close

Вход

Забыли?

вход по аккаунту

?

Microwave Assisted Synthesis of Medicinally Relevant Indole Conjugates Utilizing Modified Hemetsberger-Knittel Indole Synthesis

код для вставкиСкачать
Microwave Assisted Synthesis of Medicinally Relevant Indole Conjugates Utilizing Modified
Hemetsberger-Knittel Indole Synthesis
by Allison E. Murdza
Bachelor of Science in Chemistry, Northeastern University
A thesis submitted to
The Faculty of
The College of Science of
Northeastern University
in partial fulfillment of the requirements
for the degree of Master of Science
April 22, 2016
Thesis directed by
Dr. Graham B. Jones
Dr. Michael Pollastri
Professor of Chemistry and Chemical Biology
ProQuest Number: 10102669
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
ProQuest 10102669
Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author.
All rights reserved.
This work is protected against unauthorized copying under Title 17, United States Code
Microform Edition © ProQuest LLC.
ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, MI 48106 - 1346
© 2016
Allison Murdza
ALL RIGHTS RESERVED
ii
DEDICATION
This work is dedicated to my family for their utmost support and guidance this past year.
Without you I would not have been this successful!
Thank you!
iii
ACKNOWLEDGMENTS
This work would not have been completed without the support and guidance from all my
family, friends, colleagues, mentors, and professors. I am very grateful for each and every person
who has helped me on this adventure.
First and foremost, I would like to express my sincere gratitude to my old advisor, Dr.
Graham B. Jones for convincing me to attend Northeastern for my undergraduate career, for
teaching me Organic Chemistry 2, and for allowing me to join his laboratory my sophomore
year. Without your persistence and guidance I never would have succeeded in obtaining my
Bachelor of Science and my Master of Science degrees from Northeastern University, let alone a
year apart from each other. Thank you for encouraging me to grow as an independent researcher.
I would like to thank the members of my committee, Professors Michael Pollastri, Robert
Hanson, and George O’Doherty for their wonderful help and guidance during the preparation of
this thesis. Thank you for your valuable time and commitment towards completing my degree. I
truly appreciate all that you have done to help me write and put together this thesis this final
semester. A special thank you to Professor Pollastri, for without your wonderful advising this
process would not have been as successful. Thank you.
Thank you to the members of the Jones Lab, for not only making my graduate career a
success, but for teaching me the skills to be an excellent researcher during my undergraduate
career as well. I can’t believe my time in lab has finally come to an end. Thank you especially to
Meaghan and Enrico for all your valuable insight and help during the rough HK chemistry times,
and your help Meaghan with the peptide work. A special thanks to Chiara for helping me to
assimilate to an academic lab those early years of my undergraduate career, thank you.
iv
Additionally, I would like to send my sincere thanks to Alexandra Hendricksen for
helping me with the GC instruments, and for always putting a smile on my face when I walked in
the door looking a little more stressed out than usual. Getting to know you over the past six years
from general chemistry lab to my graduate research has been a pleasure. Thank you also to
Roger Kautz for helping me, and for fixing the NMR many a times, I truly appreciate it. This
work would not have been completed without either of your help.
Thank you to Asli Murtaugh, who was my first, and third, co-op manager. Thank you for
being an amazing mentor and friend to me these past four years. You allowed me to explore all
the areas of chemistry and business that Cubist and Merck had to offer. Working with you for
almost two years of my young professional career has taught me a wealth of knowledge that I
hope expand on with completion of this degree. Thank you for everything that you have done for
me over the past few years.
I would like to thank another Jones Lab member, Megan Sheehan, for being an amazing
friend, and all around amazing person these last six years. I can’t believe how the time has flown.
I know we will be in each other’s lives until we are old and grey, and I can’t wait to see what the
future holds. It has been such a pleasure getting to know you throughout our undergraduate and
graduate careers Meg. You will be so successful in whatever you decide to do next.
Congratulations on graduating with your Master’s degree as well! I am so proud of all we have
accomplished!
I would like to extend a very heartfelt, and very special thank you to my family. To my
mom and dad, thank you for your continued support during my undergraduate years, and
especially this last year of my graduate career. There were many times I struggled this year, and
you helped me through it. Thank you for being the best role models, and motivators, and for
v
allowing me to pursue my dreams! To Daniel, thank you for being my inspiration these past
twenty-two years. Without you I never would have become as passionate about science as I am
today. Your perseverance and determination to overcome whatever life throws your way
continues to amaze me everyday. I don’t think a big sister could be more proud of her little
brother than I am of you. Thank you for continuing to love me as much as I love you!
vi
ABSTRACT OF THESIS
Indoles are a class of nitrogen containing heterocycles first synthesized by Bayer AG in
1866 that have a broad variety of biological activities. They have widely been recognized as a
privileged scaffold and are best known for their medicinal properties in the pharmaceutical
industry. The indole nucleus signifies a vast importance in the world as indoles are built into
peptides, proteins, are the skeleton of many natural products, and are the core of many successful
marketed drugs. To date over 10,000 unique indole derivatives have been discovered as being
biologically active compounds due to their excellent binding affinity to various receptors. Upon
entering the 21st century, drug discovery and development has experienced a lack of innovative
ideas for new chemical entities. It is well known that the FDA has placed stricter safety, quality
and efficacy regulations on pharmaceutical companies over the last few decades. This, coupled
with long developmental processes and immense financial investments, has placed
pharmaceutical companies in innovative strains regarding their small-molecule pipelines.
New research over the latter part of the past century has led to new biological drug
entities that have many advantages over small molecules, but also some disadvantages as well.
Recent scientific advancements have also led to the discovery of peptide-based pharmaceuticals
and peptide-drug conjugates. These compounds fill the molecular weight gap between small
molecule and protein-based drugs while combining the advantages of both. A novel approach to
synthesizing small-molecule indole-peptide conjugates utilizing microwave-assisted
Hemetsberger-Knittel indole synthesis that builds upon previous efforts in our laboratory is
described in the remainder of this work.
vii
TABLE OF CONTENTS
Dedication
iii
Acknowledgements
iv
Abstract of Thesis
vii
Table of Contents
viii
List of Figures
x
List of Schemes
xi
List of Tables
xii
List of Abbreviations
xiii
Chapter 1: Introduction
1
1.1 Medicinal Importance of Indoles
1.1.1 Indoles in Modern Drug Discovery
1.1.1.1 Biological Activities
1.1.2 FDA Approved Indole Drugs
1.2 Indole Synthesis Methods
1
2
3
6
8
1.2.1 Fisher Indole Synthesis
8
1.2.2 Bischler Indole Synthesis
10
1.2.3 Nenitzescu Indole Synthesis
11
1.2.4 Bartoli Indole Synthesis
11
1.2.5 Hemetsberger-Knittel Indole Synthesis
12
1.3 Significance of Indole Peptide Conjugates
13
1.3.1 FDA Approved Peptide and Protein-Based Drugs
14
1.3.2 Issues with Protein, Peptide, and Small Molecule Drugs
17
viii
1.3.3 The Need for Peptide Drug Conjugates
1.4 Research Objective
1.4.1 Importance of the Nitro Group
19
21
22
Chapter 2: Efforts Towards Indole Peptide Conjugate Synthesis
24
2.1 Thermal Optimization of Indole Intermediates
24
2.1.1 Previous efforts in our Laboratory
25
2.1.2 Optimizations
27
2.2 Peptide Synthesis
33
2.3 Microwave Assisted Synthesis of 6-nitro-1H-Indole Conjugates
36
2.3.1 Previous Efforts in our Laboratory
36
2.3.2 Microwave Optimization
38
2.4 Conclusions and Future Work
Chapter 3: Experimental
41
45
3.1 General Experimental Information
45
3.2 Experimental Details of Indole Intermediates
47
3.3 Experimental Details of Peptide Synthesis
52
3.4 General Methods
54
3.4.1 Thermal Amino Acid and Peptide Addition
54
3.4.2 Microwave Assisted Hemetsberger-Knittel Synthesis
58
References
61
Appendix
63
ix
LIST OF FIGURES
CHAPTER 1: Introduction
Figure 1.1: Indole Nucleus Structure
1
Figure 1.2: Biological Activities of the Indole Nucleus
3
Figure 1.3: Important Naturally Occurring Indoles
5
Figure 1.4: Examples of Clinically Used Indoles
7
Figure 1.5: Successful Marketed Peptide-Based Therapeutic Zoladex
16
Figure 1.6: Blockbuster Agents That Contain Fluorine
22
CHAPTER 2: Efforts Towards Indole Peptide Conjugate Synthesis
Figure 2.1 The Effect of Different Solvents on the % Conversion to Indoles
39
x
LIST OF SCHEMES
CHAPTER 1: Introduction
Scheme 1.1: Fisher Indole Synthesis
9
Scheme 1.2: Bischler Indole Synthesis
10
Scheme 1.3: Nenitzescu Indole Synthesis
11
Scheme 1.4: Bartoli Indole Synthesis
12
Scheme 1.5: Hemetsberger-Knittel Indole Synthesis
13
CHAPTER 2: Efforts Towards Indole Peptide Conjugate Synthesis
Scheme 2.1: Synthesis of methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)glycinate
24
Scheme 2.2: Synthesis of methyl azidoacetate
25
Scheme 2.3: Synthesis of methyl(Z)-2-azido-3-(4-nitrophenyl)acrylate
25
Scheme 2.4: Synthesis of (Z)-2-azido-3-(4-nitrophenyl)acrylic acid
26
Scheme 2.5: Synthesis of 2,5-dioxopyrrolidin-1-yl(Z)-2-azido-3-(4-nitrophenyl)acrylate 26
Scheme 2.6: Synthesis of methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)glycinate
27
Scheme 2.7: methyl-4-nitrobenzoate Byproduct Formation
28
Scheme 2.8: Knoevenagel Condensation Mechanism
30
Scheme 2.9: Ester Hydrolyzed Azide Byproduct
31
Scheme 2.10: Mechanism of Amino Acid Addition to an Activated Succinimide Ester
32
Scheme 2.11: Synthesis of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-Lvalinate
34
Scheme 2.12: Synthesis of methyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valylglycinate 35
Scheme 2.13: Complete Synthesis of 6-nitro-1H-indole-2-carboxamides
41
xi
LIST OF TABLES
CHAPTER 1: Introduction
Table 1.1: Indole Ring Containing Drug Molecules
6
Table 1.2: Protein, Peptide and Small Molecule Therapeutics
17
CHAPTER 2: Efforts Towards Indole Peptide Conjugate Synthesis
Table 2.1 Previous Efforts Towards Microwave Solvent Optimization
37
xii
LIST OF ABBREVIATIONS
ADCs
Antibody Drug Conjugates
ADME
Absorption, Distribution, Metabolism and Excretion
CNS
Central Nervous System
Da
Dalton
DCC
Dicyclohexylcarbodiimide
DCM
Dichloromethane
DCU
Dicyclohexylurea
DME
1,2-Dimethoxyethane
DMF
Dimethylformamide
EDC HCl
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
EMEA
European Medicines Agency
EtOAc
Ethyl Acetate
EWG
Electron-Withdrawing Group
FDA
Food and Drug Administration
Fmoc
9-fluorenyl-methyloxycarbonyl
GC/MS
Gas Chromatography-Mass Spectrometry
GPCRs
G-Protein-Coupled Receptors
INDs
Investigational New Drugs
LC/MS
Liquid Chromatography-Mass Spectrometry
MeCN
Acetonitrile
MeOH
Methanol
Mol
Mole
xiii
Na
Sodium (Solid) Metal
NCEs
New Chemical Entities
NHS
N-Hydroxysuccinimide
NMEs
New Molecular Entities
NMR
Nuclear Magnetic Resonance
PDCs
Peptide Drug Conjugates
PET
Positron Emission Tomography
RT
Room Temperature
SM
Starting Material
THF
Tetrahydrofuran
TLC
Thin Layer Chromatography
t-BuOH
tert-Butyl Alcohol
xiv
CHAPTER 1: INTRODUCTION
1.1 MEDICINAL IMPORTANCE OF INDOLES
Indoles are a class of nitrogen containing heterocycles that have a variety of biological
activities, and are best known for their medicinal properties in the pharmaceutical industry.1-12
Heterocycles are cyclic compounds where one or more of the carbons on the ring are substituted
for a heteroatom.1 Heterocyclic compounds are some of the most novel and diverse biologically
active compounds, as they have a unique ability to bind reversibly to proteins and mimic the
peptide structure.3 Indoles are classified as bicyclic heterocycles that contain a benzene ring
fused to a pyrrole ring that share a double bond as depicted in Figure 1.1.1,6,13
N
H
Figure 1.1: Indole Nucleus Structure
Heterocycles are naturally incorporated into nearly all essential biochemical processes of
life.3 It has been determined that nearly half of all therapeutic compounds used are comprised of
heterocyclic compounds,6 and the indole ring system is the most commonly found
heterocycle.7,12 Three of the twenty-three essential amino acids contain heterocyclic rings:
histidine, tryptophan, and proline, and tryptophan contains the indole nucleus.2,3,7,12-14
Tryptophan is a fundamental component of peptides and proteins due to its ability of the N-H to
serve as a hydrogen bond donor to other peptides and proteins.13 In addition to being an essential
amino acid, tryptophan is also a biosynthetic precursor for many secondary metabolites,2,3,8 and
1
is the precursor for serotonin.6 The indole nucleus signifies a large importance in the world as
indoles are built into proteins, are the skeleton of many natural products and indole alkaloids,
and are the core of many marketed drugs.1,3,8 Many reviews and journals have described the
indole ring system as one of the most important and abundant heterocycles in nature having
medicinal importance.3,5,7-10,12,13 Many intricate and uncommon molecular structures occur
among their natural derivatives.5,6,10 Indole-containing compounds are also present in textile
dyes,13 dyes for human use, agriculture, animal health, dietary supplements, flavor enhancers,
and perfumes.2 Recent scientific advancements have also led to a number of synthetic indole
drugs to be approved by the FDA.5,6,8,12 The following briefly describes the medicinal
importance of indoles, indoles in modern drug discovery, their biological activities, and FDA
approved indole drugs.
