Journal of Molecular Structure 1265 (2022) 133360

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Synthesis and characterization of new monothiooxalamides containing

pyridine nuclei with promising antiproliferative and antioxidant
activity
Carlos Eduardo Macías-Hernández a,b, María M. Romero-Chávez d,
Juan Pablo Mojica-Sánchez c, Kayim Pineda-Urbina a, Mariá Teresa Sumaya Martińez d,
Edgar Iván Jimenez-Ruiz d, Lisa Dalla Via b, Ángel Ramos-Organillo a,∗
a
Facultad de Ciencias Químicas. Universidad de Colima, Km 9 Carretera Colima-Coquimatlán, Coquimatlán, Colima. C.P. 28400, México
b
Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via F. Marzolo 5, 35131, Padova, Italy
c
Tecnológico Nacional de México. Instituto Tecnológico José Mario Molina Pasquel y Henríquez Unidad Académica Tamazula de Gordiano, Carretera
Tamazula-Santa Rosa No. 329, 49650 Tamazula de Gordiano, Jalisco, México
d
Unidad de Tecnologiá de Alimentos, Universidad Autónoma de Nayarit, Cd. de la Cultura "Amado Nervo" Boulevard Tepic-Xalisco s/n, Tepic, Nayarit, CP
63190, México

a r t i c l e i n f o a b s t r a c t

Article history: New family monothiooxalamides containing pyridine moiety were synthesized. The structures of monoth-
Received 20 March 2022 iooxalamides were confirmed by one-dimension NMR experiments 1 H and 13 C, and 2D NMR (COSY, HSQC,
Revised 3 May 2022
and HMBC). In addition, antiproliferative activity was evaluated in human cancer cell lines. One com-
Accepted 23 May 2022
pound showed antiproliferative effects against human tumor cell lines. Furthermore, the cytotoxic com-
Available online 25 May 2022
pound showed the ability to induce a Reactive Oxygen Species (ROS) production on the A2780-wt cell
Keywords: line. This study discarded the substitution pattern of non-active and allowed to identify the structure
Pyridine that could be used as a scaffold for the future design of monothiooxalamides with promising antipro-
Monothiooxalamide liferative capability. Moreover, the in vitro antioxidant activity was determined by DPPH•, ABTS•+ , FRAP,
Antiproliferative ORAC, and the Fe(II) chelating ability assays. Additionally, the antiradical potential was determined the-
Donor-Acceptor-Maps oretically employing Donor-Acceptor Maps (DAM), and the function was used to estimate the reactive
Fukui Function
regions involved in this process.
Antioxidant
© 2022 Elsevier B.V. All rights reserved.

1. Introduction research groups have reported de synthesis of monothiooxalamides

employing anilines and different kinds of aromatic amines obtain-
Sulfur-containing scaffolds are present in a wide range of drugs ing good yields [5–7], which represent an opportunity to explore
and natural products. These compounds have been synthesized the reactivity of other amines to obtain new families of compounds
for many years and have shown a broad range of biological ac- with promising applications. Monothiooxalamides and its analo-
tivities such as antibacterial, antiviral, cytotoxic, antiallergic, and gous have demonstrated the ability as a ligand to complex with
antimalaria [1]. For this reason, the presence of sulfur func- metals [8–10] and their cytotoxicity on human cancer cell lines
tional groups is of great interest in the search for new bioac- [7]. It also has shown applications in asymmetric metallocataly-
tive compounds. In recent years, different research groups have re- sis [9], in the synthesis of heterocyclic compounds [11], and as
ported the synthesis of thioamide derivatives employing potassium anti-inflammatory compounds [12]. In addition, these compounds
and sodium sulfide as a source of sulfur, obtaining good yields are used in crystal and cocrystal formation due to their ability to
[2–6], which represents a cheap and suitable alternative to obtain- produce hydrogen bonds [13]. Hydrogen bonds can be classified
ing this kind of compounds. Monothiooxalamides are analogous into intramolecular hydrogen bonds and intermolecular; they de-
compounds to oxalamides and thiooxalamides. The uses of this termine the crystalline structure, solubility, and interactions with
kind of compound have been widely studied. For years, different biological targets [14,15]. The stability of hydrogen bonds between
the N-H and carbonyl group was reported previously [15]. Further-
more, monothiooxalamides, oxalamides, and thiooxalamates are
∗
Corresponding author.
prominent ligands in coordination chemistry [16–20]. Due to the
E-mail address: [email protected] (Á. Ramos-Organillo).

https://doi.org/10.1016/j.molstruc.2022.133360
0022-2860/© 2022 Elsevier B.V. All rights reserved.
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

