Synthesis and Anti-Cancer Activity In Vitro of Synephrine Derivatives

Abstract

Glucocorticoids (GCs) are routinely used to treat hematological malignancies; however, long-term treatment with GCs can lead to atrophic and metabolic adverse effects. Selective glucocorticoid receptor agonists (SEGRAs) with reduced side effects may act as a superior alternative to GCs. More than 30 SEGRAs have been described so far, yet none of them reached clinical trials for anti-cancer treatment. In the present work, we propose a novel approach to increase the number of potential SEGRAs by obtaining derivatives of synephrine, a molecule of natural origin. We synthesized 26 novel compounds from the class of synephrine derivatives and characterized them by HRMS, and ¹H and ¹³C NMR. We evaluated in vitro anti-cancer effects in leukemia K562 and lymphoma Granta cells using the MTT assay and studied their potential affinity for the glucocorticoid receptor (GR) in silico using the molecular docking approach. The novel derivative 1-[4-(benzyloxy)phenyl]-2-(hexylamino)ethanol (10S-E2) with the highest GR affinity in silico exhibited cytotoxic activity against K562 and Granta cells after 24 h of treatment at the concentration of approximately 13 µM which correlated with its highest MolDock Score. The other compound with high GR affinity, 2-(hexylamino)-1-(4-nitrophenyl)ethanol (13S-G2), demonstrated cytotoxicity in both cell lines at concentrations of 50–70 µM. Overall, our results may provide a solid rationale for developing and further investigating synephrine derivatives as SEGRAs with anti-cancer activity.

1. Introduction

Glucocorticoids (GCs) have been remarkably popular for treating autoimmune and inflammatory diseases (rheumatoid arthritis, psoriasis, atopic dermatitis, asthma) as well as cancer for the last 75 years. Unfortunately, in most cases, chronic GC administration induces severe adverse effects including osteoporosis, steroid diabetes, water–salt imbalance, lipodystrophy, and other metabolic complications [1,2]. The biological effects of GCs depend on activating the glucocorticoid receptor (GR), a well-known transcription factor regulating gene expression through two distinct mechanisms. Therapeutic effects of GCs arise from DNA-independent transcriptional repression (TR) mediated by protein–protein interactions between the GR and other transcription factors that inhibit cancer cell survival. The side effects of GCs are associated with transcriptional activation (TA) that requires GR dimerization and dimer binding to the glucocorticoid-responsive elements in the promoters of pro-inflammatory and anti-apoptotic genes [2,3,4].

Since GCs exhibit specific cytotoxic action towards immune cells, they are routinely used to treat hematological malignancies: acute and chronic leukemia as well lymphomas of different origins. Blood malignancies have been among the most common cancers in 2024, and the number of the patients is increasing; 400,000 patients with chronic myeloid leukemia are expected in Europe by 2050 [5]. Mantle cell lymphoma is a subtype of non-Hodgkin’s lymphoma accounting for 5–7% of all NHL cases associated with a poor long-term prognosis [6]. GCs still remain the first-line therapy and are often combined with conventional and novel agents for combination chemotherapy [7,8,9].

The approaches to increase GC therapeutic activity and decrease their side effects include devising selective glucocorticoid receptor agonists (SEGRAs) that activate only GR TR [4,10,11]. Over the last decades, more than 30 SEGRAs of natural and synthetic origin have been described [4,12]. Our group and others have previously described the low-molecular-weight compound 2-(4-acetoxyphenyl)-2-chloro-N-methylammonium chloride, or CpdA, isolated from the African shrub Salsola tuberculatiformis Botschantzev, which acts as a SEGRA. CpdA selectively induced GR TR and exhibited the anti-cancer effects in models of blood and solid cancers, both in vitro and in vivo, either individually or in tandem with other anti-cancer drugs (Bortezomib, MLN4924 and some others) [4,9,13,14]. However, CpdA use in clinics is limited due to its chemical instability and decomposition to the intermediate metabolite aziridine, a well-known carcinogen [15,16]. Therefore, we designed CpdA derivatives to invent novel SEGRAs (Figure 1). 4-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenol, or CpdA-03, demonstrated superior GR affinity and stability compared to CpdA as well as anti-lymphoma properties both in vitro and in vivo [17].

In the present work, we aim to evaluate a new synthetic strategy for SEGRA development with the synephrine molecule used as a template instead of CpdA. Synephrine is the final product of CpdA metabolism, which serves as a CpdA precursor [16,18,19,20]. Synephrine derivatives may prove to be more beneficial for clinical practice as compared to the GR ligands, the selective glucocorticoid receptor agonist CpdA, which is structurally similar to synephrine but less stable as stated above. First, we designed new strategies to synthesize novel synephrine derivatives that could act as potential SEGRAs. Second, we performed in silico and in vitro screening of their biological activity to select the hit compounds for further investigation.

2. Materials and Methods

2.1. Chemistry

2.1.1. General Procedures and Reagents

All reagents were obtained from commercial sources (Merck KGaA, Darmstadt, Germany) and used without additional purification. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. (Tewkesbury, MA, USA). Silica gel 60 (Merck KGaA, Darmstadt, Germany) was used for column chromatography. Analytical thin layaer chromatography (TLC) was performed on Sorbfil PTX-AF-A-UV silica gel plates (Imid Co., Ltd., Moscow, Russia).

Solvents were removed under reduced pressure on a Rotavapor R-200 rotary vacuum evaporator using a vacuum water jet pump (Buchi, Flawil, Switzerland). ¹H and ¹³C NMR spectra were recorded on a Bruker DPX-300 instrument (300 and 75 MHz, respectively). Chemical shifts are in parts per million (ppm, δ) relative to the solvents CDCl₃ (7.25 ppm), DMSO-d₆ (2.49 ppm), and CD₃OD (4.78 ppm) for ¹H nuclear magnetic resonance (NMR) and CDCl₃ (77.2 ppm), DMSO-d₆ (39.5 ppm), and CD₃OD (49.0 ppm) for ¹³C NMR. The data are presented as follows: chemical shifts, multiplicity (s—singlet, br.s—broad singlet, m—multiplet), and the relative value of integration. The determination of solvent peaks was carried out following the literature data [21]. The spectra can be found in Supplementary Figures S1–S52. High-resolution mass spectra (HRMS) were recorded on an Agilent 6224 using electron sputtering ionization, ESI (Agilent Technologies, Santa Clara, CA, USA). HPLC–MS measurements were performed on the Agilent InfinityLab LC/MSD iQ liquid chromatograph with the Agilent Single Quadrupole LC/MSD iQ mass spectrometric detector (Agilent Technologies, Santa Clara, CA, USA). The separation was performed on the Agilent Poroshell 300SB C18 column (Agilent Technologies, Santa Clara, CA, USA), 2.1 × 75 mm. Eluent: 0.1% acetonitrile in deionized water with 0.1% trifluoroacetic acid, with consumption of 0.5 mL per minute.

2.1.2. General Procedure for the Synthesis of Compounds 1A,B,C

Bromoacetophenones 1A,B,C were obtained by α-bromination of the corresponding commercially available acetophenones according to the method [22] with comparable yields.

2.1.3. General Procedure for the Synthesis of Products 3S-C1, 6S-C4, 7S-E1, and 11S-E4

A solution of bromoacetophenone 1 in methylene chloride (1 mL of solvent per 100 mg 1) were added dropwise to a solution of amine in methylene chloride (10 mL of solvent per 1 mL of amine). The reaction was monitored by the initial bromoacetophenone 1 conversion by TLC. After bromoacetophenone 1 conversion was complete, volatile components were evaporated. The residue was dissolved in 5 mL of water and extracted with methylene chloride (3 × 10 mL). The organic layers were combined and dried with anhydrous calcium chloride, filtered off, and evaporated. The resulting oil was dissolved in 10 mL of methanol, and NaBH₄ was added in portions during cooling. After the intermediate conversion (monitored by TLC), the reaction mixture was acidified with 10% aqueous HCl solution to pH 3, and the volatile components were evaporated. The extraction and drying processes were repeated. The products 3S-C1, 6S-C4, 7S-E1, and 11S-E4 were isolated by column chromatography (methanol gradient from 0 to 15%).

