Synthesis of Pyrimidine Conjugates with 4-(6-Amino-hexanoyl)-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine and Evaluation of Their Antiviral Activity

A series of pyrimidine conjugates containing a fragment of racemic 7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine and its (S)-enantiomer attached via a 6-aminohexanoyl fragment were synthesized by the reaction of nucleophilic substitution of chlorine in various chloropyrimidines. The structures of the synthesized compounds were confirmed by 1H, 19F, and 13C NMR spectral data. Enantiomeric purity of optically active derivatives was confirmed by chiral HPLC. Antiviral evaluation of the synthesized compounds has shown that the replacement of purine with a pyrimidine fragment leads to a decrease in the anti-herpesvirus activity compared to the lead compound, purine conjugate. The studied compounds did not exhibit significant activity against influenza A (H1N1) virus.


Introduction
Pyrimidine (1,3-diazine) and its fused derivative, purine (imidazo [4,5-d]pyrimidine), are the most important class of heterocyclic scaffolds that form pyrimidine (uracil, thymine, and cytosine) and purine (adenine and guanine) nucleobases, which are structural fragments of nucleic acids. Pyrimidine-based compounds exhibit a wide range of biological and pharmacological activities, including anticancer, anxiolytic, antioxidant, antiviral, antifungal, anticonvulsant, antidepressant, antibacterial, etc. (for some recent reviews, see [1][2][3][4][5][6] and references therein). Derivatives of pyrimidine and purine, as well as nucleosides based on them, are part of efficient medicinal agents (Figure 1) used in the treatment of a wide range of diseases, primarily cancer and viral infections.
In recent years, our efforts have been focused on the search for efficient antiviral agents among purine and 2-aminopurine derivatives. For this purpose, we synthesized a number of conjugates of purine and 2-aminopurine with various N-heterocycles, including chiral ones, which are attached via a linker, the omega-aminocarboxylic acid NH(CH2)nCO residue, to position 6 of the purine nucleus ( Figure 2) [22][23][24][25][26][27][28][29][30][31]. During our investigations, we varied the structure of the heterocycle (including the configuration of the chiral center in the case of chiral compounds), the length of the polymethylene chain of the linker, and the substituent at the N 9 atom of purine. Most of the resulting compounds were tested against herpes simplex virus type 1 (HSV-1), including the acyclovir-resistant strain. Compounds containing a linker fragment with n ≥ 5 are of the greatest interest in terms of anti-herpesvirus activity. The most active compound turned out to be a purine conjugate with (S)-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine attached via a 6-aminohexanoyl residue (n = 5, compound (S)-1) [27][28][29]. We have demonstrated that it is the combination of difluorobenzoxazine and purine fragments in one molecule that leads to high anti-herpesvirus activity, including that against the acyclovir-resistant HSV-1 strain [27]. In recent years, our efforts have been focused on the search for efficient antiviral agents among purine and 2-aminopurine derivatives. For this purpose, we synthesized a number of conjugates of purine and 2-aminopurine with various N-heterocycles, including chiral ones, which are attached via a linker, the omega-aminocarboxylic acid NH(CH 2 ) n CO residue, to position 6 of the purine nucleus ( Figure 2) [22][23][24][25][26][27][28][29][30][31]. During our investigations, we varied the structure of the heterocycle (including the configuration of the chiral center in the case of chiral compounds), the length of the polymethylene chain of the linker, and the substituent at the N 9 atom of purine. Most of the resulting compounds were tested against herpes simplex virus type 1 (HSV-1), including the acyclovir-resistant strain. Compounds containing a linker fragment with n ≥ 5 are of the greatest interest in terms of anti-herpesvirus activity. The most active compound turned out to be a purine conjugate with (S)-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine attached via a 6-aminohexanoyl residue (n = 5, compound (S)-1) [27][28][29]. We have demonstrated that it is the combination of difluorobenzoxazine and purine fragments in one molecule that leads to high anti-herpesvirus activity, including that against the acyclovir-resistant HSV-1 strain [27]. In this work, in order to elucidate the role of the purine fragment in the antiviral activity exhibited by the lead compound, we synthesized a series of derivatives, in which the purine fragment was replaced by a pyrimidine one, and studied the antiviral activity of the synthesized compounds against HSV-1 and influenza A (H1N1) virus.

