# A New (3, 6)-Connected Heterometallic Coordination Polymer: Therapeutic and Protective Effect on Ischemic Cerebral Infarction

Authors: Jia Ma; Jianer Wang; Yuping He; Yiwei Huang; Yejun Chen
Journal: Journal of Chemistry
 (2021)

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## Abstract

Solvothermal reaction of 4,4’,4’-nitrilotribenzoic acid (H3TCA) with Ca(NO3)2·4H2O and Cd(NO3)2·4H2O successfully afforded a heterometallic compound, that is, [Cd2Ca(TCA)2(DMA)2(H2O)3]n·2n(DMA) (1, DMA = N,N’-dimethylacetamide). The structure of 1 was further characterized through EA, diffraction of single crystal X-ray, and thermogravimetric analysis (TGA) together with powder X-ray diffraction (PXRD). Moreover, complex 1’s luminescent performance in solid state was also discussed at environmental temperature. The applications on the ischemic cerebral infarction therapy were evaluated, and the specific mechanism was investigated at the same time. Firstly, the hypersensitivity C-reactive protein (hs-CRP) content in human brain microvascular endothelial cell (HBMEC) was measured with real-time reverse transcription-polymerase chain reaction (RT-PCR). Then, interleukin-18 and interleukin-1β content released through the HBMEC was detected with enzyme-linked immunosorbent assay (ELISA).


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## Body

## 1. Introduction

Ischemic cerebral infarction refers to the degeneration, necrosis, or transient loss of function of the local brain tissue including nerve cells, glial cells, and connecting fibers due to blood supply disorders [1]. It accounts for about 80% of vascular diseases and are common clinical diseases and frequently-occurring diseases, with high mortality and disability rates [2]. Recently, the immune inflammatory response reported playing an important role in the development of the ischemic cerebral infarction. Thus, the new candidates targeting the inflammatory response were needed to be studied [3].In the last several decades, a great deal of the coordination polymers (CPs) having fascinating topological skeletons have been triumphantly produced by the polydentate organic ligands and metal ions or polynuclear metal clusters, and these crystalline CPs also show promising application properties, such as luminescence sensing, molecular recognition, gas storage/separation, and heterogeneous catalysis that have attracted tremendous interest for the development of new inorganic-organic hybrid materials [4–8]. From these reports, it can be found that the structure and properties of CPs are intimately related with the selected organic ligands. To construct the desired CPs, the key factor is the intelligent selection of suitable organic ligands with appropriate shape, flexibility, symmetry, and coordination functional groups [9–11]. As a representative multidentate ligand, multicarboxylate ligands show outstanding coordination capacities and versatile coordination modes for the formation of CPs, and interestingly, the carboxylic acid groups can connect metal ions into a variety of chains with rod-shape or polynuclear clusters that can be served as second building subunits to direct the construction of CPs [6, 12–14]. In the reports, Yao and his coworkers found that the combination of alkaline Earth metal ions and Zn(II) or Cd(II) ions with aromatic carboxylate ligands is easy to form heterometallic CPs with different seconding building subunits [15, 16]. As a C3 symmetric ligand, 4,4’,4’’-nitrilotribenzoic acid (H3TCA) has three carboxylate groups and a large triphenylamine chromophore, which makes it a good organic building block for the construction of luminescent CPs [17–19]. As yet, in the TCA-based heterometallic CPs, as far as we know, there are still few reports. Considering this, in this work, we employed H3TCA to self-assemble with the Ca(II) and Cd(II) mixed metal ions under solvothermal conditions. Triumphantly, we obtained a fresh heterometallic compound formulated as [Cd2Ca(TCA)2(DMA)2(H2O)3]n·2n(DMA) (1). Also, the thermostability and luminescent property of 1 were investigated. Furthermore, the biological activity of the new compound was evaluated, and the related mechanism was explored as well. The results of the real-time PCR indicated that the expression level of the hs-CRP expression in HBMEC was significantly reduced by the new compound. Besides, the IL-1β and IL-18 content released by HBMEC was also reduced by the new compound, which was measured by the ELISA assay.


