Synthesis of Some New Azo Compounds of Salicylic Acid Derivatives and Determine Their In Vitro Anti-Inflammatory Activity

Introduction

Compounds containing in their structure a group or more of the AZO groups (–N=N–) called azo compounds,¹ in which the nitrogen atom hybridization is sp3.² Because of their physical and chemical properties as well as their biological efficacy, they possess several important applications in pharmaceuticals, cosmetics, textile industry, analytical chemistry and food.¹

Azo compounds are well known to have medical importance, they are recognized in many applications such as antidiabetics,¹ and they involved in many biological reactions such as inhibition RNA, DNA, protein synthesis carcinogenesis, and nitrogen fixation.³ Azo compounds are studied as HIV inhibitors of viral replications.⁴ Because of the presence of the azo moiety make these compounds possess biological efficacy as anti-bacterial,⁵ antitumor⁶ insecticide and pesticidal^7,8 activities. Salicylate compounds are widely valued because of their antipyretic, pain killing and anti-inflammation properties. 2-Hydroxybenzoic acid (called also salicylic acid) is most commonly used, and known in salicylates class compounds.⁹

The aim of study synthesis of some new azo compounds of salicylic acid derivatives by the diazo coupling reaction and determine their in-vitro anti-inflammatory activity by human red blood cell (HRBC) membrane stability method.

Experimental

Material and Methods

p-Nitroaniline, p-aminobenzoic acid, m-nitroaniline, 3-methoxysalicylic acid, 3-methylsalicylic acid and 4-methylsalicylic acid and solvents were used in this study sourced from Sigma-Aldrich company and Merck Company. The purity of prepared compounds was checked by thin layer chromatography. Melting points recorded by using Gallenkamp apparatus. FT-IR spectra (KBr) of prepared compounds determined on Shimadzu spectrometer (400-4000 cm^-1). The proton nuclear magnetic resonance (¹H-NMR) spectra determined on Bruker-NMR spectrometer at 300 MHz using an internal standard Me₄Si (TMS) and deuterated DMSO-d₆ as a solvent. Elemental analysis and ¹H-NMR spectra carried out in Al-albayt University Amman / Jordan.

General method for synthesis of diazonium salts

A solution of an aromatic amine (5 mmol), 1.5 ml of water and 1.5 ml concentrated HCl kept cooled in an ice-salt bath (0°C). A solution of sodium nitrite (5.5 mmol) in 1.5 ml of water added slowly with stirring. The mixture kept at 0°C. for the next step.^10,11 The other diazonium salts synthesized in a similar procedure. Figure (1) show synthesis of diazonium salt.

Figure 1: Synthesis of diazonium salt.  Click here to view figure

 

General Method for Synthesis of Azo Compounds

The prepared solution of diazonium salt was added portion wise to a solution prepared from salicylic acid derivatives (5.4 mmol) and 10 ml of 2.5 M aq. Sodium hydroxide. The mixture kept with stirring at (0-5^oC) for 3-5 hr. The mixture then acidified with conc. HCl (1.5 ml) up to pH ≈ 3. The precipitated compound separated and washed with H₂O. The desired product dried and recrystallized with glacial acetic acid.^10,11 Figure (2) show synthesis of azo compounds. The melting points, names and the percentage of yield are given in Table (1).

Figure 2: Synthesis of azo compounds. Click here to view figure

 

Table 1: Dames and physical properties of synthesized azo compounds.