1-(4-methoxyphenyl)-2-piperedine-1-yl ethanol ( 3S-C1 )

Reaction mixture composition: 0.63 mL (6.30 mmol) piperidine, 0.42 g (1.80 mmol) of 4-methoxybromoacetophenone 1A, and 0.07 g (1.80 mmol) NaBH₄. Product yield was 0.31 g (74%). R_f = 0.45 (10% methanol in chloroform). Based on HPLC data, the content of the target substance was 97.5%. ¹H NMR spectrum (CDCl₃) δ: 7.31–7.28 (m, 2H, Ph); 6.83–6.80 (m, 2H, Ph); 5.33–5.30 (m, 1H, -CH); 4.60 (br. s, 1H, -OH); 3.76–3.69 (m, 2H,-CH₂-OH); 3.74 (s, 1H, -O-CH₃); 3.19–2.99 (m, 2H, piperidine -CH₂-); 2.83–2.76 (m, 2H, piperidine -CH₂-); 2.29–2.16 (m, 2H, piperidine -CH₂-); 1.83–1.77 (m, 3H, piperidine 2×-CH₂-); 1.47–1.37 (m, 1H, piperidine -CH₂-). ¹³C NMR spectrum (CDCl₃) δ: 159.42; 132.18; 127.15; 114.03; 67.43; 65.52; 55.51; 55.26; 54.04; 22.72; 22.67; 21.80. For C₁₄H₂₁NO₂, the calculated and experimental [M+H]⁺ values were 236.1650 and 236.1652, respectively.

2-(2-hydroxyethyl)(methyl) amino)-1-(4-methoxyphenyl)ethanol ( 6S-C4 )

Reaction mixture composition: 0.55 mL (7.00 mmol) of N-methyl ethanolamine, 0.45 g (2.00 mmol) of 4-methoxybromoacetophenone 1A, and 0.08 g (2.00 mmol) of NaBH₄. Product yield was 0.20 g (45%). R_f = 0.75 (5% methanol in chloroform). The content of the target substance was 99% (based on HPLC). ¹H NMR spectrum (CDCl₃) δ: 7.28–7.25 (m, 2H, Ph); 6.87–6.84 (m, 2H, Ph); 4.53–4.49 (m, 1H, -CH-); 3.99–3.84 (m, 2H, -CH₂-OH); 3.77 (s, 3H,-O-CH₃); 2.88–2.72 (m, 2H, -N(CH₃)-CH₂-); 2.32 (s, 3H, -N-CH₃); 2.28–2.06 (m, 2H, -CH(OH)-CH₂-). ¹³C NMR-spectrum (CDCl₃) δ: 159.11; 132.16; 127.34; 113.61; 77.48; 66.74; 61.86; 55.15; 54.46; 45.92. For C₁₂H₁₉NO₃, the calculated and experimental [M+H]⁺ values were 226.1443 and 226.1445, respectively.

1-(4-(benzyloxy) phenyl)-2-piperidine-1-yl-ethanol ( 7S-E1 )

Reaction mixture composition: 0.63 mL (6.35 mmol) of piperidine, 0.39 g (1.27 mmol) of 4-benzyloxybromoacetophenone 1B, and 0.05 g (1.27 mmol) of NaBH₄. Product yield was 7S-E1 was 0.30 g (76%). R_f = 0.45 (5% methanol in chloroform). The content of the target substance was 98% (based on HPLC). ¹H NMR spectrum (CDCl₃) δ: 7.43–7.27 (m, 7H, Ph); 6.96–6.93 (m, 2H, Ph); 5.04 (s, 2H, -CH₂-O-); 4.91–4.86 (m, 1H, -CH-OH); 4.62 (br.s., 1H, -OH); 2.86 (br.s., 2H, -CH₂-); 2.71–2.59 (m, 4H, piperidine 2×CH₂); 1.78–1.74 (m, 4H, piperidine 2×CH₂); 1.56–1.50 (m, 2H, piperidine CH₂). ¹³C NMR spectrum (CDCl₃) δ: 158.31; 136.89; 133.80; 128.54; 127.42; 127.13; 114.77; 69.96; 67.96; 66.57; 54.62; 24.94; 23.42. For C₂₀H₂₅NO₂, the calculated and experimental [M+H]⁺ values were 312.1963 and 312.1965, respectively.

1-(4-(benzyloxy)phenyl)-2-((2-hydroxyethyl)(methyl)amino)ethanol ( 11S-E4 )

Reaction mixture composition: 0.39 mL (4.9 mmol) of N-methyl ethanolamine, 0.30 g (0.98 mmol) of 4-benzyloxybromoacetophenone 1B, and 0.04 g (0.98 mmol) of NaBH₄. Product yield was 0.18 g (61%). R_f = 0.55 (7% methanol in chloroform). The content of the target substance was 98% (based on HPLC). ¹H NMR spectrum (CDCl₃) δ: 7.43–7.27 (m, 7H, Ph); 6.96–6.93 (m, 2H, Ph); 5.04 (s, 2H, -O-CH₂-); 4.91–4.86 (m, 1H, -CH(OH)-); 4.63 (br.s., 1H, -OH); 2.87 (br.s., 2H, -CH(OH)-CH₂-) 2.71–2.59 (m, 2H, -N(CH₃)-CH₂-); 1.76 (s, 3H, -N-CH₃); 1.54–1.52 (m, 2H, -CH₂-OH). ¹³C NMR spectrum (CDCl₃) δ: 158.93; 136.88; 130.18; 128.60; 128.02; 127.44; 123.29; 114.94; 70.15; 68.97; 61.42; 58.63; 55.24; 37.62. For C₁₈H₂₃NO₃, the calculated and experimental [M+H]⁺ values were 302.1756 and 302.1760, respectively.

2.1.4. General Procedure for the Synthesis of the Compounds 4S-C2, 5S-C3, 18S-C5, 19S-C6, 10S-E2, 8S-E3, 20S-E5, 21S-E6, 13S-G2, 14S-G3, 22S-G5, 23S-G6, and 9S-G1

To a solution of bromoacetophenone 1 in methanol (for 4S-C2, 5S-C3, 18S-C5, 19S-C6, 10S-E2, 8S-E3, 20S-E5, and 21S-E6) or 1,4-dioxane (for 13S-G2, 14S-G3, 22S-G5, 23S-G6, and 9S-G1) (1 mL per 100 mg) cooled in an ice bath (or at room temperature in the case of 9S-G1 compounds), 1 equivalent (eq) of NaBH₄ was added in portions. After the conversion of bromoacetophenone 1 (monitored with TLC), a solution of 5 eq of a primary amine and 1.2 eq of KOH in methanol (1 mL per 1 mL amine) was added. After 12 h incubation with stirring, the reaction mixture was acidified with 10% aqueous solution of HCl to pH 3, the volatile components were removed under reduced pressure, and the residue was suspended in 5 mL of water and extracted with methylene chloride (3 × 10 mL). The organic layers were combined and dried using anhydrous calcium chloride, filtered off, and evaporated. The product was isolated by column chromatography on silica gel in a chloroform–methanol solvent system (a methanol gradient from 0 to 20%).

2-(hexylamino)-1-(4-methoxyphenyl)ethanol ( 4S-C2 )

Reaction mixture composition: 0.5 g (2.70 mmol) of 4-methoxybromoacetophenone 1A, 0.1 g (2.70 mmol) of NaBH₄, 1.75 mL (13.5 mmol) of hexylamine, and 0.19 g (3.30 mmol) of KOH. Product yield was 0.37 g (54%). R_f = 0.45 (10% methanol in chloroform). The content of the target substance was 98% (based on HPLC). ¹H NMR spectrum (CDCl₃) δ: 7.30–7.27 (m, 2H, Ph); 6.87–6.84 (m, 2H, Ph); 4.86–4.82 (m, 1H, -CH); 3.82 (br.s, 1H, -OH); 3.78 (s, 3H, O-CH₃); 2.94–2.67 (m, 4H, 2×CH₂); 1.59–1.52 (m, 2H, -CH₂-); 1.29–1.23 (m, 6H, 3×CH₂); 0.88–0.84 (m, 3H, -CH₃). ¹³C NMR spectrum (CDCl₃) δ: 159.13; 133.70; 127.06; 113.80; 70.24; 56.30; 55.24; 49.11; 31.47; 28.39; 26.67; 22.51; 13.99. For C₁₅H₂₅NO₂, the calculated and experimental [M+H]⁺ values were 252.1963 and 252.1966, respectively.

