Accepted Manuscript Synthesis, analgesic, anti-inflammatory and anti-ulcerogenic activities of certain novel Schiff’s bases as fenamate isosteres Ahmed M. Alafeefy, Mohammed A. Bakht, Majid A. Ganie, Nazam Ansari, Nahed N. El-Sayed, Amani S. Awaad PII: DOI: Reference:
S0960-894X(14)01295-5 http://dx.doi.org/10.1016/j.bmcl.2014.11.088 BMCL 22243
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Accepted Date:
26 October 2014 28 November 2014
Please cite this article as: Alafeefy, A.M., Bakht, M.A., Ganie, M.A., Ansari, N., El-Sayed, N.N., Awaad, A.S., Synthesis, analgesic, anti-inflammatory and anti-ulcerogenic activities of certain novel Schiff’s bases as fenamate isosteres, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.11.088
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis, analgesic, anti-inflammatory and anti-ulcerogenic activities of certain novel Schiff’s bases as fenamate isosteres Ahmed M. Alafeefy a*, Mohammed A. Bakht a, Majid A. Ganie b, Nazam Ansari b, Nahed N. El-Sayed c,d, Amani S. Awaad e a
Department of Pharmaceutical Chemistry, College of Pharmacy, Salman Bin Abdulaziz
University, P.O. Box 173, Alkharj 11942, Saudi Arabia b
Department of Pharmacology and Toxicology, College of Pharmacy, Salman Bin Abdulaziz
University, P.O. Box 173, Alkharj 11942, Saudi Arabia c
Department of Chemistry, College of Science, King Saud University, P.O. Box 22452,
Riyadh 11495, Saudi Arabia d
Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622,
Egypt e
Department of Pharmacognosy, College of Pharmacy “Girls section”, Salman Bin Abdulaziz
University, P.O. Box 173, Alkharj 11942, Saudi Arabia
Abstract: A series of certain novel Schiff bases as fenamate isosteres (VI:a-k) were synthesized to locate analgesic, anti-inflammatory agent with minimal ulcerogenic potential. The structures of the newly synthesized compounds were elucidated on the basis of their elemental analysis as well as IR, and NMR and mass spectroscopic data. All the compounds were evaluated for their anti-inflammatory activity by carrageenan induced paw oedema method. The compounds possessing good anti-inflammatory activity were further tested for analgesic, ulcerogenic, lipid peroxidation potentials and liver toxicity. Compounds (VI-c), (VI-f), (VI-h) and (VI-i) showed the best anti-inflammatory and significant analgesic activities at doses comparable to that of the standard drug Indomethacin. However, compounds (VI-c) and (VI-f) could be considered the most potent antiinflammatory and analgesic molecules with maximum reduction in gastro-intestinal ulceration with no hepatocyte necrosis or liver degeneration.
Keywords: Fenamate isostere. Schiff‟s bases. Synthesis. Analgesic. Anti-inflammatory. Antiulcerogenic.
*Corresponding authors. [email protected] Cell: + 966 50706 9896. 1
Non-steroidal anti-inflammatory drugs (NSAIDs) are successfully used to treat pain, fever and inflammation, particularly arthritis.1-3 They exert their beneficial effects mainly through inhibition of cyclooxygenases (COXs), the key enzyme in prostaglandin (PG) biosynthesis from arachidonic acid (AA). Two such isoforms, COX-1 and COX-2, are involved and are regulated differently. 4-7 COX-1 is the constitutive enzyme which provides cytoprotection in the gastrointestinal (GI) tract whereas the inducible COX-2 mediates inflammation.7,8 In spite of the successful application of NSAIDs by millions of patients worldwide, there are serious problems in their medication, since most of them show greater selectivity for COX-1 than COX-2.9 This preferential selectivity leads to grave GI effects, such as mucosal lesions, hemorrhage, and ulceration.10,11 The incidence of such clinically significant GI problems is alarming in addition to more complex adverse effect especially in patients with risk factors.12,13 N-substituted anthranilic acid derivatives, commonly known as fenamates, are interesting class of NSAIDs, they are considered as nitrogen isosteres of salicylic acid. Moreover, they are important classes of compounds in the medical field and their biological activities have attracted considerable attention towards biomedical applications. They are well-known for their importance as anticancer agents14,15 antimicrobial,16 anti-inflammatory and immune modulators17-19 as well as analgesic20 agents. However, they also suffer the same general untoward effects characteristic of this class of drugs. Conversion of the carboxylic acid moiety to hydrazide and/or Schiff base is a suitable alternative to overcome such deleterious effects. In the present context a newly synthesized N-4-toluoyl anthranilic acid hydrazide was reacted with different substituted aromatic aldehydes to produce novel Schiff‟s bases as fenamate isosteres (VI:a-k). The structures of these newly synthesized compounds were confirmed by IR, NMR, Mass spectrometry and their purity was ascertained by elemental analysis. The synthesized compounds were evaluated for their anti-inflammatory, analgesic activities, acute ulcerogenicity in addition to liver enzyme and lipid peroxidation. Molecular properties, mainly hydrophobicity, molecular size, flexibility and the presence of various pharmacophoric features influence the behaviour of molecules in the living organism, mainly bioavailability. Thus, in order to achieve orally available drugs we have subjected the newly developed Schiff bases (VI:a-k) for the prediction of some basic pharmacokinetic properties under the Lipinski „„Rule of Five‟‟ to filtered compounds for anti-inflammatory and analgesic properties. 2