1.1.1 INDOLES IN MODERN DRUG DISCOVERY
Indoles have widely been recognized as a privileged scaffold that is found in synthetic
compounds and natural products such as peptides.1,3-10,12,13 Therefore, a fundamental constituent
of higher-order protein structure scaffolds is the indole structure.12 This is due to their excellent
binding affinity to various receptors.7,12,13 Also, numerous potent indole-containing compounds
are found in FDA approved drugs.1,3,7,10,12 Indoles have been, and continue to remain an
important building block of modern day medicinal chemistry due to their excellent
pharmacodynamic and pharmacokinetic properties, and the ease of their functionalization as
demonstrated on their various synthesis methods over the past hundred years.11,12,14 To date over
10,000 unique indole derivatives have been discovered as being biologically active compounds;
over 200 of these are approved, marketed drugs or are undergoing clinical trials for approval.4
2
Over five hundred drugs are registered for use in animals, only a few containing the indole
nucleus, but many of which are used in both humans and animals such as the natural product
melatonin.2 It has been noted however, that the substitution pattern in synthetic indoles is much
less complex than in natural indole products. This is evident from the fact that as of 2013 there
was no reported indole with more that one substitution on the benzenoid ring in clinical use,4
This is a current synthetic issue, which will hopefully be addressed with future indole synthesis
development.
1.1.1.1 BIOLOGICAL ACTIVITIES
Figure 1.2 Biological Activities of the Indole Nucleus1
The indole nucleus has multiple important biological activities that Sharma et. al.
describes in Figure 1.2. According to Sharma et. al., one group synthesized a series of indole
3
derivatives and came to the conclusion that indole-2 and indole-3-carboxamides were shown to
have antioxidant properties by electron spin resonance spin trapping and from
chemoluminescence.1 Some other biological activities of indoles include plant growth regulation,
cardiovascular activity, opioid antagonist, antihistamine, photochemotherapeutic activity, kinase
inhibitors, and many receptor antagonist activity and inhibiton.1,3,6,8,12 It has also been reported
that the biologically active compounds contain stereocenters in the α or β positions of the pyrrole
ring side chains.7
This structure is found in many natural compounds such as the vasoconstrictor hormone and
neurotransmitter serotonin,13 reserpine, an antihypertensive alkaloid (nitrogen containing basic
organic molecule4), and complex alkaloid compounds such as anticancer agents mitomycin C
and vinblastine,3,5,7,8,12,14 as depicted in Figure 1.3. Vinblastine is one of the earliest anti-tumor
agents recognized as a tubulin polymerization inhibitor, and is mainly used in treatment of
advanced Hodgkin’s disease.3 Many natural products containing the indole nucleus are also
found in the plant, microbial, marine, and fungal kingdoms,2 such as the plant growth-regulating
hormone indol-3-ylactic acid.3,13 Indol-3-ylactic acid is more commonly known as LSD, and has
also been used for psychiatric activities in the treatment of alcoholism, and pain relief.3,14
4
HO
O
H 2N
OCONH2
OMe
N
Me
N
N
CO2Me
NH
H
NH
O
CO2Me
N H OH
MeO
Mitomycin C
OAc
Vinblastine
N
H
N
O
HO
N
H
H
H
Serotonin
O
O
O
H
O
NH 2
O
O
O
O
Reserpine
Figure 1.3: Important Naturally Occurring Indoles5
The indole nucleus is part of many drugs in today’s market for the treatment of cluster
headaches, chemotherapy-induced nausea, antineoplastic, and antihypertensive agents,1,3,7 which
is why it comes to no surprise that the indole nucleus has been so widely researched in industry
and academia since it first was synthesized by Bayer AG in 18667,10,12 when oxindole was
distilled with zinc dust.13 As shown in Table 1.1 below, there are many indole ring-containing
drug molecules ranging from anticancer molecules such as vinblastine, to anti-HIV and antiviral
molecules; demonstrating the diverse biological activity of the indole nucleus.3
5
Table 1.1: Indole Ring Containing Drug Molecules3
1.1.2 FDA APPROVED INDOLE DRUGS
Indole synthesis has been a topic of interest for many researchers for well over the past
century due to their therapeutic uses, and has led to vast improvements in the development of
routes towards their synthesis.3,5,7,8,12,13 It is believed that due to the importance indole ring
containing compounds have shown as therapeutics, and their wide applications, indole based
pharmaceuticals could replace many existing pharmaceuticals in the future.3,6 One example of
the vast improvements made over the past century includes the synthesis of apaziquone, which is
an indolequinone prodrug and analog of previously mentioned mitomycin C that is converted to
active metabolites in hypoxic environments and alkylate DNA leading the apoptotic cell death.3
6
O
N
H
N
O
S N
O H
N
O
N
H
N
H
Sumatriptan
Tadalafil
N
N
O
O
F
N
N
OH
N
N
H
Rizatriptan
OH OH O
Fluvastatin
Figure 1.4: Examples of Clinically Used Indoles5
Many synthetic indoles are approved for clinical use and have shown researchers that
indole synthesis is profitable. Four clinically used indoles (sumatriptan, tadalafil, rizatriptan,
fluvastatin) accounted for over $3.2 billion dollars in sales in 2010 alone. The structures of these
indoles are shown in Figure 1.4.5
Indole based pharmaceuticals not only have shown to be profitable for pharmaceutical
companies in the past, but also have shown high success in clinical trials. They have a favorable
time to approval and market compared with traditional pharmaceuticals in part because of their
diverse biological activity and reduced toxicity.6 With the vast amount of academic and industry
research being conducted on traditional pharmaceuticals, there are some disadvantages with
these small-molecule compounds that indole-based pharmaceuticals, and other types of
therapeutics, are attempting to address. Protein and peptide-based pharmaceuticals have become
of great importance in modern drug discovery, and the use of peptide-drug conjugates, and
7
indole-peptide conjugates have become of recent interest as effective therapeutics. In the past,
the trouble with these was finding an optimal chemical synthesis to first synthesize the indole
drug, and then conjugate the indole to the peptide. Many indole syntheses exist, and the research
outlined in this work discusses a way to synthesize the conjugate.
1.2 INDOLE SYNTHESIS METHODS
Throughout the history of drug discovery and development of indoles, many different
methods of synthesis have been discovered. Although not many, if any, indoles bearing
substituents at more than one of the benzenoid ring positions are currently used in clinical trial as
they are synthetically difficult to create. This demonstrates that although there are effective
methods of indole synthesis, there is still more research to be conducted in the field of indole
synthesis.5 The most popular methods of indole synthesis are discussed herein and include Fisher
indole synthesis, Bischler indole synthesis, Nenitzescu indole synthesis, Bartoli indole synthesis,
and Hemetsberger-Knittel indole synthesis.5, 7-9,12,15 There are also many variations of each
synthesis method, although they are not discussed in this work. Each is briefly described, and the
description of the Hemetsberger-Knittel indole synthesis is more in depth as the focus of the
research conducted in this work was based around a modified Hemetsberger-Knittel indole
synthesis reaction.
1.2.1 FISHER INDOLE SYNTHESIS
Fisher indole synthesis is currently the most used method, and was first reported in 1883.5
This indole synthesis method has produced a vast number of indole analogs to date, especially
compared to other indole synthesis methods.8 This method requires a tautomerization of an
8
arylhydrazone to the enehydrazine when heated under acidic conditions, and undergoes an [3,3]
rearrangement followed by further tautomerization and imine exchange as demonstrated in
Scheme 1.1.5,8,15 The byproducts of this reaction include the elimination of ammonia.5
R2
O
R2
R1
R1
NHNH 2
N
H
-H2O
R2
H
R1
N
H
[3,3]
NH
H
R2
H+
N
R2
NH 2+
-NH3
R1
NH
R2
R1
N
H
+
R1
N
H
Scheme 1.1: Fisher Indole Synthesis
Fisher synthesis requires the use of monosubstituted precursors, which can give rise to
regioselectivity issues due to the ring closing in two different directions to yield isomeric
products. While this is a possible issue with any indole synthesis of monosubstituted precursors,
this method addresses the requirements of an indole synthesis with simplicity and convenience
(can use microwave-assisted technology) although there are limited commercially available
arylhydrazines.5,8 These arylhydrazines are not only limited, but are also toxic, so there has been
drastic efforts to find safer and more reliable alternatives.5
9
1.2.2 BISCHLER INDOLE SYNTHESIS
The Bischler synthesis method is also known as the Bischer-Möhlau indole synthesis.8 It
requires an acid catalyzed ring closure preceded by an aniline alkylation with an α–haloketone as
demonstrated in Scheme 1.25,15 to produce 2-arylindoles.8 Bischler indole synthesis was first
reported in 1892, and is rather limited due to the harsh reaction conditions.5
H+
O
+
NH 2
R1
R1
O
X
R1
R2
N
H
R2
Where
R1
R2
-H2O
N
H
R1
O
R2
N
H
R2
Or Rearrangement
R1
N
H
R2
O
R1
N
H
R2
N
H
Scheme 1.2: Bischler Indole Synthesis
The Bischler indole synthesis is one method that avoids the issues associated with
arylhydrazines that the Fisher indole synthesis cannot. Complications with this synthesis method
can arise if the aniline is not protected prior to the cyclization as the α–anilinoketone can
undergo rearrangement and result in isomers. Recent scientific advancements since the Bischler
indole synthesis was first reported have led to the development of hydroamination reactions that
has allowed the use of alkynes and aldehydes to be used in the reaction along with ketones.5 This
10
synthesis method can also be conducted with microwave-assisted technology using solvent free
conditions to produce the product.8
1.2.3 NENITZESCU INDOLE SYNTHESIS
The Nenitzescu indole synthesis method consists of the reaction of enamines and
quinones to produce 5-hydroxyindoles. This reaction is typically poor in yield, and usually only
results in the synthesis of 4-substituted indoles due to an electron-withdrawing group (EWG)
where R3 is located in Scheme 1.3. The starting materials for this reaction are inexpensive, and
thus has allowed for large scale-up quantities to be used for indole synthesis.5
EWG
O
NHR1
+
R3
O
R2
EWG
H+
HO
R2
R3
N
R1
Scheme 1.3: Nenitzescu Indole Synthesis
1.2.4 BARTOLI INDOLE SYNTHESIS
Bartoli indole synthesis (Scheme 1.4) involves the reaction of ortho-substituted
nitroarenes with vinyl Grignard reagents. Due to the necessary excess of the Grignard reagent
needed, only indoles with minimal substitution on the 2- and 3-positions of the pyrrole ring, and
those with multiple substitutions on the benzenoid ring, can be produced.5 Recent scientific
advancements have also shown that a solid-phase Bartoli indole synthesis method can be
conducted and is very valuable due to the direct use of ortho,ortho-unsubstituted nitroarenes and
the overall decrease in chromatographic steps.9 Only ortho-substituted nitroarenes successfully
11
produce indole product as the nucleophilic attack occurs at the nitrogen rather than the oxygen
atoms of the nitro group without them.5
BrMg
N+
O
O-
N
O
O-
N
O
BrMg
[3,3]
N
H
O
O
NH 2
N
H
Scheme 1.4: Bartoli Indole Synthesis
1.2.5 HEMETSBERGER-KNITTEL INDOLE SYNTHESIS
The Hemetsberger-Knittel synthesis includes the thermal decomposition of β-styryl
azides followed by the electrocyclization around the aromatic benzene ring.5 The HemetsbergerKnittel reaction is limited to indole-2-carboxylate synthesis as the azide intermediates are
prepared through a base-mediated Knoevenagel condensation of azidoacetate esters and a
benzaldehyde as demonstrated in Scheme 1.5.5,8 The indole products are then achieved through
thermal cyclization of the 2-azidocinnamates. This reaction has been accepted as one of the most
significant indole synthesis methods for the synthesis of indole-2-carboxylates, and can utilize
microwave-assisted technology to achieve the products in good to excellent yields.8
12
O
R
CO2R
R
N3
CO2R
CO2R
R
Δ
N3
-N2
CO2R
R
N
R
N
N
H
CO2R
Scheme 1.5: Hemetsberger-Knittel Indole Synthesis
This reaction involves the use of azides, but despite the potential hazards associated with
them, the Hemetsberger-Knittel reaction is able to be scaled up to approximately 90 g. This
reaction is exceptionally appropriate to the regioselective synthesis of 4- or 6-substituted indoles
from either para or ortho-substituted benzaldehydes as the C3-C3a bond is present in the starting
material.5 The C3 position is also the most reactive position on the indole for electrophilic
aromatic substitution.7 It is also a useful synthesis method for the synthesis of highly substituted
indole products, and indole natural products.5
1.3 SIGNIFICANCE OF INDOLE PEPTDE CONJUGATES
Since the beginning of the surge of drug discovery and development in the 20th century,
or the ‘chemistry era’,19 drug candidates have had to follow Lipinski’s ‘rule-of-five’:
characteristics for molecular weight, H-bonding abilities, lipophilicity, and oral
bioavailability.16,17 These new small molecule chemical entities have since dominated the drug
pipelines,16-19 and have many benefits including oral bioavailability, membrane permeability,
metabolic stability, cost, and conformational restriction, but also some drawbacks.16,17 New
research over the latter part of the past century has led to new biological drug entities, or
biologics, that have many advantages over small molecules, but also some disadvantages.16
13
Recent scientific advancements have also led to the discovery of peptide-based pharmaceuticals
and peptide-drug conjugates. These compounds fill the molecular weight gap between small
molecule and protein-based drugs while combining the advantages of both.
