Synthesis and Anti-hypoxic Activity Research of Daidzein Derivatives

Xiaohan Liu; Xinru Liu; Wenteng Zheng; Jing Xu; Tingting Chen; Tao Peng; Ruixin Liu; Shuchen Liu; Lin Wang; Shouguo Zhang

doi:10.1248/cpb.c25-00014

Abstract

To search for safe and efficient anti-hypoxia active molecules, 27 derivatives were synthesized by introducing aminoalkyl groups at daidzein’s position-7 and position-8. The structures of these derivatives were confirmed by ¹H-NMR, ¹³C-NMR, and mass spectrometry. The anti-hypoxia activity was evaluated in vitro using a cell hypoxia model established with the AnaeroPack-anaero. The results showed that 9 compounds significantly enhanced cell viability under hypoxic conditions, with compounds 2a, 2b, 4d, 5a, and 5d exhibiting in vitro anti-hypoxia activity significantly superior to daidzein. And the drug-like properties prediction results of the target compounds indicated that compounds 2a, 2b, 4d, 5a, and 5d may also demonstrate favorable pharmacokinetic properties. Further, the anti-hypoxia activity in vivo of these 5 derivatives were evaluated via normobaric hypoxia and hypobaric hypoxia models. The results indicated that all of the 5 compounds extended the survival time of mice under normobaric hypoxia to varying degrees, and they also alleviated oxidative stress damage to the brain and heart of mice under hypobaric hypoxia. Among these, compound 2a demonstrated superior anti-hypoxia activity both in vitro and in vivo compared to daidzein, making it worthy of further study as a potential candidate for an anti-hypoxia drug.

Introduction

Hypoxia is a pathological condition characterized by abnormal changes in tissue metabolism, function, and structure due to inadequate oxygen supply or impaired oxygen-utilization. It is particularly common in high-altitude areas. Hypoxia can lead to cellular damage due to oxygen deficiency, and oxidative stress in tissues and organs serves as the pathophysiological basis for many diseases. Both acute and prolonged exposure to hypoxia can diminish the activity of endogenous antioxidant enzymes, disrupting the delicate balance between the production and elimination of free radicals. When excessive free radicals accumulate and cannot be cleared in time, they can cause damage to various biomolecules and lead to a series of oxidative injuries, such as lipid peroxidation and enzyme inactivation. This is evidenced by an increase in the levels of malondialdehyde (MDA), the product of lipid peroxidation, and a decrease in the activity of antioxidant enzymes, such as glutathione (GSH) and superoxide dismutase (SOD).^1–4) Currently, the primary medicines used to prevent and treat high-altitude hypoxia include acetazolamide (ACE), dexamethasone, ibuprofen, and so on. While these drugs are effective in preventing high-altitude hypoxic diseases, they can also lead to significant adverse reactions.^5–10) Research indicates that various Tibetan medicines, such as Rhodiola, Radix Asteris Tatarici, and Dracocephalum tanguticum Maxim, show considerable potential in treating high-altitude hypoxic encephalopathy. However, their complex compositions pose challenges for research.¹¹⁾ As a result, natural medicines with high effects and low toxicity have become a key focus of scientific research.

Daidzein, also known as 4′, 7-dihydroxy isoflavone (Fig. 1), is an isoflavone compound primarily present in leguminous plants like soybeans and kudzu (Pueraria lobata).¹²⁾ It have displayed an extensive range of bioactivities, such as anti-hypoxic, anti-inflammatory, antioxidant, and anti-cardiovascular disease effects, as well as anti-osteoporosis.^13–18) However, the presence of the isoflavone skeleton in the structure results in poor water and lipid solubility of daidzein, restricting its widespread application.¹⁹⁾ Furthermore, daidzein requires a substantial dosage to exhibit its anti-hypoxia effects. Therefore, it is a significant focus in this research field to use daidzein as a lead compound, modify its chemical structure, and explore derivatives with enhanced pharmacological activity and biological efficacy.

Fig. 1. Daidzein’s Structure

Most drugs contain amine groups, which are functional groups that play a crucial role in their activity and are significant in organic synthesis.^20,21) The incorporation of aminoalkyl group into the structure of isoflavones can significantly improve the water solubility of these compounds. This enhancement is attributed to the presence of lone-pair electrons on nitrogen atoms, which facilitate the formation of intermolecular hydrogen bonds with water. At the same time, by changing the number of carbon atoms in the alkyl chain of the amine alkyl group, the liposolubility of the compounds can be adjusted, and the structural diversity of isoflavones can be increased. The Hofmann alkylation and Mannich reactions are widely utilized for introducing aminoalkyl into isoflavones.^22,23) These 2 methods are cost-effective and simple to implement.

The 7th and 8th positions of daidzein are associated with anti-hypoxia effects.^24,25) Therefore, alkylamine groups were introduced at position-7 of daidzein via the Hofmann alkylation reaction, and aminomethyl groups were introduced to the C-8 position of daidzein via the Mannich reaction. A total of 27 target compounds were synthesized, 20 of which have not been reported in the literature. Their structures were confirmed using ¹H-NMR, ¹³C-NMR, and mass spectrometry. The initial tests on rat H9C2 cardiomyocytes showed that 9 compounds significantly improved cell viability after hypoxia. Furthermore, 5 compounds exhibited better anti-hypoxic activity than daidzein and were chosen for further evaluation under normobaric and hypobaric hypoxia conditions.

Results and Discussion

Chemistry

The synthetic route of the daidzein derivatives was outlined in Chart 1. First, under the catalysis of potassium carbonate, daidzein underwent nucleophilic substitution with 1,3-dibromopropane, 1,4-dibromobutane, and 1,5-dibromopentane to produce intermediates 1a–1c, yielding between 50.00 and 56.00%. Subsequently, compounds 1a–1c were reacted with various secondary amines in ethanol to yield compounds 2a–2f, 3a–3d, and 4a–4h, with yields ranging from 51.20 to 97.50%. Furthermore, daidzein was reacted with different secondary amines and formaldehyde in N,N-dimethylformamide (DMF) to synthesize compounds 5a–5i, with yields ranging from 51.20 to 78.70%. All target compounds were purified by recrystallization or column chromatography, and their structures were confirmed through ¹H-NMR, ¹³C-NMR, and mass spectrometry spectral analysis.

Chart 1. Synthesis Route of Daidzein Derivatives

Reagents and conditions: (i) K₂CO₃, DMF, room temperature (r.t.), 12 h (ii) EtOH, 90°C, 8 h (iii) HCHO, DMF, 65°C, 4–6 h.

Cytotoxicity Assay

The Cell Counting Kit-8 (CCK-8) assay was used to evaluate the cytotoxicity of the 27 compounds, including 2a–2e, 3a–3d, 4a–4h, and 5a–5i, at various concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μM) on rat H9C2 cardiomyocytes after 24 h of treatment, as observed from the data in Table 1. Compounds 3a, 4a, 4b, 4c, and 4e exhibited IC₅₀ values of less than 50 μM, indicating significant toxicity to H9C2 cells. Conversely, compounds 3b, 3c, and 3d showed IC₅₀ values greater than 70 μM, suggesting relatively lower toxicity to H9C2 cells. The other 19 target compounds showed no cytotoxicity at 100 μM. These 19 compounds were selected for further evaluation of their anti-hypoxic activity.

