (254 nm) and developed with 15% solution of sulfuric acid in methanol. Column chromatography was performed on silica gel 60Å, 40-63µM (230 X 400 mesh, Sorbent Technologies). Preparative normal phase chromatography was performed on a CombiFlash Rf 150 (Teledyne Isco) with pre-packed RediSep Rf silica gel cartridges or on RediSep® gold C18 reverse phase columns. Melting points were recorded on a Mel-temp II laboratory device and are uncorrected. Nuclear magnetic spectra were recorded on Varian Inova 500 spectrometer at 500 MHz for 1H NMR, 202 MHz for 31P NMR, and 125 MHz for 13C NMR with tetramethylsilane as an internal standard. Chemical shifts (δ) are quoted as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet) and dt (double triplet). Optical rotations were measured on a JASCO DIP-370 digital polarimeter. High-resolution mass spectroscopy (HRMS) spectra were measured on Bruker Ultra-high resolution QTOF MS Impact II spectrometer. Samples were infused at 3 μL/min, and spectra were obtained in the positive or negative ionization mode with a typical resolution of 20,000 or greater. Purity of all tested compounds are ≥95%, as determined by their elemental analysis or by HPLC/UV. Elemental analysis were performed by the Atlantic Microlab Inc. Norcross, GA. HPLC/UV were determined with a Waters HPLC coupled to a photodiode array.5 μL of sample 0.5 mg/mL in methanol, or in acetonitrile or in mixture of DMSO/MeOH (0.5:10) were injected, using an XBrigde C18, 3.5 μm, (4.6 X 150) mm column at 25 °C with flow rate 0.8 mL/min or with UPLC BEH C18, 1.7 μm (100 X 2.1) mm at 50 °C with a flow rate 0.55 mL/min. The mobile phases were a mixture of A = 10 mM ammonium acetate in water and B = acetonitrile (ACN), and A = 0.05% formic acid (FA) in water and B = 0.05% in acetonitrile (ACN). Purity is given as % of absorbance at Max plot.
Optical purity of chiral intermediates and final compound were determined by the chiral HPLC. Chiral HPLC/UV were determined with a Waters HPLC coupled to a photodiode array.10 μL of samples 0.5 mg/mL in methanol, were injected, using an CHIRALCEL OX- H, 5µmm (4.6 X 250mm) column at 30 °C with flow rate 3.0 mL/min. [063] Methyl (4R)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylate (8): To a solution of 2-(benzyloxy)acetaldehyde (100 g, 665.91 mmol) and methyl (R)-2,2-dimethyl-1,3- dioxolane-4-carboxylate (7, 106.65 g, 665.91 mmol) in toluene (700 mL), p-toluene sulfonic acid or Dowex 50W X8was added (5.06 g, 26.63 mmol) and the mixture was stirred at 80 ℃ for 2 h. (Reaction mixture turned to dark black color). The reaction mixture was cooled to room temperature and the volatiles were evaporated and the crude mixture was purified by the flash silica gel column chromatography (12-18% EtOAc/hexane) as eluent to obtain an inseparable mixture of methyl (4R)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylate 8 as light-yellow oil. Yield (122.5 g, 72%); 1H NMR (500 MHz, CDCl3) δ 7.37-7.32 (m, 11H), 7.30-7.27 (m, 2H), 5.33 (t, J = 4.0 Hz, 1H), 5.22 (t, J = 4.5 Hz, 1H), 4.68-4.58 (m, 7H), 4.31 (dd, J = 8.2, 7.3 Hz, 1H), 4.24 (dd, J = 8.6 & 3.9 Hz, 1H), 4.13-4.09 (m, 1H), 3.99 (dd, J = 8.3 & 5.4 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 3.71 (d, J = 3.8 Hz, 1H), 3.67-3.61 (m, 2H), 3.59 (d, J = 4.1 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 171.35, 171.1, 128.49, 128.46, 127.91, 127.83, 127.81, 127.79, 104.87, 104.33, 74.10, 73.90, 73.80, 73.74, 70.87, 70.30, 68.71, 68.31, 52.49; HRMS-ESI (m/z): [M +Na]+ Calcd for C13H16O5Na 275.0895; Found 275.0883. [064] (4R)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylic acid (9): To