1.3.1 FDA APROVED PEPTIDE AND PROTEIN-BASED DRUGS
Protein and peptide-based therapeutics have emerged within the past 50 years due to the
advancements in scientific research that led to new analytical and biological tools such as protein
purification, synthetic protein synthesis, sequencing technologies, high throughput peptide
sequencing protocols, and recombinant protein expression.16,17 Over the past 30 years these
therapeutics have become promising drug candidates. These therapeutics are much larger than
traditional small molecule drugs as their molecular weights are much greater than 500 Dalton
(Da). They contain over 50 amino acids, and their molecular weights are over 5000 Da.16-18
These therapeutics have also been found to have higher target selectivity and potency, leading
them to produce less side effects in humans,16,18 and tend to be administered intravenously.16-18
These biological therapeutics have paved the way for humanized antibody drug therapy, which
has become a successful therapeutic class both in disease targets and economics.16 The protein
drug market has amassed over 55 billion dollars in 2011 alone and has only continued to grow
the past five years by well over 10% per year.16,18 Some of the most successful antibody
therapies that are currently on the market include Humira for treatment of rheumatoid arthritis,
Avastin for treatment of non-Hodgkin’s B-cell lymphoma, and Avonex for treatment of multiple
sclerosis, and a novel monoclonal antibody, Adcetris, was recently synthesized for targeted
anticancer therapy.
14
In addition to newer peptide therapeutics, there have been over 100 new peptide drugs to
fill the molecular weight gap between protein drugs and small molecule drugs.16,17 These new
peptides have molecular weights between 50 and 5000 Da,17 and can contain up to 100 amino
acids according to the FDA,19 but generally contain less than 50 amino acids to distinguish them
from other biologics.17 Peptides have long been of interest as therapeutic agents, but small
molecules took preference over early peptides in the drug discovery and development industry
for their ease of production, pharmacodynamic and pharmacokinetic properties, and their ability
to be administered orally.19 It is easy to see why until recently peptides were not thought to be
good drug candidates.
Peptide-based therapeutics can be synthesized in a variety of ways including chemical
synthesis through a solution phase, through solid phase synthesis, production through
recombinant microorganisms or by extraction from natural sources.19 Through chemical
synthesis scientists can incorporate the use of unnatural amino acids and pseudo-peptide bonds to
allow for a wider chemical diversity. With the recent advancements, chemical synthesis of
peptides is now often considered the best technology over biological technologies (biocatalysis
or recombinant DNA) for the synthesis of medium sized (5-50 amino acids) peptides that
encompass most of the pharmaceutically active molecules.20 They can also be designed to allow
inhibition or stimulation of proteins involved in any cellular process.21 Peptide-based
therapeutics have been found to be highly selective and potent towards their broad range of
intended targets, with low tissue accumulation due to their short half-life and the fact that
degraded peptides are simply amino acids.16,20,22,23 This leads to less toxicity than traditional
small molecule drugs, and is thought to be a foundation for personalized medicine,16,17 as one of
the drivers is the need for more targeted therapy both effectively and safely.17 Peptide-based
15
therapeutics also have advantages over protein-based therapies including longer shelf life, more
cost effective, higher stability, less potential for immune-system interaction, and better tumor
and organ penetration.16,21 Peptides also have large surface areas and are suitable for array
technologies as targets of shallow protein-protein interaction sites.17
Zoladex
OH
O
H
N
N
H
O
N
H
O
N
H
N
O
NH
NH
O
N
H
OH
H
N
O
O
N
H
O
H
N
O
O
N
H
N
O
N
H
NH 2
O
HN
H 2N
NH
Figure 1.5: Successful Marketed Peptide-Based Therapeutic Zoladex
Some of the most successful marketed peptide-based therapeutics include treatment for
multiple sclerosis (Copaxone), hormone therapies (Zoladex, shown in Figure 1.5), and insulin
derivatives for diabetes treatment (Lantus), among many other applications.16,24 Protein and
peptide-based therapeutics have been shown to have a broad range of applications. These include
treatment of cancer, hypertension, autoimmune diseases, metabolic diseases, cardiovascular
diseases, infectious diseases, and allergies, among others.18,19,22 Peptide therapeutics in clinical
trials and in the pipeline show that more than half have unique targets. Of those in the pipeline,
approximately 10% target viral, fungal or bacterial organisms, and approximately 40% target Gprotein-coupled receptors (GPCRs).22 It is suggested that due to their broad range of applications
in the pharmaceutical industry, protein and peptide-based therapeutics could soon replace small
16
molecule drug therapies, as there is a continued growth in medicinal peptide patent applications
and approvals.18,19 The approval rate in the United States for peptide therapeutics has
considerably increased after the six diverse peptide marketed approvals in 2012. This approval
rate increased from approximately 1.3 approvals per year in the 2000s to three approvals per year
for the 2010s.22
1.3.2 ISSUES WITH PROTEIN, PEPTIDE, AND SMALL MOLECULE DRUGS
Protein, peptide, and small molecule drugs all have unique benefits, but also some drastic
differences and drawbacks as summarized in Table 1.2. Small molecule drugs, which have been
the typical new drug entity since the 20th century, have reduced target selectivity and thus result
in various side effects in patients.16 This is possibly the biggest drawback of small-molecule
drugs.16,17 Protein-based therapeutics produce less side effects than small molecule drugs due to
their higher target selectivity and potency, but cannot be administered orally due to their
metabolic instability, poor membrane permeability, and size.16,17,21 Proteins must also be highly
purified and concentrated to be effective treatments, and they have very short half-lives leading
to shelf lives of less than two years.18
Table 1.2: Protein, Peptide and Small Molecule Therapeutics
Protein-Based
Therapeutics
Advantages
Target selectivity,
higher potency
Disadvantages
Cost, poor oral
bioavailability, poor
permeability,
metabolic instability
Peptide-Based Therapeutics
Target selectivity, higher
permeability, low toxicity, more
cost efficient, less immunesystem interactions, stability
Poor oral bioavailability, poor
permeability, poor
pharmacokinetics, enzymatic
degradation
Small Molecule
Therapeutics
Oral
bioavailability,
permeability, cost,
metabolic stability
Slow identification
and optimization,
target selectivity
17
The disadvantages of peptide-based therapeutics include high manufacturing costs, poor
membrane permeability, poor oral bioavailability, high hepatic and renal clearance. They are also
easily degraded in the human body resulting in poor pharmacokinetic properties.16,20-23 Peptides
composed of natural amino acids also do not make good drug candidates due to their
pharmacokinetic and intrinsic properties.20 Nonetheless, with roughly 60 FDA market-approved
peptide drugs,20,23 140 candidates in clinical trials, and over 500 therapeutic peptides in
preclinical development,23 peptide-based pharmaceuticals have continued to enter clinical trials
at a rapid rate since first emerging over the last few decades.16 Although they currently only
represent a small percentage of all drug sales, their annual growth rate is drastically increasing at
25%.25 In fact, global peptide drug sales have been predicted to increase from US$14.1 billion
dollars in 2011 to approximately US$25.4 billion dollars in 2018. Novel peptide drug sales are
also expect to increase from 60% (US$8.6 billion dollars) to 66% (US17.0 billion dollars) in
2018.23
Some barriers that prevent protein and peptide-based drugs from being as effective as
some small molecule drugs include enzymatic, intestinal epithelial, capillary endothelial, and
blood brain barriers. These barriers respectfully limit the absorption of the drugs from the
gastrointestinal tract, transportation of the drug across the intestinal and capillary epithelium, and
the transportation of the drug to the brain.18,20 To overcome the oral bioavailability issues,
protein and peptide drugs are most effectively delivered via intravenous, intramuscular, and
subcutaneous methods, with intravenous delivery as the delivery method of choice.18,21-23 With
their high success rates in clinical trials and short time to market, protein and peptide-based
drugs are playing a crucial role in the treatment of unmet medical needs, and have the possibility
18
to replace small molecule drugs in the near future, although there are still many issues to
address.18
1.3.3 THE NEED FOR PEPTIDE DRUG CONJUGATES
Upon entering the 21st century, drug discovery and development has experienced a lack
of innovative ideas for new chemical entities. It is well known that the FDA has placed stricter
safety, quality and efficacy regulations on pharmaceutical companies over the last few decades.20
This, coupled with long developmental processes and immense financial investments, has placed
innovative pressure on pharmaceutical companies regarding their pipelines.19,20 The last decade
especially has seen a larger focus on orphan dugs, repurposed drugs, and next generation drugs
to reduce development and manufacturing costs,19 and only approximately 20 new chemical
entities (NCEs) have been registered each year since 1980.16 As more than 90% of new drugs
candidates fail before market approval, and the patent protections of blockbuster drugs is
expiring, innovative ideas for new drug candidates are in high demand.19
In actuality, 38% of the drug candidates in phase I clinical trials are discarded due to
toxicity issues, and 63% of those that reach phase II clinical trials are abandoned due to poor
bioavailability and lack of efficacy. Of those that reach phase III, 45% fail, and of those that are
finally submitted as NMEs and INDs to the FDA and EMEA, 23% are not approved.20 Given the
increasing difficulties arising with these small molecule pipelines, many drug development
companies are turning back to peptide-based therapeutics.19,20
Many of the challenges that peptide drugs face in discovery and development occur in the
preclinical phases in regards to the pharmacological and pharmacokinetic properties including
absorption, distribution, metabolism and excretion (ADME) as it is difficult to determine these
19
parameters.19,21 They differ from small molecules as they exert effects through cell surface
receptor binding rather than by diffusion into cells.19 Modifications must be made to peptide
therapeutics to overcome the many obstacles to safe and effective drug delivery. Currently in
research, small-molecules and peptides are being conjugated to antibodies, PEG chains and
lipids, and carbohydrates to improve targeting, uptake and permeability, and solubility,
respectively, to try to overcome these issues.17 Also, it is thought that a small molecule-peptide
conjugate system may be an innovative approach to overcome these drug discovery and
development obstacles.
For example, there are currently more than 20 peptide-drug conjugates under evaluation
in clinical trials as various oncology treatments.23 Peptide drug conjugates (PDCs) have been
found to be as potent as antibody drug conjugates (ADCs), but have better efficacy in animals
and humans.26 PDCs have also been found to have better tissue penetration compared to ADCs
due to their smaller size (higher activity per mass unit20), and ability to overcome interstitial
tumor pressure to reach the interior of the tumor.26 The hope of these peptide-drug conjugates is
for increased efficacy and safety for patients so that a higher and more potent dose of anticancer
therapies can be administered with improved target selectivity to improve patients’ lives.23
Peptide conjugates continue to be a topic of interest in scientific research, and a novel approach
to synthesizing small-molecule indole-peptide conjugates is described in the remainder of this
work.
20
1.4 RESEARCH OBJECTIVE
As protein, and especially peptide-drug conjugates have recently become of interest in
the drug discovery and development of medicinal therapeutics, it has come to be of recent
interest to our academic laboratory to investigate peptide conjugation. Previous research in our
laboratory pointed to the indole nucleus as the desired nucleus to be conjugated to a peptide.
Indoles and other N-heterocycles have previously been researched in our laboratory due to their
medicinal importance.13 Indoles specifically, as discussed in the above work, have a broad range
of biological activities and many successful indole drugs are currently on the market. Previously
in our laboratory, a modified Hemetsberger-Knittel synthesis was investigated as a way of
synthesizing 2-substituted carboxyl esters via a nitrene insertion intermediate using microwave
technology. This novel indole synthesis method was the method used to create the indoles
discussed in this work herein.
It was presumed that a simple indole-peptide conjugate may serve as the basis for more
complex peptide-indole drug conjugates in the future to help address the gap between small
molecule and protein drugs while combining the advantages of both therapeutics. Previous
efforts in our laboratory in the synthesis of medicinally relevant indoles led to the focus of this
work to be on 6-nitro-1H-2-carboxamides due to the possibility of an indole based tag being used
as a fluorophore for PET (positron emission tomography) imaging because of the advances in
protein labeling and proteomics.13
21
1.4.1 IMPORTANCE OF THE NITRO GROUP
The introduction of fluorine onto an aromatic moiety has led to numerous blockbuster
drugs for treatment as antifungals, antimicrobials, antimetabolites, statins, and CNS treatments as
shown in Figure 1.6.27 It has previously been researched that one of the most convenient routes
to selectively introduce fluorine onto an aromatic moiety is nucleophilic aromatic fluorination.
One of the most widely used leaving groups for nucleophilic aromatic fluorination is the nitro
group, and it is especially active in the ortho or para positions of a strong electron-withdrawing
group. The conditions for fluorination are much milder with the nitro group than a chloro group,
and with higher yields, especially in the para position.28
HN
N
N O
N
O
OH
F
O
O
N
Ciprofloxacin
(Antimicrobial)
Risperidone
(CNS agent)
F
OH OH O
O
F
N
N
N
N
F
N
N
N
O-
N
NH
Ca2+
3 H 2O
OH
F
N
Diflucan
Antifungal)
Lipitor
(Statin)
Figure 1.6: Blockbuster Agents That Contain Fluorine
22
In addition to new drug entities, the continued research and development of fluorination
methods has led to 18F labeled ligands for use in PET imaging.27 The nitro group is the most
widely used leaving group used as a nucleofuge for fluorination with 18F as evident by the
synthesis of two PET imaging agents ([18F]Flumazenil and [18F]Altanserin) that have been
synthesized from the corresponding nitroarene precursors.28 This is of particular importance to
the research conducted in our laboratory and is the reason that the nitro group was the only group
investigated in the following research. It has been demonstrated that microwave methods can be
used to convert nitro indoles to the corresponding fluoro indoles, and radiolabeled 18F to be used
for PET imaging.7,27,28 It is hoped that the nitro indoles synthesized would not only have the
above biological activities of indoles, but if converted to the corresponding fluoro indoles in
future investigations, could be used as PET imaging agents as well, and have biological activities
of similar fluoropharmaceuticals. The work described herein demonstrates the efforts taken to
synthesize medicinally relevant 6-nitro-1H-indole-2-carboxamides, a peptide, and an indolepeptide conjugate utilizing microwave technology.