2-((2-hydroxyethyl)amino)-1-(4-methoxyphenyl)ethanol ( 5S-C3 )

Reaction mixture composition: 0.5 g (2.70 mmol) of 4-methoxybromoacetophenone 1A, 0.1 g (2.70 mmol) of NaBH₄, 0.82 mL (13.5 mmol) of ethanolamine, and 0.19 g (3.30 mmol) of KOH. Product yield was 0.33 g (58%). Product R_f = 0.40 (20% methanol in chloroform). The content of the target substance was 99% (based on HPLC). ¹H NMR spectrum (DMSO-d₆) δ: 7.44–7.41 (m, 2H, Ph); 6.96–6.93 (m, 2H, Ph); 5.40 (br.s., 1H, -CH₂-OH-); 5.02 (br.s., 1H, -CH-OH-); 4.11–4.07 (m, 1H, -CH-); 3.74 (s, 3H, -O-CH₃); 3.71–3.32 (m, 4H, -CH₂-CH₂-OH); 2.75–2.56 (m, 2H, -CH₂-NH-). ¹³C NMR spectrum (DMSO-d₆) δ: 159.30; 129.63; 127.28; 113.96; 62.92; 57.21; 55.11; 47.79. For C₁₁H₁₇NO₃, the calculated and experimental [M+H]⁺ values were 212.1286 and 212.1290, respectively.

2-((2-hydroxy-2-(4-methoxyphenyl)ethyl)amino)propane-1,3-diol ( 18S-C5 )

Reaction mixture composition: 0.5 g (2.20 mmol) of 4-methoxybromoacetophenone 1A, 0.08 g (2.20 mmol) of NaBH₄, 1.0 g (11.0 mmol) of 2-aminopropane-1,3-diol, and 0.15 g (2.60 mmol) of KOH. Product yield of 18S-C5 was 0.07 g (13%). Product R_f = 0.55 (15% methanol in chloroform). The content of the target substance was 96%. ¹H NMR spectrum (DMSO-d₆) δ: 7.26–7.21 (m, 2H, Ph); 6.87–6.82 (m, 2H, Ph); 4.72 (br.s., 1H, -CH-OH); 4.31 (br.s., 1H, -CH₂-OH); 4.21 (br.s., 1H, -CH-OH); 3.78–2.74 (m, 1H, -CH-NH-); 3.71 (s, 3H, -O-CH₃); 3.42–3.23 (m, 5H, -CH₂-NH- and -CH-(CH₂-OH)₂); 2.36–2.31 (m, 1H, -CH₂-NH-). ¹³C NMR spectrum (DMSO-d₆) δ: 158.17; 134.47; 128.42; 113.43; 66.90; 61.89; 61.35; 57.69; 54.93. For C₁₂H₂₀NO₄, the calculated and experimental [M+H]⁺ values were 242.1392 and 242.1395, respectively.

3-((2-hydroxy-2-(4-methoxyphenyl)ethyl)amino)propane-1,2-diol ( 19S-C6 )

Reaction mixture composition: 0.5 g (2.20 mmol) of 4-methoxybromoacetophenone 1A, 0.08 g (2.20 mmol) of NaBH₄, 0.85 mL (11.0 mmol) of 3-aminopropane-1,2-diol, and 0.15 g (2.60 mmol) of KOH. Product yield was 0.05 g (9%). Product R_f = 0.46 (13% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (CD₃OD) δ: 7.27–7.22 (m, 2H, Ph); 6.90–6.86 (m, 2H, Ph); 3.76 (s, 3H, -O-CH₃); 3.61–3.43 (m, 4H, -CH₂-CH(OH)-CH₂-OH); 3.30–3.28 (m, 1H, -CH(OH)-CH₂-OH); 2.65–2.40 (m, 2H, -CH₂-NH-). ¹³C NMR spectrum (CD₃OD) δ: 160.78; 132.86; 132.76; 129.92; 129.89; 115.06; 115.04; 67.41; 67.27; 66.26; 66.13; 66.03; 65.07; 55.73; 51.53; 50.75. For C₁₂H₂₀NO₄, the calculated and experimental [M+H]⁺ values were 242.1392 and 242.1394, respectively.

1-(4-(benzyloxy)phenyl)-2(hexylamino)ethanol ( 10S-E2 )

Reaction mixture composition: 0.5 g (1.92 mmol) of 4-benzyloxybromoacetophenone 1B, 0.07 g (1.92 mmol) of NaBH₄, 1.25 mL (9.6 mmol) of hexylamine, and 0.17 g (3.0 mmol) KOH. Product yield was 0.17 g (27%). R_f = 0.55 (20% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (CDCl₃) δ: 7.43–7.33 (m, 7H, Ph); 6.94–6.92 (m, 2H, Ph); 5.38 (br. s, 1H, -CH-OH-); 5.03 (s, 2H, -CH₂-O-); 4.23 (br.s., 1H, -OH); 3.16–3.03 (m, 4H, -CH₂-NH-CH₂); 1.90 (br.s., 2H, -CH₂-(CH₂)₃-CH₃); 1.35–1.28 (m, 6H, 3×CH₂); 0.89–0.85 (m, 3H, -CH₃). ¹³C NMR spectrum (CDCl₃) δ: 158.66; 136.76; 132.37; 128.56; 127.96; 127.41; 127.18; 69.97; 68.69; 55.11; 48.65; 31.13; 26.36; 25.82; 22.38; 13.92. For C₂₁H₂₉NO₂, the calculated and experimental [M+H]⁺ values were 328.2277 and 328.2281, respectively.

1-(4-(benzyloxy)phenyl)-2-((2-hydroxyethyl)amino)ethanol ( 8S-E3 )

Reaction mixture composition: 0.65 g (2.50 mmol) of 4-benzyloxybromoacetophenone 1B, 0.09 g (2.50 mmol) NaBH₄, 0.75 mL (12.5 mmol) ethanolamine, and 0.17 g (3.0 mmol) KOH. Product yield was 0.16 g (22%). R_f = 0.35 (20% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (DMSO-d₆) δ: 7.45–7.29 (m, 7H, Ph); 7.03–7.00 (m, 2H, Ph); 5.27 (br.s, 1H, -CH-OH); 5.09 (s, 2H, -O-CH₂-); 4.89 (br.s, 1H, -CH₂-OH); 4.04–4.00 (m, 1H, -CH-); 3.64–3.55 (m, 4H, -CH₂-CH₂-OH); 2.75–2.54 (m, 2H, -CH₂-CH(OH)). ¹³C NMR spectrum (DMSO-d₆) δ: 158.28; 135.95; 129.42; 128.37; 127.78; 127.59; 114.73; 88.84; 69.18; 69.46; 63.00; 57.65; 48.01. For C₁₇H₂₁NO₃, the calculated and experimental [M+H]⁺ values were 288.1599 and 288.1602, respectively.

2-((2-(4-(benzyloxy)phenyl)-2-hydroxyethyl)amino)propane-1,3-diol ( 20S-E5 )

Reaction mixture composition: 1.0 g (3.30 mmol) of 4-benzyloxybromoacetophenone 1B, 0.12 g (3.30 mmol) NaBH₄, 1.5 g (16.5 mmol) 2-aminopropane-1,3-diol, and 0.22 g (4.0 mmol) KOH. Product yield was 0.27 g (27%). R_f = 0.67 (13% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (DMSO-d₆) δ: 7.45–7.35 (m, 5H, Ph); 7.25–7.22 (m, 2H, Ph); 6.96–6.91 (m, 2H, Ph); 5.18 (br.s., 1H, -CH-OH); 5.07 (s., 2H, -O-CH₂-); 4.53–4.49 (m., 1H, -CH-OH); 4.44–4.26 (m, 2H, 2×-CH₂-OH-); 3.44–3.24 (m, 5H, -CH-(-CH₂-OH)₂); 2.67–2.51 (m, 2H, -CH₂-NH-). ¹³C NMR spectrum (DMSO-d₆) δ: 157.25; 137.24; 136.87; 128.43; 127.77; 127.63; 127.07; 114.25; 71.61; 69.12; 61.36; 61.24; 61.08; 55.64. For C₁₈H₂₄NO₄, the calculated and experimental [M+H]⁺ values were 318.1705 and 318.1707, respectively.

