An Efficient Route to Novel Uracil-Based Drug-Like Molecules

Abstract

In order to identify new antiretroviral agents, a series of novel uracil derivatives have been synthesized. Optimized conditions for coupling of Weinreb amides with aromatic Grignard reagents allow the convenient preparation of key benzophenone intermediates in high yields and purities. The use of a modified silyl Hilbert–Johnson reaction affords the target compounds under mild conditions.

Despite the wide arsenal of antiviral drugs currently available to fight HIV infection, the search for new, safer and more accessible alternatives remains a critical goal for medicinal chemists. Previously, we have reported a series of 1-[2-(2-benzoylphenoxy)ethyl]uracils (Figure [1]) that exhibited significant inhibitory activity against HIV-1 reverse transcriptase (RT) in both wild and mutant cell lines.[1] As part of our ongoing structure–activity studies to increase the activity of these analogues, we have designed a series containing a shorter linker between the benzophenone and the uracil nucleus of the pharmacophore. These aspects of the scaffold are well known to be important for the biological activity exhibited by a number of known HIV RT inhibitors.[2] We performed a series of docking experiments to probe the fit of the newly designed scaffold to the HIV-1 RT non-nucleoside binding pocket. For this purpose, the four most relevant HIV RT crystallographic complexes were selected (PDB codes 3DYA,[3] 3DOK,[4] 4H4M,[5] 2YNG[6]) (Figure [2]) and predicted binding modes were analyzed with emphasis on known key protein–ligand interactions[7] (Figure [3]). Preliminary evaluation of the proposed structures indicated their potential, thus we proceeded with their synthesis.

Retrosynthetic analysis (Scheme [1]) suggested the optimal pathway to the targets focused on two key steps: a) synthesis of a series of substituted 3-methylbenzophenones, and b) coupling of the benzophenones with an appropriate N-heterocycle.

Coupling N-methoxy-N-methylamides (also known as Weinreb amides) with the appropriate Grignard reagents resulted in benzophenones 3a–f (Scheme [2]). The original procedure was described by Nahm and Weinreb,[8] however, a number of approaches have subsequently been used to obtain ketones starting from various organometallic reagents.[9] Despite this, Weinreb’s original method was selected since it is facile and does not require catalysis by transition metals. Moreover, the absence of undesirable tertiary alcohol by-products, which have been shown to have formed in analogous methods by addition of two equivalents of Grignard reagent to the target ketone, is an inherent feature rendering it an attractive approach.

The carbonyl groups of the starting benzoic acids 1a–f were activated by conversion into their corresponding acid chlorides, which readily formed Weinreb amides 2a–f upon treatment with N,O-dimethylhydroxylamine hydrochloride under phase-transfer catalysis conditions (potassium carbonate, water–toluene). Subsequent coupling with m-tolylmagnesium bromide was successfully performed in absolute tetrahydrofuran at –5 °C under an argon atmosphere (Scheme [2]). The original procedure proposed three equivalents of a Grignard reagent to be reacted with one equivalent of an amide. To investigate whether this was really necessary we undertook a series of experiments. The subsequent coupling reactions were performed under identical conditions with the exception of the molar ratio of Grignard to Weinreb amide, which was varied from 1.3 to 1.5 equivalents (Table [1]). The results revealed that while indeed an excess of the organometallic reagent did improve the yield of the ketone, it became quantitative at 1.5-fold excess, so a three-fold excess is unnecessary. It should also be noted that the reduced yield of compound 3e is likely due to addition of the Grignard to the cyano substitutent.

Table 1 Effect of Excess Grignard Reagent on the Yield of Ketones 3

Entry	R	Grignard reagent (mol equiv)	Product	Yield (%)a
1	H	1.50	3a	97
2	3-Cl	1.39	3b	89
3	3-Br	1.32	3c	82
4	3-F3C	1.41	3d	85
5	3-NC	1.40	3e	46
6	3,5-Cl2	1.41	3f	83

^a Yield of isolated product.

Next, we sought the most convenient method for the preparation of the 3-bromomethylbenzophenones 4a–f. This initially appeared to be the use of free-radical bromination, however, initial attempts using molecular bromine in 1,2-dibromoethane[10] resulted in poor yields. Additionally, one major drawback of using 1,2-dibromoethane is its relatively high boiling point, which then makes it difficult to remove upon completion of the reaction.

A literature search revealed a number of commonly used bromination procedures that had been previously employed for methylbenzophenones: molecular bromine, N-bromosuccinimide (NBS), and N-bromosaccharin (among others) as the brominating agent, and carbon tetrachloride or 1,2-dibromoethane as the solvent. In addition, elevated temperatures are known to facilitate bromination of unreactive substrates.

As a result, we opted to attempt the reaction under melt conditions. It was observed (by TLC) that the reaction took place when the temperature reached 130 °C, which is close to the boiling point of 1,2-dibromoethane, however, with no improvement in yield. Slightly better results were obtained with N-bromosuccinimide in carbon tetrachloride.[11] [12] [13] Moreover, only the bromine/carbon tetrachloride[14] conditions allowed for the convenient preparation of the target 3-bromomethylbenzophenones 4a–f with average isolated yields of around 70% (Table [2], entries 5–8). All the products were purified by short-column flash chromatography on silica, and NMR spectroscopy was used to confirm their purity, since nuclear bromination products are typical by-products of this method.

The final step of the synthetic sequence involved condensation of 3-bromomethylbenzophenones 4a–f with a range of pyrimidine bases using a modified Vorbrüggen procedure (a silyl Hilbert–Johnson reaction). Silylated bases (uracil, thymine, isoorotic acid, cytosine) all reacted smoothly with bromides 4a–f in refluxing 1,2-dichloroethane in the absence of Lewis acids. Target compounds 5a–i were obtained in moderate to high yields, with no detectable amounts of N³-substitution products (Table [3]). It should be noted that we have previously demonstrated the effectiveness of this particular method for regioselective N¹-alkylation of pyrimidine bases.[15] [16]

Table 2 Optimization of the Bromination Reaction

Entry	R	Conditions	Product	Yield (%)a
1	3-Br	Br2, Br(CH2)2Br, 130 °C	4c	37
2	3-Br	Br2, neat, 130 °C	4c	38
3	3-Cl	Br2, neat, 130 °C	4b	45
4	H	NBS, CCl4, Bz2O2, 77 °C	4a	54
5	3-Cl	Br2, CCl4, 77 °C	4b	64
6	3,5-Cl2	Br2, CCl4, 77 °C	4f	68
7	3-F3C	Br2, CCl4, 77 °C	4d	71
8	3-NC	Br2, CCl4, 77 °C	4e	75

^a Yield of isolated product.

