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Heterocycle-Assisted Synthesis of Biologically Significant Systems

Permanent Link: http://ufdc.ufl.edu/UFE0024386/00001

Material Information

Title: Heterocycle-Assisted Synthesis of Biologically Significant Systems
Physical Description: 1 online resource (130 p.)
Language: english
Creator: Meher, Geeta
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aminothiatriazoles, arginine, benzotriazole, cxcr3, dipeptides, glioma, lysine, nbi, peptides, rgd, tripeptides
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The thesis is concerned with synthesis of lysine and arginine containing peptides, 5-(substituted amino)-1,2,3,4-thiatriazoles and 8-azaquinazolinone analogue, NBI-74330. Lysine and arginine are essential alpha-amino acids and are constituent of various biologically active peptides. 5-(Substituted amino)-1,2,3,4-thiatriazoles show interesting biological properties. NBI-74330 is the CXCR3 receptor antagonist. Benzotriazole methodology enables convenient syntheses of natural and unnatural di- and tripeptides derived from L-lysine by extension at N(alpha)-, N(epsilon)-, and/or C- termini, under mild reaction conditions. Extensions at N-termini have been carried out by acylation with N-(Cbz-or Fmoc-alpha-aminoacyl)benzotriazoles or N-protected dipeptidoylbenzotriazoles. Extensions at C-terminii were performed similarly by coupling the N-protected acylbenzotriazole derivative of lysine and its dipeptides with unprotected alpha-amino acids. These reactions are performed in aqueous acetonitrile solution, require short reaction times; involve simple work up procedures and form enantiopure products in high yields. Arginine containing di- and tripeptides are synthesized by chain elongation at either the N- or C-terminus of N(omega)-L-nitroarginine or L-arginine. Coupling of N(omega)-L-nitroarginine with N-(Cbz-or Fmoc-alpha-aminoacyl)benzotriazoles or N-protected dipeptidoylbenzotriazoles form di- or tripeptides in good yields. The acylbenzotriazole derivative of Cbz(alpha)-N(omega)-L-nitroarginine is coupled with free amino acids to obtain the corresponding dipeptides. All isolated peptides were enantiopure and obtained in good yields. The protected RGD peptide sequence was synthesized using benzotriazole methodology. A convenient one-pot protocol for the synthesis of 5-(substituted amino)-1,2,3,4-thiatriazoles from bis(1H-benzotriazol-1-yl)methanethione is described. Reactions of amines or amino acid esters with bis(1H-benzotriazol-1-yl)methanethione followed by addition of aqueous sodium azide at room temperature resulted with formation of corresponding 5-(monosubstituted amino)-1,2,3,4-thiatriazoles in 73-97% yields, in short reaction times, without the use of column chromatographic purification. The CXCR3 receptor antagonist NBI-74330 was synthesized and tested for its biological activity by Dr. Jeffrey K. Harrison group for treatment of malignant gliomas.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 Geeta Meher.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Katritzky, Alan R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024386:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024386/00001

Material Information

Title: Heterocycle-Assisted Synthesis of Biologically Significant Systems
Physical Description: 1 online resource (130 p.)
Language: english
Creator: Meher, Geeta
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aminothiatriazoles, arginine, benzotriazole, cxcr3, dipeptides, glioma, lysine, nbi, peptides, rgd, tripeptides
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The thesis is concerned with synthesis of lysine and arginine containing peptides, 5-(substituted amino)-1,2,3,4-thiatriazoles and 8-azaquinazolinone analogue, NBI-74330. Lysine and arginine are essential alpha-amino acids and are constituent of various biologically active peptides. 5-(Substituted amino)-1,2,3,4-thiatriazoles show interesting biological properties. NBI-74330 is the CXCR3 receptor antagonist. Benzotriazole methodology enables convenient syntheses of natural and unnatural di- and tripeptides derived from L-lysine by extension at N(alpha)-, N(epsilon)-, and/or C- termini, under mild reaction conditions. Extensions at N-termini have been carried out by acylation with N-(Cbz-or Fmoc-alpha-aminoacyl)benzotriazoles or N-protected dipeptidoylbenzotriazoles. Extensions at C-terminii were performed similarly by coupling the N-protected acylbenzotriazole derivative of lysine and its dipeptides with unprotected alpha-amino acids. These reactions are performed in aqueous acetonitrile solution, require short reaction times; involve simple work up procedures and form enantiopure products in high yields. Arginine containing di- and tripeptides are synthesized by chain elongation at either the N- or C-terminus of N(omega)-L-nitroarginine or L-arginine. Coupling of N(omega)-L-nitroarginine with N-(Cbz-or Fmoc-alpha-aminoacyl)benzotriazoles or N-protected dipeptidoylbenzotriazoles form di- or tripeptides in good yields. The acylbenzotriazole derivative of Cbz(alpha)-N(omega)-L-nitroarginine is coupled with free amino acids to obtain the corresponding dipeptides. All isolated peptides were enantiopure and obtained in good yields. The protected RGD peptide sequence was synthesized using benzotriazole methodology. A convenient one-pot protocol for the synthesis of 5-(substituted amino)-1,2,3,4-thiatriazoles from bis(1H-benzotriazol-1-yl)methanethione is described. Reactions of amines or amino acid esters with bis(1H-benzotriazol-1-yl)methanethione followed by addition of aqueous sodium azide at room temperature resulted with formation of corresponding 5-(monosubstituted amino)-1,2,3,4-thiatriazoles in 73-97% yields, in short reaction times, without the use of column chromatographic purification. The CXCR3 receptor antagonist NBI-74330 was synthesized and tested for its biological activity by Dr. Jeffrey K. Harrison group for treatment of malignant gliomas.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 Geeta Meher.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Katritzky, Alan R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024386:00001


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1 HETEROCYCLE -ASSISTED SYNTHESIS OF BIOLOGICALLY SIGNIFICANT SYSTEMS By GEETA MEHER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF D OCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Geeta Meher

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3 To my parents, my daughter Prachi Meher and my husband, Nabin K. Meher without their love and support it would never had been possible

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4 ACKNOWLEDGMENTS I would like to express my deep sense of gratit ude to my advisor, Prof. Alan Roy Katritzky for his constant encouragement and guidance. I would like to thank the me mbers of my committee Dr. Lisa McElweeWhite, Dr Sukwon Hong, Dr. Ion Ghivi riga and Dr. Hartmut D erendor f for their advices and availability. I am very grateful to Dr. Benjamin Smith, graduate coordinator, for giving me opportunity to study in the department of chemistry, and Lori Clark for all the guidance and their tireless as sistance throughout my graduate program. I would like to thank Dr. Jeffrey K. Harrison from Department of Pharmacology and Therapeutics, University of Florida for his collaboration and valuable suggestions. I would like to thank Dr. Parul Angrish, Dr. Tam ari Narindoshvi li, Dr. C. Dennis Hall for their useful suggestions during my research work Many thanks to P rof. Katritzky research group members, friends and colleagues : M r. Zouquan Wang, Dr. Kou, Dr. Prabhu Mohapatra, Dr. Minati Ku a na r, Dr. Sha ilendra S ingh, Dr. Neelam Bharti, Dr. Srinivasa R. Tala Dr. Rajeev Sakhuja, Dr. Kiran Bajaj, Dr. Anamika Singh, Dr. Maia Tsikolia, Dr. Ekatrina Todadze, Dr. Kapil Gyanda, Reen a Gyanda Janet Cusido, Megumi Yoshioka, Davit Jishkariani, Danni e belle Hasse, Longchuan Huang ( Kristin ), Bogdan Dragichi, Bahaa El -Dien El Gendy, Claudia El Nachef, Mirna El Khatib, Mrs. Elizabeth Sheppa rd and Mrs. Galina Vakulenko. I would like to thank my best friend Dr. Namrata Rastogi for her support and encouragement. I am very deeply i ndebted to my daughter s loving babysitter Mrs. Sai for taking excellent care of her during my work time. I would like to express my deep love for my little daughter Prachi Meher for being very patient and cooperative throughout my work and sleeping we ll in nights.

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5 I want to express my profound sense of emotion for my parents, sisters, brother and all the rest members of my family. In the end, with all my love, I would like to thank my husband, Dr. Nabin K. Meher for his immense love, unlimited patien ce and unconditional support in my life. His presence in my life is truly a blessing of Almighty Father I would never have been able to achieve this goal without his presence in my life.

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6 TABLE OF CONTENTS ACKNOWLED GMENTS .................................................................................................................... 4 page LIST OF TABLES .............................................................................................................................. 10 LIST OF FIGURES ............................................................................................................................ 11 LIST OF SCHEMES .......................................................................................................................... 12 LIST OF ABBREVIATIONS ............................................................................................................ 15 ABSTRACT ........................................................................................................................................ 18 CHAPTER 1 BENZOTRIAZOLE: P ROPERTIES AND SYNTHETIC UTILITY ..................................... 20 1.1 Benzotriazole and its Properties ........................................................................................... 20 1.2 Synthetic Utility of Benzotriazole ....................................................................................... 21 1.2.1 N-Acylbenzotriazoles and its application in Acylation and Peptide Synthesis ... 21 1.2.2 Bis(1H -Benzotriazol 1 yl)methanethione and its A pplication in Thiocarbamoylation and Related Reactions ....................................................... 26 2 SYNTHESIS OF PEPTIDES BY EXTENTIONS AT THE N OR C TERMINII OF LYSINE ....................................................................................................................................... 29 2.1 Introduction ........................................................................................................................... 29 2.2 Results and Discussion ......................................................................................................... 32 2.2.1 Preparation of Dipeptides 2.3a -g and the Diastereomeric Mixtures (2. 3a+2.3a' ), (2. 3d+2.3d' ) Using the NTerminus of NCbz L -Lys 2.2a ...... 32 2.2.2 Preparation of Unnatural Dipeptides 2.3h -j Using NTerminus of N-Cbz L Lys 2.2b ............................................................................................................. 35 2.2.3. Preparation of Dipeptides 2.3k n and the Diastereomeric mixture (2.3l+2.3l' ) by Chain Elongation at the C Terminus of Lysine Using NFmoc NCbz L -Lys Bt 2.1h ............................................................................... 36 2.2.4. Preparation of N -Protected Dipeptidoylbenzotriazoles 2.5a -c and the Diastereomeric Mixture ( 2.5b+2.5b' ) from N -Protected Dipeptides 2.4a,b, 2.3b and ( 2.4b+2.4b' ) ........................................................................................... 37 2.2.5 Preparation of Tripeptides 2.6a -e and the Diastereomeric Mixtures (2.6b+2.6b' ), (2.6e+2.6e' ) .................................................................................... 38 2.2.6. Preparation of Unnatural Tripeptide 2.6f by Extension from the NTerminus of NCbz L -Lys 2.2b .......................................................................... 40 2.3 Conclusion ............................................................................................................................. 41 2.4 Experimental Section ............................................................................................................ 41 2.4.1 General Methods ...................................................................................................... 41

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7 2.4.2 General Procedure for the Preparation of Cbz DL -Met Bt (2. 1d+ 2. 1d' ) and N-Fmoc NCbz L -Lys -Bt 2. 1h .......................................................................... 41 2.4.3 General Procedure for the Preparation of LLDipeptides 2.3a -g and the Diastereomeric Mixture ( 2.3b+2.3b' ) and ( 2.3d+2.3d' ) .................................... 42 2.4.4 General Procedure for the Preparation of unnatural LLDipeptides 2.3h -j and the Diastereomeric Mixture ( 2.3h+2.3h' ) .................................................... 47 2.4.5 General Procedure for the Preparation of LLDipeptides 2.3k -n and the Diastereomeric Mixture ( 2.3l+2.3l' ) from Extension at the C Terminus of Benzotriazole derivative, N-Fmoc -N-Cbz -L Lys Bt 2. 1h ............................... 49 2.4.6 General Procedure for Preparation of Dipeptidoylbenzotriazoles 2.5a -c and the Diastereomeric Mixture ( 2.5b+2.5b' ) ........................................................... 52 2.4.7 General Procedure for Preparation of LLL Tripeptides 2.6a,b and the Diastereomeric Mixture ( 2.6b+2.6b' ) ................................................................. 54 2.4.8 General Procedure for Preparation of LLL Tripeptides 2.6c -e and the Diastereomeric Mixture ( 2.6e+2.6e' ) .................................................................. 56 2.4.9 Procedure for Preparation of unnatural LLL Tripeptide 2.6f ................................ 58 3 SYNTHESIS OF PEPTIDES BY EXTENTION AT N OR C TERMINUS OF ARGININE .................................................................................................................................. 60 3.1 Introduction ........................................................................................................................... 60 3.2 Results and Discussion ......................................................................................................... 64 3.2.1 Preparation of LL Dipeptides 3.9a -e and the Diastereomeric mixtures (3.9b+3.9b' ), (3.9c+3.9c' ) by Extension at the NTerminus of NNO2-L Arg OH 3.8 ........................................................................................................... 64 3.2.2 Preparation of LL Dipeptides 3.11a d and the Diastereomeric mixture (3.11b+3.11b' ) by Chain Elongation at the NTerminus of L -Arg OH 3.10 ........................................................................................................................ 66 3.2.3 Preparation from N-Cbz NNO2-L -Arg Bt 3.13 of Arginine LL -dipeptides 3.15a -c and the Diastereomeric mixture ( 3.15a+3.15a ) by Extension at the C Terminus of NCbz NNO2L -Arg -OH 3.14 ......................................... 67 3.2.4 Preparation of C Terminal Arginine Tripeptides 3.22a -c and ( 3.22a+3.22a ) by Extension at the NTerminus of NNO2-L -Arg OH 3.8 ............................. 69 3.2.5 Application of Benzotriazole Methodology in the Preparation of the Protected RGD Peptide NCbz NNO2-L -Arg Gly L -Asp (OH)2 3.26 .......... 72 3.3 Conclusion ............................................................................................................................. 73 3.4 Experimental Section ............................................................................................................ 73 3.4.1 General Methods ...................................................................................................... 73 3.4.2 General Procedure for the Prepara tion of LLDipeptides 3.9a -e and the Diastereomeric mixture ( 3.9b+3.9b' ), ( 3.9c+3.9c' ) ........................................... 73 3.4.3 General Procedure for the Preparation of LLDipeptides 3.11a d and the Diastereomeric Mixture ( 3 .11b+3.11b' ) ............................................................. 76 3.4.4 Preparation of NCbz NNO2-L -Arg Bt, 3.15..................................................... 79 3.4.5 General Procedure for the Preparation of LLDipeptide s 3.17a d and the Diastereomeric mixture ( 3.17a+3.17a' ) .............................................................. 79

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8 3.4.6 Procedure for the Preparation of N Cbz Dipeptidoylbenzotriazole Derivatives 3.22a and ( 3.22a+3.22a' ) from Cbz -L -Asp(Obz) -OH 3.1 9 ........... 82 3.4.6.1 Procedure for the preparation of Cbz L -Asp(OBz) -Bt, 3.20 ................................................................................................................. 82 3.4.6.2 Procedure for preparation of Cbz -L -Asp(OBz) -L P he OH 3.21a and the diastereomeric mixture Cbz -L -Asp(OBz) DL Phe OH ( 3.21a+3.21a' ) .......................................................................... 83 3.4.6.3 General procedure for preparation of Cbz -L -Asp(OBz) -L Phe Bt, 3.22a and the diasteromeric mixture (3.22a+3.22a' ) ............................................................................................... 84 3.4.7 General Procedure for Preparation of Arginine LLL Tripeptides 3.23a c and the Diastereomeric Mixture ( 3.23a+3.23a' ) ....................................................... 85 3.4.8 Preparation of NCbz NNO2-L -Arg Gly Bt, 3.24 ............................................. 88 3.4.9 Preparation of NCbz NNO2-L -Arg Gly L -Asp (OH)2, 3.26 ........................... 88 4 CONVENIENT ONE POT SYNTHESIS OF 5 (SUBSTITUTED AMINO) -1,2,3,4 THIATRIAZOLES ..................................................................................................................... 90 4.1 Introduction ........................................................................................................................... 90 4.2 Results and D iscussion ......................................................................................................... 93 4.2.1 Two-Step Synthesis of 5 (Substituted amino) 1,2,3,4 -thiatriazoles 4.1a-i .......... 93 4.2.2 One Pot Synthesis of 5(Sub stituted amino) 1,2,3,4 thiatriazoles 4.1a g,i,l ....... 95 4.2.3. Attempted Synthesis of 5(Disubstituted amino) 1,2,3,4 thiatriazoles ............... 98 4.3 Conclusion ............................................................................................................................. 99 4.4 Experimental ......................................................................................................................... 99 4.4.1 General Methods ...................................................................................................... 99 4.4.2 Synthesis for the Preparation of Bis(1H benzotrizol 1 yl)methanethione 4.15 ...................................................................................................................... 100 4.4.3 General Procedure for the Preparation of Thiocarbamoylbenzotriazoles 4.20a -g,k ............................................................................................................. 100 4.4.4 General Procedure for the Preparation of Thiocarbamoylbenzotriazoles 4.20h,i .................................................................................................................. 102 4.4.5 General Procedure for the Preparation of 5(Substi tuted amino) 1,2,3,4 thiatriazoles 4.1a i from Thiocarbamoylbenzotriazloes 4.20a -i ...................... 103 4.4.6 General One -Pot Procedure for the Preparation of 5 (Substituted amino) 1,2,3,4 thiatriazoles 4.1a -g ................................................................................. 105 4.4.7 One Pot Procedure for the Preparation of 5 (Substituted amino) 1,2,3,4thiatriazoles 4.1i .................................................................................................. 106 4.4.8. One Pot Procedur e for the Preparation of 5 (Substituted amino)bis(1,2,3,4thiatriazole) 4.1l .................................................................................................. 106 5 SYNTHESIS OF 8 -AZAQUINAZOLINONE ANALOGUE, NBI 74330 AS CXCR3 RECEPTOR ANTAGONIST AND ITS APPLICATION IN TREAT MENT OF MALIGNANT GLIOMAS ....................................................................................................... 108 5.1 Introduction ......................................................................................................................... 108 5.2 Results and Discussion ....................................................................................................... 110

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9 5.2.1 Procedure for the Preparation of 8-Azaquinazolinone Analogue, NBI 74330 5.2 ........................................................................................................................ 110 5.2.2 Role of NBI 74330 as CXCR3 Receptor Antagonist for Treatment of Malignant Gliomas ............................................................................................. 113 5.3 Conclusion ........................................................................................................................... 114 5.4 Experimental Section .......................................................................................................... 114 5.2.1 Genera l Methods .................................................................................................... 114 5.2.2 Procedure for Preparation of Compound 5.10 ..................................................... 114 5.2.3 Procedure for the Preparation of Compound 5.12 ............................................... 116 5.2.5 Procedure for the Preparation of 8-Azaquinazolinone Analogue NBI 74330 5.2 ........................................................................................................................ 117 LIST OF REFERENCES ................................................................................................................. 119 BIOGRAPHICAL SKETCH ........................................................................................................... 130

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10 LIST OF TABLES Table page 2 1 Preparation of dipeptides 2.3a -g and the diastereomeri c mixtures ( 2.3a+2.3a' ), (2.3d+2.3d' ) ............................................................................................................................ 34 2 2 Preparation of unnatural dipeptides 2.3h j and the diastereomeric mixture (2. 3h + 2.3h' ) ............................................................................................................................ 36 2 3 Preparation of dipeptides 2.3k -n and the diastereomeric mixture ( 2.3l+2.3l' ) from N N-Fmoc -N-Cbz -L Lys Bt 2.1h ........................................................................................... 37 2 4 Preparation of N -Cbz -dipeptidoylbenzotriazoles 2.5a -c and the diastereomeric mixture ( 2.5b+2.5b' ) .............................................................................................................. 38 2 5 Preparation of tripeptides 2.6a,b and the diastereomeric mixture ( 2.6b+2.6b' ) ................ 39 2 6 Preparation of tripeptides 2.6c -e and the diastereomeric mixture ( 2.6e+2.6e' ) ................. 40 3 1 Preparation of arginine dipeptides 3.9a -e ( 3.9b+3.9b' ) and ( 3.9c+3.9c' ) from NNO2-L -Arg OH 3.8 ................................................................................................................ 65 3 2 Preparation of arginine dipeptides 3.11a d and ( 3.11b+3.11b' ) from L -Arg -OH 3.10. .... 67 3 3 Preparation of N-Cbz NNO2-L -Arg Bt 3 .15 and arginine dipeptides 3.17a-d (3.17a+3.17a' ) ........................................................................................................................ 68 3 4 Preparation of L arginine tripeptides 3.23a -c and the diastereomeric mixture (3.23a+3.23a ........................................................................................................................ 71 4 1 Preparation of thiocarbamoylbenzotriazoles 4.20a k .......................................................... 94 4 2 Preparation of 5 (substituted amino) 1,2,3,4 thiatriazoles 4.1a-i from thiocarbamoylbenzotrizoles 4.20a -i ...................................................................................... 95 4 3 One pot synthesis of 5 (substituted amino) 1,2,3,4 thiatriazoles 4.1a -g,i,l from 4.15...... 97

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11 LIST OF FIGURES Figure page 1 1 Benzotriazole and its properties ............................................................................................ 20 2 1 Chemical structure and coupling sites of L lysine ............................................................... 29 2 2 Peptide coupling reagents ...................................................................................................... 30 2 3 1H NMR spectrum for 2.3a .................................................................................................... 33 2 4 1H NMR spectrum for (2.3a+2.3a' ) ...................................................................................... 33 3 1 NNO2-L arginine scaffold .................................................................................................. 60 4 1 5 (Monosubstituted amino) 1,2,3,4 -thiatriazole 4.1 and heterocycles derived from 4.1 ............................................................................................................................................ 90 4 2 Structure of bis(1 H -benzotriazol 1 -yl) and bisimidazol 1 -yl reagents ............................... 92 5 1 Structure of chemokines receptors AMG 487 5.1 and NBI 74330 5. 2 ............................ 108 5 2. High temperature 1H NMR for the compound 5.2 at increasing temperature up to 120 oC ........................................................................................................................................... 112 5 3 Reverse -phase HPLC for th e compound 5.2 ...................................................................... 113

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12 LIST OF SCHEMES Scheme page 1 1 Typical reactions of N -substituted benzotriazoles ............................................................... 21 1 2 N C and O -Acylation with N acylbenzotriazoles ............................................................. 22 1 3 General scheme for peptide synthesis ................................................................................... 22 1 4 Synthesis of N -protected dipeptides ..................................................................................... 23 1 5 Dipeptides by extension at N terminus of aspartic and glutamic acids .............................. 24 1 6 Dipeptides by extension at C terminus of aspartic and glutamic acids .............................. 24 1 7 Preparation of N -protected pyroglutamyl amino acids ........................................................ 25 1 8 Stepwise and fragment coupling for synthesis of peptides .................................................. 25 1 9 Reations of bis(1H -benzotriazol 1 -yl)methanethione .......................................................... 27 1 10 Formation of isothiocyanates ................................................................................................ 27 1 11 Synthetic applications of thiocarbamoylbenzotriazoles ...................................................... 28 2 1 Literature procedures f or lysine peptide using DCC/HOBt ................................................ 30 2 2 Formyl -substituted nitrophenylthioester method ................................................................. 30 2 3 Literature procedure for lysine peptides using mixed anhydride method ....................... 31 2 4 Literature procedure for lysine peptides using Cu -complex. ........................................... 31 2 5 Preparation of dipeptides 2.3a -g and the diastereomeric mixtures (2.3a+2.3a' ), (2.3d+2.3d' ) ............................................................................................................................ 32 2 6 Preparation of unnatural dipeptides 2.3h j and the diastereomeric mixture (2. 3h + 2.3h' ) ............................................................................................................................ 35 2 7 Preparation of dipeptides 2.3k -n and the diastereomeric mixture ( 2.3l + 2.3l' ) from N-Fmoc -N-Cbz -L Lys Bt 2.1h ........................................................................................... 36 2 8 Preparation of N -Cbz -dip eptidoylbenzotriazoles 2.5a -c and the diastereomeric mixture ( 2.5b+2.5b' ) .............................................................................................................. 38 2 9 Preparation of tripeptides 2.6a,b and the diastereomeric mixture ( 2.6b+2.6b' ) ................ 39 2 10 Preparation of tripeptides 2.6c -e and the diastereomeric mixture ( 2.6e+2.6e' ) ................. 39

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13 2 11 Preparation of unnatural tripeptide 2. 6f ................................................................................ 40 3 1 Literature method for preparation of arginine peptides ....................................................... 62 3 2 -lactam formation during carboxyl activation of arginine ....................... 62 3 3 -lactam formation during carboxyl activation with EDC/HOBt .............. 63 3 4 Chemo -enzymatic synthesis of the protected RGD pept ide ................................................ 63 3 5 Synthesis step of the free RGD peptide ................................................................................ 63 3 6 Preparation of NNO2L -Arginine dipeptides 3.9a-e and diastereomeric mixtures (3.9b+3.9b' ) and ( 3.9c+3.9c' ) ............................................................................................... 64 3 7 Preparation of arginine dipeptides 3.11a d and ( 3.11b+3.11b' ) from L -Arg -OH 3.10. .... 66 3 8 Attempted synthesis of benzotriazole derivative of N-Cbz -L -Arg OH 3.12 .................... 67 3 9 Preparation of N-Cbz NNO2-L -Arg Bt 3.15 and arginine dipeptides 3.17a-d (3.17a+3.17a') ........................................................................................................................ 68 3 10 Preparation of Cbz L -Asp(OBz) -Bt 3.20 .............................................................................. 70 3 11 Synthe sis of N -Cbz -dipetidoylbenotriazole 3.22 and ( 3.22a+3.22a' ) ................................ 70 3 12 Synthesis of NNO2-L arginine tripeptides 3.23a -c and ( 3.23a+3.23a' ) .......................... 70 3 13 Preparation of protected RGD peptide 3.26 ......................................................................... 72 4 1 Literature methods for synthesis of monosubstituted aminothiatriazoles 4.1 .................... 91 4 2 Literature procedure for synthesis of substituted aminothiatriazoles 4.12 ......................... 91 4 3 Two step synthesis of 5 (substituted amino) 1,2,3,4 thiatriazoles 4.1a-i from bis(1 H benzo triazol 1 yl)methanethione 4.15 .................................................................................. 94 4 4 Literature report for reaction of bis(1 H -benzotriazol 1 -yl)methanethione 4.15 and p anisidine 4.19g........................................................................................................................ 94 4 5 One pot synthesis of 5 (substituted amino) 1,2,3,4 thiatriazoles from bis(1 H benzotriazol 1 yl)methanethione 4.15 and primary amines 4.19a -g,l ................................ 96 4 6 One pot synthesis of 5 ( substituted amino) 1,2,3,4 thiatriazoles 4.1i from bis(1 H benzotriazol 1 yl)methanethione 4.15 and amino acid ester hydrochloride 4.19i ............. 96 4 7 Attempted synthesis of 5 -(disubstituted amino) 1, 2,3,4 -thiatriazole 4.1j .......................... 98 4 8 Attempted synthesis of 5 -(disubstituted amino) 1,2,3,4 -thiatriazole 4.1k ......................... 98

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14 5 1 Synthesis of CXCR 3 receptor NBI 74330 5.2 ................................................................... 111

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15 LIST OF ABBREVIATION S Ala Alanine Anhyd Anhydrous Asp Aspartic acid Arg Arginine D Op t ical rotation Bn Benzyl Boc t ert -Butoxycarbonyl BOP Benzotriazol 1 -yl -oxy -tris (dimethylamino) -phosphon ium hexafluorophosphate Bt Benzotriazol 1 -yl Cbz Carbobenzyloxy CH3CN Acetonitrile CNS Central nervous system Cu Copper Cys Cysteine DCC N, N Dicyclohexylcarbodiimides DIPEA Diisopropylethylamine DMF Dimethylformamide DNP 2,4 Dinitrophenol DPPA Diphenyl p hosphoryl azide ECM Extracellular matrics EDC 1 Ethyl 3 (3 dimethylaminopropyl) carbodiimide hydrochloride Et3N Triethyl amine EtOAc Ethyl acetate eNOS Endothelia l n itric oxide sny t h ase

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16 Fmoc Fluorenylmeth y l oxycarbonyl GBM Glioblastoma multiforme Glu Gluta mic acid Gly Glyc ine HOBt 1 Hydroxybenzotriazole HPLC High performance liquid c hromatography HRMS H igh resolution mass spectrometry J Coupling constants in proton NMR Lit. Literature Lys Lysine MCA Mixed carboxylic -carbonic anhydride MeOH Methanol Met Meth ionine NCA N -carboxyanhydride NMM N-Methylmorpholine NMR Nuclear magnetic resonance NOS Nitric oxide synthase nNOS Ne uronal n itric oxide synthase NOC NNitroso compounds Pd Palladium Pfp Pentafluorophenyl Pg Protecting group Phe Phenylalanine RT Room tempe rature RGD Arginine -glycine aspartic

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17 SOCl2 Thionyl chloride Ser Serine tR Retention time Trp Tryptophan Tyr Tyrosine THF Tetrahydrofuran TFA Trifluoroacetic acid TFFH N, N' Tetramethylfluoroformamidinium hexafluorophosphate TMS Tetramethylsilane Val Valine

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18 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy HETEROCYCLE -ASSISTED SYNTHESIS OF BIOLOGICALLY SIGNIFICANT SYSTEMS By Geeta Meher May 2009 Chair: Alan R. Katritzky Major: Chemistry The thesis is concerned with synthesis of lysine and arginine containing peptides, 5 (substituted amino ) 1,2,3,4 thiatriazoles and 8 azaquinazolinone analogue, NBI74330. Lysine and argi nine are essential alpha amino acids and are constituent of various biologically active peptides. 5 -(Substituted amino ) 1,2,3,4 thiatriazoles show interesting biological properties. NBI 74330 is the CXCR3 receptor antagonist. Benzotriazole methodology enables c onvenient syntheses of na tural and unnatural di and tripeptides derived from L lysine by extension at N (alpha) N (epsilon) and /or C termini, under mild reaction conditions. Extensions at N termin i have been carried out by acylation with N (Cbz -or Fmoc alpha aminoacyl)benzotriazoles or N -protected dipeptidoylbenzotriazoles E xtensions at C -terminii were performed similarly by coupling the N -protected acylbenzotriazole derivative of lysine and its dipeptides with unprotected alpha amino acids These reactions are performed in aqueous acetonitrile solution, require short reaction time s ; involve simple work up procedure s and form enantiopure products in high yields. A rginine containing d i and tripeptides are synthesized by chain elongation at either t he N or C -terminus of N(omega) L -nitroarginine or L arginine. Coupling of N(omega) -L -nitro arginine with N (Cbz or Fmoc alpha aminoacyl)benzotriazoles or N -protected dipeptidoylbenzotriazoles

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19 form di or tri peptides in good yields. The acylbenzotriazole de rivative of Cbz (alpha) N(omega) L nitro arginine is coupled with free amino acids to obtain the corresponding dipeptides. All isolated peptides were enantiopure and obtained in good yields. T he protected RGD peptide sequence was synthesized using benzotriaz ole methodology. A convenient one -pot protocol for the synthesis of 5 ( substituted amino) 1,2,3,4 thiatriazoles from bis(1 H -benzotriazol 1 yl)methanethione is described. Reactions of amines or amino acid esters with bis(1 H -benzotriazol 1 yl)methanethione f ollowed by addition of aqueous sodium azide at room temperature resulted with formation of corresponding 5 ( monosubstituted amino ) 1,2,3,4 -thiatriazoles in 73 97% yields, in short reaction times, without the use of column chromatographic purification. The CXCR3 receptor antagonist NBI 74330 was synthesized and tested for its biological activity by Dr. Jeffrey K. Harrison group for treatment of malignant gliomas.

