Sage Journals: Discover world-class research

Abstract

Phorbol esters and their derivatives, such as TPA, have been reported as potential natural antitumor products, with some derivatives entering clinical research for leukemia treatment. In this study, 16 phorbol derivatives were synthesized from phorbol, and their anti-leukemia activity against Jurkat, HL-60, and K562 were tested. The results showed that several derivatives had anti-leukemia activity, with 5B demonstrating the strongest cytotoxic activity against the K562 cell line (IC₅₀ = 0.24 ± 0.04 μM). Based on the IC₅₀ values, a structure-activity relationship was established. When the substituent contained an aromatic group, phorbol derivatives esterified with long-chain organic acids at three reactive hydroxyl groups (C12-OH, C13-OH, and C20-OH) had better activity. When aryl groups were absent from the substituent groups, phorbol derivatives esterified with short-chain organic acids at two hydroxyl groups had better activity. Derivatives esterified with heterocyclic acids, either tri-substituted or di-substituted, showed a significant decrease in cytotoxicity. The mechanism of anti-leukemia activity was explored through flow cytometry, which indicated that phorbol esters inhibited the growth of leukemia cells by inducing apoptosis, leading to cell death. Molecular docking data of compound 5B docking to PKCδC1B suggested that substituent groups should not be too large, and caution should be taken when substituting C20-OH. Overall, these findings provide valuable insights into the design of more effective phorbol esters as anti-leukemia agents, but further research is needed to confirm their safety and efficacy in clinical settings.

Keywords

anti-leukemia apoptosis flow cytometry phorbol ester PKC

Introduction

Acute myeloid leukemia (AML) is the most common form of leukemia among adults,¹ characterized by the malignant proliferation and accumulation of immature myeloid cells, resulting in failure of normal hematopoiesis.² Intensive chemotherapy (IC) and allogeneic stem cell transplantation (HSCT) have been the primary treatment strategies for AML for many years.^3,4 Despite these efforts, the prognosis for individuals with AML remains poor due to high relapse rates and therapy resistance, which can further impair the immune system. Therefore, the development of new compounds with high efficacy against leukemia and low toxicity to normal cells is urgently needed.

The conventional chemotherapy drug cytarabine is derived from a natural product found in a marine sponge.⁵ In addition, numerous other natural products have exhibited antitumor properties, including callyaerins, gallic acid, and shikonin.^5–8 Recently, TPA (12-O-tetradecanoyl-phorbol-13-acetate, see Figure 1) has garnered attention from researchers as a promising anti-leukemia drug. TPA is a phorbol ester which was first isolated from the seed oil of croton tree, and it has shown tumor-promoting properties.⁹ Weber and Hecker¹⁰ subsequently isolated several phorbol esters from the roots of Croton flavens L., and confirmed their skin-irritating effects on mice. In addition, numerous phorbol esters have been isolated from the leaves and seed oil of Jatropha curcas L., which belongs to the same family as croton (Euphorbiaceae).^11–13

Figure 1.

Phorbol and phorbol derivatives.

J. curcas is an energy crop which is capable of withstanding drought, and it contains high oil content in its seeds. However, the utilization of the seeds and seed oil is hindered by the presence of toxic phorbol esters, which would cause skin irritation and promote tumor growth.^14,15 To address this issue, many studies have been conducted in recent years to detoxify J. curcas.^16–18

However, phorbol esters exhibit a dual nature, as they have both tumor-promoting and promising antitumor properties.^16,19,20 The protein kinase C (PKC) family plays key roles in regulating various cellular responses, such as cell growth, differentiation, secretion, survival, and apoptosis, and is an attractive drug target for treating cancer. The diacylglycerol (DAG) within cells could activate PKC.²¹ The search for drugs that could activate PKC for antitumor purposes is a promising direction. The natural product bryostatins is a typical PKC activator with good antitumor activity.²² Phorbol esters act at the same site as DAG within cells, and both compounds activate PKC.²³ The main difference between them is that phorbol esters are not as easily metabolized as diglycerides. As a result, they can rapidly activate PKC in a short term, but prolonged exposure to phorbol esters can reduce or even inactivate PKC activity, leading to the promotion of tumor growth. Due to their unique effect on PKC, phorbol esters can not only promote the growth of tumor cells, but also exhibit potential in fighting against multiple cancers such as leukemia and viral conditions, such as AIDS.

In the treatment of acute myeloid leukemia, there are many studies about TPA, and it has entered the clinical trial stage.^24,25 Various tumor-promoting phorbol esters, including TPA, also induce differentiation in certain leukemia cell lines. Takeda et al.²⁶ induced differentiation of ML-1 cells from patients with acute myeloid leukemia into cells with monocyte and macrophage characteristics by treatment with TPA combined with vitamin D3 analog KH1060. In addition, Zheng et al.²⁷ combined TPA with capsaicin on HL-60 and HL525 cells, demonstrating that capsaicin could improve the efficacy of TPA treatment and overcome the resistance of some myeloid leukemia patients to TPA. Ma et al.²⁸ combined DTC (diethyldithiocarbamate) and TPA in animal experiments to more effectively inhibit the growth of HL-60 tumors.

Although TPA can effectively induce leukemia cell differentiation and apoptosis, it has strong tumor-promoting activity, and some studies have proved that TPA can induce apoptosis of myeloid progenitor cells while inducing apoptosis of leukemia cells.²⁹ Therefore, we still need new drugs that exert low toxicity to normal cells and increase or maintain the activity against anti-leukemia cells in the meantime. Structural modifications of discovered natural compounds, especially those with promising activity, are an important tool for the discovery of new drugs. In order to optimize the synthesis strategy, researchers devoted to summarizing the relationships between the structures and biological activities of phorbol esters, including antitumor activity, tumor-promoting activity, and anti-HIV activity.

Wender et al.³⁰ designed and synthesized novel phorbol ester dimers and presented that the affinity of these dimers for PKC binding is related to their tether length. Among the synthesized dimers, the inhibition constant for the binding of 4b to PKC was lowest (Ki = 0.1 nM) (see Figure 2(a)). El-Mekkawy et al.³¹ evaluated the anti-HIV-1 and tumor-promoting activities of 48 synthesized phorbol esters with phorbol and isophorbol as substrates. The inhibition of human immunodeficiency virus (HIV)-1 induced cytopathic effects (CPE) on MT-4 cells indicating anti-HIV-1 activity and the activation of PKC as indices of tumor-promoting activities. Compared to 1 and 1a, the hydroxyl acylation at position C-13 resulted in an increase in anti HIV activity. Compared to 8 and 8a, both CPE inhibition and PKC activation were lost after the hydroxyl group at C-20 was acylated. Compound 10 shown the strongest activity of PKC activation (see Figure 2(b)). Pagani et al.³² synthesized a series of aminoacylphorbol derivatives and evaluated their bioactivities. Among all compounds, 3b and 3d have the best activation effect on PKC. The results showed that the ability to activate PKC was closely associated with N,N-dimethylation of the C-12 position and the side chain R-substitution (see Figure 2(c)). As PKC is present in almost all cells, treating cancer with phorbol ester may damage normal cells. Theoretically, utilizing proteases present in tumors to design targeted drugs could target cancer cells and little impact on normal cells. Tarvainen et al.³³ attempted to implement targeted therapy by conjugating a 4β-pborbol derivative with the peptides that are cleaved by proteases, prostate-specific antigen (PSA), and prostate-specific membrane antigen (PSMA), which may be expressed by the prostate cancers.