Different analogues of the desired scaffold (5j,k) were obtained (Scheme [3]) since C5 of the uracil ring reacts readily with many electrophilic agents.[17] In particular, the 5-bromo derivative 5j was readily obtained upon treatment of 5f with bromine in glacial acetic acid in the presence of sodium acetate as a hydrogen bromide acceptor.

Table 3 Properties of the Target Products

Product	R1	R2	Yield (%)a	Mp ( °C)	Rf b
5a	H	H	69	158–159.5	0.45
5b	3-Cl	H	63	174–175	0.44
5c	3-Br	H	75	185.5–186.5	0.46
5d	3-F3C	H	87	167–168.5	0.44
5e	3-NC	H	73	208.5–210	0.28
5f	3,5-Cl2	H	77	155.5–157	0.51
5g	3,5-Cl2	Me	81	193.5–195.5	0.58
5h	3,5-Cl2	CO2H	36	217–219	0.57c
5i d	–	–	59	281.5–283	0.50
5j	3,5-Cl2	Br	92	186–187.5	0.77
5k e	–	–	77	200–201	0.16f
5l g	–	–	55	173–175	0.70
5m g	–	–	77	195.5–197.5	0.47
5n g	–	–	74	141.5–143	0.50

^a Yield of isolated product.

^b EtOAc.

^c i-PrOH–EtOAc–NH₄OH_aq (9:6:5).

^d Structure is shown above.

^e Structure is shown in Scheme [3].

^f Hexane–EtOAc (1:1).

^g Structures are shown in Scheme [5].

In the case of the silylated 6-substituted uracils, for example, 6-chlorouracil, both steric and electronic effects render the N¹- and N³-substitution rates nearly equivalent (Scheme [4]).[18] [19] As a result, in order to obtain the 6-chlorouracil derivative, we used the method proposed by Ogura et al.[20] Thus alkylation of the potassium salt of 6-chlorouracil in dimethyl sulfoxide afforded compound 5l in 55% yield (Scheme [5]).

A similar procedure[21] was employed for the synthesis of the hydantoin derivative 5n and the 1-[3-(3,5-dichlorobenzoyl)benzyl]uracil 5m (Scheme [5]). The benzyloxymethyl (BOM) protecting group was subsequently cleaved in a facile manner upon reflux in a mixture of hydrochloric acid and ethanol.[22]

Once in hand, the target compounds were tested for inhibition of the RNA-dependent DNA polymerase (RDDP) activity of HIV-1 reverse transcriptase. Unfortunately, despite promising computational results, only two compounds exhibited activity, albeit moderate (5j and 5l, IC₅₀ 7.6 μM and 7.3 μM, respectively). Assays against other viral targets are currently underway.

Inspection of the binding mode predicted for compound 5j revealed a set of valuable interactions, including hydrogen bonds with the Lys103 and Pro236 backbone, as well as π-stacking with aromatic residues buried inside the binding pocket (Figure [3]). Noticeably, 5-bromo and 6-chloro substituents in derivatives 5j and 5l occupy a space next to the Leu234 side chain and favor orientation of the uracil amide group toward formation of hydrogen bonds, which apparently contributes to their potency. In re-examining the modeling in light of the lack of activity for most of the series however, we saw very little difference between the conformations of the two active compounds in comparison to all of the inactive compounds. This is depicted in Figure [4] for 5j (active) and 5f (inactive), however, the results were the same for all of the compounds. The only difference we observed was the placement of the carbonyl groups of the bottom ring, which were slightly different than the inactive compounds. We also considered the possibility of weak halogen bonds, however there are no hydrogen bond acceptors within 4 Å. As a result, we can only speculate that the differences in activity may be due to small changes in the overall tertiary structure, which in turn, would affect the binding site orientation. In general, however, it is likely that the overall low potency of the series could be attributed to conformational restrictions imposed by a carbonyl linker.

In conclusion, we have developed a convenient procedure to prepare novel uracil derivatives possessing a benzophenone moiety. This route might prove useful for future work focused on the design and synthesis of other similar antiviral agents.

All reagents were obtained at the highest grade available from Sigma and Acros Organics, and were used without further purification unless otherwise noted. Anhydrous DMF and isopropyl alcohol were purchased from Sigma-Aldrich Co. Anhydrous acetone, DCE, and EtOAc­ were obtained by distillation over P₂O₅. TLC was performed on Merck TLC Silica gel 60 F₂₅₄ plates eluting with the specified solvents and samples were made visual with a UV lamp, VL-6.LC (France). Acros Organics (Belgium) silica gel (Kieselgur 60–200 μm, 60A) was used for column chromatography. Yields refer to spectroscopically (¹H and ¹³C NMR) homogeneous materials. Melting points were determined in glass capillaries on a Mel-Temp 3.0 (Laboratory Devices Inc., US). NMR spectra were obtained using Bruker Avance 400 (400 MHz for ¹H and 100 MHz for ¹³C) and Bruker Avance 600 (600 MHz for ¹H and 150 MHz for ¹³C) spectrometers in DMSO-d ₆, CDCl₃, D₂O or CD₃CN with tetramethylsilane as an internal standard. High-resolution mass spectra were measured on Bruker micrOTOF II instruments using electrospray ionization (HRESIMS). The measurements were run in positive ion mode (interface capillary voltage –4500 V) in a mass range from m/z 50 to m/z 3000 Da; external or internal calibration was performed with ESI Tuning MixTM (Agilent Technologies). A syringe injection was used for solutions in MeCN (flow rate = 3 μL/min). N₂ was applied as a dry gas; the interface temperature was set at 180 °C.

