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Nonclassical Antifolate Compounds as Dihydrofolate Reductase Inhibitors: Synthesis and Biological Evaluation المركبات المضادة للفولات غير التقليديه كمثبطات.

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Presentation on theme: "Nonclassical Antifolate Compounds as Dihydrofolate Reductase Inhibitors: Synthesis and Biological Evaluation المركبات المضادة للفولات غير التقليديه كمثبطات."— Presentation transcript:

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2 Nonclassical Antifolate Compounds as Dihydrofolate Reductase Inhibitors: Synthesis and Biological Evaluation المركبات المضادة للفولات غير التقليديه كمثبطات لثنائي هيدروالفولات المختزل : التشييد والتقييم الحيوي

3 INTRODUCTION

4 Dihydrofolate Reductase (DHFR)  DHFR is an enzyme which play an important role in human physiology.  Blocking of its enzymatic activity is a key element in treatment of many diseases; including cancer, AIDS related infections, bacterial, and parasitic infections. 1-2  Many useful drugs have been developed to date, however, issue of toxicity, and drug resistance make it imperative that new inhibitors of DHFR be designed that have increased selectivity and lower toxicity. 1.Masur, H.; Kaplan, J.E.; Holmes, K.K. Guidelines for preventing opportunistic infections among HIV- infected persons. Ann. Intern. Med. 2002, 137, Sparano, J.; Sara, C. Infection prophylaxis and antiretroviral therapy in patients with HIV infection and malignancy. Curr. Opin. Oncol. 1996, 8, 392  399.

5 Figure 2: Sites of action of antifolates. The Folate Pathway ▲ ▲

6 Inhibitors of Dihydrofolate Reductase

7 LITERATURE BACKGROUND

8  Quinazolines such as raltitrexed (7) and thymitaq (8) are used for treatment of advanced colorectal cancer. 1,2  Analogues of (arylthio)quinazolines (9) are known DHFR inhibitors used as antimalarial agents. 3 1.Bavetsias, V.; Jackman, A.L.; Marriott, J.H.; Kimbell, R.; Gibson, W.; Boyle, F.T.; Bisset; G.M. Folate-based inhibitors of thymidylate synthase. J. Med. Chem. 1997, 40, 1495  Bavetsias, V.; Marriott, J.H.; Melin, C.; Kimbell, R.; Matusiak, Z.S.; Boyle, F.T.; Jackman, A.L. Design and synthesis of cyclopenta[g]quinazoline-based antifolate as inhibitors of thymidylate synthase and potential antitumor agents. J. Med. Chem. 2000, 43, Werbel, L.M.; Degnan, M.J. Synthesis and antimalarial and antitumor effects of 2-amino-4-(hydrazino and hydroxyamino)-6-[(aryl)thio]quinazolines. J. Med. Chem. 1987, 30, 2151  2154.

9  Recently, Brassinin (10) was reported to have cancer chemopreventive activity.  Its structure modification led to series of 4(3 H )-quinazoline derivatives with dithiocarbamate (11) with IC 50 value of 0.5  M as antileukemic Sheng ‑ Li, C.; Yu ‑ Ping, F.; Yu-Yang, J.; Shi ‑ Ying, L.; Guo ‑ Yu, D.; Run ‑ tao, L. Synthesis and in vitro antitumor activity of 4(3H) quinazolione derivatives with dithiocarbamate side chains. Bioorganic and Medicinal Chemistry Letters 2005, 15,

10  Tricyclic pyrimidonaphthridones (12) were synthesized as conformationally restricted inhibitors of DHFR and as antitumor and/or anti-infectious agents. 2 2.Gangjee, A.; Shi, J.; Queener, S.F. Synthesis and biological activities of conformationally restricted, tricyclic nonclassical antifolates as inhibitors of dihydrofolate reductases. J. Med. Chem., 1997, 40, 1930  1936.

