European Journal of Pharmaceutical Sciences 140 (2019) 105101

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European Journal of Pharmaceutical Sciences

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Synthesis and biological evaluation of new prodrugs of etodolac and T with reduced ulcerogenic potential ⁎ Sonia T. Hassiba, Ghaneya S. Hassana, Asmaa A. El-Zahera, Marwa A. Fouada, , Omnia A. Abd El-Ghafarb, Enas A. Tahac a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, b Pharmacology and Toxicology Department, Faculty of Pharmacy, Nahda University, Beni-Suef, Egypt c Pharmaceutical Chemistry Department, Faculty of Pharmacy, 6th October University, Cairo, Egypt

ARTICLE INFO ABSTRACT

Keywords: Gastric irritation and ulcerogenic effect of the acidic NSAIDs are of the most challenging problems in designing Etodolac novel anti-inflammatory agents. In this study, the new prodrugs were prepared through Steglich esterification Tolfenamic acid reaction between the carboxylic acid functional group of etodolac or tolfenamic acid and thymol. The structures Thymol were confirmed by IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. Their chemical stability in Prodrug addition to a kinetic study of their hydrolysis in 20% liver homogenate and 10% buffered plasma were in- Anti-inflammatory vestigated. In vitro enzymatic hydrolysis showed half-life times 88.84 and 106.61 min for the prodrugs of eto- Ulcerogenicity dolac and tolfenamic acid, respectively. Their ability to inhibit paw edema and their ulcerogenic potential were assessed in rats and compared to their parent drugs. the prodrugs were found to be stable in different at room and body temperatures. Both prodrugs proved to possess high percentage of inhibition of paw edema (94.68 & 97.1%) in rats comparable to that of the parent drugs (90.33 & 93.23%) and, most importantly with lower ulcerogenic potential. The prodrugs are expected to be converted to their parent drugs rapidly in plasma and liver in vivo and proved to be safer than their parent drugs. The study opens a perspective chance that can be a backbone for further investigations.

1. Introduction The use of NSAIDs can be associated with variable side effects such as renal dysfunction, cardiovascular adverse events, triggering Non-steroidal anti-inflammatory drugs (NSAIDs) represent an im- and can significantly increase clotting times (Graham et al., 2005). As portant therapeutic class of drugs which in addition to their anti-in- well, it has been demonstrated that NSAIDs exhibit adverse effects on flammatory effects, may possess both and antipyretic activ- the gastrointestinal tract including nausea, vomiting and diarrhea. ities (Mehanna, 2003). The mechanism of action of NSAIDs involves These later adverse effects are exhibited through two different me- inhibition of (COX) that initiate the formation chanisms: the first action exerted locally by the direct effect oftheor- of (Warner and Mitchell, 2003). There are 3 subtypes of ganic acid moiety of the drug with gastric mucosa (Foye et al., 2008) COX enzymes: COX-1 (constitutive), COX-2 (inducible in inflammatory and a general systemic mechanism involving E2 (PGE2) processes), and the isozyme COX-3 (Katzung et al., 2019). A drug must and (PGI2), which protect gastric mucosa by stimulating be highly lipophilic and acidic as to competitively bicarbonate and mucin secretion and increasing mucosal blood flow inhibit the natural substrate (Brune, 2007). (Brown et al., 2012; Wallace and Vong, 2008). Etodolac (1) (Fig. 1) is an derivative, which is mainly Prodrugs are transformed enzymatically and/or chemically in vivo used in postoperative pain and rheumatic diseases. It undergoes rapid in order to exert the required pharmacological effect by releasing the metabolism in the liver, and is eliminated primarily by the kidney active parent drug (Foye et al., 2008). This approach is widely used in (Sharav and Benoliel, 2008). Tolfenamic acid (2) (Fig. 1) is a fenamate medicinal chemistry as it can improve the undesirable properties and NSAID, used for pain and management, also in decreasing sometimes decrease the clinical usefulness of an existing drug. Ester the intensity and duration of migraine (Colon et al., 2011; Smith et al., prodrugs for NSAIDs containing carboxylic acid group can be helpful to 2008). depress the GIT irritation and (Sheha et al., 2002). One of the

⁎ Corresponding author. E-mail address: [email protected] (M.A. Fouad). https://doi.org/10.1016/j.ejps.2019.105101 Received 10 July 2019; Received in revised form 22 September 2019; Accepted 5 October 2019 Available online 19 October 2019 0928-0987/ © 2019 Elsevier B.V. All rights reserved. S.T. Hassib, et al. European Journal of Pharmaceutical Sciences 140 (2019) 105101

Fig. 1. Chemical structures of etodolac, tolfenamic acid, thymol and some reported synthesized prodrugs.