Comp.	X	X/	Name	M. p. (°C)	Appearance	Yield (%)
A	3-CH3	4-NO2	2-Hydroxy-3-methyl-5-(4-nitro-phenylazo)-benzoic acid	273-275 dec.	Yellow	77
B	3-CH3	3-NO2	2-Hydroxy-3-methyl-5-(3-nitro-phenylazo)-benzoic acid	243-245	Light yellow	70
C	3-CH3	4-COOH	5-(4-Carboxy-phenylazo)-2-hydroxy-3-methyl-benzoic acid	265-267 dec.	Red	83
D	3-OCH3	4-NO2	2-Hydroxy-3-methoxy-5-(4-nitro-phenylazo)-benzoic acid	245-247 dec.	Deep red	78
E	3-OCH3	3-NO2	2-Hydroxy-3-methoxy-5-(3-nitro-phenylazo)-benzoic acid	236-238	Yellow	89
F	3-OCH3	4-COOH	5-(4-Carboxy-phenylazo)-2-hydroxy-3-methoxy-benzoic acid	253-255 dec.	Brown	80
G	4-CH3	4-NO2	2-Hydroxy-4-methyl-5-(4-nitro-phenylazo)-benzoic acid	235.238 dec.	Orange	91
H	4-CH3	3-NO2	2-Hydroxy-4-methyl-5-(3-nitro-phenylazo)-benzoic acid	240-242 dec.	Deep yellow	79
I	4-CH3	4-COOH	5-(4-Carboxy-phenylazo)-2-hydroxy-4-methyl-benzoic acid	248-250 dec.	Deep red	88

 

In Vitro Anti-Inflammatory Activity¹²

HRBC method was used to estimation in vitro anti-inflammatory activity. Blood was collected from healthy volunteers; the blood was mixed with equal volume of sterilized Alsever’s solution. The blood solution centrifuged at 3000 rpm and the packed cells were separate. The packed cells were washed with isosaline solution and 10% v/v suspension was prepared by complete the volume with isosaline. Alsever’s solution were prepared of 2.05% glucose, 0.42% NaCl, 0.8% trisodium citrate, 0.055% citric acid, all dissolved in water. This solution was using for storage RBC. Other solution were using in this method Hyposaline (0.7% NaCl), Isosaline (0.9% NaCl), phosphate buffer (pH 7.4) and ethanol.

Tested compounds (75 mg) was dissolve in 1ml of ethanol. Samples of each compound, control and sodium diclofenac were separately mixed with (1ml) phosphate buffer, (2ml) of hyposaline and (0.5ml ) of HRBC suspension. All the assay mixtures were incubate at 36.5ºC for 30 minutes and centrifuged at 3000 rpm for 10 minutes. The supernatant liquid was decanted and haemoglobin content was estimate by spectrophotometer at 560 nm. The percentage of haemolysis protection was estimate by assuming the haemolysis produced in the control as 100%, according to following equation.

Percentage protection=100-(Ac-As/Ac)

Were Ac= Absorption of control and As= Absorption of sample

Statistical Analysis

The results of anti-inflammatory activity were analysis in one way analysis of variance (ANOVA). Value with probability (p<0.01) was considered significant.

Results and Discussion

Table (2) show CHN analysis of synthesized azo compounds (A-I) and the practical results support the structure of synthesized compounds.

Table 2: CHN analysis of azo compounds.

Compd.	Mol. formula	Mol. weight	Elemental analysis
			C%		H%		N%
			Cal.	Found	Cal.	Found	Cal.	Found
A	C14H11N3O5	301.25	55.82	55.53	3.68	3.90	13.95	13.57
B	C14H11N3O5	301.25	55.82	55.90	3.68	3.63	13.95	14.20
C	C15H12N2O5	300.27	60.00	60.17	4.03	4.14	9.33	9.62
D	C14H11N3O6	317.25	53.00	52.81	3.49	3.57	13.24	13.35
E	C14H11N3O6	317.25	53.00	52.75	3.49	3.41	13.24	12.51
F	C15H12N2O6	316.27	56.96	57.13	3.82	4.02	8.86	9.10
G	C14H11N3O5	301.25	55.82	55.61	3.68	3.45	13.95	13.68
H	C14H11N3O5	301.25	55.82	56.03	3.68	3.51	13.95	13.73
I	C15H12N2O5	300.27	60.00	59.83	4.03	3.85	9.33	9.49

 

FT-IR Spectra

The FT-IR spectra (KBr disc) of prepared compound show a strong absorption at (1654-1693 cm^-1) for carbonyl carboxylic group.^1,13 All compounds exhibit absorption bands at ranges (1604-1616 cm^-1), (1523-1577cm^-1) and (1438-1477 cm^-1) for the stretching vibrations of -N=N- and C=C groups respectively because are superimposed in the same ranges.^1,13 The spectra of the azo compounds show strong absorption bands at ranges (1273-1288 cm^-1), (1195-1222 cm^-1), (1249-1265 cm^-1) and (1300-1354 cm^-1) due to the stretching vibrations for (C-O, carboxylic), (C-O, phenolic), (C-N) and (NO₂) respectively.^1,13,14 The other FT-IR vibrations of the synthesized compounds are shown in Table (3).