a solution of methyl (4R)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylate 8 (60 g, 237.84 mmol) in THF (250 mL) at 0 ℃ 1 M aqueous LiOH was added (198 mL) dropwise and the mixture was slowly brought to room temperature and stirred for for 36 h. THF was removed under reduced pressure and mixture was washed with EtOAc (3 X 75 mL). The separated aqueous layer was cooled to 0 ℃ and acidified with 28% aq. HCl to pH ~3, and extracted with DCM (3 X 125 mL). The combined organic layer was washed with brine and dried over Na2SO4, filtered, and concentrated to obtain racemic acid (4R)-2-((benzyloxy)methyl)-1,3-dioxolane- 4-carboxylic acid 9 as a light brown thick liquid. Yield (52.5 g, 92%); 1H NMR (500 MHz, CDCl3) δ 7.40-7.32 (m, 10H), 7.31-7.27 (m, 1H), 5.34 (t, J = 3.6 Hz, 1H), 5.20 (s, 1H), 4.74 (d, J = 12.1 Hz, 1H), 4.70-4.64 (m, 3H), 4.61 (s, 2H), 4.42-4.34 (m, 2H), 4.15-4.09 (m, 2H), 4.04 (dd, J = 8.4 & 5.4 Hz, 1H), 3.80 (dd, J = 11.4 & 1.7 Hz, 1H), 3.76 (d, J = 11.4 Hz, 1H),
3.61 (dd, J = 5.9 & 3.6 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 174.76, 173.56, 137.68, 136.02, 128.77, 128.53, 128.28, 127.91, 127.85, 104.44, 104.25, 74.28, 73.83, 73.65, 70.48, 70.19, 68.27, 67.50; HRMS-ESI (m/z): [M +Na]+ Calcd for C12H14O5Na 261.0739; Found 261.0739. [065] (2S,4R)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylate-(S)-1-phenylethan-1- aminium (10): A solution of methyl (4R)-2-((benzyloxy)methyl)-1,3-dioxolane-4- carboxylate 9 (25 g, 105.0 mmol) in ACN (50 mL) was heated to 70 ℃ for 20 minutes. After that, (S)-(-)-1-phenylethylamine (10.80 g, 89.25 mmol) was dropwise. The mixture was heated at same temperature at 70 °C for 2 h then slowly cool to room temperature and stirred for 3 h to give white solid precipitate .The precipitate was filtered. To remove the undesired D-dioxolane salt (15%) from the precipitate, filtered solid was resuspended in a mixture of EtOAc: isopropanol (50 mL, 3:1) and stirred for 4 h at rt and filtered. The diastereomeric purity of 10 was monitored by TLC and NMR, and the process was repeated 3 times until the undesired isomer (D dioxolane salt) was completely removed from the solid precipitate. Compound 10 was obtained as a white amorphous solid. Yield (11.5 g, 32%); 1H NMR (500 MHz, CDCl3) δ 8.37 (bs, 3H), 7.35-7.25 (m, 10H), 5.03 (t, J = 3.9 Hz, 1H), 4.57-4.50 (m, 2H), 4.27-4.21 (m, 2H), 4.04 (t, J = 7.7 Hz, 1H), 3.63 (t, J = 7.1 Hz, 1H), 3.46- 3.43 (m, 2H), 1.49 (d, J = 6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 176.74, 139.31, 137.81, 128.91, 128.50, 128.47, 127.89, 127.86, 126.63, 103.01, 75.66, 73.373, 70.84, 68.69, 50.93, 21.13. [066] (2S,4R)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylic acid (11): (2S,4R)-2- ((benzyloxy)methyl)-1,3-dioxolane-4-carboxylate-(S)-1-phenylethan-1-aminium, 10 (11.2 g, 31.18 mmol) was dissolved in DCM (200 mL), and sequentially washed with cold 1 N aqueous HCl (20 mL), water (100 mL), and finally with brine (100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give (2S,4R)-2- ((benzyloxy)methyl)-1,3-dioxolane-4-carboxylic acid 11 as a thick colorless liquid. Yield (7.1 g, 95%); [^]25 D = +20.82 (c 0.5, CHCl3) 1H NMR (500 MHz, CDCl3) δ 7.61 (bs, 1H), 7.36-7.31 (m, 4H), 7.30-7.25 (m, 1H), 5.32 (t, J = 3.5 Hz, 1H), 4.69-4.66 (m, 1H), 4.60 (s, 2H), 4.34 (t, J = 7.9 Hz, 1H), 4.02 (dd, J = 8.4 & 5.4 Hz, 1H), 3.60 (dd, J = 5.9 & 3.6 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 176.74, 139.31, 137.81, 128.91, 128.50, 128.47, 127.89, 127.86, 126.63, 103.01, 75.66, 73.373, 70.84, 68.69, 50.93, 21.13; HRMS-ESI (m/z): [M +H]+ Calcd for C12H14O5Na 261.0739; Found 261.0729.