23
CHAPTER 2: EFFORTS TOWARDS INDOLE PEPTIDE CONJUGATE
SYNTHESIS
2.1 THERMAL OPTIMIZATION OF INDOLE INTERMEDIATES
Preliminary efforts in our laboratory led to a complete synthesis (Scheme 2.1) of
methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)glycinate (5a), although there was much need for
improvement. As this synthesis uses commercially available sodium azide as a starting reagent, it
is to be noted that one must be very careful when handling and using azides. The synthesis of
methyl azidoacetate must be performed behind a blast shield, and concentration via distillation
on the rotary evaporator must be limited and monitored carefully. Almost all of the reactions
performed to synthesize the indole intermediates were optimized from previous laboratory
investigations to achieve better yields and conversions, cleaner products, and to increase safety.
O
NaN 3
O
Br
+
MeOH, H 2O
O
H
O
N3
O
O2N
1
O
O
NaOMe
O
N3
O2N
O
NHS, DCC
OH
THF
N3
O2N
2
THF, EtOAc
3
O
O
O2N
2 M LiOH
O
N
H 2N
O
N3
4
O
H Cl
O
O2N
N3
DME,KHCO 3 ,H 2O
O
O
N
H
5a
Scheme 2.1: Synthesis of methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)glycinate
24
2.1.1 PREVIOUS EFFORTS IN OUR LABORATORY
As mentioned above, a complete synthesis of 5a was previous reported from our
laboratory, but there was significant need for improvement in order to successfully synthesize
indole-2-carboxamindes and a possible indole-peptide conjugate.
NaN 3
O
Br
THF, H 2O
O
O
N3
O
1
Scheme 2.2: Synthesis of methyl azidoacetate
In the synthesis of methyl azidoacetate (1) (Scheme 2.2), initial methods required the use
of methanol as the organic reaction solvent, and the work-up entailed concentrating the biphasic
reaction mixture (methanol/water) on the rotary evaporator prior to a work-up with diethyl ether
and water.13
O
O
N3
O
O
H
+
O2N
NaOMe
O
O2N
N3
2
Scheme 2.3: Synthesis of methyl(Z)-2-azido-3-(4-nitrophenyl)acrylate
Synthesis of 2 (Scheme 2.3) was previous conducted in our laboratory with 1:4:4 molar
equiv of 4-nitrobenzaldehyde:sodium metal: methyl azidoacetate.13 This ratio provided
conversion to product, but in poor yield, and provided mixed fractions with 4-nitrobenzaldehyde
due to similar structures. There were column purification conditions used in previous efforts in
our laboratory, but when replicated, those conditions did not provide clean product even after the
25
material was ran through another column. These reaction conditions never provided clean
material in order to get an accurate yield.
O
O
O
O2N
2M LiOH
THF
N3
OH
O2N
N3
1
3
Scheme 2.4: Synthesis of (Z)-2-azido-3-(4-nitrophenyl)acrylic acid
Upon obtaining clean 2, the methyl ester was hydrolyzed to a carboxylic acid with a 2 M
solution of LiOH in a biphasic mixture of THF and water at room temperature overnight by ester
hydrolysis (Scheme 2.4). The work-up consisted of acidifying the reaction mixture with 10%
HCl to a pH of 4 before it was extracted with EtOAc and water, dried, and concentrated to afford
3 in good yield. The material was then used directly in the next reaction to obtain the
succinimide ester (Scheme 2.5).
O
O
OH
O2N
N3
NHS, DCC
THF
O
O
O2N
N3
N
4
O
Scheme 2.5: Synthesis of 2,5-dioxopyrrolidin-1-yl(Z)-2-azido-3-(4-nitrophenyl)acrylate
For the synthesis of 4, 1 equiv of both NHS and DCC was used with respect to 3. Both
NHS and DCC were added at 0 ºC and stirred for 30 minutes before the solution was warmed to
room temperature and stirred for 2h. In the previous efforts, the crude was washed with EtOAc
prior to filtration of the DCU byproduct, but large amounts of DCU were still present in 4.13
26
There was no further purification conducted before the material was used directly to synthesize
the glycine conjugate.
O
O
O
O2N
O
H 2N
N
N3
O
H Cl
O
O
DME,KHCO 3 ,H 2O
O2N
N3
O
O
N
H
5a
Scheme 2.6: Synthesis of methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)glycinate
The synthesis of 5a (Scheme 2.6) consisted of using crude 4 from the previous step, and
dissolving it in DME (1,2-dimethoxyethane). Glycine methyl ester HCl and potassium
bicarbonate dissolved in water were added to the solution. This was stirred at room temperature
overnight and showed high conversion to the desired product, and 5a was isolated in good yield.
The crude was then purified on a neutralized preparative TLC plate to afford an oil, although it
was still impure by NMR. The material was then placed in the microwave with toluene and the
synthesis of the glycine product was attempted with no success.
2.1.2 OPTIMIZATIONS
Upon the beginning of synthesizing the indole intermediates, the first reaction that
included the synthesis of methyl azidoacetate (1) needed optimization. Distilling off the
methanol in the biphasic reaction mixture as mentioned in the procedure above was not the safest
way to work up this reaction. This reaction also did not provide consistently good yields, as they
ranged from 33-72% of a mostly pure product. Upon conducting some research into recent
literature, a new procedure was found that used THF as the organic reaction solvent in place of
27
methanol.29 This procedure was similar to the one preciously used in our laboratory, but by
switching the organic solvent in the reaction to THF, it eliminated the need to remove MeOH
from the biphasic reaction mixture prior to the work-up. This also allowed the work-up to be
conducted in EtOAc and water, versus the diethyl ether/water work-up that was previously used.
The new procedure gave reliable yields ranging from 60-88%, with pure product by NMR and
GCMS.
In the Knoevenagel condensation of 2, much time was spent developing a new procedure
to obtain clean product in good yield. It was very difficult to isolate the product due to coelution
with the benzaldehyde starting material. After the previous efforts in our laboratory did not
provide clean product, a new procedure was found that required the reagents be added at 0 ºC,
and the reaction to be warmed to room temperature after 30 minutes at 0 ºC and stirred for 2
hours. These reaction conditions also required a molar equiv ratio of 1:1:5 of
aldehyde:azidoacetate:sodium methoxide.30 This is very different from the previous efforts in the
molar equiv ratio and temperature where the reagents (a molar equiv ratio of 1:4:4) were added
to the freshly prepared anhydrous sodium methoxide at -20 ºC, and then the reaction was run at 10 ºC for 3-5h or until most of the starting material was consumed. The room temperature
procedure did not require careful monitoring which was optimal, however this reaction only
results in synthesis of methyl-4-nitrobenzoate (Scheme 2.7), which is a Cannizzaro product.
O
O
N3
O
O
H
+
O2N
NaOMe
RT
O
O2N
Scheme 2.7: methyl-4-nitrobenzoate Byproduct Formation
28
A new procedure was then developed after discovering a comment made in an article in
The Journal of Medicinal Chemistry in March of 2014. Khurana et. al stated that when they
attempted the Hemetsberger-Knittel synthesis that is suspected to proceed via a nitrene insertion,
sometimes the Knoevenagel condensation required highly excessive quantities (> 10 equiv) of
the catalytic base and azidoacetate.31 This was the case with our methyl azidoacetate and methyl4-nitrobenzaldehyde, as excessive quantities of sodium methoxide and methyl azidoacetate were
required to push the reaction to give almost full conversion. This procedure also allowed for a
shortened reaction time (2-3h), purer product without purification, and moderate yields (3567%). Upon using the new procedure with 10 equiv of sodium methoxide and methyl
azidoacetate with respect to the 4-nitrobenzaldehyde, 2 cleanly precipitated out of the reaction
solution within 2 hours of stirring at -10 ºC, and was used directly without further purification. It
was also noted in the literature that the yield is very dependent on the temperature, and was
found to give the best yields when the temperature was first kept at -20 ºC for the first 30
minutes then warmed to -5 ºC – 0 ºC.31 In our case, it was found that a temperature above -5 ºC
led to byproduct formation, and zero product formation. The mechanism of this Knoevenagel
condensation, which is a modification of aldol condensation, is shown in Scheme 2.8.
29
O
H
O
N3
O2N
O
O
H
O
N3
+ MeOH
Na +
OMe-
O
H
OH O
H
O
O
O2N
MeO-H
N3
O
H+
N3
O2N
OMe-
azide alcohol intermediate
O
OH O
O
O2N
N3
+ MeOH
+ H 2O
O
O2N
N3
β-elimination product
Scheme 2.8: Knoevenagel Condensation Mechanism
It should be noted that these yields are only for synthesis with the 4-nitrobenzaldehyde.
Other aldehydes, such as simpler, non–nitrogen-containing aldehydes were synthesized in
literature with slightly higher yields. The low yields associated with this reaction (typical yields
of 40-50%) are due in part to temperature control issues, not using dry methanol, oxidized
sodium metal, and ester hydrolysis of the azide alcohol intermediate (Scheme 2.9). Low yields
can also be attributed to inactivity of the methyl azidoacetate, as it has been found that the
material needs to be quickly used (within 24h) or it starts to decompose due to the inherent
instability of the azide. From here 2 can undergo microwave assisted Hemetsberger-Knittel
thermolysis to create the methyl 6-nitro-1H-indole-2-carboxylate (2a) as described in a later
section, or it can be converted into the acrylic acid (3) as described next.
30
O
O
O
O2N
N3
OH
O2N
+
MeOH
N3
Scheme 2.9: Ester Hydrolyzed Azide Byproduct
Due to the previous efforts in our laboratory providing an efficient, scalable synthesis for
3, there was no need for optimization as there was complete conversion and high yields varying
from 90-98% yield of pure product, and the characteristic IR peaks of the carboxylic acid and
azide were present. Pure 3 from the ester hydrolysis was used directly without further
purification to synthesize the succinimide ester. The synthesis of 4 was conducted according to
the previous efforts in our laboratory, but with a modified work-up to increase the purity. Instead
of washing the crude with EtOAc, MeCN was added to the reaction mixture and the mixture was
aged at 0 ºC for 30 minutes prior to filtration. The filtrate was then concentrated to afford a
yellow solid with full conversion and excellent yield (90-99%). This reaction was attempted with
EDC HCl to improve purity, but there was zero conversion to product (sticky mixture). This
material was then used directly with no further purification to synthesize 5a (acryloyl glycinate),
5b (acryloyl valinate), and 5c (acryloyl glycinate).
Previous efforts in our laboratory produced an efficient, synthesis for the amino acid
addition to 4 with excellent conversion, and good yield. This synthesis method was used to make
5a, 5b, and 5c with the proposed mechanism shown in Scheme 2.10.
31
O
O
N
O
O2N
O
O
O
N3
O
H 2N
O
O2N
N3
NH
R
O
H Cl
N
O
O
O
R
O
O2N
N3
O
O
H
N
H
R
O
O
O
N
O
O2N
N3
O
O
N
H
R
Scheme 2.10: Mechanism of Amino Acid Addition to an Activated Succinimide Ester
The glycinate product was produced with good yields ranging from 60-91%, depending
on the purity of 4. The phenylalaninate product (5c) was produced with moderate crude yields of
80% (50% pure), and the valinate product (5b) was obtained with moderate yields of 40-64%
pure product. Glycine, phenylalanine, and valine were chosen as the first amino acids to be
conjugated to the indole intermediates as glycine is a small amino acid that was used in previous
laboratory efforts, and was used to test the synthesis method and check for repeatability. Valine
was chosen as it is a small aliphatic amino acid that is larger than glycine but would not cause
any steric hindrance or side interactions. Finally, phenylalanine was chosen as it was necessary
to demonstrate that a bulky aromatic group addition would still be tolerated. These three amino
acids were also chosen as none needed protecting groups to be attached to 4. More amino acids
would need to be attached with this synthesis method in the future to demonstrate versatility, and
so that it can be determined if all the amino acids, especially charged species and bulky groups,
32
can tolerate these conditions to successfully synthesize the indole intermediates, and future
indole-peptide conjugates.
2.2 PEPTIDE SYNTHESIS
In order to synthesize a peptide that would hopefully be attached to the activated
succinimide ester indole intermediate to form an indole-peptide conjugate, it was first necessary
to make the succinimide ester of the first amino acid. Valine was chosen as it was previously
researched in our laboratory that this material could easily be made when using Fmoc-protected
L-valine, and valine was successfully used to make 5b as shown in Scheme 2.11. Synthesis of
the succinimide ester was first attempted on an unprotected valine by dissolving L-valine in THF
at 0 ºC with NHS (1 equiv) and DCC (1 equiv) added and stirred overnight at room temperature.
This was attempted a second time but only letting the reaction stir at room temperature for 2h
after stirring at 0 ºC for 30 minutes. Upon filtration of the DCU byproduct, a colorless glassy
solid was afforded, although a large quantity of DCU was still present in the material. NMR and
LCMS both showed complete conversion to the desired 2,5-dioxopyrrolidin-1-yl-L-valinate. The
DCU was attempted to be removed by first adding cold THF to the solid, and placing it in the
freezer for 1-2h, followed by filtration again. This was repeated twice, but DCU was still present.
MeCN was added as DCU is also insoluble in the solvent, and the round bottom was placed in
the freezer again for 1h. DCU was still present in a large quantity. It was thought to purify the
material by column chromatography, but the material was not UV active.
