Synthesis and characterization of Mn(I) complexes and their larvicidal activity against Aedes aegypti, vector of dengue fever

Accepted Manuscript Synthesis and characterization of Mn(I) complexes and their larvicidal activity against Aedes aegypti, vector of dengue fever

Inara de Aguiar, Edjane Rocha dos Santos, Ana Carolina Mafud, Vinicius Annies, Mario Antonio Navarro-Silva, Valeria Rodrigues dos Santos Malta, Maria Teresa do Prado Gambardella, Francisco de Assis Marques, Rose Maria Carlos PII: DOI: Reference:

S1387-7003(17)30083-7 doi: 10.1016/j.inoche.2017.07.018 INOCHE 6715

To appear in:

Inorganic Chemistry Communications

Received date: Revised date: Accepted date:

27 January 2017 10 May 2017 13 July 2017

Please cite this article as: Inara de Aguiar, Edjane Rocha dos Santos, Ana Carolina Mafud, Vinicius Annies, Mario Antonio Navarro-Silva, Valeria Rodrigues dos Santos Malta, Maria Teresa do Prado Gambardella, Francisco de Assis Marques, Rose Maria Carlos , Synthesis and characterization of Mn(I) complexes and their larvicidal activity against Aedes aegypti, vector of dengue fever, Inorganic Chemistry Communications (2017), doi: 10.1016/j.inoche.2017.07.018

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ACCEPTED MANUSCRIPT Synthesis and characterization of Mn(I) complexes and their larvicidal activity against Aedes aegypti, vector of Dengue fever

Inara de Aguiara*, Edjane Rocha dos Santosa, Ana Carolina Mafudb, Vinicius Anniesc, Mario Antonio Navarro-Silvad, Valeria Rodrigues dos Santos Maltae, Maria Teresa do

Departamento de Química, Universidade Federal de São Carlos, 13565-905, São Carlos,

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a

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Prado Gambardellaf, Francisco de Assis Marquesc, Rose Maria Carlosa

SP, Brasil.

Instituto de Física de São Carlos, Universidade de São Paulo, 13560-905, São Carlos, SP,

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b

Brasil

Departamento de Química, Universidade Federal do Paraná, 81531-980, Curitiba, PR,

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c

Brasil.

Departamento de Zoologia, Universidade Federal do Paraná, 81531-980, Curitiba, PR,

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d

Brasil.

Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, 57072-900,

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e

Maceió, AL, Brasil.

Instituto de Química de São Carlos, Universidade de São Paulo, 13566-590, São Carlos,

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f

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SP, Brasil.

Abstract 1

ACCEPTED MANUSCRIPT This article describes the structure (X-ray), spectroscopic properties (FTIR, UV-vis, 1

H NMR) and larvicidal activity of Mn(I) complexes, fac-[Mn(CO)3(phen)(L)]+, where

phen = 1,10'-phenanthroline and L = 2-methyl-imidazole, 4-methyl-imidazole, and 2phenyl-imidazole. The mode of action on the cholinesterase enzyme activity and the Cytochrome-C (P450) inhibition are also reported. The complexes were highly effective against A. aegypti larvae causing up to 90% mortality at 0.033 to 0.046 g/L concentration in

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4 days, showing their great potential to control mosquito proliferation. The complexes do

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not interact with acetylcholinesterase enzyme from the A. aegypti mosquito, but react

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strongly with the P450 enzyme under light irradiation, reducing the Fe(III) to Fe(II). This is very important since P450 has implications on the insecticidal resistance of the mosquitoes.

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In addition, manganese is one of the most abundant metals on earth and an essential element for life; therefore, no harmful effect on humans or environment is expected with

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the use of these compounds.

