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Review Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Ahmed MA, IJPRBS, 2014; Volume 3(3): 20-55

ISSN: 2277-8713 IJPRBS

INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH AND BIO-SCIENCE THE IMPORTANCE OF p38 INHIBITORS AS CHEMOTHERAPEUTIC AGENTS AHMED MA1, ABDEL NBS2, KHALED AA2, MAGED KGM1, ATEF GH1 1. Department of Chemistry of Natural Compounds, National Research Center, Dokki, 12311, Cairo, Egypt. 2. Faculty of Pharmacy, Ain Shams University, Abbasia, 11566, Cairo, Egypt. Accepted Date: 12/04/2014; Published Date: 27/06/2014 Abstract: The blooming of advanced kinase inhibitors in the past few years has urged more efforts for novel, selective drugs with potential activity in different pathological conditions. Mitogen activated protein kinases (MAPKs) are very important targets for different diseases. p38 is one of MAPKs. Since its discovery in 1994, the pivotal role of p38 in production of proinflammatory cytokines has attracted many researchers to produce a variety of p38 inhibitors with different chemical structures and binding fashions to p38. These inhibitors are mostly indicated for inflammatory diseases, such as rheumatoid arthritis. However, the role of p38 in cancer has been investigated lately. This review discusses different chemical structures of p38 inhibitors and their structure activity relationships. Also, binding fashions of these molecules to p38 are illustrated. Moreover, the role of p38 inhibitors in apoptosis and in cancer treatment is presented. Keywords: Mitogen Activated Protein Kinases, P38, Inhibitors, Structure Activity Relationships, Cancer.

Corresponding Author: MR. AHMED M. ABDELAZIZ Access Online On: www.ijprbs.com How to Cite This Article: PAPER-QR CODE

Ahmed MA, Abdel NSB, Khaled AA, Maged KGM, Atef GH; IJPRBS, 2014; Volume 3(3): 20-55

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INTRODUCTION Major characteristics of cancer and many other diseases are attributed to the disturbance in regulatory signaling pathways which is a result of mutation, deletion or amplification of specific related genes. Kinases are integral components of these regulatory signaling pathways. Kinases can transfer a phosphate group from high-energy donor molecules, such as ATP, to side chains of other proteins, thus modulating their activity status, their placement and liability to be degraded 1, 2. One of the most vital kinase families are mitogen-activated protein kinases (MAPKs). MAPKs are involved in cell proliferation, survival and differentiation. Their deregulation leads to cancer and other diseases3, 4. MAPKs have at least three major subfamilies: c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38 MAPKs. MAPK kinases (MKKs) activate MAPKs via phosphorylation on the threonine and/or tyrosine residue(s) as consequences of extracellular stimuli (Figure 1). In contrast, dual-specificity phosphatases (DUSPs) dephosphorylate MAPKs at threonine and tyrosine residues3, 5. Drugs with potential activity against different kinases are of magnificent importance, therapeutically and economically. p38 is one of kinases which are involved in different pathways and regulate different other proteins, and consequently it becomes a very important target for drug discovery 6. Rheumatoid arthritis and other inflammatory diseases used to be the main indications of p38 inhibitors. However, it was reported that p38 can play an important role in the treatment of acute promyelocytic leukemia cells 7, chronic myeloid leukemia cells 8, and multiple myeloma cells 9 through potentiating the effect of all-trans-retinoic acid, arsenic trioxide and bortezomib respectively 10. This review focuses on the MAPKs in general and p38 in specific, and presents different structural types of p38 inhibitors to detail their binding fashions and structure-activity relationships (SARs). C-Jun N-terminal kinases (JNKs) JNKs mediate different stimuli e.g., environmental stress and pro-inflammatory cytokines. These stimuli initiate the activation of mitogen activated protein kinase kinase kinases MAP3Ks (MKKKs) e.g., Ask, MKKK1 and MKKK3 (Figure 1). Activation of MKKKs leads to phosphorylation of two MKK isoforms MKK4 and MKK7, both of which finally phosphorylate and activate JNKs. MKK4 mediates environmental stress, whereas MKK7 mediates cytokines such as TNFα and IL-1 11 . JNKs are encoded by three genes; their decoding results in 10 different transcriptional homologues 12.

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JNK signaling pathway is mainly regulated by three categories of regulators: MKKKs (which are the upstream regulators), MAPK phosphatases (MKPs, which are the negative regulators of JNK signaling), and scaffold proteins which may mediate the deactivation of JNK by MKPs 11. JNKs are involved in various pathological conditions. For instance, JNKs play a clear role in insulin resistance syndrome via phosphorylation of insulin receptor substrate-1 (IRS-1) at the inhibitory site Ser30711, 13. JNKs and apoptosis It was reported that the role of JNKs in apoptosis is linked to Bcl-2 family proteins. These proteins prolong JNK activated TNF-α killing via the E3 ubiquitin ligase itch-mediated turnover of the caspase-8 inhibitor cFLIPL 14. Moreover, it was mentioned that both temporary and prolonged JNK activations induce gene decoding. Early temporary JNK activation promotes cell survival whereas sustained JNK activation mediates programmed cell death 15. Furthermore, JNKs are involved in necrosis and autophagy via contribution in TNF-dependent necrosis in cells when the NF-kB (a protein complex that controls the transcription of DNA) pathway is deactivated by enhancing the release of cytotoxic reactive oxygen species11, 16. In addition, JNKs promote the mitochondrial apoptotic pathway and pro-apoptotic members of the Bcl2-related protein family, and inhibit several anti-apoptotic members of the aforementioned family. JNKs are involved in tumor progress, however their role is debatable 11. It was reported that several tumor cell lines have active JNKs 17. Furthermore, oncogene transformation ability is inhibited when using anti-sense JNK oligonucleotides or dominant-negative JNK proteins 18. In addition, mice carrying an abnormal c-Jun that lacks the JNK activation sites are not susceptible to these changes18, 19.

