The Antioxidant and Antiproliferative Activities of 1,2,3-Triazolyl-L-Ascorbic Acid Derivatives

The novel 4-substituted 1,2,3-triazole L-ascorbic acid (L-ASA) conjugates with hydroxyethylene spacer as well as their conformationally restricted 4,5-unsaturated analogues were synthesized as potential antioxidant and antiproliferative agents. An evaluation of the antioxidant activity of novel compounds showed that the majority of the 4,5-unsaturated L-ASA derivatives showed a better antioxidant activity compared to their saturated counterparts. m-Hydroxyphenyl (7j), p-pentylphenyl (7k) and 2-hydroxyethyl (7q) substituted 4,5-unsaturated 1,2,3-triazole L-ASA derivatives exhibited very efficient and rapid (within 5 min) 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging activity (7j, 7k: IC50 = 0.06 mM; 7q: IC50 = 0.07 mM). In vitro scavenging activity data were supported by in silico quantum-chemical modelling. Thermodynamic parameters for hydrogen-atom transfer and electron-transfer radical scavenging pathways of anions deprotonated at C2-OH or C3-OH groups of L-ASA fragments were calculated. The structure activity analysis (SAR) through principal component analysis indicated radical scavenging activity by the participation of OH group with favorable reaction parameters: the C3-OH group of saturated C4-C5(OH) derivatives and the C2-OH group of their unsaturated C4=C5 analogues. The antiproliferative evaluation showed that p-bromophenyl (4e: IC50 = 6.72 μM) and p-pentylphenyl-substituted 1,2,3-triazole L-ASA conjugate (4k: IC50 = 26.91 μM) had a selective cytotoxic effect on breast adenocarcinoma MCF-7 cells. Moreover, compound 4e did not inhibit the growth of foreskin fibroblasts (IC50 > 100 μM). In MCF-7 cells treated with 4e, a significant increase of hydroxylated hypoxia-inducible transcription factor 1 alpha (HIF-1α) expression and decreased expression of nitric oxide synthase 2 (NOS2) were observed, suggesting the involvement of 4e in the HIF-1α signaling pathway for its strong growth-inhibition effect on MCF-7 cells.


Introduction
Biochemical processes in our body, including aerobic metabolism and inflammatory responses, as well as exposure to the environment, result in the generation of unstable and highly reactive free radicals, such as reactive oxygen (ROS) and nitrogen (RNS) species [1][2][3][4]. While ROS have an important biological Table 1. The scavenging activity of L-ascorbic acid derivatives assessed by DPPH test. Results are presented as IC50 values (mM) of DPPH • inhibition after 5 and 10 min treatments. Scheme 1. Reagents and conditions: (i) Cu, 1 M copper(II) sulphate (CuSO 4 ), tert-butanol, DMF, water, and a microwave reactor; (ii) 1 M boron trichloride (BCl 3 ) and dry dichloromethane; (iii) copper(II) acetate (Cu(OAc) 2 ) and methanol.
It was observed that the majority of the 4,5-unsaturated L-ASA derivatives showed a better antioxidant activity compared to their saturated counterparts ( Figure 1). Interestingly, the introduction of hydroxyl groups in 4,5-unsaturated L-ASA derivatives 7i, 7j, and 7q improved the radical scavenging activities. Besides, the electron-donating p-methyl groups in 4f/7f, the p-pentyl-substituted aryl groups in 4k/7k, and the long aliphatic chains in 4n/7n improved the antioxidant activity compared to L-ASA as well.

In Silico Analysis of Radical Scavenging Capacities
According to the pIC50 values, calculated as the negative logarithm of IC50 values (Table  1)-which are within the range of 3.55−4.22 and have the pIC50 medians of 3.7, regardless the incubation time of 5 min or 10 min-the majority of L-ASA derivatives have similar DPPH • radical scavenging capacities. The pIC50 value for the well-known antioxidant L-ASA was 3.75, demonstrating that most of compounds have a similar or somewhat higher radical scavenging capacity compared to L-ASA.