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20 CHAPTER 1 BENZOTRIAZOLE: PROPERTIES AND SYNTHETIC UTILITY 1.1 Benzotriazole and its Properties 1 H Benzotriazole 1.1 is a stable inexpensive white solid, and a readily available compound. It is almost insoluble in water, but soluble in aqueous sodium carbonate, aqueous hydrochloric acid, ethanol, benzene, toluene, chloroform, tetrahydrofuran and d imethylformamide. Benzotriazole is a good synthetic auxiliary which offers many advantages. As an aspect of a synthetic auxiliary, a group must be able to be introduced readily at the beginning of the sequence and it must be easy to remove at the end of th e synthetic sequence. It should be stable during various synthetic operations, and if possible, exert an activating influence on the other parts of molecule. B enzotriazole ( p K a 8.2) is readily removed from a reaction mixture by simply washing with aqueous base Because of its chemical properties, it is an excellent synthetic auxiliary.1 3 Facile chemical reactivity of benzotriaz ole auxiliaries arise due to activation of the carbon atom attached to the benzotriazolyl group The benzotriazolyl group can acti vate substrates in a variety of ways: as a leaving group 1.2a and 1.2 b as an electron withdrawing group assisting deprotonation 1.3 and as an electron donating group assisting ionization 1.4 (Figure 1 1) The benzotriazolyl group is also an ambident anion directing group and it can act as a radical or carbanion precursor.1 Figure 1 1. Benzotriazole and its properties

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21 N -Substituted benzotriazole s in which the N -substituent contains an -hetereoatom (N, S, O or halogen) can ionize in two ways, either (i) yielding a benzotriazole anion and a cation derived from the heteroatom 1.5 or (ii) ionizing off the -substituent 1.6 (Scheme 1 1). Scheme 1 1. Typical reaction s of N -substituted benzotriazole s N -Substituted benzotriazole s undergo several types of re action s including : N C O and S acylation;4 imidoylation;5 thioacylation;6 amino amido alkoxy and thio alkylation;7 sulfonylation;8 carbon insertion,9 benzotriazole ring cleavage reactions10 and radical reactions.1 Relevant to our present work, acylation,4 thioacylation and thiocarbamoylation6 using benzotriazole reagent s will be di scussed in this general introduction 1.2 Synthetic Utility of Benzotriazole 1.2.1 N -Acylben z otriazole s and its application in Acylation and Peptide Synthesis N-Acylbe notriazole s have been employed for: (i) N acylation of primary, secondary and tertiary amine s amino acids, sulf onamides, hydroxylamines, hydrazines;4,11 and aminosugars ;12 (ii) C acylation of ketones, cyanide s, sulf ones, heterocycles, esters;4 thioesters ,13 nitroalkanes,14 organometallics ;15 (iii) O acylation in addition to aldehydes;1617 O acylation of sugars18 and steroids (Scheme 1 2)19

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22 Scheme 1 2. N -, C and O -Acylation with N acylbenzotriazoles N acylation of amine s results in the formation of an amide bond and this methodology has been successfully extended for the synthesis of peptide s Thus, the carboxyl group of the amino acid 1.7 is activated by converting it to the corresponding N acylbenzotriazole derivative 1.8 and reacted with another amino acid 1.9 to obtain a dipeptide 1.10. The r esulting dipeptide is further activated to the corresponding benzotriazole deriv ative 1.11 and reacted with another amino acid 1.12 to form a tripeptide 1.13 (Scheme 1 3) Scheme 1 3. General scheme for peptide synthesis The first step in the peptide synthesis conversion of N protected amino acids to benzotriazole derivative s is carried out by one of the following methods: (i) by treatment with

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23 sulfonylbenzotriazole in the presence of triethyl amine or (ii) by reacting with excess of benzotriazole in the presence of thionyl chloride (Scheme 1 4). The thionyl chloride method h as been extensively used for N (Cbz -or Fmoc) amino acids It is carried out at room temperature in high yields and is more convenient than the former one which requires heating.4 Thus a wide range of N (protected amino acyl ) benzotriazoles 1.8 ha ve been prepared from the corresponding N -protected amino acids 1.7 with retention of chirality.20 Moreover, this methodology can be applied to N -protected amino acids with unprotected side chain functionality such as in Tyr, Trp, Cys, Met and Gln (con taining phenolic OH, NH of indol e, SH, SMe and amide groups);21 an d in Ser, Asn, Glu, Asp, Cys (containing aliphatic OH, primary amide and CO2H groups and the S S linkage).22 These acyl de rivatives which are stable and crystalline solids couple amino acids 1.9 with unprotected side chain functi onality in aqueous CH3CN to give the corresponding dipeptides 1.10 with no detectable ep i mer ization.2022 Scheme 1 4. Synthesis of N protected dipeptides Similarly, dipeptides 1.16 and 1.17 have been synthesized by acylation of the amino groups of fre e aspartic and glutamic acids with various N (protected aminoacyl)benzotriazoles 1.8 with complete retention of chirality (Scheme 1 5).23 Also aspartic and glutamic acids were selectively extended at each of the alternative C terminals to afford diverse natural and unnatural N -protected dipeptides 1.20, 1.21, 1.24, 1.25 by reactions between N (protected

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24 aminoacyl ) benzotriazoles 1.18, 1.19, 1.22, 1.23 and free amino acids 1.7 again with complete retention of chirality (Scheme 1 6).24 Scheme 1 5. Dipeptides by extension at N -terminus of aspartic and g lu tamic acids Scheme 1 6. Dipeptides by extension at C -terminus of aspartic and glutamic acid s However, benzotriazole derivative of N -protected glutamic ester 1.23 cyclizes to form 1.26 in aque ous CH3CN in the presence of Et3N Similarly, coupling of N -protected bis benzotriazole derivative of glutamic acid 1.27 with amino acids (Ala, Phe, Val) 1.7 in aqueous CH3CN in the presence of Et3N produce d N -protected pyroglutamyl pseudopeptides 1.28 in 55 88% yields (Scheme 1 7).25

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25 Scheme 1 7. Preparation of N -protected pyroglutamyl amino acids Tripeptides 1.13 have been synthesized by stepwise coupling utilizing N -protected dipeptidoylbenzotriazoles 1.11 with unprotected amino acids 1.12.4 Similarly, tripeptides 1.13 have also been synthesized from N (protected aminoacyl)benzotriazoles 1.8 and free dipeptides by fragme nt condensation in aqueous CH3CN solution. Scheme 1 8. Stepwise and fragment coupling for synthesis of peptides N (Fmoc aminoacyl )benzotriazoles have been employed in solid-phase pepti de synthesis under microwave irradiation to synthesize tri tetra penta hexa -, and heptapeptides (20 68%).26 Chapter 2 describe s the preparation of peptides containing lysine by extension at both C and N terminus of lysine by utilizing N (Cbz -or Fmoc aminoacyl)benzotriazoles as efficient coupling reagents.

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26 In chapter 3 the preparation of peptides containing arginine by utilizing N (Cbz aminoacyl)benzotriazoles as efficient coupling reagents will be discussed Synthesis of protected inte grin recognition sequence Arg -Gly -Asp will be described utilizing benzotriaz ole methodology. 1 .2.2 Bis (1 H -Benzotriazol -1 -yl)methanethione and i ts A pplication i n Thiocarbamoylation a nd Related Reactions Classical thioacylating agents such as thiophosgene27,28 and carbon disulfide29 have been converted to the corresponding thiocarb amoyl derivatives30 32 for further reactions with nucleophiles. Alternati vely, the thiocarbonyl group can be introduced by using thionating agents such as sulfur dihydride,33 phosphorus pentasulfide34,35 and Lawessons reagent.33 A valuable alternative rea gent to the classical thioacylating reagents is bis(1 H benzotriazol 1 yl)methanethione 1.30 (S cheme 1 9 ) which is more stable and less hygroscopic.36 This reagent has been utilized in the synthesis of thioureas,6,37 N hydroxythioureas,38 thioamides,6 thio carbamates,6 thionoesters,6 dithiocarbamates,6,39 thiocarbonates,6 dithiocarbonates,6 thiosemicarbazides,38 guanidines40 -enamino thioic acid derivatives.41 Thus, the reagent 1.30 undergo es substitution reactions with N O and S -nucleophiles with selective substitution of only one benzotriazolyl group at ambient temperature affording the corresponding benzotriazole d erivative s 1. 3 1 -1 .33 However, reactions with Grignard reagents and organolithium failed to afford thiocarbonylbenzotriazole.6 I mination with triphenylphosphine imides40 and heter o Diels Alder cycloaddition with dienes42,43 afford s 1. 34 1. 36 respectively (Scheme 1 9)

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27 Scheme 1 9. Reations of bis(1H -benzotriazol 1 yl)methanethione Enamino thiocarbonylbenzotriazoles 1.37 were synthesized by the reaction of 1.30 with ketimines ( Scheme 1 9 ).41 The second benzotriazolyl group in the intermediates obtained may be further substituted with different N -, O -, S and C nucleophiles under different reaction c onditions depending on the type of substituent present in the starting compounds.6,37 41 The benzotriazole intermediate 1. 3 1 can be used as thiocarbamoylating agent. This class of compounds is prepared by the reaction of 1. 30 and a variety of primary and secondary amines (Scheme 1 9); however the reaction with primary aryl amines is accompanied by elimination of the benzotriazole molecule and results in formati on of aryl isothiocyanates (Scheme 1 10).6 Scheme 1 10. Formation of isothiocyanates

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28 The synthetic applications of thiocarbamoyl benzotriazoles 1.31 include: nucleophilic substitutions with C N O and S -nucleophiles to obtain thiocarbamides 1. 38, t hioureas 1. 3 9 thiocarbamates 1. 4 0 and dithiocarbamates 1. 41 (Scheme 1 11) Scheme 1 11. Synthetic applications of thiocarbamoylbenzotriazoles In chapter 4 easy and convenient one -pot synthesis of 5 ( substituted amino ) 1,2,3,4 thiatriazoles will be de scribed from bis(1 H -benzotriazol 1 yl)methanethione 1.30. In chapter 5 detailed synthesis of CXCR3 antagonist 8 azaquinazolinone analogue, NBI 74330 is described. It was tested by Harrison et al for the treatment of malignant glioma s Results of the bio logical testing are summarized.

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29 CHAPTER 2 SYNTHESIS OF PEPTIDES BY EXTENTION S AT THE N OR C TERMIN II OF LYSINE 2.1 Introduction Lysine is an essential amino acid and is basic in nature. It is important for proper growth and it plays an essential role in the production of carnitine, a n utrient responsible for converting fatty acid into energy and helping low cholesterol. Lysine plays major role in calci um absorption and formation of collagens.44 The most promising role of l ysin e is its use in preventing painful herpes sores caused by herpes simplex viruses (HSV) .45 Lysine (Figure 2 1) residue occur s in various biologically active peptides, for example in the active portions of adrenocorticotropic hormones,46 melanotropic hormones,47 thrombolytically active therapeutic agents such as P6A (obtained from fibrinogen degradation),4850 adhesive proteins of marine mussel,51,52 chemotactic peptides,53 biocom patible telomers,54 hybrid peptides55 and analogues like somatostatin.56 Basic amino acid L lysine is also a constituent of sweet tasting peptide. N -A cetyl -L -p henlyalanyl -L l ysine possessed a potential sweetness 20 times stronger than sucrose.57 Figure 2 1 C hemical structure and coupling sites of L -lysine Lysine has a n amino group at the end of the side chain. So, i n addition to -peptides, it is also capable of forming alternative peptides or unnatural peptides or isopeptides

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30 Consequently, considerable efforts have been made to incorporate Lys residues in the peptide chain extension Previous preparations of peptides containing NCbz -lysine utilized coupling reagents like carbodiimides DCC ,48,52,55,58 64 and BOP.54 (Figure 2 2, Scheme 2 1). Pepti de coupling has also been achieved using the mixed anhydride method,53,56,6567 the azide method,68,69 the active ester method (formyl -subs tituted nitrophenylthio esters) in S cheme 2 2.70 Figure 2 2 Peptide coupling reagents Scheme 2 1. Literature procedure s for lysine peptide using DCC/HOBt Scheme 2 2. Formyl -substituted nitrophenylthioester method However, all these methodologies require long reaction times (18 24 h). For example, the coupling of N -protected amino acid with NCbz -L Lys or it s corresponding ester in the presence of DCC/HOBt requires 18 24 h to achieve completion .55,56,59,6365,70 In some cases, the final product is an ester and thus requires hydrolysis or hydrogenolysis as an additional step to obtain the desired free peptide.48,49,55,65,67,69,70

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31 Lysine peptide is the fundamental structural unit of clavicepamines.71 The amino group of lysine is also involved in the amide bond formation in collagen, bovine growth hormone and some natural products such as biocytin and bacitra cin.67,72 -Lysine peptides were previously prepared utilizing various procedures involving carbodiimides, mixed carboxylic -carbonic anhydrides (MCA) (Scheme 2 3) ,67,73 or using lysine Cu complex.71,73 (Scheme 2 4 ). According to Theodoropoulos in the cas e of lysine, the Cu complex formation gives the best protection for isopeptide coupling of lysine at C terminal.73 Both the carbodiimide and MCA method reportedly require reaction times of 5 12 h, and the COOH group of N-Cbz L -Lys needs to be protected and thus there is an additional hydrolysis step to obtain the free peptide. Scheme 2 3 Literature procedure for -lysine peptides using mixed anhydride method Scheme 2 4. Literature procedure for -lysine peptides using Cu-complex.

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32 In continuation of our research on peptide chain extensions, an easy and convenient preparation of diverse di and tripeptides containing NCbz -lysine and NCbz -lysine units by extension at both N and C -terminus of protected lysine is described using benzotriazole met hodology. 2.2 Results and Discussion 2.2.1 Preparation of D ipeptides 2.3a -g and the Diastereomeric M ixture s (2. 3a+2.3a'), (2. 3d+2.3d' ) Using t he NTerminus o f N -Cbz -L -Lys 2.2a Peptide coupling reactions were successfully carried out between N (Cbz or F moc aminoacyl)benzotriazoles 2.1a -g derived from L -Phe, L -Ala, L Trp, L -Met and NCbz -L -Lys 2. 2a in partially aqueous CH3CN solution in the presence of Et3N over 3045 min. ( Scheme 2 5 Table 2 1 ). By comparison, literature methods require 18 24 h for c ompletion.55,56,59,6365,70 N (Cbz or Fmoc aminoacyl)benzotriazoles 2.1a-g were prepared following literature procedure.20,21 1H NMR analysis for each compound 2.3 revealed two sets of doublets for the two NH protons ranging from 7.40 8.3 0 ppm (Figure 2 3) The methyl protons from the L -Ala fragment in 2 3a showed a clear doublet and two sets of doublet for the NH protons, supporting the enantiopurity of the LL -dipeptide. Howeve r in the case of the diastereomeric mixture (2 3a+2.3a' ), al though the meth yl protons from L -Ala fragment also showed a clear doublet but one of the NH protons showed a multiplet in the range of 8.008.15 ppm instead of a clear doublet (Figure 2 4) Scheme 2 5 Preparation of dipeptides 2.3a -g and the diastereomeric mixtures (2.3a+2.3a' ), (2.3d+2.3d' )

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33 Figure 2 3. 1H NMR spectrum for 2.3a Figure 2 4. 1H NMR spectrum for (2.3a +2.3a' )

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34 13C NMR for (2 3a+2.3a' ) showed two singlets for each aliphatic and carbonyl carbons. In the case of ( 2.3d+2.3d' ), one of the NH protons showed an apparent triplet at 7.47 ppm. 13C NMR of ( 2 3d+2.3d' ) also gave two singlets for each aliphatic and carbonyl carbons. The enantiopurity of the dipeptides 2.3a -g was further confirmed by HPLC analyses using Chirobiotic T column (detection at 254 nm, flow rate 1.0 mL/min, and MeOH as solvent). For each of the LLdipeptide 2 3a and 2.3d the HPLC results showed a single peak. By contrast two peaks were observed for the corresponding diastereomeric mixtures ( 2 3a+2.3a' ) and (2 3d+2.3d' ) confirming the enantiopurity of the LL-dipeptide 2.3a and 2.3d These results also indicated shorter retention times for the LL-configuration as compared to the LD configuration as summarized in Table 2 1 Table 2 1 Preparation of dipeptides 2.3a -g and the diastereome ric mixtures ( 2.3a+2.3a' ), (2.3d+2.3d' ) Entry Reactant Product Yield a (%) m p (C) 23 D 1 Cbz L Ala Bt 2 1a Cbz L Ala N Cbz L Lys OH 2 3a b,c 85 92 94 +4.04 2 Cbz D L Ala Bt (2.1a + 2.1a' ) Cbz D L -Ala N Cbz -L -Lys OH (2 3 a +2.3a' )d 88 105 108 1.86 3 Cbz L Phe Bt 2 1b Cbz L Phe N Cbz L Lys OH 2.3b e 95 117 119 4.53 4 Cbz L Trp Bt 2 1c Cbz L Trp N Cbz L Lys OH 2 3c 85 114 116 15.44 5 Cbz L Met Bt 2 1d Cbz L Met N Cbz L Lys OH 2 3d f 83 122 123 1.94 6 Cbz D L Met Bt (2 1d+2.1d' ) Cbz D L -Met -N -Cbz -L Lys O H (2.3d+2.3d' )g 91 45 46 0.40 7 Fmoc L Trp Bt 2.1e Fmoc L Trp N Cbz L Lys OH 2 3e 80 91 93 14.71 8 Fmoc L Met Bt 2 1f Fmoc -L Met -N Cbz -L Lys OH 2.3f 80 122 124 7.06 9 Fmoc L Phe Bt 2 1g Fmoc L Phe N Cbz L Lys OH 2 3g 92 128 132 13.03 a Isolated y ields; b Lit. data not available in 74 76 ; c Retention time = 3.07 min; d Retention time = 3.13, 3.62 min, for conditions, see experimental section; eLit. Ref 77 mp 117-121 C; [ ]25 D= 6.90 ( c 1.0, DMF); f Retention time = 2.99 min; g Retention time = 2.99, 3 .58 min.

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35 2.2.2 Preparation of Unnatural D ipeptides 2.3h -j U sing N -Terminus of N-Cbz -L -Lys 2.2b Peptide coupling reactions were carried out successfully at the side chain amino functionality of lysine using NCbz -L Lys 2.2b and N (Cbz -or Fmoc aminoacyl)benzotriazoles 2.1b c f and (2 1h+2.1h' )1 derived from L Phe, L -Trp, L -Met, D L -Phe (Scheme 2 6 Table 2 2 ) in a similar procedure as adopted for natural dipeptides. Interestingly, a shorter reaction time (<15min) sufficed compared to the natural peptides. 1H NMR and HPLC analysis showed no detectable epimer ization (<1%) for the ena ntiopure LL -dipeptides 2.3h -j. For compounds 2 3h -j 1H NMR analysis for each compound revealed three sets of doublet s for the three NH protons ranging from 7.45 8.10 ppm. The observed 1H NMR pattern was different for these unnatural dipeptides as compared to the natural dipeptides. For example, in the case of 2 3h and 2. 3 i the CH protons arising from the L -Phe and L Trp fragment were shifted upfield ( 2 3h : 3.89 ppm and 2 3 i : 3.91 ppm) as compared to those in the corresponding fragment in the natural dipeptides 2 3b (4.80 ppm) and 2 3c (4.37 ppm). The HPLC results showed a single peak for compounds 2 3h -j By contrast two peaks were observed for the corresponding diastereomeric mixture ( 2 3h+2.3h ) confirming the enantiopurity of the LL-dipeptide 2.3h Scheme 2 6 Preparation of unnatural dipeptides 2.3h j and the diastereomeric mixture (2. 3h + 2.3h' ) 1 Compound numbers written in the bracket represent racemic compound or the diastereomeric mixture.

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36 Table 2 2 Preparation of unnatural dipeptides 2.3h j and the diastereomeric mixture (2. 3h + 2.3h' ) Entry Reactant Product Yield a (%) m p (C) 23 D t R (min ) b 1 Cbz L Phe Bt 2.1b N Cbz N ( Cbz L Phe ) L Lys OH 2 3h c 79 162164 12.88 3.60 2 Cbz D L -Phe -Bt (2 1b + 2.1b' ) N Cbz N (Cbz D L Phe) -L Lys OH (2 3h + 2 3h' ) 95 93 5.00 3.16, 3.60 3 Cbz L Trp Bt 2 1c N Cbz N ( Cbz L Trp) L Lys OH 2 3i 93 87 1 7.94 3.34 4 Fmoc L Met Bt 2 1f N Cbz N (Fmoc L Met) L Lys OH 2 3j 94 83 85 9.70 2.55 a Isolated yields; b t R : Retention time ; for conditions, see the experimental section; c Physical characterization data is not available in the literature. 2.2.3. Prep aration of Dipeptides 2.3k n, and the D iastereomeric mixture (2.3l+2.3l') by Chain E longation at the C Terminus of Lysine U sing N -Fmoc -N -Cbz L -Lys Bt 2.1h Preparation of N-Fmoc -N-Cbz -L Lys Bt 2 1h was carried out using reported procedure21 from N-Fmoc -NCbz -L Lys OH 2.2c using 1H benzotriazole in the presence of SOCl2. Peptide coupling between NFmoc NCbz -L -Lys -Bt 2 1h and L amino acids 2 2a,c -e and DL -Met (2 2c+2.2c' ) proceeded in CH3CN/H2O in the presence of Et3N for 30 min ( Scheme 2 7 Table 2 3 ). The crude products were washed with 4N HCl to remove the by-product. NMR analysis showed no detectable epimerization (< 1 %) for the enantiopure LL -dipeptides 2 3k -n Scheme 2 7 Preparation of dipeptides 2.3k -n and the diastereomeric mixture ( 2.3l + 2. 3l') from N-Fmoc -N-Cbz -L Lys Bt 2.1h

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37 Table 2 3. Preparation of dipeptides 2.3k n and the diastereomeric mixture ( 2.3l+2.3l' ) from N N-Fmoc -N-Cbz -L Lys Bt 2.1h Entry Reactant Product Yield a (%) m p (C) 23 D t R (min )b 1 Cbz L -Lys 2. 2a N -Fmoc -N -Cbz -L Lys NCbz L -Lys -OH 2.3k 74 103 105 4.52 2.99 2 L Met 2.2c N Fmoc N Cbz L Lys L Met OH 2.3l 95 138 140 +2.09 2.57 3 DL Met (2.2c+2.2c' ) N -Fmoc -N -Cbz -L Lys D L Met OH ( 2.3l+2.3l' ) 85 80 81 3.94 2.57, 3.59 4 L Trp 2.2d N Fmoc N Cbz L Lys L Trp OH 2 3m 88 67 69 +2.40 2.98 5 L Ser 2.2e N -Fmoc -N -Cbz -L Lys L Ser OH 2.3n 80 85 89 1.47 3.10 a Isolated Yields; b t R : Retention time; f or conditions, see the experimental section. For compounds 2 3k -n 1H NMR of each compound revealed two sets of doublets for the two NH protons ranging from 7.5 3 8.2 5 ppm. The HPLC results showed a single peak for 2 3k n By contrast two peaks were observed for the corresponding diastereomeric mixture (2 3l+2.3l ) confirming the enantiopurity of the LL -dipeptide 2 3l 2.2.4. Preparation of N -Protected D ipeptidoylbenzotriazoles 2.5a -c and the Diastereomeric M ixture (2.5b+2.5b') from N -Protected D ipeptides 2.4a,b, 2.3b and (2.4b+2.4b') N-Cbz Dipeptides 2 4a b 2. 3b a nd the diastereomeric mixture (2.4b+2.4b' ) were successfully converted into their corresponding benzotriazole derivatives 2. 5a -c and the diastereomeric mixture (2 5b+2.5b' ) (Scheme 2 8 Table 2 4 ). The reaction was carried out at 15 C for the formation o f N protected dipeptidoylbenzotriazoles following literature procedure.22 Progress of the reaction was monitored by 1H NMR. In the case of 2. 5a after the removal of THF, the residue was treated with EtOAc, and the crystalline materi al formed was filtered to give the desired product in pure form. In the case of 2. 5c 1.5 equivalent of SOCl2 was used instead of 1 equivalent to achieve completion. In addition, 2. 5c could not be isolated by the

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38 usual acid (4N HCl) or base (dilute Na2CO3) work up and the crude product was purified by reprecipitation using EtOAc/hexanes, in order to remove excess 1 H benzotriazole. Scheme 2 8 Preparation of N Cbz -di peptidoylbenzotriazoles 2.5a -c and the diastereomeric mixture ( 2.5b+2.5b' ) Table 2 4. Preparation of N Cbz di pe ptidoylbenzotriazoles 2.5a -c and the diastereomeric mixture ( 2.5b+2.5b' ) Entry Reactant Product Yield a (%) m p (C) 23 D 1 Cbz L Ala L Trp OH 2.4 Cbz L Ala L Trp Bt 2.5 a 70 162 164 36.99 2 Cbz L Phe L Met OH 2. 4b Cbz L Phe L Met Bt 2. 5b 76 110 112 36.88 3 Cbz L Phe DL Met OH ( 2.4b+2.4b' ) Cbz L Phe DL Met Bt ( 2.5b+2.5b' ) 65 140 141 13.86 4 Cbz L -Phe -Cbz -L L ys OH 2. 3b Cbz L -Phe -Cbz -L Lys Bt 2. 5c 75 139 141 17.19 a Isolated yields 2.2.5. Preparation of T ripeptides 2. 6a -e, and the Diastereomeric M ixtures (2.6 b+ 2.6 b'), (2.6 e+ 2.6 e') Tripeptides 2.6a-e and the diastereomeric mixtures (2.6b+2.6b' ) (2.6e+2. 6e' ) were prepared by two different approaches: (i) Trip eptide s 2.6a, b and the diastereomeric mixture (2.6b+2.6b' ) by extension from the Nterminus of NCbz L -Lys 2.2a were obtained by coupling reactions between N -Cbz dipeptidoylbenzotriazoles 2.5a b and the diastereomeric mixture ( 2.5b+2.5b ') with NCbz Lys 2.2a ( Scheme 2 9 Table 2 5 ) (ii) Trip eptide s 2.6c -e and the diastereomeric mi xture (2.6e+2.6e' ), by extension at the C terminus were obtained by coupling reaction between Cbz -L -Phe -N-Cbz -L -Lys Bt 2.5c and free amino acid 2.2c,d,f and ( 2.2f+2.2f' ) ( Scheme 2 10, Table 2 6 )

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39 Scheme 2 9 Prep aration of tripeptides 2.6a, b and the di astereomeric mixture ( 2.6b+2.6b' ) Table 2 5. Preparation of tripeptides 2. 6a, b and the diastereomeric mixture ( 2.6b+2.6b' ) Entry Reactant Product Yield a (%) m p (C) 23 D t R (min ) b 1 Cbz L -Ala L Trp Bt 2.5 Cbz L -Ala L Trp -NCbz L -Lys -O H 2.6a 75 137 139 8.94 3.16 2 Cbz L -Phe -L -Met Bt 2. 5b Cbz L -Phe -L -Met NCbz L -Lys -OH 2.6b 72 135 140 7.76 3.09 3 Cbz L Phe DL Met Bt (2.5b+2.5b' ) Cbz L Phe D L Met NCbz L -Ly s -OH ( 2.6b+2.6b' ) 75 126 127 6.98 3.09, 3.60 a Isolated yields; b t R : Retention time; for conditions, see the experimental section. Scheme 2 10. Preparation of tripeptides 2.6c -e and the diastereomeric mixture ( 2.6e+2.6e' ) 1H NMR showed three separa te sets of doublets for the tripeptides 2. 6c -e ranging from 7.5 4 9.3 8 ppm. In case of 2. 6e the L -Ala fragment showed a clear doublet at 1.24 ppm, but gave a multiplet in the range of 1.201.30 ppm for the corresponding diastereomeric mixture (2. 6e+ 2. 6e' ). One of the NH protons in the diastereomeric mixture ( 2. 6e+ 2. 6e' ) showed a multiplet in the range of 8.18 8.32 ppm, instead of a clear doublet as observed in the enantiopure 2. 6e

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40 Table 2 6. Preparation of tripeptides 2.6c -e and the diastereomeric mixtu re ( 2.6e+2.6e' ) Entry Reactant Product Yield a (%) m p (C) 23 D t R (min ) b 1 L Met 2. 2c Cbz L -Phe N Cbz L Lys L Met OH 2.6c 73 137 139 10.99 2.73, 3.14c 2 L Trp 2. 2d Cbz L -Phe -N Cbz -L Lys L Trp OH 2.6d 88 71 73 2.09 3.08, 3.61c 3 L -Ala 2. 2f Cbz L Phe N Cbz L Lys L Ala OH 2.6e 74 129 131 9.60 3.37 4 D L Ala (2. 2f+ 2. 2f' ) Cbz L -Phe -NCbz -L Lys D L -Ala OH ( 2.6e+2.6e' ) 90 99101 7.35 3.37, 3.58 aIsolated Yields; btR: Retention time; for conditions, see the experimental section; c Diastereomeric ratio = 9:1 The HPLC results showed a single peak for 2.6a,b ,e By contrast two peaks were observed for the corresponding diastereomeric mixture ( 2.6b+2.6b ) and ( 2.6e+2.6e ) confirming the enantiopurity of the LL -dipeptides 2.6b and 2.6e However, two peaks were observed for 2.6c d in the ratio of 9:1 suggesting e ither epimeri zation or the existence of rotamers; however, no precise reason can yet be attributed to this observation. 2.2.6. Preparation of Unnatural Tripeptide 2.6f by E xtension from the N -Terminus of NCbz L -Lys 2.2b Peptide coupling reaction was ca rried out successfully at the side chain amino functionality of lysine using NCbz -L Lys 2 2 b and N Cbz -L -Ala -L Trp Bt ( Scheme 2 11) in a similar procedure to that adopted for the natural tripeptides. Unnatural tripeptide 2.6f was obtained in 82% yield an d enantiopurity. Scheme 2 11. Preparation of unnatural tripeptide 2. 6f

