New Scaffold for Angiogenesis Inhibitors Discovered by Targeted Chemical Transformations of Wondonin Natural Products

Abstract

The structure of wondonin marine natural products was renovated to attain new drug-like scaffolds. Wondonins have novel antiangiogenic properties without overt cytotoxicity. However, the chemical instability and synthetic complexity of wondonins have hindered their development as a new type of antiangiogenesis agent. Using a structure-based bioisosterism, the benzodioxole moiety was changed to benzothiazole, and the imidazole moiety was replaced by 1,2,3-triazole. Our efforts resulted in a new scaffold with enhanced antiangiogenic activity and minimized cytotoxicity. One compound with this scaffold effectively inhibited hyaloid vessel formation in diabetic retinopathy mimic zebrafish model. The biological findings together suggested the potential of the scaffold as a lead structure for development of antiangiogenic drugs with novel functions and as a probe to elucidate new biological mechanisms associated with angiogenesis.

Nature has provided an immense array of bioactive chemical entities across structurally diverse scaffolds.¹ Some scaffolds are suitable for direct employment as templates in the generation of compounds with drug-like properties, but other scaffolds are not directly suitable for use in drug development, mostly due to unfavorable physicochemical properties. In the latter case, transformative structural renovations are desirable to extract new scaffolds that are more relevant for drug discovery.

Wondonins and isowondonins (1 and 2, Figure 1) are structurally unique marine alkaloids that were identified from an association of the sponges Poecillatra wondoensis and Jaspis sp.^2,3 These marine natural products possess an unusual skeleton with a five-membered acetal ring. Attached vinyl sulfate and histamine moieties distinguish wondonins from other natural products. Wondonins 1 and isowondonins 2 differ in the configuration of the styryl double bond. We previously defined the relative and absolute stereochemistries of isowondonins via the asymmetric synthesis and electronic circular dichroism calculations.⁴

Figure 1.

Chemical structures of wondonins and analogues.

The family of wondonins has notable antiangiogenic properties⁵ by suppressing the expression of HIF-1α and vascular endothelial growth factor (VEGF) in endothelial cells. In contrast to other antiangiogenesis agents, a remarkable biological property of wondonins is that they inhibit angiogenesis without overt cytotoxicity.^2,3,5 Angiogenesis is the formation of new blood vessels from pre-existing ones, which occurs under normal physiological conditions such as embryonic vascular development and wound healing.⁶ Aberrant angiogenesis is observed in disease processes including diabetic ocular neovascularization, duodenal ulcers, arthritis, and cancer.⁷ VEGF is one of the most important factors regulating angiogenesis.⁸ VEGF is enriched in the endothelial cells for new blood vessels and plays roles in vascular permeability, vascular network growth, and vessel cell survival.⁸ Therefore, the modulation of the VEGF-mediated angiogenic pathway is considered an important target in drug discovery programs for diseases associated with the overactivation of angiogenesis.^9,10

Several VEGF-signaling pathway inhibitors are on the market for the treatment of cancer or are in clinical trials.⁹⁻¹¹ A large array of compounds are also currently in preclinical development. Most studies of VEGF pathway inhibitors have focused on the identification of compounds with high inhibitory effects on human umbilical vascular endothelial cell (HUVEC) proliferation that could be used for the treatment of cancer.⁹⁻¹¹ In addition to their use as anticancer drugs, VEGF pathway inhibitors have been proposed as potential therapeutic agents for obesity and diabetic retinopathy.¹²⁻¹⁴ However, few drugs that specifically inhibit the pathways for such diseases are available.

We conceived that the relevant biological properties of novel antiangiogenesis agents targeting the VEGF pathway, especially for the treatment of diabetic retinopathy, should include minimized cytotoxicity or no cytotoxicity to avoid side effects in normal cells. In this regard, we consider structurally unique wondonins a promising starting point for the generation of a new lead compound. However, the wondonin family presents challenges due to their chemical instability. The five-membered acetal ring and the vinyl sulfate moiety are unstable, particularly under acidic conditions. In our laboratory, wondonins readily decompose unless stored at freezer temperatures and remain free of acid or base contaminants. Another concern is their difficult synthesis. In our total synthesis, installation of the vinyl sulfate and histamine moieties was not trivial.⁴ In addition, the burdens associated with the introduction of two stereocenters are significant. Therefore, our primary aim in this study was to discover a new scaffold from the structures of wondonins that is chemically stable and easily accessible with drug-like properties. In this study, a series of wondonin analogues was designed, synthesized, and evaluated. Our efforts resulted in structurally distinct drug-like scaffolds with more potent antiangiogenic activity without overt cytotoxicity to HUVECs.