Figure 1. MAPK signaling pathways mediate intracellular signaling initiated by extracellular or intracellular stimuli then MKKKs phosphorylate MKKs, which in turn phosphorylate MAPKs.

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Extracellular signal-regulated kinase 1 and 2 (ERK1/2) ERK1 and ERK2 are kinases of 43 and 41 kDa respectively, and they are just 15% different in their amino acids sequences 2, 3. ERK1 and ERK2 have a Thr-Glu-Tyr motif in the activation lip of the catalytic domain that is phosphorylated by two dual threonine/tyrosine kinases MKK1 and MKK2. Upon activation, ERK1 and ERK2 are translocated to the nucleus to regulate the function of nuclear proteins (e.g., transcription factors), and then are dephosphorylated by phosphatases to return to cytoplasm. MKK1 and MKK2 are activated through phosphorylation of receptor tyrosine kinases, non-receptor tyrosine kinases and G protein-coupled receptors. Moreover, It was reported that Ras and Raf1 mediate activation of ERK1 via MKK1 activation (Figure 1) 1-3. ERK2-null mice are not vital because of impaired placental development and differentiation. However, ERK1-deficient mice display increased locomotor activity 3. ERK and apoptosis ERK activation inhibits apoptosis and protects cells against signaling from death receptors. Furthermore, ERK signaling promotes cell cycle progression and proliferation, and is important for maintaining tissue homeostasis. For instance in epidermis, ERK signaling activates proliferation of basal layer stem cells and suppresses differentiation of suprabasal layer cells. Moreover, Ras, Raf1, or MKKs signaling in the skin induces epidermal hyperplasia because of hyperproliferation and reduced differentiation 3, 20, 21. Activated ERK is observed in 50 tumor cell lines; pancreas, colon and lung show the highest frequencies. In addition, ERK activation is associated with Raf1 activation in the tumor cells and with the activation of MKKs 22. p38 MAPKs p38 MAPKs include at least four different protein isoforms p38α, p38β, p38γ and p38δ 23.These proteins are 60% identical at the amino acid level and are encoded by different genes. They can be divided into two groups according to their specificity and sensitivity to inhibitors: p38α & p38β, and p38γ & p38δ. The latter group is more tissue specific than the former. p38γ is the most common in skeletal muscle and p38δ in testes, small intestine, pancreas and kidney. They are activated by different cellular stresses, such as osmotic or heat shocks, and ultraviolet radiation. The activation of p38 is linked with the activation of a series of certain protein kinases (MKKs including MKK3, MKK4 (some situations) and MKK6, or MKKKs including .Ask, Sprk, MKKK1 and MKKK3) (Figure 1) 24, 25. Loss of p38β, p38γ or p38δ does not affect regular development, suggesting that there are compensatory pathways. In contrast, p38α gene disorder leads to embryonic death due to placental defects, and its functions cannot be compensated 25. This review focuses on p38α because of its importance as therapeutic target.

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Like other MAPKs, p38 is activated via phosphorylation of Thr and Tyr in the Thr-Gly-Tyr motif. Cdc42 and Rac (small G proteins of the Rho family) stimulate p21-activated kinases (Paks) which activate p38 MAPK. Moreover, Tak1 (an ubiquitin-dependent kinase) activates p38 MAPK after being activated by Tab1 (Tak1-binding protein) which mediates TGF-β signal; another two MKKKs that activate p38 are Sprk and Ask. Both are activated in response to TNF-α. The activation of p38 leads to the phosphorylation of heat-shock proteins via phosphorylation of MAPK-activated protein 2 and 3 (Mk2 and Mk3), MAPK-interacting kinases (Mnk1/2), transcription factors including ATF2, Elk1 (ERK substrate), CHOP (member of the C/EBP family of transcription factors), the transcription factor Max. These transcription factors are involved in many types of cancers. For example, ATF2 was shown to be associated with melanocytic oncogenesis and melanoma tumor proliferation; CHOP is important for tumor invasion. 1, 26-28. Being linked with many different substrates, p38 is involved in a variety of biological processes. It was revealed that inhibition of p38 blocks the production of cytokines including TNFα, IL-1, IL2, and IL-7. This emphasizes the important role of p38 in the production of cytokines and in cytokine-stimulated cellular proliferation 1, 29, 30. p38 and apoptosis Activation of p38α through osmotic shock and ultraviolet irradiation, sodium salicylate, overexpression of Ask (MKKK) or ligation of death receptors leads to apoptosis, which can be blocked using p38 MAPK inhibitors. However, in some cells p38 is activated without induction of apoptosis, so the role of p38 in apoptosis depends on the types of the apoptotic induction as well as on the types of the cells 1, 31, 32. p38 is activated via serine/threonine protein kinases in response to DNA double strand breaks, leading to the establishment of a G2/M cell cycle checkpoint. Cell cycle checkpoint is a mechanism by which the cell delays the progression to the next phase of the cell cycle in order to allow time for DNA repair. It should be noted that the pathways linking p38 MAPK activation to DNA damage are contingent on cell types, the specific DNA damage causative factors and the degrees of DNA damage 25, 33. There are two reported mechanisms by which p38 induces G2/M cell cycle checkpoint. The first one is via the phosphorylation, activation and stabilization of p53 by p38 which results in the synthesis of proteins such as Gadd45α, p21 and 14-3-3 that enforce a G2/M checkpoint by either directly or indirectly inactivating the Cdc2/cyclinB complex. Cdc2/cyclinB is the major engine driving the transition from G2 to M. The second mechanism is through phosphorylation and inhibition of the phosphatase Cdc25B via activation of Mk2 (a p38 substrate). Cdc25B activates the Cdc2/cyclinB complex, leading to the progression of the cell cycle 25, 34-36.