11  Host tissues can selectively be protected by the co-administration of leucovorin (13) which is a classical folate cofactor for one- carbon metabolism. 1,2 1.Smail, J.B.; Palmer, B.D.; Rewcastle, G.W., Denny, W.A.; Roberts, B.J. Tyrosine kinase inhibitors ‑ (Phenylamino)quinazoline acrylamide as irreversible inhibitors of the ATP binding site of the EGFR. J. Med. Chem. 1999, 42, Rosowsky, A.; Forsch, R.A.; Queener, S.F. Further studies on 2,4 ‑ Diamino-5-(2',5'-disubstituted benzyl)pyrimidines as inhibitors of dihydrofolate reductases. J. Med. Chem. 2003, 46,

12  Some quinazoline analogues (14) were synthesized in which the N 8 -nitrogen of the pyridopyrimidine ring was replaced with a carbon. Those compounds showed remarkable antitumor activity. 1  A series of thieno-pyrimidines (15) were synthesized and studied as inhibitors of DHFR from Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and rat liver. 2 1.Broughton, M.C.; Queener, S.F. Pneumocystis carinii dihydrofolate reductase used to screen potential antipneumocystis drugs. Antimicrob. Agents Chemother. 1991, 35, 1348 ‑ Allegra, C.J.; Kovacs, A.J. Drake, J.C.; Swan, C.J.; Chanber, B.A.; Masur, H. Activity of antifolates against Pneumocystis carinii dihydrofolate reductase and identification of a potent new agent. J. Exp. Med. 1987, 165,

13  The introduction of unsaturated alkyl group on the quinazoline nucleus (16) 1 proved also to increase the activity as tyrosine kinase inhibitor in addition to DHFR inhibition. 2,3 1.Wyss, P.C.; Gerber, P.; Hartman, P.G.; Hubschwerlen, C.; Locher, H.; Marty, H.; Stahl, M. Novel dihydrofolate reductase inhibitors. Structure-based versus diversity-based library design and high-throughput synthesis and screening. J. Med. Chem. 2003, 46, Duch, D.S.; Edelstein, M.P.; Nichol, C.A. Inhibition of histamine-metabolizing enzymes and elevation of histamine levels in tissues by lipid-soluble anticancer folate antagonists. Mol. Pharmacol. 1980, 18, Such, D.S.; Edelstein, M.P.; Bowers, S.W., Nichol, C.A. Biochemical and chemotherapeutic studies on 2,4 ‑ diamino-6-(2,5 ‑ dimethoxybenzyl)-5-methylpyrido[2,3 ‑ d]pyrimidine (BW 301V), a novel lipid-soluble inhibitor of dihydrofolate reductase. Cancer Res. 1982, 42,

14  The soft drug concept was used for the design of new DHFR inhibitors for inhalation therapy.  These drugs are intended to treat P. carinii pneumonia (PCP). Compounds 17 and 18 proved to be potent DHFR inhibitor soft drugs. 1,2 1.Nordberg, M.G.; Kolmodin, K.; Aqvist, J.; Queener, S.F.; Hallberg, P. Design, synthesis, and computational affinity prediction of ester soft drugs as inhibitors of dihydrofolate reductase from Pneumocystis carinii. Eur. J. Pharm. Sci. 2004, 22, 43  Rosowsky, A.; Bader, H.; Cucchi, C.A.; Moran, R.G.; Kohler, W.; Freisheim, J.H. Methotrexate analogues 33. N  -acyl-N  -(4-amino-4-deoxypteroyl)-L-ornithine derivatives: Synthesis and in vitro antitmor activity. J. Med. Chem. 1988, 31, 1332  1337.

15  PT523 or N α -(4-amino-4-deoxypteroyl)-N  -hemophthaloyl-L-ornithine (19) is an unusually potent antifolate which exerts its potency through a tight binding to DHFR and efficient utilization of the reduced folate carrier (RFC). 1 1.Balinska, M.; Galivan, J.; Coward, J.K. Efflux of methotrexate and its polyglutamate derivatives from hepatic cells in vitro. Cancer Res. 1981, 41,

16  8-Nitroquinazoline (20) displayed an overwhelming activity in arresting the cells at G2/M phase. 1  4 ‑ (Substituted)-aminoquinazoline derivatives such as 21a and 21b are found to be potent antitumors through DHFR inhibition. 2 1.Yin, J.; Zu-Yu, Z.; Wei, T.; Qiang, Y.; Ya ‑ Qin, L. 4 ‑ Alkoxyl substitution enhancing the anti-mitotic effect of 5- (3,4,5-substituted)aniline-4-hydroxy-8-nitroquinazolines as a novel class of antimicrotubule agents. Bioorganic and Medicinal Chemistry Letters 2006, 16, Gang, L.; De-Yu, H.; Lin-Hong, J.; Bao-An, S.; Song, Y. Ping-Shen, L.; Pinaki, S.B., Yao, M.; Luo, H.; Xian, Z. Bioorganic and Medicinal Chemistry, 2007, 15,