For some pairs of L-ASA derivatives differing in the presence of C4-C5(OH) or C4=C5 fragments, somewhat greater difference in their IC50 values has been observed ( Table 1). For these pairs of compounds-4b/7b, 4e/7e, 4g/7g, 4i/7i and 4q/7q-the extensive quantum-chemical calculations of radical scavenging capacities were performed by using an approach widely exploited for the estimation of the radical scavenging potential of polyphenols [42]. L-ASA is highly

In Silico Analysis of Radical Scavenging Capacities
According to the pIC 50 values, calculated as the negative logarithm of IC 50 values (Table 1)-which are within the range of 3.55−4.22 and have the pIC 50 medians of 3.7, regardless the incubation time of 5 min or 10 min-the majority of L-ASA derivatives have similar DPPH • radical scavenging capacities. The pIC 50 value for the well-known antioxidant L-ASA was 3.75, demonstrating that most of compounds have a similar or somewhat higher radical scavenging capacity compared to L-ASA.
For some pairs of L-ASA derivatives differing in the presence of C4-C5(OH) or C4=C5 fragments, somewhat greater difference in their IC 50 values has been observed ( Table 1). For these pairs of compounds-4b/7b, 4e/7e, 4g/7g, 4i/7i and 4q/7q-the extensive quantum-chemical calculations of radical scavenging capacities were performed by using an approach widely exploited for the estimation of the radical scavenging potential of polyphenols [42]. L-ASA is highly dissociated acidic molecule at physiological pH of 7.4 (the negative logarithms of experimental acid dissociation constants (pK a ) are pK a,1 of 4.1 and pK a,2 of 11.4) [43].
The novel 1,2,3-triazole L-ASA conjugates were shown to be acidic molecules with similar pK a values (Table 2). Accordingly, L-ASA reacts as a radical scavenger by donating an electron or a hydrogen H-atom from its anion to a free radical ( Figure 2). The first radical scavenging mechanism of L-ASA is described as a two-step sequential proton loss electron transfer (SPLET) process: after the first step of deprotonation of the most acidic OH group (described by its acidity constant), an anion (A) formed donates an electron with a capacity described by the electron transfer (free) energy (ETFE) and changes into neutral radical (NR) (cyan path in Figure 2). However, an anion may also donate a H-atom from the adjacent OH group through the hydrogen-atom transfer (HAT) mechanism which is described by the O-H bond dissociation free energy (BDFE) parameter. The product of this radical scavenging pathway is a radical anion (RA) which may release an additional electron and oxidize to a diketo form of L-ASA known as dehydroascorbic acid (DHA, green path in Figure 2). However, the neutral radical formed by SPLET, immediately releases a proton (pK a NR in Table 2) and converts to a RA. Thus, both pathways have the same final dehydroascorbic acid product. The thermodynamic preference of one of these two pathways has been estimated by comparison of reaction free energies of underlying step processes ( Table 2). The multivariate principal component analysis (PCA) analysis in terms of the experimental pIC 50 values and the calculated aqueous parameters ETFE A , BDFE A , and ETFE RA describing radical scavenging capacities of reducing anions and the intermediate radical anion, explain 87.5% variance in the DPPH • activity of modelled compounds ( Figure 3).  The novel 1,2,3-triazole L-ASA conjugates were shown to be acidic molecules with similar pKa values (Table 2). Accordingly, L-ASA reacts as a radical scavenger by donating an electron or a hydrogen H-atom from its anion to a free radical ( Figure 2). The first radical scavenging mechanism of L-ASA is described as a two-step sequential proton loss electron transfer (SPLET) process: after the first step of deprotonation of the most acidic OH group (described by its acidity constant), an anion (A) formed donates an electron with a capacity described by the electron transfer (free) energy (ETFE) and changes into neutral radical (NR) (cyan path in Figure 2). However, an anion may also donate a H-atom from the adjacent OH group through the hydrogen-atom transfer (HAT) mechanism which is described by the O-H bond dissociation free energy (BDFE) parameter. The product of this radical scavenging pathway is a radical anion (RA) which may release an additional electron and oxidize to a diketo form of L-ASA known as dehydroascorbic acid (DHA, green path in Figure 2). However, the neutral radical formed by SPLET, immediately releases a proton (pKa NR in Table 2) and converts to a RA. Thus, both pathways have the same final dehydroascorbic acid product. The thermodynamic preference of one of these two pathways has been estimated by comparison of reaction free energies of underlying step processes ( Table 2). The multivariate principal component analysis (PCA) analysis in terms of the experimental pIC50 values and the calculated aqueous parameters ETFE A , BDFE A , and ETFE RA describing radical scavenging capacities The most significant difference between saturated C4-C5(OH) and unsaturated C4=C5 derivatives is in acidity of the C2-OH group. While in saturated C4-C5(OH) L-ASA derivatives, the C3-OH group is the only one deprotonated at pH 7.4, in derivatives with the C4=C5 bond, both C2-OH group and C3-OH groups are acidic and may participate in the described DPPH • radical scavenging mechanisms (Table 2). However, in the C4=C5 derivatives, intermediate C2-Oanion is generally characterized by more favorable, lower ETFE A and BDFE A values than the C3-Oanion and it may be expected that it is a primary reactive locus for scavenging of the DPPH • radical (Table 2). Thus, the best variance explanation in the PCA analysis of DPPH • radical scavenging activity has been obtained by using the more favorable parameter values for C3-OH groups of saturated C4-C5(OH) derivatives and for the C2-OH group of the C4=C5 unsaturated derivatives, except for the compound 7e, for which C3-OH parameters were used ( Figure 3).
