Iraqi J Pharm Sci, Vol.31( 2 ) 2022 Exploring Aspirin derivatives DOI: https://doi.org/10.31351/vol31iss2pp14-32 14 Aspirin Derivatives Exploration: A Review on Comparison Study with Parent Drug Azni Izwati Hamdan*, Dike Dandari Sukmana*and Norsyafikah Asyilla Nordin*,1 *Faculty of Pharmacy, University of Sultan Zainal Abidin, Besut Campus, 22200, Besut, Terengganu, Malaysia Abstract In recent decades, drug modification is no longer unusual in the pharmaceutical world as living things are evolving in response to environmental changes. Aspirin as one of the non-steroidal anti-inflammatory drugs (NSAID) is a common over-the-counter drug due to its analgesic, antipyretic and anti-inflammatory activity. This review article highlights on the recent derivatives of aspirin, which were developed either to reduce the risk of side effects or to obtain better physicochemical properties. Aspirin derivatives can be synthesized in various pathways and have been reported to give better biological activities compared to the parent drug. Nitric oxide (NO)-aspirin gives a potent anticancer drug as it able to inhibit lung and prostate cancer cells. Meanwhile NOSH- aspirin that release hydrogen sulphide (H2S) and NO moiety is a potent anti-inflammatory agent that stimulate the gastric and colonic secretion, prevent the penetration of acid in gastrointestinal. It also has anticancer action that is effective in hindering the proliferation of pancreatic and colon cancer cells. Aspirin-thiourea has been studied its antimicrobial activity. Still, it resulted in poor inhibition due to steric hindrance of the compounds and influence its penetration into the enzyme’s active site. However, aspirin-amide has managed to inhibit the bacterial and fungal, and compound with halogen substituents is reported with the highest inhibition. Aspirin derivative linked with chalcone has poor antibacterial and antioxidant due bulky structure of the compounds, but it has a superior anticancer that induce cancer cells apoptosis by reactive oxygen species (ROS) treatment. The modification of azo-aspirin has more potential in antibacterial activity compared to ampicillin especially when the presence of halogens substituents is involved. Overall, these aspirin derivatives are safe to be considered as potential pharmaceutical agents. Keywords: Aspirin, Aspirin derivatives, Biological activities, Chemical modification, NSAIDs Introduction Drug modification is vital in drug discovery and development process as it is usually done by altering the molecular structure of the formerly characterized lead compound to improve drug potential for treatment of diseases. Some of the chemical alterations are done either by the specification of a particular body target site, modification of time course in the body, or by increasing the rate and degree of absorption. It is also able to improve the potency of drugs, provide the desired feature by decreasing the toxicity or changing the physical as well as chemical properties. Aspirin is one of the most common over- the-counter drugs, which has been widely known as a fever reducer and anti-inflammatory drug for years. At low dose (75-100 mg), aspirin is selective to inhibit COX-1 activity. As a result of that interaction, aspirin can promote antithrombotic purpose and suppressing platelet aggregation without damaging vascular endothelial cell function which express cyclooxygenase enzyme(1). Thus, it can be used prophylactically in patient with heart attack, high cardiovascular risk, and stroke by taking it in daily low dosage(2–4). However, prolonged use of the aspirin may result in vomiting, major gastrointestinal (GI) bleeding (5), and other side effects such as hypertension, renal or GI toxicity due to dosage-related(3). Therefore, aspirin derivatives are being explored in order to get better biological activity. The presence of significant moieties such as nitric oxide (NO), NOSH, thiourea, azo, amide, and chalcone on the modified aspirin plays an important role in achieving desired biological activities. The addition of the halogen in the modification has also become a preference among researchers as it also affects the actions due to its ability to hinder bacterial activity (6,7).. Aspirin Aspirin or acetylsalicylic acid (Fig. 1) is an NSAIDS approved by the FDA to be used as antipyretic, antiplatelet, and analgesic agents(8,9). Aspirin can inhibit the synthesis of prostaglandin by blocking the cyclooxygenase (COX) which contributes to its properties such as anti- inflammatory, antipyretic, antiplatelet, etc. Aspirin is also being considered as a chemopreventive agent because of its antithrombotic effects through the COX’s inhibition, (10,11) and its antioxidant action that inhibit the cancer cells growth by donating their electron to the free radicals that cause proliferation, induce apoptosis or necrosis to the cells(12,13). However, the prolonged use of aspirin can cause heartburn, ulceration, and gastro-toxicity in children and adults. 1Corresponding author E-mail: azni64@gmail.com Received: 17/10 /2021 Accepted:23 /1 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol31iss2pp14-32 Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 15 It contains an aromatic ring with carboxyl functional groups. Carboxyl group plays many important roles in pharmaceuticals like acting as solubilizer or cell permeation for antibiotic or antihistaminic drug class, prodrug and/or bio- precursor that activated at specific conditions to act as an antihypertensive, antithrombotic or antiviral(14). Aspirin is prepared by reacting acetic anhydride and salicylic acid in the presence of acid catalyst (H2SO4/H3PO4) (Scheme 1). The hydroxyl group of salicylic acid is converted to an ester, with acetic acid as a byproduct(15). Figure1. Structure of Aspirin Scheme 1. Synthesis of aspirin In the body, aspirin mainly absorbed in the stomach and upper part of small intestine after oral administration. It reacts with water in the plasma, liver and within the cells, by esterases to give salicylic acid and acetic acid (Scheme 2) (16,17). The plasma half-life of salicylic acid is 15-20 min. In the liver, most of salicylic acid is metabolized into salicyluric acid, salicylic acid and phenolic glucuronides, and a small part of it is metabolized into genistic acid. These metabolites are mainly discharge by the kidneys to the urine(18). Scheme 2. Hydrolysis of aspirin However, aspirin lead to GI side effects by reducing mucosal prostaglandin synthesis that affects leukocyte adherence and decrease in bicarbonate, mucus secretion, and blood flow(19). Mucus is mainly secreted from the surface epithelial cell and foveolar cells. The mucus bicarbonate is being used to regulate the pH gradient in the GI tract(20). It protects the stomach from a highly acidic environment. Aspirin will inhibit the synthesis of the prostaglandins and decrease gastric mucus secretion. Thus, mucosal blood flow not maintained effectively, stomach epithelium can be damaged as mucus layer is disrupted(21). Figure. 2. Mechanism of action of aspirin Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 16 Aspirin is an irreversible-COX inhibitor that causes the inhibition of prostaglandin synthesis(22). Aspirin can inhibit both COX-1 and COX-2 (Figure. 