The current compounds were subjected to computational study in order to filter the candidate compounds for biological screening. For good membrane permeability logP value should be ≤5.21 All the title compounds (VI:a-k) have logP value 4.02–6.12. High oral bioavailability is an important factor for the development of bioactive molecules as therapeutic agents. Good intestinal absorption, reduced molecular flexibility (measured by the number of rotatable bonds), low polar surface area or total hydrogen bond count (sum of donors and acceptors), are important predictors of good oral bioavailability.24 Molecular properties such as membrane permeability and bioavailability is always associated with some basic molecular descriptors such as logP (partition coefficient), molecular weight (MW), or hydrogen bond acceptors and donors counts in a molecule.23 Lipinski21 used these molecular properties in formulating his „„Rule of Five”. The rule states that most molecules with good membrane permeability have logP ≤ 5, molecular weight ≤500, number of hydrogen bond acceptors ≤10, and number of hydrogen bond donors ≤ 5. This rule is widely used as a filter for drug-like properties. Table 1 contains calculated percentage of absorption (% ABS), molecular polar surface area (TPSA) and Lipinski parameters of the investigated compounds under test. Magnitude of absorption is expressed by the percentage of absorption. Absorption percent was calculated24 using the expression: %ABS =109 - 0.345 PSA. Polar surface area (PSA) was determined by the fragment-based method of Ertl and coworkers.25,26 A poor permeation or absorption is more likely when there are more than 5 H-bond donors, 10 Hbond acceptors. Hydrogen-bonding capacity has been also identified as an important parameter for describing drug permeability.27 The results in our hands proved that compounds (VI:a-k) have hydrogen bond donor and acceptor in considerable range as shown in Table 1. The number of rotatable bonds is important for conformational changes of molecules under study and ultimately for the binding of receptors or channels. It is revealed that for passing oral bioavailability criteria no of rotatable bond should be ≤10.22 The compounds in this series (VI:a-k) in general possess acceptable of rotatable bonds (5–8) and therefore, exhibit low conformational flexibility. Molecular polar surface area (TPSA) is a very useful parameter for the prediction of drug transport properties. TPSA is a sum of surfaces of polar atoms (usually oxygen, nitrogen and attached hydrogen) in a molecule. TPSA and volume is inversely proportional to % ABS, compounds (VI-a) and (VI-e) had maximum absorption (88.98%) as their corresponding polar surface area and volume are least among the series (Table 1). All the title compounds 3
(VI:a-k) followed the Lipinski „„Rule of Five‟‟. The pharmacokinetic parameters were calculated
Online
from
Molinspiration
Chemoinformatics
(http://www.molinspiration.com/cgi-bin/properties) and are given in Table 1. Structure based drug design is now very routine work as many drug fail to reach clinical phases because of the encountered ADME/TOX problem. Therefore prediction of these problems before synthesis is rational approach to minimize the cost. The Osiris calculations are tabulated in Table 2. Toxicity risks (mutagenicity, tumorogenicity, irritation, reproduction) and physicochemical properties (miLogP, solubility, drug likeness and drug score) of compounds (VI:a-k) were calculated by the methodology developed by Osiris. The toxicity risk predictor locates fragments within a molecule, which indicates a potential toxicity risk. Based on our data (Table 2), it is obvious that, all compounds, except compound (VI-j), are supposed to be non-mutagenic, non-irritating, when run through the mutagenicity assessment system in comparison with the standard drug. The logP value of a compound is a well-established measure of the compound‟s hydrophilicity. Low hydrophilicities and therefore high logP values may cause poor absorption or permeation. It has been shown that for compounds to have a reasonable probability of good absorption, their logP value must not be greater than 5.0. On this basis, the current compounds (VI:a-k) possessed logP values in the acceptable range. Further, Table 2 shows drug likeness of compounds (VI:a-k) which is in the comparable zone with that of standard drug used for comparison. We have calculated overall drug score (DS) for the compounds (VI:a-k) and compared with that of standard drug Indomethacin. The drug score combines drug likeness, miLogP, logS, molecular weight and toxicity risks in one handy value than may be used to judge the Compound‟s overall potential to qualify for a drug. This value is calculated by multiplying contributions of the individual properties with the equation (1): DS = ∏ (1/2+1/2Si) ∏ti, where S; (1/1+eap+b) DS is the drug score. Si is the contributions calculated directly from miLogP; logS, molecular weight and drug likeness (pi) via the second equation, which describes a spline curve. Parameters a,b are (1,-5), (1, 5), (0.012, -6) and (1, 0) for miLogP, logS, molecular weight and drug likeness, respectively. The ti is the contributions taken from the four toxicity risk types and the values are 1.0, 0.8 and 0.6 for no risk, medium risk and high risk, respectively. The reported compounds (VIa-k) showed moderate to good drug score as compared with standard drug used.
