Quinazolinone Compounds Have Potent Antiviral Activity against Zika and Dengue Virus

Abstract

Dengue (DENV) and Zika (ZIKV) virus are important human pathogens, causing ~100 million symptomatic infections each year. These infections carry 20-fold increased incidence of serious neurological diseases, such as microcephaly in newborns (for ZIKV) and Guillain-Barré syndrome. Moreover, DENV can develop serious and possibly life-threatening dengue hemorrhagic fever in certain patients. Patients recovered from one of the 4 serotypes of DENV are still susceptible to other serotypes with a higher likelihood of serious disease because of antibody-dependent enhancement. Except for mosquito control, there have been no antiviral drugs to prevent and treat ZIKV/DENV infections. Phenotypic screening found 2,3,6-trisubstituted quinazolinone compounds are novel inhibitors of ZIKV replication. Fifty-four analogs were synthesized and their structure-activity relationships are discussed. Additional testing shows that compounds 22, 27 and 47 exhibited broad and potent activities against ZIKV and DENV with EC₅₀ values as low as 86 nM with no significant cytotoxicity to mammalian cells.

Graphical Abstract

Introduction

Zika virus (ZIKV) and closely related dengue virus (DENV) belong to the genus Flavivirus of the virus family Flaviviridae, which also includes other major human pathogenic viruses such as yellow fever, Japanese encephalitis and West Nile viruses. ZIKV and DENV are transmitted by Aedes mosquitoes in the tropical and subtropical regions, where approximately 3 billion people or 40% of world population live and are at risk of these infections ^{1, 2}.

ZIKV, discovered and isolated in 1947 ³, has caused three major outbreaks in the Yap Island (~7,000 cases in 2007), the French Polynesia (~28,000 cases in 2013) in the Pacific ocean, and Brazil and other American countries (2015–2016) with significantly broader impact and damages ^{4, 5}. Several millions of people in 48 Pan-American countries and territories have been infected, showing symptoms including fever, rashes and conjunctivitis. Although most people recovered in several days, ZIKV infection has been found to cause 20-fold increased incidence of serious neurological diseases, such as Guillain-Barré syndrome ^6–8 and >4,000 cases of microcephaly (small brain/head) and other neurological defects in newborns ^9–11. The prognosis of these infants to have normal brain functions is low and their life expectancy is shorter. WHO announced ZIKV is a “Public Health Emergency”. Moreover, ZIKV can be transmitted through sex or body fluids even when infected people have no symptoms ^{12, 13}. This secondary transmission route renders it more difficult to contain ZIKV infection.

DENV has been a major human pathogen for the past several decades. It is estimated that DENV infects ~400 million people per year with 100 million developing symptoms including fever, headache, rash, conjunctivitis and pain in muscle and joints, which are usually self-healing in 3–10 days ¹. However, ~500,000 cases/year develop serious and possibly life-threatening dengue hemorrhagic fever and shock syndrome. Approximately 22,000 people (mostly children) die of the disease per year ². More problematic is that there are 4 serotypes of DENV (DENV-1–4) with significantly different genomes ^{14, 15}. Patients who have recovered from one DENV serotype are still susceptible to other DENV serotypes and there is an increased likelihood to develop a more serious disease ¹⁶, because of existing antibodies against the previous DENV serotype ^{14, 17}.

Except for mosquito control, there have been no antiviral drugs to prevent and treat ZIKV and DENV infections. The only licensed DENV vaccine, Dengvaxia, has been found to have safety and efficacy concerns for DENV-naïve individuals in clinical trials ¹⁸. A limited number of compounds have been reported to have potent antiviral activities against ZIKV or DENV ^19–30. Most of these compounds are natural products with complex chemical structures that are not amenable for medicinal chemistry optimization. Only a few compounds showed broad activities against both ZIKV and DENV ^19–23. There is therefore a pressing need to find effective antiviral agents against ZIKV and DENV. Here, we report discovery, synthesis and structure-activity relationships (SAR) of a series of tri-substituted quinazolinone compounds that exhibit potent antiviral activities against both ZIKV and DENV. Interestingly, a quinazolinone compound was reported to exhibit modest activities (16.7 μM) against Venezuelan equine encephalitis virus ³¹, which is a mosquito-borne Alphavirus causing flu-like symptoms in humans.

RESULTS and DISCUSSION

Discovery of novel anti-Zika compounds

A cell-based, phenotypic anti-ZIKV activity assay was developed. Rapid replication of ZIKV (FLR strain, Colombia, 2015 ³²) in Vero (monkey kidney cells that lack interferon-mediated antiviral defense ³³) cells causes significant cytopathic effects (CPE) and eventually cell lysis, which can be clearly observed under the microscope. However, a compound that inhibits ZIKV replication can protect cells from CPE and death. If needed, an MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay may further quantitate the number of viable cells. 0.1 multiplicity of infection (MOI, the number of infectious viral particles per cell) of ZIKV was added to a monolayer of cells in 96-well plates with culturing media containing a compound at 10 μM in duplicate for 72h. Observing CPE followed by MTT assay enabled us to quickly screen compounds to identify anti-ZIKV compounds. Moreover, cytotoxic compounds (at 10 μM), showing similar phenotypes (CPE and/or cell death) in the assay, were also eliminated.

Next, activities of these hits to reduce the viral titers were further determined, using an end-point dilution assay as described in our previous publications ^34–36. Half-log (0.32×) serial dilutions of the supernatant containing newly generated ZIKV particles were added to Vero cells in quadruplicate. Upon incubation for ~5 days, ZIKV infection in each sample was determined with CPE. TCID₅₀ (tissue culture infective dose) was calculated based on the highest dilution in which ≥50% of the quadruplicate samples were infected with ZIKV. As compared to TCID₅₀ of the untreated samples, ability of a compound to reduce ZIKV replication can be determined.

Through screening of ~1,000 compounds in our in-house compound library, which were synthesized for medicinal chemistry studies targeting H3K4 demethylase LSD1, histone acetyltransferase p300 and transcription cofactor AF9/ENL ^37–39, 2,3,6-trisubstituted quinazolinone compound 1 (Chart 1) was found to be a potent anti-ZIKV agent, which can inhibit the viral replication by 99.9% (3-log reduction) at 10 μM. Interestingly, di-substituted quinazolinone compounds 2-4 can also inhibit ZIKV replication by 68%–90% (0.5–1-log reduction). Known anti-ZIKV compounds chloroquine and SYC-1307 (developed by us) ^{34, 35} were used as positive controls and exhibited expected antiviral activities (90% inhibition by chloroquine at 10 μM and 68% by SYC-1307 at 1 μM). These results show certain quinazolinone compounds are novel, potent antiviral agents against ZIKV and medicinal chemistry optimization based on these compounds are needed.

Chart 1.

Structures and anti-ZIKV (FLV strain) activities in Vero cells (at 10 μM) of compounds 1–4.

Chemical synthesis

Synthesis of quinazolinone compounds 1-54 for structure activity relationship studies is shown in Scheme 1. For compounds 2–21 and 43, isatoic anhydride compound 55 was condensed with an aniline derivative to give a benzamide compound 56, which was subjected to a cyclization reaction at 110 °C to produce 2-methyl-3-phenylquinazolinone 57. The methyl group of compound 57 was reacted with an arylaldehyde to yield target compounds 2–21 and 43.

Scheme 1:

Synthesis of compounds 1–54.^a

^aReagents and conditions: (i) an aniline, 1,4-dioxane, 90 °C, 12 h, 45–81%; (ii) 1,1,1-triethoxyethane, 110 °C, overnight, 32–94%; (iii) aryl-CHO, AcONa, AcOH, reflux, overnight, 35–91%; (iv) an amine, tris(dibenzylideneacetone)dipalladium(0), 2-dicyclohexylphosphino-2’,6’-dimethoxybiphenyl, sodium tert-butoxide, toluene, 110 °C, 24 h, 30–81%; (v) Pd/C, H₂, MeOH, 25 °C, overnight, 80–88%; (vi) tetrakis(triphenylphosphine)palladium(0), 1,4-dioxane, H₂O, 110 °C, 24 h, 45–80%.

For synthesis of compounds 1, 22-28, 35–42 and 44–52, 6-chloro-quinazolinone 20 was subjected to a Buchwald-Hartwig amination reaction with an amine to give the target compounds. Compound 25 was prepared from compound 24 by deprotection of its tert-butoxycarbonyl (BOC) group. Saturated compounds 53 and 54 were synthesized from compounds 22 and 27, respectively, by Pd/C catalyzed hydrogenation. A Suzuki coupling reaction with 6-bromo-quinazolinone 58 gave compounds 29-34.

Structure-activity relationships for inhibiting ZIKV replication

Disubstituted quinazolinone compounds 5-11 (Table 1) with a variety of R³ groups were found to be inactive at 10 μM in the compound screen. Lack of anti-ZIKV activities of these compounds, together with the high activity of compound 1, show the 3-tert-butyl R³ group in 1-3 seems to be favored. Thus, compounds 12-19 bearing a 3-tert-butyl R³ group and various R² groups were synthesized and tested for their activities to inhibit ZIKV replication. As shown in Table 1, none of these compounds exhibited significantly improved activity. SAR analysis suggested an aromatic R² group seems not to significantly affect the anti-ZIKV activity. For example, while an electron-deficient pyridin-4-yl or pyrimidine-containing R² group in compounds 13 and 14 (68% inhibition) appears to be more favorable than a phenyl in 15 (inactive), compound 12 with a pyridin-3-yl R² group was inactive. With both compounds having 90% ZIKV inhibitory activity at 10 μM, the pyridinyl R² group in compound 3 does not exhibit a superior activity to the phenyl in 18. Interestingly, the para-CF₃ group in compounds 3 and 18 appears to provide a moderate activity improvement, compared to unsubstituted or methoxy-substituted compounds 2, 11-17. A bicyclic naphthyl R² group in compound 19 (68% inhibition) does not significantly enhance anti-ZIKV activity.

Table 1:

Structures and anti-ZIKV (FLV strain) activities of compounds 2–19 in Vero cells.

Cpd #	R2	R3	% inhibition of ZIKV at 10 μM
5		H	0
6	same as above	2-CH3	0
7	same as above	3-CH3	0
4	same as above	3-OCH3	68.4%
8	same as above	4-t-Bu	0
9		same as above	0
10		same as above	0
2		3-t-Bu	68.4%
3		same as above	90.0%
11		same as above	0
12		same as above	0
13		same as above	68.4%
14		same as above	68.4%
15		same as above	0
16		same as above	0
17		same as above	0
18		same as above	90.0%
19		same as above	68.4%

Next, we focused on optimization of the R⁶ group, since compared to unsubstituted compound 11, the diethylamino group in compound 1 (99.9% inhibition at 10 μM) renders ≥1,000-fold activity enhancement to inhibit ZIKV replication. Further testing showed compound 1 can inhibit ZIKV replication in Vero cells by 68% (0.5-log reduction) at 1 μM (Table 2). With the same 3-tert-butyl R³ and 6-methoxy-pyridin-2-yl R² group, compounds 20-34 having various R⁶ groups were synthesized and their anti-ZIKV activities at 10 and 1 μM shown in Table 2. While compounds 20 and 21 with a -Cl and -OMe R⁶ group were inactive, compound 22 with a piperidin-1-yl group inhibited ZIKV replication by ≥99.9% at 10 μM and 90% at 1 μM, showing superior activities to compound 1 (68% inhibition at 1 μM). However, compound 23 with a 5-membered pyrolidin-1-yl R⁶ group is only a modest anti-ZIKV agent (68% inhibition at 10 μM). A larger, 4-BOC protected piperazin-1-yl R⁶ group in compound 24 (99% inhibition at 10 μM) is tolerable, although it is considerably less active than 1 and 22. Deprotection of BOC yielded compound 25 with a piperazin-1-yl R⁶ group, which was found to exhibit significant cytotoxicity to Vero cells, which is undesirable for this study. Similarly, compound 26 with a 4-methyl-piperazin-1-yl group is also cytotoxic, suggesting a basic N atom at the 4-position of the piperazine ring (in 25 and 26) is attributed to the toxicity, as compared to 22 and 24. Indeed, compound 27 having a morpholin-4-yl R⁶ substituent, as well as compound 28 with a larger 4-(morpholin-4-yl)piperazin-1-yl R⁶ group, is one of our most potent anti-ZIKV agents without cytotoxicity. Both compounds can inhibit ZIKV replication by 99.9% at 10 μM and 90% at 1 μM.

