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. 2022 Sep 13;13(12):1864–1869. doi: 10.1021/acsmedchemlett.2c00308

Benzo[d]isoxazole Derivatives as Hypoxia-Inducible Factor (HIF)-1α Inhibitors

Zian Xue †, Huiti Li †, Wenhao Xie †, Ying Xu ‡,*, Lu Zhou †,*, Zhi-bei Qu †,*
PMCID: PMC9743424  PMID: 36518694

Abstract

graphic file with name ml2c00308_0008.jpg

Hypoxia-inducible factor, also known as HIF, is a transcriptional factor universally found in mammalian cells. HIF-1 is one of the HIF-families and acts as a heterodimer consisting of α and β subunits. It is found to play significant roles in pathologic conditions such as tumor development and metastasis. Here, we first report benzo[d]isoxazole analogues as HIF-1α transcription inhibitors. Thereby, we designed and synthesized 26 benzo[d]isoxazole derivatives and evaluated their inhibitory activities against HIF-1α transcription in HEK293T cells by a dual-luciferase gene reporter assay. Among them, compounds 15 and 31 showed the best efficacy in a cell-based assay with an IC50 value of 24 nM and have potential antitumor effects for further development.

Keywords: Hypoxia-inducible factor (HIF)-1α, Inhibitor, Benzo[d]isoxazole, Antitumor

Cancer is the second worldwide leading cause of death just behind cardiovascular diseases.1,2 Recently, researchers noted that cancer incidence is still rising after evaluating 36 types of cancer in 185 countries and predicted that new cancer cases will increase 61.7% by 2040.3 Current cancer treatments like chemotherapeutic agents and radioactive therapy4 have been frequently used, but severe adverse effects ranging from myelosuppression,5 nausea and vomiting,6 hair loss,7 infertility,8 to organ damage,9−12 among others, were often involved. Therefore, it raises the tremendous need for developing potent anticancer drugs.13

Tumor progression is a complex process that needs diverse growth factors for invasion and expansion.14 Therefore, tumors upregulate their oncogenes such as Akt, MAP2K, VEGF, EPO, or RAS15−19 to survive. Growth factors like erythropoietin (EPO) and vascular endothelial growth factor (VEGF) are keys to the process of tumor angiogenesis,18,19 thus aiding tumor progression. Hanahan recently concluded new dimensions of cancer hallmarks in 2022 in that the tumor microenvironment can cause broad epigenetic changes.20 Hypoxia is a main characteristic in the tumor microenvironment due to a lack of vascularization, which can influence and alter the methylome21 as well as translational control22 and thus cause aberrant growth.23,24 As a transcriptional factor, hypoxia-inducible factor (HIF) is what cells mainly depend on to adapt to a hypoxia environment and plays a significant role in regulating VEGF via binding to the HRE (hypoxic response element). The upregulated VEGF can thus promote tumor vascularization and progression.25

Hypoxia-inducible factor is a transcriptional factor that is essential in tumor progression.18,19,25,26 Due to the highly active metabolism of tumors, oxygen is abnormally consumed and then causes a hypoxic microenvironment in tumor tissues.27 HIF expression is highly responsive to such hypoxia and thus gets upregulated. HIF-1 is a member of the basic helix–loop–helix PER-ARNT-SIM (bHLH-PAS) protein family with two subunits as oxygen-sensitive HIF-1α and a constituted HIF-1β unit to form a heterodimer.25,28,29 Under hypoxia, HIF-1α is stable at a low concentration of O2 without being hydroxylated by prolyl hydroxylase (PHD). The hydroxyl modification can be recognized by von Hippel-Lindau proteins for HIF degradation with a ubiquitin-proteasome system.18,25 The stabilized HIF-1 complex can work as an efficient transcription factor and anchor to DNA, thus recruiting series of cofactors and upregulating downstream gene expression such as VEGF and EPO intermediating tumor progression.25 Some researchers also found the elevated HIF-1 will lead to therapy resistance.30,31 Meanwhile, many researchers have found that HIF-1 overexpression is linked to tumor poor prognosis.32,33 Therefore, an anti-HIF-1α strategy has become a potential cancer treatment and adjuvant therapy for patients who are resistant to chemo or radiation therapy. Until now, many HIF-1 inhibitors have been developed for preclinical anticancer drugs in recent years.34