4
Based on previous work and literature survey it was expected to obtain the corresponding cyclic quinazolinone structure upon treating the oxazine (IV) with hydrazine hydrate.28-32 However, the spectroscopic data of the isolated compound were in full agreement with the proposed open structure corresponding to the formation of N-4-toluoyl anthranilic acid hydrazide (V).301,32 Condensing the later hydrazide with various aromatic aldehydes gave the corresponding Schiff‟s bases. The identity of the formed fenamate derived Schiff‟s bases (VI:a-k) is fully supported by their elemental analysis as well as IR, and NMR spectroscopic data. In general, the IR spectra showed absorption bands due to two NH groups around 3300 cm-1, in addition to other bands at 1540-1538 cm-1 due to C=N group and absorption bands at 1700-1682 cm-1 for two carbonyl groups. The 1H-NMR spectra of the current Schiff‟s bases displayed in general multiplets at δ = 6.62-8.52 ppm due to the aromatic protons. The most characteristics protons of azomethin groups (N=CH) of Schiff‟s bases appeared at δ = 8.728.84 ppm as singlets. Two broad singlets due to two amide protons appeared at δ around = 12 ppm.
13
C-NMR spectra again proved the formation of the proposed open systems. The two
carbonyl, (N=CH) as well as other aromatic and aliphatic carbons appeared at the expected δ values. Mass spectra of all the compounds revealed the parent ion peaks with a considerable relative intensity. The spectral data (IR, 1H-NMR,
13
C-NMR, MS) and C, H, N elemental
analysis of all the synthesized compounds are in accordance with the proposed structures. The compounds were tested at an equimolar oral dose relative to 10mg/kg indomethacin. The percentage inhibition was calculated both after 2 and 3h. The tested compounds showed variable degrees of anti-inflammatory activity in comparison to standard drug indomethacin after 3h (Table 3). The best activity was obtained from compounds (VI-f), (VI-h) and (VI-i), it was found to be ranging from 61.84% to 64.31%. On examining the structure of these compounds, one could conclude that compounds having more methoxy (-OCH3) functionality produced maximum anti-inflammatory inhibitory effect. It can be deduced that compound (VI-i) having 3,4,5-trimethoxy substituent exhibited more inhibitory activity than 2,4,5trimethoxy (VI-h) which is more active than monosubstituted methoxy compound (VI-f). Therefore, the number and arrangement of methoxy substituted derivatives are important for good anti-inflammatory activity. Compound (VI-c) having dihydroxy substituents exhibited moderate activity. Rest of the compounds, possessing OH (VI-b), Cl (VI-e) and unsubstituted (VI-a), were found to be weakly active as anti-inflammatory agents compared to the standard drug indomethacin (78.80% after 3h).
