Synthesis and Biological Activity of Novel Furan and 1H-Pyrrole-Containing Heterocyclic Compounds As HIV-1 Fusion Inhibitors

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Title:
Synthesis and Biological Activity of Novel Furan and 1H-Pyrrole-Containing Heterocyclic Compounds As HIV-1 Fusion Inhibitors
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1 online resource (69 p.)
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english
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Avan,Ilker
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University of Florida
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Gainesville, Fla.
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Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Katritzky, Alan R
Committee Members:
Smith, Benjamin W
Hong, Sukwon

Subjects

Subjects / Keywords:
aids -- antiretroviral -- antiviral -- fusion -- gp41 -- haart -- heterocyclic -- hiv -- inhibitor -- thiazolidin
Chemistry -- Dissertations, Academic -- UF
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Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
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Abstract:
In the present study, novel 5-((aryl-furan /-1H-pyrrol-2-yl)methylene)-2-thioxo-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-ones and 2,5-disubstituted-furan /-1H-pyrrole containing pyrrolidine, thioxooxazolidine, imidazolidine, and thiazolo3,2-b1,2,4triazine derivatives were synthesized by Knoevenagel condensation or Wittig reaction (yields 21?75%). Intermediate compounds 2-aryl-5-formyl-furans and -1H-pyrroles were obtained by Suzuki-Miyaura coupling reaction (yields 29?86%). Anti-HIV-1 activities of 5-((aryl-furan /-1H-pyrrol-2-yl)methylene)-2-thioxo-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-ones were tested. All tested compounds showed potent anti-HIV-1 activity. The compounds, 5-((5-(4-chloro-3-(1H-tetrazol-5-yl)phenyl)furan-2-yl)methylene)-2-thioxo-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-one and 5-((5-(2-fluoro-3-(1H-tetrazol-5-yl)phenyl)furan-2-yl)methylene)-2-thioxo-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-one exhibited the best anti-HIV-1 activity at low nanomolar level of EC50 with 18 and 14 nM, respectively. The docking studies of the compounds above were also able to rationalize the antiviral activities.
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In the series University of Florida Digital Collections.
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Includes vita.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Ilker Avan.
Thesis:
Thesis (M.S.)--University of Florida, 2011.
Local:
Adviser: Katritzky, Alan R.

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UFE0043135:00001


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1 S YNTHESIS AND BIOLOGICAL ACTIVITY OF NOVEL FURAN AND 1 H PYRROL E CONTAINING HETEROCYCLIC COMPOUN D S AS HIV 1 FUS I ON INHIBITORS By ILKER AVAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FU LFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Ilker Avan

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3 To my mother M r s. Fatma my father Mr. Osman and my sisters Ms. Ilkay and Ms. Ilknur f or their endless support

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4 ACKNOWLEDGMENT S I would like to thank my advisor Alan R. Katritzky for the opportunity, great understanding and support during my research I thank especially my sub group members Dr. Srinivasa R. Tala, Dr. Nader E. Abo Dya, Mr. Tarek Ibrahim Salah, Mr. Abdelmotel Abdel majeid, Mr. Suvendu Biswas, my friends in group members, Dr. C Dennis Hall, Ms. Claudia El Nachef, Mr. Zuoquan Wang, Mr. Khanh Ha Mr. Bogdan Draghici, Ms. Judit Kovacs, Ms. Rachel DeLoach, Ms. Mirna El Khatib, Mr. Davit Jishkariani I thank my friends in Gainesville, Mr. Basri Gulbakan, Mr. Muhammed Ibrahim Sukur, Mr. Ersen Gokturk, Mr. Ismail Ocsoy, Mr. Engin Kili c, Mr. Ali Ozcan, Mr. Besim Solak Dr. Sevil Ozcan, Mr. Zeynal Topalcengiz and my flatmate Mr. Bora Inci. I thank my committee members, Dr. Ben Smith, Dr. Sukwon Hong for their helpful discussion. I also would like to thank my parents and my sisters for their endless support and encourage ment

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF SCHEMES ................................ ................................ ................................ ........ 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 HIV AND HIV THERAPY ................................ ................................ ........................ 16 1.1 Introduction ................................ ................................ ................................ ....... 16 1.2 HIV Therapy ................................ ................................ ................................ ...... 17 1.2.1 Reverse transcriptase inhibitors (RTI) ................................ ..................... 19 1.2.1.1 Non nucleoside reverse transcriptase inhibitors ............ 19 1.2.1.2 Nucleoside reverse transcriptase inhibitors (NRTIs) ...................... 19 1.2.1.3 Protease inhibitors (PI) ................................ ................................ ... 20 1.2.1.4 Fusion inhibitors (Fls) ................................ ................................ ........... 21 1.3 HIV Fusion Mechanism ................................ ................................ ..................... 22 2 SYNTHESIS AND BIOLOGICAL ACTIVITY AND NOVEL FURAN OR 1 H PYRROL E CONTAINING 2 THIOXOTHIAZOLIDIN 4 ONES AS HIV 1 FUSION INHIBITORS ................................ ................................ ................................ ........... 24 2.1 Introduction ................................ ................................ ................................ ....... 24 2.2 Synthetic Approach ................................ ................................ ........................... 25 2.2.1 Synthesis of 2 aryl 5 formyl pyrroles (2.5a d) and furans (2.5e h) ....... 26 2.2.2 Synthesis of 5 ((aryl furan/ 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl) phenyl)thiazolidin 4 ones (2.4a h). ................................ ..... 27 2.2.3 Synthesis of 2 thioxo thiazolidin 4 one ring (2.10) ................................ .. 29 2.2.4 Preparation of 5 bromopyrrole 2 carboxaldehyde (2.7) ........................... 29 2.2.5 Synthesis of 5 formyl 2 furylboronic acid (2.8) [03OBC1447] .................. 30 2.2.6 Preparation of tetrazole containing bromobenzenes (2.9b d) ................. 31 2.3 Biological Activity Studies of 2.4a h ................................ ................................ 32 2.3.1 The inhibitory activity of (2.4a h) on HIV 1 IIIB replication ......................... 32 2.3.2 Molecular docking of selected inhibitors onto gp41 hydrophobic cavity .. 37 2.4 Conclusion ................................ ................................ ................................ ........ 38 2.5 Experimental Section ................................ ................................ ........................ 38 2.5.1 Synthesis of 2 ar yl 5 formyl 1 H pyrroles (2.5a d) and furans (2.5e i) ... 39

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6 2.5.1.1 General procedure for the synthesis of 2 aryl 5 formyl 1 H pyrroles (2.5a d) ................................ ................................ ..................... 39 2.5.1.2 General procedure for the synthesis of 2 aryl 5 formylfurans (2.5e i) ................................ ................................ ................................ ... 40 2.5.2 General procedure for preparation of 2.4a d, 2.4f, 2.4g .......................... 42 2.5.3 General procedure for preparation of 2.4e, 2.4h ................................ ..... 44 2.5.4 Synthesis of 2 thioxo 3 (3 (trifluorom ethyl)phenyl)thiazolidin 4 one ....... 45 2.5.5 Preparation of 5 bromopyrrole 2 carboxaldehyde (2.7) ........................... 45 2.5.6 Synthesis of 5 formyl 2 furylboronic acid (2.8) ................................ ........ 47 2.5.7 General procedure for preparation of 3 tetrazolyl bromobenzenes (2.9a c) ................................ ................................ ................................ ......... 48 3 SYNTHESIS OF SOME NOVEL FURAN OR 1 H PYRROLE CONTAINING HETEROCYCLIC COMPOUN DS ................................ ................................ ........... 50 3.1 Introduction ................................ ................................ ................................ ....... 50 3.2 Synthesis of Target Molecules (3.1a c, 3.2, 3.3, and 3.4) ................................ 51 3.2.1 Synthesis of 3 (5 ((2,5 dioxo 1 phenethylpyrrolidin 3 ylidene)methyl)furan / 1 H pyrrol 2 yl)benzoic acid (3.1a c) .......................... 52 3.2.2 Synthesis of 2 chloro 5 (5 ((4 oxo 3 phenethyl 2 thioxooxazolidin 5 ylidene)methyl)furan 2 yl)benzoic acid (3.2) ................................ ................. 54 3.2.3 Synthesis of 2 chloro 5 (5 ((2,5 dioxo 1 phenethylimidazolidin 4 ylidene)methyl)furan 2 yl)benzoic acid (3 .3) ................................ ................. 54 3.2.4 Synthesis of 2 chloro 5 (5 ((3,7 dioxo 6 phenyl 3,7 dihydro 2 H thiazolo[3,2 b ][1,2,4]triazin 2 ylidene)methyl)furan 2 yl)benzoic acid (3.4) ... 55 3.3 Conclusion ................................ ................................ ................................ ........ 56 3.4 Experimental Section ................................ ................................ ........................ 57 3.4.1 General synthesis of 3 (5 ((2,5 dioxo 1 phenethy lpyrrolidin 3 ylidene)methyl) 1 H pyrrol / furan 2 yl)benzoic acid (3.1a c) ........................ 57 3.4.2 Synthesis of 5 oxo 1 phenethyl 3 (triphenylphosphonio) 4,5 dihydro 1 H pyrrol 2 olate (3.6) ................................ ................................ ................... 58 3.4.3 Synthesis of N (2 phenylethyl)maleimide (3.9) ................................ ........ 59 3.4.4 Synthesis of 2 chloro 5 (5 ((4 oxo 3 phenethyl 2 thioxooxazolidin 5 ylidene)meth yl)furan 2 yl)benzoic acid (3.2) ................................ ................. 59 3.4.5 Synthesis of 3 phenethyloxazolidine 2,4 dione (3.10) ............................. 60 3.4.6 Synthesis of 2 chloro 5 (5 ((2,5 dioxo 1 phenethylimidazolidin 4 ylidene)methyl)furan 2 yl)benzoic acid (3.3) ................................ ................. 61 3.4.7 Synthesis of 3 phenethylimidazolidine 2,4 dione (3.11) .......................... 61 3.4.8 Synthesis of 2 chloro 5 (5 ((3,7 dioxo 6 phenyl 3,7 dihydro 2 H thiazolo[3,2 b ][1,2,4]triazin 2 ylidene)methyl)furan 2 yl)benzoic acid (3.4) ... 62 3.4.9 Synthesis of 3 mercapto 6 phenyl 1,2,4 triazin 5(4 H ) one (3.14) ........... 62 4 SUM M ARY OF ACHIEVEMEN T S ................................ ................................ .......... 64 LIST OF REFERENCES ................................ ................................ ............................... 65 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 69

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7 LIST OF TABLES Table page 2 1 Preparation of 2 aryl 5 formyl pyrroles 2.5a d and fur ans 2.5e i ..................... 27 2 2 Synthesis of 5 ((aryl furan/ 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones 2.4a h ................................ ............... 29 2 3 Synthesis of 3 tetrazolylbromobenzenes 2.9a c ................................ ............... 31 2 4 Anti HIV 1 IIIB activity, cytotoxicity and selectivity indexes of 2.4a h [11JMC572]. ................................ ................................ ................................ ....... 33 2 5 I nhibitory activity of the 2.4a h on infection by a primary HIV 1 isolate, 92US657 (clade B, R5) [11JMC572] ................................ ................................ .. 34 3 1 Preparation of 2,5 disubstituted pyrrole/fu rans 3.1a c ................................ ....... 53

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8 LIST OF FIGURES Figure page 1 1 HIV lifecycle. ................................ ................................ ................................ ....... 18 1 2 Structures of Ef avirenz 1.1 and Nevirapinez 1.2 ................................ ................. 1 9 1 3 Structures of Zidovudine 1.3 and Abacavir 1.4 ................................ ................... 20 1 4 Structures of Saquinavir 1.5 and Dar unavir 1.6 ................................ .................. 20 1 5 The process of HIV infection of CD4 + cells ................................ ......................... 21 1 6 Structures of Enfuvirtide 1.7 and Maraviroc 1.8 ................................ .................. 22 1 7 Six helix bundle formation on gp41. ................................ ................................ ... 23 2 1 Some anti HIV active compounds ................................ ................................ ...... 24 2 2 General structure of 2.4a h ................................ ................................ ................ 25 2 3 Retro synthetic route for 2.4 ................................ ................................ ............... 26 2 4 The structure of ADS J1 2.21 ................................ ................................ ............. 34 2 5 The structure of TAK779 2.22 ................................ ................................ ............. 35 2 6 Inhibition of cell cell fusion and 6 HB formation. ................................ ................. 36 2 7 CD spectra of gp41 6 HB core formation ................................ ............................ 36 2 8 Molecular docking of two highly anti HIV 1 active compounds ........................... 37 3 1 Structure of compounds 2.4a h 3.1a c 3.2 3.3 and 3.4 ................................ .. 51

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9 LIST OF SCHEME S Scheme page 2 1 Synthesis of 2 aryl 5 formyl pyrroles 2.5a d /fura ns 2.5e i .............................. 27 2 2 Synthesis of 5 ((aryl furan/ 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones 2.4a h ................................ .............. 28 2 3 Synthesis of 2 thioxo thiazolidin 4 one 2.10 ................................ ....................... 29 2 4 Synthesis 5 formyl 2 furylboronic acid 2.7 ................................ .......................... 30 2 5 Synthesis of 5 formyl 2 furylboronic acid 2.8 ................................ ...................... 31 2 6 Synthesis of 3 tetrazolylbromobenzenes 2.9a c ................................ ............... 31 3 1 Attempt to prepare 3.1a ................................ ................................ ...................... 52 3 2 Synthesis of 3.1a c ................................ ................................ ............................ 53 3 3 Preparation of Wittig r eagent 3.6 ................................ ................................ ........ 53 3 4 Synthesis of 3.2 ................................ ................................ ................................ .. 54 3 5 Synthesis of 3.3 ................................ ................................ ................................ .. 55 3 6 Synthesis of 3.11 ................................ ................................ ................................ 55 3 7 Synthesis of 3.4 ................................ ................................ ................................ .. 56 3 8 Preparation of 3.14 ................................ ................................ ............................. 56

