<i>In vivo</i> chemotherapeutic insight of a novel isocoumarin (3-hexyl-5,7-dimethoxy-isochromen-1-one): Genotoxicity, cell death induction, leukometry and phagocytic evaluation

Araújo, Flávio Henrique Souza de; Figueiredo, Débora Rojas de; Auharek, Sarah Alves; Pesarini, João Renato; Meza, Alisson; Gomes, Roberto da Silva; Monreal, Antônio Carlos Duenhas; Antoniolli-Silva, Andréia Conceição Milan Brochado; Lima, Dênis Pires de; Kassuya, Candida Aparecida Leite; Beatriz, Adilson; Oliveira, Rodrigo Juliano

doi:10.1590/1678-4685-GMB-2016-0316

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Genetics and Molecular Biology

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Mutagenesis • Genet. Mol. Biol. 40 (3) • Sept 2017 • https://doi.org/10.1590/1678-4685-GMB-2016-0316 copy

In vivo chemotherapeutic insight of a novel isocoumarin (3-hexyl-5,7-dimethoxy-isochromen-1-one): Genotoxicity, cell death induction, leukometry and phagocytic evaluation

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Chemotherapy is one of the major approaches for the treatment of cancer. Therefore, the development of new chemotherapy drugs is an important aspect of medicinal chemistry. Chemotherapeutic agents include isocoumarins, which are privileged structures with potential antitumoral activity. Herein, a new 3-substituted isocoumarin was synthesized from 2-iodo-3,5-dimethoxy-benzoic acid and oct-1-yne in a cross-coupling Sonogashira reaction followed by a copper iodide-catalyzed intramolecular cyclization as key step using MeOH/Et₃N as the solvent system. The present study also evaluated the leukometry, phagocytic activity, genotoxic potential and cell death induction of three different doses (5 mg/kg, 10 mg/kg and 20 mg/kg) of this newly synthesized isocoumarin, alone and in combination with the commercial chemotherapeutic agents cyclophosphamide (100 mg/kg) and cisplatin (6 mg/kg) in male Swiss mice. The results suggest that the isocoumarin has genotoxicity and causes cell death. Noteworthy, this new compound can increase splenic phagocytosis and lymphocyte frequency, which are related to immunomodulatory activity. When combined with either cyclophosphamide or cisplatin, chemopreventive activity led to a reduction in the effects of both chemotherapeutic drugs. Thus, the new isocoumarin is not a candidate for chemotherapeutic adjuvant in treatments using cyclophosphamide or cisplatin. Nevertheless, the compound itself is an important prototype for the development of new antitumor drugs.

Keywords:
isocoumarin synthesis; genotoxicity; splenic phagocytosis; chemotherapy

Experimental groups	Initial weight (g)	Final weight (g)
Negative control	35.8±0.66 ^a	35.0±0.75 ^a
Isocoum 5 mg/kg	34.5±0.82 ^a	33.9±0.70 ^a
Isocoum 10 mg/kg	35.6±0.54 ^a	34.8±0.66 ^a
Isocoum 20 mg/kg	34.0±0.54 ^a	33.0±0.52 ^a
Cyclophosphamide (CP)	35.2±1.02 ^a	33.0±1.05 ^a
CP + Isocoum 5 mg/kg	35.8±0.82 ^a	34.7±0.60 ^a
CP + Isocoum 10 mg/kg	35.7±0.63 ^a	34.6±0.73 ^a
CP + Isocoum 20 mg/kg	34.1±0.47 ^a	32.7±0.66 ^a
Cisplatin (CDDP)	35.5±0.96 ^a	34.2±0.64 ^a
CDDP + Isocoum 5 mg/kg	34.3±0.60 ^a	33.7±0.40 ^a
CDDP + Isocoum 10mg/kg	34.1±0.68 ^a	32.1±0.78 ^a
CDDP + Isocoum 20mg/kg	34.7±0.67 ^a	33.7±0.89 ^a

