Design, Synthesis and Biological Evaluation of Pyrazolopyrimidine Derivatives as Aryl Hydrocarbon Receptor Antagonists for Colorectal Cancer Immunotherapy

Abstract

Background: Aryl hydrocarbon receptor (AhR) is a transcription factor that is involved in the regulation of immunity. AhR inhibits T cell activation in tumors, which induces immune suppression in the blood and solid tumors. We identified effective small-molecule AhR antagonists for cancer immunotherapy. Methods: A new series of pyrazolopyrimidine derivatives was synthesized and evaluated for AhR antagonistic activity. Results: Compound 7k exhibited significant antagonistic activity against AhR in a transgenic zebrafish model. In addition, 7k exhibited good AhR antagonist activity, with a half-maximal inhibitory concentration (IC₅₀) of 13.72 nM. Compound 7k showed a good pharmacokinetic profile with an oral bioavailability of 71.0% and a reasonable half-life of 3.77 h. Compound 7k selectively exerted anti-proliferative effects on colorectal cancer cells without affecting normal cells, concurrently suppressing the expression of AhR-related genes and the PD-1/PD-L1 signaling pathway. Compound 7k exhibited potent antitumor activity in syngeneic colorectal cancer models. Importantly, the combination of anti-PD1 and compound 7k enhanced antitumor immunity by augmenting cytotoxic T lymphocyte (CTL)-mediated activity. Conclusions: Collectively, a new pyrazolopyrimidine derivative, 7k, shows promise as a potential therapeutic agent for treating colorectal cancer.

Keywords: antagonist; aryl hydrocarbon receptor; cancer; zebrafish.

Figure 1

(A) Illustration of the aryl hydrocarbon receptor (AhR) signaling pathway. Activation of AhR leads to an upregulation of programmed cell death protein-1 (PD-1) and subsequent inactivation of T cells. Figure created using BioRender.com. (B) The structure of compound 7a.

Figure 2

Reported AhR antagonists.

Scheme 1

Synthetic route for 5,7-dichloro-2-methyl-2H-pyrazolo [4,3-d]pyrimidine compounds. Reagents and conditions: (a) H₂SO₄, methanol, reflux, 12 h, 96%; (b) Bromoalkane, K₂CO₃, DMF, rt, 12 h, 56–63%; (c) Hydrogen, 10% Pd/C, methanol, rt, 12 h, 76–89%; (d) Urea, 200 °C, 12 h, 25–65%; (e) POCl₃, 100 °C, 3 h, 42–75%.

Scheme 2

Synthetic route for N-(2-(1H-indol-3-yl)ethyl)-2-isopropyl-2H-pyrazolo [4,3-d]pyrimidin-7-amine compounds. Reagents and conditions: (a) Tryptamine or serotonin hydrochloride, isopropyl alcohol, rt, 12 h, 69–80%; (b) 3-Cyanophenyl boronic acid, Pd(PPh₃)₄, NaHCO₃, 1,4-dioxane, 90 °C, 3 h, 20–66%.

Figure 3

Tg(cyp1a:egfp) zebrafish embryos at 30 h post-fertilization (hpf) were treated with TCDD (2 nM) simultaneously with either DMSO or compound 7k (100 nM). The GFP signal was captured 48 h post-treatment using a fluorescence stereomicroscope. DMSO was utilized as a vehicle control.

Figure 4

The intermolecular interactions of synthesized compound 7k with human aryl hydrocarbon receptor (AhR) reveal that compound 7k has an optimal binding with the ligand-binding pocket in the Per–Arnt–Sim (PAS)-B domain of the human AhR via cooperative halogen and hydrophobic interactions. The halogen and π–sulfur bonds with hydrophobic interactions formed by the 3,5-difluorophenyl moiety improve the binding of compound 7k with human AhR. The intermolecular interactions are displayed as dashed lines with different colors according to the interaction types (Halogen: cyan, π–Donor Hydrogen bond: Honeydew, π–Sulfur: Tangerine Yellow, π–π T-shaped: Neon Pink). The alkyl interactions between the compound 7k and AhR are not shown for clarity, but they are provided in Supplementary Information Figure S9 and Table S1.

Figure 5

The effects of 7k on the proliferation of normal cells and colorectal cancer cells. (A) Cell viability in 7k-treated normal cells, such as CHO, L929, and CCD-18Co cells, at 24 h post-treatment. (B) Effects of 7k on the proliferation of colorectal cancer cells at 24 h post-treatment. Cells were incubated with 7k for 24 h or 48 h, and cell viability was determined using a cell counting kit 8 (CCK8) cell viability assay. (C) Effects of 7k on the colony-forming ability of HCT-116 cells. Cells were seeded in 6-well plates, treated with 7k, and after 14 days, crystal violet staining was performed. Each experiment was conducted in duplicate.

Figure 6

7k-induced cell cycle arrest and apoptosis activation in colorectal cancer cells. (A,B) cell cycle analysis and (C,D) apoptosis analysis in both 7k-treated MC38 and CT26 cells. Cells were treated with 7k for 24 h before cell cycle, and apoptosis analysis was conducted. Data are represented as the mean ± SD.

Figure 7

Change in apoptosis-regulating protein expression in 7k-treated colorectal cancer cells. (A,B) Changes in the expression of apoptosis- and cell cycle–related molecules in colorectal cancer cells following 7k treatment. (C,D) Quantification of protein expression levels using ImageJ 1.54p. CT26 and MC38 cells were treated with 7k for 24 h before Western blot examination.

