What this is
- This research focuses on the design and synthesis of novel 1,2,4-triazolo[4,3-a]quinoxaline derivatives with potential anti-diabetic and anti-Alzheimer properties.
- The derivatives were evaluated for their inhibitory effects on α-amylase, α-glucosidase, and enzymes.
- Molecular docking simulations were performed to assess binding interactions and affinities of the most active compounds.
Essence
- The novel 1,2,4-triazolo[4,3-a]quinoxaline derivatives showed promising anti-diabetic activity, particularly the N-allyl derivative, which outperformed acarbose in inhibiting α-glucosidase.
Key takeaways
- The N-allyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-triazoloquinoxalin-1-amine (10a) exhibited the highest inhibitory percentages of 64.70 ± 0.02% against α-amylase and 75.36 ± 0.01% against α-glucosidase, surpassing acarbose.
- The 1-methyl-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-triazoloquinoxaline (11b) showed a maximum inhibitory percentage of 44.78 ± 0.01% against but was less effective than donepezil.
- Molecular docking simulations indicated strong binding affinities of the most active derivatives with the target enzymes, supporting their potential as dual-target therapeutic agents.
Caveats
- The in vitro activities were evaluated at a single concentration of 100 µM, which may not represent the full pharmacological profile.
- Further biological evaluations are necessary to confirm the efficacy and safety of the derivatives in vivo.
Definitions
- α-amylase: An enzyme that catalyzes the hydrolysis of starch into sugars, a target for anti-diabetic drugs.
- acetylcholinesterase: An enzyme that breaks down acetylcholine, a neurotransmitter, targeted in Alzheimer's disease therapies.
AI simplified
Introduction
Diabetes mellitus (hyperglycemia) is a long-term metabolic problem that causes blood sugar levels to be too high because of insulin deficiency (type 1) or insulin resistance (type 2). It is a significant threat to people all over the world1. A higher probability of cancer, kidney failure, blindness, and amputation2, as well as a heightened susceptibility to bone fractures3, are all associated with diabetes mellitus4. Diabetes is associated with a complex cascade of numerous metabolic and signalling pathways in its pathophysiology5. When blood sugar levels are high, auto-oxidative glycosylation occurs. It sets off protein kinase C and improves the polyol cascade. These activities generate ROS (reactive oxygen species) and RNS (reactive nitrogen species), causing oxidative stress6,7. It is imperative to develop antidiabetic medications that effectively address the significant risks associated with diabetes mellitus while minimizing adverse effects8. Two critical anti-diabetic pharmaceuticals approved by the Food and Drug Administration (FDA) that regulate hyperglycemia are acarbose and miglitol, which function by inhibiting the carbohydrate-hydrolyzing enzymes α-amylase and α-glucosidase9,10. Despite their demonstrated efficacy, these medications can elicit substantial adverse side effects, particularly gastrointestinal issues attributed to acarbose and miglitol11,12. However, the long-term use of these hypoglycemic agents has been associated with adverse and often unavoidable side effects, such as congestive heart failure, inflammation, cellular apoptosis, pancreatitis, and gastrointestinal discomfort.
On the other hand, Alzheimer’s disease (AD) is detected by the presence of dementia, which usually starts with mild difficulties in recognizing things and remembering information13. The cholinergic system in AD is particularly prone to synapse loss, notably in cortical regions that are linked to memory and executive function14. Recent research has indicated that the primary factor contributing to the decline of cognitive abilities in individuals with Alzheimer’s disease (AD) is a gradual decrease in cholinergic neurotransmission in several parts of the human brain, including the cortex15. A acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) are hydrolytic enzymes that cleave acetylcholine (ACh) into choline and acetate in the synaptic cleft to stop its effects. Both enzymes are valid targets for improving the cholinergic deficit that is believed to be the cause of the cognitive, behavioral, and global functional deterioration seen in Alzheimer’s disease16.
The triazole moiety is essential in pharmaceutical chemistry. Selective triazoles play a crucial role in the drug development process for heterocyclic bioactive compounds exhibiting a broad spectrum of activities30. When this ring is associated with other heterocyclic rings, it may lead to enhanced efficacy against α-glucosidase. Over the past two decades, researchers have synthesized numerous indole derivatives to discover novel molecules31. Also, the class of organic compounds known as substituted 1,2,4-triazoles is significant due to their broad pharmacological activities, which include antibacterial32,33, antifungal34, antimycobacterial35, anti-inflammatory32,36, and anticancer37 properties. Fluconazole38, Alprazolam, Letrozole, and Ribavirin are widely used medications in the class of 1,2,4-triazoles39. These drugs are recognized for their fluoro- and trifluoromethyl-substituted properties. On the other hand, sulfonamides are well recognized for their significant therapeutic and pharmacological importance. The sulfonamide moiety (–SO2N) is a pharmacophore that exhibits a diverse array of actions, such as antibacterial, antimalarial, anti-HIV, high ceiling diuretic, antithyroid, anticancer effects40–42, and insulin-releasing antidiabetic activity, which is particularly significant43. Pyrrolidine is classified as a nitrogenous heterocyclic compound that serves as a privileged scaffold in medicinal chemistry due to its biological activity, structural versatility, and pharmacokinetic properties44. Furthermore, its presence in numerous bioactive synthetic drugs, alkaloids, and natural products, as well as its role as a pharmaceutical intermediate, underscores its significance in drug development and discovery10. This importance may be attributed to its lipophilic properties, which contribute to membrane permeability and enhanced metabolic stability. The pyrrolidine scaffold has demonstrated a wide range of biological activities, including anticancer, antimicrobial, pesticide, antiviral, anti-diabetic, and anti-inflammatory effects45–47.
Based on the aforementioned critical features and building upon our prior research in the design and synthesis of novel bioactive agents from heterocyclic compounds48–55, this study was designed to synthesize new regioisomer sulfonamide-quinoxaline derivatives incorporating a novel hybrid core, specifically 1,2,4-triazole. The designed derivatives were evaluated for their antidiabetic properties against α-amylase and α-glucosidase, as well as for their potential as anti-Alzheimer agents against acetylcholinesterase to be used as dual-target drug therapies. Additionally, the structure-activity relationship (SAR) study provided detailed insights into the effects of different substituents at the N1 position of triazole and the placement of the pyrrolidin-1-ylsulfonyl moiety. Finally, molecular docking simulations were conducted for the most active derivatives within the active sites of three enzymes: α-amylase (PDB: 2QV4), α-glucosidase (PDB: 3W37), and acetylcholinesterase (ACHE) (PDB: 4EY7). These simulations aimed to elucidate the binding modes and types of interactions, as well as to assess the binding affinity of each derivative in comparison to positive control drugs.
The rational study focused on quinoxaline and triazole containing drugs, including our newly designed triazole-quinoxaline derivatives.