17  A series of nalidixic acid derivatives having quinazoline moiety at position ‑ 3 (22) showed an enhanced inhibitory activity against C. albicans, P. vulgaris in comparison to pure nalidixic acid. 1  Efforts still continue for the discovery of non-classical antifolates, with high selectivity toward the parasitic, the bacterial or even the tumor’s DHFR enzyme. 1.Gaurav, G.; Suvarana, G.K. Synthesis and evaluation of new quinazoline derivatives of nalidixic acid as potential antibacterial and antifungal agents. European Journal of Medicinal Chemistry 2006, 41,

18 RESEARCH OBJECTIVES AND RATIONAL DESIGN

19 Figure 3: Structures of some literature lead compounds.

20 Accordingly a new series of quinazoline analogues (A-E) is designed to possess the following structure features: On the basis of these considerations, the aim of this study is to locate novel lead compounds.

21 i.6-Methyl, 6-nitro or 6-amino functions, representing electron donating and electron withdrawing substituents. ii.A benzyl group at position 3-, based on what was concluded from our previous study. 1 iii.An alkyl, allyl or cinnamyl thioether functional groups at position 2 ‑ (A). 1.Al-Rashood, S.T.; Aboldahab, I.A.; Abouzeid, L.A.; Abdel-Aziz, A.A-M.; Nagi, M.N.; Abdul-hamide, S.G.; Youssef, K.M.; Al-Obaid, A.M.; El-Subbagh, H I. Synthesis, Dihydrofolate Reductase Inhibition, and Molecular Modeling Study of Some New 4(3H)-Quinazolinone Analogues. Bioorg. & Med. Chem. 2006, 14,

22 iv.An acryloyl or cinnamoyl thioesters at position 2-, in resemblance to the lead compounds 10 and 11 (B).

23 v.The 6 ‑ amino function of A is used to introduce an allyl and cinnamyl amines (C), or acrylamide and cinnamamide functions (D) to position 6-. These 6-amide compounds resemble the active compound 16. vi.The secondary amine C is converted to its corresponding tertiary amine (E) through its alkylation using methyl, allyl, or cinnamyl functions, to block the –NH- function to produce methotrexate (3) type analogues.

24  Those alterations and modifications are anticipated to produce active DHFR inhibitors.  Those functional groups designed to be accommodated on the quinazoline ring such as: Thioether, alkyl, aryl, arylakyl and nitro group  Those functions known to increase lipid solubility a character very much needed in DHFR inhibitors.

25 RESULTS AND DISCUSSION

26 Chemistry The synthetic strategy to synthesize the targets A-E is depicted in schemes 1-3.

27 Scheme 1: Synthesis of the new compounds ●

28 Figure 5: Structures of the two tautomeric structures of 28.

29 Scheme 1: Synthesis of the new compounds

30 Figure 7: Suggested mechanism for the formation of the side product 32.

31 Scheme 1: Synthesis of the new compounds

32 Figure 13: Suggested mechanism for the formation of the disulfide side product 35.

33 Scheme 1: Synthesis of the new compounds

34 Scheme 2: Synthesis of the new compounds

35 Figure 25: Suggested mechanism for the formation of the ester 40.

36 Scheme 2: Synthesis of the new compounds

37

38

39

40 Biological Evaluations I- Dihydrofolate Reductase (DHFR) Inhibition Assay Figure 31: Inhibition (IC 50 ) of DHFR activity by methotrexate (MTX, 3). Each point represents a mean + SD of 3 experiments.

41 Table 4: Comparative results of Bovine and hDHFR inhibition (IC 50,  M) of the active compounds.

42 Structure Activity Relationship (SAR)

43 IC 50

44

45

46

47 II- Antimicrobial Activity

48 Table 5: The in vitro antimicrobial activity results of the tested compounds -(-) Not active (8 mm), Weak activity (8-12 mm), Moderate activity (12-15 mm), Stron activity (> 15 mm). Solvent: DMSO (8 mm). - NT, Not tested - Compounds 25-27, 29-31, 33-36, 38, 39, 43, 45, 46, 48, 50, and proved inactive.

