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Abstract

A series of novel betulin derivatives containing hydrazide-hydrazone moieties were synthesized. All compounds were evaluated for their cytotoxicity against four human carcinoma cell lines (HepG2, A549, MCF-7 and HCT-116) and a normal human gastric epithelial cell line (GES-1). Among them, compound 6i was the most potent against HepG2 and MCF-7 cell lines, with IC₅₀ values of 9.27 and 8.87 μM, respectively. The results suggest that the incorporation of a hydrazide-hydrazone side chain at the C-28 position of betulin is beneficial for compounds to display significant cytotoxicity. Compound 6i may be used as a promising skeleton for antitumor agents with improved efficacy.

Keywords

betulin hydrazide hydrazone derivatives antitumor activity

Nowadays, cancer has become the second leading cause of death worldwide.¹ The most effective therapies used in cancer treatment continue to be traditional cytotoxic agents.² It is worth noting that the discovery of potent anticancer drugs from natural sources is still one of the most important directions in the field of drug research.^3–6 Betulin (lup-20(29)-ene-3β,28-diol, BE, 1) is a natural lupine-type triterpenoid widely distributed in plants, and especially abundant in the bark of birch trees (Figure 1).⁷ Betulin has been shown to exert various pharmacological and biological activities, such as antibacterial,^8,9 anti-HIV^10–12 and anti-inflammatory properties.^13–16 Recently, many studies have reported that betulin and its derivatives have significant antitumor activities against various kinds of human cancer cell lines, such as colorectal carcinoma (HT29, HCT116),^17,18 lung (A549),¹⁹ liver (SK-HEP-1, HepG2),^20,21 breast (MCF-7, MDA-MB231),²² prostate (PC3),²³ cervical (HeLa),²⁴ and leukemia (HL-60, K562, U937).^25–27 However, the high hydrophobicity of betulin hampers its further development as a cytotoxic drug.

Figure 1.

Structure of betulin (BE, 1).

It is worth noting that there are three active positions in the betulin structure, namely the isopropenyl side chain at C-19, and two hydroxyl groups at C-3 and C-28.^28–30 It is quite possible to make a chemical modification at these positions to obtain novel betulin derivatives with desired biological properties. According to the structure-activity relationship, a hydrogen donor group at C-28 can improve cytotoxic activity significantly.

Hydrazones have been demonstrated to possess antimicrobial, anti-inflammatory and antitumor activities.^31–34 Hydrazones possessing an azometine -NHN = CH- proton constitute an important class of compounds for drug development. In addition, the pharmacophore ingredient with CO-NH-N = CH (hydrazide-hydrazones) has received considerable attention due to its broad spectrum of biological applications.³⁵ Thus, hydrazide-hydrazones are known as anti-diabetic, antileishmanial, antimalarial, and anti-tumor compounds.^36–39 Hydrazide-hydrazones linked to a heterocyclic moiety such as pyrazole or pyrimidine are known to have a significant cytotoxic potential.⁴⁰ Hence, we tried to integrate the hydrazide-hydrazone moiety and heterocycles into a natural product in order to obtain new structures with remarkable cytotoxicity.

In the present work, as a part of our search for novel potential anticancer agents related to natural products, we report here on the synthesis of novel betulin derivatives containing a hydrazide-hydrazone unit as a side-chain attached to C28 of betulin. The anti-proliferative activities of the derivatives were evaluated against four human cancer cell lines in vitro.

Results and Discussion

The general procedure for the synthesis of betulin-28-hydrazide derivatives is shown in Figure 2. The 3-OH and 28-OH of betulin (1) were acetylated with acetic anhydride in the presence of DMAP (4-dimethylaminopyridine) in dry pyridine at room temperature to give compound 2. This was further reacted with titanium isopropoxide (Ti(i-PrOH)₄) in dry isopropyl alcohol (i-PrOH) for selective deacetylation at C-28 to give compound 3. Then the 28-OH of betulin was oxidized to a carbonyl group in the presence of pyridinium chlorochromate (PCC) in dry dichloromethane to give compound 4. Subsequently, this was reacted with sodium hydroxide for deacetylation at C-3 to give compound 5, which was reacted with different hydrazide substituents in ethanol to obtain target compounds 6a-i. The structures of all new compounds were characterized by MS, HRMS, and ¹H NMR and ¹³C NMR spectral methods.

Figure 2.

Reagents and conditions: (A) Ac₂O, DMAP, pyridine, room temperature, 6 h, 80%; (B) Ti(i-PrOH)₄, i-PrOH, 85°C, 5 h; 76%; (C) PCC, CH₂Cl₂, 35°C, 1 h, 80%; (D) NaOH, CH₃OH, 80°C, 2 h, 65%; (E) CH₃CH₂OH, hydrazide substituent, 35°C, 5∼7 h, 76%∼85%.