Figure 2.

Chemical structures of reported phorbol derivatives. (a) dimers (b) esters (c) aminoacylphorbol derivatives.

In this study, we describe the preparation of selected phorbol 13,20-diesters (1A-8A) and phorbol 12,13,20-triesters (1B-7B, 9B), and evaluate their potential as inhibitors of leukemia cell proliferation. Four compounds exhibiting the most promising activity were selected for molecular docking with the PKC delta C1 domain. By combining the results of molecular docking with bioactivity data, we analyzed the relationships between the structures and activities of these compounds.

Results and discussion

Although there are a number of studies about bioactivities of phorbol esters,^34,35 a lack of a structure-activity study prompted us to design and synthesize phorbol analogs, evaluate their anti-leukemia activity, and clarify the structure-activity relationships (SARs).

With the aim to analyze the effect of an extra free hydroxyl group, phorbol 13,20-diesters and phorbol 12,13,20-triesters were prepared. Furthermore, to evaluate the effect of benzene on the biological activity, phorbols bearing p-chlorobenzoyl, cinnamoyl 4-benzoylbutyl chains were prepared. We also prepared a set of phorbol esters bearing heterocycle ester chains to analyze the effects of heteroatom-bearing substituents on activity.

Chemistry

A set of phorbol esters were prepared by selective esterification with different organic acids at C-12, C-13 and C-20 positions on the phorbol (see Scheme 1).

Scheme 1.

Synthesis of phorbol esters.

Phorbol 13,20-diesters and phorbol 12,13,20-triesters could be prepared from the parent compound phorbol, which is obtainable from croton oil by reported prodecure³⁶ or from commercial sources.

Only three of the five hydroxyl groups on phorbol are reactive, namely, those at positions C-12, C-13, and C-20. Due to steric effects, the three hydroxyl groups in phorbol are acylated in the following order: C-20 > C-13 > C-12.^23,37 Therefore, in the presence of a sufficient amount of carboxylic acid or acyl chloride, the alcohol at the C-12 position of phorbol 13,20-diesters will continue to be acylated to form phorbol 12,13,20-triesters under the same conditions.

The reactivity of hydroxy with acyl chloride is higher than that with carboxylic acids. In this study, subject to the availability of commercial reagents, we gave preference to acyl chlorides for esterification. 1A, 1B, 2A, 2B, 3A, and 3B were obtained by esterification of phorbol with the p-chlorobenzoyl chloride, acetyl chloride, and cinnamoyl chloride, respectively (see Figure 3).

Figure 3.

Chemical structures of phorbol derivatives.

Without commercially available acyl chloride, phorbol diesters and triesters were prepared by using the corresponding carboxylic acid instead. 4A, 4B, 5A and 5B were prepared by treatment of phorbol with 4-benzoylbutyric acid or glutarate, 4-dimethylaminopyridine (DMAP), and N,N′-dicyclohexylcarbodiimide (DCC, an activator of carboxyl groups). However, N,N-dicyclohexylurea (DCU) produced by DCC is difficult to remove. The remaining phorbol esters (6A, 6B, 7A, 7B, 8A, and 9B) were obtained by treatment of phorbol with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) as the carboxyl activator and DMAP as the catalyst (see Figure 3).

Biology

In vitro cytotoxicity of phorbol derivatives

A previous study showed that TPA induced cytodifferentiation or apoptosis in leukemia cell lines by activating PKC.³⁸ In order to explore whether the synthesized compounds had a similar ability to induce AML cells apoptosis, we tested their cytotoxicity. We chose three leukemia cell lines (K562, Jurkat, and HL-60) and one normal human cell line (HFL1) as a comparison for any selective cytotoxicity, and the results were considered as indicators of anti-AML activity and toxicity to normal cell.

The cell proliferation or apoptosis was detected via the cell counting kit 8 (CCK8) assay, and the IC₅₀ values were calculated for phorbol (as a negative control), TPA (as a positive control), and the results are shown in Table 1.

Table 1.

Activities of phorbol esters against leukemia XXXXXs (IC₅₀).

Compound	IC₅₀ (μM)
Compound	K562	Jurkat	HL-60	HFL1
1A	0.57 ± 0.03	>10	4.72 ± 0.61	>40
1B	0.37 ± 0.02	1.30 ± 0.02	5.09 ± 0.58	>40
2A	0.44 ± 0.05	1.30 ± 0.08	5.38 ± 0.52	>40
2B	1.43 ± 0.11	>10	2.08 ± 0.23	>40
3A	2.40 ± 0.33	>10	>10	>40
3B	2.40 ± 0.28	2.50 ± 0.13	2.69 ± 0.32	>40
4A	0.77 ± 0.09	>10	>10	>40
4B	3.26 ± 0.26	>10	>10	>40
5A	0.48 ± 0.09	>10	>10	>40
5B	0.24 ± 0.04	>10	>10	>40
6A	>10	>10	>10	>40
6B	>10	>10	>10	>40
7A	>10	>10	>10	>40
7B	>10	>10	>10	>40
8A	>10	>10	>10	>40
9B	>10	>10	>10	>40
Phorbol	>10	>10	>10	>40
TPA	0.02 ± 0.01	0.25 ± 0.01	1.16 ± 0.03	>40

Activities of the synthetic phorbol derivatives varied considerably against the three test leukemia cell lines. In general, the inhibitory activities of synthesized compounds were best against K562 cells. The inhibitory activity of most of the esterified phorbol derivatives against leukemia cells increased when compared against phorbol itself as a negative control, except for the derivatives produced by esterification with heterocyclic acids.

Among all 16 phorbol derivatives, 1B, 2A, and 3B exerted higher cytotoxicity relative to other synthetic compounds against Jurkat cell line with a lower IC₅₀ value (<10 μM). 1B (IC₅₀ = 1.30 μM) and 2A (IC₅₀ = 1.30 μM) had better activities than 3B (IC₅₀ = 2.50 μM). The other 13 compounds had no apparent cytotoxicity (IC₅₀ > 10 μM).

1A, 1B, 2A, 2B, and 3B exhibited strong in vitro cytotoxicity toward the HL-60 cell line, and 2B was the most active among them (IC₅₀ = 2.08 μM), followed by 3B (IC₅₀ = 2.69 μM). The other 11 compounds had no apparent cytotoxicity (IC₅₀ > 10 μM).

For K562 cell line, compounds 1A-5A and 1B-5B exhibited strong in vitro cytotoxicity. Compound 5B had the best cytotoxicity value (IC₅₀ = 0.24 μM), followed by 1B (IC₅₀ = 0.37 μM). The comparison between 2A and 2B, and between 4A and 4B, could be used to propose some SARs. In the absence of aryl moieties in the substituent groups, the anti-leukemia activities of phorbol derivatives in which two hydroxy groups were esterified by same substituent at C-13 and C-20 position were better than those in which three hydroxy groups were esterified by the same substituent at C-12, C-13, and C-20. It was shown that the substitution of secondary alcohol on C12 affects the activity of the compounds. The anti-leukemia activities of phorbol derivatives modified by short-chain saturated alkyl groups were better than those modified by long-chain saturated alkyl groups. On the contrary, when the substituent contains an aromatic group, the anti-leukemia activities of tri-substituted phorbol derivatives were better than those of di-substituted phorbol derivatives. In addition, the activities of the aryl substituent-containing phorbol esters increase with the increase of chain length of the substituent. Besides, 1A and 1B are more cytotoxic than 3A and 3B, presumably related to the presence of halogen chlorine atoms on the benzene rings of the 1A and 1B substituents. However, the cytotoxicity of derivatives whose hydroxyl groups were esterified by heterocyclic acids (neither tri-substituted nor di-substituted) decreased significantly (the IC₅₀ of compounds 7A-9B were all greater than 10 μM).

