Synthesis, reactivity and biological activity of 5-alkoxymethyluracil analogues

This review article summarizes the results of a long-term investigation of 5-alkoxymethyluracil analogues and is aimed, in particular, at methods of syntheses. Most of the presented compounds were synthesized in order to evaluate their biological activity, therefore, a brief survey of biological activity, especially antiviral, cytotoxic and antibacterial, is also reported.


Introduction
Modifications of nucleic acid components play a significant role in the field of nucleic acids research. In particular, nucleoside analogues find broad therapeutic applications in anticancer treatments and antiviral chemotherapy. In anticancer chemotherapy the huge amount of knowledge concerning processes taking place through the cell cycle has enabled researchers to break through and to understand the mechanisms of action of many anticancer agents. 5-Fluorouracil, for instance, was one of the first [1] and most investigated anticancer drugs, either chemically or biologically, and triggered the research of 5-substituted pyrimidine analogues.
The elucidation of the life cycle of a virus is crucial in antiviral chemotherapy. Several 5-substituted pyrimidine analogues capable of affecting the life cycle of viruses were discovered as highly active antiviral agents. Two such drugs with antiviral properties are 5-iodo-2'-deoxyuridine, discovered in the 1960s as the first agent that is active against Herpes simplex and Varicella zoster viruses [2,3], and 5-vinyl-2'-deoxyuridine, exhibiting high activity against HSV [4,5], which in turn led to studies on the synthesis and biological activity of its analogues.
From these pieces of knowledge, we draw inspiration for the development of new potent biologically active compounds; Compounds that might be more selective, more specific and much less toxic for organisms.
One of those groups of investigated derivatives is a group of uracil analogues modified at the 5 position by an ether or ester moiety. Since a vast number of C-5 modified pyrimidine analogues are known, this review is focused on a group of selected compounds with specific substituents (Figure 1) and most attention is paid to the studies on synthesis of selected derivatives. A brief survey of the biological activity of investigated compounds is also reported. The following chapters concerning the synthesis are arranged according to the products of synthetic routes.

Synthesis of alkoxy-haloalkyl derivatives
The most numerous and also the most investigated group of the above mentioned derivatives is a group of alkoxy-haloalkyl compounds derived either from uracil or nucleosides ( Figure 2). With regard to the high variability of sugar moiety, the description of all the compounds is divided into sections according to the nature of the furanose present.

2'-Deoxyuridine analogues:
The earliest article describing 2'-deoxyuridine analogues was focused on uracil analogues modified at position 5 by a fluorine containing moiety [6]. Bases or nucleosides substituted by fluorine have been investigated as potent anticancer agents since the 1960s. Nevertheless, many such modified compounds were also synthesized in order to investigate their antiviral activity. As a consequence of interest in biologically active fluoro derivatives, Bergstrom and co-workers carried out the synthesis of 5-(3,3,3-trifluoro-1methoxypropyl)-2'-deoxyuridine (1) (Figure 3) which was the first perfluoro derivative from the group of aforementioned compounds.
Shortly after publishing the successful synthesis of trifluoro nucleoside 1, Bergstrom and co-workers reported a presumed mechanism for its formation (Scheme 1) [8]. In addition, they also focused their research on isopropyloxy analogue 2.
Their synthesis was based on the addition of HOX (X = Br, Cl) to the vinyl moiety of 5-vinyl-2'-deoxyuridine (9). The reaction was carried out in aqueous dioxane, and hydroxybromoethyl 10 and hydroxychloroethyl 11 derivatives were obtained in 70% and 60% yields, respectively. Subsequent treatment of hydroxyl derivatives 10 and 11 with methanolic sulfuric acid gave the corresponding desired 5-(1-methoxy-2-haloethyl) derivatives 12 and 13 in 93 and 98% yields, respectively. No details of the separation method for the two diastereomers were described in their article.
In a search for new antiviral agents, 4'-C-methylpyrimidine nucleosides were synthesized (Scheme 13) and their biological activity evaluated [22]. Firstly, the 4'-C-methyl-D-ribose 84 was prepared by a previously described procedure [23]. Next 5-bromovinyluracil (BVUr) was silylated and reacted with 84 in the presence of TMSOTf as the Lewis acid. This was followed by deacetylation with anhydrous K 2 CO 3 in MeOH to provide the di-O-benzylated nucleoside 85 in 73% yield. For the change of the configuration at 2'-C, derivative 85 was converted to its mesylate and treated with NaOH in EtOH-H 2 O to afford 4'-Cmethylnucleoside 86 in 58% yield. Finally, nucleoside 86 was debenzylated with BBr 3 in CH 2 Cl 2 at −78 °C. On quenching of the reaction with MeOH the unexpected formation of methoxy derivative 87 was observed, whilst quenching with saturated NaHCO 3 solution gave the target derivative 88.

