<%BANNER%>

Benzotriazole-Mediated Syntheses of Peptides, Peptide Conjugates and Peptidomimetics

Permanent Link: http://ufdc.ufl.edu/UFE0043652/00001

Material Information

Title: Benzotriazole-Mediated Syntheses of Peptides, Peptide Conjugates and Peptidomimetics
Physical Description: 1 online resource (121 p.)
Language: english
Creator: El Nachef, Claudia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: 1h-benzotriazole -- acylbenzotriazole -- hydrazinopeptides -- peptides -- thiadiazole -- vitamins
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: 1H-Benzotriazole has widely been studied in the Katritzky group especially as a highly effective synthetic auxiliary. The aim of this study was to expand its methodology, in particular the application of N-acylbenzotriazoles, and develop new practical synthetic routes for the construction of structural motifs that will lead to the syntheses of compounds of biological interest. The study focuses mainly on the good leaving property of 1H-benzotriazole. This thesis is divided into six parts. In Chapter 1, a general introduction of this fascinating molecule was presented. A convenient microwave assisted synthesis of chiral 1,3,4-thiadiazoles as well as their peptidyl derivatives was described in Chapter 2. This preparation was mediated by the use of bis(1H-benzotriazol-1-yl)methanethione as an alternative reagent to the classical thioacylating agent and by the use of N-acylbenzotriazoles generated from amino acids and peptides. In Chapter 3, my research studies have further investigated the application of the recently reported N-tri- and tetrapeptidoyl benzotriazoles in the synthesis of peptide scaffolds with potential biological activities. Their preparation is an extension to the O-, S-, N-, and C-acylation, already applied to the amino acids and dipeptides. A microwave assisted reaction for the preparation of peptide - vitamin conjugates was illustrated in Chapter 4. In this chapter a new stable benzotriazole derivative of biotin (vitamin H) was provided. This reagent can be further applied in the diverse biotinylation studies. Furthermore, this benzotriazole mediated synthesis under microwave irradiation showed in many cases its advantages to the general literature methods. Chapter 5 describes the use of the same methodology to form hybrid dipeptides with a a-hydrazino acid unit as well as the synthesis of a benzotriazole activated form of Nß-Cbz-protected a-hydrazino acid. This intermediate is further used in diverse chiral acylation reactions to generate esters and thioesters of a-hydrazino acids. Chapters 2 to 5 were developed in collaboration with my group leader Dr. Kiran Bajaj, and chapters 3 and 5 were also a collaborative work with Dr. Siva Panda. This joint work is clearly indicated throughout this dissertation. Finally, Chapter 6 presents a summary of achievements together with conclusions for the above discussed topics.
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 Claudia El Nachef.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Katritzky, Alan R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043652:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043652/00001

Material Information

Title: Benzotriazole-Mediated Syntheses of Peptides, Peptide Conjugates and Peptidomimetics
Physical Description: 1 online resource (121 p.)
Language: english
Creator: El Nachef, Claudia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: 1h-benzotriazole -- acylbenzotriazole -- hydrazinopeptides -- peptides -- thiadiazole -- vitamins
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: 1H-Benzotriazole has widely been studied in the Katritzky group especially as a highly effective synthetic auxiliary. The aim of this study was to expand its methodology, in particular the application of N-acylbenzotriazoles, and develop new practical synthetic routes for the construction of structural motifs that will lead to the syntheses of compounds of biological interest. The study focuses mainly on the good leaving property of 1H-benzotriazole. This thesis is divided into six parts. In Chapter 1, a general introduction of this fascinating molecule was presented. A convenient microwave assisted synthesis of chiral 1,3,4-thiadiazoles as well as their peptidyl derivatives was described in Chapter 2. This preparation was mediated by the use of bis(1H-benzotriazol-1-yl)methanethione as an alternative reagent to the classical thioacylating agent and by the use of N-acylbenzotriazoles generated from amino acids and peptides. In Chapter 3, my research studies have further investigated the application of the recently reported N-tri- and tetrapeptidoyl benzotriazoles in the synthesis of peptide scaffolds with potential biological activities. Their preparation is an extension to the O-, S-, N-, and C-acylation, already applied to the amino acids and dipeptides. A microwave assisted reaction for the preparation of peptide - vitamin conjugates was illustrated in Chapter 4. In this chapter a new stable benzotriazole derivative of biotin (vitamin H) was provided. This reagent can be further applied in the diverse biotinylation studies. Furthermore, this benzotriazole mediated synthesis under microwave irradiation showed in many cases its advantages to the general literature methods. Chapter 5 describes the use of the same methodology to form hybrid dipeptides with a a-hydrazino acid unit as well as the synthesis of a benzotriazole activated form of Nß-Cbz-protected a-hydrazino acid. This intermediate is further used in diverse chiral acylation reactions to generate esters and thioesters of a-hydrazino acids. Chapters 2 to 5 were developed in collaboration with my group leader Dr. Kiran Bajaj, and chapters 3 and 5 were also a collaborative work with Dr. Siva Panda. This joint work is clearly indicated throughout this dissertation. Finally, Chapter 6 presents a summary of achievements together with conclusions for the above discussed topics.
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 Claudia El Nachef.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Katritzky, Alan R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043652:00001


This item has the following downloads:


Full Text

PAGE 1

1 BENZOTRIAZOLE MEDIATED SYNTHESES OF PEPTIDES, PEPTIDE CONJUGATES AND PEPTIDOMIMETICS By CLAUDIA EL NACHEF A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

PAGE 2

2 2011 Claudia El Nachef

PAGE 3

3 To my beloved husband Wissam Nachef And my wonderful parents and sisters

PAGE 4

4 ACKNOWLEDGMENTS I would like to start by thanking my supervisor Professor Alan R. Katritzky for his c onsistent support, guidance and mainly for the opportunity to join his research group and pursue my doctoral studies at the University of Florida I also would like to ack nowledge my previous boss and mentor Professor Makhluf J. Haddadin of the American University of Beirut for his belief in me, his encouragement, and mainly for sharing hi s knowledge and experiences in c hemistry and life. I thank my committee members, Profe ssors Lisa McElwee White, Margaret O. James, Ron ald Castellano and Ion Ghiviriga for their valuable assistance and advice. I also would like to acknowledge Dr. Dennis Hall for hi s constan t help especially with the english correction. Very special thanks go to Dr. Kiran Bajaj and Dr. Siva Panda with whom I worked on most of the projects and to my friends at the University of Florida for their kindness, help, and love and for making my experience here in Florida easier than I could have ever imagined it would be. You became my other family and I hope to be there for all of you. Last but not least, I am so grateful and blessed to have such a loving husband and a wonderful family. Their unconditional love, support and prayers helped me to overcome my fears, purs ue my dreams and achieve my goals. You mean the world to me and I thank you for making me the woman I am and the future mother to be.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURE S ................................ ................................ ................................ .......... 9 LIST OF SCHEMES ................................ ................................ ................................ ...... 10 LIST OF ABBREVIATION S ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 GENERAL INTRODUCTION ................................ ................................ .................. 17 1.1 Benzotriazole Chemistry ................................ ................................ ................ 17 1.1.1 Introductory Remarks about Benzotriazole ................................ .......... 17 1.1.2 Synthetic Utility of 1 H Benzotriazole ................................ .................... 18 1.1.3 Some Applications of 1 H Benzotriazole ................................ .............. 21 1.1.3.1 N Acylbenzotriazoles ................................ .............................. 21 1.1.3.2 Bis(1 H benzotriazol 1 yl)methanethione ................................ 22 1.1.3.3 Benzotriazole Assisted Synthesis of Hete rocycles ................. 23 2 EFFICIENT SYNTHESES OF THIADIAZOLE PEPTI DES ................................ ...... 25 2.1 Introductory Remarks ................................ ................................ ..................... 25 2.1.1 Background ................................ ................................ ......................... 25 2.1.2 Literature Preparative Methods for 1,3,4 Thiadiazoles ........................ 27 2.1.3 Synthetic Strategy for the Preparation of Thiadiazole Peptides ........... 28 2.2 Results and Discussion s ................................ ................................ ................ 29 2.2.1 Preparation of N (Cbz aminoacyl)benzotriazoles (2.15a d, 2.15(c+c')) and N (Cbz dipeptidoyl)benzotriazoles (2.17a b, 2.17(b+b')) ................................ ................................ ........................... 29 2.2.2 Synthesis of Thiosemicarbazides 2.20a b ................................ ........... 30 2.2.3 Syntheses of Thiadiazoles (2.22a c, 2.22(c+c')) and Their Thiosemicarbazide Precursors (2.21a c, 2.21(c+c')) ........................... 31 2.3 Summary ................................ ................................ ................................ ........ 34 2.4 Experimental Section ................................ ................................ ..................... 35 2.4. 1 General Methods ................................ ................................ ................. 35 2.4.2 General Procedure for the formation of N Cbz(dipeptidoylbenzotrizole) 2.17 ................................ ....................... 35 2.4.3 General Procedure for the Preparation of 1,3,4 Thiadiazole Precursors 2.21a c and 2.21(c+c') ................................ ....................... 36

PAGE 6

6 2.4.4 General Procedure for the Preparation of 1,3,4 Thiadiazoles (2.22a c, 2.22(c+c')) ................................ ................................ ............ 38 2.4.5 General Procedure for the Preparation of 1,3,4 Thiadiazole Derivatives (2.23a c, 2.24a c, 2.24(c+c')) ................................ ............ 40 3 SYNTHESES OF CHIRAL N (PROTECTED)TRI AND TETRAPEPTIDE SCAFFOLDS ................................ ................................ ................................ .......... 44 3.1 Introductory Remarks ................................ ................................ ..................... 44 3.1.1 Background ................................ ................................ ......................... 44 3.1.2 Literature Preparative Methods ................................ ........................... 45 3.2 Results and Discussion ................................ ................................ .................. 46 3.2.1 Syntheses of Cbz Protected Tripeptidoylbenzotriazole and Tetrapeptidoylbenzotriazole ................................ ................................ 46 3.2.2 O Acylations with Tri and Tet rapeptidoylbenzotriazoles ..................... 48 3.2.3 S Acylation with Tri and Tetrapeptidoylbenzotriazoles ....................... 49 3.2.4 N Acylation with Tri and Tetrapeptidoylbenzotriazoles ....................... 51 3.2.5 C Acylation with Tri and Tetrapeptidoylbenzotriazoles ....................... 52 3.3 Summary ................................ ................................ ................................ ........ 53 3.4 Experimental Section ................................ ................................ ..................... 53 3.4.1 General Methods ................................ ................................ ................. 53 3.4.2 General Procedure for the Synthesis of N (Cbz dipeptidoyl)benzotriazoles 1a c ................................ ........................... 54 3.4.3 General Procedure for the Synthesis of N C bz tripeptides 3.2a c ....... 55 3.4.4 General Procedure for the Synthesis of N (Cbz tripeptidoyl)benzotriazoles 3.3a c ................................ ........................ 56 3.4.5 General Procedure for the Synthesis of N Cbz tetrapeptides 3.4a c ... 57 3.4.6 General Procedure for the Synthesis of N (Cbz tetrapeptidoyl)benzotriazole 3.5a c ................................ ...................... 58 3.4.7 General Procedure for O Acylation: Synthesis of Compounds 3.6a f .. 59 3.4.8 General Procedure for S Acylation: Synthesis of Compounds 3.7a d ................................ ................................ ................................ 62 3.4.9 General Procedure for N Acylation: Synthesis of Compounds 3.8a, 3.8d ................................ ................................ ................................ 63 3.4.10 General Procedure for N Acylation: Synthesis of C ompounds 3.8b c ................................ ................................ ................................ 64 3.4.11 General Procedure for C acylation: Synthesis of Compounds 3.9a b 66 4 MICROWAVE ASSISTED FORMATION O F PEPTIDE VITAMIN CONJUGATES ................................ ................................ ................................ ....... 68 4.1 Introductory Remarks ................................ ................................ ..................... 68 4.2 Results and Discussions ................................ ................................ ................ 70 4.2.1 Preparation of Benzotriazole Derivatives of Niacin and Biotin 4.3a b .. 70 4.2.2 Preparation of Peptide Niacin Bioconjugates 4.5a e ........................... 71 4.2.3 Preparation of Peptide Biotin Bioconjugates 4.6a g ............................ 72 4.2.4 Tocopherol) Peptide Conjugates 4.9a e ................ 75

PAGE 7

7 4.2.5 Preparation of Cholecalciferol Peptide Conjugates 4.11a d .............. 76 4.3 Summary ................................ ................................ ................................ ........ 77 4.4 Experimental Section ................................ ................................ ..................... 77 4.4.1 General Methods ................................ ................................ ................. 77 4.4.2 General Procedure for the Synthesis of Benzotriazole Derivatized Niacin 4.3a ................................ ................................ .......................... 78 4.4.3 General Procedure for the Synthesis of Benzo triazole Derivatized Biotin 4.3b ................................ ................................ ........................... 78 4.4.4 General Procedure for the Preparation of Peptide Niacin Bioconjugates 4.5a d ................................ ................................ ........... 79 4.4.5 General Procedure for the Preparation of Peptide Biotin Conjugates 4.6a g ................................ ................................ ............... 81 4.4.6 Tocopherol) Peptide Conjugates 4.9a f ................................ ................................ ................ 84 4.4.7 General Procedure for the Preparation of Cholecalciferol Peptide Conjugates 4.11a d ................................ ................................ ............. 87 5 BENZOTRIAZOLE ASSISTED SYNTHESES O F HYDRAZINO PEPTIDES .......... 90 5.1 Introductory Remarks ................................ ................................ ..................... 90 5.1.1 Background ................................ ................................ ......................... 90 5.1.2 Literature Preparative Methods ................................ ........................... 92 5.2 Results and Discussions ................................ ................................ ................ 94 5.2.1 Hydrazino Acids 5.3a d ................................ ............. 94 5.2.2 Hydrazino Dipeptides 5.11a f ........................ 94 5.2.3 N Cbz Hydrazino Acids and Their Benzotriazolides 95 5.2.4 Chiral Acylation of Compounds 5.13b c ................................ .............. 96 5.3 Summary and Future Prospect ................................ ................................ ...... 97 5.4 Experimental Section ................................ ................................ ..................... 97 5.4.1 General Methods ................................ ................................ ................. 97 5.4.2 Bromo Acids 5.9a d ........... 98 5.4.3 Hydrazino Acids 5.3a d ..... 98 5.4.4 General Procedure for the Synthesis of Cbz Aminoacyl benzotriazoles 5.10a e ................................ ................................ ........ 99 5.4.5 General Procedure for the Synthesis of hybrid dipeptides 5.11a f ....... 99 5.4.6 General Procedure for the Synthesis of N Cbz Hydrazino Acids 5.12b c 102 5.4.7 General Procedure fot the Chiral O Acylation of Compound 5.13c ... 103 5.4.8 General Procedure fot the Chiral S Acylation of Compounds 5.13b c ................................ ................................ .............................. 103 6 CONCLUSIONS AND SUMM ARY OF ACHIEVEMENTS ................................ ..... 105 LIST OF REFERENCES ................................ ................................ ............................. 108 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 121

PAGE 8

8 LIST OF TABLES Table page 2 1 Conversion of N Cbz amino acids to N ( Cbz dipeptidoyl)benzotriazoles ........ 30 2 2 Preparation of thiosemicarbazides 2.20a b ................................ ........................ 31 2 3 Preparation of substituted amino acids thiosemicarbazides 2.21 and 1,3,4 thiadiazoles 2.22 ................................ ................................ ................................ 32 2 4 Preparation of 1,3,4 thiad iazolo substituted amino acids and dipeptides ........... 34 3 1 Preparation of N Cbz tri an d tetrapeptidoyl benzotriazoles ............................... 48 3 2 O Acylation products using N Cbz tri and tetra peptidoylbenzotriazoles ........... 49 3 3 S Acylation by N (Cbz protected) tri and tetrapepti doylbenzotriazoles ............. 50 3 4 N Acylation by N (Cbz protected) tri and tetrapeptidoylbenzotriazoles ............. 51 3 5 C Acylated N (Cbz protected) tri and tetrapeptides. ................................ .......... 53 4 1 Preparation of benzotriazole derivatives of niacin 4.3a and biotin 4.3b ............. 71 4 2 Preparation of peptide niacin bioconjugates 4.5a d ................................ ........... 72 4 3 Preparation of biotin peptide conjugates 4.6a g ................................ ................. 73 4 4 tocopherol) peptide conjugates 4.9a f ................................ .. 75 4 5 Preparation of cholecalciferol pepti de conjugates 4.11a d ................................ 76 5 1 hydrazino acids ................................ ......... 94 5 2 Preparation of dipeptides 5.11a f containing one hydrazino acid unit ................ 95 5 3 Preparation of N Pg Hydrazino acids and their benzotriazole derivatives .......... 96 5 4 O and S acylations utilizing nucleophiles 5.15 ................................ ................. 97 5 5 Preparation of N Cbz aminoacyl)benzotriazoles 5.10a f ................................ 99

PAGE 9

9 LIST OF FIGURES Figure page 1 1 Tautomeric forms of benzotriazole ................................ ................................ ..... 17 1 2 Examples of some active 1 H benzotriazole derivatives ................................ ...... 18 1 3 Benzotriazole activation ability ................................ ................................ ............ 20 2 1 Drugs and biologically active molecules containing the 1,3,4 thiadiazole unit .... 26 2 2 Different strategies for the preparation of 1,3,4 thiadiazole ( 2.7 ) ........................ 27 4 1 Structures of the four vitamins used ................................ ................................ ... 69 5 1 Examples of biologically active hybrid peptides with a hydrazino unit ................ 91 5 2 Hydrazino and tur n conformations ................................ ................................ ... 92 5 3 hydrazino amino acids .................... 93

PAGE 10

10 LIST OF SCHEMES Scheme page 1 1 Methods for the incorporation of benzotriazole moiety ................................ ....... 19 1 2 Methods for the preparation of N a cylbenzotriazoles ................................ ......... 21 1 3 Preparation of bis(1 H benzotriazol 1 yl)methanethione ................................ ..... 23 1 4 Formation of 1,2,4 oxadiazoles from N protected aminoacylbenzotriazoles ...... 23 2 1 Synthesis of N protected(acylbenzotriazoles) 2.15 and 2.17 .............................. 29 2 2 Preparation of N substituted thiosemicarbazides 2.20 ................................ ....... 30 2 3 Synthesis of 1,3,4 thiadiazoles 2.22 and their precursors 2.21 .......................... 31 2 4 Syntheses of 1,3,4 thiadiazolo substituted amino acids and dipeptides ............. 33 3 1 Synthesis of N Pg tripeptidoyl benzotriazoles an d tetrapeptidoyl benzotriazoles ................................ ................................ ................................ .... 47 3 2 Preparation of tri and tetrapeptidoyl ester ................................ ......................... 48 3 3 Preparation of tri and tetrapeptidoyl thioester ................................ .................... 50 3 4 N Acylation with N (Cbz protected)tri and tetrapeptidoylbenzotriazoles ............ 51 3 5 C Acylation with N (Cbz protected)tri and tetrapeptidoylbenzotriazoles ............ 52 4 1 Synthesis of benzotriazole derivatized niacin 4.3a and biotin 4.3b .................... 71 4 2 Synthesis of peptide niacin conjugates 4.5a d ................................ ................... 71 4 3 Synthesis of peptide biotin bioconjugates 4.6a g ................................ ............... 73 4 4 Synthesis of peptide tocopherol conjugates 4.9a e ................................ ............ 75 4 5 Synthesis of peptide cholecalciferol conjugates 4.11a d ................................ .... 76 5 1 hydrazino acids from bromo acids ................................ .......... 94 5 2 Syntheses of hybrid dipeptides 5.11a e ................................ .............................. 95 5 3 Syntheses of N (Cbz protected)hydrazino acids 5.13b c ................................ ... 96 5 4 Displacement of benzotriazole with nucleophiles 5.15 ................................ ....... 96

PAGE 11

11 LIST OF ABBREVIATION S Alpha locant D Specific rotation Ala Alanine Beta locant Bn Benzyl Boc t Butoxycarbonyl BOP Benzotriazole 1 yl oxy tris (dimethylamino) phosphonium hexafluorophosphate b r Broad (spectral) Bt Benzotriazol 1 yl C Carbon Degree Celcius Calcd Calculated Cbz Carbo xybenzyl CDCl 3 Deuterated chloroform Cys Cysteine Chemical shift in parts per million d ownfield from tetramethylsilane d Doublet D (11 point) Dextrorotatory (right) DCC N N' Dicyclohexylcarbodiimide DCM Dichloromethane DMF Dimethylformamide DMSO Dimet hylsulfoxide D 2 O Deuterium oxide EDC 1 Ethyl 3 (3 dimethylaminopropyl ) carbodiimide equiv equivalent(s)

PAGE 12

12 Et Ethyl et al. And others Et 3 N Triethylamine EtOAc Ethyl acetate Fmoc 9 Fluorenylmethoxycarbonyl g Gram(s) Gly Glyc ine h Hour H Hydrogen HBTU O Benzotriazole N N N' N' tetrameth yl uronium hexafluoro phosphate HOBt 1 Hydroxybenzotriazole HPLC High performance liquid chromatography HRMS Hi gh resolution mass spectrometry Hz Hertz i iso (as in i Pr ) Ile I soleucine i Pr I sopropyl J Coupling constant (in NMR spectroscopy) L ( 11 point ) Levorotatory (left) Leu Leucine Lit Literature Lys lysine m Multiplet (spectral) M Molar Me Methyl

PAGE 13

13 MeCN Acetonitrile MeOH Methanol Met Methionine min Minute(s) MgSO 4 Magnesium sulfate mmol Millimole(s) mol Mole(s) mp Melting point MW Microwave m/z Mass to charge ratio N Nitrogen Na 2 CO 3 Sodiu m carbonate NMR Nuclear magnetic resonance o Ortho locant O Oxygen OH Hydroxyl group OMe M ethoxy p Para locant Pg P rotecting group Ph Phenyl Phe Phenylalanine p pm Part s per million q Quartet R Rectus (right) ref. Reference

PAGE 14

14 rt Room temperature s Singlet S Si n ister (left) SOCl 2 Thionyl chloride t Triplet t Tertiary TFA Trifluoroacetic acid TLC Thin layer chromatography TMS Trimethylsilane Trp Tryptophan Tyr T yrosine Val Valine W Watt(s) Z Carboxybenzyl same as Cbz

PAGE 15

15 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 BENZOTRIAZOLE MEDIATED SYNTHESES OF PEPTIDES, PEPTIDE CONJUGATES AND PEPTIDOMIMETICS By Claudia El Nachef December 2011 Chair: Alan R. Katritzky Major: Chemistry 1 H Benzotriazole has widely been studied in the Katritzky group especially as a highly effective synthetic auxiliary The aim of this study was to expand its methodology, in particular the applicati on of N acylbenzotriazoles and develop new practical synthetic routes for the construction of structural motifs that will lead to the syntheses of co mpounds of biological interest. The study focuses mainly on the good leaving property of 1 H b enzotriazole T his thesis is divided into six parts. In Chapter 1 a general introduction of this fascinating m olecule was presented A convenient microwave assisted synthesis of chiral 1,3,4 thiadiazoles as well as their peptidyl derivatives was described in Chapter 2. This preparation was mediated by the use of bis(1 H benzotriazol 1 yl)methan e thione as an alternative reagent to the classical thioacylating agent and by the use of N acylbenzotriazoles generated from amino acids and peptides. In Chapter 3, m y research s tudies have further investigated the application of the recently reported N tri and tetrapeptidoyl benzotriazoles in the synthesis of peptide scaffolds with potential biological activities Their preparation is an extension to the O S N and C acylat ion, already applied to the amino acids and dipeptides.

PAGE 16

16 A microwave assisted reaction for the preparation of peptide vitamin conjugates was illustrated in Chapter 4 In this chapter a new stable benzotri azole derivative of biotin (vitamin H) was provided This reagent can be further applied in the diverse biotinylation studies. Furthermore, this benzotriazole mediated synthesis under microwave irradiation showed in many cases its advantages to the general literature methods. Chapter 5 describes the use of the same methodology to form hybrid di peptides with a hydrazino acid unit as well as the synthesis of a benzotriazole activated form of N Cbz protected hydrazino acid This intermediate is further used in diverse chiral acylation reactions to generate esters and th i oesters of hydrazino acids. Chapters 2 to 5 were developed in collaboration with my group leader Dr. Kiran Bajaj, and chapters 3 and 5 were also a collaborative wo r k with Dr. Siva Panda. This joint work is clearly indicated thro ughout this dissertation. Finally, C hapter 6 presents a summary of achievements together with conclu sions for the above discussed topics

PAGE 17

17 CHAPTER 1 GENERAL INTRODUCTION 1.1 Benzotriazole Chemistry 1.1.1 Introductory Remarks about Benzotriazole Benzotriazole is a benzofused ring to 1,2,3 triazole possessing a N=N NH or =N NH N= linkage. It exists as an equilibrium mixture of 1 H benzotriazole ( 1.1 ) and 2 H benzotriazole ( 1.2 ) where the 1 H tautomer is predominant in solution and in the solid state (Figure 1 1). [91T2683, 94JOC2799, 09MRC142] This shift of equilibrium is attributed to the higher aromaticity of 1 H benzotriazole ( 1.1 ) compared to its quin oid like 2 H tautomer ( 1.2 ) Moreover, in solution there is a better solvation of the more polar methyl form of 1.1 ( 4.2 D as dip ole moment ) in comparison to the methyl form of 1.2 ( 0.4 D as dipole moment) In other words, this higher dipole moment is favored by interactions with the solvent in solution and itself in solid state. [ 94JOC2799 ] Figure 1 1. Tautomeric forms of benzotriazole 1 H Benzotriazole is an interesting motif with important chemical and biological acti vities. It is used in pharmaceutical science where some of its derivatives exhibit anticancer 1.3 antibacterial 1.4 potassium channel activator 1.5 and antiplasmodial 1 .6 activities (Figure 1 2). [11OL2102, 11JACS6868] In addition, it has also found some applications in materials science where its derivatives have proven to be efficient corrosion inhibitors 1.7 (Figure 1 2). [09JAE269]

PAGE 18

18 Figure 1 2. Examples of some active 1 H benzotriazole derivatives The interesting structure of 1 H benzotriazole ( 1.1 ) and its innate properties from its chemical stability towards heat, harsh acidic or basic conditions, an d oxidation or reduction, to its behavior as a weak acid (p K a = 8.2) and a weak Brnsted base ( p K a < 0) indicating i ts susceptibility to accept and donate electrons, [48JCS2249, 91T2683, 00JACS5849] led to the development of benzotriazole assisted synthetic methodology and to its establishment as a good synthetic auxiliary. 1.1.2 Synthetic Utility of 1 H Benzotriazole Over the last two decades, 1 H benzotriazole has been extensively studied by Katritzky and co workers and the reason is attributed to its useful physical and c hemical properties as previously mentioned. In fact, this compound ( 1.1 ) was shown to be easily incorporat ed into a substrate by addition to ketones, imines or enamines or by substitution where benzotria zole or its conjugated anion can displace a halogen in an acyl or alkyl halide, an alkoxy in a ketal or even a hydr oxyl group in an alcohol (Scheme 1 1 ). [ 98CR409 ]

PAGE 19

19 Scheme 1 1. Methods for the incorporation of benzotriazole moiety D uring various synthetic reactions the stable benzotriazole derivative that was generated will now exert some activation to its neighbor carbon This is conveyed in several ways as shown in Figure 1 3 such as: i) a proton activator or anion stabilizer [ 98CR409, 03CEJ4586 06S3231] where in the case of 1 N alkylbenzotriazole, n b utyllithium ( n BuLi ) or lithium diisopropylamide ( LDA ) can deprotonate the alkyl group and form a carbanion susceptible to electrophilic attack; ii) a cation stabilizer as in the case of N ( hydroxy alkyl) benzotriazole, where in the presence of an acidic medium or Lewis acid catalyst a benzotriazole stabiliz ed cationic intermediate is formed leading to the displacement of the hydroxyl by the nucleophile [ 91CR1809, 95SC539] ; iii) an anion director as in the case of an allylic system where the electrophilic attack is influenced by benzotriazole an d it occurs a t the carbon at the alpha position ; [90HC21 92LAC843] and i v ) an anion and radical precursor where in both cases, benzotriazole is displaced upon a reductive elimination [97JOC4148] and monoele ctronic oxidation [04CC2356 ] respectively

PAGE 20

20 A t t he end of the s ynthesis and as a good s ynthetic auxiliary this benzotri azole moiety should be easily displaced. This property is sa tisfied by the good leaving nature of 1 H benzotriazole that is compar able to phenylsulfonyl and cyano groups. Moreover, after its displacement, a fairly simple procedure for its removal by a simple basic or acidic work up is required and this is mainly accredited to its p K a This property granted this moiety its advantageous use (Figure 1 3 ) [ 91T2683, 98CR409] Figure 1 3 Benzotriazole activation ability This last property is the main focus of my thesis and indeed induced the generation of the thiadiazole precursors as well as the thiadiazole peptides in C hapter 2 the peptide scaffolds in C hapter 3, the peptide vitamin conjugates in C hap ter 4 and finally the hybrid hydrazino peptides in C hapter 5.