## 2. Experimental

### 2.1. Materials and Instrumentation

The used chemical reagents were commercially available from Shanghai Aladdin Bio-Chem Technology Co., Ltd. PXRD was analyzed through applying the PANalytical X’Pert at 0.05° step size using the Cu/Kα radiation (where λ is 1.54056 Å). The hydrogen, nitrogen, as well as carbon elements were analyzed through employing Vario EL III. With the temperature of 30–800°C, TGA was accomplished with the NETSCHZ STA-449C at 10°C per min heating rate under a flow of nitrogen. Edinburg FLS920 TCSPC was exploited to collect luminescent spectra at ambient temperature.


### 2.2. Synthesis of Compound [Cd2Ca(TCA)2(DMA)2(H2 O)3]n·2n(DMA) (1)

The mixture synthesized from 0.100 mmol Cd(NO3)2·4H2O, 0.05 mmol Ca(NO3)2·4H2O, 0.1 mmol H3TCA, 3 mL DMA, and 1.0 mL H2O was sealed into a small glass vial (20 mL), and this vial was heated for one day at 110°C temperature. After the mixture naturally cooled to environmental temperature, the colorless massive crystals were produced with 46 percent yield in accordance with H3TCA. Elemental analysis calcd. for the C58H66CaCd2 N6O19 (1416.06): N, 5.93, C, 49.15, and H, 4.66%. Found: N, 5.96, C, 49.13 and H, 4.62%. IR (KBr pallet, cm−1): 3423 (m), 3063 (m), 2934 (m), 2783 (m), 1593 (s), 1563 (s), 1489 (s), 1451 (s), 1380 (m), 1364 (m), 1338 (s), 1312 (s), 1233 (s), 1162 (m), 1150 (m), 1108 (s), 1026 (m), 1005 (w), 925 (s), 836 (m), 776 (s), 756 (s), 710 (m), 689 (s), 618 (w), 580 (w), 535 (s).


### 2.3. X-Ray Crystallography

For the compound, its data of single crystal were recorded with the diffractometer of mercury CCD controlled through computer, which was equipped with graphite monochromated Mo-Kα radiation (in which λ is 0.71073 Å) under ambient temperature. ShelXS with direct methods and ShelXL with least squares minimization were, respectively, employed to solve and refine the architecture [20]. The disordered molecules of DMA were squeezed out through exploiting PLATON [21]. The data of crystallography for the complex are illustrated in Table 1.Table 1
The compound’s crystal data.
FormulaC58H66CaCd2N6O19Fw1416.06Crystal systemOrthorhombicSpace groupPbcna (Å)24.2583 (9)b (Å)11.7412 (8)c (Å)22.5566 (12)α (°)90β (°)90γ (°)90Volume (Å3)6424.6 (6)Z4Density (calculated)1.284Abs. coeff. (mm−1)0.802Total reflections67535Unique reflections12019Goodness of fit onF20.933FinalR indices (I > 2sigma (I2))R = 0.0881, wR2 = 0.1901R (all data)R = 0.2226, wR2 = 0.2509CCDC2096366


### 2.4. Real-Time PCR

After the synthesis of the new compound, its therapeutic activity on the ischemic cerebral infarction was determined. As we all know, there was a remarkably increased hs-CRP level during the procession of cerebral infarction, which was also regarded as the indicator of acute phase inflammation of cerebral infarction. Thus, in this present research, the real-time PCR was conducted, and the hs-CRP expression level in HBMEC after compound treatment was measured following the instructions. In short, the HBMEC in the logical growth phase was harvested and seeded into a 6-well plate at a concentration of 105 cells per well. After 12 h of incubation at 37°C, 5%CO2 condition, the cells were treated with hypoxia culture, followed by the compound treatment at the concentration of 1, 2, and 5 μg/ml. Next, the cells were collected, and the total RNA was extracted with RNA isolation kit, with the ligand and metal ion used as the negative control. The spectrophotometer was used to measure the concentration of the total RNA, which was then reverse-transcripted into cDNAs using a high-capacity cDNA reverse transcription kit. Finally, the PCR detection was carried out with Step One Plus Real-time PCR systems. The hs-CRP relative expression in the HBMEC was evaluated with GADPH used as an inner reference.


