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. 2024 Apr 6;9(15):17368–17378. doi: 10.1021/acsomega.4c00068

Study of Direct N7 Regioselective tert-Alkylation of 6-Substituted Purines and Their Modification at Position C6 through O, S, N, and C Substituents

Filip Nevrlka 1, Adam Bědroň 1, Michal Valenta 1, Lenka Tranová 1, Jakub Stýskala 1,*
PMCID: PMC11024948  PMID: 38645315

Abstract

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A new N7 direct regioselective method allowing the introduction of tert-alkyl groups into appropriate 6-substituted purine derivatives is developed. This method is based on a reaction of N-trimethylsilylated purines with a tert-alkyl halide using SnCl4 as a catalyst. In this work, we study the structure and optimal reaction conditions leading to the N7 isomer and in some cases also to the N9 isomer. The main goal is devoted to preparing 7-(tert-butyl)-6-chloropurine as a suitable compound for other purine transformations. The stability of the tert-butyl group at the N7 position is tested for classic model reactions, leading to the preparation of new 6,7-disubstituted purine derivatives, which is also interesting from the point of view of possible biological activity.

Introduction

Purine is a heterocyclic compound that is most commonly found in nature and is often found in several molecules with proven biological properties.1,2 For several decades, attention has therefore been focused on the chemistry of purines to obtain biologically active compounds. New chemical reactions allowing its modification can still be found. These reactions may involve, for example, regioselective substitution at the N7 position of the purine ring, which represents a poorly explored area of purine chemistry and related biological activity.

Although N7-substituted purine derivatives are less widespread than their N9 analogues, several biologically interesting compounds can be found among them. For example, pseudovitamin B12 or raphanatin, which has cytokinin activity,3 can be considered to be an N7 nucleoside in nature. Interesting cytotoxic activity4,5 or inhibitory activity of butyrylcholinesterase has also been reported for N7 nucleosides.6−8 Compounds with antiviral activity can also be found among N7-alkylated purines.9−14 More specifically, some N7-substituted adenines showed antiviral15,16 and anticancer activities.17,18 Some N7-substituted 6-mercaptopurines showed cytostatic activity,19,20 and 7-methylxanthine was discovered to be a DNA gyrase inhibitor.21

Regarding the preparation of N7-alkylated purines, there are several ways to introduce an alkyl group at the N7 position of the purine ring. Unfortunately, the methods are complicated, or a mixture of corresponding isomers is formed, where the thermodynamically more stable N9 regioisomer usually predominates, while the N7 isomer occurs as a side product.

One method is based on alkylation of purine derivatives with alkyl halides under basic conditions (e.g., 6-chloropurine22−27). This direct alkylation usually leads to a mixture of N7/N9 derivatives and is suitable when both isomers are of interest (Scheme 1, eq 1).

Scheme 1. Preparation of N7-Alkylated 6-Chloropurines.

Scheme 1

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A second method using Grignard reagents is similar to the previous method, but N7 isomers are usually favored (Scheme 1, eq 2).28

Very unambiguous is the method conducted via 7,8-dihydropurines. This method is based on protection of the N9 position and reduction and regioselective alkylation at position N7, followed by deprotection and reoxidation (Scheme 1, eq 3).29,30

Additional methods are based on the cyclization reaction of appropriate imidazole31,32 or pyrimidine derivatives.33−35 These methods represent unambiguous regioselectivity but are of multistep and very laborious.

Other methodologies for regioselective N7 alkylation of purines are limited to allylation of 6-halopurines in the presence of a cobalt complex36 or alkylation of some N9-substituted purines, followed by selective cleavage of labile groups from formed purinium salts.10,37−40

In general, 6-chloropurine (1) is a very useful intermediate for further derivatization and biological research. We decided to expand the scope of the less common N7 purine regioselective substitution with tert-alkyl groups, which could not be introduced by the methods described in the literature. The main goal is to develop a new method enabling the easy introduction of a tert-alkyl group into 6-chloropurine at the N7 position and to verify its stability under different reaction conditions, as well as to prepare new 6,7-purine disubstituted derivatives containing different substituents connected via O, S, N, and C atoms that may be of interest in terms of possible biological activity (Scheme 1, this work).

Results and Discussion

The reported alkylation methods (Scheme 1, eqs 1–3) employ only primary or secondary alkyl halides, not tertiary. First, we successfully experimentally verified these methods from the literature for the described substances and then applied them to the tert-butyl group but without success. No desired reaction occurred.

For the introduction of the tert-butyl group, we were inspired by the Vorbrüggen (silylation) method,41 which is generally used for the preparation of purine and pyrimidine nucleosides. This method allows the preparation of mainly thermodynamically favored N9 isomers in purine chemistry. However, there are also reactions where the predicted regioselectivity decreases8,42,43 or is reversed in favor of N7 isomers.44−46

Based on previous experience with the Vorbrüggen method,46 we successfully used a tert-alkyl halide instead of the protected sugar for the reaction with the purine derivatives. Initial pilot studies were performed with 6-chloropurine (1), the most interesting derivative, and tert-butyl bromide (tert-BuBr) to obtain 7-(tert-butyl)-6-chloropurine (2) with maximum possible regioselectivity and yield.

We made several assumptions regarding how to obtain a kinetic N7 product. These included assumptions on the influence of temperature, time, solvent [1,2-dichloroethane (DCE), acetonitrile (ACN)], type of Lewis acid, and other alkyl halides (Table 1).

Table 1. Optimization of the tert-Butylation Reaction of 6-Chloropurine (1).