PAGE 21

21 1.1.3 Some Applications of 1 H Benzotriazole As illustrated in Figure 1 3 benzotriazole intermediates represent a strategy for t he introdu c tion of different functional groups into molecules. This methodology has been applied since 1980s in organic synthesis [87JCS(P T 1)805, 88JCS(P T 1)2339]. Some of the relevant applications to our present work will be further discussed. 1.1.3.1 N A cylben z otriazole s N Acylbe nzotriazoles are alternative acylating agents to the very reactive acid chlorides They are, in general, fairly stable crystalline compounds and easy to handle in the lab. Recently, these reagents were reported in a review as one of the acylazole peptide coupling reagents wit h significant advantages to other coupling reagents from the relatively moisture insensitivity to the unnecessary protection of functionalized N protected amino acids. [11CRxxx] Scheme 1 2 Me thods for the preparation of N a cylbenzotriazoles They are prepared through three different routes: (i) reacting the corresponding acid chlorides with 1 H benzotriazole (BtH) in the presence of a base or in a neat type of reaction by just heating to 80C or even reacting acid chlorides with 1 (trimethylsilyl) 1 H benzotriazole (BtSiMe 3 ) [80JOM141]; (ii) directly reacting carboxylic acids with 1 (methylsulfonyl)benzotriazole (BtSO 2 Me) as a counter attack in the presence of

PAGE 22

22 triethylamine (Et 3 N) while heating. A mixed carboxylic sulfonic anhydride and benzotriazole anion is suggested to be formed as intermediate [00JOC8210]; and finally (iii) directly reacting carboxylic acid s as well, but now with an excess of benzotriazole in the presence of thi onyl chloride (SOCl 2 ) [03S2795] (Scheme 1 2 ) All three methods provided the desired acylbenzotriazoles in good yields; however, this work focuses on the use of the last two routes for the preparation of the diverse N a cylbenzotriazoles of amino ac ids, pep tides and even vitamins and obviating the isolation of the acid chlorides. 1.1.3.2 Bis(1 H b enzotriazol 1 yl)m ethan e thione Bis(1 H b enzotriazol 1 yl)methanethione (Bt 2 CS) has been prepared as an alternative stable to storage and easy to prepare thioacylating reagent to the classical ones such as thiophosgene [78S803] and carbon disulfide [03ARK155]. It can be used for the preparation of thioamides and thioureas instead of u sing the unstable, highly toxic and air sensitive N N dimethylcarbamothioic chloride (Me 2 NCSCl) [94SL719] or thiocarbonyldiimidazole (Im 2 CS) which is also unstable and due to its high reactivity, leads to a mixture of the desired thiocarbamoyl derivat ive with isocyanate and thioureas [86JOC2613]. Bis(1 H b enzotriazol 1 yl)methanethione is prepare d in high yield through the reaction of thiophosgene with an excess of 1 H benzotriazole [06ARK226] or 1 (trimethylsilyl) 1 H benzotriazole at room temperature (S cheme 1 3). This newly developed thioacylating reagent can upon reacting with primary and secondary amines, form the corresponding crystalline, stable and easy to handle thiocarbamoylbenzotriazole which can generally replace the irritant and highly

PAGE 23

23 hazardo us thioisocyanates as it is shown in Chapter 2 during the synthesis of the thiosemicarbazides [06ARK226]. Scheme 1 3. Preparation of bis(1 H benzotriazol 1 yl)methanethione 1.1.3.3 Benzotriazole Assisted Synthesis of Heterocycles Significant amount of work has been done towar ds the development of methods to synthesize different heterocycles due to their biological importance and their vast application in medicinal chemistry. Recently, Katritzky and Rachwal combined in a review whic h is beyond the scope of this dissertation the tremendous work done using 1 H benzotriazole as either a leaving g roup or an activator of the carbon attached to it for the syntheses of heterocycles with different size and shape and sometimes for the synthes es of their precursors. [10CR1564] One particular example in significance to the w ork presented in C hapter 2 involves the expansion of the scope of N acylbenzotriazoles as activating reagents through the syntheses of 1,2,4 oxadiazoles from acylbenzotriazol es with different alkyl or aryl chain or from N protected aminoacylbenzotriazoles [05ARK36]. Both react instantaneously with amidoximes in the presence of triethylamine as a base leading to an ester which upon reflux cyclize to form these 1,2,4 oxadiazoles in 70 94% (Scheme 1 4). Scheme 1 4. Formation of 1,2,4 oxadiazoles from N protected aminoacylbenzotriazoles

PAGE 24

24 The amidoximes are usually generated from nitriles and hydroxylamine hydrochloride. In C hapter 2, I present the syntheses of 1,3,4 thiadiazoles from these N protected aminoacylbenzotriazoles and show once again the applicability of this benzotriazole mediated synthesis to the formation of a new heterocycle.

PAGE 25

25 CHAPTER 2 EFFICIENT SYNTHESES OF THIADIA ZOLE PEPTIDES 1 2.1 Introduct ory Remarks 2.1.1 Background Attempts to discover peptide analogues with increased chemical stability and oral compatibility have included replac ing peptide fragments such as NHCO CHR with a wide variety of alternative structural moieties. In particular, heterocyclic moieties amino acids have attracted considerable interest. [00JCSPT(1)2311, 06S2376, 08TL4746, 08BMC6009] Among important five membered heterocyclic synthetic building bl ocks in medicinal, agricultural and materials chemistry, 1,3,4 thiadiazoles exhibit diverse biological properties, including antibacterial [08CCL1427 11EJC94], antifungal [06EJMC531 ] antimicrobial [08EJC963], anti arrhythmic [09MC43 1], anticancer [08CL928, 09EJMC3340], anti inflammatory [07BMC5738], antidepressant [01JMC931] analgesic [07BMC57 38] and anti HIV [07HC316] activities. Some examples of these biologically active molecules (Figure 2 1) are already therapeutically important drugs such as the orally bioavailable stearoyl CoA desaturase (SCD) inhibitor MF 438 ( 2.1 ) [10BMCL499] the carbonic anhydrase inhibitor Acetazolamid e ( 2. 2 ) [09BMC1214] and the antibiotic Cefazolin ( 2.3 ) for the treatment of bacterial infections [97JOC909 9]. Other molecules ( 2.4 ) which are still under clinical trials possess remarkable analgesic effects with potency higher than both aspirin and morphine [07BMC5738]. In addition, preliminary findings showed that targets with 2 amino 1,3,4 thiadiazoles as co up ling 1 Reproduced in part with permission from The Journal of Organic Chemistry 2010 75 6009 6011 Copyright 2010 American Chemical Society

PAGE 26

26 units to peptides or peptide like molecules possess potent inhibitory activity towards metalloprotease (MMP) ( 2.5 ) [98WO9838179] and aminopeptidase N (APN) ( 2.6 ). [08BMC6663, 08APR1231] Figure 2 1. Drugs and biologically active molecules containing the 1,3,4 thiadiazole unit Moreover, reports of the synthesis of thiadiazoles from ami no acids for biological testing [ 07HC316, 07M103] disclose low toxicity, and in the case of phenylalanine derivative s, anti inflammatory activity [09M2621] In previous efforts to develop new amino acids and peptides that could be incorporated into heterocycles, we synthesized chiral 1,2,4 oxadiazoles derivatives [05ARK36] utilizing N aminoacyl )benzotriazoles [09ARK47] Enco uraged by the various pharmacological properties of compounds containing this heterocyclic unit and in continuation of our work, we were prompted to synthesize hybrid molecules that

PAGE 27

27 incorporate chiral 1,3,4 thiadiazolo substituted amino acids and peptide s caffolds in a single molecule 2.1.2 Literature Preparative Methods for 1,3,4 Thiadiazoles Figure 2 2. Different strategies for the preparation of 1,3,4 thiadiazole ( 2.7 ) Different routes are reported for the synthesis of 1,3,4 thiadiazole derivatives. The common route involves the use of thiosemicarbazides ( 2.8 ) as intermediates followed by cyclization in the presence of a dehydrating reagent such as phosphoric acid ( H 3 PO 4 ) [49JCS1163, 10JCCS1077], sulfuric acid ( H 2 SO 4 ) [02BMC2893, 07HC316, 09M2621] or phosphoryl chloride ( POCl 3 ) [08APR1231]. I ntermediates ( 2.8 ) are often generated from isothiocyanates by reaction with hydrazides. [08CCL1427, 07HC316, 07M103, 09M2621] Another method has been developed by Guha [22JA CS1510 ] by

PAGE 28

28 treatment of 2.8 with carbon disulfide (CS 2 ) and potassium hydroxide (KOH) at 140C. Other routes involve the use of : i) thiocarbazides ( 2.9 ) and formic acid [53US2623877], ii) dit h iocarbazates ( 2.10 ) in basic or acidic media [22JCS2542, 55JACS1148], iii) thioacylhydrazines ( 2.11 ) with formate derivatives [56US2733245], iv) acylhydrazines ( 2.12 ) treated with: a ) phosphorus sulfide ( P 2 S 5 ) at 160 C in xylene [10BMCL499] or b) (LR) at 55 C in THF [06OL1625, 09BMCL6632 10TL6338 ] and finally v) bithioureas ( 2.13 ) treated with hydrogen peroxide [23A1] 2.1.3 Synthetic Strategy for the Preparation of Thiadiazole Peptides To the best of our knowledge, there has been some previous work which described the preparation of chirally substituted 1,3,4 thiadiazoles [ 07HC316 09M2621 ] but none addressed their incorporation into peptides. Here in we report the use of N ( Cbz aminoacyl)benzotriazoles for the conversion of amino acid s into thiosemicarbazides under microwave irradiation using mild cond tions A fterwards t hese intermediates were cyclized to afford enantiomerically pure 1,3,4 thiadiazoles which in turn provide the first synthesis of chirally p ure peptidoylaminothiadiazoles. Chiral N (protected aminoacyl ) benzotriazoles have been studied extensively in our group and have proven to be efficient crystalline intermediates for N acylation leading to the synthesis of peptides and peptide conjugates with full retention of chirality [04S2645, 05S397, 06CBDD326, 06S411 08JOC5442 ] As mentioned earlier, t he classical starting materials for the preparation of thiosemicarbazides are the generally unstable and toxic isothiocyanates. Using the benzotriazole based chemistry earlier developed in our group [04JOC2976 06ARK226] a much milder and stable reagent, bis( 1 benzotriazolyl)methanethione ( 2.18 ) was used to generate the N substituted thiosemicarbazides. Finally, microwave irradiation was

PAGE 29

29 applied to afford the thiadiazole precursors and derivatives. It was used as an alternative to conventional he ating to shorten reaction times [02ACR717] and hence minimize any possible epimerization resulting fr om prolonged exposure to heat 2.2 Results and Discussions 2.2.1 Preparation of N (Cbz aminoacyl)benzotriazoles (2.15a d, 2.15(c+c )) and N (Cbz dipeptidoyl)benzo triazoles (2.17a b, 2.17(b+b )) Compounds ( 2.15a d 2.15 ( c+c' )) and ( 2.17a b 2.17 ( b+b' )) (Table 2 1) were prepared following established procedures. [04S2645, 09ARK47, 09S2392] Commercially available N Cbz amino acids ( 2.14 ) were converted to N (Cbz aminoacyl)benzotriazoles ( 2.15 ) in 83 88% yields upon treatment with 1 H benzotriazole an d thionyl chloride in THF at 20 C for approximately 2 hours Scheme 2 1. Synthesis of N protected(acylbenzotriazoles) 2.15 and 2.17 Compounds 2.15a,c were further coupled with free amino acids Z L Phe OH and Z L Met OH to generate dipeptides 2.16a b which were converted to their corresponding acylbenzotriazoles ( 2.17a b ) in 68 70% (Scheme 2 1, Table 2 1). Novel compounds 2.17b an d 2.17 ( b+b' ) were characterized by elemental analysis and 1 H and

PAGE 30

30 13 C NMR spectroscopy; known molecules were verified by comparison with literature melting points and spectroscopic data. Table 2 1. Conversion of N Cbz amino acids ( 2.14a d ) to N (Cbz aminoacyl)benzotriazoles ( 2.15a d ) and N Cbz dipeptides ( 2.16a b, 2.16(b+b ) ) to N (Cbz dipeptidoyl )benzotriazoles ( 2.17 a b, 2. 17 ( b + b' ) ) Entry Reactants Products d Yield a (%) Mp (C) (Lit. Mp) b 1 Z L Val OH ( 2.14a ) Z L Val Bt ( 2.15a ) 87 107 108 (73 74) 2 Z L Phe OH ( 2.14b ) Z L Phe Bt ( 2.15b ) 85 149 151 (151 152) 3 Z L Ala OH ( 2.14c ) Z L Ala Bt ( 2.15c ) 85 115 117 (114 115) 4 c Z DL Ala OH ( 2.14 ( c + c' )) Z DL Ala Bt ( 2.15 ( c + c' )) 83 116 118 (112 113) 5 Z L Trp OH ( 2.14d ) Z L Trp Bt ( 2.15d ) 88 99 100 (100 101) 6 Z L Ala L Phe OH ( 2.16a ) Z L Ala L Phe Bt ( 2.17a ) 68 148 149 (148 149) 7 Z L Val L Met OH ( 2.16b ) Z L Val L Met Bt ( 2.17b ) 70 145 147 8 c Z L Val DL Met OH ( 2.16 ( b + b' )) Z L Val DL Met Bt ( 2.17 ( b + b' )) 79 143 145 a Isolated yield. b Lit. mp [04S2645, 09S2392]. c Compound numbers with primes represent racemates or mixture of diastereomers d Compounds 2.15b and 2.17a were prepared by Dr. Bajaj. 2.2.2 Synthesis of Thiosemicarbazides 2 .20 a b Scheme 2 2. Preparation of N substituted thiosemicarbazides 2.20 Compounds 2.20a b were synthesized following established procedure s in our group. [ 04JOC2976, 06ARK226] B is( 1 benzotriazolyl)methanethione ( 2.18 ) results from the treatment of thiophosgene (CSCl 2 ) with 1 H benzotriazole, which in turn was used to generate 1 (a lkylthiocarbamoyl)benzotriazoles ( 2.19a b ) in high yields (92 83%). Finally, N substituted thiosemicarbazides were formed upon reacting compounds 2.19 with hydrazine (Scheme 2 2, Table 2 2 )

PAGE 31

31 Table 2 2. Preparation of thiosemicarbazides 2 .20 a b Products c Yield a ( % ) Mp (C) Lit Mp (C) ( 2.20a ) 50 76 78 82 83 b ( 2.20b ) 84 142 144 142 a Isolated yields. b Lit. mp [09PSSRE523] c Compound 2.20a was prepared by Dr. Bajaj. 2.2.3 Syntheses of Thiadiazoles ( 2 .22a c, 2.22(c+c )) and T heir Thiosemicarbazide Precursors (2.21a c, 2.21 (c+c')) Scheme 2 3. Synthesis of 1,3,4 thiadiazoles 2.22 and their precursors 2.21 N (Cbz aminoacyl)benzotriazoles 2.15a c and 2.15 ( c+c' ) were reacted with thiosemicarbazides ( 2.20a b ) under microwave irradiation to yield precursors 2.21a c and 2.21 ( c+c' ) (68 79%). (Scheme 2 3, Table 2 3 ). The enantiopurity of compound 2.21 c was confirm ed by chiral HPLC UV analysis; as expected, the thiosemicarbazide derivative 2.21c showed a single peak with nearly the same reten tion time (7.31 min) of one of the two equal intensity peaks (7.10 and 7.38 min) obtained from the racemic mixture 2.21 ( c+ c' ). Compounds 2.21 a c and the racemic mixture 2.21 ( c+ c' ) each underwent concurrent cyclization and deprotection of the Cbz group on treatment with

PAGE 32

32 concentrated sulfuric acid [07HC316, 07M103] to produce 1,3,4 thiadiazoles ( 2.22 a c 2.22 ( c+c' ) ) in yields of 53 73%. (Scheme 2 3, Table 2 3 ). Table 2 3. Preparation of substituted amino acids thiosemicarbazides 2.21 and 1,3,4 thiadiazoles 2.22 Compounds 2.21 a Compounds 2.22 a Reactants 2.15 R 3 Entry Yield (%) Mp, C Entry Yield (%) Mp, C 23 D Z L Val Bt Isopropyl 2.21a 68 140 142 2.22a 68 60 62 +13.0 Z L Phe Bt Cyclohexyl 2.21b 74 164 165 2.22b 53 158 159 +5.1 Z L Ala Bt Cyclohexyl 2.21c 79 193 195 2.22c 73 138 139 12.9 Z DL Ala Bt Cyclohexyl 2.21 ( c+c ) 72 189 190 2.22 ( c+c ) 67 133 135 0.0 a Compounds 2.21a and 2.22a were prepared by Dr. Bajaj. In the 1 of compound 2.21 c disappeared when thiadiazole 2.22 c was formed. The removal of the Cbz protecting group was also evident from the aromatic region of the 1 H NMR 2.21 c disappeared in the product thiadiazole 2.22 c The enantiomeric purity of c ompound 2.22 c was confirmed by HPLC studies: thus using a Chiralcel OD column, 2.22 c showed a single peak (11.47 min) whereas the racemic mixture 2.22 ( c+ c' ) showed two peaks (11.87 and 12.54 min), one of which had nearly the same retention time as the sing le peak from 2.22 c To justify the small difference in retention times, we ran the HPLC of the 1:1 ratio mixture of 2.22 c and 2.22 ( c+c' ) and observed two peaks at 11.43 and 12.26 min in ratio of 3:1, respectively, which confirmed the previous analysis. The chiral integrity of 2.22 a and 2.22 b is proved by the chiral purity of 2.24 a and 2.24 b which is discussed later in this chapter. The measu red optical rotations (Table 2 3 ) support this conclusion. 1,3,4 Thiadiazoles ( 4a c ) were each reacted with acylbenzot riazoles ( 2.15 2.17 ) under

PAGE 33

33 microwave irradiation to afford 1,3,4 thiadiazolo substituted amino acids ( 2.23a c ) and dipeptides ( 2.24a c ) (Scheme 2 4, Table 2 4). Scheme 2 4. Syntheses of 1,3,4 thiadiazolo substituted amino acids ( 2.23 ) and dipeptides ( 2.24 ) The 13 C NMR spectrum of the diastereomeric mixture 2.24 ( c+ c' ) derived from the coupling of diastereomeric mixture of dipeptides 2.17 ( b+ b' ) with enantiomerically pure thiadiazole derivative ( 2.22 c ) methionine methyl carbons, but chiral compound 2.24 c HPLC UV analysis of compound 2.24c and the diastereomeric mixture 2.24 ( c+c' ) supported the above conclusions; thus 2.24c showed a single peak with a retention time (12.17 min), nearly the same as one of the two peaks (retention times of 11.63 and 12.16 min) disclosed by the diastereomeric mixture 2.24 ( c+c' ). We further analyzed a 1:1 mixture of 2.24c and 2.24 ( c+c' ) and it was observed an intensity ratio of 1:3 for the

PAGE 34

34 two peaks at 11.72 and 12.30 min which confirms the above HPLC conclusions. The HPLC evidence of 2.24c confirms the 13 C evidence for the chiral integrity of 2.24c ; by analogy we conclude that the 13 C NMR spectra for 2.23a c and for 2.24a b are evidence of their chiral purity. Table 2 4. Preparation of 1,3,4 thiadiazolo substituted amino acids ( 2 .23 ) and dipeptides ( 2.24 ) Reactants 2.22 Reactants Products a Conditions Yield (%) Mp, C a 2 .15 b Z L Phe Bt 2.23a TEA, DMF, 2.5h 62 74 75 b 2 .15 a Z L Val Bt 2.23 b TEA, DMF, 3h 60 205 206 c 2 .15 d Z L Trp Bt 2.23 c THF, 2.5h 60 112 114 a 2 .17 a Z L Ala L Phe Bt 2.24 a THF, 2h 57 208 209 b 2 .17a Z L Ala L Phe Bt 2.24 b THF, 3h 67 229 230 c 2 .17 b Z L Val L Met Bt 2.24 c THF, 0.5h 65 237 238 (c +c') b 2 .17 ( b + b '), Z L Val D L Met Bt 2.24 ( c+ c' ) THF, 1.5h 61 233 234 a Compounds 2.23a and 2.24a were prepared by Dr. Bajaj. b Compound numbers with primes represent mixture of diastereomers. 2.3 Summary Novel N (Cbz aminoacyl)thiosemicarbazides ( 2.21a c, 2.21 ( c+ c' ) ) underwent concurrent cyclization and deprotection to give 2,5 disubstituted 1,3,4 thiadiazoles ( 2.22 a c 2.22 ( c+c' ) ) each possessing a free amino group with retention of chirality. The free amino group in the substituted thiadiazoles 2.22 coupled with diverse acylbenzotriazoles ( 2.15 2.17 ) under microwave conditions to give chirally pure thiadiazolyl amino acids 2.23 a c and dipeptides ( 2.24 a c 2.24 ( c+ c' ) ) (40 62%). In view of the high selectivity and the fact that there is no other known method to make such compounds, this method represents a promising route to the preparation of various thiadiazoles substituted pepti des.

PAGE 35

35 2.4 Experimental Section 2.4.1 General Methods Melting points were determined on a hot stage apparatus and are uncorrected. 1 H NMR (300 MHz) spectra were recorded in CDCl 3 (CD 3 ) 2 CO or DMSO d 6 with TMS as the internal standard and CDCl 3 (CD 3 ) 2 CO or DMSO d 6 as the internal standard for 13 C NMR (75 MHz) Column chromatography was conducted on flash silica gel (200 425 mesh). Visualization of TLC plates was done via UV and phosphomolybdic acid staining. Anhydrous THF was obtained by distillation immediately pri or to use, from sodium/benzophenone ketyl. Optical rotation values were measured using the sodium D line. A single mode cavity microwave synthesizer with a continuous irradiation at 2450 MHz and an infrared temperature control system was used. HPLC was don e using Chiralcel OD H and Chirobiotic T column. In the case of compound 2.21 a Chiralcel OD H column and a mixture of ethanol : hexane (8:2) was used. As per compound 2.22 Chiralcel OD H column and a mixture of 2 propanol : hexane (1:9) was used. Finally, for compound 2.24 Chirobiotic T column with a mixture of methanol : water (9:1) was used. Compounds 2.15a c 2.17a 2.18 2.19a b and 2.20a b were prepared according to literature procedure and their spectroscopic data were equivalent to those reported [ 0 4S2645 04JOC2976, 06ARK226 ]. 2.4.2 General Procedure for the formation of N Cbz(dipeptidoylbenzotrizole) 2.17 Compounds 2.1 7 b and 2.1 7 ( b+b' ) (Table 2 1 ) were prepared by literature procedures. [04S2645, 09ARK47, 09S2392 ] Benzyl ((S) 1 (((S) 1 (1H benzo[d][1,2,3]triazol 1 yl) 4 (methylthio) 1 oxobutan 2 yl)ami no) 3 methyl 1 oxobutan 2 yl)carbamate ( 2.17 b ) Recrystallization from diethyl ether yielding the product as white microcrystals (70%); mp 145 147 C; 1 H NMR

PAGE 36

36 (CDCl 3 8.25 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 8.8 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.42 7.28 (m, 5H), 7.20 (d, J = 7.2 Hz, 1H), 6.14 6.02 (m, 1H), 5.47 (d, J = 9.0 Hz, 1H), 5.20 5.08 (m, 2H), 4.21 4.10 (m, 1H), 2.71 2.60 (m, 2H), 2.54 2.40 (m, 1H), 2.40 2.12 (m, 2H), 2.06 (s, 3H), 1.00 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H); 13 C NMR (CDCl 3 128.7, 128.4, 128.2, 126.8, 120.6, 114.6, 67.4, 60.6, 53.0, 31.9, 31.2, 30.3 19.4, 18.1, 15.6; Anal. Calcd for C 24 H 29 N 5 O 4 S: C, 59.61; H, 6.04; N, 14.48; found: C, 59.85; H, 6.18; N, 14.52. Benzyl ((2S) 1 ((1 (1H benzo[d][1,2,3]triazol 1 yl) 4 (methylthio) 1 oxobutan 2 yl)amino) 3 methyl 1 oxobutan 2 yl)carbamate ( 2.17 ( b+ b' ) ) Recrystallization from diethyl ether yielding the product as white m icrocrystals (79 %); mp 143 145 C; 1 H NMR (CDCl 3 8.20 (m, 1H), 8.20 8.07 (m, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.53 (t, J = 7.4 Hz, 1H), 7.43 7.28 (m, 6H), 6.17 6.01 (m, 1H), 5.59 (d, J = 8.1 Hz, 1H), 5.20 5.09 (m, 2H), 4.32 4.14 (m, 1H), 2.75 2.59 (m, 2H), 2.54 2.38 (m, 1H), 2.30 2.10 (m, 2H), 2.04 (s, 3H), 0.98 (dd, J = 12.5, 6.8 Hz, 6H); 13 C NMR (CDCl 3 136.3, 131.2, 130.9, 128.7, 128.3, 128.1, 126.7, 12 0.5, 114.5, 67.3, 60.5, 52.9, 52.8, 31.8, 31.3, 30.3, 19.5, 19.4, 18.1, 17.9, 15.5. Anal. Calcd for C 24 H 29 N 5 O 4 S: C, 59.61; H, 6.04; N, 14.48; found: C, 59.55; H, 6.05; N, 14.43. 2.4.3 General P rocedure for the P reparation of 1,3,4 T hiadiazole Precursors 2.21a c and 2.21(c+c') N (P rotected aminoacyl)benzotriazoles ( 2.15 a c 2.15 ( c+c' ) ) (2.5 mmol) in anhydrous tetrahydrofuran (THF) (5 mL), were eac h irradiated by microwave at 70C, 65W, together with thiosemicarbazide ( 2 .20 a or 2 .20 b ) Upon completion of the reaction (monitored by TLC) (2 4 h), the solvent was evaporated and the residue was

PAGE 37

37 washed with methylene chloride or diethyl ether then filtered to afford ( 2.21 a c 2.21 ( c+c' ) ) ( S) Benzyl (1 (2 (isopropylcarbamothioyl)hydrazinyl) 3 methyl 1 oxobutan 2 yl)carbamate ( 2.21a ) Recrystallization from diethyl ether yielding the product as white microcrystals (68%); mp 140 142 C; 1 H NMR (CD 3 COCD 3 ) 9.41 (br s, 1H), 8.48 (br s, 1H), 7.40 7.31 (m, 6H), 6.90 (br s, 1H), 5.16 (d, A part of A B system, J = 12.3 Hz, 1H), 5.08 (d, B part of AB system, J = 12.3 Hz, 1H), 4.56 4.40 (m, 1H), 3.82 3.74 (m, 1H), 1.20 (d, J = 6.6 Hz, 3H), 1.13 (d, J = 6.6 Hz, 3H), 1.05 (d, J = 6.9 Hz, 3H), 1.02 (d, J = 6.9 Hz, 3H). 13 C NMR (CD 3 COCD 3 158.2, 137.8, 129.4, 128.9, 128.8, 67.3, 61.5, 47.1, 22.4, 19.5, 19.3 Anal. Calcd for C 17 H 26 N 4 O 3 S: C, 55.71; H, 7.15; N, 15.29; found: C, 56.06; H, 7.43; N, 15.13. (S) Benzyl (1 (2 (cyclohexylcarbamothioyl)hydrazinyl) 1 oxo 3 phenylpropan 2 yl)carb amate ( 2.21b ) Purified by washing with diethyl ether to yield the produ ct as white microcrystals (74 %); mp 164 165 C; 1 H NMR (CD 3 COCD 3 (br s, 1H), 7.40 7.00 (m, 11H), 6.93 (d, J = 5.4 Hz, 1H), 5.20 4.90 (m, 2H), 4.30 4.20 (m, 1H), 4.20 4.05 (m, 1H), 3.17 (dd, J = 13.8, 6.2 Hz, 1H), 3.01 (dd, J = 13.6, 8.9 Hz, 1H), 2.00 1.80 (m, 2H), 1.78 1.50 (m, 3H), 1.40 1.00 (m, 5H); 13 C NMR (CD 3 COCD 3 183.2, 1 72.1, 158.2, 138.6, 130.7, 129.8, 129.3, 129.1, 128.1, 67.7, 57.4, 54.7, 38.1, 33.6, 31.2, 26.9, 26.4. Anal. Calcd for C 24 H 30 N 4 O 3 S: C, 63.41; H, 6.65; N, 12.32; found: C, 63.37; H, 6.99; N, 12.31. ( S) Benzyl (1 (2 (cyclohexylcarbamothioyl)hydrazinyl) 1 oxo propan 2 yl)carbamate ( 2.21 c ) Purified by washing with methylene chl oride to yield the product as white microcrystals (79%); mp 193 195 C; 1 H NMR (CD 3 COCD 3

PAGE 38

38 (br s, 1H), 7.45 7.22 (m, 6H), 7.00 (br s, 1H), 5.22 5.05 (m, 2H), 4.26 4.12 (m, 1H), 4.12 3.99 (m, 1H), 2.00 1.87 (m, 2H), 1.80 1.65 (m, 2H), 1.65 1.54 (m, 1H), 1.37 (overlapped d, J = 6.6 Hz, 3H), 1.34 1.04 (m, 5H); 13 C NMR (CD 3 COCD 3 173.0, 158 .2, 138.3, 129.8, 129.3, 129.0, 67.6, 54.7, 51.6, 33.5, 26.8, 26.4, 26.3, 17.7. Anal. Calcd for C 18 H 26 N 4 O 3 S: C, 57.12; H, 6.92; N, 14.80; found: C, 57.11; H, 7.06; N, 14.92. Benzyl (1 (2 (cyclohexylcarbamothioyl)hydrazinyl) 1 oxopropan 2 yl)carbamate ( 2.21 ( c+ c' ) ) Purified by washing with methylene ch loride to yield the product as white microcrystals (72%); mp 189 190 C; 1 H NMR (CD 3 COCD 3 1H), 7.54 7.22 (m, 6H), 7.05 6.90 (m, 1H), 5.22 5.05 (m, 2H), 4.27 4.11 (m, 1H), 4.11 4.00 (m, 1H), 2.00 1.85 (m, 2H), 1.70 1.64 (m, 2H), 1.64 1.53 (m, 1H), 1.37 (overlapped d, J = 7.2 Hz, 3H), 1.35 1.04 (m, 5H); 13 C NMR (CD 3 COCD 3 129.8, 129.3, 129.1, 67.7, 54.7, 51.6, 33.6, 26.9, 26.5, 17.8. Anal. Calcd for C 18 H 26 N 4 O 3 S: C, 57.12; H, 6.92; N, 14.80; found: C, 57.17; H, 7.24; N, 15.01. 2.4.4 General P rocedure for the P reparation of 1,3,4 T hiadiazoles (2.22a c, 2.22(c+ c') ) Concentrated sulfuric acid (2 mL) was added dropwise to (2.0 mmol) of thiosemicarbazides ( 2.21 a c 2.21 ( c+ c' ) ) The resulting mixture was stirred at room temperature for 8 12 h. The solution was quenched with ice and then extracted with methylene chloride (2 x 10 mL). The aqueous portion was collected and neutralized with sodium hydroxide then extracted with methy lene chloride (3x10 mL). The organic layers were combined, washed with water (1x10 mL) and brine (1x10 mL), and then dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure to yield 1,3,4 thiadiazoles ( 2.22 a c 2.22 ( c+ c' ) ) [ 07HC316 07M103]

