Convenient Synthesis and Anticancer Activity of Methyl 2-[3-(3-Phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates and N-Alkyl 3-((3-Phenyl-quinoxalin-2-yl)sulfanyl)propanamides.

A series of methyl 2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates and their corresponding hydrazides and N-alkyl 3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamides were prepared on the basis of the chemoselective Michael reaction of acrylic acid with the parent substrate 3-phenylquinoxaline-2(1H)-thione. The parent thione was produced by a convenient novel thiation method from the corresponding 3-phenylquinoxalin-2(1H)-one. The chemical structures of the newly synthesized compounds were confirmed by elemental analyses, 1H and 13C NMR. The antiproliferative activity of the synthesized compounds was tested against human HCT-116 and MCF-7 cell lines. Out of 25 screened derivatives, 10 active compounds exhibited IC50's in the range 1.9-7.52 μg/mL on the HCT-116, and 17 active compounds exhibited IC50's in the range 2.3-6.62 μg/mL on the MCF-7 cell lines compared to the reference drug doxorubicin (IC50 3.23 μg/mL). The structure-activity relationship of the tested compounds was studied through their binding affinity to the human thymidylate synthase allosteric site in silico using molecular docking and proved the quinoxaline ring as a suitable scaffold carrying a peptidomimetic side chain in position 3.

Introduction

Malignancy is one of the significant factors behind loss of life in the developed countries.[1−3] Chemotherapy with cytotoxic medications is one of the primary approaches to dealing with established malignancy.[4,5] The primary drawbacks of the existing chemotherapy of malignancy will be the severe poisonous results such as emesis and myelosuppression and the insufficient selectivity of the drugs against the cyst tumor cellular material in comparison with the normal cellular material.[1,6] Hence, search for newer anticancer drugs is a never-ending job. Quinazolines are one of the most studied moieties in malignancy chemotherapy. Lately, the FDA has approved several quinazoline derivatives as antitumor drugs from previous 15 years such as gefitinib, erlotinib, lapatinib, and raltitrexed.[7−10]

Quinoxaline, an isostere of quinazoline, has also proven to have a good anticancer activity in different research studies. Compounds bearing quinoxaline scaffold have found great application in the discovery of novel anticancer agents.

Quinoxaline derivatives showed a good anticancer activity through different mechanisms including tyrosine kinases inhibition,[11−13] C-MET kinase inhibition,[14] induction of apoptosis,[15−17] tubulin polymerization inhibition,[18] and selective induction of tumor hypoxia.[19]

Based on the aforementioned data, the present article deals with the synthesis of a series of new compounds containing the quinoxaline moiety, a known isostere for quinazoline, coupled with amino acids or N-alkyl amines via a propanoyl spacer, to evaluate their antitumor action. The amino acids used are selected to contain different physiologically active side chains such as alkyl, carboxyl, and sulfur-containing groups (glycine, β-alanine, valine, leucine, aspartic, glutamic, and methionine). The newly synthesized derivatives were screened for their antitumor activity against HCT-116 and MCF-7 cell lines (Table ). The mechanism of the antiproliferative activity of the synthesized compounds was studied through their binding affinity to the human thymidylate synthase (hTS) allosteric site in silico using molecular docking.

Table 1

Anticancer Activity of Synthetic Compounds on Cancer Cells Using the MTT Assaya

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

The chemoselective reactions of thioamides have always attracted the attention of our research group. Earlier, we reported[20−22] the chemoselective S- and N-alkylation of the model compound 4-methyl-1-thioxo-1,2,4,5-tetrahydro[1,2,4]triazolo[4,3-a]quinazolin-5-one with different electrophiles. These results were supported by quantum-chemical calculations.[20−22] We also applied these findings to the structure modification of a number of heterocyclic thioamides, quinazolines,[23] tetrazoles,[24] triazoloquinazolines,[20−22,25] and triazoloquinoxalines.[26] Nonproteinogenic amino acids are major components in a number of drugs including β-lactam antibiotics[27] and antiviral drugs.[28] These results motivated the development of a series of N- and S-substituted amino acid esters and alkanamides of a biologically quinoxaline ring system on the basis of a chemoselective reaction of heterocyclic thioamides with electrophiles. In the present article, we report the preparation of methyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates and N-alkyl 3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamides as promising biologically active compounds.

Key interactions at protein–protein interfaces constitute important targets for small molecule inhibition because of their specific arrangements and biological importance.[29]

hTS is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs which bind to its active site. We have designed peptidomimetics that have been shown in this molecular modeling study to specifically target its dimer interface and stabilize its di-inactive form. Peptide[30] and nonpeptide[31] inhibitors were demonstrated by X-ray crystallographic studies to bind hTS at a previously unknown binding site at the homodimer interface and showed a unique mechanism for the allosteric inhibition of a homodimeric enzyme through stabilizing its inactive form. This kind of inhibition, unlike targeting the active site, inhibits intracellular hTS—and cell growth—without leading to overexpression of the protein and thereby confers more selectivity and specificity.[30]

Our compounds are peptides in nature that mimic peptide inhibitors and nonpeptide inhibitors together with being more suitable for pharmaceutical manipulations and development.

Results and Discussion

Chemistry

Recently, we introduced an interesting thiating reagent: N-cyclohexyl dithiocarbamate cyclohexyl ammonium salt 2, which was prepared by the reaction of cyclohexyl amine and carbon disulfide at room temperature for 2 h, Scheme .[32]

Scheme 1

Preparation of N-Cyclohexyl Dithiocarbamate Cyclohexylammonium Salt 2 as a Novel Thiating Agent

This reagent was used as a novel material for heterocyclic amide–thioamide transformations.[32] Thus, our target substrate 3-phenylquinoxaline-2(1H)-thione (5) could be simply prepared by thiation of the corresponding 3-phenylquinoxalin-2(1H)-one (3) in a two-step reaction: first, chlorination and second , heating chloroquinoxaline 4 with 2 in chloroform for 12 h at 61 °C to afford 5 in excellent yield, Scheme .[32]

Scheme 2

Preparation of the Starting Phenylquinoxaline-2(1H)-thione (5)

The chemical confirmation of this thiation protocol could be achieved by structure modification of quinoxaline-2(1H)-thione 5 reflecting the thioamide chemical behavior and producing a series of biologically promising compounds.

3-Phenylquinoxaline-2(1H)-thione (5) displays an interesting tautomeric equilibrium between thiol (structure 5a) and thione (structure 5b) forms, Scheme .[33−35] Therefore, quinoxaline 5 is amenable to structure modification by simple chemoselective alkylation reactions at the sulfur and nitrogen atoms. Surprisingly, the reaction of 5 with acrylic acid derivatives, methyl acrylate, acrylamide, and acrylonitrile in the presence of triethylamine repeatedly afforded S-alkylated derivatives 6–8 in good yields, Scheme . The amide derivative 7 was also formed by the ammonolysis of the ester 6.

