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DOI: 10.1055/s-0043-1775462
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Small Molecules in Medicinal Chemistry

Design, Synthesis, and Molecular Docking Studies of Sulfonyl-Substituted Chromene Derivatives as Anticancer Agents

Aman Rai
a   Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga, Punjab-142001, India
,
Arti Soni
b   Department of Pharmacy, Panipat Institute of Engineering and Technology, Samalkha, Panipat, Haryana, India
,
Rohit Bhatia  ∗
c   Chitkara College of Pharmacy, Chitkara University, Rajpura-140401, Punjab, India
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Abstract

This study explores the role of estrogen in breast cancer development through ERα and ERβ receptors and highlights the significance of aromatase in estrogen biosynthesis. Chromene-based compounds, known for their anticancer properties, were synthesized with sulfonyl substitutions to enhance their efficacy. The MTT assay on MCF-7, MDA-MB-231, and HCT-116 cancer cell lines showed that (Z)-N-(3-cyano-2H-chromen-2-ylidene)benzenesulfonamide (AN1) and (Z)-N-(6-bromo-3-cyano-2H-chromen-2-ylidene)methanesulfonamide (AN13) had strong cytotoxic activity. Aromatase inhibitory assay shows that compound AN1 and AN13 show good inhibitory activity with IC50 values 0.20 and 0.24 μM. Docking studies revealed that these compounds fit well at the active site of the aromatase enzyme, with AN1, AN2, AN3, AN7, AN8, and AN13 showing docking scores of –9.1, –9.0, –8.8, –8.0, –8.6, and –7.8, respectively, compared to Exemestane with –9.3. ADME predictions indicated good drug-like properties, suggesting that these chromene derivatives could be effective anticancer agents.


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Key words

chromene - anticancer - aromatase - breast cancer - cell lines - exemestane

Cancer, characterized by the uncontrolled division of abnormal cells, is the second leading cause of death globally, causing around 10 million deaths annually.[1] [2] [3] [4] [5] The prevalence of cancer is rising due to lifestyle choices, with the most common types being breast, lung, colon, rectum, prostate, skin, and stomach cancers (Figure [1]).[6,7] In 2020, there were 19.3 million new cancer cases worldwide, with India reporting 1,461,427 new cases.[8] Projections indicate that by 2030, new cancer cases could reach 22.2 million globally.[9] In India, one in nine people is expected to develop cancer in their lifetime, with lung cancer being most common in males and breast cancer in females.[10] For children aged 0–14, lymphoid leukemia is the most prevalent.[11] By 2040, cancer cases in India are projected to rise to 2.08 million,[12] a 57.5% increase from 2020.

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Figure 1 Percentage of new cases of the most common cancers in 2022, both sexes, of all ages

Hormones play a critical role in breast cancer development, with estrogen impacting growth through two receptors, ERα and Erβ.[13] These receptors belong to the nuclear hormone receptor family and have several key domains: DNA Binding Domain (DBD), Activator Function 1 (AF1), Activator Function 2 (AF2), and Ligand Binding Domain (LBD).[14] ERα and ERβ share high homology in their DBDs and LBDs and function in both genomic and non-genomic pathways.[15] Activated by estrogen, they move to the nucleus to regulate gene expression by interacting with DNA or transcription factors.[16]

Aromatase, an enzyme in the cytochrome P450 family, is crucial for estrogen biosynthesis,[17] as shown in Figure [2]. Found in various tissues, it converts androgens to estrogen, driving the growth of hormone receptor-positive breast cancer. The activity of this enzyme is essential for sexual development and the progression of certain breast cancers.[18] The breast cancer subtype is shown in Figure [3].

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Figure 2 Aromatase pathway
Zoom Image
Figure 3 Breast cancer subtypes

Mahapatra et al. reported the design, synthesis, and in-silico study of chromene-sulfonamide congeners as potent anticancer and antimicrobial agents. From all the synthesized series, one compound showed the best anticancer activity IC50 7.78 μM on the MDA-MB-231 cell line.[19] All the synthesized compounds follow Lipinski’s rule of five. The rationale behind the designed molecule is shown in Figure [4].

