C-5 hydroxyethyl and hydroxypropyl acyclonucleosides as substrates for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK): syntheses and biological evaluation.

The efficient syntheses of 5-(2-hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains are reported. A synthetic approach that included the alkylation of an N-anionic-5-substituted pyrimidine intermediate (method A) provided the target acyclonucleosides in significantly higher overall yields in comparison to those obtained by method B using sylilation reaction. The phosphorylation assays of novel compounds as potential substrates for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK) showed that solely pyrimidine 5-substituted acyclonucleosides with a penciclovir-like side chain acted as a fraudulent substrates of HSV-1 TK. Moreover, the uracil derivative with penciclovir-like side chain with less bulky 2-hydroxyethyl substituent at C-5 proved to be a better substrate than the corresponding one with a 3-hydroxypropyl substituent. Therefore, this acyclonucleoside was selected as a lead compound for the development of a positron emission tomography HSV-1 TK activity imaging agent.

1. Introduction

Nucleoside analogues have acquired an impon class="Chemical">rtant role as therapeutic agents in the field of chemotherapy on account of their extensive biological activities [1,2]. Thereby, modified nucleosides and nucleobases are a pharmacologically diverse family, which includes anticancer compounds, antiviral agents, and immunosuppressive molecules. Interest in acyclic nucleosides began in the mid-1970s when acyclovir was first reported as a potent anti-herpes drug [3,4]. Many variations, both of the acyclic glycone and of the heterocyclic base, have been described [5,6]. Antiviral nucleoside analogues are known to localize selectively in herpes simplex virus (HSV)-infected cells because of monophosphorylation catalyzed by virus-encoded thymidine kinase (TK) [7]. Among these, acyclovir, ganciclovir and penciclovir are reported to be potent against HSV types 1 and 2 [8,9] (Figure 1).

Figure 1

Acyclic nucleoside analogues and 2'-deoxyuridines as chemotherapeutic agents.

In another area, a great number of 5-substituted uracil derivatives, especially n class="Chemical">2'-deoxyuridines, have been investigated for the experimental and clinical treatment of neoplastic and viral diseases (Figure 1). The selective monophosphorylation of antiviral nucleoside analogues by virus-encoded thymidine kinase has been exploited in gene therapy of cancer [10]. Gene therapy offers promising new treatment modalities in oncology and other diseases [11]. The suicide gene, HSV-1 TK, has already been successfully employed in a variety of tumour models, both in vitro and in vivo [12,13]. A prodrug with low toxicity becomes toxic only in cells where the HSV-1 TK gene is expressed, thus resulting in the selective killing of transfected cells through termination of DNA synthesis. Phosphorylation of radioactive-labelled prodrugs, usually derivatives of acyclonucleosides which are selective anti-herpes agents, leads to metabolites trapped within the infected tumour cells, and the resulting accumulation of radioactivity allows monitoring of the enzyme activity with positron emission tomography (PET) [14,15,16]. A striking difference is that while all pyrimidine nucleoside analogues show low, if any, bystander killing effects, purine derivatives such as ganciclovir are endowed with bystander killer effect [17].

As found earlier, pyrimidine nucleoside analogues in which the acyclic n class="Chemical">sugar moiety is attached at the C-6 rather than at the N-1 position have the potential for the development as a new PET imaging probe. Therefore, thymines with 6-(2,3-dihydroxypropyl), 6-(1,3-dihydroxyisobutyl) and 6-(1,3-dihydroxyisobutenyl) side chains have been developed as tracer molecules for monitoring HSV-1 TK expression by means of PET [18,19,20]. Although the N-methylated thymine derivative, N-Me-[18F]FHBT, bearing a 1,3-dihydroxyisobutyl side-chain at the C-6 position was not superior to the purine analogue [18F]FHBG, an established PET reporter probe for imaging HSV-1 TK expression, N-Me-[18F]FHBT clearly showed the feasibility of its use as a PET probe to monitor HSV-1 TK gene expression in vivo [21].

In an effort to fun class="Chemical">rther explore the pyrimidine scaffold as a putative starting point for the development of new fraudulent substrates of HSV-1 TK for non-invasive imaging of HSV-1 TK gene expression with improved pharmacodynamic and pharmacokinetic profile, we have synthesized a series of novel 5-substituted acyclic nucleoside analogues. Here we report on the synthesis of target 5-(2-hydroxyalkyl)-substituted uracil derivatives bearing 2,3-dihydroxypropyl (19), acyclovir- (21), ganciclovir- (23) and penciclovir-like (31 and 32) substituents at N-1 position of the pyrimidine ring (Figure 2). In order to evaluate target nucleoside mimetics 19, 21, 23, 31 and 32 as substrates for HSV-1 TK, molecular modelling and phosphorylation assays of these compounds were performed.

Figure 2

Novel 5-(2-hydroxyalkyl)-substituted uracil derivatives bearing 2,3-dihydroxy-propyl (19), acyclovir- (21), ganciclovir- (23) and penciclovir-like (31 and 32) side chains.

2. Results and Discussion

2.1. Chemistry

5-(2-Hydroxyalkyl)uracil derivatives with n class="Chemical">2,3-dihydroxypropyl (19), acyclovir- (21), ganciclovir- (23) and penciclovir-like (31 and 32) side chains were prepared from the corresponding uracil derivatives 10 and 14–16 by the methods outlined in Scheme 1, Scheme 2, Scheme 3. The synthesis of compounds 10 and 14–16, as precursors for N-alkylation, was performed in excellent yields (80–99%) (Scheme 1). 5-(2-Hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted uracils 1 and 2 served as starting materials and their syntheses were reported earlier [22,23,24]. The synthesis of 5-substituted 2,4-dimethoxypyrimidine derivatives 10 and 11 was carried out by acetylation, chlorination at the positions C-2 and C-4 with POCl3, methoxylation with sodium methoxide and repeated acetylation [25] (Scheme 1).

Scheme 1

Synthesis of the precursors 10 and 14–16 for N-alkylation.

Scheme 2

Synthesis of 5-(2-hydroxyethyl)uracil derivatives with 2,3-dihydroxypropyl (19), acyclovir- (21) and ganciclovir-like (23) side chain.

Scheme 3

Synthesis of 5-(2-hydroxyalkyl)uracil derivatives 31 and 32 with penciclovir-like side chains.

Reagents and conditions: (i) acetic anhydride, pyridine, rt, 1 h; (ii) POCl3, N,N-diethylaniline, pyridine, reflux, 1 h; (iii) NaOMe/MeOH, rt, 20 h; (iv) AcCl, rt, 20 h; (v) silica gel column chromatography; (vi) NaOMe/MeOH, rt, 1 h.

Selective 2-dealkylation of 5-(acetoxyalkyl)-2,4-dimethoxypyrimidines [26] 10 and 11 in non-aqueous acid gave the corresponding n class="Chemical">4-methoxypyrimidin-2-one derivatives 14 and 15. Treatment of the acetyl protected 2,4-dimethoxypyrimidines 10 and 11 with acetyl chloride afforded N-acetylated 4-methoxypyrimidin-2-one derivatives 12 and 13, which after N-deprotection and purification using column chromatography on silica gel yielded 14 and 15 (Scheme 1). Final deacetylation of 14 gave 4-methoxypyrimidin-2-one 16 with a 2-hydroxyethyl substituent at C-5.