In addition, as shown in Table 1, the IC₅₀ values of phorbol derivatives 1A-9B against human lung fibroblasts (HFL-1) were greater than 40 μM, which indicated those derivatives had no cytotoxicity to normal human cells.

The mechanism of phorbol derivatives against leukemia cells

On account of TPA inhibits leukemia cells by inducing apoptosis,³⁸ we presumed that the general mechanism of the other phorbol derivatives promoting leukemia cells death is similar. The annexin V-FITC/PI double staining assay was used to explore the induction of apoptosis in K562 cells by 5B. Necrotic and late apoptotic cells are both stained by annexin V-FITC (annexin V conjugated with fluorescein isothiocyante) and PI (propidium iodide), whereas early apoptotic cells are only stained by annexin V-FITC.^39,40 The percentage of K562 cells treated by 5B in different physiological states was distinguished and quantitatively determined by flow cytometry (see Figure 4 and Table 2). The total apoptosis rate was the sum of the early and late apoptosis rates.

Figure 4.

5B promoted cell apoptosis of K562. The apoptosis rates were detected by flow cytometry analyses. (a) Control (b) 100 nM of 5B (c) A concentration of 200 nM of 5B (d) A concentration of 400 nM of 5B. Q1: the necrotic cells. Q2: the late apoptotic cells. Q4: the early apoptotic cells.

Table 2.

Apoptosis rate.

Concentration of 5B(nM)	Necrotic cells	Normal cells	Early apoptotic cells	Late apoptotic cells	Total apoptosis rate
0	0.17%	96.6%	0.94%	2.28%	3.22%
100	0.48%	81.4%	7.79%	10.3%	18.09%
200	0.74%	76.3%	7.38%	15.6%	22.98%
400	2.04%	66.0%	8.65%	23.3%	31.95%

The proportion of apoptotic cells increased as the concentration of compound 5B increased, and reached 31.95% at the concentration of 400 nM. The general mechanism of phorbol ester derivatives promoting leukemia cells death was similar to that of TPA, which both inhibit the growth of leukemia cells by inducing apoptosis, leading to cell death.

Docking analysis

According to previous studies, PKC activated by TPA generates substrate phosphorylation that induces mitogen-activated protein kinase (MAPK) cascades.⁴¹ MAPK cascades are associated with multiple physiological processes in cells, such as growth, differentiation, and apoptosis.

DAG is the natural substrate for PKC. Phorbol esters, a variety of natural products that can replace DAG binding with the PKC C1 domain, show higher affinity than DAG.⁴² This might be the reason for their ability to promote PKC activation. Phorbol derivatives were docked with PKCδC1B (PDB: 1PTR) in order to study the binding of different substitutions of phorbol to the protein.

PKCδC1B binds to phorbol 13-acetate through three residues (Gly-253, Thr-242, and Leu-251) by H-bonds. We chose the same binding groove to dock several phorbol derivatives with PKCδC1B (see Supplemental Figure S1). The docking conformation of TPA to C1B was the same with phorbol 13-acetate, the carbonyl group of C3 formed a H-bond with Gly-253, and the hydroxy groups of C20 formed two H-bonds with Gly-253, Thr-242, and Leu-251. The other compounds bind to residues differently due to the differences in position, number, and size of substituent.

Four synthesized compounds with the best activity (1B, 2A, 5A, and 5B) and TPA were docked to PKCδC1B (see supplemental Figure S1). The result of 1B docking to PKCδC1B showed the H-bond distance formed by carbonyl group of C3 with Gly-253 and by ester group of C20 with Gln-257 were 2.0 and 2.4 Å, respectively. The C9-OH of 2A formed two H-bonds with Gly-253, and their distances are 2.2 and 2.3 Å, respectively. The distance of H-bond, formed by C13-O of 2A bond with Gln-257, is 2.6 Å. The C9-OH of 5A formed two H-bonds with Gly-253, and their distances are 2.0 and 2.6 Å respectively. The docking results showed no significant H-bond between 5B and the protein because the substituent group was too large and not easily bent, making it difficult for 5B to enter the pocket and bind to the residues on PKCδC1B. However, in the previous experiments, 5B showed the best activity among the synthesized derivatives. We proposed that the reason for the better activity of 5B compared with the other phorbol esters synthesized in this study might be the more hydrophobic nature of the benzene ring, which makes it easier to enter cells and function, or might be that 5B does not work by binding to PKCδC1B, which needs to be further verified.

Conclusion

Using phorbol as a starting scaffold, 16 phorbol esters were synthesized by esterification with three types of organic acids, namely, straight chain acids, heterocyclic acids, and aromatic acids. By comparing the anti-leukemia cellular activities of the 16 compounds, some SARs can be deduced to provide a basis and direction for future study. In vitro cytotoxicity assay indicated that substituents containing benzene rings could increase the activities of the compounds, while those containing heterocycle had no cytotoxicity. The aryl substituent-containing derivatives in which all three active hydroxyl groups were esterified have stronger inhibition effect than those in which two active hydroxyl groups were esterified. Among them, 5B exhibited the best cytotoxicity. The annexin V-FITC/PI double staining assay indicated 5B inhibit the growth of leukemia cells by inducing apoptosis, leading to cell death. Docking analysis showed substituents containing bulky groups of benzene rings could make it difficult for compounds to enter the protein pocket, resulting in weak binding. This result contradicts our findings that 5B had the best overall activity, therefore it is speculated that the site or protein of action of 5B may be different from those reported previously. In addition, if we expect to achieve better anti-leukemia cell activity by improving the binding of the compound to PKC protein, the substituent group should not be too large. The above results provide some directions and references for the development of phorbol ester-based anti-leukemia drugs.