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Moreover, p38 plays an important role in G1/S cell cycle checkpoint. It decreases level of cyclin D1 either at the level of transcription, or by direct phosphorylation of cyclin D1. That results in its ubiquitination and proteosomal degradation. Cyclin D1 which binds to active cyclin dependent kinases 4 and 6 (Cdk4/6) is essential for the transition to S phase and further cell proliferation. Also, p38α inhibits the transcriptions of two different proteins p16INK4a and p19ARF. These proteins inhibit Cdk4/6 activation and regulate p53 function respectively. Also, p38 can phosphorylate and promote the degradation of Cdc25A, contributing to the establishment of a G1/S checkpoint (Figure 2) (Lavoie et al., 1996, Thornton and Rincon, 2009).

Figure 2. p38 pathway Although it was generally believed that p38 mediates cell death, it was found that in some cases activation of p38 leads to cell survival. Among these cases, the use of chemotherapeutic cancer drugs causes DNA damage and stimulates p38 which promote cell survival. Hence, p38 MAPK inhibitors can be concurrently used with anti-cancer drugs. For example, treatment of human B cell lymphoma cells with etoposide, activates p38 MAPKs. So, pharmacological and genetic inhibition of p38 MAPKs enhances the apoptotic effect of etoposide. Moreover, p38 contributes to metastasis in cancer cells. Therefore, p38 activation is required in B-cell chronic lymphocytic leukaemia for the expression of the metalloprotease MMP-9. MMP-9 is needed for survival of these cells when cultured in the presence of stromal cells. Moreover, it was reported that p38 MAPKs can promote survival of tumor cells by inhibition of autophagy which is identified as an alternative pathway for cell death 25. It can be concluded that the significance of p38 inhibitors might be in the treatment of not only inflammatory diseases but also some types of cancers via the enhancement of the activity of the chemotherapeutic agents. One of the most important points in the p38 inhibitor design is the ability of inhibitors to resemble ATP within the ATP-binding site and to resemble hydrogen-bond interactions that occur in the ATP–protein complex. When ATP binds to p38; the adenine ring of ATP forms two 25




hydrogen bonds with His107 and Met109 in the hinge region. Also, Lys53, Glu71 and Asp168 are implicated in the coordination of the triphosphate group within the phosphate-binding region (Figure 3) 37. Binding fashions of p38 inhibitors Most of p38 inhibitors bind to the ATP binding site, which is mostly conserved in all kinases. So, successful p38 inhibitors should be involved in the lipophilic pocket with the small gatekeeper Thr106, whereas in other kinases Thr106 is replaced by the bulky methionine residue. Also, Gly110 flip should be used to form another hydrogen bond as the amide bond between Met109 and Gly110 rotates and allocates the amino group of Gly110 to a favorable place to the inhibitor molecule. Therefore kinases with a larger amino acid side chain at this position cannot be interfered 38. For example, the pyridine ring of SB203580 (Figure 4), a pyridinyl imidazole inhibitor, forms a hydrogen bond with the amino group of Met109, while its fluoro-substituted aryl ring interacts with the hydrophobic pocket, however the hydrophobic area below the linker region is left unoccupied (Figure 3B) 37. A different binding fashion was shown by VX-745 (Figure 4), bicyclic heterocycle inhibitor; its carbonyl group induces re-orientation of the amide bond between Met109 and Gly110 in the hinge region in order to form two hydrogen bonds. In other kinases, amide bond re-orientation is not possible which improves selectivity of VX-745. On the other hand, difluoro aryl substituent interacts with the hydrophobic pocket, which is lined by gate-keeper Thr106 (Figure 3C) 39. In addition, it was revealed that the C-2 amino group and the N-3 imine nitrogen atom of the benzimidazole compounds interact with the hinge region in the ATP binding site of p38. In the light of these findings and the study of the X-ray structure of SB203580 bound to p38, it can be concluded that substitution of pyridyl group in SB203580 with a larger size 2-amino benzimidazole rearranges the flexible p38 hinge from His107 to Ala111 and the Lys53 side chain. For instance, N-3 imine nitrogen atom of molecule 1 (Figure 4) forms a hydrogen bond with Met109 amide N-H, and the isopropyl sulfonyl group is exposed to a small lipophilic pocket in proximity to Gly110 (Figure 3D) 40. However, the study of the crystal structure of the diarylpyrazole inhibitors in complex with the p38 at the ATP binding site indicated that one side of the pyrazole moiety is stabilized by interactions with the flexible glycine flap and the pyrazole ring forms water-bridged hydrogen bonds with Asp168 and Ser154. Also, the pyridine ring in the case of SC79659 or the pyrimidine ring in the case of SD0006 (Figure 4) interacts with Met109 through hydrogen bonding whereas 26




the piperidyl group is located between protein side chain atoms from the glycine loop and the hinge region. Meanwhile, the terminal glycolic moiety forms water mediated hydrogen bond with Asp112. In addition, the 4-cholorophenyl group is located in the hydrophobic pocket of the enzyme. This interaction determines most of the potency and selectivity. Selectivity is attributed to the presence of the small Thr106 gatekeeper residue. The corresponding residue in most other kinases is too bulky to allow access to the pocket 41.

