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Abstract

A synthesis of benzo[e][1,2,4]triazines and 1,2,4-triazolospiro[4,5]deca-2,6,9-trien-8-ones has been developed from reactions of amidrazones with 2-chloro-1,4-benzoquinone in EtOAc containing 0.5 mL of piperidine. This highly regioselective and one-pot process provided rapid access to 1,2,4-triazolospiro[4,5]deca-2,6,9-trien-8-ones (60%–70%) and benzo[e][1,2,4]triazines (11%–18%). On reacting amidrazones with 5-hydroxy-1,4-naphthoquinone in an EtOAc/piperidine mixture, the reaction proceeded to give 5-hydroxy-2-(piperidin-1-yl)naphthalene-1,4-dione. The structures of the isolated products were proved by infrared, NMR (2D-NMR), mass spectra, and elemental analyses in addition to X-ray structure analysis. The reaction mechanisms are discussed. The anticancer screening of selected compounds showed broad-spectrum anticancer activity against most melanoma cancer cell lines, ovarian cancer OVCAR-3, central nervous system cancer SF-295 and U251, non-small cell lung cancer NCI-H23, renal cancer SN12C, and colon cancer HCT-15 and HCT-116. The selected compounds exhibited moderate to weak anticancer activity to other cell lines.

Keywords

1,2,4-triazolospiro[4,5]deca-2,6,9-trien-8-ones 2-chloro-1,4-benzoquinone 5-hydroxy-1,4-naphthoquinone 5-hydroxy-2-(piperidin-1-yl)naphthalene-1,4-dione amidrazones benzo[e][1,2,4]triazines

Introduction

Amidrazones display fungistatic, bacteriostatic, and antimycotic activity.¹ In addition, they have herbicidal¹ and lipoxygenase-1 inhibitory activity.² Aly et al.³ reported that amidrazones reacted with 1,4-benzoquinone or 1,4-naphthoquinone to give, in a few minutes, benzo- and naphtho-1,2,4-triazin-6(4H)-ones. The reactions of amidrazones with 2,3,5,6-tetrachloro-1,4-benzoquinone and 2,3-dichloro-1,4-naphthoquinone in dry DMF proceed to give indazole derivatives.³ The synthesis of various 4-aryl-5-imino-3-phenyl-1H-naphtho[2,3-f]-1,2,4-triazepine-6,11-diones has also been reported⁴ by the reaction between amidrazones and 1,4-dioxo-1,4-dihydronaphthalene-2,3-dicarbonitrile, while 1,4-diphenyl-2-arylamino-2-{[1-phenylmeth-(Z)-ylidene]-hydrazino}butane-1,4-diones were obtained from the reaction of amidrazones with 1,4-diphenyl-2-butyne-1,4-dione.⁵

Quinones are widely distributed in the natural world,⁶ being found in bacteria, plants, and arthropods and are ubiquitous to living systems. Quinones play pivotal roles in biological functions including oxidative phosphorylation and electron transfer.⁷ They also have important roles as electron transfer agents in primary metabolic processes like photosynthesis and respiration which is vital to human life.⁷ A large number of chemicals with 1,4-benzoquinone as the basic subunit exhibit prominent pharmacological applications such as antibiotics,^8,9 antitumor,^10–14 antimalarial¹³ and anticoagulant compounds.¹⁵ Juglone itself shows effective anticancer activity.^16,17 However, 2-chlorocyclohexa-2,5-diene-1,4-dione (CBQ) and 2-chloro-1,4-benzoquinone are common metabolites of polycyclic aromatic hydrocarbons generated through industrial processes. Chlorobenzoquinones have remarkable effects on DNA, and a few studies are available regarding chlorobenzoquinone-induced protein modifications.^18–20 Drugs containing quinone moieties are represented by anthracyclines, anthraquinones, mitomycin C, and streptonigrin.²⁰ Doxorubicin has been used clinically to treat solid tumors²¹ and acute lymphoblastic and myeloblastic leukemia.^22–24 Anti-neoplastic agents such as actinomycin and streptonigrin as well as antibiotics such as mitamycin²⁵ (Figure 1) and rifamycin²⁶ have aminoquinone moieties in their structures. 1,4-Dioxo-3-(phenylamino)-1,4-dihydronaphthalene-2-carboxylic acid (Figure 1) shows potent cytostatic effects against both renal and melanoma cancer cell lines. Against renal cell lines, the activity (GI₅₀ = 8.38 μM) was nearly as good as that of the anticancer agent, etoposide (GI₅₀ = 7.19 μM).

Figure 1.