Though several clinical trials are ongoing (data can be found on ClinicalTrials.gov), until now, there is no drug targeting HIF-1 transcription approved for cancer treatment by the FDA (Food and Drug Administration). Due to the urgent need of HIF-1 transcription inhibitors, thousands of compounds were tested in laboratories. Naik and colleagues identified novel (E)-phenoxyacrylic amide derivatives with the best IC50 value of 0.12 μM;35 An and colleagues identified a novel scaffold through a ring-truncated deguelin strategy with the best IC50 value of 100 nM.34 Natural products strongylophorines were identified by Mohammed and colleagues to contain an anti-HIF-1 transcriptional effect with the best IC50 value of 6 μM36 (Figure 1). Here, we highlighted benzo[d]isoxazole derivatives as HIF-1α transcriptional inhibitors with a simple structure and potent activity. There are only five simple steps in the synthesis with an overall yield of about 45%. In the first four reactions, the key structure benzo[d]isoxazole-3-carboxylic acid can be easily purified without adopting chromatography and thus is promising to be scaled up. With the simple structure, benzo[d]isoxazole derivatives in our findings generally have low molecular weight (around 250) and less fancy organic groups, which suited Lipinski’s “Rule of Five”. Therefore, we totally designed and synthesized 26 benzo[d]isoxazole derivatives and found 15 and 31 have the best activities against HIF-1α transcription with remarkably low IC50 values of 24 nM. To our knowledge, among all HIF-1α transcriptional inhibitors, a simple structure compound with an IC50 value of 24 nM is hard-won and of great value.

Figure 1.

Figure 1

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Reported HIF-1 transcriptional inhibitors.

The protocompound structure of N-phenylbenzo[d]isoxazole-3-carboxamide was first synthesized by Shah and colleagues in 1971,37 but no one had reported its biological activity until now. Inspired by the previous work of Nakamura’s and Li’s groups who reported benzimidazole and benzimidazole analogues with anti-HIF-1α activities,38,39 we have been thinking if benzo[d]isoxazole derivatives would be efficient HIF-1α inhibitors. Thus, we tested a series of N-phenylbenzo[d]isoxazole-3-carboxamide compounds for HIF-1α inhibition activities using a dual-luciferase assay. We provided the synthesis route for the derivatives in Scheme 1.

Scheme 1. Synthetic Scheme for Derivatives of N-Phenylbenzo[d]isoxazole-3-carboxamide.

Scheme 1

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Reagents and conditions: (a) ethanol, sulfuric acid, reflux, 2 h; (b) Na, ethanol; hydrochloric acid; (c) NaH, diglyme, 150 °C, 5 h; (d) sulfuric acid, 85 °C, 4 h; (e) HATU, DIPEA, r.t., 6 h.

The lead compound 1 was evaluated by a dual-luciferase assay to give an IC50 value of 0.31 μM. To investigate the necessity of the benzene ring fused with isoxazole, we first designed and synthesized 2 together with amide inversion structure 9 but found a loss of anti-HIF-1α activity (Figure 2). This finding shows the structure of benzo[d]isoxazole-3-carboxamide played a crucial role in anti-HIF-1α activity.

Figure 2.

Figure 2

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Structures of compounds and modification strategy. (a) Deletion of benzene ring on benzoisoxazole; (b) amide inversion; (c) changings of formanilide group; (d) modification on formanilide.

Then, a series of compounds were synthesized to investigate whether the activities would be sustained when changing the amine condensed to benzo[d]isoxazole-3-carboxylic acid. Our study showed that only imine directly followed ring structures barely exhibited biological activities. The benzene ring next to the acylamino site could be replaced to have reserved activity such as 2-pyridine with an IC50 value of 0.94 μM (8), 4-pyridine with an IC50 value of 10 μM (7), and cyclohexane with an IC50 value of 5.4 μM (3). Since none of their activities exceeded that with benzene ring at the acylamino site, we then designed a series of compounds with substituent groups on an acylamino benzene ring to investigate substituent effects.

Surprisingly, the experimental results showed that compounds with para-substituent groups on acylamino benzene (15, 18, 21, and 31) had strongly enhanced anti-HIF capacity regardless of the electronic effect, with a best IC50 value of 24 nM (Figure 3).

Figure 3.

Figure 3

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Structures of the best two potent HIF-1α transcription inhibitors.

