Synthesis and Pharmacological Evaluation of the derivatives of

1,4-dihydropyridine: A Pleiotropic Molecule with Antiulcer activity

 

Abhinav Prasoon Mishra¹*, Rakesh Kumar Meel²

¹Associate Professor, Department of Pharmacy, Advance Institute of Biotech and Paramedical Sciences, Naramau, Kanpur, India.

²Professor, School of Pharmacy, Shridhar University, Chirawa - Pilani Rd, Pilani Rajasthan, 333031.

 

ABSTRACT:

As previously researches indicate that 1,4-Dihydropyridine is a versatile molecule and it is clearly shown that it possesses different therapeutic activity in versatile disease. It shows different activity such as anticancer, anticonvulsant, anticoagulant, anti-alzheimer, antitubercular and antiulcer. Various adverse effects of proton pump inhibitors and H2 blockers were reported by many Investigators. These are Gastric cancer, Colon cancer, Gastric carcinoid tumor, gastrointestinal infection and many others. A good antiulcer potential of 1,4 Dihydropyridine with aromatic primary amine substitution seen here, with that reason our research work to conduct synthesis of  1,4 dihydropyridine derivative by replacement with some other aromatic primary amine scaffolds, and to evaluate the antiulcer potency of 1,4 Dihydropyridine. All chemicals were purchased from verified companies. Characterization of these synthesized molecules were performed by various spectroscopic methods and evaluated for in-vivo antiulcerogenic potential by using different animal models. A series of approximately in 25 numbers 1, 4 Dihydropyridine derivatives were synthesized and characterized on their spectroscopic studies. Antiulcer activity was performed in ethanol induced gastric lesions followed with five different models. In series of approximately 25 derivatives synthesized compound A5, A6, B5 and B6 shows significant activity in comparison to standard drug i.e.Omeprazole and among all compound, A5 and B6 (30mg/kg) shown potent antiulcer activity. Our final remark clearly stated that 1,4 Dihydropyridine previously established as calcium channel blocker, must shown antiulcer potential and all synthesized compound can serve as leads for new antiulcer agents after further investigation.

 

 

 

INTRODUCTION: 

Ulcer development or mucosal lesions in the esophageal lining, stomach and the foremost part of small intestine is known as peptic ulcer. Approximately 10-15% of the population is suffered  and severe consequence of peptic ulcer about 20,000 death reported annually, it is not possible to discover any drug without its side effect or adverse event and as we know that various medications used for ulcer treatment such as proton pump inhibitor (Omeprazole), H2 receptor antagonist.

 

Clinical assessment of these medications also reveals their side effect as well as adverse effects in the long term use thus there is a need to search the safe and effective antiulcer drugs.^1-16

 

1,4-Dihydropyridine (1,4-DHP) has played an important role in synthetic, medicinal, and bioorganic chemistry [10]. 1,4-dihydropyridine possess so many therapeutic activities such as Cardiovascular activity, anti-tumour, anti-tubercular, anti-inflammatory,  anti-dyslipidemic, anti-microbial, anti-oxidant, etc.  that’s why it is called as a versatile scaffold. Previous studies of 1,4 dihydropyridine reveal it’s antiulcer property.¹⁷

 

 

 

Special Structural analysis and antiulcer potential of 1,4-Dihydropyridine derivatives give us idea to explore via their synthesis of some 1,4 Dihydropyridine derivative and screened them for their antiulcer potential. Our present study based on the development of new molecules which increase the protection mechanism of gastric mucosa and prevent the ulcer formation.

 

MATERIAL AND METHOD:

All chemicals (AR grade and LR grade) and apparatus were issued from the institute, which were purchased from Sigma Aldrich and Merck. Melting point of compounds were determined by thermionic melting point apparatus and IR spectra of compounds were recorded on PerkinElmer FTIR by Spectrum Software500MHz Jeol Delta NMR Spectrometer were used for 1H NMR of compounds. M. All the samples were taken in DMSO-d6 with standard tetramethylsilane for study of NMR of compounds. Molecular mass were measured by WATERS-Q-TOF Premier-HAB213 mass spectrometer. Elementary (Exeter Analytical Inc.model: CE 440) was used for the detection of elemental analysis for synthesized compounds.

