You are currently viewing a new version of our website. To view the old version click .
MDPIMDPI
Search all articles
Reactions
Download PDF
  • Communication
  • Open Access

6 February 2022

N-4 Alkyl Cytosine Derivatives Synthesis: A New Approach

,
,
,
,
,
,
and
1
Department of Sciences, University of Basilicata, Via dell’Ateneo Lucano 10, I-85100 Potenza, Italy
2
Glycosciences Laboratory, Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK
3
Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, I-80131 Napoli, Italy
4
Department of Pharmacy, University of Salerno, Via G. Paolo II 132, Fisciano, I-84084 Salerno, Italy
Reactions2022, 3(1), 192-202;https://doi.org/10.3390/reactions3010014
This article belongs to the Special Issue Feature Papers in Reactions in 2021
Version Notes
Order Reprints

Abstract

The selective N-4 alkylation of cytosine plays a critical role in the synthesis of biologically active molecules. This work focuses on the development of practical reaction conditions toward a regioselective synthesis of N-4-alkyl cytosine derivatives. The sequence includes a direct and selective sulfonylation at the N-1 site of the cytosine, followed by the alkylation of the amino site using KHMDS in CH2Cl2/THF mixture, providing a fast and efficient approach consistent with pyrimidine-based drug design.

1. Introduction

Integrase (IN) catalyzes the insertion of viral DNA [1] into the genome of infected cells and acts as a cofactor for reverse transcription [2].
In the context of HIV-1 infection, IN was successfully targeted for drug development [3]. Raltegravir (MK-0518) [4,5] was approved by the Food and Drug Administration in 2007, and other integrase inhibitors (INI), including Elvitegravir (GS-9137) [6,7], are progressing through clinical development [8]. The breakthrough of INI has produced a great impulse in the use of multiple drugs that act on different viral targets, known as Highly Active Antiretroviral Therapy (HAART) [9]. Important examples of this class are the lens epithelium-derived growth factor (LEDGF) inhibitors [10,11,12] (Figure 1).
Figure 1. Small molecule inhibitors of the LEDGF/p75-IN interaction.
Unfortunately, the development of resistance is a constant and inevitable threat to the application of therapies; there is always a need for new antiviral drugs with high activity and low cytotoxicity to assist and sometimes also substitute previously utilized drugs.
Molecules acting on the IN HIV-1 are not immune to this problem [13]. This has prompted the research of more efficient and inexpensive new drugs. In this context is the design and synthesis of new cytosine-based antiretroviral (ARV) compounds, which are able to inhibit IN HIV-1.
Current studies of structure–activity relationships (SAR) on the above mentioned INI structures have identified two common regions [14]: a region with two metal-binding motifs critical to all members of this class of active site binders and a region with a hydrophobic site that requires a substituted benzyl group [15,16].
Taking into consideration these findings, we exploited the commercially available cytosine scaffold to synthetize new integrase strand transfer inhibitors (INSTIs) [1,3,4,17,18]. In detail, starting from a preliminary docking analysis [19], which clarified that chelation motif N-(aryl/alkyl sulfonyl) amide could selectively fill the binding site, we set out to investigate an original and efficient strategy for the synthesis of type 1 nucleobases (Figure 2).
Figure 2. Nucleobase structure.
Cytosine derivatives are versatile intermediates in the synthesis of biologically and pharmaceutically active molecules [20,21,22,23,24,25,26,27] and are widely used as antineoplastic [6], antiviral [24], and anti-AIDS agents [27]. Some groups have very recently focused their attention on N-4 alkyl analogue, which improves uptake and bioavailability of gemcitabine, a worldwide chemotherapeutic cytidine analogue [20].
The reaction to obtain N-1 substituted cytosines has been intensively investigated [28,29,30,31,32,33]; nevertheless, to date, only a few examples describe N-4 alkyl derivatives [34,35,36,37,38,39]. One of the most useful examples involves a sodium bisulfate catalyzed transamination experiencing careful control of the pH, which is sometimes incompatible with the chemical stability of biological groups [39].
Likewise, the Borch reductive alkylation method [36,37] and the titanium (IV), which also catalyzed [35], required an excess of amine to favor the formation of the iminium intermediates, thereby hampering the dissolution in the solvent that was usually used.
The methodology described herein shows the regioselective formation of our new compounds under conditions consistent with the stability of future drug moieties.

2. Materials and Methods

All reagents (Aldrich, St. Louis, MA, USA and Merck, KGaA, Darmstadt, Germany) were acquired at the highest purity available and used without further purification. Thin-layer chromatographies were performed with silica gel plates Merck 60 F254, and the display of the products on TLC was performed with a lighting UV lamp, solutions of ninhydrin (0.2% in CH3OH mol), and molecular iodine. The column chromatographies were carried out using silica gel 70–230 mesh (Merck, KGaA, Darmstadt, Germany). Elemental analyses were performed on a FlashSmart V Elemental Analyzer (ThermoFisher Scientific, Waltham, MA, USA). The 1H and 13C NMR spectra were recorded on spectrometers: Bruker DRX (400 MHz) and Varian Inova Marker (500 MHz) in CDCl3 solution unless otherwise specified. The chemical shifts are reported in ppm (δ) and the J in Hz.