5
Compounds (VI-c), (VI-f), (VI-h) and (VI-i) which showed almost comparable antiinflammatory to that of reference drug, were further tested for their analgesic activity at an equimolar oral dose relative to 10 mg/kg indomethacin (Table 4). The tested compounds in this series showed analgesic activity ranging from 34.2% to 77.8%, compared to 81.0% inhibition displayed by the reference drug indomethacin. It was noticed that compound (VI-i) having maximum anti-inflammatory activity displayed the least analgesic properties. On the other hand, compound (VI-f) having the lowest anti-inflammatory activity (61.84%) showed significant analgesic (67.8%) activities among the current series. Compound (VI-c) showed the highest analgesic properties (77.8%) whereas compounds (VI-h) and (VI-i) showed reduced analgesic activity (46.2% and 34.2%). Consequently, compounds (VI-c and VI-f) were further screened for their acute ulcerogenic activity. The compounds were tested at an equimolar oral dose relative to 10 mg/kg indomethacin. Surprisingly, they showed significant reduction in ulcerogenic activity ranging from 1.417±0.15 to 1.750±0.11, whereas standard drug indomethacin showed high severity index of 2.750±0.44. The maximum reduction in ulcerogenic activity (1.417±0.15) was due to compound (VI-c) having dihydroxy substituent. These results clearly indicated that we succeeded to develop good anti-inflammatory and analgesic agents with reduced ulcerogenic potential. NSAIDs are associated with high incidence of GI ulceration; bleeding and kidney damage which may be linked with lipid peroxidation. So, our study aimed to examine lipid peroxidation (LP) induction capacity of compounds (VI-c and VI-f) which showed remarkable analgesic and anti-inflammatory activity. LP was measured as nanomoles of malondialdehyde (MDA)/100 mg of gastric tissue. We noticed that indomethacin exhibited maximum lipid peroxidation 6.73±0.17, whereas (VI-c and VI-f) 4.02 and 4.39, respectively (control group showed 3.14±0.14). The most potent anti-inflammatory and analgesic agents with reduced ulcerogenic and lipid peroxidation potential (VI-c and VI-f) were further examined for their hepatotoxic effects as well as their effect on serum enzymes and total albumin. Both compounds exhibited significant reduction in SGOT, SGPT and total albumin level in comparison to control as shown in Table 5. Because several studies reported that most NSAIDs induce liver damage, we studied the histopathlogy of liver samples. The results revealed that there is no indication of any
6
pathological change. Neither necrosis nor degeneration was found in comparison to the control group (Fig 1-3). In conclusion, we have accomplished the synthesis of certain novel Schiff‟s bases. The new compounds have been investigated for their anti-inflammatory and analgesic activities as well as their ulcerogenic activity, liver toxicity and lipid peroxidation. Among the synthesized Schiff‟s bases, compounds (VI-c) and (VI-f) were found to be the most potent as antiinflammatory and analgesic agents with maximum reduction in GI toxicity, no hepatocyte necrosis or degeneration in liver samples. Indeed, these two compounds could be considered as suitable candidates for bio-technologic application as anti-inflammatory and analgesic agents. Acknowledgments This research was funded by National Plan of Science, Technology and Innovation (Grant No. 12-MED2980-54), Salman bin Abdulaziz University, Alkharj, P.O.Box 173, 11942 (to AF).
References: 1. Palomer, A.; Cabré, F.; Pascual, J.; Campos, J.; Trujillo, M. A.; Entrena, A.; Gallo, M. A.; García, L.; Mauleón, D.; Espinosa, A. J. Med. Chem. 2002, 45, 1402. 2. Talley, J. J.; Brown, D. L.; Carter, J. S.; Graneto, M. J.; Koboldt, C. M.; Masferrer, J. L.; Perkins, W. E.; Rogers, R. S.; Shaffer, A. F.; Zhang, Y. Y.; Zweifel, B. S.; Seibert, K. J Med Chem. 2000, 43(5), 775. 3. Kumar H.; Javed, S. A.; Khan, S. A.; Amir, M. H.; Kumar, S. A.; Jawed, S. A.; Khan, M.; Amir, E. J. Med. Chem. 2008, 43, 2688. 4. Amir, M.; Shikha, K. M.; Amir, K.; Shikha, E. J. Med. Chem. 2004, 39, 535. 5. Dannhardt, G.; Kiefer, W.; Eur. J. Med. Chem. 2001, 36, 109. 6. Habeeb, A. G.; Praveen Rao, P. N.; Knaus, E. E. J. Med. Chem. 2001, 44, 2921. 7. Almansa, C.; Alfón, J.; de Arriba, A. F.; Cavalcanti, F. L.; Escamilla, I.; Gómez, L. A.; Miralles, A.; Soliva, R.; Bartrolí, J.; Carceller, E.; Merlos, M.; García-Rafanell, J. J. Med. Chem. 2003, 46, 3463. 8. Hinz, B.; Brune, K. Pharm. Exp. Ther. 2002, 300, 367. 9. Allison, M. C.; Howatson, A. G.; Torrance, C. J.; Lee, F. D.; Russell, R. N. Engl. J. Med. 1992, 327, 749. 10. F.K. Chan, Nat. Clin. Pract. 3 (10) (2006) 563–573. 7
