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Microwave-Accelerated Transformations in Synthetic Organic

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Microwave-Accelerated Transformations
in
Synthetic Organic & Medicinal Chemistry
A dissertation presented
by
Amy Elaine Kallmerten
to
The Department of Chemistry and Chemical Biology
In partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in the field of
Organic chemistry
Northeastern University
Boston, Massachusetts
st
October 1 , 2010
UMI Number: 3450525
All rights reserved
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UMI 3450525
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Microwave-Accelerated Transformations
in
Synthetic Organic & Medicinal Chemistry
by
Amy Elaine Kallmerten
ABSTRACT OF DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Organic Chemistry
in the Graduate School of Arts and Sciences of
Northeastern University, October 1 2010
Abstract
Microwave assisted organic syntheses have evolved from pioneering work conducted in
the 1980’s to become a central feature of contemporary organic chemistry. Though
initial research was conducted using modified domestic instruments, a variety of
sophisticated reactors are now available, and the technology has been adopted widely
throughout the pharmaceutical industry. Based on dielectric heating, microwave
mediated synthetic transformations exploit the ability to achieve very carefully controlled
yet rapid application of thermal energy, which typically results in faster reaction rates
and improved product impurity profiles. The research described herein highlights
powerful new applications of microwave assisted organic synthesis with particular
emphasis on radiolabeling methodologies. In this arena, the full benefits of fast reaction
rates are exploited as the methodology can allow practical routes to short half-life
radiopharmaceuticals otherwise inaccessible by conventional means. Following a
review of the field, Chapter 2 describes microwave mediated fluorodenitration of
nitroarenes. In addition to providing access to an array of fluoroarene building blocks for
organic and medicinal chemistry, the methodology is extended to the introduction of 18F
labeled products. The latter are of importance in the rapidly emerging field of positron
emission tomography (PET) imaging, and the reaction times are compatible with the
half-life of this positron emitting nuclide, rendering this methodology suitable for
adoption in biomedical and clinical imaging. Chapter 3 describes application of
microwave mediated cross coupling of substituted arenes with a variety of carbon based
electrophiles. Exploiting the power of microwave irradiation in the presence of transition
metal catalysts allowed access to products otherwise accessible by conventional
methods. Furthermore, by careful choice of substrate, in situ coupling followed by
tandem fluorination could be achieved. This one pot, three component methodology
provides facile access to libraries of heavily functionalized arene products, including
analogs of clinical candidates for PET imaging, where lipophilicity can be readily
modulated as desired. In Chapter 4, the application of microwave thermolysis is
harnessed in the synthesis of members of the xanthine class of heterocycles. A key
feature of the methodology involves a novel catalyzed annulation, and the process
provides access to libraries of functionalized xanthines, including inhibitors of the
adenosine A2A receptor. Chapter 5 explores the use of fluorinated tags, prepared
using microwave methodology, for the labeling of peptides, proteins, and antibodies.
Applications of these methodologies can be expected through in vivo imaging, allowing
biodistribution of the parent biogenic molecule to be studied in real time. Incorporation
of aspects of the methodology in the undergraduate chemistry laboratory is also
described, together with emerging collaborations with industrial partners.
ACKNOWLEDGMENTS
This work would not have been possible without the help, guidance, and support of
numerous people, to whom I am grateful.
First and foremost, I would like to extend my deepest gratitude to my advisor Dr.
Graham B. Jones for his guidance, mentoring, and during the course of my studies.
Thank you for your patience, training and every bit of advice, support and friendship you
gave during my seven years at Northeastern. From ACS, to recruitment and building the
program, to all of our research endeavors—it has been an honor to work with and learn
from you and I will be forever indebted for everything you have done. Thank you Boss.
To members of the Jones group, thank you for your support and advice. Especially
to my undergrads… I already miss the Jones group antics. I would especially like to
extend my sincerest gratitude to my friend and colleague Dr. Paul LaBeaume. Your
daily friendship, advise and support as well as the incredible degree of dependability
you displayed through out the years made everything that is embodied in this document
possible and I am forever indebted to you.
I would also like to warmly thank the members of my committee (Dr. Graham B.
Jones, Dr. Paul Vouros, Dr. Zhaohui Sunny Zhou, and Dr. Misha Sikovsky) for their
input, suggestions and patience during the preparation of this dissertation. Much of this
work would not have been possible without the technical aid of several people
especially Dr. James Glick for his mentoring in addition to assistance with LC-MS
analysis of products generated on the project and Dr. Roger Kautz for his help and
insight regarding NMR techniques.
I would like to extend thanks to the entire faculty, staff, and students in the
Department of Chemistry and Chemical Biology for helping make my studies and
research on this project such an enjoyable experience.
Sincerest thanks to the many friends I’ve made both at Northeastern and at UNH
Law as well from Gilford and Huckins— There are far too many people to list… but I’m
positive you know who you are and what you mean to me.
Finally, I am forever indebted to my family—Mom, Dad, Sam and Jubee… you are
my friends, biggest cheerleaders, and most important supporters and I would not be
here today without you.
DEDICATION
To my parents Dan and Pam Kallmerten;
The sacrifices you’ve made for your children are nothing short of amazing, and I am only where
I am because you dreamt of providing the means for all us to pursue ours.
And to my best friend with all my love… always.
TABLE OF CONTENTS
Abstract
3
Acknowledgments
6
Dedication
7
Table of Contents
8
List of Figures
10
List of Schemes
11
List of Tables
12
List of Abbreviations
13
Chapter 1
Chapter 2
Chapter 3
Chapter 4
Microwave accelerated radiolabeling methods and their
application in positron emission tomography imaging
15
References for Chapter 1
40
MICROWAVE ACCELERATED NUCLEOPHILIC AROMATIC SUBSTITUTIONS
48
References for Chapter 2
62
ONE POT THREE-COMPONENT FLUOROALKYLATION,
FLUOROVINYLATION AND FLUOROALKYNYLATION STRATEGIES
64
References for Chapter 3
91
MICROWAVE ASSISTED SYNTHESIS OF XANTHINE ANTAGONISTS
OF THE ADENOSINE A2A RECEPTOR
93
References for Chapter 4
102
BIOCONJUGATION OF FLUORINATED TAGS FOR 18F PET IMAGING
104
References for Chapter 5
133
Chapter 6
Experimental Procedures and Spectra
135
Appendix
Undergraduate laboratory experiments
208
Chapter 5
LIST OF FIGURES
Nucleophilic substitution pathways
51
The CEM Discover Microwave Reactor
55
Transition metal orbital geometries
66
Transition metal hybrdization
67
Syn addition / elimination pathways
73
Scope of the BDMS annulations reaction
97
Tangles / plaques involved in Alzeheimer’s Disease
106
PiB Imaging of AD plaques
109
Structure of IgG1-κ (MMHA-2 clone)
112
Structure of IgG2-ακ
113
Structure of IgG1-k
114
LIST OF SCHEMES
SnAr displacements
52
Scope of fluorodenitration
58
Haloperidol synthesis
59
Donepezil synthesis
60
Mab labeling methodology
60
Nitrodeiodination
61
Common Pd coupling methodologies
68
Castro-Stephens coupling
74
Synthesis of RP1005 analogs
89
Synthesis of nifrolidine analogs
90
BDMS route to xanthines
96
Preparation of xanthine libraries
99
Synthesis of KW6002
100
LIST OF TABLES
Incorporation of 2H labels in arenes
34
Scope of the tandem Hiyama alkylation-fluorination reaction
79
Optimized Heck cross coupling reactions
81
Scope of the Heck cross coupling reaction
83
Scope of the Sonogashira coupling reaction
87
LIST OF ABBREVIATIONS
Ac
Acetate
ACN
Acetonitrile
Ar
Argon
Bn
Benzyl
BOC
CI
Di-tert-butyl dicarbonate
Chemical ionization
DABCO 1,4-diazabicyclo[2.2.2]octane
DBU
1,8-Diazabicyclo[5.4.0]undec-7-ene
DCM
Dichloromethane
DMAP
Dimethylaminopyridine
DMF
Dimethylformamide
ESI
Electrospray ionization
HPLC
High pressure liquid chromatography
HRMS
High resolution mass spectroscopy
LAH
Lithium aluminum hydride
Ms
Methanesulphonyl
N2
Nitrogen gas
NBS
N-Bromosuccinimide
NMO
N-methylmorpholine N-oxide
NMR
Nuclear magnetic resonance
NOE
Nuclear Overhauser effect
OMe
Methoxy
PET Positron emission tomography
Ph
Phenyl
Py
Pyridine
RCY Radiochemical yield
TBAF
Tetra-butyl ammonium fluoride
TBDMS Tert-butyldimethylsilyl
Tf
Triflate
TFA
Trifluoroacetic acid
TLC
Thin layer chromatography
TMS
Tetra-methylsilyl
Ts
Toluenesulphonyl
Chapter 1: Microwave Accelerated Radiolabeling Methods
and Their Application in Positron Emission Tomography
Imaging
Positron emission tomography [PET] has become one of the most powerful in vivo
imaging modalities, capable of delivering mm3 resolution of radiotracer distribution and
metabolism. 1 When combined with anatomic imaging methods (MRI, CT) co-registered
multimode images offer the potential to track metabolic and physiologic events in
diseased states and guide and accelerate clinical trials of investigational new drugs.
Though powerful as a technique only a limited number of drugs have seen clinical use
and to date only one drug 2-fluoro-deoxy-D-glucose (FDG) has received FDA approval.
2
One of the drawbacks of PET imaging is the need for tracers labeled with an
appropriate nuclide and the half-lives of these agents places special constraints on the
chemical synthesis. Among the most popular are 11C (t ½ =20.4 min) and 18F (t ½
=109.8 min) labeled compounds and this has resulted in a resurgence of interest in
practical application of the chemistry. 3 4 This review will focus on the microwave
methods of acceleration of organic reactions for the production of PET image contrast
agents, with emphasis on the period 2006-2009.
Background on microwave acceleration of reactions
The heating properties of microwaves were observed shortly after the invention of radar
technology during world war II. The magnetic component has been shown not to
contribute to the energy transfer, so microwave acceleration studies need only to be
concerned with columbic interactions of the field with electric dipoles and charges. The
microwave region of the electro-magnetic spectrum lies in between the infrared and
radiofrequencies. Microwave dielectric heating should not be confused with microwave
spectroscopy. In this field molecules are studied in the gas phase. In the solid and liquid
phases molecules are generally not allowed to rotate independently. When energy is
applied as high frequency electro-magnetic waves the electric field exerts a force on
charged particles if the particles can move freely then a current will be induced (ionic
conduction). If the particles are bound and their movement restricted they will move until
a counter-force balances them and the net result is dielectric polarization. Both are
sources of heating. The common understanding of this absorption process is that an
oscillating microwave field results in hindered rotation of the solute and solvent
molecules, which through friction induces heating of the reaction sample.
This direct coupling of the microwave field to the molecules of the entire reaction
provides a much higher temperature. This “instantaneous” temperature is not limited by
the thermal conductivity of the vessel as they are when a conventional heat source is
applied to a substance, and thus, this results in superheating of any molecule that is
susceptible to dipole rotation or ionic conduction.
Typical microwave heating includes applying microwave power until the target
temperature is reached and then the instrument automatically shuts off. Enhanced
microwave synthetic methods include applying cooling to the system so that a constant
microwave field is applied to the reaction mixture and direct energy is applied without
degradation due to high temperatures. Lastly, unlike conventional heating in which the
upper limit temperature is the boiling point of your chosen solvent, microwave
instruments can have pressurized vessels in which reactions can be performed at
higher than the atmospheric boiling point. All of these factors can contribute observed to
higher yields and cleaner chemistries with microwave conditions.
History of Microwaves in PET labeling3
In order to achieve the most efficient incorporation of radioactivity into a tracer molecule,
PET chemistry has historically been an area that integrated the latest developments in
chemical and apparatus technique, including multi-step, remote controlled methodology
for radiolabeling with radioisotopes. In 2002 Elander and Elander published a review on
the microwave applications of radiolabeling with short-lived PET nuclides in which they
describe the field as being dependant on the creativity with which radiochemists can
find new synthetic strategies and shave minutes off total synthesis of relevant
molecules.
In 2002 household devices incorporating manipulators to remotely move vessels, open
and close the door, and for the inclusion of robotic-based automation were still routinely
used. Monomodal devices designed for the space limitations of radiolabeling
environments were just being constructed. The household devises used were highly
unstable due to variation in sample geometry and electric susceptibility. The drive for
single mode microwave cavities came from the recognition that they were more
efficient, and controllable (and therefore reproducible) because the electric field within a
reaction sample should be as strong and as homogenous as possible.
The use of microwave devices in PET radiolabeling chemistry is described as
having its own set of constraints. Lead shielded hot cells are often space-limited and
filled with various equipment and devices that restrict available working space and
accessibility. The first and second generation devices used by the Elander group are
described as being difficult in achieving comparably intense and homogenous fields and
it is pointed out that for every ten degrees increase in reaction temperature the reaction
rate should increase by a factor of two. In PET tracer synthesis, this is crucial because it
cuts down on losses due to decay of the radionuclide.
For the first ten years, microwave induced chemistry suffered from lack of
monitoring devises due to both availability and affordability. Methods of monitoring at
the time (2002) included yield driven trial and error optimization and visual inspection.
Calibrated infrared monitoring of the outside temperature of vessels was becoming
standard in microwave chemistry, but had not yet been applied to PET microwave
devices. Measuring of pressure in closed vessels is also important to maximize the
heating of volatiles solvents and also necessary to avoid exceeding the pressure
tolerances of the vessel. Devices at the time varied from pressure guages connected
via tubing through septum-capped vessels to those connected to acid digestion bombs.
The chosen vessel is an important consideration in the field of microwave
synthesis. Microwave transparency, the ability to sustain generated pressures, physical
dimensions and chemical stability are all crucial factors for general microwave
synthesis. The sample composition is also important, as solvents must be chosen
based on dielectric constant. At the time of publication, solvent free conditions for
microwave synthesis were still in its early infancy. The review concludes with reflection
on the potential growth areas for this field. Synthetic areas highlighted included metal
catalyzed carbon-carbon bond formations including ring closures, the use of bifunctional
precursors and metabolically stable position labeling. It also points out that water
approaches the dielectric constant of acetone at high temperatures, and thus could
potentially be exploited as a pseudo-organic solvent for more environmentally friendly
conditions. Lastly, the need for online monitoring methods for continuity and consistency
in synthesis is highlighted.
In 2006 Jones3 et al published a chapter in the second edition of Microwaves in
Organic Synthesis focused on the use of microwaves for PET. In 1986 the International
Isotope Association was formally organized and naturally adopted the Journal of
Labeled Compounds and Radiopharmaceuticals as its official journal. Within the
pharmaceutical industry, labeled compounds were now being used for a variety of
purposes including, screening new targets, binding experiments, identification of
metabolites, adsorption, distribution and excretion studies and for quantifying
concentrations in target organs. Despite this growth in the few years between the two
publications, many limitations were still highlighted. Most of the limitations had to do
with lack of available dedicated facilities for this type of work. Radiochemistry requires
dedicated labs, separate storage and disposal facilities and specialized training of
personnel. The scale of the reactions performed is much smaller (microgram instead of
milligram to gram scale) and thus requires ultimate precision and synthetic skill, and the
scale size limits purification to radiochromatographic methods rather than distillation and
recrystallization commonly utilized. The push at the time was to gain a degree of control
over the number of labels (mono vs. multiple) incorporated into the compounds of
interest.
In 2006, as described by Jones et al, the labeling field was described into three
basic fields—tritium/deuterium, carbon, and other positron emitters. For a more detailed
explanation of the reactions, the readers is invited to consult the 2006 review chapter,
but for the purposes of contextualization, the state of the capabilities as well as the
highlighted shortcomings will be listed. Tritium labeling was usually a one step reaction
and frequently involved a catalyst. The tremendous developments in phase transfer
catalysis, supercritical fluids, ionic liquids and catalysis in general (viz. the 2001 Nobel
prize in chemistry) benefited not only microwave chemistry as a whole but had not been
fully recognized and utilized within the labeling community. The major ways to
incorporate a tritium (or deuterium) label into a molecule of interest included base or
acid catalyzed isotope exchange, homogenous or heterogenous hydrogenation,
aromatic dehalogenation, methylation and borohydride reduction. Some of the problems
associated with isotopic exchange reactions included hesitation in the industry to use
base catalyzed exchange due to back exchange, harsh temperatures and long
reactions times needed for acid catalyzed exchanges, and general limitations due to
health and safety regulations. The highest specific activity able to be used is 2%
isotopic abundance, which limits the maximum specific activity of the product that can
be obtained. Hydrogenation with T2 requires specific commercial instruments as well as
suffering from low solubility in organic solvents leading to slow reactions. The diatomic
T2 has a dielectric constant of zero and thus is not influenced by microwave irradiation.
Aromatic dehalogenation and borohydride reduction are both very wasteful with as
much as 50% or more of the labeling going to effluent. Lastly, while tritiated ethyl iodide
can offer specific activity of up to 80 Ci in one step and is commercially available, its low
boiling point requires very careful handling and it is stable for only a short period of time.
The highlighted interest areas include a need for methylating agents with greater
flexibility, as well as alternative labeling methodologies. One alternative strategy
suggested is aromatic decarboxylations (both tritiations as well as treatment of tritiated
waste). Microwave enhanced tritiation reactions could lead to the creation of more
efficient procedures so that less waste is produced, as well as methods to convert
waste back into suitable reusable reagents.
While isotopic hydrogen labeling was by far the most in depth discussed labeling
strategy, carbon-11 labeling also gained some recognition with carbon and fluorine
being recognized as being the two most widely used radioisotopes for
radiopharmaceuticals. The short half lives are advantageous for the safety of humans
undergoing imaging studies (low radiation exposure doses) but pose enormous
challenges for chemists to produce radioligands of sufficient purity, specific activity and
quantity in severely limited periods of time. The reactions and purifications must be
both rapid and simple, and the final dose must be sterile and pyrogen free. Microwaves
were explored at an early stage but recognized by Jones as not matching the rapid
expansions in synthetic organic chemistry due to the strict radiation safety
considerations. Oxygen, sulfur and nitrogen alkylations had been achieved and
synthetic times were decreased from 5 minutes to 30 seconds, accompanied by a
twenty percent increase in radiochemical yield. Aromatic alkylation, while more
attractive, is more difficult. Classic methodology is through Stille coupling with aryl
trialkyl tin precursors. Microwave heating lead to significant increases in radiochemical
yields using Suzuki couplings from boron containing precursors, allowing the avoidance
of toxic tin-containing by products and contaminants, but more work was recognized as
being needed. Labeling with a cyanide secondary labeling agent is possible when
microwaves bring the production of this reagent from 11CO2 from ten minutes to thirty
seconds and while suffering from fluctuations in yields and impurity contamination,
reductive methylation with formaldehyde is suggested as being useful for reinvestigation
for the labeling of primary and secondary amines under enhanced conditions. Lastly,
the reaction of cyclotron generated 11CO2 with organometallic reagents to produce
carboxymagnesium halides and carboxylic acids utilizing microwaves has allowed for
more than just the previously reactive aliphatic Grignard reagents/amines to be
accessible.
Fluorine labeling is most often performed via aromatic or aliphatic nucleophilic
reactions. Electrophilic fluorination reactions, while fast, suffer from low specific
activities resulting from agents derived from F2 and thus are not frequently used. The
most widely performed reactions are displacements of leaving groups from aromatic
rings (NO2, X, or N+R3) with the nitro displacement being the most convenient and
reliable choice. These reactions require an activating group within the same aromatic
structure and while requiring high temperatures, often use polar solvents which makes
for easy conversion to microwaves. Tosylate displacement followed by Sonogashira
cross coupling reactions of fluoroiodobenzene was by far the most commonly utilized
aliphatic nucleophilic substitution process. Oxygen and nitrogen alkylations are also
relatively simple. Both of these reactions allow for use of low boiling solvents to avoid
DMSO/DMF cleanup. Under microwave optimized conditions, the synthesis time of FDA
approved FDG dropped from over an hour to only 30 minutes, and the radiochemical
yields increased from 47 to 76%.
Examples of success with other positron emitters include a procedure for
bromine-76 labeling under microwave optimized conditions via tin displacement with a
70-90% yield (up from 20% yield for conventional conditions) and the labeling of
oligonuclides with metal position emitters (Ga, Tc). By coupling with DOTA (picture)
through a hexylamine linker and labeling via microwave activation the time was cut from
twenty minutes to 60 seconds and the yields was improved from 19% to 30-52% with a
reduction of side reactions.
Surveying the literature from 2006-2010, the following highlights of microwave
accelerated synthesis of radiopharmaceuticals have been selected. Using targets
ranging from 18F, 99Tc, 124I, 11/14C, and, by analogy, 2H, all have been selected as
exemplars of progress made in labeling technology, and as a guide to where current
emphasis is being placed in the radiolabeling community.
18
F compounds
Katzenellenbogen and Welch reported the synthesis of an 18F labeled version of the
O
18 F
1
O
N
COOH
O
I
H2N
TFA
2
O
N
COOH
HN
O
O
Reaction Conditions:
(i) K18F, kryptofix, MeCN, 100° C, 10 min;
(ii) Cs2 CO3 (1.2 eq)
*iii) 18 F-iodonium compound, CuI, DMF 118° C, 90 min
18F
PPAR gamma targeting ligand 2. Synthesis utilized and Ullman type condensation of
tyrosine derivative 1 with a fluorinated iodoarene, generated itself by MW assisted
radiofluorination. Coupling of the derived iodonium salt gave 2 in good yield under MW
irradiation. PPAR gamma selective ligands, which are know regulators of lipid
metabolism, have implications in breast cancer and cardiovascular pathways, hence the
interest in ligand 2.5
A recent review by several members of the Wales Research and Diagnostic
Positron Emission Tomography (PET) Imaging Centre, discussed the many strengths
and weaknesses of various techniques for labeling both small molecules and
macromolecules. As highlighted in the review, much needs to be done to advance the
labeling capabilities before other molecules, especially macromolecules, can take their
place along well-established oncology biomarkers such as 3 and 4. 6 The review
O
OH
HN
O
N
3
HO
HO
O
18F
H
18-FLT
O
18F
4
OH
OH
18-FDG
highlights the potential of microwave technology to have a large impact in this area.
Pikes group reported an efficient synthesis of an 18F-labeled substituted hetereocycle 6
via nucleophilic displacement on bromide 5. Compound 6, which targets the glutamate
subtype 5 receptor, was used for primate brain imaging, the high efficiency and
radicochemical yield of product allowing for extensive PET imaging. 7
Another group reported a one-step method for the labeling of 9.8 These ethoxyS
18F-,
N
Br
S
87% RCY
N
18F
F
45 W X 2 min
F
6
5
CN
CN
modified compounds are less lipophilic than the alkyl-substituted metomidate tracer and
the biological evaluation has indicated that these compounds are likely candidates for
adrenal carcinoma detection.
O
O
OH
a
N
N
N
X
(1) X= H
(2) X = Cl
(3) X= Br
7
(1) X= H
(2) X = Cl
(3) X= Br
TsO
8
(a) TBAH, (b) DMF alkyl-tosyl-18F, 150° C, 15 min.
O
18
F
n
N
n
N
X
O
NBu 4
b
O
18
F
X
N
9
(1) X= H, n= 2
(2) X = Cl, n=2
(3) X= Br, n=2
(4) X=Br, n= 3
(5) X= H, n=3
Compound 10, FPhEP, is designed to target the alpha-4-beta-2 nicotinic acetylcholine
receptor, which has implications in the imaging of
Alzheimers disease. Its synthesis was effected using a
H
N
18F-
18F
N
no-carrier-added nucleophilic heteroaromatic ortho10
FPhEP-- Epibatidine analogue
for nAChR imaging
radiofluorination via a N-boc protected chloro or bromo derivative. This (epibatidinebased) antagonist allowed imaging to determine brain kinetics of the agent.9
In interesting platform for protein labeling involves the use of Cu catalyzed ‘click’
chemistry. Formation of 18F labeled azido PEG, allowed subsequent conjugation with
the alkynylated protein derivative 11 to form covalently linked agent 12. The subsequent
microPET study involved imaging of HER2 expression in vivo. 10
O
N3
N3 PEG OTs
N PEG18F
N N
N PEG
PEG18F
S
O
PE
N PEG
S
O
12
11
In an interesting study on comparative rates of nucleophilic aromatic substitution, a
H3C
F H3C
CN
Br
13
H3C
14
CN
H3C
H3C
I
Cl
15
16
CN
CN
X
K18F, K222
18F
H3C
DMF or DMSO
Microwave
17
18
CN
CN
series of meta halo 3 methyl benzonitrile derivatives (F, Br, Cl, I) 13-16 were examined
for propensity to undergo radiofluorination. Under microwave conditions (3 min), relative
yields of product 18 followed the expected trend with the fluoro compounds most
efficient F (64%) follwed by bromo (13%) and chloro (9%), with the iodo non reactive. 11
O
19
O
N
18F
N
S
N
H
Microwave induced synthesis of an 18F labeled version of the serotonin 2A receptor
targeting agent altanserin (19) was reported. The subsequent PET study allowed its
kinetic profile of metabolism to be examined, and the impact of metabolites on plasma
profiles for individual plasma input functions used in tracer modeling. 12
In a study on automation of microwave chemistry, synthesis of 18F flumazenil 21
O
N
N
O
21
20
N
DMF or DMSO
Microwave
30 min, 160 ° C
18F
N
O2N
N
K18F, K222
O
O
N
O
O
was optimized from the corresponding nitro precursor 20 using a dry K/K222+ F18complex. Following the 50-60 minute total synthesis time, the labeled flumazenil was
observed to be stable for 8 hours. 13
An authoritative micro-review on PET methods, the benefits of carrier versus no
carrier added methods, and the potential for development of automated processed was
published by Pikes group.14
Highlighting a strategy for aliphatic radiofluorination, Pike outlined used of aryl
O
O
S
O
R
X
LINK
22
O
K18F, microwave
no phase transfer catalyst
O
18F
R
sulfonate nucleophilic assisting leaving groups (NALGs). Based on the tethered
substrate 22 a metal chelating unit is attached to aryl ring via ether linker, allowing
effective conversion to the released fluoroalkyl product using radiofluoride ion under no
carrier added conditions and without phase transfer catalysts / cryptands. Significant
rate enhancements were evident in halogenation reactions using metal halides under
microwave conditions.15
O
18F
O
CHO
23
H
N
R
O
O
BOC
N
18F
O
O
R
24
R= RGD Peptide
18F labeling of cylic RGD peptides was reported using oxime conjugatation
chemistry. Various fluoroaldehydes 23 were coupled with a boc protected oxime
derivative of RGD to yield coupled products 24. Using 3 different 18F labeled aldehyde
substrates, biodistribution and tumor uptake studies were then undertaken, along with
pharmacokinetics analysis of these interesting derivatives. 16
O
25
O
O
O
O
O
O
HO
O
HO
N
N
N
K18F, K222
26
N
DMSO
microwave
N
Cl
N
18F
Microwave induced nucleophilic fluorination of heterocyclic corticosteroid analogs has
been reported. Following halogen exchange on substrate 25, the products 26 were used
as probes for the imaging of glucocorticoid receptor. 17
Microwave assisted synthesis of a series of 18F fluoro-5-deoxy-5-fluorouridine
derivatives 27-28 was reported. High radiochemical yields of products were obtained by
O
27
N
O
O
Cl
O
N
O
18F
HO
N
OH
O
O
RCY: 75-92%, <45%
N
O
28
18F
HO
simple nucleophilic displacement. 18
OH
With the long term goal of developing a fully automated radiofluorination module,
Pike reported coupling of a microwave reactor with the Synthia module to achieve an
automated system whose capabilities were demonstrated wit a series of nucleophilic
fluorination reactions. 19
Surveying a range of 18F labeled tagging agents, a thiol-reactive reagent for site
specific 18-F glycosylation of peptides (31) was developed. The reagent was reacted
with model peptides confirming chemoselectivity and excellent conjugation yields. This
opens the potential for the design and general development of 18F-labeled bioactive Sglycopepties to study their pharmacokinetics20
O
O
28
N
O
O
N
18F
N
29
N
18F
18F-FPyME
O
18F-FBAMB
O
OAc
O
O
30
O
N
N
H
O
AcO
18F
18F-FBEM
O
S
AcO
Ac3-18F-FGIc-PTS
21
S
18F
O
Microwave assisted synthesis of 18F versions of PR04MZ and LBT999 (33 and
32
18F
N
O
OMs
O
33
A
N
18F
O
O
B
A: 120° C, MeCN, K2CO3, K18F, 3 min
B: 175 °C, 18 bar, MeCN, K2CO3, K18F, 0.075 min
N
O
O
34
34), selective dopamine transporter ligands derived from cocaine, were achieved from
mesylate 32. Using TBAF in the presence KF/K222 the reactions took up to 34-40 min
under conventional conditions, but using KF under microwave acceleration products
formed within 3 min, allowing quantitative imaging to be performed.21
Davies reported synthesis of fluorinated oligosaccharides probes exemplified by
35. These proved to be potent probes of adhesion in toxoplasmosis, allowing sugarprotein binding events to be subsequently studied (di and tri saccharides were
examined).