2). As it is being administered, the aspirin transfers its acetyl group to a serine residue in the cyclooxygenase (COX) active site, making it unable for arachidonic acid to becoming prostaglandin H2, resulting in cyclic prostanoid (beckoning the molecules to mediate inflammation and other immune response) not to be synthesized(23). The pharmacological activity of aspirin is proven to be antiplatelet by inhibiting thromboxane A2 and anti-inflammatory by preventing prostaglandin I2, E2, D2, and F2a. Turning off the COX-1 enzyme can upset the stomach and cause ulcers or GI bleeding. Aspirin bearing Nitric Oxide Moiety (NO) NO-aspirin (Fig. 3) is one of gaseous mediator prodrug that is synthesized to improve the efficacy of parent aspirin, and to decrease the side effect that is associated with GI bleeding or ulcer(10). NO is needed to regulate the physiological pathways, particularly regarding the homeostasis of the GI tract. It is usually formed in esophageal, gastric, and intestinal mucosa via the enzymatic activity of NO synthases; neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) (24). Figure 3. Nitric oxide aspirin (NO-Aspirin) In various clinical conditions, NO-aspirin is a potential therapeutic agent and typically synthesized by esterification of a NO-releasing moiety to the NSAIDs (25). In addition, it has related parts in cancer biology, such as anti-inflammatory and anti-tumor properties, mainly exerted by NO- activated apoptotic pathways(26). The summary of the synthesis of NO- aspirin can be seen in Scheme 3. The halide from salicylic acid derivative react with hydroxybenzylalcohol in the presence of base, giving 2-(hydroxymethyl) phenyl 2- acetoxybenzoate continue reaction with nitric acid in organic acid. It is recrystallized using selected solvent to form final product, NO-aspirin(27). Scheme 3. Synthesis of NO-aspirin NO-Aspirin hybrids as a promising anticancer activity One of the research reviews has indicated that the biological analysis by using NO-aspirin derivative (Figure. 3) was associated with reduced GI risk and could be consider as a potentially an anticancer agent (28). NO-aspirin exhibited lower IC50 value (1 µM) in comparison to aspirin alone (>1000 µM). It was reported that proliferating cell nuclear antigen expression was reduced to 54.5%; meanwhile, at G0/G1 phases, over 83.9% of tumor cells were blocked after being treated with NO-aspirin (29). NO has dual role in anticancer activity depending on the type of cancer, the tumor microenvironment, the type of NO synthase and the concentration of NO itself. A low-rate NO donor will end up with tumorigenesis, whereas a high-rate NO can cause death to cancer cells (29). As an anticancer agent, NO has ability to sequester iron into iron-nitrosyl complexes, resulting in a loss of intracellular iron and the inhibition of mitochondrial respiration and DNA synthesis in the tumor cells. Meanwhile, aspirin is also known for its ability to bring cell cycle arrest, apoptosis, and lead to cell proliferation suppression(30,31). It can be concluded that the hybrid of aspirin and nitrate ester-based on NO donor is significantly potent anti-proliferate and apoptosis induction against the colon tumor cells compared to the aspirin itself(28,32). NO- Aspirin as a potential anti-lung cancer NO-aspirin has been studied as a highly potent in preventing lung cancer within high-risk populaces(10). When NO-aspirin (Fig. 3) was administered, the antiproliferative and apoptotic effect of erlotinib (an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor) considerably increased. The study also indicated that NO-aspirin managed to inhibit inflammation- induced lung tumorigenesis in mice (10). The drug was used to inhibit the proliferation of tumorigenic bronchial cell line (1170), non-small cell lung cancer (NSCLC), and colony formation by NSCLC cells. Effect of NO- aspirin on the 1170 and NSCLC cells was deduced by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide) assay, annexin v/propidium iodide apoptosis assay, colony formation assay, and tumor bioassay using mice (10). Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 17 Cell viability assay was used to determine the antiproliferative outcome of NO-aspirin against 1170 and NSCLC cells. The result showed that the proliferation of cells had been reduced in dose- dependent by 30%, 56%, and 71% after being compared to treatment-free cells. Furthermore, the apoptosis effect of cells increased when exposed to NO-aspirin in dose-dependent manor as 8%, 18%, and 24% using flow cytometry-based analysis of Annexin V and PI stained cells (10). Aspirin inhibited COX and platelets activation, which caused the anticancer effect. Activated platelets were not only able to activate the expression of COX-2 in epithelial cells but also capable of repressing T-cell immunity on cancer (33). It has been suggested another potential mechanism of anticancer since NO-aspirin is unable to balance the COX-2 level in mouse lung tumor tissue. Though, it was clear that phosphorylation of EGFR and the downstream effectors Akt, ERK, and STAT3 in 1170 and NSLC cells had been restricted by the presence of NO-aspirin (10). There is also a study that found NO moiety caused cell growth, apoptosis, and cancer invasion mostly over phosphorylation transition proteins, PI3K/Akt pathway, and downstream proteins (34). NO-Aspirin as a potential anticancer agent for metastatic prostate cancer One of the most prevalent malignant tumors identified in men is prostate cancer (35,36). Most cancer-related death is caused by metastatic castrate-resistant prostate cancer (CRPC) (37). Based on the phase of prostate cancer, either surgery, androgen deprivation or chemotherapy can be alternatives for the treatment (38). The influence of NO-aspirin inducing apoptosis in metastatic castration-resistant prostate cancer (CRPC) (PC3) cell via hydrogen peroxide (H2O2)-mediated oxidative stress has been reported (38). The reactive oxygen species (ROS) or oxygen radical is comprised of both radical and non- radical depend on its reactivity (39). Radicals are the species which contain at least one unpaired electron in the shells around the atomic nucleus and are capable of independent existence, such as superoxide radical (O2•–), hydroxyl (OH), nitrogen monoxide (NO), nitrogen dioxide (NO2–), and etc. While non radical species are not free radicals but can easily lead to free radical reactions in living organisms, for examples hydrogen peroxide (H2O2), hypochlorous acid (HOCl), ozone (O3), and etc. (38,40). In order to regulate normal physiological functions that are required in development, ROS is crucial. However, excessive levels of ROS harms proteins and membranes, which results in apoptosis or cell death (41). Compared to normal cells, cancer cells are more high-level in ROS, thus causing oxidative stress. Oxidative stress is an inconsistency between the output of ROS in the body that interrupts its ability to purify reactive immediate or restore the damage to the organ and cellular systems initiated by ROS (42). The free radical-induced oxidative stress can damage cellular, tissue, and organ systems, leading to several diseased conditions such as cardiovascular, asthma, and various cancers (colorectal, lung, prostate) (43,44). The majority of chemotherapeutic drugs display anticancer mechanisms by bringing free radicals into cancer cells (38,40). For example, nitric oxide as a free radical conjugated with aspirin in order to have anticancer properties to reduce the chance of proliferation of prostate cell cancer. The PC3 cells viability had been tested for antiproliferative effect by using MTT (3-(4, 5- dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, by treating it with numerous concentrations of NO-aspirin and parent compound aspirin. Untreated cells were used as control and incubated according to condition, read under spectrometer, and percent cell viability was recorded (38). Anticancer activity was investigated using three methods; colony formation assay, Annexin V- FITC/Propidium Iodide assay, and cell cycle analysis by flow cytometry (38). MTT assay showed that NO-aspirin almost completely inhibited PC3 cells at 100µM, compared to aspirin at 100mM. As anticipated, NO-aspirin is more practical in hindering PC3 cell viability compared to aspirin as an anticancer agent (38). The phosphatidylserine in the Annexin FITC/PI staining gives eat-me signals, making the identified and phagocytosis of dying cancer cells. Thus, apoptotic cells can be elucidated (45). Histogram of cell cycle analysis indicated that NO- aspirin induced G0 phase arrest at almost 90% concentration compared to untreated cells. The presence of high concentration of H2O2 also leads to cancer cell apoptosis. NO-aspirin has induced oxidative stress via NO group, which turned into H2O2, resulting in cell cancer PC3 apoptosis (38). This concluded that NO-aspirin has an anticancer effect on colon, lung, and prostate cell cancer. NOSH–Aspirin as Anti-Inflammatory and Anticancer Agent NOSH-aspirin is developed as a substitute aspirin with broader application ranges to decrease the risk of hemorrhage stroke in aspirin users (46). It is a novel hybrid between hydrogen sulphide (H2S) and NO moiety covalently bonded at 1, 2 positions of aspirin (ortho-NOSH-aspirin) (47,48) which is also known as NBS-1120 (Fig. 4) (19). The synthesis summary of the ortho NOSH-aspirin can be seen as scheme 4 below(48–50). Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 18 Fig. 4 Structure of NOSH-Aspirin releasing H2S and NO Aspirin and NOSH-aspirin had been evaluated the effect on rats’ stomachs when the drugs were being administered orally. After being treated with aspirin, the rats’ stomach showed ulceration and bleeding while NOSH-aspirin-treated was free from ulceration (47). While aspirin regulates prostaglandin, the NO and H2S donors have the same properties as prostaglandins that protect gastric mucosa (51,52). The gastric mucosa defense mechanism requires mucus to block the penetration of acid and pepsin by creating a viscous gel layer that assists a pH gradient in the epithelial surface of the stomach, thus blocking the penetration of acid and pepsin. NO, and H2S donors improve barrier function by stimulating the gastric and colonic secretion, which leads to reduce GI toxicity (51,53). Both donors also play a protective role in reducing oxidative stress, which is good in preventing cancer (54). NOSH-aspirin is a potent anti-inflammatory agent compared to aspirin parent by using Carrageenan- induced inflammation on rat paw. Inflammation is usually linked with cancer (55). As anti-inflammatory agents are capable to hinder with tumor development, they are significant in the prevention and treatment of cancer (56). Accordingly, there was a study mentioned that NOSH-aspirin showed 5 times more potency in targeting mouse model of colon cancer, which it lessens the cell proliferation and cell cycle arrest leading it to apoptosis (47). The latest study on NOSH-aspirin also stated that it was highly potent in inhibition of tumor growth in pancreatic cells. This was due to the ability of the drug to arrest cells in the G0/G1 phase transition and caused apoptosis in vitro (57). Scheme 4. The Synthesis of the ortho NOSH-Aspirin (NBS-1120) Aspirin-Thiourea Bearing Alkylated Amine Derivatives as Antimicrobial Agents Fig. 5. Thiourea Structure Thiourea (Fig. 5) is an organosulfur compound with the formula of S=C(NH2)2. This compound and its derivatives, in particular, have showed various pharmacological activities such as anti-fungal (58,59), antiviral (58,60), anticancer (61), anti- tuberculosis (62), antimicrobial (63) and anti- inflammatory (64–66). Thiourea has gained significant values since being studied for their application in commercial and industrial applications; plastics, textiles dyes, elastomer, herbicides, pesticides, rodenticides and catalytic (65,67,68). Fig. 6. Structure of Aspirin-Thiourea bearing Alkylated Amine Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 19 A study showed that compounds with two or more thiourea moiety hold better antimicrobial activity (67). Moreover, it gained much attention from researchers as it contains carbon, nitrogen, hydrogen, and sulfur elements (69). At acidic conditions, C=S and N-H functional groups can be protonated, which gives the thiourea ideal potential site for electrostatic binding on the bacterial surface, which consist of carboxyl and phosphate group (anionic), thus complementing its biological activities (67,70,71). Aspirin-thiourea (Fig. 6) with alkylated amine derivative as potential antimicrobial agents has been reported. The synthesis of aspirin-thiourea by reacting acetoxybenzoyl isothiocyanate with series of methyl, methoxy anilines, and alkylated anilines had been prepared through Williamson esterification (Scheme 5) Scheme 5. Synthesis of Aspirin-thiourea Derivatives The modification of alkylated amine on aspirin-thiourea gave various results on antibacterial activity against E. coli and S. aureus. The presence of C=O, C=S, and NH group, had increased the activities of antibacterial activities through the interaction on bacterial surface that contained carboxyl and phosphate group (72). The synthesized compounds 1-12 were studied on their antibacterial activity. However, it was found that compounds 4, 6, 10, 11, and 12 did not give any inhibition towards E. coli and S. aureus. Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 20 Table 1. Results on the alkylated amine on aspirin-thiourea based on their substituents. R’ Compounds Result (4) The presence of methyl (4) and methoxy (6) groups in the structure have reduced the biological activity due to steric hindrance (75).. (6) (10) As the alkyl chain increased from the compound (10), (11) and (12), a parabolic effect had been displayed (71) Longer the alkyl chain (>10), gave higher chance to hinder the cell membrane penetration, which prevents inhibition on bacterial growth(63). (11) (12) By comparing the result of the biological testing of the prepared derivatives on E. coli and S. aureus, it was found that E. coli was easier to be inhibited (63). This is due to the characteristic of S. aureus that is hard to penetrate because of its thick peptidoglycan layer that increases cell wall rigidity (73). N- Phenylcinnamide- Aspirin for Antimicrobial and Antifungal Activity Amide functional group contains (R-N- C=O) has been chemists’ choice since it has a lot of potentials. There are amide derivatives reported to be potent anticancer (74), anti-inflammatory (75), antioxidant (76), antibacterial, antifungal, antimalarial (77,78) and etc. As aspirin also has a lot potential, the modification of aspirin with amide is being studied, especially the antibacterial and antifungal activity. The synthesis of N-phenylcinnamamide derivatives that linked with aspirin (Scheme 6), started with dissolving the aryl aldehyde, giving substituted acetanilide chalcones compounds. The compounds were linked with aspirin by using mixed anhydride method, producing N- phenylcinnamamide-aspirin and continued with antimicrobial screening and antifungal assay (79). The final product of aspirin-N-phenylcinnamamide had three phenyl rings but was able to inhibit S. aureus and E. coli. Antimicrobial screening found that the compound 2c gave the highest inhibition against E. coli, 19 mm but 16 mm for the S. aureus, meanwhile 2a gave the highest inhibition of S. aureus, 18 mm, but against E. coli, 16 mm. As for the antifungal assay, 2c gave the largest inhibition of C. albicans, 18 mm, while 2a only gave 10 mm (79). Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 21 Scheme 6. Synthesis of N-phenylcinnamamide-Aspirin of 2a and 2c Table (2) Results on the antibacterial and antifungal of N-phenylcinnamamide-Aspirin (2a & 2c). Compounds Antibacterial Anti-fungal E. coli S. aureus C. albicans 2a 16 mm 18 mm 10 mm 2c 19 mm 16 mm 18 mm Although the aspirin with substituted amide (2a) has good antibacterial activity, the presence of –Cl substituent in the compound (2c) is slightly higher. It was determined that its high lipophilicity which penetrated the bacterial cell wall has contributed to the higher success rate of inhibition (79). This may caused by the lipophilic characteristic of N-phenylcinnamide due to interaction of its active site to the bacterial cell membrane and gain access to its target and restrained the bacteria (80,81). A study reported that halogenation also affect C. albicans virulence activity due to their steric effect that provides best fitting of small molecule to conquer the target’s binding site. The – Cl substituent also found to be the most stable halogen that tolerates a steady docking on C. albicans. As the result on these studies, -Cl substituent on the compound was found to give most stable derivatives (82). Aspirin-Chalcone Derivatives Fig. 7. Chalcone structure Chalcone (Fig. 7) comprises of two aromatic rings that are highly interconnected by three-carbon α,β-unsaturated ketones that contribute to the pharmacological activity (83,84). In medicinal chemistry, chalcone is a simple scaffold that originates in countless naturally occurring compounds and is being used widely as an efficacious model for drug discovery (85). It is stated that chalcones have many benefits, for instance, low interaction with DNA and low-risk of mutagenicity (86). Chalcone is known as