Figure 3. Different binding fashions of p38 MAPK to ATP (A), SB203580 (B), VX-745 (C), and Compound 1(D) X-ray crystallographic study of the cyanopyrimidine derivative 2 (Figure 4) bound to purified p38 showed that (1) the cyano nitrogen atom and the isopropylamino N-H form two hydrogen bonds with Met109 N-H and carbonyl groups respectively; (2) the amido group provides two hydrogen bonds with Asp168 and Glu71; and (3) there are hydrophobic interactions including the angular methylaniline group in a deep hydrophobic pocket, and the pendant isopropyl in a weaker hydrophobic interaction at the mouth of the binding site 42. Another crystallographic study on diphenylether derivatives discovered that the diphenylether moiety is buried in the hydrophobic pocket in a similar way as the aromatic ring of SB203580, and that the proximal ring of the diphenylether occupies a position in the same manner as adenine ring of ATP. In addition, depending on the derivative, polar interactions are observed either with the hinge region similar to adenine ring of ATP via two hydrogen bonds to Met109 and Gly110 (in the case of 3), or through occupying the similar position as the ribose group and triphosphate chain of ATP and forming hydrogen bonds with Asn158 and Asp168 of the DFG motif (in the case of 4) (Figure 4). Moreover, several water-mediated hydrogen bonds form a network between the inhibitor and p38 43. Similarly, the fused pyrazole-derived inhibitors show that the N-2 of the pyrazolopyridinone 5 (Figure 4) forms hydrogen bond with Met109. Also, the N-1 aryl group is projected into a hydrophobic pocket near the hinge region with Ala157 residue which is smaller than the 27




corresponding residues in other kinases, thus improving selectivity. Moreover, hydrogen bond is formed between the aniline N-H and the gate keeper Thr106. The cyclopropyl amide forms two hydrogen bonds with Asp168 and Glu71. Finally, a network water mediated hydrogen bonds are formed between the pyridinone carbonyl and Asp168 and Lys53 44. The diaryl urea p38 inhibitors indirectly compete with ATP through stabilization an unfavourable conformation for ATP binding. The conserved Asp-Phe-Gly (DFG) motif conformation is changed by diaryl urea p38 inhibitors in such way that makes Phe of DFG motif moves to a new position to reveal a hydrophobic pocket for a hydrophobic group of the inhibitor. Consequently, the side chain of Phe becomes in steric overlap with the phosphate group of ATP (DFG-out form). The crystal structure of BIRB-796 in complex with p38 reveals that the kinase exists in DFG-out form. The tolyl group causes conformational change to Glu71, which forms a hydrogen bond with only one of the two N-H groups of urea allowing the hydrophobic interaction of tolyl group with the lipophilic part of the side chain of conserved Glu71 residue. On the other hand, the morpholino group establishes interaction with ATP pocket in addition to improving the physicochemical properties of BIRB-796 45. Likewise, Sorafenib, a biaryl urea-based multi-kinase inhibitor, binds to b-Raf and p38 in the DFG-out conformation. The compounds that are associated with these conformational changes have characteristic binding parameters e.g., slow on rates (kon) and long off rates (koff). The slow rate binding is accounted for the rate-determining requirement for the change in DFG motif. It can be seen that the carboxylate oxygen atoms of Glu71 form two hydrogen bonds with both urea N-H groups of Sorafenib. Also, the presence of a glycine residue (Gly110) leads to moving the hinge region in the direction of the binding site; Phe169 side chain is rotated resulting in aromatic interactions with the pyridine ring of Sorafenib. Finally the amide nitrogen atom of the picolinamide moiety in the hinge region forms a hydrogen bond with Met109 46. To sum up, the p38 inhibitors show different binding fashions. Some compounds bind to p38 in a similar manner as ATP. Other compounds may induce conformational changes within the hinge region in variable ways. However, some p38 inhibitors show unique hydrophobic interactions with hydrophobic area/pocket and others stabilize p38 in DFG-out form.

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Figure 4. p38 inhibitors with different binding fashions Structure activity relationships (SARs) of p38 inhibitors A. Imidazole derivative inhibitors As shown in Table 1, SAR studies of 2,4,5-trisubstituted imidazole (e.g., SB 203580) revealed that the 4-pyridyl residue and the presence of another aromatic ring next to it are vital for the p38 inhibitory activity (SB 203580 vs. A-1). However, replacing the pyridine group with substituted pyrimidine (A-2) or 2-aminobenzimidazole (A-3) group improves the activity and selectivity 47, 48. On the other hand, structural changing for the R2 substituent is tolerated without loss of the activity, and using bulk groups (e.g., 2,6-difluorophenyl in A-3) decreases the inhibition of cytochrome P450 3A4 (CYP3A4) 48. Also, using the 2,6-diamino-3,5-difluoropyridine substituent at this position (A-4) increases the activity due to the formation of a salt bridge between the pyridine ring nitrogen atom and Asp168 49, 50. Moreover, it can be noticed that polar substituents at R2 position increase selectivity towards p38 over COX-1 as COX-1 activity is related to the lipophilicity of the substituents at the imidazole R2 position. Consequently, using 4-piperidinyl group (A-5) at the same position improves activity, solubility, cell permeability and protein binding. Concurrent replacement of R2 with pyrimidine and R 3 with a proper group enhances both potency and selectivity e.g., RPR203494 51, 52. Furthermore, in the 2alkylsulfanyl-5-(pyridin-4-yl)imidazole derivatives, small alkylsulfanyl groups at the imidazole R2 position e.g., ML 3375 and A-6 lead to enhanced inhibition of p38 due to the easy entry of the molecule into the ATP binding site of p38, which cannot accommodate larger substitutes, e.g., ML3163 53. The 2-alkylsulfanyl-5-(pyridin-4-yl) imidazoles compounds e.g., ML3403 are open chain analogues of the early lead compound SK&F86002. In these molecules, the hydrogen bonding between the nitrogen atom of pyridyl or pyrimidinyl residue and Met109 is enhanced 29