Representative quinone-based anticancer agents.

A series of naphthoquinone derivatives showed proteasome inhibitor activity against PI-083 (Figure 1).^27–30 In addition, they also exhibited high selectivity (twofold to fourfold more selective) for cancer cells over normal cells (L-929, IC₅₀ = 2.85 μM). The activity of 3-(4-(1-hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione against melanoma cells was comparable to that of the initial quinones, nor-ß-lapachone and β-lapachone, and better than that of doxorubicin³¹ (Figure 1).

Moreover, 3-amino-1,2,4-benzotriazine-1,4-dioxides have received considerable attention as a class of antitumor agent.³² Triazoles can easily bind with a variety of enzymes and receptors in biological systems via diverse non-covalent interactions.³³ Therefore, many triazole derivatives serve as medicinal drugs.³⁴ More specifically, compounds having the 1,2,4-triazole moiety have shown various biological activities, such as antifungal,³⁵ antimicrobial,³⁶ antitubercular,³⁷ anticancer,³⁸ anticonvulsant,³⁹ hypoglycemic,⁴⁰ anti-inflammatory, and analgesic activities.⁴¹ In this paper, we report a new straightforward reaction of amidrazones with electron-deficient naturally occurring quinones, and the new compounds with a quinone nucleus were screened for their anticancer activity.

Results and discussion

Equimolar amounts of an amidrazone 1a–d and 2-chloro-1,4-benzoquinone (2) were allowed to react in dry EtOAc containing piperidine (0.5 mL) and, after chromatographic separation and recrystallization, the products 3a–d (60%–70%) and 4a–d (11%–18%) were isolated (Scheme 1). The reactions proceeded in low yields in the absence of piperidine as shown in Table 1 (Methods a and b). Method a was established using EtOAc, piperidine, reflux for 10–14 h, whereas Method b was carried out using refluxing in EtOAc for 20–24 h. We chose amidrazones 1a–d having aryl groups with either electron-donating or electron-withdrawing substituents on the benzene ring, in order to examine the substituent effect on the reaction. Elemental analyses, infrared (IR), NMR (¹H and ¹³C), and mass spectra were in good agreement with the structures assigned to the products. For example, compound 3b was identified as 6-chloro-4-(4ʹ-chlorophenyl)-3-phenyl-1,2,4-triazaspiro[4.5]deca-2,6,9-trien-8-one. Mass spectroscopy and elemental analysis indicated the molecular formula of 3b as C₁₉H₁₃Cl₂N₃O, which corresponds to the sum of the molecular weights of the two starting materials with loss of a water molecule. The IR spectrum showed absorptions at υ = 3262 (NH), 1701 (C=O), 1613 (C=N), and 1548 cm⁻¹ (C=C). The ¹H NMR spectrum revealed a singlet at δ_H = 8.82. ¹³C NMR confirmed the structure of 3b by the appearance of the carbonyl, C=N, and spiro-carbon signals at δ_C 169.2, 161.0, and 78.12 ppm, respectively.

Scheme 1.

Reaction between amidrazones 1a–d with 2-chloro-1,4-benzoquinone (2) and synthesis of compound 6.

Table 1.

Methods and corresponding yields of compounds 3a–d and 4a–d.

Compound	Yield (%) by Method a	Yield (%) by Method b
3a/4a	65/15	35/5
3b/4b	60/13	34/2
3c/4c	61/11	35/4
3d/4d	67/18	43/6

Method a: EtOAc, piperidine, reflux for 10–14 h; Method b: EtOAc, reflux, 20–24 h.

Elemental analysis and mass spectroscopy of compound 4b showed an elimination of an HCl molecule from the two starting materials. The IR spectrum of 4b showed the OH absorption at υ = 3411, with the NH at 3270 cm⁻¹. The ¹H NMR spectrum had two singlets at δ_H = 10.05 (OH) and 7.65 ppm (NH). The aromatic protons resonated as a multiplet at δ_H = 7.55–7.40 ppm. The ¹³C NMR spectrum showed the Ar–C–OH and C=N carbons at δ_C = 156.6 and 153.5 ppm, respectively (see the experimental). The aforementioned spectroscopic data were in accordance with the absence of the two carbonyl groups with one being converted to a phenolic carbon.

The regioselectivity of the formation of compounds 3a–d was consistent with the literature previously reported by Aly et al.⁴ which described the reactions between amidrazones and 1,4-benzoquinone and/or 1,4-naphthoquinone. Benzoquinone 5 reacted with piperidine to afford initially a charge-transfer (CT) complex (Scheme 2) which gave intermediate 11. Intermediate 11 was oxidized under the reaction conditions to give 6 (Scheme 2).