OH
HO
OH
OH
OH
COO-
O
O
O
HO
O
O
AcHN
18F
HO
NHAc
OH
OH
35
22
A nanotech advion coiled tube type microreactor system has been reported, which
N
O
NH
36
O
N
18F
O
O
HO
O
O
37N
H
O
O
HO
S
38
O
N
H
S
O
18F
O
18F
allows facile conversion of tosylate precursors to 18F labeled radiofluorinated agents.
Using the CNS PET image contrast agent 18F fallypride 36 as an test bed, scale up
was easily achieved through continuously infusing reagents via precursor solutions. 23
A microwave assisted one step radiosynthesis of 37-38, derivatives of
CHS27023A has been reported. The analogs of this MMP inhibitor allowed the authors
to study tissue accumulation. 24
Using a microwave induced fluorodenitration strategy, a number of neuronal/nonN
N
39
N
K18F, K222
N
microwave, DMF
10-15 min
O
O
N
N
40
N
N
18F
O2N
neuronal alpha 7 nicotinic receptor binding agents have been prepared. The agents,
exemplified by 18F NS10743 (40) showed promising diagnostic potential in neurology
and oncology. 25
A key publication examines conventional versus microwave conditions for
exchange of 19F with F18. A series of fluorobenzophenones, 1-(4N
N
O
41
HN
HN
O
O
S
O
Lapatinib
18F
Cl
fluorophenyl)ethanones, various activated and deactivated fluorobenzenes, N(pentafluorophenyl)benzamide, (pentafluorophenyl)formamide,
(tridecafluorohexyl)benzene, tetradecafluorohexane were subjected to 19F/18F
exchange to optimize parameters. Idealized conditions were applied to several
biologically active molecules—including analogues of WAY-100635, and Lapatinib. – Of
numerous findings, multifluorinated molecules beating electron withdrawing group were
successfully labeled, whereas monofluorinated and substrates with electron donating
substituents required extended heating. 26
2H and 3H Labeling
Using arene substrates 43-46 as test cases, a series of experiments were
43
44
R'
R
N
1; R= 4-NH2
2 R= 2-NH2
3 R= 4N(CH3)2
4 R= 2-CH2NH2
5 R= 4-CH3
R
6. R= H R'= CH2
7. R= CH2 R'= H
N
NH2
45
H2N
9
NH2
8
46
NH2
N
N
OH
reported developing microwave mediated deuteration methods. The results, using
preparative scale reactions (see Table 1) were subsequently and successfully applied to
tritiation procedures on the mg scale.27
Table 1.
compou
microwave
sites
nd
time
labeled
1
1 min x 3
3,5
71
2
2 min x 5
3,5
95
3
2 min x 5
3,5
74
4
2 min x 6
CH2
100
5
2 min x 6
CH3
97
6
2 min x 6
5, CH3
92, 75
7
2 min x 6
5
97
8
2 min x 11
6,7
95, 30
9
2 min x 12
4,7
95, 95
%D
Two notable reviews on tritiation methodology appeared, one on current
developments,28 and another which focuses on C-H bond activation for 2H/3H labeling
of glycoconjugates using ultrasonic and microwave activation. 29 A series of H/D
exchange reactions was reported using hydride-activated catalysts. A number of safe,
47
N
48
HO
H
OH
OH
HN
N
H
Dextrorphan
OH
H
O
Formoterol
user friendly and efficient methods for deuterium incorporation are highlighted, with
applications in the form of bi and tricyclic aromatic compounds and chiral natural
products including dextrorphan 47 and formoterol 48. Among key findings, activation of
the Os or Rh catalysts by the NaBD4 employed proves essential. 30
The synthesis of poly deuterated di-o-sulfates of three isoflavonones (daidzein 49,
glycitein 50, and genistein 51) was reported using CF3COOD under microwave
irradiation. High yields (90%) and isotopic purities (>90%) of products were obtained. 31
HO
O
HO
O
50
49
H3CO
O
O
OH
Daidzein
OH
Glycitein
HO
O
Genistein
51
OH
O
OH
99Tc labeling
Microwave assisted synthesis of a series of eta-5 rhenium carborane complexes
has been reported. The reaction conditions proved equally effective for production of
tracer level Tc99 complexes.32
11C/14C labeling
A new strategy for isotope containment has been reported, involving use of
fluorous tags. The substrate employed was 14C bromobenzene. Subsequent Ir
catalyzed borylation and transition metal catalyzes elaboration allowed synthesis of
labeled analogs of the 4-phenyl piperidine class of CNS agents viz. 55. Using the
Br
Ge
Rf
14C
14C
53
52
Ge
Rf
14C
NBn
14C
54
55
NBn
traceless cleaver method, material loss via volatile intermediates is minimized. 33
Microwave assisted synthesis of a series of 11C labeled dopamine d3 receptor
antagonists was reported. A key step involved Pd catalyzed cross coupling with C-11
cyanide to give product, e.g. 56, which proved a potent and selective antagonist of the
d3 receptor in vitro. However, using the probe in vivo, at specific activity levels of 55.5
+/- 25.9 GBq/micromole, both in porcine and non-humane primates it was concluded
that in both species the compound does specifically populate the d3 receptor34
CN
56
O
N
N
N
CF3
Using one pot procedures under microwave enhanced conditions a series of 4aminoquinazolines and quinazolin-4 (3H)-ones were labeled with 14C in the 4-position.
Further studies were applied to 2-aminobenzonitrile-[cyano-14C], 2-aminobenzoic acid
[carboxy-14C], 2-aminobenzamide-[carboxy-14C] and 2-aminobenzamide-[carboxy14C].35
A series of 11C labeled synthetic cannabinoid receptor binders were prepared (CB-1
and CB2 type). Exemplified by 1,5-diaryl pyrazole 57, the agents were subsequently
using for in vivo imaging of the CB1 receptor. 36
Y
O
R1 = Cl, I, MeO, MeS, MeSO2, H, OH, FCH2O
R2 = Cl, H, Me, Br, CF3
R3 = H, Cl
R4 = H, Cl, MeSO2
R1
R5 = H, Cl
X = C, H, N
Y= CH2, O,
N
NH
N
57
N
R5
R2
X
R3
R4
Radiosynthesis of 11C labeled MNPA was reported. In conjunction with 18F
fallypride, the agent was used to study dopamine internalization and amphetamine
displacement. Among key findings, extracellular dopamine concentrations largely return
to baseline within 1-2 hours but radio-ligand binding remains attenuated for several
hours after. 37
Microwave synthesis was applied to the production of an 11C labeled version of the
phosphodiesterase (subtype-4) binding agent -(R)-rolipram 58. In addition to receptor
site density, the authors also studied its influence in anesthesia. 38
Microwave synthesis proved superior to classic approaches in the synthesis of a
O
NH
58
O
O
14C labeled version of the anti-malarial drug 59. 13C labeled PNDP was then used to
studying the ADME and pharmacokinetics of the drug. Its potent pharmacological
N
*
59
OH
N
HN
*
N
OCH3
* = cites of 14C labeling
Cl
N
activity is underscored by its activity against Plasmodium falciparum which is resistant
to chloroquine and other drugs. 39
A one pot microwave mediated synthesis of the versatile labeling agent methyl [14C]-
N
C
S
60
isothiocynate was reported. The six step, one pot procedure, from [14C]-KCN was
highly efficient. Subsequent oxidation of the sulfur was used to provide access to
triazole-ethers upon reaction with alcohols40
A microwave assisted synthesis of 14C labeled benzyl acetate was reported via
esterification of sodium 14C acetate with benzyl bromide in the presence of 18-crown-6ether, which gave product 62 in 97% yield. 41
O
Br
14C
O
O-
14C
O
61
62
A microwave-enhanced synthesis of a 14C labeled indole was developed. The
strategy, which potentially allows access to a broad range of substituted variants,
involves reaction of aniline with a bromoacetaldhyede acetal and subsequent ring
closure.42
Microwave assisted synthesis of [14C]-isosorbide and dimethyl isosorbide from
D-[14C]-glucose was reported, in an overall yield of 79%. Derivatives prepared include
O
OH
N
O
HO
OH
OH
O
H
-O
O
O
H
OH
63
nitrate 64. 43
64
OH
124 Iodine labeling
Microwave mediated synthesis of a series of I-124 labeled EGFR kinase specific
HN
N
N
65
radiotracers 65 was developed. The tracer was used to examine IPQA binding to the
activated kinase. 44
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CHAPTER 2: MICROWAVE ACCELERATED NUCLEOPHILIC
AROMATIC SUBSTITUTIONS
Goals and Objectives
The ultimate objective of the work described in this chapter was to demonstrate methodology for
the introduction of 18F into arenes to allow subsequent PET imaging in vivo. Given that the half life
of the nuclide is approximately 120 minutes, the methodology would need to be expeditious and
also robust, ultimately able to be employed in a clinical radiochemistry laboratory associated with a
cyclotron source. Surveying potential drug candidates on which to model the technology developed,
a large variety of arene and heterocycle drugs contain aryl fluorides, with varying degrees of
FFG1
FG2
F
MW
substitution at promixal locations on the ring. Any process to introduce the 18F nuclide into the
ring would need to be tolerant of pendant functionality and also be compatible with remote
functional groups. In terms of available nuclides from cyclotron sources for interconversion and
introduction of 18F, by far the most user friendly is 18F-. Accordingly, we set out to develop
processes that exploit the nucleophilicity of fluoride in displacement reactions. As is described
herein, fluorodenitration of aromatics is a viable reaction. However, given the timelines involved
both of the nuclide half life, and the practical limit of PET image capture [~3 half lives], traditional
thermal displacements, which often require excessive temperatures and reaction times in excess of
24h on a limited range of substrates, are not practically viable options. However, the use of
microwaves to develop such processes would seem particularly relevant, exploiting their
accelerating effects. Accordingly, a program was initiated to examine microwave assisted
fluorodenitration of nitroarenes and heterocycles for late / final stage introduction of the 18F
nuclide. Given that the nitro group would need to be installed in the molecule of interest, and the
limitations this may impose on synthetic chemistry en route [chemoselectivity, activation/
deactivation of arene ring chemistries], it was also decided to investigate means to also employ
microwave mediated nitrodehalogenation as a route to install the required precursor. Thus, the
summary objectives, as exemplified in the scheme, were to convert haloarenes (FG1) into
nitroarenes (FG2) for subsequent installation of the 18F group. Accordingly, the following
objectives were defined for the project:
1- develop a route that allows rapid installation of F2 – optimize workup to allow fast purification
3 – demonstrate compatability of the route with late stage synthesis
4 – back extrapolate the method to identify means to install the precursor leaving group (nitro
arene)
5 – demonstrate adaptation to install the active 18F nuclide
Nucleophilic substitution reactions are central features of the contemporary synthetic
arsenal and one of the first reaction mechanisms studied in organic chemistry. They are named
based on the kinetic order of the reaction thus SN1 proceeding via a two step process involving full
O
O
S
Nu
Nu
R'
Nu
Nu
S
R'
SN2
OH
O
Nu
X
R
R
R''
R''
O
NuR'
S
Nu
R'
R'
-X
R
X
+Nu
O
Nu
R
R''
SN1
R
R'
Sni:
through tightly formed, complexed
R'' internal nucleophilic substituion,
R''
ion center or through cyclic intermediate or product.
+ Nu
dissociation of the leaving group prior to attack by the
nucleophile. Likewise, SN2 reactions occur in
LG
O
O
X
Nu
R''
O
O
a concerted manner with both attack and dissociation occurring simultaneously. While these are
the more commonly discussed, there are two other mechanisms relevant to the research described
herein – the SNi and SNAr. SNi (“chemical substitution, nucleophilic, internal”) occurs with the initial
attack either produces a cyclic intermediate or an intermediate complex with tight ion pairing.
Thus, while reminiscent of the SN1, a major difference is that the ion pair is not completely
dissociated, therefore no carbocation is formed which can lead to racemisation of chiral substrates.
SNAr (aromatic substitution) describes nucleophilic displacement taking place on an aromatic or
heteroaromatic ring. In these instances the intermediates are balanced through resonance
Cl
Cl
OH
NO2
O
NO2
OH O
Cl
N
O
O
Cl
OH
O
O
O
O
N
HO
OH
NO2
O
NO2
O
Cl
O
N
O
O
O
N
O
HO Cl
O
N
O
O
O
O
Cl OH O
N
O
O
O
Nucleophilic subsitution- Aromatic, sigma complex is resonance stabilized
tautomerization, as exemplified by the hydroxy dechlorination of 2,4-dinitrochlorobenzene
depicted. Depending on the nature of the substrate, aromatic nucleophilic substitution reactions
can thus occur through several different mechanistic pathways including SNAr, SN1, SNi and also
(though not discussed herein) radical based mechanisms. Based on analysis of these pathways, we
became motivated to exploit the power of microwave mediated processes in nucleophilic aromatic
substitution reactions, with particular focus on introduction of fluoro and nitro groups.
OH
2
O2N
1
NO2
OH
O2N
NO2
O2N
NO2
OH
Nitroaromatics are among the most widely studied and storied building blocks in organic synthesis
NO2
NO2
Trinitrotoluene
(TNT)
3
Trinitroresorcinol
(styphnic acid)
O2N
4
12
nitropentadecene
(toxic termite defense chemical)
NO2
Trinitrophenol
(picric acid)
and are of historical importance in the chemical industry. Nitroarenes are commonly used solvents,
and the introduction of this potent electron withdrawing group confers numerous physico-chemical
properties which have been exploited in the pharmaceutical and commodities chemical industry. As
an example, one of the first studied common explosives 2,4,6-trinitrotoluene [TNT] dates back to
the 1800’s. The electron withdrawing capacity of the group has profound consequences on ring
substituents, including enhancing ionization potential of phenols e.g. styphnic and picric acids,
whose pKa’s are orders of magnitude less than the parent phenol. Related nitrovinyl compounds
also possess unique chemistries as a consequence of conjugation exploited in insecticides and
pesticides. The nitro group is a precursor to myriad other functional groups via reduction and
subsequent chemistries and, vide infra, as substrates for nucleophilic displacement. However,
despite their importance, reactions to produce and transform nitro compounds often remain nonselective and in some cases hazardous processes which could benefit from alternative strategies. As
an example, the classical preparation of aromatic nitro compounds, one of the most widely studied
organic reactions involves use of excess of nitric acid combined with sulfuric acid. This is a
hazardous and environmentally problematic process, generating nitrogen oxides and effluent
streams contaminated with waste acids. Such harsh conditions are also often incompatible with
existing functionalities when introduced at a late stage in a multi-step synthesis of a target
molecule. Accordingly, in addition to developing new methodology for the nucleophilic substitution
of the nitro group, we wished to investigate and developed new procedures for the introduction of
the nitro arene group itself [vide infra].
O
F
F
COOH
N
N
N
O
HN
Ciprofloxacin
OH
Cl
Haloperidol
F
N
F
N
N
F
OH
N N
O N
N
O
N
Diflucan
N
N
Risperidone
Methodology for the introduction of fluorine into heteroarenes is a significant focus of current
medicinal and bioorganic chemistry due to the large physicochemical and metabolic impact it can
have on molecule of interest in drug development. Indeed, many blockbuster drugs possess a
fluoroarene motif. 1 Examples include the antibacterial ciprofloxacin, the CNS agents haloperidol
and risperidone and the antifungal diflucan. Medicinal chemist Manfriend Schlosser of the Swiss
federal institute of chemistry perhaps summarized current thinking in medicinal chemistry stating
“smuggling fluorine into a lead structure enhances the probability of landing a hit almost 10-fold.” 2
A new and exciting application of organofluorine chemistry lies in use of 18F labeled nuclides
(generated in a cyclotron) for positron emission tomography (PET) imaging, 3 allowing in vivo
biodistribution of drugs to be determined and also for imaging of diseased states. Accordingly,
there is considerable interest in development of new highly efficient and expeditious methods for
incorporating fluorine into arenes and other building blocks, particularly methods which are also
amenable for use with 18F, whose short half life (110 min) 4 presents additional challenges and
limitations. We became specifically interested in developing expeditious methodology using a
microwave accelerated processes. 5 6Having enjoyed prior success with a variety of transformations
using microwave technology, at the outset of this project we forged a formal partnership with CEM
Corporation to explore specific applications in 18F radiochemistry which can also be applied to 19F
organic/medicinal chemistry using the CEM discover instrument platform.
Fluorine -18 is classified as a synthetic isotope—meaning that it is not found in nature. Such
can be due to a lack of natural processes or mechanisms for its production, or in the case of fluorine,
due to the extremely short half-life.
18 F
has a half life of 109.77 minutes and decays predominately
via positron emission which is a type of beta decay. Alternatively it can decay via electron capture.
This occurs in only 3% of 18F decays and generally occurs in instances where there are too many
protons in the nucleus of an atom but insufficient energy to emit a positron. Both mechanisms
decay to oxygen-18 or “heavy water.” Based on these considerations, our objective was to develop
microwave accelerated methodology for nucleophilic displacement of nitroarenes using 19F and
R
R
NO 2
5
N
F
DMF, TBAF
15 m in, 23C
CN
X
80%
NO
2
6
N
CN
X
17
7
N
18
Scheme 3: fluorodenitration
DMF, TBAF
F
1h, 23C
NO 2
64%
CN
F
N
8
CN
Scheme 1
18F fluoride sources. Fluorodenitration of di-nitrobenzene and heterocyclic variants’ has already
been reported utilizing a range of conditions. The effective reactions are limited to highly activated
substrates and often, degradation to a phenol variant is seen. Kuduk (at Merck) reported
fluorodenitration of cyanopyridines 5 and 7 using reagent grade TBAF but observed phenol
formation when the TBAF was dried. 7 Dimagno (University of Nebraska-Lincoln) has undertaken
9
11
10
NC
NC
12
NC
Cl
Cl
Cl
Cl
Cl
Cl
N
16
15
14
13
N
Cl
N
ClCl
Scheme 2: fluorination substrates
N
Cl
considerable investigation into the subtleties of the fluorodenitration process and mechanism.
8Recently
he described a remedy using anhydrous in-situ prepared TBAF that promoted facile
denitration of a range of substrates including relatively unactivated arenes 9-16. 7 Armed with this
knowledge, our goal was to study the general process for 17-18 and a) determine the impact of
microwave acceleration on the process b) explore the scope of the process c) optimize to allow
reactions to be completed in 10 min or less – compatible with radioisotope installation and d)
demonstrate its utility in the synthesis of fluorinated medicinal agents. Using a standard 10 min
reaction time, three independent methods were assessed initially: KF, with various cryptands9 10to
enhance solubility / reactivity; reagent grade TBAF11; and anhydrous TBAF. Results obtained with
substrates 19-44 are highlighted in Scheme 4. Clearly the anhydrous TBAF is most effective
[conditions C] giving very high yields in many instances. In the case of substrate 22, conditions A
were also studied using 18F labeled fluoride, giving a 50%+ radiochemical yield of product.
Application in the synthesis of haloperidol12 13 [Scheme 5] and donepezil14 [Scheme 6] was then
investigated. For the synthesis of haloperidol this involved coupling the enolate of substrate 45
with a protected iodopropanol followed by deprotection to give 46. Conversion to the mesylate 47
allowed nucleophilic displacement with substituted piperidine 48 to give coupled product 49. After
considerable optimization, synthesis of 50 was effected using a 160oC 10 min procedure, which at
75% conversion gave an effective yield of 99%. Our collaborators at Lantheus Medical Imaging have
19
20
21
NO2
22
NO2
NO2
NO2
CN
A= <5 %
B= >95 %
A= <5 %
A= <5 %
25
26
NO2
N
NO2
28
O
A= 20% NO2
B= 50%
COMe
B= <5 %
NO2
CF3
33
NO2
CO2Me
B= <5 %
NO2
NO2
B= 93 %
OCH3
Br
35
N
NO2
C= >95 %
N
36
NO2
C= > 95%
NO2
39
F
C= >95 %
37
H
40
N
CN
C= >95%
38
41
Cl
CN
C= > 95 %
N
NO2
C= > 95 %
44
NO2
A= 57 %
B= <5 %
C= >95 %
NO2
NO2
Cl
CO2Me
C= > 95 %
A= <5 %
B= > 95%
C= > 95%
CN
Cl
NO2
F
43
B= 29 %
C= >95 %
NO2
NO2
F
B= <5 %
34
NO2
B= ~5 %
CHO
A= <5 %
B= >95 %
COOH
B= <5 %
NO2
O
32
B= <5 %
30
NO2
NO2
31
H
COOMe
A= 30%
B= <5 %
O
29
NO2
NO2
N
A= >95 %
B= 85 %
A= 65%
B= 5%
27
O
CHO
O
24
NO2
N
COOH
COOMe
23
F
42
NO2
C= > 95 %
Scheme 4: Fluorodenitration scope
N
CN
Legend (all MW, 10 min)
A = KF/K222/ DMF or DMSO
B = TBAF, THF or DMSO
C= TBAF(anhyd), THF or DMSO
46
45
O
O
OTBS
1. I
Zn/Cu, Pd(PPh3)4
OH
2. TBAF,THF (74%
O2N
49
O
O2N
48
Cl
OH
OH
Ms-Cl, Et3N
0° C, (84%)
Cl
O
OMs
N
N
O2N
160 ° C, 10 min MW
99 % (75% conversion)
anhydrous TBAF, DMSO
DMF, Et3N
(55%) O2N
Cl
O
F
47
OH
50
N
Haloperidol (Haldol )
Scheme 5: Total synthesis of Haloperidol through nitro route
verified the radiochemical variant of this step using cyclotron generated 18F, suggesting the
process is amenable to use in CNS imaging. Synhesis of donepezil required coupling of the lithium
enolate of nitroindanone 52 with piperizyl aldehyde 51 which underwent in situ elimination to give
conjugated enone 53 in moderate yield. Direct fluorodenitration required considerable
optimization but none the less proved effective with a 5 min. thermolysis at 130oC to give 54 in
moderate yield based on 40% conversion. These syntheses are significant in that they demonstrate
late stage radiolabeling on heavily functionalized substrates can be effected, and on a timescale
which is compatible with required purification and formulation studies employed in in vivo
imaging. Given that 18F PET imaging can be conducted through up to three half-lives of the nuclide
(~6h) we wished to explore the potential to effect microwave accelerated fluorodenitration
followed by additional synthetic transformations of the potentially ‘labeled product’, given that
O
NO2
H
MeO
MeO
N
51
O
52
F19
O2 N
LDA, THF
HMPA
(30%)
O
MeO
N
MeO
53
O
MeO
130C 5min MW
anhydrous TBAF/DMSO
65% (40% conversion)
N
MeO
54
Scheme 6: Microwave acceleratred denitration route to fluorinated donepezil analogues
reaction times are remarkably fast for many reactions. An attractive proposition in this regard
would be to prepare a building block / substrate that could be used as a labeling tag for biomarkers.
Accordingly using substrate 29 it proved possible to effect fluorodenitration, ester
hydrolysis, and conversion to the corresponding succinate in less than 30 min, allowing
bioconjugation to an available monoclonal antibody [Scheme 7]. This strategy opened the door to
use of labeled [18F] precursors in Mab and protein labeling, which is featured in depth in Chapter 5
[vide infra].
1. TBAF19(anhyd), THF
CO2Me
O2N
55
2. LiOH, THF
3. N,N di-succinimidyl
19F
carbonate
MW<30 min (3 steps), 68%
O
O
O
O
IgG
N
O
56
pH6
N
H
57
F19
Scheme 7: Microwave expedited route to labeled monoclonal antibodies
Having addressed goals a-d initially set out, we then became interested in extending the
general nucleophilic displacement strategy for the production of the nitroarene substrates
themselves, which would then remove the requirement that the nitro group be installed in the
synthetic intermediates until late stage, expanding the versatility of the procedure. 15 Preliminary
studies were successful, giving access to these key building blocks in an effective manner [Scheme 8
and Table 1]. Specifically, using tetrabutyl ammonium nitrite in the presence of copper powder,
various iodo arenes underwent nitrodehalogenation within a 30 min. timeframe. Comparison to
conventional methods is striking – the latter giving broadly similar yields but requiring thermolysis
times of 20+h. Though electron rich substrates are more effective, the scope and limitations of this
interesting and potentially powerful transformation is currently under study and suggests that
tandem nitrodehalogeantion-fluorodenitration can become a versatile approach to the installation
H
N
57
58
N
H
nBu4NNO2, DMF
R
X
R
NO2
Cu powder, MW 110oC
Scheme 8: Microwave accelerated nitrodeiodinations
of 18F labels for PET imaging.
Table 1. Microwave accelerated Cu catalyzed nitrodehalogenations
X
R
Time (min)
% Yield
Conventional (21-27h)
I
4-MeO
17
88%
81%
I
4-Me
20
50%
25%
I
3-MeO
20
84%
65%
I
H
16
85%
65%
I
3-Me
20
84%
69%
I
H
1
58%
65%
I
3-Me
1
49%
69%
I
4-CF3
15
10%
~5%
References:
1.
Ojima, I., Fluoirine in Medicinal Chemistry. Wiley-Blackwell, Inc.: 2009.
2.
Thayer, A. M., C & EN 2006, 84 (23), 15-24.
3.
Moon, B. S., Lee, T.S., Lee, K.C., An, G.I., Cheon, G. J., Lim, S.M., Choi, C.W.,
Chi, D.Y., Chun, K.S., Bioorg. Med. Chem. Lett 2007, 17 (200).
4.
Mukherjee J., H., E., Oichika, R., Easwaramoorthy, B., Collins, D., Chen, I,
Wang, C.S., Saigal, N., Trinidad, P., Kim, K, Nguyen, V.L., J. Label. Compd.
Radiopharm. 2007, 50, 375.
5.
Roberts B.A., S., C.R.,, Acc. Chem. Res. 2005, 2004 (38), 635-661.
6.
Kaape, C. O., Angew. Chem. Int. Ed. 2004, 43, 6250-6284.
7.
Kudek, S. D., DiPardo, R.M., Bock, M.G., Org. Lett. 2005, 7 (4), 577-579.
8.
Sun, H., Dimagno, S.G., J. Am. Chem. Soc. 2005, 127, 2050.
9.
Lemaire, C., Damhaut, P. Plenevaux, A., Caintineau, R., Christiaens, L.,
Guilluaume, M. , Appl. Radiat. Isot. 1992, 43, 485-494.
10.
Atiina, M., Cacace, F., Wolf, A.P., J. Chem. Soc. Chem.Commun. 1983, 108-109.
11.
Miller, P. W., Nicholas, J.L., Vilar, R., Gee, A.D., Angew. Chem. Int. Ed. 2008, 47,
8998-9033.
12.
Dai, Y., Guo, Y., Frey, R.R., Ji, Z., Curtin, M.L., Ahmed, A.A., Albert, D.H.,
Arnold, L., Arries, S.S., Barlozzari, T., Bauch, J.L., bousaki, J.J., Bousquet, P.F., Cunha,
G.A., Glaser, K.B., Guo, J., Li, J., Marcotte, P.A., Marsh, K.C., Moskey, M.D., Pease,
L.J, Stewart, K.D., Stoll, V.S, Tapang, P., Wishart, N., Davidsen, S.K., Michaelides,
M.R., J. Med. Chem 2005, 48 (19), 6066-6083.
13.
Baldwin, J. E., Adlington, R.M., Sham, V.W.W., Marquez, R., Bulger, P.G.,
Tetrahedron 2005, 61, 2353-2363.
14.
Abe, T., Haga, T., Negi, S., Morita, Y., Takayangi, K., Hamamura, K.,
Tetrahedron 2001, 57, 2701.
15.
Saito, S., Koizumi, Y., 2005. Tetrahedron 57, 2701.
CHAPTER 3:
ONE POT THREE-COMPONENT FLUOROALKYLATION,
FLUOROVINYLATION AND FLUOROALKYNYLATION STRATEGIES
Goals and Objectives
Existing methods for the installation of 18F nuclides on aliphatic side chains of
radiopharmaceuticals largely rely on historical methods involving leaving group displacement.
While this is an effective (though often cumbersome) strategy it suffers from the drawback that
progress in identifying new lead compounds through imaging methodology is typically restricted to
a sub-set of potential compounds, identified through sequential lead optimization. A
complimentary, and potentially more attractive option would be to use in vivo imaging as an
optimization platform and screen large libraries of closely related structures within a family of
pharmacophores. This observation is particularly relevant in CNS imaging, where even very subtle
structural differences can have substantial impact on transport properties including blood-brain
barrier penetration. Given the power of metal mediated coupling reactions and the substrate
X
Hal
FG
Pd. cat
F-
LG
X
F
MW
X
F
X
F
chemoselectivity observed in these processes, a goal was therefore to develop chemistries to
assemble libraries of fluoroalkyl, fluorovinyl and fluoralkynyl derivitized arenes from common
precursors. In designing the processes described herein we wished to achieve three component
microwave accelerated synthesis of the agents, demonstrate expeditious purification, and
compatability with late stage incorporation in synthesis. The envisioned products would be
assessed for lipophilicity, and, using 18F radiolabeled variants, in vivo transport properties.