stability, monothiooxalamides can be stored for a long period, a 2.3. General procedure for synthesis of monothiooxalamides 1(a-h)
desirable characteristic in molecules with pharmacological activity.
Free radicals are highly reactive chemical species, which are To get compounds 1(a-h) (Scheme 1). First, 1.1 equivalents of
characterized by having an unpaired electron and can be stabilized the amines a-h and 10 mL of THF with 1mL of triethylamine
by both single electron transfer (SET) and hydrogen atom transfer were placed in a 100 mL ball flask, then 4.4 equivalents of el-
(HAT) [21]. Sulfur-containing compounds are well-known as good emental sulfur (0.413 g) were added, the mixture was stirred at
free-radical scavengers, specially thiocarbonyl derivatives. Taking room temperature for 30 minutes. Subsequently, 1.0 equivalent of
this into account, the incorporation of the sulfur atom in molecules chloroacetamide 1 (0.5 g) were added slowly and stirred for 72
can improve the biological effects respecting oxalamide analogs h. Monothiooxalamides were purified by column chromatography
[22–24]. Antiradical activity is related to the electron transfer phe- using toluene/ethyl acetate mixture as eluent. The products were
nomenon, in which a molecule can act as an electron donor (an- characterized by Nuclear Magnetic Resonance (1 H and 13 C NMR).
tioxidant) or electron acceptor (anti-reductant) [25]. To estimate 2-(benzylamino)-N-(pyridin-2-yl)-2-thioxoacetamide (1a)
the ability of compounds to accept or donate electrons, it is feasi- Yellow solid; yield 64% (0.319 g); m.p. 140-142 °C. 1 H-NMR
ble to determine parameters that are related to these events, such (CDCl3 , 400 MHz): δ (ppm) 10.51 (1H, s, H-7); 9.60 (1H, s, H-10);
as ionization potential (I), electron affinity (A), electron-donation 8.32 (1H, d, H-3); 8.11 (1H, d, H-6, 3 J(H6-H5) = 8.3 Hz); 7.68 (1H, m,
index (Rd ), and electron-acceptor index (Ra ). The mentioned val- H-5, 3 J(H5-H6) = 8.3 Hz); 7.31 (5H, m, H-13, H-14, H-15); 7.05 (1H,
ues could be obtained using quantum chemical Density Functional m, H-4); 4.80 (2H, d, H-11). 13 C-NMR (CDCl3 , 101 MHz): δ (ppm)
Theory (DFT). With Rd and Ra values, it is possible to construct 184.5 (C9), 155.2 (C8), 149.0 (C2), 147.5 (C3), 137.4 (C5), 134.0
a Donor-Acceptor Map (DAM), which is a plot used to find the (C12), 128.0 (C14), 127.4 (C15), 127.3 (C13), 119.7 (C4), 112.6 (C6),
better antioxidants or anti-reductants in a determined system of 49.5 (C11). MS (TOF): m/z = 270.0698 [M - H]− . E.A. C14 H13 N3 OS
molecules (Fig. 3) [26]. Moreover, the Fukui function is a suitable (%) Found: C (61.88), H (4.96), N (15.62); Calculated: C (61.97), H
tool to understand better the zones in the molecules in which the (4.83), N (15.49). HPLC purity: 99.51%.
electron-transfer phenomenon can occur [27]. In search for new N-(pyridin-2-yl)-2-((pyridin-2-ylmethyl)amino)-2-thioxoacetamide
antiradical compounds and their mechanisms, both computational (1b)
calculations were performed. Yellow solid; yield 53 % (0.264 g); m.p. 130-133 o C. 1 H-NMR
Monothiooxalamides are planar structures due to the in- (CDCl3 , 400 MHz): δ (ppm) 10.72 (1H, s, H-10); 10.49 (1H, s,
tramolecular hydrogen bonds formed between the -NH and car- H-7); 8.56 (1H, d, H-17); 8.31 (1H, m, H-3); 8.19 (1H, d, H-6,
bonyl or thiocarbonyl groups [15]. It is known that planar struc- 3J 3
(H6-H5) = 8.3 Hz); 7.67 (2H, m, H-3, H-5, J(H5-H6) = 8.3 Hz); 7.24
tures can intercalate between DNA base pairs; it leads to can- (1H, d, H-14); 7.20 (1H, d, H-16); 7.04 (1H, ddd, H-4); 4.86 (2H, d,
cer cell death. Furthermore, the incorporation of planar structures H-11). 13 C-NMR (CDCl3 , 101 MHz): δ (ppm) 184.3 (C9), 155.3 (C8),
in monothiooxalamides can improve biological activity [28–31]. 152.4 (C13), 149.2 (C2), 148.4 (C17), 147.5 (C3), 137.4 (C5), 136.0
On the other hand, pyridine moiety is usually incorporated in (C15), 122.0 (C16), 121.1 (C14), 119.6 (C4), 112.7 (C6), 49.5 (C11).
molecules with diverse biological activities such as anticancer MS (TOF): m/z = 271.0653 [M - H]− . E.A. C13 H12 N4 OS (%) Found:
[32–37], antimicrobial, and antifungal [35,38–40]. This study re- C (56.99), H (4.83), N (20.43); Calculated: C (57.34), H (4.44), N
ports the synthesis of novel monothiooxalamides with pyridine (20.57). HPLC purity: 97.17%.
moiety. Additionally, antiproliferative and antiradical capacity were (S)-2-((1-phenylethyl)amino)-N-(pyridin-2-yl)-2-thioxoacetamide
determined. (1c)
Yellow semisolid; yield 64 % (0.321 g); 1 H-NMR (CDCl3 , 400
MHz): 1 H-NMR (CDCl3 , 400 MHz): δ (ppm) 10.50 (1H, s, H-7); 9.59
2. Experimental methods (1H, s, H-10); 8.30 (1H, d, H-3); 8.11 (1H, d, H-6); 7.68 (1H, m, H-
5); 7.27 (5H, m, H-14, H-15, H-16); 7.04 (1H, m, H-4); 5.49 (1H, dq,
2.1. Materials and equipment H-11, 3 J(H11-H12) = 6.9 Hz); 1.61 (3H, d, H-12, 3 J(H12-H11) =6.9 Hz).
13 C-NMR (CDCl , 101 MHz): δ (ppm) 183.2 (C9), 155.3 (C8), 149.1
3
The reagents for the synthesis of the target compounds were (C2), 147.5 (C3), 139.3 (C13), 137.4 (C5), 127.9 (C14), 127.1 (C15),
purchased from commercial suppliers and used without further 125.5 (C16), 119.7 (C4), 112.6 (C6), 54.4 (C11), 19.1 (C12). MS (TOF):
purification. The solvents for column chromatography were dis- m/z = 284.0858 [M - H]− . E.A. C15 H15 N3 OS (%) Found: C (63.05),
tilled prior to use. A 400 MHz Bruker equipment, TMS was used H (5.11), N (14.60); Calculated: C (63.13), H (5.30), N (14.73). HPLC
as an internal reference to obtain the NMR spectra. Chemical shifts purity: 98.48%.
were reported in ppm. Melting points were obtained using a Melt- 2-(phenethylamino)-N-(pyridin-2-yl)-2-thioxoacetamide (1d)
Temp II apparatus in capillaries and were not corrected. Yellow solid; yield 42 % (0.208 g); m.p. 102-106 o C. 1 H-NMR
(CDCl3 , 400 MHz): δ (ppm) 10.50 (1H, s, H-7); 9.45 (1H, s, H-10);
8.31 (1H, d, H-3); 8.10 (1H, d, H-6, 3 J(H6-H5) = 8.3 Hz); 7.67 (1H,
2.2. Procedure for synthesis of chloroacetamide (1) td, H-5, 3 J(H5-H6) = 8.3 Hz); 7.27 (2H, m, H-14); 7.19 (3H, m, H-
15, H-16); 7.04 (1H, ddd, H-4); 3.90 (2H. td, H-11, 3 J(H11-H12) = 7.2
Chloroacetamide 1 was obtained using a method that Mantu
Hz); 2.97 (2H, t, H-12, 3 J(H12-H11) = 7.2 Hz). 13 C-NMR (CDCl3 , 101
described (Scheme 1)[41]. First, 10 mmol of amine I (0.941 g) and
MHz): δ (ppm) 184.5 (C9), 155.2 (C8), 149.0 (C2), 147.4 (C3), 137.4
12 mmol of triethylamine (1.7 ml) were placed in a 100 ml flask,
(C5), 136.6 (C13), 127.9 (C14), 127.6 (C15), 126.0 (C16), 119.7 (C4),
then was dissolved in 50 ml of tetrahydrofuran (THF). Next, the
112.6 (C6), 46.4 (C11), 32.5 (C12). MS (TOF): m/z = 284.0859 [M
reaction mixture was placed in an ice bath. Then 12 mmol of chlo-
- H]− . E.A. C15 H15 N3 OS (%) Found: C (63.24), H (5.49), N (14.96);
racetyl chloride (1 mL) was added dropwise over 30 minutes. Af-
Calculated: C (63.13), H (5.30), N (14.73). HPLC purity: 98.86%.
ter the addition, the ice bath was removed, and the reaction was
2-(cyclohexylamino)-N-(pyridin-2-yl)-2-thioxoacetamide (1e)
stirred at room temperature for 4 hours. Next, the white precip-
Yellow semisolid; yield 39 % (0.195 g); 1H-NMR (CDCl3, 400
itate (triethylamine hydrochloride) was filtered off, and the solu-
MHz): δ (ppm) 10.56 (1H, s, H-7); 9.29 (1H, s, H-10); 8.31 (1H,
tion was washed with Na2 CO3 (10%) (3×100mL) followed by water
d, H-3); 8.11 (1H, d, H-6, 3 J(H6-H5) = 8.3 Hz); 7.68 (1H, td, H-5,
(2×100mL). Finally, the solution was dried with MgSO4 , and the
3J
products were recrystallized with methylene chloride. (H5-H6) =8.3 Hz); 7.03 (1H, m, H-4); 4.18 (1H, m, H-11); 2.02 (2H,

2
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

Scheme 1. General procedure of synthesis of 1a-1h.