### 2.5. ELISA Assay

As IL-1β and IL-18 has been widely regarded as the important indicator of the cerebral infarction, the ELISA assay was next conducted, and the IL-1β and IL-18 content released by HBMEC was measured in this study. All preformation was finished according to the manufacturer’s instructions with some changes. In brief, the HBMEC in the logical growth phase was harvested and seeded into a 6-well plate (105 cells/well). After 12 h of incubation in an incubator at 37°C, 5%CO2 condition, the cells were treated with hypoxia culture as well the treatment of compound at 1 μg/ml, 2 μg/ml, and 5 μg/ml concentration, with the ligand and metal ion used as the negative control. After treated with the compound, the culture supernatant was harvested, and the IL-18 and IL-1β content was measured with ELISA assay. This research was performed for 3 times, and the results were presented as mean ± SD.


## 2.1. Materials and Instrumentation

The used chemical reagents were commercially available from Shanghai Aladdin Bio-Chem Technology Co., Ltd. PXRD was analyzed through applying the PANalytical X’Pert at 0.05° step size using the Cu/Kα radiation (where λ is 1.54056 Å). The hydrogen, nitrogen, as well as carbon elements were analyzed through employing Vario EL III. With the temperature of 30–800°C, TGA was accomplished with the NETSCHZ STA-449C at 10°C per min heating rate under a flow of nitrogen. Edinburg FLS920 TCSPC was exploited to collect luminescent spectra at ambient temperature.


## 2.2. Synthesis of Compound [Cd2Ca(TCA)2(DMA)2(H2 O)3]n·2n(DMA) (1)

The mixture synthesized from 0.100 mmol Cd(NO3)2·4H2O, 0.05 mmol Ca(NO3)2·4H2O, 0.1 mmol H3TCA, 3 mL DMA, and 1.0 mL H2O was sealed into a small glass vial (20 mL), and this vial was heated for one day at 110°C temperature. After the mixture naturally cooled to environmental temperature, the colorless massive crystals were produced with 46 percent yield in accordance with H3TCA. Elemental analysis calcd. for the C58H66CaCd2 N6O19 (1416.06): N, 5.93, C, 49.15, and H, 4.66%. Found: N, 5.96, C, 49.13 and H, 4.62%. IR (KBr pallet, cm−1): 3423 (m), 3063 (m), 2934 (m), 2783 (m), 1593 (s), 1563 (s), 1489 (s), 1451 (s), 1380 (m), 1364 (m), 1338 (s), 1312 (s), 1233 (s), 1162 (m), 1150 (m), 1108 (s), 1026 (m), 1005 (w), 925 (s), 836 (m), 776 (s), 756 (s), 710 (m), 689 (s), 618 (w), 580 (w), 535 (s).


## 2.3. X-Ray Crystallography

For the compound, its data of single crystal were recorded with the diffractometer of mercury CCD controlled through computer, which was equipped with graphite monochromated Mo-Kα radiation (in which λ is 0.71073 Å) under ambient temperature. ShelXS with direct methods and ShelXL with least squares minimization were, respectively, employed to solve and refine the architecture [20]. The disordered molecules of DMA were squeezed out through exploiting PLATON [21]. The data of crystallography for the complex are illustrated in Table 1.Table 1
The compound’s crystal data.
FormulaC58H66CaCd2N6O19Fw1416.06Crystal systemOrthorhombicSpace groupPbcna (Å)24.2583 (9)b (Å)11.7412 (8)c (Å)22.5566 (12)α (°)90β (°)90γ (°)90Volume (Å3)6424.6 (6)Z4Density (calculated)1.284Abs. coeff. (mm−1)0.802Total reflections67535Unique reflections12019Goodness of fit onF20.933FinalR indices (I > 2sigma (I2))R = 0.0881, wR2 = 0.1901R (all data)R = 0.2226, wR2 = 0.2509CCDC2096366


