Discovery of novel quinazolinones and their acyclic analogues as multi-kinase inhibitors: design, synthesis, SAR analysis and biological evaluation

Abstract

This work deals with the design and synthesis of some novel 6-iodo-2-(pyridin-3/4-yl)-3-substituted quinazolin-4-one derivatives 8a–l, 10a–h, 13–18 in addition to certain acyclic analogues thereof viz. 9a–n and 12a–h. The molecular design strategy was based on structural analogy between the new compounds and reported quinazolines and their acyclic analogues. This design scheme led to the synthesis of 8 new intermediates and 58 new final quinazolinones. The target compounds were evaluated for their antitumor activity against a panel of nine cancer cell lines viz. breast cancer (MCF-7, MDAMB-231, MDAMB-435 and HS-578T), colon cancer (HT-29 and HCC-2998) and leukemia (CCRF-CEM, K-562 and HL-60). The quinazolinones 10a–h displayed exceptional antitumor activity and compounds 12a–h showed superior potency against MCF-7. These compounds were further subjected to in vivo study. Kinase inhibitory assay was also carried out to investigate the mechanism of action of the target compounds and they displayed the highest activity against ABL, ALK and c-RAF kinases. The 3-substituted quinazolinones 10a–h showed the highest kinase activity inhibitory potency against ABL, ALK and c-RAF with the most active compound in this study being the fluoro-3-pyridyl derivative 10a. These results are in compliance with the observed antitumor activity. Finally, a molecular modeling study was performed to interpret the potential molecular interactions of these chemotypes with the most responsive biomolecular target ABL.

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Introduction

Quinazoline nucleus-based small molecules have aroused recent attention from chemical and biological view-points. This is attributed to the fact that members of this chemotype have found their way to the pharmaceutical products market after being granted FDA approval through displaying remarkable antineoplastic activity supported by a diverse array of mechanisms of action.^1–5 Among the disclosed modes of cytotoxicity activities elicited by quinazolines is their effect as dihydrofolate reductase antimetabolites,^6,7 DNA intercalators³ or protein kinase inhibitors.^8–11

The human genome contains about 500 protein kinase genes constituting about 2% of all human genes.¹² Up to 30% of all human proteins are modifiable by kinase activity where kinases are well-known regulators of many cellular pathways, especially those involved in signal transduction.¹³ Hence, it was found that protein kinases' mutation and/or over-expression play a central role in disruption of various cellular activities like growth, proliferation and migration, and thus is a hallmark of some types of cancer.¹²

Much attention has been given to exploiting small molecules that can target protein kinases as inhibitors where such chemical entities have proved effective as anti-proliferative and/or cytotoxic agents.^{10,12,14–16} Quinazoline-based molecules are one such class that act through interfering with the enzymatic phosphorylation ability of kinase enzyme family members. The antikinase activity of quinazolines is generally due to their ability to competitively occupy the adenosine triphosphate (ATP)-binding pocket with high affinity, thus acting as ATP-mimic inhibitors.^17–20

Despite extensive efforts within the oncology field to develop kinase inhibitors (KIs), uncertainty remains over the relative merits of selective compounds versus less selective or “multi-targeted” inhibitors.²¹ Targeted molecules offer the clearest indication that in vivo effects result from the intended in vitro activity. Moreover, toxicity derived from additional activity against other kinases is likely to be reduced.²² However, inhibition of a single kinase may not be sufficient to achieve a clinical benefit, either through the built-in redundancy of signaling pathways, or the ability of tumors to acquire resistance.²³ Inhibitors with activity against multiple kinases may in fact be more effective anticancer agents, and several multi-targeted kinase inhibitors are now commercially available like sorafenib.²⁴

Consequently, and motivated by the aforementioned facts, it deemed of interest to design, synthesize and evaluate novel quinazolinones as potential kinase inhibitors and cytotoxic agents.

Molecular design strategy

Design of the target compounds (Fig. 1) focused on alternating the physicochemical properties of the functional groups on the 2,3,6-trisubstituted quinazolinone backbone to target different regions of the ATP-binding pocket of the protein kinase domain. The ATP binding site has multiple regions viz. the adenine binding region which contains two key hydrogen bonds formed by the interaction of N-1 and N-6 amino group of the adenine ring; the sugar binding region which is mostly hydrophilic in nature; a hydrophobic pocket that is not used by ATP but plays a crucial role in inhibitor selectivity; hydrophobic channels that are not occupied by ATP yet may be exploited for inhibitor specificity, and finally the phosphate binding region which can be targeted for improving inhibitor selectivity.^20,25

Fig. 1 Design strategy for the target 2,3,6-trisubstituted quinazolinones and their acyclic analogs.

Molecular manipulations on the quinazoline backbone were performed according to the following rationale:

First the 2-position of the quinazolinone ring, a heteroaryl viz. 3-pyridyl or 4-pyridyl was introduced to study the effect of this positional isomerism on the enzymatic and anticancer activities of these derivatives.

For the 3-position, fragment molecular manipulation was carried out with different groups of diverse lipophilicity and length viz. substituted/unsubstituted urea or thiourea, carboxamide, carbothioamide, carbothiohydrazide, phenyl hydrazine, hydroxylamine, para-aminobenzoic acid, benzylideneamino and substituted acetylhydrazinyl to study the effect of these changes on their occupation of the lipophilic pocket of the ATP binding site of kinases and hence their associated cytotoxic activity as compared to well-known KIs.

Moreover, the 6-position of the quinazolinone was kept occupied by the bulky hydrophobic iodo substituent aiming to furnish the necessary bulkiness and hydrophobic character required at this position besides acting as a metabolic blocker to prolong the duration of action of these molecules.

Finally, modifications related to decreasing the rigidity of the structure through quinazoline ring opening were performed to assess the effect of increasing the backbone flexibility on the molecular binding interactions mode with the active site amino acids of the target enzyme and on hence on the investigated biological activity.

The newly synthesized compounds were initially screened for their cytotoxic activities against a panel of cancer cell lines. Compounds showing remarkable anticancer activity were subjected to in vivo study. The kinase inhibitory activity was also evaluated to elucidate the mechanism of action of the active derivatives. Furthermore, a molecular docking study was carried out to predict the plausible binding mode of these chemotypes with the active site of the most responsive kinase ABL.

Chemistry

The key starting materials, 5-iodoanthranilic acid 3,²⁶ nicotinoyl chloride 4a ²⁷ and isonicotinoyl chloride 4b,²⁸ were prepared as reported. 5-Iodo-2-[(pyridinyl-3/4-carbonyl)amino]benzoic acid derivatives 5a, b were prepared via the reaction of 5-iodoanthranilic acid 3 and the freshly prepared nicotinoyl chloride 4a or isonicotinoyl chloride 4b in dry DMF at 80 °C. The benzoxazinone derivatives 6a, b were synthesized through the nucleophilic reaction of the amidated anthranilic acid with acetic anhydride or via the reaction of the substituted anthranilic acid and the freshly prepared acyl chloride in dry pyridine at 5 °C. Moreover, the reaction of benzoxazinones 6a, b with hydrazine hydrate was carried out in various solvents or under various reactions conditions viz. reflux in ethanol or n-butanol or direct solvent-free fusion of the reactants to obtain the desired 3-aminoquinazolinone. In all cases, the reaction afforded the open ring structures; N-[2-(hydrazinylcarbonyl)-4-iodophenyl]pyridine-3/4-carboxamide 7a, b which were isolated and identified. Attempts to cyclize the diamides 7a, b to the corresponding 4-(3H)-quinazolin-4-one under various reaction conditions, including sodium ethoxide or polyphosphoric acid were unsuccessful (Scheme 1).

Scheme 1 Synthesis of derivatives 7a, b. Reagents and solvents: (i) KOH; (ii) I₂ KOH, glacial acetic acid; (iii) DMF; (iv) acetic anhydride; (v) dry pyridine; (vi) NH₂NH₂·H₂O, absolute ethanol.

Compounds 8a–l were prepared by refluxing the diamide derivatives 7a, b with the appropriate isocyanate or isothiocyanate in dioxane in the presence of catalytic amount of triethylamine. Synthesis of the benzylideneamino derivatives 9a–n was carried out by treating the hydrazido derivatives with the appropriate aldehyde in absolute ethanol in presence of few drops of glacial acetic acid. Cyclodehydration reaction was then carried out using sodium ethoxide to obtain the benzylidene aminoquinazolinones 10a–h from the diamide analogues. Also, the desired 2-chloroacetyl hydrazinyl derivatives 11a, b were successfully synthesized by reacting compounds 7a, b with chloroacetyl chloride in dry dimethylformamide at room temperature. The chloroacetylhydrazinyl derivative11a and its 4-pyridyl analogue 11b were refluxed with morpholine, piperidine or substituted piperazines in absolute ethanol containing anhydrous potassium carbonate to produce the desired compounds 12a–h (Scheme 2).

Scheme 2 Synthesis of derivatives 8–12. Reagents and solvents: (i) appropriate isocyanate/isothiocyanate, TEA, dioxane; (ii) p-substituted benzaldehyde, absolute ethanol, glacial acetic acid; (iii) sodium ethoxide, absolute ethanol; (iv) ClCH₂COCl, DMF; (v) secondary amines, absolute ethanol, anhydrous K₂CO₃.

The hydroxy derivatives 13a and 13b were synthesized via refluxing 6a and 6b with hydroxylamine hydrochloride in dry pyridine. Refluxing the benzoxazinone derivatives 6a, b with urea, thiourea or thiosemicarbazide in glacial acetic acid in the presence of anhydrous sodium acetate furnished the carboxamide derivatives 14a, b carbothioamides 15a, b and carbothiohydrazides 16a, b. Moreover, the phenylamino derivatives 17a–f were prepared through refluxing the various hydrazine derivatives with the benzoxazinone compounds 6a, b in absolute ethanol. Finally, reaction of a mixture of benzoxazinones 6a, b with p-aminobenzoic acid under fusion conditions at 200 °C yielded the benzoic acid derivatives 18a, b (Scheme 3).

Scheme 3 Synthesis of derivatives 13–18. Reagents and solvents: (i) NH₂OH·HCl, pyridine; (ii) urea, anhydrous CH₃COONa, glacial acetic acid; (iii) thiourea, anhydrous CH₃COONa, glacial acetic acid; (iv) thiosemicarbazide, anhydrous CH₃COONa, glacial acetic acid; (v) appropriate phenylhydrazine, absolute ethanol; (vi) p-aminobenzoic acid.

Results and discussion

(1) Biological evaluation

(a) In vitro cytotoxic activity. All the newly synthesized compounds were tested in vitro for their cytotoxic activity against a panel of 9 human cancer cell lines. This panel involved four breast cancer cell lines (MCF-7, MDAMB-231, MDAMB-435 and HS-578T), two colon cancer cell lines (HT-29 and HCC-2998) and three leukemia cell lines (CCRF-CEM, K-562 and HL-60). The obtained IC₅₀ values are displayed in Table 1.

Table 1 Cytotoxic activities of the target quinazolinones; 8a–l, 10a–h, 13a, b, 14a, b, 15a, b, 16a, b, 18a, b, 17a–f and N-(2,4-disubstituted phenyl)pyridinecarboxamides; 9a–n and 12a–h against breast, colon and leukemia cell lines

Cpd no.	IC50^a (μM)
	Breast cancer			Colon cancer			Leukemia
	MCF-7	MDA-MB-231	HS-578T	MDA-MB-435	HT-29	HCC-2998	CCRF-CEM	K-562	HL-60
a The average IC50 values were calculated from three independent tests.
8a	>100	0.0012	0.0042	>100	0.0064	0.0043	0.0575	0.0696	0.0658
8b	>100	>100	>100	>100	0.0045	0.0054	0.0484	0.087	0.0899
8c	>100	>100	>100	0.087	0.65	0.71	0.0395	0.0765	0.0640
8d	0.0009	>100	>100	0.049	0.75	0.82	0.0486	0.065	0.0273
8e	>100	0.0068	0.0065	>100	0.05	0.93	0.05679	0.054	0.0219
8f	0.00098	0.0046	0.0012	>100	0.0078	0.0065	0.0466	0.0505	0.0367
8g	0.0065	>100	>100	0.021	0.0055	0.0075	0.0579	0.0344	0.0349
8h	>100	>100	>100	0.035	0.0046	0.0070	0.0457	0.0235	0.0655
8i	>100	>100	>100	0.034	0.0057	0.0099	0.0335	0.0366	0.0894
8j	>100	>100	>100	0.023	0.0074	0.0088	0.0344	0.0478	0.0673
8k	0.0095	0.0057	0.01	>100	0.0079	0.0076	0.0455	0.0484	0.0456
8l	>100	>100	>100	0.024	0.0064	0.0034	0.0468	0.0455	0.0258
9a	0.0054	0.0047	0.0032	0.0027	0.26	0.35	0.0254	0.0268	0.3212
9b	0.0034	0.0043	0.0067	0.0016	0.15	0.24	0.0135	0.0156	0.1243
9c	0.0056	0.0078	0.0067	0.0068	0.48	0.58	0.0443	0.0467	0.0565
9d	0.0065	0.0087	0.0080	0.0045	0.65	0.36	0.0244	0.0255	0.0893
9e	0.0078	0.0090	0.0089	0.0054	0.39	0.47	0.0334	0.0366	0.0674
9f	0.0032	0.0065	0.0045	0.0039	0.57	0.46	0.0344	0.0378	0.4566
9g	0.0089	0.0053	0.0090	0.0056	0.76	0.55	0.0354	0.0755	0.0904
9h	>100	0.0065	0.0043	0.043	0.95	0.86	0.0546	0.0953	0.0566
9i	0.0065	0.0034	>100	>100	0.87	0.64	0.0455	0.0843	0.0874
9j	0.0045	0.0038	0.0046	0.034	0.06	0.97	0.0435	0.0755	0.0435
9k	>100	0.0077	45	>100	0.07	0.94	0.0565	0.0544	0.0566
9l	>100	0.0056	11	>100	0.06	0.03	0.0456	0.0654	0.0575
9m	0.0034	0.0074	0.0052	0.0043	0.05	0.96	0.0544	0.0566	0.0264
9n	>100	0.0064	67	>100	0.06	0.85	0.0478	0.043	0.0348
10a	0.000012	0.00010	0.00045	0.00028	0.0051	0.0031	0.041	0.065	0.031
10b	0.000014	0.00011	0.00057	0.00029	0.0056	0.0032	0.049	0.064	0.042
10c	0.000016	0.00012	0.00078	0.00035	0.0063	0.0033	0.058	0.076	0.053
10d	0.000018	0.00014	0.00085	0.00036	0.0072	0.0044	0.064	0.085	0.064
10e	0.000019	0.00015	0.00087	0.00044	0.0074	0.0045	0.075	0.087	0.065
10f	0.000021	0.00016	0.00095	0.00045	0.0086	0.0046	0.076	0.091	0.066
10g	0.000022	0.00017	0.00097	0.00046	0.0095	0.0048	0.087	0.096	0.067
10h	0.000023	0.00022	0.00099	0.00048	0.0099	0.0049	0.098	0.099	0.068
12a	0.00012	0.0035	0.0044	0.0035	0.025	0.035	0.0013	0.0023	0.0019
12b	0.00024	0.0043	0.0055	0.0046	0.035	0.034	0.0022	0.0034	0.0028
12c	0.00035	0.0056	0.0046	0.0047	0.026	0.057	0.0035	0.0046	0.0037
12d	0.00046	0.0078	0.0055	0.0058	0.017	0.065	0.0024	0.0035	0.0046
12e	0.00054	0.0098	0.0074	0.0067	0.036	0.034	0.0043	0.0027	0.0059
12f	0.00064	0.0054	0.0083	0.0056	0.045	0.036	0.0056	0.0038	0.0048
12g	0.00076	0.0026	0.0075	0.0045	0.054	0.055	0.0065	0.0057	0.0034
12h	0.00078	0.0045	0.0066	0.0036	0.043	0.046	0.0057	0.0066	0.0023
13a	0.0023	0.0032	0.0047	0.0078	0.034	0.054	0.0078	0.0075	0.0072
13b	0.0043	0.0045	0.0058	0.0063	0.075	0.023	0.0069	0.0084	0.0064
14a	0.0054	0.0076	0.0069	0.0052	0.086	0.014	0.0058	0.0093	0.0096
14b	0.0067	0.0053	0.0078	0.0083	0.075	0.023	0.0046	0.0084	0.0087
15a	0.0065	0.0045	0.0087	0.0091	0.098	0.054	0.0065	0.0095	0.0096
15b	0.0043	0.0067	0.0075	0.0082	0.087	0.067	0.0054	0.0076	0.0085
16a	0.0023	0.0089	0.0064	0.0093	0.058	0.056	0.0043	0.0047	0.0053
16b	0.0045	0.0087	0.0054	0.0087	0.036	0.043	0.0054	0.0056	0.0044
17a	0.0045	0.0033	0.0054	0.0078	0.054	0.067	0.0065	0.0035	0.0065
17b	0.0034	0.0044	0.004	0.0089	0.085	0.067	0.0074	0.0043	0.0076
17c	0.0056	0.0053	0.003	0.0036	0.076	0.089	0.0085	0.0054	0.0057
17d	0.0078	0.0032	0.004	0.0045	0.057	0.087	0.0095	0.0065	0.0038
17e	0.0075	0.0045	0.005	0.0054	0.068	0.053	0.0086	0.0076	0.0049
17f	0.0045	0.0054	0.004	0.0065	0.076	0.045	0.0077	0.0047	0.0059
18a	0.0034	0.0065	0.005	0.0056	0.024	0.045	0.0066	0.0035	0.0048
18b	0.0034	0.0074	0.004	0.0045	0.045	0.034	0.0055	0.0023	0.0038

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As shown in Table 1, most of the novel quinazolinones; 10a–h, 13a, b, 14a, b, 15a, b, 16a, b, 18a, b and 17a–f and N-(2,4-disubstituted phenyl)pyridine-carboxamides; 9a–n and 12a–h displayed single digit to sub-nanomolar level cytotoxic activity against the breast cancer cell lines MCF-7, MDA-MB-231, HS-578T and MDA-MB-435; the colon cancer cell lines HT29 and HCC-2998 and leukemia cell lines CCRF-CEM, K-562 and HL-60.