Compounds with meta-substituted groups (14, 17, 20, and 30), though they had lower activities than para-analogues, maintained the HIF-1α transcriptional inhibiting activities. Substitution on the ortho-position would lead to relatively reduced (16 and 19) or even loss of activities (13).

We finally synthesized compounds with larger or bulky groups adjacent to an acylamino benzene ring to evaluate the tolerability of the meta/para-position. We found compounds with large substituted groups on the meta- or para-position of acylamino benzene reserved anti-HIF-1α activity (23, 24, 25, 27, and 28), suggesting that there is still room for further modification.

All synthesized compound structures and biological activities are listed in Table 1, and their anti-HIF-1 transcriptional activities were tested by a dual-luciferase gene reporter assay, in which the florescence intensities can be the readout indicating HIF transcriptional activity. The dual-luciferase system had been testified by LW6, which was one of the most commercial available HIF-1α inhibitors applied by many researchers as a positive compound.40−42 None of our compounds showed cytotoxicity under 50 μM according to the readout of Renilla fluorescence measurements, which means benzo[d]isoxazole analogues inhibited HIF-1 transcriptional activities without killing the cells.

Table 1. Inhibitory Effects against HIF-1α Transcription of All Benzo[d]isoxazole Evaluated by Dual-Luciferase Assay In Vitro.

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To test whether the most potent compounds 15 and 31 can lead to an ultimately biological effect, VEGF and PDK1 (another downstream gene regulated by HIF-1) mRNA expressions were analyzed by using quantitative real-time PCR (Q-PCR). Eight different concentrations were selected, and concentration-dependent mRNA decreases were observed after compounds were treated for 24 h under hypoxia. The compound resulting in mRNA-decreased IC50 values showed good coincidence to the IC50s detected by a dual-luciferase gene report assay (around 25 nM). We wondered whether this trend would remain under normoxia, so we used the same concentrations above to test VEGF and PDK1 mRNA expressions under normoxia on HEK293T cells. However, under normoxia, no matter what concentration of compounds was treated, VEGF and PDK1 mRNA expressions remained almost unchanged when compared to the control group, which means compounds 15 and 31 did not downregulate VEGF and PDK1 mRNA expressions under normoxia (Figure 4). The expressions of protein VEGF were also tested. VEGF showed a 20% decrease after 24 h of being treated with compound 31 (Figure S2). To further solidify our findings, HIF-1α protein expressions were evaluated under hypoxia on HEK-293T cells. We used LW6 as our positive control, which can reduce HIF-1α protein expression, along with compounds 15 and 31 to be tested. Our results showed that when treated with different concentrations, the HIF-1α protein expressions under hypoxia were unaffected, which implies the selected compounds did not affect HIF-1α protein expressions (Figure 5). These results together are a good complement to show that benzo[d]isoxazole derivatives are on-target to inhibit HIF-1α transcriptional activity without affecting HIF-1α expressions and regulate downstream gene expression.

Figure 4.

Figure 4

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VEGF and PDK1 expression when treated with 15 and 31 in different concentrations. (A–D) Compounds 15 and 31 induced VEGF and PDK1 mRNA reduction under hypoxia. (E–H) Compounds 15 and 31 induced VEGF and PDK1 mRNA reduction under normoxia.

Figure 5.

Figure 5

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Effects of selected compounds on hypoxia-induced accumulation of HIF-1α. Expressions of HIF-1α were examined in 293T cells treated with compounds 15 and 31 at the concentrations of 2, 5, and 10 μM, respectively. LW6 was used as positive control.

The selectivity of our compound was also tested on a panel of receptors, ion channels, and enzymes (by Pharmaron Inc.), including some of the most popular druggable targets, which may result in side effects when dysregulated, to evaluate potential off-targets. Compound 15 was chosen and tested under 10 μM. Overall, 33 targets were tested using different assay formats. The detailed results are listed in Supplementary Table 2. We found that compound 15 showed no obvious effect to most of the tested targets except MAO-A, a subtype of monoamine oxidases that catalyzes the oxidation of monoamines.43 Since compound 15 contains a dimethylamino group, which is also a kind of monoamine group, this might explain that 15 can be the substrate of MAO-A. ADORA2A, also known as an adenosine A2A receptor, is a member of the G protein-coupled receptor (GPCR) family.44 It is one of the most investigated targets in cancer immunotherapy. Researchers have found mice that treated with A2AR antagonists showed significantly delayed tumor growth.45 Our studies found ADORA2A can also be slightly inhibited by 15. Although ADORA2A might be one of the potential off-targets, it seems that it is also possible that our compound could lead to a synergistic effect when treating cancer by both inhibiting HIF-1α and A2AR at the same time. In general, at this stage, our compound showed selectivity toward HIF-1α. (Table S2).

