Novel essential amino acid-sulfanilamide hybrid as safe anti-ulcerogenic agent with anti-helicobacter pylori activity.

A novel and safe essential amino acid (Leucine) incorporating sulfanilamide was synthesized, and evaluated for its anti-ulcerogenic activity and in vitro anti-Helicobacter pylori activity. The new molecule showed a dose dependent activity against absolute ethanol-induced ulcer in rats, it produced percent protection of control ulcer by 66.7 at dose 100 mg/kg. In addition it showed a potent anti-Helicobacter pylori activity in vitro against 7 clinically isolated strains. The minimum inhibitory concentration (MIC) ranged from 12.5 to 50 μg/ml. The preliminary safety studies and toxicity profile are optimistic and encouraging.

Introduction

Peptic ulcer (PU) is a major health problem which concerns the medical community all over the world. It is known that the major causative factor of a number of gastric pathologies including gastritis, peptic ulcers and certain gastric cancers is the Helicobacter pylori (H. pylori), which is well-known Gram −ve bacterial human pathogen that is responsible for type B gastritis of the stomach and can lead to duodenal ulcers and even gastric cancer. This species is typically treated with bismuth salts in combination with antibiotics (Iwahi et al., 1991). Inflammation, injury and infection with H. pylori are the main causative factors. In spite of the substantial progress in many aspects of basic and clinical research, no clear, safe remedy is available (Liou et al., 2016, Wang et al., 2015, Newman, 2008, Chimenti et al., 2007, Newman et al., 2003, Pelish et al., 2001, Sorba et al., 2001).

Sulfonamide derivatives showed many biological activities; early and recent researchers have suggested that sulfonamides are useful for the treatment of some staphylococci infections, especially against urinary infections (Blass, 2016, Bartzatt et al., 2010, Altoparlak et al., 2004). It was reported that they showed the highest inhibitory effect on gram positive bacteria, i.e. Staphylococcus aureus, Nocardia asteroides, N. farcinia and Bacillus subtilis. However, sulfonamide derivatives were also reported in treatment of Chagas disease, they showed in vitro activity against two strains of Trypanosoma cruzi (Bocanegra-Garcia et al., 2012, Genç et al., 2008).

Furthermore, sulfonamide derivatives were used as hypoglycemic agent. Sulfonamide derivatives have several clinical applications against inflammatory bowel syndrome and other related ailments in addition to their tendency to accumulate in hypoxic tumors (Ahmadi et al., 2016) (Dubois et al., 2009, Cecchi et al., 2005, Huang et al., 2001).

Sulfa drugs are well known inhibitors of dihydrofolate reductase (Bush et al., 1982). Moreover, several literatures reviews mentioned their ability to selectively inhibit the different carbonic anhydrase isoforms (Supuran, 2012). Recently, some new sulfonamide derivatives with remarkable antitumor activity were prepared in laboratory (IC50 2.5–5.5 μg/mL) (Bourais et al., 2017) (Alafeefy et al., 2013, Alafeefy et al., 2012).

These findings prompted us to hypothesize that small molecule comprising both sulfanilamide and an essential amino acid would have a beneficial effect in combating many such horrible diseases and at the same time supplying such necessary components. In this regard we synthesized sulfanilamide derivative of leucine and explored its activity against peptic ulcer, H. pylori and its effect on liver and kidney functions.

Experimental

Synthesis

4-Methyl-2-[2-oxo-2-(4-sulfamoylphenylamino) ethylamino]pentanoic acid(5)

2-Chloroacetyl chloride (1.12 g, 0.01 mol) was added drop wise with vigorous stirring to a cold suspension of sulfanilamide (1.72 g, 0.01 mol) in 10 ml dichloromethane containing 2 drops triethylamine. Stirring was continued for 1 h and the separated solid was filtered, washed with ether, dried and crystallized from aqueous-ethanol.

