2,3-Dihydroquinazolin-4(1H)-ones and quinazolin-4(3H)-ones as broad-spectrum cytotoxic agents and their impact on tubulin polymerisation

Nicholas S O'Brien; Jayne Gilbert; Adam McCluskey; Jennette A Sakoff

doi:10.1039/d3md00600j

. 2024 Mar 6;15(5):1686–1708. doi: 10.1039/d3md00600j

2,3-Dihydroquinazolin-4(1H)-ones and quinazolin-4(3H)-ones as broad-spectrum cytotoxic agents and their impact on tubulin polymerisation^†

Nicholas S O'Brien ^a, Jayne Gilbert ^b, Adam McCluskey ^a,^✉, Jennette A Sakoff ^b,^✉

PMCID: PMC11110758 PMID: 38784470

Abstract

Tubulin plays a central role in mitosis and has been the target of multiple anticancer drugs, including paclitaxel. Herein two separate families of 2,3-dihydroquinazoline-4(1H)-ones and quinazoline-4(3H) ones, comprising 57 compounds in total, were synthesised. Screening against a broad panel of human cancer cell lines (HT29 colon, U87 and SJ-G2 glioblastoma, MCF-7 breast, A2780 ovarian, H460 lung, A431 skin, Du145 prostate, BE2-C neuroblastoma, and MIA pancreas) reveals these analogues to be broad spectrum cytotoxic compounds. Of particular note, 2-styrylquinazolin-4(3H)-one 51, 2-(4-hydroxystyryl)quinazolin-4(3H)-one 63, 2-(2-methoxystyryl)quinazolin-4(3H)-one 64 and 2-(3-methoxystyryl)quinazolin-4(3H)-one 65 and 2-(naphthalen-1-yl)-2,3-dihydroquinazolin-4(1H)-one 39 exhibited sub-μM potency growth inhibition values. Of these 1-naphthyl 39 has activity <50 nM against the HT29, U87, A2780, H460 and BE2-C cell lines. Molecular modelling of these compounds, e.g. 2-(naphthalen-1-yl)-2,3-dihydroquinazolin-4(1H)-one 39, 2-(2-methoxystyryl)quinazolin-4(3H)-one 64, 2-(3-methoxystyryl)quinazolin-4(3H)-one 65, and 2-(4-methoxystyryl)quinazolin-4(3H)-one 50 docked to the known tubulin polymerisation inhibitor sites highlighted well conserved interactions within the colchicine binding pocket. These compounds were examined in a tubulin polymerisation assay alongside the known tubulin polymerisation promotor, paclitaxel (69), and tubulin inhibitor, nocodazole (68). Of the analogues examined, indoles 43 and 47 were modest promotors of tubulin polymerisation, but less effective than paclitaxel. Analogues 39, 64, and 65 showed reduced microtubule formation consistent with tubulin inhibition. The variation in ring methoxy substituent with 50, 64 and 65, from o- to m- to p-, results in a concomitant reduction in cytotoxicity and a reduction in tubulin polymerisation, with p-OCH₃50 being the least active in this series of analogues. This presents 64 as a tubulin polymerisation inhibitor possessing novel chemotype and sub micromolar cytotoxicity. Naphthyl 39, with complete inhibition of tubulin polymerisation, gave rise to a sub 0.2 μM cell line cytotoxicity. Compounds 39 and 64 induced G₂ + M cell cycle arrest indicative of inhibition of tubulin polymerisation, with 39 inducing an equivalent effect on cell cycle arrest as nocodazole (68).

Simple synthesis, binds and inhibits tubulin, and potent broad-spectrum cytotoxicity.

Introduction

The development of novel anti-cancer agents has been a priority of our team over the past two decades. Our preferred approach relies on phenotypic screening as this provides ample opportunity to identify and examine early-stage compounds that are in-cell active.¹ Our identified targets from this approach include dynamin and clathrin inhibitors,^2–5 protein phosphatase inhibitors^6,7 and most recently compounds that hijack the aryl hydrocarbon receptor pathways as potential breast cancer drugs.^8,9 As part of a current program, we sought access to a series of substituted 2,3-dihydroquinazolinones, which we consider to be a privileged scaffold in medicinal chemistry.^10,11

Tubulin polymerisation is required during cellular mitosis and is pharmacologically exploited for the treatment of cancer through either polymerisation inhibition or promotion to prevent cell division.^12,13 The binding sites of small molecules that either prevent polymerisation (colchicine,¹⁴ vinblastine¹⁵ and pironetin¹⁶) or promote polymerisation (laulimalid,¹⁷ paclitaxel¹⁸ and maytansine¹⁹) have been elucidated through X-ray and cryo-EM crystallographic studies, each possessing unique site interactions between alternating α/β tubulin subunits. Disturbingly with clinically used chemotherapeutics, resistance to common broad-spectrum chemotherapeutics is an increasing issue.^20–22 Clearly there is an urgent requirement for novel agents and novel targets to address this situation.

In the search for novel cytotoxic agents, multiple groups have investigated a wide array of chemical scaffolds, including substituted 2-aryl quinazolones with high levels of cytotoxicity noted, attributed to the inhibition of tubulin.^23–30 The related 2,3-dihydroquinazolinones are present in a range of biologically active materials spanning NCI substance T1D19143 1 (anti-cancer),¹² compound 2 (anti-cancer),³¹ aquamox 3 (antihypertensive),³² fenquizone 4 (diuretic),³³ evodiamine 5 (anti-obesity),³⁴6 (analgesic and anti-inflammatory),³⁵ and compound 7 (antileishmanial)³⁶ (Fig. 1). The diversity of their biological actions has fostered multiple synthetic routes developed to afford rapid access spanning organic catalysts: thiamine hydrochloride,¹² 2-morpholinesulfonic acid,³⁷ β-cyclodextrin-SO₃H,³⁸ ionic liquids,³⁹ and other approaches such as magnetic nanoparticles⁴⁰ and amberlyst-15.⁴¹ More recently we developed an expedient microwave and catalytic approach to obtain this scaffold which has proven to be highly robust.⁴²

Herein, we demonstrate the tubulin polymerisation inhibition of a representative sample of the analogues developed and provide biological activity studies, molecular docking, molecular dynamics simulations, tubulin polymerisation assays and cell cycle analysis that are consistent with interaction with tubulin as a mode of action.

Results and discussion

Chemistry

Access to quinazolinones has been previously achieved through a wide number of metal catalysts with several teams proposing a myriad of reasons for enhancing the green nature of this reaction.^43–45 We have recently developed a simple microwave coupling of 2-aminobenzamide (8) with benzaldehyde (9a) in the presence of 1% SnCl₂ (Scheme 1).⁴² In commencing this investigation, we developed a series of simple quinazolin-4(1H)-ones which comprised a series of aliphatic, acyclic and aromatic substituted analogues (10–17); halogenated and other functionalised simple phenyl substituents (18–33); more complex aromatic substituents (34–44); and analogues with the aromatic moiety moved more distal to the quinazoline core (43–46). In all cases the desired compounds were synthesised in moderate (17%) to excellent (99%) yields. Details of the 2,3-dihydroquinazoline R-moiety of library 1 analogues are presented in Fig. 2, which were evaluated for potential cytotoxicity against a panel of cell lines (below).

We noted that in some instances, the corresponding quinazolin-4(3H)-ones 47–53 were also generated using the synthesis method detailed in Scheme 1. On further investigation we were able to convert some 2,3-dihydroquinazoli-4(1H)-ones to the corresponding quinazolin-4(3H)-ones on treatment with KMnO₄ to generate 54–57 (Scheme 2).

Access to conjugated quinazolin-4(3H)-ones was also achieved through the reaction of 2-methylquinazolin-4(3H)-one 59 with aldehydes to yield 2-styrylquinazolin-4(3H)-ones 60–66 (Scheme 3).

Details of the quinazolin-4(3H)-one R-moiety of library 2 analogues (47–57 and 60–66) are presented in Fig. 3. These compounds were evaluated for potential cytotoxicity against a panel of cell lines (below).

Biology

The cytotoxicity of library 1 and 2 was evaluated in an MTT assay across a range of ten human cancer cell lines and one normal cell line, viz: colon (HT29), breast (MCF-7), glioblastoma (U87 and SJ-G2), ovarian (A2780), lung (H460), skin (A431), prostate (Du145), neuroblastoma (BE2-C), pancreas (MIA) and normal breast (MCF10A). The inclusion of the MCF10A cell line allows for a very crude approximation of relative toxicity, however we view this as a poor surrogate for actual toxicity studies and may fail to provide the data required to predict human toxicity.⁴⁶ Additional tiered models of toxicity are required to define and predict clinical toxicity.

Initial triage screening was conducted at 25 μM compound concentration and these data are presented in Tables S1 and S2† corresponding to library 1 and 2 (ESI†). Analogues with good broad-spectrum activity at this screening dose, defined herein as >80% growth inhibition across all cell lines or >95% against specific cell lines (to examine the potential of cancer cell line specific toxicity), were subject to a full dose response evaluation. Focusing on library 1 (10–46, Scheme 1 and Fig. 2), of the simple aliphatic and aromatic analogues (10–17) butyl 10, cyclopentyl 12 and cyclohexyl 13; the simple aromatic analogues (18–33), analogues 23–26, and 31–33; the more complex aromatic analogues (34–48); and the ‘distal’ aromatic analogues (45–46), 34, 36, 42, 43 and 45, did not proceed to full dose response evaluation (details of R-substituents are given in Fig. 2). In total 24 analogues were sufficiently active to warrant full dose response evaluation. Full dose response evaluation data is presented in Table 1.

Growth inhibition of a panel of cancer cell lines and a normal breast cell line by selected library 1–3 analogues (2,3-dihydroquinazolin-4(1H) one analogues 11, 14–22, 27–30, 35, 37–40, 43, 44 and 46). Growth inhibition, GI₅₀ values (μM) relative to untreated control (MTT assay, 72 h, n = 3).


	HT29^a	U87^b	MC-7^c	A2780^d	H460^e	A431^f	Du145^g	BE2-C^h	SJ-G2^b	MIAⁱ	MCF10A^j
11	13 ± 1.0	>50	2.9 ± 0.80	4.2 ± 0.71	14 ± 1.7	7.1 ± 1.5	>50	9.2 ± 1.5	13 ± 1.9	12 ± 3.9	11 ± 2.7
14	5.4 ± 1.4	17 ± 2.0	15 ± 3.7	3.9 ± 0.98	11 ± 3.5	3.8 ± 0.40	19 ± 3.0	5.7 ± 1.3	17 ± 4.1	12 ± 2.7	11 ± 2.6
15	2.3 ± 0.32	4.1 ± 0.00	1.3 ± 0.35	2.3 ± 0.000	2.2 ± 0.19	2.4 ± 0.34	3.0 ± 0.47	2.8 ± 0.33	2.2 ± 0.38	3.7 ± 0.26	3.7 ± 0.47
16	1.6 ± 0.32	2.6 ± 0.10	4.0 ± 0.95	2.3 ± 0.37	3.2 ± 0.27	2.3 ± 0.3	3.1 ± 0.27	1.9 ± 0.49	2.9 ± 0.18	3.4 ± 0.53	3.1 ± 0.21
17	9.7 ± 3.0	>50	8.4 ± 3.3	15 ± 4.1	>50	>50	35 ± 7.3	20 ± 3.4	>50	14 ± 6.5	19 ± 4.1
18	2.4 ± 0.26	10 ± 1.7	3.7 ± 0.28	2.5 ± 0.25	3.6 ± 0.32	2.3 ± 0.50	4.3 ± 0.58	2.5 ± 0.36	3.0 ± 0.38	4.2 ± 0.55	4.3 ± 1.5
19	2.6 ± 0.38	4.1 ± 0.12	2.0 ± 0.96	2.6 ± 0.18	1.9 ± 0.32	2.7 ± 0.52	2.9 ± 0.50	2.4 ± 0.43	1.9 ± 0.48	3.2 ± 0.13	3.9 ± 0.70
20	2.9 ± 0.49	4.7 ± 1.2	3.6 ± 1.2	3.1 ± 0.21	3.2 ± 0.21	3.1 ± 0.22	3.3 ± 0.29	2.6 ± 0.34	2.5 ± 0.29	3.2 ± 0.09	4.0 ± 0.56
21	6.3 ± 1.0	19 ± 0.33	13 ± 5.1	3.0 ± 0.47	14 ± 0.33	3.7 ± 0.03	16 ± 2.2	6.4 ± 1.3	12 ± 0.73	15 ± 0.67	14 ± 0.67
22	3.7 ± 0.56	16 ± 2.0	12 ± 6.4	3.2 ± 0.09	10 ± 3.1	3.2 ± 0.30	10 ± 2.5	4.1 ± 0.95	8.9 ± 3.0	8.8 ± 2.3	9.9 ± 3.2
27	17 ± 0.67	>50	6.7 ± 1.7	18 ± 1.5	24 ± 3.0	20 ± 2.3	45 ± 5.5	19 ± 0.88	22 ± 0.88	20 ± 1.0	21 ± 0.67
28	8.7 ± 2.2	>50	3.8 ± 0.10	11 ± 4.1	14 ± 0.88	17 ± 4.1	26 ± 1.3	10 ± 0.93	10 ± 0.00	7.5 ± 0.35	11 ± 3.5
29	1.7 ± 0.28	2.7 ± 0.26	5.8 ± 0.62	2.6 ± 0.34	3.3 ± 0.13	2.1 ± 0.4	2.3 ± 0.87	2.3 ± 0.38	3.3 ± 0.18	4.1 ± 1.0	3.2 ± 0.12
30	2.9 ± 0.07	3.2 ± 0.22	5.6 ± 0.15	2.9 ± 0.15	3.2 ± 0.22	2.7 ± 0.10	3.0 ± 0.43	3.1 ± 0.22	3.3 ± 0.30	4.4 ± 1.3	3.1 ± 0.10
35	7.6 ± 2.1	23 ± 3.6	11 ± 3.0	4.7 ± 1.1	15 ± 1.5	7.4 ± 3.3	25 ± 3.5	9.3 ± 2.2	23 ± 3.7	18 ± 2.0	15 ± 2.6
37	2.3 ± 0.49	2.2 ± 0.21	1.6 ± 0.21	3.8 ± 1.5	2.8 ± 0.18	2.4 ± 0.03	2.3 ± 0.07	2.0 ± 0.15	2.6 ± 0.21	2.3 ± 0.15	2.3 ± 0.45
38	2.7 ± 0.07	5.0 ± 0.03	2.8 ± 0.23	2.9 ± 0.10	2.9 ± 0.10	3.0 ± 0.33	4.2 ± 0.35	2.9 ± 0.13	2.8 ± 0.15	2.8 ± 0.15	3.5 ± 0.07
39	0.020 ± 0.0023	0.22 ± 0.070	0.075 ± 0.0057	0.031 ± 0.00088	0.037 ± 0.0072	0.02 ± 0.01	0.089 ± 0.037	0.027 ± 0.0052	0.050 ± 0.015	0.048 ± 0.01	0.040 ± 0.005
40	2.6 ± 0.12	4.1 ± 0.45	3.3 ± 0.40	2.9 ± 0.30	3.1 ± 0.17	2.7 ± 0.03	4.8 ± 0.61	3.1 ± 0.10	3.0 ± 0.46	3.5 ± 0.46	3.9 ± 0.10
43	3.5 ± 0.38	19 ± 0.00	14 ± 2.6	3.9 ± 0.20	12 ± 1.2	3.6 ± 0.35	17 ± 1.2	3.6 ± 0.15	16 ± 0.88	13 ± 3.9	14 ± 0.88
44	2.6 ± 0.29	5.7 ± 1.7	8.5 ± 2.8	5.0 ± 1.5	4.7 ± 0.92	8.2 ± 3.40	3.1 ± 0.17	10 ± 1.2	12 ± 1.2	12 ± 3.2	2.9 ± 0.19
46	2.2 ± 0.40	4.3 ± 0.57	7.4 ± 2.8	3.0 ± 0.20	3.3 ± 0.10	2.1 ± 0.23	4.0 ± 0.38	2.6 ± 0.37	3.0 ± 0.31	3.6 ± 0.088	3.4 ± 0.10

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Colon.

Glioblastoma.

Breast.

Ovarian.

Lung.

Skin.

Prostate.

Neuroblastoma.

ⁱ

Pancreas.

Breast (normal).

Activity against most cell lines was evident on analysis of the MTT assay data for the 2,3-dihydroquinazolin-4(1H)-one analogues examined. The phenyl 15 and tolyl 16 analogues displayed good activities (GI₅₀ 1.3 (15, MCF-7) to 4.0 μM (16, MCF-7)). Introduction of steric bulk to the aromatic moiety resulted in a 10-fold reduction in potency (17, average GI₅₀ ∼ 29 μM, across the 10 cancer cell lines examined, Table 1). In a similar manner, aliphatic substituents were less active (than the equivalent aromatic), viz11 and 14, with a 4 to 6-fold potency reduction relative to 15 and 16. Aromatic-NO₂ substitution was poorly tolerated (27 and 28), but the –CF₃ substituted 30 retained good activity suggesting that this was not an electron withdrawing effect. Bromine was generally well tolerated (18–20), but an –OH moiety resulted in a 3-fold potency decrease (21 and 22). The methoxy and phenyl analogues (29 and 37) were as active as the parent phenyl (15) suggesting that the lack of a H-bonding moiety in this region is favoured. The introduction of modified aromatic moieties was in general well tolerated, except for thiophene (35) with a 5-fold potency reduction. Most striking with these aromatic modifications was the 2-naphthyl (38) and 1-naphthyl (39). The naphthyl ring orientation has a profound effect on the observed activity, with 1-naphthyl (39) being 50-fold more potent than the equivalent 2-naphthyl (38). Across the 10 cancerous cell lines examined, 2-naphthyl (39) is sub-μM potent with GI₅₀ values of 0.02 (HT29) to 0.22 (MCF-7) μM.