2.1. Synthesis of 4-amino-1-((4-chlorophenyl)sulfonyl) pyrimidin-2(1H)-one (2)

Sodium hydride (118 mg; 4.9 mmol) at 0 °C under nitrogen atmosphere was added to a stirring solution of cytosine (500 mg, 4.5 mmol) in dry DMF (38 mL). After 2 h, 4-chlorobenzenesulfonyl chloride (1.4 g, 6.8 mmol) was added and stirring was continued over a period of 30 min. The resulting solution was then allowed to warm to room temperature. After 1.5 h, the reaction was quenched with methanol (0.60 mL). The solvent was evaporated under reduced pressure, replaced with chloroform and washed with brine, and then dried (Na2SO4). The evaporation of the solvent under reduced pressure gave a crude mixture that was purified by column chromatography (CHCl3/MeOH 95:5) to yield compound 2 (0.96 g, 75%). 1H NMR (400 MHz; DMSO-d6): δ 8.08 (d, J 7.8 Hz, 1H), 7.97 (d, J 8.5 Hz, 2H), 7.93 (s, 2H), 7.71 (d, J 8.5 Hz, 2H), 5.95 (d, J 7.8 Hz, 1H); 13C NMR (125 MHz; DMSO-d6); δ 166.5, 151.3, 140.0, 139.7, 136.5, 131.1, 129.7, 98.1. Anal. Calcd. for C10H8ClN3O3S (285.70): C, 42.04%; H, 2.82%; N, 14.71%; found C, 42.15%; H, 2.73%; N, 14.59%.

