Synthesis and pharmacological activities of azo dye derivatives incorporating heterocyclic scaffolds: a review

Nowadays, there is significant interest in the synthesis of heterocycle-incorporated azo dye derivatives as potential scaffolds in the pharmaceutical sector. The pharmaceutical or drug industries need a simplistic synthesis approach that can afford a wide range of azo dye derivatives. The incorporation of the heterocyclic moiety into the azo dye scaffold has improved the bioactive properties of the target derivatives. The various biological and pharmacological applications of drugs such as anti-fungal, anti-tuberculosis, anti-viral, anti-inflammatory, anti-cancer, anti-bacterial, DNA binding, and analgesic properties can be easily tuned by introducing heterocyclic moieties. To date, continuous efforts are being made in the search for more potent, new, and safe synthetic methodologies for azo dye derivatives. This review presents a brief discussion of the facile synthetic approaches and the relevance of the title compound and its derivatives towards various biological activities. Thus, the synthesis of azo dye derivatives incorporating heterocyclic scaffolds such as imidazole, pyrazole, thiazole, oxazolone, thiophene, pyrrole, benzothiazole and quinoline moieties and their pharmacological applications are discussed briefly.


Introduction
Azo dyes are among the most signicant classes of chromophores with diverse applications in the scientic, industrial, and pharmaceutical sectors. Researchers have explored simple and easy synthesis approaches to azo dyes and their derivatives having various potential applications. 1,2 Azo chromophores are a group of colorant organic materials characterized by the presence of azo groups in the main skeleton structure. There could be two azo groups (dis-azo), for instance, 6-hydroxy-1,4dimethyl-2-oxo-5-((4-(phenyldiazenyl)phenyl)diazenyl)-1,2dihydropyridine-3-carbonitrile (2) has two basic azo skeletons, three groups (tris-azo), four groups (tetrakis-azo), or more (polyazo) in rare cases, see Fig. 1. 3,4 In addition to their use as colorants in over 50% of all commercial dyes, they have been employed in many applications, such as in inkjet printing, thermal transfer printing, photography, color additives, the biomedical area, molecular recognition, light-controlled polymers, and in the liquid crystal industry. 4 Azo dyes are generally characterized by their nitrogennitrogen double bond (-N]N-) and this structure affords various properties in the textile industries. 4 In this sense, it is essential for azo dyes to have heterocyclic compounds containing nitrogen, oxygen or sulfur to enhance the color of the dye, leading to different shades with different intensities. Nowadays, azo dyes incorporating heterocyclic moieties exhibit enhanced coloring properties, tinctorial strength, thermal stability, and more positive solvatochromic behavior than the dyes derived from a simple aromatic amine. [5][6][7] To date, several synthetic approaches have been developed and reported for the preparation of heterocycle-incorporated azo dyes and their derivatives. The conventional synthesis procedure for the title compound is through diazonium salt coupled with one or more electron-rich nucleophile segments. 8 In the diazotization procedure, the aromatic or heterocyclic amine is initially converted into a diazonium salt. 9 The standard reaction of the diazotization reaction occurs at a low temperature in the presence of salts and acid, followed by the resulting diazonium complex interacting with various diazo coupling nucleophile components such as phenol, naphthol, or amine. 10 Although heterocycle-containing azo dye derivatives broadly contribute to pharmaceuticals and drug development, the reports are still not sufficient. 4,11 Nowadays, the synthesis of heterocycle-containing azo dyes and their derivatives has gained particular attention due to their potent bioactivities such as antimicrobial, antifungal, antiviral, anticonvulsant, antidiabetic, anti-inammatory, antitubercular, anticancer DNA binding, analgesic properties, and chemosensing activities. 12,13 Herein, we provide a brief highlight of the synthesis of various heterocycle-containing azo dyes and their derivatives with their potential pharmaceutical activities (Fig. 2).

Synthesis of heterocyclecontaining azo dyes and their derivatives
Nowadays, scholars have given much attention to the suitable design and preparation of the title compounds and their derivatives. So far, various analogs of heterocycle-containing azo dyes and their derivatives have been synthesized and reported via different methodologies. This section mainly focuses on the standard and conventional synthesis methodologies of azo dyes containing various heterocyclic moieties.