## 2.4. Real-Time PCR

After the synthesis of the new compound, its therapeutic activity on the ischemic cerebral infarction was determined. As we all know, there was a remarkably increased hs-CRP level during the procession of cerebral infarction, which was also regarded as the indicator of acute phase inflammation of cerebral infarction. Thus, in this present research, the real-time PCR was conducted, and the hs-CRP expression level in HBMEC after compound treatment was measured following the instructions. In short, the HBMEC in the logical growth phase was harvested and seeded into a 6-well plate at a concentration of 105 cells per well. After 12 h of incubation at 37°C, 5%CO2 condition, the cells were treated with hypoxia culture, followed by the compound treatment at the concentration of 1, 2, and 5 μg/ml. Next, the cells were collected, and the total RNA was extracted with RNA isolation kit, with the ligand and metal ion used as the negative control. The spectrophotometer was used to measure the concentration of the total RNA, which was then reverse-transcripted into cDNAs using a high-capacity cDNA reverse transcription kit. Finally, the PCR detection was carried out with Step One Plus Real-time PCR systems. The hs-CRP relative expression in the HBMEC was evaluated with GADPH used as an inner reference.


## 2.5. ELISA Assay

As IL-1β and IL-18 has been widely regarded as the important indicator of the cerebral infarction, the ELISA assay was next conducted, and the IL-1β and IL-18 content released by HBMEC was measured in this study. All preformation was finished according to the manufacturer’s instructions with some changes. In brief, the HBMEC in the logical growth phase was harvested and seeded into a 6-well plate (105 cells/well). After 12 h of incubation in an incubator at 37°C, 5%CO2 condition, the cells were treated with hypoxia culture as well the treatment of compound at 1 μg/ml, 2 μg/ml, and 5 μg/ml concentration, with the ligand and metal ion used as the negative control. After treated with the compound, the culture supernatant was harvested, and the IL-18 and IL-1β content was measured with ELISA assay. This research was performed for 3 times, and the results were presented as mean ± SD.


## 3. Results and Discussion

### 3.1. Crystal Structure of Compound 1

Compound1 was prepared via reaction of Cd(NO3)2·4H2O, Ca(NO3)2·4H2O and H3TCA in a mixed solvent of DMA and H2O at 110°C for one day. The H3TCA ligand could not be dissolved in water nor EtOH but could be dissolved in DMA by stirring, so we used H3TCA as the main solvent in this reaction. The compound’s architecture reveals a three-dimensional skeleton according to trinuclear subunits based on the [Cd2Ca(COO)4] cluster, which was crystallized in the space group Pbcn of the orthorhombic system. The compound’s fundamental unit is constructed from a Cd(II) ion, 0.5 Ca(II) ion, a ligand of TCA3−, a coordinated molecule of DMA, one and a half coordinated molecules of water, together with a molecule of DMA. As reflected in the Figure 1(a), the Cd1 ion is six-coordinated through 5 carboxylic acid O atoms coming from 3 ligands of TCA3− and an atom of a terminal molecule of DMA, displaying a highly distorted structure of octahedron, and the Ca1 ion is 7-coordinated via 4 carboxylic acid O atoms derived from 4 diverse ligands of TCA3− and 3 terminal ligands of water, displaying a pentagonal bipyramidal structure. The distance of Cd-O is between 2.226 (8) and 2.449 (5) Å, and the separation of Ca-O varying from 2.278(4) to 2.584(10) Å, respectively, is in a normal range, which is in accordance with the lengths of reported compounds [12]. One Ca(II) ion as well as two symmetry-related Cd(II) ions are connected through 4 carboxylic acid groups to create a trinuclear subunit based on the [Cd2Ca(COO)4] cluster, and the distance of Cd…Ca is 3.90 Å (Figure 1(b)). In the end, these trinuclear subunits based on the cluster are linked together via the ligands of TCA3−, generating the compound’s three-dimensional skeleton (Figure 1(c)). In this three-dimensional skeleton, each ligand of TCA3− connects 3 diverse trinuclear clusters [Cd2Ca(COO)4], and each of the trinuclear cluster [Cd2Ca(COO)4] is supported through 6 distinct ligands of TCA3−, By viewing TCA3− ligands and trinuclear [Cd2Ca(COO)4] clusters as three- and six-linked nodes, respectively (Figures 1(d) and 1(e)), the compound’s entire framework can be reduced topologically as a (3, 6)-linked apo net, with {4.62}2{42.69.84} point symbol (Figure 1(f)).Figure 1
(a) The coordination surroundings of Ca(II) and Cd(II) ions in complex1. (b) The heterometallic trinuclear cluster of [Cd2Ca(COO)4]. (c) The compound’s three-dimensional skeleton. (d) The defined three-linked node. (e) The defined six-linked node. (f) Complex 1’s representation of (3, 6)-linked apo topological net (green ball: [Cd2Ca(COO)4] cluster; yellow ball: TCA3- ligand).
(a)(b)(c)(d)(e)(f)


