Synthesis and Characterization of New Potential Hypoxia-Sensitive Azo-thiacalix[4]arenes Derivatives

Abstract

The subject of this article is new potential hypoxia-sensitive azo-thiacalix[4]arenes derivatives in the 1,3-alternate configuration. Previously, it was shown that azo derivatives of calix[4]arene in the cone conformation form complexes with rhodamine dyes. The present work is devoted to the synthesis of new azo derivatives using the thiacalix[4]arene platform. A new highly productive method for the synthesis of thiacalixarene with four anionic sulfonate azo fragments on the lower rim (compounds 2a–b) for further complexation with the most common cationic dyes is reported. The chemical structures of the products obtained were established based on 1H and 13C NMR, IR spectroscopy, MALDI TOF mass spectrometry, and elemental analysis.

1. Introduction

Hypoxia is a pathological process that develops as a result of an insufficient oxygen supply to tissues or a violation of its use by tissues [1,2]. It accompanies many diseases and pathophysiological states in the human body [3,4]. Particularly, hypoxia can be regarded as an indicator of tumor aggressiveness [5]. For this reason, there is a great interest in developing hypoxia visualization tools. Changes in the vascular system and blood flow resulting from tumor growth lead to the appearance of rapidly growing tumors of hypoxic regions, which in turn is accompanied by an increase in reductase activity [6]. The susceptibility of the azo bond to reduction by azoreductase formed the basis for the development of methods for visualizing hypoxia [7,8,9,10,11]. In this regard, the creation of hypoxia-sensitive systems based on complexes of azo derivatives of calixarenes seems promising [12]. Previously, it was shown that the calix[4]arenes with anionic carboxyl and sulfonate azo fragments in the cone configuration form complexes with rhodamine dyes [13]. The resulting complexes do not luminesce, but luminescence is recovered under hypoxic conditions. The present work is devoted to the synthesis of thiacalix[4]arenes 2a–b with anionic sulfonate azo groups in the 1,3-alternate conformation for further complexation with cationic dyes and the creation of signaling systems based on them for detecting hypoxia.

2. Results and Discussion

We synthesized new O-substituted thiacalix[4]arenes 2a–b in the 1,3-alternate conformation containing azo fragments linked by methylene spacers of different lengths (three and four units) (Scheme 1).

The synthesis of the initial bromopropyl thiacalixarene derivative 1a–b [14] as well as 4-[4-hydroxyphenylazo]-benzenesulfonic acid [15] was carried out using methods from the literature. We synthesized new derivatives of tetra-azo-thiacalix[4]arene 2a–b in 1,3-alternate form from the reaction of 1a–b with azo fragments. Compounds 2a–b were obtained after refluxing the reaction mixture for 5 days with 88% and 98% yield, respectively. Azo derivatives 2a–b were obtained as a result of the nucleophilic substitution reaction of the OH-nucleophile of the phenolic azo derivative with haloalkyl-thiacalixarene 1a–b in a basic medium.

The structures of 2a–b were decisively assigned based on IR, ¹H and ¹³C NMR spectroscopy, MALDI TOF mass spectrometry, and elemental analysis. The ¹H NMR spectrum of 25,26,27,28-tetrakis-4-[4-(4′-sulfonylphenyldiazinyl)phenoxy]butoxy-5,11,17,23-tetra(tert-butyl)thiacalix[4]arene 2b displayed four aromatic protons as two apparent doublet and two singlet signals at δ 7.88, 7.79, 7.41, and 7.07 and four methylene protons as multiplet signals at δ 3.96, 3.91, 1.67, and 1.34 in addition to one methyl proton signal at δ 1.24 ppm. Additionally, the substitution reaction was confirmed by the ¹³C NMR spectrum that showed the four sp³-methylene carbons at δ 69.63, 68.98, 36.77, and 34.91 (see Figures S1–S8).

3. Materials and Methods

The NMR spectra were recorded on Bruker Avance 400 and 500 Nanobay (Bruker Corporation, Billerica, MA, USA) instruments with signals from residual protons of DMSO-d6 solvent as the internal standard. MALDI mass spectra were obtained using an UltraFlex III TOF/TOF spectrometer in the linear mode; p-nitroaniline or 2,5-dihydroxybenzoic acid was used as the matrix. Elemental analysis was performed using a PerkinElmer PE 2400 CHNS/O analyzer.