Scheme 1. Reagents and reaction conditions: (a) DCC, 4-DMAP, DCM, stir, RT, 72 hr possible approaches to prevent GIT irritation problem is to hinder the stability in different temperatures and pHs. Moreover, a kinetic studyin acidic moiety by esterification without affecting its clinical effect 20% liver homogenate and 10% buffered plasma were carried out to (Kashfi et al., ).2002 evaluate their enzymatic hydrolysis. In addition, the prodrugs were Thymol (3) (Fig. 1) is the essential dietary constituent in . It evaluated for their in vivo anti-inflammatory activity and for their ul- has been used for longtime in traditional medicine as it possesses var- cerogenic effect compared to the corresponding active drugs. ious pharmacological properties such as antioxidant, anti-in- flammatory, analgesic, antispasmodic, and antitumor activ- 2. Results and discussion ities. The anti-inflammatory effect of thymol is believed to be largely attributed to inhibiting recruitment of cytokines and chemokines 2.1. Chemistry (Ojha et al., 2017). In literature, some ester and amide prodrugs of etodolac and tolfe- The target prodrugs, 2-isopropyl-5-methylphenyl-2-(1,8‑diethyl‑4,9- namic acid (as prodrugs 4&5) were prepared to increase their bioa- dihydro-3H-pyrano[3,4-b]indol-1-yl)acetate (6) and 2-isopropyl-5-me- vailability and decrease their GI tract adverse effects, Fig. 1 thylphenyl-2-[(3‑chloro‑2-methylphenyl)amino]benzoate (7) were pre- (Chaudhary et al., 2013; Paliwal et al., 2017; Pandey et al., 2018). pared via Steglich esterification (Bringmann et al., 2012; Neises and Thus, this work was aimed to synthesize new derivatives through an Steglich, 1978) as depicted in schemes 1&2. esterification reaction between the carboxylic acid functional group in Schemes 1&2: Reagents and reaction conditions: (a) DCC, 4-DMAP, etodolac or tolfenamic acid and thymol to give two new mutual pro- DCM, stir, RT, 72 hr drugs. Thymol was chosen as it has a well-known safety profile and is The well-known Steglich reaction is an esterification reaction that traditionally used for its medicinal as well as flavoring properties takes place at room temperature through the formation of the O-acy- (Ojha et al., 2017). Releasing a safe promoeity after their hydrolysis is a lisourea derivative of the carboxylic acid derivative by coupling with big challenge in designing new prodrugs. dicyclohexylcarbodiimide (DCC) in the presence of 4-N,N-dimethyla- 1 13 The new prodrugs structures were confirmed by IR, H NMR, C minopyridine (4-DMAP). or are easily added to this NMR, mass spectroscopy and elemental analysis. After synthesis and intermediate to form the ester releasing 1,3-dicyclohexylurea as by- structure confirmation, the prodrugs were evaluated for their chemical product. is the most suitable used solvent. In practice,

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Scheme 1. (continued)

Table 1 Table 2 Anti-inflammatory effect of and the tested compounds oncarra- Ulcerogenic effect of ibuprofen and the tested compounds in rats. geenan induced edema. Ulcer Index (Relative ulcerogenicity to parent drug) Groups % of inhibition of paw edema Compound After 1hr After 3hr After 3 days 1hr 2hr 3hr 4hr 5hr 6hr (3 consecutive doses) Control 0 0 0 0 0 0 Control 0 0 0 Ibuprofen 61.93 77.50 81.81 89.27 94.33 95.65 Ibuprofen 9.17 11.00 16.00 (1) 41.38 43.25 74.30 86.26 89.15 90.33 (1) 6.37 10.50 14.83 Prodrug (6) 22.35 23.52 35.17 84.54 91.50 94.68 Prodrug (6) 0 (0) 6.00 (0.57) 11.99 (0.81) (2) 36.55 40.48 75.09 86.26 92.45 93.23 (2) 6.34 6.49 15.49 Prodrug (7) 27.49 29.75 49.80 87.98 94.81 97.10 Prodrug (7) 0 (0) 3.99 (0.61) 14.32 (0.92)