Compounds 6a-i and BE (1) were evaluated for their cytotoxicity against human hepatocellular carcinoma cells (HepG2), human breast carcinoma cells (MCF-7), human lung carcinoma cells (A549), human colorectal cells (HCT-116) and normal gastric cells (GES-1) by MTT assay. Mitomycin C, a broad-spectrum antitumor drug, especially for liver, colon, lung and breast cancers, was used as the positive drug control. The anti-proliferative activity data of compounds 6a-i are summarized by IC₅₀ values in Table 1. The results suggested the following rough structure-activity relationships.

Table 1.

In Vitro Cytotoxicity Activity Data of Compounds 6a-i.

Compound	IC₅₀ (μM)¹
Compound	HepG2	A549	MCF-7	HCT-116	GES-1
6a	49.3 ± 0.49	37.5 ± 2.58	>60	36.2 ± 2.32	-²
6b	33.8 ± 2.19	>60	32.4 ± 0.50	25.8 ± 2.34	88.6 ± 3.32
6c	18.9 ± 0.88	39.6 ± 2.84	28.2 ± 1.26	18.5 ± 0.68	124.7 ± 3.60
6d	15.8 ± 0.76	29.7 ± 1.25	27.9 ± 0.93	13.7 ± 0.077	152.8 ± 4.58
6e	33.2 ± 2.24	42.2 ± 2.66	24.6 ± 1.79	25.8 ± 1.57	109.8 ± 3.63
6f	15.4 ± 0.88	37.6 ± 1.42	29.6 ± 1.45	16.7 ± 1.14	169.2 ± 4.16
6g	29.6 ± 0.98	>60	>60	56.9 ± 3.36	-
6h	48.4 ± 3.12	>60	>60	37.8 ± 2.05	-
6i	9.3 ± 0.38	20.2 ± 1.03	8.9 ± 0.65	18.0 ± 1.14	147.4 ± 4.47
1	21.0 ± 0.72	30.5 ± 1.27	18.6 ± 1.14	25.7 ± 2.03	102.5 ± 3.65
Mitomycin C	25.3 ± 1.38	11.7 ± 1.02	12.5 ± 0.88	13.0 ± 0.96	22.7 ± 1.16

IC₅₀: Concentration of the tested compound that inhibits 50% of cell growth. All data are presented as means ± SD of three independent experiments.

“-” not active.

For the HepG2 cell line, compounds 6c, 6d, 6f and 6i displayed lower anti-proliferative activities than betulin (IC₅₀ = 21.0 μM). Compound 6i, possessing an isonicotinyl hydrazide group at C-28, displayed significant cytotoxic activity with an IC₅₀ value of 9.3 μM. This is about 2.3-fold higher than that of betulin. Compounds 6c, 6d and 6f also displayed significant cytotoxic activities. The data showed that the incorporation of an electron-donating group at C-28 of betulin led to significant improvement in cytotoxic activity in comparison with a methoxy, furan or thiophene group. For the HCT-116 cell lines, compounds 6c, 6d, 6f and 6i also possessed stronger cytotoxicity than that of betulin. Among them, compound 6d (IC₅₀ = 13.68 μM) was the most active. For A549 and MCF-7 cells, most of the compounds showed no significant inhibitory activity, except 6i, which displayed significant cytotoxic activity with an IC₅₀ value of 8.9 μM against MCF-7 cells. This is about 2.1-fold higher than that of betulin. Furthermore, compound 6i was less toxic to GES-1, with an IC₅₀ value of 147.4 μM.

Experimental

General

Reagents were used without further purification. The ¹H and ¹³C NMR spectra (δ, ppm, J, Hz) were recorded using a Bruker AVANCE NEO600 spectrometer (600 and 150 MHz, respectively) in either CDCl₃ or DMSO-d₆ (Bruker, Berlin, Germany). Tetramethylsilane was used as an internal standard. Melting temperatures were determined on an MP120 auto point apparatus (Haineng, Fujian, China). The mass spectra of all compounds were tested on an Esquire 6000 mass spectrometer in ESI mode (Bruker, Berlin, Germany). HRMS were obtained using an Agilent 6250 mass spectrometer (Agilient, San Francisco, USA). Flash chromatography was performed using silica gel (400 mesh) (Biohonor, Guangzhou, China).