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Figure 4 Rationale behind the proposed compounds

SAR study revealed that substitution with substituted sulfonyl is essential for anticancer activity. Sulfonyl group increase the water solubility and improving the absorption and bioavailability of compounds. It helps in stabilizing the molecular structure and also enhance the target specificity.

Chemistry

Synthesis of 2-Imino-2H-chromene-3-carbonitrile Derivatives: Substituted salicylaldehyde (1 mmol) and malononitrile (1 mmol) in EtOH (15 mL) and water (15 mL) were taken into a well-dried round-bottom flask (RBF). The reaction mixture was continuously stirred at r.t. for 48–72 h. The reaction progress was monitored by TLC (n-hexane/EtOAc 2:1). After completion of the reaction, the mixture was poured into ice-cold water to precipitate out the solid product. Then, the solid precipitate was filtered washed with water (× 2) and dried. Purification of crude product by recrystallization (EtOH) gave pure product.

Synthesis of Sulfonyl-Containing Chromene Derivatives AN1–AN16: 2-Imino-2H-chromene-3-carbonitrile derivatives (1 eq.) and substituted sulfonamide (1 eq.) in the presence of K2CO3 as a base and EtOH (20 mL) as solvent were taken into a RBF. The reaction mixture was refluxed for 20–24 h at 40–50 °C. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was poured into ice-cold water. The precipitated crude product was filtered, washed with water, and dried. Recrystallization of the crude product (EtOH) provided AN1–AN16 in good to moderate yields (Figure [5]).

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Figure 5 Proposed synthetic scheme

The physicochemical characterization and IR data of all the compounds are given in Table [1] and the Supporting Information.


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Biological Evaluation of Synthesized Compounds

In Vitro Cytotoxicity Activity: Anticancer activity of the synthesized derivatives was performed on three cancer cell lines MCF-7, MDA-MB-231, and HCT 116 cell line. MTT assay was carried out to evaluate the activity. In this assay, all the cells were seeded into 96 well plates having a concentration of 1 × 104 cells/well. The cells were incubated for 24 h. then, different concentrations (0–10 μg/mL) of the synthesized compounds were made and added to the well plates three times; the wells were incubated for 48 h at 37 °C and 5% CO2. After incubation, cells were washed and MTT dye (20 μL) was added to each plate and allowed to stand for about 4 h. After this, the supernatant layer of the wells discarded and formazan crystals were formed. DMSO was added and incubated for 30–60 min at 37 °C to dissolve the formazan crystals. Finally, the absorbance was measured by an ELISA reader at 570 nM by experimenting three times (Table [2]). 5-Fluorouracil and Exemestane were two standard drugs that were used as standard reference compounds in cytotoxicity assays to determine the IC50 values of novel anticancer agents.

Table 1 Physiochemical Characterization and IR Data for AN1–AN16

Compound

Molecular Formula
mp
yield

Molecular Structure

FT-IR (cm–1)

AN1

C16H10N2O3S
182–184 °C
76%

1012 (C–O), 1037 (S=O), 1242 (CN stretch), 1487 (C=C), 1622 (C=N stretch), 2207 (C≡N), 3064 (Ar–CH stretch)

AN2

C11H8N2O3S
189–191 °C
78%

1012 (C–O), 1037 (S=O), 1487 (C=C), 1622 (C=N stretch), 2207 (C≡N), 3064 (CH3 stretch)

AN3

C17H12N2O3S
190–192 °C
80%

1288 (C–O stretch), 1050 (S=O), 1604 (C=C stretch), 1642 (C=N stretch), 2229 (C≡N stretch), 3049 (Ar–CH stretch)

