Synthesis and Urease Inhibition Activity of 4-hydroxy-3-methoxy benzoic acid Derivatives

 

Priyanka Sharma, Parvinder, Saloni Kakkar*, Anurag Khatkar

Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak-124001, India.

 

ABSTRACT:

A number of 4-hydroxy-3-methoxy benzoic acid (vanillic acid) compounds were prepared and evaluated for urease inhibition activity. All the prepared compounds were characterized by IR, ¹H NMR spectroscopic techniques. Synthesized compounds were screened for Urease inhibition activity against jack bean urease by using indophenols method. Results exposed that compound 3, 14 and 17 with IC₅₀ values of 33.52µg/ml , 32.41 µg/ml and 30.06µg/ml were the most active urease inhibitors comparable to standard thiourea (IC₅₀: 33.50 µg/ml). Hence newly synthesized derivatives of vanillic acid derivatives exhibits considerable urease inhibition potency.

 

 

 

INTRODUCTION:

Urease is a nickel dependent metalloenzyme which belongs to amidohydrolases and phosphotriesterases. Urease can be synthesized by plants, bacteria, fungi, algae and some invertebrates. It is also found in the natural environment (water and soil) and in human body¹. Urease catalyzes the hydrolysis of urea at physiological pH to yield carbonic acid and ammonia².  Primary cause of neurological disorders is due to excess amount of ammonia enter into the brain because of  congenital deficiencies of urea cycle enzymes, Reye syndrome, hepatic encephalopathies, atherosclerosis, rheumatoid arthritis, urinary tract infections (UTIs)  and other metabolic disorders³. Urease enzyme is a vaccine candidate, also used for taxonomic identification and for diagnosis and follow-up after treatment⁴.

 

Plants are rich sources of flavonoids, polytosterols, ellagic acid, gallotannins, hypolipidemic, hypoglycemic and antioxidant agents such as phenolic acids and other related polyphenols. Therefore, to a large extent effort has been focused on the plants to search potentially useful compounds from local medicinal plants⁵.

 

Phenolic acids are simple molecules and are easily absorbed by the human system⁶. Within all the phenolic acid contents vanillic acid is of huge significance as it possesses numerous biological activities like antioxidant⁷, antimicrobial⁸, anti-inflammatory⁹, anticancer¹⁰, antidiabetic¹¹ and antinoceceptive potency⁷. Vanillic acid was also found to possess several other activities like anticoagulant, antiulcer¹², inhibitory effect on methylglyoxal-mediated glycation in apoptotic Neuro-2A cells, nephroprotective effect¹¹ and also prevents deregulation of lipid metabolism¹³.

 

MATERIALS AND METHODS:

All solvents and reagents used in this study were procured locally and were of analytical grade. Determination of melting points was done on a Sonar melting point apparatus by using open capillary tubes and were uncorrected. Reaction improvement was checked by thin layer chromatography (TLC). Products obtained after reaction completion were recrystallized and their purity was analysed by TLC using silica gel G coated glass plates.¹H NMR spectroscopy of all the derivatives was done on Avance III 400MHz  spectrometer using DMSO as solvent. Infra-red (IR) spectroscopy was done on Bruker 12060280,software OPUS 7.2.139.1294  IR spectrophotometer in the range of 600-4000 using ATR.

 

General procedure for synthesis of 4-hydroxy-3-methoxy benzoyl chloride (2) (Scheme 1):

Thionyl chloride (0.6 mol) was mixed with 4-hydroxy-3-methoxy benzoic acid (1) (0.35 mol) in a round bottom flask. The reaction mixture was stirred for 4 hours by magnetic stirring, after addition of thionyl chloride. Mixture was heated to 80°C for 1 hour in a water bath, after completion of stirring. End of reaction was determined using TLC

 

Procedure for the synthesis of esters (1-19)(Scheme 1):

A mixture of different alcohols (Table 1) in ether (50 mL) was mixed into a solution of 4-hydroxy-3-methoxy benzoyl chloride (2) (0.05 mol) in ether (50ml). Until no further development of hydrogen chloride was pragmatic, the solution was continuously heated on a water bath. It  was cooled to room temperature and disappearance of solvent yielded the crude ester which was recrystallized with alcohol for purification of product.