Table 2:

Structures and anti-ZIKV (FLV strain) activities of compounds 1, 20–43 in Vero cells.

Cpd #	R2	R6	% inhibition of ZIKV at 10 μM	% inhibition of ZIKV at 1 μM
1			99.9%	68.4%
20	same as above	-Cl	0	NT a
21	same as above	-OCH3	0	NT a
22	same as above		99.9%	90.0%
23	same as above		68.4%	NT a
24	same as above		99.0%	NT a
25	same as above		Cytotoxic	NT a
26	same as above		Cytotoxic	NT a
27	same as above		99.9%	90.0%
28	same as above		99.9%	90.0%
29	same as above		0	NT a
30	same as above		0	NT a
31	same as above		99.0%	NT a
32	same as above		96.8%	NT a
33	same as above		96.8%	NT a
34	same as above		96.8%	NT a
35			90.0%	NT a
36		same as above	99.0%	NT a
37		same as above	68.4%	NT a
38		same as above	99.7%	NT a
39		same as above	96.8%	NT a
40	same as above		Cytotoxic	NT a
41			99.9%	68.4%
42	same as above		99.0%	NT a
43	same as above	-OCH3	0	NT a

^aNot tested.

Several compounds with an aromatic R⁶ group were investigated. While compounds 29 and 30 with a pyridine-4-yl and 3-methoxyphenyl are inactive, compounds 31-33 with a para-F, -dimethylamino and -pyrolidine substituted phenyl R⁶ group were moderately active with 99–96.8% ZIKV inhibition at 10 μM (Table 2). Compound 34 with a partially unsaturated tetrahydropyran-4-yl substituent exhibited moderate anti-ZIKV activity (96.8% inhibition at 10 μM). Collectively, SAR studies for the R⁶ show that 1) a N-atom directly attaching to the 6-position of the quinazolinone core provides a significant activity enhancement (e.g., 27 vs. 34); 2) 6-membered piperidin-1-yl or morpholin-4-yl (in 22, 27 and 28) is the most favored, while smaller 5-membered pyrrolidine ring loses most activity; and 3) a second, basic N-atom at the 4-position of the 6-membered ring is cytotoxic.

With the same 3-tert-butyl R³ and a piperidine or related R⁶ group, compounds 35-43 (Table 2) were synthesized to optimize the R² substituent. Compounds 35 and 36 (99% and 90% inhibition at 10 μM), which contain a para-CF₃-substituted pyridine and phenyl R² group showing good activities in Table 1, are less active than compound 22, indicating the 6-methoxy-pyridin-2-yl R² group in 22 is more favored. Compound 37 with a pyridine-4-yl R² substituent is a modest compound (68% inhibition at 10 μM). Compound 38 having a 2-naphthyl R² substituent exhibited a strong anti-ZIKV activity of 99.7% at 10 μM, while compound 39 bearing a 3,5-dimethoxyphenyl R² moiety is less potent (96.8% ZIKV inhibition). Its analog 40 with a 4-methyl-piperazin-1-yl R⁶ group is also cytotoxic, consistent with the results of compounds 26 and 25. With a 3-methoxyphenyl R² substituent, compound 41 is also a potent anti-ZIKV agent with 99.9% and 68% inhibition at 10 and 1 μM, but it is slightly weaker than compound 22 (90% inhibition at 1 μM), showing a pyridine-containing R² substituent is more favorable than a phenyl. Similarly, compounds 42 and 43 with a 3-methoxyphenyl R² moiety exhibited comparable anti-ZIKV activities to their pyridine-containing analogs 24 and 21. These results show the 6-methoxy-pyridin-2-yl R² group (in 22) is the most favored for this series of compounds.

Compounds 44-52 (Table 3) with the most favored R² and R⁶ groups were synthesized to further optimize the R³ substituent. 3-methyl group in compounds 44-46 (no inhibition at 1 μM) were found to be considerably less active than their 3-tert-butyl analogs 22, 27 and 28. 3-Methoxy R³ substituent in compounds 47-49 gives potent anti-ZIKV activities, but it is slightly less favored than 3-tert-butyl R³ group, when combined with a morpholine-containing R⁶ (e.g., compounds 48/49 vs. 27/28). Compounds 50-52 with an electron-withdrawing 3-CF₃ R³ substituent are considerably less active, as compared to 22, 27 and 28. These results show 3-tert-butyl or 3-OMe R³ group is more favorable.

Table 3:

Structures and anti-ZIKV (FLV strain) activities of compounds 44–52 in Vero cells.

Cpd #	R3	R6	% inhibition of ZIKV at 10 μM	% inhibition of ZIKV at 1 μM
44	3-CH3		99.9%	0%
45	same as above		90.0%	NT a
46	same as above		99.9%	0%
47	3-OCH3		99.9%	90.0%
48	same as above		99.0%	NT a
49	same as above		99.9%	68.4%
50	3-CF3		99.0%	NT a
51	same as above		0%	NT a
52	same as above		68.4%	NT a

^aNot tested.

Finally, the trans-C=C double bond in the most potent compounds 22 and 27 was hydrogenated to generate the corresponding saturated compounds 53 and 54 (Chart 2). Both compounds were found to be inactive (at 10 μM) against ZIKV replication in Vero cells. To test whether the double bond is chemically reactive in cells, which could cause covalent binding to a protein, compound 27 was tested in culture media with added glutathione (GSH), a nucleophilic thiol commonly present in cells. As shown in Supporting Information Figure S1, anti-ZIKV activity of compound 27 (as well as ZIKV replication) in Vero cells was not affected in the presence of 1 mM of GSH. These results show that the rigid, trans-C=C bond at the 2-position of the quinazolinone core is critical to the anti-ZIKV activity of this series of compounds.

Chart 2.

Structures and anti-ZIKV (FLV strain) activities of compounds 53 and 54 in Vero cells.

Antiviral activity evaluation

The most potent compounds 22, 27 and 47 in the anti-ZIKV screen were selected for dose-dependent testing against several ZIKV and DENV strains. The representative results are shown in Figure 1 and the antiviral EC₅₀ values summarized in Table 4. Compound 22 inhibited replication of the ZIKV FLR strain in Vero cells by 0%, 90% and >99.9% at 0.3, 1, 3 and 10 μM (Figure 1A) with a calculated EC₅₀ value of 900 nM (Table 4). Compound 27 exhibited ~4× more potent antiviral activity with an EC₅₀ of 180 nM, suppressing the ZIKV-FLR replication in Vero cells by 68%, 90% and >99.9% at 0.3, 1 and ≥3 μM. Compound 47 (EC₅₀ = 210 nM) was found to have similar activities to 27. Moreover, as shown in Figure 2A, treatment of ZIKV-infected Vero cells with compounds 22 and 27 can dose-dependently reduce the ZIKV RNA copies in the supernatant. Although ~1/10⁴ of these RNAs represent infectious viruses ³², the quantitative PCR results confirm the antiviral activities of these compounds. Treatment with compound 27 can also significantly reduce the cellular levels of viral NS5, NS3 and capsid proteins (Figure 2B). In addition, these three compounds inhibited the ZIKV-FLR replication in human glioblastoma U87 cells with comparable potencies, with compound 27 showing the most potent antiviral activity. It inhibited the viral replication by 90%, 97%, 99% and >99.9% at 0.3, 1, 3 and 10 μM (Figure 1B) with an EC₅₀ of 100 nM. Besides the two mammalian cells, ZIKV-FLR replication in mosquito C6/36 cells was also significantly blocked by these three compounds with EC₅₀s of 230–770 nM (Table 4 and Figure 1C).

Figure 1.

Representative antiviral activities of compounds 22, 27, and 47 in cells. (A-C, E) Treatment of Vero (A, E), U87 (B) and mosquito C6/36 cells (C) with compounds 22, 27, and 47 caused dose-dependent reduction of infectious ZIKV-FLR (A-C) and DENV-2 (E); (D, F) Treatment of Vero cells with compound 27 inhibited generation of infectious ZIKV-HN16 (D) and DENV-3 (F).

Table 4:

Antiviral activity (EC₅₀ in μM) against ZIKV and DENV.

	ZIKV-FLR in Vero cells	ZIKV-FLR in U87 cells	ZIKV-FLR in C6/36 cells	ZIKV-HN16 in Vero cells	DENV-2 in Vero cells	DENV-3 in Vero cells	CC50 (μM) in U87 cells
22	0.90 ± 0.32	0.82 ± 0.09	0.75 ± 0.40	NT a	0.16 ± 0.04	NT a	>20
27	0.18 ± 0.02	0.10 ± 0.022	0.23 ± 0.01	0.086 ± 0.057	0.21 ± 0.02	0.12 ± 0.07	>20
47	0.21 ± 0.01	0.69 ± 0.47	0.77 ± 0.41	NT a	0.56 ± 0.02	NT a	>20

^aNot tested.

Figure 2.

Treatment of Vero cells with compounds 27 and/or 22 dose-dependently reduced (A) the ZIKV RNA copies in the cell supernatant and (B) cellular viral NS5, NS3 and capsid proteins.

Next, the most potent compound 27 was tested against replication of the HN16 strain of ZIKV (Honduras, 2016 ¹⁶) in Vero cells. The compound can potently inhibit ZIKV-HN16 replication in a dose-dependent manner with an EC₅₀ of 86 nM (Table 4 and Figure 1D). Moreover, these three compounds did not significantly inhibit proliferation of human U87 cells at 20 μM, showing they are non-cytotoxic with a very high selectivity against ZIKV replication.

We next tested antiviral activity of these compounds against replication of DENV, using a similar end-point dilution assay ³⁴. We were pleased to find that compounds 22, 27 and 47 strongly inhibited replication of DENV serotype 2 virus (DENV-2, strain K0049, Thailand, 1995) in Vero cells with EC₅₀ values of 160, 210 and 560 nM, respectively (Table 4 and Figure 1E). In addition, replication of DENV serotype 3 virus (DENV-3, strain 7431, Sri Lanka, 1989) was also potently suppressed by compound 27 with an EC₅₀ of 120 nM (Table 4 and Figure 1F).

We also performed experiments to see whether delayed addition of compound 27 affects its anti-ZIKV activity in Vero cells. As shown in Figure 3A, adding compound 27 (1 μM) together with the initial virus into the cell culture media and incubating for 2 hours (2h-pretreatment), which is the regular compound treatment procedure for all of the above experiments, provided significantly higher anti-ZIKV activity, consistently reducing the viral titers by ≥90%. However, adding compound 27 (1 μM) immediately (0h-posttreatment) or 2 hours (2h-posttreatment) after removal of the inoculated virus showed reduced antiviral activity with 68% of viral titer reduction. Chloroquine, a known entry inhibitor of ZIKV, showed similar anti-ZIKV activities for these experiments (Figure 3B). These results suggest that compound 27 inhibits the attachment and/or entry of ZIKV to the cells.

Figure 3.

Anti-ZIKV (FLV strain) activities of (A) compound 27 and (B) chloroquine when added into the cell culture media at −2, 0 and 2 hours before removal of the inoculated virus (designated as 2h-pretreatment, 0h-, and 2h-posttreatment, respectively).