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41 2.3 Conclusion In summary, convenient preparations of na tural and unnatural di and tri peptides derived from lysine by extension at the N-, N, and C termini has been demonstrate d under mild reaction conditions. The reactions gave di and tri peptides in high yields with easy isolation and purification procedures. 2.4 Experimental Section 2.4.1 General M ethods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. NMR spectra were recorded in CDCl3 or DMSO d6 with TMS as an internal standard for 1H (300 MHz) or solvent as an internal standard for 13C (75 MHz). Elemental analyses were performed on a CarloErba 1106 instrument. Optical rotat ion values were measured with the use of sodium D line. N -Cbz and Fmoc amino acids were purchased from Fluka and Acros, were used without further purification. HPLC analyses were performed on Beckman system gold programmable solvent module 126 using Chi robiotic T column (4.6 x 250 mm), detection at 254 nm, flow rate of 1.0 mL/min and MeOH as an eluting solvent. 2.4.2 General P rocedure for the P reparation of Cbz -DL -Met -Bt (2. 1d+ 2. 1d' ) and N-Fmoc N -Cbz -L -Lys -Bt 2. 1h Cbz DL -Met -Bt ( 2. 1d+2.1d' ) and NFmoc NCbz -L -Lys -Bt 2.1h were prepared using the reported procedure.21 To a solution of 1 H -benzotriazole (8 mmol) in dry THF (10 mL), SOCl2 (3 mmol) was added and the reaction mixture was stirred for 20 min at room temperature. To this solution, N -protected a mino acids Cbz DL -Met OH or NFmoc NCbz L -Lys OH 2. 2 c (2 mmol) was directly added and reaction mixture was stirred for 2 h at room tempreture. Progress of the reaction was monitored by 1H NMR. The white precipitate obtained was filtered off and the filtrate was concentrated under v acuum. To the residue obtained, ethyl acetate (100 mL) was

PAGE 42

42 added and the solution was washed with 4N HCl (3 x 50 mL) solution followed by brine (25 mL) The organic layer was dried over anhydrous MgSO4 and the solvent was removed under reduced pressure. Compounds were recrystallized from CHCl3/hexane s for elemental analysis Benzyl -N -[1 -(1 H -1,2,3 -benzotriazol -1 -ylcarbonyl) -3 (methylsulfanyl)propanoyl]carbamate, Cbz -DL -Met -Bt, (2. 1d+2.1d' ) : Colorless microcrystals (93%); mp 93 94 C; 1H NMR (DMSO d6) 2.05 ( s, 3H), 2.20 2.34 (m, 2H), 2.58 2.78 (m, 2H), 5.06 (s, 2H), 5.60 5.72 (m, 1H), 7.30 7.42 (m, 5H), 7.66 (t, J = 7.6 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 8.20 8.34 (m, 3H). 13C NMR (DMSO d6) 14.2, 29.6, 29.7, 53.4, 65.9, 114.0, 120.2, 126.8, 127.9, 128.0, 12 8.4, 130.7, 131.2, 136.7, 145.4, 156.4, 171.9. Anal. Calcd for C19H20N4O3S: C, 59.36; H, 5.24; N, 14.57. Found: C, 59.22; H, 5.20; N, 14.66. 9 H -Fluoren -9 -ylmethyl -N -[(1 S )-5 benzyloxycarbonylamino-1 -(1 H -1,2,3 -benzotriazol 1 -yl -carbonyl)pentyl]carbamate, N-Fmoc -N -Cbz -L Lys Bt, 2.1h : Colorless microcrystals (70%); mp 148 149 C; [ ]23 D = 35.94 (c 1.0, DMF); 1H NMR (DMSO d6) 1.25 1.60 (m, 4H), 1.71 2.03 (m, 2H), 2.90 3.07 (m, 2H), 4.20 4.29 (m, 1H), 4.30 4.40 (m, 2H), 4.97 (s, 2H), 5.48 7.2 0 7.38 (m, 9H), 7.42 (t, J = 7.4 Hz, 2H), 7.65 (t, J = 7.7 Hz, 1H), 7.72 (d, J = 7.3 Hz, 2H), 7.82 (t, J = 7.7 Hz, 1H), 7.90 (d, J = 7.4 Hz, 2H), 8.24 (d, J = 8.5 Hz, 1H), 8.30 (d, J = 8.5 Hz, 1H). 13C NMR (CDCl3) 22.4, 29.2, 32.2, 40.1, 47.1, 54.4, 66.6, 67.1, 114.3, 119.9, 120.3, 125.0, 126.5, 127.0, 127.7, 128.0, 128.4, 130.7, 131.0, 136.4, 141.2, 143.6, 143.7, 145.9, 156.2, 156.6, 171.7. Anal. Calcd for C35H33N5O5: C, 69.64; H, 5.51; N, 11.60. Found: C, 69.39; H, 5.64; N, 11.30. 2.4.3 General Procedur e for the P reparation of LL -Dipeptides 2.3a -g and the Diastereomeric M ix ture (2.3b+2.3b') and (2.3d+2.3d') N (Protected aminoacyl)benzotriazoles 2.1a -g (0.5 mmol) were added to a solution of NCbz L -Lys 2.2a (0.5 mmol) in CH3CN /H2O ( 5 mL/ 10 mL) in the presence of Et3N (0.6

PAGE 43

43 mmol) at room temperature. The reaction mixture was then stirred at room temperature until the star ting material was completely consumed as observed on TLC using hexane s / EtOAc (2:1) as the eluent After 1 mL of 4N HCl was added, the solution was concentrated under reduced pressure to remove CH3CN The solution was extracted with EtOAc (20 mL), and the o rganic extract was washed with 4N HCl (5 mL) and saturated NaCl (10 mL), then dried over anhydrous MgSO4. Evaporation of the solvent gave the desired product in pure form, which was further recrystallized from CHCl3/hexane s unless specified, otherwise. (S )-6 -Benzyloxycarbonylamino -2 -[(S )-2 benzyl oxycarbonylaminopropanoylamino] hexanoic acid, N -Cbz L Ala N-Cbz L -Lys -OH, 2.3a: Colorless microcrystals (89%); mp 92 94 C; [ ]23 D = +4.04 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 (d, J = 7.1 Hz, 3H), 1.23 1.48 (m, 4H), 1.49 1.78 (m, 2H), 2.97 (apparent d, J = 5.8 Hz, 2H), 4.03 4.20 (m, 2H), 5.00 (s, 4H), 7.18 7.40 (m, 11H), 7.41 (d, J = 7.7 Hz, 1H), 8.03 (d, J = 7.7 Hz, 1H), 12.55 (s, 1H). 13C NMR (DMSO d6) 18.2, 22.7, 29.0, 30.8, 40.1, 49.8, 51.8, 65.2, 65.4, 127. 8, 128.4, 137.1, 137.3, 155.6, 156.1, 172.7, 173.6. Anal. Calcd for C25H31N3O7: C, 61.84; H, 6.44; N, 8.65. Found: C, 62.10; H, 6.62; N, 8.35. (S )-6 -Benzyloxycarbonylamino -2 -(2 benzyloxycarbonylaminopropanoylamino)hexanoic acid, N -Cbz D L Ala -N Cbz -L -Lys O H, (2.3a+2.3a'): Colorle ss mi crocrystals (88%); mp 105 108 C; [ ]23 D = 1.86 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 (d, J = 7.1 Hz, 3H), 1.20 1.46 (m, 4H), 1.50 1.78 (m, 2H), 2.88 3.04 (m, 2H), 4.03 4.22 (m, 2H), 4.99 (s, 4H), 7.16 7.50 (m, 12H), 8.00 8.15 (m, 1H), 12.58 (br s, 1H). 13C NMR (DMSO d6) 18.2, 18.7, 22.6, 22.7, 29.0, 29.1, 30.8, 41.0, 49.8, 50.0, 51.6, 51.8, 65.2, 65.4, 127.8, 128.4, 137.1, 137.3, 155.6, 155.7, 156.1, 172.5, 172.7, 173.6. Anal. Calcd for C25H31N3O7: C, 61.84; H, 6.44; N, 8.65. Found: C, 61.92; H, 6.50; N, 8.82.

PAGE 44

44 (S) -6 -Benzyloxycarbonylamino -2 -[(S)-2 benzyloxy carbonylamino -3 phenylpropanoyl amino]hexanoic acid, N -Cbz L -Phe -N Cbz -L -Lys -OH, 2.3b : White microcrystals (95%); mp 117 119 C; [ ]23 D = 4.53 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.50 (m, 4H), 1.50 1.82 (m, 2H), 2.61 2.80 (m, 1H), 2.88 3.15 (m, 3H), 4.11 4.23 (m, 1H), 4.23 4.36 (m, 1H), 4.92 (s, 2H), 5.00 (s, 2H), 7.08 7.44 (m, 16H), 7.48 (d, J = 8.8 Hz, 1H), 8.27 (d, J = 7.1 Hz, 1H), 12.63 (s, 1H). 13C NMR (DMSO d6) 22.9, 29.2, 30.9, 37.6, 40.2, 52.1, 56.1, 65.3, 65.4, 126.4, 127.0, 127.6, 127.8, 127.9, 128.2, 128.4, 128.5, 129.4, 137.1, 137.4, 138.2, 156.0, 156.3, 172.0, 173.7. Anal. Calcd for C31H35N3O7: C, 66.30; H, 6.28; N, 7.48. Found: C, 66.47; H, 6.47; N, 7.21. (S )-6 -Benzyloxycarbonylamino -2 -[(S )-2 benzyloxycarbonylamino-3 -(1 H -indol -3 yl)propanoylamino]hexanoic acid, N -Cbz -L -Trp -N Cbz -L -Lys -OH, 2.3c: White microcrystals (85%); mp 114 116 C; [ ]23 D = 15.44 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.50 (m, 4H), 1.53 1.83 (m, 2H), 2.83 2.99 (m, 3H), 3.04 3.18 (m, 1H), 4.13 4.28 (m, 1H), 4.28 4.41 (m, 1H), 4.92 (s, 2H), 4.99 (s, 2H), 6.97 (t, J = 7.1 Hz, 1H), 7.06 (t, J = 7.7 Hz, 1H), 7.16 (s, 1H), 7.18 7.42 (m, 13H), 7.66 (d, J = 7.4 Hz, 1H), 8.25 (d, J = 7.4 Hz, 1H), 10.81 (s, 1H), 12.61 (s, 1H). 13C NMR (DMSO d6) 22.8, 27.9, 29.1, 30.8, 40.2, 52.0, 55.3, 65.2, 65.3, 110.2, 111.3, 118.2, 118.7, 120.9, 124.0, 127.3, 127.5, 127.7, 127.8, 128.4, 136.1, 137.0, 137.3, 155.9, 156.2, 172.1, 173.7. Anal. Calcd for C33H36N4O7: C, 65.99; H, 6.04; N, 9.33. Found: C, 65.84; H, 6.10; N, 9.20. (S )-6 -Benzyloxycarbonylamino -2 -[(S )-2 benzyloxycarbo nylamino -4 methylsulfanylbutano ylamino]hexanoic acid, N -Cbz -L -Met -N Cbz L -Lys -OH, 2.3d: White microcrystals (83%); mp 122 123 C; [ ]23 D = 1.94 (c 1.0, DMF); 1H NMR (DMSO d6) 1.24 1.48 (m, 4H), 1.48 1.64 (m, 1H), 1.64 1.74 (m, 1H), 1.76 1.94 (m, 2H), 2.02 (s, 3H),

PAGE 45

45 2.42 2.54 (m, 2H), 2.92 3.04 (m, 2H), 4.05 4.20 (m, 2H), 5.00 (s, 2H), 5.01 (s, 2H), 7.18 7.45 (m, 11H), 7.48 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 7.7 Hz, 1H), 12.56 (s, 1H). 13C NMR (DMSO d6) 14.6, 22.7, 29.5 (2C), 30.6, 31.9, 51.9, 53.7, 65.1, 65.4, 127.7 (2C), 128.4 (2C), 137.0, 137.3, 155.9, 156.1, 171.6, 173.6. Anal. Calcd for C27H35N3O7S: C, 59.43; H, 6.47; N, 7.70. Found: C, 59.66; H, 6.69; N, 7.43. (S )-6 -Benzyloxycarbonylamino -2 -(2 -benzyloxycarbonylamino -4 methylsulfanylbutanoyl amino)hexanoic acid, N -Cbz -D L -Met -N Cbz L Lys -OH, (2.3d+2.3d'): Colorless microcrystals (91%); mp 45 46 C; [ ]23 D = 0.40 (c 1.0, DMF); 1H NMR (D MSO d6) 1.20 1.47 (m, 4H), 1.50 1.75 (m, 2H), 1.75 1.90 (m, 2H), 2.01 (s, 1.5H), 2.02 (s, 1.5H), 2.49 2.55 (m, 2H), 2.90 3.02 (m, 2H), 4.07 4.22 (m, 2H), 5.00 (s, 2H), 5.01 (s, 2H), 7.207.40 (m, 11H), 7.47 (t, J = 9.1 Hz, 1H), 8.14 (d, J = 7.1 Hz, 1H), 12.60 (s, 1H). 13C NMR (DMSO d6) 14.6, 14.7, 22.6, 22.8, 29.0, 29.1, 29.6, 29.7, 30.6, 30.8, 31.9, 32.0, 51.7, 51.9, 53.7, 53.8, 65.2, 65.4, 65.5, 127.7, 127.8, 127.8, 128.4, 137.0, 137.3, 156.0, 156.1, 171.5, 171.6, 173.5, 173.6. Anal. Calcd for C27H35N3O7S: C, 59.43; H, 6.47; N, 7.70. Found: C, 59.38; H, 6.48; N, 7.74. (S )-6 -Benzyloxycarbonylamino -2 -[(S )-2 -(9 H -fluoren -9 -y lmethoxycarbonylamino) -4 methyl sulfanylbutanoylamino]hexanoic acid, N Fmoc -L Trp -N Cbz -L Lys -OH, 2.3e: White microcrystals (88%); mp 91 93 C; [ ]23 D = 14.71 (c 1.0, DMF); 1H NMR (DMSO d6) 1.21 1.49 (m, 4H), 1.52 1.68 (m, 1H), 1.68 1.82 (m, 1H), 2.87 3.04 (m, 3H), 3.06 3.18 (m, 1H), 4.08 4.18 (m, 3H), 4.18 4.27 (m, 1H), 4.30 4.45 (m, 1H), 4.98 (s, 2H), 6.97 (t, J = 7.1 Hz, 1H), 7.06 (t, J = 7.7 Hz, 1H), 7.17 7.46 (m, 12H), 7.52 (d, J = 8.2 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 7.7 Hz, 1H), 7.86 (d, J = 7.4 Hz, 2H), 8.27 (d, J = 7.4 Hz, 1H), 10.82 (s, 1H), 12.62 (br s, 1H). 13C NMR (DMSO d6) 22.8, 27.8, 29.11, 30.8, 46.6, 52.0, 55.3, 65.2, 65.7,

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46 110.3, 111.3, 118.2, 118.7, 120.1, 120.9, 124.0, 124.1,125.3, 125.5, 127.1, 127.3, 127.6, 127.8, 128.4, 136.1, 137.3, 140.7, 143.8, 143.9, 155.8, 156.1, 172.2, 173.6. Anal. Calcd for C40H40N4O7: C, 69.75; H, 5.85; N, 8.13. Found: C, 69.51; H, 5.98; N, 8.01. (S )-6 -Benzyloxycarbonylamino -2 -[(S )-2 -(9 H -fluoren -9 -y lmethoxycarbonylamino) -4 methyl sulfanylbutanoylamino]hexanoic acid, N Fmoc -L -Met -N -Cbz L Lys -OH, 2.3f: White microcrystals (85%); mp 122 C; [ ]23 D = 7.06 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.48 (m, 4H), 1.50 1.63 (m, 1H), 1.63 1.76 (m, 1H), 1.76 1.97 (m, 2H), 2.03 (s, 3H), 2.43 2.54 (m, 2H), 2.90 3.02 (m, 2H), 4.08 4.19 (m, 2H), 4.19 4.31 (m, 3H), 4.98 (s, 2H), 7.20 7.36 (m 8H), 7.41 (t, J = 7.4 Hz, 2H), 7.57 (d, J = 8.2 Hz, 1H), 7.72 (t, J = 6.3 Hz, 2H), 7.88 (d, J = 7.4 Hz, 2H), 8.13 (d, J = 8.4 Hz, 1H), 12.56 (s, 1H). 13C NMR (DMSO d6) 14.7, 22.7, 29.0, 29.6, 30.6, 31.9, 46.7, 51.9, 53.6, 65.1, 65.6, 120.1, 125.3, 127. 0, 127.6, 127.7, 128.3, 137.2, 140.7, 143.7, 143.9, 156.9, 156.0, 171.6, 173.5. Anal. Calcd for C34H39N3O7S: C, 64.44; H, 6.20; N, 6.63. Found: C, 64.23; H, 6.26; N, 6.61. (S )-6 -Benzyloxycarbonylamino -2 -[(S )-2 -(9 H -fluoren -9 -ylmeth oxycarbonylamino) -3 phenyl propanoylamino]hexanoic acid, N -Fmoc -L -Phe -N Cbz L -Lys -OH, 2.3g: White microcrystals (92%); mp 128 C; [ ]23 D = 13.03 (c 1.0, DMF); 1H NMR (DMSO d6) 1.24 1.50 (m, 4H), 1.55 1.69 (m, 1H), 1.69 1.82 (m, 1H), 2.70 2.85 (m, 1H), 2.90 3.10 (m, 3H), 4.05 4.25 (m, 4H), 4.25 4.40 (m, 1H), 4.99 (s, 2H), 7.10 7.45 (m, 15H), 7.57 7.68 (m, 3H), 7.87 (d, J = 7.4 Hz, 2H), 8.28 (d, J = 7.4 Hz, 1H), 12.65 (s, 1H). 13C NMR (DMSO d6) 22.8, 29.1, 30.8, 37.5, 40.2, 46.6, 52.0, 56.0, 65.2, 65.7, 120.1, 125.3, 125.4, 126.3, 127.1, 127.7, 127.8, 128.1, 128.4, 129.2, 129.3, 137.3, 138.3, 140.7, 143.7, 143.8, 155.9, 156.2, 171.9, 173.7. Anal. Calcd for C38H39N3O7: C, 70.25; H, 6.05; N, 6.47. Found: C, 70.60; H, 6.21; N, 6.44.

PAGE 47

4 7 2.4.4 General Procedure for the Preparation of unnatural LLDipeptides 2.3h-j and the Diastereomeric M ix ture (2.3h+2.3h') N (Protected aminoacyl)benzotriazoles 2. 1h -j and (2.3h+2.3h' ) (0.5 mmol) were added to a solution of NCbz -L Lys 2.2 b (0.5 mmol) in CH3CN /H2O ( 5 mL/5 mL) in the presence of Et3N (0.6 mmol) at room temperature. The reaction mixture was then stirred at room temperature until the starting material was completely consumed as observed on TLC using hexane s /EtOAc (2:1) as the eluent After 1 mL of 4N HCl was added, the solution was conc entrated under reduced pressure to remove CH3CN The solution was extracted with EtOAc (20 mL), and the organic extract was washed with 4N HCl (5 mL) and sat urated NaCl (10 mL), then dried over anhydrous MgSO4. Evaporation of the solvent gave the desired p roduct in pure form, which was further recrystallized from EtOAc /hexane s unless specified, otherwise. (S )-2 -Benzyloxycarbonylamino -6 -[(S )-2 benzyloxy carbonylamino -3 phenylpropanoyl amino]hexanoic acid, N-Cbz -N -(Cbz L -Phe) -L -Lys -OH, 2.3h: White microcryst als (79%); mp 162164 C; [ ]23 D = 12.88 (c 1.0, DMF); 1H NMR (DMSO d6) 1.19 1.42 (m, 4H), 1.45 1.75 (m, 2H), 2.68 2.81 (m, 1H), 2.85 3.15 (m, 3H), 3.85 3.95 (m, 1H), 4.10 4.22 (m, 1H), 4.85 5.07 (m, 4H), 7.10 7.40 (m, 15H), 7.49 (d, J = 8.5 Hz, 1H), 7. 56 (d, J = 7.8 Hz, 1H), 7.99 (br s, 1H), 12.58 (s, 1H). 13C NMR (DMSO d6) 22.9, 28.6, 30.4, 37.8, 38.3, 53.8, 56.3, 65.2, 65.4, 126.2, 127.5, 127.7, 127.8, 127.8, 128.0, 128.3, 128.4, 129.2, 137.0, 137.1, 138.1, 155.8, 156.2, 171.2, 174.1. Anal. Calcd fo r C31H35N3O7: C, 66.30; H, 6.28; N, 7.48. Found: C, 66.17; H, 6.34; N, 7.59. (S )-2 -Benzyloxycarbonylamino -6 -[2 -benzyloxycarbonyl amino -3 phenylpropanoylamino] hexanoic acid N-Cbz -N -(Cbz -DL -Phe) L -Lys OH, (2.3h + 2.3h'): White microcrystals (95%); mp 93 95 C ; [ ]23 D = 5.00 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.45 (m, 4H), 1.48 1.75 (m, 2H), 2.69 2.81 (m, 1H), 2.85 3.15 (m, 3H), 3.85 3.95 (m,

PAGE 48

48 1H), 4.10 4.23 (m, 1H), 4.94 (s, 2H), 5.02 (s, 2H), 7.10 7.40 (m, 15H), 7.45 (m, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.94 (m, 1H), 12.56 (s, 1H). 13C NMR (DMSO d6) 23.0, 28.6, 28.6, 30.4, 30.5, 37.8, 38.3, 38.4, 53.8, 56.3, 65.2, 65.4, 126.3, 127.5, 127.7, 127.8, 127.9, 128.1, 128.3, 128.4, 129.3, 137.1, 137.1, 138.2, 155.9, 156.3, 171.2, 174.1, 174.1. Anal. Calc d for C31H35N3O7: C, 66.30; H, 6.28; N, 7.48. Found : C, 66.02; H, 6.38; N, 7.66. (S )-2 -Benzyloxycarbonylamino -6 -[(S )-2 benzyloxycarbonylamino-3 -(1 H -indol -3 yl)propanoylamino]hexanoic acid, N-Cbz -N -(Cbz -L -Trp) L -Lys -OH, 2.3i: White microcrystals (93%) ; mp 87 89 C; [ ]23 D = 17.94 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.45 (m, 4H), 1.48 1.75 (m, 2H), 2.83 2.97 (m, 1H), 2.97 3.11 (m, 3H), 3.85 3.97 (m, 1H), 4.15 4.27 (m, 1H), 4.94 (s, 2H), 4.98 (d, J = 12.9 Hz, 1H, A part of AB system), 5.04 (d, J = 12.6 Hz 1H, B part of AB system), 6.96 (t, J = 7.3 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 7.13 (s, 1H), 7.207.40 (m, 12H), 7.56 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.95 8.05 (m, 1H), 10.79 (s, 1H), 12.52 (br s, 1H). 13C NMR (DMSO d6) 23.0, 28.1, 28.6, 30.5, 38.4, 53.9, 55.7, 65.3, 65.5, 110.3, 111.3, 118.2, 118.6, 120.9, 123.9, 127.3, 12.5, 127.6, 127.7, 127.8, 127.9, 128.3, 128.4, 136.1, 137.1, 155.9, 156.3, 171.7, 174.1. Anal. Calcd for C33H36N4O7: C, 65.99; H, 6.04; N, 9.33. Found : C, 65.94; H, 6.10; N, 9.20. (S )-2 -Benzyloxycarbonylamino -6 -[(S )-2 -(9 H -fluoren -9 -y lmethoxycarbonylamino) -4 methyl sulfanylbutanoylamino]hexanoic acid, N-Cbz -N -(Fmoc -L -Met)L -Lys -OH, 2.3j: White microcrystals (94%); mp 83 C; [ ]23 D = 9.70 ( c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.47 (m, 4H), 1.50 1.75 (m, 2H), 1.75 1.95 (m, 2H), 2.03 (s, 3H), 2.36 2.50 (m, 2H) 2.95 3.14 (m, 2H), 3.87 3.98 (m, 1H), 3.98 4.09 (m, 1H), 4.18 4.33 (m, 3H), 5.02 (s, 2H), 7.25 7.38 (m, 6H), 7.41 (t, J = 7.4 Hz, 3H), 7.52 7.56 (m, 2H), 7.68 7.77 (m, 2H), 7.89 (d, J = 8.4 Hz, 3H), 12.56 (s, 1H). 13C NMR (DMSO d6) 14.6, 23.0, 28.6, 29.8, 30.4, 31.0, 31.7, 38.3, 46.7,

PAGE 49

49 53.9, 65. 4, 65.6, 120.1, 125.3, 127.1, 127.6, 127.7, 127.8, 128.3, 137.0, 140.8, 143.8, 143.9, 156.0, 156.2, 171.3, 174.1. Anal. Calcd for C34H39N3O7S: C, 64.44; H, 6.20; N, 6.63. Found: C, 64.54; H, 6.25; N, 6.85. 2.4. 5 General Procedure for the P reparation of LL -Dipeptides 2.3k n and the Diastereomeric M ixture (2.3l+2.3l') from Extension at the C -Terminus of Benzotriazole derivative, N-Fmoc -N -Cbz -L Lys Bt 2. 1h N-Fmoc NCbz L -Lys -Bt 2.1h (0.4 mmol) was added to a solution of NCbz -L -Lys 2.2a or unprotected ami no acids ( L -Met, DL -Met, L Trp, L -Ser) 2.2a c -e and ( 2.2c+2.2c ) (0.4 mmol) in CH3CN (5 mL)/H2O (10 mL) in the presence of Et3N (0.5 mmol) at room temperature The reaction mixture was then stirred at room temperature until the starting material was compl etely consumed as observed on TLC using EtOAc/hexane s (1:2) as the solvent. After 1 mL of 4N HCl was added, the solution was concentrated under reduced pressure to remove CH3CN The solution was extracted with EtOAc (20 mL), and the organic extract was was hed with 4N HCl (5 mL) and sat urated NaCl (10 mL), and then dried over anhydrous MgSO4. Evaporation of the solvent gave the desired product in pure forms, which were further recrystallized from CHCl3/hexane s (S )-6 -Benzyloxycarbonylamino -2 -[(S )-6 benzyloxy carbonylamino -2 -(9 H -fluoren -9 ylmetho xycarbonylamino)hexanoylamino]hexanoic acid, N -Fmoc -N -Cbz L -Lys -N -Cbz L -Lys -OH, 2.3k: White microcrystals (74%) ; mp 103 C; [ ]23 D = 4.52 (c 1.0; DMF); 1H NMR (DMSO d6) 1.20 1.48 (m, 8H), 1.48 1.75 (m, 4H), 2.88 3.05 (m, 4H), 3.90 4.15 (m, 2H), 4.18 4.32 (m, 3H), 4.90 5.10 (m, 4H), 7.20 7.44 (m, 16H), 7.48 (d, J = 8.0 Hz, 1H), 7.63 7.76 (m, 2H), 7.88 (d, J = 7.1 Hz, 2H), 8.08 (d, J = 7.9 Hz, 1H), 12.47 (br s, 1H). 13C NMR (DMSO d6) 22.7, 22.8, 22.9, 29.1, 29.2, 30.7, 31.7, 40.2, 46.7, 51.9, 53.9, 54.4, 65.2, 65.6, 120.1, 125.4, 127.1, 127.4, 127.7, 127.8, 128.4, 137.3, 140.7, 143.8, 144.0, 156.0, 156.1, 156.2,

PAGE 50

50 172.1, 173.6. Anal. Calcd for C43H48N4O9: C, 67.52; H, 6.33; N, 7.32. Found: C, 67.77; H, 6.54; N, 6.93. (S )-2 -[(S ) -6 -Benzyloxycarbonylamino -2 -(9 H -fluoren -9 ylmethoxycarbonylamino)hexanoyl amino] -4 -(methylsulfanyl) butanoic acid, N -Fmoc -N Cbz L -Lys -L -M et -OH, 2.3l: Whi te microcrystals (95%); mp 138 140 C; [ ]23 D = 2.09 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.51 (m, 4H), 1.51 1.75 (m, 2H), 1.75 1.98 (m, 2H), 2.01 (s, 3H), 2.37 2.61 (m, 2H), 2.90 3.10 (m, 2H), 3.93 4.11 (m, 1H), 4.15 4.45 (m, 4H), 5.00 ( s, 2H), 7.20 7.46 (m, 10H), 7.49 (d, J = 8.2 Hz, 1H), 7.68 J = 7.4 Hz, 2H), 8.16 (d, J = 7.7 Hz, 1H), 12.70 (br s, 1H). 13C NMR (DMSO d6) 14.6, 22.8, 29.2, 29.6, 30.8, 31.5, 40.2, 46.7, 50.9, 54.4, 65.1, 65.6, 120.1, 125.4, 127.1, 127.7, 127.8, 128.4, 137.3, 140.7, 143. 8, 143.9, 156.0, 156.1, 172.3, 173.3. Anal. Calcd for C34H39N3O7S: C, 64.44; H, 6.20; N, 6.63. Found: C, 64.25; H, 6.23; N, 6.50. 2 -[(S )-6 -Benzyloxycarbonylamino-2 -(9 H -fluoren -9 ylmethoxycarbonylamino)hexanoyl amino] -4 -(methylsulfanyl)butanoic acid, N -Fmo c -N Cbz L -Lys -D L -Met -OH, (2.3l+2.3l'): White microcrystals (90%) ; mp 80 81 C; [ ]23 D = 3.94 (c 1.0, DMF); 1H NMR (DMSO d6) 1.22 1.47 (m, 4H), 150 1.74 (m, 2H), 1.75 2.05 (m, 2H), 2.00 (s, 1.5H), 2.01 (s, 1.5H), 2.40 3.04 (m, 2H), 3.95 4.08 (m, 1H), 4.15 4.38 (m, 4H), 4.99 (s, 2H), 7.20 7.36 (m, 8H), 7.41 (t, J = 7.4 Hz, 2H), 7.49 (d, J = 7.5 Hz, 1H), 7.68 7 .76 (m, 2H), 7.89 (d, J = 7.7 Hz, 2H), 8.15 (d, J = 7.7 Hz, 0.5 H), 8.21 (d, J = 8.0 Hz, 0.5 H), 12.67 (s, 1H). 13C NMR (DMSO d6) 14.5, 14.6, 22.8, 29.1, 29.2, 29.6, 30.8, 31.5, 32.0, 40.1, 46.7, 50.8, 50.8, 54.4, 65.1, 65.6, 65.7, 120.1, 125.4, 127.1, 127.7, 127.8, 128.4, 137.3, 140.7, 143.8, 143.9, 155.9, 155.9, 156.1, 172.1, 172.2, 173.2, 173.2. Anal. Calcd for C34H39N3O7S: C, 64.44; H, 6.20; N, 6.63. Found: C, 64.43; H, 6.17; N, 6.43.