Synthesis of 3-O,28-O-acetyl-betulin (2)

To a solution of betulin (1, 3.10 g, 7.0 mmol) in pyridine (80 mL) was added DMAP (40 mg, 0.3 mmol) and acetic anhydride (Ac₂O, 2 ml, 24 mmol) at 0 °C. The reaction was stirred at room temperature for 6 h. The solution was evaporated and the residue was redissolved in CH₂Cl₂ (100 mL), and washed with sat. NaHCO₃. The combined solution was washed with brine twice and dried over anhydrous Na₂SO₄. The solvent was evaporated and purified by silica gel column chromatography (ethyl acetate/light petroleum = 1/50) to give compound 2 (2.95 g). White powder. Yield 80%. Mp 216.0 to 217.5 °C. ¹H NMR (600 MHz, CDCl₃) δ: 4.69 (1 H, s, H29), 4.59 (1 H, s, H29), 4.47 (1 H, dd, J = 10.6, 5.7 Hz, AcO-CH), 4.25 (1 H, d, J = 10.9 Hz, CH₂-O), 3.85 (1 H, d, J = 11.0 Hz, CH₂-O), 2.44 (1 H, td, J = 11.1, 5.8 Hz), 2.07 (3 H, s, CH₃-COO), 2.04 (3 H, s, CH₃-COO), 1.68 (3 H, s, CH₃), 1.03 (3 H, s, CH₃), 0.97 (3 H, s, CH₃), 0.87 to 0.82 (9 H, m, 3 × CH₃), 0.78 (1 H, d, J 9.3 Hz). ¹³C NMR (150 MHz, CDCl₃) δ: 171.6 (C = O), 171.0 (C = O), 150.2 (C20), 109.9 (C29), 80.9 (C3), 62.8 (C28), 55.4 (C5), 50.3 (C9), 48.8 (C19), 47.7 (C17), 46.3 (C18), 42.7 (C14), 40.9 (C8), 38.4 (C4), 37.8 (C1), 37.6 (C13), 37.1 (C10), 34.5 (C7), 34.1 (C22), 34.0 (C21), 29.7 (C16), 29.6 (C23), 27.9 (C2), 27.1 (C15), 25.2 (C12), 21.3 (AcO), 21.1 (AcO), 20.8 (C11), 19.1 (C30), 18.2 (C6), 16.5 (C25), 16.2 (C26), 16.0 (C24), 14.7 (C27). MS (ESI) m/z: 527.4 [M + H]⁺.

Synthesis of 3-O-acetyl-betulin (3)

To a solution of intermediate (2, 3.41 g, 6.4 mmol) in isopropyl alcohol (i-PrOH, 160 ml) was added titanium isopropoxide (Ti(i-PrOH)₄, 10 mL, 35 mmol). The reaction temperature was increased to 85°C and the mixture stirred for 5 h. When the substrates disappeared (as detected by TLC), the mixture was concentrated under vacuum. The concentrate was dissolved in dichloromethane (100 mL) and washed with brine (50 mL) for twice and dried over anhydrous Na₂SO₄. The solvent was concentrated under vacuum. The crude product was purified by flash chromatography on silica gel (ethyl acetate/light petroleum = 1/7) to give compound 3 (2.40 g). White powder. Yield 76%. Mp 256.1-257.8°C. ¹H NMR (600 MHz, CDCl₃) δ: 4.68 (1 H, d, J = 1.8 Hz, H29), 4.58 (1 H, s, H29), 4.47 (1 H, dd, J = 11.0, 5.4 Hz, AcO-CH), 3.85-3.74 (1 H, m, CH₂-O), 3.33 (1 H, d, J = 10.8 Hz, CH₂-O), 2.38 (1 H, td, J = 11.0, 5.8 Hz, H3), 2.04 (3 H, s, CH₃-COO), 1.69 (3 H, s, CH₃), 1.02 (3 H, s, CH₃), 0.97 (3 H, s, CH₃), 0.88-0.81 (9 H, m, 3 × CH₃). ¹³C NMR (150 MHz, CDCl₃) δ: 171.0 (C = O), 150.5 (C20), 109.7 (C29), 80.9 (C3), 60.6 (C28), 55.4 (C5), 50.3 (C9), 48.7 (C19), 47.8 (C17), 47.7 (C18), 42.7 (C14), 40.9 (C8), 38.4 (C4), 37.8 (C1), 37.3 (C13), 37.1 (C10), 34.7 (C7), 34.2 (C22), 34.0 (C21), 29.3 (C16), 29.2 (C23), 27.9 (C2), 27.0 (C15), 25.2 (C12), 21.3 (AcO), 20.8 (C11), 19.1 (C30), 18.2 (C6), 16.5 (C25), 16.2 (C26), 16.0 (C24), 14.7 (C27). MS (ESI) m/z: 485.4 [M + H]⁺.

