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. 2022 Sep 15;7(38):34166–34176. doi: 10.1021/acsomega.2c03522

Convenient Synthesis of N-Alkyl-2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamides and Methyl-2-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetylamino]alkanoates

Samir Mohamed El Rayes †,*, Gaber El-Enany ‡,§, Mohamed Sayed Gomaa ∥, Ibrahim A I Ali †, Walid Fathalla §, Faheem Hyder Pottoo ⊥, Firdos Alam Khan #
PMCID: PMC9520703  PMID: 36188256

Abstract

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A series of 27 new quinoxaline derivatives (N-alkyl-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)]acetamides, methyl-2-[2-(3-phenylquinoxalin-2-ylsulfanyl)-acetylamino]alkanoates, and their corresponding dipeptides) were prepared from 3-phenylquinoxaline-2(1H)-thione based on the chemoselective reaction with soft electrophiles. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to study the efficacy of 27 compounds on cancer cell viability and proliferation. A total of 13 compounds (4a–c, 5, 6, 8c, 9c, 9f, 10a, 10b, 11c, 12b, and 12c) showed inhibitory action on HCT-116 cancer cells and 15 compounds (4a–c, 5, 6, 8c, 9a, 9c, 9f, 9h, 10b, 11c, 12a, 12b, and 12c) showed activity on MCF-7 cancer cells, with compound 10b exhibiting the highest inhibitory action (IC50 1.52 and 2 μg/mL, respectively) on both cell lines. The molecular modeling studies on the human thymidylate synthase (hTS) homodimer interface showed that these compounds are good binders and could selectively inhibit the enzyme by stabilizing its inactive conformation. The study also identified key residues for homodimer binding, which could be used for further optimization and development.

1. Introduction

Quinoxaline possesses a wide variety of therapeutic properties. Many quinoxaline derivatives have been found with distinct anticancer,1 antiviral,2 anthelmintic,3 antimicrobial,4 anti-inflammatory,5 antioxidant,6 and antiprotozoal activities.7 Quinoxaline and its derivatives have recently been recognized as effective chemotherapeutic agents against a number of tumors.8−10 Earlier discussion on the feasibility of quinoxaline anticancer activity revealed a number of pathways including the inhibition of enzymes (tyrosine kinases and c-MET kinase)11−14 as well as induction of apoptosis and tumor hypoxia.15−17 Recently, we have studied the structure–activity relationship in methyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates and N-alkyl-3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamides by molecular docking via examining the binding affinity to the human thymidylate synthase (hTS) allosteric site.18 This study proved the significance of the peptidomimetic side chain at position 3 of the quinoxaline ring. These compounds were tested against human HCT-116 and MCF-7 cell lines and showed remarkable results with IC50 values in the range of 1.9–7.52 μg/mL compared to the reference drug doxorubicin (IC50 3.23 μg/mL). In continuation to this study, we found it interesting to prepare a series of N-alkyl-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)] acetamides, methyl-2-[2-(3-phenylquinoxalin-2-ylsulfanyl)-acetylamino] alkanoates, and their corresponding dipeptides as new anticancer drugs. The newly synthesized derivatives were screened for their antitumor activity against the liver carcinoma cell line (HepG2). The mechanism of the antiproliferative activity of the synthesized compounds was studied through their binding to the human thymidylate synthase (hTS) homodimer interface using molecular docking.

2. Results and Discussion

2.1. Chemistry

The feasibility of N-cyclohexyldithiocarbamatecyclohexylammonium salt 2 as an excellent thiating reagent was earlier discussed in a number of articles.18−20 3-Phenylquinoxaline-2(1H)-thione (3) could be obtained simply by the reaction of chloroquinoxaline 1 with 2 in chloroform for 12 h at 61 °C to afford 3 in excellent yield, Scheme 1.18,19

Scheme 1. Preparation of Phenylquinoxaline-2(1H)-thione (3).

Scheme 1

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The model compound 3-phenylquinoxaline-2(1H)-thione (3) as a heterocyclic thioamide is presented in a tautomeric mixture between thiol and thione forms.21,22 This ambident nucleophile behavior of 3 could be invested to modify the quinoxaline ring structure by simple chemoselective alkylation reactions at nitrogen and sulfur atoms. However, the unique structure of 3 bearing a phenyl group contributing to a continuous conjugation in the whole molecule makes the S-atom bear both soft and hard characteristics. This was practically proved in our previous research following the reaction of 3-phenylquinoxaline-2(1H)-thione (3) with hard electrophilic alkylating reagents (activated acrylic acid compounds) to give S-substituted derivatives.18 Herein, we wish to report the reaction of model quinoxaline 3 with soft electrophiles and to invest the products to prepare a number of biologically promising compounds. Thus, 3 reacted with a number of soft electrophiles (chloroacetonitrile, phenacyl chloride, allyl bromide, and ethyl chloroacetate) in the presence of triethylamine to give S-alkylated derivatives 4a–c and 5, respectively, in 73–81 and 73% yields, Scheme 2.

Scheme 2. Reaction of Phenylquinoxaline-2(1H)-thione (3) with Soft Electrophiles.

Scheme 2

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The structure assignment of the prepared S-substituted quinoxaline derivatives 4a–c and 5 is based on 1H and 13C NMR spectral and physicochemical analysis. The 1H NMR spectrum of ethyl(3-phenyl-quinoxalin-2-ylsulfanyl)acetate (5) shows an interesting singlet signal at 4.03 ppm corresponding to the SCH2CO group, which clearly confirms the site of alkylation. The 1H NMR spectrum of 5 also shows two signals at 4.25 and 1.29 ppm corresponding to ester OCH2CH3 beside several multiplet signals ranging between 8.08 and 7.51 ppm for nine aromatic protons. The 13C NMR spectrum of 5 displays an interesting signal at 33.4 ppm for SCH2CO, which once again confirms the site of alkylation. The 13C NMR spectrum of 5 also shows signals at δ 169.2, 61.6, and 14.2 ppm corresponding to C=O, OCH2, and CH3 groups, respectively.

The S-substituted ester ethyl(3-phenylquinoxalin-2-ylsulfanyl)acetate (5) is an excellent precursor for the structural modification of the quinoxaline ring system at the sulfur atom and the introduction of either amino acid or alkyl amine residues via the azide coupling method.23,24

Ester 5 was refluxed with hydrazine hydrate in ethyl alcohol to afford the corresponding hydrazides 6 in 82% yield, Scheme 3. Hydrazide 6 was converted to the corresponding carbonyl azide derivative 7 by treatment with a NaNO2 and HCl mixture in an ice bath for 15 min and was extracted with ethyl acetate. The in situ-generated ethyl acetate solution of azide 7 was used without purification and reacted with amino acid methyl ester hydrochlorides (glycine, β-alanine, and l-aspartic acid) in the presence of triethylamine to afford a series of S-substituted methyl-2-[2-(3-phenylquinoxalin-2-ylsulfanyl)acetylamino]alkanoates 8a–c in good yields, Scheme 3. Similarly, the in situ-generated ethyl acetate solution of azide 7 reacted with alkane amines at room temperature for 24 h to afford a series of N-alkyl-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)]acetamides 9a–i, Scheme 3.

Scheme 3. Preparation of Methyl-2-[2-(3-phenylquinoxalin-2-ylsulfanyl)acetylamino]alkanoates 8a–c and N-Alkyl-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)]acetamides 9a–i.

Scheme 3

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The structure assignment of the prepared methyl-2-[2-(3-phenylquinoxalin-2-ylsulfanyl)acetylamino]alkanoates 8a–c and N-alkyl-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)]acetamides 9a–i is based on 1H and 13C NMR spectral and physicochemical analysis. The 1H NMR spectrum of methyl-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetylamino]acetate (8a) showed signals at δ 7.28, 4.22, 3.96, and 3.72 ppm corresponding to NH, SCH2, NCH2, and OCH3, respectively. The 13C NMR spectrum of 8a showed signals at δ 172.12, 168.3, 52.4, 41.7, and 34.6 ppm corresponding to two C=O, OCH3, NHCH2, and SCH2, respectively.

