INTRODUCTION:

Cancer has a major influence on societies all over the world. Cancer is one of the most remarkable causes of death¹. The number of new cancer cases is expected to rise annually, it is estimated that by 2040, the number of new cases will reach 29.5 million, and cancer-related deaths will reach 16.4 million². Many procedures and medicines are available to treat cancer, such as radiotherapy, which works by damaging the DNA of cancer cells^3,4, immunotherapy⁵, hormonal therapy, surgery, and chemotherapy, which has a large number of effective synthetic and semi-synthetic drugs to treat cancer⁶, and accomplished worthy successors in new drug discovery^7,8.

Particular studies of cancer initiation, promotion, and spreading help suggest new targets to inhibit cancer growth and prevent metastasis.

The most aggressive and invasive cancer cells lean on enormous glycolysis to suffice their massive request for energy and biosynthetic precursors. The rise in glycolytic activity is often prompted by hypoxia, which is caused by high cell density combined with inadequate vascularization. The rise in glycolysis leads to high concentrations of protons and lactate, which need to be removed from cancer cells to prevent acidosis⁹.

The adaptation of cancer cells to hypoxia conditions is one of the significant factors contributing to tumor growth. Hypoxia alerts a family of transcriptional regulators such as HIF-a\PIBK\AKT\mTOR, these transcriptional regulators facilitate the proliferation, survival, and migration of cancer cells¹⁰. HIF-a (Hypoxia-induced factor-a) is a master regulator that facilitates angiogenesis through the regulation of VEGF and SDF-1, metabolism by controlling of GLUT-1, and GLUT-3, and enhances invasion ability through the upregulation of CAIX, MMP-2, and MMP-9^10,11.

The role of carbonic anhydrase IX can be summarized by the elevation of the intracellular pH to the values that permit metabolic processes, and protect cancer cells from death, CA IX also contributes to the acidification of the pericellular milieu by decreasing PH levels, which gives an advantage to cancer cells over surrounding normal cells that cannot adapt. This promotes the growth of malignant cells and protects them from death^9,12. CA IX belongs to the carbonic anhydrase family (CAs), which mediate the reversible formation of bicarbonate and protons from carbon dioxide and water^13,14. There is a great diversity of carbonic anhydrase isozymes belonging to different families, which are often denominated using Greek letters¹⁴.α–Carbonic anhydrases (α –CAs) are widespread metalloenzymes found throughout the animal kingdom including humans^14,15. (α –CAs) class includes 16 isozymes that differ in their subcellular localization and susceptibility to different inhibitors¹⁵.

These Zn²⁺ enzymes have important physiological roles. most (α –CAs) mediate the reversible hydration of carbon dioxide to produce protons and bicarbonate. Many CA isozymes are engaged in major biological operations such as respiration and acid-base regulation, electrolyte secretion, and bone resorption¹⁵. Two of α – CA isozymes, CA IX and CA XII are particularly related to and overexpressed in many cancers, acting as both the survival factors and facilitators of tumor progression¹⁶.

CA IX is responsible for the reversible conversion ofwater and carbon dioxide to bicarbonate and protons. Then the neighboring bicarbonate transporters (NBC) transmit the bicarbonate ions to the cytoplasm. Inside the cell, intracellular protons resulting from different metabolic paths react with bicarbonate ions to produce CO₂ and H₂O as a result¹².

CA IX is involved in lactate exportation in parallel with protons flux by its proteoglycan domain in cooperation with monocarboxylate transporters (MCT).

CA IX is a cell-surface glycoprotein associated with tumors, induced by hypoxia and contributes to the regulation of pH in tumor cells, cell proliferation, cell adhesion, cell invasion, and it is implicated in therapeutic resistance¹².

CA IX is commonly expressed in many types of cancers, such as breast, gastric, bladder, and oral cavity cancers^12,17. Due to the role of CAIX in cancer progression and spreading, it has been considered a suitable target for cancer treatment. Moreover, the extracellular location of this isozyme is appropriate for designing selective inhibitors which can block CA IX activity without interacting with other cytosolic and mitochondrial CAs^18,19.