N-Methoxy-N-methylbenzamides 2a–f; General Procedure

The corresponding benzoic acid (34.82 mmol) and SOCl₂ (3.30 mL, 45.24 mmol, 1.30 equiv) were heated to reflux temperature in anhydrous DCE (40 mL) for 1 h, after which the mixture was evaporated and distilled. The resulting acid chloride (31.58 mmol) was added dropwise over a period of 20 min to a stirred solution of N,O-dimethylhydroxylamine hydrochloride (3.08 g, 31.58 mmol, 1.00 equiv) and K₂CO₃ (9.51 g, 68.81 mmol, 2.18 equiv) in a mixture of H₂O–toluene (1:1, 60 mL) at 0 °C. After stirring for 2 h, the organic phase was washed with dilute aq NaOH and H₂O, and then dried over MgSO₄ and concentrated under vacuum to give 2, in quantitative yield, as a colorless oil. All the products 2 were sufficiently pure to be used directly in the next step.

Phenyl(m-tolyl)methanone (3a); Typical Procedure

A 1 M solution of m-tolylmagnesium bromide (25 mL, 25.00 mmol) in absolute THF was added dropwise over a period of 30 min to a stirred solution of 2a (2.70 g, 16.35 mmol) in THF (50 mL) under an Ar atmosphere at –5 °C, and stirring was continued for 1 h. The reaction was quenched by the addition of a cold sat. aq solution of NH₄Cl. The organic phase was washed with H₂O (2 × 50 mL), dried and evaporated. The residue was purified by flash chromatography on silica eluting with hexane to give 3a (3.87 g, 97%) as clear, yellowish oil.

R_f = 0.56 (hexane–EtOAc, 1:10).

The NMR spectra of the analytical sample were identical with the previously reported data.[23] [24]

(3-Chlorophenyl)(m-tolyl)methanone (3b)

The product was synthesized in a similar manner to 3a giving 3b (3.40 g, 89%) as yellowish crystals.

Mp 68–69.5 °C; R_f = 0.52 (hexane–EtOAc, 1:10).

¹H NMR (600 MHz, DMSO-d ₆): δ = 2.38 (s, 3 H, CH₃), 7.44 (t, J = 7.6 Hz, 1 H, H-5′), 7.50 (t, J = 6.8 Hz, 2 H, H-4′, H-6′), 7.55 (s, 1 H, H-2′), 7.57 (t, J = 7.9 Hz, 1 H, H-5), 7.63 (dt, J = 7.7, 1.3 Hz, 1 H, H-6), 7.69 (t, J = 1.8 Hz, 1 H, H-2), 7.72 (ddd, J = 7.9, 2.2, 1.1 Hz, 1 H, H-4).

¹³C NMR (150 MHz, DMSO-d ₆): δ = 25.1, 131.2, 132.4, 132.7, 133.0, 134.1, 134.7, 136.5, 137.7, 137.9, 140.7, 142.4, 143.4, 198.7.

(3-Bromophenyl)(m-tolyl)methanone (3c)

The product was synthesized in a similar manner to 3a giving 3c (6.79 g, 82%) as yellowish crystals.

Mp 66–67.5 °C; R_f = 0.59 (hexane–EtOAc, 1:10).

¹H NMR (600 MHz, DMSO-d ₆): δ = 2.37 (s, 3 H, CH₃), 7.42–7.46 (m, 1 H, H-5′), 7.48–7.53 (m, 3 H, H-4′, H-6′, H-5), 7.55 (s, 1 H, H-2′), 7.67 (dt, J = 7.7, 1.3 Hz, 1 H, H-6), 7.82 (t, J = 1.8 Hz, 1 H, H-2), 7.86 (ddd, J = 8.0, 2.0, 1.0 Hz, 1 H, H-4).

¹³C NMR (150 MHz, DMSO-d ₆): δ = 25.1, 126.1, 131.3, 132.7, 132.8, 134.1, 134.9, 135.9, 137.9, 139.4, 140.6, 142.4, 143.5, 198.7.

m-Tolyl[3-(trifluoromethyl)phenyl]methanone (3d)

The product was synthesized in a similar manner to 3a giving 3d (4.60 g, 85%) as white crystals.

Mp 55.5–57.5 °C; R_f = 0.49 (hexane–EtOAc, 1:10).

¹H NMR (400 MHz, DMSO-d ₆): δ = 2.37 (s, 3 H, CH₃), 7.41–7.48 (m, 1 H, H-5′), 7.48–7.54 (m, 2 H, H-4′, H-6′), 7.57 (s, 1 H, H-2′), 7.75–7.83 (m, 1 H, H-5), 7.97 (br s, 2 H, H-6, H-2), 8.02 (d, J = 7.8 Hz, 1 H, H-4).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 21.1, 125.91, 125.95, 127.4, 128.9, 129.2, 130.1, 130.3, 133.8, 134.1, 136.6, 138.4, 138.6, 194.9.

3-(3-Methylbenzoyl)benzonitrile (3e)

The product was synthesized in a similar manner to 3a giving 3e (3.34 g, 46%) as white crystals.

Mp 110.5–112 °C; R_f = 0.23 (hexane–EtOAc, 1:10).

¹H NMR (400 MHz, DMSO-d ₆): δ = 2.38 (s, 3 H, CH₃), 7.42–7.48 (m, 1 H, H-5′), 7.49–7.54 (m, 2 H, H-4′, H-6′), 7.57 (s, 1 H, H-2′), 7.75 (t, J = 7.8 Hz, 1 H, H-5), 7.99 (d, J = 7.8 Hz, 1 H, H-6), 8.09 (s, 1 H, H-2), 8.11 (d, J = 7.8 Hz, 1 H, H-4).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 21.2, 112.2, 118.4, 127.5, 128.9, 130.2, 130.4, 133.2, 134.2, 134.3, 136.0, 136.3, 138.5, 138.6, 194.5.

(3,5-Dichlorophenyl)(m-tolyl)methanone (3f)

The product was synthesized in a similar manner to 3a giving 3f (6.83 g, 83%) as yellowish crystals.

Mp 88–89.5 °C; R_f = 0.62 (hexane–EtOAc, 1:10).