Next, we attempted the modification of the quinoxaline residue chemical structure of our model by the attachment of the dipeptide residue to enhance the biological activity. Thus, the reactions of amino acid derivatives 8a and 8b with hydrazine hydrate in ethanol for 4 h afforded hydrazides 10a and 10b in good yields. Hydrazides 10a and 10b were first reacted with a NaNO2 and HCl mixture in an ice bath for 15 min and then extracted with ethyl acetate and reacted simultaneously with amino acid methyl ester hydrochlorides (glycine, β-alanine, and l-aspartic acid) to afford dipeptides 11–12(a–c) in good yields, Scheme 4.

Scheme 4. Preparation of Dipeptides 11–12(a–c).

Scheme 4

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2.2. Antiproliferative Activities

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to study the impact of 27 compounds on cancer cell viability and proliferation. The cytotoxic effects of the compounds were observed after 48 h of treatment. Fourteen compounds (4a–c, 5, 6, 8c, 9c, 9f, 10a–c, 11c, 12b, and 12c) showed inhibitory action on HCT-116 cancer cells, whereas the remaining 13 compounds did not show any inhibitory action on the cancerous cells. We calculated the IC50 values for these compounds, and compound 10b (β-ala hydrazide) showed the highest inhibitory action, whereas compound 9c (allylamine derivative) showed the lowest inhibitory action on HCT-116 (Table 1). We also examined inhibitory action on MCF-7 cells. We found that 15 compounds (4a–c, 5, 6, 8c, 9a, 9c, 9f, 9h, 10b, 11c, 12a, 12b, and 12c) showed inhibitory action on MCF-7 cancer cells, whereas the remaining 11 compounds did not show any inhibitory action on the cancerous cells. We calculated the IC50 values for these compounds and compound 10b showed the highest inhibitory action, whereas compound 6 showed the lowest inhibitory action on MCF-7 (Table 1).

Table 1. Impact of Synthetic Compounds on Cancer Cells Using the MTT Assaya.

2.2.

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a

NA = not active. IC50 value [μg/mL] = inhibitory concentration (IC) is expressed in μg/mL.

Next, we wanted to know whether these compounds selectively target the cancerous cells or not. We tested these compounds on normal cells (HEK-293) at the same concentrations and duration of treatments. The cell viability assay using MTT revealed null cytotoxic effects on the normal cells.

We do not know the molecular mechanism of cancer cell death, so it would be interesting to study the role of apoptotic pathways in synthetic compound-mediated cancer cell death. There are reports of nanoparticle-induced nuclear fragmentation and disintegration in cancer cells.25−29 We suggest that these synthetic compounds possess selective targeting capability to cancerous cells and could be potential candidates for cancer treatments.

2.3. Molecular Modeling

The preliminary structure–activity relationship of the synthesized peptidomimetics showed that substitution of the thiol group with a simple methyl-bearing group capable of HB formation showed good activity (4a–c, and 5). Substitution of the thiol at position 3 with a peptidomimetic side chain bearing a single peptide or amide bond gave variable results with some compounds showing as good activity as simple alkyl substitution (8c, 10b, and 9f), while others were inactive (8a, 8b, and 9d).

Further extension of the molecules through formation of dipeptides that are connected to the quinazoline scaffold through a glycine structure gave almost inactive compounds (11a, 11b, and 12a). However, when the dipeptide is connected through a β-alanine structure, the activity was regained (12b and 12c).

Molecular docking was carried out to explain the results of the antiproliferative assay further and obtain better insights into the binding requirements of these quinazoline peptidomimetics at the hTS interface.

Key interactions at protein–protein interfaces represent important targets for small molecule inhibition. This kind of inhibition, unlike targeting the active site, inhibits intracellular hTS and cell growth without leading to overexpression of the protein, thereby conferring more selectivity and specificity.30

Peptide and nonpeptide31 inhibitors were demonstrated by X-ray crystallographic studies to bind hTS at the homodimer interface and showed allosteric inhibition of the enzyme through stabilizing its inactive form. Our peptidomimetics were shown to bind the hTS dimer interface and potentially stabilize its di-inactive form. The designed peptidomimetics use their peptide-like structure for optimal binding without being peptides in nature, which makes them more suitable for pharmaceutical manipulations and development.

Upon computational docking, the inhibitors were found predominantly at the dimer interface in poses that align with the cocrystallized peptide and maintained the key interactions with the target protein (hTS, 3N5E). This was mainly through conserving H-bonding with key residues: Gln172, Arg175, Ile 190, Met191, and Cys192 from chain A and Leu204 and Pro205 from chain B (Figure 1).

Figure 1.

Figure 1

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Most active compounds 10b green, 12b red, 12c brown, 5 blue, and 9f magenta and the crystallized inhibitor light blue docked at the hTS homodimer interface and showing key binding residues. The backbone is represented as cartoons: gray for chain A and cyan for chain B.

The docking results showed that the most active compounds (5, 10b, 9f, 12b, and 12c) lie at the interface of the homodimer and established interactions with both chains of the homodimer and with the mentioned key residues (Figure 1).

The most active compounds showed good stability and affinity to the active site by holding very close conformations and key interactions for most of the provided docked conformations with a relatively low root-mean-square deviation (RMSD) value (Figure 2).

Figure 2.

Figure 2

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Docking conformations of most active compounds. 10b green, 12b red, 12c brown, 5 blue, and 9f magenta.

In these compounds, the binding affinity also correlates well with the experimental IC50 value (Table 2).

Table 2. Binding Affinities and IC50 for Most Active Compounds.

    IC50 values (μg/mL) IC50 values (μg/mL)
  binding affinity (kcal/mol) HCT-116 MCF-7
10b –10.3 1.52 2.00
12b –8.9 2.07 2.39
12c –10.7 1.79 2.24
5 –8.8 2.19 2.38
9f –9.2 1.79 2.24
crystallized inhibitor –7.6    
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Inactive compound 9d was not able to maintain the interaction with key residues in most of the docking poses and showed variable poses at the interface that could reflect the low stability and affinity of this compound, as shown in Figure 3. The long hydrophobic alkyl chain was mainly found solvent exposed, and this could have affected the stability of the compound in the binding pocket.

Figure 3.

Figure 3

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Compound 9d docking conformations.

3. Experimental Section

3.1. Chemistry

3.1.1. General Procedures

The solvent was purified and dried in the usual way. The boiling range of petroleum ether used was 40–60 °C. Thin-layer chromatography (TLC) silica gel 60 F254 plastic plates (E. Merck, layer thickness 0.2 mm) were detected by UV absorption. Elemental analyses were performed on a Flash EA-1112 instrument at the Microanalytical Laboratory, Faculty of Science, Suez Canal University, Ismailia, Egypt. Melting points were determined on a Buchi 510 melting point apparatus, and the values are uncorrected. NMR spectra were measured with a Bruker 400 MHz, and tetramethylsilane (TMS) (0.00 ppm) was used as an internal standard. 2-Chloro-3-phenylquinoxaline (1) was prepared according to the method described.32

3.1.1.1. Preparation of Phenylquinoxaline-2(1H)-thione (3)

To a solution of 2-chloro-3-phenylquinoxaline (1, 2.5 mmol) in CHCl3 (25 mL) was added N-cyclohexyldithiocarbamatecyclohexylammonium salt 2 (0.69 g, 2.5 mmol). The reaction mixture was refluxed at 61 °C for 12 h. The reaction mixture was evaporated under reduced pressure, and 25 mL of ethanol was added to the solid residue. A yellowish precipitate was filtered to give the desired product and crystallized from ethanol. Yield 69%, yellow powder, mp 224–225 °C. 1H NMR spectrum (300 MHz, dimethyl sulfoxide (DMSO)), δ, ppm (J, Hz): 14.56 (1H, bs, NH), 8.48–8.37 (1H, m, ArH), 8.18–8.01 (2H, m, ArH), 7.85–7.78 (1H, m, ArH), 7.41–7.33 (5H, m, ArH). Found, %: C, 70.13; H, 3.84; N, 11.29. For C14H10N2S (236.1). Calcd, %: C, 70.56; H, 4.23; N, 11.76.

