Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
`
An Efficient Synthetic Strategy towards
4-alkyl Piperidines
Master Thesis by
Valentinos Mouarrawis
`
An Efficient Synthetic Strategy towards
4-Alkyl Piperidines
Master thesis by Valentinos Mouarrawis
14 February 2015
Vrije Universiteit Amsterdam
&
Universiteit van Amsterdam
(10407618)
Chemistry: Molecular Design, Synthesis and Catalysis
Supervisor:
Prof. Dr. Romano Orru
Daily Supervisor:
Gydo van der Heijden
Second Reviewer:
Dr. Chris Slootweg
Research performed at:
Synthetic & Bio-organic chemistry
Division of Organic Chemistry
Vrije Universiteit Amsterdam
`
`
‘’Eὰν μὴ ἔλπηται ανέλπιστον οὐκ ἐξευρήσει,
ανεξερεύνητον ἐὸνκαὶ ἄπορον’’
-Iraklitos
`
`
i | P a g e
Summary Nowadays, organic chemists put emphasis on how environmentally friendly is the synthesis of
novel compounds due to the great importance of the environment. In the last 15 years many
developments took place towards this direction. Despite the progress there is always space
available for further developments. An attractive method to add in the synthetic chemist’s
sustainable repertoire is multicomponent reactions (MCR) methodology that surprisingly only
recently has become an important topic of research.
A step forward to MCR methodology will be the combination of more than one green synthetic
method so as to achieve even more advantages over conventional synthesis. In particular,
biocatalysis which is a powerful and ‘‘green’’ methodology for both simple and complex
transformations can be combined with MCR and serve as co-acting green synthetic step that
cooperates in an environmentally friendly manner.
In this thesis we were particularly interested in the development of an efficient and green
synthetic method towards 4-alkyl piperidines because of their great interest as inputs for the
ideal combination of enzyme-catalyzed and multicomponent reactions. These cyclic amines have
a structural motif which is present in numerous natural alkaloids and they are widely used as
building blocks in the synthesis of natural products.
Two approaches were developed for the synthesis of 4-substituted piperidines. The first method
is a three step synthetic route that includes: i) piperidone protection, ii) Wittig reaction and iii)
one-pot double bond reduction / deprotection step. However, after lack of success to meet the
criteria we set concerning the overall yields and how green is the process, we developed an
alternative approach that completely satisfies our goals. This modified strategy towards 4-alkyl
piperidines includes: i) SN2 reaction between 4-picoline anion with various alkybromides and ii)
reduction of the 4-substituted pyridine to the HCl salts of the corresponding piperidines.
A subsequent challenge that we briefly investigate includes the optimization of (i) the MAO-N
catalyzed oxidation step towards the corresponding imines in both high ee’s and conversions and
(ii) the Ugi 3 component reaction (U-3CR) that can be used to generate 4-substituted piperidyl
peptides. Furthermore, this synergistic approach can be further applied as a powerful
methodology in the synthesis of actual medicines that contain the appropriate structural
information.
`
ii | P a g e
List of abbreviations
Ac acetate mp melting point ATP adenosine triphosphate
n.c
no conversion
BIOS
biology-oriented synthesis NADP
nicotinamide adenine dinucleotide phosphate phosphate bs broad signal n-Bu n-butyl
°C degrees Celsius NMR nuclear magnetic resonance
CALB
Candida Antarctica lipase B P-3CR Petasis three component reaction
CBz carboxybenzyl ppm parts per million
CHMO
cyclohexanone monooxygenases PS
protein S
CRL
Candida rugosa q quartet
d doublet R rectus d.r.
diastereomeric ratio rt room temperature
3D three dimensional S sinister
DCM dichloromethane
s singlet
de diastereomeric exces TBDMS tert-Butyldimethylsilyl
DKR dynamic kinetic resolution
TLC thin layer chromatography
DNA deoxyribonucleic acid
TMSOTf
trimethylsilyl trifluoromethanesulfonate
DOS
Diversity-oriented synthesis U-4CR Ugi four component reaction
e.g. examplia gratia (for example) vs versus
ED enzymatic desymmetrization
ee enantiomeric excess
Eq. equivalents
Et ethyl
et al. et alii (and others)
FAD
flavin adenine dinucleotide
FTIR Fourier transform infrared spectroscopy
GC gas chromatography
GDH
glucose dehydrogenase
h hour(s)
HRMS high resolution mass spectroscopy
Hz Hertz z
i.e. id est (that is)
IMCR isocynanide multicomponent reaction
KR kinetic resolution
KRED
ketoreductase
LDA lithium diisopropylamide
m multiplet
MAO-N
Monoamine oxidase N
MCR multicomponent reaction
Me methyl
`
iii | P a g e
`
iv | P a g e
Table of Contents
Summary .......................................................................................................................................... i
List of abbreviations ........................................................................................................................ii
Table of Contents ........................................................................................................................... iv
1. Introduction ............................................................................................................................ 1
1.1 Green Chemistry ........................................................................................................................... 1
1.2 Multicomponent reactions ........................................................................................................... 4
1.3 Biocatalysis .................................................................................................................................. 10
1.4 Biocatalysis & Multicomponent Reactions ................................................................................. 21
1.5 Aim and Outline of this Thesis .................................................................................................... 26
2. Results & Discussion.............................................................................................................. 28
2.1 Synthesis of 4-substituted piperidines ........................................................................................ 28
2.1.1 Exploring the synthetic approach (i) towards 4-substituted piperidines. ........................... 28 2.1.2 Exploring the synthetic approach (ii) towards 4-substituted piperidines. .......................... 32
2.2 Monoamine Oxidase N: Biocatalytic desymmetrizations of 4-alkylpiperidines. ........................ 36
2.3 Investigation of the U-3CR towards 4-substituted piperidyl peptides. ...................................... 39
3. Conclusions ............................................................................................................................ 43
4. Future prospects.................................................................................................................... 45
4.1 Optimization and further investigations of the MAO-N catalyzed reaction. .............................. 45
4.2 Establishing U-3CR reaction as a highly diastereoselective methodology. ................................ 46
4.3 The discovery of valuable applications of the Biocatalysis/U-3CR method. ............................... 47
Acknowledgement ........................................................................................................................ 50
5. Experimental Section ............................................................................................................ 52
5.1 General remarks .......................................................................................................................... 52
5.2 Synthetic procedure (1): A synthetic strategy towards 4-substituted piperidines. .................... 52
5.2.1 Synthesis of phosphonium salts .......................................................................................... 53 5.2.2 Synthesis of CBz-protected 4-substituted piperidones ........................................................ 54 5.2.3 Synthesis of 4-alkyl piperidines ........................................................................................... 56
5.3 Synthetic procedure (2): A synthetic strategy towards 4-substituted piperidinium salts .......... 58
5.3.1 Synthesis of 4-alkylpyridines ............................................................................................... 58 5.3.2 Synthesis of 4-substituted piperidinium salts ...................................................................... 61
5.4 Synthetic procedure (3): The 3 component Ugi reaction towards 4-substituted piperidyl
peptides. .............................................................................................................................................. 64
References ..................................................................................................................................... 67
`
`
1 | P a g e
1. Introduction
1.1 Green Chemistry Organic synthesis is the science of replicating the molecules of nature and creating others like
them in the laboratory. [1] This is of great importance for the discovery of new medicines which
has served as the driving force for the development of new ways to achieve the synthesis of any
complex compound that can have potential biological activity. Over the past two centuries
fundamental theories and reactivities have been soundly established and these days the total
synthesis of natural products with very high complexity, such as vitamin B12, [1]
(Figure 1) in the laboratory are evidence of the great success in the field of organic synthesis.
Figure 1: The remarkably complex structure of vitamin B-12
However, despite the great developments in the artificial construction of natural products, we
are facing substantial challenges in future chemical synthesis. The present state-of-the-art
synthetic approaches in obtaining natural products are highly inefficient due to the large
amounts of chemical waste. Therefore, organic chemists started to develop innovative
methodologies towards green or sustainable organic synthesis. Since the early 1990’s when the
concept of green chemistry was first formulated as the design of chemical products and processes
to reduce or eliminate the use and generation of hazardous substances, the field has received
extensive attention due to its ability to connect chemical novelty and creativity with
environmental and economic goals.[2-4] Thus, green synthesis, including feedstocks, reactions,
solvents and separations, became an important topic of research. The idea of environmentally
`
2 | P a g e
friendly processes has its origins 50 years ago when Rachel Carson published Silent Spring, a book
that drew the attention to the connection between chemicals and their effects on the
environment and human health.[5] It was the birth of an environmental view in science that
captured the public’s interest and challenged the old scientific and industrial methods, and over
time, inspired significant changes in our awareness for the environment.
Since the beginning of green chemistry, it has developed into a major focus field within chemistry
and major research, education and outreach activities have been established globally. One solid
example of this development are the twelve principles of green chemistry (Table 1) which have
played a major role in promoting the subject and explaining its goals, ever since they were first
reported.[2] Specifically these principles have served as a handy assessment tool of how green a
plausible chemical route actually is.
Table 1: The twelve principles of green chemistry
1. It is better to prevent waste than to treat or clean up waste after it is formed.
2. Synthetic methods should be designed to maximize the incorporation of all materials used in the process into the final product.
3. Whenever practicable, synthetic methodologies should be designed to use and generate substances that possess little or no toxicity to human health and the environment.
4. Chemical products should be designed to preserve efficacy of function while reducing toxicity.
5. The use of auxiliary substances (e.g. solvents, separation agents, etc.) should be made unnecessary whenever possible and, innocuous when used.
6. Energy requirements should be recognized for their environmental and economic impact and should be minimized. Synthetic methods should be conducted at ambient temperature and pressure.
7. A raw material or feedstock should be renewable rather than depleting wherever technically and economically practicable.
8. Unnecessary derivatization (blocking group, protection/deprotection, and temporary modification of physical/chemical processes) should be avoided whenever possible.
9. Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.
10. Chemical products should be designed so that at the end of their function they do not persist in the environment and break down into innocuous degradation products.
11. Analytical methodologies need to be developed further to allow for real-time-in-process monitoring and control prior to the formation of hazardous substances.
12. Substances and the form of a substance used in a chemical process should be chosen so as to minimize the potential for chemical accidents, including releases, explosions, and fires.
`
3 | P a g e
Among others, synthetic chemists have an essential role in our society to find and develop ideal
synthetic methods that are able to maintain our standard of living without depleting the earth’s
resources. The concept of the ideal synthesis has its origins in 1975 when it was first addressed
by Hendrickson as one that [6] ˝creates a complex molecule in a sequence of only construction
reactions involving no intermediary refunctionalizations, and leading directly to the target, not
only its skeleton but also its correctly placed functionality.˝ In the 20th century the model of ideal
synthesis has been modernized and adjusted to the current needs of our society. P.A Wender et
al. describe this as the synthesis that should lead to the target molecule safely, from readily
available starting materials in one or two reaction steps, in good overall yield and using
environmentally benign reagents.[7] Thus, the environmentally friendly way of thinking
constitutes one of the factors of the ideal synthesis (Figure 2).
Figure 2: Ideal synthesis described by Wender and coworkers.
Surprisingly, a very essential methodology that was first introduced by Strecker in 1850 is
multicomponent reaction (MCR). This relatively old synthetic tool satisfies most of the criteria of
the ideal synthesis and can actively offers numerous advances towards the direction of a green
synthetic mentality. Therefore, MCR can play an important role in the expansion of the synthetic
chemist’s repertoire due to the high atom economy, efficiency, mild reaction conditions, step
economy, and compatibility with green solvents. Thus, MCRs nearly meet all the criteria of an
ideal synthesis and offer many advantages, if a compound allows to be synthesized in that way.[8]
IDEAL SYNTHESIS
Readily available starting materials
Safe
Resource effective
One pot
Environmentally friendly
Total conversion
100% yield
Simple
`
4 | P a g e
Despite the unique benefits that MCR offers, there is still further space in the development of a
highly valued green process that can dramatically expand the field of organic synthesis in parallel
with the 12 principles of green chemistry. A versatile method that generates useful intermediates
and at the same time enhances the dimensions of ideal synthesis is biocatalysis. In this case,
enzymes serve as the catalyst and the key feature that establishes enzymatic transformations a
green process is that enzymes can be obtained from nature without the necessity to be
synthesized. Furthermore, enzyme-catalyzed reactions can prevent waste generation with high
stereo- and regio-selectivity, and by avoiding or reducing the use of dangerous organic reagents.
In addition, one can design safe procedures with relatively high energy efficiency by conducting
reactions under ambient temperature and pressure. Also, atom economy can be increased by
avoiding protection and deprotection steps.
The benefits of MCR and biocatalysis over conventional chemical synthesis are significant due to
the high chemical and energy efficiency. In addition, the green criteria of biocatalysis are highly
unique and in combination with MCRs would represent an important step towards ideal
synthesis. In particular these two methodologies can cooperate in a synergistic manner and add
to the development of a powerful green synthetic approach.[9]
1.2 Multicomponent reactions Nowadays, the state of the environment is becoming an international issue and organic chemists
should also adapt an ecological synthetic mentality. Alongside the development of conventional
synthetic methods, multicomponent reactions strongly meet the principles of the ideal synthesis.
The MCR methodology is defined as a one-pot reaction employing more than two starting
materials where most of the atoms of the starting materials are incorporated in the final product
(Figure 3).[10]
Figure 3: Multicomponent reaction methodology (1) versus conventional multistep synthesis (2).
`
5 | P a g e
Numerous novelties have been reported in the field of MCR since its discovery in the 50s and
justifiably most of them are essentially considered as an important area of research. Specifically
this reaction methodology was first introduced in 1850 by Strecker, a reaction towards α-
aminonitriles, which are useful intermediates for the synthesis of amino acids via hydrolysis of
the nitrile. The first step is the condensation of ammonia with the aldehyde (1) for the iminium
(2) formation followed by the nucleophilic attack of the cyanide to generate the α-aminonitrile
(3). Hydrolysis forms the corresponding aminoacid (4, Scheme 1).[11]
Scheme 1: Strecker amino acid synthesis
An additional important development in the MCR field is the Mannich reaction that was first
reported in 1912.[12] It is used to convert primary or secondary amines and two carbonyl
compounds into β-amino carbonyls. The reaction mechanism starts with the formation of an
iminium ion (7). The compound with the carbonyl functional group (8) can tautomerize to the
enol form, and then attack to the iminium ion which after deprotonation provides the final β-
amino-carbonyl (also known as a Mannich base; 9) with two stereocenters (Scheme 2). The
applications of the Mannich reaction includes agro chemicals, for instance plant growth
regulators,[13] and polymer chemistry. However, the most popular use of the Mannich reaction
can be found in the pharmaceutical industry due to facile synthesis of 1, 3-amino alcohols or
Michael acceptors via the Mannich bases.[14]
Scheme 2: The Mannich reaction
Another significant MCR is the Passerini reaction which was discovered in 1921 in Florence, Italy
by Mario Passerini.[15] It is the most fundamental multi-component reaction involving
isocyanides. These compounds are chemical species with an extraordinary functional group and
scarce valence structure and reactivity. They are the only stable compounds with a formally
divalent carbon.[8] Several studies have taken place in order to grasp their electronic and
`
6 | P a g e
geometric structures [16] so that their electrophilic and nucleophilic character could be explained.
Romain Ramozzi and coworkers have showed by the means of high-level valence bond
calculations that isocyanides are dominantly of the carbenic type (Figure 4), contrasting the
usually used zwitterionic character.[17] Isocyanide based MCRs are widely compatible with a range
of functional groups not taking part in the initial MCR. This category of MCRs offers the possibility
to utilize such functional groups in a secondary reaction step in order to move beyond towards
more diverse synthetic steps and improve the current complex synthesis toolbox.
Figure 4: Sugested representation of isocynides by Romain Ramozzi et al.
The classic Passerini reaction is a three-component reaction involving a carboxylic acid (12), an
aldehyde or a ketone (10) and an isocyanide (11) to form α-acyloxycarboxamide (15). Over the
years, the scope of the reaction has broadened and now it is widely involved with accessing
biologically active molecules. The mechanism of the reaction is still a subject of uncertainty and
kinetic studies have led to different mechanistic elucidations.[18,19] However, Ugi discovered that
the reaction is accelerated in aprotic solvents indicating a non-ionic mechanism.[20] Hydrogen
bonding is believed to play an important role in the formation of the presumed cyclic transition
state for this reaction and the reaction mechanism which proceeds through an intermediate (14)
and further rearranges to the final product (15) (Scheme 3).
Scheme 3: The nonionic mechanism of the Passerini reaction.
Despite the discovery of the Passerini reaction, isocyanide chemistry started to flourish in the
late 1950s when Ugi et al. discovered the first isocyanide based 4-component reaction (U-4CR)
in 1959.[21-23] This versatile reaction is a reaction between a ketone or aldehyde (16), an amine
(19), an isocyanide (17) and a carboxylic acid (18), generating α-aminoacyl amide derivatives (23).
The Ugi reaction, compared to Passerini reaction, is much more multipurpose due to both library
size and scaffolds that can be accessed. This difference can be primarily attributed to the acid
component variability and their rearrangement options, the amines structures and to the many
intramolecular variations. The protein-like small chain products of the Ugi reaction have potential
`
7 | P a g e
pharmaceutical applications. Thus, it is particularly of great interest in diversity-oriented
synthesis, where e.g. libraries of compounds are created for screening purposes. The suggested
mechanism involves a prior formation of an imine by condensation of the amine with the
aldehyde, followed by formation of an iminium ion (20) by proton exchange with the carboxylic
acid. The next step is isocyanide addition to the iminium ion to form a nitrilium ion intermediate
(21), after which nucleophilic addition of the carboxylate ion takes place, generating an acylated
isoamide (22). The last step is a rearrangement of the acyl group to afford the desired product
(23,Scheme 4).
Scheme 4: Suggested mechanism of the U-4CR.