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Keywords: Dengue fever, Aedes aegypti, Larvicide, Manganese (I), Tricarbonyl complex

1. Introduction 2

ACCEPTED MANUSCRIPT Dengue fever is an infectious viral disease caused by the dengue virus (DENV) and transmitted to humans by the bite of the female Aedes aegypti mosquito. [1] A. aegypti is an anthropophilic mosquito which holds an intimate relationship to humans and exhibits behavioral traits such as oviposition in man-made and man-used natural and artificial containers. [2] The A. aegypti mosquito is active during the day and only the female mosquitoes feed on human blood to mature their eggs. [3] The eggs become adults after

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two weeks when the mosquito completes its metamorphosis: eggs, larvae, pupae, and

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adults. In general, the larva-to-pupa stage lasts five days and the adult life ranges from two

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weeks to one month, depending on the season of the year and environmental conditions. [4] Most insecticides are developed to act on the larval stage because it is the easiest to be

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controlled. [5]

Owing to climate conditions, dengue fever usually occurs in tropical and subtropical

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countries. [1, 5, 6] It is estimated that 50 – 100 million of dengue virus infections occur yearly. [5] Five years ago, the total cost of the dengue combat program reached 1.5 billion

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dollars in Brazil, approximately 42% of the total costs for all American countries. [7] Most of the control methods of A. aegypti mosquitoes rely on the use of chemical insecticides,

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mainly organophosphates, organochlorines, carbamates, and pyrethroids. [8, 9] Several mechanisms of action have been proposed to effectively combat dengue mosquitoes, i.e.,

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inhibition of the insect enzyme acetylcholinesterase (AChE) and GABA-gated chloride channel, sodium, and potassium ion exchange disruption, and cellular respiration. [10-13]

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Organophosphates and carbamates act as inhibitors of the insect enzyme acetylcholinesterase. [14, 15] In this mechanism, the insecticides affect the transmission of

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nerve impulses, resulting in accumulation of acetylcholine in the neuromuscular tissue, causing paralysis and death of the mosquito. [16] Most mechanisms of action using organochlorines involve GABA-gated chloride channel inhibition or sodium and potassium ion exchange disruption. The DDT (dichlorodiphenyltrichloroethane) was an organochlorine largely used in the past as insecticide. [17, 18] Although its mechanism is not entirely recognized, it is known that DDT is able to keep the sodium channel opened, which causes an imbalance of the sodium/potassium ions; consequently, damage in the transmission of nerve impulses in the insects leads to death. [19] Blockage of GABA-gated chloride channel by another class of 3

ACCEPTED MANUSCRIPT organochlorines, the cyclodienes, reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. [20] The resistance to insecticides acting on these channels is still the greatest challenge against mosquito eradication. In the case of pyrethroid insecticides, the most commonly described resistance mechanism is related to the overproduction of detoxification enzymes such as P450. [2123]

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The increase in the dengue mosquito resistance related to the uncontrolled use of

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insecticides, and the resulting environmental imbalances caused by the chemical pollution

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of surface and ground water motivates the search of new active molecules. Metal complexes comprise an emerging field in the search of larvicides against the dengue

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mosquito. [24-26] Pt(II) complexes containing acesulfame ligand have demonstrated good inhibition in the replication of virus type 2 (New Guinea C strain). [26] Penta-coordinated

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organotin (IV) and tetra-coordinated tin (II) complexes also exhibited activity against the larvae of A. aegypti, the complex dibutyltin(IV) have LC50 = 0.004 ppm compared to LC50=

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0.011 for the commercial Temephos. [25]

Despite the good profile of these metal complexes for the control/combat of A.

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aegypti mosquitoes, their use has been limited because of the high obtainment cost, the concern with environmental sustainability, and their harmful effect on human health. [5]

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The use of manganese compounds is an interesting strategy due to their reduced obtainment cost and low toxicity to human and environment. Manganese is one of the most abundant

[28, 29]

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metals on earth [27] and it is present in many natural processes, such as photosynthesis.