SAR analysis into the findings of the cytotoxicity of the synthesized compounds against breast cancer cell lines revealed that most of the quinazolinyl urea/thiourea derivatives 8a–l had weak activity on MCF-7 (>100 μM). However, most of the pyridinecarboxamides bearing benzylidene moiety 9a–n exhibited potent growth inhibitory activity. All the 3-pyridyl analogues displayed enhanced activity against all breast cancer cell lines (IC₅₀ = 0.0032–0.009 μM). Looking at the 4-pyridyl series, some congeners exhibited weak activity (>100 μM), particularly; the fluoro derivative 9h against MCF-7, the chloro derivative 9i against HS-578T and MDA-MB-435, the alkylated and nitro analogues 9l, 9m and 9n against MCF-7 and MDA-MB-435. Interestingly, the target quinazolinones 10a–h, 13–16, 18a,b & 17a–f and pyridinecarboxamides with acetyl hydrazinyl-carrying side chain12a–h displayed superior cytotoxic activity against all the breast cancer cell lines. Compounds 12a–h showed high potency, particularly against MCF-7 (IC₅₀ = 0.12–0.78 nM). Furthermore, the quinazolinones Schiff's base derivatives 10a–h displayed exceptional antitumor activity (IC₅₀ = 0.010–0.022 nM) against MCF-7 in the subnanomolar range. Regarding the cytotoxicity against colon HT29 and HCC-2998 cancer cell lines the quinazolinones Schiff's base derivatives 10a–h (IC₅₀ = 0.0031–0.049 μM) and most of the quinazolinyl urea/thiourea derivatives 8a–l (IC₅₀ = 0.0034–0.65 μM) displayed superior anticancer activity. Moreover, the target quinazolinones 13–16, 18a, b & 17a–f (IC₅₀ = 0.014–0.098 μM) and pyridinecarboxamides with acetyl hydrazinyl-carrying side chain12a–h (IC₅₀ = 0.017–0.65 μM) displayed high cytotoxicity against the colon cancer cell lines. The pyridinecarboxamides bearing benzylidene moiety 9a–n exhibited the weakest antineoplastic activity against colon cancer cell lines (IC₅₀ = 0.05–0.96 μM). Concerning leukemia CCRF-CEM, K-562 and HL-60 cell lines, all the tested compounds exhibited high growth inhibitory activity (IC₅₀ = 0.085–0.0013 μM).

(b) In vitro cytotoxicity against the non-tumorigenic MCF-10A cell line and selectivity. Before progressing into the in vivo cytotoxic screening against MCF-7 mouse xenograft model, selected compounds viz. 10a–h, 12b and 12f were subjected to in vitro evaluation against the non-tumorigenic breast cell lines MCF-10A. The cell line used was selected to represent healthy cells and to determine whether the cytotoxic properties of the tested compounds were selective for malignant cells in contrast to nonmalignant cells. Data obtained are enlisted in Table 2.

Table 2 In vitro cytotoxicity of derivatives 10a–h, 12b and 12f against MCF-10A

Cpd no.	IC50 (μM)	Selectivity^b	Cpd no.	IC50 (μM)	Selectivity
a NT: not tested.b Selectivity in fold relative to the lowest observed potency among tested breast cancer cell lines.
10a	12.09	>26 866	12a	NT^a	NT
10b	19.21	>33 701	12b	1.23	>223
10c	2.10	>2692	12c	NT	NT
10d	3.20	>3764	12d	NT	NT
10e	1.98	>2275	12e	NT	NT
10f	2.30	>2421	12f	4.30	>518
10g	2.10	>2164	12g	NT	NT
10h	3.30	>3333	12h	NT	NT

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With regards to selective cytotoxicity of the tested compounds relative to examined breast cancer cell lines, derivatives in the 10a–h series were >2164 to >33 701 fold more selective to cancer cell lines than nonmalignant cells with 10b showing the highest selectivity. On the other hand, compounds tested from other series 12b and 12f exhibited >223 and >518 fold selectivity towards the malignant breast cancer cell lines, respectively. It can be inferred from these results that the chemotypes in hand show considerable safety margin towards nonmalignant breast cells compared to malignant breast cancer cell lines when tested under the same experimental conditions.

(c) In vivo cytotoxic activity. Compounds showing higher cytotoxic activity against all breast cancer cell lines, particularly MCF-7 (10a–h and 12a–h) were selected to perform in vivo cytotoxicity and data are given in Table 3.

Table 3 MCF-7 mouse xenograft model of breast cancer for compounds 10a–h and 12a–h

Compound no.	Tumor growth Vt/Vo for compounds at time^a (days)
	0	2	4	6	8	10	12	14	16	18	20
a Tested compounds and the control drug sorafenib were administered by intraperitoneal (i.p.) injection at a dose of 5 mg kg^−1, every 48 h for 3 weeks.b Each data point is the mean ± SEM of five animals.
Vehicle control	1.0 ± 0.01^b	1.3 ± 0.07	1.8 ± 0.07	4.7 ± 0.2	9.8 ± 0.5	12.6 ± 1.4	24.7 ± 2.7	27.6 ± 1.2	29.0 ± 1.7	38.9 ± 1.2	40.2 ± 1.8
Sorafenib	1.0 ± 0.04	1.1 ± 0.06	1.2 ± 0.06	1.5 ± 0.02	2.1 ± 0.2	2.2 ± 0.1	4.5 ± 0.2	5.5 ± 0.3	6.89 ± 0.5	8.8 ± 0.7	9.1 ± 0.3
10a	1.0 ± 0.05	1.0 ± 0.08	1.0 ± 0.07	1.1 ± 0.01	1.1 ± 0.1	1.3 ± 0.2	2.0 ± 0.3	2.6 ± 0.4	3.10 ± 0.4	3.3 ± 0.3	3.5 ± 0.2
10b	1.0 ± 0.03	1.0 ± 0.09	1.0 ± 0.08	1.1 ± 0.02	1.3 ± 0.1	1.4 ± 0.1	2.1 ± 0.4	2.7 ± 0.5	3.16 ± 0.5	3.3 ± 0.2	3.8 ± 0.2
10c	1.0 ± 0.05	1.0 ± 0.08	1.0 ± 0.07	1.1 ± 0.03	1.4 ± 0.1	1.4 ± 0.1	2.2 ± 0.3	2.8 ± 0.3	3.32 ± 0.6	4.1 ± 0.3	4.5 ± 0.1
10d	1.0 ± 0.06	1.0 ± 0.07	1.0 ± 0.08	1.1 ± 0.04	1.5 ± 0.2	1.4 ± 0.2	2.3 ± 0.2	2.9 ± 0.2	3.52 ± 0.4	4.2 ± 0.4	4.8 ± 0.4
10e	1.0 ± 0.07	1.0 ± 0.06	1.0 ± 0.09	1.1 ± 0.05	1.6 ± 0.2	1.6 ± 0.1	2.3 ± 0.3	3.0 ± 0.3	3.74 ± 0.3	4.5 ± 0.3	4.9 ± 0.5
10f	1.0 ± 0.06	1.0 ± 0.07	1.0 ± 0.08	1.2 ± 0.04	1.6 ± 0.1	1.7 ± 0.1	2.5 ± 0.2	3.1 ± 0.4	3.90 ± 0.4	4.6 ± 0.5	5.0 ± 0.4
10g	1.0 ± 0.07	1.0 ± 0.06	1.0 ± 0.07	1.2 ± 0.06	1.7 ± 0.2	1.8 ± 0.1	2.6 ± 0.3	3.2 ± 0.5	4.04 ± 0.2	4.7 ± 0.6	5.0 ± 0.5
10h	1.0 ± 0.06	1.0 ± 0.07	1.1 ± 0.08	1.2 ± 0.05	1.8 ± 0.1	1.9 ± 0.1	2.7 ± 0.2	3.5 ± 0.3	4.19 ± 0.5	4.8 ± 0.7	5.1 ± 0.3
12a	1.0 ± 0.07	1.0 ± 0.06	1.1 ± 0.07	1.2 ± 0.06	1.5 ± 0.1	1.7 ± 0.1	2.0 ± 0.3	2.5 ± 0.2	2.98 ± 0.1	3.5 ± 0.8	4.3 ± 0.2
12b	1.0 ± 0.06	1.0 ± 0.07	1.1 ± 0.06	1.2 ± 0.07	1.6 ± 0.1	1.8 ± 0.2	2.1 ± 0.2	2.6 ± 0.2	3.10 ± 0.2	4.8 ± 0.8	5.4 ± 0.6
12c	1.0 ± 0.05	1.0 ± 0.06	1.1 ± 0.06	1.2 ± 0.08	1.7 ± 0.2	1.9 ± 0.2	2.2 ± 0.1	2.7 ± 0.3	3.34 ± 0.4	4.3 ± 0.7	5.8 ± 0.5
12d	1.0 ± 0.06	1.1 ± 0.05	1.1 ± 0.04	1.3 ± 0.07	1.8 ± 0.1	1.9 ± 0.2	2.3 ± 0.2	2.8 ± 0.1	3.56 ± 0.3	4.6 ± 0.4	6.3 ± 0.6
12e	1.0 ± 0.05	1.1 ± 0.04	1.2 ± 0.03	1.3 ± 0.03	1.8 ± 0.1	2.0 ± 0.3	2.5 ± 0.1	3.2 ± 0.2	4.11 ± 0.5	5.3 ± 0.5	6.5 ± 0.7
12f	1.0 ± 0.04	1.1 ± 0.06	1.2 ± 0.04	1.4 ± 0.04	1.9 ± 0.2	2.1 ± 0.2	3.0 ± 0.4	3.6 ± 0.3	4.48 ± 0.4	5.6 ± 0.6	6.8 ± 0.8
12g	1.0 ± 0.03	1.1 ± 0.07	1.2 ± 0.05	1.4 ± 0.05	1.9 ± 0.1	2.1 ± 0.3	3.2 ± 0.3	4.2 ± 0.4	5.23 ± 0.5	5.9 ± 0.7	7.1 ± 0.7
12h	1.0 ± 0.04	1.1 ± 0.06	1.3 ± 0.06	1.5 ± 0.06	2.0 ± 0.2	2.2 ± 0.2	3.5 ± 0.2	4.5 ± 0.5	5.67 ± 0.7	6.1 ± 0.8	8.1 ± 0.9

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Results showed that all compounds were 3.89–11.23 fold more potent than the vehicle control, showing tumor volume in the range of 3.58–8.12 cm³ as compared to the vehicle control which displayed a tumor volume of 40.21 cm³. This is perfectly in compliance with the in vitro cytotoxicity data where the most active compound among the pyridinecarboxamide series bearing acetyl hydrazinyl side chain 10a (IC₅₀ = 0.012 nM) was also the most active in vivo. Interestingly, the tested compounds were also 1.12–2.56 fold more potent than the positive control drug sorafenib (tumor volume 9.1 cm³).

It is worth mentioning that the in vivo experiments were tried on two doses of the tested compounds (low dose of 5 mg kg⁻¹ and high dose of 10 mg kg⁻¹, every 48 h for 3 weeks) and data reported herein are for the low dose. This is because first tumor volume changes observed at the two tested doses were not significantly different. Furthermore, there was no dose-dependent toxicity in animal groups treated at the two dose levels (data not shown).

(d) Kinases inhibition assay. To investigate the mechanism of action and the kinase inhibitory profile of the target compounds, they were tested at a single dose concentration of 10 μM over a panel of 30 kinases to determine their kinase activity percent inhibition. The synthesized compounds exerted multiple inhibitions over the tested oncogenic protein kinases at the tested concentration. The synthesized compounds completely inhibited the enzymatic activity of ABL, ALK and c-RAF kinases. On the other hand, the inhibitions were of low percent in the other tested 27 kinases.

For those kinases that were 100% inhibited at the initial high dose, viz. ABL, ALK and c-RAF, IC₅₀ values of the test compounds were determined. Kinase activity was assessed using HotSpot^SM Technology, a miniaturized radio-isotope based filter binding assay.²⁹

Abelson kinase (ABL) is a fusion tyrosine kinase playing a role in 90% of chronic myeloid leukemia (CML) cases.³⁰ Anaplastic Lymphoma Kinase (ALK) is a member of the insulin receptor tyrosine kinase family.³¹ It is constitutively active and plays an oncogenic role in 70–80% of all anaplastic large cell lymphomas.³² On the other hand, over-expression of ALK was observed in glioblastoma³³ and neuroblastoma.³⁴ Moreover, dysregulated signaling through RAF kinase isoforms has been detected in ∼30% of human cancers where wild type c-RAF is hyperactivated in a wide range of human solid tumors.³⁵

The kinase inhibitory activities (nM) of the targeted quinazolinones; 8a–l, 10a–h, (13–16, 18)a,b and 17a–f and N-(2,4-disubstituted phenyl)pyridinecarboxamides; 9a–n and 12a–h against ABL kinase (100% inhibited) are listed in Table 4.

Table 4 Kinase inhibitory activity

Compound number	IC50 (nM)			Compound number	IC50 (nM)
	ABL	ALK	c-RAF		ABL	ALK	c-Raf
8a	54	64	64	10d	0.023	0.065	0.046
8b	43	72	53	10e	0.035	0.067	0.057
8c	66	21	62	10f	0.037	0.074	0.057
8d	76	35	43	10g	0.036	0.076	0.058
8e	68	76	34	10h	0.037	0.078	0.059
8f	43	56	55	12a	0.26	0.35	0.55
8g	44	37	Inactive	12b	0.35	0.43	0.44
8h	33	25	Inactive	12c	0.44	0.65	0.35
8i	26	323	Inactive	12d	0.33	0.78	0.26
8j	32	34	Inactive	12e	0.24	0.76	0.19
8k	47	47	66	12f	0.44	0.45	0.18
8l	55	50	Inactive	12g	0.35	0.24	0.10
9a	17	84	36	12h	0.26	0.35	0.17
9b	24	93	43	13a	0.57	0.35	0.14
9c	35	65	24	13b	0.46	0.43	0.13
9d	39	46	56	14a	0.65	0.54	0.45
9e	48	54	35	14b	0.74	0.32	0.36
9f	26	75	45	15a	0.65	0.67	0.48
9g	20	37	67	15b	0.54	0.89	0.56
9h	36	59	40	16a	0.45	0.09	0.44
9i	18	28	58	16b	0.56	0.76	0.55
9j	21	30	79	17a	0.65	0.45	0.76
9k	46	58	36	17b	0.76	0.34	0.84
9l	39	19	47	17c	0.67	0.56	0.95
9m	40	20	68	17d	0.79	0.54	0.86
9n	55	67	25	17e	0.60	0.35	0.97
10a	0.011	0.050	0.042	17f	0.59	0.34	0.85
10b	0.021	0.056	0.044	18a	0.46	0.56	0.76
10c	0.022	0.068	0.045	18b	0.37	0.78	0.65
Imatinib	0.58	—	0.99	Bosutinib	0.03	—	—
Staurosporine	—	0.0022	—	Sorafenib	—	—	6

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Interestingly, the target quinazolinones; 8a–l, 10a–h and 13–18 as well as the substituted N-(2,4-disubstituted phenyl)pyridinecarboxamides; 9a–n and 12a–h exhibited a wide range of ABL, ALK and c-RAF inhibitory activity with IC₅₀ ranges 0.011–76 nM and 0.24–55 nM, 0.05–323 nM and 0.09–93 nM, 0.042–66 nM and 0.1–79 nM, respectively. The quinazolinyl-3-urea/thiourea derivatives 8a–l and the pyridinecarboxamides with benzylidene-carrying side chain 9a–n displayed moderate to very weak inhibitory activity against ABL (IC₅₀ = 17–76 nM), ALK (IC₅₀ = 19–323 nM) and c-RAF kinases (IC₅₀ = 24 nM-inactive). The pyridinecarboxamides bearing acetyl hydrazinyl-carrying side chain 12a–h and the 3-substituted quinazolin-4(3H)-one 13–18 had higher potency against ABL, ALK and c-RAF kinases (IC₅₀ = 0.24–0.79 nM, 0.09–0.89 nM and 0.1–0.97, respectively). Additionally, the 3-substituted benzylidene quinazolinones 10a–h showed exceptional ABL, ALK and c-RAF inhibitory potency (IC₅₀ = 0.011–0.037 nM, 0.05–0.074 nM, 0.042–0.057 nM, respectively).