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entry solvent SnCl4 (equiv) tert-BuBr (equiv) reaction time (h) temperature 1:2:3:byproducts %a
1 DCE 0 3 19 RT 100:0:0:0
2 DCE 1 1 3 RT 90:10:0:0
3 DCE 1 1 19 RT 83:17:0:0
4 DCE 1.2 1.5 3 RT 83:17:0:0
5 DCE 1.2 1.5 19 RT 60:40:0:0
6 DCE 1.2 3 3 RT 70:30:0:0
7 DCE 1.2 3 19 RT 40:60:0:0
8 DCE 2.1 1.5 3 RT 55:45:0:0
9 DCE 2.1 1.5 19 RT 50:50:0:0b
10 DCE 2.1 3 3 RT 25:75:0:0
11 DCE 2.1 3 19 RT 13:87:0:0c
12 DCE 2.1 3 48 RT 12:86:0:2
13 DCE 2.1d 1.5 19 RT 55:45:0:0
14 DCE 2.1d 3 19 RT 34:66:0:0
15g DCE 2.1 3 19 RT 100:0:0:0
16 ACN 2.1 1.5 3 RT 38:62:0:0
17 ACN 2.1 1.5 19 RT 37:63:0:0
18 ACN 2.1 3 3 RT 11:87:0:2h
19 ACN 2.1 3 19 RT 10:84:0:6
20 ACN 2.1 3 48 RT 15:73:2:10
21 DCE 2.1 3 3 50 °C 12:87:1:0
22 DCE 2.1 3 19 50 °C 13:79:3:5
23 ACN 2.1 3 3 50 °C 32:48:5:15
24 ACN 2.1 3 19 50 °C 15:5:30:50
25 ACN 2.1 3 5 80°C 15:0:55:30i
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a

Based on LC/MS analysis.

b

The isolated yield of 2 was 40%.

c

The isolated yield of 2 was 75%.

d

TiCl4 was used as a catalyst.

f

The isolated yield of 2 was 43%.

g

The reaction was carried out without prior silylation.

h

The isolated yield of 2 was 78%.

i

The isolated yield of N9-isomer 3 was 39%. For entries 18 and 25, see the Supporting Information.

In general, since the N7-substituted compounds are described as kinetically favorable products, the reactions were optimized by performing them under kinetically controlled conditions, i.e., mainly at room temperature, with regard to the optimal reaction time to capture the kinetic product and achieve the maximum possible conversion of the starting compounds without the formation of side products.

The Lewis acids most widely used as Vorbrüggen reaction catalysts include trimethylsilyl trifluoromethanesulfonate (TMSOTf), TiCl4, and SnCl4. The best results of the tert-butylation reaction of 6-chloropurine were obtained with the use of SnCl4 in the amount of 2.1–1 equiv of purine derivative. Similar amounts of catalyst appear in the literature for the Vorbrüggen reaction, which requires the formation of a cation from a saccharide and complexation with the purine derivative. A lower amount of catalyst had a considerable effect on the conversion of the starting material (Table 1, entries 2–7). Without a catalyst, the reaction did not occur at all (Table 1, entry 1). When TiCl4 was used, the reaction also occurred, but the conversion was lower (Table 1, entries 13–14). In contrast, when using TMSOTf or other tested Lewis acids [FeCl3, ZnCl2, AlCl3, BF3, Et2O, and Ti(iPrO)4], the reactions did not occur at room temperature. Additionally, no reaction was observed if the previous silylation with N,O-bis(trimethylsilyl)acetamide (BSA) was omitted (Table 1, entry 15).

The degree of conversion was also affected by the amount of tert-butyl bromide used. For successful reactions, 3 equiv of tert-butyl bromide was a sufficient quantity. Although the degree of conversion was not quite complete, the unreacted starting compound 1 could be easily removed by an extraction process after the reaction in the water–NaHCO3 system to obtain the desired crude product of high purity.

The solvent used had a significant effect on the rate of the kinetic tert-butylation reaction. DCE and more polar ACN, both widely used in the Vorbrüggen silylation method, were tested. The equilibrium was established faster with ACN (3 h) than with DCE (19 h) (compare Table 1, entries 10 and 11 with entries 18 and 19). In both cases, a longer reaction time (48 h) caused the gradual formation of additional isomers [detected by liquid chromatography–mass spectrometry (LC/MS)], which was significantly faster when using ACN (compare Table 1, entry 12 with entry 20).

The effect of temperature was also significant but expected in the described reactions. The reaction carried out at 50 °C for both solvents caused rapid formation of N7-isomer 2, but along with other isomers. Again, in the case of ACN, the formation of other isomers was faster, and after 19 h of heating, the portion of N7-isomer 2 started to change in favor of starting compound 1 and other isomers, where the thermodynamically more stable N9-isomer 3 predominated (compare Table 1, entry 22 with entry 24). Based on these results, we performed the silylation tert-butylation reaction with 1 in ACN at 80 °C to obtain N9-isomer 3. After 5 h of heating, no N7-isomer 2 was present, and the reaction mixture contained predominant N9-isomer 3 in addition to other byproducts (Table 1, entry 25). In a similar way, other N9-(tert-alkylated) 6-chloropurine derivatives 5 and 8 were obtained in yields of 28–39% (Scheme 2). This method can also represent the possibility of preparing tert-alkylated N9 isomers that cannot be obtained by classical direct alkylation (Scheme 1, eq 1).

Scheme 2. Preparation of N7/N9-(tert-Alkylated) Purine Derivatives.

Scheme 2

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In addition to the reaction with 6-chloropurine (1), we tested other purine derivatives to verify whether the substituent on the purine ring plays a role in the tert-butylation reaction. No reaction was observed when unsubstituted purine, 6-methylpurine, 2-chloropurine, and 6-(dimethylamino)purine were used. In contrast, 6-methoxy (9), 6-methylthiopurine (10), and 6-chloro-2-methylthiopurine (6) readily underwent N7 regioselective tert-butylation to obtain N7-isomers 7, 11, and 12 (Scheme 2). The substituents at the C6 position clearly play an important role during the regioselective reaction.