This article describes the synthesis of Mn(I) complexes with formula fac-

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[Mn(CO)3(phen)(L)]+, where phen = 1,10'-phenanthroline; L = 2-methyl-imidazole, 4methyl-imidazole, and 2-phenyl-imidazole, characterization (X-ray, H1NMR, CHN, UV-vis and FTIR), computational evaluations using DFT and TD-DFT. The protein P450 and AChE enzyme inhibition, and the larvicidal activity against A. aegypti are also reported. The presence of imidazole type ligands coordinated to Mn center may be important to improve the larvicidal activity considering that histidine is present in many biological systems, such as enzymatic reactions, acting as a proton-transfer mediator. [30] Moreover, 4

ACCEPTED MANUSCRIPT the presence of the manganese redox pair, accessible under physiological conditions, can be exploited to enhance the effectiveness of insecticidal action.

2. Experimental 2.1. Materials All reagents were purchased from Sigma-Aldrich, Strem Chemicals, or Merck and

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were used as purchased. All spectroscopic measurements were conducted in HPLC grade

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solvents.

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2.2. Instrumental methods

Crystal data collections were processed in a Nonius KappaCCD diffractometer.[31]

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Structures were solved by direct methods using the WINGX and SIR-92 [32] systems. Non-hydrogen atoms were refined anisotropically using the SHELXL97 program [33] on F2

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by the full-matrix least-squares method. Structural analysis was performed using the PLATON system. [34] Parameters in the CIF format were collected as Electronic

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Supplementary Information from Cambridge Crystallographic Data Center. Cell refinement was conducted using the program SCALEPACK. [35] The programs DENZO and

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SCALEPACK[35] were used for data reduction. SIR 92 [32] was used to solve the structure. ORTEP-3 for Windows [36] and Mercury [37] were used to prepare the material

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for publication. Optical spectra were recorded on an Agilent spectrophotometer, model 8453 A, using 1 cm path-length quartz cells. The FTIR spectra were measured in CaF2

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windows in CH2Cl2 solution on a Bomem-Michelson 102 spectrometer in the 4000-1000 cm-1 region. CHN elemental analyses were performed on an EA 1110 CHNS-O, Carlo Erba

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Instruments, in the Microanalytical Laboratory at the Federal University of Sao Carlos UFSCar.

Flash photolysis. Time-resolved optical spectra were obtained using a laser flash photolysis apparatus containing a Continuum Q-switched Nd:YAG laser (Continuum, Santa Clara, CA) with excitation provided by the third harmonic at 355 nm. The pulse length was 8 ns; the beam diameter incident on the sample was 6 mm; the repetition rate was 10 Hz. The laser pulse was set up at 8 mJ per pulse in the photobleaching studies measured with a Field Master power meter with the L-30V head. Growth-decay kinetics 5

ACCEPTED MANUSCRIPT were measured at a single wavelength using a monochromator (Bentham M300) and a photomultiplier (Hamamatsu, model R928P). Transient decays were averaged using a Tektronix TDS 340A digital oscilloscope. Digitized kinetic data were transferred to a personal computer (PC) for analysis with software supplied by Edinburgh Instruments. Continuous Photolysis. Monochromatic irradiations at 355 nm were generated using an

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RMR-600 model Rayonet photochemical reactor using RMR-3500 lamps. Experiments

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were conducted at room temperature in 1.0 cm path-length, 4-side quartz cells capped with a rubber septum. Magnetically stirred solutions (10-4 M initial complex concentration) were

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under N2. Potassium (tris-oxalate)ferrate(III), used in actinometry at 355 nm, was prepared according to Calvert and Pitts.[38] Photoreaction progress was monitored by UV-vis and

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EPR.

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2.3. Synthesis

All syntheses were performed under purified N2 atmosphere using Schlenk techniques. The fac-[Mn(CO)3(phen)(L)]+ complexes, where L = 4-methyl-imidazole (4-

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meim), 2-methyl-imidazole (2-meim), and 2-phenyl-imidazole (2-phim), were prepared

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according to the literature. [39] The complex fac-im has been previously synthesized and characterized by our research group [40, 41] and in the present study, fac-im will be used

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only for comparison.

fac-[Mn(CO)3(phen)(4-meim)]+ (fac-4meim). Anal. Calc. for C17H14F3N4S3O6Mn: C,

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43.65; H, 2.56; N, 10.18. Found: C, 44.06; H, 2.58; N, 10.21%. 1H NMR (ppm):  11.58 (bs, 1H), 9.77 (d, 2H), 8.88 (d, 2H), 8.32 (s,2H), 8.25 (dd, 2H), 7.01 (s, 1H), 6.42 (s, 1H),

