The antioxidant and antiproliferative activities of 1,2,3-triazolyl-L-ascorbic acid derivatives

International Journal of

Molecular Sciences

Article

The Antioxidant and Antiproliferative Activities of

,2,3-Triazolyl-L-Ascorbic Acid Derivatives

1

2,

3

, Marko Klobu cˇ ar ¹,

Anja Harej , Andrijana Meš cˇ i c´ Macan * , Višnja Stepani c´

4

1,

and Silvana Rai c´ -Mali c´²

Krešimir Paveli c´ , Sandra Kraljevi c´ Paveli c´

*

1

Centre for High-throughput Technologies, Department of Biotechnology, University of Rijeka, Radmile

Matej cˇ i c´ 2, 51000 Rijeka, Croatia; aharej@biotech.uniri.hr (A.H.); mklobucar@biotech.uniri.hr (M.K.)

Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb,

Maruli c´ ev trg 20, 10000 Zagreb, Croatia; sraic@fkit.hr

Division of Electronics, Ruđer Boškovi c´ Institute, Bijeni cˇ ka cesta 54, 10000 Zagreb, Croatia;

Visnja.Stepanic@irb.hr

2

3

4

Faculty of medicine, Juraj Dobrila University of Pula, Zagreba cˇ ka 30, 52100 Pula, Croatia;

kresimir.pavelic@unipu.hr

*

Correspondence: amescic@fkit.hr (A.M.M.); sandrakp@biotech.uniri.hr (S.K.P.);

Tel.: +385-1-4597-243 (A.M.M.); +385-1-5584-569 (S.K.P.)

Received: 30 August 2019; Accepted: 19 September 2019; Published: 24 September 2019

Abstract: 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

4k: IC₅₀= 26.91 M) had a selective cytotoxic eﬀect on breast adenocarcinoma MCF-7 cells. Moreover,

compound 4e did not inhibit the growth of foreskin ﬁbroblasts (IC₅₀> 100 M). In MCF-7 cells treated

with 4e, a signiﬁcant increase of hydroxylated hypoxia-inducible transcription factor 1 alpha (HIF-1

expression and decreased expression of nitric oxide synthase 2 (NOS2) were observed, suggesting

µM) and p-pentylphenyl-substituted 1,2,3-triazole L-ASA conjugate

(

µ

α

)

the involvement of 4e in the HIF-1α signaling pathway for its strong growth-inhibition eﬀect on

MCF-7 cells.

Keywords: vitamin C; 1,2,3-triazole; antioxidant; quantum-chemical modelling; antitumor; HIF-1

1

. 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 [1–4]. While ROS have an important biological

Int. J. Mol. Sci. 2019, 20, 4735; doi:10.3390 /ijms20194735

www.mdpi.com /journal /ijms

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role in cell signaling, homeostasis and defense against micro-organisms, an imbalance between ROS

production and ROS removal through the cell antioxidant protection system, comprised of endogenous

antioxidants, enzymes, dietary antioxidants, and metal-binding proteins, can induce oxidative stress

and various pathological conditions [2,5]. Free radicals may damage cellular components as they

react with membrane lipids, nucleic acids, proteins, carbohydrates, and other small molecules [6,7

].

Increasing evidence indeed, suggests that many chronic diseases, such as cancer, cardiovascular diseases,

inflammation, neurodegenerative diseases, aging, diabetes, and atherosclerosis, are associated with

high levels of ROS [8,9].

Vitamin C (L-ascorbic acid, L-ASA) or more accurately the ascorbate anion (A, Figure 2), is an

excellent reducing agent and a powerful antioxidant co-factor required for key enzyme reactions [10

–14].

Due to the low one electron reduction potential of the ascorbate radical (RA)/ascorbate (A) couple

(

Figure 2), almost every oxidizing radical formed in the biological system, including the hydroxyl

HO ), alkoxyl, peroxyl, thiol, and tocopheroxyl radicals cause one-electron oxidation of the ascorbate

•

anion, resulting in the formation of the resonance stabilized ascorbate radical which can be recycled

back to ascorbate [15 16]. Furthermore, structural modiﬁcations of hydroxyl groups of L-ASA by the

introduction of lipophilic moieties have led to L-ASA derivatives with improved radical scavenging

ability compared to L-ASA, especially in lipophilic parts of biological system [17 22]. While L-ASA

,

–

has an antioxidant property and protects the cell from damage caused by free radicals, when used

in pharmacological (millimolar) concentrations achieved through intravenous administration L-ASA

shows pro-oxidant activity [13,23–25]. The pro-oxidant activity can be explained by the ability of

3

+

2+

ascorbate to reduce catalytic metals, for instance, Fe to Fe , which than leads to the generation of the

•

superoxide anion, hydrogen peroxide and HO by Fenton type reactions [26]. The higher concentration

of ROS in this case, may lead to a cancer cell’s death.

Recent ﬁndings showed that high levels of vitamin C selectively kill colorectal cancer cells with

KRAS and BRAF mutations by increasing ROS production, which blocks glucose metabolism and

subsequently causes the energy crisis and cell death [27,28]. Additionally, high-doses of vitamin C

showed a cytotoxic eﬀect on breast adenocarcinoma (MCF7) and colon cancer (HT29) cells by inducing

metabolic changes, which caused insuﬃcient ATP formation and cell death [29]. Additionally, high

concentrations of vitamin C showed cellular toxicity on proliferating neural stem/progenitor cells [30].

Furthermore, some lipophilic ASA derivatives with modiﬁed hydroxyl groups, as well as conjugates

of ASA with pyrimidine, purine, triazole and imidazole bases, exhibited antitumor and antiviral

activity [14].

Additionally, itwasfoundthatascorbatedecreasestheactivityofthehypoxia-inducibletranscription

factor 1 (HIF-1) [31

cancers, and upregulate the expression of genes involved in angiogenesis, glucose uptake, anaerobic

metabolism, and cell proliferation, making HIF an important target for cancer therapy [13 35 37].

Under normoxic conditions, the HIF-1 subunit is continually synthesized and then degraded in

the proteasome which requires HIF-hydroxylase enzymes to hydroxylate its proline and asparagine

–34]. As a response to hypoxic stress, HIF-1 and HIF-2 are overexpressed in human

,

–

α

residues. It is presumed that ascorbate decreases the HIF-1α protein levels by acting as a co-factor for

iron-dependent HIF-hydroxylase enzymes [38,39].

Encouraged by the observed antitumor eﬀect of vitamin C and its derivatives, in our previous

work, we designed and synthesized 2,3-dibenzylated L-ASA derivatives and their C4=C5 unsaturated

analogues with a 1,4-disubstituted 1,2,3-triazole core on the C-6 atom of the lactone ring that exhibited

a pronounced and selective antitumoral and antiviral activity [40]. In continuation of our work and

with the aim to enhance the antioxidant activity of L-ASA, we have prepared a library of 1,2,3-triazole

L-ASA conjugates (Scheme 1) with free C-2 and C-3 hydroxyl groups, hypothesizing that diverse

substituted triazole moiety and hydroxyethylene and ethylidene linkers will have an impact on

free-radical-scavenging and antitumor properties.

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Scheme 1. Reagents and conditions: (i) Cu, 1 M copper(II) sulphate (CuSO ), tert-butanol, DMF, water,

4

and a microwave reactor; (ii) 1 M boron trichloride (BCl ) and dry dichloromethane; (iii) copper(II)

3

acetate (Cu(OAc) ) and methanol.

2

. Results and Discussion

2

.1. Synthesis

The target 1,2,3-triazole appended L-ASA derivatives were synthesized following Scheme 1.

The 1,2,3-triazole ring was introduced at the C-6 position of L-ascorbic acid via copper(I)-catalyzed

azide-alkyne (3 + 2) dipolar cycloaddition (CuAAC) [41] of the corresponding C-6 azido L-ascorbic acid

derivatives

3a 3h 3j 3u). Then, 6-(1,2,3-triazolyl)-4,5-didehydro-5,6-dideoxy-L-ASA (6c

were obtained [40].

The removal of the benzyl group was achieved via reaction of the 1,2,3-triazole L-ascorbic acid

derivatives 3a 3h 3j 3u 6c 6h, and 6j 6q with boron trichloride (BCl ) in dry dichloromethane,

1

and

5

with versatile terminal alkynes (2a

,

2c–

2h

,

2j

–

2u) and 6-(1,2,3-triazolyl)-6-deoxy

(

–

,

–

6h 6j 6g) derivatives

,

–

,

–

,

–

3

aﬀording compounds 4b–4q, 4i, 4k–4t, 4v, 7c–7g, and 7i–7q with yields from 42% to 86%.

•

2

.2. DPPH Radical Scavenging Activity in Vitro

Likewise, L-ASA, the 1,2,3-triazole L-ASA derivatives with free C2-OH and C3-OH groups were

expected to be potent antioxidants. Therefore, they were tested as radical scavengers by reaction

•

with the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH ). The scavenging activity data are

expressed as IC₅₀values and are summarized in Table 1.

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Table 1. The scavenging activity of L-ascorbic acid derivatives assessed by DPPH test. Results are

•

presented as IC₅₀values (mM) of DPPH inhibition after 5 and 10 min treatments.

Comp.

R

IC50 (mM) ^a

Treatm. Treatm.

min 10 min

Comp.

R

IC50 (mM) ^a

Treatm.

5 min

Treatm.

10 min

5

L-ASA

0.18 ± 0.06 0.17 ± 0.05

0.13 ± 0.05 0.13 ± 0.05

4t

0.22 ± 0.08 0.12 ± 0.02

4

b

H

4v

0.27 ± 0.01

0.27

0.28

4

c

0.26 ± 0.02

0.27 ± 0.01

0.25

0.27

7c

0.28

4

d

7d

0.23

0.28

0.23

0.28

4

e

0.20 ± 0.01 0.19 ± 0.01

0.20 ± 0.02 0.14 ± 0.01

0.19 ± 0.01 0.19 ± 0.01

7e

7f

4

f

0.10 ± 0.02 0.10 ± 0.02

> 0.28 0.26

4

g

7g

4

i

>0.28

7i

7j

0.10 ± 0.05 0.10 ± 0.04

4

k

0.21 ± 0.07 0.13 ± 0.04

0.17 ± 0.16 0.16 ± 0.14

0.06

4

l

7k

7l

0.06

0.20 ± 0.01

0.20

0.06

0.20

0.19

4

m

>0.28

4

n

0.15 ± 0.06 0.10 ± 0.03

.24 ±

7m

0

4

o

0.24 ± 0.04

7n

0.14

0.10

.028

4

p

q

0.15 ± 0.06 0.15 ± 0.06

7o

7p

7q

0.20

0.07

0.20

0.07

4

0.24 ± 0.02

0.23

4

r

0.25

0.27 ± 0.02

4

s

0.27 ± 0.02

0.25

a

•

IC50 —concentration of compound required to scavenge 50% of DPPH free radicals.