¹H-NMR Spectra

The proton-NMR analysis of prepared compounds performed by using deuterated dimethyl sulfoxide as a solvent. The ¹H-NMR spectra of all prepared azo compounds showed a singlet signal within the ranges (2.264-2.296 ppm), (3.918-3.931 ppm) and (2.681-2.725 ppm) for the groups 3-CH₃, 3-OCH₃ and 4-CH₃ respectively. Compounds A, B and C showed a singlet signals at (7.785-8.042 ppm) and (8.132-8.507 ppm) which attributed to protons H-4 and H-6 respectively. The ¹H-NMR spectra of the synthesized compounds for compounds D, E and F showed singlet signals at ranges (7.675-7.732 ppm) and (8.050-8.444 ppm) for protons H-4 and H-6 respectively with ⁴J= 2.1. In addition to that compounds G, H and I appeared singlet signals at (7.013-7.072 ppm). Proton (H-6) for compounds G and H appeared at 8.171 ppm and 8.175 ppm respectively,^15-17 the general structure of azo compounds was shown in Figure (3). Other aromatic protons of all azo synthesized compounds summarized in Table (4). The data of ¹H-NMR spectra of synthesized compounds reported in Figures (4-12).

Figure 3: General structure of azo compounds.  Click here to view figure

 

Figure 4: 1H-NMR spectrum of azo compound A. Click here to view figure

Figure 5: 1H-NMR spectrum of azo compound B. Click here to view figure

Figure 6: 1H-NMR spectrum of azo compound C. Click here to view figure

Figure 7: 1H-NMR spectrum of azo compound D.  Click here to view figure

Figure 8: 1H-NMR of azo compound E. Click here to view figure

Figure 9: 1H-NMR spectrum of azo compound F. Click here to view figure

Figure 10: 1H-NMR of azo compound G. Click here to view figure

Figure 11: 1H-NMR of azo compound H. Click here to view figure

Figure 12: 1H-NMR of azo compound I. Click here to view figure

 

Table 3: Data of FT-IR spectra (cm^-1) of synthesized azo compounds.

Compd.	C=O	N=N C=C	C=C	C-O carboxylic str.	C-O phenolic str	C-N str.	C-H Aromatic		NO2 (Str.) sym.	O-H Carboxylic	O-H phenolic	C-H aliphatic str.
							str,	bend.
A	1654	1527 1438	1608	1276	1222	1257	3039 3078	852 910	1346	2519 2600	3417 3587	2854 2989
B	1658	1531 1446	1612	1284	1222	1257	3028 3082	848 910	1354	2525 2603	3417 3475	2870 2955
C	1654 1685	1577 1446	1604	1288	1222	1265	3066 3160	867 906	1300	2596 2661	3414 3502	2866 2989
D	1678	1580 1458	1609	1261	1230	1261	3082	887 914	1342	2645	3313	2997
E	1667	1585 1469	1613	1285	1199	1267	3093 3194	879 898	1350	2603 2754	3421 3522	2870 2993
F	1658 1680	1523 1454	1608	1276	1223	1277	3035 3100	860 910	1320	2620 2730	3522 3595	2854 2962
G	1666	1527 1442	1608	1273	1219	1253	3074 3160	794 850	1342	3074 3159	3479 3552	2858 2927
H	1685	1570 1477	1616	1276	1211	1249	3101	864 894	1346	2640	3414 3482	2840 2927
I	1670 1693	1581 1485	1604	1284	1195	1250	3090	860 910	1342	2540 2700	3479 3552	2860 2910

 

Str. = stretching, bend. = bending, sym. = symmetrical.

