- Email: [email protected]
Synthesis and biological evaluation of new prodrugs of etodolac and tolfenamic acid with reduced ulcerogenic potential
European Journal of Pharmaceutical Sciences 140 (2019) 105101
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Synthesis and biological evaluation of new prodrugs of etodolac and tolfenamic acid with reduced ulcerogenic potential
T
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, Egypt Pharmacology and Toxicology Department, Faculty of Pharmacy, Nahda University, Beni-Suef, Egypt c Pharmaceutical Chemistry Department, Faculty of Pharmacy, 6th October University, Cairo, Egypt b
ARTICLE INFO
ABSTRACT
Keywords: Etodolac Tolfenamic acid Thymol Prodrug Anti-inflammatory Ulcerogenicity
Gastric irritation and ulcerogenic effect of the acidic NSAIDs are of the most challenging problems in designing novel anti-inflammatory agents. In this study, the new prodrugs were prepared through Steglich esterification reaction between the carboxylic acid functional group of etodolac or tolfenamic acid and thymol. The structures were confirmed by IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. Their chemical stability in addition to a kinetic study of their hydrolysis in 20% liver homogenate and 10% buffered plasma were investigated. In vitro enzymatic hydrolysis showed half-life times 88.84 and 106.61 min for the prodrugs of etodolac 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 pHs 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 Non-steroidal anti-inflammatory drugs (NSAIDs) represent an important therapeutic class of drugs which in addition to their anti-inflammatory effects, may possess both analgesic and antipyretic activities (Mehanna, 2003). The mechanism of action of NSAIDs involves inhibition of cyclooxygenase (COX) enzymes that initiate the formation of prostanoids (Warner and Mitchell, 2003). There are 3 subtypes of COX enzymes: COX-1 (constitutive), COX-2 (inducible in inflammatory processes), and the isozyme COX-3 (Katzung et al., 2019). A drug must be highly lipophilic and acidic as arachidonic acid to competitively inhibit the natural substrate (Brune, 2007). Etodolac (1) (Fig. 1) is an acetic acid derivative, which is mainly used in postoperative pain and rheumatic diseases. It undergoes rapid metabolism in the liver, and is eliminated primarily by the kidney (Sharav and Benoliel, 2008). Tolfenamic acid (2) (Fig. 1) is a fenamate NSAID, used for pain and inflammation management, also in decreasing the intensity and duration of migraine (Colon et al., 2011; Smith et al., 2008).
⁎
The use of NSAIDs can be associated with variable side effects such as renal dysfunction, cardiovascular adverse events, asthma triggering and can significantly increase clotting times (Graham et al., 2005). As well, it has been demonstrated that NSAIDs exhibit adverse effects on the gastrointestinal tract including nausea, vomiting and diarrhea. These later adverse effects are exhibited through two different mechanisms: the first action exerted locally by the direct effect of the organic acid moiety of the drug with gastric mucosa (Foye et al., 2008) and a general systemic mechanism involving prostaglandin E2 (PGE2) and prostacyclin (PGI2), which protect gastric mucosa by stimulating bicarbonate and mucin secretion and increasing mucosal blood flow (Brown et al., 2012; Wallace and Vong, 2008). Prodrugs are transformed enzymatically and/or chemically in vivo in order to exert the required pharmacological effect by releasing the active parent drug (Foye et al., 2008). This approach is widely used in medicinal chemistry as it can improve the undesirable properties and sometimes decrease the clinical usefulness of an existing drug. Ester prodrugs for NSAIDs containing carboxylic acid group can be helpful to depress the GIT irritation and bleeding (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.
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 acidic moiety by esterification without affecting its clinical effect (Kashfi et al., 2002). Thymol (3) (Fig. 1) is the essential dietary constituent in thyme. It has been used for longtime in traditional medicine as it possesses various pharmacological properties such as antioxidant, anti-inflammatory, analgesic, antispasmodic, antiseptic and antitumor activities. The anti-inflammatory effect of thymol is believed to be largely attributed to inhibiting recruitment of cytokines and chemokines (Ojha et al., 2017). In literature, some ester and amide prodrugs of etodolac and tolfenamic acid (as prodrugs 4&5) were prepared to increase their bioavailability and decrease their GI tract adverse effects, Fig. 1 (Chaudhary et al., 2013; Paliwal et al., 2017; Pandey et al., 2018). Thus, this work was aimed to synthesize new derivatives through an esterification reaction between the carboxylic acid functional group in etodolac or tolfenamic acid and thymol to give two new mutual prodrugs. Thymol was chosen as it has a well-known safety profile and is traditionally used for its medicinal as well as flavoring properties (Ojha et al., 2017). Releasing a safe promoeity after their hydrolysis is a big challenge in designing new prodrugs. The new prodrugs structures were confirmed by IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. After synthesis and structure confirmation, the prodrugs were evaluated for their chemical
stability in different temperatures and pHs. Moreover, a kinetic study in 20% liver homogenate and 10% buffered plasma were carried out to evaluate their enzymatic hydrolysis. In addition, the prodrugs were evaluated for their in vivo anti-inflammatory activity and for their ulcerogenic effect compared to the corresponding active drugs. 2. Results and discussion 2.1. Chemistry The target prodrugs, 2-isopropyl-5-methylphenyl-2-(1,8‑diethyl‑4,9dihydro-3H-pyrano[3,4-b]indol-1-yl)acetate (6) and 2-isopropyl-5-methylphenyl-2-[(3‑chloro‑2-methylphenyl)amino]benzoate (7) were prepared via Steglich esterification (Bringmann et al., 2012; Neises and Steglich, 1978) as depicted in schemes 1&2. Schemes 1&2: Reagents and reaction conditions: (a) DCC, 4-DMAP, DCM, stir, RT, 72 hr The well-known Steglich reaction is an esterification reaction that takes place at room temperature through the formation of the O-acylisourea derivative of the carboxylic acid derivative by coupling with dicyclohexylcarbodiimide (DCC) in the presence of 4-N,N-dimethylaminopyridine (4-DMAP). Alcohols or phenols are easily added to this intermediate to form the ester releasing 1,3-dicyclohexylurea as byproduct. Dichloromethane is the most suitable used solvent. In practice, 2
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
Scheme 1. (continued) Table 1 Anti-inflammatory effect of ibuprofen and the tested compounds on carrageenan induced edema. Groups Control Ibuprofen (1) Prodrug (6) (2) Prodrug (7)
% of inhibition of paw edema 1hr 2hr 3hr 0 0 0 61.93 77.50 81.81 41.38 43.25 74.30 22.35 23.52 35.17 36.55 40.48 75.09 27.49 29.75 49.80
4hr 0 89.27 86.26 84.54 86.26 87.98
5hr 0 94.33 89.15 91.50 92.45 94.81
Table 2 Ulcerogenic effect of ibuprofen and the tested compounds in rats. Compound
6hr 0 95.65 90.33 94.68 93.23 97.10
Control Ibuprofen (1) Prodrug (6) (2) Prodrug (7)
Ulcer Index (Relative ulcerogenicity to parent drug) After 1hr After 3hr After 3 days (3 consecutive doses) 0 0 0 9.17 11.00 16.00 6.37 10.50 14.83 0 (0) 6.00 (0.57) 11.99 (0.81) 6.34 6.49 15.49 0 (0) 3.99 (0.61) 14.32 (0.92)
was confirmed in both IR and 1H NMR spectra. In addition, 1H NMR spectrum showed a doublet equivalent to 6 protons (2 CH3) at 1.27 δ ppm and two singlets more deshielded at 2.36 and 2.40 δ ppm (2 ArCH3). Also, the integration of aromatic protons was increased including ten aromatic protons at 6.84–8.27 δ ppm and one exchangeable singlet at 9.36 δ ppm (NH). Compared to its parent drug (2), 13C NMR spectrum of (7) revealed additional three signals at 20.92 (Ar-CH3), 23.16 (2 CH3), and 27.3 δ ppm (CH) in addition to an increase in aromatic carbons by six carbons. The mass spectrum of (7) revealed the molecular ion peaks (M+) and M++2 at 393 and 395 in the ratio of 3:1 due to the presence of chlorine isotopes.