Further synthetic steps in the preparation of target compounds 19, 21 and 23 involved n class="Chemical">N-alkylation applied by two synthetic methods (A and B, Scheme 2, Table 1). In order to ensure the introduction of the substituent into position N-1 of the pyrimidine ring, the Hilbert-Johnson reactions [27] were applied in synthetic method A. These reactions involved the treatment of 5-(2-acetoxyethyl)-2,4-dimethoxypyrimidine derivative 10 with alkyl halides. Allyl bromide and 1,3-dibenzyloxy-2-chloromethoxypropane [28] were used as agents for the introduction of 2,3-dihydroxypropyl (in 19) and ganciclovir-like side chain (in 23), respectively. Furthermore, the treatment of 1,3-dioxolane with acetyl bromide gave (2-acetoxyethoxy)methyl bromide [29] as a readily distilled colourless oil, which served as the alkylating reagent for the introduction of the acyclovir-like side chain (in 21). Unlike alkylation with allyl bromide which gave N-1-substituted 4-methoxypyrimidine 17 in 73% yield, alkylation using (2-acetoxyethoxy)methyl bromide and 1,3-dibenzyloxy-2-chloromethoxypropane was accompanied by 4-demethylation and the corresponding N-1-substituted pyrimidin-2,4-dione derivatives 20 and 22 were isolated in moderate yield (43% and 36%, respectively).

Table 1

An overview of the yields in synthetic method A and B for target compounds 19, 21 and 23.

Synthetic method A		19	21	23
		Yields
1.	Acetylation	98%	98%	98%
2.	Chlorination	99%	99%	99%
3.	Methoxylation	95%	95%	95%
4.	Acetylation	90%	90%	90%
5.	Alkylation/Hydroxylation	73%/50%	43%	36%
6.	Deprotection	39%	74%	67%
	Overall yield:	12%	26%	20%
Synthetic method B		19	21	23
		Yields
1.	Acetylation	98%	98%	-
2.	Chlorination	99%	99%	-
3.	Methoxylation	95%	95%	-
4.	Acetylation	90%	90%	-
5.	Demethoxylation	80%	80%	-
6.	Deacetylation	-	93%	-
7.	Silylation/Akylation	75%	25%	8%
8.	Deprotection	37%	26%	65%
	Overall yield:	18%	4%	5%

Reagents and conditions: (i) allyl bromide; (ii) NaClO3, OsO4; (iii) 1 M NaOH, rt, 20 h; (iv) (2-acethoxyethoxy)methyl bromide, Na2CO3, rt, 24 h; (v) NaOMe/MeOH, rt, 20 h; (vi) 1,3-dibenzyloxy-2-chloromethoxypropane, K2CO3, CH2Cl2, rt, 24 h; (vii) BCl3, CH2Cl2, 3 h; (viii) NaH, DMF, 1-chloro-2,3-dihydroxypropane; (ix) BSA, CH3CN, dioxolane, KI, TMSCl, rt, 48 h; (x) 1. HMDS, TMSCl; 2. 1,3-dibenzyloxy-2-chloromethoxypropane, TBAI, CH3CN, rt, 24 h.

cis-Dihydroxylation of N-1-allyl pyrimidine derivative 17 with sodium chlorate in the presence of osmium tetroxide afforded N-1-(2,3-dihydroxypropyl)pyrimidine derivative 18 which after the 4-demethylation and deacetylation with sodium hydroxide yielded the desired 5-(2-hydroxyethyl)pyrimidine 19 bearing a 2,3-dihydroxypropyl group at N-1. The overall yield of this method was 12%. The deacetylation of the side chains at C-5 and N-1 of 20 under basic conditionsafforded the 5-(2-hydroxyethyl)pyrimidine 21 with the best overall yield of 26%. Similarly, both deacetylation in C-5 and debenzylation in N-1 side chains of 22 using boron trichloride gave the 5-(2-hydroxyethyl)-substituted pyrimidine 23 with a ganciclovir-like side chain at N-1.

In search for a more efficient synthesis of 19, an alternate method B was applied using sodium hydride as deprotonating agent and thus the n class="Chemical">uracil salt obtained in situ reacted with 1-chloro-2,3-dihydroxypropane to give trihydroxy 4-methoxypyrimidin-2-one 24. Deprotection of 24 yielded target the trihydroxypyrimidin-2,4-dione 19. In comparison to synthetic method A this method for preparation of 19 is more efficient and the use of the highly poisonous reagent osmium tetroxide for cis-hydroxylation was avoided. Similarly, compounds 21 and 23 were also prepared by silylation reactions (synthetic method B). Since silylation of 5-(2-hydroxyethyl)uracil (1) using N,O-bis-(trimethysilyl)acetamide (BSA) was unsuccessful, pyrimidin-2-one 16 was used for N-alkylation. Trimethylsilylation of 16 [26] using BSA and in situ coupling employing trimethylchlorosilane, potassium iodide and 1,3-dioxolane in dry acetonitrile was performed, and the expected product 21 was obtained after hydrolysis of 25 with sodium hydroxide (Scheme 2). Furthermore, compound 23 was prepared directly from 5-(2-hydroxyethyl)uracil (1) by the silylation of 1 and in situ reaction of O-persilylated 1 with 1,3-dibenzyloxy-2-chloromethoxypropane to give N-1-alkylated pyrimidine 26 in 8% yield (method B). The removal of the benzyl protecting groups in 26 proceeded smoothly and led to target compound 23.

For the introduction of a penciclovir-like side chain at n class="Chemical">N-1 position of the pyrimidine ring, 4-acetoxy-(3-acetoxymethyl)butyl iodide [30,31], prepared from a mesylate and the related bromide, was used. Since neither direct Hilbert-Johnson-type reaction of 2,4-dimethoxypyrimidine derivative 10 with both alkyl halides and mesylate did not yield the corresponding pyrimidine acyclonucleoside, an alternative method using 4-methoxypyrimidin-2-one derivatives 14 and 15 was applied.

Thus, the reaction of potassium salts of 14 and 15 with 4-acetoxy-(3-acetoxymethyl)butyl iodide led to the target 5-(2-hydroxyethyl)- 31 and 5-(3-hydroxypropyl)pyrimidine 32 bearing a penciclovir-like side chain (Scheme 3).

Reagents and conditions: (i) 4-Acetoxy-(3-acetoxymethyl)butyl iodide, K2CO3, DMF, 60 °C, 20 min; (ii) 1 M NaOH, rt, 20 h.