¹H NMR (600 MHz, DMSO-d ₆): δ = 2.38 (d, J = 3.8 Hz, 3 H, CH₃), 7.40–7.47 (m, 1 H, H-5′), 7.47–7.53 (m, 2 H, H-4′, H-6′), 7.55 (br s, 1 H, H-2′), 7.61 (dd, J = 4.7, 1.4 Hz, 2 H, H-2, H-6), 7.83 (d, J = 3.2 Hz, 1 H, H-4).

¹³C NMR (150 MHz, DMSO-d ₆): δ = 25.0, 131.2, 131.9, 132.8, 134.0, 135.7, 138.2, 138.7, 140.2, 142.6, 144.8.

[3-(Bromomethyl)phenyl](phenyl)methanone (4a)

Method A. NBS (2.83 g, 15.90 mmol, 1.00 equiv), Bz₂O₂ (0.19 g, 0.78 mmol, 0.05 equiv) and 3a (3.12 g, 15.90 mmol) in CCl₄ (40 mL) were heated at reflux temperature for 1 h under irradiation with a tungsten lamp (100 W). The volatiles and solvent were evaporated under vacuum, and the residue was purified by flash chromatography on silica eluting with DCE to give 4a (2.36 g, 54%) as a colorless oil.

R_f = 0.31 (hexane–EtOAc, 1:10).

The NMR spectra of the analytical sample were identical with the previously reported data.[25]

[3-(Bromomethyl)phenyl](3-chlorophenyl)methanone (4b)

Method B. Br₂ (0.45 mL, 8.73 mmol, 1.01 equiv) was added dropwise to a melt of 3b (2.00 g, 9.09 mmol) over a period of 15 min under irradiation with a tungsten lamp (100 W). After heating for 1 h at 130–140 °C, the volatiles were evaporated under vacuum, and the residue was purified as described in Method A to give 4b (1.20 g, 45%) as white crystals.

Mp 114.5–116 °C; R_f = 0.31 (hexane–EtOAc, 1:10).

¹H NMR (400 MHz, DMSO-d ₆): δ = 4.81 (s, 2 H, CH₂), 7.52–7.63 (m, 2 H, H-5′, H-4′), 7.63–7.70 (m, 2 H, H-6′, H-2′), 7.70–7.80 (m, 3 H, H-5, H-6, H-2), 7.80–7.90 (m, 1 H, H-4).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 33.4, 128.2, 128.9, 129.1, 129.6, 130.1, 130.6, 132.4, 133.0, 133.5, 133.8, 136.8, 138.8, 194.0.

[3-(Bromomethyl)phenyl](3-bromophenyl)methanone (4c)

Method C. Br₂ (0.20 mL, 3.88 mmol, 1.03 equiv) was added dropwise to a refluxing solution of 3c (1.04 g, 3.78 mmol) in 1,2-dibromo­ethane (25 mL) for 15 min under irradiation with a tungsten lamp (100 W). After heating for 1 h, the volatiles and solvent were evaporated under vacuum. The residue was purified as described in Method A to give 4c (0.48 g, 37%) as white crystals.

Mp 122–124 °C; R_f = 0.39 (hexane–EtOAc, 1:10).

¹H NMR (600 MHz, DMSO-d ₆): δ = 4.79 (s, 2 H, CH₂), 7.53 (t, J = 7.8 Hz, 1 H, H-5′), 7.56 (t, J = 7.8 Hz, 1 H, H-4′), 7.67 (d, J = 7.7 Hz, 1 H, H-6′), 7.70 (d, J = 7.7 Hz, 1 H, H-2′), 7.76 (d, J = 7.7 Hz, 1 H, H-5), 7.82–7.90 (m, 3 H, H-6, H-2, H-4).

¹³C NMR (150 MHz, DMSO-d ₆): δ = 37.6, 126.1, 132.7, 133.3, 133.7, 134.3, 135.0, 136.0, 137.9, 139.5, 141.1, 143.0, 198.1.

[3-(Bromomethyl)phenyl][3-(trifluoromethyl)phenyl]methanone (4d)

Method D. Br₂ (0.49 mL, 9.51 mmol, 1.00 equiv) was added dropwise to a refluxing solution of 3d (2.52 g, 9.54 mmol) in CCl₄ (40 mL) under irradiation with a tungsten lamp (100 W). After 20 min, the volatiles and solvent were evaporated under vacuum, and the residue was purified as described in Method A to give 4d (2.32 g, 71%) as white crystals.

Mp 74–76 °C; R_f = 0.31 (hexane–EtOAc, 1:10).

¹H NMR (400 MHz, DMSO-d ₆): δ = 4.81 (s, 2 H, CH₂), 7.51–7.62 (m, 1 H, H-5′), 7.69 (d, J = 7.8 Hz, 1 H, H-4′), 7.76–7.85 (m, 2 H, H-6′, H-2′), 7.87 (s, 1 H, H-5), 7.97–8.03 (m, 2 H, H-6, H-2), 8.05 (d, J = 7.8 Hz, 1 H, H-4).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 33.7, 126.08, 126.12, 129.4, 129.5, 130.0, 130.3, 130.7, 133.9, 134.2, 136.9, 138.1, 139.2, 194.4.

3-[3-(Bromomethyl)benzoyl]benzonitrile (4e)

The product was synthesized in a similar manner to 4d giving 4e (1.01 g, 75%) as white crystals.

Mp 109–111 °C; R_f = 0.14 (hexane–EtOAc, 1:10).

¹H NMR (400 MHz, DMSO-d ₆): δ = 4.81 (s, 2 H, CH₂), 7.52–7.61 (m, 1 H, H-5′), 7.69 (dt, J = 7.8, 1.4 Hz, 1 H, H-4′), 7.74–7.82 (m, 2 H, H-6′, H-5), 7.87 (t, J = 1.6 Hz, 1 H, H-2′), 7.97–8.05 (m, 1 H, H-6), 8.10–8.14 (m, 1 H, H-2), 8.14–8.17 (m, 1 H, H-4).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 33.7, 112.2, 118.4, 129.6, 130.2, 130.3, 130.7, 133.3, 134.2, 134.4, 136.2, 136.7, 138.2, 139.3, 194.0.

[3-(Bromomethyl)phenyl](3,5-dichlorophenyl)methanone (4f)

The product was synthesized in a similar manner to 4d giving 4f (4.36 g, 68%) as white crystals.