One of the most recent isocyanide based multicomponent reactions is the Orru reaction, a three-
component reaction developed in 2003.[24] It is a reaction between an amine (24), an aldehyde
(25) and α-acidic isocyanide (26), generating substituted 2-imidazolines (29). The first step of the
proposed mechanism is the imine formation followed by attack of the α-acidic isocyanide. The
formation of the intermediate (28) is believed to be promoted by traces of amine present in the
reaction that may act as a basic catalyst. The final step is a ring closure of 28 to afford the desired
product (29, Scheme 5 ).
Scheme 5: The mechanism of the Orru three-component reaction.
`
8 | P a g e
During the past decades, it has become clear that the aim of state-of-the-art research is the
development of green and simplified synthetic routes that give access to molecules of high
structural complexity.[10] In this perspective, MCRs have become an emerging field of research
because of their plethoric benefits for the facile and green synthesis of small-molecule libraries.
However, one of the main limitations of this methodology is the absence of stereocontrol which,
in most cases, is neither straightforward nor generally applicable. The necessity of asymmetric
synthesis stems from the high complexity of the compounds isolated from natural products which
have diverse polycyclic ring systems and significantly high 3D structural information.
Over the years there have been several developments in the direction of stereoselective MCR
that can be divided into two main categories that include (i) the use of catalysts and (ii) the use
of chiral MCR inputs. The toolbox of organocatalysis contains many promising solutions towards
asymmetric synthesis in multicomponent reaction methodology. Catalytic asymmetric
multicomponent chemistry creates a challenge in the synthesis of optically pure compounds with
structural diversity and complexity.[25]
Two examples worth mentioning are depicted in Scheme 6. Shi-Xin Wang et al. have described
an efficient enantioselective Passerini three-component reaction (P-3CR) catalyzed by Lewis acid
catalyst (34) that generates a-acyloxyamides (33) in good to excellent enantioselectivities.[26]
Another asymmetric MCR was reported by Feng shi et al. that is considered the first catalytic
asymmetric five-component reaction using chiral phosphoric acids (39), which directly assembles
aldehydes (37), anilines (35) and β-keto esters (36) into functionalized tetrahydropyridines (38)
with the creation of five s bonds and two stereocenters in high diastereo- and
enantioselectivities.[27] In particular, this is a very attractive method towards chiral
tetrahydropyridine derivatives which are core structures in a wide range of natural products and
pharmaceuticals. Thus, the development of an efficient catalytic enantioselective MCR for the
synthesis of optically pure tetrahydropyridines is highly desirable.
`
9 | P a g e
Scheme 6: Catalytic asymmetric multicomponent reactions.
Yet, the discovery of a generally applicable catalytic asymmetric Ugi reaction is a momentous
event that would open many beneficial opportunities for the future of the diverse-oriented
synthesis and it is justifiably considered as the Holy Grail in MCR chemistry.[28]
The second main approach to achieve stereocontrol in MCR methodology is the asymmetric
induction of optically pure MCR inputs. In particular, this remarkable approach provides access
to complex compounds - otherwise hard to achieve - where one stereogenic center can efficiently
control the newly formed center in a diastereoselective manner.
A representative example that uses optically active inputs is the diastereoselective Petasis 3CR,
reported by N.A Petasis in 1998.[29] It is a one-step three component reaction between an
organoboronic acid (40), an amine (41), and an R-hydroxy aldehyde (42) to give the
corresponding β-amino alcohol (43). In particular, the reaction generates only the anti-products
with de of more than 99% (Scheme 7). Interestingly the products are obtained as single
enantiomers when optically pure R-hydroxy aldehydes such as glycerinaldehyde (42) are used,
due to the absence of racemization.
`
10 | P a g e
Scheme 7: Diastereoselective Petasis 3CR with chiral aldehyde.
Despite the fact that this synthetic approach is experimentally simple and it proceeds with very
high diastereoselectivity, the limited availability of the chiral starting materials create the need
to develop simplistic, efficient and sustainable methods towards these optically active inputs. In
this perspective, biocatalysis can play an important role as an efficient and sustainable strategy
in order to gain access to highly desirable optically pure materials. For this reason the future of
asymmetric multicomponent reactions via enzyme catalyzed chiral inputs can be considered a
very promising field of research.
1.3 Biocatalysis Biocatalysis is the field that uses enzymes as catalysts for the chemical transformations of organic
compounds. A number of various reactions are feasible by means of biocatalytic processes and
can be divided into six main categories depicted in Table 2.
Table 2: Classification of enzymes
Enzyme class Reaction type
Oxidoreductases Oxidation–reduction reactions
Transferases Transfer of groups
Hydrolases Hydrolysis formation of esters, amides, lactones, lactams, epoxides,
Lyases Addition–elimination reactions
Isomerases Isomerizations
Ligases Formation-cleavage of C–O, C–S, C–N,
`
11 | P a g e
The first reported example of biocatalytic process goes back to 1858 when Louis Pasteur
described an enzyme-catalyzed kinetic resolution. This remarkable discovery has been
recognized as a Milestone in the field of biocatalysis due to the wide application in both academia
and industry.[30] In 1894, Emil Fischer introduced the lock and key hypothesis of stereoselective
enzyme catalysis model. In this model, only the substrate with the specific shape that is
complementary to the active site can bind and react. An additional keystone of biocatalysis was
described by Eduard Buchner who in 1897 reported the first successful cell-free fermentation of
sugar by yeast extracts, which establishes unquestionable evidence that biological
transformations do not necessarily need living cells.[31] This criterion alteration opened the door
to present-day biocatalysis containing fermentation technology for the production of both achiral
and chiral products. In 1913, Ludwig Rosenthaler described the preparation of (R)-mandelonitrile
(46) by treating benzaldehyde (44) with HCN (45) in the presence of emulsin extracted from bitter
almonds (Scheme 8).[32] This discovery can be considered as what we call today enzyme-catalyzed
asymmetric synthesis.
Scheme 8 : The first enzyme-mediated asymmetric catalysis described by Ludwig Rosenthaler.
Yet another landmark that likewise set the stage for present day research and technology is the
fundamental discovery in 1926 by James B. Summer in which enzymes are proven to be
proteins.[33] In 1965, Jacques Monod expanded the known information about enzymes by
proposing the allosteric model which describes the allosteric transitions of proteins made up of
identical subunits.[31] Last but not least, Whitesides et al. reported a remarkable asymmetric
example using an aldolase as biocatalyst in stereoselective aldol addition of ketone (48) to
aldehydes (47) for the synthesis of aldol adducts (49) in an asymmetric fashion (Scheme 9).[34]
Scheme 9: Biocatalytic asymmetric aldol addition of ketone to aldehydes.
`
12 | P a g e
Despite, the rapid development of enzyme catalysis, a serious obstacle persevered until the late
1970s that is how to obtain proteins in sufficient amounts for practical applications in a wide-
ranging manner. By then, enzymes were isolated from their sources like microorganisms, fungi,
insects, plants, or mammals which were often problematic and not efficient.
The revolutionary work in 1980 completed by Paul Barg, Herbert Boyer, and Stanley Cohen was
the vital step to overcome the long-standing restriction of the development of biocatalysis in
synthetic organic chemistry. Specifically they proposed the idea of recombinant DNA
methodology, according to which an enzyme occurring in one organism can be overexpressed in
a host organism, a typical host organism are Echerichia coli and Basillus subtillikl.[35] Biocatalysis
has numerous advantages compared to chemocatalysis in organic synthesis. The major
advantage of a biocatalyst is its high selectivity. This selectivity can be chiral (stereoselectivity),
positional (regioselectivity), and functional group specific (chemoselectivity). This characteristic
is very desirable in organic synthesis as it may offer numerous benefits. Opposed to conventional
chemocatalysis, biocatalysis opens the door for using protecting group free substrates. A
representative example is an aldol reaction depicted in Scheme 10 for the asymmetric synthesis
of β-hydroxy α-amino acid. Furthermore, biocatalytic processes minimize side reactions, such as
isomerization, racemization, and rearrangement compared to the classic catalysis. In addition,
due to the last 10 years of increased emphasis on developing synthetic methods that are
environmentally friendly, biocatalysis as a process is as green as catalysis gets.
Scheme 10: Conventional multistep synthesis using a chemocatalyst vs. protecting-free one-step biocatalytic approach in an aldol reaction.
`
13 | P a g e
The use of enzymes has immediate advantages in turning a chemical process green and greatly
satisfies the 12 principles of green chemistry. The mild reaction conditions, the high purity
products in one step, the low energy requirements, and both biodegradability and
biocompatibility of enzymes establishes bio-based routes an attractive sustainable tool towards
novel and complex synthesis. Biocatalysis, using either enzyme technology or whole cells, has
won numerous awards according the US Environmental Protection Agency (Table 3).[36]
Table 3: Green Chemistry Challenge Awards in biocatalysis over the past ten years
Product Technology Complany Year
Succinic acid as chemical feedstock Fermentation BioAmber 2011
1,4-butandiol for polymers and chemical feedstock Fermentation Genomatica 2011
Sitagliptin: a pharmaceutical ingredient for treatment of type 2 diabetes
Enzyme Merck and Codexis 2010
Atorvastatin intermediate for treatment of high cholesterol
Enzyme Codexis 2006
Low trans fats and oils for human nutrition Enzyme ADM and
Novozymes 2005
Taxol for treatment of breast cancer Fermentation Bristol Myers
Squibb 2004
Despite these advantages, enzymes as catalysts in synthetic organic chemistry continued to
suffer from some limitations that cannot be overlooked. In fact, the narrow substrate scope
compared to native substrate or chemocatalysis substrates prevents their establishment as a
catalyst for a wide range of inputs. The cost of enzyme including isolation and purifications
processes is a major obstacle towards commercializing biocatalysts due to the current cost-
competitive established classic methods. Furthermore, enzymes are provided by nature in only
one enantiomeric form and it is impossible to invert the chiral induction of a given enzymatic
reaction due to the limited general methods of generating mirror-image enzymes.
Another disadvantage is the insufficient stability under operating conditions due to the general
unstable nature of enzymes when their removed from their natural conditions. Even though
enzymes are very flexible in tolerating non-natural substrates, they are practically cofactor
depended in order to be functional. The most common cofactors are depicted in Figure 5.
`
14 | P a g e
Figure 5: Cofactors often applied in enzymatic organic synthesis.
Last but not least, the specificity of enzymes is not strictly limited to substrates. Frequently, the
activity of an enzyme is reduced by specific interactions with molecules termed inhibitors.
Specifically, these inhibition phenomena can occur at high substrate and/or product
concentrations and lead to low reaction rates, a factor that significantly limits the efficiency of
the enzymatic process.
Undoubtedly, the field of biocatalysis has reached a high level of sophistication through a
significant number of technological research and innovations. However, the above-mentioned
limitations in many cases dramatically prevent the revolutionary development of biocatalysis to
establish enzymatic processes a widely applicable synthetic tool. Nowadays, molecular biology
methods such as protein engineering provide the missing components to create the ideal enzyme
and to overcome these walls.[37-39] The basic goal of protein engineering is the creation of
improved versions of known enzymes in order to meet the demands in each individual case.
These improvements can include one or more properties such as, increased catalytic function
relative to the original enzyme, altered substrate specificity or stereospecificity and, increased
stability to the conditions that are required.[40] Two strategies have actively contributed to that:
(i) the so-called rational design approach and (ii) and directed evolution. [41] Rational design is the
process where enzymes are re-designed rationally at the molecular level by specifying the
sequence. It requires comprehensive understanding of structures and mechanism in order to
adjust their functions for the desired applications. Specifically, site-directed mutagenesis is used
to introduce site-specific functionalizations into the enzyme, frequently in combination with
`
15 | P a g e
computational means.[42] A noteworthy example is the development by Kazlauskas and
coworkers of an esterase with higher enantioselectivity for the hydrolysis of 3-bromo-2-
methylpropionate via targeted mutagenesis.[43] Moran Brouk et al. reported the use of a
monooxygenase variant with increased substrate specificity which resulted in 190-fold higher
reaction rate for 2-phenylethanol.[44] Another attention-grabbing example of enzyme utilization
was reported by Sneha Srikrishnan et al. whereby higher catalytic efficiency obtained via site-
directed mutagenesis.[45] The mutant displayed 2.5-fold improvements in hydrolytic activity on
cellulosic substrates, while preserving thermostability. However, its wide application site-
directed mutagenesis has two major drawbacks which cannot be ignored. The high complexity of
interpreting the structure of an enzyme and the relation in an enzyme between structure and
activity is still not a trivial. Even when the target enzyme is fully characterized, distinguishing the
amino acid residues that control the catalytic activity is a challenging assignment.
Directed evolution involves the modifications of a biocatalyst via an in vitro form of Darwinian
evolution and offers a powerful method for the growth of enzymes with unique properties. The
developed enzyme can include altered substrate specificity, thermal stability and organic solvent
resistance. The major advantage of directed evolution over rational design is that novel
properties can be induced in enzymes without the need of prerequisite knowledge of enzyme
structure and/ or catalytic mechanism. There are a considerably large number of prominent
examples of directed evolution of enzymes reported in the last 10 years. M.T. Reetz and
coworkers reported an enantioselective cyclohexanone monooxygenases (CHMO) as a catalyst
for the desymmetrization of 4-hydroxycyclohexanone (50) in Baeyer–Villiger reaction whereby
the selectivity of the oxidation inverted by using the directed evolution approach (Scheme 11).[46]
Scheme 11: CHMO-Catalyzed Oxidation of 4-Hydroxycyclohexanone.
`
16 | P a g e
Although, directed evolution has already proven its substantial role in inducing new functional
properties into an enzyme, there are a few major challenges in order to harness the advantages
of biocatalysis that trigger further technological research and innovation. For example, low
efficiency, tricky screening in complex mutant libraries for the desired functions, time-
consuming, and technically challenging. Unfortunately, there is not a single ideal method of
protein engineering due to the diversity of choosing’s observed from enzyme to enzyme and from
application to application. The growing attention paid to the cooperative method of rational
design and directed evolution towards the targeted randomization of certain areas of the protein,
enzymes can be designed in a more efficient manner in correlation with the desired property.
Biocatalytic steps are often used to introduce chirality in a reaction sequence towards the desired
product. The growing need of creating molecules with increased complexity (e.g large molecules
with many chiral centers) offers a broad spectrum of opportunities for this technology to be
further developed and established as a key component in the synthetic chemist’s arsenal. Over
the years, enormous efforts have been made to develop enantioselective methodologies for
simplistic preparation of enantiomerically pure compounds because of their significance in the
pharmaceutical, food and agricultural sectors. There are two main categories of stereoselective
biotransormations including, asymmetric synthesis and kinetic resolutions of racemic mixtures.
Kinetic resolution (KR) is a process to separate two enantiomers in a racemic mixture. In
particular, the idea is based on the different reaction rates of the two enantiomers causing an
enantioenrichement of the less reactive enantiomer.[47] The major drawbacks of this method are
the 50% maximum yield and its low atom economy. A yeast-mediated reduction of β-keto esters
illustrates representatively a kinetic resolution of racemic alcohols (Scheme 12).[48]
Scheme 12: Kinetic resolution of racemic alcohols.
Due to the limitations of this method a different approach has been developed called dynamic
kinetic resolution (DKR). In DKR, a racemization reaction takes place alongside to the enantiomer
formation, leading to the desired enantiomer in 100% theoretical yield. Specifically the
enantioselective reaction is slower than the racemization one and leads to formation of the
enantiomer with the lower activation energy. An interesting example of dynamic kinetic
`
17 | P a g e
resolution was published by Sonia Rodriguez et al. for the asymmetric synthesis of β-hydroxy
esters by recombinant Escherichia coli (Scheme 13).[49]
Scheme 13: Dynamic kinetic resolution (DKR) of β-hydroxy esters
Asymmetric synthesis, constitutes the second main class of stereoselective biotransormations,
and implies the formation of one or more chiral centers in a substrate. One of the approaches in
the field of asymmetric synthesis is the enzymatic desymmetrization (ED) of meso or prochiral
compounds and opposed to kinetic resolution a maximum yield of 100% can be achieved. Meso
and prochiral compounds have either a planar trigonal group with two enantiotopic faces or two
enantiotopic groups. The enzymatic process is faster at one of the enantiotopic groups or faces
of the substrate resulting in high enantioselectivities. Goswami et al. reported a nice example of
an industrial application of EDs for the efficient synthesis of 51 with Candida Antarctica as a
biocatalyst (Scheme 14).[50] The yield of the reaction was 99.8% and the 1S,2R-monoester (51)
was enantiomerically pure.
.
Scheme 14: An industrial enzymatic desymmetrization (ED) process.
`
18 | P a g e
Biocatalytic processes have been employed progressively towards widespread applications
especially in the environmentally benign synthesis of optically pure compounds. This is due to
the great interest stems from pharmaceutical and agrochemical industries where enantiopure
molecules are of a great importance as synthons for numerous pharmaceutically active
substances and agrochemicals. The examples depicted below are a few biocatalytic methods to
produce these highly vital synthons that illustrate the great potentials of biocatalysis in the
direction of both high enantioselectivities and conversions.
Enantiomerically enriched amines are key starting materials and intermediates for the synthesis
of a wide range of biologically active compounds. For this purpose, kinetic resolution of racemic
amine, hydrolases are the most frequently used enzymes. For example, Burkholderia plantari
lipase has been widely used for the production of enantiopure amines (54) via kinetic resolution
of racemic amines (52, Scheme 15).[51] It is worthwhile to mention the need of using an acyl donor
(53) in order to activate the carbonyl. The expected major downside of this approach is the
limitation in relation to the reaction yield of the kinetic resolution, which cannot surpass 50 %.
Scheme 15: Kinetic resolution of racemic amines with Burkholderia plantari lipase
The yield limitations of kinetic resolution can be overtaken by asymmetric synthesis approach
which is more preferred due to the maximum yield of 100%. An efficient approach to shift the
equilibrium of the reaction towards the products is to remove the co-product which is generated
from the de-amination of the amine donor. Wang et al. have reported the asymmetric synthesis
of (S)-1-phenylethan-1-amine (60) using as the amine donor 3-aminocyclohexa-1,5-
dienecarboxylic acid (57) and as the enzyme several variants of ω-transaminases (Scheme 16).