3-((2-(4-(benzyloxy)phenyl)-2-hydroxyethyl)amino)propane-1,2-diol ( 21S-E6 )

Reaction mixture composition: 1.0 g (3.30 mmol) of 4-benzyloxybromoacetophenone 1B, 0.12 g (3.30 mmol) of NaBH₄, 1.27 mL (16.5 mmol) of 3-aminopropane-1,2-diol, and 0.22 g (4.0 mmol) of KOH. Product yield was 0.12 g (12%). R_f = 0.75 (15% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (DMSO-d₆) δ: 7.45–7.29 (m, 5H, Ph); 7.25–7.20 (m, 2H, Ph); 6.95–6.92 (m, 2H, Ph); 5.05 (s, 2H, -O-CH₂-); 4.58–4.56 (m, 1H, -CH-OH); 4.34 (br.s, 4H, -CH(OH)-CH₂-OH and HO-CH-CH₂-NH-); 3.58–3.18 (m, 5H, -CH₂-CH(OH)-CH₂-OH). ¹³C NMR spectrum (DMSO-d₆) δ: 157.38; 157.33; 137.24; 137.21; 134.11; 134.06; 128.40; 128.36; 127.71; 127.58; 127.54; 127.01; 126.98; 114.39; 71.01; 70.15; 69.14; 66.75; 66.60; 64.76; 64.48; 63.89; 51.15; 50.35. For C₁₈H₂₄NO₄, the calculated and experimental [M+H]⁺ values were 318.1705 and 318.1708, respectively.

2-(hexylamino)-1-(4-nitrophenyl)ethanol ( 13S-G2 )

Reaction mixture composition: 0.85 g (3.50 mmol) of 4-nitrobromoacetophenone 1C, 0.13 g (3.50 mmol) of NaBH₄, 2.30 mL (17.5 mmol) of hexylamine, and 0.23 g (4.2 mmol) of KOH. Obtained 0.32 g (34%) of the product 13S-G2. R_f = 0.61 (15% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (DMSO-d₆) δ: 8.18–8.16 (m, 2H, Ph); 7.62–7.59 (m, 2H, Ph); 5.58 (br.s, 1H,-OH); 4.77–4.73 (m, 1H, -CH); 2.69–2.53 (m, 4H, -CH₂-NH-CH₂-); 1.40–1.35 (m, 2H, -NH-CH₂-CH₂-); 1.26–1.22 (m, 6H, -(CH₂)₃-CH₃); 0.86–0.81 (m, 3H, -CH₃). ¹³C NMR spectrum (DMSO-d₆) δ: 152.80; 146.37; 127.11; 123.08; 70.78; 57.20; 49.01; 31.25; 29.53; 26.48; 22.12; 13.94. For C₁₄H₂₃N₂O₃, the calculated and experimental [M+H]⁺ values were 267.1709 and 267.1712, respectively.

2-((2-hydroxyethyl)amino)-1-(4-nitrophenyl)ethanol ( 14S-G3 )

Reaction mixture composition: 1.25 g (5.14 mmol) of 4-nitrobromoacetophenone 1C, 0.19 g (5.14 mmol) of NaBH₄, 1.55 mL (25.7 mmol) of ethanolamine, and 0.34 g (6.17 mmol) of KOH. Obtained 0.41 g (35%) of the product 14S-G3. R_f = 0.55 (13% methanol in chloroform). The content of the target substance was 95%. ¹H NMR spectrum (DMSO-d₆) δ: 8.19–8.16 (m, 2H, Ph); 7.63–7.60 (m, 2H, Ph); 5.61 (br.s, 1H, -CH-OH); 4.78–4.74 (m, 1H, -CH-OH); 4.49 (br.s, 1H, -CH₂-OH); 3.45–3.37 (m, 2H, -CH₂- OH); 2.72–2.53 (m, 4H, -CH₂-NH-CH₂-). ¹³C NMR spectrum (DMSO-d₆) δ: 152.76; 146.39; 127.12; 123.11; 70.96; 60.42; 57.15; 51.45. For C₁₀H₁₅N₂O₄, the calculated and experimental [M+H]⁺ values were 227.1032 and 227.1034, respectively.

2-((2-hydroxy-2-(4-nitrophenyl)ethyl)amino)propane-1,3-diol ( 22S-G5 )

Reaction mixture composition: 1.7 g (7.00 mmol) of 4-nitrobromoacetophenone 1C, 0.265 g (7.00 mmol) of NaBH₄, 3.18 g (35.0 mmol) of 2-aminopropane-1,3-diol, and 0.47 g (8.4 mmol) of KOH. Obtained 0.72 g (40%) of the product 22S-G5. R_f = 0.58 (10% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (DMSO-d₆) δ: 8.18–8.15 (m, 2H, Ph); 7.64–7.61 (m, 2H, Ph); 5.51 (br.s., 1H, -CH-OH); 4.74–4.72 (m, 1H, -CH-OH); 4.31–4.27 (m, 2H, 2×-CH₂-OH); 3.43–3.24 (m, 4H, 2×-CH₂-OH-); 2.84–2.64 (m, 2H, -CH₂-NH); 2.55–2.51 (m, 1H, -CH-NH-). ¹³C NMR spectrum (DMSO-d₆) δ: 152.69; 146.39; 127.13; 123.13; 71.39; 61.39; 61.23; 60.98; 55.10. For C₁₁H₁₇N₂O₅, the calculated and experimental [M+H]⁺ values were 257.1137 and 257.1140, respectively.

3-((2-hydroxy-2-(4-nitrophenyl)ethyl)amino)propane-1,2-diol ( 23S-G6 )

Reaction mixture composition: 1.7 g (7.00 mmol) of 4-nitrobromoacetophenone 1C, 0.265 g (7.00 mmol) of NaBH₄, 2.7 mL (35.0 mmol) of 3-aminopropane-1,2-diol, and 0.47 g (8.4 mmol) of KOH. Obtained 0.52 g (29%) of the product 23S-G6. R_f = 0.49 (10% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (DMSO-d₆) δ: 8.19–8.16 (m, 2H, Ph); 7.63–7.60 (m, 2H, Ph); 5.62 (br.s, 1H, -CH-OH); 4.77–4.57 (m, 1H, -CH-OH); 4.57 (br.s, 1H, -CH₂-OH); 3.49 (br.s, 1H, -CH-OH); 3.34–3.23 (m, 3H, -CH(OH)-CH₂-OH); 2.72–2.52 (m, 3H, -CH₂-NH-CH₂-); 2.46–2.38 (m, 1H, -CH₂-NH). ¹³C NMR spectrum (DMSO-d₆) δ: 152.73; 146.41; 127.11; 123.13; 71.08; 70.68; 64.54; 57.48; 52.73. For C₁₁H₁₇N₂O₅, the calculated and experimental [M+H]⁺ values were 257.1137 and 257.1141, respectively.