the precursor for the synthesis of flavonoids, which is practical as antiplatelet(83), anticancer(84), anti-inflammatory, antioxidant, anti-diabetic, and antimicrobial (87–90). Researches on chalcone derivatives reported that chalcones have high antioxidant activity (91,92) .Since antioxidants have the ability to donate electron, it can neutralize the free radical and prevent any damage to biological compound in the body (93). As chalcones are known as minor flavonoids (94), they can scavenge free radicals (92). Excess of free radicals and ROS (reactive oxygen species) in human body may cause diseases like cancer, cardio, and cerebrovascular due to damage to lipids, proteins, and nucleic acids (95). That is the reason why the proper physiological function depend on the balance between free radicals and antioxidants (96). The accumulation of ROS in the body can be influenced by several factors including endogenous factors such as by-products of mitochondrial activity, exogenous factors including ultraviolet radiation, and even the lack of antioxidant agents in the body such as glutathione, vitamins A, C, and E (39). The accumulation of oxidative damage can be prevented by avoiding the excessive ROS formation through optimal functioning of oxygen metabolism and avoidance of environmental pollutants, as well as increasing the neutralization of ROS by having appropriate antioxidant intake (96). Therefore, a study regarding aspirin-chalcone with antibacterial effect was done. Aspirin-Chalcone with antibacterial and antioxidant activities The synthesis of aspirin-chalcone was done by the reaction of aspirin and chalcone derivatives by esterification, giving aspirin-chalcone (Scheme 7) (97). Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 22 Scheme 7. Synthesis of aspirin chalcone a-g The antibacterial evaluation of the aspirin- chalcone derivatives was analyzed against E.coli and S. aureus. However, the result indicated that most derivatives gave no inhibition against E. coli and S. aureus when compared to ampicillin (97). The similar result had been also conducted by Ngaini et al. on the previous research., in which aspirin chalcone derivatives gave no inhibition against E. coli for antibacterial assay (98). There is a high possibility it occurs due to E. coli is easier to accumulate resistance genes, making it more resistance toward older antibiotics like phenicols, sulfonamides, and trimethoprim (99). The asymmetric lipopolysaccharide (LPS)-phospholipid bilayer of the outer membrane of E. coli causes a weaker permeable barrier for both hydrophobic and hydrophilic compounds (100). Nevertheless, it was discovered that the aspirin-chalcone displayed poor antioxidant properties on 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay in comparison to ascorbic acid was due to low phenolic pharmacophore and steric hindrance which also cause bulky structures (97). The phenolic group was necessary for getting high antioxidant activity (101). The presence of phenyl ring in the compound may also contribute to the bulkiness and cause steric hindrance, making it harder for penetration into phospholipid bilayer of S. aureus and E. coli (102,103). Aspirin-chalcone with anticancer activity The studies of the aspirin-based drug for anticancer effect have been discovered and encouraged since a while back (104), including breast cancer that is highly prevalent among women worldwide (105). Anticancer drugs usually kill these cancer cells by inducing ROS generation since the high level of ROS causes cell damage as well as apoptosis, autophagic and necrotic cell death (106,107). Cancer cells have abnormal metabolism; thus, they have higher ROS compared to normal cells, making them more susceptible to ROS-induction treatment (107). Chalcone derivatives have received significant attention as they exhibited potent anticancer activity against some cancer cell lines, such as naphthalene-chalcone derivatives that displayed potent antiproliferative activity against breast cancer cells (MCF-7) (108). Chalcone inhibited proliferation in MCF-7 by inducing apoptosis and hindering cell cycle development (109) by increasing ROS (110). Aspirin also prevents breast tumor cell growth through induction apoptosis (111). Thus aspirin-conjugated chalcone polymeric micelles for anti-breast cancer activity is an interest (112). Polymeric micelle unsurprisingly is a decent delivery system for anticancer drugs with lower water solubility. At the size of 10-100 nm, it is able to elongate circulation time of the drugs in the blood (112). Aspirin also compromised the condition of chalcone which is a hydrophobic polyphenol with poor aqueous solubility (110). It has been studied that aspirin-conjugated chalcone derivative-loaded nanoparticles (AS- DK143-loaded NP) as potential chemotherapy agents with anticancer efficacy. Synthesis of AS- DK143, which is also known as (E)-2-(2,3- dimethoxyphenyl)acrylol)-4-methoxyphenyl-2- acetoxybenzoate, started with preparation on one of the chalcone derivatives from the previously reported study by the same author, which is known as (E)-3-(2,3-dimethoxyphenyl)-1-(2-hydroxy-5- methaoxyphenyl)prop-2-en-1 or DK143 (113). The method of the synthesis can be seen in Scheme 8. Then the process continued with the preparation of AS-DK143 polymeric micelles by thin-film hydration method. The AS-DK143 undergoes cell viability and animal studies for testing the anticancer effect towards nude mice (110). Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 23 Scheme 8. Synthesis of AS-DK143-loaded-NP AS-DK143 was synthesized and characterized using 1H NMR and IR spectroscopy, and the nanoparticles were tested in 4T1 cell viability. The chalcone-based compound can be used as a potent anticancer agent as it induces cancer cell apoptosis by increasing ROS production (106,110). However, due to its non-polar properties, they increased the bioavailability by interlinking –OH in DK143 with the polar group of aspirin, –COOH in the form of nanoparticles (110). AS-DK143 showed significant reduction of 4T1 cell viability to 25.97%, ±5.69% and 11.02% ± 0.01%. It was also found that the IC50 of aspirin and AS-D143 gave 4955µM and 39.61µM. Thus proving that the modification of aspirin-chalcone derivative (AS-DK143) against 4T1 cell gave greater anticancer effect at the lowest concentration compared to chalcone derivative (DK143) itself. Azo-aspirin with Antibacterial Properties In order to increase the antibacterial activity, it is recommended to introduce azo moiety into the structure as –N=N- is important in bactericidal activities (114) The study for antibacterial properties of aspirin derivatives continued on pursuing on azo-aspirin as it gives good biological activity. The synthesis started with a phenol and aniline derivatives and produced azo derivatives. The product reacted with aspirin, becoming azo-aspirin. Further explanation on synthesis can be seen in Scheme 9 (102). Scheme 9. Synthesis of Azo and Azo-Aspirin Derivatives Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 24 E. coli and S. aureus had been chosen for the antibacterial activity of azo-aspirin. Presumably, halogen being chosen as substituent, it is due to its high reactivity, which can be deadly to bacteria in a sufficient amount. The ortho-fluorine aspirin-azo derivatives (compound 2d) gave better antibacterial activity against E. coli (156.3 ppm), meanwhile meta- fluorine aspirin-azo (compound 2b) gave better results against S. aureus (194.1 ppm) in comparison to parent aspirin (>220 ppm). The result indicated that –F substituted compounds showed superior antibacterial activity compared to –Cl substituted compounds (102). This is because of larger atomic radius of –Cl atom, which creates a larger steric hindrance than –F. In other hand, although its electronegativity is less than –F atom (Pauling electronegativity of 4.0), –Cl (Pauling electronegativity of 3.2) can form very strong noncovalent interactions. However, these compounds are not superior antibacterial agents compared to the ampicillin (115). Ampicillin disrupt the bacterial cell wall synthesis during active replication and kill the bacterial, making it one of the chosen antibacterial agents used in medicine (116,117). Few