[067] (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate (12): To a stirred solution of (2S,4R)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylic acid (11, 0.5 g, 2.09 mmol) in 2- Me-THF (12 mL) was degassed with argon then added acetic acid (0.25 mL, 42.18 mmol), Mn(II)OAc (994 mg, 5.74 mmol), diacetoxy iodobenzene (2g, 6.27 mmol) and again degassed for 3 mins. The reaction mixture under argon was heated to 65 °C for 4.5h. The reaction mixture cooled to rt and filtered through a celite bed. The obtained filtrate was diluted with EtOAc (60 mL); washed sequentially with saturated Na2S2O3 (15 mL), cold 2M NaHCO3 (15 mL) water (25 mL), and finally with saturated brine (30 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by flash silica gel column chromatography using 16-19% EtOAc/Hexane as eluent to obtain a diastereomeric mixture of (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate as a colorless liquid (12). Yield (385 mg, 72%); 1H NMR (500 MHz, CDCl3) δ 7.37-7.27 (m, 8H), 6.39 (dd, J = 4.4 & 2.1 Hz, 1H), 6.33
= 3.7 Hz, 0.6H), 5.38-5.36 (m, 1H), 5.30-5.27 (m, 0.7H), 4.64-4.59 (m, 3H), 4.23 (dd, J = 9.5 & 4.7 Hz, 1H), 4.18 (d, J = 9.6 Hz, 0.6H),3.99-3.93 (m, 2H), 3.67-3.57 (m, 4H), 2.09 (s, 3H), 2.03 (s, 2H); 13C{1H} NMR (126 MHz, CDCl3) δ 170.4, 137.8, 128.5, 127.8, 105.3, 103.8, 94.6, 94.1, 73.8, 73.8, 73.7, 71.4, 71.1, 70.9, 70.2, 21.2; HRMS-ESI (m/z): [M + Na]+ calculated for [C + 13H16O5] 275.0895; found 275.0885. [068] (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate (12) (Alternative Synthesis): To a stirred solution of (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate, 11 (2.0 g, 8.39 mmol) and anhydrous pyridine (0.95 mL, 12.08 mmol) in acetonitrile (15 mL), Pb(OAc)4 (4.46 g, 1.48 mmol) was added portion wise at 0 °C and continue stirred for 16 h at rt. The reaction mixture was filtered through a celite bed. The obtained filtrate was diluted with water (100 mL) and extracted with EtOAc (2 X 50 mL), the combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by flash silica gel column chromatography using 15-18% EtOAc/hexanes as eluent to obtain (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate 12 as a colorless liquid. Yield (1.35 g, 74%); 1H NMR (500 MHz, CDCl3) δ 7.37-7.27 (m, 8H), 6.39 (dd, J = 4.4 & 2.1 Hz, 1H), 6.33 (d, J = 3.7 Hz, 0.6H), 5.38-5.36 (m, 1H), 5.30-5.27 (m, 0.7H), 4.64-4.59 (m, 3H), 4.23 (dd, J = 9.5 & 4.7 Hz, 1H), 4.18 (d, J = 9.6 Hz, 0.6H), 3.99-3.93 (m, 2H), 3.67-3.57 (m, 4H), 2.09 (s, 3H), 2.03 (s, 1.6H); 13C NMR (125 MHz, CDCl3) δ 170.04, 137.75, 128.51, 127.84, 105.29, 103.81, 94.58, 94.10, 73.82, 73.82, 73.71, 71.38,