PAGE 51

51 (S )-2 -[(S ) -6 -Benzyloxycarbonylamino -2 -(9 H -fluoren -9 ylmethoxycarbon ylamino)hexanoyl amino] -3 -(1 H -indol -3 -yl)propanoic acid, N -Fmoc -N Cbz L -Lys -L Trp -OH, 2.3m: White microcrystals (88%); mp 67 69 C; [ ]23 D = +2.40 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.75 (m, 6H), 2.90 3.04 (m, 2H), 3.00 3.25 (m, 2H), 3.85 4.08 (m, 1H) 4.15 4.36 (m, 3H), 4.40 4.55 (m, 1H), 5.00 (s, 2H), 6.96 (t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 7.15 (s, 1H), 7.20 7.59 (m, 12H), 7.64 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.1 Hz, 2H), 7.88 (d, J = 8.11, 2H), 8.11 (d, J = 7.4 Hz, 1H), 10.86 (s, 1H) 12.65 (br s, 1H). 13C NMR (DMSO d6) 22.9, 27.1, 29.2, 31.7, 40.2, 46.7, 52.9, 54.5, 65.2, 65.7, 109.6, 111.4, 118.2, 118.4, 120.2, 120.9, 123.7, 125.4, 127.1, 127.3, 127.7, 127.8, 128.4, 136.1, 137.3, 140.8, 143.8, 144.0, 156.0, 156.1, 172.2, 173.3. An al. Calcd for C40H40N4O7: C, 69.75; H, 5.85; N, 8.13. Found: C, 69.81; H, 6.14; N, 7.92. (S )-2 -[(S ) -6 -Benzyloxycarbonylamino -2 -(9 H -fluoren -9 ylmethoxycarbonylamino)hexanoyl amino] -3 -hydroxypropanoic acid, N -Fmoc -N -Cbz -L Lys -L Ser -OH, 2.3n: White microcrys t als (80%); mp 85 89 C; [ ]23 D = 1.47 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 3.65 (m, 1H), 3.65 3.78 (m, 1H), 4.00 4.15 (m, 1H), 4.15 4.35 (m, 4H), 4.99 (s, 2H), 7.20 7.36 (m, 8H), 7.41 (t, J = 7.4 Hz, 2H), 7.50 (d, J = 8.5 Hz, 1H), 7.68 7.76 (m, 2H), 7.88 (d, J = 7.4 Hz, 2H), 8.04 (d, J = 7.0 Hz, 1H), OH and COOH from -Ser missing is not observed in 1H NMR. 13C NMR (DMSO d6) 22.8, 29.2, 31.7, 40.2, 46.7, 54.4, 54.6, 61.4, 65.2, 65.7, 120.1, 125.4, 127.1, 127.7, 127.8, 128.4, 137.3, 140.7, 143.8, 144.0, 156.0, 156.1, 172.0, 172.2. Anal. Calcd for C32H35N3O8: C, 65.18; H, 5.98; N, 7.13. Found: C, 65.25; H, 6.07; N, 7.02.

PAGE 52

52 2.4.6 General P rocedure for P reparation o f D ipeptidoylbenzotriazoles 2.5a -c a nd the D iastereomeric Mixture (2.5b+2.5b ) To a solution of 1 H -benzotriazole (8 mmol) in dry THF (10 mL), SOCl2 (2.2 mmol) was added and the reaction mixture was stirred for 20 min at room temperature. It was then cooled down to 15 oC in ice salt bath. A cold solution of dipeptides 2.4 a,b, 2.3b and the diaster e omeric mixture ( 2.4b+2.4b' )(2 mmol) dissolved in dry THF (5 mL) was added drop wise, and the reaction mixture was stirred for 6 h at 15 oC. Progress of reaction was monitored by 1H NMR. The white p recipitate obtained was filtered off and the filtrate was concentrated under vacuum. To the residue obtained, ethyl acetate was added and the solution was washed with dilute Na2CO3 and brine. The organic layer was dried over anhydrous MgSO4 and the solvent was removed under reduced pressure. Compounds were recrystallized from CHCl3/hexane s for elemental analysis (S )-2 -Benzyloxycarbonylamino -N -[(1 S ) -2 -(1 H -1,2,3 -benzotriazol -1 -yl) -1 -(1 H -indol -3 yl methyl) -2 -oxoethyl]propanamide, N -Cbz -L Ala -L -Trp -Bt, 2.5a : Wh ite microcrystals (70%); mp 162 C; [ ]23 D = 36.99 (c 1.0, DMF); 1H NMR (DMSO d6) 1.24 (d, J = 7.0 Hz, 3H), 3.20 3.55 (m, 2H), 4.15 4.25 (m, 1H), 4.97 (d, J = 12.5 Hz, 1H, A part of AB system), 5.00 (d, J = 12.6 Hz, 1H, B part of AB system), 5.83 5. 95 (m, 1H), 6.91 (t, J = 7.2 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 7.20 7.40 (m, 7H), 7.46 (d, J = 7.7 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.77 (t, J = 7.7 Hz, 1H), 8.18 (d, J = 8.2 Hz, 1H), 8.23 (d, J = 8.2 Hz, 1H), 8.79 (d, J = 6.0 Hz, 1H), 10.89 (s, 1H). 13C NMR (DMSO d6) 18.1, 27.0, 49.6, 53.8, 65.4, 108.8, 111. 5, 114.0, 118.1, 118.5, 120.2, 121.1, 124.3, 126.7, 126.9, 127.9, 128.4, 130.6, 131.1, 136.1, 137.0, 145.3, 155.7, 171.5, 173.3 Anal. Calcd for C28H26N6O: C, 65.87; H, 5.1 3; N, 16.46. Found: C, 65.67; H, 5.19; N, 16.36.

PAGE 53

53 (S )-2 -Benzyloxycarbonylamino -N -[(1 S ) -1 -(1 H -1,2,3 -benzo triazol -1 -ylcarbonyl) -3 (methyl sulfanyl)propyl] -3 -phenylpropanamide, N -Cbz -L -Phe L -Met -Bt, 2.5b: Colorless microcrystals (76%); mp 110 C; [ ]23 D = 36.88 (c 1.0, DMF ); 1H NMR (DMSO d6) 2.07 (s, 3H), 2.10 2.23 (m, 1H), 2.23 2.40 (m, 1H), 2.55 2.85 (m, 3H), 2.93 3.10 (m, 1H), 4.30 4.48 (m, 1H), 4.94 (s, 2H), 5.70 7.40 (m, 10H), 7.59 (d, J = 7.5 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.8 2 (t, J = 7.8 Hz, 1H), 8.24 (d, J = 8.2 Hz, 1H), 8.31 (d, J = 8.2 Hz, 1H), 8.96 (d, J = 6.5 Hz, 1H). 13C NMR (DMSO d6) 14.4, 29.6, 30.1, 37.3, 51.9, 55.8, 65.3, 114.1, 120.2, 126.3, 126.7, 127.5, 127.7, 128.1, 128.3, 129.2, 130.7, 131.1, 136.9, 138.0, 145.4, 155.9, 171.1, 172.5. Anal. Calcd for C28H29N5O4S: C, 63.26; H, 5.50; N, 13.17. Found: C, 63.36; H, 5.45; N, 13.15. (S )-2 -Benzyloxycarbonylamino -N -[1 -(1 H -1,2,3 -benzotriazo l -1 ylcarbonyl) -3 (methylsulfan yl)propyl] -3 -phenylpropanamide, N -Cbz -L -Phe -DL -Me t-Bt, (2.5b+2.5b'): Colorless (65%); mp 140 C; [ ]23 D = 13.86 (c 1.0, DMF); 1H NMR (DMSO d6) 2.03 (s, 1.5H), 2.06 (s, 1.5H), 2.10 2.25 (m, 1H), 2.25 2.50 (m, 1H), 2.55 2.85 (m, 2H), 2.90 3.10 (m, 1H), 4.30 4.50 (m, 1H), 4.93 (s, 2H), 5.69 5.85 (m, 1H), 7.15 7.52 (m, 11H), 7.55 (t, J = 7.7 Hz, 1H), 7.65 (t, J = 7.8 Hz ,1H), 7.81 (t, J = 7.7 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.85 13C NMR (DMSO d6) 14.3, 14.4, 29.5, 29.6, 29.9, 30.1, 37.3, 38.1, 51.7, 52.0, 55.8, 65.3, 114.1, 120.3, 126.3, 126.4, 126.8 ,127.0, 127.5, 127.6, 127.8, 128.1, 128.2, 128.3, 129.3, 130.7, 131.1, 137.0, 137.8, 138.0, 145.4, 155.8, 155.9, 171.1, 171.2, 172.2, 172.5. Anal. Calcd for C28H29N5O4S: C, 63.26; H, 5.50; N, 13.17. Found: C, 62. 97; H, 5.46; N, 12.85. Benzyl -[( S )-6 benzotriazol -1 -yl -5 -((S )-2 benzyloxycarbonylamino-3 -phenylpropanoyl amino) -6 -oxohexyl]pentanoate, Cbz L -Phe -N -Cbz -L -Lys Bt, 2.5c : Colorless microcrystals

PAGE 54

54 (75%); mp 139 C; [ ]23 D = 17.19 ( c 1.0, DMF); 1H NMR (DMSOd6) 1.20 4H), 1.80 2.15 (m, 2H), 2.66 2.82 (m, 1H), 2.92 3.10 (m, 3H), 4.33 4.59 (m, 1H), 4.93 (s, 2H), 4.97 (s, 2H), 5.57 5.68 (m, 1H), 7.10 7.42 (m, 16H), 7.55 (d, J = 8.7 Hz, 1H), 7.6 5 (t, J = 7.7 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 8.24 (d, J = 8.2 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.91 (d, J = 6.2 Hz, 1H). 13C NMR (DMSO d6) 22.8, 29.1, 30.4, 37.4, 52.9, 55.7, 65.2, 65.3, 114.0, 120.3, 126.3, 126.8, 127.5, 127.8, 128.1, 128.3, 128.4, 129.2, 130.6, 131.1, 137.0, 137.3, 138.0 145.4, 155.9, 156.1, 171.44, 172.5. Anal. Calcd for C37H38N6O6: C, 67.05; H, 5.78; N, 12.68. Found: C, 66.89; H, 5.85; N, 12.32. 2.4. 7 Ge neral Procedure for P reparation of LLL-Tripeptides 2.6 a,b and the Diastereomeric M ixture ( 2. 6b+ 2. 6b') N-Cbz -dipeptidoylbenzotriazole s 2.5a, b and (2.5b+2.5b' ) (0.6 mmol) were added at 15 C to a solution of NCbz -L Lys 2.2a (0.6 mmol) in CH3CN (15 mL)/H2O (5 mL) in the presence of Et3N (0.7 mmol). The reaction mixtures were then s tirred at 15 C until the starting material was completely consumed as observed on TLC using EtOAc/hexanes (1:2) as the eluent. After 1 mL of 4N HCl was added, the solution was concentrated under reduced pressure to remove CH3CN The solution was extracte d with EtOAc (20 mL), and the organic extract was washed with 4N HCl (5 mL) and sat urated NaCl (10 mL), and then dried (anhydrous MgSO4). Evaporation of the solvent gave the desired products, which were purified by dissolving in ethyl acetate and precipitating with hexane s Compounds were recrystallized from EtOAc/hexane s for elemental analysis. (S )-6 -Benzyloxycarbonylamino -2 -[(S )-2 -(( S )-2 benzyloxycarbonylaminopropanoylamino) -3 -(1 H -indol -3 -yl)propanoylamino]hexanoic acid, N -Cbz -L Ala -L -Trp -N -Cbz -L -Lys -OH, 2.6a : Whi te microcrystals (75%); mp 137 139 C; [ ]23 D = 8.94 (c 1.0, DMF); 1H NMR (DMSO d6) 1.13 (d, J = 7.1 Hz, 3H), 1.20 1.49 (m,

PAGE 55

55 4H), 1.50 3.05 (m, 3H), 3.09 3.22 (m, 1H), 4.02 (quintet, J = 7.1 Hz, 1H), 4.11 4.23 (m, 1H), 4.50 4 .67 (m, 1H), 4.92 5.10 (m, 4H), 6.96 (t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 7.15 (s, 1H), 7.20 7.40 (m, 12H), 7.43 (d, J = 7.4 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 7.4 Hz, 1H), 10.82 (s, 1H), 12.63 (br s, 1H). 13C NMR (DMSO d6) 18.2, 22.7, 27.9, 29.1, 30.9, 40.2, 50.2, 52.0, 53.1, 65.2, 65.5, 109.9, 111. 3, 118.2, 118.5, 120.9, 123.7, 127.5, 127.8, 128.4, 136.1, 137.0, 137.3, 155.7, 156.2, 171.5, 172.3, 173.6. Anal. Calcd for C36H41N5O8: C, 64.37; H, 6.15; N, 10.43. Found: C, 64.47; H, 6.16; N, 10.38. (S )-6 -Benzyloxycarbonylamino -2 -[(S ) -2 -((S )-2 benzyloxycarbonylamino-3 phenylpropanoylamino) -4 -(methylsulfanyl)butanoylamino]hexanoic acid, N -Cbz -L Phe -L Met -N -Cbz -L Lys -OH, 2.6b : White microcrystals (72%); mp 135 1 40 C; [ ]23 D = 7.76 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.50 (m, 4H), 1.50 1.78 (m, 2H), 1.78 2.00 (m, 2H), 2.04 (s, 3H), 2.40 2.55 (m, 2H), 2.65 2.85 (m, 1H), 2.85 3.07 (m, 3H), 4.07 4.48 (m, 3H), 4.97 (s, 2H), 4.99 (s, 2H), 7.00 7.21 (m, 16H), 7.53 ( d, J = 8.5 Hz, 1H), 8.14 8.19 (m, 2H), 12.61(brs, 1H). 13C NMR (DMSO d6) 14.7, 22.8, 29.1, 29.3, 30.6, 32.4, 37.4, 51.7, 52.0, 56.1, 65.2, 65.3, 126.3, 127.5, 127.6, 127.7, 127.8, 128.1, 128.3, 128.4, 129.3, 137.0, 137.3, 138.1,155.9, 156.1, 171.0, 171.5, 173.6. Anal. Calcd for C36H44N4O8S: C, 62.41; H, 6.40; N, 8.09. Found: C, 62.07; H, 6.42; N, 8.08. (S )-6 -Benzyloxycarbonylamino -2 -[2 -((S )-2 benzyloxy carbonylamino -3 phenylpropanoyl amino) -4 -(methylsulfanyl)butanoylamino]hexanoic acid, N -Cbz -L Phe D L -Met -N -Cbz -L -Lys -OH, (2.6b+2.6b'): White microcrystals (78%); mp 126 127 C; [ ]23 D = 6.98 ( c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.51 (m, 4H), 1.51 1.78 (m, 2H), 1.78 1.98 (m, 2H), 1.97 (s, 1.5H), 2.04 (s, 1.5H), 2.45 2.55 (m, 2H), 2.67 2.90 2.99 (m 3H), 4.08 4.23 (m, 1H), 4.23 4.48 (m, 2H), 4.93 (s, 2H), 4.99 (s, 2H), 7.05 7.20 (m, 16H), 7.53 (d, J

PAGE 56

56 = 8.5 Hz, 0.6H), 7.65 (d, J = 7.7 Hz, 0.3H), 8.01 (d, J = 7.4 Hz, 0.3H), 8.05 8.29 (m, 1H), 8.31 (d, J = 8.0 Hz, 0.40H), 12.56 (br s, 1H). 13C NMR (DMSO d6) 14.5, 14.7, 22.5, 22.8, 28.9, 29.1, 29.2, 29.3, 30.5, 30.7, 31.9, 32.3, 37.3, 37.5, 51.7, 56.1, 56.3, 65.1, 65.2, 65.3,126.3, 127.4, 127.6, 127.7, 127.8, 128.1, 128.3, 128.4, 129.2, 136.9, 137.0, 137.3, 137.7, 138.1, 155.9, 156.1, 170.9, 171.0, 171. 5, 173.2, 173.4, 173.5. Anal. Calcd for C36H44N4O8S: C, 62.41; H, 6.40; N, 8.09. Found: C, 62.12, H, 6.53; N, 7.92. 2.4. 8 Ge neral Procedure for Preparation of LLL-Tripeptides 2.6 c -e and the Diastereomeric M ixture ( 2. 6 e + 2. 6 e ') N-Cbz -L Phe NCbz -L -Lys -Bt 2 .5c (0.6 mmol) was added at 15 C to a solution of free amino acid ( L -Met, L Trp, L -L -Ala, DL -Ala) 2.2c,d,f and (2.2f+2.2f ) (0.6 mmol) in CH3CN (15 mL)/H2O (5 mL) in the presence of Et3N (0.7 mmol). The reaction mixtures were then stirred at 15 C until the starting material was completely consumed as observed on TLC using EtOAc/hexane s (1:2) as the eluent. After 1 mL of 4N HCl was added, the solution was concentrated under reduced pressure to remove CH3CN The solution was extracted with EtOAc (20 mL), and the organic extract was washed with 4N HCl (5 mL) and sat urated NaCl (10 mL), and then dried (anhydrous MgSO4). Evaporation of the solvent gave the desired products, which were purified by dissolving in ethyl acetate and precipitating with hexane s Com pounds were recrystallized from EtOAc/hexane s for elemental analysis. (S )-2 -[( S )-6 -Benzyloxycarbonylamino -2 -((S )-2 benzylo xycarbonylamino-3 phenylpropano ylamino)hexanoylamino] -4 -methylsulfanylbutanoic acid, N -Cbz -L Phe -N Cbz L -Lys -L -Met -OH, 2.6c: White microcrystals (73%); mp 137 C ; [ ]23 D = 10.99 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.78 (m, 2H), 1.78 2.05 (m, 2H), 2.03 (s, 3H), 2.35 2.60 (m, 2H), 2.65 2.81 (m, 1H), 2.87 3.09 (m, 3H), 4.13 4.40 (m, 3H), 4.94 (s, 2H), 5.00 (s, 2H ), 7.00 7.40 (m, 16H), 7.50 (d, J = 8.5 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 8.26

PAGE 57

57 (d, J = 7.4 Hz, 1H), 12.66 (br s, 1H). 13C NMR (DMSO d6) 14.6, 22.5, 29.3, 29.6, 30.7, 31.9, 37.4, 50.9, 52.0, 52.4, 56.1, 65.2, 65.2, 126.3, 127.5, 127.7, 127.8, 128.1, 128. 3, 128.4, 129.2, 137.0, 137.3, 138.2, 155.9, 156.1, 171.5, 171.7, 173.2. Anal. Calcd for C36H44N4O8S: C, 62.41; H, 6.40; N, 8.09. Found: C, 62.44; H, 6.53; N, 7.90. (S )-2 -[(S ) -6 -Benzyloxycarbonylamino -2 -((S )-2 -benzyloxycarbonylamino -3 phenylpropa no ylamino) hexanoylamino] -3 -(1 H -indol -3 yl)propanoic acid, N -Cbz -L Phe N -Cbz -L -Lys -L Trp -OH, 2.6d : White microcrystals (88%); mp 71 C; [ ]23 D = 2.09 (c 1.0, DMF); 1H NMR (DMSO d6) 1.10 1.80 (m, 6H), 2.60 2.80 (m, 1H), 2.82 3.25 (m, 5H), 4.20 4.38 (m, 2H), 4.40 4.55 (m, 1H), 4.93 (s, 2H), 5.00 (s, 2H), 6.97 (t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.3 Hz, 1H), 7.05 7.20 (m, 18H), 7.48 (d, J = 8.2 Hz, 1H), 7.53 (d, J = 7.7 Hz, 1H), 8.04 (d, J = 7.7 Hz, 1H), 8.19 (d, J = 7.4 Hz, 1H), 10.85 (s, 1H), 12.65 (br s, 1H). 13C NMR (DMSO d6) 22.5, 27.0, 29.1, 29.3, 32.1, 37.4, 52.4, 53.9, 56.0, 65.2, 65.2, 109.6, 111.4, 118.2, 118.4, 120.9, 123.7, 126.2, 127.3, 127.5, 127.7, 127.8,128.0,128.3, 128.4, 129.2, 136.1, 137.0, 137.3, 138.2, 155.9, 156.1, 171.4, 171.6, 173.2. Anal Calcd for C42H45N5O8: C, 67.45; H, 6.06; N, 9.36. Found: C, 67.20; H, 6.28; N, 9.22. (S )-2 -[(S ) -6 -Benzyloxycarbonylamino -2 -((S )-2 -benzyloxycarbonylamino -3 phenylpropanoylamino)hexanoylamino]propanoic acid, N -Cbz -L Phe -N -Cbz -L -Lys -L Ala OH, 2. 6e: White microcrystals (74%); mp 129 131 C ; [ ]23 D = 9.60 (c 1.0, DMF); 1H NMR (DMSO d6) 1.20 1.80 (m, 6H), 1.27 (d, J = 7.4 Hz, 3H), 2.65 2.78 (m, 1H), 2.87 3.04 (m, 3H), 4.12 4.34 (m, 3H), 4.93 (s, 2H), 5.00 (s, 2H), 7.10 7.40 (m, 16H), 7.50 (d, J = 8.8 Hz, 1H ), 8.04 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 7.1 Hz, 1H), 12.55 (s, 1H). 13C NMR (DMSO d6) 17.1, 22.5, 29.3, 32.1, 37.5, 47.5, 52.0, 52.2, 56.1, 65.2, 65.2, 126.3, 127.4, 127.6, 127.7, 127.8, 128.0,

PAGE 58

58 128.3, 128.4, 129.2, 137.0, 137.3, 138.2, 155.9, 156.1, 17 1.3, 171.4, 174.0. Anal. Calcd for C34H40N4O8: C, 64.54; H, 6.37; N, 8.85. Found C, 64.42; H, 6.63; N, 8.68. 2 -[(S )-6 -Benzyloxycarbonylamino-2 -((S )-2 benzyloxy carbonylamino -3 phenylpropanoyl amino)hexanoylamino]propanoic acid, N -Cbz -L Phe -N -Cbz -L -Lys -D L Al a -OH, (2.6e+2.6e'): White microcrystals (90%); mp 99101 C; [ ]23 D = 7.35 ( c 1.0, DMF); 1H NMR (DMSO d6) 1.20 (m, 3H), 4.93 (s, 2H), 4.99 (s, 2H), 7.10 J = 8.6 Hz, 1H ), 8.03 (d, J = 8.3 Hz, 1H), 8.19 13C NMR (DMSO d6) 17.1, 17.4, 22.5, 22.8, 29.1, 29.2, 29.3, 30.8, 30.9, 32.1,32.3, 37.4, 47.4, 47.5, 52.0, 52.2, 56.0, 56.1, 65.1, 65.2, 126.3, 127.4, 127.5, 127.6, 127.7, 127.8, 128.1, 128.3, 128.4, 129.3, 129.3, 136.9, 137.0, 137.3, 138.2, 155.9, 156.1, 171.2, 171.3, 171.4, 171.8, 173.6, 174.0, 174.1. Anal. Calcd for C34H40N4O8: C, 64.54; H, 6.37; N, 8.85. Found : C, 64.25; H, 6.46; N, 8.46. 2.4. 9 Procedure for Preparation of unnatural LLL-Tr ipe ptide 2.6f N-Cbz -dipeptidoylbenzotriazole 2.5a (0.6 mmol) w as added at 15 C to a solution of CbzL -Lys (0.6 mmol) in CH3CN (15 mL)/H2O (5 mL) in the presence of Et3N (0.7 mmol). The reaction mixtures were then stirred at 15 C until the starting ma terial was completely consumed as observed on TLC using EtOAc/hexane s (1:2) as the eluent. After 1 mL of 4N HCl was added, the solution was concentrated under reduced pressure to remove CH3CN The solution was extracted with EtOAc (20 mL), and the organic extract was washed with 4N HCl (5 mL) and sat urated NaCl (10 mL), and then dried over anhydrous MgSO4. Evaporation of the solvent gave the desired products, which was purified by dissolving in ethyl acetate and precipitating with hexane s Compound was recr ystallized from EtOAc/hexane s for elemental analysis.

PAGE 59

59 (S )-2 -Benzyloxycarbonylamino -6 -[(S )-2 -(( S )-2 benzyloxycarbonylaminopropanoylamino) -3 -(1 H -indol -3 -yl)propanoylamino]hexanoic acid, NCbz -N -(Cbz -L Ala -L -Trp) L -Lys -OH, 2.6f: White microcrystals (82%); m p 62 C; [ ]23 D = 10.90 (c 1.0, DMF); 1H NMR (DMSO d6) 1.14 (d, J = 6.9 Hz, 3H), 1.19 (m, 4H), 1.45 4H), 6.95 (t, J = 7.3 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 7.10 (s, 1H), 7.20 J = 7.9 Hz, 1H), 7.55 (d, J = 7.7 Hz, 2H), 7.81 13C NMR (DMSO d6) 18.0, 22.9, 27.8, 28.4, 30.4, 38.3, 50.2, 53.4, 53.7, 65.3, 65.4, 110.0, 111.1, 118.1, 118.4, 120.7, 123.4, 127.3, 127.7, 127.7, 128.3, 136.0, 136.9, 137.0, 155.7, 156.1, 170.8, 172.1, 173.9. Anal. Calcd for C36H41N5O8: C, 64.37; H, 6.15; N, 10.43. Found: C, 64.03; H, 6.36; N, 10.67.

PAGE 60

60 CHAPTER 3 SYNTHESIS OF PEPTIDES BY EXTENTION AT N OR C TERMINUS OF ARGININ E 3.1 Introduction The essential amino acid L -Arginine with its guanidine group is involved in numerous diverse biological processes connected with cell division, healing wounds, removal o f ammonia from immune functions, and hormone release.78 L -Arginine i s an immediate precursor of nitric oxide (NO) in a reaction catalyzed by isoform s of NO synthase (NOS).79 Nitric oxide is a potent biological signal for diverse physiological processes within the cardiovascular, immune, and nervous systems.80 Overproduction of NO can lead to chronic neurodegenerative diseases including Alzhheimers, Parkinson81 85 and inflammatory diseases such as arthritis86 and colitis.87 On the other hand, impaired NO production is responsible for hypertension 88 and atherosclerosis.89 T herefore, many studies have been conducted on novel substrates and isoform -selective NOC inhibitors in attempt to find treatment for pathological NO production in biological systems. N-Methyl -L arginine and N-ethyl -L arginine show limited selective inhib ition of the NOC isoforms90; however, high selectivity was estimated for Nnitroarginine and phenylalanine containing dipeptides and dipeptides esters.91 Important selective inhibitors of nNOS over eNOS include nonbiological dipeptides amides and peptidom imetics, built on an NNO2-L arginine scaffold 3 1a -c (Figure 3 1).85 Figure 3 1 NNO2-L arginine scaffold

PAGE 61

61 Arginine is the preferred residue at the P1 position of substrates for serine proteases 92 such as trypsin,93 factor Xa94 and others of the co agulation cascade, and an essential residue in the integrin recognition sequence Arg -Gly -Asp.95 Arg Gly -Asp (RGD) is a well known characteristic and conservative sequence that acts as cell recognition site fo r many adhesive proteins pres e nt in extracellua r matrics (ECM) and in blood in cluding fibrinogen. RGD and some synthetic RGD containing peptides acting as competitive, reve r sible inhibitors for adhesive protein binding have been used to study adhesive interaction between cells and suppress tumor metast asis and p l atelet agreegation.95 96 The geometry, charge distribution and ability to form multiple H bonds make arginine ideal for binding negatively charged groups and preferably located on the outside of the proteins it can interact with the polar enviro nment. Certain peptides containing high percentage of cationic amino acids are known to efficiently translocate through the cell membrane. Short oligomers of arginine can migrate across the plasma membrane of a cell.97 ,9 8 Synthetic arginine rich peptides a re efficient carriers for transporting various types of biomolecules into cytoplasmic and nuclear compartments of living cells,99 including nucleic acids ,100101 peptides, proteins.102 As a result of exte nsive physiological function arginine containing pep tides and other conjugates are therapeutic agents of diverse activity103 and drugs in anti -cancer therapy.104107 Considerable effort has been devoted to the synthesis of arginine peptides and peptidomometics108,109 utilizing solutio n and solid phase metho dologies.1101 14 The highly basic nature and nucleophilic character of the guanidine moiety in arginine requires appropriate protection before chemical manipulations. Various C terminal arginine peptides have been prepared from N-Cbz NNO2-L arginine by the mixed anhydride method.11 5118 Similar

PAGE 62

62 methods gave N acetylated peptides from NNO2-L arginine ester,119 selective anticoagulant tripeptide D -Phe -Pro -Arg120 and peptides from tribenzoxycarbonyl -L arginine.121 ,12 2 Other approaches include Arg(Cbz)2OH with TFFH/collidine in CH2Cl2, 12 3 coupling agents: DCC,124 DCC/DNP, DCC/HOBt, DPPA125 and N -carboxyanhydride126 and pyrophosphate.127 Protected Leu -Arg -Pro tripeptides are prepared using NMM/picaloyl chloride/HOBt in DMF or CH2Cl2. 128 Unprotected arginine couples with activated amino acid pentafluorophenyl ester (Pfp) in DMF and utilization of orthogonal protection affords free tripeptide 3 4 ( Scheme 3 1 ).129 ,1 30 Scheme 3 1 Literature method for preparation of arginine peptides After carboxyl activat lactam f ormation competes with coupling108, 12 5,13 1,13 2 to an extent depending on the carboxyl activation and also on the amino acid component (Scheme 3 2) This side reaction is a serious problem, especially whe n the reaction involves a weak nucleophile. Mixed anh -lactam formation125,12 6 as also does EDC/HOBt/NMM in CH3CN ( Scheme 3 3 ).132 Deprotection of the guanidino function favours lactam formation,123 the side products also were minim ized by the DPPA method.125 Scheme 3 2. Intramolecular lacta m formation during carboxyl activation of arginine

PAGE 63

63 Scheme 3 3 Intramol ec u l ar lacta m formation during carboxyl activation with EDC/HOBt Because of the wide range of application for RG D and related peptide s several approaches such as mixed anhydride and DCC have been used for their synthesis. Very recently protected RGD tri peptide Bz RGD OEt was synthesized by chemo -enzymatic synthesis. First Gly -Asp was synthesized by chloroacetylati on of L aspartic acid and ammonolysis of chloroacetyl L aspartic acid. In this linkage of third amino acid Bz -Arg OEt to Gly -Asp (OEt)2 was completed by enzymatic method in organic solvent (Scheme 3 4 ).96 Scheme 3 4 Chemo enzymatic synthesis of the pr otected RGD peptide Free RGD peptide has also been synthesized by N -carboxyanhydride method. In this case first Gly -Asp was synthesized as previously. Second NCbz -Arg was reacted with Gly -Asp to yield RGD tripeptide by N c arboxyanhydride method (Scheme 3 5 ).126 Scheme 3 5 Synthesis step of the free RGD peptide

PAGE 64

64 In continuation of our extensive research on use of N acybenzotriazoles in peptide synthesis h erein, convenient procedures for preparation of arginine peptides by extens ion at C and N terminus of arginine is described In addition a novel synthesis of the protected RGD tripeptide by utilizing benzotriazole methodology is also described. 3.2 Results a nd Discussion 3.2.1 Preparation of LL-Dipeptides 3.9a -e and the D iastereomeric mixtures (3.9b+3. 9b'), (3.9c+3. 9c') by E xtension at the N-Terminus of NNO2-L Arg -OH 3.8 NNO2-L -Arg OH 3.8 couples with N ( Cbz aminoacyl)benzotriazoles 3.7a -e derived from chiral L Phe, L -Met, L -Ala, L Trp and di -Cbz -L -cystine -di -Bt, and corresponding racemic mixture s ( 3.7b+3.7b 3.7c+3.7c in aqueous acetonitrile (CH3CN/H2O, 1:2) containing Et3N in 30 min at room temperature to give dipeptides 3.9a -e and diastereomeric mixtures (3.9b+3.9b ) and (3.9c+3.9c' ) (75 95%), all isolated without column chromatograph y (Scheme 3 6 Table 3 1). Diastereomeric mixtures were prepared to compare that the original chiral i t y of amino acids and peptides used is maintained during coupling reactions by means of HPLC and NMR analysis Scheme 3 6 Preparation of NNO2-L -Argi nine dipeptides 3.9a -e and diastereomeric mixtures (3.9b+3.9b' ) and (3.9c+3.9c' ) 1H NMR analysis of dipeptides 3. 9a -e revealed that each LL -dipeptide displayed two sets of doublets for two amide NH protons ranging from 7.30 to 8.40 ppm, supporting their en antiopurity. However, for each of the diastereomeric mixtures ( 3. 9b+ 3. 9b ) and ( 3. 9c+ 3. 9c ),