Scheme 3

Chemoselective S-Alkylation Reactions of Phenylquinoxaline-2(1H)-thione (5)

Earlier reports dealing with the chemoselective behavior of heterocyclic thioamides on the model compound 4-methyl-1-thioxo-1,2,4,5-tetrahydro[1,2,4]triazolo[4,3-a]quinazolin-5-one and supported by quantum chemical calculations showed that the reaction of the model compound with acrylic acid derivatives afforded the N-substituted products because of strong Coulombic attraction between the hard part of the ambident nucleophile and the hard electrophile to finally give N-substitution.[20−22] We might conclude that the Michael reaction of 5 with acrylic acid derivatives gave chemoselective S-substitution which could be explained by a unique structure of this quinoxaline structure bearing a phenyl ring that contributes in the continuous conjugation and consequently, causes both the soft and hard character to be collected on the sulfur atom.[36]

The structure assignment of the prepared S-substituted quinoxaline derivatives 6–8 is based on 1H and 13C NMR spectral and physicochemical analysis. The 1H NMR spectrum of methyl 3-((3-phenyl-1,2-dihydroquinoxalin-2-yl)sulfanyl)propanoate (6) gave an aromatic pattern consisting of two doublet and two multiplet signals at δ 8.13, δ 8.00, δ 7.80–7.65, and δ 7.55–7.53 ppm for nine aromatic protons. The 1H NMR spectrum of ester 6 also shows two triplet signals at δ 3.58 and δ 2.89 ppm corresponding to SCH2 and CH2CO groups, respectively, which again confirms the site of alkylation. On the other hand, the 13C NMR spectrum of 7 displays two signals at 33.8 and 172.4 ppm for SCH2 and ester carbonyl groups, respectively, and also aromatic carbons at 154.7, 153.4, 141.5, 139.4, 137.2, 129.8, 129.7, 129.2, 129.0, 128.5, 128.3, and 127.6 ppm. Our experience in the chemoselective alkylation at nitrogen or sulfur of heterocyclic thioamides and chemoselective alkylation at nitrogen or oxygen of heterocyclic amides causes a dramatic change in the aromatic proton and carbon NMR patterns.[20−38] Furthermore, the expected N-substituted ester methyl 3-(3-phenyl-2-thioxoquinoxalin-1(2H)-yl)propanoate should have an extra down fielded carbon signal of about 178 ppm for the C=S group and a higher chemical shift of about 42–45 ppm for NCH2.[20,21]

The S-substituted ester methyl 3-((3-phenyl-1,2-dihydroquinoxalin-2-yl)sulfanyl)propanoate (6) is an excellent precursor for the structure 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. The azide coupling method is considered as one of the important methods to couple amino acids and amines starting from the corresponding hydrazides. It was also reported that this method decreases the degree of racemization in amino acid coupling.[39,40] Thus, the reaction of ester 6 with hydrazine hydrate in ethyl alcohol under reflux conditions afforded hydrazide 9 in 88% yield, Scheme . Hydrazide 9 was reacted with a NaNO2 and HCl mixture in an ice bath for 15 min to afford the corresponding azide derivative 10 and was extracted with ethyl acetate. The in situ-generated ethyl acetate solution of azide 10 was directly reacted with amino acid methyl ester hydrochlorides in the presence of triethylamine to afford a series of 2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates 11a–g in good yield, Scheme . Similarly, the in situ-generated ethyl acetate solution of azide 10 reacted with alkane amines at room temperature for 24 h to afford a series of N-alkyl 3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamides 12a–i, Scheme .

Scheme 4

Preparation of Methyl 2-[3-(3-Phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates 11a–g and N-Alkyl 3-((3-Phenylquinoxalin-2-yl)sulfanyl)propanamides 12a–i

The structure assignment of the prepared methyl 2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates 11a–g and N-alkyl 3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamides 12a–i is based on 1H and 13C NMR spectral and physicochemical analysis. The 1H NMR spectrum of S-substituted glycine 11a shows signals at δ 6.10, δ 3.99, δ 3.67, δ 3.51, and δ 2.70 ppm corresponding to NH, NHCH2, OCH3, SCH2, and CH2CO groups, respectively. The 13C NMR spectrum of S-substituted glycine 11a shows signals at 52.3, 41.3, 35.8, and 26.1 ppm for OCH3, NHCH2, SCH2, and CH2CO groups, respectively.

Heterocyclic carboxylic acid hydrazides are well-known biologically active compounds and were used as intermediates in amino acid coupling and heterocyclic synthesis.[41,42] The reaction of amino acid esters 11 (a, e, and g) with hydrazine hydrate in ethanol under reflux conditions afforded the hydrazides 13 (a, e, and g), respectively, in good yields, Scheme . Dihydrazide 14 was produced by hydrazinolysis of aspartic acid derivatives 11c, Scheme .

Scheme 5

Preparation of 2-[2-(3-Phenyl-quinoxalin-2-ylsulfanyl)alkanamino]alkanoic Acid Hydrazide 13 (a, e, and g)

Scheme 6

Preparation of Succinic Acid Hydrazide Derivative 14

Biological Evaluation

We have examined the impact of 25 compounds on cancer cell viability and proliferation by using the MTT assay. The cytotoxic effects of all the compounds were observed after 48 h treatment and it was found that out of 25 compounds, 10 compounds showed inhibitory action on the HCT-116 cancer cells, whereas remaining 15 compounds did not show any inhibitory action on the cancerous cells. We have calculated the IC50 values for these compounds and compound # 9 showed the highest inhibitory action, whereas compound # 11c showed the lowest inhibitory action on HCT-116 (Table ).

We have also examined inhibitory action on MCF-7 cells. We have found that out of 25 compounds, 17 compounds showed inhibitory action on the MCF-7 cancer cells, whereas remaining 8 compounds did not show any inhibitory action on the cancerous cells. We have calculated the IC50 values for these compounds and compound # 11a showed the highest inhibitory action, whereas compound # 6 showed the lowest inhibitory action on MCF-7 (Table ). Compounds (7, 8, 9, 12h, and 12i) showed a balanced good inhibitory activity on both the cell lines. There are several reports of treatment of biomaterials causing cancer cell deaths.[43−45]

Drug selectivity is the most important aspect of any treatment, and we wanted to know whether these synthetically designed compounds selectively target the cancerous cells or not. We have tested these compounds on normal healthy cells (HEK-293) using the same concentrations and durations of treatment. The cell viability assay using MTT revealed that there are no cytotoxic effects on the normal cell numbers (Supporting Information).