Yield, 69%; m.p. 270–272 °C; 1H NMR (DMSO-d6): δ 0.90 (d, 6H, J = 12.0 Hz, 2CH3), 1.49 (m, 1H, CH), 1.80 (t, 2H, J = 9.0 Hz, CH2), 2.51 (s, 1H, NH, D2O exchange.), 3.26 (s, 2H, CH2), 3.49 (t, 1H, J = 8.5 Hz, CH), 4.21 (s, 2H, NH2, D2O exchange.), 7.30 (s, 1H, NH, D2O exchange.), 7.60 (d, 2H, J = 7.5 Hz, Ar-H), 7.79 (d, 2H, J = 7.67 Hz, Ar-H), 10.55 (s, 1H, OH, D2O exchange). 13C NMR: δ 22.6 (2CH3), 24.3 (CH), 41.1 (CH2), 50.2 (CH2), 59.4 (CH), 118.6, 126.7, 138.4, 141.4 (Ar-C), 169.0, 174.7 (2C = O). MS (EI): m/z 343 [M+, %]. Anal. (C14H21N3O5S) C, H, N.

or identification of the compound spectroscopic instruments were used such as; m.p, 1H NMR, 13C NMR Later on the chiral parameters such as the chiral strength, the symmetry of response as the chiral wave vector, optical activity, configuration will be determined.

Biological activity

Animals

Swiss albino mice of both sex (26–30 g) and male Wistar rats (180–200 g) were purchased from the animal house of King Saud University, KSA. Animals were housed in standard polypropylene cages with wire mesh top and maintained under standard conditions (temperature 23 ± 1.0 °C, humidity 55 ± 10%, 12 h light/12 h dark cycle). They fed with a standard pellet diet with water ad libitum and were allowed to adapt to the laboratory environment for one week before experimentation.

Determination of median lethal dose (LD50)

The oral median lethal dose (LD50) of the target compound was determined as described by (Lorke, 1983). Swiss albino mice in groups of six, received one of 50, 100, 500, or 1000 mg/kg doses of the target compound. Control animals were received the vehicle and kept under the same conditions. Signs of acute toxicity and number of deaths per dose within 24 h were recorded.

Antiulcerogenic activity

Evaluation of the anti-ulcerogenic activity was carried out using absolute ethanol-induced ulcer model as described by (Bighettia et al., 2005). Thirty male Wistar rats were divided into 5 groups each of 6 rats. Group 1 received the vehicle and served as control, group 2 received ranitidine (100 mg/kg) and served as standard, groups 3, 4 and 5 received the synthesized compound at doses 25, 50 and 100 mg/kg respectively.

Rats of all groups were fasted for 24 h then all medications were administered orally. One hour after treatment, the animals received an oral dose of absolute ethanol (1 mL/200 g) and then sacrificed one hour later, by ether inhalation, the stomachs were rapidly removed, opened along their greater curvature and gently rinsed under running tap water.

in the glandular part of the stomach were measured under an illuminated magnifying microscope (10×). Long lesions were counted and their lengths were measured. Petechial lesions were counted, and then each five petechial lesions were taken as 1 mm of ulcer.

the mucosal lesions were quantified by the scoring system (0–5) 0 = no damage, 1 = Local edema and inflammation without ulcers; 2 = One ulcer without inflammation; 3 = one to two ulcers with inflammation & lesion diameter <1 cm; 4 = More than two ulcers with lesion diameter 1–2 cm; 5 = Sever ulceration with lesion diameter >2 cm (Morris et al., 1989).

To calculate the ulcer index (mm), the sum of the total length of long ulcers and petechial lesions in each group of rats was divided by its number. The curative ratio was determined according to the formula:

Effect on liver and kidney functions

Male Wister rats were divided into 2 equal groups each of 10 rats. The 1st group was left as a control and administrated the vehicle orally, while the 2nd group was orally administrated the synthesized compound in a dose of 100 mg/kg for 15 days. After the examination period, 6 h after the last dose blood samples were collected from the orbital plexus of rats. Samples were left to clot at room temperature for 30 min then centrifuged at 1000 rpm for 20 min.

The collected sera were used for determination of the activity of both (AST) aspirate aminotransferase and (ALT) alanine aminotransferase as liver markers. In addition, levels of blood urea, serum creatinine were also estimated as kidney markers (Awaad et al., 2013).