Table 1. IC₅₀ Values of Compounds on H9C2 Cells (μM, n = 3)

Compound	IC₅₀ value	Compound	IC₅₀ value
2a–2f	>100	4b	22.04 ± 0.54
3a	37.70 ± 1.41	4c	25.21 ± 1.46
3b	73.39 ± 2.75	4d	>100
3c	74.06 ± 1.94	4e	50.42 ± 1.02
3d	90.46 ± 2.99	4f–4h	>100
4a	12.56 ± 0.49	5a–5i	>100

In Vitro Anti-hypoxic Activity Evaluation

A hypoxia/reoxygenation model in H9C2 cardiomyocytes was created using the AnaeroPack-anaero method, and the anti-hypoxic activity of the target compounds was evaluated in the model by the CCK-8 assay.^26,27) Based on the results displayed in Table 2, the cell viability values of compounds 2a, 2b, 2d, 4d, 4g, 5a, 5d, 5f, and 5g were notably higher than those of the hypoxia model group (p < 0.05). Notably, compounds 2a, 2b, 4d, 5a, and 5d exhibited significantly higher cell viability values than the daidzein group (p < 0.05). Subsequently, the cell viability values of the 5 compounds were measured at the 5 lower concentrations. As depicted in Fig. 2, at 20 μM, compounds 2a, 2b, and 4d displayed significantly higher cell viability values than daidzein (p < 0.05). At concentrations of 5 and 2.5 μM, compound 2a showed notably higher cell viability values than daidzein (p < 0.05). When the administration concentration was reduced to 1 μM, the cell viability of the compounds showed no significant difference compared to daidzein. Neither daidzein nor the 5 compounds exhibited any discernible dose-dependency at various doses.

Table 2. Cell Viability of the Compounds at of 25 μM

Group	Cell viability (%)	Group	Cell viability (%)
Model	37.31 ± 2.71	4h	36.85 ± 1.20
Daidzein	67.00 ± 2.86****	5a	89.62 ± 7.51****^,###
2a	98.25 ± 6.41****^,####	5b	31.08 ± 6.63
2b	83.05 ± 2.90****^,#	5c	34.01 ± 3.49
2c	36.77 ± 6.73	5d	83.71 ± 4.25****^,#
2d	53.62 ± 4.79*	5e	36.08 ± 5.05
2e	35.71 ± 6.68	5f	58.42 ± 3.91***
2f	37.31 ± 4.87	5g	56.50 ± 3.22**
4d	95.58 ± 2.05****^,####	5h	36.12 ± 3.73
4f	37.05 ± 0.90	5i	39.22 ± 6.48
4g	62.17 ± 1.05***

Compared to the model group: ****p < 0.0001, ***p < 0.001, *p < 0.05; compared to the positive drug daidzein: ^####p < 0.0001, ^###p < 0.001, ^#p < 0.05.

Fig. 2. Cell Viability of the Compounds at Different Concentrations

Data were expressed as the mean ± standard deviation (S.D.) of 3 independent experiments. Compared to the positive drug daidzein: ***p < 0.001, **p < 0.01, and *p < 0.05.

Prediction for the Drug-Like Properties of Target Compounds

Log P value refers to the logarithmic ratio of the distribution coefficient of a substance in n-octanol (oil) and water, reflecting the distribution of substances between these 2 phases. A higher Log P value indicates that the substance is more lipophilic, while a lower Log P value suggests greater hydrophilicity. The 19 compounds with low toxicity in the cytotoxicity assay were predicted using the ALOGPS 2.1 and SwissADME websites. The results were presented in Tables 3 and 4. The Log P values predicted by ALOGPS 2.1 suggested that the water solubility or lipid solubility of the compounds may be enhanced. Among the compounds exhibiting higher cell viability than daidzein, the Log P values of compounds 2a, 2b, 4d, and 5d were greater than that of daidzein, indicating that they had a potential improvement in lipid solubility. Conversely, the Log P value of compound 5a was lower than that of daidzein, suggesting an enhancement in water solubility of 5a.

Table 3. The Compounds Log P Values Predicted by ALOGPS 2.1

Group	Log P	Group	Log P
Daidzein	3.30	4h	3.42
2a	3.36	5a	2.40
2b	3.50	5b	3.40
2c	4.28	5c	4.00
2d	2.94	5d	4.01
2e	3.41	5e	4.86
2f	2.58	5f	4.52
4d	4.16	5g	2.43
4f	3.81	5h	3.43
4g	4.37	5i	2.59

Table 4. Prediction Results for the Compounds by SwissADME

Compounds	MW	Rotatable bonds	H-bond acceptors	H-bond donors	cLog P	BBB permeant
Daidzein	254.24	1	4	2	2.24	Yes
2a	323.43	6	3	1	3.85	Yes
2b	381.42	6	6	1	2.86	Yes
2c	379.45	6	5	1	3.72	Yes
2d	394.46	6	6	1	2.80	Yes
2e	408.49	7	6	1	3.12	Yes
2f	424.49	8	7	2	2.38	No
4d	409.47	8	6	1	3.48	Yes
4f	422.52	8	6	1	3.45	Yes
4g	436.54	9	6	1	3.75	Yes
4h	452.54	10	7	2	3.03	No
5a	311.33	3	5	2	2.29	Yes
5b	339.39	5	5	2	3.00	Yes
5c	367.44	7	5	2	3.68	Yes
5d	367.44	5	5	2	3.50	Yes
5e	395.49	9	5	2	4.32	No
5f	395.49	7	5	2	4.17	No
5g	353.37	3	6	2	2.15	No
5h	351.40	3	5	2	3.00	Yes
5i	380.44	4	6	2	2.44	No

cLog P: octanol–water partition coefficients.

SwissADEM predicted that the number of H-bond receptors for daidzein and its derivatives was fewer than 10, the number of H-bond donors was fewer than 5, and the cLog P value was below 5. These findings aligned with the relevant values specified by Lipinski’s rules and other guidelines. Compared with daidzein, most compounds showed higher cLog P values, suggesting their lipid solubility may be enhanced. Compounds 2a, 2b, 4d, 5a, and 5d, which exhibited superior activity compared to daidzein in the in vitro anti-hypoxic activity evaluation, had the potential to penetrate the blood–brain barrier. These results indicated that compounds 2a, 2b, 4d, 5a, and 5d not only possess significant anti-hypoxia activity and cellular safety but may also demonstrate favorable pharmacokinetic properties.

Structure–Activity Relationship Analysis of the Target Compounds

When a 3-aminopropyl group was linked to the position-7 phenolic hydroxyl group of daidzein, the activity of the compound decreased as the number of amino carbon atoms increased. Compounds 2a and 2b exhibited superior efficacy compared to daidzein, enhancing cell viability by 46.63 and 23.96%, respectively. The structure with a 4-aminobutyl group showed increased toxicity. When linked to 5-aminopentyl groups, linear amines demonstrate greater cytotoxicity, whereas the activity of compounds containing cyclic amines decreased as the number of carbon atoms increased. When the amino methyl groups were introduced to the position-8 of daidzein, compounds with linear amines demonstrated superior cell viability compared to those with cyclic amines, with compounds 5a and 5d exhibiting enhanced activity over daidzein, boosting cell viability by 33.76 and 24.94%, respectively. In conclusion, attaching a propyl amino group to the position-7 phenolic hydroxyl group of daidzein enhanced the anti-hypoxia efficacy, and introducing linear amino methyl groups to position-8 was more advantageous than cyclic amino methyl groups.

Study on Anti-hypoxia Activity under Normal Pressure and Anoxic Conditions

By the close hypoxia method, an experiment on the anti-hypoxia activity under normal pressure was conducted on 5 compounds that outperformed the positive drug.^28,29) The results were detailed in Table 5. Compared to the model group, compounds 2a, 2b, 4d, 5a, and 5d significantly increased the survival time of hypoxic mice, with extension rates of 34.72, 52.55, 55.82, 36.47, and 55.12%, respectively (p < 0.05). Notably, compared to the positive drug daidzein, compounds 2b, 4d, and 5d extended the survival time of mice by 21.52, 24.13, and 23.56%, respectively (p < 0.05).