years later, another journal published by the same author reported on halogenated azo aspirin with additional procedures, diazotization followed by coupling reaction (Scheme 10). The presence of halogens, –Br and –I played a significant role in raising the antibacterial activities of the derived compounds compared with the aspirin and ampicillin (114). Scheme 10. The Synthesis of Azo-Aspirin by Diazotization and Coupling Reaction The –I substituent at ortho position gave the highest inhibition with MIC value, 74 ppm against E. coli and 64 ppm against S. aureus. Surprisingly, it showed a far superior result as antibacterial agents compared to the ampicillin. Nevertheless, –Br at ortho position also gave high MIC value, 89 ppm for both E. coli and S. aureus (114). Comparing the result from previous journals, the presence of the halogens affects the inhibition of bacteria in the rate of -Cl < -F < -Br < -I (102,114). Although the –I gave the highest inhibition, it also needs to be considered whether it will affect the other cells or not. The released of –I substance need to be regulated as it can iodinate the lipids that are main component of the cell membrane, and will oxidize various cellular components. Therefore, it can be dangerous towards human skins, or cell as well if the released of the substance is not controlled. However, it does not change the fact that halogens can be strong oxidizing substances that damage the cell wall or membrane of microorganisms which contribute to the bacteriostatic effect (118). The presence of the halogen also improved the lipophilic tendency of azo-aspirin to penetrate the microorganisms’ cell walls. The increasing levels of lipophilicity can enhance the ability of compounds to penetrate the cell membranes of gram-negative bacteria which are hydrophobic (119). That is one the reasons on why halogens are being considered to improve antibacterial effect of drugs modification. Meanwhile, the presence of –N=N- (azo) moieties that can be protonated under acidic condition and reacted with phosphate group on the peptidoglycan layer; can hinder the cell wall formation. Then hydrogen bonding will form between the azo-aspirin compound and the active site of the enzyme, causing the bacteriostatic effect (113). Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 25 Table (3) Summary of the Aspirin Derivatives and Their Related Biological Activities: Modification Authors Methods Aim Results/Finding NO-Aspirin Ding et al. (28) - Anticancer • Over 83.9 % of the tumor cells are blocked at G0/G1 phase Song et al. (10) • MTT (3-(4, 5-dimethylthiazolyl- 2)-2, 5-diphenyltetrazolium bromide) assay, • Annexin v/propidium iodide apoptosis assay, • Colony formation assay, • Tumor bioassay using mice Anti-Lung Cancer • The proliferation of the tumor cells is reduced meanwhile the frequency rate of cells’ apoptosis is increased Chinnapaka et al. (38) • MTT (3-(4, 5-dimethylthiazolyl- 2)-2, 5-diphenyltetrazolium bromide) assay, • Colony formation assay, • Annexin V-FITC/Propidium Iodide assay, • Cell cycle analysis by flow cytometry Anti-Prostate Cancer • 90% of the tumor cells are inhibited at G0 phase NOSH-Aspirin Kashfi et al.(47) • Carrageenan-induced inflammation on rat paw Anti-Inflammation & Anti-Colon Cancer • It is 5 times more potent in targeting mouse model of colon cancer, then reduce the cell proliferation and cell cycle arrest leading it to apoptosis Chattopadhyay et al.(57) • Cell growth inhibition assay, • Cell proliferation, • Flow cytometry of phase distribution in the cell cycle • Detection of apoptosis Anti-Inflammation & Anti-Pancreatic Cancer • It reduces gastric mucosa and arrests cells in the G0/G1 phase transition which caused apoptosis in vitro. Aspirin-Thiourea Nordin et al. (63) • Synthesis using Williamson esterification, • Antibacterial screening against E. Coli and S. Aureus Antimicrobial/ Antibacterial • The presence of C=O, C=S and N-H give good inhibition, however OCH3 and CH3 contribute to steric hindrance, and long alkyl chain (>10) showed parabolic effect Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 26 Aspirin-Amide Alwash et al. (79) • Synthesis of N- phenylcinnamamide using Claisen-Schmidt condensation linked with aspirin, • In-vitro antibacterial and antifungal screening against E. coli, S. aureus and C. albicans Antimicrobial & Antifungal • -Cl substituted compound gave the highest inhibition of antibacterial and antifungal. 19 mm, 16 mm and 18 mm of E. coli, S. aureus, and C. albicans • –Cl substituent also found to be the most stable halogen that tolerates a steady docking on virulence-related target Aspirin-Chalcone Nordin et al. (97) • Synthesis of hydroxylated chalcone • Synthesis of aspirin chalcone • Antibacterial screening against E. coli and S. aureus • Antioxidant evaluation using DPPH Antibacterial & Antioxidant • Poor inhibition against bacterial and fungal activity. • Bulky structures and lack of phenolic pharmacophore contribute to poor antioxidant activity Lee et al. (110) • Thin-film hydration method • Cell viability assay Anti-Breast Cancer • The cell viability of AS-DK143 against 4T1 cells reduced to 25.97%, ±5.69% and 11.02% ± 0.01%. • The IC50 of aspirin and AS-D143 gave 4955µM and 39.61µM. Azo-Aspirin Ngaini and Ho (102) • Synthesis of azo • Synthesis of aspirin-azo derivatives • Antibacterial screening against E. coli and S. aureus Antibacterial • The ortho-fluorine gave better antibacterial activity against E. coli, meanwhile meta-fluorine aspirin-azo gave better results against S. aureus. • Larger atomic radius of –Cl atom creates larger steric hindrance than –F, making –F substituted compounds good antibacterial agent Ngaini and Mortadza (114) • Synthesis of azo • Synthesis of aspirin-azo derivatives diazotization followed by coupling reaction • Antibacterial screening against E. coli and S. aureus Antibacterial • The –I substituent gave the highest inhibition with MIC value, 74 ppm against E. coli and 64 ppm against S. aureus. • –Br also gave high MIC value, 89 ppm for both E. coli and S. aureus • It gave superior result as antibacterial agents compared to the ampicillin. Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 27 References 1. Santos-Gallego CG, Badimon J. Overview of Aspirin and Platelet Biology. Am J Cardiol . 2021 Apr;144:S2–9. Available from: https://linkinghub.elsevier.com/retrieve/pii/S00 0291492031345X 2. Lourenço AL, Saito MS, Dorneles LEG, Viana GM, Sathler PC, De Aguiar LCS, et al. Synthesis and antiplatelet activity of antithrombotic thiourea compounds: Biological and structure-activity relationship studies. Molecules. 2015;20(4):7174–200. 3. Ittaman S V., VanWormer JJ, Rezkalla SH. The role of aspirin in the prevention of cardiovascular disease. Clin Med Res. 2014;12(3–4):147–54. 4. Hankey GJ, Eikelboom JW. Antiplatelet drugs. Med J Aust . 2003 Jun 2;178(11):568–74. Available from: https:// onlinelibrary .wiley.com/doi/abs/10.5694/j.1326-5377. 2003.tb05361.x 5. Huang ES, Strate LL, Ho WW, Lee SS, Chan AT. Long-term use of aspirin and the risk of gastrointestinal bleeding. Am J Med. 2011;124(5):426–33. 6. Jiang S, Zhang L, Cui D, Yao Z, Gao B, Lin J, et al. The Important Role of Halogen Bond in Substrate Selectivity of Enzymatic Catalysis. Sci Rep. 2016;6(September):1–7. 7. Saccone M, Catalano L. Halogen Bonding beyond Crystals in Materials Science. J Phys Chem B . 2019 Nov 7;123(44):9281–90. Available from: https://pubs.acs.org/doi/10.1021/acs.jpcb.9b07 035 8. Fiala C, Pasic MD. Aspirin: Bitter pill or miracle drug? Clin Biochem . 2020 Nov 1 [cited 2021 Mar 25];85:1–4. Available from: https://linkinghub.elsevier.com/retrieve/pii/S00 0991202030792X 9. Thota PNPN. Aspirin: the miracle drug? Clin Transl Gastroenterol . 2018;9(153):4–5. Available from: http://dx.doi. org/10 .1038/ s41424-018-0009-4 10. Song JM, Upadhyaya P, Kassie F. Nitric oxide- donating aspirin (NO-Aspirin) suppresses lung tumorigenesis in vitro and in vivo and these effects are associated with modulation of the EGFR signaling pathway. Carcinogenesis. 2018;39(7):911–20. 11. Jin M, Li C, Zhang Q, Xing S, Wang J, Kan X. Effects of aspirin on proliferation, invasion and apoptosis of Hep‑2 cells via the PTEN/AKT/NF‑κB/survivin signaling pathway. Oncol Lett. 2018;(15):8454–60. 