71.10, 70.87, 70.16; HRMS-ESI (m/z): [M +H]+ Calcd for C13H16O5Na 275.0895; Found 275.0884. [069] 1-((2S,4S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl)-5-((E)-2- bromovinyl)pyrimidine-2,4(1H,3H)-dione (13): To a suspension of (E)-5-bromovinyluracil (500 mg, 2.30 mmol) in dry DCM (15 mL), TBDMSOTf (1.37 mL, 5.99 mmol) and 2,4,6- collidine (0.97 mL, 5.99 mmol) were added at rt and stirred for 30 min. To the resulting solution, a solution of 12 (580 mg, 2.30 mmol) in DCM (20 mL) was slowly added dropwise, followed by addition of TMSI (0.36 mL, 2.53 mmol). The mixture was stirred at room temperature for 3 h and quenched with a saturated aqueous solution of Na2S2O3. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (1:4 EtOAc/Hexane as eluent) to give undesired α-isomer 14 (185 mg, 18%) as a white solid and desired β -isomer 13 (525 mg, 55%) as a white solid.1H NMR (500 MHz, CDCl3) δ 8.27 (brs, 1H), 7.91 (s,1H), 7.40-7.29 (m, 6H), 6.37-6.31 (m, 2H), 5.12 (s, 1H), 4.69-4.61 (m, 2H), 4.23 (d, J = 10.1 Hz, 1H), 4.15 (dd, J = 10.2 & 5.3 Hz, 1H), 3.84 (dd, J = 5.6 & 1.8 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 160.83, 149.26, 138.0, 136.93, 128.82, 128.52, 128.13, 127.94, 111.62, 110.01, 104.91, 81.44, 74.36, 71.99, 67.99; HRMS-ESI (m/z): [M +H]+ Calcd for C17H17BrN2O5Na 431.0219; Found 431.0209. [070] 5-((E)-2-bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-dioxolan-4- yl)pyrimidine-2,4(1H,3H)-dione (15): To a solution of 1-((2S,4S)-2-((benzyloxy)methyl)- 1,3-dioxolan-4-yl)-5-((E)-2-bromovinyl)pyrimidine-2,4(1H,3H)-dione, 13 (500 mg, 1.22 mmol) in DCM (2 mL) at -78 ℃, 1 M BCl3 in DCM (1.34 mL, 1.34 mmol) was added dropwise and stirred at same temperature for 30 min. After that, the reaction mixture was quenched with MeOH (1 mL) and warmed to room temperature and the inorganic salts were filtered. The organic layer was concentrated under reduced pressure and crude was purified by silica gel column chromatography (DCM/MeOH, 9:1) to give 1-((2S,4S)-2- ((benzyloxy)methyl)-1,3-dioxolan-4-yl)-5-((E)-2-bromovinyl)pyrimidine-2,4(1H,3H)-dione, 15 (235 mg, 60%) as a white solid. [^]23 1 D = -6.2 (c 0.2.7, MeOH); H NMR (500 MHz, CDCl3) δ 11.61 (brs, 1H), 8.16 (s, 1H), 7.22 (d, J = 13.6 Hz, 1H), 6.82 (d, J = 13.6 Hz, 1H), 6.21 (d, J = 5 Hz, 1H), 5.31 (t, J = 6.0 Hz, 1H), 4.97-4.94 (m, 1H), 4.31 (d, J = 9.9 Hz, 1H),
4.09 (dd, J = 9.9, 5.6 Hz, 1H), 3.74-3.67 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 162.25, 150.10, 140.10, 130.35, 110.18, 107.01, 105.83, 81.15, 71.07, 60.36; HRMS-ESI (m/z): [M +H]+ Calcd for C10H11BrN2O5Na 340.9749; Found 340.9738. [071] (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate (16): A suspension of (2S)-2- ((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate (11, 3.0 g, 12.60 mmol) and 5% Pd/C (150 mg) in MeOH (20 mL) at ambient temperature was treated with hydrogen at 8 psi in a parr hydrogenator for 3h. The mixture was diluted with methanol (20 mL) and passed through a celite bed. The filtrate was concentrated under reduced pressure to obtain (2S,4R)-2- (hydroxymethyl)-1,3-dioxolane-4-carboxylic acid 16 as a colorless thick liquid. Yield (1.72 g, 92%); 1H NMR (500 MHz, CDCl3) δ 5.26 (dt, J = 2.9, 11.0 Hz, 1H), 4.71 (ddd, J = 5.8, 7.3 & 17.4 Hz, 1H), 4.43-4.32 (m, 1H), 4.04 (ddd, J = 5.7, 8.4 & 29.7 Hz, 1H), 3.78-3.74 (m, 2H); HRMS-ESI (m/z): [M +H]+ Calcd for C5H8O5148.0372; Found 