L-ASA was used as control and its experimentally assessed IC₅₀was 0.18 mM for the 5 min

treatment. The scavenging activity data showed that conjugates having a hydroxyethylene spacer

with a 4-substituted 1,2,3-triazole moiety at the C-6 position of L-ASA (4b−4v), in general, exhibited

activity similar to that of L-ASA. Compounds 4b, 4f, 4k, 4l, 4n, 4p, and 4t from the 6-(1,2,3-triazolyl)-

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6

-deoxy-L-ASA and 7f

,

7i−7k

,

7n, and 7q from the 6-(1,2,3-triazolyl)-4,5-didehydro-5,6-dideoxy-L-ASA

series had higher antioxidant potencies in comparison with L-ASA. When a halogen (4c, 4d, or 4e)

and methoxy-substituted (4g) aryl moiety was introduced in the C-4 of 1,2,3-triazole, the antioxidant

activity was slightly lower (IC₅₀= 0.19–0.27 mM) in comparison with L-ASA and the unsubstituted

1

,2,3-triazole L-ASA derivative 4b (IC₅₀= 0.13 mM at 5 and 10 min treatments). The introduction of

an alkyl in the para position of phenyl in compounds 4f and 4k, improved the antioxidant activity

after the 10 min treatment. Among 4-alkyl-substituted triazoles, cyclopropyl in 4l, decyl in 4n, and

3

-chloropropyl substituents in 4p also improved the antioxidant activity compared to L-ASA. L-ASA

derivatives containing the benzenesulphonamide and dithiocarbamate moiety showed lower radical

scavenging activity after the 5 min treatment then L-ASA and the unsubstituted triazole 4b, but

benzenesulphonamide-substituted 1,2,3-triazole L-ASA derivative 4t had a better antioxidant activity

after the 10 min treatment then L-ASA and 4b, with an IC₅₀value of 0.12 mM.

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

activities. Besides, the electron-donating p-methyl groups in 4f

in 4k 7k, and the long aliphatic chains in 4n 7n improved the antioxidant activity compared to L-ASA

as well.

,

7j, and 7q improved the radical scavenging

/

7f, the p-pentyl-substituted aryl groups

/

Figure 1. Summarized representation of the importance of some structural features in antioxidant/radical-

scavenging activity. The arrows point to structure-activity observations.

2

.3. In Silico Analysis of Radical Scavenging Capacities

According to the pIC₅₀values, calculated as the negative logarithm of IC₅₀values (Table 1)—which

are within the range of 3.55

−

4.22 and have the pIC₅₀medians 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 pIC₅₀value 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 IC₅₀values 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

pK_a_,₁of 4.1 and pK_a_,₂of 11.4) [43].

a

) are

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

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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

NR

neutral radical formed by SPLET, immediately releases a proton (pK

a

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

A

in terms of the experimental pIC₅₀values and the calculated aqueous parameters ETFE , BDFE ,

RA

and ETFE 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).

Table 2. Calculated aqueous reaction free energy parameters (kcal/mol): acid dissociation constant

NR

of neutral specie (pK

a

) and its neutral radical (pK

a

), electron transfer free energies, and bond

A

dissociation free energy for an anion (ETFE and BDFE ), and the ETFE of an intermediate radical

anion (ETFE ) (Figure 2) for C2-OH and C3-OH groups of selected compound pairs (saturated

RA

C4-C5(OH) analogues from series 4 and unsaturated C4=C5 analogues from series 7).

ETFE^A

pKa^N^R

BDFE^A

ETFE^R^A

Comp.

L-ASA

Active OH

pKa

2-OH

10.3

3.6

7.8

72.6

79.6

73.9

81.3

−6.3

−4.4

−5.6

57.3

66.9

61.3

66.9

3

-OH

2-OH

-OH

76.6

77.9

L-ASA_C4=C5 ^a

3

3.6

4

7

b

2-OH

-OH

2-OH

-OH

10.4

3.7

7.4

3.5

72.7

79.3

75.1

82.0

−6.7

−5.0

−5.8

56.9

66.6

61.6

67.0

3

76.9

79.0

3

4

7

e

2-OH

-OH

2-OH

-OH

10.4

3.5

9.5

5.1

71.0

82.0

72.7

80.6

−5.3

−4.1

−4.2

−5.4

60.4

67.9

60.4

66.5

3

77.3

79.7

3

4

g

2-OH

-OH

2-OH

-OH

10.2

3.4

7.6

4.3

79.8

79.2

74.7

80.3

−11.0

−3.8

−4.6

−5.4

58.2

67.3

61.8

66.3

3

76.5

78.3

7

3

4

7

i

2-OH

-OH

2-OH

-OH

10.4

3.3

7.4

4.0

72.5

79.8

76.0

80.5

−6.1

−4.2

−5.1

−5.0

57.6

67.3

62.3

66.9

3

77.1

78.1

3

4

7

q

2-OH

-OH

2-OH

-OH

10.7

2.4

7.3

3.5

a

72.8

82.3

74.5

80.8

−6.5

−5.1

−4.2

−5.1

60.4

68.6

62.0

67.1

3

79.0

78.0

3

Not synthetized.

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Figure 2. Radical scavenging mechanisms and associated calculated aqueous free energies (bond

dissociation free energy (BDFE) and electron transfer free energy (ETFE) in kcal/mol) and negative

logarithm of acid dissociation constant for the most acidic OH group, pKa,1, Table 2), of L-ascorbic acid

(L-ASA) at physiological pH. The ascorbate anion deprotonated at the O-3 position may neutralize a free

radical by the donation of an H-atom (green pathway) or an electron (cyan pathway). The intermediate

radical anion transforms to the ﬁnal product, dehydroascorbic acid (DHA), by further giving an electron.

The synthesized L-ASA derivatives may follow the same radical scavenging mechanisms.

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-O anion is generally characterized by

A

–

more favorable, lower ETFE and BDFE values than the C3-O anion 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).

A

RA

By comparing ETFE , BDFE , and ETFE 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

•

A

DPPH radical scavenging, having a higher pIC₅₀value (Tables 1 and 2). ETFE and BDFE are also

A

more favorable for the stronger scavenger 7i in the 4i

/

7i pair. While BDFE 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 pIC₅₀value 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 pIC₅₀values during 5 min and 10 min time periods

of incubation.

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Figure 3. Principal component analysis (PCA) biplot for derivatives containing C4C5(OH) (series 4)

or C4=C5 (series

7) fragments obtained by the PCA analysis performed in terms of the in vitro pIC₅₀

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 pIC₅₀values and

are more on the right side of the plot.

2

.4. Biological Evaluations

2

.4.1. Antiproliferative Activities in Vitro

The C-6 substituted 1,2,3-triazole derivatives of L-ASA within the C5-OH group (4b−4q

, 4i,

4

k−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.

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

eﬀect at the highest tested concentrations (Table 1). However, except for 4e

,

4o−4q, and 4v showed an only minor antiproliferative

,

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.

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Table 3. The IC₅₀values (µM) for compounds 4b–4g, 4i, 4k–4t, and 4v for tested cell lines.

IC50 ^a(µM)

HeLa HepG2 MCF-7 SW620 WI-38 HFF-1

clogP ^b

Comp.

R

A549

CFPAC-1 HCT-116

4b

H

>100

NA

−2.89

4c

>100

0.56

4d

81.65

75.82

64.79

56.76

95.34

39.81

92.04

>100

0.12

−0.94

4

e

Br

25.44

>100

6.72

>100

73.93

>100

69.32

>100

−0.42

−0.80

−1.21

4

f

4g

4

i

>100

0.92

0.21

−1.49

4k

26.91

0.98

4

l

>100

4.61

0.12

−1.73

−1.17

4m

4n

>100

0.06

1.56

4o

>100

93.04

>100

94.95

<0.01

−1.26

4

p

>100

63.34

48.01

>100

99.59

>100

52.65

>100

6.05

>100

−1.39

−3.00

4q

4

r

>100

−1.95

4s

0.40

−1.69

4t

>100

−1.59

4

v

97.98

>100

37.33

>100

NA

−2.40

−2.46

L-ASA

Carboxyamidotriazole

-FU

>100

51.61

2.80

>100

41.55

0.14

>100

19.88

/

>100

31.84

8.81

>100

40.09

9.04

>100

14.69

0.096

>100

41.33

0.08

NA

/

0.94

>100

32.61

/

5

a

Concentration of compounds required to inhibit human tumor cell lines and normal cell line growth by 50%;

values of n-octanol/water partition coeﬃcients; clogP, were calculated by DataWarrior [44].

b

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

4

d

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 (IC₅₀= 6.72 M) and moderate activity on lung adenocarcinoma A549 cells (IC₅₀= 25.44 M).

Additionally, compound 4e did not exhibit cytotoxicity against foreskin ﬁbroblasts HFF-1 (IC₅₀> 100

M) and had some cytotoxicity on ﬁbroblasts WI-38 (IC₅₀= 73.93 M; selectivity index, SI = 11).

When comparing the activities of compounds 4e 4f, and 4g it can be concluded that the nature of

µ

,

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

Int. J. Mol. Sci. 2019, 20, 4735

10 of 26

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 (IC₅₀= 26.91 µM), albeit, it showed cytotoxicity on foreskin ﬁbroblasts (IC₅₀= 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 (IC₅₀= 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

showed themselves to be more lipophilic than L-ASA (clogP = −2.46).

−

0.42 and 0.98, respectively,

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

Supplementary Information), which implies that the linker connecting the lactone ring and the

,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].

µM (Table S1,

1

2

.4.2. 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

4

e, 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

cells was similar to the untreated MCF-7 control cells for the higher tested concentration, while the

×

IC₅₀(Table 4). After 48 h the cell death rate in the treated

percentage of cells in early apoptosis was higher by 35.2%, in treated cells at concentration 1

compared with untreated cells.

×

IC ,

50

Table 4. Results of the Annexin V assay ^a.

Duration of

Treatment

Comp.

Concentration

Early

Apoptosis (%)

Late Apoptosis Necrosis

Cell Line

(%)

Control

4e (6.72 µM)

e (13.44 µM)

18.2

30.6

5.3

4.4

6.4

38.1

0

0.8

15.9

2

4 h

8 h

4

MCF-7

Control

4e (6.72 µM)

e (13.44 µM)

11.4

46.6

9.1

10.6

4.2

2.3

0

0.8

0

4

a

Results are presented as percentages of early apoptotic cells, late apoptotic/primary necrotic cells, and necrotic

cells after 24 h and 48 h treatment. Cells were treated with 4e at 6.72

Control—untreated MCF-7 cells.

µM (1

×

IC50) and at 13.44

µM (2

×

IC50).

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 Hypoxia-

inducible factor I alpha (HIF-1

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-1 (Pro564) expression was

observed. Increased levels of hydroxylated HIF-1 (Pro564) would imply an increased form of HIF-1

α) and Nitric oxide synthase 2 (NOS2, iNOS) protein, as potential

α

for proteasomal degradation. Furthermore, 48 h after incubation of MCF-7 cells with 4e, a decreased

expression of NOS2 was observed.

Int. J. Mol. Sci. 2019, 20, 4735

11 of 26

Figure 4. Western blot analysis of predicted protein targets of compounds 4e. Representative western

blots are shown on the left panel, where cellular levels of selected proteins before and after treatment of

MCF-7 cells with compound 4e, at IC50 = 6.72

untreated) cells. Approximate molecular weights (kDa) of target proteins are presented. 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 (*).

µM for 24 and 48 h, can be observed. C denotes control

(

±

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 [46–48]. The majority of reports indicate that NO induces HIF-1

molecular mechanisms of the regulation of HIF-1 activity by NO are not yet deﬁned and are dependent

on conditions being hypoxic or normoxic, and on the concentration of NO [47 53]. Additionally, it

in endothelial cells from

α accumulation but the

α

–

was reported that vitamin C reduces the NO-induced stabilization of HIF-1

α

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-1

on MCF-7 observed.