Concerning the quinazolinone derivatives 8a–l: generally, replacement of the pyridin-3-yl moiety (8a–f) by pyridin-4-yl moiety (8g–l) caused a slight increase in ABL inhibitory activity and it did not have a significant effect on ALK inhibitory activity. On the other hand, it nearly abolished c-RAF activity. Surprisingly, side chain shortening of the phenylureido derivatives 8d, j to phenyl amino ones 17a, d enhanced the inhibitory potency by 50–100 fold. Looking at the effect of removing the nitrogen spacer between the quinazolinone core and the side chain linked to the quinazolinyl-N3, it was found that this structural modification could be beneficial for the ABL, ALK and c-RAF kinases inhibitory activity (IC₅₀ = 0.37–0.79, 0.09–0.89 and 0.13–0.97 nM, respectively) in the quinazolinones 13, 14, 15, 16, 18a, b. With respect to the pyridinecarboxamides with benzylidene-carrying side chain 9a–n, it was observed that this series displayed slightly higher activity than the quinazolinones 8a–l.

Rigidification of the Schiff's bases 9a–n through cyclodehydration afforded the most potent 3-substituted quinazolinones in this work 10a–h which showed exceptional enzyme inhibitory potency against ABL, ALK and c-RAF (IC₅₀ = 0.011–0.037 nM, 0.05–0.074 nM, 0.042–0.057 nM, respectively) which is consistent with their antitumor activity. Exploring the effect of different substituents on the benzylidene moiety, revealed that decreasing the size and increasing the electronegativity of the halogenated substituent resulted in improving the potency in case of 3-pyridyl analogues. Therefore, the fluoro derivative 10a displayed higher activity than the chloro analogue 10b which in turn exhibited slightly higher activity than the bromo derivative 10c (IC₅₀ = 0.011, 0.021, 0.022 nM, respectively in case of ABL), (IC₅₀ = 0.050, 0.056, 0.068 nM, respectively in case of ALK), (IC₅₀ = 0.042, 0.044, 0.045 nM, respectively in case of c-RAF). Similar results were observed for the 4-pyridyl derivatives. It is worth to mention that the fluoro-3-pyridyl derivative 10a exhibited the most promising kinase inhibitory activity in this study.

Finally, as evidenced from the experimental data showed, the most active kinase activity inhibiting series in this study 10a–h were equi- to many folds more potent than the reference kinase inhibitors against their target kinases viz. imatinib (ABL and c-RAF), bosutinib (ABL), sorafenib (c-RAF) and staurosporine (ALK).

(2) Molecular modelling

The docking study was initiated to predict the interaction of the newly synthesized compounds against the kinase ABL towards which they showed the highest enzymatic activity inhibition. The two PDB files 2HYY and 3UE4 representing inactive DFG-out conformations of ABL-co-crystallized with imatinib and bosutinib, respectively.^36,37 It is reported that the two conformations are distinct in the sense that the in the latter crystal structure of ABL with its inhibitor bosutinib, the activation loop adopts a conformation like that observed in active kinases, except for the conformation of the DFG motif itself. This is in contrast with the commonly known conformational orientation of the activation loop seen in case of the type II inhibitor imatinib (PDB entry 2HYY) in which the P-loop folds over the ATP binding site, and the activation-loop blocks the substrate binding site with a shift of the DFG motif nearer to the front of the active site.

Docking of the most active compounds 10a–h and 12a–h was performed on both crystal–inhibitor complexes to predict whether these derivatives elicit their activity by binding to the active pocket with a behavior compared to that of imitanib or bosutinib. Results showed that while the type II inhibitor imatinib displayed a network of hydrogen bonds to anchor itself tightly with the ABL binding cleft residues, all attempts to dock the new chemotypes in the active site of ABL DFG-out conformation were unsuccessful due to multiple steric clashes with the active site residues (data not shown). On the other hand, docking calculations based on the ABL kinase domain conformation induced by binding of bosutinib to ABL (PDB entry 3UE4, quinazoline N and Met318 amide NH hydrogen bond of 1.9 Å, S = −18.42 kcal mol⁻¹) gave rise to complexes with comparable binding modes and energetics to that of the co-crystallized ligand bosutinib. Results displayed herein are those obtained for attempted docking of the most active compound 10a (IC₅₀ = 0.011 nM) where Fig. 2 shows the respective 2D and 3D interaction contacts of the top scoring and best fitting pose obtained through this molecular modeling study. The highest ABL inhibitory activity of the Schiff's base derivative 10a might be attributed to high ABL binding, energy score (S = −21.22 kcal mol⁻¹) where the 3D interactions (Fig. 2: bottom panel) showed that the pyridyl N atom formed a hydrogen bond with the amide NH of the gatekeeper Met318 (2.4 Å). It is noteworthy that this interaction is considered the key contributor to the overall strength of drug–ABL kinase complexes.^38–40 Additionally, the carbonyl O of the quinazolin-4-one ring interacted with the amidic NH of the Asn322 through a hydrogen (2.1 Å). These results along with the other obtained but not displayed ones strongly suggest that our compounds probably act as type II kinase inhibitors by binding to the DFG out conformation of the catalytic domain of ABL.

Fig. 2 Top panel: 2D interactions; bottom panel: 3D interactions of 10a and ABL (PDB entry 3UE4).

Conclusion

The present study reports the design and synthesis of five novel series of 6-iodo-2-(pyridin-3/4-yl)-3-substituted quinazolin-4(3H)-one and acyclic analogues as multi-kinase inhibitors. The design was based on structural analogy between the new compounds, reported quinazoline derivatives and acyclic analogues. Synthesis of the target compounds was carried out employing techniques reported in the literature with some modifications when necessary. This led to the synthesis of 8 new intermediates and 58 new final compounds. The structures of the synthesized compounds were confirmed by various spectral analysis techniques as well as the elemental analyses.

The target compounds were evaluated for their antitumor activity against a panel of nine cancer cell lines. This panel involved breast cancers (MCF-7, MDAMB-231, MDAMB-435 and HS-578T), colon cancers (HT-29 and HCC-2998) and leukemia cell lines (CCRF-CEM, K-562 and HL-60). The biological results revealed that target quinazolinones 8a–l, 10a–h, 13a, b, 14a, b, 15a, b, 16a, b, 17a–f and 18a, b, and N-(2,4-disubstituted phenyl)pyridinecarboxamides 9a–n and 12a–h exhibited potent antitumor activity. Aiming to investigate their mechanism of action, the synthesized were tested over a panel of 30 kinases; ABL, ALK and c-RAF were the most responsive kinases. Molecular docking was carried out to predict the binding mode of these chemotypes with the active site amino acids of the ABL, being the most responsive kinase to the inhibitory effect of the tested compounds. Molecular modeling results showed that these derivatives recognize the active pocket of the target enzyme selectively to a more relaxed conformation of the ABL active site as induced by the known inhibitor bosutinib. In conclusion, the synthesized compounds, especially 10a, can be viewed as promising leads for further investigation and optimization as multi-kinase inhibitors.

Experimental section

Melting points were determined on Electrothermal Stuart SMP₃ digital melting point apparatus and were uncorrected. Elemental microanalyses were performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University and at the Microanalytical Centre, Faculty of Science, Cairo University. Infrared spectra were determined (KBr) using Shimadzu Infrared spectrometer (IR-435) and FT-IR 1650 (Perkin Elmer), Faculty of Pharmacy, Cairo University. ¹H-NMR spectra were performed in CDCl₃ or DMSO-d₆ using Joel, 300 MHz NMR spectrometer, Faculty of Science, Cairo University, and Bruker, 400 MHz NMR spectrometer, Microanalytical unit, Faculty of Pharmacy, Cairo University. ¹³C-NMR spectra were recorded using Bruker, 100 MHz NMR spectrometer, Microanalytical unit, Faculty of Pharmacy, Cairo University. Mass spectra were carried out using Finnigan SSQ 7000 Gas chromatograph-Mass, at the Microanalytical Centre, Faculty of Science, Cairo University. Thin layer chromatography was carried out on silica gel TLC plates with fluorescence indicator (F₂₅₄). 5-Iodoanthranilic acid (3),²⁶ nicotinoyl chloride (4a),²⁷ isonicotinoyl chloride (4b)²⁸ were prepared according to the reported method.

(1) Chemistry

5-Iodo-2-[(pyridin-3-ylcarbonyl)amino]benzoic acid (5a). A solution of 5-iodoanthranilic acid 3 (2.63 g, 10 mmol) in dry dimethylformamide (20 mL) was added to the freshly prepared nicotinoyl chloride hydrochloride (2.13 g, 12 mmol) and the mixture was heated at 80 °C for 2 h, and then allowed to cool. The reaction mixture was poured onto ice-water. The solid obtained was filtered, washed several times with water and dried to give 3.2 g (71%) which was recrystallized from acetic acid. Mp: 314–316 °C. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3435–3414 (br.) (OH), 3338 (NH), 1676, 1664 (2C=O), 1608 (C=N), 1519 (C=C), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O ppm): δ 7.66 (dd, 1H, J = 4.80, 7.80 Hz, pyridyl-H), 8.00 (dd, 1H, J = 2.10, 8.70 Hz, phenyl-H), 8.29 (t, 1H, J = 2.40 Hz, pyridyl-H), 8.32 (dd, 1H, J = 1.80, 6.30 Hz, phenyl-H), 8.40 (d, 1H, J = 8.70 Hz, phenyl-H), 8.83 (dd, 1H, J = 1.50, 4.80 Hz, pyridyl-H), 9.11 (d, 1H, J = 1.80 Hz, pyridyl-H), 9.30 (s, 1H, NH exchanged by D₂O), 12.02 (s, 1H, OH exchanged by D₂O). Mass (m/z, rel. abundance): 368 (M⁺, 44.38%), 106 (100%). Anal. calcd for C₁₃H₉IN₂O₃ (368.13): C, 42.41; H, 2.46; N, 7.61. Found: C, 42.64; H, 2.45; N, 7.70.

5-Iodo-2-[(pyridin-4-ylcarbonyl)amino]benzoic acid (5b). Compound 5b was prepared and purified by adopting the same procedure used for 5a using isonicotinoyl chloride. Mp: 290–292 °C. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3232 (OH), 3145 (NH), 1683, 1653 (2C=O), 1595 (C=N), 1573 (C=C), 526 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O ppm): δ 7.96 (d, 2H, J = 4.80 Hz, pyridyl-H), 7.99 (dd, 1H, J = 6.00, 8.10 Hz, phenyl-H), 8.29 (d, 1H, J = 1.80 Hz, phenyl-H), 8.38 (dd, 1H, J = 3.00, 8.70 Hz, phenyl-H), 8.92 (d, 2H, J = 4.80 Hz, pyridyl-H), 10.82 (s, 1H, NH exchanged by D₂O), 12.11 (s, 1H, OH exchanged by D₂O). Mass (m/z, rel. abundance): 368 (M⁺, 34.7%), 106 (100%). Anal. calcd for C₁₃H₉IN₂O₃ (368.13): C, 42.41; H, 2.46; N, 7.61. Found: C, 42.57; H, 2.48; N, 7.73.

6-Iodo-2-(pyridin-3-yl)-4H-benzo[d][1,3]oxazin-4-one (6a).
Method 1. A mixture of the amidated anthranilic acid 5a (1.84 g, 5 mmol) in acetic anhydride (15 mL) was heated under reflux for 5 h. Then the reaction mixture was poured onto ice-water. The obtained beige solid product was collected by filtration, dried to give 1.4 g (84%) and then recrystallized from methanol. Mp: 167–169 °C. Yield: 84%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3049 (CH aromatic), 1753 (C=O), 1626 (C=N), 1585 (C=C), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.53 (d, 1H, J = 8.44 Hz, phenyl-H), 7.64 (dd, 1H, J = 4.84, 8.04 Hz, pyridyl-H), 8.27 (dd, 1H, J = 2.00, 8.44 Hz, phenyl-H), 8.42 (d, 1H, J = 1.96 Hz, phenyl-H), 8.49 (t, 1H, J = 8.04 Hz, pyridyl-H), 8.83 (dd, 1H, J = 1.60, 4.80 Hz, pyridyl-H), 9.30 (d, 1H, J = 2.20 Hz, pyridyl-H). Mass (m/z, rel. abundance): 350 (M⁺, 100%). Anal. calcd for C₁₃H₇IN₂O₂ (350.11): C, 44.60; H, 2.02; N, 8.00. Found: C, 44.92; H, 1.85; N, 8.08.
Method 2. A solution of 5-iodoanthranilic acid (2.63 g, 10 mmol) in dry pyridine (30 mL) was added to the freshly prepared isonicotinoyl chloride hydrochloride (2.13 g, 12 mmol) and the mixture was stirred at 5 °C for 2 h, and then allowed to warm to room temperature. The reaction mixture was poured onto ice-water, the solid obtained was filtered, washed with diluted acetic acid, dried and recrystallized from methanol. Mp: 167–169 °C. Yield: 65%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3049 (CH aromatic), 1753 (C=O), 1626 (C=N), 1585 (C=C), 530 (C–I).

6-Iodo-2-(pyridin-4-yl)-4H-benzo[d][1,3]oxazin-4-one (6b).
Method 1. This compound was prepared according to method 1 used for preparation of 6a but using 5b as a starting material and was also recrystallized from methanol.

Mp: 185–187 °C. Yield: 75%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3057 (CH aromatic), 1764 (C=O), 1616 (C=N), 1556 (C=C), 534 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.50 (d, 1H, J = 8.40 Hz, phenyl-H), 7.99 (d, 2H, J = 6.04 Hz, pyridyl-H), 8.23 (dd, 1H, J = 2.00, 8.40 Hz, phenyl-H), 8.37 (d, 1H, J = 1.88 Hz, phenyl-H), 8.78 (d, 2H, J = 6.04 Hz, pyridyl-H). Mass (m/z, rel. abundance): 350 (M⁺, 100%). Anal. calcd for C₁₃H₇IN₂O₂ (350.11): C, 44.60; H, 2.02; N, 8.00. Found: C, 44.54; H, 1.81; N, 8.09.

Method 2. Method 2 used for preparation of 6a was also used for synthesis of 6b using 5b as a starting material.

Mp: 185–187 °C. Yield: 55%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3057 (CH aromatic), 1764 (C=O), 1616 (C=N), 1556 (C=C), 534 (C–I).

N-[2-(Hydrazinylcarbonyl)-4-iodophenyl]pyridine-3-carboxamide (7a). A mixture of benzoxazinone 6a (1.74 g, 5 mmol) and hydrazine hydrate 99% (15 mmol, 0.8 mL) in absolute ethanol (20 mL) was refluxed for 4 h, left to cool then filtered. The separated white solid was collected, washed with ethanol and recrystallized from ethyl acetate. Mp: 221–223 °C. Yield: 75%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3309 (2NH), 3213, 3151 (NH₂), 3061 (CH aromatic), 1697 (2C=O), 1631 (C=N), 1516 (C=C), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 4.70 (s, 2H, NH₂ exchanged by D₂O), 7.62 (dd, 1H, J = 4.88, 7.96 Hz, pyridyl-H), 7.90 (dd, 1H, J = 1.32, 8.80 Hz, phenyl-H), 8.11 (s, 1H, phenyl-H), 8.26 (t, 1H, J = 8.04 Hz, pyridyl-H), 8.39 (d, 1H, J = 8.8 Hz, phenyl-H), 8.80 (dd, 1H, J = 1.52, 4.80 Hz, pyridyl-H), 9.10 (d, 1H, J = 1.84 Hz, pyridyl-H), 10.29 (s, 1H, NH exchanged by D₂O), 12.49 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 382 (M⁺, 1.63%), 216 (100%). Anal. calcd for C₁₃H₁₁IN₄O₂ (382.16): C, 40.86; H, 2.90; N, 14.66. Found: C, 40.98; H, 2.96; N, 14.89.

N-[2-(Hydrazinylcarbonyl)-4-iodophenyl]pyridine-4-carboxamide (7b). Compound 7b was prepared and purified by using the same procedure adopted for the preparation of 7a but using 6b as a starting material. Mp: 238–240 °C. Yield: 75%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3250 (2NH), 3165, 3130 (NH₂), 3074 (CH aromatic), 1681 (C=O), 1638 (C=N), 1517–1434 (C=C), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 4.72 (s, 2H, NH₂ exchanged by D₂O), 7.82 (d, 2H, J = 5.96 Hz, pyridyl-H), 7.91 (dd, 1H, J = 1.56, 8.76 Hz, phenyl-H), 8.13 (s, 1H, phenyl-H), 8.40 (d, 1H, J = 8.76 Hz, phenyl-H), 8.85 (d, 2H, J = 5.96 Hz, pyridyl-H), 10.32 (s, 1H, NH exchanged by D₂O), 12.60 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₁₃H₁₁IN₄O₂ (382.16): C, 40.86; H, 2.90; N, 14.66. Found: C, 40.98; H, 2.93; N, 14.82.