By comparing ETFE A , BDFE A , and ETFE RA values for saturated and unsaturated analogues, all three parameters are clearly more favorable for both groups C2-OH and C3-OH of 7q than for the C3-OH group of its saturated analogue 4q, and it has been observed as significantly more active in DPPH • radical scavenging, having a higher pIC 50 value (Tables 1 and 2). ETFE A and BDFE A are also more favorable for the stronger scavenger 7i in the 4i/7i pair. While BDFE A values for the 4e/7e pair are comparable, the radical scavenging capacity of intermediate radical anion is somewhat better for 4e than 7e, which may explain the somewhat higher pIC 50 value of the saturated analogue 4e. However, according to the calculation, unsaturated 7g should be a stronger scavenger than the saturated 4g, which was not in agreement with the measured pIC 50 values during 5 min and 10 min time periods of incubation.   By comparing ETFE A , BDFE A , and ETFE RA values for saturated and unsaturated analogues, all three parameters are clearly more favorable for both groups C2-OH and C3-OH of 7q than for the C3-OH group of its saturated analogue 4q, and it has been observed as significantly more active in DPPH • radical scavenging, having a higher pIC50 value (Tables 1 and 2). ETFE A and BDFE A are also more favorable for the stronger scavenger 7i in the 4i/7i pair. While BDFE A values for the 4e/7e pair are comparable, the radical scavenging capacity of intermediate radical anion is somewhat better for 4e than 7e, which may explain the somewhat higher pIC50 value of the saturated analogue 4e. However, according to the calculation, unsaturated 7g should be a stronger scavenger than the saturated 4g, which was not in agreement with the measured pIC50 values during 5 min and 10 min time periods of incubation.

Antiproliferative Activities in Vitro
The C-6 substituted 1,2,3-triazole derivatives of L-ASA within the C5-OH group (4b−4q, 4i, 4k−4t, and 4v) and with the C4=C5 double bond (7c−7g and 7i−7q) were evaluated against seven malignant tumor cell lines: lung adenocarcinoma (A549), ductal pancreatic adenocarcinoma (CFPAC-1), colorectal carcinoma (HCT-116), cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2), breast adenocarcinoma (MCF-7), colorectal adenocarcinoma, and metastatic (SW620); as well as normal cell lines: lung fibroblasts (WI-38) and foreskin fibroblasts (HFF-1) ( Table 3, Table S1, Supplementary Information). L-ASA was used as a positive control, and carboxyamidotriazole, a known antiproliferative agent that inhibits tumor cell growth, invasion, and metastasis, and 5-fluorouracil (5-FU), were used as reference compounds.   50 values and three significant in silico radical scavenging parameters ( Table 2). For saturated/unsaturated derivatives, the more favorable parameter values for C3-O − /C2-O − anions have been used. The first two principal components explain 87.5% variance in the data set. Coloring is according to the active OH group in the 1st radical scavenging step. More active compounds have the higher pIC 50 values and are more on the right side of the plot.