A similar triaging exercise was performed with library 2 (47–57 and 60–66, Schemes 1 and 3, and Fig. 5), and in this case analogues 49, 53–55, 57 and 66 did not proceed to full dose response evaluation. Full dose response data is presented in Table 2.

Growth inhibition of a panel of cancer cell lines and a normal breast cell line by selected library 2 analogues (quinazolin-4(3H)-one analogues 47, 48, 50–52, 56, and 60–65). Growth inhibition, GI₅₀ values relative (μM) to untreated control (MTT assay, 72 h, n = 3).


	HT29^a	U87^b	MC-7^c	A2780^d	H460^e	A431^f	Du145^g	BE2-C^h	SJ-G2^b	MIAⁱ	MCF10A^j
47	0.39 ± 0.094	16 ± 9.4	0.28 ± 0.037	1.3 ± 0.38	3.7 ± 1.8	0.57 ± 0.076	>50	0.63 ± 0.077	>50	1.0 ± 0.15	1.2 ± 0.31
48	3.9 ± 0.20	3.2 ± 0.35	2.7 ± 0.088	2.7 ± 0.32	2.5 ± 0.30	4.9 ± 0.33	2.8 ± 0.088	3.4 ± 0.47	2.8 ± 0.57	3.5 ± 0.63	3.5 ± 0.37
50	3.8 ± 0.15	>50	2.6 ± 0.67	12 ± 4.3	11 ± 1.2	33 ± 4.4	45 ± 2.9	4.4 ± 0.81	6.4 ± 1.0	5.5 ± 1.4	11 ± 3.0
51	0.40 ± 0.042	2.7 ± 0.088	1.3 ± 0.29	0.45 ± 0.055	2.5 ± 0.10	0.64 ± 0.23	2.4 ± 0.20	0.50 ± 0.018	2.2 ± 0.15	2.3 ± 0.96	2.3 ± 0.15
52	3.2 ± 0.21	13 ± 1.2	3.1 ± 0.13	3.6 ± 0.20	11 ± 1.7	11 ± 1.92	8.9 ± 1.6	6.4 ± 0.70	15 ± 7.3	14 ± 7.3	4.6 ± 0.10
56	5.2 ± 0.78	10 ± 3.1	3.8 ± 0.52	3.9 ± 1.1	5.7 ± 0.78	5.8 ± 0.31	4.5 ± 1.1	6.4 ± 1.4	4.3 ± 0.10	8.8 ± 1.6	5.4 ± 0.40
60	3.8 ± 0.6	32 ± 4.4	2.2 ± 1.0	4.8 ± 1.1	7.2 ± 1.9	6.2 ± 1.3	16 ± 4.7	8.5 ± 3.3	5.5 ± 2.3	5.6 ± 2.3	8.5 ± 2.8
61	2.1 ± 0.38	19 ± 4.4	1.2 ± 0.46	3.7 ± 0.76	9.4 ± 2.3	4.0 ± 0.29	7.7 ± 1.0	3.6 ± 0.59	13 ± 5.5	3.3 ± 0.47	3.8 ± 0.32
62	5.9 ± 1.2	8.9 ± 2.2	1.3 ± 0.21	3.1 ± 0.033	2.6 ± 0.21	3.0 ± 0.15	1.9 ± 0.17	1.8 ± 0.24	2.5 ± 0.32	2.9 ± 0.10	3.1 ± 0.067
63	0.35 ± 0.072	0.48 ± 0.083	0.22 ± 0.049	0.30 ± 0.00	0.37 ± 0.0067	0.28 ± 0.00	0.35 ± 0.027	0.25 ± 0.031	0.29 ± 0.028	0.34 ± 0.020	0.38 ± 0.032
64	0.26 ± 0.062	1.4 ± 0.38	0.35 ± 0.088	0.33 ± 0.0067	0.42 ± 0.053	0.33 ± 0.015	0.90 ± 0.17	0.31 ± 0.0033	0.79 ± 0.26	0.37 ± 0.043	0.56 ± 0.079
65	0.90 ± 0.46	10 ± 4.7	0.47 ± 0.20	2.6 ± 0.33	2.2 ± 0.10	2.6 ± 0.31	3.9 ± 0.41	2.1 ± 0.28	2.8 ± 0.50	2.9 ± 0.66	2.2 ± 0.42

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Colon.

Glioblastoma.

Breast.

Ovarian.

Lung.

Skin.

Prostate.

Neuroblastoma.

ⁱ

Pancreas.

Breast (normal).

Examination of the MTT data obtained for the quinazolin-4(3H)-ones (Table 2) revealed slight variations in the overall levels of activity relative to the 2,3-dihydroquinazolin-4(1H)-one analogues. Within closely related analogues, e.g., indoles 47 and 48, there was a considerable variation in the cytotoxicity noted, with methyl indole 47 being 10-fold more active than the corresponding cyano indole 48 against HT29 cells, but within these two analogues, there was no consistent potency trend. Methyl indole 47 was more active against the MCF-7, A2780, A431, BE2-C and MIA cell lines, with cyano indole 48 being more active against the U87, H460, Du145 and SJ-G2 cell lines. The quinazolin-4(3H)-one analogues displayed slightly decreased overall levels of cytotoxicity with only 4-OH 63 and 2-OCH₃64 exhibiting sub-μM potent GI₅₀ values across the cell lines examined. With 4-OH 63, these values spanned 0.22 (MCF-7) to 0.48 (U87) μM.²⁴ The introduction of additional analogue conjugation was largely beneficial to cytotoxicity as evidenced from examination of the data presented for 60–65, while the 3- and 4-methoxy 65 and 50 showed decreased activity over the 2-methoxy analogue 64.

From the two classes of compounds examined, multiple analogues showed sub-μM activity, naphthyl 39 with GI₅₀ values from 0.02 μM, and the –OCH₃ containing 50, 64 and 65 with GI₅₀ values from 0.2 μM. Within the methoxy containing series, cytotoxicity has a direct relationship with the substitution pattern, decreasing from the ortho- to para-substitution (Table 2).

Mechanism of action

Modelling studies

Of known tubulin polymerisation inhibitors, two non-covalent binding sites are described as the colchicine site and vinca site. Located between alternate tubulin subunits, these pockets are highly buried within the protein (Fig. 4). Tubulin monomers are divided into three functional domains: an amino terminal domain, intermediate domain, and carboxy terminal domain. Colchicine induces assembly independent GTPase activity to promote loss of the microtubule GTP cap and disassembly resulting in curved tubulin heterodimers preventing microtubule assembly. This binding occurs mainly within the β-tubulin subunit at the α-tubulin subunit interface. In comparison, the vinca site to which vinblastine binds is located at the other end of the β-tubulin subunit which also induces this non-linear polymeric unit preventing microtubule assembly.⁴⁷

Given the known disruption of colchicine binding exhibited by 39,²⁴ docking studies were conducted into each pocket in an attempt to identify likely binding sites of 39, 50, 64, and 65. Docking was performed using Molecular Operating Environment (MOE) using existing crystallographic data with molecules bound to the site of interest (colchicine PDB: 4O2B, vinblastine PDB: 5J2T). An induced fit docking method under the AMBER forcefield was used, taking the top 15 of 500 poses for analysis.

Colchicine-site molecular modelling

Initial docking studies were performed using the pre-existing co-crystal data (PDB: 4O2B) containing colchicine (66) bound between the α/β-tubulin interface. Previously identified key interactions involve Thr179 and Val181 from the α-tubulin subunit, and Cys241 and Asn258 from the β-subunit.³⁰ The trimethoxyphenyl ring system of 66 sits deeply within the β-tubulin pocket, whilst interactions from the carbonyl group, adjacent methoxy group and acetamide form the basis of interactions with α-tubulin projecting into the adjacent subunit (Fig. 5). Amino acids are annotated to allow for precise comparison of pose positioning between species.

As shown in Fig. 6, analogues 39, 50, 64, and 65 highlight the alignment of the cyclohexanone-triene methoxy group positioning which is well conserved with respect to colchicine (66). The quinazolin-4(3H)-one core appears to sit within the β-tubulin pocket in place of colchicine's trimethoxy phenyl region, which is shown for all analogues however it is flipped 180° in the lowest energy pose for 65. The lowest energy pose scores were −6.5660 kJ mol⁻¹ (39), −6.7471 kJ mol⁻¹ (50), −6.8070 kJ mol⁻¹ (65), and −6.8436 kJ mol⁻¹ (64) which for the methoxy containing series (50, 64, 65) correlate well with the increasing cytotoxicity when moving from the 4- to 2-substitution.

Vinblastine-site molecular modelling

Next, the comparative docking studies within the vinblastine (67) binding site using the pre-existing co-crystal (PDB: 5J2T) was examined. Previously identified key residues involve Lys336 and Asn329 from the α-subunit and Lys176 and Val177 from the β-subunit which are annotated for ease of analysis. Given the size and complexity of vinblastine (67) in comparison to colchicine (66), it was predicted that this was not the site responsible for the tubulin polymerisation inhibition exhibited by the quinazolinones. Most ligand partners to this site are peptide or peptide-containing analogues, or large, polycyclic molecules with high degrees of sp³ character to enable interactions across the α/β gap.⁴⁸

The compounds were docked as the previous calculation, and images generated from the resulting docks (Fig. 7). The 3-OCH₃ (65) and 2-OCH₃ (64) analogues exhibit similar poses within the pocket with the quinazolinone region lying beneath the Val177 residue in the β-subunit. The lowest energy pose for the naphthyl analogue (50) also shows this Val177 interaction, but does not span the α/β gap. 39 however lies entirely within the α subunit groove and does not span the space.

The lowest energy pose scores were −6.0108 kJ mol⁻¹ (39), −5.9711 kJ mol⁻¹ (50), −6.3896 kJ mol⁻¹ (65), and −6.2860 kJ mol⁻¹ (64) which, unlike compounds bound to the colchicine site (Fig. 5), do not exhibit a similar energy/cytotoxicity relationship. Further, in considering the sheer volumetric differences of the quinazolinone analogues in comparison to vinblastine, it is highly unlikely that inhibition of tubulin occurs at thee binding sites. As such, it is more likely that tubulin polymerisation inhibition results from interaction at the colchicine binding site.

Molecular dynamics of 2-(2-methoxystyryl)quinazolin-4(3H)-one 64

With the colchicine site chosen as the more likely of the two, molecular dynamics simulations were performed on 64 within the colchicine binding site. A 1 ps step size was chosen for a total simulation time of 2000 ps. Poses are shown at t = 0 ps, t = 1000 ps and t = 2000 ps in Fig. 8. Previously identified amino acids are annotated for clarity of position.

Molecular dynamics at the three time points shown in Fig. 8 indicate high stability of 64 within the colchicine binding site. The quinazolin-4(3H)-one region remains stationary, corresponding to the trimethoxyphenyl region of colchicine. The styryl methoxy region also remained stable, with the terminal phenyl ring non-planar to the quinazolin-4(3H)-one region and slight rotation of the methoxy group observed. These results suggest that 64 is highly stable within the colchicine binding site.

Effect of selected analogues (15, 39, 43, 45, 47, 50, 51, 54, 64 and 65) on tubulin

The above modelling analysis supported that these analogues may be interacting with tubulin at the colchicine binding pocket. Our modelling analysis revealed that the highest incidence and most stable interactions were predicted to occur within this binding site. In an effort to validate these modelling outcomes, a selected number of analogues were examined in a tubulin binding assay. This assay was conducted as detailed in the Experimental section, with added known mode of action compounds as direct comparators. Nocodazole (68) was added as a tubulin polymerisation inhibitor control (a negative control), and paclitaxel (69) as a polymerisation promotion control (a positive control) (Fig. 9).

Selected analogues (15, 39, 43, 45, 47, 50, 51, 54, 64, and 65) were subjected to a fluorometric tubulin assay (Experimental) with nocodazole (68) and paclitaxel (69) as comparison compounds of known tubulin efficacy. Nocodazole and paclitaxel were screened at 3 μM and test compounds at 10 μM. Microtubule formation was measured by an increase in fluorescence corresponding to tubulin polymerisation. Based on the outcomes of the tubulin assay, the selected compounds have been grouped into two classes: 1. within the first grouped compound selection (15, 39, 43, 47, and 54, Fig. 10), phenyl 15 showed pronounced background fluorescence. In this instance, the background activity gives a similar signal to the known tubulin promotor, paclitaxel (69) (Fig. 10A). No background interference was noted with the equivalent 2,3-dehydro phenyl 54, and in this case, the effect was noted with the signal obtained matching that of the DMSO control (Fig. 10B). Naphthyl 39, as does nocodazole (68), completely inhibits tubulin polymerisation (Fig. 10C). The inhibition level noted with 39 remained constant throughout the entire time course of evaluation (3500 s, ∼1 h), in keeping with its reported activity.²⁴ Contrasting this, for indoles 43 and 47, a modest promotion of tubulin polymerisation is noted (Fig. 10D and E). The extent of this effect was less than that observed with paclitaxel (69). Given the smaller increase to microtubule formation when compared to the positive control paclitaxel, this may be in part due to an alternate mode of action, reduced binding affinities or increased off-rates.

Analysis of the tubulin polymerisation assay data recorded for the styryl series of compounds 45 and 51 (Fig. 11A and C) indicates that neither compound impacts tubulin polymerisation. This is despite a significant change in the cytotoxicity with 45 and 51 (GI₅₀: > 50 μM and GI₅₀: 0.40 ± 0.042–2.7 μM, respectively), indicating that this is not attributable to a tubulin effect. The decreasing tubulin effects of o-, m- and p-OCH₃-styryl analogues 64, 65, and 50 (Fig. 11B, D and E), respectively, are consistent with the decreases observed in broad spectrum cytotoxicity of these analogues. The o-CH₃-styryl 64 is an excellent tubulin inhibitor, slightly less active than nocodazole (68), with activity only decreasing (relative to 68) after 2500 s (Fig. 11E). The inclusion of the –OCH₃ group appears to be vital to affect tubulin polymerisation.

The cytotoxicity of these analogues compares very favourably with that observed with the tubulin inhibitor, nocodazole (67), at 10-fold higher potency. Nocodazole (68) is a 2.22 (HeLa), 1.81 (HCT-116), 5.09 (A549), 5.08 (HepG-2) and 7.56 (WRL-68) μM inhibitor (48 h MTT outcomes).⁴⁹

The results displayed in Fig. 11 align well with the docking studies performed on 50, 64 and 65 (Fig. 6–10), wherein it was postulated that of the colchicine and vinblastine binding sites assessed, it was more likely that these compounds would bind to the colchicine site. Nocodazole is a member of the same family and binds similarly to the colchicine site implying the same mode of action.⁵⁰ Given the strong tubulin polymerisation inhibition of 50, alongside the docking results, it is probable that 50 acts via the same mechanism as colchicine.

In order to confirm in vitro tubulin inhibition, the ability of 39 and 64 to induce cell cycle arrest at the G₂ + M phase of the cell cycle was assessed in MIA pancreatic cancer cells. The results displayed in Fig. 12 confirm that 39 and 64 induced G₂ + M cell cycle arrest, as evident from an increase in this population after 24 h exposure from 20% (untreated) to 55% (0.5 μM) and 78% (5.0 μM), respectively (Fig. 12), concomitant with a decline in the other cell populations. In line with the cytotoxicity results (GI₅₀ 0.048 μM for 39 and 0.37 μM for 64), 39 was approximately 10-fold more potent at inducing cell cycle arrest (0.5 μM) than 64 (5.0 μM) in MIA cells. The tubulin inhibitor nocodazole (68) also induced G₂ + M arrest (60%, 0.5 μM) with equivalent potency to 39.

Conclusions

A total of 57 analogues, across two libraries of 2,3-dihydroquinazolin-4(1H)-ones and quinazolin-4(3H)-ones, were synthesised and examined for their broad-spectrum cytotoxic activity. Triaging these compounds through initial screening at 25 μM compound concentration progressed 34 analogues to full dose response evaluation. Of these five, 51, 63, 64, 65, and 39 exhibited sub-μM levels of cytotoxicity. Naphthyl 39 is <50 nM potent against the HT29, U87, A2780, H460 and BE2-C cell lines. Action on tubulin was postulated as a mechanism of action, with molecular docking and molecular dynamics studies consistent with interaction with the tubulin–colchicine binding site. Subsequent evaluation in a tubulin polymerisation assay, relative to a known promoter, paclitaxel, and inhibitor, nocodazole, together with the computational and cell cycle analysis support these compounds as targeting tubulin polymerisation. Indoles 43 and 47 were modest promotors of tubulin polymerisation, but less effective than paclitaxel. With analogues 50, 64, and 65, the variation in ring substitution from o- to m- to p- saw a rank order decease in tubulin inhibition with a concomitant decrease in observed cytotoxicity. Naphthyl 39 displays effects equivalent to nocodazole (67) (Fig. 11C), and the most potent tubulin inhibitors examined which is reflected in the sub 0.2 μM cell line toxicity observed. The data observed supports the inclusion of –OCH₃-styrene as a key tubulin inhibitor pharmacophore.