4-DMAP is critical for ester formation (Bringmann et al., 2012; Neises and Steglich, 1978). IR spectrum of prodrug (6) revealed the disappearance of the carboxylic and phenolic OH band, which was also confirmed in its 1H NMR spectrum. Compared to the parent drug (1), the 1H NMR spectrum showed three additional signals each equivalent to 3 protons (3 CH3), two are doublets at 1.20 and 1.22 δ ppm referred to the nonequivalent methyl groups of the isopropyl group, and the third is a singlet of the 5 ́ methyl group which is more deshielded at 2.37 δ ppm. Also, the spectrum showed an increase in the aromatic protons due to three additional aromatic protons within the range 6.79–7.45 δ ppm and an exchangeable singlet at 9.01 δ ppm (NH). The 13C NMR spectrum revealed five signals at 7.76 - 23.21 δ ppm assigned to five CH3 groups in addition to an increase in aromatic carbons by six carbons compared to the parent drug (1). The mass spectrum of (6) revealed the parent molecular ion peak at 419 assigned to its molecular weight. For prodrug (7), the absence of both carboxylic and phenolic OH
2.2. Anti-inflammatory activity and ulcerogenic effect The new prodrugs were evaluated for their anti-inflammatory activity using carrageenan-induced rat paw edema method reported by 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. 3
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 ibuprofen as reference standard. As can be seen in Table 1 (and Table S1 in Supporting Information File), the prodrugs showed lower% of paw edema inhibition than their parent drugs in the first 3 h while after 4–6 h, the percentage increased to become comparable and even higher than the parent drugs, Fig. 2. The delayed effect can be attributed to the time required for the hydrolysis of the prodrugs to their bioactive parent drugs, while the slight increase in activity can be owing to the
additional anti-inflammatory effect of thymol, the released promoiety. In addition, the new prodrugs were estimated for their gastric ulcerogenic potential in rats and compared to their parent drugs (Etodolac and Tolfenamic acid) and ibuprofen (Reference standard). Their ulcer indices were calculated after 1 and 3 hr after drug administration, and after three consecutive daily doses. The results are presented in Table 2 and Figs. 3and 4 (Tables S2-S4 and Figures S1-S2 in Supporting Information File). None of the prodrugs at the dose of 4
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
Fig. 5. Enzymatic hydrolysis of (A) prodrug (6) and (B) prodrug (7) in 20% liver homogenate at 37 °C.
obtained by plotting the natural logarithm of the percentage of remaining concentration versus time was linear (See Supporting Information File, Figures S3 and S4).
50 mg/kg resulted in changes in the gastric mucosa after 1 h while in the same experimental conditions, the clinically used drugs, ibuprofen, etodolac and tolfenamic acid caused visible changes in the mucosa of the stomach, thereby revealing their irritant action. This could be attributed to depressing the local action due to the direct contact of the organic acid moiety. Furthermore, the ulcer index increased after 3 consecutive doses which may be mainly caused by the general systemic mechanism PGE2 and PGI2 (Brown et al., 2012; Foye et al., 2008; Wallace and Vong, 2008).
2.4. In vitro enzymatic hydrolysis To study the rate of enzymatic hydrolysis, four calibration curves were constructed for compounds (1), (6), (2), and (7) in 20% liver homogenate and in 10% buffered plasma at 37 °C as presented in Figs. 5 and 6), respectively. The complete hydrolysis profile was studied by plotting the natural logarithm of the remaining concentrations of the new prodrugs vs time in minutes. Figs. 7 and 8) show that the reactions follow pseudo first order. The half-life time of the reactions was calculated using the slope (k) of each plotted line using equation-1 (Moussa et al., 2018; Richardson et al., 2016), Table 3. As can be seen, the hydrolysis of the prodrugs is faster in liver homogenate than in plasma. Accordingly, the prodrugs may be successfully metabolized in vivo to yield their parent drugs.
2.3. Chemical stability The chemical stability of the new prodrugs was studied at three different pHs, simulating the pHs of the stomach (1.2), the colon (6.8) and the physiological pH (7.4) and at three different temperatures, body (37 °C), room (25 °C) temperatures and drastic storage conditions (60 °C). It was found that the hydrolysis rate of the two prodrugs increased with temperature. It is also obvious that prodrugs exhibited higher stability at 25 and 37 °C than 60 °C at the three studied pHs. This stability at room temperature is highly preferred as it facilitates its formulation in the required pharmaceutical dosage form. 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 stability indicates that the prodrugs will pass safely through the gastrointestinal tract without hydrolysis after oral administration, thus keeping the gastric mucosa protected. In the studied conditions, the hydrolytic reaction follows a pseudo first order as the relationship
t1/2 =
0.693/k
(1)
Where k: the specific reaction rate constant, t1/2: the half-life time 3. Conclusion Two mutual prodrugs of etodolac and tolfenamic acid with thymol were successfully synthesized in a rather simple single step scheme. Their chemical structures were confirmed by 1H NMR, 13C NMR, FT-IR, 5
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 this figure legend, the reader is referred to the web version of this article.)
Mass spectroscopy and elemental analysis. The prodrugs and their bioactive parent drugs were determined in aqueous and biological samples using a developed and validated HPLC method. The two prodrugs were found to be stable at pH 1.2, 6.8 and 7.4 in room and body temperatures. The in vitro enzymatic hydrolysis of the prodrugs was faster in human plasma and liver homogenate than chemical hydrolysis, with faster metabolic rate in liver than in plasma. Thus, it can be deduced that the prodrugs are expected to be converted in vivo to their parent drugs rapidly in plasma and liver. In addition, the new prodrugs were evaluated for their anti-inflammatory activity showing higher delayed inhibition of paw edema than their parent drugs due to the time required for their in vivo hydrolysis. By studying their ulcerogenic 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.
the reactions were followed up by TLC using Kieselgel 60 F254 sheets (Merck, Darmstadt, Germany) and chloroform: methanol 9.5:0.5 or pure chloroform as the eluting system and the spots were visualized at 254 nm by UV Vilber Lourmat, Marne La Vallee, France. IR spectra (KBr disc) were recorded on a Schimadzu FT-IR recording spectrophotometer affinity (IR-470, Schimadzu, Kyoto, Japan). The NMR spectra were recorded on Bruker AVANCE III400 MHz FT-NMR spectrometer. 1H spectra were run at 400 MHz while 13C spectra were run at 100 MHz in deuterated CDCl3. Mass spectra were recorded using Single Quadruple mass spectrometer ISQ LT (Japan). Elemental Microanalyses were carried out at the Regional Center for Mycology and Biotechnology, AlAzhar University. 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 dichloromethane (100 mL), was stirred at room temperature for 72 hr. The white precipitate formed (N,N'-dicyclohexylurea) was discarded by filtration, and the filtrate was washed with water, then with 1% diluted HCl. The organic layer was separated and concentrated under vacuum. The residue was crystallized from absolute ethanol to obtain white crystals of prodrug (6) or (7).
4. Experimental 4.1. Chemistry Solvents and chemicals were purchased from commercial sources and used without purification. Melting points were recorded using BIBBY scientific Stuart melting point apparatus, Staffordshire, UK. All 6
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 colour in this figure legend, the reader is referred to the web version of this article.)
2938, 2861 (CH aliphatic), 1693 (C = O), 1665 (NH bending). 1H NMR (400 MHz, in deuterated CDCl3) δ ppm: 1.27 (d, 6H, (CH3)2CH, J 6.88 Hz) 2.36 (s, 3H Ar-CH3,), 2.40 (s, 3H, Ar-CH3), 3.11 (m, 1H, (CH3)2CH), 6.84 (t, 1H, C5′-H-Ar, J 8.00 Hz), 6.94 (d, 1H,C4"-H Ar, J 8.00 Hz), 7.01 (s, 1H, C6"-H Ar), 7.12 (d, 1H, C3-H, J 7.68), 7.15 (t, 1H, C5-H Ar, J 7.96 Hz), 7.24 (d, 1H, C3"-H, J 7.36), 7.30 (d, 2H, C4′-H Ar and C6′-H, J 7.88 Hz), 7.39–7.43 (d,t, 1H, C4-H, J 7.16 Hz), 8.27–8.29 (dd, 1H, C6-H Ar, J 8.04 Hz) 9.36 (s, 1H, NH, exch. D2O). 13C NMR (100 MHz, CDCl3) δ ppm: 15.03, 20.92 (Ar-CH3), 23.16, (2 CH3), 27.30 (CH), 110.64, 113.99, 117.19, 123.00, 123.02, 125.85, 126.61, 126.87, 127.34, 131.70, 131.96, 134.97, 135.67, 136.74, 137.47, 140.19, 147.90, (Ar C), 149.28 (CO), 167.59 (C = O). MS (m/z,%): 393.20 (M+, 39.98), 395.13 (M+, M++2 16.59). Anal. Calcd. for C24H24ClNO2 (393.9059): C,73.18; H, 6.14; N, 3.56%. Found: C, 72.59; H, 5.96; N, 4.10%.