Fig. 1. Three-center hydrogen bond formation for compounds 1b and 1h.

dd, H-12, 3 J(H12-H13) = 9.5 Hz); 1.72 (2H, m, H-13, 3 J(H13-H12) = 9.5 C13 H18 N4 O2 S (%) Found: C (53.12), H (6.35), N (19.35); Calculated:
Hz); 1.60 (1H, m, H-14); 1.34 (4H, m, H-12, H-13); 1.20 (1H, m, C (53.04), H (6.16), N (19.03). HPLC purity: 99.78%.
H-14). 13 C-NMR (CDCl3 , 101 MHz): δ (ppm) 182.6 (C9), 155.5 (C78, 2-((3-morpholinopropyl)amino)-N-(pyridin-2-yl)-2-
149.1 (C2), 147.5 (C3), 137.3 (C5), 119.7 (C4), 112.5 (C6), 53.9 (C11), thioxoacetamide (1h)
29.9 (C12), 24.3 (C14), 23.5 (C13). E.A. C13 H17 N3 OS (%) Found: Yellow solid; yield 66 % (0.331 g); 1 H-NMR (CDCl3, 400 MHz):
C (59.61), H (6.31), N (16.12); Calculated: C (59.29), H (6.51), N δ (ppm) 11.50 (1H, s, H-10); 10.55 (1H, s, H-7); 8.31 (1H, d, H-3,
3J
(15.96). (H3-H4) = 4.9 Hz); 8.19 (1H, d, H-6); 7.69 (1H, m, H-5); 7.04 (1H,
N-(pyridin-2-yl)-2-(pyrrolidin-1-yl)-2-thioxoacetamide (1f) ddd, H-4, 3 J(H4-H3) = 4.9 Hz); 3.82 (4H, t, H-16); 3.72 (2H, dd, H-
Yellow solid; yield 74 % (0.369 g); m.p. 118-120 °C. 1H-NMR 11); 2.55 (2H, m, H-13); 2.44 (4H, d, H-15); 1.82 (2H, dt, H-12). 13 C-
(CDCl3, 400 MHz): δ (ppm) 9.90 (1H, s, H-7); 8.36 (1H, d, H-3); 8.20 NMR (CDCl3 , 101 MHz): δ (ppm) 184.4 (C9), 155.5 (C8), 149.3 (C2),
(1H, d, H-6, 3 J((H6-H5) = 8.3 Hz); 7.75 (1H, ddd, H-5, 3 J(H5-H6) = 8.3 147.4 (C3), 137.3 (C5), 119.5 (C4), 112.6 (C6), 65.5 (C16), 57.4 (C13),
Hz, 3 J(H5-H4) = 7.3 Hz); 7.10 (1H, ddd, H-4, 3 J(H4-H5) = 7.3 Hz); 4.07 52.9 (C15), 46.8 (C11), 21.6 (C12). MS (TOF): m/z = 307.1228 [M
(2H, t, H-11); 3.87 (2H, t, H-11); 2.06 (4H, m, H-11, H-12). 13 C- - H]− . E.A. C14 H20 N4 O2 S (%) Found: C (54.51), H (6.67), N (18.19);
NMR (CDCl3 , 101 MHz): δ (ppm) 184.7 (C9), 159.7 (C8), 149.6 (C2), Calculated: C (54.52), H (6.54), N (18.17). HPLC purity: 10 0.0 0%.
147.2 (C3), 137.3 (C5), 119.3 (C4), 112.8 (C6), 54.0 (C11), 52.6 (C11),
25.7 (C12), 22.6 (12). MS (ESI): m/z = 234.1493 [M - H]− . E.A.
2.3. Antiproliferative activity
C11 H13 N3 OS (%) Found: C (56.10), H (5.72), N (17.88); Calculated:
C (56.15), H (5.57), N (17.86). HPLC purity: 10 0.0 0%.
Cell culture was carried out in the appropriate culture medium
2-((2-morpholinoethyl)amino)-N-(pyridin-2-yl)-2-thioxoacetamide
for each cell line (A2780-wt, A2780-cis, HT-29, and MSTO-211H).
(1g)
A2780-wt (ovarian adenocarcinoma), A2780-Cis (ovarian adenocar-
Yellow solid; yield 68 % (0.341 g); m.p. 88-90 °C. 1 H-NMR
cinoma cisplatin-resistant), and HT-29 (colorectal adenocarcinoma)
(CDCl3, 400 MHz): δ (ppm) 10.49 (1H, s, H-7); 9.86 (1H, s, H-
cells were grown in RPMI 1640 medium (Sigma Chemical Co.).
10); 8.32 (1H, ddd, H-3, 3 J(H3-H4) = 4.9 Hz); 8.14 (1H, dt, H-6,
3J 3 3
MSTO-211H cells (human biphasic mesothelioma) were grown in
(H6-H5) = 8.3 Hz, J(H6-H4) = 1 Hz); 7.69 (1H, m, H-5, J(H5-H6) = 8.3 RPMI 1640 medium (Sigma Chemical Co.) supplemented with 2.38
Hz); 7.05 (1H, ddd, H-4, 3 J(H4-H3) = 4.9, Hz, 3 J(H4-H6) = 1 Hz); 3.68 g/L of Hepes, 0.11 g/L of sodium pyruvate, and 2.5 g/L of glucose.
(6H, m, H-11, H-15); 2.65 (2H, t, H-12); 2.47 (4H, d, H-14). 13 C- In addition, both mediums were supplemented with 10% of fetal
NMR (CDCl3 , 101 MHz): δ (ppm) 184.3 (C9), 155.3 (C8), 149.1 (C2), bovine serum (Invitrogen) inactivated by heat, 100 U/ml of peni-
147.5 (C3), 137.3 (C5), 119.7 (C4), 112.6 (C6), 65.9 (C15), 54.1 (C12), cillin, 100 μg/ml of streptomycin, and 0.25 μg/ml of amphotericin
52.2 (C14), 41.5 (C11). MS (ESI): m/z = 293.1756 [M - H]− . E.A. B. All cell lines were incubated at 37 °C with 5% CO2 for 24 hours