Experimental

Chemistry

General methods

All reactions were performed using standard laboratory equipment and glassware. Solvents and reagents were purchased from Sigma Aldrich, Alfa Aesar or AK Scientific and used as received. Organic solvents were of bulk quality, and were distilled from glass prior to use. Organic solvent extracts were dried with magnesium sulfate (MgSO₄) and dried under reduced pressure with either Büchi or Heidolph rotary evaporators. Melting points were recorded in open capillaries on a Stuart SMP11 melting point apparatus. Where available, literature values are provided and appropriately referenced. Electrospray mass spectra were recorded using 10% DMSO/H₂O or HPLC-grade methanol or acetonitrile as carrier solvent on an Agilent Technologies 1260 Infinity UPLC system with a 6120 Quadrupole LC/MS in electrospray ionization (ESI) positive and negative modes. TLC was performed on Merck silica gel 60 F254 pre-coated aluminium plates with a thickness of 0.2 mm. Column chromatography was performed under ‘flash’ conditions on Merck silica gel 60 (230–400 mesh).

Nuclear magnetic resonance (NMR) spectroscopy was performed on a Brüker Avance III 400 MHz spectrometer, where proton NMR (¹H NMR) spectra and carbon NMR (¹³C NMR) spectra were acquired at 400 and 100 MHz respectively, or a Brüker Avance III 600 MHz spectrometer, where proton NMR (¹H NMR) spectra and carbon NMR (¹³C NMR) spectra were acquired at 600 and 150 MHz respectively. All spectra were recorded in deuterated dimethyl sulfoxide (DMSO-d₆), deuterated acetone (acetone-d₆) or deuterated chloroform (CDCl₃) obtained from Cambridge Isotope Laboratories Inc. Chemical shifts (δ) were measured in parts per million (ppm) and referenced against the internal reference peaks. Coupling constants (J) were measured in hertz (Hz). NMR assignments were determined through the interpretation of one- and two-dimensional spectra. Multiplicities are denoted as singlet (s), broad singlet (bs), doublet (d), doublet of doublets (dd), triplet (t), quartet (q), triplet of doublets (td), doublet of triplets (dt) and multiplet (m). Peaks are listed in decreasing chemical shift in the following format: chemical shift (integration (1H), multiplicity (1H), coupling constant (1H). The Biotage® initiator+ was used to perform microwave reactions.

All compounds subjected to biological assessment were ≥95% pure by ¹H NMR and LCMS analysis.

Synthesis of 2,3-dihydroquinazolin-4(1H)-ones 10–46

2-Butyl-2,3-dihydroquinazolin-4(1H)-one (10)

To a microwave vial containing 2-aminobenzamide 8 (226 mg, 1.66 mmol), octanal (0.16 mL, 1.73 mmol, 1.1 equiv.) and tin(ii) chloride (3 mg, 0.015 mmol, 0.01 equiv.) were added and stirred in ethanol (3 mL) to form a suspension. The microwave vial was then irradiated at 120 °C for 20 minutes. The resultant precipitate was then collected by filtration and washed with minimal H₂O (or diethylether) affording 2-butyl-2,3-dihydroquinazolin-4(1H)-one as a white crystalline solid (136 mg, 34%).

¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (s, 1H), 7.58 (dd, J = 7.7, 1.2 Hz, 1H), 7.24–7.20 (m, 1H), 6.73 (d, J = 8.0 Hz, 1H), 6.65 (dd, J = 11.0, 3.9 Hz, 1H), 6.55 (s, 1H), 4.68 (t, J = 5.3 Hz, 1H), 1.64–1.61 (m, 2H), 1.40–1.28 (m, 4H), 0.88 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.9, 148.5, 133.0, 127.3, 116.9, 115.0, 114.4, 64.4, 34.7, 25.4, 22.1, 13.9; IR ν_max (ATR)/cm⁻¹ 3340 (NH), 3178 (NH), 2926 (CH₂), 1643 (C O); LRMS (ESI⁺): 205.2 (M + H, C₁₂H₁₇N₂O, 100%); mass 136 mg; MP: 137–143 °C.

2-Octyl-2,3-dihydroquinazolin-4(1H)-one (11)

Synthesised as per 10 from 2-aminobenzamide 8 and octanal to afford the title compound as a white crystalline solid, 113 mg, 34% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (s, 1H), 7.58 (dd, J = 7.7, 1.5 Hz, 1H), 7.22 (ddd, J = 8.3, 7.2, 1.6 Hz, 1H), 6.73 (d, 1H), 6.65 (d, 1H), 6.55 (s, 1H), 4.68 (t, J = 5.2 Hz, 1H), 1.68–1.55 (m, 2H), 1.44–1.39 (m, 2H), 1.31–1.25 (m, 8H), 0.87 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163., 148.5, 133.0, 127.3, 116.8, 115.0, 114.3, 64.4, 35.0, 31.2, 28.9, 28.7, 23.2, 22.1, 13.9; IR ν_max (ATR)/cm⁻¹ 3333 (NH), 3185 (NH), 22 925 (CH₂), 1641 (C O); LRMS (ESI⁺): 247.2 (M + H, C₁₅H₂₃N₂O, 100%); LRMS (ESI⁻): 291.2 (M⁺ formic acid–H, C₁₆H₂₅N₂O₃, 100%); MP: 160–163 °C.

2-Cyclopentyl-2,3-dihydroquinazolin-4(1H)-one (12)

Synthesised as per 10 from 2-aminobenzamide 8 and cyclopentyl carboxaldehyde to afford the title compound as a white solid, 304 mg, 87% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (s, 1H), 7.56 (dd, J = 7.7, 1.3 Hz, 1H), 7.23–7.19 (m, 1H), 6.76 (d, J = 7.9 Hz, 1H), 6.64–6.60 (m, 1H), 6.53 (s, 1H), 4.48 (d, J = 6.6 Hz, 1H), 2.25–2.10 (m, 1H), 1.65–1.43 (m, 8H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.6, 148.2, 133.0, 127.2, 116.6, 115.1, 114.3, 67.7, 44.9, 27.3, 27.2, 25.1, 25.0; IR ν_max (ATR)/cm⁻¹ 3329 (NH), 3185 (NH), 2964 (CH₂), 1635 (C O); LRMS (ESI⁺): 217.1 (M + H, C₁₃H₁₇N₂O, 100%); MP: 166–177 °C.

2-Cyclohexyl-2,3-dihydroquinazolin-4(1H)-one (13)

Synthesised as per 10 from 2-aminobenzamide 8 and cyclohexyl carboxaldehyde to afford the title compound as a white solid, 223 mg, 89% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 7.86 (s, 1H), 7.55 (dd, J = 7.7, 1.5 Hz, 1H), 7.19 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H), 6.74 (d, J = 7.7 Hz, 1H), 6.62–6.58 (m, 1H), 6.54 (s, 1H), 4.45–4.43 (m, 1H), 1.71–1.55 (m, 6H), 1.16–1.07 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.7, 148.3, 133.0, 127.2, 116.4, 114.8, 114.1, 68.6, 42.8, 27.0, 26.7, 25.9, 25.6, 25.5; IR ν_max (ATR)/cm⁻¹ 3336 (NH), 3172 (NH), 2921 (CH₂), 1642 (C O); LRMS (ESI⁺) 231.2 (M + H, C₁₅H₁₉N₂O, 100%); MP: 176–184 °C.

2-(Cyclohex-3-en-1-yl)-2,3-dihydroquinazolin-4(1H)-one (14)

Synthesised as per 10 from 2-aminobenzamide 8 and cyclohex-3-ene-1-carbaldehyde to afford the title compound as an off-white solid, 337 mg, 92% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 7.95 (d, J = 44.3 Hz, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.23–7.19 (m, 1H), 6.75 (t, J = 8.2 Hz, 1H), 6.65–6.53 (m, 2H), 5.65 (s, 2H), 4.56 (dd, J = 4.2, 1.9 Hz, 1H), 2.05–1.97 (m, 4H), 1.83–1.74 (m, 2H), 1.36–1.34 (m, 1H), ¹³C NMR (100 MHz, DMSO-d₆) δ 163.8, 163.7, 148.3, 148.3, 133.1, 133.0, 127.2, 126.8, 126.7, 126.0, 125.9, 116.6, 116.58, 114.9, 114.8, 114.2, 114.1, 67.9, 67.8, 38.9, 38.6, 25.9, 25.6, 24.70, 24.68, 23.1, 22.8; IR ν_max (ATR)/cm⁻¹ 3353 (NH), 3172 (NH), 3036 (CH₂), 160 (C O); LRMS (ESI⁺): 229.2 (M + H, C₁₄H₁₇N₂O, 100%); (ESI⁻): 273.1 (M − H + formic acid, C₁₅H₁₉N₂O₂, 100%); MP: 178–185 °C.

2-Phenyl-2,3-dihydroquinazolin-4(1H)-one (15)

Synthesised as per 10 from 2-aminobenzamide 8 and benzaldehyde to afford the title compound as a white crystalline solid, 226 mg, 80% yield.

¹H NMR: (400 MHz, DMSO-d₆) δ 8.27 (s, 1H), 7.61 (dd, J = 7.7, 1.6 Hz, 1H), 7.49 (dd, J = 8.2, 1.3 Hz, 2H), 7.43–7.31 (m, 3H), 7.24 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 7.10 (s, 1H), 6.75–6.73 (m, 1H), 6.69–6.65 (m, 1H), 5.75 (s, 1H); ¹³C NMR: (100 MHz, DMSO-d₆) δ 163.6, 147.9, 141.6, 133.3, 128.4, 128.3 (2C), 127.3, 126.8 (2C), 117.1, 114.9, 114.4, 66.5; MS: LRMS (ESI⁺): 225 (M + H, C₁₄H₁₃N₂O, 100%); (ESI⁻): 223 (M − H C₁₄H₁₁N₂O, 100%); IR ν_max (ATR)/cm⁻¹ 3308 (NH), 3169 (NH), 3061 (CH), 1649 (C O); MP: >225 °C (decomp.).

2-(p-Tolyl)-2,3-dihydroquinazolin-4(1H)-one (16)

Synthesised as per 10 from 2-aminobenzamide 8 and 4-methylbenzaldeyde to afford the title compound as an off-white solid, 141 mg, 60% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (s, 1H), 7.60 (dd, J = 7.7, 1.4 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.23 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.18 (d, J = 7.9 Hz, 2H), 7.04 (s, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.68–6.64 (m, 1H), 5.70 (s, 1H), 2.29 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.1, 148.3, 139.1, 138.2, 133.7, 129.3 (2C), 127.8, 127.2 (2C), 117.54, 115.4, 114.9, 66.8, 21.2; IR ν_max (ATR)/cm⁻¹ 3312 (NH), 2918 (CH), 1655 (C O); LRMS (ESI⁺): 239.1 (M + H, C₁₅H₁₅N₂O, 100%); LRMS (ESI⁻): 237.2 (M − H, C₁₅H₁₃N₂O₃, 100%); MP: >207 °C (decomp).

2-(4-(tert-Butyl)phenyl)-2,3-dihydroquinazolin-4(1H)-one (17)

Synthesised as per 10 from 2-aminobenzamide 8 and 4-tert-butylbenzaldehyde to afford the title compound as an off white solid, 175 mg; 38% yield.

¹H NMR (400 MHz, DMSO-d₆) 8.22 (s, 1H), 7.61 (dd, J = 7.7, 1.4 Hz, 1H), 7.42 (s, 4H), 7.24 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.06 (s, 1H), 6.73 (d, J = 7.8 Hz, 1H), 6.69–6.65 (m, 1H), 5.72 (s, 1H), 1.28 (s, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.1, 151.5, 148.4, 139.1, 133.7, 127.8, 127.1, 125.6, 117.5, 115.4, 114.8, 66.9, 31.6. IR ν_max (ATR)/cm⁻¹ 3321 (NH), 3190 (NH), 2975 (CH₂), 1632 (C O); LRMS non-ionisable; MP: > 200 °C (decomp.).

2-(2-Bromophenyl)-2,3-dihydroquinazolin-4(1H)-one (18)

Synthesised as per 10 from 2-aminobenzamide 8 and 2-bromobenzaldeyde to afford the title compound as a white solid, 187 mg; 40% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (s, 1H, NH), 7.69–7.64 (m, 3H), 7.45 (td, J = 7.5, 1.1 Hz, 1H), 7.33 (ddd, J = 7.9, 7.4, 1.8 Hz, 1H), 7.26 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 6.98 (s, 1H, NH, H1), 6.77–6.70 (m, 2H), 6.09 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.6, 147.7, 139.1, 133.4, 132.8, 130.7, 129.1, 128.1, 127.4, 122.2, 117.5, 114.7, 114.6, 66.4; MS LRMS (ESI⁺) m/z: 303 (M + H, C₁₄H₁₂⁷⁹BrN₂O, 100%), 305 (M + H, C₁₄H₁₂⁸¹BrN₂O, 95%); (ESI⁻) m/z: 301 (M − H, C₁₄H₉⁷⁹BrN₂O, 100%), 303 (M − H, C₁₄H₉⁸¹BrN₂O, 95%); IR ν_max (ATR)/cm⁻¹ 3365 (NH), 1646 (C O); MP: >173 °C (decomp.).

2-(3-Bromophenyl)-2,3-dihydroquinazolin-4(1H)-one (19)

Synthesised as per 10 from 2-aminobenzamide 8 and 3-bromobenzaldeyde to afford the title compound as a white solid, 491 mg; 99% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.39 (s, 1H), 7.65 (t, J = 1.7 Hz, 1H), 7.60 (dd, J = 7.7, 1.4 Hz, 1H), 7.52 (ddd, J = 7.9, 1.8, 1.0 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.25 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.20 (s, 1H), 6.75 (d, J = 7.8 Hz, 1H), 6.72–6.64 (m, 1H), 5.76 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.6, 147.5, 144.8, 133.7, 131.3, 130.7, 129.7, 127.5, 125.8, 121.7, 117.5, 114.9, 114.6, 65.5; IR ν_max (ATR)/cm⁻¹ 3282 (NH), 3172 (NH), 3062 (CH), 1645 (C O); LRMS (ESI⁺) m/z: 303 (M + H, C₁₄H₁₂⁷⁹BrN₂O, 100%), 305 (M + H, C₁₄H₁₂⁸¹BrN₂O, 90%); (ESI⁻) m/z: 301 (M − H, C₁₄H₁₀⁷⁹BrN₂O, 100%), 303 (M − H, C₁₄H₁₀⁸¹BrN₂O, 90%); MP: >174 °C (decomp.).

2-(4-Bromophenyl)-2,3-dihydroquinazolin-4(1H)-one (20)

Synthesised as per 10 from 2-aminobenzamide 8 and 4-bromobenzaldeyde to afford the title compound as a white solid, 418 mg; 94% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H, NH), 7.61–7.57 (m, 3H), 7.44–7.42 (m, 2H), 7.25 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 7.13 (s, 1H, NH), 6.74 (dd, J = 8.1, 0.5 Hz), 6.70–6.66 (m, 1H), 5.75 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.5, 147.6, 141.1, 133.4, 131.2 (2C), 129.1 (2C), 127.4, 121.6, 117.3, 114.9, 114.5, 65.8; MS: LRMS (ESI⁺) m/z: 303 (M + H, C₁₄H₁₂⁷⁹BrN₂O, 100%), 305 (M + H, C₁₄H₁₂⁸¹BrN₂O, 95%), 347 (M⁺ formic acid-H, C₁₄H₁₂⁷⁹BrN₂O, 100%), 349 (M⁺ formic acid-H, C₁₄H₁₂⁸¹BrN₂O, 95%); (ESI⁻) m/z: 301 (M − H, C₁₄H₁₀⁷⁹BrN₂O, 100%), 303 (M − H, C₁₄H₁₀⁸¹BrN₂O, 95%); IR ν_max (ATR)/cm⁻¹ 3308 (N–H), 1652 (C O); MP: >191 °C (decomp.).

2-(3-Hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (21)

Synthesised as per 10 from 2-aminobenzamide 8 and 3-hydroxybenzaldeyde to afford the title compound as a white solid, 260 mg; 67% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 9.52 (d, J = 1.7 Hz, 1H), 8.22 (s, 1H), 7.59 (dd, J = 7.7, 1.4 Hz, 1H), 7.27–7.19 (m, 1H), 7.16 (t, J = 8.1 Hz, 1H), 7.06 (s, 1H), 6.88 (t, J = 4.0 Hz, 2H), 6.72 (ddd, J = 7.1, 3.1, 1.8 Hz, 2H), 6.69–6.62 (m, 1H), 5.64 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.7, 157.4, 147.9, 143.3, 133.4, 129.4, 127.4, 117.5, 117.1, 115.4, 114.9, 114.4, 113.7, 66.5; IR ν_max (ATR)/cm⁻¹ 3282 (NH), 3100 (OH) 1650 (C O); LRMS (ESI⁺): 241.1 (M + H, C₁₄H₁₃N₂O₂, 100%), 481.2 (2 M + H, C₁₄H₁₃N₂O₂, 10%); (ESI⁻) 239.1 (M − H, C₁₄H₁₁N₂O₂, 100%), 479.2 (2 M + H, C₁₄H₁₁N₂O₂, 50%); MP: 206–209 °C.