4.1.1.1. 2-Isopropyl-5-methylphenyl 2-(1,8‑diethyl‑,4,9-dihydro-3Hpyrano[3,4-b]indol-1-yl)acetate (6). Rf: 0.80 (CCl4:CH3COOC2H5 9:1). Yield: (3.5 g) 83.5%. mp: 116–120 °C. IR (KBr) υmax/cm−1: 3385 (NH), 3070, 3043 (CH Ar), 2992, 2914, 2848 (CH aliphatic), 1711 (C = O), 1615 (NH bending). 1H NMR (400 MHz, in deuterated CDCl3) δ ppm: 0.99 (t, 3H, CH2CH3, J 7.36 Hz), 1.20 (d, 3H, CHCH3, J 6.92 Hz), 1.22 (d, 3H, CHCH3, J 6.88 Hz), 1.35 (t, 3H, CH2CH3, J 5.84 Hz), 2.19 (m, 1H, C4-H pyran), 2.29 (m, 1H, C4-H pyran), 2.37 (s, 3H, Ar-CH3), 2.83–2.98 (m, 5H, CH2CH3, CH2CH3 and CHCH3), 3.27 (d, 1H, CH alpha C = O, J 17.00 Hz), 3.33 (d, 1H, CH alpha C = O, J 16.96 Hz), 4.08 (m, 1H, C3-H pyran), 4.09 (m, 1H, C3-H pyran), 6.79 (s, 1H, C6′-H Ar), 7.05 (d, 1H, C4′-H Ar, J 7.08 Hz), 7.11 (d, 1H, C3′-H Ar, J 7.64 Hz), 7.15 (t, 1H, C6-H Ar, J 7.48 Hz), 7.29 (d, 1H, C5-H Ar, J 7.92 Hz), 7.45 (d, 1H, C7-H Ar, J 7.72 Hz), 9.01 (s, 1H, NH, exch. D2O). 13C NMR (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, 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). MS (m/z,%): 419.27 (M+, 100). Anal. Calcd. for C27H33NO3 (419.5558): C, 77.29; H, 7.93; N, 3.34%. Found: C,76.89; H, 7.91; N, 3.51%.
4.2. Anti-inflammatory activity and ulcerogenic effect 4.2.1. Animals Adult male albino Wistar rats, weighing 170–200 g, were obtained from Nahda University Animal House, Beni-Sueif, Egypt. Animals were acclimatized for one week in a room controlled for temperature, humidity, and light (12 h light–dark cycle). Rats were housed in well ventilated cages with free access to food and water. All experimental procedures were approved by the institutional guidelines of the Research Ethics Committee of Faculty of Pharmacy, Cairo University and the regulations of the local Animal Welfare authorities.
4.1.1.2. 2-Isopropyl-5-methylphenyl-2-[(3‑chloro‑2-methylphenyl) amino] benzoate (7). Rf: 0.85 (CCl4:CH3COOC2H5 9:1). Yield: (3 g) 76.3%. Mp: 111–114 °C. IR (KBr) υmax/cm−1: 3323 (NH), 3085, 3046 (CH Ar), 2975, 7
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 magnifying lens to check for the presence of macroscopically visible lesions. The number of lesions in each stomach, if any, was counted and recorded. 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 homogenate and (B) 10% buffered plasma at 37 °C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4.3. Prodrug chemical stability and in vitro studies 4.3.1. Method of analysis of prodrug and bioactive products a RP-HPLC method was developed and validated for the determination of each prodrug in mixture with its bioactive compounds in aqueous medium, liver homogenate and human plasma. The chromatographic separation was achieved on a Thermo RP-C18 column (250 mm x 4.6 mm, 5 µm), at ambient temperature using a mobile phase consisting of 0.2% formic acid (A) and acetonitrile (B) with a gradient mode according to Table S5 (See Supporting Information File) at a flow rate of 1 mL/min. UV detection was carried out at 274 nm for 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) (See Supporting Information File).
Table 3 The regression parameters, observed rate constants and half-lives for the hydrolysis of the new prodrugs:. Prodrug Kobs (min t½ (min)
−1
)
20% Liver homogenate (6) (7) 0.0078 0.0075 88.84 92.4
10% Buffered plasma (6) (7) 0.0065 0.006 106.61 115.5
4.2.2. Study design and procedure 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: Group 1 (control group); Group II (ibuprofen group); Group III (etodolac group); Group IV (prodrug (6) group); Group V (tolfenamic acid group); Group VI (prodrug (7) group). 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 in 2% Tween 80 and administrated orally 30 min before induction of the inflammation, while the control group received only 2% Tween 80. Subplantar injection of 0.1 mL of 1% carrageenan solution in saline (0.9%) in the right leg induced paw edema. Paw edema thickness was measured after 1, 2, 3, 4, 5 and 6 hr in the right hind paw using a screw gauge micrometer and then compared with the left hind paw thickness of each rat. The difference of average values between treated and control groups was calculated for each time interval and evaluated statistically. Quantitative variables were expressed as means ± standard error (SEM). The anti-inflammatory activity was expressed as percentage inhibition of edema volume in treated animals by comparison to the control group according to the following equation: % of edema inhibition = (VR - VL) control - (VR - VL) treated ×100 (VR - VL) controlWhere VR represents the right paw thickness, VL represents the left paw thickness, (VR - VL) control represents the mean
4.3.2. Preparation of standard and working solutions Standard stock solutions of compounds 1–3 and their corresponding prodrugs 6&7 were prepared in methanol to obtain solutions of 0.1 mg/mL (for chemical stability) and 10 mg/mL (for in vitro enzymatic hydrolysis). 4.3.3. Preparation of liver homogenate Five adult albino Wistar male and female rats, (300–400 g), free of any signs of observable clinical abnormalities were used. The rats were housed in an air-conditioned room and fed standard meals and water. To collect the livers, the rats were systemically anesthetized with ketamine hydrochloride (35 mg/kg) IM injections in combination with a muscle relaxant xylazine hydrochloride (5 mg/kg). Livers were removed by surgery, and immediately homogenized by tissue homogenizer, then centrifuged at 6000 rpm for 20 min. at 4 °C then the supernatant was separated carefully (El-Bagary et al., 2016; Moussa et al., 2019; 2018). 4.3.4. Preparation of 10% buffered plasma Pooled human plasma was donated from national blood bank (Vacsera, Egypt). Human plasma (4 mL) was diluted with 1 mL of 0.05 M phosphate buffer of pH 7.4 followed by incubation at 37 ± 1 °C in a shaking water bath for 5 min. 8
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
4.3.5. Preparation of blank buffered human plasma or liver supernatant sample An aliquot (4 mL) of the supernatant or buffered human plasma was placed in a test tube and incubated for 5 min in a shaking water bath equilibrated at 37 ± 1 °C. An aliquot of 150 µL was withdrawn and 450 µL methanol was added. The mixture was then centrifuged at 13,000 rpm for 7 min at 4 °C. From the obtained clear supernatant, 10 µL was analyzed by HPLC (See Supporting file, Figures S7 and S8).
