Synthesis, antiproliferative activity and possible mechanism of action of novel 2-acetamidobenzamides bearing the 2-phenoxy functionality

Abstract

Several new 2-(2-phenoxyacetamido)benzamides 17a–v, 21 and 22 were synthesized by stirring in pyridine the acid chlorides 16a–e and the appropriate5-R-4-R₁-2-aminobenzamide 15a–e and initially evaluated in vitro for antiproliferative activity against the K562 (human chronic myelogenous leukemia) cell line. Some of synthesized compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell line panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). The most active compounds caused an arrest of K562 cells in the G0–G1 phase of cell cycle and induction of apoptosis, which was mediated by caspase activation.

1. Introduction

Acetamidobenzamides represent a class of biologically active substances of great importance in medicinal chemistry. Despite their wide range of biological activities,^1–6 a review of the literature revealed that no anticancer activity had been described for these compounds. This omission led us to explore the potential of this class of compounds as anticancer agents by synthesizing and screening for antileukemic activity a series of novel 2-cinnamamido, 2-(3-phenylpropiolamido) and 2-(3-phenylpropanamido)benzamides.^7,8Among the synthesized compounds, 1–5 showed the best antiproliferative activity (Fig. 1).

Figure 1.

The most active compounds among the previously synthesized benzamides.^7,8

Several examples of active compounds containing the phenoxyacetamido scaffold in their structure include the kinase inhibitor 6,⁹ the carboxypeptidase R39 inhibitor 7,¹⁰ the HIF-α prolyl hydroxylase inhibitor 8,¹¹ the FLT3 inhibitor 9¹² and the HIV-protease inhibitor 10¹³ (Fig. 2).

Figure 2.

Some examples of active compounds from the literature bearing the phenoxyacetamido scaffold.

Thus, following our studies in the new anticancer agents,^14–17 and on the basis of our previous work on acetamidobenzamides,^7,8 as well as of data reported for some active compounds containing the phenoxyacetamido scaffold,^9–13 we decided to investigate the influence of the cinnamamido and 3-phenylpropiolamido scaffolds when substituted for the phenoxyacetamido scaffold. Moreover, the displacement of the 2-phenoxyacetamido moiety from the ortho to the para and meta positions was investigated.

A series of novel 2-(2-phenoxyacetamido)benzamide derivatives, including the 3-(2-phenoxyacetamido) and the 4-(2-phenoxyacetamido)benzamide derivatives, were therefore synthesized and screened for their antileukemic activity.

2. Results and discussion

2.1. Chemistry

The synthesis of 2-(2-phenoxyacetamido)benzamides 17a,¹⁸ 17b, 17c,¹⁹ 17e–f, 17g,¹⁹ and 17h–v was achieved as described in Scheme 1. A mixture of the appropriate acid chlorides 16a–e and the appropriate 5-R-4-R₁-2-aminobenzamides 15a–f in pyridine was stirred magnetically in an ice bath. Crude benzoylchlorides 16a–e, not commercially available, were obtained starting from the corresponding acid by treatment with thionyl chloride.²⁰

Scheme 1.

Synthetic pathway for the formation of 2-[(phenoxyacetyl)amino]benzamides 17a–v. Reagents and conditions: (i) SOCl₂, (ii) aqueous NH₃, (iii) I₂, aqueous NaHCO₃, (iv) SnCl₂, HCl.

The anthranilamide 15a is commercially available, while the 5-R-4-R₁-2-aminobenzamides 15b–f were obtained according to Scheme 1.

In particular, the 5-iodo-2-aminobenzamide (15c) was obtained, as reported in the literature,²¹ by treatment of anthranilamide (15a) with iodine in an aqueous solution of sodium bicarbonate.

The 5-methylanthranilamide (15d), the 5-methoxyanthranilamide (15e) and the 4,5-dimethoxyanthranilamide (15f) were obtained by reduction of 2-nitro-5-methyl benzamides 13a–c as shown in Scheme 1. Compounds 13a–c were obtained by reaction of the acids 11a–c with thionyl chloride to afford 12a–c, followed by treatment of 12a–c with aqueous ammonia.

The anthranilamide derivative 15b was obtained by stirring the 6-chloro-1H-benzo[d][1,3]oxazine-2,4-dione 14a in a 25% aqueous ammonia solution (Scheme 1).²²

The 4-(2-phenoxyacetamido)benzamide (21) and the 3-(2-phenoxyacetamido)benzamide (22) were obtained, following the same synthetic route reported for compounds 17a–v, first by transformation of the commercially available nitrobenzoyl chlorides 18a,b to nitrobenzamides 19a,b,^7,8 then by reduction with palladium on activated charcoal in a Parr apparatus (Scheme 2).

Scheme 2.

Synthetic pathway for the formation of benzamides 20a,b, 4-(2-phenoxyacetamido)benzamide (21) and 3-(2-phenoxyacetamido)benzamide (22).

The structures of the new compounds were determined by analytical and spectroscopic measurements. In particular, 17a–v, 21 and 22 showed ¹H NMR signals attributable to aromatic protons in the range 6.24–8.67 δ and a singlet in the range 11.99–12.92 δ, exchangeable with D₂O, for the amidic NH. The signals of the NH₂ benzamido protons, exchangeable with D₂O, showed a different behavior depending on the solvent. As shown in Figure 3a for 17b, taken as an example, the spectrum recorded in chloroform showed a broad signal attributable to the NH₂ moiety at 6.07 ppm. In DMSO-d₆ as solvent, the presence of an intramolecular hydrogen bond rendered the benzamido NH₂ protons diastereotopic. H_a appeared as a singlet at 7.39 δ whereas H_b was found at a lower field as a singlet at 8.25 δ (Fig. 3b). The presence of an intramolecular hydrogen bond was confirmed by performing the ¹H NMR spectrum of 17b at 90 °C. Figure 3c showed that, at 90 °C, the two singlets collapsed into a single broad signal at 7.64 δ. Furthermore, the ¹H NMR spectrum of 17b in a diluted solution of DMSO-d₆ was similar to that in a concentrated solution, proving that no intermolecular interaction occurred.

Figure 3.

¹H NMR spectrum of 17b in (a) chloroform, (b) DMSO-d₆ and (c) DMSO-d₆ performed at 90 °C.

Moreover, the ¹H NMR spectra of methyl and methoxy-substituted derivatives showed signals in the range 2.24–2.39 δ and 2.24–3.90 δ, respectively, arising from the methyl and methoxyl groups. Finally, a signal in the range 4.61–4.91 δ was found, and this was due to the methylene group of the phenoxy moiety.

2.2. Biological activity

All synthesized 2-(2-phenoxyacetamido)benzamido derivatives 17a–v, 21 and 22 were initially tested in vitro for their antileukemic activity against the K562 (human chronic myelogenous leukemia) cell line; colchicine, whose antileukemic activity is well known, was used as a reference compound.

The percent growth inhibition at a screening concentration of 10μM and the IC₅₀ values for compounds that exhibited at least 50% growth inhibition in the 10 μM screening assay are shown in Table 1.

Table 1.

Percent growth inhibition of compounds 17a–v at 10 μM and IC₅₀ values (μM) for compounds that exhibited at least 50% growth inhibition in the 10 μM screening assay against the K562 cell line

Compd	10 μM	IC50 (μM)
17a	57.4 ± 3.4	8.9 ± 0.9
17b	19.7 ± 2.0
17c	ns
17d	ns
17e	ns
17f	87.0 ± 4.3	1.3 ± 0.1
17g	75.9 ± 1.9	1.61 ± 0.7
17h	24.1 ± 0.2
17i	18.8 ± 1.0
17j	73.0 ± 0.7	0.16 ± 0.02
17k	49.8 ± 3.5
17l	74.7 ± 1.2	6.9 ± 0.3
17m	72.0 ± 1.1	4.7 ± 0.6
17n	45.6 ± 0.4
17o	30.3 ± 1.8
17p	35.4 ± 0.9
17q	30.8 ± 1.9
17r	85.3 ± 3.2	1.1 ± 0.17
17s	40.1 ± 08
17t	48.1 ± 0.7
17u	72.8 ± 3.1	0.58 ± 0.03
17v	17.7 ± 0.1
21	23.7 ± 0.8
22	ns
Colchicine	63.6 ± 1.7	0.02 ± 0.003

Values are the means of at least three independent determinations. ns = not significant (% inhibition <10).