148.0364. [072] (2S,4R)-2-((isobutyryloxy)methyl)-1,3-dioxolane-4-carboxylic acid (17): To a solution of (2S,4R)-2-(hydroxymethyl)-1,3-dioxolane-4-carboxylic acid 16 (2.0 g, 13.50 mmol) in dry pyridine (15 mL) at 0 ℃, isobutyric anhydride (3.2 mL, 20.26 mmol) was added followed by addition of DMAP (8 mg) and mixture was stirred at rt for 16 h. The reaction mixture was diluted with DCM (60 mL), washed with water (2 X 25 mL) and finally with brine (25 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude was purified by flash silica gel column chromatography using 60-65% EtOAc/Hexane as eluent to give (2S,4R)-2-[(isobutyryloxy)methyl)]-1,3-dioxolane- 4-carboxylic acid 17 as a colorless liquid. Yield (2.60 g, 88%); 1H NMR (500 MHz, CDCl3) δ 5.38 (t, J = 3.7 Hz, 1H), 4.67 (dd, J = 5.1, 7.1 Hz, 1H), 4.30-4.25 (m, 1H), 4.18 (t, J = 3.6 Hz, 2H), 4.02 (dd, J = 5.1 & 8.4 Hz, 1H), 2.60 (p, J = 7.0 Hz, 1H), 1.18 (d, J = 7.0 Hz, 6H); 13C NMR (125 MHz, CDCl3) δ 173.04, 135.94, 128.88, 128.61, 128.30, 104.21, 74.34, 70.60, 67.30; HRMS-ESI (m/z): [M +H]+ Calcd for C9H14O6Na 241.0688; Found 241.0673. [073] (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate (18): To stirred solution of (2S,4R)-2-((isobutyryloxy)methyl)-1,3-dioxolane-4-carboxylic acid (5.0 g, 2.09 mmol) in 2- Me-THF (150 mL) was degassed with argon then added acetic acid (2.7 mL, 4.58 mmol), Mn(II)OAc ( 10.7 g, 6.19 mmol), diacetoxy iodobenzene (22.15g, 6.88 mmol) and again degassed for 3 mins. The reaction mixture under argon was heated to 65 °C for 4.5h. The reaction mixture cooled to rt and filtered through a celite bed. The obtained filtrate was diluted with EtOAc (160 mL); washed sequentially with saturated Na2S2O3 (150 mL), cold 2M NaHCO3 (150 mL) water (200 mL), and finally with saturated brine (150 mL). The
organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by flash silica gel column chromatography using 16-19% EtOAc/hexane as eluent to obtain a diastereomeric mixture of (2S)-4-acetoxy-1,3-dioxolan-2-yl)methyl isobutyrate (18). Yield (4.5 mg, 84%); 1H NMR (500 MHz, CDCl3) (mix of diastereomers) δ 6.34-6.28 (m, 1H), 5.35, 5.25 (t, J = 4.0 Hz, 1H), 4.33-3.90 (m, 4H), 2.58-2.51 (m, 1H), 2.04- 2.02 (m, 3H), 1.13-1.11 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 176.5, 176.5, 170.2, 170.1, 103.6, 102.6, 102.3, 94.5, 94.0, 71.3, 70.8, 64.1, 63.5, 63.4, 63.2, 33.8, 21.1, 21.0, 18.9, 18.8; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C10H16O6Na 255.0839; found 255.0834. [074] (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate (18) (Alternative Synthesis): To a solution of (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate 17 (1.5 g, 6.87 mmol) and dry pyridine (0.78 mL, 9.90 mmol) in acetonitrile, Pb(OAc)4 (3.80 g, 8.58 mmol) was added portion wise at 0 ℃ and stirred for 16 h at rt. After that, the reaction mixture was passed through a celite bed. The filtrate was diluted with EtOAc (100 mL), washed with water (50 mL) finally with brine (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by