Scheme 2.

Plausible mechanism showing the formation of compound 6.

The structure of 6 was proved by NMR spectroscopy and has been previously reported.^42,43 Detailed NMR spectra of compound 6 are shown in Table 2. The X-ray structure of 6 is shown in Figure 2.

Table 2.

Detailed NMR spectral data of compound 6.

¹H NMR (DMSO-d₆)	¹H–¹H COSY		Assignment
11.44 (bs; 1H)			OH
7.67 (“t,” J = 7.7; 1H)	7.42, 7.21		H-7
7.42 (d, J = 7.4; 1H)	7.67, 7.21		H-8
7.21 (d, J = 8.4; 1H)	7.67, 7.42		H-6
5.97 (s; 1H)	3.46		H-2
3.46 (m; 4H)	5.97, 1.64		H-3b
1.64 (m; 6H)	3.46		H-3c, 3d
¹³C NMR (DMSO-d₆)	HSQC	HMBC	Assignment
186.73		7.21, 5.97	C-4
181.54		7.42, 5.97	C-1
160.06		11.44, 7.67, 7.42, 7.21	C-5
153.82		5.97, 3.46	C-3
136.48	7.67	7.42	C-7
132.61		7.67, 5.97	C-8a
122.26	7.42	11.44, 7.67, 7.42	C-8
116.97	7.21	11.44, 7.67, 7.42, 7.21	C-6
116.14			C-4a
109.98	5.97		C-2
50.11	3.46	3.46, 1.64	C-3b
25.33	1.64	3.46, 1.64	C-3c
23.70	1.64		C-3d
¹⁵N NMR	HSQC	HMBC	Assignment
94.3		5.97, 1.64	N-3a

Figure 2.

Molecular structure of compound 6 (displacement parameters are drawn at 50% probability level).

Biological evaluation

Screening of the in vitro anticancer activity

Among the synthesized compounds, NCI selected compounds 3c and 6. The anticancer activity of 3c and 6 was evaluated according to the protocol of the drug evaluation branch of the National Cancer Institute (NCI), Bethesda, USA, for in vitro anticancer activity (http://www.dtp.nci.nih.gov). The results for each tested compound are reported as the percentage of growth inhibition of the treated cells when compared to the untreated control cells. The compounds were tested against a panel of 60 cancer cell lines, derived from different tumors, including leukemia, melanoma, lung, colon, central nervous system (CNS), ovarian, renal, prostate, and breast cancer. The compounds were incubated with the cells at a concentration of 10 μM for 48 h. The results, in Table 3, were reported for the growth inhibition percent (GI%). Surprisingly, 3c and 6 exhibited broad-spectrum potent inhibitory effects on most of the Melanoma cell lines especially MDA-MB-435, SK-MEL-5, UASS-62, LOX IMVI, MALME-3M, M14, and UACC-257. In addition, both compounds revealed strong anticancer activity against ovarian cancer cell lines OVCAR-3, CNS cancer cell lines SF-295, SF-539 and U251, non-small cell lung cancer cell lines HOP-62, A549/ATCC and NCI-H23, renal cancer cell lines SN12C and UO31, leukemia cell lines HL-60 and K-562, and colon cancer cell lines HCT-15 and HCT-116. Moreover, compounds 3c and 6 exhibited moderate to weak anticancer activity to other cancer cell lines (Table 2).

Table 3.

Anticancer activity of compounds 3c and 6 against 60 cell lines.

