Influence of Incorporation of Different dn-Electron Metal Cations into Biologically Active System on Its Biological and Physicochemical Properties

Three new compounds, namely [HL]2+[CuCl4]2−, [HL]2+[ZnCl4]2−, and [HL]2+[CdCl4]2− (where L: imipramine) were synthesized and their physicochemical and biological properties were thoroughly investigated. All three compounds form isostructural, crystalline systems, which have been studied using Single-Crystal X-ray diffraction analysis (SC-XRD) and Fourier-transform infrared spectroscopy (FTIR). The thermal stability was investigated using thermogravimetric analysis (TGA) and melting points for all compounds have been determined. Magnetic measurements were performed in order to study the magnetic properties of the compounds. The above mentioned techniques allowed us to comprehensively examine the physicochemical properties of the newly obtained compounds. The biological activity was investigated using the number of Zebrafish tests, as it is one of the most common models for studying the impact of newly synthesized compounds on the central nervous system (CNS), since this model is very similar to the human CNS.


Introduction
Imipramine, one of the first successful antidepressant drugs for treating major depressive disorder, was discovered in 1958 [1]. Imipramine is a prototype of the tricyclic class of antidepressants [2]. Following oral administration, it is rapidly and completely absorbed (>95%), with peak plasma concentrations occurring within 2-6 h and readily crosses the blood-brain barrier. Generally, accumulation of imipramine in the brain is about 30-40 times the concentration in plasma. The absorption of imipramine takes place in the small intestine, with little or no absorption occurring in the stomach. Absorption, peak drug concentration, and time to peak do not depend on food consumption. Imipramine is subjected to extensive first-pass metabolism in the liver to yield the primary active metabolite (desipramine). Thus, the therapeutic effect of imipramine depends also on the amount of desipramine formed [1]. For a long time, imipramine was the drug of choice for treating depression, especially in the more severe forms of the disease. Unfortunately, the drug has several adverse effects, including dry mouth, constipation, urinary retention, blurred vision, palpitations, and tachycardia [2]. Despite continuous works on creating new antidepressants, side effects remain a considerable problem. Therefore, several supplementations have been employed with different antidepressants to reduce drug dose and decrease adverse effects while maintaining therapeutic effects. Such preclinical and clinical studies have also been conducted with imipramine. Recent data revealed that zinc enhances the efficacy/potency of imipramine in preclinical paradigms sensitive to antidepressants. Sub-effective doses of zinc combined with sub-effective doses of imipramine resulted in antidepressant-like effects in the forced swim test, tail suspension test, and chronic unpredictable stress model of depression [3][4][5]. Moreover, clinical studies indicate that zinc supplementation augments the efficacy and speed of onset of the therapeutic response to imipramine, especially in patients non-responsive to antidepressant pharmacotherapies [6].
The compounds that we applied in the present project are meant to influence the central nervous system (CNS) by decreasing depression and anxiety symptoms. They are derivatives of imipramine, the well-known tricyclic antidepressant that reduces the serotonin and noradrenaline reuptake, and metallic cations (Cu 2+ , Zn 2+ , and Cd 2+ ) that were supposed to enhance the imipramine activity. Since the compounds were newly synthesized, the screening of the activity towards the CNS and the toxicity test were performed using the Zebrafish model. In the present study, we have used diazepam as a reference standard for the evaluation of an anxiolytic activity. There are also studies on Zebrafish larvae proving that diazepam increases the level of serotonin and tryptophan in a dose-dependent manner after 2.5 h of bathing in the tested solution [7].
Zebrafish (Danio rerio) model has gained popularity in neuropharmacological research due to its high physiological and genetic homology to humans, as well as similar CNS morphology. Zebrafish genes show 70% correspondence to humans' nucleotide sequence and a similar number of chromosome pairs equal to 25, whereas humans have 23. That gives the advantage for Zebrafish compared to rats and mice which have 21 and 20 pairs of chromosomes, respectively. Moreover, Zebrafish possess genes that are counterparts of those in humans responsible for human diseases [8,9]. For the presented project, the Zebrafish model was used since their rapid development allows evaluating the toxicity profile and performing a fast screening of the tested compounds. The transparency of embryos in the early days and their rapid growth enable following developmental disruptions and assessing whether a newly synthesized drug is safe for the living creatures or on which stage of development it is toxic [10]. Moreover, there is high cost-efficiency as well as established anxiety-like and locomotor activity behavioral tests [11].
The amygdala and habenula, which regulate the release of serotonin and dopamine, are responsible for affective behaviors in both Zebrafish and humans. The hyperactivity of habenula was detected in patients with depression as well as in rodents and Zebrafish with anxiety/stress-like behaviors. Interestingly, reaction to stress in Zebrafish and mammals is similar and engages the cortisol mediation on the axis of the hypothalamus-pituitary gland acting on glucocorticosteroid receptors [12][13][14]. Studies show that mood disorders in the Zebrafish model were associated with reduced activity and chronic elevation of cortisol and reversed by reserpine and d-amphetamine [13,15]. We applied the locomotor protocol with the phases of light and dark in order to create stressful conditions for Zebrafish five-day larvae [16]. The measurement of locomotor activity in light/dark transition may be a useful tool for preclinical drug screening of potential anxiolytic/antidepressive drugs. The study by Giacomini showed that fluoxetine, the selective serotonin reuptake inhibitor, and tryptophan, the amino acid precursor of serotonin, decreased anxiogenic effects in adult Zebrafish by decreasing distance traveled, entries to the top of the tank, or by increasing time spent in the top of the tank and the cortisol levels compared with the control group [17]. The association of serotonin system dysfunctions and depression with a high level of cortisol and stress is well-established. Moreover, in Zebrafish, all major neuromodulatory systems similar to those in humans and rodents were found. They contain neurotransmitter receptors, transporters, and enzymes of synthesis and metabolism such as tyrosine hydroxylase essential for catecholamines synthesis [18]. It has been proved that Zebrafish have serotonin receptors such as 5-hydroxytryptamine receptor 1A (htr1aa), the expression of which can be modulated by different antidepressant agents or even therapy with supplementation of guts microbiota [19].
Taken together, it was reasonable to perform the presented study on the Zebrafish model to obtain strong grounds for the following investigations of the synthesized compounds.