The generated ketone (58) was efficiently removed by a subsequent tautomerization to 3-
hydroxybenzoic acid (59). The quantitative formation of (S)-60 (99%) in an excellent enantiomeric
excess (>99%) established this approach at least promising for the asymmetric synthesis of
amines.[52]
`
19 | P a g e
Scheme 16: Asymmetric synthesis of a chiral amine with an artificial amine donor.
Optically active aldehydes and ketones are vastly valuable intermediates in the synthesis of
complex compounds with high 3D structural information. From a chemical point of view,
enantiopure α-hydroxy ketones (also called acyloin) are highly valuable building blocks for many
applications for the fine chemistry sector as well as pharmaceuticals. Due to their significance,
several biocatalytic approaches have been developed by means of different categories of
enzymes such as lyases, oxido-reductases and hydrolases. The hydrolase-catalyzed DKR of
racemates is an important example of a well-developed methodology to afford enantioenriched
α-hydroxy ketones (62). For example a two-compartment DKR processes have been reported,
involving Candida Antarctica lipase B (CALB) as biocatalyst for the enantioselective substrate
transesterification in a first compartment, and the simultaneous racemization of the residue
alcohol facilitated by Amberlyst 15 in a separated compartment (Scheme 17).[53]
`
20 | P a g e
Scheme 17: Two compartments DKR of racemic acyloins (61).
Chiral alcohols play an essential role as reactive intermediates or starting materials in agro-,
pharma-, and fine chemical industries. In recent times, remarkable progress has been done
towards biocatalytic preparation of enantiopure alcohols. An attractive pathway towards the
synthesis of optically active alcohols is the biocatalytic reductions of ketones. Ma et al. developed
a novel green-by-design method for the synthesis of a key intermediate for atorvastatin which is
the active ingredient of LipitorR, an anti-cholesterol drug. The process includes a biocatalytic
reduction step of ethyl 4-chloro-3-oxo-butanoate (63) using a ketoreductase (KRED) combined
with glucose as a reductant and a NADP-dependent glucose dehydrogenase (GDH) for co-factor
regeneration.[54] Glucose is oxidized to gluconic acid and neutralized using sodium hydroxide. The
(S) ethyl-4-chloro-3-hydroxybutyrate (64) product was obtained in high yields and high
enantiomeric excess (Scheme 18).
Scheme 18: Biocatalytic reduction of ethyl 4-chloro-3-oxo-butanoate using a ketoreductase (KRED).
Chiral carboxylic acids can be derived from ester hydrolysis. Kazlauskas et al. reported a kinetic
resolution approach including a simple 2-propanol treatment that converts crude lipase from
Candida rugosa (CRL) to a form with an increased both activity and enantioselectivity by a factor
of 1.2-1.6 and 25 respectively. The chiral 2-substituted carboxylic acid (67) was obtained in
moderate yield and excellent enantioselectivity (Scheme 19).[55]
`
21 | P a g e
Scheme 19: Kinetic resolution of 2-substituted carboxylic acids ester by 2-propanol treated Candida rugosa lipase (CRL).
1.4 Biocatalysis & Multicomponent Reactions As mentioned in previous sections MCRs are remarkably efficient key methodology to access
highly diverse synthesis of complex molecules due to their high atom economy, mild conditions
and step economy. Yet, the not straightforward stereochemical control and the lack of catalytic
asymmetric methods create the necessity to utilized methods towards efficient and
environmentally friendly enantioselective methods. In most of the cases asymmetry is achieved
by inducing chiral inputs and the generation of those inputs is at least a significant key step
forward to MCRs stereocontrol. The wide-ranging toolbox of the biocatalytic generation of chiral
materials opens up the opportunity to control the stereochemical outcome of MCRs by
introducing a biocatalytic step throughout the synthesis.
The synergy of biocatalysis with MCRs is a handy methodology that overcomes the limiting factor
of MCRs stereoselectivity and at the same time presents a powerful process with many
advantages concerning the environment. In addition, a biocatalytic step can be combined with a
MCR step in several ways throughout the synthetic route making this method flexible and more
widely applicable. These combinations can be: (i) kinetic resolution of a racemic MCR product,
(ii) dynamic kinetic resolution in which the optically pure product reacts in MCR and (iii)
biotransformation to generate enantiopure inputs for diastereoselective MCRs.
Despite the great individual potentials of both biocatalysis and MCRs, the synergistic approach
towards stereochemical diversity in DOS/BIOS-based library design is up till now at an early stage
of development. Nevertheless, over the last years several attractive strategies of
biocatalysis/MCR were reported sowing the great interest towards the development of powerful
and generally applicable biocatalysis/MCR/ combinations. [56-58]
A very interesting example where an enzyme-catalyzed kinetic resolution was combined with
multicomponent chemistry was reported by R. Ostaszewski in 2013. They reported a simple and
efficient methodology towards enantioenriched Ugi products that could be also applied to other
type of substrates. In particular it is a very attractive approach for obtaining enantiomerically
`
22 | P a g e
pure Ugi product via enzyme mediated kinetic resolution of crude Ugi product (68) to access 1,3-
diol peptidomimetics (69) . Unfortunately the enzymatic diastereoselective acylation of hydroxyl
groups was not efficient and as a result not further pursued. On the other hand, the
enantioselective strategy was found to be very efficient and the corresponding carboxylic acid
(69) was obtained in excellent ee (Scheme 20).[59] .
Scheme 20: Enzyme mediated kinetic resolution of crude Ugi product to enantiomerically pure 1, 3-diol peptidomimetic.
Besides employing an enzymatic resolution step throughout the synthetic route, enzymes can
also be used to generate enantiomerically pure MCR adducts and therefore benefit from the
substrate-controlled diastereoselection reached through the MCR step. In addition, the
biocatalytic asymmetric synthesis of one or more MCR component would expand the input’s
diversity otherwise not easily accessible from the chiral pool, resulting in broadening the scope
of any MCR. Although the literature is still rather limited concerning this strategy, there are few
examples indicating the great potentials of this method.
One of the first examples of a MCR combined with an enzymatic desymmetrization was reported
by Larock and coworkers in 1991. They developed an efficient enzyme-desymmetrized
monoacetate (71) which was further transform to a protected diol (72) and coupled to a MCR for
the synthesis of chiral prostaglandins (75).[60] The mechanism of the MCR includes the formation
of two intermediates. The first step is the addition of the alcohol moiety in 72 to the palladium-
complex vinyl ether to afford 73. The formed complex undergoes two olefin insertions; an
intramolecular one (faster) that gives 74 and an intermolecular one that involves the unsaturated
ketone and generates the final product (75) (Scheme 21). With regard to the stereochemistry, all
`
23 | P a g e
the stereogenic centers are controlled, apart from the acetal’s one and therefore an epimeric
mixture is obtained.
Scheme 21: A 3-CR approach to prostaglandins, starting from desymmetrized diol.
Diastereoselection in isocyanide-based multicomponent reactions (IMCR) and particularly in the
Ugi reaction is very tricky. Chiral amines are the only inputs that gave good results concerning
diastereoselectivity. Though, intramolecular versions are more potentially active towards good
to excellent diastereoselectivities due to the sterics imposed by cyclic transition states. [61-63]
A representative study that highlights this approach for the synthesis of pharmacologically
relevant polyfunctionalized pyrrolidine systems (79) was described by V.Cerulli et al. in 2012.[64]
In the event, enantiomerically pure cyclic imine precursors (77), synthesized by Amano PS lipase
followed by a fully diastereoselective Ugi-Joullie reaction which gave 79, in a diastereoselective
ratio of >99:1 (Scheme 22).
`
24 | P a g e
Scheme 22: Diastereoselective Ugi reaction using chiral cyclic imine synthesized by Amano PS lipase.
Another attention-grabbing, two-step approach including the asymmetric enzyme-catalyzed
synthesis of enantiopure pyrrolines (81) combined with multicomponent chemistry was reported
by Turner and Orru.[65,66] They developed a desymmetrization method of a series of monocyclic,
bicyclic, and tricyclic meso pyrrolidines (80) by means of monoamine oxidase N (MAO-N) from
Aspergillus Niger optimized by directed evolution. What makes the biocatalytic step interesting
is the use of whole cells, thus isolation of the enzyme was not necessary. Furthermore, due to
the use of O2 as the stoichiometric oxidant, the addition of catalytic flavin adenine dinucleotide
(FAD) cofactor was needless, making this method relatively cost-effective. The MAO-N-produced
pyrrolines were used as chiral inputs in Ugi 3CR for the synthesis of 3,4-disubstituted propyl
peptides (82) with good to excellent diastereoselectivity in favor of the trans product (Scheme
23).[67] This development clearly showed in a very representative way how powerful the
combination of biocatalysis and MCR can be in the synthesis of molecules with high structural
and three dimensional information. The mild conditions, simple experiment procedures,
excellent yields, d.r and ee values also illustrate how do they synergistically facilitate the synthesis
of complex molecules in an environmentally friendly manner.
`
25 | P a g e
Scheme 23: MAO-N desymmetrization combined with U-3CR.
Most importantly, these propyl peptides were proved to hold great potentials as useful
intermediates for the efficient synthesis of molecules of highly structural complexity that can
found applications in medicinal chemistry. In this direction, a very short and efficient synthesis
of an important hepatitis C NS3 protease inhibitor, Telaprevir (IncivekTM) was developed starting
from simple biocatalytically produced pyrrolines (83). This application can be considered without
doubt as the best example of the cooperation between MCR and biocatalysis that gave access to
an actual pharmaceutical drug (84, Scheme 24).[68]
Scheme 24: Synthesis of hepatitis C NS3 protease inhibitor Telaprevir (IncivekTM).
`
26 | P a g e
In addition, the same group further expand this study towards the development of a novel Ugi
and Pictet-Spengler-type cyclization for the synthesis of polycyclic 2,5-diketopiperazines (88).[69]
This strategy constitutes the first application of MCR chemistry to generate 5-membered ring-
fused diketopiperazines establishing these compounds interesting targets for medicinal
investigations. In the event, the use of α-oxoacid (86) and an isocyanide (87) containing an
electron-rich arene promotes a subsequent MCR Pictet–Spengler (PS) reaction just by treating 88
with trimethylsilyl triflate (TMSOTf). The desired compounds (89) were obtained in moderate to
very good yields and diastereomeric ratios.
Scheme 25: Alkaloid-type compounds by the MAO-N, Joullie-Ugi and PS sequence.
1.5 Aim and Outline of this Thesis Being inspired by the work done on the MAO-N and Ugi sequence for the synthesis of 3,4-
disubstituted propyl peptides we turned our focus on developing an analogous synthetic
approach to gain access to 4-substituted piperidyl peptides. Our strategy involved the adjustment
of the method applied on the meso pyrrolidines in order to develop a green and efficient
methodology that contains all the advantages of the combination between MCR and biocatalysis.
Unfortunately, 4-substituted piperidines which are the suitable inputs of the MAO-N catalyzed
step are not commercially available and the incorporation of an additional synthetic route
towards the synthesis of these highly valuable meso 4-alkylpiperidines was more than necessary.
In fact, we wanted to develop a synthetic route that stays on the pathway of green chemistry and
can be well-combined with the follow up chemistry in an environmentally friendly manner so as
to establish an overall green methodology that includes the synthesis of MAO-N inputs,
biocatalysis and MCR chemistry. For this reason this project primarily involves the development
of an efficient and green synthetic strategy towards 4-substituted piperidines (90), which
constitute promising inputs in a MAO-N catalyzed reaction to generate optically pure 4-alkyl-
2,3,4,5-tetrahydropyridines. The first approach to synthesize these chiral 4-substituted
`
27 | P a g e
piperidines (A) included a Wittig reaction of a protected piperidone (92), followed by a one-pot
double bond reduction/deprotection step of 91 to afford 90. However, due to our continuous
effort to establish methods that stay on the path of green chemistry, we developed a different
and more efficient approach (B) that involves the one-pot 4-picoline (96) deprotonation step /SN2
nucleophilic substitution with various alkylbromides, followed by a low hydrogen pressure
pyridine reduction step to afford 94 (Scheme 26).
Scheme 26: Retrosynthetic analysis of the two developed synthetic pathways towards 4-substituted piperidines.
The second goal of this thesis involves the investigation of how enantioselective the biocatalytic
oxidation of 4-substituted piperidines is towards optically pure 4-alkyl-2,3,4,5-
tetrahydropyridines by means of MAO-N. The last part aims to describe how efficient the
combination of enzymatically produced imines with U-3CR is by investigating the stereocontrol
provided by the biocatalytically generated stereogenic center over the additional stereocenter
formed during the Ugi reaction (diastereoselectivity).
The following chapters describe all the implemented experiments in this thesis including the two
synthetic strategies to synthesize 4-substituted piperidines. In addition, we illustrate the initial
screening results related to the enantioselectivities of different 4-alkyl piperidines towards the
corresponding optically active imines by using MAO-N as the enzyme. Lastly, we focus on using
the enzymatically produced imines in a U-3CR and illustrating the observed diastereoselectivities.
Next, we describe the conclusion of this research thesis, followed by some future prospects.
Lastly, detailed experimental procedures and data are described.
`
28 | P a g e
2. Results & Discussion
2.1 Synthesis of 4-substituted piperidines: As mentioned in chapter 1, a powerful synthetic approach so as to satisfy most of the criteria of
ideal synthesis would be the combination of two efficient and at the same time environmentally
friendly methodologies. Biocatalysis and multicomponent chemistry present a real example of
such a methodology that works synergistically towards ideal synthesis. Nevertheless, the need to
expand the substrate scope of Biocatalysis-MCR methodology described by our group has
triggered us to develop an efficient and short synthetic strategy to generate a library of 4-
substituted piperidines that can be used as inputs for a MAO-N catalyzed reaction. In this section,
the two attempted approaches are described concerning the synthesis of 4-substituted
piperidines.
2.1.1 Exploring the synthetic approach (i) towards 4-substituted piperidines. Our initial strategy for the synthesis of 4-substituted piperidines includes a sequence of 3 steps.
The first step is the protection of 4-piperidone monohydrate hydrochloride (97) using
carboxybenzyl (CBz) group, a carbamate which is often used as an amine protecting group in
organic synthesis.[70] This amine protection step is highly essential in this synthetic route because
the free form of 4-piperidone is not stable due the existing of an amine and a ketone in the same
molecule that will lead to the formation of iminium. The Cbz protective group was chosen in
order to facilitate a one-pot reaction that includes the reduction of the double bond and the
deprotection of the amine in the last step of our synthetic route by using palladium on activated
carbon. In a total reaction time of 10h, full conversion was obtained. The N-Cbz protected product
98 obtained pure as a yellow liquid in an excellent yield of 99% (Scheme 27).
Scheme 27: N-CBz protection of 4-piperidone.
`
29 | P a g e
The following more complex step involves a Wittig reaction between a ketone and various alkyl
triphenylphosphonium ylides. In the event, we first synthesized the phosphonium salts, followed by
addition of n-butyl lithium (n-BuLi) so as to furnish the corresponding ylide. The compound 98 was
reacted with a number of different alkyl triphenylphosphonium ylides in order to generate a library
of 4-alkylene substituted N-CBz protected piperidines. Despite of the commercial availability of
various Wittig reagents most of them have been synthesized by us, so as to increase the cost-
effectiveness of the corresponding method. This has been done according to literature procedure by
reacting triphenylphoshine with different bromoalkanes in order to generate the desired Wittig
reagents.[70,71] Unfortunately, this SN2 nucleophilic substitution reaction was only plausible in the
case of using primary alkylhalides which was rationalized by the effects of steric hindrance present
in the secondary ones(Table 4). After all the efforts – i.e. by increasing the reaction temperature
and/or employing a halogenalkane bearing a better leaving group such as iodine – to overcome this
barrier, the conversions remained fairly low.
Table 4: Substrate scope of the Wittig reagent synthesis.[a]
[a] Reaction conditions: alkyl bromide 99 (1 eq) triphenylphoshine (1 eq) were mixed in dry toluene under N2 atmosphere, 10 h at 120
°C. Yields refer to isolated material. [b] Methylbromide (3 eq).
At this point, the Wittig reaction could be investigated by using different phosphonium salts. In the
event, a Wittig reaction between a series of different phosphonium salts and the N-protected 4-
piperidone was studied in order to get access to the corresponding N-protected 4-alkenylpiperidines
that can be further transformed to the desired products. We initially performed the Wittig reaction
by utilizing 1.5 equiv of (ethyl) triphenylphosphonium bromide in THF and 1.4 equiv of n-butyl lithium
at -78°C. After column purification this procedure led to moderate to good yields, also containing
some unreacted material. Under the conditions mentioned above, a series of different primary
phosphonium salts reacted with the N-protected piperidone, affording the desired product in
moderate to good yields (Table 5). The next step was to investigate how efficient the Wittig reaction
is by employing several secondary phosphonium salts.
`
30 | P a g e
To our disappointment, 1H NMR indicated no conversion to the desired product. We believe that the
reason behind that, is the sterically hindered proton present in the secondary alkyl
triphenylphosphonium salts, making problematic the approach of n-BuLi so as to furnish the ylide.
An additional reason when the ylide was furnished in low yields can be the difficulty of the ylide to
attack the ketone due to the effects of steric hindrance present in the secondary ylide.
Table 5: Substrate scope of the Wittig reaction.[a]
[a] Reaction conditions: (alkyl) triphenylphosphonium bromide 102 (1.5 eq) and n-BuLi (1.4 eq) were mixed in dry THF under N2
atmosphere, 1h at -78 °C. Upon completion CBz-protected piperidone (1 eq) was added at -78 °C and stirred, 12h at -78 °C to r.t. Yields
refer to isolated material after column chromatography. [b] (alkyl) triphenylphosphonium bromide 102 (1.3 eq) and n-BuLi (1.2 eq).