Conclusion

DENV and ZIKV are important human pathogens, causing ~100 million symptomatic infections each year. More significantly, these viral infections can further cause serious and life-threatening diseases, such as microcephaly (in newborns), Guillain-Barré syndrome, and dengue hemorrhagic fever and shock syndrome. In addition, patients recovered from one of 4 serotypes of DENV infection are still susceptible to other serotypes with an increased likelihood of serious disease because of antibody-dependent enhancement. Except for mosquito control, there have been no antiviral drugs to prevent and treat ZIKV/DENV infections. There is therefore a pressing need for such antiviral agents. Phenotype-based compound screening found several 2,3,6-trisubstituted quinazolinone compounds are novel inhibitors of ZIKV replication. Fifty-four analogs were synthesized and tested with their structure-activity relationships discussed above. Additional characterization shows that the most potent compounds 22, 27 and 47 are non-cytotoxic and exhibited potent activities against ZIKV and DENV with EC₅₀ values as low as 86 nM in a variety of mammalian and mosquito cells. Their structures are amenable for further medicinal chemistry optimization. Although the protein or nucleic acid target(s) that these compounds inhibit in mammalian and mosquito cells is unknown and is the subject of future investigation, our results suggest they inhibit ZIKV attachment or entry to the cell. Nevertheless, given the scarcity of potent and broad anti-Flavivirus agents, these compounds are novel pharmacological leads for drug development against ZIKV and DENV infections.

Experimental Section

All the chemicals used for synthesis were purchased from Aldrich (Milwaukee, WI) or Alfa Aesar (Ward Hill, MA). Unless otherwise stated, all solvents and reagents were used as received. All reactions were conducted with the use of a Teflon-coated magnetic stir bar at the indicated temperature and were performed under an inert atmosphere when stated. The identity of the synthesized compounds was characterized by ¹H and ¹³C NMR on a Varian (Palo Alto, CA) 400-MR spectrometer and mass spectrometer (Shimadzu LCMS-2020). Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). The identity of the potent inhibitors was confirmed with high resolution mass spectra (HRMS) using an Agilent 6550 iFunnel quadrupole-time-of-flight (Q-TOF) mass spectrometer with electrospray ionization (ESI). The purities of the final compounds were determined to be >95% with a Shimadzu Prominence HPLC using a Zorbax C18 (or C8) column (4.6 × 250 mm) at 0.7 mL/min flow rate (water:acetonitrile with 0.1% formic acid, 90:10 – 5:95 in 3 min) monitored by UV at 254 nm.

Chemical synthesis.

General synthetic methods for compounds 2–21 and 43:

A mixture of isatoic anhydride 55 (3.07 mmol, 1 eq) and an aniline (3.37 mmol, 1.1 eq) was heated in 1,4-dioxane (10 mL) at 90 °C for 12 h. The solvent was evaporated, and the crude product purified with column chromatography (silica gel, hexane/ethyl acetate, 6/1) to give compound 56 as a white or pale-yellow powder in 45–89% yield.

A mixture of 2-amino-N-phenyl-benzamide 56 (0.565 mmol, 1 eq) and 1,1,1-triethoxy-ethane (0.28 g, 1.696 mmol, 3 eq) were stirred at 110 °C for overnight. The reaction mixture was quenched with water (10 mL) and the product extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine and dried over Na₂SO₄. Upon removal of the solvent, the residue was purified with column chromatography (silica gel, hexane/ethyl acetate, 10/1) to provide compound 57 as a white or pale-yellow powder in 32–94% yield.

A mixture of 2-methyl-3-phenyl-4(3H)-quinazolinone 57 (0.14 mmol, 1 eq), an aldehyde (0.18 mmol, 1.3 eq) and NaOAc (6.8 mg, 0.08 mmol, 0.6 eq) in AcOH (1 ml) was heated at 110 °C for overnight. The reaction mixture was cooled down and the product extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine and dried over Na₂SO₄. Upon removal of the solvent, the residue was purified with column chromatography (silica gel, hexane/ethyl acetate, 5/1) to provide compounds 2–21 and 43 as a white or pale-yellow powder in 35–91% yield.

3-(3-tert-Butylphenyl)-2-[2-(2-methoxyl-3-pyridinyl)ethenyl]-4(3H)-quinazolinone (2)

¹H NMR (CDCl₃, 400 MHz) δ 8.31 (d, J = 8.3 Hz, 1H), 8.07 (dd, J = 4.9, 1.7 Hz, 1H), 7.96 (d, J = 15.6 Hz, 1H), 7.82 – 7.73 (m, 2H), 7.60 – 7.40 (m, 4H), 7.29 (d, J = 1.7 Hz, 1H), 7.18 – 7.11 (m, 1H), 6.85 (dd, J = 7.3, 4.9 Hz, 1H), 6.74 (d, J = 15.6 Hz, 1H), 3.79 (s, 3H), 1.35 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.5, 161.9, 153.4, 152.4, 147.9, 147.2, 138.7, 137.1, 134.6, 134.3, 129.5, 127.5, 127.3, 126.7, 126.2, 125.8, 123.4, 121.1, 119.1, 117.1, 53.3, 35.0, 31.4. MS (ESI) calculated for (C₂₆H₂₆N₃O₂)⁺ [M+H]⁺ 412.2, found 412.2.

3-(3-tert-Butylphenyl)-2-[2-(4-trifluoromethyl-3-pyridinyl)ethenyl]-4(3H)-quinazolinone (3)

¹H NMR (CDCl₃, 400 MHz) δ 8.64 (s, 1H), 8.33 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 15.7 Hz, 1H), 7.84 – 7.76 (m, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.56 (ddd, J = 15.5, 13.8, 8.0 Hz, 4H), 7.29 (s, 1H), 7.15 (dd, J = 7.6, 0.9 Hz, 1H), 6.52 (d, J = 15.7 Hz, 1H), 1.34 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.1, 153.8, 150.7, 149.0, 147.6, 136.4, 135.5, 134.9, 134.0, 129.8, 127.7, 127.4, 126.7, 125.8, 125.7, 124.7, 121.4, 120.6, 110.1, 35.1, 31.4. MS (ESI) calculated for (C₂₆H₂₃F₃N₃O)⁺ [M+H]⁺ 450.2, found 450.2.

3-(3-Methoxylphenyl)-2-[2-(3-pyridinyl)ethenyl]-4(3H)-quinazolinone (4)

1H NMR (CDCl₃, 400 MHz) δ 8.58 (s, 1H), 8.49 (d, J = 4.1 Hz, 1H), 8.27 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 15.6 Hz, 1H), 7.77 (d, J = 3.6 Hz, 2H), 7.56 (d, J = 8.0 Hz, 1H), 7.46 (td, J = 8.2, 5.0 Hz, 2H), 7.21 (dd, J = 7.9, 4.8 Hz, 1H), 7.07 (dd, J = 8.2, 2.2 Hz, 1H), 6.91 – 6.80 (m, 2H), 6.48 (d, J = 15.6 Hz, 1H), 3.82 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.1, 160.8, 151.0, 150.3, 149.4, 147.6, 137.9, 136.1, 134.7, 134.1, 131.1, 130.8, 127.5, 127.2, 127.0, 123.7, 121.9, 121.1, 120.8, 115.4, 114.4, 55.6. MS (ESI) calculated for (C₂₂H₁₈N₃O₂)⁺ [M+H]⁺ 356.1, found 356.2.

3-Phenyl-2-[2-(3-pyridinyl)ethenyl]-4(3H)-quinazolinone (5)

¹H NMR (CDCl₃, 400 MHz) δ 8.58 (s, 1H), 8.50 (s, 1H), 8.30 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 15.6 Hz, 1H), 7.80 (s, 2H), 7.57 (t, J = 8.3 Hz, 4H), 7.49 (s, 1H), 7.32 (d, J = 7.0 Hz, 2H), 7.24 (dd, J = 11.8, 6.8 Hz, 1H), 6.45 (d, J = 15.5 Hz, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.3, 151.1, 150.4, 149.5, 147.7, 136.9, 136.2, 134.8, 134.1, 131.2, 130.1, 129.7, 128.8, 127.6, 127.3, 127.1, 123.7, 122.0, 121.2. MS (ESI) calculated for (C₂₁H₁₆N₃O)⁺ [M+H]⁺ 326.1, found 326.1.

3-(2-Methylphenyl)-2-[2-(3-pyridinyl)ethenyl]-4(3H)-quinazolinone (6)

¹H NMR (CDCl₃, 400 MHz) δ 8.57 (s, 1H), 8.50 (d, J = 4.3 Hz, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.97 (d, J = 15.6 Hz, 1H), 7.81 (d, J = 4.2 Hz, 2H), 7.55 (d, J = 7.9 Hz, 1H), 7.51 – 7.34 (m, 4H), 7.25 – 7.18 (m, 2H), 6.38 (d, J = 15.6 Hz, 1H), 2.12 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ 161.7, 151.1, 150.4, 149.5, 147.9, 136.6, 136.2, 136.0, 134.8, 134.1, 131.7, 131.2, 130.0, 128.7, 127.8, 127.6, 127.4, 127.0, 123.8, 121.3, 121.2, 17.7. MS (ESI) calculated for (C₂₂H₁₈N₃O)⁺ [M+H]⁺ 340.1, found 340.1

3-(3-Methylphenyl)-2-[2-(3-pyridinyl)ethenyl]-4(3H)-quinazolinone (7)

¹H NMR (CDCl₃, 400 MHz) δ 8.59 (s, 1H), 8.51 (d, J = 3.9 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 15.6 Hz, 1H), 7.79 (d, J = 3.2 Hz, 2H), 7.59 (d, J = 7.6 Hz, 1H), 7.51 – 7.41 (m, 2H), 7.36 (d, J = 7.3 Hz, 1H), 7.24 (dd, J = 7.4, 4.7 Hz, 1H), 7.15 – 7.04 (m, 2H), 6.48 (d, J = 15.6 Hz, 1H), 2.44 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.3, 151.1, 150.2, 149.3, 147.7, 140.4, 136.7, 136.0, 134.8, 134.3, 131.3, 130.5, 129.9, 129.2, 127.5, 127.3, 127.0, 125.6, 123.8, 122.2, 121.2, 21.5.

134.1, 131.1, 130.8, 127.5, 127.2, 127.0, 123.7, 121.9, 121.1, 120.8, 115.4, 114.4, 55.6. MS (ESI) calculated for (C₂₂H₁₈N₃O)⁺ [M+H]⁺ 340.1, found 340.1.

3-(4-tert-Butylphenyl)-2-[2-(3-pyridinyl)ethenyl]-4(3H)-quinazolinone (8)

¹H NMR (CDCl₃, 400 MHz) δ 8.59 (s, 1H), 8.51 (s, 1H), 8.30 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 15.5 Hz, 1H), 7.79 (s, 2H), 7.59 (d, J = 7.3 Hz, 3H), 7.49 (s, 1H), 7.23 (d, J = 7.3 Hz, 3H), 6.49 (d, J = 15.6 Hz, 1H), 1.40 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.4, 152.8, 151.4, 150.3, 149.4, 147.7, 136.1, 134.8, 134.3, 134.1, 131.3, 128.2, 127.6, 127.4, 127.1, 127.0, 123.8, 122.3, 121.2, 35.1, 31.5. MS (ESI) calculated for (C₂₅H₂₄N₃O)⁺ [M+H]⁺ 382.2, found 382.2.