PAGE 65

65 one of the amide NH protons showed as a multiplet. The 13C NMR of (3. 9b+ 3. 9b ) and (3. 9c+ 3. 9c ) each showed doubled signals for each aliphatic and carbonyl carbon. The guanidine NH2 protons all appeared as broad signals ranging from 7.30 to 8.30 ppm. The NH protons of the guanidine group appear a s a broad singlet at ~ 8.50 ppm Table 3 1 Preparation of arginine dipeptides 3. 9a -e ( 3. 9b+ 3. 9b' ) and ( 3. 9c+ 3. 9c' ) from NNO2-L -Arg OH 3. 8 Reactant Product Yield a (%) m p (C) 23 D t R f (m in) Cbz L Phe Bt 3.7a Cbz L Phe N NO 2 L Arg OH 3. 9a b 90 173 175 6.20 11.16 C bz L Met Bt 3.7b Cbz L Met N NO 2 L Arg OH 3. 9b c 80 141 143 4.15 10.13 Cbz D L Met Bt (3. 7b+ 3. 7b ) Cbz D L Met N NO 2 L Arg OH ( 3.9b+3.9b ) 95 61 64 2.67 9.42, 10.03 Cbz L Al a Bt 3. 7c Cbz L Ala N NO 2 L Arg OH 3. 9c d 93 168 169 +3.84 10.79 Cbz D L Ala Bt ( 3. 7c+ 3. 7c ) Cbz D L Ala N NO 2 L Arg OH ( 3.9c+3.9c ) 92 133 134 2.33 8.64, 10.45 Cbz L Trp Bt 3. 7d Cbz L Trp N NO 2 L Arg OH 3. 9de 80 65 68 19.60 11.12 Di Cbz L Cystine di Bt 3. 7e Di Cbz L Cystine di N NO 2 L Arg OH 3. 9e 75 105 108 84.3 3.04 aIsolated yields, b,c,e Lit.11 8 mp b174 176 C c140 143 C, e68 75 C. dLit.115 mp 171172 C. f t R = Retention t ime for HPLC The enantiopurity of each of the LL-dipetides 3. 9a -e was further supported by HPLC analyses by using a Chirobiotic T column [detection at 220 nm, flow rate 0.41.0 mL/min; eluting with MeOH/H2O (1:1) for 3.9a c d and ( 3.9c + 3.9c' ), MeOH/H2O (9:1) for 3 9b and (3 9b + 3 9b' ); MeOH for 3 9e ]. Enantiopure compound 3 9b showed a single retention time at 10.13 min whereas the corresponding diastereomeric mixture ( 3.9b + 3.9b' ) showed two retention times with equal intensity at 9.42 and 10.03 min. Similarly in the case of compound 3.9c one single retention time at 10.7 9 min was observed, whereas the diastereomeric mixture

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66 (3.9c + 3.9c' ) showed two retention times at 8.64 and 10.45 min. Enantiopure compounds 3.9a 3.9d and 3.9e showed single retention times at 11.16, 11.12, and 3.04 min respectively. 3.2.2 Preparation of LL-D ipeptides 3. 11a -d an d the D iastereomeric mixture (3.11b+3.11b' ) by Chain E l ongation at the N-Terminus of L Arg -OH 3.10 L -Arginine containing dipeptides 3 11a -d and the diastereomeric mixture ( 3 11b + 3 11b' ) were prepared by extension at the N -terminus of L arginine 3. 10 by coupling with N -(Cbz aminoacyl)benzotriazoles 3 7a d and the diasteromeric mixture (3 7b + 3 7b' ) in aqueous CH3CN without Et3N at room temperature for 6 h (free arginine affords required base medium). After evaporation of solvent the residue was purified by reprecipitation from MeOH/Et2O. Repetition of this procedure three t imes afforded complete removal of byproduct 1 H Benzotriazole and gave pure dipeptides 3 11a d and the diastereomeric mixture ( 3 11b + 3 11b' ) in 75 83% yields (Scheme 3 7 Table 3 2). NMR analysis of the compounds reveale d no detectable epimeri zation (<1 %). For each of the enantiopure compounds 3 11a d, two sets of doublets were observed for the amide NH protons. For the diastereomeric mixture ( 3 11b + 3 11b' ), one of the amide NH protons appeared as a multiplet and the 13C NMR spectrum also showed doubling of the signals for the aliphatic and carbonyl carbons. Sc h e me 3 7 Preparation of arginine dipeptides 3.11a d and ( 3.11b+3.11b' ) from L -Arg OH 3.10. HPLC analyses (flow rate 1.0 mL/min with MeOH as eluent) of LL-dipeptides 3 11a 3.11b 3 11c and 3 11d each showed single retention times at 1.91, 1.84, 1.92 and 2.88 min

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67 respectively. However, in case of the diatseromeric mixture ( 3 11b + 3 11b' ), two retention time at 1.85 and 1.98 min was obtained. Table 3 2 Preparation of arginine dipeptides 3.11a -d and (3.11b+3.11b' ) from L -Arg -OH 3. 10. Reactant Product Yield a (%) Mp (C) 23 D Cbz L Phe Bt 3 7a Cbz L Phe L Arg OH 3 11a b 83 130 132 10.37 Cbz L Met Bt 3 7b Cb z L Met L Arg OH 3 11b 81 143 144 7.20 Cbz DL Met Bt ( 3 7b+7b ) Cbz D L Met L Arg OH ( 3 11b+ 3 11b ) 76 121 124 +5.86 Cbz L Trp Bt 3 7c Cbz L Trp L Arg OH c 3 11c 83 135 137 18.29 Cbz L Val Bt 3 7d Cbz L Val L Arg OH d 3 11d 75 121 123 0.99 a Isolated yields, b Lit 1 30 mp 131 133 o C, c Ref e ren ce 1 3 3 d Reference 13 4 3.2.3 Preparation from N -Cbz -N-NO2L Arg -Bt 3. 13 of A rginine LLdipeptides 3. 15a -c and the D iastereomeric mixture (3.15a+3.15a by E xtension at the C Terminus of N -Cbz N-NO2-L A rg -OH 3.14 To extend at the arginine C terminus, carboxyl group need to be activated. F irst, I tried to synthesize benzotriazole derivative 3.13 of NCbz -L -Arg OH 3.12. However the desired product 3.13 could not be isolated due to competing intramolecular cyclization product of 3.1 3 in the presence of free guanidine moiety (Scheme 3. 8 ). Next, NCbz NNO2-L -Arg -OH 3.1 4 was used inste a d of N-Cbz -L -Arg OH 3.12 for formation of ac tivated benzotriazole derivative 3.19 (Scheme 3 9 ). Scheme 3 8 Attempted synthesis of benzotriazole derivative of NCbz -L -Arg OH 3.12

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68 N-Cbz NNO2L -Arg OH 3.1 4 was treated with 1 H benzotriazole and SOCl2 in THF at 20 C to give benzotriazole deriva tive N-Cbz NNO2-L -Arg Bt 3.1 5 after acidic work up in 97% yield; this reaction was completed in 45 min without formation of side products. Coupling N-Cbz NNO2L -Arg -Bt 3.1 5 with free amino acids 3.1 6 a -d ( 3.1 6 a+3.1 6 a ) gave chiral N terminal arginine dipeptides 3.1 7 a d and the diastereomeric mixture ( 3.1 7 a+3.1 7 a' ) in yields of 6580% (Scheme 3 9 Table 3 3) Scheme 3 9 Preparation of NCbz -NNO2L -Arg -Bt 3.1 5 and arginine dipeptides 3.1 7 a d (3.1 7 a+3.1 7 a ) Table 3 3 Preparation of NCbz NNO2-L -Arg Bt 3.1 5 and arginine dipeptides 3.1 7 a d (3.1 7 a+3.1 7 a ) Reactant Product Yield a (%) m p (C) 23 D t R c (min) L Phe OH 3.1 6 a N Cbz N NO 2 L Arg L Phe OH 3.1 7 a 68 217 219 +5.32 3.01 d DL Phe OH (3.1 6 a+3.1 6 a N Cbz N NO 2 L Arg DL Phe OH ( 3.1 7 a +3.1 7 a ) 65 205 207 +8.14 3.16, 3.94d L Met OH 3. 1 6 b N -Cbz N NO2-L -Arg -Met OH 3.1 7 b 66 144 146 4.60 3.34 d L Ser OH 3.1 6 c N -Cbz N NO2L -Arg L Ser OH 3. 1 7 c 65 83 85 5.58 3.57 d L Gly OH 3.1 6 d N -Cbz N NO2-L -Arg Gly OH 3.1 7 d b 80 115 117 1.54 4.15 d aIsolated Yields; bLit.135 mp 114 117; ctR = Retention Time; dFlow rate: 1.0 mL/min, eluent MeOH.

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69 The procedure was similar to that utilized above for the preparation of 3. 9a c (Scheme 3 5 ). Purification of crude product by reprecipitation in MeOH/Et2O gave pure dipeptides 3.1 7 a -c and the diastereomeric mixture (3.1 7 a+3.1 7 a' ), while 3.1 7 d was isolated by acidifying at 15 C. 1H NMR analysis showed no detectable racemization (<5 %) for the LL-dipeptides 3.1 7 a -d and the diastereomeric mixture ( 3.1 7 a+3.1 7 a ). However monitoring by TLC, disclosed that a side product 3.1 8 (10 30%) was formed in all these reactions, and could be isolated from the filtrate. The structure of 3.1 8 was revealed (by 1H and 13C NMR) to be the intramolecular cyclization la ctam); 3.1 8 formed competitively with the expected dipeptide during coupling of benzotriazole activated nitroarginine 3.1 5 with free amino acids. In case of coupling with Gly OH 31.6d extent of intramolecular cyclization product was less as compared to ot her amino acid L Phe, L -Met and L -Ser 3.16a -c HPLC analyses for the enantiopure LL -peptides 3.1 7 a d showed single retention times, while the diastereomeric mixture ( 3.1 7 a+3.1 7 a ) showed two retention time s (Table 3 3) 3.2.4 Preparation of C-Terminal Arginine T ripeptides 3. 22a -c and (3.22a+3.22a by Extension at the N-Terminus of N-NO2L Arg -OH 3.8 N-Cbz D ipeptidoylbenzotriazoles 3.2 2 b c were obtained as reported previously.13 6 Analogs 3. 2 2 a and ( 3.2 2 a+3.2 2 a ) were prepared from Cbz -L -Asp(OBz) OH 3.1 9 (Scheme 3 10) similarly to a described procedure.23 Cbz -L -Asp(OBz) Bt 3.20 was prepared from Cbz -L Asp(OBz) OH 3.1 9 by reaction with 1 H -benzotriazole in CH2Cl2 in the presence of SOCl2. Further coupling of 3.20 with L -Phe and DL Phe yielded Cbz protected dipeptide 3.21a and the diasteromeric mixture ( 3.21a+3.21a ) which were then converted to their dipetidoylbenzotriazole derivative 3.22a and ( 3.22a+3.22a ) (Scheme 3 1 0 ).

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70 Sche me 3 10. Preparation of Cbz L -Asp(OBz) -Bt 3.20 Scheme 3 1 1 Synthesis of N -Cbz -dipetidoyl benotriazole 3.2 2 and ( 3.2 2 a+3.2 2 a ) NNO2-L -Arg OH 3.8 was coupled with 3.2 2 a -c (3.2 2 a+3.2 2 a ) in aqueous CH3CN in the presence of Et3N for 2 h at 15 oC to gi ve tripeptides 3.2 3 a -c and diastereomeric mixture (3.2 3 a+3.2 3 a ) in 66 85% yields ( Scheme 3 1 2 Table 3 4). Scheme 3 1 2 Synthesis of NNO2-L arginine tripeptide s 3.23a -c and ( 3.23a+3.23a )

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71 The 1H and 13C NMR spectra of the optically pure LLL-tripept ides 3.2 3 a -c showed the absence of epimerization (<1 %). 1H NMR showed three sets of doublets for the amide NH protons for each of the enantiopure compounds. In the case of diastereomeric mixture (3.2 3 a+3.2 3 a ), each set of amide NH protons appeared as spli t doublets and 13C NMR showed doubling of the signals for the aliphatic and carbonyl carbons. The room temperature 1H NMR for 3.22c showed the existence of two rotameric forms, which underwent coalescence in a high temperature 1H NMR experiment. Table 3 4 Preparation of L arginine tripeptides 3.2 3 a -c and the diastereomeric mixture (3.2 3 a+3.2 3 a Reactant Product Yield a (%) m p (C) 23 D Cbz L -Asp(OBz) -L -Phe Bt 3.2 2 a Cbz L -Asp(OBz) -L -Phe -NNO2-L -Arg OH 3.2 3 a 84 128 129 11.71 Cbz L -Asp(OBz) -DL -Phe -Bt (3.2 2 a+3.2 2 a ) Cbz L Asp(OBz) DL Phe N NO2-L -Arg OH ( 3.2 3 a+3.2 3 a ) 82 60.4 65.0 6.31 Cbz L -Ala L Trp -Bt 3. 2 2 b Cbz L -Ala L Trp -N-NO2-L Arg OH 3.2 3 b 66 150 151 10.24 Cbz L -Phe -L -Met Bt 3. 2 2 c Cbz L -Phe -L -Met NNO2-L Arg OH 3.2 3 c 75 77 79 7.98 a Isolated Yields HPLC analysis of tripeptide 3.2 3 b shows one retention time at 3.17 min supportin g its enantiopurity. Tripeptide 3.2 3 a is a protected analogue of H -Asp -Phe -Arg OH 3.4 recently adopted as a catalyst for asymmetric Michael addition reactions.129 The classical method12 9 for the preparation of 3.4, (Scheme 3 1) utilizing the pentafluorophenyl ester of amino acid in DMF, requires prolonged reaction times and complicated isolation procedures; our methodology advantageously includes coupling of benzotriazole activated amino acid in aqueous media, short reaction times and simple work up proced ures affording final chiral tripeptide 3.2 3 a and the diastereomeric mixture ( 3.2 3 a+3.2 3 a ) in 8284% yields.

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72 3.2.5 Application o f Benzotriazole Methodology in t he Preparation o f the P rotected RGD Peptide N-Cbz -N-NO2L Arg -Gly L Asp -(OH)2 3.26 We used be nzotriazole methodology to synthesize protected arginyl -glycyl aspartyl "RGD" tripeptide Cbz -Arg(NO2) Gly -Asp(OH)2 3.2 6 ( Scheme 3 1 3 ). A recent literature135 preparation of di -benzyl ester derivative Cbz -Arg(NO2) Gly OBz)OBz utilizes amino acid esters, requ ires low temperatures of 5 to 8 C, prolonged reaction times (14 16 h) a nd coupling reagents ( DCC/ HOBt ). Di -benzyl ester protected tripeptide was finally deprotected by conventional method to give ArgGly -Asp -(OH)2.1 35 Our methodology utili zed benzotriazole activated amino acid 3.1 5 and finally synthesized protected RGD peptide 3.26 from Cbz-L -NO2-Arg Gly Bt 3.2 4 and free aspartic acid 3.2 5 by modification of the coupling procedure adopted for the preparation of other tripeptides. First NCbz N-NO2L -Arg Gly OH 3.17d was synth e sized as described in S cheme 3 9 and then converted to its di peptidoylbenzotriazole derivative NCbz -NNO2L -Arg Gly -Bt 3.24. Further coupling of 3.24 with free aspartic acid 3.25 was achieved in CH3CN/H2O/THF in presence of Et3N at 15 oC for 3.5 h (Scheme 3 13). THF was added to reaction mixture because of very low solubility of 3.24. Scheme 3 13. Preparation of protected RGD peptide 3.2 6

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73 In comparison, our methodology offers simple preparative and workup procedures, takes less time to complete, uses inexpensive reagents, gives high yields, and allows the us e of free amino acids as coupling components affording 3.2 6 in good yield. 3.3 Conclusion In conclusion, convenient and efficient preparation of arginine di and tripeptides in short reaction times utilizing simple workup procedures, inexpensive reagents a nd free amino acids as coupling components are described The peptides can be prepared by chain elongation at either the N or C terminus of L arginine. We have successfully synthesized protected RGD peptide sequence with our benzotriazole methodology, usi ng free aspartic acid as coupling component. 3.4 Experimental Section 3.4.1 General M ethods Melting points were determined on a capillary point apparatus and are uncorrected. NMR spectra were recorded in CDCl3 or DMSO d6 with TMS as an internal standard for 1H (300 MHz) and solvent as an internal standard for 13C (75 MHz). N -Cbz and Fmoc amino acids and free amino acids were purchased from Fluka and Acros, were used without further purification. Optical rotation values were measured with the use of sodium D line. HPLC analyses were performed using Chirobiotic T column (4.6 x 250 mm), detection at 220 nm, flow rate of 0.4 1.0 mL/min and MeOH or MeOH/H2O as an eluting solvent. 3.4.2 General Procedure for the P reparation of LL -Dipeptides 3.9a -e and the D iaste reomeric mixture (3.9b+3.9b'), (3.9c+3.9c') N(Cbz aminoacyl)benzotriazoles 3.7a -d ( 3.7b+3.7b' ), (3.7c+3.7c' ) (0.5 mmol) were added at 20 C to a solution of NNO2-L -Arg OH 3.2 (0.5 mmol) in CH3CN /H2O ( 5 mL/ 10 mL) in the presence of Et3N (0.6 mmol). Th e reaction mixture was then stirred at 20 C until the starting material was completely consumed as observed by TLC using EtOAc/hexanes (1:2) as

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74 the eluent. After addition of 4N HCl (1 mL), the solution was concentrated under reduced pressure to remove CH3CN Residue was extracted with EtOAc (20 mL), and the organic extract was washed with 4N HCl (5 mL), and sat urated NaCl (10 mL) then dried over anhydrous MgSO4. Evaporation of the solvent gave the desired product in pure form, which was further recrystalli zed from MeOH/Et2O unless specified, otherwise. (S )-2 -((S ) -2 -Benzyloxycarbonylamino -3 -phenylpropanoylamino) -5 nitroguanidinopentanoic acid ( Cbz L -Phe -N-NO2L Arg -OH, 3. 9a): White microcrystals (90%); mp 173 175 C (Lit118 174 176 C); [ ]23 D = 6.2 ( c 1. 0, DMF); 1H NMR (DMSO d6) 1.40 1.70 (m, 3H), 1.70 1.87 (m, 1H), 2.60 2.80 (m, 1H), 2.90 3.05 (m, 1H), 3.05 3.25 (m, 2H), 4.10 4.35 (m, 2H), 4.92 (s, 2H), 7.00 7.40 (m, 10H), 7.51 (d, J = 8.4 Hz, 1H), 7.60 8.25 (m, 2H), 8.32 (d, J = 7.4 Hz, 1H), 8.56 (br s, 1H), 12.65 (br s, 1H). 13C NMR (DMSO d6) 24.9, 28.3, 37.4, 40.2, 51.7, 56.0, 65.2, 126.3, 127.5, 127.7, 128.1, 128.3, 129.2, 137.0, 138.1, 155.9, 159.3, 171.8, 173.4. Anal. Calcd for C23H28N6O7: C, 55.19; H, 5.64; N, 16.79. Found: C, 54.88; H, 5.76; N 16.63. (S )-2 -((S ) -2 -Benzyloxycarbonylamino -4 -methylsulfanylb utyrylamino) -5 nitroguanidinope ntanoic acid, (Cbz -L -Met -N-NO2L Arg -OH, 3. 9b): White microcrystals (80%); mp 141 143 oC (lit118 140 143 oC); [ ]23 D = 4.15 (c 1.0, DMF); 1H NMR (DMSO d6) 1.40 1.70 (m, 3H), 1.70 1.95 (m, 3H), 2.03 (s, 3H), 2.40 2.60 (m, 2H), 3.00 3.20 (m, 2H), 4.05 4.25 (m, 2H), 4.93 5.10 (m, 2H), 7.24 7.42 (m, 5H), 7.50 (d, J = 8.2 Hz, 1H), 7.60 8.16 (m, 2H), 8.21 (d, J = 7.1 Hz, 1H), 8.54 (br s, 1H). 13C NMR (DMSO d6) 14.7, 25.0, 28.2, 29.6, 31.9, 40.3, 51.7, 53.8, 65.6, 127.8, 127.9, 128.5, 137.1, 156.0, 159.4, 171.8, 173.5. Anal. Calcd for C19H28N6O7S: C, 47.10; H, 5.82; N, 17.34. Found: C, 47.23; H, 5.86; N, 17.07.

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75 (S )-2 -(2 -Benzyloxycarbonylamino-4 -methylsulfanylbutanoyla mido) -5 nitroguanidinopenta noic acid (Cbz -DL -Met -N-NO2-L Arg -OH, ( 3. 9b+ 3. 9b )): White microcrystals (95%); mp 61 64 oC; [ ]23 D = 2.67 (c 1.0, DMF); 1H NMR (DMSO d6) 1.40 1.95 (m, 6H), 2.02 (s, 3H), 2.35 2.50 (m, 2H), 3.05 3.25 (m, 2H), 4.05 4.25 (m, 2H), 5.02 (s, 2H), 7.25 7.40 (m, 5H), 7.40 7.53 (m, 1H), 7.55 8.20 (m, 2H), 8.21 (d, J = 6.9 Hz, 1H), 8.50 (br s, 1H), 12.67 (br s, 1H). 13C NMR (DMSO d6) 14.6, 24.9, 28.1, 28.4, 29.6, 29.7, 31.8, 32.0, 51.6, 51.7, 53.7, 53.9, 65.0, 65.5, 127.8, 127.8, 127.9, 128.4, 137.0, 156.0, 156.0, 159.3, 171.6, 171. 7, 173.3, 173.4. Anal. Calcd for C19H28N6O7S: C, 47.10; H, 5.82; N, 17.34. Found: C, 46.92; H, 5.87; N, 17.06. (S )-2 -((S ) -2 -Benzyloxycarbonylaminopropanoylamino) -5 -nitroguanidinopentanoic acid (Cbz L Ala -N-NO2L Arg -OH, 3. 9c): White microcrystals (93%); m p 168 169 oC (lit,116 171 172 C) ; [ ]23 D = +3.84 (c 1.0, DMF); 1H NMR (DMSO d6) 1.10 1.32 (d, J = 6.9 Hz, 3H), 1.35 1.85 (m, 4H), 3.00 3.25 (m, 2H), 4.00 4.38 (m, 2H), 4.98 (d, J = 12.2 Hz, 1H, A part of AB system), 5.37 (d, J = 12.6 Hz, 1H, B part of A B system), 7.20 7.40 (m, 5H), 7.44 (d, J = 7.2 Hz, 1H), 8.11 (d, J = 7.4 Hz, 1H), 7.50 8.20 (m, 2H), 8.52 (bs, 1H), 12.56 (s, 1H). 13C NMR (DMSO d6) 18.2, 24.8, 28.3, 49.8, 51.5, 65.4, 127.8, 128.4, 137.1, 155.6, 159.3, 172.7, 173.4. Anal. Calcd for C17H24N6O7: C, 48.11; H, 5.70; N, 19.80. found: C, 48.19; H; 5.73; N, 19.74. (S )-2 -(2 -Benzyloxycarbonylaminopropanoylamino) -5 -nitroguanidinopentanoic acid (Cbz -DL Ala -N-NO2-L Arg -OH, ( 3. 9c+ 3. 9c )): White microcryst als (92%); mp 133 134 oC; [ ]23 D = 2.33 ( c 1 .0, DMF); 1H NMR (DMSO d6) 1.21 (d, J = 6.6 Hz, 3H), 1.39 1.85 (m, 4H), 3.05 3.22 (m, 2H), 4.02 4.27 (m, 2H), 4.98 (d, J =12.6 Hz, 1H, A part of AB system), 5.03 (d, J = 12.6 Hz, 1H, B part of AB system), 7.25 7.40 (m, 5H), 7.44 (d, J = 8.0 Hz, 1H), 7.50 8.20 (m, 2H), 8.05 8.20 (m, 1H), 8.53 (br s, 1H), 12.67 (s, 1H). 13C NMR (DMSO d6) 18.2, 18.7, 24.8,

PAGE 76

76 28.3, 28.6, 40.2, 49.8, 50.0, 51.4, 51.5, 65.4, 127.8, 127.8, 128.4, 137.1, 155.7, 159.4, 172.6, 172.7, 173.4, 173.4. Anal. Calcd for C17H24N6O7: C, 48. 11; H, 5.70; N, 19.80. Found: C, 48.23; H, 5.69; N, 19.66. (S )-2 -[(S ) -2 -Benzyloxycarbonylamino -3 -(1 H -indol -3 yl) propanoylamino] -5 nitroguanidino pentanoic acid (Cbz -L -Trp -N-NO2L Arg -OH, 3. 9d): White microcrystals (80%); mp 71 73 oC (lit118 mp 68 75 oC); [ ]23 D = 19.55 (c 1.0, DMF); 1H NMR (DMSO d6) 1.45 1.85 (m, 4H), 2.84 2.97 (m, 1H), 3.05 3.33 (m, 3H), 4.18 4.40 (m, 2H), 4.92 (s, 2H), 6.94 7.00 (m, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.16 (s, 1H), 7.18 7.22 (m, 7H), 7.66 (d, J = 8.0 Hz, 1H), 7.60 8.40 (m 2H), 8.32 (d, J = 7.7 Hz, 1H), 8.55 (br s, 1H), 10.80 (s, 1H), 12.68 (s, 1H). 13C NMR (DMSO d6) 26.9, 27.8, 28.3, 40.2, 51.7 55.3, 65.3, 110.1, 11.3, 118.2, 118.6, 120.9, 124.0, 127.3, 127.5, 127.7, 128.3, 136.1, 137.0, 155.8, 159.3, 172.2, 173.5. (6 S ,9 S ,14 S ,17 S )-1 Amino -9,14-bis(benzyloxycarbonylamino) -6 -carboxy 17-(3 (nitroguani dino) propyl) -1 -(nitroimino) -8,15-dioxo -11,12-dithia -2,7,16-triazaoctadecan -18oic acid (di-Cbz -L -Cystine di -N-NO2-L Arg -OH, 3.9e): White microcrystals (75%); mp 102 108 oC; [ ]23 D = 84.22 (c 1.0, DMF); 1H NMR (DMSO d6) 1.32 1.82 (m, 8H), 2.75 2.93 (m, 2H), 3.00 3.22 (m, 6H), 4.12 4.42 (m, 4H), 5.03 (s, 4H), 7.21 7.40 (m, 10H), 7.62 (d, J = 8.5 Hz, 2H), 7.65 8.20 (m, 4H), 8.28 (d, J = 7.4 Hz, 2H), 8.53 (br s, 2H). COOH pr otons are missing. 13C NMR (DMSO d6) 24.6, 28.1, 40.2, 40.5, 51.8, 53.8, 65.6, 127.8, 127.9, 128.4, 136.9, 156.0, 159.3, 170.3, 173.2. HRMS Calcd for [C34H46N12O14S2+H]+: 911.2771. Found, 911.2721. 3.4.3 General Procedure for the P reparation of LL -Dipept ides 3.11a -d and the Diastereomeric M ixture (3.11b+3.11b') N(Cbz -A minoacyl)benzotriazoles 3.7a d and ( 3.7b+3.7b' ) (0.5 mmol) were added at 20 C to a solution of L -Arg OH 3. 10 (0.5 mmol) in CH3CN /H2O ( 5 mL / 3 mL) The reaction

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77 mixture was then stirred at room temperature until the starting material was completely consumed as observed by TLC using EtOAc/hexanes (1:2) as the eluent. The reaction mixture was evaporated under reduced pressure and the residue was dissolved in a minimum amount of MeOH and then product was reprecipitated with Et2O. This was repeated three times to remove all the 1 H Benzotriazole from the reaction mixture. (S )-2 -((S ) -2 -Benzyloxycarbonylamino -3 -phenyl -propionylamino) -5 guanidinopentanoic acid, (Cbz L -Phe -L Arg -OH, 3. 11a) : White mic rocrystals (83%); mp 131 132C ( l it130, 131133 C),: [ ]23 D = 10.37 (c 1.0, DMF); 1H NMR (DMSO d6) 1.35 1.75 (m, 4H), 1.65 1.80 (m, 1H), 2.95 3.18 (m, 3H), 3.85 4.00 (m, 1H), 4.12 4.25 (m, 1H), 4.93 (s, 2H), 7.05 7.35 (m, 10H), 7.35 7.55 (m, 3H), 7.55 7.70 (m, 2H), 9.41 (br s, 1H). 13C NMR (DMSO d6) 25.3, 29.8, 37.4, 40.4, 53.7, 56.7, 65.2, 126.2, 127.3, 127.7, 128.1, 128.3, 129.2 137.1, 138.4, 155.9, 157.4, 170.4, 175.5. (S) -2 -((S ) -2 -Benzyloxycarbonylamino -4 -methylsulfanyl butanoylamino) 5 guanidinope nta noic acid (Cbz -L -Met -L Arg -OH, 3.11b): White microcrystals (81%); mp 143 144 C; [ ]23 D = 7.20 ( c 1.0, DMF); 1H NMR (DMSO d6) 1.30 1.70 (m, 4H), 1.70 1.95 (m, 2H), 2.01 (s, 3H), 2.35 2.55 (m, 2H), 2.95 3.10 (m, 2H), 3.80 3.90 (m, 1H), 4.00 4.10 (m, 1H), 5.03 (s, 2H), 7.02 7.40 (m, 6H), 7.47 (d, J = 6.9 Hz, 1H), 7.67 (d, J = 7.2 Hz, 1H), 7.40 8.00 (m, 2H), 9.46 (br s, 1H). 13C NMR (DMSO d6) 14.6, 25.3, 29.8, 30.0, 31.8, 53.6, 54.2, 65.5, 127.0, 127.6, 127.8, 128.4, 137.0, 156.0, 157.4, 170.3, 175.3. HRMS Calcd for [C19H29N5O5S+H]+: 440.1962. Found, 440.1966. (S )-2 -(2 -Benzyloxycarbonylamino-4 -methylsulfanyl -butanoylamino) -5 -guanidino pentanoic acid (Cbz -DL -Met -L Arg -OH, (3.11b+3.11b')) : White microcrystals (76%); mp 121 124 C; [ ]23 D = +5.86 (c 1.0, DMF); 1H NMR (DMSO d6) 1.30 1.70 (m, 4H), 1.70 1.95