Synthesis of 3-O-acetyl-betulinicaldehyde (4)

To a solution of 3 (300 mg, 0.6 mmol) in CH₂Cl₂ (20 mL) was added pyridinium chlorochromate (PCC, 400 mg, 1.8 mmol). The reaction mixture was stirred at 35°C for 1 h. Then silica gel (1.50 g) was added to the mixture and concentrated to a dry powder. The crude product was purified by silica gel column chromatography to give compound 4 (240 mg). White powder. Yield 80%. Mp 270.2-271.8°C. ¹H NMR (600 MHz, CDCl₃) δ: 9.67 (1 H, d, J = 1.1 Hz, -CHO), 4.76 (1 H, d, J = 0.9 Hz, H29), 4.63 (1 H, s, H29), 4.47 (1 H, dd, J = 10.9, 5.5 Hz, AcO-CH), 2.86 (1 H, td, J = 14;H, s, CH₃-COO), 1.70 (3 H, s, CH₃), 0.97 (3 H, s, CH₃), 0.92 (3 H, s, CH₃), 0.84 (9 H, d, J = 8.5 Hz, 3 × CH₃). ¹³C NMR (150 MHz, CDCl₃) δ: 171.0 (C = O), 149.7 (C20), 110.2 (C29), 80.9 (C3), 59.3 (C28), 55.4 (C5), 50.4 (C9), 48.7 (C19), 48.1 (C17), 47.6 (C18), 42.6 (C14), 40.8 (C8), 38.4 (C4), 37.8 (C1), 37.4 (C13), 37.1 (C10), 34.3 (C7), 33.2 (C22), 32.8 (C21), 29.2 (C16), 28.8 (C23), 27.9 (C2), 27.0 (C15), 25.5 (C12), 21.3 (AcO), 20.7 (C11), 19.0 (C30), 18.2 (C6), 16.5 (C25), 16.2 (C26), 15.9 (C24), 14.2 (C27). MS (ESI) m/z: 483.4 [M + H]⁺.

Synthesis of Betulinicaldehyde (5)

A solution of 4 (200 mg, 0.4 mmol) in 2% NaOH-MeOH (10 ml) was stirred for 2 h at 80°C. The solution was evaporated and then redissolved in CH₂Cl₂ (100 mL), washed with brine twice and dried over anhydrous Na₂SO₄. The solvent was evaporated and purified by silica gel column chromatography (ethyl acetate/light petroleum = 1/10) to give compound 5 (116 mg). White powder. Yield 65%. Mp 285.3-287.2°C. ¹H NMR (600 MHz, CDCl₃) δ: 9.68 (1 H, d, J 1.3 Hz, -CHO), 4.76 (1 H, s, H29), 4.63 (1 H, s, H29), 3.18 (1 H, dd, J 11.5, 4.7 Hz, OH), 2.86 (1 H, td, J 11.2, 5.9 Hz, H3), 1.70 (3 H, s, H30), 0.97 (3 H, s, CH₃), 0.95 (3 H, s, CH₃), 0.92 (3 H, s, CH₃), 0.82 (3 H, s, CH₃), 0.75 (3 H, s, CH₃). ¹³C NMR (150 MHz, CDCl₃) δ: 149.7 (C20), 110.2 (C29), 79.0 (C3), 59.3 (C28), 55.3 (C5), 50.5 (C9), 48.9 (C19), 48.1 (C17), 47.5 (C18), 42.6 (C14), 40.8 (C8), 38.8 (C4), 38.7 (C1), 38.7 (C13), 37.2 (C10), 34.3 (C7), 33.2 (C22), 29.9 (C21), 29.3 (C16), 28.8 (C23), 28.0 (C2), 27.4 (C15), 25.5 (C12), 20.8 (C11), 19.0 (C30), 18.3 (C6), 16.1 (C25), 15.9 (C26), 15.4 (C24), 14.3 (C27). MS (ESI) m/z: 441.4 [M + H]⁺.

General Procedure for the Synthesis of Novel Derivatives 6a-i

To a solution of 5 (1 mmol) in anhydrous EtOH (20 mL) was added different hydrazide substituents (1.1 mmol). The mixture was continually stirred under 35°C for 5∼7 h until there was no starting material. Then the solvent was evaporated in a rotary evaporator. The residue was purified by flash chromatography to afford compounds 6a-i.

Betulin-28-(3-chloro)benzohydrazide (6a)