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10 LIST OF ABBREVIATION S 6 HB Six helix bundle Ac 2 O Acetic anhydride Acetone d 6 Deuterated acetone Ac OH Acetic acid ADS J1 7 [6 phenylamino 4 [4 [(3,5 disulfo 8 hydroxynaphthyl)azo] 2 methoxy 5 methylphenylamino] 1,3,5 triazin 2 yl] 4 hydroxy 3 [(2 methoxy 5 sulfophenyl)azo] 2 naphthalenesulfonic acid AIDS Acquired Immunodeficiency S yndrome Anal. Analyti cal aq. Aqueous B(OMe) 3 T rimethylborate ( n Bu) 2 SnO D ibuthyltin oxide C Celsius degree Calcd. Calculated CC 50 C oncentration causing 50% cytotoxici ty CCR5 C C C hemokine receptor type 5 CD4 C luster of differentiation 4 which is a glycoprotein expressed on the surface of T helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells. CD4 + cell T helper cell with CD4 receptor CDCl 3 Deuterated chloroform CH 2 Cl 2 Methylene chloride CH 3 CN Acetonitrile CHR C terminal heptad repeat (CH 3 ) 3 SiN 3 T rim ethylsilyl azide (COCl) 2 Oxalyl chloride

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11 CXCR4 C X C C hemokine receptor type 4 d Doublet DCM Methylene chloride dd Doublet of doublet DIEA D iisopropylethylamine DME D imethyl ether ethylene glycol Dimethoxyethane DMF Dimethylformamide DMSO d 6 Deutrated di methyl sulfoxide DNA Deoxyribonucleic acid e.g. Exempli gratia EBV Epstein Barr virus EC 50 effective concentration for 50% inhibition ELISA Enzyme linked immunosorbent assay Env Envelope glycoprotein et al And others Et Ethyl Et 3 N Triethylamine EtOAc Et hyl acetate EtOH Ethanol FBS Fetal Bovine Serum FDA Food and Drug Administration Fl Fusion inhibitors g Gram gp120 A glycoprotein with 120 kDa gp41 A glycoprotein with 41 kDa

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12 h Hour H 2 SO 4 Sulfuric acid HAART Highly Active Antiretroviral Therapy HBr Hydr obromic acid HCl Hydrochloric acid HIV Human Immunodeficiency V irus HIV 1 A subtype of HIV HIV 1 IIIB O ne of the original HIV 1 isolate HNO 3 Nitric acid HR1 H eptad repeat regions (proximal to the N terminus) HR2 H eptad repeat regions (proximal to the C terminus) Hz Hertz IgG Immunoglobulin G J Coupling constant K 2 CO 3 Potassium carbonate kDa Kilo Dalton (1 da = 1.660538782(83)10 kg.) KHCO 3 Potassium bicarbonate KOH Potassium hydroxide m Multiplet M Micromolar mAb M onoclonal antibody Me Methyl Me OH Methanol MgSO 4 Magnesium sulfate mL Milliliter

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13 mmol Mil l imole mp Melting point mRNA Messenger ribonucleic aid MT 2 cell H uman ly mphocyte cell N Normal Na 2 CO 3 Sodium carbonate Na 2 SO 4 Sodium sulfate NaCN Sodium cyanide NaHCO 3 Sodium bicarbonate NaOH Sodium hydroxide NB 2 N (4 Carboxy 3 hydroxy)phenyl 2,5 dimethylpyrrole NB 64 N (3 carboxy 4 chloro)phenylpyrrole NBS N Bromosuccini mide n BuLi n B utyllithium NHR N terminal hepta d repeat NMR Nuclear magnetic resonance NNRTI Non nucleoside reverse transcriptase inhibitors NRTI Nucleoside reverse transcriptase inhibitors p Para PDB Protein Data Bank PdCl 2 (PPh 3 ) 2 Bis(triphenylphosphine )palladium( II ) chloride pH A negative decimal logarithm of the hydrogen ion activity in a solution. Ph Phenyl PI Protease inhibitors PPh 3 T riphenylphosphine

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14 p TsOH.H 2 O p Toluenesulfonic acid mono hydrate q Quartet RCSB Research Collaboratory for Structu ral Bioinformatics RNA Ribonucleic acid rt Room temperature RTI Reverse transcriptase inhibitors s Singlet SI Selectivity index T cells A group of white blood cells known as lymphocytes t Triplet TAK779 N N dimethyl N [4 [[[2 (4 methylphenyl) 6,7 dihydr o 5 H benzocyclohepten 8 yl]carbonyl]amino]benzyl] tetrahydro 2 H pyran 4 aminium chloride TCID 50 Median tissue culture infective dose TFA d Deuterated t rifluoroacetic acid THF Tetrahydrofuran TMP 2,2,6,6 T etramethylpiperidine TMSN 3 T rimethylsilyl azide W W att WHO World Health Organization XTT S odium 30 [1 (phenyl amino)carbonyl] 3,4 tetrazolium bis(4 methoxy 6 nitro)bezenesulfonic acid hydrate Chemical shift

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science S YNTHESIS AND BIOLOGICAL ACTIVITY OF NOVEL FURAN AND 1 H PYRROL E CONTAINING HETEROCYCLIC COMPOUN D S AS HIV 1 FUS I ON INHIBITORS By Ilker Avan August 2011 Chair: Alan R. Kat ritzky Major: Chemistry In the present study, novel 5 ((aryl furan / 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones and 2,5 disubstituted furan / 1 H pyrrole containing pyrrolidine thioxooxazolidin e imidazolidine and t hiazolo[3,2 b ][1,2,4]triazine derivatives were synthesized by Knoevenagel condensation or Wittig reaction (yields 21 75%) Intermediate compounds 2 aryl 5 formyl furans and 1 H pyrroles were obtained by Suzuki Miyaura coupling reaction ( yields 29 86 % ) A n ti HIV 1 activities of 5 ((aryl furan / 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones were tested All t ested compounds showed potent anti HIV 1 activity. T he compounds 5 ((5 (4 chloro 3 (1 H tetrazol 5 yl)phenyl)furan 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one and 5 ((5 (2 fluoro 3 (1 H tetrazol 5 yl)phenyl)furan 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one exhibited the best anti HIV 1 activity at low nanomolar lev el of EC50 with 18 and 14 nM respectively. The docking studies of the compounds above were also able to ra tionalize the antiviral activities

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16 CHAPTER 1 HIV AND HIV T HERAPY 1.1 Introduction I solation of the Human Immunodeficiency Virus (HIV) in 1983 ra ised high hopes that therapies targeting the virus would be discovered and made available to infected individuals [08S7 60]. Current statistics confirm that so far 25 million people lost their lives suffering from the Acquired Immunodeficiency Syndrome (AID S) since 2008 [09UNAIDS] According to the World Health Organization (WHO), around 1.8 million people died suffering from HIV in 2009 and currently 33.3 million people are living with HIV [09WHO]. The HIV, a member of genus Lentivirus in the Retroviridae family [98HPA], represents the causative agent of AIDS [93S1273]. Lentiviruses are identified as single stranded RNA viruses, which cause characteristic long duration diseases as a result of long incubation time s A ll cell s with CD4 receptors are susceptib le to HIV infection including macrophages as well as T and B lymphocytes, and dendritic cells [10COM524]. However the primary target of HIV infection is typically CD4 + cells (known as T helper cells, a subset of T cells) in the immune system. By reducing the amount of T helper cells, HIV weakens the immune system. An HIV infected person is also more susceptible to various life threatening secondary infections (also known as opportunistic agents) like bacteria, protozoa, fungi, other viruses, and cancer, w hich may cause the death of the patient [98HPA]. Transmission of virus occurs when HIV tainted fluid from the infected person comes in direct contact with bloodstream or mucosal lining of a healthy person. Furthermore, epidemiologically, the following thr ee common ways of transmission from

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17 person to person have been pointed out: blood, sexual contact, and mother child (birth) [11ASS]. The pathogenicity and severity of HIV infection is affected by several factors, such as age and genetic differences of infe cted individuals, level and quantity of the virus, and presence of other secondary infections. 1.2 HIV Therapy S o far, there is no realistic cure for HIV/AIDS. Highly Active Antiretroviral Therapy (HAART) is recommended for the treatment of HIV [10JMC52 1]. HAART is an aggressive treatment of HIV where the combination of different antiviral drugs is used to suppress HIV replication and the progression of the disease [03NM867]. Genetically, HIV comprises of two single strand RNAs T hree diffe rent enzymes: reverse transcriptas e, integrase and protease, are responsible for the life cycle and replication of HIV. Since the HIV virus has only RNA, the genetic material replication is directed from RNA to DNA by reverse transcriptase, contrary to other living orga nisms. Similarly to other viruses, HIV also requires a host cell for its replication. HIV replication includes seven steps : 1 ) attachment of the virus to the host cell surface ; 2 ) entry of the HIV genetic material (RNA), crucial enzymes including reverse t ranscriptase, integrase, protease and other viral proteins into the host cell ; 3 ) transcription of double helix viral DNA from viral RNA by reverse transcriptase ; 4 ) transportation and integration of viral DNA into the host cell genome (DNA) by integrase, results in the production of proviral DNA in host cell ; 5 ) production of viral proteins from mRNA, which are transcripted from proviral DNA ; 6 ) fusion of viral RNA and proteins come together to form an immature HIV, which buds off from host cell by taking part of the host cell membrane ; and 7 ) virus maturation upon release of individual HIV proteins (Figure 1 1) [10JMC521] [06CBC16]. All stages of the HIV replication cycle are potential targets in HIV therapy

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18 T herapeutic agents fall into four main cate gories: non nucleoside reverse transcriptase inhibitors (PI), and fusion inhibitors (Fls) [07CR1533]. Figure 1 1. HIV lifecycle. Reprinted by permission from Macmillan P ublishers Ltd. (DeClercq,E. Strategies in the design of antiviral drugs. Nat. Rev. Drug Discovery 2002, 1, 13 25), [02NRDD13] Copyright 2002 The usual HAART therapy also consists of a combination of therapeutic agents from different classes. Many antiret roviral drugs have been developed and used in HAART (Highly Active Antiretroviral Therapy), as a combination of drugs targeting one or more steps in the viral life cycle.

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19 1.2 .1 Reverse transcriptase inhibitors (RTI) Reverse transcriptase is an essential en zyme in the HIV replication cycle. It is a DNA polymerase which transcribes the single stranded viral RNA into a double stranded proviral DNA, needed for the integration into host cell genome (DNA). RTIs block reverse transcriptase's enzymatic function pre vents the completion of double stranded viral DNA synthesis, thus preventing HIV from multiplying [09AR] [06RT]. 1.2.1 .1 Non NNRTIs are highly potent inhibitors of HIV 1 reverse transcriptase (RT). Unli ke nucleoside analogs, the NNRTIs are not incorporated in the growing strand of HIV DNA, but directly inhibit the RT by binding in a reversible and non competitive manner to the enzyme. The binding site is a hydrophobic pocket close to the polymerase catal ytic site in the p66 subunit of RT, leading to a significant slowing of the rate of polymerization catalyzed by RT [99AR37] [06RT]. Many NNRTI drugs were developed to interfere with RT functions. Efavirenz 1.1 and nevirapine 1.2 are the first examples of N NRTI drugs approved by FDA for tre ating HIV 1 infection (Figure 1 2) [10JMC521]. Figure 1 2 Structure s of Efavirenz 1.1 and Nevirapinez 1.2 1.2. 1.2 Nucleoside reverse transcriptase inhibitors (NRTIs) NRTIs are analogues of the naturally occurring deoxynucletides needed to synthesize the viral DNA, and they compete with the natural deoxynucleotides for DNA assembly. Hence, all NRTIs are classified as competitive substrate inhibitors. Unlike

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20 natural deoxynucletides, NRTIs l ack a 3 hydroxyl group in the deoxyribose moiety [09AR]. Zidovudine 1.3 is the first NRTIs drug approved by the FDA. Abacavir 1.4 is also another approved NRTIs drug (Figure 1 3) [10JMC521]. Figure 1 3 Structure s of Zidov udine 1.3 and Abacavir 1.4 1. 2.1. 3 Protease inhibitors (PI) The HIV protease is responsible for the cleavage of the longer unfunctionalized proteins into the small structural and functional proteins. HIV protease inhibitors will interfere with the late s tage of the viral replication cycle and prevent the formation of infectious virus particles [10JMC521]. PI drugs inhibit the activity of proteases that prevents viral replication. Saquanavir 1.5 and darunavir 1.6 are currently FDA approved protease inhibit ors (Figure 1 4) [10JMC521]. Figure 1 4 Structure s of Saquinavir 1.5 and Darunavir 1.6

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21 1. 2.1. 4 Fusion inhibitors (Fls) The entry of HIV 1 to a host cell begins with an interaction between glycoprotein surface envelope of virus and host cell receptor (CD4). HIV envelope protein is composed of two subunits corresponding to a 41 kDa transmembrane glycoprotein (gp41) and a noncovalently associated 120 kDa glycoprotein (gp120) [07CR1533]. Entry of HIV 1 into cells involves thre e distinct stages: 1 ) attachment of glycoprotein gp120 to CD4 receptor on the host cell surface 2 ) binding of gp120 to co receptor CCR5 or CXCR4 of host cell and 3 ) gp41 mediated fusion of HIV virus to host cell [07EIHT1] [07CR1533] (Figure 1 5). Fusion in hibitors object one of these fusion stages, for instance blocking the CD4 and gp120 interaction, preventing the binding of gp120 to co receptor and disturbing gp41 mediated fusion of HIV. Figure 1 5 The process of HIV infection of CD4 + cells includes i ) initial contact between gp120 and CD4, which causes a conformational change ii ) allowing coreceptor interactions with gp120 and gp41 and iii ) subsequent membrane fusion. Reprinted with permission from [07CR1533] Copyright 2007 American Chemical Society.