PAGE 78

78 (m, 2H), 2.01 (s, 3H), 2.40 2.60 (m, 2H), 2.90 3.12 (m, 2H), 3.80 3.95 (m, 1H), 4.00 4.15 (m, 1H), 5.03 (s, 2H), 7.20 7.40 (m, 6H), 7.40 7.90 (m, 2H), 7.49 (d, J = 5.2 Hz, 1H), 7.60 7.70 (m, 1H), 9.40 ( br s, 1H). 13C NMR (DMSO d6) 14.5, 14.6, 25.1, 25.2, 29.8, 29.8, 30.0, 31.6, 31.7, 53.5, 54.1, 65.4, 127.6, 127.8, 128.4, 137.0, 155.9, 156.0, 157.3, 170.2, 170.3, 175.1, 175.2. HRMS Calcd for [C19H29N5O5S+H]+: 440.1962. Found:440.1960. (S )-2 -[(S ) -2 -Benz yloxycarbonylamino -3 -(1 H -indol -3 yl) propanoylamino] -5 guanidinopentanoic acid, (Cbz L -Trp L Arg -OH, 3.11c): White microcrystals (83%), mp 135 137 C (lit1 33); [ ]23 D = 18.29 (c 1.0, DMF); 1H NMR (DMSOd6) 1.35 1.80 (m, 4H), 2.83 2.97 (m, 1H), 2.97 3.12 (m, 2H), 3.12 3.23 (m, 1H), 3.87 4.00 (m, 1H),,4.15 4.31 (m, 1H), 4.94 (s, 2H), 6.95 (t, J = 7.4 Hz, 1H), 7.00 7.10 (m, 1H), 7.14 (s, 1H), 7.10 7.36 (m, 7H), 7.40 8.20 (m, 5H), 9.42 (br s, 1H), 10.84 (s, 1H). 13C NMR (DMSO d6) 25.4, 27.8, 29.9, 40.5, 53.7, 56.2, 65.3, 110.5, 111.4, 118.3, 118.4, 120.9, 123.8, 127.3, 127.4, 127.5, 127.7, 128.4, 136.2, 137.1, 155.9, 157.5, 170.7, 175.5. HRMS Calcd for [C23H29N5O5+H]+: 495.2350. Found: 495.2341. (S )-2 -((S ) -2 -Benzyloxycarbonylamino -3 -methyl butanoylamino) -5 -guanidino pentanoic acid, (Cbz -L Val L Arg -OH, 3.11d): White microcrystals (75%), mp 121 123 C (lit,134); [ ]23 D = 0.99 (c 1.0, DMF); 1H NMR (DMSO d6) 0.70 0.95 (m, 6H), 1.30 1.80 (m, 4H), 1.90 2.15 (m, 1H), 2.90 3.15 (m, 2H), 3.76 3.95 (m, 2H), 5.04 ( s, 2H), 7.20 7.40 (m, 5H), 7.44 (d, J = 6.9 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.40 8.15 (m, 3H), 9.37 (br s, 1H). 13C NMR (DMSO d6) 18.0, 19.5, 25.5, 30.0, 30.3, 53.7, 60.7, 65.6, 127.7, 127.9, 128.5, 137.2, 156.4, 157.6, 170.2, 175.5. HRMS. Calcd for C19H29N5O5: 408.2241 Found: 408.2204, [M+H]+

PAGE 79

79 3.4.4 Preparation of N-Cbz -N-NO2L Arg -Bt, 3.1 5 NCbz NNO2-L -Arg Bt 3.1 5 was prepared from NCbz NNO2-L -Ar g -OH 3.14 using a reported procedure.135 NCbz -NNO2L -Arg OH (0.5 mmol) was added t o a solution of 1H benzotriazol e (2.0 mmol) in anhydrous THF (15 mL) at room temperature in the presence of SOCl2 (0.6 mmol) Recation mixture was stirred for 45 min at room temperature The white precipitate formed during the reaction was filtered off, and the filtrate was evaporated under reduced pressure. The residue was dissolv ed in ethyl acetate (75 mL) and the solution was washed with 4N HCl (3 20), brine (20 mL), and dried over MgSO4. Removal of solvent under reduced pressure gave 3. 15 which was further purified by reprecipitation from MeOH/Et2O mixture for the elemental an alysis. Benzyl N -[(1 S )-4 -{[amino(nitroimino)methyl]amino} -1 -(1 H -1,2,3 -benzotriazol -1 ylcarbon yl) butyl]carbamate ( N-Cbz -N-NO2L Arg -Bt, 3.1 5 ): White microcr ystals (97%); mp 146 148C; 23 D = 16.21 ( c 1.0, DMF); 1H NMR (DMSO d6 2.10 (m, 4H), 3.08 4.23 (m, 2H), 5.05 (s, 2H), 5.43 5.55 (m, 1H), 7.28 7.44 (m, 5H), 7.66 (t, J = 7.7 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.74 8.16 (m, 2H), 8.20 8.36 (m, 3H), 8.45 (br s, 1H). 13C NMR (DMSO d6) 24.8, 28.1, 40.2, 54.1, 65.9, 114.0, 120.3, 126.8, 127.9, 128.0, 128.4, 130.6, 131.2, 136.7, 145.4, 156.4, 159.3, 171.8. Anal. Calcd for C20H22N8O5: C, 52.86; H, 4.88; N, 24.66. Found: C, 52.96; H, 4.89; N, 24.50. 3.4. 5 General Procedure for the P reparation of LL -Dipeptides 3.17a -d and the D iastereomeric mixture (3.17a+3.17a') NCbz NNO2-L -Arg Bt 3.1 5 (0.5 mmol) was added at 20 C to a solution of free amino acid 3.16a -d and the racemic mixture ( 3.16a+3.16a' ) ( 0.5 mmol) in CH3CN /H2O ( 10 mL/10 mL) in the presence of Et3N (0.6 mmol). The reaction mixture was then stirred at 20 C until the starting material was completely consumed as observed by TLC using EtOAc/hexanes (1:2) as

PAGE 80

80 the eluent. After addition of 4N HCl (1 mL), the solution was concentrated under reduced pressure to remove CH3CN Residue was extracted with EtOAc (20 mL ), and the organic extract was washed with 4N HCl (5 mL), and sat urated NaCl (10 mL) then dried over anhydrous MgSO4. Evaporation of the solvent gave the crude mixture of the desired product and the intramolecular cyclization product 3.18. Crude mixture wa s purified by reprecipitation from MeOH/ Et2O Intramolecular cyclization product was found to be soluble in Et2O .Insoluble produc t was filterd off and washed further with Et2O to give desired product in pure form. In the case of 3.17d after completion, reaction mixture was acid ified with 4N HCl at 15 to 10 oC. Solid precipitated out was filterd off and washed with excess of Et2O to remove intramolecular cyclization product 3.18. Product 3.17d was isolated in pure form. (S )-2 -((S ) -2 -Benzyloxycarbonylami no -5 -(nitroguanidino) pentanoylamino) -3 phenylpropanoic acid, ( N-Cbz -N-NO2-L Arg -L Phe -OH, 3.1 7 a): White microcrystals (68%); mp 220 222 C (lit,118 mp 225226 C); [ ]23 D = +5.32 ( c 1.0, DMF); 1H NMR (DMSO d6) 1.30 1.80 (m, 4H), 2.80 2.98 (m, 1H), 2.98 3.22 (m, 3H), 3.90 4.15 (m, 1H), 4.37 4.54 (m, 1H), 5.02 (s, 2H), 7.07 7.50 (m, 11H), 7.60 8.25 (m, 2H), 8.12 (d, J = 7.4 Hz, 1H), 8.48 (br s, 1H), 12.78 (br s, 1H). 13C NMR (DMSO d6) 24.7, 29.2, 36.7, 53.3, 54.2, 65.5, 126.5, 127.7, 128.2, 128.4, 129.2, 137.0, 137.4, 155.9, 159.3, 171.7, 172.8. Anal. Calcd for C23H28N6O7: C, 55.19; H, 5.64; N, 16.79 Found: C, 54.84; H, 5.76; N, 16.63. 2 -((S )-2 -(Benzyloxycarbonylamino) -5 -(nitroguanidino)pentanamido) -3 phenylpropanoic acid ( N-Cbz -N-NO2L Arg -DL -Phe -OH, (3.1 7 a+3.1 7 a')): White microcrystals (65%); mp 205 207 C; [ ]23 D = +8.14 ( c 1.0, DMF); 1H NMR (DMSO d6) 1.28 1.73 (m, 4H), 2.82 3.01 (m, 1H), 3.02 3.23 (m, 3H), 3.96 4.13 (m, 1H), 4.40 4.55 (m, 1H), 5.06 (s, 2H), 7.10 7.50 (m, 11H), 7.50 8.30 (m, 2H), 8.08 8.30 (m, 1H), 8.52 (br s, 1H),

PAGE 81

81 12.82 (br, s).13C NMR (DMSO d6) 24.7, 29.2, 36.7, 37.0, 53.2, 53.3, 54.2, 65.4, 126.4, 127.0, 127.7, 127.8, 128.1, 128.4, 129.2, 137.0, 137.3, 155.8, 159.3, 171.6, 172.7. Anal. Calcd for C23H28N6O7: C, 55.19; H, 5.64; N 16.79. Found: C, 55.49; H; 5.57; N; 16.47. (S )-2 -((S ) -2 -Benzyloxycarbonylamino -5 -nitroguanidino -p entanoylamino) -4 methylsulfanyl butanoic acid, ( N-Cbz -N-NO2-L Arg -L -Met -OH, 3.1 7 b): White microcrystals (66%); mp 144 146 C; [ ]23 D = 4.60 (c 1.0, DMF); 1H NMR (DMSO d6) 1.45 1.65 (m, 4H), 1.65 2.00 (m, 2H), 2.03 (s, 3H), 2.40 2.60 (m, 2H), 3.05 3.20 (m, 2H), 3.95 4.05 (m, 1H), 4.25 4.37 (m,1H), 5.01 (s, 2H), 7.20 7.40 (m, 5H), 7.46 (d, J = 8 Hz, 1H), 7.46 8.20 (m, 2H), 8.19 (d, J = 7.7 Hz, 1H), 8.50 (b r s, 1H), 12.68 (s, 1H). 13C NMR (DMSO d6) 14.6, 24.7, 29.1, 29.6, 30.8, 50.8, 54.1, 65.4, 127.8, 127.8, 137.0, 155.9, 159.3, 171.9, 173.2. Anal. Calcd for C19H28N6O7S: C, 47.10; H, 5.82; N, 17.34. Found: C, 47.46; H, 5.74; N, 17.64. (S )-2 -((S ) -2 -Benzylo xycarbonylamino -5 -nitroguanidinopentanylamino) -3 hydroxypropano ic acid ( N-Cbz -NNO2-L Arg -L Ser -OH, 3.1 7 c): White microcrystals (65%); mp 83 85 C; [ ]23 D = 5.58 (c 1.0, DMF); 1H NMR (DMSO d6) 1.40 1.85 (m, 4H), 3.03 3.24 (m, 2H), 3.54 3.80 (m, 2H), 4.03 4.18 (m, 1H) 4.21 4.31 (m, 1H), 5.02 (s, 1H), 7.23 7.40 (m, 5H), 7.44 (d, J = 8.2 Hz, 1H), 7.50 8.20 (m, 2H), 8.05 (d, J = 7.3 Hz, 1H), 8.48 (br s, 1H), 12.61 (br s, 1H). 13C NMR (DMSO d6) 24.6, 29.3, 40.2, 54.0, 54.6, 61.3, 65.4, 127.7, 127.8, 128. 4, 137.0, 155.9, 159.3, 171.9. Anal. Calcd for C17H24N6O8: C, 46.36; H, 5.49; N, 19.08. Found: C; 46.72; H, 5.63; N, 18.77. ((S )-2 -Benzyloxycarbonylamino -5 -nitroguanidinopentanamido)ethanoic acid ( NCbz N-NO2-L Arg -Gly -OH, 3.1 7 d): White microcryst als (8 0%); mp 116 117 C (lit,136 114 117 C); [ ]23 D = 1.54 (c 1.0, DMF); 1H NMR (DMSO d6) 1.43 1.80 (m, 4H), 3.04 3.21 (m, 2H), 3.68 3.84 (m, 2H), 3.98 4.10 (m, 1H), 5.03 (s, 2H), 7.26 7.42 (m, 5H), 7.46

PAGE 82

82 (d, J = 8.0 Hz, 1H), 7.608.16 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H) 8.50 (br s, 1H). 13C NMR (DMSO d6) 24.7, 29.2, 40.7, 45.7, 54.6, 65.5, 127.8, 127.9, 128.4, 137.0, 156.0, 159.3, 171.2, 172.2. 3.4.6 Procedure for the Preparation o f N -Cbz -Dipeptid oylbenzotriazole Derivatives 3.22a a nd (3.2 2 a+3.2 2 a ') f ro m C bz -L Asp (Obz ) -OH 3. 19 Compound Cbz -L -Asp(OBz) Bt, 3. 20 was prepared from Cbz L -Asp(OBz) -OH 3.1 9 following the literature procedure.23 Cbz -L -Asp(OBz) Bt 3. 20 was coupled with L -Phe and DL Phe to afford corresponding protected dipeptides 3.21a and the diast ereomeric mixture (3.21a+3.21a ), which were finally converted to their benzotriazole derivatives 3.22a and (3.22a+3.22a ).23 3.4.6.1 Procedure for the preparation of Cbz L Asp(OBz) -Bt, 3.20 To a solution of 1 H benzotriazole (8 mmol) in anhydrous CH2Cl2 (10 mL), SOCl2 (3 mmol) was added and the reaction mixture was stirred for 20 min at room temperature. To this solution, N -protected amino acid Cbz L -Asp(OBz) -OH 3.19 (2 mmol) was directly added and reaction mixture was stirred for 2 h at room tempreture. P rogress of the reaction was monitored by 1H NMR. The white precipitate obtained was filtered off and the filtrate was concentrated under vacuum. To the residue obtained, ethyl acetate (100 mL) was added and the solution was washed with aqueous Na2CO3 (3 x 50 mL) solution followed by brine (25 mL) The organic layer was dried over anhydrous MgSO4 and the solvent was removed under reduced pressure. Compounds were recrystallized from CHCl3/hexane s for elemental analysis (S )-Benzyl 4 -(1 H benzo triazol -1 -yl) -3 -(b enzyloxycarbonylamino) -4 -oxobutanoate (Cbz -L Asp(OBz) -Bt, 3.21) White microcrystals (99%); 23 D = 9.23 ( c 1.0, DMF); 1H NMR (CDCl3J = 16.5, 4.8 Hz, 1H), 3.43 (dd, J = 16.8, 4.8 Hz, 1H), 5.07 (s, 2H), 5.13 (s, 2H), 5.88 6.00 (m, 1H), 6.06 (d, J = 8.1 Hz, 1H), 7.14 7.48 (m, 10H), 7.54 (t, J

PAGE 83

83 = 7.6 Hz, 1H), 7.68 (t, J = 8.2 Hz, 1H), 8.14 (d, J = 8.2 Hz, 1H), 8.23 (d, J = 8.2 Hz, 1H). 13C NMR (CDCl3) 37.3, 51.7, 67.0, 67.3, 114.3, 120.2, 126.5, 128.0, 128.2, 128.2, 128.3, 128.4, 130.8, 131.0, 134.9, 135.8, 145.8, 155.6, 169.2, 169.8. Anal. Calcd for C25H22N4O5: C; 65.75; H, 4.84; N, 12.22; Found: C, 65.75; H, 4.90; N, 12.00. 3.4.6.2 Procedure for preparation of Cbz-L Asp(OBz) -L Phe -OH 3.21a and the diastereomeric mixture Cbz -L Asp(OBz) -DL -Phe -OH (3.21a+3.21a') Cbz L -Asp(OBz) -Bt 3. 20 (0.5 mmol) was added at 20 C to a sol ution of L Phe 3.16a or DL Phe (3. 16a+3.16a ') (0.5 mmol) in CH3CN /H2O ( 10 mL/ 10 mL) in the presence of Et3N (0.6 mmol). The reaction mixture was then stirred at room temerature until the starting material was completely consumed as observed by TLC using Et OAc/hexanes (1:2) as the eluent. After addition of 4N HCl (1 mL), the solution was concentrated under reduced pressure to remove CH3CN Residue was extracted with EtOAc (20 mL), and the organic extract was washed with 4N HCl (5 mL), and sat urated NaCl (10 mL) then dried over anhydrous MgSO4. Evaporation of the solvent gave the desired product in pure form, which was further recrystallized from CHCl3/hexanes unless specified, otherwise (S) -2 -((S) -4 -(benzyloxy) -2 -(benzyloxycarbonylamino) -4 -oxobutanamido) -3 p henylpropanoic acid (Cbz L Asp(OBz) -L -Phe -OH, 3.2 1 a): White microcrystals (97%); mp 154 156 oC; 23 D = 5.22 (c 1.0, DMF); 1H NMR (DMSO d 2.63 (m, 1H), 2.70 2.82 (m, 1H), 2.85 2.98 (m, 1H), 2.99 3.10 (m, 1H), 4.35 4.52 (m, 2H), 5.01 (s, 2H), 5.08 (s, 2H), 7.10 7.45 (m, 15H), 7.62 (d, J = 8.2 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 12.81 (s, 1H). 13C NMR (CDCl3) 36.1, 37.3, 50.9, 53.4, 67.0, 67.4, 115.0, 126.2, 127.1, 128.1, 128.2, 128.3, 128.4, 128.5, 129.3, 135.2, 135.6, 135.8, 138.5, 156.2, 170.4, 171.5, 174.1. Anal. Calcd for C28H28N2O7: C, 66.66; H, 5.59; N, 5.55. Found: C, 66.37; H, 5.51; N, 5.62.

PAGE 84

84 2 -((S )-4 -(Benzyloxy) -2 -(benzyloxycarbonylamino) -4 -oxobutanamido) -3 phenylpropanoic acid (Cbz L Asp(OBz) -DL -Phe -OH, (3.2 1 a+3.2 1 a')): White microcrystal s (98%); mp 94 96 C; [ ]23 D = 7.67 (c 1.0, DMF); 1H NMR (DMSO d6) 2.35 2.63 (m, 1H), 2.63 2.70 (m, 1H), 2.70 2.97 (m, 1H), 2.98 3.11 (m, 1H), 4.34 4.52 (m, 2H), 4.93 5.17 (m, 4H), 7.15 7.50 (m, 15H), 7.58 (d, J = 8.8 Hz, 0.5H), 7.61 (d, J = 7.7 Hz, 0.5H), 8.09 (d, J = 6.9 Hz 0.5H), 8.18 (d, J = 7.7 Hz, 0.5 H), 12.80 (br s, 1H). 13C NMR (DMSO d6): 24.8, 29.2, 42.6, 54.2, 65.6, 113.8, 120.2, 126.7, 127.8, 127.9, 128.4, 130.6, 131.0, 137.0, 145.3, 156.1, 159.4, 168.6, 172.9. Anal. Calcd for C28H28N2O7: C, 66.66; H, 5.59; N, 5.62. Found: C, 66.81; H, 5.72; N, 5.56. 3.4.6.3 General procedure for preparation of Cbz -L Asp( OB z )-L -Phe Bt, 3.22a and the diasteromeric m ixture (3.22a+3.22a' ) To a solution of 1 H -benzotriazole (2 mmol) in anhydrous CH2Cl2 (5 mL), at room tempreture SOCl2 (0.8 mmol) was added and stirred for 20 min. The reaction mixture was cooled to 15 C and Cbz -L -Asp(OBz) L -Phe -OH 3.21a (0.6 mmol) or Cbz -L -Asp(OBz) DL -Phe OH (3.21a+3.21a' ) (0.6 mmol) was added as solid and stirred for 4 h at 15 C. The white precipita te formed during the reaction was filtered off, and the filtrate was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (75 mL) and the solution was washed with aqueous sodium carbonate (3 20), brine (20 mL), and dried over MgS O4. Removal of solvent under reduced pressure gave corresponding N (Cbz -protected)dipeptidoylbenzotriazole derivatives which were further recrystallized by reprecipitation from CHCl3/hexanes for the elemental analysis. (S) -Benzyl -4 -((S) -1 -(1 H -benzotriazol -1 yl) -1 -oxo -3 -phenylpropan-2 -ylamino) -3 (benzyloxycarbonylamino) -4 -oxobutanoate (Cbz -L Asp(OBz) L -Phe -Bt, 3.2 2 a): White microcrystals (75%); mp 140 142 C; [ ]23 D = +11.23 (c 1.0, DMF); 1H NMR (CDCl3) 2.73

PAGE 85

85 (dd, J = 17.3, 6.5 Hz, 1H), 2.96 3.15 (m, 1H), 3.20 (dd, J = 14.0, 7.8 Hz, 1H), 3.45 (dd, J = 14.1, 5.0 Hz, 1H), 4.58 4.72 (m, 1H), 5.00 5.20 (m, 4H), 5.90 (d, J = 8.5 Hz, 1H), 6.10 6.22 (m, 1H), 7.02 7.16 (m, 2H), 7.16 7.44 (m, 14H), 7.55 (t, J = 7.2 Hz, 1H), 7.68 (t, J =7.2 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H). 13C NMR (CDCl3): 36.0, 38.2, 50.7, 54.3, 66.9, 67.3, 114.2, 120.3, 126.5, 127.3, 128.1, 128.2, 128.3, 128.3, 128.5, 128.5, 128.7, 129.2, 130.7, 130.9, 134.9, 135.2, 135.9, 145.9, 156.0, 170.0, 170.3, 171.6. Anal. Calcd for C34H31N5O6, C, 67.43; H, 5.16; N, 11.56. Found: C, 67.46; H, 5.22;N, 11.45. (3 S )-Benzyl 4 -(1 -(1 H -benzotriazol -1 yl) -1 -oxo -3 -phenylpropan-2 ylamino) -3 (benzyloxycarbonylamino) -4 -oxobutanoate (Cbz -L Asp(OBz) -DL -Phe Bt, (3.2 2 a+3.2 2 a')): White microcrystals (80%); mp 129 C; [ ]23 D = 10.52 ( c 1.0, DMF); 1H NMR (CDCl3) 2.58 2.80 (m, 1H), 2.96 3.10 (m, 1H), 3.13 3.27 (m, 1H), 3.40 3.52 (m, 1H), 4.59 4.21 (m, 1H), 5.01 5.20 (m, 4H), 5.83 5.99 (m, 1H), 6.10 6.25 (m, 1H), 7.06 7.16 (m, 2H), 7.18 7.44 (m, 14H), 7.55 (t, J = 7.7 Hz, 1H), 7.69 (t, J = 7.7 Hz, 1H ), 8.16 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 8.2 Hz, 0.5 H), 8.23 (d, J = 8.2 Hz, 0.5H). 13C NMR (CDCl3) 36.0, 38.3, 38.5, 50.7, 50.8, 54.2, 54.4, 67.0, 67.3, 114.3, 120.4, 126.5, 127.4, 128.1, 128.1, 128.3, 128.3, 128.4, 128.6, 128.7, 128.7, 129.2, 130.8, 131.0, 134.9, 134.9, 135.2, 135.2, 135.8, 146.0, 156.0, 170.0, 170.2, 170.3, 171.5, 171.7. Anal. Calcd for C34H31N5O6: C, 67.43; H, 5.16; N, 11.56. Found: C, 67.11; H, 5.01; N, 11.38. 3.4.7 General Procedure f or P reparation o f Arginine LLLTripeptides 3.2 3a -c and t he Diastereomeric Mixture (3.23a+3.23a') N-Cbz -dipeptidoylbenzotriazole s 3.22a -c and (3.22a+3.22a' ) (0.5 mmol) were added at 15 C to a solution of N-NO2L -Arg OH 3.2 (0.5 mmol) in CH3CN (15 mL)/H2O (5 mL) in the presence of Et3N (0.6 mmol). The reaction mixtures were then stirred at 15 C until the starting material was completely consumed as observed on TLC using EtOAc/hexanes (1:2) as the

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86 eluent. After 1 mL of 4N HCl was added, the solution was concentrated under reduced pressure to remove CH3CN The solution was extracted with EtOAc (20 mL), and the organic extract was washed with 4N HCl (5 mL) and sat urated NaCl (10 mL), and then dried (anhydrous MgSO4). Evaporation of the solvent gave the desired products, which were purified by recprecipitaion from MeOH/Et2O. (5 S ,8S,11 S )-8 -Benzyl -5 -(2 -(benzyloxy) -2 -oxoethyl) -11-(3 -(nitroguanidino)propyl) 3,6,9 -trioxo -1 -phenyl -2 -oxa -4,7,10-triazadodecan -12-oic ac id ( N -Cbz L Asp(OBz) -L Phe N-NO2-L Arg -OH, 3. 2 3 a): White microcrystals (84%); mp 128 129 C; [ ]23 D = 11.71 (c 1.0, DMF); 1H NMR (DMSO d6) 1.35 1.85 (m, 4H), 2.50 2.62 (m, 1H), 2.65 2.90 (m, 2H), 2.90 3.21 (m, 3H), 4.10 4.20 (m, 1H), 4.22 4.70 (m, 2H), 5.00 (s, 2H), 5.07 (s, 2H), 7.00 7.50 (m, 15H), 7.50 8.20 (m, 5H), 8.54 (br s, 1H), 12.73 (br s, 1H). 13C NMR (DMSO d6) 24.8, 28.3, 36.2, 37.4, 40.2, 51.2, 51.7, 53.6, 65.6, 126.3, 127.8, 127.9, 128.0, 128.4, 129.3, 136.1, 136.8, 137.5, 155.8, 159.3, 170.1, 170.3, 170.8, 171.3. Anal. Calcd for C34H39N7O10: C, 57.87; H, 5.57; N; 13.89. Found C, 57.47; H; 5.51; N; 13.91. (5 S ,11 S )-8 -Benzyl -5 -(2 -(benzyloxy) -2 -oxoethyl) -11-(3 -(2 -nitroguanidino)propyl) -3,6,9 trioxo -1 -phenyl -2 -oxa-4,7,10-triazadodecan -12-oic a cid (N -Cbz L Asp(OBz) -D L -Phe -NNO2-L Arg -OH, ( 3. 2 3 a+ 3. 2 3 a ')): White microcrystals (82%); mp 60 65 C; [ ]23 D = 6.31 (c 1.0, DMF); 1H NMR (DMSO d6) 1.35 1.85 (m, 4H), 2.35 2.60 (m, 1H), 2.652.87 (m, 2H), 2.90 3.22 (m, 3H), 4.12 4.28 (m, 1H), 4.34 4.70 (m, 2H), 4.92 5.13 (m, 4H), 7 .00 7.50 (m, 15H), 7.50 8.40 (m, 5H), 8.53 (br s, 1H), 12.69 (br s, 1H). 13C NMR (DMSO d6) 24.8, 28.1, 28.3, 28.6, 36.2, 36.3, 37.4, 38.3, 40.2, 51.2, 51.5, 51.7, 53.7, 65.6, 65.7, 126.3, 127.7, 127.9, 128.0, 128.4, 136.0, 136.1, 136.8, 136.9, 137.5, 155.8, 159.3, 169.9, 170.1, 170.2, 170.4, 170.7,

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87 170.9, 173.3. Anal. Calcd for C34H39N7O10: C, 57.87; H, 5.57; N, 13.89. Found C, 57.47; H; 5.63, N, 13.65. HRMS Calcd for C34H39N7O10+H]+: 706.2831. Found: 706.2822. (S )-2 -[(S ) -2 -((S)-2 -Benzyloxycarbonylaminopro panoylamino) -3 -(1 H -indol -3 yl)propanoyla mino] -5 -nitroguanidinopentanoic acid (Cbz -L Ala -L -Trp N-NO2-L Arg OH, 3. 2 3 b): White microcrystals (66%); mp 150 151 C; [ ]23 D = 10.24 ( c 1.0, DMF); 1H NMR (DMSO d6) 1.13 (d, J = 7.1 Hz, 3H), 1.43 1.85 (m, 4H), 2.90 3.15 (m, 1H), 3.15 3.25 (m, 3H), 3.95 4.10 (m, 1H), 4.15 4.20 (m, 1H), 4.50 4.65 (m, 1H), 4.98 (d, J = 12.4 Hz, 1H, A part of AB system,), 5.03 (d, J = 12.6 Hz, 1H, B part of AB system), 6.96 (t, J = 7.4 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 7.15 (s, 1H), 7.20 7.40 (m, 6H), 7.41 (d, J = 7.4 Hz, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.65 8.40 (m, 2H), 7.96 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.54 (br s, 1H), 10.82 (s, 1H), 12.71 (br s, 1H). 13C NMR (DMSO d6) 18.1, 24.8, 27.5, 28.4, 50.1, 51.6, 53.1, 65.4, 109.8, 111.2, 118.2, 118.4, 120.8, 123.6, 127.4, 127.8, 128.4, 136.0, 137.0, 155.7, 159.3, 171.5, 172.3, 173.3. Anal. Calcd for C28H34N8O8H2O: C, 53.50; H, 5.77; N; 17.82. Found C, 53.18; H, 5.70; N, 17.37. HRMS calcd for [C28H34N8O8+Na] +: 633.2391. Found 633.2371. (5 S 8S ,11 S )-5 -Benzyl -8 -(2 -(methylthio)ethyl) -11-(3 -(nitroguanidino)propyl) -3,6,9 trioxo -1 -phenyl -2 -oxa-4,7,10-triazadodecan -12-oic acid (N -Cbz L -Phe -L -Met -N-NO2-L Arg -OH, 3. 2 3 c): White microcry st als (75%); mp 77 79 C; [ ]23 D = 7.98 (c 1.0, DMF); 1H NMR (DMSO d6) 1.40 1.97 (m, 6H), 1.94 2.10 (m, 3H), 2.10 2.55 (m, 2H), 2.602.84 (m, 1H), 2.85 3.05 (m, 1H), 3.05 3.23 (m, 2H), 4.11 4.45 (m, 3H), 4.93 (s, 2H), 7.00 7.40 (m, 10H), 7.52 (d, J = 8. 8 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 7.4 Hz, 1H), 7.60 8.40 (m, 2H), 8.53 (br s, 1H), 12.69 (br s, 1H). 13C NMR (DMSO d6) 14.5, 14.7, 24.8, 28.1, 29.2, 29.3, 32.3, 37.3, 51.5, 51.7, 56.0, 65.2, 65.4, 126.3, 127.4, 127.6, 127.7, 128.1, 128.3, 129.2, 136.9,

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88 137.0, 138.1, 155.9, 156.0, 159.3, 171.1, 171.5, 173.2, 173.3. HRMS. Calcd for C28H37N7O8S: 632.2497 Found: 632.2478. 3.4.8 Preparation of N-Cbz -N-NO2L Arg -Gly -Bt, 3.2 4 To a solution of 1H -benzotriazole (2.1 mmol) in anhydrous THF (5 mL) at 20 C, SOCl2 (0.8 mmol) was added and stirred for 20 min. The reaction mixture was cooled to 15 C and then added NCbz -NNO2L -Arg Gly OH 3.1 6 d (0.54 mmol) as solid and stirred for 5.5 h at 15 C. The white precipitate formed during the reaction was filtered off, and the filtrate was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (75 mL) and the solution was washed with 4N HCl (3 20), brine (20 mL), and dried over MgSO4. Removal of solvent under reduced pressure ga ve 3.2 4 which was further purified by reprecipitation from MeOH/Et2O mixture for the elemental analysis. (S )-Benzyl 1 -(2 -(1 H -benzotriazol -1 -yl) -2 -oxoethylamino) -5 -(nitroguanidino) -1 oxopentan -2 ylcarbamate ( N-Cbz -N-NO2-L Arg Gly -Bt, 3.2 4 ): White microcrystals (80%); mp 119 122 C; 23 D = 6.00 (c 1.0, DMF); 1H NMR (DMSO d6 1.90 (m, 4H), 3.08 3.27 (m, 2H), 4.11 4.25 (m, 1H), 4.92 5.05 (m, 2H), 5.06 (s, 2H), 7.26 7.45 (m, 5H), 7.58 (d, J = 8.0 Hz, 1 H), 7.64 (t, J = 7.6 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.70 8.40 (m, 2H), 8.23 (d, J = 8.2 Hz, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.55 (br s, 1H), 8.70 (t, J = 5.2 Hz, 1H). 13C NMR (DMSO d6): 24.8, 29.2, 42.6, 54.2, 65.6, 113.8, 120.2, 126.7, 127.8, 127.9, 128.4, 130.6, 131.0, 137.0, 145.3, 156.1, 159.4, 168.6, 172.9. Anal. Calcd for C22H25N9O6: C, 51.66; H, 4.99; N, 24.65. Found: C, 52.01; H, 4.99; N, 24.94. 3.4.9 Preparation of N-Cbz -N-NO2L Arg -Gly L Asp -(OH)2, 3.2 6 NCbz NNO2-L -Arg Gly Bt, 3.2 4 (0. 5 mmol) was dissolved in a minimum amount of THF and added dropwise at 15 oC to a solution of L -Asp OH 3.2 5 (0.55 mmol) in CH3CN (5

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89 mL ) / H2O ( 3 mL) in the presence of Et3N (2.1 mmol). The reaction mixture was stirred at 15 oC and progress was monitored using TLC by disappearance of the Cbz-NO2-L -Arg -Gly Bt, 3.2 4 After 3.5 h reaction mixture was concentrated under reduced pressure to remove CH3CN The reaction mixture was acidified with 4N HCl (2 mL) under cold condition, and the solution was extracted with EtOAc (100 mL) after adding sol id NaCl to the acidified solution. The organic extract was washed with 4N HCl and sat urated NaCl (10 mL) and then dried over anhydrous MgSO4. Evaporation of solvent under reduced pressure, gave the product which was purified by reprecipitation from MeOH/Et2O. (S )-2 -(2 -((S )-2 -(Benzyloxycarbonylamino) -5 -(2 nitrogua nidino)pentanamido)acetamido)succinic acid ( N-Cbz -N-NO2L Arg -Gly -L Asp (OH)2), 3.2 6 : White microcrystals (60%); mp 75 79 C; 23 D = 13.7 (c 1.0, DMF); 1H NMR (DMSO d6 1.80 (m, 4H), 2.59 (dd, J = 16.8, 6.6 Hz, 1H), 2.70 (dd, J = 16.5, 5.5 Hz, 1H), 3.03 3.22 (m, 2H), 3.65 3.83 (m, 2H), 3. 95 4.08 (m, 1H), 4.50 4.62 (m, 1H), 5.00 (d, J = 12.6 Hz, A part of AB system, 1H), 5.05 (d, J = 12.6 Hz, B part of AB system, 1H), 7.25 7.42 (m, 5H), 7.52 (d, J = 7.4 Hz, 1H), 7.55 8.10 (m, 2H), 8.07 8.30 (m, 2H), 8.48 (br s, 1H) 12.60 (br s, 2H). 13C NMR (DMSO d6): 24.7, 29.0, 36.1, 41.6, 45.6, 48.5, 54.4, 65.0, 127.8, 128.4, 136.9, 156.1, 159.3, 168.6, 171.6, 172.0, 172.3. HRMS C alcd for [C21H31N7O10+2Na]+: 570.1530. Found: 570.1531.