49 Molecular Modeling Study  Prediction of the affinity of a drug for a specific enzyme or receptor can be of a great value in the drug discovery process. Computational methods can be employed to study this affinity issue. 1,2  The target compounds (23-59) have been evaluated for their recognition profile at hDHFR binding pocket. 1.Klon, A.E.; Heroux, A.; Ross, L.J.; Pathak, V.; Johnson, C.A.; Piper, J.R.; Borhanti, D.w. Atomic resolution structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates. J. Mol. Biol., 2002, 320, Hunter, W. N. Picking pockets to fuel antimicrobial drug discovery. Biochem. Soc. Transac., 2007, 35,

50 Figure 32: Binding mode for LIH (60) docked and minimized in the hDHFR binding pocket, showing residues involved in its recognition. I. Molecular Dynamic Studies

51 Figure 33: Binding mode for compounds 45, 51, 52, 55 docked and minimized in the hDHFR binding pocket, showing residues involved in its recognition

52 The molecular docking study revealed that recognition with the key amino acid Glu30 is essential for binding and hence biological activity. The molecular docking study revealed that recognition with the key amino acid Glu30 is essential for binding and hence biological activity. Recognition with the amino acid Ser59 is important for the proper binding and the enhancement of biological activity. Recognition with the amino acid Ser59 is important for the proper binding and the enhancement of biological activity.

53 Figure 40: (a) Superposition of the most active compounds 36 (in brown), 48 (in blue), 49 (in green), 50 (in yellow), 56 (in cyan) and 59 (in red) using flexible alignments. (b) Further refinement of the overlay subjected to the constraints described in the text. (a)(b) II. Flexible Alignment

54 Figure 41: Flexible alignment of the most active compounds 36 (red), 48 (cyan), 56 (yellow) and the least active compound 40 (blue) and 41 (green).

55 III. Electrostatic and hydrophobic mappings

56 Figure 42: Electrostatic maps (right panels) and hydrophobic maps (left panels) of the lowest energy conformers for the most active compounds 48, 49, 50, and 53, maps are color coded: red for a hydrogen bond and a hydrophilic region, cyan for a medium polar region, and green for a hydrophobic region

57 Figure 44: Electrostatic maps (right panels) and hydrophobic maps (left panels) of the lowest energy conformer for the least active compounds 40 and 41, maps are color coded: red for a hydrogen bond and a hydrophilic region, cyan for a medium polar region, and green for a hydrophobic region

58 IV. Pharmacophore Prediction

59 Figure 45: Lower panel showed the most active compounds 36, 48, 49, 50, 56 and 59, mapped to the pharmacophore model for DHFRs. Upper panel showed the pharmacophoric geometry. Pharmacophore features are color coded: orange for hydrophobics aromatic, blue for a hydrogen bonds donor, and red for a hydrogen bonds acceptor feature. F5 and F6: Hydrogen bond acceptor center; F1, F4 and F7: Aromatic or hydrophobic center; F2, F3, F8 and F9: Aromatic or Pi orbital place at the receptor site; F10: H-bond acceptor place at the receptor site.

60 Figure 46: The least active compounds 40 (yellow) and 41 (pink), mapped to the pharmacophore model for DHFRs (right panel). The left panel showed the most active compounds 36 (cyan), 56 (green) and the least active 40 (yellow), 41 (pink) mapped to the pharmacophore features. Pharmacophore features are color coded: orange for hydrophobics aromatic, blue for a hydrogen bonds donor, and red for a hydrogen bonds acceptor feature.

61 CONCLUSION

62 Figure 47: Structures of the most active DHFR inhibitors.

63 Figure 48: Structures of the most active antimicrobial compounds.

64 Dr. Hussein El-Subbagh Dr. Hussein El-Subbagh Dr. Laila Abozaid Dr. Laila Abozaid Dr. Mahmoud N. Nagi Dr. Mahmoud N. Nagi Dr. El-Sayed E. Habib Dr. El-Sayed E. Habib Dr. Alaa A. M. Abdel-Aziz Dr. Alaa A. M. Abdel-Aziz Dr. Sami G. Abdel-Hamide Dr. Sami G. Abdel-Hamide KACST AT–14–19 KACST AT–14–19 Department of Pharmaceutical Chemistry, College of Pharmacy, KSU Department of Pharmaceutical Chemistry, College of Pharmacy, KSU Deanship of Graduate Studies Deanship of Graduate Studies Deanship of Scientific Research Deanship of Scientific Research Acknowledgment

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