11. Gaba, M.; Singh, D.; Singh, S.; Sharma, V.; Gaba, P. Eur. J. Med. Chem. 2010, 45, 2245. 12. Tammara, V. K.; Narurkar, M. M.; Crider, A. M.; Khan, M. A. J. Pharm. Sci. 1994, 83, 644–648. 13. Nirmal, R.; Prakash, C.; Meenakshi, K.; Shanmugapandiyan, P. J.Young. Pharm. 2010, 2, 162. 14. Chai, L. Q.; Zhang, H. S.; Huang, J. J.; Zhang, Y. L. Spectrochim Acta A Mol Biomol Spectrosc. 2014, 137C, 661. 15. Qiao, X.; Ma, Z. Y.; Xie, C. Z.; Xue, F.; Zhang, Y. W.; Xu, J. Y.; Qiang, Z. Y.; Lou, J. S.; Chen, G. J.; Yan, S. P. J. Inorg. Biochem. 2011, 5, 728. 16. Alafeefy, A. M.; Awaad, A. S.; Abdel-Aziz, H. A.; El-Meligy, R. M.; Zain, M. E.; AlOuthman, M. R.; Bacha, A. B. J. Enzyme Inhib. Med. Chem. 2014, 18, 1. 17. Kumar, K. S.; Ganguly, S.; Veerasamy, R. De Clercq, E. Eur. J. Med. Chem. 2010, 45(11), 5474. 18. Kimura, J.; Yamada, H.; Ogura, H.; Yajima, T.; Fukushima, T. Anal Chim Acta. 2009, 635(2), 207. 19. Hong, M.; Geng, H.; Niu, M.; Wang, F.; Li, D.; Liu, J.; Yin, H. Eur, J. Med Chem. 2014, 68C, 550. 20. Sasaki, J.; Takahashi, H.; Furutani, Y.; Sineshchekov, O.; A.; Spudich, J. L.; Kandori, H. Biochemistry 2014, 53(37), 5923. 21. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug. Delivery Rev. 2001, 46(1-3), 3. 22. Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D. J. Med. Chem. 2002, 45, 2615. 23. Muegge, I. Med. Res. Rev. 2003, 23, 302. 24. Zhao, Y. H.; Abraham, M. H.; Le, J.; Hersey, A.; Luscombe, C. N.; Beck, G.; Sherborne, B.; Cooper, I. Pharm. Res. 2002, 19, 1446. 25. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714. 26. . 27. Refsgaard, H. H.; Jensen, B. F.; Brockhoff, P. B.; Padkjaer, S. B.; Guldbrandt, M.; Christensen, M. S. J. Med. Chem. 2005, 48, 805. 28. Alafeefy, A. M.; Kadi, A. A.; El-Azab, A. S.; Abdel-Hamide, S. G.; Daba, M. H. Arch. Pharm. (Weinheim). 2008, 341, 377. 8
29. Alafeefy, A. M. Arzneim. Forschung. 2008, 58, 457. 30. Winter, C. W.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med.1962, 111, 544. 31. Mathew, V.; Keshavayya, J.; Vaidya, V.P. Eur. J. Med. Chem. 2006, 41, 1048. 32. Cioli, V.; Putzolu, S.; Rossi, V.; Barcellona, S. P.; Corradino, C. Toxicol. Appl. Pharmacol. 1979, 50, 283.
9
Table 1 Pharmacokinetic Properties important for good oral bioavailability of titled compounds (VI:a-k) Compounds
%ABS
Volume (A3)
Rule VI-a
88.98
355.88
58.05
5
S0960-894X(14)01295-5 http://dx.doi.org/10.1016/j.bmcl.2014.11.088 BMCL 22243
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Accepted Date:
26 October 2014 28 November 2014
Please cite this article as: Alafeefy, A.M., Bakht, M.A., Ganie, M.A., Ansari, N., El-Sayed, N.N., Awaad, A.S., Synthesis, analgesic, anti-inflammatory and anti-ulcerogenic activities of certain novel Schiff’s bases as fenamate isosteres, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.11.088
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis, analgesic, anti-inflammatory and anti-ulcerogenic activities of certain novel Schiff’s bases as fenamate isosteres Ahmed M. Alafeefy a*, Mohammed A. Bakht a, Majid A. Ganie b, Nazam Ansari b, Nahed N. El-Sayed c,d, Amani S. Awaad e a
Department of Pharmaceutical Chemistry, College of Pharmacy, Salman Bin Abdulaziz
University, P.O. Box 173, Alkharj 11942, Saudi Arabia b
Department of Pharmacology and Toxicology, College of Pharmacy, Salman Bin Abdulaziz
University, P.O. Box 173, Alkharj 11942, Saudi Arabia c
Department of Chemistry, College of Science, King Saud University, P.O. Box 22452,
Riyadh 11495, Saudi Arabia d
Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622,
Egypt e
Department of Pharmacognosy, College of Pharmacy “Girls section”, Salman Bin Abdulaziz
University, P.O. Box 173, Alkharj 11942, Saudi Arabia
Abstract: A series of certain novel Schiff bases as fenamate isosteres (VI:a-k) were synthesized to locate analgesic, anti-inflammatory agent with minimal ulcerogenic potential. The structures of the newly synthesized compounds were elucidated on the basis of their elemental analysis as well as IR, and NMR and mass spectroscopic data. All the compounds were evaluated for their anti-inflammatory activity by carrageenan induced paw oedema method. The compounds possessing good anti-inflammatory activity were further tested for analgesic, ulcerogenic, lipid peroxidation potentials and liver toxicity. Compounds (VI-c), (VI-f), (VI-h) and (VI-i) showed the best anti-inflammatory and significant analgesic activities at doses comparable to that of the standard drug Indomethacin. However, compounds (VI-c) and (VI-f) could be considered the most potent antiinflammatory and analgesic molecules with maximum reduction in gastro-intestinal ulceration with no hepatocyte necrosis or liver degeneration.