AN4

C20H18N2O3S
190–192 °C
50%

1186 (C–O stretch), 1042 (S=O), 1607 (C=C stretch), 1700 (C=N stretch), 2205 (C≡N stretch), 3063 (Ar–CH stretch), 2963 (CH3 stretch)

AN5

C25H28N2O3S
183–185 °C
50%

1012 (C–O stretch), 1038 (S=O), 1458 (C=C stretch), 1625 (C=N stretch), 2206 (C≡N stretch), 3195 (Ar–CH stretch), 2869 (CH3 stretch)

AN6

C18H13N3O4S
193–195 °C
48%

1280 (C–O stretch), 1049 (S=O), 1467 (C=C stretch), 1644 (C=N stretch), 2010 (C≡N stretch), 3069 (Ar–CH stretch), 3350 (CH3 stretch), 1741 (NH stretch)

AN7

C17H11ClN2O3S
191–193 °C
86%

688 (CCl), 1011 (C–O stretch), 1047 (S=O), 1628 (C=C stretch), 1659 (C=N stretch), 2227 (C≡N stretch), 2984 (Ar–CH stretch), 3346 (CH3 stretch)

AN8

C11H7ClN2O3S
184–186 °C
50%

836 (CCl), 1276 (C–O stretch), 1046 (S=O), 1474 (C=C stretch), 1657 (C=N stretch), 2212 (C≡N stretch), 3122 (Ar–CH stretch), 3211 (CH3 stretch)

AN9

C16H9ClN2O3S
181–183 °C
25%

753 (CCl), 1020 (C–O stretch), 1051 (S=O), 1477 (C=C stretch), 1656 (C=N stretch), 2210 (C≡N stretch), 2980 (Ar–CH stretch)

AN10

C20H17ClN2O3S
192–194 °C
25%

687 (CCl), 1011 (C–O stretch), 1036 (S=O), 1475 (C=C stretch), 1656 (C=N stretch), 2218 (C≡N stretch), 3058 (Ar–CH stretch), 2978 (CH3 stretch)

AN11

C19H15ClN3O4S
185–187 °C
76%

567 (CCl), 1007 (C–O stretch), 1053 (S=O), 1503 (C=C stretch), 1633 (C=N stretch), 1708 (C=O stretch), 2242 (C≡N stretch), 3055 (Ar–CH stretch), 3055 (CH3 stretch), 3466 (NH stretch)

AN12

C17H11BrN2O3S
180–182 °C
25%

580 (CCl), 1085 (C–O stretch), 1038 (S=O), 1477 (C=C stretch), 1646 (C=N stretch), 2223 (C≡N stretch), 2964 (Ar–CH stretch), 3066 (CH3 stretch)

AN13

C11H7BrN2O3S
188–190 °C
76%

661 (CBr), 1180 (C–O stretch), 1030 (S=O), 1475 (C=C stretch), 1592 (C=N stretch), 2260 (C≡N stretch), 2979 (Ar–CH stretch), 3075 (CH3 stretch)

AN14

C16H9BrN2O3S
182–184 °C
76%

586 (CBr), 1190 (C–O stretch), 1088 (S=O), 1476 (C=C stretch), 1650 (C=N stretch), 2247 (C≡N stretch), 2977 (Ar–CH stretch)

AN15

C20H17BrN2O3S
185–187 °C
50%

659 (CBr), 1007 (C–O stretch), 1040 (S=O), 1474 (C=C stretch), 1635 (C=N stretch), 2221 (C≡N stretch), 2962 (Ar–CH stretch), 3060 (CH3 stretch).

AN16

C18H12BrN3O4S
188–190 °C
66%

655 (CBr), 1298 (C–O stretch), 1024 (S=O), 1601 (C=C stretch), 1642 (C=N stretch), 1708 (C=O stretch), 2219 (C≡N stretch), 2993 (Ar–CH stretch), 3131 (CH3 stretch), 3437 cm–1 (NH stretch)

Aromatase Inhibitory Assay: Aromatase inhibitory assay was performed on two compounds AN1 and AN13. Here, Exemestane was used as reference drug. Results showed that two compounds AN1 and AN13 possess highest inhibitory potential having IC50 value of 0.20 μM and 0.24 μM, respectively. The inhibitory potential of reference drug Exemestane was 0.09 ± 0.02 μM (Table [3]).

