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.

1. 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.

Figure 1

Structures of selected pyrimidine-based antiviral and anticancer agents.

The mechanism of action of antiviral agents is primarily determined by selective blocking of metabolic pathways in which nucleobases are actively involved. Recently, researchers have paid great attention to the search for new efficient antiviral agents among pyrimidine [7,8,9,10,11,12,13,14,15,16] and purine [17,18,19,20,21] derivatives, which is especially important in connection with the need to create treatments for SARS-CoV2 (COVID-19) coronavirus infection.

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 N9 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].

Figure 2

Structures of the previously obtained purine conjugates with N-heterocycles.

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.

2. Results and Discussion

2.1. 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 3-methyldifluorobenzoxazine ((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), 2-amino-4,6-dichloropyrimidine (4b), 4,6-dichloropyrimidine (4c), 2-chloropyrimidine (4d), and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (or 7-deazapurine, 4e).

Scheme 1

Synthesis of the target compounds 5a–e. (a) NH2NH2 × H2O, EtOH, reflux, 2 h; (b) 4a–e, Et3N, nBuOH, reflux, 8 h.

The reaction of nucleophilic substitution of chlorine in chloropyrimidines was carried out under reflux in nBuOH in the presence of a tertiary amine, triethylamine (Et3N), 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 reversed-phase 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 highly reactive due to the influence of the electron-withdrawing substituent, chlorine, at the C6 position. Their derivatives (RS)-5b and (RS)-5c were obtained in high yields of 68% (Scheme 1, entries 3 and 4). At the same time, the presence of electron-donating substituents in structures 4a and 4e, the amino group and the conjugated π-excess pyrrole ring, respectively, reduces the reactivity of these compounds; the yields of the products of nucleophilic substitution of chlorine (compounds 5a and 5e) were moderate (29–50%; Scheme 1, entries 1, 2, 6, and 7).

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].

2.2. Antiviral Evaluation

The antiviral activity of all synthesized pyrimidine conjugates with 7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine attached via a linker (compounds 5a–e) was tested in vitro against herpes simplex virus type 1 (HSV-1), including an acyclovir-resistant strain (Table 1), and influenza A (H1N1) virus (Table 2).

Table 1

Cytotoxicity and inhibitory activity of compounds 5a–e against herpes simplex virus type 1 (HSV-1) strains in the Vero E6 cells.

Compound	CC50 ± SD (µM)	HSV-1/L2		HSV-1/L2/R
		IC50 (µM)	SI	IC50 (µM)	SI
(S)-1	293.48 ± 15.55	4.6	64	4.6	64 1
(RS)-5a	150.56 ± 8.36	18.56	8.1	18.56	8
(S)-5a	34.63 ± 0.88	9.27	3.7	9.27	<4
(RS)-5b	274.57 ± 4.30	73.38	3.7	73.38	<4
(RS)-5c	>304.25	148.44	>2.0	152.12	2
(RS)-5d	504.77 ± 17.72	296.88	1.7	296.88	<2
(RS)-5e	17.58 ± 0.85	18.56	<1	18.56	<1
(S)-5e	468.97 ± 10.83	74.22	6.3	75.22	6
Acyclovir	>444	1.73	>256	>444	1
Foscarnet	>416.61	104.15	>4	104.15	>4
Ribavirin	>4098	1025	>4	1025	>4

1 Data from [27] for comparison. Multiplicity of infection 0.1 PFU/cell; CC50 values are averages with standard deviations for three independent experiments; IC50 concentration of compounds producing 50% inhibition of the development of the virus-induced CPE relative to complete CPE in infected but untreated control cultures; SI, selectivity index calculated as CC50/IC50.

Table 2

Cytotoxicity and antiviral activity of compounds 5a–e against influenza A/Puerto Rico/8/34 (H1N1) virus in the MDCK cell culture.

Compound	CC50 ± SD (µM)	IC50 (µM)	SI
(S)-1	24 ± 1	>10	>2 1
(RS)-5a	11 ± 1	8 ± 1	>1
(S)-5a	23 ± 1.2	7 ± 1	>3
(RS)-5b	705 ± 42	92 ± 11	<8
(RS)-5c	9 ± 1	>8	1
(RS)-5d	257 ± 12	>88	3
(RS)-5e	86 ± 4	>80	1
(S)-5e	47 ± 3	>26	<2
Oseltamivir carboxylate	>200	0.3	>667

1 Data from [31] for comparison. Multiplicity of infection 0.01 TCID50/cell; CC50 values are averages with standard deviations for three independent experiments; IC50 concentration of compounds producing 50% inhibition of the development of the virus-induced CPE relative to complete CPE in infected but untreated control cultures; SI, selectivity index calculated as CC50/IC50.