3.1.2. General Procedure for Alkylation

To a mixture of quinoxaline 3 (0.24 g, 1.0 mmol) and triethylamine (0.2 mL, 2.0 mmol) in ethyl alcohol (30 mL, 95%), alkylating agents (chloroacetonitrile, 2-bromo-1-phenyl-ethanone, allyl bromide, and/or ethyl chloroacetate) (1.0 mmol) were added. The reaction mixture was heated under reflux for 12 h and concentrated under reduced pressure. The solid obtained was filtered and crystallized from ethyl alcohol.

3.1.2.1. (3-Phenyl-quinoxalin-2-ylsulfanyl)acetonitrile (4a)

From chloroacetonitrile. Yield 73%, yellow powder, mp 137–139 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.07 (1H, d, J = 8 Hz, ArH), 8.04 (1H, d, J = 8 Hz, ArH), 7.81–7.79 (2H, m, ArH), 7.71 (2H, t, J = 8.0 Hz, ArH), 7.53–7.50 (3H, m, ArH), 4.43 (2H, s, SCH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 154.7, 153.4, 141.3, 139.7, 136.3, 129.8, 129.4 129.1, 129.0, 128.5, 128.3, 127.6 (C Ar), 115.6 (CN), 25.3 (CH2). Mass spectrometry (MS) (matrix-assisted laser desorption ionization (MALDI), positive mode, matrix DHB) m/z: 300 (M + Na)+. Found, % C, 69.78; H, 3.96; N, 15.02. For C16H11N3S (277.3). Calcd % C, 69.29; H, 4.00; N, 15.15; S, 11.56.

3.1.2.2. 1-Phenyl-2-(3-phenyl-quinoxalin-2-ylsulfanyl)ethanone (4b)

From phenacyl bromide. Yield 78%, yellow powder, mp 120–122 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.09 (1H, d, J = 8 Hz, ArH), 8.07–8.03 (3H, m, ArH), 7.84–7.82 (2H, m, ArH), 7.80 (2H, d, J = 8 Hz, ArH), 7.64–7.61 (3H, m, ArH), 7.51–7.48 (3H, m, ArH), 4.98 (2H, s, SCH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 189.3 (C=O), 153.4, 153.3, 141.3, 139.7, 136.3, 135.4, 131.9, 129.8, 129.1, 129.0, 128.6, 128.4, 127.4, 118.3, 43.3 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 379 (M + Na)+. Found, % C, 74.51; H, 4.21; N, 7.77. For C22H16N2OS (356.4). Calcd % C, 74.13; H, 4.52; N, 7.86; S, 9.00.

3.1.2.3. 2-Allylsulfanyl-3-phenyl-quinoxaline (4c)

From allyl bromide. Yield 81%, yellow powder, mp 79–81 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.10 (1H, d, J = 8 Hz, ArH), 7.91 (1H, d, J = 8 Hz, ArH), 7.80–7.78 (2H, m, ArH), 7.74–7.72 (2H, m, ArH), 7.56–7.53 (3H, m, ArH), 5.89–5.86 (1H, m, CH); 5.15 (1H, d, CH2, J = 17.2 Hz), 5.03 (1H, d, CH2, J = 9.2 Hz), 4.02 (2H, d, SCH2, J = 5.6 Hz). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 154.5, 153.2, 141.8, 139.7, 136.9, 132.4 (CH), 13.1, 129.7, 129.3, 129.0, 128.6, 128.3 127.0, 117.9 (CH2), 34.8 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 302 (M + Na)+. Found, % C, 73.21; H, 5.12; N, 10.47. For C17H14N2S (278.4). Calcd % C, 73.35; H, 5.07; N, 10.06; S, 11.52.

3.1.2.4. Ethyl-(3-phenyl-quinoxalin-2-ylsulfanyl)acetate (5)

From ethyl chloroacetate. Yield 73%, yellow powder, mp 93–95 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.08 (1H, d, J = 8 Hz, ArH), 8.05 (1H, d, J = 8 Hz, ArH), 7.83–7.80 (2H, m, ArH), 7.69 (1H, t, J = 8.0 Hz, ArH), 7.64 (1H, t, J = 8.0 Hz, ArH), 7.54–7.51 (3H, m, ArH), 4.25 (2H, q, J = 7.2 Hz, OCH2), 4.03 (2H, s, SCH2), 1.29 (3H, t, J = 7.2 Hz, CH3). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 169.2 (C=O), 153.6, 153.0, 141.2, 139.6, 136.9, 129.9, 129.1, 129.0, 128.5, 128.3, 127.4, 61.6 (OCH2), 33.4 (SCH2), 14.2 (CH3). MS (MALDI, positive mode, matrix DHB) m/z: 347 (M + Na)+. Found, % C, 66.55; H, 5.05; N, 8.72. For C18H16N2O2S (324.4). Calcd % C, 66.64; H, 4.97; N, 8.64; S, 9.88.

3.1.2.5. (3-Phenyl-quinoxalin-2-ylsulfanyl)acetic Acid Hydrazide (6)

Hydrazine hydrate (80%, 2.4 mL, 5 mmol) was added to a solution of ester 5 (0.33 g, 1.0 mmol) in absolute ethanol (30 mL). The reaction mixture was refluxed for 4 h and cooled. The resultant precipitate was filtered off, washed with ethanol and diethyl ether, and then crystallized from aqueous ethanol to yield the corresponding hydrazide.

Yield 82%, yellow powder, mp 166–168 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 9.36 (1H, bs, NH), 8.05–8.02 (2H, m, ArH), 7.85–7.80 (4H, m, ArH), 7.18–7.16 (3H, m, ArH), 4.39 (2H, bs, NH2), 3.98 (2H, s, SCH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 171.2 (C=O). 153.3, 153.2, 141.7, 139.5, 136.7, 129.8, 129.3, 129.1, 128.7, 127.8, 34.5 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 333 (M + Na)+. Found, % C, 61.87; H, 4.63; N, 18.11. For C16H14N4OS (310.4). Calcd % C, 61.92; H, 4.55; N, 18.05; S, 10.33.

3.1.2.6. Preparation of Methyl-2-[2-(3-phenylquinoxalin-2-ylsulfanyl)acetylamino]alkanoates 8a–c and N-Alkyl-[2-(3-phenyl-quinoxalin-2- ylsulfanyl)]acetamides 9a–i

General method: A solution of NaNO2 (0.34 g, 5.0 mmol) in cold water (3 mL) was added to a cold solution (−5 °C) of hydrazide 6 (0.31 g, 1.0 mmol) in AcOH (6 mL), 1 N HCl (3 mL), and water (25 mL). After stirring at −5 °C for 15 min, a yellowish syrup started to form. The reaction mixture was stirred in an ice bath for another 1 h. The reaction mixture was extracted twice with ethyl acetate (30 mL). The combined organic layer was washed with 0.5 N HCl (30 mL), 3% NaHCO3 (30 mL), and H2O (30 mL) and finally dried over Na2SO4 (10 g) to give an ethyl acetate solution of azide 7. A solution of an appropriate amino acid ester hydrochloride (1.0 mmol) in ethyl acetate (20 mL) containing triethylamine (0.2 mL, 2 mmol) or the appropriate alkane amine (1.0 mmol) in ethyl acetate (20 mL) was added to the solution of azide 7. The mixture was kept at −5 °C for 24 h and then at 25 °C for another 24 h, followed by washing with 0.5 N HCl (30 mL), 3% NaHCO3 (30 mL), and H2O (30 mL), and finally dried over Na2SO4 (10 g). The solution was evaporated to dryness, and the residue was recrystallized from petroleum ether–ethyl acetate, 1:3, to give desired S-coupled products 8a–c and 9a–i.

3.1.2.7. Methyl-2-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetylamino]acetate (8a)

From GlyOMe. Yield 69%, yellow powder, mp 84–85 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.11 (1H, d, J = 8 Hz, ArH), 8.07 (1H, d, J = 8 Hz, ArH), 7.76–7.72 (2H, m, ArH), 7.69 (1H, t, J = 8.0 Hz, ArH), 7.57 (1H, t, J = 8.0 Hz, ArH), 7.48–7.45 (3H, m, ArH), 7.28 (1H, bs, NH), 4.22 (2H, s, CH2), 3.96 (2H, s, SCH2); 3.72 (3H, s, OCH3). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 172.1 (C=O), 168.3 (C=O), 153.5, 153.1, 141.2, 139.5, 137.0, 129.7, 129.3, 129.1, 128.7, 127.5, 52.4 (OCH3), 41.7 (NCH2), 34.6 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 390 (M + Na)+. Found, % C, 62.02; H, 4.43; N, 11.21. For C19H17N3O3S (367.4). Calcd % C, 62.11; H, 4.66; N, 11.44; S, 8.73.