Subpanel tumor cell lines	Growth inhibition % (GI%)
	3c NSC 800747	6 NSC 800746
Leukemia
CCRF-CEM	16.88	23.47
HL-60(TB)	76.55	70.24
K-562	74.46	71.44
MOLT-4	38.15	28.33
RPMI-8226	35.95	34.49
SR	63.21	61.47
Non-small cell lung cancer
A549/ATCC	77.69	85.16
EKVX	24.88	42.15
HOP-62	73.49	78.53
HOP-92	0	0
NCI-H226	39.48	48.09
NCI-H23	86.35	90.35
NCI-H322M	10.48	16.10
NCI-H460	35.72	65.89
NCI-H522	48.91	71.31
Colon cancer
COLO 205	5.55	19.18
HCC-2998	35.59	45.76
HCT-116	73.88	81.88
HCT-15	81.29	83.20
HT29	27.03	55.65
KM12	41.44	52.73
SW-620	47.49	65.15
CNS cancer
SF-268	42.74	50.61
SF-295	72.19	82.88
SF-539	73.42	74.57
SNB-19	65.17	67.98
SNB-75	66.93	80.11
U251	81.91	88.84
Melanoma
LOX IMVI	97.91	105.86
MALME-3M	61.23	88.35
M14	71.15	91.19
MDA-MB-435	191.54	192.36
SK-MEL-28	47.35	61.80
SK-MEL-5	170.21	191.49
UACC-257	94.15	96.88
UACC-62	133.07	150.40
Ovarian cancer
IGROV1	33.27	46.17
OVCAR-3	86.48	88.75
OVCAR-4	56.72	59.20
OVCAR-5	52.37	60.12
OVCAR-8	56.63	37.81
NCI/ADR-RES	54.95	57.77
SK-OV-3	0	8.76
Renal cancer
786-0	6.56	0
A498	8.66	0
ACHN	38.89	44.79
CAKI-1	15.97	18.44
RXF 393	0	0
SN12C	73.61	92.77
TK-10	0	8.43
UO-31	71.83	77.93
Prostate cancer
PC-3	60.86	63.96
DU-145	48.07	64.34
Breast cancer
MCF7	54.57	64.58
MDA-MB-231/ATCC	22.63	24.36
BT-549	66.49	58.90
T-47D	12.85	21.23
MDA-MB-468	52.44	63.77

CNS: central nervous system.

In vitro five-dose full NCI 60 cell panel assay

Compounds 6 were selected for five-dose testing against the full panel of 60 human tumor cell lines according to the NCI protocol (http://www.dtp.nci.nih.gov). Compound 6 exhibited noteworthy antiproliferative activity against melanoma cell lines with GI₅₀ = 1.38 µM (Table 4) and selectivity ratio = 2.10, against CNS cancer cell lines with GI₅₀ = 1.406 and selectivity ratio = 2.07 and against colon cancer cell lines with GI₅₀ = 2.06 and selectivity ratio = 1.41 (Figure 3).

Table 4.

Results of in vitro five-dose testing of nine human cancer types and selectivity for compound 6.

Panel	Cell line	GI₅₀			TGI	LC₅₀
Panel	Cell line	Concentration per cell line	MID^b	Selectivity ratio
Leukemia	CCRF-CEM	4.83	4.06	0.715	>100	>100
	HL-60(TB)	2.77			8.03	>100
	K-562	1.60			21.0	>100
	MOLT-4	6.81			>100	>100
	RPMI-8226	3.52			>100	>100
	SR	4.38			7.69	>100
Non-small cell lung cancer	A549/ATCC	1.84	4.08	0.712	5.00	>100
	EKVX	1.41			14.5	>100
	HOP-62	0.693			2.22	5.99
	HOP-92	22.8			54.2	>100
	NCI-H226	1.83			4.53	>100
	NCI-H23	0.46			2.18	8.82
	NCI-H322M	3.96			14.6	40.4
	NCI-H460	2.56			10.2	75.4
	NCI-H522	1.21			3.40	9.60
Colon cancer	COLO 205	1.89	2.06	1.41	3.83	7.76
	HCC-2998	3.83			13.0	57.3
	HCT-116	1.38			5.26	>100
	HCT-15	0.472			2.37	>100
	HT29	2.53			6.21	>100
	KM12	2.65			18.0	>100
	SW-620	1.67			3.64	7.92
CNS cancer	SF-268	1.84	1.406	2.07	14.3	>100
	SF-295	1.34			3.18	7.52
	SF-539	1.26			2.79	6.19
	SNB-19	1.59			10.8	43.5
	SNB-75	1.82			3.44	6.48
	U251	0.587			20.2	>100
Melanoma	LOX IMVI	0.275	1.38	2.10	1.28	5.75
	MALME-3M	1.28			2.58	5.20
	M14	1.30			2.60	5.22
	MDA-MB-435	0.185			0.336	0.609
	SK-MEL-2	4.60			21.5	>100
	SK-MEL-28	1.99			4.38	9.65
	SK-MEL-5	0.272			0.706	2.57
	UACC-257	1.47			2.93	5.84
	UACC-62	1.05			2.26	4.88
Ovarian cancer	IGROV1	3.19	3.225	0.90	17.1	>100
	OVCAR-3	1.08			3.70	18.6
	OVCAR-4	0.698			3.21	13.5
	OVCAR-5	1.79			3.54	>100
	OVCAR-8	2.40			>100	>100
	NCI/ADR-RES	1.62			30.9	>100
	SK-OV-3	11.8			98.0	>100
Renal cancer	786-0	6.13	4.528	0.641	43.0	>100
	A498	5.53			19.6	50.7
	ACHN	3.11			21.5	>100
	CAKI-1	5.71			50.3	>100
	RXF 393	7.36			25.0	74.0
	SN12C	1.47			2.84	5.48
	TK-10	5.46			46.0	>100
	UO-31	1.46			7.67	54.2
Prostate cancer	PC-3	3.19	2.23	1.30	>100	>100
	DU-145	1.27			2.56	5.15
Breast cancer	MCF7	1.50	2.443	1.19	3.83	9.74
	MDA-MB-231/ATCC	2.37			33.9	>100
	HS 578T	2.86			7.63	>100
	BT-549	1.13			2.69	6.36
	T-47D	4.88			37.7	>100
	MDA-MB-468	1.92			9.09	>100