Figure 8

Inhibitory effects of 7k on AHR downstream genes in colorectal cancer cells. Effects of 7k on AhR, AhRR, and IDO-1 expression in kynurenine (Kyu)-treated (A) MC38 and (B) CT26 cells. Cells were treated with various concentrations of 7k and Kyu for 24 h. RNA from treated cells were extracted, and gene expression of interest was determined using qPCR analysis. Data are presented as the mean ± SD. *, p < 0.05, **, p < 0.005, ***, p < 0.0005, and ****, p < 0.0001 compared to Kyu-treated cells; –, untreated; +, treated.

Figure 9

Effects of 7k on the cytoplasmic and nucleus localization of AhR in Kyu-treated MC38 cells. Changes in the expression of (A) cytoplasmic AhR and (B) nuclear AhR in Kyu-treated cells with or without 7k treatment. (C,D) Quantification of cytoplasmic and nuclear AhR expression using ImageJ 1.54p. Cells were treated with 7k and Kyu for 24 h. Cytosolic and nuclear protein from treated cells were isolated, and AhR expression was determined using Western blot analysis. –, untreated; +, treated.

Figure 10

Modulation of PD-1/PD-L1 signaling by 7k. (A–D) Effects of 7k on PD-L1 expression in IFNγ-treated colorectal cancer cells. Cancer cells were pre-treated with 7k for 1 h, followed by incubation with IFNγ for 24 h. PD-L1 expression levels in cancer cells were analyzed using FACS analysis with an anti-PD-L1 antibody. (E,F) Effects of 7k on PD-1 expression in PHA-stimulated Jurkat/PD-1 cells. Jurkat/PD-1 cells were pre-treated with 7k and then stimulated with PMA for 24 h. PD-1 expression in Jurkat/PD-1 cells was using FACS analysis with anti-PD1 antibody. Isotype indicated the isotype-matched control. *, p < 0.05, **, p < 0.005, ***, p < 0.0005, and ****, p < 0.0001 compared to IFNγ or PHA-treated cells; ns, not significant; –, untreated; +, treated.

Figure 11

Antitumor activity of 7k alone in immunocompetent mice with colorectal cancer Antitumor activity of 7k alone in immunocompetent mice of colorectal cancer. (A) Brief scheme for in vivo therapy. (B) Antitumor effects of 7k in CT26 tumor bearing mice. (C) Bar graph showing the tumor volume at 14 days after therapy. (D) Antitumor effects of 7k in MC38 tumor bearing mice. (E) Bar graph showing the tumor volume at 14 days after therapy. Either CT26 or MC38 cells were challenged in immunocompetent mice. (F) Comparison of antitumor effects of 3′,4′-dimethoxyflavone and 7k in MC38 tumor bearing mice. (G) Bar graph showing the tumor volume at 18 days after therapy. When tumor mass was detectable in palpation and inspection, tumor bearing mice received either 7k or 3′,4′-dimethoxyflavone via oral gavage once a day for 14 or 18 days. Data are presented as the mean ± SD. *, p < 0.05, **, p < 0.005, ***, p < 0.0005 compared to vehicle-treated cells.

Figure 12

Antitumor activity of the 7k and anti-programmed cell death protein-1 (PD1) combination in immunocompetent mice with colorectal cancer. (A) Brief scheme for in vivo therapy. (B) Enhanced antitumor effects of 7k and anti-PD1 combination in CT26 tumor-bearing mice. (C) Bar graph showing the tumor volume at 14 days after therapy. CT26 cells were challenged in immunocompetent mice. When tumor mass was detectable using palpation and inspection, a single treatment with 7k or anti-PD1 alone or in combination was initiated at the indicated times. 7k was administered via oral gavage once a day for 14 days. Anti-PD1 was injected intraperitoneally every three days, four times. Data are represented as the mean ± SD. ns, not significant. *, p < 0.05, **, p < 0.01, compared to vehicle-treated cells.

Figure 13

Antitumor activity of combination with anti-PD1 and 7k in immunocompetent mice of MC38 colorectal cancer. (A) Brief scheme for in vivo therapy. (B) Enhanced antitumor effects of combination with anti-PD1 and 7k in MC38 tumor bearing mice. (C) Bar graph showing the tumor volume at 20 days after therapy. MC38 cells were challenged in immunocompetent mice. When tumor mass was detectable in palpation and inspection, single treatment with 7k or anti-PD1 alone or combination treatment was initiated at indicated times. 7k was administered via oral gavage once a day for 20 days. Anti-PD1 was injected intraperitoneally every three days four times. Data are presented as the mean ± SD. ns, not significant. *, p < 0.05, ***, p < 0.001, compared to vehicle-treated cells.

Figure 14

Robust CTL-mediated antitumor immune response by combined anti-PD1 and 7k treatment in a syngeneic colorectal cancer model. (A) Representative flow cytometry plots showing the percentage of IFNγ-producing CD8 T lymphocytes. (B) Quantification of IFNγ-producing CD8 T lymphocytes. (C) Bioluminescence imaging to monitor the cytotoxic activity of splenocytes derived from tumor-bearing mice. (D) Quantification of luciferase signals from panel (C). MC38/luc cells were used as target cells. Data are presented as the mean ± SD. ns, not significant. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001, compared to vehicle-treated cells.