### 3.2. Powder X-Ray Diffraction Pattern (PXRD) and Thermogravimetric Analysis (TGA)

The study of PXRD was employed to demonstrate the bulk samples’ purity. As displayed in Figure2(a), there exists an excellent match between the pattern of experiment and simulation, which reflects that the as-created samples are in pure phase. Considering the following bioactivity tests, it is necessary to study the framework stability of complex 1 in the dispersing solvent DMSO and the simulated in vivo condition phosphate buffered saline (PBS). As complex 1 could not be dissolved in organic solvents and water, we used its stock solution in the following bioactivity tests according to the methods proposed in [22, 23]. With this in mind, about 100 mg of the as-prepared crystalline samples of 1 was taken in a mortar. It was then being ground manually for 30 min by using an pestle. The produced fine powders were soaked in 20 mL PBS or DMSO and subjected to the ultrasonic treatments for 2 hours to obtain the well-dispersed solution. After standing for two days, the fine powders could be recovered via centrifugation, and their PXRD measurements show that the PXRD profiles of the treated samples show good matches with those of the as-prepared samples, reflecting their good stability in the above conditions.Figure 2
(a) The compound’s manners of PXRD. (b) The1’s curve of TGA.
(a)(b)TGA experiment for1 was also carried out under nitrogen atmosphere. In the complex’s curve of TGA, the first found weightlessness appearing in the temperature of 91–152°C was caused via the departure of the coordinated molecules of DMA and water, as well as the lattice DMA molecules (obsd: 28.50%, calcd: 28.42%), and the second found weightlessness appeared between 285 and 442°C that is on account of the organic ligand combustion, resulting in the generation of CaO and CdO as the ultimate residues of 22.12 percent (with 22.10% calculated value).


### 3.3. Luminescent Property of 1

Coordination polymers based on large conjugated organic ligands usually show interesting luminescent properties and can be served as hybrid materials in the fields of chemical sensors, light-emitting diodes (LEDs), and photochemistry [24, 25]. As a result, the luminescent spectra of free ligand of H3TCA and complex 1 in solid state were detected at environmental temperature. With 330 nm excitation, free ligand of H3TCA shows intense luminescence with the maximum emission peak at 435 nm, which is probably owing to the transition of π∗⟶π of triphenylamine moiety (Figure 3). After complexation of TCA3− ligand with Ca(II) and Cd(II) ions, a more intense emission peak for 1 can be found at 452 nm excitation (with λex of 350 nm). Significantly, complex 1’s emission spectrum is similar to the emission spectrum of free ligand of H3TCA, and complex 1’s maximum peak exhibits 17 nm red-shift in comparison with that of free ligand of H3TCA. Because Ca(II) and Cd(II) ions are difficult to reduce or oxidize, the 1’s emission is probably due to the charge transfer in the ligand that are perturbed by the metal-ligand coordination interactions and the deprotonated effect of the tricarboxylate ligands [10, 26, 27].Figure 3
The luminescent emission spectra of free ligand of H3TCA and complex 1 in solid state at ambient temperature.