These data showed that the best antiproliferative agent was compound 17j, with an IC₅₀ value of 0.16 μM.

As far as structure–activity relationships are concerned, it seems that on average the substitution of the cinnamamido moiety with the phenoxyacetamido moiety had little effect on inhibition of K562 cell growth (Table 1). In particular, the average inhibitory activity at 10 μM on K562 cell growth, showed by all the previously tested compounds,^7,8 was about 46%⁷ and 44%,⁸ respectively, while with the phenoxyacetamido moiety, average inhibition was 49%. Moreover, shifting the phenoxyacetamido moiety from the ortho (compound 17a) to the para or meta position (compounds 21 and 22, Table 1) resulted in reduced activity.

As for the previously reported benzamide analogs,^7,8 our data (Table 1) showed positive effects following substitution at the 5 position of the benzamido moiety (17f, 17j, 17m, 17r and 17u, Table 1), especially when the substituent(s) was iodine or 4,5-dimethoxy (compounds 17j and 17u, Table 1). Moreover, it seems that the simultaneous introduction of a substituent into both the benzamido and phenoxyacetamido moieties is unfavourable for inhibition of K562 cell growth relative to the unsubstituted 2-cinnamamidobenzamide 4 (Fig. 1), considering that antiproliferative activity was only maintained in the ortho-phenoxyacetamido derivatives (compounds 17g, 17l, 17n, 17s and 17v, Table 1).

To obtain more insights into the cytotoxic potential of the test compounds for normal human cells, compounds 17a,17f,17g, 17j,17k,17l,17m,17r and 17u were also tested in vitro for their cytotoxicity against HuDe cells, a primary cell culture from human epidermis (Table 2).

Table 2.

Comparison of IC₅₀ values (μM) in HuDe and K562 cells

Compd	HuDe	K562	HuDe/K562
17a	523.0 ± 7.8	8.9 ± .09	58.76
17f	807.8 ± 11.7	1.3 ± 0.1	621.38
17g	27.3 ± 3.9	1.61 ± 0.7	16.96
17j	318.0 ± 15.7	0.16 ± 0.02	1987.5
17k	355.7 ± 12.1	10 ± 1.5	35.57
17l	33.8 ± 2.3	6.9 ± 0.3	4.9
17m	99.3 ± 1.4	4.7 ± 0.6	21.13
17r	941.9 ± 4.7	1.1 ± 0.17	856.28
17u	162.7 ± 1.9	0.58 ± 0.03	280.52
Colchicine	0.017 ± 0.001	0.02 ± 0.003	0.85

Values are the means of at least three independent determinations.

The data in Table 2 show that the K562-active compounds were significantly less cytotoxic against the HuDe cell line (HuDe/K562 index ranging from 21 to 1987), indicating the possibility that these compounds may have limited toxicity in vivo at doses that would limit tumor growth. This contrasts with a very low HuDe/K562 index (0.85) obtained for colchicine.

Compounds 17f, 17j, 17l, 17r and 17u were selected by the NCI to evaluate in their in vitro antiproliferative assay at a single high dose (10⁻⁵ M) against 60 human cell lines derived from nine clinically isolated human cancer types (leukemia, lung, colon, melanoma, renal, ovarian, brain, breast and prostate) according to the NCI standard protocol.²³

The antitumor activity is given by three parameters for each cell line; GI₅₀ (compound’s molar concentration inhibiting 50% net cell growth), TGI (compound’s molar concentration for total inhibition of net cell growth), and LC₅₀ (compound’s molar concentration inducing 50% net cell death).

Among these, 17f, 17j, 17r and 17u, showing the best mean graph midpoints (MG_MID) (Table 3), and therefore they were progressed to the 5-dose assay against the NCI full panel of 60 cell lines.²³

Table 3.

Overview of the results of the in vitro single high dose (10⁻⁵ M) antitumor screening for compounds 17f, 17j, 17r and 17u^a

Compd	Growth percentb MG_MIDc
17f	46.40
17j	24.31
17l	87.28
17r	25.81
17u	27.38

^aData obtained from the NCI’s in vitro disease-oriented human tumor cells screen.

^bGrowth percent is the percent of growth inhibition at the single high dose of 10⁻⁵ M.

^cMG_MID = mean graph midpoint = arithmetical mean value for all tested cancer cell lines.

The GI₅₀ values for the test compounds were reported in Table 4. All the compounds 17f, 17j, 17r and 17u tested by NCI were found to possess low cytotoxicity. On the other hand, they turn out to be inhibitor of cell growth with GI₅₀ values ranging from 0.101 to 42.8 μM. Among these, compounds 17j, 17r and 17u were particularly active, against the majority of type of tumor cell lines investigates, in the sub micromolar concentration. The best activity was showed by compound 17j that caused 50% growth inhibition at submicromolar concentrations against 44 of 56 cellular lines. Compounds 17r and 17u resulted less active with respect to 17j causing 50% growth inhibition at submicromolar concentrations against 30 of 56 and 26 of 58 cellular lines, respectively. On the contrary, compound 17f resulted less active with respect to 17j, 17r and 17u being a micro molar inhibitor with GI₅₀ values at submicromolar concentrations only against 3 of 57 cellular lines.

Table 4.

Results of multi-dose growth inhibition assay (GI₅₀, μM) for compounds 17f, 17j, 17r and 17u

Cell line		17f	17j	17r	17u
Leukemia	CCRF-CEM	3.07	0.382	0.487	0.562
	HL-60 (TB)	3.29	0.339	0.340	0.293
	K-562	0.854	0.161	0.245	0.310
	MOLT-4	8.14	1.10	2.00	2.91
	RPMI-8226	4.33	0.553	1.17	2.27
	SR	0.777	0.221	0.392	0.515
Non-small cell lung cancer	A549/ATCC	7.06	0.732	0.908	0.917
	HOP-62	5.88	0.824	1.01	1.75
	HOP-92	5.70	1.50	0.414	17.7
	NCI-H226	>100	4.94	21.5	42.8
	NCI-H23	5.90	0.657	1.38	1.32
	NCI-H322M	7.09	0.705	2.10	5.77
	NCI-H460	3.00	0.425	0.484	0.529
	NCI-H522	3.78	0.630	1.19	0.410
Colon cancer	COLO 205	2.51	0.353	0.487	0.453
	HCC-2998	12.1	2.95	2.34	3.33
	HCT-116	2.72	0.517	0.518	0.620
	HCT-15	1.57	0.253	0.432	0.804
	HT29	1.74	0.349	0.388	0.437
	KM12	2.13	0.308	0.388	0.443
	SW-620	2.58	0.454	0.448	0.646
CNS cancer	SF-268	9.89	0.934	2.42	6.77
	SF-295	1.96	0.223	0.650	0.930
	SF539	1.80	0.249	0.441	0.563
	SNB-19	11.7	0.913	3.20	3.31
	SNB75	1.81	0.215	0.245	0.547
	U251	2.34	0.396	0.664	0.777
Melanoma	LOX IMVI	8.33	0.822	0.869	1.81
	M14	2.21	0.389	0.514	4.00
	MDA-MB-435	0.356	0.101	0.121	0.614
	SK-MEL-2	5.08	0.463	0.611	0.203
	SK-MEL-28	6.25	1.79	0.502	24.2
	SK-MEL-5	7.45	0.533	1.68	0.766
	UACC-257	>100	41.7	>100	>100
	UACC-62	5.42	0.701	1.05	2.52
Ovarian cancer	IGROV1	5.60	1.37	4.75	4.07
	OVCAR-3	2.30	0.321	0.375	0.472
	OVCAR-4	>100	nt	>100	>100
	OVCAR-5	3.86	1.68	5.40	15.0
	OVCAR-8	7.06	0.904	3.13	3.01
	NCI/ADR-RES	1.43	0.344	0.418	0.487
	SK-OV-3	8.15	3.17	2.99	3.58
Renal cancer	786-0	9.54	1.07	2.10	7.32
	A498	1.41	1.02	0.304	2.77
	ACHN	10.7	1.32	2.25	8.12
	CAKI-1	4.18	0.519	0.721	3.80
	RXF 393	3.93	0.413	1.40	1.69
	SN12C	17.0	0.945	3.09	3.12
	TK-10	63.8	8.11	23.8	14.4
	UO-31	17.5	2.87	8.72	13.5
Prostate cancer	PC-3	3.22	0.428	0.464	2.23
	DU-145	6.01	0.444	1.87	0.914
Breast cancer	MCF7	1.81	0.230	0.497	0.661
	MDA-MB-231	9.01	1.28	2.03	2.39
	HS 578T	4.65	0.630	0.592	0.771
	BT-549	nt	nt	nt	0.770
	T-47D	6.87	0.556	nt	3.46
	MDA-MB-468	1.742	0.351	0.345	0.848

nt = not tested.