Experimental

Chemistry

The phorbol used in the experiment was purchased from Nanjing Spring & Autumn Biological Engineering Co., Ltd. Chromatographic analysis was performed on an Agilent 1260 UPLC-DAD-6530 ESI-QTOF MS (Agilent, USA) equipped with a ZORBAX SB C18 column (1.8 μm, 100 × 4.6 mm). Mass Hunter B0.05.0 software was used as the data processor. Mobile phase-A was 1% formic acid in water and mobile phase-B was methanol, and the flow rate was maintained at 0.3 mL/min. The gradient program was 0 min-30% A, 15 min-0% A, 30 min-0% A. The column temperature was set to 35 °C. The MS parameters were as following: Ionization mode: ESI, positive ion, Mass range: 100–1000 m/z, Nebulizer pressure: 50 psi, N₂ Drying Gas Flow: 10 mL·min⁻¹, N₂ Drying Gas Temp: 350 °C, Capillary Voltage: 4.0 kV. IR spectra were recorded on a Nexus 870 FT-IR spectrometer (Nicolet, USA) by the KBr disk method. The ¹H and ¹³C NMR spectra were measured on a Bruker DRX500 or 300 NMR spectrometer with ¹H and ¹³C NMR observed at 500 or 300 and 125 or 75 MHz (Bruker, Germany), using CDCl₃ or DMSO-d6 as solvent, and chemical shifts are given in δ ppm relative to tetramethylsilane (TMS). 2A and 2B have been reported as known compounds with synthetic methods. We have synthesized 16 phorbol esters by referring to the synthesis method of Li et al.⁴³

Synthesis of compounds 1A-3B ; general procedure

To a solution of phorbol (100 mg, 0.28 mmol, 1 equiv.) and Et₃N (163 mg, 1.62 mmol, 6 equiv.) in anhydrous CH₂Cl₂ (10 mL) was added p-chlorobenzoyl chloride (100 mg, 0.57 mmol, 2 equiv.) dropwise under nitrogen at 0 °C. After stirring for 10 min, DMAP (20 mg, 0.16 mmol, 0.6 equiv.) was added. After stirring at 25 °C for 4 h, the reaction was quenched with H₂O (20 mL). After partition, the aqueous layer was extracted with EtOAc (30 mL × 3), and the combined organic layers were washed with saturated aqueous NaHCO₃. The pH was adjusted to neutral with 1M hydrochloric acid, and then the combined organic layers were washed with brine (20 mL × 2). The solution was dried with anhydrous Na₂SO₄ and concentrated to dryness in vacuo. The residue was purified by flash column chromatography (PE/EtOAc 5:1 → 3:1) to give 1A (100 mg, 56.2%) as a white solid and 1B (46 mg, 21.9%) as a white solid.

Compound 1A white solid; 56.2%; FTIR (KBr) max: 3326, 3039, 1717, 1626, 1449, 1089, 740 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 7.92–8.07 (m, 4H, H-21, 25, 28, 32), 7.58–7.68 (m, 1H, H-1), 7.45 (m, 4H, H-22, 24, 29, 31), 5.84–5.85 (d, J = 5.5 Hz, 1H, H-7), 4.76 (s, 2H, H-20), 4.31–4.35 (m, 1H, H-12), 4.13–4.17 (d, J = 12.5 Hz, 1H, CH-OH), 3.34 (s, 1H, C-OH), 3.28 (s, 1H, C-OH), 1.83 (s, 3H, H-16), 1.14–1.33 (m, 15H, H-5, 8, 10, 11, 14, 17, 18, 19). ¹³C NMR (75 MHz, CDCl₃): δ 208.5, 168.2, 165.3, 160.7, 140.5, 139.5, 135.9, 133.3, 131.0, 129.0, 128.8, 78.3, 73.5, 70.1, 68.7, 63.2, 56.6, 45.4, 39.6, 39.12, 35.7, 29.7, 22.7, 16.8, 10.1. HRMS (ESI): m/z [M+Na]⁺ calcd for C₃₄H₃₄Cl₂O₈Na: 663.1528; found: 663.1520.

Compound 1B white solid; 21.9%; FTIR (KBr) max: 3442, 3013, 1677, 1611, 1443, 1097, 779 cm⁻¹¹; H NMR (300 MHz, CDCl₃) δ 7.92–8.08 (m, 6H, H-21, 25, 28, 32, 35, 39), 7.63–7.74 (m, 1H, H-1), 7.45 (m, 6H, H-22, 24, 29, 31, 36, 38), 5.79–5.83 (d, J = 10.0 Hz, 1H, H-7), 4.77 (s, 2H, H-20), 4.08–4.18 (m, 1H, H-12), 3.41 (s, 1H, C-OH), 3.45 (s, 1H, C-OH), 1.82 (s, 3H, H-16), 1.06–1.47 (m, 15H, H-5, 8, 10, 11, 14, 17, 18, 19). ¹³C NMR (75 MHz, CDCl₃): δ 208.8, 167.8, 165.3, 160.9, 140.4, 139.6, 135.7, 133.1, 131.0, 129.0, 128.4, 78.2, 78.0, 73.6, 70.0, 66.3, 56.2, 43.5, 39.1, 36.7, 23.9, 17.1, 14.6, 10.1. HRMS (ESI): m/z [M+Na]⁺ calcd for C₄₁H₃₇Cl₃O₉Na: 801.1401; found: 801.1401.

2A, 2B and 3A, 3B via a similar procedure, compounds 2A, 2B and 3A, 3B were prepared by phorbol with acetyl chloride and cinnamoyl chloride, respectively.

Compound 2A White solid; 52.2%; FTIR (KBr) max: 3326, 2928, 1625, 1450, 1152, 1088, 740 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.54–7.62 (m, 1H, H-1), 5.69–5.71 (d, J = 6.0 Hz, 1H, H-7), 4.52 (s, 2H, H-20), 3.99–4.02 (d, J = 5.0 Hz, 1H, C-OH), 3.48–3.49 (m, 1H, H-12), 3.23 (s, 1H, C-OH), 3.18 (s, 1H, C-OH), 1.74–2.59 (m, 15H, H-5, 8, 10, 11, 14, 16, 22, 24), 1.22–1.33 (m, 9H, H-17, 18, 19). ¹³C NMR (75 MHz, CDCl₃): δ 208.6, 174.0, 170.7, 160.4, 135.9, 133.1, 132.5, 78.3, 73.4, 69.4, 68.1, 56.7, 45.2, 39.4, 39.0, 35.3, 29.7, 23.7, 21.1, 21.0, 16.8, 15.1, 10.1. HRMS (ESI) m/z [M+Na]⁺ calcd for C₂₄H₃₂O₈Na: 471.1995; found: 471.1917.

Compound 2B White solid; 61.3%; FTIR (KBr) max: 3329, 2984, 1603, 1459, 1140, 1104, 741 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.54–7.75 (m, 1H, H-1), 5.39–5.43 (d, J = 11.5 Hz, 1H, H-7), 4.48 (s, 2H, H-20), 4.30–4.35 (m, 1H, H-12), 3.26 (m, 2H, C-OH), 1.74–2.59 (m, 18H, H-5, 8, 10, 11, 14, 16, 22, 24, 26), 1.22–1.33 (m, 9H, H-17, 18, 19). ¹³C NMR (75 MHz, CDCl₃) δ 208.5, 174.0, 170.7, 160.4, 135.9, 133.1, 132.5, 78.3, 73.4, 70.3, 69.4, 68.1, 56.7, 45.2, 39.4, 35.3, 29.7, 26.6, 23.7, 21.1, 16.8, 10.1. HRMS (ESI) m/z [M+Na]⁺ calcd for C₂₆H₃₄O₉Na: 513.2101; found: 513.2027.