We were curious if the method developed for the N7 regioselective tert-butylation of 6-chloropurine (1) would be useable for other types of alkyl halides. Primary (benzyl bromide, allyl bromide, ethyl bromide, methyl iodide), secondary (isopropyl bromide), and tertiary (neopentyl bromide) halides were tested. Unfortunately, this silylation method failed for primary and secondary alkyl halides. A very low conversion, of approximately 10%, was observed for benzyl bromide in obtaining the corresponding N7 isomer, which was identified by LC/MS analysis of the reaction mixture using appropriate N7-benzylated28 and N9-benzylated47 standards prepared by different described methods. This example suggests that other stabilized carbocations may influence purine substitution. However, by using tert-neopentyl bromide, the presented method was confirmed to be useable for other tert-alkyl halides. Under kinetically controlled conditions, the N7-isomer 4 was obtained (Scheme 2).

7-(tert-Butyl)-6-chloro-7H-purine (2) is a new compound. The position of the tert-butyl group was confirmed in several ways using 13C NMR spectroscopy, where carbon structural assignments of 2 and 3 were made based on heteronuclear multiple-bond correlation (HMBC) and heteronuclear single-quantum coherence (HSQC) experiments. To differentiate between the two isomers, the key attribute is the chemical shift of the C5 carbon atom of the 6-chloropurine ring. Based on published values, N9-alkylated or N9-glycosylated 6-chloropurine derivatives show a chemical shift for the C5 atom of approximately 132 ppm.27,48 In contrast, the C5 chemical shift of the N7 isomers of 6-chloropurine is more shielded and shows a lower value of approximately 123 ppm.28−30,35 This is in accordance with our observations for all 6-chloropurine isomers 2, 4, and 7 and 3, 5, and 8 (Figure 1).

Figure 1.

Figure 1

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Resolution of N7/N9 regioisomers based on (a) chemical shift of the C5 carbon atom, determined using HMBC and HSQC experiments; (b) difference between the chemical shifts of the C5 and C8 carbon atoms; (c) NOESY NMR experiment.

Generally, a reliable tool for the resolution of N7 and N9 purine isomers is a method based on the relative difference between the chemical shifts of C5 and C8 carbon atoms. For N7 acylpurines, the difference is large, while for N9 isomers, the difference Δδ is much smaller.49,50 This is also known to hold for N7/N9 6-chloropurine nucleosides.46 The observed difference results are similar for our prepared N7-isomer 2 (Δδ = 27) and N9-isomer 3 (Δδ = 13).

The N7-regioisomer 2 was also unambiguously determined by an NMR nuclear Overhauser effect spectroscopy (NOESY) experiment after derivatization at position C6 by a methoxy group to obtain compound 11 (Figure 1, Method A), where the interaction of tert-butyl hydrogen atoms and the methoxy group was observed. Similar cross peaks are apparent in the NOESY spectra for 6-methoxy 11 (Figure 1, Method B) and 6-methylthio derivative 12 prepared by silylation methods from the corresponding precursors 9 and 10.

In addition, the N9 position of the tert-butyl group of N9-isomer 3, which was also previously prepared by a different method via ring closure of the imidazole ring,51 was confirmed by comparing the published 1H NMR and melting point data, which were consistent with the data of our product 3 prepared by silylation tert-butylation under thermodynamically controlled conditions.

Considering the possible lability of the tert-alkyl group, we decided to perform the following tests. We found that the tert-butyl group at position N7 of compound 2 is stable in basic conditions, but in the presence of aqueous mineral acids (HCl, HCOOH) or Lewis acids, it is unstable, contrary to N9-isomer 3, which is stable in all cases. The stability of N7-isomer 2 in the presence of Lewis acid was tested with SnCl4 (1 equiv) in DCE and ACN at 50 °C. Heating in DCE caused 20% conversion to 6-chloropurine (1) after 48 h. Only traces of the N9-isomer 3 were detected. When using ACN as a solvent, a mixture of starting compound 1, N7-isomer 2, and N9-isomer 3 as major compounds was detected after 21 h. Additionally, other minor, probably N1 and N3, tert-butyl isomers of 6-chloropurine were detected by LC/MS analysis. Complete conversion of the starting N7-isomer 2 was achieved after 48 h, where 6-chloropurine (1) and the thermodynamically most stable N9-isomer 3 were observed as the predominant products. Similar results were observed using TMSOTf (2 equiv) after heating at 80 °C for 1 h in ACN, where a mixture of compounds 1 and 3 was observed at a ratio of 1:3 (Scheme 3).

Scheme 3. Instability of the N7-(tert-Butyl) Group of 2 in the Presence of Acidic Compounds.

Scheme 3

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Prepared 7-(tert-butyl)-6-chloropurine (2) was further used for the C6 modification (Scheme 4). First, we focused on the preparation of hypoxanthine analogue 13, which was prepared by the alkaline hydrolysis of 2. Because of the low stability of tert-butyl at position N7 in acidic solutions, we cannot afford to carry out this hydrolysis with HCOOH, which has been utilized for analogous 7-alkyl-6-chloropurines, where alkyl represents methyl, allyl, propargyl, ester, and keto groups.52 From this point of view, the tert-butyl group is specific. Analogically, using the corresponding alkoxides, methoxy 11 and ethoxy 14 derivatives were prepared under mild conditions and in high yields. These were used for NMR NOESY experiments to determine the position of the tert-butyl group. The sulfur analogue of hypoxanthine 15 was prepared by treating starting compound 2 with a solution of NaSH in dimethylformamide (DMF) at room temperature, indicating complete conversion after 90 min. The first chromatographic separation of 15 failed due to partial oxidation to disulfide, so rapid extraction of the product under inert conditions to obtain the solid product was preferable.

Scheme 4. Modification of 7-(tert-Butyl)-6-chloro-7H-purine (2) at Position C6.

