Synthesis and Antiangiogenic Activity of Novel Gambogic Acid Derivatives

Gambogic acid (GA) is in a phase II clinical trial as an antitumor and antiangiogenesis agent. In this study, 36 GA derivatives were synthesized and screened in a zebrafish model to evaluate their antiangiogenic activity and toxicity. Derivatives 4, 32, 35,36 effectively suppressed the formation of newly grown blood vessels and showed lower toxicities than GA as evaluated by zebrafish heart rates and mortalities. They also exhibited more potent migration and HUVEC tube formation inhibiting activities than GA. Among them, 36 was the most potent one, suggesting that it may serve as a potential new antiangiogenesis candidate with low toxicity. Additionally, 36 showed comparable antiproliferative activity to HUVECs and five tumor cell lines but low cytotoxicity to LO2 cells.


Introduction
Angiogenesis, or new blood vessel growth, is defined as a process in which a network of new blood vessels emerge from preexisting vessels. The inhibition of angiogenesis is a promising tool to fight cancer [1]. Tumor angiogenesis, which involves multiple cellular processes including endothelial cell (EC) activation, invasion, migration, proliferation, tube formation and capillary network formation, is OPEN ACCESS essential for cancer growth and metastasis [1][2][3][4]. Without the development and progression of new blood vessels, tumors can rarely grow over a few mm 3 in size or metastasize to other organs [5,6]. Therefore, inhibition of angiogenesis is an important target for cancer therapy [7,8].
The zebrafish (Danio rerio) screening model for anti-angiogenesis activity has become increasingly popular in different fields such as pharmacology, toxicology and developmental and evolutionary biology since the 1970s [9][10][11]. It is regarded as a powerful tool in the study of human diseases [9]. Indeed, zebrafish possess many advantages that make them a highly useful valid tumor model system for antiangiogenesis, including relatively small size (up to 3 cm), low cost, and being easy to house and maintain in large quantities [9,10]. The application of transgenesis to the zebrafish resulted in the ability to spatially and temporally control gene expression and to create or increase in vivo imaging capabilities [9]. Transgenic zebrafish models expressing green fluorescent protein (GFP) in vascular endothelial cells (ECs) are significantly useful for studying the formation of the vasculature in vivo [12].
Cultured ECs are also widely appreciated tools in angiogenesis research [5]. ECs commonly used for more than 30 years now are human umbilical vein ECs (HUVECs) which are easily obtained and less prone to acquire drug resistance [11]. EC migration in vivo is indispensable for wound-healing as well as for the formation of new vessels [4]. The capacity of the ECs to form tube-like structures when cultured on a semi-natural matrix is another typical in vitro angiogenesis assay [2]. Therefore, the phenotype of tumor ECs is subject to investigation to identify putative target molecules for interference to discover novel angiogenesis-inhibiting agents.
Gambogic acid is a natural product isolated from the resin of the Garcinia hurburyi tree found in Southeast Asia. It is in a phase II clinical trial in China and is widely known as an antiangiogenesis and antitumor agent [13][14][15][16][17][18][19]. However, until now, no study has focused on the structural modification of GA to evaluate antiangiogenic activity and related toxicity. In this study, the antiangiogenic activities and toxicities of GA and GA derivatives were evaluated for the first time using a zebrafish screening model.
In this paper, we synthesized and screened 36 GA derivatives. Compounds 4, 32, 35, 36 effectively suppressed the formation of newly grown segmental blood vessels in the zebrafish-based assay and were less toxic to zebrafish compared to GA. These four derivatives also exhibited more potent inhibitory potencies against the migration and tube formation of HUVECs in vitro than GA. Importantly, among them, 36 was the most potent one, with a 98.3% migration inhibition rate and 100% tube formation inhibitory rate at a concentration of 2 μM, suggesting that this derivative may serve as a potential new antiangiogenesis candidate.

