Molecular Modeling Study of Bis-sulfonamide Derivatives Synthesis Targeting Aromatase Enzyme as Anticancer

 

Mohamad M¹*, Djamila BH², Amir B³, Mustapha FC¹

¹Department of Pharmaceutical Chemistry and Drug Control,

Faculty of Pharmacy, University of Aleppo, Aleppo, Syria.

²Department of Pharmaceutical Chemistry and Drug Control Faculty of Pharmacy, Ebla University, Idlib, Syria.

³Department of Pharmaceutical Chemistry and Drug Control,

Faculty of Pharmacy, Philipps University, Marburg, Germany.

 

ABSTRACT:

Most studies indicate the important role of estrogen in the mechanism of occurrence and development of breast cancer. The importance of our research is the synthesis of bis-sulfonamide compounds that inhibit the aromatase enzyme, which is the main enzyme in the biosynthesis of estrogen. Molecular modeling of studied compounds was carried out by Molegro Virtual Docker (MVD) targeting aromatase enzyme and binding energy calculated to select the most encouraging compound. The highest binding energy among the studied compounds was -118.52 kcal/mol (compound A5) comparing with the aromatase substrate androstenedione -132.51 kcal/mol and the aromatase inhibitor letrozole -136.52 kcal/mol. Several of these compounds were synthesized in a simple way with good yields by reacting sulfonyl chloride derivatives with amino derivatives in an alkaline aqueous solution, or in a pyridine solution. The physicochemical characteristics and identification of synthesized compounds were determined by various analytical methods such as Mass spectrometry, Infrared spectroscopy and Nuclear Magnetic Resonance.

 

 

 

INTRODUCTION: 

Cancer is one of the most common diseases and cause of death in the world^1-3 and is the second leading cause of death after heart diseases^4-6. Cancer occurs when cells begin to grow randomly, form tumor metastases leading to death in case of non-treatment^7-11. Statistics indicate that there are more than 14 million cancer patient in the world and predict an increase in patient numbers in the coming years¹². Every year about 7 million people die from cancer¹³.

 

Breast cancer is the most common type of cancer after lung cancer¹⁴.

 

Estrogen plays a critical role in initiating the progression and further growth of a number of hormone-dependent benign and malignant abnormalities and the secretion of various growth factors in established cell lines such as MCF-7, T47D,^15-18 and ZR-75-1 human breast cancer lines^19-22. High levels of aromatase have been measured in affected women and this confirms the role of estrogen in the occurrence and development of these cancers^23-25. Estrogen biosynthesis is catalyzed by aromatase enzyme, which belongs to cytochrome family^26,27. The cytochrome P450arom is part of a burgeoning gene family, which at present includes more than 220 distinct individuals belonging to 36 gene families²⁸. The aromatase enzyme binds to the NADPH cytochrome P450 reductase that transfers electrons from the NADPH molecule to the aromatase enzyme for oxidation of the androgen substrate to estrogen²⁹. This reaction requires 3 mol oxygen and 3 mol NADPH. Evidence is mounting that all three oxygen molecules are exploited in the oxidation of the C19 angle methyl group to formic acid, which occurs along with A-ring aromatization to give the phenolic structure of estrogens. The first two oxygen molecules were conventionally used to oxidize the methyl group C19 and form the corresponding hydroxyl. The third oxidation reaction is carried out by ferric peroxide attack on carbon 19. After oxidative attack on the carbon-19 with the 1β hydrogen removed to give a 1,10 double bond, the protonation of the 3-oxo group by a nearby lysine or histidine, along with the provision of a leaving group for the 2β-hydrogen, as the carboxylic acid residue, will allow the formation of 3-ketone. Thus, the steps required to convert the A ring into a phenolic derivative are complete³⁰.

 

In a previous study by Ronnakorn Leechaisit and colleagues, sulfonyl chloride derivatives were reacted with meta-  or para-xylylenediamine and meta-phenylenediamine for obtaining bis-sulfonamide compounds that inhibit aromatase enzyme³¹. Several researchers in our laboratory have synthesized new sulfonamides that inhibit some enzymes such as carbonic anhydrase, tyrosine kinase³². In this research we followed up the study of sulfonamide compounds where we synthesized bis-sulfonamide compounds that inhibit the aromatase enzyme. Molecular docking indicated that both the oxygen and nitrogen atoms of such sulfonamide group can form a hydrogen bond with the aromatase enzyme and inhibit it.