Compound 3A White solid; 66.2%; FTIR (KBr) max: 3328, 3028, 1697, 1450, 1203, 1170, 768 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 7.73–7.76 (d, J = 8.1 Hz, 1H, H-27), 7.65–7.68 (d, J = 8.0 Hz, 1H, H-36), 7.55–7.57 (m, 4H, H-22, 24, 31, 33), 7.42–7.43 (m, 6H, H-21, 25, 26, 30, 34, 35), 6.43–6.51 (m, 3H, H-1, 28, 37), 5.81–5.82 (d, 1H, J = 2.3 Hz, H-7), 4.65 (s, 2H, H-20), 4.10–4.13 (d, J = 9.6 Hz, 1H, H-12), 3.27–3.30 (m, 3H, C-OH), 1.83–2.67 (m, 6H, H-5, 8, 10, 11, 14), 1.83 (s, 3H, H-16), 0.88–1.35 (m, 9H, H-17, 18, 19). ¹³C NMR (75 MHz, CDCl₃): δ 208.4, 169.4, 166.5, 159.5, 146.68, 145.14 136.0, 133.1, 130.8, 130.3, 129.0, 128.1, 117.9, 117.2, 78.3, 73.5, 69.7, 68.2, 56.7, 45.3, 39.6, 35.7, 29.6, 23.8, 16.9, 15.1, 14.0, 10.1. HRMS (ESI) m/z [M+Na]⁺ calcd for C₃₈H₄₀O₈Na: 647.2621; found: 647.2606.

Compound 3B White solid; 55.6%; FTIR (KBr) max: 3390, 3027, 1709, 1450, 1203, 1162, 766 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 7.67–7.74 (m, 3H, H-27, 36, 45), 7.53–7.58 (m, 6H, H-22, 24, 31, 33, 40, 42), 7.39–7.44 (m, 9H, H-21, 25, 26, 30, 34, 35, 43, 44, 45), 6.39–6.62 (m, 4H, H-1, 28, 37, 46), 5.85–5.87 (d, 1H, J = 5.3 Hz, H-12), 5.67–5.70 (d, 1H, J = 10.2 Hz, H-7), 4.66 (s, 2H, H-20), 3.38–3.39 (m, 2H, C-OH), 1.83–2.69 (m, 6H, H-5, 8, 10, 11, 14), 1.83 (s, 3H, H-16), 0.99–1.31 (m, 9H, H-17, 18, 19). ¹³C NMR (75 MHz, CDCl₃): δ 208.6, 169.1, 166.5, 160.8, 146.9, 145.1, 135.7, 134.4, 132.8, 130.3, 128.8, 128.4, 128.4, 124.8, 78.2, 73.7, 65.8, 56.3, 43.5, 39.6, 39.1, 36.6, 26.4, 23.9, 17.0, 14.5, 10.1. HRMS (ESI) m/z [M+Na]⁺ calcd for C₄₇H₄₆O₉Na: 777.3040; found: 777.3029.

Synthesis of compounds 4A-5B ; general procedure

To a solution of phorbol (100 mg, 0.28 mmol, 1 equiv.) and 5-oxohexanoic acid (178 mg, 1.37 mmol, 5 equiv.) in anhydrous CH₂Cl₂ (10 mL) was added DCC (20 mg, 0.12 mmol, 1.2 equiv.) dropwise under nitrogen at 0 °C. After stirring for 15 min, 4-dimethylaminopyridine (DMAP, 6 mg, 0.05 mmol, 0.5 equiv.) was added at same temperature and stirred for 10 min. After stirring at 25 °C for 4 h, the reaction was quenched with H₂O (20 mL). After partition, the aqueous layer was extracted with EtOAc (30 mL × 3), and the combined organic layers were washed with brine (20 mL × 2).

The solution was dried with anhydrous Na₂SO₄ and concentrated to dryness in vacuo. The residue was purified by flash column chromatography (PE/EtOAc 1:1 → PE/EtOAc 1:2) to give 4A (103 mg, 63.1%) as a white solid and 4B (111 mg, 56.8%) as a white solid.

Compound 4A White solid; 63.1%; FTIR (KBr) max: 3326, 2928, 1626, 1435, 1158, 1087, 737 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.53–7.62 (m, 1H, H-1), 5.69–5.71 (d, J = 6.0 Hz, 1H, H-7), 4.44–4.56 (m, 2H, H-20), 3.97–4.00 (d, J = 10.0 Hz, 1H, H-12), 3.23 (s, 1H, C-OH), 3.19 (s, 1H, C-OH), 1.92–2.57 (m, 27H, H-5, 8, 10, 11, 14, 16, 22, 23, 24, 26, 28, 29, 30, 32), 1.04–1.16 (m, 9H, H-17, 18, 19). ¹³C NMR (75 MHz, CDCl₃): δ 209.6, 208.5, 170.6, 160.4, 135.9, 133.1, 132.5, 79.3, 73.4, 70.1, 69.4, 68.1, 56.7, 45.15, 39.4, 39.0, 35.4, 31.7, 31.2, 29.3, 28.3, 20.9, 16.8, 14.1, 15.1, 10.1. HRMS (ESI) m/z [M+Na]⁺ calcd for C₃₂H₄₄O₁₀Na: 611.2832; found: 611.2838.

Compound 4B White solid; 56.8%; FTIR (KBr) max: 3326, 2927, 1626, 1448, 1152, 1085, 732 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.60–7.63 (d, J = 7.2 Hz, 1H, H-1), 5.72–5.73 (d, J = 4.3 Hz, 1H, H-7), 5.40–5.43 (d, J = 10.3 Hz, 1H, H-12), 4.40–4.43 (m, 2H, H-20), 3.25 (s, 2H, C-OH), 1.74–2.59 (m, 18H, H-5, 8, 10, 11, 14, 16, 22, 24, 26, 34, 35, 36, 38), 1.22–1.33 (m, 9H, H-17, 18, 19). ¹³C NMR (75 MHz, CDCl₃) δ 208.5, 207.5, 172.9, 160.5, 135.7, 132.9, 132.6, 78.1, 73.5, 69.1, 67.7, 65.6, 56.1, 42.1, 39.3, 36.2, 33.8, 33.4, 33.2, 29.9, 23.8, 19.1, 18.5, 14.3, 10.0. HRMS (ESI) m/z [M+Na]⁺ calcd for C₅₃H₅₈O₁₂Na: 723.3356; found: 723.3420.

5A and 5B were prepared by phorbol with glurate via a similar procedure.

Compound 5A White solid; 59.3%; FTIR (KBr) max: 3326, 3035, 1687, 1449, 1243, 1230, 740 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 7.97–7.99 (d, J = 7.0 Hz, 4H, H-21, 25, 32, 36), 7.78–7.81 (d, J = 9.0 Hz, 1H, H-1), 7.56–7.63(m, 2H, H-23, 34), 7.46–7.52 (m, 4H, H-22, 24, 33, 35), 5.7–5.72 (d, J = 7.0 Hz, 1H, H-7), 4.49–4.55 (m, 2H, H-20), 3.99–4.02 (m, 3H, OH), 4.08–4.12 (m, 1H, H-12), 3.06–3.14 (m, 4H, H-28, 39), 2.10–2.56 (m, 17H, H-5, 8, 10, 11, 14, 16, 28, 29, 40, 41), 1.06–1.10 (m, 9H, H-17, 18, 19). ¹³C NMR (75 MHz, CDCl₃): δ 208.6, 201.6, 169.1, 166.6, 146.9, 145.1, 135.7, 134.2, 128.8, 128.07, 78.2, 73.7, 69.3, 68.5, 65.8, 56.3, 47.1, 43.5, 39.6, 39.2, 36.6, 33.9, 33.7, 29.8, 26.2, 23.9, 17.0, 14.5. HRMS (ESI) m/z [M+Na]⁺ calcd for C₄₂H₄₈O₁₀Na: 735.3145; found: 735.3147.