Scheme 4

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Adenine derivative 17 was prepared by a two-step protocol from azide 16, which was smoothly prepared with sodium azide in dimethyl sulfoxide (DMSO) at 60 °C. Reduction of azide 16 was carried out by palladium-catalyzed hydrogenation, which offers a simple and efficient method under slightly elevated hydrogen pressure without the undesired influence on the tert-butyl group. Other N-substituted adenine derivatives (18–22) were prepared by classical nucleophilic substitution of the C6 chlorine atom of 2 with structurally different amines in the presence of N,N-diisopropylethylamine (DIPEA) in n-BuOH at 120 °C (Scheme 4). A similar reaction with a less nucleophilic amine (4-methoxyaniline) failed under the conditions described above, and no conversion was observed in the Buchwald–Hartwig amination. A mixture of starting compound 2, desired compound 25, de-(tert-butylated) derivative 26, and less polar N9 derivative 27 was detected by LC/MS analyses according to the described coupling protocol using InCl3 as a catalyst.53 This reaction confirmed the instability of the tert-butyl group at the N7 position using acidic compounds (Scheme 5). However, the tert-butyl group was retained under Suzuki cross-coupling conditions for two model compounds 23 and 24 during C–C bond formation from the corresponding boronic acids when Pd(PPh3)4 was used as a catalyst under microwave (MW) irradiation (Scheme 4).

Scheme 5. Illustration of the Problem of Substituting a Chlorine Atom with a Less Nucleophilic Amine.

Scheme 5

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Conclusions

By studying the reaction conditions (catalyst type, solvent, time, and temperature), we developed a new method enabling direct regioselective N7-(tert-alkylation) of C6-substituted purines under kinetically controlled conditions. This reaction is highly regioselective for the tert-alkyl group, where there is a specific substituent at position C6. The method mentioned above enables the preparation of even N9 isomers under thermodynamic conditions. Apart from the presence of acidic compounds causing cleavage of the N7-(tert-butyl) group, this group is stable, and using chlorine precursor 2, various derivatives substituted at position C6 through O, S, N, and C atoms can be obtained as new 6,7-disubstituted purines.

Experimental Section

General Methods

All reagents were purchased from commercial suppliers and used without purification. Solvents were dried according to standard procedures and stored with molecular sieves 3A. Reactions were monitored by LC/MS analyses using a UPLC Waters Acquity system equipped with PDA and QDa detectors. The system contained an XSelect HSS T3 (Waters) 3 × 50 mm C18 reverse-phase column XP (2.5 μm particles). Mobile phases: 10 mM ammonium acetate in HPLC grade water (A) and gradient grade ACN for HPLC (B). A gradient was mainly formed from 20 to 80% B in 4.5 min and kept for 1 min, with a flow rate of 0.6 mL/min. The ESI-MS system operated at a 25 V cone voltage, a 600 °C probe temperature, and a 120 °C source temperature. 1H and 13C NMR spectra were measured on a JEOL ECA 400II NMR spectrometer (1H: 399.78 MHz, 13C: 100.53 MHz). Chemical shifts (δ) are reported in ppm and referenced to the middle peak of the solvent signal (CDCl3: 7.26 ppm, 77.00 ppm; DMSO-d6: 2.49 ppm, 39.5 ppm, explicitly indicated in the spectra, presented in the Supporting Information). High-resolution mass spectrometry (HRMS) measurements were performed on a UPLC Dionex Ultimate 3000 equipped with an Orbitrap Elite high-resolution mass spectrometer, Thermo Exactive Plus. The cross-coupling reactions under MW irradiation were performed in a 10 mL glass tube sealed with polytetrafluoroethylene-coated reusable septa. All MW reactions were carried out in a CEM-Discover MW reactor operating at 2.45 GHz with continuous irradiation of 300 W of maximum power. The MW irradiation of required power was used, and the temperature was continuously raised for 2 min. Once the desired temperature was reached, the mixture was held at this temperature for a given time. Thin layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates and visualized by exposure to UV light (254 or 366 nm). Column chromatography was carried out by using silica gel grade 60, with mesh size of 230 to 400 Å. Melting points were measured on a Boetius stage apparatus and are uncorrected. Tested purines were dried at 120 °C for 45 min.

General Procedure for the Analytical Study of tert-Butylation Reactions of Purines

To a suspension of the corresponding purine derivative (0.25 mmol) in anhydrous solvent (DCE or ACN; 2 mL) was added BSA (92 μL, 0.38 mmol) under argon. This mixture was heated at 76–80 °C (temperature of the oil bath) for 30 min to obtain a clear solution. After cooling in an ice bath, a Lewis acid (Table 1) was added. Next, the ice bath was removed, and stirring was continued at room temperature for 10 min. Then, tert-butyl bromide or other studied alkyl halide was added, and the mixture was stirred (amounts, temperatures, and durations are given in Table 1). The reaction mixture was then quenched with isopropyl alcohol (0.5 mL) and analyzed by the LC/MS method. In general, N7 isomers are more polar than N9 isomers and have lower retention times on a C-18 column, similarly on silica gel TLC plates.

7-(tert-Butyl)-6-chloro-7H-purine (2)

To a suspension of 6-chloropurine (1) (775 mg, 5.0 mmol) in anhydrous DCE (40 mL) was added BSA (1.84 mL, 7.5 mmol) under argon. This mixture was heated at 76–80 °C (temperature of the oil bath) for 30 min to obtain a clear solution. After the mixture was cooled in an ice bath, SnCl4 (1.23 mL, 10.5 mmol) was added. Next, the ice bath was removed, and stirring was continued at room temperature for 10 min. Then, tert-butyl bromide (1.68 mL, 15 mmol) was added, and the mixture was stirred at room temperature for 19 h. Afterward, the reaction mixture was quenched with isopropyl alcohol (10 mL), diluted with chloroform (60 mL), and washed with water (40 mL), a saturated solution of NaHCO3 (2 × 60 mL), water (30 mL), and brine (60 mL). The organic phase was dried (MgSO4), and the solvents were evaporated under reduced pressure to obtain a yellowish crystalline compound as a crude product of sufficient purity. The yield was 792 mg (75%). A sample for analysis was obtained by crystallization from ethanol to obtain a white crystalline compound, mp: 183–185 °C (dec.). 1H NMR (400 MHz, CDCl3): δ 8.85 (s, 1H), 8.47 (s, 1H), 1.91 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 163.9, 151.8, 146.9, 142.8, 123.2, 59.1, 31.2. 1H NMR (400 MHz, DMSO-d6): δ 8.88 (s, 1H, H-8), 8.79 (s, 1H, H-2), 1.83 (s, 9H, (CH3)3). 13C{1H} NMR (101 MHz, DMSO-d6): δ 163.5 (C-4), 151.1 (C-2), 149.1 (C-8), 141.7 (C-6), 122.6 (C-5), 58.8 (C), 30.4 (CH3). HRMS (ESI, m/z): [M + H]+ calcd for C9H12ClN4, 211.0745; found, 211.0746.