1-(4-nitrophenyl)-2-piperidin-1-ylethanol ( 9S-G1 )

Reaction mixture composition: 0.5 g (2.05 mmol) of 4-nitrobromoacetophenone 1C, 0.08 g (2.05 mmol) of NaBH₄, 1.0 mL (10.2 mmol) of piperedine, and 0.14 g (2.46 mmol) of KOH. Product yield was 0.20 g (40%). R_f = 0.67 (5% methanol in chloroform). The content of the target substance was 97%. ¹H NMR spectrum (CDCl₃) δ: 8.19–8.16 (m, 2H, Ph); 7.57–7.55 (m, 2H, Ph); 5.05–5.00 (m, 1H, -CH-); 4.40 (br.s., 1H, -OH); 2.91–2.84 (m, 2H, -CH₂-CH-); 2.70–2.49 (m, 4H, piperidine 2×CH₂); 1.78–1.70 (m, 4H, piperidine 4×CH₂); 1.54–1.50 (m, 2H, piperidine CH₂). ¹³C NMR spectrum (CDCl₃) δ: 149.41; 147.30; 126.53; 123.65; 67.71; 65.99; 54.63; 25.11; 23.45. For C₁₃H₁₈N₂O₃, the calculated and experimental [M+H]⁺ values were 251.1396 and 251.1398, respectively.

2.1.5. General Procedure for the Synthesis of the Products 12S-B2, 2S-B3, 27S-B5, 17S-B6, 26S-F2, 16S-F3, 24S-F5, 25S-F6, and 15S-F1

Initially, 10% Pd on carbon (0.05 eq of Pd) was added to a solution of 12S-B2, 2S-B3, 27S-B5, 17S-B6, 26S-F2, 16S-F3, 24S-F5, 25S-F6, or 15S-F1 in ethanol and mixed in an atmosphere of H₂ at room temperature overnight. The reaction mass was filtered through a layer of celite washed with 10–15 mL ethanol, and the combined organic solutions were evaporated. The product was isolated by column chromatography on silica gel in a chloroform–methanol solvent system (methanol gradient from 0 to 35%).

4-(2-(hexylamino)-1-hydroxyethyl)phenol ( 12S-B2 )

Obtained 0.03 g (41%) of the product 12S-B2 from 0.10 g (0.3 mmol) of 10S-E2. R_f = 0.34 (20% methanol in chloroform). The content of the target substance was 97%. ¹H NMR spectrum (DMSO-d₆) δ: 7.16–7.12 (m, 2H, Ph); 6.76–6.71 (m, 2H, Ph); 4.73–4.68 (m, 1H, -CH); 3.86–2.71 (m, 4H, -CH₂-NH-CH₂-); 1.58–1.48 (m, 2H, -NH-CH₂-CH₂-); 1.30–1.25 (m, 6H, -(CH₂)₃-CH₃); 1.29–0.87–0.84 (m, 3H, -CH₃). ¹³C NMR spectrum (DMSO-d₆) δ: 156.75; 133.09; 127.06; 114.91; 69.18; 55.24; 47.78; 30.93; 26.88; 26.02; 21.98; 13.90. For C₁₄H₂₄NO₂, the calculated and experimental [M+H]⁺ values were 238.1807 and 238.1810, respectively.

4-(1-hydroxy-2-((2-hydroxyethyl)amino)ethyl)phenol ( 2S-B3 )

Obtained 0.05 g (73%) of the product 2S-B3 from 0.1 g (0.35 mmol) of 8S-E3. R_f = 0.35 (30% methanol in chloroform). The content of the target substance was 97%. ¹H NMR spectrum (CD₃OD) δ: 7.36–7.33 (m, 2H, Ph); 6.87–6.85 (m, 2H, Ph); 5.01 (br.s, 2H, 2 × OH); 4.33–4.28 (m, 1H, -CH-OH); 3.99–3.86 (m, 2H, -CH₂-OH); 3.83–3.69 (m, 2H, -CH₂-NH-); 3.05–2.91 (m, 2H, -CH₂-CH-); 2.15 (s, 1H, -OH). ¹³C NMR spectrum (CD₃OD) δ: 159.90; 130.97; 124.11; 117.02; 64.74; 63.39; 57.78; 30.76. For C₁₀H₁₅NO₃, the calculated and experimental [M+H]⁺ values were 198.1130 and 198.1134, respectively.

2-((2-hydroxy-2-(4-hydroxyphenyl)ethyl)amino)propane-1,3-diol ( 27S-B5 )

Obtained 0.03 g (25%) of the product 27S-B5 from 0.17 g (0.5 mmol) of 20S-E5. R_f = 0.23 (15% methanol in chloroform). The content of the target substance was 96%. ¹H NMR spectrum (DMSO-d₆) δ: 7.13–7.09 (m, 2H, Ph); 6.70–6.65 (m, 2H, Ph); 4.47–4.43 (m, 1H, -CH-OH); 3.44–3.20 (m, 5H, -CH-(-CH₂-OH)₂); 2.69–2.52 (m, 2H, -CH₂-NH). ¹³C NMR spectrum (DMSO-d₆) δ: 156.14; 134.72; 126.96; 114.62; 71.67; 61.29; 61.17; 60.98; 55.51. For C₁₁H₁₈NO₄, the calculated and experimental [M+H]⁺ values were 228.1235 and 228.1239, respectively.

3-((2-hydroxy-2-(4-hydroxyphenyl)ethyl)amino)propane-1,2-diol ( 17S-B6 )

Obtained 0.03 g (42%) of the product 17S-B6 from 0.1 g (0.3 mmol) of 21S-E6. R_f = 0.37 (20% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (DMSO-d₆) δ: 7.12–7.08 (m, 2H, Ph); 6.70–6.66 (m, 2H, Ph); 4.82 (br.s, 1H, -CH-OH); 4.82 (br.s, 2H, -CH(OH)-CH₂-OH); 4.06 (br.s, 1H, -CH-OH); 3.60–3.17 (m, 7H, -CH₂-CH(OH)-CH₂-OH and -CH-OH and Ph-OH); 2.47–2.19 (m, 2H, -CH₂-NH). ¹³C NMR spectrum (DMSO-d₆) δ: 156.38; 156.34; 131.28; 128.44; 128,33; 114,88; 70.70; 69.73; 64,69; 64,40; 63,84; 50,92; 50.10. For C₁₁H₁₈NO₄, the calculated and experimental [M+H]⁺ values were 228.1235 and 228.1238, respectively.

1-(4-aminophenyl)-2-(hexylamino)ethanol ( 26S-F2 )

Obtained 0.035 g (37%) of the product 26S-F2 from 0.1 g (0.4 mmol) of 13S-G2. R_f = 0.41 (10% methanol in chloroform). The content of the target substance was 95%. ¹H NMR spectrum (DMSO-d₆) δ: 6.96–6.94 (m, 2H, Ph); 6.51–6.47 (m, 2H, Ph); 4.86 (br.s, 2H,-NH₂); 4.42–4.37 (m, 1H, -CH); 2.59–2.46 (m, 4H, -CH₂-NH-CH₂-); 1.40–1.32 (m, 2H, -NH-CH₂-CH₂-); 1.27–1.22 (m, 6H, -(CH₂)₃-CH₃); 0.87–0.83 (m, 3H, -CH₃). ¹³C NMR spectrum (DMSO-d₆) δ: 147.39; 131.63; 126.53; 113.40; 71.27; 57.73; 49.02; 31.18; 29.58; 26.42; 22.02; 13.84. For C₁₄H₂₅N₂O, the calculated and experimental [M+H]⁺ values were 237.1967 and 237.1970, respectively.

1-(4-aminophenyl)-2-((2-hydroxyethyl)amino)ethanol ( 16S-F3 )

Obtained 0.062 g (79%) of the product 16S-F3 from 0.09 g (0.4 mmol) of 14S-G3. R_f = 0.31 (13% methanol in chloroform). The content of the target substance was 98%. ¹H NMR spectrum (DMSO-d₆) δ: 6.97–6.94 (m, 2H, Ph); 6.49–6.47 (m, 2H, Ph); 4.90 (br.s, 2H, 2×-OH); 4.42–4.38 (m, 1H, -CH-OH); 3.43–3.40 (m, 2H, -CH₂-OH); 2.61–2.54 (m, 4H, -CH₂-NH-CH₂-). ¹³C NMR spectrum (DMSO-d₆) δ: 147.52; 131.66; 126.64; 113.46; 71.51; 60.43; 57.75; 51.53. For C₁₀H₁₇N₂O₂, the calculated and experimental [M+H]⁺ values were 197.1290 and 197.1294, respectively.

