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. 2017 Dec 18;22(12):2265.
doi: 10.3390/molecules22122265.

Synthesis and Biocompatibility Studies of New Iminodiacetic Acid Derivatives

Magdalena Markowicz-Piasecka  1 , Piotr Dębski  2 , Elżbieta Mikiciuk-Olasik  3 , Joanna Sikora  4
Affiliations

Synthesis and Biocompatibility Studies of New Iminodiacetic Acid Derivatives

Magdalena Markowicz-Piasecka et al. Molecules. .

Abstract

Background: Iminodiacetic acid (IDA) derivatives can be used as ligands to form complexes with technetium, with potential application as hepatobiliary diagnostic agents. The aim of this study was to synthesize five novel IDA derivatives and to compare their effects on plasma haemostasis with clinically approved ligands for technetium complexation.

Methods: The influence of synthesized IDA derivatives on plasma haemostasis was evaluated spectrophotometrically by clot formation and lysis test (CL-test), coagulation assay, Prothrombin Time and Activated Partial Tromboplastin Time. The effects of the tested compounds on erythrocytes were assessed using haemolysis assays, microscopy and flow cytometry studies.

Results: Despite their significant influence on the kinetic parameters of the process of clot formation and fibrinolysis, the tested ligands, at potential diagnostic concentrations, did not alter the overall potential of clot formation and lysis (CLAUC). At potential diagnostic concentrations (0.4 μmol/mL) all the tested compounds showed no adverse effects on the membranes of RBCs (Red Blood Cells).

Conclusion: IDA derivatives with methoxy substituents in aromatic ring, exert multidirectional effects on plasma haemostasis and should be considered safe as their significant impacts were mostly observed at 4 μmol/mL, which is about 10-fold higher than the theoretical plasma concentrations of these compounds.

Keywords: biocompatibility; haemostasis; iminodiacetic acid; radiopharmaceuticals.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Synthesized derivatives of iminodiacetic acid (IDA).
Figure 2
Figure 2
Results of coagulation assay. Influence of iminodiacetic acid derivatives (A) compounds 1 and 2; (B) compounds 3–5 on thrombin generation time (TGt) (mean ± SD, n = 5) after 3 min incubation in plasma; final volume 500 μL. All compounds at the highest tested concentration 4 μmol/mL statistically significantly prolonged TGt. * p < 0.05.
Figure 2
Figure 2
Results of coagulation assay. Influence of iminodiacetic acid derivatives (A) compounds 1 and 2; (B) compounds 3–5 on thrombin generation time (TGt) (mean ± SD, n = 5) after 3 min incubation in plasma; final volume 500 μL. All compounds at the highest tested concentration 4 μmol/mL statistically significantly prolonged TGt. * p < 0.05.
Figure 3
Figure 3
The effects of iminodiacetic acid derivatives (A) compounds 1 and 2; (B) compounds 3–5 on amidolytic activity of thrombin expressed as velocity of the enzymatic reaction (dA/dt). The results are presented as mean ± SD, n = 4 after 3 min incubation in plasma; final volume 500 μL. All compounds statistically significantly decreased the velocity of the enzymatic reaction, depending on their concentration; * p < 0.05.
Figure 3
Figure 3
The effects of iminodiacetic acid derivatives (A) compounds 1 and 2; (B) compounds 3–5 on amidolytic activity of thrombin expressed as velocity of the enzymatic reaction (dA/dt). The results are presented as mean ± SD, n = 4 after 3 min incubation in plasma; final volume 500 μL. All compounds statistically significantly decreased the velocity of the enzymatic reaction, depending on their concentration; * p < 0.05.
Figure 4
Figure 4
The effects of iminodiacetic acid derivatives (A) compounds 1 and 2; (B) compounds 3–5 on Activated Partial Thromboplastin Time (APTT) (mean ± SD; n = 5) after 3 min incubation in plasma; final volume 160 μL. Exposure to the tested compounds even at the highest concentrations was shown not to significantly influence the value of APTT over the whole concentration range.
Figure 5
Figure 5
The effects of iminodiacetic acid derivatives (A) compounds 1 and 2; (B) compounds 3–5 on Prothrombin Time (PT) (mean ± SD; n = 5) after 3 min incubation in plasma; final volume 160 μL. Iminodiacetic acid derivatives did not affect in a statistically significant way the values of PT in comparison with control.
Figure 6
Figure 6
The effects of iminodiacetic acid derivatives (A) compounds 1 and 2; (B) compounds 3–5 on Thrombin Time (TT) (mean ± SD; n = 4–5) after 2 min incubation in plasma; final volume 210 μL. All synthesized compounds apart from 3 at the lowest concentration range (0.04–0.2/0.4 μmol/mL) statistically significantly shortened TT, whereas for the highest concentration (4 μmol/mL) TT prolongation was reported. * p < 0.05.
Figure 7
Figure 7
Percentage of haemolysis obtained from the interaction of iminodiacetic acid derivatives (A) compounds 1 and 2; (B) compounds 3–5 with 2% RBCs (red blood cells) suspension, compared to the positive control Triton X-100 at 0.2% (100% hemolysis) (mean ± SD; n = 5), * p < 0.05 vs. control. All newly synthesized compounds showed no adverse effect on the membrane of RBCs in the concentration range 0.04–0.4 μmol/mL. A statistically significant increase in the rate of haemolysis was documented for the highest concentrations of compounds 1–4.
Figure 8
Figure 8
(I) Effect of iminodiacetic acid derivatives on erythrocytes morphology. 2% erythrocyte suspension was treated at 37 °C for 60 min with indicated concentrations of compound 1 and 2. Representative phase-contrast images are shown (magnification of 400 times). A—concentration 0.2 μmol/mL; B—concentration 2.0 μmol/mL; (II). Effect of iminodiacetic acid derivatives on erythrocytes morphology. 2% erythrocyte suspension was treated at 37 °C for 60 min with indicated concentrations of compound 3–5. Representative phase-contrast images are shown (magnification of 400 times). A—concentration 0.2 μmol/mL; B—concentration 2.0 μmol/mL.
Figure 9
Figure 9
Effects of iminodiacetic acid derivatives on erythrocytes (flow cytometry studies). (A) On the basis of SSC-A (side scattered light A) parameter, subpopulation of erythrocytes marked in grey and separated by gate P5 was distinguished; (B) Bimodal distribution of RBCs—control and samples treated with IDA derivatives (0.2 and 2.0 μmol/mL). Erythrocytes were divided into P3 and P4 gates differing in the value of FSC (Forware Scatter) parameter corresponding to the size of measured objects. The erythrocytes of gate P5 were divided into P6 and P7 according to FSC parameter.
Figure 10
Figure 10
Effects of iminodiacetic acid derivatives on erythrocytes. (A) Percentage of all RBCs (mean ± SD, n = 3) divided into P3 and P4 gates. The highest tested concentrations (2.0 μmol/mL) of compounds 1–4 and compound 5 at concentration of 0.2 μmol/mL contributed to significant changes in the amount of RBCs within these gates; (B) Percentage of all RBCs divided into P6 and P7 gates. The highest tested concentrations (2.0 μmol/mL) of compounds 1–4 contributed to significant changes in the amount of RBCs within these gates. * p < 0.05 vs. control.
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