flash silica gel column chromatography (12-16% EtOAc/Hexane) to obtain (2S)-4-acetoxy-1,3-dioxolan-2- yl)methylisobutyrate 18 as a colorless liquid. Yield (1.23 g, 78%); 1H NMR (500 MHz, CDCl3) (mix of diastereomers) δ 6.34-6.28 (m, 1H), 5.35, 5.25 (t, J = 4.0 Hz, 1H), 4.33-3.90 (m, 4H), 2.58-2.51 (m, 1H), 2.04-2.02 (m, 3H), 1.13-1.11 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 176.5, 176.5, 170.2, 170.1, 103.6, 102.6, 102.3, 94.5, 94.0, 71.3, 70.8, 64.1, 63.5, 63.4, 63.2, 33.8, 21.1, 21.0, 18.9, 18.8; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C10H16O6Na 255.0839; found 255.0834. [075] 1-((2S,4S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl)-5-((E)-2- bromovinyl)pyrimidine-2,4(1H,3H)-dione (20): To a suspension of (E)-5-bromovinyluracil (350 mg, 1.62 mmol) in dry DCM (12 mL), TBDMSOTf (0.96 mL, 4.21 mmol) and 2,4,6- collidine (0.55 mL, 4.21 mmol) were added at room temperature. The reaction mixture was stirred for 30 min. After that, in the resulting solution, a solution of 18 (375 mg, 1.78 mmol) in DCM (10 mL), was added dropwise followed by addition of TMSI (0.25 mL, 1.78 mmol). The mixture was stirred at room temperature for 3 h and quenched with a saturated aqueous solution of Na2S2O3. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by silica gel column
chromatography (15-18% EtOAc/Hexane) to give undesired α-isomer 19 (140 mg, 22%) as a white solid and desired
-isomer 20 (340 mg, 54%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 8.27 (brs, 1H), 7.91 (s,1H), 7.40-7.29 (m, 6H), 6.37-6.31 (m, 2H), 5.12 (s, 1H), 4.69-4.61 (m, 2H), 4.23 (d, J = 10.1 Hz, 1H), 4.15 (dd, J = 10.2 & 5.3 Hz, 1H), 3.84 (dd, J = 5.6 & 1.8 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 160.83, 149.26, 138.0, 136.93, 128.82, 128.52, 128.13, 127.94, 111.62, 110.01, 104.91, 81.44, 74.36, 71.99, 67.99; HRMS-ESI (m/z): [M +H]+ Calcd for C17H17BrN2O5Na 431.0219; Found 431.0210. [076] 5-((E)-2-bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-dioxolan-4- yl)pyrimidine-2,4(1H,3H)-dione (15): To a stirred solution of 1-((2S,4S)-2- ((benzyloxy)methyl)-1,3-dioxolan-4-yl)-5-((E)-2-bromovinyl)pyrimidine-2,4(1H,3H)-dione (250 mg, 0.64 mmol) in MeOH (5 mL) in a sealed tube at 0 ℃, a 7 N solution of NH3 in MeOH was added and mixture was stirred at room temperature for 16 h. The volatiles were removed under removed pressure and the obtained residue was triturated with diethyl ether (2 X 5 mL) to give 1-((2S,4S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl)-5-((E)-2- bromovinyl)pyrimidine-2,4(1H,3H)-dione (L-BHDU, 15) (180 mg, 88%) as an off-white solid. [^]23 1 D = -6.1 (c 0.2.7, MeOH); H NMR (500 MHz, CDCl3) δ 11.61 (brs, 1H), 8.16 (s, 1H), 7.22 (d, J = 13.6 Hz, 1H), 6.82 (d, J = 13.6 Hz, 1H), 6.21 (d, J = 5 Hz, 1H), 5.31 (t, J = 6.0 Hz, 1H), 4.97-4.94 (m, 1H), 4.31 (d, J = 9.9 Hz, 1H), 4.09 (dd, J = 9.9, 5.6 Hz, 1H), 3.74- 3.67 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 162.25, 150.10, 140.10, 130.35, 110.18, 107.01, 105.83, 81.15, 71.07, 