 

In vitro evaluation of urease inhibitory potency:

The urease inhibitory potency was evaluated according to the method reported by Tanaka et al., 2003. 250µL of jack bean urease (4U) was mixed with 250µL of different synthesized test compounds and standard of different concentrations (dissolved in DMSO/H₂O mixture (1:1 v/v). The mixture was preincubated for one hour at 37°C in test tubes. 2ml of 100mM phosphate buffer (pH 6.8) containing 500mM urea and 0.002% phenol red as an indicator were added, after pre incubation and again incubated at room temperature. Absorbance of reaction mixture was calculated by UV visible spectrophotometer at 570nm. Ammonium carbonate increased the pH of phosphate buffer from 6.8 to 7.7 which was produced from urea by urease enzyme and the end peak was measured by the colour of phenol red indicator¹⁴.

 

The percentage inhibition of urease enzyme was calculated by using following formula:

                               A_control - A_sample

% Inhibition = --------------------------- X 100

                                    A_control

Where  

A_control = Absorbance of the control

A_sample = Absorbance of the sample

 

Chemistry:

Vanillic acid derivatives (1-19) were synthesized in two steps as illustrated in Scheme 1.The first step involved the synthesis of 4-hydroxy-3-methoxy benzoyl chloride. Compounds 1-19 were synthesized by the reaction of 4-hydroxy-3-methoxy benzoyl chloride (2) with alcohols (Table 1). The physicochemical properties of the synthesized derivatives have been shown in Table 2. The structures of all the lately synthesized derivatives were assigned based on their IR and¹H NMR spectral data.

 

Table 1: Arrangement of substituents used for targeted compounds

Compound	R	Compound	R
C-1		C-11
C-2		C-12
C-3		C-13
C-4		C-14
C-5		C-15
C-6		C-16
C-7		C-17
C-8		C-18
C-9		C-19
C-10

 

Scheme 1: Synthesis of vanillic acid derivatives

 

RESULTS AND DISCUSSION:

The synthesized derivatives were characterized by determination of their physicochemical properties like melting points, R_f value, % yield, ATR and¹H-NMR spectral studies and agreement was found with assigned molecular structures.  Physicochemical characteristics and spectral data of synthesized compounds have been represented in Table 2 and Table 3.

 

Table 2: Physiochemical properties of prepared compounds (1-19):

Compound	M. Formula	M. Weight.	M.P. (0C)	Rf Value	% Yield
C-1	C9H8O5	196.60	516	0.71	68
C-2	C19H14O4	306.31	353-355	0.68	74
C-3	C13H16O5	252.26	349-351	0.77	71
C-4	C15H21O4	265.32	320-323	0.69	58
C-5	C14H19O4	251.3	375-377	0.61	57
C-6	C12H14O4	222.24	362-364	0.63	61
C-7	C15H12O6	288.25	388-390	0.73	78
C-8	C15H20O9	344.11	331-333	0.70	63
C-9	C16H14O4	270.28	390-392	0.81	61
C-10	C19H14O4	306.09	393-395	0.66	67
C-11	C15H11NO6	301.25	344-346	0.69	72
C-12	C15H13NO4	271.27	332-334	0.74	81
C-13	C12H14O4	222.09	328-330	0.71	77
C-14	C15H11NO6	301.25	374-376	0.63	59
C-15	C16H15NO4	271.29	388-390	0.76	62
C-16	C13H16O4	236.26	383-385	0.64	64
C-17	C25H40O4	404.58	343-345	0.72	70
C-18	C11H13NO4	223.23	340-342	0.73	73
C-19	C12H13NO6	267.23	392-394	0.69	68

^*Hexane: Ethyl acetate: Glacial acetic acid (5:4:1)

Table 3: Spectral data of synthesized derivatives (1-19):

Comp.	IR (ATR) cm-1
	O-H str.(Ar)	C-H str.(Ar)	C-O str.(Ar)	C=O str.(ester)	C=C str.(Ar)	NH2 str.
C-1	3241	3133	1252	1841	1533	-
C-2	3390	3094	1280	1834	1546	-
C-3	3392	3137	1202	1836	1500	-
C-4	3383	3335	1289	1780	1545	-
C-5	3391	3064	1169	1783	1520	-
C-6	3383	2909	1554	1699	1478	-
C-7	3331	3099	1127	1726	1592	-
C-8	2930	3079	1080	1819	1592	-
C-9	2930	3075	1284	1717	1592	-
C-10	3396	3059	1212	1776	1559	-
C-11	3377	3060	1156	1633	1504	-
C-12	3368	3227	1153	1698	1634	1634
C-13	3375	3160	1151	1633	1446	-
C-14	3368	3054	1147	1707	1504	-
C-15	3388	3078	1283	1895	1589	-
C-16	3380	3166	1552	1630	1495	-
C-17	3350	3120	1233	1738	1606	-
C-18	2924	2954	1242	1812	1587	3525
C-19	3311	3088	1223	1729	1599	3448

NMR data of synthesized  derivatives (¹H NMR (DMSO, δppm):

 

4-Hydroxybutyl-4-hydroxy-3-methoxy-benzoate (C₃):  6.81-7.35(t, 3H, Ar-H), 4.15(s, H, Ar-OH), 2.70 (s,3H,Ar-OCH₃), 2.70(s, H, CH), 1.91(s, H, OH),1.23(m, 3H, CH₃).

