Methoxymethyl (MOM) Group Nitrogen Protection of Pyrimidines Bearing C-6 Acyclic Side-Chains

Novel N-methoxymethylated (MOM) pyrimidine (4−13) and pyrimidine-2,4-diones (15−17) nucleoside mimetics in which an isobutyl side-chain is attached at the C-6 position of the pyrimidine moiety were synthesized. Synthetic methods via O-persilylated or N-anionic uracil derivatives have been evaluated for the synthesis of N-1- and/or N-3-MOM pyrimidine derivatives with C-6 acyclic side-chains. A synthetic approach using an activated N-anionic pyrimidine derivative afforded the desired N,N-1,3-diMOM and N-1-MOM pyrimidines 4 and 5 in good yield. Introduction of fluorine into the side-chain was performed with DAST as the fluorinating reagent to give a N,N-1,3-diMOM pyrimidine 13 with a 1-fluoro-3-hydroxyisobutyl moiety at C-6. Conformational study of the monotritylated N-1-MOM pyrimidine 12 by the use of the NOE experiments revealed the predominant conformation of the compound to be one where the hydroxymethyl group in the C-6 side-chain is close to the N-1-MOM moiety, while the OMTr is in proximity to the CH3-5 group. Contrary to this no NOE enhancements between the N-1-MOM group and hydroxymethyl or fluoromethyl protons in 13 were observed, which suggested a nonrestricted rotation along the C-6 side-chain. Fluorinated N,N-1,3-diMOM pyrimidine 13 emerged as a model compound for development of tracer molecules for non-invasive imaging of gene expression using positron emission tomography (PET).


Introduction
The pyrimidine moiety is a widespread heterocyclic unit which is found in several biologically active natural products, as well as synthetic pharmacophores with biological activities that show considerable therapeutic potential [1][2][3][4][5]. The structural diversity and biological importance of pyrimidines have made them attractive targets for synthesis over the years. For this reason numerous analogues and derivatives of pyrimidines have been synthesized and developed as pharmacologically active compounds or drugs [6,7].
Many N-substituted uracil derivatives have exhibited extremely diverse physiological activity [8]. It was shown that N- 1 and/or N-3-alkylated pyrimidine derivatives had a wide range of antiviral activity [9][10][11][12][13][14]. A large number of thymidine analogues and acyclic guanosine derivatives showed antiviral activities against herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) [15]. The antiviral activity of these compounds is due to their selective and efficient in vivo activation through monophosphorylation by the viral enzyme [16,17]. The monophosphates are converted to diphosphates, and then to the corresponding triphosphates by cellular enzymes. The triphosphates prevent viral replication by inhibition of the viral DNA polymerase [18]. The molecular basis of the therapy, which uses viral thymidine kinase (TK), is the difference in substrate specificity between the herpes viral TK and the human cellular isoenzyme. Therefore, HSV-1 TK in combination with nucleoside analogue as fraudulent substrates can be used as suicide enzymes in gene therapy of cancer [19][20][21]. Furthermore, these compounds labelled with positron-emitting radioisotopes can be used as in situ reporter probes to allow non-invasive imaging of HSV-1 TK gene expression using positron emission tomography (PET) [22][23][24][25]. The pronounced biological activities exhibited by C-6 substituted pyrimidine derivatives provide a good rationale for further exploration of the chemistry and biological activities of these compounds [26][27][28][29][30]. Thus, we have synthesized nucleoside mimetics in which acyclic sugar moiety is attached at the C-6 position. Some C-6 fluoroalkylated pyrimidines exhibited pronounced cytostatic activities [31][32][33], while thymines with 6-(2,3-dihydroxypropyl) and 6-(1,3-dihydroxyisobutyl) side-chains have been developed as tracer molecules for monitoring of HSV-1 TK expression by means of PET [34][35][36]. Our investigations were prompted by the need to develop PET imaging agents which lack the disadvantages of already existing reporter probes of HSV-1 TK, such as 9-[4-[ 18 F]fluoro-3-(hydroxymethyl)butyl]guanine ([ 18 F]FHBG) which shows cytotoxicity and unfavorable pharmaco-kinetics [37]. In our previous work we described the compound 6-(1,3-dihydroxyisobutyl)thymine (DHBT) and discussed its advantages over existing compounds [35]. The molecular modeling and the X-ray structure of HSV-1 TK in complex with N-methylated DHBT gave new insights for the design and synthesis of further C-6 substituted pyrimidine derivatives with differentiated pharmacokinetics [37].
In the view of the facts mentioned above and in continuation of our previous work on development of tracer molecules for non-invasive imaging of gene expression using PET, we have now prepared new N-methoxymethylated (MOM) C-6 acyclic pyrimidine derivatives. Thus, herein we report syntheses of novel N-1and/or N-3-MOM pyrimidines 4−13 and pyrimidin-2,4-diones 15−17 bearing C-6 isobutyl side-chains, as well as a bicyclic pyrimido [1,6-c] [1,3]oxazepine derivative 14.
In our search for a more efficient synthesis of compounds 4 and 5 an alternate method was applied using activated N-anionic uracil. Potassium carbonate was used as deprotonating agent and the thus in situ obtained uracil salt reacted with MOMCl in DMF to give N,N-1,3-diMOM, N-1-MOM and N-3-MOM pyrimidine derivatives 4-6 in 28.5%, 12.1% and 15.4% yields, respectively. The yields of 4 and 5 synthesized by the various methods are sumarized in Table 1.