Moreover, a mean graph midpoint (MG_MID) is calculated for the GI₅₀, TGI and LC₅₀ parameters, giving an average activity parameter for all the cell lines. For the calculation of the MG_MID, insensitive cell lines are included and assigned as their values the highest concentration tested. Considering the MG_MID values (Table 5), the most active compound of the series was derivative 17j, at the GI₅₀, TGI and LC₅₀ levels, followed by 17r.

Table 5.

Overview of the results of the in vitro antitumor screening for compounds 17f,j,r,u^a

Compd	No. Studiede	pGI50b			pTGIc			pLC50d
		No. giving positive resultse	Range	MG_MIDf	No. giving positive resultse	Range	MG_MIDf	No. giving positive resultse	Range	MG_MIDf
17f	57	54	6.45–4.00	5.31	10	5.65–4.00	4.14	1	4.13–4.00	4.00
17j	57	57	7.00–4.38	6.16	25	6.46–4.00	4.45	8	4.84–4.00	4.05
17r	56	54	6.92–4.00	6.05	25	6.20–4.00	4.31	2	4.50–4.00	4.01
17u	58	56	6.69–4.00	5.71	23	6.06–4.00	4.31	5	4.70–4.00	4.03

^aData obtained from the NCI’s in vitro disease-oriented human tumor cell screen.

^bpGI₅₀ is the −log of the molar concentration that inhibits 50% net cell growth.

^cpTGI is the −log of the molar concentration giving total growth inhibition.

^dpLC₅₀ is the −log of the molar concentration leading to 50% net cell death.

^eRefers to the number of cell lines for which GI₅₀, TGI or LC₅₀, respectively, are ≤100 μM.

^fMG_MID = mean graph midpoint = arithmetical mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used concentration interval, the highest tested concentration was used for the calculation.

In our previous studies on the cinnamamido and 3-phenylpropiolamido benzamides (compounds 1–5, Fig. 1),^7,8 the major evidence that suggested these agents interacted with tubulin was derived from cellular studies, such as an increased proportion of cells in the G2/M phase of the cell cycle, increased proportion of apoptotic cells, disappearance of cellular microtubules and COMPARE analysis of NCI cytotoxicity data.

Therefore we first wondered whether the same mechanism of action occurred with the phenoxyacetamido scaffold and we examined the most active compounds in the series (17f, 17j, 17r and 17u) for potential effects on the assembly of purified tubulin and on the inhibition of colchicine binding to tubulin, but negligible effects were observed (data not presented). Our initial studies with the K562 cells did yield evidence for mitotic arrest following treatment with the four compounds. However further studies using compounds 17j and 17u, which were the most effective antiproliferative agents, failed to validate the initial observations.

In order to elucidate the antiproliferative effect of 17j or 17u compounds, K562 cells were treated with different doses of the agents for 48 h. Clear morphological changes were observed in cells exposed to both the compounds.

In fact, under light microscopy, control cells appeared with a regular rounded shape (Fig. 4a), whereas after incubation with 17j or 17u cells detached themselves from poly-D lysine coated plates and showed a remarkable reduction in cell number and cell size.

Figure 4.

Effects of 17j and 17u on cell morphology. Cells (8 × 10³/well) were treated with 10 μM compound for 48 h, then morphology was observed under light microscopy at 200× magnification: (a) control, (b) cells treated with 10 μM 17j, (c) cells treated with 25 μM 17j, (d) cells treated with 10 μM 17u, (e) cells treated with 25 μM 17u.

The effect, which appeared with a 10 μM dose of each compound (Fig. 4b and d), was further enhanced with 25 μM 17u (Fig. 4e), while a consistent cytotoxic effect was already reached at the lowest concentration examined of 17j (Fig. 4b).

To gain additional insight into the mechanism of action of the compounds, a 10 μM dose of 17j and 17u was used in subsequent experiments.

After propidium iodide (PI) staining of DNA, flow cytometry analyses were performed to investigate the distribution of cells in the different phases of the cell cycle. These studies showed that both compounds caused an arrest of cells in the G0/G1 phase at 24 h. As shown in Figure 5, the proportion of K562 cells in the G0/G1 phase increased from 54.9% in control cells (Fig. 5a) to 78.5% in 10 μM 17j-treated cells (Fig. 5b, upper panel) and to 80% in 10 μM 17u-treated cells (Fig. 5c, upper panel).

Figure 5.

Cell cycle distribution of K562 cells after exposure to 17j or 17u. Cells (1.5 × 10⁵) were incubated with compounds for 24 or 48 h (upper and lower panels, respectively). Then, cells were harvested, stained with PI and subjected to flow cytometry. Percentages of cells in the sub G0/G1 region was considered as an index of apoptosis. At least 10,000 events per sample were measured: (a) control, (b) cells treated with 10 μM of 17j for 24 or 48 h, (c) cells treated with 10 μM of 17u for 24 or 48 h (upper and lower panels, respectively).

Prolonging the time of incubation with the compounds to 48 h a decrease in the proportion of cells in the G0/G1 phase occurred, while the percentage of cells in sub-G0/G1 region, characteristic of apoptotic cells with fragmented DNA, increased, reaching values equal to 32.9% and 20.6% with 10 μM 17j and 17u, respectively (Fig. 5b and c, lower panels).

In order to establish whether the reduction in cell number and cell size as well as the observed DNA fragmentation were related to the apoptotic cell death, additional analyses were performed.

Apoptosis is a programmed cell death that occurs in physiological and pathological conditions.²⁴ Defects in apoptotic pathways are now to contribute to malignant transformation of cells, cancer progression and resistance to anticancer drugs.²⁴ On the other hand, many compounds exerts their cytotoxic activity in tumor cells by inducing apoptosis. Apoptosis is characterized by a series of typical morphological features, such as cell shrinkage and detachment from the surrounding tissue, nuclear condensation, fragmentation into membrane-bound apoptotic bodies which are rapidly engulfed via phagocytosis by neighboring cells. Biochemical hallmarks of apoptosis include activation of proteases termed caspases, cleavage of proteins and DNA and exposure of phosphatidylserine on the cell surface.²⁴

Therefore K562 cells were treated with 10 μM of 17j or 17u for 48 h and examined for the characteristic morphological features of apoptotic cell death under a fluorescence microscope. Using Hoechst 33342 to stain the nuclei, it was observed (Fig. 6) that, in comparison with normal cells, treatment with 10 μM 17j or 17u induced in a high percentage of cells chromatin condensation and nuclear fragmentation, which are morphological changes typical of apoptosis.²⁵

Figure 6.

Fluorescence micrographs showing the effects of 17j and 17u on K562 cells after Hoechst 33342 staining. K562 cells (8 × 10³/well) were stained with Hoechst 33342 (2.5 lg/mL medium; blue fluorescence) and then treated with 10 μM of 17j or 17u for 48 h. Cell morphology was visualized with a Leica DC 300F microscope with a fluorescence filter for DAPI. The images show details of cells with clear signs of apoptosis, such as nuclear shrinkage and condensed and fragmented chromatin: (a) control, (b) cells treated with 10 μM 17j for 48 h, (c) cells treated with 10 μM 17u for 48 h.