2.2. In Vitro Validation of Compounds 19, 21, 23, 31 and 32 as Substrates of HSV-1 TK and hTK

To determine whether compounds 19, 21, 23, 31 and 32 are substrates of n class="Species">HSV-1 TK or human TK, phosphorylation assays were performed by incubation of deoxythymidine (dT) and target compounds with recombinant HSV-1 TK and human TK (hTK) according to a previously published protocol [32]. The monophosphorylation was monitored qualitatively by the formation of new peaks corresponding to adenosine diphosphate (ADP), monophosphate derivatives of tested compounds and thymidine monophosphate (dTMP). Results of phosphorylation assays for 19, 21 and 23 indicated that these compounds are not phosphorylated by either HSV-1 TK or human TK (see Supplementary Materials). While acyclonucleosides containing dihydroxypropyl (19), acyclovir- (21) and ganciclovir-like side chain (23) were not phosphorylated, both 5-substituted acyclic pyrimidines 31 and 32 with a penciclovir-like side chain showed to be substrates of HSV-1 TK. Furthermore, small alteration in structures of 23, such as replacement of oxygen in the (1,3-dihydroxy-2-propoxy)methyl side chain at N-1 with a methylene group (compound 31), has an impact on phosphorylation reaction of these compounds. We can assume that the electron donating oxygen has an influence on the interaction of 23 and HSV-1 TK disabling the enzyme to phosphorylate fraudulent substrate 23 with a ganciclovir-like side chain. Therefore, for compounds 31 and 32 while using viral HSV-1 TK, the formation of monophosphate derivatives 31-MP (Figure 3) and 32-MP (Figure 4) as well as ADP were detected, confirming that 31 and 32 are substrates of viral HSV-1 TK. However, the size of the alkyl substituent at C-5 exhibited some effect on phosphorylation. Thus, compound 31 with a less bulky 2-hydroxyethyl substituent at C-5 had a higher ADP/ATP ratio compared to compound 32, thus proving to be a better substrate than 32. Furthermore, formation of 31-MP and 32-MP was proven by extracting the max. absorbance wavelength of the new appearing peaks which corresponds to the max. absorbance wavelength of compounds 31 and 32 (Figure 3, Figure 4). No phosphorylation was observed for compounds 31 and 32 when human TK was used, indicating that these two compounds are not substrates for hTK.

Figure 3

HPLC and DAD/HPLC chromatograms of the reaction of 31 and HSV-1 TK.

Figure 4

HPLC and DAD/HPLC chromatograms of the reaction of 32 and HSV-1 TK.

The phosphorylation patterns of compounds 31 and 32 by HSV-1 TK and hTK are presented in Figure 5a. n class="Chemical">No significant difference in ADP/ATP ratio was noticed in phosphorylation assays with hTK.

Figure 5

(a) Phosphorylation pattern of 1 mM of compounds 31 and 32, by HSV-1 TK and hTK, and of compound 23 and dT by HSV-1 TK. Blank refers to the control experiment performed in presence of the enzyme and in absence of the compound; (b) Time course for conversion of 31 and its fluorinated analogue to corresponding monophosphated derivatives.

Phosphorylation reaction of compound 31 and its fluorinated structural analogue, in which hydroxyl group in C-5 substituent is replaced with n class="Chemical">fluorine, was performed at 30, 60 and 90 min of incubation (Figure 5b). It is evident that formation of monophosphated derivatives for both compounds was increased over time. By comparison of calculated values for monophosphated derivatives which were formed at different times (30, 60 and 90 min), we can estimate that approximately 40% of 31-MP was formed in 30 min and majority of 31-MP (~80%) in 60 min of incubation. On the contrary, fluorinated derivative gave majority of corresponding monophosphate (~70%) already in 30 min and was almost completely monophosphorylated in 60 min. The synthesis of fluorinated derivative of 31 and in vivo results on small animal PET studies of 18F labeled compound 31, HHB-5-[18F]FEP, will be presented elsewhere.

(a) Phosphorylation pattern of 1 mM of compounds 31 and 32, by HSV-1 TK and hTK, and of compound 23 and n class="Chemical">dT by HSV-1 TK. Blank refers to the control experiment performed in presence of the enzyme and in absence of the compound; (b) Time course for conversion of 31 and its fluorinated analogue to corresponding monophosphated derivatives.

2.3. Cellular Activity Evaluation

Compounds 17–32 were evaluated for their cytotoxic activity against normal human fibroblasts (WI38). The results indicated that the target compounds exhibited no cytotoxic effects against normal n class="Species">human fibroblasts up to 100 µM.

3. Experimental

3.1. General

Melting points (uncorrected) were determined with a Kofler micro hot-stage (Reichert, Wien, Austria). Precoated Merck (Darmstan class="Chemical">dt, Germany) silica gel 60F-254 plates were used for thin layer chromatography and the spots were detected under UV light (254 nm). Column chromatography was performed using Fluka (Buchs, Switzerland) silica gel (0.063–0.2 mm); glass columns were slurry-packed under gravity. 1H- and 13C-NMR spectra were acquired on a Bruker 300 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) All data were recorded in DMSO-d6 at 298 K. Chemical shifts were referenced to the residual solvent signal of DMSO at δ 2.50 ppm for 1H and δ 39.50 ppm for 13C. Individual resonances were assigned on the basis of their chemical shifts, signal intensities, multiplicity of resonances and H−H coupling constants. Mass spectra were recorded on an Agilent 6410 instrument (Agilent Technologies, Wilmington, DE, USA) equipped with an 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 a Zorbax C18 reverse-phase analytical column (2.1 × 30 mm, 3.5 µm). All compounds used for biological evaluation showed >95% purity in this HPLC system.

3.2. Procedures for the Preparation of Compounds

5-(2-Hydroxyethyl)pyrimidin-2,4-dione (1), 5-(3-hydroxypropyl)pyrimidin-2,4-dione (2), 5-(2- acetoxyethyl)pyrimidin-2,4-dione (3), 5-(2-acetoxyethyl)-2,4-dichloropyrimidine (5), 5-(2-hydroxy-ethyl)-2,4-dimethoxypyrimidine (8), 5-(2-acetoxyethyl)-2,4-dimethoxypyrimidine (10), 5-(2-acetoxy-ethyl)-4-methoxypyrimidin-2-one (14) and 5-(2-hydroxyethyl)-4-methoxypyrimidin-2-one (16) were synthesized in accord with original procedures given in the literature [22,23,24,25,26].

5-(3-Acetoxypropyl)pyrimidin-2,4-dione (4). The mixture of compound 2 (30 g, 0.18 mol) and n class="Chemical">acetic anhydride (138 mL, 1.46 mol) in anhydrous pyridine (327 mL) was stirred at room temperature for 1 h. The reaction was quenched with water and the mixture was evaporated to dryness. After column chromatography (CH2Cl2–CH3OH = 8:1) white crystals of compound 4 were isolated (35.14 g, 94%, m.p.: 224–227 °C). 1H-NMR: (δ) 11.00 (1H, s, NH), 10.64 (1H, s, NH), 7.23 (1H, s, H-6), 3.97 (2H, t, H-3', J = 6.5 Hz), 2.21 (2H, t, H-1', J = 7.3 Hz), 2.00 (3H, s, COCH3), 1.77–1.64 (2H, m, H-2') ppm. 13C-NMR: (δ) 170.9 (COCH3), 164.9 (C-4), 151.8 (C-2), 138.6 (C-6), 111.5 (C-5), 63.7 (C-3'), 27.5 (C-2'), 23.2 (C-1'), 21.2 (COCH3) ppm. MS (ESI): m/z = 213.1 ([M+H]+).

5-(3-Acetoxypropyl)-2,4-dichloropyrimidine (6). The mixture of compound 4 (34 g, 0.16 mol), POCl3 (320 mL) and N,N-diethylaniline (32 mL) was refluxed for 1 h. The solvent was evaporated to dryness, the residue was diluted with CCl4 (300 mL), washed twice with iced water and dried over MgSO4. After the removal of the solvent, compound 6 (34.87 g, 87%) was isolated as a yellow oil. 1H-NMR: (δ) 8.8 (1H, s, H-6), 4.11 (2H, t, H-3', J = 6.3 Hz), 2.81 (2H, t, H-1', J = 7.7 Hz), 1.97 (2H, m, H-2'), 2.01 (3H, s, COCH3) ppm. 13C-NMR: (δ) 170.1 (COCH3), 161.2 (C-4), 161.1 (C-2), 156.5 (C-6), 132.4 (C-5), 62.8 (C-3'), 26.9 (C-2'), 25.6 (C-1'), 20.4 (COCH3) ppm. MS (ESI): m/z = 250.1 ([M+H]+).