by the electron donating substituents and is reduced by electron withdrawing substituents. For example, introduction of halogen atom at the 2-position of the 4-pyridyl moiety at R 3 significantly decreases the biological activity (ML 3375 vs. A-6) 54. Replacement of the 4-pyridyl moiety with a 4-quinoline ring (ML3375 vs. A-8) does not significantly enhance the inhibition of p38 activity because of the restricted rotation of the quinolin-4-yl group and its planarity, both of which are unfavourable for hydrogen bonding with Met109. However, amino groups at the 2-position of the pyridine improve the activity as they are able to form another hydrogen bond with the linker region and to increase the electron density which improves the interaction with the enzyme (A-9). Moreover, interaction with lower hydrophobic area is improved by lipophilic side chain substitutes 54, 55. Also, modification of the 4-aryl moiety at R1 position using suitable halogen atoms or haloalkyl groups increases selectivity as it improves the interaction with upper hydrophobic pocket (A-10 and A-11) 47. Concurrent introduction of trifluoromethylphenyl group at R 1 with arylalkylamino substituent at the R3 decreases potency as it spaces N-3 of the imidazole from Lys53 so that no hydrogen bond is formed (A-7) 54, 56. Table 1. Trisubstituted imidazole derivative inhibitors

Code SB 203580a,b

R1

R2

R3

IC50/nM 74

A-1c

2500

A-2c

57

A-3d

4.9

A-4e,f

16

A-5g

90

30




RPR203494h

9

ML 3163i

4000

ML 3375d,j

63

A-6j

372

ML3403d,j

38

A-7j

282

A-8j

41

A-9 j

9

A-10a

86

A-11a

48

SK&F86002d,j

1000

(a) Liverton et al., 1999; (b) Mader et al., 2008; (c) Wagner and Laufer, 2006; (d) Toledo et al., 1999; (e) Wilson et al., 1997; (f) Revesz et al., 2002; (g) Revesz et al., 2000; (h) Collis et al., 2001; (i) Laufer et al., 2002; (j) Laufer et al., 2008. 31




In Table 2, for various N-substituted imidazoles, it was confirmed that substitution at R3 is tolerated (A-12) but not for its regioisomer (A-13) 47. SB 235699 with a piperidine substituent at the R3 position has good bioactivity due to the hydrogen bonding interaction of the basic piperidine substituent with Asp168 47. Furthermore, inhibitory activity of N-substituted imidazoles is improved via the replacement of the 4-pyridyl at R4 position with a 2aminopyrimidinyl moiety (e.g., SB 220025). However, simultaneous presence of Nmethylpiperidin substituent at R3 position and 2-aminopyrimidinyl moiety at R 4 position decreases p38 inhibitory activity but enhances oral activity (A-14) 57. Moreover, using the electron rich substituents at R 4 position increases the electron density on the heterocyclic nitrogen, thus increasing the activity (e.g., SB 242235) 58. Table 2. Substituted imidazole derivative inhibitors

Code

R1

R2

A-12a

R3

R4

Me

A-13a

IC50/nM 0.11

1220

SB 235699b

H

60

SB 220025b

H

19

A-14c

H

86

SB 242235c

H

19

32




(a) Liverton et al., 1999; (b) Adams et al., 2001; (c) Boehm et al., 2001. As shown in Table 3, replacement of imidazole ring with pyrrole, pyrazolone, or furane in SB 203580 was attempted. L-167307 is a pyrrole analogue of SB 203580 with higher potency 59. Novartis prepared pyrrole, thiazole and oxazole analogues of the 2-piperidinol or 4-(2, 6diamino-3, 5-difluoropyridinyl) substituted imidazole (A-5 vs. A-15 and A-4 vs. A-16) and, it was found that 2, 6-diamino-3, 5-difluoropyridine pyrrole (A-16) is more potent against p38, but it is also active against COX-1. Moreover, there are different activities for regioisomers in the pyrrole series (A-17 vs. A-18), which reflects the importance of the nitrogen position. Also, 5membered triazole analogues were prepared (A-19) which show moderate activity 49. Similarly, six-membered diazines were used as scaffolds for the pyridin-4-yl-/4-fluorophenyl pharmacophore. ML 3163 analogue with pyrimidine, i.e., A-20, decreases bioactivity 60. Regiochemistry plays an important role in the bioactivity of pyridazine analogues (A-21 vs. A22) 61. Moreover, using pyrrolopyridine as a scaffold with electron donating substituents facing the nitrogen atom of the fused pyridine increases the binding affinity for p38, e.g., RWJ 68354. Also, imidazopyrimidine (A-23) and benzimidazole (A-24) were used as scaffolds for designing p38 inhibitors. However, using imidazopyridine (A-25) results in less selective p38 inhibitors which shows inhibitory activity against JNK2, EGFR, and HER-1 62. Table 3. Inhibitors with different heterocycles rather than imidazole

Code

R1

R2

R3

IC50/nM

L-167307a

5.1

A-15a

130

A-16a

4

33




A-17c

200

A-18c

1400

A-19c

210

A-20d

36000

A-21e

1000

A-22e

0.15

34




RWJ 68354b

150

A-23f

570

A-24f

22

A-25f

15

(a) de Laszlo et al. 1998; (b) Henry et al., 1998; (c) Revesz et al., 2000; (d) Laufer and Wagner, 2002; (e) McIntyre et al., 2002; (f) Wagner and Laufer, 2006. B. Pyridinone derivative inhibitors SC-25028 is one of pyridinone-based p38 inhibitors and shows double hydrogen bonding through the carbonyl group to Met109 and Gly110 of p38 which is associated with Gly110 flip. Moreover, the benzyloxyl group and the N-benzyl group interact with the upper hydrophobic pocket and the lower hydrophobic area respectively in the hinge region. Efforts have been made to improve potency and selectivity via targeting Asp112 and Asn115. For instance, meta(B-2) or para- (B-3) substitution of the N-benzyl group increases the potency but ortho- (B-1) substitution does not. Also, the carboxamide group at the para position (B-4) not only increases the activity but also improves the metabolic stability. Incorporation of chloro or bromo group at C-3 position is optimum for selectivity as the halogen atom at this position interacts with the 35