As an initial structural modification of wondonins, the imidazole moiety was replaced by its bioisostere, 1,2,3-triazole. This replacement was made because the regioselective N-alkylation of a substituted-1H-imidazole is difficult due to the rapid tautomeric equilibrium.^4,15 In addition, the preparation of a wide range of suitably substituted-1H-imidazoles is not trivial.¹⁶ By contrast, a 1,4-disubstituted 1,2,3-triazole moiety can be easily introduced via a Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC) reaction.¹⁷ Given the instability and synthetic difficulty, the vinyl sulfate moiety of wondonins was substituted for a simple ethyl group, which is neither significantly electron donating nor withdrawing. Although ethyl might not be an optimal substituent for activity, it is a good starting point for further optimization due to its unbiased physicochemical properties. Such structural modifications resulted in analogue 3 (Figures 1 and 2).

Figure 2.

Chemical structures of wondonin A (1a), analogues 3–12, and sunitinib and measurement of their effects on the VEGF-induced tube formation and cell growth of HUVECs. Tube formation was determined by counting the number of intact tubes after treatment with each compound in the absence or presence of VEGF (50 ng/mL) for 8 h. The value represents the mean IC₅₀ in triplicate experiments against VEGF-induced tube formation by HUVECs. Cell growth was measured by the MTT assay after treatment with each compound in the presence of VEGF (50 ng/mL) for 24 h. The value represents the mean IC₅₀ in triplicate experiments against VEGF-induced cell growth in HUVECs. The fold change was calculated as the ratio of the IC₅₀ values of tube formation and cell growth. For the values of IC₅₀ ± SD, see Table S2 in the Supporting Information.

The designed analogue 3 was prepared as a mixture of diastereomers that were inseparable (see Scheme S1 and synthetic details in the Supporting Information). Its antiangiogenic activity was assessed by measuring its effects on VEGF-induced tube formation and growth of HUVECs in comparison with those of natural wondonin A (1a) (see Figure S1 in the Supporting Information).¹⁸ The effect on tube formation was determined by counting the intact tube number under a microscope, and the IC₅₀ of the compound was calculated by comparison with the control group. The effect on cell growth was measured in the MTT assay. As shown in Figure 2, the triazole analogue 3 was 1.5-fold more potent than the parent natural product 1a in the tube formation assay and was nearly as inactive as the parent 1a in inhibiting the growth of HUVECs. Furthermore, analogue 3 did not show overt cytotoxicity against various cancer cell lines (see Table S1 in the Supporting Information), similar to wondonin A. These results suggested that the triazole and ethyl moieties in 3 could serve as bioisosteric replacements for the imidazole and vinyl sulfate moieties in wondonins.

The next replacement was implemented in the five-membered acetal ring. With the aim of eliminating the stereocenter at C1 and endowing stability, the benzodioxole ring of 3 was replaced with several benzo-fused heteroaromatics, including benzofuran, benzothiazole, and benzoxazole (Figures 1 and 2). The benzofuran analogue 4 displayed considerably improved potency in the inhibition of tube formation (Figure 2) and was approximately 7 and 5 times more potent than the natural product 1a and benzodioxole analogue 3, respectively. In contrast to 1a and 3, benzofuran 4 exhibited a moderate inhibitory effect on the VEGF-induced growth of HUVECs. The benzoxazole analogue 5 did not show notable activity against tube formation. By contrast, the benzothiazole analogue 6 was approximately two times more potent than the benzofuran analogue 4 and less cytotoxic to cells. Based on the selectivity index of the ratio of the IC₅₀ for tube formation to the IC₅₀ for cell growth, the benzothiazole analogue 6 had more desirable biological properties than the other heteroaromatic analogues. Thus, benzothiazole 6 was used as a new lead scaffold for further structural modification.¹⁹

To assess the importance of the catechol unit in the biological activity of benzothiazole analogue 6, two catecholic hydroxyl groups were replaced with a methoxy group (Figure 2). The methoxy analogue 7 was approximately 2–3 times less potent than the parent 6 both in the tube formation assay and the cell growth inhibition assay, and thus, the selectivity index remained nearly unchanged. Because replacement with a methoxy group did not cause a significant decrease in activity, a vicinal dimethoxy-substituted benzene ring was used in further structural modifications instead of the metabolically and chemically vulnerable catechol unit.