Azo dyes containing thiophene and its derivatives
Wei and coworkers reported the pH-induced azo-keto and azoenol tautomerism for 6-(3-methoxypropylamino)pyridin-2-one -based thiophene azo dye derivatives. 14 By linking other functional groups on the azo dye scaffold with the post-modication strategy, the bi-heterocyclic azo dyes 12 were prepared. The diazotization reaction takes place on 3-cyano-4-chloro-5formylthiophene 7, and 3-methoxypropylamino-substituted pyridine derivatives 9 are used as coupling components to produce 10. The aldehyde of the formylthiophene moiety 10 further reacted with aniline 11 to afford the Schiff base-azo dye 12 with thiophene as a bridge, as described in Scheme 1. Here, the azo dye has a stable pH regardless of the diazo components used since no proton-accepting sites could be found in the pyridine ring; both carbonyl groups are simultaneously replaced by 3-methoxypropan-1-amine.
By modifying the terminal aldehyde radical into an imine version, 2-amino-3-cyano-4-chloro-5 formylthiophene provided the basis for blue-colored heterocyclic azo dyes 15 with an enhanced p-conjugated system, solubility, and electronic spectrum properties of the synthesized compounds. 15 Azoazomethine compounds 15 were prepared through a Schiffbase condensation between 2,3-dimethylaniline 14 and the formylthiophene unit of azo dye 13 with various derivatives of aniline-coupling components. The general synthesis route of the dyes is shown in Scheme 2.

Azo dyes containing pyrrole and its derivatives
Through the diazo coupling reaction scaffold, Maruszewska and Podsiadly synthesized and report novel azo dye pyrrole derivatives 20 containing the azo-1H-pyrrole moiety. 16 During the synthesis of azo dye derivative 20, rst aniline, 4-aminobenzoic acid, N,N-diethyl-p-phenylenediamine, N-ethyl-N-2-hydroxyethyl-p-phenylenediamine, and 5-aminoisophthalic acid, respectively, reacted with sodium nitrite/aqueous HCl at 0-5 C  to afford the substituted diazonium salt, and the resulting salt reacted with 1-H-pyrrole-2-carbaldehyde 17 in ethanol neutralised with pyridine to produce 18. Finally, compounds 18 condensed with the appropriate aromatic amines 19 in ethanol to give dyes 20 (a-g) as described in Scheme 3. In these dyes, the electron-rich 1H-pyrrole moiety was assembled as p-bridges in the donor-acceptor p-conjugated dye, and aminophenylimine fragments and the carboxyl group were used as donor and anchoring acceptor groups, respectively.
Almeida et al. have also synthesized and reported pyrrole azo dye derivative 23 bearing 2-(4-dimethylaminophenylazo) benzoic acid 22, also known as Methyl Red (MR). 17 The monomer 3- (N-pyrrolyl)propyl-2-(4-dimethylaminophenylazo) benzoate (MRPy) 23 was obtained through a simple synthetic route, in mild conditions and with a good yield from 1-(3-iodopropyl) pyrrole and methyl red in the presence of triethylamine, which was added to dry CH 3 CN. The reaction mixture was stirred at 80 C for 3 h, extracted with H 2 O/CH 3 Cl (1 : 1, v/v), and the crude product was puried aer evaporation to give the nal product, as shown in Scheme 4. The formation of MRPy was successfully achieved with the addition of boron triuoride diethyl etherate (BFEE) to electrodes in (C 4 H 9 ) 4 NBF 4 /CH 3 CN in the electrolyte system.

Azo dyes containing imidazole and its derivatives
New red azo dyes containing imidazole derivative 27 were reported from previous works through the diazo-coupling reaction. The compound 27 was synthesized from the imidazole derivative 24 and passed through the diazotization step in the presence of HCl and NaNO 2 to obtain the corresponding diazonium salt. The salt was subjected to coupling with N-benzyl-N-ethyl-m-acetamide aniline 25 to afford compound 26 in good yield. 18 Compound 26 undergoes the methylation reaction through an alkylating agent to methylate the imidazole ring 27 as described in Scheme 5.
Similarly, through a convenient one-pot three-component synthesis methodology, new azo-imidazole derivatives 33 (a-h) were reported by Mahmoodi et al., 19 with moderate to excellent yields from the corresponding azo dyes 30, ammonium acetate 31, and benzyl 32 under microwave irradiation in the presence of glacial AcOH as the solvent and organocatalyst in short reaction times. Glacial AcOH is mainly used to activate and enhance the nucleophilic attack of the carbonyl group by ammonia to afford the compound 33. Aniline derivatives 28 were diazotized in the presence of NaNO 2 and HCl at 0-5 C and then coupled with the aldehyde derivatives 29 to give the precursor azo dyes 30. The resulting azo dyes 30 were subjected to ammonium acetate 31, followed by a condensation reaction with the benzyl 32, which in turn rearranged to the azoimidazole 33, as outlined in Scheme 6.