shallow hydrophobic pocket near another deep one. Moreover, replacement of benzyloxyl group with 2, 4-diflurobenzyloxyl group enhances the activity against p38 63. Furthermore, replacement of the N-benzyl group with 2, 6-disubstituted phenyl group improves the activity (B-5), but the 2, 4, 6-trisubstituted phenyl group does not offer any increase in the potency. Also, modification of C-6 substituent is tolerated (B-6), but affects the cytochrome P450mediated metabolism in a substituent-depending manner 64. On the other hand, pyrazole pyridinone was used as a scaffold for p38 inhibitors (Table 4). Substitution on the carbon atom facing the nitrogen atom of the pyridine ring with orthotolylbenzamide improves the activity (B-7). However, replacement of the amide group with a carboxyl group abolishes the activity (B-8) 44. Table 4. Pyridinone derivative inhibitors

Code

R1

R2

R3

IC50/nM

SC25028a

H

680

B-1 a

H

1200

B-2 a

H

110

B-3 a

H

190

36




B-4 a

H

22

B-5 b

Me

3

B-6 b

CH2OH

5

B-7 c

1.1

B-8 c

>1000

(a) Selness et al., 2009; (b) Selness et al., 2011; (c) Wurz et al., 2009. C. Dibenzosuberone derivative inhibitors Another approach to improve selectivity of p38 inhibitors is to design more structurally rigid molecules to prevent conformational changes associated with the activation of p38 through the interaction with p38 deep pocket. Skepinone-L is a good example of these inhibitors (Table 5). 37




Modification of Skepinone-L by introduction of a benzamide moiety to the aniline group increases the binding affinity (C-1). Hydrophilic moieties at R1 position of this series are favourable to activity. Replacement of the benzene ring of the benzamide moiety with tetrahydrofuran decreases the activity (C-2 vs. C-3). Moreover, using different heteroaromatic moieties increase the activity due to the increased hydrophilicity (C-4 vs.C-5). Another modification was carried out by adding amide (C-6) or ester (C-7) functionality at R1 position which improves the biological activities 6. Table 5. Dibenzosuberone derivative inhibitors*

NHR2

R1 O

Code

R1

R2

IC50/nM

Skepinone-L

C-1

5

1

NH F O

C-2

F O

C-3

21

NH

NH

O

2

F O

C-4

NH

1

F O

C-5

0.3

38


Review Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Ahmed MA, IJPRBS, 2014; Volume 3(3): 20-55 C-6

2

C-7

2


*Fischer et al., 2013. D. 2-Aminothiazole derivative inhibitors 2-Alkylaminothiazoles: The potency of D-1 as a p38 inhibitor is moderate (Table 6). The derivatization of the secondary amino group (at the R1 position) with a larger cyclic (D-2) or a branched alkyl (D-3) group is tolerated; the use of an aryl (D-4) or an unbranched alkyl (D-5) group significantly decreases the potency. However, substitution of the 2-chloro-6-methyl aniline moiety with a methyl group gives a more potent series of 2-alkylaminothiazole inhibitors, e.g., D-6 vs. D-3. However, modification of the thiazole scaffold by using the fused lactam decreases the inhibitory activity of the resultant compounds (D-7 vs. D-6) 65. Table 6. 2-Alkylaminothiazole derivative inhibitors*

Code

R1

R2

IC50/nM

D-1

39

D-2

36

D-3

13

39


Review Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Ahmed MA, IJPRBS, 2014; Volume 3(3): 20-55 D-4

62

D-5

140

D-6

Me


3.2

D-7

110

*Hynes et al., 2008. 2-Aminothiazol-5-yl-pyrimidines: In these inhibitors, the nitrogen atom of the thiazole ring interacts with Met109, whereas the sulphur atom forms intramolecular nonbonding interaction with one of the pyrimidine nitrogen atoms. These interactions improve the binding with p38. Substitution at R 3 position of the pyrimidine moiety with an ortho halogenated phenyl group increases the activity; using the same substitute at R2 position abolishes the activity (D-8 vs. D9) (Table 7). Also, the use of the substituted urea at R3 makes this moiety to interact with Glu71 and Asp168, thus enhancing the inhibitory activity (D-12). On the other hand, introduction of 2pyridyl at R 2 position improves the potency (D-10). Moreover, SAR studies on the substituted amino moiety revealed that small branched alkyl groups are optimum for the activity (e.g., D11) 66. Table 7. 2-Aminothiazol-5-yl-pyrimidine derivative inhibitors*

Code

R1

R2

R3

IC50/nM

40


Review Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Ahmed MA, IJPRBS, 2014; Volume 3(3): 20-55 D-8

NH2


225

D-9

H

> 30,000

D-10

52

D-11

4

D-12

2

*Lin et al., 2010. E. Indole-based heterocyclic derivative inhibitors SAR studies on indole-based heterocyclic derivatives revealed that both the indole moiety and the benzyl group are essential for the p38 inhibitory activity (E-1 and E-2) (Table 8). Also, the orientation is very important for the activity. Potency is improved by the substitution at R2 position of the indole ring with a piperazine or dimethylpiperazine ring (E-3). These substitutions restrict the conformation of the molecule which is more preferable for the binding to p38. Moreover, loss of the activity is associated with the substitution of the amide linkage with a sulfonamide, amine or amidine linkage. The carbonyl of the amide linker forms a hydrogen bond with the hinge region of p38. E-4 shows a significant decrease in activity as its carbonyl group is involved in intramolecular hydrogen bonding 67. Table 8. Indole-based heterocyclic derivative inhibitors*