Despite our unsuccessful attempts on improving the Wittig reaction to furnish better than moderate
yields we moved to the last step of this approach in order to generate an overview of this synthetic
methodology towards 4-substituted piperidines. This step involved a one-pot double bond
reduction/deprotection reaction. Specifically, catalytic transfer hydrogenation using 10% Pd/C,
reduced carbon-carbon double bond and cleaved the carbamate in a one-pot process. A series of
compounds (105a-d) were isolated after stirring for 16 h, under 1 bar of hydrogen pressure at room
temperature in excellent yields ranging between 89-97 % (Table 6). Despite that, the previous steps
were not so efficient and green, this one-pot reduction/deprotection step showed to be highly
efficient due to the simple experiment procedure, mild conditions and excellent yields in all of the
substrates.
`
31 | P a g e
Table 6: Substrate scope of the one-pot reduction/deprotection step.[a]
[a] Reaction conditions: Compound 104 (1 eq) and 10% Pd/C (0.1 eq) were mixed in methanol under N2 atmosphere. The mixture was
stirred under hydrogen atmosphere, 16 h at r.t. Yields refer to isolated material.
At this stage of the research, we were highly questioning the accessibility of these 4-substituted
piperidines through this approach due to several limitations as far as diversity of the created library
and the overall yields are concerned. In addition, the use of hazardous solvents such as toluene and
the low atom economy (Wittig reaction) are substantial factors that decrease the value of this
method as a green process. For these reasons this approach became a less attractive synthetic route
towards the generation of a representative library of 4-alkyl piperidines.
Since, the initial synthetic effort towards the desired product was not completed successfully due to
the above-mentioned reasons we turned our attention to an alternative and more environmentally
benign approach. The first stage of this alternative route development was to predetermine the
desired features that they can beneficently be combined with the low environmental impact of
Biocatalysis-MCR sequence. As mentioned in chapter one, the green criteria of this synergistic
strategy are highly unique and in combination with an efficient synthetic route towards 4-alkyl
piperidines would represent an important step towards ideal synthesis. For these reasons our major
concern was to increase yields, atom economy, step economy and the use of less hazardous solvents.
`
32 | P a g e
2.1.2 Exploring the synthetic approach (ii) towards 4-substituted piperidines. After facing the problems mentioned in the previous section of the initial synthetic approach, we
were set to move forward with an alternative strategy towards 4-substituted piperidines. In the
event, we developed a 2-step synthetic route that includes an SN2 substitution of 4-picoline with
different alkylhalides, followed by pyridine reduction.
The first step is a one-pot two-stage procedure that involves SN2 substitution of 4-picoline anion by
a series of different alkylbromides. This procedure was performed, by first deprotonating the methyl
group of 4-picoline by addition of lithium diisopropylamide (LDA) at -78°C. The 4-picoline anion was
furnished by using 1.2 equiv. of LDA which was freshly made in the same pot by reacting n-BuLi with
LDA. The required reaction time for the deprotonation step is approximately one hour and it can be
observed by the formation of a characteristic deep red color.
Next, a diverse library of 4-alkyl pyridines was generated by adding a series of different alkyl
bromides (1 equiv.) This SN2 nucleophilic substitution reaction has a remarkably broad substrate
scope and many alkylbromides could be used ranging from short alkyl chains to longer and more
complex ones such as pentyl, isopropyl, tert-butyl, cyclohexylmethyl and cyclopropylmethyl analogs.
The best yields (89-97%) were obtained with the linear straight-chain alkyl substrates (108a-c) and
decreased (36-76%) in the branched-chain ones (108d-f). (Table 7)
In addition to our efforts to further broaden the substrate scope of this reaction we employed
dibromo alkanes as electrophiles, for a secondary cyclization step towards the synthesis of cyclic
analogs. This procedure was performed, by using 2 equiv. of LDA so as to drive a second
deprotonation step, furnish the deprotonated species and complete the intramolecular cyclization.
Despite our efforts, the yields remained very low due to the formation of the double alkylated
product prior to the intramolecular ring closing step and/or the formation of the elimination
products. In spite of the formation of the above-mentioned side-products, purification by the means
of column chromatography led successfully to isolation of 108g and 108h in a yield of 25% and 18%
respectively. (Table 7).
`
33 | P a g e
Table 7: Substrate scope of the 4-alkyl pyridines synthesis.[a]
[a] Reaction conditions: 4-picoline 106 (1 eq) and LDA (1.2 eq) were mixed in dry THF under N2 atmosphere, 1h at -78 °C. Upon
completion alkyl bromide 107 (1 eq) was added at -78 °C and stirred, 12h at -78 °C to r.t. Yields refer to isolated material after column
chromatography. [b] LDA (2 eq). Dibromoalkanes (1eq.)
After having established a library of several 4-substituted pyridines we turned our attention to
develop an efficient and environmentally friendly reduction step. Although the catalytic reduction of
pyridine and pyridine derivatives has been thoroughly described in the literature, it usually involves
harsh reaction conditions such as high pressures and/or temperatures.[72-74]
We therefore started our investigations by exploring an alternative pathway towards the reduction
of these aromatic heterocycles. In the event, we developed a PtO2-catalyzed hydrogenation process
that involves an in situ generation of pyridinium salt prior to the reduction event. This salt formation
lowers the activation energy towards the reduced product making the reduction process plausible in
milder reaction conditions. As a result, pyridinium salts are in every instance more readily reduced
than pyridine and the desired 4-substituted piperidines were obtained as HCl salts in very good to
excellent yields (Table 8).
`
34 | P a g e
Table 8: PtO2-catalyzed hydrogenation of various 4-substituted pyridines.[a]
[a] Reaction conditions: 4-substituted pyridine 109 (1 eq), Hydrogen chloride / ethanol solution ( 5 eq, 1.25 N) and platinum oxide
(0.05 eq.) were mixed in methanol. The mixture stirred under 1 bar of hydrogen atmosphere, 36h at r.t. Yields refer to isolated
material.
`
35 | P a g e
Delighted by this result, we set out to further investigate how the pyridinium ring influences the
reduction of an aromatic system attached to it at the para position. Surprisingly, we were able to
fully reduce the commercially available 4-phenyl pyridine (111) to 4-cyclohexyl piperidinium salt
(112) under the same conditions mentioned above (Scheme 28). A possible explanation to this is the
in situ generation of pyridinium lowers the activation energy towards the reduced product, thus the
catalytic hydrogenation of the phenyl group is plausible under the same conditions via the effect of
conjugation with the pyridinium salt. For these reasons this process represents a powerful and
environmentally friendly reduction tool that can be widely applied as a green methodology for the
catalytic hydrogenation of pyridine derivatives with excellent yields.
Scheme 28: PtO2-catalyzed hydrogenation of 4-phenyl pyridine.
To summarize, we have developed a very short and efficient synthesis of 4-substituted piperidines
which present great potentials as inputs of enzyme-catalyzed desymmetrizations. We initial set
several criteria from an environmentally point of view for this synthetic route so as to develop an
attractive and powerful combination with the follow up enzymatic and MCR chemistry. Efficient
access to libraries of 4-substituted piperidines is essential for further development of the synergistic
approach of Biocatalysis and MCR. A powerful synthesis of these alkyl piperidines can readily expand
the dimensions of Biocatalysis-MCR methodology and can efficiently allow the rapid generation of
complex and structurally diverse molecules.
Despite the discouraging results of the initial approach, we developed an efficient synthetic route
towards the generation of the desired library that includes numerous advantages compared to our
initial efforts. In this respect, the lack of using hazardous solvents, elevated temperatures and high
pressures make our methodology attractive from an environmentally point of view. In addition, the
second tactic involves fewer synthetic steps, higher yields and it allows the in situ formation of
piperidinium salts which are stable, odorless, water-soluble and off-white solid compounds that can
be stored at r.t.
`
36 | P a g e
2.2 Monoamine Oxidase N: Biocatalytic desymmetrizations of 4-
alkylpiperidines. Enzymes proficient of oxidizing amines are abundant in organisms such as bacteria, yeasts, plans and
mammals and can be divided into two main categories, flavoprotein and qiunoprotein. The
mechanism of the qiunoprotein amine oxidases has been well described and occurs via the formation
of an enzyme-substrate covalent adduct.[75] On the other hand, amine oxidation by flavoproteins
(monoamine oxidase, amino acid oxidases and polyamine oxidases) is still debatable.[76,77] Despite
the controversy over the mechanism of flavoproteins as amine oxidizing enzymes, MAO-N presents
a versatile biocatalyst that has been extensively reported in literature. Turner and cowers were the
pioneers that have been thoroughly investigated the use of enzymes capable to oxidize amines and
significantly contribute to the design and the development of MAO-N.[78,79] Many application have
been reported by the same group and two nice representative applications are the following: (i)
deracemization of primary, secondary and tertiary amines using mutant amine oxidases in
combination with ammonia borane,[78-80] and (ii) Meso-pyrrolidines used as substrates for amine
oxidation by MAO-N D5 towards 1-pyrrolines.[81] These highly valuable applications in parallel with
the relatively high stereoselectivity and the cofactor independency establish MAO-N one of the
leading biocatalysts for amine oxidation.
Stimulated from the inspiring work of Turner and Orru on the oxidation of meso-pyrrolidines using
MAO-N D5 we turned our focus on the desymmetrization of 4-substituted piperidines which can lead
to analogous conversions and enantioselectivities. Since we developed an efficient synthetic strategy
to gain access to libraries of 4-substituted piperidines we wanted to investigate the effectiveness of
MAO-N, by incorporating it in our synthetic strategy. The aim of this section was to perform initial
set of screening reactions of different substrates and assess the trend of both conversions and
enantioselectivities towards optically active imines. In the event, we performed several enzymatic
reactions using different mutations of MAO-N (D5 and D9) so as to enzymatically desymmetrize
prochiral 4-alkyl piperidines and generate a library of optically active imines.
The MAO-N enzyme screening process included the use of freshly prepared MAO-N pellet cells of
two different mutations in order to assured that the activity of the enzyme has not been decreased.
Interestingly, the entries 1 and 6 (R= methyl and tert-butyl) furnished the highest enantioselectivities
accompanied with the highest conversions. In particular, the mutant D5 gave 100% conversion to
the entries 1 and 6 with enantioselectivities of 82% and 93% respectively. Furthermore, the
enantioselectivity observed concerning the D9 variant was 96% which is the best achieved, however
combined with 50 % conversion. Despite this promising results, it is hard to estimate any trend how
the R group of 4 substituted piperidines influence the enantioselectivities.
`
37 | P a g e
Table 9: MAO-N enzyme screening results.[a]
Entry Substrate Enzyme Conversion (%) ee (%)
1
MAO-N D5 MAO-N D9
100 1
82 n.c
2
MAO-N D5 MAO-N D9
100 30
8 93
3
MAO-N D5 MAO-N D9
100 70
8 83
4
MAO-N D5 MAO-N D9
50 50
16 >85
5
MAO-N D5 MAO-N D9
50 50
20 80
6
MAO-N D5 MAO-N D9
100 50
93 96
7
MAO-N D5 MAO-N D9
100 20
16 53
8
MAO-N D5 MAO-N D9
4 40
5 92
9
MAO-N D5 MAO-N D9
13 50
5 85
[a] The enzyme-screening reactions were done by group of Prof. Nicholas Turner from the University of Manchester as part of a joint
collaboration on this project.
`
38 | P a g e
Unfortunately, at room temperature these cyclic imines are prone to trimerization reactions that
lead to trimmers (115) formation (Scheme 29). This can lead to several problems in the GC-analysis
that imposes a few difficulties to identify the characteristic peaks of the chiral imine. In addition, we
faced various implications concerning the structural elucidation by means of NMR. For these reasons
we had to incorporate a derivatization step in our synthetic approach so as to generate additional
characteristic peaks and be able to characterize and determined the ee of the desired products.
Scheme 29: Trimmer formation proposed mechanism.
Particularly, addition of acetic anhydride (116) to these imines gives the corresponding acetylated
analogs (117) that can be straightforwardly visualized by proton NMR (Scheme 30).
Scheme 30: Imine derivatization towards acetylated analogs.
To our delight, the excellent conversions in combination with the promisingly high
enantioselectivities stimulate further optimization of the corresponding enzymatic process so as to
reach both excellent conversions and ee’s. What is highly valuable about these optically active imines
is the prospective use as inputs in a highly diastereoselective MCR.
`
39 | P a g e
2.3 Investigation of the U-3CR towards 4-substituted piperidyl peptides. As previously emphasized, we focused on the development of two green approaches that can
cooperate in a synergistic manner towards the synthesis of complex molecules. In the last two
sections we described an environmentally friendly method to generate chiral imines that can be used
as optically active inputs for the last MCR step that facilitates the one-pot formation of highly valued
molecules in a diastereoselective manner. Even though, the biocatalytic desymmetrization is at the
early stages of research, the initial screening results are very promising towards excellent ee’s and
full conversions. In this section, we primarily turned our focus on the investigation of Ugi-type 3CR
between different isocyanides, carboxylic acids and the generated chiral imines. In addition we
subsequently explored how diastereoselective is the above-mentioned multicomponent reaction
racemic imines. As mentioned in the introduction our group discovered a very diastereoselective Ugi-
3CR with 1-pyrrolines for the synthesis of enantioenriched propyl peptides (Scheme 31). The high
selectivity is due to the bulk of the R-groups and increases with the size of the R-groups.[66]
Scheme 31: Diastereoselective U-3CR towards substituted propyl peptides.
After this remarkable discovery we focused on applying the same principles towards 4-substituted
piperidyl peptides (119) by incorporating racemic six-membered ring imines (118) to MCR step.
(Scheme 32).
Scheme 32: Diastereoselective U-3CR towards 4-alkylpiperidyl peptides.
`
40 | P a g e
In our case we envisioned that the newly formed stereocenter can be controlled by the para
substituted R-groups on the imine six-membered ring. In particular, the suggested mechanism of the
Ugi reaction involves nucleophilic attack of the isocyanide (121) to the iminium ion (123) that is
formed by proton exchanged with the carboxylic acid (122). Subsequently, an acylated isoamide
(125) is formed by the nucleophilic addition of the carboxylate ion to the nitrilium anion (124). The
last step is a rearrangement, driven by the formation of the amide bond relocating the acyl group
from the oxygen to the nitrogen to afford the desired product (126) (Scheme 33).
Scheme 33: Suggested mechanism of the U-3CR.
Interestingly, the stereochemistry of the newly formed chiral center is set by the nucleophilic
addition of the isocyanide to the iminium anion making the U-3CR as a highly diastereoselective
methodology. In particular, the isocyanide addition can in principle take place either from the top or
the bottom face of the unsaturated six-membered ring which in each case provides different
stereochemical outcome. In detail, attack from the top face leads to the formation of an unstable
twist boat with high-energy transition state (128) which is less favorable than the chair conformation
transition state (127) that is formed when the attach takes place from the bottom face (Scheme
34).[70]
Scheme 34: Top face (ii) versus bottom face (i) isocyanide addition.
`
41 | P a g e
In the event, we started our investigations with the reaction between methyl substituted imine (129),
two different isocyanides (130) and four different acids (131, Table 10). The methyl analog was
selected as the components of the model reaction and we only screen the substrate tolerance in
relation to different acid and isocyanides. In analogy with the optimized reaction conditions reported
by our group in 2010 towards the synthesis of substituted propyl peptides via U-3CR we applied the
same conditions for the synthesis of 4-substituted piperidyl peptides (132). Our primary goal was to
evaluate how efficient is the corresponding reaction in relation to the yields and subsequently
establish a representative result that indicates how diastereoselective this method is based on GC
analysis. Pleasingly all the examined substrates underwent clean conversion to the desired Ugi
product in moderate to good yields (35 - 70%). Benzoic acid analog (132c) is an exception in the trend
and further purification is needed by the means of column chromatography resulting in low yield of
35 %. A plausible explanation why 132c shows such a considerable lower yield compared to the other
Ugi products is the isolated polymer-like side product which suggests oligomerization or
polymerization reactions. In addition, is known that imines are prone to trimerization reactions
suggesting that the unconverted material form a trimmer prior to the MCR step.
Table 10: U-3CR with various carboxylic acids.[a]
[a] Reaction conditions: Imine 129 (1 eq), isocyanide 130 ( 1.33) and carboxylic acid 131 (1.33 eq.) were mixed in DCM. The mixture
stirred for 48h at room temperature. Yields refer to isolated material. [b] Purified by column chromatography.
`
42 | P a g e
The next aim of the last section was to qualitative establish a representative example in relation to
the diastereoselectivity of this reaction and subsequently trigger future optimizations in order to
develop a fully diastereoselective U-3CR methodology towards 4-substituted piperidyl peptides. In
the event, we performed several GC measurements by analyzing our synthetically produced Ugi
products (Scheme 35). Based on GC analysis, we were delightful to discover that the generated
piperidyl peptides 132a-d show high d.r’s. To illustrate these high d.r’s, we chose 132a as an example
that representatively shows the high value of this method to synthesize complex molecules in an
asymmetric fashion. The compound 132a was generated in one-pot by the addition of 4-methyl-
2,3,4,5-tetrahydropyridine, tert-butyl isocyanide and acidic acid in DCM.
Scheme 35: Diastereoselectivity of the U-3CR based on GC analysis of U-3CR between 126, acidic acid and tert-butyl isocyanide. Peaks (1) and (2) show the two diastereomers.
Based on the GC analysis, illustrated in Scheme 35 we can presumably suggest that the minor peak
(2) is the minor diastereomer which indicates that the peak (1) is the major diastereomer. If this is
the case, the minor formation of the cis- product (134) clearly shows that the U-3CR towards 4-
substituted piperidyl peptides takes place in diastereoselective manner due to the formation of the
trans-product (133) as the major diastereomer. Despite this promising results, further analysis is
necessary to be done in order to clearly prove the major formation of the one diastereomer.
`
43 | P a g e
3. Conclusions. The primary aim of this research was to develop an efficient synthetic method towards the synthesis
of 4-substituted piperidines. In this research we first synthesized the alkyl piperidines through a
sequence of 3 steps and overall yields ranging between 21-57%, depending on the R-group.