Results and discussion
Chemistry
Similarly, the novel 4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline-1-thiol 9 was obtained in good yield by the ring closure reaction of the 3-hydrazino-quinoxaline 7 with carbon disulfide in refluxing pyridine. The structure of compound 9 was deduced on the basis of elemental analysis and spectral data. Also, the formation of 1,2,4-triazolo[4,3-a]quinoxaline-1-thiol 9 is assumed to be formed via the addition of the amino group of hydrazine to the activated double bond of carbon disulfide to form the intermediate dithiocarbamic acid D, which underwent cyclization with the elimination of hydrogen sulfide under the reaction conditions.
Furthermore, the novel 1,2,4-triazolo[4,3-a]quinoxalin-1-amine derivative 10a, b was achieved in a one-pot reaction by the addition of 3-hydrazino-quinoxaline derivative 5 to allyl isothiocyanate or phenyl isothiocyanate under reflux in dry pyridine. The analytical and spectral data of the isolated products completely agreed with the structure of triazolo-quinoxaline 10a, b. The infrared spectrum revealed absorption bands at 3228 and 1597 cm− 1 attributed to the NH and C = C groups, respectively. Its1H NMR spectrum showed two singlets for the protons of the pyrrolidine moieties at δ 1.68 and 3.19 ppm, a doublet for the protons of the sp3 carbon at δ 4.14 ppm, as well as multiples for the protons of the sp2 carbon and the proton of the carbon of methine at δ 5.20 and 5.88 ppm, respectively. The remaining signals represent aromatic and NH protons. Also, their13C NMR spectrum displayed signals at δ 25.67 (pyrrolidine-C), 47.03 (sp3 carbon), 49.32 (pyrrolidine -C), 102.90 (sp2 carbon), 117.62 (sp2 carbon, methine), 125.14, 129.60, 129.90, 130.29, 131.15, 135.61, 139.57, 143.49 ppm for the aromatic carbons and 155.54 related to the N = C.
Finally, the addition of 2-hydrazino-quinoxaline derivative 7 to allyl isothiocyanate or phenyl isothiocyanate in dry pyridine under reflux led to the generation of the novel [1,2,4]triazolo[4,3-a]quinoxalin-1-amine derivative 13a, b in one pot (Scheme 4). The microanalytical and spectral data of the isolated products were in complete agreement with the structure of triazolo-quinoxaline. The infrared spectrum of compound 13a showed absorption bands at 3345 and 1595 cm-1 that were attributed to the NH and C = C groups, respectively. The1H NMR spectrum showed signals at δ 1.69 and 3.17 ppm for pyrrolidine moieties, a singlet signal at δ 4.23 ppm for the protons of the sp3 carbon, and multiple two signals at δ 5.28 and 5.88 ppm for the protons of the sp2 carbon and the proton of the carbon of methine, respectively. The13C NMR spectrum additionally revealed signals at δ 25.85 (pyrrolidine-CH2), 46.47 (sp3 carbon), 49.02 (pyrrolidine-N-CH2), 101.03 (sp2 carbon), 117.45 (sp2 carbon, methine), 122.62, 127.10, 128.04, 128.44, 132.12, 134.17, 135.92, 146.48, and 157.62 (N = C). The formation of triazoloquinoxaline 13a is assumed to proceed via the addition of the amino group of hydrazine derivative 7 to the activated double bond in isothiocyanate 12a to generate the non-isolable acyclic intermediate thiosemicarbazide H, which underwent cyclo-desulfurization under the reaction conditions to yield 13a that its formation was confirmed by using a wet lead acetate paper which became black during the course reaction, suggesting the generation of hydrogen sulfide gas. Moreover, its1H NMR spectrum suggested that in addition to the expected signals, a downfield signal at δ 9.92 ppm designated for the NH group was also observed.
Synthetic strategy for synthesis of two isomer 2,3-hydrazino-6-(pyrrolidin-1-ylsulfonyl)quinoxaline derivatives 5 and 7.
Mechanismatic pathway for formation of 3-hydrazino-quinoxaline derivative 5
![Synthesis of novel 4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline derivatives,,, and, 8a b 9 10a b](https://europepmc.org/articles/PMC12134304/bin/41598_2025_3139_Sch3_HTML.jpg.jpg "Click to view full size")
Synthesis of novel 4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline derivatives,,, and, 8a b 9 10a b
![Synthesis of novel 4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline derivatives,,, and,. 11a b 12 13a b](https://europepmc.org/articles/PMC12134304/bin/41598_2025_3139_Sch4_HTML.jpg.jpg "Click to view full size")
Synthesis of novel 4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline derivatives,,, and,. 11a b 12 13a b
Biological evaluation
In-vitro anti-diabetic activity with SAR study
The two starting materials, 3-hydrazino-2-(pyrrolidin-1-yl)quinoxaline derivative 5 and 2-hydrazino-3-(pyrrolidin-1-yl)quinoxaline derivative 7 contains the 6-(pyrrolidin-1-ylsulfonyl) exhibited close inhibitory activity against the tested enzymes. For 3-hydrazino-2-(pyrrolidin-1-yl)quinoxaline derivative 5 showed inhibitory percentage of 34.81 ± 0.01 and 37.60 ± 0.01% against α-amylase and α-glucosidase enzymes, while 2-hydrazino-3-(pyrrolidin-1-yl)quinoxaline derivative exhibited inhibitory percentage of 36.17 ± 0.01 and 37.60 ± 0.01% against α-amylase and α-glucosidase enzymes indicating that these two derivatives have the same effect on α-glucosidase enzyme and very close against amylase.
Generally, for the synthesized 4-(pyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]quinoxaline derivatives 8–13, we found introducing the sulfonamide group to be 8-sulfonyl position in triazolo-quinoxaline scaffold exhibited better inhibitory activity than 7-sulfonyl that has the same nucleus and substituents, except for 1-methyl-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline 11b. The 1,2,4-triazolo[4,3-a]quinoxaline 11b revealed good inhibitory percentage against α-amylase (IP = 36.85 ± 0.01%) and α-glucosidase (IP = 39.64 ± 0.01%) compared the second isomer 8b against α-amylase (IP = 18.42 ± 0.01%) and α-glucosidase (IP = 21.90 ± 0.03%) indicating electron donating group to triazole as methyl group enhancing the activity. On the other hand, replacing the hydrogen or methyl group at position one with thiol group causes enhancing the inhibitory activity in case of 8-sulfonyl-1,2,4triazoloquinoxaline derivative 9 with inhibitory percentage of against α-amylase (IP = 30.77 ± 0.01%) and α-glucosidase (IP = 39.49 ± 0.04%) rather than 8-sulfonyl-1,2,4triazoloquinoxaline derivative 12 with inhibitory percentage against α-amylase (IP = 21.85 ± 0.01%) and α-glucosidase (IP = 23.93 ± 0.01%), but still less than 4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline (8a) α-amylase (IP = 50.46 ± 0.01%) and α-glucosidase (IP = 59.98 ± 0.01%). Additionally, the 4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline (8a) exhibited the second most active member among the synthesized derivatives and exhibited that reaction of 3-hydrazino-quinoxaline derivative 5 with triethyl orthoformate and formation of triazolo-quinoxaline is preferred.