1 1 4-DMAP is critical for ester formation (Bringmann et al., 2012; was confirmed in both IR and H NMR spectra. In addition, H NMR Neises and Steglich, 1978). spectrum showed a doublet equivalent to 6 (2 CH3) at 1.27 δ IR spectrum of prodrug (6) revealed the disappearance of the car- ppm and two singlets more deshielded at 2.36 and 2.40 δ ppm (2 Ar- 1 boxylic and phenolic OH band, which was also confirmed in its H NMR CH3). Also, the integration of aromatic protons was increased including spectrum. Compared to the parent drug (1), the 1H NMR spectrum ten aromatic protons at 6.84–8.27 δ ppm and one exchangeable singlet 13 showed three additional signals each equivalent to 3 protons (3 CH3), at 9.36 δ ppm (NH). Compared to its parent drug (2), C NMR spec- two are doublets at 1.20 and 1.22 δ ppm referred to the nonequivalent trum of (7) revealed additional three signals at 20.92 (Ar-CH3), 23.16 methyl groups of the isopropyl group, and the third is a singlet of the 5́ (2 CH3), and 27.3 δ ppm (CH) in addition to an increase in aromatic methyl group which is more deshielded at 2.37 δ ppm. Also, the spec- carbons by six carbons. The mass spectrum of (7) revealed the mole- + + trum showed an increase in the aromatic protons due to three addi- cular ion peaks (M ) and M +2 at 393 and 395 in the ratio of 3:1 due tional aromatic protons within the range 6.79–7.45 δ ppm and an ex- to the presence of chlorine isotopes. changeable singlet at 9.01 δ ppm (NH). The 13C NMR spectrum revealed five signals at 7.76 - 23.21 δ ppm assigned to five CH groups in ad- 3 2.2. Anti-inflammatory activity and ulcerogenic effect dition to an increase in aromatic carbons by six carbons compared to the parent drug (1). The mass spectrum of (6) revealed the parent The new prodrugs were evaluated for their anti-inflammatory ac- molecular ion peak at 419 assigned to its molecular weight. tivity using carrageenan-induced rat paw edema method reported by For prodrug (7), the absence of both carboxylic and phenolic OH Winter et al. (Hassanein et al., 2017; Winter et al., 1962) and compared

Fig. 2. % Inhibition of paw edema of ibuprofen and the tested compounds at different time intervals.

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Fig. 3. Ulcerogenic effect of ibuprofen and the tested compounds in rats after 1 hr of their administration.

Fig. 4. Ulcer index of ibuprofen and the tested compounds in rats. to their parent active drugs (Etodolac and Tolfenamic acid) using ibu- additional anti-inflammatory effect of thymol, the released promoiety. profen as reference standard. As can be seen in Table 1 (and Table S1 in In addition, the new prodrugs were estimated for their gastric ul- Supporting Information File), the prodrugs showed lower% of paw cerogenic potential in rats and compared to their parent drugs edema inhibition than their parent drugs in the first 3 h while after (Etodolac and Tolfenamic acid) and ibuprofen (Reference standard). 4–6 h, the percentage increased to become comparable and even higher Their ulcer indices were calculated after 1 and 3 hr after drug admin- than the parent drugs, Fig. 2. The delayed effect can be attributed to the istration, and after three consecutive daily doses. The results are pre- time required for the hydrolysis of the prodrugs to their bioactive sented in Table 2 and Figs. 3and 4 (Tables S2-S4 and Figures S1-S2 in parent drugs, while the slight increase in activity can be owing to the Supporting Information File). None of the prodrugs at the dose of

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Fig. 5. Enzymatic hydrolysis of (A) prodrug (6) and (B) prodrug (7) in 20% liver homogenate at 37 °C.

50 mg/kg resulted in changes in the gastric mucosa after 1 h while in obtained by plotting the natural logarithm of the percentage of re- the same experimental conditions, the clinically used drugs, ibuprofen, maining concentration versus time was linear (See Supporting etodolac and tolfenamic acid caused visible changes in the mucosa of Information File, Figures S3 and S4). the stomach, thereby revealing their irritant action. This could be at- tributed to depressing the local action due to the direct contact of the 2.4. In vitro enzymatic hydrolysis organic acid moiety. Furthermore, the ulcer index increased after 3 consecutive doses which may be mainly caused by the general systemic To study the rate of enzymatic hydrolysis, four calibration curves mechanism PGE2 and PGI2 (Brown et al., 2012; Foye et al., 2008; were constructed for compounds (1), (6), (2), and (7) in 20% liver Wallace and Vong, 2008). homogenate and in 10% buffered plasma at 37 °C as presented in Figs. 5 and 6), respectively. The complete hydrolysis profile was studied by 2.3. Chemical stability plotting the natural logarithm of the remaining concentrations of the new prodrugs vs time in minutes. Figs. 7 and 8) show that the reactions The chemical stability of the new prodrugs was studied at three follow pseudo first order. The half-life time of the reactions wascal- different pHs, simulating the pHs of the stomach (1.2), the colon (6.8) culated using the slope (k) of each plotted line using equation-1 and the physiological pH (7.4) and at three different temperatures, (Moussa et al., 2018; Richardson et al., 2016), Table 3. As can be seen, body (37 °C), room (25 °C) temperatures and drastic storage conditions the hydrolysis of the prodrugs is faster in liver homogenate than in (60 °C). It was found that the hydrolysis rate of the two prodrugs in- plasma. Accordingly, the prodrugs may be successfully metabolized in creased with temperature. It is also obvious that prodrugs exhibited vivo to yield their parent drugs. higher stability at 25 and 37 °C than 60 °C at the three studied pHs. This t1/2 = 0.693/k (1) stability at room temperature is highly preferred as it facilitates its formulation in the required pharmaceutical dosage form. Where k: the specific reaction rate constant, t1/2: the half-life time On the other side, the prodrugs were found to be stable to chemical hydrolysis in all the pHs at room and body temperatures. This high 3. Conclusion stability indicates that the prodrugs will pass safely through the gas- trointestinal tract without hydrolysis after oral administration, thus Two mutual prodrugs of etodolac and tolfenamic acid with thymol keeping the gastric mucosa protected. In the studied conditions, the were successfully synthesized in a rather simple single step scheme. hydrolytic reaction follows a pseudo first order as the relationship Their chemical structures were confirmed by 1H NMR, 13C NMR, FT-IR,