Compound 5B White solid; 62.9%; FTIR (KBr) max: 3326, 3035, 1626, 1436, 1243, 1230, 739 cm⁻¹;-1 ¹H NMR (300 MHz, CDCl₃): δ 7.95–8.00 (m, 6H, H-21, 25, 32, 36, 43, 47), 7.54–7.59 (m, 3H, H-23, 34, 45), 7.45–7.51 (m, 6H, H-22, 24, 33, 35, 44, 46), 5.73–5.75 (d, J = 6.0 Hz, 1H, H-7), 5.46–5.49 (d, 1H, J = 10.0 Hz, H-1), 4.45–4.55 (m, 3H, H-12, 20), 3.45–3.55 (m, 6H, H-28, 39, 50), 2.10–2.56 (m, 21H, H-5, 8, 10, 11, 14, 16, 28, 29, 40, 41, 52, 53), 1.06–1.10 (m, 9H, H-17, 18, 19). ¹³C NMR (75 MHz, CDCl₃): 208.4, 199.3, 172.8, 156.8, 136.9, 135.6, 133.1, 132.7, 128.6, 127.9, 78.5, 73.4, 68.9, 65.8, 63.7, 55.3, 49.2, 39.4, 37.3, 33.9, 33.3, 29.7, 25.6, 24.9, 19.5, 16.7, 10.0; positive-ion HRMS (ESI) m/z [M+Na]⁺ calcd for C₅₃H₅₈O₁₂Na: 909.3826; found: 909.3824.

Synthesis of compounds 6A-9B ; general procedure

A three-necked flask was charged with phorbol (100 mg, 0.28 mmol, 1 equiv.) and levulinic acid (159 mg, 1.37 mmol, 5 equiv.) and then filled with nitrogen. EDCI (86 mg, 1.5 mmol, 5.5 equiv.) was added dropwise at 0 °C. After stirring for 15 min, DMAP (20 mg, 0.16 mmol, 0.6 equiv.) was added at same temperature and stirred for 10 min. After stirring at 25 °C for 4 h, the reaction was quenched with H₂O (20 mL). After partition, the aqueous layer was extracted with EtOAc (30 mL × 3), and the combined organic layers were washed with brine (20 mL × 2). The solution was dried with anhydrous Na₂SO₄ and concentrated to dryness in vacuo. The residue was purified by flash column chromatography (PE/EtOAc 1:2) to give 6A (87.8 mg, 58.1%) and 6B (38.2 mg, 22.3%) as a white solid.

Compound 6A White solid; 58.1%; FTIR (KBr) max: 3401 2956, 2923, 1703, 1364, 1157, 1079, 767 cm⁻¹; ¹H NMR (500 MHz, DMSO-d6) δ 7.55–7.51 (s, 1H, H-1), 5.79–5.76 (s, 1H, H-7), 5.65–5.62 (s, 1H, C-OH), 5.00–4.96 (s, 1H, C-OH), 4.77–4.73 (s, 1H, C-OH), 4.47–4.42 (s, 2H, H-20), 3.78–3.73 (d, J = 9.0 Hz, 1H, H-12), 2.79–2.70 (d, 5H, H-5, 8, 11, 14), 2.56–2.46 (m, 8H, H-22, 23, 27, 28), 2.18–2.09 (m, 10H, H-10, 16, 25, 30), 1.30–1.15 (m, 9H, H-17, 18, 19). ¹³C NMR (125 MHz, DMSO-d6) δ 208.1, 206.9, 176.1, 172.2, 159.6, 135.9, 132.7, 132.4, 77.5, 75.2, 73.0, 69.2, 68.1, 56.3, 44.9, 37.9, 37.9, 37.8, 37.6, 34.7, 29.7, 28.3, 27.9, 25.3, 23.8, 17.1, 15.2, 10.3. HRMS (ESI) m/z [M+Na]⁺ calcd for C₃₀H₄₀O₁₀Na: 583.2514; found: 583.2511.

Compound 6B White solid; 22.3%; FTIR (KBr) max: 3401, 2956, 2922, 1704, 1361, 1154, 1079, 765 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.62–7.58 (s, 1H, H-1), 5.77–5.72 (dd, J = 6.0, 2.0 Hz, 1H, H-7), 5.41–5.36 (d, J = 10.0 Hz, 1H, H-12), 4.54–4.45 (m, 2H, H-20), 3.29–3.23 (m, 2H, H-5), 2.90–2.71 (dqd, J = 12.0, 7.0 Hz, 6H, H-22, 27, 32), 2.64–2.53 (m, 8H, H-8, 14, 23, 28, 33), 2.47–2.43 (s, 1H, H-10), 2.21–2.19 (d, J = 2.0 Hz, 9H, H-25, 30, 35), 1.81–1.79 (dd, J = 3.0, 1.5 Hz, 3H, H-16), 1.38–1.19 (m, 9H, H-17, 18, 19), 1.15–1.12 (d, J = 5.0 Hz, 1H, H-11). ¹³C NMR (125 MHz, CDCl₃) δ 208.5, 206.3, 175.3, 172.6, 160.5, 135.4, 132.8, 132.6, 78.1, 77.2, 73.5, 69.3, 65.6, 56.0, 42.9, 39.2, 38.7, 38.0, 37.9, 37.7, 35.8, 29.7, 29.7, 29.6, 28.0, 28.0, 27.9, 25.9, 23.6, 16.6, 14.2, 10.0. HRMS (ESI) m/z [M+Na]⁺ calcd for C₃₅H₄₆O₁₂Na: 658.2989; found: 681.2894.

Via the same procedure, 7A and 7B, 8A, and 9B were prepared by phorbol with 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid, 1-methyl-1H-pyrazole-3-carboxylic acid and 4-methyl-1,2,3-thiadiazole-5-carboxylic acid, respectively.

Compound 7A White solid; 32.7%, FTIR (KBr) max: 3407, 2950, 2924, 1693, 1542, 1337, 1258, 1161, 1126, 1069, 773 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.00–7.92 (d, 2H, H-21, 29), 7.67–7.63 (m, 1H, H-1), 7.15–7.07 (m, 1H, H-25), 7.05–6.96 (m, 1H, H-33), 5.87–5.81 (dd, J = 5.5, 2.0 Hz, 1H, H-7), 4.73–4.70 (s, 2H, H-20), 4.20–4.11 (m, 2H, H-5), 4.04–3.98 (d, J = 12.0 Hz, 6H, H-24, 32), 3.34–3.28 (t, J = 5.6 Hz, 1H, H-12), 3.28–3.23 (m, 1H, H-11), 2.66–2.53 (d, 2H, H-8, 14), 2.09–2.08 (s, 1H, H-10), 1.36–1.13 (m, 12H, H-16, 17, 18, 19). ¹³C NMR (125 MHz, CDCl₃) δ 208.7, 164.2, 161.4, 160.3, 146.6, 135.8, 135.3, 133.1, 112.1, 109.3, 107.4, 77.1, 73.4, 69.5, 68.6, 60.3, 56.6, 45.0, 39.7, 38.8, 35.3, 29.6, 26.7, 23.7, 21.0, 16.6, 14.9, 14.1, 10.0. HRMS (ESI) m/z [M+Na]⁺ calcd for C₃₂H₃₆F₄N₄O₈N: 703.2361; found: 703.2363.