### 3.4. Significantly Reduced hs-CRP Expression Levels in HBMEC after Compound Treatment

hs-CRP is a nonspecific acute phase reaction protein, which is widely regarded as the indicator of the ischemic cerebral infarction acute phase inflammation. During the procession of cerebral infarction, hs-CRP also exhibited an increased level. In this recent research, after the successful synthesis of the compound, the protective effect of the new compound against the ischemic cerebral infarction was evaluated by real-time PCR via measuring the hs-CRP expression levels in HBMEC. As the results shown in Figure4, we can see that, in contrast to the control group, there was a remarkably enhanced hs-CRP level in HBMEC of the model group, with p<0.005. However, this abnormal increased level of the hs-CRP in HBMEC was obviously reduced under the treatment of a new compound. The inhibition of the new compound revealed a time-dependent correlation. While, the ligand and the metal ion showed almost no influence on the hs-CRP expression levels in HBMEC. This data indicated the compound exhibits outstanding protective effect in cerebral infarction treatment through reducing hs-CRP expression levels in HBMEC.Figure 4
The compound could significantly reduce the hs-CRP expression levels in HBMEC.The HBMEC cells were treated with hypoxia culture as well, and the compound treatment at the concentration of 1, 2, and 5μg/ml, with the ligand and metal ion, is used as the negative control. Then, the real-time PCR was used to measure hs-CRP expression levels in HBMEC which obviously inhibited releasing of IL-18 and IL-1β by HBMEC after compound treatment.In the above research, we have revealed that the compound has excellent inhibitory effect on the hs-CRP expression in HBMEC in a dose-dependent manner. In addition to this, the increased level of IL-1β and IL-18 has been widely regarded as the important indicator of the cerebral infarction. Thus, the IL-1β and IL-18 content released by HBMEC was measured by ELISA assay. As the results shown in Figure 5, we can see that the level of IL-1β and IL-18 was significantly increased in the model group, compared with the control group. But, the levels of the inflammatory cytokines could be reversed under the treatment of the new compound in a dose-dependent manner. While, the ligand and the metal ion showed almost no influence on the IL-18 level and IL-1β level in HBMEC. This data revealed that the compound could release the IL-18 level and IL-1β level in HBMEC during cerebral infarction.Figure 5
The compound obviously inhibited the releasing of IL-1β and IL-18 by HBMEC. The HBMEC cells were treated with hypoxia culture as well the compound treatment at the concentration of 1, 2, and 5 μg/ml, with the ligand and metal ion used as the negative control.