Introducing the amino group to position one of the 1,2,4-triazole nuclei enhances the activity in the case of the 8-sulfonyl group than the 7-sulfonyl group. Moreover, the presence of allyl group attached to the amino group in the case of N-allyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxalin-1-amine (10a) exhibited the best inhibitory activity with inhibitory percentage of 64.70 ± 0.02 and 75.36 ± 0.01% against α-amylase and α-glucosidase, respectively and compared to the designed derivatives. The N-allyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxalin-1-amine (10a) showed super inhibitory activity against α-glucosidase with inhibitory percentage value of 75.36 ± 0.01% compared to acarbose (IP = 57.79 ± 0.01%) with nearly 1.3-folds higher than positive control. On the other hand, N-allyl-4-(pyrrolidin-1-yl)- 1,2,4-triazolo[4,3-a]quinoxalin-1-amine derivative 10a revealed good inhibitory activity with IP value of 64.70 ± 0.02% with a slightly lower activity than acarbose (IP = 67.33 ± 0.01%) against α-amylase. Replacing the allyl group with more hydrophobic moiety as phenyl group causes dropping in the activity as represented in N-phenyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxalin-1-amine (10b), where the inhibitory percentage values of 23.45 ± 0.01 for α-amylase and 25.75 ± 0.01% for α-glucosidase. Conversely, introducing the N-substituted-7-(pyrrolidin-1-ylsulfonyl)-triazolo[4,3-a]quinoxalin-1-aminederivatives 13a, b don’t enhance the anti-diabetic activity against the two tested enzymes.
In conclusion, it can be confirmed that the 1,2,4-triazolo-quinoxaline demonstrates anti-diabetic activity by inhibiting α-amylase and α-glucosidase, with varying percentages of inhibition influenced by the substituents on the triazole nucleus, particularly at position one. Furthermore, the presence of the sulfonyl group serves as a significant contributor to the biological activity observed in the two isomers. Notably, the isomers 8-(pyrrolidin-1-ylsulfonyl)triazolo-quinoxaline derivatives 8–10 exhibited greater activity compared to the 8-(pyrrolidin-1-ylsulfonyl)[1,2,4]-triazolo-quinoxaline derivatives 11–13 indicating the introduction of hydrophobic pyrrolidinyl at position two as electron donating group and opposite to sulfonamide moiety at position eight is preferred. Moreover, the most promising candidate, 10a, demonstrated a selective inhibition profile favoring α-glucosidase inhibition over α-amylase inhibition. This selectivity may represent a therapeutic advantage by presenting a more favorable safety and tolerability profile by mitigating gastrointestinal side effects, including flatulence, abdominal discomfort, and diarrhea, where these adverse effects primarily result from the accumulation of undigested carbohydrates in the colon, where they are fermented by gut microbiota, leading to gas production.
| CompoundsNo. | The in vitro inhibitory percentage (IP ± SE) of the designed two isomers sulfonamide quinoxaline derivatives at 100 µM | ||
|---|---|---|---|
| Anti-diabetic activitya | Anti-Alzheimer activitya | ||
| α-amylase | α-glucosidase | AChE | |
| 5 | 34.81 ± 0.01 | 37.60 ± 0.01 | 36.66 ± 0.01 |
| 7 | 36.17 ± 0.01 | 37.60 ± 0.01 | 38.61 ± 0.02 |
| 8a | 50.46 ± 0.01 | 59.98 ± 0.01 | 17.16 ± 0.01 |
| 8b | 18.42 ± 0.01 | 21.90 ± 0.03 | 14.01 ± 0.00 |
| 9 | 30.77 ± 0.01 | 39.49 ± 0.04 | 41.20 ± 0.01 |
| 10a | 64.70 ± 0.02 | 75.36 ± 0.01 | 19.26 ± 0.01 |
| 10b | 23.45 ± 0.01 | 25.75 ± 0.01 | 13.99 ± 0.01 |
| 11a | 22.28 ± 0.00 | 24.07 ± 0.01 | 12.61 ± 0.01 |
| 11b | 36.85 ± 0.01 | 39.64 ± 0.01 | 44.78 ± 0.01 |
| 12 | 21.85 ± 0.01 | 23.93 ± 0.01 | 12.44 ± 0.01 |
| 13a | 21.94 ± 0.00 | 24.08 ± 0.00 | 12.42 ± 0.03 |
| 13b | 21.90 ± 0.01 | 24.06 ± 0.00 | 12.61 ± 0.01 |
| Acarbose | 67.33 ± 0.01 | 57.79 ± 0.01 | - |
| Donepezil | - | - | 67.27 ± 0.60 |
| CompoundsNo. | The in vitro median inhibitory concentrations (IC± SE) (µM)50 | |
|---|---|---|
| Anti-diabetic activitya | ||
| α-amylase | α-glucosidase | |
| 10a | 6.89 ± 0.09 | 3.46 ± 0.06 |
| Acarbose | 5.90 ± 0.09 | 4.27 ± 0.06 |
In-vitro anti-Alzheimer activity
The most active 1-methyl-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline (11b) revealed the highest inhibitory percentage with value of 44.78 ± 0.01% compared to all the designed derivatives and the second member is [1,2,4]triazolo[4,3-a]quinoxaline derivative 9 with IP = 41.20 ± 0.01%, but both of them still showed lower inhibitory percentage for donepezil as positive control drug (IP = 67.27 ± 0.60%). The other 1,2,4-triazolo[4,3-a]quinoxaline derivatives 8–13 demonstrated low to moderate inhibitory percentage with values ranging from 12.42 ± 0.03 to 17.16 ± 0.01% compared to 3-hydrazino-quinoxaline derivatives 5 (IP = 36.66 ± 0.01%) and 7 (IP = 38.61 ± 0.02%). Moreover, modification of the parent compound 11b through the substitution of the C1 methyl group with bulkier substituents, specifically, N-allyl-1-amine in derivative 13a and N-phenyl-1-amine in derivative 13b, resulted in a notable decrease in inhibitory potency. Compounds 13a and 13b demonstrated reduced acetylcholinesterase inhibition, with IC₅₀ values recorded at 12.42 ± 0.03 µM and 12.61 ± 0.01%, respectively. This reduction in activity indicates that the introduction of larger substituents at this position may induce steric hindrance or negatively influence binding affinity at the active site of the enzyme. This observation underscores the sensitivity of enzyme interactions to subtle structural alterations within the quinoxaline framework.
In conclusion, the moderate activity exhibited by the designed derivatives against acetylcholinesterase suggests the necessity for further biological evaluations in subsequent research. This will facilitate exploring and assessing additional targets, including assays for Aβ aggregation inhibition and antioxidant studies.
Graph represented the inhibitory percentage of the designed sulfonamide-quinoxaline against acetylcholinesterase (AChE).
In-silico toxicity prediction
Furthermore, the most promising [1,2,4]triazolo[4,3-a]quinoxaline derivatives 10a and 11b were predicted to have LD50 values of 465 mg/kg and 250 mg/kg, respectively, indicating that these compounds belong to toxicity classes 4 and 3. In contrast, Acarbose belongs to class 6 with an LD50 of 24,000 mg/kg, while Donepezil is classified as class 4 (LD50 = 505 mg/kg).