Table 5. Effects of Compounds on the Survival Time of Mice under Normobaric Hypoxia

Group	Dosage (mmol/kg)	Survival time (min)	Survival time extension rate (%)
Control	—	26.87 ± 1.83	—
ACE	1.12	36.05 ± 4.15**	34.16
Daidzein	0.14	33.73 ± 3.88*	25.53
2a	0.14	36.20 ± 4.53**	34.72
2b	0.14	40.99 ± 5.42****^,#	52.55
4d	0.14	41.87 ± 4.73****^,#	55.82
5a	0.14	36.67 ± 3.53**	36.47
5d	0.14	41.68 ± 7.24****^,#	55.12

Data were expressed as the mean ± S.D., n = 8. Compared with the control group, ****p < 0.0001, **p < 0.01, *p < 0.05. Compared with the daidzein group, ^#p < 0.05.

The Protective Effect of the Target Compound on Mice Exposed to Hypobaric Hypoxia

The brain tissue contains a high concentration of unsaturated fatty acids and exhibits significant oxygen consumption. The heart is responsible for approximately 18% of the body’s total oxygen consumption, characterized by a high metabolic rate and substantial oxygen demand, yet it has limited oxygen reserves. Both the brain and heart are highly sensitive to hypoxia.^30–32) In this study, a low-pressure oxygen chamber was utilized to simulate an altitude of 7000 m. Following 24 h of hypoxia exposure in the chamber, brain and heart tissues were collected from each group of mice. Oxidative stress markers were measured in accordance with reagent kit instructions, and pathological changes were observed. The assessments of oxidative stress markers showed that (Figs. 3 and 4), in comparison to the daidzein group, MDA levels in brain tissue were diminished by 42.52, 27.62, and 53.09% in the groups administered compounds 2a, 2b, and 5d, respectively (p < 0.001). The SOD activity in the groups 2a, 4d, 5a, and 5d increased by 18.23, 20.30, 23.17, and 24.49%, respectively (p < 0.01). The GSH levels in the groups 2a, 4d, and 5a elevated by 20.61, 13.46 and 29.44%, respectively (p < 0.0001). The MDA content in cardiac tissue of group 2a was diminished by 22.49% (p < 0.05). The SOD activity in groups 2a, 4d, 5a, and 5d elevated by 23.82, 21.97, 23.06 and 38.87%, respectively (p < 0.05). The GSH levels in groups 2a and 5a elevated by 33.04 and 27.81%, respectively (p < 0.001). The aforementioned results demonstrated that compounds 2a and 5a provided substantial protective benefits in hypoxic mice, exceeding those of daidzein.

Fig. 3. Comparison of MDA and GSH Content and SOD Activity in Brain Tissue of Mice in Each Group

Data were expressed as the mean ± standard deviation (S.D.), n = 8. Compared with the model group: ****p < 0.0001 and ***p < 0.001. Compared with the daidzein group: ^####p < 0.0001,^###p < 0.001, and ^##p < 0.01.

Fig. 4. Comparison of MDA and GSH Content and SOD Activity in Heart Tissue of Mice in Each Group

Data were expressed as the mean ± standard deviation (S.D.), n = 8. Compared with the model group: ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05. Compared with the daidzein group: ^####p < 0.0001, ^###p < 0.001, and ^#p < 0.05.

The staining results of mouse brain and heart tissues indicated that the brain tissue architecture in the control/normal group (Fig. 5A) had complete structure, exhibiting densely organized cells in the hippocampus area and consistent staining, with sporadic intercellular vacuoles. In comparison to the control group, the model/hypoxia group (Fig. 5B) demonstrated a more disordered cellular arrangement in the hippocampus area, an elevation in intercellular vacuoles, and nuclear pyknosis in some neurons. Relative to the model group, the treatment groups (Figs. 5C–5I) exhibited differing levels of recovery in the hippocampus region, characterized by a decrease in intercellular vacuoles and a more organized cellular structure. The myocardial cellular architecture in the control/normal group (Fig. 6A) had complete structure, exhibiting orderly and tightly packed myocardial fibers, consistent nuclear morphology, and uniformly stained cytoplasm. Compared to the control group, the model/hypoxia group (Fig. 6B) exhibited disorganized cardiac fiber architecture, cytoplasmic contraction, and atypical nuclear morphology. In comparison to the model group, the treatment groups (Figs. 6C–6I) exhibited a propensity for a more organized cardiac fiber arrangement, and the morphology of myocardial tissue had shown some improvement.

Fig. 5. HE Staining Results of Mouse Brain Tissue

(A) Control group; (B) model group; (C) ACE group; (D) daidzein group; (E) 2a group; (F) 2b group; (G) 4d group; (H) 5a group; and (I) 5d group.

Fig. 6. HE Staining Results of Mouse Myocardial Tissue

(A) Control group; (B) model group; (C) ACE group; (D) daidzein group; (E) 2a group; (F) 2b group; (G) 4d group; (H) 5a group; and (I) 5d group.

Conclusion

In conclusion, to obtain safe and efficient anti-hypoxia active molecules, we synthesized a total of 27 derivatives by including aminoalkyl groups at position-7 and position-8 of daidzein. The anti-hypoxia efficacy of these compounds was evaluated in vitro and in vivo, including cellular assays, normobaric hypoxia, and hypobaric hypoxia evaluations. Results from the in vitro study provided evidence that 9 compounds could improve cell viability in hypoxic conditions. Compounds 2a, 2b, 4d, 5a, and 5d showed superior efficacy compared to daidzein. Additionally, results from the in vivo study indicated that all 5 compounds prolonged the survival duration of mice under normobaric hypoxia to varying extents and mitigated hypoxia-induced cerebral and cardiac injury in mice. Based on the in vitro and in vivo results, compound 2a exhibited significantly greater anti-hypoxia activity compared to daidzein. However, its effects were only observed in phenotypic assays, and the specific mechanism underlying its anti-hypoxia activity remains unclear, necessitating further investigation and validation.

Experimental

Laboratory Animals

The KM mice (half male and female, aged 6–8 weeks old; weighing 18–22 g) were acquired from Beijing Hua Fukang Biological Technology Co., Ltd. (Beijing, China; Production Licence No. SCXK (Jing) 2024-0003).

Ethical Statement

All animal procedures were performed according to a protocol approved by the Committee on the Ethics of Animal Experiments of the Animal Center at the Beijing Institute of Radiation Medicine (IACUC-DWZX-2024-P548). All operations and experimental techniques adhered to the “Regulations on the Administration of Laboratory Animals,” and all mice were exclusively utilized for animal investigations.

Materials and Instruments

Daidzein, ACE, 1,4-dibromobutane, and 1,5-dibromopentane were acquired from Anhui Zesheng Technology Co., Ltd. (Anhui, China); 1,3-dibromopropane was obtained from Beijing MREDA Technology Co., Ltd. (Beijing, China); and DMF was sourced from Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China). Silica gel for column chromatography (200–300 mesh) was procured from Qingdao Ocean Chemical Plant (Qingdao, China). Dichloromethane (DCM), methanol (MeOH), and other reagents were purchased from commercial sources and used without further purification. Assay kits for MDA, GSH, and SOD were acquired from Beijing Solarbio Technology Co., Ltd. (Beijing, China).

A RE-52A rotary evaporator from Shanghai Yarong Biochemical Instrument Co., Ltd. (Shanghai, China), a ZF-7A UV analysis cabinet from Shanghai Jiapeng Technology Co., Ltd. (Shanghai, China), a DZF6020 vacuum drying oven from Shanghai Yiheng Scientific Instrument Co., Ltd. (Shanghai, China), and an LP-1500 low-pressure oxygen chamber from Shanghai Yuyan Scientific Instrument Co., Ltd. (Shanghai, China). A tissue grinder was obtained from Beijing Xianfeng Scientific Instrument Co., Ltd. (Beijing, China), a KDC-120HR high-speed centrifuge was purchased from Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd. (Hefei, China), and a microplate reader was procured from Thermo Fisher Scientific, Massachusetts, U.S.A.

