Discovery of a Potent Anticancer Agent PVHD303 with in Vivo Activity

Abstract

As a part of our continuous structure–activity relationship (SAR) studies on 1-(quinazolin-4-yl)-1-(4-methoxyphenyl)ethan-1-ols, the synthesis of derivatives and their cytotoxicity against the human lung cancer cell line A549 were explored. This led to the discovery of 1-(2-(furan-3-yl)quinazolin-4-yl)-1-(4-methoxyphenyl)ethan-1-ol (PVHD303) with potent antiproliferative activity. PVHD303 disturbed microtubule formation at the centrosomes and inhibited the growth of tumors dose-dependently in the HCT116 human colon cancer xenograft model in vivo.

There are many types of cancer treatment such as surgery, radiation therapy, and chemotherapy. In recent years, immunotherapy has attracted considerable attention as the fourth pillar of cancer treatment in addition to the previously mentioned treatments.^1,2 Although chemotherapy receives a little less attention than it did in the previous decades, it remains one of the most powerful tools for cancer treatments. It is a common strategy to use a combination of chemotherapy and surgery/radiation therapy. In addition, successful cases of the combination of chemotherapy and immunotherapy have also been reported.^3,4 Thus there is still a significant need for the development of new small-molecule anticancer drugs and novel target biomolecules for anticancer agents.

We have previously reported the quinazoline derivative PVHD121 (1)⁵ (Figure 1) as a novel tubulin polymerization inhibitor, which exhibits antiproliferative activity through binding to the colchicine site of tubulin.^6,7 Slightly prior to our report, 4-aminoquinazolines, “aza-analogs” of 1, that exhibit antitubulin properties targeting the colchicine site were reported.^8,9 Analogous 4-aminoquinazolines had also been discovered as potent apoptosis inducers and anticancer agents.^10,11

Figure 1.

Structures of PVHD121 (1) and its derivatives.

In our subsequent study, it was disclosed that 1 selectively disturbed the microtubule formation at centrosomes during mitosis, causing aberrant spindle formation and subsequent cell death.¹² This observation indicates that 1 interacts with biomolecules other than tubulin at the colchicine site and that investigating the molecular action of 1 and its derivatives will lead to the detection of a new drug target molecule. It should be noted that 2-aminoquinazolines were found to be fluorescent during our previous structure–activity relationship (SAR) study, and one of them, 2-morpholino derivative 2, has been used as a molecular probe in the previously mentioned study.^7,12

The previous SAR study based on the structure of 1 led to the discovery of quinazoline 3a with 10 times higher activity, that is, an IC₅₀ value of 0.027 μM against the human lung cancer cell line A549.⁶ In addition, other 2-substituted quinazolines, such as 2-trichloromethyl and 2-methoxy, and 2-methythio analogs also exhibited potent activities comparable to 3a.⁶ This previous SAR study had been performed only on the substituents at the 2- and 4-positions of the quinazoline core. Therefore, we have been interested in a SAR study on the fused benzene ring and have envisioned that further study of the compounds with varied substituents at position 2 would lead to the discovery of a potential drug candidate for cancer therapy. Herein we report that this SAR study resulted in the discovery of 1-(2-(furan-3-yl)quinazolin-4-yl)-1-(4-methoxyphenyl)ethan-1-ol (PVHD303, 9e) with potent antiproliferative and in vivo antitumor activities.

Compounds 4–7 with a substituent on the benzene ring were prepared from the corresponding 4-chloroquinazolines via N-heterocyclic carbene (NHC)-catalyzed aroylation,^5,13 followed by the Grignard reaction (Scheme 1). In the same synthetic route, 2-bromo analog 3b was prepared from 2,4-dibromoquinazoline (Scheme 1).

Scheme 1. Synthesis of Common Intermediate 3a and Compounds 3b and 4–7.

Reagent and conditions: (i) N,N′-1,3-dimethylimidazolium iodide (precatalyst), p-anisaldehyde, NaH, THF, or THF/DMF; (ii) CH₃MgBr, THF, rt. (See the Supporting Information for details.)

The other 2-substituted quinazolines were prepared from 3a in one step (Scheme 2): The nucleophilic aromatic substitution of the 2-chloro group yielded 2-amino-substituted compounds 8a–o, and Suzuki–Miyaura coupling produced 2-alkenyl-, 2-cyclopropyl-, and 2-aryl-substituted quinazolines 9a–r.

Scheme 2. Synthesis of Compounds 8 and 9 from 3a.