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22 Enfuvirtide 1.7 a peptide entry inhibitor, binds to gp41 preventing the creation of an entry pore for the HIV virus. Another approved entry inhibitor, Maraviroc 1.8 is a CCR5 antagonist, which binds to CCR5 and blocks its association with gp120 (Figure 1 6) [10JMC521]. Figure 1 6. Structure s of Enfuvirtide 1.7 and Maraviroc 1.8 1. 3 HIV Fusion Mechanism HIV infection begins with the interaction between glycoprotein gp120, which is on the outer membrane of HIV virus and the CD4 receptor on the host cell surface. The viral gp120/g41 complex undergoes conformational changes which allow it to bind to a second receptor (or co receptor) CCR5 or CXCR4. The establishment of the co receptor binding leads to six helix bundle formatio n of the viral transmembrane protein gp41 (Figure 1 7). Structurally, the gp41 has two main heptad repeat regions: HR1 proximal to the N terminus) and HR2 (proximal to the C terminus). The hydrophobic region of gp41 is inserted into the cell membrane, whil e a trimeric coiled coil structure is formed by the HR1. The HR2 region then folds back within the hydrophobic grooves of the HR1 coiled coil, forming a hairpin structure containing a thermodynamically stable six helix bundle that draws the viral and cellu lar membranes together for fusion [07CR1533] [04NR215].

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23 Figure 1 7 Six helix bundle formation on gp41. Adapted with permission from [08B6802] Copyright 2008 American Chemical Society. G reat progress has been made in reducing HIV related mortality and m orbidity over the years [ 10JMC521] [05JMC1298] [03NM867]. H owever, complicated dosing regimens of HAART, have brought side effects that range in severity from skin rashes and gastrointestinal intolerance to coronary artery disease, nephrotoxicity, and bone marrow suppression. [07CR1533] Furthermore, many HIV/AIDS patients fail to respond to current antiretroviral therapeutics because of the emergence of multidrug resistance [03A2383] [04JCV1]. Therefore, it is essential to develop novel anti HIV therapeutic s targeting different steps of the HIV replication cycle, in particular the HIV fusion inhibitors as attractive candidates. In the present study, some novel furan and 1 H pyrrol e containing heterocyclic compounds were designed and synthesized as HIV 1 fusio n inhibitors targeting gp41. Most of the compounds exhibited improved anti HIV 1 activity at low nanomolar levels.

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24 CHAPTER 2 SYNTHESIS AND BIOLOG ICAL ACTIVITY AND NOVEL FURAN OR 1 H PYRROL E CONTAINING 2 THIOXOTHIAZOLIDIN 4 ONES AS HIV 1 FUSION INHIBITORS 1 2.1 Introduction T wo previously described [04AAC4349] small molecules, N (4 carboxy 3 hydroxy)phenyl 2,5 dimethylpyrrole 2. 1 (NB 2) and N (3 carboxy 4 chloro)phenylpyrrole 2.2 (NB 64) showed inhibitor activity against the HIV 1 infection at low micromol ar concentration [04AAC4349] (Figure 2 1). Molecular docking analysis on the gp41 trimer s of these two compounds indicates 2. 1 and 2. 2 fit into the deep hydrophobic pocket of gp41, but occupy only a small space [04AAC4349] [09AAC4987]. To increase the fill ing and binding activities of candidate molecules into the gp41 pocket, a bulkier series of 2 aryl 5 (4 oxo 3 phenethyl 2 thioxothiazolidinylidenemethyl)furan derivatives 2. 3a o were designed and synthesized [09JMC7631]. The stronger molecular binding aff inity on the g p41 cavity result ed in more potent HIV 1 activity with EC 50 (effective concentration for 50% inhibition) ranging from 44 to 390 nM and SI (selectivity index) in Figure 2 1 Some anti HIV active compounds The anti HIV 1 activities in the series of 2.3a o vary depending on the structural features of the molecule, ( e.g molecules containing a benzene ring has better activities 1 Reproduced in part with permission from Journal o f Medicinal Chemistry 2011 54, 572 579. Copyright 2011 American Chemical Society

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25 than molecules including a pyridine moiety) and their structural modifications are essential to improve anti HIV 1 activities. Therefore, some novel 5 ((aryl furan/ 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones 2.4a h were synthesized (Figure 2 2 ). The chemical structures of 2.3a o were modified by removing the CH 2 CH 2 side chain and also in some cases changing the carboxyl group for a tetrazolyl unit and/or by replacing a furan ring by a py rrole ring. Anti HIV 1 activities of 2.4a h were studied and all 2.4a h showed better anti HIV 1 activities than 2.3a h. Two of them, 2.3f and 2.3g, showed the best inhibitory activity against HIV 1 at low nanomolar level (EC 50 of 18nM with SI of 3686 for 2.3f EC 50 of 14nM with SI of 1989 for 2.3g ). Figure 2 2 Ge neral structure of 2.4a h 2.2 Synthetic Approach The target molecule 2.4 comprises of three main ring systems including benzene, furan/pyrrole and thiazolidinedione. Suzuki Miyaura cross coupling reaction was preferred to assemble bi aryl systems due to its versatile applicability on heterocyclic compounds and the commercial availability of the boronic acid and bromo aryl derivatives as starting materials Later, target molecules 2.4a h were obtained by linking bi aryl ring systems via Knoevenagel condens ation in basic media (Figure 2 3).

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26 Figure 2 3. Retro synthetic route for 2.4 2.2 .1 Synthesis of 2 aryl 5 formyl pyrroles (2.5a d) and furans (2.5e h) Many organic compounds, including natural products, polymers, ligands a nd drugs, contain biaryl groups [04SB] of topical interest in s in biaryl systems are mostly formed by Pd catalyzed reactions. The Suzuki Miyaura cross coupling reaction is one of the most popular reactions among the many palladium catalyzed coupling reactions, owing to mild reaction conditions, hi gh functional group tolerance and the ease of handling and separating by products [05SANROS] [04SB] Considering the commercial availability of the starti ng materials, 3 carboxyarylboronic acids 2.6 and aryl bromides 2.9 and ease of synthetic procedures outlined in Scheme 2 1, both pyrrole and furan containing biaryl systems 2.5a d and 2.5e i were prepared by following two different routes using Suzuki M iyaura cross coupling reaction In the present study, 2 a ryl 5 formylpyrroles 2.5a d were obtained from 61 to 86% yields by treatment of 3 carboxyarylboronic acids 2.6 with 5 bromopyrrole 2 carboxaldehyde 2.7 Coupling reactions of 5 formyl 2 furylboronic acid 2.8 with the corresponding aryl bromides 2.9 gave 2 aryl 5 formylfuran s 2.5e i from 29 to 60% yields (Scheme 2 1, Table 2 1). Suzuki Miyaura cross coupling reactions were accomplished using aqueous media in the presence of 5 10 mol% of PdCl 2 (PPh 3 ) 2 as catalyst.

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27 Scheme 2 1. Synthesis of 2 aryl 5 formyl pyrroles 2.5a d and furans 2.5e i Table 2 1. Preparation of 2 aryl 5 formyl pyrroles 2.5a d and furans 2.5e i Product 2.5 X Y R R 1 Reaction time (h) Yield a (%) a COOH NH H H 18 86 b COOH NH Cl H 18 61 c COOH NH F H 18 65 d COOH NH H F 18 67 b e COOH O H F 8 60 c, d f Tetrazolyl O Cl H 20 56 b g Tetrazolyl O H F 20 39 b h Tetrazolyl O F H 18 29 d i COOH O Cl H 18 56 e a Isolated yields [11JMC572] b crude yiel d; 2.5d, 2.5g, and 2.5h were used without further purification. c [ 09JMC7631] d prepared by an e used in Chapter 3. 2.2.2 Synthesis of 5 ((aryl furan/ 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl) phenyl)thiazo lidin 4 ones (2.4a h). 2 Aryl 5 formyl pyrroles 2.5a d and furans 2.5e h were treated with 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one 2.10 under basic Knoevenagel condensation condition s [05SANROS] to give 5 ((aryl furan/ 1 H pyrrol 2 yl)methy lene) 2 thioxo 3 (3

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28 (trifluoromethyl)phenyl)thiazolidin 4 ones 2.4a h ( 2, Table 2 2). All compounds 2.4a h were characterized by 1 H, 13 C NMR spectroscopy and elemental analysis. In early attempts, the condensation reactions of 2.10 with a ldehydes 2.5e and 2.5h wh ich were carried out in ethanol and catalyzed by 2,2,6,6 tetramethylpiperidine (TMP) at 80 o C gave compounds 2.4e, 2.4h in the yie lds of 22% and 44%, respectively. The reactions with aldehydes 2.5a d, 2.5f, 2.5g performed in tolue ne in the presence of 10 mol% of diisopropylethylamine resulted in better yields of 2.4a d, 2.4f, 2.4g (45 69%). Scheme 2 2. Synthesis of 5 ((aryl furan/ 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)th iazolidin 4 ones 2.4a h

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29 Table 2 2. Synthesis of 5 ((aryl furan/ 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones 2.4a h Product 2.4 X Y R R 1 Reaction time (h) Yield a (%) a COOH NH H H 16 48 b COOH NH Cl H 16 63 c COOH NH F H 16 50 d COOH NH H F 16 69 e COOH O H F 8 44 b f Tetrazolyl O Cl H 16 58 g Tetrazolyl O H F 16 45 h Tetrazolyl O F H 8 22 b a Isolated yields [ 11JMC572] ; b prepared by an 2.2.3 Synthesis of 2 thioxo thia zolidin 4 one ring (2.10) A ri ng closure reaction between 2,2' (thiocarbonylbis(sulfanediyl))diacetic acid 2.11 and 3 (trifluoromethyl)aniline 2.12 gave 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one 2.10 in 52% yield after heating under reflux fo r 6 h in water (Scheme 2 3) [56JACS384]. Scheme 2 3. Synthesis of 2 thioxo thiazolidin 4 one 2.10 2.2.4 Preparation of 5 b romopyrrole 2 carboxaldehyde (2.7) 5 Bromopyrrole 2 carboxaldehyde 2.7 was prepared starting from p yrrole a s outlined i n Scheme 2 4 [90C JC 1305] Freshly distilled pyrrole 2.13 was reacted with N N d iisopropylformamide 2.14 in the presence of oxalyl chloride in CH 2 Cl 2 to give

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30 N N diisopropylpyrrole 2 formiminium chloride 2.15 which was later treated w ith NaCN in acetonitrile to give (diisopropylamino) (pyrrol 2 yl) acetonitrile 2.16 in 65% yield. The bromination reaction of (diisopropylamino) (pyrrol 2 yl) acetonitrile 2.16 with NBS in THF produced (diisopropylamino) (4 bromopyrrol 2 yl) acetonitrile 2.17 Without isolatin g ( diisopropylamino) (4 bromopyrrol 2 yl) acetonitrile 2.17 elimination of HCN in aqueous media gave corresponding azafulven structure 2.18 in 72% yield. Finally, treatment of 2 bromo 6 diisopropylamino 1 azafulven 2.18 with NaOH in the mixture of met hanol water (1:1) gave 5 bromopyrrole 2 carboxaldehyde 2.7 in 77% yield Scheme 2 4. Synthesis 5 formyl 2 furylboronic acid 2.7 2.2.5 Synthesis of 5 formyl 2 furylboronic acid (2.8) 5 Formyl 2 furylboronic acid 2.8 was r eadily obtained by lithiation of 2 furaldehyde diethyl acetal 2.19 using n butyllithium followed by reaction with trimethylborate [03OBC1447] Acidic workup introduced the boronic acid group and released the aldehyde group to afford 5 formyl 2 furylboronic acid 2.8 in 55% yield (Scheme 2 5).

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31 Scheme 2 5. Synthesis of 5 formyl 2 furylboronic acid 2.8 2.2.6 Preparation of tetrazole containing bromobenzenes (2.9b d) ed as the interchange of functional groups leading to similar biological properties [11AAPPOC]. 5 Substituted 1 H tetrazoles are well known as a non classical bioisostere for the carboxylic acid moiety (RCO 2 H) in biologically active molecules [97PB]. Tetra zoles are often used as metabolism resistant isosteric replacements for carboxylic acids [02BMC3379]. 3 Tetrazolyl bromobenzenes 2.9a c were synthesized in 75 83% yields from the corresponding commercially available bromo cyano benzenes by a reaction with trimethylsilyl azide in the presence of dibuthyltin oxide as a catalyst (Scheme 2 6 and Table 2 3) [ 93JOC4139 ]. Scheme 2 6 Synthesis of 3 tetrazolylbromobenz enes 2.9 a c Table 2 3. Synthesis of 3 tetrazolylbromobenzenes 2 .9a c Product 2.9 R R 1 Reaction time (h) Yield (%) a Cl H 48 82 b H F 48 75 c F H 48 83

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32 2.3 Biological Activity Studies of 2.4a h 2 All biological activity studies including inhibitory activity, anti HIV 1 act ivity with cytotoxicity effects and CD spectra measurements were done by Dr. Hong Lu, and Dr. Lu Lu under supervision of Dr. Shibo Jiang M olecular docking studies were done by Dr. Asim K. Debnath at Lindsley F. Kimball Research Institute, New York Blood Center, New York 10065, United States 2.3.1 The inhibitory activity of (2.4a h) on HIV 1 IIIB replication The inhibitory activities of 2.4a h on HIV 1 replication in MT 2 cells were measured by using an enzyme linked immunosorbent assay (ELISA) [99JMC3203]. C ompounds 2.4a h significantly inhibi ted HIV 1 replication depending on applied dose with the EC 50 (effective concentration for 50% inhibition) ranging from 0.014 to 3.9 M. The cytotoxicity of these compounds on MT 2 cells was assessed using a colorimetric XTT assay as previously described [ 99JMC3203]. Their CC 50 (concentration causing 50% cytotoxicity) values ranged from 15 to 66 M and their selectivity indexes (SI) ranged from 4 to 3,686 (Table 1). Two compounds 2. 4 f and 2. 4 g exhibit the best anti HIV 1 activity, with EC 50 of 18 and 14 nM, with SI of 3,686 and 1,989, respectively (Table 2 4). Inhibitory activity of 2.4a h on infection by primary HIV 1 isolate, 92US657 (clade B, R5) were tested. C ompounds 2.4a h significantly inhibited infection of the HIV 1 92US657 isolate with EC 50 ranging from 0.3 to 21 M (Table 2 5). Two of the compounds, 2.4f and 2.4h showed the best inhibitory activity with EC 50 0.32 and 0.87 exclusively. 2 Reproduced in part with permission from Journal of Medicinal Chemistry 2011 54, 572 579. Copyright 2011 American Chemical Society.