Chemistry
Gambogic acid was isolated from the easily and widely available gamboge resin in an overall yield of approximately 5%. It was purified by changing the crude extract from the gamboge resin into a pyridine salt, followed by recrystallization [20]. There are many functional groups in the structure of GA which could potentially be modified such as 30-carboxy, 8-ketone, methyl group at C-35 or C-39, 6-hydroxy, 9,10-double bond in the α,β-unsaturated ketone, carbon-carbon double bond at C-32/33 or C-37/38. However, earlier structure activity relationship (SAR) studies had identified that the 9,10-double bond in the α,β-unsaturated ketone is critical for activity, and the modifications of 6-hydroxy and 8-ketone did not improve the activity dramatically [21,22]. Therefore, to develop novel GA derivatives as inhibitors of angiogenesis, we elected to modify the 30-carboxy and carbon-carbon double bond at C-32/33 and C-37/38.
The modification of the 30-carboxy group is depicted in Schemes 1, 2 and 3. Coupling of GA with various alcohols or phenols in the presence of DMAP and EDCI produced the corresponding GA esters 1-28 in 39%-92% yield (Scheme 1). Reaction of GA with two sulfhydryl compounds in the presence of DMAP and EDCI produced the corresponding GA thioesters 29-30 in 59%-68% yield (Scheme 2). Scheme 1. Synthesis of derivatives 1-28. Reagents  The carbon-carbon double bonds at C-32/33 and C-37/38 were modified as shown in Scheme 4 [21]. Reaction of 35 with m-CPBA for about 6 h at 25 °C, produced the GA derivative 36 containing two epoxy groups at C-32/33 and C-37/38 in 71% yield.

Antiangiogenic Activity and Toxicity in the Transgenic Zebrafish Model
As stated previously, zebrafish is regarded as a powerful tool in the study of human diseases. Transgenic zebrafish models expressing green fluorescent protein (GFP) in vascular endothelial cells (ECs) are significantly useful for studying the formation of the vasculature in vivo. In this study, all GA derivatives were incubated with the transgenic friend leukemia integration-1 (fli-1): enhanced GFP zebrafish embryos which carried a fli-1 promoter which can drive the expression of GFP in the whole endothelium [23].
At a concentration of 1 μM, compounds 16, 20, 22, 23 and 31 were inactive, the antiangiogenic rates of compound 9 was less than 25%, and the antiangiogenic rates of compounds 4, 26, 32, 35, 36 and GA were 25%-50% (Table 1). The remaining derivatives (not listed) were inactive even at concentrations exceeding 10 μM. However, GA caused zebrafish embryo death at concentrations of 2.5 μM and 10 μM. The antiangiogenic activities of 9 and 26 were moderate at 2.5 μM and not enhanced greatly when the concentration reached 10 μM. Importantly, derivatives 4, 32, 35, 36 exhibited strong antiangiogenic activities inhibiting intersomitic blood vessels from the dorsal aorta of zebrafish in a concentration-dependent manner (Figures 1 and 2).  To investigate the toxicity of these four compounds, the heart rates and the mortality rates of zebrafish embryos were recorded (Tables 2 and 3). The heart rate of zebrafish in the control group was 117 beats/min. At a concentration of 1 µM, treated with compounds 4, 32, 35, 36 respectively, the heart rates of zebrafish were slightly reduced to about 80 beats/min but the heart rate of zebrafish treated with GA decreased to 36 beats/min. Moreover, at 2.5 µM and 10 µM GA, 73.3% and 100% of zebrafish embryos, respectively, were dead, whilst when treated with each of the four compounds at the same concentrations, the mortality rates were less than 20% and the heart rates were still about half of the control group. Therefore, we suggest that compounds 4, 32, 35, 36 were less toxic than GA in zebrafish screening model.