Mp 73.5–76 °C; R_f = 0.46 (hexane–EtOAc, 1:10).

¹H NMR (400 MHz, DMSO-d ₆): δ = 4.81 (s, 2 H, CH₂), 7.52–7.61 (m, 1 H, H-5′), 7.62–7.72 (m, 3 H, H-4′, H-2, H-6), 7.75–7.82 (m, 1 H, H-6′), 7.86 (s, 1 H, H-2′), 7.90–7.96 (m, 1 H, H-4).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 33.7, 128.2, 129.6, 130.1, 130.6, 132.1, 134.5, 134.8, 136.5, 139.3, 140.5, 193.0.

1-(3-Benzoylbenzyl)pyrimidine-2,4(1H,3H)-dione (5a); Typical Procedure

A mixture of uracil (1.26 g, 11.24 mmol), HMDS (10 mL) and NH₄Cl (200 mg) was heated at reflux temperature for 10 h until a homogeneous solution was obtained. Excess HMDS was removed under reduced pressure, and the residual clear oil was dissolved in anhydrous DCE. A solution of 4a (3.09 g, 11.23 mmol, 1.00 equiv) in DCE was then added, and the mixture heated at reflux temperature for 20 h, at which point it was allowed to cool to r.t. i-PrOH was added and the resulting precipitate was filtered off and purified by flash chromatography on silica eluting with EtOAc. The residue was recrystallized from a mixture of DCE–hexane (3:2) to give 5a (2.38 g, 69%) as colorless crystals.

Mp 158–159.5 °C; R_f = 0.45 (EtOAc).

¹H NMR (600 MHz, DMSO-d ₆): δ = 4.97 (s, 2 H, CH₂), 5.62 (dd, J = 7.8, 2.2 Hz, 1 H, Ura-H-5), 7.52–7.58 (m, 3 H, H-5′, H-3, H-5), 7.61 (d, J = 7.8 Hz, 1 H, H-4′), 7.63–7.69 (m, 2 H, H-2′, H-6′), 7.70–7.75 (m, 3 H, H-4, H-2, H-6), 7.83 (d, J = 7.9 Hz, 1 H, Ura-H-6), 11.39 (s, 1 H, NH).

¹³C NMR (150 MHz, DMSO-d ₆): δ = 54.3, 105.7, 132.8, 133.2, 133.3, 133.9, 136.0, 137.0, 141.0, 141.4, 141.7, 149.8, 155.3, 167.9, 199.7.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₅N₂O₃: 307.1077; found 307.1074; m/z [M + Na]⁺ calcd for C₁₈H₁₄N₂O₃Na: 329.0897; found: 329.0892.

1-[3-(3-Chlorobenzoyl)benzyl]pyrimidine-2,4(1H,3H)-dione (5b)

The product was synthesized in a similar manner to 5a giving 5b (0.62 g, 63%) as colorless crystals.

Mp 174–175 °C; R_f = 0.44 (EtOAc).

¹H NMR (400 MHz, DMSO-d ₆): δ = 4.97 (s, 2 H, CH₂), 5.62 (dd, J = 7.8, 2.0 Hz, 1 H, Ura-H-5), 7.53–7.61 (m, 2 H, H-5′, H-4′), 7.61–7.69 (m, 3 H, H-6′, H-2′, H-5), 7.69–7.76 (m, 3 H, H-6, H-2, H-4), 7.83 (d, J = 7.8 Hz, 1 H, Ura-H-6), 11.37 (s, 1 H, NH).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 50.4, 101.8, 128.6, 129.0, 129.3, 129.4, 129.6, 130.8, 132.5, 132.8, 133.8, 136.9, 137.9, 139.1, 145.9, 151.4, 164.0, 194.4.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₄ClN₂O₃: 341.0687; found: 341.0682; m/z [M + Na]⁺ calcd for C₁₈H₁₃ClN₂O₃Na: 363.0507; found: 363.0503.

1-[3-(3-Bromobenzoyl)benzyl]pyrimidine-2,4(1H,3H)-dione (5c)

The product was synthesized in a similar manner to 5a giving 5c (0.96 g, 75%) as colorless crystals.

Mp 185.5–186.5 °C; R_f = 0.46 (EtOAc).

¹H NMR (600 MHz, DMSO-d ₆): δ = 4.97 (s, 2 H, CH₂), 5.61 (dd, J = 7.8, 2.3 Hz, 1 H, Ura-H-5), 7.51 (t, J = 7.8 Hz, 1 H, H-5′), 7.55–7.59 (m, 1 H, H-4′), 7.63 (d, J = 7.9 Hz, 1 H, H-6′), 7.66 (dt, J = 7.6, 1.4 Hz, 1 H, H-2′), 7.69 (dt, J = 7.9, 1.2 Hz, 1 H, H-6), 7.72 (m, 1 H, H-5), 7.81 (d, J = 7.9 Hz, 1 H, Ura-H-6), 7.84 (t, J = 1.7 Hz, 1 H, H-2), 7.87 (ddd, J = 8.0, 2.0, 1.0 Hz, 1 H, H-4), 11.32 (s, 1 H, NH).

¹³C NMR (150 MHz, DMSO-d ₆): δ = 54.3, 105.8, 126.1, 132.8, 133.3, 133.4, 135.0, 136.1, 136.4, 139.5, 140.8, 141.8, 143.3, 149.7, 155.3, 167.8, 198.2.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₄BrN₂O₃: 385.0182; found: 385.0179; m/z [M + Na]⁺ calcd for C₁₈H₁₃BrN₂O₃Na: 407.0002; found: 406.9998.

1-{3-[3-(Trifluoromethyl)benzoyl]benzyl}pyrimidine-2,4(1H,3H)-dione (5d)

The product was synthesized in a similar manner to 5a giving 5d (1.70 g, 87%) as colorless crystals.

Mp 167–168.5 °C; R_f = 0.44 (EtOAc).