With the mentioned method in DCE, 5 g of chloropurine was processed to obtain 4.7 g (69%) of crude compound 2 (98% purity based on 1H NMR).

The title compound 2 was prepared in a similar manner using anhydrous ACN (40 mL) instead of DCE. In this way, the reaction time was shortened to 3 h at room temperature after the addition of tert-butyl bromide. The yield was 825 mg (78%) as a crude product of sufficient purity (97% purity based on 1H NMR).

9-(tert-Butyl)-6-chloro-9H-purine (3)

This compound was prepared in a similar manner as purine 2 using 6-chloropurine (155 mg, 1 mmol), BSA (368 μL, 1.5 mmol), SnCl4 (248 μL, 2.1 mmol), and tert-butyl bromide (336 μL, 3 mmol) in ACN (8 mL). This reaction was not carried out at room temperature but at 80 °C for 5 h after the addition of tert-butyl bromide. After processing the reaction mixture and evaporating the solvents, the crude product (120 mg) was crystallized twice from isopropanol–water (1:1, v/v) to give a pale-yellow crystalline compound. The yield was 82 mg (39%), mp: 144–146 °C (lit,51 144–146 °C). 1H NMR (400 MHz, CDCl3): δ 8.72 (s, 1H), 8.20 (s, 1H), 1.83 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 152.0, 151.1, 150.9, 142.9, 132.7, 58.5, 28.9. 1H NMR (400 MHz, DMSO-d6): δ 8.76 (s, 1H, H-2), 8.68 (s, 1H, H-8), and 1.75 (s, 9H, (CH3)3). 13C{1H} NMR (101 MHz, DMSO-d6): δ 151.8 (C-4), 150.5 (C-2), 149.2 (C-6), 145.4 (C-8), 132.0 (C-5), 58.2 (C), 28.3 (CH3). HRMS (ESI, m/z): [M + H]+ calcd for C9H12ClN4, 211.0745; found, 211.0746.

7-(tert-Pentyl)-6-chloro-7H-purine (4)

This compound was prepared in a similar manner as purine 2 using 6-chloropurine (155 mg, 1 mmol), BSA (368 μL, 1.5 mmol), SnCl4 (248 μL, 2.1 mmol), and 2-bromo-2-methylbutane (383 μL, 3 mmol) in DCE (8 mL). The yield was 119 mg (53%) as a crude product. A sample for analysis was obtained by the crystallization form ethanol to obtain a white crystalline compound, mp: 95–100 °C. 1H NMR (400 MHz, CDCl3): δ 8.84 (s, 1H), 8.42 (s, 1H), 2.30 (q, J = 7.5 Hz, 2H), 1.85 (s, 6H), 0.77 (t, J = 7.5 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3): δ 163.9, 151.7, 147.6, 142.9, 123.2, 62.3, 34.5, 28.7, 8.4. HRMS (ESI, m/z): [M + H]+ calcd for C10H14ClN4, 225.0902; found, 225.0897.

9-(tert-Pentyl)-6-chloro-9H-purine (5)

This compound was prepared in a similar manner as purine 2 using 6-chloropurine (309 mg, 2 mmol), BSA (736 μL, 3 mmol), SnCl4 (496 μL, 4.2 mmol), and 2-bromo-2-methylbutane (766 μL, 6 mmol) in ACN (16 mL). This reaction was carried out not at room temperature but at 80 °C for 3 h after the addition of 2-bromo-2-methylbutane. After processing the reaction mixture and evaporating the solvents, the crude product was dried under high vacuum (10–2 Torr) for 1 h to obtain an impure product (280 mg) which was crystallized twice from isopropanol–water (1:1, v/v) to give a pale-yellow crystalline compound. The yield was 160 mg (36%), mp: 103–105 °C. 1H NMR (400 MHz, CDCl3): δ 8.70 (s, 1H), 8.14 (s, 1H), 2.24 (q, J = 7.4 Hz, 2H), 1.79 (s, 6H), 0.70 (t, J = 7.5 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3): δ 151.9, 151.0, 150.8, 143.6, 132.6, 61.6, 32.7, 26.6, 8.1. HRMS (ESI, m/z): [M + H]+ calcd for C10H14ClN4, 225.0902; found, 225.0897.

7-(tert-Butyl)-6-chloro-2-(methylthio)-7H-purine (7)

This compound was prepared in a similar manner as purine 2 using 6-chloro-2-methylthiopurine (6) (200 mg, 1 mmol), BSA (368 μL, 1.5 mmol), SnCl4 (248 μL, 2.1 mmol), and tert-butyl bromide (672 μL, 6 mmol) in ACN (8 mL). This reaction was carried out at room temperature for 4 h. After processing the reaction mixture and evaporating the solvents, the crude product (170 mg) was crystallized from isopropanol to obtain a white crystalline compound. The yield was 133 mg (52%), mp: 177–179 °C (dec). 1H NMR (400 MHz, CDCl3): δ 8.33 (s, 1H), 2.64 (s, 3H), 1.87 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 165.7, 164.8, 146.5, 142.7, 120.0, 58.9, 31.0, 14.4. HRMS (ESI, m/z): [M + H]+ calcd for C10H14ClN4S, 257.0622; found, 257.0625.