PAGE 39

39 (S) 5 (1 Amino 2 methylpropyl) N isopropyl 1,3,4 thiadiazol 2 amine ( 2.22a ) White microcrys tals (68%); mp 60 62 C; 1 H NMR (CDCl 3 J = 6.3 Hz, 1H), 3.72 3.55 (m, 1H), 2.08 1.62 (m, 3H), 1.25 (d, J = 6.6 Hz, 6H) 0.94 (overlapped d, J = 6.9 Hz, 3H), 0.91 (overlapped d, J = 6.9 Hz, 3H). 13 C NMR (CDCl 3 169.8, 164.3, 58.0, 49.4, 34.8, 22.9, 19.4, 18.0. HRMS Calcd for C 9 H 19 N 4 S [M+H] + 215.1325; found 215.1315 (S) 5 (1 Amino 2 phenylethyl) N cyclohexyl 1,3,4 thiadia zol 2 amine ( 2.22b ) White mic rocrys tals (53%); mp 158 159.0 C; 1 H NMR (CDCl 3 7.20 (m, 5H), 5.46 (br s, 1H), 4.49 (dd, J = 9.3, 4.5 Hz, 1H), 3.31 (dd, J = 13.5, 4.2 Hz, 2H), 2.87 (dd, J = 13.5, 9.3 Hz, 1H), 2.20 2.00 (m, 2H), 1.88 1.53 (m, 5H), 1.50 1.10 (m, 5H). 13 C NMR (CDCl 3 Anal. Calcd for C 16 H 22 N 4 S: C, 63.54; H, 7.33; N, 18.52; found: C, 63.17; H, 7.65; N, 18.88. (S) 5 (1 Aminoethyl) N cyclohexyl 1,3,4 thiadiazol 2 amine ( 2.22c ) White microcrystals (73%); mp 138 139 C; 1 H NMR (CDCl 3 4.30 (m, 1H), 3.29 (br s, 1H), 2.22 1.96 (m, 2H), 1.78 1.55 (m, 5H), 1.49 (dd, J = 6.6, 2.4 Hz, 3 H), 1.44 1.10 (m, 5H). 13 C NMR (CDCl 3 24.6. Anal. Calcd for C 10 H 18 N 4 S: C, 53.06; H, 8.02; N, 24.75; found: C, 52.79; H, 8.18; N, 24.51. 5 (1 Aminoethyl) N cyclohexyl 1,3,4 thiadiazol 2 amine ( 2.22 ( c+c' ) ) Whit e microcrystals (67%); mp 133 135 C; 1 H NMR (CDCl 3 4.25 (m, 1H), 3.31 3.17 (m, 1H), 2.08 1.95 (m, 2H), 1.85 1.65 (m, 4H), 1.50 1.60 (m, 1H), 1.43

PAGE 40

40 (dd, J = 6.6, 0.6 Hz, 3H), 1.38 1.08 (m, 5H). 13 C NMR (CDCl 3 48.1, 33.1, 25.6, 24.9, 24.6. HRMS Calcd for C 10 H 19 N 4 S [M+H] + 227.1325; found 227.1318. 2.4.5 General Procedure for the P reparation of 1,3,4 T hiadiazole D erivatives (2.23a c, 2.24a c, 2.24 (c+c') ) 1,3,4 Thiadiazoles 2.22 (0.3 mmol) and acylbenzotriazoles 2.15 or 2.17 (0.3 mmol) were dissolved in anhydrous THF (4 mL). The reaction mixture was exposed to microwave irradiation at 70C, 65W until completion of the reaction (monitored by TLC). After the solvent was evaporated, ethyl acetate was added (4 mL) to give a w hite precipitate. This solid was filtered and dried to afford 1,3,4 thiadiazolo substituted amino acids 2.23 a c and dipeptides 2.24 a c and 2.24 ( c+ c' ). In the case of 2.23 a and 2.23 b anhydrous DMF and triethylamine proved to be the best conditions for a sh orter reaction time and a short flash column with a gradient elution of hexanes/ethyl acetate was used to purify the product. Benzyl ((S) 1 (((S) 1 (5 (isopropylamino) 1,3,4 thiadiazol 2 yl) 2 methylpropyl)amino) 1 oxo 3 phenylpropan 2 yl)carbamate ( 2.23a ) White microcrystals (63%); mp 74 75 C; 1 H NMR (CDCl 3 J = 8.1 Hz, 1H), 7.32 7.26 (m, 5H), 7.15 7.12 (m, 3H), 7.08 7.06 (m, 2H), 6.00 5.88 (m, 1H), 5.65 (d, J = 5.1 Hz, 1H), 5.12 4.96 (m, 3H), 4.74 4.70 (m, 1H), 3.68 3.57 (m, 1H), 3.08 2.98 (m 2H), 2.28 2.18 (m,1H), 1.32 (overlapped d, J = 3.0 Hz, 3H), 1.31 (overlapped d, J = 3.0 Hz, 3H), 0.90 (d, J = 6.3 Hz, 6H). 13 C NMR (CDCl 3 128.2, 128.1, 128.0, 126.9, 67.1, 56.0, 54.3, 49.7, 38.8, 33.2, 22.9, 19.3, 18.5. Anal. Calcd for C 26 H 33 N 5 O 3 S: C, 63.01; H, 6.71; N, 14.13; found: C, 62.97; H, 6.50; N, 13.99. Benzyl ((S) 1 (((S) 1 (5 (cyclohexylamino) 1,3,4 thiadiazol 2 yl) 2 phenylethyl)amino) 4 methyl 1 oxopentan 3 yl)carbamate ( 2.23b ) White microc rystals

PAGE 41

41 (60%); mp 205 206 C; 1 H NMR (CDCl 3 J = 8.4 Hz, 1H), 7.42 7.27 (m, 5H), 7.23 7.08 (m, 5H), 6.03 (d, J = 6.9 Hz, 1H), 5.60 5.40 (m, 2H), 5.14 (d, A part of AB system, J = 12.3 Hz, 1H), 5.05 (d, B part of AB system, J = 12.0 Hz, 1H), 4.40 4.20 (m, 1H), 3.40 3.20 (m, 2H), 3.15 (br s, 1H), 2.14 1.90 (m, 3H), 1.83 1.68 (m, 2H), 1.68 155 (m, 1H), 1.41 1.10 (m, 5H), 0.85 (d, J = 6.6 Hz, 3H), 0.78 (d, J = 6.3 Hz, 3H); 13 C NMR (CDCl 3 128.1, 127.0, 67.2, 60.2, 56.8, 50.2, 40.8, 33.0, 31.5, 25.6, 24.9, 19.4, 17.8. Anal. Calcd for C 29 H 37 N 5 O 3 S: C, 65.02; H, 6.96; N, 13.07; found: C, 64.88; H, 7.25; N, 13.11. Benzyl ((S) 1 (((S) 1 (5 (cyclohexylamino) 1,3,4 thiadiazol 2 yl)ethyl)amino) 3 (1H indol 3 yl) 1 oxopropan 2 yl)carbamate ( 2.23c ) Beige microcrystals (60%); mp 112 114 C; 1 H NMR (CDCl 3 J = 7.1 Hz, 1H), 7.41 7.20 (m, 7H), 7.19 7.05 (m, 2H), 7.00 (t, J = 6.9 Hz, 1H), 6.89 (s, 1H), 5.85 (d, J = 6.0 Hz, 1H), 5.13 4.99 (m, 3H), 4.68 4.55 (m, 1H), 3.32 3.08 (m, 3H), 2.12 1.90 (m, 2H), 1.82 1.65 (m, 2H), 1.64 1.52 (m, 1H), 1.12 1.45 (m, 8H); 13 C NMR (CDCl 3 156.2, 136.5, 128.7, 128.3, 128.2, 127.5, 123.8, 122.2, 119.7, 118.8, 111.6, 109.9, 67.2, 57.0, 55.6, 45.3, 32.8, 28.9, 25.5, 24.8, 19.9. HRMS Calcd for C 29 H 35 N 6 O 3 S [M+H] + 547.2486, found 547.2500. Benzyl ((S) 1 (((S) 1 (((S) 1 (5 (isopropylamino) 1,3,4 thiadiazol 2 yl) 2 methylpropyl)ami no) 1 oxo 3 phenylpropan 2 yl)amino) 1 oxopropan 2 yl)carbamate ( 2.24a ) White microcrys tals (57%); mp 208 210 C; 1 H NMR (CDCl 3 J = 9.0 Hz, 1H), 7.40 7.28 (m, 5H), 7.20 7.10 (m, 3H), 7.10 6.98 (m, 3H), 5.71 (d, J = 6.3 Hz, 1H), 5.48 (d, J = 7.5 Hz, 1H), 5.14 4.97 (m, 3H), 4.95 4.84 (m, 1H), 4 .32 4.18 (m, 1H), 3.72 3.55 (m, 1H), 3.11 2.98 (m, 2H), 2.36 2.19 (m, 1H), 1.38 1.22 (m, 9H), 0.91 (d, J =

PAGE 42

42 7.8 Hz, 6H); 13 C NMR (CDCl 3 128.6, 128.4, 128.3, 126.9, 67.2, 54.5, 50.9, 49.7, 38.3, 33.2 23.0, 19.3, 18.8, 18.5. HRMS Calcd for C 29 H 39 N 6 O4S [M+H] + 567.2748, found 567.2736. Benzyl ((S) 1 (((S) 1 (((S) 1 (5 (cyclohexylamino) 1,3,4 thiadiazol 2 yl) 2 phenylethyl) ami no) 1 oxo 3 phenylpropan 2 yl)amino) 1 oxopropan 2 yl)carbamate ( 2.24b ) Whit e microcrys tals (67%); mp 229 230C; 1 H NMR (DMSO d 6 J = 7.5 Hz, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.60 (d, J = 6.3 Hz, 1H), 7.50 7.30 (m, 6H), 7.30 7.20 (m, 5H), 7.20 7.08 (m, 5H), 5.28 5.15 (m, 1H), 5.10 4.93 (m, 2H), 4.55 4.40 (m, 1H), 4.98 3.9 1 (m, 1H), 3.50 3.38 (m,1H), 3.30 3.18 (m, 1H), 3.16 3.02 (m, 1H), 2.96 2.83 (m, 1H), 2.80 2.70 (m, 1H), 2.03 1.86 (m, 2H), 1.77 1.64 (m, 2H), 1.62 1.50 (m, 1H), 1.39 1.14 (m, 5H), 1.08 (d, J = 6.3 Hz, 3H); 13 C NMR (DMSO d 6 168.0, 158.1, 155.6, 137.5, 137.2, 136.9, 129.1, 128.3, 128.1, 127.9, 127.7, 126.3, 126.1, 65.4, 53.6, 50.0, 37.6, 32.1, 25.2, 24.3, 18.1. Anal. Calcd for C 36 H 42 N 6 O4S: C, 66.03; H, 6.46; N, 12.83. Found: C, 66.11; H, 6.74; N, 12.78. Benzyl ((S) 1 (((S) 1 (((S) 1 (5 (cyc lohexylamino) 1,3,4 thiadiazol 2 yl)ethyl)amino) 4 (met hylthio) 1 oxobutan 2 yl)amino) 3 methyl 1 oxobutan 2 yl)carbamate ( 2.24c ) White microcry stals (60%); mp 237 238C; 1 H NMR (DMSO d 6 8.60 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 6.9 Hz, 1H), 7.50 7.22 (m, 6H), 5.17 4.94 (m, 3H), 4.48 4.26 (m, 1H), 3.96 3.80 (m, 1H), 3.52 3.38 (m, 1H), 2.49 2.30 (m, 2H), 2.02 (s, 3H), 2.00 1.86 (m, 4H), 1.86 1.74 (m, 1H), 1.74 1.61 (m, 2H), 1.61 1.50 (m, 1H), 1.44 (d, J = 6.9 Hz, 3H), 1.39 1.10 (m 5H), 0.88 0.82 (m, 6H); 13 C NMR (DMSO d 6

PAGE 43

43 53.5, 51.5, 44.3, 32.0, 30.0, 29.3, 25.2, 24.2, 19.5, 19.1, 18.0, 14.6. Anal. Calcd for C 28 H 42 N 6 O 4 S 2 : C, 56.92; H, 7.17; N, 14.22; found: C, 5 6.56; H, 7.45; N, 14.15. Benzyl ((S) 1 (((S) 1 (((S) 1 (5 (cyclohexylamino) 1,3,4 thiadiazol 2 yl) 2 phenylethyl)ami no) 1 oxo 3 phenylpropan 2 yl)amino) 1 oxopropan 2 yl)carbamate ( 2.24 ( c+ c' ) ) White micro crystals (61%); mp 233 234C; 1 H NMR (DMSO d 6 .58 (d, J = 7.5 Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.40 7.25 (m, 6H), 5.12 4.97 (m, 3H), 4.44 4.30 (m, 1H), 3.94 3.82 (m, 1H), 3.48 3.36 (m, 1H), 2.46 2.36 (m, 2H), 2.01 (s, 3H), 1.97 1.88 (m, 4H), 1.74 1.65 (m, 2H), 1.60 1.51 (m, 1H), 1.44 (d, J = 6.9 Hz, 3H), 1.34 1.14 (m, 6H), 0.85 (t, J = 7.8 Hz, 6H). 13 C NMR (DMSO d 6 171.6, 171.1, 170.6, 167.4, 160.3, 160.1, 156.4, 156.2, 137.0, 128.3, 127.7, 127.6, 65.4, 60.3, 54.0, 51.6, 44.6, 31.8, 30.1, 29.5, 25.1, 24.1, 19.3, 19.2, 18.7, 18.2, 14.7, 14.5. Anal. Calcd for C 28 H 42 N 6 O 4 S 2 : C, 56.92; H, 7.17; N, 14.22; found: C, 56.84; H, 7.45; N, 14.05

PAGE 44

44 CHAPTER 3 SYNTHESES OF CHIRAL N (PROTECTED) TRI AND TETRAPEPTIDE SCAFFOLDS 3.1 Introduct ory Remarks 3.1.1 Background Peptides and their derivatives such as hormones, neurotransmitters, and neuromodulators act as signal molecules in diverse biological and medicinal applications and thus have attracted considerable synthetic attention. [99JCC55, 96EMT162] Steroids are often attach ed to other building blocks that modulate their biological activity [06CMC813]. Peptidyl steroids, in particular, with amino acid or peptide units linked to the steroidal frame, are important i n living systems such as cholyl glycine which is found in the hu man body. [03S751] Libraries of two peptide chains on a steroid nucleus have been reported. These peptide steroid conjugates exhibited enzyme like activity [02JCC552, 00ACIE145] and some constituted synthetic receptors for oligopeptides [96JACS1813, 94JACS 7955]. Recently some new cationic steroid antibiotics with enhanced antibacterial activity were prepared by conjugating tripeptides to the steroid nuclei of a cholic acid derivative. [04OL3433] The addition of a tripeptide moiety in this case, resulted in the increase of the associative noncovalent interactions, therefore an increase of the antibiotic bacterial membrane affinity which led to an improvement of the antibacterial activity. [04OL3433] Terpenes are other scaffolds of interest since they compose one of the largest groups of natural products containing numerous medically relevant compounds. [97S1788] Amino acid esters of hydroxylic terpenes, a class of peptide conjugates, are effective medicinal agents, e.g. for atherosclerosis. [92CA67183] N (Prot

PAGE 45

45 aminoacyl) esters derived from long chain alkanols containing 12 to 22 carbon atoms amino acids or peptides possess medicinal, nutritional and industrial utility. [85JOC1457] Moreover, hybrid molecules resulting from carbohydrate conjugates di splay in general, an improved solubility with modified physicochemical properties. In the case of peptides, their combination with sugar moieties is important in biochemical processes ranging from cell growth regulation, immune response, binding of pathoge ns to intercellular communication, intercellular targeting, cancer cell metastasis and inflammation. [93G97, 96CR683, 93ACR319] We have already utilized N acylbenzotriazoles extensively for N [00JOC8210, 01ARK19] C [03JOC4932, 03JOC5720] O [99JHC777] and S acylation. Previously we reported the facile synthesis of amino acid conjugates with steroids, terpenes and sugars. [09S2392] Herein, we present the extension of our methodology to provide a convenient and effective means of preparing, in solution p hase, scaffoldings of Cbz protected tripeptide and tetrapeptide conjugates with sugars, steroids, terpenes and heterocyclic nuclei of biological importance 3.1.2 Literature Preparative Methods In most cases, the peptides are assembled using standard coupling pr ocedures. Esterification of amino acids and peptides for the protection of carboxylic acid functionality and for their activation to make peptide conjugates has been reported. This step is usually followed by hydrolysis with lithium hydroxide to regenerate the acid form. [04T2447, 05T10827, 04S1826] Preparative methods reported for peptides and their conjugates includ e: (i) activating the C terminus with coupling reagents such as carbodiimides [05T10827], 1 hydroxybenzotriazole ( HOBt ) and 1 hydroxy 7

PAGE 46

46 azabe nzotriazole ( HOAt ) [04T2447] based uro nium, [98JOC9678] phosphonium, [99TL2045] immonium [02S1388] and ammonium salts [04T2447]; and (ii) procedures involving the isolated C terminus activated intermediates such as acyl halides, [96ACR268, 90JACS9651, 02JO C6372] acylimidazoles, [89JACS4856] anhydrides, [90T7175] active alkyl and phenolic esters [76S224] and acyloxy boron intermediates [00M3511]. Peptide conjugates with sugar and steroid moieties have previously been prepared (i) by solid phase syntheses [10 JOC2111, 02BMCL3033] and (ii) in solution phase by stepwise syntheses using regular coupling reagents like 1 e thyl 3 (3 dimethyllaminopropyl)carbodiimide ( EDC ) [04BMCL801], t etramethyl O (benzotriazol 1 yl)uronium tetrafluoroborate ( TBTU ) [01EJOC1533], N,N' d icyclohexylcarbodiimide DCC [09JCR391] and benzotriazole 1 yl oxy tris (dimethylamino) phosphonium hexafluorophosphate ( BOP ) [04CRC13]. However such methods suffer from difficulties in product purification and analysis by HPLC, [10JOC2 111] since the coupling steps are sensitive to the environment and water, [10JOC2111] and often afford low yields, [01EJOC1533] especially with glycine units [09JCR391]. 3.2 Results and Discussion 3.2.1 Syntheses of Cbz Protected Tripeptidoylbenzotriazole and Tetrap eptidoylbenzotriazole The Cbz protected tripeptides 3.2a c and tetrapeptides 3.4a c were synthesized by our previously reported method [ 11S2995 ] of stepwise coupling of amino acids with subsequent benzotriazole activated N protected amino acid analogs in s olution phase in good yields (67 91%). Compounds 3.2a c and 3.4a c were further activated by

PAGE 47

47 benzotriazole to obtain known intermediates 3.3a 3.5a and novel intermediates 3.3b c and 3.5b c in good yields (60 93%). These tripeptidoyl benzotriazoles 3.3a c and tetrapeptidoyl benzotriazoles 3.5a c were used as active intermediates to prepare peptide conjugates. (Scheme 3 1, Table 3 1) Scheme 3 1. Synthesis of N Pg tripeptidoyl benzotriazoles 3.3a c and tetrapeptidoyl benzotriazoles 3.5a c

PAGE 48

48 Table 3 1. Preparation of N Cbz tri and tetra peptidoyl benzotriazoles 3.3a c and 3.5a c respectively Reactants Products d Yield a (%) Mp (C) Z L Ala L Phe Gly OH ( 3.2a ) Z L Ala L Phe Gly Bt ( 3.3a ) 92 b 180 182 Z L Val L Phe Gly OH ( 3.2b ) Z L Val L Phe Gly Bt ( 3.3b ) 93 214 215 Z L Phe Gly L Leu OH ( 3.2c ) Z L Phe Gly L Leu Bt ( 3.3c ) 84 144 146 Z L Ala L Phe Gly L Ala OH ( 3.4a ) Z L Ala L Phe Gly L Ala Bt ( 3.5a ) 60 c 200 202 Z L Ala L Phe Gly Gly OH ( 3.4b ) Z L Ala L Phe Gly Gly Bt ( 3.5b ) 62 144 145 Z L Phe Gly L Leu Gly OH ( 3.4c ) Z L Phe Gly L Leu Gly Bt ( 3.5c ) 79 280 282 a Isolated yield. b Lit. mp 186 187C [ 11S2995 ]. c Lit. mp 212 214C [ 11S2995 ] d I synthesized compounds 3.3b 3. 5a and 3.5c 3.2.2 O Acylations with Tri and Tetrapeptidoylbenzotriazoles Scheme 3 2. Preparation of tri and tetrapeptidoyl ester

PAGE 49

49 Table 3 2. O Acylation products using N Cbz tri and tetra peptidoylbenzotriazoles Products a Reactants Reactants Time Yield (%) Mp (C) 3.6a Z L Ala L Phe Gly Bt ( 3.3a ) Cholesterol 1h 45 min 45 162 163 3.6b Z L Ala L Phe Gly Bt ( 3.3a ) Nerol 1h 30 min 50 114 116 3.6c Z L Val L Phe Gly Bt ( 3.3b ) Galactopyranose 1h 30 min 47 129 132 3.6d Z L Val L Phe Gly Bt ( 3.3b ) Estrone 2h 15 min 35 151 153 3.6e Z L Ala L Phe Gly Gly Bt ( 3.5b ) Diacetone glucose 1h 45 min 58 102 104 3.6f Z L Phe Gly L Leu Gly Bt ( 3.5c ) Menthol 1h 45 min 42 104 106 a I synthesized compounds 3.6c d Compounds 3.3a c, 3.5b and 3.5c were reacted with a variety of steroids, terpenes and sugar derivatives in the presence of catalytic amount of dimethylaminopyridine (DMAP) under microwave irradiations at 70C and 65W power for 1.5h to 3h to afford novel peptide conjugates 3.6a f in mode rate to good yields ( 42 58%) (Scheme 3 2, Table 3 2). Compounds 3.6a f were fully characterized by 1 H and 13 C NMR and elemental analysis. The chiral purity of these peptide conjugates was supported by the NMR spectra of 3.6a f which did not show any duplic ation of signals. (Scheme 3 2,Table 3 2). 3.2.3 S Acylation with Tri and Tetrapeptidoylbenzotriazoles Compounds 3.3a 3.5a and 3.5c were reacted with S nucleophiles in the presence of triethylamine at room temperature for 2h to give there corresponding S acylat ed tripeptide conjugates 3.7a b (78 83%) and tetrapeptide conjugates 3.7c d (68 94%) (Scheme 3 3, Table 3 3) The chiral purity of 3.7a d was supported by their 1 H and 13 C NMR spectra which showed no evidence of epimerization in their S acylated tri and tetrapeptide conjugates. The 1 H NMR spectra of compounds 3.7a and 3.7b showed

PAGE 50

50 prominent doublets for the methyl protons of alanine unit at 1.14 ppm and 13 C NMR showed prominent signals for all carbonyls of the tripeptide unit in the range of 196.2 to 155.6 ppm. Scheme 3 3. Preparation of tri and tetrapeptidoyl thioester Duplicate signals are absent in the 13 C NMR spectra of compounds 3.7a b Compound 3.7c showed sharp singlets of CH 2 protons of mercaptoacetic acid unit and Cbz group at 3.62 and 4.99 ppm respectively with no other repetition of signals in the NMR spectrum. Compound 3.7d also showed singlet of CH 2 protons of benzyl group and Cbz group at 4.10 and 4.95 ppm respectively with no other repeated signals co nfirms t he formation of optically pure 3.7c and 3.7d Table 3 3. S Acylation by N (Cbz protected) tri and tetrapeptidoylbenzotriazoles Product a Reactant Reactant Yield (%) Mp (C) 3.7a Z L Ala L Phe Gly Bt ( 3.3a ) Thiophenol 78 146 148 3.7b Z L Ala L Phe Gly Bt ( 3.3a ) Methyl mercapto acetate 83 158 160 3.7c Z L Ala L Phe Gly L Ala Bt ( 3. 5a ) Mercapto acetic acid 68 103 105 3.7d Z L Phe Gly L Leu Gly Bt ( 3.5c ) Benzyl mercaptan 94 186 187 a I synthesized compound 3.7c

PAGE 51

51 3.2.4 N Acylation with Tri and Tetrapeptidoylbenzotriazoles Compounds 3.3a b and 3.5a 3.5c were reacted with different N nucleophiles in the presence of base to give the corresponding chirally pure N acylated tri 3.8a b and tetra peptides 3.8c d (Scheme 3 4, Table 3 4) Compound 3.3a and 3.5c were reacted with N nucleophiles in the presence of triethylamine at 20C for 1 h to give the corresponding tripeptide conjugate 3.8a (75%) and tetrapeptide conjugate 3.8d (68%). Compounds 3.3b and 3.5b reacted with heterocyclic N nucleophiles un der microwave irradiation at 65C for 30 min in DMF to give the corresponding chirally pure peptide conjugates 3.8b (68%) and 3.8c (60%). Compounds 3.8a d were fully characterized by NMR spectroscopy and elemental analysis. Scheme 3 4. N Acylation with N (Cbz protected) tri and tetrapeptidoylbenzotriazoles Table 3 4. N Acylation by N (Cbz protected) tri and tetrapeptidoylbenzotriazoles Product a Reactant Reactant Conditions Yield (%) Mp, C 3.8a Z L Ala L Phe Gly Bt ( 3.3a ) N (3 Amino propyl) imidazole THF, TEA,1h, r t 62 74 75 3.8b Z L Val L Phe Gly Bt ( 3.3b ) 2 Aminopyridine DMF, MW, 0.5h, 65 C 60 205 206 3.8c Z L Ala L Phe Gly L Ala Bt ( 3.5a ) 2 Amino 6 methoxy benzothiazole DMF, MW, 0.5h, 65 C 60 112 114 3.8d Z L Phe Gly L Leu Gly Bt ( 3.5c ) N Methylpiperazine THF, TEA,1h, r t 57 208 209 a I synthesized compounds 3.8b and 3.8d

PAGE 52

52 The chiral purity of compounds 3.8a d was supported by 1 H and 13 C NMR. Tripeptide conjugate 3.8a and tetrapeptide conjugate 3.8c showed doublets for the 3.8a ) and 1.35, 1.12 ppm ( 3.8c ) with no duplication of signals in 1 H NMR and 13 C NMR spectra. Compound 3.8b showed a prominent singlet for the NH proton of the aminopyridine unit at 10.47 ppm and compound 3.8d showed a sharp singlet for the methyl protons of the N methylpiperazin e unit at 2.15 ppm with no duplication of signals in 1 H and 13 C NMR spectra which confirms the formation of chirally pure 3.8b and 3.8d 3.2.5 C Acylation with Tri and Tetrapeptidoylbenzotriazoles Compounds 3.3a and 3.5 c were reacted with different C nucleophiles in the presence of 1 equivalent of DMAP in MW at 70 C under 50W irradiation power to give the corresponding tripeptide conjugates 3.9a (69%) and tetra peptide conjugate 3.9b (61%). (Scheme 3 5, Table 3 5) Compounds 3.9a and 3.9b were fully characterized by NMR and HRMS analysis. NMR showed the existence of compounds 3.9a and 3.9b under their enolic form with n o evidence for epimerization. Scheme 3 5. C Acylation with N (Cbz protected) tri and tetrapeptidoylbenzotriazoles

PAGE 53

53 Table 3 5 C Acylated N (Cbz protected) tri and tetrapeptides. Product a Reactant Reactant Yield(%) Mpt (C) 3.9a Z L Ala L Phe Gly Bt ( 3.3a ) Dimedone 69 135 137 3.9b Z L Phe Gly L Leu Gly Bt ( 3.5c ) 1,3 cyclohexan e dione 61 188 190 a I synthesized compound 3.9b 3.3 Summary In conclusion, N (protected tripeptidoyl)benzotriazoles and N (protected tetrapeptidoyl) benzotriazoles are convenient acylating reagents, sufficiently reactive to form amide and ester bonds at ambient temperature. They offer an efficient preparation of chirally pure N protected tri and tetra peptide conjugates with sugars, steroids, terpenes and different heterocycles by O S N and C acylations in synthetically useful yields. The chirality of starting materials was preserved (> 97%) in the products, as evidenced by NMR spectra. 3.4 Experimental Section 3.4.1 General Methods Melting points were det ermined on a hot stag e apparatus and are uncorrected. 1 H NMR (300 MHz) spectra were recorded in CDCl 3 or DMSO d 6 with TMS as the internal standard and CDCl 3 or DMSO d 6 as the internal standard for 13 C NMR (75 MHz). Column chromatography was conducted on fl ash silica gel (200 425 mesh). Visualization of TLC plates was done via UV and phosphomolybdic acid staining. Anhydrous THF was obtained by distillation immediately prior to use, from sodium/benzophenone ketyl. A single mode cavity microwave synthesizer wi th a continuous irradiation at 2450 MHz and an infrared temperature control system was used.