Organic synthesis catalyzed by transition metal complexes is widely recognized as one of
the most efficient and effective means for carbon-carbon bond formation. In the design of new
transition metal catalysts, metal oxidation states and coordinate geometry of associated ligands has
a profound impact, and has allowed chemists to exploit specific parameters to fine tune bond
forming processes. Platinum and palladium have perhaps the greatest propensity among the
transition metals for forming bonds to carbon, 1 with the IV oxidation state readily accessible and
numerous compounds (many of great significance) known. Square planar complexes of palladium
II are moderately inert as compared to the corresponding nickel species. It is notable that d8 ions
have a pronounced tendency to form square complexes even though there are no Jahn-Teller forces
operating in an octahedral d8 complex—meaning that even though small tetragonal distortions of
the octahedron are not favored, the complete removal of two trans ligands to leave a square fourcoodinated complex is often favored. The most likely reason for this is increased sigma bond and pi
bond strength. The sigma bond stability is derived from the empty dx2-y2 orbital which contributes
to the stability of the four coordinate compound. In addition the dz2 orbital which is not needed for
binding ligands along the z axis can be hybridized with the s orbital to provide a greater
d x2-y 2
d xy
Energy
d z2
dyz, dxz
contribution to the in-plane sigma bonding while the pair of nonbonding electrons occupies the
hybrid orbital which is concentrated mainly above and below the molecular planes.
A higher degree of metal-ligand out of plane pi bonding can be established in square complexes
than in octahedral ones by means of the dxz, dyz and pz orbitals. There is also evidence for axial
interactions in a number of cases.
Though the characteristic shape of the predominant complexes
of palladium 2 is square, there are indications that weaker, additional
bonds may be formed in the
vacant octahedral sites. In
solution such positions may be
occupied by solvent molecules and in catalytic reactions of these complexes initial attack
presumably occurs in the axial position.
This mechanistic, theoretical and practical knowledge of palladium chemistry propelled a new
paradigm for carbon-carbon bond formations in the last 30 years, considerably enhancing the
ability of synthetic organic chemists to assemble complex molecular frameworks. The reader is
referred to a comprehensive review focused on recent applications of the most commonly applied
palladium catalyzed carbon-carbon bond forming reactions including the Heck, Stille, Suzuki,
Sonogashira, Tsuji-Trost and Negishi reactions.1 The power of these reactions are commonly
described as a procession by activation of stable and more readily available starting materials in
situ which are both more practical and often higher yielding.
R'
X
R''
R''
R'
Mizoroki-Heck Cross-coupling
R'
Sn(R)3
R''
X
R' R''
Stille cross-coupling
R'
B(Y)3
X
R'
Suzuki cross coupling
R'
R''
X
R'
R''
Sonogashira cross coupling
R'
X
R''
ZnX
R'
R''
Negishi cross coupling
R'
Si(R)3F NBu4
R''
X
Hiyama cross-coupling
R' R''
Scheme 1: Common palladium-catalyzed Cross Coupling Reactions
In the early 1980s J. K. Stille reported and pioneered the use of what would come to be known
as the Stille reaction 2 - a cross coupling of an organostanne and an organic electrophile to form a
new carbon-carbon sigma bond. 3 4 The low nucleophilicity of the organotin reagent and high
degree of stereoselectivity and
Scheme 2: Stille Coupling
R'
Sn(R)3
R''
X
R' R''
Stille cross-coupling
PdII
R'
R''
Pd0Ln
R''
R''
PdIILm R'
X Sn(R)3
R''
X
PdIILm X
R'
Sn(R)3
regiospecificity are the hallmarks of the reactions, which offered considerable advantages over the
conventional methods of the day. Analogous organosilicon compounds were believed to be too
stable for these reactions due to the less polarized Si-C bond’s inability to disassociate as necessary
during the catalytic cycle. However, in 1988 Hiyama reported use of fluoride to active the siliconcarbon bond and facilitate similar
couplings. 5This reaction bore advantages over its organostanne counterparts including a low
environmental impact, especially important given the concern of organotin conmpounds a potential
neurotoxins. The Hiyama reaction also has a high atom efficiency. Atom efficiency (sometimes
referred to as atom economy) describes the conversion efficiency of a process. In an ideal situation,
every atom of reactant translates to an atom of product. Side products resulting in cumbersome
workup conditions lead to lower yields and are reflected in a poor degree of atom efficiency.
Finally, while tetraorgano tin compounds are stable molecules with lower toxicity, they are
metabolized to triorganotin compounds which are highly toxic. The organosilanes used in the
Hiyama reaction offer safer handling techniques due to increased stability and much lower toxicity.
Scheme 3: Hiyama Cross Coupling
R'
Si(R)3F NBu4
R''
X
R' R''
Hiyama cross-coupling
PdII
R''
Pd0Ln
R' R''
R''
PdIILm R'
X Si(R)3F NBu4
R''
X
PdIILm X
R'
Si(R)3F NBu4
In the early 1970s, shortly before Stille published his catalytic palladium coupling, Richard F.
Heck published a series of papers describing a process in which aryl, benzyl and styryl halides
reacted with olefinic compounds at elevated temperatures in the presence of a hindered amine base
and catalytic palladium zero to form coupled products. 6This reaction would come to be one of the
most widely used catalytic carbon-carbon bond forming reaction in organic synthesis. In these
reactions, the reaction rate is strongly influenced by the degree of substitution of the olefin rather
than the type of substituent. Usually the more substituted olefin reacts at a slower rate due to the
steric hindrance caused by the bulk of the groups. The electronic impact of substituents on the
olefin has limited influence on reaction. Substrates having withdrawing and donating groups work
Scheme 4: Heck Cross Coupling Reaction
R'
X
R''
R''
R'
Mizoroki-Heck Cross-coupling
PdII
R'
R''
Pd0Ln
HPdLnX
R''
HX
R'
PdIILm X
R''
R''
R''
PdIILm X
X
well, with the electron poor substrates leading to slightly higher yields. Reflecting this tolerance
wide ranging functionalities on the olefin are compatible—esters, ethers, carboxylic acids, nitriles,
phenols, dienes, all performing well as substrates. However, in the case of allylic alcohols,
rearrangements are typically encountered. In the case of unsymmetrical olefins, substitution occurs
at the least substituted olefinic carbon providing opportunity to predict and achieve control of
stereoselectivity.
The nature of the X (halogen) group on the aryl or vinyl compound is also very important and
reactions rates change accordingly. (Table I>Br~OTf>>Cl) 6. The alkyl group is most often aryl,
heteroaryl, alkenyl, benzyl and in rare cases saturated alkanes, provided the alkyl groups possesses
no hydrogen atoms in the beta position. Both electron donating and electron withdrawing
functionalities are tolerated on this locus of the substrate.
Another, important feature of the Heck reaction lies in that the active palladium catalyst is
generated in situ from a precatalyst (palladium acetate or tetrakis triphenyl phosphine palladium),
rendering the reagents relatively inexpensive and commercially available. The Heck reaction is
typically conducted in the presence of a monodente or bidente phosphine ligand and a base. Unlike
typical palladium reactions, the Heck reaction is not water sensitive and solvents need not be
thoroughly deoxygenated.
Lastly, the Heck reaction is stereospecific as the migratory insertion of the palladium
complex into the olefin and the beta hydride both proceed with syn stereochemistry. Combined
this provides myriad opportunities for application in total synthesis of complex molecules.
There exist however a few minor drawbacks to the Heck reaction. Firstly, substrates cannot
tolerate hydrogen atoms on the beta carbon because the corresponding organopalladium
Scheme 5: Syn addition and Elimination in organometallic chemistry
Y
X
R2
R4
R1
R'
syn
R3
PdIILm X
R2
R1
Syn Addition
anti
R2
R4
X
R3
R4
R1
Y
PdIILm X
R'
H
R''
R3
R'
R''
R''
syn elimination
compounds will undergo beta hydride elimination to yield olefins. Additionally, aryl
chlorides in general are not good substrates for these reactions because of their slow reaction rate,
however, this can be exploited to effect by allowing chemoselective alkylation via a pendant
bromide or iodide of the substrate, leaving the chloride open for future downstream reactions.
The rate determining step of the Heck reaction is the oxidative addition of the palladium
zero into the carbon-halogen bond - the first step of the cycle. The palladium then forms a pi
complex with the alkene which then inserts into the palladium-carbon bond via a syn addition. The
molecule then rotates to the trans isomer which relieves the torsional strain. Beta-hydride
elimination forms the new palladium-alkene pi complex which then disassociates to yield the
product and a Pd(II) compound. The palladium catalyst is regenerated by reductive elimination
with base, typically potassium carbonate. While the base is consumed stoichiometrically, the
palladium is truly catalytic.
While utilizing the Heck, Stille and Hiyama reactions provides access to both alkenes and
saturated hydrocarbon products, the work of Sonogashira introduced a version of the palladium
catalytic cycle 7which couples a terminal alkyne with an aryl or vinyl halide. This reaction is similar
to a catalytic version of the Castro-Stephens couplings. The Sonogashira reaction is typically
conducted at or slightly above room temperature which constitutes a major advantage over forcing
conditions utilized in historical versions of the transformation. The handling of shock sensitive
Scheme 6: Castro-Stephens Couplings
R X
Cu
R
R'
R'
Pd, Pyridine
R= alkyl, aryl, vinyl
X=I, Br, Cl
copper acetylides utilized in earlier reactions is also avoided by the use of a catalytic quantity of a
copper salt (CuI or CuBr).
Scheme 7: Sonogashira Cross Coupling Reaction
Sonogashira cross coupling
R'' X
R'
R'
R''
PdII
R''
Pd0Ln
HPdLnX
R''
R'
X
HX
PPh3
R
R''
PdIILm X
PPh3
R
R
Ph3P Pd PPh3
R
CuI
R'
R'
Cu
H
Et3NH I
Cu I
R'
R''
Et3N
Like the Heck reaction, the solvents and the reagents do not need to be rigorously dried,
however in this reaction thorough deoxygenation of all substrates and reagents used is crucial to
avoid poisoning of the palladium catalyst. Often in these reactions the amine base serves as the
solvent, but occasionally a co-solvent such as DMF or diethyl ether is used to solubilize reagents.
Sonogashira reactions are typically amenable to both micro and macro scales. Because the coupling
is also stereospecific, any stereochemical information in the substrate is preserved in the product
making it particularly well suited for late stage assembly / coupling of complex molecules.
The order of reactivity of the organic halide is predictable, iodide being the best leaving group
and chlorides being much less reactive. While chlorides are not typically practical for use in the
reaction, differences in reactivity between bromide and iodide substrates allows for chemoselective
coupling of bromo-iodo substrates. It is important to note also that almost all functional groups are
tolerated with a few exceptions: conjugated electron withdrawing groups on substrates often result
in Michael addition products, and propargylic substrates with electron withdrawing groups
typically rearrange to form isomeric allenes . Other substrate limitations include aryl halides
(chlorides being particularly sluggish) and bulky substrates lacking reactivity - requiring higher
temperatures to be employed. Unfortunately at such high temperatures terminal alkynes often
undergo side reactions and isomerizations.
Mechanistically, the Sonogashira follows the expected oxidative-addition reductive-elimination
pathway followed by the other types of coupling reactions. However the structure of the
catalytically active species and the exact role of the copper catalyst remains unknown. This is
unique because unlike the other palladium couplings, in this reaction there are two catalytic cycles
occurring. The palladium cycle is straightforward - the palladium catalyst oxidatively adds to the
aryl halide / triflate. This then undergoes transmetallation with the copper acetylide produced in
the copper cycle. Both of the organic substitutents add in a trans to each other, then as cis-trans
isomerization brings these two substituents in proximity, where they then reductively eliminate to
yield the product regenerate the palladium (0) catalyst. The copper cycle is less well understood
and includes the potential for a side reaction (Glaser coupling). It is generally accepted however
that copper iodide forms a pi complex with the alkyne. The complex then reacts with a base to form
the copper acetylide utilized in the palladium cycle. While the cycle involves formation of the
copper acetylide and a tetra-substituted ammonium salt, it remains unclear how deprotonation of
the terminal alkyne occurs. As the amine bases employed in the reaction are incapable of
deprotonating terminal alkynes per se, it has been theorized that the pi complex may render this
proton more acidic, facilitating the reaction. In his original paper, Sonogashira suggested that the
cuprous iodide was necessary for the substitution reaction to take place, reporting lack of
substitution in its absence. Between 2002 and 2005 several ligand, amine and copper free methods
were reported. The copper free pathway pose the same puzzling question viz. deprotonation of the
terminal alkyne. It is generally believed that in these reactions the palladium complexes to the
alkyne in a similar fashion proposed for the copper cycle—however, some skepticism has been
voiced regarding this theory, over concerns on the potential role of trace impurities / contaminants
(particularly copper) in the palladium salts used in the reactions.
With the backdrop of a maturing field of transition metal catalyzed carbon-carbon bond
formation, we were motivated to a) examine the impact of microwave heating in specific reactions
of this class b) develop efficient multi component couplings that had not been attempted /
successful prior and c) harness the benefits of a-b in the preparation of radiolabeled variants of the
substrates for in vivo imaging. A significant motivating factor for the path chosen was in a paper
from Fu’s laboratory entitled “Room-temperature Hiyama Cross-Couplings of Arylsilanes with Alkyl
Bromides and Iodides”. Fu noted that the conditions outlined in the paper were not effective for the
coupling of several substrates including alkyl-tosylates with trimethoxysilylbenzene. We
hypothesized that an α-bromo-ω-tosyloxy alkane might couple with an alkyloxysilane, and using
fluoride to activate the substrate might also allow simultaneous in situ displacement of the tosylate
to deliver a coupled fluoroalkane product. As discussed prior, in addition to its potential (in 18F
form) as a molecular imaging agent, fluorine is a bioisostere of hydrogen and has many appealing
properties including the ability to hydrogen bond, and also the improved metabolic stability when
incorporate into metabolically labile positions, thus, such a coupling methodology, if proven would
be very appealing to a wide audience of synthetic, medicinal and clinical chemists. 5a, 5c, 6, 8
Using a modified version of the Fu reaction conditions, and using triethoxysilyl benzene
with 1-tosyloxy-6-bromohexane as substrates we first examined the effect of temperature on the
yield of the reaction to produce 6-fluorohexyl benzene (Table 1). Temperatures varying from 60 to
110⁰C were examined utilizing a catalytic system that employed an air and moisture stable
phosphonium salt and either palladium bromide, iodide or acetate. Utilization of the palladium
bromide led to the highest yields with a yield of 61% after 2 minutes in a microwave at 80⁰C.
Raising the temperature to 85 ⁰ C dropped the yield to 48% and likewise dropping it to 70⁰ C also
impaired the yield (Table 1). These results were perhaps expected given the steric compression of
the palladium acetate/iodide complexes versus palladium bromide since in these reactions
oxidative insertion is the rate limiting step, thus the steric environment of the palladium complex
and its ability to achieve proximity to the substrate has a pronounced impact on the success of the
reaction. 9
It is important to note that while the yields increased in line with catalyst loading, this also
reflects the control reactions not having achieved completion within the two-minute time frame
employed. This window was chosen to be compatible with anticipated radiolabeling experiments
using 18F labeled fluoride, taking into account need to also purify product prior to in vivo imaging.
Utilizing optimal thermal and time parameters, reagents and catalyst equivalents were then varied.
Though the original conditions utilizing palladium bromide and the phosphonium salt gave the
highest yield, along the way, several problems were encountered. The major issue stemmed from
the quality of the reagents themselves. Commercial ‘anhydrous’ TBAF packaged in a sure-seal bottle
under argon reportedly contains up to 5% water (presumably for stabilization and to quench any
hydrofluoric acid that could be formed). This leads to numerous side products including bis-arylether, phenols and aryl-alkyl-alcohols (Scheme 8).
Table 1: Tandem Hiyama alkylation and fluorination reaction optimization+
Entry
Catalyst System
Temperature ⁰C
Yield#
1
PdI2/[HP(t-Bu)2Me][BF4]
60
24
2
PdI2/[HP(t-Bu)2Me][BF4]
70
25
3
PdI2/[HP(t-Bu)2Me][BF4]
80
25
4
PdI2/[HP(t-Bu)2Me][BF4]
85
27
5
PdI2/[HP(t-Bu)2Me][BF4]
90
15
6
PdI2/[HP(t-Bu)2Me][BF4]
100
12
7
PdI2/[HP(t-Bu)2Me][BF4]
110
9
8
PdOAc2/[HP(t-Bu)2Me][BF4]
60
26
9
PdOAc2/[HP(t-Bu)2Me][BF4]
70
25
10
PdOAc2/[HP(t-Bu)2Me][BF4]
80
27
11
PdOAc2/[HP(t-Bu)2Me][BF4]
85
24
12
PdOAc2/[HP(t-Bu)2Me][BF4]
90
21
13
PdOAc2/[HP(t-Bu)2Me][BF4]
100
13
14
PdOAc2/[HP(t-Bu)2Me][BF4]
110
9
15
PdBr2/[HP(t-Bu)2Me][BF4]
60
51
16
PdBr2/[HP(t-Bu)2Me][BF4]
70
55
17
PdBr2/[HP(t-Bu)2Me][BF4]
80
61
18
PdBr2/[HP(t-Bu)2Me][BF4]
85
48
19
PdBr2/[HP(t-Bu)2Me][BF4]
90
45
20
PdBr2/[HP(t-Bu)2Me][BF4]
100
42
21
PdBr2/[HP(t-Bu)2Me][BF4]
110
14
Substrates+: Triethoxysilyl benzene with 1-tosyloxy-6-bromohexane; 2 min. reaction time.
Product#: 6-fluorohexyl benzene
O
R
R
Scheme 8: Byproducts from the
fluoroalkylation process
R
OH
OH
n
Though these initial results were indeed encouraging, the requirement to utilize fluoride to
activate the Hiyama coupling reaction was ultimately deemed problematic, as when 18F labeled
versions of the process would be attempted, the opportunity for ‘leakage’ exists, which would
complicate cleanup procedures. For these reasons, we began to investigate other cross-coupling
methods.
The next cross-coupling reaction we looked to investigate was the Heck reaction. Using the
tosyloxy-5-hex-en-1-ol as substrate, preliminary reactions examined the reactivity of electron rich
and electron deficient aryl iodides, and an iodo-pyridine (Table 2). 1,4-Dioxane with DIPEA proved
the best solvent/base combination, with two combinations working modestly well (~35 percent)
with iodoanisole.
Table 2: Optimization of Heck reaction conditions—Solvent, base and catalyst+
Aryl Iodo
Solvent
Base
Catalyst
Yield (%)
4-Iodo Acetophenone
DMF
Et3N
Pd(P(Ph)3)4
0
4-Iodo Acetophenone
DMF
Et3N
Pd(OAc)2
0
4-Iodo Acetophenone
THF
Et3N
Pd(OAc)2
0
4-Iodo Acetophenone
ACN
Et3N
Pd(OAc)2I/P(o-tolyl)3
0
4-Iodo Anisole
ACN
DIPEA
Pd(OAc)2I/P(o-tolyl)3
3
4-Iodo Anisole
ACN
Et3N
Pd(P(Ph)3)2Cl2
3
4-Iodo Anisole
1,4-Dioxane
DIPEA
Pd(OAc)2I/P(o-tolyl)3
40
4-Iodo Anisole
1,4-Dioxane
DIPEA
Pd(OAc)2
0
4-Iodo Anisole
1,4-Dioxane
DIPEA
Pd(P(Ph)3)4
0
4-Iodo Anisole
1,4-Dioxane
DIPEA
Pd(P(Ph)3)2Cl2
32
3-IodoPyridine
ACN
Et3N
Pd(OAc)2I/P(o-tolyl)3
0
3-IodoPyridine
ACN
Et3N
Pd(P(Ph)3)2Cl2
0
+ tosyloxy-5-hex-en-1-ol is substrate in all tandem Heck coupling reactions unless noted
T (0C)
t (min)
Yield
100
60
45
100
40
46
100
20
48
100
10
49
100
5
54
100
3
50
80
5
4
90
5
56
110
5
52
120
5
51
OTs
Table 3: Temp and Time (4-iodoanisole)
R = COCH3, X= CH2
R= OCH3, X= CH2
R=H, X= N
F
I
R
R
X
0.10 M aryl-iodide solution,
2.00 eq alkene
2.00 eq TBAF, 1.6 eq base
X
The combination of palladium acetate and tri(o-tolyl) phosphine was selected as the most
effective catalyst (over bistriphenylphosphine-palladium chloride).
We then optimized the microwave conditions for the reaction using 4-iodoanisole as our
substrate and the conditions determined in Table 2. Our goal was to develop a method that
would produce modest yields within ten minutes (under conventional conditions many Heck
reactions take >2 hours to go to completion, with some intramolecular variants taking up to16
hours at elevated temperature). Varying temperature and reaction time (Table 3) suggests an
optimal conversion at 90oC for 5 min, extended thermolysis resulting in reduced yields due to
product decomposition.
With a reliable set of conditions in hand, we were now better prepared to examine the effect
of the electronics of the substrate on the reaction. 4-iodo-acetephenone, 3-iodopyridine and 4iodoaniline were subjected to 90/100⁰C for varying amounts of time (Table 4). Gratifyingly
both the electron withdrawing and donating substrates achieved moderate success (22-42%)
and even the 3-iodopyridine yielded 5% product. Unfortunately the reaction times were not
within our originally outlined goals. Effective reaction times for the deactivated substrate
required twenty minutes to see any product at all, and a full hour to achieve a 20% yield. Oddly,
the optimal reaction time for pyridine was 30 minutes (5%) as opposed to 60 minutes (3%).
The activated aniline achieved the best yield (43%) within 15 minutes. While these times are
clearly not within our ten minute goal, they are still acceptable for labeling and a great
improvement over the historically reported reaction times for unactivated Heck reactions.
I
F
OCH3
H3CO
OTs
0.10 mM in 1,4-Dioxane, 2.00 eq alkene,
2.00 eq TBAF, 1.6 eq (i-pr)2EtN
0.05 eq Pd(OAc)2 0.10 eq P(o-tol)3
Table 4: Scope of Heck Reaction
Aryl Iodide
4-Iodo
Acetopheneone
3-Iodopyridine
T (0C)
t (min)
Yield (%)
90
5
0
100
5
0
100
10
0
100
20
5
100
40
22
100
60
20
90
5
0
100
5
0
100
10
0
100
25
0
100
30
5
100
60
3
4-Iodo Aniline
90
5
0
100
5
0
100
10
32
100
15
43
100
20
42
The scope was further examined by looking at different substitution patterns on the aryl ring.
2,3 and 4 iodo-substituted anisole, aniline, and acetephenone as well as iodobenzene and 2, 3
and 4 iodo-pyridine were investigated (Table 5). Success was achieved on all substrates except
3-iodoacetephone and 2 and 4 iodo-pyridine. The reaction predictably yielded predominantly E
alkenes (8:1 ratio), with reaction times ranging from 5 to 40 minutes. In all cases starting
material was also recovered.
OTs
F
I
R
R
X
A
R = COCH3, X= CH2
R= OCH3, X= CH2
R=H, X= N
0.10 mM in 1,4-Dioxane, 2.00 eq alkene,
2.00 eq TBAF, 1.6 eq (i-pr)2EtN
0.05 eq Pd(OAc)2 0.10 eq P(o-tol)3
X
B
X
Table 5: Further investigation of Heck substrate scope
Conditions
Product (%)
E/Z
Aryl Iodo
t (min)
T (0C)
A
B
Ratio
Recovered
Aryl Iodo
(%)
2-Iodo Anisole
5
90
52
4
8:01
30
3-Iodo Anisole
5
90
48
5
8:01
28
4-Iodo Anisole
5
90
56
5
8:01
29
2-Iodo Aniline
15
100
47
13
8:01
29
3-Iodo Aniline
15
100
45
11
8:01
31
4-Iodo Aniline
15
100
43
9
8:01
32
IodoBenzene
2-Iodo
Acetophenone
15
100
35
9
8:01
28
40
100
24
12
8:01
56
3-Iodo
Acetophenone
40
100
0
10
NA
68
4-Iodo
Acetophenone
40
100
22
9
8:01
59
2-Iodo Pyridine
30
100
0
0
NA
91
3-Iodo Pyridine
30
100
5
0
7:02
84
4-Iodo Pyridine
30
100
0
0
NA
95
Next, we elected to investigate the effects of sterics, strain and bulk on the reaction by determining
if use of bulkier bases would cut down on side reactions. The reactions were run with iodo-anisole
under the standard conditions (tosyloxy-5-hex-en-1-ol). Interestingly, though negligible impact on
product yield was observed, use of DIPEA and DBU gave much cleaner reactions, facilitating clean
up (Table 6). DABCO is known to demethylate quaternary ammonium salts when heated, so it is not
a surprise that when subjected to the heat of the microwave that side reactions would occur. When
the halogen on the aryl-halide was varied, the expected resulting activity was seen (Table 7). Iodo
was clearly the highest yielding substrate, but bromide can be used without sacrificing much in the
way of yield or recovery. No product was recovered from the chloro substrate.
Table 6 Effect of base
Product
E/Z
Recovered
Base
A
B
Ratio
Aryl Iodo
DIPEA
56
4
8:01
29
DABCO
41
3
8:01
32
DBU
46
0
8:01
40
Table 7 Effect of halogen
Lastly, the length of the alkyl chain was varied. 4 and 3 carbon chains lengths were expected to be
incompatible due to the production of cyclopropane and ethylene respectively. This was ultimately
confirmed. However, both 5 and 6 carbon chains worked moderately well (Table 8).
Table 8: Effect of chain length
Tosylate
Carbon
Aryl Iodo
Length
Conditions
t (min)
Product
T (0C)
Conditions
A
E/Z
B
Product
Ratio
E/Z
Recovered
Aryl Iodo
t (min)
T (0C)
A
B
Ratio
Aryl Iodo
4-Iodo Anisole
5
90
56
4
8:01
29
4-Bromo Anisole
5
90
43
6
8:01
35
4-Chloro Anisole
5
90
0
0
NA
93
4-Iodo Anisole
6
5
90
56
4
8:01
4-Iodo Anisole
5
5
90
46
6
8:01
4-Iodo Anisole
4
5
90
0
0
NA
Due to the success we experienced with the tandem tosyl displacement, Heck cross couplings, we
decided to extend our methodology to sp hybridized systems and investigated the Sonogashira
cross-coupling reaction.
The initial Sonogashira reaction conditions employed, like those for the Heck reaction, came from
another project ongoing within the laboratory. The solvent system called for a 9:1 solution of THF
and Et3N and the catalysts used were bistriphenylphosphine palladium chloride and copper iodide.
Like in the Heck optimization the temperature and time were varied to determine the ideal
conditions for this reaction (ten minutes at 70⁰C). These optimization reactions performed using 4iodoacetephenone (Table 9). This portion of the optimization also included varying the equivalents
of TBAF and the catalyst as well as using exclusively TEA as the solvent. The optimal conditions in
this case called for two equivalents of TBAF (90% yield) although using a single equivalent still gave
an acceptably high yield (76%).
I
R
F
R
OTos
Table 9 :Optimization of solvent, catalyst and fluoride equivalents
Solvent
TBAF
equivalents
Catalyst
Yield (%)
THF/Et3NI
1
Pd(P(Ph)3)2Cl2
76
THF/Et3NI
2
Pd(P(Ph)3)2Cl2
91
THF/Et3NI
3
Pd(P(Ph)3)2Cl2
90
THF/Et3NI
2
Pd(P(Ph)3)4
81
Et3N
2
Pd(P(Ph)3)2Cl2
76
Table 10 (left): Substrate scope of reaction Table 11 (right): Chain length effect
Aryl Iodo
2-Iodo
Acetophenone
3-Iodo
Acetophenone
4-Iodo
Acetophenone
Yield (%)
Iodobenzene
86
2-Iodo Pyridine
90
3-Iodo Pyridine
91
4-Iodo Pyridine
89
IodoBenzene
86
2-Iodo Anisole
79
3-Iodo Anisole
4-Iodo Anisole
93
88
91
Alkyne
Carbon
Aryl Iodo
Length
Yield (%)
2-Iodo Acetophenone
6
93
82
2-Iodo Acetophenone
5
91
73
2-Iodo Acetophenone
4
59
2-Iodo Acetophenone
3
0
Next the scope of the substrate was
explored using 2, 3 and 4 subsituted iodo acetephenone, and anisole as well as iodobenzene and
2, 3 and 4 iodo-pyridine (Table 10). Gratifyingly all of these reactions gave yields of between
73% for the para-iodoanisole and 93% for 2-iodoacetephenone. It is interesting to note that
while the Heck reaction responded better to electron donating groups, the Sonogashira reaction
saw higher yields from the substrates with
Table 12: Effect of halogen
Aryl Halide
Yield (%)I
4-Iodo Anisole
73
4-Bromo Anisole
72
4-Chloro Anisole
12II
electron withdrawing functional groups. The effect of the Halogen was also examined (Table 12).