2-Methoxy-6,7-dihydro-5H-pyrano[2,3-d]pyrimidine (7) and 2,4-dimethoxy-5-(3-hydroxypropyl)-pyrimidine (9). To a solution of sodium (16.7 g, 0.72 mol) in anhydrous methanol (703 mL) compound 6 (34.87 g, 0.14 mmol) was added. The reaction mixture was stirred at room temperature for 20 h, neutralized with ether saturated with gaseous HCl at 0 °C. The solvent was evaporated and CHCl3 (560 mL) was added. The mixture was stirred at room temperature for 20 h and filtered. The filtrate was dried over MgSO4 and evaporated to dryness. After column chromatography (CH2Cl2–CH3OH = 35:1) compounds 7 (9.66 g, 42%, m.p.: 68–70 °C) and 9 (5.29 g, 19%) were isolated.

7: 1H-NMR: (δ) 8.15 (1H, s, H-6), 4.32 (2H, t, H-3', J = 5.2 Hz), 3.79 (3H, s, OCH3), 2.65 (2H, t, H-1', J = 6.4 Hz), 1.92–1.88 (2H, m, H-2') ppm. 13C-NMR: (δ) 168.5 (C-4), 164.4 (C-2), 159.2 (C-6), 109.2 (C-5), 68.3 (C-3'), 54.7 (OCH3), 21.0 (C-2'), 21.4 (C-1') ppm. MS (ESI): m/z = 167.1 ([M+H]+).

9: 1H-NMR: (δ) 8.1 (1H, s, H-6), 4.47 (1H, t, OH, J = 5.2 Hz), 3.92 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.41 (2H, q, H-3', J = 6.0 Hz), 2.47 (2H, t, H-1', J = 7.7 Hz), 1.65 (2H, k, H-2', J = 7.0 Hz) ppm. 13C-NMR: (δ) 169.3 (C-4), 164.0 (C-2), 157.5 (C-6), 115.1 (C-5), 60.5 (C-3’), 54.7 (OCH3), 54.1 (OCH3), 32.1 (C-2'), 22.9 (C-1') ppm. MS (ESI): m/z = 199.1 ([M+H]+).

5-(3-Acetoxypropyl)-2,4-dimethoxypyrimidine (11). To a stirred solution of compound 9 (5.2 g, 26.23 mmol) in dry n class="Chemical">pyridine (48 mL) acetic anhydride (20 mL, 26.4 mmol) was added and the stirring was continued for 1 h at room temperature. After the completion of reaction 15 mL of water was added. The solvent was evaporated and the residue was purified by column chromatography (CH2Cl2–CH3OH = 30:1) to yield compound 11 as a yellow oil (6.1 g, 97%). 1H-NMR: (δ) 8.12 (1H, s, H-6), 3.98 (2H, t, H-3', J = 6.5 Hz), 3.91 (3H, s, OCH3), 3.86 (3H, s, OCH3), 2.49 (2H, t, H-1', J = 7.8 Hz), 2.0 (3H, s, COCH3), 1.81 (2H, k, H-2', J = 7.0 Hz) ppm. 13C-NMR: (δ) 170.85 (COCH3), 169.29 (C-4), 162.9 (C-2), 157.7 (C-6), 114.3 (C-5), 63.6 (C-3'), 54.7 (OCH3), 54.2 (OCH3), 27.8 (C-2'), 22.9 (C-1'), 21.1 (COCH3) ppm. MS (ESI): m/z = 241.1 ([M+H]+).

5-(3-Acetoxypropyl)-4-methoxypyrimidin-2-one (15). To a dried two-necked flask compound 11 (3.23 g, 13.44 mmol) and n class="Chemical">acetyl chloride (26 mL, 0.36 mol) were added under argon atmosphere. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the solvent was evaporated at reduced pressure at 35 °C and coevaporated twice by the addition of toluene (6 mL). The residue was chromatographed (EtOAc–CH3OH = 20:1). During this step, the intermediate 13 was deacetylated to form a white crystalline product 15 (1.79 g, 59%, m.p.: 146–149 °C). Compound 4 was also isolated (966 mg, 34%). 1H-NMR: (δ) 11.14 (1H, s, NH), 7.50 (1H, s, H-6), 3.97 (2H, t, H-3', J = 6.5 Hz), 3.83 (3H, s, OCH3), 2.32 (2H, t, H-1', J = 7.4 Hz), 1.99 (3H, s, COCH3), 1.75 (2H, k, H-2', J = 6.7 Hz) ppm. 13C-NMR: (δ) 170.9 (COCH3), 170.8 (C-4), 156.6 (C-2), 143.1 (C-6), 105.8 (C-5), 63.6 (C-3'), 54.1 (OCH3), 27.8 (C-2'), 22.8 (C-1'), 21.1 (COCH3) ppm. MS (ESI): m/z = 227.2 ([M+H]+).

5-(2-Acetoxyethyl)-N-1-allyl-4-methoxypyrimidin-2-one (17). Method A: A solution of compound 10 (375 mg, 1.66 mmol) and n class="Chemical">allyl bromide (0.15 mL, 1.73 mmol) was heated under reflux for 8 h. After evaporation of the solvent and column chromatography (ethyl acetate:CH3OH = 15:1) compound 17 was obtained as a colourless oil (305.3 mg, 73%). 1H-NMR: (δ) 7.78 (1H, s, H-6), 5.90 (1H, m, H-2"), 5.16 (1H, dd, H-3", J1 = 1.3 Hz, J2 =10.3 Hz), 5.06 (1H, dd, H-3", J1 = 1.4 Hz, J2 =17.2 Hz), 4.08 (2H, t, H-2', J = 6.6 Hz), 4.36 (2H, d, H-1", J = 5.4 Hz), 3.83 (3H, s, OCH3), 2.57 (2H, t, H-1', J = 6.5 Hz), 1.95 (3H, s, COCH3) ppm. 13C-NMR: (δ) 170.7 (COCH3), 170.1 (C-4), 155.6 (C-2), 147.5 (C-6), 103.6 (C-5), 133.7 (C-2"), 117.8 (C-3"), 62.6 (C-2'), 54.5 (OCH3), 51.1 (C-1"), 26.0 (C-1'), 21.1 (COCH3) ppm. MS (ESI): m/z = 253.1 ([M+H]+).

5-(2-Acetoxyethyl)-N-1-(2,3-dihydroxypropyl)-4-methoxypyrimidin-2-one (18). Method A: n class="Chemical">OsO4 (1 mg, 0.004 mmol) was added to a solution of compound 17 (203.5 mg, 0.81 mmol), followed by addition of sodium chlorate (103 mg, 0.97 mmol) dissolved in water (2.5 mL). The reaction mixture was stirred at room temperature for 3 days. The mixture was then filtered through Celite and applied to an Amberlite IR 4 B column using water as eluens. The product was evaporated to dryness and applied on a Dowex (100–200 mesh, H+-form) column and eluted with water. The eluate was evaporated under reduced pressure and the residue was purified by column chromatography (CH2Cl2–CH3OH = 10:1) to yield compound 18 as a yellow oil (115.5 mg, 50%). 1H-NMR: (δ) 7.71 (1H, s, H-6), 4.95 (1H, d, OH, J = 5.5 Hz), 4.95 (1H, t, OH, J = 5.4 Hz), 4.02–4.11 (3H, m, H-2', H-3"), 3.84 (3H, s, OCH3), 3.70–3.78 (1H, m, H-2"), 3.42–3.48 (1H, m, H-3"), covered with water (H-1"), 2.58 (2H, t, H-1', J = 6.5 Hz), 1.98 (3H, s, COCH3) ppm. 13C-NMR: (δ) 170.8 (COCH3), 170.0 (C-4), 156.0 (C-2), 149.2 (C-6), 102.4 (C-5), 69.2 (C-2"), 64.1 (C-3"), 62.6 (C-2'), 54.4 (OCH3), 53.0 (C-1"), 25.9 (C-1'), 21.1 (COCH3) ppm. MS (ESI): m/z = 287.1 ([M+H]+).