 

MATERIAL AND METHODS:

All chemicals and solvents were obtained from a trustworthy source (Pro lab. For pharmaceutical industry). The enzyme code (PDB ID: 3EQM) has been downloaded from Protein Data Bank (PDB). The Molegro Virtual Docker (MVD) version 2022.5.5 was used to perform the docking of the compounds to be tested for their activity on the enzyme and to calculate the binding energy. The compounds were drawn and the lowest-energy conformer was selected using the Marvin Sketch version 21.2, and then applied to the Molegro program. Molinspiration software was used to investigate whether the synthetic compounds were effective orally and fulfilled Lipinski's rules. The following devices were used to identify the synthetic compounds: BÜCHI Melting Point B-540 apparatus (BÜCHI Labortechnik, Switzerland) to find out the melting point of the substance, Analytical thin layer chromatography (TLC) was performed with silica gel 60 F254 aluminum sheets (Macherey-Nagel Germany) to calculate the Retardation Factor (RF) of the material, ATR-FTIR Bruker spectrophotometer (Bruker, Billerica, Massachusetts), mass spectroscopy by using a mass spectrometer (Sciex, Framingham, USA), ¹H-NMR, ¹³C-NMR spectroscopy by using an NMR spectrometer (Joel, Tokyo, Japan), operating at 400 MHz for 1H and 13C.

 

Methods of synthesis:

Synthesis of benzenesulfonyl chloride:

An ice bath, 20ml (0.3mol) of chlorosulfonic acid was added to 9ml (0.1mol) benzene Becker. Then, the reaction was stirred at room temperature for 1 hour. the reaction mixture was poured slowly onto ice-filled beaker in order to form a suspension. The oily layer (benzenesulfonyl chloride) was extracted by chloroform, and diphenyl sulfone was formed as a byproduct.

 

Synthesis of p-methoxybenzenesulfonyl chloride:

An ice bath, 13.3ml (0.2mol) of chlorosulfonic acid was added to 10.87ml (0.1mol) anisole Becker. Then, the reaction was stirred in ice bath for 1hour, then the ice was added to the reaction to give mainly p-methoxybenzenesulfonyl chloride precipitate. The solid product was collected using vacuum filtration.

 

Synthesis of p-acetamidobenzenesulfonyl chloride:

An ice bath, 33.3ml (0.5mol) of chlorosulfonic acid was added to 13.5g(0.1mol) acetanilide Becker. Then, the reaction was stirred at 60C° for 2hour. p-acetamidobenzenesulfonyl chloride precipitate was created by pouring ice to the reaction mixture.

 

Synthesis of compound A1:

1.08g (0.01mol) phenylenediamine was dissolved in 10 ml of 10% sodium hydroxide solution, 3.5g(0.02mol) benzenesulfonyl chloride was added to the mixture with stirring at room temperature overnight until a precipitate forms. To remove diphenyl sulfone traces, the precipitate was washed by chloroform.

 

Synthesis of compound A2:

1.08g (0.01mol) phenylenediamine was dissolved in 20 ml pyridine, 4.12g (0.02mol) p-methoxybenzenesulfonyl chloride was added to the mixture with stirring at room temperature overnight. Reaction mixture was poured onto ice to produce a white precipitate.

 

Synthesis of compound B1:

0.67ml (0.01mol) ethylenediamine was mixed in 10ml 4% sodium bicarbonate solution, 3.5g (0.02mol) benzenesulfonyl chloride was added to the solution with stirring at 80C° under reflux for 1hour. The precipitate was washed by chloroform.

 

Synthesis of compound B2:

0.67ml (0.01mol) ethylenediamine was mixed in 10 ml 10% sodium hydroxide solution, 4.12g (0.02mol) p-methoxybenzenesulfonyl chloride was added to the solution with stirring at 80C° under reflux for 3 hours.

 

Synthesis of compound B3:

0.7ml (0.01mol) ethylenediamine was mixed in 10ml 10% sodium hydroxide solution, 4.67g (0.02mol) p-acetamidobenzenesulfonyl chloride was added to the solution with stirring at room temperature overnight. The precipitate was recrystallized by adding 30 ml DMSO with heating until complete dissolution, then 40 ml methanol and water was added to get the precipitate again.