Finally, based on the obtained data, the most promising [1,2,4]triazolo[4,3-a]quinoxaline derivatives 10ajiand 11b were predicted to demonstrate a more favorable safety profile in comparison to the positive control drugs acarbose and donepezil.
| Tested items | In-silico toxicity prediction of most active [1,2,4]triazolo[4,3-a]quinoxaline derivatives 10a and 11b compared to positive control drugs | |||
|---|---|---|---|---|
| 10a | 11b | Acarbose | Donepezil | |
| LD(mg/kg)50 | 465 | 250 | 24000 | 505 |
| Toxicity class | 4 | 3 | 6 | 4 |
| Hepatotoxicity | Inactive 0.63 | Inactive 0.69 | Active 0.65 | Inactive 0.98 |
| Nephrotoxicity | Inactive 0.72 | Inactive 0.77 | Active 0.80 | Inactive 0.67 |
| Cardiotoxicity | Inactive 0.90 | Inactive 0.92 | Active 0.60 | Active 0.50 |
| Immunotoxicity | Inactive 0.95 | Inactive 0.83 | Active 0.99 | Active 0.95 |
| Mutagenicity | Inactive 0.70 | Inactive 0.70 | Inactive 0.76 | Inactive 0.53 |
| Cytotoxicity | Inactive 0.53 | Inactive 0.59 | Inactive 0.70 | Active 0.63 |
| Ecotoxicity | Inactive 0.61 | Inactive 0.61 | Inactive 0.66 | Active 0.56 |
| Nutritional toxicity | Inactive 0.62 | Inactive 0.58 | Inactive 0.52 | Inactive 0.53 |
Molecular docking simulation
The molecular docking simulation was conducted to evaluate the binding affinities for the most active derivatives, N-allyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxalin-1-amine (10a) and acarbose on α-amylase (PDB: 2QV4) and α-glucosidase (PDB: 3W37) as anti-diabetic agents. Additionally, the highly active compound, 1-methyl-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline (11b) was docked within the active site of acetylcholinesterase (ACHE) (PDB: 4EY7), utilizing donepezil as a positive control.
![Graph represented the inhibitory percentage of the designed sulfonamide-quinoxaline against acetylcholinesterase (AChEGraph represented the inhibitory percentage of the designed sulfonamide-quinoxaline against acetylcholinesterase (AChE2D and 3D structure of the most active N-allyl-[1,2,4]triazolo[4,3-a]quinoxalin-1-aminederivative 10a inside the active site of α-amylase (PDB: 2QV4).](https://europepmc.org/articles/PMC12134304/bin/41598_2025_3139_Fig3_HTML.jpg.jpg "Click to view full size")
Graph represented the inhibitory percentage of the designed sulfonamide-quinoxaline against acetylcholinesterase (AChEGraph represented the inhibitory percentage of the designed sulfonamide-quinoxaline against acetylcholinesterase (AChE2D and 3D structure of the most active N-allyl-[1,2,4]triazolo[4,3-a]quinoxalin-1-aminederivative 10a inside the active site of α-amylase (PDB: 2QV4).
![2D and 3D structure of the most active N-allyl-[1,2,4]triazolo[4,3-a]quinoxalin-1-aminederivativeinside the active site of α-glucosidase (PDB: 3W37). 10a](https://europepmc.org/articles/PMC12134304/bin/41598_2025_3139_Fig4_HTML.jpg.jpg "Click to view full size")
2D and 3D structure of the most active N-allyl-[1,2,4]triazolo[4,3-a]quinoxalin-1-aminederivativeinside the active site of α-glucosidase (PDB: 3W37). 10a
![2D and 3D structure of the most active 1-methyl-4-(pyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]quinoxaline derivativeinside the active site of acetylcholinesterase (AChE) (PDB: 4EY7). 11b](https://europepmc.org/articles/PMC12134304/bin/41598_2025_3139_Fig5_HTML.jpg.jpg "Click to view full size")
2D and 3D structure of the most active 1-methyl-4-(pyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]quinoxaline derivativeinside the active site of acetylcholinesterase (AChE) (PDB: 4EY7). 11b
Conclusion
In summary, this study presented the design and synthesis of novel 1,2,4-triazolo[4,3-a]quinoxaline derivatives featuring pyrrolidin-1-ylsulfonyl as a bioactive fragment, derived from hydrazio-6-(pyrrolidin-1-ylsulfonyl)quinoxaline derivatives 5 and 7, utilizing a chemically regioselective synthesis approach. The structures of the synthesized 1,2,4-triazolo[4,3-a]quinoxaline derivatives were characterized and confirmed through various spectroscopic analyses, achieving favorable yields. The in vitro activities of these derivatives against α-amylase, α-glucosidase, and acetylcholinesterase (AChE) were evaluated and expressed as inhibitory percentages at a concentration of 100 µM. Generally, the 8-(pyrrolidin-1-ylsulfonyl) isomers 8–10 showed higher bio-evaluation than 7-(pyrrolidin-1-ylsulfonyl) isomer 11–13 cross the tested activities, with the exception of compound 11b against AChE. The N-allyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxalin-1-amine derivative 10a demonstrated exceptional inhibitory activity against α-glucosidase, with an inhibitory percentage of 75.36 ± 0.01%, surpassing that of acarbose (IP = 57.79 ± 0.01%) by nearly 1.3-fold. Conversely, the N-allyl-4-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3-a]quinoxalin-1-amine derivative 10a exhibited good inhibitory activity against α-amylase, with an inhibitory percentage of 64.70 ± 0.02%, which is slightly lower than acarbose (IP = 67.33 ± 0.01%). Furthermore, the tested derivatives demonstrated limited inhibition of acetylcholinesterase (AChE), with low inhibitory percentage values, with the exception of the 1-methyl-[1,2,4]triazolo[4,3-a]quinoxaline derivative 11b, which exhibited an inhibitory percentage of 44.78 ± 0.01% compared to donepezil, the positive control drug (IP = 67.27 ± 0.60%). Additionally, the 1,2,4-triazolo[4,3-a]quinoxaline derivative 9 displayed the second most potent activity, with an inhibitory percentage of 41.20 ± 0.01%. By determining the IC50 values for most potent derivative 10a as promising candidate for antidiabetic agent, we showed that it revealed remarkable inhibitory activity, with IC50 values of 3.46 ± 0.06 µM against α-glucosidase and 6.89 ± 0.09 µM against α-amylase. In comparison, acarbose has IC50 values of 4.27 ± 0.06 µM and 5.90 ± 0.09 µM for the same enzymes, respectively. The molecular docking simulation was conducted to elucidate the potential binding modes and affinities of the most active derivatives within the active sites of the enzymes compared to the positive control drugs acarbose and donepezil.