The next structural modification involved the N,N-dimethyl amino group of 7. Typical results are shown in Figure 2. The inhibitory effects on both tube formation and cell growth were sensitive to amino group modifications. All compounds with different amino groups (8–12) exhibited increased tube formation inhibition together with an increase in cell growth inhibition. However, the degrees of inhibition differed, indicating that these two effects were not correlated. For example, while compound 9, with a cyclopentylamino group, was approximately eight times more potent than compound 7 in tube formation inhibition and five times more potent in cell growth inhibition, compound 12, with a piperidine moiety, was approximately 13 times more potent in tube formation inhibition but showed only a 2-fold increase in cell growth inhibition. Of the analogues examined in this study, 12 exhibited the highest selectivity index, up to 75, with pronounced inhibitory activity (IC₅₀ = 0.631 μM) against tube formation. The selectivity index of the typical antiangiogenic tyrosine kinase inhibitor sunitinib²⁰ was also determined in our evaluation system, although its mechanism of action could differ from those of wondonins and the synthesized analogues. Sunitinib exhibited a much lower selectivity index of 18 compared to compound 12; it showed a similar level of inhibitory potency (IC₅₀ = 0.546 μM) against tube formation as 12 but a 4.8-fold higher inhibitory effect on the cell growth of HUVECs.

Further biological studies were performed with compound 12 to understand the molecular mechanisms of the new benzothiazole scaffold. The enhancement of endothelial cell migration and tube formation capacity by VEGF was effectively inhibited by 12 in a concentration-dependent manner (Figure 3A,B). Biomarker analysis revealed that treatment of HUVECs with VEGF (50 ng/mL) for 20 min led to the activation of PI3K (p-PI3K), AKT (p-AKT), and eNOS (p-eNOS), whereas pretreatment with 12 for 30 min significantly suppressed the activation processes in endothelial cells (Figure 3C). In addition, the levels of VEGFR2 (p-VEGFR2) activated by VEGF were also suppressed by 12, which subsequently inhibited the activation of SRC (p-SRC)/FAK (p-FAK) in VEGF-stimulated HUVECs. The phosphorylation of VEGFR2 (Tyr1175) affects the activation of PI3K signaling, and this event leads to an increase in vascular permeability.²¹ In the VEGF signaling pathway, SRC kinases are downstream of VEGFR2 and regulate vascular permeability and angiogenesis.²² The activation of FAK is also an important contributor markedly involved in endothelial cell migration, proliferation, and adherens junction integrity.²³ Therefore, the present data suggest that the inhibition of cell migration and neovascular tube formation by compound 12 is partly associated with modulation of the signaling axis of VEGFR2-mediated PI3K/AKT/eNOS and the SRC/FAK signaling pathway in HUVECs.

Figure 3.

Antiangiogenic activities of 12. (A, A-1) Inhibition of vascular tube formation in HUVECs by compound 12. The capillary structures were photographed, and the total tube length was quantified. The length of the capillary tubes formed were compared with that obtained for the VEGF-treated control groups. (B, B-1) Inhibition of cell migration in HUVECs by compound 12. The migrated cells were counted and compared with the VEGF-treated control groups. (C) Effects of compound 12 on the VEGFR2 signaling pathway. The cells were treated with various concentrations of compound 12 for 30 min and then stimulated with VEGF (50 ng/mL) for 20 min. β-Actin was used as a loading control. (D) Inhibition of hyaloid vessels in HG-treated zebrafish larvae by compound 12. The hyaloid vessel diameters were measured at a position near the optic disc (red circle) using ImageJ software (scale bar = 50 μm). Quantification of the number of primary vessel branches compared with that in the HG-treated control groups. (A, B, D) P values were calculated by ANOVA using Tukey’s test (^###P < 0.001, ^##P < 0.01; *P < 0.05, **P < 0.01, ***P < 0.001, n = 3).

Recent findings suggest the existence of cross-activation between beta-3-integrin and VEGFR-2 during angiogenesis.^24,25 In particular, integrin is associated with stimulation of the migration of endothelial cells. Although we did not directly detect the effect of wondonin analogues on integrin expression, we found that SRC/FAK activation, which is downstream of the integrin signaling pathway and correlated with cell migration, was suppressed by compound 12. This finding suggests that compound 12 is also able to affect integrin signaling indirectly.

To further determine whether compound 12 directly affects the activity of kinases that are closely associated with angiogenic processes, compound 12 was evaluated at 10 μM using KINOMEscan (DiscoverX Co., Fremont, CA, USA) against a panel of 30 kinases, including VEGFR2. Kinase activity was determined based on the binding capacity between the each kinase and the corresponding ligand. Compound 12 did not significantly inhibit the kinase activities tested (see Table S3 in the Supporting Information) and weakly inhibited only three kinases, namely, CSNK1A1, PRKCl, and MEK1 (over 40% inhibition at 10 μM). Therefore, the antiangiogenic activity of compound 12 might be more relevant in the regulation of VEGF/VEGFR2 axis suppression than in the direct inhibition of angiogenesis-associated kinase activity.