In-vitro anti-Helicobacter pylori activity

Bacterial isolates

A total of seven clinical isolates of H. pylori were isolated from 19 biopsies received from patients diagnosed with gastritis or peptic ulcer disease at Al-Kasr Al-Ainy hospital, Cairo, Egypt. Clinical isolates were symbolized from KA1 to KA7. Isolates were grown in Brucella agar plates (Difco, Detroit, Michigan, USA) containing 10% v/v sheep serum at 37 °C.

Identification was carried out using Gram stain and catalase, oxidase and urea hydrolysis activities. Helicobacter pylori ATCC 43504 was used as control.

Determination of anti-Helicobacter pylori activity

Determination of the amino acid-sulfanilamide hybrid activity against H. pylori was carried out using disk diffusion method described by McNulty et al. (2002). The amino acid-sulfanilamide hybrid compound was dissolved in 2% Tween (v/v), in order to obtain final concentration of 2 mg/ml.

Sterile 6 mm disks utilized were imbibed in 1 mL of compound solution and were deposited on the surface of the plate of Mueller–Hinton agar with 10% sheep blood inoculated with H. pylori, in a suspension of 6 × 108 CFU/mL (McFarland turbidity standard 2), using amoxicillin (30 μg) and erythromycin (15 μg) as the standard antibiotics. The plate was incubated at 37 °C under microaerophilic conditions in an atmosphere of 5–15% O2 and 5–10% CO2 for 48–72 h.

Determination of the minimum inhibitory concentration (MIC)

The Minimum inhibitory concentration (MIC) was carried out by the broth microdilution assay (European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical and Infectious, 2003). A total of 100 μL of BHI broth supplemented with 10% defibrinated sheep blood inoculated with 6 × 108 H. pylori (McFarland turbidity standard 2) and 100 μL of serial dilutions of amino acid-sulfanilamide hybrid compound dissolved in 2% Tween (v/v) was added to each well in the microplate, to reach final concentrations of 12.5; 25; 50; 100; 200; 400 and 800 μg/ml. The microplate was incubated at 37 °C under microaerophilic conditions in an atmosphere of 5–15% O2 and 5–10% CO2, for 48–72 h. After incubation, the plates were visually examined, the optical density was determined at 450 nm and each well was replicated in blood agar (Mueller–Hinton agar with 5% sheep blood), to determine the MIC.

Results and discussion

The target compound was synthesized according to a reported procedure (Saeed et al., 2015). Sulfanilamide was reacted with 2-Chloroacetyl Chloride at room temperature in dichloromethane containing triethylamine. The obtained alkyl halide was then refluxed with leucine in 1-butanol containing anhydrous potassium carbonate in the presence of catalytic amount of potassium iodide. The product was obtained in about 50% yield and purified by column chromatography. The structure was confirmed by NMR spectra. The spectrum showed the aliphatic protons at δ of 0.9 ppm, two different protons on two different tertiary carbons δ at 1.49 ppm and 3.49 ppm and the carbons bearing them at 22.6, 24.3 and 59.9 respectively. Two CH2 groups at δ 1.80, 3.26 and their carbons appeared at 41.1, 50.2, respectively. The SO2NH2 protons were found at δ 4.21. Two singlets corresponding to two NH groups were seen at δ 2.51 and 7.30, respectively. One OH appeared at δ 10.55 due to the free carboxyl group of leucine. The two carbonyl groups were seen at δ 169.0 and 174.7. The two dimensional spectrum showed that the obtained structure is in accordance with the proposed one.

The compound was identified as (see Fig. 1, Fig. 2).

Fig. 1

Chemical structure of; 4-Methyl-2-[2-oxo-2-(4-sulfamoylphenylamino) ethylamino] pentanoic acid.

Fig. 2

Scheme for synthesizing compound 5.

Determination of median lethal dose (LD50)

The target compounds in doses up to 1000 mg/kg did not produce any behavioral changes and mortality in mice. Therefore, it can be categorized as highly safe since substances possessing LD50 higher than 50 mg/kg are nontoxic (Soliman et al., 2012).