α and the antiproliferative eﬀects of 4e

2

.4.3. 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 (IC₅₀values 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 IC₅₀concentration

(

6.72

µ

M) and 2

×

IC₅₀concentration (13.44

µ

M), while it lowered cellular mitochondrial metabolism,

switching it towards protein leaking at the lower tested concentration (3.36

µM). Compound 4e did not

Int. J. Mol. Sci. 2019, 20, 4735

12 of 26

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 M).

Interestingly, 4e showed a slightly positive effect on the cellular metabolism and mitochondrial activity

of HFF-1 at a concentration of 100 M (Figure 5, panel B). The observed differences in compound

activity on MCF-7 tumor cells in comparison with normal fibroblasts HFF-1 may be due to the

µ

4e

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.

Panel A

Figure 5. Cont.

Int. J. Mol. Sci. 2019, 20, 4735

13 of 26

Panel B

Figure 5. Results of the cell metabolism analysis for cells MCF-7 and HFF1 treated for 6 and 24 h

with compound 4e having a p-bromophenyl substituent at C-4 of triazole. *The zero (0) on the X axis

denotes the starting point of measurment upon addition of oligomycine, mitochondrial oxidative

phosphorylation uncoupler carbonyl cyanide-4-(triﬂuoromethoxy)phenylhydrazone (FCCP), and

rotenone/antimycin A, respectively (marked with arrows), on cells incubated with L-ASA or 4e for 24 h.

Panel A—cells treated with L-ASA; Panel B—cells treated with compound 4e.

3

. Materials and Methods

.1. Chemistry

.1.1. 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

Int. J. Mol. Sci. 2019, 20, 4735

14 of 26

(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

1

13

silica gel (0.063–0.2 mm) (Fluka, Buchs, Switzerland). All H and C NMR spectra were recorded

in DMSO-d at 298 K on a Bruker 300 or 600 MHz NMR spectrometer (Bruker Biospin, Rheinstetten,

6

1

Germany). Chemical shifts were referenced to the signal of DMSO at

δ

: 2.50 ppm ( H NMR) and

δ:

1

3

9.50 ppm ( C NMR). High-resolution mass spectra (HRMS) of the ﬁnal compounds were recorded

on a 4800 Plus MALDI TOF/TOF mass spectrometar (Applied Biosystems, Foster City, CA, USA).

Acquisitions were performed in positive ion reﬂectron mode.

3

.1.2. Preparation of Compounds

General procedure for the removal of the benzyl groups: 1,2,3-triazole-L-ascorbic acid derivatives

3

a−3h

,

3j−3u 6c−6h, and 6j−6q were dried using a vacuum pump. Dry dichloromethane (around

,

20 mL) was added to the compound under an argon atmosphere. The reaction mixture was cooled to

◦

−

65 C and 1 M boron trichloride solution (BCl , 6 equivalents) was added dropwise to the reaction

3

◦

mixture. The reaction mixture was stirred for 3 h at

−

65 C and was left in the freezer overnight.

A mixture of dichloromethane and methanol (10 mL) was then added and the reaction mixture was

stirred for 30 min at room temperature. The solvent was evaporated and Amberlite IRA 743 was added

to neutralize the reaction mixture. The reaction mixture was ﬁltered and the solvent was evaporated.

The compounds were puriﬁed using a very short column ﬁlled with silica (CH CH :MeOH = 100:1; 50:1;

2

10:1). After trituration of the obtained product with n-hexane, diethyl ether, dichloromethane, acetone,

and methanol, compounds 4b–4q, 4i, 4k–4t, 4v, 7c−7g, and 7i−7q were isolated as white powders.

The preparation of 6-deoxy-6-(1,2,3-triazol-1-yl)-L-ascorbic acid (4b): Compound 4b (61.8 mg; 57%; m.p. =

◦

2

19–221 C) was synthesized according to the general procedure using compound 3a (194.9 mg; 0.478

1

mmol); 1 M BCl (2.9 mL) and CH Cl (20 mL). H NMR (600 MHz, DMSO)

δ

: 11.12 (bs, 1H, OH), 8.43

3

2

(

bs, 1H, OH), 8.10 (s, 1H, H-8), 7.72 (s, 1H, H-7), 5.45 (bs, 1H, OH), 4.71 (d, J = 1.4 Hz, 1H, H-4), 4.65

dd, J = 13.8 Hz, 3.9 Hz, 1H, H-6), 4.41 (dd, J = 13.8 Hz, 9.3 Hz, 1H, H-6), 4.19 (d, J = 6.4 Hz, 1H, H-5)

(

ppm. ¹³C NMR (151 MHz, DMSO)

δ

: 170.3 (C-1), 152.0 (C-3), 133.0 (C-8), 125.8 (C-7), 118.3 (C-2), 75.6

+

(

C-4), 67.2 (C-5), 52.4 (C-6) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H] = 228.0620;

8

9

3

5

found = 228.0626.

Preparation of 6-deoxy-6-[4-(3,5-bis(trifluoromethyl)phenyl)-1,2,3-triazol-1-yl]-L-ascorbic acid (4c): Compound

◦

4

3

c

(119.0 mg; 86%; m.p.

≥

250 C) was synthesized according to the general procedure using compound

1

(194.5 mg; 0.314 mmol); 1 M BCl (1.9 mL) and CH Cl (20 mL). H NMR (600 MHz, DMSO)

δ:

3

2

11.18 (s, 1H, OH), 8.99 (s, 1H, H-7), 8.53 (s, 2H, Ph-2, Ph-6), 8.46 (s, 1H, OH), 8.07 (s, 1H, Ph-4), 5.60

(

d, J = 6.9 Hz, 1H, OH), 4.79 (d, J = 1.6 Hz, 1 H, H-4), 4.74 (dd, J = 13.8 Hz, 4.0 Hz, 1H, H-6), 4.46 (dd,

J = 13.9 Hz, 9.3 Hz, 1H, H-6), 4.29–4.23 (m, 1H, H-5) ppm. ¹³C NMR (151 MHz, DMSO)

δ: 170.2 (C-1),

2

1

51.9 (C-3), 143.4 (C-8), 133.4 (Ph-1), 131.4; 131.1; 130.9; 130.7 (q, J = 32.9 Hz, Ph-3, Ph-5), 126.0; 124.1;

CF

1

22.3; 120.5 (q, J = 272.8 Hz), 125.3 (Ph), 124.5 (Ph), 121.0 (C-7), 118.3 (C-2), 75.4 (C-4), 67.0 (C-5), 53.1

CF

+

(C-6) ppm. HRMS (ESI Q-TOF): calculated for C H F N O [M + H] = 440.0681; found = 440.0671.

16

11

6

3

5

Preparation of 6-deoxy-6-[4-(3,5-diﬂuorophenyl)-1,2,3-triazol-1-yl]-L-ascorbic acid (4d): Compound 4d (44.4

◦

mg; 46%; m.p. = 189–191 C) was synthesized according to the general procedure using compound

1

3

d

(148.6 mg; 0.286 mmol); 1 M BCl (1.7 mL) and CH Cl (20 mL). H NMR (600 MHz, DMSO)

δ:

3

2

1

1.16 (s, 1H, OH), 8.72 (s, 1H, H-7), 8.45 (s, 1H, OH), 7.59–7.56 (m, 2H, Ph-2, Ph-6), 7.35–7.10 (m, 1H,

Ph-4), 5.57 (s, 1H, OH), 4.78 (d, J = 1.5 Hz, 1H, H-4), 4.71 (dd, J = 13.8 Hz, 3.8 Hz, 1H, H-6), 4.42 (dd,

13

J = 13.8 Hz, 9.4 Hz, 1H, H-6), 4.26–4.22 (m, 1H, H-5) ppm. C NMR (151 MHz, DMSO)

δ

: 170.2 (C-1),

1

3

1

63.7; 163.6; 162.1; 162.0 (dd, J = 245.6 Hz, J = 13.7 Hz, Ph-3, Ph-5), 151.9 (C-3), 144.1 (C-8), 134.4;

34.3; 134.3 (t, J = 10.7 Hz, Ph-1), 123.9 (C-7), 118.3 (C-2), 108.3–107.7 (m, Ph-2, Ph-6), 103.1; 103.0;

02.8 (t, J = 25.9 Hz, Ph-4), 75.5 (C-4), 67.0 (C-5), 53.0 (C-6) ppm. HRMS (ESI Q-TOF): calculated for

CF

3

CF

2

CF

+

C H F N O [M + H] = 340.0745; found = 340.0750.

14

11

2

3

5

Int. J. Mol. Sci. 2019, 20, 4735

15 of 26

Preparation of 6-[4-(4-bromophenyl)-1,2,3-triazol-1-yl]-6-deoxy-L-ascorbic acid (4e): Compound 4e (111.7 mg;

◦

6

(

1%; m.p. = 159–160 C) was synthesized according to the general procedure using compound 3e

1

268.5 mg; 0.477 mmol); 1 M BCl (2.86 mL) and CH Cl (20 mL). H NMR (300 MHz, DMSO)

s, 1H, OH), 8.62 (s, 1H, H-7), 8.48 (bs, 1H, OH), 7.81 (d, J = 8.5 Hz, 2H, Ph), 7.65 (d, J = 8.5 Hz, 2H, Ph),

δ

: 11.10

3

2

5

9

.55 (s, 1H, OH), 4.77 (d, J = 1.2 Hz, 1H, H-4), 4.69 (dd, J = 13.7 Hz, 3.7 Hz, 1H H-6), 4.42 (dd, J = 13.7 Hz,

.3 Hz, 1H, H-6), 4.30–4.18 (m, 1H, H-5). C NMR (151 MHz, DMSO) δ: 170.2 (C-1), 157.3 (C-3), 145.0

1

3

(

C-8), 131.8 (Ph), 130.1 (Ph-q), 127.0 (Ph), 122.8 (C-7), 120.7 (Ph-q), 118.2 (C-2), 75.5 (C-4), 67.1 (C-5), 52.9

+

C-6) ppm. HRMS (ESI Q-TOF): calculated for C H BrN O [M + H] = 382.0039; found = 382.0023.

14

12

3

5

Preparation of 6-deoxy-6-[4-tolyl-1,2,3-triazol-1-yl]-L-ascorbic acid (4f): Compound 4f (42.0 mg; 42%; m.p.

◦

=

152–154 C) was synthesized according to the general procedure using compound 3f (156.6 mg; 0.315

1

mmol); 1 M BCl (1.9 mL); CH Cl (20 mL). H NMR (300 MHz, DMSO)

δ: 11.17 (bs, 1H, OH), 8.50 (s,

3

2

1

H, H-7), 8.46 (bs, 1H, OH), 7.73 (d, J = 8.0 Hz, 2H, Ph), 7.25 (d, J = 8.0 Hz, 2H, Ph), 5.54 (d, J = 6.7 Hz,

H, OH), 4.77 (d, J = 1.0 Hz, 1H, H-4), 4.67 (dd, J = 13.6 Hz, 3.7 Hz, 1H, H-6), 4.41 (dd, J = 13.7 Hz, 9.3

13

Hz, 1H, H-6), 4.31–4.17 (m, 1H, H-5), 2.33 (s, 3H, CH ) ppm. C NMR (75 MHz, DMSO)

δ: 170.3 (C-1),

3

1

52.1 (C-3), 146.1 (C-8), 137.0 (Ph-q), 129.4 (Ph), 128.1 (Ph-q), 125.0 (Ph), 122.1 (C-7), 118.3 (C-2), 75.6

+

(C-4), 67.1 (C-5), 52.8 (C-6), 20.7 (CH ) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H]

3 15 15 3 5

=

318.1090; found = 318.1090.