¹H NMR (400 MHz, DMSO-d ₆): δ = 4.97 (s, 2 H, CH₂), 5.60 (dd, J = 7.8, 2.0 Hz, 1 H, Ura-H-5), 7.54–7.62 (m, 1 H, H-5′), 7.67 (dd, J = 12.0, 7.8 Hz, 2 H, H-4′, H-6′), 7.74 (s, 1 H, H-2′), 7.77–7.86 (m, 2 H, H-5, Ura-H-6), 7.96–8.10 (m, 3 H, H-6, H-2, H-4), 11.35 (br s, 1 H, NH).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 50.4, 101.8, 126.1, 129.0, 129.5, 129.6, 130.2, 132.7, 133.9, 136.8, 138.0, 138.1, 145.9, 151.4, 164.0, 194.5.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₁₄F₃N₂O₃: 375.0951; found: 375.0946; m/z [M + Na]⁺ calcd for C₁₉H₁₃F₃N₂O₃Na: 397.0770; found: 397.0767.

3-{3-[(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl]benzoyl}benzonitrile (5e)

The product was synthesized in a similar manner to 5a giving 5e (0.65 g, 73%) as colorless crystals.

Mp 208.5–210 °C; R_f = 0.28 (EtOAc).

¹H NMR (400 MHz, DMSO-d ₆): δ = 4.98 (s, 2 H, CH₂), 5.61 (dd, J = 7.8, 2.0 Hz, 1 H, Ura-H-5), 7.54–7.61 (m, 1 H, H-5′), 7.64 (s, 1 H, H-2′), 7.68 (d, J = 7.6 Hz, 1 H, H-4′), 7.72–7.79 (m, 2 H, H-6′, H-5), 7.83 (d, J = 7.8 Hz, 1 H, Ura-H-6), 8.01 (dt, J = 7.9, 1.4 Hz, 1 H, H-6), 8.12 (d, J = 1.2 Hz, 1 H, H-2), 8.14 (m, 1 H, H-4), 11.35 (s, 1 H, NH).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 50.4, 101.8, 112.2, 118.4, 129.1, 129.5, 129.8, 130.2, 132.8, 133.3, 134.2, 136.2, 136.5, 138.0, 138.3, 145.9, 151.4, 164.0, 194.1.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₁₄N₃O₃: 332.1030; found: 332.1027; m/z [M + Na]⁺ calcd for C₁₉H₁₃N₃O₃Na: 354.0849; found: 354.0843.

1-[3-(3,5-Dichlorobenzoyl)benzyl]pyrimidine-2,4(1H,3H)-dione (5f)

The product was synthesized in a similar manner to 5a giving 5f (0.81 g, 77%) as colorless crystals.

Mp 155.5–157 °C; R_f = 0.51 (EtOAc).

¹H NMR (600 MHz, DMSO-d ₆): δ = 4.97 (s, 2 H, CH₂), 5.61 (dd, J = 7.9, 2.3 Hz, 1 H, Ura-H-5), 7.55–7.60 (m, 1 H, H-5′), 7.63–7.70 (m, 4 H, H-4′, H-6′, H-2, H-6), 7.74 (s, 1 H, H-2′), 7.81 (d, J = 7.9 Hz, 1 H, Ura-H-6), 7.90 (t, J = 1.9 Hz, 1 H, H-4), 11.31 (d, J = 1.3 Hz, 1 H, NH).

¹³C NMR (150 MHz, DMSO-d ₆): δ = 54.3, 105.7, 132.1, 133.0, 133.4, 133.6, 135.9, 136.8, 138.7, 140.2, 141.9, 144.4, 149.7, 155.3, 167.8, 197.0.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₃Cl₂N₂O₃: 375.0298; found: 375.0294; m/z [M + Na]⁺ calcd for C₁₈H₁₂Cl₂N₂O₃Na]: 397.0117; found: 397.0114.

1-[3-(3,5-Dichlorobenzoyl)benzyl]-5-methylpyrimidine-2,4(1H,3H)-dione (5g)

The product was synthesized in a similar manner to 5a giving 5g (1.24 g, 81%) as colorless crystals.

Mp 193.5–195.5 °C; R_f = 0.58 (EtOAc).

¹H NMR (400 MHz, CDCl₃): δ = 1.74 (s, 3 H, CH₃), 4.92 (s, 2 H, CH₂), 7.57 (d, J = 7.6 Hz, 1 H, H-5′), 7.60–7.68 (m, 5 H, H-4′, H-6′, H-2, H-6, Ura-H-6), 7.70 (s, 1 H, H-2′), 7.87 (t, J = 1.8 Hz, 1 H, H-4), 11.34 (s, 1 H, NH).

¹³C NMR (100 MHz, CDCl₃): δ = 12.3, 50.2, 109.6, 128.2, 129.0, 129.5, 129.6, 132.0, 133.0, 134.8, 136.3, 138.1, 140.4, 141.5, 151.4, 164.6, 193.1.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₁₅Cl₂N₂O₃: 389.0454; found: 389.0449; m/z [M + Na]⁺ calcd for C₁₉H₁₄Cl₂N₂O₃Na: 411.0274; found: 411.0270.

1-[3-(3,5-Dichlorobenzoyl)benzyl]-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic Acid (5h)

The product was synthesized in a similar manner to 5a giving 5h (0.27 g, 36%) as colorless crystals.

Mp 217–219 °C; R_f = 0.57 (i-PrOH–EtOAc–NH₄OH_aq, 9:6:5).

¹H NMR (400 MHz, DMSO-d ₆): δ = 5.14 (s, 2 H, CH₂), 7.58 (t, J = 7.8 Hz, 1 H, H-5′), 7.66 (d, J = 2.0 Hz, 2 H, H-4′, H-6′), 7.71 (s, 2 H, H-2, H-6), 7.81 (s, 1 H, H-2′), 7.92 (t, J = 1.7 Hz, 1 H, H-4), 8.26 (s, 1 H, Ura-H-6), 8.89 (s, 1 H, NH).

¹³C NMR (100 MHz, D₂O): δ = 51.6, 102.7, 128.3, 129.5, 129.6, 129.9, 132.1, 133.3, 134.8, 136.3, 137.0, 140.5, 150.3, 153.5, 163.7, 164.5, 193.1.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₁₃Cl₂N₂O₅: 419.0196; found: 419.0191; m/z [M + Na]⁺ calcd for C₁₉H₁₂Cl₂N₂O₅Na: 441.0015; found: 441.0013.