Synthesis of Intermediates (1a–1c)

Daidzein (2.4 g, 9.6 mmol) and anhydrous potassium carbonate (0.8 g, 6.0 mmol) were dissolved in 70 mL DMF and agitated at ambient temperature for 20 min. Then, 1,3-dibromopropane (4.0 g, 20 mmol) was gradually added and kept stirring at ambient temperature for 12 h, with TLC used to monitor the reaction progress. The reaction mixture was poured into 400 mL ice water, and the pH was adjusted to 3–4 using 10% HCl. The mixture was then filtered to gain the crude product, which was purified using silica gel column chromatography with a solvent ratio of V (DCM) : V (MeOH) = 80 : 1 to yield intermediate 1a, a white solid. Intermediates 1b and 1c were synthesized by the same method.

7-(3-Bromopropoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (1a)

White solid. Yield 52.80%. m.p. 168.0–171.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.38 (s, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.42–7.39 (m, 2H), 7.19 (d, J = 2.4 Hz, 1H), 7.10 (dd, J = 8.9, 2.4 Hz, 1H), 6.84–6.79 (m, 2H), 4.25 (t, J = 6.0 Hz, 2H), 3.70 (t, J = 6.5 Hz, 2H), 2.31 (q, J = 6.3 Hz, 2H).¹³C-NMR (101 MHz, DMSO-d₆) δ: 174.66 (s), 162.64 (s), 157.32 (s), 157.27 (s), 153.10 (s), 130.03 (s), 126.99 (s), 123.68 (s), 122.32 (s), 117.71 (s), 114.94 (s), 114.85 (s), 101.12 (s), 66.27 (s), 31.55 (s), 30.94 (s). MS (electrospray ionization [ESI]) m/z = 377.01 (M + H)^+.

Based on the above ¹H-NMR result, the hydrogen signal of the –OCH₂ (δ 4.25) in 1a was selectively excited through 1D nuclear Overhauser effect (1D-NOE) NMR test, and an enhancement was observed in the signal of H-6. However, no signal enhancement was detected at H-3′ or H-5′. According to the principles of NOE, it was inferred that –OCH₂CH₂CH₂Br in 1a was attached to position-7 of daidzein, rather than position-4′. This suggested that during the preparation of 1a, the position-7 hydroxyl of daidzein participated in the reaction with priority over position-4′. Based on the analysis of the results presented above, we can infer that under these reaction conditions, bromoalkane reacts selectively with the 7-hydroxyl group.

7-(4-Bromobutoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (1b)

White solid. Yield 50.90%. m.p. 174.0–176.2°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.43–7.37 (m, 2H), 7.16 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 8.9, 2.4 Hz, 1H), 6.84–6.78 (m, 2H), 4.17 (t, J = 6.3 Hz, 2H), 3.63 (t, J = 6.6 Hz, 2H), 2.04–1.95 (m, 2H), 1.90 (dq, J = 9.4, 6.4 Hz, 2H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 175.15 (s), 163.34 (s), 157.84 (s), 157.69 (s), 153.58 (s), 130.52 (s), 127.40 (s), 124.14 (s), 122.83 (s), 118.03 (s), 115.42 (s), 101.48 (s), 68.07 (s), 35.21 (s), 29.43 (s), 27.58 (s). MS (ESI) m/z: 391.03 (M + H)⁺.

7-((5-Bromopentyl)oxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (1c)

White solid. Yield 55.60%. m.p. 141.3–143.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H) 7.42–7.38 (m, 2H), 7.15 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 6.83–6.79 (m, 2H), 4.14 (t, J = 6.4 Hz, 2H), 3.58 (t, J = 6.7 Hz, 2H), 1.89 (p, J = 6.9 Hz, 2H), 1.80 (p, J = 6.7 Hz, 2H), 1.60–1.53 (m, 2H). ¹³C-NMR (151 MHz, DMSO) δ: 175.15 (s), 163.45 (s), 157.87 (s), 157.69 (s), 153.57 (s), 130.52 (s), 127.38 (s), 124.14 (s), 122.84 (s), 117.99 (s), 115.43 (s), 101.45 (s), 68.76 (s), 35.51 (s), 32.36 (s), 28.00 (s), 24.68 (s). MS (ESI) m/z: 403.0540 (M + H)^+.

Synthesis of Compounds (2a–4h)

Compound 1a (0.37 g, 1 mmol) was added to 8 mL of absolute ethanol and stirred under reflux until the solid was completely dissolved. Then, a 40% aqueous dimethylamine solution (0.225 g, 5 mmol) was gradually added and refluxed at 90°C for a duration of 8 h, with TLC observing the reaction progress. The reaction mixture was concentrated under reduced pressure to gain the crude product, which was purified using silica gel column chromatography with a solvent ratio of V(DCM) : V(MeOH) = 20 : 1 to yield compound 2a. Compounds 2b–4h were synthesized using the same methodology.

7-(3-(Dimethylamino)propoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (2a)

Pale yellow solid. Yield 89.70%. m.p. 242.6–243.9°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 9.56 (s, 1H), 8.40 (s, 1H), 8.05 (d, J = 8.9 Hz, 1H), 7.48–7.36 (m, 2H), 7.17 (d, J = 2.4 Hz, 1H), 7.09 (dd, J = 9.0, 2.4 Hz, 1H), 6.90–6.79 (m, 2H), 4.22 (t, J = 6.1 Hz, 2H), 3.21 (t, J = 7.9 Hz, 2H), 2.79 (s, 6H), 2.21–2.11 (m, 2H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 175.15 (s), 163.01 (s), 157.79 (s), 157.72 (s), 153.66 (s), 130.52 (s), 127.48 (s), 124.18 (s), 122.76 (s), 118.22 (s), 115.45 (s), 115.41 (s),101.62 (s), 66.28 (s), 54.61 (s), 42.97 (s), 40.53 (s), 24.35 (s). MS (ESI) m/z: 340.16 (M + H)⁺.

3-(4-Hydroxyphenyl)-7-(3-morpholinopropoxy)-4H-chromen-4-one(2b)

Pale yellow solid. Yield 55.60%. m.p. 229.0–231.9°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 9.55 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.44–7.38 (m, 2H), 7.15 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 6.85–6.79 (m, 2H), 4.17 (t, J = 6.4 Hz, 2H), 3.59 (t, J = 4.6 Hz, 4H), 2.47–2.31 (m, 6H), 1.93 (p, J = 6.7 Hz, 2H).¹³C-NMR (101 MHz, DMSO-d₆) δ: 174.66 (s), 162.97 (s), 157.36 (s), 157.20 (s), 153.07 (s), 130.02 (s), 126.92 (s), 123.66 (s), 122.34 (s), 117.52 (s), 114.94 (s), 100.99 (s), 66.76 (s), 66.15 (s), 54.63 (s), 53.31 (s), 25.58 (s). MS (ESI) m/z: 382.16 (M + H)⁺.

3-(4-Hydroxyphenyl)-7-(3-(piperidin-1-yl)propoxy)-4H-chromen-4-one (2c)

Pale yellow solid. Yield 82.40%. m.p. 247.8–248.5°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.55 (s, 1H), 8.40 (s, 1H), 8.06 (d, J = 8.9 Hz, 1H), 7.43–7.38 (m, 2H), 7.18 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 8.9, 2.4 Hz, 1H), 6.85–6.79 (m, 2H), 4.23 (t, J = 6.0 Hz, 2H), 3.50 (d, J = 11.8 Hz, 2H), 3.23 (dt, J = 10.1, 5.3 Hz, 2H), 2.93 (q, J = 11.3 Hz, 2H), 2.20 (dq, J = 11.5, 6.0 Hz, 2H), 1.84 (d, J = 14.2 Hz, 2H), 1.69 (p, J = 13.6, 12.1 Hz, 3H), 1.45–1.37 (m, 1H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 175.15 (s), 162.98 (s), 157.80 (s), 157.73 (s), 153.67 (s), 130.52 (s), 127.51 (s), 124.19 (s), 122.75 (s), 118.24 (s), 115.45 (s), 115.41 (s), 101.63 (s), 66.34 (s), 53.71 (s), 52.66 (s), 23.67 (s), 23.03 (s), 21.75 (s). MS (ESI) m/z: 380.21 (M + H)⁺.