1-(6-Iodo-4-oxo-2-(pyridin-3-yl) quinazolin-3(4H)-yl)-3-substituted urea/thiourea derivatives (8a–f).
General method. A mixture of the hydrazido derivative 7a (0.72 g, 2 mmol) and the appropriate alkyl or aryl isocyanate (2.2 mmol) or isothiocyanate (3 mmol) in dioxane (20 mL) was refluxed for 6–10 h. Then allowed to cool. The precipitated solid product was collected by filtration, washed with aqueous ethanol then dried and recrystallized from absolute ethanol to give 8a–f.
1-Ethyl-3-(6-iodo-4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)thiourea (8a). Mp: 210–211 °C. Yield: 85%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3300, 3251 (2NH), 3215 (CH aromatic), 2972, 2926 (CH₂, CH₃), 1681 (C=O), 1654 (C=N), 1508 (C=C), 1215 (C=S), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 1.02 (t, 3H, J = 6.90 Hz, CH₃CH₂), 3.45 (q, 2H, J = 6.90 Hz, CH₃CH₂), 7.62 (dd, 1H, J = 4.80, 8.10 Hz, pyridyl-H), 7.98 (d, J = 2.10 Hz, quinazolinyl), 8.13–8.24 (m, 3H, quinazolinyl-H, pyridyl-H), 8.81 (dd, 1H, J = 1.8, 4.80 Hz, 1H, pyridyl-H), 9.06 (d, 1H, J = 2.40 Hz, pyridyl-H), 10.7 (s, 1H, NH exchanged by D₂O), 11.68 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 451 (M⁺, 53.61%), 171 (100%). Anal. calcd for C₁₆H₁₄IN₅OS (451.28): C, 42.58; H, 3.13; N, 15.52. Found: C, 42.33; H, 3.45; N, 15.26.
1-Cyclohexyl-3-(6-iodo-4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)urea (8b). Mp: 235–237 °C. Yield: 80%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3327, 3296 (2NH), 2935 (CH₂ aliphatic), 1687, 1660 (2C=O), 1647 (C=N), 1506–1440 (C=C), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 1.07–1.59 (m, 10H, cyclohexyl-5CH₂), 3.35 (m, 1H, cyclohexyl-CH), 7.58 (dd, 1H, J = 4.8 Hz, 7.8 Hz, quinazolinyl-H), 7.86 (s, 1H, pyridyl-H), 7.96 (d, 1H, J = 2.1 Hz, quinazolinyl-H), 8.10 (s, 1H, quinazolinyl-H), 8.19–8.22 (m, 1H, pyridyl-H), 8.80 (dd, 1H, J = 1.8, 5.1 Hz, pyridyl-H), 9.05 (s, 1H, pyridyl-H), 10.44 (s, 1H, NH exchanged by D₂O), 11.78 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 489 (M⁺, 5.6%), 106.05 (100%). Anal. calcd for C₂₀H₂₀IN₅O₂ (489.31): C, 49.09; H, 4.12; N, 14.31. Found: C, 49.16; H, 4.18; N, 14.58.
1-Cyclohexyl-3-(6-iodo-4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)thiourea (8c). Mp: 232–234 °C. Yield: 79%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3404, 3309 (2NH), 2924 (CH₂ aliphatic), 1676 (C=O), 1649 (C=N), 1516–1448 (C=C), 1201 (C=S), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 1.01–1.75 (m, 10H, cyclohexyl-5CH₂), 4.06 (m, 1H, cyclohexyl-CH), 7.52 (t, 1H, J = 6.3 Hz, pyridyl-H), 7.59 (dd, 1H, J = 4.8, 8.1 Hz, quinazolinyl-H), 7.95 (s, 1H, quinazolinyl-H), 8.14 (d, 1H, J = 6.9 Hz, pyridyl-H), 8.21 (d, 1H, J = 8.4 Hz, quinazolinyl-H), 8.79 (d, 1H, J = 3.3 Hz, pyridyl-H), 9.04 (s, 1H, pyridyl-H), 10.59 (s, 1H, NH exchanged by D₂O), 11.5 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 505.3 (M⁺, 3.49%), 106.05 (100%). Anal. calcd for C₂₀H₂₀IN₅OS (505.38): C, 47.53; H, 3.99; N, 13.86. Found: C, 47.49; H, 4.17; N, 14.11.
1-(6-Iodo-4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)-3-phenylurea (8d). Mp: 230–232 °C. Yield: 89% I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3296 (2NH), 3101 (CH aromatic), 1683, 1668 (2C=O), 1639 (C=N), 1506–1446 (C=C), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 6.99 (t, 1H, J = 7.5 Hz, phenyl-H), 7.25 (t, 2H, J = 7.5 Hz, phenyl-H), 7.45 (d, 2H, J = 8.4 Hz, phenyl-H), 7.57 (dd, 1H, J = 5.1 Hz, 7.8 Hz, pyridyl-H), 7.97 (dd, 1H, J = 2.1 Hz, 9.00 Hz, quinazolinyl-H), 8.18–8.24 (m, 2H, pyridyl-H, quinazolinyl-H), 8.78 (d, 1H, J = 4.8 Hz, pyridyl-H), 8.88 (s, 1H, pyridyl-H), 9.06 (d, 1H, J = 2.1 Hz, quinazolinyl-H), 10.66 (s, 1H, NH exchanged by D₂O), 11.73 (s, 1H, NH exchanged by D₂O). ¹³C-NMR (100 MHz DMSO-d₆, ppm): δ 88.05, 119.22, 122.60, 123.67, 123.88, 124.30 (phenyl C-2, C-6), 129.09, 130.35, 135.40, 137.07, 138.40, 139.88, 141.27, 148.77, 153.16, 155.80, 163.98 (C=O), 167.71 (C=O). Mass (m/z, rel. abundance): 483 (M⁺, 3.49%), 106.05 (100%). Anal. calcd for C₂₀H₁₄IN₅O₂ (483.26): C, 49.71; H, 2.92; N, 14.49. Found: C, 49.61; H, 2.66; N, 14.70.
1-(6-Iodo-4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)-3-phenylthiourea (8e). Mp: 270 °C. Yield: 85%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3246 (NH), 3062 (CH aromatic), 1681 (C=O), 1635 (C=N), 1521–1483 (C=C), 1180 (C=S), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 7.05 (t, 1H, J = 6.9 Hz, phenyl-H), 7.38 (t, 2H, J = 6.9 Hz, phenyl-H), 7.61–7.70 (m, 3H, phenyl-2H, pyridyl-H), 7.95 (d, 1H, J = 8.70 Hz, quinazolinyl-H), 8.11 (s, 1H, quinazolinyl-H), 8.36 (d, 1H, J = 7.80 Hz, quinazolinyl-H), 8.46 (d, 1H, J = 8.70 Hz, pyridyl-H), 8.85 (d, 1H, J = 4.2 Hz, pyridyl-H), 9.22 (s, 1H, pyridyl-H), 10.83 (s, 1H, NH exchanged by D₂O), 11.48 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 499 (M⁺, 3.49%), 106.05 (100%). Anal. calcd for C₂₀H₁₄IN₅OS (499.33): C, 48.11; H, 2.83; N, 14.03. Found: C, 47.99; H, 2.49; N, 14.33.
1-(4-Chlorophenyl)-3-(6-iodo-4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)urea (8f). Mp: 251–253 °C. Yield%: 90%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3296, 3226 (2NH), 3099 (CH aromatic), 1683, 1670 (2C=O), 1645 (C=N), 1552–1448 (C=C), 723 (C–Cl), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 7.28 (d, J = 5.1 Hz, 2H, phenyl-H), 7.48–7.58 (m, 3H, phenyl-H, pyridyl-H), 7.98 (d, 1H, J = 2.10 Hz, quinazolinyl-H), 8.18–8.23 (m, 2H, quinazolinyl-H, pyridyl-H), 8.43 (s, 1H, quinazolinyl-H), 8.78 (dd, 1H, J = 3.00, 6.30 Hz, pyridyl-H), 9.06 (s, 1H, pyridyl-H), 10.69 (s, 1H, NH exchanged by D₂O), 11.74 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 517 (M⁺, 19.22%), 519 (M⁺ + 2, 13.7%), 78 (100%). Anal. calcd for C₂₀H₁₃ClIN₅O₂ (517.71): C, 46.4; H, 2.53; N, 13.53. Found: C, 46.55; H, 2.80; N, 13.24.

1-(6-Iodo-4-oxo-2-(pyridin-4-yl)quinazolin-3(4H)-yl)-3-substituted urea/thiourea derivatives (8g–l). Compounds 8g–l were prepared and purified by applying the same method described for 8a–f starting with 7b and using the appropriate isocyanate or isothiocyanate.
1-Ethyl-3-(6-iodo-4-oxo-2-(pyridin-4-yl)quinazolin-3(4H)-yl)thiourea (8g). Mp: 229–231 °C. Yield: 83%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3321 (2NH), 3134 (CH aromatic), 2968, 2931 (CH₂, CH₃), 1683 (C=O), 1598 (C=N), 1512–1483 (C=C), 1213 (C=S), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 1.04 (t, 3H, J = 7.04, CH₃CH₂), 3.43–3.5 (q, 2H, J = 7.00, CH₃CH₂), 7.79 (d, 2H, J = 6.00 Hz, pyridyl-H), 7.98 (dd, 1H, J = 1.88, 8.68 Hz, quinazolinyl-H), 8.21 (d, 2H, J = 8.68 Hz, quinazolinyl-H), 8.86 (d, 2H, J = 6.00 Hz, pyridyl-H), 10.74 (s, 1H, NH exchanged by D₂O), 11.85 (s, 1H, NH exchanged by D₂O). ¹³C-NMR (100 MHz DMSO-d₆, ppm): δ 19.03 (CH₃), 39.05 (CH₂), 88.34, 121.32, 123.51, 123.86, 137.4, 138.26, 141.44, 141.57, 151.28, 163.76, 167.48 (C=O), 181.63 (C=S). Mass (m/z, rel. abundance): 451 (M⁺, 0.12%), 75 (100%). Anal. calcd for C₁₆H₁₄IN₅OS (451.28): C, 42.58; H, 3.13; N, 15.52. Found: C, 42.67; H, 3.33; N, 15.26.
1-Cyclohexyl-3-(6-iodo-4-oxo-2-(pyridin-4-yl)quinazolin-3(4H)-yl)urea (8h). Mp: 225–227 °C. Yield: 85%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3315 (br.) (2NH), 2924, 2850 (CH₂ aliphatic), 1687, 1660 (2C=O), 1647 (C=N), 1506 (C=C), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 1.25–1.72 (m, 10H, cyclohexyl-5CH₂), 3.39–3.46 (m, 1H, cyclohexyl-CH), 7.79 (d, 2H, J = 6.00 Hz, pyridyl-H), 7.95 (dd, 1H, J = 1.92, 8.72 Hz, quinazolinyl-H), 8.14 (d, 1H, J = 1.92 Hz, quinazolinyl-H), 8.21 (d, 1H, J = 8.68 Hz, quinazolinyl-H), 8.82 (d, 2H, J = 6.00 Hz, pyridyl-H), 10.48 (s, 1H, NH exchanged by D₂O), 11.86 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 489 (M⁺, 0.24%), 56 (100%). Anal. calcd for C₂₀H₂₀IN₅O₂ (489.31): C, 49.09; H, 4.12; N, 14.31. Found: C, 49.16; H, 3.92; N, 14.56.
1-Cyclohexyl-3-(6-iodo-4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)thiourea (8i). Mp: 254–256 °C. Yield: 70%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3325, 3230 (2NH), 2929, 2850 (CH₂ aliphatic), 1680 (C=O), 1640 (C=N), 1508 (C=C), 1116 (C=S), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 0.98–1.22 (m, 5H, cyclohexyl), 1.53–1.75 (m, 5H, cyclohexyl), 4.07 (m, 1H, cyclohexyl-CH), 7.77 (d, J = 5.40 Hz, 2H, pyridyl-H), 7.95–8.09 (m, 3H, quinazolinyl-H), 8.82 (d, 2H, J = 5.40 Hz, pyridyl-H), 10.64 (s, 1H, NH exchanged by D₂O), 11.67 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₂₀IN₅OS (505.38): C, 47.53; H, 3.99; N, 13.86. Found: C, 47.77; H, 4.08; N, 13.98.
1-(6-Iodo-4-oxo-2-(pyridin-4-yl)quinazolin-3(4H)-yl)-3-phenylurea (8j). Mp: 263–265 °C. Yield: 81% I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3304, 3211 (2NH), 3042 (CH aromatic), 1678, 1668 (2C=O), 1639 (C=N), 1558 (C=C), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 6.97 (t, 1H, J = 7.32 Hz, phenyl-H), 7.26 (t, 2H, J = 7.92 Hz, phenyl-H), 7.46 (d, 2H, J = 7.92 Hz, phenyl-H), 7.95 (d, 2H, J = 5.76 Hz, pyridyl-H), 8.00 (dd, 1H, J = 1.6, 8.72 Hz quinazolinyl-H), 8.22 (d, 1H, J = 1.64 Hz, quinazolinyl-H), 8.26 (s, 1H, J = 8.72 Hz, quinazolinyl-H), 8.79 (d, 2H, J = 5.76 Hz, pyridyl-H), 10.7 (s, 1H, NH exchanged by D₂O), 11.88 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 483 (M⁺, 0.22%), 91 (100%). Anal. calcd for C₂₀H₁₄IN₅O₂ (483.26): C, 49.71; H, 2.92; N, 14.49. Found: C, 49.65; H, 2.98; N, 14.36.
1-(6-Iodo-4-oxo-2-(pyridin-4-yl)quinazolin-3(4H)-yl)-3-phenylthiourea (8k). Mp: 227–229 °C. Yield: 78%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3340, 3300 (NH), 3062 (CH aromatic), 1693 (C=O), 1614 (C=N), 1531–1481 (C=C), 1213 (C=S), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.20 (t, 1H, J = 7.20 Hz, phenyl-H), 7.32–7.36 (m, 4H, phenyl-H), 7.40–7.55 (m, 1H, quinazolinyl-H), 7.75–7.77 (m, 2H, pyridyl-H), 7.98 (dd, 1H, J = 8.70 Hz, quinazolinyl-H), 8.23 (s, 1H, quinazolinyl-H), 8.80 (d, 2H, J = 6.30 Hz, pyridyl-H), 9.72 (s, 1H, NH exchanged by D₂O), 11.82 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 499 (M⁺, 1.55%), 364 (100%). Anal. calcd for C₂₀H₁₄IN₅OS (499.33): C, 48.11; H, 2.83; N, 14.03. Found: C, 48.32; H, 2.81; N, 14.25.
1-(4-Chlorophenyl)-3-(6-iodo-4-oxo-2-(pyridin-4-yl)quinazolin-3(4H)-yl)urea (8l). Mp: 238–240 °C. Yield%: 92%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3280 (2NH), 3107 (CH aromatic), 1666, 1649 (2CO), 1595 (C=N), 1558–1444 (C=C), 688 (C–Cl), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.31 (d, 2H, J = 8.52 Hz, phenyl-H), 7.50 (d, 2H, J = 8.52 Hz, phenyl-H), 7.78 (d, 2H, J = 4.84 Hz, pyridyl-H), 7.99 (d, H, J = 8.56 Hz, quinazolinyl-H), 8.22 (s, 1H, quinazolinyl-H), 8.26 (d, 1H, J = 8.56 Hz, quinazolinyl-H), 8.80 (d, 2H, J = 4.84 Hz, pyridyl-H), 10.72 (s, 1H, NH exchanged by D₂O), 11.87 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 517 (M⁺, 2.16%), 519 (M⁺ + 2, 1.48%), 115 (100%). Anal. calcd for C₂₀H₁₃ClIN₅O₂ (517.71): C, 46.4; H, 2.53; N, 13.53. Found: C, 46.36; H, 2.82; N, 13.37.