2.2.1. Anti-Herpesvirus Activity

The anti-herpesvirus activity of the synthesized compounds 5a–e was studied against the reference strain HSV-1/L2 and the acyclovir-resistant strain HSV-1/L2/R in the Vero E6 cells (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 (IC50 = 9.27 µM); however, this compound possesses a high cytotoxicity against Vero E6 cells (CC50 = 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.

2.2.2. 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].

The studied conjugates 5a–e showed low activity against influenza A virus and low selectivity (Table 2). The conjugate of 2-amino-6-chloropyrimidine and racemic difluorobenzoxazine (compound (RS)-5b: CC50 = 705 µM, IC50 = 92 µM, SI < 8) had the optimal parameters of activity and cytotoxicity in a series of studied compounds. Compound (RS)-5b, although it showed a lower anti-influenza activity compared to the previously obtained compound (S)-1 (IC50 > 10 µM), was less toxic to MDCK cells.

3. Materials and Methods

3.1. Chemistry General Section

Racemic 7,8-difluoro-3,4-dihydro-3-methyl-4-(6-phthalimidohexanoyl)-2H-[1,4]benzoxazine [(RS)-2] and (S)-enantiomer [(S)-2] were obtained as previously described [23]. 2-Chloropyrimidine (4d) was obtained according to the known method [36] starting from 2-aminopyrimidine. Other reagents are commercially available and were purchased from Alfa Aesar (Lancashire, UK). Solvents were purified according to traditional methods [37] and used freshly distilled. Melting points were obtained on a SMP3 apparatus (Barloworld Scientific, Staffordshire, UK) and are uncorrected. Optical rotations were measured on a Perkin Elmer M341 polarimeter. The reactions were monitored by thin layer chromatography (TLC) using silica gel precoated Sorbfil plates (Imid, Krasnodar, Russia); compounds were visualized by UV irradiation at 254 nm and iodine vapors. Flash column chromatography was performed using silica gel 60 (230–450 mesh) (Alfa Aesar, UK). The 1H, 19F, and 13C NMR spectra were recorded on a Bruker AVANCE 500 spectrometer. Chemical shifts are given in ppm and are referenced to TMS and hexafluorobenzene as internal standards, and multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). The 1H and 19F NMR spectra of compounds 5a–c,e were recorded in DMSO-d6 at 100 °C; other NMR spectra, at ambient temperature. CHN-Elemental analysis was performed using Perkin Elmer 2400 II analyser. High resolution mass spectra were obtained on a Bruker maXis Impact HD mass spectrometer (Bruker, Karlsruhe, Germany), electrospray ionization with direct sample inlet (4 L/min flow rate). Analytical chiral HPLC of compounds (RS)-5a, (S)-5a, (RS)-5e, and (S)-5e was performed on an Agilent 1100 instrument (Agilent Technologies, Santa Clara, CA, USA) using an (S,S)-Whelk-O1 column (250 × 4.6 mm, 5 µm) (Phenomenex, Torrance, CA, USA); flow rate 0.8 mL/min, detection at 280 nm.

3.2. General Procedure for the Synthesis of the Target Compounds

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 MgSO4 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 Et3N (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.