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33 Table 2 4. Anti HIV 1 IIIB activity, cytotoxicity and selectivity indexes of 2.4a h [11JMC572] Product 2.4 X Y R R 1 MW EC 50 (M) a CC 50 (M) SI b a COOH NH H H 474.5 1.2 0.1 30 2 28 b COOH NH Cl H 526.9 1.8 0. 2 5 24 31 c COOH NH F H 492.5 3.9 0. 5 16 3 4 d COOH NH H F 492.5 0.6 0.1 30 1 48 e COOH O H F 502.5 0.06 0.01 31 7 484 f Tetrazolyl O Cl H 533.9 0.0180.002 66 11 3,686 g Tetrazolyl O H F 517.5 0.0140.005 28 4 1,989 h Tetrazolyl O F H 517.5 0.0330.006 2 7 5 815 a Each compound was tested in triplicate; the data are presented as the mean SD. b SI was calculated based o n the CC 50 for MT 2 cells and EC 50 for inhibiting infection of HIV 1 The i n hibitory activity of 2.4f and 2.4g on the HIV 1 cell cell fusion and the gp41 6 HB formation w as examined. Considering the positive control of our studies, the inhibitory activiti es of 2.4f and 2.4g were compared with the effect of ADS J1 2.2 1 ( Figure 2 4 ) [99JMC3203] [09AAC4987], which is a small molecule HIV 1 fusion inhibitor, targeting gp41 and interfering with the 6 HB core formation.

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34 Table 2 5 Inhibitory activity of the 2.4a h on infection by a primary HIV 1 isolate, 92US657 (clade B, R5) [11JMC572] Product 2.4 X Y R R 1 EC 50 (M) a a COOH NH H H 21 3 b COOH NH Cl H 4.70.1 c COOH NH F H 2. 80. 6 d COOH NH H F 3.430.0 1 e COOH O H F 2.2 0.2 f Tetrazolyl O Cl H 0.320.06 g Tetrazolyl O H F 1 0 0.2 h Tetrazolyl O F H 0.9 0. 4 a Each compound was tested in triplicate and the data were presented in mean SD. Figure 2 4. The structure of ADS J1 2.2 1

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35 ADS J1 2.2 1 is effective for blocking the fusion between the H9 cells infected by HIV 1 (X4 virus) and the uninfected target (MT 2) cells. Moreover ADS J1 2.2 1 prevents the gp41 6 HB core formation between the gp41 NHR peptide N36 and CHR peptide C34, accordi ng to the ELISA measurement [98JV10213]. Similarly ADS J1 2.2 1 compounds 2.4f and 2.4g have significant inhibition on HIV 1 mediated cell cell fusion with EC 50 of 3.930.06 and 3.950.14 M. 2.4f and 2.4g also prevent ed the gp41 6 HB core formation depend ing on applied dose s with EC 50 of 1.280.39 and 4.380.39 M, respectively. Figure 2 5. The structure of TAK779 2.2 2 Negative control of our studies was done by using a CCR5 antagonist molecule N N dimethyl N [4 [[[2 (4 m ethylphenyl) 6,7 dihydro 5 H benzocyclohepten 8 yl]carbonyl]amino]benzyl]tetrahydro 2 H pyran 4 aminium chloride (TAK 779, 2.2 2 ) (Figure 2 5). H owever TAK779 exhibits no inhibition effect on HIV 1 mediated cell cell fusion and the gp41 6 HB formation [09AR] (Figure 2 6). helicity of gp41 fragments (N46 and C34 peptides) showed the mixture of the N46 and C34 peptides creates a typical helical structure with helicity (Figure 2 7 ). However, in the presence of 2.4f and 2.4g, helicity o f the N46/C34 spiral was reduced to 69% and 75%, respectively (Figure 2 7A).

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36 Figure 2 6. Inhibition of cell cell fusion and 6 HB formation, A) Inhibitory activity of 2.4f and 2.4g on HIV 1 mediated cell cell fusion and B) the gp41 6 HB formation [11JMC5 72]. Adapted with permission from [11JMC572] Copyright 2011 Americ an Chemical Society. Usually, N46 peptide alone in phosphate buffered saline (PBS) formed a typical coiled coil structure with helicity of 52% [05JBC11259], a nd addition of 2.4f or 2.4g to N46 did not significantly affect the CD spectra of N46 (Figure 2 7B). According to our results, molecules 2.4f and 2.4g inhibited HIV 1 fusion by targeting the HIV 1 gp41 and leveling gp41 6 HB core formation, without disruption of the NHR trimeric coiled coil structure of N46. Figure 2 7. CD spectra of gp41 6 HB cor e formation A) 2.4f and 2.4g inhibited HIV 1 fusion by leveling gp41 6 HB core formation B) 2.4f and 2.4g had no effect on the secondary structure the HIV 1 gp41 NHR trimer ic coiled coil Adapt ed with permission from [11JMC572] Copyright 2011 American Chemical Society.

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37 According to the our anti HIV 1 activity studies, isosteric replacement of carboxylic acid by tetrazole ring did not significantly improve their anti HIV 1 activity, but caused significant reduction of cytotoxicity, by increasing their selectivity indexes. Another interesting result was observed by replac ing the furan ring by pyrrole. Table 2 4, shows that compounds 2.4e h with furan ring (EC 50 of 0.014 0.064 M) h ave better anti HIV 1 activity than compounds 2.4a d with pyrrole ring (EC 50 of 0.6 3.9 M). 2.3.2 Molecular d ocking of selected i nhibitors ont o gp41 h ydrophobic c avity Molecular docking analysis revealed that highly anti HIV active compound 2.4g containing tetrazole moiety, and compound 2.4e containing a carboxylic acid group part filled almost perfectly the space in the deep hydrophobic pocke t of gp41 formed by the NHR trimer. Poorly active compound 2.4d containing a carboxylic acid group, docked in a different position of gp41 cavity ( Figure 2 8) Molecular docking studies are also consistent with anti HIV activity results on Table 2 4. F igure 2 8 Molecular docking of two highly anti HIV 1 active compounds, 2.4e and 2.4g and one less active compound, 2.4d in the HIV 1 gp41 cavity. A) Carboxylic acid containing inhibitor 2.4e B) Tetrazole containing inhibitor, 2.4 g C) Poorly active compo und, 2.4d Reprinted with permission from [11JMC572] Copyright 201 1 American Chemical Society. In this study, the automated docking software Glide 5.6 within Schr dinger Suite 2010 ( Schr dinger Portland, OR) was used. X ray structure of the HIV 1 gp41 core

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38 (1AIK, [97C263]) was obtained from the Protein Data Bank (PDB) a t the Research Collaboratory for Structural Bioinformatics (RCSB). 2.4 Conclusion Anti HIV active compounds 5 ((arylfuran/1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones 2.4a h were synthesized by using oss coupling, followed by Knoevenagel condensation. All 8 compounds 2.4a h significantly inhibited HIV 1 replication depending on applied dose s with the EC 50 ranging from 0.014 to 3.9 M. Two compounds 2. 4 f and 2. 4 g show best anti HIV 1 IIIB activity, with EC 50 of 18 and 14 nM and SI of 3,686 and 1,989, respectively. 2. 5 Experimental Section Melting points were determined using a capillary melting point apparatus equipped with a digital thermometer and are uncorrected. 1 H (300 MHz) and 13 C (75 MHz) NMR spec tra were recorded in DMSO d 6 (with tetramethylsilanes as the internal standard), unless otherwise stated. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br s = broad singlet, m = multiplet), coupling constants ( J values) expressed in Hz. Elemental analyses were performed on a Carlo Erba EA 1108 instrument. The solvents (ethylene glycol dimethyl ether (DME) and THF) were dried by the usual methods and distilled before use. Purity of compounds 2 .4a was determined by elemental analyses and/or HPLC; purity of target compounds was SPD 20 A using the following: column, Zorbax Rx C18 with detection at 254 nm; solvent, M eOH (100%); flow rate of 0.5 mL/min. The experimental procedures and characterization data of target molecules 2.4a and intermediates 2.5a were

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3 9 reported recently [11JMC572] 3 Compounds 2.5b 2.5d 2.5e 2.5f 2.5g and 2.5h were used without further pu rification to prepare compounds 2.4b 2.4d 2.4e 2.4f 2.4g and 2.4h respectively. Compounds 2. 9a 2. 9b and 2. 9 c were used without further purification to prepare compounds 2. 5f 2. 5g and 2.4 h respectively. 2. 5 .1 Synthesis of 2 aryl 5 formyl 1 H pyrrole s (2.5a d) and furans (2.5e i) 2. 5 .1.1 General procedure for the synthesis of 2 aryl 5 formyl 1 H pyrroles ( 2.5a d ) A mixture of aryl boronic acid 2.6a d (1.37 mmol), 5 bromo 1 H pyrrole 2 carbaxaldehyde 2.7 (0.2 g, 1.14 mmol), in DME (6.0 mL), ethanol (5. 0 mL) was flushed with nitrogen for 10 min, and then PdCl 2 (PPh 3 ) 2 (40 mg, 0.056 mmol), 2 M aqueous Na 2 CO 3 (6.9 mL, 13.8 mmol of Na 2 CO 3 ) were added. After being flushed with nitrogen for 10 more min, the reaction mixture was heated at 80 o C for 18 h under n i trogen atmosphere. The solvent was removed under reduced pressure, the residue was dissolved in water (15 mL), and the mixture obtained was filtered through celite. The filtrate was acidified with 2 N HCl to pH 3 4. T he solid that formed w as filtered, wash ed with water and recrystallized from ethanol to give 2 aryl 5 formyl 1 H pyrroles 2.5a d. 3 (5 Formyl 1 H pyrrol 2 yl)benzoic acid (2.5a). White microcrystals (86%), mp 275 276 o C; 1 H NMR (DMSO d 6 ) 6.84 6.87 (m, 1H), 7.06 7.10 (m, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.87 (d, J = 7.5 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 8.46 (s, 1H), 9.51 (s, 1H), 12.63 (s, 1H), 13.12 (s, 1H); 13 C NMR (DMSO d 6 ) 109.4, 121.5, 126.3, 128.6, 129.2, 129.8, 131.4, 131.6, 134.2, 138.5, 167.2, 179.2; Anal. Calcd for C 12 H 9 NO 3 : C, 66.97; H, 4.22, N, 6.51. Found: C, 66.68; H, 4.26; N, 6.28. 3 Reproduced in part with permission from Journal of Medicinal Chemistry 2011 54, 572 579. Copyright 2011 American Chemical Society.