Preparation of 6-deoxy-6-[4-methoxyphenyl-1,2,3-triazol-1-yl]-L-ascorbic acid (4g): Compound 4g (81.8 mg;

◦

5

0

4%; 185–187 C) was synthesized according to the general procedure using compound 3g (234 mg;

1

.456 mmol); 1 M BCl (2.74 mL) and CH Cl (20 mL). H NMR (300 MHz, DMSO)

δ

: 11.18 (bs, 1H,

3

2

OH), 8.45 (s, 2H, H-7, OH), 7.77 (d, J = 8.8 Hz, 2H, Ph), 7.01 (d, J = 8.8 Hz, 2H, Ph), 5.53 (bs, 1H, OH),

.76 (d, J = 1.5 Hz, 1H, H-4), 4.66 (dd, J = 13.7 Hz, 3.9 Hz, 1H, H-6), 4.40 (dd, J = 13.7 Hz, 9.3 Hz, 1H,

4

13

H-6), 4.28–4.19 (m, 1H, H-5), 3.79 (s, 3H, OCH ) ppm. C NMR (75 MHz, DMSO)

δ: 170.3 (C-1), 158.9

3

(

6

3

Ph-q), 152.0 (C-3), 145.9 (C-8), 126.4 (Ph), 123.4 (Ph-q), 121.5 (C-7), 118.3 (C-2), 114.3 (Ph), 75.5 (C-4),

+

7.1 (C-5), 55.1 (OCH ), 52.7 (C-6) ppm. HRMS (ESI Q-TOF): calculated for C H NO [M + H] =

3

34 45

16

34.1039; found = 334.1025.

Preparation of 6-deoxy-6-[2-hydroxyphenyl-1,2,3-triazol-1-yl]-L-ascorbic acid (4i): Compound 4i (56.5 mg;

◦

6

3%; m.p. = 129–131 C) was synthesized according to the general procedure using compound 3h (145

1

mg; 0.282 mmol); 1 M BCl (1.69 mL) and CH Cl (20 mL). H NMR (300 MHz, DMSO)

δ: 11.13 (bs,

3

2

1

(

3

H, OH), 10.20 (s, 1H, OH), 8.56–8.28 (m, 2H, H-7, OH), 8.01 (dd, J = 7.7 Hz, 1.5 Hz, 1H, Ph), 7.29–7.10

m, 1H, Ph), 7.01–6.86 (m, 2H, Ph), 5.53 (bs, 1H, OH), 4.77 (d, J = 1.4 Hz, 1H, H-4), 4.70 (dd, J = 13.7 Hz,

.7 Hz, 1H, H-6), 4.46 (dd, J = 13.7 Hz, 9.4 Hz, 1H, H-6), 4.25 (bs, 1H, H-5) ppm. ¹³C NMR (151 MHz,

DMSO) : 170.4 (C-1), 153.8; 152.1 (C-3, Ph-q), 142.7 (C-8), 128.6 (Ph), 126.4 (Ph), 124.4 (C-7), 119.3

Ph), 118.3 (C-2), 117.1 (Ph-q), 116.0 (Ph), 75.7 (C-4), 67.3 (C-5), 52.7 (C-6) ppm. HRMS (ESI Q-TOF):

δ

(

+

calculated for C H N O [M + H] = 320.0883; found = 320.0869.

14

13

3

6

Preparation of 6-deoxy-6-[4-(4-pentylphenyl)-1,2,3-triazol-1-yl]-L-ascorbic acid (4k): Compound 4k (62 mg;

◦

5

(

7%; m.p. = 237–239 C) was synthesized according to the general procedure using compound 3k

1

162.0 mg; 0.293 mmol); 1 M BCl (1.8 mL) and CH Cl (20 mL). H NMR (600 MHz, DMSO)

δ

: 11.14

3

2

(bs, 1H, OH), 8.49 (s, 1H, H-7), 8.43 (bs, 1H, OH), 7.74 (d, J = 8.2 Hz, 2H, Ph), 7.26 (d, J = 8.1 Hz, 2H,

Ph), 5.53 (bs, 1H, OH), 4.76 (d, J = 1.6 Hz, 1H, H-4), 4.67 (dd, J = 13.8 Hz, 4.0 Hz, 1H, H-6), 4.41 (dd,

0

J = 13.8 Hz, 9.3 Hz, 1H, H-6), 4.34–4.04 (m, 1H, H-5), 2.59 (t, J = 7.6 Hz, 2H, H-1 ), 1.68–1.47 (m, 2H,

0

13

CH ), 1.44–1.18 (m, 4H, CH ’), 0.87 (t, J = 7.0 Hz, 3H, CH ) ppm. C NMR (151 MHz, DMSO) δ:

2

3

1

70.2 (C-1), 152.0 (C-3), 146.1 (C-8), 142.0 (Ph-q), 128.7 (Ph), 128.3 (Ph-q), 125.0 (Ph), 122.1 (C-7), 118.3

0

(C-2), 75.5 (C-4), 67.1 (C-5), 52.7 (C-6), 34.8 (C-1 ), 30.8 (CH ’), 30.5 (CH ’), 21.9 (CH ’), 13.9 (CH ) ppm.

2

+

2

3

HRMS (ESI Q-TOF): calculated for C H N O [M + H] = 374.1716; found = 374.1700.

19

23

3

5

Preparation of 6-[4-cyclopropyl-1,2,3-triazol-1-yl]-6-deoxy-L-ascorbic acid (4l): Compound 4l (80.4 mg; 78%;

◦

m.p. = 119–121 C) was synthesized according to the general procedure using compound 3l (172.2 mg;

1

0

.385 mmol); 1 M BCl (2.3 mL) and CH Cl (20 mL). H NMR (600 MHz, DMSO)

δ: 11.13 (bs, 1H, OH),

3

2

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8

4

0

.43 (bs, 1H, OH), 7.80 (s, 1H, H-7), 5.44 (bs, 1H, OH), 4.68 (d, J = 1.2 Hz, 1H, H-4), 4.54 (dd, J = 13.8 Hz,

0

.0 Hz, 1H, H-6), 4.29 (dd, J = 13.7 Hz, 9.2 Hz, 1H, H-6), 4.17–4.12 (m, 1 H, H-5), 2.11–1.44 (m, 1H, H-1 ),

13

.96–0.82 (m, 2H, CH ’), 0.78–0.59 (m, 2H, CH ’) ppm. C NMR (75 MHz, DMSO) δ: 170.3 (C-1), 152.1

2

(

C-3), 148.5 (C-8), 121.8 (C-7), 118.3 (C-2), 75.5 (C-4), 67.1 (C-5), 52.5 (C-6), 7.6 (CH ’), 7.5 (CH ’), 6.5

2 2

0

+

C-1 ) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H] = 268.0933; found = 268.0938.

11

13

3

5

Preparation of 6-(4-butyl-1,2,3-triazol-1-yl)-6-deoxy-L-ascorbic acid (4m): Compound 4m (36.5 mg; 47%;

◦

m.p. = 98–99 C) was synthesized according to the general procedure using compound 3m (127.9 mg;

1

0

.276 mmol); 1 M BCl (1.7 mL) and CH Cl (20 mL). H NMR (300 MHz, DMSO)

δ: 11.14 (bs, 1H,

3

2

OH), 8.45 (bs, 1H, OH), 7.82 (s, 1H, H-7), 5.44 (d, J = 7.0 Hz, 1H, OH), 4.68 (d, J = 1.5 Hz, 1H, H-4),

4

2

.56 (dd, J = 13.7 Hz, 4.1 Hz, 1H, H-6), 4.32 (dd, J = 13.7 Hz, 9.0 Hz, 1H, H-6), 4.22–4.12 (m, 1H, H-5),

0

.63–2.57 (m, 2H, H-1 ), 1.65–1.49 (m, 2H, CH ’), 1.45–1.19 (m, 2H, CH ’), 0.89 (t, J = 7.3 Hz, 3H, CH )

2

3

13

ppm. C NMR (75 MHz, DMSO)

δ

: 170.3 (C-1), 152.1 (C-3), 146.5 (C-8), 122.8 (C-7), 118.3 (C-2), 75.6

0

+

(

C-4), 67.2 (C-5), 52.4 (C-6), 31.1 (C-1 ), 24.6 (CH ’), 21.7 (CH ’), 13.7 (CH ) ppm. HRMS (ESI Q-TOF):

2

3

calculated for C H N O [M + H] = 284.1246; found = 284.1250.

12

17

3

5

Preparation of 6-(4-decyl-1,2,3-triazol-1-yl)-6-deoxy-L-ascorbic acid (4n): Compound 4n (59.1 mg; 42%;

◦

m.p. = 140–142 C) was synthesized according to the general procedure using compound 3n (209.1 mg;

1

0

.382 mmol); 1 M BCl (1.6 mL) and CH Cl (20 mL). H NMR (300 MHz, DMSO)

δ: 11.14 (bs, 1H,

3

2

OH), 8.43 (bs, 1H, OH), 7.81 (s, 1H, H-7), 5.44 (d, J = 5.9 Hz, OH), 4.68 (d, J = 1.5 Hz, 1H, H-4), 4.56 (dd,

J = 13.7 Hz, 4.0 Hz, 1H, H-6), 4.32 (dd, J = 13.6 Hz, 9.1 Hz, 1H, H-6), 4.22–4.11 (m, 1H, H-5), 2.59 (t,

0

J = 7.5 Hz, 2H, H-1 ), 1.67–1.43 (m, 2H, H-2 ), 1.36–1.17 (m, 14H, CH ’), 0.85 (t, J = 6.6 Hz, 3H, CH )

2

3

ppm. ¹³C NMR (75 MHz, DMSO)

δ

: 170.3 (C-1), 152.0 (C-3), 146.6 (C-8), 122.8 (C-7), 118.3 (C-2), 75.6

0

(

2

C-4), 67.2 (C-5), 52.4 (C-6), 31.3 (C-1 ), 29.0 (CH ’), 28.8 (CH ’), 28.7 (CH ’), 28.6 (CH ’), 25.0 (CH ’),

2.1 (CH ’), 143.0 (CH ) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H] = 368.2185;

2 3 18 29 3 5

2

+

found = 368.2175.

Preparation of 6-[4-(tert-butyl)-1,2,3-triazol-1-yl]-6-deoxy-L-ascorbic acid (4o): Compound 4o (64.5 mg; 48%;

◦

m.p.

0

≥

250 C) was synthesized according to the general procedure using compound 3o (220.0 mg;

1

.475 mmol); 1 M BCl (2.9 mL) and CH Cl (20 mL). H NMR (300 MHz, DMSO)

δ: 11.17 (bs, 1H,

3

2

OH), 8.45 (bs, 1H, OH), 7.83 (s, 1H, H-7), 5.45 (bs, 1H, OH), 4.70 (d, J = 1.2 Hz, 1H, H-4), 4.56 (dd,

J = 13.6 Hz, 3.8 Hz, 1H, H-6), 4.32 (dd, J = 13.5 Hz, 9.1 Hz, 1H, H-6), 4.25–4.12 (m, 1H, H-5), 1.27 (s, 9H,

3

×

CH ) ppm. ¹³C NMR (75 MHz, DMSO)

δ

: 170.3 (C-1), 155.9 (C-8), 152.1 (C-3), 120.8 (C-7), 118.3

3

0

(

C-2), 75.6 (C-4), 67.2 (C-5), 52.4 (C-6), 30.3 (3

×

CH ), 28.9 (C-1 ) ppm. HRMS (ESI Q-TOF): calculated

3

+

for C H N O [M + H] = 284.1246; found = 284.1247.