5-(2-Hydroxyethyl)-N-1-(2,3-dihydroxypropyl)pyrimidin-2,4-dione (19). Method A: Compound 18 (92.4 mg, 0.32 mmol) was dissolved in 1 M NaOH (8 mL) and the mixture was stirred at room temperature for 20 h. Then, it was neutralized by the addition of acetic acid. After evaporation of the solvents and column chromatography (CH2Cl2–CH3OH = 5:1) white crystals of compound 19 were obtained (29 mg, 39%, m.p.: 95–98 °C).

Method B: Compound 24 (164 mg, 0.67 mmol) was dissolved in 1 M NaOH (20 mL) and the mixture was stirred at room temperature for 20 h and neutralized by the addition of n class="Chemical">HCl. After evaporation of the solvent and column chromatography (CH2Cl2–CH3OH = 5:1), compound 19 was obtained (57.7 mg, 37%). 1H-NMR: (δ) 11.18 (1H, bs, NH), 7.36 (1H, s, H-6), 4.95 (1H, d, OH, J = 5.5 Hz), 4.69 (1H, t, OH, J = 5.6 Hz), 4.53 (1H, t, OH, J = 5.6 Hz), 3.89 (1H, dd, H-3", J = 3.8 Hz, J =13.3 Hz), 3.64–3.72 (1H, m, H-2"), 3.45 (2H, q, H-2', J = 6.7 Hz), 3.36–3.41 (3H, m, H-3", H-1"), 2.29–2.34 (2H, m, H-1') ppm. 13C-NMR: (δ) 164.6 (C-4), 151.5 (C-2), 144.3 (C-6), 109.3 (C-5), 69.6 (C-2"), 64.1 (C-3"), 60.0 (C-2'), 51.4 (C-1"), 30.4 (C-1') ppm. MS (ESI): m/z = 231.1 ([M+H]+). Anal. Calcd for C9H14N2O5: C, 46.95; H, 6.13; N, 12.17. Found: C, 46.90; H, 6.15; N, 12.22.

N-1-[(2-Acetoxyethoxy)methyl]-5-(2-acetoxyethyl)pyrimidin-2,4-dione (20). Method A: To a stirred solution of compound 10 (47.2 mg, 0.21 mmol) and n class="Chemical">Na2CO3 (21.9 mg, 0.2 mmol) in anhydrous CH2Cl2 (1 mL), (2-acethoxyethoxy)methyl bromide [prepared in situ by adding acetyl bromide (0.02 mL, 0.2 mmol) to 1,3-dioxolane (0.01 mL, 0.2 mmol)] was added. The reaction mixture was stirred overnight at room temperature and evaporated to dryness. After column chromatography (CH2Cl2–CH3OH = 20:1) compound 20 was isolated as a crude product (28.2 mg, 43%, m.p.: 53–55 °C). 1H-NMR: (δ) 11.41 (1H, bs, NH), 7.64 (1H, s, H-6), 5.09 (2H, s, H-1"), 4.06–4.17 (4H, m, H-3", H-4"), 3.69 (2H, t, H-2', J = 4.6 Hz), 2.70 (2H, t, H-1', J = 6.4 Hz), 1.99 (3H, s, COCH3), 1.98 (3H, s, COCH3) ppm. 13C-NMR: (δ) 170.2 (COCH3), 163.7 (C-4), 150.9 (C-2), 142.0 (C-6), 109.4 (C-5), 76.3 (C-1"), 66.7 (C-3"), 62.9 (C-4"), 61.9 (C-2’), 25.8 (C-1'), 20.6 (COCH3), 20.6 (COCH3) ppm. MS (ESI): m/z = 315.1 ([M+H]+).

N-1-[(2-Hydroxyethoxy)methyl]-5-(2-hydroxyethyl)pyrimidin-2,4-dione (21). Method A: To a solution of sodium (2.1 mg, 0.09 mmol) in anhydrous methanol (0.5 mL) compound 20 (27 mg, 0.09 mmol) dissolved in methanol (1 mL) was added. The reaction mixture was stirred at room temperature for 1 h and neutralized with 1 M HCl/ether. After evaporation to dryness and column chromatography (CH2Cl2–CH3OH = 5:1) compound 21 was isolated (14.6 mg, 74%) as a colourless oil.

Method B: Compound 25 (37 mg, 0.15 mmol) was dissolved in 1 M NaOH (10 mL) and the mixture was stirred at room temperature for 20 h. Then, it was neutralized by the addition of n class="Chemical">acetic acid. After evaporation of the solvents and column chromatography (ethyl acetate–CH3OH = 5:1) compound 21 was obtained (9.1 mg, 26%). 1H-NMR: (δ) 11.25 (1H, bs, NH), 7.52 (1H, s, H-6), 5.08 (2H, s, H-1"), 4.60 (2H, bs, OH), 3.45–3.50 (6H, m, H-2', H-3", H-4"), 2.35 (2H, t, H-1', J = 6.6 Hz) ppm. 13C-NMR: (δ) 164.5 (C-4), 151.5 (C-2), 142.0 (C-6), 111.0 (C-5), 77.0 (C-1"), 71.0 (C-3"), 60.5 (C-4"), 60.0 (C-2'), 30.3 (C-1') ppm. MS (ESI): m/z = 231.1 ([M+H]+). Anal. Calcd for C9H14N2O5: C, 46.95; H, 6.13; N, 12.17. Found: C, 46.99; H, 6.15; N, 12.12.

5-(2-Acetoxyethyl)-N-1-[(1,3-dibenzyloxy-2-propoxy)methyl]pyrimidin-2,4-dione (22). Method A: To a stirred solution of compound 10 (114 mg, 0.50 mmol) and n class="Chemical">K2CO3 (186.4 mg, 1.35 mmol) in anhydrous CH2Cl2 (3.7 mL), 1,3-dibenzyloxy-2-chloromethoxypropane (257.6 mg, 0.8 mmol) was added under argon atmosphere. The reaction mixture was stirred overnight at room temperature and after filtration evaporated to dryness. After column chromatography (CH2Cl2–CH3OH = 35:1) compound 22 was isolated as a colourless oil (87 mg, 36%). 1H-NMR: (δ) 11.38 (1H, s, NH), 7.63 (1H, s, H-6), 7.40–7.22 (10H, m, Ph), 5.2 (2H, s, H-1"), 4.46 (4H, s, H-5"), 4.03 (2H, t, H-2', J = 6.8 Hz), 3.99–3.94 (1H, m, H-3"), 3.57–3.40 (4H, m, H-4"), 2.44 (2H, t, H-1', J = 6.8 Hz), 1.98 (3H, s, COCH3) ppm. 13C-NMR: (δ) 170. 7 (COCH3), 164.2 (C-4), 151.5 (C-2), 143.9 (C-6), 138.7 (Ph), 128.7 (Ph), 127.9 (Ph), 127.8 (Ph), 109.7 (C-5), 77.2 (C-3"), 76.5 (C-1"), 72.7 (C-5"), 70.3 (C-4"), 62.4 (C-2'), 26.2 (C-1'), 21.1 (COCH3) ppm. MS (ESI): m/z = 483.2 ([M+H]+).