Experimental
Chemistry
Materials and instrumentation
All reagents and chemicals were ordered from Aldrich Chemicals without further purification, and solvents from Fisher. Melting points (MPs) of all the newly designed compounds were recorded on a digital Gallen Kamp MFB-595 instrument using open capillaries. Within the range of 400–4000 cm− 1, IR spectra were calculated using the KBr disc methodology on a Shimadzu 440 spectrophotometer. In NMR spectra (1H / 13C), chemical shifts were calculated in δ ppm relative to TMS as an internal default (= 0 ppm) that obtained on a JOEL spectrometer 500 / 125 MHz using DMSO-d6 as solvents. The data was provided in the following format: chemical shift, multiplicity (br. = broad, m = multiplet, qu = quintet, q = quartet, t = triplet, d = doublet, and s = singlet), coupling constant (J) in Hertz (Hz), and integration. Elemental studies were carried out at Cairo University’s Micro Analytical Unit in Cairo. At Al-Azhar University’s Regional Center for Biotechnology, mass spectra were calculated at 70 eV using the DI-50 unit of a Shimadzu GC/MSQP5050A Spectrometer. Additionally, the 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride 1 was prepared and reported according to the literature methods66,67, while 6-(pyrrolidin-1-ylsulfonyl)-1,4-dihydroquinoxaline-2,3-dione 2 was prepared and reported according to the literature methods58,68,69.
Synthesis of organic materials
Synthesis of 2,3-dichloro-6-(pyrrolidin-1-ylsulfonyl)quinoxaline (3)
The key intermediate was prepared according to our previously work with slightly modification58. DMF (2 mL) was added drop by drop to a solution of 6-(pyrrolidin-1-ylsulfonyl)-1,4-dihydroquinoxaline-2,3-dione 2 (4 mmol) and POCl3 (20 mmol), the solution was stirred at 80 ºC for 6 h (monitored by TLC). After the reaction is completed, the solution is added portion wise to ice-water and neutralized with ammonia solution 30%. The formed precipitate was collected by filtration and crystallized from CH3CN to obtain the dichloro derivatives.
Grey powder (CH3CN); 85% yield; m.p. 190–192 ºC; IR (KBr): νmax = 3051(CHar), 2970, 2875 (CHalip), 1615 (C = N), 1336, 1151 (SO2) cm–1;1H NMR (δ, ppm) = 1.63 (4 H, qu, (CH2)2-pyrrolidine), 3.26 (4 H, t, N(CH2)2-pyrrolidine), 8.22 (d, 1 H, J = 8.0 Hz, H7.quinox), 8.28 (d, 1 H, J = 8.0 Hz, H8.quinox), 8.42 (s, 1 H, H5.quinox)13. C NMR (δ, ppm) = 25.10 ((CH2)2-pyrrolidine), 48.31 (N(CH2)2-pyrrolidine), 125.99, 127.68, 130.03, 138.99, 139.53 (Ar.Cs), 141.78 (C-SO2), 147.09 (N = C-Cl), 147.77 (N = C-Cl); Anal. Calcd for C12H11ClN3O2S (332.20): C, 43.39; H, 3.34; N, 12.65; Found: C, 43.46; H, 3.58; N, 12.41.
Synthesis of 3-chloro-2-(pyrrolidin-1-yl)-6-(pyrrolidin-1-ylsulfonyl)quinoxaline (4)
To a solution of 2,3-dichloroquinoxaline sulfonamide derivatives 3 (1 mmol) and a secondary amine, such as pyrrolidine (1.5 mmol) in acetonitrile was stirred for 7 h until the complete consumption of the starting materials (monitored by TLC). After evaporation of the solvent, the resulting precipitate was washed with ethanol to remove the excess of secondary amine; it did not require any further purification.
Pale-yellow powder (CH3CN); 75% yield; m.p. 214–216 ºC; IR (KBr): νmax = 3040 (CHar), 2965, 2835 (CHalip), 1639 (C = N), 1324, 1141 (SO2) cm− 1;1H NMR (δ, ppm) = 1.93–2.03 (8 H, m, (CH2)4-pyrrolidine), 3.73–3.75 (8 H, m, N(CH2)4-pyrrolidine), 7.73 (d, 1 H, J = 10.0 Hz, H7.quinox), 7.93 (d, 1 H, J = 10.0 Hz, H8.quinox), 8.07 (s, 1 H, H5.quinox);13C NMR (δ, ppm) = 25.28 ((CH2)2-pyrrolidine), 50.17 (N(CH2)2-pyrrolidine), 125.42, 127.90, 129.10, 130.29, 131.77, 135.93 (Ar.Cs), 144.59 (C-Cl), 160.57 (N = C-N); Anal. Calcd for C16H19ClN4O2S (366.86): C, 52.38; H, 5.22; N, 15.27; Found: C, 52.40; H, 5.20; N, 15.30.
Synthesis of 3-hydrazino-2-(pyrrolidin-1-yl)-6-(pyrrolidin-1-ylsulfonyl)quinoxaline (5)
To a mixture of compound 4 (1 mmol) in absolute ethanol and the appropriate hydrazine hydrate (100%) (1 mmol) was heated under reflux for 3 h at 120 OC, until the reaction completed (monitored by TLC), the solid product was precipitated on hot, collected, washed with ethanol and then recrystallized from acetonitrile to produce the following pure compound.
Orange powder (CH3CN); 67% yield; m.p. 250–252 ºC; IR (KBr): νmax = 3499, 3315, 3185 (NH2, NH), 2950 (br.CHalip), 1606 (C = N), 1325, 1136 (SO2);1H NMR (δ, ppm) = 1.60–1.63 (8 H, m, (CH2)4-pyrrolidine), 3.07 (8 H, t, N(CH2)4-pyrrolidine), 7.00 (br. s, 3 H, NH2 + NH, exchangeable with D2O), 7.35 (d, 2 H, J = 8.4 Hz, H7.quinox, H8.quinox), 7.53 (s, 1 H, H5.quinox);13C NMR (δ, ppm) 26.56 ((CH2)2-pyrrolidine), 54.76 (N(CH2)2-pyrrolidine), 125.13, 128.02, 129.62, 131.35, 133.55, 138.05, 147.21 (Ar.Cs), 156.60 (N = C-NH); Anal. Calcd for C16H22N6O2S (362.45): C, 53.02; H, 6.12; N, 23.19; Found: C, 53.05; H, 6.14; N, 23.21.
Synthesis of 3-chloro-2-hydrazino-6-(pyrrolidin-1-ylsulfonyl)quinoxaline (6)
To a mixture of the starting material 3 (1 mmol) in absolute ethanol and the appropriate hydrazine hydrate (100%) (1 mmol) was added portion-wise for 15 min. Until the addition is completed, the solution was stirred at room temperature for 3 h, (monitored by TLC). After the reaction is completed, the new product was precipitated, collected, washed with ethanol and then recrystallized from 1,4-dioxane to produce the following pure compound.