To examine morphological changes on cancerous cells because of treatments, we have also studied the cell morphology of HCT-116, MCF-7, and HEK-293 cells under a light microscope. The treatment of compound # 7 showed significant changes in the structure of the cell membrane and the cell nucleus. We observed clear indication of nuclear disintegration, nuclear condensation, and cell death as many cancer cells were found dead after the treatment (Figure b). The morphology of untreated (control group) MCF-7 cells remained normal and healthy during the testing phase (Figure c). The treatment of compound 7 showed significant changes in the structure of the cell membrane and the cell nucleus. We observed strong nuclear disintegration, nuclear condensation, and cell death as many cancer cells were found dead after the treatment (Figure d). Interestingly, the morphology of untreated (control group) HCT-116 cells remained normal and healthy during the testing phase (Figure a).

Figure 1

Cell morphology of HCT-116 and MCF-7 cells after 48 h of treatment with compound # 7: (a) HCT-116 cell control, (b) 2.19 μg/mL (HCT-116), (c) control (MCF-7), and (d) 2.65 μg/mL (MCF-7). (b,d) show significant cell death; 200 magnification.

Our study suggests that compounds # 9 and 11a showed the highest level of morphological changes in the HCT-116 and MCF-7 cancerous cells. We do not know the molecular mechanism of cancer cell death; 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.[46,47] We suggest that these synthetic compounds possess selective targeting capability to cancerous cells and could be potential candidates for cancer treatments.

All the tested quinoxaline derivatives exhibited good and similar activity that lies in the low micromolar range. This suggests that our 3-phenyl-2-sulfanyl quinoxaline scaffold which is present in all the compounds is presumably working on a specific target and therefore possesses a good and selective anticancer activity. The change in the sulfanyl peptidomimetic side chain didn’t greatly affect activity whether its size (compound 12c compared to compound 12d) or its binding groups type (compound 11c compared to compound 11f). However, the presence of this side chain is important for its activity (compound 5a compared to compound 7). It seems that the quinaxoline scaffold is establishing essential interactions with the target active site and the flexible side chain lies in a spacious channel or pocket and serves in increasing the overall compound binding and selectivity.

Molecular Modeling

Bioinformatics, including molecular modeling studies, has become useful nowadays in neuroscientific medication discovery, saving cash and effort necessary for the verification of new compounds by directing and confining the research to possible focus on/targets. The usage of docking simulation studies inside our task is quite important to assist in predicting the possible mode of action of these compounds and guiding the near future research directions in compound optimization and the biochemical enzyme assay for the possible target enzymes.[48]

The tested compounds were evaluated for their binding affinity to possible targets, namely, hTS active site (4E28) and hTS allosteric site (3N5E). The compounds were found to preferably bind to the allosteric site at the homodimer interface. Comparative docking with the target-crystallized ligand was performed to further validate the docking results and substantiate the compounds mode of action. The results were also evaluated visually through possible interaction with key residues at the active site. Upon computational docking, the inhibitors were found predominantly at the dimer interface. Although docking poses were found close to the cocrystallized peptide allosteric inhibitor in the di-inactive form, docking poses were also found on the opposite side of the dimer interface. The docking results thus support the hypothesis that inhibitors could bind to different sites at the dimer interface of the inactive conformation of hTS.

The docking results showed that the most active compounds (2, 3, 4, 5, 20, and 21) lie at the interface of the homodimer in the same pocket of the crystallized peptide inhibitor and establish interactions with both chains of the homodimer (Figure ). The less active compounds had a similar docking pose; however, they are more shifted toward chain A and therefore they predominantly established interactions with chain A rather than binding both chains as the more active compounds did (Figure ). The inactive compounds are superposed with the crystallized inhibitor chain that lies in a cleft further from the interface and deeper inside one of the homodimer.

Figure 2

Compounds 6 purple, 7 orange, 8 pink, 9 light blue, 12h green, and 12i yellow docked in the homodimer interface of hTS with the cocrystallized peptide inhibitor shown in red.

Figure 3

Inactive compounds docked in the homodimer interface of hTS.

As an example, compound 7 showed a very interesting docking pose. This compound was able to bind the homodimer at the interface and is aligned with the cocrystallized inhibitor. This means that this compound can simultaneously bind the two homodimer chains and stabilize the closed inactive conformation (Figure ).

Figure 4

Compound 7 docked in the homodimer interface and establishing interactions with residues at the allosteric site.

Two important hydrogen bonding were noted with the amide side chain and PHE154 from chain A and LEU204 from chain B. The quinoxaline ring represents an anchor group that affects the orientation of the peptidomimetic side chain in this key position. The quinoxaline ring interacts with TRP194 through hydrogen bonding and with PHE154 and TRP194 through π–π and van der Waals interactions. The phenyl ring is positioned in a lipophilic pocket between PHE154 and LEU204.

The low activity of several compounds carrying bulky and/or long peptide mimetic side chains could be because of the volume and size restrictions imposed by residues VAL170. From the abovementioned discussion, one can conclude the quinoxaline ring is a suitable scaffold for TS allosteric binding. An aromatic substituent in the position-2 increases the affinity and selectivity to the enzyme and with careful selection of the peptide mimetic chain at position-3, we could reach an active and selective candidate which is suitable for preclinical development.

Conclusions

3-Phenylquinoxaline-2(1H)-thione is an interesting parent substrate obtained by a novel thiation of 3-phenylquinoxalin-2(1H)-one and amenable of simple structure modification by the Michael reaction with acrylic acid derivatives to afford a number of S-substituted quinoxaline derivatives including the ester methyl 3-((3-phenyl-1,2-dihydroquinoxalin-2-yl)sulfanyl)propanoate. A series of amino acids and their corresponding hydrazides and N-alkyl amine attached to quinoxaline via a propanoyl spacer have been prepared by the azide coupling method from the corresponding methyl 3-((3-phenyl-1,2-dihydroquinoxalin-2-yl)sulfanyl)propanoate.

The synthesized compounds exhibited promising anticancer activity with IC50’s in the low micromolar range. The most active compounds had IC50’s of 1.9 and 2.3 μg/mL on the HCT-116 and the MCF-7 cell lines, respectively, compared to the reference drug doxorubicin (IC50 3.23 μg/mL). In silico studies suggested a possible mode of action through hTS allosteric inhibition.

Based on the obtained promising anticancer results and molecular modeling studies, the current work emphasizes the designed and synthesized quinoxaline derivatives as novel promising anticancer agents. It is necessary to extend this study by performing an enzyme-based assay and in vivo testing to proof the proposed mode of action and guide compound optimization in terms of selectivity and physicochemical properties for preclinical development.