Keywords: Fenamate isostere. Schiff‟s bases. Synthesis. Analgesic. Anti-inflammatory. Antiulcerogenic.
*Corresponding authors. [email protected] Cell: + 966 50706 9896. 1
Non-steroidal anti-inflammatory drugs (NSAIDs) are successfully used to treat pain, fever and inflammation, particularly arthritis.1-3 They exert their beneficial effects mainly through inhibition of cyclooxygenases (COXs), the key enzyme in prostaglandin (PG) biosynthesis from arachidonic acid (AA). Two such isoforms, COX-1 and COX-2, are involved and are regulated differently. 4-7 COX-1 is the constitutive enzyme which provides cytoprotection in the gastrointestinal (GI) tract whereas the inducible COX-2 mediates inflammation.7,8 In spite of the successful application of NSAIDs by millions of patients worldwide, there are serious problems in their medication, since most of them show greater selectivity for COX-1 than COX-2.9 This preferential selectivity leads to grave GI effects, such as mucosal lesions, hemorrhage, and ulceration.10,11 The incidence of such clinically significant GI problems is alarming in addition to more complex adverse effect especially in patients with risk factors.12,13 N-substituted anthranilic acid derivatives, commonly known as fenamates, are interesting class of NSAIDs, they are considered as nitrogen isosteres of salicylic acid. Moreover, they are important classes of compounds in the medical field and their biological activities have attracted considerable attention towards biomedical applications. They are well-known for their importance as anticancer agents14,15 antimicrobial,16 anti-inflammatory and immune modulators17-19 as well as analgesic20 agents. However, they also suffer the same general untoward effects characteristic of this class of drugs. Conversion of the carboxylic acid moiety to hydrazide and/or Schiff base is a suitable alternative to overcome such deleterious effects. In the present context a newly synthesized N-4-toluoyl anthranilic acid hydrazide was reacted with different substituted aromatic aldehydes to produce novel Schiff‟s bases as fenamate isosteres (VI:a-k). The structures of these newly synthesized compounds were confirmed by IR, NMR, Mass spectrometry and their purity was ascertained by elemental analysis. The synthesized compounds were evaluated for their anti-inflammatory, analgesic activities, acute ulcerogenicity in addition to liver enzyme and lipid peroxidation. Molecular properties, mainly hydrophobicity, molecular size, flexibility and the presence of various pharmacophoric features influence the behaviour of molecules in the living organism, mainly bioavailability. Thus, in order to achieve orally available drugs we have subjected the newly developed Schiff bases (VI:a-k) for the prediction of some basic pharmacokinetic properties under the Lipinski „„Rule of Five‟‟ to filtered compounds for anti-inflammatory and analgesic properties. 2
The current compounds were subjected to computational study in order to filter the candidate compounds for biological screening. For good membrane permeability logP value should be ≤5.21 All the title compounds (VI:a-k) have logP value 4.02–6.12. High oral bioavailability is an important factor for the development of bioactive molecules as therapeutic agents. Good intestinal absorption, reduced molecular flexibility (measured by the number of rotatable bonds), low polar surface area or total hydrogen bond count (sum of donors and acceptors), are important predictors of good oral bioavailability.24 Molecular properties such as membrane permeability and bioavailability is always associated with some basic molecular descriptors such as logP (partition coefficient), molecular weight (MW), or hydrogen bond acceptors and donors counts in a molecule.23 Lipinski21 used these molecular properties in formulating his „„Rule of Five”. The rule states that most molecules with good membrane permeability have logP ≤ 5, molecular weight ≤500, number of hydrogen bond acceptors ≤10, and number of hydrogen bond donors ≤ 5. This rule is widely used as a filter for drug-like properties. Table 1 contains calculated percentage of absorption (% ABS), molecular polar surface area (TPSA) and Lipinski parameters of the investigated compounds under test. Magnitude of absorption is expressed by the percentage of absorption. Absorption percent was calculated24 using the expression: %ABS =109 - 0.345 PSA. Polar surface area (PSA) was determined by the fragment-based method of Ertl and coworkers.25,26 A poor permeation or absorption is more likely when there are more than 5 H-bond donors, 10 Hbond acceptors. Hydrogen-bonding capacity has been also identified as an important parameter for describing drug permeability.27 The results in our hands proved that compounds (VI:a-k) have hydrogen