Azo dyes containing pyrazole and its derivatives
Azo dyes derived from the pyrazole and pyrazolone derivatives have potential broad spectrum biological properties such as antibacterial, anti-cancer and antimicrobial activities, and they are used in the pharmaceutical sector. 20 Demircali et al. reported the synthesis of ve new azo dyes 41 containing pyrazole derivatives, which were derived from 5-amino-4-arylazo-3-methyl-1H-pyrazoles 38, through diazotization followed by a coupling reaction in the presence of hydrazine monohydrate 37; the general route for the synthesis of the dyes is depicted in Scheme 9. 22 Aniline derivatives 34 were diazotized in the presence of NaNO 2 /HCl followed by coupling with 3-aminocrotononitrile 35 to 2arylhydrazo-3-ketiminobutyronitriles 36, as outlined in Scheme 7. 2-Arylhydrazo-3-ketiminobutyronitriles 36 were reacted with hydrazine monohydrate 37 to give 5-amino-4-arylazo-3-methyl-Scheme 4 General synthetic route towards azo dye derivatives containing pyrrole 23.
Scheme 5 General synthesis route towards azo dye derivatives containing imidazole 27.
1H-pyrazoles 38 (Scheme 8). The antibacterial activities of these dyes were evaluated against various pathogenic bacteria and exhibited good-to-excellent activities against the selected strain. 21,22 The electron-withdrawing groups in the p-position resulted in the azo dye becoming more toxic, while the substitution of the electron-donating groups caused the dye to be less toxic. 22 A new series of dispersed disazo dyes 51 containing pyrazole and isoxazole groups were synthesized by a series of synthesis processes. 23 For these ten newly synthesized disazo-dispersed dyes, one was without auxochrome groups and nine had -NO 2 , -Cl, -CH 3 auxochromes on para, meta and ortho positions. First, the different aniline derivatives 42 were diazotized and coupled with 3aminocrotononitrile 43 and coupled to result in the corresponding 2-arylhydrazono-3-ketiminobutyronitriles 44. Aer the cyclization process of 2-arylhydrazono-3-ketiminobutyronitriles 44 with hydrazine monohydrate 45, 5-amino-4-arylazo-3-methyl-1H-pyrazoles 46 were diazotised and coupled with ethyl acetoacetate and Scheme 6 General synthesis route towards azo-imidazole derivatives.

Azo dyes containing thiazole and its derivatives
According to the reports, azo dyes containing thiazole have fascinated researchers because of their broad range of pharmacological activities, such as anti-infectious, 24 antioxidant, 25 anticancer, 26 antibacterial, and antifungal. 27 There have been reports on the synthesis of azo dyes containing the thiazole ring using various methods. Keshavayya et al. synthesized three potent anticancer active azo dyes possessing the 2-aminothiazole moiety via a simple, effective, economic, and conventional diazo-coupling reaction. 28 During the reaction, 1,3thiazole-2-amine 52 in an acid mixture was reacted with nitrosyl sulphuric acid at 0-5 C to form the diazonium salt. Azo dye 54 (a & b) was formed when the diazonium salt solution was added to the well-cooled solution of coupling components 53 (a & b) in an aqueous KOH solution. The synthetic route for the preparation of azo dyes is represented in Scheme 11.
Similarly, Keshavayya et al. reported the synthesis of four new biologically active azo dyes 57 containing thiazole, which were derived from 2-amino-5-nitrothiazole 55 by the conventional diazo-coupling method in an acid condition. 29 2-Amino-5nitrothiazole 55 was diazotized in the presence of sodium nitrite in sulphuric acid and rapidly cooled in an ice bath at 0-5 C for 10 min. Cold diazonium salt solution was added dropwise with vigorous stirring to the coupling compounds 56 (a-c), which were dissolved in acetic acid, and then the whole reaction mixture was stirred at 0-5 C for 1 h to give the nal azo dyes 57 (a-d), as described in Scheme 12. All the prepared azo dyes revealed promising growth inhibitory effects against selected antibacterial strains and also showed potential antioxidant properties.