Both bromo and iodo gave nearly identical yields. The chloro-substrate substrate gave a 12 percent
yield with 74% starting material recovered. While seemingly poor in terms of product yield, the
chloro-substrate shows promise as a selective labeling agent. In situations of PET labeling with
fluorine, we are not concerned with completion of the reaction so much as we are with purity of the
reaction—being able to perform this cross coupling on a aryl-chloride and recover the starting
material means that these reactions can be done with the extraordinarily high degree of purity
required of radiolabeled compounds for imaging.
Lastly, the alkyl chain length was examined (Table 11). Success was achieved with both 5
and 6 carbon lengths. Moderate success was seen with a 4 carbon chain. Due to the conjugated
nature of the substrate however, 3 carbon chains were not expected to and did not yield any
product.
Applications of work:
The obvious extension of this project, which is currently ongoing, is to utilize this methodology to
label a molecule of interest as the last step in the total synthesis. In a collaboration with Bristol
Myers Squibb Molecular Imaging group, the Sonogashira conditions were used in the synthesis of
analogues of the BMS agent RP1005, a mitochondrial complex 1 inhibitor which is in clinical trials
as a cardiovascular imaging agent in 18F form. Given that liophilicity of the alkyl chain has an
observed impact on biodistribution of these agents, preparation of sp hybridized analogs 2 and 3
from common precursor 1 gives access to potentially useful products. The 19F versions of products
2 and 3 formed in good yield using the conditions outlined herein, and we are currently discussing
methodology for production of 18F variants for imaging. A second set of targets investigated were
analogs of nifrolidine, a selective nicotinic receptor binding agent. Which is used in 18F PET
imaging of Azheimers disease. Under the remit of a project sponsored by the ADDF, bromopyridine
intermediate 7 was formed in two steps from prolinol, then subjected to Sonogashira coupling to
give C6 analog 8 in moderate yield. In a separate study intermediate 7 was also transformed to
alkyl analog 9 using a modified Negishi coupling. Labeling of both agents is currently under
discussion with the ADDF corporate partners network.
O
O
F
O
S
O
RP1005
1
2
Br
O
O
O
S
S
F
O
3
S
F
5
6
7
8
N
H
9
F
N
Nifrolidine
In summary, the successful development of a one-pot three component coupling strategy has been
effected, allowing routine production of 19F fluoroalkyl, fluorovinyl, and fluoroalkynylated arenes
from common precursors. Application in the synthesis of medicinal agents has been demonstrated,
and the conversion to 18F variants, for use in PET imaging, is under development with
collaborators.
References
1.
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(a) Sonogashira, K.; Tohda, T.; Hagihara, N., A convenient synthesis of
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CHAPTER 4. MICROWAVE ASSISTED SYNTHESIS OF XANTHINE ANTAGONISTS OF THE
ADENOSINE A2A RECEPTOR
Goals and Objectives
The xanthine family of heterocycles enjoy a rich history in modulation of biochemical pathwways,
and heavily substituted variants have become drug candidates for a variety of indications. Based on
an active collaboration with the New England Tissue Protection Institute, we have initiated a
project engaged in the design, synthesis, and optimization of a family of aryl functionalized
O
R
N
N
O
F
N
N
O
NH2
N
O
R
N
OHC
X
NH2
F-
xanthines. The goals of the project described herein were to :
1 - develop a new and versatile synthetic route to xanthine analogs
2 – exemplify the route with a new synthesis of the Parkinsons drug KW 6002
3 – investigate the benefits of microwave acceleration and automation in the process
4 – optimize the synthetic procedures to result in improved product impurity profiles
5 – demonstrate means to diverse substitution / functionalization patterns to optimize properties
[including ligand binding and biodistribution]
6 – demonstrate a means to facilitate 18F labeling of the drugs to allow for PET imaging
The negative physical effects of too much caffeine are widely appreciated—
increased energy, flushing of the face, irritability, increased urination, and rapid
heartbeat. In very large doses, caffeine can lead to mania, depression, disorientation,
hallucinations and psychosis. It can also lead to caffeine-induced sleep disorders and
other mental disorders. Extreme overdose can lead to death by ventricular fibrillation.
However, in moderation also has many appealing health benefits. It can potentially
lower risk of diabetes, Parkinson’s disease and colon cancer. It has been proven to
increase the effectiveness of analgesics for headaches, prevent cavities, and help to
manage asthma. For this reason, the study of caffeine and its anlogs [both natural and
synthetic] has become of major interest. Caffeine belongs to the xanthine class of
heterocycles (purine based) which are found in most tissues in the body. Their
physiologic effects stem from their interaction with the adenosine receptors. In humans
there are four types of adenosine receptors; A1, A2A, A2B and A3. The A2B receptor is
involved in bronchospasm and bronchoconstriction and the A3 receptor is
cardioprotective during cardiac ischemia. However, interactions with the A2A and A1
receptors are of potentially greater significance, and form the basis of an active
collaboration between our research group and that of Professor Misha Sitkovsky,
director of the New England Inflammation and Tissue Protection Institute at
Northeastern. The Sitkovsky group have made major inroads to the A2A receptor and
its signaling pathways, and we initiated a collaboration to produce synthetic xanthines of
interest and correlate their biological function with structural motifs.
Due to pathway interactions between A2A and D2 receptors, antagonists of the
adenosine receptor have become major targets in CNS drug discovery.1 Lead
compounds based on the xanthine skeleton, including the chlorostyryl caffeine CSC,2
the thienylated xanthine DMPTX, 3 and the dimethoxystyryl xanthine KW-6002 have
been investigated. 4 Based on promising results obtained with co-administration of
levodopa, KW-6002 (Istradefylline®), is a clinical candidate for Parkinson’s disease. 5
Several annulation strategies have been explored in the construction of the xanthine
backbone, most commonly via disconnection to the corresponding aminoacylaminouracil,6 or closure of the 6-amino-5-iminouracils, typically utilizing oxidative
methods. 7
Given the importance of the xanthine class we became interested in developing a
one-pot method through direct coupling of carboxaldehydes with readily available 5,6diaminouracils 1 under mild conditions. 8
Scheme 1. BDMS accelerated route to xanthines
Under stoichiometric conditions, simple aldehydes coupled with 1 to produce high yields
of xanthines 5 presumably via the imine intermediate 3 and / or its aminal form 4
(Scheme 1). Attempts to isolate presumed intermediates 2 and 4 were unsuccessful but
at 10 mole % BDMS substantial buildup of imine 3 occurs. Nonetheless, using 0.6
equivalents of BDMS very high yields of the product xanthines 5 are attainable,
suggesting the BDMS is regenerated during the cycle. Under closer scrutiny, DMSO,
Me2S, HBr and H2O can all be detected during the reaction. DMSO, formed either
directly through oxidative elimination from 2 with BDMS or via attack of Me2S by in situ
generated HOBr, can be reconverted to BDMS by reaction with HBr/Br2/H2O. Me2S and
HBr could in turn be released by BDMS in facilitating dehydrogenation of aminal 4,
either through collapse of an N-bromo intermediate, or via bromide induced
dehydrogenation, with the aminal C-H departing as hydride to capture the bromonium
ion of the BDMS. Clearly, the intricacies of the process warrant further investigation and
thorough kinetic analysis, given the high efficiency of the method. The process proved
amenable to a broad range of substrates (Chart 1), and requires only trivial purification
of products 5.
The utility of the bromo-substituted products for subsequent transformations was
readily demonstrated through Pd mediated coupling to give analogs 7 and 9 (Scheme
2). Given the ease of coupling we were motivated to investigate the potential for in situ
tandem ring closure and bromination, and after some effort determined that utilization of
1.6 equivalents of the reagent (0.5 for 4h then and additional 1.1 eq for 10 hours)
effected conversion of p-methoxy benzaldehdye and salicylaldehyde into 10 and 11
respectively. The utility of the phenolic functionality was further demonstrated through
Scheme 2. Extending libraries of derivatives through Pd couplings
conversion to 12 and 13, and the bromo group through Suzuki coupling of 12 to produce
14 (Scheme 2). Microwave accelerated conditions to expedite the ring closing process
were also investigated, 4-bromobenzaldehyde reacting (CEM Discover, 150W, 110oC,
100 psi, CH3CN) to produce the corresponding xanthine in 72% yield within 30 min.
This result bodes well for the application of the method for the rapid production of
libraries of xanthine derivatives under automated conditions.10
In order to demonstrate the effectiveness of our approach, we conducted a comparative
synthesis of KW-6002 (18, Scheme 3) from 1. As can easily be seen the direct route to
key intermediate 17 is superior, and opens the prospect of targeted library design for
the class.
Scheme 3. Synthesis of KW 6002
Given the importance of the KW-6002 agents in modulating CNS related pathways,
methods to allow in vivo imaging will likely become of significance. A route to a 11C
labeled version has been reported, 11a and given its time-to-peak plasma concentration
of 2-5h, 11b a suitably functionalized 18F labeled derivative would presumably offer
O
H
N
N
O
NO2
N
N
19
O
1. MeI, K2CO3
DMF 99%
2. TBAF, DMSO
MW (300W, 180oC
11% (74%) on 600s
N
N
O
19F
N
N
20
Scheme 4. Fluorinated analogs via MW fluorodenitration
considerable utility in extended PET imaging studies of this class of agent.
11c
To
demonstrate proof-of-principle, nitrosubstituted xanthine 19 was N-methylated then
subjected to microwave accelerated fluorodenitration using anhydrous TBAF giving
appreciable quantities of 20 in <10 min (Scheme 4).12
Given the half life of 18F (120 min.), we expect cyclotron derived 18F variants of this
process to be useful in preparation of agents for direct PET imaging of KW-6002.13
In addition to use in CNS disorders, KW-6002 has shown promise as an antitumoral
agent, but suffers from poor water solubility in vivo. 14 Accordingly we are currently
engaged in the design and synthesis of hydrophilic variant in the form of PEGylated
conjugates. Additionally, by attaching a medium molecular weight polymer to the
template their ability to cross the blood-brain barrier will also be inhibited, resulting in
enhanced circulation and thus capacity to function as systemic/circulating antitumorals.
In summary, a direct route to the xanthine class of adenosine receptor antagonists
has been developed. The process is efficient, scalable, and can be applied to versatile
and diverse library construction. In the case of aryl aldehyde substrates tandem in situ
bromination of the products is an effective adaptation of the process, and promises to
extend the versatility in the form of heavily substituted xanthine derivatives. Both the
ring closure process and fluorodenitration of a substituted analog was demonstrated
using microwave heating. In the latter case, potential use of xanthine derived A2A
antagonists for in vivo imaging and as antitumorals may be facilitated by synthesis of
derivatives bearing 18F radiolabels using this methodology, and is currently being
explored.
References
1. Cacciari, B. ; Pastorin, G. ; Spalluto, G. Current Topics in Medicinal Chemistry, 2003,
3, 403.
2. Jacobson, K. A. ; Gallo-Rodriguez, C. ; Melman, N. ; Fischer, B. ; Maillard, M. ; van
Bergen, A. ; van Galen, P. J. M. ; Karton, Y. J. J. Med. Chem. 1993, 36, 1333.
3. Del Giudice, M. R. ; Borioni, A. ; Mustazza, C. ; Gatta, F. ; Dionisotti, S. ; Zocchi, C. ;
Ongini, E. Eur. J. Med. Chem. 1996, 31, 59.
4. Knutsen, L. J. ; Weiss, S. M. Curr. Opin. Invest. Drugs 2001, 2, 668.
5. Bara-Jimenez, W. ; Sherzai, A. ; Dimitrova, T. ; Favit, A. ; Bibbiani, F. ; Gillespie, M. ;
Morris, M. J. ; Mouradian, M. M. ; Chase, T. N. Neurology 2003, 61, 293; Koga, K. ;
Kurokawa, M. ; Ochi, M. ; Nakamura, J. ; Kuwana, Y. Eur. J. Pharmacol. 2000, 408,
249.
6. Perumattam, J. Synth. Commun. 1989, 19, 3367; Burbiel, J. C. ; Hockemeyer, J. ;
Müller, C. E. Beilstein J. Org. Chem. 2006, 2, 20.
7. de Araujo, A. D. ; Bacher, E. ; Joachim Demnitz, F. W. ; Santos, D. A. Heterocycles,
1999, 51, 29.
8. Jerchel, D. ; Kracht, M. ; Krucker, K. Liebigs Ann. Chem. 1954, 590, 232.
9. Das, B. ; Holla, H. ; Srinivas, Y. Tetrahedron Lett. 2007, 48, 61; Choudhury, L. H.
Synlett, 2006, 1619; Khan, A. T. ; Ashif, M. ; Goswami, P. ; Choudhury, L. H. J. Org.
Chem. 2006, 71, 8961; Das, B. ; Ramu, R. ; Ravikanth, B. ; Reddy, K. R. Synthesis,
2006, 1419.
10. Ma, D. ; Sitkovsky, M. ; Kallmerten, A.E. ; Jones, G.B. Tetrahedron Lett. 2008, 49,
4633.
11a. Brooks, D.J., Dooper, M., Osman, S., Luthra, S.K., Hirani, E., Hume, S., Kase, H.,
Kilborn, J., Martindill, S., Mori, A. Synapse. 2008 62 671; b. Lambertucci, C., Cristalli,
G., Dal Ben, D., Kachare, D.D., Bolcato, C., Klotz, K-N., Spalluto, G., Volpini, R.
Purinergic Signal. 2007, 3, 339; c. Mukherjee, J. ; Head, E. ; Pichika, R. ;
Easwaramoorthy, B. ; Collins, D. ; Chen, I. ; Wang, C. S. ; Saigal, N. ; Trinidad, P. ; Kim,
K. ; Nguyen, V. L. J. Label. Compd. Radiopharm. 2007, 50, 375.
12. Sun, H. ; DiMagno, S. G. J. Am. Chem. Soc. 2005, 127, 2050.
13. Miller, P.W. ; Nicholas , J. L. ; Vilar, R. ; Gee, A. D. Angew. Chem. Int. Ed. 2008, 47,
8998 .
14. Sauer, R. ; Maurinsh, J.; Reith, U.; Fülle, F. ; Klotz, K-N. ; Müller. C. E. J. Med.
Chem. 2000, 43, 440.
CHAPTER 5: BIOCONJUGATION OF FLUORINATED TAGS FOR 18F PET IMAGING
Goals and Objectives
An emerging trend in drug design is a shift from traditional ‘small molecule’ approaches in favor of
complex biopharmaceuticals. These agents often derived from cell based products include proteins,
glycoprotein’s and monoclonal antibodies. Additionally, many of these biopharmaceutical agents
have a potential use as biomarkers, capable of providing prognostic and diagnostic indicators of
diseased states. Accordingly, means to monitor and track biodistribution of the agents would be
extremely valuable, as many of the agents involve mechanisms of action at localized sites. As the
growth of these classes of drugs / agents expands, the traditional means of radiolabeling to assess
biodistribution will likely need to be refined, given the molecular complexity of the agents.
Additionally, in the case of antibody therapies, localization can take several hours, requiring careful
selection of radioimaging methodology / agent. Given the intrinsic power of PET imaging when
used in concert with the 18F nuclide, methods to achieve rapid 18F labeling of biopharmaceuticals
would be of significance. The goal of this project was therefore to investigate use of a
chemoselective protein/antibody labeling agent, specifically a fluoroaryl succinate derivative. Our
specific priorities were to:
1- identify and select an efficient protein / Mab tagging agent incorporating a fluoroarene group
2 - develop an expeditious synthetic route and purification protocol to a 19F version using
microwave chemistry
3 - demonstrate proof of principle for the agent by labeling selected protein / Mab targets
4 - apply the methodology to the production of an 18F variant, to allow subsequent PET imaging
O
O
O
18F
N
O
Given that a formal diagnosis of Alzheimer’s disease and other types dementia typically
occurs when substantive physiologic and morphologic changes in the brain present in behavioral
changes, there is considerable interest in developing methodology for the early detection and
therefore early intervention of such diseases. Such methods, in addition to having obvious
diagnostic benefits, could also be used to gauge the effectiveness of drugs as they become available
and to monitor progress and use of such drugs. PET imaging offers considerable potential given its
high resolution and the availability of designed image contrast agents which could highlight
changes in levels of key molecular targets associated with various types of dementia.
Alzheimer’s disease targeted PET imaging agents can be divided into specific categories.
Associated with the
progression of AD,
the most widely
studied are those
agents which target
beta-amyloid
plaques. These
plaques are
peptides of between
39 and 43 amino
acids. The plaques
are composed of a tangle of amyloid fibers which are normally ordered fiberrular aggregates.
Amyloid precursor protein (APP) is an integral membrane protein expressed in many tissues but
concentrated in the synapses of neurons. The
synapses are the structures that permit neurons to
pass electrical or chemical signals to other cells and
thus carry out their primary function of transmitting
information via signaling. The native biological role
of APP is unknown but its most substantiated role is
in synaptic formation and repair as its expression is
unregulated during neuronal differentiation and
after neuronal injury. Mutations to critical regions of
APP are known to cause familial susceptibility to AD. Beta-amyloids are formed by cleavage with
first a beta-secretase followed by the gamma-secretase. The beta-secretase is an aspartic acid
protease that cleaves APP extracellularly. Gamma-secretase then produces the C-terminus by
cleavage within the transmembrane region of APP. This can generate a number of isoforms from 39
to 43 residues in length. The most common
are Aβ42 and Aβ40. The Aβ40 is more
common, with the longer residue being the
most fibrilous. Therefore certain AD
therapies have considered influcing the
production of exclusively Aβ40. Mutations at
APP lead to an increased relative production
of Aβ42. The other main therapy types in this area include secretase inhibitors, immunotherapies
and anti-aggregate agents.
While the small molecular approaches are amenable to radio-labeling for diagnosis and
treatment monitoring through labeling methods previously discussed, immunotherapy via antibody
stimulation presents a different type puzzle for development of labeling methodology. Instead of
dealing with molecules of around 300 daltons, the targets range between 150 and 900 kilodaltons.
No longer are we targeting small rigid molecules with defined labeling positions, but rather large
chains with complex and highly important secondary, tertiary and quaternary structure.
One of the earliest examples of an agent targeting the beta-amyloid plaques associated with
AD is the pet tracer known as the 11C-Pittsburg Compound-B (PIB). An example of the type of
work done by groups publishing on the topic
HO
S
N
11
CH3 include investigating a hypothesis that PET
NH
imaging with 11C-PIB could differentiate between
AD and frontal-temporal lobe degeneration ( a non Aβ type of dementia). The group achieved mild
success despite setbacks by false positives. This focus laid the groundwork for this type of imaging
diagnosis. Despite its short half-life (consequence of Carbon-11 labeling) PIB has been widely used
for mapping studies and recently a 18F analogue has become available. However, several papers
published show PIB to not be as effective of a tool as originally thought. PIB is a non-specific
imaging marker. A paper published in 2007 by Lockhart, et al highlighted several problems with the
use of PIB for diagnosing alzheimers. First, high retention of PIB in areas of the brain not normally
associated with a high amount of CP cannot be readily explained by the claimed specificity of PIB.
Secondly, the concept of PIB positive of PIB negative, detracts from the focus on CP as having a
central role in disease progression. Imaging PIB basically gives a yes or no for the presense of such
proteins but does nothing to indicate the severity. And Lastly, they point out that a “PIB-positive”
retention pattern has been identified in 20-40% of normal cognitively normal individuals. This is a
significant false positive issue. They discussed the binding of PIB to areas of the brain not
associated with classical plaques as a result of PIBs affinity towards diffuse plaques as well. Though
markedly less, PIB was found to also label tangles in the brain. Their data provided a molecular
explanation for PIBs limited ability to monitor the progression of Alzheimer’s disease. While PIB is
an invaluable tool for diagnosing plaque related diseases including Alzheimer’s disease, it is not
comprehensive and tools to monitor progression of neurodegenerative disorders are needed not
only to assist in patient care and prognosis, but also to aid researchers in better understanding the
relationship between the severity of plaques and tangles and the manifestation of physical and
mental symptoms.
Accordingly, and with support from the Alzeheimers Drug Discover Foundation, we
initiated a project wherein methodology would be developed for the 18F labeling of target
monoclonal antibodies, glycoproteins, and peptides of significance to Alzeheimers disease. If
successful, the ability to tag biomolecules of interest using an 18F label, would allow real time PET
imaging to highlight the significance of a number of specific pathways, which would be of great
benefit in Alzeheimers research. Specifically, based on our microwave accelerated synthetic
chemistry protocols we had developed a route to a 19F tagging agent and demonstrated our ability
to label routine Mabs (Chapter 2, Scheme 7). An 18F variant of the tagging agent has already been
developed, thus our goal was to determine the chemical efficiency of tagging with the 19F variant
with the expectation of using the same protocols in subsequent radiotagging and imaging.
Three antibodies were chosen to label and analyze via mass spectrometry. These proteins
belong to a class of antibodies known as immunoglobulin G or IgG. They are the most abundant of
the immunogloublins, consisting of 75% of serum immunogloublins in humans. They are
approximately equally distributed between blood and tissue liquids. IgG antibodies consist of four
peptide chains and are around 150 kDa. Two identical heavy chains make up around 50 kDa and
two light chains account for 25 kDa each. At the end of each “fork” of the antibody is the site where
the antigen binds.
All of the immunoglobulins were subjected to the same reaction condtions. A solution of our
N-succinimidyl-4-fluorobenzoate (Scheme) was added to a solution of IgG (10 mg of ~150 kDa) in
phosphate buffered saline (PBS, pH 8.0) and stirred at room temperature for two hours. Prior
experiences monitoring via TLC
suggest the reaction takes only
O
O
O
F
O
N
O
15-30 minutes for the reaction to
N
H
F
complete, but for the reasons of
this experiment it was decided to let all of the reactions run for two hours and then to examine the
degree of labeling rather than if it labeled. This protocol is from a paper published by Vaidyanthan
et al in Nature Protocols in 2006. The protocol is for the synthesis of a labeling agent that is widely
used attaching a 18F atom to proteins and peptides of interest. Of note, the paper also discusses the
inability to label biologically sensitive molecules via electrophilic halogenations due to the
sensitivity of the complex 3-dimensional structure of the proteins.
The samples were analyzed by LC-MS. They were first desalted online via an LC column,
ionized via electospray ionization (ESI) then separated using a quadrupole time-of-flight mass
analyzer (Q-ToF) to gain information about the labeling of the intact antibody. Alternatively, the
samples could be reduced, alkylated or otherwise fragmented before injecting the samples into the
ESI-Q-ToF, to give more detailed information about the pieces of the antibody. In all cases, the data
was acquired in standard (20,000m/z) 1 GHz, MS only mode with a range of between 1000-5000
m/z for an intact mAb. The cappilary voltage (VCap) was held at 4000 V and the drying gas flowed
at a rate of 7 L/min at 325⁰C. The fragmentor voltage was held at 420 V for intact antibody and the
potentially labeled antibodies. In all cases, protein mass was obtained by maximum entropy
deconvolution using MassHunter BioConfirm Software.
The three antibodies selected were chosen carefully: Two of the antibodies are of direct importance
to CNS research and a mouse Mab to human interferon alpha was chosen for comparison of
methodology to a more thoroughly understood class of antibodies. All of the antibodies selected are
IgGακ’s. They are known to possess two heavy chains which consist of around 450 amino acids and
have three tandem immunoglobulin domains joined by a flexible hinge. The structure of the hinge
region is what differentiates IgG’s in terms of number. The class will be specifically mentioned later
in the context of each antibody but for the purpose of understanding we chose only to examine IgG1 and IgG-2 which together comprise of 89% of IgGs. IgG-1s are the most abundant subclass of this
class. IgG-2’s are distinguishable amongst the other subclasses in that they are the only of the IgG
class that is unable to cross the placenta (a trait unique to IgG’s). Within the context of the hinge
region, IgG-1s are much more flexible and the fab fragments can rotate about their axes of
symmetry. IgG-2s are much more rigid. Disulfide bonds within the heavy chain are largely
responsible for this rigidity. The variable domain of the heavy chain on an IgG-alpha is around 110
amino acids and differ depending on the B cell used, but is conserved among antibodies resulting
from the same B cell. The light chains of an IgG-kappa are around 215 amino acids and consist of
two tandem domains- one constant and one variable. The variable regions are responsible for
difference in antigen binding.
IgG1-κ Clone: MMHA-2
Human interferon are proteins made by our bodies’ lymphocytes in response to viruses, bacteria,
parasites, and also tumor cells. They are used for a range of purposes including use as antiviral,
antiseptic, anti-carcinogenic and to treat some autoimmune disease such as multiple sclerosis.
Through MS analysis it was found to have a molecular weight of 148.83 kDa. However, in the
spectra of the expected modified adduct we see a mass change of around 15 kDa. This corresponds
degradation of the antibody during the reaction and most likely represents the loss of a single
domain, rendering interpretation of tagging experiments impractical.
x10 4
x10 3
1.9
1.8
1.7
1.1
1.6
1.5
1
0.9
1.4
1.3
0.8
1.2
1.1
0.7
1
0.9
0.8
0.7
0.6
0.5
0.4
0.6
0.5
0.4
0.3
0.2
0.3
0.2
0.1
0.1
0
0
IgG2ακ
The second antibody is the first of two that have been designed to selectively bind to features of
specific neuro-degenerative disorders. Anti-human beta amyloid immunoglobulin monoclonal
IgG2ακ antibody. This protein was chosen both because of its similarities to another antibody- W02
that recognizes amyloid fibrils. The fab section of W02 has been studied in depth in an effort to
offer templates for the future rational design of an immunogen capable of producing the B-cell
immune response without the T-cell response. For our purposes, we wanted to provide a
complimentary method of labeling and monitoring the anti-body in vitro to gain a better
understanding of its interaction within a functioning brain. Unfortunately, the mass spectra did not
show the addition of the para-fluoro labeling agent. The expected mass of 150.51685 kDa was seen
in the spectra of the unaltered immunoglobulin. In the modified immunoglobulin spectra, there
appears to be either degradation or aggregation (which would be too large of a mass to be seen in
the spectra via this methodology). A peak at 133.11505 kDa suggests that the antibody may have
fragmented as a 17.4 kDa can correspond to a single strand amyloid fibril.
x10 3
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
The last antibody used was anti-Human PHF-Tau which is an IgG1-κ that is specific for
phosphorylated Tau. No cross ractivity with normal biopsyd or autopsyed samples of tau or fetal
tau has been observed. This suggests that this antoboody detects an abnroamly phosphyrlatino site- specifically a phosphorylated Ser212 and phosphorylated THR214 (numbering according to
IgG1-k
human tau40).
With this antibody we achieved results that bode well for the original aim of this experiment. The
unmodified IgG showed a mass of 152.93146 kDa and the modified a mass of 153. 07255 kDa. This
gives a mass change of 141.09 Da which corresponds to the para-fluoro-benzoate label. This
confirmed both that a fluoro-moiety was introduced to the antibody and preliminary results were
able to be seen using ESI-Q-TOF-MS.
x10 4
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Future [and ongoing] endeavors on Mab labeling should commence with digestion of the
antibody to smaller units to more amenable to specifically analyzing and confirming the presence of
the adducts. Based on this, other antibodies will be labeled, digested and analyzed to fully
determine the scope of the applicability of this method for labeling antibodies for use in targeted
imaging studies.
Experimental protocols for tagging with N-succidnimidyl 4-fluorobenzoate
and MS analysis
O
O
O
O
N
F
O
N
H
F
N-succidnimidyl 4-fluorobenzoate (40 μL of a 2mg/mL solution, 5eq) was added to a
solution of IgG (10 mG ~ 150 kDa) in Phosphate buffered saline (0.5 mL, pH 8.00) and stirred at 25
⁰C for 2 hours. Crude reaction mixture was kept at -78⁰C until analysis.
LC-MS Parameters: Agilent 1200 HPLC-ESI with Agilent 6520 Q-TOF LC/MS.
Analytical column: Vydac PLRP-S, 40 x 2.1 mm,
Time (minutes
B (%)
0
5
5
5
6
45
17
75
19
90
20
3
1000 Å pore size, 5 μm particle size
Flow rate: 200 μl/min from capillary pump
Solvents:
(note: a 5 minute isocratic gradient at 5% B was
utilized for online desalting of the mAb samples.
A. 0.1% formic acid in water
B. 0.1 % formic acid in acetonitrile
Sample load: 1-2 μg (estimated) on-column for
unmodified and modified mAb.
Sample analysis: gradient with cap-pump was set as detailed:
MS Parameters:
Data acquisition: Data were acquired in standard (20,000 m/z), 1 GHz, MS only mode, range: 10005000 m/z for intact mAb.
Spectra were recorded in positive ion mode and in profile mode
VCap:
4000 V and drying gas flow of 7 L/min at 325⁰C
Fragmentor voltage: 420 V for intact and modified antibody forms.