3-(4-Hydroxyphenyl)-7-(3-(4-methylpiperazin-1-yl)propoxy)-4H-chromen-4-one (2d)

Pale yellow solid. Yield 51.20%. m.p. 214.3–214.7°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 9.57 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.14 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.8, 2.4 Hz, 1H), 6.82 (d, J = 8.1 Hz, 2H), 4.15 (t, J = 6.3 Hz, 2H), 2.81 (d, J = 79.5 Hz, 8H), 2.44 (t, J = 7.2 Hz, 2H), 2.18 (s, 3H), 1.91 (p, J = 6.8 Hz, 2H).¹³C-NMR (151 MHz, DMSO-d₆) δ: 175.16 (s), 163.47 (s), 157.86 (s), 157.69 (s), 153.58 (s), 130.53 (s), 127.41 (s), 124.14 (s), 122.83 (s), 117.99 (s), 115.42 (s), 115.40 (s), 101.47 (s), 67.31 (s), 55.18 (s), 54.67 (s), 53.13 (s), 46.15 (s), 26.45 (s). MS (ESI) m/z: 395.20 (M + H)⁺.

7-(3-(4-Ethylpiperazin-1-yl)propoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (2e)

White solid. Yield 74.60%. m.p. 185.0–187.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.42–7.38 (m, 2H), 7.14 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 6.84–6.79 (m, 2H), 4.16 (t, J = 6.3 Hz, 2H), 2.45–2.24 (m, 10H), 1.91 (p, J = 6.6 Hz, 2H), 0.98 (t, J = 7.2 Hz, 3H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 175.15 (s), 163.47 (s), 157.85 (s), 157.69 (s), 153.55 (s), 130.52 (s), 127.40 (s), 124.14 (s), 122.83 (s), 117.98 (s), 115.42 (s), 115.38 (s), 101.45 (s), 67.32 (s), 54.74 (s), 53.34 (s), 52.90 (s), 52.09 (s), 26.46 (s), 12.51 (s). MS (ESI) m/z: 409.21 (M + H)⁺.

7-(3-(4-(2-Hydroxyethyl)piperazin-1-yl)propoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (2f)

White solid. Yield 62.40%. m.p. 218.1–219.5°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 9.55 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 2.3 Hz, 1H), 7.07 (dd, J = 8.9, 2.3 Hz, 1H), 6.82 (d, J = 8.5 Hz, 2H), 4.55–4.36 (m, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.50 (d, J = 6.4 Hz, 2H), 2.55 (s, 2H), 2.48–2.22 (m, 8H), 1.92 (p, J = 6.8 Hz, 2H). ¹³C-NMR (101 MHz, DMSO-d₆) δ: 174.66 (s), 162.97 (s), 157.35 (s), 157.21 (s), 153.06 (s), 130.02 (s), 126.91 (s), 123.65 (s), 122.34 (s), 117.51 (s), 114.94 (s), 114.88 (s), 100.98 (s), 66.81 (s), 60.11 (s), 58.31 (s), 54.19 (s), 53.07 (s), 52.61 (s), 25.89 (s). MS (ESI) m/z: 425.21 (M + H)⁺.

7-(4-(Dimethylamino)butoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (3a)

Pale yellow solid. Yield 86.60%. m.p. 238.3–238.9°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 9.56 (s, 1H), 8.38 (d, J = 1.8 Hz, 1H), 8.03 (dd, J = 8.9, 2.1 Hz, 1H), 7.44–7.35 (m, 2H), 7.20–7.06 (m, 2H), 6.83 (d, J = 8.5 Hz, 2H), 4.22–4.10 (m, 2H), 3.14 (d, J = 7.6 Hz, 2H), 2.78 (s, 6H), 2.03–1.73 (m, 4H). ¹³C-NMR (101 MHz, DMSO-d₆) δ: 174.67 (s), 162.78 (s), 157.34 (s) 157.21 (s), 153.10 (s), 130.01 (s), 126.93 (s), 123.67 (s), 122.30 (s), 117.59 (s), 114.96 (s), 101.04 (s), 67.76 (s), 56.24 (s), 42.16 (s), 25.42 (s), 20.63 (s). MS (ESI) m/z: 354.18 (M + H)⁺.

7-(4-(Diethylamino)butoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (3b)

White solid. Yield 52.90%. m.p. 224.1–224.8°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.55 (s, 1H), 8.39 (s, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.43–7.38 (m, 2H), 7.17 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 8.9, 2.4 Hz, 1H), 6.84–6.79 (m, 2H), 4.19 (t, J = 5.9 Hz, 2H), 3.19–3.10 (m, 6H), 1.83 (p, J = 6.8 Hz, 4H), 1.20 (t, J = 7.2 Hz, 6H). ¹³C-NMR (101 MHz, DMSO-d₆) δ: 174.66 (s), 162.78 (s), 157.35 (s), 157.22 (s), 153.10 (s), 130.01 (s), 126.95 (s), 123.67 (s), 122.30 (s), 117.59 (s), 114.96 (s), 101.05 (s), 67.79 (s), 50.39 (s), 46.31 (s), 25.51 (s), 20.03 (s), 8.51 (s). MS (ESI) m/z: 382.21 (M + H)⁺.

3-(4-Hydroxyphenyl)-7-(4-(piperidin-1-yl)butoxy)-4H-chromen-4-one (3c)

Pale yellow solid. Yield 93.00%. m.p. 208.5–211.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.38 (s, 1H), 7.42–7.38 (m, 2H), 7.15 (d, J = 2.3 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 6.84–6.80 (m, 2H), 4.16 (s, 2H), 3.10 (s, 2H), 2.30 (s, 4H), 1.78 (s, 4H), 1.51 (s, 6H). ¹³C-NMR (101 MHz, DMSO) δ: 174.66 (s), 162.79 (s), 157.34 (s), 157.22 (s), 153.09 (s), 130.01 (s), 126.94 (s), 123.67 (s), 122.30 (s), 117.59 (s), 114.96 (s), 114.93 (s), 101.03 (s), 67.79 (s), 55.49 (s), 52.05 (s), 25.62 (s), 22.49 (s), 21.35 (s), 20.19 (s). MS (ESI) m/z: 394.19 (M + H)⁺.

7-(4-(4-Ethylpiperazin-1-yl)butoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (3d)

Pale yellow solid. Yield 67.80%. m.p. 132.0–133.0°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 9.60 (s, 1H), 8.36 (s, 1H), 8.01 (d, J = 8.9 Hz, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.22–7.02 (m, 2H), 6.82 (d, J = 8.2 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H), 2.50 (s, 4H), 2.32 (t, J = 7.8 Hz, 6H), 1.76 (p, J = 7.0 Hz, 2H), 1.58 (p, J = 7.4 Hz, 2H), 0.98 (t, J = 7.2 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ: 174.65 (s), 162.97 (s), 157.36 (s), 157.21 (s), 152.98 (s), 130.00 (s), 126.87 (s), 123.66 (s), 122.33 (s), 117.46 (s), 114.94 (s), 114.90 (s), 100.90 (s), 68.29 (s), 57.20 (s), 52.59 (s), 52.29 (s), 51.55 (s), 26.27 (s), 22.57 (s), 11.84 (s). MS (ESI) m/z: 423.24 (M + H)⁺.