Azo dyes containing oxazolone and its derivatives
According to Albelwi et al., novel azo dye-containing derivatives of the oxazolone compounds were obtained via the Erlenmeyer reaction of the azo dye precursors. 30 The new 4-arylidene-5-(4H)oxazolone azo chromophore 63 was produced by the condensation of 2-(4-(4-((2-hydroxyethyl)(methyl)amino)phenyl)diazenyl) acetic acid 61 with the corresponding benzaldehydes 62 in the presence of acetic anhydride and sodium acetate, as shown in Scheme 13. Compound 61 was formed from the diazotization of compound 58 followed by the coupling of compound 60. The formation of the unsaturated 5-[4H]oxazolone was elucidated via a two-step mechanism. The rst transformed from an orange semi-solid mass into a deep red liquid. Aer cooling, the precipitated product was ltered and recrystallized in toluene to obtain the nal azo dye product 69. All synthesized compounds exhibited high tyrosinase inhibitory behavior.

Azo dyes containing benzothiazole and its derivatives
Song and co-workers reported new bi-heterocyclic dyes 72, which contained N-ethyl-3-cyano-4-methyl-6-hydroxy-2-pyridine groups from substituted benzothiazoles. 32 Under vigorous mechanical stirring at 40 C, the substituted benzothiazole 70 was mixed with concentrated phosphoric acid, concentrated sulfuric acid, and glacial acetic acid to form the diazonium salt. When the diazonium salt was added dropwise to pyridone derivatives 71 under vigorous mechanical stirring, azo dye 72 was formed. The synthesis route of designed bi-heterocyclic disperse dyes is shown in Scheme 17.
Malayappa and coworkers reported the synthesis of four benzothiazole-based dispersed azo dyes 76 derived from 2-phenyl-2,4-dihydro-3H-pyrazole-3-one 75 by diazo coupling electrophilic substitution at 0-5 C. 33 2-Amino-6-substituted benzothiazoles 73 were dissolved in a mixture of glacial acetic acid and propionic acid (2 : 1), and this solution was added dropwise to a well-cooled solution of nitrosylsulfuric acid (NaNO 2 in H 2 SO 4 ) at 0-5 C to form diazonium salt 74. The resulting diazonium salt 74 coupled with 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one 75 in acetic acid at 0-5 C. The precipitated colored product was ltered off, washed several times with distilled water, dried and recrystallized from ethanol. The general route for the synthesis of benzothiazole-azo-pyrazolone dye 76 is outlined in Scheme 18. This compound was described in US patent document 2832761 (1958) for its application in the dyeing of various textile materials.

Biological activity of heterocyclecontaining azo dyes and their derivatives
A variety of biological and pharmacological applications were explored for azo dyes that contain heterocycles. 36,37 Heterocycles are important components of the azo dyes and play an important role in increasing their pharmacological and medicinal properties, such as antibacterial, 36,38 antioxidant, 39 anticancer and antitumor, 40 and anti-inammatory activities. 37

Antifungal activity
Recently, Matada and colleagues reported new S-heterocyclic azo dyes synthesized from 1,3-benzothiazole-2-thiol with various amines by the diazo-coupling method. 43 The azo molecules derived from benzothiazole were screened for their microbial inhibition by modied tube dilution assay against two fungal strains, C. albicans, and A. avus, and the results were correlated with uconazole. The antifungal activities of compounds 92, 93 and 94 (Fig. 4) showed promising results against C. albicans and A. avus. The presence of heterocyclic rings in their structures contributed to the enhancement of antifungal activity, as described in ref. 44-46. Mallikarjuna and Keshavayya synthesized and reported bright-colored heterocyclic azo dyes from sulfamethoxazole with various coupling compounds. 47 The antifungal activity of these target compounds was studied against A. avus and C.  albicans, with the reference drug uconazole, and synthesized azo dyes 95 and 96 (Fig. 4) were proven to have antifungal properties against two pathogenic strains, viz. A. avus and C. albicans. Further, these azo dyes have shown promising antibacterial, anti-mycobacterial, and anticancer activity, which indicate that the compounds are efficient in inhibiting multiple diseases.