2-(4-Hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (22)

Synthesised as per 10 from 2-aminobenzamide 8 and 4-hydroxybenzaldeyde to afford the title compound as a white solid, 163 mg; 67% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 9.53 (s, 1H), 8.09 (d, J = 4.3 Hz, 1H), 7.61 (dd, J = 7.7, 1.5 Hz, 1H), 7.32–7.29 (m, 2H), 7.24 (ddd, J = 8.3, 7.2, 1.6 Hz, 1H), 6.94 (s, 1H), 6.79–6.66 (m, 4H), 5.66 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.2, 158.1, 148.6, 133.7, 132.1, 128.7 (2C), 127.8, 117.5, 115.4 (2C), 114.8, 67.1; IR ν_max (ATR)/cm⁻¹ 3346 (NH), 3175 (NH), 1229 (C O); LRMS (ESI⁺): 241.1 (M + H, C₁₄H₁₃N₂O₂, 100%); (ESI⁻) 239.1 (M − H, C₁₄H₁₂N₂O₂, 100%); MP: >200 °C.

2-(2-Carboxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (23)

Synthesised as per 10 from 2-aminobenzamide 8 and 2-carboxybenzaldeyde to afford the title compound as a yellow solid, 252 mg; 58% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 9.48 (s, 1H), 8.22 (s, 1H), 7.60 (dd, J = 7.7, 1.5 Hz, 1H), 7.23 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 7.06 (s, 1H), 6.89 (t, J = 4.3 Hz, 2H), 6.76–6.69 (m, 2H), 6.69–6.63 (m, 1H), 5.64 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.5, 157.3, 147.8, 143.2, 133.3, 129.3, 127.3, 117.4, 117.0, 115.3, 114.9, 114.3, 113.7, 66.5, 39.5; IR ν_max (ATR)/cm⁻¹ 3282 (NH), 3036 (NH), 1726 (C O), 1674 (C O); LRMS (ESI⁺): 251.1 (M + H–H₂O, C₁₅H₁₁N₂O₂, 100%); (ESI⁻): 249.1 (M − H-H₂O, C₁₅H₉N₂O₂, 100%); MP: >240 °C (decomp.).

2-(3-Carboxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (24)

Synthesised as per 10 from 2-aminobenzamide 8 and 3-carboxybenzaldeyde to afford the title compound as a white solid, 398 mg; 86% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 1H), 8.08 (s, 1H), 7.90 (dt, 1H), 7.71 (dt, 1H), 7.60 (dd, J = 7.8, 1.5 Hz, 1H), 7.51 (t, J = 9.6, 1H), 7.25 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 7.19 (s, 1H), 6.74 (d, 1H), 6.71–6.65 (m, 1H), 5.83 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.4, 163.8, 147.8, 142.5, 133.8, 131.5, 131.1, 129.5, 129.0, 127.9, 127.6, 117.6, 115.0, 114.7, 66.1 IR ν_max (ATR)/cm⁻¹ 3334 (NH), 3200 (NH), 1666 (C O), 750.5 (C C); LRMS (ESI⁺): 269.2 (M + H, C₁₅H₁₃N₂O₃, 100%); MP: >300 °C.

2-(4-Carboxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (25)

Synthesised as per 10 from 2-aminobenzamide 8 and 4-formylbenzoic acid to afford the title compound as a white solid, 341 mg; 96% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 1H), 7.94–7.92 (m, 2H), 7.60 (dd, J = 12.0, 4.9 Hz, 3H), 7.25 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.20 (s, 1H), 6.74 (d, J = 7.7 Hz, 1H), 6.70–6.68 (m, 1H), 5.82 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.3, 163.8, 147.7, 146.7, 133.8, 131.0 (2C), 129.6, 127.6, 127.1 (2C), 117.6, 115.0, 114.7, 66.1; IR ν_max (ATR)/cm⁻¹ 3289 (NH), 2800 (OH), 1695 (C O), 725 (C C); LRMS (ESI⁺): 269.2 (M + H, C₁₅H₁₃N₂O₃, 100%); (ESI⁻): 267.1 (M − H, C₁₅H₁₁N₂O₃, 100%); MP: >300 °C.

2-(2-Nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (26)

Synthesised as per 10 from 2-aminobenzamide 8 and 2-nitrobenzaldeyde to afford the title compound as an orange solid, 414 mg; 90% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (s, 1H), 8.06 (dd, J = 8.1, 0.9 Hz, 1H), 7.85–7.84 (m, 1H), 7.79 (t, J = 7.2 Hz, 1H), 7.66–7.61 (m, 2H), 7.28–7.24 (m, 1H), 7.00 (s, 1H), 6.77 (d, J = 8.1 Hz, 1H), 6.72 (t, J = 7.5 Hz, 1H), 6.33 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.3, 147.7, 147.1, 135.9, 133.9, 133.5, 129.9, 128.9, 127.3, 124.7, 117.7, 114.9, 114.5, 62.2; IR ν_max (ATR)/cm⁻¹ 3409 (NH), 3182 (NO), 1654 (C O); LRMS (ESI⁺): 270.1 (M + H, C₁₄H₁₁N₃O₃, 100%); MP: >194.6 °C (decomp).

2-(3-Nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (27)

Synthesised as per 10 from 2-aminobenzamide 8 and 3-nitrobenzaldeyde to afford the title compound as a yellow/orange solid, 356 mg; 78% yield.

¹H NMR R (400 MHz, DMSO-d₆) δ 8.52 (s, 1H), 8.36 (s, 1H), 8.13–8.19 (m, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.70 (t, J = 7.9 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.34 (s, 1H), 7.27 (t, 1H), 6.79 (d, J = 8.1 Hz, 1H), 6.70 (t, J = 7.4 Hz, 1H), 5.95 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.3, 147.7, 147.3, 144.3, 133.6, 133.4, 130.0, 127.4, 123.3, 121.6, 117.5, 114.9, 114.6, 65.2 IR ν_max (ATR)/cm⁻¹ 3289 (NH), 3179 (NH), 3072 (CH), 1650 (C O); LRMS (ESI⁺): 270.1 (M + H, C₁₄H₁₁N₃O₃, 100%); LRMS (ESI⁻): 268.1 (M − H, C₁₄H₁₁N₃O₃, 100%); MP: >194 °C (decomp).

2-(4-Nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (28)

Synthesised as per 10 from 2-aminobenzamide 8 and 4-nitrobenzaldeyde to afford the title compound as a yellow solid, 425 mg; 95% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (s, 1H), 8.25 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.7 Hz, 2H), 7.62–7.60 (m, 1H), 7.32 (s, 1H), 7.28–7.24 (m, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.69 (t, J = 7.4 Hz, 1H), 5.91 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.3, 149.3, 147.2, 133.6, 128.0, 127.4, 123.6, 117.5, 114.9, 114.5, 65.3; IR ν_max (ATR)/cm⁻¹ 3279 (NH), 3173 (NH), 3100 (CH), 1644 (C O) cm⁻¹; LRMS (ESI⁺): 270.1 (M + H, C₁₄H₁₁N₃O₃, 100%); LRMS (ESI⁻): 268.1 (M − H, C₁₄H₁₁N₃O₃, 100%); MP: >197 °C (decomp).

2-(3-Methoxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (29)

Synthesised as 10 from 2-aminobenzamide 8 and per 3-methoxybenzaldeyde to afford the title compound as an off-white solid, 72 mg; 17% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.29 (s, 1H), 7.61 (dd, J = 7.7, 1.5 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.28–7.22 (m, 1H), 7.12 (s, 1H), 7.09–7.04 (m, 2H), 6.91 (ddd, J = 8.2, 2.5, 0.8 Hz, 1H), 6.78–6.74 (m, 1H), 6.70–6.64 (m, 1H), 5.72 (s, 1H), 3.75 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.0, 159.7, 148.3, 143.8, 133.8, 129.9, 127.8, 119.4, 117.6, 115.4, 114.90, 114.1, 113.0, 66.5, 55.6; IR (ATR): IR ν_max (ATR)/cm⁻¹ 3288 (NH), 3053 (NH), 2916 (CH), 1645 (C O); LRMS (ESI⁺): 255.1 (M + H, C₁₅H₁₅N₂O₂, 100%); LRMS (ESI⁻): 253.1 (M − H, C₁₅H₁₃N₂O₂, 100%); MP: 140–145 °C.

2-(3-(Trifluoromethyl)phenyl)-2,3-dihydroquinazolin-4(1H)-one (30)

Synthesised as per 10 from 2-aminobenzamide 8 and 3-trifluoromethylbenzaldeyde to afford the title compound as a white solid, 138 mg; 29% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.42 (s, 1H), 7.85 (s, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.64–7.610 (m, 2H), 7.29–7.23 (m, 2H), 6.78–6.76 (m, 1H), 6.72–6.68 (m, 1H), 5.89 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.5, 147.6, 143.1, 133.5, 131.0, 129.5, 127.4, 125.20, 125.16, 123.64, 123.60, 117.4, 114.9, 114.5, 65.7; IR ν_max (ATR)/cm⁻¹ 3275 (NH), 3211 (NH), 1645 (C O); LRMS (ESI⁺): 293.1 (M + H, C₁₅H₁₂F₃N₂O, 100%); LRMS (ESI⁻): 291.1 (M − H, C₁₅H₁₀F₃N₂O, 100%); MP: 137–141 °C.

2-(3-Cyanophenyl)-2,3-dihydroquinazolin-4(1H)-one (31)

Synthesised as per 10 from 2-aminobenzamide 8 and 3-cyanobenzaldeyde to afford the title compound as a white solid, 308 mg; 72% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.42 (s, 1H), 7.91 (s, 1H), 7.85–7.82 (m, 2H), 7.62 (ddd, J = 7.8, 4.6, 3.3 Hz, 2H), 7.33–7.20 (m, 2H), 6.77 (d, J = 8.0 Hz, 1H), 6.73–6.68 (m, 1H), 5.85 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.4, 147.5, 143.3, 133.5, 132.2, 131.7, 130.6, 129.7, 127.4, 118.7, 117.5, 115.0, 114.6, 111.2, 65.5; IR ν_max (ATR)/cm⁻¹ 3372 (NH), 3178 (NH), 2232 (CN), 1659 (C O); LRMS (ESI⁺): 250.1 (M + H, C₁₅H₁₂N₃O, 100%); LRMS (ESI⁻): 248.1 (M − H, C₁₅H₁₀N₃O, 100%); MP: 224–230 °C.

2-(4-Cyanophenyl)-2,3-dihydroquinazolin-4(1H)-one (32)

Synthesised as per 10 from 2-aminobenzamide 8 and 4-cyanobenzaldeyde to afford the title compound as a white solid, 347 mg; 86% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.47 (s, 1H), 7.90–7.84 (m, 2H), 7.66 (d, J = 8.2 Hz, 2H), 7.61 (dd, J = 7.7, 1.4 Hz, 1H), 7.31–7.22 (m, 2H), 6.76 (d, J = 8.1 Hz, 1H), 6.71–6.65 (m, 1H), 5.85 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.8, 147.81, 147.79, 134.0, 132.9, 128.1, 127.9, 119.1, 117.9, 115.4, 115.0, 111.5, 66.0; IR ν_max (ATR)/cm⁻¹ 3346 (NH), 2227 (CN), 1664 (C O); LRMS (ESI⁺): 250.1 (M + H, C₁₅H₁₂N₃O, 100%); LRMS (ESI⁻): 248.1 (M − H, C₁₅H₁₀N₃O, 100%); MP: 240–247 °C.

2-(3,4,5-Trihydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (33)

Synthesised as per 10 from 2-aminobenzamide 8 and 3,4,5-trihydroxybenzaldeyde to afford the title compound as a white solid, 260 mg; 45% yield.

2-(Pyridin-3-yl)-2,3-dihydroquinazolin-4(1H)-one (34)

Synthesised as per 10 from 2-aminobenzamide 8 and 3-pyridinecarboxaldhehyde to afford the title compound as a pale orange solid, 298 mg; yield 84%.

¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (d, J = 2.1 Hz, 1H), 8.56 (dd, J = 4.8, 1.6 Hz, 1H), 8.38 (s, 1H), 7.90 (dt, J = 7.9, 1.9 Hz, 1H), 7.63 (dd, J = 7.7, 1.5 Hz, 1H), 7.43 (ddd, J = 7.9, 4.8, 0.5 Hz, 1H), 7.27 (ddd, J = 8.2, 7.3, 1.6 Hz, 1H), 7.17 (s, 1H), 6.73 (ddd, J = 15.0, 8.1, 4.3 Hz, 2H), 5.86 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.0, 150.1, 148.8, 148.2, 137.3, 135.1, 133.9, 127.9, 124.0, 118.0, 115.5, 115.0, 65.1; IR ν_max (ATR)/cm⁻¹ 3256 (NH), 2978 (CH), 1652 (C O); LRMS (ESI⁺) 226.1 (C₁₃H₁₂N₃O, 100%); LRMS (ESI⁻): 224.1 (C₁₃H₁₀N₃O, 100%); MP: > 250 °C.

2-(Thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one (35)

Synthesised as per 10 from 2-aminobenzamide 8 and thiophen-2-carboxaldehyde to afford the title compound as a white solid, 309 mg; 80% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (s, 1H), 7.62 (dd, J = 7.7, 1.3 Hz, 1H), 7.45 (dd, J = 5.0, 1.2 Hz, 1H), 7.26–7.24 (m, 2H), 7.12 (d, J = 3.0 Hz, 1H), 6.98 (dd, J = 5.0, 3.5 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H), 6.70 (t, 1H), 6.02 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.1, 147.2, 146.4, 133.4, 127.3, 126.5, 125.9, 125.7, 117.5, 115.1, 114.7, 62.6; IR ν_max (ATR)/cm⁻¹ 3295 (NH), 3172 (NH), 1649 (C O); LRMS (ESI⁺): 231.1 (M + H, C₁₂H₁₁N₂OS, 100%); LRMS (ESI⁻): 229.1 (M − H, C₁₂H₁₁N₂OS, 100%); MP: 206–207 °C.

2-(5-bromofuran-2-yl)-2,3-dihydroquinazolin-4(1H)-one (36)

Synthesised as per 10 from 2-aminobenzamide 8 and 4′-phenylbenzaldeyde to afford the title compound as a white solid, 372 mg; 74% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.35 (s, 1H), 7.70–7.67 (m, 5H), 7.58 (d, J = 8.3 Hz, 2H), 7.47 (dd, J = 10.4, 4.8 Hz, 2H), 7.39–7.37 (m, 1H), 7.26 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.17 (s, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.71–6.67 (m, 1H), 5.81 (s, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.6, 147.8, 140.9, 140.3, 139.7, 133.4, 129.0 (2C), 127.5 (2C), 127.4, 126.7 (2C), 126.65 (2C), 117.2, 115.0, 114.4, 66.2.; IR ν_max (ATR)/cm⁻¹ 3295 (NH), 3178 (NH), 1652 (C O); LRMS (ESI⁺): 301.2 (M + H, C₂₀H₁₇N₂O, 100%); LRMS (ESI⁻): 299.1 (M − H, C₂₀H₁₅N₂O, 100%). MP: 217–221 °C.

2([1,1′-Biphenyl]-4-yl)-2,3-dihydroquinazolin-4(1H)-one (37)

Synthesised as per 10 from 2-aminobenzamide 8 and 4′-phenylbenzaldeyde to afford the title compound as a white solid, 398 mg; 75% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 1H), 8.08 (s, 1H), 7.90 (dt, 1H), 7.71 (dt, 1H), 7.60 (dd, J = 7.8, 1.5 Hz, 1H), 7.51 (t, J = 9.6, 1H), 7.25 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 7.19 (s, 1H), 6.74 (d, 1H), 6.71–6.65 (m, 1H), 5.83 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.4, 163.8, 147.8, 142.5, 133.8, 131.5, 131.1, 129.5, 129.0, 127.9, 127.6, 117.6, 115.0, 114.7, 66.1; IR ν_max (ATR)/cm⁻¹ 3334 (NH), 3200 (NH), 1666 (C O), 750.5 (C C); LRMS (ESI⁻): 267.1 (M − H, C₁₅H₁₅N₂O₃, 100%), 535.2 (2 M − H, C₁₅H₁₅N₂O₃, 100%); MP: >300 °C.

2-(Naphthalen-2-yl)-2,3-dihydroquinazolin-4(1H)-one (38)

Synthesised as per 10 from 2-aminobenzamide 8 and 1-naphthylaldeyde to afford the title compound as a white solid, 130 mg; 86% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.57–8.54 (m, 1H), 8.27 (s, 1H), 8.01–7.96 (m, 2H), 7.72–7.69 (m, 2H), 7.59–7.51 (m, 3H), 7.28–7.24 (m, 1H), 7.08 (s, 1H), 6.76–6.71 (m, 2H), 6.49 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.5, 148.9, 135.6, 134.2, 133.7, 131.0, 129.8, 129.1, 128.0, 126.51, 126.50, 126.3, 125.7, 125.1, 117.7, 115.4, 115.0, 66.4; IR ν_max (ATR)/cm⁻¹, 3217 (NH), 3004, 1649 (C O); LRMS (ESI⁺) 275.2 (M + H, C₁₈H₁₅N₂O, 100%); MP: 182–186 °C.