7-((5-(Dimethylamino)pentyl)oxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (4a)

Pale yellow solid. Yield 83.80%. m.p. 243.0–246.0°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 9.56 (s, 1H), 8.38 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.47–7.36 (m, 2H), 7.20–7.03 (m, 2H), 6.88–6.79 (m, 2H), 4.15 (t, J = 6.4 Hz, 2H), 3.13–3.02 (m, 2H), 2.77 (s, 6H), 1.80 (p, J = 6.6 Hz, 2H), 1.76–1.66 (m, 2H), 1.47 (p, J = 7.8 Hz, 2H). ¹³C-NMR (101 MHz, DMSO) δ: 174.66 (s), 162.92 (s), 157.37 (s), 157.21 (s), 153.08 (s), 130.01 (s), 126.93 (s), 123.66 (s), 122.31 (s), 117.53 (s), 114.95 (s), 100.98 (s), 68.12 (s), 56.45 (s), 42.16 (s), 27.79 (s), 23.37 (s), 22.45 (s). MS (ESI) m/z: 368.18 (M + H)⁺.

7-((5-(Diethylamino)pentyl)oxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (4b)

Pale yellow solid. Yield 77.40%. m.p. 215.0–217.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.55 (s, 1H), 8.38 (s, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.43–7.38 (m, 2H), 7.16 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 6.84–6.80 (m, 2H), 4.15 (t, J = 6.3 Hz, 2H), 3.17–3.02 (m, 6H), 1.85–1.78 (m, 2H), 1.74–1.67 (m, 2H), 1.49 (h, J = 7.5, 6.6 Hz, 2H), 1.20 (t, J = 7.2 Hz, 6H). ¹³C-NMR (151 MHz, DMSO) δ: 175.15 (s), 163.41 (s), 157.86 (s), 157.71 (s), 153.60 (s), 130.52 (s), 127.43 (s), 124.15 (s), 122.80 (s), 118.01 (s), 115.44 (s), 115.42 (s), 101.46 (s), 68.67 (s), 51.14 (s), 46.71 (s), 28.35 (s), 23.32 (s), 23.16 (s), 9.03 (s). MS (ESI) m/z: 396.23 (M + H)⁺.

7-((5-(Dipropylamino)pentyl)oxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (4c)

Pale yellow solid. Yield 90.40%. m.p. 185.0–187.8°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.38 (s, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.42–7.38 (m, 2H), 7.16 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 6.84–6.80 (m, 2H), 4.16 (t, J = 6.3 Hz, 2H), 3.08 (dt, J = 9.9, 4.8 Hz, 2H), 3.02 (dt, J = 11.9, 4.8 Hz, 4H), 1.85–1.79 (m, 2H), 1.74–1.68 (m, 2H), 1.68–1.59 (m, 4H), 1.48 (p, J = 7.7 Hz, 2H), 0.91 (t, J = 7.3 Hz, 6H). ¹³C-NMR (151 MHz, DMSO) δ: 175.15 (s), 163.41 (s), 157.86 (s), 157.71 (s), 153.60 (s), 130.51 (s), 127.43 (s), 124.16 (s), 122.80 (s), 118.02 (s), 115.44, 115.41, 101.47, 68.67, 53.92, 52.37, 28.34 (s), 23.15 (s), 19.46 (s), 17.08 (s), 11.42 (s). MS (ESI) m/z: 424.25 (M + H)⁺.

3-(4-Hydroxyphenyl)-7-((5-morpholinopentyl)oxy)-4H-chromen-4-one (4d)

White solid. Yield 84.20%. m.p. 227.9–229.5°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.42–7.37 (m, 2H), 7.15 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 6.83–6.79 (m, 2H), 4.13 (t, J = 6.5 Hz, 2H), 3.58 (d, J = 6.7 Hz, 4H), 2.32 (s, 4H), 1.79 (p, J = 6.7 Hz, 2H), 1.60–1.41 (m, 4H). ¹³C-NMR (151 MHz, pyridine-d5) δ: 176.61 (s), 164.68 (s), 160.03 (s), 159.18 (s), 153.64 (s), 150.98 (s), 131.93 (s), 126.13 (s), 124.71 (s), 124.58 (s), 124.42 (s), 119.80 (s), 117.15 (s), 116.09 (s), 102.23 (s), 69.85 (s), 67.85 (s), 59.68 (s), 54.90 (s), 30.02 (s), 27.20 (s), 24.92 (s). MS (ESI) m/z: 410.20 (M + H)⁺.

3-(4-Hydroxyphenyl)-7-((5-(piperidin-1-yl)pentyl)oxy)-4H-chromen-4-one (4e)

Pale yellow solid. Yield 97.50%. m.p. 249.0–251.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.38 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.43–7.37 (m, 2H), 7.15 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 6.85–6.79 (m, 2H), 4.14 (t, J = 6.4 Hz, 2H), 3.32 (s, 4H), 1.80 (p, J = 6.7 Hz, 2H), 1.72–1.27 (m, 10H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 175.15 (s), 163.45 (s), 157.87 (s), 157.70 (s), 153.59 (s), 130.52 (s), 127.41 (s), 124.15 (s), 122.81 (s), 117.99 (s), 115.43 (s), 115.41 (s), 101.45 (s), 68.71 (s), 56.49 (s), 28.51 (s), 23.46 (s), 19.03 (s). MS (ESI) m/z: 408.23 (M + H)⁺.

3-(4-Hydroxyphenyl)-7-((5-(4-methylpiperazin-1-yl)pentyl)oxy)-4H-chromen-4-one (4f)

Pale yellow solid. Yield 75.90%. m.p. 169.0–170.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.36 (s, 1H), 8.01 (d, J = 8.9 Hz, 1H), 7.43–7.36 (m, 2H), 7.13 (d, J = 2.3 Hz, 1H), 7.06 (dd, J = 8.9, 2.3 Hz, 1H), 6.84–6.79 (m, 2H), 4.12 (t, J = 6.5 Hz, 2H), 3.44 (qd, J = 7.0, 4.9 Hz, 2H), 2.26 (t, J = 7.1 Hz, 8H), 2.13 (s, 3H), 1.77 (p, J = 6.7 Hz, 2H), 1.51–1.39 (m, 4H). ¹³C-NMR (101 MHz, DMSO-d₆) δ: 174.66 (s), 163.02 (s), 157.38 (s), 157.20 (s), 153.04 (s), 130.02 (s), 126.87 (s), 123.65 (s), 122.35 (s), 117.46 (s), 114.93 (s), 100.94 (s), 68.42 (s), 57.71 (s), 54.75 (s), 52.69 (s), 45.72 (s), 30.75 (s), 28.28 (s), 25.96 (s). MS (ESI) m/z: 423.24 (M + H)⁺.

7-((5-(4-Ethylpiperazin-1-yl)pentyl)oxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (4g)

Pale yellow solid. Yield 91.30%. m.p. 180.0–181.4°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.43–7.36 (m, 2H), 7.14 (d, J = 2.4 Hz, 1H), 7.06 (dd, J = 8.9, 2.4 Hz, 1H), 6.83–6.78 (m, 2H), 4.12 (t, J = 6.5 Hz, 2H), 3.22–2.99 (m, 2H), 2.33 (s, 10H), 1.78 (p, J = 6.7 Hz, 2H), 1.50 (p, J = 7.1 Hz, 2H), 1.47–1.39 (m, 2H), 1.00 (td, J = 7.3, 3.2 Hz, 3H). ¹³C-NMR(151 MHz, DMSO) δ: 175.15 (s), 163.49 (s), 157.87 (s), 157.70 (s), 153.57 (s), 130.51 (s), 127.38 (s), 124.14 (s), 122.82 (s), 117.96 (s), 115.43 (s), 101.43 (s), 68.86 (s), 57.72 (s), 51.78 (s), 28.70 (s), 26.04 (s), 23.75 (s), 11.77 (s). MS (ESI) m/z: 437.27 (M + H)⁺.

