Abstract
Hemslecin C, a compound with a novel skeleton, is synthesized by refluxing hemslecin A with KOH in alcohol. Hemslecin C was structurally characterized by specific optical rotation measurement, high-resolution mass spectroscopy, and nuclear magnetic resonance spectroscopic analysis. In addition, the molecular structure of hemslecin C was unambiguously determined by X-ray single-crystal determination. We found that its structure was different from that reported in the literature. The acetyl group of hemslecin A is transferred to the carbonyl α carbon of the side chain and is attacked by the 22-hydroxyl group to give a hemi-acetal that is dehydrated to give hemslecin C. The anticancer activity of this new skeleton is determined by sulforhodamine B assay method, and demonstrates excellent activity, thus providing a new framework for the development of anticancer drugs.
Introduction
With the development of traditional Chinese medicine, excellent anticancer activities in many traditional Chinese herbal medicines have been found. The extracts of many natural Chinese herbal medicines have also been proved to have anticancer activity. 1 Natural products provide various skeletons and exhibit diverse biological activities, their chemical constituents are determined and often modified by researchers to obtain more effective drugs.2,3 Hemsleya is a traditional Chinese medicinal plant which has the biological activities of detoxification and anti-inflammatory sterilization,4,5 and it has traditionally been used for treating hepatitis, coronary heart disease, tracheitis, and sore throat. 6
Hemslecin A (Figure 1), a cucurbitane-type tetracyclic triterpenoid, mainly exists in the rhizomes of Hemsleya, a cucurbitaceous plant. It demonstrates antitumor, bacteriostasis, anti-gastric ulcer, antipyretic, detoxification, pain relief, and anti-HIV activity; 7 however, hemslecin A is highly toxic to normal human cells.8,9 In order to study the structure–activity relationship of hemslecin A, researchers attempted to modify hemslecin A, obtaining new analogs, to reduce the toxicity to normal human cells while maintaining anticancer activity. In 1983, in an attempt to synthesize natural hemslecin B 7 (not detected), Wang obtained a new compound and named it hemslecin C (Scheme 1), and speculated its structure by 1H nuclear magnetic resonance (NMR) and infrared spectroscopy (IR) techniques. 10 In addition, the 13C NMR chemical shifts of hemslecin C were completely assigned by Qiu group through 13C NMR-DEPT, 1H- 1 H COSY, NOESY, 13C- 1 H COSY, and other NMR techniques in 2003. 11 However, the accurate structure of hemslecin C was not confirmed.
The structure of hemslecin A.
Synthetic route to hemslecin C in 1983: (i) KOH, EtOH, reflux, 1 h.
When we synthesized hemslecin C, the NMR data of the compound we obtained were the same as that reported in the literature,10,11 but its formula weight was different from the structure they speculated. In order to ascertain its correct structure, we analyzed it through single-crystal X-ray crystallography (Figure 2). Then we tested the cytotoxicity of hemslecin C against colon carcinoma (LOVO), ovarian carcinoma (SKOV3) and normal cardiac fibroblast cells (HCFB) by the sulforhodamine B (SRB) assay. The results showed that hemslecin C had inhibitory activity on LOVO and SKOV3, and demonstrated no cytotoxicity toward HCFB. Thus, our synthesized compound has improved selectivity against cell lines (cytotoxic to cancer cells but not to normal human cells) compared to hemslecin A and provides a good reference for future structural modifications of cucurbitacin compounds.
The revised structure of hemslecin C.
Results and discussion
Research on reaction mechanism
Hemslecin A is a tertiary alcoholic ester with a low saponification rate. However, the α-carbon of the branched carbonyl group produces carbon anions under alkaline conditions. Intramolecular attack on the carbonyl of the acetyl group (formation of five-membered ring intermediates) can far exceed the reaction rate of saponification, thus transferring the acetyl group from oxygen to carbon. Hemslecin C was synthesized by the reaction of the transferred acetyl group and the carbonyl group with the α hydroxy group (Scheme 2).