Experimental groups	Heart	Lungs	Liver	Kidneys	Spleen
Absolute weight (g)
Negative control	0.202±0.006 ^a	0.1750.005 ^a	1.650±0.053 ^a	0.490±0.015 ^a	0.1580.007 ^a
Isocoum 5 mg/kg	0.230±0.009 ^a	0.311±0.010 ^c	1.781±0.035 ^a	0.508±0.022 ^a	0.230±0.010 ^b
Isocoum 10 mg/kg	0.232±0.008 ^a	0.316±0.013 ^c	1.798±0.029 ^a	0.500±0.023 ^a	0.233±0.010 ^b
Isocoum 20 mg/kg	0.231±0.007 ^a	0.315±0.009 ^c	1.780±0.025 ^a	0.507±0.013 ^a	0.231±0.012 ^b
Cyclophosphamide (CP)	0.231±0.006 ^a	0.2240.006 ^b	1.663±0.039 ^a	0.5250.014 ^a	0.191±0.006 ^ab
CP + Isocoum 5 mg/kg	0.234±0.006 ^a	0.260±0.007 ^b	1.681±0.028 ^a	0.491±0.014 ^a	0.1950.016 ^ab
CP + Isocoum 10 mg/kg	0.235±0.011 ^a	0.230±0.009 ^b	1.650±0.059 ^a	0.490±0.023 ^a	0.199±0.009 ^ab
CP + Isocoum 20 mg/kg	0.230±0.013 ^a	0.230±0.008 ^b	1.696±0.053 ^a	0.494±0.008 ^a	0.197±0.005 ^ab
Cisplatin (CDDP)	0.234±0.013 ^a	0.2280.015 ^b	1.665±0.030 ^a	0.523±0.016 ^a	0.193±0.005 ^ab
CDDP + Isocoum 5 mg/kg	0.213±0.008 ^a	0.217±0.007 ^ab	1.693±0.073 ^a	0.500±0.017 ^a	0.198±0.008 ^ab
CDDP + Isocoum 10mg/kg	0.211±0.010 ^a	0.2180.006 ^ab	1.699±0.041 ^a	0.486±0.013 ^a	0.197±0.009 ^ab
CDDP + Isocoum 20mg/kg	0.214±0.009 ^a	0.213±0.033 ^ab	1.703±0.035 ^a	0.488±0.016 ^a	0.198±0.004 ^ab

Experimental groups		Mean±SE			DR%
	24h (T1)	48h (T2)	72h (T3)	24h (T1)	48h (T2)	72h (T3)
Negative control	9.4±0.60^a	8.4±0.67^a	8.0±0.51^a	–	–	–
Cyclophosphamide (CP)	110.4±1.27^g	97.3±0.37^e	91.51.08^d	–	–	–
Isocoum 5 mg/kg	23.4±0.96^c	16.3±0.84^b	23.7±1.06^b	–	–	–
Isocoum 10 mg/kg	33.4±1.22^d	22.6±0.76^c	27.8±0.90^b	–	–	–
Isocoum 20 mg/kg	66.1±1.51^f	29.1±0.67^d	37.1±1.31^c	–	–	–
CP + Isocoum 5 mg/kg	16.5±0.72^b	23.5±0.80^c	37.4±1.19^c	93.0	83.0	64.8
CP + Isocoum 10 mg/kg	33.2±1.14^d	23.7±0.79^c	28.4±0.98^b	76.4	82.8	75.6
CP + Isocoum 20 mg/kg	55.81.25^e	25.5±0.78^c	33.7±1.36^c	54.0	80.7	69.2
Negative control	9.4±0.60^a	8.4±0.67^a	7.8±0.49^a	–	–	–
Cisplatin (CDDP)	93.6±1.90^g	89.8±1.12^g	87.9±1.46^e	–	–	–
Isocoum 5 mg/kg	23.4±0.96^b	16.3±0.84^b	23.7±1.06^b	–	–	–
Isocoum 10 mg/kg	33.4±1.22^c	22.6±0.76^c	27.8±0.90^b	–	–	–
Isocoum 20 mg/kg	66.1±1.51^f	29.1±0.67^d	37.1±1.31^c	–	–	–
CDDP + Isocoum 5 mg/kg	49.7±1.44^e	57.5±0.97^f	38.2±1.29^c	52.1	39.7	62.0
CDDP + Isocoum 10mg/kg	41.0±1.23^d	30.4±1.54^d	48.0±1.37^d	62.5	73.0	49.8
CDDP + Isocoum 20mg/kg	49.5±1.46^e	38.5±0.99^e	52.5±1.26^d	52.4	63.0	44.2