Light-orange powder (1,4-dioxane); 80% yield; m.p. 200–202 ºC; IR (KBr): νmax = 3429, 3380, 3254 (NH2, NH), 3082 (CHar), 2990, 2946, 2891 (CHalip), 1642 (C = N), 1337, 1126 (SO2);1H NMR (δ, ppm) = 1.72–1.91 (4 H, m, ((CH2)2-pyrrolidine), 3.63–3.73 (4 H, m, N(CH2)2-pyrrolidine), 7.31 (br. s, 2 H, NH2, exchangeable with D2O), 7.43 (br. s, 1 H, NH, exchangeable with D2O), 7.90 (d, 1 H, J = 8.2 Hz, H7.quinox), 7.95–8.02 (m, 2 H, H8.quinox, H5.quinox);13C NMR (δ, ppm) = 25.57 ((CH2)2-pyrrolidine), 49.12 (N(CH2)2-pyrrolidine), 125.21, 126.36, 127.77, 129.14, 133.32, 137.99 (Ar.Cs), 143.79 (C-Cl), 153.77 (N = C-NH); Anal. Calcd for C12H14ClN5O2S (327.79): C, 43.97; H, 4.31; N, 21.37; Found: C, 43.78; H, 4.23; N, 21.59.
Synthesis of 2-hydrazino-3-(pyrrolidin-1-yl)-6-(pyrrolidin-1-ylsulfonyl)quinoxaline (7)
To a solution of compound 6 (1 mmol) and a secondary amine (pyrrolidine) (1.5 mmol) in absolute ethanol was heated under reflux for 3 h at 120 ºC, until the reaction completed (monitored by TLC), the solid product was precipitated on hot, collected, washed with ethanol and then recrystallized from acetonitrile to produce the following pure compound.
Deep-orange powder (CH3CN); 86% yield; m.p. 244–246 ºC; IR (KBr): νmax = 3422 (br. NH2, NH), 3051(CHar), 2980, 2759 (CHalip), 1598 (C = N), 1338, 1132 (SO2) cm-1;1H NMR (δ, ppm) = 1.64 (8 H, s, (CH2)4-pyrrolidine), 3.17 (4 H, s, N(CH2)2-pyrrolidine), 3.45 (4 H, s, N(CH2)2-pyrrolidine), 7.31 (br. s, 2 H, NH2, exchangeable with D2O), 7.43 (br. s, 1 H, NH, exchangeable with D2O), 7.64 (d, 1 H, J = 8.8 Hz, H7.quinox), 7.76–7.84 (m, 2 H, H8.quinox, H5.quinox);13C NMR (δ, ppm) = 25.25 ((CH2)2-pyrrolidine), 50.12 (N(CH2)2-pyrrolidine), 54.78 (N(CH2)2-pyrrolidine), 122.44, 125.54, 127.48, 129.21, 129.94, 135.96, 143.01 (Ar.Cs), 160.73 (N = C-NH); Anal. Calcd for C16H22N6O2S (362.45): C, 53.02; H, 6.12; N, 23.19; Found: C, 53.15; H, 6.18; N, 23.01.
Synthesis of 1-substituted-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline (8a, b)
To a solution of 3-hydrozienylquinoxaline derivatives 5 (1 mmol) and either triethyl orthoformate (10 mmol) or triethyl orthoacetate (10 mmol) was stirred at 100 ºC for 1 h (monitored by TLC). After the reaction is cooled, the solid product was precipitated, collected, washed ethanol and then recrystallized from acetonitrile to produce the following pure compounds.
4-(Pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxaline (8a) a
Brown powder (CH3CN); 79% yield; m.p. 344–346 ºC; IR (KBr): νmax = 3039 (CHar), 2988, 2880 (CHalip), 1633 (C = N), 1334, 1196 (SO2) cm-1;1H NMR (δ, ppm) = 1.65 (8 H, s, (CH2)4-pyrrolidine), 3.22 (4 H, s, N(CH2)2-pyrrolidine), 3.27 (4 H, s, N(CH2)2-pyrrolidine),8.08 (1 H, d, J = 8.8 Hz, H8. quinox), 8.65 (1 H, d, J = 8.8 Hz, H7. quinox), 8.85 (1 H, s, H5. quinox), 9.06 (1 H, s, CH. triazole)13. C NMR (δ, ppm) = 25.29 (4CH2.pyrrolidine), 48.52 (4 N-CH2.pyrrolidine), 123.80, 126.00, 126.61, 126.97, 135.93, 139.34, 139.39, 145.03 (Ar.CS), 159.48 (N = C-N); Anal. Calcd for C17H20N6O2S (372.45): C, 54.82; H, 5.41; N, 22.56; Found: C, 54.75; H, 5.23; N, 22.68.
1-Methyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxaline (8b) a
Light-brown powder (CH3CN); 72% yield; m.p. 310–312 ºC; IR (KBr): νmax = 3049(CHar), 2978, 2892 (CHalip), 1633 (C = N), 1337, 1196 (SO2) cm-1;1H NMR (δ, ppm) = 1.63 (8 H, qui, (CH2)4-pyrrolidine), 2.00 (3 H, s, CH3.triazole), 3.09 (4 H, s, N(CH2)2-pyrrolidine), 3.10 (4 H, t, N(CH2)2-pyrrolidine), 7.79 (1 H, d, J = 6.0 Hz, H8. quinox), 8.08 (1 H, d, J = 8.8 Hz, H7. quinox), 8.50 (1 H, s, H5. quinox)13. C NMR (δ, ppm) = 14.48 (CH3. Triazole), 25.32 ((CH2)2-pyrrolidine), 48.25 (N(CH2)2-pyrrolidine), 122.78, 125.09, 126.31, 127.41, 130.77, 133.18, 134.96, 143.53, 148.42 (Ar.CS), 157.82 (N = C-N); Anal. Calcd for C18H22N6O2S (386.47): C, 55.94; H, 5.74; N, 21.75; Found: C, 55.95; H, 5.76; N, 21.77.
Synthesis of 4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxaline-1-thiol (9). a
To a solution of compound 5 (1 mmol) and carbon disulphide (1 mmol) in pyridine (15 mL) as solvent and catalyst, the solution mixture was refluxed (until the evolution of H2S finished). The reaction mixture was then poured onto cold water, and the solid product precipitated was collected by filtration, dried, and recrystallized from 1,4-dioxane.
Deep-orange powder (1,4-dioxane); 84% yield; m.p. 288–290 ºC; IR (KBr): νmax = 3041 (CHar), 2945, 2886 (CHalip), 1633 (C = N), 1317, 1197 (SO2) cm-1;1H NMR (δ, ppm) = 1.69 (8 H, s, (CH2)4-pyrrolidine), 3.27 (8 H, s, N(CH2)4-pyrrolidine), 7.90 (1 H, d, J = 7.6 Hz, H8. quinox), 8.06 (1 H, d, J = 9.6 Hz, H7. quinox), 8.62 (1 H, s, H5. quinox), 11.11 (1 H, s, SH, D2O exchangeable)13. C NMR (δ, ppm) = 25.28 ((CH2)2-pyrrolidine), 48.42 (N(CH2)2-pyrrolidine), 124.84, 126.00, 129.04, 134.37, 137.28, 138.02, 149.05 (Ar.Cs), 164.13 (N = C-N), 164.64 (N = C-SH); Anal. Calcd for C17H20N6O2S2 (404.51): C, 50.48; H, 4.98; N, 20.78; Found: C, 50.77; H, 4.76; N, 20.60.