Data analysis:
AgilentMass Hunter Qualitative Analysis software and the MassHunter BioConfirm software
(version B.02.00) were used. Protein mass was obtained utilizing maximum entropy deconvolution
using the BioConfirm software.
Anti-Human Interferon- α monoclonal antibody. IgG1-κ Clone: MMHA-2
Fisher Scientific. Product # PI-211001. Lot # 1087(Pierce Endogen)
0.05 mL in PBS without preservatives.
Concentration: 0.5 mg/mL
Spectra 1: unmodified antiHU-IFNα mAb.
Screen shot showing
acquisition time (~15.0 min),
the spectrum preview and the
preview of the deconvoluted
mass of the molecular ion
peak at 150 kDa.
Spectra 2: “modified” antiHU-IFNα mAb. Screen
shot showing acquisition
time (~14.9min), the
spectrum preview and
deconvoluted mass at
133 kDa, which does not correspond to the expected mass of the modified mAb.
Anti-human β-amyloid monoclonal antibody: IgG-2ακ, Clone: 10H3
Fisher Scientific. Product # MN1150. Lot # EB61572(Pierce Endogen)
0.05 mL in PBS without preservatives.
Concentration: 200 μg/mL
Endotoxin content: 2e-5 ng/μg of IgG.
Spectra 1: unmodified AntiHU β-amyloid mAb. Screen
shot showing acquisition
time (~15.2 min), the
spectrum preview and the
preview of the deconvoluted
mass of the molecular ion
peak at 150 kDa.
Spectra 2: “modified” Anti-HU β-amyloid mAb
Screen shot showing acquisition time (~9.6 min), the spectrum preview and deconvoluted mass at
133 kDa, which does not correspond to the expected mass of the modified mAb.
Anti-Human Phosphorylated Tau monoclonal antibody: IgG 1/κ Clone: AT100
Fisher Scientific. Product # MN1060. Lot # DF57271(Pierce Endogen)
0.05 mL in PBS without preservatives.
Concentration: 200 μg/mL
Spectra 1: unmodified anti-HU
PHF-Tau mAb. Screen shot
showing acquisition time
(~14.9 min), the spectrum
preview and the preview of the
deconvoluted mass of the molecular ion peak at 152.98146 kDa.
Spectra 2:
x10 3
“modified” anti-
8.5
HU PHF-Tau mAb.
8
7.5
Screen shot showing
7
acquisition time
6.5
6
(~14.8 min), the
5.5
spectrum
5
preview and
4.5
deconvoluted
4
3.5
mass at 153.07255
3
kDa, which does
2.5
2
not correspond to
1.5
the expected
1
mass of the
0.5
modified mAb.
0
x10 4
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Mouse mAb
to human
x10 3
interferon-α:
1.1
IgG-1/κ.
1
Clone: MMHA-
2
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Mouse mAb to human interferon-α: IgG-1/κ. Clone: MMHA-2
x10 4
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
x10 4
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Mouse mAb to human interferon-α: IgG-1/κ. Clone: MMHA-2
x10 4
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
x10 4
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
x10 3
9
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Anti-human β-amyloid monoclonal antibody: IgG-2ακ, Clone: 10H3
x10
3
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
x10 3
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Anti-human β-amyloid monoclonal antibody: IgG-2ακ, Clone: 10H3
x10 3
1.1
1
0.9
0.8
0.7
0.6
0.5
3
x10 0.4
1.6
0.3
1.5
0.2
1.4
1.3
0.1
1.2
1.1 0
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Anti-human β-amyloid monoclonal antibody: IgG-2ακ, Clone: 10H3
x10
3
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Anti-Human Phosphorylated Tau monoclonal antibody: IgG 1/κ Clone: AT100
x10 3
9.5
9
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
x10 4
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Anti-Human Phosphorylated Tau monoclonal antibody: IgG 1/κ Clone: AT100
x10 4
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
x10 4
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Anti-Human Phosphorylated Tau monoclonal antibody: IgG 1/κ Clone: AT100
x10 4
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Anti-Human Phosphorylated Tau monoclonal antibody: IgG 1/κ Clone: AT100
x10 4
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
x10
0.6 4
0.51.3
0.4
1.2
0.3
0.21.1
0.1 1
0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
:
1. Berchtold NC, Cotman CW (1998). "Evolution in the conceptualization of
dementia and Alzheimer's disease: Greco-Roman period to the 1960s".
Neurobiol. Aging 19 (3): 173–89
2. Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.; Holt, D. P.;
Bergström, M.; Savitcheva, I.; Huang, G. F.; Estrada, S.; Ausén, B.; Debnath, M.
L.; Barletta, J.; Price, J. C.; Sandell, J.; Lopresti, B. J.; Wall, A.; Koivisto, P.;
Antoni, G.; Mathis, C. A.; Långström, B., Imaging brain amyloid in Alzheimer's
disease with Pittsburgh Compound-B. Annals of Neurology 2004, 55 (3), 306319.
3. Small, S., Imaging Alzheimer’s disease. Current Neurology and Neuroscience
Reports 2003, 3 (5), 385-392.
4. Milos D. Ikonomovic, William E. Klunk, Eric E. Abrahamson, Chester A. Mathis,
Julie C. Price, Nicholas D. Tsopelas, Brian J. Lopresti, Scott Ziolko, Wenzhu Bi,
William R. Paljug, Manik L. Debnath, Caroline E. Hope, Barbara A. Isanski,
Ronald L. Hamilton, and Steven T. DeKosky Brain (2008) 131(6): 1630-1645
5. A.J. Meulenbroek and W.P. Zeijlemaker. Human Immunoglobulin subclasses.
Useful diagnostic markers for immunocompetence. Published by CLB,
Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. CLB, 1996. ISBN 905267-011-0 Published online. http://www.researchd.com/rdikits/rdisubbk.htm.
6. Janeway CA, Jr. et al. (2001). Immunobiology. (5th ed. ed.). Garland Publishing
7. Woof J, Burton D (2004). "Human antibody-Fc receptor interactions illuminated
by crystal structures". Nat Rev Immunol 4 (2): 89–99.
8. Vaidyanathan, G., Zulutsky, M.R., Nature Protocols 2006, 1 (4), 1655-1661.
CHAPTER 6: EXPERIMENTAL DATA AND SPECTRA
General Experimental Procedures
All reactions were conducted in a 2.4 GHz CEM brand microwave reactor, using a maximum
power of 300 watts, at a maximum pressure of 300 psi. All run times were 1 minute. All reagent
solvents were purchased from the SigmaAldrich Chemical Company and dried prior to use over
sodium with a benzophenone indicator. Et3N and DIPEA were distilled prior to use1. Hex-5-en1-yl 4-methylbenzenesulfonate and pent-4-en-1-yl 4-methylbenzenesulfonate were synthesized
from the corresponding alcohols, according to literature proceedure2. All other reagents were
used without further purification. All TLC was run on Whatman UV-fluorescent 250 µm layer
plates. Preparative TLC plates were purchased from Analtech. All reactions were performed
under an Argon atmosphere and sealed with teflon tape prior to reaction. All NMR data was run
on an INOVA-500 Waters spectrometer, at the cited frequency. Portions of this work was
performed in concert with Drs Dong Ma, Dr. Paul LaBeaume and Luke Harris, whose
dissertations and notebooks are cross-referenced for additional details.
General procedure for the microwave accelerated fluorodenitration reaction using anhydrous TBAF:
Anhydrous TBAF (1M in DMSO9, 0.500 mL, 0.500 mmol) was added to a solution of the nitro arene (0.2
mmol) in THF (1 mL) and exposed to microwave irradiation. The reaction mixture was diluted with
water (10 mL), extracted with ethyl acetate or dichloromethane (3x 10 mL). The combined extracts
were washed with 10% hydrochloric acid43 (2x 10 mL), brine (10 mL), dried (MgSO4) and concentrated in
vacuo. The crude residue was analyzed by GCMS44 or purified by flash column chromatography (ethyl
acetate: hexanes) or acid base extraction.
Example experimental procedure for pyridine fluorodenitrations:
4-fluoropicolinonitrile: 4.2’
Anhydrous TBAF (1M in DMSO, 0.500 mL, 0.500 mmol) was added to a solution of 4-nitropicolinonitrile
(0.030 mg, 0.2 mmol) in THF (1 mL) and exposed to microwave irradiation at 110°C for 2 min. The
reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3x 10 mL). The
combined extracts were washed with saturated aqueous sodium bicarbonate (2x 10 mL), brine (10 mL),
dried (MgSO4) and concentrated in vacuo.
The crude residue was purified by flash column
chromatography (4:1 hexanes: ethyl acetate) to afford the title compound (0.021 g, 86%) as a light
yellow solid. mp = 74-76 1H NMR (500mHz, CDCl3): δ 8.72 (dd, J = 8, 5.5Hz, 1H), 7.48 (dd, J = 8, 2.5Hz,
1H), 7.28-7.32 (m, 1H); 13C NMR (125 mHz, CDCl3): δ 168.53 (d, J = 266.3Hz, 1C), 153.94 (d, J = 7.3Hz, 1C),
136.30 (d, J = 8.3Hz, 1C), 117.30 (d, J = 19.5Hz, 1C), 116.39 (d, J = 5.5Hz, 1C), 115.38 (d, J = 16.5Hz, 1C);
GCMS: m/z C6H3FN2 (M)+ calcd 122, obsd 122; TR = 5.22.
Example scale up experimental procedure for arene fluorodenitrations:
4-fluorobenzoic acid: 4.35’
Anhydrous TBAF (1M in DMSO, 0.500 mL, 0.500 mmol) was added to a solution of methyl 4nitrobenzoate (0.036 g, 0.2 mmol) in THF (1 mL) and exposed to microwave irradiation at 160°C for 10
min. and immediately injected with lithium hydroxide (1M aqueous solution, 0.6 mL, 0.6 mmol). The
solution was exposed to microwave irradiation at 140°C, diluted with water (10 mL), extracted with
dichloromethane (3x 10 mL) and discarded. The aqueous layer was diluted with 10% HCl (10 mL),
extracted with dichloromethane (3x 10 mL), dried (MgSO4) and concentrate in vacuo to afford the title
compound (0.016 g, 57%) as a crystalline white solid. mp = 182-184 1H NMR (500mHz, CDCl3): δ 8.5012.0 (brs, 1H), 8.15 (dd, J = 7.5, 5.5Hz, 2H), 7.16 (t, J = 9Hz, 2H); 13C NMR (125 mHz, d-DMSO): δ 171.83,
170.32 (d, J = 249.8Hz, 1C), 137.64 (d, J = 9.3Hz, 2C), 132.72 (d, J = 2.1Hz, 1C), 120.86 (d, J = 21.8Hz, 2C);
GCMS: m/z C7H5FO2 (M+H)+ calcd 141, obsd 141; TR = 14.52.
Example experimental procedure for selective fluorodenitrations:
5-bromo-3-fluoropicolinonitrile: 4.30’
Anhydrous TBAF (1M in DMSO, 0.500 mL, 0.500 mmol) was added to a solution of 5-bromo-3nitropicolinonitrile (0.046 g, 0.2 mmol) in THF (1 mL) and exposed to microwave irradiation at 110°C for
2 min. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3x 10 mL).
The combined extracts were washed with saturated aqueous sodium bicarbonate (2x 10 mL), brine (10
mL), dried (MgSO4) and concentrated in vacuo. The crude residue was purified by flash column
chromatography (4:1 hexanes: ethyl acetate) to afford the title compound (0.037 g, 81%) as a light
yellow solid. mp = 101-102 1H NMR (500mHz, CDCl3): δ 8.63-8.65 (m, 1H), 7.88 (dd, J = 7.5, 1.5Hz, 1H);
13
C NMR (125 mHz, CDCl3): δ 160.36 (d, J = 274.63Hz, 1C), 148.62 (d, J = 5.1Hz, 1C), 127.90 (d, J = 19.6Hz,
1C), 125.52 (d, J = 3.1Hz, 1C), 121.16 (d, J = 14.4Hz, 1C), 112.48 (d, J = 5.3Hz, 1C).
2-fluoro-3-methoxypyridine: 4.29’
Following the general procedure: (94%) GCMS: m/z C6H6FNO (M)+ calcd 127, obsd 127; TR = 5.83.
2-bromo-6-fluoropyridine: 4.28’
Following the general procedure: (97%) GCMS: m/z C5H3BrFN (M)+ calcd 175, obsd 175; TR = 5.19.
4-fluorobenzonitrile: 4.7’
Following the general procedure: (97%) GCMS: m/z C7H4FN (M)+ calcd 121, obsd 121; TR = 5.12.
6-fluorobenzo[de]isochromene-1,3-dione: 4.31
Following the general procedure: (97%) GCMS: m/z C12H5FO3 (M)+ calcd 216, obsd 216; TR = 15.63.
1-fluoro-2-nitrobenzene: 4.24’
Following the general procedure: (96%) GCMS: m/z C6H4FNO2 (M)+ calcd 141, obsd 141; TR = 6.82.
4-fluoroacetophenone: 4.32’
Following the general procedure: (13%) GCMS: m/z C8H7FO (M)+ calcd 138, obsd 138; TR = 6.28.
O
NO2
O
F
2-fluoroacetophenone: 4.33’
Following the general procedure: (8%) GCMS: m/z C8H7FO (M)+ calcd 138, obsd 138; TR = 5.96.
6-fluoro-1H-indazole: 4.36’
Following the general procedure: (~1%) GCMS: m/z C7H5FO2 (M+)+ calcd 136, obsd 136; TR= 6.09.
5-fluoro-1H-indole: 4.37’
Following the general procedure: (~1%) GCMS: m/z C8H6FN (M+)+ calcd 135, obsd 135; TR= 7.69.
6-fluoro-1H-indole: 4.38’
Following the general procedure: (~1%) GCMS: m/z C8H6FN (M+)+ calcd 135, obsd 135; TR= 7.72.
O
O
Cl
O
Cl
+ H3CO
H3CO
H3CO
F
F
F
NO2
Major
Minor
methyl 2-chloro-4-fluorobenzoate and methyl 2,4-difluorobenzoate: 4.39’a and 4.39’b
Following the general procedure: (93%) Major: GCMS: m/z C8H6ClFO2 (M+)+ calcd 188, obsd 188; TR=
8.59; (5%) Minor: GCMS: m/z C8H6F2O2 (M+)+ calcd 172, obsd 172; TR= 6.60.
2-chloro-4-fluorobenzonitrile and 2,4-difluorobenzonitrile: 4.40’a and 4.40’b
Following the general procedure: (74%) Major: GCMS: m/z C7H3ClFN (M+)+ calcd 155, obsd 155; TR= 7.08.
(23%) Minor: GCMS: m/z C7H3F2N (M+)+ calcd 139, obsd 139; TR= 4.86.
2-chloro-4-fluorobenzoic acid:45 4.34’
Following the general procedure: (65%) GCMS: m/z C7H4ClFO2 (M+)+ calcd 173.9, obsd 173.9; TR= 14.22.
2-fluoro-6-nitropyridine: 4.35’
Following the general procedure: (90%) GCMS: m/z C5H3FN2O2 (M+)+ calcd 142, obsd 142; TR= 6.34.
Example Procedure 1: Optimization of microwave accelerated Negishi cross coupling intermediate.
O
O
I
Cl
1-(4-(4-chlorobutyl)phenyl)ethanone50: 4.87
Iodine (0.0064 g, 0.025 mmol) and zinc dust (0.049 g, 0.75 mmol) were added to degassed
dimethylformamide (freeze-pump-thaw method 4x) in a CEM microwave tube and stirred at 25°C for ca.
10 minutes. Degassed (freeze-pump-thaw method 4x) 1-bromo-4-chloroalkane (0.058 mL, 0.50 mmol)
was added, heated to 80°C for 3.5h and cooled to 25°C. 4-iodoacetophenone (0.062 g, 0.25 mmol) and
Ni(PPh3)2Cl2 (0.0082 g, 0.0125 mmol) were added and exposed to microwave irradiation for 80°C for 10
min., poured onto 5% HCl (25 mL) and extracted with ethyl acetate (3x 25 mL). The combined organic
extracts were washed brine (50 mL), dried (MgSO4), and concentrated in vacuo. The residue was
purified by flash column chromatography (quick step gradient 49:1 to 19:1 to 9:1 hexanes: ethyl acetate)
to afford the title compound (0.033 g, 62%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.89 (d, J =
8Hz, 2H), 7.27 (d, J = 8Hz, 2H), 3.55 (t, J = 7Hz, 2H), 2.71 (t, J = 7Hz, 2H), 2.59 (s, 3H), 1.78-1.83 (m, 2H);
13
C NMR (125 mHz, CDCl3): δ 197.80, 147.65, 135.20, 128.60, 44.72, 35.10, 31.98, 28.18, 26.57.
Example Procedure 2: Optimization of microwave accelerated Negishi cross coupling fluoroalkylation.
1-(4-(4-fluorobutyl)phenyl)ethanone: 4.88
Iodine (0.0064 g, 0.025 mmol) and zinc dust (0.049 g, 0.75 mmol) were added to degassed
dimethylformamide (0.5 mL) (freeze-pump-thaw method 4x) in a CEM microwave tube and stirred at
25°C for ca. 10 minutes. Degassed (freeze-pump-thaw method 4x) 1-bromo-4-chloroalkane (0.058 mL,
0.50 mmol) was added, heated to 80°C for 3.5h and cooled to 25°C. 4-iodoacetophenone (0.062 g, 0.25
mmol) and Ni(PPh3)2Cl2 (0.0082 g, 0.0125 mmol) were added and exposed to microwave irradiation for
80°C for 10 min. Tetrabutyl ammonium fluoride (1M in THF, 1.75 mL, 1.75 mmol) was immediately
injected , exposed to microwave irradiation at 125°C for 10 min., poured onto 5% HCl (25 mL) and
extracted with ethyl acetate (3x 25 mL). The combined organic extracts were washed brine (50 mL),
dried (MgSO4), and concentrated in vacuo. The residue was purified by flash column chromatography
(quick step gradient 49:1 to 19:1 to 9:1 hexanes: ethyl acetate) to afford the title compound (0.017 g,
34%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.89 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 4.46
(d, J = 48, 6 Hz, 2H), 2.74 (t, J = 8 Hz, 2H), 2.59 (s, 3H), 1.65-1.84 (m, 4H); 13C NMR (125 mHz, CDCl3): δ
197.87, 147.87, 135.23, 128.69, 128.64, 83.85 (d, J = 164 Hz, 1C), 35.48, 29.97 (d, J = 20 Hz, 1C), 16.78 (d,
J = 5 Hz, 1C), 26.62; HRMS (ESI), m/z C12H16FO (M+H)+ calcd 195.1185, obsd 195.1179.
1-(2-(4-fluorobutyl)phenyl)ethanone: 4.89
Following example procedure 2: (0.018 g, 38%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.66 (d, J =
8 Hz, 1H), 7.40 (dt, J = 8, 1.5 Hz, 1H), 7.25-7.30 (m, 2H), 4.47 (dt, J = 48, 6 Hz, 2H), 2.89 (t, J = 8 Hz), 2.56
(s, 3H), 1.65-1.84 (m, 4H); 13C NMR (125 mHz, CDCl3): δ 202.15, 142.47, 137.85, 131.64, 131.39, 129.48,
126.05, 84.17 (d, J = 164 Hz, 1C), 33.71, 30.48 (d, J = 20 Hz, 1C), 29.98, 27.27 (d, J = 5 Hz, 1C); HRMS (ESI),
m/z C12H16FO (M+H)+ calcd 195.1185, obsd 195.1176.
1-(3-(4-fluorobutyl)phenyl)ethanone: 4.90
Following example procedure 2: (0.016 g, 32%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.75-7.81
(m, 2H), 7.36-7.42 (m, 2H), 4.47 (dt, J = 48, 6 Hz, 2H), 2.73 (t, J = 8 Hz, 2H), 2.60 (s, 3H), 1.67-1.83 (m, 4H);
13
C NMR (125 mHz, CDCl3): δ 198.48, 142.68, 137.44, 133.38, 128.74, 128.22, 126.31, 83.98 (d, J = 164
Hz, 1C), 35.42, 30.03 (d, J = 20 Hz, 1C), 27.12 (d, J = 5Hz, 1C), 26.83; HRMS (ESI), m/z C12H16FO (M+H)+
calcd 195.1185, obsd 195.1176.
1-(4-fluorobutyl)-2-methoxybenzene: 4.91
Following example procedure 2: (0.003 g, 6%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.11 (dt, J =
8, 2 Hz, 1H), 7.05 (dd, J = 8, 2 Hz, 1H), 6.81 (t, J = 8 Hz, 1H), 6.77 (d, J = 8 Hz, 1H), 4.39 (dt, J = 48, 6 Hz,
2H), 3.75 (s, 3H), 2.59 (t, J = 7.5 Hz, 2H), 1.57-1.74 (m, 4H); 13C NMR (125 mHz, CDCl3): δ 130.07, 127.32,
120.60, 110.49, 84.41 (d, J = 164 Hz, 1C), 55.48, 30.48 (d, J = 20 Hz, 1C), 29.91, 25.60 (d, J = 5 Hz, 1C);
HRMS (EI), m/z C11H15FO (M)+ calcd 182.1107, obsd 182.1105.
1-(4-fluorobutyl)-3-methoxybenzene: 4.92
Following example procedure 2: (0.008 g, 18%) as a colorless oil. 1H NMR (500 mHz, CDCl3): δ 7.17-7.23
(m, 1H), 6.76-6.79 (m, 1H), 6.71-6.75 (m, 2H), 4.45 (dt, J = 48, 6 Hz, 2H), 3.79 (s, 3H), 2.64 (t, J = 7.5 Hz,
2H), 1.64-1.79 (m, 4H); 13C NMR (125 mHz, CDCl3): δ 159.91, 143.89, 129.55, 121.09, 114.47, 111.33,
84.23 (d, J = 164 Hz, 1C), 55.39, 35.71, 30.31 (d, J = 20 Hz, 1C), 27.11 (d, J = 5 Hz, 1C); HRMS (EI), m/z
C11H15FO (M)+ calcd 182.1107, obsd 182.1107.
1-(4-fluorobutyl)-4-methoxybenzene: 4.93
Following example procedure 2: (0.005 g, 10%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.10 (d, J =
9 Hz, 2H), 6.82 (d, J = 9 Hz, 2H), 4.45 (dt, J = 48, 6 Hz, 2H), 3.79 (s, 3H), 2.60 (t, J = 8Hz, 2H), 1.64-1.79 (m,
4H); 13C NMR (125 mHz, CDCl3): δ 157.94, 134.22, 129.42, 113.91, 84.17 (d, J = 164 Hz, 1C), 55.41, 34.63,
31.10 (d, J = 20 Hz, 1C), 27.35 (d, J = 5 Hz, 1C); HRMS (EI), m/z C11H15FO (M)+ calcd 182.1107, obsd
182.1107.
Example Procedure 3: Optimization of microwave accelerated Negishi cross coupling fluoroalkylation
with basic substrates.
3-(4-fluorobutyl)pyridine: 4.95
Following example procedure 2 with the following workup: Acetic acid (ca. 0.1 mL) was added, shaken
vigorously until all the zinc was consumed, diluted with saturated sodium carbonate (25 mL or until pH ~
10) and extracted with ethyl acetate (3x 25 mL). The combined organic extracts were washed brine (50
mL), dried (MgSO4), and concentrated in vacuo.
The residue was purified by flash column
chromatography (7:1 hexanes: ethyl acetate) to afford the title compound (0.008 g, 20%) as a yellow oil.
1
H NMR (500mHz, CDCl3): δ 8.40-8.54 (m, 2H), 7.50 (dt, J = 7.5, 2 Hz, 1H), 7.19-7.25 (m, 1H), 4.47 (dt, J =
48, 6 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H), 1.67-1.82 (m, 4H); 13C NMR (125 mHz, CDCl3): δ 150.08, 147.62,
135.91, 123.49, 123.41, 83.89 (d, J = 164 Hz, 1C), 32.68, 29.98 (d, J = 20 Hz, 1C), 26.99 (d, J = 5 Hz, 1C);
HRMS (ESI), m/z C9H13FN (M+H)+ calcd 154.1032, obsd 154.1033.
2-(4-fluorobutyl)pyridine: 4.94
Following example procedure 3:51 (0.001 g, 3%) as a yellow oil. 1H NMR (500 mHz, CDCl3): δ 8.52-8.55
(m, 1H), 7.57 (dt, J = 7.5, 2Hz, 1H), 7.07-7.13 (m, 2H), 4.39 (dt, J = 47.5, 6.5Hz, 2H), 2.66-2.77 (m, 2H),
1.82-1.90 (m, 2H), 1.53-1.70 (m, 2H);
13
C NMR (125 mHz, CDCl3): δ 161.95, 149.51, 136.26, 123.84,
121.10, 84.39 (d, J = 164 Hz, 1C), 32.59, 30.97 (d, J = 20Hz, 1C), 22.38 (d, J = 5Hz, 1C); HRMS (ESI), m/z
C9H13FN (M+H)+ calcd 154.1032, obsd 154.1029.
4-(4-fluorobutyl)pyridine: 4.96
Following example procedure 3: (0.007 g, 18%) as a yellow oil. 1H NMR (500 mHz, CDCl3): δ 8.41-8.59
(brs, 2H), 7.13 (d, J = 5 Hz, 2H), 4.47 (dt, J = 47, 6.5Hz, 2H), 2.67 (t, J = 7Hz, 2H), 1.67-1.83 (m, 4H); 13C
NMR (125 mHz, CDCl3): δ 151.30, 149.85, 124.19, 83.94 (d, J = 164 Hz, 1C), 34.97, 30.09 (d, J = 20Hz, 1C),
26.25 (d, J = 5Hz, 1C); HRMS (ESI), m/z C9H13FN (M+H)+ calcd 154.1032, obsd 154.1027.
1-tert-butyl-4-(4-fluorobutyl)benzene: 4.97
Following general procedure 2: (0.011 g, 21%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.30 (d, J =
8 Hz, 2H), 7.11 (d, J = 8 Hz, 2H), 4.45 (dt, J = 48, 6 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H), 1.67-1.80 (m, 4H), 1.31
(s, 9H); 13C NMR (125 mHz, CDCl3): δ 148.89, 139.16, 128.29, 125.48, 84.30 (d, J = 164 Hz, 1C), 35.10,
34.61, 31.67, 30.29 (d, J = 20 Hz, 1C), 27.17 (d, J = 5 Hz, 1C); HRMS (EI), m/z C14H21F (M)+ calcd 208.1627,
obsd 208.1631.
1-(3-(6-fluorohexyl)phenyl)ethanone: 4.98
Following example procedure 2: (0.022 g, 40%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.88 (d, J =
8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H), 4.43 (dt, J = 48, 6 Hz, 2H), 2.67 (t, J = 8 Hz, 2H), 2.58 (s, 3H), 1.611.76 (m, 4H), 1.34-1.48 (m, 4H); 13C NMR (125 mHz, CDCl3): δ 197.99, 148.62, 135.14, 128.73, 128.64,
84.20 (d, J = 164 Hz, 1C), 35.97, 31.09, 30.43 (d, J = 20 Hz, 1C), 28.94, 26.69, 25.17 (d, J = 5 Hz, 1C); HRMS
(ESI), m/z C14H20FO (M+H)+ calcd 223.1498, obsd 223.1490.
1-(3-(5-fluoropentyl)phenyl)ethanone: 4.99
Following example procedure 2: (0.021 g, 41%) as a colorless oil. 1H NMR (500mHz, CDCl3): δ 7.88 (d, J =
8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H), 4.45 (dt, J = 48, 6 Hz, 2H), 2.69 (t, J = 8 Hz, 2H), 2.58 (s, 3H), 1.64-1.79
(m, 4H), 1.41-1.50 (m, 2H); 13C NMR (125 mHz, CDCl3): δ197.97, 148.36, 135.19, 128.73, 128.66, 84.09
(d, J = 164 Hz, 1C), 35.96, 30.82, 30.36 (d, J = 20 Hz, 1C), 26.68, 25.02 (d, J = 5 Hz, 1C); HRMS (ESI), m/z
C13H18FO (M+H)+ calcd 209.1342, obsd 209.1343.
(S)-tert-butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate42: 4.103
Boc anhydride (12.3 g, 56.4 mmol) followed by triethylamine (9.80 mL, 56.4 mmol) was added to a
solution of (S)-pyrrolidin-2-ylmethanol (4.75 g, 47.0 mmol) in dichloromethane (60 mL) at 0°C and stirred
for 30 min. The reaction mixture was diluted with dichloromethane (200 mL), washed with water (2x
100 mL), brine (100 mL), dried (MgSO4) and concentrated in vacuo. The crude residue was purified by
flash column chromatography (1:1 ethyl acetate: hexanes) to afford the title compound (9.17 g, 97%) as
a colorless oil. 1H NMR (500 mHz, CDCl3): δ 4.80-4.89 (m, 1H), 3.80-3.89 (m, 1H), 3.43-3.67 (m, 2H), 3.293.46 (m, 1H), 3.21-3.38 (m, 1H), 1.91-2.06 (m, 1H), 1.73-1.91 (m, 2H), 1.55-1.66 (m, 1H), 1.47 (s, 9H); 13C
NMR (125 mHz, CDCl3): δ 156.49, 79.76, 66.40, 63.54, 59.69, 47.23, 28.23, 23.74.