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40 2 Chloro 5 (5 formyl 1 H pyrrol 2 yl)benzoic acid ( 2.5b ). White microcrystals (61%), mp 280 285 o C; 1 H NMR (DMSO d 6 ) 6.80 6.92 (m, 1H), 7.06 7.20 (m, 1H), 7.59 (d, J = 8.4 Hz, 1H), 8.03 (dd, J = 8.1, 1.8 Hz, 1H), 8.33 (d, J = 1.8 Hz, 1H), 9.54 (s, 1H), 12.65 (br s, 1H), 13.57 (br s, 1H); 13 C NMR (DMSO d 6 ) 109.8, 121.4, 127.3, 129.1, 130.1, 130.6, 131.1, 132.4, 134.4, 137.3, 166.7, 179.4. 2 Fluoro 5 (5 formyl 1 H pyrrol 2 yl)benzoic acid ( 2.5c ). White mi crocrystals (65%), mp 278 279 o C; 1 H NMR (DMSO d 6 ) 6.84 6.92 (m, 1H), 7.12 (dd, J = 2.1, 1.5 Hz, 1H), 7.43 (dd, J = 9.0, 1.5 Hz, 1H), 8.12 8.20 (m, 1H), 8.42 (dd, J = 6.9, 2.4 Hz, 1H), 9.55 (s, 1H); 13 C NMR (DMSO d 6 ) 109.3, 117.5, 117.8, 120.1, 121.9, 1 27.6, 128.7, 131.5, 131.6, 134.2, 134.2, 137.6, 158.8, 162.2, 164.9, 179.2; Anal. Calcd for C 12 H 8 FNO 3 : C, 61.81; H, 3.46; N, 6.01. Found: C, 61.75; H, 3.23; N, 5.96. 2 Fluoro 3 (5 formyl 1 H pyrrol 2 yl)benzoic acid ( 2.5d ). Yellowish microcrystals (67%), mp 238 240 o C; 1 H NMR (DMSO d 6 ) 7.83 (s, 1H), 7.13 (d, J = 3.6 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.93 (t, J = 6.9 Hz, 1H), 8.14 (t, J = 7.2 Hz), 9.62 (s, 1H), 11.27 (br s, 1H); 13 C NMR (DMSO d 6 ) 112.4, 120.3, 124.3, 130.9, 132.0, 132.4, 133.7, 135.7, 155.8, 159.3, 164.8, 179.3. 2. 5 1.2 General procedure for the synthesis of 2 aryl 5 formylfurans ( 2.5e i ) A mixture of aryl bromide 2.9a d (0.8 mmol), 5 formyl 2 furanboronic acid 2.8 (1.2 mmol), and PdCl 2 (PPh 3 ) 2 (0.04 mmol) in DME (6.0 mL), ethanol (5.0 mL) and 0.1 M aqueous Na 2 CO 3 (6.9 mL, 13.8 mmol of Na 2 CO 3 ) was flushed with nitrogen for 5 min. and heated at 60 o C for 8 20 h (reaction time is given in Table 1) under nitrogen atmosphere. The solvents were removed under reduced pressure; the residue was dissolved in water (20 mL). The mi xture was filtered through celite and the filtrate was

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41 neutralized with 2 N HCl. The solid was filtered, washed with water, dried and recrystallized from ethanol to give 2 aryl 5 formylfurans 2.5e i 3 (5 Formylfuran 2 yl) 2 fluorobenzoic acid ( 2.5e ) Orang e microcrystals (60%), mp C; 1 H NMR (DMSO d 6 J = 3.8 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.72 (d, J 13 C NMR (DMSO d 6 J = 12.0 Hz, 1C). 118.1 (d, J = 12.0 Hz, 1C), 120.8 (d, J = 10.3 Hz, 1C), 125.0 (br s, 1C), 125.1 (d, J = 4.6 Hz, 1C), 131.0 (d, J = 2.9 Hz, 1C), 132.7 (d, J = 1.4 Hz, 1C), 151.5 (d, J = 2.3 Hz, 1C), 151.6, 157.7 (d, J = 264.5 Hz, 1C), 164.6 (d, J = 2.9 Hz, 1C), 178.3. 5 (4 Chloro 3 (1 H tetrazol 5 yl)phenyl)furan 2 carb aldehyde (2.5f). White microcrystals (56%), mp 220 222 o C; 1 H NMR (DMSO d 6 ) 7.46 (d, J = 3.6 Hz, 1H), 7.69 (d, J = 2.7 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 8.09 (dd, J = 8.4, J = 1.5 Hz, 1H), 8.30 (s, 1H), 9.64 (s, 1H); 13 C NMR (DMSO d 6 ) 110.4, 125.2, 125.4, 127.8, 128.2, 128.5, 131.5, 132.4, 132.9, 133.9, 135.2, 152.2, 155.9, 1 78.2. 5 (2 Fluoro 3 (1 H tetrazol 5 yl)phenyl)furan 2 carbaldehyde mono hydrate (2.5g) Yellowish microcrystals (39%), mp 223 225 o C; 1 H NMR (DMSO d 6 ) 7.26 (t, J = 3.9 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.74 (d, J = 3.9 Hz, 1H), 8.08 8.20 (m, 2H), 9.71 (s, 1H); 13 C NMR (DMSO d 6 ) 113.2 (m), 117.9 (d), 119.6, 124.7, 125.9 (d), 129.8 (t), 130.9, 144.7, 151.3, 151.7, 159.0, 178.3 Anal. Calcd for C 12 H 7 FN 4 O 2 .H 2 O: C, 55.82; H, 2.73, N, 21.70. Found: C, 55.54; H, 3.16; N, 19.75. 5 (4 Fluoro 3 (1 H tetrazol 5 yl)ph enyl)furan 2 carbaldehyde (2.5h) Brown microcrystals (29%), mp 115 117 o C; 1 H NMR (DMSO d 6 ) 7.44 (d, J = 3.0 Hz, 1H), 7.65 7.76 (m, 3H), 8.18 (br s, 1H), 8.51 (d, J = 4.8 Hz, 1H), 9.65 (s, 1H); 13 C NMR

PAGE 42

42 (DMSO d 6 ) 109.7, 117.9, 118.1, 119.0, 119.3, 125.4, 126.2, 130.1, 132.1, 136.0, 152.0, 156.1, 178.1 2 Chloro 5 (5 formylfuran 2 yl)benzoic acid (2.5i) Yellow microcrystals (56%), mp 174 178 o C; 1 H NMR (DMSO d 6 ) 7.46 (d, J = 3.6 Hz, 1H), 7.64 7.74 (m, 2H), 8.01 (dd, J = 8.4, J = 2.1 Hz, 1H), 8.23 (d, J = 1.5 Hz, 1H), 9.65 (s, 1H), 13.8 (br s,1H); 13 C NMR (DMSO d 6 ) 109.9, 125.0, 126.6, 127.5, 128.4, 131.5, 132.3, 151.9, 155.9, 166.0, 178.0. Anal. Calcd for C 12 H 7 ClO 4 : C, 57.51; H, 2.82. Found: C, 57.87; H, 2.74. 2. 5 .2 General procedure for preparation of 2 .4a d, 2.4f, 2.4g A mixture of aldehyde 2. 5 a d, 2. 5 f, 2. 5 g (0.5 mmol), 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one 2.10 (0.5 mmol) and 10 mol% o f DIEA in toluene (8 mL) was heated under reflux for 16 h. After cooling to room temperature, the s olid obtained was filtered and washed with diluted acetic acid, water and diethylether to give corresponding 5 ((aryl furan/1H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones ( 2. 4 a d, 2. 4 f, 2. 4 g ). 3 (5 ((4 Oxo 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 5 ylidene)methyl) 1 H pyrrol 2 yl)benzoic acid ( 2. 4 a ) Orange microcrystals (48%), mp 285 290 o C; 1 H NMR (DMSO d 6 ) 6.78 (s, 1H), 7.07 (s, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.72 7.98 (m, 6H), 8.02 (d, J = 7.8 Hz, 1H), 8.35 ( s, 1H), 12.49 (s, 1H), 13.20 (br s, 1H); 13 C NMR (DMSO d 6 ) 112.0, 115.2, 117.1, 122.4, 124.9, 126.2 (2C), 128.4, 129.0, 129.1, 129.5, 129.8, 130.5, 131.0, 131.9, 133.4, 136.4, 137.2, 166.7, 167.1, 192.9; Anal. Calcd for C 22 H 13 F 3 N 2 O 3 S 2 : C, 55.69; H, 2.76, N, 5.90. Found: C, 55.39; H, 3.25; N, 5.66. HPLC, 97.6% purity, Retention time: 3.02 min. 2 Chloro 5 (5 ((4 oxo 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 5 ylidene)methyl) 1 H pyrrol 2 yl)benzoic acid mono hydrate ( 2. 4 b ) Brownish

PAGE 43

43 microcrystal s (6 3%), mp 262 263 o C; 1 H NMR (DMSO d 6 ) 6.77 (s, 1H), 7.09 (s, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.72 (s, 1H), 7.74 7.98 (m, 5H), 8.17 (s, 1H), 12.49 (s, 1H); 13 C NMR (DMSO d 6 ) 112.4, 115.6, 117.0, 122.1, 126.2, 128.3, 129.3, 129.7, 130.4, 130.5, 131.5, 132.3, 133.4, 136.0, 136.3, 166.6, 167.0, 192.8. Anal. Calcd for C 22 H 12 ClF 3 N 2 O 3 S 2 H 2 O: C, 50.15; H, 2.68; N, 5.32. Found: C, 50.51; H, 2.85; N, 4.93. 2 Fluoro 5 (5 ((4 oxo 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 5 ylidene)methyl) 1 H pyrrol 2 yl)benzoic acid ( 2. 4 c ) Red microcrystals (50 %), mp 236 238 o C; 1 H NMR (DMSO d 6 ) 7.00 (s, 1H), 7.21 (s, 1H), 7.40 (t, J = 9.5 Hz, 1H), 7.59 (s, 1H), 7.74 8.00 (m, 5H), 8.10 8.16 (m, 1H), 12.73 (s, 1H), 13.51 (s, 1H); 13 C NMR (DMSO d 6 ) 111.9, 115.1, 117.0, 117.8, 118.2, 122.3, 126.2, 127.3, 127.4, 129.1, 130.5, 130.7, 130.8, 133.4, 136.3, 136.4, 158.7, 162.1, 164.8, 166.7, 192.8, 192.9. HRMS (ESI) [M+Na] + : Found: 515.0120, calculated for C 22 H 12 F 4 N 2 O 3 S 2 : 515.0118. HPLC, 98.8% purity, Retention time: 2.93 min. 2 Fluoro 3 (5 ((4 oxo 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 5 y lidene)methyl) 1 H pyrrol 2 yl)benzoic acid ( 2. 4 d ) Deep orange microcrystals (69%), mp 296 298 o C; 1 H NMR (DMSO d 6 ) 6.81 (br s, 1H), 7.00 (br s, 1H), 7.41 (t, J =7.5 Hz, 1H), 7.74 7.94 (m, 5H), 7.95 (s, 1H), 8.02 (t, J = 5.7 Hz, 1H), 12.29 (s, 1H), 13.45 (s, 1H); 13 C NMR (DMSO d 6 ) 114.7, 116.1, 116.4, 120.0, 120.8, 122.4, 124.8, 126.2. 129.2, 129.8, 130.2, 130.5, 13 0.8, 131.3, 131.4, 133.4, 136.3, 155.5, 159.0, 164.9, 166.7, 192.9; Anal. Calcd for C 22 H 12 F 4 N 2 O 3 S 2 : C, 53.66; H, 2.46; N, 5.69. Found: C, 53.55; H, 2.47; N, 5.55. 5 ((5 (4 Chloro 3 (1 H tetrazol 5 yl)phenyl)furan 2 yl)methylene) 2 thioxo 3 (3 (trifluorometh yl)phenyl)thiazolidin 4 one ( 2. 4 f ). O range microcrystals (58%), mp

PAGE 44

44 279 280 o C; 1 H NMR (DMSO d 6 ) 7.40 7.44 (m, 1H), 7.51 (d, J = 2.7 Hz, 1H), 7.72 7.98 (m, 6H), 8.00 8.06 (d, J = 8.4 Hz, 1H), 8.31 (s, 1H); 13 C NMR (DMSO d 6 ) 111.9, 118.1, 120.5, 123.0, 125.9, 126.1, 126.4, 127.3, 127.4, 127.7 (q, J = 298 Hz), 128.1, 130.3, 130.6, 131.7, 132.3, 133. 4, 136.1, 150.0, 154.0, 155.8, 166.5, 194.4; Anal. Calcd for C 22 H 11 ClF 3 N 5 O 3 S 2 : C, 49.49; H, 2.08, N, 13.12. Found: C, 49.66; H, 2.26; N, 12.75. 5 ((5 (2 Fluoro 3 (1 H tetrazol 5 yl)phenyl)furan 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolid in 4 one ( 2. 4 g ) Orange microcrystals (45%), mp 280 282 o C; 1 H NMR (DMSO d 6 ) 7.27 (s, 1H), 7.47 (d, J = 3.6 Hz, 1H), 7.69 (t, J =7.5 Hz, 1H), 7.76 7.88 (m, 3H), 7.90 8.00 (m, 2H), 8.08 (t, J =7.5 Hz, 2H); 13 C NMR (DMSO d 6 ) 114.8, 117.8, 120.8, 122.7, 125.4, 126.0 (2C), 128.5, 129.6, 130.2 (2C), 130.4, 133.2, 135.9, 149.5, 151 .0, 153.4, 156.8, 166.3, 194.3; Anal. Calcd for C 22 H 11 F 4 N 5 O 2 S 2 : C, 51.06; H, 2.14, N, 13.53. Found: C, 50.94; H, 2.40; N, 13.18. 2. 5 .3 General procedure for preparation of 2. 4 e, 2. 4 h A mixture of aldehyde 2. 5 e 2. 5 h (0.75 mmol), 2 thioxo 3 (3 (trifluorome thyl)phenyl)thiazolidin 4 one 2.10 (0.18 g, 0.75 mmol) and 2 drops of 2,2,6,6 tetramethylpiperidine in 7 mL of ethanol was heated under reflux for 8 h. After cooling to 40 45 o C, the solid obtained was filtered and washed with cold ethanol to give 2. 4 e 2. 4 h 2 Fluoro 3 (5 ((4 oxo 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 5 ylidene)methyl) furan 2 yl)benzoic acid mono hydrate ( 2. 4 e ) Brown microcrystals (44%), mp 220 250 o C (dec.); 1 H NMR (DMSO d 6 ) 7.32 (s, 1H), 7.50 (d, J = 3.4 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.83 7.90 (m, 3H), 7.95 8.00 (m, 3H), 8.15 (t, J = 6.5 Hz, 1H); 13 C NMR (DMSO d 6 ) 114.8, 114.9, 118.0, 118.2, 120.8, 121.0, 122.9, 125.4, 126.2, 126.4, 130.0, 130.6, 132.1, 133.4, 136.1, 149.5, 151.5, 159.1, 164.8, 166.5,