Proposed mechanism for the formation of hemslecin C: (i) KOH, EtOH, reflux, 2 h.
The spectral data of hemslecin C
White solid, high-resolution mass spectrometry (HRMS) (electrospray ionization (ESI)) calcd for C32H49O7 (M + H)+: 545.3478, found: 545.3442, 1111.6671 (2M + Na)+.
Characterization
In the IR spectrum, broad bands were observed in the regions of 3419 cm−1 which correspond to νO−H of the aliphatic hydroxy groups. The absorption frequencies around 1693 and 1618 cm−1 were attributed to νC=O of the original nucleus and νC=O of the side-chain ring, respectively. 12 Hemslecin C featured two absorption bands in the region of 200–300 nm in C2H5OH. The side-chain pentacyclic carbonyl transitions (π–π*) were noticed around 270 nm with high intensity. 13 Furthermore, the compound displayed a relatively intense band in the region of 204 nm which is ascribed to original nucleus carbonyl transitions (π–π*) (Figure 3).
Ultraviolet spectrum of hemslecin C in EtOH.
In the 1H NMR spectrum of the hemslecin C, the disappearance of the –CH2– proton signals of the methylene unit (3.37–3.28 and 3.09 ppm) revealed that the α position of the side chain is replaced, the methyl (1.91 ppm) of the acetyl group disappeared, and the chemical shift changed to a methyl at 2.28 ppm, indicating that enol isomerization took place at the acetyl group. 14 The 13C NMR spectrum of hemslecin C was also in accord with the proposed structure. The carbon signals at 186.7 and 111.3 ppm clearly indicated that new double bond had been generated on the branched chain. The carbon signal from the carbon (80.0 ppm) previously linked to the acetyl group changed to a carbon (71.5 ppm) linked to a hydroxy group. Hemslecin C is similar to the intermediate compound according to NMR, so it was easy to confuse their structures. 10 Furthermore, the molecular weight of hemslecin C was determined using ESI-MS, and the experimental m/z values were in good agreement with the theoretically calculated values. In the mass spectrum, the peak due to hemslecin C [M + H]+ occurred at m/z 545.3443 and [2M + Na]+ at m/z 1111.6671, which indicated to us that there were some discrepancies between our compound and the structure reported in the literature.
X-Ray crystal structure
The single-crystal X-ray diffraction study of hemslecin C was performed to confirm the structure of the obtained compound. The crystallographic data with refinement parameters, selected bond lengths, and bond angles are given in the Supplemental Material. The acetyl group of the side chain of hemslecin A was transferred to the α carbon of the branched chain carbonyl, and then the acetyl group attacked the branched hydroxy carbon to form a new pentacyclic compound. The X-ray crystal structure of hemslecin C is shown in Figure 4. The selected bond lengths and angles are given in Table 1. In the data, we observe that the bond angles of O(2)–C(5)–C(6), O(1)–C(4)–C(3), and O(4)–C(19)–C(20) are 107.2 (3)°, 108.9 (3)°, and 111.7 (3)°, respectively. So the chirality of hydroxy groups was not changed. The newly formed double bond C(25)–C(26) in the branched chain has a bond length of 1.344 (5) Å. The two carbon-oxygen bonds are C(22)–O(6): 1.459(4) Å and C(26)–O(6): 1.362(4) Å. The bond angle of C(23)–C(22)–C(20) is 115.7(3)° and that of C(25)–C(26)–C(27) is 132.0 (4)°. These data prove the spatial characteristics of the five-membered ring.
X-ray crystal structure of hemslecin C.
Selected bond lengths (Å) and angles (°) for hemslecin C.