3.1.2.8. Methyl-3-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetylamino]propanoate (8b)

From β-AlaOMe. Yield 73%, yellow powder, mp 77–78 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.14 (1H, d, J = 8 Hz, ArH), 8.03 (1H, d, J = 8 Hz, ArH), 7.77–7.75 (2H, m, ArH), 7.74–7.73 (2H, m, ArH), 7.55–7.53 (3H, m, ArH), 7.36–7.34 (1H, m, NH), 3.89 (2H, s, SCH2), 3.49–3.47 (2H, m, NHCH2), 3.35 (3H, s, OMe), 2.45–2.43 (2H, m, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 172.8 (C=O), 168.9 (C=O), 153.4, 153.2, 141.4, 139.6, 137.4, 129.5, 129.4, 129.2, 128.6, 127.7, 51.6 (OCH3), 36.9 (NHCH2), 34.9 (SCH2), 33.8 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 404 (M + Na)+. Found, % C, 62.86; H, 4.96; N, 11.16. For C20H19N3O3S (381.5). Calcd % C, 62.97; H, 5.02; N, 11.02; S, 8.41.

3.1.2.9. Dimethyl-2-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)-acetylamino]succinate (8c)

From l-AspOMe. Yield 64%, yellow powder, mp 132–134 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.10 (1H, d, J = 8 Hz, ArH), 7.98 (1H, d, J = 8 Hz, ArH), 7.78–7.76 (2H, m, ArH), 7.71–7.69 (2H, m, ArH), 7.55–7.52 (3H, m, ArH), 6.86–6.84 (1H, m, NH), 4.84–4.82 (1H, m, NHCH), 3.93 (2H, s, SCH2), 3.60 (3H, s, OMe), 3.32 (3H, s, OMe), 2.88–2.86 (2H, m, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 172.7 (C=O), 172.6 (C=O), 168.3 (C=O), 153.5, 153.3, 141.4, 139.4, 137.7, 129.8, 129.6, 129.3, 128.7, 127.7, 55.4 (CH), 52.4 (OCH3), 51.9 (OCH3), 34.6 (SCH2), 34.1 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 462 (M + Na)+. Found, % C, 60.07; H, 4.95; N, 9.62. For C22H21N3O5S (439.5). Calcd % C, 60.12; H, 4.82; N, 9.56; S, 7.30.

3.1.2.10. N-Propyl 2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamide (9a)

From n-propylamine. Yield 65%, yellow powder, mp 71–73 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.14 (1H, d, J = 8 Hz, ArH); 7.98 (1H, d, J = 8 Hz, ArH); 7.80–7.78 (2H, m, ArH); 7.76 (1H, t, J = 8.0 Hz, ArH); 7.73 (1H, t, J = 8.0 Hz, Ar H); 7.56–7.54 (3H, m, ArH); 7.10 (1H, bs, NH); 3.95 (2H, s, SCH2); 3.23–3.21 (2H, m, HNCH2); 1.50–1.47 (2H, m, CH2); 0.83 (3H, t, J = 7.2 Hz, CH3). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 168.6 (C=O), 153.4, 153.2, 141.3, 139.5, 137.6, 129.7, 129.5, 129.2, 128.6, 127.5, 41.8 (NHCH2), 34.9 (SCH2), 22.9 (CH2), 12.1 (CH3). MS (MALDI, positive mode, matrix DHB) m/z: 360 (M + Na)+. Found, % C, 67.55; H, 5.72; N, 12.52. For C19H19N3OS (337.4). Calcd % C, 67.63; H, 5.68; N, 12.45; S, 9.50.

3.1.2.11. N-Butyl-2-(3-phenylquinoxalin-2-ylsulfanyl)acetamide (9b)

From n-butylamine. Yield 77%, yellow powder, mp 86–87 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.11 (1H, d, J = 8 Hz, ArH), 7.96 (1H, d, J = 8 Hz, ArH), 7.79–7.77 (2H, m, ArH), 7.74 (1H, t, J = 8.0 Hz, ArH), 7.69 (1H, t, J = 8.0 Hz, Ar H), 7.55–7.53 (3H, m, ArH), 7.05 (1H, bs, NH), 3.93 (2H, s, SCH2), 3.27–3.25 (2H, m, HNCH2), 1.42–1.39 (2H, m, CH2), 1.24–1.21 (2H, m, CH2), 0.79 (3H, t, J = 7.2 Hz, CH3). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 168.5 (C=O), 153.6, 153.4, 141.4, 139.6, 137.7, 129.8, 129.6, 129.3, 128.5, 127.4, 42.1 (NCH2), 34.2 (SCH2), 31.2 (CH2), 22.3 (CH2), 17.1 (CH3). MS (MALDI, positive mode, matrix DHB) m/z: 374 (M + Na)+. Found, % C, 68.42; H, 6.11; N, 12.04. For C20H21N3OS (351.5). Calcd % C, 68.35; H, 6.02; N, 11.96; S, 9.12.

3.1.2.12. N-Allyl 2-(3-Phenyl-quinoxalin-2-ylsulfanyl)acetamide (9c)

From allylamine. Yield 68%, yellow powder, mp 67–68 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.12 (1H, d, J = 8 Hz, ArH), 7.95 (1H, d, J = 8 Hz, ArH), 7.80–7.78 (2H, m, ArH), 7.73–7.70 (2H, m, ArH), 7.55–7.52 (3H, m, ArH), 7.00 (1H, bs, NH), 5.89–5.86 (1H, m, CH), 5.12 (1H, d, J = 16.0 Hz, CH2), 5.05 (1H, d, J = 8.4 Hz, CH=CH2), 3.95 (2H, s, SCH2), 3.90–3.88 (2H, d, J = 6.4 Hz, CH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 168.5 (C=O), 153.5. 153.3, 140.9, 139.8, 136.6, 133.8 (CH), 130.1, 129.3, 129.0, 128.6, 127.0, 116.2 (CH2), 41.9 (NCH2), 34.4 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 358 (M + Na)+. Found, % C, 68.27; H, 5.10; N, 12.61. For C19H17N3OS (335.4). Calcd % C, 68.03; H, 5.11; N, 12.53; S, 9.56.

3.1.2.13. N-Octyl-2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamide (9d)

From n-octylamine. Yield 64%, yellow powder, mp 90–91 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.09 (1H, d, J = 8.0 Hz, ArH), 8.02 (1H, d, J = 8.0 Hz, ArH), 7.73–7.71 (2H, m, ArH), 7.68 (1H, t, J = 8.0 Hz, ArH), 7.54 (1H, t, J = 8.0 Hz, ArH), 7.50–7.48 (3H, m, ArH), 7.14–7.13 (1H, m, NH), 4.11 (2H, s, SCH2), 3.20–3.18 (2H, m, HNCH2), 1.63–1.61 (2H, m, CH2), 1.31–1.29 (2H, m, CH2), 1.28–1.20 (10H, m, 5CH2), 0.88 (3H, t, J = 7.2 Hz, CH3). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 168.3 (C=O), 153.5, 153.6, 141.5, 139.7, 137.8, 129.7, 129.5, 129.2, 128.6, 127.5, 42.6 (NCH2), 34.9 (SCH2), 33.2, 32.0, 31.2, 30.8, 28.1, 24.1, 18.2 (CH3). MS (MALDI, positive mode, matrix DHB) m/z: 444 (M + Na)+. Found, % C, 71.17; H, 7.32; N, 10.04. For C25H31N3OS (421.6). Calcd % C, 71.22; H, 7.41; N, 9.97; S, 7.61.