One of the earlier events of apoptosis includes translocation of phosphatidylserine (PS), a phospholipid preferentially present in the inner side of membrane, to the surface of membrane. This allows early recognition of dead cells by macrophages, resulting in phagocytosis without the release of pro-inflammatory cellular components.²⁴ In our study the effects of 17j and 17u treatment on early and late apoptosis were examined using combined annexin V/PI staining.²⁶ Annexin V, a Ca²⁺-dependent phospholipid-binding protein, has high affinity for PS, and fluorochrome-labeled annexin V can be used to specifically target and identify cells in the early stages of apoptosis.²⁶ Propidium iodide (PI) is a DNA-intercalating dye which enters in the cells only when plasma membrane integrity is lost, a typical event of late apoptotic or necrotic cells. Annexin V-FITC and PI positive populations can easily be distinguished using a flow cytometer. In the scatter plot of a double variant flow cytometry (Fig. 7), the lower left quadrant of the histograms shows the viable cells, which exclude PI and are negative for annexin V-FITC staining. The lower right quadrant represents the early apoptotic cells, which are PI negative and annexin V positive indicating that cell population endowed with cytoplasmic membrane integrity and PS externalization. The upper right quadrant represents the non-viable necrotic and late-stage apoptotic cells, which are positive for annexin V binding and PI uptake. This population has loss of membrane integrity and presents externalization of PS. Finally, the upper left quadrant provides information about necrotic cell population, which has loss of cell membrane integrity (PI positive) with no PS externalization (Annexin-V negative). In our experiments, to distinguish early from late apoptotic cells, the analysis was performed after 24 h incubation with 17j or 17u. As shown in Figure 7, after exposure to 10 μM 17j, 24.5% of the K562 cells were found to be in early apoptosis and 12.5% in late apoptosis, while no necrotic cells were detected. Similar results were obtained in cells treated with 17u.

Figure 7.

Flow cytometric analysis of apoptotic effects of 17j and 17u in K562 cells. Cells were treated with compounds, then 10⁵ cells/sample were analyzed by flow cytometry for PS externalization after double staining with annexin V and PI. To distinguish early apoptotic from late apoptotic cells, the analysis was performed after a 24 h incubation with the compounds. The different cell populations are reported in the C1–C4 areas. The C1 area represents necrotic cells, the C2 area represents late apoptotic cells, the C3 area represents viable cells and the C4 area early apoptotic cells. Analysis of fluorescence intensity of gated cells in FL-1 (annexin V-FITC) and FL-3 (PI) channels was from 10,000 events: (a) control, (b) cells treated with 10 μM 17j, (c) cells treated with 10 μM 17u. The results shown are representative of three independent experiments.

In light of these results, we focused on the possible activation of caspases, known molecular players in the process of apoptosis. Caspases are cysteine aspartyl proteases involved in the activation of apoptotic cell death, being critical for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies.²⁷ Caspases are normally present as pro-caspases, inactive proenzymes which must be cleaved and transformed in functional caspases.²⁷ There are two classes of apoptotic caspases: initiator caspases, which are associated with the initiation of apoptosis (caspases 2, 8, 9, 10), and effector caspases, which cleave cellular substrates needed for the cell’s survival (caspases 3, 6, 7).²⁷ To demonstrate the involvement of caspases in apoptosis induced by 17j and 17u in K562 cells, activation of the effector caspases 3 and 6 was evaluated by Western blotting analysis. As shown in Figure 8, with respect to the basal level, the cellular content of pro-caspase 3 protein decreased by 40% and 50% after 48 h treatments with 10 μM 17j or 17u, respectively, thus indicating activation of caspase 3.

Figure 8.

The effect of 17j and 17u on pro-caspase 3 and pro-caspase 6 expression. K562 cells (1.5 × 10⁵/well) were treated for 48 h with 10 μM 17j or 17u. Cell extracts were prepared and, following gel electrophoresis, subjected to Western blotting analysis to determine the levels of pro-caspases 3 and 6. Equal loading of the gel was verified by immunoblotting for b-actin. (a) Cells treated with 10 μM 17j. (b) Cells treated with 10 μM 17u. The results are representative of three independent experiments.

Moreover, a modest reduction in the level of pro-caspase 6 was observed in 17j-treated cells (−20%) and a greater decrease in 17u-treated cells (−50%). These results demonstrate that the cytotoxic effect exerted in K562 by both 17j and 17u was due to the induction of apoptosis mediated by caspase activation.

3. Conclusion

The data reported here show that the 2-(2-phenoxyacetamido) benzamides 17 caused growth inhibition against many tumor cell lines. The best agents inhibited proliferation at low micromolar and submicromolar concentrations in every investigated tumor cell lines. It seems that, on average, the substitution of the cinnamamido moiety with the phenoxyacetamido moiety had little positive effect on inhibition of K562 cell growth, but moving the phenoxyacetamido moiety from the ortho to the meta or para position had negative effects on activity. As in our earlier studies with this class of compounds,^7,8 we initially suspected tubulin as a target, but the most cytotoxic compounds had little antitubulin activity. In the experiments presented here, we found different behavior in compounds 17a–v, bearing a phenoxyacetamido scaffold (G0/G1 arrest of cell cycle), in comparison with previously studied cinnamamido and 3-phenylpropiolamido benzamides (G2/M arrest of cell cycle).^7,8

Compounds 17j and 17u caused a G0/G1 arrest of the cell cycle. Prolonging the treatment to 48 h demonstrated that they caused an apoptotic cell death through the activation of pro-caspase 3 and 6.

4. Experimental section

4.1. Chemistry: general procedures

Reaction progress was monitored by TLC on silica gel plates (Merck 60, F₂₅₄, 0.2 mm). Organic solutions were dried over Na₂SO₄. Evaporation refers to the removal of solvent on a rotary evaporator under reduced pressure. All melting points were determined on a Büchi 530 capillary melting point apparatus and are uncorrected. IR spectra were recorded with a Perkin Elmer Spectrum RXI FT-IR System spectrophotometer, with compound as a solid in a KBr disk. ¹H and ¹³C NMR spectra (250 and 62.90 MHz, respectively) were obtained using a Bruker AC-E 300 MHz spectrometer (tetramethylsilane as internal standard): chemical shifts are expressed in δ values (ppm). Merck silica gel (Kiesegel 60/230–400 mesh) was used for flash chromatography columns. Microanalyses data (C, H, N) were obtained by an Elemental Vario EL III apparatus and were within ±0.4% of the theoretical values. Yields refer to purified products and are not optimized. The names of the products were obtained using the ACD/I-Lab Web service (ACD/IUPAC Name Free 8.05).

4.1.1. General procedure for preparation of 5-R-4-R₁-2-nitrobenzoyl chlorides 12a–c and 2-phenoxyacetyl chlorides 16a–e²⁰

Substituted benzoyl and phenoxyacetyl chlorides 12a–c and 16a–e were obtained by refluxing for 5 h the appropriate acid derivatives (0.01 mol) with thionyl chloride (7.25 mL). After evaporation under reduced pressure, the crude liquid residue was used for subsequent reactions without purification.

4.1.2. Preparation of 5-R-4-R₁-2-nitrobenzamides 13a–c

To 0.01 mol of 5-R-4-R₁-2-nitrobenzoyl chlorides 12a–c 10 mL of an aqueous ammonia solution (25%) and 33 mL of acetonitrile were added. The solution was first refluxed for 8 h, then evaporated to give pure 13a–c.

4.1.3. Preparation of 5-R-4-R₁-2-aminobenzamides 15d,e,f

To a magnetically stirred suspension of stannous chloride (0.038 mol) in concentrated HCl (37%), 0.013 mol of 13a–c was added (15 mL) at a rate so that the temperature of the slurry was maintained below 5 °C (about 1 h). After addition was complete, the mixture was stirred for 24 h. The white slurry thus obtained was diluted with cold water (150 mL), and NaOH (40%) was added until the tin salt dissolved. The solution was extracted with ethyl acetate (3 × 150 mL), and the extracts dried and evaporated to obtain pure 15d,e,f.