2-(Naphthalen-1-yl)-2,3-dihydroquinazolin-4(1H)-one (39)

Synthesised as per 10 from 2-aminobenzamide 8 and 2-naphthylaldeyde to afford the title compound as a white solid, 374 mg; 30% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.37 (s, 1H), 7.96–7.90 (m, 4H), 7.70 (dd, J = 8.6, 1.6 Hz, 1H), 7.64 (dd, J = 7.7, 1.5 Hz, 1H), 7.53 (dd, J = 6.2, 3.3 Hz, 2H), 7.25 (ddd, J = 8.3, 7.3, 1.6 Hz, 1H), 7.19 (s, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.71–6.67 (m, 1H), 5.94 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.6, 147.9, 138.9, 133.4, 133.0, 132.5, 128.1, 128.0, 127.6, 127.4, 126.43, 126.38, 125.9, 124.8, 117.2, 114.9, 114.4, 66.8; IR ν_max (ATR)/cm⁻¹ 3275 (NH), 8978 (CH), 1646 (C O); LRMS (ESI⁺): 275.1 (M + H, C₁₈H₁₅N₂O, 100%); LRMS (ESI⁻): 273.1 (M − H, C₁₈H₁₃N₂O, 100%); MP: 202–206 °C.

2-(Benzofuran-2-yl)-2,3-dihydroquinazolin-4(1H)-one (40)

Synthesised as per 10 from 2-aminobenzamide 8 and benzofuran-2-carbaldehyde to afford the title compound as a white solid, 171 mg; 71% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (d, J = 2.9 Hz, 1H), 7.61 (ddd, J = 8.3, 7.7, 1.1 Hz, 2H), 7.52 (dd, J = 8.2, 0.6 Hz, 1H), 7.42 (s, 1H), 7.30–7.19 (m, 3H), 6.79 (d, J = 7.7 Hz, 1H), 6.72–6.67 (m, 2H), 5.93 (t, J = 2.9 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.2, 157.3, 154.2, 146.9, 133.4, 127.5, 127.3, 124.6, 123.0, 121.4, 117.4, 115.0, 114.6, 111.2, 104.0, 60.4; IR ν_max (ATR)/cm⁻¹ 3269 (NH), 3178 (NH), 3010, 1662 (C O); LRMS (ESI⁺) 265.1 (M + H, C₁₆H₁₃N₂O₂, 100%); (ESI⁻): 263.1 (M − H, C₁₆H₁₁N₂O₂, 100%); MP: 192–198 °C.

2-(1H-Indol-3-yl)-2,3-dihydroquinazolin-4(1H)-one (41)

Synthesised as per 10 from 2-aminobenzamide 8 and 1H-indole-3-carbaldehyde to afford the title compound as an orange solid, 220 mg; 53% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 11.09 (s, 1H), 8.09 (s, 1H), 7.78–7.76 (m, 1H), 7.66 (dd, J = 7.8, 1.4 Hz, 1H), 7.41–7.39 (m, 2H), 7.24 (dt, 1H), 7.11–7.10 (m, 1H), 7.01–6.98 (m, 1H), 6.92 (s, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.71–6.68 (m, 1H), 6.04 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.2, 148.8, 136.6, 133.0, 127.5, 125.4, 124.7, 121.4, 120.0, 118.8, 117.0, 115.3, 114.40, 114.41, 111.7, 61.7; MS LRMS (ESI⁺) 264.1 (M + H, C₁₆H₁₄N₃O, 100%); (ESI⁻): 262 (M − H, C₁₆H₁₂N₃O, 100%); IR ν_max (ATR)/cm⁻¹ 3579, 3398, 3185 (NH), 2991, 1641 (C O); MP: 196–216 °C.

2-(Benzo[c][1,2,5]thiadiazol-5-yl)-2,3-dihydroquinazolin-4(1H)-one (42)

Synthesised as per 10 from 2-aminobenzamide 8 and benzo[c][1,2,5]thiadiazole-5-carbaldehyde to afford the title compound as a brown solid, 112 mg; 48% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.13 (d, J = 9.2 Hz, 1H), 8.04 (s, 1H), 7.92 (dd, J = 9.1, 1.6 Hz, 1H), 7.63 (dd, J = 7.7, 1.3 Hz, 1H), 7.35 (s, 1H), 7.29–7.25 (m, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.72–6.65 (m, 1H), 6.01 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.4, 154.2, 154.0, 147.4, 143.6, 133.6, 129.2, 127.4, 121.5, 118.5, 117.4, 114.9, 114.5, 65.8; IR ν_max (ATR)/cm⁻¹ 3243 (NH), 3029, 1654 (C O) cm⁻¹; LRMS (ESI⁺) 283.1 (M + H, C₁₄H₁₁N₄OS, 100%); (ESI⁻): 281.1 (M − H, C₁₄H₉N₄OS, 100%); MP: 216–218 °C.

2-Phenethyl-2,3-dihydroquinazolin-4(1H)-one (43)

Synthesised as per 10 from 2-aminobenzamide 8 and phenylacetaldehyde to afford the title compound as a yellow solid, 159 mg; 39% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.02 (s, 1H, NH), 7.60 (dd, J = 7.7, 1.5 Hz, 1H), 7.32–7.15 (m, 6H), 6.77–6.73 (m, 1H), 6.71–6.65 (m, 2H), 4.74 (t, J = 5.0 Hz, 1H), 2.79–2.72 (m, 2H), 1.97–1.89 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.1, 148.6, 141.6, 133.1, 128.4 (2C), 128.3 (2C), 127.4, 125.8, 117.1, 115.1, 114.4, 64.0, 36.7, 29.3; IR ν_max (ATR)/cm⁻¹ 3302 (NH), 3175 (NH), 3000 (CH₂), 1647 (C O); LRMS (ESI⁺) m/z: 253.2 (M + H, C₁₆H₁₇N₂O, 100%); (ESI⁻), 251.1 (M − H, C₁₆H₁₅N₂O, 100%), 297.2 (M⁺ formic acid-H, C₁₆H₁₅N₂O, 100%); MP >128 °C (decomp).

2-(1-Bromo-2-phenylvinyl)-2,3-dihydroquinazolin-4(1H)-one (44)

Synthesised as per 10 from 2-aminobenzamide 8 and 2-bromo-3-phenylacrylaldehyde to afford the title compound as a pale yellow solid, 212 mg; 40% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (d, J = 1.3 Hz, 1H), 7.64–7.63 (m, 2H), 7.58 (dd, J = 7.7, 1.4 Hz, 1H), 7.43–7.39 (m, 2H), 7.37 (dd, J = 4.9, 3.6 Hz, 1H), 7.25–7.19 (m, 3H), 6.72 (d, J = 8.0 Hz, 1H), 6.62 (t, 1H), 5.64 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.6, 146.9, 134.3, 133.4, 129.6, 129.0 (2C), 128.6, 128.5, 128.5, 128.4 (2C), 127.0, 116.6, 113.6, 113.4, 70.6; IR ν_max (ATR)/cm⁻¹ 3263 (NH), 3178 (NH), 3053 (CH), 1646 (C O); LRMS (ESI⁺): 329.1 (M + H, C₁₆H₁₃⁷⁹BrN₂O, 100%) 331.1 (M + H, C₁₆H₁₃⁸¹BrN₂O, 95%); MP: 180–183 °C.

2-(2-Nitrostyryl)-2,3-dihydroquinazolin-4(1H)-one (45)

Synthesised as per 10 from 2-aminobenzamide 8 and (E)-3-(2-nitrophenyl)acrylaldehyde to afford the title compound as a yellow solid, 289 mg; 59% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.50 (d, J = 8.9 Hz, 1H), 8.35 (s, 1H), 8.09 (d, J = 7.0 Hz, 1H), 8.05 (dd, J = 8.1, 1.1 Hz, 1H), 7.94 (dd, J = 7.8, 1.5 Hz, 1H), 7.81–7.76 (m, 1H), 7.74 (d, J = 15.8 Hz, 1H), 7.68–7.62 (m, 2H), 7.53 (td, J = 7.7, 1.6 Hz, 1H), 7.37–7.31 (m, 2H), 7.23–7.21 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.1, 163.2, 149.0, 148.2, 139.3, 133.6, 132.4, 131.9, 130.6, 129.9, 129.7, 128.7, 128.1, 126.3, 124.6, 118.9. IR ν_max (ATR)/cm⁻¹ 3249 (NH), 2971 (NH), 1668 (C O), 1519 (NO), 1349 (NO); LRMS (ESI⁺): 296.1 (M + H, C₁₆H₁₄N₃O₃, 100%); MP: 130–134 °C.

2-(2-(Furan-2-yl)vinyl)-2,3-dihydroquinazolin-4(1H)-one (46)

Synthesised as per 10 from 2-aminobenzamide 8 and (E)-3-(furan-2-yl)acrylaldehyde to afford the title compound as a white solid, 109 mg; 28% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (s, 1H), 7.63–7.59 (m, 2H), 7.27–7.22 (m, 1H), 6.87 (s, 1H), 6.74 (d, J = 8.1 Hz, 1H), 6.71–6.65 (m, 1H), 6.54–6.48 (m, 3H), 6.12 (dd, J = 15.8, 6.5 Hz, 1H), 5.26 (d, J = 6.5 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.3, 151.1, 147.6, 143.2, 133.3, 127.3, 126.7, 119.9, 117.2, 114.9, 114.6, 111.8, 109.6, 65.0; IR ν_max (ATR)/cm⁻¹ 3236 (NH), 2971 (NH), 1634 (C O); LRMS (ESI⁺): 241.1 (M + H, C₁₄H₁₃N₂O₂, 100%); MP: 148–151 °C.

Synthesis of quinazolin-4(3H)-ones 49–57 and 60–67

Three general procedures were used to synthesise these analogues.

Procedure A: used to synthesise 47–53

Procedure B. Used to synthesise 54–57

To a round bottom flask containing 15 (159 mg, 0.71 mmol) in acetone (70 mL), KMnO₄ (355 mg, 2.24 mmol, 3 equiv.) was added and stirred at reflux for 3 hours. The reaction mixture was then removed from the heat, filtered and neutralised with concentrated sodium thiosulfate solution until the solution lost its pink colour. The reaction filtrate was then extracted with CHCl₃ (3 × 30 mL) and dried over MgSO₄. The solvent was removed in vacuo affording 2-phenylquinazolin-4(1H)-one as white crystals, 116 mg; yield 81%.

Procedure C: used to synthesise 60–66

To a microwave vial were added 2-methylquinazolin-4(3H)-one 59 (151.7 mg, 1 eq., 0.9 mol), methyl 3-formylbenzoate (222.3 mg, 1.5 eq., 1.5 mol) and 1 : 1 toluene : acetic acid (3 mL) and subsequently irradiated at 180 °C for 40 min. After completion, the resulting precipitate was collected by vacuum filtration and washed with EtOH (10 mL) and ether (10 mL) affording the title compound as a yellow solid, 88 mg; yield 31%.

2-(5-Methyl-1H-indol-3-yl)-2,3-dihydroquinazolin-4(1H)-one (47)

Synthesised as per 10 from 2-aminobenzamide 8 and 5-methyl-1H-indole-3-carbaldehyde to afford the title compound as a pink solid, 73 mg; 17% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 12.09 (s, 1H), 11.74 (s, 1H), 8.50–8.49 (m, 2H), 8.10 (dd, J = 7.9, 1.0 Hz, 1H), 7.79–7.75 (m, 2H), 7.41–7.35 (m, 2H), 7.06 (dd, J = 8.3, 1.4 Hz, 1H), 2.47 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.1, 150.4, 149.8, 135.2, 134.3, 129.6, 129.1, 127.0, 125.81, 125.80, 125.1, 124.1, 122.1, 120.4, 111.6, 108.1, 21.5; MS LRMS (ESI⁺) 276.2 (M + H, C₁₇H₁₄N₃O, 100%); (ESI⁻): 274.1 (M − H, C₁₇H₁₂N₃O, 100%); IR ν_max (ATR)/cm⁻¹ 3385 (NH), 3114 (NH), 2965, 1663 (C O); MP: >155 °C (decomp.).

3-(4-Oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-1H-indole-5-carbonitrile (48)

Synthesised as per 10 from 2-aminobenzamide 8 and 3-formyl-1H-indole-5-carbonitrile to afford the title compound as a yellow solid, 103 mg; 35% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 12.35 (s, 1H), 12.30 (s, 1H), 9.10 (s, 1H), 8.71 (d, J = 2.5 Hz, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.80 (dt, J = 14.9, 7.5 Hz, 2H), 7.67 (t, J = 7.4 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.44 (dd, J = 15.9, 9.2 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.0, 149.4, 149.3, 138.7, 134.5, 131.4, 127.6, 127.3, 125.8, 125.6, 125.5, 125.3, 120.6, 120.5, 113.6, 109.3, 103.2; IR ν_max (ATR)/cm⁻¹ 3282 (NH), 3120 (NH), 2227 (CN), 1679 (C O); LRMS (ESI⁺): 287.1 (M + H, C₁₇H₁₁N₄O, 100%); LRMS (ESI⁻): 285.1 (M − H, C₁₇H₉N₄O, 100%; MP: >250 °C (decomp.).

2-(Pyridin-4-yl)quinazolin-4(3H)-one (49)

Synthesised as per Procedure A from 2-aminobenzamide 8 and 4-pyridinecarbaldehyde to afford the title compound as a brown solid, 110 mg; yield 43%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.77 (s, 1H), 8.80 (dd, J = 4.6, 1.5 Hz, 2H), 8.19 (dd, J = 7.9, 1.1 Hz, 1H), 8.12 (dd, J = 4.6, 1.6 Hz, 2H), 7.92–7.84 (m, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.66–7.53 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.5, 151.0, 150.7, 148.7, 140.4, 135.3, 128.3, 127.9, 126.4, 122.1, 122.0. IR ν_max (ATR)/cm⁻¹ 2965 (CH), 1674 (C O) LRMS (ESI⁺): 224 (M + H, C₁₃H₁₀N₃O, 100%); LRMS (ESI⁻): 222 (M − H, C₁₃H₈N₃O, 100%); MP: > 250 °C.

2-(4-Methoxystyryl)quinazolin-4(3H)-one (50)

Synthesised as per Procedure A from 2-aminobenzamide and (E)-3-(4-methoxyphenyl)acrylaldehyde to afford the title compound as a yellow solid, 165 mg; 37% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 12.25 (s, 1H), 8.10 (dd, J = 7.9, 1.1 Hz, 1H), 7.91 (d, J = 16.1 Hz, 1H), 7.81–7.77 (m, 1H), 7.66–7.60 (m 3H), 7.47–7.43 (m, 1H), 7.02 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 16.1 Hz, 1H), 3.81 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 161.8, 160.6, 151.7, 149.2, 138.0, 134.5, 129.3 (2C), 127.6, 127.0, 125.9, 125.8, 121.0, 118.5, 114.6 (2C), 55.3; IR ν_max (ATR)/cm⁻¹ 3100 (NH), 3049, 1668 (C O); LRMS (ESI⁺) 279.1 (M + H, C₁₇H₁₅N₂O₂,100%); (ESI⁻): 277.1 (M − H, C₁₇H₁₃N₂O₂,100%); MP: >210 °C (decomp.).

2-Styrylquinazolin-4(3H)-one (51)

Synthesised as per Procedure A from 2-aminobenzamide 8 and cinnamaldehyde to afford the title compound as an off white solid, 248 mg; 54% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 12.33 (s, 1H), 8.11 (d, J = 7.4 Hz, 1H), 7.95 (d, J = 16.2 Hz, 1H), 7.81 (t, J = 7.1 Hz, 1H), 7.72–7.63 (m, 3H), 7.50–7.40 (m, 4H), 7.01 (d, J = 16.2 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 161.7, 151.4, 149.0, 138.3, 135.0, 134.5, 129.8, 129.1 (2C), 127.6 (2C), 127.1, 126.2, 125.9, 121.1, Quaternary carbon not observed IR ν_max (ATR)/cm⁻¹ 3105 (NH), 3042 (CH), 1668 (C O); LRMS (ESI⁺): 249.1 (M + H, C₁₆H₁₃N₂O, 100%); LRMS (ESI⁻): 247.2 (M − H, C₁₆H₁₁N₂O, 100%); MP: 243–256 °C.

2-(4-Bromostyryl)quinazolin-4(3H)-one (52)

Synthesised as per Procedure A from 2-aminobenzamide 8 and (E)-3-(4-bromophenyl)acrylaldehyde to afford the title compound as a white solid, 82 mg; yield 62%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.34 (s, 1H), 8.12 (dd, J = 7.9, 1.0 Hz, 1H), 7.92 (d, J = 16.2 Hz, 1H), 7.84–7.80 (m, 1H), 7.67 (dd, J = 8.1, 6.1 Hz, 3H), 7.62 (d, J = 8.6 Hz, 2H), 7.49 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 16.2 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 162.2, 151.7, 149.4, 137.4, 135.0, 134.8, 132.5 (2C), 130.0 (2C), 127.6, 126.8, 126.3, 123.4, 122.4, 121.6; IR ν_max (ATR)/cm⁻¹ 3004, 1665 (C O), 770 (C–Br); LRMS (ESI⁺) 327 (M + H, C₁₆H₁₂⁷⁹BrN₂O, 100%) 329 (M + H, C₁₆H₁₂⁸¹BrN₂O, 85%); (ESI⁻): 325 (M − H, C₁₆H₁₀⁷⁹BrN₂O, 100%), 327 (M − H, C₁₆H₁₀⁸¹BrN₂O, 95%); m.p. 296–310 °C.