Unfortunately, this approach became a less suitable synthetic route towards the generation of a
representative library of 4-alkyl piperidines due to the limited diversity of the created library, the low
overall yields and the lack to meet the principles of green chemistry. After this setback, a relatively
greener and more efficient synthetic pathway was developed towards the desired product from the
commercially available 4-picoline, in overall yields of 36-94% over 2 steps. This strategy includes
numerous advantages compared to the previous one, such as fewer synthetic steps and higher yields,
and it can be considered as a more environmentally friendly process.
After establishing the above-mentioned green synthetic route towards these highly valuable inputs,
an initial set of enzyme-catalyzed desymmetrization reactions were implemented by using MAO-N
(D5 and D9) as the enzyme. These investigations were performed by the research group led by Prof.
Nicholas Turner at the University of Manchester as part of a joint collaboration on this project. It
turned out that most of the entries converted to the corresponding imine in good to very good
enantioselectivities which in some cases reached ee’s that are greater than 90%.
The incorporation of these imines in U-3CR is of utmost importance so as to develop a powerful
methodology to gain access to structurally complex products. In this perspective, the MCR step
showed, in an initial stage of research, to be very efficient and the chosen substrates convert to the
desired Ugi product in moderate to good yields (35 - 70%) and excellent diastereoselectivities.
Nevertheless, further investigations should be done to investigate the diastereoselectivity and to
establish representative library of these 4-substituted piperidyl peptides.
The further development and the optimization of this synergistic process that includes the synthesis
of 4-substituted piperidines/ MAO-N catalyzed desymmetrization/U-3CR can give access in an
environmentally friendly manner to a highly diverse library of compounds with high molecular
complexity (Scheme 36). The most remarkable potentials about this method is that is green and
efficient and it can find many applications in to future developments in drug discovery.
`
44 | P a g e
Scheme 36: The synergistic strategy towards 4-alkylpiperidyl peptides.
`
45 | P a g e
4. Future prospects. Taking everything into account this thesis research has led to the development of an innovative
and green synthetic strategy towards 4-substituted piperidines. It further illustrated how these
highly valued inputs can incorporate in an enzyme catalyzed step for the synthesis of optically
active MCR inputs that can give access to highly complex molecular structures. However, there is
always space available for supplementary things that can be improved or studied. The main focus
of follow-up studies should involve the optimization of the biocatalytic and MCR step towards
higher yields, enantioselectivities and diastereoselectivities. In addition, further investigations
towards the desymmetrization of more diverse substrates that can be further functionalized in
follow up synthetic steps in order to gain access to molecules with higher structural complexity.
Lastly, the need to discover valuable applications in the design of pharmaceutically interesting
compounds is more than necessary so as to establish our method as a highly applicable approach
in medicinally oriented synthesis.
4.1 Optimization and further investigations of the MAO-N catalyzed
reaction. Despite the preliminary investigations done on the biocatalytic step for the generation of
optically pure imines, there is still plenty of space available towards higher conversions and ee’s.
In particular, the reaction conditions can be further optimized to fully convert the meso 4-
substituted piperidines to the corresponding optically active imines. Since, the reported
conversions in chapter 2 are the outcome of a reaction time of 24h, prolonging that time can
complete the reaction and fully convert the starting materials to the desired product. In addition,
a more extensive substrate screening can be done in order to obtain valuable insights about the
factors that influence the selectivity of MAO-N, and develop through the directed evolution
process different MAO-N variants with higher enantioselectivities. These insights can be further
combined with different protein engineering techniques such as rational design and/or in silico
studies in the direction to develop a diverse library of enantioselective MAO-N variants, suitable
for different and most exotic inputs. These inputs can be further functionalized in subsequent
steps such as several couplings and azide transformation reactions. The only limiting factor with
respect to the diversity of these promising inputs is the need to be MCR inert in order not to
interfere in the following U-3CR step (Figure 6).
Figure 6: Proposed inputs for MAO-N desymmetrization.
`
46 | P a g e
4.2 Establishing U-3CR reaction as a highly diastereoselective
methodology. Developing asymmetric MCR methods is of high importance; however, uncertainty always
remains how to efficiently control the newly formed chiral center during the process. For that
reason, inducing chirality through optically active starting materials is an alternative and robust
approach. Despite the initial studies taken place in this thesis research in respect to
diastereoselectivities, there are several investigations to be done in the direction of developing
an efficient and more diverse diastereoselective methodology. Our investigations involved
several GC measurements by analyzing our synthetically produced 4-methyl piperidyl peptides.
Despite the promising results, these MCR products were synthesized by using racemic imines
instead of the enzymatically enantioenriched ones. For that reason it would be most ideal if the
4-substituted piperidyl peptides could be diastereoselectively synthesized in an MCR of the
enzymatically produced optically active imines. Next to this, a diverse library of chiral MCR
products can be generated by inducing the MAO-N produced optically active imines in U-3CR step
(Figure 7). Subsequently, the relation between the R groups of the chiral imines and the
diastereoselectivity observed in the final product can be investigated by means of gas
chromatography and obtain valuable insights about the factors that enhance the
diastereoselectivity.
Figure 7: Proposed U-3CR library.
`
47 | P a g e
4.3 The discovery of valuable applications of the Biocatalysis/U-3CR
method. The development of a versatile and green synthetic methodology was of utmost importance in
this research thesis in order to access highly molecular structures which are otherwise hard to
achieve. However, the need to find applications in the design of pharmaceutically interesting
compounds is highly important and necessary to establish our method as a highly applicable
synthetic tool. The structural motif of the U-3CR products can be found in several medicines, thus
this can be used to either synthesize derivatives of the actual medicine and/or used as an
alternative green approach to produce the actual medicine. A very nice example is Argatropan
(153) which is an antithrombin agent and it is used for the treatment of thrombosis in patients
with heparin-induced thrombocytopenia.[82] The core-structure of Argatropan is a 4-substituted
piperidyl peptide and the synthetic route could be facilitated by an MCR step combined with a
short supplementary synthesis (Scheme 37). The suggested approach is very interesting to
explore, however, there are a few uncertainties that need to be investigated. In particular, the
use of an isocyanide (151) that can be converted to the free acid is highly important and it is a
project that is currently under investigations in our group.
Scheme 37: Argatropan proposed synthetic route.
`
48 | P a g e
A few other pharmaceutically interested target molecules that their synthesis can be simplified
by MCR step are Boceprevir (154), Asunaprevir (155) and Faldaprevir (156, Figure 8). These drugs
contain the structural information that is required in order to synthesize several derivatives of
them via U-3CR mediated step. Nevertheless, subsequent investigations should be done to
develop the entire synthetic route towards these medicines and if this methodology proved to
be efficient, it would provide a novel asymmetric procedure for these types of molecules.
Figure 8: Actual medicines containing the structural information of U-3CR product.
`
49 | P a g e
`
50 | P a g e
Acknowledgement
After 9 months of research and successfully achieving the goal of my thesis project, I would like
to thank several important people that essentially contribute to complete my internship. First of
all, I would like to thank prof. Romano Orru and dr. Eelco Ruijter for giving me the opportunity to
do my Master internship in the synthetic organic chemistry group. Most importantly, I would like
to thank Gydo van der Heijden for all his help and daily supervision that definitely helped me to
develop myself as a synthetic chemist and of course for the good times in the lab. Without this
guidance this project would undoubtedly not have been this fruitful. Also, I would like to thank
Veronica Estévez for her helpful advices and solutions to my questions. Dr. Chris Slootweg is
thanked as well for being my second reviewer. Last but not least, I would like to thank the whole
synthetic organic group for the good times in the labs and during the well-organized and nice
“borrels”. I really appreciate all the help and knowledge that I gain in the lab and I can definitely
recognize a big improvement in my synthetic organic skills.
Valentinos Mouarrawis
14 February 2015
`
51 | P a g e
`
52 | P a g e
5. Experimental Section
5.1 General remarks: Unless indicated otherwise, all reagents and solvents were purchased from Sigma Aldrich or
Acros Organics and used without any further purification. Diisopropylamine was stored in a
Schlenk flask under nitrogen atmosphere. Cyclohexane was distilled prior to use. Anhydrous THF
and Toluene were obtained by distillation using appropriate drying agents prior to use. Reactions
requiring anhydrous conditions were performed in vacuum flame – dried glassware under a
nitrogen atmosphere. Air and moisture sensitive liquids were transferred via syringe into the
reaction flasks through rubber septa. Flash chromatography was performed on silica gel (particle
size 40 - 63 μm, pore diameter 60 Å). Analytical TLC was performed on Merck Kieselgel 60F254
aluminum plates which were visualized using potassium permanganate /Δ, para – anisaldehyde/
Δ and UV light (254 nm). The 1H and 13C NMR spectra were recorded on a 400 MHz and 500 MHz
Bruker Avance spectrometer. Chemical shifts are quoted (δ) in parts per million (ppm),
referenced to the residual solvent resonance. Coupling constants are quoted in Herz (Hz).
Multiplicity is assigned as s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet
and bs for broad signal. Mass spectrometry analysis was performed on a Bruker micrOTOF-Q
instrument. Infrared spectra were recorder on a Shimadzu FTIR-8400s spectrometer and
wavelengths are reported in cm -1. Melting points were recorded on a Buchi M-565 melting point
instrument. GC-FID analysis was performed on Agilent 6850 GC.
5.2 Synthetic procedure (1): A synthetic strategy towards 4-substituted
piperidines.
Benzyl 4-oxopiperidine-1-carboxylate (1): 4-Piperidone monohydrate
hydrochloride (19.96 g, 130 mmol, 1.1 eq.) was dissolved in a mixture of THF/
water (400 mL, 1:1). K2CO3 (20, 44 g, 147.95 mmol, 2.5 eq.) was added and the
resulting mixture stirred for 20 min at 0 °C. Benzy chloroformate (8.44 mL, 59.18
mmol, 1 eq.) was added dropwise and the mixture allowed to warm to rt and
stirred for 12h. The reaction solution was quenched with NH4Cl aq. and the water layer was
extracted with ethyl acetate (3x). The combined organic layers were washed with brine, dried
over anhydrous Na2SO4. The volatiles were removed by means of a rotary evaporator yielding a
yellow liquid. Yield 46: 13.8 g, 59.18 mmol, 98 %. ; 1H-NMR (500 MHz, Chloroform-d) δ 7.45 (dd,
J = 24.6, 4.5 Hz, 5H), 5.14 (s, 2H), 3.53 (t, 4H), 2.19 (d, J = 39.1 Hz, 4H), ppm; 13C-NMR (500 MHz,
CDCl3) δ 155.14 (C), 136.87 (C), 134.82 (C), 128.35 (CH) , 127.81 (CH), 127.71 (CH), 66.89 (CH2),
35.48 (CH2), 27.58 (CH2) ppm; IR (neat): νmax (cm-1) = 2867, 1700, 1400, 1230, 1115, 730 ; HRMS
(ESI): m/z calculated for C13H15NO3 (M+H) = 233.1052, found = 234.1066
`
53 | P a g e
5.2.1 Synthesis of phosphonium salts:
General procedure: A flame-dried round-bottom flask under N2 atmosphere was charged with
alkyl bromide (60 mmol, 1 eq.) in anhydrous toluene (30ml). Triphenylphosphine (60 mmol, 1
eq.) was added and the reaction mixture refluxed for 10h. The resulting mixture was cooled to
room temperature and the precipitate was filtered, washed with toluene, and concentrated in
vacuo to afford a white solid.
(n-Ethyl)triphenylphosphonium bromide (2):
Prepared from bromoethane (13.5 mL, 180 mmol, 3.00 eq.) and triphenylphosphine
(15.74 g , 60 mmol, 1 eq.) according to the general procedure. The title compound
was isolated as a white solid. Yield 66: 15 g, 40 mmol. 67 %. m.p.: 203 – 206 °C; 1H
NMR (500 MHz, Chloroform-d): δ 7.9 – 7.86 (m, 6H), 7.79 (t, J = 7.6 Hz, 3H), 7.72 (t,
J = 8.1 Hz, 6H), 3.76 (m, 2H), 1.34 (t, J = 7.2 Hz, 3H) ppm; ; 13C-NMR (500 MHz, Chloroform-d): δ
135(CH), 133.7(CH), 130.4(CH), 118.9 (C), 23 (CH2), 15 (CH3) ppm; IR (neat): νmax (cm-1) = 2847,
1431, 1113 746, 730, 690, 518, 490; HRMS (ESI): m/z calculated for C20H20BrP (M+H) = 370.0486,
found = 291.1303
(n-Propyl)triphenylphosphonium bromide (3):
Prepared from bromopropane (5.45 mL, 60 mmol, 1,00 eq.) and
triphenylphosphine (15.74 g ,60 mmol, 1 eq.) according to the general
procedure. The title compound was isolated as a white solid. Yield 33: 15 g, 39
mmol, 65 %. m.p.: 220-223 1H NMR (500 MHz, Chloroform-d): δ 7.88– 7.83 (m, 6H), 7.81 (t, J =
7.6 Hz, 3H), 7.72 (t, J = 8.1 Hz, 6H), 3.78 (m, 2H), 2.04 (m, 2H), 1.58 (t, J = 7 Hz, 3H) ppm; ; 13C-
NMR (500 MHz, Chloroform-d): δ 138(CH), 134 (CH), 130(CH), 118 (C), 26 (CH2), 21 (CH2), 18 (CH3)
ppm; IR (neat): νmax (cm-1) = 2834, 1430, 1122 745, 735, 678, 593 565,, 572, 440; HRMS (ESI):
m/z calculated for C21H22BrP (M+H) = 384. 0642, found = 305.1344
(n-butyl)triphenylphosphonium bromide (4):
Prepared from bromobutane (6.48 mL, 60 mmol, 1,00 eq.) and
triphenylphosphine (15.74 g ,60 mmol, 1 eq.) according to the general procedure.
The title compound was isolated as a white solid. Yield 36: 20.2 g, 50 mmol, 84 %.
m.p.: 225-227 1H NMR (500 MHz, Chloroform-d): δ 7.93 – 7.89 (m, 6H), 7.81 (t, J
= 7.7 Hz, 3H), 7.76 (t, J = 8.3 Hz, 6H), 3.86 (m, 2H), 1. 63 (m, 4H), 1.12 (t, J = 6.9 Hz, 3H) ppm; ; 13C-
NMR (500 MHz, Chloroform-d): δ 136(CH), 133.9(CH), 131.4(CH), 119.2 (C), 24 (CH2), 22 (CH2),
19.8 (CH2), 11 (CH3) ppm; IR (neat): νmax (cm-1) = 2849, 1428, 1119 744, 722, 680, 605, 580, 466;
HRMS (ESI): m/z calculated for C22H24BrP (M+H) = 398.0799, found = 319.1199
`
54 | P a g e
(Isobutyl)triphenylphosphonium bromide (5):
Prepared from 1-bromo-2-methylpropane (6.52 mL, 60 mmol, 1.00 eq.) and
triphenylphosphine (15.74 g.60 mmol, 1 eq.) according to the general procedure.
The title compound was isolated as a white solid. Yield 47: 50.88 g, 14.7 mmol,
24.5 %. m.p: 194-197 °C; 1H NMR (500 MHz, Chloroform-d): δ 7.89 – 7.81 (m, 6H),
7.77 (t, J = 7.7 Hz, 3H), 7.69 (t, J = 8.0 Hz, 6H), 3.70 (dd, J = 13.3, 6.2 Hz, 2H), 2.08 – 2.02 (m,1H),
1.04(d, J = 6.7 Hz, 6H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 134.9(CH), 133.5(CH),
130.2(CH), 118.6 (C), 30.3 (CH2), 24.5 (CH), 24.2 (CH3) ppm; IR (neat): νmax (cm-1) = 2850, 1434,
1105, 748, 690, 528, 490; HRMS (ESI): m/z calculated for C22H24BrP (M+H) 398.0799, found =
319.1609
(Isopropyl)triphenylphosphonium bromide (6):
Prepared from 2-bromopropane (5.6 mL, 60 mmol, 1.00 eq.) and
triphenylphosphine (15.74 g ,60 mmol, 1 eq.) according to the general procedure.
The title compound was isolated as a white solid. Yield 38: 2.31g, 6 mmmol, 10%,
m.p.: 212-214 °C; 1H NMR (500 MHz, Chloroform-d): δ 7.89 – 7.81 (m, 6H), 7.77 (t, J = 7.7 Hz, 3H),
7.69 (t, J = 8.0 Hz, 6H), 3.70 (dd, J = 13.3, 6.2 Hz, 2H), 2.08 – 2.02 (m,1H), 1.04(d, J = 6.7 Hz, 6H)
ppm; 13C-NMR (500 MHz, Chloroform-d): δ 134.9(CH), 133.5(CH), 130.2(CH), 118.6 (C), 30.3 (CH2),
24.5 (CH), 24.2 (CH3) ppm; IR (neat): νmax (cm-1) = 2850, 1434, 1105, 748, 690, 528, 490; HRMS
(ESI): m/z calculated for C22H24BrP (M+H) 384.0642, found = 308.1466
5.2.2 Synthesis of CBz-protected 4-substituted piperidones:
General procedure: A flame-dried round-bottom flask under N2 atmosphere was charged with
phosphonium salt (1.5 eq.) in anhydrous THF and cooled to -78 °C. N-butyl lithium (1.6 N in
hexanes, 1.4 eq.) was added dropwise and the resulting solution was stirred for 1 hour. The
temperature was raised to 0 °C and the mixture stirred for 1 hour. Piperidone ( 1 eq.) was added
dropwise over a period of 20 min at - 78°C and stirred for 12h at room temperature. The reaction
mixture was then quenched with NH4Cl aq. The whole mixture was extracted with ethyl acetate
(3x), and the combined organic layers were washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by column chromatography on
silica gel using the eluent indicated below.