PAGE 54

54 3.4.2 General Procedure for the Synthesis of N (Cbz dipeptidoyl ) benzotriazoles 1a c These compounds were synthesized following our es tablished procedure. [09S2392 ] Ben zyl((S) 1 (((S) 1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxo 3 phenylpropan 2 yl)amino) 1 oxopropan 2 yl)carbamate ( 3.1a ) White microcrystals (93%); mp 148 150 o C; (Lit mp 148 149 o C ) [11S2995 ]. Benzyl((S) 1 (((S) 1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxo 3 phe nylpropan 2 yl) amino) 3 methyl 1 oxobutan 2 yl)carbamate ( 3.1b ) White microcrystals (93%); mp 188 190 o C; 1 H NMR (CDCl 3 J = 8.7 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.41 7.27 (m, 5H), 7.24 7.17 (m, 3H), 7.13 7.07 (m, 2H), 6.70 (d, J = 7.2 Hz, 1H), 6.30 6.20 (m, 1H), 5.31 (d, J = 8.4 Hz, 1H), 5.17 5.06 (m, 2H), 4.08 (t, J = 7.2 Hz, 1H), 3.47 (dd, J = 14.1, 5.1 Hz, 1H), 3.24 (dd, J = 14.0, 7.7 Hz, 1H), 2 .19 2.02 (m, 1H), 0.94 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.3 Hz, 3H); 13 C NMR (CDCl 3 128.4, 128.3, 127.7, 126.8, 120.6, 114.5, 67.4, 60.4, 54.2, 38.8, 31.1, 19.3, 17.9. Anal. C alcd for C 28 H 29 N 5 O 4 : C, 67.32; H, 5.85; N, 14.02; found: C, 67.44; H, 5.86; N, 14.09. (S) Benzyl(1 ((2 (1H benzo[d][1,2,3]triazol 1 yl) 2 oxoethyl)amino) 1 oxo 3 phenyl propan 2 yl)carbamate ( 3.1c ) White microcrystals (85%); mp 169 171 o C; 1 H NMR (DMSO d 6 ) 8.89 (t, J = 5.5 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.70 7.60 (m, 2H), 7.35 7.19 (m, 10H), 5.08 4.91 (m, 4H), 4.50 4.42 (m, 1H), 3.16 (dd, J = 13.5 3.0 Hz, 1H), 2.88 2.78 (m, 1H); 13 C NMR (DMSO d 6 ) 172.7, 168.5, 155.9, 145.3, 138.1, 137.0, 131.0, 130.6, 129.2, 128.3, 128.1, 127.6,

PAGE 55

55 127.4, 126.6, 126.3, 120.1, 113.7, 65.2, 56.1, 42.7, 37.6 Anal. Calcd for C 25 H 23 N 5 O 4 : C, 65.64; H, 5.07; N, 15.31; found: C, 65.67; H, 5.00; N, 15.10. 3.4.3 General Procedure for the Synthesis of N Cbz tripeptides 3.2a c The compounds were synthesized following our established procedure. [ 11S2995 ] (5S,8S) 8 Benzyl 5 methyl 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oic acid ( 3.2a ) White microcrystals (78%); mp 108 110 o C; 1 H NMR (DMSO d 6 8.33 (m, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.46 7.05 (m, 11H), 5.02 4.97 (m, 2H), 4.60 4.55 (m, 1H), 4.08 4.00 (m, 1H), 3.82 3.74 (m, 2H), 3.12 3.02 (m, 1H), 2.88 2.80 (m, 1H), 1.14 (d, J = 7.2 Hz, 3H)); 13 C NMR (DMSO d 6 172.2, 171.3, 171.1, 155.7, 137.6, 137.0, 129.3, 128.4, 128.0, 127.8, 126.2, 65.5, 53.5, 50.3, 40.7, 37.7, 18.2 Anal. Calcd for C 22 H 25 N 3 O 6 0.5 H 2 O: C, 60.54; H, 6.00; N, 9.63; found: C, 60.89; H, 5.83; N, 9.73. (5S,8S) 8 Benzyl 5 isopropyl 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oic acid ( 3.2b ) White microcrystals (91%); mp 212 215 o C; 1 H NMR (DMSO d 6 ) 8.36 (t, J = 5.7 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.40 7.28 (m, 5H), 7.27 7.11 (m, 6H), 5.02 (br s, 2H), 4.68 4.55 (m, 1H), 3.86 3.71 (m, 3H), 3.02 (dd, J = 13.5, 3.9 Hz, 1H), 2.78 (dd, J = 13.8, 9.9 Hz, 1H), 1.91 1.78 (m, 1H), 0.78 0.64 (m, 6H); 13 C NMR (DMSO d 6 ) 171.4, 171.0, 170.8, 156.0, 137.6, 137.0, 129.2, 128.4, 128.0, 127.8, 127.7, 126.2, 6 5.5, 60.4, 53.5, 40.7, 37.8, 30.4, 19.2, 18.1 Anal. Calcd for C 24 H 29 N 3 O 6 : C, 63.28; H, 6.42; N, 9.22; found: C, 63.48; H, 6.47; N, 9.20. (5S,11S) 5 Benzyl 11 isobutyl 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oic acid ( 3.2c ) White microcrystals (80%); mp 79 81 o C; 1 H NMR (DMSO d 6 ) 8.34 8.30 (m, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.33 7.19 (m, 10H), 4.99 4.90 (m, 2H), 4.32 4.22 (m, 2H), 3.77 (d, J = 7.8 Hz, 2H), 3.08 3.00 (m, 1H), 2.80 2.71 (m, 1H), 1.68 1.60 (m, 1H), 1.55 1 .50 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H)., 0.85 (d, J

PAGE 56

56 = 6.3 Hz, 3H); 13 C NMR (DMSO d 6 ) 174.0, 171.9, 168.6, 156.0, 138.2, 137.0, 129.2, 128.3, 128.1, 127.7, 127.5, 126.3, 65.3, 56.3, 50.2, 41.8, 40.3, 40.1, 37.3, 24.2, 22.8, 21.4. Anal. Calcd for C 25 H 31 N 3 O 6 : C, 63.95; H, 6.65; N, 8.95; found: C, 64.06; H, 6.75; N, 8.98. 3.4.4 General Procedure for the Synthesis of N (Cbz tripeptidoyl ) benzotriazole s 3.3a c The compound s were synthesized following the recent established procedure in our group [11S2995 ] Benzyl ((S) 1 (((S) 1 ((2 (1H benzo[d][1,2,3]triazol 1 yl) 2 oxoethyl)amino) 1 oxo 3 phenylpropan 2 yl)amino) 1 oxopropan 2 yl)carbamate ( 3.3a ) White microcrystals (92%); mp 182 185 o C; 1 H NMR (DMSO d 6 8.81 (m, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.25 8.20 (m, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H), 7.36 7.30 (m, 4H), 7.27 7.23 (m, 4H), 7.20 7.16 (m, 2H), 5.02 4.98 (m, 4H), 4.70 4.68 (m,1H), 4.10 4.00 (m 1H), 3.18 3.10 (m, 1H), 2.96 2.78 (m, 1H), 1.14 (d, J = 6.9 Hz, 3H); 13 C NMR (DMSO d 6 145.2, 137.5, 131.0, 130.5, 129.2, 128.3, 128.0, 127.7, 126.6, 126.2, 120.1, 113.7, 65.4, 53.5, 50.2, 42.6, 37.7, 18.2. Anal. Calcd for C 28 H 28 N 6 O 5 : C, 63.63; H, 5.34; N, 15.90; found: C, 63.22; H, 5.37; N, 15.79. Benzyl((S) 1 (((S) 1 ((2 (1H benzo[d][1,2,3]triazol 1 yl) 2 oxoethyl)amino) 1 oxo 3 phenyl propan 2 yl)amino) 3 methyl 1 oxobutan 2 yl)carbamate ( 3.3b ) White microcrystals (93%); mp 214 215 o C; 1 H NMR (DMSO d 6 ) 8.84 (br s, 1H), 8.29 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.25 7.15 (m, 11H), 5.10 4.90 (m, 4H), 4.85 4.68 (m, 1H), 3.84 (t, J = 7.5 Hz, 1H), 3.19 3.05 (m, 1H), 2.95 2.80 (m, 1H), 1.98 1.79 (m, 1H), 0.80 0.60

PAGE 57

57 (m, 6H); 13 C NMR (DMSO d 6 ) 172.1, 170.9, 168.4, 156.0, 145.3, 137, 6, 137.0, 131.0, 130.6, 129.2, 128.3, 128.0, 127.6, 126.2, 120.1, 113.7, 65.4, 60.4, 53.4, 42.6, 37.9, 30.4, 19.1, 18.1. Anal. Calcd for C 30 H 32 N 6 O 5 : C, 64.73; H, 5.79; N, 15.10; found: C, 64.60; H, 5.79; N, 15.01. Benzyl ((S) 1 ((2 (((S) 1 (1H Benzo[d][1,2,3]triazol 1 yl) 4 methyl 1 oxopentan 2 yl)amino) 2 oxoethyl)amino) 1 oxo 3 phenylpropan 2 yl)carbamate ( 3.3c ) White microcrystals (84%); mp 144 146 o C; 1 H NMR (D MSO d 6 ) 8.81 8.77 (m, 1H), 8.42 8.38 (m, 1H), 8.28 (d, J = 7.8 Hz, 1H), 8.20 (d, J = 7.2 Hz, 1H), 8.10 8.06 (m, 1H), 7.80 7.77 (m, 1H), 7.65 7.59 (m, 1H), 7.39 7.12 (m, 10H), 4.94 (s, 2H), 4.51 4.45 (m, 1H), 4.29 4.25 (m, 1H), 3.88 3.74 (m, 2H), 3.08 3.0 3 (m, 1H), 2.80 2.72 (m, 1H), 1.80 1.65 (m, 1H), 1.60 1.50 (m, 2H), 0.99 0.87 (m, 6H); 13 C NMR (DMSO d 6 ) 173.7, 172.6, 169.3, 169.1, 156.6, 146.0, 138.9, 137.6, 131.7, 129.9, 129.0, 128.7, 128.4, 128.1, 127.3, 126.9, 120.9 114.5, 65.9, 56.9, 51.5, 43.2, 42.8, 38.0, 24.8, 23.8, 22.2. Anal. Calcd for C 31 H 34 N 6 O 5 : C, 65.25; H, 6.01; N, 14.73; found: C, 65.21; H, 5.85; N, 14.56. 3.4.5 General Procedure for the Synthesis of N Cbz tetrapeptides 3.4a c The compound s were synthesized following the recent established procedure in our group [11S2995 ] (5S,8S,14S) 8 Benzyl 5,14 dimethyl 3,6,9,12 tetraoxo 1 phenyl 2 oxa 4,7,10, 13 tetraazapentadecan 15 oic acid ( 3.4a ) White microcrystals (74%); mp 184 186 o C; 1 H NMR (DMSO d 6 J = 6.0 Hz, 1H), 8.08 (d, J = 6.9 Hz, 1H), 7.98 (d, J = 6.9 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.36 7.30 (m, 5H), 7.24 7.16 (m, 5H), 5.08 4.94 (m 2H), 4.52 4.42 (m, 1H), 4.21 (quintet, J = 7.2 Hz, 1H), 4.08 3.96 (m, 1H), 3.73 (d, J = 5.7 Hz, 2H), 3.04 (dd, J = 13.5, 4.2 Hz, 1H), 2.88 2.78 (m, 1H), 1.28 (d, J = 7.2 Hz, 3H), 1.12 (d, J = 7.2 Hz, 3H); 13 C NMR (DMSO d 6 55.6, 137.6, 136.9,

PAGE 58

58 129.2, 128.3, 128.0, 127.7, 126.2, 65.4, 53.9, 50.1, 47.5, 41.7, 37.3, 18.0, 17.3 Anal. Calcd for C 25 H 30 N 4 O 7 : C, 60.23; H, 6.07; N, 11.24; found: C, 59.95; H, 6.04; N, 11.07. (5S,8S) 8 Benzyl 5 methyl 3,6,9,12 tetraoxo 1 p henyl 2 oxa 4,7,10,13 tetraazapenta decan 15 oic acid ( 3.4b ) White microcrystals (85%); mp 148 150 o C; 1 H NMR (DMSO d 6 ) 8.88 (t, J = 5.4 Hz, 1H), 8.68 (t, J = 5.8 Hz, 1H), 8.57 (d, J = 7.5 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.04 7.94 (m, 5H), 7.88 7.78 (m 5H), 5.63 (d, J = 12.6 Hz, A part of AB system, 1H), 5.57 (d, J = 12.9 Hz, B part of AB system, 1H), 5.16 5.04 (m, 1H), 4.68 4.57 (m, 1H), 4.38 4.24 (m, 4H), 3.66 3.61 (m, 1H), 3.46 3.22 (m, 1H), 1.71 (d, J = 6.9 Hz, 3H); 13 C NMR (DMSO d 6 ) 172.4, 171.1 171.1, 169.0, 155.7, 137.6, 136.9, 129.3, 128.3, 128.0, 127.8, 126.2, 65.5, 53.8, 50.2, 41.8, 40.6, 37.4, 18.1. Anal. Calcd for C 24 H 28 N 4 O 7 : C, 59.50; H, 5.82; N, 11.56; found: C, 59.12; H, 5.84; N, 11.88. (5S,11S) 5 Benzyl 11 isobutyl 3,6,9,12 tetraoxo 1 phenyl 2 oxa 4,7,10,13 tetraaza pentadecan 15 oic acid ( 3.4c ) White microcrystals (88%); mp 109 110C; 1 H NMR (DMSO d 6 8.26 (m, 2H), 7.94 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.35 7.17 (m, 10H), 5.10 4.90 (m, 2H), 4.38 4.32 (m, 1H), 4.30 4.23 (m,1H), 3.80 3.72 (m, 4H), 3.10 3.00 (m, 1H), 2.80 2.70 (m, 1H), 1.65 1.60 (m, 1H), 1.51 1.45 (m, 2H), 0.88 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H); 13 C NMR (DMSO d 6 ) 172.3, 171.8, 171.1, 168.4, 155.9, 138.2, 136.9, 129.2, 128.3, 128.0, 127.7, 127.4, 126.2, 65.3, 56.2, 50.7, 42.0, 41.0, 40.6, 37.3, 24.1, 23.1, 21.6. Anal. Calcd for C 27 H 34 N 4 O 7 : C, 61.58; H, 6.51; N, 10.64; found: C, 61.69; H, 6.62; N, 10.48. 3.4.6 General Procedure for the Synthesis of N (Cbz tetrapeptidoyl ) benzotriazole 3.5a c The compounds were synthesized following ou r established procedure. [11S2995 ] (Table 3 1)

PAGE 59

59 Benzyl ((S) 1 ((2 (((S) 1 ((2 (1H benzo[d][1,2,3]triazol 1 yl) 2 oxoethyl)amino) 4 methyl 1 oxopentan 2 yl)amino) 2 oxoethyl)amino) 1 oxo 3 phenylpropan 2 yl) carbamate ( 3.5c ) White microcrystals (79%); mp 194 195C; 1 H NMR (DMSO d 6 ) 8.80 8.70 (m, 1H), 8.35 8.30 (m, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.79 (t, J = 7.2 Hz, 1H), 7.66 7.55 (m, 2H), 7.33 7.19 (m, 10H), 4.97 4.90 (m, 4H), 4.55 4.46 (m, 1H), 4.32 4.22 (m, 1H), 3.89 3.72 (m, 2H), 3.08 3.02 (m, 1H), 2.80 2.50 (m, 1H), 1.70 1.66 (m, 1H), 1.60 1.56 (m, 2H), 0.92 (d, J = 6.3 Hz, 3H), 0.89 ( d, J = 6.3 Hz, 3H); 13 C NMR (DMSO d 6 145.2, 138.1, 136.9, 131.0, 130.5, 129.2, 128.2, 128.0, 127.6, 127.4, 126.6, 126.2, 120.1, 113.7, 65.2, 56.2, 50.7, 42.5, 42.0, 41.0, 37.3, 24.1, 23.1, 21.6. Anal. Calcd for C 33 H 37 N 7 O 6 : C, 63.14; H, 5.94; N, 15.62; found: C, 62.88; H, 5.92; N, 15.46. 3.4.7 General Procedure for O Acylation: Synthesis of Compounds 3.6a f A dried heavy walled Pyrex tube containing a small stir bar was charged with benzotriazole intermediate 3.3a c or 3.5b c (1 eq uiv .), O nucleophile (1.5 eq uiv .) and base DMAP (0.1 eq uiv .) dissolved in anhydrous THF. The reaction mixture was exposed to microwave irradiation (100W) at 70C for specified times. Each mixture was allowed to cool through an inbuilt system until the temperature had fallen below 30C (ca. 10 min). The reaction mixture was quenched with water, extracted with ethyl acetate (EtOAc) and the extracts were washed with (10%) sodium carbonate solution ( Na 2 CO 3 ) and water and dried over anhydrous magnesium sulf ate ( MgSO 4 ) The solvent was removed under reduced pressure, and the residue was subjected to silica gel column using a mixture of EtOAc/hexanes as an eluent to give the corresponding compound s 3.6a f

PAGE 60

60 (5S,8S) (8S,9S,10R,13R,14S,17R) 10,13 Dimethyl 17 ((R) 6 methylheptan 2 yl) 2,3,4,7,8,9,10,11,12,13,14,15,16,17 tetradecahydro 1H cyclopenta[a]phenanthren 3 yl 8 benzyl 5 methyl 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oate ( 3.6a ) White microcrystals (45%); mp 162 163 o C; 1 H NMR (CDCl 3 ) 7.40 7.30 (m, 5H), 7.29 7.15 (m, 5H), 6.76 (d, J = 7.2 Hz, 1H), 6.70 6.60 (m,1H), 5.38 5.34 (m,1H), 5.27 (d, J = 6.6 Hz, 1H), 5.14 5.00 (m, 2H), 4.80 4.70 (m,1H), 4.66 4.58 (m, 1H), 4.22 4.16 (m, 1H), 4.08 3.98 (m, 1H), 3.90 3.80 (m, 1H), 3.22 3.10 (m, 1H ), 3.09 3.00 (m, 1H), 2.30 (d, J = 7.8 Hz, 2H), 2.01 1.93 (m, 2H), 1.90 1.78 (m, 4H), 1.62 1.40 (m, 8H), 1.36 1.28 (m, 2H), 1.27 (d, J = 7.2 Hz, 6H), 1.17 1.07 (m, 4H), 1.06 1.00 (m, 6H), 0.91 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 7.5 Hz, 6H), 0.67 (s, 3H); 13 C NMR (CDCl 3 ) 172.4, 171.0, 169.0, 156.4, 139.5, 136.6, 136.2, 129.4, 128.8, 128.5, 128.3, 127.2, 123.2, 75.6, 67.4, 56.9, 56.4, 54.3, 51.1, 50.2, 42.5, 41.7, 39.9, 39.7, 38.2, 37.1, 36.8, 36.4, 36.0, 32.1, 29.9, 28.4, 28.2, 27.9, 24.5, 24.1, 23.0, 22.8, 21.2, 19.5, 18.9, 18.4, 12.1. HRMS Calcd for C 49 H 69 N 3 O 6 Na [M+Na] + 818.5089; found 818.5089. (5S,8S) (E) 3,7 Dimethylocta 2,6 dien 1 yl 8 benzyl 5 methyl 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oate ( 3.6b ) White microcrystals (50%); mp 114 116 o C; 1 H NMR (CDCl 3 ) 7.36 7.28 (m, 5H), 7.26 7.12 (m, 5H), 7.02 (d, J = 7.5 Hz, 1H), 6.92 6.82 (m, 1H), 5.58 (d, J = 7.2 Hz, 1H), 5.30 (t, J = 7.2 Hz, 1H), 5.12 5.00 (m, 3H), 4.80 (q, J = 7.5 Hz, 1H), 4.59 (d, J = 7.5 Hz, 2H), 4.30 4.20 (m, 1H), 4.08 3.80 (m, 2H), 3.13 (dd, J = 13.8, 6.6 Hz, 1H), 3.01 (dd, J = 13.8, 7.2 Hz, 1H), 2.14 2.00 (m, 4H), 1.74 (s, 3H), 1.67 (s, 3H), 1.58 (s, 3H), 1.26 (d, J = 6.9 Hz, 3H); 13 C NMR (CDCl 3 ) 172.7, 171.2, 169.6, 156.3, 143.4, 136.6, 136.3, 132.4, 129.4, 128.7, 128.6, 128.3, 128.2, 127.0, 123.6, 118.7, 67.2, 62.1, 54.2, 50.9, 41.4, 38.3, 32.3, 26.7, 25.8, 23.6,

PAGE 61

61 18.6, 17.8. Anal. Calcd for C 32 H 41 N 3 O 6 : C, 68.18; H, 7.33; N, 7.45; found: C, 68.28; H, 7.57; N, 7.50. (5S,8S) ((3aR,5R,5aS,8aS,8bR) 2,2,7,7 Tetramethyltetrahydro 3aH bis([1,3] dioxolo) [4,5 b:4',5' d]pyran 5 yl)methyl 8 benzyl 5 isopropyl 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oate ( 3.6c ) White microcrystals (47%); mp 129 131 o C; 1 H NMR (CDCl 3 ) 7.45 7.30 (m, 5H), 7.28 7.15 (m, 5H), 6.68 6.58 (m, 2H), 5.53 (d, J = 4.8 Hz, 1H), 5.27 (d, J = 7.5 Hz, 1H), 5.14 5.03 (m, 2H), 4.82 4.71 (m, 1H), 4.62 (dd, J = 8.0, 2.3 Hz, 1H), 4.35 4.19 (m, 4H), 4.11 3.88 (m, 4H), 3.20 3.10 (m, 1H), 3.08 2.98 (m, 1H ), 2.14 2.00 (m, 1H), 1.51 (s, 3H), 1.43 (s, 3H), 1.33 (s, 6H), 0.88 (d, J = 6.9 Hz, 3H), 0.77 (d, J = 6.6 Hz, 3H); 13 C NMR (CDCl 3 ) 171.3, 171.0, 169.4, 156.7, 136.6, 136.2, 129.4, 128.8, 128.8, 128.5, 128.4, 127.2, 109.9, 109.0, 96.4, 71.1, 70.8, 70.6, 67.5, 66.0, 64.4, 60.9, 54.2, 41.5, 38.2, 30.8, 26.3, 26.1, 25.1, 24.7, 19.4, 17.7. Anal. Calcd for C 36 H 47 N 3 O 11 : C, 61.97; H, 6.79; N, 6.02; found: C, 61.78; H, 6.94; N, 5.91. (5S,8S) (3aR,5R,6R,6aR) 5 ((R) 2,2 Dimethyl 1,3 dioxolan 4 yl) 2,2 dimethyltetra hydrofuro[2,3 d][1,3]dioxol 6 yl 8 benzyl 5 methyl 3,6,9,12 tetraoxo 1 phenyl 2 oxa 4,7,10,13 tetraazapentadecan 15 oate ( 3.6e ) White microcrystals (58%); mp 102 104 o C; 1 H NMR (CDCl 3 ) 7.40 7.29 (m, 7H), 7.26 7.09 (m, 6H), 5.86 (d, J = 3.6 Hz, 1H), 5.7 6 (d, J = 6.1 Hz, 1H), 5.27 5.25 (m, 1H), 5.10 5.00 (m, 2H), 4.78 (q, J = 7.2 Hz, 1H), 4.49 (d, J = 3.6 Hz, 1H), 4.40 4.34 (m, 1H), 4.24 4.1.8 (m, 2H), 4.16 3.99 (m, 4H), 3.98 3.80 (m, 2H), 3.12 2.95 (m, 2H), 1.50 (s, 3H), 1.38 (s, 3H), 1.30 1.25 (m, 9H), 0.92 0.82 (m, 1H); 13 C NMR (CDCl 3 ) 173.1, 171.4, 169.4, 168.8, 156.5, 136.5, 136.3, 129.4, 128.8, 128.5, 128.2, 127.3, 112.5, 109.6, 105.2, 83.3, 79.8, 72.5, 67.4, 54.8,

PAGE 62

62 51.1, 43.0, 41.4, 38.3, 27.1, 26.9, 26.4, 25.4, 18.6. Anal. Calcd for C 36 H 46 N 4 O 12 : C, 59.49; H, 6.38; N, 7.71; found: C, 59.26; H, 6.56; N, 7.35. (5S,11S) (1S,2S) 2 Isopropyl 5 methylcyclohexyl 5 be nzyl 11 isobutyl 3,6,9,12 tetra oxo 1 phenyl 2 oxa 4,7,10,13 tetraazapentadecan 15 oate ( 3.6f ) White microcrystals (42%); mp 104 106 o C; 1 H NM R (CDCl 3 ) 7.40 7.06 (m, 13H), 5.79 (d, J = 6.6 Hz, 1H), 5.06 (d, A part of AB system, J = 12 .0 Hz, 1H), 4.98 (d, B part of AB system, J = 12.3 Hz, 1H), 4.76 4.51 (m, 3H), 4.04 3.83 (m, 4H), 3.14 (dd, J = 15.0, 5.4 Hz, 1H), 3.00 2.92 (m, 1H), 2.18 2.10 ( m, 1H), 1.98 1.91 (m, 1H), 1.89 1.78 (m, 1H), 1.68 1.50 (m, 5H), 1.49 1.31 (m, 2H), 1.03 0.88 (m, 14), 0.71 (d, J = 6.9 Hz, 3H); 13 C NMR (CDCl 3 ) 172.5, 172.3, 169.7, 169.1, 156.5, 136.6, 136.2, 129.4, 128.8, 128.7, 128.4, 128.2, 127.2, 75.9, 67.3, 56.5, 51.9, 47.1, 43.4, 41.6, 41.2, 40.9, 38.4, 34.3, 31.5, 26.4, 24.9, 23.6, 23.1, 22.2, 22.1, 20.9, 16.5. HRMS Calcd for C 37 H 52 N 4 O 7 Na [M+Na] + : 687.3728; found: 687.3743. 3.4.8 General Procedure for S Acylation: Synthesis of Compounds 3.7a d Mercapto nucleophile (1 eq uiv .) was dissolved in THF and triethylamine (1.5 eq uiv .). Benzotriazole intermediate (1 eq uiv .) was added to the solution and the mixture was stirred at room temperature for 1 2 h. The mixture was then acidified with 6N HCl, concentrated under reduced pressure then diluted with ethyl acetate. The organic layer was washed with 6N HCl and dried over anhydrous MgSO 4 filtered and evaporated to give the desired compound 3.7a d (5R,8R) S Phenyl 8 benzyl 5 methyl 3,6,9 triox o 1 phenyl 2 oxa 4,7,10 triazado de cane 12 thioate ( 3.7a ) White microcrystals (78%); mp 146 147 o C; 1 H NMR (DMSO d 6 ) 8.89 8.86 (m, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.47 7.25 (m, 16H), 5.02 4.90 (m, 2H), 4.63 4.59 (m, 1H), 4.13 4.05 (m, 2H), 4.06 4.01 (m, 1H), 3.19 3.08 (m, 1H),

PAGE 63

63 2.91 2.82 ( m, 1H), 1.12 (d, J = 6.6 Hz, 3H).; 13 C NMR (DMSO d 6 ) 196.2, 172.3, 171.9, 155.6, 137.6, 137.0, 134.5, 129.5, 129.4, 129.2, 128.3, 128.1, 127.7, 126.9, 126.3, 65.4, 53.7, 50.2, 48.8, 37.3, 18.2.Anal. Calcd for C 28 H 29 N 3 O 5 S.H 2 O: C, 62.55; H, 5.81; N, 7.82; found: C, 62.85; H, 5.49; N, 7.46. (5S,8S) Methyl 8 benzyl 5 methyl 3,6,9,12 tetraoxo 1 phenyl 2 oxa 13 thia 4,7,10 triazapentadecan 15 oate ( 3.7b ) White powder (83%); mp 158 160 C; 1 H NMR (DMSO d 6 8.82 (m, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.42 7.18 (m, 11H), 5.03 4.99 (m, 2H), 4.62 4.59 (m, 1H), 4.09 3.98 (m, 3H), 3.76 (s, 2H), 3.64 (s, 3H), 3.14 3.06 (m, 1H), 2.91 2.85 (m, 1H), 1.14 (d, J = 6.9 Hz, 3H); 13 C NMR (DMSO d 6 171.9, 168.9, 155.6, 137.6, 137.0, 129.2, 128.3, 128.1, 127.7, 126.3, 65.4, 53.7, 52.4, 50.1, 48.5, 37.3, 30.2, 18.2. Anal. Calcd for C 25 H 29 N 3 O 7 S: C, 58.24; H, 5.67; N 8.15; found: C, 58.08; H, 5.63; N, 8.04. 3.4.9 General Procedure for N Acylation: Synthesis of Compounds 3.8a, 3.8d N nucleophile (1 eq uiv .) was dissolved in THF and triethylamine (1.5 eq uiv .). The benzotriazole intermediate (1 eq uiv .) was added to the solution and the mixture was stirred at room temperature for 1 h. The mixture was acidified with 6N HCl, concentrated and then diluted with ethyl acetate. The organic layer was washed with 6N HCl and dried over anhydrous MgSO 4 filtered and evaporated to give the desired compounds 3.8a 3.8d Benzyl ((S) 1 (((S) 1 ((2 ((3 (1H imidazol 1 yl)propyl)amino) 2 oxoethyl)amino) 1 oxo 3 phenylpropan 2 yl)amino) 1 oxopr opan 2 yl)carbamate ( 3.8a ) White microcrystals (75%); mp 134 137 o C; 1 H NMR (DMSO d 6 ) 8.30 8.20 (m, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.78 7.72 (m, 1H), 7.61 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.38 7.30 (m, 5H), 7.23 7.20 (m, 5H), 7.16 (s, 1H), 6.88 (s, 1H) 5.02 4.88 (m, 2H), 4.52 4.40 (m, 1H),

PAGE 64

64 4.08 3.90 (m, 3H), 3.79 3.68 (m, 1H), 3.66 3.54 (m, 1H), 3.08 2.99 (m, 3H), 2.92 2.78 (m, 1H), 1.92 1.78 (m, 2H), 1.12 (d, J = 6.9 Hz, 3H); 13 C NMR (DMSO d 6 ) 172.7, 171.2, 168.6, 155.7, 137.6, 137.3, 136.9, 129.2, 128.3, 128.0, 127.7, 126.2, 119.3, 65.5, 54.2, 50.2, 43.4, 42.2, 37.1, 35.7, 30.7, 17.9. Anal. Calcd for C 28 H 34 N 6 O 5 : C, 62.91; H, 6.41; N 15.72; found: C, 62.56; H, 6.46; N, 15.52. Benzyl ((S) 1 ((2 (((S) 4 m ethyl 1 ((2 (4 methylpiperazin 1 yl) 2 oxoethyl)amino) 1 oxopentan 2 yl)amino) 2 oxoethyl)amino) 1 oxo 3 phenylpropan 2 yl)carbamate ( 3.8d ) White microcrystals (68%); mp 181 183 o C; 1 H NMR (DMSO d 6 ) 8.29 (t, J = 5.7 Hz, 1H), 8.01 (t, J = 5.3 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.34 7.18 (m, 10H), 4.95 4.89 (m, 2H), 4.42 4.34 (m, 1H), 4.30 4.22 (m, 1H), 3.91 (d, J = 5.4 Hz, 2H), 3.76 (t, J = 5.7 Hz, 2H), 3.41 3.36 (m, 4H), 3.04 (dd, J = 13.6, 3.9 Hz, 1H), 2.75 (dd, J = 13 .5, 11.1 Hz, 1H), 2.27 2.22 (m, 4H), 2.15 (s, 3H), 1.65 1.58 (m, 1H), 1.51 1.45 (m, 2H), 0.87 (dd, J = 6.3 Hz, 3H), 0.83 (d, J = 6.3 Hz, 3H); 13 C NMR (DMSO d 6 ) 172.0, 171.8, 168.5, 166.6, 156.0, 138.2, 137.0, 129.2, 128.3, 128.1, 127.7, 127.5, 126.3, 65. 3, 56.3, 54.6, 54.2, 51.0, 45.7, 43.9, 42.1, 41.3, 41.0, 37.3, 24.1, 23.1, 21.6. Anal. Calcd for C 32 H 44 N 6 O 6 : C, 63.14; H, 7.29; N 13.81; found: C, 62.84; H, 7.38; N, 13.68. 3.4.10 General Procedure for N Acylation: Synthesis of Compounds 3.8b c A dried heavy walled Pyrex tube containing a small stir bar was charged with benzotriazole intermediate (1 eq uiv .) and N nucleophile (1 eq uiv .) dissolved in DMF. The reaction mixture was exposed to microwave irradiation (60W) for temperature of 65C for specified times. Each mixture was allowed to cool through an inbuilt system until the temperature had fallen below 30C (ca. 10 min). Each reaction mixture was

PAGE 65

65 quenched with water and the solid obtained was filtered and washed with 10% Na 2 CO 3 and water to give the d esired compounds 3.8b c Benzyl ((S) 3 methyl 1 oxo 1 (((S) 1 oxo 1 ((2 oxo 2 (pyridin 2 ylamino)ethyl) amino) 3 phenylpropan 2 yl)amino)butan 2 yl)carbamate ( 3.8b ) White microcrystals (68%); mp 214 215 o C; 1 H NMR (DMSO d 6 8.30 (m, 2H ), 8.05 (d, J = 7.8 Hz, 2H), 7.82 7.72 (m, 1H), 7.50 7.07 (m, 12H), 4.92 5.10 (m, 2H), 4.67 4.58 (m, 1H), 4.10 3.90 (m, 2H), 3.86 3.78 (m, 1H), 3.18 3.00 (m, 1H), 2.90 2.75 (m, 1H), 1.97 1.78 (m, 1H), 0.80 0.60 (m, 6H); 13 C NMR (DMSO d 6 8.3, 156.0, 151.7, 148.0, 138.2, 137.7, 137.0, 129.2, 128.3, 128.0, 127.6, 126.2, 119.4, 113.4, 65.5, 60.4, 53.6, 42.7, 37.7, 30.4, 19.1, 18.0. HRMS Calcd for C 29 H 34 N 5 O 5 [M+H] + 532.2554; found 532.2572. Benzyl ((S) 1 (((S) 1 ((2 (((S) 1 ((6 methoxybe nzo[d ]thiazol 2 yl)amino) 1 oxo propan 2 yl)amino) 2 oxoethyl)amino) 1 oxo 3 phenylpropan 2 yl)amino) 1 oxopropan 2 yl)carbamate ( 3.8c ) White microcrystals (60%); mp 148 150 o C; 1 H NMR (DMSO d 6 8.22 (m, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.58 7.52 (m, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.36 7.30 (m, 5H), 7.26 7.18 (m, 6H), 7.06 6.90 (m, 1H), 5.02 4.92 (m, 2H), 4.58 4.42 (m, 2H), 4.01 (t, J = 7.2 Hz, 1H), 3.84 3.62 (m, 5H), 3.08 2.98 (m, 1H), 2.88 2.68 (m, 1H), 1.35 (d, J = 7.2 Hz, 3H), 1.11 (d, J = 7.2 Hz, 3H); 13 C NMR (DMSO d 6 ) 172.1, 172.0, 171.2, 168.7, 156.1, 142.6, 137.6, 137.0, 132.8, 129.2, 128.3, 128.0, 127.7, 126.2, 121.1, 114.9, 104.7, 65.5, 55.6, 53.9, 50.7, 48.6, 41.7, 37.3, 22.7, 18.1, 17.6 Anal. Calcd for C 33 H 36 N 6 O 7 S: C, 59.99; H, 5.49; N 12.72; found: C, 59.61; H, 5.50; N, 12.50.