60.36; HRMS-ESI (m/z): [M +H]+ Calcd for C10H11BrN2O5Na 340.9749; Found 340.9741. [077] Bis(POM) prodrug of L-BHDU-MP (POM-L-BHDU-MP, 22): To a stirred solution of L-BHDU (15, 80 mg, 0.25 mmol) and N-methylimidazole (0.16 mL, 2.0 mmol) in dry THF (3 mL), bis(POM) phosphorochloridate25 21 (500 mg, 1.23 mmol) at 0 ℃ was added by dissolving in dry THF (3 mL) and stirred for 15 min. After that, reaction was warmed to rt and stirred for 3 h. Mixture was quenched with methanol and solvent were removed under reduced pressure. The crude was purified by silica gel column chromatography (0.5% MeOH/DCM) to give 22 as a colorless sticky oil which was crystallized in DCM-pentane to render as off-white solid. Yield: (75 mg, 47%). Mp: 80-85 ℃; 1H NMR (500 MHz, CDCl3) δ 8.97 (bs, 1H), 7.71 (s, 1H), 7.45 (d, J = 13.6 Hz, 1H), 6.79 (d, J = 13.6 Hz, 1H), 6.35 (d, J = 4.5 Hz, 1H), 5.72-5.65 (m, 4H), 5.15 (s, 1H), 4.43-4.39 (m, 1H), 4.35-4.31 (m, 1H), 4.26-4.21
(m, 1H), 4.20-4.17 (m, 1H), 1.23 (s, 18H); 31P NMR (202 MHz, CDCl ) δ 13 3 -3.02; C NMR (125 MHz, CDCl3) δ 176.9, 161.1, 149.5, 137.3, 128.4, 112.2, 110.6, 102.9, 83.2, 81.4, 71.6, 65.1, 38.9, 26.9; Anal. Calcd for C22H32BrN2O12P: C, 42.12; H, 5.14; N, 4.47. Found: C, 42.34, H, 5.23; N, 4.26; HRMS (EI) Calcd For (C + 22H32BrN2O12P+H) 627.0954, found m/z 627.0953. [078] Supporting Information [079] The supporting information may be found free of charge at the website pubs.acs.org. [080] Includes, X-ray crystallographic data and copies of 1H, 13C, & 19F NMR spectra of all intermediates and final compounds 3-28 (PDF). [081] Accession Codes [082] CCDC contains the supplementary crystallographic data for this paper. These data can be obtained free of charge at the website ccdc.cam.ac.uk/data_request/cif, by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: +441223336033. [083] References (1) Gershon, A. A.; Breuer, J.; Cohen, J. I.; Cohrs, R. J.; Gershon, M. D.; Gilden, D.; Grose, C.; Hambleton, S.; Kennedy, P. G. E.; Oxman, M. N.; et al. Varicella zoster virus infection. Nat Rev Dis Primers 2015, 1. DOI: ARTN 15016 10.1038/nrdp.2015.16. (2) Website: cdc.gov/shingles/hcp/clinical- overview.html#:~:text=The%20incidence%20for%20herpes%20zoster,per%20100%20U.S. %20population%20annually. DOI: Website: cdc.gov/shingles/hcp/clinical- overview.html#:~:text=The%20incidence%20for%20herpes%20zoster,per%20100%20U.S. %20population%20annually. (3) Breuer, J.; Whitley, R. Varicella zoster virus: natural history and current therapies of varicella and herpes zoster. Herpes 2007, 14 Suppl 2, 25-29. From NLM. (4) Sampathkumar, P.; Drage, L. A.; Martin, D. P. Herpes Zoster (Shingles) and Postherpetic Neuralgia. Mayo Clin Proc 2009, 84 (3), 274-280. DOI: Doi 10.4065/84.3.274. (5) Bharucha, T.; Ming, D.; Breuer, J. A critical appraisal of "Shingrix', a novel herpes zoster subunit vaccine (HZ/Su or GSK1437173A) for varicella zoster virus. Hum Vacc Immunother 2017, 13 (8), 1789-1797. DOI: 10.1080/21645515.2017.1317410.
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