4.1.4. Preparation of 2-amino-5-chlorobenzamide 15b¹⁸

A mixture of 0.01 mol of 6-R-7-R₁-1H-benzo[d][1,3]oxazine-2,4-diones 14a and 25 mL of aqueous ammonia solution (25%) was stirred for 1 h. The solid precipitate was removed by filtration, washed with an aqueous ammonia solution (5%) and crystallized from ethanol.

4.1.5. Preparation of 2-amino-5-iodobenzamide (15c)²¹

Powdered iodine (11.7 g, 46.2 mmol) was added portion-wise over 1 h to a stirred solution of 2-aminobenzamide (15a) (5.72, 42.0 mmol) and NaHCO₃ (3.52 g, 42.0 mmol) in water (1.3 L). The solution was stirred overnight at room temperature. Afterwards NaHSO₃ (0.87 g, 8.40 mmol) was added. The solution was extracted with ethyl acetate (3 × 800 mL). After being dried with Na₂SO₄, the organic phase was evaporated. The crude product was recrystallized with water/methanol 10:1 v/v (600 mL) to yield pure 15c; yield 95%, mp 197–198 °C.

4.1.6. General procedure for the preparation of compounds 19a, b

To 0.01 mol of 5-R-4-R₁-benzoyl chlorides 18a,b 10 mL of an aqueous ammonia solution (25%) and 33 mL of acetonitrile were added. The solution was first refluxed for 8 h, then evaporated to give pure 19a,b.²⁸

4.1.7. General procedure for the preparation of compounds 20a,b

To²⁸ a solution of 0.8 mmol of 19a,b in 20 mL of hot ethanol, 15 mg of 10% palladium on activated charcoal as a catalyst was added. The mixture was left under hydrogenation in a Parr apparatus at 40 psi for 24 h to obtain 20a,b after crystallization.^29,30

4.1.8. General procedure for preparation of phenyl 2-(2-phenoxyacetamido)benzamides 17a,¹⁸ 17b–c,¹⁹ 17d 17e–f, 17g,¹⁹ 17h–v, 4-(2-phenoxyacetamido)benzamide (21) and 3-(2-phenoxyacetamido)benzamide (22)

To a cold (0–5 °C) stirred suspension of aminobenzamides 15a–f and 20a,b (0.016 mol) in pyridine (13 mL), 0.016 mol of the appropriate phenoxyacetyl chloride 16a–e was added over 30 min. After addition was complete, the solution was stirred for 24 h and then poured onto crushed ice. The precipitate was removed by filtration, washed with water, and crystallized from the indicated solvent.

4.1.8.1. 2-(2-(o-Tolyloxy)acetamido)benzamide (17b).

White solid (1.27 g, 28% yield); mp 170–173 °C (ethanol); I.R. (KBr) cm⁻¹ 3347, 3145 (NH, NH₂), 1659, 1620 (2 × CO); ¹H NMR (DMSO) δ 2.39 (s, 3H, CH₃); 4.70 (s, 2H, OCH₂); 6.87–8.62 (a set of signals, 10H, aromatic protons and exchangeable NH₂); 12.28 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 16.68 (CH₃), 67.75 (OCH₂), 111.73 (CH_Ar), 120.85 (C_Ar), 120.88 (CH_Ar), 121.76 (CH_Ar), 123.61 (CH_Ar), 126.90 (C_Ar), 127.41 (CH_Ar), 129.01 (CH_Ar), 131.21 (CH_Ar), 132.67 (CH_Ar), 138.85 (C_Ar), 155.65 (C_Ar), 167.71 (CO), 170.91 (CO). Anal. Calcd for C₁₆H₁₆N₂O₃: C, 67.59; H, 5.67; N, 9.85. Found: C, 67.52; H, 5.60; N, 9.50.

4.1.8.2. 2-(2-(p-Tolyloxy)acetamido)benzamide (17c).

White solid (1.54 g, 34% yield); mp 205–208 °C (ethanol); I.R. (KBr) cm⁻¹ 3378, 3318, 3195 (NH, NH₂), 1694, 1651 (2 × CO); ¹H NMR (DMSO) δ 2.24 (s, 3H, CH₃); 4.63 (s, 2H, OCH₂); 6.96–8.63 (a set of signals, 10H, aromatic protons and exchangeable NH₂); 12.53 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 20.52 (CH₃), 67.88 (OCH₂), 115.18 (2 × CH_Ar), 120.38 (C_Ar), 120.43 (CH_Ar), 123.50 (CH_Ar), 129.07 (CH_Ar), 130.40 (2 × CH_Ar), 130.94 (C_Ar), 132.81 (CH_Ar), 139.05 (C_Ar), 155.56 (C_Ar), 167.73 (CO), 171.06 (CO). Anal. Calcd for C₁₆H₁₆N₂O₃: C, 67.59; H, 5.67; N, 9.85. Found: C, 67.46; H, 5.54; N, 10.10.

4.1.8.3. 2-(2-(2,4-Dichlorophenoxy)acetamido)benzamide (17d).

Sand-colored solid (1.08 g, 20% yield); mp 185–187 °C (ethanol); I.R. (KBr) cm⁻¹ 3362, 3314, 3222 (NH, NH₂), 1695, 1628 (2 × CO); ¹H NMR (DMSO) δ 4.85 (s, 2H, OCH₂); 7.15–8.56 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.23 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 68.85 (OCH₂), 116.00 (CH_Ar), 120.99 (C_Ar), 121.05 (CH_Ar), 123.41 (C_Ar), 123.74 (CH_Ar), 126.09 (C_Ar), 128.52 (CH_Ar), 128.97 (CH_Ar), 129.95 (CH_Ar), 132.63 (CH_Ar), 138.72 (C_Ar), 152.45 (C_Ar), 166.76 (CO), 170.82 (CO). Anal. Calcd for C₁₅H₁₂Cl₂N₂O₃: C, 53.12; H, 3.57; N, 8.26. Found: C, 52.95; H, 3.50; N, 7.93.

4.1.8.4. 2-(2-(2,3-Dichlorophenoxy)acetamido)benzamide (17e).

White solid (1.52 g, 28% yield); mp 202–205 °C (ethanol); I.R. (KBr) cm⁻¹ 3225–2915 (NH, NH₂), 1673 (broad, 2 × CO); ¹H NMR (DMSO) δ 4.88 (s, 2H, OCH₂); 7.16–8.58 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.24 (s, 1H, NH, exchangeable). Anal. Calcd for C₁₅H₁₂Cl₂N₂O₃: C, 53.12; H, 3.57; N, 8.26. Found: C, 52.75; H, 3.86; N, 8.63.

4.1.8.5. 5-Chloro-2-(2-phenoxyacetamido)benzamide (17f).

White solid (0.97 g, 20% yield); mp 197–200 °C (ethanol); I.R. (KBr) cm⁻¹ 3377, 3296, 3178 (NH, NH₂), 1698, 1655 (2 × CO); ¹H NMR (DMSO) δ 4.70 (s, 2H, OCH₂); 6.70–8.67 (a set of signals, 10H, aromatic protons and exchangeable NH₂); 12.51 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 67.67 (OCH₂), 115.33 (2 × CH_Ar), 122.01 (C_Ar), 122.19 (2 × CH_Ar), 127.27 (C_Ar), 128.72 (CH_Ar), 130.10 (2 × CH_Ar), 132.45 (CH_Ar), 137.94 (C_Ar), 157.55 (C_Ar), 167.70 (CO), 169.73 (CO). Anal. Calcd for C₁₅H₁₃ClN₂O₃: C, 59.12; H, 4.30; N, 9.19. Found: C, 58.90; H, 4.11; N, 9.29.

4.1.8.6. 5-Chloro-2-(2-(o-tolyloxy)acetamido)benzamide (17g).