PAGE 66

66 3.4.11 General Procedure for C acylation: Synthesis of Compounds 3. 9a b A solution of benzotriazole intermediate 3. 3a 3. 5c (1.0 eq uiv .), C nucleophile (1.0 eq uiv ) and DMAP (1.0 eq uiv .) in THF (5 mL), were added to a dried heavy walled Pyrex tube with a small stir bar. This reaction mixture was expos ed to microwave irradiation (70 C, 50W) for specified time with a simultaneous cooling. After the reaction was done (monitored by TLC), the THF was evaporated and the residue was acidified then extracted with ethyl acetate. The solvent was removed under reduced pressure and the residue was purified by recrystallization from ethyl acetate to give the corresponding product s 3. 9a b Benzyl ((S) 1 (((S) 1 ((2 (4,4 dimethyl 2,6 dioxocyclohexylidene) 2 hydroxyethyl) amino) 1 oxo 3 phenylpropan 2 yl)amino) 1 oxopropan 2 yl)carbamate ( 3. 9a ). White microcrystals (69%); mp 1 35 1 37 o C; 1 H NMR (DMSO d 6 ) 8.78 8.60 (m, 1H), 8.40 8.26 (m, 1H), 7 .84 7.55 (m, 11H), 5.38 (br s, 2H), 5.05 4.82 (m, 2H), 4.94 4.81 (m, 2H), 4.43 4.40 (m, 1H), 4.20 4.16 (m, 1H), 3.45 3.38 (m, 1H), 3.26 3.18 (m, 1H), 2.89 (s, 4H), 1.50 (d, J = 7.5 Hz 3H), 1.40 1.30 (m, 6H) ; 13 C NMR (DMSO d 6 171.3, 171.1, 155.5, 137.6, 136.9, 129.2, 128.3, 127.9, 127.7, 126.2, 111.1, 65.4, 53.5, 50.2, 47.5, 37.7, 30.7, 27.5, 18.1. HRMS Calcd for C 30 H 35 N 3 O 7 Na [M+Na] + 572.2367; found 572.2377. Benzyl ((S) 1 ((2 (((S) 1 ((2 (2,6 dioxocyclohexyl) 2 oxoethyl)amino) 4 me thyl 1 oxopentan 2 yl)amino) 2 oxoethyl)amino) 1 oxo 3 phenylpropan 2 yl)carbamate ( 3. 9b ). White microcrystals (61 %); mp 188 190 o C; 1 H NMR (DMSO d 6 ) 8.37 8.26 (m, 1H), 8.22 8.15 (m, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.36 7.18 (m, 10H), 5.02 4.88 (m, 2H), 4.50 4.20 (m, 4H), 3.88 3.67 (m, 2H), 3.07 2.98 (m, 1H), 2.80 2.65 (m, 2H), 2.55 2.27 (m, 3H), 1.95 1.44 (m, 5H), 0.89 (d, J = 6.0 Hz, 3H), 0.86 (d, J = 6.3

PAGE 67

67 Hz, 3H) ; 13 C NMR (DMSO d 6 ) 201.1, 172.3, 1 71.8, 168.5, 155.9, 138.2, 136.9, 129.2, 128.3, 128.0, 127.7, 127.4, 126.2, 112.1, 65.2, 56.2, 50.8, 47.6, 42.0, 41.0, 37.3, 24.1, 23.1, 21.6, 18.7. HRMS Calcd for C 33 H 41 N 4 O 8 [M+H] + 621.2919; found 629.2931.

PAGE 68

68 CHAPTER 4 MICROWAVE ASSISTED FORMAT ION OF PEPTIDE VITAMIN CONJUGATES 4.1 Introductory Remarks Peptides are of considerable interest as therapeutic agents. However, few possess physicochemical characteristics that allow their diffusion through the lipid bilayer surrounding a cell. [87AB99, 86DSPD13 9] The resulting low oral availability, as well as rapid biliary clearance can represent major shortcomings in the development of peptide based drugs. Common approaches to circumvent these limitations involve the use of absorption enhancers [92ADDR39], enz yme inhibitors [90JCR213], and complex emulsion systems [92ADDR253]. Over the past decades, new uptake pathways involving vitamins covalently bound to peptides were examined. Water soluble vitamins are known to be usually transported into cells via a potoc ytosis process. [90JCB637] Zhang and McCormick have proposed the use of vitamin B 6 in a co delivery strategy. In fact, having a receptor mediated transport in eukaryotic cells, with the ability to accept the amine of peptide vitamin conjugate, facilitates the cell uptake of peptide and its transport into the cytosol. [91PNAS10407, 04BC312] Moreover, cobalamin (vitamin B 12 ) another vitamin with a unique uptake system has received an increased attention. In this case, studies showed that the orally administe red peptide, which was covalently coupled to that vitamin, followed the dietary uptake pathway of B 12 and generated the highest level of peptide absorption. [00PR825, 09ACIE1022] Biotin (vitamin H) ( 4.1b ) was evaluated in a similar concept to the above, as part of a vitamin cloaking strategy; Vitamin H covalently bound to peptidic HIV 1 protease inhibitors caused an increase in serum concentration, suggesting an improvement in the resistance to clearance. [94JMC 293]

PAGE 69

69 In the present study, we attempt to couple amino acids or peptides to vitamins B 3 ( 4.1a ), H ( 4.1b ), E ( 4.7 ) and D 3 ( 4.10 ). (Figure 4 1) These vitamins were chosen to provide a range of different hydro/lipophilicity. The hydrophilic vitamins B 3 ( 4.1a ) and H ( 4.1b ) should increase the solubili ty of the bioconjugate in a physiological medium, whereas the lipophilic vitamins E ( 4.7 ) and D 3 ( 4.10 ) could ease absorption through membranes and facilitate transport into cells. [94IJC529, 02CMC683] These vitamins can all easily be incorporated into pep tides via amide or ester linkages. Figure 4 1. S tructure s of the four vitamins used The importance of the vitamin H ( 4.1b ) is due to its strong noncovalent interaction with (strept)avidin, [03JACS3452] as shown by enzymati c reactions, radiolabeling techniques, and fluorescence assays. [06BC261] Recently, novel conjugates of biotin with amino acids, exhibiting remarkable gelation properties in aqueous media, were synthesized. Preliminary studies revealed the biotinyl hydroge l potential as efficient biomaterials that can ac t as drug carriers. [05OL1741] Tocopherol (vitamin E) ( 4.7 ) is the most active lipophilic antioxidant in biological membranes. It is mainly used in the skin protection against oxidative damage. Its couplin g with amino acids resulted in the formation of pro vitamins with an ester linkage.

PAGE 70

70 The purpose was to combine antioxidant and moisturizing effect on skin. [04JCR403, 09JPS2364] Studies showed that when attached to a taxol based drug, this vitamin exhibits a therapeutic efficiency higher than the known taxol based antitumor compounds due to its better solubility in fats and better retention in membranes. [10RCB241] Furthermore, Wang et al. observed the suppression of breast cancer cells. [07CR67] We have ca rried out the preparation of a variety of peptide conjugates using benzotriazole methodology. [ 11CBDDxxx ] Herein, we are interested in expanding this approach and couple amino acids and peptides with vitamins, in an efficient way and under microwave irradi ation. This technique was applied as an alternative way to conventional heating in order to shorten reaction times [02ACR717] and hence minimize any possible epimerization resulting from prolonged exposure to heat. We believe the present work represents th e first microwave assisted formation of peptide vitamin bioconjugates. 4.2 Results and Discussions 4.2.1 Preparation of Benzotriazole Derivatives of Niacin and Biotin 4.3a b Niacin (vit B 3 ) ( 4.1a ) and Biotin (vitamin H) ( 4.1b ) were activated by treatment with 1 (methylsulfonyl) 1 H benzotriazole (BtSO 2 Me) ( 4.2 ) under microwave irradiation at 70C and 50W irradiation power in the presence of triethylamine (Et 3 N) to form from niacin and biotin the benzotriazole derivatives 4.3a and 4.3b respectively in good yield s (75 78%). (Scheme 4 1, Table 4 1) Compound 4.3a was earlier prepared under reflux conditions for 12 hours [00JOC8210] and we now found microwave irradiation shortened the reaction time to 1 hour. Compounds 4.3a b were used as active intermediates for fur ther coupling reactions.

PAGE 71

71 Scheme 4 1. Synthesis of benzotriazole derivatized niacin 4.3a and biotin 4.3b Table 4 1. Preparation of benzotriazole derivatives of niacin 4.3a and biotin 4.3b Entry Reactants 4.1 Products 4.3 b Conditions Yield % Mp (C) 1 4.1a 4.3a THF, 1 h 78 101 102 a 2 4.1b 4.3b DMF, 15 min 75 216 218 a Lit m.p. = 87 89 C [00JOC8210] b I synthesized both compounds 4.3a b 4.2.2 Preparation of Peptide Niacin Bioconjugates 4.5a e Scheme 4 2. Synthesis of peptide niacin conjugates 4.5a d Benzotriazole activated niacin 4.3a was coupled with free amino acids, dipeptides and tripeptide 4.4 under microwave irradiation at 70C in the presence of 0.5 equivalents of triethylamine to give 4 .5a d in good yields (43 81%). (Scheme 4 2, Table 4 2) The reactions of free phenylalanine (Phe) and lysine (Lys) with 4.3a take place in 5 minutes while that with the other substrates occurs in 20 minutes. All the amino acid niacin conjugates were isola ted by adjusting the reaction mixture pH to 4 5 using 4N

PAGE 72

72 solution of hydrochloric acid ( HCl ) The purity of the compounds was verified by 1 H NMR, 13 C NMR and elemental analysis. Table 4 2. Preparation of peptide niacin bioconjugates 4.5a d Products a n R 2 R 3 Yield % Mp (C) 4.5a 0 benzyl 66 183 184 4.5b 0 (CH 2 ) 4 NH(Z) 81 215 218 4.5c 0 (CH 2 ) 2 SCH 3 51 198 200 4.5 d 2 H sec butyl 43 141 143 a I prepared compound 4.5d In the literature, amino acid niacin bioconjugates were prepared either (i) by reacting nicotinyl chloride with esters of amino acids followed by hydrolysis and purification by column chromatography with overall yields from moderate to good (33 85%) [67YZ87, 09T9 702]; (ii) or by reacting the N hydroxysuccinimide (NHS) ester of nicotinic acid followed by coupling with amino a cid at room temperature (61 76% yields ) [98JMC2097, 07JAPS2989]. Our preparative methodology allows a simple procedure, less reaction time, pu rification by crystallization and avoids column chromatography. 4.2.3 Preparation of Peptide Biotin Bioconjugates 4.6a g Biotin activated with a benzotriazole moiety 4.3b coupled with free amino acids in dimethyl formamide ( DMF ) under microwave irradiation at 70 C for 30 minutes in the presence of two equivalents of Et 3 N Compounds 4.6a d were isolated in yields of 35 82 % by quenching the DMF with water and acidifying the reaction mixture to pH 2 3 with 4N HCl (Scheme 4 3, Table 4 3) Under the same reaction con ditions, free dipeptides Glycine Valine ( Gly Val OH ) and Leucine Leucine ( Leu Leu OH ) as well as free tripeptide Glycine Glycine Isoleucine ( Gly Gly Ile ) were coupled with 4.3b resulting in the formation of biotin Gly Val OH 4.6e biotin Leu Leu OH 4.6f an d biotin Gly Gly Ile bioconjugates 4.6g in 35, 75 and 48% yield respectively. The low isolated yield of 4.6e

PAGE 73

73 and 4.6g is ascribed to their hydrophilic nature. The purity of compounds 4.6a g was checked by 1 H NMR, 13 C NMR and elemental analysis. The lack of duplication of NMR peaks supports the chiral purity of these bioconjugates. Scheme 4 3. Synthesis of peptide biotin bioconjugates 4.6a g Table 4 3 Preparation of biotin peptide conjugates 4.6a g Products b n R 2 R 3 Yield % Mp (C) (Lit. Mp) a 4.6a 0 sec butyl 82 224 226 (230 233) 4.6b 0 benzyl 71 204 206 (205) 4.6c 0 (CH 2 ) 4 NH (Z) 78 148 151 4.6d 0 (CH 2 ) 2 SCH 3 70 205 208 (207) 4.6e 1 H isopropyl 35 141 144 4.6f 1 isobutyl isobutyl 75 240 243 4.6g 2 H sec butyl 4 8 156 1 59 a Lit. m p. [05OL1741] b I prepared all these compounds 4.6a g The importance of biotinylation always led previously to the development of different methods for coupling biotin to diverse scaffolds molecules via amide linkages. The most commonly involving i) the formation of N hydroxysuccinimide ester of biotin throug h its reaction with either disuccinimidyl carbonate (DSC) [01T9859] or N hydroxysuccinimide (NHS) [08T210, 09MCR538]; both requiring the furthe r use of coupling reagents such as 1 ethyl 3 (3 dimethylaminopropyl)carbodiimide hydrochloride

PAGE 74

74 ( EDC .HCl) or N dicyclohexylcarbodiimide ( DCC ) and reaction times of up to 24 hours; ii) the preparation of moisture and temperature sensitive intermediate bio tinyl chloride by the reaction of biotin with oxalyl chloride requires careful regulation of the temperature to be maintained at 0C to avoid complex by products [08GJ633]; iii) the in situ use of coupling reagents without isolation of the active intermedi ates (DCC Stegl ich conditions) [10TL6410], EDC. HCl [10BMCL6716], O (benzotriazol 1 yl) N N tetramethyluronium tetrafluoroborate ( TBTU ) or benzotriazol 1 yl oxytripyrrolidinophosphonium hexafluorophosphate ( PyBOP ) or N diisopropyl carbodiimide ( DICPDI ) generally used for solid phase synthesis of biotin peptide conjugates, always needing additives such as 1 hydroxybenzotriazole ( HOBt ) and 1 hydroxy 7 azabenzotriazole ( HOAt ) strong bases as N methylmorpholine ( NMM ) N N diisopropylethylamine ( DI EA ) and 4 dimethylaminopyridine ( DMAP ) and excesses up to 5 equivalents of biotin and coupling reagents [10JMC432, 07MCP1761, 06T6876]; iv) the formation of the pentafluorophenyl ester of biotin through coupling with DCC for 6 h ours then reacting it with a n azide under hydrogenation conditions to form the amide coupling in 56% as overall yield [08OL4453] or with an amino acid derivative in the presence of DIEA for 48 h ours in a 34 5 8% as overall yield [05CC4815]; v) the formation, in solution, of amino acid biotin conjugates from amino acid esters coupling with moisture sensitive EDC for 4 hours requiring column chromatography for purification and a further hydrolysis step to generate the product as acid. [05OL1741] As these previous methods suffer from lo ng reaction time and/or moderate yields and tedious methodology, the present procedures allow shorter reaction times and purification by simple crystallization.

PAGE 75

75 4.2.4 Tocopherol) Peptide Conjugates 4.9a e We prepared amino acid and peptide esters conjugates 4.9a e tocopherol by the O tocopherol ( 4.7 ) with Cbz protected acylbenzotriazoles ( 4.8 ) in the presence of two equivalents of potassium carbonate (K 2 CO 3 ) in anhydrous DMF and under mi crowave irradiations (50C, 20W) for 20 min (Scheme 4 4, Table 4 4) Compounds 4.9a e were isolated in moderate yields (44 68%) as microcrystals or waxes. Compounds 4.9a e were characterized by 1 H NMR, 13 C NMR and elemental analysis. Scheme 4 4. Synthesis of peptide tocopherol conjugates 4.9a e Table 4 tocopherol) peptide conjugates 4.9a f Entry Products a R 4 R 5 Yield % Mp (C) 1 4.9a (CH 3 ) 2 CH Z 68 63 64 2 4.9b benzyl Z 60 w ax 3 4.9c benzyl Z L Val 44 116 118 4 4.9d H Z L Val L Phe 55 75 78 5 4.9e H Z L Ala L Phe 51 wax a I prepared compounds 4.9a 4.9c and 4.9d tocopherol uses DCC with either DMAP [08OL4453, 98BMCL2433] or anhydrous

PAGE 76

76 pyridine as base [09JPS2364] at 20C. Here we are providing an alternative and general method for the prepa tocopherol peptide conjugates using the microwave technology. 4.2.5 Preparation of Cholecalciferol Peptide Conjugates 4.11a d Using the above similar reaction conditions, amino acid and peptide conjugates of vitamin D 3 4.11a d were prepared by O acylation of N (Cbz protected acyl ) benzotriazoles ( 4.8 ) with cholecalciferol ( 4.10 ) under microwave irradiations (70C, 50W) for 2.5 hours Compounds 4. 11a d were purified by column chromatography and characterized by 1 H NMR, 13 C NMR and HRMS analysis. No evidence of epimerization was found in the 1 H and 13 C NMR spectra of 4.11a d (Scheme 4 5, Table 4 5) Scheme 4 5. Synthesis of peptide cholecalciferol conjugates 4.11a d Table 4 5. Preparation of cholecalciferol peptide conjugates 4.11a d Entry Products a R 4 R 5 Yield % Mp (C) 1 4.11a isopropyl Z 56 o il 2 4.11b CH 2 indolyl Z 64 o il 3 4.11c benzyl Z L Ala 52 o il 4 4.11d benzyl Z L Val 58 71 73 a I synthesized compounds 4.11b and 4.11d To the best of our knowledge, the only reported method for coupling of cholecalciferol is with Boc protected aminolevulinic acid (ALA) or azido protected ALA

PAGE 77

77 by using EDC coupling reagent at room temperature resulting in a low yield (29 30% yield respectively) and for long reaction time (24 h) [08OL4453]. Our benzotriazole mediated O acylation under microwave irradiation gives in better yields (52 64%) cholecalciferol peptide conjugates after purification by column chromatography in compari son to the use of EDC as coupling reagent. 4.3 Summary In summary, we presented in this chapter a simple and alternative method with relatively short reaction time for the preparation of peptide vitamin conjugates using the microwave technology. Moreover, we p resented a new activated and fairly stable form of biotin 4.3b that could be used in the various biotinylation studies. In addition, using NMR spectroscopy, we were able to prove the retention of chirality of these conjugates. 4.4 Experimental Section 4.4.1 General Methods Melting points were determined on a hot stage apparatus and are uncorrected. 1 H NMR (300 MHz) spectra were recorded in CDCl 3 (CD 3 ) 2 CO or DMSO d 6 with TMS as the internal standard and CDCl 3 (CD 3 ) 2 CO or DMSO d 6 as the internal standard for 13 C NMR (75 MHz). Column chromatography was conducted on flash silica gel (200 425 mesh). Visualization of TLC plates was done via UV and phosphomolybdic acid staining. Anhydrous THF was obtained by distillation immediately prior to use, from sodium/benzophenone ketyl. A single mode cavity microwave synthesizer with a continuous irradiation at 2450 MHz and an infrared temperature control system was used.

PAGE 78

78 4.4.2 General Procedure for the Synthesis of Benzotriazole Derivatized Niacin 4.3a A modified procedure to the e sta blished one [00JOC8210] was employed where microwave irradiation replaced the conventional heatin g to shorten the reaction time. Nicotinic acid (2.00g, 16. 3 mmol) and BtSO 2 Me (3.20g, 16. 3 mmol) were dissolved in anhydrous THF (4 mL). Et 3 N (1.64 g, 2.28 mL, 16.3 mmol) was added to the reaction mixture and then the reaction was exposed to microwave irradiation (70C, 60W) for 1 hour until completion of the reaction (monitored by TLC). THF was then evaporated and the residue was dissolved in ethyl acetate and washed with 5% sodium carbonate solution water, and brine then dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the compound was recrys tallized from a mixture of methylene chloride: hexanes to yield the desired product 4.3a (2.73 g, 12.2 mmol ). (1H Benzo[d][1,2,3]triazol 1 yl)(pyridin 3 yl)methanone ( 4.3a ). B eige microcrystals (78%); mp 101 102 o C; 1 H NMR (CDCl 3 ) 9.34 (d, J = 2.1 Hz, 1H), 8.82 (dd, J = 4.8, 1.5 Hz, 1H), 8.48 (td, J = 8.1, 1.9 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.54 7.43 (m, 2H); 13 C NMR ( CDCl 3 ) 165.3, 153.9, 152.4, 146.0, 139.2, 132.1, 131.0, 127.9, 126.9, 123.3, 120.5, 114.9. An al. Calcd for C 12 H 8 N 4 O: C, 64.28; H, 3.60; N, 24.99; found: C, 63.95; H, 3.45; N, 24.65. 4.4.3 General Procedure for the Synthesis of Benzotriazole Derivatized Biotin 4.3b In a dried heavy walled Pyrex tube containing a s mall stir bar, D b iotin ( 4.1b ) (1 .00 g, 4.1 mmol) and BtSO 2 Me (0.81 g, 4.1 mmol) were dissolved in anhydrous DMF (4 mL). Triethylamine (0.41 g, 4.1 mmol, 0.57 mL) was added to the reaction mixture which was exposed to microwave irradiation (70C, 50W) for 15 minutes with simultaneous

PAGE 79

79 cooling until completion of the reaction. Upon cooling to room temperature, a white precipitate started to form. The solid was vacuum filtered and washed with chloroform to yield the compound 4.3 b (1.01 g, 2.9 mmol ). (3aS,4S,6aR) 4 (5 (1H Benzo[d][1,2,3]tr iazol 1 yl) 5 oxopentyl)tetrahydro 1H thieno[3,4 d]imidazol 2(3H) one ( 4.3b ). White microcrystals (75%); mp 216 218 o C; 1 H NMR (DMSO d 6 ) 8.22 (d, J = 8.7 Hz, 2H), 7.76 (t, J = 7.7 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 6.51 (s, 1H), 6.39 (s, 1H), 4.35 4.28 (m 1H), 4.20 4.12 (m, 1H), 3.45 3.40 (m, 2H), 3.18 3.10 (m, 1H), 2.89 2.80 (m, 1H), 2.59 (d, J = 12.6 Hz, 1H), 1.80 1.40 (m, 6H); 13 C NMR ( DMSO d 6 ) 172.3, 162.8, 145.4, 130.7, 130.6, 126.3, 120.0, 114.0, 61.0, 59.2, 55.4, 39.9, 34.7, 28.1, 27.9, 23.6. Ana l. Calcd for C 16 H 19 N 5 O 2 S: C, 55.63; H, 5.54; N, 20.27; found: C, 55.39; H, 5.55; N, 20.05. 4.4.4 General Procedure for the Preparation of Peptide Niacin Bioconjugates 4.5a d A dried heavy walled Pyrex tube containing a small stir bar was charged w ith nicotinyl benzotriazole (1 equ iv .) dissolved in acetonitrile and added solution of f ree amino acid or dipeptide (1 equ iv .) and base Et 3 N (0.5 equ iv .) in 1 mL water. The reaction mixture was exposed to microwave irradiation (50W) at 70C for specified time. The reac tion mixture was allowed to cool through an inbuilt system Acetonitrile was evaporated and the pH of aqueous layer was adjusted to pH 4 by adding 4N HCl. If no solid precipitates out, a back extraction using ethyl acetate is required then drying using bri ne and anhydrous magnesium sulfate ( MgSO 4 ) The solvent was removed under reduced pressure, and the residue was washed with di ethyl ether to give 4.5a d (S) 2 ( N icotinamido) 3 phenylpropanoic acid ( 4.5a ). W hite shiny crystals (66%); mp 183 184 o C; 1 H NMR ( DMSO d 6 ) 9.00 8.92 (m, 2H), 8.70 (s, 1H), 8.12 (d, J = 7.5

PAGE 80

80 Hz, 1H), 7.52 7.46 (m, 1H), 7.30 7.16 (m, 5H), 4.66 4.63 (m, 1H), 3.26 3.18 (m, 1H), 3.10 3.02 (m, 1H). 13 C NMR ( DMSO d 6 ) 173.0, 165.0, 152.1, 148.5, 138.0, 135.1, 129.4, 129.1, 128.3, 126.5, 123.5, 54.3, 36.3. Anal. Calcd for C 15 H 14 N 2 O 3 : C, 66.66; H, 5.22; N, 10.36; found: C, 66.64; H, 5.22; N, 10.35. (S) 6 (((B enzyloxy)carbonyl)amino) 2 (nicotinamido)hexanoic acid ( 4.5b ). W hite microcrystals (81%); mp 215 218 o C; 1 H NMR (DMSO d 6 ) 9.03 (s, 1H), 8.80 (d, J = 7.5 Hz, 1H), 8.72 (dd, J = 4.8, 1.5 Hz, 1H), 8.24 8.20 (m, 1H), 8.20 7.48 (m, 1H), 7.36 7.22 (m, 6H), 4.98 (s, 2H), 4.36 4.30 (m, 1H), 3.02 2.90 (m, 2H), 1.84 1.74 (m, 2H), 1.50 1.28 (m, 4H); 13 C NMR ( DMSO d 6 ) 173.6, 165.2, 156.1, 152.0, 148.6, 137.3, 135.2, 129.5, 128.4, 127.7, 123.5, 65.1, 52.7, 30.2, 29.0, 23.2. (S) 4 (M ethylthio) 2 (nicotinamido)butanoic acid ( 4.5c ). W hite microcrystals (51%); mp 198 200 o C; 1 H NMR (DMSO d 6 ) 9.03 (s, 1H), 8.87 (d, J = 7.8 Hz, 1H), 8.72 (dd, J = 4.8 Hz, 1.5 Hz, 1H), 8.24 8.19 (m, 1H), 7.55 7.50 (m, 1 H), 4.56 4.48 (m, 1H), 2.62 2.48 (m, 2H), 2.10 2.03 (m, 5H); 13 C NMR (DMSO d 6 ) 173.2, 165.2, 152.0, 148.5, 135.2, 129.4, 123.4, 51.6 30.2, 30.0, 14.6. Anal. Calcd for C 11 H 14 N 2 O 3 S: C; 51.95 H, 5.55; N, 11.02; found: C, 52.25; H, 5.29; N, 11.17. (2S,3S) 3 M ethyl 2 (2 (2 (nicotinamido)acetamido)acetamido)pentanoic acid ( 4. 5 d ). White microcrystals (43 %); mp 141 143 o C; 1 H NMR (DMSO d 6 ) 12. 62 (br s, 1H), 9.12 9.03 (m, 2H), 8.73 (dd, J = 4.8, 1.8 Hz, 1H), 8.28 8.20 (m, 2H), 7.91 (d, J = 8.4 Hz, 1H), 7.54 (ddd, J = 8. 2, 5.0 0.8 Hz, 1H), 4.19 (dd, J = 8.4, 6.0 Hz, 1H), 3.91 (d, J = 5.7 Hz, 2H), 3.79 (d, J = 5.7 Hz, 2H), 1.84 1.72 (m, 1H), 1.47 1.32 (m, 1H), 1.24 1.10 (m, 1H), 0.92 0.75 (m, 6H); 13 C NMR ( DMSO d 6 ) 172.8, 168.9, 168.7, 165.1,