Yellowish powder. Yield 81%. Mp 205-207°C. ¹H NMR (600 MHz, DMSO-d₆) δ: 11.46 (1 H, s, NH), 7.98 (1 H, s, H28), 7.92 (1 H, s, Ar), 7.84 (1 H, d, J = 7.4 Hz, Ar), 7.65 (1 H, d, J = 7.5 Hz, Ar), 7.55 (1 H, d, J = 7.6 Hz, Ar), 4.71 (1 H, s, H29), 4.60 (1 H, s, H29), 4.26 (1 H, d, J = 3.3 Hz), 2.97 (1 H, s, OH), 1.98 (1 H, d, J = 11.9 Hz, H3), 1.86 (2 H, dd, J = 14.9, 8.9 Hz), 1.78 (2 H, d, J = 11.7 Hz), 1.68 (3 H, s, CH₃), 1.66 to 1.23 (18 H, m), 1.15 (1 H, d, J = 12.0 Hz), 1.06 (1 H, d, J = 10.4 Hz), 0.97 (3 H, s, CH₃), 0.93 (3 H, s, CH₃), 0.87 (3 H, s, CH₃), 0.75 (3 H, s, CH₃), 0.65 (3 H, s, CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ: 161.5 (C = O), 157.1 (C28), 150.1 (C20), 136.0 (Ar), 133.7 (Ar), 131.9 (Ar), 130.9 (Ar), 127.6 (Ar), 126.8 (Ar), 110.5 (29), 77.2 (C3), 55.3 (C5), 50.8 (C9), 50.2 (C19), 49.3 (C17), 48.2 (C18), 42.9 (C14), 40.8 (C8), 38.9 (C4), 38.7 (C1), 38.6 (C13), 37.2 (C10), 36.8 (C7), 34.4 (C22), 32.2 (C21), 29.7 (C16), 28.6 (C23), 28.0 (C2), 27.6 (C15), 25.3 (C12), 20.8 (C11), 19.2 (C30), 18.4 (C6), 16.5 (C25), 16.4 (C26), 16.3 (C24), 14.9 (C27). HRMS (ESI): calcd for C₃₇H₅₄ClN₂O₂ [M + H]⁺: 593.3874, found: 593.3868.

Betulin-28-(4-chloro)benzohydrazide (6b)

Yellowish powder. Yield 84%. Mp 208-210°C. ¹H NMR (600 MHz, DMSO-d₆) δ: 11.43 (1 H, s, NH), 7.98 (1 H, s, H28), 7.90 (2 H, d, J = 8.2 Hz, Ar), 7.59 (2 H, d, J = 8.2 Hz, Ar), 4.71 (1 H, s, H29), 4.60 (1 H, s, H29), 4.27 (1 H, d, J = 4.8 Hz), 3.17 (1 H, d, J = 4.2 Hz, OH), 3.04-2.93 (m, 1 H), 1.68 (3 H, s, CH₃), 0.97 (3 H, s, CH₃), 0.93 (3 H, s, CH₃), 0.87 (3 H, s, CH₃), 0.76 (3 H, s, CH₃), 0.65 (3 H, s, CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ: 161.9 (C = O), 156.8 (C28), 150.1 (C20), 136.8 (Ar), 132.8 (Ar), 129.9 (Ar), 128.9 (Ar), 110.5 (C29), 77.2 (C3), 55.3 (C5), 50.8 (C9), 50.2 (C19), 49.3 (C17), 48.2 (C18), 42.9 (C14), 40.9 (C8), 39.0 (C4), 38.7 (C1), 38.6 (C13), 37.2 (C10), 36.8 (C7), 34.4 (C22), 32.2 (C21), 29.7 (C16), 28.6 (C23), 28.0 (C2), 27.6 (C15), 25.3 (C12), 20.8 (C11), 19.2 (C30), 18.4 (C6), 16.5 (C25), 16.4 (C26), 16.3 (C24), 14.9 (C27). HRMS (ESI): calcd for C₃₇H₅₄ClN₂O₂ [M + H]⁺: 593.3874, found: 593.3868.

Betulin-28-(4-bromo)benzohydrazide (6c)

Yellowish powder. Yield 76%. Mp 202-204°C. ¹H NMR (600 MHz, DMSO-d₆) δ: 11.43 (1 H, s, NH), 7.98 (1 H, s, H28), 7.82 (2 H, d, J = 8.4 Hz, Ar), 7.73 (2 H, d, J = 8.4 Hz, Ar), 4.71 (1 H, s, H29), 4.60 (s, 1 H, H29), 3.34 (1 H, s, OH), 1.68 (3 H, s, CH₃), 0.97 (s, 3 H, CH₃), 0.93 (3 H, s, CH₃), 0.87 (3 H, s, CH₃), 0.76 (3 H, s, CH₃), 0.65 (3 H, s, CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ: 162.0 (C = O), 156.9 (C28), 150.1 (C20), 133.1 (Ar), 131.9 (Ar), 130.1 (Ar), 125.8 (Ar), 110.5 (C29), 77.2 (C3), 55.3 (C5), 50.8 (C9), 50.2 (C19), 49.3 (C17), 48.2 (C18), 42.9 (C14), 40.9 (C8), 39.0 (C4), 38.7 (C1), 38.6 (C13), 37.2 (C10), 36.8 (C7), 34.4 (C22), 32.2 (C21), 29.7 (C16), 28.6 (C23), 28.0 (C2), 27.6 (C15), 25.3 (C12), 20.8 (C11), 19.2 (C30), 18.4 (C6), 16.5 (C25), 16.4 (C26), 16.3 (C24), 14.9 (C27). HRMS (ESI): calcd for C₃₇H₅₄BrN₂O₂ [M + H]⁺: 637.3369, found: 637.3363.