3.1.2.14. N-Cyclohexyl-2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamide (9e)

From cyclohexylamine. Yield 81%, yellow powder, mp 111–113 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, H): 8.10 (1H, d, J = 8 Hz, ArH), 7.95 (1H, d, J = 8 Hz, ArH), 7.79–7.77 (2H, m, ArH), 7.74 (1H, t, J = 8.0 Hz, ArH), 7.69 (1H, t, J = 8.0 Hz, ArH), 7.54–7.52 (3H, m, ArH), 7.03 (1H, s, NH), 3.87 (2H, s, SCH2), 3.76–3.74 (1H, m, CH), 1.83–1.81 (2H, m, CH2), 1.56–1.54 (2H, m, CH2), 1.42–1.21 (4H, m, 2CH2), 1.08–1.06 (2H, m, CH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 168.7, 153.6, 153.5, 141.4, 139.6, 137.9, 129.8, 129.6, 129.3, 128.4, 127.6, 45.7 (CH), 34.9 (SCH2), 33.4, 26.4, 23.1. MS (MALDI, positive mode, matrix DHB) m/z: 400 (M + Na)+. Found, % C, 70.12; H, 6.19; N, 11.07. For C22H23N3OS (377.5). Calcd % C, 70.00; H, 6.14; N, 11.13; S, 8.49.

3.1.2.15. N-Benzyl-2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamide (9f)

From benzylamine. Yield 75%, yellow powder, mp 124–125 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.11–8.09 (2H, m, ArH), 7.76–7.74 (3H, m, ArH), 7.73–7.71 (1H, m, ArH), 7.54–7.52 (3H, m, ArH), 7.41 (1H, bs, NH), 7.17–7.15 (5H, m, ArH), 4.44 (2H, d, J = 7.2 Hz, CH2), 3.97 (2H, s, SCH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 168.9 (C=O), 153.7, 153.6, 144.1, 141.5, 139.7, 137.8, 129.7, 129.6, 129.2, 128.5, 128.3, 127.9, 127.7, 127.4, 48.6 (CH2), 34.7 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 408 (M + Na)+. Found, % C, 71.76; H, 5.06; N, 10.84. For C23H19N3OS (385.5). Calcd % C, 71.66; H, 4.97; N, 10.90; S, 8.32.

3.1.2.16. 1-Morpholin-4-yl-2-(3-phenyl-quinoxalin-2-ylsulfanyl)ethanone (9g)

From morpholine. Yield 70%, yellow powder, mp 138–139 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.09 (1H, d, J = 8 Hz, ArH), 7.92 (1H, d, J = 8 Hz, ArH), 7.80–7.78 (2H, m, ArH), 7.71 (1H, t, J = 8.0 Hz, ArH), 7.67 (1H, t, J = 8.0 Hz, ArH), 7.53–7.51 (3H, m, ArH), 4.23 (2H, s, SCH2), 3.72–3.68 (8H, m, 4CH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 168.8 (C=O), 153.8, 153.7, 142.0, 139.8, 137.7, 129.6, 129.5, 129.4, 128.4, 127.8, 127.6, 64.5 (OCH2), 65.6 (OCH2), 64.2 (OCH2), 46.2 (NCH2), 45.7 (NCH2), 34.3 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 388 (M + Na)+. Found, % C, 65.81; H, 5.33; N, 11.62. For C20H19N3O2S (365.5). Calcd % C, 65.73; H, 5.24; N, 11.50; S, 8.77.

3.1.2.17. 2-(3-Phenyl-quinoxalin-2-ylsulfanyl)-1-piperidin-1-yl-ethanone (9h)

From piperidine. Yield 69%, yellow powder, mp 106–107 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.09 (1H, d, J = 8 Hz, ArH), 7.94 (1H, d, J = 8 Hz, ArH), 7.82–7.80 (2H, m, ArH), 7.70 (1H, t, J = 8.0 Hz, ArH), 7.64 (1H, t, J = 8.0 Hz, ArH), 7.52–7.49 (3H, m, ArH), 4.26 (2H, s, SCH2), 3.62–3.58 (4H, m, 2CH2); 1.68–1.64 (6H, m, 3CH2); 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 168.7 (C=O), 153.6, 153.4, 142.2, 139.7, 137.6, 129.6, 129.4, 129.4, 128.3, 127.7, 127.5, 46.2 (NCH2), 45.2 (NCH2), 34.9 (SCH2), 26.0 (CH2), 25.3 (CH2), 25.1 (CH2). MS (MALDI, positive mode, matrix DHB) m/z: 386 (M + Na)+. Found, % C, 69.42; H, 5.96; N, 11.51. For C21H21N3OS (363.5). Calcd % C, 69.39; H, 5.82; N, 11.56; S, 8.82.

3.1.2.18. N-(4-Methyl-piperazin-1-yl)-2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamide (9i)

From N-methylpiperazine. Yield 74%, yellow powder, mp 142–143 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.12 (1H, d, J = 8 Hz, ArH), 8.01 (1H, d, J = 8 Hz, ArH), 7.84–7.82 (2H, m, ArH), 7.77 (1H, t, J = 8.0 Hz, ArH), 7.69 (1H, t, J = 8.0 Hz, Ar H),7.60–7.58 (3H, m, ArH), 4.07 (2H, s, SCH2), 2.74–2.70 (4H, m, 2CH2), 2.44–2.40 (4H, m, 2CH2), 2.22 (3H, s, CH3). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 167.9 (C=O), 153.7, 153.6, 142.1, 139.7, 137.6, 129.4, 129.3, 129.3, 128.55 127.7, 127.5, 54.8 (CH2), 54.3 (CH2), 44.8 (CH2), 44.3 (CH2), 42.4 (NCH3), 34.8 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 416 (M + Na)+. Found, % C, 64.22; H, 5.91; N, 17.87. For C21H23N5OS (393.5). Calcd % C, 64.10; H, 5.89; N, 17.80; S, 8.15.

3.1.2.19. General Procedure for the Synthesis of Hydrazides 10a and 10b

Hydrazine hydrate (80%, 5 mmol) was added to a solution of esters 8a and b (1.0 mmol) in absolute ethanol (30 mL). The reaction mixture was refluxed for 4 h and cooled. The resultant precipitate was filtered off, washed with ethanol and diethyl ether, and then crystallized from aqueous ethanol to yield the corresponding hydrazide.

3.1.2.20. N-Hydrazinocarbonylmethyl-2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamide (10a)

Yield 77%, yellow powder, mp 170–171 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 9.87 (1H, bs, NH), 8.08 (1H, d, J = 8 Hz, ArH), 8.01 (1H, d, J = 8 Hz, ArH), 7.88–7.90 (2H, m, ArH), 7.71 (1H, t, J = 8.0 Hz, ArH), 7.62 (1H, t, J = 8.0 Hz, ArH), 7.44–7.41 (3H, m, ArH), 7.11–7.07 (1H, m, NH), 4.22 (2H, bs, NH2), 4.05 (2H, s, SCH2), 3.87 (2H, s, NCH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 170.8 (C=O), 168.2 (C=O), 153.4, 153.1, 141.3, 139.4, 137.1, 129.4, 129.2, 129.0, 128.8, 127.6, 45.6 (NCH2), 34.6 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 390 (M + Na)+. Found, % C, 58.76; H, 4.39; N, 19.15. For C18H17N5O2S (367.4). Calcd % C, 58.84; H, 4.66; N, 19.06; S, 8.73.

3.1.2.21. N-(2-Hydrazinocarbonyl-ethyl)-2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamide (10b)

Yield 81%, yellow powder, mp 135–136 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 9.93 (1H, bs, NH), 8.11 (1H, d, J = 8 Hz, ArH), 8.07 (1H, d, J = 8 Hz, ArH), 7.89–7.87 (2H, m, ArH), 7.80–7.78 (2H, m, ArH), 7.59–7.57 (3H, m, ArH), 7.44–7.36 (1H, m, NH), 4.03 (2H, bs, NH2), 3.87 (2H, s, SCH2), 3.57–3.55 (2H, m, NHCH2), 2.31–2.29 (2H, m, CH2CO), 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 171.9, 168.2, 153.3, 153.1, 141.3, 139.6, 137.2, 129.4, 129.3, 129.2, 128.5, 127.6, 42.4 (NCH2), 37.5 (SCH2), 34.8 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 404 (M + Na)+. Found, % C, 59.88; H, 4.94; N, 18.42. For C19H19N5O2S (381.5). Calcd % C, 59.82; H, 5.02; N, 18.36; S, 8.41.