3
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

in 24-well cell culture plates. After the incubation, the cells were results were expressed as Vitamin C equivalents Antioxidant Ca-
treated with compounds at different concentrations and then incu- pacity (VCEAC) in mg/100 mL [25,43].
bated for 72 hours in standard conditions. Cell viability was deter-
mined in a Burker chamber by trypan blue exclusion assay. 2.5.4. FRAP (Ferric Reducing Antioxidant Power) method
The FRAP method measures an antioxidant’s ability to reduce
2.4. Intracellular ROS production assay the ferric ion (Fe3+ ) to the ferrous ion (Fe2+ ) in an acid medium.
FRAP assay was carried out according to the procedure developed
ROS determination on the A2780 human cancer cell line was by Hinneburg and coworkers in 2006 with some modifications.
performed by 2’,7’-dichlorofluorescein diacetate. 10 0 0 0 cells were First, aliquots of 25 μL of each compound previously dissolved in
seeded in 100 μL complete medium in 96 well plate and incubated DMSO were mixed with 63 μL of potassium phosphate buffer (0.2
in standard conditions for 24 hours. Then, the medium was dis- M, pH 6.5) and 63 μl of an aqueous potassium ferrocyanide so-
carded, cells were washed with 100 μL of PBS (Phosphate-buffered lution [K3 Fe(CN)6 ] at 1%. This mixture was incubated at 50°C for
saline) buffer and incubated in the dark at 37 °C with dichlorofluo- 30 minutes. Afterward, 63 μL of a 10% trichloroacetic acid solution
rescein diacetate 10 μM in PBS-glucose 10 mM for 20 minutes. Af- was added, and the mixture was stirred vigorously with a vortex.
ter incubation, the solution was removed, cells were washed with From this solution, aliquots of 63 μl were taken and mixed with
PBS, treated with compound 1b or antimycin A, and measured flu- 63 μl of distilled water and 12.5 μl of 0.1% ferric chloride (FeCl3 ).
orescence for 120 minutes using a microplate reader (Victor X3 Thereupon the absorbance was read at 700 nm. All samples were
Multilabel plate reader) (Perkin Elmer) at λex=485 nm and λem= screened in triplicate, and ascorbic acid was used as a reference
527 nm. Each value was calculated as the average of three inde- compound for the standard curve. The results were presented as
pendent experiments, each with eight replicates. ROS production Vitamin C equivalents mg/100 mL (VCEAC) [44]
percentage was determined using the final values at 120 min and
fluorescence of control as 100 %. 2.5.5. ORAC (Oxygen Radical Absorbance Capacity) method
ORAC is a HAT-based assay and measures the antioxidant inhi-
2.5. Antioxidant activity bition of fluorescein oxidation induced by peroxyl radicals (ROO•).
The mixture exhibits a change of fluorescence, which is detected at
2.5.1. Equipment 485 nm of excitation and 520 nm of emission. Thermal decompo-
A Biotek Powerwave XS microplate reader was used to measure sition of 2,2 -azobis (2-amidinopropane)-2-dihydrochloride (APPH)
the absorbance and fluorescence of the samples. Micropipettes was chosen as the peroxyl radicals generating system, and analyses
(Thermo Scientific finnpipette F2 and BioPette Plus) of different ca- were conducted in phosphate buffer pH 7.0 at 37°C [45]
pacities (10, 25, 10 0, 20 0, 10 0 0 μL) were used to measure sample Monothiooxalamides ORAC determination was according to the
aliquots and prepare required solutions. Samples were prepared in method described by Huang et al. (2002) with some modifica-
Eppendorf tubes of 0.6 and 1 mL. tions. The assay began with phosphate buffer preparation (75 mM,
pH=7.0± 0.2), which was used as a medium for the fluorescein,
2.5.2. DPPH• (2,2-Diphenyl-1-picrylhydrazyl) method radical, and sample solutions. A fluorescein stock solution (44 mg
The DPPH• is a stable organic nitrogen radical, which turns a in 100 mL of buffer) was prepared and kept in the dark condition
deep purple color in solution with methanol or ethanol. Antioxi- at 4°C, then 164 μL of this solution was taken and diluted in 25 mL
dant capacity by the DPPH• method is based on the measurement of buffer to a final concentration of 78 nM. The radical was pre-
of the loss of DPPH• color at 515 nm after its reaction with test pared by dissolving 0.598 g of APPH in 10 mL of buffer (221 mM).
compounds. The underlying chemistry of this reaction involves ei- As a reference antioxidant, a Trolox stock solution was prepared
ther hydrogen atom transfer (HAT) or single electron transfer (SET) (400 μM), diluted with the same phosphate buffer to 30 0, 20 0,
[42] 100, 20, and 25 μM working solutions. Samples were dissolved in
The DPPH• antioxidant assay began with preparing a DPPH• DMSO (5 mg/100 μL), then 1:100 and 1:200 dilutions with phos-
working (7.4 mg/100 mL in ethanol), kept at room temperature phate buffer were performed. The sample’s aliquots of 75 μL of the
(25ºC ± 2) in the dark to avoid photodegradation. After that, sample were mixed with 75 μL of fluorescein (78nM) and incu-
aliquots of 15 μL of the samples dissolved in dimethyl sulfoxide bated for 30 min at 37ºC. 50 μl of APPH (221mM) were added,
(DMSO) were mixed with 75 μL of the DPPH• solution. The mix- and measurements were made at 37°C, monitoring the decrease of
ture was stirred vigorously with a vortex and left to stand at room fluorescence at 520 nm with 1-minute intervals for 150 minutes
temperature for 1h. From this solution, aliquots of 50 μL were [46]. The results were expressed as μmol Trolox equivalent /mL ac-
taken, and the absorbance was read at 515 nm. A solution of 15 μL cording to Equation 1.
of ethanol and 75 μL of DPPH• was used as a blank. The results
AUC − AUC ◦
were expressed as Trolox equivalent antioxidant capacity (TEAC) ORAC = fd (1)
AU CT rolox − AUC ◦
with values based on micromoles of Trolox L-1(μmol ET/L) [25,43]
Where AUC° is the area under the curve for control, AUC is of
2.5.3. ABTS•+ (2 2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) the sample, AUCTrolox is for Trolox, and f is the dilution factor for
method each monothiooxalamide [47].
ABTS assay is based on quantifying the radical cation ABTS•+
discoloration. This radical was obtained by reacting ABTS(NH4 )2 (7 2.5.6. Chelating activity of Fe(II)
mM in water) with a potassium persulfate (2.45 mM in water) in The chelation of ferrous ions by monothiooxalamides 2-
a 1:0.5 v/v ratio. The reaction was kept in the dark for 14-16 h at aminopyridine derivatives was estimated by their ability to inhibit
4°C before being used. After, the product was diluted with ethanol ferrozine-Fe2+ complex formation. The assay is based on reacting
(1:20) until it reached an absorbance of A=0.7 (±0.1) at 754 nm. a chelating molecule with the ferrous ion, then the ferrozine is
From solution was taken 490 μL and mixed with 10 μL of each added, which will be responsible for trapping the ferrous ion that
compound previously dissolved in DMSO, and without delay, the was not chelated by the chelating molecule. This reaction generates
reaction was stirred in a vortex and left to stand for 7 to 10 min- a blue-green color that absorbs at 562 nm. The chelating activity
utes. Aliquots of 50 μL were taken to measure its absorbance at of monothiooxalamides was according to the method described by
754 nm. Ascorbic acid was used as a reference standard, and the Gulcin et al. (2003).