Table 2 In Vitro Cytotoxicity Study Against MCF-7, MDA-MB-231, and HCT 116 Cell Lines

Compound

Cytotoxicity (IC50 ± SD) μM

MCF-7

MDA-MB-231

HCT 116

AN1

3.23 ± 0.77

3.82 ± 0.31

4.48 ± 0.22

AN2

4.23 ± 0.31

6.01 ± 0.34

>35

AN3

7.11 ± 0.81

6.58 ± 0.21

>35

AN7

4.86 ± 0.23

4.42 ± 0.19

5.51 ± 0.92

AN8

4.33 ± 0.27

4.76 ± 0.58

5.11 ± 0.10

AN12

>35

6.08 ± 0.04

>35

AN13

4.04 ± 0.51

4.21 ± 0.17

4.26 ± 0.03

Exemestame

3.40 ± 0.21

3.23 ± 0.12

5-Fluorouracil

2.12± 0.5

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In Silico Studies

Molecular Docking Studies: A series of sulfonyl containing chromene derivatives were designed. The designed compounds were sketched in Chemdraw professional. All designed compounds were investigated by AutoDock 4.2.

Protein Preparation: The structure of aromatase enzyme, (PDB ID: 3S7S) was downloaded from the protein data bank using pdb.org as the format. The selected protein was subjected to protein preparation in Autodock 4.2 software. The protein preparation includes deleting ligand (EXM); the addition of hydrogen atoms. Additionally; water molecules were deleted and polar hydrogen was added. After that Kollman charge was added and the file was saved in .pdbqt format.[20]

Table 3 Aromatase Inhibitory Activity of Potent Compounds and Reference Drug

Compound

Aromatase inhibitory assay (IC50) μM

AN1

0.20

AN13

0.24

Exemestane

0.09

Procurement of 3D Structure: The 3D X-ray structures of aromatase enzyme (PDB ID: 3S7S) was obtained from the Protein Data Bank (www.rscb.org) with a resolution of 3.21 Å as shown in Figure [6].

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Figure 6 3D X-ray structure of aromatase enzyme with EXM

Docking Protocol: AutoDock 4.2 program was used for docking. Firstly, the ligand PDB and protein PDB file were converted into ligand PDBQT and protein PDBQT formate. After that dock in command prompt by using commands. Log file and output. PDBQT file was generated. These two files were opened in Pymol and save the file. Then open the saved file in Discovery software to see the 2D-interaction.[21]

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Figure 7 Contains 2D pose of Exemestane and compound AN1 with protein (PDB ID: 3S7S)

The structure of synthesized compounds was imported into the 3D Chem software and converted into SDF format and then SDF format was imported in Pymol to convert it into PDB format. Finally, the PDB format was imported into AutoDock 4.2 for converting the PDBQT format. The ligands were docked into the cavity of the target protein. The Dock score values were calculated, and interaction poses were saved for study. Figure [7] displays the binding interactions of the standard drug exemestane with HEM 600, THR 310, and SER 478 amino acids. Compound AN1 shows the interaction with ALA 306, ILE 133, PHE 221, MET 374, HEM 600, PHE 134, VAL 373, VAL 370. Figure [8] shows the 2D pose of compound AN2 interaction with protein (3S7S).

Zoom Image
Figure 8 Contains 2D pose of compound AN2 with protein (PDB ID: 3S7S)

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Drug Likeness, ADME, and Toxicity Predictions

Drug likeness is the complex balance between various molecular and structural features such as stability, oral availability, good pharmacokinetic properties, lack of toxicity and minimum addictive potential. The ADMET prediction for anticancer agents is an essential tool to determine the safety and target reaching ability of designed compounds. Many of these properties depend on the inherent biological and physicochemical parameters of the molecule; however, the complex structure of the whole drug molecule makes correlating attempts difficult.