4-Amino-1-[3-(3,5-dichlorobenzoyl)benzyl]pyrimidin-2(1H)-one (5i)

The product was synthesized in a similar manner to 5a giving 5i (0.47 g, 59%) as colorless crystals.

Mp 281.5–283 °C; R_f = 0.50 (EtOAc).

¹H NMR (400 MHz, DMSO-d ₆): δ = 4.95 (s, 2 H, CH₂), 5.72 (d, J = 7.1 Hz, 1 H, Cyt-H-5), 6.85 (br s, 2 H, NH₂), 7.51–7.58 (m, 1 H, H-5′), 7.62 (s, 1 H, H-4′), 7.64 (d, J = 1.7 Hz, 3 H, H-6′, H-2, H-6), 7.67–7.74 (m, 2 H, Cyt-H-6, H-2′), 7.85 (t, J = 2.0 Hz, 1 H, H-4).

¹³C NMR (100 MHz, D₂O): δ = 51.5, 94.2, 128.1, 129.0, 129.2, 131.9, 133.0, 134.9, 139.1, 146.1, 156.1, 166.4, 193.1.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₄Cl₂N₃O₂: 374.0458; found: 374.0453; m/z [M + Na]⁺ calcd for C₁₈H₁₃Cl₂N₃O₂Na: 397.0277; found: 397.0273.

5-Bromo-1-[3-(3,5-dichlorobenzoyl)benzyl]pyrimidine-2,4(1H,3H)-dione (5j)

Br₂ (0.20 mL, 3.88 mmol) was added to a stirred solution of 5f (1.30 g, 3.46 mmol) and sodium acetate trihydrate (0.97 g, 7.13 mmol) in glacial AcOH (10 mL) at 60 °C. Stirring was continued for 15 h at r.t., after which the mixture was evaporated and treated with H₂O. The resulting precipitate was filtered, dried and recrystallized from a mixture of DCE–hexane (3:2) to give 5j (1.44 g, 92%) as colorless crystals.

Mp 186–187.5 °C; R_f = 0.77 (EtOAc).

¹H NMR (600 MHz, DMSO-d ₆): δ = 4.96 (s, 2 H, CH₂), 7.55–7.59 (m, 1 H, H-5), 7.64 (d, J = 1.9 Hz, 2 H, H-4′, H-6′), 7.66–7.70 (m, 2 H, H-2, H-6), 7.73 (s, 1 H, Ura-H-6), 7.86 (t, J = 1.9 Hz, 1 H, H-2′), 8.33 (s, 1 H, H-6), 11.72 (br s, 1 H, NH).

¹³C NMR (150 MHz, DMSO-d ₆): δ = 49.2, 54.8, 99.5, 132.0, 133.1, 133.4, 133.5, 135.9, 136.9, 138.7, 140.4, 141.4, 144.4, 149.2, 154.6, 197.1.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₂BrCl₂N₂O₃: 452.9403; found: 452.9401; m/z [M + Na]⁺ calcd for C₁₈H₁₁BrCl₂N₂O₃Na: 474.9222; found: 474.9222.

1-[3-(3,5-Dichlorobenzoyl)benzyl]-3-methylpyrimidine-2,4(1H,3H)-dione (5k)

A mixture of 5f (0.25 g, 0.67 mmol), Me₂SO₄ (0.25 mL, 2.64 mmol) and K₂CO₃ (0.46 g, 3.33 mmol) in acetone (10 mL) was stirred at r.t. for 8 h. Aq NaOH (50 mL) was added and stirring was continued for 30 min. The acetone was removed under vacuum, and the precipitated product was filtered and dried to give 5k (0.20 g, 77%) as white crystals.

Mp 200–201 °C; R_f = 0.16 (hexane–EtOAc, 1:1).

¹H NMR (400 MHz, DMSO-d ₆): δ = 3.32 (s, 3 H, CH₃), 5.02 (s, 2 H, CH₂), 5.75 (d, J = 7.6 Hz, 1 H, Ura-H-5), 7.53–7.62 (m, 1 H, H-5′), 7.63–7.73 (m, 4 H, H-4′, H-6′, H-2, H-6), 7.75 (s, 1 H, H-2′), 7.90 (d, J = 7.8 Hz, 1 H, Ura-H-6), 7.95 (s, 1 H, H-4).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 27.7, 51.6, 100.9, 128.3, 129.4, 129.5, 129.7, 132.1, 133.1, 134.8, 136.3, 137.7, 140.5, 144.1, 151.7.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₁₅Cl₂N₂O₃: 389.0454; found: 389.0448; m/z [M + Na]⁺ calcd for C₁₉H₁₄Cl₂N₂O₃Na: 411.0274; found: 411.0272.

6-Chloro-1-[3-(3,5-dichlorobenzoyl)benzyl]pyrimidine-2,4(1H,3H)-dione (5l)

A mixture of 6-chlorouracil (0.17 g, 1.16 mmol), 4f (0.60 g, 1.74 mmol) and K₂CO₃ (0.08 g, 0.58 mmol) in anhydrous DMSO (10 mL) was stirred for 1 h at 60 °С. The mixture was poured into a sat. aq solution of Na₂SO₄ and extracted with CHCl₃ (3 × 10 mL). The organic portions were combined, washed with H₂O (2 × 50 mL), dried over MgSO₄ and evaporated. The residue was purified by flash chromatography on silica eluting with EtOAc. The residue was recrystallized from a mixture of DCE–hexane (1:1) to give 5l (0.26 g, 55%) as colorless crystals.

Mp 173–175 °C; R_f = 0.47 (EtOAc).

¹H NMR (400 MHz, CD₃CN): δ = 5.24 (s, 2 H, CH₂), 6.01 (d, J = 2.0 Hz, 1 H, Ura-H-5), 7.55–7.61 (m, 1 H, H-5′), 7.62–7.68 (m, 4 H, H-4′, H-6′, H-2, H-6), 7.69 (s, 1 H, H-2′), 7.93 (t, J = 2.0 Hz, 1 H, H-4), 11.76 (d, J = 1.7 Hz, 1 H, NH).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 48.2, 102.9, 128.3, 129.5, 129.6, 132.1, 134.7, 136.3, 137.4, 140.4, 146.9, 150.9, 161.3, 193.0.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₂Cl₃N₂O₃: 408.9908; found: 408.9903; m/z [M + Na]⁺ calcd for C₁₈H₁₁Cl₃N₂O₃Na: 430.9727; found: 430.9725.