MID^a = 2.905; selectivity ratio = MID^a/MID^b.

Figure 3.

Five-dose diagram of compound 6.

Conclusion

Herein, we report reactions of amidrazones with two naturally occurring quinones, namely, 2-chloro-1,4-benzoquinone and 5-hydroxy-1,4-napthalene-1,4-dione (Juglone). Spiro-triazoles and 1,2,4-triazines were obtained from the reactions of amidrazones with the aforementioned quinones. The screening results were encouraging and promising, but further studies of their anticancer activities should be continued.

Experimental

Melting points were determined using open glass capillaries on a Gallenkamp melting point apparatus (Weiss Gallenkamp, Loughborough, UK), and they are uncorrected. The IR spectra were recorded from potassium bromide disks with an FT device, and Minia University NMR spectra were measured on a Bruker AV-400 spectrometer (400 MHz for ¹H, 100 MHz for ¹³C, and 40.55 MHz for ¹⁵N); chemical shifts are expressed in δ (ppm) versus internal tetramethylsilane (TMS) = 0 for ¹H and ¹³C, and external liquid ammonia = 0 for ¹⁵N. Coupling constants are stated in Hz. Correlations were established using ¹H–¹H COSY, and ¹H–¹³C and ¹H–¹⁵N HSQC and HMBC experiments. Mass spectra were recorded on a Finnigan Fab 70 eV, Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany. TLC was performed on analytical Merck 9385 silica aluminum sheets (Kieselgel 60) with Pf₂₅₄ indicator; TLCs were viewed at λ_max = 254 nm. Elemental analyses were carried out at National Research Center, Al Doki, Egypt.

Starting materials

Amidrazones 1a–d were prepared according to reported method by Müller et al.⁴⁴

Reactions of amidrazones 1a–d with 2-chloro-1,4-benzoquinone (2)

Equimolar mixtures of amidrazones 1a–d (1 mmol) and 2 (0.142 g, 1 mmol) in 50 mL of absolute EtOAc and 0.5 mL of piperidine were refluxed for 10–14 h. The reactions were followed by the TLC analysis. The mixtures were then concentrated under reduced pressure, and the resulting solids were separated by preparative PC using toluene: ethyl acetate (2:1). The faster migrating zones gave compounds 4a–d, followed by products 3a–d. The products were then recrystallized from the appropriate solvents.

6-Chloro-3,4-diphenyl-1,2,4-triazaspiro[4.5]deca-2,6,9-trien-8-one (3a)

Red solid (EtOH), yield: 0.218 g (65%), m.p. 115–117 °C. IR (KBr): ν = 3260 (NH), 3136 (Ar–CH), 1702 (C=O), 1614 (C=N), 1599 cm⁻¹ (Ar–C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 8.83 (s, 1H, NH), 8.20-8.15 (m, 2H), 7.80-7.75 (m, 2H, Ar–CH), 7.60-6.72 (m, 8H, Ar–CH), 6.60 (d, 1H, J = 0.7 Hz, Ar–CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 169.0 (C=O), 163.0 (C=N), 141.8, 134.0, 130.6 (Ar–C), 130.3, 130.0 (Ar–CH), 129.7, 129.4, 127.9, 126.4 (Ar–2CH), 126.3, 125.5, 122.6 (Ar–CH), 78.1 (Spiro-C). MS: m/z (%) = 337 (6), 335.8 (27), 256.0 (42), 225.3 (47), 185 (30), 183.6 (100), 154.0 (58), 149.2 (47), 114.1 (15), 81.0 (39), 77.1 (93). Anal. calcd for C₁₉H₁₄ClN₃O (335.79): C, 67.96; H, 4.20; Cl, 10.56; N, 12.51; found: C, 68.05; H, 4.08; Cl, 10.70; N, 12.60.