PAGE 81

81 151.7, 148 4, 135.3, 129.5, 123.5, 56.2, 42.7, 41.7 36.4, 24.6, 15.5, 11.3. HRMS Calcd for C 16 H 22 N 4 O 5 Na : [M+Na] + 37 3.1482 ; found 373.1493 4.4.5 General Procedure for the Preparation of Peptide Biotin Conjugates 4.6a g A dried heavy walled Pyrex tube containing a small stir bar was charged with a mixture of free amino acids 4.4 (1 equ iv .) in DMF (3 mL). Et 3 N (2 equ iv .) was added and 2 drops of water to dissolve the suspension. D Biotin Bt ( 4.3b ) (1 equ iv .) was added and the reaction mixture was exposed to microwave irradiation (50W) at 70C for 30 min. The reaction mixture was allowed to cool through an inbuilt system until the temperature had fallen below 40 C Once the reaction was done, the mixture was quenched in ice then acidified to pH 2 to 3 with 4N solution of HCl and a precipitate was formed. This solid was then filtered and washed with water then dried under reduced pressure to give the desired compounds 4.6 (2S) 3 Methyl 2 (5 ((3aS,4S,6aR) 2 oxohexahydro 1H thieno[3,4 d]imidazol 4 l)pentan amido)pentanoic acid ( 4.6a ). W hite microcrystals (82%); mp 224 226 o C; 1 H NMR (DMSO d 6 ) 12.50 (br s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H), 6.38 (s, 1H), 4.34 4.28 (m, 1H), 4.21 4.10 (m, 2H), 3.13 3.05 (m, 1H), 2.82 (dd, J = 12.5, 5.0 Hz, 1H), 2.58 (d, J = 12.6 Hz, 1H), 2.21 2.08 (m, 2H), 1.81 1.70 (m, 1H), 1.65 1.1 0 (m, 8H), 0.87 0.80 (m, 6H); 13 C NMR ( DMSO d 6 ) 173.2, 172.3, 162.7, 61.1, 59.2, 56.1, 55.5, 36.2, 34.7, 28.1, 28.0, 25.4, 24.7, 15.6, 11.3. Anal. Calcd for C 16 H 27 N 3 O 4 S: C, 53.76; H, 7.61; N, 11.75; found: C, 53.46; H, 7.79; N, 11.69. (S) 2 (5 ((3aS,4S,6 aR) 2 O xohexahydro 1H thieno[3,4 d]imidazol 4 yl)pentanamido) 3 phenylpropanoic acid ( 4.6b ). White microcrystals (71%); mp 204 206 o C; 1 H NMR (DMSO d 6 ) 12.62 (s, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.30 7.18 (m, 5H), 6.40 (s, 1H), 6.37 (s, 1H), 4.47 4.37 (m, 1H), 4. 33 4.27 (m, 1H), 4.12 4.07 (m, 1H),

PAGE 82

82 3.09 2.99 (m, 2H), 2.89 2.77 (m, 2H), 2.57 (d, J = 12.3 Hz, 1H), 2.04 (t, J = 7.2 Hz, 2H), 1.63 1.35 (m, 4H), 1.24 1.13 (m, 2H); 13 C NMR ( DMSO d 6 ) 137.7, 129.0, 128.1, 126.3, 61.0, 59.2, 55.4, 5 3.3, 36.8, 34.8, 28.0, 25.2. Anal. Calcd for C 19 H 25 N 3 O 4 S: C, 58.29; H, 6.44; N, 10.73; found: C, 58.20; H, 6.42; N, 10.68. (S) 6 (((B enzyloxy)carbonyl)amino) 2 (5 ((3aS,4S,6aR) 2 oxohexahydro 1H thieno[3,4 d]imidazol 4 yl)pentanamido)hexanoic acid ( 4.6c ). O ff white microcrystals (78%); mp 148 151 o C; 1 H NMR (DMSO d 6 ) 12.34 (br s, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.40 7.27 (m, 5H), 7.24 (t, J = 5.4 Hz, 1H), 6.45 6.28 (m, 2H), 5.00 (s, 2H), 4.32 4.26 (m, 1H), 4.17 4.08 (m, 2H), 3.12 3.03 (m, 1H), 3.01 2.91 (m, 2H), 2.81 (dd, J = 12.2, 5.0 Hz, 1H), 2.57 (d, J = 12.3 Hz, 1H), 2.12 (t, J = 7.4 Hz, 2H), 1.75 1.23 (m, 12H). 13 C NMR ( DMSO d 6 ) 59.2, 55.5, 51.7, 34.8, 30.7, 29.0, 28.1, 28.0, 25.2, 22.8. HRMS Calcd for C 24 H 34 N 4 O 7 SNa: [M+Na] + 529.2091 ; found 529.2091. (S) 4 (M ethylthio) 2 (5 ((3aS,4S,6aR) 2 oxohexahydro 1H thieno [3,4 d]imidazol 4 yl)pent anamido)butanoic acid ( 4. 6d ). In this case, after acidification with 4N HCl bar ely some solids started to precipitate For that reason, NaCl powder was added to saturate the aqueous solution and force the product to precipitat e ou t as white microcrystals (70 %); mp 205 208 o C; 1 H NMR (DMSO d 6 ) 12.6 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 6.42 (s, 1H), 6.37 (s, 1H), 4.35 4.25 (m, 2H), 4.16 4.09 (m, 1H), 3.09 3.05 (m, 1H), 2.82 (dd, J = 12.6, 4.8 Hz, 1H), 2.57 (d, J = 12.3 Hz, 1H), 2.48 2.38 (m, 2H), 2.12 (t, J = 7.2 Hz, 2H), 1.97 1.79 (m, 2H), 1.63 1.42 (m, 4H), 1.36 1.24 (m, 2H); 13 C NMR ( DMSO d 6 ) 173.5, 172.3, 162.7, 61.0, 59.2, 55.5, 50.8, 38.7, 34.8, 30.7, 29.8, 28.1, 25.2, 14.6. Anal. Calcd for C 15 H 25 N 3 O 4 S 2 : C, 47.98; H, 6.71; N, 11.19; found: C, 48.37; H, 6.96; N, 11.10.

PAGE 83

83 (S) 3 M ethyl 2 (2 (5 ((3aS,4S,6aR) 2 oxohexahydro 1H thieno[3,4 d]imidazol 4 yl)pentan amido)acetamido)butanoic acid ( 4.6e ). W hite microcrystals (35%); mp 141 144 o C; 1 H NMR (DMSO d 6 ) 12.7 ( s, 1H), 8.03 (t, J = 5.7 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H), 6.37 (s, 1H), 4.30 (dd, J = 7.8, 4.8 Hz, 1H), 4.18 4.10 (m, 2H), 3.75 (d, J = 5.7 Hz, 2H), 3.13 3.05 (m, 1H), 2.82 (dd, J = 12.5, 5.0 Hz, 1H), 2.57 (d, J = 12.6 Hz, 1H), 2.12 (t, J = 7.4 Hz, 2H), 2.08 1.99 (m, 1H), 1.66 1.40 (m, 4H), 1.37 1.25 (m, 2H), 0.86 (d, J = 6.9 Hz, 6H); 13 C NMR ( DMSO d 6 ) 172.9, 172.4, 169.2, 162.7, 61.0, 59.2, 57.0, 55.4, 41.7, 35.0, 30.0, 28.2, 28.0, 25.2, 19.1, 17.9. HRMS Calcd for C 17 H 28 N 4 O 5 S 2 Na: [M+Na] + 423.1667; found: 423.1673. ( S) 4 M ethyl 2 ((S) 4 methyl 2 (5 ((3aS,4S,6aR) 2 oxohexahydro 1H thieno[3,4 d]imidazol 4 yl)pentanamido)pentanamido)pentanoic acid ( 4.6f ). W hite microcrystals (75%); mp 240 243 o C; 1 H NMR (DMSO d 6 ) 8.03 (d, J = 8.1 Hz, 1H), 7 .90 (d, J = 7.8 Hz, 1H), 6.45 (s, 1H), 6.38 (s, 1H), 4.40 4.27 (m, 2H), 4.23 4.08 (m, 2H), 3.12 3.04 (m, 1H), 2.81 (dd, J = 12.6, 5.1 Hz, 1H), 2.57 (d, J = 12.3 Hz, 1H), 2.10 (t, J = 6.9 Hz, 2H), 1.68 1.38 (m, 11H), 1.35 1.24 (m, 2H), 0.88 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.3 Hz, 3H), 0.83 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H); 13 C NMR ( DMSO d 6 ) 174.0, 172.2, 171.9, 162.7, 61.1, 59.2, 55.5, 50.6, 50.2, 40.9, 35.0, 28.1, 25.4, 24.3, 24.2, 23.2, 22.9, 21.6, 21.4. Anal. Calcd for C 22 H 38 N 4 O 5 S: C, 56.15; H, 8.14; N, 11.90; found: C, 56.15; H, 8.44; N, 11.96. ( 2S) 3 M ethyl 2 (2 (2 (5 ((3aS,4S,6aR) 2 oxohexahydro 1H thieno[3,4 d]imidazol 4 yl)pentanamido)acetamido)acetamido)pentanoic acid ( 4. 6g ) This compound is water soluble. For that reason, we did not quench the reaction in acidic ice but instead we added 0.5 mL HCl 4N to neutralize the reaction mixture then diethyl ether. We collected

PAGE 84

84 the aqueous then evaporated it under reduced pressure. The resulted residue was washed with ethanol then ac etone to yield the prod uct as white microcrystals. (48 %); mp 156 159 o C; 1 H NMR (DMSO d 6 ) 12.60 (br s, 1H), 8.10 (t, J = 5.9 Hz, 1H), 8.04 (t, J = 6.3 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 6.44 (s, 1H), 6.37 (s, 1H), 4.34 4.26 (m, 1H), 4.19 4.10 (m, 2H), 3. 76 (d, J = 6.0 Hz, 2H), 3.68 (d, J = 5.7 Hz, 2H), 3.14 3.05 (m, 1H), 2.82 (dd, J = 12.5, 5.3 Hz, 1H), 2.57 (d, J = 12.3 Hz, 1H), 2.13 (t, J = 7.1 Hz, 2H), 1.85 1.72 (m, 1H), 1.66 1.10 (m, 8H), 0.90 0.80 (m, 6H); 13 C NMR ( DMSO d 6 ) 172.8, 172.6, 169.3, 168.8, 162.7, 61.0, 59.2, 56.2, 55.4, 42.1, 41.7, 36.4, 35.0, 28.2, 28.1, 25.1, 24.6, 15.6, 11.3. Anal. Calcd for C 20 H 38 N 5 O 6 S: C, 50.94; H, 7.4 5; N, 14.85; found: C, 50.90 ; H, 7.58; N, 14.80. 4.4.6 Tocopherol) Peptide Conjugates 4.9a f A dried heavy walled Pyrex tube containing a small stir bar was charged with a mixture of N (Cbz protected)acylbenzotriazoles 4.8 (1 equ iv tocopherol 4.7 (1.1 equ iv .) dissolved in anhydrous DMF (2 mL). K 2 CO 3 (2 equ iv .) was added and the mixture was exposed to microwave irradiation (20W) at 50 C for 20 min until completion of the reaction (monitored by TLC). The reaction mixture was quenched with ice then extracted with ethyl acetate (2x15 mL). The combined or ganic layers were evaporated under reduced pressure then purified by column chromatography to yield the corresponding product 4.9 (S) [(2R) 2,5,7,8 T etramethyl 2 ((4R,8R) 4,8,12 trimethyltridecyl)chroman 6 yl] 2 (((benzy loxy)carbonyl)amino) 3 methylbutan oate ( 4.9a ). Purified by column chromatography using hexanes: ethyl acetate in a 9.5:0.5 ratio yielding the product as off white microcrystals (68%); mp 63 64 C; 1 H NMR (CDCl 3 ) 7.40 7.31 (m, 5H), 5.32

PAGE 85

85 (d, J = 9.3 Hz, 1H), 5.17 5.10 (m, 2H), 4.64 (dd, J = 9.3, 3.9 Hz, 1H), 2.57 (t, J = 6.6 Hz, 2H), 2.46 2.42 (m, 1H), 2.08 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.84 1.70 (m, 2H), 1.56 1. 00 (m, 30 H), 0.87 0.83 (m, 12H); 13 C NMR ( CDCl 3 ) 171.1, 156.6, 149.8, 140.5, 136.4, 128.7, 128.4, 128.3, 126.8, 125.0, 123. 4, 117.7, 75.3, 67.3, 59.3, 39.6, 37.6, 37.5, 33.0, 32.9, 31.1, 28.2, 25.0, 24.7, 23.0, 22.9, 21.2, 20.8, 20.0, 19.9, 17.3, 13.3, 12.5, 12.1. HRMS Calcd for C 42 H 65 NO 5 Na: [M+Na] + 686.4755; found 686.4786. (S) (R) 2,5,7,8 T etramethyl 2 ((4R,8R) 4,8,12 trimethyltridecyl)chroman 6 yl 2 ((benzyl oxy) carbonyl)amino) 3 phenylpropanoate ( 4.9 b ). Wax (60%); 1 H NMR (CDCl 3 ) 7.40 7.27 (m, 10H), 5.33 (d, J = 8.4 Hz, 1H), 5.11 (s, 1H), 5.03 4.70 (m, 1H), 3.50 3.38 (m, 1H), 2.60 (t, J = 6.5 Hz, 3H), 2.11 (s, 3H), 1.96 (s, 3H), 1.93 (s, 3H), 1.90 1.70 (m, 3H), 1.6 2 1.52 (m, 2H), 1.48 1.32 (m, 14 H), 1.25 1.05 (m, 8H), 1.04 0.86 ( m, 12H); 13 C NMR ( CDCl 3 ) 170.81, 156.0, 149.8, 140.4, 136.3, 136.0, 129.5, 128.9, 128.7, 128.3, 128.2, 127.4, 126.7, 125.0, 123.3, 117.6, 75.3, 67.2, 55.1, 39.6, 38.3, 37.6, 37.5, 33.0, 32.9, 31.2, 28.2, 25.0, 24.6, 22.9, 22.9, 21.2, 20.8, 20.0, 19.9, 13.2, 12.3, 12.0. HRMS Calcd for C 46 H 65 NO 5 Na: [M+Na] + 734.4755; found 734.4775. (S) [(2R) 2,5,7,8 T etramethyl 2 ((4R,8R) 4,8,12 trimethyltridecyl)chroman 6 yl] 2 ((S) 2 (((benzyloxy)carbonyl)amino) 3 methylbutanamido) 3 phenylpropanoate ( 4.9 c ). Purified by column chromatography usin g hexanes: ethyl acetate in an 8:2 ratio yielding the product as off white microcrystals (44%) ; mp 116 118 C; 1 H NMR (CDCl 3 ) 7.35 7.25 (m, 10H), 6.38 (d, J = 7.8 Hz, 1H), 6.27 (d, J = 7.8 Hz, 1H), 5.28 5.12 (m, 2H), 5.09 5.04 (m, 2H), 4.05 3.93 (m, 1H), 3.50 3.37 (m, 1H), 3.20 3.06 (m, 1H), 2.56 (t, J = 6.5 Hz, 2H), 2.07 (s, 3H), 1.96 1.85 (m, 6H), 1.82 1.70 (m, 2H), 1.57 1.47 (m, 2 H), 1.45 1. 04 (m, 22H), 0.95 0.78 (m, 18H); 13 C NMR ( CDCl 3 ) 171.2, 170.5, 156.4, 149.8,

PAGE 86

86 140.4, 135.7, 129.5, 129.0, 128.7, 128.4, 128.3, 127.5, 125.0, 123.4, 117.7, 77.7, 77.2, 76.8, 75.3, 67.3, 60.5, 53.3, 39.6, 38.1, 37.7, 37.5, 33.0, 31.3, 28.2, 25.0, 24.7, 24.2, 23.0, 22.9, 21.3, 20.8, 20.0, 19.9, 19. 3, 19.1, 13.2, 12.4, 12.1. HRMS Calcd. for C 51 H 74 N 2 O 6 Na: [M+Na] + 833.5439; found 833.5479. (5S,8S) (R) 2,5,7,8 T etramethyl 2 ((4R,8R) 4,8,12 trimethyltridecyl)chroman 6 yl 8 benzyl 5 isopropyl 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oate ( 4.9d ). Re crystallized from diethyl ether to yield the product as off white microcrystals (55 %) ; mp 75 78 C; 1 H NMR (CDCl 3 ) 7.44 7.28 (m, 5H), 7.24 7.12 (m, 5H), 6.85 6.75 (br s, 1H), 6.67 (d, J = 8.4 Hz, 1H), 5.24 (d, J = 6.9 Hz, 1H), 5.11 4.98 (m, 3H), 4.85 4.74 (m, 1H), 4.40 4.26 (m, 1H), 4.23 4.10 (m, 1H), 3.97 (t, J = .5 Hz, 1H), 3.23 3.12 (m, 1 H), 3.10 3.00 (m, 1H), 2.62 2.50 (m, 2H), 2.07 (s, 3H), 1.98 (s, 3H), 1.94 (s 3H), 1.83 1.70 (m, 3H), 1.51 1.48 (m, 2H), 1. 45 1.02 (m, 21 H), 0.88 0.73 (m, 18H) ; 13 C NMR ( CDCl 3 ) 171. 4 171.2, 168. 6 156.9, 149.8, 140.3, 136.6, 136.1, 129.4, 128.9, 128.8, 128.5, 128.4, 127.2, 126.7, 125.0, 123.4, 117.7, 75.3, 67.5, 61.1, 54.3, 41.3, 39.6, 38.1, 37.7, 37.5, 33.0, 31.2, 30.7, 28 .2, 25.0, 24.7, 23.0, 22.9, 21.2, 20.8, 20.0, 19.9, 19.4, 17.6, 13.2, 12.4, 12.0. HRMS Calcd. for C 53 H 77 N 3 O 7 Na: [M+Na] + 890.5654 ; found 890.5667 (5S,8S) (R) 2,5,7,8 T etramethyl 2 ((4R,8R) 4,8,12 trimethyltridecyl)chroman 6 yl 8 benzyl 5 methyl 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oat e ( 4.9e ). Purified by column chromatography using hexanes: ethyl ace tate yielding the product as wax (51 %) ; 1 H NMR (CDCl 3 ) 7.35 7.28 (m, 5H), 7.20 7.16 (m, 5H), 6.82 6.58 (m, 2H), 5 .20 (d, J = 6.3 Hz, 1H), 5.11 4.96 (m, 2H), 4.81 4.72 (m, 1H), 4.40 4.26 (m, 1H), 4.20 4.10 (m, 2H), 3.18 (dd, J = 13.5, 6.0 Hz, 1H), 3.06 (dd, J = 13.8, 7.0 Hz, 1H), 2.56 (t, J = 6.2 Hz, 2H), 2.07 (s, 3H), 1.99 (s, 3H), 1.95 (s, 3H), 1.81 1.70 (m, 3H), 1.59 1.48 (m,

PAGE 87

87 3H), 1.43 1. 02 (m, 23H), 0.92 0.80 (m, 12H); 13 C NMR ( CDCl 3 ) 172.4, 171.2, 168.6, 156.4, 149.8, 140.3, 136.6, 136.1, 129.4, 128.8, 128.5, 128.3, 127.2, 126.7, 125. 0, 123.4, 117.7, 75.3, 67.5, 54.2, 51.2, 41.3, 39.6, 38.0, 37.7, 37.5, 33.0, 32.9, 31.2, 29.9, 28.2, 25.0, 24.7, 23.0, 22.9, 21.2, 20.8, 20.0, 19.9, 18.3, 13.2, 12.4, 12.0. HRMS Calcd. for C 51 H 73 N 3 O 7 Na: [M+Na] + 862.5357; found 862.5341. 4.4.7 General Procedure f or the Preparation of Cholecalciferol Peptide Conjugates 4.11a d A dried heavy walled Pyrex tube containing a small stir bar was charged with a mixture of N (Cbz protecte d acyl ) benzotriazoles 4. 8 (1 equ iv .), cholecalciferol 4. 10 (1.2 equ iv.) and DMAP (0.2 equ iv .) dissolved in freshly distilled THF (5 mL). The reaction mixture was exposed to mic rowave irradiation (50W) at 70 C for 2.5h until completion of the reaction (monitored by TLC). The reaction mixture was allowed to cool through an inbuilt system until the temperature fell belo w 30 C. Then the mixture was quenched with water and extracted with ethyl acetate. The combined organ ic layers were washed with 10% solution of Na 2 CO 3 water and dried over anhydrous MgSO 4 The solvent was removed under redu ced pressure, and the residue was subjected to silica gel column using ethyl acetate/hexanes as eluents to give the corresponding product 4. 11 (2S) (Z) 3 ((E) 2 ((1R,3aS,7aR) 7a M ethyl 1 ((R) 6 methylheptan 2 yl)hexahydro 1H ind en 4(2H) ylidene)ethylide ne) 4 methylenecyclohexyl 2 (((benzyloxy)carbonyl)amino) 3 methyl butanoate ( 4.11a ). Oil (56%) ; 1 H NMR (CDCl 3 ) 7.36 7.31 (m, 5H), 6.19 (d, J = 11.4 Hz, 1H), 6.02 (d, J = 11.4 Hz, 1H), 5.30 (d, J = 8.7 Hz, 1H), 5.11(s, 2H), 5.07 (s, 1H), 5.05 5.02 (m, 1H), 4.86 4.84 (m, 1H), 4.32 4.24 (m, 1H), 2.84 2.75 (m, 1H), 2.62 2.51 (m, 1H), 2.44 2.34 (m, 2H), 2.26 2.12 (m, 2H), 2.06 1.92 (m, 3 H), 1.86 1.75 (m, 2H), 1.68 1.60 (m, 3H), 1.58 1.42 (m, 4H), 1.38 1.20 (m,

PAGE 88

88 6H), 1.18 1.06 (m, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H), 0.89 0.85 (m, 9H), 0.54 (s, 3H); 13 C NMR (CDCl 3 ) 171.6, 156.4, 144.6, 142.8, 136.5, 133.9, 128.7, 128.3, 123.0, 117.5, 113.0, 73.0, 67.2, 59.2, 56.8, 56.5, 46.1, 42.2, 40.7, 39.7, 36.3 32.2, 32.0, 31.6, 29.2, 28.2, 27.9, 24.1, 23.8, 23.0, 22.8, 22.4, 19.2, 19.1, 17.6, 12.2. HRMS Calcd for C 4 0 H 59 NO 4 Na:[M+Na] + 640.4336; found 640.4331. (2S ) (1S,Z) 3 ((E) 2 ((1R,7aR) 7a M ethyl 1 ((R) 6 methylheptan 2 yl)hexahydro 1H inden 4(2H) ylidene)et hylidene) 4 methylenecyclohexyl 2 (((benzyloxy)carbonyl)amino) 3 (1H indol 2 yl)propanoate ( 4.11b ). Oil (64%); 1 H NMR (CDCl 3 ) 8.12 (br s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.40 7.27 (m, 5H), 7.18 (dt, J = 7.5, 1.1 Hz, 1H), 7.07 (dt, J = 7.6, 1.1 Hz, 1H), 6.98 6.96 (m, 1H), 6.14 (d, J = 11.1 Hz, 1H), 6.01 (d, J = 11.4 Hz, 1H), 5.33 (d, J = 8.4 Hz, 1H), 5.15 5.01 (m, 3H), 4.96 4.85 (m, 1H), 4.82 (d, J = 2.4 Hz, 1H), 4.72 4.62 (m, 1H), 3.34 3.17 (m, 2H), 2. 82 2.74 (m, 1H), 2.45 1.80 (m, 9 H), 1. 70 1.43 (m, 6H), 1.40 1.08 (m, 11 H), 0.92 (d, J = 6.3 Hz, 3H), 0.88 (s, 3H), 0.86 (d, J = 1.5 Hz, 3H), 0.53 (s, 3H); 13 C NMR (CDCl 3 ) 171.8, 156.0, 144.6, 142.8, 136.5, 136.3, 134.1, 128.7, 128.3, 127.8, 123.0, 122.9, 122.4, 119.8, 118.9, 117.6, 113 .0, 111.3, 110.3, 73.3, 67.1, 56.8, 56.5, 54.9, 46.1, 42.0, 40.7, 39.7, 36.3, 34.8, 32.2, 31.9, 29.2, 28.2, 27.8, 24.1, 23.8, 23.0, 22.8, 22.4, 19.0, 12.2. HRMS Calcd. for C 46 H 60 N 2 O 4 Na:[ M+Na] + 727.4445; found 727.4479. (S) ( S,Z) 3 ((E) 2 ((1R,3aS,7aR) 7a M ethyl 1 ((R) 6 methylheptan 2 yl)hexahydro 1H inden 4(2H) ylidene)ethylidene) 4 methylenecyclohexyl2 ((S) 2 (((benzyloxy)carbonyl) amino) propanamido) 3 phenylpropanoate ( 4.11c ). Oil (52 %); 1 H NMR (CDCl 3 ) 7.28 7.23 (m, 5H), 7.22 7.15 (m, 3H), 7.10 6.98 (m, 2H), 6.41 6.30 (m, 1H), 6.15 5.40 (m, 2H), 5.26 5.10 (m, 1H), 5.06 4.94 (m, 3H), 4.94 4.88 (m, 1H), 4.78

PAGE 89

89 4.70 (m, 2H), 4.20 4.08 (m, 1H), 3.06 2.94 (m, 2H), 2.78 2.60 (m, 1H), 2.49 2.36 (m, 1H), 2.30 2.0 6 (m, 2H), 1.98 1.72 (m, 3H), 1.68 1.52 (m, 2H), 1.50 1.34 (m, 3H), 1.30 1.10 (m, 9H), 1.10 1.00 (m, 3H), 0.98 0.88 (m, 2H), 0.84 (d, J = 6.1 Hz, 3H), 0.80 (d, J = 6.6 Hz., 9H), 0.46 (d, J = 7.8 Hz, 3H); 13 C NMR (CDCl 3 ) 171.9, 170.8, 156.0, 144.4, 143.2, 142.9, 136.3, 135.9, 134.2, 133.9, 129.6, 128.7, 128.4, 128.3, 127.3, 123.0, 122.7, 117.6, 113.2, 73.5, 73.2, 67.3, 56.8, 56.5, 53.3, 53.2, 50.6, 46.1, 42.1, 40.7, 39.7, 38.0, 36.3, 32.0, 31.7, 29.3, 28.2, 27.9, 24.1, 23.8, 23.0, 22.8, 22.4, 19.1, 18.7, 1 2.2. HRMS Calcd for C 47 H 64 N 2 O 5 Na: [ M+Na] + 759.4707; found 759.4738. (S) ( S,Z) 3 ((E) 2 ((1R,3aS,7aR) 7a M ethyl 1 ((R) 6 methylheptan 2 yl)hexahydro 1H inden 4(2H) ylidene)ethylidene) 4 methylenecyclohexyl 2 ((S) 2 (((benzyloxy)carbonyl) amino) 3 methylbuta namido) 3 phenylpropanoate ( 4. 11d ). White microcrystals (5 8 %); m p 71 73 C; 1 H NMR (CDCl 3 ) 7.40 7.24 (m, 5H), 7.17 6.85 (m, 5H), 6.29 (d, J = 7.8 Hz, 1H), 6.19 (d, J = 8.4 Hz, 1H), 6.15 6.08 (m, 1H), 6.00 5.90 (m, 1H), 5.22 (dd, J = 20.7, 8.1 Hz, 1H), 4.99 4.86 (m, 3H), 4.85 4.83 (m, 1H), 4.83 4.73 (m, 2H), 4.00 3.50 (m, 1H), 3.01 (d, J = 6.0 Hz, 2H), 2.77 2.67 (m, 1H), 2.51 2.40 (m, 1H), 2.30 2.17 (m, 2H), 2.16 1.75 (m, 6H), 1.69 1.54 (m,4H), 1. 50 1.35 (m, 4H), 1.34 1.15 (m, 6 H), 1.14 1.00 (m, 4H), 0.95 0.70 (m, 16 H) ; 13 C NMR (CDCl 3 ) 170.9, 156.5, 144.4, 143.0, 135.9, 133.9, 129.5, 128.7, 128.4, 128.3, 127.3, 122.9, 117.5, 113.2, 73.5, 67.3, 60.4, 56.8, 56.6, 53.3, 46.1, 42.2, 40.7, 39.7, 38.4, 36.4, 32.2, 32.0, 31.0, 29.3, 28.2 27.9, 24.1, 23.8, 23.1, 22.8, 22.4, 19.5, 19.1, 18.0, 17.5, 12.2. HRMS Calcd for C 49 H 68 N 2 O 5 Na: [M+Na] + 787.5020; found 787.5033.