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Fig. 6. Enzymatic hydrolysis of (A) prodrug (6) and (B) prodrug (7) in 10% buffered plasma at 37 °C. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)

Mass spectroscopy and elemental analysis. The prodrugs and their the reactions were followed up by TLC using Kieselgel 60 F254 sheets bioactive parent drugs were determined in aqueous and biological (Merck, Darmstadt, Germany) and : 9.5:0.5 or samples using a developed and validated HPLC method. The two pro- pure chloroform as the eluting system and the spots were visualized at drugs were found to be stable at pH 1.2, 6.8 and 7.4 in room and body 254 nm by UV Vilber Lourmat, Marne La Vallee, . IR spectra (KBr temperatures. The in vitro enzymatic hydrolysis of the prodrugs was disc) were recorded on a Schimadzu FT-IR recording spectrophotometer faster in human plasma and liver homogenate than chemical hydrolysis, affinity (IR-470, Schimadzu, Kyoto, ). The NMR spectra were with faster metabolic rate in liver than in plasma. Thus, it can be de- recorded on Bruker AVANCE III400 MHz FT-NMR spectrometer. 1H duced that the prodrugs are expected to be converted in vivo to their spectra were run at 400 MHz while 13C spectra were run at 100 MHz in parent drugs rapidly in plasma and liver. In addition, the new prodrugs deuterated CDCl3. Mass spectra were recorded using Single Quadruple were evaluated for their anti-inflammatory activity showing higher mass spectrometer ISQ LT (Japan). Elemental Microanalyses were car- delayed inhibition of paw edema than their parent drugs due to the ried out at the Regional Center for Mycology and Biotechnology, Al- time required for their in vivo hydrolysis. By studying their ulcerogenic Azhar University. effect, the prodrugs were proved to be safer than their parent drugs mostly due to masking their acidic group. The study opens a perspective chance that can be a backbone for further investigations. 4.1.1. General esterification procedure A mixture of 1 or 2 (10 mmol), 3 (1.52 g, 1.58 mL, 10.1 mmol), DCC (2.27 g, 11 mmol) and 4-DMAP (0.24 g, 2 mmol) in anhydrous di- 4. Experimental chloromethane (100 mL), was stirred at room temperature for 72 hr. The white precipitate formed (N,N'-dicyclohexylurea) was discarded by 4.1. Chemistry filtration, and the filtrate was washed with water, then with 1%diluted HCl. The organic layer was separated and concentrated under vacuum. Solvents and chemicals were purchased from commercial sources The residue was crystallized from absolute ethanol to obtain white and used without purification. Melting points were recorded using crystals of prodrug (6) or (7). BIBBY scientific Stuart apparatus, Staffordshire, UK.All

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Fig. 7. The complete hydrolysis profile of prodrug (6) in (A) 20% liver homogenate and (B) 10% buffered plasma. (For interpretation of the references to colourin this figure legend, the reader is referred to the web version of this article.)