PAGE 45

45 194.5. Anal. Cal cd for C 21 H 11 F 4 NO 4 S 2 H 2 O: C, 52.59; H, 2.41, N, 2.79. Found: C, 52.63; H, 2.40; N, 2.58. 5 ((5 (4 Fluoro 3 (1 H tetrazol 5 yl)phenyl)furan 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one ( 2. 4 h ) Brown microcrystals (22%), mp 279 28 1 o C; 1 H NMR (DMSO d 6 ) 7.46 (d, J = 3.4 Hz, 2H), 7.70 7.86 (m, 5H), 7.91 7.98 (m, 2H), 8.11 (br s, 1H), 8.54 (d, J = 6.3 Hz, 1H); 13 C NMR (DMSO d 6 ) 100.1, 111.1, 118.0, 118.2, 118.3, 120.1, 121.9, 123.1, 125.8, 126.1, 126.4, 128.9, 129.8, 130.2, 130.6, 133.4, 136.1, 149.7, 1 56.1, 157.3, 160.7, 166.5, 194.4. Anal. Calcd for C 22 H 11 F 4 N 5 O 2 S 2 : C, 51.06; H, 2.14, N, 13.53. Found: C, 50.88; H, 2.01; N, 12.93. HPLC, 97.8% purity, Retention time: 2.75 min. 2. 5 .4 Synthesis of 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one (2. 10) A suspension of 2,2' (thiocarbonylbis(sulfanediyl))diacetic acid 2.11 (2.4 g, 10.6 mmol) and 3 (trifluoromethyl)aniline 2.12 (1.7 g, 10.6 mmol) was heated under reflux for 6h in water (60 mL). The formed precipitate was filtered off and washed with co ld water to afford 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one as yellowish microc rystals (52% yield), mp 175 176 o C, (lit. [56JACS384] 176 177 C); 1 H NMR (DMSO d 6 ) 4.38 (s, 2H), 7.64 (d, J = 7.8 Hz, 1H), 7.74 7.82 (m, 2H), 7.88 (d, J = 7.8 H z, 1H); 13 C NMR (DMSO d 6 ) 37.4, 123.9 (q, J = 270.9 Hz), 126.1 (q, J = 3.8 Hz), 126.3 (q, J = 3.7 Hz), 130.1 (q, J = 32.4 Hz), 130.7, 133.5, 136.5, 174.1, 203.9. 2. 5 .5 Preparation of 5 b rom opyrrole 2 carboxaldehyde (2.7) N N Diisopropylpyrrole 2 formimi nium chloride (2.15). A solution of N,N diisopropylformamide 2.14 (1.0g, 7.74 mmol) in anhydrous CH 2 Cl 2 (10 mL) was added dropwise at 0 o C, to a solution of oxalyl chloride (1.0 g, 7.74 mmol), in CH 2 Cl 2 over 15 min. After 20 min, a solution of pyrrole 2.13 (0.52 g, 7.74 mmol) in CH 2 Cl 2 was added

PAGE 46

46 dropwise with stirring at same tem perature over 15 min. period. T he reaction mixture was stirred at room temperature for 30 min, t he solvent removed and the residue was washed with dry acetone. The residue was recry stallized from dry ethyl acetate to give N N diisopropylpyrrole 2 formiminium chloride 2.15 (1.08 g, 65%), mp 205 207 o C, (lit. [90CJC1305] 212 213 C); 1 H NMR (DMSO d 6 ) 1.41 (d, J = 4.5 Hz, 12H), 4.35 (p, J = 6.6 Hz, 1H), 4.77 (p, J = 6.6 Hz, 1H), 6.69 6 .75 (m, 1H), 7.54 (s, 1H), 7.82 (s, 1H), 9.00 (s,1H); 13 C NMR (DMSO d 6 ) 18.7, 23.4, 51.3, 54.8, 107.0, 115.3, 117.2, 121.6, 124.1, 133.1, 150.9. (Diisopropylamino) (pyrrol 2 yl) acetonitrile (2.16). A solution of N,N diisopropylpyrrole 2 formiminium chloride salt 2.15 and NaCN (0.49 g, 10 mmol) in CH 3 CN (5 mL) was stirred for 12h at room temperature. After 12h, the solvent was evaporated under vacuum. Diethyl ether (20 mL) was added and solution washed with water (2 x 15 mL) and brine (10 mL). The o r ganic phase was dried over MgSO 4 After evaporat ion of the solvent the crude product was recrystallized from ether hexane with activated carbon to give pure 2 (diisopropylamino) 2 (1 H pyrrol 2 yl)acetonitrile 2.16 (250 mg, 61%), mp 110 111 o C, (lit. [90CJC 1305] 113 115 C); 1 H NMR (CDCl 3 ) 1.06 (dd, J = 6.6, 2.4 Hz, 6H), 1.26 (dd, J = 6.6, 2.4 Hz, 6H), 3.22 (dq, J = 6.6 Hz, J = 2.4 Hz, 2H), 4.96 (s, 1H), 6.19 (t, J = 2.7 Hz, 1H), 6.37 (s, 1H), 6.78 (s, 1H), 8.28 (bs, 1H) ; 13 C NMR (CDCl 3 ) 19.5, 23.8, 45.9 47.0, 108.0, 109.0, 118.4, 120.3, 126.5. 2 Bromo 6 diisopropylamino 1 azafulven (2.18). N Bromosuccinimide (1.04 g, 5.84 mmol) was added to a stirred solution of 2 (diisopropylamino) 2 (1H pyrrol 2 yl)acetonitrile 2.16 (1.20 g, 5.84 mmol) in dry THF (60 mL) at 78C. The reaction mixture was stirred a t this temperature for 5h, unti l the temperature reached ambient.

PAGE 47

47 T h e solvent was removed in vacuum and t he residue wa s dissolved in methanol (50 mL); 0.5 M HCl (50 mL) was added and after 20 min at room temp erature the deeply colored solutio n was poured into water (50 mL). After extraction with ether (discarded) the aqueous phase was adjusted to pH 12 with 10 wt.% NaOH solution. The mixture was extracted with ether. The extract was washed with brine solution and dried over sodium sulfate. After removing the solvent the residual solid was recrystallized from hexane ether to give pure 2 bromo 6 diisopropylamino 1 azafulven 2.18 (1.08 g, 72 % yield), mp 105 106 o C, (lit [90CJC1305] 110 112 C); 1 H NMR (CDCl 3 ) 1.30 1.37 (m, 12H), 3.78 3,88 (m, 1H), 6.31 (d, J = 3.60 Hz, 1H), 6.36 6,52 (m, 1H), 6.89 (d, J = 3.9 Hz, 1H), 7.01 (s, 1H) ; 13 C NMR (CDCl 3 ) 20.0, 24.0, 47.7, 51.7, 117.8, 133.9, 142.3. 5 Bromopyrrole 2 carboxaldehyde (2.7). 10 wt.% Sodium hydroxide so lution (10 mL) was added to a solution of the azafulvene 2.18 (0.20 g, 0.78 mmol) in methanol (40 mL) and stirred a t room temperature for 5 h. T he reaction mixture was acidified with 4 N HCI (50 mL) and the product was extracted with dichloromethane. Organ ic phase was washed with brine and dried over Na 2 SO 4 Solvent was evaporated under vacuum and the residue was recrystallized from ether hexane to give 5 bromo 1 H pyrrole 2 carbaldehyde (0.105g, 77% yield), mp 94 96 o C, (lit. [90CJC1305] 91 92 C); 1 H NMR (CD Cl 3 ) 6.33 (d, J = 3.3 Hz, 1H), 6.91 (d, J = 3.3 Hz, 1H), 9.37 (s, 1H), 10.14 (s, 1H) ; 13 C NMR (CDCl 3 ) 111.4, 114.0, 122.7, 134.0, 178.2. 2. 5 .6 Synthesis of 5 formyl 2 furylboronic acid (2.8) n BuLi (1.6 M in hexane, 3.9 mL, 6.2 mmol) was added dropwise to a solution of 2 furaldehyde diethyl acetal (1.0 g, 5.87 mmol) in anhydrous THF (15 mL) at 78 o C. The reaction mixture was stirred for 5 h at the same temperature, then trimethyl borate (1.0

PAGE 48

48 g, 9.6 mmol) was added dropwise via syringe. The reaction mixt ure was allowed to warm to room temperature while stirring overnight. After removing the solvent t he r emaining oil was diluted with water (10 mL). NaOH (5%) was added to mixture to adjust to pH around 10 and washed with diethyl ether (2 x 10 mL). The pH w as set to 4 by adding slowly concentrated HBr solution. The precipitate that was formed, filtered off and washed with diethyl ether to afford 5 formyl 2 furylboronic acid as yellowish microcrystals (55%), mp 130 132 o C, (lit. [03OBC1447] 151 152 C); 1 H NMR (Acetone d 6 ) 2.99 (s, 2H), 7.20 (d, J = 3.6 Hz, 1H), 7.40 (d, J = 3.6 Hz, 1H), 7.97 (d, J = 2.1 Hz, 2H), 9.71 (s, 1H); 13 C NMR (Acetone d 6 ) 37.4, 123.9 (q, J = 270.9 Hz), 126.1 (q, J = 3.8 Hz), 126.3 (q, J = 3.7 Hz), 130.1 (q, J = 32.4 Hz), 122.3, 123.5, 156.7, 179 .2. 2. 5 .7 General procedure for preparation of 3 tetrazolyl bromobenzenes (2.9 a c ) Dibutyltin oxide (45 mg, 10 mol %) was added to a mixture of 5 bromo 2/6 halobenzonitrile 2.20 a c (6.94 mmol) and trimethylsilyl azide (13.9 mmol) in toluene (70 mL). The mi xt ure was heated for 48 h. at 110 o C, then concentrated in vacuo. The residue was dissolved in methanol and reconcentrated. The residue was dissolved in ethyl acetate (50 mL) and washed with saturated NaHCO 3 (50 mL) solution. The aqueous phase was set to pH 2 with 4N HCl solution and then extracted with ethyl acetate (3 x 25 mL). The combined organic extracts were dried over Na 2 SO 4 filtered and the solvent evaporated to give 3 tetrazolyl bromobenzenes 2. 9 a c. 5 (5 Bromo 2 chlorophenyl) 1 H tetrazole ( 2. 9 a ). Yield: 82%; white microcrystals, mp 172 173 o C (ethyl acetate hexanes); 1 H NMR (DMSO d 6 ) 7.65 (d, J = 8.4 Hz, 1H), 7.80 7.85 (m, 1H), 8.04 (d, J = 2.1 Hz, 1H), 17.0 (br s, 1H); 13 C NMR (DMSO d 6 ) 120.3, 126.3, 131.5, 132.4, 133.9, 135.2.

PAGE 49

49 5 (3 Bromo 2 fluorophenyl) 1 H tetrazole ( 2. 9 b ). Yield: 75%; white microcrystals, mp 178 180 o C (ethyl ac etate hexanes); 1 H NMR (DMSO d 6 ) 7.42 (t, J = 8.6 Hz, 1H), 7.94 8.20 (m, 1H), 8.40 8.12 (m, 1H); 13 C NMR (DMSO d 6 ) 109.4, 109.7, 114.4, 114.6, 126.7, 126.8, 129.5, 136.3, 150.8, 153.6, 156.9. 5 (5 Bromo 2 fluorophenyl) 1 H tetrazole ( 2. 9 c ). Yield: 83%; brown microcrystals, mp 122 124 o C (ethanol); 1 H NMR (DMSO d 6 ) 7.47 7.55 (m, 1H), 7.82 7.90 (m, 1H), 8.18 8.24 (m, 1H); 13 C NMR (DMSO d 6 ) 117.2, 117.8, 118.1, 132.4, 135.2, 135.3, 156.8, 160.1.

PAGE 50

50 CHAPTER 3 S YNTHESIS OF SOME NOV EL FURAN OR 1 H PYRROLE CONT AINING HETEROCYCLIC COMPOUN DS 3.1 Introduction In the previous chapter, design, s ynthesis and anti HIV activity of 5 ((aryl furan/ 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones 2.4a h were discussed. Compounds 2.4a h al l exhibit anti HIV 1 activity, with 2.4f and 2.4g showing high potency against infection by laboratory adapted and primary HIV 1 strains with EC 50 at low nanomolar level, and in inhibiting HIV 1 m ediated cell cell fusion and gp41 six helix bundle formation The docking study was also able to explain the antiviral activity of the two active compounds 2.4f and 2.4g respect ively. The anti HIV 1 activity in the series of 2.4a h depends on structural features of the molecule. HIV 1 activity studies indicate that the replacement of carboxylic acid by tetrazolyl moiety as its bi oisostere significantly improves anti HIV 1 activity and results in significant decrease of cytotoxicity. In p articular, introduction of a pyrrole ring in the plac e of furan substantially re duces the anti HIV 1 activity ( 2.4a h ). So far in Chapter 2, some fragmental changes have been made in the 2.4a h frame e xcept the thiazolidine system. In fact, thiazolidinone ring systems were reported to hav e antimicrobial, cytotoxic [09EJMC2038] and an tiproli ferative [05BMCL3930] activity Therefore, a novel series of 2,5 disubstituted furans/pyrroles containing pyrrolidine 3.1a c thioxooxazolidin e 3.2 imidazolidine 3.3 or t hiazolo[3,2 b ][1,2,4]triazine 3.4 (Figure 3 1) were synthesize d since t hese c hanges might reduce the cytotoxic properties of 2.4a h

PAGE 51

51 Figure 3 1 Structure of compounds 2.4a h 3.1a c 3.2 3.3 and 3.4 3.2 Synthesis of Target Molecules (3.1a c, 3.2, 3.3, and 3.4) The intermediate molecules 2.5a 2.5b and 2.5i were prepared by Suzuki Miyaura cross coupling, as described in Chapter 2 (Figure 2 2). Target molecules 3.1a c were obtained by a Wittig reaction of corresponding the aldehydes, 2.5a 2.5b and 2.5i with a Wittig reagent 3.5 Compound 3.2 wa s synthesized by a Knoevenagel condensation under microwave irradiation from the aldehyde 2.5i and corresponding thioxooxazolidin, 3.10 The synthesis of 3.3 was obtain ed by condensation of aldehydes 2.5i with a

PAGE 52

52 hydantoin derivative 3.11 Compound 3.4 con taining a fused ring system, was synthesized by a multi component reaction in one pot from the corresponding aldehyde 2.5i All target compounds 3.1a c 3.2 3.3 and 3.4 were characterized by 1 H, 13 C NMR spectroscopy and elemental analysis. 3.2.1 Synthesis of 3 (5 ((2,5 dioxo 1 phenethylpyrrolidin 3 ylidene)methyl)furan / 1 H pyrrol 2 yl)benzoic acid (3.1a c) The first attempt to synthesize compound 3.1a was by the Knoevenagel condensation of the corresponding aldehyde 2.5a with N (2 phenylethyl)succinimide 3.5 in the presence of DIEA in toluene. Since no reaction was observed, the same reaction was tried in NaOAc AcOH buffered media. Nevertheless, the Knoevenagel condensation of 2.5a with N (2 phenylethyl)succinimide 3.4 was un successful (Scheme3 1). Scheme 3 1 Attempt to prepare 3.1a An alternative C=C bond formation reaction is the Wittig reaction [68T2241] (Scheme3 1). The treatments of aldehydes 2.5a 2.5b and 2.5i with Wittig reagent 3.6 at room temperature in dry THF gave 3.1a c ( 2, Table 3 1).