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2.20 (s, 3H), (A isomer)  11.44 (bs, 1H), 9.74 (d, 2H), 8.90 (d, 2H), 8.34 (s,2H), 8.24 (dd, 2H), 6.89 (s, 1H), 6.46 (s, 1H), 2.56 (s, 3H) (R isomer). fac-[Mn(CO)3(phen)(2-meim)]+ (fac-2meim).

Anal. Calc. for C17H14F3N4S3O6Mn: C,

43.73; H, 2.39; N, 10.20. Found: C, 43.30; H, 2.31; N, 10.11%. 1H NMR (ppm):  11.48 (bs, 1H), 9.56 (d, 2H), 8.64 (d, 2H), 8.11 (s,2H), 8.00 (dd, 2H), 6.57 (s, 1H), 5.63 (s, 1H), 2.39 (s, 3H).

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ACCEPTED MANUSCRIPT fac-[Mn(CO)3(phen)(2-phim)]+ (fac-2phim).

Anal. Calc. for C22H16F3N4S3O6Mn: C,

49.01; H, 2.61; N, 9.15. Found: C, 50.02; H, 2.64; N, 8.95%. 1H NMR (ppm):  11.48 (bs, 1H), 9.01 (d, 2H), 8.50 (d, 2H), 8.01 (s, 2H), 7.67 (dd, 2H), 7.52 (t, 1H), 7.35 (t, 1H), 6.96 (d, 2H), 6.85 (s, 1H), 6.63 (s, 1H), 5.33 (s, 1H). 2.4. Computational studies

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DFT calculations were performed with the Gaussian 03 (G03) and Gaussian 09

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(G09) program packages [42] employing the DFT method with Becke’s three-parameter

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hybrid functional [43] and Lee-Yang-Parr’s gradient corrected correlation functional (B3LYP). [44] and LanL2DZ basis set [45]. The ground-state geometries of complexes

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were optimized in gas phase. The dichloromethane solvent was included in the calculations using the PCM system. SCF convergence criteria were used for all optimizations.

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2.5. Larvicidal activity measurement

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Electronic analysis was performed using TD-DFT calculations with 40 excited states.

Late third and early fourth-instar A. aegypti larvae were used in the bioassays.

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Larvae were from the Rockefeller-CDC (Center for Disease Control) colony reared in the insectariums of the Zoology Department of the Federal University of Parana - UFPR. 20

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larvae were used for each treatment in 20 mL of water in 330 mL disposable plastic cups. Once larvae were placed in each cup, aqueous/dimethyl sulfoxide was added to

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concentrated solutions of the manganese complexes in order to prepare five solutions with different concentrations (30, 40, 70, 100 and 150 µM) for a final volume of 100 mL per

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treatment. One control group of larvae was exposed to an aqueous solution of dimethyl sulfoxide (1 mL in 99 mL of distilled water), whereas another control group was exposed to distilled water only. Catfood (0.03 g) was added to feed the larvae. Water (to maintain 100 mL) were added as needed throughout the experiment. The cups with the larvae were kept at 25 oC with a 12:12 h photoperiod in a Model 347 CDG chamber. All assays were conducted in four replicates under identical conditions. Every 24 h, live larvae were counted in all treatments until all larvae died, or after four days. Larvae that neither swam or responded to touch using a pipette were considered dead and removed at every count. The experiment was terminated after 96 h. 7

ACCEPTED MANUSCRIPT 2.6. Enzyme Assay 2.6.1. Larval preparation for the assay Live larvae that were exposed to acute and chronic toxicity for 72 and 96 h were placed in Eppendorf tubes, frozen at - 20oC, and then stored at - 80ºC. When used, the tubes were removed from the freezer and the larvae were slowly thawed prior to use. The larvae

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were placed individually in Eppendorf tubes to which 300 µL of potassium phosphate

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buffer solution (0.1 M pH 7.4) was added. Next, the larvae were homogenized at 3000 rpm. After that, the content of the tubes was centrifuged at 12000 x g for 1 min at 4°C and then

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placed in new tubes.