configurationally unstable biaryl lactone, in. Org. Synth. 70–78. https://doi.org/10.1002/ 0471264229.os088.07. Brown, M.J., Sharma, P., Mir, F., Bennett, P.N., 2012. Clinical Pharmacology, 12th ed. Churchill Livingstone. 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:// doi.org/10.1185/030079907X242584. Chaudhary, R., Negi, V., Sharma, R., Percha, V., Dobhal, Y., Pant, R., 2013. Design, synthesis and evaluation of mutual prodrugs of etodolac with polyphenols as safer nsaids. Inven. Impact Med. Chem. 3, 152–158. 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 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. org/10.1007/s10637-009-9331-8. El-Bagary, R., Hashem, H., Fouad, M., Tarek, S., 2016. UPLC-MS-MS method for the determination of vilazodone in human plasma: application to a pharmacokinetic study. J. Chromatogr. Sci. 54, 1365–1372. https://doi.org/10.1093/chromsci/bmw084. Foye, W.O., Lemke, T.L., Williams, D.A., 2008. Foye's Principles of Medicinal Chemistry. 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 cyclo-oxygenase 2 selective and non-selective non-steroidal anti-inflammatory drugs: nested case-control study. Lancet 365, 475–481. https://doi.org/10.1016/S0140-6736(05) 17864-7. Hassan, G.S., Abou-Seri, S.M., Kamel, G., Ali, M.M., 2014. Celecoxib analogs bearing benzofuran moiety as cyclooxygenase-2 inhibitors: design, synthesis and evaluation as potential anti-inflammatory agents. Eur. J. Med. Chem. 76, 482–493. https://doi.org/10.1016/J. EJMECH.2014.02.033. Hassanein, H.H., Georgey, H.H., Fouad, M.A., El Kerdawy, A.M., Said, M.F., 2017. Synthesis and molecular docking of new imidazoquinazolinones as analgesic agents and selective COX-2 inhibitors. Future Med. Chem. 9, 553–578. https://doi.org/10.4155/fmc-2016-0240. Kashfi, K., Rayyan, Y., Qiao, L.L., Williams, J.L., Chen, J., Del Soldato, P., Traganos, F., Rigas, B., Ryann, Y., 2002. Nitric oxide-donating nonsteroidal anti-inflammatory drugs inhibit the growth of various cultured human cancer cells: evidence of a tissue type-independent effect. J. Pharmacol. Exp. Ther. 303, 1273–1282. https://doi.org/10.1124/jpet.102.042754. Katzung, B.G., Kruidering-Hall, M., Trevor, A.J., 2019. NSAIDs, Acetaminophen, & Drugs used in Rheumatoid Arthritis & Gout. Katzung & Trevor's Pharmacology: Examination & Board Review, 12e. The McGraw-Hill Companies. Mehanna, A.S., 2003. NSAIDs: chemistry and pharmacological actions. Am. J. Pharm. Educ. https://doi.org/10.5688/aj670263. Meshali, M., El-Sabbah, E., Foda, A., 1983. Effect of encapsulation of flufenamic acid with acrylic resins on its bioavailability and gastric ulcerogenic activity in rats. Acta Pharm. Technol. 29, 217–230. Moussa, B.A., El-Zaher, A.A., El-Ashrey, M.K., Fouad, M.A., 2018. Synthesis and molecular 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. org/10.1016/j.ejmech.2018.02.038. Moussa, B.A., Zaher, A.A.El, Ashrey, M.K.El, 2019. Roflumilast analogs with improved metabolic stability, plasma protein binding, and pharmacokinetic profile 1–12. doi:10.1002/ dta.2562. Neises, B., Steglich, W., 1978. Simple method for the esterification of carboxylic acids. Angew. 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 properties and molecular mechanisms of thymol: prospects for its therapeutic potential and pharmaceutical development. Front. Pharmacol. 8, 380. https://doi.org/10.3389/fphar. 2017.00380. Paliwal, M., Sucheta, Ruchita, Jain, S., Monika, Himanshu, 2017. Synthesis and biological evaluation of mutual prodrugs of carboxylic group containing some non-steroidal antiinflammatory drugs and propyphenazone. Curr. Drug Deliv. 14, 1213–1224. https://doi. org/10.2174/1567201814666170213153509. Pandey, P., Pandey, S., Dubey, S., 2018. Mutual amide prodrug of etodolac-glucosamine : synthesis, characterization and pharmacological screening synthesis of mutual prodrug : characterization and preformulation studies of the synthesized prodrug : protein binding study: in vitro hydrolysis st 10–15. Richardson, S.J., Bai, A., Kulkarni, A., F. Moghaddam, M., 2016. Efficiency in drug discovery: liver S9 fraction assay as a screen for metabolic stability. Drug Metab. Lett. 10, 83–90. https://doi.org/10.2174/1872312810666160223121836. Robert, A., Northam, J.I., Nezamis, J.E., Phillips, J.P., 1970. Exertion ulcers in the rat. Am. J. Dig. Dis. 15, 497–507. https://doi.org/10.1007/BF02283881. Sharav, Y., Benoliel, R., 2008. Pharmacotherapy of acute orofacial pain. Orofac. Pain Headache 349–376. https://doi.org/10.1016/B978-0-7234-3412-2.10015-X. Sheha, M., Khedr, A., Elsherief, H., 2002. Biological and metabolic study of naproxen-propyphenazone mutual prodrug. Eur. J. Pharm. Sci. 17, 121–130. Smith, G.W., Davis, J.L., Tell, L.A., Webb, A.I., Riviere, J.E., 2008. Extralabel use of nonsteroidal anti-inflammatory drugs in cattle. J. Am. Vet. Med. Assoc. 232, 697–701. https:// doi.org/10.2460/javma.232.5.697. Wallace, J.L., Vong, L., 2008. NSAID-induced gastrointestinal damage and the design of GIsparing NSAIDs. Curr. Opin. Investig. Drugs 9, 1151–1156. Warner, T.D., Mitchell, J.A., 2003. Nonsteroidal antiinflammatory drugs inhibiting prostanoid efflux: as easy as ABC? Proc. Natl. Acad. Sci. U. S. A. 100, 9108–9110. https://doi.org/10. 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. 3181/00379727-111-27849.
4.3.6. Chemical hydrolysis The stability of the newly synthesized prodrugs was studied in aqueous phosphate buffer solutions of different pH values (1.2, 6.8, and 7.4) and at three different temperatures (25, 37 and 60 °C). Accurately measured aliquots equivalent to (1 mg) of prodrugs 6&7 working solutions were transferred separately into three volumetric flasks (50 mL), and each flask was completed with a different phosphate buffer solution pH (1.2, 6.8 or 7.4). The solutions were well mixed using vortex for 1 min., then 10 mL of each flask was transferred into a series of 3 screw capped test tubes and kept in a temperature-controlled water bath shaker at different temperatures 25, 37 or 60 °C. At appropriate time intervals, samples of each tube were withdrawn, cooled and immediately analyzed for their content of the remaining prodrugs, and appearance of parent drugs by RP-HPLC. All the experiments were carried out in triplicate. 4.3.7. In vitro enzymatic hydrolysis For the construction of calibration curves, 80 µL from each working solution of concentrations (0.1–7 mg/mL) of 1, 2, 6 & 7 was added in a test tube with 4 mL of the prepared 10% buffered plasma or the separated liver supernatant, vortexed well for 1 min. An aliquot (150 µL) was immediately withdrawn and 450 µL methanol was added to stop the reaction and precipitate the proteins. The mixture was then centrifuged at 13,000 rpm for 7 min at 4 °C. 10 µL of the supernatant was analyzed by HPLC. Calibration curves were constructed showing good correlation of more than 0.99. Intra-day accuracy and precision were evaluated by three-replicate analysis of 1, 2, 6 & 7 at concentrations of LLOQ (0.1 mg/mL) in 10% buffered plasma or the separated liver supernatant in the same day following the preparation procedure mentioned above. The inter-day accuracy and precision were assessed by analyzing three replicates of LLOQ on three consecutive days. The precision and accuracy of the method were determined by calculating the percent coefficient of variation (% CV) and recovery for the concentrations obtained for different determinations, See Supporting Information File, Table S6. For studying in vitro enzymatic hydrolysis, 5.5 mL of the standard stock solution of each prodrug was diluted with methanol (5.5 mg/mL). An aliquot (80 µL) was added in a test tube with 4 mL of the prepared 10% buffered plasma or the separated liver supernatant and vortexed well for 1 min. The tubes were placed in a shaking water bath at 37 ± 1 °C at different time intervals ranging from 0–300 min. An aliquot (150 µL) was processed following the procedure of preparation of calibration standards and analyzed by HPLC. Then, the concentrations of each prodrug and corresponding parent drug were calculated. Supplementary materials Supplementary material associated with this article can be found, in the online version, at 10.1016/j.ejps.2019.105101. References Bringmann, G., Gulder, T.A.M., Gulder, T., 2012. Discussion addendum for: asymmetric synthesis of (M)-2-Hydroxymethyl-1-(2-Hydroxy-4,6-Dimethylphenyl)Naphthalene viaa
9
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Synthesis and biological evaluation of new prodrugs of etodolac and tolfenamic acid with reduced ulcerogenic potential
T
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, Egypt Pharmacology and Toxicology Department, Faculty of Pharmacy, Nahda University, Beni-Suef, Egypt c Pharmaceutical Chemistry Department, Faculty of Pharmacy, 6th October University, Cairo, Egypt b
ARTICLE INFO
ABSTRACT
Keywords: Etodolac Tolfenamic acid Thymol Prodrug Anti-inflammatory Ulcerogenicity
Gastric irritation and ulcerogenic effect of the acidic NSAIDs are of the most challenging problems in designing novel anti-inflammatory agents. In this study, the new prodrugs were prepared through Steglich esterification reaction between the carboxylic acid functional group of etodolac or tolfenamic acid and thymol. The structures were confirmed by IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. Their chemical stability in addition to a kinetic study of their hydrolysis in 20% liver homogenate and 10% buffered plasma were investigated. In vitro enzymatic hydrolysis showed half-life times 88.84 and 106.61 min for the prodrugs of etodolac 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 pHs 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 Non-steroidal anti-inflammatory drugs (NSAIDs) represent an important therapeutic class of drugs which in addition to their anti-inflammatory effects, may possess both analgesic and antipyretic activities (Mehanna, 2003). The mechanism of action of NSAIDs involves inhibition of cyclooxygenase (COX) enzymes that initiate the formation of prostanoids (Warner and Mitchell, 2003). There are 3 subtypes of COX enzymes: COX-1 (constitutive), COX-2 (inducible in inflammatory processes), and the isozyme COX-3 (Katzung et al., 2019). A drug must be highly lipophilic and acidic as arachidonic acid to competitively inhibit the natural substrate (Brune, 2007). Etodolac (1) (Fig. 1) is an acetic acid derivative, which is mainly used in postoperative pain and rheumatic diseases. It undergoes rapid metabolism in the liver, and is eliminated primarily by the kidney (Sharav and Benoliel, 2008). Tolfenamic acid (2) (Fig. 1) is a fenamate NSAID, used for pain and inflammation management, also in decreasing the intensity and duration of migraine (Colon et al., 2011; Smith et al., 2008).