2-(Hex-1-en-1-yl)quinazolin-4(3H)-one (53)

Synthesised as per Procedure A from 2-aminobenzamide 8 and (E)-hex-2-enal to afford the title compound as a brown solid, 102 mg; 24% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.14 (s, 1H), 7.94–7.88 (m, 2H), 7.64 (s, 1H), 2.85 (s, 1H), 2.64 (s, 1H), 1.79 (s, 1H), 1.36–1.28 (m, 4H), 0.89 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.8, 135.9, 128.1, 126.8, 120.4, 32.9, 31.2, 29.6, 28.5, 27.5, 22.3, 22.1, 14.4; IR ν_max (ATR)/cm⁻¹ 2908 (NH), 1708 (C O); LRMS (ESI⁺): 229.1 (M + H, C₁₄H₁₇N₂O, 100%); LRMS (ESI⁻): 227.1 (M − H, C₁₄H₁₅N₂O, 100%); MP: 147–176 °C.

2-Phenylquinazolin-4(1H)-one (54)

Synthesised as per Procedure B from 15 (159 mg, 0.71 mmol) to afford the title compound as a white solid, 116 mg; 81% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.34 (dd, J = 7.9, 0.9 Hz, 1H), 8.27 (m, 2H), 7.87–7.77 (m, 2H), 7.64–7.55 (m, 3H), 7.51 (dt, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 164.0, 151.9, 149.7, 135.0, 133.0, 131.8, 129.2 (2C), 128.2, 127.6 (2C), 126.9, 126.5, 121.0; IR ν_max (ATR)/cm⁻¹ 3060 (N–H stretch), 1661 (C O), 766.5 (C C); LRMS (ESI⁺): 223.2 (M + H, C₁₄H₁₁N₂O, 100%); (ESI⁻): 221.1 (M − H, C₁₄H₉N₂O, 100%); MP: >224 °C (decomp.).

2-(2-Bromophenyl)quinazolin-4(1H)-one (55)

Synthesised as per Procedure B from 18 (159 mg, 0.71 mmol) to afford the title compound as a white solid, 93 mg; 65% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.27 (s, 1H), 8.17 (dd, J = 8.0, 1.1 Hz, 1H), 7.89–7.82 (m, 1H), 7.76 (dd, J = 7.9, 1.1 Hz, 1H), 7.70 (dd, J = 8.1, 0.5 Hz, 1H), 7.65–7.45 (m, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.0, 153.9, 148.9, 136.3, 135.1, 133.1, 132.2, 131.2, 128.2, 127.9, 127.6, 126.3, 121.6, 121.4; IR ν_max (ATR)/cm⁻¹ 3282 (NH), 3159 (NNH), 1666 (C O); LRMS (ESI⁺) m/z: 301 (M + H, C₁₄H₁₀⁷⁹BrN₂O, 100%), 303 (M + H, C₁₄H₁₀⁸¹BrN₂O, 95%); (ESI⁻) m/z: 199 (M − H, C₁₄H₈⁷⁹BrN₂O, 100%), 301 (M − H, C₁₄H₈⁸¹BrN₂O, 95%); MP: >166 °C (decomp.).

2-(4-Bromophenyl)quinazolin-4(1H)-one (56)

Synthesised as per Procedure B from 20 to afford the title compound as a white crystalline solid, 147 mg; yield 84%.

¹H NMR (400 MHz, DMSO-d₆) δ 8.19–8.10 (m, 3H), 7.85–7.83 (m, 1H), 7.78–7.74 (m, 3H), 7.58–7.50 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.1, 151.5, 148.6, 134.7, 131.9, 131.6 (2C), 129.8 (2C), 127.5, 126.9, 125.8, 125.3, 121.0. IR ν_max (ATR)/cm⁻¹ 2928 (CH), 1670 (C O); LRMS (ESI⁺): 301 (M + H, C₁₄H₉⁷⁹BrN₂O, 100%), 303.1 (M + H, C₁₄H₉⁸¹BrN₂O, 100%); (ESI⁻): 299 (M − H, C₁₄H₈⁷⁹BrN₂O, 95%), 301 (M − H, C₁₄H₈⁸¹BrN₂O, 95%); MP: >300 °C.

2-(3-Hydroxyphenyl)quinazolin-4(3H)-one (57)

Synthesised as per Procedure B from 21 to afford the title compound as a white crystalline solid, 225 mg; yield 71%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.43 (s, 1H), 9.79 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.84 (t, J = 7.3 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.60 (m, 2H), 7.52 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 8.1 Hz, 1H), 6.99 (d, J = 7.3 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.0, 135.1, 135.0, 130.1, 128.0, 127.0, 126.3, 121.4, 118.0, 118.8, 115.0. IR ν_max (ATR)/cm⁻¹ 3228 (OH) 1657 (C O); LRMS (ESI⁻): 237.1 (M − H, C₁₄H₁₀N₂O₂, 100%); MP: >300 °C.

2-Methylquinazolin-4-(3H)-one (59)

To a solution of 2-aminobenzamide 8 (6.0384 g, 0.044 mol, 1.1 eq.) in 20 mL THF were added SbCl₃ (0.6122 g mg, 5 mol%) and acetaldehyde 58 (2.25 mL, 0.040 mol, 1 eq.) and the resultant mixture heated at reflux for 24 h. The resultant slurry was let cool, collected by vacuum filtration, and washed with THF (2 × 5 mL) to afford the title compound as a yellow solid (2.072 g, 33%).

¹H NMR (400 MHz, DMSO-d₆) δ_H 8.16 (dd, J = 7.9, 1.0 Hz, 1H), 7.99–7.93 (m, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 2.60 (s, 3H).

Methyl-3-(2-(4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzoate (60)

Synthesised as per Procedure C from 2-methylquinazolin-4(3H)-one 59 (151.7 mg, 1 eq., 0.9 mol), methyl 3-formylbenzoate (222.3 mg, 1.5 eq., 1.5 mol) and 1 : 1 toluene : acetic acid (3 mL) to afford the title compound as a yellow solid, 88 mg; yield 31%.

¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 8.14–8.07 (m, 1H), 7.98 (m, 2H), 7.85–7.78 (m, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.62 (t, J = 8.6 Hz, 2H), 7.53–7.46 (m, 2H), 7.11 (d, J = 16.2 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.1, 156.4, 142.4, 140.7, 140.1, 139.9, 137.5, 135.6, 135.3, 134.9, 133.0, 132.2, 131.8, 131.7, 131.2, 127.5, 126.3, 57.6. IR ν_max (ATR)/cm⁻¹ 3538 (NH), 3057 (CH), 1679 (C O); LRMS (ESI⁺): 307.1 (M + H, C₁₈H₁₅N₂O₃, 100%); LRMS (ESI⁻): 305.2 (M − H, C₁₈H₁₃N₂O₃, 100%); MP: > 183 °C (dec.)

4-(Methylstyryl)quinazolin-4(3H)-one (61)

Synthesised as per Procedure C from 2-methylquinazolin-4(3H)-one 59 (105 mg, 0.65 mmol, 1 eq.) in DMF (3 mL), 4-tolylbenzaldehyde (0.08 mL, 0.68 mmol, 1.5 eq.) to afford the title compound as a white solid, 50 mg; yield 20%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.29 (s, 1H), 8.11 (dd, J = 7.9, 1.3 Hz, 1H), 7.92 (d, J = 16.2 Hz, 1H), 7.85–7.78 (m, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.51–7.44 (m, 1H), 7.28 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 16.2 Hz, 1H), 2.35 (s, 3H). ¹³C (100 MHz, DMSO-d₆) δ 140.2, 138.8, 135.0, 132.7, 130.2, 128.1, 127.5, 126.6, 126.3, 121.5, 120.4, 21.5; IR ν_max (ATR)/cm⁻¹ 3049 (CH), 1674 (C O); LRMS (ESI⁺): 263.1 (M + H, C₁₇H₁₅N₂O, 100%); LRMS (ESI⁻): 261.1 (M − H, C₁₇H₁₃N₂O, 100%). MP: >250 °C.

2-(3-Hydroxystyryl)quinazolin-4(3H)-one (62)

Synthesised as per Procedure C from 2-methylquinazolin-4(3H)-one 59 with 3-hydroxybenzaldehyde to afford the title compound as a brown solid, 67 mg; yield 28%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.32 (s, 1H), 9.67 (s, 1H), 8.11 (dd, J = 7.9, 1.0 Hz, 1H), 7.87 (d, J = 16.1 Hz, 1H), 7.84–7.78 (m, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 7.1 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 7.7 Hz, 1H), 7.03 (s, 1H), 6.92 (d, J = 16.1 Hz, 1H), 6.83 (dd, J = 8.0, 1.8 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.2, 158.3, 151.9, 149.5, 139.0, 136.7, 135.0, 130.6, 127.6, 126.7, 126.3, 121.6, 121.2, 119.3, 117.5, 114.2. LRMS (ESI⁺): 265.1 (M + H, C₁₆H₁₃N₂O₂, 100%); LRMS (ESI⁻): 263.1 (M − H, C₁₆H₁₁N₂O₂, 100%). IR ν_max (ATR)/cm⁻¹ 3053 (OH), 1680 (C O); MP: > 250 °C.

2-(4-Hydroxystyryl)quinazolin-4(3H)-one (63)

Synthesised as per Procedure C from 2-methylquinazolin-4(3H)-one 59 and 4-hydroxybenzaldehyde to afford the title compound as white solid, 93 mg; yield 43%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.22 (s, 1H), 9.99 (s, 1H), 8.09 (dd, J = 7.9, 1.2 Hz, 1H), 7.87 (d, J = 16.1 Hz, 1H), 7.83–7.73 (m, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.45 (ddd, J = 8.1, 7.2, 1.1 Hz, 1H), 6.85 (dd, J = 9.1, 2.2 Hz, 2H), 6.78 (d, J = 16.1 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.3, 159.7, 152.4, 149.7, 139.0, 134.9, 130, 127.4, 126.5, 126.3, 121.3, 117.8, 116.4; LRMS (ESI⁺): 265.1 (M + H, C₁₆H₁₃N₂O₂, 100%); LRMS (ESI⁻): 263.1 (M − H, C₁₆H₁₁N₂O₂, 100%). IR ν_max (ATR)/cm⁻¹ 3064 (OH), 1674 (C O); MP: > 250 °C.

2-(2-Methoxystyryl)quinazolin-4(3H)-one (64)

Synthesised as per Procedure C from 2-methylquinazolin-4(3H)-one 59 with 2-methoxybenzaldehyde to afford a white solid, 89 mg; yield 34%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.35 (s, 1H), 8.17 (d, J = 16.2 Hz, 1H), 8.11 (dd, J = 7.9, 1.2 Hz, 1H), 7.85–7.75 (m, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.62 (dd, J = 7.7, 1.3 Hz, 1H), 7.50–7.44 (m, 1H), 7.44–7.37 (m, 1H), 7.16–7.09 (m, 2H), 7.07–7.01 (m, 2H), 3.92 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.2, 158.2, 152.3, 149.6, 134.9, 134.1, 131.7, 128.7, 127.6, 126.5, 126.3, 123.9, 121.9, 121.5, 121.3, 112.3, 56.1; LRMS (ESI⁺): 279.1 (M + H, C₁₇H₁₅N₂O₂, 100%); LRMS (ESI⁻): 277.1 (M − H, C₁₇H₁₃N₂O₂, 100%). IR ν_max (ATR)/cm⁻¹ 3087 (NH), 1677 (C O); MP: decomp > 225 °C.

2-(3-Methoxystyryl)quinazolin-4(3H)-one (65)

Synthesised as per Procedure C from 2-methylquinazolin-4(3H)-one 59 with 3-methoxybenzaldehyde to afford a white solid, 54 mg; yield 21%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.30 (s, 1H), 8.12 (d, J = 7.1 Hz, 1H), 7.92 (d, J = 16.2 Hz, 1H), 7.85–7.76 (m, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.24 (m, 2H), 7.01 (m, 2H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.0, 160.1, 152.1, 149.4, 138.6, 136.9, 134.9, 130.6, 127.5, 126.6, 126.3, 122.1, 121.6, 120.5, 116.1, 113.1, 55.6. LRMS (ESI⁺): 279.1 (M + H, C₁₇H₁₅N₂O₂, 100%); LRMS (ESI⁻): 277.1 (M − H, C₁₇H₁₃N₂O₂, 100%); IR ν_max (ATR)/cm⁻¹ 3022 (CH), 1674 (C O); MP: decomp > 250 °C.

Biology

Cell culture and stock solutions

Stock solutions were prepared as follows and stored at −20 °C: drugs were stored as 40 mM solutions in DMSO. All cell lines were cultured in a humidified atmosphere 5% CO₂ at 37 °C. The cancer cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Trace Biosciences, Australia) supplemented with 10% foetal bovine serum, 10 mM sodium bicarbonate, penicillin (100 IU mL⁻¹), streptomycin (100 μg mL⁻¹), and glutamine (4 mM). The non-cancer MCF10A cell line was cultured in DMEM : F12 (1 : 1) cell culture medium, 5% heat inactivated horse serum, supplemented with penicillin (50 IU mL⁻¹), streptomycin (50 μg mL⁻¹), 20 mM Hepes, l-glutamine (2 mM), epidermal growth factor (20 ng mL⁻¹), hydrocortisone (500 ng mL⁻¹), cholera toxin (100 ng mL⁻¹), and insulin (10 μg mL⁻¹).

Cell lines

The cell lines HT29 colon, U87 glioblastoma, MCF-7 breast, H460 lung, Du145 prostate, BE2-C neuroblastoma, MIA pancreas, and the non-tumour-derived MCF10A breast cancer were sourced from the American Type Culture Collection (ATCC). The A431 skin and A2780 ovarian cell lines were purchased from the European Collection of Authenticated Cell Cultures (ECACC). The SJ-G2 glioblastoma was provided by Dr. Mary Danks of St. Jude Children's Research Hospital, Memphis, TN, USA. All cell lines have been authenticated by CellBank Australia, (Children's Medical Research Institute, Westmead, Sydney, NSW, Australia).

In vitro growth inhibition assay

Cells in logarithmic growth were transferred to 96-well plates. Cytotoxicity was determined by plating cells in duplicate in 100 μL medium at a density of 2500–4000 cells per well. On day 0, (24 h after plating) when the cells were in logarithmic growth, 100 μL medium with or without the test agent was added to each well. After 72 h drug exposure growth inhibitory effects were evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay and absorbance was read at 540 nm. Percentage growth inhibition was determined at a fixed drug concentration of 25 μM. A value of 100% is indicative of complete cell growth inhibition. Those analogues showing appreciable percentage growth inhibition underwent further dose response analysis allowing for the calculation of a GI₅₀ value. This value is the drug concentration at which cell growth is 50% inhibited based on the difference between the optical density values on day 0 and those at the end of drug exposure.^46,51

Tubulin polymerization assay

Tubulin polymerization analysis was carried out using the tubulin polymerization assay kit from Cytoskeleton according to the manufacturer's protocol. Briefly, 5 mL of 10× test compound (10 μM) was added to each well of the assay plate and warmed to 37 °C for 1 minute. Tubulin solution, 50 mL, containing tubulin 2 mg ml⁻¹, 80 mM piperazine-N,N′-bis[2-ethanesulfonic acid] sequisodium salt; 2.0 mM magnesium chloride; 0.5 mM ethylene glycol-bis(β-amino-ethyl ether)-N,N,N′,N′-tetra-acetic acid pH 6.9, 5 mM fluorescent reporter, 15% v/v glycerol and 1 mM GTP was added to each assay well. The assay was conducted at 37 °C in a temperature regulated fluorescent plate reader with emission wavelength of 410–460 in kinetic mode and excitation wavelength of 340–360 nm. After shaking the plate for 5 seconds the assay plate was read at 1 minute intervals for 61 cycles. Paclitaxel was the positive microtubule enhancer control and nocodazole was the positive inhibitor control.

Cell cycle analysis

Mia pancreatic cancer cells in logarithmic growth were transferred to 6 well plates at a density of 2 × 10⁵–2.5 × 10⁵ cells per well. On day 0 (24 h after plating), the cells were treated with or without the test agent. The cells were harvested 24 h after drug treatment and washed twice in phosphate buffered saline (PBS), fixed in 70% ethanol and stored overnight at −20 °C. The cell pellet was incubated in 600 μl of PBS containing propidium iodide (40 μg mL⁻¹) and RNase (200 μg mL⁻¹) for at least 30 min at room temperature. The samples (1 × 10⁴ events) were analysed for fluorescence (FL2 detector, filter 575/30 nm band pass) using a BD FACSCanto II (Becton Dickinson). Cell cycle distribution was assessed using BD FACSDIVA software (Version 9.0.1). Experiments were each performed in duplicate and replicated on two separate occasions. Values are the percentage distribution for each phase of the cell cycle. Nocodazole was the positive control for G₂ + M cell cycle arrest.

Molecular docking and molecular dynamics studies

Molecular docking

The structures of all ligands to be docked were constructed in MOE and their conformations energy-minimized using molecular mechanics in conjunction with the AMBER force field. Docking was performed using MOE's default settings, using the triangle matcher method in combination with the London dG scoring function for the initial placement of the ligand, followed by a refinement using induced fit methods and the GBVI/WSA scoring function. Compounds were docked 500 times, and the top 15 poses subjected to energy minimisation after refinement. Analysis and visualization of the docking output, such as identification of hydrogen bonds, steric clashes, hydrophobic interactions, or π–π-interactions were performed in MOE. Protein crystal structures (PDB: 4O2B and 5J2T) were protonated and energy minimised prior to docking. Domain repeats were trimmed to include a single tubulin dimer around each binding site.