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2.6.2. Acetylcholinesterase (AChE)

An aliquot of 25 μL was transferred using a pipette (four times per treatment) to 96

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wells (transparent, flat bottom) followed by 200 μL of Ellman’s reagent, DTNB (5,5’dithiobis-(2-nitrobenzoic acid), and 50 μL of ATC (acetylcholine iodide). Afterward, this

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material was incubated for 5 min and then read using a microplate BioTek spectrophotometer at 405 nm. [46, 47]

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2.7. Cytochrome C inhibition

The stability of P450 in the presence of fac-4meim was evaluated by UV-vis

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measurement. The experiment was conducted at room temperature in a 4-side quartz cells, 1.0 cm path-length, containing a vessel for the P450. 3mL of the complex solution (10-4 M,

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buffer phosphate pH 7.4) was transferred, under N2, to the cell and 0.4 mg of P450 was added to the vessel. The complex solution was irradiated at 355 nm by 20 sec.

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Subsequently, the P450 was added to the irradiated solution and the reaction was followed by UV-vis spectrophotometer.

3. Results and Discussion 3.1. Synthesis, Characterization, and Structural Analysis of Mn(I) complexes. Single crystals of the Mn(I) complexes were grown using crystallization in CD3CN at room temperature. An ORTEP [48] view of the monomeric units of fac8

ACCEPTED MANUSCRIPT [Mn(CO)3(phen)(L)]+ is shown in Figure 1. Crystal data and structural parameters are presented in Table S1-S11. For all complexes, the manganese ion is in a distorted octahedral geometry in which two nitrogen atoms from the phen ligand and two carbon atoms from carbonyl groups define the equatorial plane. The carbon from the axial carbonyl and the nitrogen from the imidazole ligand complete the coordination sphere in apical positions, with N3(im)-Mn-

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COaxial angles between 174-178° (Figure 1A). The angles around the manganese center

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deviate from the ideal octahedral geometry as a result of the constraints imposed by the

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chelate ligand phenanthroline. The average Mn–N(L) and Mn–C(CO) bond-length values fall within the same range as those found for related complexes [40, 41, 49], supporting the

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fact that the substituent on the imidazole ring does not affect the coordinating properties on the Mn(I) center.

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The octahedral volume of fac-4meim and fac-2meim are 9.58 ų with quadratic elongation of 1.01 and angle variance in the octahedral geometry of 17.41°² and 23.24°²,

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respectively (Figures S2). Crystal packing of fac-4meim and fac-2meim (Figure S3) shows two structurally independent compounds linked via intra- and intermolecular

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hydrogen bonds between the compound and counter ion, resulting in an inversion centrosymmetric dimer [50], with graph-set motif R44(12) and symmetry [1/2-x,1/2-y,1/2-

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z]. The arrangement of the complexes fac-4meim and fac-2meim leads to a π-π stacking interaction between two independent phenanthroline ligands; this interaction is responsible

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for the stabilization of both structures in the solid state. The packing observed for fac2phim is different from the observed for fac-2meim and fac-4meim. In the crystal, two structurally independent formula units are linked via hydrogen bonds C—H…O, forming

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an inversion dimer [50], with graph-set motif R44(12) and symmetry [-x, 1-y, 2-z] (Figure S3). The interaction contacts, atomic coordinates, displacements, selected bond distances, and angles for the three compounds are summarized in Tables S2-S11. The position of the methyl group in the 4-methylimidazole ligand provides a better σ-donor character to the metal center than 2-methylimidazole and 2-phenylimidazole. As a result, complex fac4meim presents the shortest Mn-N(imidazole) bond length, 2.062Å. Compared with other Mn(I) complexes previously reported, fac-4meim also showed the shortest MnN(imidazole) length. [49, 51] 9

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Figure 1. Representative structure and ORTEP view of the molecular structure of (A) fac-

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4meim, (B) fac-2meim and (C) fac-2phim. The displacement ellipsoids are drawn at the

2phim.