Reagent and conditions: (i) amine or ammonia, 1,4-dioxane, reflux; (ii) ArB(OR)₂, K₂CO₃, PdCl₂(dppf), or Pd(PPh₃)₄, 1,4-dioxane/toluene, reflux. (See the Supporting Information for details.)

The synthesized quinazolines were tested for cytotoxicity against the human lung cancer cell line A549, and the growth inhibitions were evaluated by the MTS assay (Tables 1–3). The IC₅₀ values for the positive controls, nocodazole and vinblastine, were 0.272 ± 0.004 and 0.021 ± 0.000 μM, respectively.

Table 1. SAR of the Fused Benzene Ring.

entry	compound	R1	IC50 (μM)a
1	4a	F	12.02 ± 0.63
2	5a	F	0.35 ± 0.03
3	5b	Cl	0.08 ± 0.00
4	5c	CH3	1.10 ± 0.11
5	5d	OCH3	5.16 ± 0.62
6	6a	F	4.67 ± 0.42
7	6b	Cl	0.87 ± 0.01
8	6c	CH3	>25
9	6d	OCH3	>25
10	7a	F	0.43 ± 0.04
11	7b	Cl	0.24 ± 0.11
12	7c	CH3	1.56 ± 0.07
13	7d	OCH3	2.10 ± 0.61

^aData are presented as mean ± standard deviation (SD) (n = 3). SAR, structure–activity relationship.

Table 3. SAR of 2-Alkenyl, 2-Cyclopropyl, and 2-Aryl Derivatives.

^aData are presented as mean ± standard deviation (SD) (n = 3).

^bPreviously reported data.⁶ SAR, structure–activity relationship.

It was found that the substituents on the benzene ring tended to cause a decrease in the activity (Table 1), especially at positions 5 and 7 of the quinazoline ring (compounds 4a and 6a–d, entries 1 and 6–9). Although 6-fluoroquinazoline 5a, 6-chloroquinazoline 5b, 8-fluoroquinazoline 7a, and 8-chloroquinazoline 7b exhibited comparable activities to the initial lead compound 1 (entries 2, 3, 10, and 11), the activities generally decreased as the size of the substituent increased; for example, the IC₅₀ values increased in the order of 6-chloro (5b), 6-methyl (5c), and 6-methoxy analog 5d (entries 3–5).

A further cytotoxicity assay with quinazolines bearing various substituents at position 2 led to the discovery of compounds with potent antiproliferative activities (Tables 2 and 3). 2-Bromoquinazoline 3b showed a comparable activity to the previously optimized lead compound 3a(6) (Table 2, entry 1). Among the tested 2-N-substituted quinazolines 8a–o,¹⁴ 2-aminoquinazoline 8a, 2-methylaminoquinazoline 8b, and 2-(N-pyrazolyl)quinazoline 8h were relatively potent in activity (entries 2, 3, and 9). 2-Amino analogs bearing a smaller substituent on the nitrogen atom (a cyclic amino group with a smaller ring size) generally exhibited higher activities (entries 3–6, 17, and 18). 2-N-Thiomorpholino derivative 8j and 2-N-piperidinylquinazolines 8l–o (and 8q(6)), except for 4-fluoropiperidinyl derivative 8k, were significantly less potent compared with their oxo analog 2(6) (Table 2, entries 11–16, 18; Figure 1). The existence of π and additional lone-pair electrons in the cyclic amino groups increased the activity compared with those without them (entries 6–10 and 18). Compound 9a bearing a 2-vinyl group showed a fairly high activity (Table 3, entry 1), and compound 9b with the propenyl group exhibited comparable activity to the lead compound 1, whereas compound 9c bearing 2-cyclopropyl group was less active than 9a and 2-methyl analog 9s(6) (entries 3 and 19). Among the newly prepared 2-aryl analogs 9d–r (entries 4–18), compounds with a heteroaryl group, in general, tended to exhibit potent activities (entries 4–12, 18). 2-(Furan-3-yl)quinazoline (PVHD303, 9e) was significantly more potent (IC₅₀: 13 nM) than its regioisomer 9d, thio analog 9t, and the previously optimized lead 3a (Table 3, entries 4, 5, and 20, and Figure 1). Although the introduction of the formyl group to the furan ring decreased the activity (entry 6), additional substituents to the 2-aryl groups did not necessarily cause a decrease in the activities (entries 7–10, 13–17, 20, and 21). Quinazoline derivative 9h bearing a 5-cyanothiophen-2-yl group was as potent as PVHD303 (entries 5 and 8).