## 3.1. Crystal Structure of Compound 1

Compound1 was prepared via reaction of Cd(NO3)2·4H2O, Ca(NO3)2·4H2O and H3TCA in a mixed solvent of DMA and H2O at 110°C for one day. The H3TCA ligand could not be dissolved in water nor EtOH but could be dissolved in DMA by stirring, so we used H3TCA as the main solvent in this reaction. The compound’s architecture reveals a three-dimensional skeleton according to trinuclear subunits based on the [Cd2Ca(COO)4] cluster, which was crystallized in the space group Pbcn of the orthorhombic system. The compound’s fundamental unit is constructed from a Cd(II) ion, 0.5 Ca(II) ion, a ligand of TCA3−, a coordinated molecule of DMA, one and a half coordinated molecules of water, together with a molecule of DMA. As reflected in the Figure 1(a), the Cd1 ion is six-coordinated through 5 carboxylic acid O atoms coming from 3 ligands of TCA3− and an atom of a terminal molecule of DMA, displaying a highly distorted structure of octahedron, and the Ca1 ion is 7-coordinated via 4 carboxylic acid O atoms derived from 4 diverse ligands of TCA3− and 3 terminal ligands of water, displaying a pentagonal bipyramidal structure. The distance of Cd-O is between 2.226 (8) and 2.449 (5) Å, and the separation of Ca-O varying from 2.278(4) to 2.584(10) Å, respectively, is in a normal range, which is in accordance with the lengths of reported compounds [12]. One Ca(II) ion as well as two symmetry-related Cd(II) ions are connected through 4 carboxylic acid groups to create a trinuclear subunit based on the [Cd2Ca(COO)4] cluster, and the distance of Cd…Ca is 3.90 Å (Figure 1(b)). In the end, these trinuclear subunits based on the cluster are linked together via the ligands of TCA3−, generating the compound’s three-dimensional skeleton (Figure 1(c)). In this three-dimensional skeleton, each ligand of TCA3− connects 3 diverse trinuclear clusters [Cd2Ca(COO)4], and each of the trinuclear cluster [Cd2Ca(COO)4] is supported through 6 distinct ligands of TCA3−, By viewing TCA3− ligands and trinuclear [Cd2Ca(COO)4] clusters as three- and six-linked nodes, respectively (Figures 1(d) and 1(e)), the compound’s entire framework can be reduced topologically as a (3, 6)-linked apo net, with {4.62}2{42.69.84} point symbol (Figure 1(f)).Figure 1
(a) The coordination surroundings of Ca(II) and Cd(II) ions in complex1. (b) The heterometallic trinuclear cluster of [Cd2Ca(COO)4]. (c) The compound’s three-dimensional skeleton. (d) The defined three-linked node. (e) The defined six-linked node. (f) Complex 1’s representation of (3, 6)-linked apo topological net (green ball: [Cd2Ca(COO)4] cluster; yellow ball: TCA3- ligand).
(a)(b)(c)(d)(e)(f)


## 3.2. Powder X-Ray Diffraction Pattern (PXRD) and Thermogravimetric Analysis (TGA)

The study of PXRD was employed to demonstrate the bulk samples’ purity. As displayed in Figure2(a), there exists an excellent match between the pattern of experiment and simulation, which reflects that the as-created samples are in pure phase. Considering the following bioactivity tests, it is necessary to study the framework stability of complex 1 in the dispersing solvent DMSO and the simulated in vivo condition phosphate buffered saline (PBS). As complex 1 could not be dissolved in organic solvents and water, we used its stock solution in the following bioactivity tests according to the methods proposed in [22, 23]. With this in mind, about 100 mg of the as-prepared crystalline samples of 1 was taken in a mortar. It was then being ground manually for 30 min by using an pestle. The produced fine powders were soaked in 20 mL PBS or DMSO and subjected to the ultrasonic treatments for 2 hours to obtain the well-dispersed solution. After standing for two days, the fine powders could be recovered via centrifugation, and their PXRD measurements show that the PXRD profiles of the treated samples show good matches with those of the as-prepared samples, reflecting their good stability in the above conditions.Figure 2
(a) The compound’s manners of PXRD. (b) The1’s curve of TGA.
(a)(b)TGA experiment for1 was also carried out under nitrogen atmosphere. In the complex’s curve of TGA, the first found weightlessness appearing in the temperature of 91–152°C was caused via the departure of the coordinated molecules of DMA and water, as well as the lattice DMA molecules (obsd: 28.50%, calcd: 28.42%), and the second found weightlessness appeared between 285 and 442°C that is on account of the organic ligand combustion, resulting in the generation of CaO and CdO as the ultimate residues of 22.12 percent (with 22.10% calculated value).


## 3.3. Luminescent Property of 1

Coordination polymers based on large conjugated organic ligands usually show interesting luminescent properties and can be served as hybrid materials in the fields of chemical sensors, light-emitting diodes (LEDs), and photochemistry [24, 25]. As a result, the luminescent spectra of free ligand of H3TCA and complex 1 in solid state were detected at environmental temperature. With 330 nm excitation, free ligand of H3TCA shows intense luminescence with the maximum emission peak at 435 nm, which is probably owing to the transition of π∗⟶π of triphenylamine moiety (Figure 3). After complexation of TCA3− ligand with Ca(II) and Cd(II) ions, a more intense emission peak for 1 can be found at 452 nm excitation (with λex of 350 nm). Significantly, complex 1’s emission spectrum is similar to the emission spectrum of free ligand of H3TCA, and complex 1’s maximum peak exhibits 17 nm red-shift in comparison with that of free ligand of H3TCA. Because Ca(II) and Cd(II) ions are difficult to reduce or oxidize, the 1’s emission is probably due to the charge transfer in the ligand that are perturbed by the metal-ligand coordination interactions and the deprotonated effect of the tricarboxylate ligands [10, 26, 27].Figure 3
The luminescent emission spectra of free ligand of H3TCA and complex 1 in solid state at ambient temperature.