 

For toxicity and screening of compound Wistar rats were used and approved by college ethical committee, oral gauge needle, phosphate buffer solution, 0.5% DMSO

 

Synthesis, Purification and Characterization of Compounds:

All 1,4-DHP derivative prepared in two steps mechanism

 

A solution of aldehyde (1 mol), ethyl acetoacetate (2 mol), ammonium hydroxide (1 mol) taken in ethanol (60 ml.) was heated under reflux in a round bottom flask, for 2 hours at 140 ^oC temperature. The resulting mixture was poured into an ice bath. Separated product (A&B) was filtered off and recrystallized with 60% Alcohol. Equimolar quantities of amine group containing compounds (1 to 6) (1mol) in 10 mL of ethanol was added into a slurry containing the mixture of compound (A & B) and aqueous formaldehyde solution. The reaction mixture was heated with continuous stirring at water bath, till a clear solution was not obtained then the resulting mixture was poured into an ice bath. The products [A/B/C (1 to 6)] were separated by filtration through suction and recrystallized with 60% ethanol.^18-20

 

Evolution Of Acute Toxicity, Gastric Lesions And Antiulcer Activity In Animals:

Acute toxicity and screening of antiulcer activity was conducted at our institute. AEC clearance was done with reference no: 1273/PO/Re/S/09/CPCSEA. All Experiments performed complied with norms of CPCSCEA. Albino rats (weight from 125 to 200g) were taken from the animal house of the institute under controlled environment (23–25°C). All the animals were kept in sanitized transparent plastic cages and were fasted for 24h before conducting the experiment. Animals were served with tap water ad libitum and standard pellet diet purchased from market. All animals were randomly divided into six rats of each group. Compound A1 to B6 were given orally or intra-peritoneal. For acute toxicities the test was performed using doses (250, 500 and 1500mg/kg) of the test compounds, given orally 1ml to each animal. The animals were allowed food ad libitum and kept under observation to determine any mortality in first 24 h.²¹

 

During first phase of our pharmacological study, we evaluated all the synthesized compounds (A1 to B6) at graded doses (10, 20, and 30mg/kg,) in 99.9% ethanol induced gastric ulcer model with Omeprazole (30 mg/kg) as standard drug. The screening results are summarized in Table 1. Maximum amount of ulcer index secretion 93.20±0.37 was recorded Figure 1.²² On the bases of first screening results we further explore the anti-ulcer effects of A5, A6, B5, B6 in different anti-ulcer models. In our next model we induced gastric lesions in animals by treatment of 80% ethanol, 0.2 mol/l NaOH and 25% NaCl and by this model we calculated ulcer index.  After 1 hour, In control animals the ulcer index was 9.06±0.11, 8.25±0.07 and 7.54.± 0.16 respectively for each necrotising agent. Pretreatment of animals with compound A5 and B6 at dose of 10, 20, and 30mg/kg produce significant results in comparison to standard drug omeprazole. Compound A5 was found to be most active with ethanol, NaOH and NaCl induced gastric ulcer 5.25±0.13, 1.81±0.05 and 2.68±0.02, respectively (2A, 2B, 2C).²⁴

 

Indomethacin (30mg/Kg) induced gastric ulcer in animals and compound A5, A6, B5 and B6 shows significant result with ulcerindex of 18.70±0.17, 17.89±0.05, 22.64±0.12 and 21.68±0.20 respectively these results provide a sign of the cytoprotective nature of these compound. Indomethacin (30mg/kg) orally developed gastric damage of animals. Compound A6 was found most active antiulcer agent in this method (2D).^22,24 Ulcer formation in water-immersion restraint stress was inhibited significantly by compounds A5, A6, B5 and B6 at the dose of 30mg/kg. With ulcer index of 18.68±0.08, 21.20±0.08, 22.37±0.11, 20.79±0.12 respectively (2E).^23,24 In pylorus ligation induced gastric damage, maximum amount of gastric acid secretion were found 11.49±0.01 ml and 4.40±0.25 ulcer index was recorded. Titrable acidity in control group was found to be 186.01±1.15mEq/l.  Compound A5, A6, B5, and B6 significantly reduced gastric ulcer in comparison to reference drug omeprazole at the dose of 30mg/Kg (2F).^23,24 Ulcer formation in acetic acid induced gastric ulcer was significantly inhibited by compounds at the dose of 30mg/kg. In control animals the ulcer index was 87.51±0.11 for acetic acid. All compounds A5, A6, B5 and B6 shows significant result with ulcer index of 40.67±0.14, 42.67±0.12, 44.33±0.09 and 41.42±0.11 respectively and compound A5 shows most potent activity (2G).²⁶ Graphical represent (Figure 2) of the remaining methods were clearly indicate that Compound A5 shows most potent activity in comparison than other compound A6, B5 and B6.