PAGE 90

90 CHAPTER 5 BENZOTRIAZOLE ASSISTED SYNTHESES OF HYDRAZINO PEPTIDES 5.1 Introductory Remarks 5.1.1 Background Peptides as mentioned in the previous chapters play an important role in the design and discovery of new drugs. [09FMC361 86TCC109, 83S975 ] The se bioactive peptides are mainly examined as hormones, neuromodul ators and neurotransmitters. Upon interacting with receptor s and as a result of the signal transduction, vario us physiological processes occur [93ACIE1244] Despite the success in this area, some limitations have arisen as an outcome of the low stability towards prote olysis by peptidases, and the undesired interac tion with non target receptors due the intrinsic conformational flexibility In order to solve this problem and improve the pharmacological properties of native peptides some peptidomimetics were developed These compounds bear an identifiable resemblance to a peptide and display properties that can : (i) suppress the interaction with an alternative receptor by a conformationally restrained structure and (ii) confer a better resistance to enzymatic degradation by peptide modifica tion. [97ADD1 93ACIE 1244 ] One of the common strategies for the synthesis of peptidomimetics involves the modification of amino acids by sterically demanding entity on the C N, the side chain or even the use of D or amino a cids. [90BJ268 93ACIE1244] T he replacement amino acid units by amino acid units is a well known technique in the search for pharmacological ly active peptides [99COCB714 ] T he further replacement of the C and /or the C a tom in amino acid constituents by a hetero atom is an other attractive extension of the peptide

PAGE 91

91 concept The replacement of C atom s by nitrogen can be accomplished by hydrazino acid (H 2 N N H CH(R) COOH) units, [01JACS247 ] in a peptide leading to hydrazino pepti des i.e., peptide analogues in which one (or more) peptidic bond(s) [ HN CO CH(R) ] are replaced by one (or more) hydrazidic bond(s) [ HN HN CO CH(R) ] [04TL3569 ] L inatine 5. 1 and N egamycin 5. 2 are n atural ly occurring peptides hydrazino acid moiety (Figure 5 1) Compound 5. 1 is a vitamin B 6 antagonist and 5. 1 shows antibiotic properties [67B170, 71JACS6305 ] Figure 5 1. Examples of biologically active hybrid peptides with a hydrazino unit H ydrazino acid [ H 2 N N H CH(R) COOH] analogs resemble the corresponding amino acids [NH 2 CH(R)COOH] but can i nhibit enzymes which metabolize amino acids. [97TA1605 ] Hydrazino peptides containing an N terminal achiral hydrazinoacetic acid residue display an tica ncer properties [03BMC4881 ] and are useful intermediates in the preparat ion of lipopeptides. [01JOC443 ] H ydrazino peptides are also use ful for the synthesis of antiviral peptidomimetics [08BMCL2745 ] as substrates f or hydrazone chemical ligation [98JPR180 ] and a s solvent gelators. [01TL1887 ] Peptides containing an N alkyl an N aryl or an N acyl hydrazino group react with peptide aldehydes to give high molecular weight conjugates. [86B5774 ] From a structural point of view the introduction of hydrazino fragment in a peptide ] which folds the peptide backbone locally by way of well defined intramolecular bifurcated H bonding Q uantum

PAGE 92

92 and molecular mechanics calculations reveal that oligomers of hydraz ino acids [01JACS247 ] may adopt a wide variety of secondary structures and thus may behave as foldamers [01CR3893, 10S933 ] (Figure 5 2). This last property is similar to a turn effect resulting in a folded structure. In fact, turn is one of the appli ed strategic changes to a peptide in the synthesis of peptidomimetics that mainly deal with an imitation of a secondary structure of a peptide It is defined as a segment of four amino acids with a distance of 7 between C i and C i+3 and a turn stabilized by chelation with a cation or intramolecular hydrogen bonding and is mainly observed in a more rigid scaffol d s uch as cyclic peptides [88APC51] (Figure 5 2) Figure 5 2. Hydrazino and turn conformati ons (schemes were modified af ter references [10S933 ] 5.1.2 Literature Preparative Methods Published preparations of hydrazino acids include : (i) Hofmann rearrangement of hydantoic acids 5.4 [75ACS93 87T891] ; (ii) the asymmetric electrophilic amination of chir al enolates 5.5 [86JACS6397, 88T5525 ] ; (iii) amination of chiral amino acids with N alkoxycarbonyl 3 phenyloxaziridines [91CC435 ] ; (iv) asymmetric hydrogenation of N acylhydrazones derived from keto acids 5.6 [94T4 399 ] ;

PAGE 93

93 bromo acids 5.7 [68CJC3013, 07RJAC2165 36J PC268 ] (Figure 5 3) Moreover, recently, some racemic hydrazinopeptides motifs were reported using an intramolecular hydrazino Ugi reaction of hydrazones [10S933]. Despite their interesting biological properties a nd the diverse methods described for their preparation, hydrazino peptides have not been widely studied, probably mainly due to difficulties associated with their synthesis including tedious reaction procedures, [86 JACS6397 ] low yields, and incomplete chir al characterization of the products. [07RJAC2165 ] Figure 5 3 Literature methods for the preparation of hydrazino amino acids We recently repor a minoxy acids and p eptides using benzotri azole methodology. [09JOC8690 ] Following an analogous path, we have now hydrazino acids [68CJC3013 ] and their solution phase hydrazino hybrid dipeptides [ 09ARKIVOC47, 11ARKIVOC212 ]. Further work tow ards chiral O and S acylation of Cbz protected hydrazino acids is presented as well.

PAGE 94

94 5.2 Results and Discussions 5.2.1 Hydrazino A cids 5. 3a d Hydrazino acids 5. 3a d were prepared by the nucleophilic substitutions of the bromo ac ids 5. 8 a d [06JOC3332] with hydrazine hydrate at 70C under m icrowave irradiation for 15 min. (Scheme 5 1 Table 5 1 ) That compounds 5.3a d are optically pure is attested by the products of the coupling reactions of 5.3a d with Cbz protected aminoacylbenz otriazoles 5.10 a f (Table 5 5) as described below. Scheme 5 1 bromo acids Table 5 1. hydrazino acids Bromo acids 5.2 a Hydrazino acids 5.3 Entry d R Mp Yield (%) 22 D Entry d Mp (C) Yield (%) 22 D 5. 8 a CH 3 oil 68 + 41 5. 3a 214 215 48 32 5. 8 b CH 2 CH(CH 3 ) 2 oil 67 +15 5. 3b 214 217 43 12 5. 8 c CH 2 Ph oil 70 +20 5. 3c 203 206 b 45 22 5. 8 d CH(CH 3 ) 2 oil 60 +20 5. 3d 240 241 c 40 3 a Lit ref [06JOC3332 ]. b Lit mp 191 194 C [36JPC268 ]. c Lit mp d 250 C [68CJC3013 ] d I prepared compounds 5.8 b 5. 8 d 5.3b and 5.3d 5.2.2 Preparation Hydrazino D ipeptides 5. 11 a f hydrazino acids 5. 3a d with Cbz protected aminoacylbenzotriazoles 5. 10 a f [09S2392 ] under micro wave irradiation at 70C and 65 W power for 15 min to afford the novel hydrazino hybrid dipeptides 5. 11 a f (yields 42 71%). (Scheme 5 2 Table 5 2) The chiral integrity of compoun ds 5.11 a f were supported by NMR spectra: critically no duplication of peaks was found in 1 H and 13 C NMR spectra of 5.11 a f except 5.11 b which is prepared by the

PAGE 95

95 hydrazino valine 5.3d with benzotriazole derivative of Cbz protected phenylalani ne 5.10 c Scheme 5 2 Syntheses of hybrid dipeptides 5.11 a e Table 5 2. Preparation of dipeptides 5. 11 a f containing one hydrazino acid unit Product a R R 1 Mp (C) Yield (%) 5. 11 a CH 3 CH(CH 3 ) 2 140 141 61 5.11 b CH(CH 3 ) 2 CH 2 Ph 126 128 42 5.11 c CH 2 CH(CH 3 ) 2 CH 2 (3 indolyl) 81 83 50 5. 11 d CH 2 CH(CH 3 ) 2 (CH 2 ) 4 NH( Z ) 93 95 45 5. 11 e CH 2 Ph CH 3 113 114 68 5. 11 ( ) CH 2 Ph CH 3 145 150 71 a I prepared compounds 5.11 b 5.11 c and 5.11 d Compound 5. 11 b showed repeated peaks in 13 C NMR spectra which infer the incomplete inversion of chirality in 5. 3d Compound 5. 3c was also coupled with the racemic mixture ( 5. 10 a + 5. 10 ) gave product as the mixture of diastereomers ( 5. 11 e + 5. 11 ) for which the 13 C NMR spectrum shows expected dupli cation of all the carbonyl peaks, found in the corresponding chirally pure isomer 5. 11 e 5.2.3 N Cbz Hydrazino A cids an d Their Benzotriazolides N Cbz Hydrazino acids 5. 12 b c were prepared by reacting 1 ( t butoxycarbonyl) benzotriazole ( Cbz Bt ) [86S958 ] with unpro hydrazino acids at room temperature in the presense of triethylamine in good yields (74 92%). Compound s 5. 12 b c were further activated by benzotriazole by using thionyl chl oride at room temperature for 4 h to give benzotriazole derivatives 5. 13 b c These generated compounds 5. 13 b c are highly hygroscopic and used for next reaction without further

PAGE 96

96 characterizations (Scheme 5 3, Table 5 3) Indeed, after the general basic workup to remove the excess benzotriazole, these compounds were isolated, dried and immediately used for the next step syntheses of hydrazino dipeptides as well as different esters and thioesters. Scheme 5 3. Syntheses of N (Cbz protected)hydrazino acids 5.13b c Table 5 3. Preparation of N Pg Hydrazino acids 5. 12b c and their benzotriazole derivatives 5. 13b c Compound 5. 12 a Compound 5. 13 a Entry R 2 Mp (C) Yield % Entry R 2 Mp Yield % 5. 12 b CH 2 CH(CH 3 ) 2 119 120 74 5. 13 b CH 2 CH(CH 3 ) 2 oil 58 5. 12 c CH 2 Ph 158 160 92 5. 13 c CH 2 Ph oil 55 a I prepared compounds 5.12 b and 5. 13 b 5.2.4 Chiral A cylation of Compound s 5. 13 b c Scheme 5 4 Displacement of benzotriazole with nucleophiles 5. 15 Benzotriazole activated hydrazino acids 5.13b c were further reacted with O and S nuceophiles under different sets of condtions and found that activation of carboxylic group of Cbz protected hydrazino acids with benzotriazole could be used in any kind of chiral acylation.(Scheme 5 5, Table 5 4) The p urity of the compound s 5.15 were verified

PAGE 97

97 by 1 H NMR, 13 C NMR and elemental analysis M ore examples with O and S acylations will be synthesized from 5.13a b and C acylation reactions will be attempted as well. Table 5 4 O and S acylations utilizing nucleophiles 5. 15 Entry a Reactant Nucleophile 5. 14 Conditions Mp Yield 5. 15 a 5.13 c Menthol MW ( 70C 50W ) 2h 112 114 49 5. 15 b 5.13 b Benzyl mercaptan Et 3 N, rt, 4h oil 6 1 5. 15 c 5.13 c Methyl mercapto acetate Et 3 N, rt, 4h oil 63 a I prepared compound 5.15 b 5.3 Summary and Future Prospect Enantiomerically pure h ydrazino acid s were prepared from bromo acids through a modified procedure of the literature that involved the us e of microwave irradiation and reduced the reaction time from more than 24h at room temperature to 15 min at 70 C Chirally pure hybrid hydrazino peptides were synthesized under microwave irradiation and in 42 71% yield One example, 5.11 b showed in its NMR a diastereomeric mixture which was ascribed to the incomplete inversion of 5.9 d to 5.3d Novel N Cbz ( hydrazino aminoacyl) benzotriazoles were synthesized as precursors to different chirally pure esters and thioesters of hydrazino peptides generated from alcohols and thiols nucleophilic attack. Following these promising results, more examples of hydrazino esters and thioesters will be synthesized Moreover an attempt to C acylate d these precursors will be explored. In addition, these N Cbz hydrazino aminoacyl)benzotriazoles will serve as precursors to hydrazine dipeptides formed fro m two hydrazino acid units. 5.4 Experimental Section 5.4.1 General Methods Melting points were determined on a hot stage apparatus and are uncorrected. All reactions were carried out under nitrogen unless otherwise specified. Column

PAGE 98

98 chromatography was conducted on flash silica gel (200 425 mesh). Visualization of TLC plates was via UV and phosphomolybdic acid staining. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were determined in CDCl 3 with TMS as the internal standard, (CD 3 ) 2 CO or DMSO d 6 Anhydrous tetrahydrof uran ( THF ) was obtained by distillation immediately prior to use, from sodium/benzophenone ketyl. A single mode cavity microwave synthesizer with a continuous irradiation at 2450 MHz and an infrared temperature control system was used. Optical rotation val ues were measured using the sodium D line. 5.4.2 General Pr Bromo A cids 5. 9 a d The compounds were synthesized following our established procedure. [06JOC3332 ] 5.4.3 General Pr H ydrazino A cids 5. 3a d H ydrazine hydrate (4.7 equ iv. ) was dissolved in ethanol (3 mL) and the solution of bromo acid s 5. 9 a c in ethanol ( 1 equ iv .) was added dropwise with water cooling The reaction mixture was irradiated under MW at 70 o C, 50W for 15 min The solid thus obtained was was hed with ether and dried on vaccum pump and recrystallized to give their corresponding hydrazino acids. (S) 2 Hydrazinylpropanoic acid ( 5.3a ) Recrystallized from ethanol water to give w hite microcrystals ( 48 %); mp 2 14 2 15 C; 1 H NMR ( D 2 O ) 3.22 3.10 (m, 1H), 1.13 (d, J = 6.9 Hz, 3H) ; 13 C NMR ( D 2 O ) 176.1, 61.2, 15.7 (S) 2 H ydrazinyl 4 methylpentanoic acid ( 5.3 b ) Recrystallized from ethanol water to give w hite microcrystals ( 33 %); mp 2 14 2 17 C; 1 H NMR ( D 2 O ) 3.22 3.10 (m, 1H), 1.13 (d, J = 6.9 Hz, 3H) ; 13 C NMR ( D 2 O ) 176.1, 61.2, 15.7 Anal. Calcd for C 6 H 14 N 2 O 2 : C, 49.30 ; H, 9.65 ; N, 19.16 ; found: C, 49.29 ; H, 10.08 ; N, 19.01

PAGE 99

99 (S) 2 Hydrazinyl 3 phenylpropanoic acid ( 5.3 c ) Recrystallized from hot water to give w hite microcrystals; mp 20 3 20 6 C; 1 H NMR ( d 1 TFA ) 8.10 7.76 (m, 5H), 5.12 5.00 (m, 1H), 4.20 4.05 (m,1H), 3.88 3.72 (m, 1H) ; 13 C NMR ( TFA ) 179.1, 135.7, 122.9, 119.1, 115.4, 111.6, 64.3, 38.3 Anal. Calcd for C 9 H 12 N 2 O 2 : C, 59.99 ; H, 6.71 ; N, 15.54 ; found: C, 60.33 ; H, 6.95 ; N, 15. 73 2 Hydrazinyl 3 methylbutanoic acid ( 5. 3 d ) Recrystallized from water and ethanol to give w hite microcrystals ( 40 %); mp 2 40 2 41 C; 1 H NMR ( D 2 O ) 3.48 (d, J = 4.5 Hz, 1H), 2.26 2.15 (m, 1H), 1.00 (d, J = 7.05 Hz, 6H) ; 13 C NMR ( D 2 O ) 191.1, 71.1, 28.6, 18.0, 18.0 Anal. Calcd for C 5 H 12 N 2 O 2 : C, 45.44 ; H, 9.15 ; N, 21.20 ; found: C, 45.82 ; H, 9.44 ; N, 21.12 5.4.4 General Procedure for th e Synthesis of Cbz A minoacyl benzotriazoles 5.10 a e The compounds were synthesized following our e sta blished procedure. [10JOC3938 ](Table 5 5 ) Table 5 5 Preparation of N Cbz aminoacyl)benzotriazoles 5.10 a f Entry Products Mp (C) Lit. Mp (C) 5.10 a Z L Ala Bt 113.0 115.0 114.0 115.5 5.10a+5.10 Z DL Ala Bt 110.0 112.0 112.0 113.0 5.10 b Z L Val Bt 106.8 108.3 73.0 74.0 5.10 c Z L Phe Bt 148.0 151.0 151.0 152.0 5.10 d Z L Trp Bt 104.0 105.0 99.0 101.0 5.10 e Z L Lys(Z) Bt 80.0 82.0 74.0 83.0 5.4.5 General Procedure f or the Synthesis of hybrid dipeptides 5 .11 a f A dried heavy walled Pyrex tube containing a small stir bar wa s charged with (Cbz protected aminoacyl) benzotriazole (1.0 equ iv hydrazino acid (1.0 equ iv .) and catalytic amount of triethylamine (Et 3 N) was dissolved in THF. The reaction mixture was

PAGE 100

100 expose d to microwave irradiatio n (100 W) at 70C for 15 min. Reaction mixture was allowed to cool through an inbuilt system until the temperature had fallen below 30 C (ca. 10 min). The solvent was removed under reduced pressure, and the residue was subjected to silica gel column using dic hloromethane ( DCM ) methanol as eluent s (S) 2 (2 ((S) 2 (((Benzyloxy)carbonyl)amino) 3 methylbutanoyl)hydrazinyl) propanoic acid ( 5. 11 a ). White microcrystals (61%); mp 140 141C; 1 H NMR (DMSO d 6 ) 9.42 (s, 1H), 7.40 7.25 (m, 6H), 5.02 (br s, 2H), 3.83 3 .75 (m, 1H), 3.55 3.45 (m, 1H), 1.90 1.82 (m, 1H), 1.16 (d, J = 6.9 Hz, 3H), 0.85 0.79 (m, 6H); 13 C NMR (DMSO d 6 ) 174.4, 170.3, 156.0, 137.1, 128.3, 127.7, 127.6, 65.4, 58.8, 57.1, 30.2, 19.1, 18.3, 16.6. Anal. Calcd for C 16 H 23 N 3 O 5 : C, 56.96; H, 6.87; N, 12.45; found: C, 56.64; H, 7.05; N, 12.07. (S) 2 (2 ((S) 2 (((Benzyloxy)carbonyl)amino) 3 phenylpropanoyl)hydrazinyl) 3 methylbutanoic acid ( 5. 11 b ). White powder (42 %) ; mp 126 128 C; 1 H NMR (DMSO d 6 ) 9.53 (d, J = 6.3 Hz, 1H), 7.53 (t, J = 9.2 Hz, 1H), 7.37 7.15 (m, 10H), 4.97 4.87 (m, 2H), 4.25 4.12 (m, 1H), 3.17 (d, J = 5.1 Hz, 1H), 3.05 (d, J = 4.8 Hz, 1H), 2.93 2.81 (m, 1H), 2.80 2.67 (m, 1H), 1.95 1.80 (m, 1H), 0.96 0.87 (m, 6H) ; 13 C NMR (DMSO d 6 ) 173.6, 170.2, 155.7, 137.8, 137.0, 129.2, 128.3, 128.1, 127.7, 127.5, 126.3, 68.1, 65.2, 54.8, 37.7, 29.5, 18.7, 18.5 Anal. Calcd for C 22 H 2 7 N 3 O 5 : C, 63.91; H, 6.58; N, 10.16; found: C, 63.67; H, 6.80; N, 10.55 (S) 2 (2 ((S) 2 (((Benzyloxy)carbonyl)amino) 3 (1H indol 3 yl)propanoyl)hydrazinyl) 4 methyl pentanoic acid ( 5. 11 c ). Isolated as white microcrystals from column chromatography using ethyl acetate: hexanes as eluents in a 5:5 ratio (50%); mp 81 83 C; 1 H NMR (DMSO d 6 10.79 (s, 1H), 9.54 (s, 1H), 7.62 (d, J = 7.8

PAGE 101

101 Hz 1H), 7.38 (d, J = 8.4 Hz, 1H) 7.32 7.21 (m, 5H), 7.13 7.12 (m, 1H), 7.07 (t, J = 7.1 Hz, 1H), 6.95 (t, J = 7.4 Hz, 1H), 4.96 4.87 (m, 2H), 4.27 4.22 (m, 1H), 3.46 3.33 (m 1H), 3.00 (dd, J 14.6, 4.2 Hz 1H), 2.88 (dd, J = 14.4, 9.6 Hz, 1H), 1.75 1.69 (m, 1H), 1.40 (t, J = 6.9 Hz, 2H), 0.88 0.84 (m, 6H); 13 C NMR (DMSO d 6 ) 174.6, 170.8, 155.7, 137.0, 136.0, 128.3, 127.7, 127.5, 127.2, 123.8, 120.8, 118.5, 118.2, 111.3, 110.0, 65.2, 60.7, 54.1, 28.0, 24.4, 22.6 (S) 2 (2 ((S) 2,6 Bis(((benzyloxy)carbonyl)amino)hexanoyl)hydr azinyl) 4 methylpentanoic acid ( 5.11 d ). Isolated as white microcrystals from recrystallization from diethyl ether (45%); mp 93 95C; 1 H NMR (DMSO d 6 7.26 (m, 12H), 7.22 (t, J = 5.7 Hz, 1H), 5.05 4.91 (m, 4H), 3.96 3.83 (m, 1H), 3.44 3.37 (m, 1H), 3.00 2.90 (m, 2H), 1.78 1.66 (m, 1H), 1.55 1.44 (m, 2H), 1.43 1.15 (m, 6H), 0.87 (t, J = 6.8 Hz, 6H); 13 C NMR (DMSO d 6 ) 174.7, 171.0, 156.1, 155.9, 137.3, 137.0, 128.4, 127.8, 65.4, 65.2, 60.7, 53.3, 31.7, 29.1, 24.4, 22.8, 22.7, 22.4. Anal Calcd for C 28 H 38 N 4 O 7 : C, 61.98; H, 7.06; N, 10.32; found: C, 62.07; H, 7.31; N, 10.46. (S) 2 (2 ((S) 2 (((B enzyloxy)carbonyl)amino)propanoyl)hydrazinyl) 3 phenyl propanoic acid ( 5. 11 e ). White microcrystals (68%); mp 113 114C; 1 H NMR (DMSO d 6 ) 9.45 (br s, 1H), 7.45 (d, J = 7.2 Hz, 1H), 7.40 7.32 (m, 5H), 7.30 7.16 (m, 6H), 5.06 4.88 (m, 2H), 4.10 3.98 (m, 1H), 3.68 (t, J = 6.3 Hz, 1H), 2.87 (d, J = 6.0 Hz, 2H), 1.17 (d, J = 8.4 Hz, 3H).; 13 C NMR (DMSO d 6 ) 173.0, 171.6, 155.6, 137.5, 137.0, 12 9.3, 128.3, 128.1, 127.7, 126.3, 65.3, 63.4, 48.7, 36.0, 18.2. Anal. Calcd for C 20 H 23 N 3 O 5 : C, 62.33; H, 6.01; N, 10.90; found: C, 62.10; H, 6.14; N, 10.84. (2S) 2 (2 (2 (((B enzyloxy)carbonyl)amino)propanoyl)hydrazinyl) 3 phenyl propanoic acid ( 5.11 e + 5. 11 ). White powder (71%); mp 145 150C; 1 H NMR (DMSO

PAGE 102

102 d 6 ) 9.64 (br s, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.35 7.15 (m, 11 H ), 5.01(s, 1 H), 4.10 3.98 (m, 1H), 3.74 (t, J = 6.6 Hz, 0.5H), 3.69 (t, J = 6.3 Hz, 0.5H), 2.90 (d, J = 6.6 Hz, 2H), 1.17 (d, J = 6.9 Hz, 3H ) ; 13 C NMR (DMSO d 6 ) 171.8, 172.7, 171.6, 171.3, 155.6, 137.4, 137.3, 137.0, 129.3, 128.3, 128.1, 127.7, 127.1, 126.3, 65.4, 63.6, 63.4, 48.8, 35.9, 35.8, 18.2. Anal. Calcd for C 20 H 23 N 3 O 5 : C, 62.33; H, 6.01; N, 10.90; found: C, 62.09 ; H, 5.93 ; N, 10.74 5.4.6 General Procedure for th e Synthesis of N Cbz Hydrazino A cids 5. 12 b c To the solution of hydrazino acid (1.0 eq uiv .) in water (2 mL), Et 3 N (2.0 equ iv .) and a solution of Cbz Bt (1.0 equ iv.) in aceto nitrile (CH 3 CN) (10 mL) was added. The reaction mixture w as stirred at room temperature until completion for 3 4 h (monitored by TLC). After that, the mixture was acidified with HCl 4N (2 mL). Acetonitrile was removed under reduced pressure and the crude product obtained was subjected to column chromatography. T he column was eluted with hexanes/ethyl acetate to give the desired product. (S) 2 (2 ((Benzyloxy)carbonyl)hydrazinyl) 4 methylpentanoic acid ( 5. 12 b ). White powder (74%); mp 119 120C; 1 H NMR (DMSO d 6 ) 8.56 (br s, 1H), 7.40 7.27 (m, 5H), 5.04 (dd, J = 16.5, 16.5 Hz, 2H), 3.46 (t, J = 6.9, 6.6 Hz, 1H), 1.80 1.67 (m, 1H), 1.38 (t, J = 6.0, 7.2 Hz, 2H), 0.88 0.84 (m, 6H) ; 13 C NMR (DMSO d 6 ) 174.8, 156.8, 136.9, 128.3, 127.8, 127.6, 65.4, 24.3, 22.6, 22.3. Anal. Calcd for C 14 H 20 N 2 O 4 : C, 59.98; H, 7.19; N, 9.99; found: C, 59.91; H, 7.37; N, 9.99. (S) 2 (2 ((Benzyloxy)carbonyl)hydrazinyl) 3 phenylpropanoic acid ( 5. 12 c ). White powder (74%); mp 177 178C; 1 H NMR (DMSO d 6 ) 8.70 (br s, 1H), 7.40 7.13 (m, 11H), 5.04 (s, 2H), 3.74 (t, J = 6.3 Hz, 1H), 2.86 (d, J = 6.3 Hz, 2H) ; 13 C NMR (DMSO d 6 ) 173.2, 156.9, 137.6, 136.9, 129.3, 128.3, 128.0, 127.8, 127.7, 126.2, 65.5, 36.0.

PAGE 103

103 Anal. Calcd for C 17 H 18 N 2 O 4 : C, 64.96; H, 5.77; N, 8.91; found: C, 65.02; H, 5.70; N, 8.95. 5.4.7 General Procedure fot the Chiral O A cylation of Compound 5.13 c A dried heavy walled Pyrex tube containing a small stir bar was charged with Cbz NH Phe Bt (0.15 g, 0.4 mmol), menthol (078 g, 0.5 mmol) and 4 dimethylaminopyridine ( DMAP ) ( 0.2 equiv ) dissolved in dichloromethane ( 5 mL). The reaction mixture was exposed to microwave irradiation (50 W) at 60C for specified 2 h. The reaction mixture was allowed to cool through an inbuilt system unti l the temperature fell below 30C. DCM was evaporated and solid thus obtain ed was packed in column on silica gel and elute with the gradient of ethyl acetate and hexanes mixtur e to afford 5.15 a Benzyl 2 ((S) 1 (((1R,2S,5R) 2 isopropyl 5 methylcyclohexyl)oxy) 1 oxo 3 phenylpropan 2 yl)hydrazinecarboxylat e ( 5.15a ) White microcrysta ls (49%); oil ; 1 H NMR (CDCl 3 ) 7.33 7.18 (m, 11H), 6.40 (br s, 1H), 5.14 5.00 (m, 2H), 4.75 4.60 (m, 1H), 4.00 3.92 (m, 1H), 3.10 2.91 (m, 2H), 1.82 1.60 (m, 4H), 1.46 1.22 (m, 2H), 1.08 0.96 (m, 1H), 0.86 0.77 (m, 8H), 0.69 (d, J = 7.2 Hz, 3H) ; 13 C NMR ( CDCl 3 ) 172.4, 156.8, 136.4, 136.1, 129.4, 129.3, 128.7, 128.4, 128.2, 127.1, 75.4, 67.3, 64.2, 47.0, 40.8, 37.1, 34.3, 31.5, 26.5, 23.5, 22.2, 20.9, 16.5 HRMS Calcd for C 2 7 H 3 7 N 2 O 4 :[ M+H ] + 453.2748 ; found: 453.2744 5.4.8 General Procedure fot the Chiral S A cylation of Compound s 5.13 b c N Cbz h ydrazino a cids 5. 13 ( 1 equiv. ) was dissolved in THF ( 10 mL) with thiol as a nucleophile 5. 14 (1 equiv.). T riethyl amine (2 equiv. ) was added to the solution and the reaction mixture was stirred at room tempertature for 4h. The reaction was monitored by thin layer chromatography ( TLC ) Once the reaction was completed the solvent was

PAGE 104

104 evaporated and oil thus obtained was purified by column chromatography on silica gel with the mixture of et hyl acetate and hexanes as eluent to afford 5. 15b c ( S) B enzyl 2 (1 (benzylthio) 4 methyl 1 oxopentan 2 yl)hydrazinecarboxylate ( 5.15 b ). Oil (61%); 1 H NMR (CDCl 3 ) 7.44 7.20 (m, 10H), 6.48 (br s, 1H), 5.19 5.14 (m, 2H), 4.09 (s, 2H), 3.90 3.78 (m, 1H), 1 .88 1.73 (m, 1H), 1.49 (t, J = 6.8 Hz, 2H), 1.10 0.85 (m, 6H); 13 C NMR ( CDCl 3 ) 203.0, 157.1, 137.5, 135.9, 129.0, 128.8, 128.6, 128.4, 127.4, 69.2, 67.6, 41.0, 32.9, 25.0, 23.2, 22.2. HRMS Calcd for C 21 H 26 N 2 O 3 S Na: [ M+Na] + 409.1556 ; found: 409.1572 (S) Benzyl 2 (1 ((2 methoxy 2 oxoethyl)thio) 1 oxo 3 phenylpropan 2 yl)hydrazinecarboxylate ( 5.9c ) Oil ( 63%); 1 H NMR (CDCl 3 ) 7.35 7.20 (m, 11H), 6.68 6.60 (m, 1H), 5.08 4.96 (m, 2H), 4.20 4.02 (m, 1H), 3.71 (s, 3H), 3.65 (s, 2H) 3.14 3.06 (m, 1H), 2.84 2.78 (m, 1H) ; 13 C NMR ( CDCl 3 ) 201.0, 169.3, 156.8, 135.7, 135.2, 129.3, 128.8, 128.6, 128.4, 128.2, 127.4, 70.4, 67.4, 52.8, 37.9, 30.7 HRMS Calcd for C 2 0 H 22 N 2 O 5 S Na:[ M+Na] + 425.1142 ; found: 425.1162

PAGE 105

105 C HAPTER 6 CONCLUSION S AND SUMMARY OF AC HIEVEMENTS This work is an extension to the benzotriazole methodology and its aim was to develop new synthetic routes to form compounds with biological interest. Chapter 1 presented an overview of 1 H benzotriazole methodology and some of its applications Although an extensive work was done with this molecule, new discoveries are constantly being made due to its wide applicability in different fields. T he chapter illustrated in particular the use of 1 H benzotriazole as an acylating agent [98CR409] and an in teresting motif for the synthesis of different heterocycl es [10CR1564] Chapter 2 presents a convenient route for the synthesis of enantiomerically pure 2,5 disubstituted 1,3,4 thiadiazoles possessing a free amino g roup from n ovel N (Cbz aminoacyl)thiosemi carbazides Indeed, t hese intermediates which were synthesized from N (Cbz aminoacyl) benzotriazole underwent a concurren t cyclization and deprotection Later, t he free amino group in the substituted thiadiazoles was coupled with also N (Cbz aminoacyl)benzo triazole and N (C bz dipeptid yl)benzotriazole under microwave conditions to give chirally pure thiadiazolyl amino acids and dipeptides. In view of the high selectivity and the fact that there is no other known method to make such compounds, this method represents a promising route to the preparation of various thiadiazoles substituted peptides. In this chapter the use of chirally pure amino acids and dipeptides with the benzotriazole methodology moiety resulted in the insertion of chirality to the divers e 1,3,4 thiadiazole which exhibits diverse biological properties [08CCL1427, 11EJC94].