Betulin-28-(4-trifluoromethyl)benzohydrazide (6d)

White powder. Yield 80%. Mp 197-199°C. ¹H NMR (600 MHz, DMSO-d₆) δ: 11.57 (1 H, s, NH), 8.07 (2 H, d, J = 7.7 Hz, Ar-H), 8.00 (1 H, s, H28), 7.90 (2 H, d, J = 7.8 Hz, Ar-H), 4.72 (1 H, s, H29), 4.60 (1 H, s, H29), 4.27 (1 H, s), 2.97 (1 H, s), 1.69 (3 H, s, CH₃), 0.98 (3 H, s, CH₃), 0.93 (3 H, s, CH₃), 0.87 (3 H, s, CH₃), 0.76 (3 H, s, CH₃), 0.65 (3 H, s, CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ: 161.8 (C = O), 157.4 (C28), 150.1 (C20), 137.9 (CF₃), 131.9 (Ar), 123.0 (Ar), 128.9 (Ar), 125.3 (Ar), 123.5 (Ar), 110.6 (C29), 77.2 (C3), 55.3 (C5), 50.8 (C9), 50.2 (C19), 49.3 (C17), 48.2 (C18), 42.9 (C14), 40.9 (C8), 39.0 (C4), 38.7 (C1), 38.6 (C13), 37.2 (C10), 36.7 (C7), 34.4 (C22), 32.2 (C21), 29.7 (C16), 28.5 (C23), 28.0 (C2), 27.6 (C15), 25.3 (C12), 20.8 (C11), 19.2 (C30), 18.4 (C6), 16.5 (C25), 16.4 (C26), 16.3 (C24), 14.9 (C27). HRMS (ESI): calcd for C₃₈H₅₄F₃N₂O₂ [M + H]⁺: 627.4137, found: 627.4131.

Betulin-28-(4-methoxy)benzohydrazide (6e)

White powder. Yield 77%. Mp 191-193°C. ¹H NMR (600 MHz, DMSO-d₆) δ: 11.25 (1 H, s, NH), 7.96 (1 H, s, H28), 7.87 (2 H, t, J = 14.7 Hz, Ar), 7.04 (2 H, d, J = 8.2 Hz, Ar), 4.71 (1 H, s, H29), 4.59 (1 H, s, H29), 4.27 (1 H, d, J = 5.1 Hz, OH), 3.83 (3 H, s, OCH₃), 1.68 (3 H, s, CH₃), 0.97 (3 H, s, CH₃), 0.93 (3 H, s, CH₃), 0.87 (3 H, s, CH₃), 0.76 (3 H, s, CH₃), 0.65 (3 H, s, CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ: 162.4 (C = O), 155.7 (C28), 150.2 (C20), 129.8 (Ar), 126.1 (Ar), 114.1 (Ar), 110.5 (C29), 77.2 (C3), 55.8 (OCH₃), 55.3 (C5), 50.7 (C9), 50.2 (C19), 49.3 (C17), 48.2 (C18), 42.9 (C14), 40.9 (C8), 39.0 (C4), 38.7 (C1), 38.6 (C13), 37.2 (C10), 36.9 (C7), 34.4 (C22), 32.3 (C21), 29.8 (C16), 28.6 (C23), 28.0 (C2), 27.6 (C15), 25.3 (C12), 20.8 (C11), 19.2 (C30), 18.4 (C6), 16.5 (C25), 16.4 (C26), 16.3 (C24), 14.9 (C27). HRMS (ESI): calcd for C₃₈H₅₇N₂O₃ [M + H]⁺: 589.4369, found: 589.4378.

Betulin-28-(4-hydroxy)benzohydrazide (6f)