Synthesis of N-substituted-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxalin-1-amine (10a, b). a
To a solution of compound 5 (1 mmol) and derivatives of isothiocyanate (1 mmol) namely, ally isothiocyanate and / or phenyl isothiocyanate in pyridine (15 mL) as solvent and catalyst, the solution mixture was refluxed (until the evolution of H2S finished). The reaction mixture was then poured onto cold water, and the solid product precipitated was collected by filtration, dried, and recrystallized from 1,4-dioxane.
-Allyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxalin-1-amine (10a) N a
Brown powder (1,4-dioxane); 80% yield; m.p. 305–307 ºC; IR (KBr): νmax = 3041(CHar), 2945, 2922 (CHalip), 1634 (C = N), 1331, 1197 (SO2) cm-1;1H NMR (δ, ppm) = 1.68 (8 H, s, (CH2)4-pyrrolidine), 3.19 (8 H, s, N(CH2)4-pyrrolidine), 4.14 (2 H, d, CH2-CH.allyl), 5.20 (2 H, m, CH2 = CH-allyl), 5.88 (1 H, s, CH = CH2.allyl), 6.09 (1 H, s, NH, D2O exchangeable), 7.62 (1 H, d, J = 7.2 Hz, H8. quinox), 8.06–8.21 (1 H, m, H7. quinox), 8.70 (1 H, s, H5. quinox)13. C NMR (δ, ppm) = 25.67 ((CH2)2-pyrrolidine), 47.03 (CH2-CH.allyl), 49.32(N(CH2)2-pyrrolidine), 102.90 (CH2 = CH. allyl), 117.62 (CH2 = CH. allyl), 125.14, 129.60, 129.90, 130.29, 131.15, 135.61, 139.57, 143.49 (Ar.Cs), 155.54 (N = C-N); Anal. Calcd for C20H25N7O2S (427.53): C, 56.19; H, 5.89; N, 22.93; Found: C, 56.10; H, 5.99; N, 22.75.
-Phenyl-4-(pyrrolidin-1-yl)-8-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxalin-1-amine (10b) N a
Brown powder (1,4-dioxane); 85% yield; m.p. 319–320 ºC; IR (KBr): νmax = 3028(CHar), 2945, 2885 (CHalip), 1597 (C = N), 1338, 1197 (SO2) cm-1;1H NMR (δ, ppm) = 1.69 (8 H, s, (CH2)4-pyrrolidine), 3.27 (8 H, s, N(CH2)4-pyrrolidine), 6.92 (1 H, t, J = 7.2 Hz, ph-H), 7.28 (2 H, t, J = 8.8 Hz, ph-H), 7.39–7.41 (2 H, m, ph-H), 7.55 (1 H, d, J = 8.0 Hz, H8. quinox), 7.78 (1 H, dd, J = 7.6, 2.0 Hz, H7. quinox), 8.05 (1 H, d, J = 8.8 Hz, H5. quinox), 11.24(1 H, s, NH, D2O exchangeable).13C NMR (δ, ppm) = 25.36 ((CH2)2-pyrrolidine), 48.50 (N(CH2)2-pyrrolidine), 115.37, 117.13, 121.48, 124.44, 126.44, 128.90, 129.04, 129.87, 134.37, 136.79, 137.30, 141.67, 149.93, 156.14 (Ar.Cs), 164.34 (N = C-N); Anal. Calcd for C23H25N7O2S (463.56): C, 59.59; H, 5.44; N, 21.15; Found: C, 59.50; H, 5.26; N, 21.10.
Synthesis of 1-substituted-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxaline (11a, b)
To a solution of compound 7 (1 mmol) and either triethyl orthoformate (10 mmol) or triethyl orthoacetate (10 mmol) was stirred at 100 ºC for 1 h (monitored by TLC). After the reaction is cooled, the solid product was precipitated, collected, washed ethanol and then recrystallized from acetonitrile to produce the following pure compounds.
4-(Pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxaline (11a) a
Brown powder (CH3CN); 69% yield; m.p. 336–338 ºC; IR (KBr): νmax = 3028(CHar), 2964, 2878 (CHalip), 1597 (C = N), 1335, 1198 (SO2) cm-1 ;1H NMR (δ, ppm) = 1.68 (8 H, s, (CH2)4-pyrrolidine), 3.17–3.23 (8 H, m, N(CH2)4-pyrrolidine), 7.75 (1 H, d, J = 6.4 Hz, H8. quinox), 8.20 (1 H, d, J = 8.0 Hz, H7. quinox), 8.37 (1 H, s, H5. quinox), 8.48 (1 H, s, CH. Triazole).13C NMR (δ, ppm) = 25.25 ((CH2)2-pyrrolidine), 48.40 (N(CH2)2-pyrrolidine), 120.94, 125.10, 126.10, 129.76, 139.51, 140.90, 141.83, 146.21 (Ar.Cs), 168.34 (N = C-N); Anal. Calcd for C17H20N6O2S (372.45): C, 54.82; H, 5.41; N, 22.56; Found: C, 54.65; H, 5.54; N, 22.77.
1-Methyl-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxaline (11b) a
Light-brown powder (CH3CN); 65% yield; m.p. 324–326 ºC; IR (KBr): νmax = 3028(CHar), 2968, 2874 (CHalip), 1597 (C = N), 1337, 1198 (SO2) cm-1 ;1H NMR (δ, ppm) = 1.63 (8 H, s, (CH2)4-pyrrolidine), 1.92(3 H, s, CH3.triazole), 3.13–3.21 (8 H, m, N(CH2)4-pyrrolidine), 7.25–7.32 (1 H, m, H8. quinox), 7.64 (1 H, d, J = 8.4 Hz, H7. quinox), 7.72 (1 H, s, H5. quinox).13C NMR (δ, ppm) = 17.02 (CH3.triazole), 25.12 (4CH2.pyrrolidine), 48.49 (4 N-CH2.pyrrolidine), 122.78, 126.31, 127.41, 133.18, 140.71, 144.11, 147.81, 149.28 (Ar.Cs), 157.82 (N = C-N); Anal. Calcd for C18H22N6O2S (386.47): C, 55.94; H, 5.74; N, 21.75; Found: C, 55.76; H, 5.85; N, 21.57.
Synthesis of 4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxaline-1-thiol (12) a
To a solution of compound 7 (1 mmol) and carbon disulphide (1 mmol) in pyridine (15 mL) as solvent and catalyst, the solution mixture was refluxed (until the evolution of H2S finished). The reaction mixture was then poured onto cold water, and the solid product precipitated was collected by filtration, dried, and recrystallized from 1,4-dioxane.
Brown powder (1,4-dioxane); 77% yield; m.p. 310–312 ºC; IR (KBr): νmax = 3028(CHar), 2971, 2878 (CHalip), 1597 (C = N), 1333, 1153 (SO2) cm-1;13C NMR (δ, ppm) = 25.36 ((CH2)2-pyrrolidine), 48.50 (N(CH2)2-pyrrolidine), 122.67, 126.45, 129.04, 134.94, 137.31, 139.21, 147.60 (Ar.Cs), 164.64 (N = C-N), 167.45 (N = C-SH); Anal. Calcd for C17H20N6O2S2 (404.51): C, 50.48; H, 4.98; N, 20.78; Found: C, 50.60; H, 4.79; N, 20.81.