Synthesis
[HL] 2 + [CuCl 4 ] 2− compound: 136 mg of copper(II) chloride dihydrate (0.8 mmol) was dissolved in 5 mL of ethanol; 507 mg of imipramine hydrochloride (1.6 mmol) was dissolved in 20 mL of ethanol and slowly added to the solution of copper(II) chloride. The reaction was carried out at room temperature with constant mixing with a magnetic stirrer. After a few moments, a gold-colored, crystalline precipitate was formed. After 2 h, the precipitate was filtered and washed several times with small amounts of ethanol. Then, the product was dried in the open air and analyzed.
[HL] 2 + [ZnCl 4 ] 2− compound: 109 mg of anhydrous zinc(II) chloride (0.8 mmol) was dissolved in 5 mL of ethanol; 507 mg of imipramine hydrochloride (1.6 mmol) was dissolved in 20 mL of ethanol and slowly added to the solution of salt. The reaction conditions were the same as for the previous compound. After a few moments, a white, crystalline precipitate was formed. As previously, after 2 h, the precipitate was filtered and washed several times with small amounts of ethanol. The resulting compound was analyzed after drying in the open air.
[HL] 2 + [CdCl 4 ] 2− compound: 55 mg of anhydrous cadmium(II) chloride (0.3 mmol) was dissolved in 40 mL of ethanol; 190 mg of imipramine hydrochloride (0.6 mmol) was dissolved in 10 mL of ethanol and slowly added to the solution of cadmium(II) chloride. After 2 h of mixing at room temperature, a clear solution was left for slow solvent evaporation. As a result, colorless crystals of the product were obtained and later analyzed.