12. Sainz RM, Lombo F, Mayo JC. Radical Decisions in Cancer: Redox Control of Cell Growth and Death. Cancers (Basel) . 2012 Apr 25;4(2):442–74. Available from: http://www.mdpi.com/2072-6694/4/2/442 13. Singh K, Bhori M, Kasu YA, Bhat G, Marar T. Antioxidants as precision weapons in war against cancer chemotherapy induced toxicity – Exploring the armoury of obscurity. Vol. 26, Saudi Pharmaceutical Journal. Elsevier B.V.; 2018. p. 177–90. 14. Badea GI, Radu GL, Badea I, Radu GL, Badea GI. Introductory Chapter: Carboxylic Acids - Key Role in Life Sciences. In: Intech . InTech; 2016. p. 13. Available from: http:// www. Intechopen .com /books /carboxylic -acid - key- role -in -life-sciences/introductory-chapter- carboxylic -acids-key-role-in-life-sciences 15. Isac-García J, Dobado JA, Calvo-Flores FG, Martínez-García H. Organic Synthesis Experiments. Exp Org Chem. 2016 Jan 1;239– 89. 16. Alfonso L, Ai G, Spitale RC, Bhat GJ. Molecular targets of aspirin and cancer prevention. Vol. 111, British Journal of Cancer. Nature Publishing Group; 2014. p. 61–7. 17. Ai G, Dachineni R, Muley P, Tummala H, Bhat GJ. Aspirin and salicylic acid decrease c-Myc expression in cancer cells: a potential role in chemoprevention. Tumor Biol. 2016 Feb 1;37(2):1727–38. 18. Cai G, Zhou W, Lu Y, Chen P, Lu Z, Fu Y. Aspirin resistance and other aspirin-related concerns. Vol. 37, Neurological Sciences. Springer-Verlag Italia s.r.l.; 2016. p. 181–9. 19. Kodela R, Chattopadhyay M, Velázquez- Martínez CA, Kashfi K. NOSH-aspirin (NBS- 1120), a novel nitric oxide- and hydrogen sulfide-releasing hybrid has enhanced chemo- preventive properties compared to aspirin, is gastrointestinal safe with all the classic therapeutic indications. Biochem Pharmacol . 2015 Dec [cited 2021 Jul 4];98(4):564–72. Available from: https:// linkinghub. elsevier. com/retrieve/pii/S0006295215006243 20. Silva DA, Al-Gousous J, Davies NM, Bou Chacra N, Webster GK, Lipka E, et al. Simulated, biorelevant, clinically relevant or physiologically relevant dissolution media: The hidden role of bicarbonate buffer. Eur J Pharm Biopharm. 2019;142:8–19. 21. Clay W. Mucus Production . TeachMe Physiology. 2021 [cited 2021 Apr 12]. p. 4–6. Available from:https:// teachmephysiology .com/gastrointestinal-system/stomach/mucus- production/ #:~:text=Mucus is secreted by the,is to the functioning stomach. 22. Smolik S, Węglarz L. Aspirin – 115 years after the discovery. Ann Acad Medicae Silesiensis . 2013;67(1). Available from: https:// annales. sum.edu.pl/Aspirin-115-years-after-the- discovery- ,131430,0,2.html 23. Alegbeleye BJ, Akpoveso O-OP, Mohammed RK, Asare BY-A. Pharmacology, Pharmaceutics and Clinical Use of Aspirin: A Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 28 Narrative Review. J Drug Deliv Ther. 2020;10(5-s):236–53. 24. Danielak A, Wallace JL, Brzozowski T, Magierowski M. Gaseous Mediators as a Key Molecular Targets for the Development of Gastrointestinal-Safe Anti-In fl ammatory Pharmacology. Pharmacology. 2021;12(April):1–17. 25. Bucă BR, Mititelu-Tarțău L, Lupușoru R V., Lupușoru CE, Rezuș C. New insights into the therapeutic use of nitric oxide-donating non- steroidal anti-inflammatory drugs the. Rev Med Chir Soc Med Nat. 2018;122(2):347–51. 26. Morbidelli L, Bonavida B, Riccardi C, Cuzzocrea S. Therapeutic Applications of Nitric Oxide in Cancer and Inflammatoryrelated Disorders. In: Therapeutic applications of nitric oxide in cancer and inflammatory related disorders. Siena: Elsevier; 2018. p. 40. 27. Castaldi G, Oldani E, Razzetti G, Benedini F. Process for obtaining (nitroxymethyl)phenyl esters of salcylic acid dervatives. Vol. 1. United States; US 6,696,591 B1, 2004. p. 5–9. 28. Ding, Q, Zang J, Gao S, Gao Q, Duan W, Li X, et al. Nitric oxide donor hybrid compounds as promising anticancer agents. Drug Discov Ther . 2016;10(6):276–84. Available from: www.ddtjournal.com 29. Ghione S, Mabrouk N, Paul C, Bettaieb A, Plenchette S. Protein kinase inhibitor-based cancer therapies: Considering the potential of nitric oxide (NO) to improve cancer treatment. Biochem Pharmacol . 2020;176:113855. Available from: https:// www. Sciencedirect .com/science/article/pii/S0006295220300824 30. Shi T, Fujita K, Gong J, Nakahara M, Iwama H, Liu S, et al. Aspirin inhibits hepatocellular carcinoma cell proliferation in vitro and in vivo via inducing cell cycle arrest and apoptosis. Oncol Rep. 2020;44(2):457–68. 31. Zhang X, Feng H, Li Z, Guo J, Li M. Aspirin is involved in the cell cycle arrest, apoptosis, cell migration, and invasion of oral squamous cell carcinoma. Int J Mol Sci. 2018;19(7). 32. Huerta S. Nitric oxide for cancer therapy. Futur Sci OA. 2015;1(1). 33. Hua H, Zhang H, Kong Q, Wang J, Jiang Y. Complex roles of the old drug aspirin in cancer chemoprevention and therapy. Med Res Rev. 2019;39(1):114–45. 34. Liu X, Zhang Y, Wang Y, Yang M, Hong F, Yang S. Protein phosphorylation in cancer: Role of nitric oxide signaling pathway. Biomolecules. 2021;11(7):1–16. 35. Packer JR, Maitland NJ. Biochimica et Biophysica Acta The molecular and cellular origin of human prostate cancer. BBA - Mol Cell Res . 2016;1863(6):1238–60. Available from: http://dx.doi.org/10.1016/j.bbamcr.2016.02.01 6 36. Rawla P. Epidemiology of Prostate Cancer. World J Oncol . 2019;10(2):63–89. Available from: http://www.wjon.org/index.php/WJON/article/ view/1191 37. Hoang DT, Iczkowski KA, Kilari D, See W, Nevalainen MT. Androgen receptor-dependent and -independent mechanisms driving prostate cancer progression: Opportunities for therapeutic targeting from multiple angles. Oncotarget . 2017;8(2):3724–45. Available from: www.impactjournals.com/oncotarget/ 38. Chinnapaka S, Zheng G, Chen A, Munirathinam G. Nitro aspirin (NCX4040) induces apoptosis in PC3 metastatic prostate cancer cells via hydrogen peroxide (H 2 O 2 )- mediated oxidative stress Graphical Abstract HHS Public Access Schematic mode of action of NCX4040 in prostate cancer cells. Radic Biol Med. 2019;143:494–509. 39. Krumova K, Cosa G. Chapter 1. Overview of Reactive Oxygen Species. In 2016. p. 1–21. Available from: http:// ebook. rsc.org /?DOI =10.1039/9781782622208-00001 40. Kim SJ, Kim HS, Seo YR. Understanding of ROS-Inducing Strategy in Anticancer Therapy. Oxid Med Cell Longev. 2019;5381692. 41. Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta - Mol Cell Res . 2016;1863(12):2977–92. Available from: http://dx.doi.org/10.1016/j.bbamcr.2016.09.01 2 42. Pizzino G, Irrera N, Cucinotta M, Pallio G, Mannino F, Arcoraci V, et al. Oxidative Stress: Harms and Benefits for Human Health. 2017; Available from: https://doi.org/10.1155/2017/8416763 43. Sies H, Berndt C, Jones DP. Oxidative Stress. Annu Rev Biochem . 2017 Jun 20;86(1):715– 48. Available from: https://doi.org/10.1146/annurev-biochem- 061516-045037 44. Phaniendra A, Jestadi DB, Periyasamy L. Free Radicals: Properties, Sources, Targets, and Their Implication in Various Diseases. Ind J Clin Biochem. 2014;30(1):11–26. 45. Shlomovitz I, Speir M, Gerlic M. Flipping the dogma - Phosphatidylserine in non-apoptotic cell death. Cell Commun Signal. 2019;17(1):1– 12. 46. Hao W, Shen Y, Feng M, Wang H, Lin M, Fang Y, et al. Aspirin acts in esophageal cancer: A brief review. J Thorac Dis. 2018;10(4):2490–7. 47. Kashfi K. Development of NOSH-NSAIDs: A new class of anti-inflammatory pharmaceuticals Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 29 for the treatment of cancer. Biochem (Lond). 2017;39(4):24–9. 48. Vannini F, Mackessack-Leitch AC, Eschbach EK, Chattopadhyay M, Kodela R, Kashfi K, et al. Synthesis and anti-cancer potential of the positional isomers of NOSH-aspirin (NBS- 1120) a dual nitric oxide and hydrogen sulfide releasing hybrid. Bioorg Med Chem Lett . 2015 Oct 15;25(20):4677–82. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PM C4592841/ 49. Kodela R, Chattopadhyay M, Kashfi K. NOSH- aspirin: A novel nitric oxide-hydrogen sulfide- releasing hybrid: A new class of anti- inflammatory pharmaceuticals. ACS Med Chem Lett. 2012 Mar 8;3(3):257–62. 50. Lee M, Mcgeer E, Kodela R, Kashfi K, Mcgeer PL. NOSH-aspirin (NBS-1120), a novel nitric oxide and hydrogen sulfide releasing hybrid, attenuates neuroinflammation induced by microglial and astrocytic activation: A new candidate for treatment of neurodegenerative disorders. Glia. 2013 Oct;61(10):1724–34. 