Synthesis of-substituted-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]quinoxalin-1-amine (13a, b) N
To a solution of compound 7 (1 mmol) and derivatives of isothiocyanate (1 mmol) namely, ally isothiocyanate and / or phenyl isothiocyanate in pyridine (15 mL) as solvent and catalyst, the solution mixture was refluxed (until the evolution of H2S finished). The reaction mixture was then poured onto cold water, and the solid product precipitated was collected by filtration, dried, and recrystallized from 1,4-dioxane.
-Allyl-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxalin-1-amine (13a) N a
Deep-Brown powder (1,4-dioxane); 73% yield; m.p. 268 − 266 ºC; IR (KBr): νmax = 3345 (NH), 3028 (CHar), 2966, 2874 (CHalip), 1597 (C = N), 1335, 1199 (SO2) cm-1;1H NMR (δ, ppm) = 1.69 (8 H, s, (CH2)4-pyrrolidine), 3.17 (8 H, s, N(CH2)4-pyrrolidine), 4.23 (2 H, s, CH2-CH-allyl), 5.28–5.37 (2 H, m,CH2 = CH-allyl), 5.88(1 H, s, CH2 = CH-allyl), 6.10 (1 H, s, NH, D2O exchangeable), 8.06–8.39 (2 H, m, H8+H7. quinox), 8.70 (1 H, s, H5. quinox).13C NMR (δ, ppm) = 25.85 (4CH2.pyrrolidine), 46.47 (CH2-CH-allyl), 49.02 (4 N-CH2.pyrrolidine), 101.03 (CH2 = CH-allyl), 117.45 (CH2 = CH-allyl), 122.62, 127.10, 128.04, 128.44, 132.12, 134.17, 135.92, 146.48 (Ar.Cs), 157.62 (N = C-N); Anal. Calcd for C20H25N7O2S (427.53): C, 56.19; H, 5.89; N, 22.93; Found: C, 56.30; H, 5.99; N, 22.75.
-Phenyl-4-(pyrrolidin-1-yl)-7-(pyrrolidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-]quinoxalin-1-amine (13b) N a
Brown powder (1,4-dioxane); 75% yield; m.p. 290–292 ºC; IR (KBr): νmax = 3346 (NH), 3056 (CHar), 2925, 2869 (CHalip), 1597 (C = N), 1318, 1198 (SO2) cm-1;1H NMR (δ, ppm) = 1.67 (8 H, s, (CH2)4-pyrrolidine), 3.20 (8 H, s, N(CH2)4-pyrrolidine), 6.93 (1 H, t, J = 7.6 Hz, ph-H), 7.11 (2 H, t, J = 7.2 Hz, ph-H), 7.31–7.36 (2 H, m, ph-H), 7.44 (1 H, d, J = 7.6 Hz, H8. quinox), 7.50 (1 H, d, J = 8.0 Hz, H7. quinox), 8.62 (1 H, s, H5. quinox), 9.92 (1 H, s, NH, D2O exchangeable).13C NMR (δ, ppm) = 25.25 ((CH2)2-pyrrolidine), 48.39 (N(CH2)2-pyrrolidine), 115.44, 117.25, 118.58, 124.01, 124.82, 126.24, 128.27, 128.88, 129.43, 130.39, 137.64, 139.96, 141.68, 149.33 (Ar.Cs), 156.14 (N = C-N); Anal. Calcd for C23H25N7O2S (463.56): C, 59.59; H, 5.44; N, 21.15; Found: C, 59.39; H, 5.67; N, 21.03.
Biological evaluation
In-vitro anti-diabetic activity
This assay was conducted by calculating the percentage (%) inhibition of α-amylase and α-glucosidase enzymes at a concentration of 100 µM of the tested quinoxaline sulfonamides, by the methodologies established by Wickramaratne and the Pistia-Brueggeman method, as previously described70,71. Acarbose was employed as the standard drug. The supplementary information (SI) file provides all procedural steps and detailed information. Additionally, the values of the median inhibitory concentrations for the most active derivative 10a and acarbose as positive control were calculated from the curve plotted between the percentage of inhibition and a series of concentrations (2, 4, 6.25, 12.5, and 25 µg/mL). (all procedural steps and details are provided in the supplementary information file).
In-vitro anti-Alzheimer activity
This assay was conducted by calculating the percent inhibition (%) of the acetylcholinesterase (AChE) enzyme at a concentration of 100 µg/mL of the tested quinoxaline derivatives, following Ellman’s method72. Consequently, the active human AChE enzyme hydrolyzes the colorimetric quinoxaline sulfonamides, resulting in the generation of yellow chromophores that are detectable at 412 nm through absorbance measurement. Donepezil was used as the standard drug (all procedural steps and details are provided in the supplementary information file).
Molecular docking simulation
The molecular docking simulation was performed for the most active derivatives triazolo[4,3-a]quinoxalin-1-amine derivative 10a inside the active site of α-amylase (PDB: 2QV4) and α-glucosidase (PDB: 3W37) as anti-diabetic targets. On the other hand, the most active 1-methyl-[1,2,4]triazolo[4,3-a]quinoxaline derivative 11b on the acetylcholinesterase based on the inhibitory percentage as described on biological evaluation results was docked inside the active site of acetylcholinesterase (ACHE) (PDB: 4EY7). The docking simulation was carried out using Molecular operating Environmental (MOE) 10.2009 73–75. All the crystallography protein structures were downloaded from protein data bank (https://www.rcsb.org/↗) as pdb file and containing acarbose or donepezil as co-crystallized ligand. The structure of the most active triazolo-quinoxaline 10a and 11b was built using chembiodraw 14 as described previously5,76. The active site of α-amylase (PDB: 2QV4) and α-glucosidase (PDB: 3W37) was generated as described previously and according to standard protocol9,10. Initially, the validation process for α-amylase (PDB: 2QV4) exhibited that the acarbose as co-crystallized ligand showed binding affinity S =- 16.33 kcal/mol with RMSD = 1.36 Å through one hydrogen bond backbone acceptor with Thr163, one hydrogen bond sidechain donor with His201, and four hydrogen bond sidechain acceptors with residues (2 x Asp300, His299, and Glu233). On the other hand, for the α-glucosidase (PDB: 3W37) the acarbose as positive control and co-crystallized ligand displayed binding affinity S =- 16.82 kcal/mol with RMSD = 2.357 Å in the validation process. The acarbose bounded to the pocket through seven hydrogen bond sidechain acceptors with amino acids (Asp232, Asp568, His625, and Asp357) and one hydrogen bond sidechain donor with Asp552. For the validation process of acetylcholinesterase (ACHE) (PDB: 4EY7)14 the donepezil as co-crystallized ligand exhibited binding affinity S = -11.027 kcal/mol with RMSD = 0.8064 Å through two types of interactions as arene-arene interactions with residues (Trp286 and Trp86) and two arene-cation interaction with residues (Tyr337 and Tyr341).
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Material 1