Formation of the amide bond was attempted even though large amounts of DCU were
still present in the 2,5-dioxopyrrolidin-1-yl-L-valinate product. Glycine was chosen as the amino
acid to be part of the attempted peptide synthesis due to its size and ability to be easily attached
33
to 4 to create 6a. A similar procedure for the amino acid addition to 4 was used for the amide
bond synthesis to attempt to make a peptide between 2,5-dioxopyrrolidin-1-yl-L-valinate and
glycine methyl ester HCl.32 Here, 2,5-dioxopyrrolidin-1-yl-L-valinate was dissolved in DME,
and glycine methyl ester HCl was added to the stirred mixture at room temperature. Sodium
bicarbonate dissolved in water (0.7M) was added to the stirred mixture, and THF was added to
improve solubility while the reaction was stirred overnight at room temperature.32 A white
precipitate was formed upon completion, but NMR and LCMS of the white precipitate and
reaction solution did not show product.
H
N
O
O
Fmoc-Valine
O
NHS, DCC
OH
THF
H
N
O
O
O
O
O
N
O
7
Fmoc-Val-OSu
Scheme 2.11: Synthesis of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-Lvalinate
In order to avoid the DCU byproduct separation issues, a Fmoc (9-fluorenylmethyloxycarbonyl)-protected L-valine was used following the above procedure that was
conducted on the unprotected L-valine to created the succinimide ester (Scheme 2.11). This
reaction was allowed to stir for 2h at room temperature followed by 30 minutes at 0 ºC, before
the DCU byproduct was filtered off and the filtrate was concentrated to afford the desired
product. NMR and LCMS showed the desired product, and only a very small amount of DCU, so
the material was carried through without further purification. The Fmoc-Val-OSu product (7)
was then used to synthesize 8 following the above procedure used when attempting the peptide
34
bond with the unprotected valine succinimide ester (Scheme 2.12). A white precipitate formed
within 1h and was filtered off. LCMS and NMR showed clean peptide product.
H
N
O
O
O
O
O
Cl
N
O
H 3N
O
R
O
H
N
O
O
DME,NaHCO 3 ,H 2O, THF
O
N
H
O
O
8
Fmoc-Val-Gly
Scheme 2.12: Synthesis of methyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valylglycinate
Deprotection of the Fmoc group from 8 was attempted using piperidine. It was found that
a solution of 20% piperidine in DMF would successfully deprotect valine in ten minutes.33 The
peptide was dissolved in this freshly prepared solution, and stirred for ten minutes. TLC showed
disappearance of starting material, but the reaction mixture was allowed to stir for 1h before the
mixture was worked up with EtOAc and water, dried, and concentrated. LCMS and NMR
showed the organic layer contained the Fmoc-piperidine side product (1-((9H-fluoren-9yl)methylpiperidine) and that the desired product was in the aqueous phase. Many attempts were
made to extract the product from the water using hexanes, DCM, diethyl ether, and THF, but due
to the polarity of the product, extraction was unsuccessful. Future work will need to investigate
deprotection extraction methods in solution phase synthesis in order to extract the pure peptide
so that the indole peptide conjugate can easily be made by synthesis with 4, followed by
microwave thermolysis to give the corresponding indole-peptide conjugate.
35
2.3 MICROWAVE ASSISTED SYNTHESIS OF 6-NITRO-1H-INDOLE-2CARBOXAMIDE CONJUGATES
Due to recent scientific advancements, it is now well known that microwave-assisted
technology can be used to synthesize medicinally important indoles in a fraction of synthesis
times using conventional heating.8 In 1986 the first independent publications on microwave
assisted organic synthesis were published.12 Synthesis utilizing microwave technology is based
on the ability to efficiently heat the solvent and reagents by microwave dielectric heating effects.
The reagents and/or solvents must have the ability to efficiently absorb the microwave energy
and convert it into heat, which is the difference between microwave heating and conventional
heating.8,12 Microwave electromagnetic irradiation is in the frequency range of 300-300,000
MHz, although heating devices have a very specific and narrow frequency range. It has been
shown that microwave synthesis is favorable for drug discovery chemistry due to the reaction
times, improved conversions, clean product formations, and ability to safely and efficient
produce large numbers of compounds.8
2.3.1 PREVIOUS EFFORTS IN OUR LABORATORY
Previous efforts towards Hemetsberger-Knittel thermolysis (Scheme 1.5) of medicinally
relevant indoles led to the discovery of using copper chromite catalyst and toluene as the reaction
solvent. Various solvents were tested among different substituted indoles that resulted in the
following findings. Conventional thermal synthesis was performed by refluxing in xylene for 4h
and gave moderate yields.13 It has also been reported that refluxing the material in xylene for 1h
and refluxing in toluene for 2.5-3h yields product.34 Previous efforts in our laboratory
36
determined that toluene, quinoline, and xylene provided good conversions of product upon
microwave thermolysis at 200 ºC in 15 minutes. Although polar solvents have a high dielectric
constant that favor the absorption of microwave energy for heating, n-butanol, ethanol, THF,
MeCN, dioxane, n-hexanes and t-BuOH did not provide good conversions as shown in Table
2.1.13
Table 2.1 Previous Efforts Towards Microwave Solvent Optimization
Solvent
THF
n-Hexanes
Ethanol
n-Butanol
Dioxane
MeCN
Toluene
Xylene
Temperature
200 ºC
200 ºC
200 ºC
200 ºC
200 ºC
200 ºC
200 ºC
200 ºC
Time (minutes)
15
15
15
15
15
15
15
15
Yield (%)
25
38
39
44
49
52
95
96
It was documented that n-hexane did not perform well in the laboratory (even though it
was previously reported14 to be a good microwave solvent for indoles) due to the inability of the
microwave to reach temperature without over pressurizing the system. The maximum
temperature that was reached with n-hexane in previous laboratory efforts was 150 ºC.13 The
optimal temperature was then found to be 200 ºC as temperatures over 200 ºC led to
decomposition of the product, and temperatures below 120 ºC showed poor conversion to
product. Reactions were then run for 15 and 10 minutes at 200 ºC in toluene to determine
optimal reaction time for indole-2-carboxylates, with 10 minutes confirming suffice time as
yields were not significantly improved in 15 minutes, and yields significantly decreased at 8
minutes.11,13
37
Various catalysts including rhodium(II), copper chromite, copper chloride, copper
powder, and the copper(II) salt of indole-2-carboxylate were previously screened to assist in the
microwave thermolysis. It was shown that for the nitro indole, the cyclization proceeded much
better with copper chromite than without. Yields were less that 20% without the catalyst,
although the catalyst was not previously documented to be used for the synthesis.13 Although
there was much work done towards the ring closing Hemetsberger-Knittel synthesis of 6-nitro1H-indoles and indole conjugates, optimal synthesis conditions still needed to be found.
2.3.2 MICROWAVE OPTIMIZATION
Upon formation of 5 or 2, microwave assisted thermolysis of the azidoester in toluene at
200 ºC, 300 psi (20 bar), for 15 minutes in the presence of 12 mol% copper chromite barium
promoted (30 mol% for 2) provided the best conversion and yields from the research discussed
in this work. 5a was the first intermediate subjected to the microwave-assisted thermolysis
conditions previous determined by our laboratory. NMR and LCMS showed conversion to the
desired indole product, but with very poor conversion and many impurities. Thermolysis of 2
was conducted in both toluene and hexanes. It was decided to test n-hexanes as a reaction solvent
as it is reported in Lehmann et. al. that the indole products precipitate out in n-hexanes, and that
n-hexanes is just as good of a reaction solvent as toluene as shown in Figure 2.1 below.14
Although the reaction temperature did not exceed 150 ºC, an orange precipitate formed upon
reaction completion and LCMS of the precipitate showed both indole product and starting
material mass. The LCMS of the thermolysis experiment conducted in toluene at the same time
showed no starting material mass, and small indole product, among other impurities. Toluene
38
was determined to be better than n-hexanes for the synthesis of the 6-nitro-1H-indoles (2a and
6a-c) as it yielded a better conversion to the indole product.
Figure 2.1 The Effect of Different Solvents on the % Conversion to Indoles14
Once it was confirmed that toluene was the best solvent for thermolysis of the 6-nitro1H-indoles, copper chromite catalysts were investigated. Thermolysis experiments of 2 were
conducted with no catalyst, copper chromite powder, and copper chromite barium promoted
catalyst. It has been reported in literature that promoters in copper chromite catalysts exhibit
higher conversion to the desired products than catalysts without promoters. It is also documented
that the formation of the BrCrO4 phase contributes to prolonged catalyst activity under higher
temperature reactions.35 The reaction performed with 12 mol% copper chromite barium
promoted catalyst resulted in more product formation than the reaction with 12 mol% copper
chromite powder and the reaction with no catalyst, but significantly less than the reaction
performed with 30 mol% copper chromate barium promoted catalyst. Thus, it was determined
that 30 mol% was necessary for good conversion to the methyl 6-nitro-1H-indole-2-carboxylate
(2a). This compound can be easily synthesized, and it is possible to then synthesize 3, followed
39
by 4 and subsequent amino acid or peptide addition if the conjugate demonstrates to be unstable
during thermolysis.
Once optimal conditions were found for the synthesis of 2a, it was necessary to find
optimal conditions to perform thermolysis of 5a, along with other amino acids. Experiments
were performed with 12 mol% copper chromite barium promoted in toluene at 200 ºC for 12
minutes as a starting point, and showed good conversion to the desired glycine indole product,
but with some starting material present. The time was then increased to 15 minutes, which
showed excellent conversion to the desired glycine indole product (6a) in 30% yield. The low
yields of all the indole products was due to issues with purification as the product and starting
materials elute very close to each on the preparative TLC plates due to similar structures. This
procedure was then use to synthesize 6b and 6c, and can be used to for other amino acids and
hopefully peptide bioconjugation in the future.
40
2.4 CONCLUSIONS AND FUTURE WORK
O
NaN 3
O
Br
THF, H 2O
O
H
O
N3
+
O
O2N
1
O
O
NaOMe
N3
O2N
O
THF
2
NHS, DCC
OH
N3
O2N
THF
3
O
O
O2N
2 M LiOH
O
O
N
H 2N
O
N3
4
O
H Cl
O
R
N3
O2N
O
O
N
H
R
DME,KHCO 3 ,H 2O
O2N
Copper Chromite Barium
Promoted (12 mol%)
N
H
O
H
N
Toluene
O
5a R: H
5b R:CH(CH 3)2
5c R:CH2(Phe)
O
R
6
Scheme 2.13: Complete Synthesis of 6-nitro-1H-indole-2-carboxamides
As described in the above work, medicinally important indoles and indole-2carboxamides can safely and efficiently be synthesized utilizing microwave assisted technology
and modified Knoevenagel condensation and Hemetsberger-Knittel synthesis. The research
described in this work demonstrates the significant improvements over previous efforts in our
laboratory for the complete synthesis of 6-nitro-1H-indole conjugates (Scheme 2.13), and the
efforts towards indole-peptide conjugates. The majority of the steps were optimized, purities
were increased, conversions and yields were enhanced drastically, and safety concerns were
reduced, especially when working with sodium azide in the first step of the synthesis. The
synthesize of 1 was optimized by changing the reaction organic solvent in order to allow for
41
consistent increased yields, and safer working conditions. The Knoevenagel condensation was
drastically optimized to improve conversion, yield, purity, and reaction time was shortened to
almost half. The synthesis of 3 was optimized for improved yield and purity, and formation of 4
was optimized to improve the purity of the product, and the yield. The addition of the amino acid
was not modified although the purification was modified to give cleaner product. It was
questioned why 10 equiv of the amino acid methyl ester were required, although time did not
permit to investigate if less could be used. The modified Hemetsberger-Knittel synthesis was
optimized to include a higher conversion, higher yield, and higher purity of the indole product.
These compounds, especially the azide intermediates, were difficult to purify due to
decomposition on silica gel and could only be purified on preparative TLC plates due to the
limited exposure. Glycine, phenylalanine, and valine indole conjugates (6a-c) were also
successfully synthesized with ease following the optimized procedures in order to create a small
library of indole conjugates.
It was attempted to create an indole peptide conjugate with a synthesized dipeptide,
although issues during deprotection of the peptide did not allow for this to happen. A Fmocvaline was successfully converted to the succinimide ester, and upon coupling to glycine methyl
ester HCl, the protected peptide precipitated out cleanly. Attempts to deprotect the peptide with
20% piperidine in DMF were successful, although efforts to remove the product from the
aqueous layer, and attempts to synthesize an unprotected peptide were also unsuccessful.
Future work on this research project in the laboratory could include synthesizing possible
indole-carboxamide based tags as fluorophores for PET imaging through previous research
conducted in our laboratory.13,27,28 using compounds 6a-c, and expanding around the benzenoid
ring of the indole to expand the library. Work could also include synthesizing more indole-2-
42
carboxamides to form a larger library of indole conjugates to include more of the natural and
unnatural amino acids such as charged species, bulky species, sulfur containing species, and
polar species. Issues with some amino acid syntheses could include the oxidation of methionine
to methionine sulfoxide, although this can be easily reverted with dithiothreitol or Nmercaptoacetamide, and somewhat averted with short deprotection times. Cysteine containing
peptides are also prone to oxidative formation of disulfide bonds, which can form
intramolecularly or intermolecularly, and can lead to aggregation. This most frequently occurs
after the peptide is exposed to air after deprotection when multiple cysteines are present.
Histidine can have issues during deprotection again with the acylation of the imidiazole ring.
Tryptophan, an indole itself, is susceptible to alkylation and oxidation of the indole ring by
released protecting groups from other side chains, which can be minimized by the use of Bocprotected tryptophan. Tyrosine can also have alkylation of the side chain. Asparagine and
glutamine can be dehydrated if unprotected during coupling. Lastly, Arginine can have long
deprotection times in the resin depending on the protecting group.36
It is necessary to develop an Fmoc deprotection solution phase synthesis for these
compounds. The method tried in the above work was found in literature that also used solid
phase synthesis. One work-up was found during a recent literature search to precipitate peptides
from resin that are very hydrophobic and/or less than six residues long. The future researchers
could use this work-up to try to extract the deprotected dipeptide from the aqueous layer if
precipitation with the diethyl ether did not work. This would include dissolving the residue in
10% aqueous acetic acid and chloroform in a separatory funnel (x3), although prolonged acid
treatment may break sensitive amide bonds. The aqueous layer could then be dried under the
high vacuum, dissolved in glacial acetic acid, and lyophilized if needed.36 Had this work-up been
43
discovered earlier, it would have been attempted. Future work would then include attachment of
the unprotected dipeptide to 4, followed by microwave assisted thermolysis. If the peptide
demonstrated to be unstable during thermolysis, 2a could be synthesized directly from 2, and
then reduced to 3 and subsequent succinimide ester (4) followed by conjugation of the peptide.