Table 4: Data 1H-NMR for compounds A to I in DMSO-d₆.

1H-NMR, δ (ppm), nJ H-H (Hz)
Comp.	X’	C-H (aliphatic)	(C-H) aromatic
A	3-CH3	2.264 (3H)	7.785(H-4), 8.232(H-6), 7.952, (d, H-8, H-12, 3J8,9,3J12,10= 8.7), 8.371(d, H-9, H-11, 3J9,8= 3J11,12= 8.7)
B	3-CH3	2.296 (3H)	8.042(H-4), 8.507(H-6), 7.876(t, H-9, 3J9,8= 3J9,10= 8.1), 8.266-8.373(m, H-8,H-10,H-12)
C	3-CH3	2.294 (3H)	8.005(H-4), 8.132(H-6), 7.919(d, H-8, H-12, 3J8,9=J12,11= 8.4), 8.118(d, H-9, H-11, 3J9,8= 3J11,12= 8.4)
D	3-OCH3	3.918 (3H)	7.675(d,H-4, 4J= 2.1), 8.063(d, H-6, 4J= 2.1), 8.045(d, H-8, H-12, 3J8,9= 3J12,11= 9), 8.409(d, H-9, H-11, 3J9,8= 3J11,12= 9)
E	3-OCH3	3.931 (3H)	7.732(d,H-4, 4J= 2.1), 8.444(d,H-6, 4J= 2.1), 7.890 (t, 3J9,8= 3J9,10= 8.1), 8324-8.392(m, H-8, H-10), 8.650(d, H-12, 4J= 1.8)
F	3-OCH3	3.923 (3H)	7.690(d, H-4, 4J= 2.1), 8.050(d, H-6, 4J=2.1), 7.952(d, H-8, H-12, 3J8,9= 3J12,11= 8.7), 8.130(d, H-9, H-11, 3J9,8= 3J11,12= 8.7)
G	4-CH3	2.725 (3H)	7.072(H-3), 8.171(H-6), 8.066(d, H-8, H-12, 3J8,9= 3J12,11= 9), 8.412(d, H-9, H-11, 3J9,8= 3J11,12= 9)
H	4-CH3	2.725 (3H)	7.066(H-3), 8.175(H-6), 7.880(t, H-9, 3J9,8=3J9,10= 8.1) 8.322-8.381(m, H-8, H-10), 8.532(H-12)
I	4-CH3	2.681 (3H)	7.013(H-3), 7.913(d, H-8, H-12, 3J8,9, 3J12,11=7.5) 8.106 (3H, H-6, H-9, H-11)

 

In vitro anti-inflammatory activity

In inflammation, there is extra cellular fluid release because cell damage, lysosome preventing the inflammation response if lysosomal membrane still stable. Lysosomal contain active enzyme like bactericidal enzyme and proteases, this cause additional tissue injury. So stability of lysosomal membrane very important for inflammatory prevention. The HRBCs membrane are used as analogous to lysosomal membrane, because they have similar components, a protection of HBRCs membrane of lysis were rated as anti-inflammatory activity.¹⁸

All the synthesized azo compounds (A-I) showed a significant anti-inflammatory activity by HRBC membrane stabilization method. The results of the activity                    are listed in Table (5).

Table 5: Protection of HRBC membrane of compounds at dose 75 mg.

The compound	Protection %
A	20.16
B	69.53*
C	31.86
D	28.30
E	71.87*
F	25.15
G	23.95
H	79.76*
I	30.43
Sodium diclofenac	74.48*

 

* = p< 0.01

From the results, compounds B, E and H show high activity compared with other compounds. The active compounds B, E, and H contain a nitro group at meta position. Replacing nitro group with carboxyl group will be minimize the activity. Therefore, the activity is the best when a nitro group located at meta position. In salicylic acid moiety, the activity of protection was found increased when methyl group located at para– position compared with the meta-methyl and meta-methoxy groups. Compound E contain meta nitro group and meta methoxy group, it is the compound with meta-methoxy group has high activity than other methoxy derivatives. The results showed compounds A, D, F and I have less activity. The action of the azo compounds could be related to the binding of compounds with erythrocyte membrane, especially phospholipids. Since the compounds has polar and nonpolar groups can bonded with same groups of phospholipids. This prevents membrane damage by physical interaction of osmotic pressure differences, which is causative to hemolysis of red blood cells.¹⁹