It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.  It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a  It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a 25 It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a It can be observed that the majority of C-4 substituted 1,2,3-triazolyl-6-deoxy-L-ASA derivatives were not cytotoxic. While compound 4e showed a selective antiproliferative effect on MCF-7 and A549 cells and 4k on MCF-7 cells, compounds 4d, 4o−4q, and 4v showed an only minor antiproliferative effect at the highest tested concentrations (Table 1). However, except for 4e, 4q and 4v, other compounds exhibited cytotoxicity on foreskin fibroblasts HFF-1 as well. The compounds showed themselves to be more active than L-ASA, which showed no effects on the cell growth of the cells tested. Carboxyamidotriazole, that contains a 1,2,3-triazole ring like the other tested compounds, showed a non-selective, moderate antiproliferative effect on all evaluated cell lines.
The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a The unsubstituted 1,2,3-triazolyl derivative 4b was not cytotoxic toward any of the tested cell lines, indicating that C-4 substitution at 1,2,3-triazole moiety has an impact on growth-inhibition. Among the aryl-substituted triazole derivatives, the fluoro and bromo-substituted aromatic moieties in 4d and 4e, respectively, had influences on the antiproliferative effect. While 4d exhibited a nonspecific cytostatic effect, 4e displayed a strong and selective antiproliferative effect on breast adenocarcinoma MCF-7 (IC 50 = 6.72 µM) and moderate activity on lung adenocarcinoma A549 cells (IC 50 = 25.44 µM). Additionally, compound 4e did not exhibit cytotoxicity against foreskin fibroblasts HFF-1 (IC 50 > 100 µM) and had some cytotoxicity on fibroblasts WI-38 (IC 50 = 73.93 µM; selectivity index, SI = 11). When comparing the activities of compounds 4e, 4f, and 4g it can be concluded that the nature of the substituent in the p-position of the phenyl has an influence on the antiproliferative effect. While electron-withdrawing bromine in the structure of 4e increased the activity, compounds 4f and 4g with electron-donating groups on the 4-aryl-1,2,3-triazole units, did not show any cytotoxic effects. The only exception to the observed impact of electron-donating groups on the inhibition effect was p-pentylphenyl-1,2,3-triazolyl-6-deoxy-L-ASA derivative 4k that exerted a selective cytotoxic effect on MCF-7 cells (IC 50 = 26.91 µM), albeit, it showed cytotoxicity on foreskin fibroblasts (IC 50 = 0.21 µM). From the 4-alkyl substituted triazole derivatives, 4-tert-butyl in 4o, 3-chloropropyl in 4p, and 2-hydroxyethyl chains in 4g caused moderate to marginal cytostatic effects on cervical carcinoma HeLa, colorectal carcinoma HCT-116, and breast adenocarcinoma MCF-7 cells. Compounds 4r−4t with benzenesulphonamide substituents, did not show any cytotoxicity toward evaluated tumor cell lines. The dithiocarbamate derivative 4v showed a selective but moderate antiproliferative effect toward MCF-7 cells (IC 50 = 37.33 µM). Referring to the lipophilicity of the newly synthesized compounds, their clogP values are within the range of −3.00-2.67 (Table 3 and Table S1, Supplementary Information). Compounds 4e and 4k with the best inhibitory activity and clogP values of −0.42 and 0.98, respectively, showed themselves to be more lipophilic than L-ASA (clogP = −2.46).
The C-6 substituted 1,2,3-triazole 4,5-unsaturated L-ASA derivatives (7c−7g and 7i−7q) showed no antiproliferative effect on the tested cell panel at concentrations lower than 100 µM (Table S1, Supplementary Information), which implies that the linker connecting the lactone ring and the 1,2,3-triazole moiety has an influence on the antitumor activity of tested compounds, which is in accordance with the previously shown antitumor effects of 2,3-O,O-dibenzylated L-ASA derivatives [40].

The Detection of Apoptosis and the Validation of HIF-1α and NOS2 Protein Targets for 4e
In order to examine whether the antitumor effect of p-bromophenyl-substituted L-ASA derivative 4e, which showed a selective antiproliferative effect on MCF-7 tumor cell line, can be connected to the induction of apoptosis or any other kind of cell death, an Annexin V assay was performed following a procedure described previously [45].