3-(4-tert-Butylphenyl)-2-[2-(5-bromo-3-pyridinyl)ethenyl]-4(3H)-quinazolinone (9)

1H NMR (CDCl₃, 400 MHz) δ 8.56 (d, J = 2.1 Hz, 1H), 8.46 (d, J = 1.7 Hz, 1H), 8.35 – 8.27 (m, 1H), 7.84 (d, J = 15.6 Hz, 1H), 7.80 (dd, J = 5.8, 1.2 Hz, 2H), 7.66 (t, J = 1.8 Hz, 1H), 7.54 (dddd, J = 11.9, 8.2, 4.3, 1.8 Hz, 3H), 7.28 (t, J = 1.8 Hz, 1H), 7.19 – 7.13 (m, 1H), 6.43 (d, J = 15.6 Hz, 1H), 1.35 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.1, 153.6, 151.1, 150.8, 147.6, 147.1, 136.5, 136.4, 134.8, 134.1, 132.9, 129.8, 127.6, 127.3, 127.2, 126.7, 125.7, 123.7, 121.3, 121.1, 35.1, 31.4. MS (ESI) calculated for (C₂₅H₂₃BrN₃O)⁺ [M+H]⁺ 460.1, found 460.1

3-(4-tert-Butylphenyl)-2-[2-(5-pyrimidyl)ethenyl]-4(3H)-quinazolinone (10)

1H NMR (CDCl₃, 400 MHz) δ 9.10 (s, 1H), 8.67 (s, 2H), 8.31 (d, J = 7.9 Hz, 1H), 7.88 (d, J = 15.7 Hz, 1H), 7.79 (s, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.51 (t, J = 5.7 Hz, 1H), 7.24 (t, J = 7.8 Hz, 2H), 6.57 (d, J = 15.7 Hz, 1H), 1.40 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.2, 158.6, 155.4, 153.0, 150.7, 147.5, 134.9, 133.8, 132.1, 129.4, 128.1, 127.6, 127.4, 127.2, 124.1, 121.3, 35.1, 31.5. MS (ESI) calculated for (C₂₄H₂₃N₄O)⁺ [M+H]⁺ 383.2, found 383.2

3-(3-tert-Butylphenyl)-2-[2-(3-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (11)

¹H NMR (CDCl₃, 400 MHz) δ 8.32 (d, J = 7.7 Hz, 1H), 7.81 (d, J = 8.6 Hz, 3H), 7.60 – 7.43 (m, 4H), 7.32 (s, 1H), 7.14 (d, J = 7.2 Hz, 1H), 7.04 (d, J = 14.8 Hz, 1H), 6.89 (d, J = 7.0 Hz, 1H), 6.62 (d, J = 8.2 Hz, 1H), 3.59 (s, 3H), 1.33 (s, 9H).¹³C NMR (CDCl₃, 100 MHz) δ 163.3, 162.4, 153.5, 152.0, 150.8, 148.0, 139.1, 137.7, 137.1, 134.6, 129.5, 127.5, 127.3, 126.8, 126.2, 125.8, 125.6, 124.1, 121.3, 118.2, 112.2, 52.7, 35.0, 31.4. MS (ESI) calculated for (C₂₆H₂₆N₃O₂)⁺ [M+H]⁺ 412.2, found 412.2

3-(3-tert-Butylphenyl)-2-[2-(3-pyridinyl)ethenyl]-4(3H)-quinazolinone (12)

1H NMR (CDCl₃, 400 MHz) δ 8.57 (s, 1H), 8.51 (d, J = 4.5 Hz, 1H), 8.32 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 15.6 Hz, 1H), 7.80 (d, J = 3.6 Hz, 2H), 7.59 – 7.45 (m, 4H), 7.29 (s, 1H), 7.26 – 7.19 (m, 1H), 7.15 (d, J = 7.5 Hz, 1H), 6.44 (d, J = 15.6 Hz, 1H), 1.34 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.3, 153.6, 151.3, 150.3, 149.3, 147.7, 136.6, 135.9, 134.8, 134.1, 131.3, 129.7, 127.6, 127.3, 127.1, 126.6, 125.8, 125.7, 123.8, 122.3, 121.3, 35.1, 31.4. MS (ESI) calculated for (C₂₅H₂₆N₃O)⁺ [M+H]⁺ 382.2, found 382.2

3-(3-tert-Butylphenyl)-2-[2-(4-pyridinyl)ethenyl]-4(3H)-quinazolinone (13)

¹H NMR (CDCl₃, 400 MHz) δ 8.55 (d, J = 4.5 Hz, 2H), 8.33 (d, J = 7.6 Hz, 1H), 7.86–7.80 (m, 3H), 7.62 – 7.47 (m, 3H), 7.27 (d, J = 7.9 Hz, 1H), 7.14 (dd, J = 19.1, 5.6 Hz, 3H), 6.54 (d, J = 15.6 Hz, 1H), 1.34 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.2, 153.6, 151.0, 150.5, 147.6, 142.6, 136.6, 136.5, 134.8, 129.7, 127.7, 127.4, 127.3, 126.6, 125.8, 125.7, 124.7, 121.6, 121.4, 35.1, 31.4. MS (ESI) calculated for (C₂₅H₂₆N₃O)⁺ [M+H]⁺ 382.2, found 382.2

3-(3-tert-Butylphenyl)-2-[2-(5-pyrimidyl)ethenyl]-4(3H)-quinazolinone (14)

¹H NMR (CDCl₃, 400 MHz) δ 9.09 (s, 1H), 8.64 (s, 2H), 8.32 (d, J = 7.7 Hz, 1H), 7.92 – 7.75 (m, 3H), 7.54 (dt, J = 11.4, 7.8 Hz, 3H), 7.28 (s, 1H), 7.14 (d, J = 7.6 Hz, 1H), 6.50 (d, J = 15.7 Hz, 1H), 1.34 (s, 9H).¹³C NMR (CDCl₃, 100 MHz) δ 162.1, 158.6, 155.3, 153.8, 150.6, 147.5, 136.4, 134.9, 131.9, 129.8, 129.4, 127.7, 127.4, 126.7, 125.7, 125.7, 124.1, 121.4, 35.1, 31.4. MS (ESI) calculated for (C₂₄H₂₃N₄O)⁺ [M+H]⁺ 383.2, found 383.2.

3-(3-tert-Butylphenyl)-2-(2-phenylethenyl)-4(3H)-quinazolinone (15)

¹H NMR (CDCl₃, 400 MHz) δ 8.32 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 15.5 Hz, 1H), 7.82 – 7.78 (m, 2H), 7.60 – 7.44 (m, 3H), 7.30 (s, 6H), 7.16 (dd, J = 7.8, 1.6 Hz, 1H), 6.38 (d, J = 15.5 Hz, 1H), 1.35 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.4, 153.5, 152.1, 147.9, 139.8, 136.8, 135.5, 134.7, 129.7, 129.6, 128.9, 127.8, 127.5, 127.3, 126.7, 126.4, 125.9, 125.8, 121.2, 120.3, 35.1, 31.4. MS (ESI) calculated for (C₂₆H₂₅N₂O)⁺ [M+H]⁺ 381.2, found 381.2.

3-(3-tert-Butylphenyl)-2-[2-(2-methoxylphenyl)ethenyl]-4(3H)-quinazolinone (16)

¹H NMR (CDCl₃, 400 MHz) δ 8.31 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 16.0 Hz, 1H), 7.87 – 7.73 (m, 2H), 7.60 – 7.42 (m, 4H), 7.29 (dd, J = 6.3, 1.6 Hz, 1H), 7.24 (s, 1H), 7.15 (d, J = 7.5 Hz, 1H), 6.91 – 6.79 (m, 2H), 6.63 (d, J = 15.6 Hz, 1H), 3.71 (s, 3H), 1.35 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.5, 158.5, 153.4, 137.1, 134.6, 130.8, 130.0, 129.5, 127.3, 126.5, 126.2, 125.9, 124.5, 120.8, 111.1, 55.2, 35.1, 31.4. MS (ESI) calculated for (C₂₇H₂₇N₂O₂)⁺ [M+H]⁺ 411.2, found 411.2

3-(3-tert-Butylphenyl)-2-[2-(3-methoxylphenyl)ethenyl]-4(3H)-quinazolinone (17)

¹H NMR (CDCl₃, 400 MHz) δ 8.32 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 15.5 Hz, 1H), 7.79 (dd, J = 3.6, 1.7 Hz, 2H), 7.60 – 7.43 (m, 3H), 7.30 (s, 1H), 7.24 – 7.13 (m, 2H), 6.93 – 6.79 (m, 3H), 6.36 (d, J = 15.5 Hz, 1H), 3.76 (s, 3H), 1.35 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.4, 159.9, 153.5, 152.0, 147.9, 139.7, 136.9, 136.8, 134.7, 129.9, 129.6, 127.4, 127.3, 126.7, 126.4, 125.8, 121.1, 120.6, 120.3, 115.4, 113.0, 55.3, 35.0, 31.4. MS (ESI) calculated for (C₂₇H₂₇N₂O₂)⁺ [M+H]⁺ 411.2, found 411.2.

3-(3-tert-Butylphenyl)-2-[2-(4-trifluoromethylphenyl)ethenyl]-4(3H)-quinazolinone (18)

¹H NMR (CDCl₃, 400 MHz) δ 8.33 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 15.6 Hz, 1H), 7.81 (d, J = 3.7 Hz, 2H), 7.61 – 7.46 (m, 5H), 7.38 (d, J = 8.0 Hz, 2H), 7.30 (s, 1H), 7.17 (d, J = 7.5 Hz, 1H), 6.45 (d, J = 15.6 Hz, 1H), 1.35 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.2, 153.6, 151.5, 147.6, 138.9, 137.9, 136.6, 134.8, 129.7, 127.9, 127.5, 127.4, 127.1, 126.6, 125.9, 125.9, 125.8, 125.8, 122.6, 121.3, 35.1, 31.4. MS (ESI) calculated for (C₂₇H₂₄F₃N₂O)⁺ [M+H]⁺ 449.2, found 449.2.

3-(3-tert-Butylphenyl)-2-[2-(1-naphthyl)ethenyl]-4(3H)-quinazolinone (19)

¹H NMR (CDCl₃, 400 MHz) δ 8.77 (d, J = 15.3 Hz, 1H), 8.34 (d, J = 7.9 Hz, 1H), 8.25 (d, J = 8.3 Hz, 1H), 7.93 – 7.74 (m, 4H), 7.59 – 7.46 (m, 5H), 7.39 – 7.30 (m, 3H), 7.19 (d, J = 7.5 Hz, 1H), 6.48 (d, J = 15.3 Hz, 1H), 1.35 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 153.6, 136.9, 134.8, 133.8, 133.1, 131.6, 130.1, 129.7, 128.9, 127.5, 127.3, 126.9, 126.5, 126.3, 125.9, 125.8, 125.5, 124.8, 123.9, 122.9, 121.2, 35.1, 31.4. MS (ESI) calculated for (C₃₀H₂₇N₂O)⁺ [M+H]⁺ 431.2, found 431.2.

6-Chloro-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (20)

¹H NMR (CDCl₃, 400 MHz) δ 8.25 (d, J = 1.5 Hz, 1H), 7.79 (d, J = 14.8 Hz, 1H), 7.75 – 7.66 (m, 2H), 7.51 (ddd, J = 13.0, 12.3, 6.7 Hz, 3H), 7.31 (s, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.01 (d, J = 14.8 Hz, 1H), 6.88 (d, J = 7.1 Hz, 1H), 6.62 (d, J = 8.3 Hz, 1H), 3.58 (s, 3H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.3, 161.4, 153.6, 152.2, 150.6, 146.4, 139.0, 138.1, 136.8, 135.0, 132.4, 129.6, 129.2, 126.6, 126.3, 125.7, 125.5, 123.7, 122.2, 118.2, 112.3, 52.7, 35.0, 31.4. MS (ESI) calculated for (C₂₆H₂₅ClN₃O₂)⁺ [M+H]⁺ 446.2, found 446.1.