Synthesis of alkoxy-alkyl derivatives
The C-5 modified pyrimidine nucleosides with the short alkyl substituent have been at the center of intense interest since the early 1970s due to their potential chemotherapeutic and antiviral properties. It was reported, for instance, that 5-ethyluracil may undergo incorporation into bacterial DNA [25] and that 5-ethyl-2'-deoxyuridine readily replaces thymidine in bacteriophage DNA [26].
Bergstrom and co-workers also targeted the alkyl modification at position 5 of pyrimidine analogues [27] and synthesized 5-(1methoxyethyl)uridine (96) (Scheme 15) with a view towards transformation of the latter to the 5-ethyl analogue. In their reaction the organomercuri nucleoside 94 was converted to the organopalladium analogue via the reaction with 0.1 M palladium catalyst and ethene in methanol. Surprisingly, the major product of the reaction was the methoxy derivative 96 in 39% yield instead of the expected 5-vinyluridine (24). Similarly, the 2'-deoxyuridine organomercuri derivative 4 reacted with propene in the presence of Li 2 PdCl 4 in methanol to give 5-(1methoxypropyl)-2'-deoxyuridine (97) as one of the products but the compound was not separated from the reaction mixture.

Synthesis of acyloxy derivatives
The substitution at position 5 of the pyrimidine ring by acyloxy moiety provides another group of derivatives. Some of these compounds were synthesized as 1-(tetrahydrofuran-2-yl) pyrimidine analogues [33]. The use of such an atypical furanose ring avoids complications with the protection of hydroxyl groups of 2'-deoxyribose during the development of an appropriate method for acylation of the side chain hydroxyl group. The acyloxy derivatives 117 and 118 were synthesized in only a few steps (Scheme 19).
An oxidation of 5-vinyl-2'-deoxyuridine (9) was also studied (Scheme 20) [31]. The authors used m-chloroperbenzoic acid as an oxidizing agent and observed its influence on the reactivity of the vinyl substituent in the presence and absence of water. When the reaction is performed in the absence of water an epoxide should be obtained. Nevertheless, the authors instead observed a ring opening. However, the product was not fully characterized. As long as water was used, 2'-deoxy-5-(1,2-dihydroxyethyl)uridine (119) was obtained. This dihydroxy derivative 119 was characterized after the transformation to the acetyl analogue 120 using acetic anhydride in pyridine.
The introduction of a sugar moiety to the selected analogues 124f-i afforded 5-[alkoxy(4-nitrophenyl)methyl]uridines 126f-i and 127f-i (Scheme 22) [35]. In a first step, the alkoxy uracils 124 were silylated and then reacted with a protected ribose in