X-ray Diffraction Analysis
Tested compounds crystallized in the orthorhombic crystal system in the Pbca space group. Crystal data and structure refinement details are summarized in Table 1 Figure 1).
Unit cell parameters indicate that the structures are isostructural (the parameter indicating the similarity of cells is Π = 0.0186 [20]). The copper-containing salt exhibits a disorder at one imipramine molecule and at the copper position. However, in the case of a zinc compound, which was measured at 294 K, there is an identical disorder on the imipramine molecule and additionally on the entire anion (ZnCl 4 2− ). Both structures have analogous N-H... Cl hydrogen bonds stabilizing the cation-MCl 4 -cation fragment (Tables S1 and S2 and Figure 2).
The symbol for the hydrogen graph-set, according to Berstein's theory [21], is represented as DD. The packing of molecules in space is characterized by layers of the anioncation-cation-anion type (Figure 2b). In addition, the compound with cadmium was measured at 99.9 K. The unit cell parameters are 34.8583 Å, 19.2226 Å, 11.4967 Å, 90 • , 90 • , 90 • , and the same space group (Pbca), which may indicate isostructurality.
There is an analogous compound in the CSD crystallographic base [22] that differs by an additional methyl group (Figure 3a). The compound AFOLOW [23] adopts an analogous hydrogen bond and packing (Figure 3b).

FTIR Spectra Analysis
FTIR spectra of all three compounds ( Figure 4) are in good agreement with crystallographic data derived from SC-XRD analysis. FTIR spectrum of imipramine hydrochloride (red line) exhibits several important bands in the 3050-2450 cm −1 region. These bands can be ascribed to typical ν(NH) amine salt , as well as ν(CH) aliphatic modes: 3049, 3007, 2937, 2927, 2905, 2845, 2769, 2620, 2569, 2507, 2460 cm −1 . In all three compounds' spectra, described bands are either not present, or significantly moved, as a consequence of imipramine-metal cation interactions described in the crystallographic section, mainly hydrogen bonds. These bands, however, are altered in the same manner, which confirms that in all three cases imipramine molecule binds metal cation in an analogous way. In the 1650-1200 cm −1 region, several bands correspond to ν(CN), ν(C=C), δ(NH) and δ(CH) vibrations, with the sharpest ones: 1594, 1569, 1484, 1478, 1447, 1328, 1329, 1294, 1226 cm −1 for imipramine. All of these bands are also present in the studied compounds' spectra. In the spectra of metal(II) compounds, these bands appear at: [ In the 1650-600 cm −1 range, the observed bands clearly overlap, which is an additional confirmation of the isostructurality of described compounds.