2.2. General Procedure Synthesis of N-4 Alkyl Cytosine Derivatives

Derivative 2 (0.5 eq) was dissolved in dry CH2Cl2:THF (1:1, 5 mL), followed by the addition of 0.5 M KHMDS in THF (0.75 eq) at −40 °C under nitrogen atmosphere. After 1 h, electrophile (0.6 eq) was added and the reaction was allowed to warm to 5 °C within 24 h. TLC monitored the progress of the reaction. The mixture was then treated with methanol (0.5 mL) and further stirred for 10 min at rt. The solvent was evaporated under reduced pressure, replaced with ethyl acetate and washed with brine, and then dried (Na2SO4). The evaporation of the solvent under reduced pressure gave a crude mixture that was purified by PLC (1:1 Hexane/Ethyl Acetate) to yield the pairs 1a–3a, 1b–3b, 1e–3e, and 1h–3h.
1a. 1H NMR (400 MHz): δ 8.05 (m, 3H), 7.51 (d, J 7.2 Hz, 2H), 7.36-7.22 (m, 5H), 5.69 (d, J 7.5 Hz, 1H), 5.47 (bs, NH), 4.64 (s, 2H); 13C NMR (100 MHz, (CD3)2CO): δ 166.0, 156.9, 147.6, 139.4, 139.3, 129.9, 129.5, 128.9, 128.6, 92.6, 53.1. Anal. Calcd. for C17H14ClN3O3S (375.83): C, 54.33%; H, 3.75%; N, 11.18%; found C, 54.35%; H, 3.83%; N, 11.03%.
3a. 1H NMR (500 MHz): δ 8.07 (d, J = 8.6 Hz, 2H), 8.05 (d, J 8.1 Hz, 1H), 7.51 (d, J 8.5 Hz, 2H), 7.36–7.26 (m, 8H), 7.09 (d, J 7.3 Hz, 2H), 5.93 (d, J 8.1 Hz, 1H), 4.96 (s, 2H), 4.54 (s, 2H); 13C NMR (125 MHz): δ 164.1, 151.3, 141.5, 140.0, 136.0, 135.1, 135.0, 131.4, 129.3, 129.2, 128.8, 128.7, 128.1, 128.0, 126.2, 94.5, 50.9, 50.8. Anal. Calcd. for C24H20ClN3O3S (465.95): C, 61.87%; H, 4.33%; N, 9.02%; found C, 61.90%; H, 4.28%; N, 9.01%.
1b. 1H NMR (400 MHz): δ 8.07–8.01 (m, 3H), 7.51 (d, J 8.6 Hz, 2H), 7.30–7.20 (m, 2H), 7.00 (t, J 8.6 Hz, 2H), 5.70 (d, J 7.9 Hz, 1H), 4.60 (d, J 5.5 Hz, 2H). 13C NMR (100 MHz) δ 164.4, 160.3, 156.8, 141.4, 139.8, 135.7, 134.8, 131.3, 129.3, 127.9, 115.8, 94.3, 50.8. Anal. Calcd. for C17H13ClFN3O3S (393.82): C, 51.85%; H, 3.33%; N, 10.67%; found C, 51.96%; H, 3.35%; N, 10.76%.
3b. 1H NMR (500 MHz): δ 8.16–8.05 (m, 3H), 7.55 (d, J 8.8 Hz, 2H), 7.28 (m, 2H), 7.07–6.97 (m, 6H), 5.94 (d, J 8.2 Hz, 1H), 4.90 (s, 2H), 4.52 (s, 2H). 13C NMR (100 MHz): 164.0, 163.7, 161.2, 151.0, 141.4, 140.1, 135.0, 131.6, 131.3, 130.4, 130.3, 129.3, 129.0, 127.8, 115.7, 115.5, 94.0. 50.2, 49.9. Anal. Calcd. for C24H18ClF2N3O3S (501.93): C, 57.43%; H, 3.61%; N, 8.37%; found C, 57.37%; H, 3.67%; N, 8.35%.
1c. 1H NMR (400 MHz, (CD3)2CO): δ 8.13–8.08 (m, 3H), 8.00 (bs, 1H, NH), 7.68 (d, J 8.6 Hz, 2H), 7.35 (m, 1H), 7.17 (d, J 7.6 Hz, 1H), 7.12 (m, 1H), 7.00 (m, 1H), 6.13 (d, J 7.9 Hz, 1H), 4.62 (d, J 4.9 Hz, 2H). 13C NMR (100 MHz, (CD3)2CO): δ 169.9, 164.0, 140.9, 140.1, 138.6, 136.4, 131.0, 130.1, 128.9, 123.5, 114.4, 113.7, 97.6, 43.4. Anal. Calcd. for C17H13ClFN3O3S (393.82): C, 51.85%; H, 3.33%; N, 10.67%; found C, 51.87%; H, 3.25%; N, 10.63%.
3c. 1H NMR (400 MHz): δ 8.14–8.04 (m, 3H), 7.53 (d, J 8.4 Hz, 2H), 7.39–7.23 (m, 3H), 7.08–6.75 (m, 5H), 5.91 (d, J 8.1 Hz, 1H), 4.94 (s, 2H), 4.53 (s, 2H). 13C NMR (100 MHz): 167.8, 164.7, 159.8, 153.6, 145.2, 145.0, 144.4, 133.7, 131.9, 131.3, 128.2, 127.9, 116.7, 116.6, 112.5, 93.2, 58.6, 57.2. Anal. Calcd. for C24H18ClF2N3O3S (501.93): C, 57.43%; H, 3.61%; N, 8.37%; found C, 57.44%; H, 3.59%; N, 8.39%.
1d. 1H NMR (400MHz, DMSO-d6): δ 8.16–8.07 (m, 3H), 7.67 (d, J 10.0 Hz, 2H), 7.41 (m, 1H), 7.31 (m, 1H), 7.14–7.06 (m, 2H), 6.12 (d, J 10.0 Hz, 1H), 4.64 (d, J 6.5 Hz, 2H). 13C NMR (100MHz, (CD3)2CO): δ 163.9, 161.9, 154.7, 140.1, 138.5, 136.3, 131.0, 130.3, 129.4, 129.3, 128.9, 124.2, 115.1, 97.5, 37.9. Anal. Calcd. for C17H13ClFN3O3S (393.82): C, 51.85%; H, 3.33%; N, 10.67%; found C, 51.80%; H, 3.31%; N, 10.59%.
3d. 1H NMR (400 MHz): δ 8.06 (d, J 8.5 Hz, 2H), 7.55–7.43 (m, 3H), 7.34–7.20 (m, 2H), 7.14–6.95 (m, 6H), 5.96 (d, J 8.2 Hz, 1H), 4.99 (s, 2H), 4.66 (s, 2H). 13C NMR (100 MHz): 164.1, 163.3, 160.9, 151.3, 141.5, 140.0, 136.0, 135.1, 135.0, 131.4, 129.2, 129.1, 128.8, 128.6, 128.1, 128.0, 126.1, 115.2, 115.1, 94.5, 50.9, 50.7. Anal. Calcd. for C24H18ClF2N3O3S (501.93): C, 57.43%; H, 3.61%; N, 8.37%; found C, 57.45%; H, 3.63%; N, 8.38%.