 

2, 4, 5, 6-Tetrahydroxy-3-(hydroxymethyl)hexyl 4-hydroxy-3-methoxybenzoate (C₈): 6.96-7.21(t, 3H, Ar-H) 3.57(s, 3H, Ar-OCH₃) 1.99(m, 5H, OH) 3.57(m, 2H, CH) 3.57(s, 2H,CH₂).

 

Benzyl 4-hydroxy-3-methoxybenzoate (C₉): 6.94-7.34(t, 3H, Ar-H)  4.53(s ,H, Ar-OH)  3.64(s, 3H, Ar-OCH₃) 7.13(m, 5H, Ar-H) 4.53(s, 2H, CH₂).

 

2-Nitrophenyl 4-hydroxy-3-methoxy benzoate (C₁₁): 6.10 (t, 3H, Ar-H)  4.51(s ,H, Ar-OH)  3.61(s, 3H, Ar-OCH₃) 8.18(m, 4H, Ar-H).

 

2-Aminophenyl-4-hydroxy-3-methoxy benzoate (C₁₂): 4.54(s ,H, Ar-OH)  3.62(s, 3H, Ar-OCH₃) 6.10-6.19(m, 4H, Ar-H) 3.62(s, 2H, NH₂).

 

Propyl 4-hydroxy-3-methoxy benzoate (C₁₃): 6.87-7.12 (t, 3H, Ar-H) 3.66(s, 3H, Ar-OCH₃), 1.92(s, 4H, CH₂) , 1.92( s,3H,CH₃).

 

4-Nitrophenyl 4-hydroxy-3-methoxy benzoate (C₁₄): 6.71 (t, 3H, Ar-H) 4.50(s ,H, Ar-OH)  3.37(s, 3H, Ar-OCH₃) 8.02(m, 4H, Ar-H).

 

Sec –butyl 4-hydroxy-3-methoxy benzoate (C₁₆):

5.37(s, H, Ar-OH)  3.37(s, 3H, Ar-OCH₃) 1.38(s, 6H, (CH₃)₂) 4.00(s, H, CH) 1.92(s, 2H,CH₂).

 

Hexadecyl 4-hydroxy-3-methoxy benzoate (C₁₇):

7.00-7.27(t, 3H, Ar-H) , 5.00(s ,H, Ar-OH)  3.79(s, 3H, Ar-OCH₃) 1.90(m, 28H, CH₂) 4.77(s, 2H, CH₂) 1.90(s, 3H, CH₃).

2-Aminoethyl 4-hydroxy-3-methoxy benzoate (C₁₈): 3.81(s, 3H, Ar-OCH₃) 2.52(s, 4H, (CH₂)₂) 3.93(s, 2H, CH₂) 2.50(s, 2H, NH₂).

 

In vitro evaluation of urease inhibitory activity:

All the synthesized derivatives (1-19) were checked for in vitro urease inhibiton activity according to Tanaka et al. (2003) by indophenol method using jack bean urease and standard inhibitor thiourea and results are precised in Table 4.

 

Table 4: Urease inhibition potency of vanillic acid derivatives.

Compound	25 (µg/ml)	Inhibition (%)			IC50 (µg/ml)
		50 (µg/ml)	75 (µg/ml)	100 (µg/ml)
C-1	41.34	50.33	58.90	76.34	48.18
C-2	46.43	54.99	63.67	74.03	35.83
C-3	47.50	55.23	61.43	69.75	33.52
C-4	44.77	53.09	60.33	69.04	41.26
C-5	40.01	50.54	59.43	77.67	48.71
C-6	37.31	52.07	65.04	77.02	47.66
C-7	46.64	52.66	65.09	75.11	37.67
C-8	37.31	52.07	65.04	77.02	47.66
C-9	48.12	53.09	61.34	69.75	35.25
C-10	46.01	54.37	63.56	71.09	36.60
C-11	46.43	54.99	63.67	74.03	35.83
C-12	45.98	57.43	65.42	76.01	34.05
C-13	47.15	54.05	63.07	71.03	35.21
C-14	48.91	54.01	60.99	69.67	32.41
C-15	44.87	57.66	64.08	73.05	35.74
C-16	47.23	54.05	64.67	71.91	34.75
C-17	49.30	55.41	62.32	71.51	30.06
C-18	44.98	53.09	64.76	73.01	39.23
C-19	43.11	51.67	60.32	68.45	45.11
Thiourea*	48.01	54.33	61.89	70.03	33.50