Molecular dynamics

The stability of docking-predicted ligand poses was evaluated in molecular dynamics (MD) simulations using MOE under default settings. The system was prepared as described above and the highest scoring pose from the molecular docking study chosen for examination.

Author contributions

NSO: chemical synthesis and molecular modelling studies; JG and JAS: biological screening; AM project conception and supervision; AM, NSO and JAS: manuscript preparation.

Conflicts of interest

The authors declare no conflicts of interest.

Supplementary Material

MD-015-D3MD00600J-s001

MD-015-D3MD00600J-s001.pdf^{(9.2MB, pdf)}

Acknowledgments

N. S. O. is the recipient of a University of Newcastle Postgraduate Research Scholarship (UNPRS). This work is supported by the Australian Research Council (ARC DP180101781), project funds from the University of Newcastle Priority Research Centre for Drug Development, and equipment grants from the Australian Cancer Research Foundation and Ramaciotti Foundation.

^†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00600j

Notes and references

Moffat J. G. Rudolph J. Bailey D. Nat. Rev. Drug Discovery. 2014;13:588–602. doi: 10.1038/nrd4366. [DOI] [PubMed] [Google Scholar]
Harper C. B. Popoff M. R. McCluskey A. Robinson P. J. Meunier F. A. Trends Cell Biol. 2013;23:90–101. doi: 10.1016/j.tcb.2012.10.007. [DOI] [PubMed] [Google Scholar]
Robertson M. J. Deane F. M. Robinson P. J. McCluskey A. Nat. Protoc. 2014;9:851–870. doi: 10.1038/nprot.2014.046. [DOI] [PubMed] [Google Scholar]
Robertson M. J. Deane F. M. Stahlschmidt W. von Kleist L. Haucke V. Robinson P. J. McCluskey A. Nat. Protoc. 2014;9:1592–1606. doi: 10.1038/nprot.2014.106. [DOI] [PubMed] [Google Scholar]
von Kleist L. Stahlschmidt W. Bulut H. Gromova K. Puchkov D. Robertson M. J. MacGregor K. A. Tomilin N. Pechstein A. Chau N. Chircop M. Sakoff J. Peter von Kries J. Saenger W. Kräusslich H.-G. Shupliakov O. Robinson P. J. McCluskey A. Haucke V. Cell. 2011;146:841. doi: 10.1016/j.cell.2011.08.014. [DOI] [PubMed] [Google Scholar]
McCluskey A. Sim A. T. R. Sakoff J. A. J. Med. Chem. 2002;45:1151–1175. doi: 10.1021/jm010066k. [DOI] [PubMed] [Google Scholar]
Hizartzidis L. Gilbert J. Gordon C. P. Sakoff J. A. McCluskey A. ChemMedChem. 2019;14:1152–1161. doi: 10.1002/cmdc.201900180. [DOI] [PubMed] [Google Scholar]
Baker J. R. Sakoff J. A. McCluskey A. Med. Res. Rev. 2020;40:972–1001. doi: 10.1002/med.21645. [DOI] [PubMed] [Google Scholar]
Baker J. R. Pollard B. L. Lin A. J. S. Gilbert J. Paula S. Zhu X. Sakoff J. A. McCluskey A. ChemMedChem. 2021;16:1499–1512. doi: 10.1002/cmdc.202000721. [DOI] [PubMed] [Google Scholar]
Badolato M. Aiello F. Neamati N. RSC Adv. 2018;8:20894–20921. doi: 10.1039/C8RA02827C. [DOI] [PMC free article] [PubMed] [Google Scholar]
Mphahlele M. J. Khoza T. A. Mabeta P. Molecules. 2016;22:55. doi: 10.3390/molecules22010055. [DOI] [PMC free article] [PubMed] [Google Scholar]
Chen Y. Shan W. Lei M. Hu L. Tetrahedron Lett. 2012;53:5923–5925. doi: 10.1016/j.tetlet.2012.08.090. [DOI] [Google Scholar]
Arnst K. E. Banerjee S. Chen H. Deng S. Hwang D. Li W. Miller D. D. Med. Res. Rev. 2019;39:1398–1426. doi: 10.1002/med.21568. [DOI] [PMC free article] [PubMed] [Google Scholar]
Bhattacharyya B. Panda D. Gupta S. Banerjee M. Med. Res. Rev. 2008;28:155–183. doi: 10.1002/med.20097. [DOI] [PubMed] [Google Scholar]
Wilson L. Creswell K. M. Chin D. Biochemistry. 1975;14:5586–5592. doi: 10.1021/bi00697a008. [DOI] [PubMed] [Google Scholar]
Yang J. Wang Y. Wang T. Jiang J. Botting C. H. Liu H. Chen Q. Yang J. Naismith J. H. Zhu X. Chen L. Nat. Commun. 2016;7:12103. doi: 10.1038/ncomms12103. [DOI] [PMC free article] [PubMed] [Google Scholar]
Morris J. D. Takahashi-Ruiz L. Persi L. N. Summers J. C. McCauley E. P. Chan P. Y. W. Amberchan G. Lizama-Chamu I. Coppage D. A. Crews P. Risinger A. L. Johnson T. A. ACS Omega. 2022;7:8824–8832. doi: 10.1021/acsomega.1c07146. [DOI] [PMC free article] [PubMed] [Google Scholar]
Kampan N. C. Madondo M. T. McNally O. M. Quinn M. Plebanski M. BioMed Res. Int. 2015;2015:413076. doi: 10.1155/2015/413076. [DOI] [PMC free article] [PubMed] [Google Scholar]
Lopus M. Oroudjev E. Wilson L. Wilhelm S. Widdison W. Chari R. Jordan M. A. Mol. Cancer Ther. 2010;9:2689–2699. doi: 10.1158/1535-7163.MCT-10-0644. [DOI] [PMC free article] [PubMed] [Google Scholar]
Haider T. Pandey V. Banjare N. Gupta P. N. Soni V. Pharmacol. Rep. 2020;72:1125–1151. doi: 10.1007/s43440-020-00138-7. [DOI] [PubMed] [Google Scholar]
Lin-Rahardja K. Weaver D. T. Scarborough J. A. Scott J. G. Int. J. Mol. Sci. 2023;24:6738. doi: 10.3390/ijms24076738. [DOI] [PMC free article] [PubMed] [Google Scholar]
Gatenby R. A. Brown J. S. Cold Spring Harbor Perspect. Med. 2020;10:a040972. doi: 10.1101/cshperspect.a040972. [DOI] [PMC free article] [PubMed] [Google Scholar]
Kuo S. C. Lee H. Z. Juang J. P. Lin Y. T. Wu T. S. Chang J. J. Lednicer D. Paull K. D. Lin C. M. Hamel E. J. Med. Chem. 1993;36:1146–1156. doi: 10.1021/jm00061a005. [DOI] [PubMed] [Google Scholar]
Hamel E. Lin C. M. Plowman J. Wang H.-K. Lee K.-H. Paull K. D. Biochem. Pharmacol. 1996;51:53–59. doi: 10.1016/0006-2952(95)02156-6. [DOI] [PubMed] [Google Scholar]
Xia Y. Yang Z. Y. Hour M. J. Kuo S. C. Xia P. Bastow K. F. Nakanishi Y. Namrpoothiri P. Hackl T. Hamel E. Lee H. K. Bioorg. Med. Chem. Lett. 2001;11:1193–1196. doi: 10.1016/S0960-894X(01)00190-1. [DOI] [PubMed] [Google Scholar]
Chen K. Kuo S.-C. Hsieh M.-C. Mauger A. Lin C. M. Hamel E. Lee K.-H. J. Med. Chem. 1997;40:2266–2275. doi: 10.1021/jm960858s. [DOI] [PubMed] [Google Scholar]
Chen K. Kuo S.-C. Hsieh M.-C. Mauger A. Lin C. M. Hamel E. Lee K.-H. J. Med. Chem. 1997;40:3049–3056. doi: 10.1021/jm970146h. [DOI] [PubMed] [Google Scholar]
Li L. Wang H.-K. Kuo S.-C. Wu T.-S. Mauger A. Lin C. M. Hamel E. Lee K.-H. J. Med. Chem. 1994;37:3400–3407. doi: 10.1021/jm00046a025. [DOI] [PubMed] [Google Scholar]
Carta D. Ferlin M. Curr. Top. Med. Chem. 2014;14:2322–2345. doi: 10.2174/1568026614666141127120421. [DOI] [PubMed] [Google Scholar]
Dohle W. Jourdan F. L. Menchon G. Prota A. E. Foster P. A. Mannion P. Hamel E. Thomas M. P. Kasprzyk P. G. Ferrandis E. Steinmetz M. O. Leese M. P. Potter B. V. L. J. Med. Chem. 2018;61:1031–1044. doi: 10.1021/acs.jmedchem.7b01474. [DOI] [PubMed] [Google Scholar]
Hour M.-J. Huang L.-J. Kuo S.-C. Xia Y. Bastow K. Nakanishi Y. Hamel E. Lee K.-H. J. Med. Chem. 2000;43:4479–4487. doi: 10.1021/jm000151c. [DOI] [PubMed] [Google Scholar]
Ram V. J. Farhanullah Tripathi B. K. Srivastava A. K. Bioorg. Med. Chem. 2003;11:2439–2444. doi: 10.1016/S0968-0896(03)00142-1. [DOI] [PubMed] [Google Scholar]
Cohen E. Klarberg B. Vaughan J. R. J. Am. Chem. Soc. 1959;81:5508–5509. doi: 10.1021/ja01529a062. [DOI] [Google Scholar]
Rudolph J. Esler W. P. O'Connor S. Coish P. D. G. Wickens P. L. Brands M. Bierer D. E. Bloomquist B. T. Bondar G. Chen L. Chuang C.-Y. Claus T. H. Fathi Z. Fu W. Khire U. R. Kristie J. A. Liu X.-G. Lowe D. B. McClure A. C. Michels M. Ortiz A. A. Ramsden P. D. Schoenleber R. W. Shelekhin T. E. Vakalopoulos A. Tang W. Wang L. Yi L. Gardell S. J. Livingston J. N. Sweet L. J. Bullock W. H. J. Med. Chem. 2007;50:5202–5216. doi: 10.1021/jm070071+. [DOI] [PubMed] [Google Scholar]
Okumura K. Oine T. Yamada Y. Hayashi G. Nakama M. J. Med. Chem. 1968;11:348–352. doi: 10.1021/jm00308a036. [DOI] [PubMed] [Google Scholar]
Chinigo G. M. Paige M. Grindrod S. Hamel E. Dakshanamurthy S. Chruszcz M. Minor W. Brown M. L. J. Med. Chem. 2008;51:4620–4631. doi: 10.1021/jm800271c. [DOI] [PMC free article] [PubMed] [Google Scholar]
Labade V. B. Shinde P. V. Shingare M. S. Tetrahedron Lett. 2013;54:5778–5780. doi: 10.1016/j.tetlet.2013.08.037. [DOI] [Google Scholar]
Wu J. Du X. Ma J. Zhang Y. Shi Q. Luo L. Song B. Yang S. Hu D. Green Chem. 2014;16:3210–3217. doi: 10.1039/C3GC42400F. [DOI] [Google Scholar]
Davoodnia A. Allameh S. Fakhari A. R. Tavakoli-Hoseini N. Chin. Chem. Lett. 2010;21:550–553. doi: 10.1016/j.cclet.2010.01.032. [DOI] [Google Scholar]
Ghorbani-Choghamarani A. Norouzi M. J. Mol. Catal. A: Chem. 2014;395:172–179. doi: 10.1016/j.molcata.2014.08.013. [DOI] [Google Scholar]
Murthy P. V. Rambabu D. Krishna G. R. Reddy C. M. Prasad K. R. S. Rao M. V. B. Pal M. Tetrahedron Lett. 2012;53:863–867. doi: 10.1016/j.tetlet.2011.12.023. [DOI] [Google Scholar]
O'Brien N. S. McCluskey A. Aust. J. Chem. 2020;73:1176. doi: 10.1071/CH20101. [DOI] [Google Scholar]
Zhang J. Ren D. Ma Y. Wang W. Wu H. Tetrahedron. 2014;70:5274–5282. doi: 10.1016/j.tet.2014.05.059. [DOI] [Google Scholar]
Santra S. Rahman M. Roy A. Majee A. Hajra A. Catal. Commun. 2014;49:52–57. doi: 10.1016/j.catcom.2014.01.032. [DOI] [Google Scholar]
Chen J. Wu D. He F. Liu M. Wu H. Ding J. Su W. Tetrahedron Lett. 2008;49:3814–3818. doi: 10.1016/j.tetlet.2008.03.127. [DOI] [Google Scholar]
Baker J. R. Russell C. C. Gilbert J. Sakoff J. A. McCluskey A. ChemMedChem. 2020;15:490–505. doi: 10.1002/cmdc.201900643. [DOI] [PubMed] [Google Scholar]
Rai S. S. Wolff J. J. Biol. Chem. 1998;273:31131–31137. doi: 10.1074/jbc.273.47.31131. [DOI] [PubMed] [Google Scholar]
Chengyong W. Jinghong X. Yanyan W. Qing-Jie X. Lingling M. Yuyan L. Hai C. Qian L. Quan Z. Bo S. Yuxi W. FEBS Lett. 2021;595:195–205. doi: 10.1002/1873-3468.14003. [DOI] [PubMed] [Google Scholar]
Hasanpourghadi M. Karthikeyan C. Pandurangan A. K. Looi C. Y. Trivedi P. Kobayashi K. Tanaka K. Wong W. F. Mustafa M. R. J. Exp. Clin. Cancer Res. 2016;35:58. doi: 10.1186/s13046-016-0332-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
Nguyen T. L. McGrath C. Hermone A. R. Burnett J. C. Zaharevitz D. W. Day B. W. Wipf P. Hamel E. Gussio R. J. Med. Chem. 2005;48:6107–6116. doi: 10.1021/jm050502t. [DOI] [PubMed] [Google Scholar]
Ghods A. Gilbert J. Baker J. R. Russell C. C. Sakoff J. A. McCluskey A. R. Soc. Open Sci. 2018;5:171189. doi: 10.1098/rsos.171189. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MD-015-D3MD00600J-s001

MD-015-D3MD00600J-s001.pdf^{(9.2MB, pdf)}

[cit1] Moffat J. G. Rudolph J. Bailey D. Nat. Rev. Drug Discovery. 2014;13:588–602. doi: 10.1038/nrd4366. [DOI] [PubMed] [Google Scholar]

[cit2] Harper C. B. Popoff M. R. McCluskey A. Robinson P. J. Meunier F. A. Trends Cell Biol. 2013;23:90–101. doi: 10.1016/j.tcb.2012.10.007. [DOI] [PubMed] [Google Scholar]

[cit3] Robertson M. J. Deane F. M. Robinson P. J. McCluskey A. Nat. Protoc. 2014;9:851–870. doi: 10.1038/nprot.2014.046. [DOI] [PubMed] [Google Scholar]

[cit4] Robertson M. J. Deane F. M. Stahlschmidt W. von Kleist L. Haucke V. Robinson P. J. McCluskey A. Nat. Protoc. 2014;9:1592–1606. doi: 10.1038/nprot.2014.106. [DOI] [PubMed] [Google Scholar]

[cit5] von Kleist L. Stahlschmidt W. Bulut H. Gromova K. Puchkov D. Robertson M. J. MacGregor K. A. Tomilin N. Pechstein A. Chau N. Chircop M. Sakoff J. Peter von Kries J. Saenger W. Kräusslich H.-G. Shupliakov O. Robinson P. J. McCluskey A. Haucke V. Cell. 2011;146:841. doi: 10.1016/j.cell.2011.08.014. [DOI] [PubMed] [Google Scholar]

[cit6] McCluskey A. Sim A. T. R. Sakoff J. A. J. Med. Chem. 2002;45:1151–1175. doi: 10.1021/jm010066k. [DOI] [PubMed] [Google Scholar]

[cit7] Hizartzidis L. Gilbert J. Gordon C. P. Sakoff J. A. McCluskey A. ChemMedChem. 2019;14:1152–1161. doi: 10.1002/cmdc.201900180. [DOI] [PubMed] [Google Scholar]

[cit8] Baker J. R. Sakoff J. A. McCluskey A. Med. Res. Rev. 2020;40:972–1001. doi: 10.1002/med.21645. [DOI] [PubMed] [Google Scholar]

[cit9] Baker J. R. Pollard B. L. Lin A. J. S. Gilbert J. Paula S. Zhu X. Sakoff J. A. McCluskey A. ChemMedChem. 2021;16:1499–1512. doi: 10.1002/cmdc.202000721. [DOI] [PubMed] [Google Scholar]

[cit10] Badolato M. Aiello F. Neamati N. RSC Adv. 2018;8:20894–20921. doi: 10.1039/C8RA02827C. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit11] Mphahlele M. J. Khoza T. A. Mabeta P. Molecules. 2016;22:55. doi: 10.3390/molecules22010055. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit12] Chen Y. Shan W. Lei M. Hu L. Tetrahedron Lett. 2012;53:5923–5925. doi: 10.1016/j.tetlet.2012.08.090. [DOI] [Google Scholar]

[cit13] Arnst K. E. Banerjee S. Chen H. Deng S. Hwang D. Li W. Miller D. D. Med. Res. Rev. 2019;39:1398–1426. doi: 10.1002/med.21568. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit14] Bhattacharyya B. Panda D. Gupta S. Banerjee M. Med. Res. Rev. 2008;28:155–183. doi: 10.1002/med.20097. [DOI] [PubMed] [Google Scholar]