6-Chloro-4-(4ʹ-chlorophenyl)-3-phenyl-1,2,4-triazaspiro[4.5]deca-2,6,9-trien-8-one (3b)

Red solid (EtOH), yield: 0.222 g (60%), m.p. 204–206 °C. IR (KBr): ν = 3262 (NH), 3135 (Ar–CH), 1701 (C=O), 1613 (C=N), 1548 cm⁻¹ (Ar–C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 8.82 (s, 1H, NH), 8.40-8.35 (m, 2H, Ar–CH), 8.25-8.22 (m, 2H, Ar–CH), 7.62-7.52 (m, 7H, Ar–CH), 7.42 (d, 1H, J = 0.8 Hz, Ar–CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 169.2 (C=O), 161.0 (C=N), 141.17, 138.49, 132.48, 130.49 (Ar–C), 130.22 (Ar–CH), 129.57, 129.46, 129.12, 128.89 (Ar–2CH), 125.72, 125.65, 125.19 (Ar–CH), 78.12 (Spiro-C). MS: m/z (%) = 370.6 (63), 372.6 (42), 336.0 (14), 296.1 (18), 294.1 (59), 261.1 (6), 259.1 (26), 236.0 (36), 225.3 (47), 185.6 (29), 154.0 (59), 149.2 (47), 114.2 (4), 112.2 (14), 81.1 (39), 77.1 (93). Anal. calcd for C₁₉H₁₃Cl₂N₃O (370.23): C, 61.64; H, 3.54; N, 11.35; Cl, 19.15; found: C, 61.52; H, 3.43; Cl, 19.03; N, 11.13.

6-Chloro-4-(3ʹ-chlorophenyl)-3-phenyl-1,2,4-triazaspiro[4.5]deca-2,6,9-trien-8-one (3c)

Red solid (CH₃OH), yield: 0226 g (61%), m.p. 174–176 °C. IR (KBr): ν = 3269 (NH), 3130 (Ar–CH), 1705 (C=O), 1616 (C=N), 1548 cm⁻¹ (Ar–C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 8.73 (s, 1H, NH), 8.35 (dd, 2H, J = 1.2, 0.8 Hz, Ar–CH), 8.13-7.95 (m, 2H, Ar–CH), 7.53-7.35 (m, 7H, Ar–CH), 7.33 (d, 1H, J = 0.7 Hz, Ar–CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 169.7 (C=O), 161.8 (C=N), 141.14, 140.7, 135.8, 132.59 (Ar–C), 131.4 (Ar–CH), 131.17, 130.96, 130.45, 129.58 (Ar–2CH), 126.28, 125.22, 124.34 (Ar–CH), 78.23 (Spiro-C). MS: m/z (%) = 372.2 (16), 370.2 (53), 336.0 (15), 296.1 (20). 294.1 (62), 261.1 (9), 259.1 (27), 236.0 (37), 225.3 (47), 185.0 (100), 154.0 (60), 149.2 (47), 114.0 (7), 112.0 (26), 81.1 (39), 77.1 (70). Anal. calcd for C₁₉H₁₃Cl₂N₃O (370.23): C, 61.64; H, 3.54; Cl, 19.15; N, 11.35; found: C, 61.72; H, 3.60; Cl, 19.25; N, 11.20.

6-Chloro-3-phenyl-4(4ʹ-methoxyphenyl)-1,2,4-triazaspiro[4.5]deca-2,6,9-trien-8-one (3d)

Red solid (CH₃CN) 245 mg (67%), m.p. 254–256 C. IR (KBr): ν = 3260 (NH), 3140 (Ar–CH), 1700 (C=O), 1610 (C=N), 1550 (C=C), 1110 cm⁻¹ (OCH₃). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 8.90 (s, 1H, NH), 8.40 (dd, 2H, J = 1.2, 0.7 Hz, Ar–CH), 8.25-8.20 (m, 3H, Ar–CH), 7.52-7.45 (m, 5H, Ar–CH), 6.60 (dd, 2H, J = 0.8 Hz, Ar–CH), 3.90 (s, 3H, OCH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 169.2 (C=O), 161.0 (C=N), 150.1 (Ar–C–OCH₃), 138.2, 132.0, 130.3 (Ar–C), 129.2 (Ar–CH), 128.1, 127.5, 127.1, 126.8 (Ar–2CH), 125.7, 123.6, 122.2 (Ar–CH), 78.1 (Spiro-C), 57.6 (OCH₃). MS: m/z (%) = 367 (13), 365.0 (40), 2960 (20), 184.6 (19), 154.0 (60), 121.0 (100), 77.0 (60). Anal. calcd for C₂₀H₁₆ClN₃O₂ (365.81): C, 65.67; H, 4.41; N, 11.49; Cl, 9.69; found: C, 65.72; H, 4.43; N, 11.30; Cl, 9.60.