Code

R1

R2

R3

IC50/nM 41


Review Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Ahmed MA, IJPRBS, 2014; Volume 3(3): 20-55 E-1

H

H

N

ISSN: 2277-8713 IJPRBS 4360

N O

E-2

H

N

H

5520

H

1300

H

>30,000

N O

E-3

H

E-4

N

H

N O

*Mavunkel et al., 2003. F. Biphenyl amide derivative inhibitors F-1 is a biphenyl amide derivative and the x-ray crystal structure of this molecule in complex with p38 shows hydrogen bonding with Met109 via the amide carbonyl 68. It was reported that the cyanophenyl amide group lies in the outer lipophilic region of the ATP-binding site and pointes out towards the solvent. Modification of the cyanophenyl amide group using other substituted phenyls or benzyl amides is tolerated with the increase of the activity (e.g., F-2) (Table 9). However, activities of benzyl amides are equal to or more than those of the corresponding phenyl amides 69. F-3 is another biphenyl amide derivative. Its x-ray crystal structure in complex with p38 reveals that the oxadiazole ring occupies a hydrophobic pocket and its nitrogen atoms form hydrogen bonds with Asp168 and Phe169 of the DFG motif. So, the replacement of the oxadiazole ring with an amide (e.g., the cyclopropyl substituted amide in the case of F-4) not only keeps the hydrogen bond to Asp168 via the carbonyl group, but also forms another hydrogen bond with Glu71 via the N-H of the amide. In contrast, using isobutyl (F-5), cyclopentyl (F-6) and cyclohexyl (F-7) amides cannot form such a hydrogen bond with Glu71 duo to Steric effect , thus the activity decreased 70.

42




Table 9. Biphenyl amide derivative inhibitors

Code

R1

R2

IC50/nM

F-1a

1500

F-2 b

280

F-3 b

3000

F-4 b

75

F-5 c

11000

F-6 c

2700

F-7 c

8800

(a) Angell et al., 2008c; (b) Angell et al., 2008b; (c) Angell et al., 2008a

43




G. Aminobenzophenone derivative inhibitors SAR studies on aminobenzophenone derivatives as p38 inhibitors revealed that the carbonyl group is critical for the activity as it forms hydrogen bonding with Met109 and its replacement leads to a significant decrease in the activity (G-1 vs. G-2) (Table 10). Moreover, the small ortho substitution of ring A improves the potency, whereas larger substituents (e.g., phenyl) reduce the activity (G-3 vs. G-4). Concurrent ortho substitutions of ring A with a methyl group and ring B with a lipophilic group (e.g., methyl) restrict the rotation around the ketone bridge, thus improving the binding to p38 and enhancing the activity. On the other hand, meta substitution of ring B with a methyl group decreases the activity (G-5 vs. G-6). Also, switching the amino group on ring C to amide, urea or carbamate improves the activity (G-7 and G-8), but its modification to nitro group abolishes the activity (G-9) 71. Table 10. Aminobenzophenone derivative inhibitors*

Code

X

G-1 G-2

-CH=CH-

R1

R2

R3

IC50/nM

Me

2-Cl

NH2

10

Me

2-Cl

NH2

1550

H

NH2

811

G-3 G-4

Br

H

NH2

45

G-5

Me

2-Me

NH2

13

G-6

Me

3-Me

NH2

143

G-7

Me

2-Cl

21

G-8

Me

2-Cl

42

G-9

Me

2-Cl

NO2

437 44




*Ottosen et al., 2003 H. Diphenylether derivative inhibitors Two different series of diphenylether derivatives were prepared. In the first one, an aryl substituent is linked to the diphenylether scaffold via an amide linkage. 4-Chloro substitution at R1 position increases the activity by 4 times (H-1 vs. H-2). Also, substitution of the aryl moiety at R2 position with a more hydrophobic group improves the activity (H-3 vs. H-4). In the second series, a 4-aminopiperidine substituent is linked to the diphenylether scaffold via a sulfamide linkage. 4-Halogen substitution at R 1 position increases the potency by 3 times (H-5 vs. H-6); 2substitution decreases the potency (H-6 vs. H-7 and H-8). The presence of hydroxyl group at R2 position is important for the activity (H-5 vs. H-9) 43. Table 11. Diphenylether derivative inhibitors*

Code

R1

R2

H-1

H

1600

H-2

Cl

400

H-3

H

1500

H-4

H

450

Code

R1

R2

IC50/nM

IC50/nM

45


Review Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Ahmed MA, IJPRBS, 2014; Volume 3(3): 20-55 H-5

H

OH

16000

H-6

4-F

OH

6000

H-7

2,4-F

OH

10000

H-8

2-Cl

OH

>20000

H-9

H

H

>20000


*Michelotti et al., 2005 I. Biaryl urea derivative inhibitors Pyrazole (I-1), thiophene (I-2) and isoxazole (I-3) rings were used to prepare different biaryl urea derivative inhibitors 72, 73. Pyrazole urea derivatives: Removal of two chlorine atoms of 2, 3-dichlorophenyl as well as installation of a pyridin-3-yl-methyl group at para position in the Ar2 moiety improves the activity (I-4 and I-5) 74. Furthermore, removal of the methyl group from the pyrazole ring gives a molecule with the similar potency as the lead one (I-1 vs. I-6); replacement of the methyl group with a phenyl group leads to a more potent p38 inhibitor (I-7) 75. Also, the use of the paraaminophenyl group instead of the methyl group improves the potency and selectivity (I-8). Similarly, the use of tolyl group gives a very potent analogue BIRB-796. In BIRB-796, the morpholinoethoxy moiety improves not only the physicochemical properties but also the p38binding property of the molecule through hydrogen bonding to Met109 of the protein. The tertbutyl group interacts with a hydrophobic pocket between the two lobs of p38 76. Quinoline urea derivatives: LASSBio-998 shows a similar fashion of the binding to p38 as BIRB796, but it forms an extra hydrogen bond to Ile84 through the oxygen atoms of the methylene dioxide moiety 77. Table 12. Biaryl urea derivative inhibitors