Experimental Section

General Procedures

Solvent was purified and dried in the usual way. The boiling range of the petroleum ether used was 40–60 °C. Thin layer chromatography: silica gel 60 F254 plastic plates (E. Merck, layer thickness 0.2 mm) 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 were uncorrected. 1H and 13C NMR spectra were recorded at 400 and 100 MHz(Bruker AC 400) in CDCl3 and dimethyl sulfoxide (DMSO) solution, respectively, with tetramethylsilane as an internal standard. The NMR analyses were performed by Faculty of Science, Sohag University. The mass spectra were measured with a KRATOS analytical compact; on the MALDI-MS, the spectrometer was using 2,5-dihydroxy benzoic acid (DHB) as the matrix. 3-Phenylquinoxalin-2(1H)-one (3) and 2-chloro-3-phenylquinoxaline (4) were prepared according to the method described.[32,41]

Preparation of the Thiating Reagent N-Cyclohexyldithiocarbamate Cyclohexyl Ammonium Salt (2)[32]

To a mixture of freshly distilled cyclohexyl amine (60 mmol) and water (50 mL) was added carbon disulfide (21 mmol) dropwise. The reaction mixture was stirred overnight at room temperature. The white solid obtained was filtered, washed with water, dried, and crystallized from ethanol to provide the pure product of cyclohexyl amine cyclohexyl ammonium dithiocarbamate (2). White crystals (98%), mp 188–189 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.00–8.06 (4H, m, 3NH & NH); 4.15–3.95 (1H, m, CH); 3.05–2.96 (1H, m, CH); 1.98–0.96 (20H, m, 10CH2). 13C NMR spectrum (75 MHz, CDCl3): δ 212.4 (C=S); 55.3 (CH); 50.0 (CH); 32.3 (2CH2); 30.9 (2CH2); 25.8 (CH2); 25.5 (2CH2); 25.1 (CH2); 24.3 (2CH2). Anal. Calcd for C13H26N2S2 (274.2): C, 56.88; H, 9.55; N, 10.21. Found: C, 56.63; H, 9.28; N, 10.06.

Preparation of Phenylquinoxaline-2(1H)-thione (5)[32]

To a solution of 2-chloro-3-phenylquinoxaline (4) (2.5 mmol) in CHCl3(25 mL) was added N-cyclohexyldithiocarbamate cyclohexyl ammonium 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. The yellowish precipitate was filtered to give the desired product, crystallized from ethanol. Yellow powder (91%), mp 224–225 °C. 1H NMR spectrum (300 MHz, DMSO): δ, ppm (J, Hz): 14.56 (1H, br s, 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). Anal. Calcd for C14H10N2S (238.1): C, 70.56; H, 4.23; N, 11.76. Found: C, 70.13; H, 3.84; N, 11.29.

General Procedure for the Michael Reaction

To a mixture of quinoxaline 5 (0.24 g, 1.0 mmol) and triethylamine (0.2 mL, 2.0 mmol) in ethyl alcohol (30 mL, 95%), the appropriate acrylic acid derivative (methyl acrylate, acrylamide, and acrylonitrile) (1.0 mmol) was added. The reaction mixture was heated under reflux for 4–6 h and concentrated under reduced pressure. The solid obtained was filtered and crystallized from ethyl alcohol.

Methyl 3-((3-Phenyl-1,2-dihydroquinoxalin-2-yl)sulfanyl)propanoate (6)[36]

White crystals (91%), mp 101–103 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.13 (1H, d, J = 8 Hz, ArH); 8.00 (1H, d, J = 8.0 Hz, ArH); 7.80–7.65 (4H, m, ArH); 7.55–7.53 (3H, m, ArH); 3.74 (3H, s, OCH3); 3.58 (2H, t, J = 6.0 Hz, SCH2); 2.89 (2H, t, J = 6.0 Hz, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 172.4 (C=O); 154.7; 153.4; 141.5; 139.4; 137.2; 129.8; 129.7; 129.2; 129.0; 128.5; 128.3; 127.6 (C Ar); 51.8 (OCH3); 33.8 (SCH2); 25.6 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 347 (M + Na)+. Anal. Calcd for C18H16N2O2S (324.4) C, 66.64; H, 4.97; N, 8.64. Found: 66.57; H, 4.91; N, 8.59.

3-((3-Phenylquinoxalin-2-yl)sulfanyl)propanamide (7)

White crystals (87%), mp 213–214 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.09 (1H, d, J = 8 Hz, ArH); 8.03 (1H, d, J = 8.0 Hz, ArH); 7.75–7.56 (4H, m, ArH); 7.47–7.42 (3H, m, ArH); 5.71 (2H, br s, NH2); 3.53 (2H, t, J = 6.0 Hz, SCH2); 2.87 (2H, t, J = 6.0 Hz, CH2CONH2). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 169.8 (C=O); 155.4; 153.8; 141.1; 139.0; 137.5; 130.0; 129.6; 129.4; 127.5 (C Ar); 37.8 (SCH2); 28.7 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 332 (M + Na)+. Anal. Calcd for C17H15N3OS (309.4): C, 66.00; H, 4.89; N, 13.58. Found: C, 65.87; H, 4.69; N, 13.41.

3-((3-Phenylquinoxalin-2-yl)sulfanyl)propanenitrile (8)[36]

White crystals (84%), mp 128–130 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.05 (1H, d, J = 8 Hz, ArH); 7.98 (1H, d, J = 8.0 Hz, ArH); 7.83–7.56 (4H, m, ArH); 7.47–7.41 (3H, m, ArH); 3.65 (2H, t, J = 6.0 Hz, SCH2); 2.95 (2H, t, J = 6.0 Hz, CH2CN). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 154.6; 153.4; 141.2; 139.0; 137.5; 130.1; 129.3; 129.0; 127.6 (C Ar); 114.3 (CN); 35.8 (SCH2); 18.7 (CH2CN). MS (MALDI, positive mode, matrix DHB) m/z: 314 (M + Na)+. Anal. Calcd for C17H13N3S (291.4): 70.08; H, 4.50; N, 14.42. Found: 69.87; H, 4.39; N, 14.31.

3-((3-Phenylquinoxalin-2-yl)sulfanyl)propanoic Acid Hydrazide (9)

Hydrazine hydrate (80%) (2.4 mL, 5 mmol) was added to a solution of ester 6 (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. White crystals (88%), mp 208–209 °C. 1H NMR spectrum (400 MHz, DMSO-d6): δ, ppm (J, Hz): 9.00 (1H, s, NH); 8.04–7.99 (2H, m ArH); 7.85–7.73 (4H, m ArH); 7.57–7.47 (3H, m, ArH); 4.21 (2H, br s, NH2); 3.47 (2H, t, J = 7.4 Hz, SCH2); 2.53 (2H, t, J = 7.3 Hz, CH2CO). 13C NMR spectrum (75.0 MHz, DMSO-d6): δ, ppm: 170.3 (C=O); 155.1; 153.5; 141.2; 139.2; 137.3; 130.9; 130.2; 129.3; 129.2; 128.8; 128.4; 127.7 (C Ar); 32.9 (SCH2); 26.5 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 347 (M + Na)+. Anal. Calcd for C17H16N4OS (324.4): C, 62.94; H, 4.97; N, 17.27. Found: C, 62.85; H, 4.91; N, 17.14.