⁎
The use of NSAIDs can be associated with variable side effects such as renal dysfunction, cardiovascular adverse events, asthma triggering and can significantly increase clotting times (Graham et al., 2005). As well, it has been demonstrated that NSAIDs exhibit adverse effects on the gastrointestinal tract including nausea, vomiting and diarrhea. These later adverse effects are exhibited through two different mechanisms: the first action exerted locally by the direct effect of the organic acid moiety of the drug with gastric mucosa (Foye et al., 2008) and a general systemic mechanism involving prostaglandin E2 (PGE2) and prostacyclin (PGI2), which protect gastric mucosa by stimulating bicarbonate and mucin secretion and increasing mucosal blood flow (Brown et al., 2012; Wallace and Vong, 2008). Prodrugs are transformed enzymatically and/or chemically in vivo in order to exert the required pharmacological effect by releasing the active parent drug (Foye et al., 2008). This approach is widely used in medicinal chemistry as it can improve the undesirable properties and sometimes decrease the clinical usefulness of an existing drug. Ester prodrugs for NSAIDs containing carboxylic acid group can be helpful to depress the GIT irritation and bleeding (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.
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 acidic moiety by esterification without affecting its clinical effect (Kashfi et al., 2002). Thymol (3) (Fig. 1) is the essential dietary constituent in thyme. It has been used for longtime in traditional medicine as it possesses various pharmacological properties such as antioxidant, anti-inflammatory, analgesic, antispasmodic, antiseptic and antitumor activities. The anti-inflammatory effect of thymol is believed to be largely attributed to inhibiting recruitment of cytokines and chemokines (Ojha et al., 2017). In literature, some ester and amide prodrugs of etodolac and tolfenamic acid (as prodrugs 4&5) were prepared to increase their bioavailability and decrease their GI tract adverse effects, Fig. 1 (Chaudhary et al., 2013; Paliwal et al., 2017; Pandey et al., 2018). Thus, this work was aimed to synthesize new derivatives through an esterification reaction between the carboxylic acid functional group in etodolac or tolfenamic acid and thymol to give two new mutual prodrugs. Thymol was chosen as it has a well-known safety profile and is traditionally used for its medicinal as well as flavoring properties (Ojha et al., 2017). Releasing a safe promoeity after their hydrolysis is a big challenge in designing new prodrugs. The new prodrugs structures were confirmed by IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. After synthesis and structure confirmation, the prodrugs were evaluated for their chemical
stability in different temperatures and pHs. Moreover, a kinetic study in 20% liver homogenate and 10% buffered plasma were carried out to evaluate their enzymatic hydrolysis. In addition, the prodrugs were evaluated for their in vivo anti-inflammatory activity and for their ulcerogenic effect compared to the corresponding active drugs. 2. Results and discussion 2.1. Chemistry The target prodrugs, 2-isopropyl-5-methylphenyl-2-(1,8‑diethyl‑4,9dihydro-3H-pyrano[3,4-b]indol-1-yl)acetate (6) and 2-isopropyl-5-methylphenyl-2-[(3‑chloro‑2-methylphenyl)amino]benzoate (7) were prepared via Steglich esterification (Bringmann et al., 2012; Neises and Steglich, 1978) as depicted in schemes 1&2. Schemes 1&2: Reagents and reaction conditions: (a) DCC, 4-DMAP, DCM, stir, RT, 72 hr The well-known Steglich reaction is an esterification reaction that takes place at room temperature through the formation of the O-acylisourea derivative of the carboxylic acid derivative by coupling with dicyclohexylcarbodiimide (DCC) in the presence of 4-N,N-dimethylaminopyridine (4-DMAP). Alcohols or phenols are easily added to this intermediate to form the ester releasing 1,3-dicyclohexylurea as byproduct. Dichloromethane is the most suitable used solvent. In practice, 2
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
Scheme 1. (continued) Table 1 Anti-inflammatory effect of ibuprofen and the tested compounds on carrageenan induced edema. Groups Control Ibuprofen (1) Prodrug (6) (2) Prodrug (7)
% of inhibition of paw edema 1hr 2hr 3hr 0 0 0 61.93 77.50 81.81 41.38 43.25 74.30 22.35 23.52 35.17 36.55 40.48 75.09 27.49 29.75 49.80
4hr 0 89.27 86.26 84.54 86.26 87.98
5hr 0 94.33 89.15 91.50 92.45 94.81
Table 2 Ulcerogenic effect of ibuprofen and the tested compounds in rats. Compound
6hr 0 95.65 90.33 94.68 93.23 97.10
Control Ibuprofen (1) Prodrug (6) (2) Prodrug (7)
Ulcer Index (Relative ulcerogenicity to parent drug) After 1hr After 3hr After 3 days (3 consecutive doses) 0 0 0 9.17 11.00 16.00 6.37 10.50 14.83 0 (0) 6.00 (0.57) 11.99 (0.81) 6.34 6.49 15.49 0 (0) 3.99 (0.61) 14.32 (0.92)
was confirmed in both IR and 1H NMR spectra. In addition, 1H NMR spectrum showed a doublet equivalent to 6 protons (2 CH3) at 1.27 δ ppm and two singlets more deshielded at 2.36 and 2.40 δ ppm (2 ArCH3). Also, the integration of aromatic protons was increased including ten aromatic protons at 6.84–8.27 δ ppm and one exchangeable singlet at 9.36 δ ppm (NH). Compared to its parent drug (2), 13C NMR spectrum of (7) revealed additional three signals at 20.92 (Ar-CH3), 23.16 (2 CH3), and 27.3 δ ppm (CH) in addition to an increase in aromatic carbons by six carbons. The mass spectrum of (7) revealed the molecular ion peaks (M+) and M++2 at 393 and 395 in the ratio of 3:1 due to the presence of chlorine isotopes.