3,4-Diphenyl-1,4-dihydrobenzo[e][1,2,4]triazin-6-ol (4a)

White solid (CH₃CN), yield: 0.045 g (15%), m.p. 302–304 °C. IR (KBr): ν = 3424 (OH), 3264 (NH), 1631 (C=N), 1590 cm⁻¹ (Ar–C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 10.12 (s, 1H, OH), 8.54 (s, 1H, NH), 7.48-7.13 (m, 13H, Ar–CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 158.2 (Ar–C–OH), 154.0 (C=N), 132.0, 130.8, 130.0, 129.0 (Ar–C), 128.6, 128.4, 127.8, 127.4, 127.0 (Ar–2CH), 126.9, 126.5, 126.0 (Ar–CH). MS: m/z (%) = 301.34 (60), 225.25 (33), 156.26 (59), 133.15 (100), 77.05 (58). Anal. calcd for C₁₉H₁₅N₃O (301.34): C, 75.73; H, 5.02; N, 13.94; found: C, 75.61; H, 5.11; N, 14.02.

4-(4ʹ-Chlorophenyl)-3-phenyl-1,4-dihydrobenzo[e][1,2,4]triazin-6-ol (4b)

White solid (CH₃CN), yield: 0.044 g (13%), m.p. 264–266 °C. IR (KBr): ν = 3411 (OH), 3270 (NH), 1629 (C=N), 1593 cm⁻¹ (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 10.05 (s, 1H, OH), 8.70 (s, 1H, NH), 7.55-7.40 (m, 12H, Ar–CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 156.6 (Ar–C–OH), 153.5 (C=N), 134.2, 133.7, 130.2, 129.7, 128.5 (Ar–C), 128.3, 128.1, 127.8, 127.2, 127.0 (Ar–2CH), 126.9, 126.8 (Ar–CH). MS: m/z (%) = 337.1 (15), 335.1 (45), 261.7 (19), 259.1 (61), 225.3 (100), 149.2 (58), 133.2 (33), 114.6 (19), 112.6 (53), 77.1 (92). Anal. calcd for C₁₉H₁₄ClN₃O (335.79): C, 67.96; H, 4.20; Cl, 10.56; N, 12.51; found: C, 68.05; H, 4.08; Cl, 10.70; N, 12.77.

4-(3ʹ-Chlorophenyl)-3-phenyl-1,4-dihydrobenzo[e][1,2,4]triazin-6-ol (4c)

White solid (CH₃CN), yield: 0.037 g (11%), m.p. 262–264 °C. IR (KBr): ν = 3424 (OH), 3264 (NH), 1631 (C=N), 1584 cm⁻¹ (C=C). ¹H NMR (400 MHz, CDCl₃): δ_H = 10.22 (s, 1H, OH), 7.94 (s, 1H, NH), 7.48-7.30 (m, 12H, Ar–CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 158.2 (Ar–C–OH), 154.2 (C=N), 134.3, 133.9, 132.1, 130.3, 129.8 (Ar–C), 128.7, 128.2, 127.9, 127.3, 127.1 (Ar–2CH), 126.8, 126.5 (Ar–CH). MS: m/z (%) = 337.1 (15), 335.1 (45), 261.7 (28), 259.1 (60), 225.3 (100), 190.0 (8), 149.2 (59), 133.2 (22), 114.6 (15), 112.6 (44), 77.1 (80). Anal. calcd for C₁₉H₁₄ClN₃O (335.79): C, 67.96; H, 4.20; Cl, 10.56; N, 12.51; found: C, 68.05; H, Cl, 10.65; 4.08; N, 12.72.

4-(4ʹ-Methoxyphenyl)-3-phenyl-1,4-dihydrobenzo[e][1,2,4]triazin-6-ol (4d)

White solid (CH₃CN), yield: 0.060 g (18%), m.p. 210–212 °C. IR (KBr): ν = 3410 (OH), 3250 (NH), 1620 (C=N), 1590 cm⁻¹ (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 10.00 (s, 1H, OH), 8.80 (s, 1H, NH), 7.60 (dd, 2H, J = 1.2, 0.7 Hz), 7.30-7.10 (m, 8H, Ar–CH), 6.60 (dd, 2H, J = 1.2, 0.7 Hz), 3.90 (s, 3H, OCH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 157.8 (Ar–C–OH), 152.0 (Ar–OCH₃), 151.2 (C=N), 135.0, 130.2, 129.4, 128.6 (Ar–C), 127.6, 126.4, 125.8, 125.2, 122.0 (Ar–2CH), 121.0, 120.8 (Ar–CH), 52.0 (OCH₃). MS: m/z (%) = 331.1 (30), 225.0 (100), 149.2 (40), 133.2 (30), 77.1 (90). Anal. calcd for C₂₀H₁₇N₃O₂ (331.37): C, 72.49; H, 5.17; N, 12.68; found: C, 73.00; H, 5.00; N, 12.82.