Anti-ulcerogenic activity

The present results showed that the amino acid-sulfanilamide hybrid possessed a potent dose dependent anti-ulcerogenic activity. It produced percent protection of control ulcer ranged from 16.7% to 66.7%, while the standard drug ranitidine (100 mg/kg) produced 45.05% (Table 1). The target compound (100 mg/kg) was significantly more effective than the standard in reducing ulcer index and ulcer score.

Table 1

Anti-ulcerogenic effect of amino acid-sulfanilamide compound on absolute alcohol-induced ulcer in rats.

Groups	Dose mg/kg	Score	No of ulcers	Ulcer index	% Protection
Control	–	3.25	16.40 ± 0.5	10.92 ± 1.74	0
Ranitidine	100	2.2	6.9*** ± 1.05	6.0* ± 1.24	45.05
Compound 5	25	2.2	11.1* ± 1.79	9.1** ± 1.24	16.7
Compound 5	50	1.6	8** ± 1.58	4.48** ± 1.08	59
Compound 5	100	1.4	6.2*** ± 1.3	3.64*** ± 0.47	66.7

Data are expressed as mean ± SD, n = 6.

p ≤ 0.05.

p ≤ 0.01.

p ≤ 0.001.

Both liver and kidney functions were not affected as there is no significant difference between control and test group in all experiments, at the 0.05 level of probability (Table 2). These results showed that, the compound didn’t reveal hepatotoxic manifestation. In addition, no apparent nephrotoxic manifestations were recorded.

Table 2

Effect of amino acid-sulfanilamide compound on liver and kidney functions of rats.

Groups (100 mg/kg)	ALT (U/l)	AST (U/l)	Blood urea (mg/dl)	Creatinine (mg/dl)
Control	42.49 ± 0.37	60.77 ± 0.37	70.50 ± 1.36	0.88 ± 0.02
Compound 5	38.67 ± 0.22	63.23 ± 0.39	65.50 ± 1.9	0.85 ± 0.02

Data are expressed as mean ± SD, n = 10.

The activity of amino acid-sulfanilamide hybrid compound against H. pylori was determined (Table 3). Seven isolates of H. pylori were obtained from 19 gastric biopsies and their susceptibility to two antibiotics and amino acid-Sulfanilamide hybrid compound was determined by disk diffusion (Table 3). All the isolates of H. pylori were sensitive to amino acid-Sulfanilamide hybrid compound, amoxicillin and erythromycin.

Table 3

Activity of amino acid-sulfanilamide hybrid compound and two antibiotics against clinical isolates of H. pylori.

H. pylori strains	Inhibition zone (mm)
	Compound 5 (2 mg)	Amoxicillin (30 μg)	Erythromycin (15 μg)
KA1	18	22	21
KA2	15	23	22
KA3	16	24	24
KA4	15	23	23
KA5	15	25	21
KA6	17	26	25
KA7	19	27	24
ATCC 43504	16	33	29

The results revealed that the highest inhibition zones, 19, 18 and 17 mm, were obtained against H. pylori KA7, KA1 and KA6, respectively. On the other hand, the lowest inhibition zone 15 mm was obtained against H. pylori KA2, KA4 and KA5 (Table 3).

The minimum inhibitory concentration (MIC) was determined for the amino acid-sulfanilamide hybrid compound using broth dilution method (Table 4). The results showed that the lowest minimum inhibitory concentrations (12.5 and 25 μg/mg) were obtained against H. pylori KA7and KA6, respectively (Table 4).

Table 4

Minimum inhibitory concentration (MIC) of amino acid-sulfanilamide hybrid compound against clinical isolates of H. pylori.

H. pylori strains	Concentration (μg/ml)
KA1	50
KA2	50
KA3	50
KA4	50
KA5	50
KA6	25
KA7	12.5
ATCC 43504	50

The results of the current study confirmed the concept of Chimenti et al., 2007; they mentioned that anti-H. Pylori activity strongly depends on the presence of an acyl function like acid, ester or acyl chloride in the 3-benzamidic group and of a halogen in the 6-position of the coumarin ring.

As conclusion compound 5 is a novel compound which showed very promising anti-ulcerogenic and anti H. pylori activities with no side effects on liver and kidney functions.