Conclusion

In this study, three series of azo were prepared using three different of salicylic acid derivatives. The prepared compounds identified using the element analysis (CHN) as well as infrared and ¹H-NMR spectroscopy. The results supported the structures of prepared compounds. All compounds showed significant in vitro anti-inflammatory activity. Compounds B, E and H showed high anti-inflammatory activity compared with other prepared azo compounds.

Conflict of Interest

There is no conflict of interest.

References

1. Potey, L. C.; Urade, P.; Aate J.; Kosalge, S., Int. J. ChemTech. Res. 2017, 10, 552-556.
2. Otutu, J. O.; Okoro, D.; Ossai, E. K.; J. App. Sci. 2008, 8, 334-339.
3. Patil,C. J.; Nehete, C. A.; Int. J. Pharm. Sci. Rev. Res. 2015, 33, 248-256.
4. Swati, Ginni, Karnawat, R.; Sharma, I. K.; Verma, P. S.; Int. J. of Applied Biology and Pharmaceutical Technology, 2011, 2, 332-338.
5. Khalid, A.; Arshad, M.; Crowley, D. E.; Appl. Microbiol Biotechnol 2008,78, 361-369. DOI: 10.1007/s00253007-1302-4.
6. Farghaly, T. A.; Abdallah, Z. A.; ARKIVOC 2008, (xvii), 295-305. http://dx.doi.org/10.3998/ark.5550190.0009.h28.
7. Dardeer, H. M.; Elboray, E. E.; Mohamed, G. S.; Polycyclic Aromatic Compounds 2018, 1-11. https://doi.org/10.1080/10406638.2018.1466812
8. Fadey, O. O.; Obafemi, C. A.; Adewunmi, C. O.; lwalewa, E. O.; African Journal of Biotechnology 2004, 3, 426-431.
9. Neha, Patni, M.; Der Chemic Scienca 2016, 7, 93.
10. Ahmadi R. A.; Amani, S.; Molecules 2012, 17, 64346-448. Doi: 10.3390/molecules17066434
11. Koshti, S. M.; Sonar, J. P.; Sonawane, A. E.; Pawar, Y. A.; Nagle, P. S.; Mahulikar, P. P.; More, D. H.; Indian J. Chem. 2008, 47B, 329-331.
12. Solomon, S.; Senthamilselvi, M. M.; Muruganantham, N.; J. of Bioscience and Applied Research 2016, 2, 621-625.
13. Sahoo, J.; Paidesetty S. K.; Egypt. J. of basic and applied sciences 2015, 2, 268-280. http://dx.doi.org/10.1016/j.ejbas.2015.07.006.
14. Rathod, K. M.; Thakre, N. S.; Chem Sci Trans. 2013, 2, 25-28.
15. Zhao, Z. B.; Zheng, H. X.; Wei, Y. G.; Liu, J.; Chinese Chemical Letter 2007,18, 639-642.
16. da Silva, M.; Menezes, C. M. S.; Ferreira, E. L.; Leite, C. Q. F.; Sato, N.; Pimenta, C. P.; Botelho, K. C. A.; Chem. Biol. Drug Des. 2008, 71, 167-172.
17. A Jilani, J.; shomaf, M.; alzoubi, K. H., Drug Design, Development and Therapy, 2013, 7, 691-698.
18. De, P.; Sarkar, S.; Mukhophadhyay, M. J.; J. of Pharmacognosy and Phytochemistry 2017, 6, 103-105.
19. Oyedapo, O. O.; Akinpelu, B. A.; Akinwunmi, K. F.; Adeyinka, M. O.; and Sipeolu, F. O.; Inter. J. Plant Physiology and Biochemistry 2010, 2, 46-51.

---

This work is licensed under a Creative Commons Attribution 4.0 International License.