4-DMAP is critical for ester formation (Bringmann et al., 2012; Neises and Steglich, 1978). IR spectrum of prodrug (6) revealed the disappearance of the carboxylic and phenolic OH band, which was also confirmed in its 1H NMR spectrum. Compared to the parent drug (1), the 1H NMR spectrum showed three additional signals each equivalent to 3 protons (3 CH3), two are doublets at 1.20 and 1.22 δ ppm referred to the nonequivalent methyl groups of the isopropyl group, and the third is a singlet of the 5 ́ methyl group which is more deshielded at 2.37 δ ppm. Also, the spectrum showed an increase in the aromatic protons due to three additional aromatic protons within the range 6.79–7.45 δ ppm and an exchangeable singlet at 9.01 δ ppm (NH). The 13C NMR spectrum revealed five signals at 7.76 - 23.21 δ ppm assigned to five CH3 groups in addition to an increase in aromatic carbons by six carbons compared to the parent drug (1). The mass spectrum of (6) revealed the parent molecular ion peak at 419 assigned to its molecular weight. For prodrug (7), the absence of both carboxylic and phenolic OH
2.2. Anti-inflammatory activity and ulcerogenic effect The new prodrugs were evaluated for their anti-inflammatory activity using carrageenan-induced rat paw edema method reported by 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. 3
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 ibuprofen as reference standard. As can be seen in Table 1 (and Table S1 in Supporting Information File), the prodrugs showed lower% of paw edema inhibition than their parent drugs in the first 3 h while after 4–6 h, the percentage increased to become comparable and even higher than the parent drugs, Fig. 2. The delayed effect can be attributed to the time required for the hydrolysis of the prodrugs to their bioactive parent drugs, while the slight increase in activity can be owing to the
additional anti-inflammatory effect of thymol, the released promoiety. In addition, the new prodrugs were estimated for their gastric ulcerogenic potential in rats and compared to their parent drugs (Etodolac and Tolfenamic acid) and ibuprofen (Reference standard). Their ulcer indices were calculated after 1 and 3 hr after drug administration, and after three consecutive daily doses. The results are presented in Table 2 and Figs. 3and 4 (Tables S2-S4 and Figures S1-S2 in Supporting Information File). None of the prodrugs at the dose of 4
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
Fig. 5. Enzymatic hydrolysis of (A) prodrug (6) and (B) prodrug (7) in 20% liver homogenate at 37 °C.
obtained by plotting the natural logarithm of the percentage of remaining concentration versus time was linear (See Supporting Information File, Figures S3 and S4).
50 mg/kg resulted in changes in the gastric mucosa after 1 h while in the same experimental conditions, the clinically used drugs, ibuprofen, etodolac and tolfenamic acid caused visible changes in the mucosa of the stomach, thereby revealing their irritant action. This could be attributed to depressing the local action due to the direct contact of the organic acid moiety. Furthermore, the ulcer index increased after 3 consecutive doses which may be mainly caused by the general systemic mechanism PGE2 and PGI2 (Brown et al., 2012; Foye et al., 2008; Wallace and Vong, 2008).
2.4. In vitro enzymatic hydrolysis To study the rate of enzymatic hydrolysis, four calibration curves were constructed for compounds (1), (6), (2), and (7) in 20% liver homogenate and in 10% buffered plasma at 37 °C as presented in Figs. 5 and 6), respectively. The complete hydrolysis profile was studied by plotting the natural logarithm of the remaining concentrations of the new prodrugs vs time in minutes. Figs. 7 and 8) show that the reactions follow pseudo first order. The half-life time of the reactions was calculated using the slope (k) of each plotted line using equation-1 (Moussa et al., 2018; Richardson et al., 2016), Table 3. As can be seen, the hydrolysis of the prodrugs is faster in liver homogenate than in plasma. Accordingly, the prodrugs may be successfully metabolized in vivo to yield their parent drugs.
2.3. Chemical stability The chemical stability of the new prodrugs was studied at three different pHs, simulating the pHs of the stomach (1.2), the colon (6.8) and the physiological pH (7.4) and at three different temperatures, body (37 °C), room (25 °C) temperatures and drastic storage conditions (60 °C). It was found that the hydrolysis rate of the two prodrugs increased with temperature. It is also obvious that prodrugs exhibited higher stability at 25 and 37 °C than 60 °C at the three studied pHs. This stability at room temperature is highly preferred as it facilitates its formulation in the required pharmaceutical dosage form. 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 stability indicates that the prodrugs will pass safely through the gastrointestinal tract without hydrolysis after oral administration, thus keeping the gastric mucosa protected. In the studied conditions, the hydrolytic reaction follows a pseudo first order as the relationship
t1/2 =
0.693/k
(1)
Where k: the specific reaction rate constant, t1/2: the half-life time 3. Conclusion Two mutual prodrugs of etodolac and tolfenamic acid with thymol were successfully synthesized in a rather simple single step scheme. Their chemical structures were confirmed by 1H NMR, 13C NMR, FT-IR, 5
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 this figure legend, the reader is referred to the web version of this article.)
Mass spectroscopy and elemental analysis. The prodrugs and their bioactive parent drugs were determined in aqueous and biological samples using a developed and validated HPLC method. The two prodrugs were found to be stable at pH 1.2, 6.8 and 7.4 in room and body temperatures. The in vitro enzymatic hydrolysis of the prodrugs was faster in human plasma and liver homogenate than chemical hydrolysis, with faster metabolic rate in liver than in plasma. Thus, it can be deduced that the prodrugs are expected to be converted in vivo to their parent drugs rapidly in plasma and liver. In addition, the new prodrugs were evaluated for their anti-inflammatory activity showing higher delayed inhibition of paw edema than their parent drugs due to the time required for their in vivo hydrolysis. By studying their ulcerogenic 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.
the reactions were followed up by TLC using Kieselgel 60 F254 sheets (Merck, Darmstadt, Germany) and chloroform: methanol 9.5:0.5 or pure chloroform as the eluting system and the spots were visualized at 254 nm by UV Vilber Lourmat, Marne La Vallee, France. IR spectra (KBr disc) were recorded on a Schimadzu FT-IR recording spectrophotometer affinity (IR-470, Schimadzu, Kyoto, Japan). The NMR spectra were recorded on Bruker AVANCE III400 MHz FT-NMR spectrometer. 1H spectra were run at 400 MHz while 13C spectra were run at 100 MHz in deuterated CDCl3. Mass spectra were recorded using Single Quadruple mass spectrometer ISQ LT (Japan). Elemental Microanalyses were carried out at the Regional Center for Mycology and Biotechnology, AlAzhar University. 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 dichloromethane (100 mL), was stirred at room temperature for 72 hr. The white precipitate formed (N,N'-dicyclohexylurea) was discarded by filtration, and the filtrate was washed with water, then with 1% diluted HCl. The organic layer was separated and concentrated under vacuum. The residue was crystallized from absolute ethanol to obtain white crystals of prodrug (6) or (7).
4. Experimental 4.1. Chemistry Solvents and chemicals were purchased from commercial sources and used without purification. Melting points were recorded using BIBBY scientific Stuart melting point apparatus, Staffordshire, UK. All 6
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 colour in this figure legend, the reader is referred to the web version of this article.)
2938, 2861 (CH aliphatic), 1693 (C = O), 1665 (NH bending). 1H NMR (400 MHz, in deuterated CDCl3) δ ppm: 1.27 (d, 6H, (CH3)2CH, J 6.88 Hz) 2.36 (s, 3H Ar-CH3,), 2.40 (s, 3H, Ar-CH3), 3.11 (m, 1H, (CH3)2CH), 6.84 (t, 1H, C5′-H-Ar, J 8.00 Hz), 6.94 (d, 1H,C4"-H Ar, J 8.00 Hz), 7.01 (s, 1H, C6"-H Ar), 7.12 (d, 1H, C3-H, J 7.68), 7.15 (t, 1H, C5-H Ar, J 7.96 Hz), 7.24 (d, 1H, C3"-H, J 7.36), 7.30 (d, 2H, C4′-H Ar and C6′-H, J 7.88 Hz), 7.39–7.43 (d,t, 1H, C4-H, J 7.16 Hz), 8.27–8.29 (dd, 1H, C6-H Ar, J 8.04 Hz) 9.36 (s, 1H, NH, exch. D2O). 13C NMR (100 MHz, CDCl3) δ ppm: 15.03, 20.92 (Ar-CH3), 23.16, (2 CH3), 27.30 (CH), 110.64, 113.99, 117.19, 123.00, 123.02, 125.85, 126.61, 126.87, 127.34, 131.70, 131.96, 134.97, 135.67, 136.74, 137.47, 140.19, 147.90, (Ar C), 149.28 (CO), 167.59 (C = O). MS (m/z,%): 393.20 (M+, 39.98), 395.13 (M+, M++2 16.59). Anal. Calcd. for C24H24ClNO2 (393.9059): C,73.18; H, 6.14; N, 3.56%. Found: C, 72.59; H, 5.96; N, 4.10%.