This method may be necessary for dipeptides, and larger peptides.
Future work could entail the bioconjugation of a peptide to a successfully marketed
indole drug through the synthesis methods described in this work. It would be necessary to
compare the biological activity, oral bioavailability, permeability, selectivity, potency, toxicity,
stability, cost, and pharmacokinetics of the indole-peptide drug conjugate to the indole drug in
order to determine if indole-peptide drug conjugates are worth pursuing. This comparison is
further down the drug discovery road in terms of research and ability, but would be very
beneficial for drug companies to make in order to possibly revive their drug pipelines, and would
provide safer and more effective new drug entities.
44
CHAPTER 3: EXPERIMENTAL PROCEDURES
3.1 GENERAL EXPERIMENTAL INFORMATION
All chemical reactions were carried out in glassware that was previously dried in an oven
(160°C, for at least 12h), or flame dried under argon using Schlenk-line techniques, unless
otherwise noted. All starting materials were obtained from commercial sources, either Acros,
Aldrich, Fisher, or Sigma, and used as received. All reactions were not carried out under
anhydrous, inert argon atmospheres with freshly distilled solvents unless otherwise noted. The
solvents used including Methanol, Ethyl Acetate, Hexane, Methylene Chloride, Acetonitrile,
Chloroform, and Tetrahydrofuran were all obtained from Fisher, and any water used was
deionized. All anhydrous solvents used were purchased from commercial sources with the
exception of MeOH. Freshly distilled MeOH was prepared immediately prior to use by
distillation under reduced pressure, and was stored over 4Å molecular sieves. Reaction
temperatures of -20°C and -10°C were obtained by mixing dry ice and acetone. All microwave
thermolysis reactions were performed using a CEM-Discover monomode instrument, with
operation frequency of 2.45 GHz, pressure of 0-300psi (0-20 bar), and with continuous power
from 0 to 300W, for the desired time after the set temperature was reached (approximately 10-15
minute ramping times). All of the microwave thermolysis reactions were performed in 10mL
glass microwave tubes, with stir bars, and sealed with new Teflon caps for each reaction. All
analytical thin layer chromatography was performed using SiliaPlate TLC Aluminum Backed
pre-coated TLC plates of 200µm thickness, indicator F-254 (SiliCycle Inc). All preparative thin
layer chromatography (TLC) was performed using SiliaPlate TLC Glass Backed TLC Extra Hard
Layer, 60Å of 1000µm thickness, indicator F-254 (SiliCycle Inc). When performed, flash
45
chromatography was performed using SiliaFlash P60 (40-60µm, 230-40 mesh) silica gel
(SiliCycle Inc). FT-IR spectra were obtained from a Bruker Alpha-T FT-IR, and prepared using
Opus software. NMR spectra was obtained from a Varian Mercury 400 (400 MHz) spectrometer
and reported in ppm downfield relative to the TMS peak, and the spectra were prepared using
MestReNova. All 1H NMR are reported with ppm followed by multiplicity, coupling constant,
and number of protons. LC/MS analyses were performed on a Waters Micromass ZQ single
quadropole mass detector with a Waters e2795 Separations Module, and a Waters 2996
Photodiode Array Detector. The column used was a Waters SunFire C18 3.5µm 4.6x50mm
column. The LCMS spectra were prepared using MassLynx. GC/MS analyses were performed
on a HP GC System HP 6890 Series, with a 5973 Mass Selective Detector. The column used was
a HP-1 25m column with a 0.200 diameter and 0.11 m thickness. The GCMS spectra were
prepared using Chem Station software.
46
3.2 EXPERIMENTAL DETAILS OF INDOLE INTERMEDIATES
methyl 2-azidoacetate13 (1)
O
N3
O
This reaction should be performed behind a safety shield. To a solution of sodium azide (6.41 g,
0.099 mol) in water (12.4 mL) in a three-neck 250 mL round bottom flask equipped with a stir
bar, temperature probe, reflux condenser and drying tube in an oil bath was added a solution of
methyl bromoacetate (6.2 mL, 0.066 mol) in THF (37.2 mL). The resulting mixture was heated
to reflux for 90 minutes before being cooled to RT. The layers were separated and the top layer
washed with water, dried with MgSO4, filtered and concentrated in vacuo to afford a pale yellow
oil30 (6.65 g, 88% yield). 1H NMR (400MHz, CDCl3): δ 3.83 (s, 2H), 3.73 (s, 3H). With slight
residual solvent peaks at δ 4.11, 2.04, and 1.25. 13C NMR (100MHz, CDCl3): δ 169.01, 52.7,
50.33. GCMS: m/z C3H5N3O2 (M)+ calcd: 115.04 Obsd: 115.004
47
methyl(Z)-2-azido-3-(4-nitrophenyl)acrylate13 (2)
O
O
N3
O2N
To a flame dried three-neck 300 mL round bottom flask under argon was added anhydrous
MeOH (19 mL). The flask was equipped with a stirbar, temperature probe, and a pressureequalizing dropping funnel prior to flame drying. Na metal (1.08 g, 46.96 mmol, 10 equiv) was
dissolved in the MeOH. A solution of methyl 2-azidoacetate (5.29 mL, 46.96 mmol, 10 equiv),
4-nitrobenzaldehyde (0.7134 g, 4.7 mmol, 1 equiv), and anhydrous MeOH (30 mL) was added
via the dropping funnel to the cooled sodium methoxide mixture at -20 °C The resulting mixture
was stirred for 2-3h (until most of the SM consumed) at -10 °C. Water was added drop-wise to
the yellow mixture and the resulting yellow precipitate was filtered, and dried to afford a yellow
solid13,31 (0.780 g, 67% yield). 1H NMR (400MHz, CDCl3): δ 8.23 (d, J = 8.9 Hz, 2H), 7.97 (d, J
= 8.9 Hz, 2H), 6.90 (s, 1H), 3.95 (s, 3H). With slight residual solvent peaks at δ 1.54 2.04, and
1.25.
13
C NMR (100MHz, CDCl3): δ 163.55, 147.51, 139.59, 131.26, 129.16, 123.89, 121.99,
53.58. LCMS (ESI+): m/z C10H8N4O4 (M+H)+ calcd: 248.05 Obsd: 248.46
48
methyl 6-nitro-1H-indole-2-carboxylate13 (2a)
O2N
N
H
COOCH 3
To a flame-dried 10 mL microwave tube under argon equipped with a stir bar and new
microwave vial cap was added methyl(Z)-2-azido-3-(4-nitrophenyl)acrylate (50 mg, 0.201
mmol), and anhydrous toluene (2 mL). The tube was irradiated in the microwave in the presence
of 30 mol% copper chromite barium promoted (Strem Chemicals) for 15 minutes at 200 °C,
300W, 300 psi. The contents of the mixture were extracted with EtOAc (x3) and brine, dried
with MgSO4, filtered, and concentrated in vacuo. Prep plate purification done in 1:1
EtOAc:Hexanes to afford a light yellow solid. (42.3 mg, 95% yield). 1H NMR (400MHz,
CDCl3): δ 9.22 (s, 1H), 8.41 (s, 1H), 8.06 (dd, J = 8.9, 2.0 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.29
(d, J=1.3 Hz, 1H), 4.00 (s, 3H). With slight residual solvent peaks at δ 4.11, 2.00, 1.54, and 1.25.
LCMS (ESI+): m/z C10H8N2O4 (M+H)+ calcd: 220.05 Obsd: 220.12
49
(Z)-2-azido-3-(4-nitrophenyl)acrylic acid13 (3)
O
OH
O2N
N3
methyl(Z)-2-azido-3-(4-nitrophenyl)acrylate (350 mg, 1.41 mmol) was dissolved in 3:1
THF:H2O (10 mL) and 2 M LiOH (3 mL) was added to the round bottom flask. After stirring at
RT overnight, the THF was evaporated under reduced pressure and the resulting mixture was
acidified to pH = 4 with 10% HCl. The resulting mixture was washed with EtOAc (x3) and
brine, dried with MgSO4 and filtered before being concentrated in vacuo to afford a yellow
solid13 (0.326 g, 98% yield). FT-IR: carboxylic acid (OH – 3385.30 cm-1, C=O – 1709.17 cm-1,
C-O 1260.70 cm-1), azide (2127.14 cm-1), C-N (1341.25 cm-1), NO2 (1508.76cm-1, 1438.65cm-1).
1
H NMR (400MHz, CDCl3): δ 8.24 (d, 2H), 8.00 (d, J = 8.8 Hz, 2H), 7.03 (s, 1H). With slight
residual solvent peaks at δ 4.11, 2.05, 1.50, and 1.25. LCMS (ESI+): m/z C9H6N4O4 (M+H)+
calcd: 234.04 Obsd: 234.19
50
2,5-dioxopyrrolidin-1-yl(Z)-2-azido-3-(4-nitrophenyl)acrylate13 (4)
O
O
O
O2N
N3
N
O
(A) To a solution of (Z)-2-azido-3-(4-nitrophenyl)acrylic acid (0.396 g, 1.69 mmol) in
anhydrous THF (14 mL) was added N-hydroxysuccinimide (0.195 g, 1.69 mmol) and DCC
(0.349 g, 1.69 mmol) to the round bottom flask at 0 °C. The resulting mixture was stirred at 0 °C
for 30 minutes and then for 2h at RT.13 MeCN was added and the round bottom was placed in an
ice bath for 30 minutes. The dicyclohexylurea by-product was filtered off and the filtrate was
concentrated in vacuo to afford a yellow-orange solid (0.555 g, 99% yield). 1H NMR (400MHz,
CDCl3): δ 8.26 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 9.0 Hz, 2H), 7.16 (s, 1H), 2.94 (s, 4H). With
slight impurity peaks at δ 2.82-1.10 from DCU. 13C NMR (100MHz, CDCl3): δ 168.79, 158.69,
148.18, 138.40, 132.00, 125.86, 124.89, 124.03, 25.857. LCMS (ESI+): m/z C13H9N5O6 (M+H)+
calcd: 331.06 Obsd: 331.27
51
3.3 EXPERIMENTAL DETAILS OF PEPTIDE SYNTHESIS
2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valinate32 (7)
H
N
O
O
O
O
O
N
O
To a solution of Fmoc-L-Valine (3.0 g, 8.80 mmol) in THF (60 ml) at 0 ºC was added DCC (2.01
g, 1 equiv) and N-hydroxysuccinimide (1.12 g, 1 equiv). The resulting mixture was stirred at 0
°C for 30 minutes and then for 2h at RT. The round bottom was then placed in an ice bath for 30
minutes. The dicyclohexylurea by-product was filtered off and the filtrate was concentrated in
vacuo to afford a colorless, glassy solid that was used without purification32 (3.49 g, 91% yield).
1
H NMR (400MHz, DMSO-d6): δ 8.15 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.72 (t, J =
8.3 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.7 Hz, 2H), 4.32 (q, J = 3.8 Hz, 2H), 4.24 (m,
1H), 3.77 (m, 1H), 2.79 (s, 4H), 1.00 (dt, J = 5.3, 2.6 Hz, 6H). With slight impurity peaks at δ
2.55-1.19 from DCU, and water at δ 3.31. 13C NMR (100MHz, DMSO-d6): δ 168.66, 164.82,
156.94, 144.518, 144.29, 141.41, 128.37, 127.79, 126.04, 120.83, 66.73, 58.68, 47.27, 30.76,
26.17, 25.81, 19.26, 18.68. LCMS (ESI+): m/z C24H24N2O6 (M+Na)+ calcd: 459.16 Obsd:
459.76
52
methyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valylglycinate (8)
H
N
O
O
O
N
H
O
O
2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valinate (Fmoc-Val-NHS)
(3.40 g, 7.79 mmoles) was dissolved in DME (34 ml) and added to a solution of glycine methyl
ester HCl (1.027 g, 1.05 equiv) and sodium bicarbonate (0.687 g, 1.05 equiv) in water (34 ml).
THF (34 ml) was added to improve solubility, and the mixture was stirred at room temperature
overnight.32 The resulting white solid was collected by vacuum filtration (1.20 g, 40% yield).
1
H NMR (400MHz, DMSO-d6): δ 8.38 (s, 2H), 7.87 (dt, J = 7.0, 3.1 Hz, 2H), 7.73 (s, 2H), 7.40
(m, 2H), 7.30 (dq, J = 7.8, 3.8 Hz, 2H), 4.22 (d, J = 12.2 Hz, 2H), 3.77 (d, J = 17.3 Hz, 2H), 3.59
(m, 3H), 1.96 (s, 1H), 0.86 (d, J = 3.4 Hz, 6H). With water peak at δ 3.31. 13C NMR (100MHz,
DMSO-d6): δ 172.55, 170.89, 156.80, 144.59, 141.39, 128.33, 127.75, 126.11, 120.79, 66.41,
60.77, 52.31, 47.34, 31.00, 19.81, 18.93. LCMS (ESI+): m/z C23H26N2O5 (M+H)+ calcd: 410.18
Obsd: 411.08
53
3.4 GENERAL METHODS
All thermal amino acid additions and microwave syntheses were performed according to the
representative procedures described herein.