bond donor and acceptor in considerable range as shown in Table 1. The number of rotatable bonds is important for conformational changes of molecules under study and ultimately for the binding of receptors or channels. It is revealed that for passing oral bioavailability criteria no of rotatable bond should be ≤10.22 The compounds in this series (VI:a-k) in general possess acceptable of rotatable bonds (5–8) and therefore, exhibit low conformational flexibility. Molecular polar surface area (TPSA) is a very useful parameter for the prediction of drug transport properties. TPSA is a sum of surfaces of polar atoms (usually oxygen, nitrogen and attached hydrogen) in a molecule. TPSA and volume is inversely proportional to % ABS, compounds (VI-a) and (VI-e) had maximum absorption (88.98%) as their corresponding polar surface area and volume are least among the series (Table 1). All the title compounds 3
(VI:a-k) followed the Lipinski „„Rule of Five‟‟. The pharmacokinetic parameters were calculated
Online
from
Molinspiration
Chemoinformatics
(http://www.molinspiration.com/cgi-bin/properties) and are given in Table 1. Structure based drug design is now very routine work as many drug fail to reach clinical phases because of the encountered ADME/TOX problem. Therefore prediction of these problems before synthesis is rational approach to minimize the cost. The Osiris calculations are tabulated in Table 2. Toxicity risks (mutagenicity, tumorogenicity, irritation, reproduction) and physicochemical properties (miLogP, solubility, drug likeness and drug score) of compounds (VI:a-k) were calculated by the methodology developed by Osiris. The toxicity risk predictor locates fragments within a molecule, which indicates a potential toxicity risk. Based on our data (Table 2), it is obvious that, all compounds, except compound (VI-j), are supposed to be non-mutagenic, non-irritating, when run through the mutagenicity assessment system in comparison with the standard drug. The logP value of a compound is a well-established measure of the compound‟s hydrophilicity. Low hydrophilicities and therefore high logP values may cause poor absorption or permeation. It has been shown that for compounds to have a reasonable probability of good absorption, their logP value must not be greater than 5.0. On this basis, the current compounds (VI:a-k) possessed logP values in the acceptable range. Further, Table 2 shows drug likeness of compounds (VI:a-k) which is in the comparable zone with that of standard drug used for comparison. We have calculated overall drug score (DS) for the compounds (VI:a-k) and compared with that of standard drug Indomethacin. The drug score combines drug likeness, miLogP, logS, molecular weight and toxicity risks in one handy value than may be used to judge the Compound‟s overall potential to qualify for a drug. This value is calculated by multiplying contributions of the individual properties with the equation (1): DS = ∏ (1/2+1/2Si) ∏ti, where S; (1/1+eap+b) DS is the drug score. Si is the contributions calculated directly from miLogP; logS, molecular weight and drug likeness (pi) via the second equation, which describes a spline curve. Parameters a,b are (1,-5), (1, 5), (0.012, -6) and (1, 0) for miLogP, logS, molecular weight and drug likeness, respectively. The ti is the contributions taken from the four toxicity risk types and the values are 1.0, 0.8 and 0.6 for no risk, medium risk and high risk, respectively. The reported compounds (VIa-k) showed moderate to good drug score as compared with standard drug used.
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Based on previous work and literature survey it was expected to obtain the corresponding cyclic quinazolinone structure upon treating the oxazine (IV) with hydrazine hydrate.28-32 However, the spectroscopic data of the isolated compound were in full agreement with the proposed open structure corresponding to the formation of N-4-toluoyl anthranilic acid hydrazide (V).301,32 Condensing the later hydrazide with various aromatic aldehydes gave the corresponding Schiff‟s bases. The identity of the formed fenamate derived Schiff‟s bases (VI:a-k) is fully supported by their elemental analysis as well as IR, and NMR spectroscopic data. In general, the IR spectra showed absorption bands due to two NH groups around 3300 cm-1, in addition to other bands at 1540-1538 cm-1 due to C=N group and absorption bands at 1700-1682 cm-1 for two carbonyl groups. The 1H-NMR spectra of the current Schiff‟s bases displayed in general multiplets at δ = 6.62-8.52 ppm due to the aromatic protons. The most characteristics protons of azomethin groups (N=CH) of Schiff‟s bases appeared at δ = 8.728.84 ppm as singlets. Two broad singlets due to two amide protons appeared at δ around = 12 ppm.