N-(2-{[2-(4-Substituted benzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-3-carboxamides (9a–g).
General method. The appropriate p-substituted benzaldehyde (2.40 mmol), the hydrazido derivative 7a (0.72 g, 2 mmol) in absolute ethanol (10 mL) and in the presence of glacial acetic acid (1 mL) were refluxed for 5–8 h. The reaction mixture was left to cool; the obtained solid was filtered, washed with diethyl ether, dried and recrystallized from absolute ethanol to produce the pure compounds 9a–g.
N-(2-{[2-(4-Fluorobenzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-3-carboxamide (9a). Mp: 237–239 °C. Yield: 65%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3309 (2NH), 3100 (CH aromatic), 2920 (C–H), 1687 (C=O), 1654 (C=O), 1597 (C=N), 1506 (C=C), 1234 (C–F), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.32 (t, 2H, J = 8.76 Hz, fluorophenyl-H), 7.63 (dd, 1H, J = 4.84, 7.88 Hz, pyridyl-H), 7.82 (dd, 2H, J = 5.64, 8.76 Hz, fluorophenyl-H), 7.97 (dd, 1H, J = 1.8 Hz, 8.64 Hz, iodophenyl-H), 8.19–8.27 (m, 3H, iodophenyl-H, pyridyl-H), 8.44 (s, 1H, C–H), 8.81 (dd, 1H, J = 1.36, 4.76 Hz, pyridyl-H), 9.1 (d, 1H, J = 1.84 Hz, pyridyl-H), 11.78 (s, 1H, NH exchanged by D₂O), 12.56 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₁₄FIN₄O₂ (488.25): C, 49.20; H, 2.89; N, 11.47. Found: C, 49.29; H, 2.91; N, 11.62.
N-(2-{[2-(4-Chlorobenzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-3-carboxamide (9b). Mp: 242–244 °C. Yield: 66%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3199 (NH), 3103 (CH aromatic), 2924 (C–H), 1672, 1660 (2C=O), 1595 (C=N), 1508 (C=C), 705 (C–Cl), 514 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.54 (d, 2H, J = 8.44 Hz, chloro phenyl-H), 7.63 (dd, 1H, J = 4.84 Hz, 7.88 Hz, pyridyl-H), 7.78 (d, 2H, J = 8.44 Hz, chlorophenyl-H), 7.97 (dd, 1H, J = 1.80, 8.64 Hz, iodophenyl-H), 8.19–8.27 (m, 3H, iodophenyl-H, pyridyl-H), 8.43 (s, 1H, C–H), 8.81 (dd, 1H, J = 1.24, 4.76 Hz, pyridyl-H), 9.1 (d, 1H, J = 1.8 Hz, pyridyl-H), 11.75 (s, 1H, NH exchanged by D₂O), 12.20 (s, 1H, NH exchanged by D₂O). ¹³C-NMR 100 MHz (DMSO-d₆, D₂O, ppm): δ 88.16, 124.21, 124.37, 129.23, 129.40, 129.47, 130.36, 133.38, 135.36, 135.42, 137.09, 138.76, 141.32, 148.33, 148.79, 153.14 (C–H), 163.77 (C=O), 163.82 (C=O). Mass (m/z, rel. abundance): 504 (M⁺, 1.06%), 506 (M⁺ + 2, 0.37%), 78 (100%). Anal. calcd for C₂₀H₁₄ClIN₄O₂ (504.71): C, 47.59; H, 2.80; N, 11.10. Found: C, 47.72; H, 2.78; N, 11.24.
N-(2-{[2-(4-Bromobenzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-3-carboxamide (9c). Mp: 243–245 °C. Yield: 75%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3197 (2NH), 3059 (CH aromatic), 2922 (C–H), 1672, 1660 (2C=O), 1593 (C=N), 1508 (C=C), 610 (C–Br), 520 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.63 (dd, 1H, J = 4.92, 7.96 Hz, pyridyl-H), 7.69 (m, 4H, bromophenyl-H), 7.97 (d, 1H, J = 1.88, 6.84 Hz, iodophenyl-H), 8.19–8.28 (m, 3H, iodophenyl-H, pyridyl-H), 8.41 (s, 1H, C–H), 8.81 (dd, 1H, J = 1.28, 3.48 Hz, pyridyl-H), 9.1 (d, 1H, J = 1.84 Hz, pyridyl-H), 11.75 (s, 1H, NH exchanged by D₂O), 12.20 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₁₄BrIN₄O₂ (549.16): C, 43.74; H, 2.57; N, 10.20. Found: C, 43.79; H, 2.53; N, 10.28.
N-[2-({2-[4-(Dimethylamino)benzylidene]hydrazinyl}carbonyl)-4-iodophenyl]pyridine-3-carboxamide (9d). Mp: 255–257 °C. Yield: 80%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3253 (2NH), 3194 (CH aromatic), 2987 (CH, CH₃), 1668, 1653 (2C=O), 1589 (C=N), 1510 (C=C), 510 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 2.99 (s, 6H, 2 × CH₃), 6.67 (d, 2H, J = 8.80 Hz, N,N-dimethylaminophenyl-H), 7.56 (d, 2H, J = 8.80 Hz, dimethylaminophenyl-H), 7.64 (dd, 1H, J = 4.88, 7.88 Hz, pyridyl-H), 7.95 (dd, 1H, J = 1.92, 8.76 Hz, iodophenyl-H), 8.2 (d, 1H, J = 1.92 Hz, iodophenyl-H), 8.26–8.31 (m, 2H, iodophenyl-H, pyridyl-H), 8.31 (s, 1H, C–H), 8.82 (dd, 1H, J = 1.44, 4.8 Hz, pyridyl-H), 9.11 (d, 1H, J = 1.88 Hz, pyridyl-H), 11.87 (s, 1H, NH exchanged by D₂O), 12.01 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 513 (M⁺, 1.44%), 163 (100%). Anal. calcd for C₂₂H₂₀IN₅O₂ (513.33): C, 51.47; H, 3.93; N, 13.64. Found: C, 51.53; H, 3.98; N, 13.81.
N-[4-Iodo-2-({2-[4-(trifluoromethyl)benzylidene]hydrazinyl}carbonyl)phenyl]-pyridine-3-carboxamide (9e). Mp: 245–247 °C. Yield: 72%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3427 (2NH), 3192 (CH aromatic), 2980 (C–H), 1681 (2C=O), 1595 (C=N), 1508 (C=C), 510 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.62 (dd, 1H, J = 3.00, 4.92 Hz, pyridyl-H), 7.82 (d, 2H, J = 8.28 Hz, trifluoromethyl phenyl-H), 7.95–7.99 (m, 3H, phenyl-H), 8.18–8.27 (m, 3H, phenyl-H, pyridyl-H), 8.50 (s, 1H, C–H), 8.81 (dd, 1H, J = 3.36, 4.76 Hz, pyridyl-H), 9.1 (s, J = 1.8 Hz, 1H, pyridyl-H), 11.69 (s, 1H, NH exchanged by D₂O), 12.31 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₁H₁₄F₃IN₄O₂ (538.26): C, 46.86; H, 2.62; N, 10.41. Found: C, 46.94; H, 2.68; N, 10.54.
N-(2-{[2-(4-Hydroxybenzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-3-carboxamide (9f). Mp: 273–275 °C. Yield: 79%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3238 (OH), 3161 (2 NH), 3080 (CH aromatic), 2924 (C–H), 1718, 1680 (2C=O), 1593 (C=N), 1496 (C=C), 510 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 6.85 (d, 2H, J = 8.44 Hz, hydroxyphenyl-H), 7.59 (d, 2H, J = 8.44 Hz, hydroxyphenyl-H), 7.63 (dd, 1H, J = 2.92, 4.96 Hz, pyridyl-H), 7.95 (dd, 1H, J = 1.44, 8.76 Hz, iodophenyl-H), 8.20 (d, 1H, J = 1.56 Hz, iodophenyl-H), 8.26–8.28 (m, 2H, iodophenyl-H, pyridyl-H), 8.34 (s, 1H, C–H), 8.82 (dd, 1H, J = 3.72, 4.72 Hz, pyridyl-H), 9.10 (d, 1H, J = 1.84 Hz, pyridyl-H), 10.00 (s, 1H, OH exchanged by D₂O), 11.93 (s, 1H, NH exchanged by D₂O), 11.96 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 486 (M⁺, 1.8%), 67 (100%). Anal. calcd for C₂₀H₁₅IN₄O₃ (486.26): C, 49.40; H, 3.11; N, 11.52. Found: C, 49.56; H, 3.16; N, 11.63.
N-(4-Iodo-2-{[2-(4-nitrobenzylidene)hydrazinyl]carbonyl}phenyl)pyridine-3-carboxamide (9g). Mp: 298–300 °C. Yield: 88%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3329 (2NH), 3103 (CH aromatic), 2949 (C–H), 1683, 1660 (2C=O), 1598 (C=N), 1577 (C=C), 1517 (C–NO₂), 510 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.63 (dd, 1H, J = 4.88, 7.08 Hz, pyridyl-H), 7.97–8.07 (m, 3H, phenyl-H), 8.16–8.32 (m, 5H, phenyl-H, pyridyl), 8.53 (s, 1H, C–H), 8.80 (d, 1H, J = 3.56 Hz, pyridyl-H), 9.1 (s, 1H, pyridyl-H), 11.65 (s, 1H, NH exchanged by D₂O), 12.41 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₁₄IN₅O₄ (515.26): C, 46.62; H, 2.74; N, 13.59. Found: C, 46.80; H, 2.71; N, 13.68.

N-(2-{[2-(4-Substituted benzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-4-carboxamides (9h–n). Compounds 9h–n were prepared according to the last mentioned procedure using 7b as a starting material and were recrystallized from absolute ethanol.
N-(2-{[2-(4-Fluorobenzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-4-carboxamide (9h). Mp: 288–290 °C. Yield: 59%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3296 (NH), 3122 (CH aromatic), 2820 (C–H), 1683 (C=O), 1630 (C=N), 1508 (C=C), 1232 (C–F), 532 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.37 (t, 2H, J = 8.68 Hz, fluorophenyl-H), 7.61 (d, 1H, J = 8.56 Hz, iodophenyl-H), 7.67 (d, 2H, J = 5.92 Hz, pyridyl-H), 7.79 (d, 2H, J = 3.12 Hz, fluorophenyl-H), 8.22 (dd, 1H, J = 8.72 Hz, iodophenyl-H), 8.52 (d, 1H, J = 1.92 Hz, iodophenyl-H), 8.69 (d, 2H, J = 5.92 Hz, pyridyl-H), 9.08 (s, 1H, C–H), 11.70 (s, 1H, NH exchanged by D₂O), 12.20 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₁₄FIN₄O₂ (488.25): C, 49.20; H, 2.89; N, 11.47. Found: C, 49.29; H, 2.92; N, 11.59.
N-(2-{[2-(4-Chlorobenzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-3-carboxamide (9i). Mp: 262–264 °C. Yield: 83%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3446 (NH), 3113 (CH aromatic), 2843 (C–H), 1680 (2C=O), 1587 (C=N), 1506–1442 (C=C), 750 (C–Cl), 520 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.54 (d, 2H, J = 8.40 Hz, chloro phenyl-H), 7.78 (d, 2H, J = 8.40 Hz, chloro phenyl-H), 7.82 (d, 2H, J = 5.88 Hz, pyridyl-H), 7.99 (dd, 1H, J = 1.64, 8.72 Hz, iodophenyl-H), 8.21 (d, 1H, J = 1.60 Hz, iodophenyl-H), 8.24 (d, 1H, J = 8.72 Hz, iodophenyl-H), 8.44 (s, 1H, C–H), 8.84 (d, 2H, J = 5.88 Hz, pyridyl-H), 11.88 (s, 1H, NH exchanged by D₂O), 12.22 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 504 (M⁺, 0.23%), 506 (M⁺ + 2, 0.13%), 78 (100%). Anal. calcd for C₂₀H₁₄ClIN₄O₂ (504.71): C, 47.59; H, 2.80; N, 11.10. Found: C, 47.67; H, 2.82; N, 11.23.
N-(2-{[2-(4-Bromobenzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-4-carboxamide (9j). Mp: 283–285 °C. Yield: 70%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3423 (NH), 3076 (CH aromatic), 2843 (C–H), 1685 (2C=O), 1577 (C=N), 1506 (C=C), 526 (C–Br), 514 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.67–7.72 (m, 4H, bromophenyl-H), 7.82 (d, 2H, J = 5.80 Hz, pyridyl-H), 7.98 (dd, 1H, J = 1.60, 8.72 Hz, iodophenyl-H), 8.21 (d, 1H, J = 1.60 Hz, iodophenyl-H), 8.24 (d, 1H, J = 8.76 Hz, iodophenyl-H), 8.42 (s, 1H, C–H), 8.84 (d, 2H, J = 5.80 Hz, pyridyl-H), 11.87 (s, 1H, NH exchanged by D₂O), 12.22 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₁₄BrIN₄O₂ (549.16): C, 43.74; H, 2.57; N, 10.20. Found: C, 43.85; H, 2.53; N, 10.34.
N-[2-({2-[4-(Dimethylamino)benzylidene]hydrazinyl}carbonyl)-4-iodophenyl]-pyridine-4-carboxamide (9k). Mp: 228–230 °C. Yield: 80%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3408 (NH), 3159 (CH aromatic), 2880 (CH, CH₃(s)), 1672 (2C=O), 1610 (C=N), 1508 (C=C), 514 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 3.01 (s, 6H, 2 × CH₃), 6.77 (d, 2H, J = 8.92 Hz, aminophenyl-H), 7.56 (d, 2H, J = 8.92 Hz, aminophenyl-H), 7.83 (d, 2H, J = 5.96 Hz, pyridyl-H), 7.96 (dd, 1H, J = 1.80, 8.76 Hz, iodophenyl-H), 8.22 (d, 1H, J = 1.84 Hz, iodophenyl-H), 8.31 (d, 1H, J = 1.6 Hz, iodophenyl-H), 8.33 (s, 1H, C–H), 8.85 (d, 2H, J = 5.96 Hz, pyridyl-H), 11.89 (s, 1H, NH exchanged by D₂O), 12.43 (s, 1H, NH exchanged by D₂O). ¹³C-NMR 100 MHz (DMSO-d₆, D₂O, ppm): δ 56.49 (2 × CH₃ carbon), 88.25, 112.24, 121.36, 121.48, 123.69, 124.23, 129.24, 136.95, 138.78, 141.18, 141.70, 150.88 (C–H), 151.28, 152.25, 163.25 (C=O), 163.48 (C=O). Anal. calcd for C₂₂H₂₀IN₅O₂ (513.33): C, 51.47; H, 3.93; N, 13.64. Found: C, 51.53; H, 3.98; N, 13.81.
N-[4-Iodo-2-({2-[4-(trifluoromethyl)benzylidene]hydrazinyl}carbonyl)phenyl]-pyridine-4-carboxamide (9l). Mp: 238–240 °C. Yield: 67%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3412 (NH), 3211 (CH aromatic), 2840 (C–H), 1678 (C=O), 1651 (C=O), 1589 (C=N), 1508–1444 (C=C), 540 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.82–7.84 (m, 4H, trifluorophenyl-H), 7.97 (d, 2H, J = 7.88 Hz, pyridyl-H), 8.00 (d, 1H, J = 1.64, iodophenyl-H), 8.21–8.23 (m, 2H, iodophenyl-H), 8.51 (s, 1H, C–H), 8.84 (d, 2H, J = 7.88 Hz, pyridyl-H), 11.82 (s, 1H, NH exchanged by D₂O), 12.33 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 538 (M⁺, 0.44%), 106 (100%). Anal. calcd for C₂₁H₁₄F₃IN₄O₂ (538.26): C, 46.86; H, 2.62; N, 10.41. Found: C, 46.95; H, 2.61; N, 10.50.
N-(2-{[2-(4-Hydroxybenzylidene)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-4-carboxamide (9m). Mp: 297–299 °C. Yield: 90%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3176 (OH), 3111 (NH), 3057 (CH aromatic), 2868 (C–H), 1685 (C=O), 1643 (C=O), 1610 (C=N), 1512 (C=C), 536 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 6.86 (d, 2H, J = 8.44 Hz, hydroxyphenyl-H), 7.59 (d, 2H, J = 8.44 Hz, hydroxyphenyl-H), 7.82 (d, 2H, J = 5.68 Hz, pyridyl-H), 7.96 (d, 1H, J = 8.64 Hz, iodophenyl-H), 8.21 (d, 1H, J = 1.40 Hz, iodophenyl-H), 8.30 (d, 1H, J = 8.72 Hz, iodophenyl-H), 8.35 (s, 1H, C–H), 8.84 (d, 2H, J = 5.68 Hz, pyridyl-H), 10.01 (s, 1H, OH exchanged by D₂O), 11.98 (s, 1H, NH exchanged by D₂O), 12.07 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₁₅IN₄O₃ (486.26): C, 49.40; H, 3.11; N, 11.52. Found: C, 49.48; H, 3.14; N, 11.58.
N-(4-Iodo-2-{[2-(4-nitrobenzylidene)hydrazinyl]carbphenyl)pyridine-4-carboxamide (9n). Mp: 298–300 °C. Yield: 88%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3487 (NH), 3109 (CH aromatic), 2837 (C–H), 1689 (C=O), 1645 (C=O), 1635 (C=N), 1555 (C–NO₂), 1514 (C=C), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.82 (d, 2H, J = 5.8 Hz, pyridyl-H), 8.00–8.02 (m, 3H, phenyl-H), 8.19 (d, 1H, J = 8.7 Hz, iodophenyl-H), 8.21 (d, 1H, J = 1.64 Hz, iodophenyl-H), 8.32 (d, 2H, J = 8.44 Hz, nitrophenyl-H), 8.53 (s, 1H, C–H), 8.84 (d, 2H, J = 5.8 Hz, pyridyl-H), 11.76 (s, 1H, NH exchanged by D₂O), 12.41 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₁₄IN₅O₄ (515.26): C, 46.62; H, 2.74; N, 13.59. Found: C, 46.80; H, 2.71; N, 13.68.