`
55 | P a g e
Benzyl 4-ethylidenepiperidine-1-carboxylate (7):
Prepared from (n-Ethyl)triphenylphosphonium bromide (8.75 g, 30 mmol, 1.5
eq.) in THF (150ml), n-Butyl lithium (17.5 mL, 28 mmol, 1.4 eq.) and benzyl 4-
oxocyclohexane-1-carboxylate (4.66 g, 20 mmol, 1.00 eq.) according to the
general procedure. Column chromatography [Ethyl acetate/cyclohexane
(1:10)] gave a yellow liquid. Yield 69: 2.94 g, 12 mmol, 60%. TLC
(cyclohexane/Ethyl acetate, 10 : 1 v/v): Rf = 0.28; 1H NMR (500 MHz,
Chloroform-d): δ 7.42 – 7.28 (m, 5H), 5.29 (t, J = 7.0, 6.4 Hz, 1H), 5.15 (s, 2H), 3.47 (t, J = 6.2 Hz,
4H), 2.19 (d, J = 41.1 Hz, 4H), 1.60 (d, J = 6.7 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ
155.15 (C), 136.78 (C), 134.82 (C), 128.35 (CH), 127.81, (CH), 127.71 (CH), 118.17 (CH), 66.89
(CH2), 45.66 (CH2), 44.63 (CH2), 12.54 (CH3) ppm; IR (neat): νmax (cm-1) = 2860, 1693, 1425, 1250,
1110, 1014, 401; HRMS (ESI): m/z calculated for C15H19NO2 (M+H) 245.1416, found = 246.1477
Benzyl 4-propylidenepiperidine-1-carboxylate (8):
Prepared from (n-propyll)triphenylphosphonium bromide (12 g, 31.14 mmol, 1.5
eq.) in THF (150ml), n-Butyl lithium (18.17 mL, 29 mmol, 1.4 eq.) and benzyl 4-
oxocyclohexane-1-carboxylate (4.84 g, 20.8 mmol, 1,00 eq.) according to the
general procedure. Column chromatography [Ethyl acetate/cyclohexane (1:10)]
gave a yellow liquid. Yield 41: 3.27 g, 12.6 mmol, 61 %. TLC (cyclohexane/Ethyl
acetate, 10 : 1 v/v): Rf = 0.29; 1H NMR (500 MHz, Chloroform-d): δ 7.34 (dd, J =
24.2, 4.1 Hz, 5H), 5.23 (t, J = 6.7 Hz, 1H), 5.15 (s, 2H), 3.51 – 3.39 (m, 4H), 2.18 (d, J = 37.3 Hz, 4H),
2.01 (q, J = 7.1 Hz, 2H), 0.95 (t, J = 7.5 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ
155.14(C), 136.79 (C), 133.60 (C), 128.35 (CH), 127.81 (CH) 127.71 (CH), 126.13 (CH), 66.90 (CH2),
45.74(CH2), 44.87 (CH2), 20.26 (CH2), 14.53 (CH3) ppm; IR (neat): νmax (cm-1) = 2900, 1695, 1425,
1220, 1112, 1095, 985, 696; HRMS (ESI): m/z calculated for C16H21NO2 (M+H) 259.1572, found =
260.1634
Benzyl 4-propylidenepiperidine-1-carboxylate (9):
Prepared from (n-butyll)triphenylphosphonium bromide (20 g, 50 mmol, 1.3 eq.) in
THF (200ml), n-Butyl lithium (28.84 mL, 46.2 mmol, 1.2 eq.) and benzyl 4-
oxocyclohexane-1-carboxylate (8.97 g, 38.5 mmol, 1 eq.) according to the general
procedure. Column chromatography [Ethyl acetate/cyclohexane (1:10)] gave a
yellow liquid. Yield 44: 5.77 g, 21.12 mmol, 55 %. TLC (cyclohexane/Ethyl acetate,
10:1 v/v): Rf = 0.31; 1H NMR (500 MHz, Chloroform-d): δ 7.34 (dd, J = 24.0, 4.2 Hz,
5H), 5.23 (t, J = 7.1 Hz, 1H), 5.15 (s, 2H), 3.51 – 3.43 (m, 4H), 2.19 (d, J = 32.5 Hz, 4H), 1.98 (q, J =
7.0 Hz, 2H), 1.35 (h, J = 7.3 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-
d): δ 155.40 (C), 137.06 (C), 134.60 (C), 128.62 (CH), 128.08 (CH) 127.98 (CH), 124.54 (CH), 67.16
(CH2), 46.07(CH2), 45.15 (CH2), 29.28 (CH2), 23.20 (CH2), 13.89 (CH3) ppm; IR (neat): νmax (cm-1)
`
56 | P a g e
= 2956, 1697, 1425, 1217, 1113, 1099, 696, ; HRMS (ESI): m/z calculated for C17H23NO2 (M+H)
273.1729, found = 274.1790
Benzyl 4-propylidenepiperidine-1-carboxylate (10):
Prepared from (Isobutyl)triphenylphosphonium bromide (10 g, 25 mmol, 1.3 eq.)
in THF (150ml), n-Butyl lithium (14.45 mL, 23.12 mmol, 1.2 eq.) and benzyl 4-
oxocyclohexane-1-carboxylate (4.5 g, 19.26 mmol, 1 eq.) according to the general
procedure. Column chromatography [Ethyl acetate/cyclohexane (1:10)] gave a
yellow liquid. Yield 53: 1.23 g, 4.5 mmol, 23.4 %. TLC (cyclohexane/Ethyl acetate,
10:1 v/v): Rf = 0.34; 1H NMR (500 MHz, Chloroform-d): δ 7.30 – 7.26 (m, 5H), 5.9 (t, J = 1.6 Hz,
1H), 6.77 (s, 2H), 4.39 – 4.36 (m, 4H), 4.08 (d, J = 29.5 Hz, 4H), 2.54-2.47 (m, 1H), 1.23 (d, J = 6.8,
6H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 156 (C), 138 (C), 136 (C), 128.76 (CH), 128.18 (CH)
129.098 (CH), 124.34 (CH), 68 (CH2), 43.6 (CH2), 28.09 (CH2), 28.3 (CH), 23.98 (CH3) 23.34 (CH2)
ppm; IR (neat): νmax (cm-1) = 2956, 1697, 1425, 1210, 1114, 1101, 696; HRMS (ESI): m/z
calculated for C17H23NO2 (M+H) 273.1729, found = 274.1801
5.2.3 Synthesis of 4-alkyl piperidines:
General procedure: A flame-dried round-bottom flask under N2 atmosphere was charged with a
solution of CBz-protected piperidone (1 eq.) in methanol and 10 % Pd/C (0.1 eq.). The flask was
evacuated and flushed with H2 (3X) using a balloon. The suspension was stirred at room
temperature under hydrogen atmosphere for 16 h. Upon completion, the suspension was filtered
through celite. The celite was washed with MeOH (3x) and concentrated in vacuo to afford an oil.
4-ethylpiperidine (11):
Prepared from benzyl 4-ethylidenepiperidine-1-carboxylate (2.5 g, 10.2 mmol, 1 eq.) in
MeOH (50mL) and 10 % Pd/C (1.08 g, 1.02 mmol, 0.1 eq.) according to the general
procedure. The title compound was isolated as yellow oil. Yield 70: 1.04 g, 9.19 mmol,
90 %.1H NMR (500 MHz, Chloroform-d): δ 3.06 (d, J = 11.9 Hz, 1H), 2.94 (d, J = 11.4 Hz,
1H), 2.56 (t, J = 12.0 Hz, 1H), 2.36 (s, 1H), 1.88 (t, J = 11.1 Hz, 1H), 1.65 (t, J = 15.6 Hz, 2H),
1.30 – 1.01 (m, 5H), 0.92 – 0.82 (t, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 53.01
(CH2), 47.08 (CH2), 38.31 (CH), 32.40 (CH2), 11.47 (CH3) ppm; IR (neat): νmax (cm-1) = 2986, 2918,
2850, 1461, 1446, 466, 493; HRMS (ESI): m/z calculated for C7H15N (M+H) 113.1204, found
=114.1279
`
57 | P a g e
4-propylpiperidine (12):
Prepared from benzyl 4-propylidenepiperidine-1-carboxylate (3 g, 11.56 mmol, 1 eq.) in
MeOH (50mL) and 10 % Pd/C (1.23 g, 1.16 mmol, 0.1 eq.) according to the general
procedure. The title compound was isolated as yellow oil. Yield 42: 1.31 g, 10.28 mmol,
89 %. 1H NMR (500 MHz, Chloroform-d): δ 3.41 (d, J = 12.5 Hz, 1H), 3.09 (s,1H) 3.02 (d, J
= 10.4 Hz, 1H), 2.80 (t, J = 12.5 Hz, 1H), 2.06 (s, 1H), 1.83 (d, J = 13.6 Hz, 1H), 1.69 - 1.40
(m, 3H), 1.37 – 1.11 (m, 5H), 0.92 – 0.78 (m, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 52.68
(CH2), 44.58 (CH2), 38.34 (CH2), 34.31 (CH), 29.40 (CH2), 14.45 (CH3) ppm; IR (neat): νmax (cm-1)
= 2954, 2893, 2846, 1648, 1452, 442; HRMS (ESI): m/z calculated for C8H17N (M+H) 127.1361 ,
found = 128.1433
4-butylpiperidine (13):
Prepared from benzyl 4-butylidenepiperidine-1-carboxylate (5 g, 18.3 mmol, 1 eq.) in
MeOH (70mL) and 10 % Pd/C (1.95 g, 1.83 mmol, 0.1 eq.) according to the general
procedure. The title compound was isolated as yellow oil. Yield 49: 2.47 g, 17.5 mmol,
95 %. 1H NMR (500 MHz, Chloroform-d): δ 3.07 (d, J = 12.2 Hz, 2H), 2.77 – 2.50 (m, 3H),
1.67 (d, J = 12.9 Hz, 2H), 1.38 – 1.16 (m, 7H), 1.10 (q, J = 14.7, 13.7 Hz, 2H), 0.87 (d, J =
6.4 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 46.74 (CH2), 36.97(CH2), 36.19
(CH), 33.43 (CH2), 28.87 (CH2), 23.02 (CH2), 14.20 (CH3) ppm; IR (neat): νmax (cm-1) = 2916,
2852, 1670, 1444, 493, 468 ; HRMS (ESI): m/z calculated for C9H19N (M+H) 141.1517, found =
145.1593
4-isobutylpiperidine (14):
Prepared from benzyl 4-butylidenepiperidine-1-carboxylate (5 g, 18.3 mmol, 1 eq.) in
MeOH (50mL) and 10 % Pd/C (1.95 g, 1.83 mmol, 0.1 eq.) according to the general
procedure. The title compound was isolated as yellow oil. Yield 49: 2.47 g, 17.5 mmol,
95 %. 1H NMR (500 MHz, Chloroform-d): δ 3.65 (d, 2H), 2.74-2.61 (m, 2H), 1.72-1.54
(m, 3H), 1.42-1.48(m, 1H,), 1.11-0.98 (m, 4H), 0.86 (d, J = 6.4 Hz, 6H). ppm; 13C-NMR
(500 MHz, Chloroform-d): δ 45.9 (CH2), 33.4 (CH2), 32.3 (CH2), 28.4 (CH), 24.4 (CH3),
22.7 (CH) ppm; IR (neat): νmax (cm-1) = 2954, 2901, 2898, 1670, 1458, 476, 435 ; HRMS (ESI):
m/z calculated for C9H19N (M+H) 141.1517, found = 142.1665
`
58 | P a g e
5.3 Synthetic procedure (2): A synthetic strategy towards 4-substituted
piperidinium salts:
5.3.1 Synthesis of 4-alkylpyridines:
General procedure: A flame-dried round-bottom flask under N2 atmosphere was charged with a
solution of diisopropylamine in THF. The reaction mixture was cooled to −78°C and a solution of
n-butyllithium (1.6 M in hexanes) was added dropwise over a period of 20 min. The solution was
stirred at −78°C for 10 min, warmed to 0°C and stirred for 20 min. Subsequently, a solution of 4-
picoline in THF was added using a syringe over a period of 20 min at -78 °C. The resulting mixture
was stirred at –78°C for 20 min, warmed to -40 and stirred for 30 min. A solution of alkyl bromide
in THF was added dropwise over a period of 20 min at -78 °C. Afterwards, the reaction mixture
was stirred for 1 h at -78 °C. The temperature was allowed to reach room temperature while
stirring overnight. Subsequently, the reaction solution was quenched with NH4Cl aq. and washed
with water. The water layer was extracted with ethyl acetate (3x) and the combined organic
layers were washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The
crude product was purified by column chromatography on silica gel using the eluent indicated
below.
4-propylpyridine (15):
Prepared from 4-picoline (1.75 ml, 18 mmol, 1 eq) in THF (40 ml), lithium diisopropyl
amide (2.93 ml, 21.6 mmol, 1.2 eq) and bromoethane (1.34 ml, 18 mmol, 1 eq) according
to the general procedure. Column chromatography [Ethyl acetate/cyclohexane (1:3)]
gave a yellow liquid. Yield 127: 2.10 g, 17.33 mmol, 95 %. TLC (Ethyl acetate/
Cyclohexane/, 1:3 v/v): Rf = 0.29; 1H NMR (500 MHz, Chloroform-d): δ 8.47 (d, J = 4.8 Hz,
2H), 7.09 (d, J = 4.8 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 1.65 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.3 Hz, 3H)
ppm; 13C-NMR (500 MHz, Chloroform-d): δ 151.44 (C), 149.58 (CH), 123.93 (CH), 37.21 (CH2),
23.43 (CH2), 13.65 (CH3) ppm; IR (neat): νmax (cm-1) = 2960, 2933, 2871, 1600, 1413, 794, 626,
445; HRMS (ESI): m/z calculated for C8H11N (M+H) , 121.0891 found = 122. 0969.
`
59 | P a g e
4-butylpyridine (16):
Prepared from 4-picoline (3.21 ml, 33 mmol, 1 eq) in THF (50 ml), lithium diisopropyl
amide (5.37 ml, 39.6 mmol, 1.2 eq) and bromopropane (2.98 ml, 33 mmol, 1 eq)
according to the general procedure. The title compound was isolated as an orange
liquid. Yield 127: 4.33 g, 32.02 mmol, 97 %. 1H NMR (500 MHz, Chloroform-d): δ 8.46
(d, J = 5.1 Hz, 2H), 7.09 (d, J = 5.0 Hz, 2H), 2.59 (t, J = 7.7 Hz, 2H), 1.60 (p, J = 7.6 Hz, 2H),
1.35 (h, J = 7.4 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H ppm; 13C-NMR (500 MHz, Chloroform-d): δ 151.83
(C), 149.73 (CH), 124.03 (CH), 35.05 (CH2), 32.54 (CH2), 22.37 (CH2), 13.97 (CH3). ppm; IR (neat):
νmax (cm-1) = 2965, 2931, 2860, 1600, 1413, 630, 582, 526, 501; HRMS (ESI): m/z calculated for
C9H13N (M+H) 135.1048, found = 136.1121.
4-Pentylpyridine (17):
Prepared from 4-picoline (1.44 ml, 14.8 mmol, 1 eq) in THF (40 ml), lithium diisopropyl
amide (2.40 ml, 17.76 mmol, 1.2 eq) and bromopropane (1.6 ml, 14.8 mmol, 1 eq)
according to the general procedure. Column chromatography [Ethyl
acetate/cyclohexane (1:5)] gave a yellow liquid. Yield 127: 1.94 g, 13.05 mmol, 89%.
TLC (Ethyl acetate/ Cyclohexane/, 1:4 v/v): Rf = 0.3; 1H NMR (500 MHz, Chloroform-
d): δ 8.49 (s, 2H), 7.10 (d, J = 3.5 Hz, 2H), 2.59 (t, J = 7.7 Hz, 2H), 1.62 (p, J = 7.5 Hz,
2H), 1.32 (q, J = 11.0 Hz, 4H), 0.89 (t, J = 6.8 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ
151.87 (C), 149.75 (CH), 124.10 (CH), 35.34 (CH2), 31.47 (CH2), 30.12 (CH2), 22.58 (CH2), 14.11
(CH3) ppm; IR (neat): νmax (cm-1) = 2965, 2927, 2858, 1600, 1413, 630, 536, 503, 496; HRMS
(ESI): m/z calculated for C10H15N (M+H) 149.1204, found = 150.1278.
4-Isobutylpyridine (18):
Prepared from 4-picoline (1.44 ml, 14.8 mmol, 1 eq) in THF (40 ml), lithium diisopropyl
amide (2.40 ml, 17.76 mmol, 1.2 eq) and 2-bromopropane (1.39 ml, 14.8 mmol, 1 eq)
according to the general procedure. Column chromatography [Ethyl
acetate/cyclohexane (1:3)] gave a yellow liquid. Yield 127: 1.52 g, 11.24 mmol, 76 %.
TLC (Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.26; 1H NMR (500 MHz, Chloroform-
d): δ 8.48 (d, J = 4.7 Hz, 2H), 7.07 (d, J = 4.9 Hz, 2H), 2.46 (d, J = 7.2 Hz, 2H), 1.89 (dq, J = 13.8, 6.8
Hz, 1H), 0.91 (d, J = 6.6 Hz, 6H) ppm;13C-NMR (500 MHz, Chloroform-d): δ 150.64 (C), 149.68
(CH), 124.70 (CH), 44.79 (CH2), 29.70 (CH), 22.41 (CH3) ppm; IR (neat): νmax (cm-1) = 2965, 2925,
2898, 1602, 1413, 630, 595, 536, 490;HRMS (ESI): m/z calculated for C9H13N (M+H) 135.1048,
found = 136.1121.