[cit15] Wilson L. Creswell K. M. Chin D. Biochemistry. 1975;14:5586–5592. doi: 10.1021/bi00697a008. [DOI] [PubMed] [Google Scholar]

[cit16] Yang J. Wang Y. Wang T. Jiang J. Botting C. H. Liu H. Chen Q. Yang J. Naismith J. H. Zhu X. Chen L. Nat. Commun. 2016;7:12103. doi: 10.1038/ncomms12103. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit17] Morris J. D. Takahashi-Ruiz L. Persi L. N. Summers J. C. McCauley E. P. Chan P. Y. W. Amberchan G. Lizama-Chamu I. Coppage D. A. Crews P. Risinger A. L. Johnson T. A. ACS Omega. 2022;7:8824–8832. doi: 10.1021/acsomega.1c07146. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit18] Kampan N. C. Madondo M. T. McNally O. M. Quinn M. Plebanski M. BioMed Res. Int. 2015;2015:413076. doi: 10.1155/2015/413076. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit19] Lopus M. Oroudjev E. Wilson L. Wilhelm S. Widdison W. Chari R. Jordan M. A. Mol. Cancer Ther. 2010;9:2689–2699. doi: 10.1158/1535-7163.MCT-10-0644. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit20] Haider T. Pandey V. Banjare N. Gupta P. N. Soni V. Pharmacol. Rep. 2020;72:1125–1151. doi: 10.1007/s43440-020-00138-7. [DOI] [PubMed] [Google Scholar]

[cit21] Lin-Rahardja K. Weaver D. T. Scarborough J. A. Scott J. G. Int. J. Mol. Sci. 2023;24:6738. doi: 10.3390/ijms24076738. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit22] Gatenby R. A. Brown J. S. Cold Spring Harbor Perspect. Med. 2020;10:a040972. doi: 10.1101/cshperspect.a040972. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit23] Kuo S. C. Lee H. Z. Juang J. P. Lin Y. T. Wu T. S. Chang J. J. Lednicer D. Paull K. D. Lin C. M. Hamel E. J. Med. Chem. 1993;36:1146–1156. doi: 10.1021/jm00061a005. [DOI] [PubMed] [Google Scholar]

[cit24] Hamel E. Lin C. M. Plowman J. Wang H.-K. Lee K.-H. Paull K. D. Biochem. Pharmacol. 1996;51:53–59. doi: 10.1016/0006-2952(95)02156-6. [DOI] [PubMed] [Google Scholar]

[cit25] Xia Y. Yang Z. Y. Hour M. J. Kuo S. C. Xia P. Bastow K. F. Nakanishi Y. Namrpoothiri P. Hackl T. Hamel E. Lee H. K. Bioorg. Med. Chem. Lett. 2001;11:1193–1196. doi: 10.1016/S0960-894X(01)00190-1. [DOI] [PubMed] [Google Scholar]

[cit26] Chen K. Kuo S.-C. Hsieh M.-C. Mauger A. Lin C. M. Hamel E. Lee K.-H. J. Med. Chem. 1997;40:2266–2275. doi: 10.1021/jm960858s. [DOI] [PubMed] [Google Scholar]

[cit27] Chen K. Kuo S.-C. Hsieh M.-C. Mauger A. Lin C. M. Hamel E. Lee K.-H. J. Med. Chem. 1997;40:3049–3056. doi: 10.1021/jm970146h. [DOI] [PubMed] [Google Scholar]

[cit28] Li L. Wang H.-K. Kuo S.-C. Wu T.-S. Mauger A. Lin C. M. Hamel E. Lee K.-H. J. Med. Chem. 1994;37:3400–3407. doi: 10.1021/jm00046a025. [DOI] [PubMed] [Google Scholar]

[cit29] Carta D. Ferlin M. Curr. Top. Med. Chem. 2014;14:2322–2345. doi: 10.2174/1568026614666141127120421. [DOI] [PubMed] [Google Scholar]

[cit30] Dohle W. Jourdan F. L. Menchon G. Prota A. E. Foster P. A. Mannion P. Hamel E. Thomas M. P. Kasprzyk P. G. Ferrandis E. Steinmetz M. O. Leese M. P. Potter B. V. L. J. Med. Chem. 2018;61:1031–1044. doi: 10.1021/acs.jmedchem.7b01474. [DOI] [PubMed] [Google Scholar]

[cit31] Hour M.-J. Huang L.-J. Kuo S.-C. Xia Y. Bastow K. Nakanishi Y. Hamel E. Lee K.-H. J. Med. Chem. 2000;43:4479–4487. doi: 10.1021/jm000151c. [DOI] [PubMed] [Google Scholar]

[cit32] Ram V. J. Farhanullah Tripathi B. K. Srivastava A. K. Bioorg. Med. Chem. 2003;11:2439–2444. doi: 10.1016/S0968-0896(03)00142-1. [DOI] [PubMed] [Google Scholar]

[cit33] Cohen E. Klarberg B. Vaughan J. R. J. Am. Chem. Soc. 1959;81:5508–5509. doi: 10.1021/ja01529a062. [DOI] [Google Scholar]

[cit34] Rudolph J. Esler W. P. O'Connor S. Coish P. D. G. Wickens P. L. Brands M. Bierer D. E. Bloomquist B. T. Bondar G. Chen L. Chuang C.-Y. Claus T. H. Fathi Z. Fu W. Khire U. R. Kristie J. A. Liu X.-G. Lowe D. B. McClure A. C. Michels M. Ortiz A. A. Ramsden P. D. Schoenleber R. W. Shelekhin T. E. Vakalopoulos A. Tang W. Wang L. Yi L. Gardell S. J. Livingston J. N. Sweet L. J. Bullock W. H. J. Med. Chem. 2007;50:5202–5216. doi: 10.1021/jm070071+. [DOI] [PubMed] [Google Scholar]

[cit35] Okumura K. Oine T. Yamada Y. Hayashi G. Nakama M. J. Med. Chem. 1968;11:348–352. doi: 10.1021/jm00308a036. [DOI] [PubMed] [Google Scholar]

[cit36] Chinigo G. M. Paige M. Grindrod S. Hamel E. Dakshanamurthy S. Chruszcz M. Minor W. Brown M. L. J. Med. Chem. 2008;51:4620–4631. doi: 10.1021/jm800271c. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit37] Labade V. B. Shinde P. V. Shingare M. S. Tetrahedron Lett. 2013;54:5778–5780. doi: 10.1016/j.tetlet.2013.08.037. [DOI] [Google Scholar]

[cit38] Wu J. Du X. Ma J. Zhang Y. Shi Q. Luo L. Song B. Yang S. Hu D. Green Chem. 2014;16:3210–3217. doi: 10.1039/C3GC42400F. [DOI] [Google Scholar]

[cit39] Davoodnia A. Allameh S. Fakhari A. R. Tavakoli-Hoseini N. Chin. Chem. Lett. 2010;21:550–553. doi: 10.1016/j.cclet.2010.01.032. [DOI] [Google Scholar]

[cit40] Ghorbani-Choghamarani A. Norouzi M. J. Mol. Catal. A: Chem. 2014;395:172–179. doi: 10.1016/j.molcata.2014.08.013. [DOI] [Google Scholar]

[cit41] Murthy P. V. Rambabu D. Krishna G. R. Reddy C. M. Prasad K. R. S. Rao M. V. B. Pal M. Tetrahedron Lett. 2012;53:863–867. doi: 10.1016/j.tetlet.2011.12.023. [DOI] [Google Scholar]

[cit42] O'Brien N. S. McCluskey A. Aust. J. Chem. 2020;73:1176. doi: 10.1071/CH20101. [DOI] [Google Scholar]

[cit43] Zhang J. Ren D. Ma Y. Wang W. Wu H. Tetrahedron. 2014;70:5274–5282. doi: 10.1016/j.tet.2014.05.059. [DOI] [Google Scholar]

[cit44] Santra S. Rahman M. Roy A. Majee A. Hajra A. Catal. Commun. 2014;49:52–57. doi: 10.1016/j.catcom.2014.01.032. [DOI] [Google Scholar]

[cit45] Chen J. Wu D. He F. Liu M. Wu H. Ding J. Su W. Tetrahedron Lett. 2008;49:3814–3818. doi: 10.1016/j.tetlet.2008.03.127. [DOI] [Google Scholar]

[cit46] Baker J. R. Russell C. C. Gilbert J. Sakoff J. A. McCluskey A. ChemMedChem. 2020;15:490–505. doi: 10.1002/cmdc.201900643. [DOI] [PubMed] [Google Scholar]

[cit47] Rai S. S. Wolff J. J. Biol. Chem. 1998;273:31131–31137. doi: 10.1074/jbc.273.47.31131. [DOI] [PubMed] [Google Scholar]

[cit48] Chengyong W. Jinghong X. Yanyan W. Qing-Jie X. Lingling M. Yuyan L. Hai C. Qian L. Quan Z. Bo S. Yuxi W. FEBS Lett. 2021;595:195–205. doi: 10.1002/1873-3468.14003. [DOI] [PubMed] [Google Scholar]

[cit49] Hasanpourghadi M. Karthikeyan C. Pandurangan A. K. Looi C. Y. Trivedi P. Kobayashi K. Tanaka K. Wong W. F. Mustafa M. R. J. Exp. Clin. Cancer Res. 2016;35:58. doi: 10.1186/s13046-016-0332-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit50] Nguyen T. L. McGrath C. Hermone A. R. Burnett J. C. Zaharevitz D. W. Day B. W. Wipf P. Hamel E. Gussio R. J. Med. Chem. 2005;48:6107–6116. doi: 10.1021/jm050502t. [DOI] [PubMed] [Google Scholar]

[cit51] Ghods A. Gilbert J. Baker J. R. Russell C. C. Sakoff J. A. McCluskey A. R. Soc. Open Sci. 2018;5:171189. doi: 10.1098/rsos.171189. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

2,3-Dihydroquinazolin-4(1H)-ones and quinazolin-4(3H)-ones as broad-spectrum cytotoxic agents and their impact on tubulin polymerisation†

Nicholas S O'Brien

Jayne Gilbert

Adam McCluskey

Jennette A Sakoff

Abstract

Introduction

Results and discussion

Chemistry

Scheme 1. Reagents and conditions: (i) SnCl2 (1%), EtOH, 120 °C, μwave.42.

Fig. 2. R-substituents associated with 2,3-dihydroquinazolines 10–46 subjected to cytotoxicity screening (Table 1).

Scheme 2. Reagents and conditions: (i) SnCl2 (1%), EtOH, 120 °C, μwave; and (ii) KMnO4, acetone, Δ.

Scheme 3. Reagents and conditions: (i) SbCl3, THF Δ; (ii) R′CHO, PhCH3 : AcOH (1 : 1), 180 °C μwave.

Fig. 3. R-substituents produced as described in Schemes 2 and 3.

Biology

Growth inhibition of a panel of cancer cell lines and a normal breast cell line by selected library 2 analogues (quinazolin-4(3H)-one analogues 47, 48, 50–52, 56, and 60–65). Growth inhibition, GI50 values relative (μM) to untreated control (MTT assay, 72 h, n = 3).

Mechanism of action

Modelling studies

Fig. 4. A representative section of polymeric tubulin comprised alternating α-(blue) and β-(green) subunits. The well-hidden colchicine and vinblastine binding sites are given approximate locations on the surface of the polymer to enable simplistic discussion.

Colchicine-site molecular modelling

Fig. 6. The lowest energy poses of 39 (A), 50 (B), 65 (C) and 64 (D) within the tubulin dimer. Selected residues are annotated to show the positioning of the docked ligands (orange) with respect to colchicine (76) (blue). Image generated from MOE.

Vinblastine-site molecular modelling

Fig. 7. The lowest energy poses of 39 (A), 50 (B), 65 (C) and 64 (D) within the tubulin dimer. Selected residues are annotated to show the positioning of the docked ligands (orange) with respect to vinblastine (77) (blue). Image generated from MOE.

Molecular dynamics of 2-(2-methoxystyryl)quinazolin-4(3H)-one 64

Fig. 8. Molecular dynamics of 64 within the colchicine binding site. Relevant amino acids are labelled for comparison. Step time of 1 ps with chosen t = 0 (A), 1000 (B) and 2000 (C) ps poses are shown. Images generated from MOE.

Effect of selected analogues (15, 39, 43, 45, 47, 50, 51, 54, 64 and 65) on tubulin

Fig. 9. Control compounds used in the tubulin polymerisation assay, nocodazole (68) and paclitaxel (69).

Fig. 12. Cell cycle analysis. G2 + M cell cycle arrest was induced in MIA cells by 39, 64 and the tubulin inhibitor nocodazole 68 after 24 h exposure. A single representative set of histograms is shown.

Conclusions

Experimental

Chemistry

General methods

Synthesis of 2,3-dihydroquinazolin-4(1H)-ones 10–46

2-Butyl-2,3-dihydroquinazolin-4(1H)-one (10)

2-Octyl-2,3-dihydroquinazolin-4(1H)-one (11)

2-Cyclopentyl-2,3-dihydroquinazolin-4(1H)-one (12)

2-Cyclohexyl-2,3-dihydroquinazolin-4(1H)-one (13)

2-(Cyclohex-3-en-1-yl)-2,3-dihydroquinazolin-4(1H)-one (14)

2-Phenyl-2,3-dihydroquinazolin-4(1H)-one (15)

2-(p-Tolyl)-2,3-dihydroquinazolin-4(1H)-one (16)

2-(4-(tert-Butyl)phenyl)-2,3-dihydroquinazolin-4(1H)-one (17)

2-(2-Bromophenyl)-2,3-dihydroquinazolin-4(1H)-one (18)

2-(3-Bromophenyl)-2,3-dihydroquinazolin-4(1H)-one (19)

2-(4-Bromophenyl)-2,3-dihydroquinazolin-4(1H)-one (20)

2-(3-Hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (21)

2-(4-Hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (22)

2-(2-Carboxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (23)

2-(3-Carboxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (24)

2-(4-Carboxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (25)

2-(2-Nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (26)

2-(3-Nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (27)

2-(4-Nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (28)

2-(3-Methoxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (29)

2-(3-(Trifluoromethyl)phenyl)-2,3-dihydroquinazolin-4(1H)-one (30)

2-(3-Cyanophenyl)-2,3-dihydroquinazolin-4(1H)-one (31)

2-(4-Cyanophenyl)-2,3-dihydroquinazolin-4(1H)-one (32)

2-(3,4,5-Trihydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (33)

2-(Pyridin-3-yl)-2,3-dihydroquinazolin-4(1H)-one (34)

2-(Thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one (35)

2-(5-bromofuran-2-yl)-2,3-dihydroquinazolin-4(1H)-one (36)

2([1,1′-Biphenyl]-4-yl)-2,3-dihydroquinazolin-4(1H)-one (37)

2-(Naphthalen-2-yl)-2,3-dihydroquinazolin-4(1H)-one (38)

2-(Naphthalen-1-yl)-2,3-dihydroquinazolin-4(1H)-one (39)

2-(Benzofuran-2-yl)-2,3-dihydroquinazolin-4(1H)-one (40)

2-(1H-Indol-3-yl)-2,3-dihydroquinazolin-4(1H)-one (41)

2-(Benzo[c][1,2,5]thiadiazol-5-yl)-2,3-dihydroquinazolin-4(1H)-one (42)

2-Phenethyl-2,3-dihydroquinazolin-4(1H)-one (43)

2-(1-Bromo-2-phenylvinyl)-2,3-dihydroquinazolin-4(1H)-one (44)

2-(2-Nitrostyryl)-2,3-dihydroquinazolin-4(1H)-one (45)

2-(2-(Furan-2-yl)vinyl)-2,3-dihydroquinazolin-4(1H)-one (46)

Synthesis of quinazolin-4(3H)-ones 49–57 and 60–67

Procedure A: used to synthesise 47–53

Procedure B. Used to synthesise 54–57

Procedure C: used to synthesise 60–66

2-(5-Methyl-1H-indol-3-yl)-2,3-dihydroquinazolin-4(1H)-one (47)

3-(4-Oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-1H-indole-5-carbonitrile (48)

2-(Pyridin-4-yl)quinazolin-4(3H)-one (49)

2-(4-Methoxystyryl)quinazolin-4(3H)-one (50)

2-Styrylquinazolin-4(3H)-one (51)

2,3-Dihydroquinazolin-4(1H)-ones and quinazolin-4(3H)-ones as broad-spectrum cytotoxic agents and their impact on tubulin polymerisation^†

Scheme 1. Reagents and conditions: (i) SnCl₂ (1%), EtOH, 120 °C, μwave.⁴².

Scheme 2. Reagents and conditions: (i) SnCl₂ (1%), EtOH, 120 °C, μwave; and (ii) KMnO₄, acetone, Δ.

Scheme 3. Reagents and conditions: (i) SbCl₃, THF Δ; (ii) R′CHO, PhCH₃ : AcOH (1 : 1), 180 °C μwave.

Growth inhibition of a panel of cancer cell lines and a normal breast cell line by selected library 2 analogues (quinazolin-4(3H)-one analogues 47, 48, 50–52, 56, and 60–65). Growth inhibition, GI₅₀ values relative (μM) to untreated control (MTT assay, 72 h, n = 3).

Fig. 12. Cell cycle analysis. G₂ + M cell cycle arrest was induced in MIA cells by 39, 64 and the tubulin inhibitor nocodazole 68 after 24 h exposure. A single representative set of histograms is shown.