7B White solid; 50.3%, FTIR (KBr) max: 3408, 2958, 2924, 1715, 1546, 1342, 1259, 1163, 1125, 1076, 771 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.96–7.86 (d, J = 21.0 Hz, 3H, H-21, 27, 33), 7.70–7.64 (t, J = 2.0 Hz, 1H, H-1), 7.17–6.90 (m, 3H, H-25, 31, 36), 5.91–5.86 (m, 1H, H-7), 5.73–5.67 (d, J = 10.5 Hz, 1H, H-12), 4.77–4.68 (m, 2H, H-20), 4.04–3.98 (m, 9H, H-24, 30, 37), 3.42–3.35 (m, 2H, H-5), 2.66–2.60 (d, J = 14.5 Hz, 2H, H-8, 14), 2.39–2.31 (dt, J = 12.5, 6.5 Hz, 1H, H-11), 2.10–2.07 (s, 1H, H-10), 1.44–1.25 (m, 12H, H-16, 17, 18, 19). ¹³C NMR (125 MHz, CDCl₃) δ 208.5, 163.8, 161.5, 160.2, 146.2, 135.8, 135.2, 133.0, 112.5, 109.3, 108.8, 78.1, 77.7, 77.1, 73.5, 69.2, 66.0, 60.3, 56.0, 43.0, 39.7, 39.7, 39.6, 39.4, 38.8, 36.3, 29.5, 26.1, 23.7, 20.8, 16.7, 14.3, 10.0. HRMS (ESI) m/z [M+Na]⁺ calcd for C₃₈H₄₀F₆N₆O₉Na: 861.2653; found: 861.2654.

Compound 8A White solid; 57%; FTIR (KBr) max: 3375, 2959, 2924, 2853, 1712, 1503, 1387, 1261, 1211, 1109, 786 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) 7.46–7.40 (d, J = 9.6 Hz, 2H, H-27, 32), 7.32–7.29 (s, 1H, H-1), 6.87–6.84 (s, 1H, H-28), 6.84–6.80 (s, 1H, H-33), 5.86–5.81 (d, J = 5.5 Hz, 1H, H-7), 4.82–4.73 (d, J = 12.5 Hz, 2H, H-20), 4.22–4.16 (d, J = 10.0 Hz, 1H, C-OH), 4.05–4.01 (d, J = 3.5 Hz, 6H, H-29, 34), 3.36–3.26 (m, 2H, H-11, 12), 2.68–2.65 (s, 1H, H-8), 2.54–2.51 (s, 1H, H-14), 2.13–2.11 (s, 1H, H-10), 1.37–1.26 (m, 12H, H-16, 17, 18, 19). ¹³C NMR (125 MHz, CDCl₃) δ 208.6, 164.6, 161.8, 160.5, 142.6, 135.7, 132.8, 132.6, 131.5, 131.2, 109.6, 109.2, 73.3, 69.5, 68.7, 56.5, 44.9, 39.7, 39.5, 39.3, 38.9, 35.1, 31.3, 29.5, 26.8, 23.5, 16.8, 15.0, 9.9. HRMS (ESI) m/z [M+Na]⁺ calcd for C₃₀H₃₆N₄O₈Na: 603.2425; found: 603.2426.

Compound 9B Yellow solid; 30.2%, FTIR (KBr) max: 3390, 2955, 2917, 2849, 1708, 1537, 1312, 1236, 1160, 1077, 765 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.69–7.66 (s, 1H, H-1), 5.93–5.88 (d, J = 6.0 Hz, 1H, H-7), 5.78–5.72 (d, J = 10.5 Hz, 1H, H-12), 4.84–4.80 (s, 2H, H-20), 3.39–3.34 (s, 1H, H-10), 3.06–2.98 (m, 11H, H-5, 23, 27, 31), 2.74–2.56 (d, 2H, H-8, 14), 2.47–2.40 (dd, J = 10.5, 6.5 Hz, 1H, H-11), 1.38–1.30 (m, J = 12.0 Hz, 12H, H-16, 17, 18, 19). ¹³C NMR (125 MHz, CDCl₃) δ 208.2, 163.3, 162.7, 162.3, 159.7, 159.3, 138.3, 137.8, 135.3, 133.5, 133.2, 79.3, 73.3, 70.7, 67.7, 56.0, 42.9, 39.4, 38.8, 36.5, 31.3, 30.1, 29.5, 26.8, 23.8, 16.6, 14.4, 14.0, 13.9, 13.8, 10.0. HRMS (ESI) m/z [M+Na]⁺ calcd for C₃₂H₃₄N₆O₉S₃₂Na: 765.1442; found: 765.1312.

Biology

The CCK-8 (Cell counting kit-8) kit was used to detect the proliferation of tumor cells during the experiment. This is a WST-8 based kit for the detection of cell proliferation and cytotoxicity, which is highly sensitive and rapid. The MTT colourimetric assay was used to detect the proliferation of normal cells during the assay. Sixteen synthetic phorbol esters were tested for in vitro cytotoxicity against three leukemia cell lines (K562, Jurkat, and HL-60 cell lines).

In vitro anti-leukemia cells activity

Cells recovery: K562 cells, HL-60 cells, Jurkat cells, and HFL1 cells, which were frozen in liquid nitrogen tanks, were thawed in warm water at 37–39 °C. All resurgent cell lines were grown and maintained in RPMI-1640 medium with 10% fetal bovine serum (FBS) and with 1% penicillin/streptomycin at 37 °C in a 95% air −5% CO₂ humidified incubator. Observe the cell growth status in time, and replace the culture medium according to the situation.

Preparation of the test compound solutions: the experimental group, the blank control group, and the positive control group were set up with three replicates in each group. The sixteen phorbol esters, TPA, and phorbol were dissolved in DMSO at the concentration of 10 mmol L⁻¹ as stock solutions, and diluted in culture medium at the concentrations of 6.25, 25, 100, 400, and 1600 nM as working-solutions. TPA was used as the positive control (the experiments have been performed in triplicate).

Cell viability assay: three types of leukemia cells and HFL1 cells were seeded at a density of 5 × 10⁴ cells per well in 96-well plates and grown in 5% CO₂ at 37 °C. After 4 h of incubation, cells were treated with various concentrations of compounds or solvent control. Incubation was continued for 48 h at the same condition, and then CCK-8 (5 μL) was added to each well. After 4 h, the OD value of each well was measured on a microplate spectrophotometer at the wavelength of 450 nm.

For HFL1 cells, cells viability was detected by MTT assay. MTT dye solution (10 μL, 5 mg L⁻¹, PBS) was added to each well. After incubated in 5% CO₂ at 37 °C for 4 h, 100 μL DMSO was added to each well and the plates were shaked for 5 min. The OD value of each well was measured on a microplate spectrophotometer at the wavelength of 570 nm. The calculation formula of the cell proliferation inhibition rate is as follows:

Cell proliferation inhibition rate (%) = (1 − OD value of experimental group/OD value of blank group) × 100%

The inhibitory rate of each drug on each cell was measured under five concentration gradients, namely, 1600, 400, 100, 25, and 6.25 nmol L⁻¹, and the IC₅₀ value was calculated.