6-Methoxy-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (21)

¹H NMR (CDCl₃, 400 MHz) δ 7.80 – 7.71 (m, 2H), 7.69 (d, J = 3.0 Hz, 1H), 7.56 – 7.44 (m, 3H), 7.39 (dd, J = 8.9, 3.0 Hz, 1H), 7.30 (d, J = 2.0 Hz, 1H), 7.12 (ddd, J = 7.5, 2.2, 1.3 Hz, 1H), 7.01 (d, J = 14.8 Hz, 1H), 6.87 (d, J = 7.2 Hz, 1H), 6.60 (dd, J = 8.2, 0.9 Hz, 1H), 3.92 (s, 3H), 3.58 (s, 3H), 1.32 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 158.5, 153.3, 150.8, 149.8, 138.9, 137.0, 129.3, 128.9, 125.9, 125.6, 125.5, 124.8, 117.8, 111.8, 106.5, 55.8, 52.5, 34.8, 31.2. MS (ESI) calculated for (C₂₈H₂₈N₃O₃)⁺ [M+H]⁺ 442.2, found 442.2.

6-Methoxy-3-(3-tert-butylphenyl)-2-(3-methoxystyryl)-4(3H)-quinazolinone (43)

¹H NMR (CDCl₃, 400 MHz) δ 7.84 (d, J = 15.5 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.67 (d, J = 2.9 Hz, 1H), 7.55 (ddd, J = 8.0, 1.9, 1.2 Hz, 1H), 7.53 – 7.47 (m, 1H), 7.38 (dd, J = 9.0, 3.0 Hz, 1H), 7.28 (t, J = 1.9 Hz, 1H), 7.19 (t, J = 7.9 Hz, 1H), 7.13 (ddd, J = 7.6, 2.1, 1.2 Hz, 1H), 6.86 (ddd, J = 7.6, 1.7, 0.9 Hz, 1H), 6.84 – 6.76 (m, 2H), 6.33 (d, J = 15.5 Hz, 1H), 3.91 (s, 3H), 3.74 (s, 3H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.0, 159.7, 158.4, 153.2, 149.8, 136.9, 136.7, 129.6, 129.4, 128.9, 126.1, 125.6, 125.6, 124.9, 121.7, 120.0, 115.0, 112.8, 106.5, 55.8, 55.1, 34.8, 31.2. MS (ESI) calculated for (C₂₈H₂₉N₃O₂)⁺ [M+H]⁺ 441.2, found 441.2.

General synthetic methods for compounds 1, 22–28, 35–42, 44–52:

A mixture of a compound 20 (35 mg, 0.07 mmol), an amine (0.08 mmol), tris(dibenzylideneacetone)dipalladium(0) (4 mg, 0.004 mol), 2-dicyclohexylphosphino-2’,6’-dimethoxybiphenyl (3 mg, 0.008 mmol), sodium tert-butoxide (10 mg, 0.10 mmol) in toluene (5 mL) was heated at 110 °C for 24 h. The reaction mixture was cooled down and quenched with brine (20 mL). The product was extracted with ethyl acetate (3 × 20 mL), and the combined organic phase was washed with brine and then dried over Na₂SO₄. Upon removal of solvent, the residue was purified with column chromatography (silica gel, hexanes : ethyl acetate, 8: 1) to give the target compounds 1, 22–24, 26–28, 35–42, 44–52 as a pale yellow solid in 30–81% yield.

To the solution of compound 24 (0.1 mmol) in dichloromethane (2 mL) was added dropwise HCl (0.2 mL, 4N in p-dioxane) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 12 hours. The solid powder thus obtained was filtered and washed with in dichloromethane to give compound 25 as a hydrochloride salt.

6-Diethylamino-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (1)

¹H NMR (CDCl₃, 400 MHz) δ 7.76 – 7.59 (m, 2H), 7.54 – 7.37 (m, 4H), 7.30 (d, J = 2.0 Hz, 1H), 7.21 (d, J = 9.2 Hz, 1H), 7.11 (ddd, J = 7.4, 2.1, 1.2 Hz, 1H), 7.00 (d, J = 14.9 Hz, 1H), 6.84 (d, J = 7.2 Hz, 1H), 6.57 (d, J = 8.3 Hz, 1H), 3.57 (s, 3H), 3.46 (q, J = 7.1 Hz, 4H), 1.31 (s, 9H), 1.21 (t, J = 7.1 Hz, 6H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.0, 162.2, 153.1, 151.1, 147.3, 146.8, 138.8, 137.4, 135.1, 129.2, 128.5, 125.7, 125.5, 124.3, 122.2, 120.0, 117.5, 111.3, 105.6, 52.5, 44.6, 34.8, 31.2, 12.4. MS (ESI) calculated for (C₃₀H₃₅N₄O₂)⁺ [M+H]⁺ 483.3, found 483.2.

6-(Piperidin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (22)

¹H NMR (CDCl₃, 400 MHz) δ 7.71 (d, J = 4.2 Hz, 1H), 7.69 – 7.62 (m, 2H), 7.48 (ddd, J = 11.3, 10.7, 5.7 Hz, 4H), 7.31 (s, 1H), 7.13 (d, J = 7.3 Hz, 1H), 7.02 (d, J = 14.9 Hz, 1H), 6.85 (d, J = 7.1 Hz, 1H), 6.58 (d, J = 8.2 Hz, 1H), 3.58 (s, 3H), 3.36 – 3.26 (m, 4H), 1.72 (d, J = 4.4 Hz, 4H), 1.62 (d, J = 4.7 Hz, 2H), 1.32 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.2, 162.4, 153.3, 151.2, 150.7, 148.9, 140.7, 139.0, 137.5, 135.9, 129.4, 128.4, 125.9, 125.9, 125.7, 124.8, 124.4, 122.0, 117.8, 111.7, 110.1, 52.7, 50.3, 35.0, 31.4, 25.7, 24.3. HRMS (ESI) calculated for (C₃₁H₃₅N₄O₂)⁺ [M+H]⁺ 495.2760, found 495.2754.

6-(Pyrrolidin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (23)

¹H NMR (CDCl₃, 400 MHz) δ 7.78 – 7.66 (m, 2H), 7.55 – 7.44 (m, 3H), 7.34 – 7.28 (m, 2H), 7.15 – 7.09 (m, 2H), 7.01 (d, J = 14.9 Hz, 1H), 6.86 (d, J = 7.2 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 3.58 (s, 3H), 3.41 (d, J = 6.4 Hz, 4H), 2.07 (t, J = 6.4 Hz, 4H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.3, 153.3, 151.3, 147.0, 139.0, 137.6, 135.4, 129.4, 128.5, 125.9, 125.8, 122.3, 120.3, 117.8, 111.6, 110.2, 105.8, 52.8, 48.0, 35.0, 31.4, 25.7. MS (ESI) calculated for (C₃₀H₃₃N₄O₂)⁺ [M+H]⁺ 481.3, found 481.2

tert-Butyl-4-(3-(3-(tert-butyl)phenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4-oxo-3,4-dihydroquinazolin-6-yl)piperazine-1-carboxylate (24)

¹H NMR (CDCl₃, 400 MHz) δ 8.00 (s, 2H), 7.64 (d, J = 2.8 Hz, 1H), 7.57 – 7.44 (m, 4H), 7.29 (d, J = 1.9 Hz, 1H), 7.11 (dt, J = 7.6, 1.6 Hz, 1H), 6.99 (dd, J = 15.0, 1.4 Hz, 1H), 6.95 (d, J = 7.2 Hz, 1H), 6.62 (d, J = 8.3 Hz, 1H), 3.62 (t, J = 5.2 Hz, 4H), 3.56 (d, J = 1.3 Hz, 3H), 3.30 (t, J = 5.3 Hz, 4H), 1.46 (s, 9H), 1.32 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 161.4, 154.6, 153.5, 150.3, 149.9, 139.0, 136.5, 129.5, 129.4, 126.2, 125.5, 125.4, 124.7, 121.3, 118.7, 112.3, 110.4, 80.1, 52.5, 48.7, 43.3, 34.9, 31.2, 28.4, 28.4. MS (ESI) calculated for (C₃₅H₄₂N₅O₄)⁺ [M+H]⁺ 596.3, found 596.3.

6-(Piperazin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone hydrochloride (25)

¹H NMR (DMSO-d₆, 400 MHz) δ 9.54 (s, 2H), 7.93 – 7.83 (m, 2H), 7.72 – 7.62 (m, 2H), 7.55 (dt, J = 8.0, 1.5 Hz, 1H), 7.53 – 7.44 (m, 3H), 7.23 (dt, J = 7.9, 1.4 Hz, 1H), 7.08 (d, J = 7.2 Hz, 1H), 6.84 (d, J = 15.0 Hz, 1H), 6.72 (d, J = 8.3 Hz, 1H), 3.53 (t, J = 5.1 Hz, 4H), 3.46 (d, J = 1.1 Hz, 3H), 3.21 (s, 4H), 1.24 (d, J = 1.0 Hz, 9H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 163.0, 160.9, 153.1, 150.3, 150.2, 149.3, 140.5, 138.7, 138.0, 137.2, 129.7, 127.0, 126.3, 126.2, 126.0, 124.9, 122.5, 121.5, 119.3, 112.7, 109.9, 52.5, 45.4, 42.7, 35.0, 31.4. MS (ESI) calculated for (C₃₀H₃₄N₅O₂)⁺ [M+H]⁺ 496.3, found 496.3

6-(4-Methylpiperazin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (26)

¹H NMR (CDCl₃, 400 MHz) δ 7.75 – 7.62 (m, 3H), 7.54 – 7.41 (m, 4H), 7.29 (q, J = 3.4, 2.5 Hz, 1H), 7.14 – 7.06 (m, 1H), 7.04 – 6.95 (m, 1H), 6.84 (d, J = 7.2 Hz, 1H), 6.58 (d, J = 8.6 Hz, 1H), 3.56 (d, J = 1.4 Hz, 3H), 3.43 – 3.31 (m, 4H), 2.63 (t, J = 4.9 Hz, 4H), 2.38 (d, J = 2.6 Hz, 3H), 1.31 (d, J = 1.3 Hz, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.2, 153.2, 150.9, 149.7, 149.1, 138.8, 137.2, 136.0, 129.3, 128.4, 125.8, 125.7, 125.5, 124.2, 124.0, 121.8, 117.7, 111.6, 110.1, 54.7, 52.5, 48.5, 45.9, 34.8, 31.2. MS (ESI) calculated for (C₃₁H₃₆N₅O₂)⁺ [M+H]⁺ 510.3, found 510.3

6-Morpholino-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (27)

¹H NMR (CDCl₃, 400 MHz) δ 7.78 – 7.68 (m, 2H), 7.65 (d, J = 2.9 Hz, 1H), 7.55 – 7.38 (m, 4H), 7.29 (d, J = 2.0 Hz, 1H), 7.11 (d, J = 7.3 Hz, 1H), 7.00 (d, J = 14.9 Hz, 1H), 6.85 (d, J = 7.1 Hz, 1H), 6.59 (d, J = 8.3 Hz, 1H), 4.00 – 3.80 (m, 4H), 3.57 (s, 3H), 3.29 (dd, J = 5.9, 3.8 Hz, 4H), 1.31 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.2, 153.2, 150.8, 149.9, 149.3, 138.9, 137.1, 129.3, 128.4, 125.9, 125.6, 125.5, 123.7, 121.8, 117.7, 111.7, 109.9, 66.7, 52.5, 48.9, 34.8, 31.2; HRMS (ESI) calculated for (C₃₀H₃₃N₄O₃)⁺ [M+H]⁺ 497.2553, found 497.2536.