Synthesis of oligonucleotide intermediates
Modified oligonucleotides are powerful tools in nucleic acid research and their synthesis has become an important aspect of bioorganic and medicinal chemistry. One part of oligonucleotide chemistry associated with this review is focused on the studies of the action of 5-formyl-2'-deoxyuridine, which is one of the oxidative thymidine lesions of DNA formed by ionizing radiation. Consequently, several methods for the preparation of appropriate intermediates for the synthesis of oligodeoxynucleotides containing 5-formyl-2'-deoxyuridine have been published. Sugiyama and co-workers reported a seven-step synthesis of phosphoramidite 134 (Scheme 23, reac-tion conditions 1) starting with readily available 5-iodo-2'deoxyuridine (14) [36]. The first two steps of the synthesis involved the protection of 3',5'-dihydroxyl groups with the TBDMS group followed by a Pd-catalyzed coupling reaction with vinyl acetate to give the protected 5-vinyluridine 129 in 68% yield. Oxidation with OsO 4 with subsequent acetylation with acetic anhydride in pyridine gave nucleoside 131. The target phosphoramidite 134 was obtained after the standard phosphoramidite synthesis starting with the protection of the 5'-OH group with dimethoxytrityl chloride and final phosphitylation.
Later, Kittaka and co-workers reported the synthesis of phosphoramidite 134 under different conditions [37]. The protected 5-iodo-2'-deoxyuridine 128 was subjected to a Stille coupling reaction with tributyl(vinyl)tin using Pd(MeCN) 2 Cl 2 as a catalyst (Scheme 23, reaction conditions 2). This coupling Modified oligonucleotides can also serve as a tool for the investigation of interactions between NF-κB proteins (NF-κB is a protein complex that controls the transcription of DNA and plays a key role in regulating the immune response to infection). A study was reported by Kittaka and co-workers [38] which described an interaction between the above noted proteins and modified oligonucleotides, in which thymidine is replaced by a 5-formyl derivative. A phosphoramidite 145 for oligonucleotide synthesis was prepared from O 2 -2'-cyclouridine (135) by a multistep synthesis (Scheme 24). In a first step, O 2 -2'-cyclouridine (135) was selectively methylated at the 2'-O atom and subsequently iodinated at position 5 with CAN-I 2 in AcOH to give nucleoside 137 in 74% yield. Protection of 3',5'diol 137 by TBDMS groups (quantitative) afforded nucleoside 138 which was subsequently subjected to a Stille coupling reaction with tributyl(vinyl)tin using Pd(CH 3 CN) 2 Cl 2 as a catalyst followed by oxidation with OsO 4 /NMO to afford the dihydroxy derivative 141 in 77% yield after two steps. The desired phosphoramidite 145 was obtained in 90% yield after acetylation of the vicinal diol 141, selective deprotection of 3',5'hydroxyl groups (143 in 96% yield), dimethoxytritylation of the 5'-hydroxyl group (144 in 89% yield) and finally, 3'-Ophosphitylation.
An aryl moiety containing phosphoramidite, oligonucleotide 146 (Figure 8), was synthesized by Ding and co-workers and described its utilization of as a hole migration probe [39]. This compound should serve as a molecular probe that facilitates selective detection of excess electron transfer or hole migration in DNA using gel electrophoresis. The synthesis of the desired phosphoramidite 150 (Scheme 25) started with the Pd-catalyzed cross-coupling of 5-iodo-2'deoxyuridine (14) and a styrene to afford nucleoside 147. The oxidation of alkene function in 147 with OsO 4 led to a mixture of diastereomers of vicinal diols 148. For the introduction of the oligodeoxynucleotide 146, the dihydroxynucleoside 147 was converted to the corresponding phosphoramidite. This was carried out as follows. First, the hydroxyl groups of deoxyribofuranosyl moiety were silylated to give the protected nucleoside 149 which was then oxidized with OsO 4 to afford the protected vicinal diol. The free hydroxyl groups attached to the side chain at position 5 of the uracil ring were acetylated and the silyl protection groups at the sugar ring were removed by  the reaction with TBAF. Finally, the 5'-hydroxyl groups were tritylated and the 3'-hydroxy group converted to the corresponding phosphoramidite 150. The resulting phosphoramidite was incorporated into a 12-mer oligodeoxynucleotide via automated solid-phase synthesis.

Synthesis of bis heterocyclic derivatives
Sarfati and co-workers published an interesting and facile synthesis of C-5 alkylated 2'-deoxyuridine and uridine derivatives [40]. The C-5 position can be substituted by glycosides of either 2-acetamido-2-deoxy-β-D-glucopyranose or α-D-mannopyranose. All the products 151-154 ( Figure 9) were formed as by-products of the palladium catalyzed addition reaction of alkenes to C-5-mercuriated deoxyuridines.
The synthesis of derivatives 151 and 152 started with condensation reactions of alkenes 155 and 156 with 5-chloromercuri-2'-deoxyuridine 4 in the presence of a palladium catalyst (Scheme 26).
The vinyl derivatives 157 and 158 were obtained as major products. However, methoxy derivatives 151 and 152 were produced in modest yields. The monophosphate derivatives 153 and 154 were formed by a similar reaction with mercuriated 2'-deoxyuridine monophosphates.
Almost 10 years earlier, Bergstrom and co-workers published a synthesis based on the same reaction -the Heck cross-coupling reaction of an alkene with an organometallic derivative [41] in