White powder. Yield 84%. Mp 213-215°C. ¹H NMR (600 MHz, DMSO-d₆) δ: 11.16 (1 H, s, NH), 10.05 (1 H, s, Ar-OH), 7.95 (1 H, s, H28), 7.75 (2 H, d, J = 7.0 Hz, Ar-H), 6.83 (2 H, d, J = 6.1 Hz, Ar-H), 4.71 (1 H, s, H29), 4.59 (1 H, s, H29), 4.22 (1 H, t, J = 6.0 Hz, 3-OH), 2.97 (1 H, m, H3), 1.98 (1 H, m), 1.81 (4 H, m), 1.66 (3 H, s, CH₃), 1.64 to 1.16 (17 H, m), 0.97 (3 H, s, CH₃), 0.91 (3 H, s, CH₃), 0.89 (m, 2 H), 0.87 (3 H, s, CH₃), 0.76 (3 H, s, CH₃), 0.65 (3 H, s, CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ: 160.9 (C = O), 155.3 (C28), 150.2 (C20), 132.2 (Ar), 132.0 (Ar), 129.9 (Ar), 129.1 (Ar), 124.5 (Ar), 115.4 (Ar), 110.5 (C29), 77.2 (C3), 55.3 (C5), 50.6 (C9), 50.2 (C19), 49.3 (C17), 48.2 (C18), 42.9 (C14), 40.9 (C8), 38.9 (C4), 38.7 (C1), 38.6 (C13), 37.1 (C10), 34.4 (C7), 34.3 (C22), 30.5 (C21), 29.7 (C16), 28.5 (C23), 29.0 (C2), 27.6 (C15), 25.6 (C12), 20.8 (C11), 19.2 (C30), 18.4 (C6), 16.5 (C25), 16.4 (C26), 16.3 (C24), 14.8 (C27). HRMS (ESI): calcd for C₃₇H₅₅N₂O₃ [M + H]⁺: 575.4213, found: 575.4219.

Betulin-28-(2-furan)carbohydrazide (6 g)

Yellowish powder. Yield 75%. Mp 187-189°C. ¹H NMR (600 MHz, CDCl₃) δ: 9.19 (1 H, s, NH), 7.88 (1 H, s, H28), 7.47 (2 H, m, furan), 6.54 (1 H, dd, J = 3.3, 1.6 Hz, furan), 4.72 (1 H, s, H29), 4.62 (1 H, s, H29), 3.19 (1 H, dd, J = 11.5, 4.6 Hz, OH), 2.52 (1 H, s, H3), 2.19 (1 H, m), 2.05 (1 H, m), 2.02-1.75 (6 H, m), 1.720 (3 H, m, CH₃), 1.66-1.04 (17 H, m), 1.00 (3 H, s, CH₃), 0.99-0.92 (6 H, m, 2 × CH₃), 0.92-0.87 (2 H, m), 0.81 (3 H, s, CH₃), 0.75 (3 H, s, CH₃), 0.68 (1 H, d, J = 10.8 Hz). ¹³C NMR (150 MHz, CDCl₃) δ: 171.2 (C = O), 157.7 (C28), 149.8 (C20), 144.1 (C1’), 115. 7 (C2’, C4’), 112.4 (C3’), 110.1 (C29), 78.9 (C3), 55.3 (C5), 51.2 (C9), 50.3 (C19), 49.3 (C17), 48.1 (C18), 42.8 (C14), 40.9 (C8), 38.8 (C4), 38.7 (C1), 37.3 (C13), 37.2 (C10), 37.1 (C7), 34.2 (C22), 32.2 (C21), 29.8 (C16), 29.7 (C23), 28.0 (C2), 27.3 (C15), 25.3 (C12), 20.7 (C11), 19.1 (C30), 18.2 (C6), 16.4 (C25), 16.3 (C26), 15.4 (C24), 14.7 (C27). HRMS (ESI): calcd for C₃₅H₅₃N₂O₃ [M + H]⁺: 549.4056, found: 549.4051.

Betulin-28-(2-thiophene)carbohydrazide (6 h)

Yellowish powder. Yield 76%. Mp 176-178°C. ¹H NMR (600 MHz, CDCl₃) δ: 9.99 (1 H, s, NH), 8.18 (1 H, s, H28), 7.63 (1 H, d, J = 3.5 Hz, thio-), 7.50 (1 H, s, thio-), 7.18-7.10 (1 H, m, thio-), 4.74 (1 H, s, H29), 4.63 (1 H, s, H29), 3.18 (1 H, dd, J = 11.5, 4.6 Hz, 3-OH), 2.60 (1 H, d, J = 4.6 Hz, H3), 2.16-1.85 (7 H, m), 1.72 (3 H, s, CH₃), 1.67-1.08 (18 H, m), 1.01 (3 H, s, CH₃), 0.96 (3 H, s, CH₃), 0.90 (3 H, s, CH₃), 0.87 (2 H, m), 0.78 (3 H, s, CH₃), 0.75 (3 H, s, CH₃), 0.67 (1 H, d, J = 11.1 Hz). ¹³C NMR (150 MHz, CDCl₃) δ: 162.6 (C = O), 152.5 (C28), 149.8 (C20), 135.1 (C1’), 134.1 (C2’), 133.0 (C3’), 128.8 (C4’), 110.1 (C29), 78.9 (C3), 55.3 (C5), 51.1 (C9), 50.4 (C19), 49.4 (C17), 48.0 (C18), 42.9 (C14), 40.9 (C8), 38.8 (C4), 38.7 (C1), 38.6 (C13), 37.1 (C10), 37.0 (C7), 34.2 (C22), 32.6 (C21), 29.9 (C16), 29.8 (C23), 28.0 (C2), 27.4 (C15), 25.3 (C12), 20.7 (C11), 19.1 (C30), 18.2 (C6), 16.1 (C25), 16.0 (C26), 15.4 (C24), 14.7 (C27). HRMS (ESI): calcd for C₃₅H₅₃N₂O₂S [M + H]⁺: 565.3828, found: 565.3822.