Annexin V-FITC/PI double staining assay for apoptosis

In this experiment, annexin V-FITC apoptosis detection kit was used to detect the apoptosis of tumor cells during the experiment.

According to the test results of anti-leukemia cells activity in vitro, K562 cells and compound 5B were selected to explore apoptosis. A blank control group and three experimental groups were set up in the experiment. The concentrations of work-solutions from low to high were 100, 200, and 400 nM.

The K562 cells were adjusted to a density of 5 × 10⁴ cells mL⁻¹, and 2 mL of the cell suspension was seeded in four disposable petri dishes in each dish. The petri dishes were then incubated for 4 h in 5% CO₂ at 37 °C. The 1640 medium was addedto dilute stock solution of compound 5B mentioned above from 10 to 0.1 mmol L⁻¹. The diluted solution was taken 8, 4, and 2 μL into three petri dishes. The other group was received no drugs as a blank control. After incubation for 48 h, cells were collected and re-suspended after adding the annexin V-FITC binding solution (195 μL). Annexin V-FITC (5 μL) and PI staining solution (10 μL) were successively added and fully mixed, followed by incubation for 20 min at room temperature in the dark. The apoptosis ratio was detected by flow cytometry.

Molecular docking

Molecular docking study of PCKδC1B (PDB code: 1PTR) and phorbol derivatives was performed in AutoDock-Vina program.⁴⁴ The ligand compound was allowed to dock anywhere in a 22 Å × 22 Å × 18 Å volume centered around active site, with the Lamarckian genetic algorithm (LGA). The maximum numbers of energy evaluation and generation were set to 250,000 and 25,000, respectively. A total of 20 separate docking runs were performed with the initial population of 20 individuals, and the lowest energy structure from each run was retained. Final docked conformations were clustered by using a tolerance of 1.0 Å root mean square deviation (RMSD). The complex with the strongest binding free energy was picked out as the best conformation for further analyzed.

Supplemental Material

sj-docx-1-chl-10.1177_17475198231180835 – Supplemental material for Synthesis and anti-leukemia activity of phorbol 13,20-diesters and phorbol 12,13,20-triesters

Supplemental material, sj-docx-1-chl-10.1177_17475198231180835 for Synthesis and anti-leukemia activity of phorbol 13,20-diesters and phorbol 12,13,20-triesters by Yan Wang, Yu Shan, Rui Feng, Siyu Wang, Linwei Li, Shu Xu, Yu Chen, Xu Feng, Jinyue Luo and Fei Liu in Journal of Chemical Research

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.

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 National Natural Science Foundation of China (grant nos 32000258 and 31970375).

ORCID iD

Fei Liu

Supplemental material

Supplemental material for this article is available online.

References

Yin

, et al. Medicine 2021; 100: e27884.

Aureli

Marziani

Sconocchia

, et al. Cancers 2021; 13: 6246.

Lin

Pagano

Expert Rev Hematol 2021; 14: 303.

Andreozzi

Massaro

Wittnebel

, et al. Int J Mol Sci 2022; 23: 3887.

Naqvi

SAR

Sherazi

Hassan

, et al. Eur J Inflamm 2022; 20: 1–20.

Wang

Yin

Zhou

, et al. Expert Opin Ther Pat 2012; 22: 977.

Wang

Arch Pharm 2022; 355: 2100469.

Liao

Wen

Wang

, et al. J Ethnopharmacol 2023; 302: 115885.

Nishizuka

Cancer 1989; 63: 1892.

10.

Weber

Hecker

Experientia 1978; 34: 679.

11.

Jiang

Duan

Feng

, et al. Phytochem Lett 2022; 49: 65.

12.

Kawakami

Inagaki

Nishimura

, et al. Chem Pharm Bull 2022; 70: 286.

13.

Huang

Yuan

, et al. J Org Chem 2022; 87: 9301.

14.

Guedes

Cruz

Lima

, et al. Ind Crops Prod 2014; 52: 537.

15.

Yunping

Ngoc Ha

Eunice

, et al. Ecotoxicol Environ Saf 2012; 84: 268.

16.

Fujiki

Suttajit

Rawangkan

, et al. J Cancer Res Clin Oncol 2017; 143: 1359.

17.

Albino

Antoniassi

de Faria-Machado

, et al. J Ethnopharmacol 2019; 241: 111970.

18.

Abou-Arab

Mahmoud

Ahmed

DMM

, et al. Heliyon 2019; 5: e01689.

19.

Sieber

Stuart

Spivak

JL.

Proc Natl Acad Sci U S A 1981; 78: 4402.

20.

Oskoueian

Abdullah

Ahmad

Molecules 2012; 17: 10816.

21.

Tsai

Rédei

Hohmann

, et al. Int J Mol Sci 2020; 21: 7579.

22.

Wender

Brabander

Harran

, et al. Tetrahed Lett 1998; 39: 8625.

23.

Ezzanad

Gómez-Oliva

Escobar-Montaño

, et al. J Med Chem 2021; 64: 6070.

24.

Han

Tong

, et al. Proc Natl Acad Sci U S A 1998; 95: 5362.

25.

Schaar

Goodell

Aisner

, et al. Cancer Chemother Pharmacol 2006; 57: 789.

26.

Takeda

Minowada

Bloch

Cancer Res 1982; 42: 5152.

27.

Zheng

Ryan

Patel

, et al. Int J Oncol 2005; 26: 441.

28.

Chen

, et al. Biosci Biotechnol Biochem 2020; 84: 2069.

29.

Ozawa

Miura

Hashimoto

, et al. Cancer Res 1983; 43: 2306.

30.

Wender

Koehler

MFT

Wright

, et al. Synthesis 1999; 1999: 1401.

31.

El-Mekkawy

Meselhy

Abdel-Hafez

, et al. Chem Pharm Bull 2002; 50: 523.

32.

Pagani

Navarrete

Fiebich

, et al. J Nat Prod 2010; 73: 447.

33.

Tarvainen

Zimmermann

Heinonen

, et al. ACS Med Chem Lett 2020; 11: 671.

34.

Wang

Yang

Liu

, et al. Bioorg Med Chem Lett 2015; 25: 1986.

35.

Zheng

Chang

Cui

, et al. Clin Cancer Res 2006; 12: 3444.

36.

Mishra

Estensen

Abdel-Monem

MM.

J Chromatogr A 1986; 369: 435.

37.

Bertolini

Giorgione

Harvey

, et al. J Org Chem 2003; 68: 5028.

38.

Shimizu

Taira

Senou

, et al. Cancer Lett 2002; 186: 67.

39.

Chen

Cheng

Wang

, et al. World J Gastroenterol 2008; 14: 2174.

40.

Shang

Ding

Xiang

Eur J Med Chem 2012; 48: 305.

41.

Schaar

Liu

Sharma

, et al. Leuk Res 2005; 29: 1171.

42.

You

Mathukumali

Das

J Biomol Struct Dyn 2023; 41: 1–14.

43.

Cheng

Zhao

, et al. Bioorgan Med Chem Lett 2021; 50: 128319.

44.

Trott

Olson

AJ.

J Comput Chem 2010; 31: 455.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

4.92 MB