In silico drug likeness prediction of various designed and synthesized derivatives was carried out by using a Swiss ADME predictor tool. The results obtained from prediction data (Table [4]) revealed that the synthesized compounds follow the Lipinski’s rule of drug-like molecules. All of the synthesized compounds showed LogP value less than 5. The molecular weight is <500 and presence of HBD and HBA atoms is also favorable. The compounds showed high GI absorption only one compound showed low GI absorption. Further, all the compounds were good TPSA value. With no violation to Lipinski’s rule, it can be concluded that the synthesized compounds have optimum chemical skeleton that can be developed a potential drug molecule.

Table 4 Physiochemical Properties of Proposed Derivatives

Compound

MW

HBA

HBD

TPSA

iLogP

GI absorption

Lipinski violation

AN1

310.33

5

0

 91.81

 2.2

High

0

AN2

248.33

5

0

 91.81

 1.55

High

0

AN3

324.35

5

0

 91.81

 2.48

High

0

AN4

366.43

5

0

 91.81

 3.32

High

0

AN5

436.57

5

0

 91.81

 3.85

Low

0

AN6

367.38

6

1

120.91

 2.23

High

0

AN7

358.8

5

0

 91.81

 2.7

High

0

AN8

282.7

5

0

 91.81

 1.78

High

0

AN9

344.77

5

0

 91.81

 2.41

High

0

AN10

400.88

5

0

 91.81

 3.31

High

0

AN11

416.86

7

0

120.91

–2.27

High

0

AN12

403.25

5

0

 91.81

 2.82

High

0

AN13

327.15

5

0

 91.81

 1.9

High

0

AN14

389.22

5

0

 91.81

 2.5

High

0

AN15

445.33

5

0

 91.81

 3.34

High

0

AN16

446.27

6

1

120.91

 2.58

High

0

In conclusion, chromene-based molecules found naturally in substances, like alkaloids and flavonoids, are significant in developing anticancer therapies. Our research synthesized and evaluated chromene derivatives AN1–AN16 for anticancer activity, with AN1 and AN13 showing potent effects. Docking studies against the aromatase enzyme confirmed the efficacy of these compounds, following ADME predictions and Lipinski’s rule of five.

Sulfonyl-containing chromene derivatives, particularly AN1 and AN13, demonstrate potential as novel anticancer agents, meriting further investigation for effective cancer treatments.


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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgement

Authors greatly acknowledge Mr. Praveen Garg, Chairman, ISF College of pharmacy, Moga for providing best educational atmosphere, necessary facilities available for the research work and a platform to rise in the world of Science.

Supporting Information

    Supporting information for this article is available online at https://doi-org.accesdistant.sorbonne-universite.fr/10.1055/s-0043-1775462.
  • Supporting Information

Corresponding Author

Rohit Bhatia
Chitkara College of Pharmacy, Chitkara University
Rajpura-140401, Punjab
India   

Publication History

Received: 26 December 2024

Accepted after revision: 27 February 2025

Article published online:
04 April 2025

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Zoom Image
Figure 1 Percentage of new cases of the most common cancers in 2022, both sexes, of all ages
Zoom Image
Figure 2 Aromatase pathway
Zoom Image
Figure 3 Breast cancer subtypes
Zoom Image
Figure 4 Rationale behind the proposed compounds
Zoom Image
Figure 5 Proposed synthetic scheme
Zoom Image
Figure 6 3D X-ray structure of aromatase enzyme with EXM
Zoom Image
Figure 7 Contains 2D pose of Exemestane and compound AN1 with protein (PDB ID: 3S7S)
Zoom Image
Figure 8 Contains 2D pose of compound AN2 with protein (PDB ID: 3S7S)