All reagents and solvents were purchased from either Acros (Geel, Belgium) or Sigma-Aldrich (Burlington, VT, USA) and used without further purification. 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetrakis-[(3-bromopropoxy)]2,8,14,20-tetrathiacalix[4]arene 1a, 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetrakis-(4-bromobutoxy)-2,8,14,20-tetrathiacalix[4]arene 1b [14], and 4-[4-hydroxyphenylazo]-benzenesulfonic acid [15] were synthesized according to reported methods.

Synthesis of azo-thiacalix[4]arenes 2a–b.

25,26,27,28-Tetrakis(p-bromalkoxy)-5,11,17,23-tetra(tert-butyl)thiacalix[4]arene 1a–b (0.1 mmol) in DMF (10 mL) was added to 4-[4′-hydroxyphenylazo]benzenesulfonic acid (0.6 mmol) and Cs₂CO₃ (1 mmol). The reaction mixture was heated to 85 °C for 5 days. Then, the solvent was removed under vacuum; water was added to the residue. The precipitate formed was filtered off, washed with water, and dried in the air.

25,26,27,28-tetrakis-3-[4-(4′-sulfonylphenyldiazinyl)phenoxy]propoxy-5,11,17,23-tetra(tert-butyl)thiacalix[4]arene 2a.

Yield 88 %, orange powder, mp > 360 °C. R_f (ethyl acetate:hexane = 10:1) 0.26. IR: 1440 (N=N); 1191; 1143; 1120; 1006 (SO₂). ¹H NMR (400 MHz, DMSO-d6, 25 °C) δ, ppm: 7.90 (app d, 8H, Ar_azo-H), 7.79 (s, 16H, Ar_azo-H), 7.46 (s, 8H, ArH), 7.09 (app d, 8H, ArH_azo-H), 3.98–4.07 (m, 16H, OCH₂), 1.49 (m, 8H, CH₂), 1.23 (s, 36H, t-Bu). ¹³C NMR (101 MHz, DMSO-d6, 25 °C): δ 163.32, 162.34, 157.16, 152.73, 151.26, 147.19, 146.96, 128.35, 128.13, 127.66, 127.23, 125.70, 122.74, 120.79, 116.00, 66.08, 36.78.78, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.00, 35.78, 35.00, 35.00. 31.80, 29.28. Mass spectrum MALDI (DHB), m/z: 2028.4 [M − 4H + K]⁺. Found, %: C 58.79; H 5.71; N 6.45. C₁₀₀H₁₀₄N₈O₂₀S₈·3H₂O·2(CH₃)₂NC(O)H. Calculated, %: C 58.87; H 5.78; N 6.48.

25,26,27,28-tetrakis-4-[4-(4′-sulfonylphenyl-diazinyl)phenoxy]butoxy-5,11,17,23-tetra(tert-butyl)thiacalix[4]arene 2b.

Yield 98 %, orange powder, mp > 360 °C. R_f (ethyl acetate:hexane = 10:1) 0.56. IR: 1440 (N=N); 1237; 1194; 1120; 1007 (SO₂). ¹H NMR (500 MHz, DMSO-d6, 25 °C) δ, ppm: 7.88 (app d, 8H, Ar_azo-H), 7.79 (s, 16H, Ar_azo-H), 7.41 (s, 8H, ArH), 7.07 (app d, 8H, ArH_azo-H), 3.91–3.96 (m, 16H, OCH₂), 1.67 (m, 8H, CH₂), 1.34 (m, 8H, CH₂), 1.24 (s, 36H, t-Bu). ¹³C NMR (101 MHz, DMSO-d6, 25 °C): δ 163.31, 162.41, 157.84, 152.69, 151.29, 147.18, 146.43, 128.95, 128.71, 127.65, 125.64, 122.73, 115.92, 69.63, 68.98, 36.77, 34.91, 31.95, 31.78. Mass spectrum MALDI (p-NA), m/z: 2145.5 [M + H]⁺. Found, %: C 60.95; H 5.60; N 6.05. C₁₀₄H₁₁₂N₈O₂₀S₈·(CH₃)₂NC(O)H. Calculated, %: C 60.98; H 5.69; N 5.98.

4. Conclusions

Thus, we have demonstrated the synthesis of thiacalix[4]arenes 2a–b with anionic sulfonate azo groups in the 1,3-alternate conformation in high yields. The chemical structures of the products obtained were established based on ¹H and ¹³C NMR, IR spectroscopy, MALDI TOF mass spectrometry, and elemental analysis.