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C-NMR spectra again proved the formation of the proposed open systems. The two
carbonyl, (N=CH) as well as other aromatic and aliphatic carbons appeared at the expected δ values. Mass spectra of all the compounds revealed the parent ion peaks with a considerable relative intensity. The spectral data (IR, 1H-NMR,
13
C-NMR, MS) and C, H, N elemental
analysis of all the synthesized compounds are in accordance with the proposed structures. The compounds were tested at an equimolar oral dose relative to 10mg/kg indomethacin. The percentage inhibition was calculated both after 2 and 3h. The tested compounds showed variable degrees of anti-inflammatory activity in comparison to standard drug indomethacin after 3h (Table 3). The best activity was obtained from compounds (VI-f), (VI-h) and (VI-i), it was found to be ranging from 61.84% to 64.31%. On examining the structure of these compounds, one could conclude that compounds having more methoxy (-OCH3) functionality produced maximum anti-inflammatory inhibitory effect. It can be deduced that compound (VI-i) having 3,4,5-trimethoxy substituent exhibited more inhibitory activity than 2,4,5trimethoxy (VI-h) which is more active than monosubstituted methoxy compound (VI-f). Therefore, the number and arrangement of methoxy substituted derivatives are important for good anti-inflammatory activity. Compound (VI-c) having dihydroxy substituents exhibited moderate activity. Rest of the compounds, possessing OH (VI-b), Cl (VI-e) and unsubstituted (VI-a), were found to be weakly active as anti-inflammatory agents compared to the standard drug indomethacin (78.80% after 3h).
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Compounds (VI-c), (VI-f), (VI-h) and (VI-i) which showed almost comparable antiinflammatory to that of reference drug, were further tested for their analgesic activity at an equimolar oral dose relative to 10 mg/kg indomethacin (Table 4). The tested compounds in this series showed analgesic activity ranging from 34.2% to 77.8%, compared to 81.0% inhibition displayed by the reference drug indomethacin. It was noticed that compound (VI-i) having maximum anti-inflammatory activity displayed the least analgesic properties. On the other hand, compound (VI-f) having the lowest anti-inflammatory activity (61.84%) showed significant analgesic (67.8%) activities among the current series. Compound (VI-c) showed the highest analgesic properties (77.8%) whereas compounds (VI-h) and (VI-i) showed reduced analgesic activity (46.2% and 34.2%). Consequently, compounds (VI-c and VI-f) were further screened for their acute ulcerogenic activity. The compounds were tested at an equimolar oral dose relative to 10 mg/kg indomethacin. Surprisingly, they showed significant reduction in ulcerogenic activity ranging from 1.417±0.15 to 1.750±0.11, whereas standard drug indomethacin showed high severity index of 2.750±0.44. The maximum reduction in ulcerogenic activity (1.417±0.15) was due to compound (VI-c) having dihydroxy substituent. These results clearly indicated that we succeeded to develop good anti-inflammatory and analgesic agents with reduced ulcerogenic potential. NSAIDs are associated with high incidence of GI ulceration; bleeding and kidney damage which may be linked with lipid peroxidation. So, our study aimed to examine lipid peroxidation (LP) induction capacity of compounds (VI-c and VI-f) which showed remarkable analgesic and anti-inflammatory activity. LP was measured as nanomoles of malondialdehyde (MDA)/100 mg of gastric tissue. We noticed that indomethacin exhibited maximum lipid peroxidation 6.73±0.17, whereas (VI-c and VI-f) 4.02 and 4.39, respectively (control group showed 3.14±0.14). The most potent anti-inflammatory and analgesic agents with reduced ulcerogenic and lipid peroxidation potential (VI-c and VI-f) were further examined for their hepatotoxic effects as well as their effect on serum enzymes and total albumin. Both compounds exhibited significant reduction in SGOT, SGPT and total albumin level in comparison to control as shown in Table 5. Because several studies reported that most NSAIDs induce liver damage, we studied the histopathlogy of liver samples. The results revealed that there is no indication of any
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pathological change. Neither necrosis nor degeneration was found in comparison to the control group (Fig 1-3). In conclusion, we have accomplished the synthesis of certain novel Schiff‟s bases. The new compounds have been investigated for their anti-inflammatory and analgesic activities as well as their ulcerogenic activity, liver toxicity and lipid peroxidation. Among the synthesized Schiff‟s bases, compounds (VI-c) and (VI-f) were found to be the most potent as antiinflammatory and analgesic agents with maximum reduction in GI toxicity, no hepatocyte necrosis or degeneration in liver samples. Indeed, these two compounds could be considered as suitable candidates for bio-technologic application as anti-inflammatory and analgesic agents. Acknowledgments This research was funded by National Plan of Science, Technology and Innovation (Grant No. 12-MED2980-54), Salman bin Abdulaziz University, Alkharj, P.O.Box 173, 11942 (to AF).
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Table 1 Pharmacokinetic Properties important for good oral bioavailability of titled compounds (VI:a-k) Compounds
%ABS
Volume (A3)
Rule VI-a
88.98
355.88
58.05
5