1e. 1H NMR (400MHz, (CD3)2CO): δ 8.10–8.05 (m, 3H), 7.68 (d, J 8.8 Hz, 2H), 7.20 (d, J 8.0 Hz, 2H), 7.10 (d, J 8.0 Hz, 2H), 6.07 (d, J 7.9 Hz, 1H), 4.52 (d, J 5.7 Hz, 2H), 2.27 (s, 3H). 13C NMR (100 MHz, (CD3)2CO): δ 165.3, 152.3, 141.4, 139.9, 138.8, 135.7, 134.1, 132.0, 131.7, 130.9, 100.7, 46.9, 25.5. Anal. Calcd. for C18H16ClN3O3S (389.85): C, 55.46%; H, 4.14%; N, 10.78%; found C, 55.50%; H, 4.13%; N, 10.65%.
3e. 1H NMR (400 MHz; (CD3)2CO): δ 8.16 (d, J 8.2 Hz, 1H), 8.12 (d, J 8.8 Hz, 2H), 7.83 (d, J 8.7 Hz, 1H), 7.68 (d, J 8.8 Hz, 2H), 7.59 (d, J 8.7 Hz, 1H), 7.24-7.06 (m, 6H), 6.31 (d, J 8.2 Hz, 1H), 4.87 (s, 2H), 4.67 (s, 2H), 2.31 (s, 3H), 2.28 (s, 3H). 13C NMR (100 MHz; (CD3)2CO): δ 164.6, 154.3, 140.1, 139.2, 138.8, 138.4, 131.4, 131.2, 131.1, 129.1, 129.0, 128.7, 128.6, 127.9, 127.8, 97.8, 44.3, 43.9, 32.4, 31.7. Anal. Calcd. for C26H24ClN3O3S (494.01): C, 63.22%; H, 4.90%; N, 8.51%; found C, 63.26%; H, 4.87%; N, 8.46%.
1f. 1H NMR (400MHz): δ 8.05 (d, J 8.8 Hz, 2H), 8.02 (d, J 7.9 Hz, 1H), 7.51 (d, J 8.8 Hz, 2H), 7.23 (m, 1H), 7.14-7.03 (m, 3H), 5.72 (d, J 7.9 Hz, 1H), 5.66 (m, 1H, NH), 4.60 (d, J 5.4 Hz, 2H), 2.33 (s, 3H). 13C NMR (100 MHz): δ 163.1, 151.7, 142.7, 140.7, 138.6, 136.5, 135.1, 134.8, 131.1, 129.0, 128.7, 125.2, 123.7, 97.3, 45.3, 21.2. Anal. Calcd. for C18H16ClN3O3S (389.85): C, 55.46%; H, 4.14%; N, 10.78%; found C, 55.53%; H, 4.17%; N, 10.70%.
3f. 1H NMR (400 MHz): δ 8.10 (d, J 8.4 Hz, 2H), 8.05 (d, J 8.02 Hz, 1H), 7.53 (d, J 8.4 Hz, 2H), 7.25–7.17 (m, 2H), 7.15–7.05 (m, 4H), 6.92–6.86 (m, 2H), 5.94 (d, J 8.2 Hz, 1H), 4.94 (s, 2H), 4.50 (s, 2H), 2.4 (s, 3H), 2.3 (s, 3H). 13C NMR (100 MHz): 163.1, 151.9, 141.4, 141.2, 140.7, 138.6, 136.5, 135.1, 134.8, 131.1, 129.0, 128.7, 125.2, 123.7, 97.3, 46.1, 45.3, 30.8, 29.6. Anal. Calcd. for C26H24ClN3O3S (494.01): C, 63.22%; H, 4.90%; N, 8.51%; found C, 63.26%; H, 4.87%; N, 8.46%.
1g. 1H NMR (400 MHz, DMSO-d6): δ 8.12–8.06 (m, 3H), 7.76 (m, 1H), 7.69 (d, J 8.7 Hz, 2H), 7.27 (d, J 6.9 Hz, 1H), 7.19-7.12 (m, 3H), 6.13 (d, J 7.9 Hz, 1H), 4.58 (d, J 5.3 Hz, 2H), 2.31 (s, 3H). 13C NMR (125 MHz, (CD3)2CO): δ 164.9, 151.9, 141.0, 139.3, 137.5, 137.3, 136.4, 132.0, 131.1, 129.9, 128.5, 126.9, 98.6, 79.2, 43.3, 23.3. Anal. Calcd. for C18H16ClN3O3S (389.85): C, 55.46%; H, 4.14%; N, 10.78%; found C, 55.44%; H, 4.13%; N, 10.79%.
3g. 1H NMR (400 MHz): δ 8.08 (d, J = 8.7 Hz, 2H), 8.04 (d, J 8.2 Hz, 1H), 7.51 (d, J 8.7 Hz, 2H), 7.25–7.10 (m, 6H), 7.00 (d, J 7.9 Hz, 1H), 6.94 (d, J 6.8 Hz, 1H), 5.84 (d, J 8.2 Hz, 1H), 4.98 (s, 2H), 4.44 (s, 2H), 2.18 (s, 3H), 2.16 (s, 3H). 13C NMR (100 MHz): 162.5, 152.7, 141.4, 139.7, 136.5, 135.0, 134.9, 133.4, 132.3, 131.3, 130.7, 130.5, 129.0, 128.4, 127.7, 127.5, 126.6, 126.1, 124.5, 94.2, 52.4, 48.5, 23.2, 22.7. Anal. Calcd. for C26H24ClN3O3S (494.01): C, 63.22%; H, 4.90%; N, 8.51%; found C, 63.27%; H, 4.93%; N, 8.50%.
1h. 1H NMR (400MHz, DMSO-d6): δ 8.18 (d, J 8.7 Hz, 2H), 8.13 (d, J 7.9 Hz, 1H), 8.09 (d, J 8.6 Hz, 2H), 7.68 (d, J 8.6 Hz, 2H), 7.61 (d, J 8.7 Hz, 2H), 6.16 (d, J 7.9 Hz, 1H), 4.77 (d, J 6.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): 164.2, 150.9, 147.1, 145.9, 140.2, 138.7, 136.3, 131.1, 128.9, 128.5, 123.3, 97.5, 43.3. Anal. Calcd. for C17H13ClN4O5S (420.82): C, 48.52%; H, 3.11%; N, 13.31%; found C, 48.53%; H, 3.16%; N, 13.25%.
3h. (4%). 1H NMR (400 MHz, DMSO-d6): δ 8.25–8.09 (m, 7H), 7.70 (d, J 8.7 Hz, 2H), 7.62 (d, J 8.6 Hz, 2H), 7.56 (d, J 8.6 Hz, 2H), 6.38 (d, J 8.2 Hz, 1H), 5.14 (s, 2H), 5.08 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ 163.6, 156.6, 156.0, 149.7, 147.9, 147.8, 139.9, 139.1, 138.1, 129.8, 129.7, 128.5, 128.4, 126.8, 126.7, 95.2, 58.2, 57.4. Anal. Calcd. for C26H18ClN5O7S (555.95): C, 51.85%; H, 3.26%; N, 12.60%; found C, 51.90%; H, 3.21%; N, 12.59%.
1i. 1H NMR (400 MHz): δ 8.00 (d, J 8.7 Hz, 2H), 7.74 (d, J 8.6 Hz, 1H), 7.57 (d, J 8.7 Hz, 2H), 6.52 (d, J 8.6 Hz, 1H), 2.98 (bs, 3H). 13C NMR (100 MHz, DMSO-d6): 163.3, 156.1, 140.0, 138.3, 137.0, 130.0, 128.9, 94.7, 28.1. Anal. Calcd. for C11H10ClN3O3S (299.73): C, 44.08%; H, 3.36%; N, 14.02%; found C, 44.12%; H, 3.33%; N, 14.07%.