3-[3-(3,5-Dichlorobenzoyl)benzyl]pyrimidine-2,4(1H,3H)-dione (5m)

A mixture of 1-[(benzyloxy)methyl]uracil (0.41 g, 1.77 mmol) and K₂CO₃ (0.37 g, 2.68 mmol) in anhydrous DMF (10 mL) was stirred at 80 °C for 40 min and then allowed to cool to r.t. After the addition of 4f (0.60 g, 1.74 mmol), stirring was continued for 20 h. The mixture was poured into a sat. aq solution of Na₂SO₄ and extracted with CHCl₃ (3 × 10 mL). The organic layers were combined, washed with brine (100 mL) and H₂O (50 mL), dried over MgSO₄ and evaporated. The residue was purified by recrystallization from a mixture of EtOAc–hexane (2:3). The product (0.60 g, 1.21 mmol) was heated at reflux temperature in a mixture of HCl–EtOH (1:5) for 2 h, and then poured into sat. aq Na₂SO₄ and extracted with CHCl₃ (3 × 10 mL). The organic layers were combined, washed with brine (100 mL) and H₂O (50 mL), dried over MgSO₄ and evaporated. The residue was purified by flash chromatography on silica eluting with EtOAc and then recrystallized from a mixture of DCE–hexane (3:2) to give 5m (0.35 g, 77%) as colorless crystals.

Mp 195.5–197.5 °C; R_f = 0.47 (EtOAc).

¹H NMR (400 MHz, DMSO-d ₆): δ = 5.02 (s, 2 H, CH₂), 5.65 (dd, J = 7.5, 1.1 Hz, 1 H, Ura-H-5), 7.47 (dd, J = 7.6, 5.9 Hz, 1 H, Ura-H-6), 7.50–7.55 (m, 1 H, H-5′), 7.59–7.66 (m, 4 H, H-4′, H-6′, H-2, H-6), 7.68 (s, 1 H, H-2′), 7.90 (t, J = 2.0 Hz, 1 H, H-4), 11.24 (d, J = 5.4 Hz, 1 H, NH).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 42.7, 100.1, 128.2, 129.1, 129.2, 129.3, 132.0, 133.2, 134.7, 136.0, 138.4, 140.5, 141.4, 151.8, 163.4, 193.1.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₃Cl₂N₂O₃: 375.0298; found: 375.0294; m/z [M + Na]⁺ calcd for C₁₈H₁₂Cl₂N₂O₃Na: 397.0117; found: 397.0113.

3-[3-(3-Bromobenzoyl)benzyl]imidazolidine-2,4-dione (5n)

A 5.4 M solution of NaOMe in MeOH (0.23 mL, 1.24 mmol) was added to a stirred suspension of hydantoin (0.12 g, 1.20 mmol) in anhydrous i-PrOH (10 mL) under an Ar atm at 60 °C, followed by addition of 4c (0.47 g, 1.33 mmol) over a period of 45 min. Stirring was continued for 15 h. The mixture was then evaporated, treated with H₂O and extracted with CHCl₃ (3 × 10 mL). The combined organic layer was washed with H₂O (2 × 50 mL) and evaporated under vacuum. The residue was purified by flash chromatography on silica eluting with EtOAc and then recrystallized from a mixture of EtOAc–hexane (1:1) to give 5n (0.32 g, 74%) as colorless crystals.

Mp 141.5–143 °C; R_f = 0.50 (EtOAc).

¹H NMR (400 MHz, DMSO-d ₆): δ = 3.99 (s, 2 H, Hyd-CH₂), 4.62 (s, 2 H, CH₂), 7.53 (q, J = 7.8 Hz, 2 H, H-5′, H-4′), 7.57–7.66 (m, 2 H, H-6′, H-2′), 7.66–7.72 (m, 2 H, H-5, H-6), 7.82–7.91 (m, 2 H, H-2, H-4), 8.18 (s, 1 H, NH).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 46.4, 122.2, 129.00, 129.03, 129.2, 129.3, 131.1, 132.2, 132.6, 135.7, 136.8, 137.8, 139.3, 157.6, 172.3, 194.4.

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₇H₁₄BrN₂O₃: 373.0182; found: 373.0177; m/z [M + Na]⁺ calcd for C₁₇H₁₃BrN₂O₃Na: 395.0002; found: 394.9996.

Molecular Modeling

All protein structures followed similar preparative procedures with minimal user intervention. Cognate ligands, co-crystalized ions and solvent molecules were deleted. All hydrogen atoms were added. After computing Gasteiger charges, non-polar hydrogen atoms were merged and all atoms were AutoDock-typed to prepare the PDBQT input file. Ligand conformers were created with Marvin Sketch (ChemAxon).[26] Docking simulations were performed with AutoDock Vina 1.1.2.[27] The cubic grid box centered on the cognate ligand was adjusted for each complex to include the entire concave region around the ligand and the solvent accessible entrance of the pocket. Preliminary studies showed the reproduction of experimentally observed binding modes in employed HIV-1 RT/NNRTI complexes with an average RMSD value of 0.67 Å. Only top-score binding poses were used in subsequent analysis.

HIV-1 RT RDDP Assays

Heterodimeric HIV-1 RT was expressed in E. coli and purified as previously reported.[28] HIV-1 RT associated RDDP activity was measured as previously reported[29] using the Invitrogen EnzCheck Reverse Transcriptase Assay Kit. Briefly, in 50 μL volume containing Tris-HCl (60 mM, pH 8.1), MgCl₂ (8 mM), KCl (60 mM), dithiothreitol (13 mM), dTTP (100 μM), HIV-1 RT (2 nM) and poly(A)-oligo(dT). The reaction mixture was incubated for 30 min at 37 °C. The enzymatic reaction was stopped by the addition to EDTA and measured with a Victor3 multilabel plate reader (Perkin) at 502/523 nm after the addition to picogreen.

Acknowledgment

This work was supported by a grant from the Russian Foundation for Basic Research (13-04-91440).

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• Supplementary Material