2-((2-(4-aminophenyl)-2-hydroxyethyl)amino)propane-1,3-diol ( 24S-F5 )

Obtained 0.1 g (88%) of the product 24S-F5 from 0.14 g (0.5 mmol) of 22S-G5. R_f = 0.26 (10% methanol in chloroform). The content of the target substance is 98%. ¹H NMR spectrum (DMSO-d₆) δ: 6.97–6.94 (m, 2H, Ph); 6.49–6.46 (m, 2H, Ph); 4.92–4.90 (m, 3H, -NH₂ and -CH-OH); 4.41–4.35 (m, 3H, -CH-OH and 2×-CH₂-OH); 3.30–3.23 (m, 5H, -CH-(CH₂-OH)₂); 2.64–2.56 (m, 2H, -CH₂-NH). ¹³C NMR spectrum (DMSO-d₆) δ: 147.49; 131.67; 126.65; 113.44; 71.99; 61.37; 61.28; 61.11; 55.68. For C₁₁H₁₉N₂O₃, the calculated and experimental [M+H]⁺ values were 227.1396 and 227.1400, respectively.

3-((2-(4-aminophenyl)-2-hydroxyethyl)amino)propane-1,2-diol ( 25S-F6 )

Obtained 0.095 g (53%) of the product 25S-F6 from 0.2 g (0.8 mmol) of 23S-G6. R_f = 0.23 (10% methanol in chloroform). The content of the target substance was 95%. ¹H NMR spectrum (DMSO-d₆) δ: 6.97–6.94 (m, 2H, Ph); 6.50–6.47 (m, 2H, Ph); 4.90 (br.s, 3H, -NH₂ and -CH-OH); 4.43–4.38 (m, 1H, -CH-OH); 3.53–3.23 (m, 5H, -CH(OH)-CH₂-OH and -CH₂-NH); 2.63–2.52 (m, 3H, -CH₂-NH-CH₂-); 2.44–2.37 (m, 1H, -CH₂-NH). ¹³C NMR spectrum (DMSO-d₆) δ: 147.53; 131.66; 126.63; 113.48; 71.59; 70.65; 64.63; 58.09; 52.83. For C₁₁H₁₉N₂O₃, the calculated and experimental [M+H]⁺ values were 227.1396 and 227.1399, respectively.

1-(4-aminophenyl)-2-piperidin-1-ylethanol ( 15S-F1 )

Obtained 0.03 g (34%) of the product 15S-F1 from 0.1 g (0.4 mmol) of 9S-G1. R_f = 0.35 (10% methanol in chloroform). The content of the target substance was 95%. ¹H NMR spectrum (DMSO-d₆) δ: 7.04–7.01 (m, 2H, Ph); 6.54–6.51 (m, 2H, Ph); 5.86 (br.s., 1H, -OH); 5.09 (br.s., 2H, -NH₂); 4.95–4.91 (m, 1H, -CH-); 3.36–2.98 (m, 6H, piperidine 2×CH₂ and -CH-CH₂-); 1.77–1.76 (m, 4H, piperidine 4×CH₂); 1.51 (br.s., 2H, piperidine CH₂). ¹³C NMR spectrum (DMSO-d₆) δ: 148.24; 128.69; 126.70; 113.47; 66.72; 62.98; 52.49; 22.24; 21.46. For C₁₃H₂₁N₂O, the calculated and experimental [M+H]⁺ values were 221.1654 and 221.1656, respectively.

2.2. Biology

2.2.1. Cell Cultures

Chronic myeloid leukemia cell line K562 and mantle cell lymphoma cell line Granta were obtained from ATCC and cultured as described [9].

2.2.2. MTT Cytotoxicity Assay

Cell cultures were seeded into 96-well plates and incubated overnight under 5% CO₂ at 37 °C for 24 h. Then, cells were treated with a synthesized compound (in the concentration range of 10 mM–100 nM) or a vehicle for 24 h and 72 h. Then, 20 μL of MTT solution was added to each well and mixed. After 4 h, the supernatants were removed, and 100 μL of DMSO was added to each well to dissolve the precipitate. The cells’ viability was estimated by measuring absorbance at 570 nm using the MultiScan MCC 340 spectrophotometer (Thermo Fisher, Waltham, MA, USA). IC₅₀ values were determined with GraphPad Prism (Ver.7.0). Results are presented as mean ± standard deviation (SD).

2.2.3. Molecular Docking

Molegro Virtual Docker 6.0 software was used to perform molecular docking. The structure of GR (PDB ID: 1P93) was chosen as a target. The target structure was prepared automatically using standard procedures of the Molegro Virtual Docker package. Ligand structures were constructed and optimized by molecular dynamic methods in the MMFF94 force field using Avogadro 1.2.0. The MolDock Score was used as a scoring function; dexamethasone (CID 5743) served as a reference ligand. Molecular docking was carried out in 40 iterations. MolDock SE was used as a docking algorithm following energy minimization and optimization of hydrogen bonds.

2.2.4. Statistical Analysis

Mean and standard deviation values were calculated using Microsoft Excel and GraphPad Prism (Ver.7.0) software. Treatment effects in each experiment were compared by one-way ANOVA or t-test. The differences between groups were considered significant at p < 0.05. All experiments were repeated three times.

3. Results

3.1. Synthesis of 26 Novel Synephrine Derivatives

We aimed to evaluate the potential of 4-(1-hydroxy-2-aminoethyl)benzene derivatives substituted at the amino group and the 4-position of the benzene ring 1 as non-steroidal dexamethasone (Dex) analogs. We employed two methods to synthesize synephrine derivatives with different primary and secondary amines (Scheme 1).

The first synthetic route involved the direct alkylation of secondary amines with bromoacetophenones 1 followed by the reduction of intermediates 2a–d with NaBH₄ (Scheme 2). Bromoacetophenones 1 were obtained by α-bromination of the corresponding commercially available acetophenones.

The direct alkylation of primary amines with bromoacetophenones 1 was less preferable because of the lower selectivity of the reaction; therefore, they were synthesized by another synthetic pathway.

The second strategy was to obtain epoxides 3A,B,C in situ by the sequential reduction of bromoacetophenones 1 with NaBH₄ in methanol and the further epoxidation of the intermediates by adding 1.2 alkali equivalents. The target compounds 4S-C2, 5S-C3, 18S-C5, 19S-C6, 10S-E2, 8S-E3, 20S-E5, 21S-E6, 13S-G2, 14S-G3, 22S-G5, and 23S-G6 were obtained by adding a five-fold excess of primary amines to epoxides 3A,B,C in line with Scheme 3. The 9S-G1 analog was obtained by a similar strategy to the compounds 4S-C2, 5S-C3, 18S-C5, 19S-C6, 10S-E2, 8S-E3, 20S-E5, 21S-E6, 13S-G2, 14S-G3, 22S-G5, and 23S-G6, according to Scheme 4, except that the reaction was carried out at reflux. The first synthetic pathway for the synthesis of 3S-C1, 6S-C4, 7S-E1, and 11S-E4 (Scheme 2) was not optimal for 9S-G1 synthesis due to the low reactivity of the source bromide 1C. The compounds 12S-B2, 2S-B3, 27S-B5, and 17S-B6, as well as compounds 26S-F2, 16S-F3, 24S-F5, 25S-F6, and 15S-F, were obtained by the hydrogenation of 10S-E2, 8S-E3, 20S-E5, 21S-E6, 13S-G2, 14S-G3, 22S-G5, 23S-G6, and 9S-G1, respectively (Scheme 3 and Scheme 4) using palladium on carbon.

The compounds used for the initial scrutiny of their biological properties were synthesized as a mixture of enantiomers.

In summary, we obtained 26 synephrine analogs. Their structures were confirmed by ¹H and ¹³C NMR spectroscopy and high-resolution mass spectrometry. The purity of the obtained compounds was assessed using HPLC with a mass spectrometer. Compounds with purity greater than 95% were further subjected to biological activity evaluation.