3.1.2.22. {2-[2-(3-Phenyl-quinoxalin-2-ylsulfanyl)-acetylamino]-acetylamino}-acetic Acid Methyl Ester (11a)

Abbreviated: Gly-Gly. Yield 57%, yellow powder, mp 76–78 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.12 (1H, d, J = 8 Hz, ArH), 8.06 (1H, d, J = 8 Hz, ArH), 7.93–7.91 (2H, m, ArH), 7.67 (1H, t, J = 8.0 Hz, ArH), 7.62 (1H, t, J = 8.0 Hz, ArH), 7.36–7.34 (3H, m, ArH), 7.14–7.12 (1H, m, NH), 6.17–6.14 (1H, m, NH), 4.13–4.11 (2H, m, CH2), 4.01–3.99 (2H, m, CH2), 3.87 (2H, s, SCH2), 3.54 (3H, s, OMe). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 171.8 (C=O), 168.2 (C=O), 167.8 (C=O), 153.4, 153.3, 141.4, 139.5, 137.2, 129.5, 129.3, 129.1, 128.7, 127.5, 126.9, 52.9 (OCH3), 45.7 (NCH2), 42.6 (NCH2), 34.6 (SCH2). MS (MALDI, positive mode, matrix DHB) m/z: 447 (M + Na)+. Found, % C, 59.38; H, 4.80; N, 13.26; S, 7.59. For C21H20N4O4S (424.5). Calcd % C, 59.42; H, 4.75; N, 13.20; S, 7.55

3.1.2.23. 3-{2-[2-(3-Phenyl-quinoxalin-2-ylsulfanyl)-acetylamino]acetylamino}-propionic Acid Methyl Ester (11b)

Abbreviated: Gly-β-Ala. Yield 51%, yellow powder, mp 77–79 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.09 (1H, d, J = 8 Hz, ArH), 8.04 (1H, d, J = 8.0 Hz, ArH), 7.90–7.89 (2H, m, ArH), 7.61 (1H, t, J = 8.0 Hz, ArH), 7.58 (1H, t, J = 8.0 Hz, ArH), 7.32–7.30 (3H, m, ArH), 7.09–7.06 (1H, m, NH), 6.12–6.10 (1H, m, NH), 4.07–3.94 (4H, m, 2NCH2), 3.87 (2H, s, SCH2), 3.54 (3H, s, OMe), 2.89–2.87 (2H, m, CH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 171.9, 168.4, 167.8, 153.5, 153.3, 141.5, 139.4, 137.3, 129.6, 129.4, 129.1, 128.7, 127.4, 127.0, 52.9 (OCH3), 45.6 (NCH2), 42.5 (NCH2), 34.2 (SCH2), 32.9 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 461 (M + Na)+. Found, % C, 60.29; H, 5.11; N, 12.81. For C22H22N4O4S (438.5). Calcd % C, 60.26; H, 5.06; N, 12.78; S, 7.31.

3.1.2.24. 2-{2-[2-(3-Phenyl-quinoxalin-2-ylsulfanyl)-acetylamino]-acetyl}-malonic Acid Dimethyl Ester (11c)

Abbreviated: Gly-l-Asp. Yield 62%, yellow powder, mp 98–99 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.16 (1H, d, J = 8 Hz, ArH), 8.09 (1H, d, J = 8 Hz, ArH), 7.97–7.95 (2H, m, ArH), 7.74 (1H, t, J = 8.0 Hz, ArH), 7.61 (1H, t, J = 8.0 Hz, ArH), 7.39–7.36 (3H, m, ArH), 7.13–7.11 (1H, m, NH), 6.15–6.13 (1H, m, NH), 5.14–5.12 (1H, m, CH), 4.23–4.21 (2H, m, NCH2), 3.73 (2H, s, SCH2), 3.66 (3H, s, OMe), 3.59 (3H, s, OMe), 2.76–2.63 (2H, m, CH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 171.9 (C=O), 171.3 (C=O), 168.4 (C=O), 168.2 (C=O), 153.4, 153.1, 141.4, 139.4, 137.2, 129.5, 129.4, 129.1, 128.8, 127.5, 127.1, 56.1 (CH), 52.8 (OCH3), 52.3 (OCH3), 45.7 (NCH2), 34.4 (SCH2), 33.8 (CH2). MS (MALDI, positive mode, matrix DHB) m/z: 505 (M + Na)+. Found, % C, 57.29; H, 4.66; N, 11.67. For C23H22N4O6S (482.5). Calcd % C, 57.25; H, 4.60; N, 11.61; S, 6.65.

3.1.2.25. {3-[2-(3-Phenyl-quinoxalin-2-ylsulfanyl)-acetylamino]propionyl-amino}-acetic Acid Methyl Ester (12a)

Abbreviated: β-Ala-Gly. Yield 65%, yellow powder, mp 101–102 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.12 (1H, d, J = 8 Hz, ArH), 8.06 (1H, d, J = 8 Hz, ArH), 7.88–7.86 (2H, m, ArH), 7.59 (1H, t, J = 8.0 Hz, ArH), 7.54 (1H, t, J = 8.0 Hz, ArH), 7.29–7.27 (3H, m, ArH), 7.02–7.00 (1H, m, NH), 6.88–6.86 (1H, m, NH), 4.12–4.10 (4H, m, 2NCH2), 3.85 (2H, s, SCH2), 3.68 (3H, s, OMe), 2.27–2.25 (2H, m, CH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 171.8 (C=O), 168.4 (C=O), 168.1 (C=O), 153.5, 153.3, 141.5, 139.4, 137.3, 129.5, 129.4, 129.0, 128.6, 127.4, 127.0, 52.8 (OCH3), 45.5 (NCH2), 42.6 (NCH2), 34.3 (SCH2), 31.3 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 461 (M + Na)+. Found, % C, 60.31; H, 5.09; N, 12.84. For C22H22N4O4S (438.5). Calcd % C, 60.26; H, 5.06; N, 12.78; S, 7.31.

3.1.2.26. 3-{3-[2-(3-Phenyl-quinoxalin-2-ylsulfanyl)-acetylamino]propionyl-amino}-propionic Acid Methyl Ester (12b)

Abbreviated: β-Ala-β-Ala. Yield 62%, yellow powder, mp 84–86 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.04 (1H, d, J = 8 Hz, ArH), 8.00 (1H, d, J = 8 Hz, ArH), 7.84–7.82 (2H, m, ArH), 7.61 (1H, t, J = 8.0 Hz, ArH), 7.52 (1H, t, J = 8.0 Hz, ArH), 7.13–7.11 (3H, m, ArH), 7.04–7.02 (1H, m, NH), 6.24–6.22 (1H, m, NH), 3.91–3.72 (6H, s, SCH2, 2NCH2), 3.63 (3H, s, OMe), 2.11–2.08 (4H, m, 2CH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 172.7 (C=O), 168.9 (C=O), 168.2 (C=O), 153.6, 153.4, 141.6, 139.5, 137.4, 129.6, 129.51 129.1, 128.7, 127.3, 127.0, 52.9 (OCH3), 45.6 (NCH2), 42.4 (NCH2), 34.3 (SCH2), 31.7 (CH2CO), 31.3 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 475 (M + Na)+. Found, % C, 61.12; H, 5.39; N, 12.27. For C23H24N4O4S (452.5). Calcd % C, 61.05; H, 5.35; N, 12.38; S, 7.09.