6-(4-Morpholinopiperidin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxyl-2-pyridinyl)ethenyl]-4(3H)-quinazolinone (28)

¹H NMR (CDCl₃, 400 MHz) δ 7.70 (d, J = 3.0 Hz, 1H), 7.66 (dd, J = 5.8, 2.9 Hz, 2H), 7.54 – 7.42 (m, 4H), 7.29 (d, J = 2.0 Hz, 1H), 7.10 (dt, J = 7.2, 1.5 Hz, 1H), 6.99 (d, J = 14.8 Hz, 1H), 6.84 (d, J = 7.1 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 3.90 (d, J = 12.8 Hz, 2H), 3.74 (t, J = 4.7 Hz, 4H), 3.56 (s, 3H), 2.85 (t, J = 12.2 Hz, 2H), 2.59 (s, 4H), 2.39 (s, 1H), 1.98 (d, J = 12.4 Hz, 2H), 1.67 (q, J = 11.6 Hz, 2H), 1.31 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) 163.1, 153.1, 150.9, 149.7, 138.8, 137.2, 135.9, 129.3, 128.4, 125.8, 125.7, 125.5, 124.5, 124.2, 117.7, 111.6, 110.1, 61.9, 52.5, 49.7, 48.6, 48.5, 34.8, 31.2, 27.8. MS (ESI) calculated for (C₃₅H₄₂N₅O₃)⁺ [M+H]⁺ 580.3, found 580.3.

6-(Piperidin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(6-trifluoromethyl-3-pyridinyl)ethenyl]-4(3H)-quinazolinone (35)

¹H NMR (CDCl₃, 400 MHz) δ 8.62 (s, 1H), 7.83 (d, J = 15.7 Hz, 1H), 7.68 (dd, J = 12.1, 6.1 Hz, 3H), 7.62 – 7.45 (m, 4H), 7.29 (s, 1H), 7.16 – 7.09 (m, 1H), 6.50 (d, J = 15.7 Hz, 1H), 3.40 – 3.29 (m, 4H), 1.74 (s, 4H), 1.64 (d, J = 4.7 Hz, 2H), 1.34 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.2, 153.6, 148.8, 147.6, 136.9, 135.2, 134.4, 132.1, 129.7, 128.6, 126.5, 125.9, 125.8, 125.0, 122.2, 120.6, 110.0, 50.1, 35.1, 31.4, 25.6, 24.3. MS (ESI) calculated for (C₃₁H₃₂F₃N₄O)⁺ [M+H]⁺ 533.3, found 533.2

6-(Piperidin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(4-trifluoromethyl-3-pyridinyl)ethenyl]-4(3H)-quinazolinone (36)

¹H NMR (CDCl₃, 100 MHz) δ 7.82 (d, J = 15.6 Hz, 1H), 7.72 – 7.61 (m, 2H), 7.58 – 7.44 (m, 5H), 7.33 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 2.0 Hz, 1H), 7.13 (ddd, J = 7.5, 2.1, 1.1 Hz, 1H), 6.40 (d, J = 15.6 Hz, 1H), 3.32 (t, J = 5.4 Hz, 4H), 1.73 (t, J = 5.8 Hz, 4H), 1.62 (p, J = 5.9 Hz, 2H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.1, 153.2, 148.2, 139.0, 136.8, 135.9, 129.4, 128.3, 127.4, 126.1, 125.7, 125.6, 125.6, 122.8, 121.8, 34.8, 31.2, 29.6, 25.4, 24.1. LC-MS (M + H) = 532.3. MS (ESI) calculated for (C₃₂H₃₃F₃N₃O)⁺ [M+H]⁺ 532.3, found 532.3.

6-(Piperidin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(4-pyridinyl)ethenyl]-4(3H)-quinazolinone (37)

¹H NMR (CDCl₃, 400 MHz) δ 8.51 (d, J = 5.2 Hz, 2H), 7.77 – 7.61 (m, 3H), 7.59 – 7.44 (m, 3H), 7.26 (d, J = 1.8 Hz, 1H), 7.17 – 7.04 (m, 3H), 6.51 (dd, J = 15.6, 1.2 Hz, 1H), 3.32 (t, J = 5.4 Hz, 4H), 1.72 (t, J = 5.6 Hz, 4H), 1.61 (p, J = 5.7 Hz, 2H), 1.32 (d, J = 1.3 Hz, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.0, 153.3, 150.8, 149.9, 147.6, 143.2, 140.1, 136.7, 134.4, 129.4, 128.4, 126.2, 125.7, 125.6, 125.1, 124.4, 122.0, 121.3, 109.8, 49.9, 34.8, 31.2, 29.6, 25.4, 24.1. LC-MS (M + H) = 465.3. MS (ESI) calculated for (C₃₀H₃₃N₄O)⁺ [M+H]⁺ 465.3, found 465.3.

6-(Piperidin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(2-naphthalenyl)ethenyl]-4(3H)-quinazolinone (38)

¹H NMR (CDCl₃, 400 MHz) δ 8.00 (d, J = 15.5 Hz, 1H), 7.82 – 7.62 (m, 6H), 7.61 – 7.38 (m, 5H), 7.33 – 7.25 (m, 2H), 7.17 (dt, J = 7.5, 1.6 Hz, 1H), 6.45 (d, J = 15.5 Hz, 1H), 3.31 (t, J = 5.4 Hz, 4H), 1.74 (s, 4H), 1.62 (s, 2H), 1.34 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.1, 153.2, 136.9, 133.7, 133.3, 133.0, 129.4, 129.1, 128.4, 128.3, 127.6, 126.8, 126.5, 126.1, 125.7, 125.7, 123.1, 121.5, 34.9, 31.2, 29.6, 25.3, 23.9. MS (ESI) calculated for (C₃₅H₃₆N₃O)⁺ [M+H]⁺ 514.3, found 514.3.

6-(Piperidin-1-yl)-3-(3-tert-butylphenyl)-2-(2,6-dimethoxystyryl)-4(3H)-quinazolinone (39)

¹H NMR (CDCl₃, 400 MHz) δ 8.27 (d, J = 15.9 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.65 (d, J = 2.6 Hz, 1H), 7.55 – 7.43 (m, 3H), 7.27 (d, J = 2.0 Hz, 1H), 7.18 – 7.08 (m, 2H), 6.95 (d, J = 15.8 Hz, 1H), 6.45 (d, J = 8.4 Hz, 2H), 3.65 (s, 6H), 3.28 (t, J = 5.4 Hz, 4H), 1.73 (s, 4H), 1.61 (q, J = 5.9 Hz, 2H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.5, 159.6, 152.9, 137.7, 130.0, 129.1, 128.3, 125.8, 125.7, 125.5, 123.2, 121.3, 113.3, 103.5, 55.3, 50.5, 34.8, 31.2, 29.6, 25.4, 25.2, 24.0. LC-MS (M + H) = 524.3. MS (ESI) calculated for (C₃₃H₃₈N₃O₃)⁺ [M+H]⁺ 524.3, found 524.3.

6-(4-Methylpiperazin-1-yl)-3-(3-tert-butylphenyl)-2-(2,6-dimethoxystyryl)-4(3H)-quinazolinone (40)

¹H NMR (CDCl₃, 400 MHz) δ 8.28 (d, J = 15.8 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 2.9 Hz, 1H), 7.52 (dt, J = 8.1, 1.4 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.46 – 7.39 (m, 1H), 7.27 (d, J = 1.9 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.13 – 7.08 (m, 1H), 6.94 (d, J = 15.9 Hz, 1H), 6.45 (d, J = 8.4 Hz, 2H), 3.65 (s, 6H), 3.39 (t, J = 5.0 Hz, 4H), 2.70 (t, J = 4.9 Hz, 4H), 2.42 (s, 1H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 159.5, 152.9, 148.9, 137.7, 130.0, 129.1, 128.8, 128.5, 125.8, 125.7, 125.5, 124.2, 123.3, 121.3, 113.3, 110.3, 103.5, 55.3, 55.3, 54.6, 48.4, 45.6, 34.8, 31.2, 31.1. MS (ESI) calculated for (C₃₃H₃₉N₄O₃)⁺ [M+H]⁺ 539.3, found 539.3.

6-(Piperidin-1-yl)-3-(3-tert-butylphenyl)-2-(3-methoxystyryl)-4(3H)-quinazolinone (41)

¹H NMR (CDCl₃, 400 MHz) δ 7.78 (d, J = 15.5 Hz, 1H), 7.71 – 7.62 (m, 2H), 7.57 – 7.42 (m, 3H), 7.27 (d, J = 4.0 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.12 (ddd, J = 7.5, 2.1, 1.2 Hz, 1H), 6.89 – 6.83 (m, 1H), 6.83 – 6.76 (m, 2H), 6.32 (d, J = 15.6 Hz, 1H), 3.73 (s, 3H), 3.30 (t, J = 5.4 Hz, 4H), 1.73 (m, 4H), 1.61 (t, J = 5.3 Hz, 2H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 159.7, 153.1, 148.9, 137.8, 137.0, 137.0, 129.6, 129.3, 128.2, 126.0, 125.7, 125.7, 124.8, 121.6, 120.7, 120.0, 114.8, 112.6, 55.1, 34.8, 31.2, 29.6, 25.4, 24.1. MS (ESI) calculated for (C₃₂H₃₆N₃O₂)⁺ [M+H]⁺ 494.3, found 494.3.

tert-Butyl 4-[3-(3-tert-butylphenyl)-2-(3-methoxystyryl)-4-oxo-3,4-dihydroquinazolin-6-yl]piperazine-1-carboxylate (42)

¹H NMR (CDCl₃, 400 MHz) δ 7.82 (d, J = 15.5 Hz, 1H), 7.73 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 2.8 Hz, 1H), 7.54 (dt, J = 8.1, 1.5 Hz, 1H), 7.52 – 7.41 (m, 2H), 7.27 (d, J = 3.9 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.12 (ddd, J = 7.5, 2.0, 1.2 Hz, 1H), 6.85 (d, J = 7.7 Hz, 1H), 6.83 – 6.79 (m, 1H), 6.78 (t, J = 2.0 Hz, 1H), 6.31 (d, J = 15.5 Hz, 1H), 3.73 (s, 3H), 3.60 (t, J = 5.1 Hz, 4H), 3.26 (t, J = 5.1 Hz, 4H), 1.47 (s, 9H), 1.32 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 162.0, 159.7, 154.6, 153.2, 149.7, 149.5, 136.9, 136.8, 129.6, 129.4, 128.2, 126.1, 125.6, 125.6, 124.7, 121.5, 120.0, 115.0, 112.7, 110.5, 80.0, 55.1, 48.9, 34.8, 31.2, 28.4. MS (ESI) calculated for (C₃₆H₄₃N₄O₄)⁺ [M+H]⁺ 595.3, found 595.3.

6-(Piperidin-1-yl)-3-(m-tolyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (44)

¹H NMR (CDCl₃, 400 MHz) δ 7.75 – 7.63 (m, 3H), 7.48 (dd, J = 8.3, 7.2 Hz, 2H), 7.42 (t, J = 7.7 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.16 – 7.03 (m, 3H), 6.86 (d, J = 7.2 Hz, 1H), 6.59 (d, J = 8.3 Hz, 1H), 3.61 (d, J = 1.2 Hz, 3H), 3.31 (t, J = 5.4 Hz, 4H), 2.41 (s, 3H), 1.74 (p, J = 5.6 Hz, 4H), 1.66 – 1.60 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.2, 151.0, 139.7, 138.9, 137.4, 129.5, 129.4, 129.2, 128.3, 125.7, 124.1, 121.8, 117.6, 111.5, 52.5, 29.6, 25.5, 24.1, 21.2. MS (ESI) calculated for (C₂₈H₂₉N₄O₂)⁺ [M+H]⁺ 453.2, found 453.2.