4.1.1.1. 2-Isopropyl-5-methylphenyl 2-(1,8‑diethyl‑,4,9-dihydro-3H- 2938, 2861 (CH aliphatic), 1693 (C = O), 1665 (NH bending). 1H NMR pyrano[3,4-b]indol-1-yl)acetate (6).Rf: 0.80 (CCl4:CH3COOC2H5 9:1). (400 MHz, in deuterated CDCl3) δ ppm: 1.27 (d, 6H, (CH3)2CH, J 6.88 Hz) −1 Yield: (3.5 g) 83.5%. mp: 116–120 °C. IR (KBr) υmax/cm : 3385 (NH), 2.36 (s, 3H Ar-CH3,), 2.40 (s, 3H, Ar-CH3), 3.11 (m, 1H, (CH3)2CH), 6.84 3070, 3043 (CH Ar), 2992, 2914, 2848 (CH aliphatic), 1711 (C = O), (t, 1H, C5′-H-Ar, J 8.00 Hz), 6.94 (d, 1H,C4"-H Ar, J 8.00 Hz), 7.01 (s, 1H, 1 1615 (NH bending). H NMR (400 MHz, in deuterated CDCl3) δ ppm: C6"-H Ar), 7.12 (d, 1H, C3-H, J 7.68), 7.15 (t, 1H, C5-H Ar, J 7.96 Hz), 0.99 (t, 3H, CH2CH3, J 7.36 Hz), 1.20 (d, 3H, CHCH3, J 6.92 Hz), 1.22 7.24 (d, 1H, C3"-H, J 7.36), 7.30 (d, 2H, C4′-H Ar and C6′-H, J 7.88 Hz), (d, 3H, CHCH3, J 6.88 Hz), 1.35 (t, 3H, CH2CH3, J 5.84 Hz), 2.19 (m, 7.39–7.43 (d,t, 1H, C4-H, J 7.16 Hz), 8.27–8.29 (dd, 1H, C6-H Ar, J 13 1H, C4-H pyran), 2.29 (m, 1H, C4-H pyran), 2.37 (s, 3H, Ar-CH3), 8.04 Hz) 9.36 (s, 1H, NH, exch. D2O). C NMR (100 MHz, CDCl3) δ ppm: 2.83–2.98 (m, 5H, CH2CH3, CH2CH3 and CHCH3), 3.27 (d, 1H, CH 15.03, 20.92 (Ar-CH3), 23.16, (2 CH3), 27.30 (CH), 110.64, 113.99, alpha C = O, J 17.00 Hz), 3.33 (d, 1H, CH alpha C = O, J 16.96 Hz), 117.19, 123.00, 123.02, 125.85, 126.61, 126.87, 127.34, 131.70, 4.08 (m, 1H, C3-H pyran), 4.09 (m, 1H, C3-H pyran), 6.79 (s, 1H, C6′-H 131.96, 134.97, 135.67, 136.74, 137.47, 140.19, 147.90, (Ar C), Ar), 7.05 (d, 1H, C4′-H Ar, J 7.08 Hz), 7.11 (d, 1H, C3′-H Ar, J 7.64 Hz), 149.28 (CO), 167.59 (C = O). MS (m/z,%): 393.20 (M+, 39.98), + + 7.15 (t, 1H, C6-H Ar, J 7.48 Hz), 7.29 (d, 1H, C5-H Ar, J 7.92 Hz), 7.45 395.13 (M , M +2 16.59). Anal. Calcd. for C24H24ClNO2 (393.9059): 13 (d, 1H, C7-H Ar, J 7.72 Hz), 9.01 (s, 1H, NH, exch. D2O). C NMR C,73.18; H, 6.14; N, 3.56%. Found: C, 72.59; H, 5.96; N, 4.10%. (100 MHz, CDCl3) δ ppm: 7.76, 13.85 (2 CH3), 20.87 (C-4 pyran), 22.52, 22.98, 23.21 (3 CH3), 24.15 (CH), 27.17, 30.62 (2 CH2), 43.02 (CH2 alpha C = O), 60.78 (C-3 pyran), 74.72 (C-1 pyran), 108.61, 4.2. Anti-inflammatory activity and ulcerogenic effect 116.02, 119.77, 120.56, 122.58, 126.24, 126.71, 126.79, 127.68, 134.57, 135.83, 136.88, 137.17 (Ar C), 147.59 (CO), 172.06 (C = O). 4.2.1. Animals + MS (m/z,%): 419.27 (M , 100). Anal. Calcd. for C27H33NO3 Adult male albino Wistar rats, weighing 170–200 g, were obtained (419.5558): C, 77.29; H, 7.93; N, 3.34%. Found: C,76.89; H, 7.91; N, from Nahda University Animal House, Beni-Sueif, Egypt. Animals were 3.51%. acclimatized for one week in a room controlled for temperature, hu- midity, and light (12 h light–dark cycle). Rats were housed in well ventilated cages with free access to food and water. All experimental 4.1.1.2. 2-Isopropyl-5-methylphenyl-2-[(3‑chloro‑2-methylphenyl) amino] procedures were approved by the institutional guidelines of the benzoate (7). Rf: 0.85 (CCl4:CH3COOC2H5 9:1). Yield: (3 g) 76.3%. Mp: Research Ethics Committee of Faculty of Pharmacy, Cairo University −1 111–114 °C. IR (KBr) υmax/cm : 3323 (NH), 3085, 3046 (CH Ar), 2975, and the regulations of the local Animal Welfare authorities.