Cytotoxicity
In vitro antitumor cytotoxicity assays were performed. Briefly, the cytotoxicities of hemslecin C against SKOV3 (ovarian carcinoma), LOVO (colon carcinoma), and HCFB (human cardiac fibroblasts) cells were measured by the SRB assay with IC50 values of 8.7, 9.7, and ⩾100 μM, respectively (Table 2).
Cytotoxicity of hemslecin A and hemslecin C.
IC50: half maximal inhibitory concentration.
Conclusion
The product obtained in 1983 may be the same as the product obtained by us, because the NMR, IR, and melting point data we obtained are the same as those reported in the literature, but due to the limitations of the conditions at that time, their analysis has inevitably made mistakes. Our structure was confirmed by X-ray crystallography, and the product was characterized by ultraviolet (UV), IR, HRMS, and NMR spectroscopy. The cytotoxicity of hemslecin C was determined by the SRB method, and it not only reduces the toxicity to human normal cell (HCFB cell) but also maintains potent antiproliferative activities against SKOV3 and LOVO cells. This new skeleton provides a significant reference point for improving the selectivity of cucurbitacins, and modification with hemslecin C as a lead compound also has a significant impact on the anticancer research of natural drugs.
Experimental
Materials
Dimethyl sulfoxide, potassium hydroxide, ethanol, hexane, dichloromethane, SRB, fetal bovine serum, sodium bicarbonate, and other materials were obtained from commercial sources and used without further purification
Apparatus
HRMS was performed on an AB SCIEX X500R Accurate Mass Q-TOF by using ESI. The NMR spectra were recorded on a Bruker AM-400 spectrometer (Billerica, MA) with tetramethylsilane as the internal standard.
Synthesis
The flasks used in the reaction were heated under vacuum for 30 min and purged with N2 for 10 min. To a solution of hemslecin A (562 mg, 1 mmol) and KOH (186 mg, 3 mmol) in ethanol (50 mL) was refluxed for 2 h. A new spot appeared according to thin-layer chromatography (TLC). 10 After air-cooling, the reaction mixture was poured into a separating funnel and separated. The aqueous layer was extracted with ethyl acetate (3 × 40 mL). The combined organic layers were washed with bine and dried over Na2SO4. The organic layer was filtered and concentrated under reduced pressure to dryness to provide the crude product, which was purified by silica gel chromatography (eluted with CH2Cl2/CH3OH, 20:1) to give the product (419 mg, 77%) as a white solid.
SRB assay
The cell proliferation of adherent cells was determined by the SRB assay. Cells were seeded in 96-well plates and then treated with different concentrations of drugs. After 72 h of incubation, the cells were fixed with 10% trichloroacetic acid for 1 h at 4 °C, washed five times with tap water, and air-dried. Cells that survived were stained with 0.4% (w/v) SRB for 20 min at room temperature and washed five times with 1% acetic acid. Bound SRB was solubilized with 10 mM Tris and the absorbance was measured at 540 nm. 15
X-ray structure determination
A single crystal of hemslecin C suitable for X-ray diffraction analysis was grown by slow evaporation of CH2Cl2/hexane solutions of hemslecin C at 4 °C. X-ray diffraction was performed on a Bruker D8 Venture diffractometer. 16 Data were collected at 173 K by using a graphite monochromator with CuKα radiation (1.54178 Å) in the ω–ϕ scanning mode. Using ShelXL-2014/7, the structure was solved with the ShelXS17 structure solution program using direct methods and refined with the ShelXL-2014/7 refinement package using least squares minimization. Hydrogen and oxygen atoms were located using the geometric method.
Supplemental Material
supplementary_material – Supplemental material for Structural revision and pharmacological activity of hemslecin C
Supplemental material, supplementary_material for Structural revision and pharmacological activity of hemslecin C by Kun Yu, Xinmei Yang, Ying Li, Xue Cui, Bo Liu and Qingqiang Yao 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 (81903473) and was also supported by the Innovation Project of Shandong Academy of Medical Sciences.
References
Supplementary Material
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