Betulin-28-isonicotinohydrazide (6i)

Yellowish powder. Yield 78%. Mp 221-223°C. ¹H NMR (600 MHz, DMSO-d₆) δ: 11.58 (1 H, s, NH), 8.76 (2 H, d, J = 5.6 Hz, Py), 8.00 (1 H, s, H28), 7.78 (2 H, d, J = 5.7 Hz, Py), 4.71 (1 H, s, H29), 4.60 (1 H, s, H29), 4.27 (1 H, m, 3-OH), 3.04-2.93 (1 H, m, H3), 1.98 (1 H, d, J = 12.2 Hz), 1.86 (2 H, d, J = 9.0 Hz), 1.78 (2 H, m), 1.68 (3 H, s, CH₃), 1.67-1.00 (17 H, m), 0.98 (3 H, s, CH₃), 0.93 (3 H, s, CH₃), 0.87 (3 H, s, CH₃), 0.85 (2 H, m), 0.76 (3 H, s, CH₃), 0.74 (1 H, s), 0.65 (3 H, s, CH₃), 0.63 (1 H, s). ¹³C NMR (150 MHz, DMSO-d₆) δ: 161.4 (C = O), 157.9 (C28), 150.7 (C20), 150.1 (C1’), 141.1 (C2’), 121.9 (C3’), 110.6 (C29), 77.2 (C3), 55.3 (C5), 50.8 (C9), 50.2 (C19), 49.3 (C17), 48.2 (C18), 42.9 (C14), 40.9 (C8), 39.0 (C4), 38.7 (C1), 38.6 (C13), 37.2 (C10), 36.7 (C7), 34.5 (C22), 32.2 (C21), 29.7 (C16), 28.6 (C23), 28.0 (C2), 27.6 (C15), 25.3 (C12), 20.8 (C11), 19.2 (C30), 18.4 (C6), 16.4 (C25), 16.3 (C26), 16.2 (C24), 14.9 (C27). HRMS (ESI): calcd for C₃₆H₅₄N₃O₂ [M + H]⁺: 560.4216, found: 560.4211.

In Vitro Cytotoxic Activity

All cell lines were obtained from the Shanghai Cell Bank of the Chinese Academy of Science. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/mL of penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. Cytotoxic activities of all tested compounds against four cancer cell lines were evaluated by MTT assay. Cells were seeded into 96-well plates (1 × 10⁴ cells/well) for 24 h. Then the cells were treated with compounds at gradient concentrations from 5 to 60 μM for 48 h and then 10 μL MTT (Sigma Chemical Co., Ltd, USA) solution (5 mg/mL in PBS) was added for 2 h. The solution was replaced by 100 μL DMSO, and the absorbance was measured at 490 nm on a Spectra Max 340 microplate reader. The IC₅₀ values were derived by SPSS nonlinear regression analysis.

Conclusions

In summary, a series of novel betulin derivatives containing a hydrazide-hydrazone side-chain were synthesized by five steps. The yields of these derivatives were ideal from 76% to 85%, which indicated that the synthetic route was designed reasonably. All compounds were evaluated for their in vitro antiproliferative activities against four human carcinoma cell lines (HepG2, A549, MCF-7 and HCT-116). Among the compounds under biological study, compound 6i was the most potent against HepG2 and MCF-7 cell lines, with IC₅₀ values of 9.27 and 8.87 μM, respectively. The data showed that the incorporation of heterocycles with a hydrazide-hydrazone moiety at the C-28 position of betulin led to significant improvement in cytotoxic activity. It will be of great significance to study the anti-tumor mechanism in the future. These results indicated that compound 6i may be used as a valuable skeleton structure for developing novel antitumor agents.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211055345 - Supplemental material for Synthesis and Cytotoxic Activity of Novel Betulin Derivatives Containing Hydrazide-Hydrazone Moieties

Supplemental material, sj-docx-1-npx-10.1177_1934578X211055345 for Synthesis and Cytotoxic Activity of Novel Betulin Derivatives Containing Hydrazide-Hydrazone Moieties by Jiale Wu, Jiafeng Wang, Yinglong Han, Yu Lin, Jing Wang and Ming Bu in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical Approval is not applicable for this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Central Government Support Fund for the Reform and Development of Local Universities-Talent Training Support Program Project (grant number ZYZX2019).

Trial Registration

Not applicable, because this article does not contain any clinical trials.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iD

Ming Bu

Supplemental Material

Supplemental material for this article is available online.

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