 

Table 1. The antiulcer effect of compounds on ulcerative lesions ethanol induced ulcer (mean ± SE)

Treatment	Control (99.95 Ethanol)	Omeprazole 30mg/Kg		Compound
				10 mg/Kg		20 mg/Kg		30 mg/Kg
	Mean ± SE	Mean ± SE	% Change	Mean ± SE	% Change	Mean ± SE	% Change	Mean ± SE	% Change
A1	92.31±0.43	22.34±0.39	75.79	83.56±0.44	9.47	74.24±0.21	19.57	70.74±0.45	23.36
A2	91.93±0.36	21.33±0.34	76.79	82.50±0.44	10.25	74.71±0.4	18.73	71.71±0.35	21.99
A3	92.59±0.27	21.66±0.45	76.60	84.80±0.34	17.26	74.72±0.35	19.30	70.62±0.48	23.72
A4	92.02±0.28	22.21±0.26	75.86	83.03±0.47	9.76	73.27±0.22	20.37	72.60±0.49	21.10
A5	90.39±0.28	22.45±0.38	75.16	46.26±0.44	48.82	38.60±0.42	57.29	31.61±0.44	65.02
A6	92.57±0.37	22.22±0.28	75.99	54.80±0.38	40.80	40.89±0.41	55.82	34.56±0.10	62.66
B1	93.20±0.37	21.68±0.46	76.73	84.30±0.21	9.54	76.06±0.19	18.39	73.52±0.12	21.11
B2	91.48±0.29	22.99±0.35	74.86	84.94±0.16	7.14	78.82±0.39	13.83	71.75±0.39	21.56
B3	91.64±0.44	21.66±0.22	76.36	82.48±0.48	9.99	74.42±0.48	18.79	69.42±0.15	24.24
B4	92.11±0.37	21.66±0.28	76.48	83.61±0.37	9.22	74.84±0.17	18.74	70.41±0.49	23.55
B5	92.68±0.34	21.57±0.38	76.72	58.30±0.36	37.09	48.42±0.49	47.75	36.59±0.42	60.52
B6	90.78±0.47	21.72±0.45	76.07	51.66±0.41	43.09	42.94±0.17	52.69	32.72±0.43	63.95

Six rats were used in each group.

 *p < .05,

**p < .01,

***p < .001 vs. control group

 

 

A                                                          B

 

C                                                        D

Figure 1: (A) Mucosal ulceration with absolute ethanol treatment. (B) Treatment with Omeprazole (30 mg/kg) shows Protective effect. (c) Treatment with test compound A5 (30 mg/kg) showing mild ulceration with intact mucosa. (D) Treatment with compound B6 (30 mg/kg) showing mild ulceration

 

 

Figure 2: Graphical representation of pharmacological evaluation of compound A5, A6, B5, and B6 at dose of 30mg/Kg along with omeprazole as reference drug. Evaluation performed by necrotising agent (2A, 2B and 2C), by indomethacin (2D), Water restraints immersion (2E), Pylorus ligation method (2F), and acetic acid induced gastric ulcer method (2G) Statistical analyses were performed by using Graphpad Prism 5.0. Values are expressed as the means ± standard error of the mean (SEM). The data were analyzed using a one-way ANOVA and Two-way ANOVA. P < 0.05, P<0.001, P<0.0001 was for significant result.

 

DISCUSSION AND CONCLUSIONS:

As shown in reaction scheme the 1, 4 Dihydropyridine hybrids were synthesized by two step reaction starting from Ethyl-acetoacetate. Completion of synthesis was confirmed by TLC.  TLC of synthesized compounds clearly indicate the formation of compounds during reaction. Melting point of the synthesized compounds were determined in a one end fused capillary tube method. All compounds shows melting point above 105 ^oC. Solubility of the synthesized compounds were analysed in the different solvents and reported. On the bases of physical analysis and spectral analysis we came to this conclusion that the entire designed compound were developed and structurally corrected.

 

On 14^thday of acute toxicity studies we observe that there was no single sign of abnormalities present. Individual mortality data and necropsy were observed.  During acute toxicity study all the animals were safe and no abnormalities detected that means all synthesized compounds were safe for oral use.

 

Among all synthesized compound only four compounds A5, A6, B5 and B6 were found to most effective compound among the series. These four compounds were further investigated with different in vivo antiulcer models and compound A5 was found to be highly potent compound of the series.

 

Though our result showed the positive effect of 1,4dihydropyridine derivative as antiulcer agent but we cannot say that it is safe for human because it is well known that 1,4-dihydropyridine work as calcium channel blocker and used in treatment of hypertension so it may be harmful for hypotensive patient this is why pre-clinical trial as well as clinical trial needed. So before going any final decision we must be sure that this synthesized compound exactly possess only antiulcer effect or they also possess some calcium channel blocking activity. If we want to study only antiulcer effect of the synthesized derivative so there is need to study the activity of derivative against other In-vivo antiulcer models.

 

ACKNOWLEDGEMENT:

The author wishes to thank Advance institute of biotech and paramedical sciences, Naramau, Kanpur, Uttar Pradesh, India, for carrying out practical work and providing literature reviewing support for this study.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 04.04.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):561-565.