3.1.2.27. 2-{3-[2-(3-Phenyl-quinoxalin-2-ylsulfanyl)-acetylamino]propionyl-amino}-malonic Acid Dimethyl Ester (12c)

Abbreviated: β-Ala-l-Asp. Yield 51%, yellow powder, mp 96–97 °C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 8.11 (1H, d, J = 8 Hz, ArH), 8.05 (1H, d, J = 8 Hz, ArH), 7.84–7.82 (2H, m, ArH), 7.56 (1H, t, J = 8.0 Hz, ArH), 7.51 (1H, t, J = 8.0 Hz, ArH), 7.27–7.25 (3H, m, ArH), 7.07–7.05 (1H, m, NH), 6.97–6.95 (1H, m, NH), 4.78–4.76 (1H, m, CH), 3.93–3.82 (4H, m, SCH2, NCH2), 3.72 (3H, s, OMe), 3.68 (3H, s, OMe), 2.96–2.94 (2H, m, CH2), 2.44–2.42 (2H, m, CH2). 13C NMR spectrum (75.0 MHz, CDCl3), δ, ppm: 172.1 (C=O), 171.9 (C=O), 168.2 (C=O), 167.0 (C=O), 153.4, 153.2, 141.5, 139.4, 137.3, 129.5, 129.2, 129.0, 128.7, 127.5, 127.0, 55.8 (CH), 52.8 (OCH3), 51.3 (OCH3), 45.5 (NCH2), 34.4 (SCH2), 33.7 (CH2), 31.3 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 519 (M + Na)+. Found, % C, 58.09; H, 4.91; N, 11.32. For C24H24N4O4S (496.5). Calcd % C, 58.05; H, 4.87; N, 11.28; S, 6.46.

3.2. Pharmacological Studies

3.2.1. MTT Assay

Human embryonic kidney cells (HEK-293), human colorectal (HCT-116) carcinoma cells, and human adenocarcinoma (MCF-7) cells were cultured in the media containing Dulbecco’s modified Eagle’s medium (DMEM), (10%) l-glutamine, (10%) fetal bovine serum (FBS), (10%) selenium chloride, and (120 unit/mL) penicillin/streptomycin. The cultures were placed in a CO2 (5%) incubator (Thermo Scientific Heracell-150) at 37 °C to achieve 70–80% confluence and thereafter exposed to different concentrations (2–40 μg/mL) of 27 synthetic compounds for 48 h. After this, cell cultures were incubated with MTT (5.0 mg/mL) for 4 h. DMSO was added to each well, plates were read at 570 nm using an ELISA plate reader (Biotek Instruments, Winooski), and % cell viability was calculated.

3.2.2. Microscopic Analysis

All cells (HCT-116, MCF-7, and HEK-293) were observed under different magnifications of an inverted microscope (TS-100F-Eclipse, Nikon, Japan). The structural morphology of both treated and untreated cells was observed, and the structural morphological difference between cancerous cells (HCT-116 and MCF-7) and healthy normal cells (HEK-293) was also examined.

3.2.3. Statistical Evaluation

The mean ± standard deviation (SD) from the control and treated groups was calculated. All statistical analyses were completed with GraphPad Prism 6 (GraphPad Software). The difference between control and compound 1, 2, and 3 treated groups was calculated by one-way analysis of variance (ANOVA), and p-values were calculated by Student’s t-test (*p < 0.05, **p < 0.01).

3.2.4. Molecular Modeling

All molecular modeling studies were performed on a Hewlett–Packard Pentium Dual-Core T4300 2.10 GHz running Windows 10 using Molecular Operating Environment (AUTODOCK) 2008.10 molecular modeling software for molecular docking simulations and ligand binding energy calculations and Pymol for output data visualization and figure generation. The crystal structure of the human TS dimer bound to a short peptide LSCQLYQR (PDB code: 3N5E) was chosen as a receptor. This structure was a homodimer in its closed conformation and represented the inactive conformation of the enzyme. The putative ligand binding site was assigned based on the positions of the heavy atoms of the peptide reported.33 The selected targets were used after deleting the cocrystallized inhibitors, all hydrogens were added to the ligand PDB file, and partial charges were computed. Docking was performed using an AUTODOCK dock tool in AUTODOCK and performed with default values. Amino acid residues involved in binding the cocrystallized ligand were used to define the active site for ligand binding.

The docking results were evaluated using binding energy calculation in AUTODOCK and checking the ligand binding position through interaction with key residues and were further validated through comparative docking with the crystallized ligand position in Pymol.

4. Conclusions

The results of the biological testing and molecular docking studies showed that the designed and synthesized quinoxaline peptidomimetics possess good antiproliferative activity, particularly against breast cancer hepatic carcinoma, and apparent selectivity that is potentially mediated through binding the hTS homodimer interface and stabilizing its inactive conformation. The compounds are also peptidomimetic in nature and therefore are suitable for further pharmaceutical and preclinical development.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c03522.

  • NMR spectra for the synthesized compounds (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao2c03522_si_001.pdf (433KB, pdf)