The results obtained indicate that compound 4e induced apoptosis in MCF-7, and showed a moderate increase in early apoptotic cell populations of 12.4% and late apoptotic cell populations, 2.0%, after 24 h-treatment at concentration 1 × IC 50 (Table 4). After 48 h the cell death rate in the treated cells was similar to the untreated MCF-7 control cells for the higher tested concentration, while the percentage of cells in early apoptosis was higher by 35.2%, in treated cells at concentration 1 × IC 50 , compared with untreated cells. To further understand the observed antiproliferative and cell death effects on MCF-7 cells by p-bromophenyl 1,2,3-triazole L-ASA conjugate 4e, the analysis of the relative expressions of Hypoxiainducible factor I alpha (HIF-1α) and Nitric oxide synthase 2 (NOS2, iNOS) protein, as potential targets of 4e, was performed by western blot (Figure 4). The results showed that in MCF-7 cells treated with 4e for 48 h, a statistically significant increase of hydroxylated HIF-1α (Pro564) expression was observed. Increased levels of hydroxylated HIF-1α (Pro564) would imply an increased form of HIF-1α for proteasomal degradation. Furthermore, 48 h after incubation of MCF-7 cells with 4e, a decreased expression of NOS2 was observed. molecular mechanisms of the regulation of HIF-1α activity by NO are not yet defined and are dependent on conditions being hypoxic or normoxic, and on the concentration of NO [47][48][49][50][51][52][53]. Additionally, it was reported that vitamin C reduces the NO-induced stabilization of HIF-1α in endothelial cells from umbilical cords (HUVECs) under normoxic conditions [54].
Based on the literature overview, we can conclude that the decreased expression of NOS2 is consistent with the enhanced expression of hydroxylated HIF-1α and the antiproliferative effects of 4e on MCF-7 observed. Relative signal intensities of target proteins were normalized to the alpha-tubulin loading control and are shown in the right panel. Data are presented as mean values ± SDs. Statistically significant (p < 0.5) differences in the expression levels are marked by an asterisk (*).

Cell Metabolism Analysis-Mitochondrial Toxicity
The effects on the mitochondrial respiration after 24 h exposure of MCF-7 and HFF-1 cells to L-ASA and 4e tested at concentrations comparable to the DPPH test (IC50 values and correspondingly higher or lower concentrations) were assessed by use of the Seahorse XF Cell Mito Stress Test kit ( Figure 5). Even though no statistically relevant changes were observed, interesting trends were consistent. The basal respiration and ATP production of MCF-7 and HFF-1 cells after NOS2s are homodimer enzymes that catalyze the formation of nitric oxide radicals (NO) from L-arginine and oxygen. NO is an important signaling molecule and mediates various physiological and pathological processes, including systemic blood pressure, the upregulation of hypoxic genes, the regulation of stress response pathways, host-microbe interactions, immune signaling, and apoptosis [46][47][48]. The majority of reports indicate that NO induces HIF-1α accumulation but the molecular mechanisms of the regulation of HIF-1α activity by NO are not yet defined and are dependent on conditions being hypoxic or normoxic, and on the concentration of NO [47][48][49][50][51][52][53]. Additionally, it was reported that vitamin C reduces the NO-induced stabilization of HIF-1α in endothelial cells from umbilical cords (HUVECs) under normoxic conditions [54].
Based on the literature overview, we can conclude that the decreased expression of NOS2 is consistent with the enhanced expression of hydroxylated HIF-1α and the antiproliferative effects of 4e on MCF-7 observed.