3.4.1 THERMAL AMINO ACID ADDITION
methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)amino acid
O
R
O2N
N3
2,5-dioxopyrrolidin-1-yl(Z)-2-azido-3-(4-nitrophenyl)acrylate (0.15 g, 0.45 mmol) and amino
acid methyl ester hydrochloride (0.57 g, 4.53 mmol) were dissolved in DME (5 mL) and added
to potassium bicarbonate (0.45 g, 4.5 mmol) dissolved in water (3 mL). The solution was stirred
overnight at RT. The volatiles were removed under reduced pressure in vacuo and the resulting
mixture was poured over 0.5N H2SO4 – ice (6 mL) and extracted with EtOAc (x3), and washed
with brine, and dried with MgSO4 before being filtered and concentrated to afford the crude
product.13 The crude product was purified on a neutralized preparative TLC plate with 9:1
DCM:MeOH (glycine), 99:1 DCM:MeOH (phenylalanine), or 20:1 DCM:MeOH (valine) to
afford a yellow oil (40-91% yield). A neutralized preparative TLC was prepared by dipping it in
1% Et3N in DCM solution and allowing it to dry before loading crude product.
54
methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)glycinate13 (5a)
O
O2N
1
N3
O
O
N
H
H NMR (400MHz, CDCl3): δ 8.25 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 6.68 (s, 1H),
6.59 (s, 1H), 4.20 (d, J = 5.1 Hz, 2H), 3.84 (d, J = 3.2 Hz, 3H). With slight residual solvent peaks
at δ 2.05, 1.55, and 1.25. 13C NMR (100MHz, CDCl3): δ 170.08, 163.09, 147.31, 139.56, 133.64,
130.58, 123.96, 117.80, 52.99, 41.88. LCMS (ESI+): m/z C12H11N5O5 (M+H)+ calcd: 305.08
Obsd: 306.13
55
methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)-L-valinate (5b)
O
O2N
1
N3
O
O
N
H
H NMR (400MHz, CDCl3): δ 8.25 (d, 2H), 7.84 (d, 2H), 6.66 (s, 1H), 6.56 (s, 1H), 4.68 (m,
1H), 3.82 (s, 3H), 2.29 (m, 1H), 1.01 (ddd, J = 11.4, 7.2, 3.7 Hz, 6H). With slight residual
solvent peaks at δ 2.05, 1.55, and 1.25. 13C NMR (100MHz, CDCl3): δ 169.11, 162.83, 139.57,
130.58, 127.32, 123.94, 117.55, 94.66, 57.91, 52.78, 31.79, 19.23, 18.13. LCMS (ESI+): m/z
C15H17N5O5 (M+H)+ calcd: 347.12 Obsd: 347.99
56
methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)-L-phenylalaninate (5c)
O
O2N
1
N3
O
O
N
H
H NMR (400MHz, CDCl3): δ 8.25 (d, 2H), 7.78 (d, 2H), 7.35 (m, 3H), 7.13 (dt, J = 6.1, 1.8 Hz,
2H), 6.56 (d, J = 7.6 Hz, 1H), 6.32 (d, J = 1.5 Hz, 1H), 4.98 (m, 1H), 3.81 (d, J = 1.6 Hz, 3H),
3.25 (m, 2H). With slight residual solvent peaks at δ 2.05, 1.55, and 1.25. 13C NMR (100MHz,
CDCl3): δ 171.73, 162.48, 147.25, 139.55, 135.56, 133.88, 130.56, 129.51, 129.04, 127.76,
123.93, 117.36, 53.82, 53.01, 37.85. LCMS (ESI+): m/z C19H17N5O5 (M+H)+ calcd: 395.12
Obsd: 396.05
57
3.4.2 MICROWAVE ASSISTED HEMETSBERGER-KNITTEL SYNTHESIS
methyl(6-nitro-1H-indole-2-carbonyl)amino acid
O
O2N
N
H
R
methyl(Z)-(2-azido-3-(4-nitrophenyl)acryloyl)amino acid (0.05 g, 0.164 mmol) was placed in a
flame dried 10 mL microwave tube with a stir bar under argon and dissolved in anhydrous
toluene (2 mL). The tube was irradiated in the microwave in the presence of 12 mol% copper
chromite barium promoted (Strem Chemicals) for 15 minutes at 200 °C, 300 W, 300 psi. The
contents of the mixture were extracted with EtOAc (x3) and brine, dried with MgSO4, filtered,
and concentrated in vacuo. The crude product was purified on a preparative TLC plate in either
1:1 EtOAc:Hexanes, or 3:2 EtOAc:Hexanes to afford the desired compound as a yellow-orange
sticky solid (~30-40% yield).
58
methyl(6-nitro-1H-indole-2-carbonyl)glycinate (6a)
O2N
N
H
1
H
N
O
O
O
H NMR (400MHz, CDCl3): δ 9.79 (s, 1H), 8.31 (d, J = 9.0, 2.1 Hz, 2H), 7.98 (d, 2H), 6.75 (s,
1H), 4.28 (d, J = 4.9 Hz, 2H), 3.83 (d, J = 3.2 Hz, 3H). With slight impurity peaks at δ 2.00-0.86
from DCU. 13C NMR (100MHz, CDCl3): δ 170.39, 165.61, 152.09, 150.05, 139.40, 128.55,
124.16, 94.66, 52.98, 42.11. LCMS (ESI+): m/z C12H11N3O5 (M+H)+ calcd: 277.07 Obsd:
278.03
59
methyl(6-nitro-1H-indole-2-carbonyl)valinate (6b)
H
N
O
N
H
O2N
1
O
O
H NMR (400MHz, CDCl3): δ 10.98 (s, 2H), 8.46 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.73 (d, J =
8.8 Hz, 1H), 7.00 (m, 1H), 4.85 (dd, J = 8.6, 5.1 Hz, 1H), 3.84 (s, 3H), 2.33 (d, J = 7.6 Hz, 1H),
1.06 (t, J = 6.7 Hz, 6H). With slight impurity peaks at δ 2.02-1.22 from DCU. 13C NMR
(100MHz, CDCl3): δ 172.56, 161.16, 145.27, 135.24, 132.17, 122.47, 116.01, 109.44, 103.14,
94.66, 57.87, 52.88, 31.95, 19.27, 18.30. LCMS (ESI+): m/z C15H17N3O5 (M+H)+ calcd: 319.12
Obsd: 319.22
methyl(6-nitro-1H-indole-2-carbonyl)phenylalaninate (6c)
H
N
O2N
1
O
N
H
O
O
H NMR (400MHz, CDCl3): δ 10.70 (s, 2H), 8.44 (d, J = 5.2 Hz, 1H), 8.02 (dd, J = 8.9, 2.0 Hz,
1H), 7.70 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 7.2 Hz, 1H), 6.95 (m, 5H), 5.16 (dq, J = 12.1, 6.2 Hz,
1H), 3.83 (d, J = 4.9 Hz, 3H), 3.35 (m, 2H). With slight impurity peaks at δ 2.03-0.86 from
DCU. 13C NMR (100MHz, CDCl3): δ 171.94, 160.64, 145.29, 135.54, 135.24, 134.96, 132.15,
129.50, 129.06, 127.71, 122.53, 116.02, 109.46, 103.19, 53.43, 38.10, 29.96. LCMS (ESI+): m/z
C19H17N3O5 (M+H)+ calcd: 367.12 Obsd: 367.15
60
REFERENCES
1. Sharma V, Kumar P, Pathak D. Biological Importance of the Indole Nucleus in Recent
Years: A Comprehensive Review. J. Heterocyclic Chem. 2010; 47, 491, with permission
from Wiley.
2. Barden T. Indoles: Industrial, Agricultural and Over-the-Counter Uses. Top Heterocycl
Chem. 2011; 26, 31-46.
3. Kaushik NK, Kaushik N, Attri P, Kumar N, Kim CH, Verma AK, Choi EH. Biomedical
Importance of Indoles. Molecules. 2013; 18, 6620-6662.
4. Indoles and Indolizidines. In: Majumdar K, Chattopadhyay S, editors. Heterocycles in
Natural Product Synthesis. New Jersey (USA) Wiley-VCH; 2011; 221.
5. Inman M, Moody CJ. Indole Synthesis – Something Old, Something New. Chem. Sci., 2013;
4, 29, with permission from the Royal Society of Chemistry.
6. Biswal S, Sahoo U, Sethy S, Kumar HKS, Banerjee M. Indole: The Molecule of Diverse
Biological Activities. Asian J. Pharm and Clin. Res. 2012; 5(1): 1-6.
7. Bartoli G, Bencivenni G, Dalpozzo R. Organocatalytic Strategies for the Asymmetric
Functionalization of Indoles. Chem. Soc. Rev. 2010; 39, 4449-4465.
8. Patil SA, Patil R, Miller DD. Microwave-Assisted Synthesis of Medicinally Relevant
Indoles. Curr. Med. Chem. 2011; 18 (4): 615-637.
9. Knepper K, Vanderheiden S, Brase S. Synthesis of Diverse Indole Libraries on Polystyrene
Resin – Scope and Limitations of an Organometallic Reaction on Solid Supports. Beilstein J.
Org, Chem. 2012; 8, 1191-1199.
10. Koenig SG, Dankwardt J, Liu Y, Zhao H, Singh SP. Copper-Catalyzed Synthesis of Indoles
and Related Heterocycles in Renewable Solvents. ACS Sustainable Chem. Eng. 2014; 2,
1359-1363.
11. Ranasinghe N, Jones GB. Extending the versatility of the Hemestberger-Knittel Indole
Synthesis Through Microwave and Flow Chemistry. Bioorg. Med. Chem. Lett. 2013; 23,
1740-1742.
12. Ranasinghe N, Jones GB. Flow and Microwave Assisted Synthesis of Medicinally relevant
Indoles. Curr. Green Chem. 2015; 2 (1): 66-76.
13. Ranasinghe N. Technology Assisted Methodology in the Synthesis of Medicinally Relevant
N-Heterocycles. [Dissertation]. Northeastern University. 2014; 1-93.
14. Lehmann F, Holm M, Laufer S. Rapid and Easy Access to indoles via Microwave-Assisted
Hemetsberger-Knittel Synthesis. Tetrahedron Lett. 2009; 50, 1708-1709, with permission
from author and Elsevier.
15. Taber DF, Tirunahari PK. Indole Synthesis: A Review and Proposed Classification.
Tetrahedron. 2011; 67, 7195-7210.
16. Mezo G. Introduction. In: Farkas E, Ryadnov M. Amino Acids, Peptide and Protein Based
Pharmaceuticals. Hungary: RSC Publishing; 2013, 38, 203-252.
17. Craik D, Fairlie D, Liras S, Price D. The Future of Peptide-based Drugs. Chem Biol Drug
Des. 2013; 81, 136-147.
18. Ratnaparkhi MP, Chaudhari SP, Pandya VA. Peptides and Proteins in Pharmaceuticals. Int. J.
Curr. Pharma. Research. 2010; 3 (2), 1-9.
61
19. Uhlig T, Kyprianou T, Martinelli FG, Oppici CA, Heiligers D, Hills D, Calvo XR, Verhaert
P. The Emergence of Peptides in the Pharmaceutical Business: From Exploration to
Exploitation. EuPA Open Proteomics. 2014; 4, 58-69.
20. Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M. Synthetic Therapeutic Peptides:
Science and Market. Drug Disc. Today. 2010; 15(1/2), 40-55.
21. Bidwell III, GL. Peptides for Cancer Therapy – A Drug Development Opportunity and a
Drug Delivery Challenge. Ther. Deliv. 2012; 3(5), 609-621.
22. Kasper A, Reichert JM. Future Directions for Peptide Therapeutics Development. Drug Disc.
2013; 18, 807-817.
23. Fosgerau K, Hoffmann T. Peptide Therapeutics: Current Status and Future Directions. Drug
Disc. Today. 2015; 20 (1), 122-127.
24. Duc-Doan N, Zhang J, Traoré M, Kamdem W, Lubell WD. Solid-Phase Synthesis of CTerminal Azapeptides. J. Peptide Sci. 2015; 21(5), 387-391.
25. Hamzeh-Mivehroud M, Alizadeh AA, Morris MB, Church WB, Dastmalchi S. Phage
Display as a Technology Delivering of the Promise of Peptide Drug Discovery. Drug Disc.
Today. 2013; 18(23/24), 1144-1157.
26. Gangakhedkar A, Thudimadathil J. Peptides & Antibodies – Peptides in Antibody & Peptide
Drug Conjugates. Drug Dev. & Deliv. 2014; 14(6), 50-54.
27. LaBeaume P, Placzek M, Daniels M, Kendrick I, Ng P, McNeel M, Afroze R, Alexander A,
Thomas R, Kallmerten A, Jones GB. Microwave-Accelerated Fluorodenitrations and
Nitrodehalogenations: Expeditious routes to labeled PET Ligands and
Fluoropharmaceuticals. Tetrahedron Lett. 2010; 51, 1906-1909.
28. Placzek M. Technology Assisted Methodology in the Synthesis of Fluoropharmaceuticals.
[Dissertation]. Northeastern University. 2013; 33-38.
29. Swetha M, Ramana PV, Shirodkar SG. Simple and Efficient Method for the Synthesis of
Azides in Water-THF Solvent System. Org. Prep. and Procedures Int. 2011; 43 (4), 348353.
30. Khan AH, Chen JS. Synthesis of Breitfussin B by Late-Stage Bromination. Org. Lett. 2015;
17, 3718-3721.
31. Khurana L, Ali HI, Olszewaka T, Ahn KH, Damaraju A, Kendall DA, Lu D. Optimization of
Chemical Functionalities of Indole-2-Carboxamides to Improve allosteric Parameters for the
Cannabinoid Receptor 1 (CB1). J. Med. Chem. 2014; 57, 3040-3052.
32. Firestone RA, Dubowchik GM, inventors. 1993 May 14. Lysosomal Enzyme-Cleavable
Antitumor Drug Conjugates. United States Patent 08,062,366.
33. Fields GB. Methods for Removing the Fmoc Group. Methods in Molecular Bio. 1994; 35,
17-27.