Code

Ar1

Ar2

I-1a,b

IC50/nM 53

46


Review Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Ahmed MA, IJPRBS, 2014; Volume 3(3): 20-55 I-2a,b

76

I-3a,b

36

I-4c

42

I-5c

13

I-6c

44

I-7d

30

I-8d

29


47


Review Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Ahmed MA, IJPRBS, 2014; Volume 3(3): 20-55 BIRB-796 e

18

LASSBio-998 f

14


(a) Dumas et al., 2000b; (b) Redman et al., 2001; (c) Dumas et al., 2002; (d) Dumas et al., 2000a; (e) Regan et al., 2003, (f) de Oliveira Lopes et al., 2012. CONCLUSIONS p38 is one of the most important target kinases for treatment of inflammatory disorders; it is involved in the production of proinflammatory cytokines. These cytokines are involved in many different pathological disorders. p38 is activated in rheumatoid arthritis, Alzheimer’s disease and ischaemic heart disease, which are aggravated by the production of inflammatory cytokines. These cytokines are involved in the cancer development as there are inflammatory lesions associated with many types of cancer 5. The role of p38 in cancer therapy is still controversial. It is activated in transformed follicular lymphomas. SB203580 not only inhibits its growth but also leads to production of certain proteins. These proteins are involved in growth inhibition, cell cycle arrest, or tumour inhibition 78 . Moreover, two types of colorectal adenocarcinoma cell lines were inhibited with the use of p38 inhibitors in a dose-dependent manner 79. Also, LY2228820 dimesylate, a p38 inhibitor, decreased VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation, so it is concluded that p38 is involved in tumour angiogenesis via direct tumoural effects 80. On the other hand, activation of p38 with DNA-methylating agents leads to G2/M checkpoint. So, p38 activation is involved in the resistance of some tumours to chemotherapeutic DNA-methylating drugs 81. Consequently, p38 inhibitors can be used in combination with these chemotherapeutic agents to overcome resistance initiated by p38 activation. The role of p38 in apoptosis depends on the types of the apoptotic induction and on types of the cell. For these, huge efforts have been made for the design of more potent and selective p38 inhibitors. A wide range of p38 inhibitors were designed with different chemical scaffolds and show different fashions of the binding to p38. However, more selective and potent p38 inhibitors are needed. 48




As a future plan, novel p38 inhibitors are to be designed. Selectivity of these inhibitors will be considered through targeting different amino acids residues adjacent to the ATP binding site like Ile84 in the case of LASSBio-998. Also, the conformational changes of the ATP binding site in p38 induced by the inhibitors (e.g., in sorafenibe) should be utilized. In addition, application of p38 inhibitors in the field of cancer therapy should be increased either separately or in combination with known chemotherapeutic agents. ACKNOWLEDGEMENT Ahmed M. Abdelaziz and Atef G. Hana thank Prof. Shudong Wang, Dr. Mingfeng Yu and Sarah Diab from Centre for Drug Discovery and Development, Sansom Institute for Health Research and School of Pharmacy and Medical Sciences, University of South Australia for their efforts in this work. Also, The authors are grateful to the Egyptian National research Centre and the Egyptian Culture affairs and mission sector. REFERENCES 1. C. Widmann, S. Gibson, M. B. Jarpe and G. L. Johnson, Physiological reviews, 1999; 79: 143180. 2. G. Pearson, F. Robinson, T. Beers Gibson, B. E. Xu, M. Karandikar, K. Berman and M. H. Cobb, Endocrine reviews, 2001; 22: 153-183. 3. L. Min, B. He and L. Hui, Seminars in cancer biology, 2011; 21: 10-20. 4. Y. Son, Y. K. Cheong, N. H. Kim, H. T. Chung, D. G. Kang and H. O. Pae, Journal of signal transduction, 2011; 2011: 792639. 5. L. Hui, L. Bakiri, E. Stepniak and E. F. Wagner, Cell cycle, 2007; 6: 2429-2433. 6. S. Fischer, H. K. Wentsch, S. C. Mayer-Wrangowski, M. Zimmermann, S. M. Bauer, K. Storch, R. Niess, S. C. Koeberle, C. Grutter, F. M. Boeckler, D. Rauh and S. A. Laufer, Journal of medicinal chemistry, 2013; 56: 241-253. 7. S. Uddin, F. Lekmine, N. Sharma, B. Majchrzak, I. Mayer, P. R. Young, G. M. Bokoch, E. N. Fish and L. C. Platanias, The Journal of biological chemistry, 2000; 275: 27634-27640. 8. A. Verma, M. Mohindru, D. K. Deb, A. Sassano, S. Kambhampati, F. Ravandi, S. Minucci, D. V. Kalvakolanu and L. C. Platanias, The Journal of biological chemistry, 2002; 277: 44988-44995. 9. T. Hideshima, K. Podar, D. Chauhan, K. Ishitsuka, C. Mitsiades, Y. T. Tai, M. Hamasaki, N. Raje, H. Hideshima, G. Schreiner, A. N. Nguyen, T. Navas, N. C. Munshi, P. G. Richardson, L. S. Higgins and K. C. Anderson, Oncogene, 2004; 23: 8766-8776. 49




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