(S)-tert-butyl 2-((5-bromopyridin-3-yloxy)methyl)pyrrolidine-1-carboxylate: 4.105
DIAD (0.602 mL, 3.06 mmol) was added dropwise to a solution of (S)-tert-butyl 2(hydroxymethyl)pyrrolidine-1-carboxylate (0.560 g, 2.78 mmol), 3-bromo-5-hydroxypyridine (0.533 g,
3.06 mmol) and PPh3 (0.803 g, 3.06 mmol) in THF (12 mL) at 0°C. The solution was slowly warmed to
25°C and stirred for 16h and concentrated in vacuo. The crude residue was purified by flash column
chromatography (17:3 hexanes: ethyl acetate) to afford the title compound (0.844 g, 85%) as a colorless
oil. 1H NMR (500 mHz, CDCl3): δ 8.25 (d, J = 2.5Hz, 1H), 8.21-8.31 (m, 1H), 7.34-7.47 (m, 1H), 3.54-4.23
(m, 3H), 3.29-3.49 (m, 2H), 1.83-2.09 (m, 3H), 1.47 (s, 9H); 13C NMR (125 mHz, CDCl3): δ 155.77, 154.99,
143.11, 137.03, 124.10, 120.60, 79.83, 68.97, 56.15, 47.23, 28.75, 28.31, 24.13.52
(S)-3-(4-fluorobutyl)-5-(pyrrolidin-2-ylmethoxy)pyridine: 4.106
Dimethylacetamide (DMA) (0.5 mL) was degassed by the freeze pump thaw method (4x). Iodine (6.4mg,
0.025mmol) and zinc dust (49mg, 0.75mmol) were added at room temperature and stirred vigorously
until the solution went clear. Then the mixture was heated to 80 °Cfor 3.5h and cooled to room
temperature.
Next, a solution of DMA (0.5 mL) and (S)-tert-butyl 2-((5-bromopyridin-3-
yloxy)methyl)pyrrolidine-1-carboxylate (89mg, 0.25 mmol) was degassed by the freeze pump thaw
method (4x) and cannulated to the aforementioned solution. Ni(PPh3)2Cl2 (8.2mg, 0.0125mmol) was
then added and the mixture was exposed to microwave irradiation at 80 °C for 10 minutes. 1M TBAF in
THF (1.75 mmol, 1.75 ml) was directly injected and exposed to microwave irradiation at 125 °C for 10
minutes. TFA (0.5 mL) was added and exposed to microwave irradiation at 125 °C for 10 minutes. The
reaction mixture was diluted with DCM (15 mL) carefully poured onto a saturated solution of Na2CO3.
Solid Na2CO3 was added until pH = 10. The aqueous layer was extracted with DCM (5x 15mL), dried over
MgSO4 and concentrated in vacuo. The crude oil was purified by preparative TLC (19:1 DCM:MeOH,
neutralized plate with 9:1 hex:TEA) to afford the title compound (9.5mg, 15%) as a yellow oil. 1H NMR
(500mHz, CDCl3): δ 8.15 (d, J = 2.5 Hz, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.05 (t, J = 2.5 Hz, 1H), 4.46 (td, J = 47,
6 Hz, 2H), 3.94-4.04 (m, 2H), 3.59-3.66 (m, 1H), 3.35-3.60 (brs, 1H), 3.0-3.23 (m, 2H), 2.64 (t, J = 8 Hz,
2H), 1.59-2.06 (m, 7H); 13C NMR (125 mHz, CDCl3): δ 155.02, 142.64, 138.08, 135.54, 121.49, 83.87 (d, J
= 164 Hz, 1C), 70.98, 57.47, 46.52, 32.45, 29.93 (d, J = 20 Hz, 1C), 27.92, 26.83 (d, J = 5 Hz, 1C), 25.20;
HRMS (ESI), m/z C14H22FN2O (M+H)+ calcd 253.1716, obsd 253.1714.
(S)-methyl 1-allylpyrrolidine-2-carboxylate42: 4.110
Triethylamine (11.4 mL, 82.2 mmol) and allyl bromide (2.85 mL, 32.9 mmol) were added to a solution of
(S)-methyl pyrrolidine-2-carboxylate (2.12 g, 16.4 mmol) in dimethylformamide (40 mL) at 0°C, slowly
warmed to 25°C, stirred for 24 h, diluted with water (200 mL) and extracted with ethyl acetate (3x
100mL).
The combined organic extracts were washed with brine (150 mL), dried (MgSO4) and
concentrated in vacuo. The residue was purified by flash column chromatography (3:1 ethyl acetate:
hexanes) to afford the title compound (2.40 g, 86%) as a colorless oil. 1H NMR (500 mHz, CDCl3): δ 5.865.97 (m, 1H), 5.06-5.21 (m, 2H), 3.54 (s, 3H), 3.26-3.34 (m, 1H), 3.07-3.16 (m, 3H), 2.33-2.41 (m, 1H),
2.05-2.19 (m, 1H), 1.72-1.98 (m, 3H); 13C NMR (125 mHz, CDCl3): 174.58, 135.21, 117.41, 65.20, 57.76,
53.47, 51.74, 29.48, 23.03.
O
N
OCH3
(S)-(1-allylpyrrolidin-2-yl)methanol42: 4.111
N
OH
A suspension of lithium aluminum hydride (1.59 g, 41.9 mmol) in ether (15 mL) was added dropwise to a
solution of (S)-methyl 1-allylpyrrolidine-2-carboxylate (2.39 g, 14.1 mmol) at 0°C, slowly warmed to 25°C
and heated at reflux for 6 h. The solution was cooled to 0°C, quenched by the slow addition of water (10
mL), diluted with saturated sodium bicarbonate (50 mL) and extracted with ether (5x 100 mL). The
combined extracts were dried (MgSO4) and concentrated in vacuo to afford the title compound (1.97 g,
99%) as a colorless oil.
1
H NMR (500 mHz, CDCl3): δ 5.84-5.94 (m, 1H), 5.08-5.22 (m, 2H), 3.62 (dd, J =
10.5, 4Hz, 1H), 3.39-3.45 (m, 2H), 3.07-3.13 (m, 2H), 2.93 (dd, J = 13.5, 7.5Hz, 1H), 2.60-2.66 (m, 1H),
2.27-2.34 (m, 1H), 1.85-1.93 (m, 1H), 1.68-1.81 (m, 3H); 13C NMR (125 mHz, CDCl3): δ 136.14, 117.15,
64.39, 62.45, 57.60, 54.53, 27.97, 23.66.
O
N
OH
N
N
O
(S)-2-((1-allylpyrrolidin-2-yl)methyl)isoindoline-1,3-dione: 4.112
(S)-(1-allylpyrrolidin-2-yl)methanol (1.00 g, 7.08 mmol), phthalimide (1.14 g, 7.79 mmol) and triphenyl
phosphine (2.04 g, 7.79 mmol) were dissolved in THF (50 mL). DIAD (1.53 mL, 7.79 mmol) was added
dropwise over 2 h and the solution stirred at 25°C for 5 h. The solution was diluted with ethyl acetate
(200 mL) and washed with water (2x 100 mL). The combined aqueous layers were extracted with ethyl
acetate (2x 100 mL) and the combined organic extracts washed with brine (200 mL), dried (MgSO4) and
concentrated in vacuo. The residue was purified by flash column chromatography (4:1 hexanes: ethyl
acetate) to afford the title compound (1.20 g, 63%) as a light yellow oil. 1H NMR (500 mHz, CDCl3): δ
7.84 (dd, J = 5.5, 2.5Hz, 2H), 7.71 (dd, J = 5.5, 2.5Hz, 2H), 5.86-5.86 (m, 1H), 5.07-5.24 (m, 2H), 3.74 (dd, J
= 13.5, 4Hz, 1H), 3.66 (dd, J = 13.5, 8Hz, 1H), 3.57-3.59 (m, 1H), 3.07-3.13 (m, 1H), 3.00 (dd, J = 13, 7.5Hz,
1H), 2.24-2.30 (m, 1H), 1.64-1.87 (m, 4H); 13C NMR (125 mHz, CDCl3): δ 168.65, 136.09, 133.96, 132.16,
123.27, 117.08, 62.14, 58.02, 54.20, 41.93, 29.31, 22.87; HRMS (ESI), m/z C16H19N2O2 (M+H)+ calcd
271.1447, obsd 271.1445.
(S)-(1-allylpyrrolidin-2-yl)methanamine: 4.115
Hydrazine hydrate (32 µL, 6.66 mmol) was added to a solution of (S)-2-((1-allylpyrrolidin-2yl)methyl)isoindoline-1,3-dione (0.600 g, 2.22 mmol) in ethanol (30 mL) at 50°C, brought to reflux for 3
h, cooled to 0°C and the solid filtered and washed with ethyl acetate (5x 100 mL). The filtrate was dired
(MgSO4) and concentrated in vacuo to afford the title compound (0.295 g, 95%) as a light yellow oil. This
compound was >95% pure by NMR.
Further purification can be obtained by flash column
chromatography (careful elution using 9:1 dichloromethane: methanol, neutralized column by 19:1
dichloromethane: triethylamine) using a short pad of silica gel. 1H NMR (500 mHz, CDCl3): δ 5.85-5.95
(m, 1H), 5.06-5.21 (m, 2H), 3.38-3.44 (m, 1H), 3.05-3.11 (m, 1H), 2.83-2.89 (m, 1H), 2.66-2.77 (m, 2H),
2.40-2.48 (m, 1H), 2.19-2.26 (m, 1H), 1.85-2.05 (brs, 2H), 1.85-1.95 (m, 1H), 1.67-1.75 (m, 2H), 1.57-1.65
(m, 1H); 13C NMR (125 mHz, CDCl3): δ 136.28, 116.71, 65.38, 57.80, 54.43, 44.44, 28.20, 22.88.
5-iodo-2-methoxybenzoic acid53: 4.114
Sodium hydride (1.11 g, 46.4 mmol) was added in portions to a solution of 2-hydroxy-5-iodobenzoic acid
(5.57 g, 21.2 mmol) in dimethylformamide (20 mL) followed by the addition of methyl iodide (2.94 g,
63.3 mmol). The solution was heated to 85°C for 24 h, cooled to 25°C, diluted with ethyl acetate (100
mL) and washed with saturated sodium bicarbonate (3x 100 mL). The organic extract was dried (MgSO4)
and concentrated in vacuo. The crude residue was dissolved in tetrahydrofuran (200 mL) and lithium
hydroxide (1.52 g, 63.6 mmol) in water (15 mL) was added. The solution was heated to 60°C for 2 h, and
concentrated in vacuo to a syrup which was diluted with water (150 mL) and extracted with ethyl acetat
(2x 100 mL). The aqueous layer was acidified by the addition of hydrochloric acid until pH = ~2,
extracted with ethyl acetate (4x 100 mL), dried (MgSO4) and concentrated in vacuo. The residue was
purified by flash column chromatography (dichloromethane) to afford the title compound (4.20 g, 72%)
as a white solid. mp = 147-149°C; . 1H NMR (500 mHz, CDCl3): δ 10.40-10.55 (brs, 1H), 8.47 (d, J = 2.5Hz,
1H), 7.85 (dd, J = 8.5, 2.5Hz, 1H), 6.84 (d, J = 8.5Hz, 1H), 4.07 (s, 3H);
13
C NMR (125 mHz, CDCl3): δ
166.61, 158.60, 141.87, 139.17, 124.36, 115.94, 82.94, 56.62.
(S)-N-((1-Allylpyrrolidin-3-yl)methyl)-5-iodo-2-methoxybenzamide41: 4.116
Triethylamine (0.224 mL, 1.63 mmol) and ethyl chloroformate (0.156 mL, 1.63 mmol) were added to a
solution of 5-iodo-2-methoxybenzoic acid (0.377 g, 1.36 mmol) in dichloromethane (12 mL) at 0°C and
stirred for 15 min. Then a solution of (S)-(1-allylpyrrolidin-2-yl)methanamine (0.190 g, 1.36 mmol)
dissolved in dichloromethane (7 mL) was added dropwise and stirred at 0°C for 30 min. The reaction
mixture was diluted with dichloromethane (25 mL), washed with water (2x 25 mL) and the combined
aqueous layers extracted with dichloromethane (2x 25 mL). The combined organic extracts were
washed with brine (50 mL), dried (MgSO4) and concentrated in vacuo. The residue was purified by flash
column chromatography (slow gradient 100:0 to 24:1 dichloromethane: methanol) to afford the title
compound (0.413 g, 76%) as a yellow oil. 1H NMR (500 mHz, CDCl3): δ 8.47 (d, J = 2.5Hz, 1H), 8.20-8.35
(brs, 1H), 7.70 (dd, J = 9, 2.5Hz, 1H), 6.74 (d, J = 9Hz, 1H), 5.85-5.94 (m, 1H), 5.09-5.24 (m, 2H), 3.92 (s,
3H), 3.72 (ddd, J = 28, 7.5, 2Hz, 1H), 3.43-3.50 (m, 1H), 3.29-3.37 (m, 1H), 3.11-3.18 (m, 1H), 2.86-2.95
(m, 1H), 2.66-2.78 (m, 1H), 2.22-2.32 (m, 1H), 1.87-1.97 (m, 1H), 1.59-1.76 (m, 3H); 13C NMR (125 mHz,
CDCl3): δ 164.05, 157.39, 140.97, 140.67, 136.10, 123.94, 116.79, 113.66, 83.60, 61.83, 57.03, 55.98,
54.29, 41.33, 28.42, 23.01.
(R)-tert-Butyl (1-allylpyrrolidin-3-yl)methyl(5-iodo-2-methoxybenzoyl)carbamate: 4.117
Triethylamine (0.258 mL, 1.88 mmol) was added to a solution of (S)-N-((1-allylpyrrolidin-3-yl)methyl)-5iodo-2-methoxybenzamide (0.250 g, 0.625 mmol), Boc anhydride (0.409 g, 0.625 mmol) and DMAP
(0.008 g, 0.063 mmol) in acetonitrile (10 mL) and heated to 70°C for 5 h. The solution was concentrated
in vacuo and purified by flash column chromatography (7:3 hexanes: ethyl acetate) to afford the title
compound (0.314 g, 74%) as an orange oil.
1
H NMR (500 mHz, CDCl3): δ 7.60-7.65 (m, 2H), 6.63 (d, J =
8.5Hz, 1H), 5.88-5.88 (m, 1H), 5.21 (dd, J = 17, 1.5Hz, 1H), 5.10 (d, J = 10Hz, 1H), 3.80-3.92 (m, 2H), 3.77
(s, 3H), 3.52-3.60 (m, 1H), 3.04-3.11 (m, 1H), 2.96 (dd, J = 13.5, 8Hz, 1H), 2.79-2.87 (m, 1H), 2.25-2.34 (m,
1H), 1.66-1.94 (m, 4H), 1.23 (s, 9H); 13C NMR (125 mHz, CDCl3): δ 168.78, 155.52, 153.33, 139.65, 137.07,
136.46, 130.68, 117.08, 112.86, 83.01, 82.48, 62.49, 58.26, 55.77, 54.20, 48.67, 29.37, 27.71, 23.30;
HRMS (ESI), m/z C21H30IN2O4 (M+H)+ calcd 501.1250, obsd 501.1260.
O
F
4-Iodo anisole (0.047 g, 0.2 mmol), 5-hexene-1-tosylsulfonate (0.093 mL, 2 equiv), TBAF (0.4
mL, 2 equiv), DIPEA (0.056 mL, 1.6 equiv) and 1,4 dioxane (1 mL) were introduced to a 5 mL
round bottom flask and degassed.
1,4 dioxane (1mL) was introduced to a CEM microwave tube and degassed.
Pd(OAc)2(0.0023 g, 5 mol%) and P(o-tolyl)3(0.0061 g, 10 mol%) were introduced followed by
the initial reactant mixture. The resulting solution was exposed to microwave irradiation (300W)
for 5 minutes2 at 900C2 and cooled to 250C. The reaction mixture was diluted with
dichloromethane (6mL), filtered through a plug of silica gel and concentrated before purification
by preparative thin layer chromatography (hexanes/ ethyl acetate = 9:1) to afford the title
compound (0.0233g, 56%) as a yellow oil. TLC (hexane/ethyl acetate = 9:1): Rf = 0.37; 1H NMR
(500MHz, CDCl3)): δ 7.24-7.29(d, J = 9 Hz, 2H), 6.81-6.85(d, J = 9 Hz, 2H), 6.28-6.38(d, J =
15.5 Hz, 1H), 6.00-6.10(dt, J = 15.5, 7 Hz, 1H), 4.38-4.55(dt, J = 47.5, 6 Hz, 2H), 3.78(s, 3H),
2.20-2.26(bt, J = 7 Hz, 2H), 1.67-1.90(m, 2H), 1.53-1.62(dt, J = 14.5, 7.5 Hz, 2H); 13C NMR
(125MHz, CDCl3): δ 158.82, 136.56, 113.82, 114.20, 107.46, 84.94, 83.63, 78.43, 55.54, 32.77,
30.25, 24.58.
O
F
(E)-1-(6-fluorohex-1-en-1-yl)-2-methoxybenzene
The title compound was isolated as a yellow oil (0.0216 g, 52%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.37; 1H NMR (500MHz, CDCl3)): δ 7.39-7.43(dd, J = 8, 1.5Hz, 1H), 7.16-7.22(td, J =
7.5, 1.5Hz, 1H), 6.87-6.94(bt, J = 7Hz, 1H), 6.82-6.87(bd, J = 8Hz, 1H), 6.68-6.76(d, J = 16Hz,
1H), 6.15-6.26(dt, J = 16.5, 6.5Hz, 1H), 4.39-4.55(dt, J = 47, 6Hz, 2H), 3.84(s, 3H), 2.212.33(bt, J = 7Hz, 2H), 1.68-183(m, 2H), 1.57-1.64(dt, J = 14.5, 8 Hz, 2H); 13C NMR (125MHz,
CDCl3): δ 156.54, 131.16, 128.19, 127.01, 126.66, 125.19, 120.89, 111.05, 84.96, 55.71, 30.27,
29.74, 25.29.
O
F
(E)-1-(6-fluorohex-1-en-1-yl)-3-methoxybenzene
The title compound was isolated as a yellow oil (0.0199 g, 48%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.37; 1H NMR (500MHz, CDCl3)): δ 7.18-7.24(t, J = 8 Hz, 1H), 6.91-6.96(bd, J = 7.5
Hz, 1H), 6.87(bs, 1H), 6.73-6.78(dd, J = 8.5, 3 Hz, 1H), 6.33-6.42(d, J = 16 Hz, 1H), 6.156.25(dt, J = 16, 6.5 Hz, 1H), 4.38-4.56(dt, J = 47, 6 Hz, 2H), 3.81(s, 3H), 2.22-2.30(bt, J = 6.5
Hz, 2H), 1.68-1.82(m, 2H), 1.52-1.66(dt, J = 15, 8 Hz, 2H); 13C NMR (125MHz, CDCl3): δ
159.81, 139.17, 130.57, 130.27, 129.47, 118.64, 112.59, 111.32, 84.46, 55.20, 32.47, 29.96,
24.94.
O
F
(E)-1-(2-(6-fluorohex-1-en-1-yl)phenyl)ethanone
The title compound was isolated as a yellow oil (0.0106 g, 24%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.40; 1H NMR (500MHz, CDCl3)): δ 7.57-7.62(d, J = 8 Hz, 2H), 7.49-7.53(bd, J = 7.5
Hz, 2H), 7.39-7.44(t, J = 8 Hz, 1H), 7.26-7.31(t, J = 7 Hz, 1H), 6.85-6.92(bd, J = 16.5 Hz,1H),
6.02-6.14(dt, J = 15.5, 7 Hz, 1H) 4.40-4.55(dt, J = 47.5, 6 Hz, 2H), 2.57(s, 3H), 2.24-2.33(bt, J =
7.5Hz, 2H), 1.70-1.83(m, 2H), 1.58- 1.66(dt, J = 14.5, 7.5 Hz, 2H); 13C NMR (125MHz, CDCl3):
δ 202.68, 137.70, 133.62, 131.66, 129.32, 128.90, 128.56, 127.80, 126.94, 84.88, 32.89, 30.24,
25.03.
O
F
(E)-1-(4-(6-fluorohex-1-en-1-yl)phenyl)ethanone
The title compound was isolated as a yellow oil (0.0097 g, 22%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.40; 1H NMR (500MHz, CDCl3)): δ 7.87-7.92(d, J = 8 Hz, 2H), 7.38-7.44(d, J = 8 Hz,
2H), 6.41-6.48(d, J = 15.5 Hz, 1H), 6.32-6.40(dt, J = 15.5, 7 Hz, 1H), 4.40-4.58(dt, J = 47, 6.5
Hz, 2H), 2.59(s, 3H), 2.27-2.34(bt, J = 7Hz, 2H), 1.69-1.83(m, 2H), 1.59- 1.67(dt, J = 15.5, 7.5
Hz, 2H); 13C NMR (125MHz, CDCl3): δ 197.61, 142.39, 135.64, 133.51, 129.58, 128.77,
125.99, 84.57, 32.65, 29.99, 26.56.
N
F
(E)-3-(6-fluorohex-1-en-1-yl)pyridine
The title compound was isolated as a yellow oil (0.0018 g, 5%). TLC (hexane/ethyl acetate =
7:3): Rf = 0.31; 1H NMR (500MHz, CDCl3)): δ 8.54-8.58(d, J = 2 Hz, 1H), 8.41-8.45(dd, J = 5,
1.5 Hz, 1H), 7.62-7.67(dt, J = 8.5, 2 Hz, 1H), 7.19-7.24(dd, J = 8, 5 Hz, 1H), 6.35-6.42(d, J = 16
Hz, 1H), 6.24-6.33(dt, J = 16, 6.5 Hz, 1H), 4.40-4.58(dt, J = 47.5, 6 Hz, 2H), 2.26-2.33(bt, J = 7
Hz, 2H), 1.70-1.83(m, 2H), 1.57-1.67(dt, J = 15.5, 7.5, 2H); 13C NMR (125MHz, CDCl3): δ
148.30, 148.21, 133.11, 132.98, 132.69, 127.12, 123.65, 84.80, 32.87, 30.22, 25.04.
NH2
F
(E)-2-(6-fluorohex-1-en-1-yl)aniline
The title compound was isolated as a red oil (0.0182 g, 47%). TLC (hexane/ethyl acetate = 7:3):
Rf = 0.34; 1H NMR (500MHz, CDCl3)): δ 7.20-7.25(dd, J = 8, 1.5 Hz, 1H), 7.02-7.08(td, J = 7.5,
1.5 Hz, 1H), 6.72-6.79(bt, J = 8 Hz, 1H), 6.65-6.70(bd, J = 8.5 Hz, 1H), 6.39-6.47(d, J = 15.5
Hx, 1H), 6.02-6.10(dt, J = 15.5, 6.5 Hz, 1H), 4.40-4.56(dt, J = 47.5, 6 Hz, 2H), 3.76(bs, 2H),
2.25-2.32(bt, J = 7.5, 2H), 1.70-1.83(m, 2H), 1.57-1.65(dt, J = 15, 8 Hz, 2H); 13C NMR
(125MHz, CDCl3): δ 143.30, 132.30, 130.62, 128.01, 127.35, 125.91, 118.97, 115.93, 84.66,
32.90, 29.99, 25.04.
NH2
F
(E)-3-(6-fluorohex-1-en-1-yl)aniline
The title compound was isolated as a red oil (0.0174 g, 45%), TLC (hexane/ethyl acetate = 7:3):
Rf = 0.34; 1H NMR (500MHz, CDCl3)): δ 7.05-7.11(t, J = 8 Hz, 1H), 6.73-6.78(d, J = 8 Hz, 1H),
6.67(bs, 1H), 6.52-6.57(dd, J = 7.5, 2 Hz, 1H), 6.27-6.35(d, J = 16 Hz, 1H), 6.10-6.20(dt, J = 16,
6.5 Hz, 1H), 4.38-4.54(dt, J = 47.5, 6 Hz, 2H), 3.63(bs, 2H), 2.21-2.28(bt, J = 8 Hz, 2H), 1.681.81(m, 2H), 1.54-1.63(dt, J = 15, 7.5 Hz, 2H); 13C NMR (125MHz, CDCl3): δ 146.77, 139.04
129.34, 126.72, 116.94, 114.23, 112.78, 84.93, 78.55, 32.73, 30.21, 25.21.
H2N
F
(E)-4-(6-fluorohex-1-en-1-yl)aniline
The title compound was isolated as a red oil (0.0166 g, 43%). TLC (hexane/ethyl acetate = 7:3):
Rf = 0.34; 1H NMR (500MHz, CDCl3)): δ 7.13-7.17(d, J = 8.5 Hz, 2H), 6.60-6.65(d, J = 9 Hz,
2H), 6.25-6.33(d, J = 16, 1H), 5.96-6.04(dt, J = 16, 7 Hz, 1H), 4.39-4.53(dt, J = 47.5, 6 Hz, 2H),
3.70(bs, 2H), 2.19-2.25(bt, J = 8, 2H), 1.70-1.83(m, 2H), 1.53-1.61(dt, J = 15.5, 8 Hz, 2H); 13C
NMR (125MHz, CDCl3): δ 145.40, 135.40, 130.06, 127.02, 126.49, 115.17, 84.37, 32.48, 29.96,
25.11.
F
(E)-(6-fluorohex-1-en-1-yl)benzene
The title compound was isolated as a clear oil (0.0125 g, 35%)TLC (hexane/ethyl acetate = 9:1):
Rf = 0.59; 1H NMR (500MHz, CDCl3)): δ 7.25-7.40(m, 4H), 7.16-7.23(m, 1H), 6.36-6.43(d, J =
15.5, 1H), 6.16-6.25(dt, J = 15.5, 7 Hz, 1H), 4.40-4.54(dt, J = 47, 6.5 Hz, 1H), 2.23-2.30(bt, J =
7 Hz, 2H), 1.69-1.81(m, 2H), 1.55-1.64(dt, J = 15, 8.5 Hz, 2H); 13C NMR (125MHz, CDCl3): δ
137.94, 130.62, 130.46, 128.74, 127.18, 129.19, 84.91, 32.76, 30.22, 25.17.
O
F
(E)-1-(5-fluoropent-1-en-1-yl)-4-methoxybenzene
The title compound was isolated as a yellow oil (0.0179 g, 46%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.40; 1H NMR (500MHz, CDCl3)): δ 7.26-7.29(d, J = 9 Hz, 2H), 6.82-6.87(d, J = 9 Hz,
2H), 6.34-6.41(d, J = 16.5 Hz, 1H), 6.02-6.10(dt, J = 15.5, 7 Hz, 1H), 4.42-4.58(dt, J = 47.5, 5.5
Hz, 2H), 3.80(bs, 3H), 2.29-2.35(bt, J = 7 Hz, 2H), 1.80-1.94(m, 2H); 13C NMR (125MHz,
CDCl3): δ 159.06, 130.45, 127.49, 127.33, 127.14, 114.20, 85.54, 55.55, 30.94, 19.51.
All reactions were conducted in a 2.4 GHz CEM brand microwave reactor, using a maximum
power of 300 watts, at a maximum pressure of 300 psi. All run times were 1 minute. All reagent
solvents were purchased from the SigmaAldrich Chemical Company and dried prior to use over
sodium with a benzophenone indicator. Et3N and DIPEA were dried and distilled prior to use1.
Dichloro-bis(triphenylphosphine)palladium (II) was purchased from Strem Chemicals and used
without further purification. Copper Iodide was purified according to literature proceedures1.
Hex-5-yn-1-yl 4-methylbenzenesulfonate and pent-4-yn-1-yl 4-methylbenzenesulfonate were
synthesized from the corresponding alcohols, according to literature proceedure2. All other
reagents were used without further purification. All TLC analysis was done on Whatman UVfluorescent 250 µm layer plates. Column chromatography conducted using 230-400 mesh silica
gel. All reactions were performed under an Argon atmosphere and sealed with teflon tape. All
NMR spectra was run on an INOVA-500 Waters spectrometer, operating at the cited frequency.
O
F
1-(4-(6-fluorohex-1-yn-1-yl)phenyl)ethanone
4-Iodo Acetophenone (0.123 g, 0.5 mmol), Hex-5-yn-1-yl 4-methylbenzenesulfonate (0.127 mL,
1.1 equiv), TBAF ( 1.1 mL, 2.2 equiv), THF (0.9 mL) and Et3N (0.1 mL) were added to a CEM
microwave tube and degassed. Pd(PPh3)2Cl2 (0.0175 g, 5 mol%) and CuI (0.0048 g, 5 mol%)
were added and the resulting solution was exposed to microwave irradiation (300W) for 10
minutes at 700C and cooled to 250C. The crude product was diluted with dichloromethane(6 mL),
filtered through a plug of silica gel and concentrated before purification by column
chromatography (100% hexanes) to afford the title compound (0.099 g, 91%) as a yellow oil.