9-(tert-Butyl)-6-chloro-2-(methylthio)-9H-purine (8)

This compound was prepared in a similar manner as purine 2 using 6-chloro-2-methylthiopurine (6) (200 mg, 1 mmol), BSA (368 μL, 1.5 mmol), SnCl4 (248 μL, 2.1 mmol), and tert-butyl bromide (672 μL, 6 mmol) in ACN (8 mL). This reaction was carried out not at room temperature but at 80 °C for 5 h after the addition of tert-butyl bromide. After processing the reaction mixture and evaporating the solvents, the crude product (120 mg) was purified by column chromatography (3 cm ID, silica gel, DCM–MeOH, 40:1, v/v) and crystallized from isopropanol–water (1:1, v/v) to obtain a white crystalline compound. The yield was 72 mg (28%), mp: 144–146 °C. 1H NMR (400 MHz, CDCl3): δ 8.03 (s, 1H), 2.62 (s, 3H), 1.81 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ: 165.1, 152.7, 150.9, 141.5, 129.6, 58.2, 28.9, 14.8. HRMS (ESI, m/z): [M + H]+ calcd for C10H14ClN4S, 257.0622; found, 257.0621.

7-(tert-Butyl)-6-methoxy-7H-purine (11)

Method A

To a mixture of 7-(tert-butyl)-6-chloropurine (2) (150 mg, 0.71 mmol) in anhydrous methanol (3 mL) was added a solution of sodium methoxide in methanol (2.3 mL, 1 M MeONa). This mixture was stirred for 30 min at room temperature and then neutralized with dilute acetic acid, followed by the addition of toluene (8 mL). The organic phase was washed with brine (2 × 4 mL), dried (MgSO4), and evaporated under reduced pressure to obtain a white crystalline solid. The yield was 97 mg (66%) as a crude product. 1H NMR (400 MHz, CDCl3): δ 8.59 (s, 1H, H-2), 8.15 (s, 1H, H-8), 4.15 (s, 3H), 1.71 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 163.2, 155.8, 151.7, 143.2, 112.5, 57.9, 54.0, 30.1. HRMS (ESI, m/z): [M + H]+ calcd for C10H15N4O, 207.1240; found, 207.1243.

Method B

The title compound 11 was prepared in a similar manner as purine 2 using 6-methoxypurine (9) (150 mg, 1 mmol), BSA (368 μL, 1.5 mmol), SnCl4 (248 μL, 2.1 mmol), and tert-butyl bromide (336 μL, 3 mmol) in ACN (8 mL). The reaction time was 5 h at room temperature. The yield was 124 mg (60%) as a crude product. A sample for analysis was obtained by crystallization from hexane–EtOAc (5:2, v/v) to obtain a white crystalline compound, mp: 117–119 °C. This method gave product 11 with the same spectral characteristics as Method A.

7-(tert-Butyl)-6-(methylthio)-7H-purine (12)

This compound was prepared in a similar manner as purine 2 using 6-(methylthio)purine (10) (166 mg, 1 mmol), BSA (368 μL, 1.5 mmol), SnCl4 (248 μL, 2.1 mmol), and tert-butyl bromide (336 μL, 3 mmol) in ACN (8 mL). The reaction time was 3 h at room temperature. The yield was 150 mg (68%) as a crude product. A sample for analysis was obtained by crystallization form hexane–EtOAc (5:2, v/v) to obtain a white crystalline compound, mp: 134–136 °C. 1H NMR (400 MHz, CDCl3): δ 8.80 (s, 1H, H-2), 8.27 (s, 1H, H-8), 2.71 (s, 3H), 1.85 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 160.2, 153.3, 151.3, 144.6, 123.9, 57.9, 31.6, 14.4. HRMS (ESI, m/z): [M + H]+ calcd for C10H15N4S, 223.1012; found, 223.1008.

7-(tert-Butyl)-7H-purin-6-ol (13)

To a mixture of 7-(tert-butyl)-6-chloropurine (2) (100 mg, 0.48 mmol) in water (4 mL) was added sodium hydroxide (60 mg, 1.5 mmol). The resulting solution was heated in a sealed vial for 30 min at 100 °C and then cooled and neutralized with dilute acetic acid. After the mixture was left to stand overnight at 2 °C, the precipitated white crystalline compound was filtered off, washed with water, and dried. The yield was 60 mg (66%), mp: 268–272 °C (dec). 1H NMR (400 MHz, CDCl3): δ 11.62 (bs, 1H), 8.16 (s, 1H), 8.10 (s, 1H), 1.82 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 159.9, 155.3, 144.5, 141.2, 115.6, 58.6, 29.9. HRMS (ESI, m/z): [M + H]+ calcd for C9H13N4O1, 193.1084; found, 193.1085.

7-(tert-Butyl)-6-ethoxy-7H-purine (14)

To a mixture of 7-(tert-butyl)-6-chloropurine (2) (100 mg, 0.48 mmol) in anhydrous ethanol (2 mL) was added a solution of sodium ethoxide in ethanol (1.5 mL, 1 M EtONa). This mixture was stirred for 30 min at room temperature, neutralized with dilute acetic acid, and toluene (5 mL) was then added. The organic phase was washed with brine (2 × 3 mL), dried (MgSO4), and evaporated under reduced pressure to obtain a white crystalline solid. The yield was 98 mg (93%) as a crude product. A sample for analysis was obtained by crystallization form hexane–EtOAc (5:2, v/v) to obtain a white crystalline compound, mp: 85–87 °C. 1H NMR (400 MHz, CDCl3): δ 8.60 (s, 1H), 8.18 (s, 1H), 4.66 (q, J = 7.2 Hz, 2H), 1.76 (s, 9H), 1.51 (t, J = 7.2 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3): δ 163.3, 155.6, 151.8, 143.2, 112.6, 63.1, 58.0, 30.2, 14.4. HRMS (ESI, m/z): [M + H]+ calcd for C11H17N4O, 221.1397; found, 221.1398.