Thermogravimetric Analysis
Imipramine hydrochloride itself is stable at up to 175 • C (Figure 5a). At this temperature, it melts; the melting point was determined to be 173.7-174.9 • C, which corresponds very well with the endothermic DTA peak at 175 • C that can be ascribed to this phase change. In the temperature range of 175-340 • C, we observe one major mass loss (95%) (DTG peak at 285°C), which is a result of thermal destruction of the compound. Next, from 400 • C up to 660 • C, we observe small mass loss (5%), which can be probably ascribed to post-combustion processes of the organic molecule.  [HL] 2 + [CuCl 4 ] 2− compound begins to decompose at 130 • C (Figure 5b). Its melting point was determined to be 130.3-132.5 • C, which corresponds well with the endothermic DTA peak at 135 • C. Mass loss in the temperature range 130-420 • C is associated with almost total destruction of organic molecules (mass loss found: 68.0%, calc.: 73.27%) (DTG peak at 275 • C). Next, from 460 • C up to 680 • C, we can observe mass loss that corresponds with the destruction of [CuCl 4 ] 2− (mass loss found: 27.0%, calc.: 26.73%). It is most likely that volatile copper(II) halide derivatives are formed, the existence of which has already been well documented [24].
The determined melting point of [HL] 2 + [ZnCl 4 ] 2− was 165.2-166.6 • C (Figure 5c). Once again, it very well corresponds with the endothermic DTA peak at 170 • C. As well, in this case, the first step of thermal destruction involves almost total decomposition of organic molecules (mass loss found: 68.0%, calc. 73.09%) in the temperature range 170-390 • C. Next, up to 660 • C, we observe total destruction of the molecule (mass loss found: 30.0%, calc. 26.91%).
The [HL] 2 + [CdCl 4 ] 2− compound is stable at up to 220 • C (Figure 5d). The determined melting point (158.2-159.8 • C) corresponds very well with the observed endothermic peak (160 • C). As in all three cases, the first step of decomposition is almost total destruction of organic molecules (mass loss found: 67.0%, calc. 68.89%) that takes place in the temperature range of 220-460 • C. In the temperature range of 460-710 • C, we observe total destruction of the molecule (mass loss found: 33.0%, calc. 31.11%).

Magnetic Studies
All compounds presented a paramagnetic-like behavior, approximately following the Curie-Weiss law: where D accounts for a constant diamagnetic background, C is the Curie constant, and χ ≡ M/H is the molar magnetic susceptibility. The samples' temperature-varying component of the magnetic susceptibility, χ = χ − D, is presented in Figure 6a through the use of the reduced quantity ∆χT. Results revealed nearly temperature-independent curves above 150 K. For paramagnets, such plots are expected to converge to ∆χT = C. This allows the estimation C and its associated molecular magnetic momentum number J, through   Nonlinear fit analysis ( Figure 7B)   Two-way ANOVA analysis showed statistically significant difference in heart rate after 48 h post fertilization ( Figure 8B, left) as a response to used treatment (F (7, 85) = 86.91, p < 0.0001), drug-solvent interactions (F (7, 85) = 9.487, p < 0.0001) and non-significant difference in response to the treatment with applied solvents (F (1, 85) = 0.0004440, p = 0.9832 Two-way ANOVA analysis showed statistically significant difference in heart rate after 48 h post fertilization ( Figure 8C, left) as a response to used treatment (F (6, 51) = 42.20, p < 0.0001), used solvent (F (1, 51) = 28.14, p < 0.0001), and drug-solvent interactions (F (6, 51) = 10.07, p < 0.0001    Two-way ANOVA analysis revealed a significant difference in mortality rate (%) depending on the treatment with the drug dissolved initially in the water ( Figure 10A

Chemistry
All chemicals used for the synthesis were purchased from the companies: Sigma-Aldrich, AlfaAesar, POCH and used without further purification. FTIR spectra were recorded with an IRTracer-100 Shimadzu Spectrometer (4000-600 cm −1 ) with an accuracy of recording 1 cm −1 , using KBr pellets. The thermolysis of compounds in air atmosphere was studied by TG-DTG-DTA techniques in the range of temperature 25 to 800 • C at a heating rate of 10 • C min −1 ; TG, DTG, and DTA curves were recorded on a Netzsch TG 209 apparatus under air atmosphere, v = 20 mL min −1 , using ceramic crucibles. As a reference material, ceramic crucibles were used.

X-ray Diffraction Analysis
Crystal structures were determined by the Single Crystal X-ray analysis method. The crystals to be measured were selected from the product obtained by described synthesis. X-ray data were collected on the XtaLAB Synergy, Dualflex, Pilatus 300K diffractometer apparatus (Rigaku Corporation, Tokyo, Japan) equipped with the PhotonJet microfocus X-ray tube apparatus (Rigaku Corporation, Tokyo, Japan). Data reduction was performed with CrysAlisPro (Agilent Technologies UK Ltd., Yarnton, England) [25]. The structures were refined in ShelXL [26]. Molecular structures and packing diagrams were drawn using Mercury [27]. Molecular geometry parameters were computed with publCIF [28].
CCDC: Deposition Number 2090557-2090558 contains the supplementary crystallographic data for this paper. The data are provided free of charge by The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures. Deposited: 06/17/2021.