 

Synthesis of compound C3:

1.7g (0.01mol) sulfanilamide was dissolved in 10ml 4% sodium bicarbonate solution, 2.3g (0.01mol) p-acetamidobenzenesulfonyl chloride was added to the mixture with stirring at room temperature overnight.

All compounds A1-2, B1-3 were washed by hydrochloric acid solution to remove mono-substituted product traces, and a di-substituted product was extracted by adding ethyl acetate solvent. All synthesized compounds were recrystallized by methanol and water except compound B3.

 

Aromatase structure:

Aromatase is a monomeric enzyme consisting of a heme prosthetic group (which is the reaction center in the conversion of androgens into estrogens) and a monopeptide chain of 503 amino acid residues. Aromatase is attached to the endoplasmic reticulum by a domain called the amino terminal transmembrane domain. Ribbon diagram of the overall crystal structure of human placental aromatase is shown in (Figure 1). The tertiary structure consists of 12 major α-helices (labeled A through L) and 10 β-strands (numbered 1 through 10) distributed into 1 major and 3 minor sheets, and follows the characteristic cytochrome P450 fold. The heme is sandwiched between the L helix including its N-terminal loop and the I helix. The bound androstenedione (ASD) molecule at the heme distal site, the active site of the enzyme, and shown within its unbiased electron density, make two hydrogen bond-forming contacts - the 3-keto and 17-keto oxygen’s with Asp309 side chain and Met374 backbone amide, respectively. A striking feature of the tertiary aromatase structure is that long loops interconnect well-defined secondary structure elements, in general agreement with other P450 structures³³.

 

Figure 1: Aromatase structure cocrystallized with Androstenedione 

Molecular Docking:

The Molecular Docking was performed in MVD. The following parameters were used for docking in the Aromatase shown in (table 1).

 

Table 1: The following parameters were used for docking in the Aromatase

Parameters	Value
Cavity volume	Cavity 1: 99.84. surface:216.32
Scoring function	Modoc Score {GRID}
Grid resolution)Å(	0.30
Binding side radius	15
Searching algorithm	Modoc optimizer
Number of runs	10
Mix iteration	1500
Max population size	50
Energy threshold	100
Simplex evaluation max steps	300
Neighbor distance factor	1
Max number of poses returned	5

 

Validation docking method:

Molecular modeling was carried out to find out the possibility of inhibition of the aromatase enzyme by compounds A1-C7 (Figure 3) and to calculate the binding energy. Before conducting molecular modeling, an appropriate protocol must be selected and validated. The validity of the used protocol was verified by re-docking the androstenedione substrate with the aromatase enzyme and calculating the root mean restocking standard deviation (rmsd).  The rmsd value for the used protocol was good 0.4738A°. After verifying the validity of the protocol used, molecular modeling was carried out by this protocol for compounds A1-7/B1-7/C1-7 where they were all compound in the same active pocket as shown in the (Figure 2).

 

(a) Asd binding with aromatase binding pocket

(b) All compounds binding with aromatase binding pocket

Figure 2. Binding of all designed compunds andsubstrate Asd with the same binding pocket

 

RESULTS AND DISCUSSIONS:

Chemistry:

Benzenesulfonyl chloride derivatives have instability characteristics due to their liquid forms or low melting solids. That's why, purification methods such as recrystallization and distillation can decompose compound even under low pressure. The chlorosulfonation of benzene derivatives was carried out using chorosulfonic acid (more than one molar equivalent), where the first equivalent reacts with the aromatic compound to produce the corresponding sulfonic acid, and then the resulting sulfonic acid reacts with the excess of chlorosulfonic acid to give the corresponding sulfonyl chloride derivatives. The optimum conditions for chlorosulfonation vary widely and depend on the nature of the compound. The presence of an electron-donating group on the ring reduces the number of equivalents of chlorosulfonic acid and the reaction takes place at a low temperature. As for the presence of a group electron-withdrawing group, it makes the ring inactive and, therefore, we need larger equivalents of acid with heating. Theoretically, it takes two equivalents of chlorosulfonic acid to synthesize p-acetamidobenzenesulfonyl chloride. However, due to the viscous nature of the acid and its immiscibility with solid acetanilide, an excess of chlorosulfonic acid (five equivalents) was added. This can be dispensed with by adding an inert solvent such as dichloromethane.