6-Morpholino-3-(m-tolyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (45)

¹H NMR (CDCl₃, 400 MHz) δ 7.81 – 7.69 (m, 2H), 7.64 (d, J = 2.9 Hz, 1H), 7.51 – 7.39 (m, 3H), 7.29 (d, J = 7.7 Hz, 1H), 7.15 – 7.04 (m, 3H), 6.87 (d, J = 7.2 Hz, 1H), 6.60 (d, J = 8.3 Hz, 1H), 3.89 (t, J = 4.8 Hz, 4H), 3.61 (s, 3H), 3.38 – 3.17 (m, 4H), 2.41 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.0, 150.8, 149.9, 149.2, 139.8, 138.9, 137.2, 129.7, 129.5, 129.1, 128.2, 125.6, 123.7, 121.7, 117.9, 111.8, 109.9, 66.6, 52.5, 48.8, 21.2. MS (ESI) calculated for (C₂₇H₂₇N₄O₃)⁺ [M+H]⁺ 455.2, found 455.2.

6-(4-Morpholinopiperidin-1-yl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-3-(m-tolyl)-4(3H)-quinazolinone (46)

¹H NMR (CDCl₃, 400 MHz) δ 7.74 – 7.61 (m, 3H), 7.51 – 7.38 (m, 3H), 7.29 (d, J = 7.7 Hz, 1H), 7.15 – 7.03 (m, 3H), 6.85 (d, J = 7.1 Hz, 1H), 6.59 (d, J = 8.2 Hz, 1H), 3.90 (d, J = 12.5 Hz, 2H), 3.75 (bs, 4H), 3.60 (s, 3H), 2.91 – 2.77 (m, 2H), 2.61 (bs, 4H), 2.40 (s, 4H), 2.05 – 1.92 (m, 2H), 1.68 (d, J = 12.2 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 150.9, 149.7, 148.7, 140.8, 139.8, 138.9, 137.4, 136.0, 129.5, 129.5, 129.2, 128.4, 125.7, 124.5, 124.1, 121.8, 117.6, 111.6, 110.2, 62.0, 49.7, 48.5, 21.2. MS (ESI) calculated for (C₃₂H₃₆N₅O₃)⁺ [M+H]⁺ 538.3, found 538.2

6-(Piperidin-1-yl)-3-(3-methoxyphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (47)

¹H NMR (CDCl₃, 400 MHz) δ 7.74 – 7.62 (m, 3H), 7.53 – 7.39 (m, 3H), 7.09 (d, J = 14.9 Hz, 1H), 7.02 (ddd, J = 8.5, 2.6, 1.0 Hz, 1H), 6.90 (ddd, J = 7.7, 1.9, 0.9 Hz, 1H), 6.88 – 6.83 (m, 2H), 6.59 (d, J = 8.2 Hz, 1H), 3.80 (s, 3H), 3.63 (s, 3H), 3.31 (t, J = 5.4 Hz, 4H), 1.73 (t, J = 5.7 Hz, 4H), 1.62 (q, J = 6.0 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.1, 160.6, 151.0, 148.5, 138.9, 138.6, 136.0, 130.3, 128.3, 124.6, 124.0, 121.7, 121.0, 117.6, 115.0, 114.1, 111.5, 55.4, 52.6, 25.5, 24.1; HRMS (ESI) calculated for (C₂₈H₂₉N₄O₃)⁺ [M+H]⁺ 469.2240, found 469.2227.

6-Morpholino-3-(3-methoxyphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (48)

¹H NMR (CDCl₃, 400 MHz) δ 7.80 – 7.69 (m, 2H), 7.64 (d, J = 2.9 Hz, 1H), 7.53 – 7.39 (m, 3H), 7.09 (d, J = 14.8 Hz, 1H), 7.03 (dd, J = 8.5, 2.7 Hz, 1H), 6.94 – 6.83 (m, 3H), 6.60 (d, J = 8.2 Hz, 1H), 3.89 (t, J = 4.8 Hz, 4H), 3.81 (d, J = 1.7 Hz, 3H), 3.63 (d, J = 1.7 Hz, 3H), 3.29 (dd, J = 5.8, 3.7 Hz, 4H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 161.9, 160.7, 150.8, 149.9, 149.1, 138.9, 138.3, 130.4, 128.2, 123.7, 121.6, 120.9, 117.8, 115.1, 114.0, 111.8, 109.9, 66.6, 55.5, 52.6, 48.8. MS (ESI) calculated for (C₂₇H₂₇N₄O₄)⁺ [M+H]⁺ 471.2, found 471.2

6-(4-Morpholinopiperidin-1-yl)-3-(3-methoxyphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (49)

¹H NMR (CDCl₃, 400 MHz) δ 7.74 – 7.62 (m, 3H), 7.51 – 7.40 (m, 3H), 7.09 (d, J = 14.9 Hz, 1H), 7.03 (ddd, J = 8.5, 2.6, 1.0 Hz, 1H), 6.90 (ddd, J = 7.8, 1.9, 1.0 Hz, 1H), 6.85 (d, J = 6.8 Hz, 2H), 6.59 (d, J = 8.3 Hz, 1H), 3.91 (d, J = 12.5 Hz, 2H), 3.80 (d, J = 1.1 Hz, 3H), 3.75 (s, 4H), 3.62 (d, J = 1.1 Hz, 3H), 2.85 (td, J = 12.5, 2.2 Hz, 2H), 2.62 (d, J = 6.1 Hz, 4H), 2.07 – 1.93 (m, 2H), 1.77 – 1.62 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz)) δ 163.1, 162.3, 153.3, 151.6, 150.9, 150.6, 139.4, 138.9, 136.9, 136.7, 133.3, 129.4, 127.7, 126.7, 126.0, 125.9, 125.7, 125.5, 124.6, 121.3, 118.1, 112.0, 52.6, 34.8, 34.6, 31.3, 31.2. MS (ESI) calculated for (C₃₂H₃₆N₅O₄)⁺ [M+H]⁺ 554.3, found 554.3.

6-(Piperidin-1-yl)-3-(3-trifluoromethylphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (50)

¹H NMR (CDCl₃, 400 MHz) δ 7.83 – 7.59 (m, 6H), 7.58 – 7.42 (m, 3H), 6.97 (dd, J = 14.8, 1.7 Hz, 1H), 6.86 (d, J = 7.1 Hz, 1H), 6.60 (t, J = 6.1 Hz, 1H), 3.56 (s, 3H), 3.42 – 3.23 (m, 4H), 1.81 – 1.68 (m, 4H), 1.63 (q, J = 5.8 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.2, 162.1, 150.6, 138.9, 138.2, 136.5, 132.6, 130.3, 128.5, 126.0, 125.6, 124.6, 123.2, 121.5, 117.9, 111.9, 52.4, 25.4, 24.1. MS (ESI) calculated for (C₂₈H₂₆F₃N₄O₂)⁺ [M+H]⁺ 507.2, found 507.2.

6-Morpholino-3-(3-trifluoromethylphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (51)

¹H NMR (CDCl₃, 400 MHz) δ 7.80 – 7.67 (m, 4H), 7.65 (s, 1H), 7.62 (d, J = 2.9 Hz, 1H), 7.55 – 7.42 (m, 3H), 6.97 (d, J = 14.7 Hz, 1H), 6.86 (d, J = 7.1 Hz, 1H), 6.61 (d, J = 8.2 Hz, 1H), 4.00 – 3.82 (m, 4H), 3.56 (s, 3H), 3.38 – 3.22 (m, 4H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.2, 161.9, 150.4, 150.1, 148.3, 138.9, 138.1, 137.2, 132.5, 132.2, 130.4, 128.5, 126.0, 125.9, 125.8, 125.7, 124.7, 123.8, 122.9, 121.5, 118.0, 112.1, 109.7, 66.6, 52.4, 48.7. MS (ESI) calculated for (C₂₇H₂₄F₃N₄O₃)⁺ [M+H]⁺ 509.2, found 509.2.

6-(4-Morpholinopiperidin-1-yl)-3-(3-(trifluoromethyl)phenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (52)

¹H NMR (CDCl₃, 400 MHz) δ 7.82 – 7.56 (m, 6H), 7.56 – 7.38 (m, 3H), 6.97 (dt, J = 14.9, 3.0 Hz, 1H), 6.86 (dd, J = 7.1, 1.9 Hz, 1H), 6.60 (dd, J = 8.3, 1.9 Hz, 1H), 3.91 (d, J = 12.4 Hz, 2H), 3.74 (s, 4H), 3.56 (d, J = 1.9 Hz, 3H), 2.87 (t, J = 12.5 Hz, 2H), 2.60 (s, 4H), 2.48 – 2.35 (m, 1H), 1.99 (d, J = 12.4 Hz, 2H), 1.76 – 1.57 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.2, 162.0, 150.5, 149.9, 147.9, 140.5, 138.9, 138.2, 136.7, 132.5, 130.3, 128.6, 125.9, 125.7, 124.6, 123.2, 121.5, 117.9, 112.0, 110.0, 61.9, 52.4, 49.7, 48.4, 27.7. LC-MS (M + H) = 592.3. MS (ESI) calculated for (C₃₂H₃₃F₃N₅O₃)⁺ [M+H]⁺ 592.3, found 592.2

General synthetic methods for compounds 29–34:

A mixture of compound 58 (0.25 mmol, 1 eq), an aryl or vinyl boronic acid (0.30 mmol, 1.2 eq), tetrakis(triphenylphosphine)palladium(0) (0.029 g, 0.0125 mmol, 0.05 eq), and Na₂CO₃ (0.053 g, 0.5 mmol, 2 eq) in 1,4-dioxane/H₂O (8/2 mL) was heated to 110 °C for 24 h. The reaction mixture was cooled down and quenched with brine (20 mL). The product was extracted with ethyl acetate (3 × 20 mL), and the combined organic phase was washed with brine and then dried over Na₂SO₄. Upon removal of solvent, the residue was purified with column chromatography (silica gel, hexanes : ethyl acetate, 9:1 – 3:1) to give the target compounds as an off-white or pale yellow solid in 45–80% yield.

6-(Pyridin-4-yl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone dihydrochloride (29)

¹H NMR (CDCl₃, 400 MHz) δ 8.71 (d, J = 5.4 Hz, 2H), 8.62 (d, J = 2.2 Hz, 1H), 8.08 (dd, J = 8.6, 2.2 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.85 (d, J = 14.8 Hz, 1H), 7.77 – 7.70 (m, 2H), 7.65 (ddd, J = 12.0, 8.2, 1.4 Hz, 1H), 7.58 – 7.43 (m, 4H), 7.32 (t, J = 1.9 Hz, 1H), 7.14 (ddd, J = 7.4, 2.2, 1.2 Hz, 1H), 7.04 (d, J = 14.8 Hz, 1H), 6.91 (d, J = 7.1 Hz, 1H), 6.63 (d, J = 8.3 Hz, 1H), 3.58 (s, 3H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.2, 162.0, 153.5, 152.9, 150.4, 148.7, 148.6, 138.9, 138.5, 136.6, 135.2, 132.8, 132.1, 132.0, 132.0, 131.9, 131.9, 129.5, 128.6, 128.5, 128.4, 126.2, 125.9, 125.5, 125.4, 123.6, 121.8, 121.6, 118.3, 112.3, 52.6, 34.9, 31.2. MS (ESI) calculated for (C₃₁H₂₉N₄O₂)⁺ [M+H]⁺ 489.2, found 489.2

6-(3-Methoxyphenyl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (30)

¹H NMR (CDCl₃, 400 MHz) δ 8.56 – 8.50 (m, 1H), 8.03 (dd, J = 8.5, 2.2 Hz, 1H), 7.90 – 7.78 (m, 2H), 7.57 – 7.44 (m, 3H), 7.38 (t, J = 7.9 Hz, 1H), 7.35 – 7.26 (m, 2H), 7.23 (dd, J = 2.5, 1.6 Hz, 1H), 7.14 (ddd, J = 7.4, 2.1, 1.3 Hz, 1H), 7.04 (d, J = 14.8 Hz, 1H), 6.93 (ddd, J = 8.2, 2.6, 1.0 Hz, 1H), 6.90 (d, J = 7.1 Hz, 1H), 6.62 (dd, J = 8.3, 0.7 Hz, 1H), 3.87 (s, 3H), 3.58 (s, 3H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.3, 160.1, 153.3, 151.8, 150.6, 141.1, 139.4, 138.9, 137.7, 136.9, 133.5, 129.9, 129.4, 127.8, 126.0, 125.6, 125.5, 125.0, 123.8, 121.3, 119.6, 118.1, 113.4, 112.6, 112.0, 55.3, 52.6, 34.8, 31.2. MS (ESI) calculated for (C₃₃H₃₂N₃O₃)⁺ [M+H]⁺ 518.2, found 518.2.