Synthesis of metallocenonucleosides
The first "metallocenonucleosides" were synthesized and characterized by Meunier and co-workers in 1991 [42]. The term "metallocenonucleosides" was derived from nucleosides containing a metallocene moiety and these compounds were prepared in order to study their chemical as well as cytotoxic properties (Scheme 28). The reported work was focused on a group of nucleosides with the formula: a) Ns-CH=CH-Fc or b) Ns-CH 2 -CH 2 -Fc, where Ns (= nucleoside) is either uridine (derivatives 168, 169) or 2'-deoxyuridine (derivatives 165, 166), and Fc is the abbreviation of ferrocene of molecular formula C 5 H 4 FeC 5 H 5 . From the reaction of 5-(chloromercuri)-nucleosides 4 or 94 with ethenylferrocene, methoxyderivatives 164 or 167 were also formed along with nucleosides 165, 166, 168 and 169.
Subsequent cyclization of both isomers in hydrochloric acid gave α-and β-furanose forms of pseudouridine 174 and 175, respectively ( Figure 10). Other studies on the synthesis of pseudouridine analogues were made by Lee and co-workers 20 years later [48]. The 5'-modified pseudouridine 176 and secopseudouridines 177 and 178 were prepared via the ring cleavage of the sugar moiety ( Figure 11).

Biological activity
A number of the previously mentioned compounds were synthesized in order to evaluate their antiviral and cytotoxic activity. Moreover, antibacterial activity of some of these derivatives has also been studied. Some of the tested compounds have shown interesting results and a brief survey is given in the following section.

Cytotoxic activity
Only a few derivatives have been tested for their anticancer properties. The cytotoxic activity for derivatives 12, 13 and 28 ( Figure 12) were determined by an in vitro L1210 assay [9,10]. However, a comparison of the results for the investigated compounds with those of the reference compound melphalan showed lower activity.
Recent studies on cytotoxic activity of 5-[alkoxy-(4-nitrophenyl)methyl]uracil analogues 124, 126 and 127 ( Figure 18) have been published [34,35]. All of the prepared compounds were tested for their cytotoxic activity in vitro against different cell lines and relationships between structure and cytotoxic activity were evaluated. Although all of the tested compounds exhibited weaker activity than reference carboplatin or 6-thioguanine, interesting relationships between activity and length of alkyl chain were observed.

Antibacterial activity
The recurrence of the chronic infectious disease tuberculosis has initiated research on new classes of antimycobacterial agents. The exigency of new drugs was also caused by multidrug-resistant tuberculosis strains, which are resistant to the most widely used agents, either Isoniazid or Rifampicin, and the need for new highly active compounds is increasing. Tuberculosis is caused by species of the genus Mycobacterium, for instance, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium bovis.
Recently, a study on the effect of arabinofuranosyl analogues against Mycobacterium was published [19]. A series of 1-β-D-2'-arabinofuranosyl pyrimidine nucleosides was prepared in order to evaluate their antimycobacterial activity. Amongst others, the methoxyiodoethyl pyrimidine nucleoside 79  ( Figure 19) was synthesized. Nevertheless, this nucleoside did not show any significant antimycobacterial activity.
In addition to this, nucleosides containing a dodecynyl moiety instead of an alkoxyhaloethyl group proved to be significantly active. The introduction of longer alkynyl chains might be a successful way to obtain potentially active antimycobacterial drugs.

Conclusion
This review was an attempt to summarize all available information on the synthesis and biological activity of selected C-5 substituted pyrimidine derivatives. Many authors have reported facile and successful syntheses by a large range of methods to obtain the desired compounds, and, in addition, they have also highlighted ineffectual synthetic routes. Most of the published derivatives were biologically inactive; although some exhibited weak activity. However, all of these results have made a significant and invaluable contribution to the development of new potent antiviral, cytotoxic or antibacterial agents and have elucidated possible structure-activity relationships.