PAGE 106

106 In Chapter 3 N (Pg tripeptidoyl)benzotriazoles and N (Pg tet rapeptidoyl) benzotriazoles were synthesized [11S2995] and used as convenient acylating reagents to form biologically active peptide scaffolds with a retention of chirality These chi rally pure N protected tri a nd tetra peptide conjugates were in fact coupled with sugars, steroids, terpenes and different heterocycles by O S N and C acylations in synth etically useful yields. Chapter 4 described a new microwave assisted synthesis of peptide vitamins conjugates. Vitamins B 3 H, E and D 3 were used in this work for their diverse applicability and activity [03JACS3452, 05OL1741 04JCR403, 07CR67 ], and the e ase in linking them to peptides through amide or ester bonds. This simple method with relatively short reaction time directly related to the microwave use, has prov en to be an alternative and sometimes better way to form these building blocks in comparison to the reported methods Moreover, upon the formation of these conjugates, we introduced a new activated and fairly stable form of biotin 4.3b t hat could be used in the wide biotinylation studies. All synthesized c ompounds retained their chirality based on NMR spectroscopy. Preliminary successful results in chapter 5 showed once again the applicability of benzotriazole methodology in the syntheses in solution of hydrazino peptides with one or two units of hydrazi no acids as well as the formation of their esters and thioesters form through already established methods in our group. Examples of these interesting peptidomimetics showed their importance in exhibiting biological activities which explain the importance i n develop ing new efficient method for their preparation

PAGE 107

107 To summarize, in this study, using the synthetic auxiliary property of 1 H benzotriazole, we were able to expand the already established methodology and develop new practical synthetic routes for the construction of structural motifs that will lead to the syntheses of compounds with biological interest.

PAGE 108

108 LIST OF REFERENCES The reference citation system employed throughout this research report is taken (vol. 102) Academic Press, 2011 (Ed. A. R. Katritzky). Each time a reference is cited a number letter code is designated to the corresponding reference with the first two or four if before 1910, numbers indicating the year followed by the letter code of th e journal and the page number in the end. Additional notes to this reference system are as follows: (i) Each reference code is followed by the conventional literature citation as depicted in the Advances in Heterocyclic Chemistry instruction for authors (ii) Less (iii) The list of references are arranged according to the designated code in the order of (a) year, (b) journal in alphabetical order, (c) page number 22JACS1510 P.C. Guha, J. Am. Chem. Soc ., 44 1510 (1922). 22JCS2542 S.M. Losanitch, J. Chem. Soc. 121 2542 (1922). 23JLAC1 E. Fromm, E. Layer, and K. Nerz, Justus Liebigs Annalen der Chemie 433 1 (1923). 36JPC268 A. Darabsky, J. Loevenich, O. Creifelds, B. Wilhelm, K. Erwin, B. Viktor, W. Hans, and B. Heinz, Journal fuer Praktische Chemie 146 268 (1936). 48JCS2249 A. Albert, R. Goldacre, and J. Phillips, J. Chem. Soc ., 2240 (1948). 49JCS1163 E. Hoggarth, J. Chem. Soc. 1163 (1949). 52USP2623877 U. S. Pat. 2623877, Chem. Abstr., 47 55018 (1952). 55JACS400 R.W. Young, and K.H. Wood, J. Am. Chem. Soc. 77 400 (1955).

PAGE 109

109 56USP2733245 U. S. Pat. 2733245, Chem. Abstr., 50 64808 (1956). 67B170 H.J. Klosterman G.L. Lamoure ux, and J.L. Parsons Biochemistry 6 170 (1967). 67YZ87 U. Hitoshi, F. Hiroko, I. Akira, and Y. Yoshio, Yakugaku Zasshi 87 1293 (1967). 68CJC3013 E. Ronwin, J.V. Roach, and B.L. Tucker, Canadian J. Chem. 46 3013 (1968). 71JACS6305 S. Kondo S. Shibahara, K. Maeda, and H. Umezawa, J. Am. Chem. Soc. 97 6305 (1971). 75ACS(B)93 H. Gustafsson and U. Ragnardsson Acta. Chem. Sc and. Serie B: Org. Chem. Biochem. 29 93 (1975). 76S224 M. Bodanszky, Y.S. Klausner, and M.A Ondetti, Peptide Synthesis 2nd ed.; Wiley & Sons: New York, 224 (1976). 78S803 S. Satyavan, Synthesis 803 (1978). 80JOM141 J.P. Gasparani, and R. Gassend, J. Organometallic Chem. 188 141 (1980). 83S975 D.T. Krieger, Science 222 975 (1983). 85JOC1457 C.L. Penney, P. Shah, and S. Landi, J. Org. Chem. 50 1457 (1985). 86B5774 T.P. King, S.W. Zhao, and T. Lam, Biochemsitry 25 5774 (1986). 86DSPD139 M.J. Humphrey, Delivery Syst. Pept. Drugs 139 (1986). 86JACS6397 L.A. Trimble, and J.C. Vederas, J. Am. Chem. Soc. 108 6397 (1986). 86JOC2613 S. Kim, and K.Y. Yi, J. Org. Chem 51 2613 (1986). 86S958 E. Wunsch, Synthesis 958 (1986). 86TCC109 G. Schmidt, Top. Curr. Chem. 136 109 (1986). 87AB99 M.L.G. Gardner, Adv. Biosci. 65 99 (1987).

PAGE 110

110 87T891 J. Viret, J. Gabard, and A. Collet, Tetrahedron 43 891 (1987). 88APC51 W.F. DeGrado, Adv. Protein Chem. 39 51 (1988). 88JMC2097 H. Shinkai, K. Toi, I. Kumashiro, Y. Seto, M. Fukuma, K. Dan, and S. Toyoshima, J. Med. Chem. 31 2092 (1988). 88T5525 D.A. Evans, T.C. Brition, R.L. Dorow, and J.F. Dellaria Jr., Tetrahedron 44 5525 (1988). 89JACS4856 A.K. Saha, P. Schultz, and H. Rapoport, J. Am. Chem. Soc. 111 4856 (1989). 90BJ249 V.J. Hruby, F. Al Obeidi, and W. Kazmierski, Biochem. J. 268 249 (1990). 90HC21 A. R. Katritzky and J. N. Lam, Heteroat. Chem ., 1 21 (1990). 90JACS9651 L.A. Carpino, D. Sadat Aalaee, H.G. Chao, and R.H. Desselms, J. Am. Chem. Soc. 112 9651 (1990). 90JCB637 K.G. Rothberg, Y. Ying, J.F. Kolhouse, B.A. Kamen, and R.G.W. Anderson, J. Cell. Biol. 110 637 (1990) 90JCR213 V.H.L. Lee, Contr. Rel. 13 213 (1990). 90T7175 K. Afarinkia, C.W. Rees, and J.I.G. Cadogan, Tetrahedron 46 7175 (1990) 91B793 A. Aubry, D. Bayeul, J.P. Mangeot, J. Vidal, S. Sterin, A. Collet, A. Lecoq, and M. Marraud, Biopolymers 31 793 (1991). 91CB1809 A.R. Katritzky, X. Lan, and J.N. Lam Chem. Ber. 124 1809 (1991) 91JCS435 J. Vidal, J. Drouin, and A. Collet, J. Chem. Soc., Chem. Commun. 435 (1991). 91PNAS10407 Z. Zhang, and D.B. McCormick, Proc. Natl. Acad Sci. 88 10407 (1991). 91T2683 A.R. Katritzky, S. Rachwal, and G.J. Hitchings, Tetrahedron 47 2683 (1991).

PAGE 111

111 92ADDR39 E.S. Swenson, and W.J. Curatolo, Adv. Drug. Del. Rev. 8 39 (1992) 92ADDR253 P.L. Smith, D.A. Wall, C.H. Gochoco, and G. Wilson, Adv. Drug Del. Res. 8 253 (1992). 92FRP 2654338 FR Patent 2654338, 1991; Chem. Abstr. 116 67183 (1992) 92LAC843 A.R. Katritzky, J. Li and N. Malhotra, Liebigs Ann. Chem ., 843 (1992). 93ACR319 A. Kobata, Acc. Chem. Res. 26 319 (1993). 93ACIE1244 A. Giannis, and T. Kolter, Angew. Chem. Int. Ed. Engl. 32 1244 (1993). 93G97 A. Varki, Glycobiology 3 97 (1993). 94BC312 T. Zhu, and S. Stein, Bioconjugate Chem. 5 312 (1994). 94IJC529 S.H. Goh, N.F. Hew, A.W. Norhanom, and M. Yadav, Int. J. Cancer 57 529 (1994). 94IJPPR378 S. Zerkout, V. Dupont, A. Aubry, J. Vidal, A. Collet, A. Vicherat, and M. Marraud, Int. J. Pep. Protein Res 44 378 (1994). 94JACS7955 R. Boyce, G. Li, H.P. Nestler, T. Suenaga, and W.C. Still, J. Am. Chem. Soc. 116 7955 (1994). 94JMC293 I. Islam, K. Y. Ng, K.T. Chong, T.J. McQuade, J.O. Hui, K.F. Wilkinson, B.D. Rush, M.J. Ruwart, R.T. Borchardt, and J.F. Fisher, J. Med. Chem. 37 293 (1994). 94JOC2799 F. Tomas, J. Catalan, P. Perez, and J. Elguero, J. Org. Chem. 59 2799 (1994). 94SL719 F. Babudri, V. Fiandanese, G. Marchese, and A. Punzi, Synlett 719 (1994). 94T4399 M.J. Burk, J.P. Martinez, J.E. Feaster, and N. Cosford, Tetrahedron 50 4399 (1994). 95SC539 A.R. Katritzky, L. Xie, and D. Cundy, Synth. Commun. 25 539 (1995).

PAGE 112

112 96ACR268 L.A. Carpino, M. Beyermann, H. Wenschuh, and M. Bienert, Acc. Chem. Res 29 268 (1996). 96CR683 R.A. Dwek, Chem. Rev. 96 683 (1996). 96EMT162 I. Gill, R. Lopez Fandino, X. Jorba, and E.N. Vulfson, Enzyme Microb. Technol. 18 162 (1996) 96JACS1813 Y. Cheng, T. Seunaga, and W.C. Still, J. Am. Chem. Soc. 118 1813 (1996). 97ADD1 A. Giannis, and F. Rubsam, Advances Drug. Res. 29 1 (1997). 97TA1605 C. Cativiela, M. Diaz de Villegas, and J.A. Galvez, Tetrahedron : Asymmetry 8 1605, (1997). 97JOC4148 A.R. Katritzky, C.N. Fali and J. Li, J. Org. Chem ., 62 4148 (1997). 97JOC9099 O.H. Justiz, R. Fern, andez Lafuente, J.M. Guisan, P. Negri, G. Pagani, M. Pregnolato,, and M. Terreni, J. Org. Chem. 62 9099, (1997). 98CR409 A.R. Katritzky, X. Lan, J.Z. Yang, and O.V. Denisko, Chem. Rev. 98 409 (1998). 98BMCL2433 P. Arya, N. Alibhai, H. Qin, and G.W. Burton, Bioorg. Med. Chem. Lett. 8 2433 (1998). 98JOC9678 F. Albericio, J.M. Bofill, A. El Faham, and S.A. Kates, J. Org. Chem. 63 9678 (1998). 98JPR180 O. Melnyk, M. Bossus, D. David, C. Rommens, and H. Gras Masse, J. Peptide Res. 52 180 (1998). 98WO9838179 PCT Int. Appl. WO 9838179, Chem. Abstr. 129 216923 ( 1998). 99COCB714 K.D. Stigers, M.J. Soth, and J.S. Nowick, Curr. Opinion Chem. Bio. 3 714 (1999). 99JCC55 A.K. Ghose, V.N. Viswanadhan, and J.J. Wendoloski, J. Comb. Chem 1 55 (1999) 99TL2045 J. Klose, A. El Faham, P. Henklein, L.A. Carpino, and M. Bienert, Tetrah edron Lett. 40 2045 (1999).

PAGE 113

113 00ACIE145 H. De Muynck, A. Madder, N. Farcy, P.J. De Clercq, M.N. Perez Payan, L.M. Ohberg, and A.P. Davis, Angew. Chem., Int. Ed. 39 145 (2000). 00JACS5849 H. Wang, C. Burda, G. Persy, and J. Wirz, J. Am. Chem. Soc. 122 5849 (2000). 00JCS(PT1)2311 R.M. Adlington, J.E. Baldwin, D. Catterick, G.J. Pritchard, and L.T. Tang, J. Chem. Soc., Perkin Trans. 1 2311 (2000). 00JOC8210 A.R. Katritzky, H. Y. He, and K. Suzuki J. Org. Chem. 65 8210 (2000). 00M3511 K. Ishihara, S. Ohara, and H. Yamamoto, Macromolecules 33 3511 (2000). 00PR825 J. Alsenz, G.J. Russell Jones, S. Westwood, B. Levet Trafit, and P. Chris de Smidt, Pharm. Res. 17 825 (2000) 01ARKIVOC19 A.R. Katritzky, M. Wang S. Zhang Arkivoc ix 19 (2001). 01CR3893 D.J. Hill, M.J. Mio, R.B. Price, T.S. Hughes, and J.S. Moore, Chem. Rev. 101 3893 (2001). 01EJOC1533 I. Jeric, and S. Horvat, Eur. J. Org. Chem. 1533 (2001). 01JACS247 R. Gunther, and H. J. Hofmann, j. Am. Chem. Soc. 123 247 (2001) 01JOC443 D. Bonnet, N. Ollivier, H. Gras Masse, and O. Melnyk, J. Org. Chem. 66 443 (2001). 01JMC931 F. Clerici, and D.J. Pocar, J. Med. Chem. 44 931 (2001). 01T9859 C. Booth, R.J. Bushby, Y. Cheng, S.D. Evans, Q. Liu, and H. Zhang, Tetrahedron 57 9859 (2001) 01TL1887 A. Carre, P. Le Grel, and M. Baudy Tetrahedron Lett. 42 1887 (2001). 02ACR717 M. Lared, C. Moberg, and A. Hallberg, Acc. Chem. Res. 35 717 (2002). 02BMC2893 H.N. Dogan, A. Duran, S. Rollas, G. Sener, M.K. Uysal, and D. Gulen, Bioorg. Med. Chem. 10 2893 (2002).

PAGE 114

114 02BMCL3033 T. Yasukata, H. Shindo, O. Yoshida, Y. Sumino, T. Munekage, Y. Narukawa, Y. Nishitiani, Bioorg. Med. Chem. Lett. 12 3033 (2002). 02CMC683 D.A. Gewirtz, M.S. Gupta, and S. Sundaram, Curr. Med. Chem.: Anti Cancer Agents 2 683 (2002). 02JCC552 A. Madder, L. Li, H. De Muynck, N. Farcy, D. Van Haver, F. Fant, G. Vanhoenacker, P. Sandra, A.P. Davis, P.J. De Clercq, J. Comb. Chem. 4 552 (2002). 02JOC6372 A. DalPozzo, M. Ni, L. Muzi, A. Caporale, R. de Castiglione, B. Kaptein, Q.B. Broxterman, and F. Formaggio, J. Org. Chem 67 6372 (2002). 02S1388 C. Najera, Synlett 9 1 388 (2002). 03ARK155 C. Wang, J. Chen, Q. Song, Z. Li, and Z. Xi, Arkivoc ii 155 (2003). 03BMC4881 K. Bouget, S. Aubin, J. G. Delcros, Y. Arlot Bonnemains, and M. Baudy Bioorg. Med. Chem. 11 4881 (2003). 03CEJ4586 A. R. Katritzky and B. V. Rogovoy, Chem. Eur. J ., 9 4586 (2003). 03JACS3452 Y. Weizmann, F. Patolsky, E. Katz, and I. Willner, J. Am. Chem. Soc. 125 3452 (2003). 03JOC4932 A.R. Katritzky, A.A.A. Abdel Fattah, and M. Wang, J. Org. Chem. 68 4932 (2003). 03JOC5720 A.R. Katritzky, K. Suzuki, S.K. Singh, and H Y. He, J. Org. Chem. 68 5720 (2003). 03S751 T. Matsumoto, M. Watanabe, S. Mataka, and T. Thiemann, Steroids 68 751 (2003). 03S2795 A.R. Katritzky, Y. Zhang, and S.K. Singh, Synthesis 2795 (2003). 04BC312 T. Zhu, and S. Stein, Bioconjugate Chem. 5 312 (1994). 04BMCL801 A.V. Kachalova, D.A. Stetsenko, M.J. Gait, and T.S. Oretskaya, Bioorg. Med. Chem. Lett. 14 801 (2004).

PAGE 115

115 04CC2356 C. Galli, P. Gentili, O. Lanzalunga, M. Lucarini and G.F. Pedulli, Chem. Commun ., 2356 (2004). 04CRC3 F. Coutrot, C. Grison, and P. Coutrot, C. R. Chimie 7 3 (2004). 04JCR 403 C. Ostacolo, F. Marra, S. Laneri, A. Sacchi, S. Nicoli, C. Padula, and P. Santi, J. Controlled Release 99 403 (2004). 04OL3433 B. Ding, U. Taotofa, T. Orsak, M. Chadwell, and P.B. Savage, Org. Lett. 6 3433 (2004). 04T2447 S. Y. Han, and Y. A. Kim, Tetrahedron 60 2447 (2004). 04TL3569 I. Bouillon N. Brosse R. Vanderesse, and B. Jamart Gregoire, Tetrahedron Letters 45 3569 (2004). 04S1826 B. Iorga, and J. M. Campagne, Synlett 1826 (2004). 04S2645 A.R. Katritzky, K. Suzuki, and S.K. Singh, Synthesis 16 2645 (2004). 04JOC2976 A.R. Katritzky, S. Ledoux, R.M. Witek, and S.K. Nair, J. Org. Chem. 69 2976 (2004). 05ARK36 A.R. Katritzky, A.A. Shestopalov, and K. Suzuki, Arkivoc (vii) 36 (2005). 05 CC4815 M. Skander, C. Ma lan, A. Ivanova, and T.R. Ward, Chem. Comm. 4815 (2005). 05OL1741 S. Bhuniya, S.M. Park, and B.H. Kim, Org. Lett. 7 1741 (2005). 05S397 A.R. Katritzky, P. Angrish, D. Huer, and K. Suzuki, Synthesis 3 397 (2005). 05T10827 C.A.G.N. Montalbetti, and V. Falque, Tetrahedron 61 10827 (2005). 06ARK226 A.R. Katritzky, N.M. Khashab, and A.V. Gromova, Arkivoc iii 226 (2006). 06BC261 E. Schirrmacher, C. Beck, B. Brueckner, F. Schmitges, P. Siedlecki, P. Bartenstein, F. Lyko, an d R. Schirrmacher, Bioconjugate. Chem. 17 261 (2006).

PAGE 116

116 06CBDD326 A.R. Katritzky, G. Meher, and P. Angrish, Chemical Biol. Drug Design 68 326 (2006). 06CC2335 K.M. Bhattarai, V. Del Amo, G. Magro, A.L. Sisson, J. B. Joos, J.P.H. Charmant, A. Kantacha, and A.P. Davis, Chem. Commun. 2335 (2006). 06CMC813 D.B. Salunke, B.G. Hazra, and V.S. Pore, Curr. Med. Chem. 13 813 (2006). 06JOC3332 R. Moumne, S. Lavielle, and P. Karoyan, J. Org. Chem. 71 3332 (2006). 06OL1625 Z. Kaleta, B.T. Makowski, T. Soos, and R. Dembinski, Org. L ett. 8 1625 (2006). 06S411 A.R. Katritzky, P. Angrish, and K. Suzuki, Synthesis 411 (2006). 06S2376 U. Ursic, D. Bevk, S. Pirc, L. Pezdirc, B. Stanovnik, and J. Svete, Synthesis 2376 (2006). 06S3231 A.R. Katritzky, S.R. Tala, and S.K. Singh, Synthesis 3231 (2006). 06T6876 E. Prats Alfonso, F. Garcia Martin, N. Bayo, L.J. Cruz, M. Pla Roca, J. Samitier, A. Errachid, and F. Albericio Tetrahedron 62 6876 (2006). 07BMC5738 U. Salgin Goksen, N. Gokhan Kelekci, O. Goktas, Y. Koysal, E. Kilic, S. Isik, G. Aktay, and M. Ozalp, Bioorg. Med. Chem. 15 5738 (2007). 07CR67 X. F. Wang, M. Birringer, L. F. Dong, P. Veprek, P. Low, E. Swettenham, M. Stantic, L. H. Yuan, R. Zobalova, K. Wu, M. Ledvina, S.J. Ralph, and J. Neuzil, Cancer Res. 67 3337 (2007) 07HC316 T. Akhtar, S. Hameed, N.A. Al Masoudi, and K.M. Khan, Heteroatom Chem. 18 316 (2007). 07JAPS2989 S. H. Son, and J. Jegal, J. Appl. Polymer Sci. 106 2989 (2007). 07M103 O. Pintilie, L. Profire, V. Sunel, M. Popa, and A. Pui, Molecules 12 103 (2007). 07MCP1761 M. Fonovic, S.H.L. Verhelst, M.T. Sorum, and M. Bogyo, Molecular & Cellular Proteomics 6 1761 (2007)

PAGE 117

117 07RJAC2165 S.A. Malin, B.M. Laskin, and A.S. Malin, Russian J. Applied Chem. 80 2165 (2007). 08APR1231 S. H. Li, G. Li, H. M. Huang, F. Xiong, C. M. Liu, and G. G. Tu, Arch. Pharm. Res. 31 1231 (2008). 08BMC2745 J.T. Randolph, X. Zhang, P.P. Huang, L.L. Klein, K.A. Kurtz, A.K. Konstantinidis, W. He, W.M. Kati, and D.J. Kempf, Bioorg. Med. Chem ., 1 8 2745 (2008). 08BMC6009 B. Severino., F. Fiorino, E. Perissutti, F. Frecentese, G. Cirino, F. Roviezzo, V. Santagada and G. Caliendo, Bioorg. Med. Chem ., 16 6009 (2008). 08BMC6663 G. G. Tu, S. H. Li, H. M. Huang, G. Li, F. Xiong, X. Mai, H. W. Zhu, B. H. Kuang, and F. Wen, Bioorg. Med. Chem ., 16 6663 (2008). 08CCL928 T. Wang, Y.H. Zhang, S. Yu, H. Ji, Y.S. Lai, and S.X. Peng, Chinese Chem. Lett ., 19 928 (2008). 08CCL1427 M.R. B, and ay, and A. Rauf, Chinese Chem. Lett ., 19 1427 (2008). 08EJC963 S. Hussain, J. Sharma, and M. Amir, Eur. J. Chem ., 5 963 (2008). 08GJ6 33 F.J. Munoz, A. Rumbero, J.V. Sinisterra, J.I. Santos, S. Andre, H. J. Gabius, J. Jimenez Bsrbero, and M.J. Hernaiz, Glucoconj. J. 25 633 (2008). 08OL4453 R. Vallinayagam, J. Weber, and R. Neier, Org. Lett. 10 4453 (2008). 08T210 S. Nara, V. Tripathi, S.K. Chaube, K. Rangari, H. Singh, K.P. Kariya, and T.G. Shrivastav, Talanta 77 210 (2008). 08TL4746 J.I. Gavrilyuk, A.J. Lough, and R.A. Batey, Tetrahedron Lett ., 49 4746 (2008). 09ACIE1022 A.K. Petrus, T.J. Faichild, and R.P. Doyle, Anhew. Chem. Int. Ed. 48 1022 (2009). 09ARK47 A.R. Katritzky, A. Singh, D.N. Haase, and M. Yoshioka, Arkivoc (viii) 47 (2009).

PAGE 118

118 09BMC1214 H.C. Jeong, N.S. Kang, N. J. Park, E.K. Eul, and Y. S. Jung, Bioorg. Med. Chem ., 1 9 1214 (2009 ). 09BMCL6 632 S.H. Lee, H.J. Seo, M.J. Kim, S.Y. Kang, S. H. Lee, K. Ahn, M. Lee, H. K. Han, J. Kim, and J. Lee, Bioorg. Med. Chem. lett. 19 6632 (2009). 09EJMC3340 M. X. Wei, L. Feng, X. Q. Li, X. Z. Zhou, and Z. H. Shao, Eur. J. Med. Chem ., 44 3340 (2009). 09FMC361 A.A. Zompra, A.S. Galanis, O. Werbitzky, and F. Albericio, Future Med. Chem. 1 361 (2009). 09JAE269 D. Gopi, K.M. Govindaraju, V. Collins Arun Prakash, V. Manivannan, and L. Kavitha, J. Appl. Electrochem. 39 269 (2009). 09JCR391 T. Matsumoto K. Shiine, S. Mataka, and T. Thiemann, J. Chem. Res. 391 (2009). 09JOC8690 A.R. Katritzky, I. Avan, and S.R. Tala, J. Org. Chem. 74 8690 (2009). 09JPS2364 F. Marra, C. Ostacolo, S. Laneri, A. Bernardi, A. Sacchi, C. Padula, S. Nicoli, and P. Santi, J. Pharm aceutical Sc ience 98 2364 (2009). 09M2621 M. Moise, V. Sunel, L. Profire, M. Popa, J. Desbrieres, and C. Peptu, Molecules 14 2621 (2009). 09MC431 H.A. Abd el Aziz, B.F. Abdel Wahab, M.A.M.Sh. El Sharief, and M.M. Abdulla, Monatsh. Chem. 140 431 (2009). 09MCR538 J. H. Shi, J. H. Xiao, and D. Z. Wei, Med. Chem. Res. 18 538 (2009). 09MRC142 L.I. Larina, and V. Milata, Magn. Reson. Chem. 47 142 (2009). 09PSSRE523 B.X. Hu, Z.L. Shen, J. Lu, X.Q. Hu, W.M. Mo, N. Sun, and D. Xu, Phosphorus, Sulfur Silicon Relat. Elem. 184 523 (2009). 09S1708 A.R. Katritzky, S.R. Tala, N.E. Abo Dya, and Z.K. Abdel Samii, Synthesis 10 1708 (2009). 09S2392 A.R. Katritzky, P. Angrish, E. Todadze, Synlett, 2329 (2009).

PAGE 119

119 09T9702 K. Harju, N. Manevski, and J. Yli Kauhaluoma, Tetrahedron 65 9702 (2009). 10BMCL499 S. Leger, W.C. Black, D. Deschenes, S. Dolman, J. P. Falgueyret, M. Gagnon, S. Guiral, Z. Huang, J. Guay, Y. Leblanc, et al., Bioorg. Med. Chem. Lett. 20 499 (2010). 10BMCL6716 N.C. Ulrich, C.H. Kuder, R.J. Hohl, and D.F. Wiemer, Bioorg. Med. Chem. Lett. 20 6716 (2010) 10CR1564 A.R. Katritzky, and S. Rachwal, 110 1564 (2010). 10JCCS1077 J. Tao, D. Z. Wang, and L. H. Cao Jing, J. Chin. Chem. Soc. 57 1077 (2010). 10JOC2111 J.J. Reczek, E. Rebolini, and A.R. Urbach, J. Org. Chem. 75 2111 (2010). 10JOC3938 A.R. Katritzky, K. Bajaj, M. Charpentier, and E.G. Zadeh, J. Org. Chem. 75 3938 (2010). 10JMC432 A. Pratesi, F. Bucelli, I. Mori, M. Chinol, A. Verdoliva, G. Paganelli, V. Rivieccio, L. Gariboldi, and M. Ginanneschi. J. Med. Chem. 53 432 (2010). 10RCB241 A.Y. Spivak, R.R. Mufazzalova, E.R. Shakurova, V.N. Odinokov, and U.M. Dzhemilev, Russian Chem. Bull. 59 241 (2010). 10S933 M. Krasavin, E. Bushkova, V. Parchinsky, and A. Shumsky, Synthesis 933 (2010) 10S1337 A.R. Katritzky, N.E. Abo Dya, S.R. Tala, E.H. Ghazvini Zadeh, K. Bajaj, S.A. El Feky, Synlett 9 1337 (2010). 10TL 6410 M. Pretze, F. Wuest, T. Peppel, M. Kockerling, and C. Mamat, Tetrahedron Lett. 51 6410 (2010). 11ARK212 S. Biswas, S.R. Tala, A. Kalatozishvili, and A.R. Katritzky, Arkivoc iii 212 (2011). 11CBDDxxx K. Bajaj, S.S. Panda, C. El Nachef, and A.R. Katrit zky, Chemical Biol. Drug Design (2011) manuscript submitted 11CRxxx A. El Faham, and F. Albericio, Chem. Rev. xxx (2011)

PAGE 120

120 11EJC94 R. Kruszynski, and T. Sieranski, Eur. J. Med. Chem. 9 94 (2011). 11JACS6868 J. Zhou, J. He, B. Wang, W. Yang, and H. Ren, J. Am. Chem. Soc. 133 6868 (2011). 11OL2102 R.K. Kumar, M.A. Ali, and T. Punniyamurthy, Org. Lett. 13 2102 (2011). 11S 2995 A. Abdelmajeid, S.R. Tala, M.S. Amine, and A.R. Katritzky, Synthesis 2995 ( 2011 )

PAGE 121

121 BIOGRAPHICAL SKETCH Claudia El Nachef was born in Saida, Lebanon and was raised in Beirut. She received her Bachelor of S cience in c hemistry in 2003 from Saint Joseph University, Le banon, and her master in chemistry of b iomolecules in 2005 from the University of Montpellier II, France. For the subsequent two years, Claudia worked under the mentoring of Professor Makhluf J. Haddadin as his research assistant at the American University of Beiru t. She has since joined the Ph .D. program at the Chemistry Department at the University of Florida under the supervision of Professor Alan R. Katritzky. During her course of study at the University of Florida, Claudia was awarded the Proctor and Gamble Award for Excellence in Graduate Research and the Department of Chemistry Teac hing Award. She receive d her Ph.D. from the University of Florida in the fall of 2011.