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30% probability level for fac-4meim and 50% probability level for fac-2meim and fac-

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3.1.2. Theoretical DFT analysis of complexes A general view of the optimized structures is shown in Figure S4. The geometries were optimized using B3LYP/LanL2DZ in gas phase. Harmonic vibrational frequencies were computed at the same level of theory to characterize the stationary points as true minima, representing equilibrium structures on the potential energy surfaces and ensuring that imaginary frequencies were not generated in the minimum structures. The optimized structures reproduce the X-ray crystal structural data of Mn(I) complexes quite closely. Selected bond lengths for the optimized geometry in gas phase are shown in Table 1 and 10

ACCEPTED MANUSCRIPT the XYZ coordinates of the singlet ground state are given in the Supporting Information, Tables S12-S15. Table 1. Theoretical and experimental bond length (Å) and angles (◦) obtained for the

fac-2meim

DFT

X-ray

DFT

X-ray

DFT

X-ray

Mn-C (eq1)

1.815

1.805(2)

1.814

1.811(3)

1.823

1.802(4)

Mn-C(eq2)

1.815

1.820(2)

1.811

1.792(3)

1.811

1.803(5)

Mn-C(axial)

1.826

1.813(2)

1.813

1.807(3)

1.822

1.814(5)

Mn-N1

2.075

2.055(17)

2.090

2.053(2)

2.076

2.050(3)

Mn-N2

2.075

2.055(18)

2.089

2.062(2)

2.081

2.062(3)

Mn-N3

2.088

2.062(18)

2.150

2.093(2)

2.114

2.090(3)

N1-Mn-N2

80.09

80.04(6)

79.04

79.37(9)

80.02

79.82(11)

N1-Mn-N3

88.81

87.21(7)

86.64

84.27(9)

87.64

90.72(12)

Cax-Mn-Ceq2

89.64

89.83(11)

91.47

89.39(14)

91.55

89.16(2)

Ceq2-Mn-Ceq1

91.34

89.01(10)

90.64

87.50(14)

92.46

87.90(2)

Cax-Mn-Ceq1

89.65

90.21(11)

90.90

88.44(14)

88.69

89.02(19)

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fac-2phim

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fac-4meim

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complexes fac-4meim, fac-2meim, and fac-2phim

3.1.3. Spectroscopic properties

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Electronic absorption spectra of fac-[Mn(CO)(phen)(L)]+ complexes in CH2Cl2 solution are shown in Figure S5A and Table S16 summarizes the absorption maximum

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and absorptivities for the three complexes. In addition to ligand localized absorption bands (<300 nm), the electronic spectra of these complexes are dominated by relatively intense MLCT bands (dMn→*phen, dMn→*CO ) in the visible region (330–450 nm). [39, 40, 49] The HOMO is constituted by dMn orbitals and the LUMO by * orbital of the phen ligand. The theoretical and experimental spectra and assignments are shown in Figure S5 and Table S16-20. Considering the spectroscopic properties of the Mn compounds and the fact that they will be exposed to sunlight during their use as larvicidal agents, studies in the presence 11

ACCEPTED MANUSCRIPT of light were performed. The photochemical behavior of complexes fac-2meim, fac-4meim, and fac-2phim was very similar to the described for fac-im in CH2Cl2 . [40] After light irradiation (355 nm), the species {MnII(phen•–)}* is generated and it was detected by UVvis, time-resolved flash photolysis, and EPR, Figures 2 and 3. The UV-vis spectra (Figure 2A and S6) show the decrease of the MLCT (at 380 nm due to the increase of Mn(II), and the formation of a new broad absorption band at 540 nm. [52, 53] Flash photolysis

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experiments also showed the consumption of the MLCT band and formation of a new band

1.5

0.3

A

B

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1.0

0.0

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A

0.1

-0.1

0.5

-0.2

400

500 600 700 Wavelength, nm

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0.0 300

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Absorbance

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at 500-650 nm assigned to ILCT (πphen→π*phen), Figure 2B. [52, 53]

-0.3 350 400 450 500 550 600 650 700 Wavelength, nm

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Figure 2. UV-vis spectra of fac-4meim during irradiation (measured each 10s of irradiation) (A), transient absorption spectrum of fac-4meim (B) Conditions: λirr= 355 nm,

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CH2Cl2 (1×10-4 M, I0 = 1×10-9einstein s-1).