Preparation of Methyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoates 11 and N-Alkyl 3-((3-Phenylquinoxalin-2-yl)sulfanyl)propanamide 12 (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 9 (0.32 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, yellowish syrup started to form. The reaction mixture was stirred in an ice bath for further 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 10. 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 10. The mixture was kept at −5 °C for 24 h, 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 the desired S-coupled products 11 and 12.

Methyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]acetate (11a)

White crystals (69%), mp 149–150 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.04 (1H, d, J = 8 Hz, ArH); 7.92 (1H, d, J = 8.0 Hz, ArH); 7.71–7.56 (4H, m, ArH); 7.47–7.44 (3H, m, ArH); 6.10 (1H, br s, NH); 3.99 (2H, d, J = 7.2 Hz, NHCH2); 3.67 (3H, s, OCH3); 3.51 (2H, t, J = 7.2 Hz, SCH2); 2.70 (2H, t, J = 7.2 Hz, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 171.2 (C=O); 170.3 (C=O); 154.9; 153.5; 141.4; 139.5; 137.2; 129.9; 129.8; 129.3; 129.0; 128.4; 128.3; 127.5 (C Ar); 52.3 (OCH3); 41.3 (NHCH2); 35.8 (SCH2); 26.1 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 404 (M + Na)+. Anal. Calcd for C20H19N3O3S (381.5): C, 62.98; H, 5.02; N, 11.02. Found: C, 62.85; H, 4.93; N, 10.89.

Methyl-3-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]propanoate (11b)

White crystals (64%), mp 165–166 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.09 (1H, d, J = 8.0 Hz, ArH); 8.01 (1H, d, J = 8.0 Hz, ArH); 7.79–7.64 (4H, m, ArH); 7.53–7.48 (3H, m, ArH); 5.67 (1H, br s, NH); 3.68 (3H, s, CH3); 3.60–3.56 (4H, m, NHCH2, SCH2); 2.70 (2H, t, J = 7.2 Hz, CH2COOCH3); 2.57 (2H, t, J = 7.2 Hz, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 173.0 (C=O); 171.0 (C=O); 154.9; 153.5; 141.4; 139.5; 137.2; 129.9; 129.8; 129.3; 129.0; 128.5; 128.3; 127.5 (C Ar); 51.7 (OCH3); 36.0 (NHCH2); 34.9 (SCH2); 33.8 (CH2COOCH3); 26.4 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 318 (M + Na)+. Anal. Calcd for C21H21N3O3S (395.5): C, 63.78; H, 5.35; N, 10.63. Found: C, 63.65; H, 5.27; N, 10.54.

Dimethyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]succinate (11c)

White crystals (72%), mp 117–118 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.08 (1H, d, J = 8 Hz, ArH); 7.95 (1H, d, J = 8.0 Hz, ArH); 7.71–7.57 (4H, m, ArH); 7.47–7.43 (3H, m, ArH); 6.53 (1H, d, J = 7.2 Hz, NH); 4.83–4.79 (1H, m, CH); 3.66 (3H, s, OCH3); 3.57 (3H, s, OCH3); 3.55–3.39 (2H, m, SCH2); 2.97–2.66 (4H, m, CH2, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 172.9 (C=O); 172.4 (C=O); 168.5 (C=O); 154.5; 153.2; 141.3; 139.1; 137.1; 130.0; 129.8; 129.6; 129.5; 129.0; 128.5; 128.3; 127.6 (C Ar); 56.8 (CH); 52.3 (OCH3); 51.9 (OCH3); 36.0 (SCH2); 35.8 (CH2COOCH3); 26.4 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 476 (M + Na)+. Anal. Calcd for C23H23N3O5S (453.5): C, 60.91; H, 5.11; N, 9.27. Found: C, 60.82; H, 5.03; N, 9.18.

Dimethyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]pentandioate (11d)

White crystals (78%), mp 140–141 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.05 (1H, d, J = 8.0 Hz, ArH); 7.95 (1H, d, J = 8.0 Hz, ArH); 7.71–7.54 (4H, m, ArH); 7.47–7.41 (3H, m, ArH); 5.37 (1H, br s, NH); 4.81–4.69 (1H, m, CH); 3.68 (3H, s, OCH3); 3.62 (3H, s, OCH3); 3.55 (2H, t, J = 7.2 Hz, CH2CO); 3.44 (2H, m, SCH2); 2.68 (2H, t, J = 7.2 Hz, CH2CO); 2.38 (2H, t, J = 7.2 Hz, CH2). MS (MALDI, positive mode, matrix DHB) m/z: 490 (M + Na)+. Anal. Calcd for C24H25N3O5S (467.5): C, 61.65; H, 5.39; N, 8.99. Found: C, 61.57; H, 5.26; N, 8.81.

Methyl-3-methyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propan-amido]butanoate (11e)

White crystals (79%), mp 118–119 °C. 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 8.07 (1H, d, J = 8.0 Hz, ArH); 7.97 (1H, d, J = 8.0 Hz, ArH); 7.71–7.54 (4H, m, ArH); 7.47–7.43 (3H, m, ArH); 6.08 (1H, br s, NH); 4.60–4.52 (1H, m, CH); 3.72 (3H, s, OCH3); 3.62–3.56 (2H, m, SCH2); 2.76 (2H, t, J = 7.2 Hz, CH2CO); 2.14 (1H, m, CH); 0.93 (3H, s, CH3); 0.89 (3H, s, CH3). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 172.4 (C=O); 170.9 (C=O); 154.9; 153.5; 141.5; 139.2; 136.9; 130.0; 129.8; 129.1; 128.5; 127.5 (C Ar); 57.1 (CH); 52.1 (OCH3); 36.0 (SCH2); 31.4 (CH); 26.4 (CH2CO); 18.9 (CH3); 17.8 (CH3). MS (MALDI, positive mode, matrix DHB) m/z: 446 (M + Na)+. Anal. Calcd for C23H25N3O3S (423.5): C, 65.23; H, 5.95; N, 9.92. Found: C, 65.23; H, 5.95; N, 9.92.