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90 CHAPTER 4 CONVENIENT ONE POT SYNTHESIS OF 5 (SUBSTITUTED AMINO) -1,2,3,4 THIATRIAZOLES 4.1 Introduction Azoles are one important family of heterocycles; important as biologically active compounds and synthetic intermediates.137 My research interest focused on thiatriazoles, particularly monosubstituted aminothiatriazoles, i e 5 -subst itu ted amino 1,2,3,4 thiatriazole 4.1 ,138141 because of their interesting biological properties including antihypertensive,142 antibacterial,143 antitubercular,144 antiviral,146 fungicidal,147 anticancer,11 and central nervous sy st em stimulant and muscle relaxant activities.148 Monosubstituted aminothiatriazoles 4.1 are also synthetic intermediates for the preparation of 3 oxo1,2,4 thiadiazolin 5 yl ureas 4.2 ,149 fused thiazolidine 4.3,150 fused thiazoline 4.4150and fused 1,2,4 thiadiazoles 4.5 -4.7 (Fi gure 4 1).151 Figure 4 1. 5 (Monosubstituted amino ) 1,2,3,4 -thiatriazole 4.1 and het e rocycles derived from 4.1 Reported synthesis of 5 ( monosubstituted amino ) 1,2,3,4 thiatriazoles 4.1 include: (i) reaction of thiosemicarbazides with nitrous acid,143 144 ,1 46157 which is the most widely used method, (ii) reaction of isothiocyanates with hydrazoic acid,1 55, 156, 158 161 trimethylsilyl azide159,162 or sodium azide163,164 that proceed through 1,3-dipolar cycloadditions and electrocyclizations,165

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91 (iii) aza transfer procedure with diazonium salts (Scheme 4 1).166 The disadvantages associated with the thiosemicarbazide method are: use of strong acid which may affect the acid sensitive functional group, if present in the molecule, and use of hazardous reagent ( hydrazine) during the preparation of thiosemicarbazide.150 ,15 1 Similarly, the hydrazoic acid method accomplish with moderate yields and formation of side product. Though, the reaction conditions using trimethylsilyl azide and sodium azide methods are milde r, there is no detail synthetic study to show the generality of the method. Scheme 4 1. Literature methods for synthesis of monosubstituted aminothiatriazoles 4.1 Recently Batey et al reported synthesis of substituted aminothiatriazoles 4.12 from cor responding thiocarbamoylimidazolium salts 4.11 by treatment with sodium azide followed by electrocyclization in 50 96% yield (Scheme 4 2).167 Thiocarbamoylimidazolium salts 4.11 were synthesized from corresponding amines 4.8 with thiocarbonyldiimidazole 4. 9 fo llowed by methylation with iodo methane. Scheme 4 2. Literature procedure for synthesis of substituted aminothiatriazoles 4.12

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92 Though this procedure is milder as compared to previously describ ed classical methods, it suffers from disadvantages such as: utilization of excess (10 equivalents) of methyl iodide and long reaction time (2743 h). From the foregoing account, it is obvious that, mild, efficient and convenient methods are limited in number, and there is a great demand for such a reaction. Th erefore, in continuation of our group extensive research on the utility of benzotriazole functionalized reagents, I was interested to find the possibility of such a reagent that could fulfill the demand. In earlier work from our group, 1,1 -carbonylbisbenz otriazole 4. 13,168 di(1 H benzotriazol 1 -yl)methanimine 4.14,169 and bis(1H benzotriazole 1 -yl)methanethione 4.1541, (Figure 4 2) have provided convenient synthes i s of substituted ureas from 4.13,168 of di and trisubstitu t ed thioureas from 4.15,37 of tri and tetrasubstituted guanidines from 4.14,169 and of 1,2,3 trisubstitu t e d guanidines from 4.15.40 Analogous to this, Batey and c oworkers have utilized the imidazol yl reagents 4.16 and 4.17 for the synthesis of ureas170 and thioureas171 in comparable y ield s. However, the use of imidazole methodology needed an extra step of conversion in situ into quaternary derivatives 4.11, 4.18 as compared to the benzotriazole methodology for the synthesis of ureas and thioureas. The synthesis of substituted guanidines fr om both the reagents 4.14169 and 4.9172 are comparable in yields and number of steps. Figure 4 2. Structure of bis( 1 H -benzotriazol 1 yl) and bisimidazol 1 -yl reagents

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93 Herein, I describe a convenient method for synthesis of 5 ( substituted amino)1,2,3,4 thiatriazoles 4.1 from bis(1 H -benzotriazol 1 yl)methanethione 4.15, which is superior to Batey s thiocarbonyldiimidazole method.167 4.2 Results and Discussion 4.2.1 Two Step Synthesis o f 5 -( Substituted a mino )-1,2,3,4 -thiatriazole s 4.1a -i Bis(1 H -benzotriaz ol 1 -yl)methanethione 4.15 was prepared in 99% yield from thiophosgene by modification of the literature procedure.41 The r eaction was carried out at room temperature for 3 h instead at 0 oC. Yield was considerably improved from 80 to 99%. Thiocarbamoylben zotriazoles 4.20a -g,j,k were easily prepared from 4.15 and amines 4.19a -g,j,k in anhydrous CH2Cl2 at 20 C in shorter duration of time, 1 4 h (Scheme 4 3, Table 4 1) as compared to literature procedure.37,40 According to the literature37 reaction of aromat ic amine, p anisidine 4.19g with 4.15 did not yield the corresponding thiocarbamoyl derivative 4.20g, only corresponding isothiocyanate was obtained (Scheme 4 4) However, i n my case, foll o wing same literature procedure ,37 corresponding thiocarbamoyl derivative 4.20g was obtained in 40% yield Optimizing the reaction condition, by decreasing reaction time from 18 h to 45 min yielded desired product 4.20g in 80% yield. Structure of the product was established from 1H NMR, 13C NMR and elemental analysis. Thio carbamoylbenzotriazoles 4.20i,j were prepared from bis(1 H benzotriazol 1 yl)methanethione 4.15 a nd amino acid ester hydrochlorides 4.19h,i i n aqueous CH3CN in the presence of Et3N (Scheme 4 3, Table 4 1) Subsequent treatment of 4.20a -i with NaN3 in aqueou s CH3CN at room temperature for 1 4 h yielded 5 ( substituted amino )1,2,3,4 thiatriazoles 4.1a -i after 1,3 dipolar cycloaddition followed by electrocyclization in 65 96% yields and overall yields of 50 94% starting from bis(1 H -benzotriazol 1 -yl)methane thione 4.15 (Scheme 4 3, Table 4 2). Monosubstituted amino thiatriazoles 4.1a -i were isolated without any

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94 chromatographic purification. It was observed that these compounds 4.1a i were decomposed slowly at room temperature. Scheme 4 3. Two step synthesi s of 5 (substituted amino) 1,2,3,4 thiatriazoles 4.1a-i from bis(1 H benzotriazol 1 -yl)methanethione 4.15 Scheme 4 4. Literature report for reaction of b is(1 H benzotriazol 1 -yl)methanethione 4.15 and p anisidine 4.19g T able 4 1 Preparation of thiocar bamoylbenzotriazoles 4.20a k aFor structure of product s 4.20 see Table 4 2.bIsolated Yields. Entry Amines 4.19 ( R ) Product a 4.20 Yield b (%) Time (h) 1. n Propyl 4.20a 89 4 2. Benzyl 4.20b 95 4 3. Methylbenzyl 4.20c 93 4 4. (Furan 2 yl)methyl 4.20d 91 1 5. Cyclohexyl 4.20e 95 3 6. Allyl 4.20f 93 3 7. 4 Met hoxyphenyl 4.20g 90 0.8 8. CH 2 COOCH 3 4.20h 70 5 9. ( R ) CH(CH 3 )COOCH 3 4.20i 68 3.5 10 CH 2 CH 2 CH 2 CH 2 4.20j 97 3.5 11. (CH 3 ) 2 4.20k 68 2

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95 Table 4 2 Preparation of 5 -(substituted amino ) 1,2,3,4 thiatriazoles 4.1a -i from thiocarbamoylbenzotrizoles 4.20a -i Entry Thiocarbamoylben zotriazoles 4.20a -i Product 4.1a -i Time (h) Yield a (%) Overall yieldb(%) 1 4.20a 4.1a 4 84 88 2 4. 20b 4.1b 3 96 94 3 4.20c 4.1c 2 94 78 4 4 .20d 4.1d 2 91 83 5 4.20e 4.1e 2.5 87 87 6 4.20f 4.1f 1 78 73 7 4.20g 4.1g 2.5 65 63 8 4.20h 4.1h 3 86 60 9 4. 20i 4.1 i 4 74 50 aIsolated yields. bOverall yield after two steps in scheme 4 3 cLit. references for known compounds are indicated in experimental section. 4.2.2 One -Pot S ynthes is of 5 -(Substituted amino) -1,2,3,4 -thiatriazoles 4.1a -g,i,l Success of this two step reaction scheme, and generation of a stable byproduct (1 H benzotriazole) during the first step led me to att empt one -pot synthesis of aminothiat riazole s

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96 4.1a -g, i,l In the one -pot reactions, in case of amines 4.19a -g,l after completion of the first step, CH2Cl2 was removed under reduced pressure and the residue (a crude mixture of 4.20a -g, l and by -product, 1 H b enzotriazole), was treated with NaN3 in aqueous CH3CN solution for 1 4 h to obtain 5 ( substituted amino ) 1,2,3,4 thiatriazoles 4.1a -g,i,j in 73 97% yields (Scheme 4 5 Table 4 3). 1 H Benzotriazole, the only by product of the conversion was removed by simpl y washing the organic layer with saturated aqueous Na2CO3. All products 4.1a-g, l were isolated after one pot reaction without chromatographic purification. In the case of 4.1i the first step of coupling 4.15 with D -Ala OMe ester hydrochloride 4.19i was ca rried in CH3CN/H2O in presence of Et3N instead of CH2Cl2 (Scheme 4 6, Table 4 3) After completion of first step, an aqueous solution of NaN3 was directly added to the reaction mixture, without evaporation of CH3CN. In the case of 4.1l after evaporating CH3CN from the reaction m ixture, EtOAc was added for work up. Since the product was insoluble, it was directly filtered off to give 4.1l. Since trace of 1 H -benzotriazole was evident from 1H NMR, the crude product was further stirred in MeOH for 1 h and fil tered off to give 4.1l in pure form. Scheme 4 5 One -pot synthesis of 5 ( substituted amino ) 1,2,3,4 -thiatriazoles from bis(1 H benzotriazol 1 yl)methanethione 4.15 and primary amines 4.19a -g,l Scheme 4 6. One -pot synthesis of 5 ( substituted amino ) 1 ,2,3,4 -thiatriazoles 4.1i from bis(1 H benzotriazol 1 yl)methanethione 4.15 and amino acid ester hydrochloride 4.19i

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97 Table 4 3 One -pot synthesis of 5 ( substitut ed amino ) 1,2,3,4 thiatriazoles 4.1a -g,i,l from 4.15 Entry Amine 4.19 Product 4.1 Time (h) Yie ld a (%) Lit Yieldb(%) (% 1 4.19a 4.1a 7 92 65 2 4.19b 4.1b 5 96 77 3 4.19c 4.1c 5 97 4 4.19d 4.1d 3 93 5 4.19e 4.1e 5 93 84 6 4.19f 4.1f 5 85 62 7 4.19g 4.1g 4 81 81 8 4.19i 4.1i 7 74 9 4.19l 4.1l 8 73 aIsolated yields after one pot reaction. bLit. r eference s for known compounds are indicated in experimental section This represents a general method for the synthesis of 5 -( monosubstituted amino ) 1,2,3,4 thiatriazoles since a variety of substrat -

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98 amino acid esters have been utilized (Table 4 3). Amines 4.19a -e undergo one -pot reaction to the corresponding aminothiatriazoles 4.1a -e in excellent yields (9297%). Similarly, the allylamine 4.19f h as been used to prepare 4.1f in 85% yield. Interestingly, aromatic amine 4.19g can also be converted to the corresponding aminothiatriazole 4.1g in 81% yield. In the case of amino acid, D -Ala OMe ester hydrochloride 4.19i the product 4.1i was obtained in 74% yield. The substituted bis aminothiatriazole 4.1 l was also prepared in 73% yield from 4.19 l Yields obtained from one -pot procedure were higher than the two step reaction scheme. 4.2.3. Attempted Synthesis of 5 -(Disubstituted a mino )-1,2,3,4 -t hiatriazol e s In atte mpt to synthesize 5 (disubstituted amino ) 1,2,3,4 thiatriazole, thiocarbamoylbenzotriazole 4.20j was treated with NaN3 in aqueous CH3CN at room temperature under the same reaction condition (Scheme 4 7 ). No product was obtained. Continuing the re action for 48 h yielded no desired product. All the starting material was recovered from reaction mixture. Refluxing the reaction mixture for 12 h did not indicate any product formation by TLC. Changing the solvent from CH3CN/H2O to THF/H2O did not show an y product formation at room temperature for 28 h and refluxing for 24 h. Also changing the substrate from 4.20j to 4.20k did not yield the desired product under room temperature for 48 h or refluxing for 24 h (Scheme 4 8 ). Scheme 4 7 Attempted synthes is of 5 (disubstituted amino ) 1,2,3,4 thiatriazole 4.1j Scheme 4 8 Attempted synthesis of 5 (disubstituted amino ) 1,2,3,4 thiatriazole 4.1k

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99 Failure of the formation of disubstituted aminothiatriazole may be explained by the lack of formation of isothi ocyanate intermediate. However, in the cases of imidazole derivatives, disubstituted aminothiatriazoles have been reported to form in about 50% yields.167 Thus, the formation of isothiocyanate intermediate may not be the correct explanation. When thiocarba moylimidazole 4.10 (Scheme 4 2) including disubstituted amine derivatives were converted to its imidazolium salt 4.11, those underwent facile reactions with sodium azide to form the corresponding thiatriazole compounds. This may be explained by the gain of electrophilicity of the thiocarbonyl carbon atom due to the positive charge on the nitrogen atom of the imidazole ring. This may also be true for benzotriazole derivatives. Thus, in the cases of 4.20j and 4.20k the thiocarbonyl carbon atoms are not suffic iently electrophilic for the attack of the azide nucleophile to displace the benzotriazole group. 4.3 Conclusion In conclusion, a simple, mild and convenient novel one -pot synthetic route for preparation of 5 ( monosubstituted amino ) 1,2,3,4 thi atriazoles has been developed. This method is versatile, general and involves easily accessible starting materials, and can be carried out at room temperature to obtain products in 73 97% yields without the necessity of column purification. Moreover, these yields are better than the reported yields. Thus, it represents a superior method than the reported methods. 4.4 Experimental 4.4.1 General Method s Melting points were determined on a capillary point apparatus equipped with a digital thermometer. 1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded in CDCl3 or DMSO d6. 1H chemical shifts in CDCl3

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100 Solvent peaks were used as internal references for all 13C NMR. Solvent peaks were used as internal refe rences for all 13C NMR. All the amines were purchased from Fluka or Aldrich, and were used without further purification. Elemental analyses were performed on a Carlo Erba 1106 instrument. 4.4.2 Synthesis for the Preparation of Bis (1 H benzotrizol -1 yl)metha nethione 4.15 Thiophosgene (1 mmol) was dropw i s e added to a solution of 1 H -benzotriazole (6.3 mmol) dissolved in CH2Cl2 (100 mL) at room temperature The reaction mixture was stirred for 3 h. After completion, the reaction mixture was diluted with CH2Cl2 (100 mL) and washed with saturated sodium carbonate solution (3 x 100 mL) in order to remove excess of 1H -benzotrizole. The organic layer was dried over anhydrous Na2SO4, and evaporated under vacuum to give bis (1 H benzotrizol 1 yl)methanethione in pure form Bis (1 H -benzotrizol -1 -yl)methanethione 4.15: Yellow microcrystals (99%); mp. 172173 oC (Lit.173 mp 169170 oC); 1H NMR (CDCl3) 7.61 (t, J = 7.4 Hz, 2H), 7.75 (t, J = 7.5 Hz, 2H), 8.23 (d, J = 8.2 Hz, 2H), 8.28 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3) 113.9, 121.0, 126.9, 130.6, 133.1, 146.8, 169.6. 4.4.3 General Procedure for the Preparation of Thiocarbamoylbenzotriaz ole s 4.20a-g,k Thiocarbamylbenzotriazole s 4.20a -g,j,k were synthesized by adding bis(1H -benzotriazol 1 yl)methanethione 4.15 (1 mmol) to ap propriate amine (1 mmol) 4.19a -g,j,k dissolved in CH2Cl2 (10 mL) at room temperature for 1 5 h according to reported procedure.37,40 Reaction mixture was diluted with CH2Cl2, washed with saturated Na2CO3 (3 x 25 mL) followed by brine (25 mL) and dried over anhydrous Na2SO4 and evaporated under reduced pressure to give desired thiocarbamoylbenzotriazoles in pure form. It was further recrystallized from CHCl3/hexane s for elemental analysis. M elting point and spectral data was used to characterize

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101 known 4.20b, d -f,j and were found to be identical to reported values. 4.20b : m p 108109 oC (Lit.172 m p 108109 oC); 4.20d : m p 118 oC (Lit.40 m p 117 oC); 4.20e : m p 72 73 oC (Lit.40 m p 72 73 oC); 4.20f : m p 5758 oC (Lit.37 m p 56 57 oC); 4.20j m p 144145 oC (Lit.40 m p 144145 oC); Lit data not availab le for 4.20k. Compounds 4.20a, c ,g are novel and has been fully characterized by 1H and 13C NMR spectroscopy and elemental analysis. Compound 4.20k was also fully characterized. N -Propyl -1 H -benzotriazole -1 -carbothioamide 4.20a : White microcrystals (89%); m p 89 90 C; 1H NMR (CDCl3) 1.09 (t, J = 7.4 Hz, 3H), 1.85 (sextet, J = 7.4 Hz, 2H), 3.83 (q, J = 6.7 Hz, 2H), 7.49 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 8.94 ( d, J = 8.5 Hz, 1H), 9.11 (br s 1H). 13C NMR (CDCl3) 11.5, 21.4, 46.7, 116.0, 120.1, 125.6, 13 0.2, 132.3, 147.0, 174.3. Anal Calcd for C10H12N4S: C, 54.52; H, 5.49; N, 25.43. Found: C, 54.77; H, 5.50; N, 25.36. N -(1 -Phenylethyl) -1 H -benzotriazole -1 -carbothioamide 4.20c: White M icroc rystals (98%); mp 95 oC; 1H NMR (CDCl3) 1.77 (d, J = 6.9 Hz, 1H), 5.79 (quintet, J = 7.1 Hz, 1H), 7.287.52 (m, 6H), 7.64 (t, J = 7.8 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 8.91 (d, J = 7.5 Hz, 1H), 9.32 (d, J = 6.6 Hz, 1H).13C NMR (CDCl3) 21.0, 54.0, 116. 0, 120.2, 125.6, 126.4, 127.9, 128.8, 130.2, 132.3, 141.0, 147.0, 173.3. Anal. Calcd for C15H14N4S: C, 63.80; H, 5.00; N, 19.84. Found: C, 63.45; H, 4.59; N, 19.63. N -(4 -Methoxyphenyl) -1 H benzotriazole -1 -carbothioamide 4.20g: White Microcrystals (80%); mp 101 oC;1H NMR (CDCl3) 3.87 (s, 3H), 7.00 (d, J = 8.8 Hz, 2H), 7.52 ( t J = 7.7 Hz, 1H), 7.63 (d, J = 9.1 Hz, 2H), 7.68 (t, J = 7.4 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 8.96 (d, J = 8.4 Hz, 1H), 10.60 (br s, 1H). 13C NMR (CDCl3) 112.5, 114.6, 119.6, 126.2, 127.5, 128.8, 129.4,

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102 131.7, 145.2,159.3, 170.2. Anal. Calcd for C14H12N4O S : C, 59.14; H, 4.25; N, 19.70. Found: C, 59.02; H, 4.00; N, 19.45. N N -D imethyl -1 H -benzotriazole -1 -carbothioamide 4.20k: Yellow Microcrystals (68%); mp 4143 oC ; 1H NMR (CDCl3) 1 .32 (t, J = 6.7 Hz, 3H), 1.48 (t, J = 6.7 Hz, 3H), 3.51 (q, J = 6.9 Hz, 2H), 4.12 (q, J = 6.7 Hz, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1H), 7.948.12 (m, 2H). 13C NMR (CDCl3) 10.6, 13.7, 47.9, 48.0, 113.1, 119.4, 124.7, 128.5, 132.9, 174.4. Anal. C alcd for C9H10N4S: C, 52.41; H, 4.89; N, 27.16. Found: C, 52.11; H, 4.80, N, 27.30. 4.4.4 General Procedure for the Preparation o f Thiocarbamoylbenzotriazole s 4.20h, i : Thiocarbamylbenzotriazole s 4.20h,i were synthesized by adding bis(1 H -benzotria zol 1 yl)methanethione 4.15 (1 mmol) to appropriate amino acid ester hydrocholride (1 mmol) 4.19h,i dissolved in CH3CN/H2O (4 mL/1 mL) in the presence of Et3N (1.1 mmol) at room temperature for 1 5 h. After completion of reaction, CH3CN was evaporated from the reaction mixture, EtOAc (75 mL) was added and the resulting solution was washed with saturated Na2CO3 (3 x 25 mL) followed by brine (25 mL) and dried over anhydrous Na2SO4 and evaporated under reduced pressure to give desired thiocarbamoylbenzotriazol es 4.20h,i in pure form. Compounds were further recrystallized from CHCl3/hexane s for elemental analysis. Methyl 2 -[(1 H -1,2,3 -benzotriazol -1 ylcarbothioyl)amino]acetate 4.20h : White microcrystals (70%); m p 120 oC; 1H NMR (CDCl3) 3.87 (s, 3H), 4.62 (d, J = 4.8 Hz, 2H), 7.67 (t, J = 7.8 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 8.12 (d, J = 8.3 Hz, 1H), 8.87 (d, J = 8.5 Hz, 1H), 9.50 (br s, 1H). 13C NMR (CDCl3) 46.1, 52.8, 115.7, 120.3, 125.8, 132.3, 146.9, 168.5, 174.7. Anal. C alcd for C10H10N4O2S: C, 47.99; H, 4.03; N, 22.39. Found: C, 47.63; H, 4.01; N, 22.25. Methyl (2 R)-2 -[(1 H -1,2,3 -benzotriazol -1 -ylcarbothioyl)amino]propanoate. 4.20i: White microcrystals (68%); mp 103 105 oC; 1H NMR (CDCl3) 1.71 (d, J = 7.1 Hz, 3H), 3.85

PAGE 103

103 (s, 3H), 5.12 5.30 (m, 1H), 7.50 ( t, J = 7.2 Hz, 1H), 7.66 (dt, J = 8.2, 0.8 Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 8.88 (d, J = 8.5 Hz, 1H), 9.45 (br s, 1H). 13C NMR (CDCl3) 17.3, 52.9, 53.0, 115.9, 120.3, 125.8, 130.4, 132.4.147.1, 171.9, 174.0. Anal. C alcd for C11H12N4O2S: C, 49.99; H, 4.58; N, 21.20. Found: C, 49.76; H, 4.49; N, 21.05. 4.4.5 General Procedure for the Preparation o f 5 -(S ubstituted a mino )-1,2,3,4-thiatriazoles 4.1a -i f rom Thi o carbamoylbenzotriazloe s 4.20a -i Thiocarbamoylbenzotriazoles (1 mmol) 4.20a -i w ere added to a solution of NaN3 (2.5 mmol) dissolved in CH3CN/H2O (12 mL/2 mL) at room temperature. Reaction mixture was stirred at room temperature. When TLC (15% EtOAc/hexane s ) indicated completion of reaction, CH3CN was evaporated under vacuum and EtOAc (100 mL) was added. The solution was washed with saturated Na2CO3 (3 x 50 mL) followed by brine (50 mL) and dried over anhydrous Na2SO4. Evaporation under vacuum yielded the corresponding monosubstituted aminothiatriazole s 4.1a -i which were recrystallized from CH2Cl2/hexane s or CHCl3/hexane s N -Prop yl -1,2,3,4 -thiatriazol -5 amine 4.1a : Colorless microcrystals (84%); m p 59 60 oC, (Lit.157 m p 58 59 oC); 1H NMR (CDCl3), 1.05 (t, J = 7.4 Hz, 3 H ); 1.80 (sextet, J = 7.3 Hz, 2H); 3.27 3.42 (m, 2 H); 7.18 (brs, 1 H). 13C NMR (CDCl3) 11.3, 22.0, 52.0, 179.6. N -Benz yl -1,2,3,4 -thiatriazol -5 amine 4. 1b : Colorless microcrystals (96%); m p 78 80 oC, (Lit.166 m p 78 80 oC); 1H NMR (CDCl3) 4.60 (d, J = 5.4 Hz, 2 H), 7.30 7.45 (m, 6 H). 13C NMR (CDCl3) 52.5, 127.9, 128.5, 129.0, 134.7, 179.1. Anal. C alcd for C8H8N4S: C, 49.98; H, 4.19; N, 29.14. Found: C, 50.18; H, 4.13; N, 29.00. N -(1 -Phenylethy l) -1,2,3,4 -thiatriazol -5 -amine 4.1c : Colorless m icrocrystals (94%); mp 120 121 oC; 1H NMR (CDCl3) 1.79 (d, J = 6.9 Hz, 3H); 4.45 (quintet, J = 6. 5 Hz, 1 H), 7.28 7.46 (m, 5 H), 8.32 8.74 (m, 1 H). 13C NMR (CDCl3) : 23.8, 59.7, 126.7, 128.5, 129.1, 140.0,

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104 178.5. Anal. C alcd for C9H10N4S : C, 52.41; H, 4.89; N, 27.16. Found : C, 52.35; H, 4.86 N, 26.91. N -(Furan -2 ylmethyl) -1,2,3,4 -thiatriazol -5 amine 4.1d : Colorless microc rystals (91%); mp 68 69 oC; 1H NMR (CDCl3) 4.59 (d, J = 4.4 Hz, 2H), 6.25 6.40 (m, 1 H), 6.42 (d, J = 2.6 Hz, 1 H), 7.25 7.45 (m, 1 H), 7.80 8.00 (m, 1 H). 13C NMR (CDCl3) : 44.8, 109.9, 110.5, 143.3, 148.0, 178.5. Anal. C alcd for C6H6N4OS: C, 39.55; H, 3.32; N, 30.75. Found: C, 39.69; H, 3.12; N, 30.48. N -Cyclohexy l -1,2,3,4 -thiatriazol -5 -amine 4.1e: Colorless microcrystals (87%); m p 118 oC (Lit.166 mp 113 115 oC); 1H NMR (CDCl3) 1.20 1.72 (m, 6 H); 1.74 1.92 (m, 2 H); 2.04 2.20 (m, 2 H), 3.12 3.30 (m, 1 H), 6.88 7.30 (m, 1 H). 13C NMR (CDCl3) 24.4, 25.1, 31.9, 59.8, 178.9. Anal. C alcd for C7H12N4S: C, 45.63; H, 6.56; N, 30.41. F ound: C, 45.99; H, 6.4; N, 30.54. N -All yl -1,2,3,4 -thiatriazol -5 -amine 4.1f : Colorless microcrystals ( 78%); m p 62 64 oC (Lit.155 mp 53 53.5 oC). 1H NMR (CDCl3) 4.03 (t, J = 5.7 Hz, 2H); 5.31 5.45 (m, 2 H), 5.82 5.98 (m, 1 H), 7.10 7.40 (m, 1H). 13C NMR (CDCl3) 51.3, 119.5, 130.8, 179.2. N -(4 -Methoxyphenyl) -1,2,3,4 -thiatriazol -5 amine 4.1g: Colorless m icrocrystal s (65%); m p 142 oC (Lit.142 m p 140 oC); 1H NMR (CDCl3) 3.85 (s, 3 H), 7.01 (dd, J = 8.9, 2.1 Hz, 2 H), 7.23 7.40 (m, 2 H), 10.31 (br s, 1 H). 13C NMR (CDCl3) 55.6, 115.3, 120.7, 132.8, 157.5, 176.5. Methyl 2 -(1,2,3,4 -thiatriazol -5 -ylamino)ac etate 4.1h : Colorless microcrystal s (86%), m p 142 oC; 1H NMR (CDCl3) 3.86 (s, 3H), 4.33 (d, J = 4.3 Hz, 2H), 6.90 (br s, 1H). 13C NMR (CDCl3) 47.8, 53.0, 169.3, 177.1. Anal. C alcd for C4H6N4O2S: C, 27.58; H, 3.47 ; N, 32.17. Found: C, 27 45; H, 3.40; N, 32.01.