Magnetic Studies
Magnetization measurements were carried out using a superconducting quantum interference device (SQUID) magnetometer with Quantum Design 7T magnetic property measurement system (MPMS XL-7) in the magnetic field range 0.0 T < H < 7.0 T and temperature interval 2.0 K < T < 300.0 K. The magnetic susceptibility χ of the samples was calculated by normalizing the measured magnetic moment by the applied magnetic field.

Biological Assays
Zebrafish Husbandry and Egg Collection: Danio rerio (Zebrafish, wild type zebrafish strain (AB strain)) stocks from the local husbandry in the Experimental Medicine Centre in Lublin, (Medical University of Lublin, Poland) were housed at 28.  (3 mM) in E3 were prepared for locomotor activity assay. The dose of diazepam (10 µM) for behavioral tests was chosen based on literature data [11].
Fish Embryo Acute Toxicity Test (FET) and Lethal Concentration 50 (LC 50) determination: The FET was performed according to the requirements described by Busquet et al., (2013) in the OECD guidelines for the testing of chemicals 236-Fish Embryo Acute Toxicity (FET) Test with minor modifications. On the first day, newly fertilized zebrafish eggs were exposed to the tested chemicals' solutions for 3 h. After this time, the mortality was analyzed and only well-developed embryos were selected (20 per group/5 per well in 24-well plaque). For the next 96 h, the malformations appearance, heart rate, hatching, and mortality rate were observed. Every 24 h, all solutions were replaced by freshly prepared dilutions of the tested compounds. Two control groups (20 eggs/group) were immersed in the E3 solution and ethanol in the E3 solution (0.2%). The maximum tolerated concentration of ethanol was established in the preliminary studies (0.2%). The test was considered valid when less than 90% of embryos were found dead until the end of 96 h of exposure. The maximum tolerated doses and LC 50 were calculated.
Locomotor activity and anxiety protocol: 24 5 dpf-larvae were used for each concentration (8 larvae/plague, 3 repetitions). Larvae were bathed in the freshly prepared solutions (3 µM, 1.5 µM, 0.5 µM) of tested compounds and imipramine or diazepam (10 µM) for 30 min before the test in the dark (28 • C). Twenty-four multi-well plates were used for the assays and only one larva was placed in each well in the 1 mL of tested solution. Plates were situated in the zebra box for 95 min; that included the acclimatization part (min 0-10), with the light on, and the visual motor challenge part lasting from 11 min to 95 min. The second part was divided into the continuous illumination phase lasting for 40 min (evaluation of the spontaneous locomotor activity and thigmotaxis behavior, before applying the factor causing the anxiety), and light/dark transition where larvae were subjected to three cycles (the 10-min continuous light and the 5-min dark phase (anxiogenic factor)).
Statistical Analysis: GraphPad Prism (8.0.1) software was used to perform oneway ANOVA analyses with posthoc Tukey's test to evaluate spontaneous locomotor activity (light-dark phase) and the hatching rate. For two-way ANOVA analysis, two main contributing factors were used: treatment (