4
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

Fig. 2. ROS production percentage of compound 1b, and antimycin A, taken as reference, on A2780 cell line. The symbol ∗ refers to the pairwise comparison with the control
with a significative difference according to the Tukey test (p < 0.05). The letters “ns” indicate non-significative difference with control.

Samples were dissolved in DMSO, then aliquots of 15 μL from

this solution were taken into an Eppendorf tube and mixed with
7.5 μL of a solution of ferrous sulfate (2 mM in water) and 67.5
μL methanol. The mixture was stirred in a vortex and kept for
five minutes in incubation at room temperature, then 50 μL of a
ferrozine solution (5 mM in water) were added, the reaction was
stirred and kept for 10 minutes at room temperature. Subsequently,
aliquots of 100 μL were taken, and the absorbance was read at
562 nm. A standard curve was constructed using different concen-
trations of ethylenediaminetetraacetic acid (EDTA) as a reference
compound. The results were expressed as millimoles of EDTA L-1
(mM EDTA L-1) [48].

2.6. Computational details

2.6.1. Donor-Acceptor Maps

The molecules were optimized without symmetry restrictions
at a Density Functional Theory level implemented in ORCA soft-
ware [49]. The all-electron def2-SVP basis set [50] was employed
with the PBEh-3c hybrid functional [51]. Frequency calculations
verified all minima energy states found through optimizations.
The optimized structures were used to calculate each system’s
anion and cation energies. The results were used to compute the Fig. 3. Distinct regions in a Donor-Acceptor Map (DAM) [25].

vertical ionization potential (I) and electron affinity (A) from

Donor and acceptor indexes are used to normalize the values of
I = E (N − 1 ) − E (N )
electron-donating and electron-accepting powers for the values of
and the Na and F atoms, respectively. The donor index is defined as:
A = E (N ) − E (N + 1 ) ω−
Rd =
Where E(N) is the energy in its neutral state, E(N-1) is the energy ωNa
−

of its cation, and E(N+1) is the energy of the corresponding anion. While the acceptor index is:
With these values, the electron-donating (ω− ) and electron-
accepting (ω+ ) powers [52] were determined for the 12 systems
ω+
Ra =
by: ωF+
In this sense, any molecule with a donor index value of 1
(3I + A)2
ω− = should have a similar performance to the sodium atom, a well-
16(I − A )
known electron donor. The acceptor index is employed likewise,
and using the fluor atom as reference instead. Suitable electron donors
(I + 3A)2 are characterized by donor index values below 1, while good
ω+ = electron acceptors have acceptor index values above 1. A donor-
16(I − A )
acceptor map can be constructed with both indexes’ values to clas-

5
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

Table 1
Optimization process for synthesizing monothiooxalamides 1(a-h).

Yield
Compound Amine State
Without base (72h) Triethylamine (24h)

1a Yellow powder 41 % 64 %

1b Yellow powder 31 % 53 %

1c Yellow semisolid 56 % 64 %

1d Yellow powder 0% 42 %

1e Yellow semisolid 0% 39 %

1f Yellow powder 41 % 74 %

1g Yellow powder 36 % 68 %

1h Yellow powder 33 % 66 %

Fig. 4. Optimized structures of the molecules considered in DAM.

sify a set of molecules as good or bad electron donors or acceptors 2.6.2. Fukui function f − (r)
(Fig. 3). The Fukui functions describe changes in the electron density of
A donor-acceptor map was done with the Ra and Rd val- a system after a change in its number of electrons. These functions
ues for the molecules under study, where they were compared can predict the regioselectivity of nucleophilic and electrophilic re-
to well-known antioxidants: Vitamin C, Trolox and Resveratrol actions. In the context of this work, the Fukui function can be used
[25–27,53,54]. to predict the radical-molecule interaction site [55–59]. Due to the

6
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

Table 2 using Lawessonś reagent under reflux conditions is reported by Pi-

Chemical shift in ppm (13 C-NMR) for monothiooxalamides 1(a-h) and
otrkowska et al. in 2007, obtaining good yields [62]. Nevertheless,
chloroacetamide (1) in CDCl3 .
the reagents are more expensive than Zavaarzin and coworkerś al-
Chemical shift (δ in ppm) ternative. Additionally, the fact that our method does not require
Compound
C= O C=S (1a-1h) or CH2 (1) reflux conditions gives it a suitable alternative.
1 164.6 42.8
1a 155.2 184.5 3.2. Structural elucidation
1b 155.3 184.3
1c 155.3 183.2
1d 155.2 184.5 In 1 H NMR for compounds 1(a-h), the characteristics broad sig-
1e 155.5 182.6 nals for amide and thioamide groups appear at around 10 and 9
1f 159.7 184.7 ppm, respectively, except for compounds 1b and 1h. Thioamide
1g 155.3 184.3
proton is the most downfield signal (See supporting information).
1h 155.5 184.4
This effect could be associated with deprotection of this position
Table 3 due to any possible interaction of thioamide proton with picolyl
Antiproliferative activity of monothiooxalamides on human cancer cell lines and morpholine group nitrogen. In both cases, the chemical shift
expressed as a mean of at least three experiments in duplicate ± standard for amide proton is deshielded, respecting the rest of compounds
deviation.
more than 1 ppm (10.72 and 11.50 ppm, respectively). This behav-
GI50 (μM) ior in 1 H NMR was reported previously by Martínez and cowork-
Compound ers in oxamides when three-center intramolecular hydrogen bonds
A2780-wt A2780-Cis MSTO-211H HT-29
are happening [63]. The three-center hydrogen bonds proposed are
1a >20 >20 >20 >20
shown in Fig. 1. On compound 1b, this intramolecular hydrogen
1b 0.19 ±0.02 2.77±1.30 >20 18.8±4.6
1c >20 >20 >20 >20 bond is possible due to the presence of N-atom of the picolyl group
1d >20 >20 >20 >20 and produces a downfield effect on thioamide proton.
1e >20 >20 >20 >20 On the other hand, for compound 1a, this hydrogen bond is not
1f >20 >20 >20 >20 possible. On compound 1g, if we considered the morpholyl group
1g >20 >20 >20 >20
1h >20 >20 >20 >20
as a chair conformation, the steric hindrance makes the formation
of the three-center hydrogen bond impossible. However, for com-
pound 1h, this is feasible. This is supported by an optimized struc-
ture for compound 1h (Fig. 4).
characteristics of the systems under study, the Fukui function for
For compounds 1(e-h), aliphatic protons appeared between
the removal of an electron was calculated as:
1.20-4.07 ppm. On compound 1f, pyrrolyl group protons are not
f − (r ) = ρN (r ) − ρN−1 (r ) equivalent as it was expected (See supporting information). It can
suggest a possible orientation of the ring protons to a thiocarbonyl
Where ρN (r ) is the electron density of the system in its neu-
group.
tral state and ρN−1 (r ) is the electron density of the same system
In 13 C-NMR for compounds 1(a-h), the thiocarbonyl group’s
after removing an electron. These calculations were done with the
characteristic signal is the most downfield (Table 2). It appears be-
Chemcraft software [60] using electron density cubes.
tween 182.3-184.5 ppm. On the other hand, the carbonyl signal ap-
peared between 155.2-159.7 ppm. The deshielded effect presented
3. Results and discussions
in the thiocarbonyl group is more intense than carbonyl, it is be-
cause the C=S bond is longer than the C=O bond [64].
3.1. Synthesis