51. Chattopadhyay M, Kodela R, Duvalsaint PL, Kashfi K. Gastrointestinal safety, chemotherapeutic potential, and classic pharmacological profile of NOSH-naproxen (AVT-219) a dual NO- and H2S-releasing hybrid. Pharmacol Res Perspect. 2016;4(2):1– 15. 52. Ornelas A, Zacharias-Millward N, Menter DG, Davis JS, Lichtenberger L, Hawke D, et al. Beyond COX-1: the effects of aspirin on platelet biology and potential mechanisms of chemoprevention. Cancer Metastasis Rev. 2017;36(2):289–303. 53. Tavares L, Renan L, Silva O, Paula A, Santana M, Melo B De, et al. Nitric Oxide and Hydrogen Sulfide Interact When Modulating Gastric Physiological Functions in Rodents. Dig Dis Sci. 2017;62:93–104. 54. Shen F, Zhao C-S, Shen M-F, Wang Z, Chen G. The role of hydrogen sulfide in gastric mucosal damage. Med Gas Res . 2019;9(2). Available from: http://www.medgasres.com/text.asp?2019/9/2/ 0/260650 55. Munn LL. Cancer and inflammation. WIREs Syst Biol Med . 2017 Mar 12;9(2). Available from: https://onlinelibrary.wiley.com/doi/10.1002/ws bm.1370 56. Zappavigna S, Cossu AM, Grimaldi A, Bocchetti M, Ferraro GA, Nicoletti GF, et al. Anti-inflammatory drugs as anticancer agents. Int J Mol Sci. 2020 Apr 1;21(7):1–29. 57. Chattopadhyay M, Kodela R, Santiago G, Le TTC, Nath N, Kashfi K. NOSH-aspirin (NBS- 1120) inhibits pancreatic cancer cell growth in a xenograft mouse model: Modulation of FoxM1, p53, NF-κB, iNOS, caspase-3 and ROS. Biochem Pharmacol . 2020 Jun;176(3):113857. Available from: https://linkinghub.elsevier.com/retrieve/pii/S00 0629522030085X 58. Wang H, Zhai Z-W, Shi Y-X, Tan C-X, Weng J-Q, Han L, et al. Novel Trifluoromethylpyrazole Acyl Thiourea Derivatives: Synthesis, Antifungal Activity and Docking Study. Lett Drug Des Discov . 2019 Jun 27;16(7):785–91. Available from: http://www.eurekaselect.com/163481/article 59. Campo R del, Criado JJ, Gheorghe R, González FJ, Hermosa MR, Sanz F, et al. N-benzoyl-N′- alkylthioureas and their complexes with Ni(II), Co(III) and Pt(II) – crystal structure of 3- benzoyl-1-butyl-1-methyl-thiourea: activity against fungi and yeast. J Inorg Biochem . 2004 Aug;98(8):1307–14. Available from: https://linkinghub.elsevier.com/retrieve/pii/S01 62013404001102 60. Ravichandran V, Shalini S, Kumar KS, Rajak H, Agrawal RK. Design, Synthesis and Evaluation of Thiourea Derivatives as Antimicrobial and Antiviral Agents. Lett Drug Des Discov . 2019 May 24;16(6):618–24. Available from: http://www.eurekaselect.com/164242/article 61. Alimohammadi A, Mostafavi H, Mahdavi M. Thiourea Derivatives Based on the Dapsone‐ Naphthoquinone Hybrid as Anticancer and Antimicrobial Agents: In Vitro Screening and Molecular Docking Studies. ChemistrySelect . 2020 Jan 16;5(2):847–52. Available from: https://onlinelibrary.wiley.com/doi/10.1002/slc t.201903179 62. Konduri S, Pogaku V, Prashanth J, Siva Krishna V, Sriram D, Basavoju S, et al. Sacubitril‐Based Urea and Thiourea Derivatives as Novel Inhibitors for Anti‐Tubercular against Dormant Tuberculosis. ChemistrySelect . 2021 Apr 28;6(16):3869–74. Available from: https://onlinelibrary.wiley.com/doi/10.1002/slc t.202004724 63. Nordin NA, Chai TW, Tan BL, Choi CL, Abd Halim AN, Hussain H, et al. Novel Synthetic Monothiourea Aspirin Derivatives Bearing Alkylated Amines as Potential Antimicrobial Agents. J Chem. 2017;2017:2378186. 64. Mumtaz A, Arshad J, Saeed A, Azhar M, Nawaz H, Iqbal J, et al. Synthesis, characterization and urease inhibition studies of transition metal complexes of thioureas bearing ibuprofen moiety. J Chil Chem Soc. 2018;63(2):3934–40. 65. Naz S, Zahoor M, Umar MN, Alghamdi S, Sahibzada MUK, UlBari W. Synthesis, characterization, and pharmacological evaluation of thiourea derivatives. Open Chem . 2020 Jun 29;18(1):764–77. Available from: Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 30 https://www.degruyter.com/document/doi/10.1 515/chem-2020-0139/html 66. Rivera A, Maldonado M, Ríos-motta J. A Facile and Efficient Procedure for the Synthesis of New Benzimidazole-2-thione Derivatives. Molecules. 2012;17:8578–86. 67. Ngaini Z, Wan Zulkiplee WSH, Abd Halim AN. One-Pot Multicomponent Synthesis of Thiourea Derivatives in Cyclotriphosphazenes Moieties. J Chem . 2017;2017:1–7. Available from: https://www.hindawi.com/journals/jchem/2017 /1509129/ 68. Shakeel A. Thiourea Derivatives in Drug Design and Medicinal Chemistry: A Short Review. J Drug Des Med Chem. 2016;2(1):10. 69. Khairul WM, Ariffin AA, Ismail N, Daud AI. Synthesis, Spectroscopic Studies, and Biological Activities of Acylthiourea Derivatives as Potential Anti-Bacteria Agents. Educ J Sci Math Technol . 2016 Jun 6;3(1 SE- Articles):13–9. Available from: https://ejournal.upsi.edu.my/index.php/EJSMT /article/view/40 70. Khairul WM, Daud AI, Ismail N. Understanding the properties of chitosan aryl substituted thioureas in their role and potential as antibacterial agents. In 2018. p. 020002. Available from: http://aip.scitation.org/doi/abs/10.1063/1.5023 936 71. Abd Halim AN, Ngaini Z. Synthesis and Bacteriostatic Activities of Bis(thiourea) Derivatives with Variable Chain Length. J Chem . 2016;2016:1–7. Available from: https://www.hindawi.com/journals/jchem/2016 /2739832/ 72. Ngaini Z, Mohd Arif MA, Hussain H, Mei ES, Tang D, Kamaluddin DHA. Synthesis and antibacterial activity of acetoxybenzoyl thioureas with aryl and amino acid side Chains. Phosphorus, Sulfur Silicon Relat Elem. 2012;187(1):1–7. 73. Mashuri NF, Tan HL, Lim YP, Maqsood-Ul- Haque SNS. Isolation of Antimicrobial Peptide from Food Protein Hydrolysates: An Overview. Key Eng Mater . 2019 Mar;797(April):168–76. Available from: https://www.scientific.net/KEM.797.168 74. Shahinshavali S, Sreenivasulu R, Guttikonda VR, Kolli D, Rao MVB. Synthesis and Anticancer Activity of Amide Derivatives of 1,2-Isoxazole Combined 1,2,4-Thiadiazole. Russ J Gen Chem. 2019;89(2):324–9. 75. Zhang Z, Hao K, Li H, Lu R, Liu C, Zhou M, et al. Design, synthesis and anti-inflammatory evaluation of 3-amide benzoic acid derivatives as novel P2Y14 receptor antagonists. Eur J Med Chem . 2019;181:111564. Available from: https://www.sciencedirect.com/science/article/ pii/S0223523419306889 76. Malki F, Touati A, Hamza K, Moulay S, Baltas M. Antioxidant activity of a series of amides. J Mater Environ Sci. 2016;7(3):936–41. 77. Wu W, Chen M, Wang R, Tu H, Yang M, Ouyang G. Novel pyrimidine derivatives containing an amide moiety: design, synthesis, and antifungal activity. Chem Pap . 2019;73(3):719–29. Available from: https://doi.org/10.1007/s11696-018-0583-7 78. Bhatt A, Kant R. Synthesis of Some Bioactive Sulfonamide and Amide Derivatives of Piperazine Incorporating Imidazo[1,2- B]Pyridazine Moiety. Med Chem (Los Angeles). 2016;06(04). 79. Alwash AH, Mahdi AM, Al-Karagully HJ. Synthesis, characterization, and antimicrobial evaluation of new n-phenylcinnamamide derivatives linked to aspirin and ibuprofen. Asian J Pharm Clin Res. 2018 Oct 1;11(10):443–6. 80. Echeverría J, Opazo J, Mendoza L, Urzúa A, Wilkens M. Structure-activity and lipophilicity relationships of selected antibacterial natural flavones and flavanones of Chilean flora. Molecules. 2017;22(4). 81. Constantinescu T, Lungu CN, Lung I. Lipophilicity as a central component of drug- like properties of chalchones and flavonoid derivatives. Molecules. 2019;24(8):1–11. 82. Garcia C, Burgain A, Chaillot J, Pic É, Khemiri I, Sellam A. A phenotypic small-molecule screen identifies halogenated salicylanilides as inhibitors of fungal morphogenesis, biofilm formation and host cell invasion. Sci Rep. 2018;8(1):1–16. 83. Tekale S, Mashele S, Pooe O, Thore S, Kendrekar P, Pawar R. Biological Role of Chalcones in Medicinal Chemistry. Vector- Borne Dis - Recent Dev Epidemiol Control. 2020;1–25. 84. Salehi B, Quispe C, Chamkhi I, El Omari N, Balahbib A, Sharifi-Rad J, et al. Pharmacological Properties of Chalcones: A Review of Preclinical Including Molecular Mechanisms and Clinical Evidence. Front Pharmacol. 2021;11(January). 85. Zhuang C, Zhang W, Sheng C, Zhang W, Xing C, Miao Z. Chalcone: A Privileged Structure in Medicinal Chemistry. Chem Rev . 2017 Jun 28;117(12):7762–810. Available from: https://doi.org/10.1021/acs.chemrev.7b00020 86. Das M, Manna K. Chalcone Scaffold in Anticancer Armamentarium: A Molecular Insight. J Toxicol . 