Since diabetic retinopathy (DR) is associated with the angiogenic processes,²⁶ we evaluated the effects of compound 12 on neovascular formation in a DR-mimic animal model. In general, DR leads to progressive vascular occlusions, ultimately resulting in blindness with pericyte loss and vessel thickening.²⁷ DR also increases vascular permeability, which leads to macula edema.²⁸ Zebrafish larvae treated with high levels of glucose (HG) were employed as a mimic of mammalian DR models.²⁹ Transgenic zebrafish (flk:EGFP) embryos (3 dpf) were treated with 130 mM glucose for 3 days to induced DR-mimic hyaloid vessels in the larval eye lenses. As shown in Figure 3D, HG-treated zebrafish larvae exhibited significantly increased hyaloid vessel diameters in isolated eye lenses. However, treatment with 12 effectively suppressed the increase in hyaloid vessel diameters induced by HG in a concentration-dependent manner.³⁰ In particular, treatment with 2.5 μM compound 12 markedly reduced the hyaloid vessel diameters in the optic disc area. These results suggest that the reduction in HG-induced hyaloid vessel diameters by 12 might also be associated with antiangiogenic activity in the in vivo animal model.

To evaluate pharmaceutical properties of compound 12, the aqueous solubility, permeability, plasma stability, and metabolic stability were examined (Table 1). A notable property of 12 is its high plasma stability that is important for the achievement of in vivo activity. Compound 12 showed plasma stability of 95% remaining intact after 30 min incubation with human plasma. In the metabolic stability study using human liver microsomes, a modest microsomal stability was observed. Compound 12 exhibited high MDCK cell permeability but low aqueous solubility (Biopharmaceutics Classification System Class 2), probably due to its high lipophilicity. Our theoretical calculations indicated that the physicochemical properties of 12 were in compliance with Lipinski’s rule of five and Veber’s rule for druglikeness except a logP value (5.955) (see Table S4 in the Supporting Information). Functional group modifications of 12 are in progress in our laboratory, such as replacement of a methoxy group with a more polar bioisosteric moiety, to improve solubility and metabolic stability by lowering the lipophilicity.

Table 1. Preliminary ADME Study of 12.

pKa	solub (mM)a	MDCK Papp,A-B (10–6 cm/s)b	plasma stability (%)c	microsomal stability (%)d
9.24	0.024	11	95	31

^aMeasured by the CheqSol method at 25 °C.

^bApparent permeability coefficients (P_app,A-B) across MDCK cell monolayers.

^cThe percentage of parent compound remaining after 30 min incubation in human plasma.

^dThe percentage of parent compound remaining after 30 min incubation in human liver microsomes.

In summary, using the structure of wondonin marine natural products, we created a novel lead scaffold for the development of a new type of antiangiogenesis agent that possesses potent antiangiogenic activity with minimized cytotoxicity to avoid side effects. The analogues with benzothiazole and 1,2,3-triazole rings, in place of benzodioxole and imidazole moieties, respectively, displayed the desired biological properties. The representative compound 12, which was more easily synthesized than the parent natural product, exhibited an excellent selectivity index with pronounced inhibitory activity. The in vivo antiangiogenic activity was also assessed using DR-mimic zebrafish, which demonstrated the high potential of the new scaffold for the treatment of diseases requiring antiangiogenesis agents with minimized cytotoxicity. The precise target of this new class of compounds remains unclear. Further elucidation of the specific molecular target of compound 12 is in progress. In addition, we are currently investigating the structure–activity relationship in this series of compounds in more detail to elucidate the minimal pharmacophore required for the selectivity between antiangiogenic activity and cytotoxicity to HUVECs. The results of these efforts will be reported in due course.

Glossary

ABBREVIATIONS

ADME

absorption distribution metabolism excretion

AKT

protein kinase B

CSNK1A1

casein kinase 1 isoform alpha

EGM-2

endothelial cell growth medium-2

eNOS

endothelial NOS

FAK

focal adhesion kinase

FBS

fetal bovine serum

HIF-1α

hypoxia inducible factor 1-alpha

MDCK

Madin–Darby canine kidney

MEK1

mitogen-activated protein kinase 1

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PI3K

phosphoinositide 3-kinase

PRKCl

protein kinase C iota

PVDF

polyvinylidene difluoride

qPCR

quantitative PCR

VEGFR

vascular endothelial growth factor receptor

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.7b00281.

• Synthetic Schemes S1 and S2, experimental procedures and spectroscopic data for all new compounds, supplementary Tables S1–S4, Figures S1 and S2, and HPLC analysis of biologically tested compounds (PDF)

Author Contributions

^† S.Y. and J.O. contributed equally to this work. All authors have given approval to the final version of the manuscript.

This work was supported by the Mid-Career Researcher Program (NRF-2016R1A2A1A05005375) of the National Research Foundation (NRF) grant funded by the Korea government (MSIP). This work was also supported by NRF (Grant 2009-0083533) grant funded by the Korean Government (MEST).

The authors declare no competing financial interest.