3.2. The Evaluation of Cytotoxicity of Novel Synephrine Derivatives

The cytotoxic effects of new synthetic synephrine derivatives were evaluated using the MTT assay in chronic myelocytic leukemia K562 cells and mantle cell lymphoma Granta cells, well-characterized cell lines used in our previous studies [9,14,17,23]. Cells were treated with synephrine derivatives in a wide concentration range of 10 mM–100 nM for 24 h. Ultimately, 8S-E3, 10S-E2, 13S-G2, and 26S-F2 exhibited the strongest cytotoxic effect in K562 cells, with IC₅₀ values of 120 ± 20 µM, 13.1 ± 1.5 µM, 184 ± 95 µM, and 670 ± 148 µM, respectively (Figure 2A). Granta-519 cells were more sensitive to 4S-C2, 8S-E3, 10S-E2, 12S-B2, 13S-G2, 21S-E6, and 26S-F2, with IC₅₀ values of 201 ± 32 µM, 89.1 ± 22.4 µM, 13 ± 0.7µM, 246 ± 6 µM, 26.8 ± 1.2 µM, 725 ± 392 µM, and 215 ± 71 µM, respectively (Figure 2B). The other tested synephrine derivatives did not have IC₅₀ values, which most likely exceeded 1 mM. The highest cytotoxicity in both cell lines was demonstrated for the compound 1-[4-(benzyloxy)phenyl]-2-(hexylamino)ethanol (10S-E2) at the concentration of 13 µM. Taken together, these results and the data on 10S-E2’s affinity for the GR make this synephrine derivative an attractive candidate for further investigation. Another potential target is 2-(hexylamino)-1-(4-nitrophenyl)ethanol (13S-G2), with the IC₅₀ value of 26.8 ± 1.2 µM in Granta cells.

After 72 h incubation with the compounds in the same concentration range (100 nM–10 mM), the IC₅₀ values for 12S-B2, 20S-E5, 21S-E6, and 26S-F2 in K562 cells reached 77.3 ± 34.1 µM, 31.0 ± 0.4 µM, 130 ± 30 µM, and 56.1 ± 0.9 µM, respectively. In Granta cells, 20S-E5 did not reach IC₅₀, while the IC₅₀ values for 12S-B2, 21S-E6, and 26S-F2 were 53.1 ± 18.9 µM, 80.1 ± 41.7 µM, and 108 ± 14.1 µM, respectively (Figure 3). During a prolonged treatment, 4-(2-(hexylamino)-1-hydroxyethyl)phenol (12S-B2) exhibited cytotoxic effects within 3 days after the treatment at concentrations of 50–70 µM. This may be associated with a gradual induction of the mechanisms underlying cell survival inhibition. Our findings implicate a new dosage regimen for in vivo experiments that does not require daily administration. Detailed stability and pharmacokinetic studies will be required to further validate this approach.

All findings on the cytotoxic effects of the synephrine derivatives in K562 and Granta cells after 24 and 72 h incubations are summarized in Table 1.

3.3. The Evaluation of GR Affinity In Silico

To rank the tested compounds based on their affinity for the GR, we performed molecular docking studies, in order to predict their binding to the ligand binding domain (LBD) of the GR structure (PDB ID: 1P93) using Dex (CID 5743) as a reference ligand. According to the obtained results, the greatest potential affinity for the GR was shown for the derivative 10S-G2. The MolDock Score, which correlates to the steric orientation and hydrogen bonds in the active site of 10S-E2, was similar to the score of Dex (−143 and −146, correspondingly; Table 2 and Table S1). The following top five compounds included 21S-E6, 8S-E3, 20S-E5, 7S-E1, and 13S-G2. All mentioned compounds occupied a sterically advantageous location at the GR binding site formed by the amino acid residues Thr 739, Asn564, and Gln642 (see Figure 4 and Supplementary Figure S53 for the representative images of molecular docking of novel compounds compared to the Dex background). Dex binding to the LBD mostly involved Thr 739, Asn564, and Gln642 residues (Figure 3 and Supplementary Figure S53).

The affinity of the other synephrine derivatives for the GR in silico was more than 1.2-fold lower (Table 2 and Supplementary Table S1). Of note, the template molecule synephrine demonstrated the weakest MolDock Score and potentially the lowest GR affinity.

4. Discussion

The biological effects of synephrine, its molecular targets, and a safe pharmacological profile along with the possible mechanisms of action indicate that it can act as a potential SEGRA and can be used as a template to obtain novel SEGRAs [15]. SEGRAs could serve as safer alternatives to GCs for cancer therapy [4,9,10,13,15,17,18,24,25,26]. Therefore, searching for synephrine derivatives opens up new avenues for the development of novel SEGRAs.

Synephrine has itself exhibited anti-cancer and anti-inflammatory properties in several studies [27,28,29,30]. Synephrine is a small molecule with a hydroxyl group located at one terminus lacking a methyl group on the side chain, which affects its stereochemistry and impairs the interaction with proteins [15]. In the present work, we proposed modifications of synephrine with bulky substituents and synthesized 26 synephrine derivatives that could be utilized as potential SEGRAs by two synthetic routes: the direct alkylation of the secondary amines with bromoacetophenones or by interaction between alcohol-derived epoxides that corresponded to bromoacetophenones and primary amines. We estimated the cytotoxic effects of the compounds in K562 and Granta cells as well as their potential affinity for the glucocorticoid receptor (GR) in silico. The non-steroidal GR structures were designed in order to preserve the dimensional arrangement of the functional groups in the molecule and remove the dexamethasone (Dex) sterane core, if possible. We varied the lipophilicity of the designed compounds using different substituents at the aromatic ring and the amino group (Supplementary Table S2).

The novel derivative 1-[4-(benzyloxy)phenyl]-2-(hexylamino)ethanol (10S-E2) exhibited micromolar-range cytotoxicity in both cell lines after 24 h of treatment, which correlated with the highest affinity for the GR in silico. This implies the GR-dependency of 10S-E2’s cytotoxic effects. The synephrine derivative 2-(hexylamino)-1-(4-nitrophenyl)ethanol (13S-G2) with high GR affinity exerted cytotoxic activity at concentrations of 50–70 µM. Furthermore, we revealed a positive correlation between the lipophilicity of the novel synephrine analogs and their biological activity, with the most lipophilic compounds exhibiting higher activity. However, it should be noted that hydroxyl groups in the substituent at the amino group improve the interaction with the GR as the derivatives of aminoethanol (8S-E3) and aminopropanediols (20S-E5, 21S-E6) also demonstrate affinity for the GR.

These results imply that synephrine derivatives could serve as promising anti-cancer agents of the SEGRA class. Further investigation is required to elucidate the detailed mechanism of action of the compounds with the highest cytotoxicity in vitro, such as their direct interaction with the GR as well as in vivo anti-cancer activity. It is especially important to scrutinize the safety of the obtained compounds, including the absence of atrophic or metabolic GC-related side effects.

5. Conclusions

In the present work, we synthesized 26 synephrine derivatives that could be used as potential selective glucocorticoid receptor agonists (SEGRAs) via two synthetic routes: the direct alkylation of secondary amines with bromoacetophenones or the interaction between alcohol-derived epoxides corresponding to bromoacetophenones with primary amines. We investigated the in vitro anti-cancer effects of the obtained compounds against leukemia and lymphoma cells as well as their potential affinity for the glucocorticoid receptor (GR) in silico.

The novel derivative 1-[4-(benzyloxy)phenyl]-2-(hexylamino)ethanol (10S-E2) exhibited cytotoxicity in the micromolar range for both cell lines after 24 h of treatment. These effects positively correlated with the highest affinity for the GR in silico, implying the GR-dependency of 10S-E2’s activity. The synephrine derivative 2-(hexylamino)-1-(4-nitrophenyl)ethanol (13S-G2) with high GR affinity demonstrated cytotoxicity in vitro at concentrations of 50–70 µM.

Overall, our findings indicate a high anti-proliferative activity of the synephrine derivatives 1-[4-(benzyloxy)phenyl]-2-(hexylamino)ethanol (10S-E2) and 2-(hexylamino)-1-(4-nitrophenyl)ethanol (13S-G2) and their possible application for anti-cancer treatment strategies that employ SEGRA class drugs.

