2.1. Chemistry
3-Amino-1,2,4-triazole is an interesting substrate for the reaction with triethyl orthoformate and diethyl phosphite because it possesses multiple groups that can serve as the amino component in the reaction—namely, the amino moiety in the side-chain and the triazole amine. Moreover, due to tautomerism [
16,
17,
18,
19,
20], three positions of the triazole ring can react.
If two amino groups of the substrate are considered reactive, then the required stoichiometric amount of the amine to phosphite to orthoformate is 1:4:2. NMR analysis of the crude reaction mixture, obtained after hydrolysis, indicated the formation of the expected mono-substituted 1,2,4-triazolyl-3-yl-aminomethylenebisphosphonic acid (compound
1) as the major product and a mixture of its
N-ethylated compounds (likely
2,
3,
4 and
5;
Figure 1). The
N-ethylated products are easily observed in the
1H-NMR spectra of the crude reaction mixture as well-separated ethyl groups (see
Supplementary Materials). Their formation was additionally confirmed by mass spectrometry (see
Supplementary Materials). The
N-ethylation of bisphosphonate 1 was not surprising, considering that diethyl phosphite is known to act as an alkylating agent [
6,
7].
We succeeded in isolating of one of these compounds (compound
2). Its structure was determined by X-ray analysis (
Figure 2 and
Figure 3). Interestingly, the disappearance of the signal related to the proton at position 5 in compound
2 after prolonged (several days) storage in D
2O/NaOD solution was also observed (see
Supplementary Materials). This was due to the exchange of this proton with deuterium.
For this reaction, the influence of reaction conditions was studied in some detail, and the obtained results are collected in
Table 1. As seen from
Table 1, in all reaction conditions, compound
1 was produced as the predominate product with the formation of the
N-ethylated products dependent on the ratio of the substrates.
2.3. Potentiometry
The acid-base properties of compounds
1 and
2 were determined to understand their behaviour in aqueous solutions. These studies were performed using potentiometry prompted by pH-UV titration studies and, for compound
1, pH*-NMR titration studies (pH* = pH uncorrected for the isotopic effect). Calculated protonation values (p
Ks) are given in
Table 2.
The fully protonated forms of both compounds possess six dissociable protons (H
6L): four on the bisphosphonic functional group, one on the 1,2,4-triazole ring and one located on the central tertiary N amine atom. However, we were only able to determine five protonation constants (p
K1–p
K5) because p
K6, which corresponds to the dissociation of one proton from the PO
3H
2 group, is strongly acidic and could not be determined under the conditions of our pH measurements (pH 2–11). Species distribution diagrams are depicted in
Figure 4. For the case of both ligands, three protonation steps of phosphonic groups were found from the p
Ks values, which agree well with previous studies on
N-(pyridinyl)bisphosphonates [
21,
22] that are only slightly lower in value. Based on the comparison of the preliminary assignments, which assume that the highest estimated p
K1 value for compound
1 belongs to the amine, the next three (p
K2 = 9.62, p
K3 = 6.54 and p
K5 = 1.48) values correspond to the bisphosphonic functional group, and the remaining value (p
K4 = 4.22) belongs to the 1,2,4-triazole ring. A significant reduction of the p
K value of the triazole functional group (p
K4 values of 4.52 and 3.51, respectively, for compounds
1 and
2) in comparison to the unsubstituted triazole (p
K = 9.26 for 1,2,3-triazole [
23] and p
K = 9.95 [
24] for 1,2,4-triazole) was also observed previously for substituted triazoles. The previously reported values were 4.20 for 3-amino-1,2,4-triazole [
19,
24] and approximately 3 for the whole family of 1,2,3-triazole-pyridines (for example, p
K = 3.40 was found for 3-(4-(pyridine-2-yl)-1
H-1,2,3-triazol-1-yl-propan-1-ol) [
25]. Species distribution diagrams (
Figure 4) indicate that the monoprotonated phosphonic groups (forms H
3L and H
2L) dominate in a broad range of pH values.
The results of the potentiometric studies are supported by spectroscopic determination of the p
K values of the studied compounds. The results, obtained from electronic spectra generated in the HypSpec program for the detected species [
26], are presented in
Table 2 and
Figure S1. Unsubstituted 1,2,4-triazole shows a very weak absorption at 205 nm in the ultraviolet absorption spectrum, which shifts bathochromically with triazole substitution, e.g., to 221 nm for
N-acetyl-1,2,4-triazole [
27]. For compound
1, the band at approximately 215 nm was already present under a very acidic pH value (pH 1.57) (
Figure 4). This band is assigned to the protonated 1,2,4-triazole. The band underwent a bathochromic shift to 230 nm when the pH rises, which is assigned to the H
4L → H
3L
− + H
+ deprotonation process at a p
K4 of 4.22 or 4.16 (estimated potentiometrically or spectrophotometrically, respectively). This was the biggest change in absorption for the spectroscopic determination by UV spectrophotometry. This change was the reason for assigning the p
K4 value to the deprotonation of 1,2,4-triazole. Adequate changes occur for the spectra of compound
2. The triazole band was located at approximately 230 nm (represented by the H
3L species) and underwent a bathochromic shift to 245 nm at slightly lower pH than was observed for compound
1 (p
K4 = 3.51 or 3.32 by potentiometric or spectrophotometric estimation, respectively). Generally, spectroscopic titrations (see
Figure S23 Supplementary Materials) are in a good agreement with potentiometric titrations and provide similar p
K values.
To comprehend the solution behaviour of the compounds, NMR spectra of compound
1 were monitored over a broad range of pH* (the results are presented in
Figure S24, Supplementary Materials). The studied compounds can be considered a family, in which, for both compounds, each of the phosphonate groups can accept two protons, and the heterocyclic nitrogen atom can accept one proton. The goal of these experiments was not to determine all protonation constants but only to confirm the protonation scheme. The shifts of the
31P phosphorus nuclei reflected the protonation process for almost all groups (
Figure S24A) and revealed the protonation processes at a pH below 2, in the pH range of 4–6 and in the pH range of 10–12. For the
1H nuclei, we followed the shifts on the aromatic ring of compound
1 (
Figure S24B). The biggest change fell in the pH range of 4–5, confirming the acidic deprotonation of the triazole ring. Although the concentration of the studied compound was far higher in the NMR studies than in the potentiometric studies, which caused precipitation to occur above pH 10, the obtained p
K values are in good agreement with those obtained by potentiometry.
2.4. In Vitro Evaluation
To screen for potential antiosteoporotic activity of the bisphosphonates, their antiproliferative activity towards in vitro cell cultures was determined. J774E macrophages and osteoclasts are both derived from haematopoietic lineage and are highly endocytic and capable of demineralizing bone particles [
15]. Therefore, they are models for studies on the influence of bisphosphonates on the proliferation and activity of tumour-associated macrophages (TAMs) [
28]. For this purpose, mouse macrophage-like J774E cells, originating from the same precursors as the osteoclasts, were used [
29,
30]. Such cells are well recognized for being sensitive to bisphosphonates, which likely act by inducing apoptosis in the cells. The J744E cell line was obtained from a cell bank at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences.
Both compounds were moderately active, with IC50 values of 24.88 ± 5.17 (for compound 1) and 30.58 ± 4.64 (for compound 2) μM. Comparing the activities of compounds 1 and 2 with the activities of control samples, zoledronic acid (24.51 ± 2.23 μM) and incadronic acid (47.85 ± 1.39 μM), indicates that compounds 1 and 2 are promising antiosteoporotic drug candidates.