5-(2-Hydroxyethyl)-N-1-[(1,3-dihydroxy-2-propoxy)methyl]pyrimidin-2,4-dione (23). Method A: A solution of compound 22 (63 mg, 0.13 mmol) in anhydrous CH2Cl2 (5 mL) was cooled to −68 °C and 1 M solution of BCl3 in CH2Cl2 (0.45 mL, 0.45 mmol) was added under argon atmosphere. The reaction mixture was stirred at −68 °C for 3 h, quenched by the addition of CH2Cl2–CH3OH (1:1) solution (15 mL) and allowed to warm to room temperature. The solvent was then evaporated and the residue chromatographed (CH2Cl2–CH3OH = 25:1). Compound 23 was isolated as a white solid (22.7 mg, 67%, m.p.: 118–120 °C).

Method B: In the same way as described for 22, the deprotection of 26 was performed. The reagents used were: compound 26 (106 mg, 0.24 mmol), anhydrous CH2Cl2 (5 mL) and n class="Gene">BCl3 (0.7 mL, 0.7 mmol). After column chromatography (CH2Cl2–CH3OH = 10:1) compound 23 (40.9 mg, 65%) was isolated. 1H-NMR: (δ) 11.27 (1H, s, NH), 7.53 (1H, s, H-6), 5.16 (2H, s, H-1"), 4.64–4.59 (3H, m, 3×OH), 3.52–3.40 (7H, m, H-2', H-4", H-3") 2.35 (2H, t, H-1', J = 6.6 Hz) ppm. 13C-NMR: (δ) 164.5 (C-4), 151.4 (C-2), 142.1 (C-6), 110.7 (C-5), 80.9 (C-3"), 76.3 (C-1"), 61.4 (C-4"), 59.9 (C-2'), 30.3 (C-1') ppm. MS (ESI): m/z = 261.1 ([M+H]+). Anal. Calcd for C10H16N2O6: C, 46.15; H, 6.20; N, 10.76. Found: C, 46.10; H, 6.22; N, 10.72.

5-(2-Hydroxyethyl)-N-1-(2,3-dihydroxypropyl)-4-methoxypyrimidin-2-one (24). Method B: A mixture of compound 14 (194.4 mg, 0.91 mmol) and n class="Chemical">sodium hydride (90.9 mg, 2.27 mmol, 60% dispersion in oil) in DMF (6 mL) was stirred at 60 °C for 1 h. To the resulting suspension glycerol α-chlorohydrine (0.6 mL, 6.82 mmol) was added dropwise and the mixture was stirred at 70 °C overnight. The solvent was evaporated under reduced pressure and after column chromatography (CH2Cl2–CH3OH = 5:1) compound 24 (166.9 mg, 75%) was obtained. 1H-NMR: (δ) 7.63 (1H, s, H-6), 4.92 (1H, d, OH, J = 5.8 Hz), 4.71 (1H, t, OH, J = 5.8 Hz), 4.53 (1H, t, OH, J = 5.5 Hz), 4.05 (1H, dd, H-3", J = 3.3 Hz, J = 13.0 Hz), 3.83 (3H, s, OCH3), 3.69–3.71 (1H, m, H-2"), 3.5–3.4 (2H, m, H-2'), covered with water (H-3", H-1"), 2.40 (2H, t, H-1', J = 6.4 Hz) ppm. 13C-NMR: (δ) 170.2 (C-4), 156.9 (C-2), 148.8 (C-6), 103.6 (C-5), 69.3 (C-2"), 64.1 (C-3"), 60.1 (C-2'), 54.3 (OCH3), 53.0 (C-1"), 30.0 (C-1') ppm. MS (ESI): m/z = 245.1 ([M+H]+).

N-1-[(2-Hydroxyethoxy)methyl]-5-(2-hydroxyethyl)-4-methoxypyrimidin-2-one (25). Method B: To a stirred solution of compound 16 (110 mg, 0.65 mmol) in n class="Chemical">acetonitrile (6 mL) under exclusion of moisture N,O-bis-trimethysilylacetamide (BSA: 0.8 mL, 3.2 mmol) was added. The mixture was stirred until a clear solution appeared (about 10 min). Dioxolane (0.12 mL, 1.66 mmol), KI (139 mg, 0.83 mmol) and TMSCl (0.1 mL) were added sequentially. The stirring was continued for 48 h at room temperature. The volatile materials were evaporated under reduced pressure and the residue was purified using column chromatography (CH2Cl2–CH3OH = 5:1) to give compound 25 (39.1 mg, 25%). 1H-NMR: (δ) 7.78 (1H, s, H-6), 5.17 (2H, s, H-1"), 4.62 (1H, t, OH, J = 5.4 Hz), 4.59 (1H, t, OH, J = 5.4 Hz), 3.45–3.56 (4H, m, H-3", H-4"), 3.84 (3H, s, OCH3), 3.62 (2H, t, H-2', J = 6.4 Hz), 2.45 (2H, t, H-1', J = 6.6 Hz) ppm. 13C-NMR: (δ) 171.7 (C-4), 155.8 (C-2), 146.8 (C-6), 105.1 (C-5), 78.3 (C-1"), 71.3 (C-3"), 60.5 (C-4"), 59.8 (C-2'), 54.7 (OCH3), 29.9 (C-1') ppm. MS (ESI): m/z = 245.1 ([M+H]+).

5-(2-Hydroxyethyl)-N-1-[(1,3-dibenzyloxy-2-propoxy)methyl]pyrimidin-2,4-dione (26) and 5-(2-chloroethyl)-N-1-[(1,3-dibenzyloxy-2-propoxy)methyl]pyrimidin-2,4-dione (27). Method B: A mixture of compound 1 (1.3 g, 8.33 mmol), hexamethyldisilazane (30 mL), trimethylsilyl chloride (0.58 mL, 4.57 mmol) and (NH4)2SO4 (416 mg, 3.15 mmol) was refluxed for 24 h under argon atmosphere. The resultant solution was evaporated in vacuum using oil pump. To the residual oil in acetonitrile (50 mL), TBAI (73.7 mg, 0.2 mmol) and 1,3-dibenzyloxy-2-chloromethoxypropane (4 g, 12.45 mmol) were added under argon atmosphere. The reaction mixture was stirred at reflux overnight. After the reaction was complete the solvent was evaporated and the residue purified using column chromatography (CH2Cl2–CH3OH = 35:1) to obtain compounds 26 (289.6 mg, 8%) and 27 (87.3 mg, 2%).

26: 1H-NMR: (δ) 11.30 (1H, s, NH), 7.53 (1H, s, H-6), 7.34–7.28 (10H, m, Ph), 5.21 (2H, s, H-1"), 4.56 (1H, t, OH, J = 5.4 Hz), 4.46 (4H, s, H-5"), 4.0−3.9 (1H, m, H-3") 3.50−3.40 (6H, m, H-2', H-4"), 2.30 (2H, t, H-1', J = 6.7 Hz) ppm. 13C-NMR: (δ) 164.4 (C-4), 151.6 (C-2), 142.1 (C-6), 138.7 (C-Ph), 128.7 (C-Ph), 127.8 (C-Ph), 127.8 (C-Ph), 111.0 (C-5), 77.1 (C-3"), 76.5 (C-1"), 72.7 (C-5"), 70.3 (C-4"), 60.0 (C-2'), 30.3 (C-1') ppm. MS (ESI): m/z = 441.3([M+H]+).