Chemistry
To obtain the target pyrimidine conjugates, we used a synthetic sequence developed earlier for the preparation of purine and 2-aminopurine conjugates [27,30,31]. The starting compounds were amides of N-phthalimidohexanoic acid and racemic 3methyldifluorobenzoxazine ((RS)-2) and its (S)-enantiomer ((S)-2) [23] (Scheme 1). The removal of the phthaloyl protecting group was carried out according to the standard method of hydrazinolysis in boiling ethanol; the resulting compounds (RS)-3 or (S)-3 with a free amino group were introduced into the reaction of nucleophilic substitution of chlorine in the corresponding chloropyrimidines: 2-amino-4-chloropyrimidine (4a), 2amino-4,6-dichloropyrimidine (4b), 4,6-dichloropyrimidine (4c), 2-chloropyrimidine (4d), and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (or 7-deazapurine, 4e). In this work, in order to elucidate the role of the purine fragment in the antiviral activity exhibited by the lead compound, we synthesized a series of derivatives, in which the purine fragment was replaced by a pyrimidine one, and studied the antiviral activity of the synthesized compounds against HSV-1 and influenza A (H1N1) virus.
The reaction of nucleophilic substitution of chlorine in chloropyrimidines was carried out under reflux in nBuOH in the presence of a tertiary amine, triethylamine (Et 3 N), as an HCl acceptor (Scheme 1). The reaction products were purified by silica gel flash chromatography to afford the desired conjugates 5a-e in moderate to high yields. The enantiomeric purity (>99% ee) of conjugates (S)-5a and (S)-5e was determined by reversedphase chiral HPLC on a (S,S)-Whelk-O1 column (see the Supplementary Materials).
It is known that the presence of electron-donating or electron-withdrawing substituents determines the reactivity of pyrimidines [32]. 4,6-Dichloropyrimidines 4b and 4c are During the nucleophilic substitution of chlorine in dichloropyrimidines 4b and 4c, we did not observe the formation of by-products of double substitution, despite the excess of nucleophile 2. This is due to the fact that the substitution of one of the chlorine atoms with an electron-donating alkylamino group deactivates another chlorine atom, and its substitution requires more drastic conditions [32]. The second chlorine atom can also be substituted in the presence of electron-withdrawing substituents in the pyrimidine ring [33,34].

Anti-Herpesvirus Activity
The anti-herpesvirus activity of the synthesized compounds 5a-e was studied against the reference strain HSV-1/L 2 and the acyclovir-resistant strain HSV-1/L 2 /R in the Vero E6  (Table 1) by suppressing the virus-induced cytopathogenic effect (CPE) as described previously [27]. Acyclovir, Foscarnet (sodium phosphonoformate hexahydrate), a drug of choice in clinical practice in case of inefficiency of acyclovir and related compounds, including cases of drug resistance, and Ribavirin, were used as reference drugs. To assess the cytotoxicity of the studied compounds, the percentage of viable and non-viable cells was determined using Trypan blue exclusion assay.
As can be seen from the data in Table 1, compounds 5a-e exhibit low or very moderate antiviral activity against HSV-1. The most active is the 2-aminopyrimidine derivative (S)-5a containing the (S)-enantiomer of difluorobenzoxazine (IC 50 = 9.27 µM); however, this compound possesses a high cytotoxicity against Vero E6 cells (CC 50 = 34.63 µM), which results in a low antiviral selectivity (SI < 4). Compounds (RS)-5a and (S)-5e showed the highest selectivity of anti-herpesvirus activity (SI = 8 and 6, respectively); moreover, these indicators are preserved against the acyclovir-resistant HSV-1 strain. However, all the studied pyrimidine derivatives are inferior in antiviral activity and selectivity to the previously identified lead compound (S)-1. In general, it can be concluded that the replacement of a purine fragment with a pyrimidine one does not lead to an enhancement of anti-herpesvirus activity.

Anti-Influenza Activity
The study of anti-influenza activity of the synthesized compounds 5a-e was carried out against the reference strain of influenza virus A/Puerto Rico/8/34 (H1N1) using the test for the suppression of virus-induced CPE in the MDCK cell culture (Table 2), as described earlier [31]. The reference drug was Oseltamivir carboxylate, a drug used to treat influenza. The viability of infected and healthy cells was assessed using the MTT-assay [35].

General Procedure for the Synthesis of the Target Compounds 5a-e
Hydrazine monohydrate (0.22 mL, 4.50 mmol) was added to a solution of compound (RS)-2 or (S)-2 (1.07 g, 2.50 mmol) in ethanol (25 mL). The reaction mixture was refluxed for 2 h and then evaporated to dryness under reduced pressure. The residue was stirred in 2 M HCl (25 mL) at +4 • C for 12 h. The precipitate was filtered off, washed with 1 M HCl (2 × 3 mL). The filtrate and washings were combined and neutralized with solid NaOH to pH 9, then extracted with chloroform (3 × 5 mL). The organic layers were dried with MgSO 4 and evaporated to dryness under reduced pressure to afford compound (RS)-3 or (S)-3 (0.56 g, 75%). The residue was dissolved in nBuOH (8 mL). The appropriate chloropyrimidine 4a-e (1.25 mmol) and Et 3 N (0.17 mL, 1.25 mmol) were added to the resulting solution. The reaction mixture was refluxed for 8 h, cooled to room temperature, diluted with nBuOH (12 mL), washed with 1 M HCl (in the case of pyrimidines 4a,b, with 10% aqueous citric acid) (3 × 5 mL), saturated aqueous NaCl solution (3 × 5 mL), and water (3 × 3 mL), then evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography on silica gel using the appropriate eluent.  13

Conclusions
In summary, we have synthesized a series of pyrimidine conjugates containing a fragment of both racemic 7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine and its (S)-enantiomer, which are attached via a 6-aminohexanoyl fragment. The structure of the synthesized compounds was confirmed by 1 H, 19 F, and 13 C NMR spectroscopy data. Chiral HPLC was used to confirm the enantiomeric purity of the optically active derivatives. Evaluation of the antiviral activity of the synthesized compounds has shown that the replacement of purine with a fragment of pyrimidines and aminopyrimidines leads to a decrease in the anti-herpesvirus activity compared to the lead compound. The studied compounds did not exhibit significant activity against influenza A (H1N1) virus.