In summary, we elucidated benzo[d]isoxazole derivatives as potent anti-HIF-1α transcription compounds using a dual-luciferase report assay. Among them, six compounds showed IC50 values below 100 nM. Analogues 15 and 31 with dimethylamino and acetyl groups substituted on the para-position respectively were identified as highly potent HIF-1α transcriptional inhibitors, both with the best IC50 value of 24 nM. After HEK293T cells were treated with 15 and 31, VEGF and PDK1 mRNA expression showed a concentration-dependent decrease. For our off-target evaluation, our lead compound 15 led to great selectivity. Overall, benzo[d]isoxazole derivatives, showing structural simplicity and synthesis convenience, are promising to serve as antitumor progression agents and have potentials for cancer treatment and reducing cancer therapy resistance.

Experimental Procedures

Chemistry

The synthesis route for N-phenylbenzo[d]isoxazole-3-carboxamide derivatives is given in Scheme 1, and the key structure of benzo[d]isoxazole-3-carboxylic acid follows the patent reported by He and co-workers. (Patent number EP3456711A1). Compounds 2 and 9 can be easily synthesized by condensation reaction, and all their substrates are commercially available. The key compounds in context can be prepared easily by five-step synthesis including esterification, carbonation affinity, ring-forming condensation, hydrolysis, and amide condensation reactions. The synthetic details along with compound spectra and high-resolution mass spectra can be found in Supporting Information.

Dual-Luciferase Assay

The readout assay followed Promega manufacturer’s introduction, and all reagents were produced by Promega (Madison, WI, USA). HEK293T cells were transfected with HIF-1α (P2A), Renilla, and HRE-firefly luciferase for 24 h, and then, the testing compounds were added for another 24 h incubation. The readout of luciferin (the substrate of firefly luciferase) fluorescence intensity is the indicator of the HIF-1 inhibition ratio. The readout of coelenterazine (the substrate of Renilla) is for homogenization. The formula that illustrates how to calculate HIF-1α transcriptional activity can be found in Supporting Information.

VEGF Expression Testing

RNA was isolated by Trizol kit (Invitrogen, Carlsbad, CA). RNA was treated with DNase (Promega, Madison, WI). Complementary DNA was synthesized using the cDNA synthesis kit (Takara, Dalian, Liaoning, China) according to the manufacturer’s instructions. Fluorescence real-time PCR was performed with the double-stranded DNA dye SYBR Green PCR Core Reagents (PE Biosystems, Warrington, UK) using the ABI PRISM 7300 system (Perkin–Elmer, Torrance, CA).

Forward and reverse primes of VEGF are 5′-GCAGAATCATCACGAAGTGG-3′ and 5′-GCATGGTGATGTTGGACTCC-3′ respectively. Forward and reverse primes of PDK1 are 5′-GAGAGCCACTATGGAACACCA-3′ and 5′-GGAGGTCTCAACACGAGGT-3′ respectively. PCR was performed in triplicate, and standard deviations representing experimental errors were calculated. All data were analyzed using ABI PRISM SDS 2.0 software (Perkin–Elmer).

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 91853206, 21472026, 21877014, 22004058, 81972615) and the Science & Technology Commission of Shanghai Municipality (No. 21XD1420600).

Glossary

Abbreviations

HIF

hypoxia-inducible factor

IC50

concentration for 50% of maximal inhibition

EPO

erythropoietin

VEGF

vascular endothelial growth factor

HRE

hypoxic response element

PHD

hydroxylated by prolyl hydroxylase

SAR

structure–activity relationship

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00308.

  • Materials and reagents, characterization and instruments, synthetic experimental details (1H NMR, 13C NMR, and HRMS), compound purity, and target selectivity (PDF)

Author Contributions

Z.X. and H.L. contributed equally.

The authors declare no competing financial interest.

Supplementary Material

ml2c00308_si_001.pdf (420.9KB, pdf)

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