7-(tert-Butyl)-1,7-dihydro-6H-purin-6-thione (15)

To a solution of 7-(tert-butyl)-6-chloropurine (2) (150 mg, 0.71 mmol) in DMF (4.3 mL) was added a solution of NaSH in water (893 μL, 4 M NaSH) under argon. The resulting mixture was stirred for 90 min at room temperature, then neutralized with dilute acetic acid and EtOAc (80 mL), and bubbled with argon. The organic phase was washed with brine (3 × 30 mL), dried (MgSO4), and evaporated under reduced pressure to obtain a white solid as a crude product. The yield was 104 mg (70%). 1H NMR (400 MHz, DMSO-d6): δ 13.60 (bs, 1H), 8.58 (s, 1H), 8.15 (d, J = 4.0 Hz, 1H), 1.92 (s, 9H). 13C{1H} NMR (101 MHz, DMSO-d6): δ 169.9, 156.1, 146.1, 144.8, 127.3, 59.5, 30.8. HRMS (ESI, m/z): [M + H]+ calcd for C9H13N4S, 209.0855; found, 209.0854.

6-Azido-7-(tert-butyl)-7H-purine (16)

To a solution of 7-(tert-butyl)-6-chloropurine (2) (300 mg, 1.43 mmol) in DMSO (5.7 mL) was added sodium azide (279 mg, 4.3 mmol). The resulting mixture was heated for 4 h at 60 °C, diluted with EtOAc (100 mL), and the organic layer was washed with brine (3 × 40 mL). Then, the organic phase was dried (MgSO4) and concentrated under reduced pressure to obtain a white crystalline compound as a crude product. The yield was 282 mg (91%). 1H NMR (400 MHz, CDCl3): δ 9.57 (s, 1H), 8.34 (s, 1H), 1.97 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 152.8, 143.2, 142.0, 133.6, 110.7, 59.4, 29.2. HRMS (ESI, m/z): [M + H]+ calcd for C9H12N7, 218.1149; found, 218.1149.

7-(tert-Butyl)-7H-purin-6-amine (17)

To a solution of azide 16 (100 mg, 0.46 mmol) in ethanol (12 mL) was added palladium on charcoal (25 mg, 10% Pd/C). This mixture was hydrogenated under a hydrogen pressure of 5 bar for 24 h, filtered through a syringe microfilter (0.22 μm, 2.5 cm ID), and evaporated under reduced pressure. The residue was crystallized from water (about 2 mL) to obtain a white crystalline compound. The yield was 66 mg (75%), mp: 287–290 °C. 1H NMR (400 MHz, DMSO-d6): δ:8.35 (s, 1H), 8.20 (s, 1H), 6.67 (s, 2H), 1.69 (s, 9H). 13C{1H} NMR (101 MHz, DMSO-d6): δ 161.5, 151.8, 150.7, 143.5, 110.7, 56.2, 30.9. HRMS (ESI, m/z): [M + H]+ calcd for C9H14N5, 192.1244; found, 192.1243.

N-Benzyl-7-(tert-butyl)-7H-purin-6-amine (18)

To a solution of 7-(tert-butyl)-6-chloropurine (2) (105 mg, 0.5 mmol) in butanol (2 mL) were added benzylamine (81 μL, 0.75 mmol) and DIPEA (174 μL, 1 mmol). The reaction mixture was heated for 6.5 h at 120 °C. After evaporation of the volatiles under reduced pressure, the residue was purified by column chromatography (4 cm ID) on silica gel using DCM–methanol (80:1, v/v) to obtain a light orange colored compound. The yield was 90 mg (64%). 1H NMR (400 MHz, CDCl3): δ 8.57 (s, 1H), 8.12 (s, 1H), 7.30–7.40 (m, 5H), 5.34 (s, 1H), 4.88 (d, J = 5.2 Hz, 2H), 1.75 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 161.6, 152.8, 149.5, 142.1, 138.3, 128.9, 127.70, 127.68, 111.8, 56.1, 45.9, 31.7. HRMS (ESI, m/z): [M + H]+ calcd for C16H20N5, 282.1713; found, 282.1711.

7-(tert-Butyl)-N-cyclohexyl-7H-purin-6-amine (19)

To a solution of 7-(tert-butyl)-6-chloropurine (2) (110 mg, 0.52 mmol) in butanol (2 mL) were added cyclohexylamine (120 μL, 1.04 mmol) and DIPEA (182 μL, 1.02 mmol). The reaction mixture was heated for 13 h at 120 °C. After that time, additional cyclohexylamine (60 μL) was added to increase the conversion, and heating was extended for another 2 h. Next, the volatiles were evaporated under reduced pressure, and the residue was purified by column chromatography (4 cm ID) on silica gel using DCM–methanol (80:3, v/v) to obtain a yellowish colored compound. The yield was 108 mg (76%). 1H NMR (400 MHz, CDCl3): δ 8.45 (s, 1H), 8.04 (s, 1H), 4.99 (d, J = 7.0 Hz, 1H), 4.20–4.29 (m, 1H), 2.07–2.11 (m, 2H), 1.75 (s, 9H), 1.58–1.72 (m, 3H), 1.41–1.51 (m, 2H), 1.19–1.32 (m, 3H). 13C{1H} NMR (101 MHz, CDCl3): δ 161.2, 152.7, 149.1, 141.7, 111.6, 55.9, 49.5, 33.0, 31.7, 25.5, 24.5. HRMS (ESI, m/z): [M + H]+ calcd for C15H24N5, 274.2026; found, 274.2028.