3. Results and Discussion

We started with the synthesis of the suitable precursor of our target, namely the cytosine sulfonylate derivate 2, obtained by exploiting the well-known good reactivity of the N-1 site [31,32,40,41,42]. Indeed, as shown in Scheme 1, the commercially available cytosine was selectively sulfonylated with 4-chlorobenzene-1-sulfonyl chloride in DMF, the solvent required to overcome the known low solubility of the starting material. It is noteworthy that a temperature of 0 °C was mandatory to avoid a competitive reaction in favor of the exocyclic amine group. Under these conditions, compound 2 was obtained with 78% yield, as confirmed by NMR.
Scheme 1. N-4 alkyl model reaction.
Then, in our exploratory studies, we experimented with the representative N-4 alkylation (Scheme 1) with benzyl bromide as an electrophile under different conditions in terms of base, time, solvent, and temperature.
Firstly, the mixture of 2 and benzyl bromide was dissolved in DMSO and left at 25 °C for 4 h, then it was allowed to reach 80 °C and was kept under these conditions for a further 20 h, but no reaction took place and the starting materials remained completely unconsumed (Table 1, entry 1). Next, the exploitation of non-nucleophilic bases was investigated. In detail, pyridine (Pyr) and triethylamine (TEA) were found to be ineffective (Table 1, entries 2–4) and only a trace of the desired product was achieved when bicyclic amide (1,8-diazabiciclo(5.4.0)undec-7ene, (DBU)) [43,44]—which is able to form a charge transfer complex—was employed. The low nucleophilicity of the nitrogen atom, as well as the steric hindrance on the same nitrogen, resulting in a stalled reaction, could be clarified by the supposed complex reported in Figure 3.
Table 1. Reaction condition screening.
Figure 3. DBU Complex.
When the concept of strongest base (lithium diisopropylamide (LDA), hexamethyldisilazane lithium (LiHMDS), and hexamethyldisilazane potassium (KHMDS)) was explored, positive results were produced.
Remarkably, the N-1 substituted cytosines that participated as an acidic compound (with pKa lower than that of KHMDS) reacted with the base. Thus, the formed anion of the substrate could act as a nucleophile in reaction with benzyl halides. In fact, as reported in Table 1, when using KHMDS in DMF at −40 °C (entry 10) the reaction was completed within 4 h and workup afforded the expected monobenzylated product 1a as the major compound with 40% yield, together with a minor side-product 3a (with 30% yield, Scheme 1). As is well known for enolates, our products increased the separation of the metal cation from the anion with the larger alkali metals, which leads to a more reactive but less stable anionic intermediate.
Attempts to optimize the reaction through modification of the ratio between 2 and BnBr proved unsuccessful, and we did not find any effects of the ratio between 2 and bases on the reaction in terms of yield.
Therefore, the promising approach of the protocol prompted us to evaluate the substrate scope. As shown in Table 2 (entries b–h), a wide range of benzyl bromides containing both electron-donating (EDG) and electron-withdrawing (EWG) substituents were well tolerated with good conversion. However, at this stage, the results are difficult to rationalize. In relation to entry i, the reactivity of bromomethane is definitely higher compared to that of primary alkyl bromides, and the fact that there is more than one nucleophilic center on the cytosine substrate results in byproduct formation that is not valuable.
Table 2. Substrate scope.
However, as in the model reaction, a mixture of two different N-alkylated products, namely 1 and 3, were obtained. The mono/di-alkylation ratio ranged from 6:4 to 7:3, as determined by the integration of characteristic protons for each product in the 1HNMR spectra of the concentrated reaction mixtures.
A combination of homo- and heteronuclear 2D NMR experiments (DQF-COSY, 13C-1H HSQC, and HMBC, NOESY) were used to assign all the spin systems of 1a and 3a. In detail, the proton resonances of all systems were obtained by the COSY technique and were used to assign the carbon resonance in the HSQC spectra. The 13C-1H HMBC spectrum of 3a (see Supplementary Materials) shows a correlation between CH2 at δ = 4.54 ppm and the nitrogen-bearing carbon C4 signal at δ = 164.1 ppm, as well as a comparable correlation between CH2 at δ = 4.96 ppm and the same C4. However, the first CH2 is also correlated to carbons at δ = 126.2 ppm and at δ = 135.0 ppm, whereas the second CH2 shows a correlation to carbons at δ = 128.7 ppm and δ = 136.0 ppm. These values, together with the NOE contact, are diagnostic of benzyl groups on different nitrogen atoms, as depicted in the structure of 3a.
Based on the entire experimental outcome and the reported literature [45,46,47], we postulated that the undesired dibenzylated byproduct 3a might be due to the competitive pathway illustrated [47] in Scheme 2, where the nucleophilic substitution of benzyl bromide first occurs by the NH2(N4) group and then by the cytosine N3 site of the bidentate nucleophile.
Scheme 2. Postulated mechanism.
DMSO and DMF have large dielectric constants (47.24 and 38.25, respectively), large dipole moments (3.96 and 3.82 D, respectively), and they do not participate in hydrogen bonding. Their high polarity allows them to dissolve charged species such as various anions used as nucleophiles. The lack of hydrogen bonding in the solvent means that the latter is relatively “free” in the solution, making it more reactive.
Moreover, THF and CH2Cl2 as borderline polar aprotic solvents have moderately higher dielectric constants (7.52 and 8.93, respectively) and small dipole moments (1.75 and 1.60 D, respectively). The intermediate polarity makes them good “general purpose” solvents for a wide range of reactions where they serve only as the medium (for example, in the Grignard reaction and for enolate formation).
Thus, to reduce the reactivity of the nitrogen ring in an attempt to increase the efficiency of the behavior, the model reaction was carried out using DMF in a 1:1 mixture with THF as co-solvent [48,49]; we obtained an interesting result, which drove us to perform the reaction in a new 1:1 mixture of CH2Cl2/THF. As we postulated, these conditions led to very efficient results (Table 3, entry 4).
Table 3. Solvent effects.
In our mind, the CH2Cl2-THF mixture could synergistically ensure the solubility of our polar substrate, resulting in the best interaction of the latter with the base as well as the electrophile.
CH2Cl2 did not change the regiochemical outcome (N-enamine type versus N-imine type) of mono-alkylation.
Under the optimized reaction conditions, the scope of various para-substituted benzyl bromides was again investigated, and the results are summarized in Table 4.
Table 4. Optimized reaction conditions on different substituted benzyl bromides.