Reaction of piperidine with 5-hydroxy-1,4-naphthaquinone (5)

An equimolar mixture of 5 (0.161 g, 1 mmol) and piperidine (0.085 g, 1 mmol) in 100 mL of absolute EtOAc was stirred at room temperature for 4 h. Blue precipitate was formed, which by time (1 h) dissolved and a green precipitate of compound 6 was formed. The green precipitate was then dissolved in acetone and purified on applying to preparative PC using toluene: ethyl acetate (10:1). The formed product 6 was then recrystallized from EtOH.

8-Hydroxy-2-(piperidin-1-yl)naphthalene-1,4-dione (6) [42 and 43]

Green solid (EtOH), yield: 0.206–0.219 g (80%–85%), m.p. 159–160 °C (rep. 158 °C). IR (KBr): ν = 3440 (OH), 1700 (C=O), 1590 cm⁻¹ (C=C). NMR (see Table 2). MS: m/z (%) = 257 (M⁺, 100). Anal. calcd for C₁₅H₁₅NO₃ (257.28): C, 70.02; H, 5.88; N, 5.44; found: C, 70.20; H, 5.98; N, 5.60.

Crystal structure determinations of 6

The single-crystal X-ray diffraction study was carried out on a Bruker D8 Venture diffractometer with Photon100 detector at 123(2) K using Mo-Kα radiation (λ = 0.71073 Å). Dual space methods (SHELXT)⁴⁵ were used for structure solution, and refinement was carried out using SHELXL-2014 (full-matrix least squares on F²).⁴⁶ Hydrogen atoms were localized by difference electron density determination and refined using a riding model (H(O) free). A semi-empirical absorption correction and an extinction correction were applied. 6: red crystals, C₁₅H₁₅NO₃, M_r = 257.28, crystal size 0.236 mm × 0.24 mm × 0.16 mm, monoclinic, space group P2₁/c (no. 14), a = 10.1353(5) Å, b = 12.0295(5) Å, c = 11.1132(7) Å, β = 116.045(2)°, V = 1217.35(11) Å³, Z = 4, ρ = 1.404 Mg/m⁻³, µ(Mo-K_α) = 0.10 mm⁻¹, F(000) = 544, 2θ_max = 55.0°, 32,736 reflections, of which 2810 were independent (R_int = 0.031), 176 parameters, 1 restraint, R₁ = 0.038 (for 2480 I > 2σ(I)), wR₂ = 0.101 (all data), S = 1.03, largest difference peak/hole = 0.37/−0.19 e Å⁻³.

CCDC 1968210 (6) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

NCI screening assay

The methodology of the NCI procedure for primary anticancer assay is detailed on their site (http://www.dtp.nci.nih.gov), but briefly, the protocol was performed using the 60 human tumor cell lines panel derived from nine different neoplastic diseases. NCI-60 testing is performed in two parts: first, a single concentration is tested in all 60 cell lines at a single dose of 10⁻⁵ M or 15 μg/mL in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda, USA. If the results obtained meet selection criteria, then the compound is tested again in all 60 cell lines in 5 × 10 folds of dilution.

Supplemental Material

supporting_file – Supplemental material for Synthesis of quinone-based heterocycles of broad-spectrum anticancer activity

Supplemental material, supporting_file for Synthesis of quinone-based heterocycles of broad-spectrum anticancer activity by Ashraf A Aly, Alaa A Hassan, Nasr K Mohamed, Mohamed Ramadan, Amal S Abd El-Aal, Stefan Bräse and Martin Nieger in Journal of Chemical Research

Footnotes

Acknowledgements

The authors thank DFG Collaborative Center “3MET,” Karlsruhe Institute of Technology, Karlsruhe, Germany, for financial support to Professor Aly enabling him to carry out analyses in the aforesaid Institute. Purchase of the NMR spectrometer at the Florida Institute of Technology was assisted by the US National Science Foundation (CHE 03-42251).

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Ashraf A Aly

Supplemental material

Supplemental material for this article is available online.

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