2-(Heptylthio)pyrimidine-4,6-diamine

Abstract

Thiopyrimidines represent one of the most active classes of compounds, possessing a wide spectrum of biological activities. Herein, we report the synthesis of 2-(heptylthio)pyrimidine-4,6-diamine (HPDA) via S-alkylation. The structure of HPDA was elucidated using ¹H and ¹³C nuclear magnetic resonance (NMR), heteronuclear multiple bond correlation (HMBC), high resolution mass (HRMS), and infrared (IR) spectroscopies.

1. Introduction

Heterocyclic compounds hold significant importance for the pharmaceutical industry, as many crucial life-science products contain them. Derivatives of pyrimidine show various biological activities [1] such as antidiabetic [2], anti-inflammatory [3], antitumor [4], antibacterial [5], antihypertensive [6], antifungal [7], antioxidant [8], and antitubercular [9]. Among these, diaminomercaptopyrimidine (DAMP) has a broad range of synthetic applications arisen from pyrimidine-conjugated amino and mercapto groups [10,11]. The structural feature of DAMP also facilitates cyclization reactions, leading to the formation of expanded heterocycles such as 5-(buthylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidine-7-amine, 2-(buthylthio)-9-(4-nitrobenzyl)-9H-purine-6-amine, 2-butyloxy-9-(2,6-difluorobenzyl)-N-(morpholinoethyl)-9H-purine-6-amine (purine derivatives) [12], and 8-benzyl pteridine-6,7-diones [13]. Additionally, DAMP can undergo condensation reactions on its amino group(s) with carbonyl compounds to form Schiff bases [14]. On the other hand, the mercapto group of DAMP reacts with alkyl halides to form thioethers [10,15]. In this work, the heptylation of DAMP was examined to synthesize a novel S-alkylated derivative as one of the candidates to subject to our future drug discovery project.

2. Results and Discussion

A reaction of n-heptyl chloride with sodium salt of DAMP (1) in DMF at 50 °C furnished a single solid product (2) in 67% yield just by washing the crude material with water (Scheme 1). Although previous alkylation reactions of DAMP in the literature reported the formation of S-alkylated products [10,15,16,17], there were few pieces of spectroscopic evidence. Since there are three possible mono-alkylated products on a nitrogen or a sulfur atom (2, 3, and 4, respectively, Chart 1), the structure of our product was carefully elucidated by considerable spectroscopic analyses. First, an IR spectrum of compound 2 showed overlapped absorption bands at ν = 3470, 3320 and 3181 cm⁻¹ which were assigned to the N-H stretching from two amino groups. It also indicated the existence of an alkyl sulfide moiety attached to the C2 atom in a pyrimidine ring by peaks at ν = 1076, 1236, and 1280 cm⁻¹ (Figure S1) [18]. The HRMS (ESI-TOF) of the obtained compound showed m/z = 241.1500 which matched the calculated value for [M+H]⁺ ([C₁₁H₂₀N₄S]⁺, 241.1481) (Figure S2). These results indicated that the mono-alkylation occurred onto the mercapto group of DAMP, but to obtain more robust evidence, we further analyzed NMR.

The ¹H NMR spectrum of the isolated compound exhibited a triplet signal for the methyl group at the terminal position of the heptyl chain (H7′) at δ 0.87 ppm. Among fourteen methylene protons of the n-heptyl group, ten (H2′–H6′) resonated at δ 1.22–1.32, 1.36–1.41 and 1.60–1.65 ppm, while the two methylene protons next to a sulfur atom (H-1’) gave a triplet at δ 3.01 ppm. A singlet at δ 5.26 ppm corresponded to an aromatic proton (H5) on the pyrimidine ring (Figure S3).

In the ¹³C NMR spectrum, the terminal methyl carbon C7′ appeared at δ 13.2 ppm, and the methylene carbons C2′–C6′ resonated between δ 22.4 and 29.9 ppm, which were consistent with their aliphatic environment. The chemical shift of the carbon (C1′) adjacent to the sulfur atom showed a signal at δ 31.7 ppm, which is also consistent with the S-alkylation. In contrast, if compounds were N-alkylated products, it would exhibit the N-CH₂- peak at around δ 43 ppm according to the literature [19]. The aromatic carbons were identified at δ 79.2 (C5), 163.8 (C4 and C6) [17] and 170.0 ppm (C2) [20], respectively (Figure S4). These assignments were also evidenced by COSY and HSQC experiments.