Methyl-4-methyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propan-amido]pentanoate (11f)

White crystals (77%), mp 125–126 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.03 (1H, d, J = 8 Hz, ArH); 7.92 (1H, d, J = 8.0 Hz, ArH); 7.70–7.55 (4H, m, ArH); 7.47–7.43 (3H, m, ArH); 5.98 (1H, d, J = 7.2 Hz, NH); 4.62–4.57 (1H, m, CH); 3.63 (3H, s, OCH3); 3.53–3.47 (2H, m, SCH2); 2.68 (2H, t, J = 7.2 Hz, CH2CO); 1.58–1.39 (3H, m, CH2, CH); 0.85 (3H, d, J = 7.2 Hz, CH3); 0.81 (3H, d, J = 7.2 Hz, CH3). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 172.4 (C=O); 170.9 (C=O); 155.0; 153.5; 141.5; 139.2; 136.9; 130.0; 129.8; 129.6; 129.1; 128.5; 128.4; 127.5 (C Ar); 57.1 (NHCH); 52.1 (OCH3); 38.9 (CH2); 36.0 (SCH2); 26.4 (CH2CO); 21.3 (CH); 17.8 (CH3). MS (MALDI, positive mode, matrix DHB) m/z: 460 (M + Na)+. Anal. Calcd for C24H27N3O3S (437.6): C, 65.88; H, 6.22; N, 9.60. Found: C, 65.67; H, 6.18; N, 9.49.

Methyl-4-methylsulfanyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanamido]butanoate (11g)

White crystals (68%), mp 118–120 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.05 (1H, d, J = 8 Hz, ArH); 7.93 (1H, d, J = 8.0 Hz, ArH); 7.71–7.56 (4H, m, ArH); 7.45–7.43 (3H, m, ArH); 6.08 (1H, br s, NH); 4.62–4.57 (1H, m, CH); 3.67 (3H, s, OCH3); 3.52–3.50 (2H, m, SCH2); 2.72–2.69 (4H, m, CH2, CH2CO); 2.18–1.97 (5H, m, CH2, SCH3). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 171.4 (C=O); 168.7 (C=O); 155.3; 153.5; 141.2; 139.2; 137.3; 130.2; 129.6; 129.4; 128.8; 128.4; 127.7 (C Ar); 56.5 (CH); 52.3 (OCH3); 38.5 (CH2); 35.7 (SCH2); 34.5 (CH2); 26.1 (CH2CO); 21.8 (SCH3). MS (MALDI, positive mode, matrix DHB) m/z: 478 (M + Na)+. Anal. Calcd for C23H25N3O3S2 (455.6): C, 60.63; H, 5.53; N, 9.22. Found: C, 60.54; H, 5.48; N, 9.16.

N-Propyl-3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamide (12a)

White crystals (78%), mp 151–153 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.05 (1H, d, J = 8 Hz, ArH); 7.99 (1H, d, J = 8.0 Hz, ArH); 7.71–7.57 (4H, m, ArH); 7.48–7.42 (3H, m, ArH); 5.75 (1H, br s, NH); 3.56 (2H, t, J = 7.2 Hz, SCH2); 3.19–3.15 (2H, m, NHCH2); 2.66 (2H, t, J = 7.2 Hz, CH2CO); 1.50–1.37 (2H, m, CH2); 0.87 (3H, d, J = 7.2 Hz, CH3). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 171.1 (C=O); 155.0; 153.5; 141.4; 139.4; 137.2; 129.9; 129.8; 129.3; 129.0; 128.4; 128.3; 127.4 (C Ar); 41.3 (NHCH2); 36.0 (SCH2); 26.5 (CH2CO); 22.8 (CH2); 11.3 (CH3). Anal. Calcd for C20H21N3OS (351.5): C, 68.35; H, 6.02; N, 11.96. Found: C, 68.25; H, 5.94; N, 11.84.

N-Butyl 3-((3-Phenylquinoxalin-2-yl)sulfanyl)propanamide (12b)

White crystals (68%), mp 170–171 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.06 (1H, d, J = 8 Hz, ArH); 7.91 (1H, d, J = 8.0 Hz, ArH); 7.71–7.57 (4H, m, ArH); 7.45–7.41 (3H, m, ArH); 5.64 (1H, m, NH); 3.52 (2H, t, J = 7.0 Hz, SCH2); 3.20–3.17 (2H, m, NHCH2); 2.62 (2H, t, J = 7.0 Hz, CH2CO); 1.41–1.38 (2H, m, CH2); 1.28–1.23 (2H, m, CH2); 0.85 (3H, t, J = 7.0 Hz, CH3). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 171.0 (C=O); 155.2; 153.3; 141.4; 139.0; 136.6; 130.1; 130.0; 129.1; 128.5; 127.3 (C Ar); 39.4 (NHCH2); 36.1 (SCH2); 31.7 (CH2); 26.6 (CH2CO); 20.0 (CH2); 16.7 (CH3). MS (MALDI, positive mode, matrix DHB) m/z: 388 (M + Na)+. Anal. Calcd for C21H23N3OS (365.5): C, 69.01; H, 6.34; N, 11.50. Found: C, 68.87; H, 6.21; N, 11.42.

N-Allyl-3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamide (12c)

White crystals (75%), mp 176–178 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 7.99 (1H, d, J = 8 Hz, ArH); 7.87 (1H, d, J = 8.0 Hz, ArH); 7.68–7.54 (4H, m, ArH); 7.44–7.41 (3H, m, ArH); 5.78–5.69 (2H, m, NH, CH); 5.09 (1H, d, J = 20.0 Hz, CH2); 5.03 (1H, d, J = 8.0 Hz, CH2); 3.82 (2H, t, J = 7.0 Hz, NHCH2); 3.52 (2H, t, J = 7.0 Hz, SCH2); 2.62 (2H, t, J = 7.0 Hz, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 171.5 (C=O); 154.7; 153.8; 141.2; 139.4; 137.1; 133.5 (CH); 130.0; 129.8; 129.5; 129.4; 128.6; 128.5; 127.6; 118.1 (CH2); 41.6 (NHCH2); 36.8 (SCH2); 26.4 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 372 (M + Na)+. Anal. Calcd for C20H19N3OS (349.4): C, 68.74; H, 5.48; N, 12.02. Found: C, 68.69; H, 5.42; N, 11.97.

N-Tetradecyl-3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamide (12d)

White crystals (65%), mp 126–128 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 7.96 (1H, d, J = 8 Hz, ArH); 7.85 (1H, d, J = 8.0 Hz, ArH); 7.66–7.51 (4H, m, ArH); 7.44–7.41 (3H, m, ArH); 5.68 (1H, br s, NH); 3.82 (2H, t, J = 7.0 Hz, NHCH2); 3.52 (2H, t, J = 7.0 Hz, SCH2); 2.62 (2H, t, J = 7.0 Hz, CH2CO); 1.25–1.08 (24H, m, 12CH2); 0.80 (3H, d, J = 7.2 Hz, CH3). MS (MALDI, positive mode, matrix DHB) m/z: 528 (M + Na)+. Anal. Calcd for C31H43N3OS (505.8): C, 73.62; H, 8.57; N, 8.31. Found: C, 73.47; H, 8.49; N, 8.21.