TLC (hexane/ethyl acetate = 9:1): Rf = 0.41; 1H NMR (500MHz, CDCl3): ä 7.86-7.90 (d, J = 8.5
Hz, 2H), 7.44-7.48 (d, J = 8Hz, 2H), 4.45-4.60 (dt, J = 5.5Hz, 47.5Hz, 2H), 2.59 (s, 3H), 2.492.53 (t, J = 6.5Hz, 2H), 1.83-2.05 (m, 2H) 1.70-1.81(dt, 6.5Hz, 15.5Hz, 2H); 13C NMR
(125MHz, CDCl3): δ 197.63, 131.92, 128.44, 93.58, 84.50, 80.87, 29.92, 26.85, 24.61.
O
F
1-(2-(6-fluorohex-1-yn-1-yl)phenyl)ethanone:
The title compound was isolated as a yellow oil (0.104 g, 93%).TLC (hexane/ethyl acetate =
9:1): Rf = 0.41;1H NMR (500MHz, CDCl3): δ 7.64-7.70 (dd, J = 1 Hz, 8 Hz, 1H), 7.46-7.52 (dd,
J = 1Hz, 8Hz, 1H), 7.38-7.43(td, J = 1.5Hz, 7.5Hz, 1H), 7.30-7.36 (td, J = 1.5Hz, 7Hz, 1H), 4.454.60 (dt, J = 6Hz, 47Hz, 2H), 2.70(s, 3H), 2.50-2.56(t, J = 7Hz, 2H), 1.83-1.95 (m, 2H) 1.731.82(dt, 7Hz, 15Hz, 2H); 13C NMR (125MHz, CDCl3): δ 201.09, 141.24, 134.30, 131.36,
128.61, 127.93, 122.42, 95.94, 84.51, 80.41, 30.22, 29.81, 24.61.
O
F
1-(3-(6-fluorohex-1-yn-1-yl)phenyl)ethanone:
The title compound was isolated as a yellow oil (0.096 g, 88%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.41;1H NMR (500MHz, CDCl3): δ 7.96 (bs,1H), 7.84-7.89 (bd, J = 8Hz, 1H), 7.557.60(bd, J = 8Hz, 1H), 7.36-7.42 (t, J = 8Hz, 1H), 4.42-4.60 (dt, J = 6Hz, 47.5Hz, 2H), 2.60(s,
3H), 2.46-2.52(t, J = 7Hz, 2H), 1.82-2.06 (m, 2H) 1.72-1.82(dt, 6.5Hz, 15.5Hz, 2H); 13C NMR
(125MHz, CDCl3): δ 197.79, 137.36, 136.13, 131.79, 128.82, 127.54, 124.71, 90.95, 84.53,
78.45, 32.54, 29.77, 26.91.
N
F
2-(6-fluorohex-1-yn-1-yl)pyridine
The title compound was isolated as a yellow oil (0.080 g, 90%). TLC (hexane/ethyl acetate =
7:3): Rf = 0.34; 1H NMR (500MHz, CDCl3): δ 8.52-8.58 (bd, 5Hz, 1H), 7.58-7.65 (td, J = 2Hz,
8Hz, 1H), 7.35-7.40(d, J = 7.5Hz, 1H), 7.16-7.23 (dd, J = 5Hz, 7.5Hz, 1H), 4.42-4.60 (dt, J =
5.5Hz, 47.5Hz, 2H), 2.49-2.55(t, J = 7.5Hz, 2H), 1.83-2.02 (m, 2H) 1.73-1.83(dt, 7Hz, 14.6Hz,
2H); 13C NMR (125MHz, CDCl3): δ 150.12, 144.01, 136.31, 127.06, 122.64, 84.51, 78.50,
76.99, 31.83, 29.91, 24.39.
N
F
3-(6-fluorohex-1-yn-1-yl)pyridine
The title compound was isolated as a yellow oil (0.081 g, 91%). TLC (hexane/ethyl acetate =
7:3): Rf = 0.34; 1H NMR (500MHz, CDCl3): δ 8.60-8.65 (bd, 1.5Hz, 1H), 8.46-8.52 (dd, J =
2Hz, 4.5Hz, 1H), 7.62-7.7.72(dt, J = 2Hz, 7.5Hz, 1H), 7.18-7.24 (dd, J = 5Hz, 7.5Hz, 1H), 4.424.60 (dt, J = 6Hz, 47.5Hz, 2H), 2.47-2.53(t, J = 7Hz, 2H), 1.82-2.00 (m, 2H) 1.72-1.81(dt, 6.5Hz,
15.5Hz, 2H); 13C NMR (125MHz, CDCl3): δ 152.57, 148.29, 138.71, 123.17, 121.19, 93.36,
84.48, 78.50, 29.90, 29.75, 24.64.
N
F
4-(6-fluorohex-1-yn-1-yl)pyridine
The title compound was isolated as a yellow oil (0.079 g, 89%). TLC (hexane/ethyl acetate =
7:3): Rf = 0.34; 1H NMR (500MHz, CDCl3): δ 8.51-8.55 (bd, 5Hz, 2H), 7.23-7.26(bd, J = 5Hz,
1H), 4.42-4.60 (dt, J = 6Hz, 46.5Hz, 2H), 2.46-2.54(t, J = 6.5Hz, 2H), 1.81-2.04 (m, 2H) 1.701.81(dt, 6.5Hz, 14Hz, 2H); 13C NMR (125MHz, CDCl3): δ 149.92, 126.03, 95.22, 84.41, 79.15,
31.83, 29.88, 24.48.
O
F
1-(6-fluorohex-1-yn-1-yl)-2-methoxybenzene
The title compound was isolated as a yellow oil (0.081 g, 79%)TLC (hexane/ethyl acetate = 9:1):
Rf = 0.39; 1H NMR (500MHz, CDCl3): δ 7.34-7.39 (dd, 1.5Hz, 6Hz, 1H), 7.21-7.27 (dd, J =
6.5Hz, 1H), 6.79-6.91 (m, 2H), 4.43-4.59 (dt, J = 5.5Hz, 47.5Hz, 2H), 3.86(s, 3H), 2.49-2.57(t, J
= 7Hz, 2H), 1.83-1.97 (m, 2H) 1.71-1.79(dt, 6.5Hz, 16Hz, 2H); 13C NMR (125MHz, CDCl3): δ
160.13, 133.84, 129.29, 120.66, 110.79, 107.74, 93.92, 84.63, 78.44, 56.51, 30.94, 29.91, 24.79.
O
F
1-(6-fluorohex-1-yn-1-yl)-3-methoxybenzene
The title compound was isolated as a yellow oil (0.085 g, 82%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.39;1H NMR (500MHz, CDCl3): δ 7.16-7.22 (bt, 8Hz, 1H), 6.96-7.01(bd, J = 7.5Hz,
1H), 6.92 (bs, 1H), 6.81-6.86 (dd, 3Hz, 8.5Hz, 1H), 4.41-4.60 (dt, J = 5.5Hz, 47Hz, 2H), 3.79(s,
3H), 2.44-2.51(t, J = 7Hz, 2H), 1.82-1.95 (m, 2H) 1.70-1.78(dt, 7Hz, 15Hz, 2H); 13C NMR
(125MHz, CDCl3): δ 159.50, 129.48, 125.04, 124.31, 116.63, 114.40, 89.52, 84.53, 78.41, 55.45,
30.87, 29.87, 24.71.
O
F
1-(6-fluorohex-1-yn-1-yl)-4-methoxybenzene
The title compound was isolated as a yellow oil (0.075 g, 73%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.39; 1H NMR (500MHz, CDCl3): δ 7.28-7.36(d, J = 7Hz, 2H), 6.78-6.84(d, J = 6.5Hz,
2H), 4.40-4.60 (dt, J = 6Hz, 47.5Hz, 2H), 3.79(s, 3H), 2.40-2.48(t, J = 7Hz, 2H), 1.80-2.05 (m,
2H) 1.66-1.79(dt, 7.5Hz, 14.5Hz, 2H); 13C NMR (125MHz, CDCl3): δ 159.37, 133.14, 116.23,
114.08, 88.04, 84.61, 81.09, 55.51, 32.97, 29.93, 24.82.
F
(6-fluorohex-1-yn-1-yl)benzene
The title compound was isolated as a clear oil (0.076 g, 86%)TLC (hexane/ethyl acetate = 9:1):
Rf = 0.56; 1H NMR (500MHz, CDCl3)): δ 7.36-7.42(m, 2H), 7.24-7.31(m, 3H), 4.42-4.58(dt, J =
47.5, 6Hz, 2H), 2.44-2.50(t, 7Hz, 2H), 1.81-1.95(m, 2H), 1.68-1.78(dt, 15, 7.5Hz, 2H); 13C NMR
(125MHz, CDCl3): δ 131.79, 128.47, 127.89, 124.09, 89.67, 84.57, 81.39, 31.17, 29.92, 24.78.
O
F
1-(2-(5-fluoropent-1-yn-1-yl)phenyl)ethanone
The title compound was isolated as a yellow oil (0.074 g, 91%). TLC (hexane/ethyl acetate =
9:1): Rf = 0.41;1H NMR (500MHz, CDCl3)): δ 7.65-7.71(dd, J = 10, 1Hz, 1H), 7.48-7.55(dd, J =
7.5, 0.5Hz, 1H), 7.39-7.44(td, J = 7.5, 1.5Hz, 1H), 7.32-7.39(td, J = 7.5, 1Hz, 1H), 4.55-4.70(dt,
J = 47, 5.5Hz, 2H), 2.70(s, 1H), 2.60-2.66(t, J= 6.5Hz, 2H), 1.96-2.07(m, 2H); 13C NMR
(125MHz, CDCl3): δ 201.22, 137.70, 133.91, 132.36, 132.22, 129.83, 128.81, 90.07, 84.32,
80.94, 29.51, 29.62, 14.39.
Procedure 1: synthesis of Bromodimethylsulfonium Bromide (BDMS):
Br
Br2. CH2Cl2
S
S
Br
Dimethyl sulfide (1.5 g, 25 mmol) was dissolved in freshly distilled dichloromethane (5 mL). A
solution of bromine (1.997 g, 25 mmol) in dichloromethane (5 mL) and added into the above
solution at 0oC over 5 min. The orange precipitate was collected by vacuum filtration washed
with hexane and dried in vacuo to yield the title compound (4.3 g, 77%) as a yellow solid. mp
80-83 °C.1
O
O
O
NH2
N
N
NH2
BDMS
N
O
N
H
N
N
Procedure 2: synthesis of xanthines, 1,3-diethyl-8-phenyl-1H-purine-2,6(3H,7H)-dione :
BDMS (0.25 mmol) was added to a solution of benzaldehyde (0.053 g, 0.25 mmol) and 1, 3diethyl-5,6-aminouracil (1) (0.025 g, 0.25 mmol) in freshly distilled acetonitrile (5 mL). The
reaction mixture was stirred at 25°C for 4h. The precipitate was collected via filtration, washed
1
Khan, A.T., Ali, M.A. Goswami, P., Choudhury L.H., J. Org. Chem. 2006, 71, 8961.
with ethyl acetate ( 25 mL) and dried in vacuo. The crude solid was purified by recrystallization
from DMSO and water to yield the title compound (0.050 g, 70%) as a white solid. mp > 300
ºC ; 1H NMR (500 MHz, DMSO-d6): δ 1.16 (t, J = 7Hz, 3H), 1.26 (t, J = 7Hz, 3H), 3.97 (q, J =
7Hz, 2H), 4.10 (q, J = 7Hz, 2H), 7.44-56 (m, 3H), 8.14 (d, J = 8Hz, 2H), 13.90 (br, 1H). HRMS
(ESI), C15H17N4O2 m/z (M+H)+: calcd 284.1273, obsd 284.1277
Procedure 3: microwave accelerated coupling of xanthines: 8-(4-bromophenyl)-1,3-diethylO
N
O
O
O
NH2
N
H
N
NH2
Br
O
H
N
Br
N
N
1H-purine-2,6(3H,7H)-dione:
(Bromodimethyl) sulfonium bromide (0.010 g, 0.045 mmol) was added to a mixture of 4bromobenzaldehyde (0.0466 g, 0.25 mmol), 1,3-diethyl-5,6-diaminouracil (0.4955 g, 0.25 mol)
and anhydrous acetonitrile (500 µL) in a 14 x 86 mm (o.d.) glass microwave tube. The tube was
capped with a CEM Corp. PL cap, the atmosphere was flushed with argon gas and then the tube
was placed in the cavity of a CEM discover Lab Mate reactor. The solution was subjected to
microwave irradiation while stirring, the temperature was brought to 110 °C over 15 min. and
then held for 10 min (150 W, 100 psi). The resulting precipitate was filtered, washed with ethyl
acetate (5 mL) and methanol (2.5 mL) and then recrystallized from ethyl acetate to yield the title
compound (0.065g, 72%) as a yellow solid. mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ
1.13 (t, J = 7Hz, 3H), 1.26 (t, J = 7Hz, 3H), 3.94 (q, J = 7Hz, 2H), 4.08 (q, J = 7Hz, 2H), 7.01 (d,
J = 8.5Hz, 2H), 8.05 (d, J = 8.5Hz, 2H), 13.89 (br, 1H). HRMS (ESI), C15H16BrN4O2 m/z
(M+H)+: calcd 362.0378, obsd 362.0369.
O
O
NH2
N
O
N
NH2
O
H
HO
N
O
N
Br
H
N
N
HO
Procedure 4 : 8-(5-bromo-2-hydroxyphenyl)1,3-diethyl-1H-purine-2,6(3H,7H)-dione :
(Bromodimethyl) sulfonium bromide (0.879 g, 4.0 mmol) was added (in two portions) to a
mixture of p-methoxybenzaldehyde (0.3053 g, 2.5 mmol) and 1,3,-diethyl-5,6-diaminouracil
(0.495 g 2.5 mmol) in acetonitrile (5 mL). The reaction mixture was stirred at room temperature
for 14 hours. The precipitate formed was collected by vacuum filtration, washed with ethyl
acetate and dried in vacuo. The crude solid was purified by recrystallization from DMSO and
water to yield the title compound (0.66 g, 73%) as a white solid. mp 315-317 °C; TLC
(dichloromethane/ methanol = 19:1): Rf 0.30; 1H NMR (500 MHz, d6-DMSO): δ 13.80-14.20
(brs, 1H,), 11.75-12.10 (brs, 1H), 8.29 (d, J = 2.5 Hz, 1H), 7.80 (dd, J = 8.5, 2.5 Hz, 1H), 6.97 (d,
J = 8.5 Hz, 1H), 4.08 (q, J = 7.5 Hz, 2H), 3.96 (q, J = 7.5 Hz, 2H), 1.28 (q, J = 7.5 Hz, 3H), 1.15
(q, J = 7.5 Hz, 3H); 13C NMR (125 mHz, d6-DMSO): δ 156.6, 154.4, 150.7, 150.6, 148.1, 135.1,
129.7, 120.2, 118.0, 115.3, 111.3, 55.6, 39.2, 36.7, 13.9; HRMS (ESI) m/z C15H16BrN4O3
(M+H)+: calcd 379.0406, obsd 379.0401.
Procedure 5: 4,5-Dimethoxy-cinnimaldehyde:
O
H3CO
H3CO
OH
1. SOCL2, MeOH
2. 2.2 eq DIBAL-H
3. PCC
99% (3 steps_
O
H3CO
H
H3CO
4,5-Dimethoxycinnamic acid (2.96 g, 11.7 mmol) was dissolved in 60 mL dry methanol. Thionyl
Chloride (0.893 mL, 1.05 eq) added dropwise. Mixture refluxed for 30 minutes. 1.7 mL
triethylamine added to neutralize. Reaction allowed to reflux for an additional 30 minutes,
cooled and the solvent was removed in vacuo. Purification via flash chromatography provided
3.06 g (98%) as a yellow solid.
4,5-Dimethoxy-methylcinnimate (3.0g , 11.23 mmol) dissolved in anhydrous Dichloromethane
(180 mL). Reaction cooled to -78°C and Diisobutyl aluminum hydride (28 mL, 1M in Hex, 2.5
eq) was added over one hour. Reaction quenched with 1.2 mL of methanol then 1 mL of water at
-60°C. Precipitated salts filtered off, then dissolved in 1% Hydrochloric acid. Extracted with
dichloromethane (4 x 200 mL). All organic layers collected and washed with water (100 mL)
and brine (100 mL) then concentrated in vacuo and used directly in the next step without
purification. 1H NMR (500 MHz, CDCl3): 2.2 (s, broad, 1H) 3.99 (s., 3H), 4.0 (s, 3H), 4.38 (d,
2H) 6.25 (dt, 1H) 6.9 (s, 1H), 7.2 (d, 1H) 7.47 (s, 1H); The crude reaction mixture was dissolved
in 110 mL of dry dichloromethane, this solution was added dropwise to a solution of pyridinium
chlorochromate (2.5231 g) in 170 mL of dry dichloromethane. The mixture was stirred
overnight. An additional 20 mol % of PCC was added and allowed to stir an additional hour. The
solution was filtered through a plug of fluoricil, concentrated in vacuo and purified via flash
chromatography to provide 2.037 g (73.4%) of a yellow solid. m.p. 82-83°C 1H NMR (300 MHz,
CDCI,) 9.67 (d, J = 7.8 Hz, CHO), 7.42 (d, J = 15.8 Hz, 1 H), 7.17-6.91 (m, 3 H) 6.62 (dd, J =
15.8, 7.7 Hz, 1H) 3.94-3.93 (s, 6 H); 13C (300 MHz, CDC1,) 6 193.6, 152.8, 152.5 127.1, 126.7,
123.4, 111.1, 110.1, 109.0, 56.0, 55.9 .2
Procedure 6: (E)-1,3-diethyl-8-(4-hydroxystyryl)-7-methyl-1H-purine-2,6(3H,7H)-dione:
OCH3
O
N
N
O
N
N
N
N
OH
O
O
N
N
A 1M BBr3 in CH2Cl2 (170 μl, 0.85 mmol) was added to a solution of (E)-1,3-diethyl-8-(4methoxystyryl)-7-methyl-1H-purine-2,6(3H,7H)-dione3 (100 mg, 0.28 mmol) in dry CH2Cl2
(0.35 mL). The reaction mixture was stirred at room temperature for 3 h and diluted with water
(0.3 mL). The formed precipitate collected via vacuum filteration, washed with excess water and
dried in vacuo to give the title compound as a yellow solid. (90 mg, 95%) mp > 300 °C; TLC
(MeOH: CH2Cl2 = 1: 9): Rf 0.2 1H NMR (500 MHz, DMSO-d6): δ 1.12 (t, J = 7 Hz, 3H), 1.25
(t, J = 7 Hz, 3H), 3.91 (q, J = 7.5 Hz, 2H), 3.99 (s, 3H), 4.06 (q, J = 7.5 Hz, 2H), 6.80 (d, J = 9
Hz, 2H), 7.11 (d, J = 16 Hz, 1H), 7.58 (d, J = 16 Hz, 1H), 7.62 (d, J = 9 Hz, 2H). 13C NMR (300
MHz, DMSO-d6): δ 51.1, 69.2, 73.3, 75.6, 144.9, 147.1, 153.6, 164.6, 167.3, 174.8, 185.4,
187.9, 188.3, 191.8, 196.7 HRMS (ESI), m/z C18H21N4O3 (M+H)+: calcd 340.1535, obsd
340.1549
2
J. Org. Chem, 1990 55 , 3679
3
Synthesized according to EP 0590919.
N
N
O
N
O
O
O
N
N
N
Br
O
N
N
Procedure 7: 8(3'-acetylbiphenyl-4-yl)-1,3-diethyl-1H-purine-2,6(3H,7H)-dione
Pd(PPh3)4 (0.0017 g, 3mol%) was added to a degassed solution of 8-(4-bromophenyl)1,3-diethyl-1H-purine-2,6(3H,7H)-dione (0.020 g, 0.053 mmol), 4-acetylphenylboronic acid
(0.0095 g, 1.1 equiv) and barium hydroxide octahydrate (0.025 g, 1.5 equiv) in dimethoxyethane
(DME) (2 mL) and water (0.5 mL) in a CEM microwave tube. The resulting solution was
exposed to microwave irradiation (300W) for 10 minutes at 100 °C and cooled to 25 °C. The
reaction mixture was diluted with dichloromethane (10 mL) and filtered through a plug of silica
gel. The filtrate was diluted with water (10 mL) and extracted with dichloromethane (3x, 10
mL). The combined extracts were washed with brine (20 mL), dried with MgSO4 and
concentrated in vacuo. The crude product was purified by preparative thin layer chromatography
(hexanes/ ethyl acetate = 3:2) to afford the title compound (0.016 g, 73%) as a white solid. Mp =
182-184 °C; TLC (hexane/ ethyl acetate = 1:1): Rf 0.36; 1H NMR (500mHz, CDCl3): δ 8.22-8.25
(m, 1H), 7.97-8.01 (m, 1H), 7.76-7.87 (m, 5H), 7.60 (t, J = 7.5 Hz, 1H), 4.24 (q, J = 7 Hz, 2H),
4.13 (q, J = 7 Hz, 2H), 4.12 (s, 3H), 2.68 (s, 3H), 1.40 (t, J = 7 Hz, 3H), 1.29 (t, J = 7 Hz, 3H);
13
C NMR (125 mHz, CDCl3): δ 198.90 155.6, 151.7, 150.9, 148.1, 142.2, 140.6, 138.0, 131.8,
129.9, 129.5, 128.13, 128.09, 127.8, 127.0, 109.1, 77.4, 38.6, 36.5, 34.1, 26.9, 13.6, 13.5; HRMS
(ESI) m/z C24H25N4O3 (M+H)+: calcd 417.1927, obsd 417.1919.
Procedure 8: 8-(5-bromo-2-methoxyphenyl)-1,3-diethyl-7-methyl-1H-purine-2,6(3H,7H)dione
O
N
O
N
O
Br
H
N
N
N
HO
O
Br
N
N
N
H3CO
Methyl iodide (36.5 µL, 3 equiv) was added to a solution of 8-(5-bromo-2hydroxyphenyl)-1,3-diethyl-1H-purine-2,6(3H,7H)-dione (0.074 g, 0.194 mmol) and potassium
carbonate (0.085 g, 3 equiv) in dimethylformamide (4 mL) at 25 °C (Ar atmosphere) then heated
to 60°C overnight. The resulting solution was diluted with water (20 mL) and acidified to pH ~2
by the slow addition of 5% HCl. The aqueous layer was extracted with chloroform (3x 20 mL),
dried over MgSO4 and concentrated in vacuo to afford the title compound (0.065 g, 82%) as a
yellow solid. Mp = 159-161 °C; TLC (Hexane/ ethyl acetate = 1:1): Rf 0.28; 1H NMR (500mHz,
CDCl3): δ 7.57-7.63 (m, 2H), 6.92 (d, J = 8.5 Hz, 1H), 4.20 (q, J = 7 Hz, 2H), 4.11 (q, J = 7 Hz,
2H), 3.85 (s, 3H), 3.80 (s, 3H), 1.37 (t, J = 7 Hz, 3H), 1.28 (t, J = 7 Hz, 3H); 13C NMR (125
mHz, CDCl3): δ 156.6, 155.4, 150.8, 148.7, 147.8, 134.96, 134.76, 119.7, 113.4, 113.1, 108.8,
55.1, 38.6, 36.5, 33.3, 13.6, 13.4; HRMS (ESI) m/z C17H20BrN4O3 (M+H)+: calcd 407.0719,
obsd 407.0714.
Procedure 9: 8-(5-bromo-2-hydroxyphenyl)-1,3-diethyl-7-methyl-1H-purine-2,6(3H,7H)dione
O
N
N
O
O
Br
N
N
H3CO
N
O
Br
N
N
N
HO
Boron tribromide (32
µL, 3 equiv) was added to a solution of 8-(5-bromo-2-methoxyphenyl)-1,3-diethyl-7-methyl-1Hpurine-2,6(3H,7H)-dione (0.046g, 0.113mmol) in dichloromethane (2 mL) at 25°C and stirred
for 2h. The resulting solution was diluted with water (10 mL) and extacted with
dichloromethane (3x 10mL). The combined extract were washed with brine (10 mL), dried with
MgSO4, and concentrated in vacuo to afford the title compound (0.0417g, 94%) as a yellow
solid. Mp = 176-177 °C; TLC neutralized with triethylamine (ethyl acetate): Rf 0.22; 1H NMR
(500mHz, CDCl3): δ 10.8-11.8 (brs, 1H), 7.72 (d, J = 2 Hz, 1H), 7.47 (dd, J = 9, 2 Hz, 1H), 7.02
(d, J = 9 Hz, 1H), 4.27 (s, 3H), 4.18 (q, J = 7 Hz, 2H), 4.11 (q, J = 7 Hz, 2H), 1.37 (t, J = 7 Hz,
3H), 1.28 (t, J = 7 Hz, 3H); 13C NMR (125 mHz, CDCl3): δ 157.18, 155.14, 150.46, 147.94,
145.73, 135.15, 129.41, 120.30, 113.87, 111.18, 108.23, 38.97, 36.84, 35.22, 13.53, 13.40;
HRMS (ESI) m/z C16H18BrN4O3 (M+H)+: calcd 393.0562, obsd 393.0548.
Procedure 10: 8-(2-(benzyloxy)-5-bromophenyl)-1,3-diethyl-7-methyl-1H-purine2,6(3H,7H)-dione
O
N
O
N
HO
Br
N
N
N
N
O
O
Br
N
N
O
Benzyl bromide (7.7
µL, 1.5 equiv) was added to a solution of 8-(5-bromo-2-hydroxyphenyl)-1,3-diethyl-7-methyl1H-purine-2,6(3H,7H)-dione (0.017g, 0.0432mmol) and potassium carbonate (0.018g, 3 equiv)
in dimethylformamide (2 mL) at 25°C. The resulting solution was heated to 70°C for 3h and
cooled to 25°C. The solution was diluted with water (10 mL) and brought to pH ~ 2 by the slow
addition of 5% HCl. The aqueous layer was extracted with dichloromethane (3x 10mL) and the
combined extracts washed with brine (10 mL), dried with MgSO4 and concentrated in vacuo.
The crude residue was purified by preparative thin layer chromatography (hexanes/ ethyl acetate
= 3:1) to afford the title compound (0.0194g, 93%) as a yellow solid. Mp = 56-57 °C; TLC
(hexane/ ethyl acetate = 1:1): Rf 0.48; 1H NMR (500mHz, CDCl3): δ 7.59 (d, J = 2Hz, 1H), 7.55
(dd, J = 8.5, 2 Hz, 1H), 7.23-7.39 (m, 5H), 6.96 (d, J = 8.5 Hz, 1H), 5.11 (s, 2H), 4.20 (q, J = 7
Hz, 2H), 4.10 (q, J = 7 Hz, 1H), 3.80 (s, 3H), 1.38 (t, J = 7 Hz, 3H), 1.27 (t, J = 7 Hz, 3H); 13C
NMR (125 mHz, CDCl3): δ 155.94, 155.43, 150.87, 148.70, 147.86, 135.68, 135.01, 134.91,
128.93, 128.56, 127.32, 120.44, 115.01, 113.80, 108.81, 71.38, 38.65, 36.56, 33.52, 13.67,
13.49; HRMS (ESI) m/z C23H24BrN4O3 (M+H)+: calcd 483.1032, obsd 483.1026.
O
N
O
O
N
N
N
N
N
NO2
O
N
N
F
Procedure 11: 1,3-diethyl-8-(4-fluorophenyl)-7-methyl-1H-purine-2,6(3H,7H)-dione
Anhydrous TBAF (1M in DMSO, 3 equiv, 262 µL) was added to a solution of 1,3-diethyl-7methyl-8-(4-nitrophenyl)-1H-purine-2,6(3H,7H)-dione (0.030g, 0.0873mmol) in DMSO (0.5
mL) and exposed to microwave irradiation (300W) at 180 °C for 10 minutes and cooled to 25
°C. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane
(3x 10 mL). The combined extracts were washed with brine (10 mL), dried over MgSO4 and
concentrated in vacuo. The crude residue was purified by preparative thin layer chromatography
(hexanes/ ethyl acetate = 3:1) to afford the title compound (0.0031g, 11%) as a white solid and
recovered starting material (0.0189g, 63%). Mp = 162-165 °C; TLC (hexane/ ethyl acetate =
4:1): Rf 0.27; 1H NMR (500mHz, CDCl3): δ 7.65-7.71 (m, 2H), 7.23 (t, J = 8.5 Hz, 2H), 4.21 (q,
J = 7 Hz, 2H), 4.11 (q, J = 7 Hz, 2H), 4.05 (s, 3H), 1.37 (t, J = 7 Hz, 3H), 1.27 (t, J = 7 Hz, 3H);
13
C NMR (125 mHz, CDCl3): δ 164.96, 162.96, 155.52, 151.20, 150.85, 147.91, 131.47, 131.40,
124.84, 116.39, 116.22, 108.92, 38.59, 36.56, 33.93, 13.61, 13.48; HRMS (ESI) m/z
C16H18FN4O2 (M+H)+: calcd 317.1414, obsd 317.1402.