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The presence of Mn(II) was confirmed by EPR after in situ irradiation of fac-4meim in CH3CN. The typical signal of Mn(II) in a distorted octahedral geometry containing the six hyperfine lines (5/2, A=540G and g=2.003) was detected, Figure 3. [54, 55]

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2700

3000

3300

3600

3900

4200

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Field (G)

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Figure 3. EPR spectra of the fac-4meim complex after irradiation at 355 nm in CH3CN (10M, I0 = 1 x 10-9einstein s-1).

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Considering the possible interaction sites, the charge distribution on the Mn

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complexes was evaluated by DFT through NBO analysis. The most positive charge was located on the carbon of the carbonyl group (axial position, +0.8) for the three compounds, Figure S7. The manganese center retains the higher electron density (-1.1), which explains

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the photochemical behavior previously described.

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3.2. Biological evaluations

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3.2.1. Larvicidal activity against Aedes aegypti Figure 4 shows the mortality curves for the A. aegypti larvae that had been feeding with the complexes. The lethal concentration (LC99) values suggested that the A. aegypti larvae were susceptible to all complexes at 0.0882 g/L in 4 days. Noteworthy was the fact that the complexes had a major influence on A. aegypti metamorphosis by a mechanism that seemed to interrupt the development from larvae into pupae. The organophosphate temephos and the entomopathogenic bacterium Bacillus thuringiensis israelensis demonstrated a better insecticide profile for A. aegypti control. [56-58] According to the 13

ACCEPTED MANUSCRIPT WHO, the concentration of 0.012 mg/L of temephos is capable of causing 100% mortality of A. aegypti larvae.[59] However, both insecticides have shown mosquito resistance. [5962] Depending on the region, 0.012 mg/L of temephos was unable to cause more than 5% of mosquito mortality. [60] The Mn(I) complexes also caused 90% mortality in the range of concentration from 0.033 to 0.046 g/L in 4 days, thereby showing their great potential to control mosquito

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proliferation. The same effect was observed for the fac-im complex at a much higher

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concentration (0.146 g/L) (Table 3). All the substituents herein studied improved the

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larvicidal activity against A. aegypti.

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100

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80 70 60

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% Mortality

90

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50

0.04 0.06 0.08 Concentration (g/L)

0.10

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0.02

Figure 4. Mortality (%) of A. aegypti larvae (n = 60) varying the concentration of Mn(I)

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complexes for 96 h (4 days), at 25 ºC ± 1, relative humidity of 70% ± 10%, and 12:12 h

line).

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photoperiod. fac-im (black line), fac-4meim (red line), fac-2phim (green line) and fac-2meim (blue

Table 3. Lethal concentrations (LC) after 96 h exposures of A. aegypti larvae to manganese complexes (g/L) Complex

LC50

LC90

LC95

LC99

fac-im

0.018

0.146

0.264

0.799

fac-4meim

0.011

0.046

0.069

0.148

fac-2phim

0.007

0.042

0.070

0.186

fac-2meim

0.009

0.033

0.047

0.092 14

ACCEPTED MANUSCRIPT 3.2.2. Mode of Action of the complexes Acetylcholinesterase enzyme. Acetylcholinesterase is the primary enzyme on the autonomic nervous system and it has been the target of many insecticides. For this reason, the interaction between the fac-4meim and the acetylcholinesterase enzyme of the A. aegypti mosquito was investigated. Although all Mn(I) complexes containing substituted imidazole ring exhibit similar larvicidal activity, the complex fac-4meim presented the

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higher larvae mortality at concentration 0.08g/L, being chosen to the next studies. No