Monothiooxalamides 1(a-h) were obtained using the method 3.3. Antiproliferative activity
described by Zavarzin and coworkers with some modifications[5,6].
The reactions were carried out in two steps described in Scheme 1, Antiproliferative effect of monothiooxalamides 1(a-h) was stud-
employing S8 as a source of sulfur and chloroacetamide 1 as a sub- ied in A2780 (human ovarian adenocarcinoma), A2780-Cis (human
strate. Nevertheless, N, N-dimethylformamide (DMF) was initially ovarian adenocarcinoma resistant to Cis-Platin), MSTO-211H (hu-
changed for tetrahydrofuran (THF) due to better yields. The better man biphasic mesothelioma), and HT-29 (colorectal adenocarci-
yields obtained with THF are related to the fact that this solvent noma). The obtained results, expressed as GI50 , values are shown
can be easier removed than DMF at reduced pressure under a ro- in Table 3. The most active compound was 1b, while the strictly
tative evaporator, employing a temperature lower than 40o C and structurally related 1a and all other compounds (1c-1h) showed
reducing the risk of thermal degradation of products during this GI50 values higher than 20 μM. A2780 cell line is the most sensi-
step. In addition, the use of THF also facilitates the purification tive, followed by A2780 cis-platinum resistant cells; nevertheless,
process. Furthermore, to improve the chemical yield, triethylamine 1b is almost 15 times more active on A2780 wild-type cells. 1b
was added as a base, increasing the yield and reducing the reaction also exerts a slight activity on the HT-29 cell line. Instead, on the
time. Table 1 shows that the reaction conditions for compounds MSTO-211 H cell line, 1b appears ineffective in our experimental
1(a-h) are shown without using any base and triethylamine. In all conditions.
cases, reaction times are reduced from 72 hours (without any base) According to the structure, it is possible to establish that the
to 24 hours with base, with a marked improvement in yields. The substitution of the picolyl group in the R position (compound 1b)
yields improvement can be explained because triethylamine in- improves the cytotoxic activity concerning the benzyl group (com-
creases the reactivity of amine, reducing their protonation. Accord- pound 1a). The presence of N-atom in the picolyl group of 1b
ing to Oosthoek-de Vries et al., the role of tertiary amines such as seems to be crucial for intramolecular hydrogen bond formation
triethylamine as a base is crucial to increasing the reaction rate. In (Fig. 1). This interaction can reduce the rotability of methylene
addition, triethylamine neutralizes the HCl produced during de re- fragment, improving cytotoxicity. Moreover, according to Matsson
action, reducing the probability of acidic hydrolysis of monothioox- et al. in 2026, intramolecular hydrogen bonds can improve the cell
alamides [61]. Another method for monothiooxalamides synthesis permeability of compounds; it can also explain the good uptake of

7
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

Table 4
OSIRIS physicochemical and toxicological prediction. 2-aminopyridine (2-AMP), low risk (green), high risk (red).

Compound
Property
1a 1b 1c 1d 1e 1f 1g 1h 2-AMP

Toxicity Risks Mutagenic

Tumorigenic
Irritant
Reproductive
Physicochemical cLogP 1.28 0.34 1.57 1.71 1.42 0.92 -0.47 -0.01 0.33
Properties LogS (Solubility) -3.41 -2.64 -3.49 -3.53 -3.67 -2.42 -1.58 -1.85 -1.42
Molecular Weight 271 272 285 285 263 235 294 308.6 94.0
TPSA 86.11 99.0 86.11 86.11 86.11 77.32 98.58 98.58 38.91
Druglikeness 2.7 2.75 3.33 3.44 -2.72 4.49 4.46 4.37 -1.63
Drug Score 0.85 0.90 0.85 0.85 0.46 0.56 0.94 0.93 0.34

Table 5
Monothiooxalamides results were obtained by DPPH•, ABTS•+, FRAP, ORAC and Fe(II) chelation methods.

Compounds DPPH• (TEAC∗ ) ABTS•+ (VCEAC∗ ∗ ) FRAP (VCEAC∗ ∗ ) ORAC (μM ET/mL) Chelating Activity (mM EDTA/L)

1a 252.56 ± 10.05
bc
5.49 ± 0.16
b
242.71 ± 2.44
bc
254.6 ± 57.36
a
NA
1b 398.11a ± 19.89 11.28b ± 0.38 177.54g ± 1.42 NA 0.02c ± 0.074
1c 880.56e ± 6.74 23.49b ± 0.24 215.49d ± 1.42 137.68ab ± 23.81 0.34c ± 0.074
1d 273.11b ± 9.77 5.57b ± 1.07 263.56a ± 1.72 51.86b ± 15.10 0.17c ± 0.532
1e 1000de ± 4.41 10.84b ±3.94 234.22c ± 6.07 184.67ab ± 54.09 NA
1f 1144.44cd ± 8.39 24.73b ±3.95 193.02f ± 7.44 NA NA
1g 1116.67cd ± 9.28 76.37a ± 30.94 194.64ef ± 0.37 NA NA
1h 244.78bc ± 5.36 17.35b ± 7.15 205.01de ± 2.66 NA 1.85b ± 0.68

NA (Not Active); ∗ TEAC: μmol Trolox/L; ∗ ∗ VCEAC: mg Vitamin C/100 mL

The letters (a-f) in each column refer to the pairwise comparisons between groups with a significant difference according to Tukey’s HSD
test (p < 0.05).