White solid (0.51 g, 10% yield); mp 218–220 °C (ethanol); I.R. (KBr) cm⁻¹ 3411, 3182 (NH, NH₂), 1665 (broad, 2 × CO); ¹H NMR (DMSO) δ 2.38 (s, 3H, CH₃); 4.71 (s, 2H, OCH₂); 6.90–8.64 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.21 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 16.72 (CH₃), 67.73 (OCH₂), 111.80 (CH_Ar), 121.65 (CH_Ar), 122.26 (CH_Ar), 126.85 (C_Ar), 127.17 (C_Ar), 127.36 (CH_Ar), 128.71 (CH_Ar), 130.61 (C_Ar), 131.20 (CH_Ar), 132.34 (CH_Ar), 137.98 (C_Ar), 157.63 (C_Ar), 167.22 (CO), 169.48 (CO). Anal. Calcd for C₁₆H₁₅ClN₂O₃: C, 60.29; H, 4.74; N, 8.79. Found: C, 60.34; H, 5.03; N, 8.95.

4.1.8.7. 5-Chloro-2-(2-(p-tolyloxy)acetamido)benzamide (17h).

White solid (1.12 g, 22% yield); mp 225–227 °C (ethanol); I.R. (KBr) cm⁻¹ 3390, 3253, 3248 (NH, NH₂), 1660 (broad, 2 × CO); ¹H NMR (DMSO) δ 2.24 (s, 3H, CH₃); 4.65 (s, 2H, OCH₂); 6.95–8.66 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.48 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 20.51 (CH₃), 67.86 (OCH₂), 115.19 (2 × CH_Ar), 122.01 (C_Ar), 122.17 (CH_Ar), 127.25 (C_Ar), 128.71 (CH_Ar), 130.40 (2 × CH_Ar), 131.00 (C_Ar), 132.45 (CH_Ar), 137.94 (C_Ar), 155.52 (C_Ar), 167.85 (CO), 169.70 (CO). Anal. Calcd for C₁₆H₁₅ClN₂O₃: C, 60.29; H, 4.74; N, 8.79. Found: C, 60.39; H, 4.50; N, 8.53.

4.1.8.8. 5-Chloro-2-(2-(2,4-dichlorophenoxy)acetamido)benzamide (17i).

White solid (0.65 g, 11% yield); mp 215–218 °C (ethanol); I.R. (KBr) cm⁻¹ 3378, 3254, 3246 (NH, NH₂), 1684, 1644 (2 × CO); ¹H NMR (DMSO) δ 4.91 (s, 2H, OCH₂); 6.24–8.64 (a set of signals, 8H, aromatic protons and exchangeable NH₂); 12.19 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 63.83 (OCH₂), 116.10 (CH_Ar), 122.72 (C_Ar), 122.82 (CH_Ar), 123.43 (C_Ar), 126.17 (C_Ar), 127.54 (C_Ar), 128.53 (CH_Ar), 128.60 (CH_Ar), 129.95 (CH_Ar), 132.96 (CH_Ar), 137.57 (C_Ar), 152.40 (C_Ar), 166.88 (CO), 169.45 (CO). Anal. Calcd for C₁₅H₁₁Cl₃N₂O₃: C, 48.22; H, 2.97; N, 7.50. Found: C, 48.38; H, 2.65; N, 7.66.

4.1.8.9. 5-Iodo-2-(2-phenoxyacetamido)benzamide (17j).

Sand-colored solid (2.41 g, 38% yield); mp 206-209 °C (ethanol); I.R. (KBr) cm⁻¹ 3377, 3295, 3178 (NH, NH₂), 1694, 1652 (2 × CO); ¹H NMR (DMSO) δ 4.69 (s, 2H, OCH₂); 6.99–8.46 (a set of signals, 10H, aromatic protons and exchangeable NH₂); 12.50 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 67.73 (OCH₂), 87.03 (C_Ar), 115.13 (2 × CH_Ar), 122.20 (CH_Ar), 122.50 (C_Ar), 122.57 (CH_Ar), 130.11 (2 × CH_Ar), 137.17 (CH_Ar), 138.74 (C_Ar), 141.19 (CH_Ar), 157.55 (C_Ar), 167.70 (CO), 169.62 (CO). Anal. Calcd for C₁₅H₁₃IN₂O₃: C, 45.47; H, 3.31; N, 7.07. Found: C, 45.40; H, 3.59; N, 7.25.

4.1.8.10. 5-Iodo-2-(2-(p-tolyloxy)acetamido)benzamide (17k).

White solid (1.57 g, 24% yield); mp 220–222 °C (ethanol); I.R. (KBr) cm⁻¹ 3386, 3297, 3195 (NH, NH₂), 1693, 1651 (2 × CO); ¹H NMR (DMSO) δ 2.24 (s, 3H, CH₃); 4.63 (s, 2H, OCH₂); 6.94–8.44 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.44 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 20.53 (CH₃), 67.85 (OCH₂), 87.05 (C_Ar), 115.16 (2 × CH_Ar), 122.42 (C_Ar), 122.53 (CH_Ar), 130.41 (2 × CH_Ar), 130.98 (C_Ar), 137.16 (CH_Ar), 138.74 (C_Ar), 141.18 (CH_Ar), 155.47 (C_Ar), 167.96 (CO), 169.86 (CO). Anal. Calcd for C₁₆H₁₅IN₂O₃: C, 46.85; H, 3.69; N, 6.83. Found: C, 47.00; H, 3.98; N, 6.79.

4.1.8.11. 2-(2-(2,4-Dichlorophenoxy)acetamido)-5-iodobenzamide (17l).

White solid (1.04 g, 14% yield); mp 213–215 °C (ethanol); I.R. (KBr) cm⁻¹ 3376, 3261, 3144 (NH, NH₂), 1670, 1665 (2 × CO); ¹H NMR (DMSO) δ 4.84 (s, 2H, OCH₂); 7.17–8.38 (a set of signals, 8H, aromatic protons and exchangeable NH₂); 12.11 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 68.93 (OCH₂), 87.38 (C_Ar), 116.10 (CH_Ar), 122.92 (C_Ar), 123.02 (CH_Ar), 123.41 (C_Ar), 126.07 (C_Ar), 128.50 (CH_Ar), 129.96 (CH_Ar), 137.10 (C_Ar), 138.66 (CH_Ar), 140.99 (CH_Ar), 152.47 (C_Ar), 166.84 (CO), 169.35 (CO). Anal. Calcd for C₁₅H₁₁Cl₂IN₂O₃: C, 38.74; H, 2.38; N, 6.02. Found: C, 38.37; H, 2.59; N, 5.88.

4.1.8.12. 5-Methyl-2-(2-phenoxyacetamido)benzamide (17m).

White solid (1.91 g, 42% yield); mp 199–202 °C (ethanol); I.R. (KBr) cm⁻¹ 3381, 3302, 3182 (NH, NH₂), 1693, 1652 (2 × CO); ¹H NMR (DMSO) δ 2.31 (s, 3H, CH₃); 4.67 (s, 2H, OCH₂); 6.98–8.52 (a set of signals, 10H, aromatic protons and exchangeable NH₂); 12.43 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 20.84 (CH₃), 67.73 (OCH₂), 115.38 (CH_Ar), 120.23 (C_Ar), 120.31 (CH_Ar), 122.02 (CH_Ar), 129.44 (CH_Ar), 130.05 (CH_Ar), 132.33 (C_Ar), 133.16 (CH_Ar), 136.92 (C_Ar), 157.00 (C_Ar), 167.14 (CO), 171.08 (CO). Anal. Calcd for C₁₆H₁₆N₂O₃: C, 67.59; H, 5.67; N, 9.85. Found: C, 67.45; H, 5.76; N, 9.90.

4.1.8.13. 5-Methyl-2-(2-(o-tolyloxy)acetamido)benzamide (17n).

White solid (0.72 g, 15% yield); mp 160–162 °C (ethanol); I.R. (KBr) cm⁻¹ 3385, 3344, 3186 (NH, NH₂), 1693, 1652 (2 × CO); ¹H NMR (DMSO) δ 2.03 (s, 3H, CH₃); 2.38 (s, 3H, CH₃); 4.67 (s, 2H, OCH₂); 6.87–8.50 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.14 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 16.78 (CH₃), 20.82 (CH₃), 67.78 (OCH₂), 111.79 (CH_Ar), 120.70 (CH_Ar), 120.78 (C_Ar), 121.63 (CH_Ar), 126.91 (CH_Ar), 127.36 (C_Ar), 129.37 (CH_Ar), 131.15 (CH_Ar), 132.44 (C_Ar), 133.00 (CH_Ar), 136.72 (C_Ar), 155.74 (C_Ar), 167.28 (CO), 170.92 (CO). Anal. Calcd for C₁₇H₁₈N₂O₃ : C, 68.44; H, 6.08; N, 9.39. Found: C, 68.65; H, 6.34; N, 9.78.