With the full assignment of ¹H and ¹³C NMR in hand, we turned our attention to the unambiguous determination of the alkylation site via an HMBC experiment. Our careful analysis of the HMBC chart revealed significant cross peaks, that is, it provided the most critical ⁴J_CH interaction between H1′ and C2, which confirmed the attachment of the alkyl group to the sulfur atom, consistent with the structure of 2 (Figure S5). As shown above, the combined spectral analyses firmly corroborate the structural assignment of compound 2 [21]. This selectivity might be explained by the so-called hard and soft acids and bases (HSAB) rules in which soft bases (such as sulfur anions) tend to react with soft acids (such as alkyl halides).

3. Materials and Methods

3.1. Instrumentation

IR spectrum was recorded on a JASCO FT/IR-4100. NMR spectra were recorded on a JEOL JNM-ECA 600 spectrometer. Chemical shifts are reported in ppm from tetramethysilane (TMS) with reference to an internal residual solvent [¹H NMR: CD₃OD (3.31); ¹³C NMR: CD₃OD (47.3–48.1)]. The following abbreviations are used to designate the multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad. High resolution mass spectrum (HRMS) was recorded on a Bruker microTOFII.

3.2. 2-(Heptylthio)pyrimidine-4,6-diamine (2)

A three-necked round-bottomed flask, equipped with a thermometer, a reflux condenser and a magnetic stirrer was set up. DAMP (1, 50.0 mg, 0.351 mmol) was dissolved in a mixture of NaOH aq. (1.0 N, 0.35 mL, 0.35 mmol) and methanol (5.0 mL). The reaction mixture was stirred at room temperature for 1 h and evaporated to give a tan solid. To a solution of the tan solid in DMF (5.0 mL) was added n-heptyl chloride (0.050 mL, 0.35 mmol) and stirred at 50 °C for 16 h. After the completion of the reaction (as monitored by TLC), DMF was evaporated. The obtained residue was treated with water (50 mL) and extracted with chloroform (3 × 50 mL). The combined organic layer was dried over Na₂SO₄, filtered, evaporated under reduced pressure, and the residue was dried under vacuum to provide compound 2 as a brown amorphous solid (56.9 mg, 0.237 mmol, 67%, over 2 steps).

R_f = 0.62 (CHCl₃/MeOH = 5/1); IR (neat), 3470, 3320, 3181, 1610, 1457, 1303, 1280, 1235, 1076, 974, 805, 668 cm⁻¹; ¹H NMR (600 MHz, CD₃OD) δ: 0.87 (t, J = 12.0 Hz, 3H, H7′), 1.22–1.32 (m, 6H, H4′–6′), 1.36–1.41 (m, 2H, H3′), 1.60–1.65 (m, 2H, H2′), 3.01 (t, J = 7.8 Hz, 2H, H1′), 5.26 (s, 1H, H5) ppm; ¹³C NMR (150 MHz, CD₃OD) δ: 13.2 (C7′), 22.4, 28.6, 28.8, 29.5, 29.9 (C2′–6′), 31.7 (C1′), 79.2 (C5), 163.8 (C4, C6), 170.0 (C2) ppm; HRMS (ESI-TOF) m/z [M+H]⁺ calcd for C₁₁H₂₀N₄S = 241.1481, found 241.1500.

4. Conclusions

In conclusion, we have shown that S-heptylated 4,6-diamino-2-mercapto-pyrimidine (DAMP) (2) could be synthesized from the sodium salt of DAMP (1) with n-heptyl chloride in DMF. The structure of 2 was unambiguously elucidated using ¹H-, ¹³C-, and HMBC-NMR, as well as HRMS, and IR spectroscopies to find the alkylation mainly occurred on a sulfur atom, probably because of the HSAB rules. Biological assays of 2 are now underway in our laboratory.