34. Henn L, Hickey DMB, Moody C, Rees C. Formation of Indoles, Isoquinolines, and Other
Fused Pyridines from Azidoacrylates. J. Chem. Soc. 1984; 2189-2196.
35. Mane RB, Ghalwadkar AA, Hengne AM, Suryawanshi YR, Rode CV. Role of Promoters in
Copper Chromite Catalysts for Hydrogenolysis of Glycerol. Catalysis Today. 2011; 164,
447-450.
36. Applied Biosystems. Cleavage, Deprotection, and Isolation of Peptides after Fmoc Synthesis.
Applied Biosystems. Thermo Fisher Scientific. 2007; 1-12.
62
APPENDIX
63
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
ROYAL SOCIETY OF CHEMISTRY LICENSE
TERMS AND CONDITIONS
Apr 16, 2016
This Agreement between Allison Murdza ("You") and Royal Society of Chemistry ("Royal Society of Chemistry") consists of your license details
and the terms and conditions provided by Royal Society of Chemistry and Copyright Clearance Center.
License Number
3851051234100
License date
Apr 16, 2016
Licensed Content Publisher
Royal Society of Chemistry
Licensed Content Publication
Chemical Science
Licensed Content Title
Indole synthesis – something old, something new
Licensed Content Author
Martyn Inman,Christopher J. Moody
Licensed Content Date
Sep 6, 2012
Licensed Content Volume Number
4
Licensed Content Issue Number
1
Type of Use
Thesis/Dissertation
Requestor type
academic/educational
Portion
figures/tables/images
Number of figures/tables/images
2
Format
electronic
Distribution quantity
1
Will you be translating?
no
Order reference number
None
Title of the thesis/dissertation
Microwave Assisted Synthesis of Medicinally Relevant
Indole Conjugates Utilizing Modified HemetsbergerKnittel Indole Synthesis
Expected completion date
Apr 2016
Estimated size
100
Requestor Location
Allison Murdza
72 Hillside Street #2
ROXBURY CROSSING, MA 02120
United States
Attn: Allison Murdza
Billing Type
Invoice
Billing Address
Allison Murdza
72 Hillside Street #2
ROXBURY CROSSING, MA 02120
United States
Attn: Allison Murdza
Total
0.00 USD
Terms and Conditions
This License Agreement is between {Requestor Name} (“You”) and The Royal Society of
Chemistry (“RSC”) provided by the Copyright Clearance Center (“CCC”). The license consists of
your order details, the terms and conditions provided by the Royal Society of Chemistry, and the
payment terms and conditions.
RSC / TERMS AND CONDITIONS
INTRODUCTION
The publisher for this copyrighted material is The Royal Society of Chemistry. By clicking “accept” in connection with completing this licensing
transaction, you agree that the following terms and conditions apply to this transaction (along with the Billing and Payment terms and conditions
established by CCC, at the time that you opened your RightsLink account and that are available at any time at .
LICENSE GRANTED
The RSC hereby grants you a non-exclusive license to use the aforementioned material anywhere in the world subject to the terms and conditions
indicated herein. Reproduction of the material is confined to the purpose and/or media for which permission is hereby given.
RESERVATION OF RIGHTS
The RSC reserves all rights not specifically granted in the combination of (i) the license details provided by your and accepted in the course of this
licensing transaction; (ii) these terms and conditions; and (iii) CCC’s Billing and Payment terms and conditions.
REVOCATION
The RSC reserves the right to revoke this license for any reason, including, but not limited to, advertising and promotional uses of RSC content,
third party usage, and incorrect source figure attribution.
THIRD-PARTY MATERIAL DISCLAIMER
If part of the material to be used (for example, a figure) has appeared in the RSC publication with credit to another source, permission must also
be sought from that source. If the other source is another RSC publication these details should be included in your RightsLink request. If the other
source is a third party, permission must be obtained from the third party. The RSC disclaims any responsibility for the reproduction you make of
items owned by a third party.
PAYMENT OF FEE
If the permission fee for the requested material is waived in this instance, please be advised that any future requests for the reproduction of RSC
materials may attract a fee.
ACKNOWLEDGEMENT
The reproduction of the licensed material must be accompanied by the following acknowledgement:
Reproduced (“Adapted” or “in part”) from {Reference Citation} (or Ref XX) with permission of The Royal Society of Chemistry.
If the licensed material is being reproduced from New Journal of Chemistry (NJC), Photochemical & Photobiological Sciences (PPS) or Physical
Chemistry Chemical Physics (PCCP) you must include one of the following acknowledgements:
For figures originally published in NJC:
Reproduced (“Adapted” or “in part”) from {Reference Citation} (or Ref XX) with permission of The Royal Society of Chemistry (RSC) on behalf
of the European Society for Photobiology, the European Photochemistry Association and the RSC.
For figures originally published in PPS:
Reproduced (“Adapted” or “in part”) from {Reference Citation} (or Ref XX) with permission of The Royal Society of Chemistry (RSC) on behalf
of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
For figures originally published in PCCP:
Reproduced (“Adapted” or “in part”) from {Reference Citation} (or Ref XX) with permission of the PCCP Owner Societies.
HYPERTEXT LINKS
With any material which is being reproduced in electronic form, you must include a hypertext link to the original RSC article on the RSC’s
website. The recommended form for the hyperlink is http://dx.doi.org/10.1039/DOI suffix, for example in the link
http://dx.doi.org/10.1039/b110420a the DOI suffix is ‘b110420a’. To find the relevant DOI suffix for the RSC article in question, go to the
Journals section of the website and locate the article in the list of papers for the volume and issue of your specific journal. You will find the DOI
suffix quoted there.
LICENSE CONTINGENT ON PAYMENT
While you may exercise the rights licensed immediately upon issuance of the license at the end of the licensing process for the transaction,
provided that you have disclosed complete and accurate details of your proposed use, no license is finally effective unless and until full payment is
received from you (by CCC) as provided in CCC's Billing and Payment terms and conditions. If full payment is not received on a timely basis,
then any license preliminarily granted shall be deemed automatically revoked and shall be void as if never granted. Further, in the event that you
breach any of these terms and conditions or any of CCC's Billing and Payment terms and conditions, the license is automatically revoked and shall
be void as if never granted. Use of materials as described in a revoked license, as well as any use of the materials beyond the scope of an
unrevoked license, may constitute copyright infringement and the RSC reserves the right to take any and all action to protect its copyright in the
materials.
WARRANTIES
The RSC makes no representations or warranties with respect to the licensed material.
INDEMNITY
You hereby indemnify and agree to hold harmless the RSC and the CCC, and their respective officers, directors, trustees, employees and agents,
from and against any and all claims arising out of your use of the licensed material other than as specifically authorized pursuant to this licence.
NO TRANSFER OF LICENSE
This license is personal to you or your publisher and may not be sublicensed, assigned, or transferred by you to any other person without the
RSC's written permission.
NO AMENDMENT EXCEPT IN WRITING
This license may not be amended except in a writing signed by both parties (or, in the case of “Other Conditions, v1.2”, by CCC on the RSC's
behalf).
OBJECTION TO CONTRARY TERMS
You hereby acknowledge and agree that these terms and conditions, together with CCC's Billing and Payment terms and conditions (which are
incorporated herein), comprise the entire agreement between you and the RSC (and CCC) concerning this licensing transaction, to the exclusion of
all other terms and conditions, written or verbal, express or implied (including any terms contained in any purchase order, acknowledgment, check
endorsement or other writing prepared by you). In the event of any conflict between your obligations established by these terms and conditions
and those established by CCC's Billing and Payment terms and conditions, these terms and conditions shall control.
JURISDICTION
This license transaction shall be governed by and construed in accordance with the laws of the District of Columbia. You hereby agree to submit
to the jurisdiction of the courts located in the District of Columbia for purposes of resolving any disputes that may arise in connection with this
licensing transaction.
LIMITED LICENSE
The following terms and conditions apply to specific license types:
Translation
This permission is granted for non-exclusive world English rights only unless your license was granted for translation rights. If you licensed
translation rights you may only translate this content into the languages you requested. A professional translator must perform all translations and
reproduce the content word for word preserving the integrity of the article.
Intranet
If the licensed material is being posted on an Intranet, the Intranet is to be password-protected and made available only to bona fide students or
employees only. All content posted to the Intranet must maintain the copyright information line on the bottom of each image. You must also fully
reference the material and include a hypertext link as specified above.
Copies of Whole Articles
All copies of whole articles must maintain, if available, the copyright information line on the bottom of each page.
Other Conditions
v1.2
Gratis licenses (referencing $0 in the Total field) are free. Please retain this printable license for your reference. No payment is required.
If you would like to pay for this license now, please remit this license along with yourpayment made payable to "COPYRIGHT CLEARANCE
CENTER" otherwise you will be invoiced within 48 hours of the license date. Payment should be in the form of a check or money order
referencing your account number and this invoice number {Invoice Number}.
Once you receive your invoice for this order, you may pay your invoice by credit card.
Please follow instructions provided at that time.
Make Payment To:
Copyright Clearance Center
Dept 001
P.O. Box 843006
This License Agreement is between {Requestor Name} (“You”) and The Royal Society of
Chemistry (“RSC”) provided by the Copyright Clearance Center (“CCC”). The license consists of
your order details, the terms and conditions provided by the Royal Society of Chemistry, and the
payment terms and conditions.
RSC / TERMS AND CONDITIONS
INTRODUCTION
The publisher for this copyrighted material is The Royal Society of Chemistry. By clicking “accept” in connection with completing this licensing
transaction, you agree that the following terms and conditions apply to this transaction (along with the Billing and Payment terms and conditions
established by CCC, at the time that you opened your RightsLink account and that are available at any time at .
LICENSE GRANTED
The RSC hereby grants you a non-exclusive license to use the aforementioned material anywhere in the world subject to the terms and conditions
indicated herein. Reproduction of the material is confined to the purpose and/or media for which permission is hereby given.
RESERVATION OF RIGHTS
The RSC reserves all rights not specifically granted in the combination of (i) the license details provided by your and accepted in the course of this
licensing transaction; (ii) these terms and conditions; and (iii) CCC’s Billing and Payment terms and conditions.
REVOCATION
The RSC reserves the right to revoke this license for any reason, including, but not limited to, advertising and promotional uses of RSC content,
third party usage, and incorrect source figure attribution.
THIRD-PARTY MATERIAL DISCLAIMER
If part of the material to be used (for example, a figure) has appeared in the RSC publication with credit to another source, permission must also
be sought from that source. If the other source is another RSC publication these details should be included in your RightsLink request. If the other
source is a third party, permission must be obtained from the third party. The RSC disclaims any responsibility for the reproduction you make of
items owned by a third party.
PAYMENT OF FEE
If the permission fee for the requested material is waived in this instance, please be advised that any future requests for the reproduction of RSC
materials may attract a fee.
ACKNOWLEDGEMENT
The reproduction of the licensed material must be accompanied by the following acknowledgement:
Reproduced (“Adapted” or “in part”) from {Reference Citation} (or Ref XX) with permission of The Royal Society of Chemistry.
If the licensed material is being reproduced from New Journal of Chemistry (NJC), Photochemical & Photobiological Sciences (PPS) or Physical
Chemistry Chemical Physics (PCCP) you must include one of the following acknowledgements:
For figures originally published in NJC:
Reproduced (“Adapted” or “in part”) from {Reference Citation} (or Ref XX) with permission of The Royal Society of Chemistry (RSC) on behalf
of the European Society for Photobiology, the European Photochemistry Association and the RSC.
For figures originally published in PPS:
Reproduced (“Adapted” or “in part”) from {Reference Citation} (or Ref XX) with permission of The Royal Society of Chemistry (RSC) on behalf
of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
For figures originally published in PCCP:
Reproduced (“Adapted” or “in part”) from {Reference Citation} (or Ref XX) with permission of the PCCP Owner Societies.
HYPERTEXT LINKS
With any material which is being reproduced in electronic form, you must include a hypertext link to the original RSC article on the RSC’s
website. The recommended form for the hyperlink is http://dx.doi.org/10.1039/DOI suffix, for example in the link
http://dx.doi.org/10.1039/b110420a the DOI suffix is ‘b110420a’. To find the relevant DOI suffix for the RSC article in question, go to the
Journals section of the website and locate the article in the list of papers for the volume and issue of your specific journal. You will find the DOI
suffix quoted there.
LICENSE CONTINGENT ON PAYMENT
While you may exercise the rights licensed immediately upon issuance of the license at the end of the licensing process for the transaction,
provided that you have disclosed complete and accurate details of your proposed use, no license is finally effective unless and until full payment is
received from you (by CCC) as provided in CCC's Billing and Payment terms and conditions. If full payment is not received on a timely basis,
then any license preliminarily granted shall be deemed automatically revoked and shall be void as if never granted. Further, in the event that you
breach any of these terms and conditions or any of CCC's Billing and Payment terms and conditions, the license is automatically revoked and shall
be void as if never granted. Use of materials as described in a revoked license, as well as any use of the materials beyond the scope of an
unrevoked license, may constitute copyright infringement and the RSC reserves the right to take any and all action to protect its copyright in the
materials.
WARRANTIES
The RSC makes no representations or warranties with respect to the licensed material.
INDEMNITY
You hereby indemnify and agree to hold harmless the RSC and the CCC, and their respective officers, directors, trustees, employees and agents,
from and against any and all claims arising out of your use of the licensed material other than as specifically authorized pursuant to this licence.
NO TRANSFER OF LICENSE
This license is personal to you or your publisher and may not be sublicensed, assigned, or transferred by you to any other person without the
RSC's written permission.
NO AMENDMENT EXCEPT IN WRITING
This license may not be amended except in a writing signed by both parties (or, in the case of “Other Conditions, v1.2”, by CCC on the RSC's
Документ
Категория
Без категории
Просмотров
0
Размер файла
16 447 Кб
Теги
sdewsdweddes
1/--страниц
Пожаловаться на содержимое документа