2016;2016:1–14. Available from: http://dx.doi.org/10.1155/2016/7651047 87. Lin Y, Zhang M, Lu Q, Xie J, Wu J, Chen C. A novel chalcone derivative exerts anti- inflammatory and anti-oxidant effects after Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 31 acute lung injury. Aging (Albany NY). 2019;11(18):7805–16. 88. Zhu H, Tang L, Zhang C, Wei B, Yang P, He D, et al. Synthesis of Chalcone Derivatives: Inducing Apoptosis of HepG2 Cells via Regulating Reactive Oxygen Species and Mitochondrial Pathway. Front Pharmacol . 2019 Nov 15;10. Available from: www.frontiersin.org 89. Gaonkar SL, Vignesh UN. Synthesis and pharmacological properties of chalcones: a review. Res Chem Intermed. 2017;43(11):6043–77. 90. Syahri J, Yuanita E, Nurohmah BA, Armunanto R, Purwono B. Chalcone analogue as potent anti-malarial compounds againstPlasmodiumfalciparum: Synthesis, biological evaluation, and docking simulation study. Asian Pac J Trop Biomed . 2017;7(8):675–9. Available from: http://dx.doi.org/10.1016/j.apjtb.2017.07.004 91. Shaik A, Bhandare RR, Palleapati K, Nissankararao S, Kancharlapalli V, Shaik S. Antimicrobial, antioxidant, and anticancer activities of some novel isoxazole ring containing chalcone and dihydropyrazole derivatives. Molecules. 2020;25(5). 92. Bale AT, Salar U, Khan KM, Chigurupati S, Fasina T, Ali F, et al. Chalcones and Bis- Chalcones Analogs as DPPH and ABTS Radical Scavengers. Lett Drug Des Discov. 2020;18(3):249–57. 93. Shalaby E, Azzam GM. Antioxidants in Foods and Its Applications. In: Shalaby E, editor. Food and Nutrition . London: IntechOpen; 2018. Available from: https://www.intechopen.com/books/6678 94. Kłósek M, Kuropatnicki AK, Szliszka E, Korzonek-Szlacheta I, Król W. Chalcones Target the Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Signaling Pathway for Cancer Chemoprevention . Nutrition and Functional Foods for Healthy Aging. Elsevier Inc.; 2017. 233–244 p. Available from: http://dx.doi.org/10.1016/B978-0-12-805376- 8.00020-4 95. Bajpai VK, Baek KH, Kang SC. Antioxidant and free radical scavenging activities of taxoquinone, a diterpenoid isolated from Metasequoia glyptostroboides. South African J Bot . 2017;111:93–8. Available from: http://dx.doi.org/10.1016/j.sajb.2017.03.004 96. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn Rev . 2010 Jul;4(8):118–26. Available from: https://pubmed.ncbi.nlm.nih.gov/22228951 97. Nordin NA, Ibrahim AR, Ngaini Z. Biological Studies of Novel Aspirin-Chalcone Derivatives bearing Variable Substituents. J Agrobiotechnology . 2020 Mar 8;11(1):20–31. Available from: https://journal.unisza.edu.my/agrobiotechnolog y/index.php/agrobiotechnology/article/view/18 5 98. Ngaini Z, Hui DHA, Hussain H, Wan Zulkiplee WSH, Tay MG, Sahari N, et al. Synthesis and Antibacterial Study of Aspirin-Chalcone Derivatives. Borneo J Resour Sci Technol . 2013 Jan 1;3(1):52–7. Available from: http://publisher.unimas.my/ojs/index.php/BJR ST/article/view/256 99. Poirel L, Madec J-Y, Lupo A, Schink A-K, Kieffer N, Nordmann P, et al. Antimicrobial Resistance in Escherichia coli. Aarestrup FM, Schwarz S, Shen J, Cavaco L, editors. Microbiol Spectr . 2018 Jul 27;6(4). Available from: https://journals.asm.org/doi/10.1128/microbiol spec.ARBA-0026-2017 100. Krishnamoorthy G, Wolloscheck D, Weeks JW, Croft C, Rybenkov V V., Zgurskaya HI. Breaking the permeability barrier of Escherichia coli by controlled Hyperporination of the outer membrane. Antimicrob Agents Chemother. 2016;60(12):7372–81. 101. San Miguel-Chávez R. Phenolic Antioxidant Capacity: A Review of the State of the Art. In: Phenolic Compounds - Biological Activity . InTech; 2017. p. 13. Available from: http://www.intechopen.com/books/phenolic- compounds-biological-activity/phenolic- antioxidant-capacity-a-review-of-the-state-of- the-art 102. Ngaini Z, Kui HB. Synthesis and Antibacterial Activity of Azo and Aspirin-azo Derivatives. Malaysian J Anal Sci. 2017;21(5):1183–94. 103. Nowok A, Dulski M, Grelska J, Szeremeta AZ, Jurkiewicz K, Grzybowska K, et al. Phenyl Ring: A Steric Hindrance or a Source of Different Hydrogen Bonding Patterns in Self- Organizing Systems? J Phys Chem Lett. 2021;12(8):2142–7. 104. Tran PHL, Wang T, Yin W, Tran TTD, Nguyen TNG, Lee BJ, et al. Aspirin-loaded nanoexosomes as cancer therapeutics. Int J Pharm. 2019 Dec 15;572:118786. 105. Amaral MEA, Nery LR, Leite CE, de Azevedo Junior WF, Campos MM, Eduarda Azambuja Amaral M, et al. Pre-clinical effects of metformin and aspirin on the cell lines of different breast cancer subtypes. Invest New Drugs . 2018;36(5):782–96. Available from: https://doi.org/10.1007/s10637-018-0568-y 106. Zhang S, Li T, Zhang L, Wang X, Dong H, Li L, et al. A novel chalcone derivative S17 induces apoptosis through ROS dependent DR5 up-regulation in gastric cancer cells OPEN. Sci Iraqi J Pharm Sci, Vol.31(2) 2022 Exploring Aspirin derivatives 32 Rep . 2017;7(9873). Available from: www.nature.com/scientificreports/ 107. Zou Z, Chang H, Li H, Wang S. Induction of reactive oxygen species: an emerging approach for cancer therapy. Apoptosis . 2017;22(11):1321–35. Available from: http://dx.doi.org/10.1007/s10495-017-1424-9 108. Wang G, Liu W, Gong Z, Huang Y, Li Y, Peng Z. Synthesis, biological evaluation, and molecular modelling of new naphthalene- chalcone derivatives as potential anticancer agents on MCF-7 breast cancer cells by targeting tubulin colchicine binding site. 2019; Available from: https://www.tandfonline.com/action/journalInf ormation?journalCode=ienz20 109. Hsu YL, Kuo PL, Tzeng WS, Lin CC. Chalcone inhibits the proliferation of human breast cancer cell by blocking cell cycle progression and inducing apoptosis. Food Chem Toxicol. 2006 Jun 1;44:704–13. 110. Lee DY, Lee KP, Beak S, Park JS, Kim YJ, Kim KN, et al. Antibreast Cancer Activity of Aspirin-Conjugated Chalcone Polymeric Micelles. Macromol Res . 2021;29(1):105–10. Available from: www.springer.com/13233pISSN1598- 5032eISSN2092-7673 111. Maity G, De A, Das A, Banerjee S, Sarkar S, Banerjee SK. Aspirin blocks growth of breast tumor cells and tumor-initiating cells and induces reprogramming factors of mesenchymal to epithelial transition. Lab Investig . 2015;95:702–17. Available from: www.laboratoryinvestigation.org 112. Zhang Y, Huang Y, Li S. Polymeric Micelles: Nanocarriers for Cancer-Targeted Drug Delivery. AAPS PharmSciTech . 2014 Aug 4;15(4):862–71. Available from: http://link.springer.com/10.1208/s12249-014- 0113-z 113. Shin SY, Lee JM, Lee MS, Koh D, Jung H, Lim Y, et al. Targeting Cancer Cells via the Reactive Oxygen Species-Mediated Unfolded Protein Response with a Novel Synthetic Polyphenol Conjugate. 2014 [cited 2021 May 4]; Available from: http://clincancerres.aacrjournals.org/ 114. Ngaini Z, Mortadza NA, Zainab N, Arif MN. Synthesis of halogenated azo-aspirin analogues from natural product derivatives as the potential antibacterial agents. Nat Prod Res . 2019;33(24):3507–14. Available from: https://doi.org/10.1080/14786419.2018.148631 0 115. Zhao Q, Qu J, He F. Chlorination: An Effective Strategy for High-Performance Organic Solar Cells. Adv Sci. 2020;7(14):1–25. 116. Mahdi BM. Chapter 7 - Role of Antimicrobial Agents in the Management of Perianal Abscess. In: Hasan RM, Mahdi BMBT-NC in the M of SPC, editors. Academic Press; 2018. p. 71–7. Available from: https://www.sciencedirect.com/science/article/ pii/B9780128161111000070 117. Kuswandi B, Futra D, Heng LY. Chapter 15 - Nanosensors for the Detection of Food Contaminants. In: Oprea AE, Grumezescu AMBT-NA in F, editors. Academic Press; 2017. p. 307–33. Available from: https://www.sciencedirect.com/science/article/ pii/B9780128119426000157 118. Yoo J. Review of Disinfection and Sterilization – Back to the Basics. Infect Chemother . 2018;50(2):101. Available from: https:// icjournal. org/ DOIx.php? id=10.3947 /ic. 2018.50.2.101 119. Cherdtrakulkiat R, Boonpangrak S, Sinthupoom N. Derivatives ( halogen , nitro and amino ) of 8-hydroxyquinoline with highly potent antimicrobial and antioxidant activities. Biochem Biophys Reports . 2016;6:135–41. Available from: http:// dx.doi. org/10.1016 /j.bbrep .2016.03.014 This work is licensed under a Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/