4.1.8.14. 5-Methyl-2-(2-(p-tolyloxy)acetamido)benzamide (17o).

White solid (1.72 g, 36% yield); mp 216–218 °C (ethanol); I.R. (KBr) cm⁻¹ 3390, 3262, 3222 (NH, NH₂), 1665, 1660 (2 × CO); ¹H NMR (DMSO) δ 2.24 (s, 3H, CH₃); 2.30 (s, 3H, CH₃); 4.61 (s, 2H, OCH₂); 6.95–8.51 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.39 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 20.65 (CH₃), 20.81 (CH₃), 67.90 (OCH₂), 115.22 (2 × CH_Ar), 120.88 (C_Ar), 120.23 (CH_Ar), 129.42 (CH_Ar), 130.35 (2 × CH_Ar), 130.76 (C_Ar), 132.30 (C_Ar), 133.14 (CH_Ar), 136.91 (C_Ar), 155.66 (C_Ar), 167.28 (CO), 171.05 (CO). Anal. Calcd for C₁₇H₁₈N₂O₃: C, 68.44; H, 6.08; N, 9.39. Found: C, 68.46; H, 6.19; N, 9.76.

4.1.8.15. 2-(2-(2,4-Dichlorophenoxy)acetamido)-5-methylbenzamide (17p).

White solid (1.02 g, 18% yield); mp 214–216 °C (ethanol); I.R. (KBr) cm⁻¹ 3391, 3320, 3201 (NH, NH₂), 1665, 1660 (2 × CO); ¹H NMR (DMSO) δ 2.30 (s, 3H, CH₃); 4.82 (s, 2H, OCH₂); 7.16–8.44 (a set of signals, 8H, aromatic protons and exchangeable NH₂); 12.07 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 20.83 (CH₃), 68.92 (OCH₂), 116.08 (CH_Ar), 120.84 (C_Ar), 120.90 (CH_Ar), 123.41 (C_Ar), 125.99 (C_Ar), 128.51 (CH_Ar), 129.32 (CH_Ar), 129.95 (CH_Ar), 132.59 (C_Ar), 132.96 (CH_Ar), 136.57 (C_Ar), 152.57 (C_Ar), 166.42 (CO), 170.81 (CO). Anal. Calcd for C₁₆H₁₄Cl₂N₂O₃: C, 54.41; H, 4.00; N, 7.93. Found: C, 54.09; H, 4.20; N, 7.58.

4.1.8.16. 2-(2-(2,3-Dichlorophenoxy)acetamido)-5-methylbenzamide (17q).

White solid (0.56 g, 10% yield); mp 223–225 °C (ethanol); I.R. (KBr) cm⁻¹ 3310–2910 (NH, NH₂), 1670 (broad, 2 × CO); ¹H NMR (DMSO) δ 2.30 (s, 3H, CH₃); 4.85 (s, 2H, OCH₂); 7.14–8.44 (a set of signals, 8H, aromatic protons and exchangeable NH₂); 12.07 (s, 1H, NH, exchangeable). Anal. Calcd for C₁₆H₁₄Cl₂N₂O₃: C, 54.41; H, 4.00; N, 7.93. Found: C, 54.68; H, 3.74; N, 7.47.

4.1.8.17. 5-Methoxy-2-(2-phenoxyacetamido)benzamide (17r).

White solid (0.96 g, 20% yield); mp 178–181 °C (ethanol); I.R. (KBr) cm⁻¹ 3391, 3313, 3164 (NH, NH₂), 1698, 1654 (2 × CO); ¹H NMR (DMSO) δ 3.80 (s, 3H, OCH₃); 4.66 (s, 2H, OCH₂); 7.00–8.56 (a set of signals, 10H, aromatic protons and exchangeable NH₂); 12.27 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 55.93 (OCH₃), 67.69 (OCH₂), 113.99 (CH_Ar), 115.38 (2 × CH_Ar), 118.18 (CH_Ar), 121.64 (C_Ar), 121.86 (CH_Ar), 122.01 (CH_Ar), 130.05 (2 × CH_Ar), 132.58 (C_Ar), 154.82 (C_Ar), 157.71 (C_Ar), 166.79 (CO), 170.69 (CO). Anal. Calcd for C₁₆H₁₆N₂O₄: C, 63.99; H, 5.37; N, 9.33. Found: C, 64.05; H, 5.72; N, 9.64.

4.1.8.18. 5-Methoxy-2-(2-(o-tolyloxy)acetamido)benzamide (17s).

Brown solid (0.65 g, 13% yield); mp 165–168 °C (ethanol); I.R. (KBr) cm⁻¹ 3347, 3289, 3186 (NH, NH₂), 1672 (broad, 2 × CO); ¹H NMR (DMSO) δ 2.39 (s, 3H, OCH₃); 3.90 (s, 3H, OCH₃); 4.67 (s, 2H, OCH₂); 6.87–8.53 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 11.99 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 16.42 (CH₃), 55.38 (OCH₃), 67.39 (OCH₂), 111.62 (CH_Ar), 113.52 (CH_Ar), 118.07 (CH_Ar), 121.98 (CH_Ar), 122.96 (C_Ar), 123.11 (CH_Ar), 126.85 (C_Ar), 127.46 (CH_Ar), 131.03 (C_Ar), 131.30 (CH_Ar), 155.26 (C_Ar), 155.40 (C_Ar), 167.71 (CO), 170.81 (CO). Anal. Calcd for C₁₇H₁₈N₂O₄: C, 64.96; H, 5.77; N, 8.91. Found: C, 65.16; H, 6.16; N, 9.19.

4.1.8.19. 5-Methoxy-2-(2-(p-tolyloxy)acetamido)benzamide (17t).

Yellow solid (0.85 g, 17% yield); mp 238–240 °C (ethanol); I.R. (KBr) cm⁻¹ 3353, 3245, 3191 (NH, NH₂), 1669, 1667 (2 × CO); ¹H NMR (DMSO) δ 2.24 (s, 3H, CH₃); 3.79 (s, 3H, OCH₃); 4.61 (s, 2H, OCH₂); 6.95–8.55 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.24 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 20.55 (CH₃), 55.91 (OCH₃), 67.85 (OCH₂), 113.93 (CH_Ar), 115.20 (CH_Ar), 118.18 (CH_Ar), 121.70 (C_Ar), 121.92 (CH_Ar), 130.38 (CH_Ar), 130.82 (C_Ar), 132.47 (C_Ar), 154.84 (C_Ar), 155.64 (C_Ar), 167.98 (CO), 170.69 (CO). Anal. Calcd for C₁₇H₁₈N₂O₄: C, 64.96; H, 5.77; N, 8.91. Found: C, 65.21; H, 5.69; N, 9.17.

4.1.8.20. 4,5-Dimethoxy-2-(2-phenoxyacetamido)benzamide (17u).

White solid (2.38 g, 45% yield); mp 219–222 °C (ethanol); I.R. (KBr) cm⁻¹ 3367, 3289, 3173 (NH, NH₂), 1693, 1652 (2 × CO); ¹H NMR (DMSO) δ 3.81 (s, 6H, 2 × OCH₃); 4.67 (s, 2H, OCH₂); 6.98–8.44 (a set of signals, 9H, aromatic protons and exchangeable NH₂); 12.92 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 55.58 (OCH₃), 56.37 (OCH₃), 67.76 (OCH₂), 103.33 (CH_Ar), 111.21 (C_Ar), 112.13 (CH_Ar), 115.38 (2 × CH_Ar), 121.98 (CH_Ar), 130.03 (2 × CH_Ar), 135.01 (C_Ar), 144.08 (C_Ar), 151.88 (C_Ar), 157.75 (C_Ar), 167.15 (CO), 170.84 (CO). Anal. Calcd for C₁₇H₁₈N₂O₅: C, 61.81; H, 5.49; N, 8.48. Found: C, 62.06; H, 5.18; N, 8.83.