PAGE 53

53 Scheme 3 2. Synthesis of 3.1a c Wittig reagent 3.6 was prepared according to the outlined procedure in Scheme 3 3. N (2 Phenylethyl)maleimide 3.9 was obtained from maleic anhydride 3.7 usi ng a Dean Stark apparatus [73OS944]. Then, the nucleophilic addition of triphenylphosphine (PPh 3 ) to 3.9 in acetic acid gave Wittig reagent 3.6 in 72% yield [68T2241]. Table 3 1 Preparation of 2,5 disubstituted pyrrole/furans 3.1a c Product 3.1 R Y Rea ction time (h) Yield (%) a a H NH 8 39 b Cl NH 8 48 c Cl O 6 57 b a Isolated yields, b Prepared by an other group member. Scheme 3 3. Preparation of Wittig reagent 3.6

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54 3.2.2 Synthesis of 2 chloro 5 (5 ((4 oxo 3 phenethyl 2 thioxooxazolidin 5 ylidene)methyl)furan 2 yl)benzoic acid (3.2) It was a challenging task to prepare compound 3.2 because of the decreased stability of 2 thioxooxazolidin 4 one ring 3.10 under the Knoevenagel condensation condition. Several attempts we re mad e for instance, ethanol/TMP (80 o C), toluene/DIEA (120 o C), NaH/THF (room temp.), AcOH (130 o C), NaOAc/AcOH (130 o C). Attempts at room temperature resulted in no reaction; however, using elevated temperature caused decomposition of the 2 thioxooxazolidin 4 one ring 3.10 and left 2 chloro 5 (5 formylfuran 2 yl)benzoic acid 2.5i unreacted. Therefore, a reaction under microwave irradiation was suggested. 2 Chloro 5 (5 formylfuran 2 yl)benzoic acid 2.5i was treated with excess amount of 3 phenethyloxazolidine 2,4 dione 3.10 in the presence of acetic anhydride, acetic acid and sodium acetate in the microwave at 50 W. After complete reaction (1h, 120 o C), 2 chloro 5 (5 ((4 oxo 3 phenethyl 2 thioxooxazolidin 5 ylidene)methyl)furan 2 yl)benzoic acid 3.2 was obtaine d in 21% yield (Scheme 3 4). Scheme 3 4. Synthesis of 3.2 3.2.3 Synthesis of 2 chloro 5 (5 ((2,5 dioxo 1 phenethylimidazolidin 4 ylidene)methyl)furan 2 yl)benzoic acid (3.3) Another pharmacologically attractive ring syste m is hydantoin, which occurs in many natural products and exhibits a diverse range of biological activity such as

PAGE 55

55 anticonvulsant, neuroprotective, antiarrhythmic, antihypertensive, antidiabetic and analgesic properties [62JPS74] [04OPPI391]. Scheme3 5 Synthesis of 3.3 In the present study, 2 chloro 5 (5 formylfuran 2 yl)benzoic acid 2.5i was treated with 3 phenethylimidazolidine 2,4 dione 3.11 in the presence of pyrrolidine in ethanol. The desired product 3.3 was obtaine d in low yield (5%) after heating under reflux for several hours. Later, the reaction was catalyzed by hexamethyldisilazane, [(CH 3 ) 3 Si] 2 NH, activat ing hydantoin derivatives 3.11 as silyl ether s in situ and followed by condensation reaction to form 3.3 in b etter yield (75%). 3 p henethylimidazolidine 2,4 dione 3.1 1 was derives from hydantoin 3.1 2 by a nucleophilic substitution (Scheme 3 6). Scheme 3 6 Synthesis of 3.11 3.2.4 Synthesis of 2 chloro 5 (5 ((3,7 dioxo 6 phenyl 3, 7 dihydro 2 H thiazolo[3,2 b ][1,2,4]triazin 2 ylidene)methyl)furan 2 yl)benzoic acid (3.4) Another idea to increase the stability of the ring 2 thioxothiazolidin 4 ones in 2.4a h was to fuse it with another ring system, 1,2,4 triazine 5 (4 H ) one. In the lit erature [05SC333], the some t hiazolo[3,2 b ][1,2,4]triazine derivatives were obtained by a multicomponent reaction, including appropriate aldehyde, 3 mercapto 5 triazinone and monochloroacetic acid in acetic acid and acetic anhydride in the presence of sodi um

PAGE 56

56 acetate. 2 Chloro 5 (5 formylfuran 2 yl)benzoic acid 2.5i was treated with 3 mercapto 6 phenyl 1,2,4 triazin 5(4 H ) one 3.14 and chloroacetic acid 3.15 in the presence of sodium acetate in acetic acid and acetic anhydride to afford 2 chloro 5 (5 ((3,7 di oxo 6 phenyl 3,7 dihydro 2 H thiazolo[3,2 b ][1,2,4]triazin 2 ylidene)methyl)furan 2 yl)benzoic acid 3.4 in 69% yield (Scheme 3 7). Scheme 3 7. Synthesis of 3.4 3 Mercapto 6 phenyl 1,2,4 triazin 5(4 H ) one 3.14 was prepared in 81% yield according to a literature [84LAC1302] procedure by a reaction of phenylglyoxalic acid 3.16 and thiosemicarbazid e 3.17 (Scheme 3 8). Scheme 3 8. Preparation of 3.14 3.3 Conclusion A novel series of 2,5 disubs tituted furan / 1 H pyrrole containing pyrrolidine 3.1a c thioxooxazolidin 3.2 imidazolidine 3.3 t hiazolo[3,2 b ][1,2,4]triazine 3.4 derivatives were synthesized by Knoevenagel condensation or Wittig reactions.

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57 3.4 Experimental Section Melting points we re determined using a capillary melting point apparatus equipped with a digital thermometer and are uncorrected. 1 H (300 MHz) and 13 C (75 MHz) NMR spectra were recorded in DMSO d 6 (with tetramethylsilanes as the internal standard), unless otherwise stated. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br s = broad singlet, m = multiplet), coupling constants ( J values) expressed in Hz. Elemental analyses were performed on a Carlo Erba EA 1108 instrument. The solvents (ethylene glycol dimethyl ether (DME) and THF) were dried by the usual methods and distilled before use. 3.4.1 General synthesis of 3 (5 ((2,5 dioxo 1 phenethylpyrrolidin 3 ylidene)methyl) 1 H pyrrol / furan 2 yl )benzoic acid (3.1 a c) A mixture of aldehyde compound 2.5a, 2.5b, 2.5i (0.2 mmol) and pho s phorous ylide 3.5 (0. 2 mmol) was stirred for 24 h in dry THF at room temperature. The precipitate formed was filtered and washed with CH 2 Cl 2 to give pure 3.1a c 3 (5 ((2,5 Dioxo 1 phe nethylpyrrolidin 3 ylidene)methyl) 1 H pyrrol 2 yl)benzoic acid (3.1a) Yellowish microcrystals (39%), mp 292 293 o C; 1 H NMR (DMSO d 6 ) 2.85 (t, J = 7. 7 Hz, 2H), 3.47 (s, 2H), 3.70 (t, J = 7.7 Hz, 2H), 6.45 (s, 1H), 6.8 8 (s, 1H), 7.16 7.36 (m, 5H), 7.46 (s, 1H), 7.5 4 (t, J =7.8 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 8.31 (s, 1H), 12.06 (s, 1H), 13.09 (br s, 1H); 13 C NMR (DMSO d 6 ) 33.2, 33.7, 39.3, 110.1, 114.8, 118.3, 122.1, 124.5, 126.4, 127.5, 128.4, 128.6, 129.2, 129.6, 131.7, 131.8, 134.3, 138.3, 167.2, 170.3, 174.0; Anal. Calcd for C 24 H 20 N 2 O 4 : C, 71.99; H, 5.03, N, 7.00. Found: C, 72.34; H, 5.08; N, 6.85. 2 C hloro 5 (5 ((2,5 dioxo 1 phenethylpyrrolidin 3 ylidene)methyl) 1 H pyrrol 2 yl)benzoic acid (3.1b). Yellowish microcrystals (48%), mp 286 287 o C; 1 H NMR

PAGE 58

58 (DMSO d 6 ) 2.84 (t, J = 6.9 Hz, 2H), 3.46 (s, 2H), 3.69 (t, J = 8.4 Hz, 2H), 6.63 (s, 1H), 6.89 (s, 1H), 7.15 7.3 6 (m, 5H), 7.44 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 7.5 Hz, 1H), 8.11 (s, 1H), 12.17 (s, 1H); 13 C NMR (DMSO d 6 ) 33.2, 33.8, 39.3, 110.6, 114.8, 118.7, 122.0, 125.7, 126.5, 127.7, 128.5, 128.6, 129.2, 130.0, 131.1, 132.4, 133.1, 138.3 166.8, 170.3, 174.1; HRMS (ESI) [M+Na] + : Found: 457.0936, calculated for C 24 H 19 ClN 2 O 4 : 457.0926. 2 Chloro 5 (5 ((2,5 dioxo 1 phenethylpyrrolidin 3 ylidene)methyl)furan 2 yl)benzoic acid (3.1c). Yellowish microcrystals (57%), mp 204 206 o C; 1 H NMR (DMSO d 6 ) 2.93 (t, J = 7.8 Hz, 2H), 3.75 3.80 (m, 4H), 7.12 (d, J = 3.9 Hz, 1H ) 7.22 7.35 (m, 6H), 7.65 (d, J = 8.4 Hz, 1H), 7.86 (br s, 1H), 7.99 (d, J = 8.1 Hz, 1H), 8.23 (s, 1H); 13 C NMR (DMSO d 6 +Aceton d 6 ) 34.07, 110.6, 119.3, 119.8, 123.4, 127.1, 128.3, 12 9.2, 129.4, 132.2, 132.6, 133.3, 139.2, 152.0, 155.2, 167.0, 170.7; Anal. Calcd for C 24 H 18 ClNO 5 : C, 66.14; H, 4.16, N, 3.21. Found: C, 65.82; H, 4.17; N, 2.96. 3.4.2 Synthesis of 5 o xo 1 phenethyl 3 (triphenylphosphonio) 4,5 dihydro 1 H pyrrol 2 olate (3.6) A mixture of N (2 p henylethyl)maleimide 3.9 (300 mg, 1.65 mmol) and triphenyphosphine (460 mg, 1.65 mmol ) w as stirred in acetic acid (5 mL) for 24 h at 100 o C. Then the reaction mixture cool ed to room temperature, and the solvent was evaporated. Diethyl ether (8 mL) was added and to the residue was stirred for 20 min. at 40 o C. A white precipitate was collected and washed with diethyl ether to afford 1 phenethyl 3 (triphenylphosphoranylidene)pyrrolidine 2,5 dione as y ellowish microcrystals (500 mg, 72% y ield) m p 155 160 o C; 1 H NMR (DMSO d 6 ) 2.83 (t, J = 7.2 Hz, 2 H), 2.87 (s, 2H), 3.57 (t, J = 7.2 Hz, 2H), 7.16 7.34 (m, 5H), 7.48 7.64 (m, 12 H), 7.66 7.74 (m, 3H); 1 H NMR (TFA d ) 2.55 2.65 (m, 2H), 3.11 (t, J = 18.9 Hz, 1H),

PAGE 59

59 3.59 (dd, J =19.2, J = 9.3 Hz, 1H), 3.69 (t, J = 7.2 Hz, 2H), 6.9 6 7.04 (m, 2H), 7.14 7.24 (m, 3H), 7.64 7.82 (m, 12H), 7.84 7.96 (m, 3H); 13 C NMR (TFA d ) 33.7, 35.0, 43.9, 117.2, 118.3, 129.4, 130.5, 131.0, 133.1, 133.3, 135.9, 136.1, 138.6, 138.8, 138.9, 173.8, 178.8; Anal. Calcd for C 30 H 26 NO 2 P: C, 77.74; H, 5.65, N 3.02. Found: C, 77.40; H, 5.76; N, 3.01. 3.4.3 Synthesis of N (2 p henylethyl)maleimide (3.9) Maleic anhydride (1.08 g, 11 mmol) and 2 phenylethylamine (1.21 g, 10 mmol) were stirred for 2 h in diethyl ether at room temperature. The precipitate that for med was filtered and washed with water and diethylether to afford N phenylethyl maleamic acid. After N phenylethyl maleamic acid was dried under vacuum, it was dissolved in toluene (20 mL) and p TsOH.H 2 O (3.8 g, 20 mmol) was added The reaction mixture was heated under reflux for 5h using Dean Stark apparatus. After cool ing to room temperature the solvent was evaporated. The residue was dissolved in EtOAc (25 mL) and washed with 1N NaHCO 3 and water. The o rganic layer was dried over MgSO 4 and the solvent wa s evaporated under vacuum. The crude product was recrystallized from EtOAc hexanes to give N phenyethyl maleimide as light y ellowish microcrystals (1.67 g, 83% overall yield) mp 102 103 o C (lit. [05JMC7503] 109 110 o C); 1 H NMR (CDCl 3 ) 2.89 (t, J = 7. 8 Hz, 2H), 3.75 (t, J = 7.8 Hz, 2H), 6.64 (s, 2H), 7.15 7.40 (m, 5H).; 13 C NMR (CDCl 3 ) 34.7, 39.3, 126.9, 128.7, 129.0, 134.2, 138.0, 170.7. 3.4.4 Synthesis of 2 chloro 5 (5 ((4 oxo 3 phenethyl 2 thioxooxazolidin 5 ylidene)methyl)furan 2 yl)benzoic acid (3.2) A mixture of 3 phenethyl 2 thioxooxazolidin 4 one 3.10 (70 mg, 0.3 mmol), 2 chloro 5 (5 formylfuran 2 yl)benzoic acid 2.5i (50 mg, 0.2 mmol) and NaOAc (150 mg) in acetic anhydride (1mL) and acetic acid (1 mL) was stirred for 1h und er microwave (50