4. Conclusions

Cytosine derivatives have recently gained great interest as bioactive molecules. This work explores the synthesis and characterization of new cytosine-based potential ARV compounds, exploiting a route via the new N-1 sulfonylate precursor 2. The latter, containing one of the groups useful for the targeted application, avoids any expensive protection–deprotection steps. Moreover, it prevents the use of excess reactant. The scope of the approach was explored, which provided a good tolerance and satisfactory yields. Further studies are currently underway in order to exploit the new methodology for other nucleoside analogues.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/reactions3010014/s1.

Author Contributions

Conceptualization, M.D.N. and S.P.; methodology, M.D.N. and A.D.M.; formal analysis, C.O., A.B., M.M. and S.P.; resources, I.M.G.-M. and P.C.; writing—original draft preparation, S.P.; writing—review and editing, M.D.N. and M.M.; project administration, I.M.G.-M.; funding acquisition, I.M.G.-M. and P.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the Italian Ministry of Education (MIUR) (PRIN n° 2010-11E61J12000210001: Bloccare la replicazione di HIV-1 attraverso un approccio rivolto verso diversi bersagli molecolari), Italy.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Espeseth, A.S.; Felock, P.; Wolfe, A.; Witmer, M.; Grobler, J.; Anthony, N.; Egbertson, M.; Melamed, J.Y.; Young, S.; Hamill, T.; et al. HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase. Proc. Natl. Acad. Sci. USA 2000, 97, 11244–11249. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Zhu, K.; Dobard, C.M.; Chow, S.A. Requirement for Integrase during Reverse Transcription of Human Immunodeficiency Virus Type 1 and the Effect of Cysteine Mutations of Integrase on its Interactions with Reverse Transcriptase. J. Virol. 2004, 78, 5045–5055. [Google Scholar] [CrossRef] [Green Version]
  3. McColl, D.J.; Chen, X. Strand transfer inhibitors of HIV-1 integrase: Bringing IN a new era of antiretroviral therapy. Antivir. Res. 2010, 85, 101–118. [Google Scholar] [CrossRef]
  4. Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi, M.; Ferrara, M.; Fiore, F.; Gardelli, C.; Gonzalez Paz, O.; Hazuda, D.J.; et al. Discovery of Raltegravir, a Potent, Selective Orally Bioavailable HIV-Integrase Inhibitor for the Treatment of HIV-AIDS Infection. J. Med. Chem. 2008, 51, 5843–5855. [Google Scholar] [CrossRef] [PubMed]
  5. Anker, M.; Corales, R.B. Raltegravir (MK-0518): A novel integrase inhibitor for the treatment of HIV infection. Expert Opin. Investig. Drugs 2008, 17, 97–103. [Google Scholar] [CrossRef] [PubMed]
  6. Sax, P.E.; Dejesus, E.; Mills, A.; Zolopa, A.; Cohen, C.; Wohl, D.; Gallant, J.E.; Liu, H.C.; Zhong, L.; Yale, K.; et al. Co-formulated elvitegravir, cobicistat, emtricitabine, and tenofovir versus co-formulated efavirenz, emtricitabine, and tenofovir for initial treatment of HIV-1 infection: A randomised, double-blind, phase 3 trial, analysis of results after 48 weeks. Lancet 2012, 379, 2439–2448. [Google Scholar] [CrossRef]
  7. Shimura, K.; Kodama, E.; Sakagami, Y.; Matsuzaki, Y.; Watanabe, W.; Yamataka, K.; Watanabe, Y.; Ohata, Y.; Doi, S.; Sato, M.; et al. Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS-9137). J. Virol. 2008, 82, 764–774. [Google Scholar] [CrossRef] [Green Version]
  8. Sato, M.; Motomura, T.; Aramaki, H.; Matsuda, T.; Yamashita, M.; Ito, Y.; Kawakami, H.; Matsuzaki, Y.; Watanabe, W.; Yamataka, K.; et al. Novel HIV-1 Integrase Inhibitors Derived from Quinolone Antibiotics. J. Med. Chem. 2006, 49, 1506–1508. [Google Scholar] [CrossRef]
  9. De Clercq, E. Anti-HIV drugs: 25 compounds approved within 25 years after the discovery of HIV. Int. J. Antimicrob. Agents 2009, 33, 307–320. [Google Scholar] [CrossRef]
  10. Cuzzucoli Crucitti, G.; Pescatori, L.; Messore, A.; Madia, V.M.; Pupo, G.; Saccoliti, F.; Scipione, L.; Tortorella, S.; Di Leva, F.S.; Cosconati, S.; et al. Discovery of N-aryl-naphthylamines as in vitro inhibitors of the interaction between HIV integrase and the cofactor LEDGF/p75. Eur. J. Med. Chem. 2015, 101, 288–294. [Google Scholar] [CrossRef]
  11. Engelman, A.; Kessl, J.J.; Kvaratskhelia, M. Allosteric inhibition of HIV-1 integrase activity. Curr. Opin. Chem. Biol. 2013, 17, 339–345. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. De Luca, L.; Ferro, S.; Morreale, F.; De Grazia, A.; Chimirri, S. Inhibitors of the Interactions between HIV-1 IN and the Cofactor LEDGF/p75. ChemMedChem 2011, 6, 1184–1191. [Google Scholar] [CrossRef]