Sulfonamides derivatives are commonly used as antimicrobial, antifungal, and recently as anti-cancer agents.^20,21

muliple sulfonamide/sulfamate/sulfamide and coumarin derivatives are known as selective CA IX inhibitors. Ureido-substituted benzenesulfonamides have shown powerful anticancer effects against both primary tumors and metastases (in animal models)^22,23,24.

Ureido-substituted benzenesulfonamides are competitive inhibitors that target the active site of CA IX by binding to the cofactor which is Zn²⁺, this binding leads to the blockage of the bicarbonate obtaining process, and the suppression of the catalytic activity of CA IX^19,25,²⁶.

Ureido-substituted benzenesulfonamides are obtained by coupling sulfonamide with aryl-isocyanates²⁷. Traditional synthetic pathways prepare aryl-isocyanates in the presence of phosgene which is highly toxic²⁸, while other pathways prefer using triphosgene²⁹, despite its toxicity because it is solid at room temperature.³⁰

In our lab, at the University of Aleppo, department of pharmaceutical chemistry, several novel sulfonamide derivatives were prepared to target EGFR TK³¹ and carbonic anhydrase IX.

The aim of this current study is to discover new methods to prepare ureido-substituted benzenesulfonamides as CA IX blockers.

2. MATERIALS AND METHODS:

The synthesized compounds were identified by:

· A BUCHI Melting Point B-540 apparatus (BUCHI Labortechnik, Switzerland) was used for melting point determination.

· An 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 500 MHz for ¹H and ¹³C.

2.1. General procedures for the synthesis of (2-b), (SLC-0111), and (2-d) compounds:

· Synthesis procedures according to method A:

1. Sulfanilamide (10 mmol) was dissolved in a mixture of Conc. HCl (0.9 ml), and water (3 ml) in a 25-ml round flask.

2. Substituted-phenylurea (10 mmol) was added to the above mixture. Next, the reaction mixture was heated under reflux for 6 hours with continuous stirring.

3. After two hours, the resulting compound crystals begin to separate, in absence of the corresponding impurity.

4. The progress of the reaction is monitored by TLC using a mixture of 8:2 ethyl acetate:petroleum ether respectively as a mobile phase.

5. The resulting compound and a little of corresponding impurity crystals will be deposited at the bottom of the flask after the last four hours have passed. These crystals are collected before the mixture is cooled and washed with hot water.

6. finally, Recrystallization can be performed using aqueous ethanol to remove impurities from the compound crtystals. The resulting compound crystals are collected and dried with a yield up to 30%.

· Synthesis procedures according to method B:

1. Ureido benzenesulfonamide (10mmol) was dissolved in a mixture of Conc. HCl (0.9ml) and water (6 ml) in a 25-ml round flask.

2. Substituted-Aromatic amine (10mmol) was added to the above mixture, then, The reaction mixture was heated under reflux in an oil bath for 6 hours with continuous stirring.

3. After two hours, the desired crystals begin to separate, with absence of impurity.

4. The progress of the reaction is monitored by TLC using a mixture of 7:2:1 Ethyl acetate: petroleum ether:methanol respectively as a mobile phase.

5. The resulting compound is deposited at the bottom of the flask after the last four hours have passed, these crystals are collected before the mixture is cooled and washed with hot water.

6. The quantity of secondary resulting compound (2-f) is less than 1%, and it can be removed by recrystallization using aqueous ethanol. Then the compound crystals are collected and dried with a yield up to 50%.

· Synthesis procedures according to method C:

1. Ureido benzenesulfonamide (10 mmol) was dissolved in 15 ml of boiling butanol in a 25-ml round flask with continuous stirring.

2. Aromatic amine derivative (10 mmol) was added to the above solution.

3. The reaction mixture is exposed to heat under reflux in an oil bath overnight with continuous stirring.

4. The progress of the reaction is monitored by TLC using a mixture of 7:2:1 Ethyl acetate: petroleum ether:methanol respectively as a mobile phase.

5. When the reaction is completed, butanol can be removed in a rotary evaporator.

6. Recrystallization can be performed by aqueous ethanol. Compound crystals are collected and dried with a yield up to 80%.