(RS)-4-[6-(2-Aminopyrimidin-4-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine Sesquihydrate [(RS)-5a]. White powder m.p. 59–60 °C (yield 0.22 g, 44%) after flash column chromatography, chloroform–EtOH 85: 15 as an eluent. HPLC ((S,S)-Whelk-O1, MeOH–0.2% aqueous Et3N 9: 1, 0.8 mL/min; detection at 280 nm): τ( 8.0 min, τ( 9.8 min. 1H NMR (500 MHz, DMSO-d6, 100 °C) δ (ppm): 1.13 (d, J = 6.9 Hz, 3H, Me), 1.34–1.40 (m, 2H, CH2 hexanoyl), 1.52–1.57 (m, 2H, CH2 hexanoyl), 1.59–1.66 (m, 2H, CH2 hexanoyl), 2.44–2.50 (m, 1H, 2-HB hexanoyl, partly overlapped with DMSO signal), 2.58–2.64 (m, 1H, 2-HA hexanoyl), 3.28 (td, J = 6.7, 6.2 Hz, 2H, 6-H hexanoyl), 4.14 (dd, J = 10.9, 2.8 Hz, 1H, 2-HB benzoxazine), 4.33 (d, J = 11.1 Hz, 1H, 2-HA benzoxazine), 4.74 (q, J = 6.1 Hz, 1H, 3-H benzoxazine), 5.94 (d, J = 6.7 Hz, 1H, 5-H pyrimidine), 6.59 (br. s, 2H, NH2), 6.85 (dd, J = 9.7, 8.5 Hz, 1H, 6-H benzoxazine), 7.54–7.57 (m, 1H, 5-H benzoxazine), 7.59 (br. s, 1H, NH), 7.60 (br. s, 1H, 6-H pyrimidine). 19F NMR (470 MHz, DMSO-d6, 100 °C) δ (ppm): 2.05 (dd, J = 20.8, 6.7 Hz, 1F, 8-F), 20.18 (br. s, 1F, 7-F). 13C NMR (126 MHz, DMSO-d6) δ (ppm): 15.10, 24.33, 25.95, 28.35, 33.37, 39.6 (overlapped by DMSO signal), 44.75 (br. s), 69.85, 96.95 (br. s), 106.77 (d, J = 17.9 Hz), 119.25, 121.78, 135.69 (br. d, J = 9.7 Hz), 138.91 (dd, J = 243.3, 15.3 Hz), 145.52 (br. s), 146.49 (br. d, J = 241.6 Hz), 158.50, 162.67, 170.91. Calcd (%) for C19H23F2N5O2 × 1.5H2O: C 54.54, H 6.26, N 16.74, F 9.08. Found (%): C 54.33, H 6.13, N 16.37, F 9.03.

(S)-4-[6-(2-Aminopyrimidin-4-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine Hydrate [(S)-5a]. White powder m.p. 78–80 °C (yield 0.18 g, 36%) after flash column chromatography, chloroform–EtOH 85: 15 as an eluent. [α]D20 +60.5 (c 0.5, EtOH). >99% ee. HPLC ((S,S)-Whelk-O1, MeOH–0.2% aqueous Et3N 9: 1, 0.8 mL/min; detection at 280 nm): τ( 8.0 min. 1H, 19F, and 13C NMR spectra were identical to those of compound (RS)-5a. Calcd (%) for C19H23F2N5O2 × 1.25H2O: C 55.13, H 6.21, N 16.92. Found (%): C 55.07, H 6.29, N 16.62.

(RS)-4-[6-(2-Amino-6-chloropyrimidin-4-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine [(RS)-5b]. Yellowish powder m.p. 92–94 °C (yield 0.36 g, 68%) after flash column chromatography, chloroform–EtOH 97: 3 as an eluent. HPLC ((S,S)-Whelk-O1, MeOH–H2O 75: 25, 0.8 mL/min; detection at 280 nm): τ1 23.7 min, τ2 31.5 min. 1H NMR (500 MHz, DMSO-d6, 100 °C) δ (ppm): 1.12 (d, J = 6.9 Hz, 3H, Me), 1.33–1.38 (m, 2H, CH2 hexanoyl), 1.48–1.54 (m, 2H, CH2 hexanoyl), 1.57–1.66 (m, 2H, CH2 hexanoyl), 2.44–2.52 (m, 1H, 2-HB hexanoyl, partly overlapped with DMSO signal), 2.57–2.64 (m, 1H, 2-HA hexanoyl), 3.20 (td, J = 6.7, 5.9 Hz, 2H, 6-H hexanoyl), 4.14 (dd, J = 10.9, 2.8 Hz, 1H, 2-HB benzoxazine), 4.33 (dd, J = 11.0, 1.5 Hz, 1H, 2-HA benzoxazine), 4.73 (q, J = 6.9 Hz, 1H, 3-H benzoxazine), 5.74 (s, 1H, 5-H pyrimidine), 5.88 (br. s, 2H, NH2), 6.69 (br. s, 1H, NH), 6.85 (ddd, J = 9.8, 8.3, 5.7 Hz, 1H, 6-H benzoxazine), 7.56 (ddd, J = 11.0, 6.8, 2.8 Hz, 1H, 5-H benzoxazine). 19F NMR (470 MHz, DMSO-d6, 100 °C) δ (ppm): 2.01 (ddd, J = 21.0, 8.2, 2.1 Hz, 1F, 8-F), 20.14 (br. s, 1F, 7-F). 13C NMR (126 MHz, DMSO-d6) δ (ppm): 15.10, 24.38, 25.98, 28.67, 33.40, 39.6 (overlapped by DMSO signal), 45.12 (br. s), 69.84, 92.58 (br. s), 106.77 (d, J = 17.9 Hz), 119.26, 121.78, 135.68 (br. d, J = 9.7 Hz), 138.92 (dd, J = 243.3, 15.3 Hz), 146.47 (br. d, J = 241.6 Hz), 157.05 (br. s), 162.88, 164.00, 170.93. Calcd (%) for C19H22ClF2N5O2: C 53.59, H 5.21, N 16.45, Cl 8.33, F 8.92. Found (%): C 53.53, H 5.33, N 16.32, Cl 8.10, F 9.27.