Author Contributions

Conceptualization, A.V.M., E.M.Z., M.G.Y. and E.A.L.; methodology, E.S.O., E.A.M., E.D.S., D.D.G., D.V.S., K.V.G. and L.E.G.; investigation, E.M.Z., E.S.O., E.A.M., E.D.S., D.D.G., D.V.S., K.V.G. and L.E.G.; resources, A.V.M., K.V.G., E.M.Z. and E.A.L.; writing—original draft preparation, A.V.M., K.V.G., E.M.Z., M.G.Y. and E.A.L.; writing—review and editing, A.V.M., E.M.Z., M.G.Y. and E.A.L.; visualization, A.V.M., K.V.G., E.M.Z., L.E.G. and E.A.L.; supervision, A.V.M., E.M.Z., M.G.Y. and E.A.L.; project administration, E.A.L.; funding acquisition, E.A.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

This study was performed using the equipment of the Shared Science and Training Center for Collective Use RTU MIREA and supported by the Ministry of Science and Higher Education of the Russian Federation. K.V.G., E.M.Z. and E.A.L. would like to thank the participants of the Telegram-chat Nenauchiye Razgovory (non-scientific talks), where their fruitful collaboration started.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures, Schemes and Tables

Figure 1. Structure of synephrine and described SEGRAs.

Scheme 1. Two synthetic routes to non-steroid Dex analogues.

Scheme 2. Synthesis of the compounds 3S-C1, 6S-C4, 7S-E1, and 11S-E4.

Scheme 3. Synthesis of the compounds 4S-C2, 5S-C3, 18S-C5, 19S-C6, 10S-E2, 8S-E3, 20S-E5, 21S-E6, 13S-G2, 14S-G3, 22S-G5, 23S-G6 and 12S-B2, 2S-B3, 27S-B5, 17S-B6, 26S-F2, 16S-F3, 24S-F5, 25S-F6, and 15S-F1.

Scheme 4. Synthesis of the compounds 9S-G1 and 15S-F1.

Figure 2. The cytotoxic effects of synephrine derivatives on K562 (A) and Granta (B) cells after 24 h incubation. Chronic myeloid leukemia cell line K562 (A) and mantle cell lymphoma cell line Granta (B) were seeded into 96-well plates and incubated overnight under 5% CO2 at 37 °C for 24 h. Next, cells were treated with 3S-C1, 6S-C4, 7S-E1, 11S-E4, 4S-C2, 5S-C3, 18S-C5, 19S-C6, 10S-E2, 8S-E3, 20S-E5, 21S-E6, 13S-G2, 14S-G3, 22S-G5, 23S-G6, 12S-B2, 2S-B3, 27S-B5, 17S-B6, 26S-F2, 16S-F3, 24S-F5, 25S-F6, 15S-F, 9S-G1 (100 nM–10 mM), or vehicle for 24 h. Then, 20 μL of MTT solution was added to each well and mixed. After 4 h, the supernatants were removed and 100 μL DMSO was added to each well to dissolve the precipitate. Cell viability was estimated by measuring absorbance at 570 nm using the MultiScan MCC 340 spectrophotometer (Thermo Fisher, Waltham, MA, USA). IC50 values were determined using GraphPad Prism, Ver.7.0. Results are presented as mean ± standard deviation (SD).

Figure 3. The cytotoxic effects of synephrine derivatives on K562 (A) and Granta (B) cells after 72 h incubation. Chronic myeloid leukemia cell line K562 (A) and mantle cell lymphoma cell line Granta (B) were seeded into 96-well plates and incubated overnight under 5% CO2 at 37 °C for 72 h. Next, cells were treated with 3S-C1, 6S-C4, 7S-E1, 11S-E4, 4S-C2, 5S-C3, 18S-C5, 19S-C6, 20S-E5, 21S-E6, 14S-G3, 22S-G5, 23S-G6, 12S-B2, 2S-B3, 27S-B5, 17S-B6, 16S-F3, 24S-F5, 25S-F6, 15S-F, 9S-G1 (100 nM–10 mM), or vehicle for 72 h. Then, 20 μL of MTT solution was added to each well and mixed. After 4 h, the supernatants were removed and 100 μL DMSO was added to each well to dissolve the precipitate. Cell viability was estimated by measuring absorbance at 570 nm using the MultiScan MCC 340 spectrophotometer (Thermo Fisher, Waltham, MA, USA). IC50 values were determined using GraphPad Prism, Ver.7.0. Results are presented as mean ± standard deviation (SD).

Figure 4. The structure of the GR active site interacting with the analogs of synephrine 10S-C2 (C), 13S-G2 (D), and synephrine (B) compared to Dex (A). Molegro Virtual Docker 6.0 software was used for molecular docking. The GR structure (PDB ID: 1P93) was chosen as a target. The target structure was processed automatically by standard procedures of the Molegro Virtual Docker package. Ligand structures were constructed and optimized by molecular dynamic methods in the MMFF94 force field using Avogadro 1.2.0. The MolDock Score was chosen as the scoring function; dexamethasone (CID 5743) served as the reference ligand. Molecular docking was carried out in 40 iterations. MolDock SE was chosen as the docking algorithm following energy minimization and the optimization of hydrogen bonds.

Table 1

The cytotoxic effects of the synephrine derivatives in K562 and Granta cells after 24 and 72 h incubations.

Code	IC50, µM
	K562		Granta
	24 h	72 h	24 h	72 h
4S-C2	N/I	N/I	201 ± 32.1	N/I
8S-E3	120 ± 20.4	N/I	89.1 ± 22.41	N/I
10S-E2	13.1 ± 1.51	N/I	13.0 ± 0.73	N/I
12S-B2	N/I	77.3 ± 34.1	246 ± 6.4	53.1 ± 18.92
13S-G2	184 ± 91.2	36.0 ± 14.0	26.8 ± 1.24	N/I
20S-E5	N/I	31.0 ± 0.4	N/I	N/I
21S-E6	N/I	130 ± 30	725 ± 392.3	80.1 ± 41.73
26S-F2	670 ± 148.0	56.1 ± 0.93	215 ± 71.1	108 ± 14.4

N/I—no inhibition

Table 2

The in silico synephrine derivatives’ affinity for the GR.

Compound	MolDock Score	H-Bond	Compound	MolDock Score	H-Bond
Dex	−146.169	Asn564, Thr739, Tyr735, Gln642, Arg611	19S-C6	−102.139	Asn564, Gln570, Arg611, Leu563
10S-E2	−142.988	Asn564, Thr739, Gln642	9S-G1	−99.2892	Gln570, Arg611
21S-E6	−136.472	Asn564, Thr739, Gln642, Leu732	18S-C5	−99.2424	Asn564, Gln570, Arg611, Met560
8S-E3	−125.395	Asn564, Thr739, Gln642, Leu732	17S-B6	−95.9832	Asn564, Leu732, Cys643
20S-E5	−123.111	Thr739, Gln642	25S-F6	−95.955	Asn564, Leu563, Gln570
7S-E1	−118.911	Thr739, Leu732	5S-C3	−95.2164	Thr739, Cys643, Leu732
13S-G2	−117.383	Asn564, Gln642, Arg611	3S-C1	−94.9736	Thr739, Leu732
11S-E4	−116.985	Thr739, Tyr735, Gln642	6S-C4	−94.1991	Met604, Gln570, Arg611, Leu563
4S-C2	−106.142	Thr739	24S-F5	−93.3857	Asn564, Gln642, Cys643
26S-F2	−105.588	Asn564, Thr739, Leu732	2S-B3	−90.1894	Asn564, Cys643
12S-B2	−103.893	Asn564, Thr739, Gln642	15S-F1	−85.8472	Asn564, Met604
23S-G6	−103.408	Thr739, Gln642, Cys643, Leu732	16S-F3	−89.1399	Asn564, Leu732, Cys643
14S-G3	−102.897	Asn564, Gln570, Arg611, Cys736	27S-B5	−89.0255	Asn564, Gln570, Arg611

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom15010002/s1, Figures S1–S52: copies of NMR spectra; Figure S53: Structure of the GR active site with analogue of synephrine; Table S1: Synephrine derivatives affinity for GR in silico; Table S2: Calculated lipophilicity of structures.

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