A Comparison of the three methods:

The method used to prepare the synthesized compounds has a very important effect, not only on yields but also on impurities formation and the time needed for the reaction to end. Method C shows the highest isolated yields in comparison with A and B methods. The differences between tested methods are displayed in (Table 1).

Table 1: shows the variation between preparation methods.

	Method A	Method B	Method C
Time	6h	6h	Overnight
Impurity existence	Yes	Less than 1%	No
Isolated yield	30%	50%	80%

3. RESULTS AND DISCUSSION:

Synthesis mechanism:

Compounds (2-b) (SLC-0111) (2-d) were synthesized according to three new methods (A, B, C) in this study.

Method (A) allows obtaining ureido-substituted benzenesulfonamides through the reaction of phenylurea derivatives (3) and sulfanilamide (1) using water as a solvent in the presence of hydrochloric acid as shown in figure 1. The mechanism of reaction suggests that substituted-phenylurea (3) is converted to substituted-phenyl isocyanate (4), and ammonia (5) when exposed to heat. Thus, substituted-phenylurea could be used as a substituted-phenyl isocyanate source through the reaction³². Adding HCl to sulfanilamide results in the conversion of sulfonamide into sulfonamidium chloride (2). The formed ammonia may react with substituted-phenyl isocyanate to produce substituted-phenylurea once again. This process is blocked by the presence of sulfaniamidium chloride, Because the Pka of the conjugate acid of ammonia is 9.2 ³³, while the pka value of the conjugate acid of sulfanilamide is 2.2. This means that ammonia is more basic than sulfanilamide. So, sulfaniamidium chloride reacts with ammonia instead, yielding ammonium chloride (6) and sulfanilamide.

The carbon atom in the isocyanate group (-N=C=O) has a positive charge, making it vulnerable to being attacked by nitrogen atom of the aromatic amine of sulfanilamide. Meanwhile, the active hydrogen in the isocyanate group is added to the negatively charged nitrogen, (nucleophilic addition to C=N bond) to obtain ureido-substituted benzenesulfonamides³⁴.

Figure 1: Mechanism of synthesis according to method A.

Method A has two disadvantages. First, the isolated yield is limited to only 30%. Second, substituted-diphenylurea is formed as the result of the acidic hydrolysis of substituted-phenylurea, which in turn leads to the production of corresponding aromatic amines (7) ³⁵(aniline, p-fluoro aniline, p-hydroxy aniline), which are all stronger than sulfanilamide as nucleophiles, and react with phenyl isocyanate to produce the corresponding diphenylurea derivatives (8) as impurities figure 2.

Figure 2: Mechanism of impurity formation.

Method B is implemented to enhance the quantity of yield, while maintaining the same reaction conditions as method A. This is achieved by starting with ureido benzenesulfonamide (9) and substituted aromatic amines (7). This modification is necessary because the lone pair on the nitrogen atom of the substituted aromatic amine acts as a better nucleophile than sulfanilamide. So, at the beginning of the reaction, the aromatic amine molecules are charged positively (11) due to the presence of hydrochloric acid. Then, ureido benzenesulfonamide (9) is converted to 4-isocyanato benzenesulfonamide, (10) and ammonia during the reaction. Ammonium chloride is formed by the reaction between positively charged aromatic amine (11) and ammonia. Then, the corresponding resulting compound is obtained from the reaction between the aromatic amine derivatives and 4-isoctanato-benzenesulfonamide (figure 3).

igure 3: Synthetic route according to method B.

The hydrolysis of ureido benzenesulfonamide leads to sulfanilamide formation, that reacts with 4-isocyanato-benzenesulfonamide to produce 4,4-Bis-sulfonamide diphenylurea (2-f) as a secondary compound (figure 4).These procedures are useful for obtaining the desired compound in a yield up to 50%, with a low amount of the (2-f) compound due to the weakness of sulfanilamide as a nucleophile.

Figure 4: The mechanism of (2-f) formation.

Hydrolysis reactions occur due to the presence of water and hydrochloric acid, leading to the formation of corresponding impurities.