27: 1H-NMR: (δ) 11.43 (1H, s, NH), 7.69 (1H, s, H-6), 7.38–7.2 (10H, m, Ph), 5.20 (2H, s, H-1"), 4.46 (4H, s, H-5"), 4.04–3.95 (1H, m, H-3"), 3.62 (2H, t, H-2', J = 6.9 Hz), 3.55–3.40 (4H, m, H-4"), 2.58 (2H, t, H-1', J = 7.0 Hz) ppm. 13C-NMR: (δ) 164.1 (C-4), 151.5 (C-2), 143.0 (C-6), 138.7 (C-Ph), 128.7 (C-Ph), 127.8 (C-Ph), 127.7 (C-Ph), 109.8 (C-5), 77.2 (C-3"), 76.6 (C-1"), 72.7 (C-5"), 70.3 (C-4"), 43.5 (C-2'), 30.2 (C-1') ppm. MS (ESI): m/z = 459. 2 ([M+H]+).

5-(2-Chloroethyl)-N-1-[(1,3-dihydroxy-2-propoxy)methyl]pyrimidin-2,4-dione (28). Method B: In the similar way as described for compound 23, the synthesis of 28 was performed. The reagents used were: compound 27 (43.7 mg, 0.1 mmol), anhydrous CH2Cl2 (8 mL) and BCl3 (0.7 mL, 0.7 mmol). After column chromatography (CH2Cl2–CH3OH = 5:1) compound 28 (15 mg, 57%) was isolated. 1H-NMR: (δ) 11.36 (1H, s, NH), 7.65 (1H, s, H-6), 5.13 (2H, s, H-1"), 4.58 (2H, t, 2×OH, J = 5.5 Hz), 3.66 (2H, t, H-2', J = 6.9 Hz), 3.51–3.32 (5H, m, H-3", H-4"), 2.62 (2H, t, H-1', J = 6.9 Hz) ppm. 13C-NMR: (δ) 164.2 (C-4), 151.3 (C-2), 143.2 (C-6), 110.7 (C-5), 81.0 (C-3"), 76.4 (C-1"), 61.4 (C-4"), 43.6 (C-2'), 30.3 (C-1') ppm. MS (ESI): m/z = 279.2([M+H]+).

N-1-[4-Acetoxy-(3-acetoxymethyl)butyl]-5-(2-acetoxyethyl)-4-methoxypyrimidin-2-one (29). To a stirred solution of compound 14 (76.3 mg, 0.36 mmol) and n class="Chemical">K2CO3 (74.5 mg, 0.54 mmol) in anhydrous DMF (7.6 mL), 4-acetoxy-(3-acetoxymethyl)butyl iodide (192 mg, 0.61 mmol) was added under argon atmosphere. The reaction mixture was stirred at 60 °C for 20 min and then evaporated to dryness. After column chromatography (CH2Cl2–CH3OH = 30:1) compound 29 was isolated as a colourless oil (87.1 mg, 60%). 1H-NMR: (δ) 7.89 (1H, s, H-6), 4.12 (2H, t, H-2', J = 6.6 Hz), 4.03 (4H, d, H-4", J = 5.8 Hz), 3.85 (3H, s, OCH3), 3.83 (2H, t, H-1", J = 7.7 Hz), 2.60 (2H, t, H-1', J = 6.6 Hz), 2.02 (6H, s, 2×COCH3), 1.99 (3H, s, COCH3), within COCH3 signal (H-3") 1.67 (2H, q, H-2", J = 7.1 Hz) ppm.13C-NMR: (δ) 170.8 (COCH3), 169.9 (C-4), 155.6 (C-2), 147.7 (C-6), 103.3 (C-5), 64.0 (C-4"), 62.6 (C-2'), 54.3 (OCH3), 47.2 (C-1"), 34.9 (C-3"), 27.9 (C-1') 26.0 (C-2"), 21.1 (COCH3) ppm. MS (ESI): m/z = 399.2 ([M+H]+).

N-1-[4-Acetoxy-(3-acetoxymethyl)butyl]-5-(3-acetoxypropyl)-4-methoxypyrimidin-2-one (30). The synthesis of 30 was performed as described for 29. The reagents used were: compound 15 (1.54 g, 6.81 mmol), n class="Chemical">K2CO3 (1.41 g, 10.2 mmol), anhydrous DMF (40 mL) and 4-acetoxy-(3-acetoxymethyl)butyl iodide (3.63 g, 11.6 mmol). After column chromatography (CH2Cl2–CH3OH = 50:1) compound 30 was isolated as a yellow oil (1.15 g, 41%). 1H-NMR: (δ) 7.83 (1H, s, H-6), 4.04–3.98 (6H, m, H-4", H-3'), 3.84–3.80 (5H, m, OCH3, H-1"), 2.34 (2H, t, H-1', J = 7.5 Hz), 2.02 (6H, s, 2×COCH3), 2.00 (3H, s, COCH3), within COCH3 signal (H-3"), 1.78 (2H, k, H-2', J = 7.0 Hz), 1.67 (2H, q, H-2", J = 7.2 Hz) ppm. 13C-NMR: (δ) 170.9 (COCH3), 170.1 (C-4), 155.7 (C-2), 146.6 (C-6), 106.5 (C-5), 64.1 (C-4"), 63.6 (C-3'), 54.4 (OCH3), 47.2 (C-1"), 34.9 (C-3"), 27.9 (C-2'), 27.7 (C-2"), 22.9 (C-1'), 21.1 (COCH3), 21.1 (COCH3) ppm. MS (ESI): m/z = 413.2 ([M+H]+).

5-(2-Hydroxyethyl)-N-1-[4-hydroxy-(3-hydroxymethyl)butyl]pyrimidin-2,4-dione (31). The synthesis of 31 was performed as described for 19. The reagents used were: compound 29 (62.4 mg, 0.16 mmol) and 1 M n class="Chemical">NaOH (5 mL). After column chromatography (CH2Cl2–CH3OH = 5:1) compound 31 was obtained as a white solid (28.2 mg, 70%, m.p.: 83–84 °C). 1H-NMR: (δ) 11.15 (1H, bs, NH), 7.46 (1H, s, H-6), 4.54 (1H, t, OH, J = 5.5 Hz), 4.40 (2H, t, 2×OH, J = 5.2 Hz), 3.69 (2H, t, H-1", J = 7.6 Hz), 3.45 (2H, q, H-2', J = 6.3 Hz), 3.42–3.32 (4H, m, H-4"), 2.32 (2H, t, H-1', J = 6.7 Hz), 1.55 (2H, q, H-2", J = 7.3 Hz), 1.47 (1 H, m, H-3", J = 5.9 Hz) ppm. 13C-NMR: (δ) 164.5 (C-4), 151.2 (C-2), 142.9 (C-6), 109.3 (C-5), 61.9 (C-4"), 59.9 (C-2'), 46.2 (C-1"), 41.2 (C-3"), 30.4 (C-1'), 28.2 (C-2") ppm. MS (ESI): m/z = 259.1 ([M+H]+). Anal. Calcd for C11H18N2O5: C, 51.15; H, 7.02; N, 10.85. Found: C, 51.10; H, 6.99; N, 10.89.