Anti-tuberculosis activity
In recent years, tuberculosis has become one of the most dangerous infectious diseases and a leading cause of death worldwide, 48 and it is a challenge for researchers to design effective anti-TB drugs. An azo dye based on coumarinbenzothiazole was synthesized by Bodke and co-workers. 49 The effectiveness of the synthesized dyes was tested against Mycobacterium tuberculosis (H37 RV strain) and compared to the standard drugs using the microplate Alamar Blue Assay method. Among the azo dyes, 3-(6-chloro-benzothiazol-2-ylazo)-4-hydroxy-chromen-2-one (97) and 4-hydroxy-3-(6-nitrobenzothiazol-2-ylazo)-chromen-2-one (98) (Fig. 5) exhibited similar excellent sensitivities (MIC ¼ 1.6 mg mL À1 ) relative to the standard streptomycin (MIC ¼ 6.24 mg mL À1 ).
In a recent paper, Unnisa and co-workers reported and synthesized pyrimidine azo dyes by coupling phenylpyrimidine 2-amine with different aromatic amines. 52 The synthesized compounds were screened for their anti-inammatory activities through the heat-induced hemolysis method. Most of the synthesized compounds exhibited a membrane stabilization effect by inhibiting the lysis of the erythrocyte membrane. Compounds 105 and 106 (Fig. 7) showed maximum inhibitory activities of 71.08%, and 71.91%, respectively, which are closer to the standard aspirin (72.91%).

Antioxidant activity
A series of novel bioactive disperse dyes (Fig. 8) consisting of thiazolyl and piperazine moieties were reported by Mohammadi and co-workers via azo coupling reactions. 53 The antioxidant activities of the newly synthesized compounds were evaluated by FRAP. All of the compounds displayed signicant antioxidant activity. Among the tested dyes, azo dye 107 and 108 exhibit good radical scavenging activity. The activities of these compounds were attributed to the presence of thiazolyl derivatives and piperazine moieties as bioactive components in the structures of synthesized dyes. 53 Abu-Melha and co-workers reported the synthesis of novel bioactive thiazolyl-curcumin azo dyes in which curcumin was coupled with different aromatic diazonium salts of 2-amino thiazole derivatives, such as 2-aminobenzothiazole, 2-amino-5phenylthiazole, 2-amino-5-methylthiazole and 2-amino-5-nitrothiazole. 54 All synthesized compounds were tested, and their antioxidant activities reected the ability to inhibit oxidation. The antioxidant activities of the synthesized compounds were examined by ABTS inhibition, and compounds 109 and 110 showed higher antioxidant activity, comparable to ascorbic acid as a standard. Furthermore, the synthesized thiazolyl-curcumin derivatives exhibited promising antimicrobial, anticancer and antioxidant activities.

Antiviral activity
Yellow-colored heterocyclic azo dye derivatives (1Hbenzoimidazol-2-yl)-(4-ethyl-phenyl)-diazene (111) and (1H-benzoimidazol-2-yl)-o-tolyl-diazene (112), which are antiviral in nature, were synthesized and reported by Mohammad Ashfaq. 55 The compounds were tested in vivo against viruses in developing chick embryos. Labels were applied to nine-day-old embryonated chicken eggs based on the compound used. As a result of the hemagglutination test in the case of the anti-NDV potential of the compounds for 100% at 0.1 mg/100 ml, both compounds inhibited 50% of NDV and AIV (H9N2) viral growth (Fig. 9).

Conclusion
Azo dyes incorporating heterocyclic scaffolds generate the largest volume of dye production, and they are regularly used in the food, pharmaceutical, paper, cosmetics, textile, and leather industries, among others. Nowadays, researchers are exploring the biological activities of various azo dyes by incorporating heterocyclic components in the synthesis, and the resulting dyes have enhanced applications in a wide range of elds, especially pharmaceuticals. Previously, azo dyes were synthesized through diazotization but nowadays, researchers are synthesizing various azo dyes and incorporating heterocyclics through diazotization coupling reactions followed by post-modication methods, thus improving their biological and pharmaceutical activities. Due to the potential chemistry of azo dyes and their derivatives that incorporate heterocyclic scaffolds, there is much to contribute toward the discovery of new, potent and bioactive drugs with a broad spectrum of activities. Therefore, the synthesis of azo dyes with heterocyclic moieties requires further investigation to enhance the pharmacological activities, leading to the development of new drugs.

Conflicts of interest
There are no conicts to declare.