4-isocyanato benzenesulfonamide has several resonance forms, given that the carbon atom of the isocyanate group is positively charged, while the negative charge is delocalized on oxygen, nitrogen, aromatic ring, and sulfonamide group (figure 5). This means when water is used as a solvent, there is a partial hindrance to the formation of transition state due to the high electric constant value of water (ε = 78.4) at 25 C⁰³⁶. This phenomenon is known as water shield (figure 6).

Three advantages make butanol a much more suitable solvent than Water:

· The first is the boiling point of butanol which is (117.7) C⁰ at 760 mm Hg, which is significantly higher than that of water. This high boiling point allows for the conversion phenylurea to phenyl isocyanate.

· The second is that butanol has a low ε value (17.90 at 20 C⁰), which helps form the transition state and prepares the desired compound.

· The third is the low solubility of ammonia in butanol especially under reflux conditions.

The evaporation of ammonia makes the addition of hydrochloric acid not necessary. This helps to avoid hydrolysis reaction and prevent the formation of the corresponding impurity.

Method C states that using butanol helps in a selective preparation of ureido-substituted benzenesulfonamides, starting with ureido benzenesulfonamide and aromatic amine derivatives, in good yields up to 80% (figure 7, 8, 9).

Figure 5: Transition state hindrance due to water shield phenomenon.

Figure 6: Resonance forms of 4-isocyanato benzenesulfonamide

Figure 7: Ureido-substituted benzenesulfonamides preparation according to method C.

Figure 8: Transition state formation and obtaining ureido-substituted benzenesulfonamides.

Figure 9: Ureido-substituted benzenesulfonamides preparation mechanism following method C

Analytical data:

The chemical structures of synthesized compounds were verified, according to spectroscopic analyses.

(2-b): 4-(3-Phenyl-ureido)-benzenesulfonamide white powder, mp 230-231 C⁰.

TLC used mobile phase consists of acetate ethyl:petrolatum ether 8:2, where the Rf values are as follow: sulfanilamide 0.46, phenylurea 0.25, compound (2-b) 0.428, and diphenylurea is 0.85.

The used mobile phase consists of acetate ethyl:petrolatum ether:methanol 7:2:1, where the Rf values are as follow: ureido benzenesulfonamide 0.20, aniline: 0.92, and (2-b) compound 0.73.

IR spectrum: (νmax, cm−1): 3402.61, 3338.17 (N–H), 3237.95, 3136.09 (C–H aromatic), 1685.24 (C=O), 1541.85, 1523.24, 1486.56 (C=C), 1144.95 (S=O).

¹H-NMR spectrum (DMSO-d6,δ, ppm): 6.96 (t, 1H, Ar-H), 7.15 (s, 2H, SO2NH2), 7.25 (t, 2H, Ar-H), 7.42 (s, 2H, Ar-H), 7.57 (d, 2H, Ar-H), 7.70 (d, 2H, Ar-H) 8.73 (s, 1H, -CONH), 9.00 (s, 1H, -CONH).

¹³C-NMR (DMSO-d6, δ, ppm): 117.96 (CH in the aromatic ring), 118.99 (CH in the aromatic ring), 122.77 (CH in the aromatic ring), 127.35 (CH in the aromatic ring), 129.36 (CH in the aromatic ring), 137.35 (CH in the aromatic ring), 139.82 (CH in the aromatic ring), 143.40 (CH in the aromatic ring) 152.69 (C=O).

Mass spectrum (m/z, ESI): showed molecular ion [M + H]⁺ peak at 292.04 corresponding to the molecular formula of C13H13N3O3S, and its fragments.

(SLC-0111): 4-[3-(4-Fluoro-phenyl)-ureido]-benzene- sulfonamide pinkish white powder, mp 233-236 C⁰.

TLC used mobile phase consists of acetate ethyl:petrolatum ether 8:2, where the Rf values are as follow: sulfanilamide 0.48, compound (SLC-0111) 0.46, 4-fluoro phenylurea: 0.32, and 4,4\-Bis fluoro diphenylurea 0.89.

TLC mobile phase consists of acetate ethyl:petrolatum ether:methanol 7:2:1, where the Rf values are as follow: ureido benzenesulfonamide 0.21, 4-fluoro aniline: 0.90, (SLC-0111) compound 0.69.