 

CONCLUSION:

All the synthesized derivatives (1-19) were evaluated for in vitro urease inhibition activity by indophenol method using jack bean urease and standard inhibitor thiourea and results are precised in Table 4. The compounds 3, 14 and 17 were established to possess highest urease enzyme inhibition potency amongst all the prepared derivatives having IC₅₀ value of 33.52µg/ml, 32.41 µg/ml and 30.06µg/ml respectively compared with thiourea with IC₅₀ value of  33.50µg/ml. Hence, these derivatives may be further taken as lead compound as novel urease inhibitors.

 

REFERENCES:

1.      Jodi LB. Scott BM. and Robert PH. Nickel-Dependent Metalloenzymes. Archives of Biochemistry and Biophysics. 2014; 142–152.

2.      Harry LT. Mobley HLT. and Robert P. Microbial Ureases: Significance, Regulation and Molecular Characterization. Journal of American Society for Microbiology. 1989; 85-108.

3.      Chawla J. Hyperammonemia, Medscape; 2016.

4.      Iwonaq K. Paulina Z. Marek K.  Beata K.  Justyna F. Zbigniew K. and Wiesław K. Bacterial Urease and its Role in Long-Lasting Human Diseases. Current Protein and Peptide Science. 2012; 13(8): 789–806.

5.      Kumarappan CT. Rao TN. and Mandal SC. Polyphenolic extract of Ichnocarpus frutescens modifies hyperlipidemia status in diabetic rats. Journal of Molecular and Cellular Biology. 2007; 6(22): 175-187.

6.      Maksymiak M. Kowalczuk M. and Adamus G. Electrospray tandem mass spectrometry for the structural characterization of p-coumaric acid–oligo (3-hydroxybutyrate) conjugates. International Journal of Mass Spectrum. 2014; 359: 6-11.

7.      Zhu Y. Li T. Fu X. Abbasi AM. Zheng B. and Liu RH. Phenolics content, antioxidant and antiproliferative activities of dehulled highland barley (Hordeum vulgare L.). Journal of Functional Foods. 2015; 19: 439-450.

8.      Falleh H. Ksouri R. Chaieb K. Bouraoui NK. Trabelsi N. Boulaaba M. and Abdelly C. Phenolic composition of Cynara cardunculus L. organs, and their biological activities. Comptes Rendus Biologies. 2008; 331(5): 372-379.

9.      Prince PSM. Dhanasekar K. and Rajakumar S. Vanillic acid prevents altered ion pumps, ions, inhibits FAS-receptor and caspase mediated apoptosis-signaling pathway and cardiomyocyte death in myocardial infarcted rats. Chemical and Biological Interactions. 2015; 232: 68-76.

10.   Dziki UG. Swieca M. Sułkowski M. Dziki D. Baraniak B and Czyz J. Antioxidant and anticancer activities of Chenopodium quinoa leaves extracts–in vitro study. Food and Chemical Toxicology. 2013; 57: 154-160.

11.   Huang SM. Hsu CL. Chuang HC. Shih PH. Wu CH. and Yen GC. Inhibitory effect of vanillic acid on methylglyoxal-mediated glycation in apoptotic Neuro-2A cells, Neurotoxicology. 2008; 29(6): 1016-1022.

12.   Malairajan P. Gopalakrishnan G. Narasimhan S. Veni KJK. and Kavimani S. Anti-ulcer activity of crude alcoholic extract of Toona ciliata Roemer (heart wood), Journal of Ethnopharmacology. 2007; 110(2): 348-351.

13.   Kumar S. Prahalathan P. Saravanakumar M. and Raja B. Vanillic acid prevents the deregulation of lipid metabolism, endothelin 1 and up regulation of endothelial nitric oxide synthase in nitric oxide deficient hypertensive rats. European Journal of Pharmacology. 2014; 743: 117-125.

14.   Tanaka T. Kawase M. and Tani S. Urease inhibitory activity of simple α,β-unsaturated ketones. Life Sciences. 2003; 73(23): 2985-2990.

 

Accepted on 27.09.2019                                 © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(3): 1453-1456.