`
60 | P a g e
4-(cyclohexylmethyl)pyridine (19):
Prepared from 4-picoline (1.44 ml, 14.8 mmol, 1 eq) in THF (30 ml), lithium
diisopropyl amide (2.40 ml, 17.76 mmol, 1.2 eq) and bromocyclohexane (1.88 ml,
14.8 mmol, 1 eq) according to the general procedure. Column chromatography
[Ethyl acetate/cyclohexane (1:5)] gave a yellow liquid. Yield 127: 1.15 g, 6.6 mmol,
44%. TLC (Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.32;1H NMR (500 MHz,
Chloroform-d): δ 8.46 (d, J = 4.8 Hz, 2H), 7.05 (d, J = 4.9 Hz, 2H), 2.46 (d, J = 7.1 Hz,
2H), 1.73 – 1.59 (m, 5H), 1.58 – 1.48 (m, 1H), 1.26 – 1.08 (m, 3H), 0.93 (q, J = 11.9 Hz, 2H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 150.37 (C), 149.58 (CH), 124.76 (CH), 43.49 (CH2), 39.14
(CH), 33.12 (CH2), 26.47 (CH2), 26.27 (CH2) ppm; IR (neat): νmax (cm-1) = 2923, 2850, 1600, 1448,
1413, 804, 592,499; HRMS (ESI): m/z calculated for C12H17N (M+H) 175.1361, found = 176.1438.
4-(cyclopropylmethyl)pyridine (20):
Prepared from 4-picoline (3.21 ml, 33 mmol, 1 eq) in THF (50 ml), lithium diisopropyl
amide (5.37 ml, 39.6 mmol, 1.2 eq) and bromocyclopropane (2.64 ml, 33 mmol, 1 eq)
according to the general procedure. Column chromatography [Ethyl
acetate/cyclohexane (1:2)] gave an orange liquid. Yield 103: 1.57 g, 11.8 mmol, 36%.
TLC (Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.3; 1H NMR (500 MHz, Chloroform-
d): δ 8.49 (d, J = 4.9 Hz, 2H), 7.19 (d, J = 4.9 Hz, 2H), 2.53 (d, J = 6.9 Hz, 2H), 0.98 (dt, J
= 12.5, 5.3 Hz, 1H), 0.57 (d, J = 7.7 Hz, 2H), 0.21 (d, J = 4.7 Hz, 2H) ppm; 13C-NMR (500 MHz,
Chloroform-d): δ 151.20 (C), 149.69 (CH), 123.92 (CH), 39.69 (CH2), 10.83 (CH), 4.86 (CH2) ppm;
IR (neat): νmax (cm-1) = 2999, 1601, 1415, 815, 588 ,495, 487, 418, 406; HRMS (ESI): m/z
calculated for C9H11N (M+H) 133.0891, found =134.0969.
4-cyclohexylpyridine (21):
Prepared from 4-picoline (1.75 ml, 18 mmol, 1 eq) in THF (40 ml), lithium diisopropyl
amide (4.88 ml, 36 mmol, 2 eq) and 1,5-dibromopentane (2.45 ml, 18 mmol, 1 eq)
according to the general procedure. Column chromatography [Ethyl
acetate/cyclohexane (1:2)] gave a yellow liquid. Yield 125: 0.7 g, 4.34 mmol, 25%. TLC
(Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.34; 1H NMR (500 MHz, Chloroform-d): δ
8.47 (d, J = 4.7 Hz, 2H), 7.11 (d, J = 4.8 Hz, 2H), 2.48 (s, 1H), 1.85 (s, 4H), 1.75 (d, J = 12.5
Hz, 1H), 1.40 (q, J = 11.3, 10.1 Hz, 4H), 1.31 – 1.18 (m, 1H) ppm; 13C-NMR (500 MHz, Chloroform-
d): δ 156.64 (C), 149.85 (CH), 122.46 (CH), 43.92 (CH), 33.62 (CH2), 33.50 (CH2), 26.64 (CH2) ppm;
IR (neat): νmax (cm-1) = 2923, 2850, 1595, 1448, 811, 622 ,555, 493, 420; HRMS (ESI): m/z
calculated for C11H15N (M+H) 161.1204, found =162.1278.
`
61 | P a g e
4-cyclopentylpyridine (22):
Prepared from 4-picoline (1.75 ml, 18 mmol, 1 eq) in THF (40 ml), lithium diisopropyl
amide (4.88 ml, 36 mmol, 2 eq) and 1, 4-dibromobutane (1.83 ml, 18 mmol, 1 eq)
according to the general procedure. Column chromatography [Ethyl
acetate/cyclohexane (1:2)] gave a yellow liquid. Yield 128: 0.477 g, 3.24 mmol, 18%. TLC
(Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.33; 1H NMR (500 MHz, Chloroform-d): δ
8.47 (d, J = 4.7 Hz, 2H), 7.14 (d, J = 4.8 Hz, 2H), 2.97 (p, J = 8.6 Hz, 1H), 2.12 – 2.00 (m,
2H), 1.84 – 1.77 (m, 2H), 1.74 – 1.66 (m, 2H), 1.58 (p, J = 9.3 Hz, 2H) ppm; 13C-NMR (500 MHz,
Chloroform-d): δ 155.69 (C), 149.72 (CH), 122.76 (CH), 45.24 (CH), 34.04 (CH2), 25.63 (CH2) ppm;
IR (neat): νmax (cm-1) = 2948, 2916, 2867, 1596, 1409, 813, 630 ,545, 493; HRMS (ESI): m/z
calculated for C10H13N (M+H) 147.1048, found =148.1120.
5.3.2 Synthesis of 4-substituted piperidinium salts:
General procedure: To a solution of 4-alkylpyridine in ethanol in a 2-neck round bottom flask,
HCl/methanol (1.25 N) and platinum oxide were respectively added. Subsequently, the flask was
evacuated and flushed with H2 (3X) using a balloon. The mixture was stirred at room temperature
under hydrogen atmosphere for 36 h. Upon completion, the reaction mixture filtered through
Celite with thorough washing (EtOAc) and concentrated in vacuo to afford a solid.
4-ethylpiperidinium chloride (23):
Prepared from 4-ethylpyridine (0.57 ml, 5 mmol, 1 eq) in ethanol (30 ml), HCl/methanol
(20 ml, 25 mmol, 5 eq) and platinum oxide (0.057 g, 0.25 mmol, 0.05 eq) according to the
general procedure. The title compound was isolated as an off-white solid Yield 123: 0.735
g, 4.91 mmol, 98%. m.p.: 170 – 176.1 °C; 1H NMR (500 MHz, Chloroform-d): δ 9.37 (d, J =
158.3 Hz, 2H), 3.45 (d, J = 8.5 Hz, 2H), 2.83 (s, 2H), 1.88 (d, J = 13.2 Hz, 2H), 1.59 (d, J = 11.1
Hz, 2H), 1.46 – 1.25 (m, 3H), 0.88 (t, J = 7.0 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-
d): δ 44.30 (CH2) , 35.91 (CH2), 28.59 (CH) , 28.57 (CH2), 11.10 (CH3) ppm; IR (neat): νmax (cm-1)
= 2973, 2794, 2727, 2497, 1456, 1396, 993, 574, 441; HRMS (ESI): m/z calculated for C7H16ClN
(M+H) 149.0971, found = 148.1113.
4-propylpiperidinium chloride (24):
Prepared from 4-propylpyridine (1 g, 8.26 mmol, 1 eq) in ethanol (50 ml), HCl/methanol
(33.04 ml, 41.3 mmol, 5 eq) and platinum oxide (0.096 g, 0.42 mmol, 0.05 eq) according
to the general procedure. The title compound was isolated as an off-white solid. Yield
133: 1.34 g, 8.19 mmol, 99%. m.p.: 189 – 196.8 °C; 1H NMR (500 MHz, Chloroform-d): δ
9.35 (d, J = 149.9 Hz, 2H), 3.47 (d, J = 8.3 Hz, 2H), 2.87 (s, 2H), 1.93 (d, J = 12.9 Hz, 2H),
1.64 (d, J = 11.4 Hz, 2H), 1.46 – 1.28 (m, 3H), 1.13 (m,2H), 0.84 (t, J = 6.9 Hz, 3H) ppm;
`
62 | P a g e
13C-NMR (500 MHz, Chloroform-d): δ 44.37 (CH2) , 35.98 (CH2), 28.69 (CH) , 28.64 (CH2), 20.36
(CH2) 11.14 (CH3) ppm; IR (neat): νmax (cm-1) = 2941, 2923, 2796, 2718, 1456, 574; HRMS (ESI):
m/z calculated for C8H18ClN (M+H) 163.1128, found = 156.1740
4-butylpiperidinium chloride (25):
Prepared from 4-butylpyridine (1.35 g, 10 mmol, 1 eq) in ethanol (55 ml),
HCl/methanol (40 ml, 50 mmol, 5 eq) and platinum oxide (0.114 g, 0.5 mmol, 0.05 eq)
according to the general procedure. The title compound was isolated as an off-white
solid. Yield 116: 1.65 g, 9.26 mmol, 93%. m.p.: 180 – 193 °C; 1H NMR (500 MHz,
Chloroform-d): δ 9.37 (d, J = 151.5 Hz, 2H), 3.44 (d, J = 11.3 Hz, 2H), 2.82 (d, J = 10.3
Hz, 2H), 1.85 (d, J = 13.6 Hz, 2H), 1.59 (q, J = 12.2, 11.6 Hz, 2H), 1.45 (s, 1H), 1.26 (s,
6H), 0.85 (t, J = 6.7 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.21 (CH2), 35.47 (CH2),
34.20 (CH), 28.88 (CH2), 28.67 (CH2), 22.76 (CH2), 14.09 (CH3) ppm; IR (neat): νmax (cm-1) = 2947,
2920, 2844, 2792, 2769, 2719, 1448, 576 ; HRMS (ESI): m/z calculated for C9H20ClN (M+H)
177.1244, found = 176.1421
4-pentylpiperidinium chloride (26):
Prepared from 4-pentylpyridine (1 g, 6.7 mmol, 1 eq) in ethanol (35 ml),
HCl/methanol (27ml, 33.5 mmol, 5 eq) and platinum oxide (0.08 g, 0.335 mmol, 0.05
eq) according to the general procedure. The title compound was isolated as an off-
white solid. Yield 134: 1.16 g, 6.05 mmol, 90%. m.p.: 178 –191 °C; 1H NMR (500
MHz, Chloroform-d): δ 9.39 (d, J = 160.0 Hz, 2H), 3.45 (d, J = 8.9 Hz, 2H), 2.91 – 2.73
(m, 2H), 1.87 (d, J = 13.0 Hz, 2H), 1.60 (d, J = 11.3 Hz, 2H), 1.47 (s, 1H), 1.26 (d, J =
14.6 Hz, 8H), 0.86 (t, J = 6.6 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.28 (CH2), 35.76
(CH2), 34.26 (CH), 31.91 (CH2), 28.94 (CH2), 26.17 (CH2), 22.65 (CH2), 14.14 (CH3) ppm; IR (neat):
νmax (cm-1) = 2945, 2910 2846, 2792, 2767, 2734, 1448, 1074, 576, 437 ; HRMS (ESI): m/z
calculated for C10H22ClN (M+H) 191.1441, found = 200.1999
4-(isobutyl)piperidinium chloride (27):
Prepared from 4-isobutylpyridine (1.03 g, 7.61 mmol, 1 eq) in ethanol (38 ml),
HCl/methanol (30.44ml, 38.05 mmol, 5 eq) and platinum oxide (0.09 g, 0.381 mmol,
0.05 eq) according to the general procedure. The title compound was isolated as an
off-white solid. Yield 131: 1 g, 7.03 mmol, 92%. m.p.: 175 – 195 °C; 1H NMR (500
MHz, Chloroform-d): δ 9.41 (d, J = 161.8 Hz, 2H), 3.46 (d, J = 9.9 Hz, 2H), 2.84 (s, 2H),
1.85 (d, J = 9.6 Hz, 2H), 1.61 (dd, J = 17.0, 10.0 Hz, 4H), 1.17 (s, 2H), 0.86 (d, J = 6.2 Hz,
6H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 45.11 (CH2), 44.28 (CH2), 31.84 (CH), 29.07 (CH2),
24.52 (CH), 22.75 (CH3) ppm; IR (neat): νmax (cm-1) = 2890, 2796, 2769, 2736, 1448, 595, 499;
HRMS (ESI): m/z calculated for C9H20ClN (M+H) 177.1284, found = 176.1423
`
63 | P a g e
4-(tert-butyl)piperidinium chloride (28):
Prepared from 4-(tert-butyl)pyridine (1.35 g, 10 mmol, 1 eq) in ethanol (55 ml),
HCl/methanol (40 ml, 50 mmol, 5 eq) and platinum oxide (0.114 g, 0.5 mmol, 0.05 eq)
according to the general procedure. The title compound was isolated as an off-white
solid. Yield 130: 1.73 g, 9.73 mmol, 98%. m.p.: 247 – 293 °C; 1H NMR (500 MHz,
Chloroform-d): δ 9.39 (d, J = 150.6 Hz, 2H), 3.53 (d, J = 12.2 Hz, 2H), 2.79 (q, J = 11.9 Hz,
2H), 1.85 (d, J = 13.8 Hz, 2H), 1.70 (q, J = 13.0 Hz, 2H), 1.21 (t, J = 12.0 Hz, 1H), 0.87 (s,
9H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.95 (CH), 44.85 (CH2), 32.42 (C), 27.15 (CH3),
24.02 (CH2) ppm; IR (neat): νmax (cm-1) = 2960, 2837, 2798, 2777, 2761, 2744, 2719, 1448, 1396,
1361, 1078, 568, 509; HRMS (ESI): m/z calculated for C9H20ClN (M+H) 177.1284, found = 176.1424
4-(cyclohexylmethyl)piperidinium chloride (29):
Prepared from 4-(cyclohexylmethyl)pyridine (0.75 g, 4.8 mmol, 1 eq) in ethanol (30
ml), HCl/methanol (19.2 ml, 24 mmol, 5 eq) and platinum oxide (0.055 g, 0.24
mmol, 0.05 eq) according to the general procedure. The title compound was
isolated as an off-white solid. Yield 93: 0.68 g, 3.73 mmol, 78 %. m.p.: 267 – 283
°C; 1H NMR (500 MHz, Chloroform-d): δ 9.40 (d, J = 164.6 Hz, 2H), 3.59 – 3.37 (m,
2H), 2.93 – 2.73 (m, 2H), 1.85 (d, J = 8.7 Hz, 2H), 1.65 (q, J = 18.2 Hz, 8H), 1.36 – 1.06
(m, 6H), 0.85 (q, J = 11.0 Hz, 2H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.31
(CH2), 43.66 (CH2), 34.11 (CH), 33.56 (CH2), 31.09 (CH), 29.22 (CH2), 26.66 (CH2), 26.37 (CH2) ppm;
IR (neat): νmax (cm-1) = 2945, 2849, 2821, 2785, 2763, 2729, , 1446, 1074, 950, 597; HRMS (ESI):
m/z calculated for C12H24ClN (M+H) 217.1597, found = 216.1896
4-(cyclopropylmethyl)piperidinium chloride (30):
Prepared from 4-(propylmethyl)pyridine (1.33 g, 10 mmol, 1 eq) in ethanol (55 ml),
HCl/methanol (40 ml, 50 mmol, 5 eq) and platinum oxide (0.114 g, 0.5 mmol, 0.05
eq) according to the general procedure. The title compound was isolated as an off-
white solid. Yield 115: 1.52 g, 8.65 mmol, 86 %. m.p.: 170 – 174.8 °C; 1H NMR (500
MHz, Chloroform-d): δ 9.40 (d, J = 163.6 Hz, 2H), 3.47 (d, J = 10.5 Hz, 2H), 2.96 –
2.78 (m, 2H), 1.96 (d, J = 9.9 Hz, 2H), 1.63 (s, 3H), 1.21 (s, 2H), 0.63 (s, 1H), 0.43 (d,
J = 7.6 Hz, 2H), 0.33 (d, J = 4.2 Hz, 2H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.31 (CH2),
40.86 (CH2), 35.23 (CH), 28.95 (CH2), 8.37 (CH), 4.62 (CH2) ppm; IR (neat): νmax (cm-1) = 2939,
2839, 2794, 2769, 2733, 2700, 1591, 1454, 1074, 1010, 819, 590, 497; HRMS (ESI): m/z calculated
for C9H18ClN (M+H) 175.1128, found = 174.1424
`
64 | P a g e
4-cyclohexylpiperidinium chloride (31):
Prepared from 4-cyclohexylpyridine (0.776 g, 5 mmol, 1 eq) in ethanol (30 ml),
HCl/methanol (20 ml, 25 mmol, 5 eq) and platinum oxide (0.057 g, 0.25 mmol, 0.05 eq)
according to the general procedure. The title compound was isolated as an off-white
solid. Yield 129: 0.91 g, 4.5 mmol, 89 %. m.p.: 180 – 222 °C; 1H NMR (500 MHz,
Chloroform-d): δ 9.43 (d, J = 163 Hz, 2H), 3.47 (d, J = 9.8 Hz, 2H), 2.78 (m, 2H), 1.88 –
1.63 (m, 10H), 1.26 – 1.20 (m, 4H), 1.22 (q, J = 10.3 Hz, 2H) ppm; 13C-NMR (500 MHz,
Chloroform-d): δ 44.97 (CH), 44.86 (CH2), 36.23 (CH), 29.34 (CH2), 27.32 (CH2) 13.65
(CH2), 11.65 (CH2) ppm; IR (neat): νmax (cm-1) = 2964, 2848, 2794, 2777, 2727, 1450, 541; HRMS
(ESI): m/z calculated for C11H22ClN (M+H) 203.1441, found = 202.1745
4-cyclopentylpiperidinium chloride (32):
Prepared from 4-cyclopentylpyridine (0.2 g, 1.36 mmol, 1 eq) in ethanol (10 ml),
HCl/methanol (5.44 ml, 6.8 mmol, 5 eq) and platinum oxide (0.015 g, 0.07 mmol, 0.05
eq) according to the general procedure. The title compound was isolated as an off-
white solid. Yield 136: 0.247 g, 1.3 mmol, 96 %. m.p.: 259 – 298 °C; 1H NMR (500
MHz, Chloroform-d): δ 9.29 (d, J = 213.0 Hz, 2H), 3.49 (s, 2H), 2.85 (s, 2H), 2.04 – 1.44
(m, 11H), ), 1.27 (m, 1H) 1.09 (s, 2H).ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.88
(CH), 44.77 (CH2), 36.22 (CH), 29.45 (CH2), 27.55 (CH2), 11.65 (CH2) ppm; IR (neat): νmax (cm-1)
= 2966, 2850, 2792, 2765, 2738, 2704, 2495, 1589, 1444, 1076, 522; HRMS (ESI): m/z calculated
for C10H20ClN (M+H) 189.1284, found = 188.1588
5.4 Synthetic procedure (3): The 3 component Ugi reaction towards 4-
substituted piperidyl peptides.