IR spectrum: (νmax, cm−1): 3374.64, 3294.21 (N–H), 3237.95, 3136.09 (C–H aromatic), 1732.99 (C=O), 1533.16, 1516.17, 1505.18 (C=C), 1169.47 (S=O),

1230.84(C-F).

¹H-NMR spectrum (DMSO-d6,δ, ppm): 7.07 (t, 2H , Ar-H), 7.15 (s, 2H, SO2NH2), 7.42 (dd, 2H, Ar-H) 7.55 (d, 2H, Ar-H), 7.69 (d, 2H, Ar - H), 8.73 (s, 1H, -CONH), 8.97 (s, 1H, -CONH).

¹³C-NMR spectrum (DMSO-d6, δ, ppm): 115.75 (d, CH in the aromatic ring), 117.90 (CH in the aromatic ring), 120.78 (d, CH in the aromatic ring), 127.29 (CH in the aromatic ring), 136.12 (d, CH in the aromatic ring), 137.38 (CH in the aromatic ring), 143.35 (CH in the aromatic ring), 152.89 (C=O), 156.93 (CH in the aromatic ring).

Mass spectrum (m/z, ESI): showed molecular ion [m + H]⁺ peak at 309.95 corresponding to the molecular formula of C13H12FN3O3S, and its fragments.

(2-d):4-[3-(4-Hydroxy-phenyl)-ureido]-benzene- sulfonamide, reddish Brown powder, mp 235-238 C⁰.

TLC used mobile phase consists of acetate ethyl:petrolatum ether 7:3, in where the Rf values are as follow: sulfanilamide is 0.41, phenylurea is 0.22, compound(2-d) is 0.39, and 4.4-Bis-hydroxy diphenylurea is 0.80.

TLC used mobile phase consists of acetate ethyl:petrolatum ether:Methanol 3:2:1, where the Rf values are as follow: Ureido benzenesulfonamide is 0.19, (2-d) 0.73, and aniline 0.93.

IR spectrum (νmax, cm⁻¹): 3312.51 (O–H), 3229.09, 3120.59 (N–H), 1715.36 (C=O), 1595.15, 1538.66, 1512.07 (C=C), 1163.31 (S=O).

¹H-NMR spectrum (DMSO-d6,δ, ppm): 6.65(d, 2H , Ar-H), 7.14 (s, 2H, SO2NH2), 7.18 (d, 2H, A-H) 7.56 (d, 2H, Ar-H), 7.68 (d, 2H, Ar- H), 8.39 (d, 1H, -CONH). 8.87 (s, 1H, -CONH), 9.94 (s, 1H, -OH,).

¹³C-NMR spectrum (DMSO-d6, δ, ppm): 117.74 (CH in the aromatic ring), 118.05 (CH in the aromatic ring), 121.17 (CH in the aromatic ring), 127.29 (CH in the aromatic ring), 131.20 (CH in the aromatic ring) 137.93 (CH in the aromatic ring), 142.86 (CH in the aromatic ring), 153.39 (C=O), 154.00 (CH in the aromatic ring).

Mass spectrum (m/z, ESI): showed molecular ion [M + H]⁺ peak at 308.06 corresponding to the molecular formula of C13H13N3O4S, and its fragment.

4. CONCLUSION:

Novel phosgene-free methods were implemented to prepare different aryl ureido benzenesulfonamide derivatives (2-b), (SLC-0111), and (2-d) as carbonic anhydrase IX inhibitors with a high yield up to 80%.

butanol is a better solvent than water for synthesizing the UBSs and preventing hydrolysis reaction.

5. ACKNOWLEDGMENTS:

We are grateful to Philipps University-Marburg, Institute of Pharmacy, Department of Pharmaceutical Chemistry, for providing technical support in identifying the synthesized compounds through IR, Mass, ¹H-NMR, ¹³C-NMR spectroscopies.

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Received on 25.11.2022 Modified on 06.01.2023

Research J. Pharm. and Tech 2023; 16(8):3884-3890.

DOI: 10.52711/0974-360X.2023.00640