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increase in paw thickness in the control group of rats and (VR -VL) treated represents the mean increase in paw thickness in rats treated groups.

4.2.2.2. Ulcerogenic effect. After one-week adaptation, three sets of experiments were performed. Each set was containing twenty-four healthy rats that were randomly divided into 6 groups, each of 4 rats as follow: Group 1 (control group, 2% Tween 80+saline); Group II (ibuprofen group, 25 mg/kg); Group III (etodolac group, 50 mg/kg); Group IV (prodrug (6) group, 50 mg/kg); Group V (tolfenamic acid group, 50 mg/kg); Group VI (prodrug (7) group, 50 mg/kg). All rats were deprived of food but not water for 18 h before drug administration. Then, each of ibuprofen, etodolac, prodrug (6), tolfenamic acid and prodrug (7) at the specified doses was suspended in 2% Tween 80 and administrated orally to animals. One hr after drug administration, animals in the first set of experiment were sacrificed by decapitation; then the stomachs were removed, opened along the greater curvature, washed with distilled water and cleaned in normal saline 0.9%. Animals in the second set of experiment were sacrificed after 3 h, while another two doses of drugs (once daily) were orally administered to the animals in the third set for the following two days, then sacrificed in the fourth day after 24 hr of the last dose of the drugs. The mucosal damage of each stomach was examined using a mag- nifying lens to check for the presence of macroscopically visible lesions. The number of lesions in each stomach, if any, was counted and re- corded. The ulcerogenic effect was evaluated according to Meshali's method (Hassan et al., 2014; Meshali et al., 1983) and ulcer index was calculated according to the method of Robert et al. (Hassan et al., 2014; Robert et al., 1970).

Fig. 8. The complete hydrolysis profile of prodrug (7) in (A) 20% liver 4.3. Prodrug chemical stability and in vitro studies homogenate and (B) 10% buffered plasma at 37 °C. (For interpretation ofthe references to colour in this figure legend, the reader is referred to theweb version of this article.) 4.3.1. Method of analysis of prodrug and bioactive products a RP-HPLC method was developed and validated for the determi- nation of each prodrug in mixture with its bioactive compounds in Table 3 aqueous medium, liver homogenate and human plasma. The chroma- The regression parameters, observed rate constants and half-lives for the hy- tographic separation was achieved on a Thermo RP-C18 column drolysis of the new prodrugs:. (250 mm x 4.6 mm, 5 µm), at ambient temperature using a mobile 20% Liver homogenate 10% Buffered plasma phase consisting of 0.2% formic acid (A) and acetonitrile (B) with a Prodrug (6) (7) (6) (7) gradient mode according to Table S5 (See Supporting Information File) K (min −1) 0.0078 0.0075 0.0065 0.006 obs at a flow rate of 1 mL/min. UV detection was carried out at 274nmfor t½ (min) 88.84 92.4 106.61 115.5 prodrug (6) and 280 nm for prodrug (7), each in the presence of its bioactive prodrugs with good resolution as shown in Figures S5 and S6) 4.2.2. Study design and procedure (See Supporting Information File). 4.2.2.1. Anti-inflammatory activity. After one-week adaptation, healthy rats were randomly divided into six groups, each of 4 rats as follow: 4.3.2. Preparation of standard and working solutions Group 1 (control group); Group II (ibuprofen group); Group III Standard stock solutions of compounds 1–3 and their corresponding (etodolac group); Group IV (prodrug (6) group); Group V (tolfenamic prodrugs 6&7 were prepared in methanol to obtain solutions of 0.1 mg/mL acid group); Group VI (prodrug (7) group). (for chemical stability) and 10 mg/mL (for in vitro enzymatic hydrolysis). Each of ibuprofen (25 mg/kg), etodolac (50 mg/kg), prodrug 6 (50 mg/ kg), tolfenamic acid (50 mg/kg) and prodrug 7 (50 mg/kg) was suspended 4.3.3. Preparation of liver homogenate in 2% Tween 80 and administrated orally 30 min before induction of the Five adult albino Wistar male and female rats, (300–400 g), free of inflammation, while the control group received only 2% Tween 80. any signs of observable clinical abnormalities were used. The rats were Subplantar injection of 0.1 mL of 1% carrageenan solution in saline housed in an air-conditioned room and fed standard meals and water. To (0.9%) in the right leg induced paw edema. Paw edema thickness was collect the livers, the rats were systemically anesthetized with measured after 1, 2, 3, 4, 5 and 6 hr in the right hind paw using a screw hydrochloride (35 mg/kg) IM injections in combination with a muscle gauge micrometer and then compared with the left hind paw thickness relaxant xylazine hydrochloride (5 mg/kg). Livers were removed by of each rat. The difference of average values between treated and surgery, and immediately homogenized by tissue homogenizer, then control groups was calculated for each time interval and evaluated centrifuged at 6000 rpm for 20 min. at 4 °C then the supernatant was statistically. Quantitative variables were expressed as means ± separated carefully (El-Bagary et al., 2016; Moussa et al., 2019; 2018). standard error (SEM). The anti-inflammatory activity was expressed as percentage inhibition of edema volume in treated animals by compar- 4.3.4. Preparation of 10% buffered plasma ison to the control group according to the following equation: Pooled human plasma was donated from national blood bank % of edema inhibition = (VR -VL) control - (VR -VL) treated ×100 (Vacsera, Egypt). Human plasma (4 mL) was diluted with 1 mL of (VR -VL) controlWhere VR represents the right paw thickness, VL 0.05 M phosphate buffer of pH 7.4 followed by incubation at 37±1°C represents the left paw thickness, (VR -VL) control represents the mean in a shaking water bath for 5 min.