  13. Hare, S.; Vos, A.M.; Clayton, R.F.; Thuring, J.W.; Cummings, M.D.; Cherepanov, P. Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance. Proc. Natl. Acad. Sci. USA 2010, 107, 20057–20062. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Kawasuji, T.; Johns, B.A.; Yoshida, H.; Taishi, T.; Taoda, Y.; Murai, H.; Kiyama, R.; Fuji, M.; Yoshinaga, T.; Seki, T.; et al. Carbamoyl pyridone HIV-1 integrase inhibitors. 1. Molecular design and establishment of an advanced two-metal binding pharmacophore. J. Med. Chem. 2012, 55, 8735–8744. [Google Scholar] [CrossRef]
  15. Pendri, A.; Meanwell, N.A.; Peese, K.M.; Walker, M.A. New first and second generation inhibitors of human immunodeficiency virus-1 integrase. Expert Opin. Ther. Pat. 2011, 21, 1173–1189. [Google Scholar] [CrossRef] [PubMed]
  16. Johns, B.A.; Svolto, A.C. Advances in two-metal chelation inhibitors of HIV integrase. Expert Opin. Ther. Pat. 2008, 18, 1225–1237. [Google Scholar] [CrossRef]
  17. Di Santo, R. Inhibiting the HIV Integration Process: Past, Present, and the Future. J. Med. Chem. 2014, 57, 539–566. [Google Scholar] [CrossRef]
  18. Baumann, R.; Baxendale, I.R. An overview of the synthetic routes to the best selling drugs containing 6-membered heterocycles. Beilstein J. Org. Chem. 2013, 9, 2265–2319. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Tintori, C. University of Siena, Siena, Italy. Unpublished work, 2022.
  20. Cesar Gonzalez, C.; de Cabrera, M.; Wnuk, S.F. Gemcitabine analogues with 4-N-alkyl chain modified with fluoromethyl ketone group. Nucleosides Nucleotides Nucleic Acids 2018, 37, 248–260. [Google Scholar] [CrossRef]
  21. Quercia, R.; Perno, C.F.; Koteff, J.; Moore, K.; McCoig, C.; St. Clair, M.; Kuritzkes, D.J. Twenty-Five Years of Lamivudine: Current and Future Use for the Treatment of HIV-1 Infection. Acquir. Immune Defic. Syndr. 2018, 78, 125–135. [Google Scholar] [CrossRef] [PubMed]
  22. Keane, S.J.; Ford, A.; Mullins, N.D.; Maguire, N.M.; Legigan, T.; Balzarini, J.; Maguire, A.R. Design and Synthesis of α-Carboxy Nucleoside Phosphonate Analogues and Evaluation as HIV-1 Reverse Transcriptase-Targeting Agents. J. Org. Chem. 2015, 80, 2479–2493. [Google Scholar] [CrossRef]
  23. Masaki; K.; Takayuki, S.; Isamu, K. Bicyclic cytosine derivative-containing double strand nucleic acids interfering HIV replication. JP 2012179027 A, 20 September 2012.
  24. Hossain, N.; Rozenski, J.; Clercq, E.D.; Herdewijn, P. Synthesis and Antiviral Activity of the α-Analogues of 1,5-Anhydrohexitol Nucleosides (1,5-Anhydro-2,3-dideoxy-d-ribohexitol Nucleosides). J. Org. Chem. 1997, 62, 2442–2447. [Google Scholar] [CrossRef] [PubMed]
  25. Hattori, H.; Tanaka, M.; Fukushima, M.; Sasaki, T.; Matsuda, A. Nucleosides and Nucleotides. 158. 1-(3-C-Ethynyl-β-d-ribo-pentofuranosyl)-cytosine, 1-(3-C-Ethynyl-β-d-ribopentofuranosyl)uracil, and Their Nucleobase Analogues as New Potential Multifunctional Antitumor Nucleosides with a Broad Spectrum of Activity. J. Med. Chem. 1996, 39, 5005–5011. [Google Scholar] [CrossRef] [PubMed]
  26. Badawey, E.; Kappe, T. Synthesis of some new imidazo[1,2-a]pyrimidin-5(1H)-ones as potential antineoplastic agents. J. Heterocycl. Chem. 1995, 32, 1003–1006. [Google Scholar] [CrossRef]
  27. Joseph, H.; Burke, J.M. Optimization of an Anti-HIV Hairpin Ribozyme by in Vitro Selection. J. Biol. Chem. 1993, 268, 24515–24518. [Google Scholar] [CrossRef]
  28. Güzel-Akdemir, Ö.; Akdemir, A.; Pan, P.; Vermelho, A.B.; Parkkila, S.; Scozzafava, A.; Capasso, C.; Supuran, C.T. A Class of Sulfonamides with Strong Inhibitory Action against the α-Carbonic Anhydrase from Trypanosoma cruzi. J. Med. Chem. 2013, 56, 5773–5781. [Google Scholar] [CrossRef] [PubMed]
  29. Tayebee, R.; Nehzat, F. A Simple and Effective Methodology for the Sulfonylation of Alcohols and Aniline under Solvent Free Condition at Room Temperature. Am. J. Med. Sci. 2012, 2, 36–39. [Google Scholar] [CrossRef] [Green Version]
  30. Barcelò-Oliver, M.; Baquero, B.A.; Bauzà, A.; Garcìa-Raso, A.; Terròn, A.; Mata, I.; Molinsb, E.; Frontera, A. Experimental and theoretical study of thymine and cytosine derivatives: The crucial role of weak noncovalent interactions. CrystEngComm 2012, 14, 5777–5784. [Google Scholar] [CrossRef]
  31. Kašnar-Šamprec, J.; Ratkaj, I.; Mišković, K.; Pavlak, M.; Baus-Lončar, M.; Kraljević Pavelić, S.; Glavaš-Obrovac, L.; Žinić, B. In vivo toxicity study of N-1-sulfonylcytosine derivatives and their mechanisms of action in cervical carcinoma cell line. Investig. New Drugs 2012, 30, 981–990. [Google Scholar] [CrossRef]