General procedure: Imine was dissolved in 9 ml of DCM together with the substituent addition
of isocyanide and carboxylic acid. The mixture was stirred at r.t for 48 h and upon completion 9
ml of DCM were added. The resulting mixture was washed with Na2CO3 (2x12ml), dried over
Na2SO4, filtered and concentrated in vacuo.
1-acetyl-N-(tert-butyl)-4-methylpiperidine-2-carboxamide (33):
Prepared from 4-methyl-2,3,4,5-tetrahydropyridine (0.097 g, 1 mmol, 1 eq), tert-
butyl isocyanide (0.15 ml, 1.33 mmol, 1.33 eq) and acetic acid (0.077 g, 1.33 mmol,
1.33 eq) in DCM (9 ml) according to the general procedure. The title compound
was isolated as yellow solid. Yield 109: 0.177 g, 0.74 mmol, 74%. m.p.: 85.4 - 91
°C; 1H NMR (500 MHz, Chloroform-d): δ 5.96 (s, 1H), 5.13 (d, J = 5.8 Hz, 1H), 3.71
(d, J = 13.6 Hz, 1H), 3.13 (t, J = 13.4 Hz, 1H), 2.28 – 2.07 (m, 3H), 1.62 (s, 4H), 1.33 (d, J = 20.4 Hz,
`
65 | P a g e
9H), 0.92 (d, J = 6.6 Hz, 3H). ppm; 13C-NMR (500 MHz, Chloroform-d): δ 171.04 (C), 170.58(C),
52.88 (C), 44.72 (CH), 40.00 (CH2) 34.31 (CH2) , 33.93 (CH2), 29.15 (CH3), 27.14 (CH), 22.43 (CH3),
22.20 (CH3) ppm; IR (neat): νmax (cm-1) = 2988.42, 1683.74, 1558.38, 1541.02, 1458.08, 1419.51;
HRMS (ESI): m/z calculated for C13H24N2O2 (M+H) 240.1838, found = 240.1911
1-acetyl-N-isopropyl-4-methylpiperidine-2-carboxamide (34):
Prepared from 4-methyl-2,3,4,5-tetrahydropyridine (0.097 g, 1 mmol, 1 eq),
isopropyl isocyanide (0.13 ml, 1.33 mmol, 1.33 eq) and acetic acid (0.077 g, 1.33
mmol, 1.33 eq) in DCM (9 ml) according to the general procedure. The title
compound was isolated as yellow oil. Yield 110: 0.116 g, 0.52 mmol, 52%. 1H NMR
(500 MHz, Chloroform-d): δ 5.90 (d, J = 7.9 Hz, 1H), 5.17 (d, J = 5.9 Hz, 1H), 4.01
(dt, J = 13.8, 6.9 Hz, 1H), 3.71 (d, J = 13.7 Hz, 1H), 3.13 (t, J = 13.4 Hz, 1H), 2.27 – 2.03 (m, 4H),
2.03 – 1.87 (m, 1H), 1.66 (d, J = 13.4 Hz, 1H), 1.10 (qd, J = 14.2, 12.9, 6.1 Hz, 7H), 0.92 (d, J = 6.5
Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 170.83 (C), 170.07 (C), 52.20 (CH), 44.46 (CH2),
41.39 (CH), 34.02 (CH2), 33.73 (CH2), 26.91 (CH), 22.72 (CH3) 22.15 (CH3), 21.95 (CH3) ppm; IR
(neat): νmax (cm-1) = 2925.81, 1697.33, 1533.30, 1429.15 1365.51, 981.70; HRMS (ESI): m/z
calculated for C18H26N2O2 (M+H) 226.1681, found = 227.1751
1-benzoyl-N-(tert-butyl)-4-methylpiperidine-2-carboxamide (35):
Prepared from 4-methyl-2,3,4,5-tetrahydropyridine (0.097 g, 1 mmol, 1 eq),
tert-butyl isocyanide (0.15 ml, 1.33 mmol, 1.33 eq) and benzoic acid (0.17 g,
1.33 mmol, 1.33 eq) in DCM (9 ml) according to the general procedure. Column
chromatography [Ethyl acetate/cyclohexane (1:3)] gave a white solid. Yield
111: 0.24 g, 0.35 mmol, 35%. m.p.: 81 – 89.5 °C; 1H NMR (500 MHz,
Chloroform-d): δ 7.42 (q, J = 7.9 Hz, 5H), 6.42 (s, 1H), 5.19 (d, J = 5.5 Hz, 1H),
3.68 (d, J = 13.7 Hz, 1H), 3.03 (t, J = 13.4 Hz, 1H), 2.26 (d, J = 14.1 Hz, 1H), 1.65 (s, 1H), 1.36 (s,
9H), 1.27 – 1.04 (m, 2H), 0.96 (d, J = 6.6 Hz, 3H). ppm; 13C-NMR (500 MHz, Chloroform-d): δ 172.15
(C), 170.05 (C), 135.31 (C), 130.17 (CH), 128.58 (CH), 126.96 (CH), 53.31 (CH), 50.97 (C), 45.87
(CH2), 34.00 (CH2), 33.47 (CH2), 28.79 (CH3), 26.94 (CH), 22.05 (CH3) ppm; IR (neat): νmax (cm-1)
= 2954.33, 1672.17, 1630.23, 1533.30, 1450.37, 1413.72, 1321.15, 1207.36; HRMS (ESI): m/z
calculated for C12H22N2O2 (M+H) 302.1994, found = 303.2064.
N-(tert-butyl)-4-methyl-1-pivaloylpiperidine-2-carboxamide (36):
Prepared from 4-methyl-2,3,4,5-tetrahydropyridine (0.097 g, 1 mmol, 1 eq),
tert-butyl isocyanide (0.15 ml, 1.33 mmol, 1.33 eq) and pivalic acid (0.14 g, 1.33
mmol, 1.33 eq) in DCM (9 ml) according to the general procedure. The title
compound was isolated as white solid. Yield 112: 0.20 g, 0.67 mmol, 67 %. m.p.:
88 – 99.3 °C; 1H NMR (500 MHz, Chloroform-d): δ 6.05 (s, 1H), 5.07 (d, J = 5.4
Hz, 1H), 4.15 (d, J = 13.7 Hz, 1H), 2.91 (t, J = 13.4 Hz, 1H), 2.35 – 2.13 (m, 1H),
1.62 (d, J = 13.1 Hz, 1H), 1.29 (s, 18H), 1.11 – 0.98 (m, 2H), 0.90 (d, J = 6.5 Hz, 3H) ppm; 13C-NMR
`
66 | P a g e
(500 MHz, Chloroform-d): δ 178.28 (C), 170.45 (C), 50.98 (C), 44.44 (C), 39.00 (CH2), 33.93 (CH2),
33.63 (CH2), 31.05 (CH) 28.87 (CH3), 28.32 (CH3), 27.15 (CH), 22.15 (CH3) ppm; IR (neat): νmax
(cm-1) = 2867.33, 1687.21, 1602.74, 1514.02, 1473.51, 1458.08, 1363.58; HRMS (ESI): m/z
calculated for C16H30N2O2 (M+H) 282.2307, found = 305.2198.
`
67 | P a g e
References
[1] K. C. NIcolaou, E. J. Sorensen in Classics in Total Synthesis: Targets, Strategies, Methods, Vol. 1, Wiley-VCH, New York, 1996.
[2] P. T. Anastas, J. C. Warner in Green Chemistry: Theory and Practice, Vol. 1, Oxford University Press, New York, 1998.
[3] N. Eghbali, P. T Anastas, Chem. Soc. Rev. 2010, 39, 301.
[4] P. T. Anastas, T. C. Williamson in Green Chemistry: Designing Chemistry for the Environment, Vol. 1, American Chemical Series Books, Washington DC, 1996.
[5] R. Carson in Silent Spring, Vol. 1, Houghton Mifflin, United States, 1962.
[6] J. B. Hendrickson, J. Am. Chem. Soc. 1975, 20, 5784.
[7] P. A. Wender, S. T Handy, D. L Wright, Chemistry & Industry. 1997, 765.
[8] I. Ugi, A. Dömling, Angew. Chem. Int. Ed. 2000, 39, 3168.
[9] S. Wenda, S. Illner, A. Annett, U. Kragl, Green Chem. 2011, 13, 3007.
[10] I. Ugi, A. Dömling, H. Werner, Endeavour. 1994, 18, 115.
[11] A. Strecker, Justus Liebigs Ann. Chem. 1850, 75, 27.
[12] C. Mannich, W. Krösche, Arch. Pharm. (Weinheim). 1912, 250, 647.
[13] A. F. da RosaI, A. Ricardo, M. G. NascimentoII, J. Braz. Chem. Soc. 2003, 14, 11.
[14] M. Arend, Angew. Chem. Int. Ed. 1998, 37, 1044.
[15] M. Passerini, Gazz. Chim. Ital. 1921, 51, 126.
[16] I. Ugi in Isonitrile Chemistry, Vol. 20, Academic Press, New York, 1971.
[17] R. Ramozzi, New J. Chem. 2012, 5, 1137.
[18] I. Hagedorn, U. Eholzer, Chem. Ber. 1965, 98, 936.
[19] I. Ugi, R. Meyr, Chem. Ber. 1961, 94, 2229.
[20] I. Ugi, Angew. Chem. Int. Ed. Engl. 1962, 1, 8.
`
68 | P a g e
[21] I. Ugi, Angew. Chem. 1959, 71, 386.
[22] I. Ugi, C. Steinbrückner, Angew. Chem. 1960, 72, 267.
[23] I. Ugi, F. Rosendahl, F. Bodesheim, Liebigs Ann. Chem. 1963, 54, 666.
[24] R. S. Bon, C. Hong, M. J. Bouma, R. F. Schmitz, Org. Lett. 2003, 5, 3759.
[25] R. C. Wende, P. R. Schreiner, Green Chem. 2012, 14, 1821.
[26] S. Wang, Angew. Chem. Int. Ed. 2008, 47, 388.
[27] F. Shi, Adv. Synth. Catal. 2013, 355, 1605.
[28] C. Razvan, E. Ruijter, R.V.A. Orru, Green Chem. 2014, 16, 2958.
[29] N. A. Petasis, I. A. Zavialov, J. Am. Chem. Soc. 1998, 45, 11798.
[30] J. Gal, Chirality. 2008, 20, 5.
[31] J. Monod, J. Mol. Biol. 1965, 12, 88.
[32] L. Rosenthaler, Biochem. Z. 1913, 14, 238.
[33] J. B. Summer, J. Biol. Chem. 1926, 69, 435.
[34] G. M. Whitesides, C. Wong, Angew. Chem. Int. Ed. Engl. 1985, 24, 617.
[35] S. S. Hughes, Isis. 2001, 92, 541.
[36] United States environmental protection agency. 2014, Presidential green chemistry challenge winners, Retrieved from http://www.epa.gov/
[37] H. Hailes, P. Dalby, P. A Woodley, J. Chem. Technol. Biotechnol, 2007, 82, 1063.
[38] A. Schmid, J. S. Dordick, Nature. 2001, 409, 258.
[39] M. T. Reetz, J. Am. Chem. Soc. 2013, 34, 12480.
[40] S. Lutz, U. Bornscheuer in Protein engineering handbook, Vol. 3, Wiley-VCH Verlag GmbH & Co. KGaA, Atlanta, 2012.
[41] R. K. Singh, Int. J. Mol. Sci. 2013, 14, 1232.
[42] Y. Watanabe, Curr. Opin. Chem. Biol. 2002, 6, 208.
`
69 | P a g e
[43] S. Park, K. Morley, G. Horsman, M. Holmquist, K. Hult, R. Kazlauskas, Chem. Biol. 2005, 12, 45.
[44] M. Brouk, Y. Nov, A. Fishmam, Appl. Environ. Microbiol. 2010, 76, 6397.
[45] S. Srikrishnan, A. Randall, P. Baldi, N. A. Da Silva, Biotechnol. Bioeng. 2012, 109, 1595.
[46] M. T. Reetz, B. Brunner, T. Schneider, F. Schulz, C. M. Clouthier, M. M. Kayser, Angew. Chem. Int. Ed. Engl. 2004, 43, 4075.
[47] E. L. Ernest, W. H. Samuel in Topics in stereochemstry, Vol. 18, John Wiley and Sons, New York, 1988.
[48] T. Hudlicky, T. Tsunoda, K. G. Gadamasetti, J. A. Murry, G. E. Keck, J. Org. Chem. 1991,11, 3619.
[49] S. Rodríguez, K. T. Schroeder, M. M. Kayser, J. D. Stewart, J. Org. Chem. 2000, 8, 2586.
[50] A. Goswami, T. P. Kissick, Org. Process Res. Dev. 2009, 3, 483.
[51] F. Balkenhohl, K. Ditrich, B. Hauer, W. Ladner, Adv. Synth. Catal. 1997, 339, 381.
[52] B. Wang, H. Land, P. Berglund, Chem. Commun. 2013, 49, 161.
[53] P. Odman, J. Org. Chem. 2005, 70, 9551.
[54] G. DeSantis, J. Am. Chem. Soc. 2002, 124, 9024.
[55] I. J. Colton, S. N. Ahmed, R. J. Kazlauskas, J. Org. Chem. 1995, 1, 212.
[56] X. Liu, D. S. Clark, J. S. Dordick, Biotechnol. Bioeng. 2000, 69, 457.
[57] B. Schnell, W. Krenn, K. Faber, C. O. Kappe, J. Chem. Soc. , Perkin Trans. 1. 2000, 24, 4382.
[58] R. Kourist, G. Nguyen, D. Strübing, D. Böttcher, K. Liebeton, C. Naumer, J. Eck, U. T. Bornscheuer, Asymmetry. 2008, 19, 1839.
[59] S. Klossowski, A. Redzej, S. Szymkuc, R. Ostaszewski, ARKIVOC. 2013, 4, 134.
[60] R. C. Larock, N. H. Lee, J. Am. Chem. Soc. 1991, 113, 7815.
[61] B. V. S. Reddy, N. Majumder, R. T. Prabhakar, B. Bridhar, Tetrahedron Lett. 2012, 53, 2273.
`
70 | P a g e
[62] D. C. Dylan, M. Turner, A. Ciufolini, Org. Lett. 2012, 14, 4970.
[63] L. Banfi, A. Basso, C. Chiappe, F. De Moliner, R. Riva, L. Sonaglia, Org. Biomol. Chem. 2012, 10, 3819.
[64] V. Cerulli, L. Banfi, A. Basso, V. Rocca, R. Riva, Org. Biomol. Chem. 2012, 10, 1255.
[65] V. Köhler, K. R. Bailey, A. Znabet, J. Raftery, M. Helliwell, N. J. Turner, Angew. Chem. 2010, 122, 2228.
[66] A. Znabet, E. Ruijter, F. J. Kanter, V. Köhler, M. Helliwell, N. J. Turner, R. V. A. Orru, Angew. Chem. 2010, 122, 5417.
[67] A. Znabet, E. Ruijter, F. J Kanter, V. Köhler, M. Helliwell, N. J Turner, R. V. A. Orru, Angew. Chem. Int. Ed. 2010, 49, 5289.
[68] A. Znabet, P. M. Marloes, J. Elwin, F. J. Kanter, N. J. Turne, R. V. A. Orru, E. Ruijter, Chem. Commun. 2010, 46, 7918.
[69] A. Znabet, P. M. Marloes, E. Janssen, F. J. Kanter, N. J Turner, R. V. A. Orru, E. Ruijter, Chem. Commun. 2010, 46, 7706.
[70] J. Clayden, N. Greeves, S. Warren, P. Wothers in Organic Chemistry, Vol. 1, OUP Oxford, Oxford, 2001.
[71] Y. Li, M. Hesse, Helv. Chim. Acta. 2003, 86, 310.
[72] J. Ambler, L. Brown, X. Cockcroft, M. Grütter, Bioorg. Med. Chem. Lett. 1999, 9, 1317.
[73] C. Bright, T. J. Brown, P. Cox, F. Halley, P. Lockey, M. McLay, U. Moore, B. Porter, R. J. Williams, Bioorg. Med. Chem. Lett. 1998, 8, 771.
[74] J. M. Dener, V. R. Wang, K. D. Rice, A. R. Gangloff, K. Y. Elaine, N. S. William, D. Putnam, M. Wong, Bioorg. Med. Chem. Lett. 2001, 11, 2325.
[75] M. Mure, S. A. Mills, J. P. Klinman, Biochemistry. 2002, 41, 9269.
[76] R. J. Rohlfs, R. Hille, J. Biol. Chem. 1994, 269, 30869.
[77] J. M. Kim, M. A. Bogdan, P. S. Mariano, j. Am. Chem. Soc. 1993, 115, 10591.
[78] R. Carr, M. Alexeeva, M. J. Dawson, V. Gotor-Fernández, C. E. Humphrey, N. J. Turner, ChemBioChem. 2005, 6, 637.
`
71 | P a g e
[79] R. Carr, M. Alexeeva, A. Enright, T. S. Eve, M. J. Dawson, N. J. Turner, Angew. Chem. Int. Ed. 2003, 42, 4807.
[80] J. Dunsmore, R. Carr, T. Fleming, N. J. Turner, J. Am. Chem. Soc. 2006, 128, 2224.
[81] V. Köhler, K. R. Bailey, A. Znabet, J. Raftery, M. Helliwell, N. J. Turner, Angew. Chem. Int. Ed. 2010, 49, 2182.
[82] J. Fareed; W. P. Jeske, Baillieres Clin. Haematol. 2004, 17, 127.