3-[(4-Substituted benzylidene)amino]-6-iodo-2-(pyridin-3-yl)quinazolin-4(3H)-one (10a–d). A mixture containing equimolar amounts of sodium ethoxide and the diamide 9a, 9b, 9c or 9g (1 mmol) was heated under reflux for 3 h. The reaction mixture was cooled to room temperature. The separated solid was filtered, washed with water and recrystallized from ethanol.
3-[(4-Fluorobenzylidene)amino]-6-iodo-2-(pyridin-3-yl)quinazolin-4(3H)-one (10a). Mp: 337–339 °C. Yield: 80%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3190 (CH aromatic), 2922 (C–H), 1681 (C=O), 1593 (C=N), 1506 (C=C), 1226 (C–F), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.23 (t, 2H, J = 8.64 Hz, fluorophenyl-H), 7.59 (d, 2H, J = 7.44 Hz, fluorophenyl-H), 7.64 (d, 1H, J = 8.68 Hz, quinazolinyl-H), 7.70 (t, 1H, J = 7.00 Hz, pyridyl-H), 8.18 (s, 1H, C–H), 8.32 (d, 1H, J = 7.76 Hz, quinazolinyl-H), 8.47 (d, 1H, J = 8.64 Hz, pyridyl-H), 8.51 (s, 1H, quinazolinyl-H), 8.77 (d, 1H, J = 3.76 Hz, pyridyl), 9.17 (s, 1H, pyridyl). Mass (m/z, rel. abundance): 470 (M⁺, 9.28%), 78 (100%). Anal. calcd for C₂₀H₁₂FIN₄O (470.24): C, 51.08; H, 2.57; N, 11.91. Found: C, 51.19; H, 2.55; N, 12.04.
3-[(4-Chlorobenzylidene)amino]-6-iodo-2-(pyridin-3-yl)quinazolin-4(3H)-one (10b). Mp: 238–340 °C. Yield: 77%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3290 (CH aromatic), 2922 (C–H), 1645 (C=O), 1577 (C=N), 1504 (C=C), 750 (C–Cl), 520 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.44 (d, 2H, J = 7.76 Hz, chloro phenyl-H), 7.82 (d, 2H, J = 8.54 Hz, chloro phenyl-H), 7.89 (d, 1H, J = 5.88 Hz, pyridyl-H), 8.20 (dd, 1H, J = 1.58, 8.70 Hz, quinazlinyl-H), 8.24–8.35 (m, 3H, quinazolinyl-H, pyridyl-H), 8.44 (d, 1H, J = 8.72 Hz, quinazolinyl-H), 8.65 (s, 1H, C–H), 8.84 (d, 1H, J = 6.00 Hz, pyridyl-H). Anal. calcd for C₂₀H₁₂ClIN₄O (486.69): C, 49.36; H, 2.49; N, 11.51. Found: C, 49.08; H, 2.60; N, 11.77.
3-[(4-Bromobenzylidene)amino]-6-iodo-2-(pyridin-3-yl)quinazolin-4(3H)-one (10c). Mp: 335–337 °C. Yield: 81%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3284 (CH aromatic), 2951 (C–H), 1678 (C=O), 1587 (C=N), 1506 (C=C), 526 (C–Br), 514 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.56–7.62 (m, 4H, bromophenyl-H), 7.65 (d, 2H, J = 2.76 Hz, pyridyl-H, quinazolinyl-H), 8.13 (s, 1H, C–H), 8.32 (d, 1H, J = 7.84 Hz, quinazolinyl-H), 8.47 (d, 2H, J = 8.72 Hz, quinazolinyl-H, pyridyl-H), 8.50 (s, 1H, pyridyl-H), 8.77 (d, 1H, J = 4.44 Hz, pyridyl-H). Mass (m/z, rel. abundance): 531 (M⁺, 19.59%), 532 (M⁺ + 1, 13.21%), 78 (100%). Anal. calcd for C₂₀H₁₂BrIN₄O (531.14): C, 45.23; H, 2.28; N, 10.55. Found: C, 45.31; H, 2.26; N, 10.63.
6-Iodo-3-((4-nitrobenzylidene)amino)-2-(pyridin-3-yl)quinazolin-4(3H)-one (10d). Mp: 315–317 °C. Yield: 70%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3064 (CH aromatic), 2954 (CH), 1654 (C=O), 1629 (C=N), 1512 (C–NO₂), 1506 (C=C), 514 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.60–7.73 (m, 4H, nitrophenyl-H), 8.22 (d, 2H, J = 8.28 Hz, pyridyl-H, quinazolinyl-H), 8.30–8.34 (m, 2H, pyridyl-H, quinazolinyl-H), 8.46 (d, 1H, J = 7.36 Hz, quinazolinyl-H), 8.53 (s, 1H, C–H), 8.80 (d, 1H, J = 4.64 Hz, pyridyl-H), 9.17 (s, 1H, pyridyl-H). Mass (m/z, rel. abundance): 497 (M⁺, 37.31%), 78 (100%). Anal. calcd for C₂₀H₁₂IN₅O₃ (497.25): C, 48.31; H, 2.43; N, 14.08. Found: C, 48.50; H, 2.46; N, 14.17.

3-[(4-Substituted benzylidene)amino]-6-iodo-2-(pyridin-4-yl)quinazolin-4(3H)-one (10e–h). Compounds 10e–h were similarly prepared and purified adopting the method described for compounds 10a–f.
3-[(4-Fluorobenzylidene)amino]-6-iodo-2-(pyridin-4-yl)quinazolin-4(3H)-one (10e). Mp: 304–306 °C. Yield: 59%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3111 (CH aromatic), 2921 (C–H), 1645 (C=O), 1598 (C=N), 1506 (C=C), 1230 (C–F), 536 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.32 (t, 2H, J = 7.60 Hz, phenyl-H), 7.82 (d, 2H, J = 7.60 Hz, phenyl-H), 7.98 (d, 2H, J = 8.76 Hz, quinazolinyl-H), 8.21 (d, 2H, J = 5.44 Hz, pyridyl-H), 8.21–8.26 (m, 2H, quinazolinyl-H), 8.45 (s, 1H, C–H), 8.84 (d, 2H, J = 5.44 Hz, pyridyl-H). Mass (m/z, rel. abundance): 470 (M⁺, 0.46%), 75 (100%). Anal. calcd for C₂₀H₁₂FIN₄O (470.24): C, 51.08; H, 2.57; N, 11.91. Found: C, 51.22; H, 2.62; N, 12.07.
3-[(4-Chlorobenzylidene)amino]-6-iodo-2-(pyridin-4-yl)quinazolin-4(3H)-one (10f). Mp: 331–333 °C. Yield: 65%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3111 (CH aromatic), 2920 (C–H), 1654 (C=O), 1597 (C=N), 1508 (C=C), 750 (C–Cl), 520 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.44 (d, 2H, J = 8.28 Hz, chloro phenyl-H), 7.64 (dd, 1H, J = 1.76, 8.76 Hz, quinazolinyl-H), 7.67 (d, 2H, J = 8.40 Hz, chloro phenyl-H), 7.88 (d, 2H, J = 5.44 Hz, pyridyl-H), 8.16 (s, 1H, C–H), 8.45 (d, 1H, J = 1.60 Hz, quinazolinyl-H), 8.50 (d, 1H, J = 8.72 Hz, quinazolinyl-H), 8.78 (d, 2H, J = 5.44 Hz, pyridyl-H). Anal. calcd for C₂₀H₁₂ClIN₄O (486.69): C, 49.36; H, 2.49; N, 11.51. Found: C, 49.43; H, 2.54; N, 11.67.
3-[(4-Bromobenzylidene)amino]-6-iodo-2-(pyridin-4-yl)quinazolin-4(3H)-one (10g). Mp: 327–329 °C. Yield: 72%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3203 (CH aromatic), 2968 (C–H), 1649 (2C=O), 1577 (C=N), 1506 (C=C), 526 (C–Br), 512 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.57–7.65 (m, 5H, bromophenyl-H, quinazolinyl-H), 7.88 (d, 2H, J = 5.84 Hz, pyridyl-H), 8.14 (d, 1H, J = 3.60 Hz, quinazolinyl-H), 8.46 (d, 1H, J = 8.60 Hz, quinazolinyl-H), 8.51 (s, 1H, C–H), 8.79 (d, 2H, J = 5.84 Hz, pyridyl-H). Anal. calcd for C₂₀H₁₂BrIN₄O (531.14): C, 45.23; H, 2.28; N, 10.55. Found: C, 45.34; H, 2.31; N, 10.73.
6-Iodo-3-((4-nitrobenzylidene)amino)-2-(pyridin-4-yl)quinazolin-4(3H)-one (10h). Mp: 333 °C. Yield: 84%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3070 (CH aromatic), 2960 (CH), 1657 (C=O), 1610 (C=N), 1518 (C–NO₂), 1508 (C=C), 514 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 7.68 (d, 1H, J = 8.36 Hz, quinazolinyl-H), 7.88–7.90 (m, 4H, nitrophenyl-H), 8.20 (d, 2H, J = 5.28 Hz, pyridyl-H), 8.29 (s, 1H, C–H), 8.47 (d, 1H, J = 8.40 Hz, quinazolinyl-H), 8.53 (s, 1H, quinazolinyl-H), 8.82 (d, 2H, J = 5.28 Hz, pyridyl-H). Mass (m/z, rel. abundance): 497 (M⁺, 1.4%), 59 (100%). Calcd for C₂₀H₁₂IN₅O₃ (497.25): C, 48.31; H, 2.43; N, 14.08. Found: C, 48.39; H, 2.41; N, 14.19.

N-(2-{[2-(Chloroacetyl)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-3-carboxamide (11a). The hydrazido derivative 7a (10 mmol, 3.64 g) and chloroacetyl chloride (15 mmol, 1.69 g) were stirred overnight at room temperature in dimethylformamide (10 mL). The reaction mixture was poured onto ice-cold water and the separated solid was collected, dried and recrystallized from methanol. Mp: 276–278 °C. Yield: 83% I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3479 (NH), 3176 (CH aromatic), 2953 (CH₂), 1707, 1687 (3C=O), 1651 (C=N), 1516 (C=C), 785 (C–Cl), 524 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 4.24 (s, 2H, CH₂), 7.62 (dd, 1H, J = 3.64, 7.88 Hz, pyridyl-H), 7.98 (dd, 1H, J = 1.72, 8.76 Hz, phenyl-H), 8.19 (d, 1H, J = 1.76 Hz, phenyl-H), 8.24 (d, 1H, J = 7.96 Hz, pyridyl-H), 8.29 (d, 1H, J = 8.76 Hz, phenyl-H), 8.80 (dd, 1H, J = 1.04, 4.68 Hz, pyridyl-H), 9.1 (d, 1H, J = 1.72 Hz, pyridyl-H), 10.56 (s, 1H, NH exchanged by D₂O), 10.98 (s, 1H, NH exchanged by D₂O), 11.81 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₁₅H₁₂ClIN₄O₃ (458.64): C, 39.28; H, 2.64; N, 12.22. Found: C, 39.41; H, 2.68; N, 12.38.

N-(2-{[2-(Chloroacetyl)hydrazinyl]carbonyl}-4-iodophenyl)pyridine-4-carboxamide (11b). This compound was prepared and purified as per the previously mentioned procedure, using the hydrazido derivative 7b. Mp: 289–291 °C. Yield: 80%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3468, 3448 (NHs), 3178 (CH aromatic), 2974 (CH aliphatic), 1695, 1685 (3C=O), 1631 (C=N), 1508–1436 (C=C), 524 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 4.24 (s, 2H, CH₂), 7.79 (d, 2H, J = 5.48 Hz, pyridyl-H), 7.99 (d, 1H, J = 8.68 Hz, phenyl), 8.20 (s, 1H, phenyl), 8.30 (d, 1H, J = 8.76 Hz, phenyl-H), 8.83 (dd, 2H, J = 5.48 Hz, pyridyl-H), 10.58 (s, 1H, NH exchanged by D₂O), 11.00 (s, 1H, NH exchanged by D₂O), 11.89 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 460 (M⁺ + 2, 1.10%), 458 (M⁺, 2.37%), 56 (100%). Anal. calcd for C₁₅H₁₂ClIN₄O₃ (458.64): C, 39.28; H, 2.64; N, 12.22. Found: C, 39.38; H, 2.68; N, 12.28.

N-(4-Iodo-2-{[2-(substituted acetyl)hydrazinyl]carbonyl}phenyl)pyridine-3-carboxamide (12a–d). Equimolar amounts of chloroacetyl hydrazido derivative 11a (0.45 g, 1 mmol) and the appropriate secondary amine (1 mmol) in absolute ethanol (15 mL) containing anhydrous potassium carbonate (1.5 mmol, 0.2 g), were refluxed for 12 h. The reaction mixture was filtered while hot and the clear solution was evaporated under reduced pressure. The remaining residue was collected, washed with water and recrystallized from ethanol affording the corresponding derivatives 12a–d.
N-(4-Iodo-2-{[2-(piperidin-1-ylacetyl)hydrazinyl]carbonyl}phenyl)pyridine-3-carboxamide (12a). Mp: 200–202 °C. Yield: 55%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3419 (NHs), 3132 (CH aromatic), 2941 (CH₂), 1670 (C=O), 1653 (C=N), 1541 (C=C), 516 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 1.39 (d, J = 4.44 Hz, 2H, piperidinyl-H), 1.55 (t, J = 5.36 Hz, 4H, piperidinyl-H), 3.09 (s, 2H, CH₂), 3.34 (s, 4H, piperidinyl-H), 7.60 (dd, 1H, J = 4.84, 7.92 Hz, pyridyl-H), 7.95 (dd, 1H, J = 1.48, 8.72 Hz, phenyl-H), 8.20 (d, 1H, J = 1.68 Hz, phenyl-H), 8.30 (d, 1H, J = 7.60 Hz, pyridyl-H), 8.36 (d, 1H, J = 8.76 Hz, phenyl-H), 8.79 (dd, 1H, J = 1.04, 4.72 Hz, pyridyl-H), 9.07 (d, 1H, J = 1.64 Hz, pyridyl-H), 9.93 (s, 1H, NH exchanged by D₂O), 10.74 (s, 1H, NH exchanged by D₂O), 12.07 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₀H₂₂IN₅O₃ (507.32): C, 47.35; H, 4.37; N, 13.80. Found: C, 47.54; H, 4.44; N, 13.96.
N-(4-Iodo-2-{[2-(morpholin-4-ylacetyl)hydrazinyl]carbonyl}phenyl)pyridine-3-carboxamide (12b). Mp: 268–270 °C. Yield: 53%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3251 (NHs), 3182 (CH aromatic), 2856 (CH₂), 1700, 1681 (3C=O), 1641 (C=N), 1556 (C=C), 514 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 2.52 (t, 4H, J = 4.04 Hz, morpholinyl-H), 3.11 (s, 2H, CH₂), 3.63 (t, 4H, J = 4.52 Hz, morpholinyl-H), 7.61 (dd, 1H, J = 4.80, 7.88 Hz, pyridyl-H), 7.97 (dd, 1H, J = 1.73, 8.80 Hz, phenyl-H), 8.18 (d, 1H, J = 1.44 Hz, phenyl-H), 8.25 (d, 1H, J = 7.88 Hz, pyridyl-H), 8.35 (d, 1H, J = 8.76 Hz, phenyl-H), 8.81 (dd, 1H, J = 1.12, 4.68 Hz, pyridyl-H), 9.06 (d, 1H, J = 1.76 Hz, pyridyl-H), 9.98 (s, 1H, NH exchanged by D₂O), 10.74 (s, 1H, NH exchanged by D₂O), 11.90 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 509 (M⁺, 1.04%), 60 (100%). Anal. calcd for C₁₉H₂₀IN₅O₄ (509.30): C, 44.81; H, 3.96; N, 13.75. Found: C, 44.89; H, 3.99; N, 13.89.
Ethyl 4-({N′[(5-iodo-2-pyridine-3-amido)benzoyl]hydrazinecarbonyl}methyl)-piperazine-carboxylate (12c). Mp: 237–239 °C. Yield: 78%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3244 (NHs), 3111 (CH aromatic), 2927 (CH₂ aliphatic), 1701, 1681, 1654 (3C=O), 1597 (C=N), 1508 (C=C), 516 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 1.18 (t, 3H, J = 7.08 Hz, CH₃CH₂), 3.15 (s, 2H, CH₂), 3.41 (s, 8H, piperazinyl-H), 4.04 (q, 2H, J = 7.04 Hz, CH₃CH₂), 7.61 (dd, 1H, J = 4.84, 7.84 Hz, pyridyl-H), 7.97 (dd, 1H, J = 1.68, 8.76 Hz, phenyl-H), 8.18 (d, 1H, J = 1.76 Hz, phenyl-H), 8.24 (d, 1H, J = 7.96 Hz, pyridyl-H), 8.35 (d, 1H, J = 8.80 Hz, phenyl-H), 8.80 (dd, 1H, J = 3.56, 4.76 Hz, pyridyl-H), 9.06 (d, 1H, J = 1.68 Hz, pyridyl-H), 10.01 (s, 1H, NH exchanged by D₂O), 10.80 (s, 1H, NH exchanged by D₂O), 11.89 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 580 (M⁺, 0.17%), 165 (100%). Anal. calcd for C₂₂H₂₅IN₆O₅ (580.38): C, 45.53; H, 4.34; N, 14.48. Found: C, 45.58; H, 4.36; N, 15.55.
N-(4-Iodo-2-{[2-(4-phenylpiperazin-1-ylacetyl)hydrazinyl]carbonyl}phenyl)pyridine-3-carboxamide (12d). Mp: 213–215 °C. Yield: 62%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3219 (NHs), 3188 (CH aromatic), 2823 (CH aliphatic), 1683, 1658 (3C=O), 1597 (C=N), 1506–1452 (C=C), 514 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 2.51 (t, 4H, J = 1.68 Hz, piperazinyl), 2.69 (s, 4H, piperazinyl), 3.19 (s, 2H, CH₂), 6.78 (t, 1H, J = 7.2 Hz, phenyl-H), 6.95 (d, 2H, J = 7.44 Hz, phenyl-H), 7.22 (t, 2H, J = 7.44 Hz, phenyl-H), 7.60 (dd, 1H, J = 4.84, 7.88 Hz, pyridyl-H), 7.97 (dd, 1H, J = 1.80, 8.72 Hz, iodophenyl-H), 8.19 (d, 1H, J = 1.88 Hz, iodophenyl-H), 8.25 (d, 1H, J = 7.96 Hz, pyridyl-H), 8.35 (d, 1H, J = 8.76 Hz, iodophenyl-H), 8.80 (dd, 1H, J = 1.32, 4.76 Hz, pyridyl-H), 9.07 (d, 1H, J = 1.80 Hz, pyridyl-H), 10.01 (s, 1H, NH exchanged by D₂O), 10.82 (s, 1H, NH exchanged by D₂O), 11.90 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₅H₂₅IN₆O₃ (584.41): C, 51.38; H, 4.31; N, 14.38. Found: C, 51.54; N, 4.36; N, 14.48.