12

17

3

5

Preparation of 6-[4-(3-chloropropyl)-1,2,3-triazol-1-yl]-6-deoxy-L-ascorbic acid (4p): Compound 4p (104.0 mg;

◦

8

3%; m.p. = 86–88 C) was synthesized according to the general procedure using compound 3p (200.0

1

mg; 0.413 mmol); 1 M BCl (2.5 mL) and CH Cl (20 mL). H NMR (300 MHz, DMSO)

δ: 11.17 (s, 1H,

3

2

OH), 8.47 (s, 1H, OH), 7.88 (s, 1H, H-7), 5.47 (d, J = 7.2 Hz, 1H, OH), 4.71 (d, J = 1.4 Hz, 1H, H-4), 4.58

dd, J = 13.7 Hz, 3.9 Hz, 1H, H-6), 4.33 (dd, J = 13.7 Hz, 9.2 Hz, 1H, H-6), 4.26–4.11 (m, 1H, H-5), 3.68 (t,

(

0

13

J = 6.5 Hz, 2H, H-3 ), 2.75 (t, J = 7.4 Hz, 2H, H-1 ), 2.08–2.01 (m, 2H, H-2 ) ppm. C NMR (151 MHz,

DMSO)

δ

0

: 170.3 (C-1), 152.1 (C-3), 145.2 (C-8), 123.2 (C-7), 118.3 (C-2), 75.6 (C-4), 67.2 (C-5), 52.5 (C-6),

0

+

4

4.7 (C-3 ), 31.8 (C.-1 ), 22.3 (C-2 ) ppm. HRMS (ESI Q-TOF): calculated for C H ClN O [M + H] =

11

14

3

5

3

04.0700; found = 304.0697.

Preparation of 6-deoxy-6-(4-(2-hydroxyethyl)-1,2,3-triazol-1-yl)-L-ascorbic acid (4q): Compound 4q (59.5 mg;

◦

7

(

0%; m.p. = 184–186 C) was synthesized according to the general procedure using compound 3q

1

140.3 mg; 0.311 mmol); 1 M BCl (1.9 mL) and CH Cl (20 mL). H NMR (600 MHz, DMSO)

δ

: 11.17

3

2

(

bs, 1H, OH), 8.46 (bs, 1H, OH), 7.85 (s, 1H, H-7), 5.46 (bs, 1H, OH), 4.73–4.66 (m, 2H, H-4, OH), 4.57

(

3

dd, J = 13.8 Hz, 4.0 Hz, 1H, H-6), 4.33 (dd, J = 13.8 Hz, 9.2 Hz, 2H, H-6), 4.25–4.10 (m, 2H, H-5),

.64–3.60 (m, 2H, H-2 ), 2.76 (t, J = 7.0 Hz, 2H, H-1 ) ppm. C NMR (151 MHz, DMSO) δ: 170.4 (C-1),

0

13

Int. J. Mol. Sci. 2019, 20, 4735

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0

1

52.1 (C-3), 144.1 (C-8), 123.5 (C-7), 118.3 (C-2), 75.6 (C-4), 67.2 (C-5), 60.4 (C-2 ), 52.4 (C-6), 29.2 (C-1 )

+

ppm. HRMS (ESI Q-TOF): calculated For C H N O [M + H] = 272.0883; found = 272.0895.

10

13

3

6

Preparation of 6-deoxy-6-[(4-(4-methylbenzenesulphonamide)methyl-1,2,3-triazol-1-yl]-L-ascorbic acid (4r):

◦

Compound 4r (115.9 mg; 85%; m.p. = 125–127 C) was synthesized according to the general procedure

1

using compound 3r (196.0 mg; 0.330 mmol); 1 M BCl (2.0 mL) and CH Cl (20 mL). H NMR (600 MHz,

3

2

DMSO) δ: 11.15 (bs, 1H, OH), 8.44 (bs, 1H, OH), 8.03 (t, J = 6.0 Hz, 1H, NH), 7.83 (s, 1H, H-7), 7.68 (d,

J = 8.2 Hz, 2H, Ph), 7.38 (d, J = 8.0 Hz, 2H, Ph), 5.44 (d, J = 7.4 Hz, 1H, OH), 4.64 (d, J = 1.5 Hz, 1H,

H-4), 4.55 (dd, J = 13.8 Hz, 4.2 Hz, 1H, H-6), 4.32 (dd, J = 13.8 Hz, 9.1 Hz, 1H, H-6), 4.16–4.11 (m, 1H,

13

H-5), 4.01 (d, J = 6.0 Hz, 2H, CH NH), 2.38 (s, 3H, CH ) ppm. C NMR (75 MHz, DMSO)

(

δ

: 170.3

C-1), 152.0 (C-8), 143.13 (C-3), 142.65 (Ph-q), 137.5 (Ph-q), 129.6 (Ph), 126.6 (Ph), 124.2 (C-7), 118.3 (C-2),

5.5 (C-4), 67.2 (C-5), 52.4 (C-6), 38.1 (CH NH), 21.0 (CH ) ppm. HRMS (ESI Q-TOF): calculated for

2

3

7

2

3

+

C H N O S [M + H] = 411.0974; found = 411.0961.

16

18

4

7

Preparation of 6-[(4-(4-chlorobenzenesulphonamide)methyl-1,2,3-triazol-1-yl]-6-deoxy-L-ascorbic acid (4s):

◦

Compound 4s (87.0 mg; 80%; m.p. = 132–134 C) was synthesized according to the general procedure

1

using compound 3s (154.0 mg; 0.252 mmol); 1 M BCl (1.5 mL) and CH Cl (20 mL). H NMR (300 MHz,

3

2

DMSO)

δ

: 11.17 (bs, 1H, OH), 8.45 (bs, 1H, OH), 8.26 (t, J = 5.9 Hz, 1H, NH), 7.88 (s, 1H, H-7), 7.81–7.71

m, 2H, Ph), 7.67–7.61 (m, 2H, Ph), 5.46 (d, J = 7.4 Hz, 1H, OH), 4.67 (d, J = 1.5 Hz, 1H, H-4), 4.56

dd, J = 13.7 Hz, 4.0 Hz, 1H, H-6), 4.32 (dd, J = 13.6 Hz, 9.1 Hz, 1H, H-6), 4.18–4.09 (m, 1H, H-5), 4.06

(

13

d, J = 6.0 Hz, 2H, CH NH) ppm. C NMR (75 MHz, DMSO)

δ: 170.1 (C-1), 152.0 (C-3), 142.9 (C-8),

2

1

39.3 (Ph-q), 137.2 (Ph-q), 129.2 (Ph), 128.5 (Ph), 124.3 (C-7), 118.4 (C-2), 75.5 (C-4), 67.2 (C-5), 52.5

+

(C-6), 38.0 (CH NH) ppm. HRMS (ESI Q-TOF): calculated for C H ClN O S [M + H] = 431.0428;

2 15 5 4 7

found = 431.0412.

Preparation of 6-[(4-(2-chloro-4-ﬂuorobenzenesulphonamide)methyl-1,2,3-triazol-1-yl]-6-deoxy-L-ascorbic acid

◦

(

4t): Compound 4t (88.7 mg; 59%; m.p. = 198–200 C) was synthesized according to the general

1

procedure using compound 3t (210.0 mg; 0.334 mmol); 1 M BCl (2.0 mL) and CH Cl (20 mL). H NMR

3

2

(300 MHz, DMSO)

δ: 11.18 (bs, 1H), 8.45 (t, J = 5.9 Hz, 1H, NH), 7.96 (dd, J = 8.9, 6.0 Hz, 1H, Ph), 7.83

(

s, 1H, H-7), 7.62 (dd, J = 8.7, 2.5 Hz, 1H, Ph), 7.36 (td, J = 8.6, 2.6 Hz, 1H, Ph), 4.65 (d, J = 1.3 Hz, 1H,

H-4), 4.54 (dd, J = 13.8 Hz, 4.0 Hz, 1H, H-6), 4.30 (dd, J = 13.7 Hz, 9.1 Hz, 1H, H-6), 4.17 (d, J = 5.9 Hz

,

2

H, CH₂NH), 4.14–4.06 (m, 1H, H-5) ppm. ¹³C NMR (75 MHz, DMSO)

δ

: 170.2 (C-1), 165.3; 161.9

(

d, ¹J_C_F= 254.3 Hz, Ph-4), 151.9 (C-3), 142.9 (C-8), 134.8; 137.7 (d, ⁴J_C_F= 3.6 Hz, Ph-1), 132.8; 132.6 (d,

³J = 10.1 Hz, Ph-6), 132.6; 132.4 (d, J_C_F= 10.1 Hz, Ph-2), 124.2 (C-7), 119.2; 118.9 (d, J_C_F= 25.9 Hz

3

2

,

CF

2

Ph), 118.3 (C-2), 114.8; 114.5 (d, J = 21.8 Hz, Ph), 75.5 (C-4), 67.2 (C-5), 52.4 (C-6), 37.8 (CH NH)

CF

2

+

ppm. HRMS (ESI Q-TOF): calculated for C H ClFN O S [M + H] = 449.0334; found = 449.0326.

15

14

4

7

Preparation of 6-[4-((tert-butyl 1-carboxylate)-4-(methylthiocarbothionyl)piperazine))-1,2,3-triazol-1-yl]-6-deoxy-

◦

L-ascorbic acid (4v): Compound 4v (31.2 mg; 35%; m.p.

≥

250 C) was synthesized according to the

general procedure using compound 3u (147.7 mg; 0.221 mmol); 1 M BCl (1.3 mL) and CH Cl (15 mL).

3

2

1

H NMR (600 MHz, DMSO)

δ

: 9.74 (bs, 1H, NH), 8.52 (bs, 1H, OH), 8.08 (s, 1H, H-7), 5.46 (bs, 1H, OH),

4

.71 (d, J = 1.5 Hz, 1H, H-4), 4.67–4.54 (m, 3H, H-6, CH S), 4.39–4.11 (m, 4H, H-6, H-5, 2

×

CH ), 3.2–3.1

2

13

(m, 4H, 2

×

CH ) ppm. C NMR (151 MHz, DMSO)

δ

: 195.8 (CS), 170.1 (C-1), 151.7 (C-3), 141.2 (C-8),

2

124.8 (C-7), 118.3 (C-2), 75.6 (C-4), 67.2 (C-5), 52.7 (C-6), 42.3 (CH ’), 31.9 (CH S) ppm. HRMS (ESI

2

+

Q-TOF): calculated for C H N O S [M + H] = 402.0906; found = 402.0891.

14

19

5

5 2

Preparation of 4,5-didehydro-5,6-dideoxy-(4-(3,5-di-(triﬂuoromethyl)phenyl)-1,2,3-triazol-1-yl)-L-ascorbic acid

◦

(

7c): Compound 7c (72.1 mg; 52%; m.p.