5-(3-Hydroxypropyl)-N-1-[4-hydroxy-(3-hydroxymethyl)butyl]pyrimidin-2,4-dione (32). The synthesis of 32 was performed in the same way as described for 31. The reagents used were: compound 30 (204 mg, 0.49 mmol) and 1 M n class="Chemical">NaOH (5 mL). After column chromatography (CH2Cl2–CH3OH = 5:1) compound 32 was obtained as white crystals (110 mg, 82%, m.p.: 113–114 °C). 1H-NMR: (δ) 11.14 (1H, s, NH), 7.46 (1H, s, H-6), 4.43–4.38 (3H, m, OH), 3.70 (2H, t, H-1", J = 7.3 Hz), 3.46–3.30 (6H, m, H-4", H-3'), 2.22 (2H, t, H-1', J = 7.4 Hz), 1.63–1.42 (5H, H-2', H-2", H-3") ppm. 13C-NMR: (δ) 164.4 (C-4), 151.2 (C-2), 141.9 (C-6), 113.0 (C-5), 61.9 (C-4"), 60.6 (C-3'), 46.2 (C-1"), 41.2 (C-3"), 31.9 (C-2'), 28.3 (C-2"), 23.3 (C-1') ppm. MS (ESI): m/z = 273.2 ([M+H]+). Anal. Calcd for C12H20N2O5: C, 52.93; H, 7.40; N, 10.29. Found: C, 52.97; H, 7.43; N, 10.33.

3.3. Phosphorylation Assay of 19, 21, 23, 31 and 32

3.3.1. Protein Expression

The HSV-1 TK was expressed in E. coli as a GST-TK fusion protein and further purified as HSV-1 TK-GST protein by using glutathione affinity chromatography following protocols that have been reported previously [33]. The human thymidine kinase 1 (hTK) was expressed in E. coli as a 6xHisTagged protein and purified following the previously published protocol [34].

3.3.2. HPLC System and Conditions

The high liquid performance chromatography system used Merck HITACHI LaChrom (Gynkotek HPLC, Münich, Germany) equipped with a pump (L-7100), autosampler (L-7200) and a UV detector (L-7400). The analyses were performed on an LiChroCART® 250-4 can class="Chemical">rtridge column packed with a stationary phase made of spherical partical of silica (LiCrospher® 100 RP-18 endcapped, (5 µm)) and coupled with a guard column LiChroCART® 4-4 of the same type (Merck, KGaA, Darmstadt, Germany). Mobile phase consisted of 200 mM NaH2PO4, 25 mM tetrabutylammonium hydrogen sulfate (TBAHS) as the ion-pairing reagent and 0.5% methanol (HPLC grade). All aqueous solutions were prepared using de-ionized water from Millipore. The column was kept at a constant temperature of 20 °C. The separation was performed isocratically at a flow-rate of 1.1 mL/min and the UV detection was done at 254 nm [32].

3.3.3. The Phosphorylation Reaction

Phosphorylation of dT and compounds 19, 21, 23, 31, fluorinated derivative of 31 and 32 was monitored by HPLC. Reactions were carried out in a final volume of 70 µL containing 50 mM n class="Chemical">HEPES pH 7.5, 5 mM ATP, 5 mM MgCl2, 1 mM solution of dT and tested compounds and either 11 µg of purified HSV-1 TK-GST or 0.8 µg of hTK. As the solutions of dT and tested compounds were prepared with 100% DMSO attention was paid for not adding more than 5% DMSO in the sample reaction (the final concentration of DMSO was 1%). The reaction was started with the addition of HSV-1 TK-GST. The reaction was done at 37 °C and was stopped after 30, 60 and 90 min by a 10-fold dilution with 50 mM EDTA (final concentration 5 mM) used to chelate the cofactor Mg2+. Each reaction was done in triplicate. Finally, a volume of 40 µL of the sample reaction was injected for analysis. The formation of the nucleotide monophosphate was monitored qualitatively. Two different blank reactions (no enzyme or no compound) were performed under the same experimental conditions to account for the background ATP hydrolysis. Control reactions were stopped after 30, 60 and 90 min (Supplementary Materials). The resulting ADP/ATP ratios were calculated and used as a measure for protein activity. The mean of at least triplicate measurements are reported.

3.3.4. Cellular Activity Evaluations

The WI38 (normal diploid human fibroblasts) cell lines were cultured as monolayers and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal n class="Species">bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. The panel cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0, at 3000 cells to 6000 cells per well according to the doubling times of specific cell line. Test agents were then added in five, 10-fold dilutions (1 × 10−8 to 1 × 10−4 M) and incubated for further 72 h. Working dilutions were freshly prepared on the day of testing in the growth medium. The solvent (DMSO) was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in the working concentrations (DMSO concentration never exceeded 0.1%). After 72 h of incubation, the cell growth rate was evaluated by performing the MTT assay: experimentally determined absorbance values were transformed into a cell percentage growth (PG) using the formulas proposed by NIH and described previously [35].This method directly relies on control cells behaving normally at the day of assay because it compares the growth of treated cells with the growth of untreated cells in control wells on the same plate—the results are therefore a percentile difference from the calculated expected value. The IC50 and LC50 values for each compound were calculated from dose-response curves using linear regression analysis by fitting the mean test concentrations that give PG values above and below the reference value. If, however, all of the tested concentrations produce PGs exceeding the respective reference level of effect (e.g., PG value of 50) for a given cell line, the highest tested concentration is assigned as the default value (in the screening data report that default value is preceded by a “>” sign). Each test point was performed in quadruplicate in three individual experiments. The results were statistically analyzed (ANOVA, Tukey post-hoc test at p < 0.05). Finally, the effects of the tested substances were evaluated by plotting the mean percentage growth for each cell type in comparison to control on dose-response graphs.

4. Conclusions

5-(2-Hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted 4-methoxypyrimidin-2-ones (10 and 14–16) were prepared by an efficient synthetic route and subjected to N-1 alkylation to obtain target compounds bearing 2,3-dihydroxypropyl (19), acyclovir- (21), ganciclovir- (23), and penciclovir-like side chains (compounds 31, 32). The two synthetic methods A and B that were applied for N-alkylation indicated that method A using activated N-anionic 5-substituted pyrimidine derivatives was more successful than the silylation reaction method.

The influence of the acyclic glycone in target compounds 19, 21, 23, 31 and 32 on their phosphorylation ability was observed. Thereby, n class="Chemical">5-substituted pyrimidine derivatives 31 and 32 bearing N-1 penciclovir-like side chain revealed to be substrates of HSV-1 TK. The structural congener of 31 bearing an oxygen isosteric replacement of methylene group in side chain of 23 was not phosphorylated by HSV-1 TK. Moreover, 5-(2-hydroxyethyl)pyrimidin-2,4-dione 31 showed to be a better substrate when compared to compound 32 with a longer substituent bearing an additional methylene group.

Besides, both compounds 31 and 32 exhibited no cytotoxic effect against normal human fibroblasts up to 100 µM. Compound 31 has been selected as a lead compound for development as a new PET probe for imaging n class="Species">HSV-1 TK expression. Therefore, the fluorine-18 radiolabeling of the corresponding pyrimidine precursor of 31 and the subsequent in vivo PET studies are currently underway.