NMR Studies
The chemical identities of compounds 2−17 were confirmed by 1 H-, 13 C-and 19 F-NMR measurements. Proton and fluorine NMR chemical shifts, as well as carbon NMR chemical shifts are reported in the Experimental section. N-1 and N-3 regioisomers were identified on the basis of heteronuclear 1 H− 13 C correlation signals in 2D HMBC spectra and homonuclear 1 H− 1 H correlations in NOESY spectra. NOESY cross-peaks demonstrate dipole-dipole interactions of nearby protons that are spatially within ca. 5 Å. Correlation signals were observed between methylene protons of MOM group (δ 5.19 ppm) and H-1' (δ 2.75 ppm) as well as H-2' (δ 2.20 ppm) in 5, which suggested that MOM group is bound

General
All solvents were dried/purified following recommended drying agents and/or distilled over 3 Å molecular sieves. For monitoring the progress of a reaction and for comparison purposes, thin layer chromatography (TLC) was performed on pre-coated Merck silica gel 60F-254 plates using an appropriate solvent system and the spots were detected under UV light (254 nm). For column chromatography silica gel (Fluka, 0.063-0.2 mm) was employed, glass column was slurry-packed under gravity and eluents were CH 2 Cl 2 /MeOH mixtures. Melting points (uncorrected) were determined with Kofler micro hot-stage (Reichert, Wien). 1 H-, 13 C-and 19 F-NMR spectra were acquired on a Bruker 300 MHz and Varian Unity Inova 300 MHz NMR spectrometers. All data were recorded in DMSO-d 6 at 298 K. Chemical shifts were referenced to the residual solvent signal of DMSO at δ 2.50 ppm for 1 H and δ 39.50 ppm for 13 C. 19 F-NMR chemical shifts were referenced externally with respect to CCl 3 F (δ 0.0 ppm). Individual resonances were assigned on the basis of their chemical shifts, signal intensities, multiplicity of resonances and H−H coupling constants. NOESY spectra were acquired with mixing time of 150 ms. Mass spectra were recorded on an Agilent 6410 instrument equipped with electrospray interface and triple quadrupole analyzer (LC/MS/MS). High performance liquid chromatography was performed on an Agilent 1100 series system with UV detection (photodiode array detector) using Zorbax C18 reverse-phase analytical column (2.1 × 30 mm, 3.5 µm). 6-[(3-Benzyloxy-2-benzyloxymethyl-2-hydroxy)propyl]-5-methyl-2,4-dimethoxypyrimidine (1) and 6-[(3-benzyloxy-2benzyloxymethyl)propyl]-5-methyl-2,4-dimethoxypyrimidine (2) were synthesized using analogous procedures as described previously [35,38].

Conclusions
In summary, we have adopted simple and efficient methods for the protection and deprotection of the carbonyl and nitrogen moieties in a pyrimidine ring, as well as hydroxyl groups in a C-6 isobutyl side-chain under mild conditions in moderate to excellent yields. The methoxymethyl (MOM) moiety as protecting group was introduced using different synthetic methods. Two methods performed by silylation of uracil and in situ reaction of O-persilylated uracil with MOMCl gave N-1and/or N-3-MOM pyrimidine derivatives 4−6. A synthetic approach using activated an N-anionic pyrimidine derivative afforded desired N,N-1,3-diMOM and N-1-MOM pyrimidines 4 and 5 in good yield. N-1 and N-3 regioisomers were assigned on the basis of heteronuclear 1 H− 13 C correlation signals in 2D HMBC spectra and homonuclear 1 H− 1 H correlations in NOESY spectra. Thus, NOE interactions between the methylene protons of a MOM group and H-1' as well as H-2' in 5 revealed that the MOM group is bound to N-1 of the pyrimidine ring. The removal of benzyl protecting groups in 4 and 5 was accomplished using boron trichloride to give 6-(1,3-dihydroxyisobutyl)-N-MOM pyrimidines 7 and 8 as a major products. Pyrimidine derivatives 7, 8 and 15 with free hydroxyl functionalities were subsequently converted to ditritylated (compounds 10 and 16) and monotritylated (compounds 11, 12 and 17) derivatives. It is interesting to note that debenzylation of 4 and 5 and tritylation of 7 was accompanied with removal of the N-MOM protecting group. For preparation of precursor for 18 F radiolabelling that contains appropriate leaving groups, introduction of mesylate, instead of tosylate, as less bulky group is foreseen.