Procedure 12: (E)-8-[2-(3,4-Dimethoxyphenyl) vinyl]-1,3-diethyl-7-methyl-3, 7
O
N
O
N
O
H
N
N
N
N
OCH3
OCH3
O
N
N
OCH3
OCH3
dihydropurine-2,6-dione (KW 6002). 4
K2CO3 (1.35g, 9.43mmol) was added to a solution of compound 3-5 (2.02g, 5.45mmol) in dry
DMF (27mL). Iodomethane (0.68mL, 10.77mmol) was added and the reaction mixture was
stirred at room temperature for 1h. The formed precipitate was filtered off. The filtrate was
diluted with water (30 mL) and the resulting mixture was extracted with chloroform (3°—100
mL). The organic extracts were washed with water (100 mL) and brine (100 mL), dried with
MgSO4 and evaporated in vacuo. The residue was purified via flash chromatography to give the
title compound as a yellow solid. (2.0g, 95%) mp=191-195 °C; TLC (hexanes: ethyl acetate = 3:
2): Rf 0.22 1H NMR (500 MHz, CDCl3): R 1.23 (t, J = 7 Hz, 3H), 1.36 (t, J = 7 Hz, 3H), 3.90 (s,
3H,), 3.93 (s, 3H), 4.03 (s, 3H), 4.07 (q, J = 7 Hz, 2H), 4.18 (q, J = 7 Hz, 2H), 6.74 (d, J = 15.8
Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 7.06 (d, J = 1.9 Hz, 1H), 7.15 (dd, J = 8.2, 1.9 Hz, 1H), 7.70
(d, J = 15.8 Hz, 1H) 13C NMR (300 MHz, CDCl3): R 13.3, 13.4, 31.5, 36.3, 38.4, 55.9, 56.0,
108.0, 109.3, 109.5, 111.2, 121.2, 128.6, 138.1, 148.2, 149.2, 150.2, 150.4, 150.7, 155.0 HRMS
(ESI), m/z (M+H)+: calcd 384.1798, obsd 384.1789. Elemental Analysis: (C20H24N4O4) calcd
(%): C, 62.48; H, 6.29; N, 14.57; found (%): C, 62.45; H, 6.39; N, 14.55
4
EP 0590919.
O
H
N
N
O
Br
N
N
8-(4-bromophenyl)-1,3-diethyl-1H-purine-2,6(3H,7H)-dione:
Procedure 2: yellow solid. (72 mg, 79%) mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): d 1.13
(t, J = 7Hz, 3H), 1.26 (t, J = 7Hz, 3H), 3.94 (q, J = 7Hz, 2H), 4.08 (q, J = 7Hz, 2H), 7.01 (d, J =
8.5Hz, 2H), 8.05 (d, J = 8.5Hz, 2H), 13.89 (br, 1H). HRMS (ESI), m/z (M+H)+: calcd 362.0378,
obsd 362.0369.
O
H
N
N
O
F
N
N
1,3-diethyl-8-(4-fluorophenyl)-1H-purine-2,6(3H,7H)-dione:
Procedure 2: white solid. (53 mg, 70%) mp > 300 ºC ;1H NMR (500 MHz, DMSO-d6): δ 1.14 (t,
J = 7Hz, 3H), 1.27 (t, J = 7Hz, 3H), 3.95 (q, J = 7.5Hz, 2H), 4.08 (q, J = 7Hz, 2H), 7.37 (m, 2H),
8.18 (m, 2H), 13.85 (br, 1H). HRMS (ESI), m/z (M+H)+: calcd 302.1179, obsd 302.1159.
O
N
O
H
N
NO2
N
N
1,3-diethyl-8-(4-nitrophenyl)-1H-purine-2,6(3H,7H)-dione:
Procedure 2: yellow solid. (57 mg, 79%) mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ 1.15
(t, J = 7Hz, 3H), 1.29 (t, J = 7Hz, 3H), 3.96 (q, J = 7.5Hz, 2H), 4.11 (q, J = 7Hz, 2H), 8.37 (s,
2H) HRMS (ESI), m/z (M+H)+: calcd 329.1124, obsd 329.1124
O
N
O
N
H
N
N
O 2N
1,3-diethyl-8-(2-nitrophenyl)-1H-purine-2,6(3H,7H)-dione :
Procedure 2: yellow solid. (59 mg, 72%) mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ 1.15
(t, J = 7Hz, 3H), 1.21 (t, J = 7Hz, 3H), 3.95 (q, J = 7.5Hz, 2H), 3.99 (q, J = 7.5Hz, 2H), 7.74-7.76
(m, 1H), 7.83-7.86 (m, 1H), 7.93 (d, J = 7.5Hz, 1H), 8.03 (d, J = 8Hz, 1H) HRMS (ESI), m/z
(M+H)+: calcd 329.1124, obsd 329.1111.
O
N
O
H
N
OCH3
N
N
1,3-diethyl-8-(4-methoxyphenyl)-1H-purine-2,6(3H,7H)-dione:
Procedure 2: yellow solid. (56 mg, 71%) mp > 300 ºC ;
1
H NMR (500 MHz, DMSO-d6): δ
1.14 (t, J = 7Hz, 3H), 1.27 (t, J = 7Hz, 3H), 3.82 (s, 1H), 3.95 (q, J = 7Hz, 2H), 4.09 (q, J =
7.5Hz, 2H), 7.06 (d, J = 9Hz, 1H), 8.08 (d, J = 9Hz, 1H), 13.61 (br, 1H) HRMS (ESI), m/z
(M+H)+: calcd 314.1379, obsd 314.1392.
O
N
O
H
N
N
N
N
8-(4-(dimethylamino) phenyl)-1,3-diethyl-1H-purine-2,6(3H,7H)-dione:
Procedure 2: yellow solid (53 mg, 66%). mp > 300 ºC
1
H NMR (500 MHz, DMSO-d6): δ 1.14
(t, J = 7Hz, 3H), 1.27 (t, J = 7Hz, 3H),2.98 (s, 3H), 3.33 (s, 3H), 3.94 (q, J = 7Hz, 2H), 4.08 (q, J
= 7.5Hz, 2H), 6.77 (d, J = 9.5Hz, 1H), 7.96 (d, J = 9.5Hz, 1H), 13.35 (br, 1H) HRMS (ESI), m/z
(M+H)+: calcd 327.1695, obsd 327.1678.
O
H
N
N
O
O
N
N
O
8-(3,4-dimethoxyphenyl)-1,3-diethyl-1H-purine-2,6(3H,7H)-dione:
Procedure 2: Yellow solid (54mg, 63%). mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ 1.14
(t, J = 7.5Hz, 3H), 1.27 (t, J = 7.5Hz, 3H), 3.82 (s, 3H), 3.84 (s, 3H), 3.95 (q, J = 7.5Hz, 2H),
4.09 (q, J = 7Hz, 2H), 7.08 (d, J = 8Hz, 1H), 7.71-7.74 (m, 2H) HRMS (ESI), m/z (M+H)+:
calcd 344.1485, obsd 344.1475.
O
N
O
N
H
N
N
1,3-diethyl-8-(naphthalen-2-yl)-1H-purine-2,6(3H,7H)-dione:
Procedure 2: yellow solid (61mg, 74%). mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ 1.16
(t, J = 7.5Hz, 3H), 1.31 (t, J = 7.5Hz, 3H), 3.97 (q, J = 6.5Hz, 2H), 4.14 (q, J = 7Hz, 2H), 7.08 (d,
J = 8Hz, 1H), 7.58-7.61 (m, 2H), 7.96-7.99 (m, 1H), 8.01-8.06 (m, 2H), 8.25 (dd, J = 10, 2Hz,
1H), 8.73 (s, 1H), 14.01 (br, 1H) HRMS (ESI), m/z (M+H)+: calcd 334.1430, obsd 334.1449.
O
N
O
O
H
N
CH
N
N
(E)-1,3-diethyl-8-(4-methoxystyryl)-1H-purine-2,6(3H,7H)-dione:
Procedure 2: yellow solid (54mg, 63%). mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ 1.13
(t, J = 7.5Hz, 3H), 1.25 (t, J = 7.5Hz, 3H), 3.79 (s, 1H), 3.93 (q, J = 7Hz, 2H), 4.06 (q, J = 7Hz,
2H), 6.89 (d, J = 17Hz, 1H), 6.98 (d, J = 9Hz, 1H), 7.56-7.62 (m, 2H), 13.46 (br, 1H) HRMS
(ESI), m/z (M+H)+: calcd 340.1535, obsd 340.1521.
O
H HO
N
N
N
N
O
1,3-diethyl-8-(2-hydroxyphenyl)-1H-purine-2,6(3H,7H)-dione:
Procedure 2: yellow solid (50mg, 66%). mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ 1.14
(t, J = 7Hz, 3H), 1.27 (t, J = 7Hz, 3H), 3.95 (q, J = 7Hz, 2H), 4.08 (q, J = 7Hz, 2H), 6.62-6.98
(m, 2H), 7.30-7.33(m, 1H), 8.06 (d, J = 7.5Hz, 1H). HRMS (ESI), m/z (M+H)+: calcd 300.1222,
obsd 300.1220.
O
HOOC
H
N
N
N
N
O
2-(1,3-diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)benzoic acid:
Procedure 2: yellow solid (56mg, 68%). mp > 300 ºC; 1H NMR (300 MHz, DMSO-d6): δ 1.07
(t, J = 7Hz, 3H), 1.14 (t, J = 7Hz, 3H), 3.80 (q, J = 7.5Hz, 2H), 3.92 (q, J = 7Hz, 2H), 7.32 (d, J =
7.2H, 1H), 7.41-7.54 (m, 2H), 8.86 (d, J = 7.2Hz, 1H), 8.80 (s, 1H) HRMS (ESI), m/z (M+H)+:
calcd 328.1172, obsd 328.1188.
O
H
N
N
O
N
O
N
1,3-diethyl-8-(furan-2-yl)-1H-purine-2,6(3H,7H)-dione:
Procedure 2: white solid (41mg, 60%). mp > 300 ºC ; 1H NMR (500 MHz, CDCl3): δ 1.32 (t, J =
7Hz, 3H), 1.40 (t , J = 7Hz, 3H), 4.18 (q, J = 7Hz, 2H), 4.26 (q, J = 7Hz, 2H), 6.59-6.60 (m, 1H),
O
N
O
N
H
N
N
7.24-7.26 (m, 1H), 7.58-7.59 (m, 1H), 11.58 (br, 1H) HRMS (ESI), m/z (M+H)+: calcd
274.1066, obsd 274.1085.
8-cyclohexyl-1,3-diethyl-1H-purine-2,6(3H,7H)-dione:
Procedure 2: white solid (49mg, 68%).mp > 300 ºC ;
1
H NMR (500 MHz, DMSO-d6): δ 1.10
(t, J = 7Hz, 3H), 1.20 (t, J = 7Hz, 3H), 1.27-1.36 (m, 2H), 1.50-1.58 (m, 2H), 1.62-1.68 (m, 2H),
1.72-1.78 (m, 2H), 1.86-1.90 (m, 2H), 2.69-2.75 (m, 1H), 3.90 (q, J = 7Hz, 2H),3.99 (q, J = 7Hz,
2H), 13.05 (br, 1H)HRMS (ESI), m/z (M+H)+: calcd 290.1740, obsd 290.1755.
O
H
N
N
O
N
N
1,3-diethyl-8-pentyl-1H-purine-2,6(3H,7H)-dione:
Procedure 2: white solid (47mg, 68%).mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ 0.85 (t,
J = 7Hz, 3H), 1.10 (t, J = 7.5Hz, 3H), 1.20 (t, J = 7Hz, 3H), 1.21-1.31 (m, 4H), 1.64-1.70 (m,
2H), 2.65 (t, J = 7.5Hz, 2H), 3.90 (q, J = 7Hz, 2H), 4.00 (q, J = 7Hz, 2H) HRMS (ESI), m/z
(M+H)+: calcd 278.1743, obsd 278.1744.
O
N
O
N
H
N
N
(E)-1,3-diethyl-8-(pent-1-enyl)-1H-purine-2,6(3H,7H)-dione:
Procedure 2: white solid (48mg, 70%). mp > 300 ºC ; 1H NMR (500 MHz, DMSO-d6): δ 0.91
(t, J = 7Hz, 3H), 1.11 (t, J = 7.5Hz, 3H), 1.21 (t, J = 7Hz, 3H), 1.40-1.52 (m, 2H), 1.17-1.24 (m,
2H), 3.91 (q, J = 7Hz, 2H), 4.01 (q, J = 7Hz, 2H), 6.28 (d, J = 15.9Hz, 1H), 6.76-6.87 (m, 1H)
HRMS (ESI), m/z (M+H)+: calcd 276.1586, obsd 276.1602.
O
N
O
H
N
N
N
OCH3
OCH3
(E)-8-(3-4,-dimethoxystyryl)-1,3-diethyl-1H-purine-2,6(3H,7H)-dione:
Procedure 2: yellow solid (3.27 mg, 58%) . m.p. 260-262°C 5
O
N
O
N
OCH3
H
N
N
OCH3
O2N
(E)-8-[2-(4, 5-Dimethoxy-2-nitro-phenyl) vinyl]-1, 3-diethyl- 3, 7-dihydropurine-2, 6-Dione:
Procedure 2: Yellow solid. (3.39g, 60%) mp=268-269 °C; TLC (MeOH: CH2Cl2 = 05:9.5): Rf
0.63
5
1
H NMR (500 MHz, CDCl3): δ 13C NMR (300 MHz, CDCl3): δ 13.4, 13.5, 36.9, 39.0,
EP 0590919.
55.8, 56.0, 107.3, 109.1, 111.2, 113.4, 121.2, 128.6, 136.8, 149.3, 149.6, 150.4, 150.5, 151.7,
155.6. HRMS (ESI), m/z (M+H)+: calcd 370.1641, obsd 370.1647.
O
N
O
OCH3
H
N
N
N
(E)-8-[2-(4-Dimethoxyphenyl) vinyl]-1, 3-diethyl-3, 7-dihydropurine-2, 6-Dione:
Procedure 2: yellow solid (189mg, 62%).mp=244-246oC ; TLC (MeOH: CH2Cl2 = 0.5:9.5): Rf
0.43 1H NMR (300 MHz, DMSO-d6): δ 1.17 (t, J = 7 Hz, 3H), 1.29 (t, J = 7 Hz, 3H), 3.84 (s,
3H), 3.97 (q, J = 7 Hz, 2H), 4.10 (q, J = 7 Hz, 2H), 5.79(s, 1H), 6.94 (d, J = 16.2 Hz, 1H), 7.03(d,
J = 9Hz, 2H), 7.61 (d, J = 9Hz, 2H), 7.65 (d, J = 16.2 Hz, 1H) 13C NMR (300 MHz, DMSO-d6):
δ 14.1, 36.5, 38.9, 39.6, 56.1, 107.9, 114.2, 115.3, 128.9, 129.5, 128.6, 135.8, 148.9, 149.6,
150.9, 151.1, 154.5, 161.0.
HRMS (ESI), m/z (M+H)+: calcd 340.1535, obsd 340.1551.
O
H3CO
H3CO
H
NO2
4,5-Dimethoxy-2-nitro-cinnamaldeyde
Procedure 5 yellow solid (2.4 mg, 75% ). 1H NMR (500 MHz, CDCl3): 9.78-9.72 (d, 1H) 8.178.14 (d, 1H), 7.68 (s, 1H), 7.02 (s, 1H) 6.6-6.56 (q, 1H) 4.01 (d, 6H
O
N
O
N
Cl
H
N
N
CF3
8-(2'-chloro-5'-(trifluoromethyl)biphenyl-4-yl)-1,3-diethyl-1H-purine-2,6(3H,7H)- dione
Procedure 7: white solid (0.0204g, 81%) M.p. = 195-197 °C; TLC (Hexane/ ethyl acetate = 4:1):
Rf 0.37; 1H NMR (500mHz, CDCl3): δ 7.80 (d, J = 8 Hz, 2H), 7.57-7.67 (m, 5H), 4.24 (q, J = 7
Hz, 2H), 4.13 (s, 3H), 4.12 (q, J = 7 Hz, 2 Hz), 1.40 (t, J = 7 Hz, 3H), 1.29 (t, J = 7 Hz, 3H); 13C
NMR (125 mHz, CDCl3): δ 155.58, 151.49, 150.88, 148.04, 140.30, 140.06, 136.46, 130.92,
130.07, 129.90, 129.64, 129.30, 128.64, 128.07-128.25 (m, 1C), 125.90-126.07 (m, 1C), 124.82,
122.65, 109.13, 38.64, 36.60, 34.09, 13.64, 13.50; HRMS (ESI) m/z C23H21N4O2ClF3 (M+H)+:
calcd 477.1305, obsd 477.1313.
F3C
O
O
Cl
N
N
N
N
O
8-(2'-chloro-4-methoxy-5'-(trifluoromethyl)biphenyl-3-yl)-1,3-diethyl-7-methyl-1H-purine2,6(3H,7H)-dione
Procedure 7: white solid.(0.015 g, 78%) Mp = 119-120 °C; TLC (hexane/ ethyl acetate = 1:1):
Rf 0.32; 1H NMR (500mHz, CDCl3): δ 7.52-7.65 (m, 5H), 7.12 (d, J = 8.5 Hz, 1H), 4.20 (q, J = 7
Hz, 2H), 4.12 (q, J = 7 Hz, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 1.36 (t, J = 7 Hz, 3H), 1.28 (t, J = 7
Hz, 3H); 13C NMR (125 mHz, CDCl3): δ 157.63, 155.65, 151.04, 149.79, 148.05, 139.98,
136.66, 133.47, 133.33, 131.18, 130.93, 129.94, 129.69, 128.40-128.61 (m, 1C), 126.60-51 (m,
1C), 118.13, 111.35, 108.95, 56.13, 38.76, 36.65, 33.49, 13.78, 13.63; HRMS (ESI) m/z
C24H23ClF3N4O3 (M+H)+: calcd 507.1411, obsd 507.1410.
1,3-diethyl-7-methyl-8-(4-nitrophenyl)-1H-purine-2,6(3H,7H)-dione
O
N
N
O
N
N
NO2
Procedure 9: white solid (0.071g, 99%). Mp = 222-224 °C; TLC (hexane/ ethyl acetate = 4:1):
Rf 0.25; 1H NMR (500mHz, CDCl3): δ 8.39 (d, J = 9 Hz, 2H), 7.94 (d, J = 9 Hz, 2H), 4.22 (q, J =
7 Hz, 2H), 4.15 (s, 3H), 4.11 (q, J = 7 Hz, 2H), 1.38 (t, J = 7 Hz, 3H), 1.28 (t, J = 7 Hz, 3H); 13C
NMR (125 mHz, CDCl3): δ 155.40, 150.59, 149.17, 148.54, 147.84, 134.57, 130.09, 124.08,
109.61, 38.56, 36.57, 34.13, 13.46, 13.32; HRMS (ESI) m/z C16H18N5O4 (M+H)+: calcd
344.1359, obsd344.1364.
Appendix: Undergraduate laboratory module on the BDMS mediated synthesis of
xanthines
Designed Organic chemistry for Majors lab, March 2009
In March of 2009 I was asked to design a three party lab for Northeastern’s research
intensive, dedicated section of organic chemistry. I designed a three-part lab for the students (all
chemistry majors) when they were in the second semester of Organic Chemistry. Each of the 45
students synthesized their own derivative of the KW-6002 drug utilizing microwave acceleration.
The students were taught to use the microwave oven and optimize their conditions. This allowed
them to see first hand the power of a microwave to cut down on reaction time and improve
efficiency.
This two-week project (12 hours total laboratory time) divided into groups of six. Each
group was assigned a graduate student or advanced undergraduate to assist. The students were
responsible for using the formula weight to determine the correct stoichiometry for their
substrate. They then performed a BDMS-mediated ring closure. They filtered their solid product
and stored it for the week. They were responsible during the time between the two sessions to
come to the lab to perform characterization (Mass spec and NMR) on their compounds. The first
step of the next week was to alkylate their newly formed xanthine ring with one of several alkyl
chains ranging from methyl-iodide to large fluorinated alkyl iodides(vida infra). They then
purified their compound by loading their reaction mixture onto preparative-scale TLC plates
(including learning how to develop and use their own TLC solvent system). After purification,
they again were characterized then given to a senior undergraduate to continue to work on in
concert with her own independent research project.
Majors Lab Handouts and Directions:
Week 1:
1. Uracil: __________________________
2. Aldehyde: ________________________
Reaction Structure:
O
R
NH2
O
NH2
R
Compound
FW
mmol
1.
0.25
2.
0.25
3. BDMS
221.94
0.25
mg
density
mililiters
50
n/a
n/a
Procedure:
Week 1:
Measure out 0.25 mmol of uracil and transfer to a microwave tube. Add 0.25 mmol of your
aldehyde. Add 1 mL of dry acetonitrile. Add 0.25 mmol of Bromodimethylsulfonium Bromide.
Irradiate in
O
Br
microwave
S
reactor for 15
NH2
N
N
O
NH2
Br
min at 110 °C,
Chemical Formula: C2H6Br2S
Molecular Weight: 221.94
Chemical Formula: C10H18N4O2
Molecular Weight: 226.28
300 psi. Remove
CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CHO
Chemical Formula: C12H24O
Molecular Weight: 184.32
microwave.
O
When cool,
O
NH2
N
collect precipitate
H
O
N
tube from
NH2
by vacuum
filtration. Wash
Chemical Formula: C8H14N4O2
Molecular Weight: 198.22
O
the filtrate with
ethyl acetate (5
O
H
mL). Place your
NH2
N
O
Chemical Formula: C11H8O
Molecular Weight: 156.18
crude compound
N
NH2
Chemical Formula: C6H10N4O2
Molecular Weight: 170.17
in labeled glass
Chemical Formula: C11H8O
Molecular Weight: 156.18
vial and stored in desicator.
Week 2 handout
Group 1:
F
F
F
F
F
F
F
F
F
F
I
F
F
F
F F
F
F
1. Start kw-alkylation.
a. Dissolve 100 milligrams of KW analogue in 5 mL of dry DMF. Add two
equivalents of K2CO3. Add 1.1 equivalent of alkyl iodide. Stir for an hour at
room temperature.
2. Workup KW-coupling reaction.
a. Add approximately 10mL of dichloromethane to your crude reaction mixture and
ensure all contents have gone into solution. After standing for a few minutes,
small granular crystals should precipitate, collect these on a Hirsh funnel. Wash
with cold ethyl acetate (3x3mL).
3. Work up KW-alkylation reaction
a. Filter off precipitate. Dilute the filtrate (liquid) with 10 mL of water. Perform an
aqueous workup as such: Add water/DMF reaction to sep. funnel. Add 20 mL of
chloroform. Shake up then allow layers to separate. Put organic layer in a beaker.
Add more chloroform (20 mL). Shake again; then again separate the layers,
adding the organic layer to the beaker containing the chloroform from before. Do
this once more. Put the aqueous layer into another beaker. Put chloroform back
into sep funnel. Add 20 mL water. Shake and allow to separate. Drain organic
layer into organic beaker and aqueous into aqueous beaker. Add organic back to
sep. funnel. Add 20 mL Brine (saturated sodium chloride). Shake, and allow
layers to separate. Drain organic layer into organic beaker. Add magnesium
sulfate to dry. Filter off magnesium sulfate. Dry on rotary evaporator.
4. Load Prep Plate.
a. Figure out appropriate solvent system for your product based on TLC. Dissolve
your crude product in as little (insert solvent from Ian) as possible. Load prep
plate according to demo from your assigned TA. Place prep plate in chamber with
solvent. Check prep plate periodically to be sure there is still enough solvent in
chamber (so that it doesn’t run dry). If adding more solvent, be sure to add slowly
down the side of the chamber so that it doesn’t splash the prep plate.
5. Get spectra on KW coupling reaction from last week
a. Get melting point (check twice for accuracy).
b. Place one milligram (using Jones Lab scale for accuracy) of product into a labeled
vial to send out for high-resolution mass spectrometry. On an index card write—
compound name, molecular weight, and your names. Label your vial with your
initials-molecular weight of your compound.
c. Weigh your remaining product on Jones lab scale. Dissolve the rest of your
compound in 500 µL of deuterated DMSO. And collect a proton NMR. On your
index card write how many milligrams of compound is dissolved in the DMSO.
Label your NMR tube the same way you labeled your High-Res Mass Spec vial.
d. Give your labeled High-Res MS vial, labeled DMSO NMR tube, and index card
to your TA.
6. Work up Prep Plate.
a. Use UV lamp to identify bands on your prep plate. Scrape your prep plate
according to your TA. Filter your reaction through a plug of silica gel. You should
have two bands for the two different isomers.
7. Get spec data on alkylation compound.
a. Get melting point (check twice for accuracy).
b. Place one milligram (using Jones Lab scale for accuracy) of product into a labeled
vial to send out for high-resolution mass spectrometry. On an index card write—
compound name, molecular weight, and your names. Label your vial with your
initials-molecular weight of your compound.
c. Weigh your remaining product on Jones lab scale. Dissolve the rest of your
compound in 500 µL of deuterated DMSO. And collect a proton NMR. On your
index card write how many milligrams of compound is dissolved in the DMSO.
Label your NMR tube the same way you labeled your High-Res Mass Spec vial.
Group 2:
1. Workup KW coupling reaction.
a. Add approximately 10mL of dichloromethane to your crude reaction mixture and
ensure all contents have gone into solution. After standing for a few minutes,
small granular crystals should precipitate, collect these on a Hirsh funnel. Wash
with cold ethyl acetate (3x3mL).
2. Start KW alkylation.
a. Dissolve 100 milligrams of KW analogue in 5 mL of dry DMF. Add two
equivalents of K2CO3. Add 1.1 equivalent of alkyl iodide. Stir for an hour at
room temperature.
3. Get spectra on KW coupling reaction from last week
a. Get melting point (check twice for accuracy).
b. Place one milligram (using Jones Lab scale for accuracy) of product into a labeled
vial to send out for high-resolution mass spectrometry. On an index card write—
compound name, molecular weight, and your names. Label your vial with your
initials-molecular weight of your compound.
c. Weigh your remaining product on Jones lab scale. Dissolve the rest of your
compound in 500 µL of deuterated DMSO. And collect a proton NMR. On your
index card write how many milligrams of compound is dissolved in the DMSO.
Label your NMR tube the same way you labeled your High-Res Mass Spec vial.
d. Give your labeled High-Res MS vial, labeled DMSO NMR tube, and index card
to your TA.
4. Work up KW alkylation reaction
a. Filter off precipitate. Dilute the filtrate (liquid) with 10 mL of water. Perform an
aqueous workup as such: Add water/DMF reaction to sep. funnel. Add 20 mL of
chloroform. Shake up then allow layers to separate. Put organic layer in a beaker.
Add more chloroform (20 mL). Shake again; then again separate the layers,
adding the organic layer to the beaker containing the chloroform from before. Do
this once more. Put the aqueous layer into another beaker. Put chloroform back
into sep funnel. Add 20 mL water. Shake and allow to separate. Drain organic
layer into organic beaker and aqueous into aqueous beaker. Add organic back to
sep. funnel. Add 20 mL Brine (saturated sodium chloride). Shake, and allow
layers to separate. Drain organic layer into organic beaker. Add magnesium
sulfate to dry. Filter off magnesium sulfate. Dry on rotary evaporator.
5. Load Prep Plate.
a. Figure out appropriate solvent system for your product based on TLC. Dissolve
your crude product in as little (insert solvent from Ian) as possible. Load prep
plate according to demo from your assigned TA. Place prep plate in chamber with
solvent. Check prep plate periodically to be sure there is still enough solvent in
chamber (so that it doesn’t run dry). If adding more solvent, be sure to add slowly
down the side of the chamber so that it doesn’t splash the prep plate.
6. Work up Prep Plate.
a. Use UV lamp to identify bands on your prep plate. Scrape your prep plate
according to your TA. Filter your reaction through a plug of silica gel. You should
have two bands for the two different isomers.
7. Get spec data on alkylation compound.
a. Get melting point (check twice for accuracy).
b. Place one milligram (using Jones Lab scale for accuracy) of product into a labeled
vial to send out for high-resolution mass spectrometry. On an index card write—
compound name, molecular weight, and your names. Label your vial with your
initials-molecular weight of your compound.
c. Weigh your remaining product on Jones lab scale. Dissolve the rest of your
compound in 500 µL of deuterated DMSO. And collect a proton NMR. On your
index card write how many milligrams of compound is dissolved in the DMSO.
Label your NMR tube the same way you labeled your High-Res Mass Spec vial.
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