PAGE 60

60 Watt) at 120 o C. The reaction mixture was poured onto crushed ice and stirred for 3 h. The brown solid formed was filtered and recrystallized from methanol to give the product 3.2 as brownish microcrystals (26 mg, 20% yield ), mp 187 189 o C; 1 H NMR (DMSO d 6 ) 3.01 (t, J = 7.5 Hz, 2H), 4.0 1 (t, J = 7. 5 Hz, 2H), 6.99 (s, 1H), 7.18 7.3 6 (m, 6H), 7. 40 7.47 (m, 1 H), 7.7 1 (d, J = 8.4 Hz, 1H), 7.90 7.9 8 (m, 1H), 8. 18 8.22 (m, 1H); 13 C NMR (DMSO d 6 ) 31.9, 43.7, 100.8, 111.1, 121.3, 126.0, 126.7, 12 7.5, 128.0, 128. 5 128.7, 131.6, 132.5, 136.7, 137.5, 147.5, 154.7, 161.1, 166.3, 172.0, 182.7; Anal. Calcd for C 23 H 16 ClNO 5 S: C, 60.86; H, 3.55, N, 3.09. Found: C, 60.63; H, 3.86; N, 3.26. 3.4.5 Synthesis of 3 p henethyloxazolidine 2,4 dione (3.10) Carbon disulfide (2.6 g, 0.04 mol) was added to a mixture of glycol ic acid (2.5 g, 0.032 mol), KOH (4.5 g, 0.08 mol) and water (5 mL). The solution was stirred for 24 h at room temperature. Then, acetonitrile (5 mL) and benzyl chloride (5 g, 0.04 mol) were added. The reaction mixture was heated under refl ux for 5 hours. The precipitate formed was filtered and washed with benzene until it turned white The precipitate was dissolved in water (30 mL), and 2 phenylethylamine (4.0 g, 0.032 mol) was added. After stirr in g at room temperature for 3 h, oil was formed as a by product. Concentrated sulfuric acid was added to the aqueous layer until pH 1, when a precipitate formed during acidification. The precipitate was filtrat ed and it was dissolved in ethyl acetate (30 mL) and washed with saturated Na 2 CO 3 and brine. After it dried over MgSO 4 solvent was evaporated. The solid was recrystallized from EtOAc hexanes to give 3 phenethyl 2 thioxooxazolidin 4 one as yellowish microcrystals (4.6 g, 65% yield), mp 95 96 o C (lit. [81 JP56034675] 110 111 o C); 1 H NMR (CDCl 3 ) 3.00 (t, J = 7.8 Hz, 2H), 4.00 (t, J = 9.3 Hz, 2H), 4.70 (s, 2H), 7.05 7.60 (m, 5H); 13 C NMR (CDCl 3 ) 32.7, 44.1, 69.8, 127.1, 128.8, 129.0, 137.1, 170.8, 190.6.

PAGE 61

61 3.4.6 Synthesis of 2 chloro 5 (5 ((2,5 dioxo 1 phenethylimidazolidin 4 ylidene)methyl)furan 2 yl)benzoic acid (3.3) A mixture of the corresponding aldehyde 2.5i (50 mg, 0.2 mmol) and 3 phenethylimidazolidine 2,4 dione 3.1 1 (45 mg, 0.2 mmol) in hexamethyldisilazane [(CH 3 ) 3 Si] 2 NH (3mL) was stirred for 1h at 90 o C. After cooling to room temperature pyrrolidine (0.5 mL) was added and stirring continued for 4h at 90 o C. T he solvent was evaporated under vacuum to give an oily residue. The residue was diluted with water (5 mL) and acidified w ith concentrated HCl until pH 1 while a precipitate formed. Th e precipitate was collected, filtered and washed with water to give crude product. The crude was crystallized from methanol to give 3.3 as brown microcrystals (65 mg, 75% yield) mp 204 205 o C; 1 H NMR (DMSO d 6 ) 2.92 (t, J = 7.2 Hz, 2H), 3.74 (t, J = 7.2 H z, 2H), 6.46 (s, 1H), 7.11 (d, J = 3.6 Hz, 1H), 7.16 7.24 (m, 3H), 7.27 (s, 1H), 7.28 7.34 (m, 2H), 7.5 8 7.66 (m, 1H), 8.0 4 8.1 4 (m, 1H), 8.25 (s, 1H), 10.71 (s, 1H), 13.60 (s, 1H); 13 C NMR (DMSO d 6 ) 33.2, 33.7, 110.5, 114.7, 118.7, 121.9, 125.6, 126. 4 127.7, 128.4, 128.6, 129.2, 129.9, 130.4, 131.1, 132.4, 133.1, 138.3, 166.7, 170.3, 174.0; Anal. Calcd for C 23 H 17 ClN 2 O 5 : C, 63.24; H, 3.92, N, 6.41. Found: C, 62.93; H, 3.91; N, 6.14. 3.4.7 Synthesis of 3 phenethylimidazolidine 2,4 dione (3.11) Phenylethy l bromide 3.13 (1.85 g, 10 mmol) was added to a solution of hydantoin 3.12 (1 g, 10 mmol) and K 2 CO 3 (1.38 g, 10 mmol) in DMF (10 mL) The mixture was stirred at 90 o C for 10 h. Solvent was evaporated under reduced pressure. The residue was poured onto ice w ater mixture and stirred for 1h at room temperature.. The precipitate that formed was collected by filtration The solid was recrystallized from ethanol to give 3 phenethylimidazolidine 2,4 dione as white microcrystals 3.11 (1.75 g, 82 % yield), ), mp 145 147 o C (lit. 1 47 1 48 o C [6 1 JSIR145]); 1 H NMR (DMSO d 6 ) 2.84

PAGE 62

62 (t, J = 7.2 Hz, 2H), 3.58 (t, J = 7.2 Hz, 2H), 3.86 (s, 2H), 7.02 7.46 (m 5H), 8.04 (s, 1H); 13 C NMR (DMSO d 6 ) 32.8, 33.9, 42.2, 126.6, 128.4, 128.6, 137.8, 171.8. 3.4.8 Synthesis of 2 c hloro 5 (5 ((3,7 dioxo 6 phenyl 3,7 dihydro 2 H thiazolo[3,2 b ][1,2,4]triazin 2 ylidene)methyl)furan 2 yl)benzoic acid (3.4) A mixture of mercapto triazinone 3.14 (125 mg, 0.5 mmol), chloroacetic acid 3.15 (100 mg, 1.0 mmol), anhydrous sodium acetate (200 mg), glacial acetic acid (2 mL), acetic anhydride (1.5 mL), and 2 chloro 5 (5 formylfuran 2 yl)benzoic acid 2.5i (105 mg, 0.05 mmol) was heated to reflux for 6 h. The reaction mixture was cooled and poured o nto crushed ice with vigorous stirring. The solid obtained was filtered, washed with water, and dried under vacuum. The product was recrystallized from ethanol to obtain pure 2 c hloro 5 (5 ((3,7 dioxo 6 phenyl 3,7 dihydro 2 H thiazolo[3,2 b ][1,2,4]triazin 2 ylidene)methyl)furan 2 yl)benzoic acid 3.4 (165 mg, 69% yield) mp >300 o C; 1 H NMR (DMSO d 6 ) 7.45 (s, 1H), 7.48 7.62 (m, 4H), 7.70 (dd, J = 8.4, 1.5 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 6.9 Hz, 2H), 8.11 (d, J = 1.5 Hz, 1H), 8.2 4 (s, 1H), 13.69 (br s, 1H) ; 13 C NMR (DMSO d 6 ) 111.6, 113.3, 123.3, 124.2, 126.4, 127.1, 127.9, 128.2 (2C), 129.0 (2C), 1 31.1, 131.4, 131.8, 132.2, 148.8, 149.2, 156.2, 158.9 (2C), 163.9, 165.9; Anal. Calcd for C 23 H 12 ClN 3 O 5 S: C, 57.81; H, 2.53, N, 8.79. Found: C, 57.95; H, 2.60; N, 8.40. 3.4.9 Synthesis of 3 m ercapto 6 phenyl 1,2,4 triazin 5(4 H ) one (3.14) A mixture of phen ylglyoxalic acid 3.16 (1.83 g, 12 mmol), thiosemicarbazite 3.17 (1.14 g, 12.6 mmol) and KHCO 3 (2.40 g, 24 mmol) w as boiled in water (30 mL). After 5 h, the reaction mixture was cooled to room temperature and acidified with 6N HCl until pH 3. The formed pre cipitate was collected and recrystallized from EtOH water to afford 3 mercapto 6 phenyl 1,2,4 triazin 5(4 H ) one 3.14 as white microcrystals (2.0 g, 81%

PAGE 63

63 yield), mp 237 239 o C (Lit. [84LAC1302] 260 264 o C) ; 1 H NMR (DMSO d 6 ) 7.42 7.54 (m, 3H), 7.90 7.98 (m, 2 H), 13.26 (s, 1H), 13.70 (s, 1H); 13 C NMR (DMSO d 6 ) 128.1 (4C), 130.0, 131.7, 145.6, 152.7, 173.1. Anal. Calcd for C 9 H 7 N 3 OS: C, 52.67; H, 3.44, N, 20.47. Found: C, 52.38; H, 3.43; N, 20.43.

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64 CHAPTER 4 SUM M ARY OF ACHIEVEMEN T S In the present study, novel 5 ((aryl furan / 1 H pyrrol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones and 2,5 disubstituted furan / 1 H pyrrole containing pyrrolidine thioxooxazolidin e imidazolidine and t hiazolo[3,2 b ][1,2,4]triazine derivatives were synt hesized by Knoevenagel condensation or Wittig reaction (yields 21 75%) Intermediate compounds 2 aryl 5 formyl furans and 1 H pyrroles were obtained by using Suzuki Miyaura coupling reaction ( yields 29 86 % ). A nti HIV 1 activities of 5 ((aryl furan / 1 H pyr rol 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 ones were tested. All tested compounds showed potent anti HIV 1 activity. T he compounds 5 ((5 (4 chloro 3 (1 H tetrazol 5 yl)phenyl)furan 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl )phenyl)thiazolidin 4 one and 5 ((5 (2 fluoro 3 (1 H tetrazol 5 yl)phenyl)furan 2 yl)methylene) 2 thioxo 3 (3 (trifluoromethyl)phenyl)thiazolidin 4 one showed the best anti HIV 1 activity, with EC 50 of 18 and 14 nM and SI of 3,686 and 1,989, respectively. A s detected by CD spectroscopy, helicity of the N46/C34 complex was significantly decreased ( 69 75 % ) in the presence of both highly active compounds. The docking studies of the compounds above were also able to ra tionalize the antiviral activities.

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65 LIST OF REFERENCES The reference citation system employed throughout this thesis is from Katritzky, A. R.; Rees, C. W.; Scriven, E.). Each time a reference is cited, a number letter code is designated to the corresponding reference with the first two or four if the of the journal and the page number in the end. Additional notes to this re ference system are as follow: 1) Journals which are published in more than one part including in abbreviation cited the appropriate part. 2) Less commonly used books and journals are still abbreviated as using initials of the journal name. 3) Patents are given by t heir application number. 4) The list of the references arranged according to the designated code in the order of (i) year, (ii) journal/book in alphabetical order, (iii) part number or volume number if it is included in the code, and (iv) page number. REFEREN CES 56JACS384 F. C. Brown, C. K. Bradsher, E. C. Morgan, M. Tetenbaum, and P. Wilder, J. Am. Chem. Soc. 78 384 (1956) 62JPS74 H. H. Wolf, E. A. Swinyard, and L. S. Goodman J. of Pharm. Sci., 51 74 ( 1962). 61 JSIR145 D. N. Dhar, S. P. Popli and M. L. Dhar, J. Sci. Ind. Res., 20C 145 (1961). 68T2241 E. Hedaya, and S. Theodoropulos, Tetrahedron 24 2241 (1967). 73OS944 M. N. Cava, A. A. Deana, K. Muth, and M. Mitchell, Org Synt Coll ., 5 944 (1973)

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69 BIOGRAPHICAL SKETCH Ilker Avan was born and raised i n Eskisehir, Turkey. He re ceived his Master of Science degree from Anadolu University in August 2006. During his study, he worked as graduate assistant in Chemistry Department at the same university. In 2008, he joined the research group of Professor A. R. Katritzky at University o f Florida as a research scholar. He received his second Master of Science degree from the University of Florida in August 2011. His research focuses on the synthesis of drug candidates in heterocyclic systems and peptidomimetic compounds in synthetic organ ic chemistry.