Effects on the HUVECs Migration
The migration of ECs is an important step of angiogenesis [16,24]. The continuity of vascular ECs is the most important factor in preserving vessel wall function. Migration of ECs is essential to the recovery of this continuity after vascular ECs injury [24]. Since compounds 4, 32, 35 and 36 presented more antiangiogenic potency than GA, a would-healing migration assay was therefore applied to evaluate their ability to inhibit HUVECs migration [8]. As illustrated in Figure 3(I), the HUVECs migrated into the wound area actively (between the two white dash lines) under the compound-free condition (control).  At a concentration of 0.25 μM, these four compounds showed significant differences compared with control, but only compounds 35, 36 manifested more potency compared with GA, and stronger inhibition than those of 4 and 32. At a concentration of 0.5 μM, derivatives 35, 36 had similar potency to GA. At a concentration of 1 μM, compounds 35, 36 were nearly equal to GA and the migratory rates were 3.6, 1.7 and 2.2% respectively [ Figure 3(II)]. The effects of these compounds were not due to cell death, because the HUVECs did not show any morphological changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation.

Effects on the HUVECs Tube Formation
In the later stages of angiogenesis, tube formation of ECs is also an important process [16]. Thus, a Matrigel tube formation assay was applied to assess the ability of HUVECs to form ECs vascular structures which is believed to be significant in new vessel formation [24].
In the control, HUVECs showed the highest mobility on Matrigel and formed an intact tubular network in 6 h ( Figure 4I). In comparison with the control group, the inhibitory rates of samples treated with 4, 32, 35, 36 and GA at a concentration of 0.5 μM were 43.5, 39.1, 37.0, 56.5 and 26.1%, respectively. Among them, 36 had the strongest inhibitory activity with a 2-fold improvement in the inhibition of tube formation when compared with GA. Most significantly, at concentrations exceeding 1 μM, derivative 36 could completely inhibit tubular structures ( Figure 4II), demonstrating that 36 was the most potent antiangiogenesis agent.

Antiproliferative and Anticancer Activity
Inhibiting the proliferation of ECs is another important strategy for antiangiogenesis [25]. Therefore, this experiment was designed to demonstrate the effects of the compounds on antiangiogenesis, HUVEC migration and tube formation. As the exposure time of these derivatives was 24 h in the migration assay and 6 h in the tube formation assay, the effect of the incubation for 24 h in the presence of these compounds on HUVECs proliferation was then tested. Cell viability was assessed by the MTT method [25]. We can see from Table 4 that these compounds had different antiproliferative activities. in DMEM containing each compound (0.5 or 1 or 2 μM) were added to the Matrigel. Control was treated with DMEM alone. After incubation for 6 h at 37 °C, capillary networks were photographed and quantified (Olympus CKX41, magnification: 100×); (II) Rates of the HUVECs tube formation. The number of intact tubes was counted in five randomly chosen regions and expressed as the percentage of the control. The results were expressed as mean ± SD. ** p < 0.01; *** p < 0.005 compared with control.

Structure-Activity Relationship
As a result of zebrafish embryos assay, wound-healing assay, tube formation assay and MTT assay, we found that compared to the unsubstituted GA, introduction of electron-withdrawing groups (EWG; e.g., nitro for 1, 5, 8, 14; halogenations including chlorine, bromine and fluorine atoms for 2, 17, 19, 24; cyano for 12; carbonyl for 5, 11, 15, 16, 20, 23) and electron-donating groups on the phenyl ring (EDG; e.g., methoxyl for 3, 13, 22, 31; 2-methoxyethyl for 18; ethyl for 21; 2,4,4-trimethylpentan-2-yl for 25) failed to improve their antiangiogenic potencies in contrast to GA. The acetamido group at the para position of the phenyl ring (para-acetamido for 4 and 35) improved the antiangiogenic activity. A heterocyclic ring substituent (thiazole for 32) showed more potency compared with GA. These properties may contribute to the basic groups which can improve the solubility of the compounds in acidic media [22]. It has been reported that epoxidation of the carboncarbon double bond at C-32/33, C-37/38 could improve antiangiogenic activity [21]. Therefore, we modified the carbon-carbon double bond on the structure of 35 to produce derivative 36. We found that it indeed improved antiangiogenic activity.