4.1.1.1. 2-Isopropyl-5-methylphenyl 2-(1,8‑diethyl‑,4,9-dihydro-3Hpyrano[3,4-b]indol-1-yl)acetate (6). Rf: 0.80 (CCl4:CH3COOC2H5 9:1). Yield: (3.5 g) 83.5%. mp: 116–120 °C. IR (KBr) υmax/cm−1: 3385 (NH), 3070, 3043 (CH Ar), 2992, 2914, 2848 (CH aliphatic), 1711 (C = O), 1615 (NH bending). 1H NMR (400 MHz, in deuterated CDCl3) δ ppm: 0.99 (t, 3H, CH2CH3, J 7.36 Hz), 1.20 (d, 3H, CHCH3, J 6.92 Hz), 1.22 (d, 3H, CHCH3, J 6.88 Hz), 1.35 (t, 3H, CH2CH3, J 5.84 Hz), 2.19 (m, 1H, C4-H pyran), 2.29 (m, 1H, C4-H pyran), 2.37 (s, 3H, Ar-CH3), 2.83–2.98 (m, 5H, CH2CH3, CH2CH3 and CHCH3), 3.27 (d, 1H, CH alpha C = O, J 17.00 Hz), 3.33 (d, 1H, CH alpha C = O, J 16.96 Hz), 4.08 (m, 1H, C3-H pyran), 4.09 (m, 1H, C3-H pyran), 6.79 (s, 1H, C6′-H Ar), 7.05 (d, 1H, C4′-H Ar, J 7.08 Hz), 7.11 (d, 1H, C3′-H Ar, J 7.64 Hz), 7.15 (t, 1H, C6-H Ar, J 7.48 Hz), 7.29 (d, 1H, C5-H Ar, J 7.92 Hz), 7.45 (d, 1H, C7-H Ar, J 7.72 Hz), 9.01 (s, 1H, NH, exch. D2O). 13C NMR (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, 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). MS (m/z,%): 419.27 (M+, 100). Anal. Calcd. for C27H33NO3 (419.5558): C, 77.29; H, 7.93; N, 3.34%. Found: C,76.89; H, 7.91; N, 3.51%.
4.2. Anti-inflammatory activity and ulcerogenic effect 4.2.1. Animals Adult male albino Wistar rats, weighing 170–200 g, were obtained from Nahda University Animal House, Beni-Sueif, Egypt. Animals were acclimatized for one week in a room controlled for temperature, humidity, and light (12 h light–dark cycle). Rats were housed in well ventilated cages with free access to food and water. All experimental procedures were approved by the institutional guidelines of the Research Ethics Committee of Faculty of Pharmacy, Cairo University and the regulations of the local Animal Welfare authorities.
4.1.1.2. 2-Isopropyl-5-methylphenyl-2-[(3‑chloro‑2-methylphenyl) amino] benzoate (7). Rf: 0.85 (CCl4:CH3COOC2H5 9:1). Yield: (3 g) 76.3%. Mp: 111–114 °C. IR (KBr) υmax/cm−1: 3323 (NH), 3085, 3046 (CH Ar), 2975, 7
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
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 magnifying lens to check for the presence of macroscopically visible lesions. The number of lesions in each stomach, if any, was counted and recorded. 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 homogenate and (B) 10% buffered plasma at 37 °C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4.3. Prodrug chemical stability and in vitro studies 4.3.1. Method of analysis of prodrug and bioactive products a RP-HPLC method was developed and validated for the determination of each prodrug in mixture with its bioactive compounds in aqueous medium, liver homogenate and human plasma. The chromatographic separation was achieved on a Thermo RP-C18 column (250 mm x 4.6 mm, 5 µm), at ambient temperature using a mobile phase consisting of 0.2% formic acid (A) and acetonitrile (B) with a gradient mode according to Table S5 (See Supporting Information File) at a flow rate of 1 mL/min. UV detection was carried out at 274 nm for 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) (See Supporting Information File).
Table 3 The regression parameters, observed rate constants and half-lives for the hydrolysis of the new prodrugs:. Prodrug Kobs (min t½ (min)
−1
)
20% Liver homogenate (6) (7) 0.0078 0.0075 88.84 92.4
10% Buffered plasma (6) (7) 0.0065 0.006 106.61 115.5
4.2.2. Study design and procedure 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: Group 1 (control group); Group II (ibuprofen group); Group III (etodolac group); Group IV (prodrug (6) group); Group V (tolfenamic acid group); Group VI (prodrug (7) group). 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 in 2% Tween 80 and administrated orally 30 min before induction of the inflammation, while the control group received only 2% Tween 80. Subplantar injection of 0.1 mL of 1% carrageenan solution in saline (0.9%) in the right leg induced paw edema. Paw edema thickness was measured after 1, 2, 3, 4, 5 and 6 hr in the right hind paw using a screw gauge micrometer and then compared with the left hind paw thickness of each rat. The difference of average values between treated and control groups was calculated for each time interval and evaluated statistically. Quantitative variables were expressed as means ± standard error (SEM). The anti-inflammatory activity was expressed as percentage inhibition of edema volume in treated animals by comparison to the control group according to the following equation: % of edema inhibition = (VR - VL) control - (VR - VL) treated ×100 (VR - VL) controlWhere VR represents the right paw thickness, VL represents the left paw thickness, (VR - VL) control represents the mean
4.3.2. Preparation of standard and working solutions Standard stock solutions of compounds 1–3 and their corresponding prodrugs 6&7 were prepared in methanol to obtain solutions of 0.1 mg/mL (for chemical stability) and 10 mg/mL (for in vitro enzymatic hydrolysis). 4.3.3. Preparation of liver homogenate Five adult albino Wistar male and female rats, (300–400 g), free of any signs of observable clinical abnormalities were used. The rats were housed in an air-conditioned room and fed standard meals and water. To collect the livers, the rats were systemically anesthetized with ketamine hydrochloride (35 mg/kg) IM injections in combination with a muscle relaxant xylazine hydrochloride (5 mg/kg). Livers were removed by surgery, and immediately homogenized by tissue homogenizer, then centrifuged at 6000 rpm for 20 min. at 4 °C then the supernatant was separated carefully (El-Bagary et al., 2016; Moussa et al., 2019; 2018). 4.3.4. Preparation of 10% buffered plasma Pooled human plasma was donated from national blood bank (Vacsera, Egypt). Human plasma (4 mL) was diluted with 1 mL of 0.05 M phosphate buffer of pH 7.4 followed by incubation at 37 ± 1 °C in a shaking water bath for 5 min. 8
European Journal of Pharmaceutical Sciences 140 (2019) 105101
S.T. Hassib, et al.
4.3.5. Preparation of blank buffered human plasma or liver supernatant sample An aliquot (4 mL) of the supernatant or buffered human plasma was placed in a test tube and incubated for 5 min in a shaking water bath equilibrated at 37 ± 1 °C. An aliquot of 150 µL was withdrawn and 450 µL methanol was added. The mixture was then centrifuged at 13,000 rpm for 7 min at 4 °C. From the obtained clear supernatant, 10 µL was analyzed by HPLC (See Supporting file, Figures S7 and S8).