N-Cyclohexyl-3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamide (12e)

White crystals (71%), mp 190–192 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.11 (1H, d, J = 8 Hz, ArH); 7.99 (1H, d, J = 8.0 Hz, ArH); 7.79–7.53 (4H, m, ArH); 7.51–7.44 (3H, m, ArH); 5.56 (1H, br s, NH); 3.81–3.79 (1H, m, NHCH); 3.60 (2H, t, J = 7.0 Hz, SCH2); 2.67 (2H, t, J = 7.0 Hz, CH2CO); 1.92–1.69 (2H, m, CH2); 1.41–1.26 (4H, m, 2CH2); 1.15–1.06 (4H, m, 2CH2). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 170.0 (C=O); 155.0; 153.6; 141.4; 139.5; 137.2; 129.9; 129.8; 129.3; 129.0; 128.4; 128.3; 127.4 (C Ar); 48.3 (NHCH); 36.2 (SCH2); 33.2 (CH2); 26.5 (CH2CO); 25.5 (CH2); 24.8 (CH2). MS (MALDI, positive mode, matrix DHB) m/z: 414 (M + Na)+. Anal. Calcd for C23H25N3OS (391.5): C, 70.56; H, 6.44; N, 10.73. Found: C, 70.48; H, 6.34; N, 10.65.

N-Benzyl-3-((3-phenylquinoxalin-2-yl)sulfanyl)propanamide (12f)

White crystals (79%), mp 176–178 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 7.97 (1H, d, J = 8.0 Hz, ArH); 7.76 (1H, d, J = 8.0 Hz, ArH); 7.67–7.54 (4H, m, ArH); 7.44–7.42 (3H, m, ArH); 7.20–7.15 (5H, m, ArH); 5.97 (1H, br s, NH); 4.37 (2H, d, J = 7.2 Hz, NHCH2); 3.52 (2H, t, J = 7.2 Hz, SCH2); 2.65 (2H, t, J = 7.2 Hz, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 170.8 (C=O); 154.9; 153.5; 141.3; 139.3; 138.1; 137.0; 129.9; 129.8; 129.2; 129.0; 128.6; 128.4; 128.3; 127.8; 127.5; 127.2 (C Ar); 43.7 (NHCH2); 36.0 (SCH2); 26.3 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 422 (M + Na)+. Anal. Calcd for C24H21N3OS (399.5): C, 72.15; H, 5.30; N, 10.52. Found: C, 72.03; H, 5.22; N, 10.48.

1-Morpholino-3-((3-phenylquinoxalin-2-yl)sulfanyl)propan-1-one (12g)

White crystals (70%), mp 183–185 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 7.98 (1H, d, J = 8 Hz, ArH); 7.84 (1H, d, J = 8.0 Hz, ArH); 7.69–7.53 (4H, m, ArH); 7.43–7.41 (3H, m, ArH); 3.67–3.50 (4H, m, 2CH2); 3.51 (2H, t, J = 7.0 Hz, SCH2); 3.48–3.41 (2H, m, CH2); 2.86 (2H, t, J = 7.2 Hz, CH2); 2.75 (2H, t, J = 7.2 Hz, CH2CO). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 169.0 (C=O); 154.1; 152.5; 140.4; 138.5; 137.2; 129.9; 129.8; 129.3; 128.9; 128.4; 128.3; 127.3 (C Ar); 66.6 (OCH2); 65.4 (OCH2); 45.1 (NCH2); 44.9 (NHCH2); 36.1 (SCH2); 26.1 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 402 (M + Na)+. Anal. Calcd for C21H21N3O2S (379.5): C, 66.47; H, 5.58; N, 11.07. Found: C, 66.34; H, 5.41; N, 11.01.

3-((3-Phenylquinoxalin-2-yl)thio)-1-(piperidin-1-yl)propan-1-one (12h)

White crystals (84%), mp 200–201 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.04 (1H, d, J = 8 Hz, ArH); 7.90 (1H, d, J = 8.0 Hz, ArH); 7.69–7.56 (4H, m, ArH); 7.47–7.44 (3H, m, ArH); 3.51 (2H, t, J = 7.0 Hz, SCH2); 3.57 (4H, m, ring 2NCH2); 2.75 (8H, m, CH2CO, ring 3CH2). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 169.0 (C=O); 154.1; 152.5; 140.4; 138.5; 136.2; 128.9; 128.8; 128.4; 128.0; 127.4; 126.3 (C Ar); 46.3 (NCH2); 43.1 (NCH2); 34.1 (SCH2); 25.9 (CH2); 25.2 (CH2); 25.1 (CH2CO); 23.9 (CH2). MS (MALDI, positive mode, matrix DHB) m/z: 400 (M + Na)+. Anal. Calcd for C22H23N3OS (377.5): C, 70.00; H, 6.14; N, 11.13. Found: C, 69.88; H, 6.01; N, 11.07.

1-(4-Methylpiperazin-1-yl)-3-((3-phenylquinoxalin-2-yl)thio)propan-1-one (12i)

White crystals (69%), mp 207–208 °C. 1H NMR spectrum (400 MHz, CDCl3): δ, ppm (J, Hz): 8.01 (1H, d, J = 8 Hz, ArH); 7.89 (1H, d, J = 8.0 Hz, ArH); 7.71–7.57 (4H, m, ArH); 7.46–7.43 (3H, m, ArH); 3.53–3.48 (2H, m, SCH2); 3.06–2.97 (8H, m, 4CH2); 2.71–2.66 (2H, m, CH2CO); 1.95 (3H, s, NCH3). 13C NMR spectrum (75.0 MHz, CDCl3): δ, ppm: 170.0 (C=O); 154.8; 153.5; 141.3; 139.4; 137.1; 137.0; 129.9; 129.8; 129.3; 129.0; 128.4; 128.3; 127.3; 54.2 (NCH2); 52.0 (NCH2); 45.5 (NCH2); 43.4 (NCH2); 35.2 (SCH2); 25.9 (CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 415 (M + Na)+. Anal. Calcd for C22H24N4OS (392.5): C, 67.32; H, 6.16; N, 14.27. Found: C, 67.18; H, 6.11; N, 14.09.

Preparation of 2-[3-(3-Phenyl-quinoxalin-2-ylsulfanyl)propanamido]alkanoic Acid Hydrazides

To a solution of 2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanoylamino]alkanoates 11a, c, e, and g (Gly, l-Asp, l-Val, and l-Meth) (1.0 mmol) in ethyl alcohol 95% (15 mL) was added hydrazine hydrate (60%) (0.2 mL, 2.0 mmol). The reaction mixture was refluxed for 6 h and kept in the fridge for 12 h, and the formed crystals were filtered and crystallized from ethanol 95%.