3-(((7-(tert-Butyl)-7H-purin-6-yl)amino)methyl)phenol (20)

This compound was prepared in a similar manner as adenine 18 using 7-(tert-butyl)-6-chloropurine (2) (110 mg, 0.52 mmol), 3-hydroxybenzylamine (128 mg, 1.04 mmol), and DIPEA (182 μL, 1.04 mmol) in butanol (2 mL). The reaction time was 3 h at 120 °C; mobile phase: DCM–methanol (80:3, v/v) for column separation. The yield was 73 mg (47%) as a light yellow crystalline compound. 1H NMR (400 MHz, CDCl3): δ 9.79 (bs, 1H), 8.40 (s, 1H), 8.03 (s, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 1.8 Hz, 1H), 6.89 (dd, J = 8.1, 2.3 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 5.48 (t, J = 5.2 Hz, 1H), 4.81 (d, J = 5.2 Hz, 2H), 1.71 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 160.4, 158.0, 152.3, 149.8, 142.0, 139.5, 129.9, 118.5, 115.3, 114.8, 111.7, 56.5, 45.7, 31.7. HRMS (ESI, m/z): [M + H]+ calcd for C16H20N5O, 298.1662; found, 298.1662.

7-(tert-Butyl)-N-(furan-2-ylmethyl)-7H-purin-6-amine (21)

This compound was prepared in a similar manner as adenine 18 using 7-(tert-butyl)-6-chloropurine (2) (110 mg, 0.52 mmol), furfurylamine (100 μL, 1.04 mmol), and DIPEA (182 μL, 1.04 mmol) in butanol (2 mL). The reaction time was 3 h at 120 °C; mobile phase: DCM–methanol (40:2, v/v) for column separation. The yield was 106 mg (75%) as a light yellow crystalline compound. 1H NMR (400 MHz, CDCl3): δ 8.56 (s, 1H), 8.11 (s, 1H), 7.37 (d, J = 0.8 Hz, 1H), 6.33–6.34 (m, 1H), 6.30 (d, J = 4.0 Hz, 1H), 5.44 (bs, 1H), 4.86 (d, J = 5.2 Hz, 2H), 1.77 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 161.6, 152.6, 151.3, 149.2, 142.3, 142.2, 112.0, 110.6, 107.5, 56.2, 38.7, 31.6. HRMS (ESI, m/z): [M + H]+ calcd for C14H18N5O, 272.1506; found, 272.1505.

7-(tert-Butyl)-6-(piperidin-1-yl)-7H-purin (22)

This compound was prepared in a similar manner as adenine 18 using 7-(tert-butyl)-6-chloropurine (2) (110 mg, 0.52 mmol), piperidine (256 μL, 2.6 mmol), and DIPEA (182 μL, 1.04 mmol) in butanol (2 mL). The reaction time was 3 h at 120 °C. The reaction mixture was worked up by dilution with toluene (5 mL) and extraction with brine (2 × 8 mL). The organic phase was dried (MgSO4) and evaporated under reduced pressure to obtain a light orange crystalline compound. The yield was 118 mg (87%) A sample for analysis was obtained by crystallization form ethanol–water, mp: 167–169 °C. 1H NMR (400 MHz, CDCl3): δ 8.89 (s, 1H), 8.33 (s, 1H), 3.04 (t, J = 5.3 Hz, 4H), 1.83 (s, 9H), 1.71–1.76 (m, 4H), 1.65–1.67 (m, 2H). 13C{1H} NMR (101 MHz, CDCl3): δ 164.8, 158.7, 152.7, 146.3, 119.6, 59.0, 53.1, 30.0, 25.6, 23.8. HRMS (ESI, m/z): [M + H]+ calcd for C14H22N5, 260.1870; found, 260.1873.

7-(tert-Butyl)-6-(4-methoxyphenyl)-7H-purine (23)

A MW vial was charged with a magnetic stirring bar, 7-(tert-butyl)-6-chloropurine (2) (100 mg, 0.48 mmol), 4-methoxyphenylboronic acid (109 mg, 0.72 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(PPh3)4 (26.7 mg, 0.023 mmol), and toluene (6 mL). The vial was sealed and inserted into a MW reactor, where it was irradiated at variable power, so the temperature was maintained at 140 °C for 30 min. After that time, the volatiles were removed under reduced pressure, and the residue was purified by column chromatography (4 cm ID, silica gel, DCM–MeOH, 80:3, v/v) to yield the title product as a white crystalline compound. The yield was 88 mg (66%). 1H NMR (400 MHz, CDCl3): δ 9.05 (s, 1H), 8.44 (s, 1H), 7.37 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 3.87 (s, 3H), 1.47 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 162.8, 160.3, 153.2, 151.7, 147.5, 132.6, 130.3, 124.1, 113.8, 58.7, 55.4, 30.8. HRMS (ESI, m/z): [M + H]+ calcd for C16H19N4O, 283.1553; found, 283.1551.

7-(tert-Butyl)-6-(thiophen-3-yl)-7H-purine (24)

This compound was prepared in a similar manner as purine derivative 23 using 7-(tert-butyl)-6-chloropurine (2) (100 mg, 0.48 mmol), 3-thienylboronic acid (92 mg, 0.72 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(PPh3)4 (26.7 mg, 0.023 mmol), and toluene (6 mL). The yield was 103 mg (84%), as a light orange colored compound. 1H NMR (400 MHz, CDCl3): δ 9.06 (s, 1H), 8.45 (s, 1H), 7.47 (dd, J = 8.1, 3.2 Hz, 2H), 7.24 (d, J = 4.6 Hz, 1H), 1.50 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 162.9, 151.7, 149.0, 147.5, 140.1, 128.6, 126.5, 125.9, 124.5, 58.6, 30.6. HRMS (ESI, m/z): [M + H]+ calcd for C13H15N4S, 259.1012; found, 259.1012.

Acknowledgments

This work was supported by the Czech Science Foundation (21-06553S) and the Internal Grant Agency of Palacký University (IGA_PrF_2023_20).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c00068.

  • Copies of 1H and 13C NMR spectra (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao4c00068_si_001.pdf (4.4MB, pdf)

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