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substantial variation was observed in the AChE activity compared with the control when

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mosquitoes were chronically exposed to fac-4meim for 72 h, at concentrations of 0.016 g/L and 0.083 g/L, Figure 5. At 96 h and 30 µM, the enzyme activity increased somewhat

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compared with the control. Therefore, the larvicidal activity of the fac-4meim complex does not involve AChE inhibition, indicating that the mechanism of action for fac-4meim is

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different from those observed for other products usually employed in the control of this

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vector, including organophosphates and carbamates. [63]

30 M

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Control

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AChE activity (mmol mg -1 ptn min -1)

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0

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AChE activity (mmol mg -1 ptn min -1)

a 100

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150

Figure 5. Acetylcholinesterase activity (AChE, mean + standard error) after 72 h (A) and 96 h (B) of the A. aegypti larvae exposed to the fac-4meim complex. N = 20 for each treatment. Analyses were performed using ANOVA and Tukey contrasts.

P450 inhibition. Considering that Cytochrome P450 monooxygenase of A. aegypti presents important functional roles and implications on the resistance to insecticides [21, 64], the effect of the complexes on P450 stability in aqueous solution were evaluated. P450 acts in the detoxification of enzymes, increasing the insecticidal resistance of dengue 15

ACCEPTED MANUSCRIPT mosquitoes. [22] The catalytic cycle of P450 may be interrupted by molecules that bind to the reduced form of the iron metal center. [65]. In the presence of light, the changes in its UV-vis, EPR, and transient absorption spectrum at 355 nm light irradiation are consistent with the formation of the species Mn(II) species, as previously discussed, Figures 2 and 3. The electronic spectrum of P450 in phosphate buffer (pH 7.4) exhibits absorption maxima at 408 nm and 500 nm. [65] When

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P450 was mixed to the irradiated solution of fac-4meim, Figure 6, the bands at 418 nm in

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the Soret band region, and at 536 and 567 nm, in the Q-band region were formed. The

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spectra showed in Figure 6 is typical of the formation of Fe(II) species.

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Figure 6. UV-vis spectra of fac-4meim (10-5 M) in the presence of P450 after 10 sec of

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irradiation at 355 in a phosphate buffer solution (pH 7.4) (I0 = 1 x 10-9 einstein s-1).

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Although the involvement of P450s in insecticide resistance has been known since the 1980's, the exact target sites effective against resistant insects are unknown to date. [21, 22, 64] Through this experiment was possible to certify that fac-4meim is able to reduce P450, which would avoid high levels of this protein during the day and consequently, the insecticidal resistance of dengue mosquitoes.

4. Conclusion In summary, three Mn complexes were synthesized, characterized by X-ray, NMR, UV-vis, FTIR, and DFT. The photochemical studies revealed the formation of the Mn(II) 16

ACCEPTED MANUSCRIPT species, which is probably the larvicidal active species against A. aegypti. These results, together with the selectivity to P450, show the promising potential of these Mn complexes for the control of the dengue fever vector.

Supplementary Material Crystallographic data for structural analysis have been deposited with the

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Cambridge Crystallographic Data Center, CCDC 957966-4meim 986965-RS03, and

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1005953-RS05. Copies of this information may be obtained free of charge on application to

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CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-336033; e-mail:

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[email protected] or http://www.ccdc.cam.ac.uk).

Acknowledgments

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The authors are grateful to FAPESP (Proc. 09/08218-0; 14/02282-6), CAPES, and CNPq for the financial support. Prof. A. B. P. Lever, Department of Chemistry, York

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University, Canada, is acknowledged for granting computational time at the Shared Hierarchical Academic Research Computing Network (SHARCNET: www.sharcnet.ca),

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Ontario, Canada.

3. 4. 5. 6. 7. 8.

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Graphical Abstract (Figure)

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Manganese (I) complexes and their X-ray crystal structure

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Potent larvicidal activity against Aedes aegypti with up to 90 % of mortality

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Inhibition of P450 by the Mn(I) compounds aiming to avoid insecticidal resistance

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