(RS)-4-[6-(6-Chloropyrimidin-4-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine [(RS)-5c]. White powder m.p. 101–103 °C (yield 0.35 g, 68%) after flash column chromatography, EtOAc as an eluent. HPLC ((S,S)-Whelk-O1, MeOH–H2O 75: 25, 0.8 mL/min; detection at 254 nm): τ1 21.6 min, τ2 28.7 min. 1H NMR (500 MHz, DMSO-d6, 100 °C) δ (ppm): 1.12 (d, J = 6.9 Hz, 3H, Me), 1.34–1.40 (m, 2H, CH2 hexanoyl), 1.51–1.57 (m, 2H, CH2 hexanoyl), 1.59–1.64 (m, 2H, CH2 hexanoyl), 2.44–2.50 (m, 1H, 2-HB hexanoyl, overlapped with DMSO signal), 2.57–2.63 (m, 1H, 2-HA hexanoyl), 3.27 (td, J = 6.7, 6.2 Hz, 2H, 6-H hexanoyl), 4.13 (dd, J = 10.9, 2.9 Hz, 1H, 2-HB benzoxazine), 4.33 (dd, J = 10.9, 1.4 Hz, 1H, 2-HA benzoxazine), 4.74 (qd, J = 6.9, 2.9 Hz, 1H, 3-H benzoxazine), 6.47 (s, 1H, 5-H pyrimidine), 6.84 (ddd, J = 9.9, 9.6, 8.3 Hz, 1H, 6-H benzoxazine), 7.31 (br. s, 1H, NH), 7.55 (ddd, J = 9.3, 5.4, 2.4 Hz, 1H, 5-H benzoxazine), 8.19 (s, 1H, 2-H pyrimidine). 19F NMR (470 MHz, DMSO-d6, 100 °C) δ (ppm): 2.02 (ddd, J = 21.0, 8.1, 2.3 Hz, 1F, 8-F), 20.09–20.17 (m, 1F, 7-F). 13C NMR (126 MHz, DMSO-d6) δ (ppm): 15.17, 24.40, 25.96, 28.53, 33.42, 39.6 (overlapped by DMSO signal), 44.88 (br. s), 69.91, 103.53, 106.83 (d, J = 17.9 Hz), 119.33, 121.85, 135.75 (br. d, J = 9.7 Hz), 138.99 (dd, J = 243.3, 15.4 Hz), 146.55 (br. d, J = 243.9 Hz), 156.86 (br. s), 158.56, 163.19, 170.97. Calcd (%) for C19H21ClF2N4O2: C 55.54, H 5.15, N 13.64, Cl 8.63, F 9.25. Found (%): C 55.44, H 5.01, N 13.58, Cl 8.61, F 8.89.

(RS)-7,8-Difluoro-3,4-dihydro-3-methyl-4-[6-(pyrimidin-2-yl)aminohexanoyl]-2H-[1,4]benzoxazine [(RS)-5d]. White powder m.p. 106–108 °C (yield 0.41 g, 87%) after flash column chromatography, benzene–EtOAc–MeOH 7: 3: 0.2 as an eluent. 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.11 (d, J = 5.8 Hz, 3H, Me), 1.28–1.40 (m, 2H, CH2 hexanoyl), 1.49–1.64 (m, 4H, CH2–CH2 hexanoyl), 2.43-2.50 (m, 1H, 2-HB hexanoyl, partly overlapped with DMSO signal), 2.62-2.68 (m, 1H, 2-HA hexanoyl), 3.23 (td, J = 6.6, 6.5 Hz, 2H, 6-H hexanoyl), 4.14 (d, J = 10.3 Hz, 1H, 2-HB benzoxazine), 4.35 (d, J = 10.7 Hz, 1H, 2-HA benzoxazine), 4.71 (br. s, 1H, 3-H benzoxazine), 6.51 (dd, J = 4.7, 4.7 Hz, 1H, 5-H pyrimidine), 6.92 (dd, J = 9.5, 8.8 Hz, 1H, 6-H benzoxazine), 7.09 (t, J = 5.5 Hz, 1H, NH hexanoyl), 7.40-8.10 (m, 1H, 5-H benzoxazine), 8.23 (d, J = 4.6 Hz, 2H, 4-H and 6-H pyrimidine). 19F NMR (470 MHz, DMSO-d6,) δ (ppm): 1.85 (br. s, 1F, 8-F), 19.53 (br. s, 1F, 7-F). 13C NMR (126 MHz, DMSO-d6,) δ (ppm): 15.07, 24.41, 25.97, 28.68, 33.39, 40.36, 44.73 (br. s), 69.83, 106.73 (d, J = 17.9 Hz), 109.54, 119.24, 121.78, 135.66 (dd, J = 9.8, 2.4 Hz), 138.90 (dd, J = 243.2, 15.4 Hz), 146.45 (dd, J = 242.0, 10.5 Hz), 157.73 (2C), 162.27, 170.93. Calcd (%) for C19H22F2N4O2: C 60.63, H 5.89, N 14.88. Found (%): C 60.60, H 6.02, N 14.71.