7-((5-(4-(2-Hydroxyethyl)piperazin-1-yl)pentyl)oxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (4h)

White solid. Yield 73.10%. m.p. 196.0–198.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.54 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.43–7.37 (m, 2H), 7.14 (d, J = 2.3 Hz, 1H), 7.06 (dd, J = 8.9, 2.4 Hz, 1H), 6.84–6.78 (m, 2H), 4.44 (s, 1H), 4.12 (t, J = 6.5 Hz, 2H), 3.50 (t, J = 5.5 Hz, 2H), 2.48–2.17 (m, 8H), 1.78 (p, J = 6.8 Hz, 2H), 1.50 (q, J = 7.7 Hz, 2H), 1.43 (hept, J = 6.7, 6.0 Hz, 2H), 1.24 (d, J = 9.5 Hz, 2H). ¹³C-NMR (151 MHz, DMSO) δ: 174.07 (s), 162.39 (s), 156.79 (s), 156.63 (s), 152.50 (s), 129.43 (s), 126.31 (s), 123.06 (s), 121.72 (s), 116.89 (s), 114.35 (s), 100.36 (s), 67.73 (s), 51.40 (s), 44.90 (s), 27.5. MS (ESI) m/z: 453.24 (M + H)⁺.

Synthesis of Compounds (5a–5i)

Daidzein (1.00 g, 3.90 mmol) was placed into a 50 mL reaction flask, followed by the addition of 10 mL of DMF. The solution was agitated at ambient temperature until it turned transparent. Subsequently, a 37% formaldehyde aqueous solution (0.50 mL) and a 40% aqueous dimethylamine solution (0.50 g, 3.90 mmol) were introduced.³³⁾ The reaction was subjected to reflux at 65°C for a duration of 4–6 h. The reaction mixture was introduced into freezing water and filtered to gain the crude product, which was purified using silica gel column chromatography with a solvent ratio of [V(DCM) : V(MeOH) = 25 : 1] to yield compound 5a. Compounds 5b–5i were synthesized with the same methodology.

8-((Dimethylamino)methyl)-7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (5a)

White solid. Yield 53.05%. m.p. 136.0–138.3°C. ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.29 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.42–7.35 (m, 2H), 6.89–6.78 (m, 3H), 3.94 (s, 2H), 2.35 (s, 6H). ¹³C-NMR (101 MHz, DMSO) δ: 174.74 (s), 163.85 (s), 157.14 (s), 154.99 (s), 152.33 (s), 130.04 (s), 125.72 (s), 123.26 (s), 122.51 (s), 115.83 (s), 115.17 (s), 114.91 (s), 108.25 (s), 53.34 (s), 43.96 (s). MS (ESI) m/z: 312.14 (M + H)⁺.

Based on the mass spectrometry data for compound 5a, there was a molecular ion peak at 312.14 [M + H]⁺, which indicated that the compound was a monoaminemethyl daidzein derivative. In the ¹H-NMR spectrum of daidzein, the chemical shift at δ 6.46 (s, 1H) corresponded to hydrogen atom H-8 on the A ring. In contrast, the ¹H-NMR spectrum of compound 5a showed the disappearance of the H-8 peak and the emergence of a methylene peak at δ 3.94 (s, 2H). In the ¹³C-NMR spectrum, the chemical shift for carbon C-8 on the A ring of daidzein was observed at δ 101.4. However, in compound 5a, the chemical shift of C-8 moves downfield by 6.85 ppm to δ 108.25, while the chemical shifts of adjacent carbons remain relatively unchanged. These findings were evident in the spectra of compounds 5b–5i. Through a comprehensive analysis of ¹H-NMR, ¹³C-NMR, and mass spectrometry data, we concluded that the site of methylation on daidzein occurred at position-8, demonstrating significant regioselectivity.

8-((Diethylamino)methyl)-7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (5b)

White solid. Yield 51.20%. m.p. 147.0–149.1°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 8.25 (s, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.40–7.31 (m, 2H), 6.83–6.78 (m, 2H), 6.76 (d, J = 8.8 Hz, 1H), 4.04 (s, 2H), 2.67 (q, J = 7.1 Hz, 4H), 1.08 (t, J = 7.1 Hz, 6H). ¹³C-NMR (101 MHz, DMSO) δ: 174.69 (s), 164.60 (s), 157.15 (s), 154.82 (s), 152.23 (s), 130.03 (s), 125.54 (s), 123.28 (s), 122.50 (s), 115.68 (s), 115.39 (s), 114.91 (s), 107.71 (s), 48.76 (s), 46.19 (s), 10.71 (s). MS (ESI) m/z: 340.15 (M + H)⁺.

8-((Dipropylamino)methyl)-7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (5c)

White solid. Yield 78.70%. m.p. 160.0–163.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.52 (s, 1H), 8.30 (s, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.41–7.34 (m, 2H), 6.84–6.78 (m, 3H), 4.08 (s, 2H), 2.58–2.52 (m, 4H), 1.60–1.50 (m, 4H), 0.86 (t, J = 7.3 Hz, 6H). ¹³C-NMR (151 MHz, DMSO) δ: 175.22 (s), 164.55 (s), 157.64 (s), 155.28 (s), 152.82 (s), 130.54 (s), 126.01 (s), 123.76 (s), 122.97 (s), 116.39 (s), 115.71 (s), 115.40 (s), 108.75 (s), 55.49 (s), 50.39 (s), 19.32 (s), 12.03 (s). MS (ESI) m/z: 368.19 (M + H)⁺.

8-((Diisopropylamino)methyl)-7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (5d)

White solid. Yield 74.20%. m.p. 191.2–193.3°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.52 (s, 1H), 8.30 (s, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.40–7.35 (m, 2H), 6.83–6.79 (m, 2H), 6.74 (d, J = 8.8 Hz, 1H), 4.16 (s, 2H), 3.21 (p, J = 6.6 Hz, 2H), 1.13 (d, J = 6.6 Hz, 12H). ¹³C-NMR (101 MHz, DMSO) δ: 174.67 (s), 165.31 (s), 157.13 (s), 154.46 (s), 152.16 (s), 130.02 (s), 125.11 (s), 123.27 (s), 122.56 (s), 115.62 (s), 115.40 (s), 114.91 (s), 108.07 (s), 54.86 (s), 48.91 (s), 18.99 (s). MS (ESI) m/z: 368.19 (M + H)⁺.

8-((Dibutylamino)methyl)-7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (5e)

White solid. Yield 58.40%. m.p. 196.0–198.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.52 (s, 1H), 8.30 (s, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.40–7.35 (m, 2H), 6.84–6.78 (m, 3H), 4.08 (s, 2H), 2.61–2.55 (m, 4H), 1.55–1.48 (m, 4H), 1.28 (h, J = 7.4 Hz, 4H), 0.87 (t, J = 7.4 Hz, 6H). ¹³C-NMR (101 MHz, DMSO) δ: 174.72 (s), 164.13 (s), 157.16 (s), 154.80 (s), 152.26 (s), 130.02 (s), 125.52 (s), 123.28 (s), 122.48 (s), 115.87 (s), 115.21 (s), 114.91 (s), 108.15 (s), 52.75 (s), 49.92 (s), 27.67 (s), 19.89 (s), 13.71 (s). MS (ESI) m/z: 396.21 (M + H)⁺.

8-((Diisobutylamino)methyl)-7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (5f)

White solid. Yield 52.40%. m.p. 197.0–199.0°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.52 (s, 1H), 8.30 (s, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.41–7.37 (m, 2H), 6.89 (d, J = 8.7 Hz, 1H), 6.83–6.78 (m, 2H), 3.93 (s, 2H), 2.26 (d, J = 7.1 Hz, 4H), 1.92 (hept, J = 6.7 Hz, 2H), 0.86 (d, J = 6.6 Hz, 12H). ¹³C-NMR (101 MHz, DMSO) δ: 174.81 (s), 162.60 (s), 157.16 (s), 155.17 (s), 152.33 (s), 130.02 (s), 125.61 (s), 123.27 (s), 122.43 (s), 116.31 (s), 114.92 (s), 114.74 (s), 109.55 (s), 62.93 (s), 49.89 (s), 25.30 (s), 20.73 (s). MS (ESI) m/z: 396.21 (M + H)⁺.