 

After the synthesis of benzenesulfonyl chloride derivatives (1-7), these compounds were reacted with many families of diamine compounds like phenylenediamine (A), ethylenediamine (B), sulfanilamide (C) for obtaining bis-sulfonamide compounds that inhibit aromatase enzyme A1-7, B1-7, C1-7 respectively, as shown in (figure 3).

 

This reaction is carried out in an alkaline medium such as an aqueous solution of sodium hydroxide, an aqueous solution of sodium bicarbonate, pyridine, while the acidic medium leads to the protonation of the amino group and the weakening of its nucleophiles. Most kinetic studies indicated that these reactions follow an addition-elimination reaction mechanism (S_AN mechanism)³⁴.

 

The attack of the nucleophile was considered to be the rate-determining step as it forms a trigonal bipyramidal transition state. It contains both nucleophiles and halides, which makes the bond between sulfur and halogen weak, so that the elimination reaction occurs when the halogen group dissociates.

 

By calculating the reaction yield, it was found that the use of pyridine medium gives the highest yield because conducting the reaction in an aqueous solution leads to the hydrolysis of a derivative of benzenesulfonyl chloride and a loss of part of the yield. After filtration and obtaining compounds A1-7-B1-7, a solution of hydrochloric acid was added to purify them from the mono-substituted product which has good solubility in acid due to its basic property. As for the bis-substituted target compound, it remains a precipitate due to its weak basic properties.

 

Figure 3. Synthesis of bis-sulfonamide derivatives

 

Docking:

The compounds referred to in the (figure 3) were drawn using the Marvin Sketch program and saved in Mol format, and then applied to the Molegro Virtual Docker program.

 

By Looking at A1-7 group compounds and interpreting the results of the binding energies shown in the (table 2), we find that the highest binding energy was of the A5 compound with R = 4-OHC₆H₄ (Energy = -118.52) due to its ability to form a hydrogen bond with His480-Leu477 to mimic steroidal backbone of the natural substrate (ASD), it can also form hydrogen bonds with amino acids that did not appear with other compounds such as Gln218-Glu483 as shown in (figure 4).

 

Compound A3 with R = 4-NHCOCH₃C₆H₄ (Energy = -112.85) showed a bond energy close to that of A5 due to the formation of a hydrogen bond with Lu372. Also compound A1 with R = C₆H₅ (Energy = -110.65) gave a high binding energy to form a hydrogen bond with Leu 477. We conclude from the above that compounds which are able to form a bond with the amino acid Leu 477- leu 372 give high binding energy.

 

By looking at B1-7 group compounds, we find thatthese compounds gave lower binding energies compared to A1-7, and this indicates the importance of the presence of a benzene ring to create a π-interaction with the iron Fe³⁺ in the heme structure, some compounds, such as B2 with R = 4-OCH₃C₆H₄ (Energy = -108.14), B4 with R = 4- CH₃C₆H₄ (Energy = -104.38) and B5 with R = 4-OHC₆H₄ (Energy = -105.45) gave high bonding energies due to the formation of hydrogen bonds between SO₂ and the amino acids His480-Ser478-Asp309.

 

Conversely, C1-7 group compounds gave the highest binding energy due to the presence of a free group SO₂NH₂ capable of forming hydrogen bonds with His480-Ser478-Asp309. All compounds' interactions with amino acids are indicated in the (table 3).

 

Figure 4: Binding compound A5 with the aromatase amino acids

 

Table 2: Binding energies (Kcal/mol) of compounds designed compared to letrozole (-136.52 Kcal/mol) and Androstenedione (-132.51 Kcal/mol). The energy of both the enzyme and the compound before binding is higher than their energy after binding, so the negative sign expresses the energy difference before and after binding.

compound	Binding energies (Kcal/mol)	compound	Binding energies (Kcal/mol)	compound	Binding energies (Kcal/mol)
A1	-110.65	B1	-92.29	C1	-108.81
A2	-74.40	B2	-108.14	C2	-111.60
A3	-112.85	B3	-61.55	C3	-108.02
A4	-99.22	B4	-104.38	C4	-109.13
A5	-118.52	B5	-105.45	C5	-115.27
A6	-62.90	B6	-99.78	C6	-106.56
A7	-71.42	B7	-70.09	C7	-118.14

 

Table 3: The interaction of the compounds designed with amino acids of aromatase enzyme.