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4.3.5. Preparation of blank buffered human plasma or liver supernatant configurationally unstable biaryl lactone, in. Org. Synth. 70–78. https://doi.org/10.1002/ sample 0471264229.os088.07. Brown, M.J., Sharma, P., Mir, F., Bennett, P.N., 2012. Clinical Pharmacology, 12th ed. Churchill An aliquot (4 mL) of the supernatant or buffered human plasma was Livingstone. placed in a test tube and incubated for 5 min in a shaking water bath Brune, K., 2007. Persistence of NSAIDs at effect sites and rapid disappearance from side-effect compartments contributes to tolerability. Curr. Med. Res. Opin. 23, 2985–2995. https:// equilibrated at 37 ± 1 °C. An aliquot of 150 µL was withdrawn and doi.org/10.1185/030079907X242584. 450 µL methanol was added. The mixture was then centrifuged at Chaudhary, R., Negi, V., Sharma, R., Percha, V., Dobhal, Y., Pant, R., 2013. Design, synthesis 13,000 rpm for 7 min at 4 °C. From the obtained clear supernatant, and evaluation of mutual prodrugs of etodolac with as safer nsaids. Inven. Impact Med. Chem. 3, 152–158. 10 µL was analyzed by HPLC (See Supporting file, Figures S7 and S8). Colon, J., Basha, M.R., Madero-Visbal, R., Konduri, S., Baker, C.H., Herrera, L.J., Safe, S., Sheikh-Hamad, D., Abudayyeh, A., Alvarado, B., Abdelrahim, M., 2011. Tolfenamic acid 4.3.6. Chemical hydrolysis decreases c-Met expression through sp proteins degradation and inhibits lung cancer cells growth and tumor formation in orthotopic mice. Invest. New Drugs 29, 41–51. https://doi. The stability of the newly synthesized prodrugs was studied in aqu- org/10.1007/s10637-009-9331-8. eous phosphate buffer solutions of different pH values (1.2, 6.8, and7.4) El-Bagary, R., Hashem, H., Fouad, M., Tarek, S., 2016. UPLC-MS-MS method for the determi- nation of vilazodone in human plasma: application to a pharmacokinetic study. J. and at three different temperatures (25, 37 and 60 °C). Accurately Chromatogr. Sci. 54, 1365–1372. https://doi.org/10.1093/chromsci/bmw084. measured aliquots equivalent to (1 mg) of prodrugs 6&7 working solu- Foye, W.O., Lemke, T.L., Williams, D.A., 2008. Foye's Principles of Medicinal Chemistry. tions were transferred separately into three volumetric flasks (50 mL), Graham, D.J., Campen, D., Hui, R., Spence, M., Cheetham, C., Levy, G., Shoor, S., Ray, W.A., 2005. Risk of acute myocardial infarction and sudden cardiac death in patients treated with and each flask was completed with a different phosphate buffer solution cyclo-oxygenase 2 selective and non-selective non-steroidal anti-inflammatory drugs: pH (1.2, 6.8 or 7.4). The solutions were well mixed using vortex for nested case-control study. Lancet 365, 475–481. https://doi.org/10.1016/S0140-6736(05) 1 min., then 10 mL of each flask was transferred into a series of 3screw 17864-7. Hassan, G.S., Abou-Seri, S.M., Kamel, G., Ali, M.M., 2014. analogs bearing benzo- capped test tubes and kept in a temperature-controlled water bath shaker furan moiety as cyclooxygenase-2 inhibitors: design, synthesis and evaluation as potential at different temperatures 25, 37 or 60 °C. At appropriate time intervals, anti-inflammatory agents. Eur. J. Med. Chem. 76, 482–493. https://doi.org/10.1016/J. EJMECH.2014.02.033. samples of each tube were withdrawn, cooled and immediately analyzed Hassanein, H.H., Georgey, H.H., Fouad, M.A., El Kerdawy, A.M., Said, M.F., 2017. Synthesis and for their content of the remaining prodrugs, and appearance of parent molecular docking of new imidazoquinazolinones as analgesic agents and selective COX-2 drugs by RP-HPLC. All the experiments were carried out in triplicate. inhibitors. Future Med. Chem. 9, 553–578. https://doi.org/10.4155/fmc-2016-0240. 