Chemistry
All commercial chemicals and solvents were reagent grade and were used without further treatment unless otherwise noted. Chemistry reagents of analytical grade were purchased from Chengdu Changzheng Chemical Factory, Sichuan, China. Analytical thin-layer chromatography was performed on 0.20 mm Silica Gel 60 F 254 plates (Qingdao Ocean Chemical Factory, Shandong, China). Nuclear magnetic resonance spectra (NMR) were recorded at 400 MHz on a Varian model Gemini 400 spectrometer (Varian, Palo Alto, CA, USA) and peaks are reported in parts per million. Mass spectra (MS) were measured by a Q-TOF Premier mass spectrometer (Micromass, Manchester, UK).
General procedure for the synthesis of gambogates, gambogthioesters and gambogamides 1-35. A mixture of gambogic acid (0.18 mmol), R-OH or R-SH or R-NH 2 (0.20 mmol), DMAP (0.10 mmol) and EDCI (0.30 mmol) in CH 2 Cl 2 (10 mL) was stirred at 25 °C overnight. The solution was washed by hydrochloric acid solution (2 × 10 mL) and water (2 × 10 mL). The combined organic layer was dried and concentrated to yield the crude product, which was purified by silica gel column chromatography. (1)        gelatin-coated culture flasks in M-199 medium with 20% fetal bovine serum (FBS), 2 ng/mL VEGF, 10 ng/mL basic FGF, 100 IU/mL penicillin, and 100 μg/mL streptomycin; all of them were obtained from the American Type Culture Collection (ATCC) and all cell lines were incubated in an atmosphere of 5% CO 2 at 37 °C.

Wound-Healing Assay
HUVECs were seeded in 6-well plates precoated with 0.1% gelatin, and grown overnight to confluence. The monolayer cells were wounded by scratching with 10 μL pipet tips and washed twice with serum-free DMEM (Dulbecco's modified eagle medium) to remove the nonadherent cells and then replaced by serum-free DMEM with the indicated concentrations of compounds for 24 h. Images were taken at 0 h and 24 h independently after incubation at 37 °C, 5% CO 2 environment. The migrated HUVECs were manually counted. The values were observed from four randomly selected fields.

Tube Formation Assay
Matrigel was dissolved at 4 °C overnight. Each well of prechilled 96-well plates was coated with 50 μL of Matrigel, incubated and solidified at 37 °C for 45 min. After removing the unsolidified fluid, HUVECs at the density of 1 × 10 4 were cultured in DMEM containing the indicated concentrations of compounds for 6 h. Controls were treated with the DMEM alone. Images were digitally captured and quantitatively analyzed (Olympus). The numbers and line lengths of the circular tubules formed by the cells were calculated manually. The values were observed from five randomly selected fields.

MTT Assay
Cells were treated with various concentrations of compound in 96-well culture plates for 24 h in final volumes of 200 μL (5 × 10 3 cells/well). Then 20 μL of MTT solution (5 mg/mL) was added to each well, and cells were incubated for an additional 3 h. Then the medium was carefully removed, and precipitates were dissolved in 150 μL of DMSO, shaken mechanically for 30 min, and then absorbance values at a wavelength of 570 nm were taken on a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Values were calculated using percentage of growth versus untreated control. The IC 50 was defined as the concentration that caused 50% inhibition of cell proliferation, and was calculated by SAS statistical software.

Conclusions
In conclusion, 36 GA derivatives possessed different antiangiogenic activities. Derivatives 4, 32, 35, 36 blocked the new blood vessels from the dorsal aorta in the zebrafish embryos assay, exhibited strong inhibitory effects on the migration, tube formation and proliferation of HUVECs, additionally they showed relatively lower toxicities to zebrafish and LO2 cell line than GA. The four compounds were also capable to effectively inhibit the five tumor cell lines tested (A549, HepG2, HCT116, K562 and HeLa). Given the results of this comprehensive research, we found that compound 36 was the most potent one and may serve as a potential new antiangiogenesis candidate.