Ligand	Residue	Hydrogen bond	Distance (A°)	Energy
A1	Thr310 Leu477	OH….SO (Ligand) CO….NH (Ligand)	2.15 2.56	+1.27 -2.17
A2	Arg115 Met374 Ser478	NH….SO (Ligand) NH….SO (Ligand) OH….OCH3 (Ligand)	2.67 3.09 3.45	-2.50 -1.85 -0.73
A3	Leu372 Ser478	CO….NH (Ligand) OH….SO (Ligand)	2.95 1.91	-1.51 +3.39
A4	Ser478	OH….NH (Ligand)	2.69	-0.97
A5	Gln218 Leu477 Ser478 His480 Glu483	NH….OH (Ligand) CO…..OH (Ligand) OH…..SO (Ligand) NH…..SO (Ligand) CO…..OH (Ligand)	3.42 2.79 1.78 3.12 2.98	-0.88 -2.50 +4.52 -0.51 -2.50
A6	Arg115 Met374	NH….SO (Ligand) NH….SO (Ligand)	2.53 3.16	-1.94 -0.83
A7	Val370 Met374 Ser478	CO…..OH (Ligand) NH….SO (Ligand) OH….OH (Ligand)	3.14 2.84 2.14	-1.62 -2.36 +1.39
B1	Ser478 His480	OH….SO (Ligand) NH….SO (Ligand)	2.27 2.61	+0.27 -0.87
B2	Asp309 Ser478 His480	CO….NH (Ligand) OH….SO (Ligand) NH….SO (Ligand)	3.12 2.52 2.50	-1.64 -1.82 -0.35
B3	Arg115 Glu302 Thr310 Leu477	NH….SO (Ligand) CO…..NH (Ligand) OH….SO (Ligand) CO….NH (Ligand)	3.10 2.69 3.28 2.76	-2.50 -2.41 -1.61 -2.50
B4	Asp309 Ser478 His480	CO….NH (Ligand) OH….SO (Ligand) NH….SO (Ligand)	3.03 2.48 2.50	-1.71 -1.49 -0.34
B5	Arg115 Leu372 Ser478 His480	NH….SO (Ligand) CO….OH (Ligand) OH….OH (Ligand) NH….OH (Ligand)	2.99 2.84 2.98 3.28	-0.24 -2.50 -2.50 -0.20
B6	Asp309 Ser478 His480	CO….NH (Ligand) OH….SO (Ligand) NH….SO (Ligand)	3.02 2.50 2.53	-1.74 -1.70 -0.39
B7	Arg115 Asp309 Asp309 Thr310 Ser478 His480	NH….OH (Ligand) CO….NH (Ligand) CO….OH (Ligand) OH….CO (Ligand) OH….SO (Ligand) NH….SO (Ligand)	2.99 3.13 3.30 2.60 2.58 2.16	-2.50 -1.68 -1.50 -2.50 -2.30 -0.64
C1	Asp309 Ser478 His480	CO….NH (Ligand) OH….SO (Ligand) NH….SO (Ligand)	2.73 1.21 3.15	-2.10 9.49+ -1.14
C2	Asp309 Ser478 His480	CO….NH (Ligand) OH….SO (Ligand) NH….SO (Ligand)	2.76 1.51 3.06	-2.50 10.02+ -1.28
C3	Asp309 Thr310 Ser478 His480	CO….NH (Ligand) OH….SO (Ligand) OH….SO (Ligand) NH….SO (Ligand)	3.52 2.91 1.54 3.08	-0.27 -2.50 +6.62 -0.87
C4	Arg115 Thr310 Met 374	NH….SO (Ligand) OH….SO (Ligand) NH….SO (Ligand)	2.43 3.39 3.17	-1.06 -1.05 -2.01
C5	Asp309 Leu372 Met374 Ser478 His480	CO….NH (Ligand) CO….OH (Ligand) NH….OH (Ligand) OH….SO (Ligand) NH….SO (Ligand)	2.71 3.14 3.22 1.50 2.99	-2.28 -2.30 -1.20 +6.93 -0.87
C6	Asp309 Ser478 His480	CO….NH (Ligand) OH….SO (Ligand) NH….SO (Ligand)	2.66 1.30 3.13	-2.35 +8.70 -1.07
C7	Asp309 Met374 Ser478 His480	CO….NH (Ligand) NH….OH (Ligand) OH….SO (Ligand) NH….SO (Ligand)	2.49 2.70 1.76 2.95	-1.61 -2.50 +4.67 -0.61
Letrozole	Arg115 Met374 Ser478	NH….CN (Ligand) NH….CN (Ligand) OH…. CN (Ligand)	3.17 2.96 2.48	-0.10 -2.50 -1.50
Androstenedione	Arg115 Asp309 Met374	NH….OH (Ligand) CO….OH (Ligand) NH….OH (Ligand)	3.44 2.72 2.84	-0.20 -2.05 -2.50