N-(4-Iodo-2-{[2-(substituted acetyl)hydrazinyl]carbonyl}phenyl)pyridine-4-carboxamide (12e–h). Derivatives 12e–h were prepared by applying the procedure used for the preparation of the nicotinamido analogues 12a–d and were recrystallized from ethanol.
N-(4-Iodo-2-{[2-(piperidin-1-ylacetyl)hydrazinyl]carbonyl}phenyl)pyridine-4-carboxamide (12e). Mp: 204–206 °C. Yield: 45%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3412 (NHs), 3118 (CH aromatic), 2926 (CH₂ aliphatic), 1668 (3C=O), 1629 (C=N), 1577 (C=C), 518 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 1.40 (s, 2H, piperidinyl-H), 1.57 (s, 4H, piperidinyl-H), 3.08 (s, 2H, CH₂), 3.32 (s, 4H, piperidinyl-H), 7.83 (d, 2H, J = 5.68 Hz, pyridyl-H), 7.96 (d, 1H, J = 8.92 Hz, phenyl-H), 8.22 (s, 1H, phenyl-H), 7.36 (d, 1H, J = 8.84 Hz, phenyl-H), 8.82 (d, 2H, J = 5.68 Hz, pyridyl-H), 9.93 (s, 1H, NH exchanged by D₂O), 10.78 (s, 1H, NH exchanged by D₂O), 12.02 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 507 (M⁺, 1.32%), 85 (100%). Anal. calcd for C₂₀H₂₂IN₅O₃ (507.32): C, 47.35; H, 4.37; N, 13.80. Found: C, 47.50; H, 4.39; N, 13.89.
N-(4-Iodo-2-{[2-(morpholin-4-ylacetyl)hydrazinyl]carbonyl}phenyl)yridine-4-carboxamide (12f). Mp: 229–231 °C. Yield: 70%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3444 (NHs), 3120 (CH aromatic), 2927 (CH₂ aliphatic), 1697, 1681, 1651 (3C=O), 1598 (C=N), 1510–1440 (C=C), 520 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 2.51 (t, 4H, J = 1.72 Hz, morpholinyl-H), 3.12 (s, 2H, CH₂), 3.63 (t, 4H, J = 4.52 Hz, morpholinyl-H), 7.79 (d, 2H, J = 5.96 Hz, pyridyl-H), 7.98 (dd, 1H, J = 1.92, 8.76 Hz, phenyl-H), 8.20 (d, 1H, J = 1.96 Hz, phenyl-H), 8.35 (d, 1H, J = 8.76 Hz, phenyl-H), 8.82 (dd, 2H, J = 1.40, 4.52 Hz, pyridyl-H), 9.99 (s, 1H, NH exchanged by D₂O), 10.80 (s, 1H, NH exchanged by D₂O), 11.96 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₁₉H₂₀IN₅O₄ (509.30): C, 44.81; H, 3.96; N, 13.75. Found: C, 44.89; H, 3.98; N, 13.79.
Ethyl 4-({N′[(5-iodo-2-pyridine-4-amido)benzoyl]hydrazinecarbonyl}methyl)-piperazinecarboxylate (12g). Mp: 176–178 °C. Yield: 60%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3479, 3419 (NHs), 3228 (CH aromatic), 2980 (CH₂ aliphatic), 1683, 1653 (3C=O), 1591 (C=N), 1508 (C=C), 538 (C–I). ¹H-NMR 400 MHz (CDCl₃, D₂O, ppm): δ 1.30 (t, 3H, J = 7.08 Hz, CH₃CH₂), 2.69 (s, 4H, piperazinyl-H), 3.32 (s, 2H, CH₂), 3.62 (s, 4H, piperazinyl-H), 4.18 (q, 2H, J = 7.08 Hz, CH₃CH₂), 7.60 (d, 1H, J = 8.72 Hz, phenyl-H), 7.84 (d, 2H, J = 5.36 Hz, pyridyl-H), 8.05 (s, 1H, phenyl-H), 8.53 (d, 1H, J = 8.72 Hz, phenyl-H), 8.85 (d, 2H, J = 5.36 Hz, pyridyl-H), 9.38 (s, 1H, NH exchanged by D₂O), 10.53 (s, 1H, NH exchanged by D₂O), 12.14 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₂H₂₅IN₆O₅ (580.38): C, 45.53; H, 4.34; N, 14.48. Found: C, 45.70; H, 4.19; N, 14.50.
N-(4-Iodo-2-{[2-(4-phenylpiperazin-1-ylacetyl)hydrazinyl]carbonyl}phenyl)pyridine-3-carboxamide (12h). Mp: 240–242 °C. Yield: 68%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3277 (NHs), 3103 (CH aromatic), 2891 (CH₂ aliphatic), 1695, 1678, 1647 (2C=O), 1593 (C=N), 1556–1452 (C=C), 511 (C–I). ¹H-NMR 400 MHz (CDCl₃-d₆, D₂O, ppm): δ 2.88 (t, 4H, J = 4.56 Hz, piperazinyl-H), 3.33 (t, 4H, J = 4.6 Hz, piperazinyl-H), 3.36 (s, 2H, CH₂), 6.93 (t, 1H, J = 7.4 Hz, phenyl-H), 6.97 (d, 2H, J = 8.32 Hz, phenyl-H), 7.32 (t, 2H, J = 8.32 Hz, phenyl-H), 7.77 (dd, 1H, J = 1.80, 8.88 Hz, iodophenyl-H), 7.84 (dd, 2H, J = 1.48, 4.48 Hz, pyridyl-H), 8.04 (d, 1H, J = 1.72 Hz, iodophenyl-H), 8.61 (d, 1H, J = 8.88 Hz, iodophenyl-H), 8.83 (d, 2H, J = 6.00 Hz, pyridyl-H), 9.58 (s, 2H, NH exchanged by D₂O), 11.99 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₂₅H₂₅IN₆O₃ (584.41): C, 51.38; H, 4.31; N, 14.38. Found: C, 51.46; N, 4.16; N, 14.46.

3-Hydroxy-6-iodo-2-(pyridin-3-yl)quinazolin-4(3H)-one (13a). A mixture of (pyridin-3-yl)-benzoxazin-4-one 6a (3.5 g, 10 mmol) and hydroxylamine hydrochloride (0.69 g, 10 mmol) in dry pyridine (10 mL) was heated under reflux for 8 h. The product obtained upon cooling was filtered off, washed with diluted acetic acid and recrystallized from ethanol. Mp: 317–319 °C. Yield: 48%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3151 (OH), 3082 (CH aromatic), 1666 (C=O), 1608 (C=N), 1517 (C=C), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 7.64 (t, J = 5.10 Hz, 1H, pyridyl-H), 7.99 (dd, J = 1.80, 8.70 Hz, 1H, quinazolinyl-H), 8.26–8.29 (m, 2H, pyridyl-H), 8.40 (d, J = 8.7 Hz, 1H, quinazolinyl-H), 8.83 (s, 1H, quinazolinyl-H), 9.12 (s, 1H, pyridyl-H), 12.04 (s, 1H, OH exchanged by D₂O). Mass (m/z, rel. abundance): 365 (M⁺, 0.15%), 350 (100%). Anal. calcd for C₁₃H₈IN₃O₂ (365.13): C, 42.76; H, 2.21; N, 11.51. Found: C, 42.90; H, 2.29; N, 11.66.

3-Hydroxy-6-iodo-2-(pyridin-4-yl)quinazolin-4(3H)-one (13b). Compound 13b was similarly prepared and purified as compound 13a but using (pyridin-4-yl)-benzoxazin-4-one (6b) as a starting material. Mp: 297–299 °C. Yield: 42%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3431 (OH), 3068 (CH aromatic), 1681 (C=O), 1593 (C=N), 1496 (C=C), 534 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.82 (d, 2H, J = 5.32 Hz, pyridyl-H), 8.01 (d, 1H, J = 8.64 Hz, quinazolinyl-H), 8.30 (d, 1H, J = 1.20 Hz, quinazolinyl-H), 8.42 (d, 1H, J = 8.80 Hz, quinazolinyl-H), 8.85 (d, 2H, J = 5.32 Hz, pyridyl-H), 12.19 (s, 1H, OH exchanged by D₂O). Mass (m/z, rel. abundance): 365 (M⁺, 9.23%), 350 (100%). Anal. calcd for C₁₃H₈IN₃O₂ (365.13): C, 42.76; H, 2.21; N, 11.51. Found: C, 42.87; H, 2.24; N, 11.67.

6-Iodo-4-oxo-2-(pyridin-3-yl)quinazoline-3(4H)-carboxamide (14a). To a suspension of the benzoxazinone 6a (3.5 g, 10 mmol) in glacial acetic acid (10 mL), anhydrous sodium acetate (1.64 g, 20 mmol) and urea (0.6 g, 10 mmol) were added. The mixture was heated under reflux for 2 h, cooled to room temperature and poured on ice/water. The separated solid was filtered, washed with water and recrystallized from methanol. Mp: 307–309 °C. Yield: 71%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3153, 3107 (NH₂), 3080 (CH aromatic), 1660 (2C=O), 1606 (C=N), 1516 (C=C), 530 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 7.63 (dd, 1H, J = 4.8, 8.1 Hz, pyridyl-H), 7.99 (dd, 1H, J = 2.10, 8.10 Hz, quinazolinyl-H), 8.26–8.29 (m, 2H, pyridyl-H, quinazolinyl-H), 8.42 (d, 1H, J = 8.7 Hz, quinazolinyl-H), 8.81 (d, 1H, J = 4.8 Hz, pyridyl-H), 9.11 (s, 1H, pyridyl-H), 12.08 (s, 2H, NH₂ exchanged by D₂O). Mass (m/z, rel. abundance): 394 (M⁺ + 2, 85%), 393 (M⁺ + 1, 1.68%), 392 (M⁺, 93.42%), 76 (100%). Anal. calcd for C₁₄H₉IN₄O₂ (392.15): C, 42.88; H, 2.31; N, 14.29. Found: C, 43.01; H, 2.28; N, 14.54.

6-Iodo-4-oxo-2-(pyridin-4-yl)quinazoline-3(4H)-carboxamide (14b). Compound 14b was synthesized according to the adopted procedure for 14a using (pyridin-4-yl)-benzoxazin-4-one (6b) and was recrystallized from methanol. Mp: 330–332 °C. Yield: 70%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3099, 3066 (NH₂), 3043 (CH aromatic), 1681 (2C=O), 1597 (C=N), 1496 (C=C), 534 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.83 (d, 2H, J = 5.80 Hz, pyridyl-H), 8.01 (dd, 1H, J = 1.72, 8.72 Hz, quinazolinyl-H), 8.30 (d, 1H, J = 1.88 Hz, quinazolinyl-H), 8.42 (d, 1H, J = 8.80 Hz, quinazolinyl-H), 8.76 (d, 2H, J = 5.80 Hz, pyridyl-H), 12.17 (s, 1H, NH₂ exchanged by D₂O). Mass (m/z, rel. abundance): 392 (M⁺, 0.14%), 78 (100%). Anal. calcd for C₁₄H₉IN₄O₂ (392.15): C, 42.88; H, 2.31; N, 14.29. Found: C, 43.07; H, 2.32; N, 14.51.

6-Iodo-4-oxo-2-(pyridin-3-yl)quinazoline-3(4H)-carbothioamide (15a). A mixture of benzoxazinone 6a (3.5 g, 10 mmol) and thiourea (0.76 g, 10 mmol) in glacial acetic acid (15 mL) containing sodium acetate anhydrous (1.64 g, 20 mmol), was refluxed for 4 h. Upon pouring on crushed ice/water, beige powder was obtained, filtered, washed with water and recrystallized from ethanol. Mp: 303–305 °C. Yield: 70%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3365 (NH₂), 1666 (C=O), 1606 (C=N), 1516 (C=C), 1159 (C=S), 530 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.54 (d, 1H, J = 8.56 Hz, quinazolinyl-H), 7.58 (dd, 1H, J = 4.84, 7.88 Hz, pyridyl-H), 8.12 (dd, 1H, J = 1.36, 8.48 Hz, quinazolinyl-H), 8.42–8.48 (m, 2H, quinazolinyl-H, pyridyl-H), 8.76 (d, 1H, J = 4.28 Hz, pyridyl-H), 9.28 (d, 1H, J = 1.44 Hz, pyridyl-H), 12.86 (s, 2H, NH₂ exchanged by D₂O). Mass (m/z, rel. abundance): 408 (M⁺, 44.59%), 78 (100%). Anal. calcd for C₁₄H₉IN₄OS (408.22): C, 41.19; H, 2.22; N, 13.72. Found: C, 41.30; H, 2.18; N, 13.85.

6-Iodo-4-oxo-2-(pyridin-4-yl)quinazoline-3(4H)-carbothioamide (15b). This compound was prepared and purified as per the previously mentioned procedure.

Mp: 326–328 °C. Yield: 66%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3400 (NH₂), 3043 (CH aromatic), 1680 (C=O), 1597 (C=N), 1496 (C=C), 1085 (C=S), 534 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.83 (d, 2H, J = 5.84 Hz, pyridyl-H), 8.00 (dd, 1H, J = 1.76, 8.76 Hz, quinazolinyl-H), 8.30 (d, 1H, J = 1.84 Hz, quinazolinyl-H), 8.42 (d, 1H, J = 8.80 Hz, quinazolinyl-H), 8.84 (d, 2H, J = 5.84 Hz, pyridyl-H), 12.24 (s, 2H, NH₂ exchanged by D₂O). Mass (m/z, rel. abundance): 408 (M⁺, 0.97%), 350 (100%). Anal. calcd for C₁₄H₉IN₄OS (408.22): C, 41.19; H, 2.22; N, 13.72. Found: C, 41.34; H, 2.24; N, 13.80.

6-Iodo-4-oxo-2-(pyridin-3-yl)quinazoline-3(4H)-carbothiohydrazide (16a). A solution of the benzoxazine derivative 6a (3.5 g, 10 mmol) and thiosemicarbazide (0.75 g, 10 mmol) in glacial acetic acid (15 mL) in the presence of sodium acetate (1.64 g, 20 mmol) was refluxed for 6 h. The reaction was poured onto ice/water and the obtained product was filtered off, washed with water and recrystallized from ethanol to give white crystals. Mp: 230–232 °C. Yield: 71%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3290 (NH₂), 3176 (NH), 3088 (CH aromatic), 1685 (C=O), 1591 (C=N), 1498 (C=C), 540 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 7.55 (d, 1H, J = 8.40 Hz, quinazolinyl-H), 8.05 (d, 1H, J = 7.80 Hz, pyridyl-H), 8.12–8.20 (m, 1H, pyridyl-H), 8.22 (d, 1H, J = 2.10 Hz, pyridyl-H), 8.46 (d, 1H, J = 4.00 Hz, quinazolinyl-H), 8.69 (dd, 1H, J = 4.8, 12.90 Hz, quinazolinyl-H), 8.85 (d, 1H, J = 20.40, pyridyl-H), 10.17 (s, 2H, NH₂ exchanged by D₂O), 10.43 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 423 (M⁺, 0.67%), 349 (100%). Anal. calcd. for C₁₄H₁₀IN₅OS (423.23): C, 39.73; H, 2.38; N, 16.55. Found: C, 39.89; H, 2.41; N, 16.79.