Experimental groups	Total of cells	Number of dead cells
		Liver			Kidneys
		AV	Mean±SE	(%)	AV	Mean±SE	(%)
Negative control	1000	147	14.7±0.56 ^a	14.7	164	16.4±0.62 ^a	16.4
Cyclophosphamide (CP)	1000	363	36.3±1.13 ^d	36.3	273	27.3±0.73 ^b	27.3
Isocoum 5 mg/kg	1000	264	26.4±1.16 ^b	26.4	274	27.4±0.65 ^bc	27.4
Isocoum 10 mg/kg	1000	259	25.9±1.20 ^b	25.9	260	26.0±0.77 ^b	26.0
Isocoum 20 mg/kg	1000	361	36.1±0.75 ^d	36.1	267	26.7±0.73 ^b	26.7
CP + Isocoum 5 mg/kg	1000	368	36.8±0.70 ^d	36.8	307	30.7±0.98 ^cd	30.7
CP + Isocoum 10 mg/kg	1000	310	31.0±0.54 ^c	31.0	313	31.3±0.77 ^d	31.3
CP + Isocoum 20 mg/kg	1000	360	36.0±0.54 ^d	36.0	310	31.0±0.68 ^d	31.0
Negative control	1000	147	14.7±0.56 ^a	14.7	164	16.4±0.62 ^a	16.4
Cisplatin (CDDP)	1000	307	30.7±1.50 ^c	30.7	289	28.9±0.81 ^bcd	28.9
Isocoum 5 mg/kg	1000	264	26.4±1.16 ^bc	26.4	274	27.4±0.65 ^bcd	27.4
Isocoum 10 mg/kg	1000	259	25.9±1.20 ^b	25.9	260	26.0±0.77 ^b	26.0
Isocoum 20 mg/kg	1000	361	36.1±0.75 ^d	36.1	267	26.7±0.73 ^bc	26.7
CDDP + Isocoum 5 mg/kg	1000	340	34.0±1.01 ^cd	34.0	304	30.4±0.76 ^d	30.4
CDDP + Isocoum 10 mg/kg	1000	334	33.4±1.13 ^cd	33.4	286	28.6±0.62 ^bcd	28.6
CDDP + Isocoum 20 mg/kg	1000	350	35.0±1.02 ^cd	35.0	298	29.8±0.77 ^cd	29.8

Experimental groups	Total of cells	Cells without phagocytosis evidence			Cells with phagocytosis evidence
Experimental groups	Total of cells	AV	Mean±SE	(%)	AV	Mean±SE	(%)
Negative control	1000	495	49.5±0.40 ^d	49.5	505	50.5±0.40 ^a	50.5
Cyclophosphamide (CP)	1000	344	34.4±0.79 ^c	34.4	656	65.6±0.79 ^b	65.6
Isocoum 5 mg/kg	1000	144	14.4±0.65 ^a	14.4	856	85.6±0.65 ^d	85.6
Isocoum 10 mg/kg	1000	134	13.4±0.40 ^a	14.0	866	86.6±0.40 ^d	86.6
Isocoum 20 mg/kg	1000	138	13.8±0.83 ^a	13.8	862	86.2±0.83 ^d	86.2
CP + Isocoum 5 mg/kg	1000	261	26.1±0.64 ^b	26.1	739	73.9±0.64 ^c	73.9
CP + Isocoum 10 mg/kg	1000	250	25.0±0.39 ^b	25.0	750	75.0±0.39 ^c	75.0
CP + Isocoum 20 mg/kg	1000	260	26.0±0.76 ^b	26.0	740	74.0±0.76 ^c	74.0
Negative control	1000	495	49.5±0.40 ^d	49.5	505	50.5±0.40 ^a	50.5
Cisplatin (CDDP)	1000	403	40.3±0.42 ^c	40.3	597	59.7±0.42 ^b	59.7
Isocoum 5 mg/kg	1000	144	14.4±0.66 ^a	14.4	856	85.2±0.66 ^d	85.2
Isocoum 10 mg/kg	1000	134	13.4±0.40 ^a	13.4	866	86.6±0.40 ^d	86.6
Isocoum 20 mg/kg	1000	138	13.8±0.83 ^a	13.8	862	86.2±0.83 ^d	86.2
CDDP + Isocoum 5 mg/kg	1000	361	36.1±0.64 ^b	36.2	639	63.9±0.64 ^c	63.9
CDDP + Isocoum 10 mg/kg	1000	366	36.6±0.75 ^b	36.6	634	63.4±0.75 ^c	63.4
CDDP + Isocoum 20 mg/kg	1000	358	35.8±0.88 ^b	35.8	642	64.2±0.88 ^c	64.2