Conclusions
In this paper, we have thoroughly investigated the influence of incorporation of different cations into the imipramine system on its physicochemical and biological properties. We have synthesized three new compounds, which have been structurally and thermally described. All three compounds are isostructural with approximately the same lattice parameters. FTIR spectra are in good agreement with structures measured using the SC-XRD method. All compounds are stable at room temperature and decompose gradually along with the increasing temperature. Results suggest that [ applied at the doses of 6.25 µM in water and 3.125 µM in ethanol caused disruptions in the heart rate compared with the control groups which were stabilized after 96 hpf. This may suggest the influence of these compounds on the heart activity and developing abilities in the early stages of the development, but it requires confirmation in further studies.
[HL] 2 + [CuCl 4 ] 2− affected the hatching rate stronger than the other complexes and inhibited it in a dose equal to and higher than 0.125 µM in ethanol and 1.25 µM in water after 96 hpf. Moreover, there were abnormalities in the heart activity associated with the decrease of the heart rate only after 96 hpf when [HL] 2 + [CuCl 4 ] 2− was applied, suggesting the developing cardiotoxicity of the complex. The solvent-dependent differences in the heart rate were also visible.
Water solutions caused deaths of embryos in the early stage of development when compared with ethanol solutions. We may suggest that ethanol formulates safer compoundsolvent complexes than water. Interestingly, the deaths of dividing cells appeared also after 3 h after [HL] 2 + [CuCl 4 ] 2− application in the doses 100-6.25 µM in water and 25-12.5 µM in ethanol and 3 h after application of 100 µM of [HL] 2 + [ZnCl 4 ] 2− in water, which may suggest the embryotoxic effect of these compounds in specified concentrations. This confirms the toxic effect of [HL] 2 + [CuCl 4 ] 2− compounds and suggests the influence of mentioned above doses on the developing cell processes. We may claim that higher doses of the tested compounds could have a teratogenic effect on developing organisms.
All tested compounds presented a dose-dependent decrease in the spontaneous locomotor activity where the highest dose 3 µM decreased and the lowest 0.5 µM increased the locomotor activity when compared with control groups after 30-min bathing. The manner of changes was similar for each compound and was not solvent-dependent.
After the application of 3, 1.5, and 0.5 µM of [HL] 2 + [CuCl 4 ] 2− in ethanol, locomotor activity in the light phase stayed on the same level which was lower than the control groups. Moreover, the overall locomotor activity in the dark phase after dissolving [HL] 2 + [CuCl 4 ] 2− in water was lower than in ethanol and lower than in the control group. Studies showed that the acute application of small doses of ethanol influences the level of anxiety-like behavior in Zebrafish [29,30]. In our study, imipramine-metallic ion compounds were also dissolved in ethanol. For this reason, the activity of these solutions could result in either higher dissolving abilities or applied solvent. This may suggest that ethanol as a solvent for this compound enhances the anxiogenic activity of the [HL] 2 + [CuCl 4 ] 2− in a dose-dependent manner, while water intensifies the anxiolytic activity of the tested compounds. The increase in locomotor activity in the dark phase of the experiment by [HL] 2 + [CuCl 4 ] 2− compound is not only dose-dependent, but also solvent-dependent. Taken together, we can suggest that the highest doses (3 µM) may have anxiolytic activity associated with the higher locomotor activity when compared with the control group in the dark phase versus the lowest doses (0.5 µM) of anxiogenic activity. The activity of 3 µM of [HL] 2 + [ZnCl 4 ] 2− and [HL] 2 + [CdCl 4 ] 2− complexes in water and ethanol were similar to the effect obtained after 10 µM of diazepam (the anxiolytic agent). That may confirm our conclusions about the anxiolytic activity of this dose without solvent dependence. Each compound caused a dose-dependent manner of changes in the level of locomotor activity presented in the dark phase without a solvent dependence.
The activity of each dose of complexes differed from the effect caused by the imipramine which suggests that the addition of the cations of Cu 2+ , Zn 2+ , or Cd 2+ modified imipramine's influence on the CNS and anxiety-like behavior. The highest safe doses of the tested compounds and [HL] 2 + [CuCl 4 ] 2− in water showed similar effects to diazepam (10 µM) what may suggest their anxiolytic effect. However, the underlying mechanism of this activity associated with a dose-and/or solvent-dependent manner of changes remains unknown. Thus, further biochemical studies are required to investigate the level of neurotransmitters after complex application.