6-Iodo-4-oxo-2-(pyridin-4-yl)quinazoline-3(4H)-carbothiohydrazide (16b). Compound 16b was prepared by applying the same procedure used for preparation of 16a and was recrystallized from ethanol to give beige crystals. Mp: 210–212 °C. Yield: 60%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3290 (NH₂), 3242 (NH), 3062 (CH aromatic), 1681 (C=O), 1591 (C=N), 1504 (C=C), 540 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.55–7.60 (m, 1H, quinazolinyl-H), 7.70 (d, 2H, J = 5.12 Hz, pyridyl-H), 8.22 (d, 1H, J = 8.52 Hz, quinazolinyl-H), 8.48 (s, 1H, quinazolinyl-H), 8.75 (d, 2H, J = 5.12 Hz, pyridyl-H), 10.16 (s, 1H, NH₂ exchanged by D₂O), 10.43 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 423 (M⁺, 0.61%), 64 (100%). Anal. calcd. for C₁₄H₁₀IN₅OS (423.23): C, 39.73; H, 2.38; N, 16.55. Found: C, 39.85; H, 2.37; N, 16.68.

6-Iodo-3-(4-un/substituted phenylamino)-2-(pyridin-3-yl)quinazolin-4(3H)-one (17a–c). To a suspension of benzoxazinone 6a (3.5 g, 10 mmol) in absolute ethanol (20 mL), the required hydrazine derivative (10 mmol) was added and the reaction mixture was heated under reflux for 8–12 h. Then concentrated. Upon cooling the separated solid mass was filtered, washed with water and recrystallized from absolute ethanol to afford the titled derivatives 17a–c.
6-Iodo-3-(phenylamino)-2-(pyridin-3-yl)quinazolin-4(3H)-one (17a). Mp: 176–178 °C. Yield: 55%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3325 (NH), 2922 (CH aromatic), 1681 (C=O), 1645 (C=N), 1598 (C=C), 520 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 6.74 (t, 1H, J = 7.28 Hz, phenyl-H), 6.84 (d, 2H, J = 7.72 Hz, phenyl-H), 7.16 (t, 2H, J = 7.72 Hz, phenyl-H), 7.59 (dd, 1H, J = 4.84, 7.92 Hz, pyridyl-H), 7.98 (d, 1H, J = 8.80 Hz, quinazolinyl-H), 8.20–8.23 (m, 1H, pyridyl-H), 8.30–8.32 (m, 2H, quinazolinyl-H), 8.78 (dd, 1H, J = 1.16, 4.68 Hz, pyridyl-H), 9.05 (d, 1H J = 1.76 Hz, pyridyl-H), 12.03 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 440 (M⁺, 100%). Anal. calcd for C₁₉H₁₃IN₄O (440.24): C, 51.84; H, 2.98; N, 12.73. Found: C, 51.99; H, 3.03; N, 12.94.
3-[(4-Fluorophenyl)amino]-6-iodo-2-(pyridin-3-yl)quinazolin-4(3H)-one (17b). Mp: 310–312 °C. Yield: 52%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3325 (NH), 2924 (CH aromatic), 1681 (C=O), 1645 (C=N), 1598 (C=C), 501 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 7.00 (dd, 2H, J = 4.52, 8.88 Hz, phenyl-H), 7.17 (t, 2H, J = 4.72, 7.76 Hz, phenyl-H), 7.65 (dd, 1H, J = 4.72, 7.76 Hz, pyridyl-H), 8.01 (dd, 1H, J = 1.88, 8.68 Hz, quinazolinyl-H), 8.27–8.30 (m, 2H, pyridyl-H, quinazolinyl-H), 8.42 (d, J = 8.76 Hz, 1H, quinazolinyl-H), 8.82 (d, J = 3.80 Hz, 1H, pyridyl-H), 9.11 (s, 1H, pyridyl), 12.06 (s, 1H, NH exchanged by D₂O). ¹³C-NMR (100 MHz DMSO-d₆, ppm): δ 79.46, 87.38, 116.10, 116.33, 117.07, 117.15, 120.06, 123.14, 124.72, 135.95, 139.56, 140.22, 142.95, 148.18, 152.71, 162.21 (C–F), 168.70 (C=O). Mass (m/z, rel. abundance): 458 (M⁺, 8.24%), 78 (100%). Anal. calcd for C₁₉H₁₂FIN₄O (458.23): C, 49.80; H, 2.64; N, 12.23. Found: C, 49.95; H, 2.68; N, 12.40.
3-[(4-Chlorophenyl)amino]-6-iodo-2-(pyridin-3-yl)quinazolin-4(3H)-one (17c). Mp: 254–256 °C. Yield: 65%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3265 (NH), 3082 (CH aromatic), 1681 (C=O), 1583 (C=N), 1487 (C=C), 520 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, D₂O, ppm): δ 6.67 (d, 2H, J = 8.70 Hz, phenyl-H), 7.15 (d, 2H, J = 8.70 Hz, phenyl-H), 7.47 (d, 1H, J = 6.78 Hz, pyridyl-H), 7.68 (d, 1H, J = 8.40 Hz, quinazolinyl-H), 8.11 (d, 1H, J = 7.80 Hz, pyridyl-H), 8.22 (d, 1H, J = 8.70 Hz, quinazolinyl-H), 8.24 (s, 1H, quinazolinyl-H), 8.65 (s, 1H, pyridyl-H), 8.92 (s, 1H, pyridyl-H), 9.30 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 474 (M⁺, 49.72%), 476 (M⁺ + 2, 17.35%), 349 (100%). Anal. calcd for C₁₉H₁₂ClIN₄O (474.68): C, 48.07; H, 2.55; N, 11.80. Found: C, 48.28; H, 2.61; N, 11.94.

6-Iodo-3-(4-un/substituted phenylamino)-2-(pyridin-4-yl)quinazolin-4(3H)-one (17d–f). The quinazolinones 17d–f were prepared and purified according to the previously mentioned procedure starting from the benzoxazinone 6b.
6-Iodo-3-(phenylamino)-2-(pyridin-4-yl)quinazolin-4(3H)-one (17d). Mp: 239–241 °C. Yield: 40%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3304 (NH), 3057 (CH aromatic), 1672 (C=O), 1647 (C=N), 1519 (C=C), 534 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, D₂O, ppm): δ 6.74 (t, 1H, J = 7.28 Hz, phenyl-H), 6.84 (d, 2H, J = 7.88 Hz, phenyl-H), 7.16 (t, 2H, J = 7.64 Hz, phenyl-H), 7.77 (d, 2H, J = 5.68 Hz, pyridyl-H), 7.99 (s, 1H, quinazolinyl-H), 8.31–8.33 (m, 2H, quinazolinyl-H), 8.80 (d, 2H, J = 5.68 Hz, pyridyl-H), 10.77 (s, 1H, NH exchanged by D₂O). Anal. calcd for C₁₉H₁₃IN₄O (440.24): C, 51.84; H, 2.98; N, 12.73. Found: C, 51.97; H, 2.96; N, 12.91.
3-[(4-Fluorophenyl)amino]-6-iodo-2-(pyridin-4-yl)quinazolin-4(3H)-one (17e). Mp: 234–236 °C. Yield: 42%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3240 (NH), 2922 (CH aromatic), 1678 (C=O), 1645 (C=N), 1598 (C=C), 1230 (C–F), 501 (C–I). ¹H-NMR 400 MHz (DMSO-d₆, ppm): δ 6.05 (d, 1H, J = 8.76 Hz, quinazolinyl-H), 7.09 (t, 2H, J = 8.80 Hz, phenyl-H), 7.30 (dd, 2H, J = 4.72, 8.84 Hz, phenyl-H), 7.52 (d, 2H, J = 5.92 Hz, pyridyl-H), 7.56 (d, 1H, J = 1.72 Hz, quinazolinyl-H), 8.19 (d, 1H, J = 1.76 Hz, quinazolinyl-H), 8.52 (d, 2H, J = 5.92 Hz, pyridyl-H), 10.06 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 458 (M⁺, 30.88%), 110 (100%). Anal. calcd for C₁₉H₁₂FIN₄O (458.23): C, 49.80; H, 2.64; N, 12.23. Found: C, 49.98; H, 2.67; N, 12.39.
3-[(4-Chlorophenyl)amino]-6-iodo-2-(pyridin-4-yl)quinazolin-4(3H)-one (17f). Mp: 259–261. Yield: 51%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3290 (NH), 3180 (CH aromatic), 1656 (C=O), 1643 (C=N), 1483 (C=C), 755 (C–Cl), 520 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, ppm): δ 7.35 (d, 2H, J = 7.80 Hz, phenyl-H), 7.45 (d, 2H, J = 7.80 Hz, phenyl-H), 7.55 (d, 1H, J = 8.70 Hz, quinazolinyl-H), 8.01 (d, 2H, J = 6.00 Hz, pyridyl-H), 8.20 (s, 1H, quinazolinyl-H), 8.69 (d, 2H, J = 6.00 Hz, pyridyl-H), 9.27 (s, 1H, quinazolinyl-H), 10.84 (s, 1H, NH exchanged by D₂O). Mass (m/z, rel. abundance): 474 (M⁺, 35.35%), 476 (M⁺ + 2, 11.89%), 126 (100%). Anal. calcd for C₁₉H₁₂ClIN₄O (474.68): C, 48.07; H, 2.55; N, 11.80. Found: C, 48.31; H, 2.60; N, 11.94.

4-(6-Iodo-4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)benzoic acid (18a). Equimolar amounts of benzoxazin-4-one 6a (0.70 g, 2 mmol) and p-aminobenzoic acid (0.28 g, 2 mmol) were fused together at 200 °C in an oil bath for 1 h. The mixture was cooled, dissolved in boiling glacial acetic acid (30 mL) and filtered; the filtrate was concentrated up to (10 mL) in vacuo and cooled. The resulting solid was filtered, washed with water, dried and recrystallized from glacial acetic acid. Mp: 307–309 °C. Yield: 50%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3292 (OH), 1685 (2C=O), 1635 (C=N), 1508 (C=C), 540 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, ppm): δ 7.58 (dd, 1H, J = 4.80, 7.80 Hz, pyridyl-H), 7.82 (d, 2H, J = 8.70 Hz, phenyl-H), 7.92 (d, 2H, J = 8.70 Hz, phenyl-H), 7.99 (d, 1H, J = 2.10 Hz, quinazolinyl-H), 8.20–8.23 (m, 1H, pyridyl-H), 8.29 (d, 1H, J = 1.80 Hz, quinazolinyl-H), 8.40 (s, 1H, quinazolinyl-H), 8.76 (d, 1H, J = 4.20 Hz, pyridyl-H), 9.04 (s, 1H, J = 1.76 Hz, pyridyl-H), 11.23 (s, 1H, OH exchanged by D₂O). Mass (m/z, rel. abundance): 469 (M⁺, 1.01%), 78 (100%). Anal. calcd for C₂₀H₁₂IN₃O₃ (469.23): C, 51.19; H, 2.58; N, 8.96. Found: C, 51.34; H, 2.60; N, 9.08.

4-(6-Iodo-4-oxo-2-(pyridin-4-yl)quinazolin-3(4H)-yl)benzoic acid (18b). This compound was prepared and purified as per the previously mentioned procedure, using the benzoxazinone derivative 6b. Mp: 244–246 °C. Yield: 55%. I.R. (KBr, cm⁻¹): [small nu, Greek, tilde]_max 3334 (OH), 1681 (2C=O), 1589 (C=N), 1506 (C=C), 519 (C–I). ¹H-NMR 300 MHz (DMSO-d₆, ppm): δ 7.35 (d, 2H, J = 7.80 Hz, phenyl-H), 7.45 (d, 2H, J = 7.80 Hz, phenyl-H), 7.55 (d, 1H, J = 8.70 Hz, quinazolinyl-H), 8.01 (d, 2H, J = 6.00 Hz, pyridyl-H), 8.20 (d, 1H, J = 8.64 Hz, quinazolinyl-H), 8.69 (d, 2H, J = 6.00 Hz, pyridyl-H), 9.27 (s, 1H, quinazolinyl-H), 12.12 (s, 1H, OH exchanged by D₂O). Mass (m/z, rel. abundance): 469 (M⁺, 42.74%), 78 (100%). Anal. calcd for C₂₀H₁₂IN₃O₃ (469.23): C, 51.19; H, 2.58; N, 8.96. Found: C, 51.31; H, 2.62; N, 9.03.

(2) Biology

(a) In vitro cytotoxicity. The in vitro cytotoxicity of the newly synthesized compounds against a panel of nine cancer cell lines and one non-tumorigenic cell line was assessed using the MTT assay according to Mosmann's method.⁴¹ The MTT assay which was developed in 1983, is based on the reduction of the soluble 3-(4,5-methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) into a blue-purple formazan product, mainly by mitochondrial reductase activity inside living cells. Mosmann showed that MTT assay has very wide applicability for measuring survival and/or proliferation of various cells and can potentially be applied to any assay in which living cells must be distinguished from dead cells or a lack of cells. The assay also has potential value for quantitative and rapid measurement of cell death.

The cells used in cytotoxicity assay were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. Cells suspended in the medium (2 × 10⁴/mL) were plated in 96-well culture plates and incubated at 37 °C in a 5% CO₂ incubator. After 12 h, the test sample (2 μL) was added to the cells in 96-well plates and cultured at 37 °C for 3 days.

The cultured cells were mixed with 20 μL of MTT solution and incubated for 4 h. at 37 °C. The supernatant was carefully removed from each well and 100 μL of DMSO were added to each well to dissolve the formazan crystals which were formed by the cellular reduction of MTT. After mixing with a mechanical plate mixer, the absorbance of each well was measured by a microplate reader using a test wavelength of 570 nm.

(b) In vivo cytotoxicity. Female athymic pathogen-free nude mice (nu/nu, 4–6 weeks) were purchased and maintained in accordance with the guidelines of the so adopted Guide for the Care and Use of Laboratory Animals 8th Edition 2011, and as approved by the Research Ethics Committee (REC) of Faculty of Pharmacy, Cairo University. To establish MCF-7 human breast cancer xenografts, each of the female nude mice was first implanted with a 60 day subcutaneously (s.c.) slow release estrogen pellet (SE-121, 1.7 mg 17β-estradiol/pellet). The next day, cultured MCF-7 cells were harvested from confluent monolayer cultures, washed twice with serum-free medium, resuspended and injected s.c. (5 × 10⁶ cells, total volume 0.2 mL) into the left inguinal area of the mice. All animals were monitored for activity, physical condition, body weight, and tumor growth. Tumor size was determined by caliper measurement in two perpendicular diameters of the implant every other day. Tumor volume was calculated by the formula, 1/2a × b² where “a” is the long diameter and “b” is the short diameter (in cm).

The animals bearing human cancer xenografts were randomly divided into various treatment groups (of 5 animals each) and a control group (7–10 mice/group). The untreated control group received the vehicle only. For the MCF-7 xenograft model, tested compounds and the reference control drug sorafenib were dissolved in PEG400 : ethanol : saline (57.1 : 14.3 : 28.6, v/v/v), and were administered by intraperitoneal (i.p.) injection at doses equivalent of 5 or 10 mg kg⁻¹, every 48 h for 3 weeks were dosing and dose regimen were selected based on previous reports of sorafenib.⁴²

(c) Kinase inhibitory assay. In a final reaction volume of 25 μL, kinase (5–10 mU) was incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA, 0.66 mg mL⁻¹ myelin basic protein, 10 mM magnesium acetate and [γ³³P-ATP] (specific activity approximately 500 cpm pmol⁻¹, concentration as required). The reaction was initiated by the addition of the Mg–ATP mixture. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of 5 μL of a 3% phosphoric acid solution. 10 μL of the reaction was then spotted onto a P30 ltermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.

(3) Molecular docking

The molecular modeling study was carried out using Molecular Operating Environment (MOE 2009.10) software provided by chemical computing group, Canada. The energy minimization for the compounds was performed with MOE with MMFF94X force field and the partial charges were automatically calculated.^43,44

Crystallographic complexes of ABL with its respective inhibitors imatinib and bosutinib (PDB IDs: 2HYY and 3UE4) were downloaded from the protein data bank available at http://www.rcsb.org/pdb. The protein-ligand complex obtained from the protein data bank was prepared for docking as follows: the enzyme was 3D protonated, where hydrogen atoms were added at their standard geometry, the partial charges were computed and the system was optimized.

Deletion of water of crystallization together with extra chains of the protein was then done. Finally, isolation of the active site and recognition of the involved amino acids was carried out then the backbone was hidden.

Docking was performed using Triangle Matcher placement method, poses were prioritized based on London dG scoring and refinement of the results was done using forcefield. The most stable docking model was selected per the best scored conformation predicted by the MOE scoring function.

Conflict of interest

The authors declare no conflict of interest.

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