≥

200 C) was synthesized according to the general procedure

1

using compound 6c (200 mg; 0.33 mmol); 1 M BCl (1.98 mL) and CH Cl (20 mL). H-NMR (300 MHz,

3

2

DMSO) δ: 11.21 (s, 1H, OH), 9.95 (s, 1H, OH), 9.01 (s, 1H, H-7), 8.54 (s, 2H, Ph-2, Ph-6), 8.07 (s, 1H,

1

3

Ph-4), 5.61 (t, J = 7.6 Hz, 1H, H-5), 5.30 (d, J = 7.7 Hz, 2H, H-6) ppm. C-NMR (151 MHz, DMSO)

δ

:

2

1

64.6 (C-1), 145.8 (C-3), 143.8 (C-4), 142.9 (C-8), 133.2 (Ph-1), 131.3; 131.1; 130.9; 130.7 (q, J = 33.0 Hz,

Ph-3, Ph-5), 125.9; 124.1; 122.3 (m, J = 272.8 Hz, CF ), 125.3 (Ph-2, Ph-6), 123.3 (C-7), 122.1 (C-2),

CF

1

CF

3

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+

1

21.1 (Ph-4), 98.2 (C-5), 44.5 (C-6) ppm. HRMS (ESI Q-TOF): calculated for C H F N O [M + H] =

16

9

6

3

4

22.0576; found = 422.0580.

Preparation of 4,5-didehydro-5,6-dideoxy-(4-(3,5-diﬂuoromethyl)phenyl)-1,2,3-triazol-1-yl)-L-ascorbic acid (7d):

◦

Compound 7d (61.1 mg; 48%; m.p.

≥

200 C) was synthesized according to the general procedure

using compound 6d (200 mg; 0,39 mmol); 1 M BCl (2.27 mL) and CH Cl (20 mL): Compound 7d was

isolated as white powder. H-NMR (600 MHz, DMSO)

3

2

1

δ: 11.31 (s, 1H, OH), 9.83 (s, 1H, OH), 8.75 (s,

1

H, H-7), 7.59 (d, J = 6.6 Hz, 2H, Ph-2, Ph-6), 7.31–7.03 (m, 1H, Ph-4), 5.58 (t, J = 7.7 Hz, 1H, H-5), 5.26

13

(

d, J = 7.7 Hz, 2H, H-6) ppm. C-NMR (151 MHz, DMSO)

J_C_F= 245.6 Hz, J = 13.7 Hz, Ph-3, Ph-5), 145.7 (C-3), 144.6 (C-8), 142.9 (C-4), 134.2; 134.2; 134.1 (t,

δ

: 164.6 (C-1), 163.7; 163.6; 162.1; 162.0 (dd,

1

3

CF

2

4

J = 10.5 Hz, Ph-1), 122.7 (C-7), 122.1 (C-2), 108.2; 108.1; 108.0; 108.0 (dd, J = 21.1 Hz, J = 5.5 Hz,

Ph-2, Ph-6), 103.2; 103.1; 102.9 (t, J = 26.0 Hz, Ph-4), 98.4 (C-5), 44.5 (C-6) ppm. HRMS (ESI Q-TOF):

calculated for C H F N O [M + H] = 322.0639; found = 322.0642.

CF

2

+

14

9

2

3

4

Preparation of 6-[4-(4-bromophenyl)-1,2,3-triazole-1-yl]-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (7e):

◦

Compound 7e (24 mg; 21%; m.p. = 219–222 C) was synthesized according to the general procedure

1

using compound 6e (177 mg; 0.324 mmol); 1 M BCl (1.94 mL) and CH Cl (20 mL). H NMR (300 MHz,

3

2

DMSO)

δ

: 10.48 (s, OH), 8.67 (s, 1H, H-7), 7.82 (d, J = 8.5 Hz, 2H, Ph), 7.64 (d, J = 8.6 Hz, 2H, Ph), 5.58

t, J = 7.5 Hz, 1H, H-5), 5.25 (d, J = 7.5 Hz, 2H, H-6). ¹³C NMR (75 MHz, DMSO-d6)

: 164.7 (C-1),

45.5 (C-3), 143.0 (C-8, C-4), 131.8 (Ph), 129.9 (Ph-q), 127.1 (Ph), 122.0 (C-7), 121.8 (C-2), 120.8 (Ph-q),

(

1

δ

+

9

8.7 (C-5), 44.4 (C-6) ppm. HRMS (ESI Q-TOF): calculated for C H BrN O [M + H] = 363.9933;

14 10 3 4

found = 363.9915.

Preparation of 6-[4-tolyl-1,2,3-triazole-1-yl]-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (7f): Compound 7f

◦

(

6

20 mg; 15%; m.p. = 188–192 C) was synthesized according to the general procedure using compound

1

f

(203 mg; 0.407 mmol); 1 M BCl (2.45 mL) and CH Cl (20 mL). H NMR (600 MHz, DMSO)

δ: 11.40

3

2

(

5

s, 1H, OH), 9.78 (s, 1H, OH), 8.56 (s, 1H, H-7), 7.74 (d, J = 8.0 Hz, 2H, Ph), 7.25 (d, J = 7.9 Hz, 2H, Ph),

.59 (t, J = 7.6 Hz, 1H, H-5), 5.24 (d, J = 7.6 Hz, 2H, H-6), 2.33 (s, 3H, CH ) ppm. C NMR (151 MHz,

3

13

DMSO)

δ: 164.7 (C-1), 146.5 (C-3), 145.3 (C-4), 143.0 (C-8), 137.2 (Ph-q), 129.4 (Ph), 127.9 (Ph-q), 125.0

(

Ph), 121.9 (C-7), 120.9 (C-2), 98.8 (C-5), 44.3 (C-6), 20.7 (CH ) ppm. HRMS (ESI Q-TOF): calculated for

3

+

C H N O [M + H] = 300.0984; found 300.0974.

15

13

3

4

Preparation of 4,5-didehydro-5,6-dideoxy-6-[4-(4-methoxyphenyl)-1,2,3-triazole-1-yl]-L-ascorbic acid (7g):

◦

Compound 7g (58.1 mg; 28%; m.p. = 198–199 C) was synthesized according to the general procedure

1

using compound 6g (328 mg; 0.662 mmol); 1 M BCl (3.96 mL) and CH Cl (25 mL). H NMR (600 MHz,

3

2

DMSO)

d, J = 8.7 Hz, 2H, Ph), 5.58 (t, J = 7.6 Hz, 1H, H-5), 5.23 (d, J = 7.6 Hz, 2H, H-6) ppm. C NMR (151

MHz, DMSO) : 164.7 (C-1), 159.0 (Ph-q), 146.5 (C-3), 145.3 (C-8), 126.5 (Ph), 123.3 (Ph-q), 121.9 (C-2),

20.4 (C-7), 114.3 (Ph), 99.0 (C-5), 55.1 (OCH ), 44.3 (C-6) ppm. HRMS (ESI Q-TOF): calculated for

δ: 11.05 (bs, 1H, OH), 9.95 (bs, 1H, OH), 8.49 (s, 1H, H-7), 7.78 (d, J = 8.7 Hz, 2H, Ph), 7.01

13

(

δ

1

3

+

C H N O [M + H] = 316.0933; found = 316.0930.

15

13

3

5

Preparation of 4,5-didehydro-5,6-dideoxy-6-[4-(2-hydroxyphenyl)-1,2,3-triazole-1-yl]-L-ascorbic acid (7i):

◦

Compound 7i (64.2 mg; 56%; m.p. = 167–169 C) was synthesized according to the general procedure

1

using compound 6h (188 mg; 0.379 mmol); 1 M BCl (2.28 mL) and CH Cl (20 mL). H NMR (300 MHz,

3

2

DMSO)

.7 Hz, 1H, Ph), 7.43–7.05 (m, 1H, Ph), 6.99–6.78 (m, 2H, Ph), 5.58 (t, J = 7.5 Hz, 1H, H-5), 5.25 (d,

J = 7.5 Hz, 2H, H-6) ppm. 13C NMR (75 MHz, DMSO) : 164.6 (C-1), 153.9 (Ph-q), 145.1 (C-3), 143.1

C-8), 143.0 (C-4), 128.7 (Ph), 126.5 (Ph), 123.1 (C-7), 121.9 (C-2), 119.2 (Ph), 116.9 (Ph-q), 115.9 (Ph),

δ: 11.13 (bs, 1H, OH), 10.15 (s, 1H, OH), 9.74 (bs, 1H, OH), 8.41 (s, 1H, H-7), 7.97 (dd, J = 7.7 Hz,

1

δ

(

9

+

9.3 (C-5), 44.2 (C-6) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H] = 302.0777;

14 11 3 5

found = 302.0782.

Preparation of 4,5-didehydro-5,6-dideoxy-6-[4-(3-hydroxyphenyl)-1,2,3-triazole-1-yl]-L-ascorbic acid (7j):

◦

Compound 7j (45.3 mg; 41%; m.p. = 155–157 C) was synthesized according to the general procedure

1

using compound 6j (175 mg; 0.363 mmol); 1 M BCl (2.18 mL) and CH Cl (20 mL). H NMR (600 MHz,

3

2

Int. J. Mol. Sci. 2019, 20, 4735

19 of 26

DMSO)

.72 (d, J = 7.0 Hz, 1H, Ph), 5.59 (t, J = 7.6 Hz, 1H, H-5), 5.23 (d, J = 7.6 Hz, 2H, H-6) ppm. ¹³C NMR

151 MHz, DMSO) : 164.7 (C-1), 157.8 (Ph-q), 146.7 (C-3), 145.3 (C-4), 143.0 (C-8), 131.9 (Ph-q), 130.0

Ph), 122.0 (C-7), 121.4 (C-2), 116.1 (Ph), 114.9 (Ph), 111.9 (Ph), 99.0 (C-5), 44.4 (C-6) ppm. HRMS (ESI

δ: 11.33 (s, 1H, OH), 9.90 (s, 1H, OH), 9.55 (s, 1H, OH), 8.55 (s, 1H, H-7), 7.54–7.02 (m, 3H, Ph),

6

(

δ

+

Q-TOF): calculated for C H N O [M + H] = 302.0777; found = 302.0765.

14

11

3

5

Preparation of 4,5-didehydro-5,6-dideoxy-6-[4-(4-pentylphenyl)-1,2,3-triazole-1-yl]-L-ascorbic acid (7k):

◦

Compound 7k (31 mg; 24%; m.p. = 230–232 C) was synthesized according to the general procedure

1

using compound 6k (195 mg; 0.364 mmol); 1 M BCl (2.18 mL) and CH Cl (20 mL). H NMR (300 MHz,

3

2

DMSO)

δ

: 11.10 (bs, 1 H, OH), 9.98 (bs, 1H, OH), 8.55 (s, 1H, H-7), 7.75 (d, J = 8.1 Hz, 2H, Ph), 7.26 (d,

0

J = 8.1 Hz, 2H, Ph), 5.59 (t, J = 7.6 Hz, 1H, H-5), 5.24 (d, J = 7.6 Hz, 1H, H-6), 2.59 (t, J = 7.6 Hz, 2H, H-1 ),

13

1

.65–1.51 (m, 2H, CH ), 1.38–1.17 (m, 4H, CH ), 0.86 (t, J = 6.8 Hz, 3H, CH ) ppm. C NMR (75 MHz,

2 2 3

DMSO) δ: 164.7 (C-1), 146.6 (C-3), 145.3 (C-4), 143.0 (C-8), 142.1 (Ph-q), 128.8 (Ph), 128.1 (Ph-q), 125.1

0 0

Ph), 122.0 (C-2), 121.0 (C-7), 99.0 (C-5), 44.3 (C-6), 34.8 (C-1 ), 30.9 (C-2 ), 30.5 (CH ), 21.9 (CH ), 13.9

2 2

(

+

CH ) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H] = 356.1610; found = 356.1598.