Cell type	Lymphocytes	Neutrophils	Monocytes	Eosinophils	Basophils
Reference values	55-95%	10-40%	0.1-3.5%	0-0.4%	0-0.3%
Experimental groups
Negative control	49.2±2.10 ^a	45.8±1.62 ^e	3.2±0.42 ^abc	1.8±0.63 ^ab	0.0±0.00 ^a
Cyclophosphamide (CP)	54.2±0.79 ^b	39.7±0.82 ^d	5.2±0.42 ^d	0.9±057 ^a	0.0±0.00 ^a
Isocoum 5 mg/kg	52.2±1.81 ^ab	33.71.25 ^c	4.2±0.42 ^cd	9.9±0.99 ^b	0.0±0.00 ^a
Isocoum 10 mg/kg	62.3±2.00 ^c	28.0±1.33 ^b	4.5±0.71 ^cd	5.2±0.63 ^b	0.0±0.00 ^a
Isocoum 20 mg/kg	67.8±1.47 ^d	27.0±2.11 ^b	3.9±0.74 ^bcd	1.3±1.16 ^a	0.0±0.00 ^a
CP + Isocoum 5 mg/kg	76.5±6.96 ^e	21.4±6.52 ^a	1.4±0.51 ^a	0.7±0.48 ^a	0.0±0.00 ^a
CP + Isocoum 10 mg/kg	68.5±1.08 ^d	28.2±1.13 ^b	2.2±0.79 ^ab	1.1±0.31 ^a	0.0±0.00 ^a
CP + Isocoum 20 mg/kg	79.3±1.83 ^e	17.9±1.85 ^a	1.8±0.63 ^a	1.0±0.00 ^a	0.0±0.00 ^a
Negative control	49.2±2.10 ^a	45.8±1.62 ^e	3.2±0.42 ^bcd	1.8±0.63 ^ab	0.0±0.00 ^a
Cisplatin (CDDP)	56.3±2.16 ^c	37.7±2.83 ^d	4.6±1.26 ^d	1.50.71 ^a	0.0±0.00 ^a
Isocoum 5 mg/kg	52.2±1.81 ^b	33.71.25 ^c	4.2±0.42 ^d	9.9±0.99 ^b	0.0±0.00 ^a
Isocoum 10 mg/kg	62.3±2.00 ^d	28.0±1.33 ^b	4.5±0.71 ^d	5.2±0.63 ^b	0.0±0.00 ^a
Isocoum 20 mg/kg	67.8±1.47 ^e	27.0±2.11 ^b	3.9±0.74 ^cd	1.3±1.16 ^a	0.0±0.00 ^a
CDDP + Isocoum 5 mg/kg	81.0±0.81 ^f	16.8±0.92 ^a	2.1±0.57 ^abc	0.1±0.31 ^a	0.0±0.00 ^a
CDDP + Isocoum 10 mg/kg	66.2±2.04 ^e	33.2±1.87 ^c	0.1±0.31 ^a	0.5±0.53 ^a	0.0±0.00 ^a
CDDP + Isocoum 20 mg/kg	80.7±1.50 ^f	18.61.58 ^a	0.6±0.51 ^ab	0.1±0.31 ^a	0.0±0.00 ^a

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[1] Send correspondence to Rodrigo Juliano Oliveira. Medicine College, Federal University of Mato Grosso do Sul. Cidade Universitária, S/N. 79070-900 Campo Grande, MS, Brazil. E-mail: rodrigo.oliveira@ufms.br.