8-Hydroxy-3-(4-hydroxyphenyl)-8-(morpholinomethyl)-4H-chromen-4-one (5g)

White solid. Yield 66.50%. m.p. 239.5–241.0°C. ¹H-NMR (600 Hz, DMSO-d₆) δ: 9.52 (s, 1H), 8.34 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.41–7.37 (m, 2H), 6.94 (d, J = 8.8 Hz, 1H), 6.83–6.78 (m, 2H), 3.90 (s, 2H), 3.61 (t, J = 4.6 Hz, 4H), 2.54 (t, J = 4.5 Hz, 4H). ¹³C-NMR (101 MHz, DMSO) δ: 174.82 (s), 162.14 (s), 157.16 (s), 155.34 (s), 152.55 (s), 130.02 (s), 125.83 (s), 123.28 (s), 122.43 (s), 116.50 (s), 114.93 (s), 114.74 (s), 108.68 (s), 66.03 (s), 52.67 (s), 51.44 (s). MS (ESI) m/z: 354.14 (M + H)⁺.

8-Hydroxy-3-(4-hydroxyphenyl)-8-(piperidin-1-ylmethyl)-4H-chromen-4-one (5h)

White solid. Yield 55.20%. m.p. 206.0–208.0°C. ¹H-NMR(600 MHz, DMSO-d₆) δ: 9.50 (s, 1H), 8.30 (s, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.40–7.35 (m, 2H), 6.85 (d, J = 8.8 Hz, 1H), 6.83–6.78 (m, 2H), 3.99 (s, 2H), 2.58 (s, 4H), 1.58 (p, J = 5.6 Hz, 4H), 1.50–1.42 (m, 2H). ¹³C-NMR (101 MHz, DMSO) δ: 174.73 (s), 163.72 (s), 157.15 (s), 154.90 (s), 152.33 (s), 130.03 (s), 125.57 (s), 123.28 (s), 122.47 (s), 115.99 (s), 115.15 (s), 114.91 (s), 107.71 (s), 53.12 (s), 53.08 (s), 25.24 (s), 23.22 (s). MS (ESI) m/z: 352.16 (M + H)⁺.

8-((4-Ethylpiperazin-1-yl)methyl)-7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (5i)

White solid. Yield 56.70%. m.p. 214.0–214.8°C. ¹H-NMR (600 MHz, DMSO-d₆) δ: 8.33 (s, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.41–7.35 (m, 2H), 6.89 (d, J = 8.8 Hz, 1H), 6.83–6.77 (m, 2H), 3.97 (s, 2H), 2.59 (s, 4H), 2.33 (q, J = 7.1 Hz, 4H), 0.99 (t, J = 7.2 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ: 174.77 (s), 162.82 (s), 157.16 (s), 155.01 (s), 152.44 (s), 130.02 (s), 125.69 (s), 123.30 (s), 122.43 (s), 116.30 (s), 114.94 (s), 114.92 (s), 108.18 (s), 52.14 (s), 52.06 (s), 51.87 (s), 51.37 (s), 11.91 (s). MS (ESI) m/z: 381.18 (M + H)⁺.

Cytotoxicity Assay

H9C2 cardiomyocytes in the logarithmic growth phase were seeded in 96-well plates at a density of 0.8 × 10⁵ cells/well and incubated overnight. Each compound was assigned 6 concentration groups: 3.125, 6.25, 12.5, 25, 50, and 100 μM, with 3 replicate wells per group. Following 24 h of pharmacological intervention, 10 μL of CCK-8 solution was introduced to each well and incubated at 37°C for 1–3 h until orange coloration was observed. The optical density of each well was assessed at a wavelength of 450 nm using a microplate reader. The IC₅₀ value was subsequently computed.

Evaluation of Anti-hypoxia Activity in Vitro

H9C2 cardiomyocytes in the logarithmic growth phase were inoculated into 96-well plates at a density of 0.8 × 10⁵ cells/well and incubated overnight. The cells were categorized into the following groups: normal control group (cells without hypoxia), blank group (no cells seeded), hypoxia model group (cells subjected to hypoxia), daidzein group, and daidzein derivatives groups. The original culture medium was removed and replaced with either drug-containing (treatment group) or drug-free DMEM medium that was glucose-free and serum-free. All groups, excluding the normal control, were placed in a sealed chamber with AnaeroPack for hypoxia treatment at 37°C in a 5% CO₂ incubator for 24 h. Subsequently, the medium was replaced with a standard medium for reoxygenation for 6 h. Next, 10 μL of CCK-8 solution was introduced into each well and incubated at 37°C for 1–3 h until orange coloration was observed. The absorbance at 450 nm was measured using a microplate reader.

Prediction of Drug-Like Properties

The Log P value of the compound was calculated using the website https://vcclab.org/lab/alogps/. The structural formula of the compound was created using ChemDraw and saved in MDL SDF (*.sdf) format. Log in to the website, import the saved files sequentially, and click “Calculate.” The medicinal properties of the compounds were predicted using http://www.swissadme.ch/. After accessing the website, the chemical structure formulas of daidzein and its derivatives were drawn using the toolbar below. Click in the toolbar on the right to initiate the analysis, and the results were exported to Excel.

Normobaric Hypoxia Experiment

Following 3 d of adaptive feeding, the mice were randomly divided into 8 groups (n = 8 per group). These 8 groups were designated as a control group, an ACE group (250 mg/kg), a daidzein group (35 mg/kg), and daidzein derivatives groups (dosed according to the molar equivalent of daidzein). Each group was administered the corresponding dose by intraperitoneal injection, with the control group receiving saline. Thirty minutes after administration, the mice were placed in a 250 mL sealed plastic container containing 10 g of soda lime. The bottle cap was secured, and the interval from cap tightening to the mouse’s demise was recorded as the hypoxic survival duration. The rate of survival time extension in the mice was subsequently calculated.

Low-Pressure Hypoxia Experiment

Following 3 d of adaptive feeding, the mice were randomly divided into 9 groups (n = 11 per group). These 9 groups were designated as a control group, a hypoxia model group, an ACE group (250 mg/kg), a daidzein group (35 mg/kg), and daidzein derivatives groups (dosed based on the molar equivalent of daidzein). The control group and the hypoxia model group were given saline through intraperitoneal injection, whilst the other groups received the appropriate doses via the same method. Thirty minutes after administration, all groups, excluding the control group, were placed in a low-pressure oxygen chamber, and the height was artificially elevated to 7000 m, sustained for 24 h. Next, the brain and cardiac tissues were promptly harvested from the mice. Three samples of heart and brain tissue from each group were washed with pre-cooled saline to eliminate bloodstains and then immersed in a 10-fold volume of general-purpose fixative (formaldehyde) for fixation at ambient temperature for 24 h. Following fixation, the tissues were embedded, sectioned, stained, and scanned to examine the pathological alterations in the hippocampus area of the brain and cardiac tissues of the mice. The remaining samples were utilized to quantify the amounts of MDA, GSH, and the activity of SOD in cerebral and cardiac tissues, in accordance with the assay kit protocols.

Statistical Analysis

The experimental results were statistically analyzed and plotted using GraphPad Prism 9.5.1 (GraphPad, San Diego, CA, U.S.A.), and the data were presented as mean ± standard error of mean. The statistical significance of differences between groups was obtained by the ANOVA multiple comparisons. p-Value less than 0.05 (p < 0.05) was considered indicative of significance.

Acknowledgments

This study was supported by the Beijing Natural Science Foundation of China (Project No. 7242207).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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