  32. Spijker, H.J.; van Delft, F.L.; van Hest, J.C.M. Atom Transfer Radical Polymerization of Adenine, Thymine, Cytosine, and Guanine Nucleobase Monomers. Macromolecules 2007, 40, 12–18. [Google Scholar] [CrossRef]
  33. Glavaš-Obrovac, L.; Karner, I.; Štefanic, M.; Kašnar-Šamprec, J.; Žinic, B. Metabolic effects of novel N-1-sulfonylpyrimidine derivatives on human colon carcinoma cells. Il Farmaco 2005, 60, 479–483. [Google Scholar] [CrossRef] [PubMed]
  34. Plitta, B.; Adamska, E.; Giel-Pietraszuk, M.; Fedoruk-Wyszomirska, A.; Naskret-Barciszewska, M.; Markiewicz, W.T.; Barciszewski, J. New cytosine derivatives as inhibitors of DNA methylation. Eur. J. Med. Chem. 2012, 55, 243–254. [Google Scholar] [CrossRef] [PubMed]
  35. Mattson, R.J.; Pham, K.M.; Leuck, D.J.; Cower, K.A. An improved method for reductive alkylation of amines using titanium(IV) isopropoxide and sodium cyanoborohydride. J. Org. Chem. 1990, 55, 2552–2554. [Google Scholar] [CrossRef]
  36. Borch, R.F. Reductive Amination with sodium cyanoborohydride: N,N-dimethylcyclohexylamine. Org. Synth. 1972, 52, 124–125. [Google Scholar]
  37. Borch, R.F.; Hassid, A.I. New method for the methylation of amines. J. Org. Chem. 1972, 37, 1673–1674. [Google Scholar] [CrossRef]
  38. Borch, R.F.; Bernstein, M.D.; Durst, H.D. Cyanohydridoborate anion as a selective reducing agent. J. Am. Chem. Soc. 1971, 93, 2897–2904. [Google Scholar] [CrossRef]
  39. Shapiro, R.; Weisgras, J.M. Bisulfite-catalyzed transamination of cytosine and cytidine. Biochem. Biophys. Res. Commun. 1970, 40, 839–843. [Google Scholar] [CrossRef]
  40. Kasnar-Samprec, J.; Glavas-Obrovac, L.; Pavlak, M.; Mihaljevic, I.; Mrljak, V.; Stambuk, N.; Konjevoda, P.; Zinic, B. Synthesis, Spectroscopic Characterization and Biological Activity of N-1-Sulfonylcytosine Derivatives. Croat. Chem. Acta 2005, 78, 261–267. [Google Scholar]
  41. Sun, T.; Darbre, F.; Keese, R. Synthesis of vitamin B12 derivatives incorporating peripheral cytosine and N-acetylcytosine. Tetrahedron 1999, 55, 9777–9786. [Google Scholar] [CrossRef]
  42. Kašnar, B.; Krizmanić, I.; Žinića, M. Synthesis of the Sulfonylpyrimidine Derivatives as a New Type of Sulfonylcycloureas. Nucleosides Nucleotides 1997, 16, 1067–1071. [Google Scholar] [CrossRef]
  43. Nand, B.; Khanna, G.; Chaudhary, A.; Lumb, A.; Khurana, J.M. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU): A Versatile Reagent in Organic Synthesis. Curr. Org. Chem. 2015, 19, 790–812. [Google Scholar] [CrossRef]
  44. Jagrut, L.B.; Waghmare, R.A.; Mane, R.A.; Jadhav, W.N. An Improved Synthetic Route for the Synthesis of Sulfonamides. Int. J. ChemTech Res. 2011, 3, 1592–1595. [Google Scholar]
  45. Timmerman, J.C.; Widenhoefer, R.A. Gold-Catalyzed Intermolecular anti-Markovnikov Hydroamination of Methylenecyclopropanes with 2-Pyridones. Adv. Synth. Catal. 2015, 357, 3703–3706. [Google Scholar] [CrossRef]
  46. Tsuyoshi, M.; Gunzi, S.; Kazukuni, N.; Yuichiro, E.; Genki, H.; Yasuhito, S.; Shogo, M.; Masafumi, S.; Drozdova, O.O.; Kyuya, Y. Complex Formation between a Nucleobase and Tetracyanoquinodimethane Derivatives: Crystal Structures and Transport Properties of Charge-Transfer Solids of Cytosine. Bull. Chem. Soc. Jpn. 2008, 81, 331–344. [Google Scholar]
  47. Freccero, M.; Di Valentin, C.; Sarzi-Amade, M. Modeling H-Bonding and Solvent Effects in the Alkylation of Pyrimidine Bases by a Prototype Quinone Methide:  A DFT Study. J. Am. Chem. Soc. 2003, 125, 3544–3553. [Google Scholar] [CrossRef]
  48. Kawabata, T.; Majumdar, S.; Tsubaki, D.; Monguchi, D. Memory of chirality in intramolecular conjugate addition of enolates: A novel access to nitrogen heterocycles with contiguous quaternary and tertiary stereocenters. Org. Biol. Chem. 2005, 3, 1609–1611. [Google Scholar] [CrossRef] [PubMed]
  49. Moon, K.Y.; Moschel, R.C. Effect of Ionic State of 2’-Deoxyguanosine and Solvent on Its Aralkylation by Benzyl Bromide. Chem. Res. Toxicol. 1998, 11, 696–702. [Google Scholar] [CrossRef] [PubMed]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Article Metrics

Citations

Article Access Statistics

Journal Statistics
Multiple requests from the same IP address are counted as one view.
Download PDF
Synthesis and Reactivity of Cyclic Oxonium Derivatives of nido-Carborane: A Review
Previous
Linear-Rate Reactions for the Thermal Devolatilization of Wheat Straw Based on Pseudo-Components
Next