1b by cancer cells and consequently their prominent antiprolifer- aminopyridine); however, only compound 1f has a high risk of ir-
ative effect in comparison to the rest of compounds [65]. Taking ritant effect due to the presence of the pyrrolidine group.
into account the structural characteristics of 1b, this must be con- Finally, the drug score expresses the potential of compounds to
sidered in later designs of a promising antitumor compound. qualify as a drug. This parameter combines physicochemical pa-
rameters with toxicity risk to obtain a summary. Osiris has been
used widely to select suitable compounds [73–76]. In this study,
3.4. Intracellular ROS production assay
compounds 1g, 1h, and 1b have the best drug-score values; com-
pound 1b is the active compound on the human cancer cell line,
One of the events that can induce programmed cell death is
suggesting that 1b can be a promising drug candidate. On the
the increase of Reactive Oxygen Species (ROS)[66–70]. To explore
other hand, compounds 1f and starting material (2-AMP) are not
the cell dead mechanism by which the active compounds can lead
good drug candidates.
to cell death, ROS production was evaluated on the most sensi-
tive A2780 cell line. Compound 1b was also evaluated for com-
3.6. Antioxidant activity
parison, and antimycin A was used as a reference for ROS produc-
tion (Fig. 2). The results are expressed as a percentage of the con-
The results were expressed as a mean ± standard deviation
trol. After two hours in the presence of test compounds, 1b (10μM)
(SD) and evaluated by one-way ANOVA with a α =0.05, using Rstu-
produced 63 % more ROS on the A2780 cell line than non-treated
dio (Version 4.1.4). In addition, the Tukey’s honestly significant
cells, and this could be retained consistent with the antiprolif-
difference test (Tukey’s HSD) was applied for each assay (DPPH•,
erative effect exerted on cells. Moreover, as expected, antimycin
ABTS•+ , FRAP, ORAC, and chelating ability). All values achieved are
A was significantly effective in ROS production even at low
shown in Table 5.
concentration.
3.6.1. Antioxidant activity by DPPH• method
3.5. Physicochemical and toxicologic properties prediction In the DPPH• assay, all monothiooxalamides showed significant
activity (p<0.05). Compound 1f reached the highest value with
In order to do a good design of new active compounds, the 1144.44 μmol ET/L, a result close to 1g (1116.67 μmol ET/L) and
physicochemical properties were estimated using OSIRIS Property 1e (10 0 0 μmol ET/L). These derivatives showed better antioxidant
Explorer (Table 4). Log P, a coefficient of partition n-octanol/water, activity than 1c (880.56 μmol ET/L) and 1b (398.11 μmol ET/L). On
is a parameter that describes the hydrophobicity of compounds; the other hand, the lowest antiradical activity was observed for 1a,
this parameter must be less than 5 to achieve a good absorption 1d, and 1h with 273.11, 252.56, and 244.78 μmol ET/L, respectively
process in the live organisms [71]. All compounds are in a range (Table 5).
between -0.47 to 1.71. LogS expresses the drug solubility, which is According to a report by Pior et al. (2005) [42], various an-
a parameter for predicting the absorption process [72]. The LogS tioxidants with the ability to donate electrons or hydrogen atoms
values must be greater than -4 to achieve a good absorption, dis- can decrease their reaction times or be inert towards DPPH• due
tribution, and elimination process. All compounds have good solu- to steric hindrance. That is consistent with the antiradical activity
bility; nevertheless, the bests are 1g and 1h. Finally, OSIRIS Prop- difference observed between 1f and 1h, the smallest and largest
erty Explorer estimated toxicity risks. All compounds are suitable monothiooxalamide, respectively [42]. Another factor involved in
in terms of toxicity respecting starting material 2-AMP (2- the reaction is the presence of intramolecular hydrogen bonds.

8
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

Table 6
Molecular descriptors were calculated to obtain the gas phase DAM. Ionization potential (I), electron
affinity (A), electron-donation power (ω− ), and electron-acceptor power (ω+ ) are in kcal/mol.

Compound I A ω− ω+ Ra Rd A symbol used in DAM

1a 184.79 -4.94 99.43 9.51 0.11 1.25

1b 180.78 -7.35 95.08 8.36 0.10 1.19

1c 179.31 -8.42 93.34 7.89 0.10 1.17

1d 191.84 5.66 113.39 14.64 0.18 1.43

1e 183.33 -4.29 99.19 9.67 0.12 1.25

1f 179.74 -14.88 88.29 5.86 0.07 1.11

1g 179.56 -1.70 99.42 10.49 0.13 1.25

1h 184.07 -9.43 95.15 7.83 0.09 1.20

Trolox 159.05 -13.36 77.97 5.12 0.06 0.97

Ascorbic acid 195.24 -6.12 104.26 9.70 0.11 1.31

Resveratrol 159.20 8.28 97.76 14.02 0.15 1.22

− −
Fig. 5. Fukui function f (r) mapped over an isodensity. n f (r) mapped over an isodensity.

Fig. 6. Gas-phase Donor-Acceptor Map.

9
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

Fig. 7. Proposed mechanism for free-radical stabilization by monothiooxalamides.

It can be observed when structures of 1g and 1h are compared the monothiooxalamides exhibited a significant capacity to reduce
(Fig. 1). The additional methylene (-CH2 -) of 1h allows the forma- the Fe+3 to Fe+2 , with values from 177 until 264 VCEAC (P<0.05).
tion of a three-center hydrogen bond with the thioamide group, Compound 1d (263.56 VCEAC) showed the highest reducing ca-
which decreases the molecule possibility to donates both hydrogen pacity, with a measurement close to 1a (242.71 VCEAC). The final
and electrons of nitrogen in the reaction with DPPH•. ranking was: 1e> 1c> 1h> 1g> 1f>1b (Table 5).
One of the most important differences for future structural The presence of electron-donating atoms can explain monoth-
modifications is the presence of the methyl near the thioamide iooxalamides behavior as the sulfur atom of the thiocarbonyl
group. Since in the monothiooxalamides 1c and 1a, the methyl group, which, according to Fukui functions, results in the most
group presence causes an increase of 628 μmol ET/L in the an- rich-electron site in the molecule (Fig. 5).
tioxidant activity. In contrast, 1g did not present this structural ar-
rangement and showed three times more activity than 1h.
3.6.4. Oxygen Radical Absorbance Capacity (ORAC)
3.6.2. Antioxidant activity by ABTS•+ method Exploring different methods to measure the antioxidant activity
ABTS•+ method is considered an electron transfer method; its allows proposing a possible mechanism of antioxidant action, ei-
mechanism is influenced by the chemical characteristics of the an- ther by donating electrons, hydrogen atoms, or both. ORAC assay
tioxidant to be evaluated and the pH of the medium [42]. Antioxi- measures the antioxidant capacity of substances by a purely HAT
dant activity results of 1(a-h) compounds by ABTS•+ assay was re- mechanism. Monothiooxalamide results were reported in μmol
ported as mg Vitamin C/100 mL, see Table 5. Monothiooxalamide Trolox/mL see Table 5. Active compounds in this test were: 1a
1g showed the highest antiradical activity (76.37 VCEAC), with an (254.60 TEAC), 1e (184.67 TEAC), 1c (137.68 TEAC) and 1d (51.86
amount three times greater than 1f (24.73 VCEAC) and 1c (23.49 TEAC). Concerning monothiooxalamides 1b, 1f, 1g, and 1h, they
VCEAC). The remaining compounds (1a, 1b, 1d, 1e, and 1h) exhib- did not show the ability to donate hydrogen atoms by the ORAC
ited results between 5-18 VCEAC (P<0.05). Previous data confirm method.
the trend observed in the DPPH• assay, except for compound 1e, Compound 1f lacks the thioamide proton, and it does not show
which in DPPH• was one of the most active. That suggests that the ability to donate the amidic proton or any other hydrogen
1e might have antiradical activity via hydrogen atom transfer [42]. atom to stabilize peroxyl radicals. The same behavior is observed
Meanwhile, monothiooxalamides 1c, 1g, and 1f showed significant in molecules where the N-H (thioamide) forms three-center in-
ability in electron donation to neutralize ABTS•+ radical. tramolecular hydrogen bonds (1b and 1h, Fig. 1). The aforemen-
tioned allows proposing that compounds 1a, 1c, 1d, and 1e neu-
3.6.3. Ferric Reducing Antioxidant Power (FRAP) tralize peroxyl radicals by the thioamide protons donation. These
FRAP is one of the most commonly used method to assess the protons in the active molecules appear in a range of 9.29-9.60
electron-donating ability of natural and synthetic compounds. All ppm in NMR, while compounds where the N-H protons (amide