Cell Metabolism Analysis-Mitochondrial Toxicity
The effects on the mitochondrial respiration after 24 h exposure of MCF-7 and HFF-1 cells to L-ASA and 4e tested at concentrations comparable to the DPPH test (IC 50 values and correspondingly higher or lower concentrations) were assessed by use of the Seahorse XF Cell Mito Stress Test kit ( Figure 5). Even though no statistically relevant changes were observed, interesting trends were consistent. The basal respiration and ATP production of MCF-7 and HFF-1 cells after sequential injection of the standard assay compounds, as described in the Material and methods section, was affected by higher concentrations of L-ASA and compound 4e that both induced a decrease in the cellular-metabolism rate. A lower L-ASA concentration (50 mM) did not act on the mitochondrial activity (dashed lines in Figure 5, panel A). In MCF-7 tumor cells, it was clearly visible that higher L-ASA concentrations decreased the basal cell respiration and ATP production in the way that the whole cell metabolism switched towards non-mitochondrial respiration while this effect was not observed in the normal HFF-1 fibroblasts. Compound 4e increased the basal respiration and ATP production of MCF-7 cells at its IC 50 concentration (6.72 µM) and 2 × IC 50 concentration (13.44 µM), while it lowered cellular mitochondrial metabolism, switching it towards protein leaking at the lower tested concentration (3.36 µM). Compound 4e did not affect the basal cell respiration or ATP production of HFF-1 cells which correlates with MTT-results where 4e showed no cytotoxicity on this cell line at micromolar concentrations (Table 3; IC 50 > 100 µM). Interestingly, 4e showed a slightly positive effect on the cellular metabolism and mitochondrial activity of HFF-1 at a concentration of 100 µM ( Figure 5, panel B). The observed differences in compound 4e activity on MCF-7 tumor cells in comparison with normal fibroblasts HFF-1 may be due to the specific metabolic status of tumor cells, in which case an antioxidant compound with ROS scavenging properties, such as L-ASA, may have a cytotoxic effect; i.e., through a pro-oxidant effect on cancer cells associated with increase of ROS in tumor cells [55]. Particularly in MCF-7 cells, a small amount of an important cellular antioxidant enzyme manganese-dependent superoxide dismutase (MnSOD) was found in comparison with other cell lines [56]. It is therefore, possible that the compound 4e exerts a pro-oxidant activity in MCF-7 cells, which elevates the levels of ROS and induces cell death. This hypothesis should be further studied, however, in more experimental detail. mitochondrial activity (dashed lines in Figure 5, panel A). In MCF-7 tumor cells, it was clearly visible that higher L-ASA concentrations decreased the basal cell respiration and ATP production in the way that the whole cell metabolism switched towards non-mitochondrial respiration while this effect was not observed in the normal HFF-1 fibroblasts. Compound 4e increased the basal respiration and ATP production of MCF-7 cells at its IC50 concentration (6.72 μM) and 2 × IC50 concentration (13.44 μM), while it lowered cellular mitochondrial metabolism, switching it towards protein leaking at the lower tested concentration (3.36 μM). Compound 4e did not affect the basal cell respiration or ATP production of HFF-1 cells which correlates with MTT-results where 4e showed no cytotoxicity on this cell line at micromolar concentrations (Table 3; IC50 > 100 μM). Interestingly, 4e showed a slightly positive effect on the cellular metabolism and mitochondrial activity of HFF-1 at a concentration of 100 μM ( Figure 5, panel B). The observed differences in compound 4e activity on MCF-7 tumor cells in comparison with normal fibroblasts HFF-1 may be due to the specific metabolic status of tumor cells, in which case an antioxidant compound with ROS scavenging properties, such as L-ASA, may have a cytotoxic effect; i.e., through a pro-oxidant effect on cancer cells associated with increase of ROS in tumor cells [55]. Particularly in MCF-7 cells, a small amount of an important cellular antioxidant enzyme manganese-dependent superoxide dismutase (MnSOD) was found in comparison with other cell lines [56]. It is therefore, possible that the compound 4e exerts a pro-oxidant activity in MCF-7 cells, which elevates the levels of ROS and induces cell death. This hypothesis should be further studied, however, in more experimental detail.

General
The melting points of novel compounds were determined using a Kofler micro hot-stage (Reichert, Wien, Austria) apparatus. The progress of all reactions was monitored by thin-layer chromatography (TLC) on silica gel 60F-254 plates (Merck, Darmstadt, Germany) and the spots were observed under UV light (254 nm). Purification of compounds using column chromatography was carried out with silica gel (0.063-0.2 mm) (Fluka, Buchs, Switzerland). All 1 H and 13 C NMR spectra were recorded in DMSO-d6 at 298 K on a Bruker 300 or 600 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany). Chemical shifts were referenced to the signal of DMSO at δ: 2.50 ppm ( 1 H NMR) and δ: 39.50 ppm ( 13 C NMR). High-resolution mass spectra (HRMS) of the final compounds

General
The melting points of novel compounds were determined using a Kofler micro hot-stage (Reichert, Wien, Austria) apparatus. The progress of all reactions was monitored by thin-layer chromatography