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105 (R)-Methyl 2 -(1,2,3,4 -th iatriazol -5 ylamino)propanoate 4.1i : Colorless microcrystals (74%); m p 88 91 oC; [ ]23 D = 3.31 (c 1.0, CHCl3);1H NMR (CDCl3) 1.65 (d, J = 7.1 Hz, 3H), 3.83 (s, 3 H), 4.22 4.62 (m, 1 H), 7.32 (s, 1 H). 13C NMR (CDCl3) : 17.8, 53.0, 55.3, 172.6, 176.7. Anal. Calcd for C5H8N4O2S: C, 31.91; H, 4.28; N, 29.77. Found: C, 32.32; H, 4.14; N, 29.39. 4.4.6 General One -Pot Procedure for the Preparation of 5-(Substituted amino) -1,2,3,4 thiatriazoles 4.1a -g To a solution of corresp onding amine 4.19a -g (1 mmol) in CH2Cl2 (10 mL) was added bis(1 H benzotriazol 1 -yl)methanethione 4.15 (1 mmol) and the reaction mixture was stirred at room temperature for 1 3 h. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was evaporated to remove CH2Cl2. NaN3 (2.5 mmol) dissolved in CH3CN/H2O (12 mL/2 mL) was directly added to the residue obtained. The reaction mixture was stirred for 1 4 h at room temperature. When TLC (15% EtOAc/hexane) indicated comp lete reaction, CH3CN was evaporated under vacuum and EtOAc (100 mL) was added. The solution was washed with saturated Na2CO3 (3 x 50 mL) followed by brine (50 mL) and dried over anhydrous Na2SO4. Evaporation under vacuum yielded the corresponding mono subst ituted aminothiatriazole s which were recrystallized from CH2Cl2/hexane s or CHCl3/hexane s Yields and melting point are indicated. Spectral characterization is same as for product obtained from two step synthesis. N -Prop yl -1,2,3,4 -thiatriazol -5 amine 4.1a : Colorless microcrystals (92%); m p 59 60 oC (Lit.157 mp 58 59 oC). N -Benz yl -1,2,3,4 -thiatriazol -5 amine 4.1b : Co lorless microcrystals (96%); m p 78 80 oC (Lit.166 m p 78 80 oC). N -(1 -Phenylethy l) -1,2,3,4 -thiatriazol -5 -amine 4.1c : Colorless microcrystals (97 %); m p 120 121 oC.

PAGE 106

106 N -(Furan -2 ylmethyl) -1,2,3,4 -thiatriazol -5 amine 4.1d : Co lorless microcrystals (93%); m p 68 69 oC. N -Cyclohexyl -1,2,3,4 -thiatriazo l -5 -amine 4.1e : Colorless microcrystals (93%); m p 118 oC (Lit.166 m p 113 115 oC). N -All yl -1,2,3,4 -thiatr iazol -5 -amine 4.1f : Colorless microcrystals (85%); m p 62 64 oC (Lit.155 m p 53 53.5 oC). N -(4 -Methoxyphenyl) -1,2,3,4 -thiatriazol -5 amine 4.1g: Colorless microcrystals (81%); m p 142 oC (Lit.142 m p 140 oC). 4.4.7 One -Pot Procedure for the Preparation of 5 -( Substituted a mino) -1,2,3,4 -thiatriazoles 4.1 i To a solution of D alanine methyl ester hydrochloride 4.19i (1 mmol) in CH3CN/H2O (6 mL/1 mL) and Et3N (1.1 mmol) was added bis(1H -benzotriazol 1 yl)methanethione 4.15 (1 mmol) and the reaction mixture was stir red at room temperature for 3 h. Progress of the reaction was monitored by TLC. After completion of reaction, a solution of NaN3 (2.5 mmol) in H2O (2 mL) was added directly to reaction mixture. The reaction mixture was stirred for 3.5 h at room temperature When TLC (15% EtOAc/hexane s ) indicated complete reaction, CH3CN was evaporated under vacuum and EtOAc (100 mL) was added. The solution was washed with saturated Na2CO3 (3 x 50 mL) followed by brine (50 mL) and dried over anhydrous Na2SO4. Evaporation und er vacuum yielded the corresponding 5 -( monosubstituted amino ) 1,2,3,4 thiatriazole 4.1 i which was recrystallized from CHCl3/hexane s (R)-Methyl 2 -(1,2,3,4 -thia triazol -5 ylamino)propanoate 4.1i Colorless microcryst als (74%); mp 88 91 oC. 4.4.8 One -Pot Pr ocedure for the Preparation of 5 -(Substituted a mino )bis(1,2,3,4 thiatriazole ) 4.1 l To a solution of amine 4.19l (1 mmol) in CH2Cl2 (35 mL) was added bis(1 H -benzotriazol 1 yl)methanethione 4.15 (2 mmol) and the reaction mixture was stirred at room temperatu re for 3 h Progress of the reaction was monitored by TLC (20% EtOAc/hexane s After completion of

PAGE 107

107 reaction, the reaction mixture was evaporated to remove CH2Cl2. and to the residue was added a solution NaN3 (5 mmol) in CH3CN/H2O (60 mL/10 mL). The reaction mixture was stirred for 5 h at room temperature. When TLC (40% EtOAc/hexane s ) indicated complete reaction, CH3CN was evaporated under vacuum and EtOAc (100 mL) was added. The p roduct was insoluble in EtOAc. It was directly filtered off and dried to give 5 ( substituted amino) bis(1,2,3 ,4 aminothiatriazole ) 4.1l The p roduct was insoluble in MeOH, so it was further stirred in MeOH for 1 h and filter e d off to get pure 4.1l without trace of 1 H benzotriazole. N N -(1,4 -Phenylenemethylene)bis(l,2,3,4 -thiatriazol -5 -amine) 4.1 l Colorless microcrystal s (75%); m p 141 oC; 1H NMR (DMSO d6) 4.59 (s, 4H), 7.36 (s, 4H), 9.38 (br s, 2H) 13C NMR (DMSO d6) 49.3, 127.9, 136.6, 177.2. HRMS C a lcd for [C10H10N8S2+Na]+: 329.0362. Found: 329.0355.

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108 CHAPTER 5 SYNTHESIS OF 8 -A ZAQUINAZOLINONE ANALOGUE, NBI 74330 AS CXCR3 RECEPTOR ANTAGONIST AND ITS APPLICATION IN TREATMENT OF MALIGNANT GLIOMAS 5.1 Introduction Chemokines are a family of small proteins secreted by leukocytes or tissue cells that induce chemotaxis of responsive ce lls and are attractive molecules to mediate the migration of immune cells into tumor. In addition to these chemoattractive functions, chemokines also exert direct effects on tumor growth, angiogenesis, and metastasis.174,175 Depending on the number and spa cing of conserved cysteine residues, chemokines are classified into four major groups CC, CXC, CX3C and XC.176,177 Like all the chemokine receptors, CXC chemokine receptor 3 (CXCR3) belongs to super family of G -protein coupled receptor predominantly expres sed on activated inflammatory T lymphocytes that promote Th1 response.178 More recently, the related 3 H pyrido[2,3 d] pyrimidin 4 -one compounds AMG 487, 5.1 and NBI 74330, 5.2 (Figure 5 1 ) have been reported as nanomolar CXCR3 antagonists and these ligands are currently under clinical investigation .179 These ligands are the most potent CXCR3 ligands reported to date, showing affinity values for the human CXCR3 receptor in nanomolar range. Figure 5 1. Structure of chemokines receptors AMG 487 5.1 and N BI 74330 5.2 Malignant gliomas are the most frequent and lethal type of brain c ancer originating in the central nervous system (CNS). Human glioblastoma multiform e (GBM) [World Health

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109 Organization (WHO) g rade IV astrocytoma], the primary brain tumor associ ated with dismal prognosis, is the most biologically aggressive subtype of malignant gliomas .180 The c urrent standard for the treatment of GBM patients is surgical resection of the tumor mass, followed by adjuvant radiation therapy and chemotherapy with or al alkylating agent temozolomide .180 Due to relative ineffectiveness of these traditional treatments, immunotherapy is currently being evaluated as better alternative treatment for GBM.181 Microglia are macrophage like cell s present within the central nerv ous system ( CNS ) .182 Macrophage/microgia l cells play a critical role in mediating potent immune response to infectious challenges in the huma n brain and are the largest immune cell population infiltrating human glioma. The marked presence of glioma infiltr ating microglia and lymphocytes suggests that targeting immune system could be one way to treat GBM.175 A number of recent studies have demonstrated that multiple toll like receptors expressed in murine microglial cells are very crucial in detecting and ge nerating innate immune response in CNS.182 Murine microglia/macrophages are also able to activate CD4+ receptor and helper T cells.183 The mechanism by which the various immune cells grow into t umor is not yet very clear. Very recently, Liu et al studied effect of chemokine receptor CX3CR 1 and its ligands CX 3 CL1 in gliomagenesis, using GL261 murine model of glioma .174 Based on the techniques of in situ hybridization analysis, 184 CX3CR1 and CX3CL1 expression in GL 261 glioma was established in vivo from wi ld type C57BL/6 mice. Tumor growth in CX3CR1 deficient mice indicated larger tumor size in homozygous animal ( / ) as comp ared to heterozygous animal (+/ ) .175 The se results indicated that CX3CR1 has little or no impact on the glioma formation or migratio n of microglia and lymphocytes into gliomas.

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110 The present study was undertaken to evaluate the role of various chemokine systems in gliomagenesis utilizing CXCR3 gene disruption and with CXCR3 antagonism. Herein synthesis of 8 azaquinazolinone analogue NBI 74330 5.2 is describe d by literature method185 with some modification s and i ts biological application as CXCR3 receptor antagonist in treatment of human glioma s is briefly described 5.2 Results and Discussion 5.2.1 Procedure for the Preparation of 8 -Aza quinazolinone A nalogue NBI -74330 5.2 Synthesis of desired compound NBI 74330 5.2 was done following literature method185 (Scheme 5 1). Boc D -Ala 5.3 was activated with iso -butylchloroformate 5.4 under basic conditions in anhydrous CH2Cl2 under nitrogen at mosphere at 20 oC, and then allowed to react with 2 aminonicotinic acid 5.6 Intermediate 5.7 was isolated after acidic work up with saturated citric acid. Crude intermediate was immediately reacted with p phenitidine in anhydrous CH2Cl2 under nitrogen at mosphere at 20 oC for 1.5 h. Intermediate 5.9 was isolated after acidic work with saturated citric acid and used in next step without any further purification. Subsequent ring closure of intermediate 5.9 was achieved in presence of N -methylmorpholine (NMM ) and iso butylchloroformate in anhydrous CH2Cl2 under nitrogen atmosphere, at 20 oC for 4 h. Crude product 5. 10 was purified by reprecipitation in tert-butyl methyl ether/heptane solvent mixture. Compound 5.10 was isolated in overall yield of 22% after t hree steps. Deprotection of Boc group with trifluoroacetic acid (TFA) 5.11 afforded intermediate compound 5.12 in 94 % yields Reductive amination of intermediate 5.12 with pyridine 3 -carboxaldehyde and sodium triacetoxyborohydride at room temperature for 18 h afforded crude product 5.15 in 97% yield which was further used in the final step without any purification.

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111 Scheme 5 1. Synthe sis of CXCR3 receptor NBI 74330 5. 2 In the final step coupling of crude product 5.15 with 4 -fluoro 3 -trifluroacetic acid 5.16 with 1 -ethyl 3 (3 dimethylaminopropyl)carbodiimide hydrochloride (EDC ), hydroxybenzotr i azole (HOBt), and NMM in anhydrous DMF at room temperature for 22 h afforded the final crude product 5.2 as sticky solid. Crude product could not be purified by t riturating with tert -butyl methyl ether as reported.181 So, it was purified by flash colum n chromatography (10% MeOH/EtOAc ) on silica gel. Pure product after column was dissolved in

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112 minimum amount of tert butyl methyl ether, and precipitated by adding hexa ne to the solution White solid precipitated was filtered off from solution to give final pure compound 5.2 in 43% yield. Procedure described in the patent185 did not indicate the yield for final ste p. However, according to literature,186 fin al step was ob tained in only 19% yield. Overall yield for product 5.2 was 8.6 %. Room temperature 1H NMR for final compound was very complicated due to presence of rotamers. Peak splitting due to presence of rotamers were confirmed by high temperature 1H NMR measurement (F igure 5 2) in DMSO d6 at 120 oC. Compound 5.6 under reverse phase HPLC shows 100% purity (Figure 5 3). Figure 5 2. High temperature 1H NMR for the compound 5.2 at increasing temperature up to 120 oC

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113 Figure 5 3. Reverse -p hase HPLC for the compound 5.2 5.2.2 Role of NBI -74330 as CXCR3 Receptor Antagonist for Treatment of Malignant Glioma s This work was done by Harrison et al .1 1 Biological studies were carried out by Dr. Jeffrey K. Harrison, Defang Luo and Che Liu in the Department of Pharmacology and Therapeutics, College of Medicine, University of Florida. The r ole of the CXCR3 chemokine receptor system in gliomagenesis and associated immune response was studied using murine GL 261 model of malignant gliomas. Gliomas were established from GL261 cells grown as adherent (GL261-AD) monolayers A prominent microglia infiltration and various types of tumor infiltrated lymphocytes (CD4+, CD8+, Foxp3+ and Ly49G2+ ) were present in both G L261-AD and GL261 NS derived tumors. In situ hybridization analysis determined that chemokine s, CXCL9/MIG and CXCL10/IP 10 (c hemokines ligands of CXCR3 receptor), are expressed by the tumors in vivo The role of CXCR3 in GL261 gliomagenesis was therefore evaluated in

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114 CXCR3 defecient mice as well as glioma bearing mice treated with CXCR3 receptor antagonist, NBI74330. A b rief summary of results are as follows CXCL9 and CXCL 10 are expressed in murine GL261 gliomas estab lished in C57BL/6 wild type CXCR3 defic ient and NBI 74330 treated mice CXCR3 deficient mice displayed reduced tumor growth and animal survival. Reduced level s of NKT cells were found wit hin GL261 tumors established in CXCR3 deficient mice. CXCR3 antagonist NBI 74330 reduced tumor growth and prolonged survival in tumor bearing mice in a manner independent of host CXCR3. 5.3 Conclusion In conclusion CXCR3 antagonism is a potential therapeutics approach for treatment of malignant gliomas. 5.4 Experimental Section 5.2.1 General M ethods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. NMR spectra were recorded in CDCl3 or DMSO d6 with TMS as an internal standard for 1H (300 MHz) and solvent as an internal standard for 13C (75 MHz) Boc D -Ala was p urchased from Fluka. All other chemicals were purchased from Fluka or Aldrich and were used without further purification. Elemental analyses were performed on a Carlo Erba 1106 instrument. 5.2.2 Procedure for Preparation of C ompound 5. 10 Synthesis of compo und 5.10 was performed in three steps, starting from Boc D -Ala -OH 5.3 Step 1 N -Methylmorpholine (NMM) 5.5 (25 mmol) was added to a solution of Boc D Ala OH 5.3 (10 mmol) in CH2Cl2 (40 mL) under nitrogen atmosphere and the reaction mixture

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115 was stirred for 15 min. The solution was cooled to 20 oC and iso -butylchloroformate 5.4 (22 mmol) dissolved in CH2Cl2 (10 mL) was added dropwise. After stirring the r eaction mixture for 45 min at 20 oC, 2 a minonicotinic acid 5. 6 (10 mmol) was added and the reaction mix ture was allowed to warm to room temperature for 18 h. The r eaction mixture was diluted with CH2Cl2 (100 mL) and washed with saturated citric acid (3 x 50 mL), and brine (75 mL). The o rganic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give 88% of yellow orange oil 5.7 The crude product from step 1 was used in step 2 without any further purification. Step 2 Crude product 5.7 (8.72 mmol) was dissolved in CH2Cl2 (60 mL) and cooled to 20 oC under a nitrogen atmosphere and p phenitidine 5. 8 (8.72 mmol) was added dropwise. The reaction mixture was stirred for 1.5 h at 20 oC to 0 oC. After completion the reaction mixture was diluted with CH2Cl2 (75 mL) and washed with saturated citric acid solution (3 x 75 mL) and saturated so dium bicarbonate solution (3 x 50 mL). The o rganic phase was dried over anhydrous Na2SO4, filtered and co ncentrated in vacuum to give 65% of crude bis amide product 5. 9 The c rude product from step 2 was used in step 3 without any further purification. Ste p 3 N -Methylmorpholine ( NMM ) (0.58 mL) and iso -butylchloroformate w ere added to the crude bis amide product 5. 9 (5.3 mmol) from step 2 dissolved in anhydrous CH2Cl2 (60 mL) under nitrogen atmosphere at 20 oC. The r eaction mixture was stirred at 20 oC to 15 oC for 4 h. The r eaction mixture was diluted with of CH2Cl2 (250 mL) and washed successively with satura ted citric acid (3 x 75 mL) saturated sodium bicarbonate solution (3 x 75 mL) and brine (50 mL). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give the crude product as brown viscous oil. The crude product was dissolved in tert butyl methyl ether (50 mL) and stirred at room temperature. The product started precipitating out

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116 of the solution. Heptane (50 mL) was then added and stirred at 0 oC. The resulting precipitate was collected by vacuum filtration, washed with heptane and dried to afford 5. 1 0 as off white solid in overall yield of 22% after three steps. (R)-tert-B utyl 1 -(3 -(4 -ethoxyphenyl) -4 -oxo -3,4 -di hydropyrido[2,3-d]pyrimidin-2 yl)ethylcarbamate, 5. 10: Colorless microcrystals (22% ); mp 152 oC; [ ]23 D = +3.90 (c 1.0, CHCl3); 1H NMR ( CDCl3) 1.31 (d, J = 6.7 Hz, 3H), 1.40 (s, 9H), 1.46 (t, J = 7.0 Hz, 3H), 4.10 (q, J = 6.9 Hz, 2H), 4.58 4.73 (m, 1H), 5.80 (d, J = 9.2 Hz, 1H), 7.04 (dd, J = 8.7, 2.7 Hz, 1H), 7.10 (dd, J = 8.8, 2.8 Hz, 1H), 7.16 (dd, J = 8.8, 2.7 Hz, 1H), 7.30 7.38 (m, 1H), 7.45 (dd, J = 8.0,4.6 Hz, 1H), 8.61 (dd, J = 7.9, 2.0 Hz, 1H), 8.99 (dd, J = 4.4, 2.0 Hz, 1H). 13C NMR (CDCl3) 1 4.7, 20.7, 28.2, 47.7, 63.7, 79.5, 115.5, 115.8, 116.2, 122.4, 127.4, 129.3, 136.8, 154.9, 156.0, 157.2, 159.7, 162.6, 162.8. Anal C alcd for C22H26N4O4: C, 64.37; H, 6.38; N, 13.65. Found: C, 64.64; H, 6.60; N, 13.58. 5.2.3 Procedure for the Preparation o f Compound 5.12 (R)-2 -(1 -A minoethyl) -3 -(4 -ethoxyphenyl)pyrido[2,3-d]pyrimidin-4(3 H )one, 5.12: Colorless microcrystals (94% ); mp 185190 oC; [ ]23 D = 14.15 (c 1.0, CHCl3); 1H NMR (CDCl3) 1.18 (d, J = 6.5 Hz, 3H), 1.38 (t, J = 6.9 Hz, 3H), 1.94 (br s, 2H), 3.53 (q, J = 6.4 Hz, 1H), 4.11 (q, J = 6.7 Hz, 2H), 6.97 7.15 (m, 2H), 7.30 7.45 (m, 2H), 7.55 (dd, J = 7.8, 4.5 Hz, 1H), 8.49 (dd, J = 8. 0, 2.0 Hz, 1H), 8.99 (dd, J = 4.5, 1.9 Hz, 1H). 13C NMR (CDCl3) 14.6, 23.1, 48.8, 63.8, 115.6, 115.7, 115.9, 122.2, 127.9, 128.9, 129.3, 136.7, 156.1, 157.4, 159.6, 162.8, 165.8. 5.2. 4 Procedure for the Preparation of C ompound 5. 15 To a solution of 5. 12 (5.17 mmol) in dic hl oroetha ne (100 mL), was added pyridine 3 carboxaldehyde 5. 13 (5.43 mmol) followed by sodium triacetoxy borohydride ( 7.24 mmol). The

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117 Reaction mixture was allowed to stir at room temperature for 18 h. The reaction mixture was diluted wi th CH2Cl2 (250 mL) and washed with 1M ammonium hydroxide (2 x 250 mL). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated i n vacuum to afford 97% of (R ) 3 (4 E thoxyphenyl) 2 (1 (pyridin 3 ylmethylamino)ethyl)p yrido[2,3 d]pyrimidin 4(3 H )one, 5. 15 as white microcrystal s (R)-3 -(4 -Ethoxyphenyl) -2 -(1 -(pyridin-3 -ylmethylamino)ethyl)pyrido[2,3-d]pyrimidin 4(3 H )-one 5.15: White microcrystals ( 97% ); mp 61 63 oC; [ ]23 D = +9.96 ( c 1.0, CHCl3); 1H NMR ( DMSO d6) 1.20 (d, J = 6.5 Hz, 3H) 1.35 (t, J = 6.9 Hz, 3H), 3.23 3.40 (m, 1H), 3.54 (d, J = 14.0 Hz, 1H, A part of AB system), 3.75 (d, J = 14.0 Hz, 1H, B part of AB system), 3.974.12 (m, 2H), 6.86 (dd, J = 8.8, 2.7 Hz, 1H), 7.02 (dd, J = 8.7, 2.6 Hz, 1H), 7.17 (dd, J = 8.7, 2.3 Hz, 1H) 7.29 (dd, J = 7.7, 4.8 Hz, 1H), 7.36 (dd, J = 8.8, 2.3 Hz, 1H), 7.57 (dd, J = 7.8, 4.3 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 8.41 (d, J = 3.5 Hz, 1H), 8.46 (s, 1H), 8.51 (dd, J = 7.8, 1.7 Hz, 1H), 9.00 (dd, J = 6.7, 1.7 Hz, 1H). 13C NMR (CDCl3) 14.6, 21.4, 48.8, 54.2, 63.7, 115.5, 115.6, 116.0, 122.3, 123.3, 127.5, 128.7, 128.9, 135.0, 135.8, 136.8, 148.2, 149.3, 156.1, 157.5, 159.6, 162.7, 164.7. HRMS C alcd for [C23H23N5O2+H]+: 402.1925, found : 402.1939. 5.2. 5 Procedure for the Preparation of 8 -Azaquinazolinone Analogue NBI 74330 5. 2 To a solution of 4 -f luoro 3 -trifluoroacetic, 5.16 (16.4 mmol) in anhydrous DMF (45 mL) was added 1 -ethyl 3 (3 -dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (21.3 mmol), hydroxybezotriazole ( HOBt ) (16.4 mm ol), N -methylmorpholine (NMM) (24.6 mmol). Afte r stirring for 30 min, ( 8.2 mmol) of ( R ) 3 (4 ethoxyphenyl) 2 (1 (pyridin 3 ylmethylamino)ethyl)pyrido[2,3 d]pyrimidin 4(3 H ) one 5.15 was added. The reaction mixture was allowed to st ir at room temperature for 22 h The reaction mixture was diluted with CH2Cl2 (500 mL), and washed with water (3 x 500 mL), saturated sodium bicarbonate solution (3 x 300 mL) and brine (300 mL). The organic extract was dried over anhydrous Na2SO4, filtered and

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118 concentrated under va cuum. Crude product obtained was purified by flash colum n chromatography (10% MeOH/EtOAc ) on silica gel. Pure product after column was dissolved in minimum amount of tert butyl m ethyl ether, and precipitated by adding hexane to the solution. White solid pr ecipitated was filtered of f from solution to give 43% of (R ) -N -(1 (3 (4 ethoxyphenyl) 4 -oxo 3,4 -dihydropyrido[2,3 d]pyrimidin 2 -yl)ethyl) 2 -(4 -fluoro 3 (trifluoromethyl)phenyl) N (pyridin 3 -ylmethyl)acetamide 5. 2 The p roduct obtained was further recrysta llized from EtOAc/ hexanes for elemental analysis. (R)-N -(1 -(3 -(4 -Ethoxyphenyl) -4 -oxo -3,4 -dihydropyrido[2,3 -d]pyrimidin -2 -yl)ethyl) -2 (4 -fluoro -3 -(trifluoromethyl)phenyl) -N -(pyridin-3 -ylmethyl)acetamide, 5. 2: White microcrystal ( 43%); mp 186 oC; [ ]23 D = 42.22 (c 1.0, CHCl3); 1H NMR ( DMSO d6) [High temperature NMR at 120 oC, Figure 5 2 1.33 (d, J = 6.9 Hz, 3H), 2.89 (d, J = 6.7 Hz, 3H), 2.85 (s, 1H), 3.60 (d, J = 16.4 Hz, 1H), 4.09 (q, J = 6.9 Hz, 2H), 4.75 (br s, 2H), 5.20 5.35 (m, 1H), 7.00 7.22 (m, 4H), 7.23 7.34 (m, 1H), 7.34 7.48 (m, 3H), 7.49 7.60 (m, 2H), 8.29 8.40 (m, 2H), 8.41 8.40 (m, 1H), 8.96 9.04 (m, 1H) Anal Calcd for C32H27F4N5O3: C, 63.47; H, 4.49; N, 11.41. Found: C, 63.23; H, 4.40; N, 11.41. HRMS C alcd for [C32H27F4N5O3+H]+: 606.21 23, f ound : 606.2129.

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119 LIST OF REFERENCES The references citation system employed throughout this dissertation is from J ournal of Org anic Chem istry from American Chemical Society ( ACS ) publication. Standard reference format is as follows: All the authors name s are given, last name followed by first name and middle name if any Abbreviated journal name is listed in italics. Year of publication is given in bold. Volume or page number is given but not bolded. Volume number is italicized Page number range is at the end. Complete format : Author of Article, A. A.; Author of Article, B B.; Author of Article, C. C. Abbreviated Title of Journal Year Volume page number ( xx -xx ).

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120 LIST OF REFERENCES 1 Katritzk y, A. R.; Lan, X.; Yang, J.; De n i sko, O. V. Chem Rev 1998, 98, 409548. 2 Katritzky, A. R.; Rogovoy, B. Chem Eur J 2003, 9 45864593. 3 Carvalho, L. C. C. R. Synlett 2008, 28952896. 4 Katritzky, A. R.; Suzuki, K.; Wang, Z. Synlett 2005, 16561665. 5 Katritzky, A. R.; Rogovoy, B. V.; Cai, X.; Kirichenk o, N.; Kovalenko, K. V. J Org Chem 2004 6 9 3 0 9 313. 6 Katritzky, A. R.; Witek, R. M.; Rodriguez Garcia, V.; Mohapatra, P. P.; Rogers, J. W.; Cusido, J.; Abdel -Fattah, A. A. A.; Steel, P. J. J Org Chem 2005, 70, 7866 7881. 7 Katritzky, A. R.; Manju, K.; Singh, S. K.; Meher, N. K. Tetrahedron 2005, 61, 25552581. 8 Katritzky, A. R.; Abdel Fattah, A. A. A.; Vakulenko, A. V.; Tao, H. J Org Chem 2005, 70, 91919197. 9 Katritzky, A. R.; Bobrov, S.; Khashab, N.; Kirichenko, K. J Org Chem 2004 6 9 42694271 10. Katritzky, A. R.; Khelashvili, L.; Le, K. N. B.; Mohapatra, P. P.; Steel, P. J. J Org Chem 2007 72, 58055808. 11. Gilley, C. B.; Kobayashi, Y J Org Chem 2008 73, 4 1 984204. 12. Katritzky, A. R.; Narindoshvili, T.; Draghici, B.; Angrish, P. J Org Che m 2008 73, 511516. 13. Zhou, G.; Lim, D.; Coltart, D. M. Org. Lett. 2008, 10, 38093812. 14. Katritzky, A. R.; Abdel Fattah, A. A. A.; Gromova, A. V.; Witek, R.; Steel, P. J. J Org. Chem 2005, 70, 92119214. 15. Katritzky, A. R.; Le, K. N. B.; Khelashvili, L.; Mo hapatra, P. P. J Org Chem 2006 71, 98619864. 16. Katritzky, A. R.; Pastor, A.; Voronkov, M. V. J Heterocycl. Chem 1999, 36, 777781. 17. Katritzky, A. R.; Deni sko, O. V.; Fang, Y.; Zhang, L.; Wang, Z. Arkivoc 2001, xi 41 4 8. 18. Katritzky, A. R.; Cusido, J.; N arindoshvili, T. Bioconjugate Chem 2008, 19 14711475. 19. Katritzky, A. R.; Angrish, P. Steroids 2006, 71, 660669.

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128 BIOGRAPHICAL SKETCH Geeta Meher (Geeta) daughter of Uma Shanker Ram Prajapati and Lalit a Prajapati was born in 1978 in Poonapar, Uttar Pradesh India. She received her Bachelor of Science degree in 1998 from Udai Pratap College, Varanasi and obtained her Master of Science degree in Chemistry from Banaras Hindu University, Varan a si India in 2000. After completing her Master in Technology degree in Modern Methods of Chemical Analysis from Indian Institute of Technology, Delhi, India in 2001, s he joined in a p olymer laboratory at Indian Institute of Technology, Bombay and worked there from 2002 to 2004. Later, s he joined Dr. Alan R. Katritzky Research group in March 2005 and there on worked with him towards he r Doctor of P hilosophy degree in the field of peptides and heterocyclic chemistry.