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An aliquot (150 µL) https://doi.org/10.5688/aj670263. was immediately withdrawn and 450 µL methanol was added to stop Meshali, M., El-Sabbah, E., Foda, A., 1983. Effect of encapsulation of with the reaction and precipitate the proteins. The mixture was then cen- acrylic resins on its bioavailability and gastric ulcerogenic activity in rats. Acta Pharm. Technol. 29, 217–230. trifuged at 13,000 rpm for 7 min at 4 °C. 10 µL of the supernatant was Moussa, B.A., El-Zaher, A.A., El-Ashrey, M.K., Fouad, M.A., 2018. Synthesis and molecular analyzed by HPLC. Calibration curves were constructed showing good docking of new roflumilast analogues as preferential-selective potent pde-4b inhibitors with improved pharmacokinetic profile. Eur. J. Med. Chem. 148, 477–486. https://doi. correlation of more than 0.99. org/10.1016/j.ejmech.2018.02.038. Intra-day accuracy and precision were evaluated by three-replicate Moussa, B.A., Zaher, A.A.El, Ashrey, M.K.El, 2019. Roflumilast analogs with improved meta- analysis of 1, 2, 6 & 7 at concentrations of LLOQ (0.1 mg/mL) in 10% bolic stability, , and pharmacokinetic profile 1–12. doi:10.1002/ dta.2562. buffered plasma or the separated liver supernatant in the sameday Neises, B., Steglich, W., 1978. Simple method for the esterification of carboxylic acids. Angew. following the preparation procedure mentioned above. The inter-day Chemie Int. Ed. English 17, 522–524. https://doi.org/10.1002/anie.197805221. Ojha, S.K., Al Taee, H., Azimullah, S., Javed, H., Nagoor Meeran, M.F., 2017. Pharmacological accuracy and precision were assessed by analyzing three replicates of properties and molecular mechanisms of thymol: prospects for its therapeutic potential and LLOQ on three consecutive days. The precision and accuracy of the pharmaceutical development. Front. Pharmacol. 8, 380. https://doi.org/10.3389/fphar. method were determined by calculating the percent coefficient of var- 2017.00380. 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Mutual amide prodrug of etodolac- : stock solution of each prodrug was diluted with methanol (5.5 mg/mL). synthesis, characterization and pharmacological screening synthesis of mutual prodrug : An aliquot (80 µL) was added in a test tube with 4 mL of the prepared characterization and preformulation studies of the synthesized prodrug : protein binding study: in vitro hydrolysis st 10–15. 10% buffered plasma or the separated liver supernatant and vortexed Richardson, S.J., Bai, A., Kulkarni, A., F. Moghaddam, M., 2016. Efficiency in drug discovery: well for 1 min. The tubes were placed in a shaking water bath at liver S9 fraction assay as a screen for metabolic stability. Drug Metab. Lett. 10, 83–90. 37 ± 1 °C at different time intervals ranging from 0–300 min. Anali- https://doi.org/10.2174/1872312810666160223121836. Robert, A., Northam, J.I., Nezamis, J.E., Phillips, J.P., 1970. Exertion ulcers in the rat. Am. 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NSAID-induced gastrointestinal damage and the design of GI- the online version, at 10.1016/j.ejps.2019.105101. sparing NSAIDs. Curr. Opin. Investig. Drugs 9, 1151–1156. Warner, T.D., Mitchell, J.A., 2003. Nonsteroidal antiinflammatory drugs inhibiting efflux: as easy as ABC? Proc. Natl. Acad. Sci. U. S. A. 100, 9108–9110. https://doi.org/10. References 1073/pnas.1733826100. Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenin-Induced edema in hind paw of the rat as an assay for antiinflammatory drugs. Exp. Biol. Med. 111, 544–547. https://doi.org/10. Bringmann, G., Gulder, T.A.M., Gulder, T., 2012. Discussion addendum for: asymmetric 3181/00379727-111-27849. synthesis of (M)-2-Hydroxymethyl-1-(2-Hydroxy-4,6-Dimethylphenyl)Naphthalene viaa

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