## 3.4. Significantly Reduced hs-CRP Expression Levels in HBMEC after Compound Treatment

hs-CRP is a nonspecific acute phase reaction protein, which is widely regarded as the indicator of the ischemic cerebral infarction acute phase inflammation. During the procession of cerebral infarction, hs-CRP also exhibited an increased level. In this recent research, after the successful synthesis of the compound, the protective effect of the new compound against the ischemic cerebral infarction was evaluated by real-time PCR via measuring the hs-CRP expression levels in HBMEC. As the results shown in Figure4, we can see that, in contrast to the control group, there was a remarkably enhanced hs-CRP level in HBMEC of the model group, with p<0.005. However, this abnormal increased level of the hs-CRP in HBMEC was obviously reduced under the treatment of a new compound. The inhibition of the new compound revealed a time-dependent correlation. While, the ligand and the metal ion showed almost no influence on the hs-CRP expression levels in HBMEC. This data indicated the compound exhibits outstanding protective effect in cerebral infarction treatment through reducing hs-CRP expression levels in HBMEC.Figure 4
The compound could significantly reduce the hs-CRP expression levels in HBMEC.The HBMEC cells were treated with hypoxia culture as well, and the compound treatment at the concentration of 1, 2, and 5μg/ml, with the ligand and metal ion, is used as the negative control. Then, the real-time PCR was used to measure hs-CRP expression levels in HBMEC which obviously inhibited releasing of IL-18 and IL-1β by HBMEC after compound treatment.In the above research, we have revealed that the compound has excellent inhibitory effect on the hs-CRP expression in HBMEC in a dose-dependent manner. In addition to this, the increased level of IL-1β and IL-18 has been widely regarded as the important indicator of the cerebral infarction. Thus, the IL-1β and IL-18 content released by HBMEC was measured by ELISA assay. As the results shown in Figure 5, we can see that the level of IL-1β and IL-18 was significantly increased in the model group, compared with the control group. But, the levels of the inflammatory cytokines could be reversed under the treatment of the new compound in a dose-dependent manner. While, the ligand and the metal ion showed almost no influence on the IL-18 level and IL-1β level in HBMEC. This data revealed that the compound could release the IL-18 level and IL-1β level in HBMEC during cerebral infarction.Figure 5
The compound obviously inhibited the releasing of IL-1β and IL-18 by HBMEC. The HBMEC cells were treated with hypoxia culture as well the compound treatment at the concentration of 1, 2, and 5 μg/ml, with the ligand and metal ion used as the negative control.


## 4. Conclusion

In conclusion, we synthesized a new heterometallic compound of [Cd2Ca(TCA)2(DMA)2(H2O)3]n•2n(DMA) (1) via the solvothermal self-assembly of H3TCA, Ca(NO3)2·4H2O and Cd(NO3)2·4H2O. Compound 1 exhibits a three-dimensional skeleton with heterometallic trinuclear clusters building subunits of [Cd2Ca(COO)4] and reflected a (3, 6)-linked apo topological net. Under the environmental temperature, 1 reveals strong luminescence centered at ligand. The results of the real-time PCR indicated that the expression level of the hs-CRP expression in HBMEC was significantly reduced by the new compound. Besides, the IL-1β and IL-18 content released by HBMEC was also reduced by the new compound, which was measured by the ELISA assay. In the end, we draw this conclusion. The new compound reveals outstanding application values against the ischemic cerebral infarction therapy by reducing the content of hypersensitivity C-reactive protein.



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*Source: 5685596-2021-10-31.xml*