4.1.8.21. 2-(2-(2,3-Dichlorophenoxy)acetamido)-4,5-dimethoxybenzamide (17v).

Yellow solid (0.96 g, 15% yield); mp 248–250 °C (ethanol); I.R. (KBr) cm⁻¹ 3363, 3259, 3178 (NH, NH₂), 1664, 1616 (2 × CO); ¹H NMR (DMSO) δ 3.80 (s, 6H, 2 × OCH₃); 4.85 (s, 2H, OCH₂); 7.13–8.35 (a set of signals, 7H, aromatic protons and exchangeable NH₂); 12.60 s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 55.87 (OCH₃), 56.28 (OCH₃), 68.96 (OCH₂), 104.33 (CH_Ar), 111.70 (C_Ar), 111.79 (CH_Ar), 113.11 (CH_Ar), 121.66 (C_Ar), 123.48 (CH_Ar), 128.92 (CH_Ar), 132.90 (C_Ar), 134.43 (C_Ar), 144.25 (C_Ar), 151.67 (C_Ar), 154.87 (C_Ar), 166.44 (CO), 170.63 (CO). Anal. Calcd for C₁₇H₁₆Cl₂N₂O₅: C, 51.14; H, 4.04; N, 7.02. Found: C, 51.21; H, 4.05; N, 6.77.

4.1.8.22. 4-(2-Phenoxyacetamido)benzamide (21).

Sandcolored solid (2.81 g, 65% yield); mp 216–218 °C (ethanol); I.R. (KBr) cm⁻¹ 3379, 3323-3175 (NH, NH₂), 1681, 1645 (2 × CO); ¹H NMR (DMSO) δ 4.74 (s, 2H, OCH₂); 6.96–7.88 (a set of signals, 11H, aromatic protons and exchangeable NH₂); 10.24 (s, 1H, NH, exchangeable). ¹³C NMR (δ) (DMSO) 67.48 (OCH₂), 115.08 (2 × CH_Ar), 119.22 (2 × CH_Ar), 121.68 (CH_Ar), 128.84 (2 × CH_Ar), 129.64 (C_Ar), 129.99 (2 × CH_Ar), 141.48 (C_Ar), 158.19 (C_Ar), 167.46 (CO), 167.89 (CO). Anal. Calcd for C₁₅H₁₄N₂O₃: C, 66.66; H, 5.22; N, 10.36. Found: C, 66.34; H, 4.93; N, 10.32.

4.1.8.23. 3-(2-Phenoxyacetamido)benzamide (22).

Sandcolored solid (1.12 g, 26% yield); mp 168–170 °C (ethanol); I.R. (KBr) cm⁻¹ 3400–3212 (NH, NH₂), 1678, 1652 (2 × CO); ¹H NMR (DMSO) δ 4.72 (s, 2H, OCH₂); 6.96–8.13 (a set of signals, 11H, aromatic protons and exchangeable NH₂); 10.24 (s, 1H, NH, exchangeable). Anal. Calcd for C₁₅H₁₄N₂O₃: C, 66.66; H, 5.22; N, 10.36. Found: C, 66.91; H, 5.26; N, 10.24.

4.2. Biology

4.2.1. Percent growth inhibition assay

Human chronic myelogenous leukemia cell line K562 was maintained in suspension culture in RPMI1640 medium (SIGMA–Aldrich, cat. #R8758) supplemented with 5% fetal bovine serum (FBS: SIGMA–Aldrich, cat. #F6178), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 lg/mL amphotericin B (complete medium) at 37 °C in a 5% CO₂ humidified atmosphere.

The trypan blue exclusion assay was performed to assess the effect of all test compounds on the viability of K562 cells, as described previously.³¹ Briefly, about 2 × 10⁵ cells/mL were cultured for 2 4 h, and compounds were added at 10 μM (or at different concentrations to determine the IC₅₀). The effects of test compounds on cell viability were assayed on a portion of the cell suspension after 24 h. The cell number was determined with a hemocytometer, and viability was estimated by trypan blue dye exclusion.

4.2.2. MTT assay

The MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed as described earlier³² and was used to determine the viability of HuDe cells.

MTT is a yellow-colored tetrazolium salt that is taken up and cleaved only by metabolically active cells, which reduce it to a colored, water-insoluble formazan salt. The solubilized formazan product can be quantified via absorbance at 570 nm (690 nm for blank), which is measured using a 96-well-format spectrophotometer (ELx800-BioTek instruments). The absorbance correlates directly with cell number.

Briefly, about 2 × 10⁵ cells/mL (2 × 10⁴ cells/mL for HuDe) were cultured; different concentrations of test drugs or 0.1% DMSO were added to the wells 24 h after seeding. Cells were collected after 72 h and evaluated by the MTT assay. Cells were incubated with 10 μl of MTT (5 mg/mL in PBS) at 37 °C for 3 h. The tetrazolium crystals were solubilized by the addition of 4.5 mL isopropyl alcohol, 0.5 mL triton X-100 and 150 μL of 37% HCl. Cells grown in culture media alone or with appropriate concentrations of DMSO as controls.

4.2.3. Hoechst staining and cell cycle distribution

For these experiments, after plating on poly-(d-lysine) (Sigma–Aldrich, Milan, Italy) coated 96 or 6 well plates, K562 cells were allowed to adhere overnight and then treated with chemicals or vehicle only.

In order to determine either changes in nuclear morphology or plasma membrane damage, the cells were stained with Hoechst 33342 (Sigma–Aldrich, Milan, Italy), a fluorescent cell permeant dye. Specifically, cells (8 × 10³/well) were stained with Hoechst 33342 (2.5 lg/mL medium) for 30 min, washed with PBS, resuspended in culture medium and treated for various times with compounds. Cell morphology was visualized using a Leica DC 300F microscope (Leica Microsystems, Wetzlar, Germany), and all images were acquired by Leica Q Fluoro Software using appropriate filters to examine Hoechst 33342 (DAPI filter with excitation wavelength of 372 nm and emission wavelength of 456 nm).

Cell cycle distribution was analyzed by flow cytometry using PI staining. Cells were seeded in 6 well plates (1.5 × 10⁵/well), treated with compounds and harvested for flow cytometric analysis. Fluorescence of the cells was analyzed by a FACscan (Beckman Coulter Inc., Brea, CA). The proportion of cells with fragmented DNA was estimated by evaluating the percentage of events accumulated in the hypodiploid sub G0/G1 area of each scan. Data were analyzed using Expo32 software (Beckman Coulter Inc., Brea, CA).

4.2.4. Annexin V and PI staining

Cell apoptosis was detected using an Annexin V-FITC Detection Kit (Biotool, Munich, Germany). Briefly, after treatment for 24 h with compounds, K562 cells were harvested, centrifuged at 800 rpm for 10 min and washed in PBS. Then 10⁵ cells/sample were incubated in the dark with annexin V-FITC (5 ll) and PI (5 ll) for 15 min according to the manufacturer’s instructions. After that, stained cells were immediately analyzed by flow cytometry (Beckman Coulter Inc., Brea, CA) measuring the fluorescence emission at 530 nm (FL1) and >575 nm (FL3).

4.2.5. Western blotting analysis

Cell lysates were prepared as previously reported.³³ Protein concentration was determined by Lowry assay, 50 lg protein/lane were subjected to SDS polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane for detection with specific antibodies conjugated with alkaline phosphatase. Analyses of pro-caspase 3 and pro-caspase 6 were performed by using specific antibodies produced by Santa Cruz Biotechnology (Santa Cruz, CA) while antibody for b actin was purchased from Sigma Aldrich (Milan, Italy). The correct protein loading was ascertained by means of both red Ponceau staining and immunoblotting for b-actin. The densitometric analysis of western blot bands was performed by Image J software. All the blots shown are representative of at least three separate experiments.