 

Analytical data:

N‐(4‐benzenesulfonamidophenyl) benzene sulfonamide (A1):

White powder, Yield: 60%, melting point 255-257℃, TLC: mobile phase was used acetate ethyl: petrolatum ether: 7.5:2.5, which the Rf for compound A1 is 0.68. IR spectrum (νmax, cm^-1): 3217 (N–H),3060, 3003 (C–H aroma), 1319 (S=O). ¹H-NMR spectrum (DMSO-d6, δ, ppm): 6.87 (d, 4H, Ar–H), 7.45 (t, 4H, Ar–H), 7.56 (t, 2H, Ar–H), 7.60 (d, 4H, Ar–H), 10.10 (s, 2H, 2NH). ¹³C-NMR (DMSO-d6, δ, ppm): 122.21, 127.12, 129.65, 133.36, 134.52, 139.85. Mass spectrum (m/z, ESI): showed molecular ion [M + H]⁺ peak at 389.06 corresponding to the molecular formula of C₁₈H₁₆N₂O₄S₂, and [M + Na]⁺peak at 411.09, and its fragments are 248.07, 214.3, 141.2.

 

4-methoxy-N-[4-(4-methoxybenzenesulfonamido) phenyl] benzene-1-sulfonamide (A2):

White powder, Yield: 82%, melting point 245-246℃, TLC: mobile phase was used acetate ethyl: petrolatum ether: 7.5:2.5, which the Rf for compound A2 is 0.7. IR spectrum (νmax, cm-1): 3230 (N–H), 3050 (C–H aroma), 1321 (S=O), 1147 (C–O). 1H-NMR spectrum (DMSO-d6, δ, ppm): 3.73 (s, 6H, 2CH3), 6.86 (d, 4H, Ar–H), 6.95(d, 4H, Ar–H), 7.53 (d, 4H, Ar–H), 9.89 (s, 2H, 2NH). 13C-NMR (DMSO-d6, δ, ppm): 56.11, 114.75, 122, 129.39, 131.42, 134.55, 162.95. Mass spectrum (m/z, ESI): showed molecular ion [M + H]⁺peak at 449.08 corresponding to the molecular formula of C20H20N2O6S2, and [M + Na]⁺peak at 471.11, and its fragments are 278.03, 107.07.

 

N-(2-benzenesulfonamidoethyl) benzene sulfonamide (B1):

White powder, Yield: 74%, melting point 162-163℃, TLC: mobile phase was used acetate ethyl: petrolatum ether: 7.5:2.5, which the Rf for compound B1 is 0.64. IR spectrum (νmax, cm-1): 3317, 3270 (N– H),3065 (C–H aroma), 2943 (C–H), 1302 (S=O). 1H-NMR spectrum (DMSO-d6, δ, ppm):2.72 (q, 4H, CH2–CH2), 7.54 (t, 4H, Ar–H), 7.60 (t, 2H, Ar–H), 7.65 (t, 2H, 2NH), 7.69 (d, 4H, Ar–H). 13C-NMR (DMSO-d6, δ, ppm): 42.68, 126.29, 129.81, 133.06, 140.66. Mass spectrum (m/z, ESI): showed molecular ion [M + H] +peak at 341.12 corresponding to the molecular formula of C14H16N2O4S2, and [M + Na]+ peak at 363.08, and its fragments are 184.08, 154.05, 124.98.