configurationally unstable biaryl lactone, in. Org. Synth. 70–78. https://doi.org/10.1002/ 0471264229.os088.07. Brown, M.J., Sharma, P., Mir, F., Bennett, P.N., 2012. Clinical Pharmacology, 12th ed. Churchill Livingstone. 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:// doi.org/10.1185/030079907X242584. Chaudhary, R., Negi, V., Sharma, R., Percha, V., Dobhal, Y., Pant, R., 2013. Design, synthesis and evaluation of mutual prodrugs of etodolac with polyphenols as safer nsaids. Inven. Impact Med. Chem. 3, 152–158. 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 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. org/10.1007/s10637-009-9331-8. El-Bagary, R., Hashem, H., Fouad, M., Tarek, S., 2016. UPLC-MS-MS method for the determination of vilazodone in human plasma: application to a pharmacokinetic study. J. Chromatogr. Sci. 54, 1365–1372. https://doi.org/10.1093/chromsci/bmw084. Foye, W.O., Lemke, T.L., Williams, D.A., 2008. Foye's Principles of Medicinal Chemistry. 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 cyclo-oxygenase 2 selective and non-selective non-steroidal anti-inflammatory drugs: nested case-control study. Lancet 365, 475–481. https://doi.org/10.1016/S0140-6736(05) 17864-7. Hassan, G.S., Abou-Seri, S.M., Kamel, G., Ali, M.M., 2014. Celecoxib analogs bearing benzofuran moiety as cyclooxygenase-2 inhibitors: design, synthesis and evaluation as potential anti-inflammatory agents. Eur. J. Med. Chem. 76, 482–493. https://doi.org/10.1016/J. EJMECH.2014.02.033. Hassanein, H.H., Georgey, H.H., Fouad, M.A., El Kerdawy, A.M., Said, M.F., 2017. Synthesis and molecular docking of new imidazoquinazolinones as analgesic agents and selective COX-2 inhibitors. Future Med. Chem. 9, 553–578. https://doi.org/10.4155/fmc-2016-0240. Kashfi, K., Rayyan, Y., Qiao, L.L., Williams, J.L., Chen, J., Del Soldato, P., Traganos, F., Rigas, B., Ryann, Y., 2002. Nitric oxide-donating nonsteroidal anti-inflammatory drugs inhibit the growth of various cultured human cancer cells: evidence of a tissue type-independent effect. J. Pharmacol. Exp. Ther. 303, 1273–1282. https://doi.org/10.1124/jpet.102.042754. Katzung, B.G., Kruidering-Hall, M., Trevor, A.J., 2019. NSAIDs, Acetaminophen, & Drugs used in Rheumatoid Arthritis & Gout. Katzung & Trevor's Pharmacology: Examination & Board Review, 12e. The McGraw-Hill Companies. Mehanna, A.S., 2003. NSAIDs: chemistry and pharmacological actions. Am. J. Pharm. Educ. https://doi.org/10.5688/aj670263. Meshali, M., El-Sabbah, E., Foda, A., 1983. Effect of encapsulation of flufenamic acid with acrylic resins on its bioavailability and gastric ulcerogenic activity in rats. Acta Pharm. Technol. 29, 217–230. Moussa, B.A., El-Zaher, A.A., El-Ashrey, M.K., Fouad, M.A., 2018. Synthesis and molecular 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. org/10.1016/j.ejmech.2018.02.038. Moussa, B.A., Zaher, A.A.El, Ashrey, M.K.El, 2019. Roflumilast analogs with improved metabolic stability, plasma protein binding, and pharmacokinetic profile 1–12. doi:10.1002/ dta.2562. Neises, B., Steglich, W., 1978. Simple method for the esterification of carboxylic acids. Angew. 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 properties and molecular mechanisms of thymol: prospects for its therapeutic potential and pharmaceutical development. Front. Pharmacol. 8, 380. https://doi.org/10.3389/fphar. 2017.00380. Paliwal, M., Sucheta, Ruchita, Jain, S., Monika, Himanshu, 2017. Synthesis and biological evaluation of mutual prodrugs of carboxylic group containing some non-steroidal antiinflammatory drugs and propyphenazone. Curr. Drug Deliv. 14, 1213–1224. https://doi. org/10.2174/1567201814666170213153509. Pandey, P., Pandey, S., Dubey, S., 2018. Mutual amide prodrug of etodolac-glucosamine : synthesis, characterization and pharmacological screening synthesis of mutual prodrug : characterization and preformulation studies of the synthesized prodrug : protein binding study: in vitro hydrolysis st 10–15. Richardson, S.J., Bai, A., Kulkarni, A., F. Moghaddam, M., 2016. Efficiency in drug discovery: liver S9 fraction assay as a screen for metabolic stability. Drug Metab. Lett. 10, 83–90. https://doi.org/10.2174/1872312810666160223121836. Robert, A., Northam, J.I., Nezamis, J.E., Phillips, J.P., 1970. Exertion ulcers in the rat. Am. J. Dig. Dis. 15, 497–507. https://doi.org/10.1007/BF02283881. Sharav, Y., Benoliel, R., 2008. Pharmacotherapy of acute orofacial pain. Orofac. Pain Headache 349–376. https://doi.org/10.1016/B978-0-7234-3412-2.10015-X. Sheha, M., Khedr, A., Elsherief, H., 2002. Biological and metabolic study of naproxen-propyphenazone mutual prodrug. Eur. J. Pharm. Sci. 17, 121–130. Smith, G.W., Davis, J.L., Tell, L.A., Webb, A.I., Riviere, J.E., 2008. Extralabel use of nonsteroidal anti-inflammatory drugs in cattle. J. Am. Vet. Med. Assoc. 232, 697–701. https:// doi.org/10.2460/javma.232.5.697. Wallace, J.L., Vong, L., 2008. NSAID-induced gastrointestinal damage and the design of GIsparing NSAIDs. Curr. Opin. Investig. Drugs 9, 1151–1156. Warner, T.D., Mitchell, J.A., 2003. Nonsteroidal antiinflammatory drugs inhibiting prostanoid efflux: as easy as ABC? Proc. Natl. Acad. Sci. U. S. A. 100, 9108–9110. https://doi.org/10. 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. 3181/00379727-111-27849.
4.3.6. Chemical hydrolysis The stability of the newly synthesized prodrugs was studied in aqueous phosphate buffer solutions of different pH values (1.2, 6.8, and 7.4) and at three different temperatures (25, 37 and 60 °C). Accurately measured aliquots equivalent to (1 mg) of prodrugs 6&7 working solutions were transferred separately into three volumetric flasks (50 mL), and each flask was completed with a different phosphate buffer solution pH (1.2, 6.8 or 7.4). The solutions were well mixed using vortex for 1 min., then 10 mL of each flask was transferred into a series of 3 screw capped test tubes and kept in a temperature-controlled water bath shaker at different temperatures 25, 37 or 60 °C. At appropriate time intervals, samples of each tube were withdrawn, cooled and immediately analyzed for their content of the remaining prodrugs, and appearance of parent drugs by RP-HPLC. All the experiments were carried out in triplicate. 4.3.7. In vitro enzymatic hydrolysis For the construction of calibration curves, 80 µL from each working solution of concentrations (0.1–7 mg/mL) of 1, 2, 6 & 7 was added in a test tube with 4 mL of the prepared 10% buffered plasma or the separated liver supernatant, vortexed well for 1 min. An aliquot (150 µL) was immediately withdrawn and 450 µL methanol was added to stop the reaction and precipitate the proteins. The mixture was then centrifuged at 13,000 rpm for 7 min at 4 °C. 10 µL of the supernatant was analyzed by HPLC. Calibration curves were constructed showing good correlation of more than 0.99. Intra-day accuracy and precision were evaluated by three-replicate analysis of 1, 2, 6 & 7 at concentrations of LLOQ (0.1 mg/mL) in 10% buffered plasma or the separated liver supernatant in the same day following the preparation procedure mentioned above. The inter-day accuracy and precision were assessed by analyzing three replicates of LLOQ on three consecutive days. The precision and accuracy of the method were determined by calculating the percent coefficient of variation (% CV) and recovery for the concentrations obtained for different determinations, See Supporting Information File, Table S6. For studying in vitro enzymatic hydrolysis, 5.5 mL of the standard stock solution of each prodrug was diluted with methanol (5.5 mg/mL). An aliquot (80 µL) was added in a test tube with 4 mL of the prepared 10% buffered plasma or the separated liver supernatant and vortexed well for 1 min. The tubes were placed in a shaking water bath at 37 ± 1 °C at different time intervals ranging from 0–300 min. An aliquot (150 µL) was processed following the procedure of preparation of calibration standards and analyzed by HPLC. Then, the concentrations of each prodrug and corresponding parent drug were calculated. Supplementary materials Supplementary material associated with this article can be found, in the online version, at 10.1016/j.ejps.2019.105101. References Bringmann, G., Gulder, T.A.M., Gulder, T., 2012. Discussion addendum for: asymmetric synthesis of (M)-2-Hydroxymethyl-1-(2-Hydroxy-4,6-Dimethylphenyl)Naphthalene viaa
9