Table 2. SAR of 2-Bromo and 2-Amino Derivatives.

^aData are presented as mean ± standard deviation (SD) (n = 3).

^bPreviously reported data.⁶ SAR, structure–activity relationship.

To examine the mechanisms behind the antimitotic activity of 9e (PVHD303), which exhibited the highest in vitro activity, we observed the spindles and the centrosomes in the 9e-arrested mitotic cells. HeLa cells were treated with various concentrations around IC₅₀ of 9e for 16–18 h and double-immunostained with anti-α-tubulin and anti-γ-tubulin antibodies (Figure 2 and Table 4). At 8 nM, half of the mitotic cells showed normal bipolar spindles, whereas most of the other half exhibited irregular spindles, including multipolar or radially arranged spindles. In mitotic cells treated with 12 nM, we observed randomly arranged microtubule bundles around the condensed chromosomes and unseparated centrosomes in more than half of the mitotic cells. The appearance of unseparated centrosomes is one of the characteristics of 1-arrested mitotic cells,¹² suggesting that 9e and 1 share the same mechanism of antimitotic activity other than binding to the tubulin dimer. Even at 16 nM (higher concentration than the IC₅₀), fewer than half of 9e-arrested mitotic cells exhibited diffuse α-tubulin distribution without microtubules.

Figure 2.

Microtubules and centrosomes in 9e-arrested mitotic cells. HeLa cells were treated with 9e for 16–18 h and were immunostained with anti-α-tubulin (microtubules, red) and with anti-γ-tubulin (the centrosome, green) antibodies. Chromosomes were stained with DAPI (blue). (a) Normal bipolar spindles observed in cells treated with 9e. (b) Multipolar spindles observed in cells treated with 9e. (c) Cells treated with 10 μM of 9e contained unseparated centrosomes (arrow), whereas they possessed microtubule bundles around the chromosomes. (d) Diffuse staining for α-tubulin in 9e-arrested cells at 16 μM.

Table 4. Concentration-Dependent Phenotypes of 9e-Arrested Mitotic Cells.

concentration (nM)	4	8	12	16
bipolar spindle (%)	100	50	0	0
irregular spindle (%)	0	44.2	0	0
with unseparated centrosome (%)	0	5.8	67	57
diffuse α-tubulin (%)	0	0	33	43
number of cells observed	50	52	52	60

The antitumor activity of compound 9e was evaluated in the HCT116 human colon cancer xenograft model in vivo; the in vitro antiproliferative activity against the cell line (IC₅₀: 13 nM) was confirmed by the MTS assay prior to the in vivo study. The intravenous administration of compound 9e at 10 and 20 mg/kg significantly inhibited the growth of tumors dose dependently, with tumor growth inhibition rate (IR) values of 24.6 (P < 0.05 vs control) and 62.9% (P < 0.001 vs control), respectively (Figure 3). Compound 9e inhibited tumor growth dose-dependently.

Figure 3.

Antitumor activity of compound 9ein vivo. Mice were inoculated subcutaneously with HCT116 cells. Following tumor formation, mice were treated intravenously on days 1, 3, 5, 8, 10, 12, 15, 17, and 19 with 10 or 20 mg/kg of compound 9e or with vehicle only (control). Tumor volumes were measured as described in the Supporting Information. Data are presented as means ± SD (n = 5).

In conclusion, through SAR studies and lead optimization, quinazoline derivative PVHD303 (9e) with a potent antiproliferative activity and an IC₅₀ of 13 nM was discovered. SARs were investigated by introducing substituents into the quinazoline core at positions 2, 5, 6, 7, and 8. Although the substituents on the benzene ring did not positively affect the activity, analogs with small-sized substituents and heteroaryl groups at position 2 generally exhibited potent activities. In mitotic HeLa cells treated with PVHD303, the disturbed microtubule formation at unseparated centrosomes was also observed, as seen in the case with 1, suggesting that both compounds have the same antimitotic mechanism, which presumably involves the interaction with biomolecules other than tubulin. In addition, PVHD303 exhibited antitumor activity in vivo using the HXT116 human colon cancer xenograft model. Further derivatizations, SAR studies, and the search for the target molecule of this class of compounds are underway in our laboratories.

Glossary

Abbreviations

SAR

structure–activity relationship

MTS

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

IR

tumor growth inhibition rate

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00119.

• Experimental procedures of synthesis, analytical data, and experimental methods of assays (PDF)

The authors declare no competing financial interest.