10
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

and thioamide) have a higher chemical shift than 9.60 ppm did not group and optimized structures suggests an intramolecular hydro-
show activity in the ORAC method, as in the case of compound 1g. gen bond between the amide proton and the thiocarbonyl group.
Antiproliferative activity of compounds was studied against four
3.6.5. Chelating activity of Fe(II) human cancer cell lines. Compound 1b exerted an interesting an-
Previous studies have shown that both antioxidants and metal tiproliferative effect on A2780-wt cells. The compounds 1a and 1c-
chelating compounds can be beneficial in the treatment of neu- h were not active at the evaluated concentrations. The intracellu-
rodegenerative diseases. This is due to metals such as Fe’s role lar ROS-production assay suggests that 1b could induce cell death
in ROS production, increasing oxidative stress [77]. In addition, by oxidative damage through an intracellular increase in ROS pro-
it has been reported that the antibacterial activity of thioamide duction. A comparison between the cytotoxicity and the molecu-
complexes with Fe(II) is higher than those of Co(II), Ni(II), Cu(II), lar structure allows to hypothesize a crucial role for the picolyl
and Zn(II) complexes [78]. Chelating ability of the ferrous ion group on monothiooxalamides, and this should be taken into ac-
by monothiooxalamides was reported as mM EDTA/L (Table 5). count for the future design of promising anticancer monothioox-
Monothiooxalamide 1h displayed the best chelating activity with alamides containing the pyridine moiety. In future designs of an-
1.85 mM EDTA/L at a concentration of 32.4 μM, followed by 1b, titumor compounds based on monothiooxalamides, the addition of
1c, 1d, which exhibited values of 0.02, 0.34, and 0.17 mM EDTA/L, substituents on the picolyl group of 1b must be considered to es-
respectively. On the other hand, the compounds 1a, 1e, 1f, and 1g tablish a structure-activity relationship (SAR).
did not display the ability to trap ferrous ions at higher concentra- Regarding in vitro antioxidant activity, compounds 1a, 1c, 1d,
tions. and 1e are suggested as candidates for future studies due to their
significant capacity to donate electrons to the Fe(III) and ABTS•+,
3.7. Donor-Acceptor Maps and Fukui function as well as hydrogen atoms to the oxygen radicals. Under this,
monothiooxalamides may react with DPPH• by either two mech-
Donor-Acceptor Map (DAM) is widely used to predict the abil- anisms (HAT or SET). About 1b, 1f, 1g, and 1h compounds only
ity of compounds to stabilize free radicals by donating electrons showed the ability to donate electrons. Of which, 1f and 1g were
(antioxidant), accepting electrons (anti-reductant), or both [79]. better. On the other hand, 1h was the best Fe(II) chelating com-
These mechanisms stabilize free radicals avoiding that it reacts pound.
with biomolecules. The medical importance of free radical scaveng- DAM suggests that compounds 1d, 1e, and 1g are the best an-
ing is to prevent oxidative stress, producing neurodegenerative dis- tiradical species in the evaluated system, while active compound
eases and cell damage [27]. DAM values are shown in Table 6. On 1b is not a good antiradical compound. Additionally, according to
the studied system (Fig. 6), it is possible to observe good acceptor Fukui functions, the region that could be involved in free-radical
molecules (1d and 1g and 1e), which could be good free-radical scavenging seems to be the thiocarbonyl group of monothioox-
scavengers by accepting an electron, oxidizing the free-radical, as alamides.
well as resveratrol, which is a well-known as a good anti-reductant
agent (Upper-right quadrant). Trolox is the best electron-donor Credit Author Statement
compound (Lower-left quadrant) in the system known to act as an
antioxidant to stabilize free radicals [20]. On the other hand, in the Carlos Eduardo Macías-Hernández: Synthesis, Characterization,
upper left quadrant is possible to identify most of the compounds, Antiproliferative activity and Writing.
which are the worst electron donor and acceptors in the system María M. Romero-Chávez: Antioxidant activity, Chelating activ-
(1a, 1b, 1c, 1e, 1f, and 1h) like ascorbic acid. ity and Writing.
The major ROS present in biological systems are electrophiles. Juan Pablo Mojica-Sánchez: DAM analysis, Fukui functions and
Comparing DAM results with intracellular ROS production on Writing
A2780-wt cells, it is possible to infer that prooxidant 1b (a very Kayim Pineda-Urbina: DAM analysis, Fukui functions and Writ-
active compound on human cancer cell lines) is also poor radi- ing.
cal scavenging on exploring the system. Fukui functions can also Mariá Teresa Sumaya Martińez: Antioxidant and Chelating ac-
predict the local reactivity of different regions in the molecules tivity - Review.
against nucleophile or electrophile species. Then, they prefer to Edgar Iván Jimenez-Ruiz: Antioxidant and Chelating activity -
interact with rich-electron regions in the molecules [27]. In the Review.
case of monothiooxalamides 1(a-h), Fukui functions establish that Lisa Dalla Via: Antiproliferative activity - Review & Editing.
the most rich-electron site is the sulfur atom of the thiocarbonyl Ángel Ramos-Organillo: Conceptualization, Formal analysis, Su-
group (Fig. 5). This suggests that the region involved in free- pervision - Review & Editing.
radical scavenging is the thioamide group, and this is consistent
with the antiradical capability reported previously in literature for
thioamides [80]. A proposal of a free-radical scavenging mecha- Declaration of Competing Interest
nism for monothiooxalamide is shown in Fig. 7. An electron is
transferred to a free-radical by thioamide group stabilizing radical The authors declare that they have no known competing finan-
with disulfide bond formation. cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
4. Conclusions
Acknowledgments
Compounds 1(a-h) were synthesized using chloroacetamide 1,
elemental sulfur, triethylamine, and corresponding amines. An op- The student Carlos Eduardo Macías Hernández thanks CONA-
timization process to get the best reaction condition was carried CYT for a PhD. Scholarship 631257. Authors thanks to Universi-
out. The best results were obtained with stirring at room temper- dad de Colima, Università degli Studi di Padova, and Universidad
ature for 72 hours using triethylamine as a base. The structures Autónoma de Nayarit for the used infrastructure and equipment.
of compounds 1(a-h) were unambiguously confirmed using 1 H, Authors thanks to Unidad Profesional Interdiciplinaria de Biotec-
13 C, COSY, HSQC, HMBC NMR spectroscopy, and mass spectrome- nología of Instituto Politécnico Nacional and PhD. Itzia Irene Padilla
try. The multiplicity pattern of neighboring protons to the amide Martínez for mass spectrometry and HPLC analysis.

11
C.E. Macías-Hernández, M.M. Romero-Chávez, J.P. Mojica-Sánchez et al. Journal of Molecular Structure 1265 (2022) 133360

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