2-[3-(3-Phenyl-quinoxalin-2-ylsulfanyl)propanoylamino]acetic Acid Hydrazide (13a)

White crystals (85%), mp 185–186 °C. 1H NMR spectrum (400 MHz, DMSO-d6): δ, ppm (J, Hz): 9.01 (1H, br s, NH); 8.12 (1H, br s, NH); 8.04–7.99 (2H, m, ArH); 7.87–7.72 (4H, m, ArH); 7.69–7.55 (3H, m, ArH); 4.23 (2H, br s, NH2); 3.68 (2H, s, NHCH2); 3.46 (2H, t, J = 7.0 Hz, SCH2); 2.67 (2H, t, J = 7.0 Hz, CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 404 (M + Na)+. Anal. Calcd for C19H19N5O2S (381.5): C, 59.82; H, 5.02; N, 18.36. Found: C, 59.71; H, 4.94; N, 18.29.

3-Methyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propanoylamino]butanoic Acid Hydrazide (13e)

White crystals (78%), mp 251–253 °C. 1H NMR spectrum (400 MHz, DMSO-d6): δ, ppm (J, Hz): 9.10 (1H, br s, NH); 8.12 (1H, br s, NH); 8.06–7.99 (2H, m, ArH); 7.79–7.67 (4H, m, ArH); 7.58–7.55 (3H, m, ArH); 4.21 (2H, br s, NH2); 4.18–4.11 (1H, m, CH); 3.46 (2H, t, J = 7.0 Hz, SCH2); 2.67 (2H, t, J = 7.0 Hz, CH2CO); 1.88–1.85 (1H, m, CH); 0.81 (6H, d, J = 7.0 Hz, 2CH3). MS (MALDI, positive mode, matrix DHB) m/z: 446 (M + Na)+. Anal. Calcd for C22H25N5O2S (423.5): C, 62.39; H, 5.95; N, 16.54. Found: C, 62.31; H, 5.89; N, 16.43.

4-Methylsulfanyl-2-[3-(3-phenyl-quinoxalin-2-ylsulfanyl)propan-amido]butanoic Acid Hydrazide (13g)

White crystals (81%), mp 121–122 °C. 1H NMR spectrum (400 MHz, DMSO-d6): δ, ppm (J, Hz): 9.00 (1H, br s, NH); 8.11–8.00 (3H, m, 2ArH, NH); 7.82–7.69 (4H, m, ArH); 7.59–7.56 (3H, m, ArH); 4.34 (2H, br s, NH2); 3.75–3.68 (1H, m, CH); 3.46 (2H, t, J = 7.0 Hz, SCH2); 2.82–2.66 (4H, m, CH2, CH2CO); 2.05–1.92 (5H, m, CH2, SCH3). MS (MALDI, positive mode, matrix DHB) m/z: 478 (M + Na)+. Anal. Calcd for C22H25N5O2S2 (455.6): C, 58.00; H, 5.53; N, 15.37. Found: C, 57.93; H, 5.49; N, 15.21.

2-[3-(3-Phenyl-quinoxalin-2-ylsulfanyl)propanamido]succinic Acid Hydrazide (14)

White crystals (76%), mp 176–177 °C. 1H NMR spectrum (400 MHz, DMSO-d6): δ, ppm (J, Hz): 9.00 (1H, br s, NH); 8.88 (1H, br s, NH); 8.05–8.01 (2H, m, ArH); 7.85–7.75 (5H, m, ArH, NH); 7.69–7.56 (3H, m, ArH); 4.68–4.57 (1H, m, CH); 4.19 (4H, br s, 2NH2); 3.48–3.46 (2H, m, SCH2); 2.63–2.33 (4H, m, 2CH2CO). MS (MALDI, positive mode, matrix DHB) m/z: 476 (M + Na)+. Anal. Calcd for C21H23N7O3S (453.5): C, 55.62; H, 5.11; N, 21.62. Found: C, 55.49; H, 5.03; N, 21.53.

In Vitro Antiproliferative Activity

Cell Culture & In Vitro Drug Treatments

Human embryonic kidney cells (HEK-293), human colorectal (colon cancer) carcinoma cells (HCT-116) and human adenocarcinoma (breast cancer) cells (MCF-7) were cultured in the media containing DMEM; (10%) l-glutamine; (10%) fetal bovine serum; (10%) selenium chloride; (120 unit/mL) penicillin/streptomycin. The cells were cultured in the CO2 (5%) incubator (Thermo Scientific Heracell-150) at 37 °C. Then, the cells (HCT-116, MCF-7, and HEK-293) with 70–80% confluence were treated with different concentrations (2–40 μg/mL) of 38 synthetic compound treatments. The treated (HCT-116, MCF-7, and HEK-293) cells were analyzed after 48 h intervals.

Microscopic Analysis

All the 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 we also examined the structural morphological difference between cancerous cells (HCT-116 & MCF-7) and healthy normal cells (HEK-293).

Cell Viability by the MTT Assay

All the cells (HCT-116, MCF-7, and HEK-293) were grown in 96-well cell culture plates, and once they were 70–80% confluence, they were treated with 38 synthetic compounds. The cells were treated with different concentrations of (2–40 μg/mL) of 38 different compounds. In the control group, synthetic compounds were not added. After 48 h of treatment, MTT (5.0 mg/mL) was exposed to control and treated cells and was kept under incubation for 4 h. Then, DMSO was added to each well and plates were measured at 570 nm wavelength using the ELISA Plate Reader (Biotek Instruments, Winooski, USA). We have calculated the percentage (%) of cell viability (%).

Statistical Evaluation

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

Molecular Modeling and Docking

All the molecular modeling studies were performed on a Hewlett-Packard Pentium Dual-Core T4300 2.10 GHz running Windows 10 using autodock 4.3 for molecular docking simulation and ligand binding energy calculation and Molsoft ICM-Pro 3.5-0 for output data visualization. The crystal structure of the human TS dimer bound to a short peptide LSCQLYQR (PDB code: 3N5E) has been chosen as a receptor. This structure is a homodimer, in its closed conformation, and represents the inactive conformation of the enzyme. The putative ligand binding site has been assigned based on the positions of the heavy atoms of the peptide reported.[30] The selected targets were used after deleting the cocrystallized inhibitors. Docking calculations were carried out using the AutoDock 4.3 software (La Jolla, CA).[31] First, all hydrogens were added to the ligand PDB file and Gasteiger charges were computed and all the torsion angles of the ligand were defined with the autodock tools program so they could be explored during molecular modeling. A grid box of 50 × 25 × 25 Å with a grid spacing of 0.375 Å, centered at the crystallized ligand (X = −43.413 Y = 0.0164 Z = 11.8350) to cover all the homodimer interface, was used to calculate the atom types needed for the calculation. The Lamarckian genetic algorithm was used as a search method with a total of 30 runs (maximum of 20 000 000 energy evaluations; 27 000 generations; initial populations of 150 conformers).

The docking results were evaluated visually through interaction with key residues and have been calibrated using crystallized ligands by checking the ligand binding position in Molsoft ICM-Pro 3.5-0.