3

19 21

3

4

Preparation of 6-[4-cyclopropyl-1,2,3-triazole-1-yl]-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (7l): Compound

◦

7

l

(52.5 mg; 53%; m.p. = 176–178 C) was synthesized according to the general procedure using

1

compound 6l (170 mg; 0.396 mmol); 1 M BCl (2.38 mL) and CH Cl (20 mL). H NMR (300 MHz,

3

2

DMSO)

δ: 11.10 (bs, 1H, OH), 9.88 (bs, 1H, OH), 7.83 (s, 1H, H-7), 5.50 (t, J = 7.5 Hz, 1H, H-5), 5.11

(

d, J = 7.6 Hz, 2H, H-6), 1.98–1.86 (m, 1H, CH), 0.95–0.84 (m, 2H, CH ), 0.73–0.65 (m, 2H, CH ) ppm.

C NMR (75 MHz, DMSO) δ: 164.6 (C-1), 149.2 (C-8), 145.0 (C-3), 143.0 (C-4), 121.9 (C-2), 120.6 (C-7),

2

1

3

9

9.2 (c-5), 44.0 (C-6), 7.5 (CH ), 6.4 (CH) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H]

2

11 11

3

4

+

=

250.0828; found = 250.0826.

Preparation of 6-[4-butyl-1,2,3-triazole-1-yl]-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (7m): Compound

◦

7

m

(66.8 mg; 47%; m.p. = 180–183 C) was synthesized according to the general procedure using

1

compound 6m (237 mg; 0.532 mmol); 1 M BCl (3.19 mL) and CH Cl (20 mL). H NMR (600 MHz,

3

2

DMSO)

δ

: 11.32 (s, 1H, OH), 9.76 (s, 1H, OH), 7.88 (s, 1H, H-7), 5.52 (t, J = 7.6 Hz, 1H, H-5), 5.14 (d,

0

J = 7.6 Hz, 2H, H-6), 2.60 (t, J = 7.6 Hz, 2H, H-1 ), 1.68–1.42 (m, 2H, CH ), 1.41–1.26 (m, 2H, CH ), 0.89

2

13

(t, J = 7.4 Hz, 3H, CH ) ppm. C NMR (151 MHz, DMSO)

δ

0

: 164.7 (C-1), 147.2; 145.0; 143.1 (C-3, C-4,

3

C-8), 121.9 (C-2), 121.8 (C-7), 99.4 (C-5), 44.0 (C-6), 31.1 (C-1 ), 24.7 (CH ), 21.7 (CH ), 13.7 (CH ) ppm.

2

3

Preparation of 6-(4-decyl-1,2,3-triazol-1-il)-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (7n): Compound 7n

◦

(

6

55 mg; 42%; m.p. = 169–171 C) was synthesized according to the general procedure using compound

1

n

(200 mg; 0.38 mmol); 1 M BCl (2.28 mL) and CH Cl (20 mL). H-NMR (600 MHz, DMSO)

δ:

3

2

1

2

1.28 (s, 1H, OH), 9.73 (s, 1H, OH), 7.86 (s, 1H, H-7), 5.52 (t, 1H, J = 7.5 Hz, H-5), 5.14 (s, 2H, H-6),

0

.60–2.57 (m, 2H, H-1 ), 1.58–1.56 (m, 2H, H-2 ), 1.33–1.20 (m, 14H, CH ), 0.84 (t, J = 7.0 Hz, 3H, CH )

2

3

13

ppm. C-NMR (151 MHz, DMSO)

δ

: 164.6 (C-1), 147.2 (C-3), 144.9 (C-4), 143.0 (C-8), 121.9 (C-2), 121.7

0

(

C-7), 99.3 (C-5), 43.9 (C-6), 31.2 (C-1 ), 28.9 (CH ’), 28.7 (CH ’), 28.6 (CH ’), 28.5 (CH ’), 24.9 (CH ’),

2

+

2

2.1 (CH ’), 13.9 (CH ) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H] = 350.2080;

2 3 18 27 3 4

found = 350.2086.

Preparation of 6-[4-tert-butyl-1,2,3-triazole-1-yl]-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (7o): Compound

◦

7

o

(30 mg; 24%; m.p. = 182–184 C) was synthesized according to the general procedure using

1

compound 6o (203 mg; 0.455 mmol); 1 M BCl (2.72 mL) and CH Cl (20 mL). H NMR (300 MHz,

3

2

DMSO)

δ

: 11.31–9.54 (bs, 2H, OH

×

2), 7.88 (s, 1H, H-7), 5.53 (t, J = 7.5 Hz, 1H, H-5), 5.14 (d, J = 7.5 Hz,

13

2

H, H-6), 1.27 (s, 9H, CH₃

×

3). C NMR (75 MHz, DMSO)

δ: 164.6 (C-1), 144.9 (C-3), 143.0 (C-4), 121.9

0

(C-2), 119.7 (C-7), 99.3 (C-5), 43.9 (C-6), 30.2 (C-1 ), 30.2 (CH ) ppm. HRMS (ESI Q-TOF): calculated for

3

+

C H N O [M + H] = 266.1141; found = 266.1129.

12

15

3

4

Preparation of 6-[4-chloropropyl-1,2,3-triazole-1-yl]-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (7p): Compound

◦

7

p

(53.2 mg; 38%; m.p. = 160–162 C) was synthesized according to the general procedure using

1

compound 6p (230 mg; 0.494 mmol); 1 M BCl (2.96 mL); CH Cl (20 mL). H NMR (300 MHz, DMSO)

3

2

Int. J. Mol. Sci. 2019, 20, 4735

20 of 26

δ

: 11.18–9.67 (bs, 2H, OH

×

2), 7.93 (s, 1H, H-7), 5.53 (t, J = 7.5 Hz, 1H, H-5), 5.15 (d, J = 7.5 Hz, 2H,

0

13

H-6), 3.67 (t, J = 6.5 Hz, 2H, H-3 ), 2.82–2.66 (m, 2H, H-1 ), 2.10–1.98 (m, 2H, H-2 ) ppm. C NMR

(

4

[

75 MHz, DMSO)

δ

: 164.6 (C-1), 145.7; 145.0; 143.0 (C-3, C-4, C-8), 122.1 (C-7), 121.9 (C-2), 99.2 (C-5),

0

4.6 (C-1 ), 44.0 (C-6), 31.7 (C-1 ), 22.3 (C-3 ) ppm. HRMS (ESI Q-TOF): calculated for C H ClN O

M + H] ⁺= 286.0595; found = 286.0599.

11

12

3

4

Preparation of 6-[4-(2-hydroxyethyl)-1,2,3-triazole-1-yl]-4,5-didehydro-5,6-dideoxy-L-ascorbic cid (7q): Compound

◦

7

q

(81.2 mg; 70%; m.p. = 172–173 C) was synthesized according to the general procedure using

1

compound 6q (200 mg; 0.461 mmol); 1 M BCl (2.77 mL); CH Cl (20 mL). H NMR (300 MHz, DMSO)

3

2

δ

: 11.21 (bs, 1H, OH), 9.87 (bs, 1H, OH), 7.87 (s, 1H, H-7), 5.52 (t, J = 7.6 Hz, 1H, H-5), 5.15 (d, J = 7.6 Hz,

H, H-6), 4.65 (bs, 1H, OH), 3.62 (t, J = 6.9 Hz, 2H, H-2 ), 2.76 (t, J = 6.9 Hz, H-1 ) ppm. C NMR (75

0

13

2

MHz, DMSO)

δ

: 164.6 (C-1), 145.0 (C-3), 144.7 (C-8), 143.0 (C-4), 122.3 (C-7), 121.9 (C-2), 99.3 (C-5),

0

+

6

2

0.3 (C-2 ), 43.9 (C-6), 29.1 (C-1 ) ppm. HRMS (ESI Q-TOF): calculated for C H N O [M + H]

=

10

11

3

5

54.0777; found = 254.0770.

.2. Biological Evaluation

.2.1. DPPH Assay

3

Antioxidant capacity of selected compounds was assessed by 2,2-diphenyl-1-picrylhydrazyl assay

(

DPPH, Sigma Aldrich, Darmstadt, Germany), in which the antioxidant activity of compounds is

•

measured by the reaction between a stable radical of 1,1-diphenyl-2-picylhydrazil (DPPH ) and a

selected compound according to the manufacturer recommendations. Brieﬂy, compounds 4b–4e

,

4

f–4h 4k 4l 4o 4p–4s 4t 4v 7c–7f 7i–7n 7q 7p, and 7o were tested at 0.06, 0.12, 0.18, 0.23, 0.25,

,

and 0.28 mM. L-ascorbic acid was used as the reference compound. Absorbance was measured 5

and 10 min after incubation of the compounds with the DPPH reagent. The reduction of DPPH with

selected compounds was monitored by measuring the absorbance at a wavelength of 517 nm on the

monochromator (Inﬁnite M200 PRO, TECAN, Männedorf, Switzerland).

3

.2.2. In Silico Approaches—DFT Calculations and PCA Analysis

The gas-phase (g) reaction parameters for radical scavenging models: bond dissociation enthalpy

(

BDE), basicity, and electron transfer enthalpy (ETE), and their aqueous free energy (FE) counterparts

BDFE, pK , and ETFE, respectively, were calculated by using thermochemical cycles [42] in the density

functional theory (DFT) model (U)M052X/6-311++G** implemented in Gaussian 09 [57 58]. The

a

,

gas-phase equilibrium geometries were optimized for species in neutral and anion closed-shell singlet

and neutral monoradical and radical anion open-shell doublet ground electronic states. The minima

were conﬁrmed by absence of imaginary vibrational frequencies. Calculations were done for the

compounds with a free C5OH group in the 4R,5S-conﬁguration as a starting L-ASA (series

compounds with a C4=C5 double bond were determined in Z-conﬁguration (series ) (Scheme 1) [59].

The free energies of hydratation G*_h_y_dwere determined at the gas phase geometries by using the

4). The

7

∆

SMD method [60]. The gas-phase thermodynamic reaction parameters were calculated in terms of the

sums of electronic and thermal enthalpies or free energies estimated at temperature of 298.15 K and

pressure of 1 atm. For the electron and the proton, values reported in the reference [61] were used.

◦

•

◦

+

◦

−

(e ), the experimental

hyd

Regarding free energies of hydratation

∆

G

(H ),

∆

G

(H ), and

∆

G

hyd

values of 27.8 kJ/mol [62], −1104.62 kJ/mol [63] and −148.5 kJ/mol [64] were used, respectively.

The PCA analyses were done by R package princomp [65]. The values of partition n-octanol/water

coeﬃcients—clogP were calculated by DataWarrior by using SMILES as inputs [44].