(RS)-7,8-Difluoro-3,4-dihydro-3-methyl-4-[6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)aminohexanoyl]-2H-[1,4]benzoxazine Hemihydrate [(RS)-5e]. White powder m.p. 86–88 °C (yield 0.26 g, 50%) after flash column chromatography, EtOAc–EtOH 9: 1 as an eluent. HPLC ((S,S)-Whelk-O1, MeOH–H2O 75: 25, 0.8 mL/min; detection at 280 nm): τ( 22.4 min, τ( 29.0 min. 1H NMR (500 MHz, DMSO-d6, 100 °C) δ (ppm): 1.12 (d, J = 6.8 Hz, 3H, Me), 1.39–1.44 (m, 2H, CH2 hexanoyl), 1.60–1.68 (m, 4H, CH2–CH2 hexanoyl), 2.45–2.51 (m, 1H, 2-HB hexanoyl, partly overlapped with DMSO signal), 2.58–2.64 (m, 1H, 2-HA hexanoyl), 3.47 (td, J = 6.9, 6.1 Hz, 2H, 6-H hexanoyl), 4.13 (dd, J = 10.9, 2.8 Hz, 1H, 2-HB benzoxazine), 4.32 (dd, J = 10.9, 1.3 Hz, 1H, 2-HA; benzoxazine), 4.73 (q, J = 5.6 Hz, 1H, 3-H benzoxazine), 6.50 (d, J = 3.3 Hz, 1H, 5-H pyrrole), 6.83 (dd, J = 9.8, 8.4 Hz, 1H, 6-H benzoxazine), 6.87 (br. s, 1H, NH hexanoyl), 6.97 (d, J = 3.4 Hz, 1H, 4-H pyrrole), 7.54-7.57 (m, 1H, 5-H benzoxazine), 8.05 (s, 1H, 2-H pyrimidine), 11.05 (s, 1H, NH pyrrole). 19F NMR (470 MHz, DMSO-d6, 100 °C) δ (ppm): 1.99 (ddd, J = 21.1, 8.3, 2.1 Hz, 1F, 8-F), 20.09 (br. s, 1F, 7-F). 13C NMR (126 MHz, DMSO-d6) δ (ppm): 15.09, 24.47, 26.08, 29.09, 33.43, 39.6 (overlapped by DMSO signal), 44.87 (br. s), 69.84, 98.48, 102.37, 106.75 (d, J = 17.9 Hz), 119.26, 120.45, 121.79, 135.68 (dd, J = 9.7, 1.9 Hz), 138.92 (dd, J = 243.2, 15.3 Hz), 146.50 (br. d, J = 241.8 Hz), 149.99, 151.41, 156.09, 170.95. Calcd (%) for C21H23F2N5O2 × 0.5H2O: C 59.43, H 5.70, N 16.50, F 8.95. Found (%): C 59.59, H 5.76, N 16.40, F 8.58.

(3S)-7,8-Difluoro-3,4-dihydro-3-methyl-4-[6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)aminohexanoyl]-2H-[1,4]benzoxazine [(S)-5e]. White powder m.p. 87–89 °C (yield 0.15 g, 29%) after flash column chromatography, EtOAc–EtOH 9: 1 as an eluent. [α]D20 +57.5 (c 0.5, EtOH). >99% ee. HPLC ((S,S)-Whelk-O1, MeOH–H2O 75: 25, 0.8 mL/min; detection at 280 nm): τ( 22.3 min. 1H, 19F and 13C NMR spectra were identical to those of compound (RS)-5e. HRMS (ESI): m/z [M+H]+ calcd for [C21H24F2N5O2]+: 416.1898, found: 416.1897.

4. 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 1H, 19F, and 13C 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.