References

  1. Harmenberg J.; Akesson-Johansson A.; Graslund A.; Malmfors T.; Bergman J.; Wahren B.; Akerfeldt S.; Lundblad L.; Cox S. The mechanism of action of the anti-herpes virus compound 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline. Antiviral Res. 1991, 15, 193–204. 10.1016/0166-3542(91)90066-z. [DOI] [PubMed] [Google Scholar]
  2. Naylor M. A.; Stephen M. A.; Nolan J.; Sutton B.; Tocher J. H.; Fielden E. M.; Adams J. E.; Strafford I. Heterocyclic mono-N-oxides with potential applications as bioreductive antitumour drugs: Part 1. 8-Alkylamino-substituted phenylimidazo [1,2-a] quinoxalines. Anticancer Drug Des. 1993, 8, 439–461. [PubMed] [Google Scholar]
  3. Hui X.; Desrivot J.; Bories C.; Loiseau C. P. M.; Franck X.; Hocquemiller R.; Figadere B. Synthesis and antiprotozoal activity of some new synthetic substituted quinoxalines. Bioorg. Med. Chem. Lett. 2006, 16, 815–820. 10.1016/j.bmcl.2005.11.025. [DOI] [PubMed] [Google Scholar]
  4. Kim Y. B.; Kim Y. H.; Park J. Y.; Kim S. K. Synthesis and biological activity of new quinoxaline antibiotics of echinomycin analogueS. Bioorg. Med. Chem. Lett. 2004, 14, 541–544. 10.1016/j.bmcl.2003.09.086. [DOI] [PubMed] [Google Scholar]
  5. El-Sabbagh O. I.; El-Sadek M. E.; Lashine S. M.; Yassin S. H.; El-Nabtity S. M. Synthesis of new 2 (1H)-quinoxalinone derivatives for antimicrobial and antiinflammatory evaluation. Med. Chem. Res. 2009, 18, 782–797. 10.1007/s00044-009-9203-y. [DOI] [Google Scholar]
  6. Settypalli T.; Chunduri V. R.; Maddineni A. K.; Begari N.; Allagadda R.; Kotha P.; Chippada A. R. Design, synthesis, in silico docking studies and biological evaluation of novel quinoxaline-hydrazide hydrazone-1,2,3-triazole hybrids as _-glucosidase inhibitors and antioxidants. New J. Chem. 2019, 43, 15435–15452. 10.1039/c9nj02580d. [DOI] [Google Scholar]
  7. Hui X.; Desrivot J.; Bories C.; Loiseau P. M.; Franck X.; Hocquemiller R.; Figadere B. Synthesis and antiprotozoal activity of some new synthetic substituted quinoxalines. Bioorg. Med. Chem. Lett. 2006, 16, 815–820. 10.1016/j.bmcl.2005.11.025. [DOI] [PubMed] [Google Scholar]
  8. Kotb E. R.; Anwar A. M.; Soliman S. M.; Salama A. M. Synthesis and reactions of some novel quinoxalines foranticancer evaluation. Phosphorus, Sulfur Silicon Relat. Elem. 2007, 182, 1119–1130. 10.1080/10426500601142114. [DOI] [Google Scholar]
  9. Carta A.; Loriga M.; Piras S.; Paglietti G.; La Colla P.; Busonera B.; Collu G.; Loddo R. Synthesis of variously substituted 3-phenoxymethyl quinoxalin-2-ones and quinoxalines capable to potentiate in vitro the antiproliferative activity of anticancer drugs in multi-drug resistant cell lines. Med. Chem. 2006, 2, 113–122. 10.2174/157340606776056197. [DOI] [PubMed] [Google Scholar]
  10. Piras S.; Loriga M.; Paglietti G. Quinoxaline chemistry. Part XVII. Methyl [4-(substituted 2-quinoxalinyloxy) phenyl] acetates and ethyl N-{[4-(substituted 2-quinoxalinyloxy) phenyl] acetyl} glutamates analogues of methotrexate: Synthesis and evaluation of in vitro anticancer activity. Farmaco 2004, 59, 185–194. 10.1016/j.farmac.2003.11.014. [DOI] [PubMed] [Google Scholar]
  11. El Newahie A. M. S.; Ismail N. S. M.; Abou El Ella D. A.; Abouzid K. A. M. Quinoxaline-Based Scaffolds Targeting Tyrosine Kinases and Their Potential Anticancer Activity. Arch. Pharm. 2016, 349, 309–326. 10.1002/ardp.201500468. [DOI] [PubMed] [Google Scholar]
  12. Ingle R.; Marathe R.; Magar D.; Patel H. M.; Surana S. J. Sulphonamido-quinoxalines: Search for anticancer agent. Eur. J. Med. Chem. 2013, 65, 168–186. 10.1016/j.ejmech.2013.04.028. [DOI] [PubMed] [Google Scholar]
  13. Noolvi M. N.; Patel H. M.; Bhardwaj V.; Chauhan A. Synthesis and in vitro antitumor activity of substituted quinazoline and quinoxaline derivatives: Search for anticancer agent. Eur. J. Med. Chem. 2011, 46, 2327–2346. 10.1016/j.ejmech.2011.03.015. [DOI] [PubMed] [Google Scholar]
  14. Abbas H.-A. S.; Al-Marhabi A. R.; Ammar Y. A.; Ammar Y. A. Molecular modeling studies and synthesis of novel quinoxaline derivatives with potential anticancer activity as inhibitors of c-Met kinase. Bioorg. Med. Chem. 2015, 23, 6560–6572. 10.1016/j.bmc.2015.09.023. [DOI] [PubMed] [Google Scholar]
  15. El Newahie A.; Nissan Y.; Ismail N.; Abou El Ella D.; Khojah S.; Abouzid K. Design and Synthesis of New Quinoxaline Derivatives as Anticancer Agents and Apoptotic Inducers. Molecules 2019, 24, 1175 10.3390/molecules24061175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tseng C.-H.; Chen Y.-R.; Tzeng C.-C.; Liu W.; Chou C.-K.; Chiu C.-C.; Chen Y.-L. Discovery of indeno[1,2-b]quinoxaline derivatives as potential anticancer agents. Eur. J. Med. Chem. 2016, 108, 258–273. 10.1016/j.ejmech.2015.11.031. [DOI] [PubMed] [Google Scholar]
  17. Ismail M. M. F.; Amin K. M.; Noaman E.; Soliman D. H.; Ammar Y. A. New quinoxaline 1, 4-di-N-oxides: Anticancer and hypoxia-selective therapeutic agents. Eur. J. Med. Chem. 2010, 45, 2733–2738. 10.1016/j.ejmech.2010.02.052. [DOI] [PubMed] [Google Scholar]
  18. El Rayes S. M.; Aboelmagd A.; Gomaa M. S.; Ali A. I. A.; Fathalla W.; Pottoo F. H.; Khan F. A. Convenient synthesis and antiproliferative activity of methyl 2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates and N-Alkyl 3-((3-phenylquinoxalin-2-yl)sulfanyl) propanamides. ACS Omega 2019, 4, 18555–18566. 10.1021/acsomega.9b02320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fathalla W.; Ali I. A. I.; Pazdera P. A novel method for heterocyclic amide–thioamide transformations. Beilstein J. Org. Chem. 2017, 13, 174–181. 10.3762/bjoc.13.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Megahed M. I.; Fathalla W. Synthesis and antimicrobial activity of methyl (2-(2-(2-arylquinazolin-4-yl)sulfanyl)acetyl)amino alkanoates. J. Heterocycl. Chem. 2018, 55, 2809. 10.1002/jhet.3348. [DOI] [Google Scholar]
  21. El Rayes S. M.; Ali I. A. I.; Fathalla W. Convenient Synthesis of Some Novel Pyridazinone Bearing Triazole Moieties. J. Heterocycl. Chem. 2019, 56, 51. 10.1002/jhet.3369. [DOI] [Google Scholar]
  22. Fathalla W.; El Rayes S. M.; Ali I. A. I. Convenient synthesis of 1-substituted-4-methyl-5-oxo[1,2,4]triazolo[4,3-a]quinazolines. Arkivoc 2007, 173. 10.3998/ark.5550190.0008.g18. [DOI] [Google Scholar]
  23. Han S.-Y.; Kim Y.-A. Recent Development of Peptide Coupling Reagents in Organic Synthesis. Tetrahedron 2004, 60, 2447. 10.1016/j.tet.2004.01.020. [DOI] [Google Scholar]
  24. Ali I. A. I.; Al-Masoudi I. A.; Saeed B.; Al-Masoudi N. A.; La Colla P. Amino Acid Derivatives, Part 2: Synthesis, Antiviral, and Antitumor Activity of Simple Protected Amino Acids Functionalized at N-terminus with Naphthalene Side Chain. Heteroatom Chem. 2005, 16 (2), 148–155. 10.1002/hc.20082. [DOI] [Google Scholar]
  25. Baharara J.; Ramezani T.; Divsalar A.; Mousavi M.; Seyedarabi A. Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells. Avicenna J. Med. Biotechnol. 2016, 8, 75–83. [PMC free article] [PubMed] [Google Scholar]
  26. Mytych J.; Lewinska A.; Zebrowski J.; Wnuk M. Gold nanoparticles promote oxidant-mediated activation of NF-κB and 53BP1 recruitment-based adaptive response in human astrocytes. BioMed Res. Int. 2015, 2015, 304575 10.1155/2015/304575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wang L.-W.; Ai-Ping Q.; Wen-Lou L.; Jia-Mei C.; Jing-Ping Y.; Han W.; Yan L.; Juan L. Quantum dots-based double imaging combined with organic dye imaging to establish an automatic computerized method for cancer Ki67 measurement. Sci. Rep. 2016, 6, 20564 10.1038/srep20564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Irani S.; Shahmirani Z.; Atyabi S. M.; Mirpoor S. Induction of growth arrest in colorectal cancer cells by cold plasma and gold nanoparticles. Arch. Med. Sci. 2015, 11, 1286–1295. 10.5114/aoms.2015.48221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Khan F. A.; Akhtar S.; Almofty S. A.; Almohazey D.; Alomari M. FMSP-Nanoparticles Induced Cell Death on Human Breast Adenocarcinoma Cell Line (MCF-7 Cells): Morphometric Analysis. Biomolecules 2018, 8, 32 10.3390/biom8020032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Fletcher S.; Hamilton A. D. Targeting protein–protein interactions by rational design: mimicry of protein surfaces. J. R. Soc. Interface 2006, 3, 215–233. 10.1098/rsif.2006.0115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Carosati E.; Tochowicz A.; Marverti G.; Guaitoli G.; Benedetti P.; Ferrari S.; Stroud R. M.; Finer-Moore J.; Luciani R.; Farina D.; Cruciani G.; Costi M. P. Costi, Inhibitor of ovarian cancer cells growth by virtual screening: a new thiazole derivative targeting human thymidylate synthase. J. Med. Chem. 2012, 55, 10272–10276. 10.1021/jm300850v. [DOI] [PubMed] [Google Scholar]
  32. Aggarwal R.; Eakta M.; Garima S. Facile and Efficient Regioselective Synthesis of 1-(3′-Substituted Quinoxalin-2′-yl)-3-aryl/heteroaryl-5-methylpyrazoles. Synth. Commun. 2013, 43, 1842–1848. 10.1080/00397911.2012.674168. [DOI] [Google Scholar]
  33. Cardinale D.; Guaitoli G.; Tondi D.; Luciani R.; Henrich S.; Frassineti O.; Salo-Ahen M.; Ferrari S.; Marverti G.; Guerrieri D.; A Ligabue C. C.; Pozzi S.; Mangani D.; Fessas R.; Guerrini G.; Ponterini R. C.; Wade M. P.; Costi Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, E542–E549. 10.1073/pnas.1104829108. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

ao2c03522_si_001.pdf (433KB, pdf)

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