 

4-methoxy-N-[2-(4-methoxybenzenesulfonamido) ethyl] benzene-1-sulfonamide (B2):

White powder, Yield: 70%, melting point 149-150℃, TLC: mobile phase was used acetate ethyl: petrolatum ether: methanol 6.8:2.3:0.9, which the Rf for compound B2 is 0.53. IR spectrum (νmax, cm^-1): 3282 (N–H),3086 (C–H aroma), 2921, 2830(C–H), 1319 (S=O), 1136 (C–O). ¹H-NMR spectrum (DMSO-d6, δ, ppm): 2.67 (q, 4H, CH₂–CH₂ ,3.8 (s, 6H, 2CH₃), 7.03(d, 4H, Ar–H), 7.60 (d, 4H, Ar–H), 7.65 (t, 2H, 2NH). ¹³C-NMR (DMSO-d6, δ, ppm): 42.66, 56.15, 115.03, 129.29, 132.36, 162.69. Mass spectrum (m/z, ESI): showed molecular ion [M + H]⁺peak at 401.11 corresponding to the molecular formula of C₁₆H₂₀N₂O₆S₂, and [M + Na]⁺ peak at 423.07, and its fragments are 213.11, 184.06,155.03.

 

N-(4-{[2-(4-acetamidobenzenesulfonamido) ethyl] sulfamoyl} phenyl) acetamide (B3):

White powder, Yield: 65%, melting point 315-317℃, TLC: mobile phase was used acetate ethyl: petrolatum ether: methanol 6.8:2.3:0.9, which the Rf for compound B3 is 0.48. IR spectrum (νmax, cm^-1): 3330, 3190 (N–H),3051, 3000 (C–H aroma), 2873 (C–H), 1676 (C=O), 1319 (S=O). ¹H-NMR spectrum (DMSO-d6, δ, ppm): 2.06 (s, 6H, 2CH₃), 2.68 (q, 4H, CH₂-CH₂), 7.47 (t, 2H, 2NH), 7.62 (d, 4H, Ar–H), 7.69 (d, 4H, Ar–H), 10.24 (s, 2H, 2NH).). ¹³C-NMR (DMSO-d6, δ, ppm): 24.65, 42.27, 119.19, 128.12, 134.35, 143.37, 169.45. Mass spectrum (m/z, ESI): showed molecular ion [M + H]⁺ peak at 455.12 corresponding to the molecular formula of C₁₈H₂₂ N₄O₆S₂, and [M + Na]⁺ peak at 477.18, and its fragments are 413.30, 258.18, 214.10.

 

N‐{4‐[(4‐sulfamoylphenyl) sulfamoyl] phenyl} acetamide (C3):

White powder, Yield: 58%, melting point 260-262℃, TLC: mobile phase was used acetate ethyl: petrolatum ether: 7.5:2.5, which the Rf for compound C3 is 0.29. IR spectrum (νmax, cm^-1): 3326, 3260 (N–H),3195, 3117, 3034 (C–H aroma), 2925, 2860 (C–H), 1681 (C=O), 1314 (S=O). ¹H-NMR spectrum (DMSO-d6, δ, ppm): 2.06 (s, 3H, CH₃), 7.14 (s, 2H, SO₂NH₂), 7.19(d, 2H, Ar–H), 7.63 (d, 2H, Ar–H), 7.67 (d, 2H, Ar–H), 7.71 (d, 2H, Ar–H), 10.25 (s, 1H, NH), 10.65 (s, 1H, NH). ¹³C-NMR (DMSO-d6, δ, ppm): 24.63, 118.93, 119.24, 127.64, 128.5, 133.12, 139.31, 141.38, 143.91, 169.58. Mass spectrum (m/z, ESI): showed molecular ion [M + H]⁺ peak at 370.04 corresponding to the molecular formula of C₁₄H₁₅N₃O₅S₂, and [M + Na]⁺ peak at 392.07, and its fragments are 353.09, 213.70, 198.40.

 

CONCLUSION:

Six bis-sulfonamide compounds were synthesized in a simple way by reacting benzenesulfonyl chloride derivatives with amine compounds in different alkaline media. The efficacy of the compounds was confirmed by molecular modeling. Compound A5 had the best binding energy with the enzyme. Molecular docking suggests that the hydroxyl group may play a crucial role in the hydrophobic interaction with Leu477 of the aromatase to mimic steroidal backbone of the natural substrate (ASD). The C1-7 compounds gave the high binding energies due to their formation of bonds with three amino acids His480-Ser478-Asp309. In summary, this study demonstrated the integrative approach towards discovering a novel class of aromatase inhibitors where compounds (A5 and C1-7) were highlighted as promising compounds for further development.

 

ACKNOWLEDGEMENTS:

We thank Dr. A. Balash, Philips University, Marburg, Germany for help us in the NMR spectra inThis research and their interpretation.

 

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Accepted on 18.07.2023          © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):43-50.