3

.2.3. Cell Culturing

Human tumor cell lines HeLa (cervical carcinoma), SW620 (colorectal adenocarcinoma, metastatic),

A549 (lung adenocarcinoma), MCF

metastatic), HCT-116 (colorectal carcinoma), and HepG2 (hepatocellular carcinoma), and normal cell

−

7 (breast adenocarcinoma), CFPAC-1 (pancreatic adenocarcinoma,

Int. J. Mol. Sci. 2019, 20, 4735

21 of 26

lines HFF-1 (foreskin ﬁbroblasts) and WI-38 (immortalized human lung ﬁbroblast), were from the

US American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in a liquid

nutrient medium (Dulbecco’s Modiﬁed Eagle Medium, LONZA, Basel, Switzerland) supplemented

with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 2 mM L-glutamine (GIBCO, Grand

Island, NY, USA), 100

µ

g/mL penicillin, and 100

µ

g/mL streptomycin (LONZA, Basel, Switzerland), in

◦

an incubator (NÜVE, Ankara, Turkey), at a humid atmosphere with 5% CO at 37 C. Trypsin/EDTA

2

Solution (Lonza, Basel, Switzerland), was used for cell passaging and cell detachment.

3

.2.4. Proliferation Assay- MTT Assay

Adherent cells were seeded on 96 well microtiter plates (Falcon, New York, NY, USA) at a seeding

density of 5000 cells per well, added in 150

seeding, cells were treated with compound 4b–4g

of 0.01 M to 100 M. L-ascoric acid (Sigma Aldrich, Darmstadt, Germany) and Carboxyamidotriazole

Sigma Aldrich, Darmstadt, Germany) were used as control compounds. Incubation of the cells

µL of cell suspension in a nutrient medium. 24 h after

,

4i 4k–4t 4v 7c–7g, and 7i–7q dilutions in the range

,

µ

(

◦

with the compounds were performed for 72 h in a 5% CO , in humid atmosphere at 37 C. After

2

incubation, nutrient medium was removed and 40

diphenyltetrazole bromide (Invitrogen, Carlsbad, CA, USA) was added to each well. 3 h after incubation,

60 L of DMSO (VWR CHEMICALS, Radnor, PA, USA) were added to each well to dissolve the crystals

µL of the MTT reagent (3-(4-dimethylthiazol-2-yl)-2,5-

1

µ

of purple formazane. The absorbance was measured at 570 nm (TECAN SUNRISE Microtiter plate

reader (Männedorf, Switzerland). The experimentally measured optical densities (OD) were converted

to the PG (percentage of growth), using the formulas according to the protocol assumed by NIH

(National Institute of Health). The IC₅₀values (concentration causing 50% cell growth inhibition) for

each compound were calculated by regression analysis of the survival count curve of the concentrations

of the tested compounds. The results were processed in Excel 2010 (Microsoft, Redmond, WA, USA).

3

.2.5. Apoptosis Detection—Annexin V Assay

4

A total of 2

×

10 MCF-7 cells were seeded into 8-well Nunc Lab-Tek II Chamber Slide system

(

Thermo Fischer Scientiﬁc, Waltham, MA, USA), and treated with compound 4e at 1

×

IC₅₀and 2

×

IC₅₀for 24 and 48 h. The assay (Annexin-V-FLUOS Staining kit, Roche Applied Science, Penzberg,

Germany) was preformed according to the manufacturer protocol. Slides were analyzed by ﬂuorescence

microscopy (Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescent Microscope, Jena, Germany)).

3

.2.6. Western Blot Analysis

6

MCF-7 cells were seeded in Petri dishes, at 1

µ

×

10 cells, and treated with compound 4e at IC₅₀

6

.72

M for 24 and 48 h. Protein lysates were prepared using RIPA buffer containing cocktail of protease

g of proteins were resolved on

2% SDS polyacrylamide gels using the Mini-protean cell (Bio-Rad, Hercules, CA, USA). The membranes

were incubated with primary antibodies raised against hydroxy-HIF-1 (Pro564) (HIF-1 , 1:1000, rabbit

and phosphatase inhibitors (Roche, Basel, Switzerland). Amounts of 50

µ

1

α

mAb, Cell Signaling Technology, NL) and NOS2 (1:500, mouse mAb, SantaCruzBiotechnology, Dallas,

◦

TX, USA) at 4 C overnight. A secondary antibody linked to anti-mouse (1:1000, Dako, Santa Clara, CA,

USA) was used. The signal was visualized by the Western Lightening Chemiluminescence Reagent

Plus Kit (Perkin Elmer, Massachusetts, MA, USA) on the ImageQuant LAS500 (GE Healthcare, Chicago,

IL, USA), and as a loading control, α-tubulin (1:1000, mouse mAb, Sigma, Sigma Aldrich, Darmstadt,

Germany) was used. The signal intensities of bands were normalized and compared in Quantity One

software (Bio-Rad, Hercules, CA, USA). Differences in protein relative expression status obtained by

western blot analysis were analyzed by two-tailed, paired t-tests (p < 0.05) in the Statistica software

package (v.12.0).

3

.2.7. Seahorse XF Cell Myto Stress Test

The Seahorse XF Cell Mito Stress Test was used for measurements of mitochondrial respiration

by direct assessment of the oxygen consumption rate (OCR), of treated and untreated cells on the

Int. J. Mol. Sci. 2019, 20, 4735

22 of 26

Seahorse XFe24 Analyzer, (Agilent, Santa Clara, CA, USA). Key parameters of the mitochondrial

function were measured by direct assessment of the OCR, by use of the Mito Stress Test (Agilent,

Agilent, Santa Clara, CA, USA). The Mito Stress Test uses modulators of respiration that target

components of the electron transport chain in the mitochondria. The compounds added to the

reaction were: oligomycin, mitochondrial oxidative phosphorylation uncoupler carbonyl cyanide-4-

(

triﬂuoromethoxy)phenylhydrazone (FCCP), and the mix of rotenone and antymicin A. These were

directly injected to the cell growth medium containing cells treated with selected L-ASA derivatives to

determine the production of ATP, respiration at maximal level, and the non-mitochondrial respiration.

Measured parameters were used for calculation of the proton leakage, spare respiratory capacity, and

basal respiration. The assay was performed according to the manufacturer’s instructions (SeaHorse—

Mito Stress Test: Agilent, Santa Clara, CA, USA). Brieﬂy, selected compound 4e was tested due to its

selective antiproliferative eﬀect on MCF-7 cells. MCF-7 cells were treated for 24 h with compound 4e

at 3 diﬀerent concentrations (at 12.72

µM, 25.44

µM and 50.88

µM) while the cells HFF-1 were treated

for 24 h with compound 4e at concentrations 50

µ

M, 100 M, and 200 µ

µ

M. L-ascorbic acid, was used as

control compound and tested at concentrations 50, 100, and 150 mM (same as those used for DPPH

assay) on MCF-7 and HFF-1 cells. The experiment was performed with biological duplicates.

4

. Conclusions

With the aim to ﬁnd compounds with superior antioxidant and antiproliferative activity compared

to L-ASA, two novel classes of L-ASA derivatives, 6-(1,2,3-triazolyl)-6-deoxy-L-ASA (series ) with

the C4-C5(OH) group, and their analogues (series ) with the C4=C5 double bond were synthesized.

4

7

•

The assessment of the antioxidant activity by DPPH radical scavenging assay showed that the majority

of the 4,5-unsaturated L-ASA derivatives (series ) showed better antioxidant activity compared to

their saturated analogues (series ). Moreover, the introduction of an additional hydroxyl group at

,2,3-triazole of the 4,5-unsaturated L-ASA derivatives (in 7i

7

4

1

,

7j, and 7q) resulted in improvement

•

of the radical scavenging activity. An explanation of the observed diﬀerences in the DPPH radical

scavenging activities of the L-ASA triazole conjugates has been obtained using in silico analysis,

−

which showed that the triazole conjugates have an analogous radical scavenging mechanism to

L-ASA and that the most signiﬁcant diﬀerence between saturated C4-C5(OH) and unsaturated C4=C5

derivatives is in acidity of C2-OH group. The active radical scavenging center in L-ASA derivatives

with a C4-C5(OH) fragment is the C3-OH group, while for majority unsaturated C4=C5 derivatives,

the radical scavenging center can be ascribed to C2-OH group. Furthermore, the antiproliferative

evaluation on seven malignant tumor cell lines proved that the linker connecting the lactone ring

and the 1,2,3-triazole moiety has an inﬂuence on the antitumor activity. Hence, 1,2,3-triazole L-ASA

derivatives with a hydroxyethylene C4-C5(OH) unit showed better growth-inhibition eﬀects on tumor

cells compared to their 4,5-unsaturated C4=C5 analogues. Additionally, unsubstituted 1,2,3-triazolyl

derivative 4b was not cytotoxic toward any of the tested cell lines, which shows that C-4 substitution

at the 1,2,3-triazole moiety has an impact on growth-inhibition related eﬀects. Compound 4e with a

p-bromophenyl and 4k with a p-pentylphenyl-substituted 1,2,3-triazole L-ASA both exerted a selective

cytostatic eﬀect on MCF-7 cells (IC₅₀= 6.72

of the relative expression of HIF-1 and NOS2 proteins showed that compound 4e strongly increased

the expression of hydroxylated HIF-1 and somewhat decreased the expression of NOS2, indicating its

µM; IC₅₀= 26.91 µM, respectively). Western blot analysis

α

role in the HIF-1-triggered response of MCF-7 cells to hypoxia. Cellular-metabolism analysis showed

that compound 4e increased the basal respiration and ATP production of MCF-7 cells, while it did

not aﬀect the basal cell respiration and ATP production of HFF-1 cells, which is in agreement with

MTT-results, in which 4e showed no cytotoxicity on HFF-1 cells. Overall, 4-substituted 1,2,3-triazole

L-ASA conjugates were identiﬁed as a promising chemical entity that aﬀected the HIF-1α signaling

pathway, and further structure optimization of the saturated C4-C5(OH) derivatives could lead to a

more potent and selective growth-inhibition eﬀect on breast adenocarcinoma (MCF-7) cells.

Int. J. Mol. Sci. 2019, 20, 4735

23 of 26

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com /1422-0067 /20 /19 /

735 /s1 .

4

Author Contributions: conceptualization, S.R.-M., K.P., and S.K.P.; methodology, S.R.-M. and S.K.P.; writing—

original draft preparation, writing—review and editing, A.H., A.M.M, S.R.-M., V.S., and S.K.P.; investigation, A.H.,

A.M.M., V.S., and M.K.; supervision, A.M.M., K.P., S.K.P., and S.R.-M.; project administration, S.R.-M. and S.K.P.;

funding acquisition, S.R.-M. and S.K.P.

Funding: Financial support from the Croatian Science Foundation under the project HRZZ-IP-2018-01-4682 is

gratefully acknowledged. We greatly appreciate the access granted to equipment owned by the University of

Rijeka within the project “Research Infrastructure for Campus-based Laboratories at University of Rijeka,” ﬁnanced

by the European Regional Development Fund (ERDF). We also acknowledge the University of Rijeka’s research

support, uniri-biomed-18-133. We would like to thank Croatian Government and the European Union (European

Regional Development Fund—the Competitiveness and Cohesion Operational Programme—KK.01.1.1.01) for

funding this research through project Bioprospecting of the Adriatic Sea (KK.01.1.1.01.0002) granted to The

Scientiﬁc Centre of Excellence for Marine Bioprospecting—BioProCro.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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