6-(4-fluorophenyl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (31)

¹H NMR (CDCl₃, 400 MHz) δ 8.47 (d, J = 2.2 Hz, 1H), 7.99 (dd, J = 8.5, 2.2 Hz, 1H), 7.90 – 7.78 (m, 2H), 7.70 – 7.61 (m, 2H), 7.57 – 7.45 (m, 3H), 7.32 (d, J = 2.0 Hz, 1H), 7.20 – 7.11 (m, 3H), 7.04 (d, J = 14.8 Hz, 1H), 6.89 (d, J = 7.1 Hz, 1H), 6.62 (d, J = 8.3 Hz, 1H), 3.58 (s, 3H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 153.4, 151.8, 150.6, 138.9, 138.5, 136.8, 133.2, 129.4, 128.8, 128.7, 127.9, 126.1, 125.6, 125.5, 124.8, 121.3, 118.1, 116.0, 115.8, 112.1, 52.6, 34.8, 31.2. MS (ESI) calculated for (C₃₂H₂₉N₃O₂)⁺ [M+H]⁺ 506.2, found 506.2

6-(4-dimethylaminophenyl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (32)

¹H NMR (CDCl₃, 400 MHz) δ 8.49 (d, J = 2.3 Hz, 1H), 8.02 (ddd, J = 8.5, 2.3, 0.9 Hz, 1H), 7.85 – 7.74 (m, 2H), 7.68 – 7.59 (m, 2H), 7.56 – 7.44 (m, 3H), 7.32 (d, J = 2.0 Hz, 1H), 7.18 – 7.11 (m, 1H), 7.04 (d, J = 14.8 Hz, 1H), 6.88 (d, J = 7.1 Hz, 1H), 6.83 (d, J = 8.3 Hz, 2H), 6.60 (d, J = 8.2 Hz, 1H), 3.58 (d, J = 1.0 Hz, 3H), 3.01 (s, 6H), 1.32 (d, J = 1.0 Hz, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.4, 153.2, 151.0, 150.8, 146.0, 139.6, 138.9, 137.1, 137.0, 132.6, 129.3, 127.7, 127.7, 125.9, 125.7, 125.5, 124.1, 123.3, 121.3, 117.9, 112.8, 111.8, 52.6, 40.5, 34.8, 31.2, 29.6. MS (ESI) calculated for (C₃₄H₃₅N₄O₂)⁺ [M+H]⁺ 531.3, found 531.3.

6-[4-(Pyrrolidin-1-yl)phenyl]-3-(3-tert-butylphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (33)

¹H NMR (CDCl₃, 400 MHz) δ 8.48 (d, J = 2.2 Hz, 1H), 8.02 (dd, J = 8.6, 2.2 Hz, 1H), 7.85 – 7.74 (m, 2H), 7.63 (d, J = 8.6 Hz, 2H), 7.56 – 7.44 (m, 3H), 7.32 (d, J = 1.9 Hz, 1H), 7.14 (dt, J = 7.4, 1.7 Hz, 1H), 7.03 (d, J = 14.8 Hz, 1H), 6.88 (d, J = 7.1 Hz, 1H), 6.66 (d, J = 8.3 Hz, 2H), 6.60 (d, J = 8.3 Hz, 1H), 3.58 (s, 3H), 3.34 (t, J = 8.0, 4H), 2.13 – 1.91 (m, 4H), 1.33 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.4, 153.2, 150.8, 150.8, 147.6, 139.9, 138.9, 137.1, 132.5, 129.3, 127.8, 127.6, 125.9, 125.7, 125.5, 124.1, 123.1, 121.3, 117.9, 111.8, 52.6, 47.6, 34.8, 31.2, 25.4. MS (ESI) calculated for (C₃₆H₃₇N₄O₂)⁺ [M+H]⁺ 557.3, found 557.3.

6-(3,6-dihydro-2H-pyran-4-yl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxypyridin-2-yl)vinyl]-4(3H)-quinazolinone (34)

¹H NMR (CDCl₃, 400 MHz) δ 8.27 (d, J = 2.1 Hz, 1H), 7.88 (dd, J = 8.6, 2.2 Hz, 1H), 7.84 – 7.72 (m, 2H), 7.56 – 7.44 (m, 3H), 7.30 (d, J = 2.0 Hz, 1H), 7.12 (ddd, J = 7.4, 2.2, 1.3 Hz, 1H), 7.02 (d, J = 14.8 Hz, 1H), 6.88 (d, J = 7.2 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H), 6.32 (tt, J = 3.2, 1.5 Hz, 1H), 4.37 (q, J = 2.8 Hz, 2H), 3.96 (t, J = 5.4 Hz, 2H), 3.57 (s, 3H), 2.69 – 2.51 (m, 2H), 1.32 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 163.1, 162.2, 153.3, 151.6, 150.6, 138.9, 138.4, 136.8, 133.0, 131.0, 129.4, 127.4, 126.0, 125.6, 125.4, 123.8, 122.4, 120.9, 118.1, 112.0, 65.8, 64.3, 52.6, 34.8, 31.2, 26.9. MS (ESI) calculated for (C₃₁H₃₂N₃O₃)⁺ [M+H]⁺ 494.2, found 494.3.

General synthetic methods for compounds 53–54:

A mixture of a compound (22, 27) (0.25 mmol, 1 eq) and palladium on activated charcoal (10% w/w) in methanol was stirred under hydrogen gas. Upon completion, the reaction mixture was filtered using Celite bed, and solvent was removed under reduced pressure. Obtained residue was purified with column chromatography (silica gel, hexanes : ethyl acetate, 9:1 – 3:1) to give the target compounds as an off-white or orange solid in 80–88% yield.

6-(Piperidin-1-yl)-3-(3-tert-butylphenyl)-2-[2-(6-methoxypyridin-2-yl)ethyl]-4(3H)-quinazolinone (53)

¹H NMR (CDCl₃, 400 MHz) δ 7.68 – 7.52 (m, 2H), 7.52 – 7.28 (m, 4H), 7.28 – 7.17 (m, 1H), 7.06 – 6.95 (m, 1H), 6.61 (d, J = 7.2 Hz, 1H), 6.46 (d, J = 8.2 Hz, 1H), 3.70 (s, 3H), 3.26 (t, J = 5.5 Hz, 4H), 3.08 (t, J = 7.7 Hz, 2H), 2.88 – 2.68 (m, 2H), 1.71 (q, J = 5.7 Hz, 4H), 1.59 (t, J = 6.2 Hz, 2H), 1.31 (s, 9H).¹³C NMR (100 MHz, cdcl₃) δ 162.5, 157.8, 153.1, 138.6, 137.2, 129.3, 125.9, 125.3, 125.2, 121.3, 115.3, 107.9, 52.9, 34.8, 34.6, 34.6, 31.2, 25.4, 24.0. MS (ESI) calculated for (C₃₁H₃₇N₄O₂)⁺ [M+H]⁺ 497.3, found 497.3.

6-Morpholino-3-(3-tert-butylphenyl)-2-[2-(6-methoxypyridin-2-yl)ethyl]-4(3H)-quinazolinone (54)

¹H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 8.9 Hz, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.52 – 7.34 (m, 4H), 7.24 (d, J = 2.8 Hz, 1H), 7.01 (dt, J = 7.6, 1.6 Hz, 1H), 6.62 (d, J = 7.2 Hz, 1H), 6.47 (d, J = 8.2 Hz, 1H), 3.87 (t, J = 4.8 Hz, 4H), 3.69 (s, 3H), 3.25 (t, J = 4.8 Hz, 4H), 3.09 (t, J = 7.7 Hz, 2H), 2.85 (t, J = 7.5 Hz, 2H), 1.31 (s, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 163.4, 153.2, 149.8, 138.6, 129.3, 126.1, 125.2, 125.2, 123.8, 115.3, 109.9, 108.0, 66.7, 52.9, 49.0, 34.8, 34.6, 31.2, 29.6. MS (ESI) calculated for (C₃₀H₃₅N₄O₃)⁺ [M+H]⁺ 499.3, found 499.3.

Cellular antiviral assays.

The cellular antiviral activities of these compounds were determined in Vero, U87 glioma or mosquito C6/36 cells, following our previous methods ³⁴. Briefly, 2 × 10⁴ cells/well were seeded in 96-well plates and cultured in DMEM media with 2% FBS to form a monolayer of cells. For regular compound treatment (2h-pretreatment), 0.1 MOI (multiplicity of infection) of ZIKV or DENV, together with various concentrations of a compound, was added to each well. After incubation for 2h, the supernatant was removed and cells were washed with PBS to remove unattached viral particles. Fresh medium (150 μL/well) containing various concentrations of a compound in duplicate were added. For 0h-post-treatment, a compound was added into cell culture media right after removal of the unattached viruses. For 2h-post-treatment, a compound was added into cell culture media 2 hours after removal of the unattached viruses. Upon incubation at 37 °C for 48h, aliquots of the supernatant from each well were used to determine viral TCID₅₀ (tissue culture infectious dose) using end-point dilution assay. Half-log serial dilution of the viral supernatant (50 μL) was added to a monolayer of Vero cells in quadruplicate in 96-well plates and cultured for 5–7 days. CPE or cell lysis was determined with microscope followed by MTT assay. TCID₅₀ was calculated based on the highest dilution in which ≥50% (i.e., ≥2 out of the 4 quadruplicate wells) of Vero cells were infected with ZIKV. Compared to controls, the ability for a compound to reduce TCID₅₀ can be determined. The results were from at least 2 independent experiments. Inhibition data were imported into Prism (version 5.0) and EC₅₀ values with standard deviation were obtained by using a standard dose-response curve fitting.

Quantitative RT-PCR to determine ZIKV RNA copies.

Viral RNA was extracted from the supernatant (50 μL) using TRIzol (ThermoFisher). qPCR is based on amplification of ZIKV envelope gene region 3, using ZIKV-specific primers and probes, following our previous method ³⁴. PCR was performed using a TaqMan Fast Virus 1-step Master Mix kit on a StepOnePlus RT-PCR system (Applied Biosystems). Concentrations of ZIKV RNA (copies/mL) were calculated by using a standard curve.

Western blot.

8 × 10⁵ Vero cells in petri dishes were infected with ZIKA (MOI = 0.1) for 2h. Upon removal of the virus, the cells were cultured with fresh media contain 2% FBS and compound 27 for 48h. The cells were harvested and lysed using ice-cold radioimmunoprecipitation assay buffer (Invitrogen). Equal amounts of total proteins were then separated on SDS-PAGE and transferred to PVDF membranes. The blots were probed and visualized with primary antibodies against Zika virus NS3 (GTX133309, GeneTex), Zika virus capsid (GTX133317, GeneTex), Zika virus NS5 (GTX133327, GeneTex), and human β-Actin (4967S, Cell Signaling).

Abbreviation:

SAR

structure–activity relationships

DENV

Dengue virus

ZIKV

Zika virus

RNA

Ribonucleic acid

EC₅₀

half maximal effective concentration

WHO

World Health Organization

CPE

cytopathic effects

MOI

multiplicity of infection

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

TCID₅₀

half maximal tissue culture infectious dose

LCMS

liquid chromatography–mass spectrometry

ESI

electrospray ionization

HPCL

high performance liquid chromatography

DMF

N,N-dimethylformamide